Synthesis, antimalarial activity in vitro, and docking studies of novel neolignan derivatives

Article abstract of DOI:10.1111/cbdd.12968

The absence of effective vaccines against malaria and the difficulties associated with controlling mosquito vectors have left chemotherapy as the primary control measure against malaria. However, the emergence and spread of parasite resistance to conventional antimalarial drugs result in a worrisome scenario making the search for new drugs a priority. In the present study, the activities of nine neolignan derivatives were evaluated as follows: (i) against blood forms of chloroquine-resistant Plasmodium falciparum (clone W2), using the tritiated hypoxanthine incorporation and anti-HRPII assays; (ii) for cytotoxic activity against cultured human hepatoma cells (HepG2); and (iii) for intermolecular interaction with the P. falciparum cysteine protease of falcipain-2 (F2) by molecular docking. The neolignan derivatives 9 and 10 showed activity against the blood form of the chloroquine-resistant P. falciparum clone W2 and were not cytotoxic against cultured human hepatoma cells. A molecular docking study of these two neolignans with FP2 revealed several intermolecular interactions that should guide the design of future analogs.

Full text of DOI:10.1111/cbdd.12968

DR. CLÁUDIO NAHUM ALVES (Orcid ID : 0000-0001-6576-4229)
Received Date : 19-Oct-2016
Revised Date : 15-Feb-2017
Accepted Date : 19-Feb-2017
Article type : Research Article
Synthesis, antimalarial activity in vitro and docking studies of novel neolignan
derivatives
Glaécia A. N. Pereira1,4,*, Gisele C. Souza2, Lourivaldo S. Santos2, Lauro E. S. Barata5, Carla
C. F. Meneses1, Antoniana U. Krettli3, Cláudio Tadeu Daniel-Ribeiro4, Cláudio Nahum
Alves1*
1 Laboratório de Planejamento e Desenvolvimento de Fármacos, Instituto de Ciências Exatas
e Naturais, Universidade Federal do Pará, PA, Brasil.
2
Laboratório de Síntese Orgânica, Instituto de Ciências Exatas e Naturais, Universidade
Federal do Pará, Belém, PA, Brasil.
3
Laboratório de Malária, Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte,
MG, Brasil.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12968
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4Laboratório de Pesquisa em Malária, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio
de Janeiro, RJ, Brasil.
5Laboratório de Síntese Orgânica, Instituto de Química, UNICAMP, Campinas, SP, Brasil.
Keywords: neolignan derivatives, anti-plasmodial activity, falcipain-2, docking.
Corresponding author:
*Phone: (+55) 91-3201-8235. Fax: (+55) 91-3201-8235. E-mail: nahum@ufpa.br
*Phone: (+55) 91-3295-3115. Fax: (+55) 91-3201-7363. E-mail: glaecia@gmail.com
Running Title: Synthesis and docking studies of new neolignan derivatives with
antimalarial activity.
Abstract
The absence of effective vaccines against malaria and the difficulties associated with
controlling mosquito vectors has left chemotherapy as the primary control measure against
malaria. However, the emergence and spread of parasite resistance to conventional
antimalarial drugs result in a worrisome scenario making the search for new drugs a priority.
In the present study, the activities of nine neolignan derivatives were evaluated as follows: (i)
against blood forms of chloroquine-resistant Plasmodium falciparum (clone W2), using the
tritiated hypoxanthine incorporation and Anti-HRPII assays; (ii) for cytotoxic activity against
cultured human hepatoma cells (HepG2); and, (iii) for intermolecular interaction with the P.
falciparum cysteine protease of falcipain-2 (F2) by molecular docking. The neolignan
derivatives 9 and 10 showed activity against the blood form of the chloroquine-resistant P.
falciparum clone W2 and were not cytotoxic against cultured human hepatoma cells. A
molecular docking study of these two neolignans with FP2 revealed several intermolecular
interactions that should guide the design of future analogs.
