Synthesis and evaluation of new heteroaryl nitrones with spin trap properties

Article abstract of DOI:10.1039/d0ra07720h

A new series of heteroaryl nitrones were synthesized and evaluated as free radical traps due to the results showed in our previous report. The physicochemical characterization of these new nitrones by electron spin resonance (ESR) demonstrated their high capability to trap and stabilize different atom centered free radicals generated by the Fenton reaction. Additionally, we intensely studied them in terms of their physicochemical properties. Kinetic studies, including the use of a method based on competition and the hydroxyl adduct decay, gave the corresponding rate constants and half-lives at the physiological pH of these newly synthesized nitrones. New nitrones derived from quinoxaline 1,4-dioxide heterocycles were more suitable than DMPO to trap hydroxyl free radicals with a half-life longer than two hours. We explain some of the results using computational chemistry through density functional theory (DFT).

Full text of DOI:10.1039/d0ra07720h

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Synthesis and evaluation of new heteroaryl nitrones
with spin trap properties†
Cite this: RSC Adv., 2020, 10, 40127
a
bc
d
C. Olea-Azar,e
*
G. Barriga-Gonzalez,
C. Aliaga,
E. Chamorro,
´
E. Norambuena,a W. Porcal,f M. Gonzalezf and H. Cerecetto
´
f
A new series of heteroaryl nitrones were synthesized and evaluated as free radical traps due to the results
showed in our previous report. The physicochemical characterization of these new nitrones by electron
spin resonance (ESR) demonstrated their high capability to trap and stabilize different atom centered free
radicals generated by the Fenton reaction. Additionally, we intensely studied them in terms of their
physicochemical properties. Kinetic studies, including the use of a method based on competition and
the hydroxyl adduct decay, gave the corresponding rate constants and half-lives at the physiological pH
of these newly synthesized nitrones. New nitrones derived from quinoxaline 1,4-dioxide heterocycles
were more suitable than DMPO to trap hydroxyl free radicals with a half-life longer than two hours. We
explain some of the results using computational chemistry through density functional theory (DFT).
Received 9th September 2020
Accepted 9th October 2020
DOI: 10.1039/d0ra07720h
rsc.li/rsc-advances
attack or other degradation reactions, and low stability of the
generated spin adduct. Some new spin traps with better trap-
ping abilities like BMPO, EMPO, and DEPMPO (Fig. 1B)9,10 have
been developed. However, despite the efforts, there are still
limitations in their employment. In the past years, we found
signicant advances in the development of new free radical spin
traps. Some new free radical spin traps, such as isoindole-based
nitrones, have shown they can trap the nitric oxide radical
(TMINO11–13 and 3-TF-TMINO14).
We have described a new series of heteroaryl nitrones in
previous work, bearing furoxanyl and thiadiazolyl moieties with
the capability to trap and stabilize oxygen-, carbon-, sulfur-, and
nitrogen-centered free radicals.15 The spin trapping properties
Introduction
Due to natural metabolic activity in the human body, some of
the essential by-products found are reactive oxygen and
nitrogen species,1,2 which mediate various physiological oxida-
tive processes. The ability to reliably identify and quantify such
short-lived oxidant species is critical to determine their role in
human disease. In the past three decades, spin trapping, in
conjunction with electron spin resonance spectrometry (ESR),
has proven to be an essential experimental technique to
accomplish this goal. In the last years, the spin-trapping tech-
nique was used to deeply study the behavior and role played by
oxygen and nitrogen centered free radicals.3,4 The most widely
used spin trap has been the non-cyclic nitrones a-phenyl-N-t-
butylnitrone and a-4-pyridyl-1-oxide-N-t-butylnitrone (PBN and
POBN, respectively, Fig. 1A) and the cyclic one 5,5-dime-
thylpyrroline-N-oxide (DMPO, Fig. 1B).5–8 In general terms, these
available spin traps present many disadvantages such as low
water solubility (in the case of PBN), sensitivity to nucleophilic
a
´
´
Departamento de Quımica, Facultad de Ciencias Basicas, Universidad Metropolitana
˜
´
´
˜
de Ciencias de la Educacion, Av. Jose Pedro Alessandri 774, Nunoa, Santiago, Chile.
E-mail: german.barriga@umce.cl
b
´
´
Facultad de Quımica y Biologıa, Universidad de Santiago de Chile, Av. Bernardo
O'Higgins 3363, Santiago, Chile
c
´
Centro para el Desarrollo de la Nanociencia y la Nanotecnologıa, CEDENNA, Chile
d
´
Departamento de Ciencias Quımicas, Facultad de Ciencias Exactas, Universidad
´
´
Andres Bello, Avenida Republica 275, 8370146 Santiago, Chile
e
´
´
´
´
Departamento de Quımica Inorganica y Analıtica, Facultad de Ciencias Quımicas y
´
Farmaceuticas, Universidad de Chile, Santiago, Chile
f
´
´
´
´
Grupo de Quımica Organica Medicinal, Laboratorio de Quımica Organica, Facultad de
Fig. 1 Structures of most used nitrones as spin trap agents today. (A)
PBN and POBN, (B) DMPO, BMPO, EMPO, and DEPMPO, and (C)
furoxanyl nitrone (FxBN) described by us.
