870 Chem.

Biodiversity 2016, 13, 870 – 874

FULL PAPER

Synthesis and Antileishmanial Activity of Natural Dehydrodieugenol and Its

Mono- and Dimethyl Ethers
by Luis Cezar Rodriguesa), Jos
e Maria Barbosa-Filhob), Marcia Rosa de Oliveirac), Patr
ıcia Lima do Nascimento N

erisd),
d e
Fl
avio Valadares Pereira Borges ), and Roberto Mioso* )
a
) Department of Biotechnology, Biotechnology Center, Federal University of Para
ıba, Cidade Universitaria, Jo~ao Pessoa,
PB, 58051-970 Brazil
b
) Department of Pharmaceutical Sciences, Biotechnology Center, Federal University of Para
ıba, Cidade Universitaria, Jo~ ao
Pessoa, PB, 58051-970 Brazil
c
) Department of Molecular Biology, Federal University of Para
ıba, Cidade Universitaria, Jo~ ao Pessoa, PB, 58051-970 Brazil
d
) Post-Graduate Program in Natural Products and Bioactives, Federal University of Para
ıba, Cidade Universitaria, Jo~
ao
Pessoa, PB, 58051-970 Brazil
e
) Department of Biochemistry, Federal University of Pernambuco, Av. Prof. Moraes Rego, 1235 – Cidade Universitaria,
Recife, PE, 50670-901 Brazil (phone: +55-81-21268576/8547; fax: +55-81-21268576; e-mail: robertomioso@yahoo.co.uk)

The study of chemistry of naturally occurring compounds and the synthesis of their derivatives is fundamentally important
for the development of new drugs. In this work, dehydrodieugenol (DHDE) was obtained through oxidative coupling of
eugenol, promoted by an aqueous mixture of potassium ferricyanide (K3[Fe(CN)6]) and NH3
H2O. The partial
methoxylation of DHDE with MeI and K2CO3 mainly resulted in the molecular-shaped monomethyl ether (DHDE-1MeO)
and its dimethyl ether derivative (DHDE-2MeO). The products from the reactions were characterized by 1H- and 13C-NMR
spectroscopy. Additionally, these studies have reported the antileishmanial activity of DHDE against Leishmania amazonensis
(IC50 value of 42.20 lg ml1) and shown that partial methoxylation of DHDE results in a significant increase in its
antiparasitic activity (IC50 value of 13.68 lg ml1). Based on in vitro bioassays, DHDE-1MeO has shown the highest
leishmanicidal activity in promastigota form. Production by direct one-step synthesis of this monomethoxylated compound can
be considered to be a cost-effective and environmentally friendly method with a short reaction time.

Keywords: Dehydrodieugenol, Eugenol dimer, Oxidative coupling, Lignans, Leishmanicidal activity.

[9][10], anti-inflammatory [11], antiarthritic [12], and

Introduction
Dehydrodieugenol (DHDE) is a neolignan type o-biphe-
nyl phenol derived from lignin found in some higher
plants that has been little researched – a circumstance
which contrasts with the relevance and multiplicity of
health-promoting activities reported for the well-known
eugenol [1]. As its natural monomer, eugenol is the major
constituent of the essential oil obtained from the flowers,
stems, and leaves of the clove tree Eugenia aromatica
(Syzygium aromaticum) or Eugenia caryophyllata [2][3],
and it is a valuable and useful compound for starting scaf-
folds in medicinal chemistry programs for developing
multifunctional drugs [4].
The o-hydroxylated biphenyl structure found in
DHDE also exists in a large number of naturally occur-
ring compounds, such as magnolol and honokiol, which
have important pharmacological properties [5 – 7]. Fur-
thermore, many studies have shown that this small biphe-
nolic compound exhibits antineoplasic [8], antimicrobial
antiproliferative activities [4], as well as functioning as an
apoptosis-inducing agent [13][14].
Bioactivity assays of the DHDE extracted from clove
(S. aromaticum) buds indicate antimutagenic activity [15],
induction of cytotoxicity and apoptosis, inhibition of
COX-2 gene expression [16], the ability to treat cancer
and other neoplasms [17], and possible inhibition of NF-
jB [18].
As a result of these pharmacological applications, a
range of efforts has been undertaken to produce DHDE,
which can be prepared by oxidative coupling reaction of
eugenol [19], biotransformation in vegetal cell cultures
[20], radical reactions [21], conventional synthesis [22],
and enzymatic biotransformation using peroxidases [23].
Chemically, DHDE (I) can be partially or totally
methylated to provide other compounds such as DHDE
monomethyl ether (II) and dimethyl ether (III). Although
the first synthesis of DHDE was done by Farias Dias [24]
and Fujisawa et al. [19], until now there are no records

