Evaluation of the antitrypanosoma activity and SAR study of novel LINS03 derivatives

Article abstract of DOI:10.1016/j.bioorg.2019.102996

Chagas’ disease is a parasitic infection caused by Trypanosoma cruzi that is still treated by old and toxic drugs. In the search for novel alternatives, natural sources are an important source for new drug prototypes against T. cruzi to further structural

Full text of DOI:10.1016/j.bioorg.2019.102996

BioorganicChemistry89(2019)102996
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Evaluation of the antitrypanosoma activity and SAR study of novel LINS03
derivatives
⁎
Marina T. Varelaa,1, Thais A. Costa-Silvab,1, João Henrique G. Lagob, Andre G. Temponec,
,
⁎
João Paulo S. Fernandesa,
a Departamento de Ciências Farmacêuticas, Universidade Federal de São Paulo, Rua São Nicolau 210, 09913-030 Diadema, SP, Brazil
b Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Av. dos Estados 5001, 09210-580 Santo André, SP, Brazil
c Centre for Parasitology and Mycology, Instituto Adolfo Lutz, Av. Dr. Arnaldo 351, 01246-000 São Paulo, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Chagas’ disease is a parasitic infection caused by Trypanosoma cruzi that is still treated by old and toxic drugs. In
the search for novel alternatives, natural sources are an important source for new drug prototypes against T. cruzi
to further structural exploitation. A set of natural-based compounds (LINS03) was designed, showing promising
antitrypanosoma activity and low cytotoxicity to host cells. In this paper, nine novel LINS03 derivatives were
evaluated against T. cruzi trypomastigotes and amastigotes. The selectivity was assessed through cytotoxicity
assays using NCTC mammalian cells and calculating the CC50/IC50 ratio. The results showed that compounds 2d
and 4c are noteworthy, due their high activity against amastigotes (IC50 13.9 and 5.8 µM) and low cytotoxicity
(CC50 107.7 µM and > 200 µM, respectively). These compounds did not showed alteration on plasma membrane
permeability in a Sytox green model. SAR analysis suggested an ideal balance between hydrosolubility and
lipophilicity is necessary to improve the activity, and that insertion of a meta-substituent is detrimental to the
activity of the amine derivatives but not to the neutral derivatives, suggesting different mechanisms of actions.
The results presented herein are valuable for designing novel compounds with improved activity and selectivity
to be applied in future studies.
Anti-Trypanosoma cruzi
Antiparasitic compounds
Hydrophilic lipophilic balance
Natural product analogues
SAR analysis
1. Introduction
variety of plants, animals and microorganisms providing a great deal of
chemical diversity which no chemical library is capable of generating
Chagas’ disease (CD) is a neglected tropical parasitic infection
caused by the trypanosomatid parasite Trypanosoma cruzi. The natural
life cycle of T. cruzi depends on an invertebrate host, most commonly
Triatoma spp. which transmits the parasite through the infected feces
eliminated alongside the blood feeding. The transmission can also occur
via blood transfusion, organ transplant and contaminated food [1]. This
alternative transmission led to a disease spreading to non-endemic re-
gions (such as Europe, US, Canada and Japan) due to the migration of
infected individuals [2]. Despite this, no drug has recently been ap-
proved for the treatment of CD and only two drugs are available,
benznidazole and nifurtimox, which are nitroheterocyclic molecules
that are frequently associated with many harmful side effects that im-
pair the patient’s compliance to the treatment.
[3]. These molecules can reach the market as found in natura, such as
paclitaxel, morphine and digoxin, or they can even be used as proto-
types for designing new and improved molecules, such as captopril
(inspired from a peptide isolated from the venom of Bothrops jararaca)
[4].
To obtain active compounds against T. cruzi, a series of analogues
were designed from gibbilimbols A and B (Fig. 1), natural molecules
with simple motifs isolated from the leaves of Piper malacophyllum
[5–7]. Previous results showed that the insertion of a functional group
into the side chain led to important changes in the antitrypanosoma and
cytotoxic activity [5]. while the substitution of the phenol for a benzene
or methoxybenzene lowered the cytotoxic profile [6]. Recently, an
exploratory data analysis using several molecular descriptors was per-
formed in order to better understand which characteristics play the role
in the biological activity and selectivity profile [7]. The results showed
that there are two different subsets of compounds: a group of basic
Natural sources are frequently a great inspiration for new medicinal
entities. The interest on such sources resides on the fact that natural
molecules come from several different metabolic pathways from a
⁎ Corresponding authors.
E-mail addresses: andre.tempone@ial.sp.gov.br (A.G. Tempone), joao.fernandes@unifesp.br (J.P.S. Fernandes).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2019.102996
Received 27 November 2018; Received in revised form 25 March 2019; Accepted 17 May 2019
Availableonline18May2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

M.T. Varela, et al.
BioorganicChemistry89(2019)102996
gibbilimbol A
gibbilimbol B
NATURAL PROTOTYPES
LINS03003 (1a)
LINS03017 (1b)
LINS03008 (1g)
LINS03016 (2e)
LINS03007 (2g)
LINS03001 (3a)
LINS03002 (4a)
Fig. 1. Molecular structures of the natural prototypes and the previously tested analogues from the LINS03 series.
amines (1), where ionization (represented by distribution coefficient at
pH 5.0, logD5.0) is associated to the activity and neutral compounds
(2–4) in which hydrophilic lipophilic balance (HLB) is the most im-
portant feature to increase the activity.
Purity of the tested compounds was determined using gas chromato-
graphy coupled with a mass spectrometer (GC-LRMS) on a Shimadzu
QP2010 Plus model with helium as carrier gas and silica gel capilar
column (RESTEK, model Rtx-5 ms, 30 m × 0.25 mm × 0.25 μM).
