Comparative spectroscopic and electrochemical study of nitroindazoles: 3-alcoxy, 3-hydroxy and 3-oxo derivatives.
ABSTRACT Cyclic voltammetry and electron spin resonance techniques were used in the investigation of novel 3-alkoxy- and 3-hydroxy-1-[omega-(dialkylamino)alkyl]-5-nitroindazole derivatives. A self-protonation process involving the protonation of the nitro group was observed. The reactivity of the nitro-anion radical for these derivatives with glutathione, a biological relevant thiol, was also studied by cyclic voltammetry. These studies demonstrated that glutathione could react with radical species from 5-nitroindazole system. Also we demonstrated that nitro-anion radicals show three different patterns of delocalization where the indazole 1-lateral chain does not have major influence.
Comparative spectroscopic and electrochemical study of nitroindazoles:
3-Alcoxy, 3-hydroxy and 3-oxo derivatives
Jorge Rodr´ ıgueza,b, Claudio Olea-Azara,∗, German Barrigaa,
Christian Folcha, Alejandra Gerpec, Hugo Cerecettoc,
Mercedes Gonz´ alezc
aDepartamento de Qu´ ımica Inorg´ anica y Anal´ ıtica, Facultad de Ciencias Qu´ ımicas y Farmac´ euticas,
Universidad de Chile, Chile
bDepartamento de Qu´ ımica, Facultad de Ciencias B´ asicas, Universidad Metropolitana de
Ciencias de la Educaci´ on, Chile
cLaboratorio de Qu´ ımica Org´ anica, Facultad de Qu´ ımica-Facultad de Ciencias,
Universidad de la Rep´ ublica, Uruguay
Cyclic voltammetry and electron spin resonance techniques were used in the investigation of novel 3-alkoxy- and 3-hydroxy-1-[?-
(dialkylamino)alkyl]-5-nitroindazole derivatives. A self-protonation process involving the protonation of the nitro group was observed. The
reactivity of the nitro-anion radical for these derivatives with glutathione, a biological relevant thiol, was also studied by cyclic voltammetry.
These studies demonstrated that glutathione could react with radical species from 5-nitroindazole system. Also we demonstrated that nitro-anion
radicals show three different patterns of delocalization where the indazole 1-lateral chain does not have major influence.
Keywords: 5-Nitroindazole; Cyclic voltammetry; ESR; Trypanosoma cruzi
American Trypanosomiasis, or Chagas’ disease, is one of
the most relevant endemic trypanosomiasis in Central and
South America. Chagas’ disease is caused by the protozoa Try-
panosoma cruzi (T. cruzi). Latest data from WHO indicates that
over 24 million of people, 8% of Latin-American population,
for Chagas’ disease is usually effective when it is given during
the acute stage of infection. No medication has been proven
to be effective once the disease has progressed to later stages.
Moreover, this pathology is treated with synthetic drugs such
as Nifurtimox®(Nfx) and Benznidazole®, two nitro-containing
heterocycles. The chemotherapy is still inadequate due to its
undesirable side effects, including cardiac and/or renal toxicity.
This explains the constant need for discovering and to inves-
∗Corresponding author. Tel.: +56 2 9782834; fax: +56 2 7370567.
E-mail address: email@example.com (C. Olea-Azar).
tigate new effective chemotherapeutic and chemoprophylactic
agents against T. cruzi [1,2]. In this sense, we have synthesized
a series of indazoles as potential anti-T. cruzi agents [3–5]. In
these studies, the 5-nitroindazole derivatives were identified as
good in vitro antiparasites, being compound 1 (Scheme 1) the
most selective trypanosome/mammal agent. Recently, efforts
directed to obtain a novel series of 3-alkoxy or 3-hydroxy-1-[?-
(dialkylamino)alkyl]-5-nitroindazole and evaluate its antiproto-
zoa properties have been performed. Two groups of compounds
have been prepared (5-NI, Scheme 1), the 3-alkoxy- and 3-
hydroxy-1-[?-(dialkylamino)alkyl]-5-nitroindazoles, series A,
and the corresponding indazole[1,2-a]1,2-diazepines, series B
Little information has been gathered related to the mecha-
nism of action of these compounds. However, the reduction of
the nitro group may be a key step in its biological activitiy, as is
observed for other nitro-heterocyclic systems. In this paper the
family of the 5-NI indazole derivatives was electrochemically
studied in aprotic solvent using cyclic voltammetry (CV) tech-
nique. The nitro-anion radical species were characterized using
J. Rodr´ ıguez et al. /
Scheme 1. Chemical structure of 5-nitroindazole derivatives.
the interaction between the radical species generated from 5-NI
and glutathione (GSH).
