Electrochemical behaviour of dipyrone (metamizole) and others pyrazolones

Article abstract of DOI:10.1016/j.electacta.2018.04.082

The electrochemical oxidation of dipyrone (MTM) in aqueous medium was characterized using antipyrine (AA), 4-aminoantipyrine (4AA), 4-methyl-aminoantipyrine (MAA) and 4-dimethyl-aminoantipyrine (DMAA) as model molecules for the elucidation of all MTM voltammetric signals. The MTM and the other pyrazolones show up to four oxidation electrochemical processes. The voltammograms obtained in AA solutions presented an irreversible electrochemical oxidation process involving one electron at Eap_3, which is common to all pyrazolone derivatives, while the amino pyrazolones present electrochemical oxidation processes at Eap₀ or Eap₁. The stabilization of the oxidation products depends on different effects: the proton release added to the thermodynamic stability, in the case of the imine formation at Eap₀ (4AA and MAA) and the hyperconjugation (σ-stabilization) in the case of iminium formation (DMAA and MTM) at Eap₁. The process observed at Epa₀ corresponds to the pH-dependent oxidation of the primary and secondary enamines, while the process observed at Eap₁ occurs in the tertiary enamines, is pH independent. The oxidation peak potential follows the order: MAA < 4AA < DMAA < MTM and it was demonstrated that DMAA in an aqueous medium can simulate the MTM in an aprotic medium; therefore, the analytical MTM determination can be performed using the DMAA aqueous analytical curve. DMAA and MTM analytical curves, presented a linear range from 10 μmol L^?1 to 100 μmol L^?1 with a LOD of 1.94 and 2.97 μM for DMAA and MTM, respectively, LOQ of 6.48 and 9.91 μM (n = 10) and, sensitivity of 0.96 μA/μM for DMAA and 0.92 μA/μM; with a recoveries of 95–105% for MTM.

Full text of DOI:10.1016/j.electacta.2018.04.082

Accepted Manuscript
Electrochemical behaviour of dipyrone (metamizole) and others pyrazolones
Raphael P. Bacil, Rafael M. Buoro, Othon S. Campos, Matesa A. Ramos, Caroline G.
Sanz, Silvia H.P. Serrano
PII:
S0013-4686(18)30826-0
10.1016/j.electacta.2018.04.082
EA 31649
DOI:
Reference:
To appear in: Electrochimica Acta
Received Date: 24 January 2018
Revised Date: 9 April 2018
Accepted Date: 11 April 2018
Please cite this article as: R.P. Bacil, R.M. Buoro, O.S. Campos, M.A. Ramos, C.G. Sanz, S.H.P.
Serrano, Electrochemical behaviour of dipyrone (metamizole) and others pyrazolones, Electrochimica
Acta (2018), doi: 10.1016/j.electacta.2018.04.082.
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ACCEPTED MANUSCRIPT
Electrochemical behaviour of dipyrone (metamizole) and others
pyrazolones
a
b
a
a
Raphael P. Bacil , Rafael M. Buoro , Othon S. Campos , Matesa A. Ramos ,
a
a
Caroline G. Sanz , Silvia H. P. Serrano *.
a
Department of Fundamental Chemistry, Institute of Chemistry – University of
São Paulo, CEP: 05508-000, São Paulo (SP), Brazil
b
Department of Physics and Molecular Chemistry, São Carlos Institute of
Chemistry – University of São Paulo, CEP: 13566-590 - São Carlos (SP), Brazil
Keywords: Dypirone; Metamizole; Antipyrines; Mechanism; Voltammetry;
*
Email: shps@iq.usp.br

