Design and evaluation of non-carboxylate 5-arylidene-2-thioxo-4-imidazolidinones as novel non-competitive inhibitors of protein tyrosine phosphatase 1B

Article abstract of DOI:10.1016/j.bioorg.2019.103211

Protein tyrosine phosphatase 1B (PTP1B) acts as a negative regulator of insulin and leptin signalling and is crucially involved in the development of type 2 diabetes mellitus, obesity, cancer and neurodegenerative diseases. Pursuing our efforts to identify PTP1B inhibitors endowed with drug-like properties, we designed and evaluated 3-aryl-5-arylidene-2-thioxo-4-imidazolidinones (7) as a novel class of non-carboxylate PTP1B inhibitors. In agreement with our design, kinetic studies demonstrated that selected compounds 7 act as reversible, non-competitive inhibitors of the target enzyme at low micromolar concentrations. Accordingly, molecular docking experiments suggested that these inhibitors can fit an allosteric site of PTP1B that we previously individuated. Moreover, cellular assays demonstrated that compound 7e acts as a potent insulin-sensitizing agent in human liver HepG2 cells. Taken together, our results showed that these non-competitive PTP1B inhibitors can be considered promising lead compounds aimed to enhance druggability of the target enzyme and identify novel antidiabetic drugs.

Full text of DOI:10.1016/j.bioorg.2019.103211

BioorganicChemistry92(2019)103211
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and evaluation of non-carboxylate 5-arylidene-2-thioxo-4-
imidazolidinones as novel non-competitive inhibitors of protein tyrosine
phosphatase 1B
⁎
Rosaria Ottanàa,1, , Paolo Paolib,1, Giulia Lorib, Ilenia Adornatoa, Santo Previtia, Alexandra Naßc,
Gerhard Wolberc, Rosanna Maccaria,1
a Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Polo Universitario Annunziata, Viale SS. Annunziata, 98168
Messina, Italy
b Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Firenze, Viale Morgagni 50, 50134 Firenze, Italy
c Institute of Pharmacy, Computer-Aided Molecular Design, Freie Universitaet Berlin, Koenigin-Luisestr. 2+4, 14195 Berlin, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
Protein tyrosine phosphatase 1B (PTP1B) acts as a negative regulator of insulin and leptin signalling and is
crucially involved in the development of type 2 diabetes mellitus, obesity, cancer and neurodegenerative dis-
eases. Pursuing our efforts to identify PTP1B inhibitors endowed with drug-like properties, we designed and
evaluated 3-aryl-5-arylidene-2-thioxo-4-imidazolidinones (7) as a novel class of non-carboxylate PTP1B in-
hibitors. In agreement with our design, kinetic studies demonstrated that selected compounds 7 act as reversible,
non-competitive inhibitors of the target enzyme at low micromolar concentrations. Accordingly, molecular
docking experiments suggested that these inhibitors can fit an allosteric site of PTP1B that we previously in-
dividuated. Moreover, cellular assays demonstrated that compound 7e acts as a potent insulin-sensitizing agent
in human liver HepG2 cells. Taken together, our results showed that these non-competitive PTP1B inhibitors can
be considered promising lead compounds aimed to enhance druggability of the target enzyme and identify novel
antidiabetic drugs.
3-aryl-5-arylidene-2-thioxo-4-imidazolidinones
Protein tyrosine phosphatase 1B
Non-competitive inhibitors
Insulin-sensitizing agents
Cellular assays
1. Introduction
has been showed that the inhibition or genetic ablation of PTP1B can
improve insulin and leptin signalling pathways, resulting in improved
Protein tyrosine phosphatase 1B (PTP1B) is an intracellular enzyme
critically involved in the regulation of specific signalling pathways re-
lated to cell growth, metabolism, and energy homeostasis [1]. In par-
ticular, PTP1B acts as a negative regulator of insulin signalling by de-
phosphorylating specific phosphotyrosine (pTyr) residues on the
activated insulin receptor (IR) and IR substrate proteins, thus control-
ling the cascade of cellular responses to the hormone [2]. Moreover, in
the hypothalamus, PTP1B downregulates the signal of leptin by de-
phosphorylating JAK2, a kinase associated to leptin receptor, and
consequently interrupting downstream events that lead to the tran-
scription of genes involved in feeding and energy homeostasis [3,4].
The overexpression of PTP1B is strictly correlated with development
of both insulin and leptin resistance, which are important pathogenic
mechanisms underlying the onset and progression of type 2 diabetes
mellitus (T2DM) and obesity [5–7]. In accord with this hypothesis, it
sensitivity to both hormones in specific tissues, such as skeletal muscle,
liver, adipose tissue and hypothalamus, without producing significant
toxic effects [3,8–12].
In addition, chronic inflammation, which is a subclinical condition
generally associated to both T2DM and obesity, can positively up-reg-
ulate PTP1B expression in adipocytes, hepatocytes, and hypothalamus,
thus contributing to exacerbate resistance to both insulin and leptin
[13,14]. Recently, PTP1B was also found to function as a positive
regulator of neuroinflammation, which is considered to be a distinctive
feature of neurodegenerative diseases including Alzheimer’s disease,
Parkinson’s disease, and multiple sclerosis [15,16]. Compelling evi-
dence demonstrated that insulin-resistance in brain tissues can im-
portantly contribute to cognitive impairment and neurodegenerative
processes [17–19].
Furthermore, numerous studies demonstrated that upregulated
⁎ Corresponding author.
E-mail address: rottana@unime.it (R. Ottanà).
1 These Authors contributed equally to the work.
https://doi.org/10.1016/j.bioorg.2019.103211
Received 17 May 2019; Received in revised form 7 August 2019; Accepted 18 August 2019
Availableonline21August2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

R. Ottanà, et al.
BioorganicChemistry92(2019)103211
PTP1B expression is significantly associated with the progression of
several human tumor types, such as colon and breast cancers, and,
accordingly, the inhibition of the enzyme results in better prognostic
outcome [20–24].
Therefore, on the whole, numerous biochemical and pharmacolo-
gical findings validate PTP1B as a promising drug target not only for
T2DM and obesity, but also for neurodegenerative diseases and cancer,
and support the search for PTP1B inhibitors as novel drug candidates
for the treatment of these serious human diseases [25–30].
The development of drug-like inhibitors targeted to the active site of
PTP1B as therapeutics has proven to be challenging, mainly due to
intrinsic features of the catalytic region of the enzyme.
The catalytic domain of PTP1B contains a high-affinity phosphate-
binding loop (P-loop) which includes the signature motif CX5R(S/T). In
this short amino acid sequence, the cysteine and arginine residues are
essential for the catalytic dephosphorylation mechanism common to all
protein tyrosine phosphatases (PTPs) and, thus, are conserved in all
members of the PTP family. The flexible WPD loop, containing the
catalytic residue Asp181, and the YRD loop, including Tyr46 critical for
the recognition of pTyr, surround the catalytic site of PTP1B and cru-
cially participate to the binding of ligands. In particular, upon the
binding of substrate, the WPD loop shifts over the pTyr-binding loop,
thus allowing the enzyme to assume a “closed” catalytically competent
form.
