Searching for new drugs for Chagas diseases: triazole analogs display high in vitro activity against Trypanosoma cruzi and low toxicity toward mammalian cells

Article abstract of DOI:10.1007/s10863-018-9746-z

Chagas disease is one of the most relevant endemic diseases in Latin America caused by the flagellate protozoan Trypanosoma cruzi. Nifurtimox and benzonidazole are the drugs used in the treatment of this disease, but they commonly are toxic and present severe side effects. New effective molecules, without collateral effects, has promoted the investigation to develop new lead compounds with to advance for clinical trials. Previously, 3-nitro-1H-1,2,4-triazole-based amines and 1,2,3-triazoles demonstrated significant trypanocidal activity against T. cruzi. In this paper, we synthesized a new series of 92 examples of 1,2,3-triazoles. Six compounds exhibited antiparasitic activity, 14, 25, 27, 31 and 40, 43 and were effective against epimastigotes of two strains of T. cruzi (Y and Dm28-C) and 25, 27 and 31 exhibited trypanocidal activity similar to benzonidazole. Notably, the compound 25 compared to benzonidazole increase the toxicity against T. cruzi, with no apparent toxicity to the cell line of mice macrophages or primary mice peritoneal macrophages. As results, we calculated selectivity indexes up to 2000 to 25 and 31 in both T. cruzi strains. Derivative 14 caused a trypanostatic effect because it did not damage external epimastigote membrane. Triazoles 40 and 43 impaired parasites viability using a pathway not dependent on ROS production.

Full text of DOI:10.1007/s10863-018-9746-z

Journal of Bioenergetics and Biomembranes
https://doi.org/10.1007/s10863-018-9746-z
Searching for new drugs for Chagas diseases: triazole analogs display
high in vitro activity against Trypanosoma cruzi and low toxicity
toward mammalian cells
Robson Xavier Faria1 & Daniel Tadeu Gomes Gonzaga2 & Paulo Anastácio Furtado Pacheco2
André Luis Almeida Souza3 & Vitor Francisco Ferreira4 & Fernando de Carvalho da Silva2
&
Received: 6 November 2017 /Accepted: 7 February 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Chagas disease is one of the most relevant endemic diseases in Latin America caused by the flagellate protozoan Trypanosoma
cruzi. Nifurtimox and benzonidazole are the drugs used in the treatment of this disease, but they commonly are toxic and present
severe side effects. New effective molecules, without collateral effects, has promoted the investigation to develop new lead
compounds with to advance for clinical trials. Previously, 3-nitro-1H-1,2,4-triazole-based amines and 1,2,3-triazoles demon-
strated significant trypanocidal activity against T. cruzi. In this paper, we synthesized a new series of 92 examples of 1,2,3-
triazoles. Six compounds exhibited antiparasitic activity, 14, 25, 27, 31 and 40, 43 and were effective against epimastigotes of two
strains of T. cruzi (Y and Dm28-C) and 25, 27 and 31 exhibited trypanocidal activity similar to benzonidazole. Notably, the
compound 25 compared to benzonidazole increase the toxicity against T. cruzi, with no apparent toxicity to the cell line of mice
macrophages or primary mice peritoneal macrophages. As results, we calculated selectivity indexes up to 2000 to 25 and 31 in
both T. cruzi strains. Derivative 14 caused a trypanostatic effect because it did not damage external epimastigote membrane.
Triazoles 40 and 43 impaired parasites viability using a pathway not dependent on ROS production.
.
.
.
Keywords 1,2,3-triazoles Epimastigotes strains Y strain Dm28-C strain
Introduction
the American continent in wild mammalian host, and dissemi-
nated by the triatomine bug insect vector. T. cruzi infects human
host through vectorial transmission, ingestion of food and
drinks infected with live parasites, from mother to child during
pregnancy, and contaminated blood transfusion or organ trans-
plantation (Kinoshita-Yanaga et al. 2009). Latin Americans ex-
tensive migration, in the last decades, has transported a crescent
number of infected individuals to non-endemic regions such as
Europe, North America, Asia and Australia. In this area, T. cruzi
transmission can occur through the nonvectorial pathway
(Gascon et al. 2010; Bern and Montgomery 2009). Although
this disease possesses high percussion in Latin America, a re-
gion with millions of people infected and thousands die annu-
ally, only nifurtimox and benzonidazole are the drugs available
to treat this disease. Inconveniences associated to use these
drugs comprise severe side effects and low efficacy, essentially
in the chronic phase of the disease. In consequence, there is a
crucial hunt for new drugs with improving efficacy, tolerability,
and safety (Field et al. 2017).
