Probing acid replacements of thiophene PTP1B inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.02.043

The following account describes our systematic effort to replace one of the carboxylate groups of our diacid thiophene PTP1B inhibitors. Active hits were validated using enzymatic assays before pursuing efforts to improve the potency. Only when the C2 car

Full text of DOI:10.1016/j.bmcl.2007.02.043

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2913–2920
Probing acid replacements of thiophene PTP1B inhibitors
Zhao-Kui Wan,a Bruce Follows,a,* Steve Kirincich,a Douglas Wilson,a Eva Binnun,a
Weixin Xu,a Diane Joseph-McCarthy,a Junjun Wu,a Michael Smith,a Yan-Ling Zhang,b
May Tam,a David Erbe,b Steve Tam,a Eddine Saiaha and Jinbo Leea
aChemical and Screening Sciences, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
bCardiovascular and Metabolic Diseases, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
Received 18 January 2007; revised 15 February 2007; accepted 16 February 2007
Available online 20 February 2007
Abstract—The following account describes our systematic effort to replace one of the carboxylate groups of our diacid thiophene
PTP1B inhibitors. Active hits were validated using enzymatic assays before pursuing efforts to improve the potency. Only when the
C2 carboxylic acid was replaced with another ionizable functional group was reversible and competitive inhibition retained. Use of a
tetrazole ring or 1,2,5-thiadiazolidine-3-one-1,1-dioxide as a carboxylate mimetic led to the discovery of two unique starting series
that showed improved permeability (PAMPA) and potency of the order of 300 nM. The SAR from these efforts underscores some of
the major challenges in developing small molecule inhibitors for PTP1B.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Protein tyrosine phosphatase 1B (PTP1B) plays a criti-
cal role in the signal transduction of both insulin and
leptin pathways.1–4 As a therapeutic target, PTP1B has
received considerable attention from the drug industry
as a potential treatment for diabetes mellitus.5 Two-
independent studies demonstrated that PTP1B deficient
mice show greater insulin sensitivity, prolonged insulin
receptor autophosphorylation, and maintained lower
glucose and insulin levels.6,7 More recently, anti-sense
oligonucleotides (ASOs) have been shown to effectively
block PTP1B activity in vivo to control glucose levels
and promote insulin sensitivity in ob/ob mice.8 ASOs
were also found to prolong the lives of Zucker rats with
diabetes further validating PTP1B as a potential thera-
peutic target.9
and sulfotyrosines.20 While potency has been achieved
for many of these phosphate bioisosteres, the cell perme-
ability has been compromised significantly in most
cases. Several groups have reported the acid replacement
and acid mimetics approaches in an effort to improve
the PK of their lead series. Replacing the carboxylate
group of O-carboxymethyl salicylic acid with a tetra-
zole21 led to improvements in membrane permeability.
Burke and coworkers investigated the use of monocarb-
oxy-phosphotyrosine mimetics leading to a series with
poor to moderate potency.22 Abbott has reported a
(2-hydroxyphenoxy) acetic acid based phosphotyrosyl
mimetic linked with a previously optimized ligand which
resulted in a Caco-2 cell permeable albeit less potent
PTP1B inhibitor.23 An extension of this work involved
the design of an isoxazole carboxylic acid with a single
negative charge. While this monoacid showed only mod-
erate potency (double digit micromolar), cell based
activity was detected as changes in the phosphorylation
levels in COS 7 cells.24 These examples illustrate the
challenges in developing a potent, drug-like PTP1B
inhibitor.
Designing phosphate mimetics with desirable drug-like
properties has been a significant challenge. Most of the
reported competitive, reversible PTP1B inhibitors have
polar, charged phosphate mimetics such as O-carb-
oxymethyl salicylic acids,10 2-(oxalylamino)-benzoic
acids,11 diaryloxamic acids,12 difluoro-b-ketophospho-
nates,13 fluoro O-malonyltyrosines,14 cinnamic acids,15
oxa-acetic acids,16 difluoromethylphosphonates,17–19
Our initial efforts combined high-throughput screening
(HTS) and structure based drug design to discover the
pyridylthiophene and benzothiophene series of PTP1B
inhibitors.25 Further medicinal chemistry effort on these
scaffolds culminated in the discovery of a thiophene
Keyword: Protein tyrosine phosphatase 1B (PTP1B).
