α-aryl-N-alkyl nitrones, as potential agents for stroke treatment: Synthesis, theoretical calculations, antioxidant, anti-inflammatory, neuroprotective, and brain-blood barrier permeability properties

Article abstract of DOI:10.1021/jm201105a

We report the synthesis, theoretical calculations, the antioxidant, anti-inflammatory, and neuroprotective properties, and the ability to cross the blood-brain barrier (BBB) of (Z)-α-aryl and heteroaryl-N-alkyl nitrones as potential agents for stroke trea

Full text of DOI:10.1021/jm201105a

Article
pubs.acs.org/jmc
α-Aryl-N-alkyl Nitrones, as Potential Agents for Stroke Treatment:
Synthesis, Theoretical Calculations, Antioxidant, Anti-inflammatory,
Neuroprotective, and Brain−Blood Barrier Permeability Properties
Mourad Chioua,†,▽ David Sucunza,†,▽ Elena Soriano,† Dimitra Hadjipavlou-Litina,*,‡ Alberto Alcazar,*,§
́
Irene Ayuso,§ María Jesus Oset-Gasque,*,∥,⊥ María Pilar Gonzalez,∥ Leticia Monjas,#
́
́
María Isabel Rodríguez-Franco,# Jose Marco-Contelles,*,† and Abdelouahid Samadi†
́
†Laboratorio de Radicales Libres y Química Computacional, Instituto de Química Organ
28006-Madrid, Spain
‡Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
́
ica General (CSIC), Juan de la Cierva, 3,
§Departamento Investigacion
́ ́
, IRYCIS, Hospital Ramon y Cajal, Madrid, Spain
∥Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, Universidad Complutense de Madrid (UCM), 28040
Madrid, Spain
⊥Instituto Universitario de Investigacion
Spain
́
en Neuroquímica (IUIN), Universidad Complutense de Madrid (UCM), 28040 Madrid,
#Instituto de Química Med
́
ica (IQM-CSIC), Juan de la Cierva, 3, 28006-Madrid, Spain
ABSTRACT: We report the synthesis, theoretical calculations, the antioxidant, anti-inflammatory, and
neuroprotective properties, and the ability to cross the blood−brain barrier (BBB) of (Z)-α-aryl and
heteroaryl-N-alkyl nitrones as potential agents for stroke treatment. The majority of nitrones compete with
DMSO for hydroxyl radicals, and most of them are potent lipoxygenase inhibitors. Cell viability-related
(MTT assay) studies clearly showed that nitrones 1−3 and 10 give rise to significant neuroprotection. When
compounds 1−11 were tested for necrotic cell death (LDH release test) nitrones 1−3, 6, 7, and 9 proved to
be neuroprotective agents. In vitro evaluation of the BBB penetration of selected nitrones 1, 2, 10, and 11
using the PAMPA-BBB assay showed that all of them cross the BBB. Permeable quinoline nitrones 2 and 3 show potent
combined antioxidant and neuroprotective properties and, therefore, can be considered as new lead compounds for further
development in specific tests for potential stroke treatment.
permanent focal ischemia and stroke model in rodents, which
has been launched several times in different programmes in
advanced clinical studies, although with limited success.5
However, the efforts devoted to improving previous results,
or to finding new nitrones for the treatment of ischemic stroke,
showed that the neuroprotective strategy is still a choice for the
development of new drugs for stroke.6−12
INTRODUCTION
■
It is well-established that oxidative stress represents one of the
most important molecular events after ischemic damage of
membrane lipids, which leads to the death of cerebral tissue.1
As a consequence, most of current research efforts in this area
are focused on blocking and scavenging oxygenated free
radicals.2
The case of edaravone, a potent free radical scavenger
recently approved for the treatment of patients with acute
stroke,13 also confirms the viability of this approach. Among the
nitrones, (Z)-α-phenyl-N-tert-butylnitrone (PBN), for instance,
inhibits the oxidation of lipoproteins,14 reduces the oxidative
damage to erythrocytes, the peroxidation of lipids due to
phenylhydrazine,15 and protects gerbils from brain stroke and
mice against MPTP toxicity.16
However, PBN mechanism of action is still not clear. It does
not seem to be related to its ability to act as ROS trap but to
the suppression of inducible nitric oxide synthase expression,
cytokine accumulation, and apoptosis.17 In fact, the formation
of nitric oxide (NO) from PBN spin adducts may play a role in
Neuronal membranes are rich in polyunsaturated fatty acids,
which are particularly susceptible to free radicals attack at
carbons adjacent to double bonds. The produced lipid
hydroperoxides are not completely stable in vivo and, in the
presence of metals or metal complexes, can be decomposed to
reactive radicals, which will trigger the chain reactions. Potential
consequences of damage to membrane lipids include changes in
fluidity, in permeability, and in the orientation of proteins
embedded in the bilayer of the plasma membrane, along with
other cellular endomembranes.
