Design, synthesis and antitrypanosomal activity of some nitrofurazone 1,2,4-triazolic bioisosteric analogues

Article abstract of DOI:10.1016/j.ejmech.2016.04.065

Chagas disease, caused by Trypanosoma cruzi, is a parasitosis that predominates in Latin America. It is estimated that 25 million people are under the risk of infection and, in 2008, more than 10 thousand deaths were registered. The only two drugs available in the therapeutics, nifurtimox and benznidazole, showed to be more effective in the acute phase of the disease. However, there is no standard treatment protocol effective for the chronic phase. Nitrofurazone (NF), an antimicrobial drug, has activity against T. cruzi, although being toxic. Considering the need for new antichagasic drugs, the existence of promising new therapeutic targets, as 14α-sterol demethylase and cruzain, and employing the bioisosterism and molecular hybridization approaches, four novel compounds were synthesized, characterized by melting point range, elemental analysis, IR and NMR spectroscopy. The compounds were tested against T. cruzi amastigotes in infected U2OS cells. All compounds showed selectivity towards T. cruzi and showed trypanomicidal activity in low micromolar range. The compound 3 showed potency similar to benznidazole, but lower efficacy. These results highlight the importance of the 1,2,4-triazole, thiosemicarbazonic and nitro group moieties for designing new efficient compounds, potentially for the chronic phase of Chagas disease.

Full text of DOI:10.1016/j.ejmech.2016.04.065

Accepted Manuscript
Design, synthesis and antitrypanosomal activity of some nitrofurazone 1,2,4-triazolic
bioisosteric analogues
Fredson T. Silva, Caio H. Franco, Denize C. Favaro, Lucio H. Freitas-Junior, Carolina
B. Moraes, Elizabeth I. Ferreira
PII:
S0223-5234(16)30358-0
10.1016/j.ejmech.2016.04.065
EJMECH 8580
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 January 2016
Revised Date: 25 April 2016
Accepted Date: 26 April 2016
Please cite this article as: F.T. Silva, C.H. Franco, D.C. Favaro, L.H. Freitas-Junior, C.B. Moraes,
E.I. Ferreira, Design, synthesis and antitrypanosomal activity of some nitrofurazone 1,2,4-
triazolic bioisosteric analogues, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.04.065.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and antitrypanosomal activity of
some nitrofurazone 1,2,4-triazolic bioisosteric
analogues
Fredson T. Silva1, Caio H. Franco2, Denize C. Favaro3,4, Lucio H. Freitas-Junior2, Carolina B.
Moraes2, Elizabeth I. Ferreira1*
1. School of Pharmaceutical Sciences, University of São Paulo, Avenida Prof. Lineu Prestes,
580, Bl. 13, São Paulo, São Paulo, Brazil
2. National Laboratory of Biosciences, National Center for Research on Energy and Materials,
Rua Giuseppe Máximo Scolfaro, 10000, Campinas, São Paulo, Brazil
3. Institute of Chemistry, University of São Paulo, Avenida Prof. Lineu Prestes, 748, São Paulo,
São Paulo, Brazil
4. Department of Chemistry, Institute of Exact Sciences, Federal University of Minas Gerais,
Belo Horizonte, Brazil.
*Corresponding author: hajudan@usp.br
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HIGHLIGHTS
•
•
•
Four novel trypanomicidal non-toxic compounds are proposed.
A novel compound showed promising EC50 value.
The ratio of Z isomer of compounds increases with time due to mesomeric stabilization.
ABSTRACT
Chagas disease, caused by Trypanosoma cruzi, is a parasitosis that predominates in Latin
America. It is estimated that 25 million people are under the risk of infection and, in 2008, more
than 10 thousand deaths were registered. The only two drugs available in the therapeutics,
nifurtimox and benznidazole, showed to be more effective in the acute phase of the disease.