A less conserved low-affinity non-catalytic aryl-phosphate
binding site was individuated near the catalytic region and can be
successfully exploited to enhance the selectivity of inhibitors towards
PTP1B over other PTPs [31–33].
The polar nature of the highly conserved catalytic site of PTP1B
represents the primary obstacle to the identification of inhibitors en-
dowed with appropriate drug-like properties. This could explain, at
least in part, why only few PTP1B inhibitors have been evaluated as
drug candidates in clinical trials, so far, and no PTP1B-based drug is
currently available for therapy. In the early stage of the search for
PTP1B inhibitors, extensive efforts to achieve an efficient inhibition of
the target enzyme resulted in the identification of numerous potent
inhibitors directed to the catalytic site, mainly through the insertion of
anionic pTyr-mimetic moieties on suitable scaffolds. In order to im-
prove the bioavailability of these polar molecules, different design ap-
proaches has been exploited, i.e. designing less ionisable and/or more
lipophilic inhibitors bearing monoanionic pTyr-mimetics and hydro-
phobic moieties in their structures [27,28].
Fig. 1. Structure of trodusquemine and general structures of 5-arylidene-4-
thiazolidinones 1–6 [37,39–43] and of 3-aryl-5-arylidene-2-thioxo-4-imidazo-
lidinones 7.
between the β-sheet including Leu71 and Lys73 and a loop consisting of
aminoacids Leu204-Pro210, together forming a lipophilic pocket. This
pocket is connected to the catalytic site by a short β-strand, thus it can
be inferred that ligand binding to this allosteric region is capable to
impair the catalytic functions of PTP1B by influencing the conformation
of the active site [37,38].
An alternative strategy to circumvent the issues associated with
inhibitors directed to the PTP1B catalytic centre is the design of ligands
able to target non-conserved and less polar allosteric sites. Indeed, al-
losteric modulation has been proposed as an approach that can sig-
nificantly improve the druggability of PTP1B [34,35]. It is worth noting
that the natural aminosterol trodusquemine (MSI-1436, Fig. 1), a pro-
mising PTP1B inhibitor which successfully passed phase I clinical trials
as an antidiabetic and antiobesity candidate, primarily acts as a non-
competitive PTP1B inhibitor capable to control insulin-resistance and
leptin-resistance [36].
In the last few years, we investigated 5-arylidene-4-thiazolidinone
derivatives (1–6, Fig. 1), which were shown to be potent PTP1B in-
hibitors [37–43]. Kinetic studies highlighted that the substitution pat-
tern in positions 2 and 5 of the thiazolidinone scaffold can markedly
affect the inhibition mechanism of compounds 1–6. Most of them act as
competitive PTP1B inhibitors, whereas 2-thioxo-4-thiazolidinone ana-
logues 2a-c (Fig. 1) behaved as mixed type non-competitive inhibitors,
being able to bind two not mutually exclusive sites, namely the catalytic
centre and the newly-identified allosteric site described above [37]. In
particular, the thiocarbonyl group of the 2-thioxo-4-thiazolidinone
scaffold (compounds 2) appeared to be more suitable for fitting into the
allosteric site than the more bulky 4-fluorophenylimino substituent
(compounds 6) [37]. Accordingly, different SARs were observed for 2-
thioxo-4-thiazolidinones 2 compared to the other 4-thiazolidinone
analogues (1, 3–6, Fig. 1) that act via a competitive inhibition me-
chanism [37,39–43]. In any case, compounds 2 maintained the residue
of p-methylbenzoic acid as a pTyr-mimetic moiety that makes them
capable to bind also the PTP1B catalytic site [37].
A PTP1B allosteric site was first identified by Weissman and coll. in
a region between helices α3 and α6 of the enzyme. It has been shown
that, upon the binding of non-pTyr-mimetic inhibitors to this site, the
enzyme is prevented from assuming the “closed” catalytically compe-
tent form. In fact, the closure of the WPD loop, which allows Asp181 to
assume a position suitable to carry out its role as acid-base catalyst
essential for the dephosphorylation mechanism, in part depends on a
series of H-bond interactions between helices α3, α6 and α7. In the
presence of an allosteric inhibitor bound in the region between helices
α3 and α6, the helix α7 results to be displaced and disordered and,
consequently, the WPD loop is blocked in an “open” inactive con-
formation [34].
The potential of PTP1B allosteric modulators as therapeutics en-
couraged us to pursue this approach in order to identify new inhibitors
capable to control the catalytic function of the target enzyme through a
Recently, we have identified a second allosteric site, positioned
2

R. Ottanà, et al.
BioorganicChemistry92(2019)103211
non-competitive inhibition mechanism. Therefore, starting from our
previous studies, we designed and synthesised 3-aryl-5-arylidene-2-
thioxo-4-imidazolidinones (7, Fig. 1) as bioisosteres of 5-arylidene-2-
thioxo-4-thiazolidinones 2. The 4-methylbenzoic acid residue on N-3 of
4-thiazolidinones 2 was replaced by an aromatic moiety, in order to
obtain more lipophilic non-pTyr-mimetic compounds able to interact
with the allosteric site, without binding to the catalytic centre. Besides,
the replacement of the sulfur atom in position 1 of the 4-thiazolidinone
scaffold with the imidazolidinone NH makes possible the exploitation of
further favourable interactions, such as H-bonds, with the target en-
zyme. The 5-arylidene portion was maintained, since it was able to fit
the lipophilic cavity of the newly discovered allosteric site, and dif-
ferent substituents were inserted on the 5-benzylidene ring in order to
evaluate the influence of this moiety in the modulation of the inhibitory
effects.
2. Results and discussion
2.1. Chemistry
The synthesis of (5Z)-3-aryl-5-arylidene-2-thioxo-4-imidazolidi-
nones (7a-w) was performed as depicted in Scheme 1, according to the
procedure that we reported recently [44].
3-Aryl-2-thioxo-4-imidazolidinones 8a-e were obtained by the re-
action of glycine with the appropriate aryl isothiocyanates, in hydro-
alcoholic solution under reflux for 24 h. The subsequent Knoevenagel
condensation of compounds 8a-e with the appropriate arylaldehydes,
in the presence of piperidine as base in refluxing ethanol, afforded 3-
aryl-5-arylidene-2-thioxo-4-imidazolidinones 7a-w (Scheme 1).
Analytical, spectroscopic data (1H and 13C NMR) and X-ray dif-
fraction study confirmed the structure of the newly-synthesized com-
pounds. X-Ray crystallographic study of the 5-(4-phenoxybenzylidene)-
3-phenyl-2-thioxo-4-imidazolidinone (7g) [44], unambiguously attrib-
uted the Z configuration at the chiral axis of derivatives 7. This result is
consistent with previously reported 5-arylidene-2,4-thiazolidinediones
[45] and 5-arylidene-2-phenylimino-4-thiazolidinones [46].
