Evaluation of novel chalcone-thiosemicarbazones derivatives as potential Anti-leishmania amazonensis agents and its HSA binding studies

Article abstract of DOI:10.3390/biom9110643

A series of seven chalcone-thiosemicarbazones (5a–5g) were synthesized and evaluated as potential new drugs (anti-leishmanial effect). Although four of the chalcone-thiosemicarbazones are already known, none of them or any compound in this class has been previously investigated for their effects on parasites of the Leishmania genus. The compounds were prepared in satisfactory yields (40–75%) and these compounds were evaluated against promastigotes, axenic amastigotes and intracellular amastigotes of L. amazonensis after 48 h of culture. The half maximal inhibitory concentration (IC50) values of the intracellular amastigotes were determined to be in the range of 3.40 to 5.95 μM for all compounds assayed. The selectivity index showed value of 15.05 for 5a, whereas pentamidine (reference drug) was more toxic in our model (SI = 2.32). Furthermore, to understand the preliminary relationship between the anti-leishmanial activity of the chalcone-thiosemicarbazones, their electronic (σ), steric (MR) and lipophilicity (π) properties were correlated, and the results indicated that moieties with electronic withdrawing effects increase the anti-leishmanial activity. The preliminary pharmacokinetic evaluation of one of the most active compound (5e) was studied via interaction to human serum albumin (HSA) using multiple spectroscopic techniques combined with molecular docking. The results of antiparasitic effects against L. amazonensis revealed the chalcone-thiosemicarbazone class to be novel prototypes for drug development against leishmaniasis.

Full text of DOI:10.3390/biom9110643

biomolecules
Article
Evaluation of Novel Chalcone-Thiosemicarbazones
Derivatives as Potential Anti-Leishmania amazonensis
Agents and Its HSA Binding Studies
Edinéia Pastro Mendes 1 , Carla Marins Goulart , Otávio Augusto Chaves 1,3,
,2
1
4
4
4
Viviane dos S. Faiões , Marilene M. Canto-Carvalho , Gerzia C. Machado ,
Eduardo Caio Torres-Santos * and Aurea Echevarria *
4
,
1,
1
Instituto de Química, Departamento de Química Orgânica, Universidade Federal Rural do Rio de Janeiro,
Seropédica 23.890-000, Rio de Janeiro, Brazil; edinea@bio.fiocruz.br (E.P.M.);
cmarinsgoulart@gmail.com (C.M.G.)
Programa de Pós-graduação em Ciência, Tecnologia e Inovação em Agropecuária, Universidade Federal
2
Rural do Rio de Janeiro, Seropédica, Seropédica 23890-000, Rio de Janeiro, Brazil
Instituto SENAI de Inovação em Química Verde, Maracanã 20271-030, Rio de Janeiro, Brazil;
3
oachaves@firjan.com.br
Laboratório de Bioquímica de Tripanosomatídeos, Instituto Oswaldo Cruz, FIOCRUZ,
4
Rio de Janeiro 21040-900, Brazil; faioes@gmail.com (V.d.S.F.); mcantoca@ioc.fiocruz.br (M.M.C.-C.);
machado@ioc.fiocruz.br (G.C.M.)
*
Correspondence: ects@ioc.fiocruz.br (E.C.T.-S.); echevarr@ufrrj.br (A.E.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 22 September 2019; Accepted: 21 October 2019; Published: 23 October 2019
Abstract: A series of seven chalcone-thiosemicarbazones (5a–5g) were synthesized and evaluated
as potential new drugs (anti-leishmanial effect). Although four of the chalcone-thiosemicarbazones
are already known, none of them or any compound in this class has been previously investigated
for their effects on parasites of the Leishmania genus. The compounds were prepared in satisfactory
yields (40–75%) and these compounds were evaluated against promastigotes, axenic amastigotes
and intracellular amastigotes of L. amazonensis after 48 h of culture. The half maximal inhibitory
concentration (IC ) values of the intracellular amastigotes were determined to be in the range of 3.40
50
to 5.95 µM for all compounds assayed. The selectivity index showed value of 15.05 for 5a, whereas
pentamidine (reference drug) was more toxic in our model (SI = 2.32). Furthermore, to understand the
preliminary relationship between the anti-leishmanial activity of the chalcone-thiosemicarbazones,
their electronic (σ), steric (MR) and lipophilicity (π) properties were correlated, and the results
indicated that moieties with electronic withdrawing effects increase the anti-leishmanial activity.
The preliminary pharmacokinetic evaluation of one of the most active compound (5e) was studied
via interaction to human serum albumin (HSA) using multiple spectroscopic techniques combined
with molecular docking. The results of antiparasitic effects against L. amazonensis revealed the
chalcone-thiosemicarbazone class to be novel prototypes for drug development against leishmaniasis.
Keywords:Leishmaniaamazonensis; promastigotes; intracellularamastigotes; chalcone-thiosemicarbazones;
human serum albumin; spectroscopy; molecular docking
1
. Introduction
Leishmaniasis is caused by more than 20 Leishmania species belonging to the Trypanosomatidae
family. These parasites are transmitted to humans by the bite of female sand flies. There are three
principal forms of the disease: cutaneous, mucocutaneous and visceral. This disease is prevalent in
Biomolecules 2019, 9, 643; doi:10.3390/biom9110643
www.mdpi.com/journal/biomolecules

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3
8 countries on five continents and is estimated to cause 700,000 to 1 million new cases and 20,000 to
0,000 deaths annually [1].
The chemotherapeutic treatments for leishmaniasis remain not satisfactory due to their limited
effectiveness, long timeline of treatment, high cost and severe secondary effects [
2,3]. The first-choice
treatment involves the use of pentavalent antimonials (SbV) such as sodium stibogluconate
®
®
(
Pentostam ) and meglumine antimoniate (Glucantime ) [
4]. The second choice includes pentamidine
and amphotericin B, which are highly toxic and cause serious side effects [5,6
]. More recently, the oral
drug miltefosine has been used for the treatment of visceral leishmaniasis, but it presents a high
cost and a low therapeutic ratio and, in addition, has mild-to-severe gastrointestinal side effects and
teratogenicity [7].
Thus, due to these serious disadvantages of the current treatment, the development of safer
and more effective drugs for the treatment of the leishmaniasis is a priority. Currently, our research
group has been developing novel compounds based on natural products, such as chalcones and
their derivatives.
Chalcones are an important class of natural products with widespread biological activities [8–11].
This molecular class is present in several vegetal species, and its synthesis is easy, representing a great
opportunity to generate several new derivatives. Chalcones have the ability to coordinate metals and
exhibit a diversified range of biological activities, e.g., antiparasitic [8,9], anticancer [10] and antioxidant
activity [11]. The thiosemicarbazones derivatives also present interesting structural properties due to
their several pharmacological effects such as antiparasitic [12–14], antimicrobial [15,16] and anticancer
activities [17,18].
The molecular composition from hybridizing chalcones and thiosemicarbazones may result in
new and interesting molecules with favorable biological activities. In the last few years, some
chalcone-thiosemicarbazones have been reported with promising biological activities, such as
anticancer [19] and anti-tyrosinase activities [20]. Furthermore, several metal complexes of
chalcone-thiosemicarbazones with biological activity have been reported [21,22].
Studies on molecular interactions of drugs with macromolecules have significantly contributed to
the understanding of preliminary pharmacodynamics and/or pharmacokinetics evaluation of potential
drugs. According to this, human serum albumin (HSA) is the most abundant globular protein in the
human bloodstream, having attracted great interest from the pharmaceutical industry because it can bind
to a remarkable variety of endogenous and exogenous molecules, impacting their delivery and efficacy
and altering mainly their pharmacokinetic properties [23,24]. The HSA is a globular protein consisting
of a single polypeptide chain of 585 amino acid residues and is composed of three structurally similar
domains (I, II and III). The amino acid residue Trp-214 is the main fluorophore of HSA and generally its
fluorescence is used to obtain parameters which are related to the binding ability [25,26].
Based on the background described above, the main goal of the present work was the synthesis
and characterization of a novel series of seven chalcone-thiosemicarbazones: 1-phenyl-3-(4-X-phenyl)-2-
ylprop-2-en-1-one thiosemicarbazones, where X=H (5a), CH3 (5b), CN (5c), F (5d), Br (5e), Cl (5f) and
NO (5g) (Figure 1), as well as the biological evaluation of each compound toward promastigotes,
2
axenic and intracellular amastigotes of Leishmania amazonensis. For the compound which presented the
best biological activity, its preliminary pharmacokinetic parameter was evaluated via ability to interact
to HSA (spectroscopic and theoretical methods).
Figure 1. Synthetic hybrid chalcone-thiosemicarbazone structures under study.

