Synthesis, characterization and antichagasic evaluation of thiosemicarbazones prepared from chalcones and dibenzalacetones

Article abstract of DOI:10.1016/j.molstruc.2021.130014

Chagas disease is a neglected disease, being one of the leading causes of death from infectious diseases. In view of the severity of this pathology, this work describes the synthesis of new thiosemicarbazones derived from chalcones and dibenzalacetones as potential drugs for the treatment of this disease. The structures of all compounds were elucidated by infrared (IR) and nuclear magnetic resonance (¹H and ¹³C NMR) spectroscopies. The chalcone derived thiosemicarbazones 10-14 were tested against the intracellular amastigote form of the protozoan Trypanosoma cruzi and had their cytotoxicity assessed using LLC-MK2 cells. The compound 10 (IC₅₀ = 12.25 μM) presented the best activity when compared with the standard drug benznidazole (IC₅₀ = 5.64 μM).

Full text of DOI:10.1016/j.molstruc.2021.130014

Journal of Molecular Structure 1232 (2021) 130014
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Synthesis, characterization and antichagasic evaluation of
thiosemicarbazones prepared from chalcones and dibenzalacetones
Aline Alves da Silvaa, Pedro Ivo da Silva Maiab, Carla Duque Lopesc,d, Sergio de
Albuquerquec, Marcelo Siqueira Vallea,∗
a Departamento de Ciências Naturais, Universidade Federal de São João del-Rei, 36301-160, São João del-Rei, MG, Brazil
b Departamento de Química, Universidade Federal do Triângulo Mineiro, 38025-440, Uberaba, MG, Brazil
c Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto e FCFRP-USP, Universidade de
São Paulo, Avenida do Café s/n, 14040-903, Ribeirão Preto, SP, Brazil
d Centro Universitário Estácio de Ribeirão Preto, Abraão Issa Halach Street, 980, 14096-160, Ribeirão Preto, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Chagas disease is a neglected disease, being one of the leading causes of death from infectious diseases.
In view of the severity of this pathology, this work describes the synthesis of new thiosemicarbazones
derived from chalcones and dibenzalacetones as potential drugs for the treatment of this disease. The
structures of all compounds were elucidated by infrared (IR) and nuclear magnetic resonance (1H and
13 C NMR) spectroscopies. The chalcone derived thiosemicarbazones 10-14 were tested against the intra-
cellular amastigote form of the protozoan Trypanosoma cruzi and had their cytotoxicity assessed using
LLC-MK2 cells. The compound 10 (IC50 = 12.25 μM) presented the best activity when compared with the
standard drug benznidazole (IC50 = 5.64 μM).
Received 13 August 2020
Revised 10 November 2020
Accepted 24 January 2021
Available online 28 January 2021
Keywords:
Chagas disease
Thiosemicarbazones
Trypanosoma cruzi
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
over, BZN present limitations due to side effects, long treatment
regiments [5,7–9]. In terms to improve the treatment options for
Chagas disease (CD) is a neglected disease, caused by hemoflag-
ellate protozoan Trypanosoma cruzi (TC) [1,2]. This disease is en-
demic in countries of Central and South America, but, in the last
decades the number of cases of CD increase in other countries in
the South of United States of America, Canada, West Mediterranean
and Western Pacific [2,3]. It is estimated that approximately 6 to 7
million people are potentially infected with TC which causes ap-
proximately 20,000 deaths per year and is the leading cause of
infectious myocarditis [1,2]. Acute infections are usually asymp-
tomatic but one third of chronically infections progress to death,
showing diverse manifestations affecting the heart, intestines, and
nervous systems with determines the debilitating character of this
disease. Although Chagas disease was identified more than 100
years ago, current therapeutic options are limited only two nitro-
heterocyclic drugs: Benznidazole (BZN) and Nifurtimox (NFX). In
some countries, NFX was discontinued due to serious side effects,
such as neuropathy and anorexia, among others. BZN is only ef-
fective in the acute stage, with low cure infection but its efficacy,
in the chronic phase of disease, remain questionable [4–6]. More-
the CD, many therapeutic options have been tested. Recently, the
inhibitor of ergosterol synthesis, Posoconazol, was promising in re-
duce the infection. Unfortunately it failed in the clinical trial [5,10].
Therefore, one effective therapeutic option for patients with Cha-
gas’ disease are clearly needed.
With this concern in mind, the search for new drug candi-
dates for the treatment of Chagas disease patients is urgent. Many
molecules having thiosemicarbazones moiety has shown a broad
pharmacological applications that include antineoplastic, antibac-
terial, antiviral, antiprotozoal and antifungal activity [11–15]. Re-
garding the antichagasic activity, a series of thiosemicarbazones,
semicarbazones and aminoguanidine derivatives (1-3, Fig. 1) have
shown trypanocidal activity which is supposedly related to their
capacity in inhibiting essential enzymes for parasite survival in-
side the host cells [16,17]. Also interesting, the coordination of
thiosemicarbazones to metal ions have shown to be a good strat-
egy to potentialize their activity. For example, organometallic
gold(III) complexes, such as 4, were evaluated both by in vitro and
in vivo tests, being more effective than benznidazole in eliminat-
ing both the extracellular trypomastigote and intracellular amastig-
ote forms of the parasite without cytotoxic effects on mammalian
cells [18].
∗
Corresponding author.
E-mail address: marcelovalle@ufsj.edu.br (M.S. Valle).
https://doi.org/10.1016/j.molstruc.2021.130014
0022-2860/© 2021 Elsevier B.V. All rights reserved.

A.A. da Silva, P.I.d.S. Maia, C.D. Lopes et al.