Compounds with > 95% purity were considered pure for biological
testing.
Among the promising compounds previously reported, the vanillic
acid ester (LINS03016, 2e) was the first with additional substituent in
the meta position of the aromatic ring, showing good activity against
both trypomastigotes and amastigotes forms of T. cruzi [7], when
compared to the standard drug benznidazole. In addition, analogues
with shorter side chain showed reduced activity against T. cruzi in
comparison to the longer analogues. Only the ester LINS03007 (2g)
presented moderate activity against the amastigote form [5], reinfor-
cing the hypothesis of different mechanisms of action between basic
and neutral analogues. Additionally, none of the compounds showed
important interference on plasma membrane permeability assessed
through Sytox Green method.
2.2. Synthesis of compounds 1
In a flask, 2 mmol of the corresponding aldehyde and 2 mmol
(0.258 g) of 1-octylamine were dissolved in 15 mL of MeOH and stirred
for 3h (Scheme 1). Afterwards 6 mmol (0.227 g) of sodium borohydride
were added and the reaction was kept at 25 °C overnight. The solvent
was then evaporated, and the residue was dissolved in 15 mL of EtOAc
and washed with 3 × 15 mL of distilled H2O. The organic layer was
dried over anhydrous Na2SO4 and evaporated. The crude material was
purified through silica gel chromatography, using hexane:EtOAc (1:3)
as eluent.
Considering the progress made so far, this work proposes a new
series of analogues (Fig. 2) exploiting different properties of these
molecules: (1) the addition of another substituent in the aromatic ring
on meta position (1d–f and 2d) in order to improve hydrosolubility and
volume, both associated to high activity; (2) the modification of the
substituent of the α,β-unsaturated alcohols (4b and 4c); (3) three novel
α,β-unsaturated ketones with different aromatic rings and side chain
lengths (3c, 3g and 3h) to understand the impact of the side chain
length in these neutral analogues on biological activity.
4-[(Octylamino)methyl]benzene-1,2-diol (1d). 0.2773 g of proto-
catechuic aldehyde yielded 0.2411 g (0.96 mmol, 48%) of 1d as
greenish solid (mp 85–87 °C). 1H NMR (300 MHz, CDCl3): δ = 6.64 (d,
J = 8.0 Hz, 1H), 6.54 (d, J = 7.0 Hz, 2H), 4.47 (br.s, 3H), 3.57 (s, 2H),
2.67 (t, J = 7.4 Hz, 2H), 1.60–1.45 (m, 2H), 1.35–1.15 (m, 10H), 0.87
(t, J = 6.6 Hz, 3H).13C NMR (75 MHz, DMSO): δ = 145.40, 144.35,
131.96, 119.24, 115.99, 115.58, 53.17, 48.97, 31.73, 29.78, 29.46,
29.17, 27.34, 22.57, 14.42.
2. Materials and methods
4-[(Octylamino)methyl]-2-methoxyphenol (1e). 0.3475 g of vanillin
yielded 0.2852 g (1.1 mmol, 53%) of 1e as white solid (mp 68–70 °C).
1H NMR (300 MHz, CDCl3): δ = 6.85 (d, J = 8.6 Hz, 2H), 6.78 (d,
J = 8.0 Hz, 1H), 3.89 (s, 3H), 3.71 (s, 2H), 2.62 (t, J = 7.2 Hz, 2H),
1.56–1.44 (m, 2H), 1.38–1.14 (m, 10H), 0.88 (t, J = 6.6 Hz, 3H). 13C
NMR (75 MHz, CDCl3): δ = 146.64, 144.70, 132.27, 121.00, 114.20,
110.85, 55.88, 53.99, 49.47, 31.84, 30.00, 29.54, 29.28, 27.40, 22.67,
14.11.
2.1. Chemicals and equipment
All the reagents and solvents were acquired from commercial
sources in adequate purity to be used without further purification. The
progress of the reactions was monitored through TLC in silica gel plates
with fluorescence indicator and visualized in UV at 254 nm. The hy-
drogen (1H) and carbon (13C) NMR data were acquired on a Bruker
Advance 300, operating at 300 MHz (1H) and 75 MHz (13C) in CDCl3.
Chemical shifts (δ, in ppm) were measured using TMS as internal
standard. Coupling constants are reported in hertz (Hz), if applicable.
[(3,4-Dimethoxyphenyl)methyl]octylamine (1f). 0.3401 g of vera-
traldehyde yielded 0.240 g (0.86 mmol, 43%) of 1f as orange oil. 1H
NMR (300 MHz, CDCl3): δ = 6.83–6.71 (m, 3H), 3.81 (s, 3H), 3.79 (s,
3H), 3.66 (s, 2H), 2.55 (t, J = 7.2 Hz, 2H), 1.50–1.40 (m, 2H),
2

M.T. Varela, et al.
BioorganicChemistry89(2019)102996
LINS03019 (1d)
LINS03021 (1f)
LINS03020 (1e)
LINS03022 (2d)
LINS03023 (4b)
LINS03024 (4c)
LINS03027 (3h)
LINS03026 (3g)
LINS03025 (3c)
Fig. 2. The new set of LINS03 analogues.
1.37–1.16 (m, 10H), 0.80 (t, J = 6.6 Hz, 3H).13C NMR (75 MHz,
CDCl3): δ = 148.95, 147.96, 133.17, 120.19, 111.37, 110.98, 55.91,
55.85, 53.89, 49.50, 31.84, 30.10, 29.54, 29.28, 27.40, 22.66, 14.10.
water. The organic layer was then dried over anhydrous Na2SO4 and
evaporated. The crude material was purified through silica gel chro-
matography, using hexane:EtOAc (9:1) as eluent.