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2
. Materials and Methods
.1. Organic Synthesis
.1.1. Chemicals and Instruments
All reagents (hydrochloric acid, acetophenone, X-benzaldehydes, NaOH) and solvents (DMSO-d6,
ethanol, hexane, ethyl acetate)used in the organic synthesis were purchased from Sigma Aldrich,
St. Louis, MO, USA and used without further purification. The Nuclear Magnetic Resonance (NMR)
1
13
spectra ( H and C) were obtained using a Bruker NMR Ultrashield 400 MHz spectrometer with
tetramethylsilane as internal reference and deuterated dimethylsulfoxide (DMSO-d ) as the solvent;
6
the chemical shifts were reported in ppm. The infrared (IR) spectra were recorded on a Bruker Vertex
70 spectrometer using potassium bromide (KBr) tablets. Elemental analyses were performed on a
PerkinElmer 2400 CHN in the Laboratory of Environmental Science in the State University of Northern
Rio de Janeiro (UENF). The melting points were recorded on a Gehaka (PF 1500 Farma) capillary
melting points apparatus and are uncorrected. Reactions were monitored by thin layer chromatography
(
TLC) on Merck silica gel 60 F245 aluminium sheets. TLC spots were visualized by inspection of the
plates under ultraviolet (UV) light (254 and 365 nm).
2
.1.2. (2E)-3-(4-X-phenyl)-1-phenylprop-2-en-1-one (3a–g)
The chalcone derivatives (3a
literature reported [27,28].
–
g, Figure 2) were synthesized and characterized according to the
O
X = H (3a); CH (3b); CN (3c)
3
;
X
F (3d); Cl (3e); Br (3f); NO2 (3g)
Figure 2. Chemical structure for the synthetic chalcones 3a–g.
2
.1.3. General Procedure for the Preparation of the (1E,2E)-3-(4-X-phenyl)-1-phenylprop-2-en-1-one
Thiosemicarbazones (5a–g)
A mixture of chalcones (3a
–g) (2 mmol) and thiosemicarbazide (4) (2.4 mmol) in hot ethanol
(50 mL) had a few drops of concentrated hydrochloric acid added. The reaction mixture was stirred
at reflux temperature for 2–6 h, and monitored by TLC using hexane:ethyl acetate (8:2) as the eluent.
Afterwards, the precipitate was filtered off and the crude product purified by recrystallization from
ethanol, resulting in the target compounds (5a–g, Figure 3).
H
H
N
S
X = H (5a); CH (5b); CN (5c)
3
;
H
N
N
1
F (5d); Cl (5e); Br (5f); NO2 (5g)
2
''
3
2'
1
''
1'
3'
3
''
2
4
'
6
''
6'
4
''
X
5
''
5'
Figure 3. Chemical structure for the synthetic chalcones 3a–g.
(1E,2E)-3-(phenyl)-1-phenylprop-2-en-1-one thiosemicarbazone (5a)
◦
◦
−1
Pale yellow amorphous solid; yield 82%; m.p. 133–135 C (135–137 C [29]). IR (KBr, cm ):
3
469, 3287, 3187 and 3140 (N-H), 3036 (C-H), 1585 (C=N), 1498 and 1444 (C=C), 1128 (C=S), 968 (C=C
1
trans-substituted). H NMR (DMSO-d )
δ
: 8.65 and 8.50 (2H, 2s, NH ), 8.16 (1H, s, NH), 7.67–7.60
6
2
00 00 00 00 00 0
(3H, m, H-3 , H-4 and H-5 ), 7.47–7.46 (2H, d, J = 7.0 Hz, H-2 , H-6 ), 7.38-7.29 (5H, m, H-2 ,
0
0
0
0
H-3 , H-4 , H-5 and H-6 ), 7.20–7.17 (1H, d, J = 16.0 Hz, H-3), 6.45–6.42 (1H, d, J = 16.0 Hz, H-2).