In this regard, free radical scavengers, such as nitrones, have
demonstrated to be effective as neuroprotective tools in
experimental ischemia studies.3 However, to date, nitrones
have not been successful in human trials. This is the case of
nitrone NXY-059,4 a well-known free radical scavenger with
good neuroprotective profile in rat models of transient and
Received: August 18, 2011
Published: November 29, 2011
© 2011 American Chemical Society
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Article
the observed effects in the central nervous system.18 On the
basis of these grounds, we have recently started a project
targeted to the synthesis and biological evaluation of α-aryl, N-
alkyl nitrones as potential drugs for cerebral ischemia
Table 1. Structure of the Nitrones 1−11, Synthetic Method
(Scheme 1), and Chemical Yields
treatment.19
In this work, we report the synthesis, theoretical calculations,
antioxidant capacity, in vivo antinflammatory properties, and
neuroprotective properties as well as the ability of nitrones 1−
25 to cross the blood−brain barrier (BBB). From these studies,
we conclude that the quinoline motif is a simple, readily
available privileged heterocyclic ring system, leading to (Z)-N-
((2-chloro-6-methylquinoline-3-yl)methylene)-2-methylpro-
pan-2-amine oxide (nitrone 2) and (Z)-N-((2-chloro-6-
methylquinoline-3-yl)methylene)-1-phenylmethanamine oxide
(nitrone 3) as potent neuroprotective agents with strong
antioxidant capacity, able to cross the BBB, for the potential
treatment of stroke.
RESULTS AND DISCUSSION
■
1. Chemistry. (Z)-α-Aryl and heteroaryl N-alkyl nitrones
1−11 (Table 1) have been synthesized from the corresponding
commercial [3-(trifluoromethyl)benzaldehyde, 2-chloro-6-
methylquinoline-3-carbaldehyde, 3,5-di-tert-butyl-4-hydroxy-
benzaldehyde; 5-bromofuran-2-carbaldehyde] or from the
readily available aldehydes [3-(4-formylphenoxy)propyl ni-
trate20 and 2-(4-formylphenoxy)ethyl nitrate]20 with N-tert-
butyl- or N-benzylhydroxylamine, as shown in Scheme 1. The
aldehyde choice was based on the previous literature,12 our
results and experience in the topic,19 which prompted us to use
benzaldehydes substituted with strong electron-withdrawing
groups or free phenol highly hindered moieties or with 2(3)-
(nitrooxy)alkoxy) chains at position C-4 and heterocyclic
(quinoline, furan) carbaldehydes containing halogen substitu-
ents in appropriate positions.
All new or known (1,21 4,22 522) nitrones have been
characterized by their analytical and spectroscopic data (see
Experimental Section). The stereochemistry at the double bond
has been assigned as Z by comparing NMR data with those
analyzed and reported for known nitrones.
With these nitrones in hand, first of all, we carried out some
theoretical calculations in order to predict the permeability of
these compounds to the BBB, and their neuroprotective
capacity.
2. Theoretical Calculations. Because of the high attrition
rate of central nervous system drug candidates during clinical
trials, the assessment of BBB penetration in early research is
particularly important. The BBB is the most important barrier
between systemic circulation and the central nervous system
(CNS) due to its substantially larger surface area. It constitutes
a major challenge to the treatment of most brain disorders.23 In
the majority of the cases, the access of medicines to the brain is
restricted at the level of the brain capillary endothelial wall,
which forms the BBB. Therefore, potential CNS drugs
frequently do not achieve sufficient drug concentration in the
brain to show pharmacological activity.