´
´
Ciencias/Facultad de Quımica, Universidad de la Republica, Montevideo, Uruguay
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra07720h
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internal standard. Shimadzu DI-2010 was used to determine the
mass spectra.
2-Formylquinoxaline 1,4-di-N-oxide (V). A mixture of SeO2
(0.09 g, 0.80 mmol) in dioxane (5 mL)/H2O (0.1 mL) was heated
at reux. Then 2-methylquinoxaline 1,4-di-N-oxide (IV) (0.1 g,
0.57 mmol) was added, and the nal mixture was stirred for two
hours. The resulting dispersion was then ltered through
a short pad of Celite, the organic phase concentrated in vacuo,
and the residue puried by column chromatography (SiO2,
AcOEt), yielding derivative V as a yellow solid (100 mg, 86%). 1H-
NMR (CDCl3) d: 8.00 (m, 2H), 8.64 (s, 1H), 8.69 (m, 2H), 10.65 (s,
1H).
Fig. 2 Structures of the heteroaryl nitrones described in the present
work.
of the nitrones depends in great part on the connectivity and the
nature of substituents on the nitrone group. As part of our
ongoing investigations in the development of new nitrone-free
radical trapping, we set out to design and synthesizer new
heteroarylnitrones starting from suitable precursors available
in our heterocyclic library. Notably, the 4-furoxanyl nitrone
FxBN (Fig. 1C) showed good solubility in aqueous solution, and
FxBN adducts formed with hydroxyl and superoxide radicals
exhibited distinct and characteristic ESR spectral patterns.
Herein, we study and assay the spin trapping capabilities of
three new heteroaryl nitrones (nitrone 1, nitrone 2, and nitrone
3, Fig. 2) against oxygen-, carbon-, sulfur-, and nitrogen-
centered free radicals, i.e., hydroxyl-, superoxide-, 1-hydrox-
yethyl-, methyl-, sulfur trioxide anion-, and azidyl-free radicals.
Thus, kinetic constants and decay rates were obtained for the
corresponding hydroxyl adduct at pH 7.4 in aqueous solution.
Additionally, we performed assays for the trap of nitric oxide-
free radicals and the behavior of these heteroaryl nitrones in
biological models.
Synthetic procedure of a-heteroaryl-N-alkylnitrone deriva-
tives (1–3). A mixture of the corresponding aldehyde (1.0
equivalent), N-alkylhydroxylamine hydrochloride or the corre-
sponding acetate (1.2 equivalent), and sodium bicarbonate (1.2
equivalent) in absolute ethanol (5 mL mmolꢁ1) as solvent was
ꢂ
heated at 60 C until the carbonyl compound was not present
(checked by TLC). The solvent was removed in vacuo, and the
residue was puried by column chromatography (SiO2, mixtures
of petroleum ether/EtOAc).
a(Z)-(3-Methylfuroxan-4-yl)-N-cyclohexylnitrone (1). Pale yellow
solid (60%); mp 118.0–120.0 ꢂC. Found: C, 52.9; H, 6.5; N, 18.6.
C
10H15N3O3 requires C, 53.3; H, 6.7; N, 18.7. 1H-NMR (CDCl3) d:
1.21 (m, 1H, CH), 1.40 (m, 2H, CH2), 1.73 (m, 1H, CH), 1.95 (m,
4H, CH2), 2.15 (m, 2H, CH2), 2.41 (s, 3H, CH3), 4.01 (m, 1H, CH–
N), 7.57 (s, 1H, CH]N). 13C-NMR (CDCl3) d: 10.3 (CH3), 24.9
(cyclohexyl-CH2), 25.1 (cyclohexyl-CH2), 31.2 (cyclohexyl-CH2),
76.1 (cyclohexyl-CH), 112.5 (furoxanyl-C]N/O), 119.8 (nitrone-
CH]N/O), 150.6 (furoxanyl-C]N). ESI-MS, m/z: 248 (M+c +
Na), 226 (M+c + H), 144, 100.
