Synthesis, structure, theoretical and experimental in vitro antioxidant/pharmacological properties of α-aryl, N-alkyl nitrones, as potential agents for the treatment of cerebral ischemia

Article abstract of DOI:10.1016/j.bmc.2010.11.053

The synthesis, structure, theoretical and experimental in vitro antioxidant properties using the DPPH, ORAC, and benzoic acid, as well as preliminary in vitro pharmacological activities of (Z)-α-aryl and heteroaryl N-alkyl-nitrones 6-15, 18, 19, 21, and 23, is reported. In the in vitro antioxidant activity, for the DPPH radical test, only nitrones bearing free phenol groups gave the best RSA (%) values, nitrones 13 and 14 showing the highest values in this assay. In the ORAC analysis, the most potent radical scavenger was nitrone indole 21, followed by the N-benzyl benzene-type nitrones 10 and 15. Interestingly enough, the archetypal nitrone 7 (PBN) gave a low RSA value (1.4%) in the DPPH test, or was inactive in the ORAC assay. Concerning the ability to scavenge the hydroxyl radical, all the nitrones studied proved active in this experiment, showing high values in the 94-97% range, the most potent being nitrone 14. The theoretical calculations for the prediction of the antioxidant power, and the potential of ionization confirm that nitrones 9 and 10 are among the best compounds in electron transfer processes, a result that is also in good agreement with the experimental values in the DPPH assay. The calculated energy values for the reaction of ROS (hydroxyl, peroxyl) with the nitrones predict that the most favourable adduct-spin will take place between nitrones 9, 10, and 21, a fact that would be in agreement with their experimentally observed scavenger ability. The in vitro pharmacological analysis showed that the neuroprotective profile of the target molecules was in general low, with values ranging from 0% to 18.7%, in human neuroblastoma cells stressed with a mixture of rotenone/oligomycin-A, being nitrones 18, and 6-8 the most potent, as they show values in the range 24-18.4%.

Full text of DOI:10.1016/j.bmc.2010.11.053

Bioorganic & Medicinal Chemistry 19 (2011) 951–960
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, structure, theoretical and experimental in vitro
antioxidant/pharmacological properties of
a-aryl, N-alkyl nitrones,
as potential agents for the treatment of cerebral ischemia
Abdelouahid Samadi a, , Elena Soriano a, Julia Revuelta a, Carolina Valderas a, Mourad Chioua a,
⇑
Ignacio Garrido b, Begoña Bartolomé b, Isabelle Tomassolli c, Lhassane Ismaili c, Laura González-Lafuente d,
Mercedes Villarroya d, Antonio G. García d,e, María J. Oset-Gasque f, José Marco-Contelles a,
⇑
a Laboratorio de Radicales Libres y Química Computacional (IQOG, CSIC), 3, Juan de la Cierva, 28006 Madrid, Spain
b Instituto de Fermentaciones Industriales, 3, Juan de la Cierva, 28006 Madrid, Spain
c Equipe 2SBP EA4627, Laboratoire de Chimie Thérapeutique, UFR Sciences Médicales et Pharmaceutiques, 4 Place de Saint Jacques, 25030 Besançon, Cedex, France
d Instituto Teófilo Hernando, Departamento de Farmacología y Terapéutica, Facultad de Medicina, C/ Diego de León 62, 28006 Madrid, Spain
e Servicio de Farmacología Clinica, Hospital Universitario de la Princesa, C/Diego de León 62, 28006 Madrid, Spain
f Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, Universidad Complutense de Madrid (UCM), 28040 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, structure, theoretical and experimental in vitro antioxidant properties using the DPPH,
Received 5 October 2010
Revised 22 November 2010
Accepted 23 November 2010
Available online 7 December 2010
ORAC, and benzoic acid, as well as preliminary in vitro pharmacological activities of (Z)-a-aryl and het-
eroaryl N-alkyl-nitrones 6–15, 18, 19, 21, and 23, is reported. In the in vitro antioxidant activity, for the
DPPH radical test, only nitrones bearing free phenol groups gave the best RSA (%) values, nitrones 13 and
14 showing the highest values in this assay. In the ORAC analysis, the most potent radical scavenger was
nitrone indole 21, followed by the N-benzyl benzene-type nitrones 10 and 15. Interestingly enough, the
archetypal nitrone 7 (PBN) gave a low RSA value (1.4%) in the DPPH test, or was inactive in the ORAC
assay. Concerning the ability to scavenge the hydroxyl radical, all the nitrones studied proved active in
this experiment, showing high values in the 94–97% range, the most potent being nitrone 14. The theo-
retical calculations for the prediction of the antioxidant power, and the potential of ionization confirm
that nitrones 9 and 10 are among the best compounds in electron transfer processes, a result that is also
in good agreement with the experimental values in the DPPH assay. The calculated energy values for the
reaction of ROS (hydroxyl, peroxyl) with the nitrones predict that the most favourable adduct-spin will
take place between nitrones 9, 10, and 21, a fact that would be in agreement with their experimentally
observed scavenger ability. The in vitro pharmacological analysis showed that the neuroprotective profile
of the target molecules was in general low, with values ranging from 0% to 18.7%, in human neuroblas-
toma cells stressed with a mixture of rotenone/oligomycin-A, being nitrones 18, and 6–8 the most potent,
as they show values in the range 24–18.4%.
Keywords:
Nitrones
Synthesis
Structure
Computational chemistry
ROS
Antioxidant activity
DPPH
ORAC
Peroxyl radical
Hydroxyl radical
Neuroprotection
Cerebral ischemia
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
1. Introduction
ischemia, and stroke model in rodents, that has been launched sev-
eral times in different programmes in advanced clinical studies,
For years, the ability of nitrones to act as good radical traps has
been in the origin of a number of basic and clinical studies aimed at
the investigation of their potential therapeutical application for the
treatment of cerebral ischemia, or neurodegenerative diseases
where reactive oxygen species (ROS) are implicated.1 This is the
with limited success, but always with renewed interest.4 However,
there is still a large consensus on the convenience of the neuropro-
tective strategy according to the recently published papers on the
area, and the efforts devoted to improving previous results or find-
ing new nitrones for the treatment of ischemic stroke.5 The case of
edaravone, a potent free radical scavenger, recently approved for
treatment of patients with acute stroke in Japan,6 also confirms
the viability of this approach. Between the nitrones, PBN, for in-
stance, inhibits the oxidation of lipoproteins,7 reduces oxidative
damage to erythrocytes and peroxidation of lipids due to phen-
ylhydrazine,8 and protects gerbils from brain stroke and mice
against MPTP toxicity.9 However, the mechanism of action of
case of (Z)-a
-phenyl-N-tert-butylnitrone (PBN),2 or nitrone
NXY-059,3 a well known free radical scavenger with high neuro-
protective profile in rat models of transient and permanent focal
⇑
Corresponding authors. Tel.: +34 91 562 29 00; fax: 34 91 564 48 53.
E-mail addresses: samadi@iqog.csic.es (A. Samadi), iqoc21@iqoqg.csic.es
(J. Marco-Contelles).
0968-0896/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.053

952
A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 951–960
PBN is not clear. In addition, the neuroprotective properties of nit-
rones seem to be related not to the simple ability to act as ROS
traps, but to the suppression of iNOS expression, cytokine accumu-
lation, and apoptosis.10 The formation of nitric oxide (NO) from
PBN spin adducts may play a role in the observed effects in the cen-
tral nervous system.11
Based on these grounds, we have recently started a project in
the context of a virtual research network (‘Retic RENEVAS’),
supported by the Spanish government (Instituto de Salud Carlos III,
Ministerio de Investigación, Ciencia e Innovación), and targeted to
(Br) substituents in the aromatic ring, respect to the parent PBN
compound. Heteroaryl nitrones 18, 19; 21, and 23 bear a 2-ha-
lo(Br, Cl) substituted pyridine, and an indole ring, respectively
(Table 1). Known nitrones 6,19 7,2,19 819 showed spectroscopic
data in good agreement with those reported in the literature,
while new ones (9–15, 18, 19, 21, and 23) have analytical and
spectral data in accordance with their structure (see Section 4).