© 2016 Wiley-VHCA AG, Z€

urich DOI: 10.1002/cbdv.201500280
 Chem. Biodiversity 2016, 13, 870 – 874
about the synthesis of its monomethyl ether. Nevertheless,
the exhaustive methylation of DHDE provides its
dimethyl ether form [25], a secondary metabolite found in
Nectandra polita that is a common Amazon Lauraceae
from the Andes region [26]. This compound attributes to
a broad spectrum of biological activities, such as the anal-
gesic and depressant effects of the central nervous system
[27], antiproliferative and apoptotic activities on malig-
nant human melanoma cells [17], and antimutagenic activ-
ities [15].
The DHDE monomethyl ether arrangement is a form
of neolignan that is found in certain species of Virola
[28], Magnolia [29], and in plants of the Lauraceae family
[30][26]. Nevertheless, except for the study on tyrosinase
inhibitory activity reported by Nguyen et al. [31], little
information about this neolignan has been reported to
date. This shortage of information contrasts with the great
number of articles published on its phenolic congeners.
However, an explanation for this situation can be attribu-
ted to its low natural abundance and the absence of any
synthetic chemical proposal.
Thus, this article reports the synthesis of DHDE and
its derivatives, in particular, its monomethyl ether deriva-
tive (DHDE-1MeO), through a simple procedure involv-
ing oxidative coupling of the eugenol moiety.
Subsequently, effective antipromastigote activity is
demonstrated for Leishamania amazonensis, particularly
in relation to the DHDE-1MeO produced. The analysis
of antipromastigote activity is a widely used method for
investigating potential new drugs for the treatment of
leishmaniasis [32 – 34].
Leishmaniasis is a complex, infectious, and parasitic
tropical diseases caused by protozoa of the genus Leish-
mania. These parasites are unicellular organisms which
have a heteroxenic life cycle, characterized in two alternate
forms of morphological development, promastigotes, and
amastigotes. These protozoans have a considerable diver-
sity, with at least 22 species pathogenic to man [35].
Results and Discussion
DHDE monomethyl ether (1.65 g, 48.5%) was obtained
as an oil, and DHDE dimethyl ether (0.5 g, 14.1%) was
obtained as a pasty yellowish oil. The reaction products
were characterized by 1H- and 13C-NMR spectroscopy
(see Supporting Information).
© 2016 Wiley-VHCA AG, Z€
urich
871
The free rotation between the aromatic cycles causes
the input of the first MeO group, which impedes the entry
of the next group. This is a possible explanation for the
greater production of the monomethoxylated compound.
However, when this same reaction is carried out ther-
mally at higher temperatures, it results in complete
methoxylation, which demonstrates the influence that this
variable has on the mono- and dimethyl ether ratio.
Due the significance of these scaffolds in drug discov-
ery and medicinal chemistry, efficient synthesis of DHDE
and its derivatives continues to attract the interest of syn-
thetic chemists [21]. However, most classical methods for
the synthesis of the biphenyl compounds and their corre-
sponding monomers need expensive reagents and cata-
lysts, as well as the use of high temperatures or extended
reaction times [36][37]. Thus, to overcome these limita-
tions, a cost-effective and environmentally friendly
method with a short reaction time is always welcome.
In Vitro Antileishmanial Activity
