In vitro and in vivo antiplasmodial activity of novel quinoline derivative compounds by molecular hybridization

Article abstract of DOI:10.1016/j.ejmech.2021.113271

Chloroquine (CQ) has been the main treatment for malaria in regions where there are no resistant strains. Molecular hybridization techniques have been used as a tool in the search for new drugs and was implemented in the present study in an attempt to pro

Full text of DOI:10.1016/j.ejmech.2021.113271

European Journal of Medicinal Chemistry 215 (2021) 113271
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
In vitro and in vivo antiplasmodial activity of novel quinoline
derivative compounds by molecular hybridization
,
1
,
*
b, 1
Juliane Aparecida Marinho a , Daniel Silqueira Martins Guimaraes
,
~
Nícolas Glanzmann c 1, Giovana de Almeida Pimentel c
,
,
, 1
Izabelle Karine da Costa Nunes d 1, Henrique Marcelo Gualberto Pereira d
,
,
,
, 1
Maribel Navarro c 1, Fernando de Pilla Varotti b 1, Adilson David da Silva c
,
,
, 1
,
1
Clarice Abramo a
a
^
ꢀ
ꢀ
Departamento de Parasitologia, Microbiologia e Imunologia, Instituto de Ciencias Biologicas, Universidade Federal de Juiz de Fora, Campus Universitario,
Juiz de Fora, Minas Gerais, CEP: 36036-900, Brazil
b
ꢀ
~
~
~
Núcleo de Pesquisa Em Química Biologica, Universidade Federal de Sao Joao Del Rei e Campus Centro Oeste, 400 Sebastiao Gonçalves Coelho Street,
ꢀ
Divinopolis, MG, 35501-296, Brazil
c
^
ꢀ
Departamento de Química, Instituto de Ciencias Exatas, Universidade Federal de Juiz de Fora, Campus Universitario, Juiz de Fora, Minas Gerais, CEP:
36036-900, Brazil
d
ꢀ
ꢀ
ꢀ
Laboratorio de Apoio Ao Desenvolvimento Tecnologico, LADETEC/IQ, Universidade Federal Do Rio de Janeiro, Av. Horacio Macedo, 1281 - Polo de Química,
ꢀ
~
Cidade Universitaria, Ilha Do Fundao, RJ, 21941-598, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Chloroquine (CQ) has been the main treatment for malaria in regions where there are no resistant
strains. Molecular hybridization techniques have been used as a tool in the search for new drugs and was
implemented in the present study in an attempt to produce compound candidates to treat malarial
infections by CQ-resistant strains. Two groups of molecules were produced from the 4-aminoquinoline
ring in conjugation to hydrazones (HQ) and imines (IQ). Physicochemical and pharmacokinetic prop-
erties were found to be favorable when analyzed in silico and cytotoxicity and antiplasmodial activity
were assayed in vitro and in vivo showing low cytotoxicity and selectiveness to the parasites. Candidates
IQ5 and IQ6 showed important values of parasite growth inhibition in vivo on the 5th day after infection
(IQ5 15 mg/kg ¼ 72.64% and IQ6 15 mg/kg ¼ 71.15% and 25 mg/kg ¼ 93.7%). IQ6 also showed interaction
with ferriprotoporphyrin IX similarly to CQ. The process of applying condensation reactions to yield
imines is promising and capable of producing molecules with antiplasmodial activity.
Received 27 November 2020
Received in revised form
31 January 2021
Accepted 1 February 2021
Available online 8 February 2021
Keywords:
Chloroquine derivatives
Malaria
Hydrazones
Imines
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
related to acute paroxysmal attacks, associated with intense fever
and weariness. P. falciparum is responsible for severe malaria,
Malaria is a worldwide tropical disease and the majority of
malaria cases are in the African continent. The most frequent spe-
cies are Plasmodium falciparum, P. vivax and P. ovale (Howes et al.,
2015). Other species are also related to human infection such as:
P. malariae and P. knowlesi [1]. The critical events of infection are
including cerebral malaria that can lead to death [2].
Malaria parasites metabolize the heme group of host hemo-
globin to survive. The free heme (Hydroxyferriprotorporphyrin IX-
Fe (III) PPIX) is oxidized to haematin, which is a toxic metabolite for
the parasite and red blood cells [3,4]. Free heme can also generate
* Corresponding author.
~
E-mail addresses: jumarinho28@yahoo.com.br, julianemarinhofarm@gmail.com (J.A. Marinho), danielmartinsguimaraes@gmail.com (D.S. Martins Guimaraes),
nicolasglanz@gmail.com (N. Glanzmann), gapimentel@gmail.com (G. de Almeida Pimentel), isabelle.nunes@iq.ufrj.br (I. Karine da Costa Nunes), henriquemarcelo@iq.ufrj.
br (H.M. Gualberto Pereira), maribel.navarro@ufjf.edu.br (M. Navarro), varotti@ufsj.edu.br (F. de Pilla Varotti), david.silva@ufjf.edu.br (A. David da Silva), clarice.abramo@
ufjf.edu.br (C. Abramo).
1
The authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113271
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

~
J.A. Marinho, D.S. Martins Guimaraes, N. Glanzmann et al.
