Synthetic thiosemicarbazones as a new class of Mycobacterium tuberculosis protein tyrosine phosphatase A inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.10.030

Mycobacterium tuberculosis secretes two protein tyrosine phosphatases as virulence factors, PtpA and PtpB. Inhibition studies of these enzymes have shown significant attenuation of the M. tuberculosis growth in vivo. As PtpA mediates many effects on the r

Full text of DOI:10.1016/j.bmc.2018.10.030

Accepted Manuscript
Synthetic thiosemicarbazones as a new class of Mycobacterium tuberculosis
protein tyrosine phosphatase A inhibitors
Larissa Sens, Ana Caroline Arruda de Souza, Lucas Antonio Pacheco, Angela
Camila Orbem Menegatti, Mattia Mori, Alessandra Mascarello, Ricardo José
Nunes, Hernán Terenzi
PII:
S0968-0896(18)31299-9
https://doi.org/10.1016/j.bmc.2018.10.030
BMC 14589
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Accepted Date:
20 July 2018
1 October 2018
26 October 2018
Please cite this article as: Sens, L., de Souza, A.C.A., Pacheco, L.A., Menegatti, A.C.O., Mori, M., Mascarello, A.,
Nunes, R.J., Terenzi, H., Synthetic thiosemicarbazones as a new class of Mycobacterium tuberculosis protein
tyrosine phosphatase A inhibitors, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2018.10.030
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Bioorganic & Medicinal Chemistry
Synthetic thiosemicarbazones as a new class of Mycobacterium tuberculosis protein
tyrosine phosphatase A inhibitors
Larissa Sensa, Ana Caroline Arruda de Souzab, Lucas Antonio Pachecoa, Angela Camila Orbem
Menegattib, Mattia Moric, Alessandra Mascarelloa, Ricardo José Nunesa and Hernán Terenzib
aLaboratório Estrutura e Atividade, Departamento de Química, LEAT-CFM-UFSC, Universidade Federal de Santa Catarina, Campus Trindade, 88040-900
Florianópolis, SC, Brazil
bCentro de Biologia Molecular Estrutural, Departamento de Bioquímica, CEBIME-UFSC, Universidade Federal de Santa Catarina, Campus Trindade, 88040-
900, Florianópolis, SC, Brazil
CDepartment of Biotechnology, Chemistry and Pharmacy, University of Siena. Via Aldo Moro 2, 53100 Siena, Italy
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Mycobacterium tuberculosis secretes two protein tyrosine phosphatases as virulence factors,
PtpA and PtpB. Inhibition studies of these enzymes have shown significant attenuation of the M.
tuberculosis growth in vivo. As PtpA mediates many effects on the regulation of host signaling
ensuring the intracellular survival of the bacterium we report, for the first time,
thiosemicarbazones as potential novel class of PtpA inhibitors. Several compounds were
synthesized and biologically evaluated, revealing interesting results. Enzyme kinetic assays
showed that compounds 5, 9 and 18 are non-competitive inhibitors of PtpA, with Ki values
ranging from 1.2 to 5.6 µM. Modeling studies clarified the structure–activity relationships
observed in vitro and indicated a possible allosteric binding site in PtpA structure. To the best of
our knowledge, this is the first disclosure of potent non-competitive inhibitors of PtpA with
great potential for future studies and development of analogues.
Available online
Keywords:
Tuberculosis
PtpA
Inhibitors
Thiosemicarbazones
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB),
an infectious-contagious disease that is one of the top 10 causes
of death worldwide. In 2017, 1.3 million people died from TB
and 10 million became ill.1 For many years, the same first-line
anti-TB drugs have been used to combat the disease, but there are
numerous reports of strains resistant to this treatment. An
alternative is to use a second-line of antibiotics, which has
disadvantages, such as major side-effects, long duration, and high
costs. In addition, cases of strains resistant to second-line anti-TB
drugs have already been reported. 1-3
proteins that modulate the immune response of the host, among
which are protein kinases and phosphatases, such as protein
tyrosine phosphatases (PTPs). 6-9
Two tyrosine phosphatases, PtpA and PtpB, were shown to be
involved in the survival of the bacterium10-12 and that their
inhibition attenuates the growth of Mtb and the infection in
animal models, thus becoming targets for the development of
new anti-TB drugs.8,13-20
Our research group has been focusing efforts on the
development of new, more potent and selective inhibitors of
PtpA and PtpB. Several chalcones have been studied by our
group; some naphthyl chalcones were found to be excellent
inhibitors of PtpA or PtpB15,21,22 and some sulfonyl hydrazones
excellent competitive inhibitors of PtpB.23
As a result, the scientific community has turned its attention to
the research of new TB treatments with different mechanisms of
action from those already in use. Mtb resides within
macrophages, which are the cells responsible for the activation of
the immune response necessary to control and eliminate the
invasion of microorganisms.4,5 Mtb has developed a strategy to
remain alive within macrophages, secreting several immunogenic
———
From this came the interest in studying a novel class of
compounds as PtpA inhibitors. Thiosemicarbazones constitute an
 Corresponding authors. Tel.: +55-48-3721-6426 (HT) +55-89-981450839 (AM); e-mail: hernan.terenzi@ufsc.br,
angelamenegatti@yahoo.com.br

important class of compounds whose properties have been
extensively studied in Medicinal Chemistry,24 displaying a wide
range of biological activities, such as antifungal,25 antitumor,26,27
antiviral,28,29 antimicrobial,30,31 and antibacterial properties.32
enzyme kinetic assays. To provide new insights into the
mechanism of inhibition and binding of PtpA inhibitors we
performed molecular docking simulations.
This class of compounds has been shown to comprise good
enzyme inhibitors, acting, for example, on the enzyme tyrosinase,
which is involved in the development of melanomas,33 and on
cysteine proteases, which are essential for several functions of
the malaria parasite.34 Moreover, the thiosemicarbazone triapine,
a very potent inhibitor of ribonucleotide reductase (RNR), is a
promising molecule for anticancer therapy.35 In more recent
2. Results and discussion
A chemical library of 49 thiosemicarbazones (4–52) was
produced in two stages, according to the scheme on Table 1.
First, the synthesis of thiosemicarbazides 1–3 was performed by
the reaction between different isothiocyanates and hydrazine
hydrate in isopropanol.37 The thiosemicarbazides were used in
subsequent reactions with different aldehydes, using acetic acid
as a catalyst in a solvent mixture of water/ethanol.30 The
synthesized compounds presented yields in the range of 60–90%,
studies,
iminodiacetate−thiosemicarbazones
and
their
dicopper(II) complexes have also been found to be RNR
inhibitors.36
were characterized by melting point (mp), H and 13C nuclear
1
Based on these observations, the aim of this study was to
synthesize thiosemicarbazones as potential PtpA inhibitors that
could be used in TB therapy. The compounds were assayed to
investigate their PtpA inhibitory activity, potency, and
selectivity. In addition, we investigated the inhibition mechanism
involved, using the four most potent thiosemicarbazone in
magnetic resonance (NMR) spectroscopy, and had their
structures confirmed by high-resolution mass spectrometry
(HRMS). Thiosemicarbazones 6, 8, 9, 11–18, 44, 45, 47, 48, 50,
and 51 have not been reported in the literature.
Table 1. Synthesis of thiosemicarbazones 4–52.
Compound
R1
R2
Compound
R1
R2
4
5
6
7
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
Ph
29
30
31
32
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
1-naphthyl
2-naphthyl
3,4-diCl-Ph
2,5-diOCH3-Ph
4-NO2-Ph
4-Cl-Ph
4-OCH3-Ph
8
9
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
3,4-OCH2O-Ph
4-Ph-Ph
3-OCH3,4-OH-Ph
3,4,5-triOCH3-Ph
1-naphthyl
2-naphthyl
3,4-diCl-Ph
2,5-diOCH3-Ph
3,4-diOCH3-Ph
2-NO2-Ph
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
3,4-diOCH3-Ph
2-NO2-Ph
5-(2-Cl,5-CF3)-Ph-furan-2-yl
4-N(CH3)-Ph
Ph
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
4-CH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-CH3-Ph
4-NO2-Ph
4-Cl-Ph
4-OCH3-Ph
3,4-OCH2O-Ph
4-Ph-Ph
3-OCH3,4-OH-Ph
3,4,5-triOCH3-Ph
1-naphthyl
2-naphthyl
3,4-diCl-Ph
2,5-diOCH3-Ph
3,4-diOCH3-Ph
2-NO2-Ph
5-(2-Cl,5-CF3)-Ph-furan-2-yl
4-N(CH3)-Ph
Ph
4-CH3-Ph
4-NO2-Ph
4-Cl-Ph
4-OCH3-Ph
3,4-OCH2O-Ph
4-Ph-Ph
3-OCH3,4-OH-Ph
3,4,5-triOCH3-Ph
4-N(CH3)-Ph
(i) Isopropanol, r.t.; (ii) ethanol/water, acetic acid, reflux; Ph = phenyl.