The drugs used in leishmaniasis treatment are highly tox-
ic; therefore, it is necessary to search for more efficient
antileishmanial compounds [38]. For this reason, the
antileishmanial activity of DHDE and its methyl deriva-
tives was investigated in vitro against the promastigote
stages of L. amazonensis.
DHDE and its mono- and dimethyl ether derivatives
showed antipromastigote activity by inhibiting parasite
growth at all the concentrations tested (Fig. 1), resulting
in IC50 values of 42.2, 13.68, and 47.75 lg ml1, respec-
tively. Although total methylation of the DHDE (which
produces the DHDE dimethyl ether) does not improve its
leishmanicidal activity, it was observed that its partial
methylation (as a monomethyl ether) results in a signifi-
cant increase in antiparasitic activity.
The antiparasitic activity of DHDE is little explored
in the literature. It is reported that DHDE isolated in
nature presents antipromastigote activity against Leishma-
nia major with an IC50 value of 13.6 lg ml1 [30], and
L. amazonensis and L. braziliensis with the IC50 values of
148 and 150 lg ml1, respectively [39]. The results
obtained from both studies confirm the antileishmanial
activity of DHDE, which raises new possibilities for the
use of its mono- and dimethyl ether derivatives as
antiparasitic agents.
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872 Chem. Biodiversity 2016, 13, 870 – 874
Fig. 1. Growth inhibition of Leishmania amazonensis promastigotes in the presence of DHDE and its mono- and dimethyl ether derivatives.
The graph shows the mean  SEM for three independent experiments performed in duplicate (**P ≤ 0.01, ***P ≤ 0.001).
Table. Sensitivity of the promastigote forms of Leishmania amazo-
nensis to DHDE and its mono- and dimethyl ether derivatives, and
meglumine antimoniate and amphotericin B as active control groups
Compound Leishmania
amazonensis (IC50)
Dehydrodieugenol (I) 42.20 lg ml1
Dehydrodieugenol monomethyl ether (II) 13.68 lg ml1
Dehydrodieugenol dimethyl ether (III) 47.75 lg ml1
Meglumine antimoniate > 4.00 mg ml1
Amphotericin B 0.13 lg ml1
The results observed for the IC50 values presented by
DHDE and its mono- and dimethyl ether derivatives
show that these compounds are more active against
L. amazonensis promastigotes when compared to meglu-
mine antimoniate (SbV) (IC50 > 4.0 mg ml1); however,
they are less effective than amphotericin B
(IC50 = 0.13 lg ml1) (Table). It was reported in the litera-
ture that SbV has lower activity against Leishmania pro-
mastigotes when compared to amastigote forms [33], and
also that amphotericin B is a second choice drug in the
treatment of leishmaniasis because its high toxicity causes
serious renal side effects [40].
The studies done as part of this work have reported the
antileishmanial activity of DHDE against L. amazonensis
(IC50 42.20 lg ml1) and shown that partial methoxylation
of DHDE results in a significant increase in its antiparasitic
activity, with an IC50 value of 13.68 lg ml1. Based on
in vitro bioassays, DHDE-1MeO has shown the highest
leishmanicidal activity in promastigote form.
Conclusions
The study of the chemistry of naturally occurring com-
pounds and the synthesis of their derivatives is fundamen-
tally important for creating chemical entities suitable for
www.cb.wiley.com
the development of new drugs to combat the etiological
agents of human leishmaniasis, which is one of the
world’s most devastating but neglected tropical diseases.