European Journal of Medicinal Chemistry 215 (2021) 113271
List of abbreviations
IC50
IPM
kg
inhibitory concentration 50%
inhibition of parasite multiplication
kilogram
mM
micromolar
Abs
ACT
BSA
CC50
CQ
CQDP
DMSO
DNDi
d.p.i
ELISA
FBS
Absorbance
Log P
mg
mL
m.p.
MTT
partition coefficient
milligram
milliter
melting point
3-(4, 5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide
molecular weight
number of hydrogen acceptors
number of hydrogen donors
Phosphate buffered saline
pharmacokinetic/pharmacodynamic
standard deviation
Artemisinin-based Combination Therapy
bovine serum albumin
citotoxicity concentration 50%
chloroquine
chloroquine diphosphate
dimethyl sulfoxide
Drugs for Neglected Diseases initiative
days pos-infection
enzyme-linked immunosorbent assay
fetal bovine serum
MW
nON
nOHNH
PBS
PK/PD
SD
Fe(III)PPIX ferriprotoporphyrin IX
H
Hypochromism
SI
selectivity index
HEPES
HRPII
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
histidine-rich protein II
TPSA
UVevis
WHO
topological polar surface area
Ultravioletevisible
world health organization
HR-ESI-MS High Resolution Electrospray Ionization Mass
Spectrometry
free radicals that are potentially toxic to parasite cells [5]. Plasmo-
dium has a differentiated mechanism to reduce this toxicity,
through the crystallization of up to 95% of this toxic product, con-
verting it into hemozoin [6]. In an aqueous environment, together
with a lipid fraction [3], Fe (III) PPIX dimerizes and, as the final
product, forms the hemozoin dimer [7], which becomes innocuous
to the parasite, but toxic to the host. Chemical compounds that
interfere with the formation of this crystal, such as chloroquine, for
example, have been highly successful in combating plasmodium for
many years [8].
Due to the emergence of strains resistant to chloroquine treat-
ment, currently the use of Artemisinin-based Combination Therapy
(ACT) was recommended by WHO; however, there are already
cases of resistance to this therapy reported [9e11]. Efforts have
been made to contain the problem, studying new compounds with
antimalarial property [8,12].
From the discovery of quinine and the potent antimalarial ac-
tivity of its quinolinic nucleus, many researchers synthesized new
quinoline containing compounds that gave rise to new drugs, such
as chloroquine and mefloquine. The compounds derived from 8-
substituted quinolines such as primaquine and mefloquine are
still used, but none of them have the same properties and advan-
tages as those of chloroquine, such as low cost, low toxicity, long
half-life and high effectiveness [13]. Thus, in order to deal with the
chloroquine resistant strains, it became necessary to search for new
effective antimalarials, that also present low toxicity and low cost.
Molecular hybridization techniques have been widely reported to
generate new bioactive compounds. In these techniques, two or
more chemical groups with known pharmacological activity are
linked by means of chemical reactions. The purpose of this is to form
a new compound, which may exhibit the activity of each bioactive
group or even a synergistic activity [14]. Using molecular hybridi-
zation to combine quinolinic compounds containing hydrazine
groups and phenolic compounds containing hydroxyl, aldehyde and
carboxylic groups has previously exhibited antiparasitary activity,
more specifically in some species of leishmania [15].
hydrazones or imines.
2. Experimental section
2.1. General
All chemicals were purchased from Sigma-Aldrich. N-(7-
chloroquinolin-4-yl)-ethanediamine was prepared according to
literature methods [16]. For the determination of the melting
ranges of the synthesized compounds
a
MQAPF-301-
Microchemical digital apparatus was used. 1H and 13C Nuclear
Magnetic Resonance spectra were obtained on a BRUKER AVANCE
III 500 MHz spectrometer. Chemical shifts (d) are expressed as ppm
in relation to TMS. HRMS analysis were performed on a Q-Exactive
Plus Orbitrap high resolution mass spectrometer (ThermoFisher
Scientific, Bremen, Germany) using the electrospray ionization
method (ESI) operating in positive and negative mode through
direct injection analysis. UVevis spectra for the study of interaction
with Fe(III)PPIX were acquired in a Shimadzu UV-1800 spectro-
photometer using 1 cm quartz cuvettes.
2.1.1. Synthesis
The hydrazone products (Antinarelli et al., 2016) and compound
IQ3 [17] were synthesized according to the literature.
All imine products were synthesized through the same pro-
cedure as described to compound IQ1, varying only by the aromatic
aldehydes used as starting material. Hence, for compounds IQ2,
IQ4, IQ5 and IQ6, only the aldehyde used, NMR data and HRMS data
were presented. Completely assigned 1H and 13C spectra can be
found in the supplementary information file.
The purity of all chemical compounds was assessed through 1H
and 13C NMR spectroscopy, thin layer chromatography and melting
point prior to their use in the biological assays.
(E)-4-(((2-((7-chloroquinolin-4-yl)amino)ethyl)imino)
methyl)phenol (IQ1). 4-hydroxybenzaldehyde 0.146 g (1.2 mmol)
was dissolved in ethanol (5 mL). Subsequently, 0.221 g (1.0 mmol)
of N-(7-chloroquinolin-4-yl)-ethanediamine was solubilized in
ethanol (25 mL) in a beaker. This solution was poured into the re-
action flask at room temperature and moderate stirring. The color
of the solution quickly changed to yellow and then the reaction
As a result of what has been exposed, this work proposes to test
the antiplasmodial activity of new compounds elaborated using the
molecular hybridization technique, attaching the main pharmaco-
phore group, quinolinic ring, to aromatic groups through
2

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J.A. Marinho, D.S. Martins Guimaraes, N. Glanzmann et al.
