2-Benzazepine nitrones protect dopaminergic neurons against 6-hydroxydopamine-induced oxidative toxicity

Article abstract of DOI:10.1002/ardp.201200007

A number of C-3 spirocyclic 2-benzazepine analogs of α-phenyl-N-tert- butyl nitrone (PBN) were synthesized and tested for their activity in protecting rat brain mitochondria and dopaminergic (DA) neurons against 6-hydroxydopamine (6-OHDA), a toxin inducin

Full text of DOI:10.1002/ardp.201200007

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
1
Full Paper
2-Benzazepine Nitrones Protect Dopaminergic Neurons
against 6-Hydroxydopamine-Induced Oxidative Toxicity
1
Ramon Soto-Otero , Estefanıa Mendez-Alvarez , Sofıa Sanchez-Iglesias ,
1
1
´
´
´
2
´
´
´
2
3
´
´
´
´
Jose Luıs Labandeira-Garcıa , Jannette Rodrıguez-Pallares , Fedor I. Zubkov ,
Vladimir P. Zaytsev3, Leonid G. Voskressensky3, Alexey V. Varlamov3, Modesto de Candia4,
Filomena Fiorella4, and Cosimo Altomare4
1
´
´
Facultad de Medicina, Departamento de Bioquımica y Biologıa Molecular, Universidad de Santiago de
Compostela, Santiago de Compostela, Spain
Facultad de Medicina, Departamento de Ciencias Morfologicas, Universidad de Santiago de Compostela,
2
´
Santiago de Compostela, Spain
3 Organic Chemistry Department of the Russian Peoples Friendship University, Moscow, Russia
4
`
Facolta di Farmacia, Dipartimento Farmaco-Chimico, Universita degli Studi di Bari ‘‘Aldo Moro’’, Bari, Italy
`
A number of C-3 spirocyclic 2-benzazepine analogs of a-phenyl-N-tert-butyl nitrone (PBN) were
synthesized and tested for their activity in protecting rat brain mitochondria and dopaminergic
(DA) neurons against 6-hydroxydopamine (6-OHDA), a toxin inducing destruction of the DA nigro-
striatal pathway in rodent models of Parkinson’s disease. The newly synthesized nitrone derivatives
were firstly investigated for their activity in decreasing the level of hydroxyl radicals generated during
6-OHDA oxidation, and inhibit lipid peroxidation (TBARS assay) and protein carbonyl content (PCC) in
rat brain mitochondria. Most of the studied 2-benzazepine nitrones showed inhibitory potencies in
both TBARS and PCC assays at least two magnitude orders higher than that of PBN. The data obtained
usefully complemented the known structure–activity relationships. In particular, 5 and 10, bearing
C-3 spiro cyclopentyl and tetrahydropyranyl moieties, respectively, at 8 mM concentration proved to
be significantly more effective than PBN in protecting cultured DA neurons exposed to 6-OHDA, which
alone causes about 45% cell loss in 24 h. In addition, we found that 5 inhibited butyrylcholinesterase
with an IC50 value of 16.8 mM, which would enhance its potential as neuroprotective agent in
Alzheimer’s neurodegeneration. These findings extend the utility of benzazepine-based PBN
analogs in the treatment of age-related free radical-mediated disorders.
Keywords: 2-Benzazepine nitrones / Cholinesterase inhibition / Dopaminergic neurons / 6-Hydroxydopamine /
Neuroprotection
Received: January 5, 2012; Revised: March 8, 2012; Accepted: March 9, 2012
DOI 10.1002/ardp.201200007
Supporting information available online
:
Introduction
among the most commonly used spin-trapping reagents, which
not only have contributed to the understanding of free radical-
mediated processes in biochemical systems, but have found
applications as therapeutic agents in the treatment of patho-
logical disorders caused by unregulated production of reactive
oxygen species (ROS) [1], including ischemia-reperfusion injury
[2], neurodegeneration, and aging processes [3, 4].
Linear and cyclic nitrones (Fig. 1), such as a-phenyl-N-tert-butyl
nitrone (PBN, 1), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and 5-
diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), are
PBN has been used in the treatment of age-related disorders,
such as stroke, Parkinson’s (PD), and Alzheimer’s disease (AD)
[1, 5, 6]. Disodium-[(tert-butylimino)-methyl]benzene-1,3-disul-
fonate N-oxide (NXY-059), which was the first neuroprotective
Correspondence: Prof. Cosimo Altomare, Dipartimento Farmaco-
`
Chimico, Facolta di Farmacia, Universita degli Studi di Bari, Via E. Orabona
4, I-70125 Bari, Italy.
`
E-mail: altomare@farmchim.uniba.it
Fax: þ39 080 5442230
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2
R. Soto-Otero et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
R1
HO
R2
N
O
N
O
N
N
O
O
PBN (1)
DMPO
2
3
R1, R2 = H, Me
O
P
SO3Na
OEt
OEt
N
O
N
O
R1
R1
NaO3S
R2
R2
X
n
NXY-059
DEPMPO
N
N
O
O
Figure 1. Structures of PBN, 1 and other linear and cyclic nitrones.
4-9
n = 1-3
10-12
X = O, NCHMe2, NBn
agent that entered phase-3 clinical trials [7, 8], showed efficacy
in the acute treatment of ischemia injury due to stroke. The
cyclic derivative DMPO has shown cardioprotective proper-
ties [9]. Recently, new amphiphilic amide conjugates of cyclic
and linear nitrones have been reported which protect the
cell against oxidative insults, acting not only as radical
scavengers but also as bioenergetic agents targeted to the
mitochondrial electron and proton transport chain [10, 11].
Based on Floyd’s seminal work [1], PBN and analogs are
more than just radical scavengers. Mechanisms other than
spin-trapping, including antioxidant properties, action on
membrane enzymes and ion transport proteins, and anti-
inflammatory activity, have been investigated. With respect
to the neuroprotection properties, PBN has been also shown
to reversibly and dose-dependently inhibit acetylcholinester-
ase (AChE, EC 3.1.1.7) from mouse brain, with a competitive
inhibition constant (Ki) of 0.58 mM [12], whereas it does not
interfere with muscarinic or glutamate receptors [13, 14].
Among the cyclic nitrones, a number of 3,3-dimethyl-4,5-
dihydro-3H-2-benzazepine 2-oxides, such as compound 2
(Fig. 2), and its spiro C-3 cycloalkyl analogs, have been found
out as inhibitors of lipid peroxidation. As the electron para-
magnetic resonance (EPR) spectroscopy demonstrated, 2-ben-
zazepine nitrone analogs are able to trap radicals, forming
more stable spin adducts than PBN in both lipid and apo-
protein fractions of low density lipoproteins [15, 16]. A number
of 2-benzazepine nitrone derivatives have been also evaluated
by us [17] for their activity in vitro as protective agents against
oxidative stress induced in rat brain mitochondria by
6-hydroxydopamine (6-OHDA), a toxin inducing degeneration
of dopaminergic (DA) neurons in the nigro-striatal tract, which
is widely used to reproduce a rodent model of PD [18–20].
Once inside DA neurons, 6-OHDA readily oxidizes to form
ROS, such as H2O2 [21], and the corresponding p-quinone, to
reduce striatal levels of antioxidant enzymes, such as total
glutathione (GSH) [22] or superoxide dismutase [23], to
increase iron levels in the substantia nigra [24], and to inter-
Figure 2. Structures of 3,3,6,8-tetramethyl-4,5-dihydro-3H-2-ben-
zazepin-7-ol 2-oxide (2) [16] and benz-2-azepine nitrones (3–12)
investigated in this study.
act directly with complexes I and IV of the mitochondrial
respiratory chain [25, 26], leading as a consequence to respir-
atory inhibition and further oxidative stress. H2O2 may gen-
erate hydroxyl radicals (ꢀOH) [27], and ultimately lipid
peroxidation products. The 6-OHDA quinone triggers a cas-
cade of oxidative reactions resulting in the formation of an
insoluble polymeric pigment related to neuromelanin [28].
ROS may be central to the etiology of PD and other disorders
with a-synuclein pathology [29]. Transition metals (e.g., iron)
are able to catalyze the production of ROS [30], which could
induce a-synuclein aggregation, suggesting a possible link
between oxidative stress and a-synuclein fibril formation [31].
Besides the age-related alterations in the ubiquitin–protea-
some system [32], impaired antioxidative mechanisms may
result in oxidative stress. Indeed, human brain copper–zinc
superoxide dismutase (SOD1) has been proven to be a major
target of oxidative damage in PD and AD [33].
ROS in neuronal cell membranes can initiate lipid peroxi-
dation of polyunsaturated fatty acids (PUFAs). PUFAs, follow-
ing free radical attacks under oxidative stress conditions, can
produce lipid peroxides, which would be fragmented to form
reactive carbonyl species (RCS), such as acrolein, malondial-
dehyde, 4-oxo-2-nonenal (ONE), and 4-hydroxy-2-nonenal
(HNE) [34, 35]. These extremely reactive carbonyl compounds,
in addition to being cytotoxic themselves, are capable of
covalently modifying proteins, altering their normal struc-
ture and function. PCC is an irreversible oxidative damage
that often leads to formation of high-molecular-weight aggre-
gates, which are resistant to degradation and accumulate as
damaged or unfolded proteins [36]. RCS, formed during lipid
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Neuroprotection by New 2-Benzazepine Nitrone Derivatives
3
peroxidation and sugar glycoxidation, accumulate with age-
ing and oxidative stress-related diseases, such as atheroscle-
rosis, diabetes, or neurodegenerative diseases [37]. Acrolein-
modified a-synuclein was found in PD substantia nigra [38];
increased levels of HNE-modified proteins have been observed
in affected areas of PD, dementia with Lewy bodies (DLB), and
multiple-system atrophy (MSA) brains [39–41]. Moreover,
increased levels of HNE and malondialdehyde have been
detected in plasma from AD patients [42].
comparison, the previously reported compounds 5 and 7
were included in the study. Besides the measurements of
their ability to antagonize 6-OHDA-induced lipid peroxi-
dation (TBARS assay) and increase of PCC in rat brain mito-
chondria, the most active compounds were tested for their
protective effects in primary mesencephalic DA neurons
exposed to 6-OHDA. Furthermore, some of them were studied
in vitro for the cholinesterase (ChE) inhibition, in order to
assess any additional neuroprotective properties not directly
related to their radical-trapping activity.
