Amidinoquinoxaline N-oxides: Spin trapping of O- and C-centered radicals

Article abstract of DOI:10.1039/c7ob01387f

Amidinoquinoxaline N-oxides represent a novel family of heterocyclic spin traps. In this work, their ability to trap O- and C-centered radicals was tested using selected derivatives with different structural modifications. All the studied nitrones were ab

Full text of DOI:10.1039/c7ob01387f

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Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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Amidinoquinoxaline N-oxides: spin tapping of O- and C-centered
radicals
Received 00th January 20xx,
Accepted 00th January 20xx
Nadia Gruber,a,b Liliana R. Orelli,*b Roberto Cipolletti,a and Pierluigi Stipa,*a
Amidinoquinoxaline N-oxides represent a novel family of heterocyclic spin traps. In this work, their ability to trap O- and
C-centered radicals was tested using selected derivatives with different structural modifications. All the studied nitrones
were able to trap radicals forming persistent spin adducts, also in the case of OH and OOH radicals which are of wide
biological interest as examples of ROS. Adducts stability was mainly attributed to the wide delocalization of the unpaired
electron over the whole quinoxaline moiety. The nitroxides spectral parameters (hfccs and g-factors) were analyzed and the
results were supported by DFT calculations. N-19 hfccs and g-factors were characteristic of each aminoxyl and could aid in
DOI: 10.1039/x0xx00000x
www.rsc.org/
the identification of the trapped radical. The enhanced stability of OH adducts in the employed reaction conditions could be
.
ascribed to their possible stabilization by IHB with two different acceptors: the N-O moiety or the amidine functionality.
DFT calculations indicate that the preferred IHB is strongly conditioned by the amidine ring size. While five membered
.
homologues show a clear preference for the IHB with the N-O group, in six membered derivatives this stabilizing
interaction is preferentially established with the amidine nitrogen as IHB acceptor.
centered Free Radicals (OFR) such as superoxide, peroxy,
hydroperoxy, alkoxy, and hydroxy radicals, with respect to a
series of human diseases such as, for example, ischemia-
reperfusion syndrome, Friedreich’s ataxia, atherosclerosis,2
Introduction
Electron paramagnetic resonance (EPR) spin trapping
represents one of the most specific and reliable techniques for
neurodegenerative diseases,3 and cellular aging.4 In particular,
detecting and identifying transient free radicals, such as those liposoluble spin traps, are interesting because they are able to
penetrate biological barriers like cell membranes,5 or to reach
produced in chemical and biological processes, whose lifetime
the inner layers of the skin.6
is too short in the EPR spectroscopic timescale. This technique,
widely used since its introduction in the 1970’s,1 is based upon
the fast reaction between a suitable diamagnetic molecule (a
spin trap) and short-lived free radicals with formation of
relatively long-lived radicals (spin adducts), whose EPR signals
are persistent enough to be recorded and studied.
Additionally, the analysis of hyperfine coupling constants
(hfccs) and g-factors allows to go back to the radical initially
trapped.
Nitrones (N-oxides) act as very efficient spin traps,7 being able
to undergo fast radical additions with C- and O-centered
radicals to yield aminoxyls (nitroxides) as spin adducts, the
most persistent organic free radicals in liquid solutions. Among
all
the
commercially
available
nitrones,
N-tert-
butylbenzylideneamine N-oxide (PBN) and 5,5-dimethyl-3,4-
dihydro-2H-pyrrole N-oxide (DMPO) are probably the most
popular, but their use is not without limitations.8 For these
reasons a continuous effort has been devoted to the synthesis
of new nitrones9,10 to be used in spin trapping experiments,
with the aim to overcome the typical drawbacks affecting spin
trapping rate, adduct lifetime and the possibility of recognizing
the trapped radical from the spectral parameters of the
adduct.11
Amidinoquinoxaline N-oxides represent a heterocyclic core of
interest due to its pharmacological properties. Some suitably
substituted derivatives possess antineoplastic activity,12
especially against hypoxic tumors. Others have been employed
as antiamoebic13 and antianaerobic agents.14 As part of our
research on nitrogen heterocycles, we reported a novel
methodology for their synthesis,15 and investigated some of
their chemical,16 stereochemical17 and pharmacological18
properties. In previous work19 we studied nitrones derived
This technique has been successfully used in biological systems
recording a considerable increase in the number of its
applications. Many of them are directed toward the study of
the role played by Reactive Oxygen Species (ROS) and Oxygen
a. SIMAU Dept. - Chemistry Division, Università Politecnica delle Marche, Via Brecce
Bianche 12, Ancona (I-60131) Italy.
b. Departamento de Química Orgánica. Facultad de Farmacia
y Bioquímica.
Universidad de Buenos Aires. CONICET. Junín 956, (1113) Buenos Aires, Argentina.
*
Corresponding authors: E-mail: lorelli@ffyb.uba.ar (L. R. O.) and
pstipa@univpm.it (P. S.).
Electronic Supplementary Information (ESI) available: Scheme for the Synthesis of
all nitrones; selected bond distances and dihedral angles for nitrones 1-5; NMR
spectra of compounds
4, 8, 9; cartesian coordinates and thermochemical
parameters from frequency calculations of all compounds; selected EPR
parameters and Energies and B3LYP/6-31G(d) optimised geometries of isomeric 1b
spin adducts from relaxed PES scan; relative energies and IHB distances and angles
for all OH· and OOH· adducts; IHB distances and angles in 3c and 3e adducts. See
DOI: 10.1039/x0xx00000x
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from the pyrimidoquinoxaline nucleus as novel spin traps, and 1.275 (± 0.006) Å, respectively, for all nitrones, in goVoidewaAgrtriecleemOnelinnet
described their ability to trap methyl radicals produced in a with literature reports.20 In addition, the N(19)-C(8) double bond is
DOI: 10.1039/C7OB01387F
Fenton reaction in DMSO, to yield very persistent nitroxides as almost coplanar with the aromatic ring of the quinoxaline moiety:
spin adducts in the reaction milieu. At the same time, the the C(8)-N(19)-C(3)-C(4) angle is close to 4° and to 1° for derivatives
importance of steric factors has been outlined in such a study: 1-2 and 3-5 respectively (data reported in SI). An excellent
in general, the more hindered nitrones showed lower addition coplanarity with the above-mentioned ring exists also for N(20):
reaction rates, but the corresponding nitroxides decompose dihedral angles close to 179° are in general formed between N(20),
with lower rates too. These features prompted us to C(4), C(5) and C(6), allowing delocalization of N(20) nonbonding
investigate their trapping ability toward other radicals electrons, as shown by the HOMO plot in Figure 1b. That picture
including O-centered ones (ROS) due to their relevance in also shows the dihedral angle N(19)-C(8)-C(23)-C(25), which exerts
autooxidation processes, in polymers as well as in biological in fact a great influence on the radical addition rates at C(8),
systems (ROS). We have also explored the effect of relevant mainly ascribed to steric factors.19 For this reason, in order to
structural modifications in the heterocyclic nitrones.