Introduction
Malaria is the leading public health problem among transmissible diseases in the
world. According to the 2015 World Malaria Report, there were around 214 million cases of
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human malaria with 438.000 reported deaths (1). Despite various attempts not completely
successful to produce a vaccine, chemotherapy remains the main control tool against the
disease (2). However, multi-resistance of Plasmodium falciparum against antimalarial
compounds and the lower sensitivity of P. vivax to chloroquine have been important obstacles
to preventing spread of the disease (3). Many efforts have been made towards identifying
novel molecular targets for development of new compounds against malaria. One attractive
target is a cysteine-protease of P. falciparum, falcipain-2 (FP2), an essential enzyme for
parasite development (4). FP2 is particularly suited for the hydrolysis of native hemoglobin in
the acidic food vacuole, and several studies reveal that cysteine protease inhibitors such as
chalcones block globin hydrolysis by inhibition of FP2 (5, 6), however most chalcones
exhibit high toxicity (7, 8)
The neolignans correspond to a kind of lignoids derived from the oxidative homo- or
cross-coupling of alylphenols and propenyl phenols (9). They are found in plants from the
Miristicaceae family (10), and have demonstrated antibacterial (11), anti-Schistosoma (12,
13), antifungal (14), trypanosomicidal , anti-Plasmodium (15,16) and leishmanicida (17)
activities. Neolignan derivatives are active against Plasmodium at nanomolar concentrations,
and are considered the most active lignoids against malaria, thus promising antimalarial
prototypes (15,16, 18).
In an attempt to identify non-toxic FP2 inhibitors retaining good activity, novel
neolignan derivatives were synthesized based on their structural similarity with chalcones.
Nine neolignan derivatives were tested against chloroquine-resistant P. falciparum blood
forms (clone W2). The inhibitory drug concentration that eliminates 50% of the parasites was
determined using the Anti-HRPII (histidine-rich protein II) and hypoxanthine-tritiated
incorporation assays, in parallel with tests of drug cytotoxicity to HepG2 cells (MDL50) to
calculate their selectivity indexes (SI). The best compounds were subjected to theoretical
docking studies with the FP2.
Methods and Material
Experimental Section
Material
The reactions were monitored by thin-layer chromatography performed on TLC plates
with silica gel 60 F254 (Merck®). The spectra data 1H (62.5, 400 and 500 MHz) and 13C (50,
100 and 125 MHz) were obtained by Bruker AC 250/P, Bruker Avance-400 and Varian
Inova-500, using CDCl3 and DMSO-d6 as solvents and TMS as the internal standard
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(chemical shift δ in ppm). The infrared absorption spectra were obtained by using a
spectrophotometer Bomen Model MB Series II, and mass spectra (MS) were evaluated on
VG Auto Spec High Resolution Mass Spectrometer (Micromass Company).
Synthesis
Compound preparation (4, 5, 7-10)
A solution of 1.02 equivalent of phenol derivative in anhydrous ethyl methyl ketone
(4.5 mL of solvent/mmol of α-bromoketone) and 1.80 equivalent of anhydrous K2CO3 was
stirred for 10 min at room temperature, and then a solution of α-bromoketone in anhydrous
ethyl methyl ketone (1.5 mL of solvent/mmol of α-bromoketone) was added dropwise. The
reaction mixture was stirred and refluxed for 12 h. After completion, the mixture was
concentrated under vacuum, diluted with H2O, and extracted with CHCl3 (3x). The organic
layer was washed with water, 5% NaOH solution, brine, and dried over Na2SO4. The solvent
was evaporated to result in a crude product which was purified by crystallization from
ethanol.
2-oxo-2-phenylethyl
(2E)-3-[4-(2-oxo-2-phenylethoxy)phenyl]prop-2-enoate
4:
This
compound was obtained from 0.42 g (2.56 mmol) of 4-hidroxycinnamic acid, 0.50 g (2.51
mmol) of phenacyl bromide and 0.625 g (4.52 mmol) of K2CO3. The product obtained was a
1
colorless crystalline solid (0.160 g). Yield 32%. M.P. 145-147 ºC. H NMR (400 MHz,
DMSO) δ: 7.99 (d, J=7.4 Hz, 2H), 8.04 (d, J =7.4 Hz, 2H), 7.70 (d, J = 16.0 Hz, 1H), 7.60-
7.73 (m, 4H), 7.59-7.55 (td, J =7.4 Hz and J=2.7 Hz, 4H), 7.04 (d, J = 9.0 Hz, 2H), 6.64 (d, J
=16.0 Hz, 1H), 5.68 (s, 2H), 5.60 (s, 2H).13C NMR (100 MHz, DMSO) δ: 194.2, 193.0,
165.9, 160.0, 145.1, 134.3, 134.0, 130.2, 128.9, 128.9, 127.9, 127.8, 126.9, 115.1, 114.8,
70.2, 66.4. HRMS (ESI): calcd for C25H21O5 [M+H]+ 401.1384, found 401,1389. IR (KBr,
+
cm-1): 1721 (C=O), 1706 (C=O).
