Synthesis and in vitro and in vivo biological evaluation of substituted nitroquinoxalin-2-ones and 2,3-diones as novel trichomonacidal agents

Article abstract of DOI:10.1371/journal.pone.0085706

Two series of ten novel 7-nitroquinoxalin-2-ones and ten 6-nitroquinoxaline-2,3-diones with diverse substituents at positions 1 and 4 were synthesized and evaluated against the sexually transmitted parasite Trichomonas vaginalis. Furthermore, diverse mole

Full text of DOI:10.1371/journal.pone.0085706

A Quinoxaline Derivative as a Potent Chemotherapeutic
Agent, Alone or in Combination with Benznidazole,
against Trypanosoma cruzi
1
2
3
ˆ
ˆ
Jean Henrique da Silva Rodrigues , Tania Ueda-Nakamura , Arlene Gonc¸alves Correa ,
Diego Pereira Sangi3, Celso Vataru Nakamura2*
´
˜
ˆ
´
´
´
´
1 Programa de Pos-Graduac¸ao em Ciencias Biologicas – Biologia Celular e Molecular, Universidade Estadual de Maringa, Maringa, Parana, Brazil, 2 Departamento de
ˆ
´
´
´
˜
´
´
´
Ciencias Basicas da Saude - Laboratorio de Inovac¸ao Tecnologica no Desenvolvimento de Farmacos e Cosmeticos, Universidade Estadual de Maringa, Maringa, Parana,
´
´
´
´
´
Brazil, 3 Departamento de Quımica - Laboratorio de Sıntese de Produtos Naturais, Universidade Federal de Sao Carlos, Sao Carlos, Sao Paulo, Brazil
´
˜
˜
˜
Abstract
Background: Chagas’ disease is a condition caused by the protozoan Trypanosoma cruzi that affects millions of people,
mainly in Latin America where it is considered endemic. The chemotherapy for Chagas disease remains a problem; the
standard treatment currently relies on a single drug, benznidazole, which unfortunately induces several side effects and it is
not successful in the cure of most of the chronic patients. In order to improve the drug armamentarium against Chagas’
disease, in the present study we describe the synthesis of the compound 3-chloro-7-methoxy-2-(methylsulfonyl)
quinoxaline (quinoxaline 4) and its activity, alone or in combination with benznidazole, against Trypanosoma cruzi in vitro.
Methodology/Principal Findings: Quinoxaline 4 was found to be strongly active against Trypanosoma cruzi Y strain and
more effective against the proliferative forms. The cytotoxicity against LLCMK2 cells provided selective indices above one for
all of the parasite forms. The drug induced very low hemolysis, but its anti-protozoan activity was partially inhibited when
mouse blood was added in the experiment against trypomastigotes, an effect that was specifically related to blood cells. A
synergistic effect between quinoxaline 4 and benznidazole was observed against epimastigotes and trypomastigotes,
accompanied by an antagonistic interaction against LLCMK2 cells. Quinoxaline 4 induced several ultrastructural alterations,
including formations of vesicular bodies, profiles of reticulum endoplasmic surrounding organelles and disorganization of
Golgi complex. These alterations were also companied by cell volume reduction and maintenance of cell membrane
integrity of treated-parasites.
Conclusion/Significance: Our results demonstrated that quinoxaline 4, alone or in combination with benznidazole, has
promising effects against all the main forms of T. cruzi. The compound at low concentrations induced several ultrastructural
alterations and led the parasite to an autophagic-like cell death. Taken together these results may support the further
development of a combination therapy as an alternative more effective in Chagas’ disease treatment.
ˆ
Citation: Rodrigues JHdS, Ueda-Nakamura T, Correa AG, Sangi DP, Nakamura CV (2014) A Quinoxaline Derivative as a Potent Chemotherapeutic Agent, Alone or
in Combination with Benznidazole, against Trypanosoma cruzi. PLoS ONE 9(1): e85706. doi:10.1371/journal.pone.0085706
´
Editor: M. Carolina Elias, Instituto Butantan, Laboratorio Especial de Toxinologia Aplicada, Brazil
Received September 28, 2013; Accepted December 2, 2013; Published January 17, 2014
Copyright: ß 2014 Rodrigues et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
´
´
˜
Funding: This work was supported by the Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq), Coordenac¸ao de Aperfeic¸oamento de Pessoal
`
de N´ıvel Superior (CAPES), Financiadora de Estudos e Projetos (FINEP), Fundac¸a˜o de Aux´ılio a Pesquisa do Estado de Sao Paulo (FAPESP), Trust in Science Project –
˜
´
GlaxoSmithKline (GSK), Programa de Pos-Graduac¸ao em Ciencias Biologicas and Complexo de Centrais de Apoio a Pesquisa (COMCAP-UEM) da Universidade
Estadual de Maringa. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
˜
ˆ
´
´
Competing Interests: The authors have the following interests. Funding for this study was received from GlaxoSmithKline (GSK). There are no patents, products
in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as
detailed online in the guide for authors.
* E-mail: cvnakamura@uem.br
variable presentation and progression. [2] Most infected patients
will never develop any symptoms, characterizing the indetermi-
nate form of chronic Chagas’ disease. Approximately 30% of
Introduction
The American trypanosomiasis, also known as Chagas’ disease,
is a major public health problem that affects more than 10 million
infected individuals will develop severe digestive or cardiac
people worldwide, mostly in Latin America where it is considered
symptoms, [3] making this infection the most common cause of
endemic. [1] The causative agent of Chagas’ disease is the
myocarditis worldwide. [4].
hemoflagellate protozoan Trypanosoma cruzi that belongs to
Trypanosomatidae family. This taxon also includes other parasites
The available treatment for Chagas’ disease is restricted to only
two nitroheterocyclic compounds, benznidazole and nifurtmox,
that are responsible for relevant diseases, such as Trypanosoma brucei
(Human African Trypanosomiasis) and Leishmania spp. (leishman-
iasis). With regard to its clinical aspects, Chagas’ disease has a
that have been used to treat Chagas’ disease for more than 40
years. [5] Although both drugs are trypanocidal against all three
forms of the parasite, their high systemic toxicity is important to
PLOS ONE | www.plosone.org
1
January 2014 | Volume 9 | Issue 1 | e85706