2.2. Antioxidant evaluation
the synthesis and biological evaluation of a series of
(methyl, tert-butyl, benzyl) hetero(aryl) or nitrones, as potential
drugs for the treatment of cerebral ischemia.
a
-aryl, N-alkyl
The in vitro antioxidant activity of these nitrones was initially
determined by the use of the DPPH and ORAC tests.
In this work, we report our results in this area, based on the syn-
thesis, structure, and in vitro antioxidant/pharmacological proper-
ties of nitrones 6–15, 18, 19, 21, and 23, shown in Chart 1.
2.2.1. DPPH
The stable free radical 2,2-diphenyl-1-picylhydrazyl (DPPH) is a
useful reagent to investigate the scavenger properties of phenols,
catechols and anilines. It is now widely accepted that the reaction
between phenols and DPPH proceeds through two different mech-
anisms: (a) the direct hydrogen atom transfer (HAT) and (b) the
sequential proton loss electron transfer (SPLET) (Scheme 2).20
A freshly prepared DPPH solution exhibits a deep purple colour
with an absorption maximum at 517 nm. This purple colour gener-
ally disappears when an antioxidant is present in the medium. Thus,
antioxidant molecules can quench DPPH free radicals (by providing
hydrogen atoms or by electron donation, conceivably via a free-rad-
ical attack on the DPPH molecule) and convert them to colourless/
bleached product. In this assay, we measure the DPPH initial absor-
bance, and the absorbance once the potential antioxidant has been
added. The reduction of absorbance is a measure of the free DPPH
due to the action of the antioxidant. We have used curcumin as
the reference compound.21 The antioxidant activity was expressed
as the RSA% (Radical Scavenging Activity), calculated as follows:
2. Results and discussion
2.1. Synthesis
The selected nitrones 6–15, 18, 19, 21, and 23 in this work have
been synthesized using three different methods, as shown in
Scheme 1, by reacting the corresponding commercial (1, 4, 17,
and 18) or easily available aldehydes by the reported procedures,
(2,12 3,13 5,14 20,15 and 2216), with the corresponding N-substituted
hydroxylamines (method A,5d B17) or with the appropriate nitro
compounds (method C) (Table 1).18
Nitrones 6 and 8 (Chart 1) are N-alkyl analogues of the well
known nitrone 7 (PBN),2 which has been used here as a reference
compound. Nitrones 9–15 (Table 1) have been synthesized and
investigated in order to evaluate the effect of different electron
donor, protected (OMe) or free (OH), and electron withdrawing
RSA% ¼ 100½ðAo ꢀ AiÞ=AoÞ ꢁ 100
where Ao, and Ai, are the DPPH absorbance in absence and in pres-
ence of added nitrone, respectively.
O
The RSA (%) for nitrones 7 (PBN), 8–12, 18, 19, 21, and 23, at
0.5 mM, has been incorporated in Table 2. As shown, and as ex-
pected, nitrones 9, 10, and 13–15 bearing free phenol groups pro-
vided the best RSA (%) values. Comparing the RSA (%) values for
nitrone 10 (25.9 1.8) to compound 15 (40.7 2.5), it is clear that
the para is preferred to the meta for the position of the free
Ar
R
O
Methods
N
Ar
H
H
Scheme 1. Methods used for the synthesis of the nitrones. Reagents: RNHOHꢂHCl,
Na2SO4, Et3N, THF, MW (method A); RNHOHꢂHCl, NaOAc, MW (method B); RNO2,
Zn, EtOH (method C).
R1
R2
R1
R2
R3
O
N
O
N
O
O
X
N
X
X
N
X
16
17
6 R1, R2, X= H, R3= Me
X = Br
X = Cl
18 X = Br
X = Cl
1 R1, R2, X= H
19
7 R1, R2, X= H, R3= t-Bu
2 R1= OH, R2= OMe, X= Br
3 R1, R2= OMe, X= Br
8 R1, R2, X= H, R3= Bn
9 R1= OH, R2= OMe, X= Br, R3= t-Bu
4 R1= OMe, R2= OH, X= H
1
2
3
1
2
10
11
12
R = OH, R = OMe, X= Br, R = Bn
5
R = OMe, R = OH, X= Br
1
2
3
R , R = OMe, X= Br, R = t-Bu
1
2
3
R , R = OMe, X= Br, R = Bn
13 R1= OMe, R2= OH, X= H, R3= Me
14 R1= OMe, R2= OH, X= H, R3= Bn
15 R1= OMe, R2= OH, X= Br, R3= Bn
O
N
MeO
O
MeO
N
O
N
N
O
N
N
H
Me
Me
H
21
22
23
20
Chart 1.

A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 951–960
953
Table 1
O2N
O2N
Synthesis of nitrones 6–15, 18, 19, 21, and 23
H
Nitrone
Structure
Time
Yield (method)
75% (B)
N
N
NO2
N
N
NO2
Me
O2N
DPPH-H
(HAT)
O2N
N
O
6
5 min
DPHP
+
N
O
7 (PBN)
6 h
48% (C)
84% (B)
Ar-O + DPPH-H
(A) Ar-OH
DPPH
N
O
8
3 min
Ar-O
+
H
(SPLET)
(B) Ar-OH
Ar-O
HO
Ar-O
+
DPPH
+
DPPH
N
O
9
3 h
65% (A)
91% (A)
42% (B)
DPPH-H
MeO
HO
DPPH +
H
Br
Br
N
O
Scheme 2. Reaction of phenols with DPPH. Reagents: (A) HAT and (B) SPLET
10
11
55 min
2 min
mechanisms.
MeO
MeO
N
O
capability, using fluorescein (FL) as the fluorescent probe. The
peroxydation of FL gives nonfluorescent products at 520 nm.
Consequently, the remaining fluorescence of FL in presence of
the free radical scavenger nitrone must be a measure of the anti-
oxidant activity of the compound under study. There are three
ORAC types of assays: hydrophilic ORAC, (ORAC-H), lipophilic
ORAC (ORAC-L), and total ORAC. In our work, we used the
ORAC-H, using trolox as a reference compound.23 In Table 2,
the values obtained in this experiment, expressed as micromol
trolox/micromol compound, are shown. We conclude from these
data that most of the nitrones showed a significant antioxidant
activity regarding peroxyl radicals, the most potent being nitrone
indole 21 (7.98) followed by the phenol bearing N-benzyl ben-
zene-type nitrones 10 (7.36), and 15 (6.40). Interestingly, the
comparison between nitrone 13 (or 14) to 10 highlights the
importance of a bromine atom in the ortho position respect to
the nitrone moiety to give nitrones with an important free radi-
cal scavenger activity in this assay. This is just the contrary of
what we observed in the structure–activity relationship for the
bromine motif in the DPPH test (see above). Concerning the type
of the N-substituent, the benzyl bearing nitrones seem more po-
tent than those bearing a t-butyl group (compare nitrones 9 and
10). Notice, also, that the known nitrone 7 (PBN), was inactive in
this assay.
MeO
MeO
Br
Br
N
O
12
13
14
15
3 min
3 h
64% (B)
60% (A)
55% (A)
70% (A)
MeO
MeO
Me
N
O
HO
MeO
N
O
4 h
HO
MeO
N
O
3 h
HO
Br
N
O
18
19
21
3 h
76% (A)
N
Br
Cl
N
O
1 h 45 min
99% (A)
N
MeO
6 h
20% (56%)a (C)
N
Me
N
O
+
In view of these results, we have investigated next the ability of
these nitrones to trap hydroxyl free radicals.
N
23
33 h
76% (91%)a (A)
O
N
H
Å
2.2.3. Free radical OH scavenger activity
a
Taking into account the recovered unreacted starting product.
