5-Arylidene-2-phenylimino-4-thiazolidinones as PTP1B and LMW-PTP inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.01.044

As part of a project aimed at identifying effective low molecular weight nonphosphorus monoanionic inhibitors of PTPs, we have synthesized 4-[(5-arylidene-4-oxo-2-phenyliminothiazolidin-3-yl)methyl]benzoic acids (4) and evaluated their inhibitory activity

Full text of DOI:10.1016/j.bmc.2009.01.044

Bioorganic & Medicinal Chemistry 17 (2009) 1928–1937
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
5-Arylidene-2-phenylimino-4-thiazolidinones as PTP1B
and LMW-PTP inhibitors
a
a
b
b
Rosaria Ottanà a, , Rosanna Maccari , Rosella Ciurleo , Paolo Paoli , Michela Jacomelli ,
*
Giampaolo Manao b, Guido Camici b, , Christian Laggner c, Thierry Langer d
a Dipartimento Farmaco-chimico, University of Messina, Polo Universitario dell’Annunziata, Vl. SS. Annunziata, 98168 Messina, Italy
b Dipartimento di Scienze Biochimiche, University of Firenze, Vl. Morgagni 50, 50134 Firenze, Italy
c Department of Pharmaceutical Chemistry, Institute of Pharmacy and Center for Molecular Biosciences (CMBI), University of Innsbruck, Innrain 52c, A-6020 Innsbruck, Austria
d Prestwick Chemical Inc., Bld. Gonthier d’Andernach, 67400 Strasbourg-Illkirch, France
a r t i c l e i n f o
a b s t r a c t
Article history:
As part of a project aimed at identifying effective low molecular weight nonphosphorus monoanionic
inhibitors of PTPs, we have synthesized 4-[(5-arylidene-4-oxo-2-phenyliminothiazolidin-3-yl)methyl]-
benzoic acids (4) and evaluated their inhibitory activity against human PTP1B and LMW-PTP enzymes.
The introduction of a 2-phenylimino moiety onto the 4-thiazolidinone ring was designed to enhance
the inhibitor/enzyme affinity by means of further favourable interactions with residues of the active site
and the surrounding loops. Some of the compounds (4a–d, f) showed interesting inhibition levels in the
low micromolar range. The 5-arylidene moiety of acids 4 proved to markedly influence the potency of
these inhibitors. Molecular modeling experiments inside the binding sites of both enzymes were
performed.
Received 25 November 2008
Revised 14 January 2009
Accepted 20 January 2009
Available online 25 January 2009
Keywords:
Protein tyrosine phosphatases
PTP1B
LMW-PTP
Ó 2009 Elsevier Ltd. All rights reserved.
5-Arylidene-2-phenylimino-4-
thiazolidinones
Inhibitors
1. Introduction
PTP1B is the prototypical member of the PTP superfamily. It is
an intracellular enzyme that acts as a key negative regulator of
Protein tyrosine phosphorylation is the predominant post-
translational modification that cells utilize to regulate signal trans-
duction and to control a wide variety of cellular functions, such as
growth, differentiation, survival, apoptosis, metabolism and gene
transcription. In vivo it is a reversible and dynamic process which
is governed by the coordinated activities of protein tyrosine ki-
nases (PTKs), which catalyse protein phosphorylation, and protein
tyrosine phosphatases (PTPs), which are responsible for dephos-
phorylation.1–4
both insulin and leptin signalling pathways. This is achieved by
catalysing the dephosphorylation of specific phosphotyrosine
(pTyr) residues on insulin receptor, insulin receptor substrate pro-
teins and on Janus kinase 2, a protein tyrosine kinase which is asso-
ciated with leptin receptors.7–10
Inhibition of PTP1B results in both increased insulin sensitivity
and resistance to obesity, without producing abnormalities in
growth or fertility or other pathogenetic effects in mice.11,12 This
makes PTP1B an exemplary target for obesity and type 2 diabetes
(DM2).7,9,10,13 These two related metabolic pathologies can also
be part of the so-called ‘metabolic syndrome’ which currently rep-
resents a serious health threat.14 Increasing concern about the
widespread diffusion of these diseases has led to intensive efforts
to develop new drugs that are able to counteract insulin resistance,
which is the biochemical defect underlying both DM2 and meta-
bolic syndrome.14 Indeed, insulin resistance is not sufficiently tar-
geted by the current therapies. Therefore, inhibition of PTP1B is
predicted to provide a novel strategy to fill this therapeutic gap.
There is evidence that PTP1B can also play an oncogenic role. It
is overexpressed in certain types of human cancer, such as breast
tumours, ovarian and epithelial carcinomas, and can act as a
positive regulator of tumour onset and progression. PTP1B has
been shown to play an important role as a positive regulator of
The PTP superfamily comprises more than 100 enzymes.1–5
PTPs can exert both positive and negative critical effects on various
signalling pathways, thus playing complex and important roles in
many pathophysiological processes. The deregulation of their
activity leads to aberrant tyrosine phosphorylation and is linked
to the development of many human pathologies, such as diabetes,
obesity, cancer, inflammation and neurodegenerative diseases.1,3,6
Thus, there is a great interest in PTPs as molecular targets for the
development of novel therapeutic agents.
* Corresponding author. Fax: +39 90 6766402.
E-mail address: ottana@pharma.unime.it (R. Ottanà).