Quinoxaline 4 as an Anti-Trypanosoma cruzi Agent
consider, which can be expressed in a variety of symptoms, such as
anorexia, nausea, headache, psychological disorders, polyneurop-
athies, and dermatitis. [6] Their efficacy is still considered low and
restricted to acute manifestations of the disease. These drugs
successfully cure approximately 20% and 80% of chronic and
acute Chagas’ disease patients, respectively. [7] Thus, new drugs
and therapies should be sought for the treatment of Chagas’
disease.
60, 230–400 mesh, E. Merck). Gas chromatography was
performed using a Shimadzu GC-17A with N2 as the carrier
and a DB-5 column. Melting points were performed in Micro-
quimica MQAPF-301.
3-Methoxy-N-[1-(methylthio)-2-nitroethenyl]-
benzenamine (2) [26]
Nitroethene 1 (0.182 mmol), m-methoxyaniline (0.182 mmol),
and ethanol (1 mL) were placed in a glass tube, purged with
oxygen-free nitrogen for 10 min, sealed, and irradiated for 90 min
in a CEM Discovery focused microwave oven at 110uC and 70 W.
The crude product was purified by flash chromatography using
hexane:ethyl acetate (4:1 ratio) as the eluent, furnishing nitroke-
tene N,S-acetal 2 in 91% yield (melting point: 125–126uC). 1H
NMR (400 MHz, CDCl3) d: 2.39 (s, 3H), 3.83 (s, 3H), 6.70 (s, 1H),
6.90 (t, 1H, J 2.15 Hz), 6.92–6.87 (m, 2H), 7.33 (t, 1H, J 8.26 Hz),
11.82 (s, 1H). 13C NMR (100 MHz, CDCl3) d: 14.71, 54.01,
111.44, 113.76, 117.89, 130.09, 137.19, 160.25, 163.28. IR (KBr)
Several studies have been conducted to find new active
compounds against T. cruzi. The search based on natural products
is promising, with the possibility of screening a high number of
plants and isolate active compounds with low toxicity. [8–9]
Another approach that shows potential is the design and
evaluation of synthetic compounds, either alone or in combina-
tion, against the parasite. [10–12] Among the groups of synthetic
molecules, quinoxalines are a class of heterocyclic compounds that
have been intensively studied for their biological activities, showing
promising effect against bacteria, fungi, [13] tumors, [14] viruses,
[15] and protozoa. [16–17] A library of new quinoxaline
derivatives was recently tested against Leishmania peruviana and T.
cruzi and showed interesting results. [18].
n
max/cm21
: 3159.17, 3070.45, 2995.23, 1602.73, 1546.80,
1465.79, 1417.58, 1330.79, 1269.07, 1222.78, 1157.20, 958.55,
848.62, 684.68, 570.89, 532.31. GC-MS (70 eV) m/z (%): 240
(M+, 12), 206 (41), 159 (38), 147 (64), 107 (100), 77 (99).
Combination therapy is an important way to improve the
effectiveness of available drugs with different mechanisms of action
in the treatment of several disorders, especially in the control of
infectious diseases. [19] It is an approach that is currently used for
the treatment of cancer, [20] osteoarthits, [21] tuberculosis, [22]
acquired immune deficiency syndrome, [23] and opportunistic
infections. [24] Compared with monotherapy, combination
therapy presents higher cure rates, reduced treatment times, lower
side effects, and delays in the selection of resistant parasites. [19].
Combination therapies based on the available drugs for Chagas’
disease treatment, benznidazole and nifurtimox, and novel uses of
old drugs, such as allopurinol, itraconazole, ketoconazole, and
fluconazole, have been proposed as a promising therapeutic
alternative for patients with acute and chronic Chagas’ disease.
[7,25].
2-Chloro-7-methoxy-3-methylsulfanylquinoxaline (3) [27]
To a suspension of nitroketene N,S-acetal 2 (0.208 mmol) in
acetonitrile (1 mL), POCl3 (0.625 mmol) was added dropwise at
0uC for 15 min. The mixture was stirred at 80uC for 4 h and
cooled to room temperature, and a saturated solution of NaHCO3
(2.5 mL) was added. The aqueous solution was extracted with
CHCl3 (362 mL). The combined organic phases were washed
with water (262 mL) and brine (2 mL) and dried over anhydrous
Na2SO4. The solvent was removed under vacuum, and the crude
product was purified with column chromatography using silica gel
and hexane:ethyl acetate (9:1) as the eluent, affording compound 3
in 54% yield (melting point: 109–111uC. 1H NMR (400 MHz,
CDCl3) d: 7.83–7.80 (m, 1H), 7.28–7.26 (m, 2H), 3.96 (s, 3H), 2.67
(s, 3H). 13C NMR (100 MHz, CDCl3) d: 161.08, 157.10, 142.94,
134.68, 129.08, 121.18, 105.95, 99.99, 55.80, 13.76. IR (nmax,
KBr): 3006, 1616, 1494, 1213 cm21. GC-MS (70 eV) m/z: 240
(M+, 100), 205 (88), 190 (35), 159 (40), 63 (29).
Considering that no effective vaccine against Chagas’ disease
exists and the controversial and inefficient chemotherapy available
to patients, new therapies are required. [5] Thus, the present study
investigated the anti-protozoan activity and the main alterations
induced by
a quinoxaline derivative, 3-chloro-6-methoxy-2-
(methylsulfonyl)quinoxaline (4), against the three main forms of
T. cruzi and assessed the safety of this compound by testing its
toxicity against cultured mammalian cells and human red blood
cells. We also verified the in vitro activity of quinoxaline 4 and
benznidazole combined against T. cruzi and mammalian cells to
determine whether this combination has potential as a future
multi-drug therapy.
3-Chloro-7-methoxy-2-(methylsulfonyl)quinoxaline (4)
[27]
To a solution of m-chloroperbenzoic acid (0.25 mmol) in
dichloromethane (1 mL) was added a solution of quinoxaline 3
(0.11 mmol) in dichloromethane (1 mL) at 0uC for 2 h. Ice water
(2 mL) was then added, and the organic layer was washed with a
10% aqueous solution of NaHCO3 (2 mL), water (2 mL), and
brine (2 mL). It was dried over anhydrous Na2SO4 and
concentrated under vacuum. The crude product was purified
with column chromatography in silica gel and hexane:ethyl acetate
(3:2) as the eluent, affording quinoxaline 4 in 87% yield. 1H NMR
(400 MHz, CDCl3) d: 7.98 (d, J = 9.26 Hz, 1H), 7.59 (dd,
J = 9.26; 2.85 Hz, 1H), 7.40 (d, J = 2.85 Hz, 1H), 4.01 (s, 3H),
3.53 (s, 3H). 13C NMR (100 MHz, CDCl3) d: 162.19, 150.00,
140.33, 139.07, 138.67, 129.23, 127.39, 106.68, 56.17, 40.27. CG-
MS (70 eV) m/z: 272 (M+, 63), 210 (68), 193 (90), 158 (100), 117
(50), 77 (36).
Methods
Synthesis of 3-Chloro-7-methoxy-2-
(methylsulfonyl)quinoxaline (4)
Unless otherwise noted, all of the commercially available
reagents were purchased from Aldrich Chemical Co. and used
without purification. 1H and 13C nuclear magnetic resonance
(NMR) spectra were recorded with a Bruker ARX-400 (400 and
100 MHz, respectively). The infrared (IR) spectra refer to tablets
in KBr and were measured with a Bomem M102 spectrometer.
Mass spectra were recorded with a Shimadzu GCMS-QP5000.
Stock solutions of quinoxaline 4 were prepared aseptically in
dimethylsulfoxide (DMSO; Sigma, St. Louis, MO, USA) at the
time of use, with the final concentration of the experiments not
exceeding 0.5%. All of the negative controls received the same
concentration of DMSO as the treatment.
Analytical thin-layer chromatography was performed on
a
0.25 mm film of silica gel that contained the fluorescent indicator
UV254 supported on an aluminum sheet (Sigma-Aldrich). Flash
column chromatography was performed using silica gel (Kieselgel
PLOS ONE | www.plosone.org
2
January 2014 | Volume 9 | Issue 1 | e85706

