Synthesis, antioxidant activity, acetylcholinesterase inhibition and quantum studies of thiosemicarbazones

Full text of DOI:10.21577/0103-5053.20170146

http://dx.doi.org/10.21577/0103-5053.20170146
J. Braz. Chem. Soc., Vol. 29, No. 2, 343-352, 2018.
Printed in Brazil - ©2018 Sociedade Brasileira de Química
Article
Synthesis, Antioxidant Activity, Acetylcholinesterase Inhibition and Quantum
Studies of Thiosemicarbazones
Larissa Sens,a Aldo S. de Oliveira,*,b Alessandra Mascarello,a Inês M. C. Brighente,a
Rosendo A. Yunesa and Ricardo J. Nunesa
aDepartamento de Química, Centro de Ciências Físicas e Matemáticas,
Universidade Federal de Santa Catarina, 88040-900 Florianópolis-SC, Brazil
bDepartamento de Ciências Exatas e Educação, Universidade Federal de Santa Catarina,
89065-300 Blumenau-SC, Brazil
Thiosemicarbazones are a class of compounds of interest for Medicinal Chemistry, as they are
structurally diverse and have numerous biological activities reported in the literature. This study
describes the synthesis of seventeen thiosemicarbazones, which were investigated as potential
therapeutic agents for the treatment of Alzheimer’s disease through antioxidant tests and an
inhibitory assay of the acetylcholinesterase enzyme. All compounds showed excellent inhibition
of acetylcholinesterase and exhibited excellent antioxidant action when compared to the standards.
In addition, a quantum study was carried out, in which the HOMO (highest occupied molecular
orbital) and LUMO (lowest unoccupied molecular orbital) energy values of each compound were
obtained. From these theoretical data, chemical properties were calculated and correlated with
the experimental data.
Keywords: antioxidants, acetylcholinesterase, Alzheimer’s disease, thiosemicarbazone
Introduction
as cancer,5 cardiovascular diseases,6 alcohol-induced
liver disease,7 atherosclerosis,8 neural disorders,9 and
Alzheimer’s disease,10,11 and also aging processes.12
Oxidative stress appears as a risk factor for age-
associated cognitive decline and is considered to be a
critical aspect in the pathogenesis of Alzheimer’s disease
(AD).13 AD is a progressive neurodegenerative disorder
of the central nervous system characterized by mental
deterioration.14 The disease is not completely understood,
different hypotheses try to explain its causes and form the
basis of current therapies.15 Treatment strategies employ the
use of acetylcholinesterase inhibitors, glutamate receptor
modulators, and antioxidants and anti-inflammatory
agents.16,17 Nowadays, the cholinergic hypothesis is the
most accepted explanation of the cause of AD13 since
the disease is associated with a reduction in the levels
of the acetylcholine (ACh) neurotransmitter in areas of
the brain that deal with behavior, learning, memory and
emotional responses.18 Acetylcholinesterase (AChE) is
one of the enzymes responsible for ACh hydrolysis, thus
controlling ACh concentration in the organism.15 For this
reason, current drugs for AD aim to inhibit AChE, such
as galantamine, rivastigmine, tacrine, ensaculin, and
donepezil.18
Free radicals are reactive molecules containing an
unpaired electron or an odd number of electrons.1 They
can be formed by redox reactions, the cleavage of a radical
to form another or the breaking of a chemical bond, each
of these conditions being able to generate a fragment that
maintains an unpaired electron. Examples of free radicals
are hydroxyl (OH•), nitric oxide (NO•), superoxide (O2•ˉ),
nitrogen dioxide (NO2•), peroxyl (ROO•) and lipid peroxyl
(LOO•).2 In addition, there are other reactive species that
can oxidize compounds, such as reactive oxygen species
(ROS) and reactive nitrogen species (RNS), and species
that can easily form radicals, such as singlet oxygen (1O2),
hydrogen peroxide (H2O2), ozone (O3), hypochlorous acid
(HOCl) and peroxynitrite (ONOO−).3
Free radicals and ROS are present in biological systems
and can be generated from a variety of sources. These
reactive species are able to oxidize proteins, lipids or
nucleic acids, and, thereby, have the potential to initiate
oxidative stress and cause damage to cell structures, being,
in turn, linked to chronic and degenerative diseases,4 such
*e-mail: aldo.sena@ufsc.br

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Synthesis, Antioxidant Activity, Acetylcholinesterase Inhibition and Quantum Studies of Thiosemicarbazones
J. Braz. Chem. Soc.
Another interesting hypothesis proposes that AD is
without additional purification. The obtained compounds
were analyzed by thin layer chromatography (TLC)
using aluminum plates coated with silica gel on TLC Al
foils (Sigma-Aldrich). All synthesized compounds were
characterized by melting points (mp), infrared (IR) and
1H and 13C nuclear magnetic resonance spectroscopy
(NMR). The structures of all compounds were confirmed
by mass spectrometry. Melting points were determined
with a Microquimica MGAPF-301 apparatus. Infrared
spectra were obtained on FTIR spectrophotometer with a
Fourier transform and accessory attenuated total reflection
(FTIR-ATR) PerkinElmer Spectrophotometer Spectrum
100 with a crystal of ZnSe (45°) and a TGS (triglycine
sulfate) detector, the samples were analyzed directly on
the crystal by the attenuated total reflection, averaging
18 scans in the range 4000-400 cm-1 and a resolution
related to oxidative stress, with several studies showing
that oxidative damage is linked to AD, and more recent
studies suggesting that it is associated with the earlier
stages of the disease.19,20 The brain is especially susceptible
to oxidative damage because it has limited antioxidant
capacity, high lipid content and high oxygen consumption
rate when compared to other organs.11,20 Thereby, the
use of antioxidant compounds have produced promising
results in relation to AD.21 The main characteristic of an
antioxidant is its ability to trap free radicals; antioxidant
compounds scavenge free radicals and thus inhibit the
oxidative mechanisms that lead to degenerative diseases.4
The existence of different possible hypotheses to explain
the onset and progression of AD directs the development
of new drugs toward molecules with multiple actions that
are able to act in a complementary manner and could bring
satisfactory results for the treatment of the disease.11,22
There is considerable interest in thiosemicarbazones
because they constitute an important class of compounds
whose properties have been extensively studied in
Medicinal Chemistry,23 displaying a wide range of
biological activities, such as antiviral,24 antitumor,25
antimicrobial,26 antibacterial and antifungal27 properties.