In a previous study [17], we investigated a number of 4,5-
dihydro-3H-2-benzazepine N-oxide derivatives for their poten-
tial as neuroprotective agents, by assessing inhibition of both Results and discussion
6-OHDA-induced lipid peroxidation (formation of thiobarbi-
turic acid (TBA) reactive substances, TBARS) and protein
oxidation (assessed as the increase of PCC) in rat brain
mitochondria. New 2-benzazepine nitrones were identified
which exhibited in vitro activity in protecting rat brain
mitochondria from oxidative injury triggered by 6-OHDA.
In particular, compound 7 (Fig. 2; Table 1), bearing 5-gem-
dimethyl and spiro C-3 cyclohexyl groups, proved to be orders
of magnitude more effective than PBN against 6-OHDA-
induced oxidative damage.
We evaluated a number of new derivatives of 4,5-dihydro-3H-
2-benzazepine N-oxide, bearing some diverse spiro C-3 cyclo-
alkyl groups, for their ability to protect DA neurons against
the oxidative injury induced by 6-OHDA, a neurotoxin pro-
ducing experimental model of PD [18, 43–46]. It has been
shown that 6-OHDA, under physiological conditions, under-
goes rapid oxidation with formation of H2O2, which in turn
ꢀ
produce cytotoxic species like OH radicals, and the corre-
sponding oxidation product, namely the deprotonated p-qui-
none, which is the only species detected at pH >5 [47, 48].
Evidence has been provided that 6-OHDA reacts releasing
Fe(II) from the iron storage protein ferritin [49], whereas
the 6-OHDA quinone intermediate yields dopaminechrome,
which is not stable and further reacts to form an insoluble
polymeric material known as neuromelanin [50]. The for-
mation of the ꢀOH radical occurs without involvement of
the Fe ion, or any other transition metal ion, in Fenton-type
reactions [51].
With the aim of extending our structure–activity relation-
ship (SAR) study, we synthesized a number of new 2-benza-
zepine nitrones (Fig. 2), focusing our investigation in
particular on spiro C-3 cycloalkyl analogs. For the sake of
Table 1. Lipophilicity (log k0w) and activity (IC50) of PBN and related
2-benzazepine nitrones against 6-OHDA-induced hydroxyl radical
formation
The effects of the newly synthesized nitrones, the reference
compound PBN and the already reported 5 and 7 (positive
controls) on the ꢀOH generated during 6-OHDA (10 mM)
oxidation were firstly assessed by measuring the relative
fluorescence after 10 min of incubation, using THA as a
chemical dosimeter [17, 20, 48, 52, 53]. The IC50 values are
listed in Table 1.
a)
Compounds
R1
R2
n or X
log k0w
IC50 (mM)b)
1 (PBN)
3
4
5
6
7
8
9
1.77
3.17
2.80
3.12
3.40
3.26
3.52
3.32
2.28
1.72
3.30
785 ꢁ 19
24 ꢁ 0.8ꢂ
135 ꢁ 7.2ꢂ
8.1 ꢁ 0.9ꢂ
9.0 ꢁ 1.9ꢂ
22 ꢁ 1.2ꢂ
11 ꢁ 3.2ꢂ
249 ꢁ 9.5ꢂ
16 ꢁ 0.5ꢂ
22 ꢁ 2.9ꢂ
157 ꢁ 7.8ꢂ
Me
Me
H
Me
Me
Me
H
Me
Me
Me
Me
H
H
H
H
Me
Me
H
H
H
1
2
2
3
3
3
O
The data show that all the test nitrones inhibited 6-OHDA-
dependent ꢀOH production more potently than PBN. The
activities of the analogs 3, 5–8, 10, and 11, which displayed
30–100-fold improvement over PBN, are worthy of note. The
10
11
12
NCHMe2
NBn
ꢀ
bulkiness of the spiro C-3 cyclic group affects the OH-scav-
a)
Log of RP-HPLC polycratic capacity factor, that is capacity factor
extrapolated at 100% aqueous mobile phase (see Experimental
section).
Concentration of the test nitrone required to inhibit by 50%
the ꢀOH formation, fluorimetrically monitored using tereph-
enging activity. Indeed, their optimal size should be between
those of the cyclopentyl (5 and 6) and cyclohexyl (7 and 8)
groups, whereas the bulkiest N-benzylpiperidine group (12)
causes a sharp drop in activity. It was previously observed
that, in general, 5,5-dimethyl-substituted 2-benzazepine
nitrones are more active than the respective monomethyl
derivatives [17]. In this study, this is particularly evident with
the spiro C-3 cyclohexyl derivatives (8 vs. 9), but not with the
cyclopentyl ones (6 vs. 5).
b)
thalic acid (THA) as
a chemical dosimeter. Values are
means ꢁ SEM from four independent determinations.
ꢂ
Statistically significant at p <0.05 (one-way ANOVA and
Bonferroni post hoc test) when compared to the control
(BPN, 1). Previously reported IC50 values [17] for compounds 5
and 7 were 7.0 ꢁ 1.2 and 19 ꢁ 2.6, respectively.
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4
R. Soto-Otero et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
The oxidative stress induced by 6-OHDA, which causes lipid
11, are more lipophilic than the reference PBN. Compound 11
(calculated pKa ¼ 9.33), which should be predominantly pro-
tonated at the piperidine nitrogen at physiological pH (like
12, pKa ¼ 8.22), was nearly isolipophilic with PBN. The whole
spread of the measured log k0w is 1.80 log units, but most of
the examined compounds (i.e., the spiro C-3 cycloalkyl ana-
logs 4–9) vary in just 0.7 log units. Homologation of the spiro
C-3 cyclobutyl group (4) to the cyclopentyl one (6) results in
log k0w increment of 0.6 units, whereas lipophilicity of the
higher cyclohexyl homolog (7 vs. 6) no longer increases. The
addition of a second CH3 group at C-5 (compare 6 with 5, and
8 with 9), as well as the grafting of a CH3 at the C-7 position
(e.g., 8 vs. 7), results in a modest log k0w increment (0.2–
0.3 units).
peroxidation and protein damage, is thought to be involved
in neuronal cell death associated to PD. Using this model, we
investigated the effects of compounds 3–12 on 6-OHDA-
induced oxidative damage in rat brain mitochondrial mem-
brane homogenates [17, 54], assessing both the formation of
TBARS and increase of PCC. With the exception of derivatives
9 and 12, which did not attain 50% TBARS inhibition at the
maximum concentration tested (1 mM), all the assayed ben-
zazepine nitrones showed reduction in the TBARS level, with
inhibitory potencies 1–2 orders of magnitude higher than
that of PBN (Table 2).
The C-3 spirocyclic benzazepine nitrone derivatives 5–8, 10,
and 11, with IC50 values ranging from 17 to 45 mM, showed
the most remarkable effects against lipid peroxidation, dis-
playing 150–400-fold increase of the TBARS inhibition
potency over PBN. The most lipophilic compound 8 was
the most potent one.
Our data suggest that all the 2-benzazepine nitrones
studied should be lipophilic enough to have a good affinity
for and great residence time in the lipid phase, allowing
the lipids to be protected from peroxidation in brain mito-
Previous findings of others [55, 56] and ourselves [17]
proved that lipophilicity contributes to increase the inhi-
bition of lipid peroxidation. We measured the relative lip-
ophilicity (log k0w) of the 2-benzazepine nitrones through a
reversed-phase (RP)-HPLC technique [17, 57] (Table 1), showing
that all the investigated compounds, with the exception of
chondria. While no statistically significant relationship was
TBARS
found between pIC
and log k0w, the lipid peroxidation
50
inhibition potency was linearly correlated with the ꢀOH-
scavenging activity in vitro, as demonstrated by the following
regression equation:
TBARS
50
OH
50
pIC
¼ 1:29ðꢁ0:093ÞpIC ꢃ 1:71ðꢁ0:411Þ
(1)
n ¼ 11; r2 ¼ 0:955; s ¼ 0:197; F ¼ 192
Table 2. Inhibitory effect (IC50) of PBN and related 2-benzazepine
nitrones on lipid peroxidation (TBARS) and protein carbonylation
(PCC) induced by 6-OHDA in rat brain mitochondria
TBARS
50
In the above regression analysis a truncated pIC
value
of 3.00 for compounds 9 and 12 (IC50 >1 mM) was used to
retain SAR information.
Compounds
IC50 (mM)a)
As for the inhibition of PCC increase, PBN exhibited very
low activity (IC50 ¼ 17.3 mM). In contrast, the examined 2-
benzazepine nitrones, with the exception of 9, significantly
reduced the carbonyl content in rat brain mitochondrial
proteins with IC50 values in the micromolar range (Table 2).