(1E)-1-(4-Methoxyphenyl)dec-1-en-3-ol (4b). 0.2604 g of (1E)-1-(4-
methoxyphenyl)dec-1-en-3-oneyielded 0.2151 g (0.81 mmol, 82%) of
4b as colorless oil. 1H NMR (300 MHz, CDCl3): δ = 7.34–7.28 (m, 2H),
6.88–6.81 (m, 2H), 6.50 (d, J = 15.9 Hz, 1H), 6.07 (d, J = 15.9 Hz,
1H), 4.23 (q, J = 7.1 Hz, 1H), 3.80 (s, 3H), 1.68–1.50 (m, 2H),
1.48–1.20 (m, 10H), 0.88 (t, J = 6.7 Hz, 3H). 13C NMR (75 MHz,
CDCl3): δ = 159.26, 130.46, 129.81, 129.48, 127.64, 114.00, 73.35,
55.31, 37.45, 31.83, 29.57, 29.27, 25.51, 22.67, 14.10.
2.3. Synthesis of compound 2d
In a microwave flask, 1.5 mmol of 3,4-dihydroxybenzoic acid and
1 mmol of 1-octanol were dissolved in 3 mL of tetrahydrofuran and then
0.2 mL of concentrated sulfuric acid was added (Scheme 2). The reac-
tion proceeded in a microwave reactor (Discovery, CEM Inc.) for 1 h at
100 °C. Solvent was removed and the residue dissolved in 15 mL of
CH2Cl2 and washed with 5x15 mL of NaHCO3 saturated solution. The
organic layer was dried over anhydrous Na2SO4 and evaporated to give
the product 2d with adequate purity.
(1E)-Phenyldec-1-en-3-ol (4c). 0.2301 g of (1E)-phenyldec-1-en-3-
one (7) yielded 0.2092 g (0.9 mmol, 90%) of 4c as colorless oil. 1H NMR
(300 MHz, CDCl3): δ = 7.44–7.17 (m, 5H), 6.57 (d, J = 15.9 Hz, 1H),
6.25 (d, J = 15.9 Hz, 1H), 4.28 (q, J = 7.2 Hz, 1H), 1.72–1.51 (m, 2H),
1.49–1.15 (m, 10H), 0.88 (t, J = 6.7 Hz, 3H). 13C NMR (75 MHz,
CDCl3): δ = 136.76, 132.62, 130.21, 128.57, 127.60, 126.45, 73.15,
37.39, 31.81, 29.55, 29.25, 25.46, 22.65, 14.09.
Octyl 3,4-dihydroxybenzoate (2d). 0.231 g of 3,4-dihydroxybenzoic
acid and 0.1301 g of 1-octanol yielded 55% of the ester as a white
powder (mp 94–96 °C). 1H NMR (300 MHz, CDCl3): δ = 7.67 (d,
J = 1.7 Hz, 1H), 7.57 (dd, J = 8.3, 1.7 Hz, 1H), 6.91 (d, J = 8.3 Hz,
1H), 6.21 (br.s, 1H), 6.05 (br.s, 1H), 4.28 (t, J = 6.6 Hz, 2H), 184–164
(m, 2H), 1.47–1.18 (m, 10H), 0.88 (t, J = 6.7 Hz, 3H). 13C NMR
(75 MHz, CDCl3): δ = 167.22, 148.90, 143.20, 123.71, 122.70, 116.67,
114.81, 65.36, 31.80, 29.30, 29.20, 28.69, 26.04, 22.65, 14.09.
2.5. Synthesis of compound 3c
In a mortar, 2 mmol of benzaldehyde and 2 mmol of 2-nonanone
and 2 mmol of solid NaOH were grinded (Scheme 4) for 15 min and
afterwards neutralized with 1 M HCl. The crude material was re-
crystallized from EtOH:water giving the product 7 with adequate
purity.
2.4. Synthesis of compounds 4
To a solution of 1 mmol of the corresponding ketone in MeOH,
3 mmol of sodium borohydride were added (Scheme 3). The mixture
was stirred at 25 °C for 3 h and the solvent was evaporated. The residue
was dissolved in 15 mL of EtOAc and washed with 3x15 mL of distilled
(1E)-phenyldec-1-en-3-one (3c). 0.2130 g of benzaldehyde and
0.2845 g of 2-nonanone yielded 0.2074 g (1.8 mmol, 90%) of 3c as a
white powder (mp 50–52 °C). 1H NMR (300 MHz, CDCl3):
NaBH4
1d: R1 = R2 = OH (48%)
1e: R1 = OH; R2 = OMe (53%)
1f: R1 = R2 = OMe (43%)
Scheme 1. Synthetic scheme for the preparation of the analogues 1d-f.
3

M.T. Varela, et al.
BioorganicChemistry89(2019)102996
H2SO4
2d (55%)
Scheme 2. Preparation of the ester derivative 2d.
δ = 7.62–7.50 (m, 3H), 7.43–7.37 (m, 2H), 6.75 (d, J = 16.3 Hz, 1H),
2.66 (t, J = 7.4 Hz, 2H), 1.71–1.60 (m, 2H), 1.42–1.10 (m, 10H), 0.87
(t, J = 6.6 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ = 170.58, 147.61,
142.31, 134.64, 128.95, 128.26, 126.30, 41.00, 31.72, 29.32, 27.44,
24.42, 22.64, 14.10.