Between the ROS, the ÅOH free radical is possibly the most toxic,
as it reacts with a number of biological important molecules such
as DNA, lipids or carbohydrates. In order to asses the capacity of
our molecules to trap this radical species, we have used the benzo-
ate hydroxylation method.24,25 The hydroxyl radical ÅOH generated
in a Fenton reaction (Scheme 3) reacts with benzoate to produce
the fluorescent hydroxybenzoates, which can be measured spec-
trofluorimetrically with excitation at 305 nm and emission at
407 nm. However, the hydroxylation of benzoate could be inhib-
ited by ÅOH-radical scavengers such as nitrones, and then, the
fluorescence generally decrease.
hydroxyl group, respect to the C@N(O)Bn moiety, in order to have
the best free radical scavenger activity. Similarly, the elimination of
the bromine atom at the ortho position in nitrone 15 affords a two-
fold more potent nitrone 14 (88.2% RSA), the most potent in this
series of compounds for this assay. Notice, also, that changing
the benzyl (in nitrone 14) for a methyl (in nitrone 13) group in
the alkyl substituent, has no influence on the RSA(%) values. Final-
ly, the archetypal nitrone 7 (PBN) gave a low RSA(%) value under
our experimental conditions, rising to 16% at 10 mM, a result that
is in good agreement with the reported 20% at the same concentra-
tion.5c The other reference molecules used in this assay, such as
curcumin and quercetin gave RSA, 35.0% and 94.2%, values,
The fluorescence of the hydroxylated benzoate products was
measured after 2 h of incubation at 37 °C, in absence and in
presence of the nitrones. The RSA (%) values are shown in Table 2.
All the nitrones studied were active in this experiment, showing
high values, in the 94–97% range, the most potent corresponding
to nitrone 14 that shows a 96.9%. This activity is in the same range
as the reference compounds curcumin (96.8%) and querecetin
(96.6%).
respectively, at 15 lM.
2.2.2. ORAC
The ORAC (Oxygen Radical Absorbance Capacity) assay22
measures the peroxyl (ROOÅ) free radical scavenger compounds

954
A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 951–960
Table 2
Fe2+ + H2O2
Fe3+
+
OH + OH
Antioxidant activity of nitrones 6–15, 18, 19, 21, and 23 determined by ORAC, DPPH,
and OHÅ assaysa
OH + C6H5CO2
HOC6H4CO2
Nitrone Structure
DPPH
[(RSA
(%)]b
ORACe
OHÅ [(RSA
(%)]f
OH
R2
R2
R1
N
O
+
OH
R1
N
O
Me
N
O
6
0.8 0.7
1.4 0.9
0.6 0.3
0.0 0.0
95.3 0.8
94.9 1.0
95.7 0.5
Å
Scheme 3. Fenton’s reaction and free radical reaction of radical OH with a nitone.
N
O
7 (PBN)
0.0 0.0
N
O
8
0.01 0.00
HO
N
O
9
31.1 2.1
25.9 1.8
0.8 0.9
5.58 0.33
7.36 0.32
0.03 0.00
96.3 0.5
94.6 0.9
96.3 0.8
O
MeO
HO
Br
N
N
O
N
O
10
(Z)-7
MeO
Br
(E)-7
MeO
Chart 2. Isomers (E)- and (Z)-7.
N
O
11
MeO
MeO
Br
Br
N
O
that the Z-isomer is 15.9 kcal/mol more stable than the E-isomer,
due to a strong steric repulsion between the phenyl and the t-Bu
or Bn groups in the E-isomer. To minimize these repulsions, the
C@N bond is elongated from 1.317 Å in the Z-isomer to 1.329 Å
in isomer E-, and the CCN and CNC bond angles open from
126.8° and 121.9° in isomer Z- to 138.5°, and to 127.6° in isomer
E-, respectively. This conclusion is supported by the chemical shifts
(dH) in the 1H NMR spectra, and the published crystallographic data
for C-aryl-nitrones.26
12
0.2 0.2
87.6 1.2
88.2 1.0
40.7 2.5
0.03 0.002 95.1 1.3
MeO
MeO
Me
N
O
13
3.35 0.07
2.93 0.07
6.40 0.35
96.5 0.7
96.9 0.7
95.7 1.0
HO
MeO
N
O
14
HO
MeO
N
O
15
We also evaluated the relative population of syn/anti rotamers
around the C–C bond for nitrone 19 (Chart 3). The repulsion be-
tween the aromatic ring and the nitroxyl moiety in the syn rotamer
induces a distortion of the C–C bond (Table 3) that prevents the
aryl-nitrone co-planarity. Obviously, this is a destabilizing effect,
absent in the anti rotamer, and that contributes to the higher sta-
bility of the anti-nitrone rotamers.
HO
Br
N
O
18
3.9 3.8
1.6 1.5
4.8 1.8
0.01 0.00
0.01 0.00
7.98 0.21
95.1 1.1
94.9 1.3
ndd
N
Br
Cl
N
O
19
21
N
MeO
2.3.2. Antioxidant capacity
First of all, we calculated the potential of ionization of nitrones
7–12, 18, 19, 21, 23, and curcumin as the reference compound
(Table 4).
N
Me
N
O
This parameter detected that nitrones 9 and 10 were the best
compounds in order to participate in electron transfer processes,
a result that is also in good agreement with the experimental val-
ues in the DPPH assay (see Table 2), and the fact that these nitrones
are phenol derivatives, well known for their ability to transfer
hydrogen atoms.27 These values also compare very favourably with
the potential of ionization obtained in the same experiment for
curcumin.
+
N
23
1.2 1.1
3.69 0.15
ndd
O
N
H
Curcumin
Quercetin
Trolox
35.0 0.3c ndd
96.8 0.6
96.6 0.5
94.3 1.6
94.2 0.9c 10.49 0.45
ndd
1.00 0.0
a
Data are expressed as mean values ( SEM) of at least three different experi-
ments in quadruplicate.
The determination of the physicochemical parameters impli-
cated in the reaction of a radical with a substrate may help in
b
Determined at 0.5 mM.
c
Determined at 15 lM.
nd, not determined.
d
e
f
micromol trolox/micromol compound.
Benzoic acid methods: determined at 50
l
M.
O
N
N
O
2.3. Computational chemistry. Theoretical calculations
N
Cl
N
Cl
2.3.1. Structural analysis of the nitrones
The most stable geometry for the C@N double bond has been
investigated in nitrone 7 (‘PBN’) (Chart 2). These calculations show
syn-19
anti-19
Chart 3.

A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 951–960
955
Table 3
Torsion of the CC bond, and energy differences between the anti and syn rotamers in
OH
nitrones 9, 11, 18, 19, and 21 (kcal/mol)a
N
O
9
11
18
19
21
Dihedral angle NCCC in A (°)
Eanti-syn (kcal/mol)
35.1
ꢀ0.7
55.5
ꢀ4.2
56.7
ꢀ6.0
54.6
ꢀ8.3
55.3
ꢀ4.5
D
Chart 4. Spin-density in the adduct 7 (PBN)-OH.
a
Geometry optimization using B3LYP/6-31G*.
In Table 4, we have included the calculated values for all the
spin-adducts nitrone-OHÅ. We did not calculate the correspon-
dence for curcumin, because, in this case, the position of the rad-
ical attack is unknown. These data show that the reaction is
strongly exothermic, and consequently, this is a very favourable
thermodynamic process.30 The energy differences between the
different nitrones were small, taking into account the high exo-
thermicity, a result that is in agreement with the experimental
values (see Table 2), showing that all these molecules are good
Table 4
The potential of ionization, Energies for the formation of adducts-spin nitrone-OHÅ
and nitrone-OOHÅ of nitrones 7–12, 18, 19, 21, 23, and curcumin
b
c
Nitrone Structure
Potential of
ionizationa
(eV)
D
(kcal/
mol)
Erxn
DErxn
(kcal/
mol)
N
O
7 (PBN)
7.10
7.45
ꢀ54.1
ꢀ50.7
ꢀ18.3
ꢀ19.8
Å
trapping agents, and react similarly to radical OH.