The synthesised compounds (7a-w) were evaluated for their in vitro
inhibitory activity towards recombinant human PTP1B. Furthermore,
representative inhibitors were tested in cultures of human liver HepG2
cells. Kinetic studies were performed in order to determine the inhibi-
tion mechanism of compounds 7. In addition, in silico investigation
supported our design and the rationalization of results.
1H NMR spectra of 3-aryl-2-thioxo-4-imidazolidinones 8 showed a
diagnostic singlet at 4.28–4.33 ppm attributable to 5-CH2 protons as
Scheme 1. Synthesis of 3-aryl-5-arylidene-2-thioxo-4-imidazolidinones 7a-w. Reagents and conditions: i, EtOH/H2O, Δ. ii, arylaldehyde, piperidine, EtOH, Δ.
3

R. Ottanà, et al.
BioorganicChemistry92(2019)103211
Table 1
decreased as a result of the introduction of either an oxygen bridge
(compound 7g IC50 = 15.3 μM) or an oxymethylene bridge (compound
7i IC50 = 16.8 μM). The displacement of the phenoxy group from the
para position (compound 7g) to the meta one (compound 7f
IC50 = 2.7 μM) proved to be favourable, by reducing the IC50 value
almost six-fold. The replacement of the benzyloxy group of compounds
7h and 7i with the methoxy one provided compounds 7j and 7k, re-
spectively, which showed to be inactive. The replacement of the 4-
methoxy group with the 4-thiomethyl isostere gave rise to compound 7l
(IC50 = 5.6 μM), markedly improving the inhibitory ability which, in-
stead, proved to be halved in the case of 4-trifluoromethylthio sub-
stituted analogue (compound 7n IC50 = 10.2 μM).
In vitro inhibitory activity of compounds 7a-w against human PTP1B, ex-
pressed as IC50 (μM).
Compd
R
Ar
IC50 (μM)
7a
7b
7c
7d
7e
7f
C6H4-4-F
C6H4-4-OCH3
C6H4-4-SCH3
1-naphthyl
C6H5
C6H4-4-OC6H5
C6H4-4-OC6H5
C6H4-4-OC6H5
C6H4-4-OC6H5
C6H4-4-C6H5
3.5
0.3
0.4
0.2
Among methoxybenzylidene substituted compounds 7j, 7k, 7o, and
7q-t, the only active compound was shown to be the 5-(3-hydroxy-4-
methoxy)benzylidene substituted derivative 7r (IC50 = 12.2 μM).
Among 5-(4-phenoxybenzylidene) substituted derivatives 7a-d and
7g, the presence of a substituent of different electronic nature on the 3-
aryl group (compounds 7a-c) was beneficial for the inhibition of the
target enzyme compared to 3-phenyl analogue 7g, whereas the in-
hibitory potency appeared to be indifferent to the extension of the
aromatic system on N-3 (7d vs 7g).
7.4
3.5
15.2
2.9
1.0
0.2
C6H5
C6H4-3-OC6H5
C6H4-4-OC6H5
C6H4-3-OCH2C6H5
C6H4-4-OCH2C6H5
C6H4-3-OCH3
2.7
0.24
0.5
7g
7h
7i
C6H5
15.3
12.1
16.8
> 50
> 50
5.6
C6H5
0.90
C6H5
1.70
7j
C6H5
7k
7l
C6H5
C6H4-4-OCH3
C6H5
C6H4-4-SCH3
0.1
Recently, we have selected some of these 3-aryl-2-thioxo-4-imida-
zolidinones (7d, 7f, 7g, 7j, 7k, 7l, 7o, 7q, 7r, 8a and 8e) in order to
evaluate them in the context of a search for novel non-covalent in-
hibitors of human proteasome and immunoproteasome, which are po-
tential targets for the treatment of haematological malignancies [44]. It
is worth noting that the SARs that emerged from the PTP1B inhibition
assay results are clearly different from those revealed by the evaluation
towards proteasome. Indeed, compound 7f, which is the most effective
in vitro PTP1B inhibitor of the series, was shown to be almost inactive
against human proteasome enzymes; conversely, compound 7o ex-
hibited significant inhibitory properties against human im-
munoproteasome [44], without affecting the activity of PTP1B at the
tested concentrations.
7m
7n
7o
7p
7q
7r
C6H5
C6H4-3-OCF3
49.7
10.2
> 50
> 50
> 50
12.2
> 50
> 50
> 50
10.6
6.0
1.3
0.2
C6H5
C6H4-4-SCF3
C6H5
C6H3-3,4-(OCH3)2
C6H3-3,4-(OH)2
C6H3-3-OCH3-4-OH
C6H3-3-OH-4-OCH3
C6H2-3,4,5-(OCH3)3
C6H3-3-OCH3-4-F
C6H4-4-NHCOCH3
1-naphthyl
C6H5
C6H5
C6H5
0.40
0.5
7s
7t
C6H5
C6H5
7u
7v
7w
C6H5
C6H5
C6H5
2-naphthyl
0.4
aIC50 values were determined by regression analyses and expressed as
means
SE of three replicates.
In order to define the action mechanism of PTP1B inhibitors 7, we
performed additional tests to verify if representative compounds 7d,
7e, 7f, 7h, 7i, and 7r behave as reversible or irreversible inhibitors.
Data reported in Fig. 2 show that the recovery of PTP1B activity was
almost complete in all cases, demonstrating that these compounds be-
have as reversible inhibitors.
well as the NH proton of the imidazolidinone ring which resonated at
9.99–10.40 ppm. 13C NMR spectra contributed to the unambiguous
attribution of compound structure due to the signals of 5-CH2 group
(49.7–55.1 ppm) along with the resonance of 2-thiocarbonyl
(183.3–183.8 ppm) and 4-carbonyl (171.8–174.8 ppm) groups.
In 1H NMR spectra of compounds 7a-w, the presence of a singlet
attributable to the 5-methylidene proton at 6.53–7.23 ppm along with
the disappearance of the 5-CH2 signal of intermediate 8 confirmed the
structure of compounds. Moreover, the presence of only one set of
signals in the NMR spectra of compounds 7 indicated that only the Z
isomer was obtained.
2.3. Kinetic studies
Kinetic studies were carried out to determine the type of inhibition
of selected compounds 7d, 7e, 7f, 7h, 7i, and 7r. The double reciprocal
plots (Fig. 3) obtained using increasing concentrations of inhibitors
demonstrated that compounds 7d, 7f, 7h, and 7r behave as pure non-
2.2. PTP1B inhibition
120
100
80
60
40
20
0
The in vitro evaluation of the PTP1B inhibitory ability of 3-aryl-5-
arylidene-2-thioxo-4-imidazolidinones (7a-w) was carried out using the
human recombinant enzyme and p-nitrophenylphosphate as substrate.
