Electrochemical characterization of para- and meta-nitro substituents in aqueous media of new antichagasic pharmaceutical leaders

Article abstract of DOI:10.1016/j.electacta.2020.137582

The electrochemical reduction mechanism of two isomers of position drug leaders against Chagas disease, p-nitrosulfonylhydrazine derivative (p-NSF), and m-nitrosulfonylhydrazine derivative (m-NSF), was investigated in aqueous media and in the absence of oxygen using voltammetric techniques. The main reduction process was attributed to the reduction of the nitro group (E_pc (p-NSF) = ?0.58 V and E_pc (m-NSF) = ?0.62 V), generating nitroso derivative in intermediate and higher values of pH and hydroxylamine in lower values of pH. Another reduction process in a less negative potential value was only observed for p-NSF and attributed to the generation of the nitro radical anion. The difference in the reduction potentials between both compounds and the presence of another reduction process for p-NSF was associated with the position of the nitro group, due to the distinct stabilization of the reduction intermediates by the aromatic ring. A catalytic response for the reduction process of the nitro anion radical was observed for p-NSF in the presence of oxygen, given that its magnitude of current increased when increasing the oxygen availability. Also, for m-NSF, this reduction process could be detected, even though it was not observed in the voltammograms in the absence of oxygen. This further confirmed the generation of the nitro radical anion, since it reacts with molecular oxygen and regenerates the initial nitro compound. The interaction with cysteine was also evaluated, which favored the reduction process towards the generation of the nitroso derivative due to adduct formation, destabilizing the reduction process regarding the nitro radical anion. Therefore, electrochemical experiments can evaluate how different isomers of position and other coupled functional groups affect the reduction of the nitro group and, consequently aid in the design of new classes of antichagasic pharmaceuticals, with improved stability of reactive intermediates that are often correlated with the degree of injury towards the parasite.

Full text of DOI:10.1016/j.electacta.2020.137582

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Electrochemical characterization of para- and meta- nitro substituents
in aqueous media of new antichagasic pharmaceutical leaders
Caroline G. Sanz , Kevin A. Dias , Raphael P. Bacil ,
Ricardo A.M. Serafim , Leandro H. Andrade , Elizabeth I. Ferreira ,
Silvia H.P. Serrano
PII:
S0013-4686(20)31975-7
DOI:
Reference:
https://doi.org/10.1016/j.electacta.2020.137582
EA 137582
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12 November 2020
28 November 2020
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Caroline G. Sanz ,
Kevin A. Dias ,
Raphael P. Bacil ,
Ricardo A.M. Serafim ,
Leandro H. Andrade ,
Elizabeth I. Ferreira ,
Silvia H.P. Serrano ,
Electrochemical characterization of para- and meta- nitro substituents in aqueous me-
dia of new antichagasic pharmaceutical leaders, Electrochimica Acta (2020), doi:
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Highlights

Electrochemical characterization of these two synthesized antichagasic leaders
was performed in aqueous media using cyclic, differential, and square wave
voltammetry


Mesomeric correlation towards the electrochemical stabilization of the nitro
radical anion was addressed using electronic flux of both radicals formed
Interaction with oxygen and cysteine further confirmed the electrochemical
detection of the nitro radical anion and other reactive intermediates

Electrochemical characterization of para- and meta- nitro substituents
in aqueous media of new antichagasic pharmaceutical leaders
a
a
a
b
Caroline G. Sanz , Kevin A. Dias , Raphael P. Bacil , Ricardo A.M. Serafim , Leandro
a
b
a
H. Andrade , Elizabeth I. Ferreira , Silvia H. P. Serrano *
a
Department of Fundamental Chemistry, Institute of Chemistry, University of São
Paulo, 05508-000 São Paulo/SP, Brazil
b
Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of São
Paulo, 05508-900 São Paulo/SP, Brazil.
*
Corresponding Author
Tel: +551130912687
e-mail: shps@iq.usp.br

Abstract
The electrochemical reduction mechanism of two isomers of position drug leaders
against Chagas disease, p-nitrosulfonylhydrazine derivative (p-NSF), and m-
nitrosulfonylhydrazine derivative (m-NSF), was investigated in aqueous media and in
the absence of oxygen using voltammetric techniques. The main reduction process was
attributed to the reduction of the nitro group (E (p-NSF) = -0.58 V and E (m-NSF) = -
pc
pc
0
.62 V), generating nitroso derivative in intermediate and higher values of pH and
hydroxylamine in lower values of pH. Another reduction process in a less negative
potential value was only observed for p-NSF and attributed to the generation of the nitro
radical anion. The difference in the reduction potentials between both compounds and
the presence of another reduction process for p-NSF was associated with the position of
the nitro group, due to the distinct stabilization of the reduction intermediates by the
aromatic ring. A catalytic response for the reduction process of the nitro anion radical
was observed for p-NSF in the presence of oxygen, given that its magnitude of current
increased when increasing the oxygen availability. Also, for m-NSF, this reduction
process could be detected, even though it was not observed in the voltammograms in the
absence of oxygen. This further confirmed the generation of the nitro radical anion,
since it reacts with molecular oxygen and regenerates the initial nitro compound. The
interaction with cysteine was also evaluated, which favored the reduction process
towards the generation of the nitroso derivative due to adduct formation, destabilizing
the reduction process regarding the nitro radical anion. Therefore, electrochemical
experiments can evaluate how different isomers of position and other coupled functional
groups affect the reduction of the nitro group and, consequently aid in the design of new
classes of antichagasic pharmaceuticals, with improved stability of reactive
intermediates that are often correlated with the degree of injury towards the parasite.
Keywords: Chagas disease; nitro compounds; electrochemical characterization; nitro
anion radical; voltammetry