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Abstract
The electrochemical oxidation of dipyrone (MTM) in aqueous medium was
characterized using antipyrine (AA), 4-aminoantipyrine (4AA), 4-methyl-
aminoantipyrine (MAA) and 4-dimethyl-aminoantipyrine (DMAA) as model molecules
for the elucidation of all MTM voltammetric signals.
The MTM and the other pyrazolones show up to four oxidation electrochemical
processes. The voltammograms obtained in AA solutions presented an irreversible
electrochemical oxidation process involving one electron at Eap3, which is common to
all pyrazolone derivatives, while the amino pyrazolones present electrochemical
oxidation processes at Eap
on different effects: the proton release added to the thermodynamic stability, in the case
of the imine formation at Eap (4AA and MAA) and the hyperconjugation (σ-
stabilization) in the case of iminium formation (DMAA and MTM) at Eap . The process
observed at Epa corresponds to the pH-dependent oxidation of the primary and
secondary enamines, while the process observed at Eap occurs in the tertiary enamines,
0
or Eap
1
. The stabilization of the oxidation products depends
0
1
0
1
is pH independent. The oxidation peak potential follows the order: MAA < 4AA <
DMAA < MTM and it was demonstrated that DMAA in an aqueous medium can
simulate the MTM in an aprotic medium; therefore, the analytical MTM determination
can be performed using the DMAA aqueous analytical curve. DMAA and MTM
–
1
–1
analytical curves, presented a linear range from 10 µmol L to 100 µmol L with a
LOD of 1.94 and 2.97 ꢀM for DMAA and MTM, respectively, LOQ of 6.48 and 9.91
ꢀ
M (n = 10) and, sensitivity of 0.96 ꢀA/ꢀM for DMAA and 0.92 ꢀA/ꢀM; with a
recoveries of 95 to 105% for MTM.

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1
. Introduction
The antipyrines or pyrazolones are a group of the first synthesized
pharmaceuticals to work as analgesics and antipyretics. The antipyrine (1,5-dimethyl-2-
phenylpyrazol-3-one), AA, was the first one made by Knorr, thus, getting the group
name. Some time after, other drugs were made, until they reached the aminoantipyrine
(4-amino-1,5-dimethyl-2-phenylpyrazol-3-one), 4AA; the dimethylaminoantipyrine,
a.k.a. pyramidon, (4-(dimethylamino)-1,5-dimethyl-2-phenylpyrazol-3-one), DMAA
and finally, [(1,5-dimethyl-3-oxo-2-phenylpyrazol-4-yl)- ethylamino] methanesulfonic
acid], known as metamizole or dipyrone, MTM, an injectable formulation with
antipyretic and analgesic effects [1].
MTM undergoes a hydrolysis reaction and generates the 4-methylamino
antipyrine, MAA [2–4], which is enzymatically metabolized in the liver producing other
metabolites, such as 4-amino-2,3-dimethyl-1-phenyl-3-pyrazol-5-one, FAA and 4-
dimethylamino-1,5-dimethyl-2-phenylpyrazol-3-one, AAA[4, 5], Schemes 1 and 2.
INSERT SCHEME 1
INSERT SCHEME 2
The analgesic effect observed for MTM, as well as for other pyrazolones, is
attributed to the inhibition of the cyclooxygenase enzyme (COX), in the liver, by the
pharmacologically active metabolite, MAA [6, 7]; despite that, the inhibition
mechanism of cyclooxygenase by the MTM is still unknown. Besides the COX
hindering, the MTM and other pyrazolones can induce circumstantial allergic responses,
such as Stevens–Johnson’s syndrome [8], rhinoconjunctivitis [9–11] and cause
agranulocytosis[2, 8]. Still, MTM is in worldwide use [2, 3, 12–14].

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Most studies dedicated to pyrazolones aim at their analytical detection. MTM
gets more attention due to its high utilization, being determined and quantified by a
variety of techniques, such as HPLC [15, 16], iodometry [17], FIA systems with
amperometric detection [18–20], spectrophotometry, also associated with FIA systems
by the addition of various reagents [21–23], capillary electrophoresis [24], NMR [16]
and voltammetry [25–29]. AA is determined by HPLC [30–32], gas chromatography
[
33], NMR [34] and voltammetry [35–37]. Pyramidon also gets attention in some
papers, such as HPLC with amperometric detection [30,38], voltammetry [36], NMR
39] and a CG method utilized to detect DMAA in apprehended samples of cocaine, in
[
which it is used as adulterant [40].
On an electrochemical overview, several studies were carried out with different
types of electrodes, our group utilized a glassy carbon electrode in previous studies [2],
Marcolino et al. [25] utilized carbon paste electrodes as working electrode, as well as,
Teixeira et al. [35], which modifies the carbon paste with N,N′-
ethylenebis(salicylideneaminato)oxovanadium(IV), and Cumba et al. [41], who utilized
a carbon paste modified with a composite titanium phosphate/nickel hexacyanoferrate.
Metallic electrodes were also utilized, Basaez et al. [29] used platinum electrodes and
Munoz et al. [42] disposable gold electrodes obtained from recordable CDs. The
difference between carbon-based electrodes and metallic or metallic composites
electrodes are the different potential windows and the electrochemical processes
definitions. Therefore, carbon-based, such as glassy carbon electrodes, present a most
adequate potential window to the electrochemical pyrazolones’ oxidation mechanism
study providing a constant surface for the electrochemical oxidation without surface
passivation problems due to oxide formations in multiple scans, which can give an