Once again, compounds 5–8, 10, and 11 (IC50 values ranging
from 45 to 53 mM) showed more than 300-fold increase of
the PCC inhibition potency over PBN. The scatter plot of the
TBARS
PCC
1 (PBN)
3
4
5
6
7
8
9
7245 ꢁ 211
43 ꢁ 6.7ꢂ
369 ꢁ 9.4ꢂ
29 ꢁ 1.8ꢂ
27 ꢁ 2.6ꢂ
25 ꢁ 3.7b)
17 ꢁ 2.9ꢂ
17 329 ꢁ 351
55 ꢁ 6.1ꢂ
117 ꢁ 8.1ꢂ
49 ꢁ 2.0ꢂ
46 ꢁ 1.3ꢂ
51 ꢁ 3.4ꢂ
45 ꢁ 4.4ꢂ
PCC
50
OH
50
pIC versus pIC (not shown) suggested a trend of nonlinear
PCC
50
b)
–
b)
–
(quadratic or bilinear) correlation; pIC
OH
linearly increases
10
11
12
32 ꢁ 1.5ꢂ
49 ꢁ 2.8ꢂ
53 ꢁ 3.4ꢂ
276 ꢁ 3.7ꢂ
45 ꢁ 2.3ꢂ
along with the increase of pIC
up to the value of ca. 4
50
b)
–
(compounds 1, 4, 9, and 12), while remaining quite constant
PCC
50
for the compounds showing pIC >4 (3, 5–8, 10, and 11).
a)
Concentration of the test nitrone required to reduce the
formation of thiobarbituric acid reactive substances (TBARS)
and increase of protein carbonyl content (PCC). Values are
means ꢁ SEM from four independent determinations.
Overall, the inhibition data in Table 2 show that, with two
exceptions (9 and 12), the investigated compounds are able to
efficiently inhibit TBARS formation and PCC increase in rat
brain mitochondria preparations, with IC50 values orders of
magnitude lower than those of PBN. The observed corre-
lations between pIC50 values in TBARS (linear Eq. 2) and
PCC (trend of quadratic or bilinear relationship) assays and
pIC50OH values would suggest that the neuroprotective prop-
erties of the 2-benzazepine nitrones should depend, at least
b)
No statistically significant inhibition activity was attained up
to the maximum tested concentration (1 mM).
ꢂ
Statistically significant at p <0.05 (one-way ANOVA and
Bonferroni post hoc test) when compared to the control (PBN).
The following IC50 values were previously reported [17] for com-
pounds 5 and 7: 36 ꢁ 6.2 and 20 ꢁ 3.5 (TBARS), and 53 ꢁ 3.9 and
48 ꢁ 4.0 (PCC), respectively.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Neuroprotection by New 2-Benzazepine Nitrone Derivatives
5
ꢀ
in part, upon their ability to trap damaging OH radicals or
other free radical species. However, the formation of nitroxyl
radical species, which act as chain-breaking antioxidants [58],
thus preventing propagation of either lipid peroxidation and
PCC, may be supposed as additional mechanism accounting
for the observed activities.
The differences in cell viability maintenance between the
test compounds are not correlated with the differences in
OH
their IC values. This lack of correlation could be attributed
50
to the existence of other factors involved in their pharmaco-
logical action and/or accumulation and localization of the
nitrone compounds in the cell, which could be nonlinearly
related to lipophilicity as our data suggest.
In terms of SARs, the data obtained in this study support
the importance of the size of the spirocycle moiety at C-3,
which must be as large as the cyclopentyl (5 and 6) and
cyclohexyl (7 and 8) groups. The spiro C-3 cyclohexyl group
may be replaced by saturated six-membered heteroyclic
groups of comparable molar volume, such as the tetrahy-
dro-2H-pyran-4-yl group (10), without significantly decreasing
the protective activity in brain mitochondria. In contrast, the
replacement of the cyclohexyl group with the 1-alkylpiper-
idin-4-yl groups (11 and 12), predominantly in protonated
form at pH 7.4, can be tolerated for relatively small alkyl
groups, like the isopropyl one (11), whereas the alkylation of
the piperidine nitrogen with the bulky benzyl group (12) does
sharply decrease the activity.
Previously reported evidence, showing that the beneficial
effects of chronic PBN treatment in retarding of aging and
age-related cognitive deficits could be related, besides to its
capacity of removing ROS, to its ability of inhibiting AChE
[12], prompted us to study the ChE (AChE and BuChE) inhibi-
tory properties of compounds 5–7 and 10. The potential for
ChE inhibition by our compounds is somehow supported by
the evidence that also chlordiazepoxide, a benzodiazepine
containing a nitrone group, is a reversible mixed-type inhibi-
tor of AChE and BuChE, with Ki values of 50 and 0.30 mM,
respectively [59].
The effects in vitro on the ChE activity of PBN 1 and 2-
benzazepine nitrones 5–7 and 10 were studied in concen-
tration up to 2 mM, using the spectrophotometric kinetic
assay of Ellman et al. [60] with AChE from electric eel
(Electrophorus electricus) and BuChE from equine serum, as
model ChEs. The mean IC50 values are given in Table 3.
The ChE inhibition assay revealed that the tested 2-benza-
zepine nitrones act as BuChE-selective inhibitors, with a
potency higher than (or at least comparable with) that of
PBN. Compound 5 showed about 70-fold selectivity for
BuChE over AChE, with IC50 values of 16.8 mM and 1.15 mM,
respectively. A comparison between BuChE inhibition data and
log k0w values suggests that the inhibition potency depends
largely on the lipophilicity, since 5–7, with log k0w ranging
between 3.1 and 3.4, are one order of magnitude more potent
than 1 and 10, having log k0w values of 1.8 and 2.3, respectively.
While a more in-depth SAR study on cyclic nitrones could
help in the understanding of the molecular factors modulat-
ing their ChE inhibition potency and selectivity, our prelimi-
nary data highlighted the anti-BuChE activity as a possible
additional mechanism contributing to improve the potential
of the benzazepine nitrone analogs for the treatment of AD
neurodegeneration. Indeed, it is known that in healthy
human brain AChE predominates over BuChE activity, but,
as AD progresses, the levels of AChE in the brain decrease by
as much as 90%, whilst the levels of BuChE, mainly in the G1
form (i.e., globular form of monomer structure), increase [61–63].
This suggests that the inhibition of BuChE may be useful in
ameliorating the cholinergic transmission, which likely wor-
sen in AD due to the BuChE increased activity [64].
The protective effects of PBN (1) and the most active 2-
benzazepine nitrones in the TBARS and PCC assays, namely 5,
6, 7, and 10 (and not 8, which exhibited the lowest solubility
in the assay conditions), were tested and compared to PBN in
cultured primary mesencephalic DA neurons exposed to 6-
OHDA. Cells were incubated with the test compounds, each at
three concentrations (8, 40, or 200 mM), 30 min before being
exposed to 20 mM 6-OHDA for 24 h. In the cell cultures, DA
neurons were identified by TH immunohistochemistry.
Control cultures contained numerous TH-immunoreactive
(ir) cells showing long and branching processes (Fig. 3A).
The ability of the test compounds to prevent the 6-OHDA-
induced degeneration and death of the TH-ir cells is shown in
Fig. 4; the photomicrographs of TH-ir neurons showing the
effects of compounds 5 and 7 at 200 mM are reported in Fig. 3
(C and D).
Twenty-four hours after treatment with 20 mM 6-OHDA,
cultures showed a marked reduction (about 45%) in the
number of TH-ir neurons relative to the control cultures
(Figs. 3B and 4).
Treatment with PBN at 8 and 40 mM concentrations (but
not 200 mM) significantly decreased the 6-OHDA-induced DA
neuron degeneration (around 25% reduction), but the num-
ber of TH-ir neurons was significantly lower than in the
control cultures. In contrast, the loss of TH-ir neurons
induced by the toxin was blocked (i.e., levels not significantly
different to controls) by simultaneous treatment with 5 and
10 at all the concentrations tested. Compound 6 showed
significant protective effects only at the highest concen-
tration (200 mM), whereas compound 7 did not significantly
reduce the 6-OHDA-induced cell loss even at 200 mM concen-
tration (Figs. 3D and 4).
In conclusion, this study led us to identify some new
nitrone-based compounds with good activity, much stronger
than that exerted by PBN, in protecting rat brain mitochon-
dria from lipid peroxide formation and protein oxidative
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6
R. Soto-Otero et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Figure 3. Photomicrographs of representative TH-immunoreactive (-ir) neurons: control culture (A); culture treated with 20 mM 6-OHDA (B);
cultures simultaneously treated with 6-OHDA (20 mM) and compounds 5 (C), or 7 (D) at 200 mM concentration. Scale bar: 100 mm.
Louis, MO, USA). Avidin–biotin-peroxidase complex and biotiny-
damage induced by the neurotoxin 6-OHDA. A major out-
lated secondary antibody were purchased from Vector
come was the demonstration that two compounds, namely 5
(Burlingame, CA, USA). Guanidine hydrochloride was purchased
from Aldrich Chemical Co. (Milwaukee, WI, USA). Dulbecco’s
and 10, bearing as spirocyclic moieties at C-3 the cyclopentyl
and tetrahydropyranyl groups, respectively, are significantly
more effective than PBN in protecting cultured DA neurons
against 6-OHDA-induced toxicity, maintaining their function
and viability. The observed inhibitory effects on BuChE
activity, with IC50 in the micromolar range, may contribute
to improve the therapeutic potential of 2-benzazepine nitro-
nes in age-related neurodegenerative diseases.
modified Eagle’s medium (DMEM) and Ham’s nutrient mixture
F12 (HAMS F12) were obtained from Gibco (Paisley, UK). Fetal
bovine serum was from Biochrom KG (Berlin, Germany). The
water used for the preparations of solutions was of 18.2 MV
(Milli-RiOs/QA10 grade, Millipore Corp., Bedford, MA, USA). All
remaining chemicals used were of analytical grade and were
purchased from Fluka Chemie AG (Buchs, Switzerland).