However, there is no standard treatment protocol effective for the chronic phase. Nitrofurazone
(NF), an antimicrobial drug, has activity against T. cruzi, although being toxic. Considering the
need for new antichagasic drugs, the existence of promising new therapeutic targets, as 14α-
sterol demethylase and cruzain, and employing the bioisosterism and molecular hybridization
approaches, four novel compounds were synthesized, characterized by melting point range,
elemental analysis, IR and NMR spectroscopy. The compounds were tested against T. cruzi
amastigotes in infected U2OS cells. All compounds showed selectivity towards T. cruzi and
showed trypanomicidal activity in low micromolar range. The compound 3 showed potency
similar to benznidazole, but lower efficacy. These results highlight the importance of the 1,2,4-
triazole, thiosemicarbazonic and nitro group moieties for designing new efficient compounds,
potentially for the chronic phase of Chagas disease.
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KEYWORDS: Chagas disease; molecular hybridization; bioisosterism; nitroheterocyclic
compounds.
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INTRODUCTION
Chagas disease, also known as American trypanosomiasis, is an infectious disease caused by the
protozoan Trypanosoma cruzi1,2. Predominantly present in Latin America, it is estimated to cause
7 thousand deaths yearly and approximately 7 million people are estimated of being infected
worldwide. However, some studies have suggested that this number is underestimated due to
failures on diagnosis and notification3,4.
The acute stage of this illness is virtually asymptomatic and often difficult to detect,
depending of the host’s immune system5. The therapy mainly relies on nitroaromatic drugs,
nifurtimox and benznidazole, which are responsible for 50 to 60% of parasitological cure of
adults in the chronic stage and show considerable toxicity6. Therefore the search for new and
more effective drugs is of utmost importance.
Finding an appropriate treatment for the chronic phase of Chagas disease has been a
complex and difficult challenge. Despite many molecular targets have been explored, and high
potency and low toxicity molecules are being found, the clinical trials did not succeed (Merck,
ClinicalTrials.gov ID NCT01377480, Hospital Universitari Vall d’Hebron Research Institute,
ClinicalTrials.gov ID NCT01162967). The differences among strains of the parasite and the
complexity of the interactions between parasite and host can be responsible for those
drawbacks7.
It is worth mentioning nitrofurazone, a nitroheterocyclic topic antiseptic, which is known
to have antitrypanosomal activity by generating oxygen reactive species, which interferes with
trypanothione reductase8,9 a specific parasite detoxifying enzyme, and also a inhibition of
,
cruzipain, the main parasite protease10. The exploration of this dual mechanism is a strategy to
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overcome the problems of low efficacy and parasite resistance during the chronic phase. Another
approach includes the 1,2,4-triazole scaffold, that can interact with the parasitic CYP51 by
coordinating the heterocyclic nitrogen to the iron atom of the CYP51 prosthetic heme group, thus
inhibiting the synthesis of the parasitic cellular membranes11,12,13
.
O2N
O
N NH
NH2
O
1
(caption removed)
Molecular modification is considered the most effective approach for developing new
molecular entities14. Bioisosterism and molecular hybridization are among methods for achieving
better pharmacokinetic and pharmacodynamic properties, and have been widely used on the
search for novel bioactive compounds15,16
.
Considering the promising activity of triazolic CYP51 inhibitors as potential antichagasic
agents, the use of nitroheterocyclic compounds on the Chagas disease treatment and the reported
function of the thiosemicarbazone group as a potent cruzain inhibitor17, we decided to use the
mentioned moieties in a molecular hybridization approach, designing molecules similar to
nitrofurazone for optimizing the biological profile.
In this work, we report the design, synthesis and the biological evaluation of four new
compounds resulted from bioisosteric modifications of the furane ring and the hydrazone lateral
chain of nitrofurazone. In addition, two intermediate synthesis have been submitted to the same
biological evaluation.
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RESULTS AND DISCUSSION
Synthesis
The designed molecules present a bioisosteric replacement of the furane ring to a 1,2-4-
triazole system. The 1-H-1,2,4-triazole chemistry, especially the substitution on nitrogen 1, is
well described in the literature due to its function in the pharmacophore group of azolic
antifungals, such as fluconazole, itraconazole and voriconazole18,19
.