The spectral characterization of the compounds (4–19 and 37–
52) is detailed in the Experimental and Supplementary
Information Sections. 1H NMR spectra revealed that all structures
had an E configuration, with the N−H chemical shifts appearing
as two singlets at around 10–11 ppm38 and the iminic hydrogen
as a singlet at 7–8 ppm. The synthesis of thiosemicarbazones 20–
36 was previously reported by the authors.39
The inhibitory activity of the thiosemicarbazone derivatives
was evaluated in vitro against recombinant PtpA from Mtb, using
p-nitrophenyl phosphate (pNPP) as substrate. The percentage of

inhibition was assessed spectrophotometrically at a single
concentration (25 μM) for each compound of the series. As
shown in Table 2, several compounds were active against the
enzyme, with the percentage of inhibition reaching 87%.
Specifically, the results indicate that 22 out of 50 compounds (4–
6, 8, 9, 13–19, 22, 25, 26, 29, 30, 35, 36, 39, and 43) exhibited
inhibitory activity against PtpA (inhibition > 25%).
In addition, it is interesting to note the results found for
compounds 12 and 13, both having 1-naphthyl substituent at R1,
but in R2 the groups are 1-naphthyl and 2-naphthyl, respectively.
Compound 12 showed 10% inhibition of PtpA activity while the
thiosemicarbazone 13 showed an inhibition of 68%. In previous
studies of our group it was reported that a chalcone derived from
2-naphthyl was five times more potent than the same derivative
of 1-naphthyl, due to the 2-naphthyl positioning which favored
the pi-stacking interaction.15,40 Observing compounds 30 and 47,
which also have a 2-naphthyl at R2, we did not observe
significant inhibition of PtpA activity, at most 30% inhibition,
this finding indicates that for the thiosemicarbazones studied the
1-naphthyl in R1 is extremely important for the inhibitory
activity.
Among these 22 active compounds, only those that reached
inhibition results higher than 60% (5, 8, 9, 13, 14, 16, 18, 35, and
39) were selected for IC50 assays, which were performed to better
understand the potency involved in the inhibition of PtpA (Table
2). The compounds showed substantial PtpA inhibition, with IC50
values lower than 24 μM (Table 2).
The best PtpA inhibition result was achieved with compound
18 (IC50 = 2.9 ± 1.7 μM), derived from 1-naphthyl and 5-(2-Cl,5-
Table 2. Percentage of PtpA inhibition at the single concentration of 25 μM
and IC50 of the most active compoundsa
CF3)-Ph-furan-2-yl.
A study has previously reported that
Compd
PtpA
IC50
Compd
PtpA
IC50
inhibition (%)
(µM)
inhibition (%)
(µM)
benzanilide derivatives with electron-withdrawing groups at the
meta and para positions result in a substantial improvement in
PtpA inhibition, and that the presence of a trifluoromethyl group
(CF3) leads to higher binding affinity, which is explained by the
pi-stacking with Trp48 of the enzyme’s active site. 41 In addition,
the most potent and selective PtpA inhibitor described in the
literature19 presents a scaffold similar to that described by
Rawls41, with electron-withdrawing groups in its structure.
Compound 18 resembles these inhibitors by the presence of
electron withdrawing groups, but our work differs in that it has
an apolar group that is important for inhibition of PtpA. In that
work, the most active compounds presented hydrophobic groups
in R1 and polar substituents in R2.
4
5
6
7
58.1 ± 5.3
77.7 ± 5.1
0
nd
9.5 ± 3.3
nd
29
30
31
32
33.7 ± 1.0
29.3 ± 1.2
23.4 ± 4.2
2.8 ± 2.2
nd
nd
nd
nd
22.6 ± 3.9
nd
8
62.6 ± 3.0
79.7 ± 1.8
0
17.8 ± 0.6
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
6.6 ± 1.8
nd
nd
9
7.4 ± 2.8
21.6 ± 2.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
nd
66.0 ± 0.6
9.1 ± 5.0
nd
0
nd
35.4 ± 3.4
10.3 ± 1.6
67.7 ± 5.2
83.7 ± 3.5
44.2 ± 2.1
64.5 ± 4.6
52.4 ± 2.0
86.7 ± 0.8
51.6 ± 1.2
16.6 ± 2.3
0.7 ± 7.5
37.2 ± 1.8
4.9 ± 5.6
8.9 ± 2.9
25.9 ± 1.5
26.3 ± 3.3
8.8 ± 1.6
0
nd
0
nd
23.7 ± 2.9
0
nd
13.4 ± 2.2
64.4 ± 3.5
15.1 ± 5.8
nd
nd
1.0 ± 9.3
18.2 ± 2.2
0
nd
Selectivity is a great challenge in the development of PTP
inhibitors since the active site is highly conserved across the
family and most PTPs of pharmaceutical interest have close
human homologs, which would cause adverse effects if inhibited
by non-specific drugs.42 In view of this, the scientific community
has been looking for selective PTP inhibitors through novel
strategies to attack these enzymes, as orthosteric, allosteric, and
oligomerization-inhibiting.13 The four most potent PtpA
inhibitors (compounds 5, 9, 18, and 35) were further evaluated
against PtpB from Mtb, human PTP1B, YopH from Yersinia
enterocolitica, human PTP-PEST, and human LYP (Table 3). In
the selectivity assay, the IC50 of the compounds against the
different phosphatases was determined. The selectivity index (SI)
was represented by the IC50PTP/IC50PtpA ratio.
nd
2.9 ± 1.7
nd
0
41.2 ± 8.4
6.6 ± 1.8
0
nd
nd
nd
nd
nd
nd
22.7 ± 1.8
23.7 ± 7.0
21.4 ± 3.7
0
nd
nd
nd
nd
nd
nd
nd
nd
0
nd
nd
0
nd
nd
3.9 ± 4.0
nd
nd
aData are expressed as means ± SD of three independent experiments. Compd
= compound; nd = not determined.
As shown in Table 3 all evaluated compounds inhibited to
some extent the PTPs assessed. Among Mtb PTPs, the
thiosemicarbazones appear to preferentially inhibit PtpA over
PtpB (SI ≥ 1.9). Regarding human PTP1B the result was similar
to PtpB (SI ≥ 1.8), they preferentially inhibit PtpA, compound 9
showed the most promising specificity result over PTP1B. For
human PTP-PEST and LYP, we noted the most satisfactory
selectivity results, with SI values higher than 6.8. Concerning
YopH, the analyzed thiosemicarbazones had an SI ≥ 0.9,
indicating that they were non-selective over this enzyme. This
does not represent a problem as this phosphatase is involved in
diseases caused by Yersinia spp.43 Compound 9 presented the
most attractive results regarding its specificity for PtpA over
human PTPs, among the other inhibitors highlighted (5, 9, 18 and
35), it is the only one that presents in its structure an apolar group
in R2, which might be the factor that contributes to its specificity
compared to other thiosemicarbazones.
The series that presented
a higher number of active
compounds had a 1-naphthyl substituent in R1, thus this group
seems to be important for thiosemicarbazones activity against
PtpA. This was an interesting result, in a previous study by our
research group, it was observed that hydrophobic groups appear
to be promising for the inhibition of PtpA by chalcone
derivatives, a chemical class different from that presented in this
work.40 For chalcones, this effect was explained by the presence
of π−π interactions between the 2-naphthyl substituent (at ring B)
of competitive inhibitors and the side chain residue Trp48 of the
PtpA binding site.15
By comparing the three different R1 substituents (1-naphthyl,
4-CH3-Ph, and 4-OCH3-Ph), we noted that when the electron-
donor effect of the substituent increases the inhibitory activity
tends to decrease. Regarding the R2 substituents of the
compounds with marked PtpA inhibition, electron-withdrawing
groups tend to enhance the inhibitory activity. This effect was
most evident for compounds 5, 9, 14, 18, 35, and 39.

