Reactivities of MeO-substituted PBN-type nitrones

Article abstract of DOI:10.1039/c9nj03805a

In this work, α-phenyl-N-tert-butylnitrone (PBN), N-benzylidene-1-diethoxyphosphoryl-1-methylethylamine N-oxide (PPN) and N-benzylidene-1-ethoxycarbonyl-1-methylethylamine N-oxide (EPPN) derivatives bearing methoxy groups (MeO-) on the phenyl ring were sy

Full text of DOI:10.1039/c9nj03805a

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Reactivities of MeO-substituted PBN-type Nitrones
Anaïs Deletraz,a Kamal Zéamari,a Florent Di Meo,b Paul-Louis Fabre,c Karine Reybier,c Patrick
Trouillas,b Béatrice Tuccio,d and Grégory Duranda*
Received 00th January 20xx,
Accepted 00th January 20xx
In this work, α-Phenyl-N-tert-butylnitrone (PBN), N-benzylidene-1-diethoxyphosphoryl-1-methylethylamine N-oxide (PPN)
and N-benzylidene-1-ethoxycarbonyl-1-methylethylamine N-oxide (EPPN) derivatives bearing methoxy groups (MeO-) on
the phenyl ring were synthesized. Their electrochemical properties were studied by cyclic voltammetry and showed that the
position and the number of methoxy substituents influence the redox potential of the nitronyl group. The spin-trapping
ability of the derivatives was next investigated by EPR spectroscopy for hydroxymethyl radical (•CH2OH). The ortho, meta
and para mono-substituted-PBN derivatives exhibited similar trapping rates than the parent PBN while surprisingly, nitrones
with two MeO-substituents on the ortho-position failed to trap •CH2OH. The trapping rates of the parent compounds were
ranked as follows: PPN > EPPN > PBN, indicating that the presence of diethoxyphosphoryl and ethoxycarbonyl electron
withdrawing groups on the N-tert-butyl group significantly enhanced the reactivity towards hydroxymethyl radicals. The
effect of the substitutions on the atomic partial charges of the nitronyl-moieties and on their ionization potentials was
computationally rationalized and showed a strong correlation between the ionization potential and the experimentally
measured oxidation potential.
DOI: 10.1039/x0xx00000x
Keywords : Nitrones, Antioxidants, Electrochemistry, Electron Paramagnetic Resonance (EPR) Spectroscopy, Spin-Trapping.
developed as very promising therapeutic agents.3,4 Indeed,
PBN-type nitrones have shown protective properties in animal
models of neurodegenerative diseases,5 stroke,6 cardiovascular
diseases7 and cancer.8 The PBN derivative disodium [(tert-
butylimino)methyl]-benzene-1,3-disulfonate N-oxide (NXY-059)
was the first neuroprotective agent to reach phase III clinical
trials in the USA for the treatment of acute stroke.9,10
In this context, several PBN derivatives have been synthesized
and studied over the past decades with the aim to improve their
biological and/or spin-trapping activities.11–19 With regards to
the spin-trapping properties, excellent results have been
Introduction
Nitrones have been initially designed as probes for the indirect
detection of free radical species in chemical and biological
systems.1 The addition of a free radical onto the nitronyl-carbon
atom yields a persistent aminoxyl spin adduct, which can be
detected and characterized in spin-trapping experiments by
electron paramagnetic resonance (EPR) spectroscopy. This has
allowed the identification of several carbon-, oxygen- or sulfur-
centered biologically relevant free radicals.2 The cyclic 5,5-
dimethyl-1-pyrrolidine N-oxide (DMPO) and the linear α-
phenyl-N-tert-butyl nitrone (PBN) have been the most
commonly used spin traps. DMPO-type nitrones have shown
better spin-trapping properties with more distinctive EPR
spectra, faster kinetics of trapping and longer half-lives of their
spin adducts whereas PBN-type nitrones exhibited a better
distribution within tissues and cells and have been largely
obtained
with
N-benzylidene-1-diethoxyphosphoryl-1-
phosphorylated
methylethylamine N-oxide (PPN),20–22
a
analogue of PBN and with N-benzylidene-1-ethoxycarbonyl-1-
methylethylamine N-oxide (EPPN),13,23 an ester analogue of
PBN. These derivatives, bearing an electron-withdrawing group
in α-position of the nitronyl-function have been shown to
efficiently trap superoxide in aqueous media and to significantly
increase stability of the superoxide adduct. Our previous work
on para-substituted PBNs has identified 4-MeO-PBN as a potent
nitrone derivative with low oxidation potential and efficient
trapping of phenyl radical.24
The current work aims at studying the influence of the electron-
donating methoxy (MeO-) substituent on the reactivity of the
nitronyl group of three classical nitrones, that is, α-phenyl-N-
tert-butylnitrone (PBN), N-benzylidene-1-diethoxyphosphoryl-
1-methylethylamine N-oxide (PPN), and N-benzylidene-1-
ethoxycarbonyl-1-methylethylamine N-oxide (EPPN) (Figure 1).
A series of methoxy-substituted phenyl nitrones were thus
a.Institut des Biomolécules Max Mousseron, UMR 5247 CNRS-Université
Montpellier-ENSCM & Avignon Université, Equipe Chimie Bioorganique et
Systèmes Amphiphiles, 301 rue Baruch de Spinoza, BP 21239, Avignon 84916
Cedex 9, France ;
b.INSERM UMR 850, Université de Limoges, Faculté de Pharmacie, 2 rue du Dr.