Moreover, studies show that these compounds have
excellent antioxidant activities27,28 and other authors have
reported interesting results of thiosemicarbazones in
combatingAD.29-31 Thiosemicarbazones are reported to act as
enzyme inhibitors by complexing endogenous metals or by
redox reactions and as inhibitors of DNA synthesis through
DNA interactions.23 Furthermore, thiosemicarbazones have
been proposed to inhibit cysteine proteases, which are
essential to several functions of the malaria parasite.32
Based on these observations, the aim of this study
was to synthesize a series of thiosemicarbazones, four
of which had not been reported in the literature before,
as potential therapeutic agents for the treatment of
AD. The compounds were assayed to investigate their
ability to inhibit acetylcholinesterase (AChE), and two
in vitro antioxidants tests were performed, evaluating the
thiosemicarbazones’nitric oxide (NO) scavenging activity
and hydrogen peroxide (H2O2) scavenging activity. In
addition, quantum studies were performed to theoretically
analyze the compounds’ antioxidant activity.
1
13
of 4 cm-1. H and C NMR spectra were obtained using
1
a Bruker Ac-200F (operating at 200 MHz for H and
50 MHz for 13C) or a BrukerAvance Drx 400 spectrometer
(operating at 400 MHz for 1H and 100 MHz for 13C), with
tetramethylsilane as internal standard.
High-resolution mass spectra (HRMS) were recorded
on a micrOTOF-QII (Bruker Daltonics) mass 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).
Spectrophotometric measurements were performed on a
DU640 spectrophotometer from Beckman Instruments,
Inc. (Fullerton, CA).
Procedure for the synthesis of N-(p-tolyl)hydrazinecarbo-
thioamide (1)
Compound 1 was synthesized by reacting
1-isothiocyanato-4-methylbenzene (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.33
Experimental
General procedure
N-(p-Tolyl)hydrazinecarbothioamide (1)
Yield 52%; white solid; mp 137.7-138.3 °C; IR (ATR)
ν / cm-1 3294 (NH), 3242 and 3190 (NH2), 1200 (C=S);
1H NMR (200 MHz, CDCl3) d 7.39 (d, 2H, J 7.8 Hz), 7.17
All reagents were obtained commercially (Sigma-
Aldrich), and all solvents used were of analytical grade,

Vol. 29, No. 2, 2018
Sens et al.
345
(d, 2H, J 8.3 Hz), 3.99 (s, 2H, NH2), 2.34 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 181.0, 135.9, 135.1, 129.4, 124.6,
21.0; HRMS (ESI-TOF) m/z: 182.0745 [M + H]+, calculated
for C8H11N3S, 182.0746.
HRMS (ESI-TOF) m/z: 315.0913 [M + H]+, calculated for
C15H14N4O2S, 315.0910.
(E)-2-(4-Chlorobenzylidene)-N-(p-tolyl)hydrazinecarbo-
thioamide (2d)
General procedure for the synthesis of thiosemicarbazones
Yield 93%; white solid; mp 172.5-174.4 °C; IR (ATR)
ν / cm-1 3328 and 3306 (NH), 1593 (C=N), 1192 (C=S);
1H NMR (200 MHz, CDCl3) d 9.99 (s, 1H, NH), 9.07 (s, 1H,
NH), 7.88 (s, 1H, -NCH), 7.62 (d, 2H, J 8.6 Hz), 7.49 (d,
2H, J 8.3 Hz), 7.39 (d, 2H, J 8.6 Hz), 7.22 (d, 2H, J 8.2 Hz),
2.38 (s, 3H); 13C NMR (50 MHz, CDCl3) d 176.1, 141.4,
136.6, 136.4, 135.1, 131.6, 129.4, 129.2, 128.5, 124.9, 21.0;
HRMS (ESI-TOF) m/z: 304.0672 [M + H]+, calculated for
C15H14ClN3S, 304.0670.
(2a-q)
Thiosemicarbazones 2a-q were prepared by the
stirring and refluxing of a solution containing: N-(p-tolyl)
hydrazinecarbothioamide (1.19 mmol), 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 down to ambient
temperature, and the precipitate formed was collected with
vacuum filtration and washed with water.26
(E)-2-(4-Methoxybenzylidene)-N-(p-tolyl)hydrazinecarbo-
thioamide (2e)
Data referencing the characterization of compounds
2a-2q can be analyzed subsequently.