study the role played by the attacking radical, none of the
nitrones chosen for the present study bears an ortho-
substituted aromatic ring in that position.
Results and discussion
The studied spin traps, together with the corresponding spin
adducts, are reported in Scheme 1. All radical species have
been produced in organic solvents as previously reported.10
Attempts to use other superoxide radical sources were
hampered by the water insolubility of the nitrones.‡ In the
present study, 2-cyano-2-propyl and benzyl radicals have been
chosen as representative of electrophilic and nucleophilic C-
centered radicals respectively, while hydroxyl, hydroperoxyl
and tert-butoxyl radicals for ROS. Five model nitrones have
been selected, comprising structural variations such as the size
Figure 1: Tube-type optimized geometry of nitrone
1 computed at the B3LYP/6-
31G(d) level (a) showing the arbitrary atom labelling, and HOMO plot (b)
computed at the B3LYP/6-31+G(d,p) level: positive values in red and negative in
green.
All compounds were able to trap the C- and O-centered radicals
employed in this study, giving persistent aminoxyls as spin adducts
whose EPR signals were detectable for several hours. Although we
did not carry out kinetic measurements, hence our findings are only
qualitative, it has to be outlined that in our spin trapping
experiments the radicals to be scavenged are not continuously
generated. Thus, the persistence of the EPR signals reflects the
stability of the adducts.
of the amidine ring, substitution at the
carbon and inclusion
of an electron withdrawing nitrogen atom in the fused ring.
Scheme 1: Spin trapping reactions
The EPR parameters (hfccs and g-factors) of the adducts have been
collected in Table 1. For simplicity, only the largest hfccs have been
reported with the arbitrary numbering shown in Figure 2. The
experimental values arise from the computer simulation of the
corresponding spectra carried out by means of the Winsim
program,21 while the computed ones have been obtained from DFT
calculations as previously described.22 In general, a good agreement
between the calculated coupling constants and the experimental
ones was found. In the case of superoxide trapping, only the
.
-
.
replacement of OO with HOO in the corresponding DFT
calculations gave hfcc values in satisfactory agreement with the
experimental results. This implies that protonation could in some
way occur in the reaction medium, as suggested for the
corresponding DMPO analogue.23 For nitroxides arising from
nitrones 1, 2 and 5, in which the amidine function is part of a
tetrahydropyrimidine ring, two minima were found in all cases.
Their geometries were labelled as “syn” and “anti” when methylene
C(10) (amidine ring) and substituent R at C(8) (Scheme 1) are
located on the same or on the opposite face, respectively. Aminoxyl
2a, which represents the only achiral adduct, shows two minima
corresponding to the axial benzyl substituent located on the same
Compounds 1-5 were obtained as previously reported,15,19 as well
as their computed geometries.19 As a representative view, the tube-
type molecular geometry of 1 computed at the B3LYP/6-31G(d)
level, together with the corresponding highest occupied molecular
orbital (HOMO) plot, are reported in Figure 1, showing the arbitrary
atom numbering. The oxygen atoms were coloured in red, nitrogen
in blue, carbon in grey and hydrogen in white: such a rendering will
be used throughout the whole paper. These calculations gave
N(19)-C(8) and N(19)-O(22) bond distances of 1.349 (± 0.02) and
2 | J. Name., 2012, 00, 1-3
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(Figure 2a - syn) or opposite face (Figure 2b - anti) as methylene Finally, adducts derived from OH ad OOH radicals (1-5c and 1-5e)
View Article Online
DOI: 10.1039/C7OB01387F
C(10).
represent a special case, and their geometries will be treated
separately in this paper. Aminoxyls 1, 2, 5 show in fact four energy
minima, arising from the relative disposition of the substituent R at
C8 (Scheme 1) with respect to C-10 (syn, anti) and from the
formation of Intramolecular Hydrogen Bonds (IHBs) with O(22) of
the N-O. moiety, or with the amidine nitrogen N(21). Two energy
minima were located for adducts derived from nitrones 3, 4,
corresponding to IHBs with both possible acceptors.
Figure 2: Tube-type B3LYP/6-31G(d) optimised structures for spin adducts 2a
:
the axial benzyl substituent is located on the same (a) or on the opposite face (b)
of C(10) methylene, named as syn and anti, respectively.
Table 1. Selected experimental (exp - in italic) and computed22 hyperfine coupling constants (hfccs, in Gauss) and g-factors of
spin adducts 1-5(a-e) in benzene solutiona,b according to the arbitrary atom labelling of Figure 2. The negative sign of the reported
experimental values have been attributed on the basis of DFT calculations and maintained during the optimization process in
spectral simulatione.