Marcland, 87025 Limoges, France ;
c. Pharma-Dev, UMR 152, Université de Toulouse, IRD, UPS, 35 chemin des
Maraîchers, 31400 Toulouse, France ;
d.Aix-Marseille Université, CNRS, ICR UMR 7273, Avenue Escadrille Normandie
Niemen, 13397 Marseille Cedex 20, France.
*Corresponding Author. Grégory Durand. E-mail: gregory.durand@univ-avignon.fr;
Phone: +33 (0)4 9014 4445
Electronic Supplementary Information (ESI) available. See DOI: 10.1039/x0xx00000x
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diethyl [1-(hydroxyamino)-1-methylethyl]phosphonate, which
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DOI: 10.1039/C9NJ03805A
was obtained by the method described by Petrov et al. with p-
anisaldehyde in EtOH. For 4-MeO-EPPN ( ), ethyl 2-methyl-2-
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nitropropanoate was first synthesized as previously reported28
and was reduced in situ in hydroxylamine and condensed with p-
anisaldehyde and NH4Cl, following the procedure described by
Stolze et al.23
Calculated octanol-water partition coefficients (ClogP) of the
nitrones were determined using ALOGPS 2.1 software and are
listed in Table 1, as well as experimental values found in the
literature. The introduction of a methoxy substituent on the
phenyl ring decreases very slightly the lipophilicity compared to
the parent nitrones (~ 0.10), with no influence of the position of
the substituent. This is in good agreement with the experimental
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Figure 1. Chemical structures of nitrones used in this study.
synthesized and their electrochemical properties were first
studied. Using EPR competition kinetic experiments, the
relative rate constant of hydroxymethyl radical trapping was
determined. Finally, the effect of methoxy substituents on the
nitronyl atomic charge densities and on the Ionization Potential
(IP) were described, as supported by Density Functional Theory
(DFT) calculations.
values found in the literature where PBN, 3-MeO-PBN (
MeO-PBN (
) exhibited similar LogP values.29 The polar effect of
the methoxy groups on the lipophilicity seems to be additive with
nitrone and being slightly less lipophilic than the
monosubstituted derivatives 1-3. This does not perfectly fit with
2) and 4-
3
4
5,
the experimental value for nitrone
5 that was found to be similar
to PBN.29 With regards to PPN and EPPN, more pronounced
differences were observed between the calculated and
Results and Discussion
Synthesis. The MeO-PBN derivatives 1-4 were obtained by a one-
pot reduction/condensation of 2-methyl-2-nitropropane onto the
appropriate benzaldehyde in the presence of zinc powder,25 as
shown in Scheme 1. The 2,4,6-tri-MeO-PBN compound (5) was
synthesized through direct condensation between 2,4,6-
trimethoxybenzaldehyde and N-(tertbutyl)hydroxylamine acetate
in CH2Cl2, using pyrrolidine as catalyst, following the procedure
described by Morales et al.26 PPN21 and EPPN23 were synthesized
as previously described and the methoxy derivatives 4-MeO-PPN
and 4-MeO-EPPN were obtained using similar synthetic routes.
The 4-MeO-PPN compound (6) was prepared by condensing
Scheme 1. Synthesis of nitrones by (A) a one-pot reduction/condensation of nitro-
derivatives onto the appropriate benzaldehyde or by (B) direct condensation of N-
substituted hydroxylamines with aromatic aldehydes.a
aReagents and conditions: (i) AcOH, EtOH, 60 °C for compounds 1-4; (ii) NH4Cl,
H2O/MeOH (6/4, v/v), 60 °C for compound 7; (iii) pyrrolidine, DCM, rt for compound 5;
(iv) EtOH, 60 °C for compound 6.
Figure 2. Cyclic voltammograms of PBN-derivatives in acetonitrile containing 0.1 M of
TBAP at a Cv electrode, potential scan rate ʋ = 0.1 V/s vs. Ag: (A) reduction and (B)
oxidation.
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Table 1. Physicochemical, Electrochemical and Spin-Trapping Properties of MeO-Derivatives.
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6
Voltammetryf
EPR Spin Trapping
7
8
9
Lipophilicity
ClogPa
LogP
2.67
1.2b
in CH3CNg, compound (1 mM)
CH2OH
j
Compounds
PBN
Ep(c) (V)
-2.12 (-2.12)i
-2.15
Ep(a) (V)
1.60 (1.53)i
1.46
Stability domain (V)h
aN (G)
14.3
14.1
14.0
14.1
ndc,k
ndc,k
13.5
13.5
14.2
13.7
aH (G)
2.7
aP (G)
kN/kPBN
1
3.72
3.61
3.68
3.62
3.80
3.77
3.60
3.52
3.63
3.57
-
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2-MeO-PBN (1)
3-MeO-PBN (2)
4-MeO-PBN (3)
2,6-di-MeO-PBN (4)
2,4,6-tri-MeO-PBN (5)
PPN
2.57
2.57
2.57
2.46
2.36
3.01
2.90
2.20
2.10
ndc
1.4b
1.2
3.2
-
1.026
-2.11
1.57
1.40 (1.26)i
3.2
-
0.935
-2.22 (-2.23)i
3.1
-
1.128 (0.832)g
ndc,k
ndc
-2.41
1.39
ndc,k
ndc,k
2.7
-
1.2b
1.0d
ndc
1.5e
ndc
-2.56
1.21
-
38.5
38.8
-
ndc,k
-1.96
1.64
6.369
4-MeO-PPN (6)
EPPN
-2.08
1.44
2.9
5.882
-1.95
1.68
3.0
1.498
4-MeO-EPPN (7)
-2.11
1.46
3.0
-
0.891
a Calculated octanol/water partition coefficient values obtained using ALOGPS 2.1 software (http://www.vcclab.org/lab/alogps/).