Table 3. Selectivity index (SI) values of compounds 5, 9, 18, and 35 measured against some PTP representativesa
Compd
PTP
5
9
18
35
IC50
(µM)
SI
IC50
(µM)
SI
IC50
(µM)
SI
IC50
(µM)
SI
PtpA
PtpB
PTP1B
YopH
9.5 ± 3.3
18.4 ± 0.9
17.0 ± 3.5
7.1 ± 0.8
Ref value
1.9
7.4 ± 2.8
17.6 ± 2.7
>50
Ref value
2.4
> 6.8
0.9
2.9 ± 1.7
6.2 ± 1.5
9.4 ± 0.8
2.6 ± 1.5
Ref value
2.1
9.1 ± 5.0
30.3 ± 1.1
38.2 ± 8.0
6.9 ± 1.2
Ref value
3.3
1.8
0.7
3.2
0.9
4.2
0.8
6.5 ± 2.3
PTP-PEST
> 50
> 5.3
> 50
> 6.8
15.7 ± 3.1
5.4
> 50
> 50
> 5.5
> 5.5
LYP
> 50
> 5.3
> 50
> 6.8
> 50
> 10.3
aData are expressed as means ± SD of three independent experiments. The selective index (SI) is given by IC50PTP/IC50PtpA.
Figure 1. Michaelis–Menten plots representing the inhibitory profile of compounds 5, 9, 18 and 35 against PtpA.
The thiosemicarbazones derivatives that were most potent
against PtpA (5, 9, 18 and 35) were selected for further enzyme
kinetic studies. To determine the mechanism of inhibition and Ki
values, we tested three different inhibitor concentrations and
seven concentrations of pNPP. The preliminary kinetic analysis
suggested that compounds 5, 9 and 18 presents a non-competitive
mode of inhibition, on the other hand, the compound 35 showed
a competitive mode of inhibition, as presented in the Michaelis–
Menten plot in Figure 1.
remains unaffected for compound 5, 9 and 18, suggesting a non-
competitive mechanism of inhibition, with α = 1. The kinetic
parameter Ki calculated by nonlinear fit to the non-competitive
inhibition equation, resulted in Ki values for thiosemicarbazones
5 of 1.2 ± 0.3 µM, for 9 of 5.6 ± 1.0 µM, and for 18 of 1.7 ± 0.3
µM for PtpA.
For the inhibitor 35, it was observed that the Vmax is not
affected by the increase in inhibitor concentration, only the Km
values, the analysis revealed that compound 35 is a competitive
inhibitor, with Ki = 12.0 ± 5.3 µM for PtpA. Accordingly, we
found two different modes of inhibition of thiosemicarbazones
against PtpA. Interestingly, these data indicate that the structural
variation of these thiosemicarbazones significantly influences the
mechanism of inhibition for this enzyme. Furthermore, all the
three inhibitors (5, 9 and 18) that displayed the non-competitive
kinetic profile have 1-naphthyl substituent in R1 and showed the
best Ki values. The competitive inhibitor identified here,
compound 35, derived from 4-CH3-Ph in R1 and 5-(2-Cl,5-CF3)-
To the best of our knowledge, only competitive inhibitors
were described for PtpA to date, therefore, this work describes
the first non-competitive PtpA inhibitors. The search for
inhibitors that avoid the PTPs catalytic pocket is of upmost
importance, given that allosteric sites are not well conserved
among PTPs, which offers a promising approach for more
selective and bioavailable inhibitors. 44,45
Analyzing the kinetic parameters, we observed that Vmax
decreases when the inhibitor concentration increased, and that Km

Ph-furan-2-yl in R2 (the same substituent on compound 18 in R2),
thus the absence of the 1-naphthyl group in R1 affected the
potency and mechanism of inhibition.
binding in this particular region of the adjacent site by means of
electrostatic complementarity. Notably, 35 is docked with
opposed polarity in the adjacent site compared to the naphthyl
derivatives, with the 2-Cl-5-CF3-phenyl ring being docked in the
hydrophobic groove (Supplementary Information Figure S2),
which may explain the lower affinity of 35 for the adjacent site
compared to the catalytic site, as also highlighted by the docking
scores (Table 4). Indeed, docking into the catalytic site revealed a
noticeable fitting by compound 35, which is able to bind deeper
in the pocket and to mask the catalytic Cys11 residue from the
solvent area (Figure 3) with a higher docking score than 5, 9, and
18 (see Table 4 and Supplementary Information Figure S3).
To rationalize kinetics inhibition profiles, and to propose a
possible binding mode of the most potent PtpA inhibitors 5, 9,
18, and 35, molecular docking simulations were carried out. A
preliminary blind docking experiment (data not shown) revealed
that compounds are able to bind within the catalytic site of Mtb
PtpA, as well as in a site that is herein referred to as ‘adjacent
site’ formed by residues Ala94, Arg98, Val107, Arg108, Met109,
Ser112, Phe113, Pro115, His120, Leu122, Gly148, and Trp152
(see Supplementary Information Figure S1). Notably, this site is
separated from the catalytic site by the flexible D-loop (Arg111-
Asp131), and was identified as a druggable site by pocket finder
tools such as fpocket 1.0.46 Accordingly, molecular docking of 5,
9, 18, and 35 was performed within the catalytic site and the
adjacent site described above in two independent runs.
Table 4. PLP score of most potent PtpA inhibitors in the two binding sites
studied herein by molecular docking
Compound
PLP score – adjacent site
PLP score – catalytic site
5
9
18
35
64.78
62.39
65.72
57.92
51.29
57.08
50.91
61.16
Results of these theoretical investigations showed that PtpA
inhibitors bearing a naphthyl moiety, namely 5, 9, and 18, bind
preferentially in the adjacent site, whereas compound 35 binds
preferentially within the catalytic site, in good agreement with
Overall, these theoretical results substantiate that
thiosemicarbazones might bind preferentially to different binding
sites of Mtb PtpA. Compound 35 showed a tighter binding to the
catalytic site, whereas naphthyl derivatives 5, 9, and 18 showed a
remarkable scoring preference for the adjacent site. The
agreement between experimental and theoretical data reinforces
the reliability of molecular modeling findings, and paves the way
to further rational optimization of thiosemicarbazones as Mtb
PtpA inhibitors.
experimental results. This preference was defined by
a
combination of scores and docking poses. In detail, the naphthyl
ring of 5, 9, and 18 was docked in a hydrophobic groove of the
adjacent site in correspondence of Phe113, Pro115, Gly148, and
Trp152, while the variable aromatic portion is docked in a
positively charged cavity over Met109 that is delimited by
Arg98, Arg108, and His120 (Figure 2). It is worth noting that the
most potent PtpA inhibitor 18 bears a phenyl ring substituted
with two electron-withdrawing groups that could reinforce the
Figure 2. Docking-based binding mode of thiosemicarbazones 5 (panel A), 9 (panel B), and 18 (panel C) within the adjacent binding site of PtpA.
Compounds are showed as green sticks, the NMR solution structure of PtpA (PDB 2LUO)47 is shown as cyan cartoon in the top panels, residues within 5 Å from
the ligand are showed as lines. Residues contacted by the ligands are labeled. In the bottom panels, PtpA electrostatic surface calculated by APBS is shown.

4. Experimental
All reagents were obtained commercially (Sigma-Aldrich),
and all solvents used were analytical grade, without additional
purification.