Gd
Spin Adduct N(19) H(12) H(N)(14)
H(13)
H(15)
N(21) H(16)c H(36)c
H-(O)OH
g
1a,syn
1a,anti
exp
1b,syn
1b,anti
exp
11.61 1.18
9.72 1.10
10.45 1.13
11.66 1.12
11.33 1.21
10.17 1.07
10.70 1.19
0.98
-2.96
-2.92
0.51
1.30
0.12
2.00558
5.45
0.96
0.78
0.95
1.02
0.85
1.09
-3.06
-3.11
-2.83
-2.58
-3.02
-3.32
-3.05
-2.86
-2.83
-2.55
-2.73
-3.39
0.19
0.40
0.76
0.74
0.37
0.32
0.59
0.47
1.52
0.91
0.54
1.20
0.10
0.20
0.21
0.40
0.23
0.17
2.00572
2.00552
2.00563
2.00571
2.00569
2.00540
1.66
1c,syn(N-O)
1c,anti(N-O) 10.95 1.23
1c,syn(amid) 9.97 1.18
-0.25
-0.24
-0.18
-0.39
-0.52
1.35
0.31
1.10
1.00
-3.19
-2.93
-3.26
-2.90
0.32
0.40
1.05
1.22
0.36
0.16
2.00545
2.00587
1c,anti(amid) 10.41 1.24
1.00
0.98
0.97
0.85
0.79
1.12
0.99
0.99
1.10
0.89
-2.83
-3.01
-3.01
-2.59
-3.04
-3.31
-2.85
-2.96
-2.88
-2.94
-2.78
-2.82
-3.00
-2.54
-2.68
-3.45
-2.85
-2.95
-2.95
-2.79
0.40
0.39
0.32
0.77
0.43
0.30
0.19
0.35
0.49
0.39
0.94
0.66
1.12
2.12
0.48
1.30
0.88
1.14
0.98
0.67
0.33
0.25
0.12
0.27
0.19
0.20
0.26
0.20
0.39
0.24
2.00585
2.00548
2.00580
2.00597
2.00583
2.00541
2.00590
2.00585
2.00562
2.00560
exp
1d,syn
1d,anti
exp
10.82 1.11
10.19 1.12
1.41
9.05
1.03
9.40
1.12
1e,syn(N-O)
1e,anti(N-O)
1e,syn(amid)
11.08 1.16
-0.29
-0.04
-0.13
-0.44
-0.54
0.03
2.47
9.58
9.68
1.15
1.13
1e,anti(amid) 11.33 1.20
exp
10.70 1.10
2a,syn
10.14 1.16
1.03
-3.24
-3.24
0.26
0.68
0.11
2.00555
2a,anti
exp
2b,syn
2b,anti
exp
11.61 1.17
10.23 0.91
10.61 1.15
10.11 1.16
1.00
0.75
1.05
1.06
0.87
1.09
-3.13
-3.34
-3.00
-3.22
-2.74
-3.20
-3.12
-3.46
-3.06
-3.26
-2.99
-3.30
0.19
0.22
0.31
0.22
0.48
0.37
0.48
0.65
0.59
0.81
0.49
1.50
0.09
0.12
0.13
0.15
0.22
0.35
2.00563
2.00547
2.00562
2.00559
2.00562
2.00545
0.54
1.59
9.76
1.27
2c,syn(N-O)
2c,anti(N-O) 11.02 1.12
2c,syn(amid)
10.58 1.14
-0.80
-0.85
-0.51
-0.47
-0.51
0.03
3.95
1.02
1.05
-3.06
-3.14
-3.10
-3.14
0.42
0.20
1.42
0.85
0.34
0.23
2.00546
2.00583
9.74
1.17
1.15
2c,anti(amid) 9.52
1.06
1.01
0.91
0.89
0.79
0.90
1.02
1.00
0.99
0.98
0.97
0.77
0.91
-3.15
-3.14
-2.63
-2.76
-2.75
-2.58
-2.94
-2.86
-3.01
-3.31
-3.11
-3.08
-2.74
-3.16
-3.29
-2.58
-2.79
-3.08
-2.55
-2.98
-2.88
-3.08
-3.42
-3.15
-3.23
-2.77
0.17
0.40
0.67
0.51
0.41
0.71
0.19
0.23
0.46
0.39
0.32
0.27
0.57
0.79
0.88
1.57
1.74
0.46
1.63
0.97
0.78
1.82
0.89
0.40
0.67
0.54
0.17
0.23
0.52
0.28
0.20
0.55
0.19
0.18
0.41
0.17
0.59
0.39
1.02
2.00577
2.00547
2.00580
2.00576
2.00569
2.00546
2.00580
2.00584
2.00570
2.00546
2.00554
2.00554
2.00563
exp
2d,syn
2d,anti
exp
10.44 1.21
9.83
9.58
9.47
9.61
8.72
8.79
1.08
0.97
1.14
1.07
1.08
1.08
1.05
1.72
2e,syn(N-O)
2e,anti(N-O)
2e,syn(amid)
0.46
2.02
0.37
-0.43
-0.37
0.06
2e,anti(amid) 9.47
exp
3a
exp
3b
10.23 1.01
11.11 1.12
10.59 0.81
11.86 1.08
-0.24
-
-
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exp
10.47 0.98
0.89
-2.88
-3.04
0.31
0.76
0.40
2.00565
View Article Online
3c,(N-O)
10.72 1.14
1.04
-3.18
-3.29
0.40
0.68
0.98
-0.30
-0.49
-0.55
2.00543
DOI: 10.1039/C7OB01387F
1.50
3c,(amid)
9.87
1.16
0.98
0.86
0.83
1.00
1.02
0.96
0.89
-2.97
-2.82
-2.63
-2.76
-2.92
-2.89
-2.75
-2.95
-3.00
-2.62
-2.91
-3.03
-2.91
-2.86
0.35
0.34
0.73
0.30
0.51
0.37
0.22
0.76
0.60
1.49
0.70
1.14
0.62
0.68
0.57
0.24
1.21
0.29
0.68
0.81
0.32
2.00578
2.00552
2.00592
2.00571
2.00559
exp
10.77 1.09
3d
9.00
9.55
11.04 1.11
0.95
1.10
-
exp
3e,(N-O)
-0.40
-0.13
-0.36
0.12
3e,(amid)
exp
9.65
1.10
2.00584
2.00557
10.68 1.01
4a
11.39 1.15
0.95
-2.87
-2.85
0.48
0.86
0.40
2.00563
-
-
exp
10.41 0.87
0.775
-3.06
-3.14
0.27
0.66
0.43
2.00554
4b
exp
4c,(N-O)
11.51 1.13
0.93
0.89
1.03
0.95
0.84
0.83
1.04
1.05
-2.28
-2.70
0.49
0.24
0.38
0.38
0.34
0.73
0.40
0.61
0.36
0.48
0.46
0.59
0.24
0.71
0.18
0.62
0.49
0.25
0.32
0.36
0.55
0.27
0.40
0.73
0.93
0.84
0.65
1.48
0.97
1.41
0.77
0.72
1.18
0.81
0.70
1.42
0.78
1.53
1.75