European Journal of Medicinal Chemistry 215 (2021) 113271
mixture was monitored by thin layer chromatography (TLC) for
24 h. After this period, the formation of a precipitate in the solution
was observed. The solid formed was filtered on a buchner funnel
with a filter paper and washed several times with ethanol to
remove any starting material residue that could remain in the
product. Then, the solid was taken to the oven at 80 ꢀC for 2 h. The
product was obtained as yellow solid. (0.293 g, yield 90%). m.p.
can be administered orally. The software provides useful informa-
tion to understand the nature of the molecule and its pharmaco-
kinetic properties.
In the analysis by the Lipinski rule, the number of violations is
evaluated when considering the following conditions: the molec-
ular mass should be ꢁ 500 g/mol, log P ꢁ 5, number of hydrogen
donors ꢁ5, number of hydrogen acceptors ꢁ10 and the polar sur-
face area ꢁ140 Å2 [18].
265 ꢀC. 1H NMR (500 MHz, DMSO‑d6)
d
8.40 (d, 1H, J ¼ 5.5 Hz), 8.27
(d, 1H, J ¼ 9.0 Hz), 8.19 (s, 1H), 7.79 (d, 1H, J ¼ 2.0 Hz), 7.55 (d, 2H, J ¼
8.5 Hz), 7.43 (dd, 1H, J ¼ 2,0 Hz and 9.0 Hz), 6.82 (d, 2H, J ¼ 8.5 Hz),
6.59 (d, 1H, J ¼ 5.5 Hz), 3.81 (t, 4H, J ¼ 6.0 Hz). 13C NMR (126 MHz,
2.2. Biological experimental
DMSO‑d6),
d
162.1,160.3,151.9,149.0,134.0,130.2,127.7,127.5,124.6,
2.2.1. Assessment of the hemolytic activity of compounds
124.5, 117.8, 115.8, 99.4, 59.1, 43.8. HR-ESI-MS: m/z calculated for
C
The test of hemolytic capacity of the compounds was performed
as proposed by Ref. [19] with modifications. Blood was collected
from a healthy donor (approved by the Committee on Ethics in
Human Research, No. 2.160.565). To perform the test, a red cell
pellet was obtained, diluted with PBS (Phosphate-buffered saline)
to the 1% haematocrit and stored in micro tubes. The compounds
18H17ClN3O [MþH]þ 326.1055, found 326.10504.
(E)-5-(((2-((7-chloroquinolin-4-yl)amino)ethyl)imino)
methyl)benzene-1,3-diol
dihydroxybenzaldehyde (yield 82%). m.p. 270 ꢀC. 1H NMR
(500 MHz, DMSO‑d6)
(IQ2).
Aldehyde
used:
3,5-
d
8.40 (d, 1H, J ¼ 5,5 Hz), 8.24 (d, 1H, J ¼
9.0 Hz), 8.14 (s, 1H), 7.79 (d, 1H, J ¼ 2.0 Hz), 7.44 (dd, H-6, J ¼ 2.5 Hz
and 9.0 Hz), 6.59 (d, 3H, J ¼ 2.0 Hz), 6.29 (s, 1H), 3.82 (t, 2H, J ¼
6.5 Hz), 3.58 (t, 2H J ¼ 6.0 Hz). 13C NMR (126 MHz, DMSO‑d6)
diluted at different concentrations (4.68e150 mM) were added to
the micro tubes with the red blood cells solution. Chloroquine was
used as the standard drug in the same concentrations as the test
compounds. 1% saponin solution was used as a positive control and
a PBS solution was used as a negative control. The tubes were
incubated at 37 ꢀC for 16 h and then centrifuged (Thermo Scientific
Heraues Megafuge 16R) at 2000 rpm for 3 min. The absorbance data
of the obtained supernatant were measured in the spectropho-
tometer at 540 nm (Thermo Multiskan EX). The percentage of he-
d
162.9, 158.9, 152.3, 150.5, 149.4, 138.3, 133.9, 127.9, 124.6, 124.4,
117.9, 106.5, 105.4, 99.5, 59.0, 43.6. HR-ESI-MS: m/z calculated for
C
18H17ClN3O2 [MþH]þ 342.1004, found 342.10019.
(E)-4-(((2-((7-chloroquinolin-4-yl)amino)ethyl)imino)
methyl)benzaldehyde
(IQ4).
Aldehyde
used:
tereph-
thalaldehyde(yield 78%). m.p. 234 ꢀC. 1H NMR (500 MHz, DMSO‑d6)
d
8.39 (d, 1H, J ¼ 5.5 Hz), 8.36 (s, 1H), 8.22 (d, 1H, J ¼ 9.0 Hz), 8.80 (d,
molysis was calculated (100
m
L) using the formula:
%
1H, J ¼ 1.0 Hz), 7.82 (dd, 1H, J ¼ 7.5 Hz and 1.0 Hz), 7.76 (d, 2H, J ¼
5.5 Hz), 7.43 (m, 2H), 6.59 (d, 1H, J ¼ 5.5 Hz), 3.88 (t, 2H, J ¼ 6.0 Hz),
Hemolysis ¼ [(AT-AN)/(AP-AN)].100, where AT ¼ mean absorbance
of the test compound; AN ¼ mean absorbance of the negative
control (PBS); AP ¼ mean absorbance of the positive control
(saponin solution).