Yield 82%; white solid; mp 183.2-185.0 °C; IR (ATR)
ν / cm-1 3317 and 3142 (NH), 1607 (C=N), 1194 (C=S);
1H NMR (200 MHz, CDCl3) d 9.98 (s, 1H, NH), 9.10 (s,
1H, NH), 7.88 (s, 1H, -NCH), 7.63 (d, 2H, J 8.7 Hz), 7.51
(d, 2H, J 8.2 Hz), 7.22 (d, 2H, J 8.1 Hz), 6.94 (d, 2H,
(E)-2-Benzylidene-N-(p-tolyl)hydrazinecarbothioamide (2a)
Yield 81%; white solid; mp 171.0-172.0 °C; IR (ATR)
ν / cm-1 3300 and 3148 (NH), 1589 (C=N), 1201 (C=S);
1H NMR (200 MHz, CDCl3) d 10.15 (s, 1H, NH), 9.12 (s,
1H, NH), 7.95 (s, 1H, -NCH), 7.68 (m, 2H), 7.50 (d, 2H,
J 8.2 Hz), 7.42 (m, 3H), 7.22 (d, 2H, J 8.2 Hz), 2.37 (s,
13
J 8.7 Hz), 3.86 (s, 3H, p-OCH3), 2.38 (s, 3H); C NMR
(50 MHz, CDCl3) d 175.8, 161.7, 142.9, 136.1, 135.3,
129.4, 129.1, 125.8, 124.9, 114.3, 55.4, 21.0; HRMS
(ESI-TOF) m/z: 300.11653 [M + H]+, calculated for
C16H17N3OS, 300.11651.
13
3H); C NMR (50 MHz, CDCl3) d 176.1, 142.9, 136.2,
135.2, 133.1, 130.6, 129.4, 128.8, 127.4, 124.8, 21.0;
HRMS (ESI-TOF) m/z: 270.1063 [M + H]+, calculated for
C15H15N3S, 270.1059.
(E)-2-(Benzo[d][1,3]dioxol-5-ylmethylene)-N-(p-tolyl)
hydrazinecarbothioamide (2f)
(E)-2-(4-Methylbenzylidene)-N-(p-tolyl)hydrazinecarbo-
thioamide (2b)
Yield 60%; white solid; mp 183.0-184.8 °C; IR (ATR)
ν / cm-1 3305 and 3178 (NH), 1591 (C=N), 1184 (C=S);
1H NMR (200 MHz, DMSO-d6) d 11.66 (s, 1H, NH),
10.02 (s, 1H, NH), 8.05 (s, 1H, -NCH), 7.83 (s, 1H),
7.39 (d, 2H, J 8.2 Hz), 7.18-7.12 (m, 3H), 6.94 (d, 1H,
J 7.5 Hz), 6.08 (s, 2H), 2.31 (s, 3H); 13C NMR (50 MHz,
DMSO-d6) d 176.3, 149.5, 148.5, 143.0, 137.0, 134.9,
129.1, 128.9, 126.4, 124.7, 108.6, 106.1, 101.9, 21.0;
HRMS (ESI-TOF) m/z: 314.0956 [M + H]+, calculated
for C16H15N3O2S, 314.0958.
Yield 80%; white solid; mp 195.8-196.5 °C; IR (ATR)
ν / cm-1 3302 and 3127 (NH), 1602 (C=N), 1190 (C=S);
1H NMR (200 MHz, CDCl3) d 9.73 (s, 1H, NH), 9.11 (s,
1H, NH), 7.87 (s, 1H, -NCH), 7.57 (d, 2H, J 8.1 Hz), 7.50
(d, 2H, J 8.1 Hz), 7.22 (d, 4H, J 8.0 Hz), 2.39 (s, 3H), 2.37
(s, 3H); 13C NMR (50 MHz, CDCl3) d 176.0, 143.2, 141.1,
136.2, 135.2, 130.4, 129.6, 129.4, 127.4, 124.9, 21.5, 21.0;
HRMS (ESI-TOF) m/z: 284.1213 [M + H]+, calculated for
C16H17N3S, 284.1216.
(E)-2-([1,1’-Biphenyl]-4-ylmethylene)-N-(p-tolyl)hydrazine-
carbothioamide (2g)
(E)-2-(4-Nitrobenzylidene)-N-(p-tolyl)hydrazinecarbo-
thioamide (2c)
Yield 82%; white solid; mp 165.9-167.8 °C; IR (ATR)
ν / cm-1 3303 and 3146 (NH), 1591 (C=N), 1187 (C=S);
1H NMR (200 MHz, CDCl3) d 10.77 (s, 1H, NH), 9.15 (s,
1H, NH), 8.03 (s, 1H, -NCH ), 7.72 (d, 2H, J 8.4 Hz), 7.63-
7.58 (m, 4H), 7.49 (m, 4H, J 8.4 and 8.4 Hz), 7.41-7.36
Yield 97%; yellow solid; mp 207.5-209.5 °C; IR (ATR)
ν / cm-1 3306 and 3127 (NH), 1592 (C=N), 1177 (C=S);
1H NMR (200 MHz, CDCl3) d 9.99 (s, 1H, NH), 9.08 (s,
1H, NH), 8.29 (d, 2H, J 8.8 Hz), 7.96 (s, 1H, -NCH), 7.85
(d, 2H, J 8.8 Hz), 7.50 (d, 2H, J 8.3 Hz), 7.22 (d, 2H), 2.39
13
(m, 1H), 7.21 (d, 2H, J 8.0 Hz), 2.36 (s, 3H); C NMR
13
(s, 3H); C NMR (50 MHz, DMSO-d6) d 177.0, 148.1,
(50 MHz, CDCl3) d 175.9, 143.3, 142.9, 140.1, 136.2,
141.0, 140.4, 136.8, 135.2, 129.0, 128.9, 126.5, 124.2, 21.0;
135.2, 132.1, 129.4, 128.9, 127.9, 127.5, 127.0, 125.0, 21.1;

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Synthesis, Antioxidant Activity, Acetylcholinesterase Inhibition and Quantum Studies of Thiosemicarbazones
J. Braz. Chem. Soc.
HRMS (ESI-TOF) m/z: 346.1369 [M + H]+, calculated for
C21H19N3S, 346.1372.
