5-Arylidene-2,4-thiazolidinediones as inhibitors of protein tyrosine phosphatases

Article abstract of DOI:10.1016/j.bmc.2007.05.027

4-(5-Arylidene-2,4-dioxothiazolidin-3-yl)methylbenzoic acids (2) were synthesized and evaluated in vitro as inhibitors of PTP1B and LMW-PTP, two protein tyrosine phosphatases (PTPs) which act as negative regulators of the metabolic and mitotic signalling

Full text of DOI:10.1016/j.bmc.2007.05.027

Bioorganic & Medicinal Chemistry 15 (2007) 5137–5149
5-Arylidene-2,4-thiazolidinediones as inhibitors of protein
tyrosine phosphatases
b
a
b
Rosanna Maccari,a, Paolo Paoli, Rosaria Ottana, Michela Jacomelli,
*
`
Rosella Ciurleo,a Giampaolo Manao,b Theodora Steindl,c Thierry Langer,c
Maria Gabriella Vigoritaa and Guido Camicib,
a
`
Dipartimento Farmaco-chimico, Universita di Messina, Vl. SS. Annunziata, 98168 Messina, Italy
Dipartimento di Scienze Biochimiche, Universita di Firenze, V.le Morgagni 50, 50134 Firenze, Italy
b
`
cInstitute of Pharmacy, Department of Pharmaceutical Chemistry, University of Innsbruck, Innrain 52, A-6020 Innsbruck, Austria
Received 17 November 2006; revised 4 May 2007; accepted 11 May 2007
Available online 17 May 2007
Abstract—4-(5-Arylidene-2,4-dioxothiazolidin-3-yl)methylbenzoic acids (2) were synthesized and evaluated in vitro as inhibitors of
PTP1B and LMW-PTP, two protein tyrosine phosphatases (PTPs) which act as negative regulators of the metabolic and mitotic
signalling of insulin. The synthesis of compounds 2 represents an example of utilizing phosphotyrosine-mimetics to identify effective
low molecular weight nonphosphorus inhibitors of PTPs. Several thiazolidinediones 2 exhibited PTP1B inhibitory activity in the low
micromolar range with moderate selectivity for human PTP1B and IF1 isoform of human LMW-PTP compared with other related
PTPs.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
is critical to glucose uptake and metabolism in insulin-
sensitive tissues.4,9–11
Reversible protein tyrosine phosphorylation regulates a
variety of cellular functions, including proliferation,
differentiation, migration, metabolism, gene transcrip-
tion, cell–cell communication, ion channel activity,
immune response and apoptosis/survival decisions. It is
controlled by the coordinated action of protein tyrosine
kinases (PTKs) and protein tyrosine phosphatases
(PTPs).1–6 Some members of the PTP family, which
comprises more than 100 enzymes, exhibit substrate
specificity. They have also been recognized to exert
positive and negative critical effects on specific signalling
pathways and thus play complex and crucial physiolog-
ical roles in most mammalian cells.1–4,6–8 As observed
with PTKs, the deregulation of PTP activity contributes
to the development of many human pathologies, includ-
ing cancer, diabetes, inflammation and neurodegenera-
tive diseases.1–4,9 Moreover, tyrosine phosphorylation
PTP1B is an intracellular PTP and is a key negative reg-
ulator of the insulin signalling pathway. It acts by
dephosphorylation of specific phosphotyrosine (pTyr)
residues on the insulin receptor and insulin receptor sub-
strate proteins.1–3,9–12 A high correlation between cellu-
lar levels of PTP1B and insulin resistance has been
found. In fact, PTP1B knockout mice displayed both
increased insulin sensitivity in liver and in skeletal mus-
cles and resistance to obesity, without abnormalities in
growth or fertility or increased incidence of cancer. This
indicates that PTP1B functions are tissue specific.9,13
Other PTPs, such as leukocyte antigen related PTP
(LAR-PTP), SHP-2, PTPa and low molecular weight
PTP (LMW-PTP), appear to be involved in the regula-
tion of insulin signalling. It is probable that they control
this pathway in cell types which are unaffected by
PTP1B.9 However, for some their physiological role
remains unclear.
Keywords: Protein tyrosine phosphatases; Diabetes mellitus; PTP1B
and LMW-PTP inhibitors; Molecular modelling.
*
Corresponding author.
rmaccari@pharma.unime.it
Fax:
+39
90
355613; e-mail:
One of these, LMW-PTP, possesses a phosphate binding
loop structurally identical to that of PTP1B and has
been recognized as a negative regulator of insulin-medi-
Center of Excellence for Scientific Research DENOTHE, Viale
Morgagni 44, 50134 Florence, Italy.
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.027

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R. Maccari et al. / Bioorg. Med. Chem. 15 (2007) 5137–5149
ated mitotic and metabolic signalling.14,15 It has also
been reported that LMW-PTP can interact with other
receptor tyrosine kinases, including EGFR,16
PDGFR,17 fibroblast growth factor receptor18 and Eph-
rin receptor.19
interactions of pTyr residue with the active site of the
enzyme onto different optimal templates.26g,26i,26j,32–37
Replacing the phosphate group of pTyr with bioisosteric
monoanionic groups, such as carboxylates, is considered
a valid method to obtain inhibitors with low polar-
ity.26c,30,31,34 Indeed, several carboxylic acids with in vitro
PTP1B inhibitory properties and good cellular activity or
oral bioavailability have been reported.26b,26d,30,31,36
Allosteric PTP1B inhibitors are also known.38
Other reports demonstrated that the overexpression of
PTP1B and LMW-PTP is sufficient to transform non-
transformed cells. Indeed, high levels of these enzymes
have been detected in some types of human
tumours.4,20–22 The overexpression of PTPa also pro-
duces cell transformation in rat embryonic fibroblasts.23
Tartaglia identified some PTPN11 mutations that are
associated with some forms of leukaemia,24 while Saha
found that PRL-3 PTP is clearly associated with colo-
rectal cancer metastasis.25 Thus, like PTKs, PTPs can
also play an oncogenic role.