4.1. General procedure for the synthesis of thiosemicarbazides
Compounds 1–3 were synthesized by reacting the different
isothiocyanates (10 mmol) and hydrazine hydrate (50%, 20
mmol) in 20 mL of isopropanol under magnetic stirring for 3 h at
room temperature. The precipitate formed was separated by
vacuum filtration and washed with isopropanol to obtain the pure
product. 37
4.2. General procedure for the synthesis of thiosemicarbazones
Thiosemicarbazones 4–52 were prepared by stirring and
refluxing a solution containing thiosemicarbazide (1.19 mmol),
the appropriately substituted benzaldehyde (1.25 mmol), 11 mL
of ethanol, and 22 mL of water with a catalytic amount of acetic
acid. After one hour, the mixture was cooled to ambient
temperature, and the precipitate formed was collected by vacuum
filtration and washed with water.30
4.3. General procedure for compound characterization
The obtained compounds were analyzed by thin-layer
chromatography (TLC), using aluminum plates coated with silica
gel on TLC Al foils. All synthesized compounds were
1
characterized by H and 13C nuclear magnetic resonance (NMR)
spectroscopy and melting points (mp). Melting points were
1
Figure 3. Docking-based binding mode of thiosemicarbazone 35 within
the catalytic site of PtpA. The compound is shown as green sticks, the NMR
solution structure of PtpA (PDB 2LUO)47 is shown as cyan cartoon in the top
panel, residues within 5 Å from the ligand are showed as lines. The catalytic
cysteine residue (Cys11) is shown as stick and is labeled. In the bottom panel,
PtpA electrostatic surface calculated by APBS is shown (transparent surface).
determined with a Microquimica MGAPF-301 apparatus. H and
13C NMR spectra were obtained using a Bruker Ac‑ 200F
(operating at 200 MHz for H and 50 MHz for 13C) or a Bruker
1
1
Avance Drx 400 spectrometer (operating at 400 MHz for H and
100 MHz for 13C), with tetramethylsilane as internal standard.
The structures of all compounds were confirmed by mass
spectrometry. High-resolution mass spectra (HRMS) were
recorded on
a micrOTOF-QII (Bruker Daltonics) mass
3. Conclusions
spectrometer, equipped with an automatic syringe pump (KD
Scientific) for sample injection (constant flow of 3 μL min−1), by
positive mode of electron spray ionization (ESI-MS) technique
(4.5 kV and 180 °C) using acetonitrile as solvent. The instrument
was calibrated in the range m/z 50–3000 using an internal
calibration standard (low concentration tuning mix solution),
supplied by Agilent Technologies. Data was processed via
Bruker Data Analysis software (version 4.0). When the
calculated and experimental masses were compared, the error
was as expected (< 2 ppm).
We synthesized 49 thiosemicarbazones that were evaluated in
vitro against recombinant PtpA from Mtb. Four compounds
displayed reasonable inhibition, with IC50 lower than 9.5 µM (5,
9, 18, and 35) and satisfactory specificity for PtpA. Our kinetic
findings revealed two different inhibition mechanism for this
class of molecules, interestingly, for the first-time non-
competitive
inhibitors
for
PtpA
were
identified,
thiosemicarbazones 5, 9 and 18 that present a naphthyl moiety at
R1 showed a non-competitive mode of inhibition with a Ki of 1.2
- 5.6 µM. Molecular modeling highlighted the structural features
important for potency and mechanism of inhibition, although all
inhibitors may interact with the active site of PtpA, those with the
naphthyl moiety preferably bind to an adjacent site in the
structure of the enzyme. Further, this finding corroborates the
modest results of selectivity found for these compounds. More
importantly, our study indicates a possible allosteric binding of
PtpA, some of the amino acid residues involved in the binding
were in previously hot spots reported in the structure of PtpA by
Silva and Tabernero.48 Our data support that thiosemicarbazone
derivatives are a novel class of potential lead compounds to
further development of PtpA inhibitors and, consequently, for
anti-TB drugs.
The synthesis and characterization compound 2 has been
previously reported by the authors.39 Data on the characterization
of compounds 1 and 3 are shown below.
N-(naphthalen-1-yl)hydrazinecarbothioamide (1) Yield: 77%;
white solid; mp 134,9-136,8 ºC; H NMR (400 MHz, DMSO-d6)
1
δ/ppm: 9.25 (s, 1H, NH), 7.96-7.94 (m, 1H), 7.90-7.88 (m, 1H),
7.81 (d, 1H, J 8.1 Hz), 7.67 (d, 1H, J 6.8 Hz), 7.54-7.49 (m, 3H).
13C NMR (100 MHz, DMSO-d6) δ/ppm: 181.7 (C=S), 135.7,
134.2, 130.2, 128.5, 126.4, 125.8, 125.2, 123.1. HRMS (ESI-
TOF) m/z: 240.05656 [M+H]+, calculated for C11H11N3S,
240.05659.
N-(4-methoxyphenyl)hydrazinecarbothioamide (3) Yield:
86%; white solid; mp 138.2-139.6 ºC; 1H NMR (200 MHz,
DMSO-d6) δ/ppm: 8.98 (s, 1H, NH), 7.44 (d, 2H, J 8.7 Hz), 6.86

(d, 2H, J 8.7 Hz), 4.72 (s, 2H, NH2), 3.73 (s, 3H). 13C NMR (50
MHz, DMSO-d6) δ/ppm: 180.4, 156.6, 132.7, 126.0, 113.7, 55.6.
HRMS (ESI-TOF) m/z: 198.0695 [M + H]+, calculated for
C8H11N3OS, 198.0696.
(ESI-TOF) m/z: 382.1375 [M + H]+, calculated for C24H19N3S,
382.1372.
(E)-2-(4-hydroxy-3-methoxybenzylidene)-N-(naphthalen-1-
yl)hydrazinecarbothioamide (10) Yield: 69%; yellow solid; mp
1
Data on the characterization of compounds 4–19 and 37–52
are shown below. The synthesis of thiosemicarbazones 20–36
was previously reported by the authors. 30
210.0-212.0ºC; H NMR (400 MHz, pyridine-d6) δ/ppm: 13.07
(s, 1H, NH), 11.14 (s, 1H, NH), 8.61 (s,1H, -NCH), 8.41 (d, 1H,
J 7.9 Hz), 8.02 (d, 1H, J 6.8 Hz), 7.89 (d, 1H, J 7,9 Hz), 7.81 (m,
2H), 7.52-7.47 (m, 3H), 7.38 (d, 1H, J 8,1 Hz), 7.25-7.23 (m,
2H), 3.53 (s, 3H). 13C NMR (50 MHz, DMSO-d6) δ/ppm: 177.8,
149.5, 148.5, 144.0, 136.2, 134.2, 131.1, 128.4, 127.3, 126.9,
126.5, 126.4, 125.9, 125.8, 123.8, 123.2, 115.8, 110.3, 56.4.
HRMS (ESI-TOF) m/z: 352.1112 [M + H]+, calculated for
C19H17N3O2S, 352.1114.
(E)-2-benzylidene-N-(naphthalen-1-yl)hydrazinecarbothio-
1
amide (4) Yield 76%; white solid; mp 217.0-218.0 ºC; H NMR
(200 MHz, DMSO-d6) δ/ppm: 11.93 (s, 1H, NH), 10.41 (s, 1H,
NH), 8.22 (s,1H, -NCH), 8.00-7.84 (m, 5H), 7.57-7.52 (m, 4H, J
8.2 Hz), 7.43-7.40 (m, 3H). 13C NMR (50 MHz, CDCl3) δ/ppm:
177.8, 143.0, 134.3, 133.6, 133.1, 130.8, 129.7, 129.0, 128.6,
127.7, 127.5, 126.8, 126.3, 125.4, 125.2, 121.8. HRMS (ESI-
TOF) m/z: 306.10588 [M + H]+, calculated for C18H15N3S,
306.10594.
(E)-N-(naphthalen-1-yl)-2-(3,4,5-trimethoxybenzylidene)hy-
drazinecarbothioamide (11) Yield: 38%; yellow solid; mp 202.0–
203.0 °C; 1H NMR (200 MHz, CDCl3) δ/ppm: 10.75 (s, 1H, NH),
9.36 (s, 1H, NH), 8.02 (dd, 1H), 7.95 (s,1H, -NCH), 7.92–7.86
(m, 3H), 7.61–7.49 (m, 3H), 6.98 (s, 2H), 3.89 (s, 3H); 3.87 (s,
6H). 13C NMR (50 MHz, DMSO-d6) δ/ppm: 178.2, 153.6, 143.3,
139.7, 136.2, 134.2, 131.2, 130.0, 128.5, 127.5, 127.0, 126.6,
126.5, 125.9, 123.8, 105.4, 60.5, 56.6. HRMS (ESI-TOF) m/z:
396.1376 [M + H]+, calculated for C21H21N3O3S, 396.1377.