2-oxo-2-phenylethyl 4-(2-oxo-2-phenylethoxy)benzoate 5: This compound was obtained from
0.35 g (2.56 mmol) of 4-hydroxybenzoic acid, 0.50 g (2.51 mmol) of phenacyl bromide and
0.62 g (4.52 mmol) of K2CO3. The product obtained was a white crystalline solid (0.16 g).
1
Yield 34%. M.P. 146-148 ºC. H NMR (400 MHz, DMSO) δ: 8.07 (d, J = 9.0 Hz, 2H), 7.98
(d, J = 8.5 Hz, 2H), 7.94 (d, J = 8.5 Hz, 2H), 7.46-7.52 (m, 4H), 6.97 (d, J = 9.0 Hz, 2H),
5.52 (s, 2H), 5,50 (s, 2H). 13C NMR (100 MHz, DMSO) δ: 193.6, 192.3, 165.5, 162.0, 134.4,
134.3, 134.1, 133.8, 132.1, 128.9, 128.9, 128.1, 127.8, 122.7, 114.5, 70.5, 66.3. HRMS (ESI):
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calcd for C23H18NaO5 [M+Na]+ 397.1046, found 397.1089. IR (KBr, cm-1): 1716 (C=O),
+
1698 (C=O).
2-oxo-2-phenylethyl (2E)-3-(1,3-benzodioxol-5-yl)prop-2-enoate 7: This compound was
obtained from 0.50 g (2.56 mmol) of 3,4-(methylenedioxy)cinnamic acid, 0.50 g (2.51
mmol) of phenacyl bromide and 0.62 g (4.52 mmol) of K2CO3.The product obtained was a
1
colorless crystalline solid (0.54 g). Yield 69%. M.P. 144-146 ºC. H NMR (400 MHz,
CDCl3) δ: 5.46 (s, 2H), 6.01 (s, 2H), 6.42 (d, J = 16.0 Hz, 1H), 6.81 (d, J = 7.9 Hz, 1H), 7.01-
7.05 (m, 2H), 7.49 (t, J = 7.9 Hz, 2H), 7.61 (t, J = 7.3 Hz, 1H), 7.82 (d, J = 16.0 Hz,1H), 7.95
(d, J = 7.3 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ: 66.0, 101.6, 106.6, 108.5, 114.8, 124.7,
127.8, 128.7, 128.8, 129.2, 133.8, 134.3, 145.8, 148.3, 149.8, 166.4, 192.4. HRMS (ESI):
calcd for C18H14KO5 [M+K]+ 349.0473, found 349.0478. IR (KBr, cm-1): 1715 (C=O), 1694
+
(C=O).
2-(4-chlorophenyl)-2-oxoethyl (2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoate 8: This
compound was obtained from 1.04 g (4.36 mmol) of 3,4,5-trimethoxycinnamic acid, 1.0 g
(4.28 mmol) of 2-bromo-4'-chloroacetophenone and 1.1 g (7.74 mmol) of K2CO3. The
1
product obtained was a colorless crystalline solid (0.75 g). Yield 45%. M.P. 117-119 ºC. H
NMR (400 MHz, CDCl3) δ: 7.90 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 16.0 Hz, 1H), 7.48-7.46 (m,
2H), 6.78 (s, 2H), 6.50 (d, J = 16.0 Hz, 1H), 5.44 (s, 2H), 3.89 (s, 9H). 13C NMR (100 MHz,
CDCl3) δ: 191.2, 166.1, 153.3, 146.2, 140.3, 140.3, 132.5, 129.6, 129.1, 115.9, 105.4, 65.8,
60.9, 56.1.HRMS (ESI): calcd for C20H20ClO6 [M+H]+ 391.0943, found 391.0948. IR (KBr,
+
cm-1): 1714 (C=O), 1629 (C=O).