One of the most commonly used parameters to quantify
brain penetration is the ratio of concentration measured in the
brain to that in the blood at steady state, expressed as logBB
(log [brain]/[blood]). Although the extent of brain exposure is
affected by various factors including influx and efflux trans-
porters at the BBB, most compounds do cross the BBB via
passive diffusion.
Scheme 1. Methods for the Synthesis of Nitrones 1−11
Because of our insufficient understanding of the molecular
recognition processes involved in various active transport
systems, the time-consuming, expensive, and difficult exper-
imental in vivo determination of logBB, the in silico prediction
of BBB permeation, is a research area of enormous interest.
Most of the developed computational approaches are based on
physicochemical properties, such as molecular weight, n-
octanol−water partition coefficient, molecular surface area,
and Gibbs free energy. Herein, we have applied the prediction
software CSBBB24 based on topological structure descriptors
and developed by the use of artificial neural networks.
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The results, which are summarized in Table 2, suggest that all
the nitrones, except for those bearing a 3-(nitrooxy) alkoxy)
concentration, significantly delay or prevent oxidation of easily
oxidizable substrates. The formation of Reactive Oxygen
Species (ROS) is characteristic of aerobic organisms, as an
unavoidable consequence of cell metabolism. Their involve-
ment in myocardial and CNS ischemia is under intensive study.
The potential role of free radicals in cerebral ischemia is due to
their extreme reactivity and their tendency to initiate and
participate in chain reactions. Normally, defense against these
highly reactive species can be accomplished by the organism
using enzymes and naturally occurring antioxidants. For the
estimation of the antioxidative potential of chemical compo-
nents, different experimental approaches were used. The most
popular screening assays use commonly available instrumenta-
tion. They have been developed to be fast and easy. Most of
them require a spectrophotometric measurement and a certain
reaction time in order to obtain reproducible results.26 The
used assays do not measure the same chemistry. In this way,
factors such as solubility or steric hindrance, which may be of
overriding importance in one environment but not in another,
can be varied and the antioxidant ability of a compound in a
variety of milieus may be evaluated.
3.1. Estimation of Inhibition of Lipid Peroxidation (LP)
Induced by AAPH. AAPH induced linoleic acid oxidation has
been developed as a quick and reliable method for measuring
the antioxidant activity. The water-soluble azo compound
AAPH [2,2′-azobis(2-amidinopropane) dihydrochloride] has
been extensively used as a clean and controllable source of
thermally produced alkyl peroxyl free radicals. Azo compounds
generating free radicals through spontaneous thermal decom-
position are useful for in vitro studies of free radical
production.27 The use of the free radical reactions initiator
AAPH is recommended as more appropriate to measure
radical-scavenging activity in vitro because the activity of the
peroxyl radicals produced by the action of AAPH shows a
greater similarity to cellular activities such as lipid perox-
idation.28 Oxidation of sodium linoleate by a thermal free
radical initiator (AAPH) is followed by UV spectrophotometry
in a highly diluted sample.27 In the AAPH assay, the highly
reactive alkylperoxyl radicals are intercepted mainly by a
hydrogen atom transfer (HAT) from the antioxidant.29
Therefore, particularly effective HAT agents are compounds
with high hydrogen atom donating ability, that is, compounds
with low heteroatom-H bond dissociation energies and/or
compounds from which hydrogen abstraction leads to sterically
hindered radicals as well as compounds from which abstraction
of hydrogen leads to C-centered radicals stabilized by
resonance.
Table 2. Estimated logBB and Percentage of
Neuroprotection for Nitrones 1−11
a
b
nitrone
logBB (sd)
neuroprotection (%)
1
0.51 (0.12)
0.62 (0.17)
0.52 (0.14)
0.55 (0.16)
0.64 (0.23)
0.29 (0.14)
0.11 (0.18)
−0.26 (0.23)
−0.25 (0.23)
−0.28 (0.18)
−0.15 (0.19)
12
70
72
82
80
42
37
30
18
31
22
2
3
4
5
6
7
8
9
10
11
a
b
Standard deviation shown in parentheses. See ref 25 for details.
benzylidene substituent (nitrones 8−11), should present a
good brain penetration profile with logBB values in the range
0.11−0.64, the most permeable being nitrones 2 (logBB =
0.62) and 5 (logBB = 0.64).