In this work, DHDE and its mono- (DHDE-1MeO) and
dimethyl (DHDE-2MeO) ethers were obtained syntheti-
cally from oxidative coupling of eugenol, and they were
characterized by NMR spectroscopy. The bioassay done
with DHDE and DHDE-2MeO resulted in weak antileish-
manial activity against L. amazonensis promastigote forms.
However, it was observed that partial methoxylation of
DHDE, which produces DHDE-1MeO, results in a signifi-
cant increase in its antiparasitic activity, with an IC50 value
of 13.68 lg ml1. Thus, based on in vitro bioassays, the syn-
thetic DHDE monomethyl derivative had the highest leish-
manicidal activity in promastigote form, and it may offer an
alternative to the usual leishmaniasis treatment.
Direct one-step synthesis of this monomethoxylated
compound can be considered to be a cost-effective and
environmentally friendly production method with a short
reaction time. These structures may also be useful
moieties when added to existing structures to improve the
pharmacokinetic properties of drugs that are currently
being used in the clinical environment or are under devel-
opment.
The authors would like to thank the Brazilian National
Council for Scientific and Technological Development
(CNPq) for the ProDoc fellowship granted to L. C. R.,
and the doctoral fellowships granted to P. L. N. N. and F.
V. P. B. We also would like to thank the Centre for Char-
acterization and Analysis of the Federal University of
Para
ıba (NUCAL) for technical assistance and NMR
spectroscopy.
Supporting Information
Additional Supporting Information may be found in the
online version of this article.
© 2016 Wiley-VHCA AG, Z€
urich
 Chem. Biodiversity 2016, 13, 870 – 874
Experimental Part
Synthesis of Dehydrodieugenol
Commercial eugenol was used without any further purifi-
cation, and DHDE was synthesized via the oxidative cou-
pling reaction of it. Thus, to a flask containing 32.9 g of
K3[Fe(CN)6] (100 mmol, 1 equiv.), dist. H2O was added
to boil the minimum amount necessary to completely dis-
solve the heat-formed 145 ml of sat. solution, which was
kept on a hot plate. The dissolution was then transferred
slowly into a flask containing a solution of 16.4 g of euge-
nol (100 mmol, 1 equiv.) in 160 ml of acetone, 80 ml of
dist. H2O, and 250 ml of NH3
H2O (25%). The mixture
was kept under magnetic stirring for 5 h. Then,
NH3
H2O was neutralized with conc. HCl (220 ml) and
observed until the formation of a precipitate, which was
separated by a B€ uchner funnel and washed three times
with dist. H2O. Recrystallization from absolute EtOH
resulted in colorless crystalline plates, Dehydrodieugenol
(= 3,30 -Dimethoxy-5,50 -di(prop-2-en-1-yl)biphenyl-2,20 -diol
(I); 11.5 g, yield 70%) with a melting point of 105 °C. 1H-
NMR (500 MHz, CDCl3): 6.7 (dd, J = 6.5, 2.0, arom. H);
6.0 (m, CH=CH2); 5.0 – 5.2 (m, CH=CH2); 3.9 (s, MeO);
3.3 (d, J = 6.5, CH2). 13C-NMR (50.3 MHz, CDCl3): 39.9
(CH2); 56.1 (MeO); 110.6 (CH); 115.7 (CH=CH2); 123.0
(CH); 124.4 (Ar–Cq); 131.9 (Cq–CH2); 137.7 (CH=CH2);
140.8 (Cq–OMe); 147.2 (Cq–OH). Values are consistent
with the data described in the literature [24].
Syntheses of Dehydrodieugenol Monomethyl and
Dimethyl Ether
A mixture of DHDE (3.26 g, 10 mmol), DMF (30 ml),
and anh. K2CO3 (2.76 g, 20 mmol) was stirred at r.t. for
15 min. Then, a solution of MeI (2.84 g, 20 mmol) in
DMF (10 ml) was added drop by drop, and the mixture
was kept at r.t. for 4 h under magnetic stirring. The mix-
