Thymol and eugenol derivatives as potential antileishmanial agents

Article abstract of DOI:10.1016/j.bmc.2014.08.020

In Northeastern Brazil visceral leishmaniasis is endemic with lethal cases among humans and dogs. Treatment is toxic and 5-10% of humans die despite treatment. The aim of this work was to survey natural active compounds to find new molecules with high activity and low toxicity against Leishmania infantum chagasi. The compounds thymol and eugenol were chosen to be starting compounds to synthesize acetyl and benzoyl derivatives and to test their antileishmanial activity in vitro and in vivo against L. i. chagasi. A screening assay using luciferase-expressing promastigotes was used to measure the growth inhibition of promastigotes, and an ELISA in situ was performed to evaluate the growth inhibition of amastigote. For the in vivo assay, thymol and eugenol derivatives were given IP to BALB/c mice at 100 mg/kg/day for 30 days. The thymol derivatives demonstrated the greater activity than the eugenol derivatives, and benzoyl-thymol was the best inhibitor (8.67 ± 0.28 μg/mL). All compounds demonstrated similar activity against amastigotes, and acetyl-thymol was more active than thymol and the positive control drug amphotericin B. Immunohistochemistry demonstrated the presence of Leishmania amastigote only in the spleen but not the liver of mice treated with acetyl-thymol. Thus, these synthesized derivatives demonstrated anti-leishmanial activity both in vitro and in vivo. These may constitute useful compounds to generate new agents for treatment of leishmaniasis.

Full text of DOI:10.1016/j.bmc.2014.08.020

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Thymol and eugenol derivatives as potential antileishmanial agents
a
a,c
Selene Maia de Morais a,b, , Nadja Soares Vila-Nova , Claudia Maria Leal Bevilaqua
,
⇑
Fernanda Cristina Rondon c, Carlos Henrique Lobo d, Arlindo de Alencar Araripe Noronha Moura d,
Antônia Débora Sales a, Ana Paula Ribeiro Rodrigues a, José Ricardo de Figuereido a,
Claudio Cabral Campello a,e, Mary E. Wilson f,g, Heitor Franco de Andrade Jr. h
a Programa de Pós-Graduação em Ciências Veterinárias, Universidade Estadual do Ceará, Avenida Paranjana 1700, Campus do Itaperi, 60740-000 Fortaleza, Ceará, Brazil
b Curso de Quimica, Centro de Ciências e Tecnologia, Universidade Estadual do Ceara, Avenida Paranjana 1700, Campus do Itaperi, 60740-000 Fortaleza, Ceará, Brazil
c Laboratório de Doenças Parasitárias, Universidade Estadual do Ceará, Avenida Paranjana 1700, Campus do Itaperi, 60740-000 Fortaleza, Ceará, Brazil
d Laboratório de Biologia da Reprodução, Universidade Federal do Ceará, Av. Mister Hull s/n, Campus do Pici, 60021-970 Fortaleza, Ceará, Brazil
e Faculdade de Veterinária – FAVET, Avenida Paranjana 1700, Campus do Itaperi, 60740-000 Fortaleza, Ceará, Brazil
f Department of Internal Medicine, University of Iowa and the VA Medical Center, Iowa City, IA 52242, USA
g Department of Microbiology, University of Iowa and the VA Medical Center, Iowa City, IA 52242, USA
h Laboratório de Protozoologia, Universidade de São Paulo, Avenida Eneas de Carvalho Aguiar, 470, 05403-000 São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In Northeastern Brazil visceral leishmaniasis is endemic with lethal cases among humans and dogs. Treat-
ment is toxic and 5–10% of humans die despite treatment. The aim of this work was to survey natural
active compounds to find new molecules with high activity and low toxicity against Leishmania infantum
chagasi. The compounds thymol and eugenol were chosen to be starting compounds to synthesize acetyl
and benzoyl derivatives and to test their antileishmanial activity in vitro and in vivo against L. i. chagasi. A
screening assay using luciferase-expressing promastigotes was used to measure the growth inhibition of
promastigotes, and an ELISA in situ was performed to evaluate the growth inhibition of amastigote. For
the in vivo assay, thymol and eugenol derivatives were given IP to BALB/c mice at 100 mg/kg/day for
30 days. The thymol derivatives demonstrated the greater activity than the eugenol derivatives, and ben-
Received 4 February 2014
Revised 10 August 2014
Accepted 18 August 2014
Available online xxxx
Keywords:
Leishmania infatum chagasi
Thymol
Eugenol
zoyl-thymol was the best inhibitor (8.67 0.28 lg/mL). All compounds demonstrated similar activity