The treatment of Chagas disease stills is a major challenger since
its discovery more than a century ago. It is occasioned by the
protozoan parasite Trypanosoma cruzi, distributed throughout
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10863-018-9746-z) contains supplementary
material, which is available to authorized users.
* Robson Xavier Faria
robson.xavier@gmail.com
1
Laboratory of Toxoplasmosis and other Protozoans, Oswaldo Cruz
Institute - Fiocruz, Rio de Janeiro, Brazil
2
Departamento de Química Orgânica, Instituto de Química,
Universidade Federal Fluminense, Campus do Valonguinho,
Niterói, RJ CEP 24020-150, Brazil
3
Laboratory of Biochemistry of Peptides, Oswaldo Cruz Institute –
Fiocruz, Rio de Janeiro, Brazil
Novel chemical entities (NCEs) for chemotherapy of Chagas’
disease have been increased in the last years. Posaconazole, a
4
Departamento de Tecnologia Farmacêutica, Faculdade de Farmácia,
Universidade Federal Fluminense, Niterói, RJ 24241-002, Brazil

J Bioenerg Biomembr
1,2,4-triazole, commonly used to treat systemic fungal infections,
administrated against T. cruzi caused trypanocidal activity with
satisfactory efficacy in vitro and in vivo (Urbina et al. 1998;
Molina et al. 2000a, b; Urbina et al. 2003a, b; Ferraz et al.
2007; Olivieri et al. 2010; Veiga-Santos et al. 2012). However,
T. cruzi strains exhibit different levels of natural susceptibility (or
resistance) to benzonidazole and nifurtimox over time (Filardi
and Brener 1987; Camandaroba et al. 2003; Teston et al.
2013). Therefore, other new drugs studied in clinical trials
(Bahia et al. 2012; Bustamante et al. 2013) or in vitro assays
(Moraes et al. 2014) exhibited poor activity due to resistant
strains. More studies are required to discover selective drugs
for each strain or a more efficient to six discrete typing units
(DTUs) (Zingales et al. 2009).
Analogs of 1,2,3-Triazole present potent antiprotozoal
activity and confer metabolic stability when administrated
in vivo (Richardson et al. 1988; Troke et al. 1988; Van den
Bossche et al. 1988; Van den Bossche et al. 1987).
Peculiarly, 1,2,4-triazoles as fluconazole and itraconazole
exhibited antiproliferative action to T. cruzi, both in vivo
and in vitro assays (Goad et al. 1989; McCabe et al. 1986).
ICI 195,739, which is a 3′-styryl-substituted bistriazolyl
tertiary alcohol demonstrated high activity contra T. cruzi
parasitemia when it was orally administrated in a murine
model (Boyle et al. 1988; Ryley et al. 1988).
Other several 1,2,4-triazole analogues also were tested
(Buckner et al. 2012), including D0870 (Molina et al.
2000a, b), posaconazole (POS) (Molina et al. 2000a, b;
Urbina et al. 2000), ravuconazole (Urbina et al. 2003a, b),
albaconazole (Guedes et al. 2004; Urbina et al. 2003a, b),
and TAK-187 (Corrales et al. 2005; Urbina et al. 2003a, b).
3-nitro-1H-1,2,4-triazole-based derivatives manifested signif-
icant and selective action against T. cruzi amastigotes in in-
fected L6 cells activating a type I nitroreductase, selective to
trypanosomatids (Papadopoulou et al. 2013a, b).
Posaconazole is another antifungal drug of the triazole
class used to treat invasive fungal infection in humans
(Kwon and Mylonakis 2007). Additionally, it has been proven
potent trypanocidal activity in both in vitro and in vivo
models. As an example, in a murine model of acute Chagas’
disease, it recovered more than 90% of animals infected with
susceptible and partially susceptible T. cruzi strains, whereas
only 76% were rehabilitated with benzonidazole. In a chronic
model, posaconazole restored up to 60% in animals infected
with susceptible and partially susceptible T. cruzi strains,
whereas benzonidazole treatment was ineffective (Urbina
et al. 1998; Molina et al. 2000a, b). However, in a clinical trial
in patients with chronic Chagas’ disease, the posaconazole
presented a minor trypanocidal activity than benzonidazole
group (Molina et al. 2014). Based on this sensibility the
T. cruzi to triazole derivatives, we tested a new series of
1-Aryl-1H- and 2-aryl-2H-1,2,3-triazole against trypano-
somes viability in vitro.
