Design, synthesis and in vitro trypanocidal and leishmanicidal activities of novel semicarbazone derivatives

Article abstract of DOI:10.1016/j.ejmech.2015.05.046

Trypanosomatids are protozoan parasites that cause various diseases in human, such as leishmaniasis, Chagas disease and sleeping sickness. The highly syntenic genomes of the trypanosomatid species lead the assumption that they can encode similar proteins,

Full text of DOI:10.1016/j.ejmech.2015.05.046

European Journal of Medicinal Chemistry 100 (2015) 24e33
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and in vitro trypanocidal and leishmanicidal
activities of novel semicarbazone derivatives
,
,
,
c
Marina A. Alves a b, Aline C. de Queiroz a c, Magna Suzana Alexandre-Moreira a
,
d
d
d
a
,
e
ꢀ
Javier Varela , Hugo Cerecetto , Mercedes Gonzalez , Antonio C. Doriguetto
,
Iara M. Landre a e, Eliezer J. Barreiro a b, Lídia M. Lima a
,
,
, b, *
a
^
ꢀ
ꢀ
~
Instituto Nacional de Ciencia e Tecnologia de Farmacos e Medicamentos, Universidade Federal do Rio de Janeiro, Laboratorio de Avaliaçao e Síntese de
Substancias Bioativas, CCS, Cidade Universitaria, P.O. Box 68024, 21941-971, Rio de Janeiro, RJ, Brazil
Programa de Pos-Graduaçao em Química, Instituto de Química, Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, RJ, Brazil
LaFI e Laboratorio de Farmacologia e Imunidade, Instituto de Ciencias Biologicas e da Saúde, Universidade Federal de Alagoas, Maceio, AL, Brazil
Grupo de Química Medicinal, Laboratorio de Química Organica, Facultad de Ciencias, Universidad de la República, Igua 4225, Montevideo, 11400, Uruguay
Laboratorio de Cristalografia, Instituto de Química, Universidade Federal de Alfenas, 37130-000, Alfenas, MG, Brazil
1, 2
^
ꢀ
b
ꢀ
~
c
ꢀ
^
ꢀ
ꢀ
d
e
ꢀ
ꢀ
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a r t i c l e i n f o
a b s t r a c t
Article history:
Trypanosomatids are protozoan parasites that cause various diseases in human, such as leishmaniasis,
Chagas disease and sleeping sickness. The highly syntenic genomes of the trypanosomatid species lead
the assumption that they can encode similar proteins, indicating the possibility to design new anti-
trypanosomatid drugs with dual trypanosomicidal and leishmanicidal activities. In this work a series of
compounds (6aeh and 7aeh), containing a semicarbazone scaffold as a peptide mimetic framework, was
designed and synthesized. From this series compound 7g (LASSBio-1483) highlighted, showing dual
in vitro trypanosomicidal and leishmanicidal activities, with potency similar to the standard drugs
nifurtimox and pentamidine. This data, taken together with its good in silico druglikeness profile and its
great chemical and plasma stability, make LASSBio-1483 (7g) a new antitrypanosomatid lead-candidate.
© 2015 Published by Elsevier Masson SAS.
Received 13 November 2014
Received in revised form
28 May 2015
Accepted 30 May 2015
Available online 2 June 2015
Keywords:
Neglected diseases
Trypanosomiasis
Leishmaniasis
Protease
Semicarbazone
Peptide mimetic
1. Introduction
cruzi, and Trypanosoma brucei [3]. The highly syntenic genomes of
the trypanosomatid species lead the assumption that they can
Neglected diseases (DN) represent a set of parasitic illnesses that
primarily affect poor people in developing countries. Those caused
by Trypanosomatidae protozoans include Chagas disease and
sleeping sickness, produced by Trypanosoma species, and leish-
maniasis, caused by different species belonging to the genus
Leishmania [1]. Affording to World Health Organization (WHO),
trypanosomiasis and leishmaniasis are the most challenging among
the neglected tropical diseases [2]. A comparative genomics of
trypanosomatid parasitic protozoa revealed a conserved core pro-
teome of about 6200 genes among Leishmania major, Trypanosoma
encode similar proteins and drugs designed against conserved core
processes should have the advantage of being potentially useful
against all three protozoa [4e7]. Among the possible drug targets in
trypanosomatids, the peptidases or proteases have concerned
attention due their many roles in highly specific functions to the
parasites’ life cycles [8e10]. Considering the ability of these en-
zymes to catalyze the hydrolysis of peptide bonds [11e13] com-
pounds containing amide or amide-mimetic frameworks can be
designed as proteolytic inhibitors with antitrypanosomatid activity,
as exemplified by compounds 1e4 (Chart 1) [13e16]. In similar
manner, the ortho-hydroxyphenyl group linked to the imine sub-
unit of a hydrazone functional group is believed to be an interesting
scaffold for cysteine proteases inhibition. This statement is based
on a theoretical proposed mechanism involving the nucleophilic
attack of sulfhydryl group of a cysteine-protease on a reactive
ortho-quinonemethyde intermediate, generated from the
* Corresponding author. Universidade Federal do Rio de Janeiro, Centro de
^
Ciencias da Saúde, Bloco B, Sala 14, P.O. Box 68024, Rio de Janeiro, RJ, 21941-971,
Brazil.
E-mail addresses: lidia@lassbio.icb.ufrj.br, lmlima23@gmail.com (L.M. Lima).
1
INCT-INOFAR; http://www.inct-inofar.ccs.ufrj.br/.
LASSBio®, http://www.farmacia.ufrj.br/lassbio/.
2
http://dx.doi.org/10.1016/j.ejmech.2015.05.046
0223-5234/© 2015 Published by Elsevier Masson SAS.

M.A. Alves et al. / European Journal of Medicinal Chemistry 100 (2015) 24e33
25
tautomeric equilibrium of ortho-hydroxyarylaldehydehydrazone
moiety (e.g. compound 5, Chart 2) [17].
details of all hydrogen bond contacts involved in its networks can
be found in the Supplementary Material (Fig. 29 and Table 1S)
In order to design new peptide mimetic derivatives enclosing
frameworks able to be recognized by trypanosomatids proteases, a
series of semicarbazone derivatives (6aeh and 7aeh) were plan-
ned by molecular modification on prototype 5 (LASSBio-1022) [18].
These modification were based on ring replacement between qui-
noxaline nucleus and 1,3-benzodioxole system (a, Chart 2); mo-
lecular simplification represented by elimination of methyl group
(b, Chart 2); followed by aza-homologation strategy (c, Chart 2),
2.3. Cytotoxic studies
Before starting the evaluation of the trypanosomicidal and
leishmanicidal activities of semicarbazones 6aeh and 7aeh, the
eventual cytotoxic profile of these compounds against mammalian
cells was investigated by MTT assay [26]. In this study murine
macrophages cell line J774.A1 was treated with compounds 6aeh
converting the N-acylhydrazone subunit in
a
semicarbazone
and 7aeh at serial concentrations (0.1e100 mM) and the half
framework. The congeners series was further designed by classical
isosterism replacement on 2-hydroxyphenyl subunit, varying the
electronic nature of the monovalent group (a-f) and by isosteric
ring replacement of phenyl group by a substituted furan system (g)
and its phenylogous analogue (h) (Chart 2) [19]. In this paper we
described the synthesis of the designed compounds 6aeh and
7aeh and their trypanosomicidal and leishmanicidal activities.
maximal inhibitory concentration (IC50) was determined as illus-
trated in Table 2. Only compounds 7b and 7e showed cytotoxic
activity to mammalian cell with IC50 ¼ 71.2 and 35.7
mM, respec-
tively (Table 2).