against amastigotes, and acetyl-thymol was more active than thymol and the positive control drug
amphotericin B. Immunohistochemistry demonstrated the presence of Leishmania amastigote only in
the spleen but not the liver of mice treated with acetyl-thymol. Thus, these synthesized derivatives dem-
onstrated anti-leishmanial activity both in vitro and in vivo. These may constitute useful compounds to
generate new agents for treatment of leishmaniasis.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
development follows three lines: first, exploration of the parasite
metabolic pathways to find targets and develop synthetic com-
The treatment of leishmaniasis has been carried out with pen-
tavalent antimonials such as antimoniate N-methyl glucamine
(GlucantimeÒ) and sodium stibogluconate (PentostanÒ) for more
than sixty years. These are the drugs of first choice for the treat-
ment of visceral leishmaniasis in Latin America and the Mediterra-
nean region. The antimonials drugs are toxic, not always effective,
and used in prolonged.1 In geographical regions where there is
documented Leishmania to antimony compounds, alternate drugs
such as pentamidine, amphotericin B, liposomal formulation are
used in the treatment of these patients.2 Antileishmanial drug
pounds; second, study of other drugs that are already on the mar-
ket with hitherto unknown antileishmanial activity (e.g., cancer
drugs), and third, focus on the use of medicinal plants as a source
of anti-protozoan molecules.3 As part of the search for new and
better drugs with improved activity and low toxicity compared
to existing regimens, the Programme for Tropical Diseases of the
World Health Organization (WHO) has targeted research using
plants to treat leishmaniasis essential and high priority.4
Plants are important sources of compounds for drug discovery,
especially for antiparasitic drugs due to the occurrence of human
parasitic infections in regions where medicinal plants are in use.5
Research using herbal resources can lead to the identification of
secondary metabolites that can either serve as valuable drugs in
⇑
Corresponding author. Tel.: +55 85 3101 9933.
E-mail address: selenemaiademorais@gmail.com (S.M. de Morais).
http://dx.doi.org/10.1016/j.bmc.2014.08.020
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: de Morais, S. M.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.08.020

2
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
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S.M. de Morais et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
incubator for an additional 72 h. Stock solutions of all of the
compounds were prepared at a concentration of 0.2% in ethanol.
An additional five concentrations were obtained by two-fold
serial-dilution. The compound solutions were added to microplates
containing the confluent layer of cells with amastigotes for 48 h at
37 °C in a humidified 5% CO2 incubator. Macrophages incubated
without drugs were used as the control, and amphotericin B
(Sigma-Aldrich, St. Louis, MO, USA) was used as the positive
control drug at the same concentration as the other compounds.
Murine macrophage RAW 264.7 cells were incubated with 0.01%
saponin in PBSA, containing 1% bovine serum albumin, for
30 min. The wells were then blocked for 30 min with 5% nonfat
milk (Nestlé) in PBS. In the next step, the cells were incubated in
serum from a rabbit immunized with a saline extract of L. i. chagasi
promastigotes, collected 30 days after the infection, at 37 °C for
1 h. The serum employed was diluted 1:500 and pre-absorbed with
10% foetal calf serum (FCS) at 22 °C for 1 h. The wells were washed
three times with 0.05% Tween-20 in PBSA, and peroxidase-
conjugated goat anti-rabbit IgG (Sigma Chemical Co.) diluted
1:5000 in 5% nonfat milk was added and incubated at 37 °C
for l h. After the wells were washed, o-phenylenediamine
(0.4 mg/mL) and 0.05% H202 were added. The reaction was stopped
by the addition of 1M HC1. The plates were read at 492 nm in a
Multiskan MS (UniScience) ELISA reader.
None of the animals died during the experiment, and the vol-
ume injected was 0.5 mL. After the treatment period all animals
were euthanized and their liver and spleen collected to verify the
parasite burden.
All procedures involving animals in this study were reviewed
and approved by the Ceará State University Ethics Committee
(CEUA-UECE).