1
. INTRODUCTION
Neglected diseases, by definition, are those caused by parasites or infectious
agents, and considered endemic in underdeveloped and developing countries where
there is a lack of interest in the optimization of drugs towards treatment, prevention,
cure, or even their diagnosis [1,2]. These fall into a comprehensive group of endemics
that remains a neglected target in research regarding new drug development, where only
1
.15% of new drugs, registered between 1975 and 2004, were intended for its control,
even though the global health burden surpasses 10% of all disease occurrence [3–5].
Chagas disease, a parasitic tropical disease of the American continent, caused by the
protozoan Trypanosoma cruzi, and discovered by Carlos Chagas in 1909, belongs to
this neglected endemic group [6,7]. The mechanism of transmission of the parasite is
primarily through a mechanical vector, nocturnal insects of the Triatominae family [8],
which mainly affects low-income populations susceptible to poor sanitation conditions,
and induces characteristic symptoms that may take up to 30 years to manifest [9]. The
rate of morbidity and mortality is significant, given that the disease entails a chronic
evolutionary heart condition, victimizing up to 20% or more of infected individuals
[10].
Trypanocidal drugs such as benznidazole and nifurtimox, introduced in 1971 and
965, respectively, were and still are used to treat Chagas disease, acting mainly by
1
inhibiting enzymes necessary to the defense mechanism of the parasite cells, however
also cytotoxically affecting the individual [11]. Although effective, the use of these
drugs is linked in 40% of cases to side effects such as skin diseases, brain toxicity, and
digestive system irritation [12,13].
One of the routes in the development of drugs with antichagasic activity is based
on their ability to affect the Trypanosoma cruzi detoxification mechanism, making it

deficient to combat oxidative stress of the cell. The parasite mechanism of defense is
sustained by a specific enzyme called trypanothione reductase (TR), given that it lacks
the enzymes catalase, glutathione peroxidase, and glutathione reductase, responsible for
a significant portion of oxidative control in mammals [14]. Certain antichagasic drugs
act indirectly to inhibit TR, making it difficult to reduce trypanothione disulfide
(physiological substrate of the enzyme) to its thiol component, which in turn is
responsible for the control of oxidative stress [15]. Therefore, inhibiting the action of
the enzyme increases the parasite's vulnerability to the drug, causing its death through
the increase in the concentration of reactive oxygen species in the intracellular
environment [15,16].
Both benznidazole and nifurtimox are part of an antichagasic class of drugs that
presents a nitro (-NO ) functional group usually linked to aromatic rings. Its
2
cytotoxicity is associated with the formation and stabilization of the anionic nitro
●
─
radical (R-NO2 ), in which the reduction potential corresponding to the redox pair R-
●
─
NO /R-NO2 is usually an indicator of drug efficiency [17]. The anionic nitro radical
2
and its derivatives (RNO, nitrous derivative and RNHOH, hydroxylamine) can interact
toxically in two ways: acting directly to interfere with the parasite's oxygen metabolism,
or interacting directly with DNA by destabilizing the double helix through reactions
with nitrogenous bases (depending on pH, specifically thymine and adenine), besides
additionally affecting lipids and proteins in a less significant way [18]. The stability of
the nitro compound reduction intermediates is related to the degree of injury it may
promote to biomolecules, or else, the slower the rate of decomposition of the nitro
radical anion and the easier it is to be generated (both correlated to its reduction
potential), the larger and faster will be the damage caused [19]. The bioactive
compound crosses the cell membrane by passive diffusion, and its concentration in the

intracellular environment increases while reactive species, coming from the
bioreduction of these nitro compounds destabilize the membrane, Figure 1.
FIGURE 1
Electrochemical measurements have been used to correlate the mechanism of
action of drugs with their redox potential and biological efficiency in biomedical
applications, however, one must address the complexity of these systems regarding
stereochemistry, diffusion, solubility, and membrane permeability, amongst others [20].
Still, given that most important physiological processes occur through charge transfer
reactions, potentially induced heterogeneous processes in aqueous or organic media can
be used to clarify redox processes and reactive intermediate stabilization. Linear and
pulse voltammetry are often used to elucidate the biological process of interest without
the necessity of spectroscopic and/ or chromatographic methods and gather further
evidence towards biological electron-transfer reactions [21].
A variety of aromatic nitro compounds has been electrochemically characterized,
such as nitrofuran [22–25] and nitroimidazole derivatives [26–29]. Still, mostly all
experiments were performed in aprotic or organic media, to further aid in the
stabilization of intermediate radicals [30,31] and access the reduction potential of
pharmaceutical compounds and mechanism of action. The reduction processes were
also evaluated at different electrode materials, such as mercury [32], carbon [33], boron-
doped diamond [34,35], and modified electrodes [36], in which the properties of the
transducer material improved the stabilization of reactive intermediates. Still, the main
interest regarding the electrochemical study of this class of compounds are either
biological [37–40] or their potential damage to DNA [28,41–48].