Experimental
Materials
a(Z)-(1,4-Dioxide-2,3-dimethylquinoxalin-6-yl)-N-t-butylnitrone
ꢂ
(2). Yellow solid (35%); mp 209.4–212.3 C. Found: C, 62.0; H,
The newly developed heteroaryl nitrones studied as spin traps
are summarized in Fig. 2. 5,5-Dimethyl-1-pyrroline-N-oxide
(DMPO, Fig. 1), dimethylsulfoxide, anhydrous sodium sulte,
monobasic potassium phosphate, sodium hydroxide, N-methyl-
D-glucamine (MGD), diethylenetriaminepentaacetic acid,
xanthine, and xanthine oxidase were purchased from Sigma-
Aldrich. Sodium azide, hydrogen peroxide aqueous solution
(30%), anhydrous ethanol, dibasic sodium phosphate hepta-
hydrate, methanol, carbon disulde, and sodium nitrite were
purchased from Merck. Ferrous ammonium sulfate hexahy-
drate was purchased from Mallinckrodt Baker.
1
6.7; N, 14.3. C15H19N3O3 requires C, 62.3; H, 6.6; N, 14.5. H-
NMR (CDCl3) d: 1.67 (s, 9H, (CH3)3C), 2.75 (s, 6H, CH3), 7.85
(s, 1H, quinoxalinyl-C5H), 8.62 (d, 1H, J ¼ 8.8 Hz, quinoxalinyl-
C7H), 9.03 (d, 1H, J ¼ 8.8 Hz, quinoxalinyl-C8H), 9.13 (s, 1H,
CH]N). 13C-NMR (CDCl3) d: 14.7 (CH3-quinoxalinyl), 28.4
((CH3)3C), 72.3 ((CH3)3C), 119.7 (nitrone-CH]N/O), 120.4
(quinoxalinyl-C]N/O), 128.2 (quinoxalinyl 7-C), 130.5 (qui-
noxalinyl 6-C), 130.6 (quinoxalinyl 5-C), 133.9 (quinoxalinyl 8-C),
136.3 (quinoxalinyl quaternary-C), 141.6 (quinoxalinyl quater-
nary-C). EI-MS m/z (%): 289 (M+c, 83), 273 (21), 257 (4), 233 (100),
216 (86), 199 (33).
a(Z)-(1,4-Dioxidequinoxalin-2-yl)-N-t-butylnitrone (3). Yellow
solid (95%); mp 191.8–193.5 ꢂC. Found: C, 59.6; H, 5.9; N, 15.8.
Synthesis
Before using, all solvents were distilled, and experimental
C
10H15N3O3 requires C, 59.8; H, 5.8; N, 16.1. 1H-NMR (CDCl3) d:
conditions for analytical TLC and column chromatography 1.67 (s, 9H, (CH3)3C), 7.86 (m, 2H, quinoxalinyl-C6H and qui-
purication was used according to previous work. The electro- noxalinyl-C7H), 8.61 (m, 2H, quinoxalinyl-C5H and quinoxalinyl-
thermal 9100 apparatus was used to determining melting C8H), 8.70 (s, 1H, quinoxalinyl-C3H), 10.30 (s, 1H, CH]N). 13C-
points, and they are uncorrected. Microanalyses were per- NMR (CDCl3) d: 28.3 ((CH3)3C), 74.4 ((CH3)3C), 119.8 (nitrone-
formed on a Fisons EA 1108 CHNS-O instrument and were CH]N/O), 120.3 (quinoxalinyl-C]N/O), 120.6 (quinoxalinyl-
within ꢀ0.4% of the calculated compositions. Bruker DPX-400 C]N/O), 129.9 (quinoxalinyl 6-C and quinoxalinyl 7-C), 131.6
spectrometer was used to record NMR spectra. The assign- (quinoxalinyl 8-C), 132.2 (quinoxalinyl 5-C), 135.8 (quinoxalinyl
ment of chemical shis is based on standard NMR experiments quaternary-C), 137.6 (quinoxalinyl quaternary-C). EI-MS m/z (%):
(1H, 13C, 1H-COSY, HSQC, HMBC, and NOE). The chemical shi 261 (M+c, 23), 245 (13), 205 (19), 189 (37), 172 (35), 129 (26), 105
values are expressed in ppm relative to tetramethylsilane as an (24), 57 (100).
40128 | RSC Adv., 2020, 10, 40127–40135
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concentration of the generated free radical species. The decay
rate was also calculated from the double integral of the ESR
spectra. Therefore, it may differ from the rate calculated from
the concentration. The relative spin trapping rate for hydroxyl
radical was determined using a competitive-trapping method.21
In this method, two spin traps are mixed (DMPO and nitrones
studied), and the ESR signal intensities are compared using the
value of the double integral of the recorded spectra. The reac-
tion scheme for spin trapping in the competition method for
the hydroxyl radical with both spin traps compounds is:
Free radical generation and ESR measurements
Radicals were generated by employing a Fenton type reaction.
Aqueous solutions of Fe2+ (1.0 mM of ferrous ammonium
sulfate hexahydrate) and hydrogen peroxide (1%) in phosphate
buffer (pH 7.4) was loaded into the ESR cell. Thus, for 1-
hydroxyethyl, methyl, sulfur trioxide anion, and azidyl radicals,
we employed as generation source the following reactants:
anhydrous ethanol (200 mM), anhydrous dimethylsulfoxide
(200 mM), anhydrous sodium sulte (200 mM), and anhydrous
sodium azide (200 mM), respectively. POBN (50 mM) and DMPO
(50 mM) were used as a control to check the generation of 1-
hydroxyethyl, hydroxyl, and superoxide free radical,
respectively.16
hn
H2O2 2OH
ꢃ
!