Experimental section
Trypanosoma cruzi culture Y and DM28c strains of T. cruzi
epimastigotes were cultured in medium LIT (Tryptose Liver
infusion) supplemented with 10% fetal calf serum veal, a tem-
perature of 28 °C, with strong agitation (100 rpm). Live par-
asites were counted daily using a Neubauer chamber. The
initial parasite concentration was 106 parasites/ml.
Mammalian cells culture J774.G8 cells and mice peritoneal
macrophages were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM), supplemented with 10% inactivated FBS,
along with 1% of 10,000 units/mL penicillin and 10 mg/mL
streptomycin. Jurkat cells were cultured in RPMI 1640 medi-
um supplemented with 10% inactivated FBS, along with 1%
of 10,000 units/mL penicillin and 10 mg/mL streptomycin.
Evaluation of the triazoles-induced antitrypanosomal activity
Epimastigotes toxicity was determined using Alamar Blue.
Cells seeded into 96-well flat-bottom microplates at a density
of 1 × 105 cells/200 μl in the presence of benzonidazole and
1,2,3-triazole derivatives, serially diluted to concentrations
from 1 nM to 10 mM, and incubated for 72 h at 37 °C in a
humidified atmosphere of 5% CO2. Drugs or vehicle (dimeth-
yl sulfoxide [DMSO]) was added after 24 h under each con-
dition. At least three independent experiments were performed
for each drug, and the 50% effective concentration (IC50) was
determined using Prisma GraphPad 5.0. Twenty microliters of
10% Alamar Blue® (AbD Serotec, Raleigh, North Carolina)
were added to wells after 56 h, and fluorescence determined at
the 72 h point using a Spectramax M5 microplate fluorometer
(Molecular Devices) with a 530 nm excitation and 590 nm
emission wavelengths.
Evaluation of mitochondrial membrane potential and cell
membrane integrity Parasitic forms of T. cruzi (1 × 106
parasites/mL for epimastigotes and 1 × 107 parasites/mL for
amastigotes) treated or not with the IC90 of selected triazoles,
and incubated with 10 μM Rh123 for 15 min at 37 °C at room
temperature. Parasites were washed, suspended in PBS, and
kept on ice until analysis using a FACSCalibur flow cytometer
(Becton-Dickinson, Rutherford, NJ, USA), and CellQuest soft-
ware (Joseph Trotter, The Scripps Research Institute, La Jolla,
CA, USA). Ten thousand events were acquired in a region pre-
viously established with epimastigote. Alterations in Rh123
fluorescence quantified using an index variation obtained by
the eq. (MT − MC) / MC, where MT is the median fluorescence

J Bioenerg Biomembr
Fig. 1 Synthetic routes for
obtainment 1,2,3-triazole series
Synthetic routes used to prepare 1,2,3-triazole series. The reagents and
conditions were as follows: i) NaNO2, HCl(aq) 50 %, 0-5 ºC then NaN3, H2O; ii)
Propargylic alcohol or ethyl propiolate (for preparation of 7), CuSO4, ascorbic
acid, tBuOH, H2O; iii) RCOCl, CH2Cl2, Pyridine, DMAP, rt; iv) R-Br, THF, NaH,
reflux; v) PhNHNH2.HCl, H2O, reflux; vi) CuSO4, H2O, reflux; vii) NaIO4, H2O, rt;
viii) IBX, DMSO, rt; ix) NaBH4, CH3OH, rt; x) Ph3CH3Br, NaH, THF; xi)
2NHR7, EtOH, H2SO4 (cat) and xii) NH2OH.HCl, CH2Cl2, pyridine, rt
OH
Fig. 2 Chemical structures of the
selected triazoles and % viability
against T. cruzi (Y strain)
N
O
O
H
N
N
N
N
N
N
N
N
N
Cl
Cl
N
Cl
Cl
NO2
Cl
N
H
N
14, 34 0.79
25, 26 3.76
27, 29 4.99
O
O
O
O
Benznidazole
33 5.67
O
X
O
X
X
N
Y
N
Y
N
N
Y
N
N
Cl
Cl
OMe
Cl
31, 45 5.09
40, 39 5.80
43, 52 6.20

J Bioenerg Biomembr
Table 1 Biological effects of the
selected Triazole derivatives
Drugs
Y strain MTT
EC50 (uM)
Dm28-C strain
MTT EC50 (uM)
Peritoneal Macrophages
LDH CC50 (mM)
J774.G8 LDH
CC50 (mM)
40
103.8
0.185
6.11
104
0.017
0.03
0.008
0.02
27
0.3
14
11.39
0.246
7.96
88.72
19.86
0.165
0.529
0.254
0.087
0.037
0.153
0.450
0.358
0.024
0.036
25
0.209
7.28
31
43
24.08
7.85
Benzonidazole
LD50: Median lethal dose calculated with 95% Confidence interval capable to death 50% of the population; IC50:
Median inhibitory concentration
for treated parasites and MC is the median fluorescence of con-
trol parasites. Negative index variation values corresponded to
depolarization of the mitochondrial membrane. FCCP (100 μM)
was used as a positive control for the Rh123 experiments.