127.4, 126.8, 125.0, 122.4, 21.1; HRMS (ESI-TOF) m/z:
320.1214 [M + H]+, calculated for C19H17N3S, 320.1216.
(E)-2-(4-Hydroxy-3-methoxybenzylidene)-N-(p-tolyl)
hydrazinecarbothioamide (2h)
(E)-2-(3,4-Dichlorobenzylidene)-N-(p-tolyl)hydrazine-
carbothioamide (2l)
Yield 83%; white solid; mp 157.5-159.0 °C; IR (ATR)
ν / cm-1 3515 (OH), 3394 and 3250 (NH), 1602 (C=N),
1192 (C=S); 1H NMR (200 MHz, CDCl3) d 10.37 (s, 1H,
NH), 9.05 (s, 1H, NH), 7.89 (s, 1H, -NCH), 7.47 (d, 2H,
J 8.4 Hz ), 7.24-7.14 (m, 4H), 6.93 (d, 1H, J 8.0 Hz), 5.93
(s, 1H), 3.94 (s, 3H, m-OCH3), 2.37 (s, 3H); 13C NMR
(50 MHz, CDCl3) d 175.8, 148.3, 146.9, 143.6, 136.3,
135.3, 129.4, 125.6, 125.2, 122.7, 114.7, 108.5, 56.1, 21.0;
HRMS (ESI-TOF) m/z: 316.1113 [M + H]+, calculated for
C16H17N3O2S, 316.1114.
Yield 76%; white solid; mp 206.4-208.0 °C; IR (ATR)
ν / cm-1 3345 and 3127 (NH), 1590 (C=N), 1270 (C=S);
1H NMR (200 MHz, CDCl3) d 10.54 (s, 1H, NH), 9.04
(s, 1H, NH), 7.89 (s, 1H, -NCH), 7.77 (s, 1H), 7.50-7.45
(m, 4H), 7.23 (d, 2H, J 8.2 Hz), 2.38 (s, 3H); 13C NMR
(50 MHz, CDCl3) d 176.2, 140.2, 136.6, 134.9, 134.6,
133.4, 133.2, 130.9, 129.5, 128.6, 126.5, 125.1, 21.1;
HRMS (ESI-TOF) m/z: 338.02801 [M + H]+, calculated
for C15H13Cl2N3S, 338.02800.
(E)-2-(2,5-Dimethoxybenzylidene)-N-(p-tolyl)hydrazine-
carbothioamide (2m)
(E)-N-(p-Tolyl)-2-(3,4,5-trimethoxybenzylidene)hydrazine-
carbothioamide (2i)
Yield 87%; white solid; mp 179.0-181.2 °C; IR (ATR)
ν / cm-1 3294 and 3143 (NH), 1594 (C=N), 1217 (C=S);
1H NMR (200 MHz, CDCl3) d 9.20 (s, 1H, NH), 9.08
(s, 1H, NH), 8.22 (s, 1H, -NCH), 7.51 (d, 2H, J 8.4 Hz),
7.38 (d, 1H, J 2.9 Hz), 7.20 (d, 2H, J 8.2 Hz), 6.97 (dd,
1H, J 9.0 and 3.0 Hz), 6.87 (d, 1H, J 9.1 Hz), 3.84 (s, 3H,
Yield 73%; white solid; mp 167.8-169.9 °C; IR (ATR)
ν / cm-1 3298 and 3140 (NH), 1577 (C=N), 1193 (C=S);
1H NMR (200 MHz, CDCl3) d 10.36 (s, 1H, NH), 9.04
(s, 1H, NH), 7.88 (s, 1H, -NCH), 7.48 (d, 2H, J 8.2 Hz),
7.23 (d, 2H, J 8.4 Hz), 6.88 (s, 2H), 3.91 (s, 6H, m-OCH3),
13
13
3.89 (s, 3H, p-OCH3), 2.38 (s, 3H); C NMR (50 MHz,
o-OCH3), 3.82 (s, 3H, m-OCH3), 2.36 (s, 3H); C NMR
CDCl3) d 176.1, 153.6, 143.1, 140.6, 136.5, 135.2, 129.5,
128.4, 125.3, 104.7, 61.0, 56.3, 21.0; HRMS (ESI-TOF)
m/z: 360.1373 [M + H]+, calculated for C18H21N3O3S,
360.1376.
(50 MHz, CDCl3) d 176.1, 153.7, 153.1, 138.8, 136.0,
135.3, 129.3, 124.7, 122.1, 117.7, 112.6, 110.6, 56.2, 55.9,
21.0; HRMS (ESI-TOF) m/z: 330.1274 [M + H]+, calculated
for C17H19N3O2S, 330.1271.
(E)-2-(Naphthalen-1-ylmethylene)-N-(p-tolyl)hydrazine-
carbothioamide (2j)
(E)-2-(3,4-Dimethoxybenzylidene)-N-(p-tolyl)hydrazine-
carbothioamide (2n)
Yield 83%; light yellow solid; mp 173.0-175.0 °C; IR
(ATR) ν / cm-1 3305 and 3206 (NH), 1593 (C=N), 1189
(C=S); 1H NMR (200 MHz, CDCl3) d 10.28 (s, 1H, NH),
9.18 (s, 1H, NH), 8.70 (s, 1H, -NCH), 8.41 (dd, 1H, J 9.0
and 2.5 Hz), 7.97-7.89 (m, 3H), 7.60-7.48 (m, 5H), 7.21
(d, 2H, J 8.2 Hz), 2.37 (s, 3H);13C NMR (50 MHz, CDCl3)
d 176.1, 141.9, 136.2, 135.5, 133.8, 131.2, 130.9, 129.4,
129.0, 128.8, 127.4, 126.9, 126.3, 125.3, 124.7, 123.2, 21.0;
HRMS (ESI-TOF) m/z: 320.12157 [M + H]+, calculated for
C19H17N3S, 320.12159.
