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S.M. de Morais et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
themselves, or lead to the development of new therapeutic sub-
stances with similar structure.6 Many studies have validated the
effect of natural products as potential sources of new and selective
agents for the treatment of tropical diseases caused by protozoa
and other parasites.7
Thymol and eugenol are largely used as flavoring agents for
food because they are the main constituents of oregano and cloves,
respectively, and they display many pharmacological actions
including antimicrobial,8,9 anti-inflammatory,10,11 fungicidal,12,13
and antioxidant activity.14,15 Common medicinal plants used by
local populations include Ocimum gratissimum, whose essential
oil and main constituent eugenol displays anthelmintic and anti-
microbial activities,16,17 and Lippia sidoides, which is rich in the
general antiseptic thymol.18
The cytotoxicities and antileishmanial activities of thymol and
its hemisynthetic derivatives, as well as quinoline–triclosan and
quinoline–eugenol hybrids have been demonstrated both in vitro
and in vivo.19,20 Lippia sidoides Cham. Essential oil was shown to
display in vitro cytotoxicity and antileishmanial activity,21 and
the antileishmanial activity of Eugenol-rich essential oil from Oci-
mum gratissimum was also reported.22
In Northeastern Brazil leishmaniasis is an endemic disease,
causing lethal cases among humans and dogs. The Brazilian Health
Care Ministry cites the incidence of VL as 2 cases per 100,000 habi-
tants.23 The incidence of canine leishmaniasis is underestimated
due to the incomplete communication between the private clinics
vets and the authorities. Due to concern about the lack of effica-
cious antileishmanial drugs, we surveyed natural active com-
pounds to find new molecules with enhanced activity against
Leishmania and low toxicity for host cells. Based on this, thymol
and eugenol were chosen as lead compounds from which acetyl
and benzoyl derivatives could be synthesized and tested for their
antileishmanial activity in vitro and in vivo against Leishmania
infantum chagasi life stages.
polarity. The fractions were collected and compared by TLC. The
chemical structures of the purified compounds were confirmed
by spectroscopic analysis of the nuclear magnetic resonance spec-
tra recorded on a Bruker Avance DRX-500 spectrometer, in CDCl3.
O-acetyl-thymol (AT): 1H NMR (d, ppm, J Hz): 7.25 (H-3, d,
J = 7.7 Hz), 7.02 (H-4, d, J = 7.7), 6.85 (H-6, s), 3.02 (H-7, heptete,
J = 6.9 Hz), 1.29 (H-8, d, J = 6.9 Hz), 1.29 (H-9, d, J = 6.9 Hz), 2.34
(H-10, s). 13C NMR (d, ppm, CHCl3): 148.8 (C-1), 116.5 (C-2),
126.0 (C-3), 126.0 (C-4), 134.9 (C-5), 122.1 (C-6), 34.4 (C-7), 24.1
(C-8), 24.1 (C-9), 21.3 (C-10), 170.3 (C-11), 20.0 (C-12).
O-benzoyl-thymol (BT); 1H NMR (d, ppm, CHCl3): 7.28 (H-3, d,
J = 7.7 Hz), 7.12 (H-4, d, J = 7.7 Hz), 7.0 (H-6, s), 8.29 (H-13, m),
7.59 (H-14, m), 7.68 (H-15, m), 7.59 (H-16, m), 8.29 (H-17, m).
13C NMR (d, ppm, CHCl3): 135.5 (C-1), 138.6 (C-2), 126.3 (C-3),
126.0 (C-4), 134.9 (C-5), 122.1 (C-6), 34.4 (C-7), 24.1 (C-8), 24.1
(C-9), 21.3 (C-10), 166.8 (C-11), 148.5 (C-12), 129.7 (C-13), 128.4
(C-14), 133.3 (C-15), 128.4 (C-16), 129.7 (C-17).
O-acetyl-eugenol (AE): 1H NMR (d, ppm, CHCl3): 6.9 (H-3, s), 6.7
(H-5, d, J = 7.9 Hz), 6.9 (H-6, d, J = 7.9 Hz), 3.4 (H-7, d, J = 6.7), 5.9
(H-8, m), 5.1 (H-9, m), 3.8 (H-10, s, OCH3), 2.3 (H-12, s, CH3). 13C
NMR (d, ppm, CCCl3): 138 (C-1), 150.8 (C-2), 112.7 (C-3), 138.9
(C-4), 120.6 (C-5), 122.5 (C-6), 40 (C-7), 137 (C-8), 116.1 (C-9),
55.7 (C-10), 169.1 (C-11), 20.60 (C-12).