Quinoxaline 4 as an Anti-Trypanosoma cruzi Agent
number of infected cells and amastigotes was determined under a
light microscope by counting randomly 200 cells in duplicate
cultures. The results are expressed as the survival index (%). The
survival index was obtained by multiplying the percentage of
infected cells by the number of amastigotes per infected LLCMK2
cell. The survival index observed in the control without treatment
was considered as 100%, the results for treated groups were
comparatively evaluated.
Parasites and Cell Cultures
All of the experiments were performed with the Y strain of
Trypanosoma cruzi. Epimastigote forms were cultivated in Liver
Infusion Tryptose (LIT) medium [28] supplemented with 10%
heat-inactivated fetal bovine serum (FBS; Gibco Invitrogen,
Grand Island, NY, USA), kept at 28uC, and maintained by
weekly transfers. Trypomastigotes were collected from the
supernatant of LLCMK2 cells (Macaca mulatta epithelial kidney
cells) preinfected by bloodstream trypomastigotes aseptically
obtained by heart puncture of infected Swiss mice at the peak of
parasitaemia. LLCMK2 cells, infected or not, were maintained in
Dulbecco’s modified Eagle medium (DMEM; Gibco Invitrogen),
pH 7.4, supplemented with 2 mM L-glutamine, 10% FBS, and
Cytotoxicity Assay
To evaluate the cytotoxicity of the quinoxaline derivate, the
MTT assay was used. This colorimetric assay is based on the
ability of viable mitochondria to convert MTT, a water-soluble
tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazo-
lium bromide), into an insoluble purple-colored formazan
precipitate. [32] LLCMK2 cells were collected from confluent
cultures, plated in 96-well plates, and incubated at 37uC in a
humid 5% CO2 atmosphere. After 24 h, the medium was replaced
with new DMEM that contained concentrations of quinoxaline 4
that ranged from 3.7 to 73.6 mM. Following 96 h incubation, the
cells were washed in PBS, and 50 mL of MTT (2 mg/mL) was
added to each well. The formazan crystals were solubilized in
DMSO, and absorbance was read at 570 nm in a microplate
reader (Bio Tek – Power Wave XS). The concentration that
diminished 50% of the absorbance value observed in the control
represented the CC50 (cytotoxic concentration for 50% of the
cells).
50 mg/L gentamicin at 37uC in
a humidified 5% CO2
atmosphere. [29] Amastigotes and trypomastigotes present on
the supernatant of infected LLCMK2 cells were separated by
differential centrifugation at 8506g for 5 min. The elongated
forms were harvested from the supernatant, and the round forms
were collected from the pellet.
Anti-proliferative Activity against Epimastigote Forms
Epimastigotes (16106 parasites/mL) in the exponential phase of
growth (96 h) were harvested and incubated in the presence of
LIT supplemented with 10% FBS added or not to increasing
concentrations of quinoxaline 4 (0.37–37.0 mM). Parasites were
incubated at 28uC in 24-well flat-bottom plates, collected
aseptically every 24 h, and counted in a Neubauer hemocytom-
eter. Benznidazole (Laborato´rio Central de Medicamentos,
Pernambuco, Brazil) was used as the reference drug and counted
after 96 h. The IC50 (concentration that inhibited 50% of parasite
growth) and IC90 (concentration that inhibited 90% of parasite
growth) were determined by regression analysis of the data.
Hemolytic Activity
Another way to assess the safety of a drug is to measure its
hemolytic activity. Blood (A+ type) from a healthy human donor
was collected, defibrinated, and washed in glycosylated saline to
remove any free hemoglobin from the defibrinization process. Red
blood cells were inoculated in 96-well plates at 3% in glycosylated
saline with different concentrations of quinoxaline 4 (3.7–
1000 mM). The plates were incubated for 3 h at 37uC, and the
supernatant was read at 550 nm. To calculate the percentage of
hemolysis, 1% Triton X-100 was used as a positive control.
Activity against Trypomastigote Forms
Trypomastigotes, obtained from the supernatant of infected
LLCMK2 cells, were inoculated (16107 parasites/mL) in DMEM
and added to 96-well plates in the presence and absence of
increasing concentrations of quinoxaline
4 (1.8–360.0 mM).
Parasites were incubated for 24 h at 37uC in a 5% CO2
atmosphere. After incubation, the viability of the parasites was
examined by mobility under a light microscope (Olympus CX31)
using the Pizzi-Brener method. [30] The same experiment was
performed three times with supplementation with 20% FBS, Swiss
mouse blood, and Swiss mouse plasma. The concentrations that
inhibited 50% (EC50) and 90% (EC90) of parasite viability were
determined by regression analysis of the data.
Drug Combination Assay
To verify the effect of the combination of quinoxaline 4 and
beznidazole on epimastigotes, trypomastigotes, and LLCMK2
cells, we applied the Combination Index method proposed by
Chou and Talalay [33] and reviewed by Zhao. [34] The
experimental design consists of combinations of at least four
concentrations of each drug arranged in a checkerboard at a 1:2
concentration ratio. Briefly, 16106 epimastigotes/mL were
resuspended in LIT in the presence of different concentrations
of both drugs and counted after 96 h incubation at 28uC. For the
trypomastigote inhibition assay, cells were collected (16107
parasites/mL) and exposed to different concentrations of each
drug in combination. After 24 h incubation, the parasites were
analyzed for viability. In the cytotoxicity experiment, LLCMK2
cells were plated as described and exposed to different concentra-
tions of both drugs. After 96 h, cell viability was quantified using
the MTT method. The data were calculated and mathematically
Activity against Intracellular Amastigotes
To assess the activity of quinoxaline 4 against the intracellular
form of the parasite, the assay was carried out as described
previously [31] with slight modifications. For that, LLCMK2 cells
were harvested, resuspended in DMEM plus 10% FBS and plated
(2.56105 cells/mL) in 24-well plates that contained round glass
coverslips. When confluent growth was achieved, the cells were
infected with trypomastigotes obtained from pre-infected cultures
at a ratio of 10 parasites per 1 mammalian cell. After 24 h, the
medium that contained the parasites was removed, the cells were
washed in phosphate-buffered saline (PBS), and DMEM with or
without increasing concentrations of quinoxaline 4 (1.8–18.0 mM)
was added. The cells were maintained for 96 h at 37uC in a 5%
CO2 atmosphere. After the incubation period, the glass coverslips
were subjected to fixation with methanol and Giemsa staining and
permanently prepared with Entellan (Merck, Germany). The
expressed as the Combination Index (CI = [IC50benzo
/
combined
IC50benzo alone]+[IC50quinoxaline 4 combined/IC50quinoxaline 4 alone]; the
numerators are the concentrations of each drug that in combina-
tion are active against 50% of the cells, and the denominators are
the concentrations that have this same effect for each drug alone).
The interpretation of the CI was based on the broadly used
specifications established by Chou and Talalay, [33,35] in which
CI values less than, equal to, and more than 1 indicate synergism,
PLOS ONE | www.plosone.org
3
January 2014 | Volume 9 | Issue 1 | e85706