(E)-N-(naphthalen-1-yl)-2-(4-nitrobenzylidene)hydrazine-
carbothioamide (5) Yield 71%; yellow solid; mp 229.0-230.0 ºC;
1H NMR (400 MHz, DMSO-d6) δ/ppm: 12.25 (s, 1H, NH), 10.66
(s, 1H, NH), 8.30 (s,1H), 8.24 (m, 4H), 8.01-7.98 (dd, 1H, J 6.0
Hz and J 3.0 Hz), 7.93 (d, 1H, J 8.5 Hz), 7.88-7.85 (dd, 1H, J 6,4
Hz and J 3,4 Hz), 7.60-7.50 (m, 4H). 13C NMR (50 MHz,
DMSO-d6) δ/ppm: 178.8, 148.1, 141.2, 140.5, 136.0, 134.2,
131.0, 128.9, 128.5, 127.6, 127.0, 126.6, 126.5, 125.9, 124.2,
123.8. HRMS (ESI-TOF) m/z: 351.0909 [M + H]+, calculated for
C18H14N4O2S, 351.0910.
(E)-N-(naphthalen-1-yl)-2-(naphthalen-1-ylmethylene)hydra-
zinecarbothioamide (12) Yield: 78%; yellow solid; mp 199.0–
1
201.0 °C; H NMR (200 MHz, DMSO-d6) δ/ppm: 11.98 (s, 1H,
NH), 10.47 (s, 1H, NH), 9.13 (s, 1H, -NCH), 8.50 (d, 1H, J 7.2
Hz); 8.38 (d, 1H, J 8.0 Hz); 8.05–7.90 (m, 5H), 7.69–7.56 (m,
7H). 13C NMR (50 MHz, DMSO-d6) δ/ppm: 178.3, 141.4, 136.1,
134.2, 133.8, 131.1, 130.9, 130.8, 129.7, 129.3, 128.5, 127.6,
127.4, 126.8, 126.5, 126.0, 125.9, 123.8, 123.0. HRMS (ESI-
TOF) m/z: 356.12160 [M + H]+, calculated for C22H17N3S,
356.12159.
(E)-2-(4-chlorobenzylidene)-N-(naphthalen-1-yl)hydrazine-
carbothioamide (6) Yield: 44%; white solid; mp 206.0–208.0 °C;
1H NMR (200 MHz, DMSO-d6) δ/ppm: 11.69 (s, 1H, NH), 10.17
(s, 1H, NH), 7.89 (s,1H, -NCH), 7.70 (d, 2H, J 8.3 Hz), 7.62–
7.54 (m, 2H), 7.30–7.16 (m, 7H). 13C NMR (50 MHz, DMSO-d6)
δ/ppm: 178.5, 141.7, 136.1, 134.8, 134.1, 133.6, 131.0, 129.7,
129.1, 128.4, 127.4, 126.9, 126.5 126.5, 125.9, 123.9. HRMS
(ESI-TOF) m/z: 340.0674 [M + H]+, calculated for C18H14ClN3S,
340.0670.
(E)-N-(naphthalen-1-yl)-2-(naphthalen-2-ylmethylene)hydra-
zinecarbothioamide (13) Yield: 63%; white solid; mp 198.0-
1
200.0ºC; H NMR (400 MHz, DMSO-d6) δ/ppm: 12.07 (s, 1H,
NH), 10.51 (s, 1H, NH), 8.40-8.38 (m, 2H), 8.21 (s, 1H, -NCH),
8.01-7.90 (m, 6H), 7.60-7.53 (m, 6H). 13C NMR (100 MHz,
DMSO-d6) δ/ppm: 178.4, 143.3, 136.3, 134.2, 133.4, 132.5,
131.2, 129.6, 128.7, 128.6, 128.5, 128.2, 127.5, 127.4, 127.1,
127.0, 126.6, 126.5, 125.9, 124.0. HRMS (ESI-TOF) m/z:
356.1218 [M + H]+, calculated for C22H17N3S, 356.1216.
(E)-2-(4-methoxybenzylidene)-N-(naphthalen-1-yl)hydrazine-
carbothioamide (7) Yield: 69%; white solid; mp 203.0-205.0 ºC;
1H NMR (200 MHz, DMSO-d6) δ/ppm: 11.83 (s, 1H, NH), 10.34
(s, 1H, NH), 8.16 (s,1H, -NCH), 8.00-7.88 (m, 5H), 7.56-7.54
(m, 4H), 6.98 (d, 2H, J 8.4 Hz), 3.80 (s, 3H, OCH3). 13C NMR
(100 MHz, DMSO-d6) δ/ppm: 178.0, 161.3, 143.2, 136.2, 134.2,
131.1, 129.7, 128.4, 127.3, 127.2, 126.9, 126.5, 126.4, 125.9,
123.8, 114.6, 55.8. HRMS (ESI-TOF) m/z: 336.1161 [M + H]+,
calculated for C19H17N3OS, 336.1165.
(E)-2-(3,4-dichlorobenzylidene)-N-(naphthalen-1-yl)hydra-
zinecarbothioamide (14) Yield: 75%; white solid; mp 206.0–
1
207.0 °C; H NMR (200 MHz, DMSO-d6) δ/ppm: 12.10 (s, 1H,
NH), 10.56 (s, 1H, NH), 8.42 (s, 1H), 8.17 (s, 1H, -NCH), 8.01–
7.82 (m, 4H); 7.68 (d, 1H, J 8.3 Hz); 7.62–7.48 (m, 4H). 13C
NMR (50 MHz, DMSO-d6) δ/ppm: 178.6; 140.4; 136.1; 135.5;
134.1; 132.4; 132.2; 131.2; 131.1; 128.7; 128.4; 127.5; 127.1;
126.6; 126.5; 125.9; 123.9. HRMS (ESI-TOF) m/z: 374.0284 [M
+ H]+, calculated for C18H13Cl2N3S, 374.0280.
(E)-2-(benzo[d][1,3]dioxol-5-ylmethylene)-N-(naphthalen-1-
yl)hydrazinecarbothioamide (8) Yield: 60%; white solid; mp
208.0–209.0 °C; H NMR (200 MHz, DMSO-d6) δ/ppm: 11.84
(s, 1H, NH), 10.40 (s, 1H, NH), 8.12 (s, 1H, -NCH), 8.00–7.84
(m, 4H), 7.60–7.48 (m, 4H), 7.17 (d, 1H J 8.1 Hz), 6.95 (d, 1H, J
7.6 Hz), 6.06 (s, 2H).13C NMR (50 MHz, DMSO-d6) δ/ppm:
178.1, 149.4, 148.5, 142.8, 136.2, 134.1, 131.1, 129.2, 128.4,
127.3, 127.0, 126.5, 125.9, 124.7, 123.9, 108.6, 106.0, 101.9.
HRMS (ESI-TOF) m/z: 350.0957 [M + H]+, calculated for
C19H15N3O2S, 350.0958.
1
(E)-2-(2,5-dimethoxybenzylidene)-N-(naphthalen-1-yl)hydra-
zinecarbothioamide (15) Yield: 69%; white solid; mp 200.0–
1
202.0 °C; H NMR (200 MHz, DMSO-d6) δ/ppm: 11.92 (s, 1H,
NH), 10.39 (s, 1H, NH), 8.55 (s, 1H, -NCH), 7.98 (dd, 1H J 3.3
and 6.0 Hz), 7.91-7.84 (m,3H), 7.58-7.52 (m, 4H), 7.04-6.97 (m,
2H), 3.82 (s, 3H), 3.72 (s, 3H). 13C NMR (50 MHz, DMSO-d6)
δ/ppm: 178.3, 153.8, 152.9, 138.7, 136.3, 134.1, 131.1, 128.4,
127.3, 127.0, 126.5, 125.9, 123.8, 123.3, 117.6, 113.5, 111.4,
56.7, 56.1. HRMS (ESI-TOF) m/z: 366.1270 [M + H]+,
calculated for C20H19N3O2S, 366.1271.
(E)-2-([1,1'-biphenyl]-4-ylmethylene)-N-(naphthalen-1-yl)hy-
drazinecarbothioamide (9) Yield: 42%; white solid; mp 204.0–
1
206.0 °C; H NMR (200 MHz, DMSO-d6) δ/ppm: 11.99 (s, 1H,
NH), 10.48 (s, 1H, NH), 8.26 (s, 1H, -NCH), 8.06–7.96 (m, 3H),
7.93–7.86 (m, 2H), 7.73 (d, 4H, J 7.8 Hz), 7.57–7.38 (m, 7H).