2.4. Trypanosomicidal activity
Semicarbazone derivatives 6aeh and 7aeh were evaluated
in vitro against epimastigote forms of Trypanosoma cruzi, Tulahuen
2 strain, discrete typing unit, DTU, Tc VI [27] in a screening con-
2. Results and discussion
2.1. Chemistry
centration of 100
superior than 50% at 100
m
M. Compounds which presented an inhibition
M were selected to determine their IC50
m
Compounds 6aeh and 7aeh were synthesized in three linear
steps from the amines 8 and 9, obtained commercially (Scheme 1).
In the first step the amines were condensed with phenyl chlor-
oformate in chloroform at room temperature in order to furnish the
carbamates 10 and 11 [20,21]. These compounds were treated with
hydrazine monohydrate in ethanol to provide the semicarbazide
derivatives 12 and 13 [22]. These key-intermediates were finally
condensed with appropriated aldehydes [23], selected based on the
design concept depicted in Chart 2, to obtain the semicarbazones
6aeh and 7aeh in good overall yields (Scheme 1). The chemical
structure of the compounds 6aeh and 7aeh was elucidated by 1H
and 13C NMR, IR and mass spectrometry. The analysis of the 1H and
13C NMR spectra of these compounds revealed the presence of only
one signal relative to the hydrogen and carbon of imine double
bond (N]CH), suggesting that all compounds were synthesized as
a single diastereoisomer. The unequivocal characterization of the
relative configuration of imine double bond (E or Z) was performed
using X-ray diffraction study. However, considering the difficulty of
getting compounds 6aeh and 7aeh in crystalline form, only de-
rivative 7g (LASSBio-1483), obtained as crystal solid, was used in X-
ray experiment. As shown in Fig. 1, this experiment revealed that
compound 7g was obtained as diastereoisomer E. Based on these
data and considering the similarity in chemical shifts of imine
hydrogen in 1H NMR spectra of compounds 6aeh and 7aeh, is
reasonable to propose that all semicarbazone derivatives (6aeh
and 7aeh) were obtained with the same stereochemistry (N]CH;
configuration E).
values, and their ability to inhibit the parasite growth was tested in
comparison to the standard drug nifurtimox [28]. As shown in
Table 2, compounds 7a (IC50 ¼ 21
m
M), 7g (IC50 ¼ 11.9
mM), 6d
(IC50 ¼ 8.5 M) and 6g (IC50 ¼ 11.5
m
mM) presented the better try-
panosomicidal profile being equipotent to the standard drug
nifurtimox (IC50 ¼ 7.7 M).
m
2.5. Leishmanicidal activity
The ability of compounds 6aeh and 7aeh to inhibit the growth
of promastigotes forms of L. major were investigated, using pent-
amidine as standard [29]. Compounds with IC50 values <100 mM
were selected to study their cytotoxic activity against amastigostes
forms of L. major. As exemplified in Table 2, all compounds con-
taining the 1,3-benzodioxole system (6aeh) were inactive as
leishmanicide. In contrast, compounds 7c, 7d, 7f, 7g and 7h showed
cytotoxic activity against promastigotes of L. major, although with
potency inferior than pentamidine. Among these compounds only
the semicarbazones 7d (IC50 ¼ 74.0
(IC50 ¼ 0.6 M) were active against amastigote forms of L. major;
being compounds 7g and 7h more potent than pentamidine
(IC50 ¼ 17.1 M).
mM), 7g (IC50 ¼ 1.5
mM) and 7h
m
m
Considering the aim of identify a new antitrypanosomatid, the
analysis of the results depicted in Table 2, allowed the selection of
compound 7g (LASSBio-1483) as a dual trypanosomicidal and
leishmanicidal agent. Therefore, the in silico prediction of physi-
cochemical, ADME and toxicity properties of LASSBio-1483 (7g)
were calculated using the ACD/Labs Percepta Platform (License#
56950) and the results were compared to those obtained for
nifurtimox and pentamidine.
2.2. X-ray diffraction analyses
Fig. 1 is a structure representation of 7g crystallized in the P21/c
space group. Table 1 present its main crystallographic data. The
geometric features were studied with the software MOGUL [24]
and this analysis showed that all bond lengths and angles were in
agreement with the expected statistical values when compared
with similar fragments of structures deposited in Cambridge
Structural Database (CSD) [25]. The leastesquare plane through the
non-hydrogen atoms of the 5-nitro-2-furaldehyde semicarbazone
moiety shows a high planarity (r.m.s ¼ 0.0358). This molecular
moiety forms an angle of 10.78(6)o with that one through the
benzoic ring (r.m.s ¼ 0.0281).
As demonstrated in Table 3, the druglikeness of compound 7g
was very similar to nifurtimox and different from pentamidine,
with no violations of Lipinsky's rule of 5 [30]. Regardless of the poor
solubility (predicted in buffer at pH of 6.5), compound 7g (LASSBio-
1483) was showed to be highly permeable based on predicted
permeability across Caco-2 monolayers (Pe) and human intestinal
absorption (HIA) test. These results were similar to nifurtimox and
opposite to pentamidine, that was demonstrated to be a poorly
permeable drug (Pe ꢀ 1 * 10-6 cm/s and HIA < 30%) with zero oral
bioavailability (F ¼ 0%, Table 3). LASSBio-1483 (7g) was expected to
have an oral bioavailability (F) of 39% (Table 3). The drug safety
profile of these compounds was also projected using Program ACD/
Information about intermolecular geometry of 7g and the

26
M.A. Alves et al. / European Journal of Medicinal Chemistry 100 (2015) 24e33
Scheme 1. Synthesis of semicarbazone derivatives 6aeh and 7aeh from the amines 8 and 9.
in agreement with the presence of the toxicophoric 5-nitrofuranyl
subunit.
Bearing in mind the possibility of the chemical instability of
the imine function, present in the semicarbazone framework,
the stability profile of compound 7g (LASSBio-1483) was
investigated in buffer solution in pH ¼ 2.0 and 7.4 (Fig. 2A).
Moreover, in view of the peptide mimetic profile of semi-
carbazone scaffold, the plasma stability of LASSBio-1483 was
also studied (Fig. 2B).
As demonstrated in Fig. 2A, compound 7g (LASSBio-1463) pre-
sented high stability in buffer solution, either in pH value that
simulate gastric juice (pH ¼ 2) or either in pH value that mimic
serum content (pH 7.4). The aqueous solubility of LASSBio-1483
(7g) was determined using UV-spectroscopic method [31]. Com-
pound 7g presented a low aqueous solubility, with experimental
value (0.0028 mg/mL) similar to that predicted by Program ACD/
Percepta 14.0.0 (0.004 mg/mL; Table 3).
Fig. 1. View of representative semicarbazone derivative 7g with ellipsoids represent
50%-probability level. H atoms are shown as small spheres of arbitrary radii.
Percepta 14.0.0, based on probabilistic predictors. The metabolic
stability in human liver microsomes (HLM), the inhibition of hERG
ꢁ
(the human Ether-a-go-go-Related Gene) and the mutagenic profile
The plasma stability of compound 7g (LASSBio-1483) was
determined following the methodology adapted from Konsoula and
Jung (2008) [32]. The rat plasma sample was validated using methyl
biphenyl-4-carboxylate as standard. As indicated in Fig. 2B, this
standard was completely metabolized by plasma enzymes at time
of 240 min, resulting in the formation of the biphenyl-4-carboxylic
acid (data not shown). In contrast, LASSBio-1483 (7g) was not
(i.e. probability of positive Ames test) were calculated and the re-
sults converted in the so called classification scores (Table 4). As
depicted in Table 3 compound 7g (LASSBio-1483) was predicted as
stable in HLM (ꢀ0.33) and its ability to inhibit hERG was undefined
(score > 0.33 and ꢀ 0.67). However, similar to nifurtimox, LASSBio-
1483 (7g) was predictable to be mutagenic (score > 0.67), which is

M.A. Alves et al. / European Journal of Medicinal Chemistry 100 (2015) 24e33
27
Table 1
Crystal data and the structure refinement for 7g (LASSBio-1483).