*
Corresponding author. Tel.: +1 617 665 5653; fax: +1 617 665
5682; e-mail: bfollows@wyeth.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.043

2914
Z.-K. Wan et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2913–2920
series as potent PTP1B inhibitors.26 While our goal of
HO
HO
O
O
achieving high potency had been met, we anticipated
compromised permeability associated with the polar
head group and therefore embarked on a study to
replace one of the acid groups with an appropriate acid
mimetic Scheme 1.
O
O
Br
OH
O
OH
O
R1
S
S
X
1, X = C
2, X = N
Several key binding interactions between the diacid (3)
and PTP1B are evident from the co-crystal structure.26
The two acidic side chains, C2 and C3, extend into the
phosphate oxygen binding region of the catalytic site;
the C3 carboxylate oxygen and Arg221 form a critical
electrostatic interaction which is further strengthened
by several putative hydrogen bonds between the oxygen
atom of the carboxylate and the peptide backbone while
the C2 acid contacts Lys120 through a water mediated
electrostatic interaction.
R2 =
3
H
4
5
R1
NHR2
OR2
0.024
O
S
N
Ki (μM)
3.2 0.005
O
Scheme 1. Pyridylthiophene, benzothiophene, and thiophene series.
O
O
C3
OH
The benzylsulfonamide side chain, appended to C5, im-
proved the potency of our PTP1B inhibitors by roughly
650-fold. We hoped that this improved affinity would
compensate for the removal of an acid group. However,
in a preliminary experiment, removing the C3 carboxylate
group (4–6, Scheme 2) led to a complete loss in activity.
This finding exemplified the significance of the interac-
tion between the C3 carboxylate group of our scaffold
and the catalytic site of PTP1B.
Br
H
N
O
S
O
OH
C2
N
S
O
4, Ki = 5 nM
Br
O
We began our synthetic efforts by exploring a mimetic
for the C3 carboxylate group. In the second phase, we
focused on probing acid replacement groups for the
C2 carboxylate group.
H
N
O
S
OH
N
S
O
O
6, Ki = > 2500 μM
Scheme 2. Potency loss from cleaving C3 carboxylate group.
The C3 analogs were prepared from alkylation of
the hydroxyl group of methyl 4,5-dibromo-3-hydroxy-
thiophene-2-carboxylate 7 (Scheme 3). The strategy
of incorporating 1,2,5-thiadiazolidine-3-one-1,1-dioxide
R1
9, R1 = CHCH2
10, R1 = CN
Br
Br
O
R1
OH
OH
Br
b
11, R1 = CF3
OH
Br
Br
S
S
Br
O
a
O
O
Br
O
R1
S
Br
OMe
f, g
S
8
7
OMe
R2
O
12, R1 = -CH2OH
13, R1 = tetrazole
i. c, d or e
ii. b
14, R1 =
OH
OH
O
O
O
OR2
Br
O
Br
O
h, b
OH
O
O
Br
Br
S
S
OMe
15, R2 = tBu
17, R2 = NHR3
18, R2 = NHOH
19, R2 = OH
16, R2 = H
Scheme 3. Reagents and condition: (a) RCH2Br, K2CO3, DMF, 85–95%; (b) LiOH, THF–H2O; HCl, 98%; (c) DIBAL, THF, 30%; (d) NaN3, heat,
45%; (e) OsO4, NMO, THF–H2O, 15%; (f) tert-butylbromoacetate, K2CO3, DMF, 96%; (g) TFA, CH2Cl2, 98%; (h) RNH2, DMF, BOP, (i-Pr)2NEt,
90–99%.