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1
3
00
0
00
C NMR (DMSO-d )
δ
: 179.3 (C=S), 148.6 (C=N), 140.1 (C-3), 137.3 (C-1 ), 136.2 (C-1 ), 130.2 (C-4 ),
6
00
00
0
0
0
0
00
00
0
1
1
29.7 (C-2 and C-6 ), 129.1 (C-2 and C-6 ), 128.7 (C-3 and C-5 ), 128.4 (C-3 and C-5 ), 127.3 (C-4 ),
19.2 (C-2).
0
(
1E,2E)-3-(4 -methylphenyl)-1-phenylprop-2-en-1-one thiosemicarbazone (5b)
◦
◦
−1
Yellow amorphous solid; yield 51%; m.p. 138–140 C (145 C [29]). IR (KBr, cm ): 3408, 3341, 3233
and 3143 (N-H), 3037 (C-H), 1598 (C=N), 1477 and 1440 (C=C), 1126 (C=S), 966 (C=C trans-substituted).
1
H NMR (DMSO-d ) δ: 11.08 and 8.10 (1H, s, NH), 8.59 and 8.44 (2H, 2s, NH ), 7.67–7.62 (3H, m,
2
6
00
00
00
00
00
H-3 , H-4 and H-5 ), 7.37–7.34 (2H, dd, J = 7.5 and 1.5 Hz, H-2 and H-6 ), 7.23–7.22 (2H, d, J =
8
H-3), 6.42–6.39 (1H, d, J = 16.4 Hz, H-2), 2.30 (3H, s, CH ). C NMR (DMSO-d ) δ: 179.3 (C=S), 148.9
0 0 0 0
.0 Hz, H-2 and H-6 ), 7.18–7.17 (2H, d, J = 8.0 Hz, H-3 and H-5 ), 7.14–7.10 (1H, d, J = 16.4 Hz,
13
3
6
0 00 0 00 00 0 0
(C=N), 140.1 (C-3), 139.6 (C-1 ), 137.4 (C-1 ), 133.6 (C-4 ), 129.7 (C-2 and C-6 ), 129.5 (C-2 and C-6 ),
00
00
0
0
00
1
28.7 (C-3 and C-5 ), 128.4 (C-3 and C-5 ), 127.3 (C-4 ), 118.1 (C-2), 21.5 (CH ).
3
0
(
1E,2E)-3-(4 -cyanophenyl)-1-phenylprop-2-en-1-one thiosemicarbazone (5c)
◦
−1
Pale yellow amorphous solid; yield 75%; m.p. 195–196 C. IR (KBr, cm ): 3422, 3341, 3246
and 3146 (N-H), 3040 (C-H), 2233 (C
trans-substituted). H NMR (DMSO-d )
≡
N), 1599 (C=N), 1549 and 1476 (C=C), 1131 (C=S), 951 (C=C
: 8.72 and 8.67 (2H, 2s, NH ), 8.19 (1H, s, NH), 7.83–7.81 (2H,
1
δ
6
2
0
0
0
0
00
00
d, J = 8.5 Hz, H-3 and H-5 ), 7.70–7.68 (2H, d, J = 8.5 Hz, H-2 and H-6 ), 7.67–7.62 (3H, m, H-3 , H-4
00
00
00
and H-5 ), 7.38–7.36 (2H, dd, J = 7.3 and 1.7 Hz, H-2 and H-6 ), 7.35–7.31 (1H, d, J = 16.5 Hz, H-3),
13
0
.56–6.52 (1H, d, J = 16.5 Hz, H-2). C NMR (DMSO-d ) δ: 178.4 (C=S), 150.1 (C=N), 140.9 (C-1 ), 134.7
6
6
0
0
00
00
00
00
00
(
C-3), 133.2 (C-3 and C-5 ), 132.3 (C-2), 130.5 (C-4 ), 130.5 (C-1 ), 130.3 (C-2 and C-6 ), 128.7 (C-3
and C-5 ), 128.0 (C-2 and C-6 ), 119.2 (CN), 111.0 (C-4 ). Anal. Calcd. for C H N S: C, 66.64, H,
00
0
0
0
17
14
4
4
.61, N, 18.29. Found: C, 66.68, H, 4.57, N, 18.32.
0
(
1E,2E)-3-(4 -fluorphenyl)-1-phenylprop-2-en-1-one thiosemicarbazone (5d)
◦
−1
Yellow amorphous solid; yield 37%; m.p. 152–154 C (without m.p., [29]). IR (KBr, cm ): 3413,
3
9
7
342, 3256 and 3154 (N-H), 3043 (C-H), 1598 (C=N), 1476 and 1443 (C=C), 1229 (C-F), 1127 (C=S),
1
67 (C=C trans-substituted). H NMR (DMSO-d )
δ
: 8.63 and 8.50 (2H, 2s, NH ), 8.12 (1H, s, NH),
6
2
0
0
00
00
0
0
.68–7.62 (3H, m, H-3 , H-4 and H-5 ), 7.57–7.53 (2H, dd, J = 8.8 and 3.2 Hz, H-2 and H-6 ), 7.37–7.34
00 00 0 0
(2H, dd, J = 8.1 and 1.5 Hz, H-2 and H-6 ), 7.23–7.18 (2H, t, J = 8.8 Hz, H-3 and H-5 ), 7.15–7.11
13
(1H, d, J = 16.4 Hz, H-3), 6.48–6.44 (1H, d, J = 16.4 Hz, H-2). C NMR (DMSO-d )
δ
: 179.3 (C=S),
6
0
00
164.1 and 162.1 (1C, d, 1J = 248.0 Hz, C-4 ), 148.4 (C=N), 138.8 (C-3), 137.2 (C-1 ), 132.9 and 132.9 (1C,
0
0
0
00
00
d, 4J = 2.5 Hz, C-1 ), 130.6 and 130.5 (2C, d, 3J = 8.5 Hz, C-2 and C-6 ), 129.7 (C-2 and C-6 ), 129.5
00
00
00
0
0
(C-4 ), 128.7 (C-3 and C-5 ), 119.0 (C-2), 116.1 and 115.9 (2C, d, 1J = 21.5 Hz, C-3 and C-5 ). Anal.
Calcd. for C H FN S: C, 64.19, H, 4.71, N, 10.04. Found: C, 64.25, H, 4.68, N, 10.09.
16
14
3
0
1E,2E)-3-(4 -chlorophenyl)-1-phenylprop-2-en-1-one thiosemicarbazone (5e)
(
◦
◦
−1
Pale yellow amorphous solid; yield 57%; m.p. 163–164 C (164-166 C [29]). IR (KBr, cm ): 3413,
3
9
7
341, 3256 and 3154 (N-H), 3043 (C-H), 1598 (C=N), 1476 and 1441 (C=C), 1127 (C=S), 1093 (C-Cl),
1
68 (C=C trans-substituted). H NMR (DMSO-d )
δ
: 8.63 and 8.53 (2H, 2s, NH ), 8.12 (1H, s, NH),
6
2
0
0
00
00
0
0
.67–7.60 (3H, m, H-3 , H-4 and H-5 ), 7.52–7.50 (2H, d, J = 8.5 Hz, H-2 and H-6 ), 7.42–7.41 (2H,
0
0
00
00
d, J = 8.5 Hz, H-3 and H-5 ), 7.37–7.35 (2H, dd, J = 6.9 and 1.0 Hz, H-2 and H-6 ), 7.20–7.16 (1H,
13
d, J = 16.4 Hz, H-3), 6.47–6.44 (1H, d, J = 16.4 Hz, H-2). C NMR (DMSO-d ) δ: 178.3 (C=S), 150.7
6
00 0 0 00 00 00
C=N), 135.5 (C-3), 135.2 (C-1 ), 133.6 (C-1 ), 130.6 (C-4 ), 130.5 (C-4 ), 130.2 (C-2 and C-6 ), 129.6
C-2), 129.3 (C-2 and C-6 ), 129.0 (C-3 and C-5 ), 128.7 (C-3 and C-5 ).
(
(
0
0
0
0
00
00