13C NMR (50 MHz, DMSO-d6) δ/ppm: 178.4, 142.8, 141.9,
139.8, 136.2, 134.2, 133.8, 131.1, 129.4, 128.7, 128.4, 128.3,
127.3, 127.3, 127.1, 126.9, 126.5, 126.4, 125.9, 123.8. HRMS
(E)-2-(3,4-dimethoxybenzylidene)-N-(naphthalen-1-yl)hydra-
zinecarbothioamide (16) Yield: 87%; white solid; mp 217.0–

1
218.0 °C; H NMR (200 MHz, DMSO-d6) δ/ppm: 11.85 (s, 1H,
DMSO-d6) δ/ppm: 177.3, 157.6, 148.1, 141.1, 140.3, 132.2,
128.8, 128.2, 124.2, 113.8, 55.7. HRMS (ESI-TOF) m/z:
331.0857 [M + H]+, calculated for C15H14N4O3S, 331.0859.
NH), 10.29 (s, 1H, NH), 8.15 (s,1H, -NCH), 7.99 (dd, 1H, J 6.1
Hz and J 2.8 Hz), 7.92-7.87 (m, 2H), 7.63-7.53 (m, 5H), 7.27 (d,
1H, J 7.9 Hz), 6.99 (d, 1H, J 7.9 Hz), 3.80 (s, 6H). 13C NMR (50
MHz, DMSO-d6) δ/ppm: 177.9, 151.2, 149.6, 143.6, 136.3,
134.1, 131.2, 128.4, 127.4, 127.2, 127.0, 126.5, 125.9, 123.9,
123.0, 111.7, 109.3, 56.2, 56.0. HRMS (ESI-TOF) m/z: 366.1273
[M + H]+, calculated for C20H19N3O2S, 366,1271.
(E)-2-(4-chlorobenzylidene)-N-(4-methoxyphenyl)hydrazine-
carbothioamide (40) Yield: 89%; white solid; mp 174.0-175.7 ºC;
1H NMR (200 MHz, CDCl3) δ/ppm: 10.08 (s, 1H, NH), 9.00 (s,
1H, NH), 7.89 (s,1H, -NCH), 7.61 (d, 2H, J 8.7 Hz), 7.48 ( 2H, J
8.9 Hz), 7.38 (d, 2H, J 8.4 Hz), 6.40 (d, 2H, J 8.9 Hz), 3.83 (s,
3H). 13C NMR (50 MHz, CDCl3) δ/ppm: 176.5, 158.1, 141.7,
136.5, 131.6, 130.5, 129.2, 128.6, 126.9, 114.1, 55.4. HRMS
(E)-N-(naphthalen-1-yl)-2-(2-nitrobenzylidene)hydrazine-
carbothioamide (17) Yield: 72%; yellow solid; mp 202.0–204.0
°C; H NMR (200 MHz, DMSO-d6) δ/ppm: 12.26 (s, 1H, NH),
10.56 (s, 1H, NH), 8.66 (s,1H, -NCH), 8.06 (d, 1H, J 8.1 Hz);
7.99 (dd, 1H, J 6.2 Hz and J 3.0 Hz); 7.92 (d, 1H, J 8.0 Hz); 7.88
(dd, 1H, J 6.3 Hz and J 3.0 Hz); 7.74 (dd, 1H, J 7.6 Hz); 7.66–
7.51 (m, 6H). 13C NMR (50 MHz, DMSO-d6) δ/ppm: 178.8,
148.8, 138.1, 136.0, 134.1, 133.7, 130.9, 130.8, 129.0, 128.9,
128.4, 127.5, 126.9, 126.6, 126.5, 125.9, 125.0, 123.8. HRMS
1
(ESI-TOF) m/z: 320.0617 [M
C15H14ClN3OS, 320.0619.
+
H]+, calculated for
(E)-2-(4-methoxybenzylidene)-N-(4-methoxyphenyl)hydra-
zinecarbothioamide (41) Yield: 83%; white solid; mp 159.5-
1
161.0 ºC; H NMR (200 MHz, CDCl3) δ/ppm: 10.21 (s, 1H,
NH), 9.02 (s, 1H, NH), 7.91 (s,1H, -NCH), 7.61 (d, 2H, J 8.7
Hz), 7.48 ( 2H, J 8.7 Hz), 6.99- 6.90 (m, 4H, overlapping peaks
with J 8.7 Hz), 3.84 (s, 3H), 3.83 (s, 3H). 13C NMR (50 MHz,
CDCl3) δ/ppm: 176.2, 161.7, 158.0, 142.9, 130.8, 129.1, 126.8,
125.8, 114.4, 114.0, 55.4 (2). HRMS (ESI-TOF) m/z: 316.1113
[M + H]+, calculated for C16H17N3O2S, 316.1114.
(ESI-TOF) m/z: 351.09101 [M
C18H14N4O2S, 351.09102.
+
H]+, calculated for
(E)-2-((5-(2-chloro-5-(trifluoromethyl)phenyl)furan-2-
yl)methylene)-N-(naphthalen-1-yl)hydrazinecarbothioamide (18)
Yield: 82%; yellow solid; mp 213.0–214.0 °C; H NMR (200
1
(E)-2-(benzo[d][1,3]dioxol-5-ylmethylene)-N-(4-methoxy-
phenyl)hydrazinecarbothioamide (42) Yield: 88%; white solid;
mp 189.4-191.3 ºC; 1H NMR (200 MHz, DMSO-d6) δ/ppm:
11.64 (s, 1H, NH), 10.00 (s, 1H, NH), 8.04 (s,1H, -NCH), 7.83
(s, 1H), 7.37 ( d, 2H, J 8.9 Hz), 7.13 (d, 1H, J 8.3 Hz), 6.93 (m,
3H), 6.07 (s, 2H), 3.77 (s, 3H). 13C NMR (50 MHz, DMSO-d6)
δ/ppm: 176.6, 157.4, 149.4, 148.5, 142.8, 132.5, 129.1, 128.1,
124.6, 113.6, 108.6, 106.0, 101.9, 55.7. HRMS (ESI-TOF) m/z:
330.0902 [M + H]+, calculated for C16H15N3O3S, 330.0907.
MHz, DMSO-d6) δ/ppm: 12.12 (s, 1H, NH), 10.32 (s, 1H, NH),
8.23 e 8.21 (s, s, 2H, overlapping peaks), 7.99 (dd, 1H, J 7.3 Hz
and J 3.3 Hz), 7.93-7.82 (m, 3H), 7.71 (dd, 1H, J 1.9 Hz and J
8.5 Hz), 7.59–7.52 (m, 4H), 7.47 ( d, 1H, J 3.7 Hz), 7.37 (d, 1H,
J 7.8 Hz). 13C NMR (50 MHz, DMSO-d6) δ/ppm: 178.1; 150.9;
149.5; 135.8; 134.1; 133.6; 132.6; 132.4; 130.7; 129.2; 129.1;
128.6; 128.5; 127.3; 126.6; 126.5; 125.9; 125.8; 124.9; 124.9;
123.5; 115.3; 114.5. HRMS (ESI-TOF) m/z: 474.0650 [M + H]+,
calculated for C23H15ClF3N3OS, 474.0649.
(E)-2-([1,1'-biphenyl]-4-ylmethylene)-N-(4-methoxyphenyl)-
hydrazinecarbothioamide (43) Yield: 97%; white solid; mp
188.1-189.3 °C; H NMR (200 MHz, CDCl3) δ/ppm: 10.23 (s,
1H, NH), 9.08 (s, 1H, NH), 7.99 (s,1H, -NCH), 7.75 (d, 2H, J 8.7
Hz); 7.66–7.60 (m, 4H); 7.53–7.38 (m, 5H); 6.95 (d, 2H, J 8.7
Hz); 3.83 (s, 3H). 13C NMR (50 MHz, CDCl3) δ/ppm: 176.5,
158.1, 143.4, 142.6, 140.0, 132.0, 130.7, 128.9, 127.9, 127.5,
127.0, 126.8, 114.1, 55.5. HRMS (ESI-TOF) m/z: 362.1316 [M +
H]+, calculated for C21H19N3OS, 362.1322.