2.9. Determination of the 50% inhibitory concentration (IC50)
Extracellular trypomastigotes. Trypomastigotes were counted in a
hemocytometer chamber and seeded at 1x106 cells/well in 96-well
microplates. The compounds were diluted in RPMI-1640 medium and
incubated for 24 h at 37 °C in a 5% CO2 humidified incubator. The
trypomastigote viability was determined by the colorimetric resazurin
assay (0.011% in PBS) in a spectrophotometer microplate reader
(FilterMax F5, Molecular Devices, USA) at 570 nm. Benznidazole was
used as a standard drug. Compounds were tested to the highest con-
centration of 150 µM and were reported as not active when the IC50
value was above this concentration [8].
2.6. Synthesis of compounds 3g and 3h
In a flask, 5 mmol of 4-hydroxybenzaldehyde were dissolved in
10 mL of the corresponding ketone (acetone or 2-pentanone) and 5 mL
of EtOH (Scheme 5). Afterwards, 2 mL of a 40% NaOH solution to the
stirred solution and reacted for 2 h. The solvent was evaporated, and
the crude material was recrystallized from EtOH:water giving the pro-
ducts with adequate purity.
Intracellular amastigotes. Peritoneal macrophages were seeded at
1x105/well in 16-well chamber slides (NUNC, Thermo, USA) and
maintained for 24 h in the same medium at 37 °C in a 5% CO2 humi-
dified incubator for attachment. Non-adherent cells were removed by
two step washings with medium. After 24 h, these cells were infected
with 1x106 culture trypomastigote forms for 2 h. Subsequently, infected
cells were incubated with the compounds for 48 h. Finally, the slides
were fixed with MeOH, stained with Giemsa, and observed in light
microscopy. The parasite load was defined by counting 200 macro-
phages/well by evaluating the infection index defined by the equation
(number of infected macrophages)x(number of intracellular amasti-
gotes)/number of total macrophages (out of 200 macrophages).
Benznidazole was used as the standard drug [8].
(1E)-1-(4-Hydroxyphenyl)but-1-en-3-one (3g). 0.6272 g of 4-hydro-
xybenzaldehyde yielded 0.502 g (3.1 mmol, 62%) of 3g as yellowish
solid (mp 109–112 °C).1H NMR (300 MHz, CDCl3): δ = 7.55–7.36 (m,
3H), 6.87 (d, J = 8.7 Hz, 2H), 6.61 (d, J = 16.1 Hz, 1H), 5.55 (br. s,
1H), 2.37 (s, 3H).13C NMR (75 MHz, CDCl3): δ = 199.38, 158.51,
144.08, 130.35, 126.81, 124.69, 116.13, 27.33.
(1E)-1-(4-Hydroxyphenyl)hex-1-en-3-one (3h). 0.6305 g of 4-hydro-
xybenzaldehyde yielded 0.542 g (2.85 mmol, 57%) of 3h as yellowish
solid (mp 116–121 °C).1H NMR (300 MHz, CDCl3): δ = 7.58–7.40 (m,
3H), 6.86 (d, J = 8.62 Hz, 2H), 6.63 (d, J = 16.1 Hz, 1H), 5.26 (br. s,
1H), 2.63 (t, J = 7.3 Hz, 2H), 1.81–1.61 (m, 2H), 0.98 (m, 3H).13C NMR
(75 MHz, CDCl3): δ = 201.20, 158.06, 142.54, 130.24, 127.21, 124.01,
116.02, 42.70, 18.02, 13.89.
2.10. Cytotoxicity in mammalian cells
NCTC cells were counted in a hemocytometer chamber, seeded at
6 × 104/well and incubated in with the compounds (200–1.56 µM) for
48 h at 37 °C in a 5% CO2-humidified incubator. The cell viability was
determined using the MTT assay [9]. Benznidazole was used as a
standard drug. The selectivity index (SI) was determined using the re-
lationship between CC50 against NCTC cells/IC50 against parasites.
2.7. Biological assays
BALB/c mice were obtained from the animal breeding facility at the
Instituto Adolfo Lutz-SP, Brazil. The animals were maintained in ster-
ilized cages under a controlled environment, and received water and
food ad libitum. Animal procedures were performed with the approval
of the Research Ethics Commission, in agreement with the Guide for the
Care and Use of Laboratory Animals from the National Academy of
Sciences.
2.11. Evaluation of plasma membrane permeability
Compounds 4 and 6 were incubated with trypomastigotes of T. cruzi
aiming to study the plasma membrane permeability using the fluor-
escent probe Sytox Green (Molecular Probes Inc.). Sytox Green is a
high-affinity nucleic acid stain that penetrates cells with compromised
plasma membranes and enhances its fluorescence by more than 500-
fold upon nucleic acid binding. The parasite cells were washed in
phosphate-buffered saline, suspended at 2x106 cells/well and incubated
for 15 min with 1 µM of Sytox Green in Hanks' balanced salts solution
(HBSS; Sigma-Aldrich) supplemented with 10 mM
2.8. Trypanosoma cruzi parasites and mammalian cell maintenance
Trypomastigotes of T. cruzi (Y strain) were maintained in Rhesus
monkey kidney cells (LLC-MK2-ATCC CCL 7), cultivated in RPMI-1640
medium supplemented with 2% FBS at 37 °C in a 5% CO2-humidified
incubator. Macrophages were collected from the peritoneal cavity of
BALB/c mice by washing them with RPMI-1640 medium supplemented
with 10% FBS and were maintained at 37 °C in a 5% CO2-humidified
incubator. The murine conjunctive cells (NCTC clone 929, ATCC) and
LLC-MK2 were maintained in RPMI-1640 supplemented with 10% FBS
at 37 °C in a humidified atmosphere containing 5% CO2.