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indirect response to the analyte through interactions with the formed oxide that are
independent of the molecule itself, a problem in both platinum and gold electrodes.
The aim of this work was to study the electrochemical oxidation of MTM in an
aqueous medium, using AA, 4AA, MAA and DMA as model molecules for the
elucidation of all MTM voltammetric signals and to understand why the oxidation peak
potentials are different although the oxidation sites are the same in all molecules, except
AA.
Using this strategy, it was possible not only to understand but also to describe,
the electrochemical oxidation mechanism and reactivity of MTM and other biologically
active pyrazolones, as well as the probable inhibition mechanism of the COX enzyme.
2
. Experimental
2
.1. Reagents and Solutions
All reagents were of analytical grade without any previous purification. The
solutions were prepared using deionized water from a reverse osmosis device (Gehara
Co., model OS10LX ultra-pure system, water resistivity ≥ 18 MΩ cm). Phosphate
–
1
buffer solutions (PBS), 0.1 mol L , were prepared by mixing appropriate amounts of
H PO and NaH PO (Merck) in deionized water. Similar procedures were used to
3
4
2
4
prepare the AA (Sigma Aldrich, code D-8890), 4AA (Sigma Aldrich, code A-4382),
DMAA (Sigma Aldrich, code D8015) and MTM (Sigma Aldrich, code D-8890)
solutions. The pH adjustments to the desired pH were performed by the addition of 4.0
–
1
mol L NaOH solution. The supporting electrolyte used in the organic medium (DMF -
–
1
Sigma Aldrich, code 227056) was 0.1 mol L tetrabutylammonium tetrafluoroborate
TFBTBA – Merck code 8.18244.0025). All experiments were performed at room
(

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temperature (25 ± 1 °C) and micro volumes were measured using EP-10 and EP-100
Unipette microliter pipettes (Uniscience, Brazil).
2
.2. Obtainment and Characterization of MAA
–
1
MAA was obtained by hydrolysis of 10 mmol L MTM solutions at 80 °C for 2 h under
vigorous stirring. The MAA was extracted from the solution with chloroform and
obtained in the solid form, after rotation evaporation of the solvent. It was resolubilized
in CDCl to perform the NMR essays.
3
2
.3. Apparatus
All
voltammograms
were
obtained
using
a
PGSTAT
101
potentiostat/galvanostat, Metrohm AUTOLAB, connected to an IME663 interface
stirrer device. The data treatment was performed with NOVA software version 1.10.4.
and Origin 8.0. Glassy carbon electrode (GCE – 3 mm φ), Ag/AgCl, KCl(sat) and a
platinum wire were used as working, reference and auxiliary electrodes, respectively, in
a 20 mL electrochemical cell. All pH measurements were performed using a pH meter
model 654 and a combined glass electrode, model 6.0203.100 (OE), both from
Metrohm.
The spectrophotometric measurements were performed with an Agilent 8453
spectrophotometer with the Chem Station software from Agilent Technologies. The
high-performance liquid chromatograms were obtained using a Shimadzu HPLC system
LC 10 with UV–vis detection and the data treatment performed with a software HPLC
class LC 10. NMR spectra were obtained in a Bruker 500 MHz equipment with ACS
labs software. All data were obtained at room temperature, 25 ± 3 °C.
2
.3.1. UV–vis Spectra