O-benzoyl-eugenol (BE): 1H NMR (d, ppm, CHCl3): 6.8 (H-3, m),
6.8 (H-5, m), 7.1 (H-6, d, J = 7.6 Hz), 3.4 (H-7, d, J = 6.2 Hz), 6.0 (H-8,
m), 5.2 (H-9, m), 3.8 (H-10, OCH3), 8.2 (H-13, m), 7.5 (H-14, m), 7.6
(H-15, m), 7.5 (H-16, m), 8.2 (H-17, m). 13C NMR (d, ppm, CCCl3):
129.44 (C-1), 151.03 (C-2), 112.78 (C-3), 138.93 (C-4), 120.62
(C-5), 122.55 (C-6), 40.00 (C-7), 137.00 (C-8), 116.02 (C-9), 55.74
(C-10), 164.75 (C-11), 138.14 (C-12), 130.16 (C-13), 128.3 (C-14),
133.28 (C-15), 128.3 (C-16), 130.16 (C-17).
2.4. Antileishmanial activity
A Brazilian strain of L. infantum chagasi (Lic; MHOM/BR/00/
1669) was originally isolated from a patient with visceral leish-
maniasis. A strain expressing luciferase (Lic-luc), was generated
from a hamster-passaged clinical isolate in the lab of MEW as
described by Thalhofer et al.25, using the integrating vector pIR1-
SAT kindly provided by Dr. Steve Beverley (Washington University,
St. Louis). The promastigote form of Lic-Luc was cultured in a
hemoflagellate-modified MEM (HOMEM), supplemented with
10% fetal bovine serum at 26 °C. Thymol, acetyl-thymol, benzoyl-
thymol, eugenol, acetyl-eugenol and benzoyl-eugenol were dis-
solved in DMSO at a concentration of 0.2% and diluted in HOMEM
in 96-well microplates. The assay was performed at concentrations
2. Materials and methods
2.1. Acetylation reaction
Eugenol and thymol were purchased from VETEC, in Fortaleza,
Brazil. A mixture of acetic anhydride (6 g) and pyridine (2 g) was
added to eugenol or thymol (3 g). The mixture was left stirring
for 24 h at room temperature, and then cold water was added
(20 mL). The solution was neutralized to pH 7.0. The reaction mix-
ture was transferred to a separating funnel and washed three times
with chloroform (20 mL). The chloroform layer containing acety-
lated material was washed with water and then dried with anhy-
drous sodium sulfate. The solvent was evaporated under reduced
pressure.
of 100, 50, 25, 12.5 and 6.25 lg/mL. Control wells contained DMSO
or no additives. Promastigotes were counted in a Neubauer hemo-
cytometer and seeded at 1 Â 106/well. After 24 h at 26 °C incuba-
tion in drug or control conditions, the number of remaining
viable promastigotes was assessed using a luciferase assay. Tripli-
cate samples from each condition were tested for luciferase activ-
ity in a 96-well plate (adapted methodology from Lang et al.26).
Pentamidine (Sigma-Aldrich, St. Louis, MO, USA) was used as the
positive control drug. The optical density (OD) was determined
in a Fluostar Omega (BMG Labtech) at 620 nm.
The drug activity against amastigotes was measured using a
modified ELISA to detect intracellular parasites as a measure of
the growth of L. i. chagasi in the murine macrophage RAW 264.7
cell line (ATCC TIB-71) in the absence or presence of drug (method
adapted from Piazza et al.27). 4 Â 105 RAW 264.7 cells in
RPMI-1640, 10% foetal bovine serum, were added to each well of
a 96-well plate. After 24 h they adhered to plates, and L. i. chagasi
promastigotes were added at a 1:10 (macrophage/promastigote)
ratio at 37 °C, 5% CO2. No adherent promastigotes were removed
by rinsing after 2 h, and macrophages were returned to the CO2
2.2. Benzoylation reaction
Eugenol (8.2 g or 0.05 mol) or thymol (7.5 g or 0.05 mol) was
dissolved in 40 mL of 5% NaOH in the cold, and benzoyl chloride
(7 g/5.8 ml, i.e., 0.05 mol) was added. The mixture was vigorously
mixed until the odor of benzoyl chloride disappeared (20–
25 min). The solid product was filtered on a Buchner funnel,
washed with cold water, recrystallized with 60 mL of rectified
spirit, and collected on a filter.24
2.3. Analysis of synthesized derivatives
The crude synthesized derivatives were subjected to silica gel
column chromatography and eluted with mixtures of hexane,
dichloromethane, ethyl acetate and methanol with increasing