Methyl-(2E)-3-[3-methoxy-4-(2-oxo-2-phenylethoxy)phenyl]prop-2-enoate 9: This compound
was obtained from 0.77 g (3.71 mmol) of methylferulate, 0.70 g (0.35 mmol) of 8-
bromoacetophenone and 0.87 g (6.33 mmol) of K2CO3. The product obtained was a brown
1
crystalline solid (0.37 g). Yield 32%. M.P. 105-107 ºC. H NMR (500 MHz, CDCl3) δ: 8.00
(d, J = 8.0 Hz, 2H), 7.63-7.59 (m, 2H), 7.50 (t, J1 = 8.0 Hz, 2H), 7.07 (s, 1H), 7.02 (d, J1 =
8.0 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.31 (d, J = 16.0 Hz, 1H), 5.41 (s, 2H), 3.92 (s, 3H),
3.79 (s, 3H). 13C NMR (125 MHz, CDCl3) δ: 193.7, 167.5, 149.5, 149.3, 144.5, 134.2, 133.9,
128.8, 128.5, 127.9, 122.0, 115.9, 113.6, 110.4, 71.3, 55.9, 51.6. HRMS (ESI): calcd for
C19H19O5 [M+H]+ 327.1227, found 327.1233. IR (KBr, cm-1): 1721.4 (C=O), 1689.7 (C=O).
+
1-(4-methoxyphenyl)-1-oxopropan-2-yl-(2E)-3-(4-{[1-(4-methoxyphenyl)-1-oxopropan-2-
yl]oxy}phenyl)prop-2-enoate 10: This compound was obtained from 0.18 g (1.05 mmol) of 4-
hydroxycinnamic acid, 0.25 g (1.03 mmol) of 4-methoxy-8-bromopropiophenone and 0.27 g
(1.98 mmol) of K2CO3. The product obtained was a white crystalline solid (0.18 g). Yield
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71%. M.P. 142-144 ºC. 1H NMR (400 MHz, DMSO) δ: 8.12 (d, J = 8.8 Hz, 2H), 8.05 (d, J =
8.8 Hz, 2H), 7.70 (d, J = 8.8 Hz, 2H), 7.64 (d, J = 16.0 Hz, 1H), 7.19-7.08 (m, 4H), 6.94 (d, J
= 8.8 Hz, 2H), 6.61 (d, J = 16.0 Hz, 1H), 6.16-6.06 (m, 2H), 3.91 (s, 3H), 3.90 (s, 3H), 1.59
13
(d, J = 7.0 Hz, 3H), 1.52 (d, J = 7.0 Hz, 3H). C NMR (100 MHz, DMSO) δ: 196.1, 195.1,
165.8, 163.8, 163.6, 159.2, 144.9, 130.9, 130.8, 130.3, 126.9, 126.7, 126.7, 115.3, 115.0,
114.3, 114.2, 74.3, 71.2, 55.7, 18.5, 17.2. 13C NMR – (63 MHz, DMSO) δ: 196.1, 195.1,
165.8, 163.8, 163.6, 159.2, 144.9, 131.0, 130.8, 130.3, 126.9, 126.7, 126.7, 115.3, 115.0,
114.3, 114.3 , 74.3, 71.2, 55.7, 55.6, 18.5, 17.3. HRMS (ESI): calcd for C29H29O7 [M+H]+
+
489.1941, found 489.1942. IR (KBr, cm-1): 1712.6 (C=O), 1699 (C=O), 1685.5 (C=O).
Continuous in vitro culture of P. falciparum blood forms
The chloroquine-resistant and mefloquine-sensitive W2 clone of P. falciparum was
cultivated as described by Trager and Jensen (19), with some modifications. The
antiplasmodial activity of neolignan derivatives was evaluated using tritiated hypoxanthine
and anti-HRPII (Histidine Rich Protein) assays. At least, two assays for each technique were
performed. Each assay was performed in triplicates and chloroquine (as a reference drug) was
used in each experiment as a control antimalarial.
Briefly, a 2% hematocrit of human red blood cells type A+ suspended in RPMI 1640
medium supplemented with 10% of Human inactivated-sera type A+ was maintained in
culture plates at 37 °C in a gas mixture (5% CO2, 5% O2 and 90% N2 atmosphere). The test
of hipoxantine incorporation described previously (20) was used to evaluate drug activity,
with some modifications. Briefly, before the test, the blood cultures were kept for at least
three days in medium without hypoxanthine. The test was performed after sorbitol-
synchronization with ring stages (21); then adjusted for 1% both parasitemia and hematocrit.