3.62 (t, 2H, J ¼ 6.5 Hz). 13C NMR (126 MHz, DMSO‑d6)
d 193.5, 162.2,
152.2, 150.5, 149.4, 133.9, 130.4, 130.2, 128.9, 128.6, 127.8, 124.6,
124.4, 117.8, 99.5, 59.3, 43.5. HR-ESI-MS: m/z calculated for
C
19H17ClN3O [MþH]þ 338.1055, found 338.10547.
2.2.2. Cytotoxicity test
(E)-4-(((2-((7-chloroquinolin-4-yl)amino)ethyl)imino)
Cytotoxicity was evaluated in human lung fibroblast cells WI 26
VA4. Cells were cultured in vented bottles with RPMI1640 medium
supplemented with 10% fetal bovine serum and 0.5% antibiotic
(penicillin and streptomycin). Cytotoxicity was assessed by the MTT
test (Sigma-Aldrich) [20]. Compounds were diluted (5% DMSO in
methyl)benzoic acid (IQ5). Aldehyde used: 4-formylbenzoic acid
(yield 82%). m.p. 305 ꢀC. 1H NMR (500 MHz, DMSO‑d6)
d
8.40 (d, 1H,
J ¼ 5.5 Hz), 8.27 (d, 1H, J ¼ 9.0 Hz), 8.19 (s, 1H), 7.79 (d, 1H, J ¼
2.0 Hz), 7.55 (d, 2H, J ¼ 8.5 Hz), 7.43 (dd, 1H, J ¼ 2.0 Hz and 9.0 Hz),
682 (d, 2H, J ¼ 8.5 Hz), 6.59 (d,1H, J ¼ 5.5 Hz), 3.80 (t, 4H, J ¼ 6.0 Hz).
culture medium) at various concentrations (0.019e337 mM). Chlo-
13C NMR (126 MHz, DMSO‑d6)
d
167.7, 162.2, 151.9, 150.8, 149.0,
roquine, at the same concentrations as the other compounds, was
used for comparison purposes. The cells were plated in 96 wells
(106 cells per well) along with the compounds and incubated for
48 h under the same conditions of temperature and humidity. The
final DMSO concentration in the plates did not exceed 0.05%.
The absorbance results were calculated as percentage of alive
cells in relation to the positive control. Growth inhibition was
calculated by the formula: % Inhibition ¼ 1- (Absorbance of
Duplicate Mean x 100)/Absorbance of Negative Control). With the
values of the applied concentrations and their percentages of in-
hibition, log dose response curves were performed in the Origin 8.0
program to calculate the value of CC50 (toxic concentration to 50%
of cells). The CC50 mean of 2 or more experiments plus the standard
deviation was performed.
139.7, 134.1, 133.9, 130.0, 128.3, 124.7, 124.5, 117.7, 99.5, 59.3, 43.5.
HR-ESI-MS: m/z calculated for C19H17ClN3O2 [MþH]þ 354.1004,
found 354.09985.
(E)-4-(((2-((7-chloroquinolin-4-yl)amino)ethyl)imino)
methyl)benzene-1,3-diol
dihydroxybenzaldehyde (yield 84%). m.p. 236 ꢀC. 1H NMR
(500 MHz, DMSO‑d6)
(IQ6).
Aldehyde
used:
2,4-
d
8.41 (d, 1H, J ¼ 4.5 Hz), 8.33 (s, 1H), 8.25 (d,
1H, J ¼ 9.0 Hz), 7.79 (s, 1H), 7.46 (dd, 1H, J ¼ 1.5 Hz and 8.5 Hz), 7.13
(d, 2H, J ¼ 8.5 Hz). 6.60 (d, 1H, J ¼ 5.0 Hz), 6.24 (dd, 2H, J ¼ 2.0 Hz ad
8.5 Hz), 6.16 (d, 1H, J ¼ 1.5 Hz), 3.80 (t, 3H J ¼ 6.0), 3.59 (t, 2H, J ¼
5.5). 13C NMR (126 MHz, DMSO‑d6),
d 166.3, 165.2, 162.2, 152.2,
150.5, 149.3, 134.0, 133.8,127.8, 124.6, 124.4, 117.9, 111.7, 107.2, 103.0,
99.4, 55.6, 43.6. HR-ESI-MS: m/z calculated for C18H17ClN3O2
[MþH]þ 342.1004, found 342.10013.
2.2.3. Evaluation of in vitro antiplasmodial activity
2.1.2. Analysis of physico-chemical properties
The continuous culture of P. falciparum clone W2 (chloroquine
resistant) was maintained, according to the protocol of [21] with
Physico-chemical properties were analyzed in silico in relation
to Lipinski’s rule of 5. The Molsinspiration program was used, a free
platform available on the Internet through the site: http://www.
molinspiration.com/. For each compound, the software returned
values of molecular mass, partition coefficient (log P), TPSA (topo-
logical polar surface area), number of hydrogen bond acceptors
(N þ O) and number of hydrogen bond donors (NH þ OH), which
are the parameters used to predict whether the studied molecule
modifications, in RPMI 1640 medium supplemented with HEPES,
D-
glucose, -glutamine, hypoxanthine, sodium bicarbonate, genta-
L
mycin, inactivated human A þ serum, and human A or O red blood
cells with 5% hematocrit. The culture was maintained in plaque at
37 ꢀC in an environment with the appropriate atmosphere of ox-
ygen obtained by the combustion of a candle.