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The UV and visible spectrophotometric measurements were performed using
deuterium and tungsten lamps, respectively, and a quartz cuvette with 1.0 cm optical
path. The blank spectra were performed in PBS, pH 7.4.
2
.3.2. High-Performance Liquid Chromatography (HPLC)
The chromatograms were obtained with the Shimadzu Diode Array at 241 nm
fixed wavelength using a reversed phase technique and analytical column of C18 (5.0
m, 15 cm and 4.7 mm), previously coupled to a precolumn with a 1.0 ꢀL loop. The
mobile phase was methanol/PBS in a 55%/45% (V/V) proportion at pH 2.5 with a flow
ꢀ
–
1
rate of 1.0 mL min .
2
.3.3. Nuclear Magnetic Resonance (NMR)
1
3
The 1H and C NMR spectra (500 MHz frequency) were obtained using a
Brucker equipment, http://ca.iq.usp.br. The samples were prepared in CDCl , a
3
1
sequence pulse zg30 was utilized in both spectra. To compare quantitatively the H and
1
3
C NMR spectra, a computational simulation was obtained using the software
ChemBioDraw 2010.
2
.3.4. Electrochemical Measurements
2
.3.4.1. Glassy Carbon Surface Pretreatment
The GCE was polished using 1.0/0.1 µm size particle diamond spray (Buehler, US) in a
metallographic felt (Buehler, US) to avoid superficial adsorption effects.
2
.3.4.2. Cyclic Voltammetry (CV)
The voltammograms were performed using the following experimental conditions:
–
1
Einitial = 0.0 V; E = 1.4 V, Efinal = 0.0 V, step potential = 2.0 mV and ν = 100.0 mV s .
λ
The solutions were stirred before each measurement. Distinct conditions in any assay
are specified in the figure caption.

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2
.3.4.3. Square Wave Voltammetry (SWV)
The SW voltammograms were obtained using the following experimental conditions:
Einitial = 0.0 V; Efinal = 1.4 V; step potential = 2.0 mV, pulse amplitude = 50.0 mV,
–1
frequency = 25.0 Hz, resulting in scan rate ν = 50.0 mV s . The solutions were stirred
before each measurement.
2
.3.4.4. Differential Pulse Voltammetry (DPV)
The experimental conditions for DPV experiments were: Einitial = 0.0 V, Efinal = 1.4 V;
pulse amplitude = 50 mV; pulse width = 100.0 ms; step potential = 2.5 mV and interval
–1
time = 0.5 s resulting in scan rate of 5 mV s . The solutions were stirred before each
measurement.
3
. Results and Discussion
3
.1. Electrochemical Characterization of MTM and the Other Pyrazolones
Cyclic voltammograms obtained in the antipyrine solutions show up to four
oxidation processes (Eap /Eap , Eap , Eap and Eap ), Figure 1. The voltammograms
0
1
2
2'
3
obtained in AA solutions presented only Eap process because this molecule has no
3
substituents on the pyrazolone ring; therefore, the most plausible oxidation possibility is
in the nitrogen atom, adjacent to the aromatic ring, in a similar mechanism observed for
acetaminophen [43], Figure 1A. The other molecules (4-AA, MAA, DMA and MTM)
have an enamine moiety (group –C=C–N–) as a substituent of the pyrazolone ring,
Scheme 1. The comparison between the voltammograms presented in Figure 1A with
those on Figures 1B–1E permit the conclusion that the oxidation processes in these
molecules involve the enamine moiety.
Insert Figure 1

ACCEPTED MANUSCRIPT
Probably, the oxidation processes Eap (4AA and MAA) and Eap (DMAA and
0
1
MTM) occur in the enamine itself; however, the oxidation products obtained in Eap0
and Eap differ in stability and the more stable the product, the less positive is the
1
oxidation peak potential, Table 1.
Insert Table 1
Table 1 shows that the oxidation peak potential follows the order MAA < 4AA
<
DMAA < MTM. The first oxidation processes of 4AA and MAA (at Eap ) are
0
irreversible and pH dependent, Figures 1 and 2, while for DMAA and MTM, the first
oxidation process at Eap is pH independent and shows a small cathodic component in
1
the reverse scan, Figure 1E. According to the slope of the Pourbaix diagrams, 0.054
V/pH and 0.055 V/pH for 4AA and MAA, respectively, both oxidation processes occur
involving the same number of electrons and protons and, the experimental pK value for
a
both molecules is 7.4, Figure 2.
Insert Figure 2
To estimate the number of electrons involved in the oxidation processes,
differential pulse voltammograms were obtained with GCE in aqueous and nonaqueous
media and the data are presented in Figures 3, 4 and Table 2. The Eap peak obtained
0
for 4AA (Figure 3B) and MAA (Figure 3C) can be mathematically deconvoluted to
distinguish the involved processes. Two distinct peaks were obtained: Eap the first one,
0
which corresponds to the oxidation of the enamine (red line, Figure 3B) to the
corresponding imine, which can undergo a hydrolysis, resulting in a product that can
also be oxidized [44] (green line, Figure 3B and red line on Figure 3C).
Insert Figure 3
To avoid water-dependent coupled chemical reactions, differential pulse
–
1
voltammograms were also obtained in DMF containing 0.1 mol L TFBTBA solution.