Quinoxaline 4 as an Anti-Trypanosoma cruzi Agent
additivity, and antagonism, respectively. The data were also
graphically expressed as isobolograms.
principles for medical research involving human subjects) last
reviewed in 2008. The donors received an explanation about the
purpose of the study and provided the written consent before the
blood collection. The blood was collected by brachial vein
puncture by a trained professional with appropriate material and
medical support. All procedures were conducted as described in
Electron Microscopy
To evaluate the effect of the compound on the morphology of
the parasite by SEM, epimastigotes (16106 parasites/mL) and
trypomastigotes (16107 parasites/mL) were treated as described
previously with the compound at the concentrations that
corresponded to the IC50 and IC90. After incubation, they were
harvested, washed twice in PBS, and fixed with 2.5% glutaralde-
hyde in 0.1 M sodium cacodylate buffer at 4uC for 24 h. The
parasites were then placed on a glass support covered with poly-L-
lysine, dehydrated in crescent grades of ethanol, dried by the
critical-point method with CO2, coated with gold, and observed
on a Shimadzu SS-550 SEM. For the ultrastructure analysis,
epimastigotes fixed as described above were postfixed in a solution
of 1% OsO4, 0.8% potassium ferrocyanide, and 10 mM CaCl2 in
0.1 M cacodylate buffer. The samples were then dehydrated in an
increasing acetone gradient and embedded in Polybed 812 resin.
Ultrathin sections were then obtained, stained with uranyl acetate
and lead citrate, and observed on a JEOL JM 1400 TEM. The
parasites were analyzed and compared with controls without any
treatment.
´
specific protocol approved by the ‘‘Comiteˆ de Etica em Pesquisa
com Seres Humanos of the Universidade Estadual de Maringa´’’
(acceptance 293/2006 COPEP-UEM). For the assays that
involved mouse blood, Swiss and BALB/c mice were obtained
from the Central Animal Facility of the Universidade Estadual de
Maringa´. All procedures were carried out in accordance with the
guidelines established by the Committee on Ethics of Animal
Experiments of the Universidade Estadual de Maringa´, as stated in
the detailed protocol approved for this experiment (acceptance
058/2010).
Results
Synthesis of 3-chloro-7-methoxy-2-
(methylsulfonyl)quinoxaline (4)
The synthesis of quinoxaline 4 was based on the procedure
described by Venkatesh et al. [27] The first step was improved by
employing microwave irradiation. Thus, the reaction of ni-
troethene 1 with p-nitroaniline using ethanol as a solvent was
irradiated at 70 W and 110uC for 90 min, furnishing nitroketene
N,S-arylaminoacetal 2 in 91% yield.27 The cyclization of 2 using
POCl3 yielded quinoxaline 3, which was oxidized with m-
chloroperbenzoic acid to furnish quinoxaline 4 in 87% yield
(Fig. 1).
Cell Volume Determination
Epimastigotes (16106 parasites/mL) treated for 24 h with
different concentrations of quinoxaline 4 (1.8, 3.7, and 7.4 mM)
were collected by centrifugation, washed twice in PBS, resus-
pended in PBS, and directly analyzed by fluorescence-activated
cell sorting using a BD FACSCalibur flow cytometer (Becton-
Dickinson, Rutherford, NJ, USA). A total of 10,000 events were
acquired in a region previously established for the parasites.
Parasites treated with actinomycin D (20.0 mM) were used as a
positive control. Histograms were generated and the analysis was
performed using CellQuest software (Joseph Trotter, The Scripps
Research Institute, La Jolla, CA, USA), considering the FSC
parameter that represents the cell volume.
Activity against Epimastigote Forms
Quinoxaline 4 showed effects against all three forms of T. cruzi.
Against epimastigotes (i.e., the form present in the midgut vector),
the drug dose-dependently inhibited parasite growth, with an IC50
of 1.160.04 mM for 96 h, which was even greater (p#0.05) than
the result found for benznidazole (IC50: 8.860.04 mM). By
monitoring the number of parasites every 24 h, we noticed that
a large proportion of the effect was reached after 48 h incubation
(Table 1). The same IC50 was obtained 1 month later with stock
solutions of the drug that were kept in a freezer, showing stability
of the drug’s activity (data not shown). The morphological
alterations induced by the treatment were assessed by scanning
electron microscopy (SEM). The parasites treated with quinoxa-
line 4 (IC50: 1.1 mM; IC90: 3.4 mM) showed severe alterations in
cell shape, exhibiting a wrinkled cell surface (Fig. 2B), distortion of
the flagellum, a reduction of body size (Fig. 2D), and cell
membrane swelling (Fig. 2C). The ultrastructural changes induced
by quinoxaline treatment for 96 h against epimastigote forms were
verified by transmission electron microscopy. The untreated cells
presented a normal organelle ultrastructure, such as a prominent
nucleus, a ramified mitochondrion, reservosomes, and cellular
membranes with preserved structures (Fig. 3A). Treated parasites
displayed an altered ultrastructure, showing well-developed
profiles of the endoplasmic reticulum (ER) that surrounded
organelles, mainly reservosomes (Fig. 3B–C). Diverse autophago-
some-like structures could also be seen, such as vacuoles that
contained cellular remains (Fig. 3B–D), the formation of myelin-
like structures (Fig. 3E), and membranous structures that involved
acidocalcisomes (Fig. 3C). These autophagic vacuoles occupied
almost all the cytoplasm in epimastigotes treated with the IC90 of
quinoxaline 4 (Fig. 3F). Parasites treated with quinoxaline 4 also
showed an increase in the number of vesicular structures within
the cytosol (Fig. 3B–F), intense disorganization of the Golgi
Cell Membrane Integrity Assay
Epimastigotes (16106 parasites/mL), trypomastigotes (16107
parasites/mL), and amastigotes (16107 parasites/mL) were
incubated in the presence or absence of different concentrations
of quinoxaline 4 (1.8, 3.7, and 7.4 mM) for 24 h. After incubation,
the cells were harvested, washed twice in PBS, and marked with
0.2 mg/mL propidium iodide for 10 min. Digitonin (40.0 mM) was
used as a positive control for the loss of cell membrane integrity.
Data acquisition and analysis were performed using a FACSCa-
libur flow cytometer equipped with CellQuest software. A total of
10,000 events were acquired in the region previously established as
the one that corresponded to the parasites.
Statistical Analysis
All of the quantitative experiments were conducted in at least
three independent experiments in duplicate. The statistical
analyses were performed using GraphPad Prism 5.0 software.
The data were analyzed using one-way analysis of variance
(ANOVA), and the Tukey post hoc test was used to compare means
when appropriate. Values of p,0.05 were considered statistically
significant.
Ethics Statement
For the hemolytic assay, the blood was obtained from healthy
volunteer donors according to Declaration of Helsinki (Ethical
PLOS ONE | www.plosone.org
4
January 2014 | Volume 9 | Issue 1 | e85706