b Data from Janzen et al.29
c Not determined.
d Data from Roubaud et al.21
e Data from Roubaud et al.13
f The peak potentials are given versus a silver wire electrode for a potential scan rate 0.1 V/s.
g Containing 0.1 M of TBAP with reduction Ep(c) and oxidation Ep(a) at vitreous carbon Cv electrode.
h The stability domain of potentials is given as: Ep(a)-Ep(c).
i Data from Rosselin et al.24
j Ratio of the second-order rate constants for the hydroxymethyl radical trapping by various nitrones (kN) and by PBN (kPBN) in methanol, calculated with a ratio
kPBN/kTN = 0.069.
k The EPR signal of the adduct was too weak to allow a reliable determination of the ratio.
experimental values, this, however, remains within the same that the position of the substituent slightly influences the
range.13,21
electrochemical properties of nitrones. The effect was more
pronounced when increasing the number of substituents. The
Cyclic Voltammetry. The electrochemical properties of the oxidation and reduction potentials were respectively decreased
seven MeO-derivatives were investigated using cyclic and increased, suggesting an additive effect of the substituents.
voltammetry, and redox potentials are reported in Table 1. The When comparing to the parent compounds, the presence of the
experiments were performed in acetonitrile containing tetra- diethoxyphosphoryl or the ethoxycarbonyl electron-
butylammonium perchlorate (TBAP) as electrolyte. For the sake withdrawing groups facilitates reduction (~ 0.2 V) while slightly
of comparison, the parent compounds were also analysed. PBN impairing oxidation (~ 0.05 V).
was oxidized through one-electron irreversible transfer and was Table 1 shows the values of the stability domains of the
reduced through a one-step two-electron reduction as derivatives, which corresponds to the differences between the
previously reported,30–32 the same behaviour being observed reduction and oxidation potentials, namely Ep(a)-Ep(c). These
for the other derivatives.
values represent the windows of electrochemical potentials
Oxidation and reduction of nitrones were clearly observed, with over which spin-trapping can be employed. This is of
values ranging from 1.21 to 1.68 V and from -2.56 to -1.95 V, importance when detecting electro-chemically generated
respectively (Figure 2). The oxidation potentials measured for radical species. Indeed, inverted spin–trapping may occur and
the MeO-derivatives were always lower than that of their lead to incorrect conclusions. In the inverted spin–trapping
parent compounds, showing that the presence of an electron- process,33,34 the nitronyl group is oxidized to a radical cation or
donating substituent facilitates the oxidation of the nitronyl reduced to a radical anion, which can then react with a
group in agreement with the literature.24,30–32 An opposite nucleophile or an electrophile, respectively, both yielding an
effect was noted for the reduction, with reduction potentials aminoxyl radical which does not come from the radical addition
being higher than that of the parent compound, indicating onto the nitronyl group. For PBN, the stability domain value was
harder reduction.
3.72 V, as previously reported.24 The mono-substituted
derivatives ( ) exhibited a slightly smaller potential window
For the mono-substituted-PBNs, the para derivative (
exhibited the lowest oxidation potential and the highest whereas the stability domain of the di- and tri-substituted
reduction potential while the meta derivative ( ) exhibited the derivatives ( and ) was slightly larger. For both PPN and EPPN
highest oxidation potential and the lowest reduction potential, derivatives, the stability domain remains in the same range.
however the differences remained below 0.2 V. This indicates
3
)
1-3
2
4
5
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Hydroxymethyl radical trapping by Electron Paramagnetic different [
Resonance. The spin-trapping properties of the derivatives order to compare the spin trapping of DMOeI:O10-d.1e03ri9v/aCt9iNveJ0s3w80i5thA
were studied by evaluating their capacity of trapping of the PBN for nitrones , EPPN and nitrone , a kPBN/kTN ratio of
hydroxymethyl radical (•CH2OH), which is representative of the 0.069 was determined using commercially available PBN
formation of alcohol-derived radicals during oxidative stress. instead . From these results, the kN/kPBN ratio was calculated
N]/[TN] ratios kept between 2 and 5 were used. In
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-
3
7
N
Oxidative stress leads to overproduction of several reactive and the values obtained are reported in Table 1.
•-
oxygen species (O2 , H2O2, HO•) which further react with sugars, All the derivatives tested exhibited lower •CH2OH radical
proteins, lipids and DNA thereby yielding carbon-centered trapping efficiency than TN, which bears three nitronyl groups.
