Microwave-assisted synthesis of hydroxyphenyl nitrones with protective action against oxidative stress

Article abstract of DOI:10.1016/j.ejmech.2012.09.044

Oxidative stress plays an important role in neuronal death in neurodegenerative disorders such as Parkinson's disease (PD). Hydroxyphenyl nitrones, derivatives of the nitrone spin trap alpha-phenyl-N-tert-butylnitrone (PBN), were synthesized and their antioxidant, anti-inflammatory and neuroprotective activity in neural cells evaluated. These hydroxyphenyl nitrones 5-7 were synthesized by reaction of the corresponding hydroxybenzaldehyde with N-tert-butyl hydroxylamine under microwave irradiation. They showed good peroxyl free radical scavenger capacities, analyzed by oxygen radical absorbance capacity (ORAC). Also inhibited peroxynitrite-mediated tyrosine nitration of alpha-synuclein in vitro and protected human neuroblastoma (SH-SY5Y) cells against SIN-1 and 6-OHDA toxicity when micromolar concentrations were used. Besides, the hydroxyphenyl nitrones evaluated showed anti-inflammatory activity modulating nitrite production in primary neural cell cultures of astrocytes and microglia treated with lipopolysaccharide (LPS), a potent inflammatory agent. These experimental data suggest a potential therapeutic use of these hydroxyphenyl nitrones against oxygen and nitrogen reactive species involved in neurodegenerative pathology.

Full text of DOI:10.1016/j.ejmech.2012.09.044

European Journal of Medicinal Chemistry 58 (2012) 44e49
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Microwave-assisted synthesis of hydroxyphenyl nitrones with protective action
against oxidative stress
,
,
,e
Cecilia Chavarría a b, Daniel I. Perez c, Concepción Pérez c, Jose A. Morales Garcia d e, Sandra Alonso-Gil d
**
,
Ana Pérez-Castillo d e, Carmen Gil c, José M. Souza b , Williams Porcal
,
,
a,
*
a Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay
b Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, 11800 Montevideo,
Uruguay
c Instituto de Química Médica (IQM-CSIC), Juan de La Cierva 3, 28006 Madrid, Spain
d Instituto de Investigaciones Biomédicas “Alberto Sols” (CSIC), Arturo Duperier 4, 28029 Madrid, Spain
e Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Oxidative stress plays an important role in neuronal death in neurodegenerative disorders such as
Parkinson’s disease (PD). Hydroxyphenyl nitrones, derivatives of the nitrone spin trap alpha-phenyl-N-
tert-butylnitrone (PBN), were synthesized and their antioxidant, anti-inflammatory and neuroprotective
activity in neural cells evaluated. These hydroxyphenyl nitrones 5e7 were synthesized by reaction of the
corresponding hydroxybenzaldehyde with N-tert-butyl hydroxylamine under microwave irradiation.
They showed good peroxyl free radical scavenger capacities, analyzed by oxygen radical absorbance
capacity (ORAC). Also inhibited peroxynitrite-mediated tyrosine nitration of alpha-synuclein in vitro and
protected human neuroblastoma (SH-SY5Y) cells against SIN-1 and 6-OHDA toxicity when micromolar
concentrations were used. Besides, the hydroxyphenyl nitrones evaluated showed anti-inflammatory
activity modulating nitrite production in primary neural cell cultures of astrocytes and microglia
treated with lipopolysaccharide (LPS), a potent inflammatory agent. These experimental data suggest
a potential therapeutic use of these hydroxyphenyl nitrones against oxygen and nitrogen reactive species
involved in neurodegenerative pathology.