Culture of P. falciparum was adjusted to the hematocrit of 1.5%
3

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J.A. Marinho, D.S. Martins Guimaraes, N. Glanzmann et al.
European Journal of Medicinal Chemistry 215 (2021) 113271
and parasitemia of 0.05%. A volume of 180
m
L of the culture was
2.3. Statistical analysis
added to the wells, along with 20 mL of the compounds already
diluted in the plate (KASVI). To evaluate the antimalarial activity of
the compounds, the parasite histidine rich protein II (HRPII) was
measured through ELISA assay [22]. For the ELISA assay, a 96-well
Statistical analysis was performed using GraphPad Prism 5
(GraphPad InC, USA). The variation between the means of hemo-
lysis percentages followed by the Tukey test. For the data obtained
in the cytotoxicity and antimalarial activity experiments in vitro,
the average of two or three experiments in triplicate was calculated
and the standard deviation was calculated. Analysis of variance
(ANOVA) was performed, with Tukey’s post-test, to compare
cytotoxicity data (CC50) and in vitro activity (IC50). In vivo results
were verified by Student’s t-test, followed by Mann Whitney test.
Values of p < 0.05 were considered significant.
plate (KASVI) was sensitized with 100 mL of solution with the pri-
mary anti-HRPII monoclonal antibody (MPFM-55A, ICLLAB®). The
plate was incubated at 4 ꢀC accompanied by a blocking solution
(PBS/BSA2%). Plates were then washed with 0.05% PBS-Tween 20. In
the next step, 100 mL of the product from each well of the test plate
(culture with the compounds) was added and blocked. This was
then incubated for 1 h, and then washed.
The secondary antibody at 0.05
added and the incubation was performed for 1 h, was washed and,
then, 100 L of the TMB solution (Tetrametylbenzidine) was added
mg/mL (MPFG559, ICCLAB®) was
3. Results and discussion
m
in each well. The plate remained at rest approximately for 10 min
and the reaction was stopped by the addition of 1 M sulfuric acid.
The test was read in a spectrophotometer (Biotek Power Wave XS2).
The viability of the parasite in relation to the control was calculated
according to the following formula: % viability ¼ 100 x [(AC - AW -
AB)/(A72- AB)], where AC ¼ mean absorbance of compound,
AW ¼ mean white absorbance, AB ¼ mean background absorbance,
A72 ¼ mean control absorbance of 72 h.
All the 7-chloro-4-quinolinylhydrazone derivatives HQ1 to HQ5
were synthesized by our research group (Scheme 1). 7-chloro-4-
hydrazinequinoline was synthesized by reaction between 4,7-
dichloroquinoline and hydrazine hydrate (80%) in ethanol under
reflux. The derivatives were obtained through the reaction of 7-
chloro-4-hydrazinequinoline with different aromatic aldehydes
using ethanol as solvent as described in our previous work [27,28].
The structure of these compounds was characterized by 1H and 13
C
The viability values were analyzed in the Origin 8.0 program, for
the calculation of IC50 values and the standard deviation.
NMR and melting point and are in agreement with the literature
[15,29].
The N-(7-chloroquinolin-4-yl)-ethanediamine was synthesized
through the reaction between 4,7-dichloroquinoline and ethyl-
enediamine as described by Refs. [30,31]. All the imines IQ1 to IQ6
were synthesized through the reaction between N-(7-
chloroquinolin-4-yl)-ethanediamine and different aromatic alde-
hydes using ethanol as the solvent (Scheme 2). Except for derivative
IQ3 [32] all other compounds are unpublished and have been
characterized by 1H and 13C NMR, HRMS and melting point.
Some physicochemical properties of the compounds were
evaluated in silico, using the Molispiration software, to verify if they
meet the requirements to comply with the Lipinski rule. According
to the data obtained in the analysis (Table 1), only HQ2, HQ3 and
HQ5 have minimally exceeded the partition coefficient values. All
too often, this has not been found to be a severe violation, so they
may still be good candidates for oral administration and have good
bioavailability when evaluated in vivo.
2.2.4. Evaluation of the antimalarial activity in vivo
Female Swiss mice from the Center of Reproduction Biology
(UFJF) were used in the in vivo experiments, after approval of the
ethics committee on animal research (UFJF, numbers 042/2016 and
043/2016). The suppressive test with modifications [23] was per-
formed. The treatments were performed by gavage and the com-
pounds were solubilized (5% DMSO) and diluted in water at 15 or
25 mg/kg. Blood smears were stained by Giemsa and inhibition of
parasite multiplication was calculated by the formula: [(A- B)/A] x
100, where A ¼ parasitemia of the group treated with water þ 5%
DMSO, and B ¼ parasitemia of the group treated with the com-
pounds [24,25].