The xanthine/xanthine oxidase couple was used for super-
oxide anion radical, and the detection procedure was modied
from literature17 with the following conditions: the assayed
nitrone (50 mM) was incubated with xanthine (0.4 mM) and
xanthine oxidase (0.06 units per mL) in phosphate-buffered
saline (PBS, 100 mM, pH 7.4, containing 1.0 mM DTPA). In
order to verify the proper generation of superoxide anion free
radical, DMPO was used as spin trap control.
The following procedure was used for nitric oxide radical
generation: MDG (50 mM),18 Fe2+ (10 mM), NaNO2 (100 mM),
and the corresponding nitrone (50 mM). In order to verify the
proper generation of nitric oxide free radical, MGD–Fe2+
complex was used as spin trap control.
k1
!
ꢃ
ꢃ
ꢃ
DMPO þ OH
DMPO ꢁ OH
k2
!
ꢃ
Nitrone þ OH
nitrone ꢁ OH
First, we assumed rst-order rates for the formation of the
two spin adducts, and the intensities found can be related as
follows:
IDMPO
k1DMPO
¼
Initrone
k2nitrone
The value of kDMPO has been previously determined.22
Bruker ECS 106 ESR (X-band) spectrometer equipped with
a rectangular cavity was used to record the ESR spectra at room
temperature. The spectrometer conditions were microwave
frequency, 9.80 GHz; modulation frequency, 50 kHz; microwave
power, 20 mW; modulation amplitude, 0.9 G; time constant,
81.92 ms; the number of scans, 12.
Spin trapping in biological systems studies
The spin trap capability of the new nitrones was assessed in
mouse mammary adenocarcinoma TA3 cell line exposed to 5-
nitroindazole, a free radical producer agent. ESR spectra were
obtained using the following conditions: studied nitrones (50
mM), mouse mammary adenocarcinoma TA3 cells (4.0 mg
mLꢁ1), and 5-nitroindazole (25 mM), in phosphate buffer, pH
7.4. The mixture was transferred to a 100 mL capillary. A Bruker
ECS 106 spectrometer (rectangular cavity and 50 kHz eld
modulation) was used to record ESR spectra at the X band (9.80
GHz). Aer 12 scans, all the spectra were registered on the same
scale.23
ESR simulation
The NIEHS WinSim soware was used for the ESR spectra
simulation.19 The simulations of the spectra give us a systematic
study based on the dependencies of certain spectral features
with some magnetic parameters and accurate parameter
extraction from obtained experimental data.
Computational calculations
Rate constant and half-life estimation for cnitrone–OH adduct
In order to explain results obtained in the trapping of the other
radicals using these new heteroaryl nitrones, we used Density
Functional Theory (DFT)24,25 calculations using Jaguar so-
ware.26 We used the B3LYP/6-31++G** level for optimization of
the geometries and to obtain the spin density isosurfaces.
Solvent effects were investigated using the polarizable
continuum model (PCM).27–29
The measure of the decay constant for the nitrone–hydroxyl
adduct used the same procedure described above for the
generation of hydroxyl radical via Fenton reaction. Following
the decrease of an appropriate ESR line was used to monitoring
the decay of the spin adduct.20,21 Pseudo-rst-order decay of the
spin adduct was measured by continuously recording the ESR
signal intensity. Although the decay of a spin adduct should
follow second-order kinetics, under the experimental condi-
tions used in the present work, it can be treated as pseudo-rst-
Results and discussion
order due to the spin trap concentration used. This was in The previous results with the furoxanyl nitrone FxBN (Fig. 1C)
a signicant excess compared to the radical concentration and conduct us to use it as a basic template to produce new analogs
making us consider the concentration of radical trapping with modied physicochemical properties. In this sense, in
constant throughout the experiment. The only variable that order to study the stability of the generated spin adduct, we
changes through the duration of the experiment was the designed and synthesized nitrone 1 (Fig. 2), where FxBN t-butyl
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Scheme 1 Synthesis of heteroarylnitrones 1–3. Experimental condi-
tions are: (A) (1) NaNO2/AcOH, (2) C6H11–NHOH$HCl/NaHCO3/EtOH/
60 ꢂC. (B) (3) (i) butanone/NH3 (g), (ii) HCl (c), (4) (CH3)3–NHOH$AcOH/
NaHCO3/EtOH/60 ꢂC. (C) (5) SeO2/dioxane/H2O/reflux, (6) (CH3)3C–
NHOH$HAc/NaHCO3/EtOH/60 ꢂC.