Address: Centro di Ricerca, Trasferimento ed Alta Formazione DENOThe, University
of Firenze, Italy.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.044

R. Ottanà et al. / Bioorg. Med. Chem. 17 (2009) 1928–1937
1929
COOH
O
N
S
O
Ar
1
COOH
O
COOH
O
N
N
S
O
S
O
O
O
1a
1b
Figure 1. Structures of previously reported inhibitors.32
the Erb2-PTK, which is overexpressed in several human breast
cancers that are difficult to treat. PTP1B deficiency or inhibition
delays Erb2-induced tumourogenesis and protects from lung
metastasis.3,10,15,16
interactions of pTyr residue with the catalytic sites of both PTP1B
and LMW-PTP.32
The 5-arylidene moiety of acids 1, which was inserted in order
to improve the stability of the inhibitor/enzyme complex through
contacts with residues bordering the active site, proved to mark-
edly influence the potency and selectivity of these inhibitors. In
particular, inhibitory effectiveness, especially towards PTP1B and
IF1, was shown to be enhanced by a larger lipophilic 5-arylidene
moiety containing two aromatic rings rather than by a smaller
one composed of a hydroxysubstituted benzylidene ring.32
Compounds 1a and 1b (Fig. 1) were found to be the most effec-
tive inhibitors of both human PTP1B and IF1. Moreover, molecular
docking experiments into the PTP1B active site indicated that the
4-phenoxybenzylidene and 4-benzyloxybenzylidene moieties of
compounds 1a and 1b fitted a second noncatalytic arylphosphate
binding site very well by establishing favourable hydrophobic
interactions and hydrogen bonds. This secondary pocket is not con-
served among all PTPs and thus provides a structural basis for
selective PTP1B inhibition.10,33,34 These findings were in agreement
with our in vitro inhibition results. In particular 1a and 1b exhib-
ited good activity towards PTP1B as well as towards other related
PTPs containing this pocket (such as TC-PTP, YopH, PTPb), whereas
their inhibitory effect was lower (10–20-fold) against LAR-PTP, a
transmembrane receptor-like PTP which does not contain the sec-
ond arylphosphate binding site.32
Low molecular weight PTP (LMW-PTP), which possesses a phos-
phate binding loop structurally identical to that of PTP1B, also acts
as a negative regulator of insulin mediated mitotic and metabolic
signalling.10,17,18 Recently, Pandey reported that the reduction of
LMW-PTP expression (both in vitro and in vivo), using a specific
antisense oligonucleotide, induces increased phosphorylation and
activity of key insulin signalling intermediates in response to insu-
lin stimulation.19 Analogously to PTP1B, it has also been shown to
be a possible positive factor in tumour development. High levels of
this PTP have been detected in breast and colon tumours and neu-
roblastoma.20–22 Consequently, both PTP1B and LMW-PTP are
attractive targets for the development of new antidiabetic and
antitumour agents.
Although a variety of PTP1B inhibitors have been reported in
the last decade,7,10,13,23 the identification of selective, safe and or-
ally available inhibitors of these PTPs has proven difficult and it
still represents a challenge for medicinal chemists.
In the search for active site PTP inhibitors, a valid method to ob-
tain compounds with low polarity is to replace the phosphate
group of pTyr with nonhydrolysable monoanionic bioisosteres,
such as carboxylates.13,24–26 This may prevent the pharmacokinetic
limitations of certain highly charged pTyr mimetics, such as phos-
phonate, which have often been utilized to obtain potent in vitro
PTP1B inhibitors.3,7,10,13 Indeed, several carboxylic acids with good
PTP1B inhibitory profiles and cellular activity or oral bioavailability
have been reported.25,27–31
In continuing our search for PTP1B and LMW-PTP inhibitors, we
designed analogous 4-[(5-arylidene-4-oxo-2-phenyliminothiazoli-
din-3-yl)methyl]benzoic acids (4) (Fig. 2) with the aim of improv-
ing the inhibitory effects of acids 1.
We wanted to test the hypothesis whether replacing the car-
bonyl group in position 2 of the 2,4-thiazolidinedione scaffold with
a phenylimino moiety could enhance the inhibitor/enzyme affinity
by means of further favourable interactions with residues of the
active site and the surrounding loops, particularly with the WPD
loop (residues 179–189). This flexible loop, which forms one edge
of the PTP1B catalytic pocket, contributes to substrate and inhibi-
tor affinity and specificity. In the absence of substrate the WPD
loop is in an ‘open’ conformation and closes down on the side chain
of pTyr residue upon substrate binding. This conformational
change is required for catalysis. Thus, inhibitors which reduce
We have recently reported a series of 4-[(5-arylidene-2,4-
dioxothiazolidin-3-yl)methyl]benzoic acids (1) (Fig. 1) that we
have designed and evaluated as PTP1B and LMW-PTP inhibitors.
They were shown to be competitive and reversible PTP inhibitors,
with an appreciable selectivity towards human PTP1B and IF1 iso-
form of human LMW-PTP compared with other related PTPs.32
In the structure of compounds 1, the p-methylbenzoic acid
residue was inserted as a nonphosphorus monoanionic pTyr-
mimic onto N-3 of the 2,4-thiazolidinedione ring. It proved to
be an effective pTyr-mimetic group that is able to replicate the

1930
R. Ottanà et al. / Bioorg. Med. Chem. 17 (2009) 1928–1937
COOH
N
N
S
O
Ar
4 a-k
O
O
OH
OH
Ar =
g
h
a
b
c
OH
O
i
j
OMe
OH
O
d
OMe
OH
e
f
k
COOH
Figure 2. Structures of compounds 4a–k.
the mobility of WPD loop may prevent substrate binding especially
when they bind the enzyme in the closed conformation.4,10
The insertion of the phenylimino moiety in position 2 of the pent-
atomic scaffold also enhances the hydrophobic character of the com-
pounds. Considering that it is important to include at least one
monoanionic functionality in the structure of PTP inhibitors in order
to obtain electrostatic driving forces for efficacious binding with the
catalytic phosphate binding site of these enzymes, the introduction
of lipophilic moieties in the structure of such inhibitors can repre-
sent a good general approach to improve their cell permeability.10,13
Compounds 4a–k were evaluated for their in vitro inhibitory
activity against recombinant human PTP1B as well as the two ac-
tive isoforms of human LMW-PTP (IF1 and IF2) and Saccharomyces
cerevisiae LMW-PTP (Ltp1).
conditions compound 3 is the only isomer obtained from N-aryl-
N’-alkyl-substituted thioureas, as previously reported.35 In fact,
when the reaction was carried out in CHCl3 at room temperature
both 2-phenylimino and 2-benzylimino isomers were obtained;
they arose from the condensation of chloroacetyl chloride and
two possible intermediate ene-thiols generated by the delocaliza-
tion of the lone pairs of the two different nitrogen atoms on the
adjacent thiocarbonyl group.35
The condensation of compound 3 with the appropriate aromatic
aldehydes, using piperidine as base in refluxing ethanol for 96 h,
provided compounds 4a–k. The reaction time was significantly re-
duced (6 h) by using microwave irradiation (200 psi, 300 W maxi-
mum power) at 140 °C.