3. Experimental section
3.1. Chemistry
Parameters
7g (LASSBio-1483)
Empirical formula
Formula weight
Wavelength, Å
Crystal system
Space group
C12H9ClN4O4
308.68
1.5418
Monoclinic
P21/c
Reagents and solvents were purchased from commercial sup-
pliers. The reactions were monitored by thin layer chromatography,
which was performed on aluminum sheets pre-coated with silica
gel 60 (HF-254, Merck) to a thickness of 0.25 mm. The chromato-
grams were viewed under ultraviolet light (254e265 nm). 1H NMR
and 13C NMR spectra were determined in deuterated dimethyl
sulfoxide using a Bruker DPX-200 at 200 MHz, Varian Mercury-300
(300 MHz), Varian MR-400 (400 MHz). Signal multiplicities are
represented by: s (singlet), d (doublet), t (triplet), q (quadruplet), m
(multiplet) and br (broad signal). Infrared (IR) spectra were ob-
tained with a FTLA 2000e100 spectrophotometer using potassium
bromide plates. Melting points of final products were determined
with a Quimis 340 apparatus and are uncorrected. The purity of
compounds were determined by HPLC (>95%) using the Shimadzu
eLC20AD apparatus, a Kromasil 100-5C18 (4.6 mm ꢁ 250 mm)
column and the SPD-M20A detector (Diode Array) at 254 nm for
quantification of analyte in a 1 mL/min constant flux. The injector
ꢂ
Cell parameters, Å and
a ¼ 8.8854(2), b ¼ 13.8588(3),
c ¼ 13.5620(3)
b
¼ 129.229(2)
V, Å3
Z
1293.65(6)
4
m
r
, mmꢃ1
2.855
1.585
calc., Mg mꢃ1
q
-range for data collection
5.283e62.270ꢂ
ꢃ10 ꢀ h<¼9, ꢃ14 ꢀ k<¼15,
ꢃ15 ꢀ l<¼15
Index ranges
Refl. Collected/Unique
Completeness to theta ¼ 67.680ꢂ
Data/restraints/parameters
R indices [I > 2sigma(I)]
R indices (all data)
3977/2005 [R(int) ¼ 0.0198]
85.6%
2005/0/190
R1 ¼ 0.0349, wR2 ¼ 0.0886
R1 ¼ 0.0479, wR2 ¼ 0.0964
1.042
0.200 and ꢃ0.258
GooF on F2
Residual density, eÅꢃ3
was programmed to inject a volume of 20 mL. The mobile phases
used were: CH3CN:H2O 1:1; 6:4 and 7:3. The results of elemental
analysis were obtained FlashEA 1112 Series instrument (Thermo
Scientific) from samples previously dried under vacuum. Ultravio-
let spectroscopy was performed using Femto spectrophotometer.
metabolized during all the analysis time (0, 30, 60, 120, 180,
240 min). The data revealed the great plasma stability of LASSBio-
1483 (Fig. 2B).
The wavelength used in solubility assay was determined by the
l
max characteristic of each compound. Spectra were analyzed in
FemtoScan software. Mass spectrometry was obtained by positive
and negative ionization at Esquire 6000- ESI Ion Trap MSn System
Bruker Daltonics and data analyzed in Compass 1.3.SR2 software.
In summary a series of semicarbazone derivatives (6aeh and
7aeh), containing structural modifications on the rings linked to
the amine (NH) and imine (N]CH) groups, were designed and
synthesized. From this series compound 7g (LASSBio-1483) high-
lighted, showing dual in vitro trypanosomicidal and leishmanicidal
activities with potency similar to the standards drugs nifurtimox
and pentamidine. This data, taken together with its good in silico
druglikeness profile and its great chemical and plasma stabilities,
3.1.1. Procedure for the preparation of intermediate carbamates 10
and 11 (adapted from references [20,21])
In 30 mL of chloroform was added 7.1 mL (56 mmol) of phenyl
chloroformate and allowed to stir. Then it was added slowly with a
pipette, a solution of 6g of the functionalized aniline 8 or 9
(47 mmol) in 50 mL of chloroform. The end of the reaction was
make LASSBio-1483 (7g)
a new antitrypanosomatid lead-
candidate. The in vivo trypanosomicidal and leishmanicidal activ-
ities of 7g will be study in our labs.
Table 2
Determination of the cytotoxicity of semicarbazone derivatives against macrophages (J774.A1), epimastigotes of T. cruzi and promastigote and amastigote forms of L. major.
Compounds
J774.A1 cell line IC50
(m
M)a
Epimastigote T. cruzi IC50
(m
M)b
Promastigote L. major IC50
(m
M)c
Amastigote L. majorIC50 (m
M)c
Nifurtimox
Pentamidine
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
71.2
>100
>100
35.7
>100
>100
>100
7.7
0.9
0.8
44.1
17.1
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
>100
74.0
N.D.
>100
1.5
N.D.
>100
>100
>100
8.5
>100
>100
11.5
50.0
21.0
89.1
>100
>100
29.0
86.7
11.9
>100
6a (LASSBio-1200)
6b (LASSBio-1205)
6c (LASSBio-1201)
6d (LASSBio-1203)
6e (LASSBio-1206)
6f (LASSBio-1210)
6g (LASSBio-1302)
6h (LASSBio-1303)
7a (LASSBio-1487)
7b (LASSBio-1701)
7c (LASSBio-1490)
7d (LASSBio-1489)
7e (LASSBio-1486)
7f (LASSBio-1488)
7g (LASSBio-1483)
7h (LASSBio-1699)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
6.9
9.4
>100
12.6
18.5
9.7
0.6
N.D. ¼ not determined.
a
IC50 is the concentration required to give 50% death of cells, calculated by linear regression analysis from the Kc values at employed concentrations (100, 10, 1 and 10-1
mM).
b
IC50 is the concentration required to give 50% death of T. cruzi epimastigotes, calculated by linear regression analysis from the Kc values at employed concentrations (100,
50, 25, 10, 5, 1 M).
IC50 is the concentration required to give 50% death of L. major parasites, calculated by linear regression analysis from the Kc values at employed concentrations (100, 10, 1,
10ꢃ1, 10ꢃ2 and 10-3
M).
m
c
m

28
M.A. Alves et al. / European Journal of Medicinal Chemistry 100 (2015) 24e33
Table 3
Physico-chemistry properties and ADMET profile of compounds 7g (LASSBio-1483),
nifurtimox and pentamidine calculated using the Program ACD/Percepta 14.0.0.
Predicted
properties
Compounds
7g (LASSBio-1483)
Nifurtimox
Pentamidine
MW (g/mol)
H-Donors
H-Acceptors
Rotatable Bonds
LogP
308.68
2
8
4
287.25
0
8
3
340.42
6
6
10
3.03
0.04
2.09
Solubility
Caco-2
0.004 mg/ml
Pe ¼ 204 ꢁ 10ꢃ6
cm/s
100%
39%
0.33
0.40
0.74
2.39 mg/mL
Pe ¼ 88 ꢁ 10ꢃ6
cm/s
100%
99%
0.39
0.42
0.86
0.61 mg/mL
Pe ¼ 0.6 ꢁ 10ꢃ6
cm/s
16%
0%
0.33
0.34
0.16
HIA
% F (oral)
HLM
hERG
AMES
Chart 1. Examples of compound containing amide mimetic framework able to inhibit
proteases of parasites.