ACCEPTED MANUSCRIPT
It would have been expected that, in the absence of water, the pH-dependent oxidation
peak potentials, which occur in the same moiety of the molecule, to have similar
oxidation potentials, as in fact occurred for Eap and Eap peaks, Figure 4.
0
1
Insert Figure 4
Insert Table 2
Table 2 shows the peak width at half height (w ) values obtained in nonaqueous
1
/2
media for all oxidation processes and, according to these data, Eap (4AA and MAA)
0
corresponds to a process involving one electron, given that the w1/2 values were 89 and
1
07 mV, respectively. In the same medium, the w1/2 values for DMAA and MTM at
Eap were 91 and 89 mV, respectively, which are close to the theoretical value (90 mV)
1
for a charge transfer reaction involving one electron, typical of processes in which the
oxidized products are radical species.
It is important to note that the Eap value in Figure 3E is much less positive
0
than the Eap value in Figure 4E; this great difference can be explained by the number
0
of protic species in the reaction medium in which the measurements were taken. Eap in
0
Figure 3E corresponds to the MAA oxidation from MTM hydrolysis; the oxidation
process is proton-dependent and facilitated in an aqueous medium. DMF always
contains a small amount of water and the MTM hydrolysis process is expected to occur,
although to a small extent, but due to the low water content, the pH-dependent oxidation
process will be difficult and observed at more positive potentials.
To evaluate the reversibility of the Eap and Eap processes, square wave
0
1
voltammograms were obtained and data are presented in Figure 5.
Insert Figure 5
As can be seen from Figure 5, only Eap for DMAA has reversible character and
1
this result can be associated with the difference in the substituents bonded to the

ACCEPTED MANUSCRIPT
pyrazolones ring: a tertiary enamine in the case of DMAA and a secondary enamine in
the case of MAA.
In the first case, the iminium cation radical formed at Eap can be stabilized by
1
the hyperconjugation effect, improved by the hydrogens of two methyl groups (σ-
stabilization) and therefore, can be reversibly reduced at Ecp. However, in the MAA,
the electrochemical oxidation process is favored by the loss of one proton and enhanced
by the thermodynamic stability of the product, which confers a less positive oxidation
peak potential to the MAA. The Eap oxidation process for MAA (Figure 3C), shows
0
that the broad peak can be mathematically deconvoluted into two peaks: Eap the first
0
•
one, corresponds to the oxidation of the enamine to the respective imine radical ( C–
C=N–R), (red line, Figure 3B) [43], similarly to that observed during the formation of
the Schiff’s bases [45,46] and a small peak, corresponding to the oxidation of the
hydrolysis imine radical product, formed in a coupled chemical reaction (red line on
Figure 3C). This can be the main reason for the irreversibility of the process observed
at Eap in the MAA molecule.
0
Note that a similar effect was observed for 4AA (green line, Figure 3b) and
therefore, the product formed at Eap for 4AA suffers hydrolysis reaction and is
0
subsequently oxidized, following a similar oxidation mechanism to MAA. The primary
enamine in 4AA has no methyl substituent to stabilize the oxidation product, and then,
the Eap has a more positive value than those observed for DMAA and MAA.
0
Finally, it is important to understand why the Eap , Figure 3, for the MTM has
1
the most positive value of all molecules. This fact can be explained taking into account
the presence of the sulfoxide group (an electronic acceptor) as a substituent in the
enamine group, in relation to the other studied compounds, the stabilization of its
oxidation product is difficult. The Eap , Figure 4, corresponds to the oxidation of MAA
0