ROS detection with DHE We evaluated ROS generation label-
ing epimastigotes with 10 μM dihydroethidium (DHE)
(Molecular Probes) for 30 min at 28 °C. Our positive control
was 25 μM antimycin A (AA) (Sigma-Aldrich). The samples
were analyzed in a FACSCalibur flow cytometer (Becton
Dickinson, CA, USA) equipped with the Cell Quest software
(Joseph Trotter, Scripps Research Institute, La Jolla, USA). A
total of 10,000 events were acquired in the region previously
established as that of the parasites.
Alterations in the fluorescence of propidium iodide (PI) PI (10
uM) fluorescence was quantified as the percentage of altered
plasma membrane cells. PI was added in the last 5 min of
incubation for all tubes. Digitonin (40 μM) used as a positive
control for the PI experiments.
Fig. 4 Benzonidazole and selected triazole effects on the plasmatic
membrane integrity on mammalian cells using LDH assay. a Mice
peritoneal macrophages and (b) J774.G8 cell line. All data are
represented as Mean S.D. These graphics are generated for 4
experiments conducted in distinct days. % LDH release was normalized
for 0.1% Tx 100 effect. These graphics were generated with data from 3
experiments conducted in distinct days
Fig. 3 Benzonidazole and triazole effects on epimastigotes of T. cruzi
viability using MTT assay. a Y strain and (b) Dm28-C strain. All data
are represented as Mean S.D. Percentage of trypanocidal activity was
normalized for 0.1% Tx 100 effect. These graphics were generated with
data from 4 experiments conducted in distinct days

J Bioenerg Biomembr
Table 2 Selective index of selected Triazole derivatives
Drugs
Selectivity Index (S.I.) Peritoneal
macrophages/ Y strain
Selectivity Index (S.I.) Peritoneal
macrophages/ Dm28-C strain
Selectivity Index (S.I.)
J774.G8 cells/ Y strain
Selectivity Index (S.I.)
J774.G8 cells/ Dm28-C strain
40
0,16
0,16
0,07
0,07
27
162,16
27,02
2531,1
34,89
366,25
4,71
100
108,1
25,04
2153,1
49,17
9,96
66,6
14
14,48
2550,4
31,90
99,09
1,86
13,43
1829,2
44,97
2,7
25
31
43
Benzonidazole
4,58
1,81
Results
In vitro Antiparasitic activity and cytotoxicity
Chemistry
Epimastigotes of Yand Dm28-c strains treated with a fixed dose
of 10 μM triazoles for 72 h, Triton X 100 detergent (0.1%) in the
last 10 min (positive control), and no treated epimastigotes (neg-
ative control). Among 92 triazole analogs, 14, 25, 27, 31 and 40,
43 reduced epimastigote viability in values comparable to
benzonidazole treatment (Fig. 2, Table 1). Other triazoles did
not cause a trypanocidal effect (Supplemental Fig. 1).