(HBSS + Glu) as described [10]. The compounds were addDe-dGlautcothsee
previously determined IC50 and the fluorescence was measured every
10 min for a total period of 120 min. Maximum fluorescence was de-
termined by the addition of 0.5% of the non-ionic surfactant Triton X-
100 (positive control). The control group consisted of untreated
Scheme 3. Preparation of the α,β-un-
saturated alcohols 4b and 4c.
NaBH4
3b: R1 = OMe
3c: R1 = H
4b: R1 = OMe (82%)
4c: R1 = H (90%)
4

M.T. Varela, et al.
BioorganicChemistry89(2019)102996
Scheme 4. Preparation of the α,β-un-
saturated ketone 3c.
NaOH
3c (90%)
Scheme 5. Preparation of the α,β-unsaturated
ketones 3g and 3h.
NaOH
3g: n = 0 (62%)
3h: n = 2 (57%)
trypomastigotes. Fluorescence was measured using a fluorimetric mi-
croplate reader (FilterMax F5, Molecular Devices, USA) with excitation
and emission wavelengths of 485 and 520 nm, respectively [10,11].
neutralization with HCl, afforded 3c with high yield and purity [14].
This reaction is a nice example of a green chemistry approach, since no
solvent, heating or electric energy were necessary.
2.12. Group efficiency analysis
3.2. Biological evaluation
Group efficiency (GE) values were calculated using the Eq. (1):
The antiparasitic activity of the compounds 1–4 was determined in
vitro against trypomastigote (extracellular) and amastigote (in-
tracellular) forms of T. cruzi (IC50) and mammalian NCTC cells in order
to determine cytotoxicity (CC50) and calculate the selectivity index of
the analogues, following the same methodology described in Varela
et al. [7] (Table 1).
GE = (1.37 × pIC50)/ HA
(1)
where ΔpIC50 is the variation of the negative logarithm of IC50 value in
molar between the substituted and non-substituted compound and ΔHA
is the variation of heavy (non-hydrogen) atoms in the specific sub-
stituent [12].
As can be noted in Table 1, all the long side chain compounds (a-f)
showed activity against the trypomastigote forms of T. cruzi; compound
1d was the most potent (IC50 4.8 μM), followed by compounds 2d and
4b (IC50 5.3 and 9.4 μM, respectively). Two compounds were active
against the replicative form (amastigotes) of T. cruzi (2b and 4c),
showing IC50 values of 13.9 μM and 5.8 μM, respectively. Additionally,
the α,β-unsaturated alcohol 4c showed the highest SI (34.5) among the
27 analogues in the LINS03 series tested so far considering the amas-
tigote form of the parasite [5–7]. Intracellular amastigotes are the
clinical relevant form of the parasite and persist in the chronic phase of
the disease [15]. Additionally, amastigotes are responsible for the
amplification of the parasitic load and thus the anti-amastigote activity
could reduce the parasitic burden in chronic patients. This compound
also showed an IC50 value of 9.8 μM against the trypomastigotes. Try-
pomastigotes can also be found in humans, causing an elevated para-
sitemia in the acute phase of the disease; drugs lacking efficacy against
this parasite form, may leave trypomastigotes alive, resulting in the
recrudescence of the disease after treatment [16]. The results obtained
for compound 4c are comparable to the standard drug benznidazole.
The promising results in both parasite forms suggest that this compound
should be further evaluates as lead compound. Additionally, com-
pounds with antitrypomastigote activity can help avoiding the spread of
the infection [17]. The selectivity of 4c against both forms of the
parasite were at least 20-fold higher than the cytotoxicity to mamma-
lian cells, fulfilling the requirements of the DNDi for a hit compound
against CD [16].
2.13. Descriptor calculations
Molecular surface area (MSA), logD5.0 and HLB descriptors were
calculated using Marvin Sketch 16.1.8.0 (ChemAxon Inc.) using the
default configuration in the software [7].
3. Results and discussion
3.1. Chemistry
The amines 1d–1f were prepared through reductive amination
method, i.e. by the formation of the imine in situ that is further reduced
to its amine analogue, without isolation of the imine [7]. This led to
moderate yields possibly due to competition with the reduction of the
aldehyde to its corresponding alcohol. Ester 2d was prepared through
classic Fischer esterification adapted to a microwave reactor. This
adaptation was done due to the possible oxidation of the catechol
moiety, especially when high concentration of the catalyst is employed.
By using the microwave instead of the conventional method (using
Dean-Stark apparatus and reflux) previously reported [7], it was pos-
sible to keep a more uniform and stable temperature (100 °C) which
facilitates the dehydration step and thus less acid may be used [13].
This led to better yield than the previous report.
The α,β-unsaturated alcohols 4b and 4c were obtained from the
reduction of their corresponding carbonyl analogues 3 by using sodium
borohydride as reductive agent, giving excellent yields [5]. The analysis
of the NMR spectra showed that only the trans isomer is obtained and
that the reduction was selective to the carbonyl group (1,2-reduction).
This may be explained by the stabilization of the C]C bond by con-
jugation to the aromatic ring, making the 1,4-reduction less feasible.
The α,β-unsaturated ketones 3 were synthesized following base-cata-
lyzed aldol condensation between and aromatic aldehyde and alkyl
ketone using sodium hydroxide as a basic catalyst. Compound 3c was
not isolated when the conventional methodology was used (i.e. with the
reagents dissolved in ethanol). As alternative, a solvent-free reaction
was carried out by grinding the reactants benzaldehyde and 2-non-
anone with solid sodium hydroxide (1 equivalent each) in a mortar.