Table 4
The meaning of classification score range values for ADMET properties using Pro-
gram ACD/Percepta 14.0.0.
C6), 120.5 (C4); purity calculated by elemental analysis: analysis
calculated (C 63.04%, H 4.07%, N 5.66%), experimental analysis (C
62.99%, H 4.04%, N 5.50%); MS: m/z ¼ 246.0 [M-H]-.
Classification scores
Predicted ADMET properties
HLM
hERG
AMES
ꢀ0.33
Stable
Undefined
Unstable
Non-inhibitor
Undefined
Inhibitor
Non-mutagenic
Undefined
Mutagenic
3.1.2. Procedure for the preparation of semicarbazides 12 and 13
(adapted from reference [22])
>0.33 and ꢀ0.67
>0.67
In a solution containing 1g (4 mmol) of carbamate derivative (10
or 11) and 40 mL of ethanol was added 2.3 mL (15 eq) of hydrazine
hydrate 80%. After 12 h at room temperature, it was checked the
end of the reaction by TLC (dichloromethane/5% methanol), and the
solvent volume was reduced and added ice checking the precipi-
tation of product that was vacuum filtered.
monitored by TLC after 4 h of reaction, with total consumption of
the starting material (eluent: dichloromethane/methanol 5%). 2/3
of the solvent volume was reduced in vacuum being subsequently
added 50 mL of hexane and left under stirring for 10 min. The
mixture was vacuum filtered, and washed with hexane.
3.1.2.1. N-(benzo[1,3]dioxol-5- yl) hydrazinecarboxamide (12; LASS-
Bio-1212). Yield: 85%, beige solid, m.p. 215e217 ꢂC; I.R. (KBr)
(cmꢃ1): 3356 (ѵNH2), 3102e3216 (ѵNH), 1634 (ѵCO), 1634 (ѵC]N);
3.1.1.1. Phenyl benzo[d][1,3]dioxol-5-ylcarbamate (10; LASSBio-1213).
Yield: 82%, black solid, m.p. >250 ꢂC; I.R. (KBr) (cmꢃ1): 3390 (ѵNH),
1H NMR (400 MHz, DMSO-d6)
d (ppm): d 8.50 (s, 1H, ArNH), 7.30 (s,
1717 (ѵCO); 1H NMR (300 MHz, DMSO-d6)
d (ppm): d 10.59 (s, 1H,
1H, CONH), 7.25 (s, 1H, H4), 6.84 (d,1H, H7), 6.78 (d,1H, H6), 5.90 (s,
2H, H2), 4.29 (s, 1H, NH2), 98% purity in HPLC (R.T. ¼ 2.9 min,
CH3CN:H2O (6:1)); MS: m/z ¼ 196.1 [MþH]þ.
AreNH) 8.73 (s,1H,C]NH), 7.94 (s, 1H,CONH), 7.82 (d, 2H, H2' &
H60), 7.39 (m, 3H, H30, H4' & H50), 7.28 (s, 1H, H4), 7.15 (d, 1H, H6),
6.83 (d, 1H, H7), 5.97 (s, 2H, H2); 99% purity in HPLC (R.T. ¼ 7.3 min,
CH3CN:H2O (6:1)); MS: m/z ¼ 258.1 [MþH]þ.
3.1.2.2. N-(4-chlorophenyl)hydrazinecarboxamide (13; LASSBio-
1482). Yield: 61%, white solid, m.p. >250 ꢂC; I.R. (KBr) (cmꢃ1): 3337
(ѵNH), 1668 (ѵC]O), 1011 (ѵC-Cl); 1H NMR (200 MHz. DMSO-d6)
3.1.1.2. Phenyl 4-chlorophenylcarbamate (11; LASSBio-1481).
Yield: 72%, white solid, m.p. 190e192 ꢂC; I.R. (KBr) (cmꢃ1): 3311
d
(ppm):
CONH), 7.25 (d, 2H, H3 & H5), 4,35 (s, 2H, NH2); 13C NMR (200 MHz.
DMSO-d6) (ppm): 157.2 (CO), 138.9 (C1), 128.2 (C3 & C5),
d8.76 (s, 1H, AreNH), 7.58 (d, 2H, H2 & H6), 7.46 (s, 1H,
(ѵNH). 1716 (ѵC]O); 1H NMR (200 MHz. DMSO-d6)
d (ppm): d 10.38
(s, 1H, AreNH), 7.54 (d, 2H, H2 & H6), 7.47e7.36 (m, 5H, H20, H30,
H40, H50 & H60), 7.28 (d, 2H, H3 & H5); 13C NMR (200 MHz. DMSO-
d
d
124.8(C4), 119.6 (C2 & C6); purity calculated by elemental analysis:
calculated (C 45.30%, H 4.34%, N 22.64%), experimental (C 45.36%, H
4.37%, N 22.70%); MS: m/z ¼ 184.0 [M-H]-.
d6)
d (ppm): d
152.2 (C]O), 150.9 (C]NH), 138.1 (C1), 129.9 (C20,
C60, C3 & C5), 129.3(C3' &C50), 127.2 (C10), 126.0 (C4), 122.4 (C2 &
Fig. 2. A) Chemical stability of the compound LASSBio-1483 (7g) at pH 2 and 7.4; B) Plasma stability of LASSBio-1483 (7g) and the standard methyl biphenyl-4-carboxylate.

M.A. Alves et al. / European Journal of Medicinal Chemistry 100 (2015) 24e33
29
Chart 2. Design concept of semicarbazone derivatives (6aeh and 7aeh) from molecular modifications on prototype 5.
3.1.3. Procedure for the preparation of semicarbazones 6aeh and
7aeh (adapted from reference [23])
147.50 (N]CH), 142.94 (C3), 141.59 (C1), 134.08 (C5), 129.22 (C20&
C60), 126.03 (C10), 116.00 (C30& C50), 113.21 (C6), 108.28 (C7), 102.82
(C4), 101.33 (C2); 98% purity in HPLC (R.T. ¼ 3.6 min, CH3CN:H2O
(6:1)); MS: m/z ¼ 300.0 [MþH]þ.
Semicarbazide 12 or 13 (0.25 g, 1.35 mmol) was added in 10 mL
ethanol and 1.35 mmol of aldehyde, at room temperature, followed
by addition of 1 drop of concentrated HCl. The solution remained
under stirring for 30e240 min until TLC (dichloromethane/meth-
anol 5e10%) indicated completion of reaction. The volume of
ethanol was reduced, and after addition of ice, was observed pre-
cipitation of the product that was filtered and dried under vacuum.
Yields and characterization pattern are described below.
3.1.3.3. (E)-N-(benzo[d][1,3]dioxol-5-yl)-2-(2-nitrobenzylidene)hy-
drazine-carboxamide (6c; LASSBio-1201). Yield: 89%, yellow solid,
m.p.203e205 ꢂC; I.R. (KBr) (cmꢃ1): 3201e3094 (ѵNH), 1689 (ѵCO),
1554 (ѵC]N), 1522 & 1335 (ѵNO2); 1H NMR (300 MHz, DMSO-d6)
d
(ppm): d 10.96 (s, 1H, AreNH), 8.78 (s, 1H, N]CH), 8.44 (d, 1H,
H20), 8.34 (s,1H, CONH), 8.02 (d,1H, H50), 7.77 (t,1H,H30), 7.62 (t,1H,
H40), 7.27 (s, 1H, H4), 7.11 (d, 1H, H6), 6.84 (d, 1H, H7), 5.97 (s, 2H,
H2); 99% purity in HPLC (R.T. ¼ 6.0 min, CH3CN:H2O (6:1)); MS: m/
z ¼ 329.1 [MþH]þ.