Z.-K. Wan et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2913–2920
2915
R2
NH
R2
R2
NH
O
O
O
R1
R1
R1
NH2
R1
NH
b
a
c
R3
R3
O
O
O
O
(Cl)Br
S
(Cl)Br
S
S
S
O
O
O
O
22, R3 = H or NH2
20, R1 = Me, Br, Cl, H
23a-i, R3 = H or NHAlkyl
21, R2 = CH3 or CF3
O
Boc
HN
O
R1
O
NH2
e, f
NH
S
d
O
R3
O
g, i
R1
R1
N
S
N
O
O
R3
S
R3
S
S
25a-i
24a-i
26a-h, R3 = NHAlkyl
R3 = N(SO2NH2)Alkyl
or H
h, g
O
NH
S
O
N
Cl
26i
O
Cl
S
Scheme 4. General scheme for the synthesis of 1,2,5-thiadiazolidine-3-one-1,1-dioxide derivatives. Reagents and conditions: (a) Ac2O or TFAA,
K2CO3, DMF, 99%; (b) ArB(OH)2, Pd(PPh3)4, KF, THF, 67–92%; (c) ketone, NaCNBH3, HOAc, MeOH, 36–98%; (d) i—NaOH/EtOH; ii—HCl,
heat, 66–91%; (e) methyl bromoacetate, K2CO3, DMF, 72–98%; (f) chlorosulfonyl isocyanate, t-BuOH, DIPEA, DCM, 78–89%; (g) i—TFA, DCM;
ii—NaH, THF, 37–75% (two steps); (h) SO2Cl2, CHCl3, reflux, 76%; (i) THF–MeOH, 2 N NaOH, 60 ꢁC, 74%.
O
OR2
29a-e, R2 = Me
O
Br
Br
30a-e, R2 = H
O
S
NHR3
c, d
MeO
HO
HO
MeO
O
O
O
O
O
OH
O
g, h
O
Br
Br
i
a, b
e, f
Br
Br
O
O
Br
Br
Br
O
Br
O
O
O
TMSO OTMS
OTMS
O
O
S
S
Br
Br
O
S
S
S
R1
OH
OH
OH
27, R1 = OH
28, R1 = Cl
31
19
32
33
l
j, k, d
O
O
HO
OH
O
OMe
m, n, d
O
Br
O
O
Br
Br
O
Br
Br
Boc
Br
N
H
N
S
S
H
S
36
35
34
R3O
O
OH
Br
Br
OH
Br
Br
O
O
Br
o
d, p
k, d
OMe
OMe
Br
Br
S
S
R'
S
S
O
O
7
38
39, R3 = t-Bu
40, R3 = H
37
Scheme 5. Reagents and condition: (a) HCl, MeOH, 98%; (b) SOCl2, 99%; (c) amine, THF–H2O, 64%; (d) LiOH, THF–H2O; HCl, 78%; (e) SnCl2,
CH2Cl2;29 (f) HCl, dioxane, 71% (two steps); (g) isobutylene, H2SO4, CH2Cl2, 87%; (h) LiOH, THF–H2O, 64%; (i) TFA, CH2Cl2, 38%; (j) DMSO,
NaOH, heat; 60%; (k) tert-butylbromoacetate, K2CO3, DMF, 96%; (l) DPPF, t-BuOH, 60%; (m) Ac2O, Et3N, DCM, 60%; (n) TFA, CH2Cl2, 95%;
(o) allylbromide, K2CO3, DMF, 89%; (p) heat, xylenes, 94%.30

2916
Z.-K. Wan et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2913–2920
heterocycle into our scaffold was inspired by previously
reported work (Scheme 4).27 The acetate protecting
group was introduced in the first step. The tailpiece
was introduced through a cross coupling step with
3-aminophenyl boronic acid followed by a reductive
amination with a ketone (22–23). Under basic condi-
tions, the methyl ester was hydrolyzed, the resulting acid
was lost as carbon dioxide, and the acetate group was
cleaved to give amino thiophene 24. Interestingly, the
de-carboxylation step could not be performed on sub-
strate 20, presumably due to the donating effect of the
3-amino group. Amine 24 was alkylated with methyl-
bromoacetate and then treated with chlorosulfonyl iso-
cyanate in the presence of tert-butanol to afford 25a–i.