The parasite suspension was placed in a plate containing the test compounds and incubated
in culture conditions for 24 h when3H-hypoxanthine was added, (0.5 μCi) (PerkinElmer, MA,
EUA) to each well. Plates were incubated at 37°C for 18 h and then frozen for at least 24 h at
-20°C and thawed to allow cell lysis. The plates were harvested [Tomtec 96-Harvester
(Tomtec Inc., Handem, CT, USA)] in glass-fiber filters (WallacOu, Turku, Finland), which
were placed in sample bags (Wallac) and subsequently immersed in scintillation fluid
(Optiphase super mix, Wallac). The [3H] uptake was measured in a 1450 Microbeta reader
(Wallac).
An anti-HRPII immunoenzymatic assay was performed as described (22), using
0.05% parasitemia and 1.5% hematocrit. The compounds were incubated at 37º C with
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parasite cultures for 72h. The culture was frozen and thawed for cell lysis to occur and
release the HRPII protein present in live parasites. Parasite growth was evaluated by specific
interactions between the histidine- and alanine-rich parasite protein (HRPII) and
commercially available monoclonal antibodies (MPFM ICLLAB-55A® and MPFG55P
ICLLAB®, USA), which can be measured at 450 nm using SpectraMax340PC384
(Molecular Devices).
The anti-P. falciparum activity of neolignans was evaluated using curve-fitting
software (Microcal Origin Software 5.0, Inc.) by comparing parasite growth in relation to
drug-free control cultures, considered as 100% growth, and drug test-cultures. A sigmoidal
dose-response curve was generated and the half-maximal drug inhibitory concentration (IC50)
of the parasite growth was determined. A molecule was considered active when the IC50
value was lower than 30 μM.
Cytotoxicity Assays and determination of drug selectivity indexes
The in vitro cytotoxicity assays were performed with cells derived from the hepatoma
cell line HepG2A16 (ATTC, Manassas, VA, USA), cultivated in 75 cm2 sterile culture flasks
(Nunc) at 37°C in a 5% CO2 atmosphere. The cells were maintained in RPMI 1640 culture
medium (Sigma-Aldrich Co., St. Louis, MO) supplemented with 40 mg/L gentamicin and
10% fetal bovine serum (FBS) (Invitrogen, Life Technologies, Carlsbad, CA, USA). When
cell confluence reached 80%, they were trypsinized (0.25% trypsin-EDTA) (Gibco, Life
Technologies, Carlsbad, CA, USA), washed, counted, diluted in complete medium at 104
cells/well, and placed in a flat-bottomed 96-well plate (Corning, Santa Clara, CA, EUA), then
incubated for 18 h at 37 °C to allow cell adhesion. The test and control compounds were
added to the plates at various concentrations (up to 1.000 µg/mL) and incubated for another
24 h. MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] at 5 mg/mL in
water, was added to each well, followed by another 3 h of incubation at 37 ºC (23, 24). The
supernatant was removed, and 100 μL of DMSO were added to each well, the reactions were
read in a spectrophotometer (Spectra Max 340PC384, Molecular Devices) with a 570nm filter,
and a 630 nm filter for background. The cell viability was expressed as the percentage of the
absorbance compared to untreated cells, subtracted from the appropriate background. The
minimum lethal dose (MLD50) of the test compounds was determined as previously described
(25); each test was performed in duplicates. The selectivity index (SI), or therapeutic activity,
was calculated only for the active compounds, as the ratio between cytotoxicity and P.
falciparum activity (MLD50/IC50), as described (20).
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Molecular Docking
All structures were subjected to the geometry optimization using the hybrid density
functional (B3LYP) (26, 27) and a basis set 6-31G* using the Gaussian03 package (28). The
3D structure of enzyme FP2 complexed with the E64 (L-trans-Epoxysuccinyl-leucylamido(4-
guanidino)butane) were obtained from the Protein Data Bank (PDB accession code 3BPF).
All compounds (neolignan derivatives and inhibitor E64) were submitted to docking studies
using Molegro Virtual Docker (MVD) software. The enzyme cavity was detected with an
active site radius of 6.0 Å from the side chain sulfur atom of residue Cys42, according to the
literature (29). Docked results were visually inspected to ensure an acceptable drug/enzyme
interaction was present. The following parameters were used for the guided differential
evolution algorithm: population size = 50, crossover rate = 0.9, scaling factor = 0.5 and max
evaluations = 100000. The algorithm used in the docking studies was MolDock Score, an
adaptation of the Differential Evolution (DE) algorithm. The results of the molecular docking
for neolignan derivatives were compared with the experimental data (29).