The results shown in Table 4 confirm the hypothesis that differ-
ent mechanisms account for trapping the different ROS, and
suggest that the ability to transfer hydrogen atoms has no relation
with the stability of the hydroxyl radical.31
N
O
8
HO
N
O
9
6.61
6.70
7.42
7.49
7.10
ꢀ51.0
ꢀ49.8
ꢀ53.4
ꢀ50.6
ꢀ52.7
ꢀ22.4
ꢀ23.7
ꢀ20.7
ꢀ18.3
ꢀ20.5
On the other hand, the computational calculation of the energy
reaction in the formation of the adduct-spin between nitrones and
the hydroperoxyl ÅOOH radical, has previously been used to predict
its reactivity.32 In the case of nitrones 7–12, 18, 19, 21, and 23, we
have observed that the most stable conformation for the adduct-
spin has an intramolecular OO–Hꢂ ꢂ ꢂO–N hydrogen bond (for
nitrone 21, see Chart 5) with bond lengths of 1.84 and 2.19 Å. Sim-
ilarly to the nitrone adducts-spin with radical OHÅ, the spin density
is now transferred and concentrated over the nitroxyl group N–O.
In nitrone 7 (PBN), the N and O atoms show values of 39% and 56%,
respectively.
The calculated values range from ꢀ16.8 to ꢀ23.7 kcal/mol
(Table 4), showing also an exothermic reaction between OOHÅ
and nitrones, although less strong than in the case of the OHÅ addi-
tion. These values predict that the most favourable adduct-spin
will take place between nitrones 9, 10, and 21, a fact that would
be in agreement with their experimentally observed scavenger
ability (Table 2); conversely, nitrone 23 would not be a good radi-
cal trap agent.
The capacity to cross the brain–blood–barrier (BBB) is of para-
mount importance for a drug in order to reach its biological target
and act as an active agent in the CNS. The experimental determina-
tion of the BBB parameter is not very easy, and this is the reason
why the theoretical prediction, using CSBBB (software ChemSili-
co)33 is an alternative path, although the method is not very reli-
able yet. As shown in Table 5, and taking into account that the
higher the log BBB is, the higher the ability to cross the BBB, the
predicted capacity in decreasing order would be as follows:
8 ꢃ 21 P 12, 11 > 10.
MeO
HO
Br
Br
N
O
10
MeO
MeO
N
O
11
MeO
MeO
Br
Br
N
O
12
MeO
N
O
18
N
B
N
O
19
21
7.39
7.02
ꢀ51.8
ꢀ50.9
ꢀ16.8
ꢀ23.7
N
MeO
Cl
N
N
O
Me
+
N
23
7.12
6.38
ꢀ48.3
ꢀ19.0
O
N
H
Curcumin
ndd
ndd
a
b
c
The potential of ionization of nitrones 7–12, 18, 19, 21, 23, and curcumin.
Energies for the formation of adducts-spin nitrone-OHÅ.
Energies for the formation of adducts-spin nitrone-OOHÅ.
nd, not determined.
d
the analysis of the antioxidant activity of our nitrones. In this con-
text, the determination of the reaction free energy in the formation
of the adduct-spin gives a good correlation between the values of
the potential of ionization and the experimental kinetic con-
stants.28 Accordingly, this theoretical approach can be useful in or-
der to predict the respective reactivities.
The NPA (Natural Population Analysis) proves that the spin den-
sity in the adduct is mainly delocalized over the N–O group.29 In
the case of nitrone 7 (PBN), for instance, the N and O atoms showed
values of 355% and 63%, respectively. In Chart 4 we have depicted
the calculated spin-density for the nitrone 7 and hydroxyl radical
adduct.
Chart 5. Hydrogen-bond in the adduct-spin nitrone 21-OOHÅ.

956
A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 951–960
Table 5
Then, we investigated the neuroprotective effect of nitrones
6–15, 18, 19, 21, and 23 against rotenone/oligomycin-A-induced
death of human neuroblastoma SH-SY5Y cells. The mixture of rote-
none plus oligomycin-A blocks mitochondrial electron transport
chain complexes I and V, respectively,36 inducing cell death by oxi-
dative stress.
Theoretical determination of log P, neuroprotection and log BBB, in nitrones 7–12, 18,
19, 21, 23, and curcumin
Nitrone Structure
log P Neuroprotection5g log BBBa
(%)
0.16
(0.15)
The neuroprotective effect of nitrones 6–15, 18, 19, 21, and 23
against this toxic stimulus was evaluated by the method of MTT
7 (PBN)
N
O
1.86
2.21
73
metabolic reduction,37a at the concentration of 1
l
M, in SH-SY5Y
N
O
0.55
(0.22)
8
111
neuroblastoma cells exposed to 30 lM rotenone plus 10 lM oligo-
mycin-A for 24 h, as described.37b As shown in Table 6, the neuro-
protective profile of the target molecules was in general low, with
HO
0.01
(0.19)
N
O
9
2.26
2.63
2.45
2.82
1.71
87
MeO
HO
Br
Br
Table 6
N
O
0.23
(0.10)
Neuroprotection induced by nitrones 6–15, 18, 19, 21, and 23 (1
lM) in human
10
111
70
neuroblastoma cells stressed with a mixture of rotenone/oligomycin-Aa
MeO
Nitrones Structure
MTT reduction (%
vehicle)
%
MeO
0.34
(0.23)
N
O
Protection
11
MeO
MeO
Me
Br
Br
N
O
6
64.7 5.4
69.6 2.9
70.8 7.1
18.7
18.6
18.4
N
O
0.36
(0.12)
12
116
76
MeO
7 (PBN)
N
O
0.00
(0.15)
N
O
18
N
O
8
N
B
0.03
(0.15)
HO
N
O
19
21
1.55
2.36
74
50
N
O
9
65
3
11
8.7
0
N
MeO
Cl
MeO
HO
Br
0.39
(0.17)
N
O
10
64.2 2.5
63.1 3.3
N
N
O
Me
MeO
Br
+
MeO
N
O
N
ꢀ0.05
11
23
2.02
3.16
60
(0.18)
MeO
MeO
Br
Br
O
N
H
N
O
Curcumin
106
ꢀ0.95
12
65.3 6.9
60.9 4.7
63.9 6.1
62.8 4.4
3
(0.34)
MeO
a
Standard shifts in parenthesis.
MeO
Me
N
O
13
9.7
16.9
13.4
HO
MeO
N
O
The determination of log P (partition coefficient in octanol–
14
water) has been carried out with VCCLAB software.34 The values
in table 5 suggest that these nitrones are soluble in lypophilic med-
ia, and, consequently, they should be active in the CNS. According to
the Lipinski rules, the log P should be less than 5,35 but others have
suggested that the optimal value must be between 1.5 and 2.0.5g
Goldstein and colleagues used QSAR for the determination of
the neuroprotection of nitrones using the log P values and the
HOMO orbital energies,5g and Eq. 1:
HO
MeO
N
O
15
HO
Br
N
O
18
60.1 7.3
61.4 5.5
24
0
N
Br
Cl
N
O
Neuroprotection ð%Þ ¼ 33:2 ꢄ log P ꢀ 2:15 ꢄ EðHOMOÞ ꢀ 379:43
ð1Þ
One of the most interesting results is that nitrones bearing the
N-benzyl group must show better neuroprotective profile than
those with a N-t-butyl substituent [12 vs 11; 8 vs 7; 10 vs 9]. In
addition, it seems that nitrones bearing the N-benzyl group show
better capacity to cross the BBB, and the type of the halogen (Br
vs Cl in nitrones 18 and 19) atom has no effect on these properties.
19
21
N
MeO
62.8
3
4.4
N
Me
N
O
+
N
23
64.1 2.1
8
O
N
H
NAC (1 mM)
Melatonin (10 nM)
63.1 3.7
59 2.4
25
17
2.4. In vitro neuroprotection assay
a
Next, the in vitro pharmacological analysis was carried out in
order to determine the neuroprotective capacity of these nitrones.
Data are expressed as the means SEM of at least three different cultures in
quadruplicate.