Journal of Molecular Structure 1232 (2021) 130014
perimental procedure used for the synthesis of these compounds
was based on the work of Aljamali and collaborators [21]. Briefly,
5.0 mmol of acetophenone and 20 mL of ethanol were added to
a round-bottom flask in the presence of sodium hydroxide (5.0
mmol). The reaction system was kept under magnetic stirring un-
til the mixture was completely solubilized. Immediately thereafter,
the substituted aromatic aldehyde (5.0 mmol) was added, and the
system remained under magnetic stirring until the precipitation of
crystals. The reaction time varied from 1 to 4 hours at room tem-
perature. The end of the reaction was verified by TLC (thin layer
chromatography) (eluent: 7:3 hexane/ethyl acetate; developer: ul-
traviolet light and iodine vapor). The generated solids were vac-
uum filtered and left in a desiccator till dryness. The same proce-
dure was made to the synthesis of dibenzalacetones but using ace-
tone instead of acetophenone and 2 equivalents of aldehyde were
required.
Fig. 1. Selected semicarbazones, thiosemicarbazones and aminoguanidine deriva-
tives with properties against Chagas disease (1-4).
In view of the promising antichagasic properties of thiosemi-
carbazones, we propose the synthesis of new derivatives of this
class of substances by conjugation with chalcones and its structural
analogues dibenzalacetones (dbas). Chalcones are known for their
varied medicinal activities [19,20]. Since chalcones and dbas are
α,β-unsaturated ketones, they are liable to react with thiosemicar-
bazides to provide the respective thiosemicarbazones.
The corresponding thiosemicarbazones (10-14, 18-20, 22) were
synthesized from the chalcones and dibenzalacetones previously
obtained (Scheme 1). The procedure was performed as described
by Góes et al. [22] 5.0 mmol of the chalcones (5-9) or dibenzalace-
tones (15-17, 21) were subjected to a condensation reaction with
thiosemicarbazide (5.0 mmol) in ethanol in the presence of cat-
alytic amounts of sulfuric acid (H2SO4). The reaction time varied
from 1 to 4 hours at room temperature. The end of the reaction
was verified by TLC (eluent: 7:3 hexane/ethyl acetate; developer:
ultraviolet light and iodine vapor). The generated solids were fil-
tered off and dried under vacuum.
2. Experimental section
2.1. Materials and instruments
All reagents were purchased from commercial sources and
used without further purification. The melting point measurements
were performed on the MICRO-CHEMICAL equipment, model
MQAPF-302. The FT-IR spectra were obtained using the BRUKER
ALPHAFT-IR MB 102 spectrophotometer, in the 4000-400 cm−1
region on KBr pellets. The 1H (500 MHz) and 13C (125 MHz)
NMR spectra were performed on the BRUKER ADVANCE spec-
trometer, using deuterated chloroform (CDCl3) or dimethylsulfox-
ide ((CD3)2SO).
2.3. Spectroscopic data
(2E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (5). Pale yellow
solid; Yield: 96%; mp: 108–110°C (lit. 108.0 – 110.0°C) [23]; FT-IR
(KBr) 1/λ (cm−1): 1660 (C=O), 1591 (C=C olefin), 1465 and 1402
(C=C aromatic), 1090 (C-Cl), 3241 (=C-H sp2), 813 (p-substituted),
2356 (p-substituted harmonic); 1H NMR (500 MHz, CDCl3) δ 7.36
– 7.59 (m, 8H, H and aromatic hydrogens), 7.75 (d, 1H, J 15.7 Hz,
α
2.2. Synthesis
H ), 7.98 (d, 2H, J 7.5 Hz, H3, H5); 13C NMR (125 MHz, CDCl3) δ
β
122.5 (C ), 128.5 (C , C ), 128.7 (C , C ), 129.2 (C , C ), 129.6 (C ,
α
2’
6’
3
5
3’
5’
2
The α,β-unsaturated ketones (5-9, 15-17, 21) were obtained us-
C6), 132.9 (C1), 133.4 (C1’), 136.4 (C4), 138.0 (C4’), 143.3 (C ), 190.2
β
ing Claisen-Schmidt aldolic condensation (Scheme 1) and the ex-
(C=O).
Scheme 1. Synthetic route for the synthesis of thiosemicarbazones 10-14, 18-20 and 22. Reactional conditions: a) NaOH, EtOH/H2O, rt; b) NH2NHCSNH2, H2SO4(cat), EtOH, rt.
2

A.A. da Silva, P.I.d.S. Maia, C.D. Lopes et al.
Journal of Molecular Structure 1232 (2021) 130014
(2E)-3-(4-bromophenyl)-1-phenylprop-2-en-1-one (6). Pale yellow
solid; Yield: 94%; mp: 121–122°C (lit. 121.0 – 123.0°C) [24]; FT-IR
(KBr) 1/λ (cm−1): 3419 (=C-H sp2), 1662 (C=O), 1598 (C=C olefin),
1482 and 1444 (C=C aromatic), 1069 (C-Br), 813 (p-substituted); 1H
H
stretch), 2364 (C-H sp3), 1637 (C=N), 1618 (C=C olefin), 1512
(N-H folding), 1402 and 1383 (C=C aromatic), 1258 (C-O), 804 (p-
substituted); 1H NMR (500 MHz, CDCl3) δ 3.92 (s, 3H, CH3), 6.31
(d, 1H, J 16.3 Hz, H ), 6.42 (d, 1H, J 16.3 Hz, H ), 6.92 (s, 1H,
α
β
NMR (500 MHz, CDCl ) δ 7.47–7.59 (m, 8H, H and aromatic hy-
α
3
NH), 7.00 – 7.01 (d, 2H, H3, H5), 7.24 – 8.08 (m, 7H, aromatic
hydrogens), 8.55 (s, 2H, NH2); 13C NMR (125 MHz, CDCl3) δ 55.4
(C ), 114.4 (C , C ), 119.8 (C ), 125.6 (C ), 127.6 (C , C ), 128.4
drogens), 7.73 (d, 1H, J 15.7 Hz, H ), 7.98 (d, 2H, J 7.3 Hz, H3, H5);
β
13C NMR (125 MHz, CDCl ) δ 122.5 (C ), 124.8 (C ), 128.5 (C , C ),
128.7 (C2, C6), 129.8 (C3’, C5’), 132.2 (C3, C5), 132.9 (C1), 133.8 (C4’),
α
3
4
2’
6’
α
7
3
5
1
3’
5’
(C2’, C6’), 128.6 (C2, C6), 130.2 (C4’), 132.5 (C ), 138.5 (C1’), 144.7
β
138.0 (C1’), 143.3 (C ), 190.2 (C=O).