The methods used to prepare the 1-aryl-1H-1,2,3-triazoles and
2-aryl-2H-1,2,3-triazoles (92 examples) involved classical
chemical transformations widely described in the literature
and previously explored by our research group (Boechat
et al. 2011; Gonzaga et al. 2014; Kolb et al. 2001) (Fig. 1).
Fig. 5 Selected triazole effects on
ΔΨm of T.cruzi strains. a Y strain
treated for 3 h, b Dm28c treated
for 3 h, c Y strain treated for 24 h
and Dm28c strain treated for 24 h.
All data are represented as Mean
S.D. Percentage of
fluorescence, respective to
Rhodamine- 1,2,3 fluorescence,
was normalized for FCCP
(100 μM) effect. These graphics
are generated with data from 4
experiments conducted in distinct
days. *** for a p < 0.05 compared
with the control bar

J Bioenerg Biomembr
Trypanocidal effect of dose- response triazoles was inves-
tigated against Yand Dm28-C strains (Fig. 3a, b). In compar-
ison to benzonidazole, derivatives 40 and 43 exhibited high
EC50 values for Yand Dm28-C strains, respectively. Triazoles
14 and 31 demonstrated activity similar to benzonidazole
(EC50) and the triazoles 27 and 25 obtained EC50 values more
than 30 times inferior to benzonidazole for both strains (Fig.
3a, b, Table 1).
Selected triazoles effect in mouse J774.G8 cell lineage
and mice peritoneal macrophages monitored for 72 h of
continuous exposition. The CC50 values in both cell types
are represented in Fig. 4 and Table 1. Triazole 25 and 31 in
particular displays a remarkably reduced toxicity against
both cell mammalian types with CC50 values in high mil-
limolar concentrations (Table 1). Additionally, triazole 14
also did not exhibit appreciable toxicity (Fig. 4 and
Table 1). In contrast, triazoles 27, 40 and 43 demonstrated
toxicity similar to benzonidazole (Fig. 4 and Table 1).
Selectivity indices (S.I.) calculated for triazole analogs
indicate an excellent value for 25 and 27. Especially, 25
exhibited a value above to 2 thousand for both T. cruzi
strains (Table 2). Triazole 27 demonstrated a high S.I.
with values above to 1 hundred for both strains.
Triazoles 14 and 31 reached S.I. values superior to
benzonidazole for all cell types and 40 displayed S.I.
worse than benzonidazole. In addition, undoubtedly the
derivative 25 was the most promisor (Table 2).
We used Rhodamine 123 to evaluate mitochondrial mem-
brane potential (ΔΨm) in treated epimastigotes. Parasites
treated with IC90 reduced ΔΨm after 3 h of treatment in both
strains (Fig. 5a, b) when compared to no treated strains.
Triazoles profile activity was similar between both strains
with the following order of activity: 25 > 27 > 31 > 14 >
43 > 40. After 24 h of treatment, 40 and 43 were not respon-
sive (Fig. 5c, d) for both T. cruzi strains. In relation to other
triazoles, the order above was the same for both strains after
24 h, 25 > 27 > 31 > 14.
All these triazoles promoted more mitochondrial membrane
disruption when compared with 3 h of treatment. A possible
consequence of disrupting ΔΨm is generated ROS and this
agent may act on intracellular enzymes of the parasites. Then,
we used DHE to measure intracellular ROS production in the
parasite strains. Triazoles 40 and 43 did not produce ROS in
both strains independent of time of treatment (Fig. 6). Triazole
25 produced highest ROS quantity after 3 h, this effect was
followed by compound 27, 31, and 14 in both strains (Fig.
Fig. 6 Selected triazole effects on
ROS production of T. cruzi
strains. a Y strain treated for 3 h, b
Dm28c treated for 3 h, c Y strain
treated for 24 h, and d Dm28c
strain treated for 24 h. All data are
represented as Mean S.D.
Percentage of fluorescent cells,
respective to DHE fluorescence,
were normalized for 25 μM
antimycin (AA) effect. These
graphics are generated with data
from 4 experiments conducted in
distinct days. *** for a p < 0.05
compared with the control bar

J Bioenerg Biomembr
6a, b). Similar result occurred after 24 h of treatment. When
compared to Antimycin A, compound 25 almost reach the
maximal ROS production in 24 h in both strains (Fig. 6c, d).
This augments in the triazole-induced ROS production
may cause external membrane destruction in the parasites.