Yield 83%; white solid; mp 182.5-184.5 °C; IR (ATR)
ν / cm-1 3329 and 3138 (NH), 1601 (C=N), 1135 (C=S);
1H NMR (200 MHz, CDCl3) d 9.93 (s, 1H, NH), 9.04 (s,
1H, NH), 7.86 (s, 1H, -NCH), 7.49 (d, 2H, J 8.2 Hz), 7.25-
7.16 (m, 4H), 6.89 (d, 1H, J 8.2 Hz), 3.94 and 3.93 (s, 6H,
m-OCH3 and p-OCH3), 2.37 (s, 3H); 13C NMR (50 MHz,
CDCl3) d 175.9, 151.5, 149.4, 143.3, 136.3, 135.3, 129.4,
126.0, 125.1, 122.4, 111.0, 109.0, 56.0, 21.0; HRMS (ESI-
TOF) m/z: 330.1270 [M + H]+, calculated for C17H19N3O2S,
330.1271.
(E)-2-(Naphthalen-2-ylmethylene)-N-(p-tolyl)hydrazine-
carbothioamide (2k)
(E)-2-(2-Nitrobenzylidene)-N-(p-tolyl)hydrazinecarbo-
thioamide (2o)
Yield 73%; white solid; mp 199.3-200.3 °C; IR (ATR)
ν / cm-1 3311 and 3140 (NH), 1593 (C=N), 1196 (C=S);
1H NMR (200 MHz, DMSO-d6) d 10.25 (s, 1H, NH),
9.18 (s, 1H, NH), 8.10 (s, 1H, -NCH), 7.96-7.82 (m, 5H),
7.55-7.50 (m, 4H), 7.23 (d, 2H, J 8.6 Hz), 2.38 (s, 3H);
13C NMR (50 MHz, CDCl3) d 176.0, 143.2, 136.3, 135.2,
134.4, 133.1, 130.7, 129.8, 129.4, 128.7, 128.4, 127.9,
Yield 90%; yellow solid; mp 194.3-195.7 °C; IR
(ATR) ν / cm-1 3281 and 3125 (NH), 1535 (C=N), 1188
(C=S); H NMR (200 MHz, CDCl3) d 9.72 (s, 1H, NH),
9.06 (s, 1H, NH), 8.42 (s, 1H, -NCH), 8.09-8.00 (m, 2H),
7.74-7.50 (m, 4H), 7.21 (d, 2H, J 8.4 Hz), 2.37 (s, 3H);
13C NMR (50 MHz, CDCl3) d 176.4, 148.2, 137.6, 136.3,
135.0, 133.3, 130.6, 129.4, 128.7, 128.1, 125.0, 124.4, 21.0;
1

Vol. 29, No. 2, 2018
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HRMS (ESI-TOF) m/z: 315.0907 [M + H]+, calculated for
C15H14N4O2S, 315.0910.
whereAcontrol is the absorbance without the sample;Asample is
the absorbance of the sample.
(E)-2-((5-(2-Chloro-5-(trifluoromethyl)phenyl)furan-2-yl)
methylene)-N-(p-tolyl)hydrazinecarbothioamide (2p)
Yield 92%; light yellow solid; mp 207.5-209.5 °C;
IR (ATR) ν / cm-1 3344 and 3140 (NH), 1621 (C=N),
Hydrogen peroxide (H2O2) scavenging activity
The H2O2 scavenging ability of the test compounds
was determined spectrophotometrically, according to the
method described by Ruch et al.35 A solution of H2O2
(40 mM) was prepared in phosphate buffer (pH 7.4). 25, 50,
75 and 100 mg mL-1 concentrations of the test compounds
in 3.4 mL phosphate buffer were added to the H2O2 solution
(0.6 mL, 40 mM). The absorbance value of the reaction
mixture was recorded at 230 nm. The percentage of H2O2
scavenging was calculated using equation 1.34 The results
were expressed as EC50.
1
1266 (C=S); H NMR (200 MHz, CDCl3) d 9.96 (s, 1H,
NH), 9.19 (s, 1H, NH), 8.20 (s, 1H, -NCH), 7.83 (s, 1H),
7.62-7.46 (m, 4H), 7.33 (d, 1H, H3’, J 3.7 Hz), 7.23 (d,
2H, J 8.5 Hz), 6.92 (d, 1H, H2’, J 3.7 Hz), 2.38 (s, 3H);
13C NMR (50 MHz, CDCl3) d 176.1, 150.7, 149.5, 136.8,
134.9, 133.6, 132.6, 132.1, 129.9, 129.3, 129.0, 128.6,
128.0, 126.7, 126.0, 125.4, 125.0, 121.3, 115.2, 114.9, 21.0;
HRMS (ESI-TOF) m/z: 438.0650 [M + H]+, calculated for
C20H15ClF3N3OS, 438.0649.
Acetylcholinesterase inhibitory activity
(E)-2-(4-(Dimethylamino)benzylidene)-N-(p-tolyl)hydrazine-
carbothioamide (2q)
The enzymatic inhibition was measured using the method
described by Canto et al.11 with modifications. Briefly
were mixed: 90 μL of Tris-HCl buffer (of concentration
50 mmol L−1, pH 8.0), 30 μL of methanolic solution of the
compound (0.1 mg mL−1) and 15 μL of an AChE solution
(0.25 U mL−1). The AChE solution was prepared by
dissolving 0.1% of bovine serum albumin in 50 mmol L−1 of
Tris-HCl pH 8 buffer. The mixture was incubated for 15 min.