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•
radicals, like hydroxyalkyl radicals.35 The CH2OH radical was For derivatives
1
-
3
, kN/kPBN values ranged from 0.935 to 1.128,
generated in situ by a standard Fenton system in the presence indicating similar rates of trapping than PBN. This suggests that
of the nitrone derivatives noted
N
and the competitive for a given nitrone, the position of the MeO-substituent on the
•
scavenger 1,3,5-tri[(N-(1-diethylphosphono)-1-methylethyl) N- phenyl ring has no significant effect on the trapping of CH2OH.
oxy-aldimine] noted TN.16 For 2,6-di-MeO-PBN (
MeO-PBN (
) only background noise was observed indicating •CH2OH radicals 1.5 times faster than PBN, while PPN exhibited
4) and 2,4,6-tri- With regards to the α-substituted derivatives, EPPN trapped
5
that these nitrones did not efficiently trap hydroxymethyl even more improved reactivity, that is, 6.4 times faster
radicals. Although we have no strong evidence to discard side- trapping. This demonstrates that the introduction of an
•
•
reactions between OH or CH2OH radicals with the methoxy electron-withdrawing group in α-position of the nitronyl-
groups of these nitrones, the steric hindrance due to the ortho function improves the spin-trapping properties.
substituents very likely explains the inefficiency of (4) and (5) to
act as spin traps. The five mono-substituted derivatives gave Calculated Nitronyl Atomic Partial Charge and Ionization
rise to a standard six-line EPR spectrum due to hyperfine Potentials. The electronic population analysis within the natural
couplings of the unpaired electron with nitrogen and
β
-
bond orbitals (NBO) framework37,38 on the nitronyl atoms (H, C,
hydrogen nuclei. For PPN and 4-MeO-PPN ( ), additional lines N and O) as well as the IPs of PBN derivatives were calculated in
6
due to the coupling with the phosphorous atom were observed. water using the DFT formalism (Table 2). The electronic
The hyperfine coupling constants (hfcc) are reported in Table 1 densities on the nitronyl atoms depend on the position and on
and are consistent with PBN-type spin adducts.36
The CH2OH spin trapping rate was assessed by measuring the an electron-withdrawing group in α-position of the nitronyl
the number of MeO substituents as well as on the presence of
•
relative intensity (as the signal area) of the EPR signals of the
CH2OH and TN-CH2OH adducts (Figure 3). PPN and 4-MeO-PPN affecting the nitronyl atomic partial charges of the derivatives,
) were directly tested versus PBN instead of TN. The standard the inductive effect of the diethoxyphosphoryl and carbonyl
N-
function. Therefore, two electronic effects are operatively
(
6
kinetic competition model described by Roubaud et al.16 was groups at the α position and the mesomeric effect of the
employed (equation 1):
[ ]⁄
methoxy substituents. Using 1H NMR spectroscopy, the
chemical shifts of the nitronyl-H in CDCl3 were measured and
⁄
푅 푟 = 1 + 푘N
[
푘TN TN
]
N
(1)
Where, kN and kTN correspond to the second-order rate showed a good correlation with the nitronyl H-atom partial
•
constants for CH2OH trapping by the nitrones
N
and TN atomic charges (Figure 4A). It is worth mentioning that only 2,6-
) and 2,4,6-tri-MeO-PBN ( ) were out of the
respectively, while R and r represent the trapping rate by both di-MeO-PBN (
4
5
TN and
N, and by TN only, respectively.
correlation. This correlation confirms the polar effect of the
By plotting the R/r ratio as a function of [
N]/[TN] ratio, a straight substituents both on the aromatic ring and in α-position of the
line was obtained for each derivative (Figure 3) and five nitronyl function, however with subtler effects in case of poly-
Figure 3. (A)-(B) EPR signals of TN and 2-MeO-PBN (1) hydroxymethyl radical adducts, respectively. Hydroxymethyl radical was generated by a Fenton system and the ratio [1]/[TN]
was: (A) [1]/[TN] = 2; (B) [1]/[TN] = 5. The peaks topped by a cross (×) correspond to the hydroxymethyl radical adduct of TN and those topped by a round (○) to the hydroxymethyl
radical adduct of 1. (C) Determination of the relative rate constant kN/kTN of •CH2OH trapping by nitrone 1 (R² = 0.999).
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effect over the whole charge delocalization.
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Table 2. Calculated Atomic Partial Charges of the Nitronyl Atoms and Ionization
Potentials of MeO-Derivatives at the (CPCM)/M06-2X/6-31+g(d,p) Level of Theory.