2. Materials and methods
to methods optimized in our previous works [5,6].
2.2. Cyclic voltammetry
Dimethylsulfoxide (DMSO) (spectroscopy grade) was
obtained from Aldrich. Tetrabutylammonium perchlorate
(TBAP), used as supporting electrolyte, was obtained from
Fluka. CV was carried out using a Metrohm 693VA instru-
ment with a 694VA Stand convertor and a 693VA Processor, in
DMSO (ca. 1.0×10−3molL−1), under a nitrogen atmosphere
electrode cell. A hanging mercury drop electrode was used as
and saturated calomel as the reference electrode.
2.3. ESR spectroscopy
ESR spectra were recorded in the X band (9.7GHz) using
a Bruker ECS 106 spectrometer with a rectangular cavity and
50kHz field modulation. The hyperfine splitting constants were
estimated to be accurate within 0.05G. The anion radicals were
generated by electrolytic reduction in situ using DMSO as sol-
vent and TBAP as supporting electrolyte. All experiments were
carried out at room temperature and under nitrogen atmosphere.
The ESR spectra were simulated using the program WINEPR
Simphonia 1.25 version.
2.4. Theoretical calculations
Density functional theory as implemented in the Spartan 04
 computational package was used to calculated and display
spin density maps. The compounds were built with standard
bond lengths and angles implemented in the program and the
J. Rodr´ ıguez et al. / 559
Fig. 1. Cyclic voltammograms of the isolated RNO2/RNO2•−couple of JNI 6 (1mM) derivative in aprotic medium (DMSO+0.1M TBAP) and at different sweep
rates (between 5000 and 100mVs−1). Insets: (a) current ratio vs. log(sweep rate); (b) cathodic peak current vs. log(sweep rate).
geometry of each molecule was fully optimized by applying
semiempirical AM1 method in gas phase from the most sta-
ble conformer obtained using molecular mechanics (MMFF)
methods. Then single point calculation using density func-
tional methodology (Becke’s three parameter exact exchange
functional, B3)  combined with gradient corrected correla-
tion functional of Lee–Yang–Parr (LYP)  of DFT method
(U)B3LYP/6–31G* and calculations of electronic properties at
3. Results and discussion
3.1. Cyclic voltammetry
In order to achieve the best experimental conditions that
warranty the nitro-anion radical stability, an aprotic medium
Under these conditions, all 5-NI derivatives displayed com-
parable voltammetric behaviour showing a very well-defined
derivatives JNI 2–6 showed a one electron reversible transfer-
ence process (peak Ic/Ia, around −1.0V, Fig. 1) corresponding
moiety, in R2substituent, is unable to protonate the free radical
under the experimental conditions used. From the study of the
(b), Fig. 1) showing that the one electron reversible transference
corresponds to diffusion controlled process without adsorption
the sweep rate increase up to 1.0 (inset (a), Fig. 1). This result
could indicate a typical variation of an ECi mechanism ,
showing values lower than 1.0 at low sweep rates and values
around 1.0 at higher sweep rates. In this sense, the electrochem-
ically generated nitro-anion radical could undergo two different
decay paths, disproportionation or dimerization . Table 1
lists the values of voltammetric cathodic and anodic peaks for
all the compounds studied. All derivatives exhibited more nega-
tive potential values than Nfx (−0.91V ) indicating a lesser
capacity to be reduced.
Fig. 2 shows the typical voltammogram of 3-hydroxy-5-
nitroindazole derivative JNI 1. Two reduction waves appeared,
one cathodic peaks Ic (around −1.1V) corresponding to nitro-
anion radical RNO2•−and a new wave at higher cathodic
potential peak (IIc/IIa, around −1.4V) corresponding to the
reduction of the anion –ORNO2 specie generated through a
self-protonation reactions (C1). This process corresponds to
an acid–base equilibrium in aprotic media, a typical behaviour
of self-protonation phenomenon displayed by nitro-compounds
with acidic moieties in their structures [4,15–17]. It is prob-
ably that the higher negative potential (couple IIc/IIa) of the
3-hydroxylate derivative corresponds to a diminish capacity to
accept electrons due to its negative charge:
HORNO2+ e → HORNO2•−
HORNO2•−+ HORNO2→ HORNO2•H +−ORNO2
−ORNO2+ e →−ORNO2•−
Characteristic CV parameters in DMSO vs. saturated calomel electrode (sweep
J. Rodr´ ıguez et al. /
Fig. 2. CV of JNI 1 derivative (1mM) in DMSO (0.1M TBAP) for different amounts of aqueous NaOH (0.1M), sweep rates 1Vs−1. Inset: current ratio (peak II)
vs. the amount of NaOH (0.1M) in ?L.