ACCEPTED MANUSCRIPT
coming from the MTM hydrolysis process, while the Eap oxidation process
2
corresponds to the oxidation of the product formed at Eap , as previously demonstrated
1
for MTM [2].
3
.2.Mechanism Evaluation
The MTM first electrochemical oxidation occurs in the tertiary enamine without
the presence of proton to produce an iminium cation radical, which can suffer a
nucleophilic attack by water to produce another reactive intermediate. Thus, to evaluate
the oxidation mechanism in the absence of a possible coupled reaction, cyclic
voltammograms were obtained in DMF solutions containing TFBTBA as a supporting
electrolyte, Figure 6A (full window) and Figure 6B (restricted window).
INSERT FIGURE 6
If the potential window is restricted to +0.7 V, where the electrochemical
process at Eap does not occur, it was observed that ipa /ipc increases with increasing
2
1
–
1
scan rate, reaching a stable value of 0.75 at 100 mV s . These results suggest that the
product formed at Eap can, at high scan rate, be reduced at Ep or be consumed, at low
1
c
scan rates, by a coupled chemical reaction in an EC mechanism, Figure 7.
The process occurring at Epa is diffusion controlled throughout the scan range
1
2
studied (log iap = 0.483 log v + 0.730, R = 0.985), while that occurring at Epa is
1
2
2
diffusion controlled at higher scan rate [log iap = 0.443 log v + 0.974, R = 0.982 (for
2
–
1
–1
9
0
0 mV s ≤ v ≤ 1000 mV s )] and adsorption controlled at low scan rate [log Iap =
2
2
–1
–1
.893 log v + 0.091, R = 0.947 (for 10 mV s ≤ v ≤ 90 mV s )], data not shown.
INSERT FIGURE 7
The diffusion regions, in both Eap and Eap , as well as the constant ratio
1
2
–
1
iap /icp, observed at high scan rates (>100 mV s ), indicate an electrochemical–
1

ACCEPTED MANUSCRIPT
electrochemical (EE) mechanism, which was previously studied [2]. The
electrochemical mechanism at Eap changes as a function of the scan rate, which is
1
characteristic of electrochemical processes followed by a coupled chemical reaction.
Figure 8 is a representation of the first MTM electrochemical process in
nonaqueous medium. Eap increases linearly with increasing scan rate, ν, from 10 to
1
–
1
1
00 mV s and the slope of the linear portion is 30 mV/decade ν, which is expected for
an EC process involving one electron, Eap /Ecp. It is important to note that there is a
1
region in the plot where Eap is constant and near of 0.56 V. Below this value, for low
1
scan rates, the peak potential values are lower, showing that the chemical coupled
reaction is facilitating the electrochemical process.
INSERT FIGURE 8
The chemical step after the electrochemical process is most likely a dimerization
because a nucleophilic attack in an organic medium is highly unlikely to occur. In
addition, the slope obtained in the Eap vs log v plot is very close to the documented
1
theoretical value, 29.9 mV/log v [47] as expected for a process involving a dimerization
step. Thus, it is suggested that the coupled chemical reaction is that represented by
equation 1, where the oxidation product of the MTM formed in Eap undergoes a
1
dimerization process and, apparently, this species is inactive.
.
.
MTM + M TM → MTM -M TM
The possible reaction pathways after the electrochemical oxidation of MTM are
illustrated in Scheme 3.
INSERT SCHEME 3
Equation (1)

ACCEPTED MANUSCRIPT
Table 3 compiles the voltammetric data for Eap , Eap , Eap and Eap in AA,
0
1
2
3
4
AA, MAA, DMAA and MTM.
INSERT TABLE 3
Based on the data presented in Table 3, an oxidation mechanism, Scheme 4, was
proposed for the electrochemical oxidations of all pyrazolones studied.
INSERT SCHEME 4
3
.3. Analytical Application
As observed in the S1 and S2 Figures, MTM’s hydrolysis occurs with fast
kinetics; thus, it is difficult to use MTM as a standard to obtain an analytical curve.
However, if the proposed mechanism is correct, it should be possible to use DMAA as a
model molecule to substitute MTM, because both have the same Eap process.
1
To validate this hypothesis, an analytical curve was constructed utilizing DMAA
in an aqueous medium and compared with another MTM curve in an organic medium.
–
1
DMAA and MTM analytical curves, present a linear range from 10 µmol L to 100
–
1
µmol L with a limit of detection (LOD) of 1.94 and 2.97 µM for DMAA and MTM,
respectively, limit of quantitation (LOQ) of 6.48 and 9.91 µM (n = 10) [48] and a
sensitivity of 0.96 µA/µM for DMAA and 0.92 µA/µM for MTM, Table 4 and Figure
9
. Using this approach, MTM was quantified in commercial formulations with a
recovery of 95 to 105%, Table 4.
INSERT FIGURE 9
INSERT TABLE 4
The obtained recoveries are satisfactory, implying that the proposed mechanism
is correct.