Then, we measured PI entry after a possible membrane
disruption. Y strain treatment with triazoles 25, 27, and
31 caused PI labeling after 3 h (Fig. 7a), in this order of
activity. Dm28c strain treatment with triazoles 14, 25, 27,
and 31 exhibited PI uptake (Fig. 7b). After 24 h of treat-
ment, triazoles 25, 27 and 31 augmented their trypanocide
action and 14 caused a modest PI elevation in both
epimastigote strains (Fig. 7c) in both strains.
Based on results above, we considered triazole 25 the more
active trypanocide agent for T. cruzi epimastigotes. Moreover,
triazoles 27 and 31 also exhibited promisor trypanocide action.
Another manner of triazole analogs eliminate epimastigote
forms is augmenting redox mammalian cell metabolism. All
analogs induced ROS in mice peritoneal macrophages, and
J774 cells incubated with DHE after 3 h (Fig. 8a, b).
Triazoles 40, and 43 sustain ROS production until 24 h of
treatment (Fig. 8c, d). However, 14, 25, 27, and 31 molecules
returned to basal ROS production levels after 24 h (Fig. 8c, d).
Discussion
Since 1976, Novinsion and collaborates synthetized several
pyrazolo[1,5-alpha]pyrimidines, pyrazolo[1,5-alpha]-1,3,5-
triazines, s-triazolo[1,5-alpha]pyrimidines and imidazo[1,2-
alpha]pyrimidines condensed with 5-nitrofurfural against
T. cruzi (Novinsion et al. 1976). A large diversity of triazole
classes have been tested against T. cruzi (Silva et al. 2016;
Carvalho et al. 2014; da Silva Júnior et al. 2012), but the
majority is of 3-Nitro-1H-1,2,4-triazole analogs exhibiting ef-
fects in vitro and in vivo. However, there are few studies
testing 1-Aryl-1H- and 2-Aryl-2H-1,2,3-triazole.
Fig. 7 Selected triazole effects on
PI cell entry in T. cruzi strains. a Y
strain treated for 3 h, b Dm28c
treated for 3 h, c Y strain treated
for 24 h and Dm28c strain treated
for 24 h. All data are represented
as Mean S.D. Percentage of
fluorescent cells, respective to PI
fluorescence, were normalized for
Digitonin (40 μM) effect. These
graphics are generated with data
from 4 experiments conducted in
distinct days. *** for a p < 0.05
compared with the control bar

J Bioenerg Biomembr
Fig. 8 Selected triazole effects on
ROS production of mammalian
cells. a Mice peritoneal
macrophages treated for 3 h, b
J774 cells treated for 3 h, c Mice
peritoneal macrophages treated
for 24 h, and (d) J774 cells strain
treated for 24 h. All data are
represented as Mean S.D.
Percentage of fluorescent cells,
respective to DHE fluorescence,
were normalized for 25 μM
antimycin (AA) effect. These
graphics are generated with data
from 3 experiments conducted in
distinct days. *** for a p < 0.05
compared with the control bar
After a screening evaluating MTT reduction of Y strains
treated with 92 triazoles analogs, six molecules were selected
for additional assays. They were evaluated against no infected
mice peritoneal macrophages and J774.G8 cells to calculate
CC50 values. Moreover, this action against two strains of
T. cruzi (Y and Dm28-C) for calculating the EC50 values.
Urbina and collaborates developed a bis-triazole deriva-
tive, ICI 195,739 with inhibitory action against epimastigote
growth in 100% for a concentration of 100 nM (Urbina et al.
1991). In 2000, Urbina’s group exhibited UR-9825
[(1R,2R)-7-chloro-3-[2-(2,4-difluoropenyl)-2-hydroxyl-1-
methyl-3-(1H-1,2,4-triazol-1-yl)propyl]quinazolin-4(3H)-
one] (Uriach and collaborates) with a new triazole inhibiting
epimastigote MIC with 30 nM after 144 h (Urbina et al.
2000). This result was considered excellent in comparison
to other selective inhibitors acting against parasite’s sterol
C14α demethylase, an enzyme related to T. cruzi growth
(Urbina et al. 1998).
against T. cruzi in vitro are posaconazole and ravuconazole.