Subsequent treatment of 25a–i with TFA to cleave the
Boc group followed by treatment with NaH to induce
cyclization of the ring provided the desired analogs
26a–i.
gion of the enzyme active site, proved most sensitive, as
most of the C3 analogs had no activity (>2500 lM).
Efforts to modify the C2 position led, in many instances,
to complete losses in activity with the exception of three
analogs: allylthiophene 40, morpholine amide 30c, and
tetrazole 47 (165, 210, and 430 lM, respectively).28
Replacement of both acids with a thiazolidine heterocycle,
26a, revealed a more promising result with Ki =
18 lM. With the active C2 and C3 analogs in hand,
we directed our next effort toward validating these
inhibitors.
The active compounds were tested for enzyme reversibil-
ity (Rev) and enzyme concentration dependence ([E]).
In the first validation assay, PTP1B and the inhibitor
were incubated, diluted 10-fold, and then the enzymatic
activity was assessed. Typically, inhibition due to an
irreversible process displayed partial or reduced recov-
ery of enzymatic activity. In the second assay, the IC50
was measured at two separate concentrations of PTP1B,
10 and 100 nM. As expected, well-behaved inhibitors
showed virtually no change in activity at the two enzyme
concentrations, while inhibitors that operate through
aggregation, non-specific binding or through an impu-
rity,32 show enzyme concentration dependence. Use of
these assays provided a means of validating the hits
and eliminating undesired inhibitors. Analogs 47 and
26a showed reversible inhibition as well as no enzyme
concentration dependence. The morpholine analog 30c
and allyl thiophene 40 were irreversible and displayed
enzyme concentration dependence. Hydroxy ketone 33
initially looked promising, however, derivatives made
The preparation of the C2 analogs is shown in Schemes
5 and 6. The analogs in Scheme 5 were synthesized from
either the de-carboxylated intermediate 34 or from the
C3 methyl ester 27, an intermediate prepared by treat-
ment of the diacid 11 with dilute sulfuric acid in metha-
nol. Acidic esterification of the C3 acid 19 enabled
selective activation of the C2 acid in the preparation
of these C2 analogs. Scheme 6 shows the preparation
of heterocycles at C2.
A variety of C3 and C2 analogs were tested as potential
inhibitors of the PTP1B enzyme (Tables 1 and 2). The
C3 position, corresponding to the phosphate-binding re-
HO
O
HO
O
j
42, R3 = Me
Br
O
Br
O
43, R3 = Et
44, R3 = i-Pr
N
N
OH
Br
O
Br
S
S
N
41
NH2
R3
h,i
HO
R4
B(OH)2
O
O
R2O
O
O
O
O
N
R1
O
R1
g
R1
e, f
a-d
O
O
R4
R1
O
R4
N
O
NH
S
Br
N
S
S
Br
N
N
S
O
15
45, R2 = Me
49a-h
48a-h
R4 = NH2 or NHR5
R1 = Br, Me or H
g
46, R2 = H
HO
O
R
O
47
NH
N
N
Br
S
N
Scheme 6. Reagents and conditions: (a) TFA–CH2Cl2; (b) NH3, MeOH; (c) H2SO4, MeOH, 80% (three steps); (d) cyanuric chloride, DMF, 78%; (e)
Pd(PPh3)4, KF, DME–H2O, microwave heating; (f) NaBH(OAc)3, AcOH, aldehyde, 45–65% (two steps); (g) ZnBr2, NaN3, i-PrOH–H2O, reflux,
80%;31 (h) LiOH, THF–H2O, HCl; (i) NH2OH, EtOH, K2CO3, 80 ꢁC, 95% (two steps); (j) RCOCl, heat, 20–40%.