3.1.3.1. (E)-N-(benzo[d][1,3]dioxol-5-yl)-2-(2-hydroxybenzylidene)
hydrazine carboxamide (6a; LASSBio-1200). Yield: 95%, brown solid,
m.p. 215e217 ꢂC; I.R. (KBr) (cmꢃ1): 3334e2779 (ѵOH), 3190e3042
(ѵNH), 1651 (ѵCO), 1576 (ѵC]N); 1H NMR (300 MHz, DMSO-d6)
d
(ppm): 10.48 (s, 1H, AreNH), 10.02 (s, 1H, AreOH), 8.72 (s, 1H, N]
CH), 8.25 (s, 1H, CONH),7.87 (d, 1H, H20), 7.29 (s, 1H, H4), 6.84e7.24
(m, 6H, H4, H6, H7, H30, H4΄& H50), 5.97 (s, 2H, H2); 99% purity in
HPLC (R.T. ¼ 4.8 min, CH3CN:H2O (6:1)); MS: m/z ¼ 300.1 [MþH]þ.
3.1.3.4. (E)-N-(benzo[d][1,3]dioxol-5-yl)-2-(4-nitrobenzylidene)hy-
drazine-carboxamide (6d; LASSBio-1203). Yield: 88%, orange solid,
m.p.>250 ꢂC; I.R. (KBr) (cmꢃ1): 3196e3085 (ѵNH), 1682 (ѵCO), 1549
(ѵC]N), 1507 & 1339 (ѵNO2), 1H NMR (300 MHz, DMSO-d6)
3.1.3.2. (E)-N-(benzo[d][1,3]dioxol-5-yl)-2-(4-hydroxybenzylidene)
hydrazine-carboxamide (6b; LASSBio-1205). Yield: 84%, beige solid,
m.p. 202e204 ꢂC; I.R. (KBr) (cmꢃ1): 3389e2894 (ѵOH), 3200e3088
(ѵNH), 1659 (ѵCO), 1523 (ѵC]N);1H NMR (300 MHz, DMSO-d6)
d (ppm): d5.97 (s, 2H, H2), 6.83 (d, 1H, H7), 7.02 (d, 1H, H6), 7.27 (s,
1H, H4), 8.05 (s,1H, CONH), 8.09 (d, 2H, H20& H60), 8.23 (d, 2H, H30&
H50), 8.92 (s, 1H, N]CH), 10.96 (s, 1H, AreNH); 13C NMR (300 MHz,
DMSO-d6)
d (ppm): d 101.44 (C2), 103.35 (C4), 108.29 (C7), 113.92
d
(ppm):
d
10.38 (AreNH), 9.73 (s, 1H, AreOH), 8.64 (s, 1H, N]CH),
(C6), 124.30 (C20& C60), 128.39 (C30& C50), 133.64 (C5), 138.73 (C10),
141.47 (C1), 143.32 (C3), 147.52 (N]CH), 147.90 (C]O), 153.49 (C40).
99% purity in HPLC (R.T. ¼ 6.0 min, CH3CN:H2O (6:1)); MS: m/
z ¼ 329.1 [MþH]þ.
7.82 (s, 1H, CONH), 7.63 (d, 2H, H20& H60), 7.29 (d, 1H, H6), 7.03 (d,
1H, H7), 6.82 (s, 1H, H4), 6.77 (d, 2H, H30& H50), 5.95 (s, 2H, H2); 13C
NMR (300 MHz, DMSO-d6) d (ppm): d 159.35 (COH), 153.78 (C]O),

30
M.A. Alves et al. / European Journal of Medicinal Chemistry 100 (2015) 24e33
3.1.3.5. (E)-N-(benzo[d][1,3]dioxol-5-yl)-2-(4-isopropylbenzylidene)
hydrazinecarboxamide (6e; LASSBio-1206). Yield: 75%, beige solid,
m.p.136e138 ꢂC; I.R. (KBr) (cmꢃ1): 3193e1117(ѵNH), 1686 (ѵCO),
H5), 6.80 (d, 2H, H30 & H50); 13C NMR (200 MHz. DMSO-d6)
d (ppm):
d
158.87 (COH), 153.05 (C]O), 141.44 (CN]CH), 138.25 (C1), 128.74
(C4), 128.22 (C3 & C5), 125.89 (C10), 125.36 (C20 & C60), 121.17 (C2 &
C6), 115.44 (C30 & C50); 98% purity in HPLC (R.T. ¼ 3.9 min
CH3CN:H2O (7:1)), MS: m/z ¼ 288.1 [M-H]-.
1549 (ѵC]N), 740 ѵ(iPr); 1H NMR (300 MHz, DMSO-d6)
d (ppm):
d
10.50 (s, 1H, AreNH), 8.67 (s, 1H, N]CH), 7.89 (s, 1H, CONH), 7.71
(d, 2H, H20, H60),7.28 (d, 2H, H30, H50), 7.25 (s, 1H H4), 7.03 (d, 1H,
H6), 6.80 (d, 1H, H7), 5.95 (s, 2H, H2), 2.85 (m, 1H,CH), 1.20 (d, 6H,
3.1.3.11. (E)-N-(4-chlorophenyl)-2-(2-nitrobenzylidene)hydrazine-
carbox amide (7c; LASSBio-1490). The melting point, 1H NMR, 13C
NMR and IR data are in agreement with previous reports [33].
Yield: 85%, yellow solid, m.p. 200e202 ꢂC; I.R. (KBr) (cmꢃ1): 3390
(ѵNH), 1709 (ѵC]O), 1537 e 1344 (ѵAr-NO2), 1013 (ѵAr-Cl); 1H NMR
(CH3)2); 13C NMR (300 MHz, DMSO-d6)
d (ppm): d24.25 (CH3)2,
33.88 (CH), 101.36 (C2), 102.96 (C4), 108.29 (C7), 113.40 (C6), 127.06
(C30& C50), 127.62 (C20& C60), 132.68 (C5), 133.98 (C10), 141.30 (C1),
143.04 (N]CH), 147.51 (C3), 150.47 (C40), 153.72 (C]O); 98% purity
in HPLC (R.T. ¼ 13.4 min, CH3CN:H2O (6:1)); MS: m/z ¼ 326.2
[MþH]þ.
(200 MHz. DMSO-d6)
d (ppm): d 11.15 (s, 1H, AreNH), 9.08 (s, 1H,
CONH), 8.47 (d, 1H, H20), 8.37 (s, 1H, N]CH), 8.03 (d, 1H, H50),
7.78e7.58 (m, 2H, H30 & H40), 7.69 (d, 2H, H2 & H6), 7.35 (d, 2H, H3
3.1.3.6. (E)-N-(benzo[d][1,3]dioxol-5-yl)-2-(4-hydroxy-3-methoxy
benzylidene)hydrazine carboxamide (6f; LASSBio-1210). Yield: 75%,
white solid, m.p.198e200 ꢂC; I.R. (KBr) (cmꢃ1): 3193e3100 (ѵNH),
3349e2841 (ѵOH), 1666 (ѵCO), 1549 (ѵC]N); 1H NMR (300 MHz,
& H5); 13C NMR (200 MHz. DMSO-d6)
d (ppm): d152.6 (C]O), 147.8
(C60), 137.8 (N]CH), 136.0 (C1), 133.2 (C30), 129.9 (C4), 128.4 (C40),
128.3 (C20), 128.2 (C3 & C5), 126.3 (C10), 124.3 (C50), 121.4 (C2 & C6);
purity calculated by elemental analysis: analysis calculated (C
52.76%, H 3.48%, N 17.58%), experimental analysis (C 52.54%, H
3.41%,N 17.34%); MS: m/z ¼ 317.1 [M-H]-.