Analysis of the molecular electrostatic potential
The maps of electrostatic density can be indicators of nucleophilic and electrophilic
centers which control the strength of the connections, and of the unbound interactions and
molecular reactivity. With the Adaptive Poisson-Boltzmann Solver (APBS) methodology
(30) it was possible to construct around the structure of the FP2 enzyme (Figure 1). The
three-dimensional surfaces of the molecular electrostatic potential (MEP) were generated
using the software Chimera and APBS (31), with an ionic strength of 12 mM. Mapping of the
electrostatic potential onto the molecular surface of the protein was performed with a
potential range from −8 eV to 8 eV.
Results
Chemistry
Compounds 2 (32), 3 (33) and 6 (34) were synthesized as previously described.
Compounds 7 and 8 were commercial, but their synthesis and spectral data are not available
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in the literature, therefore we described these in the experimental section. Neolignans
derivatives 4, 5, 9 and 10 are new compounds and were synthesized as shown in scheme 1.
The commercially available aromatic ketones were converted to the corresponding α-
bromoketones 1a–c (Scheme 1, step i) in one-step according to the methodology (35) and
used without purification due to their strong tear-gas properties. All coupling reactions
between α-bromoketones and phenoxy or carboxy nucleophiles generated in situ with
anhydrous K2CO3 were performed in reflux of butanone as solvent. Compounds 7 (69%
yield) and 8 (45% yield) were synthesized through the reaction of the appropriate α-
bromoketone with cinnnamic acid, 3,4-(methylenedioxy)cinnamic acid and 3,4,5-
trimethoxycinnamic acid, respectively (Scheme 1, step iii). Compound 9 was obtained via
reaction of phenacyl bromide with methyl ferulate in 32% yield (Scheme 1, step ii).
Treatment of 4-hydroxycinnamic acid and 4-hydroxybenzoic acid with anhydrous K2CO3
(Scheme 1, step iv) led to the formation of bi-nucleophiles (carboxy and phenoxy) in situ.
Nucleophilic reactions of bi-nucleophile generated from 4-hidroxycinnamic acid with
phenacyl bromide and 4-methoxy-8-bromopropiophenone afforded, respectively, compounds
4 (32% yield) and 10 (71% yield). Nucleophilic reactions of bi-nucleophile generated from 4-
hydroxybenzoic acid with phenacyl bromide resulted in compound 5 at 34% yield (Scheme 1,
step v). The reactions were monitored using TLC, and the products were purified by
1
crystallization from ethanol. All compounds were fully characterized using IR, H and 13C
NMR and EIMS, where their spectra were consistent with the assigned structures.
Pharmacology
Among the nine neolignan derivatives evaluated in vitro against the W2 P. falciparum
resistant clone, only derivatives 9 and 10 exhibited IC50 of 26.6 and 12.1µM, respectively, by
3H-hypoxanthine incorporation assay (Table 1).
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Table 1. Cytotoxicity assay (MLD50) against human hepatoma cells (line HepG2), in vitro
activity against P. falciparum based on IC50 values and selectivity index (SI) of the neolignan
derivatives.
IC50
MLD50
x ± SD (µM)
*SI
x ± SD(µM)
3H-hypoxantine Anti-HRPII
̅
Compound
̅
O
O
> 4498.0
> 3755.3
> 2497.4
108.0 80.5 130.4 9.7
-
O
(2)
O
O
-
> 187.8
> 124.9
> 187.8
> 124.9
O
(3)
O
O
O
-
-
O
O
(4)
O
O
O
O
> 2671.0
> 133.6
> 133.6
O
(5)
O
O
-
-
> 3325.1
> 3222.7
> 166.3
> 161.1
> 166.3
> 161.1
O
Cl
(6)
O
O
O
O
O
(7)
OCH3
OCH3
O
O
-
O
> 2558.8
> 127.9
> 127.9
OCH3
O
Cl
(8)
OCH3
O
>3064.3
>1545.5
26.6 18.8 39.5 11.2 >115.2
O
O
(9)
O
O
O
12.1 0.0
NT
>128.8
2750
O
H3CO
OCH
O
(10)
CH3
N
HN
N
330 28
0.12 0.01 0.18 0.37
Cl
Chloroquine
*SI = MLD50/ IC50 obtained by [3H]hypoxantine method which is a gold-test among
semi-automated techniques. MLD, minimum lethal dose. IC50, half-maximal drug
inhibitory concentration. SD, standard deviation. NT, not tested.