Then, 25 μL of an acetylthiocholine iodide solution (15 mmol
in water) and 140 μL of Ellman’s reagent (3 mmol L-1 in
Tris-HCl pH 8.0 buffer containing 0.1 mol L-1 NaCl and
0.02 mol L-1 MgCl2) were added, and the final mixture was
incubated for another 30 min at 28 °C. The absorbance of the
mixture was measured at 405 nm. The same solvent in which
the sample was dissolved, considered to have 100% AChE
activity, was used as negative control. The inhibition (%) was
calculated as follows, in which Asample is the absorbance of
the sample and Acontrol is the absorbance without the sample:
Yield 89%; yellow solid; mp 195.2-196.4 °C; IR (ATR)
ν / cm-1 3290 and 3140 (NH), 1588 (C=N), 1265 (C=S);
1H NMR (200 MHz, CDCl3) d 9.45 (s, 1H, NH), 9.09 (s,
1H, NH), 7.77 (s, 1H, -NCH), 7.57-7.50 (m, 4H), 7.20 (d,
2H, J 8.3 Hz), 6.69 (d, 2H, J 8.9 Hz), 3.03 (s, 6H), 2.36 (s,
13
3H); C NMR (50 MHz, CDCl3) d 175.4, 152.0, 143.8,
135.8, 135.6, 129.3, 129.0, 124.7, 120.6, 111.8, 40.1, 21.0;
HRMS (ESI-TOF) m/z: 313.1479 [M + H]+, calculated for
C17H20N4S, 313.1481.
Nitric oxide (NO) scavenging activity
Nitric oxide scavenging activity was measured by the
methodology described by Chandrashekaraiah et al.34 In
this reaction, nitric oxide radicals (NO•) are generated
from sodium nitroprusside and interact with oxygen
to produce nitrite ions that can be estimated using
a Griess reagent (1% sulfanilamide, 2% H3PO4 and
0.1% naphthylethylenediamine dihydrochloride). To
measure the nitric oxide scavenging activity, 1 mL of
sodium nitroprusside (10 mM) and 1.5 mL of phosphate
buffer saline (0.2 M, pH 7.4) were added to different
concentrations (25, 50, 75 and 100 mg mL-1) of the test
compounds and incubated for 150 min at 25 °C, and
then 1 mL of the reaction mixture was treated with 1 mL
of Griess reagent. The absorbance (A) was measured at
546 nm and nitric oxide scavenging activity was calculated
using equation 1.34 The results of antioxidant assays were
expressed as EC50.
(2)
The tests were performed in triplicate, and a blank
containing Tris-HCl buffer was used instead of the
enzyme solution. The concentration required to inhibit
enzyme activity by 50%, IC50, was determined from the
graph-plotted concentration versus absorbance. Remynil®
(0.1 mg mL-1) was used as the positive control, which
contains 50% of galantamine.
Statistical analysis
(1)
All experiments were performed three times and the

348
Synthesis, Antioxidant Activity, Acetylcholinesterase Inhibition and Quantum Studies of Thiosemicarbazones
J. Braz. Chem. Soc.
mean results were calculated. Data was expressed as mean
SD. Linear regression analysis was used to calculate EC50
(for antioxidant assays) or IC50 (for acetylcholinesterase
inhibitory activity) for each compound.
Antioxidant activity
The evaluation of a compound’s antioxidant activity
should not be solely based on a single method.10 Several
in vitro antioxidant tests are reported in the literature, and
in this study, the antioxidant activity of the compounds
2a-q was analyzed using two in vitro assays, which were
correlated with the quantum assays.
Nitric oxide (NO) has a role in the regulation of various
physiological processes and is an important chemical
mediator. Moreover, the NO pathway is involved in various
types of disorders, as Alzheimer’s disease.36
Quantum studies
The molecular representation sketch of the reference
compound was plotted using ChemBioOffice 2010
software. All quantum chemical calculations were
performed using the MM2 methodology.
Results and Discussion
Hydrogen peroxide (H2O2) is rapidly decomposed into
oxygen and water, and this can produce hydroxyl radicals
(OH•) that initiate lipid peroxidation and cause DNA
damage in the body, thus becoming a target for the research
of new compounds.10
The results of nitric oxide scavenging activity and
hydrogen peroxide scavenging activity of compounds 2a-q
are shown in Table 1. All analyzed compounds were more
active than the standard (ascorbic acid). Compound 2j
was the most active and compound 2o the less active from
this series. The results of both experimental activities are
linearly correlated (y = 0.96810x + 2.07336; R2 = 0.99995),
indicating consistent results between both tests.
The synthesis of thiosemicarbazones 2a-q was
performed in two stages, according to Scheme 1. First,
the synthesis of thiosemicarbazide 1 was performed by the
reaction between 1-isothiocyanato-4-methylbenzene and
hydrazine hydrate in isopropanol.33 Compound 1 was used
in subsequent reactions with different aldehydes using acetic
acid as a catalyst in a solvent mixture of water/ethanol26
to yield thiosemicarbazones 2a-q. Thiosemicarbazones
2g, 2i, 2k and 2l have not been reported in the literature.
In general, the synthesis of all compounds showed similar
yields (ranging 60-97%).
All synthesized compounds were unequivocally
1
characterized by their melting points, IR, H NMR, and
Acetylcholinesterase inhibitory activity
13C NMR, and the structures were confirmed by HRMS.