2.2.5. Interaction with ferriprotoporphyrin (Fe(III)PPIX)
The association constant of IQ6 with ferriprotoporphyrin IX
(Fe(III)PPIX) was measured as described previously [26]. A stock
solution of hemin was prepared by dissolving 3.5 mg of hemin in
10 mL DMSO. The solution of Fe(III)PPIX (pH ¼ 7.5) in aqueous-
The cytotoxicity assays in red blood cells (Fig. 1A and B) showed
that no compound even at the highest concentration tested
(150
mM) caused more than 10% hemolysis, which shows these
DMSO (40% v/v) was prepared with 140
mL hemin stock solution,
compounds are not toxic [33]. Among the imines, a slight per-
centage of hemolysis was observed for compound IQ3, when
compared to the others in the same group.
However, when compared to the hemolysis value of the positive
control, the value of IQ3 was much lower. The data suggest that the
studied compounds do not provoke hemolysis and that any
observed antiplasmodial activity may be directly related to the
pharmacological performance of the compound and not due to lysis
of the erythrocytes in a non-specific manner.
3.86 mL DMSO, 1 mL of 0.2 M Tris buffer ((Tris(hydroxymethyl)
aminomethane), pH 7.5) and distilled deionized water to a total
volume of 10 mL. A 0.3 mM solution of IQ6 was prepared using the
same water:buffer:DMSO ratio. The blank solution was also pre-
pared using the same water:buffer:DMSO ratio but no hemin or
compound were added. The titration was performed adding 20
aliquots of the IQ6 solution to 2 mL of the Fe(III)PPIX solution and
measuring the UVevis spectra after each addition. Aliquots of 20
mL
mL
of IQ6 were also added to the blank solution at each step, in order to
subtract the absorbance of the compound. The absorbance readings
were recorded at 402 nm (Soret band) in the presence and absence
of the IQ6. The data were fitted to the equation A¼(A0þA∞K[C])/
(1þK[C]) for a 1:1 complexation model using nonlinear least
squares fitting, The same procedure used for IQ6 was used to
calculate the association constant for chloroquine diphosphate
(CQDP) with Fe(III)PPIX. A0 is the absorbance of hemin before
addition of IQ6 or CQDP, A∞ is the absorbance for the drugehemin
adduct at saturation, A is the absorbance at each point of the
titration, and K is the association constant. The experiment mea-
surements were made in triplicate.
To assess the CC50 values, the compounds were tested against
human lung fibroblasts, WI26VA-4 cells. All compounds were
active in vitro against P. falciparum. The compounds under study
were active at concentrations lower than 1
presented in Table 2. However, no compound tested achieved the
IC50 value of chloroquine (0.053 M). Even so, the reported in vitro
mM. These results are
m
antiplasmodial activity reaches a recently established set of criteria
for the development of compounds with activity against malaria
[34]. These criteria include: (i) knowledge of the structureeactivity;
(ii) an in vitro potency around 1e2 mM; (iii) SI greater than 10-fold
against a human cell line. All compounds tested in this work met
these criteria.
4

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J.A. Marinho, D.S. Martins Guimaraes, N. Glanzmann et al.
European Journal of Medicinal Chemistry 215 (2021) 113271
Scheme 1. Synthetic pathway for hydrazones.
Scheme 2. Synthetic pathway for imines.
Table 1
Among the hydrazones, the most selective compound was HQ5.
As for the imines, compound IQ6 obtained the best SI (989.62),
which demonstrates that it is a highly selective compound. The
compounds IQ1, IQ2, IQ3 and IQ4, also stood out for their high SI on
chloroquine-resistant P. falciparum.
In this work, the compounds were divided in two main groups:
imines and hydrazones. Comparing these two groups, the main
difference is the linking group between the quinolinic ring and the
phenolic ring. For the imine derivatives a 1,2-ethanediamine group
is present between the aromatic groups, providing a bigger dis-
tance between these groups and no conjugation. On the other side,
for the hydrazone derivatives, a hydrazine group is present instead
In silico evaluation of physicochemical properties of molecules.
Compound code
Log P
MW
nON
nOHNH
Violations
HQ1
HQ2
HQ3
HQ4
HQ5
IQ1
IQ2
IQ3
IQ4
IQ5
4,99
5,09
5,17
4,62
5,56
3,67
3,12
4,09
3,94
4,06
3,58
327,77
313,74
297,75
313,74
325,75
325,80
341,80
325,80
337,81
353,81
341,80
5
5
4
5
5
4
5
4
4
5
5
2
3
2
3
2
2
3
2
1
2
3
0
1
1
0
1
0
0
0
0
0
0
IQ6
Fig. 1. In vitro hemolysis after treatment with group A (hydrazones), and group B (imines). The mean absorbance of the compounds tested at their highest concentration were
expressed as mean standard deviation. ANOVA was used followed by the Tukey test to compare the means. The means with different letters are statistically different from each
other (p < 0.05). Compound IQ4 has not been tested.
5

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J.A. Marinho, D.S. Martins Guimaraes, N. Glanzmann et al.
European Journal of Medicinal Chemistry 215 (2021) 113271
Table 2
Evaluation of the cytotoxicity of the quinoline derivatives (CC50) on WI 26VA-4 cells and in vitro activity (IC50) and selectivity index (SI).