Synthesis of the aromatic hydrazones 4a, 4b, 7a and 7b (Chart 1) required the preparation
of the alkyl-1,2,4-triazoles 3 and 6, respectively. The alkyl-1,2,4-triazoles were obtained by the
nucleophile substitution of 2-Chloro-2’,4’-difluoroacetophenone by the appropriate triazole in
basic conditions and purified by chromatographic column. Ketones were mixed in reflux with the
hydrazides in acidic conditions to afford the desired hydrazones. Nitrotriazole 2 was prepared by
the oxidation of commercially available 3-amino-1,2,4-triazole, using the procedure described in
literature20.
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Chart 1. Synthetic route for obtaining nitrofurazone analogues. Reactions conditions: a) triazole
sodium salt, acetonitrile, 2-Chloro-2′,4′-difluoroacetophenone, reflux; b) semicarbazide or
thiosemicarbazide, ethanol, HCl, reflux.
All compounds were characterized by melting point, TLC, IR and NMR. The hydrazones
were found to form a time dependent E/Z-equilibrium in solution and once formed could not be
separated from the crude products by crystallization or column chromatography. Experimentally,
it is unequivocal that the E-isomer is almost the only compound when the solution is fresh. In
order to make the analysis of the diastereoisomeric population easier, the compound 4b was used
as reference (NMR experiments are presented in the Supporting Information).
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Figure 1. 1H NMR spectra of compound 4b using a fresh solution and 6 days later.
It was observed in the NOESY experiment that a distinct NOE interaction occurs between
H-2 and H-3 for only one of the isomers, what is expected for the isomer that possesses shorter
intramolecular distances (Table 1). For correct peak assignment and E/Z proportion calculation,
the interatomic distance between H-2 and H-3 was calculated for both isomers of molecules 4a,
4b, 7a and 7b using the most stable conformers (see Computational analysis section).
Table 1. Interatomic distances between H-1 and H-3 on the studied hydrazones.
Compound
E (Å)
4.4
Z (Å)
2.7
4a
4b
7a
7b
4.5
2.3
4.4
2.7
4.4
2.7
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Figure 2. NOESY spectrum for compound 4b. Distinctive correlations are depicted by
dashed lines.
The formation of isomers during hydrazone synthesis is common and well described in the
literature21. Separation of the isomers is often achieved using chromatography column, and
structural characterization is performed separately when possible. However, some hydrazones
present time-dependent interconversion when dissolved. Those compounds, even when isolated,
may interconvert to the other configuration in biological fluids. This phenomenon is of particular
interest of nanotechnology, using this conversion as molecular switches of molecular motors.21
Conversely, regarding to small bioactive molecules, such behavior can difficult the
determination of the favored bioactive configuration since the rate of isomerization may be
dependent on biologic assay conditions.
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Computational analysis
All energy minimizations calculations were performed using B3LYP/cc-pVTZ considering
both an isolated molecule and taking into account the solvent effect. The most stable conformer
of each isomer is shown in Table 2. Surprisingly, although E is the main reaction product, the
calculations indicate that, thermodynamically, the Z-isomer is the most stable (Table 2). In this
way, we left the fresh solution of 4b in DMSO-d6 for 6 days at room temperature, and after that
1
we acquired the H spectrum again. Unexpectedly, we observed that the population of the E-
isomer has decreased while the population of the Z-isomer has increased along time (Figure 1).
In the literature, we found that this fact is well documented for some semicarbazones; In 2013,
Jakusová and collaborators22 showed that, for isatin-3-(4-phenyl)semicarbazone and N-
methylisatin-3-(4-phenyl)semicarbazone, the type and initial concentration of the reactants and
the solvent of the reaction could affect the E/Z isomeric ratio. Besides, depending on the
thermodynamic stability of the product a thermally or a photo chemically initiated isomerization
can occur after its re-dissolution. In the occasion, the authors assumed, without a deep
investigation, that the Z-isomer is more stable thermodynamically compared to E due to the
existence of intramolecular hydrogen bonds.