(C=N), 161.7 (C4), 178.3 (C=S).
β
(2E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (7). Pale yel-
low solid; Yield: 97%; mp: 64–66°C (lit. 65.0 – 67.0°C) [25]; FT-
IR (KBr) 1/λ (cm−1): 3261 (=C-H sp2), 2834 (C-H sp3), 2048 (p-
substituted harmonic), 1602 (C=O), 1511 (C=C olefin), 1471 and
1422 (C=C aromatic), 1252 (C-O), 822 (p-substituted); 1H NMR (500
MHz, CDCl3) δ 3.83 (s, 3H, CH3), 6.92 (d, 2H, J 8.5 Hz, H2, H6),
(Z)-2-((E)-3-(3,4-dimethoxyphenyl)-1-phenylallylidene)
hydrazinecarbothioamide (13). Orange solid; Yield: 75 %; mp:
245–249°C; FT-IR (KBr) 1/λ (cm−1): 3470 (=C-H sp2), 2826 (C-H
sp3), 1645 (C=N), 1500 (C=C olefin), 1467 (N-H folding), 1410 and
1329 (C=C aromatic), 1265 (C-O); 1H NMR (500 MHz, CDCl3) δ 3.97
(s, 6H, CH ), 6.32 (d, 1H, J 16.3 Hz, H ), 6.35 (d, 1H, J 16.3 Hz, H ),
α
3
β
7.37–7.59 (m, 5H, aromatic hydrogens), 7.41 (d, 1H, J 15.7 Hz, H ),
α
6.83 (s, 1H, NH), 6.93–8.04 (m, 8H, aromatic hydrogens), 8.52 (s,
2H, NH2); 13C NMR (125 MHz, CDCl3) δ 56.0 (C7, C8), 110.1 (C2),
7.78 (d, 1H, J 15.6 Hz, H ), 7.98 (d, 2H, J 7.6 Hz, H3, H5); 13C NMR
β
(125 MHz, CDCl ) δ 55.4 (C ), 114.4 (C , C ), 119.8 (C ), 127.6 (C ),
α
3
7
3
5
1
111.1 (C ), 120.1 (C ), 123.2 (C ), 127.9 (C ), 128.4 (C , C ), 128.6
α
5 6 1 3’ 5’
128.4 (C2’, C6’), 128.6 (C3’, C5’), 130.2 (C2, C6), 132.6 (C1’), 138.5
(C2’, C6’), 129.9 (C4’), 132.6 (C1’), 138.5 (C4), 145,0 (C ), 149.3 (C3),
β
(C4’), 144.7 (C ), 161.7 (C4), 190.6 (C=O).
151.5 (C=N), 178.3 (C=S).
β
(2E)-3-(3,4-dimethoxyphenyl)-1-phenylprop-2-en-1-one (8). Pale
yellow solid; Yield: 98%; mp: 81–83°C (lit. 83.0 – 84.0°C) [26]; FT-
IR (KBr) 1/λ (cm−1): 3344 (=C-H sp2), 2968 (C-H sp3), 1604 (C=O),
1519 (C=C olefin), 1468 and 1420 (C=C aromatic), 1261 (C-O); 1H NMR
(500 MHz, CDCl3) δ 3.00 (d, 6H, CH3), 5.95 (d, 1H, J 8.4 Hz, H5),
6.21 (s, 1H, H2), 6.26 (t, 1H, H4’), 6.29 (t, 1H, H6), 6.45 (d, 1H, J
(Z)-2-((E)-1-phenyl-3-(3,4,5-trimethoxyphenyl)allylidene)
hydrazinecarbothioamide (14). Orange solid; Yield: 71 %; mp:
247 – 251°C; FT-IR (KBr) 1/λ (cm−1): 3425 (=C-H sp2), 3154 (N-H
stretch), 2826 (C-H sp3), 1645 (C=N), 1601 (N-H), 1504 (C=C olefin),
1473 and 1419 (C=C aromatic), 1123 (C-O); 1H NMR (500 MHz,
CDCl ) δ 3.87 (s, 9H, CH ), 6.38 (d, 1H, J 16.2 Hz, H ), 6.55 (s,
α
3
3
15.6 Hz, H ), 6.55 (t, 2H, H H5’), 6.82 (d, 1H, J 15.6 Hz, H ), 7.06
(d, 2H, J 7.3 Hz, H2’, H6’); 13C NMR (125 MHz, CDCl3) δ 56.0 (C7),
α
3’,
1H, NH), 7.01 (d, 1H, J 16.2 Hz, H ), 7.27–7.29 (d, 2H, H2, H6),
β
7.58–7.61 (m, 3H, H3’, H4’, H5’), 8.02–8.04 (d, 2H, H2’, H6’), 8.52 (s,
2H, NH2); 13C NMR (125 MHz, CDCl3) δ 56.2 (C7, C9), 61.0 (C8),
β
56.1 (C ), 110.1 (C ), 111.1 (C ), 120.1 (C ), 123.2 (C ), 127.9 (C ),
α
8
2
5
6
1
128.4 (C2’, C6’), 128.6 (C3’, C5’), 132.6 (C4’), 138.5 (C1’), 145.0 (C ),
β
104.2 (C , C ), 105.7 (C ), 121.5 (C ), 128.5 (C , C ), 128.6 (C ,
α
2
6
1
3’
5’
2’
149.2 (C4), 151.4 (C3), 190.6 (C=O).
C6’), 130.0 (C4’), 130.4 (C1’), 132.7 (C ), 138.3 (C4), 140.5 (C3, C5),
β
(2E)-3-(3,4,5-trimethoxyphenyl)-1-phenylprop-2-en-1-one
(9).
145.0 (C=N), 177.8 (C=S).