Received 28 July 2012
Received in revised form
26 September 2012
Accepted 27 September 2012
Available online 5 October 2012
Keywords:
Hydroxyphenyl nitrones
Microwave-assisted synthesis
Alpha-synuclein
SH-SY5Y human neuroblastoma cells
Primary neural cell
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
6]. It is suggested that biological oxidation in the human body
generates highly pathogenic ROS anꢀꢁd RNS such as hydroxyl free
Parkinson’s disease (PD) is
a
neurodegenerative disorder
radical (ꢀOH), superoxide anion (O2 ), nitric oxide (ꢀNO), perox-
ynitrite (ONOOꢁ) and lipid peroxide free radicals (ROOꢀ), causing
cellular injury [7e9]. Nitric oxide also reacts with superoxide anion
radical to produce significant amounts of peroxynitrite (ONOOꢁ),
a potent oxidizing agent that can cause DNA fragmentation, protein
oxidation and nitration and lipid peroxidation [10]. Although the
body has developed several defense mechanisms to counteract
oxidative stress, the brain appears to be more susceptible to this
damage than any other organ. Studies in Parkinson proposed
a possible role for ROS and/or RNS in selective loss of SN dopami-
nergic neurons in the disease [11,12]. Enhancing antioxidant and
radical scavenger capabilities in the SN therefore constitutes
a rational approach to prevent or slow ongoing damage of dopa-
involving the progressive degeneration of dopamine neurons
arising in the substantia nigra (SN). Although PD has been heavily
researched in the last decades, the precise etiology of the disease is
still unknown. However, research in recent years has provided
substantial evidence supporting the hypothesis that oxidative
stress plays a major role in disease pathogenesis [1e3]. The
generation of reactive oxygen (ROS) and nitrogen species (RNS),
and oxidative damage in the central nervous system (CNS) are
major events occurring in many neurodegenerative disorders,
besides Parkinson’s disease (PD) including Alzheimer’s disease,
amyotrophic lateral sclerosis, as well as in the aging processes [4e
minergic neurons. In the last decade, nitrone spin traps, a-phenyl-
N-tert-butylnitrone (PBN, Fig. 1) and structurally related
compounds, have shown their utility in the treatment of neuro-
degenerative diseases as well as in the prolongation of life span due
to their neuroprotective effects [13,14]. Neuroprotective
* Corresponding author. Tel.: þ598 2525 8618/216; fax: þ598 2525 0749.
** Corresponding author. Tel.: þ598 2924 9562; fax: þ598 2924 9563.
E-mail addresses: jsouza@fmed.edu.uy (J.M. Souza), wporcal@fq.edu.uy
(W. Porcal).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.044

C. Chavarría et al. / European Journal of Medicinal Chemistry 58 (2012) 44e49
45
widely used especially applicable to small organic molecules
synthesis [23,24]. Frequent issues such as time consumption and
excess use of reagents found during traditional synthesis, can be
solved with microwave heating application. Based on this,
a comparison between yields obtained with conventional proce-
dure and microwave heating were done (Table 1). In both cases the
yields obtained for the desired nitrones were similar, but under
microwave irradiation the reactions time were shorter. Aldehydes 3
and 4 (Scheme 1) were less reactive and the formation of the
nitrones 6 and 7 required reflux in EtOH for several hours (Table 1,
entries e and g). With the procedure here described, the reactions
studied were very fast, with no need to use an excess of the
expensive hydroxylamines and several hours refluxing to obtain
good yields. A significant problem associated with many reactive
nitrones is dimerization. No dimerization products were obtained
under our reaction conditions. Therefore, the procedure is signifi-
cantly more energy-efficient than conventional heating with
enhanced reaction rates and higher purity of final products. Known
nitrones 5, 6 and PBN showed spectroscopic data in good agree-
ment with those reported in the literature [20e22], while struc-
tures of new nitrone 7 were confirmed unequivocally using
analytical and spectroscopic techniques. The proton and carbon
assignment of the different nitrones was confirmed by 1H NMR,
COSY, and HSQC, HMBC spectra. The 1H NMR analysis confirmed
that only one stereoisomer was formed in all cases. The Z-config-
uration of nitrones synthesized was confirmed by NOE
experiments.