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Table 2. Free energy of the most stable conformers in DMSO for compound 4b, isomers E and
Z, calculated at the B3LYP/cc-pVTZ theoretical level.
E-isomer
+7.7
Z-isomer
0.00
Isolated molecule
DMSO
∆G (kcal/mol)
+2.49
0.00
*
According to results presented on Table S1 (Supporting Information), the Z-isomer is by far the most stable in the
vacuum (∆G > 5kcal/mol) and this difference decreases when the solvent effect is included.
Although this kind of isomerization has been reported previously, considering the practical
application of our compounds, we decided to investigate the influence of stereoeletronic
interactions [attractive (delocalization) or repulsive (steric interactions)] on this time dependent
equilibrium and using the compound 4b as reference, we performed Natural Bond Orbital
(NBO)23 analyses to explain the Z-isomer stability.
First, the NBO analysis of a full wave function was performed. The most important orbital
interactions from the NBO analysis, responsible for the stabilization of the Z-isomer, are
presented at Table 3. Surprisingly, the NBO analysis pointed out that only for the Z-isomer the
mesomeric effect is effective. For the E-isomer the hyperconjugative interactions involving the
nitrogen’s lone pairs (LP) and the sigma antibonding orbital (σ*CS) constitute the most important
interaction. So, in order to analyse the influence of the attractive and repulsive interactions on the
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stability of the isomers, only the attractive delocalization interactions were deleted (NBOdel) and
the energy of the isomers were recalculated. The energy change presented at Table 3 is the
difference between the full molecular electronic energy and the molecular electronic energy
calculated disregarding the attractive interactions. These values can be used to compute the
amount of attractive or repulsive interactions in each isomer. The higher energy change for the Z-
isomer demonstrates that for this isomer the repulsive interactions are bigger than in the E-
isomer; meanwhile the attractive interaction, larger in the Z-isomer, overcomes the repulsive
ones.
Table 3 displays the most important interactions from the NBO analysis involving the
semicarbazone moiety of the two isomers. From this Table, the attractive interactions involving
the semicarbazone moiety are larger for the Z-isomer. It is worth to mention that other
interactions also contribute for the Z-isomer stabilization.
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Table 3. Orbital interactions energies (kcal/mol) from NBO of a full wave function analysis and
energies obtained from the NBO deletion calculation for Z- and E-isomer at the B3LYP/cc-
pVTZ level.
Orbital Interactions
LP(N11)→π*C5-S12
LP(N10)→π*C5-S12
LP(N10)→π*C4-N9
LP(N10)→σ*C5-S12
LP(N11)→σ*C5-S12
LP(N2)→σ*N10-H3
LP(N9)→σ*N11-H7
Z
E
-
78.99
56.45
36.04
-
6.56
33.18
32.59
44.72
-
0.55
2.79
1.13
-
LP(N9)→σ*N10-H3
Sum
8.28
184.23
9.28
126.33
Total SCF energy (a.u)
Deletion energy (a.u)
Energy change (a.u)*
∆E (kcal/mol)**
-1556.474193519
-1553.488265196
2.985928
-1556.460129916
-1553.703426618
2.756703
143.8
0.0
* Energy change = Total SCF energy - Deletion energy (a.u). ** ∆E (kcal/mol) = [Energy change(Z) - Energy
change(E)] x 627.5095.