Fig. 3 (A) The left spectrum corresponds to the trap of 1-hydroxyethyl
radical, nitrone 1 was used as a spin trap. The center spectrum showed
the hyperfine splitting pattern for the trap of sulfur trioxide anion
radical when nitrone 2 was used. It is possible to observe some minor
artifacts in the spectra. The right spectrum is nitrone 3 trapping azidyl
radical. (B) Experimental spectra are shown in blue, and the corre-
sponding simulations are remarked with red color. From left to right,
simulations of the trap of methyl, hydroxyl, and azidyl radical with
nitrones 1, 2, and 3, respectively.
moiety was substituted by a cyclohexyl group. On the other
hand, due to FxBN possesses a cyclic nitrone group in the
furoxanyl ring, we aimed to analyze the effect of other nitrone
containing cyclic N-oxides. To do this, we selected the well-
known quinoxaline 1,4-dioxide heterocycle, which involves
two cyclic-nitrone moieties, and additionally, we selected two
different positions for the non-cyclic extra nitrone (nitrone 2,
and nitrone 3, Fig. 2). The different non-cyclic nitrone positions
and substituents on the quinoxaline nucleus could modify the
physicochemical properties of these compounds.
Each of the nitrones tested gave an ESR-detectable spin
adduct with other radicals (Fig. 3A and B).
The new nitrones were synthesized through the condensa-
tion between heteroaromatic aldehydes and N-t-butylhydroxyl-
amine or N-cyclohexylhydroxylamine with moderate to excellent
yields (Scheme 1).30
The obtained spin adducts spectrum was composed as
follows: for hydroxyl and 1-hydroxyethyl radicals, the pattern
obtained was composed of six-line (triplet of doublet) due to
coupling of the unpaired electron with nitrogen and hydrogen
at a-carbon, except for nitrone 1, which the obtained pattern
was composed by nine lines (triplet of triplet, Fig. 3A) probably
due to the extra hydrogen located in the cyclohexyl moiety. In
order to explain this different behavior, we proceeded to
perform the corresponding computational calculations using
DFT level, explaining the hyperne pattern found. From the
analysis of the spectrum, it was obtained that a second H atom
with a smaller hyperne coupling constant would explain the
observed hyperne pattern. The spin density reveals that this is
The formylfuroxan I was obtained from crotonaldehyde
through reaction with NaNO2 in AcOH by intramolecular cycli-
zation (Scheme 1A).30 We employed the synthetic route showed in
Scheme 1B to obtain the desired formylquinoxaline 1,4-dioxide
III. Beirut reaction of bezofuroxan II with butanone in the pres-
ence of NH3 (g) gave the compound III in good yield.31 Using the
Beirut reaction, the 2-methylquinoxaline 1,4-di-N-oxide interme-
diate IV (Scheme 1C) was synthesized, from benzofuroxan react-
ing with acetone in the presence of pyrrolidine.32 Then we
proceeded to the oxidation of the methyl group in position 2 of
the quinoxaline ring, using SeO2 as the oxidant agent in order to
obtain the carboxaldehyde derivative V. Finally, the desired het-
eroaryl nitrones, 1–3, were obtained for the reaction between the
corresponding aldehydes I, III, V and N-substituted hydroxyl-
amine in the presence of NaHCO3 (Scheme 1).
Spin trapping assays
Firstly, the spin trapping properties of these new nitrones were
investigated against different centered free radicals (O, C, N,
and S atoms, respectively). The radicals generated were
hydroxyl, 1-hydroxyethyl, azidyl, and sulfur trioxide anion. All
radicals mentioned above were generated through Fenton's
reagents, using aqueous solutions of Fe2+ and hydrogen
peroxide (1%), ethanol, sodium azide, or sodium sulte,
respectively.
Fig. 4 Spin density isosurface for cnitrone 1–OH spin adduct using
DFT calculus. The arrows indicate the atoms in which the unpaired
electron is delocalized and forms the hyperfine pattern are observed
experimentally.
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Table 2 The half-life for nitrones 1, 2, and 3, PBN, DMPO, and FxBN
with hydroxyl radical at pH 7.4.
delocalized to a part of the cyclohexyl group. Fig. 4 shows the
atoms contributing to the hyperne pattern found, conrming
that this substituent group inuences the spectrum pattern.