Most of compounds 7a-w showed to be noteworthy inhibitors of the
target enzyme (Table 1). Compounds 7a-c, 7e, 7f, 7l, and 7w were the
best PTP1B inhibitors, with IC50 values in the low micromolar range
(< 10 μM). Analogues 7d, 7g-i, 7n, 7r and 7v also showed appreciable
activity, with IC50 values slightly higher than 10 μM (Table 1).
The 5-arylidene moiety showed to play a critical role in the in-
hibition of PTP1B. In fact, the inhibitory effectiveness of 3-phenyl
substituted derivatives 7e-w showed to be sensitive to the nature and
position of substituents on the 5-benzylidene ring. The introduction of
an additional aryl ring on the 5-benzylidene moiety (compounds 7e-7i)
or an extended aromatic group (compounds 7v, 7w) on C-5 of the
imidazolidinone scaffold generally proved to enhance the affinity to-
ward the target enzyme. The interesting inhibitory potency of 5-(4-
phenyl)benzylidene substituted compound 7e (IC50 = 2.9 μM)
Ctr 7d 7e
7f
7h
7i
7r
Fig. 2. Inhibition reversibility assay of compounds 7d, 7e, 7f, 7h, 7i and 7r.
Control experiments were carried out by adding DMSO without inhibitor. Data
obtained were normalized respect to the control. Data reported in the fig. re-
present the mean value
S.E.M. of three replicates. Additional details are
described in the experimental section.
4

R. Ottanà, et al.
BioorganicChemistry92(2019)103211
A (7d)
C (7f)
E (7i)
B (7e)
D (7h)
F (7r)
60
50
40
30
20
10
10
8
6
4
2
-0.5
0.0
0.5
1.0
1.5
2.0
-0.5
0.0
0.5
1.0
1.5
2.0
1/pNPP (mM-1)
1/[pNPP] mM-1
30
25
20
15
10
5
20
16
12
8
4
-0.5
0.0
0.5
1.0
1.5
2.0
-0.5
0.0
0.5
1.0
1.5
2.0
1/[pNPP] (mM-1)
1/pNPP (mM-1)
25
16
12
8
20
15
10
5
4
-0.5
0.0
0.5
1.0
1.5
2.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
1/pNPP (mM-1)
1/pNPP (mM-1)
Fig. 3. Double reciprocal plots obtained using PTP1B. (A) compound 7d: ■, 0 μM; ○, 6 μM ; ▴, 12 μM; ∇, 24 μM. (B) compound 7e: ■, 0 μM; ○, 1 μM ; ▴, 3 μM; ∇,
5 μM. (C) compound 7f: ■, 0 μM; ○, 2.5 μM ; ▴, 5 μM; ∇, 10 μM. (D) compound 7 h: ■, 0 μM; ○, 3 μM ; ▴, 5 μM; ∇, 7 μM. (E) compound 7i: ■, 0 μM; ○, 1 μM ; ▴,
2 μM; ∇, 4 μM. (F) compound 7r: ■, 0 μM; ○, 2.5 μM ; ▴, 5 μM; ∇, 7.5 μM.
Scheme 2. (A) non-competitive inhibition; (B)
mixed type non-competitive inhibition. E, free
enzyme; ES, enzyme-substrate complex; EI, en-
zyme-inhibitor complex; ESI, enzyme-substrate-
inhibitor ternary complex; P, reaction product; Ks,
dissociation constant of enzyme-substrate com-
plex; Ki, dissociation constant of EI complex; αKs
and αKi, dissociation constants of enzyme-sub-
strate-inhibitor complex (α > 1).
competitive inhibitors (see Scheme 2A). In fact, experimental data ob-
tained using increasing inhibitor concentrations describe straight lines
intersecting one each other in a point on the x axis (Fig. 3A, C, D and F).
Moreover, the tested compounds determine the decrease of Vmax,
without affecting Km values appreciably (see Supplementary material).
On the other hand, we found that compounds 7e and 7i show a dif-
ferent behaviour, since they affect both Km and Vmax parameters (see
Supplementary material). Moreover, double reciprocal plot analysis
revealed that data points described straight lines intersecting one each
other in a point located in the left quadrant (Fig. 3B and E). Together,
these evidences showed that compounds 7e and 7i behave as mixed-
type non-competitive inhibitors (Scheme 2B).
Among the pure non-competitive inhibitors, compounds 7f, 7h, and
7r bear a substituent in the meta position of the 5-benzylidene ring,
while the mixed-type inhibitors 7e and 7i are para substituted analo-
gues. This might suggest that the substitution pattern of the 5-arylidene
5

R. Ottanà, et al.
BioorganicChemistry92(2019)103211
confirmed that compounds 7e and 7f did not impair cell viability (data
not shown).
Then, we analysed the effect of 7e and 7f on insulin cell signalling
pathway. HepG2 cells were incubated with both compounds for 1 h,
and then stimulated (lanes 3 and 4) or not (lanes 1 and 2) with insulin
(Fig. 9). After treatment, the phosphorylation levels of Akt, which is a
donwnstream protein of the insulin signalling pathway, was determined
using specific antibodies (Fig. 9). We found that: (i) in the absence of
insulin, treatment with compound 7e slightly increases the phosphor-
ylation levels of Akt (lanes 9 and 10); (ii) compound 7e enhances the
effectiveness of insulin, as demonstrated by the strong increase of
phosphorylation levels of Akt in cells pre-incubated with compound 7e
and then stimulated with insulin (lanes 11 and 12). These results clearly
indicate that, in HepG2 cells, compound 7e acts as a potent insulin-
sensitizing agent, also showing weak insulin mimetic activity. Con-
versely, no relevant increase of phosphorylation levels of Akt was de-
tected after treatment of the tested liver cells with compound 7f, either
when it was administrated alone (lanes 5 and 6) or in combination with
insulin (lanes 7 and 8) (Fig. 9).
Fig. 4. PTP1B structure (backbone) based on a crystal structure (PDB code
1Q6T) [32]; catalytic loop highlighted in violet, lipophilic loop belonging to the
recently suggested allosteric site in yellow, connecting beta strand in pink.
3. Conclusions
PTP1B plays critical roles in controlling insulin and leptin signaling
pathways and it has been demonstrated that its inhibition or deletion
can improve tissue response to these hormones, providing a promising
therapeutic strategy aimed to counteract the development of diabetes,
obesity, neuroinflammation and cancer.
moiety represents a structural feature that is able to influence the action
mechanism of these inhibitors. The aryl group on N-3 of the imidazo-
lidinone scaffold might also play a role in determining the inhibition
mechanism. In fact, compound 7d, although bearing a phenoxy group
in the para position of the 5-benzylidene ring, behaved as a pure non-
competitive inhibitor. It could be inferred that the 1-naphtyl group on
N-3 may affect the different interaction mode with the enzyme, com-
pared to the phenyl one.
Considering that the highly conserved polar catalytic site poses
challenges in the identification of drug-like inhibitors, the identification
of a new non-catalytic site represents an intriguing opportunity for the
design of non-competitive PTP1B inhibitors potentially endowed with
better bioavailability, compared to pTyr-mimetic inhibitors directed to
the active site.