Pale yellow solid; Yield: 98%; mp: 135–137°C (lit. 135.0 – 136.0°C)
[27]; FT-IR (KBr) 1/λ (cm−1): 3344 (=C-H sp2), 2827 (C-H sp3),
1581 (C=O), 1504 (C=C olefin), 1473 and 1419 (C=C aromatic), 1114
(C-O); 1H NMR (500 MHz, CDCl3) δ 3.89 (s, 9H, CH3), 6.84 (s, 2H,
(1E,4E)-1,5-bis(4-chlorophenyl)penta-1,4-dien-3-one (15). Pale yel-
low solid; Yield: 93 %; mp: 190–193°C (lit. 193°C) [28]; FT-IR (KBr)
1/λ (cm−1): 3043 (=C-H sp2), 1650 (C=O), 1588 (C=C olefin), 1492
and 1405 (C=C aromatic), 1087 (C-Cl), 821 (p-substituted); 1H NMR
H , H ), 7.24 – 7.99 (m, 7H, H , H and aromatic hydrogens); 13C
(500 MHz, CDCl ) δ 7.08 (d, 2H, J 15.6 Hz, H , Hα’), 7.43 (d, 4H,
α
α
2
6
β
3
NMR (125 MHz, CDCl3) δ 56.2 (C7, C9), 61.0 (C8), 105.7 (C2, C6),
H3, H5, H3’, H5’), 7.58 (d, 4H, H2, H6, H2’, H6’), 7.73 (d, 2H, J 15.6
Hz, H , Hβ’); 13C NMR (125 MHz, CDCl ) δ 125.7 (C , Cα’), 129.3
(C3, C5, C3’, C5’), 129.5 (C2, C6, C2’, C6’), 133.2 (C1, C1’), 136.5 (C4,
121.5 (C ), 128.5 (C ), 128.6 (C , C ), 130.4 (C , C ), 132.7 (C ),
α
α
1
2’
6’
3’
5’
4’
3
β
138.3 (C1’), 140.5 (C4), 145.0 (C ), 153.5 (C3, C5), 190.6 (C=O).
β
(Z)-2-((E)-3-(4-chlorophenyl)-1-phenylallylidene)
C4’), 142.0 (C , Cβ’), 188.3 (C=O).
β
hydrazinecarbothioamide (10). Pale yellow solid; Yield: 81%; mp:
218–220°C; FT-IR (KBr) 1/λ (cm−1): 3416 (=C-H sp2), 3230 (N-H
stretch), 1639 (C=N), 1619 (C=C olefin), 1479 and 1400 (C=C aromatic),
1253 (N-H folding), 1085 (C-Cl), 815 (p-substituted); 1H NMR (500
MHz, CDCl ) δ 6.38 (d, 1H, J 16.2 Hz, H ), 6.47 (s, 1H, NH), 7.03 (d,
(1E,4E)-1,5-bis(4-bromophenyl)penta-1,4-dien-3-one (16). Pale
yellow solid; Yield: 95 %; mp: 209–212°C (lit. 211.0 – 213.0°C)
[29]; FT-IR (KBr) 1/λ (cm−1): 2930 (=C-H sp2), 1653 (C=O), 1592
(C=C olefin), 1490 and 1402 (C=C aromatic), 1076 (C-Br), 822 (p-
substituted); 1H NMR (500 MHz, CDCl3) δ 7.07 (d, 2H, J 15.6 Hz,
α
3
1H, J 16.2 Hz, H ), 7.39 – 7.79 (m, 7H, aromatic hydrogens), 8.03 –
8.04 (d, 2H, H3, H5), 8.54 (s, 2H, NH2); 13C NMR (125 MHz, CDCl3)
H , Hα’), 7.50 (d, 4H, H3, H5, H3’, H5’), 7.58 (d, 4H, H2, H6, H2’,
α
β
H6’), 7.70 (d, 2H, J 15.6 Hz, H , Hβ’); 13C NMR (125 MHz, CDCl3)
β
δ 124.9 (C , C ), 125.8 (C , Cα’), 129.7 (C2, C6, C2’, C6’), 132.2 (C3,
δ 122.5 (C ), 128.2 (C , C ), 128.3 (C , C ), 129.0 (C , C ), 129.2
α
4
4’
α
3’
5’
3
5
2
6
C5, C3’, C5’), 133.6 (C1, C1’), 142.1 (C , Cβ’), 188.3 (C=O).
(C2’, C6’), 130.0 (C4’), 130.4 (C1), 134.7 (C1’), 137.4 (C4), 143.3 (C ),
β
β
(1E,4E)-1,5-bis(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one (17).
Orange solid; Yield: 96 %; mp: 124–127°C (lit. 123.0 – 124.0°C)
[29]; FT-IR (KBr) 1/λ (cm−1): 3000 (=C-H sp2), 2830 (C-H sp3),
1591 (C=O), 1510 (C=C olefin), 1464 and 1415 (C=C aromatic), 1128
(C-O); 1H NMR (500 MHz, CDCl3) δ 3.93 (s, 18H, H7, H8, H9, H7’,
152.0 (C=N), 178.5 (C=S).
(Z)-2-((E)-3-(4-bromophenyl)-1-phenylallylidene)
hydrazinecarbothioamide (11). Pale yellow solid; Yield: 79 %;
mp: 228–230°C; FT-IR (KBr) 1/λ (cm−1): 3407 (=C-H sp2), 3234
(N-H stretch), 1661 (C=N), 1639 (C=C olefin), 1484 and 1392 (C=C
aromatic), 1221 (N-H), 1071 (C-Br), 812 (p-substituted); 1H NMR
(500 MHz, CDCl ) δ 6.30 (d, 1H, J 16.3 Hz, H ), 6.40 (s, 1H, NH),
H , H ), 6.87 (s, 4H, H , H , H , H ), 6.98 (d, 2H, J 15.0 Hz, H ,
α
8’
9’
2
6
2’
6’
H
α’), 7.66 (d, 2H, J 15.0 Hz, H , Hβ’); 13C NMR (125 MHz, CDCl3)
β
α
3
δ 56.2 (C7, C9, C7’, C9’), 61.0 (C8, C8’), 105.6 (C2, C6, C2’, C6’), 124.8
7.00 (d, 1H, J 16.3 Hz, H ), 7.40–7.70 (d, 2H, H3, H5), 7.40–8.00 (m,
β
7H, aromatic hydrogens), 8.50 (s, 2H, NH2); 13C NMR (125 MHz,
(C , Cα’), 130.2 (C1, C1’), 140.4 (C4, C4’), 143.3 (C , C ), 153.5 (C3,
α
β
β’
C5, C3’, C5’), 188.5 (C=O).