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Biological evaluation (In vitro anti-T. cruzi evaluation)
The antiparasitic activity of the synthetized nitrofurazone analogs was determined against
intracellular amastigotes of the Trypanosoma cruzi Y strain in a High Content Screening (HCS)
assay7 in two sets of experiments. The results are shown in Table 4. The mean EC50 value of the
reference compound benznidazole, in the low micromolar range (approximately 4 ꢀM), and
efficacy (maximum activity) of approximately 100%, are within the expected values, as
previously reported, while the reference compound nifurtimox was slightly more potent, with a
mean EC50 value of 0.39 ꢀM7,24,25. Compounds 4b and 7b showed low potency against
amastigotes, with EC50 values of approximately 22 and 15 ꢀM, respectively, but were efficacious
in reducing infection, with maximum activity values of 92 – 95%. Compound 3 was similar to
benznidazole in potency, with an EC50 in the low micromolar range (5.53 ꢀM), however it was
not as efficacious, with a maximum activity of approximately 87%. Compound 4a was far less
potent than the others, with an EC50 value of approximately 100 ꢀM, but efficacious nonetheless.
Compounds 7a was only moderately active, with a mean EC50 greater than 60 ꢀM, but with
levels of efficacy (max. active. of 96%) comparable to values observed for compounds 4a and
7b. Compound 6 was poorly active against T. cruzi, with an EC50 of 144 ꢀM a maximum
efficacy of approximately 68. None of the tested compounds displayed overt cytotoxicity on the
host cells under the tested conditions, and all were selective towards T. cruzi, as suggested by the
selectivity indexes obtained. The compounds were also assayed for cytotoxicity against non-
infected U2OS, however no differences were observed in comparison with infected U2OS cells
(data not shown).
It can be observed that the absence of the nitro group in compound 6 significantly impacted
trypanocidal activity, as its nitro analogue 3 is 26-fold more potent. This indicates that the ROS
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releasing mechanism predominates over the CYP51 inhibition in that kind of scaffold,
independently on its resemblance to the antifungal azoles structure. Compound 3 can be
considered a bioisoster of benznidazole, possibly acting via similar mechanisms. It has similar
potency, but it is 3-fold less selective and its efficacy is 16% lower.
Otherwise, comparing the hydrazones 4 and 7, it is observed that the removal of the nitro
group resulted in a slightly enhanced activity. That difference may be related to the influence of
hydrazone lateral chain on the nitro group oxidation potential. Thiosemicarbazone compounds
4b and 7b showed to be about 4-fold more potent than the semicarbazonic bioisosteres 4a and
7a.
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Table 4. EC50, CC50 and Selectivity Index (S.I.) values for nitrofurazone analogues and
reference compounds against T. cruzi Y strain intracellular amastigotes
Compounds
EC50 (µM)
3.96
CC50 (µM)
N.D.
S.I.
> 101
78.9
Max. Actv. (%)
103.5
Benznidazole
Nifurtimox
0.34
26.8
101.5
3
5.53
N.D.
> 36
86.99
4a
4b
6
101.30
21.57
144
N.D.
> 2
95.23
N.D.
> 9.27
> 1.4
> 3.1
> 13.01
92.75
N.D.
68.7
7a
7b
63.1
N.D.
96.6
15.37
N.D.
95.22
Data obtained from three independent experiments. N.D. indicates that the mean value could not
be calculated. S.I., selectivity index. Max. Actv., maximum normalized activity.
Figure 3. Positive control for activity.
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Figure 4. Negative control for activity (cells infected with T. cruzi amastigotes).
Figure 5. Non-infected U2OS cells after 96 h of exposure of compound 3.
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CONCLUSION
Four novel compounds were designed, synthesized and more two intermediates of synthesis
had their antitrypanosomal activity evaluated in comparison with standard drugs benznidazole
and nifurtimox. Due to the nature of the general scaffold, a triple mechanism of action: CYP51
inhibition, cruzain inhibition and ROS releasing, was expected.
The presence of thiosemicarbazonic group is considered important for the biological activity
of the synthesized hydrazones, possibly by the inhibition of cruzain. The removal of nitro group
did not cause total loss of activity, evidencing that this group of compounds acts by other
mechanisms beyond the production of reactive oxygen species.
Compound 3 has potency similar to benznidazole, which has a very similar chemical
structure, possibly acting by the same ROS releasing mechanism. This compound could be used
as a scaffold for the design of new, more active and selective compounds. Enzymatic studies
should be performed to elucidate the exact mechanism of action for better understanding the
relationships between different targets and further structural optimizations.