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0
(
1E,2E)-3-(4 -bromophenyl)-1-phenylprop-2-en-1-one thiosemicarbazone (5f)
◦
−1
Pale yellow amorphous solid; yield 43%; m.p. 166–168 C (without m.p. [19]). IR (KBr, cm ):
3
9
7
414, 3341, 3256 and 3154 (N-H), 3043 (C-H), 1598 (C=N), 1476 and 1441 (C=C), 1127 (C=S), 1072 (C-Br),
1
69 (C=C trans-substituted). H NMR (DMSO-d )
δ
: 8.65 and 8.54 (2H, 2s, NH ), 8.15 (1H, s, NH),
6
2
0
0
00
00
0
0
.68–7.62 (3H, m, H-3 , H-4 and H-5 ), 7.57–7.55 (2H, d, J = 8.5 Hz, H-2 and H-6 ), 7.46–7.44 (2H,
0
0
00
00
d, J = 8.5 Hz, H-3 and H-5 ), 7.37–7.35 (2H, dd, J = 7.0 and 1.5 Hz, H-2 and H-6 ), 7.22–7.18 (1H,
13
d, J = 16.5 Hz, H-3), 6.46–6.41 (1H, d, J = 16.5 Hz, H-2). C NMR (DMSO-d ) δ: 178.3 (C=S), 150.7
6
00 0 0 0 00 00 00
C=N), 135.5 (C-3), 135.5 (C-1 ), 132.3 (C-3 and C-5 ), 130.6 (C-1 ), 130.5 (C-4 ), 130.2 (C-2 and C-6 ),
(
0
0
00
00
0
1
29.6 (C-2), 129.3 (C-2 and C-6 ), 128.7 (C-3 and C-5 ), 122.3 (C-4 ). Anal. Calcd. for C H BrN S: C,
16
14
3
5
3.34, H, 3.92, N, 11.66. Found: C, 53.39, H, 3.88, N, 11.72.
0
(
1E,2E)-3-(4 -nitrophenyl)-1-phenylprop-2-en-1-one thiosemicarbazone (5g)
◦
◦
−1
Orange amorphous solid; yield 66%; m.p. 188–190 C (184-186 C [30]). IR (KBr, cm ): 3482,
3
9
337, 3249 and 3152 (N-H), 3057 (C-H), 1594 (C=N), 1512 and 1471 (C=C), 1341 (N=O), 1131 (C=S),
1
66 (C=C trans-substituted). H NMR (DMSO-d )
δ
: 8.70 and 8.69 (2H, 2s, NH ), 8.20–8.19 (3H, m, NH,
6
0
2
0
0
0
00
00
00
H-3 and H-5 ), 7.76–7.64 (2H, d, J = 8.5 Hz, H-2 and H-6 ), 7.68–7.61 (3H, m, H-3 , H-4 and H-5 ),
00 00
.38–7.35 (3H, m, H-3, H-2 and H-6 ), 6.61–6.58 (1H, d, J = 16.4 Hz, H-2). C NMR (DMSO-d ) δ:
6
13
7
0 0 00 00 00
79.5 (C=S), 147.7 (C-4 ), 147.5 (C=N), 143.1 (C-1 ), 137.3 (C-3), 136.9 (C-1 ), 129.7 (C-2 and C-6 ),
1
1
00
00
0
0
00
0
0
29.4 (C-3 and C-5 ), 128.8 (C-2 and C-6 ), 128.3 (C-4 ), 124.2 (C-3 and C-5 ), 123.4 (C-2).
2
.2. Biologic Assays
2
.2.1. Chemicals
The chalcone-thiosemicarbazones (5a-g) were solubilized in dimethylsulphoxide (DMSO; Sigma
Aldrich, St. Louis, MO, USA) to obtain a stock solution of 10 mM.
2
.2.2. Parasite Cultures
Leishmania amazonensis (MHOM/BR/77/LTB0016) were regularly isolated from infected mice and
maintained in culture as promastigotes through weekly passages in Schneider’s medium (Sigma Aldrich,
St. Louis, MO, USA) supplemented with 10% (v/v) heat-inactivated fetal calf serum, penicillin (100
UI/mL), and streptomycin (100 µg/mL) at 299K. Parasites were maintained until the 10th passage [31].
The Animal Ethics Committee of the Oswaldo Cruz Foundation (license number L026/2015) approved
this study.
2
.2.3. Promastigote Assays
6
Promastigotes of L. amazonensis (1
×
10 promastigotes/mL) in the 4th day on culture in
the conditions described above were incubated with the chalcones serially diluted in culture
medium (0.032% v/v DMSO at the highest concentration tested) on a 96-well cell culture plate
(TPP™
test plate, Switzerland). The assayed concentrations ranged from 50 to 1.56
µM for all
chalcone-thiosemicarbazones (5a
–
g) and for pentamidine (Sigma Aldrich, St. Louis, MO, USA).
The plates were incubated in the presence of the compounds for 24 h at 299K. All compounds were
analyzed in at least three experiments performed in triplicate. Pentamidine was used as the reference
drug and diluted in the same concentrations used for 5a–g. The vehicle (DMSO) had no effect on
the parasites.
The antileishmanial activity was evaluated by adding 22
µL of 3-(4,5-dimethylthiazol-2-yl)
-2-5diphenyltetrazolium bromide (MTT) at 5 mg/mL (Sigma Aldrich, St. Louis, MO, USA) to each
well. After 2 h, 80
µ
L of DMSO was added and the optical density was determined at a wavelength of
®
570 nm in a microplate reader (
µQuant Bio-Tek Instruments , Winooski). The assays were carried out
in triplicate in 96-well plates (Costar, New York, USA). The inhibition percentage was estimated by the

Biomolecules 2019, 9, 643
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comparison with the non-treated control. The values for the inhibitory concentration for 50% of the
promastigotes (IC ) were calculated by a sigmoidal model.
50
2
.2.4. Macrophage Cytotoxicity
Peritoneal macrophages from albino laboratory-bred strain (BALB/c) mice were obtained
in RPMI 1640 medium at pH 7.2 (Gibco, New York, NY, USA) supplemented with 10% (v/v)
heat-inactivated fetal bovine serum, 100 U/mL penicillin and 0.1 mg/mL of streptomycin and
6
distributed (1 × 10 macrophages/well) in triplicate in 96-well plates (Falcon Co., Franklin Lakes,
USA) and incubated for 1 h in a 5% CO atmosphere incubator at 310K. The supernatant was removed,
2
and the compounds were added to the plate at concentrations from 10
µM to 50 µM. Next, the cultures
were incubated for 48 h in a CO incubator at 310K. After incubation, the supernatant was removed
2
and a resazurin solution (50
the fluorescence was measured using a wavelength of 560 nm for excitation and 590 nm for detection
Molecular Devices, Silicon Valley, USA). Results were expressed as the percentage of viable cells
compared to the control cells treated with the highest DMSO dose used to dissolve the drugs. The 50%
cytotoxic concentration (CC ) was calculated using GraphPad Prism 6.0 software (GraphPad Company,
µM) was added to each well and incubated at 310K for 4 h. Furthermore,
(
50
San Diego, CA, USA).
2
.2.5. Intracellular Amastigotes Assays
Peritoneal macrophages were obtained BALB/c mice with cold Roswell Park Memorial Institute
(
RPMI) 1640 medium (Sigma Aldrich, St. Louis, MO, USA) supplemented as above. The cell suspension
6
was adjusted to 1
×
10 macrophage/mL and incubated in a LAB-TEK 8-chamber slide (Nunc, Roskilde,
Denmark) at 310K and 5% CO for 1 h. Non-adherent cells were removed and stationary-phase
2
L. amazonensis promastigotes were added at a 3:1 parasite/macrophage ratio. The cultures were
incubated for a further 4 h, and free parasites were removed. The chambers were washed, and the
monolayers were incubated with the compounds for 48 h. Infected macrophages incubated only
in the culture medium were used as controls to compare with the infected treated macrophages.
The concentration of DMSO in the wells did not exceed 0.001% (v/v), which is not toxic to the
parasites. The cultures were fixed in methanol and stained with the hematological system Instant
®
Prov (Newprov , Curitiba, Brazil). The slides were examined by light microscopy, and the number
of infected macrophages and the mean number of parasites per macrophage were determined in
100 cells counted. The experiments were performed twice in triplicate. The percentage of infected
macrophages was calculated by dividing the number of infected macrophages by the total number
of macrophages (infected and uninfected) counted on the slides, multiplied by 100. To determine
the infection index, the percentage of infected macrophages was multiplied by the mean number of
parasites per macrophage. The percentage inhibition of infection was calculated by multiplying the
difference between the infection index of the control and the compound by 100 and dividing by the
infection index (IF) of the control using the Equation (1):
IF = % infected cells × amastigotes number/total number of macrophages
(1)
The IC50
±
SD (SD: standard deviation) values were obtained by a sigmoidal model using Origin 6.0
with statistical error limits up to 10%. All tests were conducted in duplicate for each concentration,
and two independent assays were performed.
2
.2.6. Axenic Amastigotes Assays
Promastigote forms of L. amazonensis (MHOM/BR/77/LTB0016) were grown to a stationary phase,
6
washed once in phosphate buffer solution (PBS) (pH = 7.4) and adjusted to 5
×
10 parasites/mL in
Schneider’s medium, supplemented with 20% fetal bovine solution (FBS) pH = 5.5 and maintained at
05K. After 5 days of cultivation, the rounded forms without free flagella were obtained and, after heat
3