Fig. 1. Chemical structure of PBN, NXY-059 and Nitrone-TDZ.
mechanism of action postulated for PBN involved its ability to
inhibit signal transduction processes such as suppression of
proinflammatory cytokines, genes, and mediators associated with
enhanced neuroinflammatory processes [15]. In an effort to opti-
mize the biological profile of PBN, a wide structural diversity of
PBN-like nitrones has been designed and synthesized [16]. The
nitrone NXY-059 (Fig. 1) was evaluated in clinical trials phase III but
unfortunately no positive effects was found in ischemic stroke
patients [17]. Despite this clinical result, the hypothesis of using
nitrones as neuroprotective agents remains viable. In particular,
a phenolic nitrone-TDZ (Fig. 1) showed excellent free radical scav-
enger capacity and good neuroprotective effects without cellular
toxicity [15]. Furthermore, recently experimental in vitro antioxi-
dant properties, as well as preliminary in vivo pharmacological
activities of nitrone derivatives of Trolox (a-tocoferol derivative)
were reported, showing that nitrones bearing free phenol groups
exerted the best antioxidant values [17,18]. Following our ongoing
research on nitrones with neuroprotective properties, we proposed
the synthesis of new molecules keeping the nitrone moiety, but
combining it with antioxidant fragment able to stabilize the
generated free radical [15,19]. Thus we described here an efficient
and rapid synthetic route to phenolic PBN analogs based on
microwave irradiation. The hydroxyphenyl nitrones developed
were evaluated as antioxidant and neuroprotective agents on
a human neuroblastoma (SH-SY5Y) cell model as well as anti-
inflammatory agents in primary cultures of neural cells.
3. Results and discussion
3.1. Antioxidant capacity of hydroxyphenyl nitrones
The antioxidant activity of the hydroxyphenyl nitrones 5e7
synthesized was determined in vitro by using the oxygen radical
absorbance capacity (ORAC) assay. This assay measures peroxyl free
radical scavenger compounds capability and uses fluorescein (FL) as
the fluorescent probe [15]. Results of the experiment were
expressed as Trolox equivalents (mmol of Trolox equivalents per
m
mol of tested compound) (Table 2). The peroxyl free radical
2. Chemistry
scavenger properties of the hydroxyphenyl nitrones 5e7 were
better than Trolox. The 4-hydroxyphenyl nitrone 6 showed the
highest ORACFL value (3.2 Trolox equiv), whereas the parent nitrone
PBN showed the lowest ORACFL value (0.014 Trolox equiv).
Although several methods have been reported to prepare
nitrones [19,20], the selected hydroxyphenyl nitrones 5e7
(Scheme 1) and PBN were synthesized through the condensation
between the corresponding phenolic aldehyde and N-tert-butyl
hydroxylamine. Previous studies indicate that aromatic aldehydes
with electron donors groups react slower than those substituted by
electron withdrawing groups with N-tert-butyl hydroxylamine
[20e22]. The main disadvantage of this synthesis is the use of 2
equiv of N-tert-Butyl hydroxylamine, an expensive reagent, and
long reaction times to go to completion [21,23]. In this context, in
recent years the microwave assisted organic synthesis has been
3.2. Hydroxyphenyl nitrones prevent protein nitration
Due to the ability of the hydroxyphenyl nitrones 5e7 to act as
scavengers of free radicals, we next evaluated their ability to
prevent protein nitration. The formation of 3-nitrotyrosine (3-NT)
in proteins occurs as an in vivo posttranslational modification with
Table 1
Synthesis of nitrones 5e7 and PBN, conditions and yields.
Entry
Nitrone
Conditions
Yields (%)
Temperature (ꢂC)
Time (min)
a
b
c
d
e
f
PBN
5
60
180
10
180
10
1800
10
30
64
72
78
90
42
35
100a
78
100a
78
6
i) 100a
ii) 150a
78
g
h
7
180
20
49
47
110e120a
a
Scheme 1. Synthetic methodology of hydroxyphenyl nitrones 5-7 and PBN.