Synthesis
Experimental
All solvents were distilled and dried before use. Melting points
(mp) were determined in a capillary tube and are uncorrected.
1H and 13C NMR spectra were recorded in CDCl3 or in DMSO-d6
solutions using a Bruker WM 400 or Jeol JNM-ECA 600 NMR
spectrometers. Chemical shift values are expressed in d (ppm)
and the coupling constants J in Hertz. Abbreviations used for
signal patterns are as follows: s, singlet; d, doublet; t, triplet; q,
quartet, dd, doublet of doublets; m, multiplet; brd, broad; signals
due to NH and OH protons were located by deuterium exchange
with D2O. Mass spectra were obtained by the EI technique
(Finnigan-MAT 95 XL engine), ESI method (Agilent 1100 Series
Chemicals and reagents
PBN, 6-OHDA hydrobromide, terephthalic acid (THA), TBA, buty-
lated hydroxytoluene crystalline, 2,4-dinitrophenylhydrazine
hydrochloride, desferrioxamine, 1,1,3,3-tetraethoxypropane,
5,50-dithiobis-(2-nitrobenzoic acid), sodium dodecylsulfate
(SDS), EDTA, hydrogen peroxide, bovine serum albumin (BSA),
mouse monoclonal antibody to tyrosine hydroxylase (TH), 3,30-
diaminobenzidine, trypsin, DNase, poly-L-lysine, laminin, and
Triton X-100 were purchased from Sigma Chemical Co. (St.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Neuroprotection by New 2-Benzazepine Nitrone Derivatives
7
2-benzazepines were synthesized from readily available imines.
They were then converted into 2-benzazepine nitrones 3–12 via
hydrogen peroxide oxidation in the presence of catalytic
amounts of sodium tungstate, under mild conditions.
Compounds 5 and 7 have been reported earlier [17, 65].
Compounds 3–10 and 12 were obtained and tested as hydrochlo-
rides. Chiral compounds 5 and 9 were tested as racemic mixtures.
General synthesis procedures and/or suitable references, along
with selected spectral and physical data, for the newly synthes-
ized target nitrone derivatives (3, 4, 6, and 8–12) are given below.
Data for the respective homoallylamine and 2,3,4,5-tetrahydro-
1H-2-benzazepine intermediates are reported in Supporting
Information.
Homoallylamines
2,4-Dimethyl-N-(4-methylbenzyl)pent-4-en-2-amine (for 3), 1-(2-
methylprop-2-en-1-yl)-N-(phenylmethyl)cyclobutanamine (for 4),
1-(2-methylprop-2-en-1-yl)-N-(phenylmethyl)cyclopentanamine
(for 6), N-(4-methylbenzyl)-1-(2-methylprop-2-en-1-yl)cyclohexan-
amine (for 8), 1-allyl-N-(4-methylbenzyl)cyclohexanamine
(for 9), 4-(2-methylprop-2-en-1-yl)-N-(phenylmethyl)tetrahydro-
2H-pyran-4-amine (for 10), 1-isopropyl-4-(2-methylprop-2-en-1-yl)-
N-(phenylmethyl)piperidin-4-amine (for 11), and 4-(2-methylprop-
2-en-1-yl)-N-di(phenylmethyl)piperidin-4-amine (for 12) were
obtained in 51–71% yields, according to reported procedures
[65–67] from the corresponding imines and allyl(methallyl) mag-
nesium bromide (chloride).
Figure 4. Effects of treatment with 6-OHDA (20 mM) and PBN or
compounds 5–7 and 10 (8, 40, and 200 mM) on the number of TH-
immunoreactive (-ir) cells. The data are expressed as percentages
of the number of TH-ir cells obtained in the respective control cul-
tures (100%) and represent means ꢁ SEM; ꢂp <0.05 versus control
group (untreated cells), #p <0.05 versus 20 mM 6-OHDA group
(one-way ANOVA and Bonferroni post hoc test).
Tetrahydrobenz-2-azepines
3,3,5,5,7-Pentamethyl-2,3,4,5-tetrahydro-1H-2-benzazepine (for
3), 5,5-dimethyl-1,2,4,5-tetrahydrospiro[2-benzazepine-3,10-cyclo-
butane] (for 4), 5,5-dimethyl-1,2,4,5-tetrahydrospiro[2-benzaze-
pine-3,10-cyclopentane] (for 6), 5,5,7-trimethyl-4,5-dihydrospiro-
[2-benzazepine-3,10-cyclohexane] (for 8), 5,7-dimethyl-4,5-dihy-
drospiro[2-benzazepine-3,10-cyclohexane] (for 9), 5,5-dimethyl-
1,2,20,30,4,5,50,60-octahydrospiro[2-benzazepine-3,40-pyran] (for
10), 10-isopropyl-5,5-dimethyl-1,2,4,5-tetrahydrospiro[2-benzaze-
pine-3,40-piperidine] (for 11), and 10-benzyl-5,5-dimethyl-1,2,4,5-
tetrahydrospiro[2-benzazepine-3,40-piperidine] (for 12) were pre-
pared according by analogy to reported procedures [17, 65–68]
and obtained in 33–82% yields.
LC/MSD Trap System VL) or GC-MS method (Thermo Focus DSQ II,
EI, 70 eV, ion source temperature 2008C, gas chromatographic
inlet probe with Varian FactorFour VF-5ms column). IR spectra
were recorded using a Perkin-Elmer Spectrum One FT-IR spectro-
photometer, and the most significant absorption bands
expressed in cmꢃ1 are listed. Elemental analyses (C, H, and N)
were performed on an Euro EA3000 analyzer, and the results
agreed to within 0.4% of the theoretical values.
3,3-Dimethyl-4,5-dihydro-3H-2-benzazepine N-oxide derivatives
and their spiro C-3-cycloalkyl analogs were prepared through the
reactions shown in Scheme 1. Accordingly, 2,3,4,5-tetrahydro-1H-
Oxidation of benz-2-azepines to nitrones 3, 4, 6, and 8–12:
Typical procedure
Table 3. Cholinesterase (ChE) inhibition data of PBN and selected
2-benzazepine nitrones
To a solution of 50 mmol of benz-2-azepine and Na2WO4 ꢄ 2H2O
(0.83 g, 2.5 mmol) in acetone/water mixture (9:1 v/v, 100 mL)
50% H2O2 (12 mL, 200 mmol) was added dropwise at 08C in
half an hour. The resulting mixture was stirred at room
temperature for 1–4 days (TLC monitoring). The reaction
mixture was then poured into water (300 mL) and extracted
with CH2Cl2 (5 ꢄ 30 mL). The combined organic layers were
washed with H2O (2 ꢄ 50 mL), dried over MgSO4 and concen-
trated in vacuo. Crystallization of the resulting solid from
hexane/ethyl acetate mixture gave white crystals of nitrones
(4, 8, 10, and 12). In case of compounds 6 and 11 the oily residue
was purified by column chromatography on Al2O3 (using
hexane/ethyl acetate mixture, 5:1 v/v, as eluent). In case of
poorly crystallizing nitrones, oils obtained after the workup
were converted into hydrochloride salts by mixing their ether
solutions (ca. 1 g of nitrone in 50 mL of dry diethyl ether) with
saturated HCl/Et2O solution until pH 5 was achieved. Resulting
Compounds
ChE inhibition, IC50 (mM)a)
AChE
BuChE
1 (PBN)
5
6
7
10
>2000 (12%)
1147 ꢁ 34
925 ꢁ 42
478 ꢁ 38
16.8 ꢁ 0.54
27.5 ꢁ 3.3
73.0 ꢁ 1.0
418 ꢁ 33
>1000 (6%)b)
>2000 (21%)
a)
IC50 values (means ꢁ SEM of at least three experiments) of
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE),
or % inhibition (average value of three measurements in paren-
theses) at the maximum inhibitor concentration assayed (2 mM).
b)
Due to its limited solubility, compound 7 was tested up to
1 mM concentration.
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8
R. Soto-Otero et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
R2
R1
R1
R2
R2
H
N
(i)
(ii)
R1
NH
N
O
R1, R2 = H or Me
_
Reagents and conditions: (i) H2SO4, 70°C, 1 4 h; (ii) 50% v/v H2O2, Na2WO4×2H2O, Me2CO/H2O
_
(9:1), 0°C, 30 min, then r.t. 1 4 d (TLC monitoring).
Scheme 1. General synthesis scheme of the examined spiro C-3 cycloalkyl 2-benzazepine nitrones.
white precipitates were filtered off, washed with absolute ether,
and dried on air.
124.58C; 1H NMR (CDCl3, 400 MHz) d 1.39 (s, 6H), 1.43–1.75
(m, 8H), 2.21–2.27 (m, 2H), 2.27 (s, 2H), 2.35 (s, 3H), 7.02
(m, 2H), 7.16 (d, J ¼ 1.3 Hz, 1H), 8.00 (s, 1H) ppm; 13C NMR
(CDCl3, 150.9 MHz) d 21.5, 22.5 (2C), 24.9, 31.7 (2C), 35.9 (2C),
37.4, 54.4, 73.6, 126.4, 126.6, 127.2, 129.8, 137.8, 138.9,
146.9 ppm; GC-MS (70 eV) m/z (rel. intensity): 271 (38, Mþ), 239
(40), 197 (15), 183 (16), 176 (29), 171 (25), 158 (89), 144 (91), 143 (91),
128 (66),115 (55), 105 (22), 95 (34), 91 (47), 77 (37), 67 (48), 55 (40),
3,3,5,5,7-Pentamethyl-4,5-dihydro-3H-2-benzazepine
2-oxide 3
White crystals, 64% yield, mp 62–638C (from hexane/ethyl acetate
mixture); hydrochloride – white crystals, mp 107–1088C; 1H NMR
(CDCl3, 400 MHz) d 1.42 (s, 6H), 1.55 (s, 6H), 2.22 (s, 2H), 2.35 (s, 3H),
7.06 (m, 2H), 7.18 (brs, 1H), 8.00 (s, 1H) ppm; 13C NMR (CDCl3,
150.9 MHz) d 21.6, 29.1 (2C), 31.7 (2C), 38.2, 54.6, 71.0, 125.2, 126.9,
127.3, 131.7, 139.2, 139.4, 147.6 ppm; GC-MS (70 eV) m/z (rel.
intensity): 231 (33, Mþ), 215 (5), 199 (13), 176 (23), 158 (100),
143 (86), 128 (66), 115 (65), 91 (34), 77 (19), 65 (16), 55 (20);
41 (100); IR (KBr, n cmꢃ1) 1549 (C N); Anal. Calc. for C18H25NO: C,
–
–
79.66; H, 9.28; N, 5.16. Found: C, 79.62; H, 9.37; N, 5.36.