EXPERIMENTAL SECTION
All starting materials were purchased from Sigma-Aldrich and were used without further
purification. The progress of all reactions was monitored by Analytical thin-layer
chromatography (TLC), which was performed on silica-gel 60 GF (5-40 µM thickness) plates.
Visualization was accomplished with UV light. Melting points and decomposition temperatures
were measured with an eletrothermal melting-point apparatus (Büchi, M-565 model) in open
capillary tubes and are uncorrected. Automated flash column chromatography was carried out
using a Biotage Isolera Prime (Biotage GB Limited, Hengoed, UK) with 25 g and 100 g SNAP
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cartridges. Infrared experiments were performed on a Shimadzu FTIR spectrophotometer, using
5 mg samples for the preparation of KBr pellets. Wavelengths of maximum absorbance are
1
quoted in wavenumbers (cm-1). The H, 13C, HSQC, HMBC and NOESY experiments were
performed on a Bruker Avance III NMR spectrometer in DMSO-d6 at 300 MHz and 500 MHz.
Chemical shifts (δ) are reported in ppm and are referenced to Me4Si. Elemental analyses (C, H
and N) were performed on a Perkin-Elmer model 2400 analyser, and the data were within 0.4%
of the theoretical values.
General procedure for the synthesis of the alkyl-triazoles
The triazoles were converted to their sodium salts by treatment with an aqueous solution of
sodium hydroxide followed by precipitation, according to the previous literature26. The reaction
proceeded according to the procedure described by Papadopoulou et al.27 After dried, the sodium
salt of the respective triazole was solubilized in acetonitrile and added to an equimolar solution
(0,5 M) of 2-Chloro-2’,4’-difluoroacetophenone in acetonitrile for a nucleophilic substitution,
which occurred under refluxing conditions (8 h). The resulting suspension was filtered, and the
liquid phase was evaporated. The resulting solid was purified using automated flash column
chromatography.
1-(2,4-difluorphenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethane-1-one (3): Off-white powder,
1
Yield, 46%. mp 113-114 ºC. IR (cm-1): 1697 (C=O), 1609 (C=C), 1508 (Ar-NO2). H NMR
(DMSO-d6): 8.81 (s, 1H); 8.03-8.11 (m, 1H); 7.51-7.59 (m, 1H); 7.30-7.36 (m, 1H); 6.03 (s, 2H).
13C NMR (DMSO-d
): 187.96; 167.51; 164.37; 160.77; 148.31; 132.60; 119.23; 112.71; 105.40;
6
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59.27. Anal. Calcd for C10H6F2N4O3: C, 45.01; H, 2.30; N, 20.39. Found: C, 44.96; H, 2.32; N,
20.51.
General procedure for the synthesis of the hydrazones
The adequate ketone (10 mmol) and the respective hydrazide (10 mmol) were solubilized
in anhydrous ethanol (10 mL) and mixed with 0,15 mL of hydrochloric acid. The mixture was
stirred under reflux (12 h). The resulting solution was evaporated and the solid was purified
using automated flash column chromatography.
2-(1-(2,4-difluorophenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethylidene)hydrazine-1-
carboxamide (4a): (E:Z proportion = 87:13) Off-white powder, Yield: 10%, temperature of
decomposition: 140 ºC. IR (cm-1): 1670 (C=O), 1617 (C=N), 1504 (C=C Ar). 1H NMR (DMSO-
d6), 9.32/10.27 (s, 1H); 8.82/8.88 (s, 1H); 7.74-7.82 (m, 1H); 7.30-7.38 (m, 1H); 7.02-7.19 (m,
1H); 6.61 (s, 2H); 5.40/5.62 (s, 2H). Anal. Calcd for C11H9F2N7O3: C, 40.12; H, 3.07; N, 28.91.
Found: C, 40.18; H, 3.00; N, 29.00.