2.7. Qualitative immunohistochemistry
The presence or absence of Leishmania after treatment (qualita-
tive histological events) was determined by the presence of amas-
tigote forms, Leishmania antigens detected by immunohisto
chemistry.
Silanized slides containing sections of fragments from BALB/c
liver and spleen obtained after the treatment of leishmaniasis were
submitted to immunohistochemistry for the detection of amastig-
otes forms. The tissues were deparaffinized in histological sections
(4 mm) with xylene, rehydration in a decreasing ethanol series.
Immunohistochemistry was performed with anti-Leishmania anti-
bodies produced in rabbits reacted with peroxidase-conjugated
goat anti-rabbit IgG (Sigma Chemical Co.). All reactions were devel-
oped in the same way using a diaminobenzidine chromogen solu-
tion (Sigma Chemical Co., MO, USA-D5637), which precipitates a
brown product and counterstaining was performed with Harris
hematoxylin. The slides were dehydrated in a growing ethanol ser-
ies and mounted with Permount resin (Fisher Chemicals, NJ, USA).
2.5. Macrophage cytotoxicity assay
A MTT assay adapted from Tempone et al.28 was used. Murine
macrophages-RAW 264.7 cells were seeded at 4 Â 104/well in
96-well microplates and incubated at 37 °C for 48 h in the presence
of the compounds, dissolved previously in ethanol at a concentra-
tion of 0.2% and diluted with M199 medium to the highest concen-
2.8. Statistical analysis
The number of living promastigotes was determined using the
luciferase assay (OD—620 nm) representing the percentage of sur-
vival. The EC50 values were calculated using a nonlinear regression
curve using the statistical software GhaphPad Prism 4.0. The data
were initially tested for Shapiro–Wilk and Bartlett for confirmation
of normal distribution and homogeneity of variance between treat-
ments, respectively. As they were not verified for homoscedastic-
ity, the results were compared using the nonparametric Kruskal–
Wallis test, using statistical software SAS (2002). Differences were
considered significant when p < 0.05 and the results were
expressed as mean standard deviation.
tration of 100 lg/mL. The microplates were incubated for 48 h at
37 °C in a 5% CO2 humidified incubator. Control cells were incu-
bated in the presence of DMSO without the drugs, or DMSO plus
amphotericin B or pentamidine (standard drugs). The viability of
the macrophages was determined with the MTT assay, as described
above, macrophage morphology remained intact by light
microscopy.
2.6. Animals and infection
This study involved 35 21-day-old male BALB/c mice, kept
according to institutional guidelines, which were inoculated intra-
peritoneally (given IP) with 107 infective promastigotes of L. i.
chagasi. Promastigotes were cultured in M199 medium, supple-
mented with 10% foetal bovine serum and 5% human male urine
at 24 °C. Metacyclic promastigotes were obtained from cultured
stationary phase promastigotes by density sedimentation on a
Ficoll gradient.29 Metacyclic promastigotes were centrifuged
(3000 rpm, 10 min, Beckman tabletop centrifuge), the supernatant
was discarded and the pellet was resuspended and washed in
M199 medium, then 1 Â 107 metacyclic promastigotes were used
for the animal infection. Thirty five days post infection one animal
from each group (5 animal total) was euthanized and the liver and
spleen were imprinted to confirm infection and verify the parasite
burden, and then the treatment was started.
The animals were treated once a day for 30 days. The groups
(n = 5) were formed by the compounds acetyl-thymol, benzoyl-
thymol, acetyl-eugenol, benzoyl-eugenol (100 mg/kg intra perito-
neal), to reach this dose an acute toxicity was preciously conducted
(data not shown). The animal during the acute toxicity started to
show signs of toxicity such as disorientations, bristly hair and quiet
behavior at 500 mg/Kg. The controls groups were glucantime
(80 mg/Kg intra muscular) and water treated group.
3. Results and discussion
In the search for new compounds with lesser toxicity and
greater activity, the compounds thymol and eugenol were chemi-
cally modified by acetylation and benzoylation processes. At the
end of the process four derivatives were obtained, acetyl-thymol,
benzoyl-thymol, acetyl-eugenol and benzoyl-eugenol (Fig. 1). Both
thymol (C10H14O2) and eugenol (C10H12O2) have ten carbon atoms.