On other hand, it is well-established that organic molecules
incorporating a nitrone moiety can act as free radical trapping
agents (spin traps) and are capable of opposing oxidative
challenges, although the operational mechanism of nitrones
might not exclusively reside in their capacity to directly
scavenge free radicals. Thus, the addition of free radicals to the
carbon−nitrogen double bond of such molecules occurs very
easily, yielding nitroxide radical species, which are generally
much more stable and biochemically less harmful than the
original free radicals. Because free radicals react via their
SOMO (single occupied molecular orbital), improved spin
trapping properties are expected for heteroatom radicals
formed from nitrones (SOMO−HOMO control). The
presence of heterocyclic ring and extended conjugation with
the nitrone function would improve the bioavailability and
reduce the toxicity characteristics of the nitrones in comparison
to compounds described in the literature. As a matter of fact,
high-level theoretical calculations indicate that these molecules
possess a higher energy HOMO (highest occupied molecular
orbital) compared to the nitrones with reduced conjugation.
With these observations in mind, we have estimated the
neuroprotective effects (as % neuroprotection) following the
approach by Goldstein et al.25 based on the calculation of
orbital energies and lipophilicity. The results suggest that the
presence of a p-hydroxyl group in nitrones 4 and 5 should lead
to excellent levels of neuroprotection. In addition, a quinolinyl
group (nitrones 2 and 3), which gives rise to a high energy
HOMO, would also exhibit good levels of protection. On the
other hand, the presence of a 3-(nitrooxy) alkoxy) benzylidene
group (compounds 8−11) would lead to a poor level of
neuroprotection, mainly for short alkyl moieties (nitrones 9 and
11).
In Tables 3 and 4 we show the rate of increase in lipid
peroxidation by measuring the formation of conjugated dienes
as an indirect measure of lipid peroxidation (% inhibition). The
induction period time tinh produced by the tested nitrones
representing the period where the added antioxidant inhibits
lipid peroxidation by reacting with the AAPH derived peroxyl
radical, itself being consumed by this reaction, is, in brackets,
given in minutes. Thus, nitrone 4 with tinh 88.2−83.0 min is
very interesting and close to the inhibition time of trolox used
as a reference compound. Compounds 1, 6, 7, and 9 did not
show any inhibition value, whereas 5 (9%) presented very
limited value (Table 3). The remaining nitrones within this
subgroup exhibited significant inhibition of lipid peroxidation,
the most potent being nitrones 2 (85% and 56.7 min) and 10
(81% and 36.6 min) which, despite the different substructural
characteristics, inhibited similarly the lipid peroxidation. It is
Taking into consideration the theoretical results above, we
carried out the corresponding in vitro antioxidant and
neuroprotection analyses on these nitrones.
3. Antioxidant Evaluation. In this investigation, α-aryl-N-
alkyl (methyl, tert-butyl, benzyl) and α-hetero (aryl)-N-alkyl
(tert-butyl, benzyl) nitrones, have been tested as antioxidants.
Antioxidants are defined as substances that, even at low
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Table 4. In Vitro Antioxidant Activities for Nitrones 12−25
a
b
c
d
0 at 0.01 mM, 0 at 0.05 mM. 0 at 0.01 mM, 0 at 0.05 mM. 0 at 0.01 mM, 0 at 0.05 mM: these results concern the % LOX inhibition. Inhibition
e
f
g
h
at 0.1 mM. Inhibition at 0.1 mM. Induction time tinh produced by the tested nitrones. Inhibition at 0.1 mM. These results concern the ability of
nitrones to compete with DMSO for hydroxyl radicals. 0.1 mM. 0.1 mM + 1 mM cysteine. nd: not determined. Nitrate reductase/NADPH
system.
i
k
l
m
worth pointing out that nitrones 3 and 4, with close
lipophilicity values, have high tinh values. Nitrones 11 (64%),
4 (61%), 8 (49%), and 4 (37%) follow.