2-(1-(2,4-difluorophenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethylidene)hydrazine-1-
carbothioamide (4b): (E:Z proportion = 79:31) Off-white powder, Yield: 60%, Temperature of
1
decomposition: 211 ºC. IR (cm-1): 1605 (C=N), 1508 (Ar-NO2), 1365 (Ar-NO2). H NMR
(DMSO-d6): 10.31/11.17 (s, 1H); 8.85/8.91 (s, 1H); 8.43/8.56 (s, 1H); 7.83/8.06 (s, 1H); 7.06-
7.45 (m, 3H); 5.47/5.75 (s, 2H). 13C NMR (DMSO-d
), 75 MHz: 179.48; 165.31; 162.00;
6
157.99; 147.63; 138.17; 131.32/132.44; 114.94/119.88; 112.60; 109.94; 55.47/48.08. Anal.
Calcd for C11H9F2N7O2S: C, 38.63; H, 2.64; N, 28.95. Found: C, 38.72; H, 2.56; N, 28.81.
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2-(1-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethylidene)hydrazine-1-carboxamide (7a):
1
(E:Z proportion = 82:18) Off-white powder, Yield, 19%, Hygroscopic. H NMR (DMSO-d
6
):
10.20/9.21 (s, 1H); 8.55/8.44 (s, 1H); 7.91/7.94 (s, 1H); 7.65-7.73 (m, 1H); 7.08-7.34 (m, 2H);
6.57/6.41 (s, 2H); 5.51/5.28 (s, 2H). Anal. Calcd for C11H10F2N6O: C, 45.99; H, 3.88; N, 28.65.
Found: C, 46.24; H, 3.81; N, 28.72.
2-(1-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethylidene)hydrazine-1-carbothioamide
(7b): (E:Z proportion = 58:42) Brown powder, Yield, 19%, Temperature of decomposition: 130
1
ºC. H NMR (DMSO-d6): (E) 10,24/11,15 (s, 1H); 8,49/8,59 (s, 1H); 8,48/8,56 (s, 1H);
7,96/8,37 (s, 1H); 7,92/8,01 (s, 1H); 7,78-7,86 (m, 1H); 7,08-7,39 (m, 2H); 5,36/5,63 (s, 2H).
.Anal. Calcd for C11H10F2N6S: C, 47.62; H, 4.45; N, 22.72. Found: C, 47.13; H, 4.33; N, 23.13.
Computational method
Geometry optimizations and energy calculations of compounds 4a, 4b, 7a and 7b were
carried out at B3LYP and MP2 level applying cc-pVTZ28 as basis set using the Gaussian09 suit
of programs29. In order to study the solvent influence in the isomers energy, geometry
optimizations were performed using SMD model30 at MP2/cc-pVTZ and B3LYP/cc-pVTZ28
level.
T. cruzi in vitro assay
The assay was performed as previously reported7. Briefly, on day 1 of the experiment,
U2OS cells were seeded in black ꢀClear 384-well tissue culture treated polystyrene plates
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(Greiner Bio-One) at ratio of 700 cells in 40 ꢀl of high glucose DMEM media (Hyclone)
supplemented with 10% of heat inactivated fetal bovine serum, 100 µg/mL penicillin and 100
U/mL streptomycin (all reagents supplied by Gibco). Dispensing of cell suspension was
performed with the aid of a Wellmate Liquid Handler (Thermo-Scientific). Plates were incubated
for 24 h at 37 ºC/5% CO2.