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Atomic partial charges of the
nitronyl atoms (e)
Compounds
PBN
H
C
N
O
IP (eV)
6.0
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0.254
0.262
0.255
0.253
0.261
0.259
0.258
0.254
0.250
0.248
0.019
0.009
0.021
0.028
0.018
0.036
0.033
0.056
0.020
0.031
0.080
0.084
0.081
0.069
0.085
0.075
0.073
0.056
0.084
0.072
-0.636
-0.633
-0.635
-0.651
-0.638
-0.644
-0.628
-0.644
-0.610
-0.626
2-MeO-PBN (1)
3-MeO-PBN (2)
4-MeO-PBN (3)
2,6-di-MeO-PBN (4)
2,4,6-tri-MeO-PBN (5)
PPN
5.6
6.0
5.7
5.7
5.4
6.0
4-MeO-PPN (6)
EPPN
5.6
6.0
4-MeO-EPPN (7)
5.7
Since nitrones have a similar reactivity than carbonyl groups
allowing nucleophilic addition reactions, the electronic
population of the nitronyl-carbon provides hints about the
reactivity of the nitronyl function. For the ortho-derivative (1),
the nitronyl C-atomic partial charge is close to zero, being the
lowest over the series. This suggests that this position would be
mildly reactive toward a nucleophilic attack. Conversely, for 4-
MeO-PPN (6), both para electron-donating MeO substituent
and α-position electron-withdrawing diethoxyphosphoryl group
lead to synergic electronic effects. This suggests that it would
be highly reactive toward a nucleophilic attack, as shown by the
nitronyl-C atomic partial charge which was the most positive of
the series. While plotting the relative rate constants of •CH2OH
addition to nitrones versus the nitronyl-C atomic partial charge
(Figure 4B), a good correlation for PBN, PPN and EPPN was
observed (R² = 0.9996), showing that the more positive the
nitronyl-C atomic partial charge, the faster the addition
reaction. Our experimental values therefore indicate that
electron withdrawing inductive effect on the N-tert butyl group
•
increases the reactivity of the nitronyl group towards CH2OH
radical. This is supported by the nucleophilic nature of the
•CH2OH radical.39 However when adding the MeO-derivatives,
the positive mesomeric effects on the aromatic ring tend to
decrease the reactivity and no correlation with the nitronyl-C
atomic partial charge was observed, suggesting that not only
electronic factors are involved in the trapping reaction of
•CH2OH with the nitronyl group. This points out the need for
further studies to determine how these two effects alter the
reactivity of the nitronyl group and which of the two is more
prominent.
Figure 4. (A) Correlation of nitronyl-H chemical shifts with nitronyl-H charge densities
(R2 = 0.852 excluding the outliers 2,6-di-MeO-PBN and 2,4,6-tri-MeO-PBN marked as
(○)); (B) Plots of experimental relative rate constants of hydroxymethyl addition to
nitrones with nitronyl-C charge density; (C) Correlation of ionization potential with
oxidation potential of nitrones (R2 = 0.926).
Given the chemical accuracy of IP calculations at the DFT level
of theory (ca. 0.1 eV), the following ranking was obtained (See
Table 3): 2,4,6-tri-MeO-PBN (
4-MeO-PBN ( 2,6-di-MeO-PBN (
EPPN PPN 3-MeO-PBN ( ). This ranking is in excellent
5
) < 4-MeO-PPN (
6
)
 2-MeO-PBN
(
1
)

3

)

3
)

4-MeO-EPPN (
7
) <
(See Figure 4C); the lower the IP, the lower the oxidation
potential. For instance, the highest calculated IP value was
observed for EPPN, which also exhibits the highest oxidation
PBN


2
agreement with experimentally observed oxidation potentials
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potential. Such strong correlation between IP and oxidation the reduction of the nitronyl function. With regard to the
potential (R² = 0.926, Figure 4C) clearly suggests that a number of substituents, the electronic effects appeared
View Article Online
DOI: 10.1039/C9NJ03805A
computational approach might (i) predict the electrochemical additive. The computed Ionisation Potentials were found to
properties of nitrones but also (ii) provide hints in order to tune correlate with the experimental oxidation potentials,
oxidation properties of nitronyl derivatives. For example, the suggesting that a computational approach may predict the
presence of electron-withdrawing group in α-position of the electrochemical properties of nitrones. The reactivity towards
nitronyl moiety does not modify the stability of the radical •CH2OH radical trapping was correlated with the electron-
cation formed after electron transfer event, as depicted by withdrawing nature of the substituent in α-position on the
similar spin density distributions (Figure 5). Likewise, meta- nitronyl function, with the PPN being the most effect trap,
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•
substitution by donating moieties (e.g., MeO) does not stabilize supporting the nucleophilic nature of CH2OH. However, when
radical cation forms. However, the significant increase of

-
adding MeO-substituents, the rates of trapping were
delocalization over the aromatic ring strongly stabilizes the significantly lowered, with an absence of correlation with the
radical cation, that in turn decreases IP and thus the oxidation nitronyl-C atomic partial charges. In addition, with the di-ortho
potential.
substituted derivatives no trapping was observed. This suggests
that not only electronic factors are involved in the spin trapping
reaction and therefore this warrants further investigation.
Experimental Section
Synthesis. All reagents were from commercial sources and were
used as received. All solvents were distilled and dried according
to standard procedures. The TLC analysis was performed on
aluminium sheets coated with silica gel (40−63 µm). Compound
detection was achieved either by exposure to UV light (254 nm)
and/or by spraying a 5% sulfuric acid solution in ethanol or a 2%
ninhydrin solution in ethanol and then by heating up to
∼150
°C. Flash column chromatography was carried out on silica gel
(40−63 μm). Melting points have not been corrected. NMR
spectra were recorded on a Bruker AC400 at 400, 100 and 162
1
MHz for H, 13C and 31P experiments, respectively. Chemical
shifts are given in ppm relative to the solvent residual peak as a
1
heteronuclear reference for H and 13C. Abbreviations used for
signal patterns are as follows: s, singlet; d, doublet; dd, doublet
of doublets; t, triplet; td, triplet of doublets; q, quartet; m,
multiplet. HR-MS spectra were recorded on
spectrometer equipped with a TOF analyzer for ESI+
experiments.
a mass
Figure 5. Mulliken spin density distribution of the radical cations formed after single
electron abstractions.