A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 951–960
957
nitrones 18, and 6–8, the most potent, showing values in the range
24–18.4%. Note also that phenol nitrone 14 showed a significant
neuroprotective value (16.9%). N-Acetylcisteine (NAC) at 1 mM
and melatonin at 10 nM were used as positive controls.
using Silica Gel 60 (230–400 mesh, Merck). Melting points were
determined on a Kofler block and are uncorrected. IR spectra were
obtained on a Perkin–Elmer Spectrum One spectrophotometer. 1H
NMR spectra were recorded with a Varian VXR-200S spectrometer,
using tetramethylsilane as internal standard and 13C NMR spectra
were recorded with a Bruker WP-200-SY. All the assignments for
protons and carbons were in agreement with 2D COSY, HSQC,
HMBC, and 1D NOESY spectra. Values with (*) can be interchanged.
Elemental analyses were conducted on a Carlo Erba EA 1108 appa-
ratus. 1,1-Diphenyl-2-picrylhyrazyl (DPPH) radical, sodium benzo-
ate, FeSO4ꢂ7H2O, EDTA, 30% H2O2, nitro blue tetrazolium chloride,
NADH and phenazine methosulfate were purchased from Sigma–
Aldrich. Phosphate buffer (0.1 M and pH 7.4) was prepared mixing
an aq KH2PO4 solution (50 mL, 0.2 M), and an aq of NaOH solution
(78 mL, 0.1 M); the pH (7.4) was adjusted by adding a solution of
KH2PO4 or NaOH.
3. Conclusions
In the context of our current project targeted to the develop-
ment of new drugs for the treatment of cerebral ischemia, we have
synthesized 11 new nitrones (9–15, 18, 19, 21, and 23), and three
known nitrones (6–8) for comparative purposes. The archetypal
nitrone 7 (PBN) has been used as internal reference compound.
Regarding the in vitro antioxidant activity, for the DPPH radical
test, and as expected, nitrones 13 and 14 bearing free phenol
groups gave the best RSA (%) values in this assay. In the ORAC anal-
ysis, the most potent radical scavenger was nitrone indole 21, fol-
lowed by the N-benzyl benzene-type nitrones 10, and 15. Notice,
also, that out of the five nitrones bearing a phenol group, the most
potent in the DPPH test was nitrone 14, bearing an hydroxyl group
and no bromine atom in para and ortho positions, respectively, re-
spect to the nitrone moiety. Conversely, the most potent in the
ORAC assay was nitrone 10, bearing an hydroxyl group and a bro-
mine atom in meta and ortho positions, respectively, respect to the
nitrone group. Very interestingly, the archetypal nitrone 7 (PBN)
gave a low RSA value (1.4%) in the DPPH test, or was inactive in
the ORAC assay. Concerning the ability to scavenge the hydroxyl
radical, we were aware that, since the hydroxyl radical is able to
react with everything just limited by its diffusion speed, the hydro-
xyl scavenging assay cannot make differences between the com-
pounds. Consequently, and not surprisingly, the nitrones studied
proved active in this experiment, showing high values in the
94–97% range, the most potent being nitrone 14.
4.2. General procedure for the synthesis of nitrones
Method A:5d In a 20 mL glass tube equipped with septa, the
aldehyde, dry Na2SO4 (2.81 equiv) and triethylamine (2 equiv)
were suspended in dry THF. Then, the hydroxylamine hydrochlo-
ride (2 equiv) was added. The mixture was stirred for 30 s, and
then exposed to MWI (250 W) at 80 °C during the time indicated
for each compound. When the reaction was over (TLC analysis),
the reaction mixture was diluted with water, extracted with
CH2Cl2, dried over anhydrous sodium sulphate, filtered and the
solvent was evaporated. The resultant solid was purified by col-
umn chromatography to give pure compounds. Method B:17 In a
20 mL glass tube equipped with septa, the aldehyde, the hydrox-
ylamine hydrochloride (1 equiv) and NaOAc (1.2 equiv) were ex-
posed to MWI (250 W) at 80 °C during the time indicated for
each compound. The formed solid was washed with CH2Cl2, the
solvent was evaporated and the resultant solid was purified by
column chromatography to give pure compounds. Method C:18
To a solution of the aldehyde in EtOH (0.14 M) was added
methyl-2-nitropropane (2 equiv) and zinc (3 equiv). The reaction
mixture was cooled to 0 °C, and glacial acetic acid (6 equiv) was
added dropwise. The reaction mixture was stirred at room tem-
perature for 6 h, and then was kept at 0 °C for 12 h. The precipi-
tate was filtered, washed with EtOH. The solvent was evaporated
and the resultant solid was purified by column chromatography
to give pure compounds
Regarding the prediction of the antioxidant power using theo-
retical calculations, the potential of ionization confirms that nitro-
nes 9 and 10 are the best compounds in electron transfer
processes, a result that is also in good agreement with the experi-
mental values in the DPPH assay. The calculated energy values for
the reaction of ROS (hydroxyl, peroxyl) with the nitrones predict
that the most favourable adduct-spin will take place between nit-
rones 9, 10 and 21, a fact that would be in agreement with their
experimentally observed scavenging ability. However, nitrone 23
would not be a good radical trap agent.
Finally, the in vitro pharmacological analysis, the neuroprotec-
tive profile of the target molecules was in general low, with values
ranging from 0% to 18.7%, in human neuroblastoma cells stressed
with a mixture of rotenone/oligomycin-A, the most potent nitrones
(6–8 and 18) showing values in the range 24–18.4%.38
4.2.1. (Z)-a
-Phenyl-N-methylnitrone (6)19
Following the general procedure (method B), reaction of benzal-
dehyde (1) (0.38 mL, 3.76 mmol), N-methylhydroxylamine hydro-
chloride (0.25 g, 3.13 mmol) and NaOAc (0.308 g, 3.76 mmol),
after 5 min and column chromatography (hexane/EtOAc, 1:1, v/
v), gave compound 6 (0.317 g, 75%): 1H NMR (300 MHz, CDCl3) d
8.26–8.18 (m, 2H), 7.46–7.40 (m, 3H), 7.38 (s, 1H), 3.85 (s, 3H);
MS (EI) m/z (%): 135 [M]+ (66), 134 [MꢀH]+ (66). Anal. Calcd for
C8H9NO: C, 71.09; H, 6.71; N, 10.36. Found: C, 70.87; H, 6.82; N,
10.24.
Overall, the nitrones studied in this work39 show a good antiox-
idant and neuroprotective profile, as the first step in our projected
investigation of their potential use as drugs for the treatment of
cerebral ischemia. Other studies and analyses are now in progress
and will be reported in due course.
4. Experimental part
4.1. General methods
4.2.2. (Z)-a
-Phenyl-N-tert-butylnitrone (7)2,19
Following the general procedure (method C), reaction of benzal-
dehyde (1) (0.48 mL, 4.71 mmol), 2-methyl-2-nitropropane
(1.02 mL, 9.42 mmol), Zn (924 mg, 14.13 mmol) and acetic acid
(1.61 mL, 28.26 mmol) in EtOH (35 mL), and column chromatogra-
phy (0.5% of MeOH in DCM), gave compound 7 (400 mg, 48%) as a
solid: mp 72–4 °C; 1H NMR (300 MHz, CDCl3) d 8.40–8.12 (m, 2H),
7.53 (s, 1H), 7.44–7.32 (m, 3H), 1.60 (s, 9H); MS (EI) m/z (%): 177
[M] (33), 121 (25), 57 (100). Anal. Calcd for C11H15NO: C, 74.54;
H, 8.53; N, 7.90. Found: C, 74.29; H, 8.71; N, 8.02.
Reactions were monitored by TLC using precoated silica gel alu-
minium plates containing a fluorescent indicator (Merck, 5539).