The ESR spectrum when methyl radical was trapped was
composed of a triplet of doublet due to coupling with a nitrogen
atom (triplet) and b-hydrogen atom (doublet) at the a-carbon
atom, except for nitrone 1 (Fig. 3B). The ESR spectrum for
nitrone 1 trapping methyl radical was composed again by nine
lines. The spin adducts with the azidyl radical exhibit twelve-
line (Fig. 3A and B) attributed to a triplet of triplet of
doublets. This pattern is explained due to two different nitrogen
atoms, one atom from the nitrone group and the other atom
from the azidyl radical. To complete the pattern explanation, we
considered the contribution of the b-hydrogen atom to this
splitting. For nitrone 1, the intensity of the signal is weak, and
the spectrum is not well resolved. Finally, the spin trapping
properties of these new nitrones were investigated against
sulfur-centered free radical produced by sulfur trioxide anion
free radical. The spin adducts from these nitrones showed
symmetrical patterns in all the cases. The ESR spectrum for
nitrone 1 was composed of nine lines (triplet of triplet). For
nitrones 2 and 3, the hyperne pattern is similar, and it can be
observed in the value of the hyperne splitting constant
(Fig. 3A). Fig. 3B showed the ESR simulations for all data
correlated well with experimental ESR spectra.
Half-life
Spin adduct
(s)
cDMPO–OH
cPBN–OH
cFxBN–OH
cNitrone 1–OH
cNitrone 2–OH
cNitrone 3–OH
3300
ꢄ38
7567
3398
8923
7270
Determination of spin adduct stability and the decay rate
constant for cnitrone–OH spin adducts
Regardless of this, all nitrones under study showed a behavior
as spin traps. To get insight into the physicochemical properties
of cnitrone–OH spin adducts, stability, and kinetic studies in
PBS (pH ¼ 7.4) were performed. The stability of spin adducts
was studied through the monitoring of the disappearance of the
ESR signal intensity of cnitrone–OH spin adducts (see example
in Fig. 5a).
It could be attributed to a solvolysis process as the mecha-
nism of decay.7,33,34 The half-life of cnitrone–OH spin adduct was
determined, assuming that the decay is of rst-order. Both
nitrones 2 and 3 showed excellent stability with a t1/2 more than
100-fold higher the value for cDMPO–OH adduct3 (Table 2).
We also included the values for the previously reported FxBN
nitrone15
Until this stage of experiments, we discussed some aspects
concerning the structural characteristics of these new nitrones.
First, unlike that, it was observed in our previous work furoxanyl
substituent did not exert an excellent stabilizing effect in the
trapped radicals as it happens with the nitrone 1 (compare t1/2
for cFxBN–OH and cnitrone 1–OH in Table 2). However, the use
of a cyclohexyl substituent gives us the possibility to obtain
Table 1 resumes the hyperne constants coupling for the
different generated spin adducts.
In the case of nitrones 2 and 3, the nitrogen hyperne
constants between the trapping of the hydroxyl, methyl, 1-
hydroxyethyl, and sulte radical have the same values. For the
hydrogen atom, these hyperne constants showed a slight
difference. It can be appreciated that nitrone 1 has very different
hyperne coupling constants compared with nitrones 2 and 3.
Among the other radicals trapped, the hyperne splitting
constant showed similar values for nitrogen atom and the same
behavior for the two hydrogen atoms (except for azidyl radical).
Table 1 Hyperfine constant coupling values of the different nitrones–radicals spin adducts and the corresponded g value
CH3ꢃ CHOH
cN3
cOH
cSO3
ꢁ
cCH3
aN
aH
aH
aN
aH
aH
aN
aN
aH
aN
aH
aH
aN
aH
aH
14.7
5.0
4.1
14.7
5.1
3.1
4.0
15.0
5.5
3.9
14.8
5.1
3.9
14.6
5.2
4.2
g value
g value
2.00925
15.1
2.00919
15.5
2.01435
14.3
2.01119
15.1
2.01135
15.0
2.8
2.2
nsa
ns
ns
ns
2.1
2.3
1.6
2.5
2.8
2.2
ns
ns
1.9
2.0
ns
ns
2.00953
2.00702
2.00841
2.00879
2.00885
14.4
14.4
2.1
14.4
14.4
14.5
g value
2.00829
2.00863
2.00840
2.00846
2.00834
a
No signal.
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Fig. 6 ESR spectra obtained after the generation of hydroxyl radical
using Fenton reaction, cDMPO–OH spin adduct, was marked with red
dots. (a) Nitrone 3 competitive spectrum obtained versus DMPO
trapping hydroxyl radical. (b) Nitrone 1 competitive spectrum obtained
versus DMPO for the same radical mentioned above.
Fig. 7 Hyperfine patterns for the trapping of nitrogen dioxide radical
(cNO2). (a) Nitrone 2 spectrum of cNO2 radical trapping process, in blue
experimental results and in red the corresponding simulation. (b) The
spectrum for FxBN trapping cNO2 radical process, in blue experimental
results and in red the corresponding simulation.
Fig. 5 (a) The time course of the ESR signal intensity for cnitrone 2–
OH adduct, in PBS (pH ¼ 7.4). The decay agrees with a first-order
reaction. (b) The time course of the ESR signal intensity is expressed as
the natural logarithm of intensity for cnitrone 2–OH adduct, con-
firming a process of first-order decay.
previously reported nitrone FxBN and DMPO (Table 3), being for
DMPO k1 ¼ 3.4 ꢅ 109 dm3 molꢁ1 sꢁ1
.