Afterward, we carried out similar antioxidant studies with
nitrones 12−25, previously prepared in our laboratory (Table
4).19 With the exception of compounds 13 and 18, which did
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once the potential antioxidant had been added. The reduction
of absorbance is a measure of the free DPPH due to the action
of the antioxidant. The antioxidant activity was expressed as the
RSA% (Radical Scavenging Activity). We used curcumin and
quercetin as reference compounds. The RSA(%) values for
nitrones 1, 2, and 6−9, at 0.5 mM, are very low (Table 3). As
shown, and as expected, nitrones 4 and 5 bearing free phenol
groups provided the best RSA(%) values, in the 79−82% range.
Quinoline 3 is a moderate antioxidant agent with a RSA value
(42%) 24-fold more potent than the analogous quinoline 2,
stressing the critical importance of the N-benzyl group
compared to the N-tert-butyl, in order to have higher
RSA(%) values. Not surprisingly, nitrone 4 bearing the benzyl
moiety is slightly more potent than nitrone 5.
3.3. Competition of the Tested Compounds with DMSO
for Hydroxyl Radicals. Superoxide (O2−•) anion and hydroxyl
radical (•OH) are free radical species of potential importance in
cerebral ischemia. In the acidic conditions of ischemic brain,
O2−• is probably protonated to give HO2−• species. Iron
released from damaged brain cells is more likely to be readily
available to catalyze the generation of OH radicals. Between the
ROS, the hydroxyl (•OH) free radical is possibly the most toxic,
as it reacts with a number of biological important molecules
such as DNA, lipids, or carbohydrates. Polyunsaturated fatty
acids are found in high concentrations in the CNS and are
particularly vulnerable by free radicals. Thus, we tried to test
the ability of our compounds to scavenge hydroxyl radicals. The
competition of the synthesized nitrones with DMSO for HO
radicals, generated by the Fe3+/ascorbic acid system, expressed
as percent inhibition of formaldehyde production, was used for
the evaluation of their hydroxyl radical scavenging activity.32 In
this experiment, the majority of the tested nitrones showed
remarkable activity at 100 μM, with values higher than the well-
known antioxidant trolox (see Tables 3 and 4). Within the
subgroup 1−11 (Table 3), nitrones 2, 11, 9, 10, 7, and 3
exhibited 95−100% scavenging activity, whereas 4 with 77%, 1
with 60%, and 8 with 54% seemed to follow. Two nitrones,
namely 5 and 6, did not present any result under the
experimental conditions. Derivatives with low lipophilicity
values, such as nitrones 2, 3, 7, 9, 10, and 11 (clogP = 2.60−
3.74), showed high antioxidant activity. It is evident that the
type and substitution pattern in the aromatic rings is important
for the HO radical scavenging ability of the tested nitrones, e.g.,
the presence of the quinolinyl group or of the 3-(nitrooxy)-
propoxy)benzylidene moiety.
Table 3. In Vitro Antioxidant Activities for Nitrones 1−11
b
•
LOX
a
AAPH (%)
DPPH
O
c
d
f
2 g
nitrone
(%)
(min)
(RSA) (%)
OH
60
(%)
clogP
1
2
3
4
5
6
7
8
9
65
no
no
no
no
17
39
57
19
30
21
no (0)
0.9
1.7
29
12
23
8
3.44
3.74
4.27
5.87
5.34
3.13
2.60
3.29
3.04
3.83
3.57
85 (56.7)
37 (78)
61 (83)
9 (17.4)
no (0)
100
95
42.3
81.9
78.6
8.6
77
no
no
95
no
no
no
2
no (0)
0.0
49 (47)
no (0)
0.4
54
0.6
98
19
no
no
15
h
10
81 (36.6)
64 (46)
nd
98
h
11
nd
100
caffeic acid
curcumin
quercetin
trolox
e
35.0
e
94.2
63 (62.1)
a
b
c
Determined at 0.1 mM. Determined at 0.1 mM. Induction time tinh
d
produced by the tested nitrones. Determined at 0.5 mM.
e
f
g
Determined at 15 μM. Determined at 0.1 mM. Determined at 0.1
h
mM. nd: not determined.
not show any inhibition, all other compounds caused inhibition
of LP. Nitrone 18 did not present any inhibition at zero time.