Biomolecules 2019, 9, 643
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shock, were used in the appropriate assays [32]. To evaluate the activity against axenic amastigotes,
the parasites were incubated with increasing concentrations of the prototypes (diluted in DMSO)
for 72 h at 305K. These assays were performed in triplicate in 96-well flat bottom plates (Falcon Co,
Franklin Lakes, USA). The leishmanicidal activity was evaluated by adding 22 µl of resazurin to each
well. After 4 h, the cell viability was measured as described above. Calculation of the IC50 values was
determined in relation to the control by a sigmoidal model using GraphPad Prism 6.0 software.
2
.2.7. Statistical Analysis
Significance was determined using a non-paired Student’s t-test. Differences were considered
significant when p < 0.05. Each experiment was, at minimum, performed in triplicate.
2
.3. HSA Binding Studies
2
.3.1. Spectroscopic Analysis
The chemicals HSA and PBS buffer were purchased from Sigma Aldrich, St. Louis, MO, USA
and the pH of buffer solution was checked (pH = 7.40). Jasco J-815 optical spectrometer (Jasco Easton,
MD, USA) was used as equipment for steady-state fluorescence and circular dichroism (CD) spectra
measurements and a thermostatic cuvette holder Jasco PFD-425S15F (Jasco Easton, MD, USA) was
applied for control of temperature in the quartz cell (1.0 cm optical path). All spectra were recorded as
the average of three scans with appropriate background corrections. For steady-state fluorescence
measurements it was applied 305–450 nm range as emission wavelength (λexc = 295 nm) at 289K, 296K,
−
5
3
03K and 310K. First, it was recorded spectra for HSA solution (1.00
×
10 M, in PBS) and then it was
added manually the compound 5e, achieving final ligand concentrations of 0.35; 0.70; 1.05; 1.40; 1.74;
−
5
2
.09; 2.44; and 2.78 × 10 M.
In order to delete the possible absorption contribution of the compound 5e in the steady-state
fluorescence results, inner filter effect was applied, according to the Equation 2 [33]:
Aex+Aem
Fcor = FObs10[
]
(2)
2
where Fcor and Fobs are the corrected and observed fluorescence intensity values, while Aex and Aem
−
1
−1
M in PBS) and emission
represent the absorbance value at the excitation (
wavelengths (λ = 340 nm: ε = 4,550 cm
λ
= 295 nm:
−1
M in PBS).
ε
= 6,235 cm
−1
The steady-state fluorescence results for the interaction HSA:5e were analyzed according to
Stern-Volmer (Equation 3), double logarithmic (Equation 4), van’t Hoff (Equation 5) and Gibbs’ free
energy (Equation 6) approximations [25,26]:
F0
F
=
1 + Ksv[Q] = 1 + kqτ [Q]
(3)
(4)
0
ꢀ
ꢁ
F − F
0
log
= log Ka + n log[Q]
F
◦
◦
∆
H
∆S
R
ln Ka = −
+
(5)
(6)
RT
◦
◦
◦
∆
G = ∆H − T∆S
where F and F are the steady-state fluorescence intensities of HSA in the absence and presence of
0
5
e
, respectively. [Q], KSV and k
q
are the 5e concentration, Stern-Volmer quenching constant and
is the experimental fluorescence lifetime of HSA
bimolecular quenching rate constant, respectively.
in the absence 5e (5.98
fluorescence intensity corresponding to the fluorophore which is accessible by the quencher (f
respectively.
τ
0
−9
±
0.15)
×
10 s. K
a
and
ƒ
are the binding constant and fraction of the initial
1.00),
G are the enthalpy, entropy and Gibbs’ free energy change, respectively.
≈
◦
◦
◦
∆
∆
H ,
∆
S and

Biomolecules 2019, 9, 643
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−
1
−1
T and R are the temperature (289 K, 296 K, 303 K and 310 K) and gas constant (8.3145 Jmol K ),
respectively. All quantitative values were expressed as “± SD” (obtained through three scans).
The fluorimeter model FL920 CD from Edinburgh Instruments (Edinburgh, UK), equipped with
an electrically pumped laser (EPL, with λexc = 280
of 1.8 W/pulse; monitoring emission at 340 nm) was used for time-resolved fluorescence decays
measurement. The experiment was conducted for a 3.0 mL HSA solution (1.00
±
10 nm; a pulse of 850 ps with the energy
µ
−
5
×
10 M, in PBS)
−5
without and in the presence of 5e (2.78 × 10 M) at room temperature.
The fluorimeter model Xe900 from Edinburgh Instruments (Edinburgh, UK) was used for
synchronous fluorescence (SF) measurement in the 240–320 nm range by setting ∆λ = 60 nm and
∆
λ = 15 nm for tryptophan and tyrosine residues, respectively. The experiment was conducted for a
−
5
3
.0 mL HSA solution (1.00
×
10 M, in PBS) without and in the presence of successive additions of 5e (in
the same concentration range used in the steady-state fluorescence measurement) at room temperature.
Circular dichroism (CD) spectra were measured in the same equipment used in the steady-state
fluorescence analysis, using 200–250 nm range at 310K (human body temperature). The experiment
−6
was conducted for a 3.0 mL HSA solution (1.00
×
10 M, in PBS) in the absence and presence of 5e
10 M). Each spectrum obtained was the average of three scans. The intensity of the signal
from the CD spectra was expressed as mean residue ellipticity (MRE), defined according to Equation
−5
(2.78
×
(
7) [34]:
θ
MRE =
(7)
1
0 × n × l × Cp
p
where θ, n, l and C are the observed CD (in millidegrees), number of amino acid residues (585
for HSA) [35], optical pathlength of the cell (1.00 cm) and molar concentration of HSA (1.00
×
−6
1
0
M), respectively.
In order to calculate quantitative information on the percentage of changes in the
α
-helix content
upon 5e binding, the resulting MRE at 208 and 222 nm (far-UV region) were analyzed according to
Equations (8A) and (8B) [36]:
(
−MRE208 − 4000)
α − helix% =
× 100
(8A)
(8B)
(
33000 − 4000)
(
−MRE222 − 2340)
α − helix% =
× 100
30300
2
.3.2. Molecular Docking Analysis for the Interaction HSA:5e
The chemical structure 5e was built and energy-minimized with Spartan’14 program
Wavefunction, Inc., Irvine, CA, USA). The Density Functional Theory (DFT) method was used,
(
with B3LYP potential and basis set 6-31G* for the minimization of 5e structure. The crystallographic
structure of HSA was obtained in the Protein Data Bank, with access code 1N5U [35]. Molecular docking
studies were performed with GOLD 5.6 program (CCDC, Cambridge Crystallographic Data Centre).
The GOLD 5.6 program added the hydrogen atoms to the protein structure according to the
ionization and tautomeric states inferred by the program. Molecular docking calculations were
explored for the three main protein binding pocket. Thus, docking interaction cavity in the protein
was established with a 10 Å radius from the Trp-214, Tyr-411, and Tyr-161 residues, for sites I, II and III,
respectively. These amino acid residues were chosen according to the crystallographic structure of
each site probe inside HSA (warfarin, ibuprofen and camptothecin) [37,38]. The number of genetic
operations (crossover, migration, mutation) in each docking run used in the search procedure was set
to 100,000. The scoring function used was ‘ChemPLP’, which is the default function of the GOLD 5.6
program [39]. Figures of the best docking pose were generated with the PyMOL program (DeLano
Scientific LLC).