Cytotoxic Activity and Selectivity Index
The nine neolignans tested showed MLDs above 1,500 μM, thus indicating that they
are non-toxic to HepG2 cells (Table 1). The active derivate 10 (MLD50 above 1,545.5 μM)
and 9, (MLD50 above 3,064.3 μM) showed selectivity indexes above 128.8 and 115.2,
respectively.
Molecular Docking
Co-crystallized ligand (E64) was re-docked to their target protein (FP2) to validate the
docking protocol. When compared with the crystal structure, the docked conformation of E64
gave RMSD of 0.15Å and had a MolDock score of -133.2 kcal.mol-1, shown in Figure 2.
These results illustrate a good adjustment between the theoretical and experimental data (29).
Docked conformations of derivatives 9 and 10 were ranked based on their MolDock
score and the best conformation (Table 2). The RMSD value obtained for the two complexes
was 0.19 Å and 0.12 Å, and the score values -102.2 and -111.7 kcal.mol-1, respectively.
These values are relatively close to the score value of inhibitor E64 (-133.2 kcal.mol-1).
Interestingly, the observed activities against clone W2 were 26.6 μM, 12.1 μM and 3.0 μM,
for 9, 10 and E64, respectively.
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Table 2. Molecular docking studies for E64 and neolignan derivatives.
MolDock Score Hbond score Number of H-
Compound
(kcal.mol-1)
(kcal.mol-1)
Bonds
- 82.3
-2.2
2
2
-77.9
-90.1
-90.5
-80.4
-91.5
-82.6
-102.2
-111.7
-133.2
-2.0
0
2
0
1
2
2
1
1
4
4
3
4
-0.4
-2.3
-3.2
-1.9
-4.1
-7.3
-8.5
5
6
7
8
9
10
E64*
* Experimental value obtained by Kerr and Lee et al. (27).
In this study, the carbonyl group (L-trans-Epoxysuccinyl) and the amide group
(leucylamido) of E64 inhibitor were located near the catalytic cysteine residue (Cys42),
tryptophan (Trp43) and glycine (Gly83) of the FP2. In addition, the oxyanion hole located in
the S2 cavity was occupied by the carbonyl group (L-trans-Epoxysuccinyl), which interacts
with the thiol group of Cys42 at a distance of 2.5 Å. This carbonyl group also interacts
through hydrogen bonds with the aromatic ring of Trp43 at 2.9 Å. For the amide group
(leucylamido), two interactions with amine and carboxyl group of Gly83 at 3.2 Å and 2.9 Å,
respectively, were observed (Figure 3 A).
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Figure 3B shows the predicted binding site of compound 10 in the F2 catalytic site
suggested by molecular docking. In this complex (FP2-10), the ester group of derivate 10 also
occupies the oxyanion hole of the cavity S2, interacting with amine and thiol groups of Cys42
at bond lengths of 3.0 Å and 3.1 Å, respectively, same as for the E64 inhibitor. Moreover, a
series of hydrogen bonds can be observed between methoxy groups of derivate 10 and the
residues of His174, Ser149 and Ile85 situated within cavities S1’ and S2, similar to previous
reports (36, 37), being the bond length 3.1 Å, 2.9 Å and 2.9 Å respectively (Figure 3B).
Lastly, derivate 9 showed only one interaction at a distance of 2.9 Å with residue Ile85
(Figure 3C) evidencing the low potency against FP2 as well as its IC50 value.
Discussion
The neolignans constitute a class of chemical substances with described biological
actions against leishmaniasis, cancer and other diseases (12-14, 15, 38). Some neolignans and
their derivatives have shown antimalarial activity at low concentration (39, 40). Derivate 9 in
this study exhibited in vitro activity, with IC50 of 12.1 µM, similar to other neolignan
derivatives such as Virolongin A (IC50 of 14.9 μM) against chloroquine-resistant P.
falciparum (clone Dd2) (36). These derivatives have a similar structure to compound 10 with
the presence of three benzene nuclei. Furthermore, the presence of methoxyl and the absence
of chlorine as substituents in the neolignan derivatives is important for good antimalarial
activity (37, 40, 41). In previous work (18) where active lignans were evaluated against a
chloroquine-resistant P. falciparum strain, the importance of the methoxy substituent for
good antiparasitic activity was demonstrated. Most lignoids were not cytotoxic (MLD50>
1,500 μM), and are therefore good new antimalarial candidates as suggested previously (17).