In the compound’s H NMR spectra, a singlet signal at
the 2.30-2.39 region is attributed to a CH3 moiety, and
aromatic hydrogens appear at 6.5-8.5 ppm, depending
on the substituent in the aromatic (Ar) group. The iminic
hydrogen is assigned as a singlet at 7-8 ppm and the N−H
chemical shifts appear as two singlets around 9-12 ppm.
1
All studied compounds were evaluated as AChE
inhibitors. The concentration of test compounds that
inhibited substrate hydrolysis by 50% (IC50, Table 1) was
determined by plotting the inhibition percentage against
the sample solution concentrations.
Thiosemicarbazones 2a-2q showed a high percentage
of inhibition of the enzyme acetylcholinesterase, being as
active as the galantamine standard (active drug used for the
treatment of Alzheimer’s disease, Remynil®). The result
obtained for the standard is in accordance with literature
data.11 The most active compounds in the series were 2c,
13
The C NMR spectra of the compounds showed signals
between 100.0 and 160.0 ppm due to the resonance of aryl
and unsaturated carbons. The signals at the region between
170 and 180 ppm, attributed to the carbon resonance of the
C=S group, are consistent with the expected structures.
Scheme 1. Synthesis of thiosemicarbazide 1 and thiosemicarbazones 2a-q. (i) Isopropanol, r.t.; (ii) ethanol/water, acetic acid, reflux.

Vol. 29, No. 2, 2018
Sens et al.
349
Table 1. Antioxidant activity and acetylcholinesterase inhibitory assays of compounds 2a-2q
Nitric oxide (NO) scavenging
activity EC50 / (μmol L-1)
Hydrogen peroxide (H2O2)
scavenging activity EC50 / (μmol L-1)
Compound
AChE inhibition IC50 / (μmol L-1)
2a
8.856
8.758
9.681
8.714
8.788
8.649
8.784
8.862
8.778
8.631
8.753
8.646
8.645
8.855
10.203
8.632
8.726
14.75
−
10.654
10.556
11.483
10.512
10.585
10.450
10.583
10.661
10.571
10.429
10.551
10.448
10.443
10.653
12.006
10.431
10.528
16.35
6.12
6.36
1.98
9.44
8.01
10.33
4.66
9.86
8.94
2.26
4.45
4.16
7.28
8.56
2.04
6.54
14.26
−
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
Ascorbic acid
Galantamine
−
17.05
2o and 2j. These compounds have a NO2 group (2c, 2o) or
a naphthyl group (2j) in their structures.
molecular orbital) and LUMO (lowest unoccupied
molecular orbital) energy values were calculated and are
shown in Table 2. The HOMO and LUMO band gap (energy
difference between HOMO and LUMO) was determined
for the compounds, as illustrated in Figure 1. Thus, it was
possible to search for relations between the in vitro tests
results and the theoretical data.
EHOMO was a better indicator of antioxidant activity
than ELUMO. In general, it is possible to relate HOMO
energy values and scavenging activities. Compound 2a,
however, did not show a direct relationship between EHOMO
and antioxidant activity, presenting a high HOMO energy
value and a low NO and H2O2 scavenging activity when
compared to the other compounds. This case aside, EHOMO
was considered a good indicator of antioxidant activity for
the tested series in this study.
Byanalyzingthedataobtainedfortheacetylcholinesterase
enzyme inhibition assay it is possible to perceive a strong
correlation between the IC50 value and the substituent groups
present on the ring. The two compounds that presented lower
IC50 values, therefore more active, have in their structure
groups that withdraw electrons, especially the nitro group for
compounds 2c and 2o. Similarly, the compounds for which
the lowest enzymatic inhibition activity, 2q and 2f, were
observed, present groups capable of increasing the electronic
density of the aromatic ring, by donor effect.
Compounds 2j and 2k, which have a naphthyl
substituent, can be highlighted since the results can be
considered to be good for both enzymatic inhibition and
antioxidant assays. As well as, a thiosemicarbazone 2l,
which have a 3,4-diCl-Ph substituent, also showed a good
ratio for the biological assay. Clearly, the ability to act as
donors or as electron withdrawals contributes to the good
results mentioned.
ELUMO was a better indicator than EHOMO regarding
acetylcholinesterase inhibitory activity.
Other properties can also be deducted from the
analysis of HOMO and LUMO energy values, such as
a compound’s ionization potential (IP), electron affinity
(EA), electronegativity (μ), hardness (η) and softness (S).37
The ionization potential (IP) of thiosemicarbazones
2a-q was obtained using equation 3.37 Therefore, the lower
the energy required to remove an electron, higher is the
Quantum studies
Quantum studies was employed to analyze
thiosemicarbazones 2a-q. The HOMO (highest occupied

350
Synthesis, Antioxidant Activity, Acetylcholinesterase Inhibition and Quantum Studies of Thiosemicarbazones
J. Braz. Chem. Soc.
Table 2. Energy values of HOMO, LUMO and band gap of
thiosemicarbazones 2a-q
EA = –ELUMO
(4)
Electronegativity (μ) of thiosemicarbazones 2a-q was
obtained using equation 5.37 Thus, a compound with a lower
electronegativity is expected to have a higher antioxidant
activity.