However, it showed 67% after 13 min and tinh value = 53.7 min.
Compounds 12, 17, 22, 23, and 25 showed inhibition values
(5−57%) lower than the common standard trolox (63%).
Obviously, the absence of hydrogen atoms (phenolic hydroxyl
groups) has a direct impact on these results. The standard
inhibitor trolox exerts its inhibitory effect on LP mainly through
the ability of its 6-hydroxy-5, 7,8-trimethylchromane moiety to
break the radical chain. On the other hand, nitrones 14−16,
19−21, and 24 gave higher values (67−90%) than trolox, the
most potent inhibitors being the compounds 19 and 20 (90%).
Nitrones 15, 16, 19, 20, and 21 might exert their antioxidative
effect through their easily oxidizable hydroxyl unit. The
antioxidative effect of nitrone 24 should be attributed through
interception of the alkylperoxyl radicals mainly by the indole
moiety and possible formation of oxidized derivatives. Lip-
ophilicity, as clogP theoretically calculated values using the
CLOG P program,30 does not seem to influence their
interaction.
3.2. Antioxidant Activity of Nitrones Determined by DPPH.
The in vitro antioxidant activity for nitrones 1−11 was
determined by the use of the 2,2-diphenyl-1-picrylhydrazyl
(DPPH) test and is given in Table 3. The results referred to
nitrones 12−25 have been recently described.19 The stable free
radical 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 mechanisms: (a) the direct
hydrogen atom transfer (HAT) and (b) the sequential proton
loss electron transfer (SPLET).31 A freshly prepared DPPH
solution exhibits a deep-purple color with an absorption
maximum at 517 nm. This purple color generally 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 radical attack on the DPPH molecule, and convert
them into colorless/bleached product. In this assay, we
measured the DPPH initial absorbance and the absorbance
Regarding nitrones 12−25, (previously prepared in our
laboratory19), the data are indicated in Table 4. Nitrones 16,
18, and 19 did not show any result at 0.1 mM concentration,
whereas compound 23 presented 30% scavenging activity. All
the other compounds significantly compete with DMSO at 0.1
mM, giving, in some cases, values higher than trolox (73%). It
seems that lipophilicity does not play any significant role.
However, antioxidants with hydrophilic or lipophilic character
are both needed to act as radical scavengers in the aqueous
phase or as chain-breaking antioxidants in biological mem-
branes.
3.4. Superoxide Anion Radical (O2−•) Scavenging Activity.
The superoxide anion radical (O2−•) is less toxic than the
hydroxyl radical but still one of the most known toxic ROS. In
addition, this radical suffers dismutation by the superoxide
dismutase enzyme (SOD), producing H2O2, the precursor for
the formation of hydroxyl radicals (see above) in the presence
of iron ions.33 Superoxide anion radicals O2• and H2O2 are
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reactive oxygen species produced in vivo, through a one-
electron reduction process of molecular oxygen. The charged
superoxide anion probably escapes via an anion channel in the
cell membrane to enter the cerebrospinal fluid (CSF) and brain
extracellular fluid. The evaluation of superoxide anion radical
scavenging activity can be determined using assays involving
nonenzymatic or enzymatic production of superoxide anions.
Herein, enzymatic superoxide anion radicals were generated by
a hypoxanthine and xanthine oxidase (XOD) reaction system.
The majority of the nitrones of both groups did not even
present any, or presented very low, scavenging activity at 0.1
mM compared to caffeic acid (15%), used as a standard
reference compound. Only nitrone 1 showed 29% scavenging
activity. At pH 7.4, superoxide anion reduces the tetrazolium
blue into formazan blue (λmax = 560 nm). The production of
superoxide radical was estimated by the nitroblue tetrazolium
method.34 The superoxide anion scavenging activity (O2−•) (%)
of the tested nitrones has been spectrophotometrically
measured.
replacement of N-tert-butyl by N-methyl or N-benzyl groups
diminishes/suppresses inhibition (e.g., 12 with N-methyl and
14 with N-benzyl group). Nitrones 12, 18, 19, 21, and 23−25
exhibit weaker inhibition. Nitrones 14−17 and 20 do not
present any biological activity under the reported experimental
conditions. Furthermore, it seems that within the pyridinyl
analogues, the physicochemical properties of substituent at C-2
must be taken under consideration. Bulkier and more lipophilic
substituents lead to more potent LOX inhibitors. Thus, the 2-
Br derivative 22 (π-Br = 0.86, MR-Br = 0.888) exhibits higher
activity than the 2-Cl substituted analogue 23 (π-Cl = 0.71,
MR-Cl = 0.63).