This alternative methodology furnished a solid mixture, that after
Interestingly, the 4-methoxybenzenic α,β-unsaturated alcohol, 4b
was inactive against the amastigotes of the parasite but it showed ac-
tivity against the trypomastigote form, similarly to its non-substituted
analogue 6. The 4-hydroxy analogue of these structures (4a) was also
active against trypomastigotes with an IC50 value of 6.1 μM but was
inactive against the amastigotes [5]. This suggests for these reduced
analogues, the absence of a substituent in the aromatic ring favors the
antiparasitic activity for both forms of the parasite. Moreover, the
substitution of the hydroxyl group for a methoxyl or hydrogen im-
proved significantly the selectivity towards the parasitic cells in com-
parison to the NCTC cells. On the other hand, this profile seems op-
posite to the observed activity of the α,β-unsaturated ketones.
Compounds 3c, 3g and 3h were inactive against amastigotes and only
compound 3c showed comparable activity (IC50 16.5 μM, SI 12.1) to its
5

M.T. Varela, et al.
BioorganicChemistry89(2019)102996
Table 1
Antiparasitic activity of the compounds against trypomastigote and amastigote forms of T. cruzi and cytotoxicity against mammalian NCTC cells.
Code
Structure
T. cruzi IC50 μM ( SD)
CC50 μM ( SD)
SI
Trypomastigotes (TCT)
Amastigotes (TCA)
> 100
NCTC
TCT
2.1
TCA
–
1d
1e
1f
4.8
2.4
0.3
2.8
3.8
9.9
0.6
4.6
16.4
12.2
5.3
> 100
> 100
53.0
21.7
107.7
3.2
–
16.1
7.0
1.8
–
2d
13.9
3.2
20.3
7.7
4b
4c
3c
9.4
1.7
1.5
> 100
5.8
> 200
> 200
> 200
> 21.3
> 20.4
> 12.2
–
9.8
0.1
> 34.5
–
16.5
3.7
> 100
3g
> 100
> 100
> 200
–
–
3h
> 100
16.4
> 100
5.2
> 200
> 200
–
–
BZN
4.1
3.1
> 12.2
> 38.5
BZN: benznidazole; ND: not determined; IC50: 50% effective concentration; CC50: 50% cytotoxic concentration; SI: Selectivity Index calculated from CC50/IC50
.
phenolic analogue 3a (IC50 11.8 μM, SI 7.0), but with higher selectivity.
However, the 4-hydroxy and 4-methoxy analogues (3a and 3b) pre-
sented moderate activity against amastigotes, suggesting that the pre-
sence of these polar groups in the aromatic ring increased the activity
against this intracellular form.
hydroxy derivative 1a, which was considerably active against amasti-
gotes (IC50 5.5 μM) but was also cytotoxic to mammalian cells (CC50
23.5 μM) [5]. Since the 4-methoxy and the non-substituted analogues
showed lower cytotoxicity [6,7], the higher toxicity of 1a was corre-
lated to the presence of the known toxicophoric phenol group [18,19].
In fact, all the phenolic analogues showed higher cytotoxicity than the
non-phenolic, corroborating to the hypothesis. However, all the amine
derivatives previously reported also showed high cytotoxicity that
could be associated with the characteristic of this functional group [7],
although they presented high activity against T. cruzi amastigotes in-
deed. Considering that the HLB is a key factor to achieve high activity
with lower cytotoxicity, the analogues 1d-1f were designed to detect if
an additional substitution in meta-position would improve the activity
against the parasite without increasing the cytotoxicity.
A recent work [7] reported that the 3-methoxy-4-hydroxy sub-
stitution pattern on the aromatic ring led to improvement on the anti-
trypanosoma activity (IC50 4.4 μM and 6.8 μM for TCT and TCA, re-
spectively) and selectivity (SI 28.0 and 18.8, respectively) for the ester
derivative 2e. In the present work, it was found that the 3,4-dihydroxy
substitution pattern of compound 2d gives comparable results against
the trypomastigote form (IC50 5.3 μM, SI 20.3) but it led to lowered
activity in a 2-fold order against the amastigote form (IC50 13.9 μM)
and decreased selectivity (SI 7.8). The 3-methoxy-4-hydroxy substitu-
tion pattern also led to lowered lipophilicity, leading to the improve-
ment the biological activity and reduced cytotoxicity as well, which is
corroborated by Varela et al. [7]. This also reinforces that the phenolic
hydroxyl group is intrinsically linked to cytotoxicity as well, whereas
compound 2d is the most cytotoxic among the neutral compounds in
this series.
Curiously, this behavior was not observed for these amine deriva-
tives. The insertion of a meta-substituent led to inactivity against the
amastigote form of the parasite, but retained the activity against try-
pomastigote forms. Accordingly, the high cytotoxicity of such com-
pounds virtually confirm the toxicophoric characteristic of both phenol
and amine groups. As can be noted in the compound 1d, the insertion of
an additional hydroxyl in the 3-position increased significantly the
cytotoxicity to mammalian cells, while the presence of the 3-methoxy
group in compound 1e led to lower cytotoxic activity than the 1a [5].
On the other hand, the insertion of this 3-methoxy group in compound
1b (CC50 31.9 μM) [7] resulted in compound 1f which present increased
cytotoxicity (CC50 21.7 μM). This suggests that the insertion of the
additional substituent in the 3-position of the amine derivatives is
detrimental to the activity against TCA and decreases the selectivity of
such compounds. Moreover, this also reinforces the hypothesis that the
amine derivatives may act through a different mechanism of action
from the non-ionizable molecules 2–4 [7].