1.17
0.89
1.12
1.13
1.08
0.87
0.49
0.64
0.48
0.48
1.20
0.70
1.25
0.57
0.29
0.15
0.44
0.34
0.25
0.24
1.533
0.46
0.25
0.37
0.18
0.55
0.18
2.00567
2.00576
2.00543
2.00587
2.00548
2.00579
2.00569
2.00539
2.00583
2.00553
2.00555
2.00570
2.00553
2.00561
2.00569
2.00561
2.00586
2.00546
2.00579
2.00583
2.00543
2.00578
9.98
1.21
-3.014 -2.886
10.68 1.14
-3.20
-3.29
-0.30
-0.16
-0.55
1.71
4c,(amid)
10.14 1.14
10.67 1.00
-2.88
-2.67
-2.64
-2.65
-3.13
-2.91
-3.1
-3.27
-3.18
-3.62
-3.09
-3.42
-2.92
-3.19
-3.67
-3.24
-3.21
-3.15
-3.36
-2.87
-2.86
-2.62
-2.78
-3.33
-2.92
-3.19
-3.31
-3.22
-3.79
-3.17
-3.51
-3.42
-3.28
-3.84
-3.29
-3.26
-3.55
-3.43
exp
4d
8.98
0.96
-
exp
9.76
1.17
4e,(N-O)
10.74 1.06
-1.57
-0.12
-0.58
0.78
4e,(amid)
exp
9.69
1.10
0.95
0.78
10.89 0.926
11.34 1.14
10.96 1.15
10.22 1.02
11.44 1.08
5a,syn
5a,anti
exp
-0.78
-0.78
0.86
0.22
5.45
5b,syn
-0.75
5b,anti
exp
5c,syn(N-O)
5c,anti(N-O) 10.12 1.16
5c,syn(amid)
9.28
1.13
-0.85
-0.78
-0.77
-0.89
-0.83
-0.80
-0.81
-0.80
-0.68
-0.99
-0.82
-0.83
-0.84
10.85 1.08
10.04 0.99
-0.83
-0.29
-0.41
-0.20
-0.32
2.32
0.42
9.68
1.18
1.15
5c,anti(amid) 9.71
exp
5d,syn
5d,anti
exp
10.31 1.28
9.85
1.09
1.21
8.95
1.00
-2.85
-2.91
-3.29
-3.34
-3.20
-3.26
-2.79
-2.86
-3.33
-3.45
-3.51
-3.29
-3.32
-2.79
0.74
0.36
0.48
0.44
0.19
0.33
0.10
2.07
0.70
0.99
1.33
0.92
1.10
0.72
0.36
0.50
0.49
0.23
0.37
0.24
0.28
2.00595
2.00617
2.00543
2.00555
2.00583
2.00583
2.00554
9.21
1.11
5e,syn(N-O)
10.71 1.11
-0.40
-0.41
-0.07
-0.12
-0.29
0.26
5e,anti(N-O) 10.63 1.12
5e,syn(amid)
5e,anti(amid) 9.44
exp
8.68
1.09
1.11
2.33
-0.81
-0.57
10.70 1.02
a: dioxane for OH trapping; b: improved spectral resolution after dilution with acetonitrile for benzyl adducts; : H(16) and H(36) are
c
d
nonequivalent, hence their hfccs are different; :Differences in Reaction Gibbs Free Energy ( G, in Kcal/mol) between couples of isomers
computed at the M062X/6-31+G(d,p) level; e: Spectral simulations were carried out by means of the Winsim program.21
Data reported in Table 1 show that the EPR spectra recorded are the relatively large spin density values observed on N(21),
characteristic of the trapped radical. These spectra were explaining its relatively large hfcc; similarly, the relatively large spin
interpreted, and hence simulated, on the basis of hfccs typical for density found on the substituent oxygen atoms could explain why
this kind of radicals, in which the nitrogen three lines are mainly aminoxyls 1-5d show the smallest N(19) hfccs of the series. Finally,
split by two different couples of aromatic hydrogens of the in Figure 4 the experimental EPR spectra of some adducts, together
quinoxaline moiety: H(13) and H(15) with the larger hfccs (ca. 3 with the corresponding simulations, are shown as typical examples.
Gauss) and H(12) and H(14) with the smaller ones (ca. 1 Gauss). In
addition, the splitting of the two non equivalent protons H(16) and
H(36) is clearly visible, while a further splitting of ca. 0.4 Gauss was
assigned to N(21). In aminoxyls from nitrone 5, the H(14) splitting is
replaced by the N(14) one, while in the 5-membered amidine
derivatives 3 and 4 further couplings, attributable to the two
remaining methylenic protons of the amidine ring, are observed on
the basis of DFT calculations (data not shown). These results could
be easily explained considering a wide delocalization of the
unpaired electron over the whole quinoxaline moiety, in line with
what observed in indolinonic22 and benzoxazinic nitroxides:10 as a
Figure 3: Single occupied molecular orbital (SOMO) (a) and
-
spin density
distributions (b) of 1d computed at the unrestricted B3LYP/ EPR-III level. Positive
spin densities are in red (a) and blue (b), while negative ones are always in green.