DMSO-d6)
d (ppm): d3.85 (s, 3H, CH3), 5.96 (s, 2H, H2), 6.78e6.83
(m,2H,H50&H7), 7.04 (d,1H, H6), 7.13 (d, 1H, H60), 7.31 (d, 1H, H4),
7.43 (s, 1H, H20), 7.83 (s, 1H, CONH), 8.68 (s, 1H, N]CH), 9.28 (s, 1H,
OH), 10.39 (s, 1H, AreNH); 13C NMR (300 MHz, DMSO-d6)
d
(ppm):
3.1.3.12. (E)-N-(4-chlorophenyl)-2-(4-nitrobenzylidene)hydrazine-
56.36 (CH3), 101.35 (C2), 102.94 (C4), 108.31 (C50), 110.48 (C6),
113.39 (C7), 115.89 (C20), 121.95 (C50), 126.46 (C5), 134.07 (C10),
141.79 (C1), 142.98 (C]NH), 147.50 (C40), 148.50 (C30), 148.9 (C3),
153.80 (C]O); 99% purity in HPLC (R.T. ¼ 3.7 min, CH3CN:H2O
(6:1)); MS: m/z ¼ 330.2 [MþH]þ.
carbox amide (7d; LASSBio-1489). The melting point, 1H NMR, 13
C
NMR and IR data are in agreement with previous reports [33].Yield:
66%, yellow solid, m.p. 248e250 ꢂC; I.R. (KBr) (cmꢃ1): 3086 (ѵNH),
1684 (ѵC]O), 1541 e 1318 (ѵAr-NO2), 1009 (ѵAr-Cl); 1H NMR
(200 MHz. DMSO-d6)
d (ppm): d11.16 (s, 1H, AreNH),9.19 (s, 1H,
CONH), 8.25 (d, 2H, H30 & H50), 8.15 (s, 1H, N]CH), 8.07 (d, 2H, H20
& H60), 7.70 (d, 2H, H2 & 6), 7.36 (d, 2H, H3 & H5); 13C NMR
3.1.3.7. (E)-N-(benzo[d][1,3]dioxol-5-yl)-2-((5-nitrofuran-2-yl)meth-
ylene) hydrazine carboxamide (6g; LASSBio-1302). Yield: 90%, or-
ange solid, m.p.221e223 ꢂC; I.R. (KBr) (cmꢃ1): 3155(ѵNH), 1676
(200 MHz. DMSO-d6)
d (ppm): d
152.80 (C]O), 147.41 (C40), 140.78
(N]CH), 138.64 (C10), 137.91(C1), 128.30 (C3 & C5), 127.91 (C20 &
C60), 126.40 (C4), 123.74 (C30 & C50), 121.67 (C2 & C6); 98% purity in
HPLC (R.T. ¼ 5.6 min CH3CN:H2O (6:1)), MS: m/z ¼ 317.1 [M-H]-.
(ѵCO), 1551 & 1325 (ѵNO2); 1H NMR (300 MHz, DMSO-d6)
d (ppm):
d
5.96 (s, 2H, H2), 6.82 (d, 1H, H7), 6.97 (d, 1H, H6), 7.23 (s, 1H, H4),
7.34 (d,1H, H40), 7.78 (d,1H, H30), 7.88 (s,1H, CONH), 8.78 (s,1H, N]
CH), 11.08 (s, 1H, AreNH); 98% purity in HPLC (R.T. ¼ 4.4 min,
CH3CN:H2O (6:1)); MS: m/z ¼ 318.9 [MþH]þ.
3.1.3.13. (E)-N-(4-chlorophenyl)-2-(4-isopropylbenzylidene)hydra-
zine carboxamide (7e; LASSBio-1486). Yield: 62%, white solid, m.p.
138e140 ꢂC; I.R. (KBr) (cmꢃ1): 3375 (ѵNH), 1694 (ѵC]O), 1012 (ѵAr-
3.1.3.8. (E)-N-(benzo[d][1,3]dioxol-5-yl)-2-((5-(4-nitrophenyl)furan-
Cl); 1H NMR (200 MHz. DMSO-d6)
d (ppm): d10.77 (s, 1H, AreNH),
2-yl)methylene)hydrazine
carboxamide
(6h;
LASSBio-1303).
9.01 (s, 1H, CONH), 7.93 (s, 1H, N]CH), 7.76 (d, 2H, H2 & H6), 7.73
(d, 2H, H3& H5), 7.34 (d, 2H, H20 & H60), 7.29 (d, 2H, H30 & H60), 2.94
(s, 1H, CH), 1.21 (d, 6H, (CH3)2); 13C NMR (200 MHz. DMSO-d6)
Yield: 95%, orange solid, m.p.226e228 ꢂC; I.R. (KBr) (cmꢃ1):
3095(ѵNH), 1698 (ѵCO), 1501 & 1355 (ѵNO2),1H NMR (300 MHz,
DMSO-d6)
d
(ppm):
d5.96 (s, 2H, H2), 6.83 (d, 1H, H7), 7.03 (d, 1H,
d (ppm): d
153.0 (C]O), 150.0 (C40), 141.1 (N]CH). 138.1 (C1), 132.0
H6), 7.12 (d, 1H, H30), 7.29 (s, 1H, H4), 7.41 (d, 1H, H40), 7.9 (s, 1H,
CONH), 8.02 (d, 2H, H3” & H500), 8.25 (d, 2H, H2” & H6”), 8.65 (s, 1H,
N]CH), 10.77 (s,1H,AreNH); 95% purity in HPLC (R.T. ¼ 8.4 min,
CH3CN:H2O (6:1)); MS: m/z ¼ 395.1 [MþH]þ.
(C4), 128.2 (C3 & C5), 127.1 (C20 & C60), 126.5 (C30 & C50), 126.0 (C10),
121.3 (C2 & C6), 33.31 (CH), 23.66 (CH3)2; purity calculated by
elemental analysis: analysis calculated (C 52.72%, H 3.24%, N
12.30%), experimental analysis (C 52.52%, H 2.9%, N 12.31%); MS: m/
z ¼ 314.1 [M-H]-.
3.1.3.9. (E)-N-(4-chlorophenyl)-2-(2-hydroxybenzylidene)hydrazine
carboxamide (7a; LASSBio-1487). Yield: 89%, white solid, m.p.
197e199 ꢂC; I.R. (KBr) (cmꢃ1): 3435 (ѵNH), 1696 (ѵ C]O), 1492 (ѵO-
3 .1. 3 .14 . ( E ) - N - ( 4 - c h l o r o p h e n y l ) - 2 - ( 4 - h y d r o x y - 3 -
methoxybenzylidene)hydrazine carboxamide (7f; LASSBio-1488).
Yield: 90%, white solid, m.p. 218e220 ꢂC; I.R. (KBr) (cmꢃ1):
3410e2835 (ѵAr-OH), 3334 (ѵNH), 1677 (ѵC]O), 1092 (ѵAr-Cl); 1H
H), 1013 (ѵAr-Cl); 1H NMR (200 MHz. DMSO-d6)
d (ppm): d 10.70 (s,
1H, AreNH), 10.10 (s, 1H, OH), 9.02 (s, 1H, CONH), 8.21 (s, 1H, N]
CH), 7.95 (d, 1H, H20), 7.68 (d, 2H, H2 & H6), 7.33 (d, 2H, H3 & H5),
7.21 (t, 1H, H40); 6.86 (m, 2H, H30 & H50); 13C NMR (200 MHz.