The first publication with these compounds [5] reported that lower
homologues 1g and the 2g, both containing a side chain with 6-atom
length, showed poor activity against T. cruzi. Only the compound 2g
presented moderate activity against the amastigotes of the parasite
(IC50 26.4 μM), while 1g did not showed significant activity. Com-
pounds 3g and 3h were tested to determine whether the length of the
side chain could affect the antiparasitic activity and cytotoxicity in the
α,β-unsaturated series. This modification produced again inactive
analogues against both forms of the parasite (Table 1). However, they
also showed low cytotoxicity to the mammalian cells. This may be due
to the lower lipophilicity of these compounds, which impairs the per-
meation through the cell membrane. Given this, it seems that the re-
duction of the side chain length is detrimental to the activity of such
compounds, corroborating to the hypothesis that there is an optimal
lipophilicity to achieve highly active compounds.
3.3. Membrane disruption activity
Oliveira et al. [11] reported that the natural product gibbilimbol B
had a membrane disruption activity, possibly due to the structural
The first amine derivative evaluated by our group was the 4-
6

M.T. Varela, et al.
BioorganicChemistry89(2019)102996
Table 3
Calculated values of logD5.0 and HLB for the basic analogues.
Compound Chemotype
R
logD5.0 HLB
HLB/
IC50 TCA
(μM)
logD5.0
1a
1b
1c
1d
1e
Amine
Amine
Amine
Amine
Amine
4-OH
4-OMe
H
1.11
1.26
1.41
8.75
8.82
7.14
7.88
7.00
5.06
5.5a
1.33b
13.3c
> 100
> 100
3,4-di-OH 0.84
3-OMe-4- 0.96
OH
10.30 12.26
10.31 10.74
Fig. 3. Effects on plasma membrane permeabilization of compounds 2d and 4c
in trypomastigotes of T. cruzi. Sytox Green dye (1 µM) fluorescence was spec-
trofluorometrically measured every 10 min. Minimum (untreated) and max-
imum permeabilization (TX-100) were obtained. Fluorescence was quantified
by calculating the mean percentages of untreated (0%) and TX-100%-treated
(100%) parasites.
1f
Amine
3,4-di-
OMe
4-OH
4-OMe
H
1.11
10.25 9.23
> 100
5a
5b
5c
Imine
Imine
Imine
1.39
1.53
1.65
3.34
3.43
1.72
2.40
2.24
1.04
> 100a
> 100c
50c
a
b
c
similarity between this alkylphenol and the membrane phospholipids
(polar head and apolar tail). This surfactant-like characteristic could to
favor the interaction between the gibbilimbol and the ergosterol of the
membrane giving to this molecule the capacity of forming a pore, like
amphotericin B for Leishmania (L.) infantum [20], a parasite of the same
family of T. cruzi. The assay uses the Sytox Green dye, which is not able
to cross intact plasma membrane. If the membranes’ stability is com-
promised, this dye becomes able to enter into the cells and bind to the
nucleic acid, becoming fluorescent. Previous work7 reported the eva-
luation of the most active compounds in this model and only two (1b
and 2e, amine and ester, respectively) showed to promote a small in-
crease (< 20%) in the intracellular fluorescence, suggesting that these
compounds may led to some alteration in the plasma membrane in-
tegrity. This behavior could also justify the cytotoxicity to mammalian
cells, since the mammalian plasma membrane could also be affected as
well.
Available from Varela et al. [5].
Available from Varela et al. [7].
Available from Varela et al. [6].
become protonated, accumulating inside this compartment and ex-
erting their activity against the amastigotes. The compounds 1d-1f are
considerably more hydrophilic than the former amine analogues due to
the presence of the oxygenated substituents, which lead the
logD5.0 < 1.1 (Table 3). This data was confirmed by the lower values
of the calculated logD5.0 for these compounds.
However, the corresponding imines are also basic, with similar
logD5.0 values to the amines and unfortunately they showed poor ac-
tivity against the amastigote forms of T. cruzi. The explanation to this
different activity profile may be correlated to the HLB values. The
imines present lower HLB values (< 4.0) than the amines (> 7.0),
suggesting that the hydrophilicity may also be a key factor related to
the activity. On the other hand, the compounds 1d–1f are more hy-
drophilic than the mono-substituted amines (such as 1b) as expressed
by the higher HLB values, which can difficult their penetration in the
parasite cells and their antiparasitic activity indeed.
The two active derivatives of this set (2d and 4c) were submitted to
the plasma membrane assay for a period of 120 min incubation (Fig. 3).
None of the both compounds increased significantly (p > 0.05) the
fluorescence levels when compared to positive control (TX-100) in a
similar manner than observed with the former derivatives [7] (Table 2).
This indicates that the lethal action of both compounds is other than
causing a membrane disruption and should be evaluated for future
optimization studies.
When the information given by these two descriptors are putted
together, it can be suggested that there is an optimal value for the
logD5.0 and HLB values, which can be described as the HLB/logD5.0
ratio. The amines with anti-amastigote activity present HLB/logD5.0
ratio values between 5 and 8, while the more lipophilic imines present
HLB/logD5.0 ratio value < 3. The di-substituted amines, in counterpart,
present HLB/logD5.0 ratio values > 9. Considering this, further amine
analogues should be designed considering the HLB/logD5.0 ratio in this
ideal range. Possibly this range indicates the optimal lipophilicity value
to reach the intracellular environment adequately.
3.4. Ionization and hydrophilic lipophilic balance
Even though the amines of this set proved to be inactive against
amastigotes after the addition of a substituent in meta-position of the
aromatic ring, the most active derivative against this form is still 1b
(methoxy group in the para-position) [7]. This conducted to a more
systematic investigation on why the amines presented herein showed
low activity against the amastigotes.