We have been searching for new 2,4-thiazolidinediones
(2,4-TZDs) as potential agents for the treatment of
DM2 and its complications over the last few years.39
2,4-TZDs have excited considerable interest as antihy-
perglycaemic compounds40 and aldose reductase inhibi-
tors.41 Some of them (pioglitazone, rosiglitazone) are
insulin-sensitizing agents acting as peroxisome prolifera-
tor-activated receptor c (PPAR c) agonists,40d and have
recently been marketed for the treatment of DM2. In
addition some 2,4-TZDs have proved to be PTP1B
inhibitors.42
In light of the above, the inhibition of PTP1B, as well as
of LMW-PTP, can be considered an attractive approach
in the design of new therapeutic agents for the treatment
of type 2 diabetes (DM2) and obesity. These two related
pathologies are often associated with complex metabolic
disorders known as metabolic syndrome (MS). Given
the epidemic proportions of these diseases, it is crucial
to discover new drugs that are able to counteract both
insulin resistance and weight gain, as these are closely
connected with the onset and progression of both
DM2 and MS.1,4
In this paper, we report on the synthesis and biological
activity of a series of 4-(5-arylidene-2,4-dioxothiazoli-
din-3-yl)methylbenzoic acids (2a–j) which have been de-
signed as PTP1B and LMW-PTP inhibitors.
The starting point for our PTP1B inhibitor design was
to insert the p-methylbenzoic acid residue onto N-3 of
the thiazolidinedione scaffold. It has already been sug-
gested that benzoic acid could act as a nonphospho-
rus-containing pTyr-mimic.43 It is likely that its
carboxylic group mimics the interactions of the phos-
phate group of pTyr with Arg221 in the PTP1B catalytic
site while the benzene ring might interact with Phe182
and Tyr46.
At the same time, PTP1B and LMW-PTP are of interest
as novel targets in the search for new antitumoral drugs.
Indeed, there is a great deal of research in progress
exploring PTP1B and LMW-PTP inhibitors as potential
therapeutic agents.26,27 Over the last decade, numerous
different PTP1B inhibitors have been reported. How-
ever, none have passed clinical trials to date. This failure
in trials is generally due either to side effects or a low
rate of in vivo efficacy. These could be the consequence
of either scarce selectivity or limited cell permeability.4
The lipophilic 5-arylidene moiety of acids 2 containing
one or two aromatic rings was also inserted in an
attempt to improve the stability of the enzyme/inhibitor
complex. This may occur due to hydrophobic contacts
with the lipophilic residues which surround the active
pTyr-binding site.
The specific inhibition of a single enzyme of the PTP
family is often difficult to achieve because the active site
is highly conserved among such enzymes. However, in
the case of PTP1B, the identification of a second
noncatalytic arylphosphate binding site, which is not
present in all PTPs, has provided a further structural
basis for targeted drug design.11,28,29 On the other hand,
although cell permeable inhibitors of PTP1B have been
reported, 26d,26g,30,31 efforts to obtain selective inhibitors
of this phosphatase have often resulted in the synthesis
of peptidic or large highly charged molecules with high
in vitro potency but potentially low bioavailability.
Thus the discovery of pharmaceutically appropriate
PTP1B inhibitors is still a great challenge.
Compounds 2a–j were tested for their in vitro inhibitory
activity not only against human PTP1B but also the two
active isoforms of human LMW-PTP (IF1 and IF2) as
well as the S. cerevisiae LMW-PTP (Ltp1). Compounds
2b and 2c were also selected to be assayed against other
related PTPs (TC-PTP, LAR-PTP, PTPb, YopH).
Docking simulations into the PTP1B active site allowed
the results obtained to be rationalised.
2. Chemistry
The current thrust of research in this field is the identifica-
tion of low molecular weight, nonphosphorus and nonpep-
tide inhibitors thatarepotentially endowed with favourable
pharmacokinetics. A useful and commonly adopted strat-
egy in the design of PTP1B inhibitors is to insert nonhydro-
lysable pTyr-mimetic groups that are able to replicate the
The general method for the synthesis of (Z)-4-(5-arylid-
ene-2,4-dioxothiazolidin-3-yl) methylbenzoic acids (2) is
depicted in Scheme 1.
(Z)-5-Arylidene-2,4-thiazolidinediones 1 were obtained
by the condensation of commercially available

R. Maccari et al. / Bioorg. Med. Chem. 15 (2007) 5137–5149
5139
O
H
N
O
H
N
a
S
+
Ar CHO
O
S
O
Ar
1
O
O
Ar =
a
b
c
b
O
O
COOH
O
N
S
O
d
Ar
2 a-i
e
a = C5H11N, C2H5OH, Δ
b = 1) BrCH2C6H4COOH, K2CO3, acetone, Δ
2) HCl
f
OH
g
OMe
OH
h
i
OH
Scheme 1.
COOH
COOH
O
O
N
N
a
S
S
O
O
R
R
R = 4-OCH2C6H5 2c
R = 3-OCH2C6H5 2d
R = 4-OH 2h
R = 3-OH 2i
COOH
O
N
b
S
O
2h
a = HCl/AcOH, Δ
b = 1) BrCH2COOCH3, K2CO3, acetone, Δ
2) HCl/AcOH, Δ
HOOC
O
2j
Scheme 2.