In continuing our search for PTP1B inhibitors, we designed and
synthesized novel 3-aryl-5-arylidene-2-thioxo-4-imidazolidinones (7) as
isosteres of selected 4-[(5-arylidene-4-oxo-2-thioxothiazolidin-3-yl)
methyl]benzoic acids 2, which had proved to be effective allosteric
inhibitors of PTP1B [37]. 4-imidazolidinone derivatives 7 were de-
signed as non-carboxylate analogues of 2 lacking of the benzoic acid
moiety responsible for active site-directed enzyme inhibition. More-
over, the calculated Lipinski “rule of five” [48] parameters of com-
pounds 7 (see Supplementary material) appear to be consistent with
suitable drug-like properties.
2.4. Molecular modelling study
The main structural difference between the previously investigated
PTP1B inhibitors (1–6) and compounds 7 is the lack of a carboxyl pTyr-
mimetic moiety. Due to the polar properties of the PTP1B active site
[47], the lack of a terminal ionisable group in the new compounds
should direct preferentially to an allosteric mechanism of inhibition.
Accordingly to our design hypothesis and kinetic study reported above,
docking experiments revealed that, for compounds 7, consistent poses
can only be found in the allosteric site positioned between Leu71-Lys73
and Leu204-Pro210 that we reported in our previous studies (Fig. 4)
[37,38]. In this site, the aromatic moiety in position 3 of the 2-thioxo-4-
imidazolidinone core is buried into the lipophilic pocket delimitated by
Pro206 and Pro210, while the 4-carbonyl group forms a hydrogen bond
with the side chain of Ser80 and Lys73. The 2-thiocarbonyl group in-
teracts with the lipophilic pocket and could form weak hydrogen bonds
with the side chain of Gln78. Figs. 5 to 8 depict this interaction mode
for compounds 7f, 7h, 7i and 7r. Compound 7f shows a slightly more
compact binding mode than compounds 7h and 7i, due to the shorter
substituent, resulting in higher shape complementarity of the protein
and the bound ligand, which could explain its higher activity.
The results of inhibition assays indicated that most of compounds 7
were interesting inhibitors of PTP1B. Compounds 7a-c, 7e, 7f, 7l, and
7w proved to inhibit PTP1B in the low micromolar range. The nature
and position of substituents on 5-arylidene moiety as well as on N-3
were shown to play a critical role in the interaction with PTP1B en-
zyme. The presence of an additional aryl ring on the 5-benzylidene
group or a naphthyl moiety generally proved to improve enzyme in-
hibition. Moreover, the substituents on the 5-arylidene moiety of
compounds 7 displayed to influence the mechanism of PTP1B inhibi-
tion.
Notably, kinetic studies highlighted that compounds 7f, 7h and 7r
act as reversible, pure non-competitive inhibitors binding to the allos-
teric site positioned between Leu71-Lys73 and Leu204-Pro210 that we
identified in a previous study [37]. Compounds 7e and 7i behave as
reversible, mixed-type non-competitive inhibitors.
2.5. Ex vivo assay
Compounds 7e and 7f, which were shown to be the most potent in
vitro PTP1B inhibitors among compounds 7, were selected to be eval-
uated in human liver HepG2 cell cultures. First, we evaluated the cy-
totoxicity of these compounds using MTT assay. To this aim, HepG2
cells were incubated in the presence of both compounds for 24 h, and
then analysed to evaluate cells viability. The results of this assay
Tests carried out on human liver HepG2 cells showed that 3-phenyl-
5-(4-phenylbenzylidene)-2-thioxo-4-imidazolidinone (7e) possesses
significant insulin-sensitizing activity. Indeed, when compound 7e was
administered in combination with insulin, a very strong increase of Akt
phosphorylation levels was observed. Conversely, treatment of HepG2
cells with compound 7e alone causes only a slight increase in Akt
6

R. Ottanà, et al.
BioorganicChemistry92(2019)103211
Fig. 5. Docking pose of compound 7f in an allosteric binding site of PTP1B. Surface coloring by hydrophilicity (cyan)/lipophilicity (sand); hydrogen bonds depicted
as yellow lines.
phosphorylation. Taken together, these results suggested that com-
pound 7e behaves mainly as a potent insulin-sensitizing agent. Notably,
the insulin-sensitizing activity of compound 7e was shown to be
markedly higher than those of thiazolidinone analogues that we pre-
viously investigated [37,42,43]. This finding suggests that compound
7e can be assumed as a promising lead compound of a new class of non-
competitive inhibitors of PTP1B.
starting point to further develop novel drug candidates allosterically
targeted to this phosphatase.
4. Materials and methods
4.1. Chemistry
Since non-competitive inhibition of PTP1B is considered an attrac-
tive approach to be exploited with the aim of improving the drugg-
ability of this target enzyme, this result represents an interesting
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). Rf values were determined by using a mixture of
Fig. 6. Docking pose of compound 7h in an allosteric binding site of PTP1B. Surface coloring by hydrophilicity(cyan)/lipophilicity(sand); hydrogen bonds depicted
as yellow lines.
7

R. Ottanà, et al.
BioorganicChemistry92(2019)103211
Fig. 7. Docking pose of compound 7i in an allosteric binding site of PTP1B. Surface coloring by hydrophilicity (cyan)/lipophilicity (sand); hydrogen bonds depicted
as yellow lines.
diethyl ether/n-hexane (6/4) as eluent. Combustion analyses (C, H, N),
determined by means of a C. Erba mod. 1106 elem. Analyzer, were
within 0.4% of the theoretical values. 1H and 13C NMR spectra were
recorded on a Varian 300 MHz spectrometer operating at frequencies of
300.13 and 75.47 MHz, respectively, or on a Varian 500 MHz spectro-
meter operating at 499.74 and 125.73 MHz, respectively. Chemical
shifts δ are given in ppm and coupling constants are expressed in Hz. All
the spectra were phased, baseline was corrected where necessary and
DMSO‑d6 signals were used as reference for both 1H and 13C spectra. All
exchangeable protons were confirmed by addition of D2O. Unless stated
otherwise, all materials were obtained from commercial suppliers and
used without further purification. The purity of synthetic compounds
was established as ≥95% by combustion analysis.
synthesised according to our previously reported procedure and their
chemical-physical data are reported in Ref. 44.
Compounds 7e, 7i, 7p, 7s, 7u, 7v, 7w are commercially available
and CAS numbers are assigned; however, their synthetic procedures,
chemical properties and NMR characterization are not available in lit-
erature (except the synthesis and 1H NMR data of 7p [49]).
4.1.1. General procedure for the synthesis of 3-aryl-2-thioxo-4-
imidazolidinones (8b-d)
A mixture of the appropriate aryl isothiocyanate (5.32 mmol) and
glycine (6.66 mmol) in hydroalcoholic (35% water vol/vol) solution
was heated under reflux for 24 h. The solvent was then evaporated
under reduced pressure. The solid residue was washed, dried and re-
crystallized in methanol providing pure compounds 8.