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3
. Results and Discussion
3
.1. Synthesis of Chalcone-Thiosemicarbazones
A series of seven 1-phenyl-3-(4-X-phenyl)-2-ylprop-2-en-1-one thiosemicarbazones (5a
–g) were
prepared using an adapted pathway [40] involving substituted chalcones (3a
). The substituted chalcones were synthetized by a traditional Claisen-Schmidt condensation
reaction [27] using acetophenone ( ) and X-benzaldehydes (2a ) in the presence of NaOH and
–g) and thiosemicarbazide
(4
1
–g
ethanol. After the purification process with ethanol, it was obtained the synthetic compounds in good
yields (75–93%). Then, the chalcone-thiosemicarbazones were obtained from a mixture of previously
synthesized substituted chalcones and thiosemicarbazide using a few drops of concentrated HCl and
ethanol as a solvent at the reflux temperature within 6 h (Scheme 1). The products were purified by
recrystallization from ethanol in reasonable yields (40–75%).
O
O
O
NaOH (10%)
EtOH
H
+
X
2a-g
X
1
3a-g
+
S
N
NH2
H
H
N
N
HCl drops
NH2
H N
2
EtOH, reflux, 6 h
S
X
5a-g
4
5a
5b
5c
X = H
X = CH3
X = CN
5d X = F
5e X = Cl
5f X = Br
5g X = NO2
Scheme 1. Synthetic pathways for chalcone-thiosemicarbazones.
All spectra for the characterization of the synthetic compounds 5a–5b are shown as Figures S1–S19
in the supplementary material. The infrared spectra of the chalcone-thiosemicarbazones showed
−
1
the disappearance of the carbonyl band (
to the precursor chalcones [41], and a new band (
was evidenced, being in accordance with literature [22]. Additionally, the
ν C=O) at approximately 1640 cm , which corresponds
−1
ν
C=N) at 1585–1598 cm , resulting from imine,
C=S absorption was
ν
−1
observed in the range of 1126–1131 cm
.
1
13
The H and C chemical shift values were used to characterize the proposed structures of 5a
–g (the
spectra are shown in the Supplementary data). The chemical shift values of NH were absorbed as two
2
singlets in the range of
δ 8.50–8.72 due to the resonance effect from the thiocarbamoyl moiety, and the
hydrogen bond between one of the hydrogens to a sulphur atom led to diastereotopic hydrogens, as
indicated in Scheme 2a,b, respectively. On the other hand, the resonance effect caused by electron
acceptor groups in the aromatic ring caused a more upfield absorption in the range of
singlet to HN.
δ 8.12–8.19 as a

Biomolecules 2019, 9, 643
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(
a)
H
H
H
N
H
N
S
S
HN
HN
N
N
X
X
(b)
H
S
N
H
HN
N
X
Scheme 2. Effects involving the hydrogens H N: (
a) resonance effect with thiocarbamoyl group;
2
(
b) intramolecular hydrogen bond between the hydrogen and azomethine nitrogen.
The 13C NMR spectra showed the chemical shifts in the typical range of
C=S. The chemical shifts of C=N were observed at 147.5–150.7 due to the resonance effect with a
δ 178.3–179.5 assigned to
δ
conjugated double bond enhancing the electronic density of this carbon atom. The other carbon atoms
showed chemical shift values as expected.
3
.2. Anti-Leishmanial Effects
The treatment of the parasites with the chalcone-thiosemicarbazones 5a–g against L. amazonensis
promastigotes resulted in a dose-dependent response after 48 h of culture. The values of 50% of growth
inhibitory activity (IC ) was assessed by the MTT method [42], and the values were obtained by a
50
sigmoidal curve, relating the percentage of growth inhibition and the log of drug concentration in
M, as shown in Table 1. The results revealed a good inhibitory effect, with the exception of 5c and
µ
5
g
which showed no significant activity. The most active compound (5e) showed an IC50 value of
5
.22 ± 0.75 µM, and the positive control pentamidine had an IC value of 4.90 ± 0.60 µM.
5
0
The assays with axenic amastigotes of L. amazonensis were performed with 5a, 5b, 5d, 5e and 5f
over 72 h of culture. The results showed good growth inhibitory effects for all assayed compounds
with IC50 in the range of 3.19 to 7.08 M, with the chalcone-thiosemicarbazone chloro-substituted (5e
µ
)
the most active compound. On the other hand, pentamidine showed IC50 = 12.29 ± 1.17 µM.
Table 1. In vitro anti-leishmanial activity of chalcone-thiosemicarbazones against promastigotes, axenic
amastigotes, and intracellular amastigotes of L. amazonensis after 48 h of culture, and their macrophage
cytotoxicity and selectivity index.
Promastigotes
Axenic Amastigotes
Intracellular Amastigotes Macrophages
Selectivity
Index (SI)
Compound
IC50 a (µM)
b
IC50 (µM)
IC50 (µM)
LD50 (µM)
5
a
14.68 ± 0.31
14.80 ± 1.92
6.88 ± 1.09
7.08 ± 1.03
3.40 ± 0.28
5.95 ± 0.61
n.d.
40.98 ± 1.12
56.35 ± 0.78
n.d.
35.47 ± 1.38
32.48 ± 1.81
44.24 ± 4.21
n.d.
15.05
9.47
-
5b
c
d
5
c
n.a.
n.d.
5
5
d
e
13.09 ± 1.85
5.22 ± 0.75
12.52 ± 0.99
n.a.
5.57 ± 1.15
3.19 ± 1.20
4.24 ± 1.07
n.d.
> 3.13
-
4.47 ± 0.42
3.88 ± 0.24
n.d.
7.27
11.40
-
5
f
5g
Pentamidine
4.90 ± 0.60
12.29 ± 1.17
11.12 ± 1.98
25.85 ± 4.06
2.32
a
IC50: half maximal inhibitory concentration; b LD50: median lethal dose; c n.a.: no active; d n.d.: not determined.
Thus, after the promising results against promastigotes and axenic amastigotes of L. amazonensis,
the cytotoxicity and activity against intracellular amastigotes were evaluated for 5a 5b 5d 5e
,
,
,
and 5f. The cytotoxic effects were assessed using murine peritoneal macrophages after 48 h of
culture. The pentamidine, used as a positive control, was also assayed under the same conditions.
The macrophages in the presence of the chalcone-thiosemicarbazones showed LD50 in the range of
3
2.48 to 56.35 µM, and the pentamidine with LD50 = 25.85 ± 4.06 µM.

Biomolecules 2019, 9, 643
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The treatment of L. amazonensis-infected macrophages with 5a
,
5b
,
5d
,
5e and 5f after 48 h of
culture reduced the infection, with IC50 in the range of 3.40 to 5.95 µM, as shown in Table 1, whereas
the reduction was 11.12 µM for pentamidine.
Thus, the selectivity index (SI = LD /IC intracellular amastigotes) showed values in the range
5
0
50
of 7.27 and 15.05, whereas pentamidine was found to be more toxic in our model (SI = 2.32). It is
important to highlight that some guidelines suggest an SI higher than 10 and IC50 against intracellular
amastigotes below 10 µM as thresholds for an anti-leishmanial hit before proceeding with further
studies [43,44]. The chalcone-thiosemicarbazones 5a and 5f met both criteria.
It is interesting that the growth inhibitory effects of chalcone-thiosemicarbazones against
L. amazonensis axenic and intracellular amastigotes were observed in the same numerical range
with lower values when compared to pentamidine but also with a lower toxicity, thus, consequently, it
can be considered to be safer.
To understand which parameters are involved in the antileishmanial activity of the
chalcone-thiosemicarbazones, their electronic (σ) [45], steric (MR) and lipophilicity (π) properties [46]
(see Table 2) were correlated with log IC50 or 1/IC50 values of L. amazonensis axenic amastigotes
by linear regression models. After analysis, it was observed that the best correlation was with the
2
electronic parameter (Hammett constant, σp) in the polynomial model with R = 0.9998 (coefficient of
determination), SD = 0.0033 (standard deviation), n = 5 and p = 0.00024. Thus, this result indicates that
moieties with electronic withdrawing effects increase their anti-leishmanial activity.
Table 2. Electronic, steric and lipophilic parameters: Hammett constant (σp), Taft constant (Es),
molecular refractivity (MR), and Hansh constant (πm).
a
Es b
MR b
b
Compound
4-X
σp
π
5
a
H
CH3
CN
F
Cl
Br
0.00
−0.14
0.71
0.15
0.34
0.26
0.81
0.00
0.00
0.57
0.63
0.10
0.60
0.89
0.74
0.00
0.56
5
b
−0.25
−0.51
−0.46
−0.97
−1.16
−1.01
5
c
−0.57
5
d
0.14
5
e
0.71
0.86
−0.28
5
f
5
g
NO2
a
Isaacs, N. [45]; b Hoekman, D. [46].
3
.3. Evaluation on the Interaction between HSA and 5e
Among the synthetic organic compounds which presented the highest toxic activity against
L. amazonensis, the chalcone-thiosemicarbazone chloro-substituted (5e) was selected as a model to study
the interaction with the main carrier protein in the human bloodstream (HSA) [35]. The steady-state
fluorescence emission of HSA (mainly due to the presence of Trp-214 residue) and its quenching upon
5
e
addition is showed in Figure 4. Since, about 50% of HSA fluorescence emission decreased and
there is not any significant blue or red shift in the maximum fluorescence emission of the albumin
−
5
(
5
λem = 340 nm) upon maximum addition of the ligand (2.78
probably can interact with HSA in the region next to Trp-214 residue and does not perturb the
microenvironment around the main albumin’s fluorophore [47].
×
10 M), there is an indication that
e