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R. Maccari et al. / Bioorg. Med. Chem. 15 (2007) 5137–5149
Table 1. In vitro inhibitory activity of compounds 2a–j against human PTP1B, human LMW-PTP (IF1 and IF2 isoforms) and S. cerevisiae LMW-
PTP (Ltp1)a
O
COOH
N
S
O
Ar
H
Ar
Compound
IC50 (lM)
IF2
PTP1B
IF1
37.0 11.0
Ltp1
O
O
2a
2b
18.0 1.0
130.0 17.0
483.0 132.0
2.8 0.2
1.6 0.2
1.1 0.1
5.5 1.0
6.5 0.3
2.6 0.4
10.0 1.0
10.2 1.1
85.0 24.0
0.9 0.3
2.5 0.2
13.0 4.0
5.6 0.4
34.0 4.0
15.0 1.0
19.7 4.5
99.0 7.0
149.0 8.0
389 107
188.0 15.0
1395 220
>450
CH2O
CH2O
2c
2d
2e
2f
2g
2h
2i
7.1 1.4
18.4 1.0
138.0 37.0
92.0 5.0
26.6 1.5
85.0 8.0
122.0 37.0
12.0 1.0
9.1 1.6
HO
CH3O
30.0 2.0
85.7 21
219.0 50.0
HO
HO
112.4
9
O
2j
1642.0 75.0
HOOC
a IC50 values were determined by regression analyses and expressed as means SE of three replicates.
2,4-thiazolidinedione with suitable aromatic aldehydes
in refluxing ethanol in the presence of piperidine, as
already reported39a,b (Scheme 1).
48 h and crystallization from methanol the desired acid
2g was obtained as a single product.
4-[5-(4-Hydroxybenzylidene)-2,4-dioxothiazolidin-3-yl]
methylbenzoic acid (2h) and its 5-(3-hydroxybenzylidene)
substituted isomer 2i were preparedby the acidic hydrolysis
of 5-(4-benzyloxybenzylidene)- and 5-(3-benzyloxybenzy-
lidene) substituted analogues 2c and 2d, respectively
(Scheme 2). As for 2g, acids 2h and 2i were also obtained
by the reaction of 1h and 1i with 4-(bromomethyl)benzoic
acid (Scheme 1), but with slightly lower yields.
The reaction between 2,4-thiazolidinediones 1 and 4-
(bromomethyl)benzoic acid, using potassium carbonate
as base in refluxing acetone, followed by a workup in
acidic medium and recrystallization from methanol,
provided pure acids 2 (Scheme 1).
The synthesis of 4-[5-(4-hydroxy-3-methoxybenzylid-
ene)-2,4-dioxothiazolidin-3-yl] methylbenzoic acid (2g)
was carried out by adding 4-(bromomethyl)benzoic acid
in small amounts to a solution of 1g and potassium car-
bonate in acetone over a 2-h period in order to prevent
the nucleophilic attack of 4-(bromomethyl)benzoic acid
by the hydroxy group. After refluxing the mixture for
The O-alkylation of 2h with methyl bromoacetate in the
presence of potassium carbonate in refluxing acetone
followed by acidic hydrolysis provided 4-[5-(4-carboxy-
methoxybenzylidene)-2,4-dioxothiazolidin-3-yl]methyl-
benzoic acid (2j) (Scheme 2).

R. Maccari et al. / Bioorg. Med. Chem. 15 (2007) 5137–5149
5141
The structures of compounds 2 were assigned on the ba-
The results of the in vitro inhibition assays, reported in
Table 1, did in fact indicate that the inhibitory effects
and selectivity of acids 2 differed markedly depending
on the 5-arylidene moiety.
sis of their analytical and spectroscopic data (IR, 1H and
1
13C NMR). The H NMR spectra showed a singlet in
the range from 4.75 to 4.93 ppm attributable to N–
CH2 resonance, which was diagnostic. In 13C NMR
spectra, the carbon atom of the same methylene group
resonated at 44.0–44.9 ppm as a triplet. In addition, in
13C NMR spectra, besides two signals due to the reso-
nances of 2- and 4-carbonylic groups of the thiazolidin-
edione ring at 164.8–168.9 ppm, another singlet
attributable to the resonance of the carboxylic carbon
was present in the same range.
Acids 2b and 2c, bearing a phenoxy and a benzyloxy
group in the para position of the 5-benzylidene ring,
respectively, performed well as inhibitors of both hu-
man PTP1B and human LMW-PTP isoforms and
exhibited IC50 values in the low micromolar range. In
contrast, they were poor Ltp1 inhibitors (Table 1).
Compond 2b in particular was found to be fairly selec-
tive, since it was from 5- to 38-fold more effective
Analogously to the NMR spectra of parent compounds
against IF1 (IC50 = 0.9 lM) and PTP1B (IC50 =
2.8 lM) than against IF2 (IC50 = 13 lM) and Ltp1
(IC50 = 34 lM).
1
1, which were obtained only as Z isomers,39a the H
NMR spectra of acids 2 showed only one singlet attrib-
utable to the resonance of the 5-methylidene proton in
the range from 7.88 to 8.22 ppm. In 13C NMR spectra
a doublet at 131.0–134.2 ppm was due to the 5-methyli-
dene carbon, while thiazolidinedione C-5 resonated as a
singlet at 116.4–124.7 ppm.
The displacement of the 4-benzyloxy group to position 3
of the 5-benzylidene moiety yielded compound 2d and
this exhibited an appreciable PTP1B inhibitory effect,
similar to that of 2c, but with lower activity levels
against IF1, IF2 and Ltp1 (Table 1). In contrast, the dis-
placement of the 4-phenoxy group to meta position re-
sulted in a reduction of activity. In fact, acid 2a was
shown to be about 6-fold less active than its isomer 2b
towards PTP1B. The decrease in inhibitory effectiveness
was even more significant (from 10 to 40 times) against
human LMW-PTP isoenzymes (Table 1).
The structure of acids 2 was also confirmed by means of
their IR spectra. These showed a very broad band in the
region of 3450–2450 cmÀ1 attributable to the stretching
of carboxylic OH.
3. Results and discussion
The condensation of a further benzene ring to the
5-benzylidene group led to 5-naphthalen-1-ylmethylene
and 5-naphthalen-2-ylmethylene substituted derivatives
(compounds 2e and 2f). These were only fairly selective
micromolar inhibitors of PTP1B, and their inhibitory
effects against human LMW-PTP isoforms as well as
Ltp1 were moderate or poor (Table 1). In particular,
2e and 2f proved to be 18- to 25-fold and 2- to 23-fold
more selective for PTP1B, respectively (Table 1).