Quinoxaline 4 as an Anti-Trypanosoma cruzi Agent
Figure 1. General procedure for the synthesis of 3-chloro-7-methoxy-2-(methylsulfonyl)quinoxaline (4).
doi:10.1371/journal.pone.0085706.g001
complex (Fig. 3E), and the formation of abnormal structures nears
the flagellum (Fig. 3D).
cells were exposed to different concentrations of quinoxaline 4.
After 96 h incubation, the concentration that was cytotoxic to
50% of the cells (CC50) was 23.361.97 mM. By comparing the
CC50 with the concentration of the drug that inhibited 50% of the
parasites, the drug was found to exhibit values of Selective Indices
(SI) above one for the clinical relevant forms of T. cruzi (SItrypo: 3.3;
SIama: 8.9), indicating that quinoxaline 4 is more active against the
parasite than toxic to mammalian cells in vitro. Another way to
estimate the toxicity of a compound is to evaluate its hemolytic
potential. Red blood cells were incubated in the presence of
different concentrations of quinoxaline 4 for 3 h. After incubation,
the maximum concentration tested (1000 mM) induced less than
3% hemolysis (data not shown).
Activity against Trypomastigote Forms
When quinoxaline 4 was tested for 24 h against trypomasti-
gotes, a dose-dependent inhibition of the viability of the parasites
was found, with an EC50 of 7.161.3 mM. We also assessed the
effect of the compound in an experiment with the addition of
mouse blood. The results (Fig. 4) showed a striking reduction of the
activity of the compound in the presence of mouse blood (EC50[
:
109.562.5 mM). To determine whether this reduction was related
to plasma components or the blood cells themselves, the assay was
repeated by adding 20% of mouse plasma. No inhibition was
observed with the addition of mouse plasma, and the EC50 was
statistically the same as the one obtained in the regular experiment
without blood. To verify morphologic alterations, trypomastigotes
were treated with the EC50 (7.1 mM) and EC90 (15.4 mM) of
quinoxaline 4 for 24 h and then analyzed by SEM. The treated
parasites showed distortions in cell shape, with shortening and
winding of the cell body (Fig. 4).
Drug Combination Assay
The combination of benznidazole and quinoxaline 4 demon-
strated promising results (Fig. 6). Strong synergism against
epimastigotes was observed. The combination was below the
additivity line on the isobolograph, with a combination index (CI)
of 0.69. Similar results were obtained in the experiment with
trypomastigotes with regard to the profile on the isobolograph and
CI of 0.66, confirming the synergistic interaction. Finally, in
LLCMK2 cells, the drug combination had a CI of 1.04, with all
points above the additivity line, indicating an antagonist effect.
Activity against Intracellular Amastigotes
In the present study, quinoxaline 4 also exerted a strong
inhibitory effect against intracellular amastigotes. After 96 h
incubation in the presence of drug, the number of infected cells
and amount of amastigotes inside each cell were reduced
compared with the untreated control (Fig. 5). Both parameters
are expressed together as the survival index. The compound
inhibited the growth of 50% of the amastigotes when the
concentration was 2.661.2 mM.
Cell Volume Determination
Epimastigote forms treated as previously described were
analyzed for Forward Scatter (FSC) by flow cytometry. The
histograms in Figure 7 show a dose-dependent reduction of FSC in
the cell population treated with quinoxaline 4. The higher
concentration tested (7.4 mM) had an effect similar to actinomycin
D treatment (i.e., the positive control).
Toxicity Assays
The first experiment that was performed to evaluate the safety
of the drug was a cytotoxicity assay in LLCMK2 cells. Mammalian
Table 1. Antiproliferative activity of quinoxaline 4 against epimastigotes of Trypanosoma cruzi.
Quinoxaline 4 (mM)
Time (h)
0.37
1.84
3.68
18.38
36.76
24
48
72
96
1.0261.77a
3.4665.99
0.7661.31
11.38610.58
35.4668.13
54.3963.99
53.63624.26
63.41628.07
42.8563.33
72.28610.86
83.07612.64
82.97619.97
64.86611.86
91.2960.66
97.7260.28
99.8260.09
81.16618.07
96.2062.78
10060.00
10060.00
agrowth inhibition (%) of epimastigotes compared to control. Results presented as median 6 standard deviation of three independent experiments.
doi:10.1371/journal.pone.0085706.t001
PLOS ONE | www.plosone.org
5
January 2014 | Volume 9 | Issue 1 | e85706

Quinoxaline 4 as an Anti-Trypanosoma cruzi Agent
Figure 2. Morphological alterations (SEM) in T. cruzi epimastigotes treated with quinoxaline 4. Parasites were treated for 72 h. Control
parasites exhibited a normal elongated body (a). The epimastigotes treated with the IC50 (1.1 mM) presented a wrinkled cell surface (b). The
epimastigotes treated with the IC90 (3.4 mM) showed distortions in the flagellum with swelling of the membrane (c) and a reduction of the cell body
(d). Bars = 2 mm.
doi:10.1371/journal.pone.0085706.g002
trypanocidal activity but none of the tested compounds presented
activity higher than quinoxaline 4.
Cell Membrane Integrity
Cell membrane integrity in all three parasite forms was
unaffected by incubation with quinoxaline 4. The low number
of high fluorescence events (upper left and upper right quadrants)
shows cells with abnormal permeability, allowing the entrance of
propidium iodide inside the cells. In the three experiments with
each parasite form, the values were similar in quinoxaline-treated
and untreated parasites (Fig. 8).
Another important consideration is the in vitro safety of the drug
revealed by cytotoxicity and hemolysis tests. Comparing the
toxicity against LLCMK2 cells with the trypanocidal activity of
quinoxaline 4, it is possible to observe that the drug is clearly
selective against all three parasite forms, with higher SIs for the
proliferative forms (epimastigote and amastigote). Hemolytic
activity is another problem that some toxic drugs present.
Amphotericin B is a commercial drug used against fungi and
some protozoa, but it is also known to have hemolytic activity,
inducing hemolysis in 50% of red blood cells at 50.8 mM. [36]
Quinoxaline 4 did not present significant hemolysis at the highest
concentration tested (1000 mM).
When the drug was tested in the presence of blood, a substantial
reduction of activity against trypomastigotes was found. Both a
decrease and complete inhibition of the activity of a trypanocidal
compound with the addition of blood were observed previously,
explained by an interaction between the drug and serum proteins
or oxyhaemoglobin. [37–38] To verify whether this inhibition was
related to an interaction with serum or the blood cells themselves,
the same experiment was conducted using mouse plasma. In the
Discussion
Quinoxaline derivatives are a group of heterocyclic compounds
characterized by a broad spectrum of biological activity. They are
used as therapeutic agents in the treatment of several infections.
[13,16,17] In the present study, quinoxaline 4 exhibited strong
anti-Trypanosoma cruzi activity against all of the main forms of the
parasite, which was even greater than the activity against
epimastigotes determined in vitro for benznidazole, the standard
drug for treating Chagas’ disease. In a previous study, Estevez [18]
synthesized and evaluated the anti-Trypanosoma properties of a
library of 11 quinoxaline derivatives, all the compounds exhibits
PLOS ONE | www.plosone.org
6
January 2014 | Volume 9 | Issue 1 | e85706

Quinoxaline 4 as an Anti-Trypanosoma cruzi Agent
PLOS ONE | www.plosone.org
7
January 2014 | Volume 9 | Issue 1 | e85706

Quinoxaline 4 as an Anti-Trypanosoma cruzi Agent
Figure 3. Ultrastructural alterations (TEM) in T. cruzi epimastigotes treated with quinoxaline 4. Parasites were treated for 72 h. Control
parasites (A) exhibited organelles with normal morphology. The epimastigotes treated with the IC50 (1.1 mM) (B–D) exhibited ER profiles that
surrounded organelles (black arrows), autophagosome-like structures (asterisk), and membrane extensions (black arrow head). The parasites treated
with the IC90 (3.4 mM) (E–F) showed severe alterations in their ultrastructure, exhibiting concentric myelin-like membrane structures (white arrow),
distortions in the structure of the Golgi complex, and autophagosome-like structures that comprised a great part of the cytoplasm (F). N, nucleus; R,
reservosomes; K, kinetoplast; F, flagellum; M, mitochondrion; G, Golgi complex. Bars = 0.2 mM (A, C–F); Bar = 0.5 mM (B).
doi:10.1371/journal.pone.0085706.g003
presence of mouse serum, the same EC50 as the one found in the
regular experiment with FBS was found, indicating that the drug
may interact with blood cells through incorporation into red blood
cells or linking to surface proteins, [39] such that the drug was
unable to act against the free trypomastigotes during the 24 h
incubation. The scenario for this interaction in vivo may be
different when the exposure time is longer than in in vitro
experiments and the storage and transport of the drug by
erythrocytes may extend the half-life and decrease the toxicity
profile. [40].
combination of benznidazole with various drugs against T. cruzi.
[12,41] Based on these results, one question that arises is the
mechanism by which the drug combination is synergistic.
According to Chou, [42] predicting the mechanism of action of
two drugs based on their synergistic interaction is difficult. The
mechanism of such a combination can be one specific point of
action or innumerable steps. However, several studies have
demonstrated that compounds with different mechanisms of
action can cooperate to enhance the final effect observed.
[43–45] Combination therapy with new drugs has been shown
to be an important approach against T. cruzi. The combination of
different enzymatic inhibitors of the ergosterol biosynthesis
pathway, a specific target for fungi and protozoa, can promote
the cure of infected mice at low concentrations of both drugs. [44]
A promising interaction between quinoxaline 4 and benznida-
zole was also found. The combination was synergistic against
epimastigotes and trypomastigotes and antagonistic against
LLCMK2 cells. Several studies demonstrated the potential of the
Figure 4. Activity of quinoxaline 4 against trypomastigote forms of T. cruzi. (A) Effective concentration (EC50) of quinoxaline 4 that inhibited
the viability of 50% of the trypomastigotes after 24 h incubation, assays conducted with the addition of FBS, mouse blood or mouse plasma. The
experiment was repeated three times. The bars show the median 6 standard deviation. *** p#0.01 (B–D) Effects of quinoxaline 4 on the morphology
of trypomastigotes. Control parasites (B) exhibited a normal elongated body. The parasites treated with the IC50 (C) and IC90 (D) showed alterations in
regular morphology, expressing shortening and widening of the cell body. Bars = 1 mm.
doi:10.1371/journal.pone.0085706.g004
PLOS ONE | www.plosone.org
8
January 2014 | Volume 9 | Issue 1 | e85706