In the acetylation process, two carbon atoms are introduced (CH3-
C@O) which corresponds to carbons 11 (carbonyl) and 12 (methyl).
Benzoyl derivatives gave an additional seven carbons (carbonyl
and benzene ring) to this moiety. Thymol is a p-cymene type
monoterpene phenol with many pharmaceutical properties includ-
ing antimicrobial,8 anti-inflammatory and healing10, fungicidal12
,
and antioxidant.14 It has been found that some p-cymene deriva-
tives have anti-leishmanial activity with low toxicity and greater
inhibition than thymol.19,30
The thymol derivatives demonstrated better activity against
promastigotes, between the tested compounds. Benzoyl-thymol
showed the highest degree of inhibitor of promastigoten growth,
with an EC50 of 8.67
lower with an EC50 of 23.21
when compared to the control Pentamidine, were statistically
lg/mL (SD 0.28), and acetyl-eugenol was
l
g/mL (SD 3.46). All compounds,
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4
S.M. de Morais et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Eugenol is a phenylpropanoid with antifungal,13 anthelmintic,16
insecticide,31 and anti-leishmanialactivity.22 This compound is the
main constituent of essential oil of Ocimum gratissimum and Euge-
nia caryophyllus.32,33 Many phenylpropanoids demonstrate growth
inhibition34,35 but there are no studies on derivatives of eugenol.
Similar to the thymol derivatives, we hypothesized that the euge-
nol derivative could also provide greater antileishmanial activity
than nonderivatized compound.
O
O
O
O
CH3
O-Acetyl-thymol
O-Benzoyl-thymol
Comparing the two eugenol derivatives, benzoyl-eugenol had
the greatest antileishmanial activity against promastigotes
O
O
(EC50 = 10.58
(EC50 = 23.21
l
l
g/mL, SD 0.18), followed by acetyl-eugenol
g/mL, SD 3.46) All compounds presented similar
O
CH3
O
inhibitory capacities against the amastigote form. Ueda-Nakamura
et al.22 tested the eugenol-rich essential oil of Ocimum gratissimum
and its main constituent against L. amazonensis amastigotes and
promastigotes, and observed that the purified eugenol had a greater
inhibition than the essential oil. Pessoa et al.16 measured the per-
centage of the constituents in O. gratissimum essential oil and found
that only 43.7% is eugenol. Comparing the activity of O. gratissimum
essential oil and pure eugenol, the eugenol derivatives tested in this
study revealed a greater activity than the eugenol-rich essential oil
and the pure compound. Arango et al.20 evaluated the ant-leish-
manial activity of six quinolone-eugenol hybrids and pure eugenol
and demonstrated that the hybrids had a greater inhibition than
eugenol, indicating that derivatives can potentiate the inhibition
activity. Other studies demonstrate that parasitic activity of euge-
nol is superior to eugenol-rich essential oil. Santoro et al.36 reported
that the trypanocidal activity of Ocimum basilicum essential oil was
lower than its main constituent, eugenol; another study measured
the anti-giardia activity of Syzygium aromaticum and its major com-
pound eugenol, and observed that the eugenol itself had a greater
growth inhibition of Giardia lamblia,37 and these activities might
improve with the use of compound derivates.
The amastigote is the parasitic form persisting in the host and
causing symptomatic disease. Thus this form should be the major
chemotherapeutic target in studies of new antileishmanial agents,
although promastigotes and amastigotes share many metabolic
pathways making it valid to screen both parasite forms.19 All the
compounds tested in this study were statistically similar to the
positive control drug Amphotericin B against amastigotes, with
the exception of acetyl-thymol which had a greater activity than
amphtericin B. Additionally, only the acetyl-eugenol was signifi-
cantly different between its activity against the promastigote ver-
sus the amastigote forms. In this case the inhibition of
promastigotes was greater than inhibition of amastigotes. The
activity of the other compounds was statistically similar against
either promastigotes or amastigotes. Thus, these compounds are
active against both the intracellular and the extracellular parasite
stages (Table 1).
OCH3
OCH3
O-Acetyl-eugenol
O-Benzoyl-eugenol
Figure 1. Representation of chemical structures of thymol and eugenol derivatives.
different. When compared to the pure substances, acetyl-thymol
demonstrated better antileishmanial activity against promastig-
otes than thymol, and eugenol presented an activity that was
worse than both derivatives (Table 2).