Compounds 7d, 7f, 7g, 7j, 7k, 7l, 7o, 7q, 7r, 8a and 8e were
Fig. 8. Docking pose of compound 7r in an allosteric binding site of PTP1B. Surface coloring by hydrophilicity (cyan)/lipophilicity (sand); hydrogen bonds depicted
as yellow lines.
8

R. Ottanà, et al.
BioorganicChemistry92(2019)103211
with water and dried. The crystallization from methanol of the crude
A
1
2
3
4
5
6
7
8
9
10 11 12
lane
pAkt
product provided pure compounds 7.
4.1.2.1. (5Z)-3-(4-Fluorophenyl)-5-(4-phenoxybenzylidene)-2-thioxo-4-
imidazolidinone (7a). Yield 54%; Rf 0.85; mp 219–222 °C; 1H NMR
(DMSO‑d6): δ 6.69 (s, 1H, CH methylidene); 7.04 (m, 2H, arom); 7.11
(m, 2H, arom); 7.22 (m, 1H, arom); 7.35 (m, 2H, arom); 7.43–7.47 (m,
4H, arom); 7.85 (m, 2H, arom). 13C NMR (DMSO‑d6): δ 113.4, 116.4
(J = 23.9 Hz), 118.8, 120.1, 124.9, 126.1, 127.9, 130.2, 130.9, 131.7
(J = 8.8 Hz), 133.1, 156.2, 158.7, 162.5 (J = 245.2 Hz), 164.5, 178.8.
Anal. (C22H15FN2O2S) calcd: C 67.68; H 3.87; N 7.18; found: C 68.01; H
3.99; N 6.98.
Akt
Acꢀn
Insulin
7e
-
-
-
+
-
-
-
+
-
-
+
-
+
+
-
7f
-
+
+
4.1.2.2. (5Z)-3-(4-Methoxyphenyl)-5-(4-phenoxybenzylidene)-2-thioxo-4-
imidazolidinone (7b). Yield 30%; Rf 0.73; mp 227–230 °C; 1H NMR
(DMSO‑d6): δ 3.80 (s, 3H, OCH3); 6.67 (s, 1H, CH methylidene); 7.04
(m, 4H, arom); 7.10 (m, 2H, arom); 7.22 (m, 1H, arom); 7.26 (m, 2H,
arom); 7.45 (m, 2H, arom); 7.85 (m, 2H, arom). 13C NMR (DMSO‑d6): δ
55.4, 112.4, 114.0, 118.1, 119.4, 124.3, 125.5, 125.8, 127.4, 129.9,
130.3, 132.4, 155.6, 158.0, 159.3, 164.1, 178.8. Anal. (C23H18N2O3S)
calcd: C 68.64; H 4.51; N 6.96; found: C 68.50; H 4.27; N 7.14.
B
14000
12000
10000
8000
6000
300
200
100
0
4.1.2.3. (5Z)-3-(4-Methylthiophenyl)-5-(4-phenoxybenzylidene)-2-thioxo-
4-imidazolidinone (7c). Yield 63%; Rf 0.77; mp 239–242 °C; 1H NMR
(DMSO‑d6): δ 2.53 (s, 3H, SCH3); 6.68 (s, 1H, CH methylidene); 7.03
(m, 2H, arom); 7.10 (m, 2H, arom); 7.22 (m, 1H, arom); 7.29–7.39 (m,
4H, arom); 7.45 (m, 2H, arom); 7.85 (m, 2H, arom). 13C NMR
(DMSO‑d6): δ 15.1, 113.0, 118.6, 119.9, 125.9, 126.5, 127.8, 129.4,
129.9, 130.4, 131.0, 132.7, 139.8, 151.3, 158.5, 164.3, 178.8. Anal.
(C23H18N2O2S2) calcd: C 66.00; H 4.33; N 6.69; found: C 66.19; H 4.18;
N 6.88.
+
+
-
+
-
-
-
-
+
-
-
+
-
-
-
Insulin 10 nM
Compound 7f
Compound 7e
+
-
+
Fig. 9. Phosphorylation levels of Akt in human liver HepG2 cells treated with
compounds 7e and 7f. HepG2 cells were starved for 20 h, and incubated in the
presence of 50 μM of compounds 7e and 7f for 1 h at 37 °C. After, cells were
stimulated with 10 nM insulin, and then lysed to evaluate phosphorylation le-
vels of Akt. (A), western blot; (B), quantification of western blot. Data reported
in the fig. represent the mean values
S.E.
4.1.2.4. (5Z)-3-Phenyl-5-(4-phenylbenzylidene)-2-thioxo-4-
imidazolidinone (7e). (CAS 740814–34-0) Yield 60%; Rf 0.78; mp
273–275 °C; 1H NMR (DMSO‑d6): δ 6.69 (s, 1H, CH methylidene);
7.35–7.40 (m, 2H, arom); 7.46–7.53 (m, 3H, arom); 7.74–7.78 (m, 5H,
arom); 7.93 (m, 2H, arom); 8.16 (m, 2H, arom); 12.67 (brs, 1H, NH).
13C NMR (DMSO‑d6): δ 112.6, 126.3, 126.8, 127.0, 128.1, 128.9, 129.1,
131.1, 131.5, 133.3, 139.2, 140.9, 164.0, 178.6. Anal. (C22H16N2OS)
calcd: C 74.13; H 4.52; N 7.86; found: C 73.97; H 4.38; N 7.98.
4.1.1.1. 3-(4-Fluorophenyl)-2-thioxo-4-imidazolidinone (8b). Yield 54%;
Rf 0.25; m.p. 227–230 °C; 1H NMR (DMSO‑d6): δ 4.28 (s, 2H, CH2); 7.32
(m, 2H, arom); 7.34 (m, 2H, arom); 10.46 (brs, 1H, NH). 13C NMR
(DMSO‑d6):
δ
49.7, 116.2 (J = 23.25), 130.3, 131.6, 162.3
(J = 243.75), 172.8, 183.8. Anal. (C9H7FN2OS) calcd: C 51.42; H
3.36; N 13.33; found: C 51.74; H 3.51; N 13.17.
4.1.2.5. (5Z)-5-(3-Benzyloxybenzylidene)-3-phenyl-2-thioxo-4-
4.1.1.2. 3-(4-Methoxyphenyl)-2-thioxo-4-imidazolidinone
(8c). Yield
imidazolidinone (7h). Yield 57%; Rf 0.80; mp 197–200 °C; 1H NMR
(DMSO‑d6): δ 5.19 (s, 2H, OCH2), 6.65 (s, 1H, CH methylidene); 7.09
(m, 1H, arom); 7.38–7.53 (m, 13H, arom). 13C NMR (DMSO‑d6): δ 69.5,
113.0, 116.3, 116.4, 123.30, 126.7, 128.0, 128.1, 128.7, 128.9, 129.1,
130.2, 133.4, 133.7, 137.0, 158.8, 164.1, 178.9. Anal. (C23H18N2O2S)
calcd: C 71.48; H 4.69; N 7.25; found: C 71.65; H 4.84; N 7.03.