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Figure 4. Steady-state fluorescence emission for HSA without and in the presence of successive
−5
additions of 5e at 310K and pH = 7.4. [HSA] = 1.00
2
×
10 M and [5e] = 0.35; 0.70; 1.05; 1.40; 1.74; 2.09;
.44; and 2.78 × 10− M.
5
It is very well known that two main different mechanisms account for the interaction
fluorophore/quencher (e.g., serum albumin/ligand): dynamic and/or static process. Dynamic and
static quenching can be distinguished by Stern-Volmer analysis (inset in the Figure 4), as well as the
known relationship between Stern-Volmer quenching constant (KSV) and bimolecular quenching rate
constant (k
inside the experimental error (Table 3); however, the k
larger than the diffusional collision quenching constant (kdiff ~7.40
Smoluchowski-Stokes-Einstein theory at 298 K) [49], indicating that the potential anti-Leishmania
amazonensis agent (5e) can interact to HSA via a ground state association (static process).
q
) at different temperatures [47
,
48]. The calculated KSV values are practically the same
q
values are about three orders of magnitude
9
−1 −1
×
10 M s , according to the
Table 3. Binding constant values for the interaction between HSA and 5e at four temperatures.
4
12
4
◦
◦
−2
K
◦
∆G
KSV (×10 )
kq (×10
)
Ka (×10 )
∆H
∆S (×10
)
)
nb
T (K)
c
c
c
−
1
a
−1 −1
) a
−1
b
−1
−1 −1
−1
(kJmol )
(
M
)
(M
s
(M
)
(kJmol
)
(kJmol
289
296
303
310
2.48 ± 0.52
2.49 ± 0.50
2.59 ± 0.51
2.75 ± 0.33
4.15
4.16
4.34
4.60
5.16 ± 0.04
4.85 ± 0.06
4.57 ± 0.06
4.39 ± 0.04
1.05 ± 0.01
1.07 ± 0.01
1.06 ± 0.01
1.05 ± 0.01
−26.1
−5.79 ±
−26.6
−27.1
−27.6
7
.02 ± 0.09
0.27
a
r : 0.9996–0.9987; r : 0.9998–0.9996; r2: 0.9978.
2
b
2
c
In order to further confirm if this fluorescence quenching mechanism really occurs via a static
or dynamic process, time-resolved fluorescence decays were measured for HSA in the absence and
presence of 5e (Figure 5). The HSA solution exhibited two fluorescence lifetimes (
second one the largest contribution (~78.0%): = 1.80 0.16 ns and = 5.98
These results are in agreement with those in the literature [50]. Upon the maximum addition of
τ
), having the
2
τ
1
±
τ
±
0.15 ns −χ = 1.102.
2
the compound 5e to the protein solution, the new fluorescence lifetimes data are the same inside
2
the experimental error:
τ
1
= 1.77
±
0.12 ns and
τ
2
= 5.91
±
0.16 ns −χ = 1.110. These results are a
clear indication that really the static fluorescence process is the main quenching mechanism for the
interaction between HSA and 5e [36].

Biomolecules 2019, 9, 643
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Figure 5. Time-resolved fluorescence decays for human serum albumin (HSA) without and in the
−
5
−5
presence of 5e at room temperature and pH = 7.4. [HSA] = 1.00
IRF: Instrument response function.
×
10 M and [5e] = 2.78
×
10 M.
a
The evaluation of binding constant (K ) between serum albumin and ligands is important to
understand the biodistribution of a potential drug in the plasma, body tissues and organs. A too weak
binding can lead to a poor distribution of the molecule in the body, while strong binding can decrease
the concentrations of free molecules in the bloodstream [24]. For static quenching, the steady-state
fluorescence quenching data could be analyzed to calculate the K
a
and number of binding sites (n) values
values
for the interaction HSA:5e are in the order of 10 M (Table 3), which indicates a moderate binding
affinity between the potential anti-Leishmania amazonensis agent and albumin [24 26]. In addition,
by double logarithmic approximation (Figure S20 in the supplementary material) [25]. The K
a
4
−1
,
n ~ 1 indicates that just one 5e can interact to one main binding site in the albumin structure [48].
The chalcone-thiosemicarbazone chloro-substituted under study showed the same binding trend
toward HSA compared to the interaction of some chalcone-thiosemicarbazone derivatives and serum
albumin [21].
When small molecules bind to proteins, intermolecular interactions, such as hydrogen bounding,
hydrophobic, electrostatic and van der Waals forces, can be involved. The thermodynamic analysis
◦
◦
(
enthalpy and entropy change − ∆H and
∆S , respectively—calculated according to the van’t Hoff
plot in the Figure S21 in the supplementary material) can be applied to identify the main interaction
S > 0 (Table 3) can be related to hydrophobic forces and/or desolvation process and
H < 0 (Table 3) can be related to hydrogen bonding and/or electrostatic forces [51]. In addition, the
◦
forces [51]. The
∆
negative
∆
◦
◦
∆
G values are consistent with the spontaneity of the binding process in all temperatures
◦
under study (Table 3). Since the negative ∆G value for the complete association process is primarily
determined by the positive entropy change accompanying the first step and the negative enthalpy
change of the second step, the association HSA:5e is enthalpically and entropically driven [52].
3
.4. Evaluation on the Microenvironment and Structure of HSA upon 5e Binding
Synchronous fluorescence (SF) spectroscopy is a common technique used to study the changes in
the microenvironment of tyrosine and tryptophan amino acid residues when the difference between
excitation and emission wavelength (∆λ) is 15 and 60 nm, respectively [25]. Figure 6 shows the effect of

Biomolecules 2019, 9, 643
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increasing the concentration of 5e on the synchronous spectra of HSA at ∆λ = 15 nm and ∆λ = 60 nm.
In all cases, a decrease in emission intensity of serum albumin with increasing in ligand concentration
was observed; however, there is not any significant blue or red shift in the maximum fluorescence
emission of albumin upon ligand binding, indicating that 5e can bind to serum albumin without affect
significantly the microenvironment of both tyrosine and tryptophan residues. These results are different
from those observed for the interaction between serum albumin and some chalcone-thiosemicarbazone
derivatives [21].
Figure 6. Synchronous fluorescence (SF) spectra for HSA without and in the presence of 5e at
−5
(
2
A) ∆λ = 15 nm and (
B
)
∆λ = 60 nm. [HSA] = 1.00
×
10 M and [5e] = 0.35; 0.70; 1.05; 1.40; 1.74; 2.09;
.44; and 2.78 × 10− M.
5
Circular dichroism (CD) spectroscopy is a widely technique employed to evaluate the interaction
between exogenous agents and macromolecules (e.g., proteins). Thus, CD measurement allows
obtaining information on perturbation of the albumin upon ligand binding. More specifically,
far-UV CD spectra can offer information regarding the protein secondary structure. The CD spectrum
of HSA displays two negative bands: one at 208 nm and other at 222 nm, which are characteristic of
the α-helix structure [52]. Figure 7 shows the CD spectra for HSA in PBS medium, without and in
the presence of 5e at 310K. In this figure, the ellipticity signal was less negative after the addition of
the potential anti-Leishmania amazonensis agent, suggesting a possible perturbation on the secondary
structure of the albumin. Taking into account the quantitative values at 208 and 222 nm for HSA (57.1%
and 53.5%, respectively) as well as for HSA:5e (55.2% and 52.2%, respectively), a discrete variation
in the α-helix percentage for HSA can be observed. These results demonstrated that 5e is able to
induce slight conformational changes on HSA [53], which is consistent with the results described above
employing SF spectroscopy.
Figure 7. CD spectra for HSA without and in the presence of 5e at 310K and pH = 7.4.
HSA] = 1.00 × 10− M and [5e] = 2.78 × 10−5 M.
5
[