The introduction of 5-arylidene moiety provided only Z isomers,
as already demonstrated by X-ray diffraction studies.35,36
The structure of all compounds was unambiguously assigned by
means of analytical and 1H and 13C NMR spectroscopy data.
In 1H NMR spectrum of compounds 3, the signal of NCH2 pro-
tons as well as the analogy of previously reported compounds32,35
allowed us to identify the structure of the 3-(4-carboxy)benzyl-2-
phenylimino isomer. The above mentioned protons appeared as a
singlet resonating at chemical shift ranging between 5.26 and
4.92 ppm, due to the deshielding effect generated by the extended
electronic delocalization of the 3-N lone pair on the highly conju-
gated system. In fact, in the case of the 2-(4-carboxy)benzyl-3-phe-
nylimino isomer the N–CH2 protons should resonate at lower
chemical shifts.35
2. Chemistry
(Z)-4-[(5-Arylidene-4-oxo-2-phenyliminothiazolidin-3-yl)met-
hyl]benzoic acids (4) were synthesized following the synthetic
pathway described in Scheme 1.
The intermediate 4-(3-phenylthioureidomethyl)benzoic acid
(2) was obtained by the reaction of phenylisothiocyanate and 4-
(aminomethyl)benzoic acid in ethanol at reflux.
The subsequent synthesis of 4-[(4-oxo-2-phenyliminothiazoli-
din-3-yl)methyl]benzoic acid (3) was performed by condensation
of compound 2 with chloroacetyl chloride in the presence of trieth-
ylamine as base in ethanol at reflux for 24 h. In these experimental

R. Ottanà et al. / Bioorg. Med. Chem. 17 (2009) 1928–1937
1931
S
a
COOH
NCS
+
COOH
N
N
H
H
H2N
2
b
c
N
S
N
COOH
COOH
N
N
S
O
O
Ar
4 a - k
3
Scheme 1. Reagents and conditions: (a) EtOH,
D
; (b) ClCH2COCl, Et3N, EtOH, D; (c) ArCHO, C5H11N, EtOH, D.
The disappearance of the 5-CH2 signal of compound 3 as well as
the presence of the methylidene proton at 7.70–8.56 ppm were
determinant in assigning the structure of compounds 4. 13C spectra
showed a clear change in the splitting pattern of 5-C which reso-
nated as a triplet at 32.70 ppm in 3 and as a singlet in the range
118.3–126.4 ppm in compounds 4.
Compound 4a was shown to be 10-fold more active than 4-{[2,-
4-dioxo-5-(3-phenoxybenzylidene)thiazolidin-3-yl]methyl}benzoic
acidtowardshuman PTP1Bandfrom 5- to 7-foldagainstIF1 and IF2.32
A 5-naphthalenylmethylene group was inserted in order to ex-
pand the 5-arylidene moiety, thereby providing compounds 4e and
4f (5-naphthalen-1-yl)methylene and (5-naphthalen-2-yl)methy-
lene substituted. Compound 4f maintained good inhibition levels
against both human PTP1B and IF1 LMW-PTP and proved to be
more active than both the corresponding 2,4-thiazolidinedione
and compound 4e against all tested PTPs. The latter compound
was shown to be 7-fold less active against PTP1B and 4-fold against
IF1 than 4f.
3. Results and discussion
Table 1 shows the results of inhibition levels of 4-[(5-arylidene-
4-oxo-2-phenyliminothiazolidin-3-yl)methyl]benzoic acids (4a–
k), expressed as IC50 (lM), against recombinant human PTP1B,
Molecular docking experiments as well as the PTP inhibitory
activity of 4-[(5-arylidene-2,4-dioxothiazolidin-3-yl)methyl]ben-
zoic acids (1) had indicated that a second aromatic ring on 5-ary-
lidene moiety promoted the activity.32 This beneficial effect most
likely derived from its ability to interact with the secondary pocket
of PTP1B by enabling it to reach the most remote aminoacids
(Ala27, Met258, Gly259, Arg24). In fact, the replacement of the
phenoxy and benzyloxy groups on 5-arylidene with a hydroxy
group that is able to establish different interactions resulted in
lower inhibitory activity.32
A similar pattern was observed in compounds 4g–k, which
were shown to be significantly less active than both analogous
compounds 4a–d and corresponding 5-arylidene-2,4-thiazolidin-
ediones 1. Indeed, 3-hydroxy- and 4-hydroxybenzylidene deriva-
tives (4g and 4h, respectively) were from 10-fold to 30-fold less
active towards PTP1B and IF1 and 30-fold less active towards IF2
and Ltp1 compared with derivatives 4a–d.
The introduction of a methoxy group in the para or meta posi-
tions of hydroxybenzylidene moiety of compounds 4g and 4h pro-
vided derivatives 4i and 4j, respectively; these modifications did
not result in any change in the levels of inhibitory activity.
Among compounds 4g–k bearing a hydroxy group on 5-benzyli-
dene moiety, compound 4k, 5-(3-carboxy-4-hydroxy)benzylidene
substituted, was shown to be the most active against both PTP1B
the two active IF1 and IF2 isoenzymes of human LMW-PTP as well
as S. cerevisiae LMW-PTP (Ltp1). The p-nitrophenylphosphate was
used as substrate.
All compounds proved to be good reversible inhibitors of hu-
man PTP1B and LMW-PTP, exhibiting IC50 values in the micromolar
range. We determined also the inhibition type analyzing experi-
mental data of some effective inhibitors of PTP1B (compounds
4b, 4d and 4f) by the double reciprocal plot method. The initial
hydrolysis rate using eight substrate final concentrations (0.5–
40 mM range) was measured in the presence of increasing concen-
trations of each compound. All reciprocal plots display straight
lines that intersect each other in the 1/v axis, suggesting that they
are competitive type inhibitors. In fact the increasing inhibitor con-
centration is accompanied by the increase of Km values and by very
close values of Vmax (see Supplementary data).