Heating under microwave irradiation.

46
C. Chavarría et al. / European Journal of Medicinal Chemistry 58 (2012) 44e49
Nitration of tyrosine primarily occurs via attack by the ꢀNO2
Table 2
Antioxidant capacity and permeability assay.
radical that originates from the decomposition of peroxynitrite [9].
Hydroxyphenyl nitrone 6 can prevent peroxynitrite-dependent
protein nitration through different mechanisms. It could react
and form a spin adduct with the radical derived from the decom-
position of ONOOꢁ, react directly with ONOOꢁ or reduce the tyrosyl
radical in proteins. We assumed that the first hypothesis is the most
plausible because the inhibition of protein tyrosine nitration by the
nitrone it is similar in different proteins and phenolic groups did
not react with ONOOꢁ directly, they react with the radicals gener-
ated by ONOOꢁ homolysis [28]. Through westernꢀꢁblot analysis we
Nitrone
ORAC (Trolox equiv)a
Permeability (Pe)b
PBN
5
6
0.014 ꢃ 0.001
2.6 ꢃ 0.2
Nd
37.5 ꢃ 1.3 (CNSþ)
0.2 ꢃ 0.1 (CNSꢁ)
10.2 ꢃ 0.9 (CNSþ)
3.2 ꢃ 0.3
7
2.0 ꢃ 0.1
nd: not determinate.
a
Data are expressed as
mmol of Trolox equiv/
m
mol of tested nitrones and are the
mean (n ¼ 3) ꢃ SD.
b
Prediction of the brain penetration using PAMPA-BBB assay (CNSþ: compounds
able to cross the BBB).
ꢀ
ꢀ
demonstrate that nitrone 6 reacts with OH, CO3 and NO2 radi-
cals (not shown). To investigate the possible mechanism by which
hydroxyphenyl nitrones prevent peroxynitrite-dependent protein
nitration compound 6 was treated with peroxynitrite (ONOOꢁ) and
the product analyzed by HPLC. Peroxynitrite homolysis (in the
absence of bicarbonate) results in equals amounts of ꢀOH and ꢀNO2.
To especifically evaluated the reaction of nitrone 6 and ꢀNO2 radical
the reaction mixture was preincubated with an excess of sodium
nitrite (4 mM), which was rapidly oxidized by ꢀOH to ꢀNO2 [29]. An
additional peak when nitrone 6 reacts with ONOOꢁ as an indicator
of a modified structure was observed (Fig. 4). This was studied by
mass spectrometry analysis, nitrone 6, presents a unique peak with
important pathophysiological consequences. Elevated levels of 3-
NT have been detected in a myriad of pathological conditions
including inflammatory, neurodegenerative, and cardiovascular
disorders, among others [25,26]. Western blot analysis was
employed to demonstrate the ability of these nitrones to prevent
peroxynitrite-dependent tyrosine nitration of alpha-synuclein (
S is a major fibrillar component of Lewy bodies and the aggrega-
tion of this protein represents a critical step in the pathogenesis of
Parkinson’s disease (PD) and other -synucleinopathies. It has been
demonstrated that exposure of human recombinant S to nitrating
agents induces formation of nitrated S monomers and oligomers
aS).
a
a
a
a
a retention time of 18.7 min (m/z 194.4 Da, Mþ þ H) (Fig. 4A).