35
Table 3 Comparison and relationship of the constants for the trapping
of hydroxyl radical. DMPO was used as a pattern
It was possible to observe that nitrone 2 and 3 exhibited the
fastest rate in trapping hydroxyl radical when was compared
with DMPO and the previously reported FxBN (see example in
Fig. 7a and results in Table 3). However, when the same kind of
competitive experiment was performed with nitrone 1, a slower
trapping process compared to DMPO occurred (Fig. 6b and
Table 3).
Nitrone
kST (dm3 molꢁ1 sꢁ1
)
k2/k1
DMPO
FxBN
Nitrone 1
Nitrone 2
Nitrone 3
3.4 ꢅ 109
1.22 ꢅ 1010
1.17 ꢅ 109
1.70 ꢅ 1010
1.61 ꢅ 1010
1.00
3.59
0.34
5.00
4.74
It could be correlated with the different structural moieties
with the different reactivity and stability between these
a hyperne pattern. Although it was similar in all cases, it was
more sensitive to changes in the hyperne coupling constants
compared with t-butyl substituent used in the nitrones 2 and 3.
Instead, the usage of a quinoxalinyl substituent conferred an
essential aid in stabilizing the other radicals trapped (compare
t1/2 for cFxBN–OH and cnitrone 2–OH in Table 2). Probably this
substituent imparts a planar structure that helps the electronic
delocalization and the consequent stabilization of the corre-
sponding spin adduct.
Competitive studies for cOH-trapping
We determined the ratio of constants through a competitive
Fig. 8 Spin density isosurface for cnitrone 2–NO spin adduct using
study, (k2/k1) of hydroxyl radical for the studied nitrones, the DFT.
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Table 4 The hyperfine coupling constant for the trapping of nitrogen
dioxide radicals
cNO2
aN
aH
Nitrone 1
Nitrone 2
Nitrone 3
FxBN
ns
ns
15.0
15.0
15.0
2.6
2.0
2.3
Fig. 10 ESR spectra for the cnitrone–OOH adducts were obtained
with nitrone 2 (left) and nitrone 3 (right).
nitrones36 claiming the relevance of the quinoxaline 1,4-dioxide
Table 5 Hyperfine splitting for the trap of superoxide anion radical
substructure on nitrone 2 and 3 in the spin adduct stabilization.
with studied nitrones
ꢁ
cO2
Trapping of nitric oxide and superoxide anion radicals
aN
aH
aH
Nitric oxide (NO) is a signaling and regulatory molecule in many
physiological and pathological processes.37 Several attempts
have been performed to detect NO in aqueous solutions and
biological systems by using spin trapping methodology.38
However, the traditional nitrone spin traps such as PBN and
DMPO appeared to be unsuitable as they did not form stable
and characteristic spin adducts with NO.38 These limitations
suggest the need to develop new spin trapping agents, which
can specically trap NO generated under physiological condi-
tions. In this context, the ability of the newly synthesized
nitrones to trap the nitric oxide radical was studied. Thus, we
performed the same test with the FxBN nitrone, in which it was
clear that it can trap the cNO2 radical (Fig. 7).
The analysis of the intensities of the lines by double inte-
gration allows us to point out that the nitrone 2 is better
compared to FxBN.
The spectra obtained were composed of six lines (triplet of
doublet) for nitrones 2 and 3. FxBN nitrone exhibited the same
spectrum as described above. Because the obtained pattern was
not than expected, computational calculations were performed
in order to determine the trapped species. Structures were
optimized for nitrones 2 and 3, simulating the trapping of NO
and cNO2 free radicals. If the nitrones trap NO free radical
Nitrone 1
Nitrone 2
Nitrone 3
FxBN
ns
ns
ns
ns
ns
1.2
15.3
15.3
15.6
2.9
2.0
1.9
(Fig. 8 for nitrone 2), according to the calculus, it should be
observed a hyperne pattern composed by two nitrogen atoms,
one from the trapped radical and the other from the nitrone
group, with no signal being observed for the b-position
hydrogen (Table 4).
The isosurface showed that the spin density is mainly
located on each of the mentioned nitrogen atoms.
When we performed the same analysis using cNO2 as trapped
free radical, it was observed that the spin density was primarily
located between the nitrogen and hydrogen in the b-position of
the nitrone group (Fig. 9).
This result showed that the observed spectra would corre-
spond to a triplet of doublet as observed in experimental spectra
(Fig. 7) with some minor inuences from the nitrogen atom
from the nitrogen dioxide radical. Therefore, under the condi-
tions in which was generated the NO radical, these nitrones are
not capable of trapping the NO free radical in contrast to that
occurs when using the complex MGD–Fe2+ as spin traps.