CDCl ) δ 122.5 (C ), 124.8 (C ), 128.2 (C , C ), 128.7 (C , C ),
129.8 (C2’, C6’), 130.4 (C4’), 132.0 (C3, C5), 132.2 (C1), 134.7 (C1’),
α
3
4
2
6
3’
5’
2-((1E,4E)-1,5-bis(4-chlorophenyl)penta-1,4-dien-3-ylidene)
hydrazinecarbothioamide (18). Orange solid; Yield: 77 %; mp:
226–228°C; FT-IR (KBr) 1/λ (cm−1): 3435 (=C-H sp2), 3236 (N-H
stretch), 1627 (C=N), 1601 (C=C olefin), 1482 and 1402 (C=C aromatic),
1318 (N-H folding), 1084 (C-Cl), 814 (p-substituted); 1H NMR
143.4 (C ), 152.0 (C=N), 178.5 (C=S).
β
(Z)-2-((E)-3-(4-methoxyphenyl)-1-phenylallylidene)
hydrazinecarbothioamide (12). Orange solid; Yield: 72 %; mp:
226–228°C; FT-IR (KBr) 1/λ (cm−1): 3435 (=C-H sp2), 3236 (N-
3

A.A. da Silva, P.I.d.S. Maia, C.D. Lopes et al.
Journal of Molecular Structure 1232 (2021) 130014
(500 MHz, CDCl ) δ 7.04 (d, 2H, J 16.2 Hz, H , Hα’), 7.24 (s, 1H,
control and the culture media as negative control. The concentra-
tion of the compound corresponding to 50% trypanocidal activity
in amastigote forms was expressed as the IC50 amastigote.
α
3
NH), 7.29 – 7.58 (m, 8H, aromatic hydrogens), 7.72 (d, 2H, J 16.2
Hz, H , H ), 9.08 (s, 2H, NH2); 13C NMR (125 MHz, CDCl3) δ 125.7
β
β’
(C , Cα’), 126.4 (C4, C4’), 128.3 (C1, C1’), 128.5 (C2, C6, C2’, C6’),
α
129.3 (C3, C5, C3’, C5’), 129.5 (C , Cβ’), 142.1 (C=N), 178.7 (C=S).
β
2.5. In vitro cytotoxicity assay
2-((1E,4E)-1,5-bis(4-bromophenyl)penta-1,4-dien-3-ylidene)
hydrazinecarbothioamide (19). Orange solid; Yield: 75 %; mp:
230–232°C; FT-IR (KBr) 1/λ (cm−1): 1655 (C=N), 1596 (C=C olefin),
1487 and 1398 (C=C aromatic), 1202 (N-H), 1076 (C-Br), 822 (p-
substituted); 1H NMR (500 MHz, CDCl3) δ 7.03 (d, 2H, J 15.3 Hz,
The LLC-MK2 cell viability was assessed using the clas-
sical [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide] (MTT) colorimetric assay to determine selectivity against T.
cruzi. For this propose, the same protocol established to trypanoci-
dal activity was maintained. Briefly, 2.5 × 104 cells were incubated
for 48 h in 96-well microplate for cell culture. After this incubation
period, the compounds or BZN was added (concentrations 500 to
3.9 mM in serial dilution / compounds solubilized in DMSO 0.5%)
in a final volume of 200 mL. Cells were incubated for 72 hours at
37 °C. After incubation with the ligands and their complexes, the
H , Hα’), 7.31 (s, 1H, NH), 7.45 – 7.53 (m, 8H, aromatic hydrogens),
α
7.66 (d, 2H, J 15.3 Hz, H , H ), 8.95 (s, 2H, NH2); 13C NMR (125
β
β’
MHz, CDCl ) δ 124.9 (C , Cα’), 125.8 (C4, C4’), 128.7 (C1, C1’),
α
3
129.7 (C2, C6, C2’, C6’), 132.2 (C3, C5, C3’, C5’), 133.6 (C , Cβ’), 142.1
β
(C=N), 188.3 (C=S).
2-((1E,4E)-1,5-bis(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-
ylidene)hydrazinecarbothioamide (20). Pale red solid; Yield: 80
%; mp: 250–252°C; FT-IR (KBr) 1/λ (cm−1): 2919 (C-H sp3), 1582
(C=N), 1500 (N-H), 1456 (C=C olefin), 1339 and 1237 (C=C aromatic),
1128 (C-O); 1H NMR (500 MHz, CDCl3) δ 3.87 (s, 18H, H7, H8, H9,
medium was removed and added 50 mL of MTT (5.0 mg mL–1
)
diluted in phosphate buffered saline (PBS). The precipitated blue
MTT formazan was then dissolved in 50 mL of DMSO, and the
absorbance was measured at 570 nm in a VARIAN CARY-50 plate
reader MPR multiwell. Cell viability was expressed as the percent-
age of absorption values in treated cells compared with untreated
(control) cells. CC50 (cytotoxic concentration of 50% of the cells)
H , H , H ), 6.37 (d, 2H, J 15.3 Hz, H , Hα’), 6.74 (d, 4H, H2, H6,
α
7’
8’
9’
H2’, H6’), 6.95 (s, 1H, NH), 7.35 (d, 2H, J 15.3 Hz, H , Hβ’), 9.01
β
(s, 2H, NH2); 13C NMR (125 MHz, CDCl3) δ 56.2 (C7, C9, C7’, C9’),
61.0 (C8, C8’), 104.3 (C2, C6, C2’, C6’), 104.6 (C1, C1’), 114.9 (C4, C4’),
125.5 (C , Cα’), 131.5 (C , C ), 137.4 (C3, C5, C3’, C5’), 153.6 (C=N),
was also calculated. Thus, SI is defined by the ratio of CC50 to IC50
.
α
β
β’
187,0 (C=S).