5,7-Dimethyl-4,5-dihydrospiro[2-benzazepine-3,10-
cyclohexane]-2-oxide 9
IR (KBr, n cmꢃ1) 1565, 1548 (C N); Anal. Calc. for C15H21NO: C,
White crystals, 68% yield, mp 111–111.58C (from hexane/ethyl
acetate mixture); hydrochloride – white crystals, mp 146–
147.58C; 1H NMR (CDCl3, 400 MHz) d 1.40 (d, J ¼ 6.8 Hz, 3H),
1.32–1.92 (m, 8H), 1.85 (dd, J ¼ 10.1, 15.3 Hz, 1H), 2.32 (m, 1H),
2.33 (s, 3H), 2.42 (dd, J ¼ 2.5, 15.3 Hz, 1H), 2.51 (m, 1H), 3.07
(ddq, J ¼ 2.5, 6.8, 10.1 Hz, 1H), 7.00 (dd, J ¼ 1.0, 7.6 Hz, 1H), 7.03
(d, J ¼ 7.6 Hz, 1H), 7.05 (d, J ¼ 1.0 Hz, 1H), 7.94 (s, 1H) ppm;
13C NMR (CDCl3, 150.9 MHz) d 20.7, 21.3, 22.1, 22.7, 24.8, 31.7,
32.4, 36.3, 43.8, 75.4, 126.13, 126.03, 126.9, 130.6, 138.93, 139.0,
144.4 ppm; GC-MS (70 eV) m/z (rel. intensity): 257 (1, Mþ), 240 (6),
224 (15), 215 (17), 198 (7), 173 (6), 162 (41), 144 (100), 130 (21),115
–
–
77.88; H, 9.15; N, 6.05. Found: C, 78.19; H, 9.09; N, 5.88.
5,5-Dimethyl-4,5-dihydrospiro[2-benzazepine-3,10-
cyclobutane]-2-oxide 4
White crystals; hydrochloride – white crystals, 85% yield,
mp 97–988C; 1H NMR (CDCl3, 400 MHz) d 1.39 (s, 6H), 1.60 (m,
2H), 1.70 (m, 2H), 2.53 (m, 2H), 2.55 (s, 2H), 7.13 (dd, J ¼ 7.6,
1.0 Hz, 1H), 7.23 (dt, J ¼ 7.6, 1.0 Hz, 1H), 7.28 (dt, J ¼ 7.8, 1.3 Hz,
1H), 7.32 (brdd, J ¼ 7.8 Hz, 1H), 7.78 (s, 1H) ppm; 13C NMR (CDCl3,
100.6 MHz) d 12.8, 31.8 (2C), 32.7 (2C), 36.9, 56.6, 72.1, 125.5,
126.3, 129.0, 129.2, 129.6, 134.8, 146.8 ppm; EI-MS (70 eV)
m/z (rel. intensity): 229 (78, Mþ), 212 (100), 184 (36), 162 (69),
156 (93), 143 (53), 128 (87), 115 (52), 91 (38), 77 (26); IR (KBr,
(37), 95 (30), 91 (26), 77 (23), 67 (27); IR (KBr, n cmꢃ1) 1529 (C N);
–
–
Anal. Calc. for C17H23NO: C, 79.33; H, 9.01; N, 5.44. Found: C,
79.14; H, 9.20; N, 5.69.
n cmꢃ1) 1550 (C N); Anal. Calc. for C15H19NO: C, 78.56; H, 8.35; N,
–
–
6.11. Found: C, 79.08; H, 8.16; N, 6.38.
0
0
5,5-Dimethyl-20,3,4,5,5,60-hexahydrospiro[2-
benzazepine-3,40-pyran]-2-oxide 10
White crystals, 48% yield, mp 109–1108C (from hexane/ethyl
acetate mixture); 1H NMR (CDCl3, 400 MHz) d 1.39 (s, 6H), 1.52
(m, 2H), 2.20 (m, 2H), 2.33 (s, 2H), 3.60 (dt, J ¼ 12.0, 4.4 Hz, 2H),
3.77 (ddd, J ¼ 12.0, 9.4, 2.7 Hz, 2H), 7.07 (dd, J ¼ 7.6, 1.3 Hz, 1H),
7.23 (dt, J ¼ 7.6, 1.3 Hz, 1H), 7.27 (ddd, J ¼ 7.9, 7.6, 1.3 Hz, 1H),
7.37 (brdd, J ¼ 7.8 Hz, 1H), 7.95 (s, 1H) ppm; 13C NMR (CDCl3,
100.6 MHz) d 32.2 (2C), 36.1 (2C), 37.2, 58.5, 64.2 (2C), 70.4,
126.0, 126.7, 128.9, 129.0, 129.6, 136.6, 146.2 ppm; EI-MS
(70 eV) m/z (rel. intensity): 259 (26, Mþ), 242 (37), 198 (80),
186 (78), 156 (27), 143 (50), 129 (100), 115 (79), 81 (99), 77 (28);
5,5-Dimethyl-4,5-dihydrospiro[2-benzazepine-3,10-
cyclopentane]-2-oxide 6
Pale-yellow viscous oil, 80% yield; hydrochloride – white crystals,
mp 171–1728C; 1H NMR (DMSO-d6, 400 MHz) d 1.40 (s, 6H),
1.77–1.63 (m, 8H), 2.38 (s, 2H), 7.43 (brdt, J ¼ 7.3 Hz, 1H),
7.55–7.52 (m, 2H), 7.63 (brdd, J ¼ 7.8 Hz, 1H), 8.49 (s, 1H) ppm;
13C NMR (DMSO-d6, 100.6 MHz) d 25.9 (2C), 29.4 (2C), 37.8, 39.9
(2C), 51.8, 80.8, 124.4, 125.2, 127.0, 134.2, 137.0, 152.2,
155.1 ppm; EI-MS (70 eV) m/z (rel. intensity): 243 (47, Mþ), 226
(97), 211 (45), 162 (100), 144 (57), 128 (98),115 (44), 91 (35), 77 (24);
IR (KBr, n cmꢃ1) 1546 (C N); Anal. Calc. for C16H21NO2: C, 74.10;
–
–
H, 8.16; N, 5.40. Found: C, 74.28; H, 8.37; N, 5.14.
IR (KBr, n cmꢃ1) 1559 (C N); Anal. Calc. for C16H21NO: C, 78.97; H,
–
–
8.70; N, 5.76. Found: C, 79.13; H, 8.47; N, 5.63.
10-Isopropyl-5,5-dimethyl-4,5-dihydrospiro[2-
5,5,7-Trimethyl-4,5-dihydrospiro[2-benzazepine-3,10-
cyclohexane]-2-oxide 8
White crystals, 76% yield, mp 117–1188C (from hexane/ethyl
acetate mixture); hydrochloride – white crystals, mp 123–
benzazepine-3,40-piperidine]-2-oxide 11
Pale-yellow oil, 32% yield (ꢅ10% impurities of the corresponding
N,N-dioxide, 10-isopropyl-5,5-dimethyl-4,5-dihydrospiro[2-benza-
zepine-3,4’-piperidine]-10,2-dioxide, as determined by 1H NMR).
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Neuroprotection by New 2-Benzazepine Nitrone Derivatives
9
1H NMR (CDCl3, 400 MHz) d 1.34 (d, J ¼ 6.5 Hz, 6H), 1.42 (s, 6H),
2.15 (m, 2H), 2.39 (s, 2H), 2.52 (m, 2H), 2.97 (m, 2H), 3.33 (spt,
J ¼ 6.5 Hz, 1H), 3.54 (m, 2H), 7.08 (dd, J ¼ 7.5, 1.2 Hz, 1H), 7.25
(ddd, J ¼ 8.1, 7.5, 1.2 Hz, 1H), 7.32 (dt, J ¼ 7.5, 1.2 Hz, 1H), 7.44
(dd, J ¼ 8.1, 1.2 Hz, 1H), 7.92 (s, 1H) ppm; 13C NMR (CDCl3,
100.6 MHz) d 16.7 (2C), 30.7 (2C), 32.8 (2C), 37.7, 57.9 (2C),
58.9, 69.7, 70.7, 127.0 (2C), 129.5 (2C), 129.8, 137.6, 146.9 ppm;
4
3
2
1
IR (KBr, n cmꢃ1) 1545 (C N); Anal. Calc. for C19H28N2O: C, 75.96;
–
–
H, 9.39; N, 9.32. Found: C, 75.59; H, 9.58; N, 9.43.