(E)-2-(4-(dimethylamino)benzylidene)-N-(naphthalen-1-
yl)hydrazinecarbothioamide (19) Yield 78%; white solid; mp
195.0-197.0 ºC; 1H NMR (400 MHz, DMSO-d6) δ/ppm: 11.70 (s,
1H, NH), 10.22 (s, 1H, NH), 8.10 (s,1H, -NCH), 7.99-7.96 (dd,
1H, J 6.3 Hz and J 3.6 Hz), 7.90- 7.86 (m, 2H), 7.73 (d, 2H, J 8.7
Hz), 7.57-7.52 (m, 4H), 6.72 (d, 2H, J 9.1 Hz), 2.97 ( s, 6H). 13C
NMR (50 MHz, DMSO-d6) δ/ppm: 177.4, 151.9, 144.3, 136.3,
134.2, 131.1, 129.4, 128.4, 127.2, 126.8, 126.5, 126.4, 125.9,
123.9, 121.9, 112.1, 40.2. HRMS (ESI-TOF) m/z: 349.1482 [M +
H]+, calculated for C20H20N4S, 349.1481.
1
(E)-2-(4-hydroxy-3-methoxybenzylidene)-N-(4-methoxy-
phenyl)hydrazinecarbothioamide (44) Yield: 86%; white solid;
mp 193.1-195.0 ºC; 1H NMR (200 MHz, acetone-d6) δ/ppm:
10.48 (s, 1H, NH), 9.64 (s, 1H, NH), 8.16 (s,1H, -NCH), 7.58-
7.52 (m, 3H), 7.23 (dd, 1H, J 8.1 and 1.9 Hz), 6.93-6.87 (m,
3H), 3.88 (s,3H), 3.81 (s, 3H). 13C NMR (50 MHz, acetone-d6)
δ/ppm: 176.7, 157.5, 149.1, 147.9, 143.1, 132.1, 126.6, 126.1,
122.5, 115.0, 113.3, 109.4, 55.6, 54.8. HRMS (ESI-TOF) m/z:
332.1060 [M + H]+, calculated for C16H17N3O3S, 332.1063.
(E)-2-benzylidene-N-(4-methoxyphenyl)hydrazinecarbo-
thioamide (37) Yield: 79%; white solid; mp 192.2-193.8 ºC; H
1
NMR (200 MHz, CDCl3) δ/ppm: 10.21 (s, 1H, NH), 9.05 (s, 1H,
NH), 7.95 (s,1H, -NCH), 7.70-7.65 (m, 2H), 7.51-7.40 (m, 5H),
6.95 (d, 2H, J 8.9 Hz), 3.83 (s, 3H). 13C NMR (50 MHz, CDCl3)
δ/ppm: 176.4, 158.1, 143.2, 133.2, 130.7, 130.6, 128.8, 127.5,
126.9, 114.1, 55.4. HRMS (ESI-TOF) m/z: 286.1006 [M + H]+,
calculated for C15H15N3OS, 286.1009.
(E)-N-(4-methoxyphenyl)-2-(3,4,5-trimethoxybenzylidene)-
hydrazinecarbothioamide (45) Yield: 86%; white solid; mp
176.3-177.5 °C; 1H NMR (200 MHz, CDCl3) δ/ppm: 9.92(s, 1H,
NH), 8.96 (s, 1H, NH), 7.83 (s,1H, -NCH), 7.47 (d, 2H, J 8.9
Hz); 6.95 (d, 2H, J 8.9 Hz); 6.88 (s, 2H); 3.91 (s, 6H); 3.89 (s,
3H); 3.83 (s,3H). 13C NMR (50 MHz, CDCl3) δ/ppm: 176.6,
158.2, 153.6, 143.0, 140.7, 130.6, 128.3, 127.0, 114.1, 104.7,
60.9, 56.3, 55.4. HRMS (ESI-TOF) m/z: 376.1323 [M + H]+,
calculated for C18H21N3O4S, 376.1326.
(E)-N-(4-methoxyphenyl)-2-(4-methylbenzylidene)hydrazine-
carbothioamide (38) Yield: 85%; white solid; mp 174.9-175.4 ºC;
1H NMR (200 MHz, CDCl3) δ/ppm: 10.00 (s, 1H, NH), 9.04 (s,
1H, NH), 7.90 (s,1H, -NCH), 7.57 (d, 2H, J 8.3 Hz), 7.49 ( 2H, J
8.9 Hz), 7.22 (d, 2H, J 8.0 Hz), 6.94 (d, 2H, J 8.7 Hz), 3.83 (s,
3H), 2.39 (s, 3H). 13C NMR (50 MHz, CDCl3) δ/ppm: 176.4,
158.0, 143.2, 141.1, 130.7, 130.4, 129.6, 127.4, 126.8, 114.0,
55.4, 21.5. HRMS (ESI-TOF) m/z: 300.1166 [M + H]+,
calculated for C16H17N3OS, 300.1165.
(E)-N-(4-methoxyphenyl)-2-(naphthalen-1-ylmethylene)-
(E)-N-(4-methoxyphenyl)-2-(4-nitrobenzylidene)hydrazine-
carbothioamide (39) Yield: 44%; yellow solid; mp 200.9-
202.7ºC; H NMR (200 MHz, DMSO-d6) δ/ppm: 12.01 (s, 1H,
hydrazinecarbothioamide (46) Yield: 88%; yellow solid; mp
1
192.8-193.5 ºC; H NMR (200 MHz, CDCl3) δ/ppm: 10.16 (s,
1
1H, NH), 9.11 (s, 1H, NH), 8.68 (s,1H, -NCH), 8.40 (d, 1H, J 7.3
Hz), 7.98-7.89 (m, 3H), 7.63-7.49 (m, 5H), 6.94 (s, 2H, J 9,2
NH), 10.23 (s, 1H, NH), 8.27-8.21 (m,5H), 7.40 (d, 2H, J 8.8
Hz), 6.95 (d, 2H, J 9.0 Hz), 3.78 (s, 3H). 13C NMR (50 MHz,

Hz), 3.83 (s, 3H). 13C NMR (50 MHz, CDCl3) δ/ppm: 176.5,
158.0, 141.8, 133.8, 131.2, 130.9, 130.7, 129.0, 128.7, 127.4,
126.9, 126.6, 126.3, 125.3, 123.1, 114.1, 55.4. HRMS (ESI-TOF)
m/z: 336.1168 [M + H]+, calculated for C19H17N3OS, 336.1165.
PtpA and PtpB from Mycobacterium tuberculosis, YopH from
Yersinia enterocolitica, and human PTP1B, LYP, and PTP-PEST
were expressed and purified as previously described.22,49, 50
(E)-N-(4-methoxyphenyl)-2-(naphthalen-2-ylmethylene)hy-
drazinecarbothioamide (47) Yield: 89%; white solid; mp 204.6-
206.7 °C; H NMR (200 MHz, DMSO-d6) δ/ppm: 11.85 (s, 1H,
4.5. Measurement of PtpA Activity (%) and Inhibition (IC50)
1
The phosphatase assays were carried out as previously
described.21 First, the compounds were screened in a 200 μL
reaction in 96-well plates by adding 5 μL of the compounds
diluted to 1 mM in dimethyl sulfoxide (100% DMSO) (final
concentration of 25 μM), 20 mM imidazole pH 7.0, 50 nM PtpA
(2 μL in 20 mM Tris–HCl pH 8.0, 50 mM NaCl, 5 mM EDTA,
20% glycerol, and 5 mM DTT), 10 mM p-nitrophenyl phosphate
(pNPP), and Milli-Q water to complete the volume. The
compound was premixed with PtpA for 10 min at 37 °C, and the
reaction was started by the addition of pNPP. Absorbance was
measured by UV−VIS spectrophotometry (TECAN Magellan
Infinite M200) for 10 min at 37 °C at 410 nm with readings every
1 min. Negative controls were performed in the absence of
enzyme and compound, and positive controls in the presence of
enzyme and 2.5% DMSO for screening e 4% DMSO for IC50.
The percentage of residual activity was calculated as the
difference in absorbance between times 7 min and 2 min.
Compounds that reduced more than 60% of the enzyme activity
were selected for the IC50 assay. The IC50 was determined in the
same manner as described above, but the solvent concentration
was adjusted to not exceed 4% (v/v) in the final reaction mixture
and values were determined with increasing concentrations of
inhibitor (0.5−50 μM) versus % of inhibition. The enzymatic
activity was expressed in % of phosphatase residual activity
compared with the results of wells without inhibitor.