Compounds
WI26VA-4 cells CC50
(m
M)
P. falciparum W2 IC50
(
mM)
Selectivity index (SI)
HQ1
HQ2
HQ3
HQ4
HQ5
IQ1
IQ2
IQ3
IQ4
IQ5
10.77 1.58 **
7.27 0.544 **
2.42 0.48 **
12.89 0.261 **
82.04 12.87 **
130.07 55.91 **
79.28 3.72 **
25.33 10.22 **
137.79 46.88 **
>28.26 **
0.235 0.007 *
0.235 0.021 *
0.215 0.064 *
0.625 0.007 *
0.335 0.289 *
0.162 0.06 *
0.205 0.106 *
0.26 0.014 *
0.160 0.01 *
0.145 0.021 *
0.172 0.003 *
0.053 0.016 *
45.82
30.93
11.25
20.62
24.89
802.46
386.73
97.42
861.18
>194.89 ***
989.62
>365.66 ***
IQ6
Chloroquine
170.7 14.14 **
>19.38 **
CC50 and IC50 values were expressed as mean standard deviation. Indeterminate values of CC50 and IC50 (represented by ***) were not statistically evaluated. The * and **
were used for comparative statistics between the CC50 or IC50 values with the respective controls for each type of assay. SI: selectivity index ¼ CC50 WI26VA-4/IC50 P. falciparum
W2.
of the 1,2-ethanediamine group and this provides a smaller dis-
tance between the aromatic groups and allows conjugation be-
tween the quinolinic and phenolic rings. Our results showed higher
SI values for the imine derivatives than for the hydrazone de-
rivatives, demonstrating that the imine containing compounds,
in vitro, can be more selective to parasite and less harmful to fi-
broblasts tested. This is a result of the higher cytotoxicity presented
by the hydrazone derivatives and slightly lower antiplasmodial
activity values.
Inside each group, the substituents in the phenolic ring were
varied in order to include electron donating or electron with-
drawing groups in different positions. No linear relation between
the activity and the electron donating/withdrawing profile was
observed for the compounds. In fact, for the Hydrazone derivatives
a higher antiplasmodial activity was observed for the compounds
containing electron donating groups, while for the imine de-
rivatives a higher antiplasmodial activity was observed for the
compounds containing electron withdrawing groups.
It is also interesting to compare compounds IQ1 and IQ3, which
differ only in the position of a hydroxyl group but present very
different SI values. This could be related to hydrogen bond forma-
tion that is possible between the hydrogen from the hydroxyl group
and the nitrogen from the imine group of compound IQ3, which is
impossible for IQ1, indicating that the free hydroxyl group could be
important to the selectivity once compound IQ1 showed higher SI
value than IQ3. Indeed, compound IQ6, which presented the higher
SI of the series, contains a free hydroxyl group in the same position
as IQ1.
presented a result of inhibition of parasite multiplication compa-
rable to chloroquine at 10 mg/kg.
Global antimalarial discovery projects, like Drugs for Neglected
Diseases initiative (DNDi), suggest that a new potential molecule
should have a high in vivo efficacy (90% in parasite reduction), with
an oral and single dose [35]. Nevertheless, parasite reduction is just
one criterion adopted during the development of a new antima-
larial. In this process, it is necessary to observe other parameters
like: i) low-cost synthesis, ii) good oral bioavailability, iii) drug
metabolism, and iv) good membrane permeability. These proper-
ties, combined with parasite reduction, can lead a potential com-
pound to final phases of clinical trials [36].
Additionally, compound IQ6 was chosen to be further studied
regarding heme as a possible target. It is known that malaria par-
asites convert heme (Fe(III)PPIX) into the insoluble and inert
hemozoin crystals in its digestive vacuole. This vital process for the
parasite survival stands out as a selective target to be attacked by
antimalarial drugs, in fact, this is the most accepted mechanism of
action of chloroquine [8,37].
Based on this, the interaction of IQ6 with ferriprotoporphyrin IX
was studied by UVevis spectrophotometric titration at the Soret
band of porphyrin (402 nm) in order to determine the association
constant (log K) of this compound with Fe(III)PPIX (Fig. 2). Fig. 2
shows the perturbation of the band at 402 nm, when the concen-
tration of compound IQ6 was increased to saturation, causing a
reduction in the absorption intensity (hypochromism) of 50.4%.
Following the procedure described in the experimental section and
data fitting for
a 1:1 association model, the value of log
Two promising compounds were chosen for in vivo testing, IQ5
was tested in vivo at 15 mg/kg, and compound IQ6 at 15 and 25 mg/
kg (Table 3). These compounds had important antiplasmodial ac-
tivity observed on the 5th d.p.i and reduced activity on the 9th dpi.
The molecules were active against P. berghei NK65, with inhibition
values higher than 70% on the 5th day after infection. A reduction in
activity was observed on the 9th dpi. At 25 mg/kg, compound IQ6
K ¼ 4.77 0.01 was determined for compound IQ6. Using the same
procedure, the value of log K ¼ 4.97 0.04 was obtained for CQDP,
which is in good agreement with the values reported in the liter-
ature for CQDP [26,38]. These results indicate that IQ6 interacts
with Fe(III)PPIX similarly to CQDP under these conditions, which
would suggest that our compound may have the ability to inhibit
the hemozoin formation, and therefore could cause the parasite’s
death. Due to the structural similarities with the remaining com-
pounds studied in this work, it can also be suggested that they have
potential to present a similar interaction with Fe(III)PPIX.