82%; Rf 0.23; m.p. 222 °C; 1H NMR (DMSO‑d6): 3.87 (s, 3H, OCH3);
4.33 (s, 2H, CH2); 7.09 (m, 2H, arom); 7.26 (m, 2H, arom); 10.40 (brs,
1H, NH). 13C NMR (DMSO‑d6): δ 55.1, 55.6, 113.9, 129.8, 130.0, 159.1,
172.3, 183.7. Anal. (C10H10N2O2S) calcd: C 54.04; H 4.53; N 12.60;
found: C 53.89; H 4.44; N 12.78.
4.1.1.3. 3-(4-Thiomethylphenyl)-2-thioxo-4-imidazolidinone (8d). Yield
75%; Rf 0.24; m.p. 232–235 °C; 1H NMR (DMSO‑d6): 2.32 (s, 3H,
SCH3); 4.28 (s, 2H, CH2); 6.53 (m, 2H, arom); 7.04 (m, 2H, arom); 9.93
(brs, 1H, NH). 13C NMR (DMSO‑d6): δ 15.9, 55.0, 122.0, 130.5, 135.9,
139.1, 173.5, 182.5. Anal. (C10H10N2OS2) calcd: C 50.40; H 4.23; N
11.75; found: C 50.28; H 4.33; N 11.88.
4.1.2.6. (5Z)-5-(4-Benzyloxybenzylidene)-3-phenyl-2-thioxo-4-
imidazolidinone (7i). (CAS 469896–60-4) Yield 56%; Rf 0.75; mp
233–235 °C; 1H NMR (DMSO‑d6): δ 5.19 (s, 2H, OCH2), 6.67 (s, 1H,
CH methylidene); 7.10 (m, 2H, arom); 7.39–7.49 (m, 10H, arom); 7.82
(m, 2H, arom); 12.51 (brs, 1H, NH). 13C NMR (DMSO‑d6): δ 69.4, 113.5,
115.3, 124.5, 125.1, 127.9, 128.0, 128.5, 128.9, 132.4, 133.4, 136.7,
159.5, 164.0, 178.0. Anal. (C23H18N2O2S) calcd: C 71.48; H 4.69; N
7.25; found: C 71.73; H 4.89; N 7.12.
4.1.2. General procedure for the synthesis of (5Z)-3-aryl-5-arylidene-2-
thioxo-4-imidazolidinones (7a-w)
A mixture of 3-aryl-2-thioxo-4-imidazolidinone 8 (2.6 mmol), the
appropriate arylaldehyde (2.6 mmol) and piperidine (2.1 mmol) in
ethanol (20 ml) was refluxed for 4–5 h until the reagent disappearance.
The reaction mixture was cooled and poured into water acidified with
AcOH (pH 3–4). The precipitate was separated by filtration, washed
4.1.2.7. (5Z)-5-(3-Trifluoromethoxybenzylidene)-3-phenyl-2-thioxo-4-
imidazolidinone (7m). Yield 38%; Rf 0.76; mp 140–143 °C; 1H NMR
(DMSO‑d6): δ 6.71 (s, 1H, CH methylidene); 7.36–7.39 (m, 3H, arom);
7.44–7.47 (m, 1H, arom), 7.49–7.52 (m, 2H, arom), 7.54–7.58 (m, 1H,
arom), 7.79–7.81 (m, 1H, arom), 7.84 (s, 1H, arom). 13C NMR
9

R. Ottanà, et al.
BioorganicChemistry92(2019)103211
(DMSO‑d6): δ 111.1, 120.7 (J = 256.6 Hz), 122.0, 122.9, 128.0, 129.3,
129.4, 130.0, 131.2, 133.7, 135.2, 149.3, 164.4, 179.5. Anal.
(C17H11F3N2O2S) calcd: C 56.04; H 3.04; N 7.69; found: C 55.81; H
2.79; N 7.92.
NH). 13C NMR (DMSO‑d6): δ 113.0, 126.7, 126.8, 127.4, 127.5, 127.7,
128.4, 128.6, 128.9, 129.0, 130.0, 130.1, 133.0, 133.1, 133.4, 164.0,
178.9. Anal. (C20H14N2OS) calcd: C 72.70; H 4.27; N 8.48; found: C
72.51; H 4.09; N 8.60.
4.1.2.8. (5Z)-5-(4-Trifluoromethylthiobenzylidene)-3-phenyl-2-thioxo-4-
imidazolidinone (7n). Yield 25%; Rf 0.87; mp 186–189 °C; 1H NMR
(DMSO‑d6): δ 6.69 (s, 1H, CH methylidene); 7.37 (m, 2H, arom);
7.48–7.51 (m, 3H, arom); 7.75 (m, 2H, arom), 7.91 (m, 2H, arom),
12.70 (brs, 1H, NH). 13C NMR (DMSO‑d6): δ 110.9, 123.9, 128.5, 129.2,
129.3, 130.0 (J = 309.3 Hz), 131.7, 133.6, 135.8, 136.6, 164.2, 179.7.
Anal. (C17H11F3N2OS2) calcd: C 53.68; H 2.91; N 7.36; found: C 53.95;
H 3.06; N 7.52.
4.2. Docking studies
In order to clarify the preferred inhibition mode and binding in-
teractions of the tested 5-arylidene-2-thioxo-4-imidazolidinones 7,
compounds 7f, 7g-i, 7l, 7o, 7r, 7v, and 7w were docked into a crystal
structure of PTP1B (PDB code 2VEV) [50].
Docking was performed with the software GOLD [51] version 5.2
allowing binding to the whole protein surface. The protein and ligand
structures were prepared prior to docking using MOE [52] 2015.10. For
each ligand 25 poses were generated and analyzed after energy mini-
mization of the ligand in the respective pose together with the sur-
rounding protein residues with MOE using the Amber12:EHT forcefield.
3D representations were generated with MOE.
4.1.2.9. (5Z)-5-(3,4-Dihydroxybenzylidene)-3-phenyl-2-thioxo-4-
imidazolidinone (7p). (CAS 897340–70-4) Yield 21%; Rf 0.13; mp
186–189 °C; 1H NMR (DMSO‑d6): δ 6.53 (s, 1H, CH methylidene),
6.80 (s, 1H, arom), 7.17–7.21 (m, 2H, arom); 7.33–7.36 (m, 2H, arom),
7.43–7.52 (m, 3H, arom); 9.20 and 9.79 (2 brs, 2H, OH); 12.40 (s br,
1H, NH). 13C NMR (DMSO‑d6): δ 115.4, 116.5, 118.5, 123.9, 124.3,
124.4, 129.2, 129.3, 129.4, 134.0, 146.0, 148.5, 164.4, 178.3. Anal.