Compounds 4b and 4d, 4-phenoxy- and 4-benzyloxybenzylid-
ene substituted, were the most effective inhibitors as they
achieved interesting inhibition levels against both human PTP1B
(IC50 = 1.1
lV) and IF1 isoform of LMW-PTP (IC50 = 3.1 and
2.7 V, respectively) while their inhibitory effectiveness was found
l
to be lower towards IF2 isoform and Ltp1 (Table 1). They were
slightly more active towards PTP1B in comparison with com-
pounds 1a and 1b (IC50 = 2.8 and 1.6 l
V, respectively).32
The displacement of both 4-phenoxy and 4-benzyloxy groups
to position 3 of the 5-benzylidene moiety gave access to com-
pounds 4a and 4c, respectively, which exhibited an interesting
(IC50 = 19.5 lV) and IF1 (IC50 = 43.1 lV). However, it was less active
than compounds bearing two aromatic rings inthe 5-arylidene moiety.
The generally increased affinity of compounds 4 towards Ltp1 in
comparison with compounds 1 may be determined by the 2-phe-
nylimino moiety which extends the surface contact between the
inhibitors and hydrophobic residues lining the deep active site cav-
ity of the enzyme.37 In fact, among compounds 4, the most lipo-
philic derivatives (4a–d, 4f) reached good inhibition levels
towards Ltp1.
enzymatic inhibition profile particularly against PTP1B (IC50
=
1.9 and 3.8 V) and IF1 (IC50 = 5.4 and 3.1 V) without reaching
l
l
the inhibition levels of the 4-substituted analogues. In compari-
son with the 5-arylidene-2,4-thiazolidinedione analogues they
proved to be generally more active towards all enzymes, except
in the case of 4c that was about 3-fold less effective against
PTP1B.

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R. Ottanà et al. / Bioorg. Med. Chem. 17 (2009) 1928–1937
Table 1
In vitro inhibition activity of compounds 4a–k against human PTP1B, IF1 and IF2 isoforms of human LMW-PTP and LMW-PTP (Ltp1) from S. cerevisiaea
Ar
Compound
IC50 (lM)
PTP1B
IF1
IF2
Ltp1
O
4a
4b
4c
4d
4e
4f
1.9 1.0
5.4 0.5
26.1 1.7
11.1 0.6
O
1.1 0.1
3.8 0.1
1.1 0.1
14.0 1.0
1.9 0.1
35.4 3.0
3.1 0.4
3.1 0.5
2.7 0.2
16.1 1.8
3.7 0.2
62.5 1.6
47.7 1.6
87.1 9.2
8.2 0.3
12.1 0.5
21.5 2.2
1004 99
13.8 0.8
>250
6.3 0.2
12.9 0.1
15.9 2.2
>500
CH2O
CH2O
27.6 3.2
>250
4g
4h
4i
HO
HO
30.1
3
269.7 1.5
>250
195.4 5.6
>250
MeO
32.1 3.6
33.7 8.0
HO
HO
MeO
4j
71.2 1.5
43.1 3.1
>250
>250
4k
19.5 0.8
389.0 35
344.9 38
HO
COOH
a
IC50 values were determined by regression analyses and expressed as means SE of three replicates.
For our docking studies, PDB structures 1G7F, 1G7G38 and
We selected compounds 4a, 4b, 4e, 4f and 4h, a diverse sample
of compounds with different shapes and polarities, for docking
experiments. In addition, their respective analogues from series 1
were docked in order to detect any possible differences between
the two series. All compounds were drawn and minimized using
Maestro. The carboxylic acid groups of the ligands were always
drawn in their deprotonated form.
Docking in 1G7F resulted in poses that were largely in agree-
ment with our previous predictions, with the carboxylate group
positioned at the phosphate binding region, and the distal groups
stretching into the secondary non-catalytic hydrophobic pocket.
1XBO29 were selected as templates for human PTP1B. PDB struc-
tures 5PNT39 and 1XWW40 were selected as templates for human
LMW-PTP IF1 and IF2, respectively. Structures 1G7F and 1G7G
have similar ligands, but in 1G7F the WPD loop is in its open
form, whereas in 1G7G, as like in most of the reported protein–li-
gand complex structures for PTP1B, it is closed. In both structures,
the main axis of the ligand is oriented towards the wall formed
by residues Asn42 to Arg47, whereas in 1XBO, which is also a
‘closed’ structure, the main axis of the ligand reaches deeper into
the secondary pocket lined by residues Arg24 to Lys36 and this is
the one we previously predicted to be the relevant one for com-
pounds 1.32 The selected protein structures were processed in
Maestro (Schrödinger, LLC)41 using the Protein Preparation Wiz-
ard. All water molecules were removed from the protein struc-
ture. The Induced Fit Docking (IFD) protocol developed by
Schrödinger, LLC, which takes into account the flexibility of the
binding site, was used for docking our compounds.42,43 This ap-
proach seemed reasonable because in both PTP1B and LMW-PTP
flexible side chains of multiple residues reach into the binding
site. Based on the ligand from 5PNT, identical box sizes were cho-
sen for LMW-PTP.39 The enclosing box size was enlarged to 30 Å
for all investigated structures. All other values of the IFD protocol
were kept at their default values.