With regard to drug resistance, the occurrence of cross-
resistance in 4-aminoquinolines is not observed in all strains. For
example, P. falciparum strains may be resistant to chloroquine, but
are sensitive to amodiaquine or other related compounds [39].
Some structural modifications in the quinolinic nucleus can
maintain the activity without causing resistance, as has been
observed in some studies [40e42].
Table 3
Evaluation of in vivo suppressive antimalarial activity of compounds IQ5 and IQ6:
In vivo antimalarial evaluation by the modified Peters suppressive test. The value of
inhibition of parasite multiplication was calculated based on the mean values of
parasitemias, considering as 100% of parasitemia the values obtained in the un-
treated group. Compounds with IPM values above 30% were considered active [24]].
d.p.i
CQ
ART 5 mg/kg
MF 10 mg/kg
IQ5
IQ6
IQ6
10 mg/kg
15 mg/kg
15 mg/kg
25 mg/kg
5
9
94.04%
41.27%
94.90%
96.64%
72.64%
21.67%
71.15%
40.13%
93.70%
41.01%
Many strategies have been tested in order to enable novel
aminoquinoline derivatives to have good antimalarial activity, even
6

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J.A. Marinho, D.S. Martins Guimaraes, N. Glanzmann et al.
European Journal of Medicinal Chemistry 215 (2021) 113271
Fig. 2. Spectroscopic titration of Fe(III)PPIX with IQ6. Data from experiment 1. Data from experiments 2 and 3 can be found in the supplementary material. Abs. ¼ Absorbance.
H ¼ hypochromism.
against resistant parasites. For example, the use of substituents that
prolong the half-life in the host, such as the quaternary carbons,
which help reduce their biodegradation [39].
compounds had in vivo activity on the 5th dpi which was reduced
on the 9th dpi. Compound IQ6 showed interaction with Fe(III)PPIX
inhibiting the formation of hemozoin, which could be associated
with the mechanism of action.
Some medicinal chemistry studies have been reported using
techniques that allow modification in the side chain of the 7-
chloro-4-aminoquinoline group and molecular hybridization in
order to maintain antimalarial activity [30]. The application of this
technique, connecting two bioactive pharmacophoric groups, im-
proves the efficacy of these molecules, as has been observed in
many studies of new agents for cancer chemotherapy [14]; Kerru
et al., 2017).
Based on this knowledge, our research group has used this
concept to synthesize hybrid molecules. In this study, an initial
screening with two chemical groups was carried out, referred to
here as groups HQ and IQ (hydrazone ligands and imine ligands),
already tested in Leishmania [15]. The compounds of this study
were neither hemolytic or toxic (WI26VA-4 human fibroblasts)
presenting SI higher than 10.
4. Conclusions
In conclusion, we synthesized a set of quinoline derivative
compounds by molecular hybridization.
Compounds IQ5 and IQ6 were the most active in vitro, with high
SI values. Compound IQ6 presented the highest inhibition against
P. berghei in vivo development. We also determined that compound
IQ6 interacts with Fe(III)PPIX forming a 1:1 complex, therefore
inhibiting the hemozoin formation, which could be related to the
mechanism of action. Thus, compound IQ6 presents itself as a
promising molecule for optimization as a new antimalarial drug.
Ongoing studies are now focused on establishing the PK/PD profile
of compound IQ6.
Considering the data of in vitro activity, all compounds of both
HQ and IQ groups had high levels of selectivity, higher than 10.
According to Ref. [34]; compounds showing IC50 values below 1 mM
Declaration of competing interest
to P.falciparum resistant strains, and with 10-fold selectivity over
the CC50 value in a mammalian cell, can be considered "hit com-
pounds”. Other features related to a compound that could be
considered as a hit would be: identified chemical structure, high
purity, in vitro activity, preliminary knowledge on its structure ac-
tivity relation, compliance with the rule of 5, and synthesis that
should take a maximum of 5 steps [34]. All compounds of this study
met these criteria.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgement
Molecules containing hydrazone groups have already been
analyzed in other studies that have also observed antimalarial ac-
tivity in vitro [43,44], corroborating the fact that hybridization with
the hydrazone group maintains the antimalarial activity of the
molecule.
~
ꢁ
This research was funded by FAPEMIG (Fundaçao de Amparo a
Pesquisa do Estado de Minas Gerais, Project n. APQ-02065-14) and
~
CAPES (Coordenaçao de Aperfeiçoamento de Ensino Superior) and
was supported by UFJF (Universidade Federal de Juiz de Fora).
In silico studies have helped to predict the behavior of com-
pounds that have not yet undergone in vivo testing. Most com-
pounds did not violate any rules and a minority of the compounds
presented values very close to the maximum required.
Regarding the antimalarial evaluation, it was seen that the
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113271.
7

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J.A. Marinho, D.S. Martins Guimaraes, N. Glanzmann et al.
European Journal of Medicinal Chemistry 215 (2021) 113271
[22] H. Noedl, C. Wongsrichanalai, R.S. Miller, K.S.A. Myint, S. Looareesuwan,
Y. Sukthana, V. Wongchotigul, H. Kollaritsch, G. Wiedermann,
W.H. Wernsdorfer, Plasmodium falciparum: effect of anti-malarial drugs on
the production and secretion characteristics of histidine-rich protein II, Exp.
Parasitol. 102 (3e4) (2002) 157e163, https://doi.org/10.1016/s0014-4894(03)
00051-1.
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