3.5. Group efficiency
It was observed that the ionization capability and the HLB play
important roles in the activity of these amine analogues [7]. Regarding
the ionization, the logD5.0 showed important correlation to the activity
of such basic compounds, possibly indicating that these molecules
should penetrate the macrophages in the unionized form, and when
reaching the more acidic pH in the intracellular environment they
To evaluate the efficiency of the inserted groups in the amine de-
rivatives 1, group efficiency (GE) analysis was carried out. The GE
analysis is a measurement of the binding efficiency for an added
functional group in the parent molecule [12]. Accordingly, it represents
how much the added group contributed to overall affinity of the mo-
lecule, considering the number of heavy atoms [21]. This only makes
sense when a given set of compounds act binding in the same molecular
target and accordingly it was assumed that these amine derivatives act
through the same mechanism of action [22].
Table 2
Relative permeability to the Sytox Green probe in T. cruzi trypomastigotes.
Compound
Relative permeability to TX-100 (mean
SD, n = 3)
The GE analysis (Table 4) indicated that the insertion of substituents
on the 3-position of the aromatic ring play different roles in the activ-
ities against trypomastigotes, amastigotes and mammalian cells. Con-
sidering the activity against trypomastigotes, the insertion of the 3-OH
group led to important improvement against the parasite, with a GE
2d
0.8
2.4
0.6
0.8
4c
−0.5
0.5
untreated
7

M.T. Varela, et al.
BioorganicChemistry89(2019)102996
Table 4
ketones, but not of the corresponding alcohols.
GE analysis of the substituent in the 3-position of the amine analogues (1)
calculated using Eq. (1).
As can be seen in Table 5, the active analogues 4c, 3a and 3b have
logD5.0 values ranging from 4.98 to 5.23. This suggests that the optimal
logD5.0 value seems around 5.00. More hydrophilic compounds (such as
4a, 4b, 3g and 3h) and more lipophilic compounds (3c) showed to be
inactive. However, this data alone could not explain the role of the
substituents in the aromatic ring.
3-R
GE pIC50 TCT
GE pIC50 TCA
GE pCC50
OH
0.75a
< −1.73a
< −0.86a
< −1.29b
0.51a
OMe
0.01a
−0.24a
0.11b
−0.31b
The presence of substituents increases the MSA values, but as can be
seen in Table 5, there is no correlation between MSA and IC50 values.
By weighting the MSA values with the logD5.0, it can be seen that a
MSA/logD5.0 ratio around 85 seems optimal. This suggests that the
insertion of a hydroxyl or methoxyl groups (a and b, respectively) leads
to higher MSA values, but is also decreases the logD5.0, impairing the
biological activity of the alcohols 4 (which are more hydrophilic) and
improving the activity of the ketones 3 (which are more lipophilic). In
summary, the substituents on the aromatic ring seem to contribute to
optimize the lipophilicity of the molecule instead of performing a
specific interaction with the biological target.
a
b
Calculated based on compound 1a.
Calculated based on compound 1b.
Table 5
Lipophilicity and volume descriptors calculated for the α,β-unsaturated com-
pounds.
Compounds
X
n
R
logD5.0 MSA
MSA/
IC50 TCA (μM)
3.7. Conclusions
logD5.0
In summary, nine LINS03 analogues with antitrypanosoma activity
were reported, and compounds 2d and 4c showed a promising effect
due to their potency against T. cruzi amastigotes with high selectivity.
Additionally, none of these compounds induced alteration of plasma
membrane of T. cruzi. The balance between lipophilicity and hydro-
solubility showed to be the key factors associated with the activity of
the analogues, and must be considered in a novel set of compounds.
These SAR results will certainly aid the design of future analogues of
the LINS03 series with improved activity.
3a
3b
3c
3g
3h
4a
4b
4c
C]O
C]O
C]O
C]O
C]O
CHOH
CHOH
CHOH
6
6
6
0
2
6
6
6
OH
5.09
422.33 82.97
459.81 87.92
411.53 76.35
236.60 109.54
299.77 90.56
432.51 92.61
470.11 97.53
421.99 84.73
22.5a
OMe 5.23
26.2b
> 100
> 100
> 100
> 100a
> 100
5.8
H
5.39
2.16
3.31
4.67
OH
OH
OH
OMe 4.82
H
4.98
a
Available from Varela et al. [5].
Available from Varela et al. [6].
b
Declaration of Competing Interest
The authors declare none.
Acknowledgements
value of 0.75. The insertion of the 3-OMe group did not contribute or
even worsen the efficacy of the compounds 1e and 1f in comparison to
the prototypes 1a and 1b. Curiously, the analysis indicates that this role
is totally different regarding the anti-amastigote activity. The insertion
of any substituent in the 3-position led to loss of efficacy against the
intracellular form of T. cruzi (negative GE values). However, the results
suggest that the role of the 3-substituents is more important in the
cytotoxicity than in the antiparasitic activity. The insertion of the 3-OH
group led to increased cytotoxicity with considerable efficiency, sug-
gesting that this group contributes more significantly to the cytotoxicity
of compound 1d instead to increase the antitrypanosomal activity and
corroborating to the hypothesis that the phenol is a toxicophore [18].
Accordingly, the insertion of the 3-OMe group did not contribute to the
cytotoxicity or even decreased this effect, as can be verified by the small
or negative GE values for the 3-OMe group.
The authors are grateful to São Paulo Research Foundation
(FAPESP) [Grants 2016/25028-3; 2018/03918-2; 2018/07885-1,
2015/23403-9] and to National Council for Scientific and
Technological Development (CNPq) [Grant 455411/2014-0] for fi-
nancial support to this work and to the Scientific award to JHGL, AGT
and JPSF [Grant 306355/2018-3].
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