Only weak interactions of the phenylimino group with Arg24 (
p–
p
stacking) and Thr263 (hydrophobic) could be detected. No
favourable interaction with the open WPD loop were found. In fact,
the open conformation of 1G7F moves the lipophilic side chain of
Phe182 away from the position where it can set up hydrophobic
interactions with the phenylimino group of the ligands. In 1G7G,
we found many poses where the caboxylate group interacts with
the basic center formed by Arg24, Arg254 and Tyr20, leaving the
primary phosphate binding region occupied by the distal ligand
residues. The analysis of available X-ray structures of PTP1B shows
these poses to be unlikely, since the presence of an acidic group at
the phosphate binding region seems to be a prerequisite for ligand-
binding. In 1XBO (Fig. 3), 4b is more consistently placed in a posi-

R. Ottanà et al. / Bioorg. Med. Chem. 17 (2009) 1928–1937
1933
tion where the phenoxyphenyl residue is placed in the hydropho-
bic secondary pocket. The phenylimino group makes hydrophobic
interactions with either Phe182 or with Tyr 46. In the first case, a
Asn50 and the carbonyl group in position 4 (Fig. 4). The aromatic
residues of the 5-arylidene and phenylimino groups fold slightly
around the indole ring of Trp49, making hydrophobic and maybe
p-cation interaction can be perceived between Arg24 and the three
also slightly off-plane p–p interactions. Again, additional hydro-
phenyl groups from the phenylimino and phenoxybenzylidene res-
idues, which surround at a distance of about 4–6 Å, whereas in the
second proposed orientation, the phenylimino group appears to
stack with an intramolecular salt-bridge formed by side-chain res-
idues of Arg47 and Asp48. For shorter molecules 4e, 4f and 4h, no
consistent pose preferences were obtained.
Docking into the two LMW-PTP binding sites gave more consis-
tent results over the series of investigated compounds, especially
for IF2 (1XWW). The preferred pose for all compounds, both from
series 1 and 4, is the one in which the carboxylic group is placed in
the phosphate-binding region through charged interactions with
Arg18 and also by receiving hydrogen bonds from Cys12, Leu13,
Cys17 and Arg18. The thiazolidinone ring is stacked with the in-
dole ring of Trp49, and is held by hydrogen bonds between the
phenolic group of Tyr132 and the oxygen of the carbonyl group
in position 2 (compounds 1) or the 2-imino nitrogen atom (com-
pounds 4), and between the amide side chain nitrogen atom of
phobic interactions of the phenylimino group contribute to their
improved affinity (Fig. 4, right side). IF1 has Glu instead of Asn at
position 50, leading to the loss of one possible hydrogen bond do-
nor. The other difference in the binding site is the replacement of
Trp with Tyr at position 49, which again is embraced by the thia-
zolidine residue and its double-bonded phenylimino and 5-arylid-
ene moieties in poses obtained for 4a and 4b (Fig. 4, left side).
However, for the shorter molecules (4e, 4f, 4h) as well as for the
2,4-thiazolidinediones Tyr49 is moved outwards, placing the thia-
zolidinone ring between the aromatic ring of Tyr49 and the isobu-
tyl chain of Leu13. However, since the arylidene part does not
interact with the protein this pose appears as unlikely. A hydro-
gen-bond is detected between Tyr131 and the carbonyl group in
position 4 of the ligand, while the 2-phenylimino group is too far
away from Tyr132 for a hydrogen bond to form. In both isoforms,
the distal phenoxy group interacts only weakly, if at all, with the
protein surface.
Figure 3. Main docking poses received for 4b in PTB1B (PDB structure 1XBO.39 Top: 3D representation, bottom: 2D representation. Pharmacophoric interactions were
automatically detected by LigandScout.44 Yellow sphere: hydrophopic feature; red arrow: hydrogen bond acceptor; red star: negative ionizable feature.

1934
R. Ottanà et al. / Bioorg. Med. Chem. 17 (2009) 1928–1937
Figure 4. Main docking poses received for 4b in LMW-PTP IF1 (PDB code 5PNT,39 left) and IF2 (PDB code 1XWW,40 right). Top: 3D representation, bottom: 2D representation.
Pharmacophoric interactions were automatically detected by LigandScout.44 Yellow sphere: hydrophopic feature; red arrow: hydrogen bond acceptor; red star: negative
ionizable feature; purple ring and arrow: aromatic feature. For IF2, the surface accessibility threshold value was lowered from 250 to 130 to detect the hydrophobic
interaction of the benzylidene ring.
On the whole, comparing the inhibitory effects of these 2-phe-
nylimino-4-thiazolidinones with those of their 2,4-thiazolidinedi-
one analogues highlighted that some phenylimino derivatives
(4a, 4b, 4f) with a 5-arylidene moiety containing two aromatic
rings, had higher potency against PTP1B and/or IF1. In contrast,
hydroxybenzylidene substituted analogues were found to be less
effective than the corresponding thiazolidinediones.
In our docking experiments, we were able to detect additional
hydrophobic interactions of the introduced phenylimino group in
PTP1B, IF1 and IF2, which is consistent with the improved affinities
of derivatives 4a and 4b in comparison with the corresponding
analogues 1. For PTP1B, the possible ligand orientation presented
in our previous paper still appears plausible, while other orienta-
tions—especially for smaller compounds 4e, 4f, 4h— cannot be ru-
led out. The binding site of PTP1B is large, open and lined with
flexible side chains, making it difficult to predict the binding pose.
Although we obtained poses that look very plausible for LMW-PTP
IF2 we are unable to explain the generally higher affinity of our
compounds towards IF1.
4. Conclusions
The design and the synthesis of 4-[(5-arylidene-4-oxo-2-phe-
nyliminothiazolidin-3-yl)methyl]benzoic acids (4) are part of a
project that aims to identify new nonphosphorus monoanionic
PTP1B and LMW-PTP inhibitors.
Compounds 4a–d, 4f appeared to possess the highest inhibitory
activity levels against both human PTP1B and IF1 isoform of
human LMW-PTP in the low micromolar range, while they were
relatively moderate inhibitors of IF2 isoform and Ltp1 from
S. cerevisiae.
The SARs that were previously acquired with 2,4-thiazolidined-
iones derivatives were found to be retained in compounds 4. A
large lipophilic arylidene moiety in position 5 particularly favoured
the activity; phenoxy and benzyloxy groups both in the para and
meta positions of the 5-benzylidene group gave the best levels of
enzyme inhibition. The removal of the second aromatic ring and
its replacement with hydrophilic groups led to a significant de-
crease in the inhibitory potency.