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4
. Conclusions
The pyrazolones, depending on their structure, may exhibit up to four
electrochemical processes, Eap or Eap , Eap , Eap and Eap . The Eap process occurs
0
1
2
2'
3
3
in the pyrazolone ring. It involves an irreversible electron charge transfer and is
common to all compounds of this class. The molecules with the enamine moiety present
three electrochemical processes, in addition to Eap3.
The processes Eap or Eap , both involving one electron, occur in the same
0
1
electroactive moiety, the enaminic nitrogen, substituent of the pyrazolone ring, and
these processes depend on two different effects: the hyperconjugation (σ-stabilization)
in the case of iminium cation radical formation (DMAA and MTM) at Eap , which
1
electrochemical processes are pH independent and the proton release, added to the
thermodynamic stability, in the case of the imine radical formation at Eap (4AA and
0
MAA).
The Eap process, which occurs after the Eap or Eap processes, is irreversible,
2
0
1
involves one electron and is not pH dependent. In MTM, the electrochemical oxidation
at Eap competes with a chemical dimerization reaction, just after Eap . The
2
1
–
1
electrochemical process is favored at high scan rates (>90 mV s ), when the chemical
step is not favored, Scheme 3. The complete electrochemical oxidation mechanism for
several pyrazolones, which data were compiled in Table 3, is illustrated in Scheme 4.
Pharmaceutical samples of MTM were prepared in an organic medium and the
drug was quantified using an aqueous DMAA analytical curve. Satisfying recoveries
were obtained, which corroborates the proposed oxidation mechanism for these
molecules.

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The voltammetric study of the pyrazolone group allowed understanding the
reactivity of the molecules and shows that at physiological pH, MAA (secondary
enamine) is easily oxidized and can act as a good reducing agent, which is the probable
reason for which the COX enzyme, with iron(III) in its peroxidase active site, is
inhibited by the dipyrone.
5
. Acknowledgements
The authors would like to thank São Paulo Foundation Research (FAPESP),
CNPq (Process 140833/2016-1), CAPES for the financial support and to Kevin Dias,
PhD student, for the valuable discussions. This work is dedicated to Professor Dr. P.V.
Antonio William Oliveira Lima (in memorian).
6
. References
[
[
1] K. Brune, The early history of non-opioid analgesics, Acute Pain. 1 (1997) 33–
0.
4
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Figure 1. Cyclic voltammograms obtained with GCE in 0.1 M PBS, pH = 7.4,
containing 1 mM of each of the compounds: AA (A); 4AA (B); MAA (C); DMAA (D);
MTM (E). Experimental conditions: as described in the experimental section.
Figure 2. Pourbaix diagrams: Eap from MAA (red line) and 4AA (black line).
0
–
1
Figure 3. Differential pulse voltammograms obtained with GCE in 0.1 mol L PBS,
–
1
pH 7.4, containing 1.0 mmol L of AA (A); 4AA (B); MAA (C); DMAA (D) and
MTM (E). The peak width at half height values (w ) were obtained after the
1
/2
voltammogram deconvolution processes (colored lines). Experimental conditions: as
described in the experimental section.
Figure 4. Differential pulse voltammograms obtained with GCE in DMF containing 0.1
–
1
M of TFBTBA and 1.0 mmol L of A) AA; B) 4AA; C) MAA; D) DMAA; E) MTM.
Deconvolution processes were performed, when necessary, to obtain the values of w .
1
/2
Experimental data are shown as black lines while deconvolutions are displayed as
colored lines. Experimental conditions: as described in the experimental section.
Figure 5. Square wave voltammograms (SWV) obtained with GCE in 0.1 M PBS, pH =
7
.4, containing 1.0 mM of each of the compounds: AA (A); 4AA (B); MAA (C);
DMAA (D); MTM (E). (1) Total current; (2) forward current; (3) backward current.
Experimental conditions: as described in the experimental section.
–
1
Figure 6. Cyclic voltammograms obtained with GCE in DMF containing 0.1 mol L of
–
1
–1
–1
–1
TFBTBA and 1.0 mmol L of MTM: 10 mV s (1); 50 mV s (2); 100 mV s (3); 500
–
1
–1
–1
mV s (4) and 1000 mV s (5); E = 0.0 V; E = 1.4 V; E = 0.0 V (A) and 10 mV s
0
λ
f
–
1
–1
–1
–1
(
1); 50 mV s (2); 100 mV s (3); 500 mV s (4) and 1000 mV s (5); E = 0.0 V; E
0 λ
=
0.7 V; E = 0.0 V (B)
f
Figure 7. iap /icp ratio as a function of scan rate (v). Applied potential window
1
restricted to the interval in which only the first electrochemical process occurs.