(C16H12N2O3S) calcd: C 61.53; H 3.87; N 8.97; found: C 61.78; H 3.64;
N 9.09.
4.3. Biology
4.3.1. Expression and purification of recombinant human PTP1B
Recombinant, human PTP1B enzyme was expressed as fusion pro-
tein in E. coli and purified as previously described [37]. Briefly, E. coli
TB1 strain was transformed with pGEX-2T bacterial expression vector
harbouring the complete sequence of PTP1B cloned downstream the
Glutathione-S-Transferase (GST) sequence. Transformed bacteria were
grown until 0.8 O.D. and then stimulated for two hours with 0.1 mM
isopropyl-thiogalactoside (IPTG) to induce expression of fusion protein
(GST-PTP1B). Then, bacteria were collected by centrifugation and
stored at −20 °C. The fusion protein was purified from bacterial lysate
by affinity chromatography, using a column loaded with glutathione-
agarose resin. After elution from column, fractions containing fusion
protein were collected, concentrated and incubated with 5 U.I. of bo-
vine thrombin (Sigma Aldrich T7513) for 3 h at 37 °C. Finally, PTP1B
was purified from GST and thrombin by gel filtration using a Superdex
G75 column. The enzyme purity of purified protein was determined by
SDS-PAGE.
4.1.2.10. (5Z)-5-(3,4,5-Trimethoxybenzylidene)-3-phenyl-2-thioxo-4-
imidazolidinone (7s). (CAS 94452–09-2) Yield 59%; Rf 0.39; mp
186–189 °C; 1H NMR (DMSO‑d6): δ 3.70 (s, 3H, OCH3), 3.86 (s, 6H,
OCH3), 6.63 (s, 1H, CH methylidene), 7.03 (s, 2H, arom), 7.35–7.37 (m,
2H, arom); 7.44–7.47 (m, 1H, arom), 7.49–7.52 (m, 2H, arom); 12.66
(brs, 1H, NH). 13C NMR (DMSO‑d6): δ 56.3, 60.2, 108.1, 113.8, 125.6,
127.9, 128.9, 133.4, 139.0, 153.1, 164.0, 178.5. Anal. (C19H18N2O4S)
calcd: C 66.61; H 4.90; N 7.56; found: C 66.90; H 5.11; N 7.68.
4.1.2.11. (5Z)-5-(4-Fluoro-3-methoxybenzylidene)-3-phenyl-2-thioxo-4-
imidazolidinone (7t). Yield 55%; Rf 0.73; mp 217–220 °C; 1H NMR
(DMSO‑d6): δ 3.94 (s, 3H, OCH3); 6.68 (s, 1H, CH methylidene);
7.27–7.55 (3 m, 8H, arom). 13C NMR (DMSO‑d6): δ 56.4, 112.4,
115.7, 116.4, 116.5, 123.7, 126.5, 128.9, 129.0, 129.6, 133.4, 147.5,
151.2, 153.2, 164.1, 178.8. Anal. (C17H13FN2O2S) calcd: C 62.18; H
3.99; N 8.53; found: C 61.95; H 3.87; N 8.69.
4.3.2. Enzymatic assays
The assays were carried out at 37 °C in 0.075 M β,β-dimethylglutaric
acid buffer pH 7.0 containing 1 mM EDTA and 0.1 mM DTT. The p-
nitrophenylphosphate (p-NPP) was used as substrate; the final volume
of tests was 1 ml. Reactions were started by diluting an aliquot of en-
zyme in the assay solution, and stopped after an appropriate time di-
luting reaction solution with 2 ml of 0.1 M KOH. The amount of p-NPP
released was determined using a spectrophotometer, reading the ab-
sorbance of solutions at 400 nm (ε = 18000 M−1 cm−1).
4.1.2.12. (5Z)-5-(4-Acetylaminobenzylidene)-3-phenyl-2-thioxo-4-
imidazolidinone (7u). (CAS 41534–94-5) Yield 51%; Rf 0.05; mp
277–280 °C; 1H NMR (DMSO‑d6): δ 2.06 (s, 3H, CH3), 6.62 (s, 1H, CH
methylidene); 7.37 (m, 2H, arom), 7.49 (m, 2H, arom), 7.65 (m, 2H,
arom), 7.77 (m, 2H, arom), 10.17 (brs, 1H, NH), 12.49 (brs, 1H, NH).
13C NMR (DMSO‑d6): δ 20.6, 101.5, 118.9, 125.4, 127.3, 128.9, 129.6,
129.7, 131.5, 141.0, 164.4, 169.2, 178.6. Anal. (C18H15N3O2S) calcd: C
64.08; H 4.48; N 12.45; found: C 64.21; H 4.36; N 12.63.
Preliminary screening and the IC50 values were both carried out
using a fixed substrate concentration (2.5 mM) corresponding to the Km
value of PTP1B. The IC50 values were determined measuring residual
activity of enzyme in the presence of increasing inhibitor concentration.
All tests were carried out in triplicate. Data obtained were fitted using
the following equation:
4.1.2.13. (5Z)-5-(Naphtalen-1-ylmethylidene)-3-phenyl-2-thioxo-4-
imidazolidinone (7v). (CAS 61442–32-8) Yield 72%; Rf 0.82; mp
108–110 °C; 1H NMR (DMSO‑d6): δ 7.23 (s, 1H, CH methylidene);
7.37–7.61 (m, 8H, arom); 7.86 (m, 1H, arom); 7.97 (m, 2H, arom); 8.10
(m, 1H, arom). 13C NMR (DMSO‑d6): δ 109.5, 123.8, 125.9, 126.5,
127.2, 128.6, 128.8, 128.9, 129.0, 129.7, 131.2, 133.4, 163.6, 178.8.
Anal. (C20H14N2OS) calcd: C 72.70; H 4.27; N 8.48; found: C 72.93; H
4.50; N 8.31.
Vi
Max Min
=
slope
x
V 0
1 +
(
)
IC
50
where Vi is the activity measured in the presence of the inhibitor, V0 the
activity of enzyme measured in the absence of inhibitor, and “x” is the
concentration of inhibitor.
4.1.2.14. (5Z)-5-(Naphtalen-2-ylmethylidene)-3-phenyl-2-thioxo-4-
imidazolidinone (7w). (CAS 61442–35-1) Yield 52%; Rf 0.82; mp
210–213 °C; 1H NMR (DMSO‑d6): δ 6.80 (s, 1H, CH methylidene);
7.36–7.57 (m, 10H, arom); 7.83–7.98 (m, 2H, arom); 12.74 (brs, 1H,
Dilution assays were carried out to evaluate the action mechanism
of inhibitors [37]. The enzyme was incubated in the presence of
10

R. Ottanà, et al.
BioorganicChemistry92(2019)103211
saturating amount of each inhibitor for 1 h at 37 °C. Then, aliquot of
samples was diluted 400 folds in the assay buffer to evaluate residual
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Ex vivo tests were carried out using human liver HepG2 cells ob-
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This work was supported by University of Messina and University of
Florence.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103211.
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