N-tert-butyl-α-(2-methoxy)phenylnitrone (1). In an argon
atmosphere and under stirring, o-anisaldehyde (1.22 g, 9.0
mmol, 1.0 equiv), 2-methyl-2-nitropropane (1.9 mL, 17.9 mmol,
2.0 equiv) and AcOH (3.1 mL, 54.0 mmol, 6.0 equiv) were
dissolved in dry EtOH. The mixture was cooled down to 0 °C,
then zinc powder (2.4 g, 36.0 mmol, 4.0 equiv) was slowly added
in order to keep the temperature below 15 °C. The mixture was
stirred at room temperature for 30 min then heated at 60 °C in
the dark for 22h in the presence of molecular sieves (4Å). The
reaction mixture was filtered off through a pad of Celite, and
the solvent was removed under vacuum. The crude mixture was
purified by flash chromatography (EtOAc/cyclohexane, 2/8)
followed by two successive crystallisations from EtOAc/n-
hexane to afford 1.70 g of the title compound (8.2 mmol, yield
92%) as a white powder. Rf (EtOAc/cyclohexane, 2/8) = 0.16. 1H
NMR (400 MHz, CDCl3,) δ 9.36 (1H, dd, J = 8.0 and 1.2 Hz), 8.05
(1H, s), 7.35 (1H, td, J = 8.4 and 2.0 Hz), 7.02 (1H, t, J = 7.6 Hz),
6.88 (1H, d, J = 8.2 Hz), 3.86 (3H, s), 1.61 (9H, s); 13C NMR (100
Conclusion
In this work, we have studied the effect of the position and the
number of the electron-donating MeO-substituents on the
reactivity of phenyl nitrones. A series of mono-substituted
(ortho-, meta- and para-) and di- and tri-substituted derivatives
of PBN were synthesized as well as the 4-MeO-derivatives of
PPN and EPPN. The substituent effect was also demonstrated
by a computational approach, from which a correlation
1
between H NMR chemical shifts and nitronyl-H atomic partial
charges of the derivatives was obtained. Cyclic voltammetry
experiments showed that the redox potentials depend on the
position and on the number of substituents. While electron-
donating groups on the phenyl ring tend to favour oxidation
compared to unsubstituted derivatives, electron-withdrawing
groups in α-position on the nitronyl function slightly impair
oxidation. An opposite trend was obviously demonstrated for
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MHz, CDCl3) δ 157.2, 131.3, 128.8, 124.5, 120.9, 120.2, 109.7, hexane to afford 0.81 g of the title compound (3.03 mmol, yield
View Article Online
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DOI: 10.1039/C9NJ03805A
71.1, 55.7, 28.5. The spectral data was in agreement with the 59%) as a white powder. Rf (EtOAc/MeOH, 95/5) = 0.26. H NMR
literature.40
(400 MHz, CDCl3,) δ 7.47 (1H, s), 6.12 (2H, s), 3.81 (3H, s), 3.80
(6H, s), 1.59 (9H, s); 13C NMR (100 MHz, CDCl3) δ 162.6, 159.7,
N-tert-butyl-α-(3-methoxy)phenylnitrone (2). The synthetic 124.7, 102.4, 91.0, 69.8, 56.0, 55.6, 28.6. The spectral data were
procedure was essentially the same as for compound
. m- in agreement with the literature.42
1
anisaldehyde (1.39 g, 10.2 mmol) and 2-methyl-2-nitropropane
(2.2 mL, 20.4 mmol) were used as starting materials. The N-2-(diethoxyphosphoryl-propyl)-α-(4-
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reaction time was 24h. The crude mixture was purified by flash methoxy)phenylnitrone (6). Under argon atmosphere and
chromatography (EtOAc/cyclohexane, 4/6) followed by two stirring, p-anisaldehyde (565 mg, 4.15 mmol, 1.0 equiv) and
successive crystallisations from EtOAc/n-hexane to afford 1.76 diethyl [1-(hydroxyamino)-1-methylethyl]phosphonate20 (876
g of the title compound (8.5 mmol, yield 83%) as a white mg, 4.15 mmol, 1.0 equiv) were dissolved in dry EtOH. The
powder. Rf (EtOAc/cyclohexane, 4/6) = 0.28. 1H NMR (400 MHz, mixture was stirred at 60 °C for 22h. The solvent was removed
CDCl3,) δ 8.34 (1H, s), 7.54 (1H, s), 7.48 (1H, d, J = 7.6 Hz), 7.31 under vacuum and the crude mixture was purified by flash
(1H, t, J = 8.0 Hz), 6.97 (1H, dd, J = 8.0 and 2.4 Hz), 3.86 (3H, s), chromatography (EtOAc/cyclohexane, 9/1) to afford 1.27 g of
1.62 (9H, s); 13C NMR (100 MHz, CDCl3) δ 159.7, 132.4, 130.3, the title compound (3.86 mmol, yield 93%) as a colorless oil. Rf
1
129.3, 122.1, 117.6, 112.3, 71.1, 55.5, 28.5. The spectral data (EtOAc/cyclohexane, 9/1) = 0.22. H NMR (400 MHz, CDCl3,) δ
were in agreement with the literature.41
8.27 (2H, d, J = 8.8 Hz), 7.67 (1H, d, J = 2.8 Hz), 6.92 (2H, d, J =
9.2 Hz), 4.23-4.19 (4H, m), 3.84 (3H, s), 1.82 (6H, d, J = 14.8 Hz),
N-tert-butyl-α-(4-methoxy)phenylnitrone (3). The synthetic 1.32 (6H, t, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) δ 161.3, 133.1
procedure was essentially the same as for compound 1. p- (d, J = 4 Hz), 131.2, 123.9 (d, J = 2 Hz), 113.9, 72.3 (d, J = 153 Hz),
anisaldehyde (1.42 g, 10.4 mmol) and 2-methyl-2-nitropropane 63.4 (d, J = 7 Hz), 55.5, 23.4, 16.6 (d, J = 6 Hz); 31P NMR (162
(2.3 mL, 20.8 mmol) were used as starting materials. The MHz, CDCl3) δ 23.38. HR-MS (ESI+, m/z) calcd for C15H25NO5P
reaction time was 26h. The crude mixture was purified by flash [(M + H)+] 330.1470, found 330.1469.