Spin densities on H(13), H(15), N(21) and on the t-BuOO oxygens are indicated by
arrows (see text).
typical example, in Figure 3 the SOMO (a) and Spin density (b)
distribution plots for compound 1d have been reported. Figure 3a
clearly shows the SOMO marked -character and the unpaired
electron delocalization. Figure 3b displays the negative spin
densities found for H(13) and H(15), justifying the negative sign of
the corresponding hfcc values. Moreover, that picture clearly shows
4 | J. Name., 2012, 00, 1-3
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View Article Online
DOI: 10.1039/C7OB01387F
Figure 5: (a) 1b,syn plot of relaxed PES simultaneous scan of C(7)-C(8)-C(23)-
C(25) and N(19)-C(8)-C(37)-C(46) dihedral angles at the B3LYP/6-31G(d) level; the
arrow indicates isomer 1b,syn-1, whose molecular geometry is shown in (b)
(details in SI).
Concerning Nitrogen hyperfine coupling in aminoxyls, it is worth
recalling that its value is determined by spin density on the atom
.
and by hybridization. Spin density is mainly located on the N-O
group25 with typical values for alkyl aminoxyls of about 14-15 G but,
if conjugation with an aromatic system is possible, the
corresponding hfccs are somewhat smaller due to spin
delocalization.26 This is the case for N(19) hyperfine couplings
determined for the aromatic aminoxyls obtained in the present
work, ranging from 9.21 (5d) to 10.89 G (4e), as well as for other
aromatic aminoxyls described in the literature.22,26,27 Regarding
N(19) hybridization, its pyramidalization degree (Table
4 SI)
Figure 4: Experimental (upper trace) and simulated (lower trace) EPR spectra of
indicates a planar geometry (sp2), showing no significant variation
among the individual geometries.
selected spin adducts.
Since EPR parameters are very sensitive to geometry, their DFT
computation was performed taking into account the possibility of
different geometries (syn and anti), as mentioned before. However,
a comparison of these data with the experimental ones does not
allow to establish the predominant isomer. In addition, proper
frequency calculations at the M062X/6-31+G(d,p) level24 upon the
corresponding B3LYP/6-31G(d) optimised geometries show, besides
a very few examples (1a and 5b), quite small energy differences
Moreover, N(19) hfccs may also be affected by the nature of the
radical added to the a-carbon C(8), since it may contribute both to
the unpaired electron delocalization and to the geometry of the
whole system. In particular, geometric factors such as bond
distances and out of plane angles10 could influence the value of
nitrogen hyperfine coupling. Data reported in Table 1 indicate a
variation in N(19) hfccs depending on the particular radical trapped,
with the highest values for HO and HOO adducts (1-5c and 1-5e
respectively) and the lowest ones for tert-butoxyl radicals (spin
adducts 1-5d). From the above discussion, it arises that analysis of
.
.
(
G in Table 1) between pairs of isomers, suggesting that the
recorded spectra should be considered as a dynamic average of
their signals.
aminoxyl N-coupling could be
a useful approach for the
In order to get a deeper insight on the possible structures arising
from radical scavenging, proper relaxed (i.e. with geometry
optimization at each step) Potential Energy Surface (PES) scans at
the B3LYP/6-31G(d) level have been performed. In these studies,
the possible contribution of the substituents at C(8) due to steric
factors have been also considered. As a typical example, different
conformations of aminoxyl 1b,syn chosen for the relative bulkiness
of the 2-cyano-2-propyl group, have been investigated. The results
indicate the presence of 6 minima (Figure 5), showing that the C(8)-
phenyl substituent always prefers the axial orientation, and that the
energy differences among the resulting minima are extremely small.
A typical plot resulting from a simultaneous relaxed PES scan of
both C(7)-C(8)-C(23)-C(25) and N(19)-C(8)-C(37)-C(46) dihedral
angles is shown in Figure 5 (optimised geometries and calculations
reported in SI).
identification of the species trapped by these nitrones.
Moreover, in organic free radicals the isotropic g-factor deviates
from the corresponding value of the free electron (2.0023) due to
spin-orbit coupling effects arising from the contribution of each
individual atom. As a consequence, the contribution of a specific
group results from all its atoms, and will be larger the more the odd
electron is delocalized onto that group, producing the
corresponding deviation of g from the free electron value. These
substituent shifts of g in -type radicals (including aminoxyls) are
quite characteristic of the radical structure.28 Analysis of data
collected in Table 1 reveals g changes with the trapped radical, the
maximum values being usually found for 1-5d, where the tert-
butoxyl group participates in the delocalization of the unpaired
electron, as previously stated and as shown in Figure 3. This last
finding represents another important feature of the studied
nitrones, because the g-value estimation of a spin adduct, together
with N(19) hfccs and with the specific pattern of the EPR spectrum
could represent an aid for the identification of the trapped radical.
A plot of the experimental values of N(19) hfccs versus g-factors for
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all detected spin-adduct indicates a possible relationship between
aminoxyl nitrogen coupling and g-value for each radical trapped
(Figure 6): higher g-factors are associated with smaller N(19)-hfccs.
View Article Online
DOI: 10.1039/C7OB01387F
Figure 7: B3LYP/6-31G(d) optimized geometries of 1c and 3c. Dotted lines
indicate possible IHBs.
Figure 6: Experimental N-19 hfcc (aN) versus g-factor plot for adducts 1-5 (a-e)
.
As previously mentioned, aminoxyls derived from our nitrones are
stabilized by delocalization of the unpaired electron in the
system, and show unusual persistence in organic solvents
presumably due to the absence of hydrogens
to the N-oxide
function.19 Additionally, in hydroxyl and hydroperoxyl adducts, the
possibility of Intramolecular Hydrogen Bond (IHB) formation could
contribute to stabilization. Such interactions had also been
proposed in alcoxyamine derived nitroxides29 and in benzoxazinic
Figure 8: B3LYP/6-31G(d) optimized geometries of 1e and 3e. Dotted lines
indicate possible IHBs.
analogues originated by OH radical trapping,30 in which
.
unexpectedly high hfccs (0.5-0.7 Gauss) were found for the -OH
proton, with an increase of the nitroxide nitrogen coupling as well.§
Accordingly, the EPR spectra of aminoxyls 1-5c, but also those
derived from OOH radical trapping (1-5e), showed the highest N(19)
couplings of the series, together with others of about 0.5 Gauss
attributable to the -OH or -OOH proton (Table 1), thus reinforcing
our hypothesis. Furthermore, our system has another potential H
bond acceptor, represented by the amidine nitrogen N(21): these
possibilities could contribute to adducts stabilization, accounting for
their enhanced persistence in organic solvents, as for derivatives 1-
5e, which showed no appreciable decrease of the EPR signal
intensity after several hours.