The thymol derivatives acetyl-thymol and benzoyl-thymol
exhibited an EC50 = 9.07 lg/mL (SD 0.06) and EC50 = 8.67 lg/mL
(SD 0.28) against promastigote forms, respectively. Acetyl-
thymol demonstrated the greatest activity against intracellular
amastigote forms (EC50 = 10.95
was benzoyl-thymol (EC50 = 14.93
l
g/mL, SD 3.0). The next highest
g/mL, SD 4.5) (Table 1).
l
Medeiros et al.21 reported the antileishmanial activity of thymol-
rich essential oil from Lippia sidoides and pure thymol against
promastigote of Leishmania amazonensis, demonstrating an EC50 of
44.3 and 19.4 lg/mL, respectively. The magnitude of inhibition in
these reported studies was lower than that observed using
benzoyl-thymol or acetyl-thymol in this study. Another study
demonstrated inhibition of Leishmania panamensis promastigote
growth by thymol and derivatives, with five of the eight derivatives
showing significantly more activity than thymol.19 The difference
between the activity of essential oil and the pure substance can
be explained by the fact that the essential oil has other elements
and the concentration of the constituents is not equivalent to the
pure substance. Thus, the greater inhibition using the thymol
derivatives compared to thymol is an indication that the acetylation
and benzoylation can improve the growth inhibition and lower the
toxicity.
Table 1
Anti-leishmanial activity of thymol or eugenol derivatives: Activity against axenically cultured promastigotes or intracellular amastigotes in RAW 264.7 cells, and toxicity of
compounds for RAW 264.7 cells
Compounds
Promastigote EC50
(
lg/mL)
Amastigote EC50
(l
g/mL)
p valueb
Toxicity % RAW 264.7 cells survival at 100 lg/mL ( SD)
Acetyl-Eugenol
Acetyl-Thymol
Benzoyl-Eugenol
Benzoyl-Thymol
Eugenol
23.21 3.46*Ab
9.07 0.06*Ae
10.58 0.18*Ad
8.67 0.28*Af
56.13 2.09*Aa
12.85 1.61*Ac
Nd
18.53 4.79 Aab
10.95 3.00*Ab
14.93 4.50 Aab
15.09 3.65 Bab
20.81 1.59 Ba
23.93 6.29Ba
20.44 0.98
p < 0.3796
p < 0.4687
p < 0.3454
p < 0.0437
p < 0.0027
p < 0.0319
Nd
>100
>100
97.7 5.6
63.6 5.3
29 1.3
36.5 0.5
98.5 7.5
85.6 6.9
Nd
Thymol
Amphotericin B
Pentamidine
p valueb
5.30 0.81
p < 0.001
Nd
p < 0.0476
Nd
Nd
Nd, not determined.
*
Represents significant difference in relation to the control. Different capital letters represent differences between columns (action of each drug on different forms).
Lowercase letters represent significant differences between different lines (action of different drugs within each form).
b,c
Significance level of the test (probability of Type I error, i.e., rejection of a true null hypothesis).
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S.M. de Morais et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Table 2
Leishmanicidal activity of thymol and eugenol derivatives and toxicity against L. i. chagasi and RAW 264.7 cells
Compounds
Promastigote LC50
(l
g/mL)
Amastigote (
lg/mL)
Toxicity% RAW 264.7 cells survival at 100 lg/mL ( SD)
Acetyl eugenol
Acetyl thymol
Benzoyl eugenol
Benzoyl thymol
Eugenol
Thymol
Amphotericin B
pentamidine
23.21 3.46*Ab
9.07 0.06*Ae
10.58 0.18*Ad
8.67 0.28*Af
56.13 2.09*Aa
12.85 1.61*Ac
Nd
18.53 4.79 Aab
10.95 3.00*Ab
14.93 4.50 Aab
15.09 3.65 Bab
20.81 1.59 Ba
23.93 6.29Ba
20.44 0.98
>100
>100
97.7 5.6
63.6 5.3
29 1.3
36.5 0.5
98.5 7.5
85.6 6.9
5.30 0.81
Nd
Nd, not determined.
*
Represents significative difference in relation to the control. Different capital letters represent differences between columns (action of each drug on different forms).
Lowercase letters represent significant differences between different lines (action of different drugs within each form).