ꢀ
[26]. The nitrated oligomers are highly stabilized due to covalent
cross-linking via the oxidation of tyrosine to form o,o-dityrosine
[26,27]. An in vitro screening of hydroxyphenyl nitrones 5e7 was
performed to evaluate them as potential inhibitors of the nitration
Whereas a new peak appeared after the reaction between nitrone 6
and ꢀNO2 with a retention time of 26.9 min (m/z 239.4 Da, Mþꢀ þ H)
(which presents absorbance at 300 and 360 nm, not shown) asso-
ciated with a mono nitrated form of nitrone 6 (Fig. 4B). In the
absence of NaNO2 there are several peaks including the peak with
a retention time of 26.9 min. The other peaks could correspond to
hydroxylated and nitrated forms of nitrone 6. These peaks appear in
small amount and are difficult to characterize.
and oxidation of
aS (Fig. 2). The bands at about 15 kDa correspond
to S monomers whereas the 34 kDa bands indicate the presence of
a
nitrated and aggregated forms of the protein. Derivatives of
phenolic nitrones with the hydroxyl group in para position to the
nitrone moiety (compounds 6e7), show the best behavior pre-
venting
molecular weight aggregates of
anti-nitrotyrosine western blots a doseeresponse curve of inhibi-
tion of nitration of S by nitrone 6 was built. The treated S with
compound 6 markedly inhibited the nitration in a dose-dependent
manner (see Supplementary data). However, these properties of
a
S tyrosine nitration and also the formation of high
a
S. Using densitometry analysis in
3.3. Neuroprotective studies in vitro
a
a
The hydroxyphenyl nitrones were then evaluated for their
activity in vitro as protectants against induced damage in human
neuroblastoma (SH-SY5Y) cells by 6-hydroxydopamine (6-OHDA)
and the peroxynitrite donor 3-morpholinosydnonimie (SIN-1)
[30,31]. 6-OHDA is known to destroy dopaminergic neurons
through free radical-mediated mechanisms and it is used as
nitrones as agents that prevent aS nitration could be extended to
other proteins. Thus the activity of hydroxyphenyl nitrones 6e7 to
prevent peroxynitrite-dependent tyrosine nitration was performed
using western blot analysis. Small proteins, with tyrosine residues
exposed to the solvent and accessible for modification by nitrating
agents, like cytochrome c and RNAse A were used (Fig. 3). Results
shown that, the hydroxyphenyl nitrones also prevent
peroxynitrite-dependent protein tyrosine nitration of cytochrome c
and RNAse A. Based on these results, we can extrapolate the anti-
oxidants properties of the hydroxyphenyl nitrones 6e7 to other
neurotoxin producing experimental model of Parkinson’s disease
ꢀ
(PD). SIN-1 generates superoxide anion (O2 ꢁ) and nitric oxide
(ꢀNO) resulting in constant production of ONOOꢁ, and therefore
serves as a model for physiological ONOOꢁ production [32]. The
rate of ONOOꢁ formation by SIN-1 (0.5 mM) was measured
following the DHR method obtaining ONOOꢁ at a rate of 9
mM per
minute [33]. To explore the effects of hydroxyphenyl nitrones 5e7
protein and not only to aS.
on 6-OHDA-injured SH-SY5Y cells, cell viability and cytotoxicity
Fig. 2. Immunochemical analysis of peroxynitrite-treated
nitrotyrosine (B). Lines 2e6: 5 S treated with authentic 200
Nitrone 6. Reverse addition of ONOOꢁ is similar to lane 1 (not shown).
a
S and the hydroxyphenyl nitrones effects. 12% SDS-PAGE and western blot using antibodies anti-
aS (A) and anti-3-
mM
a
m
M ONOOꢁ. 1:
a
S control (not treated), 3: þDMSO 1%, 4: þ50 M Nitrone 7, 5: þ50 M Nitrone 5, 6: þ50
m
m
mM

C. Chavarría et al. / European Journal of Medicinal Chemistry 58 (2012) 44e49
47
after 25
mM 6-OHDA treatment (Fig. 5A). Nitrones 5e7 showed
significative protective effect at 10
mM as the viability of cells
treated with these was approximately 100% (Fig. 5A). Cytopro-
tective properties of the phenolic nitrones against SIN-1 damage
were evaluated using the MTT assay. The effects of the hydrox-
yphenyl nitrones 5e7 were evaluated on oxidative injury in SH-
SY5Y cells using SIN-1 to generate ONOOꢁ in vitro. Nitrones 5 and
7 did not show any significant protective effect (data not shown). In
these conditions, only compound 6 showed protective effects
against human neuroblastoma cell injury by SIN-1. Although the
oxidative stress induced by 0.5 mM SIN-1 caused cell death of about
21%, treating cells with nitrone 6 markedly reverted cell death in
a dose-dependent manner. On the other hand, at concentrations of
100 and 500 mM, the viability of cells treated with nitrone 6 was
91% and 100%, respectively (Fig. 5B).