3. Results and discussion
(1E,4E)-1,5-di(thiophen-2-yl)penta-1,4-dien-3-one (21). Brown
solid; Yield: 92 %; mp: 110–113°C (lit. 113.0 – 114.0°C) [30]; FT-IR
(KBr) 1/λ (cm−1): 3073 (=C-H sp2), 1609 (C=O), 1555 (C=C olefin),
1420 and 1370 (C=C aromatic); 1H NMR (500 MHz, CDCl3) δ 6.80
3.1. Synthesis
For the synthesis of thiossemicarbazones, we started prepar-
ing the α,β-insaturated ketones chalcones and dibenzalacetones by
Claisen-Schmidt aldolic condensation of acetophenone or acetone
with different aromatic aldehydes in basic medium (Scheme 1).
The reaction time varied from 1 to 4 hours and the solids gener-
ated were filtered under vacuum and washed with cold water and
left in a desiccator till dryness. The products were obtained in ex-
cellent yields (94-98% for the chalcones 5-9 and 92-96 % for the
dibenzalacetones 15-17, 21). The melting range values correspond-
ing to these compounds were between 2 to 3°C, which indicates
a relatively high degree of purity and are in accordance with the
values described in the literature (see Experimental Section).
From the chalcones and dbas obtained, the synthesis of
thiosemicarbazones in good yields (71-83 %) was possible by re-
actions with the desired thiosemicarbazide in ethanol under acid
catalysis (Scheme 1), providing the chalcones (10-14) and dibenza-
lacetones (18-20 and 22) derivatives.
(d, 2H, J 15.7 Hz, H , Hα’), 7.05 (t, 2H, H3, H3’), 7.31 (d, 2H, H2,
α
H2’), 7.38 (d, 2H, H4, H4’), 7.82 (d, 2H, J 15.7 Hz, H , Hβ’); 13C NMR
β
(125 MHz, CDCl ) δ 124.4 (C , Cα’), 128.3 (C3, C3’), 128.8 (C2, C2’),
α
3
131.8 (C , C ), 135.6 (C4, C4’), 140.3 (C1, C1’), 187.7 (C=O).
β
β’
2-((1E,4E)-1,5-di(thiophen-2-yl)penta-1,4-dien-3-ylidene)
hydrazinecarbothioamide (22). Dark brown solid; Yield: 73 %;
mp: 217–220°C; FT-IR (KBr) 1/λ (cm−1): 3422 (=C-H sp2), 3264
(N-H), 1610 (C=N), 1484 (C=C olefin), 1420 and 1360 (C=C aromatic),
1094 (N-H folding); 1H NMR (500 MHz, CDCl3) δ 6.80 (d, 2H, J 15.3
Hz, H , Hα’), 7.05–7.42 (m, 5H, H3, H3’, H2, H2’, NH), 7.82 (d, 2H, J
α
15.3 Hz, H , H ), 8.99 (s, 2H, NH2); 13C NMR (125 MHz, CDCl3) δ
β
β’
114.2 (C , C ), 127.8 (C , C ), 128.0 (C , Cα’), 128.5 (C2, C2’), 128.8
α
3
3’
β
β’
(C4, C4’), 135.7 (C1, C1’), 145.7 (C=N), 178.6 (C=S).
2.4. Biological assays
2.4.1. In vitro trypanocidal activity assay
Trypanocidal activity of the thiossemicarbazones complexes and
benznidazole (BZN) was evaluated against amastigotes forms of
the Tulahuen strain expressed beta-galactosidase. Briefly, LLCMK-
2 cells (2.5 × 104 cells. mL–1) were ressuspended in RPMI-1640
medium without phenol red (Sigma-Aldrich), supplemented with
5% bovine foetal serum (GIBCO, Grand Island, NY, USA), 100 IU
mL−1 penicillin G, and 100 mg mL−1 streptomycin (Gibco-BRL,
Grand Island, NY, USA) and seeded in 96-well microplates. After 3
h, the cells were infected with 5.0 × 105 trypomastigotes forms of
T. cruzi Tulahuen strain stably expressing the β-galactosidase gene
from Escherichia coli and incubated for 48 h. After the infection pe-
riod, the plates were washed to remove the free trypomastigotes
forms of T. cruzi. The infected cells with the intracellular amastig-
otes forms were incubated with the compounds or benznidazole
(BZN), in serial concentrations between 500 to 3.9 mM. After 72
h, 50 μL of PBS (phosphate buffered saline) containing 0.3% of Tri-
ton X-100 and 400 mM chlorophenol Red-β-D-galactopyranoside
(CPRG) were added. Plates were incubated at 37°C for 4 h and the
absorbance was read at 570 nm. The BZN was used as positive
3.2. Spectroscopic analysis
The characterization of all compounds was performed by FTIR
and multinuclear NMR (1H and 13C) spectroscopies and by compar-
ison of values of melting points with those from literature for the
compounds previously described. The IR spectra analysis of chal-
cones (5-9) and dbas (15-17, 21) showed the main bands of absorp-
tion related to the desired products. All of them showed asymmet-
ric intense stretching bands for typical unsaturated carbonyl group
(C=O) absorptions at 1660 cm−1 or close to this region. The band
referring to the olefin C=C double bond was found close to 1500
cm−1, while the stretches associated with the aromatic ring (C=C)
showed two absorptions between 1480 and 1402 cm−1. The for-
mation of the products was also verified by the analysis of the 1H
NMR spectra, from which it’s possible to observe a doublet (J 15.7
Hz) related to the β-H in the 7.40 to 7.80 ppm region, belonging to
the trans isomer of the olefin. In the 13C NMR spectra, we highlight
the signal at around 190.0 ppm which is attributed to carbonyl
4

A.A. da Silva, P.I.d.S. Maia, C.D. Lopes et al.
Journal of Molecular Structure 1232 (2021) 130014
Table 1
by including other withdrawing and donating groups in the aro-
matic rings or their coordination to metal ions must be performed
in order to establish a better structure-activity relationship of this
class of compounds in order to fine tune their antiparasitc activity,
which is an ongoing subject.
IC50 and CC50 values of the trypanocidal activity and cyto-
toxicity of the chalcone derived thiosemicarbazones 10-14.