Since the possibility of an IHB could affect the spin adducts
properties, their bond distances and O-H….O or O-H….N angles
(Figure 8 SI) for “N-O” or “amid” isomers respectively have been
considered as an indication of the corresponding strength.31 In
particular, their possible correlation with adducts stabilities,
estimated from the corresponding computed reaction Gibbs Free
Energy changes ( G) have been investigated. In addition, the
possibility of different formation rates has been considered by
computing the corresponding Activation Gibbs Free Energies ( G#).
Results obtained on representative derivatives 1c,e and 3c,e have
been collected in Table 2.
Table 2: IHB distances (in Ångström, Å), IHB angles (in degrees, º), Reaction Gibbs
Both possibilities to yield IHBs were studied by DFT calculations
(details in the Experimental section) upon OH and OOH radical
adducts from nitrones 1-5. Four energy minima were located for
adducts arising from nitrones 1, 2 and 5 and two for those arising
from nitrones 3 and 4. The resulting optimized geometries of 1e
Free Energies ( G) and Activation Gibbs Free Energies ( G#) for OH (1c and 3c
and OOH (1e and 3e) spin adducts.
)
Nitroxide
G (Kcal/mol)
G# (Kcal/mol)
IHB distance
IHB angle
geometry
1c,anti(N-O)
1c,anti(amid)
1c,syn(N-O)
1c,syn(amid)
1e,anti(N-O)
1e,anti(amid)
1e,syn(N-O)
1e,syn(amid)
3c,(N-O)
.
.
-44.79
-46.46
-46.14
-46.77
-16.63
-15.29
-16.66
-17.76
-43.87
-42.37
-15.00
6.09
7.05
2.04
1.93
2.04
1.92
2.10
1.85
1.89
2.04
2.09
2.11
2.15
114
123
115
123
123
139
136
127
112
119
122
and 3e (OOH adducts) and of 1c and 3c (OH adducts) are reported
in Figures 7 and 8 as typical examples, labelled as syn/anti as
already described, and as “N-O” or “amid” depending on the two
possible IHBs.
6.62
6.62
14.59
17.28
16.49
16.08
5.75
3c,(amid)
6.30
3e,(N-O)
14.34
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known that the basic site in cyclic amidines is the formally sp2
3e,(amid)
-14.88
16.26
1.91
138
View Article Online
nitrogen, acting in our case as IHB acceptor.DHOeI:n1c0e.1,0n3i9tr/oCx7iOdBe0s1w38it7hF
more basic amidine nitrogens would preferentially establish IHBs
with this function.
In line with the results previously obtained with benzoxazine
nitrones,32 the energies involved in the spin trapping reactions
.
show significantly more negatives values for OH adducts formation
.
with respect to OOH trapping, in which IHB brings about the
formation of a 6-membered ring. The reported values indicate that
1e,syn(amid) represents the most stable structure for OOH radical
addition to nitrone 1, despite its much longer IHB distance and
smaller angle with respect to the corresponding anti analogue
which, in turn, is the less stable. In addition, no significant energy
differences were found between the two remaining isomers, in
.
which the N-O moiety is involved in IHB, regardless to the
corresponding differences found in bond distances and angles. In
fact, the structure with the best IHB distance and angle
(1e,anti(amid)) is the less stable. It could therefore be concluded
that the possibility of hydrogen bonding in these nitroxides does
not play by itself a dominant role in their stabilisation, which is
mainly determined by the extensive delocalization of the unpaired
.
Figure 10: Computed Gibbs Free Energies (Kcal/mol) vs. IHB distances for OH
adducts of nitrones 1-5.
electron.19 The same analysis of the remaining OOH adducts is in
.
line with these results (Table 3 SI). In addition, by comparing the
corresponding activation parameters, it appears that shorter IHB
distances decrease OOH radical addition rates (Figure 9), suggesting
that IHB formation should not be a relevant stabilizing factor in the
corresponding transition states as well.
Summarizing, IHB would be a relevant stabilizing factor only in the
case of OH adducts. Besides, a change in the preferential IHB
interaction with the size of the amidine ring is observed: while the
.
five membered homologues 3 and 4 show a clear preference for the
.
IHB with the N-O functionality, in the six membered ones an IHB
involving the amidine nitrogen would be preferred.
Conclusions
Amidinoquinoxaline nitrones are able to trap O- and C-
centered radicals to form persistent nitroxides. Their
stabilization could be at least in part attributed to the wide
delocalization of the unpaired electron over the whole
.
system. In the case of OH adducts, DFT calculations indicate
that additional stabilization in organic solvents would result
.
from IHB interaction between the trapped radical and the N-O
moiety or the amidine nitrogen. The preferred IHB is strongly
conditioned by the amidine ring size, suggesting that there is a
determinant difference in the basicity of the amidine nitrogens
that define their ability as IHB acceptors. Besides, the N-19
hfccs and g-factors are characteristic for each nitroxide and
could contribute, together with the specific pattern of the EPR
spectrum, to the identification of the radical trapped.
In summary, the high persistence of the spin adducts in
organic solvents, including those derived from ROS, together
with the possibility of identifying the radical trapped by means
of their EPR spectral parameters makes these new spin traps
of potential interest for their applications in other chemical or
suitable biological systems.
Figure
9: Computed Gibbs Free Energies (Kcal/mol) of Activation vs. IHB
.
distances for OOH spin adducts of nitrones
1
and 3.
On the other hand, as the OH radical trapping is concerned, IHB
formation proceeds always through a 5-membered ring formation.