10-Benzyl-5,5-dimethyl-4,5-dihydrospiro[2-benzazepine-
3,40-piperidine]-2-oxide 12
White crystals (hydrochloride), 60% yield; mp 1798C (with
decomposition); 1H NMR (CDCl3, 400 MHz) 1.37 (s, 6H), 2.11
(m, 2H), 2.80 (m, 2H), 2.94 (s, 2H), 2.6 (m, 2H), 3.65 (m, 2H), 4.89
(s, 2H), 7.16–7.21 (m, 3H), 7.23 (m, 1H), 7.02 (brdt, J ¼ 7.3 Hz, 2H),
7.42 (brdt, J ¼ 7.3 Hz, 1H), 7.47 (brdd, J ¼ 7.3 Hz, 2H), 8.00 (s, 1H)
ppm; 13C NMR (CDCl3, 100.6 MHz) d 29.1, 30.6, 31.7, 36.7 (2C), 38.9,
50.8, 58.7 (2C), 74.3, 88.3, 126.4, 126.5, 128.0 (2C), 128.9 (2C), 130.5,
133.2, 140.2, 141.3, 162.8, 178.6 ppm; IR (KBr, n cmꢃ1) 1541
(C ¼ N); EI-MS (70 eV) m/z (rel. intensity): 350 (10, Mþ), 327 (10),
312 (29), 186 (14), 145 (21), 131 (7), 115 (8), 96 (15), 91 (100), 77 (17),
65 (8), 51 (6); Anal. Calc. for C23H28N2O: C, 79.27; H, 8.10; N, 8.04.
Found: C, 78.99; H, 9.27; N, 8.31.
1
2
3
4
5
clog P
Figure 5. Graphical comparison between RP-HPLC lipophilicity
parameter log k0w and 1-octanol/water log P calculated by the
ACDLabs software. For the basic compounds 11 and 12, which
should be predominantly in the protonated form at physiological
pH (7.4), the calculated log D values were used.
equation, and F is the statistical significance of fit; 95% confi-
dence intervals of the regression coefficient are given in
parentheses.
Determination of log k0w values by RP-HPLC
Lipophilicity parameters of the examined derivatives were
measured by an RP-HPLC technique [17, 57]. Methanol solutions
of the 2-benzazepine nitrones (0.25 mg/mL) were injected
Animals
Sprague–Dawley rats (250–300 g) obtained from the breeder of
the Universidad de Santiago de Compostela were used for the
studies in accordance with the NIH Guide for the Care and Use
of Laboratory Animals. All the procedures were approved by
the Animal Ethics Committee of the Universidad de Santiago
de Compostela. Rats were housed at 228C on a 12-h light–dark
cycle with food and water available ad libitum. For sacrifice,
animals were stunned with carbon dioxide and killed by
decapitation.
into
a HPLC equipped with a Symmetry C18 column
(150 mm ꢄ 3.9 mm id, 5 mm) from Waters Assoc. (Milford, MA,
USA). The nitrones were eluted at different mobile phase compo-
sitions (0.05 increments of MeOH volume fraction in 40 mM
phosphate buffer at pH 5, ranging between 0.75 and 0.45). All
the RP-HPLC measurements were carried out at 25 ꢁ 18C, flow-
rate of 1.0 mL/min, at both 254 and 320 nm on a Waters HPLC
1525 multisolvent delivery system, equipped with a Waters 2487
variable wavelength UV detector (Waters Assoc., Milford).
The capacity factors (k0) at different mobile phase compositions
were calculated as: k0 ¼ (tR ꢃ t0)/t0,where tR is the retention time of
the solute and t0 is the elution time of MeOH, which is not retained
on the column. For each nitrone compound a linear regression
analysis (r2 >0.96) was performed on at least five data points (the
lowest MeOH fractions) and the resulting line extrapolated to 100%
aqueous mobile phase to give the log k0w values (Table 1).
Lipophilicity was also calculated using the ACDLabs software,
release 9.0 (Advanced Chemistry Development, Inc., Toronto,
Canada). The comparison between log k0w and calculated log P
(clog P values of the basic compounds 11 and 12, with
calculated pKa of 9.33 and 8.22, respectively, were corrected
for ionization) showed that a linear relation between the two
sets of parameters holds up to clog P of ca. 3.5, a value beyond
which log k0w no longer increases (Fig. 5).
Rat brain mitochondrial preparations
After sacrifice, brains were immediately removed and washed in
ice-cold isolation medium (pH 7.4, Na2PO4/KH2PO4 isotonized
with sucrose). Then, brain mitochondria were obtained by differ-
ential centrifugation with minor modifications to a previously
published method [54]. After removing blood vessels and pial
membranes, the brains were manually homogenized with four
volumes (w/v) of the isolation medium. Then, the homogenate
was centrifuged in an Avanti J-25 centrifuge (Beckman
Instruments, Palo Alto, USA) at 1000 ꢄ g for 5 min at 48C.
The supernatant was centrifuged at 12 500 ꢄ g for 15 min.
The mitochondrial pellet was then washed once with isolation
medium and recentrifuged under the same conditions. Finally,
the mitochondrial pellet was reconstituted in a buffer solution
(Na2PO4/KH2PO4 isotonized with KCl, pH 7.4) and stored in
aliquots under liquid nitrogen. The protein concentration of
mitochondrial preparations was determined according to the
method of Markwell et al. [69], using BSA as the standard.
Omitting from the regression analysis the most lipophilic
derivatives 7–9, the following linear equation was obtained:
logk0w ¼ 0:79ðꢁ0:080Þ clog P þ 0:78ðꢁ0:204Þ
(2)
n ¼ 8; r2 ¼ 0:942; s ¼ 0:178; F ¼ 97:1
Monitoring of hydroxyl radical formation
Hydroxyl radical (ꢀOH) formation was investigated using a pre-
viously reported method [17], in which THA is used as a chemical
where n represents the number of data points, r2 the squared
correlation coefficient, s the standard deviation of the regression
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10
R. Soto-Otero et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
ꢀ
ꢀ
dosimeter of OH in vitro. The formation of OH during 6-OHDA
autoxidation was monitored fluorimetrically using a lumines-
cence spectrometer Model LS50B (Perkin-Elmer, Norwalk, CT,
USA). Briefly, a buffer solution (Na2PO4/KH2PO4 isotonized with
KCl, pH 7.4) containing 10 mM THA was incubated for 5 min to
reach the working temperature (378C). Then, the test 2-benzaze-
pine nitrone (final concentration ranging from 1 mM to 1 mM) or
water was incorporated into the incubation followed by addition
of 6-OHDA (10 mM) and 1 mM KCl (pH 2.0). Monitoring of
ꢀOH formation was performed for 10 min. The excitation and
emission wavelengths used were 312 and 426 nm, respectively.
Fluorescence measurements were all relative to the initial read-
ing and the peak of relative fluorescence (DFmax) used to express
the amount of the produced ꢀOH. The test compound concen-
tration that inhibited ꢀOH formation by 50% (IC50) was calculated
from a concentration-response curve generated with seven differ-
ent concentrations of the 2-benzazepine nitrone, using the pro-
gram OriginPro 8.0 (OriginLab Corporation, Northampton, MA,
USA). The data are reported in Table 1.
A sample aliquot (200 mL) was then immediately submitted to
protein precipitation, by addition of trichloroacetic acid (TCA
20% w/v), followed by centrifugation at 15 000 ꢄ g for 5 min.
The resulting pellet was reconstituted in 0.5 M NaOH with soni-
cation (Branson Sonic Corp., Danbury, CT, USA) for 5 s. Then,
10 mM 2,4-dinitrophenylhydrazine in 2 M chloric acid was
added and the mixture was incubated at room temperature
for 1 h, in darkness with continuous agitation. After the addition
of TCA (20% w/v), the mixture was centrifuged at 15 000 ꢄ g for
5 min. The resulting pellet was washed twice with ethyl acetate/
EtOH (1:1 v/v). Then, the washed pellet was reconstituted with
6 M guanidine in a 20 mM KH2PO4 buffer (pH 2.3) and centri-
fuged at 15 000 ꢄ g for 5 min. The absorbance of the resulting
solution was measured at 370 nm. The carbonyl content was
calculated from the absorbance data and expressed as nmol
carbonyls/mg protein. Because of the numerous washing steps,
protein content in the final pellet was estimated on HCl blank
pellet processed simultaneously, using a BSA standard curve in
6 M guanidine, and reading the absorbance at 280 nm. The IC50
values are listed in Table 2.
Lipid peroxidation assay
Primary mesencephalic cell cultures
The inhibitory effects of test 2-benzazepine nitrones on the lipid
peroxidation were determined by monitoring the formation of
TBARS, using a spectrophotometric method [17].
Ventral mesencephalic tissue was dissected from rat embryos of
14 days gestation (E14). The tissue was incubated in 0.1% trypsin,
0.05% DNase and DMEM for 20 min at 378C and then washed in
DNase/DMEM and mechanically dissociated. The resulting cell
suspension was centrifuged at 50 ꢄ g for 5 min, the supernatant
was removed carefully and the pellet was resuspended in 0.05%
DNase/DMEM to the final volume required. The number of viable
cells in the suspension was estimated by acridine orange/ethi-
dium bromide staining, and cells were plated onto 35-mm cul-
ture dishes (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA)
previously coated with poly-L-lysine (100 mg/mL) and laminin
(4 mg/mL). The cells were seeded at a density of 1.5 ꢄ 105 cells/
cm2 and maintained under control conditions (DMEM/HAMS
F12, 1:1, containing 10% fetal bovine serum). The cell cultures
were maintained in a humidified CO2 incubator (5% CO2, 378C)
for 7 days in vitro. The medium was totally removed on day 2 and
replaced with fresh culture medium.