On day 2, trypomastigotes were harvested from the supernatant of LLC-MK2 cell cultures
infected with T. cruzi Y strain and added to the U2OS-containing 384 microplate at 2800
trypomastigotes in 10 ꢀl of low glucose DMEM media (supplemented with 2% of heat
inactivated fetal bovine serum – Gibco). On day 3, compounds were serially diluted by a factor
of 2 (i.e. in 2-fold serial dilutions) in 100% DMSO in 15 dilution points in a polypropylene 384
well plate (Greiner BioOne) using a 16-channel manual pipette equipped with disposable tips
(ThermoScientific), followed by transfer of 10 ꢀL of compound solution onto assay plates,
yielding a final concentration of 1% DMSO and a final volume of 60 ꢀl/well. After 96 h, the
plates were fixed for 15 min with 4% paraformaldehyde followed by 3 times washing with PBS
and stained with 5 ꢀM Draq5 (Biostatus) in DPBS. The highest compound concentration tested
was 200 ꢀM for test compounds, 400 ꢀM for benznidazole and 100 ꢀM for nifurtimox. The
compounds were tested in two sets of experiments. In the first experiment, benznidazole was
used as a control compound for evaluation of antiparasitic activity of compounds 3, 4a, 4b, and
7b, while nifurtimox was used as control for the test of compounds 6 and 7a in the second set of
experiments. Each compound concentration was tested in doublets (i.e.. two wells per plate) and
each experiment was performed in triplicate (i.e.. three independent experiments).
The plates were imaged in the High Content Analysis System Operetta (Perkin Elmer) with a
20x WD objective and images were analyzed with the High Content Analysis (HCA) software
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Harmony (Perkin Elmer). This automated HCA protocol identifies, segment and quantitate for
identification, segmentation and quantitation of host cell nuclei, cytoplasm and intracellular
parasite based on the DNA staining. The HCA provides as output data for all images from one
well: the total number of cells, total number of infected cells, total number of intracellular
parasites and average number of parasite per infected cell.
Data analysis was performed as described elsewhere7. The infection ratio (IR) is the ratio of
infected cells in a given population, and was determined as the ratio between the total number of
infected cells and the total number of cells in in the test condition. The raw data for IR values
was normalized to negative (infected cells, DMSO-treated) and positive (not infected cells,
DMSO-treated) controls to determine the normalized antiparasitic activity.
Normalized activity values were processed with the Graphpad Prism software – version 6,
for generation of sigmoidal dose-response (variable slope) nonlinear curve fitting and
determination of EC50 and CC50 values by interpolation. The software also outputs maximum
compound activity (Max. Actv.), calculated based on mean top normalized activity values
provided by sigmoidal-curve fitting. For the purpose of this study, EC50 was defined as the
compound concentration corresponding to 50% normalized activity after 96 h of compound
incubation. Potency is used in reference to EC50 values (ie., the concentration of compound that
reduces infection by 50% in comparison to controls), whereas efficacy relates to the maximum
observed activity of a compound, regardless of the concentration. Cytotoxicity was measured by
the CC50 value, defined as the compound concentration corresponding to reduction of 50% in the
number of cells in comparison with negative controls (DMSO-treated, infected cells). A plate
was assayed only with non-infected U2OS cells and compounds in order to compare CC50 values
with those observed in infected cells. The selectivity index (S.I.) is a ratio between the value of
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CC50 and EC50 and indicates the selectivity of biological activity towards the parasite; whenever
CC50 cannot be calculated, the S.I. value was estimated as a ratio between the highest compound
concentration tested and the EC50.
AUTHOR CONTRIBUTIONS
Fredson T. Silva – Design, synthesis, chemical analysis of the compounds, molecular modeling
calculations, text elaboration
Caio H. Franco – Anti-trypanosomal and cytotoxicity assays, text elaboration
Denize C. Favaro – Nuclear Magnetic Resonance and Molecular modeling calculations
Carolina B. Moraes - Anti-trypanosomal and cytotoxicity experiments supervisor, data analysis,
text review
Lucio H. Freitas-Junior - Anti-trypanosomal and cytotoxicity experiments supervisor
Elizabeth I. Ferreira – General supervisor, MSc. student F. T. Silva supervisor, overall
manuscript review, corresponding author
CONCFLICT OF INTEREST
All authors declare there is no conflict of interest at all.
ACKNOWLEDGEMENT
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The authors thank FAPESP for F. T. Silva scholarship and funding of computational analysis
facilities (2011/11499-0, 2011/17357-3, 2012/21865-7), and also to CNPq for E. I. Ferreira
research fellowship.
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