Compound
IC50 (μM)
CC50 (LLC-MK2) (μM)
SI
10
12.25
46.42
31.96
30.40
40.59
5.64
39.89
45.12
65.17
80.49
36.03
204.00
3.25
0.97
2.03
2.64
0.88
36.00
11
12
Declaration of Competing Interest
13
14
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper. The authors declare the
following financial interests/personal relationships which may be
considered as potential competing interests
BZN
carbon. In the range of 122.5 to 143.3 ppm the aromatic carbon
signals and α and β carbonyl signals are observed.
The structures of thiosemicarbazones were also confirmed by
NMR and FTIR techniques. By analyzing their IR spectra, we could
observe the expected stretching bands associated with the groups
N-H and C=N in the region of 3400 and 1630 cm−1, respectively.
In general, for the 1H NMR spectra, the structures of thiosemicar-
bazones were confirmed by observing the presence of a wide inte-
grated singlet peak with chemical shift close to 6.4 ppm, referring
to the hydrogen atom from the NH group, as well as another sin-
glet in the region of 8.6 ppm referring to the hydrogen atoms from
the amine group (NH2). Likewise, the signals arising in their 13C
NMR spectra at around 152.0 ppm and 187 ppm can be attributed
to the carbon atoms from the C=N and C=S groups, respectively,
which confirm the expected structures.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130014.
CRediT authorship contribution statement
Aline Alves da Silva: Visualization, Investigation, Data curation,
Writing - original draft. Pedro Ivo da Silva Maia: Writing - re-
view & editing, Data curation, Writing - original draft. Carla Duque
Lopes: Data curation, Writing - original draft, Validation. Sergio
de Albuquerque: Data curation, Writing - original draft, Valida-
tion. Marcelo Siqueira Valle: Conceptualization, Methodology, Vi-
sualization, Investigation, Writing - review & editing, Supervision,
Data curation, Writing - original draft.
3.3. Evaluation of the antichagasic activity
References
To evaluate the potential of these compounds as therapeutic
agents for Chagas disease treatment, preliminary biological tests
were performed on the intracellular form of Trypanosoma cruzi
and determined their cytotoxicity using the LLC-MK2 (Macaca mu-
latta) cell line. The data obtained are shown in Table 1.
[1] D.H. Molyneux, L. Savioli, D. Engels, Neglected tropical diseases: progress to-
wards addressing the chronic pandemic, Lancet 389 (10066) (2017) 312–325,
doi:10.1016/S0140-6736(16)30171-4.
[2] WHO, “What is Chagas disease?,” Who, 2020. [Online]. Available: http://www.
who.int/chagas/disease/en/. [Accessed: 26-Jul-2020].
From the data shown in Table 1, it is possible to notice that
thiosemicarbazone 10 showed a remarkable IC50 value when com-
pared to benznidazole (standard drug used to treat the disease).
When comparing compounds 10 and 11, structural analogues sub-
stituted at the same position by different halogen atoms, it is ob-
served that compound containing the more electronegative halo-
gen atom, 10, is about three times more effective than compound
11. Similarly, the replacement of the chlorine atom by a methoxy
group, compound 12, did not improve the activity. Furthermore, by
increasing the number of substitutions by methoxy groups, com-
pounds 13 and 14, the activity was kept or decreased when com-
pared to the monosubstituted 12. Regarding the CC50 values, it is
possible to verify that the peripheral groups of the thiosemicar-
bazones play a role on the cytotoxicity. Therefore, although the
compounds studied here showed low selectivity indexes, it was
possible to verify that the activity and cytotoxicity may be tuned
by changes in their structure.
[3] M. Castillo-Riquelme, Chagas disease in non-endemic countries, Lancet Glob.
Heal. 5 (4) (2017) e379–e380, doi:10.1016/S2214-109X(17)30090-6.
[4] J.A. Urbina, Recent clinical trials for the etiological treatment of chronic chagas
disease: advances, challenges and perspectives, J. Eukaryot. Microbiol. 62 (1)
(2015) 149–156, doi:10.1111/jeu.12184.
[5] I. Molina, et al., Randomized trial of posaconazole and benznidazole for
chronic Chagas’ disease, N. Engl. J. Med. 370 (20) (2014) 1899–1908, doi:10.
1056/NEJMoa1313122.
[6] F. Villalta, G. Rachakonda, Advances in preclinical approaches to Chagas dis-
ease drug discovery, Expert Opin. Drug Discov. 14 (11) (2019) 1161–1174,
doi:10.1080/17460441.2019.1652593.
[7] R. Viotti, et al., Towards a paradigm shift in the treatment of chronic chagas
disease, Antimicrob. Agents Chemother. 58 (2) (2014) 635–639, doi:10.1128/
AAC.01662-13.
[8] J. Bermudez, C. Davies, A. Simonazzi, J. Pablo Real, S. Palma, Current drug ther-
apy and pharmaceutical challenges for Chagas disease, Acta Trop. 156 (2016)
1–16, doi:10.1016/j.actatropica.2015.12.017.
[9] J.A. Urbina, Specific chemotherapy of chagas disease: relevance, current limi-
tations and new approaches, Acta Trop. 115 (1–2) (2010) 55–68, doi:10.1016/j.
actatropica.2009.10.023.
[10] C.A. Morillo, et al., Benznidazole and posaconazole in eliminating parasites in
asymptomatic T. Cruzi carriers: the Stop-Chagas trial, J. Am. Coll. Cardiol. 69
(8) (2017) 939–947, doi:10.1016/j.jacc.2016.12.023.
4. Conclusions
[11] L. Feun, et al., Phase
I
and pharmacokinetic study of 3-aminopyridine-2-
single intravenous dose
carboxaldehyde thiosemicarbazone (3-AP) using
a
schedule, Cancer Chemother. Pharmacol. 50 (3) (2002) 223–229, doi:10.1007/
s00280-002-0480-0.