R. Ottanà et al. / Bioorg. Med. Chem. 17 (2009) 1928–1937
1935
1H, CH); 8.08 (m, 2H, arom); 13C NMR (CDCl3): d 46.0 (NCH2),
118.5, 119.4, 119.5, 120.9, 123.9, 124.3, 124.8, 128.4, 129.2,
129.7, 129.9, 130.2, 130.5 (CH arom and CH methylidene); 122.2
(5-C); 130.3, 135.1, 140.4, 147.6, 155.9, 158.1 (Cq arom); 149.5
(C@N); 166.3, 166.9 (C@O). Anal. Calcd for C30H22N2O4S: C,
71.13; H, 4.38; N, 5.53. Found: C, 71.27; H, 4.52; N, 5.38.
5. Experimental
5.1. Chemistry
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), deter-
mined by means of a C. Erba mod. 1106 elem. Analyzer, were with-
in 0.4% of theory. 1H and 13C NMR spectra were recorded on a
Varian 300 magnetic resonance spectrometer (300 MHz for 1H
and 75 MHz for 13C). Chemical shifts are given in d units (ppm) rel-
ative to internal standard Me4Si and refer to DMSO-d6 solutions.
Coupling constants (J) are given in hertz (Hz). 13C NMR spectra
were determined by Attached Proton Test (APT) experiments and
the resonances were always attributed by proton–carbon hetero-
nuclear chemical shift correlation.
5.1.5. 4-{[4-Oxo-5-(4-phenoxybenzylidene)-2-
phenyliminothiazolidin-3-yl]methyl}benzoic acid (4b)
Yield 43%; mp 249–252 °C; 1H NMR (CDCl3): d 5.26 (NCH2);
7.01–7.42 (m, 14H, arom); 7.64 (m, 2H, arom); 7.78 (s, 1H, CH);
8.11 (m, 2H, arom); 13C NMR (DMSO-d6): d 46.0 (NCH2); 118.9,
120.1, 121.4, 124.9, 125.3, 128.1, 129.9, 130.0, 130.7, 130.8, 132.5
(CH arom and CH methylidene); 119.7 (5-C); 128.4, 130.5, 141.4,
148.0, 150.0, 158.9 (Cq arom); 150.0 (C@N); 166.2, 167.5 (C@O).
Anal. Calcd for C30H22N2O4S: C, 71.13; H, 4.38; N, 5.53. Found: C,
71.02; H, 4.28; N, 5.67.
Unless stated otherwise, all materials were obtained from com-
mercial suppliers and used without further purification.
5.1.6. 4-{[5-(3-Benzyloxybenzylidene)-4-oxo-2-
phenyliminothiazolidin-3-yl]methyl}benzoic acid (4c)
Yield 23%; mp 256–258 °C; 1H NMR (CDCl3): d 4.92 (s, 2H, NCH2);
5.07(s,2H,OCH2);6.85–7.25(m,14H,arom);7.42(m,2H,arom);7.57
(s, 1H, CH); 7.88 (m, 2H arom); 13C NMR (CDCl3): d 44.7 (NCH2); 68.7
(OCH2); 114.8, 115.6, 119.8, 121.3, 123.8, 126.2, 126.8, 127.0, 127.3,
128.2, 128.7, 129.0, 129.9 (CH arom and CH methylidene); 120.4 (5-
C); 129.3, 133.6, 135.3, 139.4, 146.5, 157.7 (Cq arom); 148.3 (C@N);
165.0, 166.5 (C@O). Anal. Calcd for C31H24N2O4S: C, 71.52; H 4.65;
N, 5.38. Found: C, 71.36; H, 4.51; N, 5.54.
5.1.1. Synthesis of 4-[(3-phenyltioureido)methyl]benzoic acid (2)
A
mixture of phenylisothiocyanate (0.35 g, 2.6 mmol) and
4-(aminomethyl)benzoic acid (0.5 g, 3.3 mmol) in ethanol was
refluxed for 24 h. The solvent was evaporated under vacuo and
the solid residue was crystallized from methanol providing com-
pound 2.
Yield 100%; mp 223–225 °C; 1H NMR (CDCl3): d 4.80 (d,
J = 5.7 Hz, 2H, NCH2); 7.07 (m, 2H, arom); 7.32–7.42 (m, 5H, arom);
7.89 (m, 2H, arom); 8.30 (br s, 1H, CH2NH); 9.75 (br s, 1H, NH);
11.00 (br s, 1H, COOH); 13C NMR (DMSO-d6): d 47.4 (CH2); 124.0,
124.9, 127.8, 129.2, 129.9 (CH arom); 130.0, 139.7, 144.9 (Cq
arom); 167.8 (C@O); 181.6 (C@S). Anal. Calcd for C15H14N2O2S: C,
62.92; H, 4.93; N, 9.78. Found: C, 63.18; H, 4.73; N, 10.01.
5.1.7. 4-{[5-(4-Benzyloxybenzylidene)-4-oxo-2-
phenyliminothiazolidin-3-yl]methyl}benzoic acid (4d)
Yield 42%; mp 276–279 °C; 1H NMR (CDCl3): d 4.90 (s, 2H,
OCH2); 5.02 (s, 2H, NCH2); 6.77–7.22 (m, 14H, arom); 7.37 (m,
2H, arom); 7.52 (m, 2H, CH); 7.82 (m, 2H, arom); 13C NMR
(DMSO-d6): d 45.9 (NCH2); 70.0 (OCH2); 116.1, 121.5, 125.3,
128.1, 128.2, 128.4, 128.9, 129.9, 130.0, 131.3, 132.3 (CH arom
and CH methylidene); 118.3 (5-C); 126.4, 130.5, 136.9, 141.4,
148.1, 160.3 (Cq arom); 150.1 (C@N); 166.3, 167.5 (C@O). Anal.
Calcd for C31H24N2O4S: C, 71.52; H, 4.65; N, 5.38. Found: C,
71.41; H, 4.68; N, 5.24.