Compounds 2a–j were evaluated in vitro for their
inhibitory activity against recombinant human PTP1B
as well as the two active isoforms of human LMW-
PTP (IF1 and IF2) and S. cerevisiae LMW-PTP
(Ltp1). p-Nitrophenylphosphate (p-NPP) was used as a
substrate.
It is known that interactions between pTyr-flanking res-
idues and sub-pockets bordering the PTP1B catalytic
site contribute to high affinity substrate binding.1,44
Thus, it has been suggested that interactions with these
adjacent subsites could also enhance inhibitor affinity
and selectivity.1,29 We therefore decided to vary the
5-arylidene moiety in order to evaluate its influence on
the inhibitory properties of acids 2, whilst keeping the
2,4-TZD framework and the pTyr-mimetic group on
N-3 constant.
The removal of the additional aromatic ring and its
replacement with a hydrophilic group (compounds
2g–j) resulted in either a slight or a marked reduction
of the inhibitory effect depending on the substituents
on the 5-benzylidene ring. The removal of the benzyl
group converted the most potent PTP1B inhibitors
among the tested acids 2, that is, 5-(4-benzyloxybenzy-
lidene)- and 5-(3-benzyloxybenzylidene) substituted
acids 2c and 2d, into the hydroxybenzylidene substituted
analogues 2h and 2i, respectively. These were about 10-
fold less active against PTP1B and from 6 to 70 times
less effective towards the other PTPs tested. In fact, 2h
and 2i proved to be 3- to 19-fold and 8- to 137-fold more
selective for PTP1B, respectively (Table 1).
We first synthesized 4-(5-arylidene-2,4-dioxothiazoli-
din-3-yl)methylbenzoic acids with a 5-arylidene moiety
containing two aromatic rings (compounds 2a–f). It
was thought this might stabilize the complex with
the enzyme through hydrophobic interactions with
the active site and surrounding subpockets, as this
has often been observed in PTP1B/inhibitor com-
plexes.26c,31,34,36,43 Subsequently, we also prepared
analogues which had a 5-benzylidene moiety with
hydrophilic H-donor substituents (compounds 2g–j).
This was done because the enzyme/inhibitor complex
stability might be increased by means of further inter-
actions via hydrogen bonds due to the presence of
hydrophilic residues in both catalytic and vicinal bind-
ing sites of PTP1B.26c,31,36,44,45
The insertion of a methoxy group in the meta position of
the 5-(4-hydroxybenzylidene) moiety of 2h led to deriv-
ative 2g and provided a 4-fold gain in the PTP1B inhib-
itory effect. Compound 2g was also about 3 times more
active against both human LMW-PTP isoforms and two
fold less effective against Ltp1 than 2h (Table 1).
In contrast, the replacement of the 4-hydroxy group
with a carboxymethoxy one (compound 2j) resulted in

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R. Maccari et al. / Bioorg. Med. Chem. 15 (2007) 5137–5149
a significant reduction of activity against all screened
PTPs. In fact, acid 2j was the least effective PTP inhibi-
tor among the tested compounds 2.
The capability of compounds 2 to inhibit peptide
dephosphorylation by PTP1B was assessed by using a
peptide corresponding to the sequence 1142–1153 of
the insulin receptor. The IC50 values of the most active
PTP1B inhibitors 2b (23.0 1.3 lM), 2c (7.6
1.7 lM), and 2d (28.8 2.4 lM) were calculated
measuring the dephosphorylation rates at increasing
inhibitor concentrations, in the presence of 170 lM
phosphopeptide substrate (a concentration correspond-
ing to the Km value for PTP1B).
A kinetic study was performed in order to shed light on
the inhibitory mechanism of compounds 2. It revealed
that acids 2 behaved as competitive and reversible inhib-
itors of all the PTPs tested. This finding was to be
expected given that they all are nonhydrolysable pTyr-
mimetic compounds. Figure 1 shows the double recipro-
cal plots of the three most active PTP1B inhibitors (2b,
2c, 2d) out of acids 2.
The most effective inhibitors of both human PTP1B and
LMW-PTP out of the tested acids 2, compounds 2b and
2c, were further assayed against certain other related
PTPs (Table 2).
They proved to be equally effective as inhibitors of both
PTP1B and T-cell protein tyrosine phosphatase (TC-
PTP) (Table 2). These two enzymes are highly homolo-
gous (74% sequence identity) and selectivity for only one
of them is generally difficult to achieve.26k,29,37,45
In contrast, the inhibitory effects of 2b and 2c markedly
decreased (from 10 to 20 times) against LAR, a trans-
membrane receptor-like PTP. LAR has also been recog-
nized as a negative regulator of insulin signalling.
However, its deficiency leads to neuronal defects and im-
paired development of mammary glands in mice.9 Thus,
an inhibitor that is selective to PTP1B rather than LAR
would be desirable. Moreover, since LAR does not con-
tain the second arylphosphate binding site of PTP1B,
these inhibition data seem to suggest that the residues
lining this secondary pocket should play a role in the
binding mode of inhibitors 2. This hypothesis was sup-
ported by the significant inhibitory activity of 2b and
2c against YopH (Table 2). The latter PTP is a virulence
factor that is essential for the pathogenicity of Yersinia
genus and it possesses a second phosphate binding site,
just as found in PTP1B.46
We also observed different inhibitory effects of com-
pounds 2b and 2c towards the human LMW-PTP isoen-
zymes which differ only in the sequence 40–73.47 In
particular, the inhibitory activity of 2b towards IF1
was 14-fold higher than for IF2, while inhibitory activity
of 2c towards IF1 was only 2-fold higher than for IF2.