The molecular design of isomers of position in ring systems of compounds
containing sulfonylhydrazone scaffold can be used as a strategy to generate new
antichagasic leaders since the presence of the nitro functional group (R-NO ) in the
2
benzene ring at the para- and meta- positions promotes, in the intracellular
environment, major reactivity of the intermediates from the nitro bioreduction process.
According to a previous study with acylhydrazone derivatives, its bioisostere
sulfonylhydrazone group may also act directly on the active site of cruzain, an
endoprotease vital to the parasite's nutrition, consequently increasing the selectivity in
comparison to the current benznidazole treatment [49]. Also, the presence of nitric
oxide in the intracellular environment aids the rupture of the plasma membrane of the
etiological agent, although its excessive release may also damage the host cells. Nitric
oxide release occurs in neutral and alkaline medium, in which the furoxan ring from
both leaders undergoes hydrolysis [50].
Therefore, the objective of this work was to describe the synthesis procedure and
electrochemical reduction mechanisms of two pharmaceutical leaders against Chagas
disease:
nitrophenyl)sulfonyl)hydrazone)methyl) -1,2,5-oxadiazole) and (2-Oxide of (E)-3-
methyl-4-((2-((meta-nitrophenyl)sulfonyl) hydrazone)methyl)-1,2,5-oxadiazole),
(2-Oxide
of
(E)-3-methyl-4-((2-((para-
denominated p-NSF and m-NSF, respectively. The stabilization of the nitro radical
anion from the reduction processes of the nitro group was evaluated in aqueous media at
bare glassy carbon electrode in the absence and the presence of oxygen. Given that
compounds containing thiol groups on their structure can form adducts with one of the
intermediates from the nitro group reduction process, this interaction was evaluated.
Their redox potentials and intermediate stabilization were used to establish the degree of
injury between both leaders and compared with similar compounds that present the nitro

group at the same position and that were already tested in vitro against the protozoan
49].
[
2
. EXPERIMENTAL
2
.1 Reagents
Britton-Robinson (BR) buffer 0.04 M was prepared by dissolving the appropriate
amount of sodium borate and diluting the appropriate volume of phosphoric acid and
acetic acid in deionized water. The pH adjustment was made with small volume
additions of 6.0 M sodium hydroxide solution in a pH range between 4.0 and 10. All
reagents employed were of analytical grade and obtained from Merck, Rio de Janeiro,
Brazil. Nitrogen and oxygen gases used in the experiments were of R grade and
purchased from Air Products, São Paulo, Brazil. All buffer solutions were prepared
using Millipore Direct-Q ultrapure water with resistivity ≥ 18 MΩ cm. For the synthetic
section of this work, reagents and solvents were purchased from commercial suppliers
and used as received.
2
.2 Instrumentation
Buffer solution’s pH measurements were performed with pH-meter model 654
and a combined glass electrode (Ecotrode Plus, model 60262100, Metrohm).
Electrochemical measurements were performed using a computer-controlled μ-Autolab
Type III potentiostat-galvanostat running with software NOVA for Windows version
1
.11. For the organic synthesis of p-NSF and m-NSF, the progress of all reactions was
monitored by analytical thin-layer chromatography, which was performed on silica-gel
0 GF (5-40 µM thickness) plates. Visualization was accomplished with UV light.
Melting points were measured with an electrothermal melting-point apparatus (Büchi,
6
1
13
M-565 model) in open capillary tubes and are uncorrected. H and proton decoupled C
™
NMR spectra were scanned on Bruker AVANCE NMR spectrometer in DMSO-d at
6

1
13
3
00 MHz ( H) and 75 MHz ( C). The chemical shifts (δ) are reported in ppm. High-
resolution mass spectrometry (HRMS) was performed using electrospray ionization
+
+
(
ESI). The parent ions ([M - H ]) or ([M + Na ]) are quoted.
2
.3 Intermediates (p-NS, m-NS and p-MS) and Antichagasic Leaders (p-NSF and
m-NSF) Synthesis
A straightforward convergent synthetic pathway was chosen for p-NSF and m-
NSF synthesis (Scheme 1). In the superior branch, the sulfonyl hydrazides intermediates
p-NS, m-NS, and p-MS was achieved by treating the correspondent sulfonyl chlorides
(1a-c) with hydrazine hydrate (step a). Concerning the lower branch, the 3-methyl-4-
furoxancarbaldehyde (3) was obtained by the reaction between crotonaldehyde (2) and
aqueous sodium nitrite in the presence of acetic acid, following the methodology
described by Fruttero et al. [51]. Finally, in the convergent step, the sulfonyl hydrazones
p-NSF and m-NSF were obtained by the acid-mediated condensation of p-NS and m-NS
with 3, respectively. The purity of all target compounds was found to be >95%.
SCHEME 1
2
.3.1 Procedure ‘a’: synthesis of sulfonyl hydrazides (Scheme 1; intermediates p-
NS, m-NS, and p-MS)
The respective sulfonyl chloride (1a-c) (10 mmol) was added slowly in a solution
of hydrazine hydrate 80% (25 mmol) and THF (4 mL) over stirring at 0 °C. After the
total consumption of the starting material, water was added and the resulting solid was
filtered and washed with cold water to obtain the respective pure sulfonyl hydrazides.
2
.3.1.1 p-Nitrobenzenesulfonylhydrazide (p-NS)
1
Pale yellow solid; Yield 91%. H NMR (300 MHz, DMSO-d ), δ (ppm): 8.43 (d,
6
J = 9.0 Hz, 2H), 8.13 (d, J = 9.0 Hz, 2H), 7.96 (s, 1H), 3.45 (s, 2H).