chromatography (EtOAc/cyclohexane, 5/5) followed by two
successive crystallisations from EtOAc/n-hexane to afford 1.70 N-2-(ethoxycarbonyl-propyl)-α-(4-methoxy)phenylnitrone (7).
g of the title compound (8.2 mmol, yield 79%) as a white Under argon atmosphere and stirring, ethyl 2-methyl-2-
powder. Rf (EtOAc/cyclohexane, 5/5) = 0.18. 1H NMR (400 MHz, nitropropanoate23 (2.00 g, 12.42 mmol, 1.0 equiv), p-
CDCl3,) δ 8.27 (2H, d, J = 9.2 Hz), 7.45 (1H, s), 6.91 (2H, d, J = 8.0 anisaldehyde (1.8 mL, 14.90 mmol, 1.2 equiv) and NH4Cl (930
Hz), 3.83 (3H, s), 1.59 (9H, s); 13C NMR (100 MHz, CDCl3) δ 160.9, mg, 17.39 mmol, 1.4 equiv) were dissolved in H2O/MeOH (6/4,
130.8, 129.5, 124.2, 113.8, 70.2, 55.4, 28.4. The spectral data v/v). The mixture was cooled down to 0 °C, then zinc powder
were in agreement with the literature.40
(1.61 g, 24.84 mmol, 2.0 equiv) was slowly added to keep the
temperature below 15°C. The mixture was stirred at room
The temperature for 1h then heated at 60 °C in the dark for 18h. The
N-tert-butyl-α-(2,6-dimethoxy)phenylnitrone
(4).
synthetic procedure was essentially the same as for compound reaction mixture was filtered off through a pad of Celite, and
. 2,6-dimethoxybenzaldehyde (890 mg, 5.36 mmol) and 2- the solvent was removed under vacuum. The crude mixture was
1
methyl-2-nitropropane (1.2 mL, 10.72 mmol) were used as purified by flash chromatography (EtOAc/cyclohexane, 5/5)
starting materials. The reaction time was 21h. The crude followed by two successive crystallisations from EtOAc/n-
mixture was purified by flash chromatography (EtOAc/MeOH, hexane to afford 1.49 g of the title compound (5.62 mmol, yield
98/2) followed by two successive crystallisations from EtOAc/n- 45%) as a white powder. Rf (EtOAc/cyclohexane, 5/5) = 0.26. 1H
hexane to afford 944 mg of the title compound (3.98 mmol, NMR (400 MHz, CDCl3,) δ 8.27 (2H, d, J = 8.8 Hz), 7.40 (1H, s), δ
yield 74%) as a white powder. Rf (EtOAc/MeOH, 95/5) = 0.32. 1H 6.93 (2H, d, J = 8.8 Hz), 4.25 (2H, q, J = 7.2 Hz), 3.85 (3H, s), 1.81
NMR (400 MHz, CDCl3,) δ 7.54 (1H, s), 7.27 (1H, t, J = 8.4 Hz), (6H, s), 1.27 (3H, t, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) δ
6.55 (2H, d, J = 8.4 Hz), 3.82 (6H, s), 1.61 (9H, s); 13C NMR (100 170.8, 161.4, 131.2, 131.0, 123.5, 114.0, 76.5, 62.2, 55.5, 24.6,
MHz, CDCl3) δ 158.9, 130.7, 124.9, 109.1, 104.0, 70.1, 56.1, 28.5. 14.1. HR-MS (ESI+, m/z) calcd for C14H20NO4 [(M + H)+] 266.1392,
HR-MS (ESI+, m/z) calcd for C13H20NO3 [(M + H)+] 238.1443, found 266.1398.
found 238.1449.
Determination of ClogP Values. The partition coefficient
N-tert-butyl-α-(2,4,6-trimethoxy)phenylnitrone (5). Under octanol/water (ClogP) was determined by using ALOGPS 2.1
argon atmosphere and stirring, 2,4,6-trimethoxybenzaldehyde software, which is available at www.vcclab.org/lab/alogps/.