For the generation of superoxide anion radical was used as
the system xanthine/xanthine oxidase. The results showed that
Fig. 9 Spin density isosurface for cnitrone 2–NO2 spin adduct using
DFT calculus. In this model, we could corroborate that the nitrones Fig. 11 Spin density isosurface for cnitrone 2–OOH spin adducts using
were not capable of trapping nitric oxide radical, and the experimental DFT calculus. The experimental spectra correspond to a delocalization
spectra corresponded to the trap of nitrogen dioxide radical.
of the spin density over the atoms indicated with arrows.
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stabilize O-, C-, S-, and N-centered radicals. Nitrone 1 showed
different ESR-spectra, and the hyperne coupling constant
allows us to distinguish the different radicals trapped easily, but
with lower half-life compared to nitrone 2 and 3. On the other
hand, nitrone 2 and 3 showed better spin traps capabilities due
to the high stability of the spin adduct formed. In some cases, it
was not so easy to distinguish the radical trapped using nitrones
2 and 3 (specically hydroxyl and methyl radicals), but the g
value helped us to differentiate the trapped species. We found
that these new nitrones were capable of trapping superoxide
anion radical generated using xanthine/xanthine oxidase
couple. Thus, we performed the trap of nitric oxide radical, in
which the results obtained showed that these new nitrones are
not capable of trapping this radical, but they can trap nitrogen
dioxide radicals. In aqueous solution, we found that nitrones 2
and 3 can form a spin adduct with hydroxyl radical. The half-life
for these spins adducts shows a value higher than two hours. In
the competition assays, the results indicated that these new
nitrones (2 and 3) are more sensitive than DMPO to trap
hydroxyl free radicals. Computational calculations supported
the experimental data and allowed us to explain the different
phenomena observed in this study.
Fig. 12 (Left) The ESR-spectrum obtained in the absence of NI.
Center: the ESR-spectrum obtained when nitrone 2 traps exclusively
hydroxyl free radical produced by NI in the presence of mouse
mammary adenocarcinoma TA3 cells. (Right) The ESR-spectrum ob-
tained when FxBN was used in the same biological system. Both
spectra were presented in the same conditions.
the nitrone 1 was not able to trap this radical. Instead, the
nitrones 2 and 3 showed similar hyperne patterns for this
radical trapping. Fig. 10 shows the experimental spectra of the
hyperne patterns displayed by nitrones 2 and 3. Hyperne
coupling constants for nitrones 2 and 3 are summarized in
Table 5.
In order to corroborate the obtained spectra, we made the
computational calculations for the cnitrone–OOH spin adduct
for nitrone 2 and 3. In Fig. 11, it is observed that the spin
densities were located mainly between the nitrogen atom and
the hydrogen atom located in b-position from the nitrone
group.
Conflicts of interest
In this sense, the theoretical results conrm that the exper-
imental spectra correspond to the trapping of the superoxide
anion radical.
There are no conicts to declare.
Acknowledgements
Spin trapping in biological systems studies
´
´
German Barriga-Gonzalez thanks nancial support by FONDE-
As it was described in our previous work,15 the new nitrones
were tested in biological models to corroborate their trapping
capacity. We used mouse mammary adenocarcinoma TA3 cells
and a nitroindazole (NI, 5-nitroindazole) derivative. The main
characteristic of this nitro derivative is its capability to be
a substrate of nitroreductases and undergoing a reduction in
the presence of oxygen by one or two-electron transfer mecha-
nism.39 The mono electronic reductions catalyzed by nitro-
reductases in the presence of molecular oxygen generate
superoxide anion radical, and the nitro compounds are regen-
erated to their initial state. This redox cycle can cause oxidative
stress due to the production of superoxide anion radical.
Finally, the superoxide anion radical decays to hydroxyl radical,
which may trap by using spin traps. Our results showed that
nitrone 1 was not able to trap any free radicals in this model.
Instead, nitrones 2 and 3 showed sharp spectra (Fig. 12).
The analysis of the hyperne coupling constant points out
that the radical trapped corresponds to hydroxyl generated by
the NI. Additionally, as Fig. 12 shows, nitrone 2 was more
sensitive to hydroxyl radical trapping in this biological model
than FxBN.
CYT grant no. 3120241 and UMCE APIX 2019 (second semester).
´
´
German Barriga Gonzalez also thanks to Patricio Canales Vol-
pone from Unidad Segundo Idioma at UMCE for correcting the
manuscript. Carolina Aliaga thanks CEDENNA AFB180001
project. Eduardo Chamorro thanks nancial support by FON-
DECYT no. 1100277 and UNAB NUCLEO DI-219-12/N. Eduardo
Chamorro acknowledges the continuous support provided by
´
Fondo Nacional de Ciencia y Tecnologıa (FONDECYT-Chile)
through Project No. 1181582. We also thank Cecilia Chavarria
and Marcos Nieves for their collaboration in the synthetic
procedures.
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