2
.3.1.2 m-Nitrobenzenesulfonylhydrazide (m-NS)
1
White solid; Yield 88%. H NMR (300 MHz, DMSO-d ), δ (ppm): 8.75 (bs, 1H),
6
8
.54-8.48 (m, 2H), 8.20 (ddd, J = 7.8 Hz, J = 2.4 Hz, J = 0.6 Hz, 1H), 7.91 (dt, J = 7.8
Hz, J = 0.6 Hz, 1H), 4.34 (bs, 2H).
2
.3.1.3 p-Methylbenzenesulfonylhydrazide (p-MS)
1
Colourless crystals; Yield 89%. H NMR (300 MHz, DMSO-d ), δ (ppm): 8.20
6
(s, 1H), 8.43 (d, J = 8.1 Hz, 2H), 8.13 (d, J = 8.1 Hz, 2H), 4.00 (bs, 2H).
2
.3.2 Procedure ‘b’: synthesis of 3-methyl-4-furoxancarbaldehyde (Scheme 1,
intermediate 3)
Compound 3 was synthesized from 2 according to the methodology previously
described by Fruttero et al. [51].
2
.3.3 Procedure ‘c’: synthesis of sulfonyl hydrazones (Scheme 1, final compounds
p-NSF and m-NSF)
The synthesis of p-NSF and m-NSF has already been described [50]. The
intermediate 3 (2 mmol) was reacted with the corresponding sulfonyl hydrazides (p-NS,
m-NS) (2 mmol) using acid catalysis (0.1 mL of concentrated HCl) in anhydrous
methanol (50 mL) as a solvent. The reaction was kept stirring under reflux until the total
consumption of the starting materials. After cooling to room temperature, cold water
was added, and the resulting solid was filtered and washed with cold water to obtain the
pure final product.
2
.3.3.1 (E)-3-methyl-4-((2-((4-nitrophenyl)sulfonyl)hydrazono)methyl)-1,2,5-
oxadia-zole 2-oxide (p-NSF)
1
Pale yellow solid; Yield 87%; m.p.: 160-162 °C. H NMR (300 MHz, DMSO-
d6), δ (ppm): 8.45 (d, J = 8.7 Hz, 2H), 8.11 (d, J = 8.7 Hz, 2H), 7.98 (s, 1H), 2.19 (s,

1
3
3
H). C NMR (75 MHz, DMSO-d ), δ (ppm):153.0, 150.1, 143.5, 136.4, 128.8, 124.7,
6
1
11.2, 9.0. HRMS (ES-, TOF-MS): calculated for C H N O S [M - H+]: 326.0195;
1
0
9
5
6
found: 326.0206.
2
.3.3.2 (E)-3-methyl-4-((2-((3-nitrophenyl)sulfonyl)hydrazono)methyl)-1,2,5-
oxadia-zole 2-oxide (m-NSF)
Pale yellow solid; Yield 91%; m.p.: 167-169 °C. H NMR (300 MHz, DMSO-
d6), δ (ppm): 8.54 (d, 2H), 8.28 (d, J = 8.7 Hz, 1H), 7.95 (t, J = 8.7 Hz, 2H), 2.18 (s,
1
1
3
3
H). C NMR (75 MHz, DMSO-d ), δ (ppm): 153.0, 147.9, 139.5, 136.7, 133.1, 131.6,
6
1
28.1, 122.0, 111.2, 8.9. HRMS (ES+, TOF-MS): calculated for C H N O S [M +
1
0
9
5
6
Na+]: 350.0171; found: 350.0180.
2
.4 Electrode Surface Cleaning Procedure
The bare glassy carbon electrode (GCE) surface was polished with a diamond
spray down to 1.0 μm particle size from Kemet, United Kingdom, and rinsed thoroughly
with deionized water and ethanol. Cyclic voltammograms were recorded in 0.04 M BR
-1
buffer pH 7.0, in a potential range from 0.0 V to 1.0 V and scan rate of 100 mV s for 5
cycles, to stabilize the baseline.
2
.5 Electrochemical measurements
All electrochemical measurements were conducted on a one-compartment 10 mL
2
electrochemical cell containing a 1.5 mm diameter (geometric area 0.071 cm ) glassy
carbon electrode (GCE), platinum wire, and Ag/AgCl (saturated KCl) as a working,
auxiliary, and reference electrodes, respectively. The experiments were performed at
room temperature (25 ± 1 ºC) and, to simulate anaerobic conditions, nitrogen gas was
purged in the solution with a flow rate of 0.5 L/min for 15 min before performing the
electrochemical measurements. The electrolyte was indicated as predominantly aqueous

(95% aqueous and 5% organic) due to the presence of DMSO from the stock solution of
the compounds. The following experimental conditions were used to perform the cyclic
voltammetry measurements: E = 0.0 V, E = -1.4 V, E = 1.0 V and E = 0.0 V, scan
i
λ1
λ2
f
-1
rate of 100 mV s , step potential of 3 mV, recording 3 cycles in each analysis, unless
indicated otherwise. Differential pulse voltammetry measurements were performed
using the following experimental conditions: E = 0.0 V, E = -1.4 V, pulse amplitude of
i
f
2
5 mV, step potential of 2.5 mV, the interval time of 0.5 s, resulting in a scan rate of 5
-1
mV s . Square wave voltammetry measurements were performed using the following
experimental conditions: E = 0.0 V, E = -1.4 V pulse amplitude of 25 mV, step
i
f
-1
potential of 3 mV, frequency of 33 Hz, resulting in a scan rate of 100 mV s . The
influence of oxygen and cysteine on the electrochemical profile of p-NSF and m-NSF
was evaluated as follows: oxygen was bubbled for 10, 20, and 30s in the
electrochemical cell before each measurement; cysteine was added to the
electrochemical cell in 1:2, 1:1, and 2:1 molarity ratio cysteine:p-NSF (increasing
concentration of 0.25 mM, 0.50 mM, and 1.0 mM of cysteine and fixed concentration of
0
.50 mM of p-NSF).
3
3
. RESULTS AND DISCUSSION
.1 Voltammetric Characterization
To obtain an overview of the electrochemical profile of the m-NSF and p-NSF
antichagasic leaders, cyclic voltammograms were obtained at GCE in 0.04 M BR buffer
pH 7.0 containing 1.0 mM of each compound and in the absence of O , Figure 2A and
2
2
B.
FIGURE 2
As can be seen from Figure 2A and 2B, both compounds presented the same
number of reduction and oxidation processes, most likely due to the structural similarity