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–
1
Figure 8. Eap vs log v plot obtained in DMF containing 0.1 mol L TFBTBA and 1.0
1
–
1
2
mmol L MTM. (Ep = 0.030log v + 0.495, R = 0.975)
1
Figure 9. DMAA and MTM analytical curves obtained in PBS pH 7.4 (black line) and
–
1
DMF containing 0.1 mol L of TFBTBA (red line), respectively. DMAA solutions in
PB, pH = 7.4 (Black). Data obtained from square wave voltammograms in 10 to 100
–
1
µmol L range (average of five measurements).
2
ip(DMAA)/µA = 0.96[DMAA]µM – 0.45 (R = 0.998) and ip(MTM)/µA = 0.92[MTM] µM –
2
0
.59 (R = 0.998).
Scheme 1. The MTM hydrolysis and its generated products.
Scheme 2. Structure of the pyrazolones studied in this work. The abbreviations, as well
as, the letter on the molecule were maintained throughout the work.
Scheme 3. Schematic representation of the possible reaction pathways after the Eap1
process.
Scheme 4. Electrochemical oxidation mechanism proposed for dipyrone and antipyrine
molecules.
Table 1. Compilation of the results obtained from the cyclic voltammograms presented
in Figure 1.
Table 2. Peak width half height (w ) values obtained from the differential pulse
1
/2
voltammograms presented in Figure 4.
Table 3. Compilation of reversibility, number of electrons and pH-dependency of the
electrochemical oxidations processes for the pyrazolone derivatives.
Table 4. Analytical parameters obtained for MTM quantification in commercial
pharmaceutical samples using DMAA and MTM analytical curves.

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Table 1: Oxidation peak potentials obtained from the cyclic
voltammograms presented in Figure 1.
Process/ Molecule Eap / V
Eap / V
Eap / V
Eap3/ V
0
1
2
AA
-
-
-
1.25
1.22
1.24
1.25
1.27
4AA
0.552
-
-
MAA
0.245
-
-
DMAA
MTM
-
-
0.444
0.578
0.67
0.903

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Table 2: Peak width at half height (w ) obtained from the
1
/2
differential pulse voltammograms presented in Figure 4.
w1/2
AA
Eap / mV
Eap / mV
Eap / mV
Eap / mV
0
1
2
3
-
-
-
-
-
96±6
91±5
4AA
89±3
99±3
99±6
99±4
99±3
MAA
107 ± 7
110±7
102±5
107±6
DMAA
MTM
-
-
91±
3
89±4

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Table 3: Reversibility, number of the electrons and pH
dependency of the electrochemical oxidation processes for the
pyrazolone derivatives.
o
Process
Eap0
Molecule
4AA, MAA
DMAA, MTM
AA, 4AA, MAA,
MTM
Reversibility n of electrons pH dependency
No
Yes
No
1
1
1
Yes
No
No
Eap1
Eap2
Eap3
AA, 4AA, MAA,
MTM
No
1
No

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Table 4: Analytical parameters obtained for MTM Quantification
in pharmaceutical samples using DMAA and MTM analytical
curves
Analytical Curve
Limit of detection
Limit of quantification
-1
-1
(
µmol L )
(µmol L )
DMAA(PBS)
MTM (DMF)
Sample
1.94
6.48
2.97
9.91
Recovery - DMAA (%)
Recovery - MTM (%)
TM
Novalgina
95.0
96.5
86.2
87.6
Dipyrone
(Medley)
TM
Dorflex
98.0
89.0
95.2
TM
Neosaldina
105.0