5.1.2. Synthesis of 4-[(4-oxo-2-phenyliminothiazolidin-3-
yl)methyl]benzoic acid (3)
A mixture of 2 (0.5 g, 1.7 mmol) and triethylamine (0.52 g,
5.1 mmol) in ethanol (50 ml) was maintained at reflux for
30 min. A solution of chloroacetic chloride (0.28 g, 2.5 mmol) in
ethanol (10 ml) was added and the mixture was refluxed for
24 h. After evaporation of the solvent under reduced pressure,
the crude solid was dissolved in CHCl3 and the solution was
washed with water. The evaporation of the solvent under reduced
pressure and the recrystallization from methanol provided pure
compound 3. Yield 70%; mp 165 °C; 1H NMR (CDCl3): d 3.88 (s,
2H, 5-CH2); 5.14 (s, 2H, NCH2); 6.96 (d, J = 7.5 Hz, 2H, arom);
7.18 (dd, J = 7.5 and 7.5 Hz, 1H, arom); 7.37 (dd, J = 7.5 and
7.5 Hz, 2H, arom); 7.60 (m, 2H, arom); 8.09 (m, 2H, arom); 13C
NMR (CDCl3): d 32.7 (5-CH2); 46.0 (NCH2); 120.9, 124.8, 128.9,
129.3, 130.4 (CH arom); 141.6, 147.5 (Cq arom); 153.8 (C@N);
171.0, 171.6 (C@O). Anal. Calcd for C17H14N2O3S: C, 62.56; H,
4.32; N, 8.58. Found: C, 62.68; H, 4.17; N, 8.71.
5.1.8. 4-[(5-Naphthalen-1-ylmethylene-4-oxo-2-
phenyliminothiazolidin-3-yl)methyl] benzoic acid (4e)
Yield 28%; mp 252–255 °C; 1H NMR (CDCl3): d 5.32 (s, 2H,
NCH2), 6.99–8.19 (m, 16H, arom); 8.56 (s, 1H, CH); 13C NMR
(DMSO-d6): d 46.2 (NCH2); 121.4, 123.8, 125.4, 126.0, 126.6,
127.2, 127.8, 128.1, 128.2, 129.3, 129.9, 130.1, 130.9 (CH arom
and CH methylidene); 125.0 (5-C); 130.5, 130.9, 131.3, 133.7,
141.3, 147.9 (Cq arom); 150.2 (C@N); 165.7, 167.4 (C@O). Anal.
Calcd for C28H20N2O3S: C, 72.39; H, 4.34; N, 6.03. Found: C,
72.53; H, 4.48; N, 5.89.
5.1.9. 4-[(5-Naphthalen-2-ylmethylene-4-oxo-2-
5.1.3. General method for the synthesis of 4-[(5-arylidene-4-
oxo-2-phenyliminothiazolidin-3-yl)methyl]benzoic acids (4a–k)
A mixture of 4-[(4-oxo-2-phenyliminothiazolidin-3-yl)methyl]-
benzoic acid 3 (0.5 g, 1.5 mmol), appropriate aldehyde (1.5 mmol)
and piperidine (0.26 g, 3 mmol) in ethanol (50 ml) was refluxed for
96 h. The solution was poured in water acidified with glacial acetic
(pH 3–4); the crude solid was washed with H2O and recrystallized
from methanol providing pure acids 4.
phenyliminothiazolidin-3-yl)methyl] benzoic acid (4f)
Yield 56%; mp 264–266 °C; 1H NMR (CDCl3): d 5.12 (s, 2H, NCH2);
6.92–7.97(m, 17H, aromandCHmethylidene); 13CNMR(DMSO-d6):
d 46.1 (NCH2), 121.5, 125.6, 126.1, 127.6, 128.2, 128.3, 129.1, 139.4,
130.0, 131.1, 131.2, 131.5 (CH arom and CH methylidene); 121.7 (5-
C); 130.7, 131.3, 133.2, 133.6, 141.3, 147.9 (Cq arom); 149.4 (C@N);
166.2, 167.3 (C@O). Anal. Calcd for C28H20N2O3S: C, 72.39; H, 4.34; N,
6.03. Found: C, 72.18; H, 4.21; N, 6.21.
5.1.4. 4-{[4-Oxo-5-(3-phenoxybenzylidene)-2-
5.1.10. 4-{[5-(3-Hydroxybenzylidene)-4-oxo-2-
phenyliminothiazolidin-3-yl]methyl}benzoic acid (4a)
Yield 19%; mp 219–223 °C; 1H NMR (CDCl3): d 5.23 (s, 2H,
NCH2), 6.94–7.43 (m, 14H, arom); 7.61 (m, 2H, arom); 7.72 (s,
phenyliminothiazolidin-3-yl]methyl}benzoic acid (4g)
Yield 67%; mp 280–282 °C; 1H NMR (DMSO-d6): d 5.16 (s, 2H,
NCH2); 6.81–7.41 (m, 9H, arom); 7.44 (m, 2H, arom); 7.70 (s, 1H,

1936
R. Ottanà et al. / Bioorg. Med. Chem. 17 (2009) 1928–1937
CH); 7.94 (m, 2H, arom); 9.80 (br s, 1H, OH); 13C NMR (DMSO-d6): d
46.1 (NCH2); 115.9, 118.0, 121.5, 121.9, 125.5, 128.1, 130.0, 130.1,
130.9, 131.6 (CH arom and CH methylidene); 121.2 (5-C); 130.5,
134.9, 141.4, 148.0, 158.3 (Cq arom); 150.1 (C@N); 166.3, 167.5
(C@O). Anal. Calcd for C24H18N2O4S: C, 66.96; H, 4.21; N, 6.51.
Found: C, 70.15; H, 4.13; N, 6.41.