between them. To elucidate and distinguish the nitro group reduction processes from
others that might interfere or overlap with them, a comparison with two structurally
similar compounds was performed.
In the direct potential sweep, which primarily runs towards negative potential
values, the main reduction process can be observed for both compounds, process 2c,
Ep2c (p-NSF) = -0.62 V and Ep2c (m-NSF) = -0.64 V. Process 2c was also observed in a
solution containing 1.0 mM of nitrobenzene, which indicates that it corresponds to the
main reduction of the nitro group attached to the benzene ring, Figure 2C. This process
is usually registered for other nitro compounds in aqueous media, but at peak potentials
around -0.40 V to -0.70 V. The change in the peak potential values is usually associated
with the position of the nitro group and the aromatic structure attached to it (often a
furan or imidazole ring), which are responsible for the stability of the reduction
products [30]. Also, the transducer material can split an electrochemical process into
two or more components and, in this case, changes in the reduction or oxidation peak
potentials for each intermediate can be noticed. Commonly, the electrochemical
characterization of these nitro compounds is conducted at carbon-based electrodes
(glassy carbon electrodes, boron-doped diamond electrodes, carbon paste electrodes)
given their chemical compatibility with aromatic structures.
For both compounds (p-NSF and m-NSF), changes in the peak potentials and
depletion of the magnitude of current were observed for all processes, in the second
cycle, most likely due to adsorption of reduction products on the electrode surface.
Process 2a appears on the cyclic voltammograms for p-NSF and m-NSF,
however, it does not appear at the measurements conducted in the presence of
nitrobenzene, which suggests that this oxidation process is unrelated with the nitro
group present in the benzene ring, in either meta- or para- position. Cyclic

voltammograms obtained at GCE in 0.04 M BR buffer solutions, pH 7.0, containing 1.0
mM of p-MS, Figure 2D, also present the process 2a. Given the absence of the
sulphonylhydrazide moiety in nitrobenzene, and presence in p-NSF, m-NSF, and p-MS,
process 2a can be associated with the oxidation of the nitrogen close to the sulfone
moiety. The oxidation peak potential for process 2a is similar to other compounds that
share this functional group [52].
According to the reduction mechanism for the nitro group in the p-NSF and
m-NSF, it was expected that its free radical reactive intermediate could not be distinctly
observed due to their short lifetime in an aqueous medium. Besides, in the cyclic
voltammograms obtained for p-NSF, another reduction process is present, process 1c,
which does not appear on the voltammograms obtained with m-NSF. The peak potential
of process 1c and its absence on the measurements obtained for m-NSF indicates that
this might be related to one of the reactive intermediates, therefore, the voltammograms
obtained on the negative potential region were chosen as the focus of this study.
Besides cyclic voltammetry, relevant results can be also obtained by differential
pulse and square wave voltammetry since both techniques present major sensitivity than
the cyclic voltammetry. The first one, performed at a slow scan rate, gives an estimative
of the number of electrons involved in each electrochemical process, an important step
to understanding electron transfer reaction mechanisms, while the last one allows
evaluation of the reversibility of the processes at faster scan rates without losing
sensitivity.
Differential pulse voltammograms were obtained in 1.0 mM p-NSF (BR buffer
solution) at GCE in the pH range from 4.0 to 10 in the absence of oxygen. Figure 3A,
3B and 3C shows DP voltammograms in pH 4.0, 7.0 and 10, respectively. The reduction
process 3c only occurs at higher pH values (pH > 8.0) and process 4c only occurs at

lower pH values (pH < 7.0). However, process 2c occurs in a wide range of pH values,
although at higher pH only one shoulder can be detected (process 1c). Figure 3D shows
the E vs. pH plots, and in which is possible to observe that only the processes E and
p
p2c
Ep4c are pH-dependent.
FIGURE 3
In lower pH values (4.0 < pH < 6.0), Ep2c shifts towards more negative values as
the pH value increases, according to (equation 1),
2
Ep2c (p-NSF) / mV = -0.185 - 0.065 pH (R = 0.991)
(equation 1)
in which the slope of the curve (close to the theoretical value of 59 mV/pH) indicates
that the number of protons and electrons involved in this process is the same. In pH
values higher than 6.0, the shift in peak potential no longer occurs, and possibly a
different reduction product is formed, and the redox reaction no longer involves
protons. At pH ≥ 6.0, a reduction shoulder at Ep1c becomes more distinctive. The peak
potential for this process remains constant and indicates that this electrochemical step
also does not involve protons, as well as process 4c at higher pH values (pH > 7.0).
However, in lower values of pH (4.0 < pH < 7.0), Ep4c shifts to a less negative potential
when the pH value decreases according to equation (equation 2), showing that the
reduction process is facilitated in acidic media.
2
Ep4c (p-NSF) / mV = -0.890 -0.055 pH (R = 0.990)
(equation 2)
The slope of the curve also indicates that the number of protons and electrons involved
in this step is the same, similar to that observed in lower pH values for process 2c.
Process 3c does not suffer any change in the peak potentials in the wide range of pH
studied.