In order to obtain suitable conditions for the nitro-anion
radical from the 3-hydroxylate derivative and verify the self-
protonation mechanism proposed, we have worked in presence
of increasing amounts of aqueous NaOH (0.1M). Fig. 2
shows the typical voltammograms obtained for 3-hydroxy-5-
nitroindazole derivatives in the presence of different amounts
of base. The electroreduction wave Ic gradually disappears with
(Fig. 2). The calculated Ipa/Ipc ratio using the Nicholson and
NaOH for peak IIc/IIa in the case of 3-hydroxy-5-nitroindazole
derivative (inset Fig. 2). We confirm the mechanism ECErev
proposed for this 3-hydroxy-5-nitroindazole derivative given by
the increment in the Ipa/Ipc ratio toward the reversibility of its
3.2. Reactivity of the nitro-anion radical anion
electrochemically generated from 5-NI with GSH
In order to study the capacity of nitro-anion radical of 5-NI
to react with a natural antioxidant such as GSH, we studied by
CV the effect of the GSH concentration on the Ipa/Ipc ratio
or IIpa/IIpc ratio for the JNI 6 derivative. The concentration of
in buffer phosphate pH 7.4) to the medium until the complete
absence of the anodic peak. Fig. 3 shows the isolated couple of
the typical 5NI CV in DMSO in the absence and in the presence
of increasing amounts of GSH. This couple change when GSH
nificantly. In the case of 3-hydroxy-5-nitroindazole derivative,
JNI 1, a large increase in the 1-electron HORNO2reduction
step corresponding to the electroreduction of uncharged species
(E 1) and the absence of the return oxidation step are observed
when GSH was added to the medium. On the other hand, the
other cathodic and anodic peaks disappeared (data not shown),
which could indicate that the oxidation of GSH is faster than
5-nitroindazole derivative was previously treated with NaOH,
the addition of GSH to the medium produces a large increase
in the 1-electron –ORNO2reduction step corresponding to the
electroreduction of charged species (E 2). The absence of the
return oxidation step is also observed. This could indicate that
the GSH reacts with both radical species ((E 1) and (E 2)). The
radical detection at the studied concentrations [20–22]. These
results indicated that the electrochemically obtained nitro-anion
rial by the action of GSH. The effect is essentially catalytic,
since the nitro-voltammetric changes were virtually complete.
The species responsible for redox cycling were not identified,
but it is possible that the –S•radical (produced via the 1 elec-
tron oxidation of GSH) be the oxidizing agent for the RNO2−•.
The fact that in biological medium high thiol levels is present,
this kind of process could take place in the parasite explain-
Fig. 3. CV of JNI 6 (1mM) in DMSO (0.1M TBAP) for different amount of
GSH (0.1M in buffer phosphate pH 7.4), sweep rates 1Vs−1.
J. Rodr´ ıguez et al. / 561
Hyperfine coupling constants and g value of the simulated 5-NI free radical
ing the observed biological activity for 5-NI derivatives against
3.3. Electron spin resonance
5-NI free radicals characterized by ESR were prepared in
situ by electrochemical reductions in DMSO by applying the
potential corresponding to peak Ic or IIc obtained from the CV
experiments. The interpretation of the ESR by means of a sim-
ulation process confirmed the stabilities of these radical species
Fig. 4. ESR experimental spectrum of the dianion radical of compound JNI 1
in DMSO and computer simulation of the same spectrum. Spectrometer condi-
tions: microwave frequency, 9.72GHz; microwave power, 20mW; modulation
amplitude, 0.98G; receiver gain, 59db. Spectrum was simulated using the fol-
hyperfine constants are included in Table 2.