Quinoxaline 4 as an Anti-Trypanosoma cruzi Agent
Figure 5. Activity of quinoxaline 4 against intracellular amastigotes of T. cruzi. Infected LLCMK2 cells were incubated in the presence of
different concentrations of quinoxaline 4 for 96 h. Cells were stained with Giemsa, and the number of infected cells and amastigotes inside the cells
were counted and used to calculate the survival index (%). (A) Control cells. (B) Quinoxaline 4, 1.8 mM. (C) Quinoxaline 4, 3.6 mM. (D) Quinoxaline 4,
9.2 mM. (E) Quinoxaline 4, 18.4 mM. (F) Inhibition curve of increasing concentrations of quinoxaline 4 against the survival index (%) of intracellular
amastigotes. The experiment was repeated three times. The bars show the median 6 standard deviation.
doi:10.1371/journal.pone.0085706.g005
Amiodarone, an ergosterol biosynthesis blocker, exerted an
intrinsic synergistic effect when combined with Posaconazole
against T. cruzi. [45] Another study performed with Chagas’
disease patients found that combinatory therapy with benznida-
zole and allopurinol was promising, reflected by a reduction of
parasite burden. [25].
Figure 6. Combination effect of benznidazole and quinoxaline 4. Isobolographs describes the effect of the combination of quinoxaline 4 and
benznidazole against epimastigotes (A), trypomastigotes (B), and LLCMK2 cells (C). The dotted lines correspond to the additivity effect. Points below
the line indicate a synergistic effect. Points above the line indicate an antagonistic effect. The experiment was repeated three times. The points show
median values.
doi:10.1371/journal.pone.0085706.g006
PLOS ONE | www.plosone.org
9
January 2014 | Volume 9 | Issue 1 | e85706

Quinoxaline 4 as an Anti-Trypanosoma cruzi Agent
Figure 7. Cell volume alteration of epimastigotes treated with quinoxaline 4. Parasites were treated for 24 h and analyzed in flow
cytometer. The histograms show the relationship between the number of cells (counts) and Forward Scatter (FSC) considered as a function of cell
size. Gray-filled areas represent untreated cells. Unfilled areas represent treated parasites. (A) Actinomycin D, 20 mM. (B) Quinoxaline 4, 1.8 mM. (C)
Quinoxaline 4, 3.7 mM. (D) Quinoxaline 4, 7.4 mM. Typical histograms of at least three independent experiments are shown.
doi:10.1371/journal.pone.0085706.g007
The combination of benznidazole, nifurtimox, and posacona-
zole was found to be effective in the complete cure of mice that
were chronically or acutely infected by the Tulahuen strain of T.
cruzi, but the same effect was not observed for the Y strain. This
synergy was explained by the authors as cooperation between the
mechanisms of action of the drugs. [46] Benznidazole and
nifurtimox are prodrugs that induce oxidative stress and generate
electrophilic metabolites in the parasite through different path-
ways. [6] Posaconazole acts by inhibiting lanosterol demethylase in
the parasite, preventing the de novo synthesis of ergosterol. [47]
Thus, the effect of the combination of quinoxaline 4 and
benznidazole may also involve different cooperative mechanisms
of action.
DNA, proteins, and lipids, and induce the death of parasite. [6,47]
The mechanism of action of quinoxaline 4, in contrast, has not
been elucidated.
A previous docking study with different
quinoxaline derivatives demonstrated a strong interaction with
the poly (ADP-ribose) polymerase of T. cruzi, [18] an enzyme
implicated in the DNA-damage response and signaling of different
cell death pathways. [48] Several quinoxaline derivatives are also
known to have strong anti-tumor activity. XK469 (2-{4-[(7-chloro-
2-quinoxalinyl)oxy]phenoxy}propionic acid) and SH80 ([2-{4-[(7-
bromo-2-quinolinyl)oxy]phenoxy}]propionic acid) are two che-
motherapeutic agents that are effective against a broad range of
human tumors and related to the induction of cell cycle arrest and
a mixed autophagy and apoptosis profile. [49] In the present
study, the treatment with quinoxaline 4 led to a reduction of the
cell volume of the epimastigotes and did not induce severe
alterations in the cell membrane integrity of the three parasite
forms at the maximum concentration tested (7.4 mM). This was
also confirmed by the TEM analysis of the epimastigotes, showing
that the majority of the cells had a well-preserved external
The mechanism of action of benznidazole is well established.
The nitro group of the drug is reduced by nitroreductases of the
protozoan, with the formation of various free radical intermediates
that induce oxidative stress and electrophilic metabolites. These
metabolites are responsible for the main mechanism of action of
benznidazole. They covalently bond to macromolecules, such as
Figure 8. Analysis of cell membrane integrity of parasites treated with quinoxaline 4. Epimastigotes (A–E), amastigotes (F–G), and
trypomastigotes were untreated (A, F, K) or treated for 24 h with quinoxaline 4 at 1.8 mM (C, H, M), 3.7 mM (D, I, N), and 7.4 mM (E, J, O) and 40 mM
digitonin as a positive control (B, G, L). Events in the upper right and left quadrants represent cells with abnormal membrane permeability. Typical
histograms of at least three independent experiments are shown.
doi:10.1371/journal.pone.0085706.g008
PLOS ONE | www.plosone.org
10
January 2014 | Volume 9 | Issue 1 | e85706