The values for W1/2 obtained from the DP voltammograms in 0.04 M BR buffer
pH 7.0 are -72 and -96 for processes 1c and 2c, respectively. Within the margin of error,
these values indicate that this reduction processes occur with the transfer of one
electron, which is equivalent to 90 mV per electron transferred [53]. Process 3c and 4c
presented a higher W1/2 value, in which more than one electrochemical reduction
processes might be overlapped.
At the second DP measurements (data not shown), depletion in the current levels
was not observed, which differs from the second cycle registered at the cyclic
voltammograms from Figure 2A and 2B. There are two reasons for this difference.
-1
Cyclic voltammetry was carried out at 100 mV s , therefore, in the second reduction
cycle, the sweep is fast enough, that it prevents the electrode surface from being
completely renewed with nitro compound, which must arrive by diffusion from within
the solution. Also, cyclic voltammograms were obtained after reversing the potential
from the positive potential region, where the generated electrochemical oxidation
products could be adsorbed on the electrode surface. Differential pulse voltammetry, on
-1
the other hand, was carried out at a speed of 5 mV s , which infers that a higher amount
of time takes place between measurements and, at the second cycle, the surface has been
renewed. In this case, given that the DP voltammograms were conducted only in the
negative potential sweep, the generated electrochemical products in the first
measurement do not interfere with charge transfer for the subsequent measurements.
DP voltammograms were also obtained at GCE in 0.04 M BR buffer, containing
1
.0 mM m-NSF in a range from pH 4.0 to 10 and in the absence of oxygen. Figure 4A-
C, shows DP voltammograms in pH 4.0, 7.0, and 10, respectively.
FIGURE 4

The main processes (2c and 4c) observed for p-NSF were present, however,
process 1c did not appear even at pH > 6.0. Process 3c was only detected in lower pH
than those observed with p-NSF, even though it is detected at intermediate and higher
pH values (pH > 7.0).
The E vs. pH plot, Figure 4D, indicates a similarity regarding the reduction
p
processes that the molecules share, 2c, 3c, and 4c. Processes 2c and 4c suffer a shift in
peak potentials according to (equation 3) and (equation 4), as also observed for the p-
NSF.
2
Ep2c (m-NSF) / mV = -0.2890 - 0.056 pH (R = 0.945)
(equation 3)
(equation 4)
2
Ep4c (m-NSF) / mV = -0.8438 - 0.065 pH (R = 0.985)
Thus, the conclusions about the involvement of protons and electrons are the
same already discussed for the p-NSF. Process 3c does not suffer any change in peak
potentials, similar as observed at the DP voltammograms obtained with p-NSF. The
estimative of the number of electrons involved in the reductions process was not
performed given that W1/2 values indicated that process 3c can contain more than one
reduction process overlapped.
If Figures 3B and 4B are compared, it is easy to note that p-NSF suffers another
reduction process (1c), Ep1c (p-NSF) = -0.457 V at 0.04 M BR buffer pH 7.0, which is
absent in the m-NSF compound, as previously seen in cyclic voltammograms. It is also
important to highlight that the reduction process 2c occurs at less negative potential
value than for that observed for the m-NSF leader, Ep2c (p-NSF) = -0.58 V, and Ep2c (m-
NSF) = -0.62 V at 0.04 M BR buffer pH 7.0.
The change in peak potentials (less energy to promote the reduction for p-NSF)
and the presence of another reduction process (more stable intermediate product), again,

could be associated with the difference in the position of the nitro group in the aromatic
structure as can be seen on Scheme 2, which shows a straightforward analogy of the
●
─
electronic flux involved in the formation of the nitro anion radical (RNO2 ) in the
single electron reduction of p-NSF and m-NSF.
SCHEME 2
As a first step, the single electron reduction of p-NSF and m-NSF takes place,
inducing the homolyses of the π bond between the nitrogen and oxygen present in the
●
─
●─
●─
nitro moiety, yielding the nitro anion radicals p-NSF and m-NSF (RNO2 ) [54].
●
─
Structure-wise, p-NSF possesses the ability to be stabilized by the resonance of the
lone pair allocated in the nitrogen, disrupting the aromaticity of the benzenic ring as
well as the double bond between the sulfur and oxygen in the sulfonyl portion. Thus, the
●
─
presence of resonance structures of p-NSF could considerably escalate its lifetime
during the electrochemical measurements.
To evaluate the reversibility of the generated reduction products, square wave
voltammograms (SWVs) were obtained at GCE in 0.04 M BR buffer with different
values of pH (4.0, 7.0, and 10) containing 1.0 mM p-NSF and m-NSF and in the
absence of oxygen, Figure 5.
FIGURE 5
Data indicate that process 2c is irreversible at pH 4.0 for p-NSF and m-NSF,
given that the backward current (Ibackward) does not present any anodic components.
With the increase in pH value (7.0 and 10), the reversibility of process 2c appears, with
the presence of the anodic component on the backward current, with values of
Ip, forward/Ip, backward closer to 1 at pH 10 for both compounds. Again, process 1c is only
detected at the measurements conducted for p-NSF, and the anodic current component