Detection was done by UV (254 nm) followed by charring with sul-
furic-acetic acid spray, 1% aqueous potassium permanganate solu-
tion or 0.5% phosphomolybdic acid in 95% EtOH. Anhydrous
Na2SO4 was used to dry organic solutions during work-ups and
the removal of solvents was carried out under vacuum with a ro-
tary evaporator. Flash column chromatography was performed

958
A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 951–960
4.2.3. (Z)-
a
-Phenyl-N-benzylnitrone (8)19
(841 mg, 64%): mp 164–6 °C; IR (KBr) 3068, 3010, 2951, 1592,
1497, 1443, 1387, 1277, 1218, 1171 cmꢀ1 1H NMR (300 MHz,
Following the general procedure (method B), reaction of benzal-
dehyde (1) (0.38 mL, 3.76 mmol), N-benzylhydroxylamine hydro-
chloride (0.5 g, 3.13 mmol) and NaOAc (0.308 g, 3.76 mmol), after
3 min, and column chromatography (hexane/EtOAc from 5:1 to
2:1, v/v), gave compound 8 (668 mg, 84%) as a solid: mp 84–6 °C;
1H NMR (300 MHz, CDCl3) d 8.25–8.12 (m, J = 9.8 Hz, 2H), 7.54–
7.30 (m, 9H), 5.01 (s, 2H); MS (EI) m/z (%): 211 [M] (31), 91
(100). Anal. Calcd for C14H13NO: C, 79.59; H, 6.20; N, 6.63. Found:
C, 79.31; H, 6.42; N, 6.72.
;
CDCl3) d 9.08 (s, 1H), 7.78 (s, 1H), 7.57–7.26 (m, 5H), 6.98 (s,
1H), 5.00 (s, 2H), 3.84 (s, 3H), 3.81 (s, 3H); 13C NMR (75 MHz,
CDCl3) d 150.5, 147.6, 133.1, 133.07, 129.3, 129.0 (2 C), 122.3,
115.3, 115.0, 111.5, 71.7, 56.1, 56.0; MS (EI) m/z (%): 349
[Mꢀ1] (4), 270 (54), 91 (100). Anal. Calcd for C16H16BrNO3: C,
54.87; H, 4.61; N, 4.00; Br, 22.82. Found: C, 54.87; H, 4.55; N,
4.02; Br, 22.68.
4.2.8. (Z)-a
-(40-Hydroxy-30-methoxyphenyl)-N-methylnitrone
(13)
4.2.4. (Z)-a
-(20-Bromo-50-hydroxy-40-methoxyphenyl)-N-tert-
butylnitrone (9)
Following the general procedure (method A), reaction of com-
mercial 3-hydroxy-4-methoxybenzaldehyde (4) (0.15 g, 1 mmol),
dry Na2SO4 (0.39 g, 2.8 mmol), N-methylhydroxylamine hydro-
chloride (0.16 g, 2 mmol) and triethylamine (0.28 mL, 2 mmol) in
THF (1.35 mL), after 3 h, and column chormatography (2% of MeOH
in DCM), gave nitrone 13 (0.18 mg, 60%): mp 177–9 °C; IR (KBr)
2992, 2963, 2938, 2744, 2585, 1594, 1528, 1423, 1403, 1289,
Following the general procedure (method A), reaction of 2-bro-
mo-5-hydroxy-4-methoxybenzaldehyde (2)12 (44 mg, 0.192 mmol),
Na2SO4 (77 mg, 0.54 mmol), Et3N (50 lL, 0.38 mmol) and N-tert-
butylhydroxylamine hydrochloride (48 mg, 0.384 mmol) in THF
(1.3 mL), after 3 h, and column chromatography (hexane/EtOAc
from 2:1, v/v), nitrone 9 (38 mg, 65%) as a solid: mp 186–8 °C; IR
(KBr) 2975, 2937, 2762, 1598, 1498, 1428, 1392, 1362, 1274,
1261, 1153, 1124 cmꢀ1 1H NMR (400 MHz, DMSO) d 9.63 (br s,
;
1098, 1028 cmꢀ1 1H NMR (300 MHz, CDCl3) d 9.18 (s, 1H), 8.77
;
1H, OH), 8.11 (d, J = 1.6 Hz, 1H, CH-2), 7.64 (s, 1H), 7.54 (dd,
J = 8.4 and 1.6 Hz, 1H, CH-6), 8.21 (d, J = 8.4 Hz, 1H, CH-5), 3.74
(s, 3H, OCH3), 3.68 (s, 3H, NCH3); 13C NMR (100 MHz, CDCl3) d
148.3 (C-3), 146.8 (C-4), 133.8 (CH@N), 122.9 (C-1), 122.3 (C-6),
115.2 (C-5), 111.4 (C-2), 55.4 (OCH3), 53.5 (NCH3); MS (ES) m/z
(%): 204 [M+Na]+ (30), 182.2 [M+H]+ (100). Anal. Calcd for
C9H11NO3: C, 59.66; H, 6.12; N, 7.73. Found: C, 59.45; H, 6.31; N,
7.84.
(s, 1H), 8.00 (s, 1H), 7.01 (s, 1H), 3.85 (s, 3H), 1.62 (s, 9H); 13C
NMR (75 MHz, CDCl3) d 149.7, 145.6, 131.8, 121.9, 116.2, 115.6,
114.6, 71.3, 56.0, 28.6; MS (EI) m/z (%): 302 [M] (5), 222 (19),
166 (100), 57 (40). Anal. Calcd for C12H16BrNO3: C, 47.70; H,
5.34; N, 4.64; Br, 26.44. Found: C, 47.54; H, 5.21; N, 4.72; Br, 26.45.
4.2.5. (Z)-a
-(20-Bromo-50-hydroxy-40-methoxyphenyl)-N-
benzylnitrone (10)
Following the general procedure (method A), reaction of 2-bromo-
5-hydroxy-4-methoxybenzaldehyde (2)12 (200 mg, 0.866 mmol),
Na2SO4 (344 mg, 2.42 mmol), Et3N (0.24 mL, 1.73 mmol) and N-ben-
zylhydroxylamine hydrochloride (276 mg, 1.73 mmol) in THF
(5.8 mL), after 55 min, and column chromatography (hexane/AcOEt
1:1, v/v), gave nitrone 10 (266 mg, 91%) as a solid: mp >230 °C; IR
(KBr) 3082, 2836, 1592, 1501, 1454, 1439, 1413, 1277, 1207, 1174,
1144, 1026 cmꢀ1; 1H NMR (300 MHz, DMSO) d 9.50 (s, 1H), 8.79 (s,
1H), 7.94 (s, 1H), 7.60–7.27 (m, 5H), 7.20 (s, 1H), 5.13 (s, 2H), 3.80
(s, 3H); 13C NMR (75 MHz, DMSO) d 149.3, 145.20, 134.6, 131.4,
129.0, 128.4, 128.3, 122.1, 116.1, 114.9, 111.8, 70.3, 55.9; MS (EI)
m/z (%): 336 [M] (1), 256 (54), 91 (100). Anal. Calcd for C15H14BrNO3:
C, 53.59; H, 4.20; N, 4.17; Br, 23.77. Found: C, 53.38; H, 4.43; N, 4.33;
Br, 23.66.
4.2.9. (Z)-a
-(40-Hydroxy-30-methoxyphenyl)-N-benzylnitrone
(14)
Following the general procedure (method A), reaction of com-
mercial 4-hydroxy-3-methoxybenzaldehyde (4) (0.15 g, 1 mmol),
dry Na2SO4 (0.39 g, 2.8 mmol), N-benzylhydroxylamine hydro-
chloride (0.32 g, 2 mmol) and triethylamine (0.28 mL, 2 mmol)
in THF (1.35 mL), after 4 h, and column chromatography (1% of
MeOH in DCM), gave compound 14 (0.14 g, 55%): mp 160–2 °C;
IR (KBr)
;
m 3226, 2956, 2934, 2556, 1625, 1580, 1464, 1288 cm
ꢀ1
1H NMR (400 MHz, CDCl3) d 8.52 (d, J = 2 Hz, 1H, CH-2),
7.48–7.42 (m, 2H), 7.41–7.35 (m, 3H), 7.31 (s, 1H, CH@N), 7.19
(dd, J = 8.4 and 2 Hz, 1H, CH-6), 6.90 (d, J = 8.8 Hz, 1H, CH-5),
6.07 (br s, 1H, OH), 5.01 (s, 2H, CH2), 3.84 (s, 3H, OCH3); 13C
NMR (100 MHz, CDCl3) d 148.4 (C-4), 146.3 (C-3), 135.2 (CH@N),
133.0 (C-Ph); 129.1 (2 ꢁ CH), 128.8 (2 ꢁ CH), 124.1 (C-6), 122.6
(C-1), 114.4 (C-5), 110.7 (C-2), 70.3 (CH2), 55.8 (OCH3); MS (ES)
m/z (%): 258.2 [M+H]+ (100), 280.2 [M+Na]+ (30). Anal. Calcd for
4.2.6. (Z)-a-(2-Bromo-4,5-dimethoxyphenyl)-N-tert-butylnitrone
(11)
Following the general procedure (method B), reaction of 2-bro-
mo-4,5-dimethoxybenzaldehyde (3)13 (0.47 g, 1.92 mmol) with
N-tert-butylhydroxylamine hydrochloride (0.2 mg, 1.6 mmol) and
NaOAc (157 mg, 1.92 mmol), after 2 min, and column chromatog-
raphy [hexane/AcOEt, from 5:1 to 2:1, v/v], gave compound 11
(213 mg, 42%) as a solid: mp 123–5 °C; IR (KBr) 2971, 1502,
C15H15NO3: C, 70.02; H, 5.88; N, 5.44. Found: C, 69.88; H, 5.75;
N, 5.31.