Biomolecules 2019, 9, 643
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3
.5. Molecular Docking Analysis for HSA:5e
HSA is a globular protein composed of three homologous
α
-helical domains (I-III), and each
domain contains two subdomains (A and B). There are three main binding pockets in the albumin
structure: two located in hydrophobic cavities in subdomains IIA and IIIA (sites I and II, respectively)
and one located in an external region in subdomain IB (site III) [35]. In order to suggest the main
binding site for the compound 5e toward serum albumin’s binding pockets, as well as to offer a
molecular description (calculate the possible conformation of each ligand) on the binding ability,
molecular docking calculations were carried out with Gold 5.6 program. Figure 8 depicts the best
docking pose for the ligand inside the three main possible binding sites (I, II and III), and Table 4 shows
the main amino acid residues, which can interact with the potential anti-Leishmania amazonensis agent.
The docking score of each pose was calculated as the negative of the sum of a series of energy
terms involved in the protein-ligand interaction process, so that the more positive the score, is the
better the interaction. The highest docking score value for the docking pose of 5e in the sites I, II and
III was 81.5, 59.6 and 65.6, respectively. Since the docking score value was positive for all possible
binding sites, there is an indication that the ligands could bind in the three subdomains (not at the same
time, according to the experimental number of binding sites: n ~ 1.0); however, the highest docking
score value was obtained for site I, suggesting subdomain IIA, where Trp-214 residue can be found,
as the main binding site. This result is in accordance with the experimental data, which indicated
a ground state association between Trp-214 residue and 5e (see Section 3.3). From the literature,
some thiosemicarbazone derivatives were also able to interact inside subdomain IIA (site I) [54,55].
Molecular docking results suggested hydrogen bonding and van der Waals forces as the main
intermolecular interaction between 5e and the amino acid residues presented in the three different
binding sites (Table 4). These results are in accordance with the experimental thermodynamic
parameters once the cited two main binding forces were detected (see Section 3.3). In addition, it is
◦
important to note that according to the experimental
∆
S value, the positive parameter could be related
to hydrophobic forces and/or desolvation process. According to the docking results, hydrophobic
forces were not contributing to the binding ability of compound 5e, thus, the desolvation process
◦
is the phenomenon that could explain the experimental
corroborated with the experimental data.
∆
S value. Overall, the theoretical analysis
Figure 8. Best docking pose (ChemPLP function) for the interaction HSA:5e inside three main binding
pockets: ( ) site I, ( ) site II and ( ) site III. Selected amino acid residues are represented as stick in
A
B
C
blue, while 5e structure is represented in pink. Hydrogen, oxygen, nitrogen, sulfur and chloro atoms
are represented in white, red, dark blue, yellow and green, respectively.

Biomolecules 2019, 9, 643
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Table 4. Molecular docking results for the interaction HSA:5e in the sites I, II and III.
Sites
Amino Acid Residues
Interaction
Distance (Å)
Phe-210
Trp-214
Ser-201
His-241
Val-343
Leu-346
Leu-480
Val-481
Van der Waals
Van der Waals
Hydrogen bonding
Van der Waals
Van der Waals
Van der Waals
Van der Waals
Van der Waals
2.60
3.00
2.80
3.60
3.50
2.40
2.70
2.70
I
Leu-386
Lys-413
Val-414
Val-425
Leu-429
Leu-452
Ser-488
Van der Waals
Van der Waals
Van der Waals
Van der Waals
Van der Waals
Van der Waals
Hydrogen bonding
3.20
3.10
2.90
2.20
2.90
3.60
1.80
II
Arg-113
Leu-114
Arg-116
Tyr-137
Ile-141
Hydrogen bonding
Van der Waals
Van der Waals
Van der Waals
Van der Waals
Van der Waals
Hydrogen bonding
3.30
2.40
2.40
2.40
2.70
3.70
3.80
III
Phe-156
Arg-185
4
. Conclusions
A novel series of chalcone-thiosemicarbazones were synthesized in reasonable yields.
All compounds tested against intracellular amastigotes of L. amazonensis were more potent and
selective than the reference drug (pentamidine). The most active compound (5e) showed an IC50 value
of 5.22 ± 0.75 µM and the compounds 5a and 5f had a selectivity index higher than 10 and an IC50 value
below of 10 µM, showing that both are promising anti-leishmanial hits. The interaction between HSA
and 5e (the selected compound as a model to study preliminary pharmacokinetic evaluation) indicated
a spontaneous, moderate, enthalpically and entropically driven association. The binding occurs via a
ground state interaction (static process) and does not perturb significantly both microenvironment
and secondary structure of albumin. According to the thermodynamic parameters and molecular
docking results, the main intermolecular forces involved in the binding process towards serum albumin
are hydrogen bonding and van der Waals interactions. Overall, the results of the study of their
antiparasitic effects against L. amazonensis, as well as the binding ability toward HSA revealed the
chalcone-thiosemicarbazone class as a novel prototype for drug development against leishmaniasis.
Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/9/11/643/s1,
Figures S1–S19: all spectra for the characterization of the synthetic compounds 5a–5g. Figure S20: Double
logarithmic plots for the interaction between HSA:5e at 289 K, 296 K, 303 K, and 310 K. Figure S21: Van’t Hoff plot
for the interaction between HSA:5e at four different temperatures.
Author Contributions: A.E. and E.C.T.-S. conceptualized the research; E.P.M. and C.M.G., synthesized and
characterized the chalcone-thiosemicarbazone series; V.d.S.F., M.M.C.-C. and G.C.M. conducted the biological
assays (promastigotes, axenic amastigotes and intracellular amastigotes of L. amazonensis); E.C.T.-S. was responsible
for the critical analysis of the L. amazonensis assays; A.E. was responsible for the critical analysis of the organic
synthesis and conducted the QSAR studies; O.A.C. conducted the HSA binding studies (spectroscopic and
theoretical analysis). All authors contributed to the writing of the manuscript.
Funding: This research was funded by the following Brazilian agencies: Coordenaç
de Pessoal de N vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cient
CNPq–Process: 310345/2015-4), Fundaç o de Amparo Pesquisa do Estado do Rio de Janeiro (FAPERJ–Process:
E-26/202.918/2018 Cientista do Nosso Estado) and Fundação do Instituto Oswaldo Cruz (FIOCRUZ).
ã
o de Aperfeiçoamento
í
í
fico e Tecnol gico
ó
(
ã
à

Biomolecules 2019, 9, 643
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Acknowledgments: C.M.R. Sant’Anna from Laborat
ório de Modelagem Molecular (Institute of Chemistry-UFRRJ)
for the molecular docking facilities and N.C.L. Garden (Institute of Chemistry—UFRJ) for the time-resolved and
synchronous fluorescence facilities.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(