Although no clear correlation between computed activation
parameters and IHB distances have been found in this case, the
reaction Gibbs Free Energies ( G) correlate in general well with IHB
distances (Figure 10) and angles (Table 3 SI), outlining the
importance of such interaction in adduct stabilisation. Noteworthy,
an IHB with the amidine moiety is preferred in the additions to
nitrones 1, 2 and 5, while adducts derived from 3 and 4 show the
opposite behaviour. This amidine ring size effect could be a
consequence of the different basicity of both amidine moieties,
which in turn reflects their ability as H bond acceptors. In fact, it has
been reported33 that 1,2-diaryltetrahydropyrimidines are
Experimental
Melting points were determined with a Büchi capillary apparatus
1
and are uncorrected. H and 13C NMR spectra were recorded on a
considerably more basic than the homologous imidazolines.§ It is
§
Bruker Bio Spin Avance III 600 MHz spectrometer, a Bruker Avance
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II 500 MHz spectrometer or a Bruker MSL 300 MHz spectrometer, N-Phenylacetyl-N'-(2-nitrophenyl)ethylenediamine View8.Article OTnlhinies
using deuteriochloroform as the solvent. Chemical shifts are compound was obtained as an orange solidD(0O,I2: 2104.1g0,379/5C%7)O, Bm0p138777F-
1
reported in parts per million (ppm) relative to TMS as an internal 79 °C (from hexane/chloroform). H NMR (500 MHz, CDCl3): 8.14
standard. Coupling constants are reported in Hz. D2O was employed (1H, d, J = 8.6), 7.39-7.44 (1H, m), 7.27-7.33 (3H, m), 7.18-7.22 (2H,
to confirm exchangeable protons (ex). Splitting multiplicities are m), 6.92 (1H, d, J = 8.6), 6.63-6.68 (1H, m), 5.90 (1H, bs ex), 3.58
reported as singlet (s), broad signal (bs), doublet (d), double doublet (2H, s), 3.44-3.50 (4H, m). 13C NMR (125 MHz, CDCl3): 172.1,
(dd), doublet of double doublets (ddd), and multiplet (m). HRMS 145.4, 136.5, 134.5, 129.54, 129.47, 129.2, 127.6, 127.0, 115.9,
(ESI) were performed with a Bruker MicroTOF-Q II spectrometer. 113.8, 43.7, 42.0, 39.0. HRMS (ESI) m/z: [M + H]+ calcd for
Reagents, solvents, and starting materials were purchased from C16H18N3O3: 300.1343. Found: 300.1332.
standard sources and purified according to literature procedures.
N-(4-Methoxyphenyl)acetyl-N'-(2-nitrophenyl)ethylenediamine 9.
Synthesis of N-oxides 1-5. General procedure.
This compound was obtained as an orange solid (0,237 g, 72%), mp
N-oxides 1-5 were prepared by cyclodehydration of aminoamides 6- 95-97 °C (from hexane/chloroform). 1H NMR (300 MHz, CDCl3):
10 (Scheme 1 SI).15 The mixture of the aminoamide (1 mmol) and 8.15 (1H, dd, J = 8.6, 1.7), 8.08 (1H, bs ex), 7.43 (1H, ddd, J = 8.6,
ethyl polyphosphate (PPE, 1 mL/0.05 g) was refluxed for 5 h in an oil 6.9, 1.7), 7.11 (2H, d, J = 8.6), 6.93 (1H, d, J = 8.6), 6.84 (2H, d, J =
bath. After reaching room temperature, the resulting solution was 8.6), 6.66 (1H, ddd, J = 8.6, 6.9, 1.2), 5.87 (1H, bs ex), 3.78 (3H, s),
extracted with water (5 x 6 mL). The aqueous phases were pooled, 3.52 (2H, s), 3.44-3.50 (4H, m). 13C NMR (75 MHz, CDCl3) δ 172.4,
filtered and made alkaline with 10% aqueous NaOH. The mixture 159.0, 145.4, 136.5, 132.2, 130.6, 127.0, 126.5, 115.8, 114.6, 113.8,
was extracted with chloroform (3 x 15 mL). The organic phases 55.4, 42.9, 42.1, 39.0. HRMS (ESI) m/z: [M + H]+ calcd for
were washed with water, dried over sodium sulphate and filtered. C17H20N3O4: 330.1448. Found: 330.1453.
The chloroformic solution was left at r.t. until no further conversion
to compounds 1-5 was evidenced by TLC (silica gel, EPR measurements
chloroform:methanol 9:1). The solvent was then removed in vacuo Isotropic X-band EPR spectra were recorded on a Bruker EMX
and the crude product was purified by column chromatography spectrometer system equipped with
a microwave frequency
(silica gel, chloroform:methanol 10:0-9:1).
counter and an NMR Gaussmeter for field calibration; for g-factor
determination the whole system was standardized with a sample of
and 5 were described in the literature. perylene radical cation in concentrated sulfuric acid (g=2.00258).
34
19
Compounds 1, 2,15
3
Yield and analytical data of compound 4 are as follows.
General EPR spectrometer settings: microwave power 5 mW,
modulation amplitude 0.2 Gauss, time constant 0.64 ms, receiver
4-(4-Methoxyphenyl)-1,2-dihydroimidazo[1,2-a]quinoxaline 5-oxide gain 4.48x104, sweep time 1342.177 s, conversion time 1310.720
4. This compound was obtained as an orange solid (0.208, 71%), mp ms. EPR spectra simulations were carried out by means of the
168–170 °C (from EtOH). 1H NMR (600 MHz, CDCl3, TMS): 8.28 Winsim program, freely available from NIEHS.21 EPR spectral
(1H, dd, J=8.4, 1.4), 7.97 (2H, d, J=8.9), 7.45 (1H, ddd, J=8.1, 7.3, parameters for all nitroxide spin adducts are collected in Table 1.