This behaviour could be due to the structural differences
Table 2. Comparison of the inhibitory activity of 2b and 2c against
protein phosphatasesa
PTPs
2b
2.8 0.2
2c
1.6 0.2
PTP1B
IF1
IF2
0.9 0.3
13.0 4.0
34.0 4.0
2.8 0.3
24.6 4.1
1.1 0.1
1.0 0.1
2.5 0.2
5.6 0.4
15.0 1.0
1.3 0.2
27.0 6.6
0.4 0.1
0.3 0.1
Ltp1
Figure 1. Lineweaver–Burk plots for the three best PTP1B inhibitors.
The inhibitor concentration was as follows: top panel, (d) no
inhibitor; (h) 1lM; (ꢀ) 2 lM; (j) 3 lM. Middle panel, (d) no
inhibitor; (n) 0.5 lM; (ꢀ) 0.75 lM; (h) 1 lM. Bottom panel, (d)
no inhibitor; (h) 1lM; (m) 2 lM; (ꢁ) 3 lM. All tests were performed
with the enzyme PTP1B. The symbol v in the ordinate axis indicates
the initial substrate hydrolysis rate expressed as arbitrary units.
TC-PTP
LAR-PTP
PTPb
YopH
a IC50 values were determined by regression analyses and expressed as
means SE of three replicates.

R. Maccari et al. / Bioorg. Med. Chem. 15 (2007) 5137–5149
5143
in the alternative sequences which are known to partic-
ipate in the formation of a loop situated very close to the
active site.11,29 In addition, greater differences are
observed when comparing the human isoenzymes with
the yeast Ltp1 that shares about 40% sequence identity
with the mammalian enzymes.48
PDB entry 1XBO31 was chosen as a starting point for
the docking process. Analysis of the cocrystallized
ligand (CAS 745079-21-4) guided this choice. 745079-
21-4 is similar to acids 2b and 2c in size and topology
(Fig. 2) and is also active at low micromolar doses.
˚
The resolution of this complex is 2.5 A. In LigandFit
the protein was prepared for docking (removal of ligand
and water molecules, addition of hydrogens) using the
cff1.02 force field. Compounds 2b, 2c and 2j were im-
ported as minimised structures from the software Cata-
lyst50 as sd files.
4. Molecular docking
Compounds 2b and 2c were selected for docking exper-
iments into the PTP1B active site. Compound 2j was
also included in this investigation in order to find a pos-
sible explanation for its lower inhibitory activity.
On completion of the flexible docking process, the
resulting conformations/poses of the ligands in the bind-
ing site of 1XBO were studied (Figs. 3–5). A dense net-
work of hydrogen bonds between the carboxylic group
of inhibitors and Arg221, Gly220, Ser216, Cys215,
Gly218, Ala 217 was observed as well as hydrophobic
interactions between the phenyl rings of the ligands
and surrounding lipophilic amino acid residues (e.g.,
with Phe182). A strong driving force for binding was
the charge-charge interaction between the carboxylate
group of the ligand and Arg221. This interaction pattern
is common in many known PTP1B/inhibitor
complexes.26d,28,29,31,33,34,43
The LigandFit module implemented in the Cerius2 soft-
ware package49 was used for the docking procedure.
Furthermore, the ligands reached into the secondary
noncatalytic binding pocket and therefore appeared
to be dual site inhibitors. In particular, 2b and 2c
fitted this secondary site very well because of their
larger 5-arylidene moieties and they were able to
perform favourable hydrophobic interactions with a
number of lipophilic amino acid side chains lining
the pocket: Ala27, Met258, Gly259 and Arg24 (side
chain centrepiece). An additional interaction via
hydrogen bond between Arg24 and the ether oxygen
connecting the terminal phenyl rings of compounds
2b and 2c was possible (Figs. 3 and 4). On the other
hand, compound 2j (for which docking positions with
the opposite carboxylic group facing Arg221 were
also found) achieved positions where hydrogen bonds
COOH
N
O
O
COOMe
CH2OH
HO
CAS 745079-21-4
Figure 2. Compound 2c (yellow) overlaid with bound ligand (red)
from 1XBO.
Figure 3. Compound 2b docked in the PTP1B active site.

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R. Maccari et al. / Bioorg. Med. Chem. 15 (2007) 5137–5149
Figure 4. Compound 2c docked in the PTP1B active site.
Figure 5. Compound 2j docked in the PTP1B active site.
and charge–charge interactions between the second
carboxylic group and Arg24, Arg254 were possible
(Fig. 5). However, 2j was not able to fill the
secondary binding site as well as 2b and 2c, due to
the smaller size of its 5-arylidene moiety. At the same
time, the removal of the second aromatic ring
brought about a significant decrease in lipophilicity
and in capability to perform additional favourable
hydrophobic interactions. This might explain the low-
er activity of this compound.
Compounds 2b–g were shown to possess PTP1B inhibi-
tory activity in the low micromolar range. Out of these,
acids 2b and 2c were the most effective inhibitors of both
human PTP1B and IF1 isoform of human LMW-PTP,
showing from 3- to 38-fold selectivity for PTP1B/IF1
over IF2, Ltp1 and LAR-PTP. In contrast, their inhibi-
tion levels against PTP1B, TC-PTP, YopH and PTPb
were very similar.
All tested acids 2 proved to inhibit preferentially PTP1B
and IF1 isoform rather than IF2 and Ltp1.
5. Conclusions
The PTP inhibition data here reported confirmed the ini-
tial hypothesis that p-methylbenzoic acid residue
inserted onto N-3 of a 5-arylidene-2,4-thiazolidinedione
scaffold can act as a monoanionic pTyr-mimetic group.
The docking simulations into the PTP1B active site indi-
cated that this moiety can replicate the interactions of
pTyr with the catalytic site of the enzyme.
The design and synthesis of 4-(5-arylidene-2,4-dioxo-
thiazolidin-3-yl)methylbenzoic acids 2 are an example
of utilizing pTyr-mimetics to identify effective low
molecular weight nonphosphorus monoanionic PTP
inhibitors.