(
Ibackward) increases with the increase in the pH of the buffer solution for both 2c and 1c.
Process 3c and 4c do not appear to be reversible in any of the pH range evaluated.
Given the differences in the electrochemical characterization for both
compounds, regarding the reduction processes 1c and 2c, the evaluation of the reduction
pathway aids in gathering information regarding their proposed biological action.
Process 2c can be attributed to the main reduction of the nitro group, according to
(reaction 1),
-
+
RNO + 4e + 4H  RNHOH + H O
(reaction 1)
2
2
4
.0 < pH < 6.0
in which, in lower values of pH, the hydroxylamine derivative is formed in a unique
step of reduction involving 4 electrons and 4 protons, favored by the availability of
protons in the electrolyte solution, Ep2c peaks in figures 3A and 4A. The irreversibility
of this reduction process was confirmed using square wave voltammetry, Ep2c peaks in
Figure 5A, and 5D.
On the other hand, the reduction of nitro compounds in intermediate and high
values of pH favors the stabilization of the reactive intermediate such as nitro radical
anion and nitroso derivative, which significantly slow down further reduction to
hydroxylamine, Figure 3B and 5B. Given that the availability of protons decreases, the
formation of the nitroso derivative is favored, according to (reaction 2),
-
+
RNO + 2e + 2 H  RNO + H O
(reaction 2)
2
2
7
.0 < pH < 10
in which two electrons and two protons are involved. The slope of the E vs pH for both
p
compounds does not indicate that there is an involvement with protons for this reduction

process. However, the reduction pathway of nitro compounds towards the formation of
the nitroso derivative occurs through a cascade reaction, in which there is the formation
of the nitro anion radical. Probably, it is possible that the last step of the reduction
process is seen, which involves the transfer of one electron from the nitro anion radical
to generate the nitroso derivative, in a process which also involves the loss of a water
molecule. This hypothesis agrees with the data for W obtained for p-NSF.
1
/2
As previously mentioned, the molecular design of p-NSF and m-NSF drug
leaders aims towards the interference with the parasite metabolism in 3 different ways.
The furoxan ring attached to both molecules is unstable in aqueous media, especially in
higher values of pH, in which the hydrolysis of the furoxan ring is facilitated and
introduces nitric oxide in the cell of the parasite, further inducing apoptosis and cell
death [49]. In the electrochemical measurements conducted for both compounds,
process 3c is observed, however, it is not present in the voltammograms of nitrobenzene
and p-MS, which do not have the furoxan ring in their molecule structure. This process
could be associated with the reduction process of a product generated from the
hydrolysis of the furoxan ring, in which its magnitude of current increases whenever the
pH value of the media increases [55]. Moreover, process 4c could be attributed to the
reduction process of the sulfone moiety, another functional group present in both
molecules and introduced to improve membrane permeability within the parasitic cell.
Still, given that neither process 3c nor process 4c is directly related to the nitro group
and also it appears that there is no influence from these processes on the electrochemical
direct response of the nitro group reduction, no assumptions were further verified.
Process 1c occurs at less negative potential values than process 2c and its
occurrence can be associated with the reduction of the nitro group in the formation of its
first unstable intermediate, the nitro anion radical, according to (reaction 3),

-
●─
RNO + 1e  RNO
(reaction 3)
2
2
6
.0 < pH < 10
It is usually difficult to detect the free nitro anion radical at glassy carbon
electrodes and in aqueous media, as well as any reactive oxygen or nitrogen species,
given its short lifetime and high reactivity [56,57]. Most studies regarding the
characterization of the nitro anion radical are performed in organic or aprotic media
[58], in which its reversibility is easily seen, or at a boron-doped diamond electrode,
where the reduction process is observed whenever a high boron doping content is
ensured [35]. In biological systems, the futile cycle of nitro anion radical in the presence
of oxygen usually entails competition between the radical’s natural decay mechanism
and the one-electron transfer to oxygen generating superoxide radicals [17]. Therefore,
to further confirm if process 1c could be correctly attributed to the formation of the nitro
radical anion in aqueous media and simulate biological conditions, pulse
voltammograms for both compounds were obtained in the presence of oxygen.
Moreover, DP voltammograms were also obtained with p-NSF in the presence of
cysteine, which has been proved to induce the direct reduction of the nitro group
towards the nitroso derivative by adduct formation [34]. For both cases, further
reduction processes, characteristics of the nitro group reduction, are hindered.
Influence of O and cysteine
2
DP voltammograms in the presence of oxygen were obtained at GCE in 0.04 M
BR buffer pH 7.0 containing 1.0 mM of p-NSF and m-NSF. With the increase in the
amount of oxygen inside of the electrochemical cell (bubbling oxygen gas during 10,
2
0, and 30s) the magnitude of current for process 1c increases as well, Figures 6A and

6B. This process is unrelated to the oxygen reduction process itself at -0.63 V, as
illustrated in Figures S9 and S10.
FIGURE 6
A simple comparison between figures 6A and 6B shows that the process
detected at Ep is initially present only in the voltammograms obtained in the solutions
1
c
containing p-NSF. It appears in solutions containing m-NSF, only after there is the
availability of O , and, for both cases, the current levels increased with the increase of
2
oxygen amongst the electrochemical cell. These results agree with the hypothesis that
process 1c can be attributed to the formation of the nitro anion radical, according to
(reaction 4)[35],
●
─
●─
RNO2 + O  RNO + O
2
(reaction 4)
2
2
in which the intermediate reduction product interacts with oxygen, generates superoxide
radicals, and regenerates the initial nitro compound in an electrocatalytic cycle.
DP voltammograms were also obtained at GCE in 0.04 M BR buffer pH 7.0
containing 0.5 mM of p-NSF and increasing concentration of cysteine (0.25 mM, 0.5
mM, and 1.0 mM), in the presence of oxygen, Figure 7A, and an absence of oxygen,
Figure 7B. The interaction of the nitroso derivative with thiol molecules has already
been reported [34,45], in which, in acidic and neutral media and under no applied
potential, there is the formation of an adduct between both species, which is further
chemically reduced to hydroxylamine derivative, (reaction 5),
RNO + 2RSH  RNHOH + RS-SR
(reaction 5)
There is a depletion in current levels for process 1c whenever cysteine is present
until it is entirely hindered at ratios equal or higher than 1:1 cysteine:p-NSF. In the