3.4. Anti-inflammatory studies in vitro
Fig. 3. Protein tyrosine nitration assessed in different proteins exposed to peroxyni-
trite. 12% SDS-PAGE and western blot using anti-3-nitrotyrosine antibody. Lines 1e6:
different proteins (5
Our next step was to explore whether the hydroxyphenyl
nitrones present neuroprotective effects in different cell based
assays. In this context, activation of astrocytes and microglia are
involved on the neuronal death [34,35]. Thus, we used primary
cultures of astrocytes and microglia treated with lipopolysaccha-
ride (LPS), a potent inflammatory agent. The anti-inflammatory
activity of the hydroxyphenyl nitrones was tested by evaluating
the production of nitrites from primary cultured glial cells
m mM). 1: RNase
M) treated with authentic ONOOꢁ (200
A þ Nitrone 6, 2: RNase A þ Nitrone 7, 3: RNase A control, 4: Cyt c þ Nitrone 6, 5: Cyt
c þ Nitrone 7, 6: Cyt c control.
was determined using the MTT assay. First, SH-SY5Y cytotoxicity of
the hydroxyphenyl nitrones 5e7 was evaluated. Different nitrones
doses (10e100
mM) do not alter SH-SY5Y cell viability at 24 h (data
not shown). The basal percentage of viable neurons was 75% at 24 h
Fig. 4. Chromatograms of Nitrone 6 treated with peroxynitrite. (A) 50
mM Nitrone 6 (untreated) as a control. (B) 50 mM of Nitrone 6 was preincubated with an excess of sodium
nitrite (4 mM NaNO2) and then treated with 500
m
M ONOOꢁ in potassium phosphate buffer (50 mM KPO4) at pH 7.4.
Fig. 5. (A) Neuroprotective effect of nitrones 5e7 on 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were pre-incubated for 1 h with the
indicated concentration of the test compounds and then stimulated with 6-OHDA (25
m
M) or vehicle (mean ꢃ SD values from three independent experiments). *, p ꢄ 0.01, **,
p ꢄ 0.001. (B) Neuroprotective effect of nitrone 6 on SIN-1 induced neurotoxicity in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were pre-incubated for 1 h with the
indicated concentrations of test compound and then stimulated with SIN-1 (0.5 mM) or vehicle (mean ꢃ SD values from three independent experiments). *, p ꢄ 0.01, **, p ꢄ 0.001.

48
C. Chavarría et al. / European Journal of Medicinal Chemistry 58 (2012) 44e49
Fig. 6. (A) Effect of hydroxyphenyl nitrones on the inflammatory response of microglial cultures. (B) Effect of hydroxyphenyl nitrones on the inflammatory response of astrocyte
cultures. Rat primary microglial and astrocyte cultures were treated for 24 h with LPS (10 g/mL) in the absence or presence of nitrones 5e7 and the production of nitrite was
evaluated by the Griess reaction. Values represent the means ꢃ SD from three different experiments. ***, p ꢄ 0.001, **, p ꢄ 0.01, *, p ꢄ 0.05.
m
(astrocytes and microglia) [36]. Cultures were incubated with the
indicated concentration of the nitrones 5, 6 and 7 for 1 h, and then
cells were cultured for another 24 h with LPS. When primary
microglial and astrocytes cells were stimulated with LPS (Fig. 6) we
observed a significant induction of nitrite production in the culture
medium (1.7- and 4.0-fold, respectively), which was significantly
diminished by hydroxyphenyl nitrones treatment. Nitrite produc-
tion is mainly due to induction of inducible nitric oxide synthase
(iNOS) enzyme which produces nitric oxide in LPS-treated cells.