1.4), 7.10 (1H, ddd, J=8.4, 7.3, 1.2), 6.99 (2H, d, J=8.9), 6.79 (1H, dd,
J=8.1, 1.2), 4.16-4.21 (2H, m), 4.03-4.08 (2H, m), 3.85 (3H, s). 13C Spin trapping experiments
NMR (151 MHz, CDCl3): δ 161.0, 153.1, 135.8, 133.2, 132.2, 131.9, Spin trapping experiments were performed by generating the
131.2, 121.4, 121.3, 120.7, 113.5, 111.9, 55.5, 54.2, 46.7. HRMS radical to be trapped directly in the sample tube in the presence of
(ESI) m/z: [M + H]+ calcd for C17H16N3O2: 294.1237. Found: the nitrone under investigation in argon deaerated benzene (0.1
294.1231.
mM) solutions. A different solvent was used in the trapping of HO
radicals in the Fenton system: aqueous FeSO4 (10 M) was added to
Synthesis of N-arylacetyl-N´-(2-nitroaryl)-1,3-propanediamines a [1,4]dioxane degassed solution of the nitrone (0.1 mM) in the
and
N-arylacetyl-N´-(2-nitroaryl)-1,2-ethanediamines
6-10. presence of 20 M hydrogen peroxide. EPR spectra were recorded
40 s after the addition of FeSO4. A 10 M benzene solution of
potassium superoxide (KO2) was used as source of superoxide anion
General Procedure.
The acyl chloride (1 mmol) was added to a chloroformic solution of prepared by adding the minimum amount of 18-crown-6 necessary
the corresponding N-(o-nitrophenyl)-1,n-diamine (1 mmol in 15 to ensure complete solubility of KO2 in benzene. tert-Butylperoxyl
mL), followed by 4% NaOH aqueous solution (1 mL). The mixture radicals were generated by adding traces of solid lead dioxide
was shaken for 15 min, after which the organic layer was separated, (PbO2) to a degassed benzene solution containing 0.1 mM of the
washed with H2O, dried (Na2SO4) and filtered. The solvent was nitrone and 10
M
of
a
commercially available tert-
removed in vacuo. The crude product was purified by flash butylhydroperoxide nonane solution. Benzyl radicals were
chromatography on silica gel using mixtures of chloroform:ethyl produced by in situ PbO2 oxidation of the corresponding Grignard
acetate as eluent.
reagent from commercial ethereal solutions as previously
described.35 2-Cyano-2-propyl radicals were generated by thermal
Compounds 6, 7,15 10 19 were described in the literature. Yields and decomposition of 2,2’-azobisisobutyronitrile (AIBN), added as a
analytical data of new compounds are as follows.
solid to the starting nitrone solution.
Computational details
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Density Functional Theory36 calculations were carried out using the
GAUSSIAN 09 suite of programs37 on the GALILEO cluster at Cineca
Supercomputing Center.38 The spin adducts (nitroxides)
conformations were systematically screened by means of
appropriate relaxed (i.e., with optimization at each point) Potential
Energy Surface (PES) Scans at the B3LYP/6-31G(d) level to ensure
that species were global minimum energy structures. In particular,
in the study upon 1b conformers, a 360° relaxed PES scan was
performed simultaneously of the C(7)-C(8)-C(23)-C(25) and N(19)-
C(8)-C(37)-C(46) dihedral angles at the same time at the B3LYP/6-
31G(d) level. Each conformer characterized by a relative Energy
Inamoto, Bull. Chem. Soc. Jpn., 1967, 40, 702; (c) G. R.
View Article Online
Chalfont, M. J. Perkins and A. HorsfieDldO,I:J1.0A.1m03.9C/Ch7eOmB.01S3o8c7.F,
1968, 90, 7141; (d) E. G. Janzen and B. J. Blackburn, J. Am.
Chem. Soc., 1968, 90, 5909; (e) C. Lagerscrantz and S.
Forshult, Nature, 1968, 218, 1247.
2
3
J. A. Berliner and J. W. Heinecke, Free Radical Biol. Med.,
1996, 20, 707.
(a) R. T. Dean, S. Fu, R. Stocker and M. J. Davies, Biochem. J.,
1997, 324, 1; (b) S. Fu, M. J. Davies, R. Stocker and R. T.
Dean, Biochem. J., 1998, 333, 519.
(a) J. Moskovitz, M. B. Yim and P. B. Chock, Arch. Biochem.
Biophys., 2002, 397, 354; (b) R. A. Floyd and K. Hensley,
Neurobiol. Aging, 2002, 23, 795.
4
5
6
7
A. Iannone, A. Marconi, G. Zambruno, A. Giannetti, V.
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minimum was submitted to
a further optimization without
constraints. In addition, the geometry optimization of all aminoxyls
was carried out with the unrestricted formalism, giving <S2>=0.7501
± 0.0003 for spin contamination (after annihilation). Moreover, in
frequency calculations on all conformers characterized by an Energy
minimum in the corresponding PES, imaginary (negative) values
were never found, confirming that the computed geometries were
always referred to a minimum. Thermodynamic quantities were
computed at 298 K by means of frequency calculations performed
employing the M06-2X24 functional in conjunction with the 6-
31+G(d,p) basis set. EPR parameters calculations were performed
following the multistep procedure previously described.22 Transition
state optimizations were performed employing the MPW1K
functional39 in conjunction with the 6-31G(d) basis set for
optimizations and 6-31+G(d,p) for frequency calculations; in these
last runs, all optimized stationary points were found to have the
appropriate number of imaginary frequencies, and the imaginary
modes (negative sign) correspond to the correct reaction
coordinates, also confirmed by their visualization with appropriate
programs.
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Acknowledgements
Università Politecnica delle Marche and University of Buenos
Aires (UBACyT 20020130100466BA) are kindly acknowledged
for financial support, as well as Cineca Supercomputing Center
for Computational Resource Allocation (ISCRA grant ANTIOX-D,
code: HP10CZ6SCE).
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‡
Attemps of superoxide generation with the xanthine-xanthine
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13 P. C. Parthasarathy, B. S. Joshi, M. R. Chaphekar, D. H.
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§ Nitroxides have two different resonance structures. Hydrogen
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14 (a) G. J. Ellames, K. R. Lawson, A. A. Jaxa-Chamiec and R. M.
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§§
Reported
pKA
values
7.65 and
for
for
1-phenyl-2-(4-
1-phenyl-2-(4-
nitrophenyl)imidazoline:
nitrophenyl)-1,4,5,6-tetrahydropyrimidine: 10.51 (From Ref. 33).
1
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