4.2.10. (Z)-a
-(20-Bromo-40-hydroxy-5-methoxyphenyl)-N-
benzylnitrone (15)
1285, 1217, 1168, 1023 cmꢀ1 1H NMR (300 MHz, CDCl3) d 9.18
;
Following the general procedure (method A), reaction of of
2-bromo-4-hydroxy-5-methoxybenzaldehyde
(5)14
(0.044 g,
(s, 1H), 7.95 (s, 1H), 7.04 (s, 1H), 3.91 (s, 3H), 3.86 (s, 3H), 1.58
(s, 9H); 13C NMR (75 MHz, CDCl3) d 150.3, 147.8, 129.1, 122.9,
115.6, 115.4, 111.7, 71.6, 56.2, 56.1, 28.4; MS (EI) m/z (%): 315
[M] (4), 236 (13), 180 (100), 57 (34). Anal. Calcd for C13H18BrNO3:
C, 49.38; H, 5.74; N, 4.43. Found: C, 49.61; H, 5.92; N, 4.48.
0.19 mmol), dry Na2SO4 (0.077 g, 0.54 mmol), triethylamine
(0.050 mL, 0.38 mmol) and N-benzylhydroxylamine hydrochloride
(0.12 g, 1 mmol) in THF (1 mL), after 3 h, and column chromatogra-
phy (3% of MeOH in DCM), gave nitrone 15 (0.12 g, 70%). mp 206–
208 °C; IR (KBr)
1505, 1478, 1415, 1391, 1295, 1209, 1169, 1113, 875, 704 and
508 cmꢀ1 1H NMR (300 MHz, CDCl3) d 9.15 (s, 1H, CH-6), 7.85 (s,
m 3114, 3002, 2961, 2936, 2906, 2694, 2518, 1579,
4.2.7. (Z)-a
-(20-Bromo-40,50-dimethoxyphenyl)-N-benzylnitrone
(12)
;
1H, CH@N), 7.53–7.37 (m, 5H), 7.16 (s, 1H, CH-3), 6.09 (br s, 1H,
OH), 5.06 (s, 2H, CH2), 3.91 (s, 3H, OCH3); 13C NMR (300 MHz,
CDCl3) d 148.0, 145.3, 133.8, 132.9, 129.3, 129.1, 129.0, 121.5,
118.7, 115.7, 111.3, 71.5, 56.1; EM (ES) m/z (%): 336.0 [M+H]+
(100), 360.0 [M+Na+H]+ (17), 695.3 [2M+Na]+ (1);. Anal. Calcd for
Following the general procedure (method B), reaction of 2-
bromo-4,5-dimethoxybenzaldehyde (3)13 (1.1 g, 4.5 mmol), with
N-benzylhydroxylamine hydrochloride (0.6 g, 3.8 mmol) and
NaOAc (0.369 g, 4.5 mmol), after 3 min, and column chromatog-
raphy (hexane/EtOAc from 5:1 to 2:1, v/v), gave nitrone 12

A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 951–960
959
C15H14BrNO3: C, 53.59; H, 4.20; N, 4.17; Br, 23.77. Found: C, 53.37;
H, 4.08; N, 4.23; Br, 23.49.
4.3. Determination of antioxidant activity
4.3.1. DPPH radical
4.2.11. (Z)-
a
-(20-Bromo-3-pyridyl)-N-tert-butylnitrone (18)
The assay was carried out in 96-well microplates and the final
Following the general procedure (method A), reaction of com-
mercial 2-bromonicotinaldehyde (16) (100 mg, 0.54 mmol),
Na2SO4 (216 mg, 1.52 mmol), Et3N (0.15 mL, 1.08 mmol) and N-
t-butylhydroxylamine hydrochloride (135 mg, 1.08 mmol) in THF
(4 mL), after 3 h, and column chromatography (hexane/EtOAc
from 4:1, v/v), gave compound 18 (106 mg, 76%) as yellow oil:
1H NMR (300 MHz, CDCl3) d 9.56 (dd, J = 8.0, 2.0 Hz, 1H), 8.24
(dd, J = 4.7, 2.0 Hz, 1H), 8.02 (s, 1H), 7.27 (dd, J = 7.9, 4.7 Hz,
1H), 1.57 (s, 9H); 13C NMR (75 MHz, CDCl3) d 149.6, 143.0,
136.7, 128.1, 127.2, 123.0, 72.6, 28.3; MS (EI) m/z (%): 257 [M]
(23), 201 (11), 177 (16), 121 (75), 57 (100). Anal. Calcd for
reaction mixture was 200 lL per well. A methanol DPPH solution
(1.35 ꢁ 10ꢀ4 M, final concentration) (2.7 mL) was mixed with dif-
ferent concentrations of the nitrones (0, 100, 200, 300, 400, and
500
methanolic stock solution (10ꢀ3 M). The resulting mixture was
incubated for 2 h at rt in the dark. After this time, 200 L was
lM, final concentration) (0.3 mL) prepared freshly from a
l
placed in a 96-well microplate and the absorbance of DPPH rad-
ical was measured at 517 nm in a spectrophotometric plate read-
er (FluoStar OPTIMA, BMG Labtech). All reaction mixtures were
prepared in quadruplicate and at least three independent runs
were performed for each sample. The antioxidant activity was
determined as the RSA% (sadical scavenging activity), calculated
as follows: RSA% = 100[(Ao ꢀ Ai)/Ao) ꢁ 100; where Ao and Ai is
the DPPH absorbance in absence and in presence of added nit-
rone concentration i, respectively. Data are expressed as means
SEM.
C10H13BrN2O: C, 46.71; H, 5.10; N, 10.89; Br, 31.08. Found: C,
47.00; H, 5.39; N, 10.73; Br, 30.87.