Quinoxaline 4 as an Anti-Trypanosoma cruzi Agent
membrane, even when the inside of the cell was completely
altered. The SEM analysis showed almost no cells with leakage of
cellular content. Altogether, these results exclude the possibility
that death occurred through classic necrosis, which is well
characterized by an increase in cell volume, swelling of the
organelles, plasma membrane rupture, and subsequent loss of
intracellular content. [50–51].
is a controlled process of death characterized by a lack of tissue
inflammatory response in the host. [56].
Conclusion
The quinoxaline derivative tested in the present study represents
a possible scaffold in the complex process of development of a new
drug against T. cruzi. The results achieved in the drug combination
assay with quinoxaline 4 and benznidazole are also promising.
The syntheses of new derivates could result in enhanced molecules
with still higher selective indices. Further studies are necessary to
elucidate how exactly autophagic cell death is involved in the
mechanism of action of quinoxaline 4 and how both drugs can
interact synergistically and result in an enhanced final effect.
The ultrastructure of quinoxaline-treated epimastigotes also
revealed signs of the classic programmed cell death type II (PCD
II; i.e., autophagic cell death), which is morphologically charac-
terized by autophagosome formation and the appearance of
membranes that surround organelles and concentric cytosolic
membrane structures. [36,52,53] The well-developed ER profiles
that surround organelles, especially reservosomes, observed in
quinoxaline-treated epimastigotes was also found in T. cruzi after
treatment with a triazolic naphthoquinone. [54] Another alter-
ation found was secretory pathway stress, expressed as numerous
vesicles in the cytoplasm with severe disturbance of the
ultrastructure of the Golgi complex, which may indicate that this
organelle was acting as a source of the membranes for the
expansion of phagophores, a process already verified in yeast
autophagosome formation. [55] In summary, our results strongly
indicate parasite PCD through an autophagic pathway, which is
promising for possible future therapeutic approaches because this
Acknowledgments
We thank all the staff of the ‘‘Laborato´rio de Inovac¸a˜o Tecnolo´gica no
Desenvolvimento de Fa´rmacos e Cosme´ticos’’ for their collaboration.
Author Contributions
Conceived and designed the experiments: JHDSR AGC CVN. Performed
the experiments: JHDSR DPS. Analyzed the data: JHDSR AGC DPS
CVN. Contributed reagents/materials/analysis tools: AGC TUN CVN.
Wrote the paper: JHDSR TUN AGC DPS CVN.
References
17. Barea C, Pabo´n A, Galiano S, Pe´rez-Silanes S, Gonzalez G, et al. (2012)
Antiplasmodial and leishmanicidal activities of 2-cyano-3-(4-phenylpiperazine-1-
carboxamido) quinoxaline 1,4-dioxide derivatives. Molecules 7: 9451–9461.
18. Estevez Y, Quiliano M, Burguete A, Cabanillas B, Zimic M, et al. (2011)
Trypanocidal properties, structure-activity relationship and computational
studies of quinoxaline 1,4-di-N-oxide derivatives. Exp Parasitol 127: 745–751.
19. Barrett MP, Croft SL (2012) Management of trypanosomiasis and leishmaniasis.
Br Med Bull 104: 175–196.
1. WHO (2012) Chagas disease (American trypanosomiasis). WHO: Fact Sheet nu
340: 1–4.
2. Macedo AM, Machado CR, Oliveira RP, Pena SD (2004) Trypanosoma cruzi:
genetic structure of populations and relevance of genetic variability to the
pathogenesis of chagas disease. Mem Inst Oswaldo Cruz 1: 1–12.
3. Rassi Jr A, Rassi A, Marcondes-de-Rezende J (2012) American trypanosomiasis
(Chagas disease). Infect Dis Clin North Am 26: 275–291.
4. Feldman AM, McNamara D (2000) Myocarditis. N Engl J Med Nov 343: 1388–
1398.
20. Jeon YW, Suh YJ (2012) Synergistic apoptotic effect of celecoxib and luteolin on
breast cancer cells. Oncol Rep 29: 812–825.
5. Wilkinson SR, Kelly JM (2009) Trypanocidal drugs: mechanisms, resistance and
new targets. Expert Rev Mol Med 29: 11–31.
21. Park KS, Choi JJ, Kim WU, Min JK, Park S, et al. (2012) The efficacy of
tramadol/acetaminophen combination tablets (UltracetH) as add-on and
maintenance therapy in knee osteoarthritis pain inadequately controlled by
nonsteroidal anti-inflammatory drug (NSAID). Clin Rheumatol 31: 317–23.
22. Nahid P, Pai M, Hopewell PC (2006) Advances in the diagnosis and treatment of
tuberculosis. Proc Am Thorac Soc 3: 103–110.
6. Maya JD, Cassels BK, Iturriaga-Va´squez P, Ferreira J, Fau´ndez M, et al. (2007)
Mode of action of natural and synthetic drugs against Trypanosoma cruzi and their
interaction with the mammalian host. Comp Biochem Physiol, Part A Mol
Integr Physiol 146: 601–620.
7. Coura JR (2009) Present situation and new strategies for Chagas disease
chemotherapy: a proposal. Mem. Inst. Oswaldo Cruz 104: 549–554.
8. Veiga-Santos P, Pelizzaro-Rocha KJ, Santos AO, Ueda-Nakamura T, Dias
Filho BP, et al. (2010) In vitro anti-trypanosomal activity of elatol isolated from
red seaweed Laurencia dendroidea. Parasitology 137: 1661–1670.
23. Simon V, Ho DD, Abdool Karim
Q (2006) HIV/AIDS epidemiology,
pathogenesis, prevention, and treatment. Lancet 368: 489–504.
24. Loyse A, Wilson D, Meintjes G, Jarvis JN, Bicanic T, et al. (2012) Comparison of
the early fungicidal activity of high-dose fluconazole, voriconazole, and
flucytosine as second-line drugs given in combination with amphotericin B for
the treatment of HIV-associated cryptococcal meningitis. Clin Infect Dis 54:
121–128.
25. Perez-Mazliah DE, Alvarez MG, Cooley G, Lococo BE, Bertocchi G, et al.
(2012) Sequential combined treatment with allopurinol and benznidazole in the
chronic phase of Trypanosoma cruzi infection: a pilot study. J Antimicrob
Chemother 1: 1–14.
9. Izumi E, Ueda-Nakamura T, Dias Filho BP, Veiga Ju´nior VF, Nakamura CV
(2011) Natural products and Chagas’ disease: a review of plant compounds
studied for activity against Trypanosoma cruzi. Nat Prod Rep 28: 809–823.
10. Leite Silva CMB, Garcia FP, Rodrigues JHS, Nakamura CV, Ueda-Nakamura
T, et al. (2012) Synthesis, antitumor, antitrypanosomal and antileishmanial
activities of benzo[4,5]canthin-6-ones bearing the N -(substituted benzylidene)-
carbohydrazide and N-alkylcarboxamide groups at C-2. Chem Pharm Bull 60:
1372–1379.
26. Sangi DP, Correa AG (2010) Microwave-assisted synthesis of nitroketene N,S-
arylaminoacetals. J Braz Chem Soc 21: 795–799.
11. Becerra MC, Guin˜azu´ N, Hergert LY, Pellegrini A, Mazzieri MR, et al. (2012)
In vitro activity of N-benzenesulfonylbenzotriazole on Trypanosoma cruzi
epimastigote and trypomastigote forms. Exp Parasitol 131: 57–62.
12. Valdez RH, Tonin LT, Ueda-Nakamura T, Silva SO, Dias Filho BP, et al.
(2012) In vitro and in vivo trypanocidal synergistic activity of N-butyl-1-(4-
dimethylamino)phenyl-1,2,3,4-tetrahydro-b-carboline-3-carboxamide associated
with benznidazole. Antimicrob. Agents Chemother 56: 507–512.
27. Venkatesh C, Singh B, Mahata PK, Ila H, Junjappa H (2005) Heteroannulation
of nitroketene N,S-arylaminoacetals with POCl3: A novel highly regioselective
synthesis of unsymmetrical 2,3-substituted quinoxalines. Org Lett 7: 2169–2172.
28. Camargo EP (1964) Growth and differentiation in Trypanosoma cruzi. I. Origin of
metacyclic trypanosomes in liquid media. Rev Inst Med Trop Sao Paulo 6: 93–
100.
29. Andrews NW, Colli W (1982) Adhesion and interiorization of Trypanosoma cruzi
in mammalian cells. J Protozool 29: 264–269.
30. Brener Z (1962) Therapeutic activity and criterion of cure on mice
13. Ishikawa H, Sugiyama T, Kurita K, Yokoyama A (2012) Synthesis and
antimicrobial activity of 2,3-bis(bromomethyl)quinoxaline derivatives. Bioorg
Chem 41: 1–5.
experimentally infected with Trypanosoma cruzi. Rev Inst Med Trop Sa˜o Paulo
4: 389–396.
14. Kim YB, Kim YH, Park JY, Kim SK (2004) Synthesis and biological activity of
new quinoxaline antibiotics of echinomycin analogues. Bioorg Med Chem Lett
19: 541–544.
31. Izumi E, Morello LG, Ueda-Nakamura T, Yamada-Ogatta SF, Dias Filho BPD,
et al. (2008) Trypanosoma cruzi: Antiprotozoal activity of parthenolide obtained
from Tanacetum parthenium (L.) Schultz Bip. (Asteraceae, Compositae) against
epimastigote and amastigote forms. Exp Parasitology 118: 324–330.
32. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J Immunol Meth 16: 55–63.
33. Chou TC, Talalay P (1984) Quantitative analysis of dose-effect relationships: the
combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22:
27–55.
15. Font M, Monge A, Alvarez E, Cuartero A, Losa MJ, et al. (1997) Synthesis and
evaluation of new Reissert analogs as HIV-1 reverse transcriptase inhibitors. 1.
Quinoline and quinoxaline derivatives. Drug Des Discov 14: 305–332.
16. Espinosa A, Sosha AM, Ryke E, Rowley DC (2012) Antiamoebic properties of
the actinomycete metabolites echinomycin A and tirandamycin A. Parasitol Res
111: 2473–2477.
PLOS ONE | www.plosone.org
11
January 2014 | Volume 9 | Issue 1 | e85706