NMR (200 MHz. DMSO-d6)
d (ppm): d10.63 (s, 1H, AreNH), 9.45 (s,
1H,OH), 9.01 (s, 1H, CONH), 7.87 (s, 1H, N]CH), 7.73 (d, 2H, H2 &
DMSO-d6) d (ppm): d156.1 (COH), 152.9 (CO), 139.0 (CN]CH), 138.2
H6), 7.49 (s, 1H, H20), 7.35 (d, 2H, H3 & H5), 7.16 (d, 1H, H60), 6.82 (d,
(C1), 130.7 (C4), 128.3 (C3 & C5), 127.1 (C40), 126.0 (C20), 121.3 (C2 &
C6), 120.3 (C30), 119.2 (C10), 116.0 (C50); 99% purity in HPLC
(R.T. ¼ 11.7 min CH3CN:H2O (1:1)), MS: m/z ¼ 288.1 [M-H]-.
1H, H50), 3.37 (s, 3H, OCH3); 13C NMR (200 MHz. DMSO-d6)
d
d
(ppm):
153.6 (C]O), 148.9 (N]CH), 148.5 (C40), 142.2 (C30), 138.8 (C1),
128.8 (C3 & C5), 126.52(C4), 126.3 (C10), 122.1 (C60), 121.8 (C2 & C6),
115.9 (C20), 110.5 (C50), 56.3 (CH3); purity calculated by elemental
analysis: analysis calculated (C 56.35%, H 4.41%, N 13.14%), experi-
mental analysis (56.35%, H 4.67%, N 12.92%); MS: m/z ¼ 318.1 [M-
H]-.
3.1.3.10. (E)-N-(4-chlorophenyl)-2-(4-hydroxybenzylidene)hydrazi-
necarbox amide (7b; LASSBio-1701). Yield: 67%, white solid, m.p.
204e206 ꢂC; I.R. (KBr) (cmꢃ1): 3301 (ѵNH), 1617 (ѵC]O), 1488
(ѵOH); 1015(ѵAr-Cl); 1H NMR (200 MHz. DMSO-d6)
d (ppm): d 10.59
(s, 1H, AreNH), 9.83 (s, 1H, OH), 8.93 (s, 1H, CONH), 7.86 (s, 1H, N]
3.1.3.15. (E)-N-(4-chlorophenyl)-2-((5-nitrofuran-2-yl)methylene)
hydrazine carboxamide (7g; LASSBio-1483). Yield: 80%, yellow
CH), 7.71 (d, 2H, H20 & H60), 7.67 (d, 2H, H2 & H6), 7.33 (d, 2H, H3 &

M.A. Alves et al. / European Journal of Medicinal Chemistry 100 (2015) 24e33
31
crystal, m.p. 182e184 ꢂC; I.R. (KBr) (cmꢃ1): 3372 (ѵNH), 1694 (ѵ C]
O),1532 e 1327 (ѵAr-NO2),1013 (ѵAr-Cl); 1H NMR (200 MHz. DMSO-
d6) (ppm): 11.28 (s, 1H, AreNH), 9.09 (s, 1H, CONH), 7.91 (s, 1H,
N]CH), 7.82 (d, 1H, H30), 7.66 (d, 2H, H2 & H6), 7.35 (m, 3H, H3,H5
& H40); 13C NMR (200 MHz. DMSO-d6)
(ppm): 152.6 (C]O),152.3
5.2. Culture of Trypanosoma cruzi epimastigotes
d
d
T. cruzi epimastigotes (Tulahuen 2 strain) were cultured at 28 ꢂC
for 5e7 days (exponential phase of growth) under aerobiosis in
axenic BHI-tryptose milieu (33 g/L brain-heart infusion, 3 g/L
tryptose, 0.02 g/L hemin, 0.3 g/L D-(þ)-glucose, supplemented with
10% (v/v) calf serum, 200,000 units/L penicillin and 0.2 g/L
streptomycin).
d
d
(C20), 151.3 (N]CH), 137.6 (C10), 129.0 (C1), 128.3 (C3 & C5), 126.4
(C4), 121.4 (C2 & C6), 115.1 (C30), 112.9 (C40); purity calculated by
elemental analysis: analysis calculated (C 46.69%, H 2.94%, N
18.15%), experimental analysis (C 46.93%, 2.95%, N 18.28%); MS: m/
z ¼ 307.1 [M-H]-.
5.3. Culture of L. major promastigotes of L. major
Promastigotes of L. major IOC/L0581 (MHOM/SU/1973/5-ASKH)
were obtained from Leishmania collection of the Oswaldo Cruz
Institute - Fiocruz. Promastigotes of L. amazonensis (MHOM/BR/77/
LTB0016) were obtained from Dr. Eduardo Caio Torres dos Santos at
Oswaldo Cruz Institute - Fiocruz. The parasites were maintained
in vitro in Schneider's medium, supplemented with 10% FBS and 2%
human urine at 27 ꢂC in BOD incubator.
3.1.3.16. (E)-N-(4-chlorophenyl)-2-((5-(4-nitrophenyl)furan-2-yl)
methylene)hydrazine carboxamide (7h; LASSBio-1699). Yield: 64%,
yellow solid, m.p. 248e250 ꢂC; I.R. (KBr) (cmꢃ1): 3365
(ѵNH),1688 (ѵ C]O), 1532 e 1326 (ѵAr-NO2), 1010 (ѵAr-Cl); 1H
NMR (200 MHz. DMSO-d6)
d (ppm): d10.98 (s, 1H, AreNH), 8.98
(s, 1H, CONH), 8.28 (d, 2H, H3” & H500), 8.05 (d, 2H, H2” & H600),
7.92 (s, 1H, N]CH), 7.72 (d, 2H, H2 & H6), 7.45 (d, 1H, H30), 7.35
(d, 2H, H3 & H5), 7.17 (d, 1H, H4); 13C NMR (200 MHz. DMSO-d6)
5.4. Cytotoxicity against host cells
d
(ppm): d
152.65 (C]O), 151.80 (C20), 151.26 (N]CH), 146.13
(C400), 137.96 (C10), 135.30 (C1), 130.56 (C10), 128.32 (C3 & C5),
126.18 (C4), 124.40 (C200,C6”, C300 & C500), 121.23 (C2 & C6),
114.31(C30), 112.40 (C40); 98% purity in HPLC (R.T. ¼ 8.7 min
CH3CN:H2O (7:1)), MS: m/z ¼ 383.1 [M-H]-.
To evaluate the cytotoxic activity against the J774.A1 cell line,
the host cells were plated in 96-well vessels at 2 ꢁ 105 cells per well
in complete culture medium 10% FBS at 37 ꢂC. After 1 h wells were
washed with warm HBSS to remove non-adherent cells, leaving
approximately 105 adherent macrophages. All cultures were done
in DMEM complete supplemented with 10% FBS. The compounds
and pentamidine were added at serial concentrations
4. Methodology for single crystal X ray diffraction
measurements
(0.1e100 mM). The cells were also cultured with medium free from
compounds or vehicle (basal growth control) or in media with
DMSO 0.1% (vehicle control). Positive control (dead cells) was ob-
tained by cellular lysis with 1% of Triton 100X in DMEM complete.
After 48 h, the cytotoxicity was evaluated by the MTT assay [26].
Data obtained from experiments were expressed as the
mean standard error of the mean (Mean S.E.M.) and statistical
differences between the treated and the vehicle groups of experi-
ments were evaluated by ANOVA and Dunnett hoc tests. IC50
(concentration required to give 50% death of cells) was calculated
by linear regression analysis from the Kc values at employed
concentrations.