4.2.12. (Z)-a
-(20-Chloro-3-pyridyl)-N-t-butylnitrone (19)
Following the general procedure (method A), reaction of com-
mercial 2-chloronicotinaldehyde (17) (100 mg, 0.71 mmol),
Na2SO4 (283 mg, 1.99 mmol), Et3N (0.19 mL, 1.4 mmol) and N-t-
butylhydroxylamine hydrochloride (177 mg, 1.4 mmol) in THF
(5 mL), after 105 min and column chromatography (hexane/EtOAc
from 4:1, v/v), gave compound 19 (154 mg, 99%): mp 36–8 °C; IR
4.3.2. ORAC assay
The radical scavenging activity of the nitrone was determined
by the ORAC method using fluorescein as a fluorescence probe.23
Briefly, the reaction was carried out at 37 °C in 75 mM phosphate
(KBr) 2975, 1556, 1377, 1140 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d
buffer (pH 7.4) and the final assay mixture (200
rescein (70 nM), 2,20-azobis(2-methyl-propionamidine)-dihydro-
chloride (12 mM), and antioxidant [Trolox (1–8 M) or nitrone
lL) contained fluo-
9.67 (d, J = 7.9 Hz, 1H), 8.30 (d, J = 4.2 Hz, 1H), 8.05 (s, 1H), 7.29
(dd, J = 7.9, 4.2 Hz, 1H), 1.61 (s, 9H); 13C NMR (75 MHz, CDCl3) d
149.7, 149.2, 136.9, 125.9, 124.8, 122.8, 72.5, 28.3; MS (ES) m/z
(%): 213 [M++1], 235 [M+Na]+, 447 [2M+Na]+. Anal. Calcd for
l
(at different concentrations)]. A Polarstar Galaxy plate reader
(BMG Labtechnologies GmbH, Offenburg, Germany) with 485-P
excitation and 520-P emission filters was used. The equipment
was controlled by the Fluostar Galaxy software version (4.11–0)
for fluorescence measurement. Black 96-well untreated micro-
plates (Nunc, Denmark) were used. The plate was automatically
shaken before the first reading and the fluorescence was recorded
every minute for 98 min. 2,20-Azobis(2-methyl-propionamidine)-
dihydrochloride and Trolox solutions were prepared daily and fluo-
rescein was diluted from a stock solution (1.17 mM) in 75 mM
phosphate buffer (pH 7.4). All reaction mixtures were prepared
in duplicate and at least three independent assays were performed
for each sample. Fluorescence measurements were normalized to
the curve of the blank (no antioxidant). From the normalized
curves, the area under the fluorescence decay curve (AUC) was cal-
culated as:
C10H13ClN2O: C, 56.47; H, 6.16; N, 13.17; Cl, 16.67. Found: C,
56.16; H, 6.40; N, 12.99; Cl, 16.35.
4.2.13. (Z)-a-N-(5-Methoxy-1-methyl-1H-2-indolyl)-N-tert-
butylnitrone (21)
Following the general procedure (method C), reaction of 5-meth-
oxy-1-methyl-1H-indol-2-carbaldehyde (20)15 (303 mg, 1.6 mmol),
2-methyl-2-nitropropane (0.34 mL, 3.2 mmol), Zn (314 mg,
4.8 mmol) and acetic acid (0.55 mL, 9.6 mmol) in EtOH (12 mL), after
column chromatography (hexane/EtOAc, from 4:1 to 1:1, v/v), gave
unreacted starting material 20 (189 mg), and nitrone 21 (87 mg,
56%): mp 182–4 °C; IR (KBr) 2953, 1561, 1510, 1484, 1339, 1212,
1144, 1028 cmꢀ1 1H NMR (300 MHz, CDCl3) d 8.11 (s, 1H), 7.73 (s,
;
1H), 7.15 (d, J = 8.9 Hz, 1H), 7.06 (d, J = 2.3 Hz, 1H), 6.92 (dd, J = 8.9,
2.3 Hz, 1H), 3.83 (s, 3H), 3.70 (s, 3H), 1.62 (s, 9H); 13C NMR
(75 MHz, CDCl3) d 154.4, 133.9, 130.8, 128.0, 120.0, 115.2, 109.8,
107.6, 102.8, 70.6, 55.9, 30.0, 28.5; MS (EI) m/z (%): 260 [M] (100),
229 (9), 204 (35), 187 (94). Anal. Calcd for C15H20N2O2: C, 69.20;
H, 7.74; N, 10.76. Found: C, 69.31; H, 7.83; N, 10.60.
AUC ¼ 1 þ i¼98 fi=f0
X
i¼1
where f0 is the initial fluorescence reading at 0 min and fi is the fluo-
rescence reading at time i. The net AUC corresponding to a sample
was calculated as follows:
4.2.14. (Z)-a-(1H-3-Indolyl)-N-tert-butylnitrone (23)
Following the general procedure (method A) reaction of 1-H-in-
dol-3-carbaldehyde (22)16 (100 mg, 0.69 mmol), Na2SO4 (172 mg,
1.38 mmol), Et3N (0.20 mL, 1.38 mmol) and N-t-butylhydroxyl-
amine hydrochloride (172 mg, 1.38 mmol) in THF (5 mL), after
33 h, and column chromatography (11% of MeOH in EtOAc) gave
unreacted 22 (16 mg) and compound 23 (114 mg, 91%): mp 209–
211 °C; IR (KBr) 3066, 2873, 1602, 1505, 1442, 1362, 1233,
net AUC ¼ AUCantioxidant ꢀ AUCblank
The net AUC was plotted against the antioxidant concentration and
the regression equation of the curve was calculated. The ORAC value
was obtained by dividing the slope of the latter curve between the
slope of the Trolox curve obtained in the same assay. Final ORAC
values were expressed as
lmol of Trolox equivalents/lmol of nit-
1097 cmꢀ1 1H NMR (300 MHz, CDCl3) d 9.99 (s, 1H), 9.01 (d,
;
rone. Data are expressed as means SEM.
J = 2.7 Hz, 1H), 7.98 (s, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.39 (d,
J = 8.8 Hz, 1H), 7.28–7.06 (m, 2H), 1.64 (s, 9H); 13C NMR (75 MHz,
CDCl3) d 135.9, 129.6, 126.7, 124.8, 123.0, 120.7, 117.7, 112.2,
107.9, 68.6, 28.5; MS (EI) m/z (%): 216 [M] (100), 160 (98), 117
(93), 57 (48). Anal. Calcd for C13H16N2O: C, 72.19; H, 7.46; N,
12.95. Found: C, 72.01; H, 7.62; N, 12.77.
4.3.3. The benzoic acid method for the hydroxyl radical scavenging
activity
In a screw-cap test tube, sodium benzoate (10 mmol), FeS-
O4ꢂ7H2O (10 mmol), and EDTA (10 mmol) were added. Then, differ-
ent concentrations of nitrone (0, 10, 25, 50, 75, 100, 200, 300, 400,

960
A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 951–960
and 500 lM) prepared freshly from a methanolic stock solution
References and notes
(10ꢀ3 M) and a phosphate buffer (pH 7.4) (0.1 mol) were mixed
to give a total volume of 1.8 mL. Finally, H2O2 (10 mmol) was
added, and the whole incubated at 37 °C for 2 h. After incubation,
the fluorescence was measured at 407 nm emission with excitation
at 305. All reaction mixtures were prepared in quadruplicate and at
least three independent runs were performed for each sample.
Data are expressed as means SEM.
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Cell death and neuroprotection were studied in the human
neuroblastoma cell line SH-SY5Y, a kind gift from Dr. F. Valdivieso
(Centro de Biología Molecular, CSIC, Madrid, Spain). SH-SY5Y cells
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(Sigma–Aldrich, Madrid, Spain) supplemented with 15 non-essen-
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Acknowledgments
36. Egea, J.; Rosa, A. O.; Cuadrado, A.; García, A. G.; López, M. G. J. Neurochem. 2007,
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The present work has been carried out in the context of the
‘NITROSTROKE’ project, funded by ISCIII [Retic RENEVAS (RD06/
0026/1002); IP: José Marco-Contelles]. M.C. thanks Instituto de Sa-
lud Carlos III (ISCIII, MCINN) for a post-doctoral ‘Sara Borrell’ con-
tract. A.S. thanks CSIC for a I3P Post-doctoral Contract. J.M.-C.
thanks MICINN (SAF2006-08764-C02-01; SAF2009-07271) and
Comunidad de Madrid(S/SAL-0275-2006). A.G.G. thanks ISCIII [Re-
tic RENEVAS (RD06/0026/0009)], and M.J.O.-G. thanks MICINN
(SAF2009-11219, SAF2010-20337), and ISCIII [Retic RENEVAS
(RD06/0026/012)] for financial support. E.S. thanks CSIC for Grant
200880I221 and MICINN for Grant CTQ2009-10478. J.R. thanks
CSIC for Grant 200980I152.
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the solvents used. We thank one of referees for comments on this regard.
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clinical assays on PBN. Thus, in our case, it is important to highlight that in the
experimental conditions of the antioxidant assays (see Section 4), the new
nitrones were stable in the solvents used. We thank one the reviewers for
attracting our attention to this point.
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