Band gap
Compound
EHOMO / eV
ELUMO / eV
(EHOMO – ELUMO
3.714
3.941
1.421
4.416
4.366
4.844
2.520
4.546
4.396
1.815
2.391
4.337
4.044
4.424
1.960
3.957
5.658
9.171
8.265
)
2a
−6.228
−6.219
−6.874
−6.187
−6.240
−6.142
−6.238
−6.293
−6.233
−6.099
−6.215
−6.140
−6.138
−6.282
−7.245
−6.129
−6.197
−10.474
−8.968
−2.514
−2.278
−5.453
−1.771
−1.874
−1.298
−3.718
−1.747
−1.837
−4.284
−3.824
−1.803
−2.094
−1.858
−5.285
−2.172
−0.539
−1.303
0.703
2b
2c
(5)
2d
2e
2f
The interaction of valence shell electrons with the
nucleus and intervening filled shells also affects the
polarizability of the valence shell electrons. Polarizability
can be described in terms of hardness and softness. A
relatively large atom or ion with a small effective nuclear
charge is relatively easily distorted (polarized) by an
external charge and is called soft.38 A more compact
electron distribution resulting from a higher net nuclear
charge and less effective screening is called hard. The η
and S of thiosemicarbazones 2a-q were obtained using
equations 6 and 7.37
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
(6)
(7)
Ascorbic acid
Galantamine
The values obtained for IP, EA, μ, η and S can be seen
in Table 3.
The chemical properties that were theoretically
calculated from the LUMO values did not present a good
correlation with the antioxidant activities, which was
already expected, since the ELUMO values were not good
indicators. However, a linear correlation was observed
between the IP and the nitric oxide (NO) scavenging
activity (y = 1.3981x + 0.0719; R2 = 0.99699) and between
the IP and the hydrogen peroxide (H2O2) scavenging
activity (y = 1.40210x + 1.84593; R2 = 0.99705).
The EA presented a better correlation with the results
of acetylcholinesterase inhibition assays, but it was not
as linear as the antioxidant activity. In general, the higher
the electron affinity value, the higher the inhibition of
acetylcholinesterase. Furthermore, a relation with hardness
and softness can be found considering only the best and the
worst results of the enzymatic inhibition test. In this study,
it was possible to observe that the softer compound was the
one that presented the better results (2c), and the harder,
the worse results (2q). The correlations found involving
quantum studies, in relation to biological activities
evaluated at this work, are of a qualitative nature.
Figure 1. Band gap energy for compound 2j.
compound’s antioxidant activity, or, in other words, the
higher the EHOMO, smaller is the IP.
IP = –EHOMO
(3)
The EA of thiosemicarbazones 2a-q was obtained using
equation 4.37 High EA values represent that an electron can
be easily added to the species, being directly related to the
compound’s antioxidant capability.

Vol. 29, No. 2, 2018
Sens et al.
351
Table 3. Chemical properties of thiosemicarbazones 2a-q
Compound
Ionization potential / eV
6.228
Electron affinity / eV
Hardness (η)
1.857
1.970
0.7105
2.208
2.183
2.422
1.260
2.273
2.198
0.9075
1.195
2.168
2.022
2.212
0.980
1.978
2.829
4.585
4.132
Softness (S)
0.5385
0.5075
1.407
Electronegativity (μ)
2a
2.514
2.278
5.453
1.771
1.874
1.298
3.718
1.747
1.837
4.284
3.824
1.803
2.094
1.858
5.285
2.172
0.539
1.303
−0.703
4.371
4.248
6.163
3.979
4.057
3.720
4.978
4.020
4.035
5.191
5.019
3.971
4.116
4.070
6.265
4.150
3.368
5.888
4.835
2b
6.219
2c
6.874
2d
6.187
0.4529
0.4581
0.4129
0.7936
0.4399
0.4549
1.102
2e
6.240
2f
6.142
2g
6.238
2h
6.293
2i
6.233
2j
6.099
2k
6.215
0.8365
0.4611
0.4945
0.4521
1.020
2l
6.140
2m
6.138
2n
6.282
2o
7.245
2p
6.129
0.5054
0.3535
0.2181
0.2420
2q
6.197
Ascorbic acid
Galantamine
10.47
8.968
For the antioxidant activity, the bulky groups of
compounds 2j and 2p (1-naphthyl and 5-(2-Cl-5-CF3)-Ph-
furan-2yl, respectively) appear to be important for the assays
performed in this study. In addition, thiosemicarbazones
2c and 2o presented the worst results of the series; both
compounds have a nitro group (NO2) in their structure,
concluding that this electron-withdrawing group decreases
antioxidant activity.
and the results showed good antioxidant activity.
Compound 2j showed multiple action, a property that
makes it a potential therapeutic agent for AD. In addition,
quantum studies showed that EHOMO values were a better
indicator of antioxidant activity than ELUMO values, while
ELUMO was a better indicator of AChE inhibition.
Supplementary Information
Regarding the inhibitory activity of acetylcholinesterase,
the nitro group (NO2) appeared to be important for a higher
rate of inhibition, a fact that differs from the antioxidant
activity. However, the 1-naphthyl group presented good
results for both activities, thus thiosemicarbazone 2j is
a potential therapeutic agent for AD treatment, since it
has multiple actions, acting as an efficient inhibitor of
acetylcholinesterase and as a potent antioxidant.
Supplementary information with analytical data (1H and
13C NMR and HOMO and LUMO of the surface) is available
free of charge at http://jbcs.sbq.org.br as a PDF file.
Acknowledgments
The authors thank the Chemistry Department’s Center
of Analysis of the Federal University of Santa Catarina
(UFSC) and CNPq (Brazil) for the scholarship.
Conclusions
In this study, the synthesis of seventeen
thiosemicarbazones was performed and the compounds
were analyzed forAChE inhibition potency and antioxidant
activity. All compounds showed excellent activity as
inhibitors of AChE, being more active than the standard
drug. Nitric oxide (NO) scavenging activity and hydrogen
peroxide (H2O2) scavenging activity assays were employed
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Submitted: May 9, 2017
Published online: August 4, 2017