Quinoxaline 4 as an Anti-Trypanosoma cruzi Agent
34. Zhao L, Au JL, Wientjes MG (2010) Comparison of methods for evaluating
drug-drug interaction. Front Biosci 1: 241–249.
35. Chou TC (2010) Drug combination studies and their synergy quantification
using the Chou-Talalay method. Cancer Res 2: 440–446.
46. Cencig S, Coltel N, Truyens C, Carlier Y (2012) Evaluation of benznidazole
treatment combined with nifurtimox, posaconazole or AmBisome(H) in mice
infected with Trypanosoma cruzi strains. Int J Antimicrob Agents 40: 527–532.
47. Urbina JA (2010) Specific chemotherapy of Chagas disease: relevance, current
limitations and new approaches. Acta Trop 115: 55–68.
36. Izumi E, Ueda-Nakamura T, Veiga VF, Pinto AC, Nakamura CV (2012)
Terpenes from Copaifera demonstrated in vitro antiparasitic and synergic activity.
J Med Chem 12: 2994–3001.
37. Menna-Barreto RF, Goncalves RL, Costa EM, Silva RS, Pinto AV, et al. (2009)
The effects on Trypanosoma cruzi of novel synthetic naphthoquinones are
mediated by mitochondrial dysfunction. Free Radic Biol Med 47: 644–653.
38. Lopes JN, Cruz FS, Docampo R, Vasconcellos ME, Sampaio MC, et al. (1978)
In vitro and in vivo evaluation of the toxicity of 1,4-naphthoquinone and 1,2-
naphthoquinone derivatives against Trypanosoma cruzi. Ann Trop Med Parasitol.
72: 523–531.
48. Vilchez-Larrea SC, Alonso GD, Schlesinger M, Torres HN, Flawia´ MM, et al.
(2011) Poly(ADP-ribose) polymerase plays a differential role in DNA damage-
response and cell death pathways in Trypanosoma cruzi. Int J Parasitol 41: 405–
416.
49. Reiners JJ, Kleinman M, Joiakim A, Mathieu PA (2009) The chemotherapeutic
agents XK469 (2-{4-[(7-chloro-2-quinoxalinyl)oxy] phenoxy}propionic acid)
and SH80 (2-{4-[(7-bromo-2-quinolinyl)oxy]phenoxy} propionic acid) inhibit
cytokinesis and promote polyploidy and induce senescence. J Pharmacol Exp
Ther 328: 796–806.
39. Schrijvers
D (2003) Role of red blood cells in pharmacokinetics of
50. Chaabane W, User SD, El-Gazzah M, Jaksik R, Sajjadi E, et al. (2013)
Autophagy, Apoptosis, Mitoptosis and Necrosis: Interdependence Between
Those Pathways and Effects on Cancer. Arch Immunol Ther Exp. 61: 43–58.
51. Schweichel JU, Merker HJ (1973) The morphology of various types of cell death
in prenatal tissues. Teratology 7: 253–266.
chemotherapeutic agents. Clin Pharmacokinet. 42: 779–791.
40. Krantz A (1997) Red cell-mediated therapy: opportunities and challenges. Blood
Cells Mol Dis 23: 58–68.
41. Pelizzaro-Rocha KJ, Tiuman TS, Izumi E, Ueda-Nakamura T, Dias Filho BP,
et al. (2010) Synergistic effects of parthenolide and benznidazole on Trypanosoma
cruzi. Phytomedicine 18: 36–39.
42. Chou TC (2006) Theoretical basis, experimental design, and computerized
simulation of synergism and antagonism in drug combination studies. Pharmacol
Rev 58: 621–681.
43. Chen L, Dou J, Su Z, Zhou H, Wang H, et al. (2011) Synergistic activity of
baicalein with ribavirin against influenza A (H1N1) virus infections in cell culture
and in mice. Antiviral Res. 91: 314–320.
44. Maldonado RA, Molina J, Payares G, Urbina JA (1993) Experimental
chemotherapy with combinations of ergosterol biosynthesis inhibitors in murine
models of Chagas’ disease. Antimicrob Agents Chemother. 37: 1353–1359.
45. Benaim G, Sanders JM, Garcia-Marcha´n Y, Colina C, Lira R, et al. (2006)
Amiodarone has intrinsic anti-Trypanosoma cruzi activity and acts synergisti-
cally with posaconazole. J Med Chem 49: 892–899.
52. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, et al. (2008)
Guidelines for the use and interpretation of assays for monitoring autophagy in
higher eukaryotes. Autophagy 4: 151–175.
53. Bursch W, Karwan A, Mayer M, Dornetshuber J, Fro¨hwein U, et al. (2008) Cell
death and autophagy: cytokines, drugs, and nutritional factors. Toxicology 254:
147–157.
54. Fernandes MC, Da Silva EM, Pinto AV, De Castro SL, Menna-Barreto RF
(2012) A novel triazolic naphthofuranquinone induces autophagy in reservo-
somes and impairment of mitosis in Trypanosoma cruzi. Parasitology 139: 26–36.
55. van der Vaart A, Reggiori F (2010) The Golgi complex as a source for yeast
autophagosomal membranes. Autophagy 6: 800–801.
56. Levine B, Mizushima N, Virgin HW (2011) Autophagy in immunity and
inflammation. Nature 20: 323–335.
PLOS ONE | www.plosone.org
12
January 2014 | Volume 9 | Issue 1 | e85706