Z.-K. Wan et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2913–2920
2917
Table 1. C3 acid-mimetic analogs37
Table 2. C2 acid-mimetic analogs37
R1
HO
Br
O
R3
S
R2
R2
Br
O
Compound
R1
R3
Br
IC50 (lM)
R1
Br
R1
S
O
19
–CO2H
29a
O
Compound
IC50 (lM)
OH
N
15c
>2500
OH
O
O
9
–CO2H
–CO2H
Br
Br
>2500
>2500
H
N
10
30a
30b
30c
>2500
NH2
O
F
F
F
11
12
13
14
–CO2H
–CO2H
–CO2H
–CO2H
Br
Br
Br
Br
>2500
>2500
>2500
>2500
O
O
NH2
>2500
210a
O
OH
NH
O
S
N
N
N
O
O
O
N
H
N
OH
O
30d
30e
>2500
>2500
O
O
OH
OH
N
18
–CO2H
Br
Ph
>2500
29a
H
N
N
H
O
O
O
HN
N
26a
H
NH
O
N
S
OH
330b
O
33
34
36
40
42
a Ki values for validated hits: 19 = 18 lM, 26a = 18 lM.
H
>2500
>2500
165a
O
from this core were also irreversible and enzyme concen-
tration dependent.
N
H
With compounds 47 and 26a validated, we set out to im-
prove their potencies using some of the side chains dis-
covered in a previous effort.25,33 The results are shown
in Table 3. Substitution on the thiadiazolidine scaffold
resulted in modest improvements in potency where the
most active analog had a Ki = 1.7 lM (26f). Substitution
on the tetrazole scaffold had a greater impact on the po-
tency where meta-substituted cyclic compounds such as
49d and 49e showed 15- and 160-fold improvements,
respectively. The benzylsulfonyl amide 49g was the most
potent inhibitor with a Ki = 300 nM.
N
O
>2500
N
N
N
N
O
43
>2500
N
O
44
46
47
>2500
>2500
430b
Both tetrazole 49g and thiadiazolidine 26c have been
crystallized with PTP1B (Figs. 1 and 2). The WPD
loop is in the closed conformation similar to the mode
shared by many other PTP1B inhibitors reported in the
literature.10 The key interactions highlighted by the co-
crystal structure of the tetrazole acid with PTP1B are
maintained: the thiophene core is anchored to the A
site via electrostatic interactions between the C3 acid
and the Arg residue and the tetrazole ring interacts
with the Lys120. The key interactions of 26c indicate
that the sulfonyl oxygen atoms interact with Arg221
and Lys 120.
N
N
N
NH
N
a Compounds were found to have one or more of the following:
enzyme concentration dependence, irreversible inhibition, dynamic
light scattering.
b Ki values for validated hits: 33 = 210 lM, 47 = 270 lM.
c Side product from DIBAL reduction (Scheme 3, step c).

2918
Z.-K. Wan et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2913–2920
Table 3. SAR of C2 tetrazole and C3 thiazolidine
HO
O
O
NH
R1
R1
O
N
S
O
H
R2
R2
N
O
N
N
R3
S
S
N
Compound
R1
R2
Ki (lM)
Compound
R1
R2
R3
Ki (lM)
49a
49b
49c
Br
Me
H
H
H
H
20
26a
26b
26c
Br
Me
Cl
H
H
H
H
H
H
18
41
16
170
120
H
N
H
N
49d
49e
Br
Br
18
26d
26e
Me
Cl
H
H
12
14
H
N
H
N
1.6
O
NH2O2S
N
N
49f
Br
4.6
26f
Cl
H
H
1.7
15
H
N
H
N
49g
49h
Br
0.30
1.3
26g
Me
N
BnO2S
BnO2S
H
N
O
Me
26h
26i
Me
Cl
H
4.3
16
N
N
BnO2S
H
Cl
Table 4. PAMPA values for selected tetrazoles and TDDs; PAMPA = Pe · 10–6 s/cm; R1 = cyclic benzylsulfonamide shown in Table 3
HO
O
HO
O
O
O
O
O
NH
NH
O
Br
Cl
N
S O
O
N
S
O
Cl
R1O
R1HN
R1O
H
N
OH
N
N
S
S
S
S
26j
O
N
26i
51h
5
PAMPA (Pe · 10–6 cm/s)
0.00
0.22
0.19
3.54
Table 4 shows the PAMPA34,35 permeability data for
some of the best compounds selected from each series.