The toxicity of drugs against the murine RAW 264.7 macro-
phage-like cell line at revealed that 100 g/mL acetyl-thymol
was not toxic, although 100 g/mL benzoyl-thymol decreased
macrophage survival to 63.6%, a low but worrisome level of toxic-
ity. There was no detectable toxicity of either eugenol derivative
against RAW 264.7 macrophages in this study. All derivatives
had lower toxicity than their parent compounds eugenol or thymol
(Table 1). This is an excellent demonstration that the acetylation
and benzoylation processes can decrease the toxicity of chemical
compounds for mammalian cells.
The antileishmanial mechanism of these compounds could be
explained by the ability of ergosterol to interact with the Leish-
mania membrane,38,39 causing an increase in cell permeability
and loss of small cations such as K+. Eugenol also induces apopto-
sis, which could inhibit both stages of Leishmania parasites,7 an
observation that could be related to the membrane effects.
A few published studies have demonstrated that essential oils
rich in thymol and eugenol have anti-parasitic activity as well as
toxicity for mammalian cells. However, no studies were found that
tested the activity of derivatives of these compounds, such as the
compounds studied in the current report, acetyl-thymol, benzoyl-
thymol, acetyl-eugenol and benzoyl-eugenol.
not be visualized in spleens of mice treated with acetyl-eugenol,
benzoyl-eugenol or benzoyl-thymol. Amastigotes were visualized
in the livers of mice that did not receive drug treatment (water
treated animals), but not in the livers of mice treated with any of
the drugs. The histopathological findings of the liver and spleen
samples stained with HE showed no significant alterations in the
spleen, all the livers presented moderate hydropic degeneration.
L. i. chagasi infection in mice leads to an initial rise of parasites in
the liver followed by local resolution, after which parasites expand
in the spleen during chronic infection. It is difficult to discern
where in the kinetics of liver growth the treated mice are, although
lower parasite loads the spleens would suggest an overall decrease
in parasite load rather than merely a change in infection kinetics.
Data presented in this study documented antileishmanial activ-
ities of thymol, eugenol or their derivatives both in vivo and
in vitro. The data shows the derivatives are active against the par-
asite in vitro, and demonstrate the ability of derivatives to reduce
the parasite loads in the livers and spleens of mice. The modifica-
tions of thymol or eugenol to yield benzoylated or acetylated
derivatives most likely was responsible for the decreased toxicity
and increased antileishmanial activities of these compounds., The
data suggest these modified compounds are promising candidates
for further studies of antileishmanial drug development.
l
l
Most reports measure in vitro growth inhibition and toxicity of
essential oils and their major compounds, but most have not been
followed up with in vivo studies, with the exception of a few that
evaluate Leishmania species leading to cutaneous disease. In the
current study we evaluated the activity of thymol and eugenol
derivatives in vivo in BALB/c mice infected with L. i. chagasi, the
main cause of visceral leishmaniasis in Latin America. Preliminary
acute toxicity studies were conducted and the optimal safe treat-
ment dose was reached (data not shown). Subsequently male
BALB/c mice were infected with 107 promastigotes IP, and the treat-
ment started 30 days post-infection. Thymol and eugenol deriva-
tives were administered at 100 mg/kg dose once a day for an
additional 30 days. Control groups of mice received either Glucan-
time or water in the same volume as drugs. All groups of animals
were euthanatized, and spleen and liver tissue samples were col-
lected for histopathology and immunohistochemistry analyses.
Immunohistochemical stains with rabbit polyclonal antisera to
total Leishmania lysate were performed to detect qualitatively
whether Leishmania amastigote forms were present or absent. All
stains were compared with control animals that received either
no drug or Glucantime, the standard therapy for visceral leishman-
iasis. The data revealed that amastigotes were only visible in the
spleens of animals treated with no drug (water), Glucantime, or
acetyl-thymol. Even though the immunohistochemical assay is a
qualitative assay, it was possible to notice the difference between
the amount of amastigotes present in the Glucantime and the
acetyl-thymol and water treated stains. The first one, only a few
amastigotes was found, and the last two it was possible to find a
regular amount of parasites spread in the stain. Parasites could
Acknowledgements
We are grateful to the CENAUREMN (Northeastern Center for
the Application and Use of Nuclear Magnetic Resonance) of the
Federal University of Ceará for the NMR spectra of the compounds.
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