R. Maccari et al. / Bioorg. Med. Chem. 15 (2007) 5137–5149
5145
In line with the in vitro inhibition and selectivity results,
the computational study highlighted that the 5-arylidene
moiety of compounds 2b and 2c could further interact
with aminoacidic residues lining the PTP1B secondary
noncatalytic binding pocket. In contrast, 5-(4-carboxy-
methoxybenzylidene) substituted analogue 2j was un-
able to fit this site successfully.
in vacuo, the crude solid was washed with H2O and
recrystallized from methanol providing pure acid 2.
6.3. 4-[2,4-Dioxo-5-(3-phenoxybenzylidene)thiazolidin-3-
yl]methylbenzoic acid (2a)
1
Yield 54%; mp 207 °C; H NMR (DMSO-d6): d 4.90
(s, 2H, CH2);7.11(m, 2H, arom); 7.15–7.26(m, 3H,arom);
7.42–7.49 (m, 5H, arom); 7.60 (m, 1H, arom); 7.93 (m, 2H,
arom); 7.98 (s, 1H, CH); 13C NMR (DMSO-d6): d 44.4
(CH2); 119.2 (5-C); 118.9, 120.3, 122.0, 124.1, 124.8,
127.5, 129.6, 130.2, 131.0 (CH arom); 132.8 (CH methyli-
dene); 134.7, 140.1, 155.7, 157.5 (Cq arom); 165.2, 166.8,
167.0 (CO); Anal. (C24H17NO5S) C, H, N.
The investigation of structure/activity relationships rele-
vant to this new class of PTP inhibitors has so far been
limited to a small library of analogues. However, it
emerged that the 5-arylidene moiety markedly influ-
enced their potency and selectivity. A larger lipophilic
arylidene moiety containing two aromatic rings
appeared to be more favourable than a smaller one
composed of a benzylidene ring with hydrophilic
substituents.
6.4. 4-[2,4-Dioxo-5-(4-phenoxybenzylidene)thiazolidin-3-
yl]methylbenzoic acid (2b)
1
In conclusion, compounds 2b and 2c, which proved to
be effective micromolar in vitro inhibitors of both
human PTP1B and LMW-PTP, can be used as good
low molecular weight, nonpeptide and nonphosphorus
starting points for a drug discovery programme aimed
at enhancing their potencies and selectivity.
Yield 50%; mp 252 °C; H NMR (DMSO-d6): d 4.91 (s,
2H, CH2); 7.11–7.15 (m, 4H, arom); 7.23 (m, 1H, arom);
7.41–7.49 (m, 4H, arom); 7.67 (m, 2H, arom); 7.92 (m,
2H, arom); 7.97 (s, 1H, CH); 13C NMR (DMSO-d6): d
44.4 (CH2); 119.3 (5-C); 118.2, 119.8, 124.6, 127.5,
129.6, 130.2, 132.4 (CH arom); 133.0 (CH methylidene);
130.1, 140.2, 155.0, 159.1 (Cq arom); 165.5, 166.9, 167.3
(CO); Anal. (C24H17NO5S) C, H, N.
6. Experimental
6.1. Chemistry
6.5. 4-[5-(4-Benzyloxybenzylidene)-2,4-dioxothiazolidin-
3-yl]methylbenzoic acid (2c)
1
Melting points were recorded on a Kofler hot-stage
apparatus and are uncorrected. TLC controls were
carried out on precoated silica gel plates (F 254 Merck).
Elemental analyses (C, H, N), determined by means of a
C. Erba mod. 1106 elem. Analyzer, were within 0.4%
of theory. IR spectra were obtained with a Perkin-Elmer
683 spectrophotometer as Nujol or esachlorobutadiene
Yield 90%; mp 250 °C (dec); H NMR (DMSO-d6): d
4.92 (s, 2H, NCH2); 5.21 (s, 2H, OCH2); 7.21 (m, 2H,
arom); 7.38–7.50 (m, 7H, arom); 7.62 (m, 2H, arom);
7.93 (m, 2H, arom); 7.95 (s, 1H, CH); 13C NMR
(DMSO-d6): d 44.3 (NCH2); 69.5 (OCH2); 115.7,
127.5, 127.8, 128.0, 128.5, 129.6, 132.3 (CH arom);
117.8 (5-C); 133.5 (CH methylidene); 125.5, 130.2,
136.4, 140.3, 160.3 (Cq arom); 165.6, 166.9, 167.4
(CO); Anal. (C25H19NO5S) C, H, N.
1
mulls. H and 13C NMR spectra were recorded on a
Varian 300 magnetic resonance spectrometer (300 MHz
1
for H and 75 MHz for 13C). Chemical shifts are given
in d units (ppm) relative to internal standard Me4Si
and refer to DMSO-d6 solutions. Coupling constants
(J) are given in hertz (Hz). 13C NMR spectra were deter-
mined by Attached Proton Test (APT) experiments and
the resonances were always attributed by proton–carbon
heteronuclear chemical shift correlation.
6.6. 4-[5-(3-Benzyloxybenzylidene)-2,4-dioxothiazolidin-
3-yl]methylbenzoic acid (2d)
1
Yield 30%; mp 222 °C (dec); H NMR (DMSO-d6): d
4.84 (s, 2H, NCH2); 5.17 (s, 2H, OCH2); 7.15–7.25
(m, 5H, arom); 7.36–7.51 (m, 6H, arom); 7.89 (m, 2H,
arom); 7.96 (s, 1H, CH); 13C NMR (DMSO-d6): d
44.0 (NCH2); 68.9 (OCH2); 115.6, 121.0, 121.8, 127.1,
127.3, 127.8, 128.8, 132.7, 135.7 (CH arom); 117.0
(5-C); 133.7 (CH methylidene); 126.0, 129.9, 136.1,
138.1, 158.1 (Cq arom); 164.8, 166.6, 168.9 (CO); Anal.
(C25H19NO5S) C, H, N.
Unless stated otherwise, all materials were obtained
from commercial suppliers and used without further
purification.