ture was transferred to a separatory funnel containing
100 ml of an aq. 5% HCl solution, extracted with 30 ml
of CHCl3 (39), and the org. phases were dried (MgSO4),
filtered into a flask, and finally, the solvent was removed
under rotary evaporator at reduced pressure. After sol-
vent evaporation, the crudes compounds were purified by
Scheme 1. Synthesis of Deydrodieugenol (I), Deydrodieugenol monomethyl ether (II) and Deydrodieugenol dimethyl ether (III)
© 2016 Wiley-VHCA AG, Z€
urich
873
flash chromatography (silica gel, hexane/AcOEt 90:10),
affording DHDE-2MeO (= 2,20 ,3,30 -Tetramethoxy-5,50 -di
(prop-2-en-1-yl)biphenyl; III) as yellowish pasty oil (0.5 g,
14.1% yield). 1H-NMR (500 MHz, CDCl3): 6.73 (d, J = 2,
arom. H); 6.68 (d, J = 2, arom. H); 5.97 (ddt, J = 16.8,
10.0, 6.8, CH=CH2); 5.03 – 5.12 (m, CH=CH2); 3.87 (s, 2
MeO); 3.62 (s, 2 MeO); 3.35 (d, J = 6.7, CH2=CH). 13C-
NMR (50.3 MHz, CDCl3): 40.0 (CH2); 55.8, 60.6 (4
MeO); 111.9 (CH=CH2); 115.8 (CH=CH2); 118.8, 123.1,
129.7, 137.3 (arom. CH); 132.6, 135.0, 145.0, 152.5 (Cq).
Values are consistent with the data described in the
literature [25].
Then, using a mixture of hexanes/AcOEt 90:20 as
eluent furnished DHDE-1MeO (= 20 ,3,30 -Trimethoxy-5,50 -
di(prop-2-en-1-yl)biphenyl-2-ol; II) as pasty oil (1.65 g,
48.5% yield). 1H-NMR (500 MHz, CDCl3): 6.74 (dd,
J = 6.5, 2.0, arom. H); 6.71 (s, arom. H); 6.39 (s, arom.
H); 5.97 (m, CH=CH2); 5.05 – 5.15 (m, CH2=CH); 3.89 (s,
MeO); 3.88 (s, MeO); 3.64 (s, MeO); 3.4 – 3.3 (d, J = 7,
CH2–CH). 13C-NMR (50.3 MHz, CDCl3): 39.9, 40.1 (2
CH2); 55.9, 56.0, 61.2 (3 MeO); 111.0, 112.0 (2 CH=CH2);
115.6, 116.0 (2 CH=CH2); 122.9, 123.4, 137.2, 137.7 (arom.
CH); 125.5, 131.5, 132.0, 136.3, 141.4, 144.2, 148.0, 152.5
(Cq). Values are consistent with the data described in the
literature [24]. The reactions are shown in the Scheme 1
Parasites and Antipromastigote Activity Bioassay
The promastigote forms of Leishmania amazonensis
(IFLA/BR/67/PH8) were cultivated in vitro at 25  1 °C
in ‘Novy & MacNeal-Nicolle (NNN)/Schneider’ (Sigma-
Aldrich, St. Louis, MO, USA) biphasic culture media
supplemented with fetal bovine serum (20%), together
with streptomycin (100 lg ml1) and penicillin
1
(100 UI ml ), which were purchased from Cultilab,
Campinas, SP, Brazil.
The antipromastigote activity assay was done by fol-
lowing a previously described protocol [27], which
involves L. amazonensis promastigotes in logarithmic
growth phase (1 9 106 cells ml1) being incubated in
complete Schneider’s medium, in both the absence and
presence of different concentrations of the tested sub-
stances. After 72 h at 25 °C, the cultures were analyzed
and quantified in a Neubauer chamber under an optical
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874 Chem. Biodiversity 2016, 13, 870 – 874
microscope. Three independent experiments were per-
formed in duplicate.
Statistical Analysis
The results are presented as a mean  standard deviation
(SD) using the Student’s t-test for data analysis. The
GraphPad Prism 5.0 software for Windows (GraphPad
Software, San Diego, CA, USA) was used for this statisti-
cal analysis. Only P ≤ 0.05 values will be considered to be
significant. The half maximal inhibitory concentration
(IC50) was calculated by Probit analysis (SPSS for Win-
dows 14.0).
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Received September 3, 2015
Accepted February 29, 2016
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