In this work, 18 compounds were synthesized, from which 9
are thiosemicarbazones, and all of them were characterized by FT-
IR and 1H/13C NMR analysis. The products were obtained with
global yields (two steps) that varied from 70 to 78%. The thiosemi-
carbazones derived from chalcones 10-14 showed activity against
the protozoan Trypanosoma cruzi, the etiological agent of CD, being
compound 10 the most effective (IC50 = 12.25 μM). Furthermore,
it was observed that the cytotoxicity on LLC-MK2 cells is depen-
dent on the substituents on the thiosemicarbazone moiety. These
preliminary results show that the investigated compounds present
potential as basis for the development of new drugs for Chagas
disease treatment. However, more modifications in their structure
[12] N.C. Kasuga, et al., Synthesis, structural characterization and antimicrobial ac-
tivities of 12 zinc(II) complexes with four thiosemicarbazone and two semi-
carbazone ligands, J. Inorg. Biochem. 96 (2–3) (2003) 298–310, doi:10.1016/
S0162-0134(03)00156-9.
[13] Y. Teitz, D. Ronen, A. Vansover, T. Stematsky, J.L. Riggs, Inhibition of human im-
ꢀ
ꢀ
munodeficiency virus by N-methylisatin-β4 :4 -diethylthiosemicarbazone and
ꢀ
ꢀ
N-allylisatin-β-4 :4 -diallythiosemicarbazone, Antiviral Res. 24 (4) (1994) 305–
314, doi:10.1016/0166-3542(94)90077-9.
[14] D.X. West, et al., Thiosemicarbazone complexes of copper(II): structural and
biological studies, Coord. Chem. Rev. 123 (1–2) (1993) 49–71, doi:10.1016/
0010-8545(93)85052-6.
[15] N. Bharti, K. Husain, M.T.G. Garza, D.E. Cruz-vega, B.D. Mata-cardenas, A. Azam,
Synthesis and in vitro antiprotozoal activity of thiosemicarbazone derivatives,
Bioorg. Med. Chem. Lett. 12 (2002) 3475–3478.
5

A.A. da Silva, P.I.d.S. Maia, C.D. Lopes et al.
Journal of Molecular Structure 1232 (2021) 130014
[16] D.G. Vital, et al., Application of bioisosterism in design of the semicar-
bazone derivatives as cruzain inhibitors: a theoretical and experimental study,
J. Biomol. Struct. Dyn. 35 (6) (2017) 1244–1259, doi:10.1080/07391102.2016.
1176603.
[23] S.R. Sarda et al., “Solvent-free NaOH-Al 2 O 3 supported synthesis of Ar,” vol.
1, no. 2, pp. 265–269, 2009.
[24] A. Stroba, et al., 3,5-Diphenylpent-2-enoic acids as allosteric activators of the
protein kinase PDK1: structure-activity relationships and thermodynamic char-
acterization of binding as paradigms for PIF-binding pocket-targeting com-
pounds, J. Med. Chem. 52 (15) (2009) 4683–4693, doi:10.1021/jm9001499.
[25] T.P. Acids, et al., Synthesis of some benzalacetophenones and their imino
derivatives, J. Cryst. Growth 310 (10) (2008) 4702–4708, doi:10.1055/
sos-sd-039-00764.
[17] L. Blau, et al., Design, synthesis and biological evaluation of new aryl thiosemi-
carbazone as antichagasic candidates, Eur. J. Med. Chem. 67 (2013) 142–151,
doi:10.1016/j.ejmech.2013.04.022.
[18] C.D. Lopes, et al., Organometallic Gold(III) complex [Au(Hdamp)(L14)]+
(L1 = SNS-donating thiosemicarbazone) as a candidate to new formulations
against Chagas disease, ACS Infect. Dis. 5 (10) (2019) 1698–1707, doi:10.1021/
acsinfecdis.8b00284.
[26] V.D. Silva, B.U. Stambuk, M.da G. Nascimento, Efficient chemoselective biohy-
drogenation of 1,3-diaryl-2-propen-1-ones catalyzed by Saccharomyces cere-
visiae yeasts in biphasic system, J. Mol. Catal. B Enzym. 63 (3–4) (2010) 157–
163, doi:10.1016/j.molcatb.2010.01.010.
[19] R. Arif, et al., Facile synthesis of chalcone derivatives as antibacterial agents:
synthesis, DNA binding, molecular docking, DFT and antioxidant studies, J. Mol.
Struct. 1208 (2020) 127905, doi:10.1016/j.molstruc.2020.127905.
[20] M. Mustafa, Y.A. Mostafa, A facile synthesis, drug-likeness, and in silico molec-
ular docking of certain new azidosulfonamide–chalcones and their in vitro an-
timicrobial activity, Monatshefte fur Chemie 151 (3) (2020) 417–427, doi:10.
1007/s00706-020-02568-8.
[27] S. Ducki, et al., Combretastatin-like chalcones as inhibitors of microtubule
polymerization. Part 1: synthesis and biological evaluation of antivascular ac-
tivity, Bioorganic Med. Chem. 17 (22) (2009) 7698–7710, doi:10.1016/j.bmc.
2009.09.039.
[28] H.J. Ravindra, et al., Synthesis, crystal growth and characterization of a phase
matchable nonlinear optical single crystal: p-chloro dibenzylideneacetone, J.
Cryst. Growth 310 (10) (2008) 2543–2549, doi:10.1016/j.jcrysgro.2007.12.066.
[29] H. Ohori, et al., Synthesis and biolgical analysis of new curcumin analogues
bearing an enhanced potential for the medicinal treatment of cancer, Mol. Can-
cer Ther. 5 (10) (2006) 2563–2571, doi:10.1158/1535- 7163.MCT-06-0174.
[30] T.P. Acids, Studies the xvii, 1931 no. 232.
[21] A.M. Jawad, M.N.M. Salih, T.A. Helal, Review on chalcone (preparation, reac-
tions, medical and bio applications), Int. J. Chem. Synth. Chem. React. 5 (1)
(2019) 16–27.
[22] R.P. Tenório, A.J.S. Góes, J.G. De Lima, A.R. De Faria, A.J. Alves, T.M. De
Aquino, Tiossemicarbazonas: Métodos de obtenção, aplicações sintéticas
e
importância biológica, Quim. Nova 28 (6) (2005) 1030–1037, doi:10.1590/
S0100-40422005000600018.
6