A direct comparison of these compounds has some lim-
itations, as other structural changes are present. Note
that 5 and 49h have oxygen atoms connecting the piper-
idine sulfonamide group to the phenyl ring groups and
the C4 position varies from methyl, to chloro to bromo.
Despite these differences, some interesting observations
between the series can be made. None of the diacids
had PAMPA values above 0.0, whereas a number of
tetrazoles showed moderate permeability and the thi-
adiazolidines, in general, showed improvements over
the tetrazoles. Thus, an overall permeability of the series
based on PAMPA data gives the following trend:
thiadiazolidine > tetrazole > diacid.
The SAR in this study underscores the challenge in
designing ligands for phophatase targets. The polar rec-
ognition elements required for the ligand to bind with
high affinity for the active site often have poor perme-
ability and consequently limited bioavailability. As
shown in the SAR tables of the C2 and C3 analogs,
replacement of either acid with a neutral functional
group or even groups designed to replace the electro-

Z.-K. Wan et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2913–2920
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Figure 2. X-ray co-crystal structure of sulfonyl urea 26c with PTP1B
(PDB code: 2NTA36).
static interactions of the acid functional group led to
dramatic losses in potency (Ki > 2500 lM). Several com-
pounds including the morpholine amide 30c and allyl
thiophene 40 initially looked promising, however,
detailed enzymology studies of each analog revealed
distinct characteristics consistent with non-classical
inhibition. Ultimately, potency was maintained when
the C2 or C3 groups in our scaffold were replaced with
an ionizable group, the tetrazole ring or the thiadiazoli-
dine ring. Both templates were shown to give improve-
ments in permeability based on PAMPA. Further
experiments with these two series will be reported in
due course.
19. Burke, T. R., Jr.; Kole, H.; Roller, P. Biophys. Res.
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Acknowledgments
21. Liljebris, C.; Larsen, S. D.; Ogg, D.; Palazuk, B. J.;
Bleasdale, J. E. J. Med. Chem. 2002, 45, 1785.
22. Gao, Y.; Wu, L.; Luo, J. H.; Guo, R.; Yang, D.; Zhang,
Z.-Y.; Burke, T. R., Jr. Bioorg. Med. Chem. Lett. 2000, 10,
923.
The authors thank Drs. Nelson Huang, Walt Massefski,
Ying Ge, Patrick Stenberg, and Ms. Ning Pan for pro-
viding analytical data.
23. Xin, Z.; Liu, G.; Abad-Zapatero, C.; Pei, Z.; Szczepan-
kiewicz, B. G.; Li, X.; Zhang, T.; Hutchins, C. W.;
Hajduk, P. J.; Ballaron, S. J.; Stashko, M. A.; Lubben, T.
H.; Trevillyan, J. M.; Jirousek, M. R. Bioorg. Med. Chem.
Lett. 2003, 13, 3947.
24. Zhao, H.; Liu, G.; Xin, Z.; Serby, M. D.; Pei, Z.;
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Hutchins, C. W.; Lubben, T. H.; Ballaron, S. J.; Haasch,
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37. The Enzymatic assay was carried out at room temperature
in 96-well plates in DMG buffer, pH 7.0. The reaction was
initiated by addition of the enzyme at a final concentration
of 10 or 100 nM for PTP1B. The initial rate of PTPase-
catalyzed hydrolysis of p-nitrophenol phosphate (pNPP)
was measured by following the absorbance change at
405nm. IC50 value was determined under fixed pNPP
concentration of 1 mM. All the assays were carried out in
duplicate or triplicate and the average results are pre-
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