Experimental data were analyzed with GraphPad Prism 5.0 and
the IC50 values determined by linear regression. All assays were
performed in triplicate.
NH), 10.10 (s, 1H, NH), 8.34 (d, 2H, J 7.5 Hz); 8.17 (s,1H, -
NCH), 8.00–7.92 (m, 3H); 7.58–7.53 (m, 2H); 7.43 (d, 2H, J
8.7); 6.95 (d, 2H, J 8.7); 3.78 (s,3H). 13C NMR (50 MHz,
DMSO-d6) δ/ppm: 176.9, 157.4, 143.1, 134.2, 133.3, 132.5,
132.4, 129.5, 128.7, 128.6, 128.1, 127.5, 127.1, 123.8, 113.7,
55.7. HRMS (ESI-TOF) m/z: 336.1169 [M + H]+, calculated for
C19H17N3OS, 336.1165.
(E)-2-(3,4-dichlorobenzylidene)-N-(4-methoxyphenyl)hydra-
zinecarbothioamide (48) Yield: 86%; white solid; mp 185.5-
186.5 °C; 1H NMR (200 MHz, CDCl3) δ/ppm: 10.52 (s, 1H, NH),
8.98 (s, 1H, NH), 7.89 (s,1H, -NCH); 7.77 (s, 1H); 7.49–7.44 (m,
4H); 6.95 (d, 2H, J 8.8 Hz); 3.84 (s,3H). 13C NMR (50 MHz,
CDCl3) δ/ppm: 176.9, 158.3, 140.1, 134.6, 133.4, 133.1, 130.9,
130.4, 128.6, 126.9, 126.5, 114.1, 55.4. HRMS (ESI-TOF) m/z:
354.0227 [M + H]+, calculated for C15H13Cl2N3OS, 354.0229.
(E)-2-(2,5-dimethoxybenzylidene)-N-(4-methoxyphenyl)hy-
drazinecarbothioamide (49) Yiel 92%; white solid; mp 194.8-
195.6 ºC; 1H NMR (200 MHz, CDCl3) δ/ppm: 9.10 (s, 1H, NH),
9.01(s, 1H, NH), 8.21 (s,1H, -NCH), 7.50 (d, 2H, J 8.9 Hz), 7.38
(d, 1H, J 2.8 Hz),7.00-6.85 (m, 4H), 3.84, 3.83 and 3.82 (s, 9H,
overlapping peaks). 13C NMR (50 MHz, DMSO-d6) δ/ppm:
176.8, 157.4, 153.8, 152.9, 138.7, 132.5, 128.2, 123.2, 117.5,
113.7, 113.5, 111.4, 56.7, 56.1, 55.7. HRMS (ESI-TOF) m/z:
346.1221 [M + H]+, calculated for C17H19N3O3S, 346.1220.
(E)-2-(3,4-dimethoxybenzylidene)-N-(4-methoxyphenyl)hy-
drazinecarbothioamide (50) Yield: 81%; yellow solid; mp 175.3-
177.8 °C; 1H NMR (200 MHz, CDCl3) δ/ppm: 10.09 (s, 1H, NH),
8.98 (s, 1H, NH), 7.88 (s,1H, -NCH); 7.48 (d, 2H, J 8.9 Hz); 7.24
(d, 1H, J 2.0); 7.17 (dd, 1H, J 8.3 and 2.0 Hz); 6.95 (d, 2H, J 8.9
Hz); 6.88 (d, 1H, J 8.3 Hz); 3.94 and 3.93 (s, 6H); 3.83 (s, 3H).
13C NMR (50 MHz, CDCl3) δ/ppm: 176.3, 158.1, 151.5, 149.5,
143.4, 130.8, 127.0, 126.0, 122.4, 114.1, 111.0, 108.6, 56.1, 56.0,
55.4. HRMS (ESI-TOF) m/z: 346.12203 [M + H]+, calculated for
C17H19N3O3S, 346.12199.
4.6. Selectivity assay
The selectivity assays were carried out using 2 µL of several
recombinant PTPs: LYP (169 nM), PTP-PEST (205 nM), PTP1B
(38 nM), and PtpB (50 nM). Assays were carried out as described
above.
(E)-N-(4-methoxyphenyl)-2-(2-nitrobenzylidene)hydrazine-
carbothioamide (51) Yield: 67%; yellow solid; mp 209.3-211.6
°C; H NMR (200 MHz, CDCl3) δ/ppm: 9.45 (s, 1H, NH), 8.98
4.7. Enzyme Kinetics
1
The kinetic parameters of compounds were determined by
varying the concentrations of pNPP (0.2–20 mM) for each
concentration of compound through PtpA-catalyzed hydrolysis.
Vmax and Km were determined by fitting of the data to the
Michaelis–Menten equation. Reaction rates were expressed as
specific activity of the protein (μmol pNPP min−1 mg−1). The
kinetic parameter Ki was calculated by nonlinear fits of the data
to the competitive or non-competitive inhibition equation. Ki
values were calculated using at least three independent
experiments. Data were plotted using GraphPad Prism software.22
(s, 1H, NH), 8.40 (s,1H, -NCH); 8.06 (dd, 2H, J 9.2 and 7.6 Hz);
7.73–7.59 (m, 2H); 7.51 (d, 2H, J 8.8 Hz); 6.94 (d, 2H, J 8.8 Hz);
3.83 (s, 3H). 13C NMR (50 MHz, DMSO-d6) δ/ppm: 177.3,
157.5, 148.8, 137.9, 133.7, 132.2, 130.9, 129.0, 128.8, 128.0,
124.9, 113.8, 55.7. HRMS (ESI-TOF) m/z: 331.0863 [M + H]+,
calculated for C15H14N4O3S, 331.0859.
(E)-2-(4-(dimethylamino)benzylidene)-N-(4-methoxyphenyl)
hydrazinecarbothioamide (52) Yield 79%; yellow solid; mp
180.1-181.8 ºC; 1H NMR (200 MHz, CDCl3) δ/ppm: 9.89 (s, 1H,
NH), 9.02 (s, 1H, NH), 7.82 (s,1H, -NCH), 7.52 (m, 4H
overlapping peaks with J 8.9 Hz), 6.93 (d, 2H, J 8.9 Hz), 6.68
(d, 2H, J 8.9 Hz), 3.83 (s, 3H), 3.02 (s, 6H). 13C NMR (50 MHz,
CDCl3) δ/ppm: 175.7, 157.8, 152.0, 144.0, 131.0, 129.0, 126.7,
120.5, 114.0, 111.7, 55.4, 40.1. HRMS (ESI-TOF) m/z: 329.1435
[M + H]+, calculated for C17H20N4OS, 329.1431.
4.8. Molecular Modeling.
The first model of the NMR solution structure of Mtb PtpA
coded by PDB ID: 2LUO was used as rigid receptor in molecular
docking simulations,47 which were carried out by the GOLD
docking program.51 The CHEMPLP scoring function was used
with default parameters. Docking efficiency was increased up to
200%. While docking within the adjacent site, the binding site
was centered on the side chain of Ser112 (carbon beta) and had a
4.4. PTPs expression and purification

radius of 12 Å; while docking in the catalytic site, the binding
site was centered on the catalytic cysteine Cys11 (S atom) and
had a radius of 14 Å. Before molecular docking simulations,
ligands were energy minimized by Szybki version 1.9.0.3
(OpenEye) at the MMFF94S force field.52
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Acknowledgments
19. Dutta, N. K.; He, R.; Pinn, M. L.; He, Y.; Burrows, F.;
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The authors thank the Chemistry Department of the Federal
University of Santa Catarina (UFSC) and Central Laboratory of
Structural Biology for providing the equipment and some of the
facilities. We thank CNPq and CAPES (Brazil) for the
scholarship grant and financial support.
Supplementary Material
Supplementary data associated with this article can be found
in the online version.
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Synthetic thiosemicarbazones as a new class
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tyrosine phosphatase A inhibitors
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Larissa Sens, Ana Caroline Arruda de Souza, Lucas Antonio Pacheco, Angela Camila Orbem Menegatti, Mattia
Mori, Alessandra Mascarello, Ricardo José Nunes and Hernán Terenzi
UFSC, Campus Trindade, 88040-900 Florianópolis, SC, Brazil
Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy