In silico evaluation and in vitro growth inhibition of Plasmodium falciparum by natural amides and synthetic analogs

Article abstract of DOI:10.1007/s00436-020-06681-9

Malaria, caused by protozoa of the genus Plasmodium, is a disease that infects hundreds of millions of people annually, causing an enormous social burden in many developing countries. Since current antimalarial drugs are starting to face resistance by the

Full text of DOI:10.1007/s00436-020-06681-9

Parasitology Research
https://doi.org/10.1007/s00436-020-06681-9
PROTOZOOLOGY - ORIGINAL PAPER
In silico evaluation and in vitro growth inhibition of Plasmodium
falciparum by natural amides and synthetic analogs
Minelly Azevedo da Silva1,2,3 & Márcia Paranho Veloso4,5 & Kassius de Souza Reis4,5
&
Guilherme de Matos Passarini1,3,6 & Ana Paula de Azevedo dos Santos1,3,6 & Leandro do Nascimento Martinez1,3,6
Harold Hilarion Fokoue7 & Massuo Jorge Kato7 & Carolina Bioni Garcia Teles1,3,6,8 & Christian Collins Kuehn1,3
&
Received: 23 January 2019 /Accepted: 26 March 2020
#
Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
Malaria, caused by protozoa of the genus Plasmodium, is a disease that infects hundreds of millions of people annually, causing
an enormous social burden in many developing countries. Since current antimalarial drugs are starting to face resistance by the
parasite, the development of new therapeutic options has been prompted. The enzyme Plasmodium falciparum enoyl-ACP
reductase (PfENR) has a determinant role in the fatty acid biosynthesis of this parasite and is absent in humans, making it an
ideal target for new antimalarial drugs. In this sense, the present study aimed at evaluating the in silico binding affinity of natural
and synthetic amides through molecular docking, in addition to their in vitro activity against P. falciparum by means of the SYBR
Green Fluorescence Assay. The in vitro results revealed that the natural amide piplartine (1a) presented partial antiplasmodial
activity (20.54 μM), whereas its synthetic derivatives (1m—IC50 104.45 μM), (1b, 1g, 1k, and 14f) and the natural amide
piperine (18a) were shown to be inactive (IC50 > 200 μM). The in silico physicochemical analyses demonstrated that compounds
1m and 14f violated the Lipinski's rule of five. The in silico analyses showed that 14f presented the best binding affinity (−
13.047 kcal/mol) to PfENR and was also superior to the reference inhibitor triclosan (− 7.806 kcal/mol). In conclusion, we found
that the structural modifications in 1a caused a significant decrease in antiplasmodial activity. Therefore, new modifications are
encouraged in order to improve the activity observed.
.
.
Keywords Malaria Fatty acids Plasmodium falciparum
.
Section Editor: Tobili Sam-Yellowe
enoyl-ACP reductase Piperidine alkaloids
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00436-020-06681-9) contains supplementary
material, which is available to authorized users.
Introduction
* Minelly Azevedo da Silva
minelly.silva@ifro.edu.br
Malaria is a disease that constitutes a serious public health
problem worldwide, with an estimated 219 million cases in
2017 (WHO 2018). The disease is caused by protozoa of
the genus Plasmodium, of which five species are known to
cause disease in humans (Cox 2010; Amir et al. 2018).
Plasmodium falciparum, alongside Plasmodium vivax, are
the species that cause most of the cases and deaths annu-
ally, making them the most relevant species both clinically
and epidemiologically (Hay et al. 2010). These species can
cause severe form of malaria in some cases (Mackintosh
et al. 2004; Alexandre et al. 2010). For P. falciparum, this
severe form may cause serious organ damage, resulting in
respiratory and neurological problems, in addition to fetal
loss in pregnant women (Mackintosh et al. 2004). For
P. vivax, common clinical manifestations are
1
Programa de Pós graduação em Biologia Experimental, PGBIOEXP,
Porto Velho, Brazil
2
Instituto Federal de Rondônia, IFRO, Porto Velho / Rondônia, Brazil
3
Universidade Federal de Rondônia –UNIR, Porto Velho, Brazil
4
Laboratório de Avaliação e de Síntese de Substâncias Bioativas,
Alfenas - MG, Brazil
5
Universidade Federal de Alfenas, Alfenas - MG, Brazil
6
Fundação Oswaldo Cruz, Rondônia (FIOCRUZ –RO) / EpiAmO,
Porto Velho, Brazil
7
Instituto de Química da Universidade de São Paulo, IQ-USP, São
Paulo, Brazil
8
Centro Universitário São Lucas, UniSL, Porto Velho, Brazil

Parasitol Res
thrombocytopenia, and renal, hepatic, cerebral, and pulmo-
nary involvement (Zubairi et al. 2013).
extracted with a mixture of methanol/chloroform 1:2 (1 L)
for 72 h. The mixture was rotary evaporated under reduced
pressure to yield a white solid which, after recrystallization in
MeOH, produced pure piplartine (1a) (PubChem CID:
637858), which was identified by comparison of established
NMR and MS data with reported data (Araújo-Vilges et al.
2017).
Fruits of Piper nigrum Koehler, 1887 were purchased from
a local market in São Paulo. Piperine (18a) was purified from
250 g of dried P. nigrum fruits. The fruits were ground into a
powder and extracted twice with 2 L of methanol/chloroform
(1:2). The extract was filtered and concentrated in a rotary
vacuum evaporator. The crude extract was subjected to silica
column chromatography eluted with hexanes/ethyl acetate
mixtures of increasing polarity. Fractions containing piperine
(18a) were pooled and recrystallized in methanol yielding a
yellow crystal. Piperine (18a) (PubChem CID 638024) was
identified by NMR and MS analyses and comparison with
reported data (Parmar et al. 1997; Araújo-Júnior et al. 1997).
The chemotherapy currently used in the treatment of ma-
laria, which is based on artemisinin combination therapy
(ACT), has started to face resistance by the parasite in some
regions of the globe, especially in Southeast Asia (Ashley
et al. 2014), which prompts the development of drugs
targeting specific pathways of the parasite.
In this sense, dissociative or type II fatty acid synthase,
named FAS II, constitutes a promising molecular target for
potential antimalarial drug candidates, since it is absent in
humans (Qidwai and Khan 2012). Plasmodium falciparum
enoyl-ACP reductase (PfENR), which belongs to this path-
way, is involved in the final step of fatty acid chain elongation,
catalyzing the reduction of enoyl-ACP to acyl-ACP using
NADH as an electron donor, and is also the rate-limiting en-
zyme in this cycle (Tasdemir 2006).
Regarding potential compounds for antiparasitic agents,
natural products derived from plant secondary metabolism
possess a plethora of potential antiparasitic molecules (Wink
2012). With respect to antimalarial therapy, the alkaloid qui-
nine originated a series of analogs of great relevance for the
treatment of disease (Krafts et al. 2012). In this context, plants
that are rich in alkaloids—which encompasses the species of
the genus Piper (Gutiérrez et al. 2013)—constitute a great
potential to develop new compounds against the disease.
Piplartine and piperidine, two alkaloids isolated from Piper
species, have been shown to present various biological activ-
ities. Piplartine presents activity against various parasitic spe-
cies (Leishmania amazonensis Lainson & Shaw, 1972;
Leishmania infantum Cunha & Chagas, 1937; Leishmania
donovani Laveran & Mesnil, 1902 and P. falciparum), espe-
cially Trypanosoma cruzi Chagas, 1909 (Araújo-Vilges et al.
2017; Bodiwala et al. 2007; Moreira et al. 2018; Cotinguiba
et al. 2009; Gomes et al. 2014; Silva-Jardim et al. 2004). In
this light, this study aimed at evaluating the in silico binding
affinity of two natural amides and five synthetic analogs with
PfENR, their in silico physicochemical properties, and their
in vitro antiplasmodial activity.
Synthesis of the synthetic analogs
Compound 1b (PubChem CID: 53440296) was obtained by
the catalytic hydrogenation (4 atm of hydrogen, Pd–C) of
piplartine (1a) for 4 h and was identified by comparison of
NMR and MS analyses with reported data (Fokoue et al.
2018).
Compounds 1g and 1k were synthesized by adding
triethylamine (3 equivalents) and the appropriate amines
(i.e., pyrrolidine and n-pentylamine) to a methylene chloride
solution of various acyl chlorides (1.0 equivalent). The reac-
tion mixtures were stirred overnight at room temperature,
quenched with a saturated aqueous ammonium chloride solu-
tion and then extracted with methylene chloride 3 times.
Combined organic phases were washed with brine and dried
over anhydrous magnesium sulfate. After filtration and con-
centration, the residues were purified by flash chromatogra-
phy over a silica gel using hexanes-ethyl acetate (typically 30–
50%) as an eluent, yielding the desired amides. Compounds
1g (PubChem CID: 121169) and 1k (PubChem 19173974)
were identified by comparison of NMR and MS analyses with
reported data (Campelo et al. 2018; Fokoue et al. 2018).
Compounds 1m and 14f were synthesized by adding 2E-
3 , 4 , 5 - t r i m e t h o x y c i n n a m i c a c i d a n d 2 E - 3 , 4 -
methylenedioxycinnamic acid (1 equivalent) to a solution of
THF 0.9 equivalent of N,N′-dicyclohexylcarbodiimide
(DCC). The reaction mixture was stirred overnight at room
temperature and dried on a rotary evaporator to remove any
remaining solvent. The product was dissolved in dichloro-
methane (DCM), a saturated solution of NaHCO3 was added,
and the resulting solution was washed three times with DCM.
The organic phase was combined, dried with MgSO4, filtered,
and concentrated under pressure. The obtained crystalline
Materials and methods
All the compounds were obtained using the following
methodology
Isolation of the natural compounds (piplartine and piperine)
Roots of Piper tuberculatum Jacq, 1897 were harvested on the
University of São Paulo (USP) Campus, São Paulo, Brazil. A
voucher specimen (Kato-0169) has been deposited at the
Institute of Biosciences Herbarium. For the isolation of
piplartine, 100 g of ground roots of P. tuberculatum were

Parasitol Res
product was then recrystallized on hot methanol or purified by
chromatography over silica-gel using hexanes-ethyl acetate
(typically 30–50%). Compound 1m was identified by com-
parison of NMR and MS analyses with reported data
(Campelo et al. 2018).
Compound 14f was characterized according to the follow-
ing data: 3-[(2E)-3-(2H-1,3-benzodioxol-5-yl)prop-2-enoyl]-
1,3-dicyclohexylurea (Fokoue 2015).
high risk, yellow indicates moderate risk, and green indicates
no risk. The software also evaluated the druglikeness and
drugscore of the respective compounds, which aim to measure
whether or not a compound qualifies as a drug by considering
its physicochemical properties, structural similarity with com-
mercially available drugs, and toxicity risks.
The software also shows the druglikeness, a parameter that
evaluates how structurally similar the compound is to com-
mercially available drugs. Its values range from negative to
positive values, with positive values indicating that the com-
pound is structurally similar to commercially available drugs,
whereas negative values indicate otherwise. Finally, the soft-
ware calculates the drugscore, a final parameter which is
based on (1) pharmacokinetic and physicochemical features,
(2) toxicological prediction, and (3) druglikeness. The
drugscore values range from 0 to 1, with values close to 1
indicating that the compound may qualify as a drug (https://
www.organic-chemistry.org/prog/peo/).
1H NMR (300 MHz; CDCl3): δ 7.57 (d; J = 15.0 Hz; 1H,
H2); 7.13 (sl, NH); 6.97 (dd; J = 8.5 and 1.5 Hz; 1H, H9); 6.95
(d; J = 1.5 Hz; 1H, H5); 6.80 (d; J = 8.0 Hz; 1H, H8); 6.55 (d;
J = 15.0 Hz; 1H, H3); 6.01 (s, 2H, OCH2O); 4.13–4.06 (m;
1H, H1a); 3.78–3.73 (m; 1H, H1b); 1.99–1.19 (m; 20H, H2a,
H2b, H3a, H3b, H4a, H4b, H5a, H5b, H6a, H6b). 13C NMR
(75 MHz; CDCl3): δ 166.64 (C1); 154.07 (C*); 149.34 (C6);
148.23 (C7); 143.14 (C3); 129.11 (C4); 124.20 (C9); 117.26
(C2); 108.51 (C8); 106.28 (C5); 101.51 (OCH2O); 55.90
(C1a); 49.92 (C1b); 34.90 (C2b); 33.95 (C6b); 32.78 (C2a);
30.92 (C6a); 26.25 (C4b); 25.61 (C4a); 25.45 (C3b); 25.37
(C5b); 24.93 (C3a); 24.71 (C5a). EI-MS (m/z) (%): 273 (35).
257 (25). 241 (25). 190 (90). 175 (55). 145 (75). 118 (60). 98
(72). 89 (100). 63 (35). 44 (50). ESI-MS: calculated for
Molinspiration
The evaluated compounds were also analyzed in the server
Molinspiration, which calculated the number of violations to
the Lipinski’s rule of 5 according to their: molecular weight (≤
500 μM), clogP (≤ 5), hydrogen bond acceptors (≤ 10), and
hydrogen bond donors (≤ 5) (Lipinski 2004). The server also
evaluated two additional properties: the total polar surface
area (TPSA) and the number of rotatable bonds. According
to Veber et al. (2002), it is desirable that drug candidates
should have a TPSA ≤ 140 Å2 as well as a number of rotatable
bonds ≤ 10.
C23H31N2O4 [M + H]+: 399.2278; found: 399.2210. IR
+
(KBr, ѵmáx/cm−1): 3256. 3047. 2931. 2855. 2118. 1704.
1646. 1542. 1503. 1491. 1448. 1388. 1344. 1255. 1229.
1042. 989. 936. 810.
Virtual screening
OSIRIS property explorer and Molinspiration
Initially, a virtual screening of the natural compounds and
their derivatives was performed. This analysis allows for a
rational choice of drugs by selecting those that present ade-
quate physicochemical properties, which might, in turn, trans-
late into good oral bioavailability. For these analyses, we used
the software OSIRIS property explorer (https://www.organic-
chemistry.org/prog/peo/) and the server Molinspiration (http://
www.molinspiration.com).
Molecular modeling and molecular docking studies
The molecular modeling study was conducted in partnership
with the Laboratory of Molecular Modeling and
Computational Simulation of the Federal University of
Alfenas – Minas Gerais, Brazil. All computer applications
were run on OpenSUS Tumbleweed. Structures of the ligands
1a, 1b, 1g, 1k, 1m, 14f, 18a, and triclosan (ligand used in the
redocking analysis) were constructed using the software
Maestro 10.2.010. The software LigPrep 3.4 with OPLS_3
force field and ionization state for pH 7.0 2.0 was used to
prepare the ligands involved in the studies. The crystallo-
graphic structures of PfENR with triclosan (PDB ID: 1UH5)
from P. falciparum were obtained from the Protein Data Bank
(PDB) database and the software Protein Preparation Wizard
was used for the preparation of these receptors. Co-
crystallized NAD and triclosan present in 1UH5 were re-
moved from the structure and chain A alone was selected for
docking studies. The OPLS3 force field in MacroModel 9.9
was used for optimization. Molecular docking studies be-
tween PfENR and the ligands were performed using the
OSIRIS
The substances were drawn in the software OSIRIS property
explorer (https://www.organic-chemistry.org/prog/peo/) and
compared with a list of approximately 5300 different
fragments in respect to their druglikeness properties. These
fragments are obtained from 3300 commercial drugs and
15,000 chemicals available from the company Fluka®.
The software OSIRIS computes theoretical physicochemi-
cal and toxicological properties of compounds, aiming to eval-
uate some properties and toxicological assessments such as
mutagenic, tumorigenic, irritant, and reproductive risks. The
toxicological results are color-coded, wherein red indicates

Parasitol Res
Induced Fit Docking protocol, in which the program Prime
was used for refinement of the compounds and the program
Glide provided the scoring considering the proteins and the
flexible ligand. The grid box area was defined with the amino
acids Tyr 277, Ile 323 and Phe 368. All computer programs
used belong to the Schrödinger suite (Pidugu et al. 2004;
Schrödinger, Small-Molecule Drug Discovery Suite 2015;
Schrödinger release 2015a, b, c).
lysis buffer solution. The mixture was homogenized and
200 μL of it was transferred to the reading plate where
200 μL of PBS 1× was previously added. The plate was in-
cubated for 30 min at room temperature. Subsequently, a UV/
visible spectrophotometer reading (Synergy HT (BioTek))
with an excitation of 485 nm and emission of 535 nm was
performed. The concentration at which the compounds caused
50% of parasite growth inhibition (IC50) was determined by
the dose-response curve of the triplicate mean for each evalu-
ated compound (Smilkstein et al. 2004).
In vitro culture of P. falciparum
Erythrocytic forms of the W2 strain of P. falciparum were
used in the antiplasmodial assays. The parasites were cultured
in O+ red blood cells in RPMI 1640 medium (Gibco) supple-
mented with 25 mM HEPES, 300 μM hypoxanthine, 11 mM
glucose, 40 μg/ml gentamicin 10% (v/v), and O+ plasma or
5% albumax (Gibco) under the conditions established by
Trager and Jensen (1976). The erythrocytes added to the cul-
ture originated from a single volunteer donor, with a final
hematocrit of 4%. The culture flasks were kept in an incubator
at 37 °C, with the addition of a composite gaseous mixture
(5% CO2, 5% O2, and 90% N2). The medium was changed
every 48 h. The project was approved by the local Ethics and
R e s e a r c h C o m m i t t e e ( C E P ) u n d e r n u m b e r :
83791418.8.0000.5300, case: 2.541.143.
Cultivation of mammal cell lines
VERO cells (renal cells isolated from the kidneys of the
African monkey Cercopithecus aethiops) and HepG2 cells
(cell line derived from human hepatocarcinoma) were used
for the cytotoxicity assays. These cell lines are related to the
excretion of substances and to drug metabolism, respectively.
Cells were cultured in RPMI medium supplemented with 10%
fetal bovine serum (FBS) (Gibico) and 40 mg/L gentamicin
(complete medium) in an incubator with 5% CO2 at 37 °C.
The culture was monitored daily by optical microscopy, and
the medium was changed whenever necessary until 90% con-
fluence was reached, indicating favorable conditions for the
cytotoxicity assay (Calvo-Calle et al. 1994).
Determination of parasite growth inhibition (IC50)
Cytotoxicity assay using the MTT method
The substances were diluted in dimethylsulfoxide (DMSO), at
concentrations up to 0.5%. The serial dilution started from an
initial concentration of 2000 μM in the first well (20 μL).
Cultures were synchronized until they reached the desired
parasitemia of 8% young trophozoites, as described by
Lambros and Vanderberg (1979). Subsequently, the parasite
culture with a predominance of ring forms was adjusted to
0.5% parasitemia and 2% hematocrit. Thus, all compounds
were diluted 10×, at concentrations from 200 to 1.56 μM.
Three controls were used: artemisinin (170 nM) was used as
the positive control, untreated red blood cells infected with
P. falciparum constituted the negative control, and uninfected
red blood cells were used as a blank (whose fluorescence was
discounted from all values obtained).
Plates were incubated at 37 °C with 5% CO2 for 48 h.
Thereafter, the supernatant was discarded without suspending
the red cells. To the latter, 1× PBS was added (200 μL/well)
and centrifuged at 1500 rpm for 10 min. In parallel, 200 μL of
PBS 1× was added to a flat-bottom 96-well culture plate for
fluorescence reading. After centrifugation of the plate, the
supernatant was discarded again. In the plate containing the
culture, 200 μL of lysis buffer (20 mM Tris-HCl, 5 mM
EDTA, 0.08% Triton X-100, 0.008% saponin in 1× PBS,
pH 7.5) containing SYBR Green I (Invitrogen) was added in
the following ratio: 2 μL of SYBR Green I dye to 10 ml of the
After reaching the desired confluence, the mammal cell lines
were detached from the bottom of the flasks with the aid of
trypsin 1× (Sigma-Aldrich) and centrifuged (1500 rpm for
10 min). The supernatant was removed and the pellet resus-
pended in 1 ml of complete medium. An aliquot of 10 μL of
this volume was removed so that the cells could be counted in a
Neubauer chamber using an optical microscope. Afterwards,
the culture was adjusted to 1 × 104 cells/well. The cells were
then plated in 96-well plates and incubated for 24 h at 36 °C.
All compounds were distributed separately on the plate, in trip-
licate, using the serial dilution method (200 to 1.56 μM). The
culture was incubated again (48 h). Untreated cells were con-
stituted as the positive control, whereas the cells treated with
lysis buffer containing Tris at 20 mM (Sigma-Aldrich), EDTA
at 5 mM (Dinâmica), saponin at 0.008%, and Triton X-100 at
0.008% (v/v) (Sigma-Aldrich) constituted the negative control.
After the treatment period, the plates were revealed using
the MTT technique ([3-(4,5-dimethylthiazol-2yl)-2,5-
diphenyl tetrazolium bromide]) (Mossmann 1983). This tech-
nique evaluates cell viability through mitochondrial dehydro-
genase, present only in metabolically viable cells. After the
addition of MTT (Sigma-Aldrich) at a concentration of
5 mg/ml, the plate was incubated for 4 h, the supernatant
discarded immediately, and 100 μL of dimethyl sulfoxide
(DMSO) (Dynamic) was added to dissolve the formazan

Parasitol Res
crystals (violet coloration). A spectrophotometer (Biochrom,
Asys Expert Plus) was used to read the absorbance (570 nm)
of the test plate. Afterwards, the software Origin 9.1 (Origin
Lab Corporation, Northampton, MA, USA) was used to de-
termine the cytotoxic concentration for 50% of the cell popu-
lation (CC50).
compounds, while negative values indicate a hydrophilic na-
ture. Compounds 14f and 1m presented cLogP values = 4.7
and 4.38, respectively, indicating that these molecules are hy-
drophobic/lipophilic. The cLogP of compounds 1a and 18a
were = 1.78 and 3.6, which means that these compounds are
hydrophobic/lipophilic. The compounds also presented desir-
able values for druglikeness (0.115 3.30), drugscore (0.385
0.19), and solubility (− 3.04 1.35). The drugscore values
can range from 0 to 1 (a compound that completely qualifies
as a drug). The positive values of druglikeness observed mean
that the evaluated compounds possess fragments that are com-
monly found in commercially available drugs (Magalhães
2009).
Selectivity index (SI)
The SI of the evaluated compounds was obtained using the
ratio: CC50/IC50. Compounds whose SI were ≥ 10 were con-
sidered to be selective, whereas compounds with SI < 10 were
considered non-selective (Katsuno et al. 2015; Gomes et al.
2014; Nogueira and Rosário 2010).
The S (solubility—Table 1) scale classifies the compounds’
solubility in water as being insoluble for values lower than 10;
slightly soluble < 6; moderately soluble < 4; soluble < 2; very
soluble < 0 (Guerra 2019). The S (solubility) values for the
natural compounds 1a (piplartine) and 18a (piperine) were
2.39 and − 3.61, respectively. These results underpin their
hydrophobic/lipophilic attributes. Since they are poorly solu-
ble in water, these compounds have limited biological appli-
cability. Aqueous solubility has a determinant role in the ab-
sorption and distribution process to reach the blood circula-
tion. Most commercial compounds have S (mol/L) greater
than − 4.00 (OSIRIS Property explorer). In general, poorly
water-soluble compounds are incompletely absorbed in the
body. However, water solubility can be increased and/or im-
proved by the addition of hydrochlorides, sodium salts, etc.
The total polar surface area (TPSA), hydrogen bond do-
nors, hydrogen bond acceptors and rotatable bonds were with-
in the desired thresholds for all compounds in Table 2, except
14f, which presented more than five hydrogen bond donors.
Compounds 1g and 1a presented the best druglikeness and
drugscores (Tables 1 and 2), indicating that these compounds
are the ones that best qualify as drugs. Therefore, we infer that
the modifications made at the dihydropiperidinone moiety of
the ring of compound 1a, substituted by a piperidinyl ring (1g)
contributed to these results.
Results and discussion
Piplartine, piperine, and analogs
For the present study, five analogs (1b, 1g, 1k, 1m, and 14f)
were synthesized from the natural amide piplartine following
previously published literature, without major changes, and
characterized by spectrometric and spectroscopic data. The
yields were not optimized since we focused on obtaining a
sufficient amount of pure compounds for the assays.
In silico prediction of physicochemical and toxicological
properties of the evaluated compounds.
Due to the importance of some physicochemical features
for the biological activity of chemicals (Lipinski 2004), the
virtual prediction of parameters such as molecular weight,
hydrogen bond acceptors and donors and cLogP (computed
partition coefficient), as well as possible biological activities
(drugscore), were calculated using the software OSIRIS
Property explorer and the server Molinspiration.
Most of the compounds in Tables 1 and 2 are within the
limits established by Lipinski’s rule of five, with some obser-
vations. Compounds 1m and 14f presented a red alert for mu-
tagenicity and reproductive toxicity (the latter was also present-
ed by compounds 14f and 18a), which can be explained by the
presence of groups with a large volume in their structures.
Compounds 1m and 14f presented one violation to the
Lipinski’s rule of five. It is important to stress, however, that
these alerts do not necessarily exclude compounds 14f and 18a
from being safe drugs, but rather indicate a probable risk.
The molecular weights of the compounds averaged 303.36
58.15 g/mol; the cLogP values averaged 2.87 1.09 and
were below 5 for all compounds. This parameter is related to
lipophilicity, in that compounds with cLogP values < 5 present
inadequate oral bioavailability, owing to the difficulty of pass-
ing through biological membranes (Oliveira 2013).
Inhibitory concentration (IC50) and cytotoxic
concentration (CC50)
The compounds 1a and 1m obtained IC50 = 20.54 μM and
104.45 μM respectively. The compounds 1b, 1g, 1k, 14f,
and 18a obtained IC50 values > 200 μM (Table 3). The control
with the solvent DMSO did not affect cell viability.
The cytotoxicity assays demonstrated that piplartine was
toxic on the evaluated mammal cell lines and on
P. falciparum, which suggests that the compound might be
exerting its effects through non-specific mechanisms, involv-
ing enzymes, receptors and/or pathways that are present in
both protozoa and mammal cells. Some studies show that
the biological and toxicological activity of piplartine may be
According to Freitas (2015), high and positive cLogP
values indicate a hydrophobic and lipophilic nature of the

Parasitol Res
Table 1 Physicochemical properties, druglikeness and toxicity alert of compounds 1a, 1b, 1g, 1k, 1m, 14f, and 18a
Toxicity alert
Drug-score
Compounds
MW
MUT TUM IRRI
REP
CP
S
DL
3.8
DS
317.34
1.78
-2.39
-2.49
-2.37
-3.16
-5.29
-5.95
-3.61
0.39
1a
321.37
291.35
307.39
444.57
398.50
285.34
2.17
2.28
2.99
4.38
4.7
2.89
5.41
-2.77
0.45
-2.42
0.6
0.14
0.72
0.44
0.24
0.09
0.39
1b
1g
1k
1m
14f
18a
3.6
: non-toxic;
: slightly toxic;
: toxic
MUT mutagenic, TUM tumorigenic, IRRI irritant, REP reproductive, CP cLogP, S solubility, DL druglikeness, DS drug-score.
attributed to the presence of α,β-unsaturated carbonyl groups,
in which the substitution of any of these unsaturated groups
led to a loss of biological activity as well as a decrease in
cytotoxicity (Campelo et al. 2018; Bezerra et al. 2013;
Bezerra et al. 2008; Ponte-Sucre et al. 2015).
Selectivity Indexes were not obtained for the compounds
1b, 1g, 1k, 14f, and 18a, since neither the CC50 nor the IC50
values could be determined for these compounds.
Molecular docking studies on a series of molecules
against P. falciparum P fENR
The molecular docking results revealed that the compounds
that presented the highest binding affinities for the enzyme
were 1m and 14f (Supplementary material). However, the rest
of the evaluated compounds also obtained binding energies
that were higher than the reference inhibitor triclosan.
All the evaluated compounds presented superior affinity
values for PfENR (GScore (Glide Score)) when compared to
the reference inhibitor triclosan. This might be explained by
the higher volume of the compounds when compared to tri-
closan, along with the contribution of steric effects, thus
Table 2 Physicochemical properties of compounds 1a, 1b, 1g, 1k, 1m,
14f, and 18a
Table 3 Values of growth inhibition (IC50), cytotoxic concentration
(CC50), and selectivity index (IS) of the evaluated compounds
Physicochemical properties
Compounds IC50 (μM) CC50
Compounds
TPSA
HBA
HBD
VIO
ROT
VOL
HepG2 (μM) VERO (μM) IS(a) IS(b)
1a
65.08
65.08
48.01
54.80
77.11
67.88
38.78
6
6
5
5
7
4
4
0
0
0
1
1
6
0
0
0
0
0
1
1
0
5
6
5
9
7
4
3
289.03
301.40
276.23
303.25
431.24
378.53
267.74
1a
20.54
> 200
> 200
> 200
104.45
> 200
> 200
14.98
> 500
> 500
> 500
> 500
> 500
> 500
23.37
> 500
> 500
> 500
> 500
> 500
> 500
0.73
1.14
1b
1b
–
–
–
–
–
–
1g
1g
1k
1k
1m
14f
18a
1m
14f
18a
> 4.79 > 4.79
–
–
–
–
TPSA total polar surface area, HBA hydrogen bond acceptors, HBD hy-
drogen bond donors, VIO number of violations, ROT rotatable bonds,
VOL volume
IS(a) selectivity index in relation to HepG2 cells, IS(b) selectivity index in
relation to VERO cells

Parasitol Res
Table 4 Affinity values between the ligands and the enzyme PfENR (GScore), number of hydrogen bonds (Hbond), and van der Waals (good vdW)
interactions between the ligands and PfENR (Schrödinger Suite, Induced Fit Docking Program)
Ligands
GScore (kcal mol−1
)
Hbond
Amino acids that form hydrogen bonds
Good vdW
1a
− 10.762
− 8.794
− 8.229
− 9.659
− 10.981
− 13.047
− 9.900
− 7.806
1
2
2
3
1
2
2
0
Tyr277
404
334
310
440
546
483
313
183
1b
Tyr277, Lys285
Lys285, Leu315
Tyr111 (2), Tyr277
Ala319
1g
1k
1m
14f
Tyr111, Tyr277
Ala219, Tyr277
–
18a
Triclosan
increasing their affinity for the enzyme, which indicates that
the molecules may present higher inhibitory activities than the
reference compound (Table 4).
presented a completely different hydrophobicity value
(cLogP) when compared to the analogs. All structural modi-
fications performed in 1a were shown to substantially de-
crease its antiplasmodial activity. Fokoue (2015) reported that
the solubility of drugs in a hydrophobic medium decreases
their biological activities, which was observed in the present
study for the compound’s analogs.
It is important to stress that the IC50 values of 1a and 1k
against P. falciparum were previously reported in a study by
Araújo-Vilges et al. (2017), being 19.5 μM and 79.1 μM,
respectively (48 h of incubation). The strain and the culture
used in the study are different from those of the present study,
which might explain the differences between the results found
in each study.
The compound 14f (Table 4) presented the highest
affinity value (GScore = − 13.047 kcal mol−1), with 2
interactions per hydrogen bond (HBond) between the
carbonyl oxygen atoms and the Tyr111 and Tyr277 res-
idues, in addition to displaying a high number of favor-
able van der Waals (vdW) contacts (483). No parasite
growth inhibition was observed for the compound 14f at
200 μM, which might be explained by steric effects
relative to the molecule’s size or other physicochemical
properties.