6.2. General method for the synthesis of 4-(5-arylidene-
2,4-dioxothiazolidin-3-yl) methylbenzoic acids 2a–g
6.7. 4-(5-Naphthalen-1ylmethylene-2,4-dioxothiazolidin-
3-yl)methylbenzoic acid (2e)
A
mixture of 5-arylidene-2,4-thiazolidinedione
1
(10 mmol), 4-(bromomethyl)benzoic acid (4.30 g,
20 mmol) and potassium carbonate (5.53 g, 40 mmol)
in acetone (150 ml) was refluxed for 48 h. The solvent
was evaporated under reduced pressure and the solid
residue was dissolved in methanol; the solution was
acidified (pH 3) with HCl and was stirred at room
temperature for 30 min. After evaporation to dryness
1
Yield 60%; mp 268 °C; H NMR (DMSO-d6): d 4.93
(s, 2H, CH2); 7.45 (m, 2H, arom); 7.57–7.73 (m, 4H,
arom); 7.92 (m, 2H, arom); 8.02–8.14 (m, 3H, arom);
8.59 (s, 1H, CH); 13C NMR (DMSO-d6): d 44.3 (CH2);
123.4, 125.5, 126.4, 126.8, 127.4, 127.6, 128.8, 129.6,
130.5 (CH arom); 124.7 (5-C); 131.0 (CH methylidene);

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R. Maccari et al. / Bioorg. Med. Chem. 15 (2007) 5137–5149
130.1, 130.2, 130.8, 133.2, 140.1 (Cq arom); 166.9, 167.1,
167.6 (CO); Anal. (C22H15NO4S) C, H, N.
s, 1H, OH); 13C NMR (DMSO-d6): d 44.6 (NCH2);
115.8, 117.9, 121.2, 127.3, 129.6, 130.5 (CH arom);
120.8 (5-C); 133.7 (CH methylidene); 130.4, 133.8,
140.4, 157.9 (Cq arom); 165.5, 166.9, 167.4 (CO); Anal.
(C18H13NO5S) C, H, N.
6.8. 4-(5-Naphthalen-2ylmethylene-2,4-dioxothiazolidin-
3-yl)methylbenzoic acid (2f)
Yield 72%; mp 288–290 °C (dec); 1H NMR (DMSO-d6):
d 4.92 (s, 2H, CH2); 7.44 (m, 2H, arom); 7.59–7.76 (m,
3H, arom); 7.92–8.09 (m, 4H, arom); 8.14 (m, 2H,
arom); 8.26 (s, 1H, CH); 13C NMR (DMSO-d6): d
44.5 (CH2); 121.3 (5-C); 126.2, 127.2, 127.6, 127.7,
128.6, 128.7, 129.7, 130.1, 133.6 (CH arom); 131.3
(CH methylidene); 130.5, 131.6, 132.8, 132.9, 140.3
(Cq arom); 165.6, 167.0, 167.5 (CO); Anal.
(C22H15NO4S) C, H, N.
6.12. 4-[5-(4-Carboxymethoxybenzylidene)-2,4-dioxo-
thiazolidin-3-yl]methylbenzoic acid (2j)
A mixture of 4-[5-(4-hydroxybenzylidene)-2,4-dioxo-
(2h) (3.55 g,
thiazolidin-3-yl]methylbenzoic acid
10 mmol), methyl bromoacetate (3.06 g, 20 mmol) and
potassium carbonate (2.76 g, 20 mmol) in acetone
(120 ml) was refluxed for 24 h. After cooling, the inor-
ganic salts were filtered off; then the solvent was evapo-
rated under reduced pressure. A mixture of the solid
residue (3.93 g), glacial AcOH (120 ml) and HCl 12 N
(30 ml) was refluxed for 2 h. After evaporation in vacuo,
the residue was refluxed again with AcOH (120 ml) and
HCl (30 ml) for 2 h. After evaporation to dryness in va-
cuo, the crude solid was washed with H2O and recrystal-
lized from ethanol providing pure acid 2j. Yield 84%;
mp 280 °C; 1H NMR (DMSO-d6): d 4.75 (s, 2H,
NCH2); 4.90 (s, 2H, OCH2); 7.09 (m, 2H, arom); 7.42
(m, 2H, arom); 7.61 (m, 2H, arom); 7.91 (m, 2H, arom);
7.94 (s, 1H, CH); 13C NMR (DMSO-d6): d 44.9 (NCH2);
65.2 (OCH2); 116.1, 128.1, 130.2, 132.8 (CH arom);
118.7 (5-C); 134.0 (CH methylidene); 126.4, 130.8,
140.9, 160.3 (Cq arom); 166.1, 167.5, 167.8 (CO); Anal.
(C20H15NO7S) C, H, N.
6.9. 4-[5-(4-Hydroxy-3-methoxybenzylidene)-2,4-dioxo-
thiazolidin-3-yl]methylbenzoic acid (2g)
This was synthesized as above described, adding 4-(bro-
momethyl)benzoic acid in small amounts over 2 h to a
solution of compound 1g and potassium carbonate in
1
acetone. Yield 34%; mp 242 °C; H NMR (DMSO-d6):
d 3.80 (s, 3H, OCH3); 4.88 (s, 2H, CH2); 6.92 (d
J = 8.4, 1H, arom); 7.11 (dd J = 8.4 and 1.8, 1H, arom);
7.20 (d J = 1.8, 1H, arom); 7.39 (m, 2H, arom); 7.88 (s,
1H, CH); 7.90 (m, 2H, arom); 10.04 (br s, 1H, OH); 13C
NMR (DMSO-d6): d 44.3 (NCH2); 55.7 (OCH3); 113.7,
124.3, 127.6, 129.3, 130.3 (CH arom); 116.6 (5-C); 134.2
(CH methylidene); 126.1, 140.4, 141.6, 148.1, 149.9 (Cq
arom); 165.6, 167.0, 167.5 (CO); Anal. (C19H15NO6S) C,
H, N.