Fig. 5. ESR experimental spectrum of the anion radical of compound JNI 2
in DMSO and computer simulation of the same spectrum. Spectrometer condi-
tions: microwave frequency, 9.70GHz; microwave power, 20mW; modulation
amplitude, 0.98G; receiver gain, 30db. Spectrum was simulated using the fol-
lowing parameters: line width=0.3G, Lorentzian/Gaussian ratio=0.6001 and
hyperfine constants are included in Table 2.
of the spectra was made by using hyperfine coupling constants
ulation amplitude and Lorentzian/Gaussian component until the
resulting spectra reached the greatest similarity with the exper-
imental ones. Table 2 presents the hfccs obtained for JNI 1,
Fig. 6. Spin density surfaces (blue, isovalue 0.003, AM1/6–31G*) for com-
pounds JNI 1, JNI 2 and JNI 6. The arrows show N1-indazole atoms spin
density. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of the article.)
J. Rodr´ ıguez et al. /
Fig. 7. ESR experimental spectrum of the anion radical of compound JNI 6 in
microwave frequency, 9.75GHz; microwave power, 20mW; modulation ampli-
tude, 0.98G; receiver gain, 30db. Spectrum was simulated using the following
parameters: line width=0.95G, Lorentzian/Gaussian ratio=0.6 and hyperfine
constants are included in Table 2.
JNI 3 and JNI 4 free radicals. The simulated spectrum of JNI
1 radical (Fig. 4) corresponds to one triplet of nitrogen from
nitro group and three doublets from H-4, H-6 and H-7. Fig. 5
displays a simulated spectrum for JNI 2, in terms of two triplets
corresponding to the nitrogen atoms of the nitro group and the
N1 indazole and three doublets assigned to nuclei H-4, H-6 and
H-7. This hyperfine pattern shows that only one nitrogen of the
cate that the nitrogen N-1 shows a larger electronic density than
the nitrogen N-2 adjacent to carbonyl group. The spin maps are
completely in agreement with these results (Fig. 6). JNI 4 free
radical spectra was simulated in terms of one triplet assigned
to nitro-nuclei N and three doublets assigned to nuclei H-4, H-
6 and H-7 of the indazole moiety. Derivatives JNI 4, 5 and 6
showed the same pattern (Fig. 7). The hyperfine coupling con-
stants (hfccs) obtained from three pattern observed in the ESR
study of the family of the 5-NI are shown in Table 2.
Our CV results show that 3-alkoxy- or 3-hydroxy-1-[?-
(dialkylamino)alkyl]-5-nitroindazole derivatives are electro-
chemically reduced via formation of a nitro-anion radical. The
reduction mechanism depends on the acidic moieties in their
structures. A self-protonation process involving the protonation
of the nitro group was also observed. The reduction mecha-
nism proposed to 3-hydroxy-5-nitroindazole derivatives is an
ECErev corresponding to the generation of the nitro-anion rad-
ical from uncharged species, followed by a self-protonation
process from an hydroxyl moiety and the generation of a nitro-
anion radical from negative charged species. On the other hand,
GSH was capable of acting as an oxidizing agent for the 5-NI,
regenerating the starting material from the nitro-anion radi-
cal. The oxidizing effect of GSH is supported by the parallel
decrease of the anodic peak current and the increase of the
wave from uncharged species with the addition of GSH. Stable
free radicals were generated using electrochemical reductions
at potentials corresponding to the first wave and characterized
by ESR spectroscopy. The 5-NI compounds studied showed
three different patterns of spectrum depending on its struc-
tural characteristics. All these results indicated that the lateral
chain does not have major influence on the electron delocal-
ization. The ESR spectra pattern was similar for compounds
JNI 4 and JNI 5 which correspond to 3-alkoxyindazole and 3-
benzyloxyindazoles derivatives. In the case of compounds JNI
2 and JNI 3, the different ESR pattern respect to the other
5-NI derivatives could be explained in terms of the molecu-
lar structure due to that they show a structure which facilitates
the delocalization of the unpaired electron in the heterocyclic
system. This is reflected in the major hyperfine coupling con-
This research was supported by FONDECYT 1071068 grant
(Chile), MECESUP UMC-0204 grant (Chile), RTPD NET-
WORK,Comisi´ onSectorialdeInvestigaci´ onCient´ ıfica–UdelaR
CSIC 16/07-08 grant (Uruguay) and PEDECIBA (Uruguay).
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