Orange plate-like crystals of 7g were obtained by slow evap-
oration of methanol at room temperature. A well-shaped and
suitably-sized single crystal (0.15 ꢁ 0.10 ꢁ 0.04 mm) was selected
for the X-ray diffraction structure determination experiment. The
X-ray intensity data were collected at 150 K on a Gemini-Oxford
Diffractometer, using CuK
a graphite monochromated radiation.
The programs CrysAlis CCD and CrysAlis RED [34] were used for
data collection, cell refinement and data reduction. The structure
was solved by direct methods using the software Sir-92 [35] and
the refinement was carried out using SHELXL-2013 [36]. The
non-hydrogen atoms were clearly solved and full matrix least-
squares refinement of these atoms with anisotropic thermal pa-
rameters was carried on. All hydrogen atoms were positioned
stereochemically and were refined with fixed individual
displacement parameters [Uiso (H) ¼ 1.2 Ueq] using a riding
group model with CeH and NeH bond lengths of 0.95 and 0.88 Å,
respectively. WINGX software was used to analyze and prepare
the data for publication [37]. Molecular graphics were prepared
using ORTEP-3 for Windows [38] and Mercury [39]. Crystallo-
graphic data for the structural analysis of the compound dis-
cussed here has been deposited at the Cambridge
Crystallographic Data Centre as a supplementary publication
under number CCDC 1031516. Copies of the data can be obtained,
free of charge, on application to CCDC, 12 Union Road, Cambridge
CB12 1EZ, UK [fax: þ44 1223 336033 or e-mail: deposit@ccdc.
cam.ac.uk].
5.5. In vitro anti-T. cruzi activity
Parasites were harvested in the late log phase, resuspended in
fresh medium, counted in a Neubauer chamber, and placed in 24-
well plates (3
ꢁ
106/mL). Parasite growth was followed
measuring the absorbance of the culture at 610 nm [28]. Before
inoculation, the media were supplemented with the indicated
amount of the studied compound from a stock solution in DMSO.
The final concentration of DMSO in the culture media never
exceeded 0.4% and the control was run in the presence of 0.4%
DMSO and in the absence of any compound. No effect on epi-
mastigote growth was observed in the presence of up to 1%
DMSO in the culture medium. The percentage of growth inhibi-
tion at 100
mM determined at day 5th was calculated as:
PGI ¼ {1-[(Ap - A0p)/(Ac - A0c)]} x 100, where Ap ¼ A600 of the
culture containing the compound at day 5; A0p ¼ A600 of the
culture containing the compound right after addition of the
inocula (day 0); Ac ¼ A600 of the culture in the absence of any
compound (control) at day 5; A0c ¼ A600 in the absence of the
compound at day 0. To determine IC50, the parasite growth was
followed in the absence (control) and presence of increasing
5. Biology
5.1. Culture of J774.A1 murine macrophages
These adherent-phenotype macrophage line was cultured in
Dulbecco's Modified Eagle's medium (DMEM, Sigma) supple-
mented with 10% FBS at 37 ꢂC with 95% humidity and 5% CO2.
concentrations of the corresponding compound (1e100 mM). The
IC50 value was taken as the concentration of compound needed
to reduce the absorbance ratio to 50%.

32
M.A. Alves et al. / European Journal of Medicinal Chemistry 100 (2015) 24e33
5.6. In vitro activity against promastigote forms of Leishmania
major
5.9. In vitro plasma stability assay (adapted from reference [32])
The in vitro plasma stability of compound 7g (LASSBio-1483)
was performed using a pool of rat plasma. Heparinized blood was
centrifuged at 2000 rpm for 15 min at 10 ꢂC to obtain plasma.
Plasma was diluted to 64% (v/v) with phosphate buffered saline
Stock solutions of novel semicarbazone derivatives as well as
pentamidine were prepared in DMSO immediately before use. The
cytotoxicity of novel semicarbazone derivatives and pentamidine
against promastigotes was determined [29,40,41]. Stationary phase
L. major promastigotes were plated in 96-well vessels (Nunc) at 105
cells per well, in Schneider's medium, supplemented with 10% FBS
and 2% human urine. Each derivatives solution was added at
(PBS, pH ¼ 7.4) at 37 ꢂC. The reaction was started by adding 1
m
m
L
L
(0.01 mM), of a stock solution 25 mM in DMSO, of sample to 249
of plasma. The plasma samples remained in the water bath at 37 ꢂC
under constant agitation at 0 and 240 min. After each reaction time
increasing concentrations (10ꢃ3 e 100
mM). Cells were also cultured
was added 500 mL of cold methanol, 500 mL of acetonitrile and then
in a medium free of compounds or vehicle (basal growth control) or
with DMSO 0.1% (vehicle control). After 48 h, extracellular load of
L. major promastigotes was estimated by counting the parasites in
Schneider's medium in a CELM automatic cell counter (model
CC530) [41]. Data obtained from experiments were expressed as
the mean S.E.M. and statistical differences between the treated
and the vehicle groups of experiments were evaluated by ANOVA
and Dunnett hoc tests. IC50 (concentration required to give 50%
death of cells) was calculated by linear regression analysis from the
Kc values at employed concentrations.
left microtube on ice for 10 min. After agitation, samples were
centrifuged at 13,000 rpm for 15 min at room temperature. The
supernatant was analyzed by HPLC-PDA (mobile phase: 60%
acetonitrile, 40% water), in the equipment Shimadzu LC-20AD, 100-
5 Kromasil C18 column (4.6 mm ꢁ 250 mm) detector SPD-M20A
(Diode Array) and performed to quantify the analyte in wave-
length 340 nm, flow rate 1 ml/min with 20 mL injection. Data were
acquired by LC solution software, version 4.0, using as standard
methyl biphenyl-4-carboxylate. Experiments were performed with
incubation of plasma with 0.5% DMSO without sample (blank) and
validating the approach used was performed using standard methyl
biphenyl-4-carboxylate using the same conditions described for
the analyzed sample (i.e. compound 7g).
5.7. In vitro activity against amastigote forms of Leishmania major
To assess the activity of the test derivatives against the amas-
tigote stage of L. major were realized model of infection in cover-
glass [42]. The murine macrophages (J774.A1 cell line) were
prepared in 24-well vessels (Corning) at 2 ꢁ 105 adherent cells/
well, infected with 2 ꢁ 106 promastigotes in glass coverslips placed
inside 1 ml medium culture. The cultures were cultured or not with
Acknowledgment
The authors would like to thank Leishmania collection of the
Oswaldo Cruz Institute for having provided L. major IOC/L0581
(MHOM/SU/1973/5-ASKH). The authors would like to thank CNPq
(BR), FAPERJ (BR), FAPEAL (BR) and INCT-INOFAR (BR, 573.564/
2008-6 and E-26/170.020/2008), PEDECIBA, CSIC-UdelaR (UR, 661)
for fellowship and financial support. This work was done under the
auspices of the Collaborative Network RIDIMEDCHAG-CYTED.
the test derivatives or reference drugs (10ꢃ3 -100
mM), and kept for
24 h at 37 ꢂC, 5% CO2. After 24 h, coverslips were washed, stained
with Giemsa-MayGrünwald, and intracellular amastigotes were
counted in 100 macrophages. Data obtained from in vitro experi-
ments were expressed as the Mean S.E.M. of duplicate cultures of
representative assays. Statistical differences between the treated
and the control groups were evaluated by ANOVA and Dunnett hoc
tests. Differences with a p value <0.05 or lower were considered
significant. IC50 (concentration required to give 50% death of cells)
was calculated by linear regression analysis from the Kc values at
employed concentrations.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.05.046.
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