A brain mitochondrial preparation (1 mg protein/mL), in a
Na2PO4/KH2PO4 buffer (pH 7.4) isotonized with KCl, was incu-
bated at 378C for 5 min to reach the working temperature. Then,
the test 2-benzazepine nitrone (final concentration ranging from
1 mM to 1 mM) or water was incorporated into the incubation
cell, followed by addition of 6-OHDA (10 mM) or 1 mM KCl
(pH 2.0), and the mixture incubated for 20 min exactly.
Butylated hydroxytoluene (20 mM) and desferrioxamine
(20 mM) were immediately added, in order to prevent amplifica-
tion of the lipid peroxidation during the assay.
A sample aliquot (200 mL) was treated with SDS (8.1%, w/v),
followed by addition of acetic acid (20%), and the mixture vor-
texed for 1 min. Then, TBA (0.8%) was added and the resulting
mixture incubated at 958C for 60 min. After cooling to room
temperature, 3 mL of n-butanol was added and the mixture shaken
vigorously. After centrifugation at 2500 ꢄ g for 10 min, the
absorbance of the supernatant (organic layer) was measured at
532 nm using an Ultrospec III spectrophotometer (Pharmacia
Biotech, Uppsala, Sweden). For calibration, a standard curve (5–
150 nM) was generated using the malondialdehyde (MDA), pre-
pared via acid-catalyzed hydrolysis (H2SO4; 1.5% v/v) of 1,1,3,3-
tetraethoxypropane. The protein concentration of the samples
was determined according to a previously reported method [69],
using BSA as the standard, and the TBARS results were expressed as
nmol MDA/mg protein. The IC50 values are listed in Table 2.
Treatment and immunohistochemistry of cell cultures
Cultures were treated with the test 2-benzazepine nitrone (8, 40,
or 200 mM concentrations) 30 min before the treatment with 6-
OHDA (20 mM in 0.02% saline ascorbate) on 6 days in vitro for
24 h. Cultures were fixed with 4% p-formaldehyde in DPBS at pH
7.4 for 20 min, and the endogenous peroxidase activity was
quenched by incubation for 5 min with 3% hydrogen peroxide
in DPBS. Cultures were preincubated with a blocking solution
containing 10% normal serum in DPBS with 1% BSA and 0.3%
Triton X-100 for 1 h, and then incubated overnight at 48C with a
mouse monoclonal anti-tyrosine hydroxylase (anti-TH; 1:30,000)
as the DA marker. Cultures were then washed and incubated for
1 h with the corresponding biotinylated secondary antibody
(horse anti-mouse) diluted 1:500. The cultures were washed
and incubated for 90 min with avidin–biotin-peroxidase com-
plex (1:500). Finally, the labeling was revealed with 0.04% H2O2
and 0.05% 3,30-diaminobenzidine as the chromogen.
Assessment of protein carbonyl content
The ability of the test 2-benzazepine nitrones to inhibit the
protein oxidative damage caused by the autoxidation of 10 mM
6-OHDA in the rat brain mitochondrial preparation was assayed
by determining PCC, according to the previously reported method
[17]. Brain mitochondria (1 mg protein/mL) were incubated in
Na2PO4/KH2PO4 buffer (pH 7.4, isotonized with KCl), at 378C for
5 min. Then, the test nitrone compound (final concentrations
ranging from 1 mM to 1 mM) or water was incorporated into
the incubation, followed by 6-OHDA (10 mM) or 1 mM KCl
(pH 2.0), and the mixture was incubated for exactly 20 min.
Cell counting in primary mesencephalic cultures
The cultured cells, observed with phase contrast microscopy (TH-
immunoreactive (ir) cells; Eclipse, Nikon; ꢄ100 magnification),
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Neuroprotection by New 2-Benzazepine Nitrone Derivatives
11
were counted in five randomly chosen longitudinal and trans-
verse microscopic fields along the diameter of the culture dish
away from the curve edge. The operator was blind to the treat-
[2] J. L. Zweier, M. A. H. Talukder, Cardiovasc. Res. 2006, 70, 181–
190.
[3] E. R. Stadtman, Free Radical Res. 2006, 40, 1250–1258.
ment condition. The microscopic field was defined by
a
[4] S. Mandel, E. Gru¨nblatt, P. Riederer, M. Gerlach, Y. Levites,
M. B. H. Youdim, CNS Drugs 2003, 17, 729–762.
0.5 cm ꢄ 0.5 cm reticule (i.e., a total of 1.25 cm2). The average
number of TH-positive cells in a control culture dish was
2321 ꢁ 165. The results from at least three different exper-
iments were recorded, with a minimum sample size of four wells
per group and per run. The results were normalized to the counts
of the control group in the same batch (i.e., expressed as the
percentage over the control group counts), in order to account
for possible variability among batches.
[5] R. A. Floyd, K. Hensley, M. J. Forster, J. A. Kelleher-Andreson,
P. L. Wood, Mech. Ageing Dev. 2002, 123, 1021–1031.
[6] B. Moosmann, C. Behl, Expert Opin. Invest. Drugs 2002, 11,
1407–1435.
[7] K. R. Maples, A. R. Green, R. A. Floyd, CNS Drugs 2004, 18,
1071–1084.
[8] C. X. Wang, A. Shuaib, Int. J. Clin. Pract. 2004, 58, 964–969.
Cholinesterase inhibition assay
[9] L. Zuo, Y. R. Chen, L. A. Reyes, H. L. Lee, C. L. Chen, F. A.
Villamena, J. L. Zweier, J. Pharmacol. Exp. Ther. 2009, 329, 515–
523.
Compounds were evaluated using AChE (EC 3.1.1.7) from electric
eel (Electrophorus electricus), and butyrylcholinesterase (BuChE,
EC 3.1.1.8) from equine serum (Sigma) following the method of
Ellman et al. [60]. The AChE activity was determined in a reaction
mixture containing 200 mL of a solution of AChE (0.415 U/mL in
0.1 M phosphate buffer, pH 8.0), 100 mL of a solution of 5,50-
dithio-bis(2-nitrobenzoic) acid (DTNB 3.3 mM in 0.1 M phosphate
buffered solution, pH 7.0, containing 6 mM NaHCO3), 100 mL of a
solution of the inhibitor (0.25–2.0 mM), and 500 mL of phosphate
buffer, pH 8.0. After incubation for 20 min at 258C, acetylthio-
choline iodide (100 mL of 0.05 mM water solution) was added as
the substrate, and AChE activity was determined by UV spectro-
photometry from the absorbance changes at 412 nm for 3.0 min
at 258C. The concentration of compound which produced 50%
inhibition of the AChE activity (IC50) was calculated by nonlinear
regression of the response–concentration (log) curve. BuChE
inhibitory activity determinations were carried out similarly
using butyrylthiocholine iodide (0.05 mM) as the substrate.
IC50 values are reported in Table 3 as means ꢁ SEM of at least
three independent measurements.
[10] G. Durand, B. Poeggeler, S. Ortial, A. Polidori, F. A.
Villamena, J. Bo¨kler, R. Hardeland, M. A. Pappolla, B.
Pucci, J. Med. Chem. 2010, 53, 4849–4861.
[11] G. Durand, R. A. Prosak, Y. Han, S. Ortial, A. Rockenbauer, B.
Pucci, F. A. Villamena, Chem. Res. Toxicol. 2009, 22, 1570–1581.
[12] D. Milatovic, Z. Radic, M. Zivin, W.-D. Dettbarn, Free Radical
Biol. Med. 2000, 28, 597–603.
[13] D. Milatovic, M. Zivin, E. Hustedt, W.-D. Dettbarn, Neurosci.
Lett. 2000, 278, 25–28.
[14] M. Zivin, D. Milatovic, W.-D. Dettbarn, Brain Res. 1999, 850,
63–72.
[15] C. E. Thomas, D. F. Ohlweiler, A. A. Carr, T. R. Nieduzak, D. A.
Hay, G. Adams, R. Vaz, R. C. Bernotas, J. Biol. Chem. 1996, 271,
3097–3104.
[16] C. E. Thomas, D. F. Ohlweiler, B. Kalyanaraman, J. Biol. Chem.
1994, 269, 28055–28061.
´
´
´
[17] R. Soto-Otero, E. Mendez-Alvarez, S. Sanchez-Iglesias, F. I.
Zubkov, L. G. Voskressensky, A. V. Varlamov, M. de Candia,
C. Altomare, Biochem. Pharmacol. 2008, 75, 1526–1537.
Statistical analysis
Multiple comparisons were analyzed by one-way ANOVA fol-
lowed by Bonferroni’s post hoc test. The normality of popu-
lations and homogeneity of variances were tested before each
ANOVA. Differences at p <0.05 were considered as statistically
significant. Statistical analyses were carried out with SigmaStat
3.0 (Jandel Scientific, San Rafael, CA, USA).
[18] S. Duty, P. Jenner, Br. J. Pharmacol. 2011, 164, 1357–1391.
[19] N. Simola, M. Morelli, A. R. Carta, Neurotoxicol. Res. 2007, 11,
151–167.
´
[20] R. Soto-Otero, E. Mendez-Alvarez, A. Hermida-Ameijeiras,
´
´
A. M. Lopez-Real, J. L. Labandeira-Garcıa, Biochem. Pharmacol.
´
2002, 64, 125–135.
´
Supporting Information
[21] E. A. Mazzio, R. R. Reams, K. F. Soliman, Brain Res. 2004, 1004,
29–44.
Supplementary data to this article (physicochemical and spec-
tral data for homoallylamine and 2,3,4,5-tetrahydro-1H-2-benza-
zepine intermediates of the synthesis of nitrone derivatives 3, 4,
6, and 8–12) can be found online.
[22] J. Sian, D. T. Dexter, A. J. Lees, S. Daniel, Y. Agid, F. Javoy-
Agid, P. Jenner, C. D. Marsden, Ann. Neurol. 1994, 36, 348–355.