presence of oxygen, Figure 7B, where the nitro anion radical is easily seen due to the
previous electrocatalytic mechanism illustrated, the same depletion is observed,
however, the cysteine concentration needed to hinder its current levels completely is
higher than 2:1 cysteine:p-NSF. This mainly occurs given that oxygen and cysteine
have opposite effects over the nitro anion radical production. The adduct formed
between the nitroso derivative and thiol molecules may affect the cascade reaction for
the generation of the nitro anion radical, destabilizing it (process 1c), and instead, drives
the reduction process under applied potential towards the direct formation of the nitroso
derivative. In fact, in square wave voltammograms obtained for p-NSF in the absence
and presence of cysteine in pH 7.0 and pH 10.0, Figure S11 and S12, respectively, the
backward current component for the generation of the nitroso derivative shows a
depletion in current values whenever cysteine is present, further confirming the
chemical reaction described in (reaction 5). Moreover, the reduction of the nitro
compound to hydroxylamine in acid or neutral media containing cysteine is inhibited
and the reduction step to the nitroso derivative is favored due to the presence of the
coupled chemical reaction.
●─
3
.2
Stabilization of RNO2 between p-NSF and m-NSF and comparison with
other antichagasic pharmaceuticals
As previously mentioned, the reduction potential of the nitro group can be
associated with the degree of injury within the parasite’s cell, since less negative
potentials are required to generate reactive intermediates such as the nitro anion radical,
nitroso derivative, and hydroxylamine. p-NSF presents a less negative reduction
potential for the nitro group when compared with m-NSF, which indicates that this
molecular structure, where the nitro group is in para- position, is preferred to incite
higher biological activity. Moreover, the lower reduction potential and the higher

stability of the nitro anion radical due to a higher number of resonance structures also
increase the reactivity of p-NSF when compared to m-NSF. Table 1 shows a
comparison of reduction potential observed in carbon-based electrodes of nitroaromatic
compounds in protic media. For these compounds, the generation of the nitro anion
radical is often only observed in aprotic or organic media, while the nitro group
reduction in aqueous media produces the nitroso derivative or still hydroxylamine. Both
compounds, p-NSF and, m-NSF presents reduction potentials close to pharmaceuticals
already used in the treatment of Chagas disease, which, alongside their other structural
designed moieties, indicates their potential to be investigated as pharmaceutical
antichagasic candidates. The in vitro biological experiments to confirm this hypothesis
will be performed.
TABLE 1
Thus, the electrochemical characterization of these compounds is particularly
important to understand more deeply the mechanism of action of bioactive compounds,
as well as to aid in the structural and configuration design of their functional groups in
aromatic rings towards the development of new pharmaceutical molecules.
4
. CONCLUSIONS
The electrochemical reduction mechanism of two candidates of drug leaders against
Chagas disease, p-NSF, and m-NSF, were investigated in the presence and absence of
oxygen using cyclic, differential pulse, and square wave voltammetry. The detection of
the nitro anion radical from the reduction processes of the nitro group was evaluated in
aqueous media at a bare glassy carbon electrode previously polished and stabilized in a
buffer solution. Both compounds presented similar oxidation and reduction processes in
the absence of oxygen given their similar molecular structure (reduction to the
formation of nitroso derivative in intermediate and higher values of pH and

hydroxylamine in lower values of pH). However, p-NSF presented one reduction
process that was not observed in the voltammograms for m-NSF, 1c, the generation of
the nitro anion radical. The higher stability of this radical intermediate (and an increased
lifetime to be registered using voltammetry in aqueous media) was associated with the
●
─
electronic flux of the p-NSF . This compound presents more resonance structures
●
─
than m-NSF , hypothesis further confirmed with data obtained using differential pulse
voltammetry in the absence and presence of cysteine and oxygen. Cysteine (thiol) forms
an adduct with nitroso derivatives, which favors the reduction process in a step
involving 2 electrons and 2 protons, even in an acidic medium, where hydroxylamine is
normally produced. In the presence of oxygen, the nitro anion radical is consumed to
generate superoxide and the original nitro compound, in a catalytic cycle. Therefore, the
reduction potential of the nitro group of p-NSF, when compared with m-NSF, alongside
the higher stabilization of the nitro anion radical due to a higher number of resonance
structures, indicates that this molecular structure can be chosen for the design of new
antichagasic drugs since that inhibit detoxifying enzymes of the parasite, and further
aids the beginning steps of the structural design of new antichagasic pharmaceuticals
before bioassays in vitro against the parasite.
Acknowledgments
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior - Brasil (CAPES) - Finance Code 001. The authors would like to thank
São Paulo Foundation Research (FAPESP) (Project #2012/06240; #2017/02854-8,
#
2019/10762-1), CNPq (Project #303206/2019-5 and #140828/2016-8).

Credit Author Statement
Caroline G. Sanz: First author and main researcher involved in voltammetric
measurements, data analysis, original draft preparation, writing, reviewing and editing.
Kevin A. Dias: Organic synthesis characterization of nitro compounds and draft
preparation.
Raphael P. Bacil: Data and paper discussion and draft preparation.
Ricardo A.M. Serafim: Organic synthesis of antichagasic leaders, their
characterization and draft preparation.
Leandro A. Helgueira: Supervisor of organic synthesis.
Elizabeth I. Ferreira: Supervisor of organic synthesis of antichagasic leaders
draft preparation.
Silvia H.P. Serrano: Corresponding author, main supervisor, data discussion
analysis and conceptualization of the article, draft preparation, reviewing and editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

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