S-transferase (GST) in the pGEX-2T bacterial expression vector. En-
zyme expression and purification were achieved in the Escherichia
coli TB1 strain. Briefly, the recombinant fusion proteins were puri-
fied from bacterial lysate using a single step affinity chromatogra-
phy on glutathione-Sepharose. The solution contained purified
fusion proteins were treated with thrombin for 3 h at 37 °C. Then
the enzymes were purified from GST and thrombin by gel filtration
on Superdex G-75. The purity of proteins preparations were ana-
lyzed by SDS–polyacrylamide gel electrophoresis according to
Laemmli.45
5.1.11. 4-[5-(4-Hydroxybenzylidene)-4-oxo-2-
phenyliminothiazolidin-3-ylmethyl]benzoic acid (4h)
Yield 62%; mp 260–262 °C; 1H NMR (CDCl3): d 5.08 (s, 2H,
NCH2); 6.75 (m, 2H, arom); 6.85 (m, 2H, arom); 7.06–7.23 (m,
5H, arom); 7.43 (m, 2H, arom); 7.58 (s, 1H, CH); 7.89 (m, 2H,
arom); 13C NMR (DMSO-d6): d 46.1 (NCH2); 116.8, 121.4, 125.3,
128.0, 129.9, 130.0, 131.3, 132.7 (CH arom and CH methylidene);
124.6, 130.5, 141.5, 148.1, 160.1 (Cq arom); 150.2 (C@N); 166.4,
167.4 (C@O). Anal. Calcd for C24H18N2O4S: C, 66.96; H, 4.21; N,
6.51. Found: C, 70.08; H, 4.33; N, 6.37.
5.3.1. Phosphatase assay and inhibition experiments
Phosphatase assay was carried out at 37 °C using p-nitrophenyl-
phosphate as substrate; the final volume was 1 ml. The assay buf-
fer (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-
nitrophenolate ion was determined by reading the absorbance at
5.1.12. 4-[5-(3-Hydroxy-4-methoxybenzylidene)-4-oxo-2-
phenyliminothiazolidin-3-ylmethyl]benzoic acid (4i)
400 nm (e
= 18,000 MÀ1 cmÀ1). The main kinetic parameters (Km
Yield 48%; mp 213–216 °C; 1H NMR (CDCl3): d 3.93 (s, 3H, OCH3);
5.17 (s, 2H, NCH2); 6.94–7.36 (m, 8H, arom); 7.56 (m, 2H, arom); 7.65
(s, 1H, CH);7.97(m, 2H, arom);9.81(s, 1H, OH);13C NMR(DMSO-d6):
d 46.1 (NCH2); 56.2 (OCH3); 113.0, 116.0, 121.5, 124.0, 125.4, 128.1,
130.0, 130.1, 131.9 (CH arom and CH methylidene); 126.4 (5-C);
117.9, 130.7, 135.7, 141.4, 147.4, 148.2 (Cq arom); 150.4 (C@N);
166.5, 167.6 (C@O). Anal. Calcd for C25H20N2O5S: C, 65.2; H, 4.38;
N, 6.08. Found: C, 65.35; H, 4.19; N, 5.97.
and Vmax) were determined by measuring the initial rates at differ-
ent substrate concentrations. Experimental data were analysed
using the Michaelis equation and a non-linear fitting program (Fig-
Sys). Inhibition constants were determined measuring initial
hydrolysis rates at differing substrate and inhibitor concentrations.
The apparent Km values measured at the various inhibitor concen-
trations were plotted against concentration of the inhibitor to cal-
culate the Ki values. All initial rate measurements were carried out
in triplicate. For each inhibitor, IC50 was determined by measuring
the initial hydrolysis rate under fixed p-nitrophenylphosphate con-
centration, 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 rate when the inhibitor
concentration is [I], V0 is the reaction rate with no inhibitor, and
IC50 = Ki + Ki[S]/Km. Therefore, when the substrate concentration
[S] is equal to Km, IC50 = 2Ki.
5.1.13. 4-[5-(4-Hydroxy-3-methoxybenzylidene)-4-oxo-2-
phenyliminothiazolidin-3-ylmethyl]benzoic acid (4j)
Yield 43%; mp 214–218 °C; 1H NMR (CDCl3): d 3.89 (s, 3H,
OCH3); 5.26 (s, 2H, NCH2); 6.93–7.39 (m, 8H, arom); 7.63 (m, 2H,
arom); 7.73 (s, 1H, CH); 8.06 (m, 2H, arom); 13C NMR (DMSO-d6):
d 42.5 (NCH2); 56.2 (OCH3); 116.0, 117.2, 121.6, 123.7, 125.6,
127.6, 129.0, 130.1, 132.3 (CH arom and CH methylidene); 124.7
(5-C); 132.1, 135.5, 139.2, 148.3, 161.3, 161.6 (Cq arom); 148.6
(C@N); 166.5, 168.8 (C=O). Anal. Calcd for C25H20N2O5S: C, 65.2;
H, 4.38; N, 6.08. Found: C, 65.28; H, 4.44; N, 5.89.
Acknowledgements
Work
supported
in
part
by
FIRB
2003-project
5.1.14. 4-[5-(3-Carboxy-4-hydroxybenzylidene)-4-oxo-2-
phenyliminothiazolidin-3-ylmethyl]benzoic acid (4k)
RBNE03FMCJ_007, by ‘Ente Cassa di Risparmio di Firenze’ and by
University of Messina (PRA-2005).
Yield 20%; mp 280–284 °C; 1H NMR (DMSO-d6): d 5.16 (s, 2H,
NCH2); 6.97 (m, 3H, arom); 7.21 (m, 1H, arom); 7.38–7.65 (m,
5H, arom); 7.77 (s, 1H, CH); 7.95 (m, 3H arom); 13C NMR (DMSO-
d6): d 46.2 (NCH2); 118.8, 121.4, 125.4, 128.1, 129.9, 130.1, 131.0,
132.8, 136.8, (CH arom and CH methylidene); 115.4, 118.4, 124.3,
130.5, 141.4, 148.0, 163.3 (Cq arom + 5-C); 150.0 (C@N); 166.4,
167.4, 171.2 (C@O). Anal. Calcd for C25H18N2O6S: C, 63.28; H,
3.82; N, 5.9. Found: C, 63.12; H, 4.00; N, 5.77.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.01.044.
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