[23] G. Kunikowska, G. P. Jenner, Brain Res. 2001, 922, 51–64.
C. A. acknowledges the financial support by the Italian Ministry for Education
Universities and Research (MIUR, Rome, Italy; PRIN 2007, Grant No.
2007T9HTFB_003). The Spanish authors (R. S.-O. and E. M.-A.) thank the
Ministerio de Ciencia e Innovacio´n and the European Regional Development
Fund (Madrid, Spain, Grant SAF2007-66114) for financial support.
[24] E. Oestreicher, G. J. Sengstock, P. Riederer, C. W. Olanow,
A. J. Dunn, G. W. Arendash, Brain Res. 1994, 660, 8–18.
[25] Y. Glinka, M. Gassen, M. B. Youdim, J. Neural Transm. Suppl.
1997, 50, 55–66.
[26] Y. Y. Glinka, M. B. H. Youdim, Eur. J. Pharmacol. 1995, 292, 329–
332.
The authors have declared no conflict of interest.
[27] A. Pezzella, M. d’Ischia, A. Napolitano, G. Misuraca, G. Prota,
J. Med. Chem. 1997, 40, 2211–2216.
References
[28] P. Gee, A. J. Davison, Free Radical Biol. Med. 1989, 6, 271–284.
[1] R. A. Floyd, R. D. Kopke, C.-H. Choi, S. B. Foster, S. Doblas,
R. A. Towner, Free Radical Biol. Med. 2008, 45, 1361–1374.
[29] L. M. Sayre, G. Perry, M. A. Smith, Chem. Res. Toxicol. 2008, 21,
172–188.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

12
R. Soto-Otero et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
´ ´ ´
[52] R. Soto-Otero, C. Sanmartın-Suarez, S. Sanchez-Iglesias, A.
[30] L. M. Sayre, G. Perry, C. S. Atwood, M. A. Smith, Cell. Mol. Biol.
2000, 46, 731–741.
´
Hermida-Ameijeiras, I. Sanchez-Sellero, E. Mendez- Alvarez,
´
J. Biochem. Mol. Toxicol. 2006, 20, 209–220.
´
[31] M. Hashimoto, L. J. Hsu, Y. Xia, A. Takeda, A. Sisk, M.
Sundsmo, E. Masliah, NeuroReport 1999, 10, 717–721.
[53] J. C. Barreto, G. S. Smith, N. H. Strobel, P. A. McQuillin, T. A.
Miller, Life Sci. 1995, 56, PL89–96.
[32] J. N. Keller, J. Gee, Q. Ding, Ageing Res. Rev. 2002, 1, 279–293.
´
[54] A. Hermida-Ameijeiras, E. Mendez- Alvarez, S. Sanchez-
´
´
Iglesias, C. Sanmartın-Suarez, R. Soto-Otero, Neurochem.
´
[33] J. Choi, H. D. Rees, S. T. Weintraub, A. I. Levey, L. S. Chin, L. Li,
J. Biol. Chem. 2005, 280, 11648–11655.
´
Int. 2004, 45, 103–116.
´
[34] S. H. Lee, I. A. Blair, Chem. Res. Toxicol. 2000, 13, 698–720.
[55] T. L. Fevig, S. M. Bowen, D. A. Janowick, B. K. Jones, H. R.
Munson, D. F. Ohlweiler, C. E. Thomas, J. Med. Chem. 1996, 39,
4988–4996.
[35] H. Esterbauer, R. J. Schaur, H. Zollner, Free Radical Biol. Med.
1991, 11, 81–128.
[36] I. Delle-Donne, G. Aldini, M. Carini, R. Colombo, R. Rossi,
A. Milani, J. Cell. Mol. Med. 2006, 10, 389–406.
[56] C. E. Thomas, P. Bernardelli, S. M. Bowen, S. F. Chaney,
D. Friedrich, D. A. Janowick, B. K. Jones, F. J. Keeley, J. H.
Kehne, B. Ketteler, D. F. Ohlweiler, L. A. Paquette, D. J. Robke,
T. L. Fevig, J. Med. Chem. 1996, 39, 4997–5004.
[37] A. Negre-Salvayre, C. Coatrieux, C. Ingueneau, R. Salvayre,
Br. J. Pharmacol. 2008, 153, 6–20.
[38] M. Shamoto-Nagai, W. Maruyama, Y. Hashizume, M.
Yoshida, T. Osawa, P. Riederer, M. Naoi, J. Neural. Transm.
2007, 114, 1559–1567.
[57] M. de Candia, P. Fossa, S. Cellamare, L. Mosti, A. Carotti,
C. Altomare, Eur. J. Pharm. Sci. 2005, 26, 78–86.
[58] J. Skolimowski, A. Kochman, L. G˛ebicka, E. Metodiewa,
Bioorg. Med. Chem. 2003, 11, 3529–3539.
[39] N. Shibata, Y. Inose, S. Toi, A. Hiroi, T. Yamamoto, M.
Kobayashi, Acta Histochem. Cytochem. 2010, 43, 69–75.
[59] S.-Y. Chiou, G.-W. Lai, Y.-T. Tsai, L.-Y. Lin, G. Lin, Med. Chem.
Res. 2005, 14, 297–308.
[40] R. J. Castellani, G. Perry, S. L. Siedlak, A. Nunomura, S.
Shimohama, J. Zhang, T. Montine, L. M. Sayre, M. A.
Smith, Neurosci. Lett. 2002, 319, 25–28.
[60] G. L. Ellman, K. D. Courtney, V. Anres, R. M. Featherstone,
Biochem. Pharmacol. 1961, 7, 88–95.
[41] A. Yoritaka, N. Hattori, K. Uchida, M. Tanaka, E. R. Stadtman,
Y. Mizuno, Proc. Natl. Acad. Sci. USA 1996, 93, 2696–2701.
[61] E. Giacobini, Int. J. Geriatr. Psychiatry 2003, 18, S1–S5.
[62] Z. Rakonczay, Acta Biol. Hung. 2003, 54, 183–189.
[42] K. Chen, J. Maley, P. H. Yu, J. Neurochem. 2006, 99, 1413–1424.
´
[43] S. Sanchez-Iglesias, P. Rey, E. Mendez-Alvarez, J. L. Labandeira-
[63] T. Arendt, M. K. Bru¨ckner, M. Lange, V. Bigl, Neurochem. Int.
1992, 21, 381–396.
´
Garcıa, R. Soto-Otero, Neurochem. Res. 2007, 32, 99–105.
´
´
[64] N. H. Greig, T. Utsuki, D. K. Ingram, Y. Wang, G. Pepeu,
C. Scali, Q. S. Yu, J. Mamczarz, H. W. Holloway, T. Giordano,
D. Chen, K. Furukawa, K. Sambamurti, A. Brossi, D. K. Lahiri,
Proc. Natl. Acad. Sci. USA 2005, 102, 17213–17218.
[44] W. Dauer, S. Przedborski, Neuron 2003, 39, 889–909.
[45] Z. I. Alam, S. E. Daniel, A. J. Lees, D. C. Marsden, P. Jenner,
P. Halliwell, J. Neurochem. 1997, 69, 1326–1329.
[46] D. T. Dexter, A. E. Holley, W. E. Flitter, T. F. Slater, F. R. Wells,
S. E. Daniel, A. J. Lees, P. Jenner, C. D. Marsden, Mov. Disord.
1994, 9, 92–97.
[65] A. V. Varlamov, V. V. Kouznetsov, F. I. Zubkov, A. I.
Chernyshev, G. G. Alexandrov, A. R. Palma, L. Vargas, S.
Salas, Synthesis 2001, 6, 849–854.
´
[47] R. Soto-Otero, E. Mendez-Alvarez, A. Hermida-Ameijeiras,
´
A. Mun˜oz-Patin˜o, J. L. Labandeira-Garcıa, J. Neurochem.
2000, 74, 1605–1612.
´
´
[66] J. M. G. Urbina, J. C. Cortes, A. Palma, S. N. Lopez, S. A.
Zacchino, D. R. Enriz, J. C. Ribas, V. V. Kouznetzov, Bioorg.
Med. Chem. 2000, 8, 691–698.
´
[48] G. N. L. Jameson, A. B. Kudryavtsev, W. Linert, J. Chem. Soc.,
Perkin Trans. 2 2001, 2, 557–562.
[67] V. V. Kouznetsov, A. R. Palma, S. Salas, L. Y. Vargas, F. I.
Zubkov, J. R. Martinez, A. V. Varlamov, J. Heterocyclic Chem.
1997, 34, 1591–1595.
[49] G. N. L. Jameson, R. F. Jameson, W. Linert, Org. Biomol. Chem.
2004, 2, 2346–2351.
[68] F. I. Zubkov, J. D. Ershova, A. A. Orlova, V. P. Zaytsev, E. V.
Nikitina, A. S. Peregudov, A. V. Gurbanov, R. S. Borisov, V. N.
Khrustalev, A. M. Maharramov, A. V. Varlamov, Tetrahedron
2009, 65, 3789–3803.
[50] W. Linert, E. Herlinger, R. F. Jameson, E. Kienzi, K. Jellinger,
M. B. H. Youdim, Biochim. Biophys. Acta 1996, 1316, 160–168.
´
[51] E. Mendez-Alvarez, R. Soto-Otero, A. Hermida-Ameijeiras,
´
M. E. Lopez-Martın, J. L. Labandeira-Garcıa, Free Radical
´
Biol. Med. 2001, 31, 986–998.
´
´
[69] M. A. K. Markwell, S. M. Haas, L. L. Bieber, N. E. Tolbert, Anal.
Biochem. 1978, 87, 206–210.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com