7. Molecular modelling
6.10. 4-[5-(4-Hydroxybenzylidene)-2,4-dioxothiazolidin-
3-yl]methylbenzoic acid (2h)
Docking calculations were performed on an SGI Octane
double processor R10K workstation using the Ligand-
Fit module in the Cerius2 software package version
4.1049 and the cff1.02 forcefield. For ligand minimisation
the software tool Catalyst version 4.1050 was used. Site
definition resulted from the cocrystallized ligand in the
PDB complex 1XBO.31 Flexible docking runs were car-
ried out without interaction filters and all scoring func-
tions implemented in Cerius2 served for ranking
analyses. For site definition, docking, and scoring calcu-
lations, default parameter settings were kept, if not men-
tioned otherwise in the text. In the docking preferences a
fixed number of Monte Carlo trials of 5000, a maximum
number of 20 saved poses, and 500 iterations in the final
rigid body minimisation were required, the force field
selection box in the energy preferences was set to CFF.
A mixture of 4-[5-(4-benzyloxybenzylidene)-2,4-dioxo-
thiazolidin-3-yl]methylbenzoic
acid
(2c)
(4.45 g,
10 mmol), glacial AcOH (120 ml) and HCl 12 N
(30 ml) was refluxed for 2 h. After evaporation in vacuo,
the residue was refluxed again with AcOH (120 ml) and
HCl (30 ml) for 2 h. After evaporation to dryness in
vacuo, the crude solid was washed with H2O and recrys-
tallized from ethanol providing pure acid 2h. Yield 85%;
1
mp 290 °C; H NMR (DMSO-d6): d 4.89 (s, 2H, CH2);
6.92 (m, 2H, arom); 7.40 (m, 2H, arom); 7.52 (m, 2H,
arom); 7.88 (s, 1H, CH); 7.92 (m, 2H, arom); 10.40 (br
s, 1H, OH); 13C NMR (DMSO-d6): d 44.2 (NCH2);
116.4 (5-C); 116.5, 127.5, 129.7, 132.7 (CH arom);
134.1 (CH methylidene); 123.8, 130.3, 140.3, 160.3 (Cq
arom); 165.6, 166.9, 167.5 (CO); Anal. (C18H13NO5S)
C, H, N.
8. Enzyme section
6.11. 4-[5-(3-Hydroxybenzylidene)-2,4-dioxothiazolidin-
3-yl]methylbenzoic acid (2i)
The complete coding sequences of IF1, IF2, Ltp1
LMW-PTPs and PTP1B were cloned in frame with the
sequence of the glutathione S-transferase (GST) in the
pGEX-2T bacterial expression vector. Enzyme expres-
sion and purification were achieved in the E. coli TB1
strain.51 Briefly, the recombinant fusion proteins were
purified from bacterial lysate using a single-step affinity
chromatography. The solution containing purified
fusion proteins was treated with thrombin for 3 h at
The procedure was the same as reported for compound
2h, starting from 4-[5-(3-benzyloxybenzylidene)-2,4-
dioxothiazolidin-3-yl]methylbenzoic acid (2d). Yield
1
38%; mp 260 °C (dec); H NMR (DMSO-d6): d 4.86
(s, 2H, CH2); 6.90 (m, 1H, arom); 7.01 (s, 1H, arom);
7.08 (m, 1H, arom); 7.34 (m, 1H, arom); 7.36 (m, 2H,
arom); 7.87 (s, 1H, CH); 7.88 (m, 2H, arom); 9.89 (br

R. Maccari et al. / Bioorg. Med. Chem. 15 (2007) 5137–5149
5147
37°C. Then the enzymes were purified from GST and
thrombin by gel filtration. The purity of proteins’
preparations was analysed by SDS–polyacrylamide gel
electrophoresis according to Laemmli.52 TC-PTP, LAR-
PTP, PTPb and YopH were purchased from SIGMA.
amount of inorganic phosphate released from the pep-
tide was determined reading the absorbance at 595 nm.
Control assays without PTP1B were also performed.
The Km and Vmax of PTP1B for the phosphopeptide
were determined measuring the rate of inorganic phos-
phate release at increasing substrate concentration rang-
ing from 10 to 400 lM.
9. Enzymatic assay and inhibition experiments
Acknowledgments
The enzymatic assay was carried out at 37 °C using
p-nitrophenylphosphate as substrate; the final volume
was 1 ml. The assay buffer (pH 7.0) contained 0.075 M
of b,b-dimethylglutarate buffer, 1 mM EDTA and
5 mM dithiothreitol. The reactions were initiated by
addition of aliquots of the enzyme preparations and
stopped at appropriate times by adding 4 ml of 1 M
KOH. The released p-nitrophenol was determined by
reading the absorbance at 400 nm (e = 18,000 MÀ1
cmÀ1). The main kinetic parameters (Km and Vmax) were
determined by measuring the initial rates at different sub-
strate concentrations. Experimental data were analysed
using the Michaelis equation and a nonlinear fitting pro-
gram (FigSys).53
This work was supported in part by FIRB 2001 Grant to
G. Camici and by ‘‘Ente Cassa di Risparmio di
Firenze’’.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2007.05.027.
References and notes
Inhibition constants were determined measuring initial
hydrolysis rates at differing substrate and inhibitor concen-
trations. The apparent Km values measured at the various
inhibitor concentrations were plotted against concentra-
tion of the inhibitor to calculate the Ki values. All initial rate
measurements were carried out in triplicate. For each inhib-
itor, IC50 was determined by measuring the initial hydroly-
sis rate under fixed p-nitrophenylphosphate concentration,
equal to the Km value of the considered PTP. Data were
fitted to the following equation using the FigSys program:
Vi/V0 = IC50/(IC50 + [I]), where Vi is the reaction velocity
when the inhibitor concentration is [I], V0 is the reaction
velocity with no inhibitor and IC50 = Ki + Ki[S]/Km.
Therefore, when the substrate concentration [S] is equal
to Km, IC50 = 2Ki.
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