The selectivity index was above 100 μM for two new compounds tested, the neolignan
derivatives 9 and 10.
The active site of FP2 can be divided into four cavities denominated S1, S1’, S2 and
S3 (Figure 1), and is located in a cleft between the two structurally distinct domains, with
catalytic triad of Cys42, Asn173 and His174 (29). Derivate 10 is a potential inhibitor of FP2,
and as can be seen in Figure 1, this derivative effectively occupied cavities S1 and S2.
Furthermore, it establishes several interactions with catalytic FP2 residues localized in cavity
S2 (Figure 3), commonly observed among cysteine protease inhibitors (42). The S2 site is a
predominantly hydrophobic region where residues were shown to be preferentially linked to
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groups of the substrate (43), but can also interact with hydrophilic groups. This fact explains
the interactions of the methoxy groups of derivates 9 and 10 in this cavity (44).
Conclusions
Nine neolignans tested were not toxic to HepG2 cells exhibiting minimal lethal doses
(MLD50) greater than 1,500 μM. Only derivatives 9 and 10 showed activity against
chloroquine-resistant P. falciparum clone W2. Interactions and score values obtained by
molecular docking techniques for complexes formed with neolignans and FP2 were
satisfactory when compared to the crystallographic E64-FP2 complex. These results show
that neolignans are promising prototypes to be considered in the search of potent new
derivatives against malaria parasites, as well as less toxic products for safe therapy.
Author contributions
CNA and CCFM designed the theoretical study and analyzed the data. GANP performed
the in vivo and in vitro experiments under the supervision of CTDR and AUK. GCS, LSS and
LESB designed and performed the synthesis of compounds. Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES) for financial support. The manuscript was written and
revised by all authors.
Conflict of interest
The authors have declared no conflict of interest.
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Supplementary Information
1
13
Supplementary data related to this article include representative H and C NMR and HRMS
spectra of new compounds.
Figure Legends
Figure 1. Electrostatic density map around of 3D structures of FP2 with E64 (pink) and 10
(yellow) ligands.
Figure 2. Superposition of the docked (blue) and crystal structure (pink) conformations of
E64 in the active site of FP2.
Figure 3. Hydrogen bonds formed by the E64 (pink) and derivatives 10 (yellow) and 9
(orange) with the active site residues of the Falcipaína-2 (green).
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Supplementary Figure Legends
Figure S1: 1H NMR (400 MHz, DMSO) spectrum of compound 4
Figure S2: 13C NMR (100 MHz, DMSO) spectrum of compound 4
Figure S3: HRMS spectrum of compound 4
Figure S4: 1H NMR (400 MHz, DMSO) spectrum of compound 5
Figure S5: 13C NMR (100 MHz, DMSO) spectrum of compound 5
Figure S6: HRMS spectrum of compound 5
Figure S7: 1H NMR (400 MHz, CDCl3) spectrum of compound 7
Figure S8: 13C NMR (100 MHz, CDCl3) spectrum of compound 7
Figure S9: HRMS spectrum of compound 7
Figure S10: 1H NMR (400 MHz, CDCl3) spectrum of compound 8
Figure S11: 13C NMR (100 MHz, CDCl3) spectrum of compound 8
Figure S12: HRMS spectrum of compound 8
Figure S13: 1H NMR (500 MHz, CDCl3) spectrum of compound 9
Figure S14: 13C NMR (125 MHz, CDCl3) spectrum of compound 9
Figure S15: HRMS spectrum of compound 9
Figure S16: 1H NMR (400 MHz, DMSO) spectrum of compound 10
Figure S17: 13C NMR (63 MHz, DMSO) spectrum of compound 10
Figure S18: HRMS spectrum of compound 10
Scheme Legend
Schemes 1. Synthesis of neolignan 8.O.4’ derivatives. Reagents and conditions:(i) CHCl3,
Br2, 0.5 h, reflux; (ii) Methyl ferulate; K2CO3, butanone, 12 h, reflux (iii) Cinnamic acid or
derivatives, K2CO3, butanone, 12 h, reflux. (iv) 4-Hydroxycinnamic acid, K2CO3, butanone,
12 h, reflux. (v) 4-Hydroxybenzoic acid, K2CO3, butanone, 12 h, reflux.
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