The treatment of primary cultured glial cells with phenolic nitrones
5, 6 and 7, resulted in a significant reduction in nitrite production.
Additionally hydroxyphenyl nitrones cytotoxicity in astrocytes and
microglia cultures was evaluated in vitro for 24 h. All the nitrones
did not displayed cytotoxicity in primary glial cells, at assayed
4. Conclusions
We have reported a series of hydroxyphenyl nitrones, designed
as multifunctional agents, since they combine hydroxyphenyl
antioxidant with a neuroprotective and spin-trapping profile of
nitrone moiety. Nitrones 5e7 were prepared in short time by
condensation between the corresponding phenolic aldehyde and
N-tert-butyl hydroxylamine under microwave irradiation. The
biological evaluation showed that the hydroxyphenyl nitrones
were more potent antioxidant than the reference compound PBN,
probably due to the combination of aromatic hydroxy function and
nitrone moiety. Hydroxyphenyl nitrones 6 and 7 avoid protein
nitration and also the presence of high molecular aggregates forms
of alpha-synuclein (aS). Nitrones 5e7 were also able to improve cell
neuroprotective doses of 10
m
M (data not shown).
viability of SH-SY5Y cells damaged with 6-OHDA, whereas
compound 6 was able to exhibited antioxidant effects against
ONOOꢁ-induced oxidative stress. Moreover, these nitrones also
showed significant anti-inflammatory effect, modulating nitrite
production in neural cells. These results indicate that the hydrox-
yphenyl nitrones evaluated are promising anti-inflammatory and
neuroprotective agents, with a potential use as therapeutic agents
in neurodegenerative diseases.
3.5. Blood brain barrier permeation prediction
Early assessment of compound availability in the central
nervous system (CNS) is essential for CNS drugs and useful for
optimizing the toxicity profile of non CNS drugs. Drug’s penetration
through the blood brain barrier (BBB) is one of the major obstacles
for the treatment of diseases in the CNS. For the hydroxyphenyl
nitrones 5e7 the capacity to cross the bloodebrain barrier (BBB)
was evaluated employing a parallel artificial membrane perme-
ation assay for BBB (PAMPA-BBB) using a lipid extract of porcine
brain [37]. The assay validation was made by comparing experi-
mental permeabilities of 10 commercial drugs to reported values
[30,36]. A good correlation between experimental-described values
was obtained Pe (exp) ¼ 0.9479 (bibl) þ 0.7528 (R2 ¼ 0.9723) (see
Supplementary data). From this equation and following the
pattern established in the literature for BBB permeation prediction
we could classify compounds as CNS þ when they present
a permeability 4.54 ꢅ 10ꢁ6 cm sꢁ1. Based on these results we can
consider that nitrones 5 and 7 are able to cross the BBB by passive
permeation (Table 2).
Acknowledgments
This work was supported by Agencia Nacional de Investigación
e
Innovación (ANII, Fondo Clemente Estable 491), Comisión
Sectorial de Investigación Científica (CSIC)-UdelaR (CSIC-Nꢂ 417),
PEDECIBA-Química and project agreement UdelaR (Uruguay)-CSIC
(Spain) (2009UY0006) and Ministerio de Ciencia y Tecnología
(Spain) (SAF2010-16365, SAF2009-13015-C02-01). The authors
acknowledge Mr. Horacio Pezzaroglo for recording the NMR
spectra, Drs. Madia Trujillo and Gonzalo Peluffo for assistance with
some experiments. We thank ANII for scholarship to CC. DIP
acknowledges postdoctoral fellowship from the CSIC (JAE
program).

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