The molecule with the second highest binding affinity was
1m (− 10.981 kcal mol−1) (Table 4), also presenting a hydro-
gen bond interaction with the residue Ala319 and a superior
number of favorable vdW contacts (546) when compared to
14f. These data indicate that trimethoxyphenyl groups favor
more hydrophobic interactions. The compound 1m presented
the second best IC50 value (104.45 μM), which may be related
to its molecular volume and to its access to the enzyme’s
active site. Other features that could possibly explain such
differences between 1m and 14f are the number of hydrogen
bond acceptors and rotatable bonds, which can influence the
flexibility of the molecule and consequently, its interaction
with the enzyme’s active site (Veber 2002).
The compound that presented the third highest GScore value
was 1a (− 10.762 kcal mol−1) (Table 4), with the presence of
one hydrogen bond with the Tyr277 residue and 404 favorable
vdW contacts, which are related to the small size of this mole-
cule when compared to the derivatives 14f and 1m. Despite the
satisfactory binding affinity values of these two compounds,
they did not display in vitro antiplasmodial activity.
When comparing the in vitro IC50 values, it was observed
that 1a presented the best results (20.54 μM) against
P. falciparum owing to its molecular volume, which may fa-
cilitate its access to the active site of the enzyme. Regarding
the in silico results displayed in Table 1, compound 1a
Gomes et al. (2014) when citing Ryckmans et al. (2009)
state that it is desirable that the compounds evaluated for
antiplasmodial activity present low IC50 values in in vitro as-
says. In this perspective, Boechat et al. (2014) consider com-
pounds with IC50 < 10 μM to be active; with IC50 > 10 and <
50 μM to be partially active; and compounds with IC50
>
50 μM to be inactive. It is also noteworthy that substances
with high molecular weights and high lipophilicity are more
likely to present inadequate oral bioavailability. These features
may minimize the risk for lack of specificity of the drug for its
respective target, besides enabling one to administer lower
doses, thus reducing the occurrence of adverse effects
(Lipinski 2004).
The structural modifications of piplartine (1a) were per-
formed at the dihydropiridinone moiety, generating the com-
pounds 1b, 1g, 1k, and 1m. These alterations caused changes
in the physicochemical properties of the in silico study, as well
as in the in vitro growth inhibition assay against P. falciparum,
rendering the molecules inactive (according to Boechat et al.
2014) against the parasite at concentrations up to 200 μM
when compared to the natural compound piplartine (1a). In
14f, the modifications occurred both at the dihydropiridinone
and trimethoxybenzene moieties, which significantly altered
its antiplasmodial activity and physicochemical properties.

Parasitol Res
Araújo-Vilges KM, de Oliveira SV, Couto SCP, Fokoue HH, Romero
GAS, Kato MJ, Romeiro LAS, Leite JRSA, Kuckelhau SAS
(2017) Effect of piplartine and cinnamides on Leishmania
amazonensis, Plasmodium falciparum and on peritoneal cells of
Swiss mice. Pharm Biol 55:601–1607
Significantly, in the molecular docking studies, only affin-
ity values were observed, i.e., whether or not energetically
favorable interactions (electronic and steric effects) between
ligand and enzyme were formed. Favorable affinity values
may lead to intrinsic activity by the molecule, wherein the
intrinsic activity depends on the affinity. The opposite, how-
ever, is not true: one can find favorable affinity values without
the desired biological activity, since this activity is also related
to other factors, such as bioavailability, metabolic stability,
and others (Piccirillo and Amaral 2018).
It is important to highlight that the theoretical data (in
silico) that present some violation do not eliminate the possi-
bility of being evaluated in in vitro or in vivo tests; they only
indicate which substances are valid and can guide investiga-
tors on whether or not to proceed with experimental analyses.
In relation to toxicity characteristics (mutagenic, tumorigenic,
genotoxic, etc.), there are in vitro and in vivo tests that allow
for the evaluation of these properties.
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However, the modifications caused a significant decrease
in in vitro antiplasmodial activity in all analogs and even
caused one compound (14f) to generate a toxicological alert.
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Acknowledgments We are thankful to the Instituto Federal de Rondônia,
campus of Porto Velho – Calama; Plataforma de Bioensaios de Malária e
Leishmaniose – FIOCRUZ-RO; Institute of Chemistry of the Federal
University of São Paulo – USP; the Instituto Nacional de Ciência e
Tecnologia em Fármacos e Medicamentos (INCT-INOFAR), Rede
Mineira de Química (RQ-MG), FAPEMIG and CNPq; and Programa
de Pós-Graduação em Biologia Experimental – PGBIOEXP/UNIR.
Compliance with ethical standards
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18:703–711
Conflict of interest The authors declare that they have no conflict of
interest.
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