(1.01 g, 5.17 mmol, 1.0 equiv) and N-(tert-butyl)hydroxylamine
acetate (770 mg, 5.17 mmol, 1.0 equiv) were dissolved in dry Cyclic Voltammetric Measurements. The electrochemical
DCM (0.33 M). Pyrrolidine (0.5 mL, 6.20 mmol, 1.2 equiv) was experiments were carried out using a three-electrode cell under
added and the mixture was stirred at room temperature for 1h. a dry argon atmosphere at room temperature. A silver wire
The solvent was removed under vacuum and the crude mixture electrode was used as the reference electrode and a platinum
was purified by flash chromatography (EtOAc/MeOH, 95/5) wire as the auxiliary electrode. The working electrode (glassy
followed by two successive crystallisations from EtOAc/n-
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carbon) was polished prior to each experiment using a 0.04 μm compounds. A frequency analysis was systematically performed
View Article Online
DOI: 10.1039/C9NJ03805A
aqueous alumina slurry on a wetted polishing cloth.
at 298 K and 1 atm, at the M06-2X/6-31+G(d,p) level of theory
confirming that optimized geometries at in a potential energy
EPR Measurements and Spin-Trapping Kinetics. EPR surface minimum owing to the absence of any imaginary
measurements were carried out on a Bruker EMX spectrometer frequency. Solvent (water) effects were considered by using the
operating at X-band with 100 kHz modulation frequency. The C-PCM (conductor-like polarizable continuum model) solvation
general instrument settings used for spectral acquisition were model.46,47 In continuum models, the solute is embedded into a
as follows: microwave power, 20 mW; modulation amplitude, 1 shape-adapted cavity surrounded by a dielectric continuum
G; received gains, 9 × 101 to 9 × 102; scan time, 5 s; sweep width, characterized by its dielectric constant. Calculations were
100 G. Spectra were recorded at room temperature, and performed in water (ε = 78.35). The atomic charge analysis was
measurements were performed using a capillary tube. The performed using the natural population analysis (NPA) within
spectrum simulation was carried out using the WINSIM the NBO (Natural Bond Orbital) framework. The IP was
program,43 available as free software from Public Electron calculated as follows:
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Paramagnetic
Resonance
Software
Tools IP = E(ArNO•+) – E(ArNO), where ArNO•+ is the radical cation
(http://www.niehs.nih.gov/research/resources/software/tox-
pharm/tools/).
obtained after electron withdraw from the neutral form of the
PBN derivatives, named here ArNO. It is worth mentioning that
The solvents were of the highest purity grade and used without IP corresponds to the adiabatic energy of electron withdraw
further purification. The trinitrone TN was synthesized and energy, i.e., considering the radical cation energy using the
purified as previously described.16 To generate the neutral form geometry. All calculations were performed with
hydroxymethyl (•CH2OH) radical, nitrone (100 mM) was Gaussian 09 Rev. A2.48
dissolved in a Fenton system containing hydrogen peroxide (6%)
and iron (II) sulfate (6 mM) in methanol. The method of kinetic
Conflicts of interest
There are no conflicts to declare.
competition permitted to evaluate the ratio of the second-
•
order rate constants for the trapping of CH2OH by one of the
nitrone N of interest (kN, corresponding to the compounds 1–7)
and TN (kTN) or PBN (kPBN) used as competitive inhibitors. Then,
the commercially available PBN was tested versus TN to
determine the ratio of the rate constants for the trapping of
Acknowledgements
Anaïs Deletraz was the recipient of a fellowship from the
“Région Provence Alpes Côte d’Azur”. We thank the company
Neuro-Sys (www.neuro-sys.com) for supporting this work.
Thanks are also due: to Fédération RENARD IR-RPE CNRS 3443
for providing funds for the EPR studies; to CALI for
computational facilities and the National Program of
Sustainability I from the Ministry-of-Youth, Education and
Sports of the Czech Republic (LO1305); and to Miss Jamila
Chetouani for her excellent technical assistance in acquiring
computational data. This study was conducted with the
financial support of the European Regional Development Fund,
the French Government, the “Région Provence Alpes Côte
d'Azur”, the Departmental Council of Vaucluse and the Urban
Community of Avignon.
•CH2OH by PBN and by TN: i.e., kPBN/kTN for nitrones
1-5 and 7.
The concentrations of the various nitrones were varied to keep
the [N]/[TN] ratio between 2 and 5. For each nitrone, at least
five experiments were repeated twice. In each case, a series of
10 EPR spectra was then recorded (scan time for a single
spectrum; 20 s). The signal-to-noise ratio was improved using a
SVD procedure, as described elsewhere.44 The signal recorded
exactly 2 min after the beginning of the reaction was then
simulated using the WinSim software to determine the relative
areas of the
N-CH2OH and TN-CH2OH adducts. In this approach,
the R/r ratio was evaluated as follows:
푹
풓
풂풓풆풂 풐풇 푵−푹 풔풊품풏풂풍+풂풓풆풂 풐풇 푻푵−푹 풔풊품풏풂풍
=
(2)
풂풓풆풂 풐풇 푻푵−푹 풔풊품풏풂풍
General Computational Methods. Geometries and energies
were optimized using DFT formalism with the hybrid meta-GGA
(Generalized Gradient Approximation) M06-2X functional and
the 6-31+G(d,p) basis set. Three other hybrid functionals were
tested on PBN, taken as a reference compound, namely B3LYP,
B3P86 and PBE1PBE (data not shown). No differences were
observed in terms of atomic charges, one of the major
parameters under scrutiny in this work, hence the M06-2X
functional was chosen for all compounds, as a good standard.45
The use of larger basis set (namely 6-311+g(2d,3pd)) did not
exhibit significant differences in terms of atomic charges with
respect to 6-31+G(d,p), suggesting the absence of basis set
effect owing to the relatively small molecular size of the series.
Therefore, the 6-31+G(d,p) basis set was used for all
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DOI: 10.1039/C9NJ03805A
MeO-derivatives of phenyl nitrones were synthesized and their electrochemical and spin-
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