Commonly, they have sterol 14alpha-demethylase enzyme
(also known as CYP51), required for ergosterol biosynthesis
as a target (Urbina et al. 1998; Buckner 2011). Becerra and
collaborates synthetized N-benzenesulfonylbenzotriazole
(BSBZT) against epimastigote forms of T. cruzi. BSBZT and
benzotriazole (BZT) inhibited in vitro growth with an IC50
value of 81.07 μM and caused a reduced toxicity against hu-
man red blood cells and mouse macrophage-derived RAW
264.7 cell line (Becerra et al. 2012).
Moraes and colleagues in 2014 tested these molecules
against 8 T. cruzi clones (Dm28c, Y, ARMA13, cl1, ERA
cl2, 92–80 cl2, CL Brener, and Tulahuen). Posaconazole
inhibited epimastigote activity in concentrations of 1–11 nM
for all clones, and Ravuconazole inhibited in the range from
0.62–3.6 nM for all clones (Moraes et al. 2014).
Pertino and collaborates tested 34 triazole derivatives of
dehydroabietic acid and oleanolic Acid against T. cruzi for
72 h of treatment. All derivatives inhibited epimastigote activ-
ity in micromolar concentrations (Pertino et al. 2017).
Selected triazoles 25 and 27 were more potent than all other
triazoles tested against epimastigote T. cruzi form described
above and low toxicity to mammalian cells. Both factors
highlighted 25 and 27 as a trypanocidal agent.
ICI 195,739 possess an R(+) enantiomer, named D0870,
this compound inhibited epimastigote growth with IC50 of
0.1 μM, in contrast, its S(−) enantiomer with higher IC50
value, 3 μM (Liendo et al. 1998).
Other triazoles used for treating systemic fungal infections
and have shown potent trypanocidal activity and efficacy

J Bioenerg Biomembr
Author contributions Robson Xavier Faria and André Luis Souza de-
signed the research; Fernando de Carvalho da Silva, Vitor Francisco
Ferreira e Robson Xavier Faria provided the space and the reagents;
Robson Xavier Faria and André Luis Souza performed the experiments;
Daniel Tadeu Gomes Gonzaga synthetized the triazoles, Fernando de
Carvalho da Silva and Robson Xavier Faria wrote the manuscript. All
authors have read and approved the final version of the manuscript.
Selected triazoles 14, 25, 27, 31 and 40, 43 were in-
vestigated in relation to the mechanism to cause a
trypanocidal effect in Y and Dm28c strains. Triazoles
14, 25, 27 and 31 altered ΔΨm in both epimastigote
strains after brief and sustained stimulation (Fig. 5). This
effect is associated with ROS augment by 25 and 27 after
3 or 24 h, in contrast to 14 and 31 with elevating ROS
production after 24 h (Fig. 6). ROS increase essentially
caused for 25 and 27 treatment may react with
epimastigote membrane to promote damage. Triazoles 25
and 27 induced external membrane disruption after 3 h,
and this effect increased after 24 h for them and 31 ana-
logs. Therefore, these triazoles generated a pronounced
trypanocidal action, as previously observed for other
triazoles (Urbina et al. 1998). However, triazole 14 ele-
vate ROS production after 24 h did not cause external
membrane damage, thus 14 effects may be related to other
pathway associated cytochrome P-450-dependent enzyme
CYP51 (Buckner et al. 2012; Keenan et al. 2013). Other
derivatives 40 and 43 only caused a restrict increase in the
ROS production in 3 h. This may be an indication of
action on pathways related to glycolytic pathway, poly-
amine biosynthesis or microtubule stability in T. cruzi.
Triazoles, 25, 27, and 31 caused plasma membrane
disruption after 24 h in both T.cruzi strains. However,
14, 40, and 43 did not promote PI entry. Surprisingly,
LDH release assay in mammalian cell types indicates
low toxicity for molecules 25, and 31. Thus, these
triazoles may exhibit selectivity for parasites. In relation
to derivative 27, it also was potent to cause mammalian
cell toxicity. Therefore, this triazole should be discarded
for a hypothetical treatment in vivo. Analog 40 did not
possess trypanocidal action and high toxicity in mam-
malian cells. Additionally, 14, and 43 exhibited
trypanostatic action with low, and moderate, respective-
ly, toxicity to mammalian cells. However, these triazoles
inhibited T. cruzi proliferation only in micromolar
concentrations.
Compliance with ethical standards
Conflicts of interests The author(s) declare that they have no competing
interests.
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