Compounds incorporating phenol moieties do not seem to
be particularly effective agents. Although lipophilicity is referred
to as an important physicochemical property for LOX
inhibitors,42 among the tested nitrones, the most potent
compound 22 has a % inhibition value of 88% and clogP 2.01,
which does not follow this trend. On the contrary, nitrone 18,
with the higher lipophilicity value, shows low antilipoxygenase
activity (21%, Table 4).
3.5. In Vitro Inhibition of Soybean Lipoxygenase. Cerebral
ischemia-reperfusion (IR) triggers lipid peroxidation and
inflammation, which exacerbate injury. Recognition of inflam-
matory components involved in stroke has expanded the list of
potential targets for therapy, including inducible nitric oxide
synthase (iNOS), nuclear factor kappa B (NF-κB), and 5-
lipoxygeanse (5-LOX).35 LOX is the key enzyme in leukotriene
biosynthesis.36 Leukotrienes, derived from the biotransforma-
tion of arachidonic acid catalyzed by 5-lipoxygenase (5-LOX),
cause inflammation and are thus involved in the pathobiology
of stroke injury. LOXs play a role in membrane lipid
peroxidation by forming hydroperoxides in the lipid bilayer.37
Inhibitors of LOX have attracted attention initially as potential
agents for inflammatory diseases treatment, but their
therapeutic potential has now been expanded to certain types
of cardiovascular diseases.38 Inhibition of LOX was performed
by the UV absorbance based enzyme assay.32 While one may
not extrapolate the quantitative results of this assay to the
inhibition of mammalian LOX, it has been shown that
inhibition of plant LOX activity by nonsteroidal anti-
inflammatory agents (NSAIDs) is qualitatively similar to their
inhibition of the rat mast cell LOX and may be used as a simple
qualitative screen for such activity.39 The four classes of direct
5-lipoxygenase inhibitors encompass: (a) redox-active com-
pounds keeping the active site iron in the ferrous state, thereby
uncoupling the catalytic cycle of the enzyme, (b) iron ligand
inhibitors, (c) nonredox type inhibitors that compete with
arachidonic acid or lipid hydroperoxides to bind to 5-LOX,40
and (d) a novel class of inhibitors that may act in an allosteric
manner.41
Among the nitrones 1−11, the most potent representative is
compound 1 followed by 8, whereas nitrones 6, 7, 9, 10, and 11
showed moderate inhibition. In compounds 2 and 3, which
share the common structural feature of a quinolinyl substituent,
as well as in nitrones 4 and 5, in which a 3,5-di-tert-butyl-4-
hydroxybenzylidene group is incorporated, no inhibitory
activity was observed.
Perusal of the % inhibition values in Table 4 shows that the
most potent inhibitors are the N-tert-butyl nitrones 22 (88%)
and 13 (83%), presenting higher activity than the reference
compound NDGA (40%). We were not able to determine their
IC50 values under our experimental conditions. Thus, it seems
that the inhibitory effect can mainly be attributed to the
presence of the N-tert-butyl moiety (23−25), as the
In general, the inhibitory activity of the nitrones does not
seem to be correlated with their antioxidant activity. Because
these molecules do not bear a close and chemical similarity to
the arachidonic acid (the substrate of LOX), it is possible that
they might bound not to the active site but to a separate
enzyme site. Further studies will be necessary to confirm their
mechanism of action.
3.6. NO Donating Activity. Nitric oxide (NO) is a radical
acting as a bioregulator in many cells and tissues, a key inter-
and intracellular messenger molecule involved in a variety of
pathophysiological processes,43 such as maintenance of the
vascular tone, neuronal signaling, and host response to
infection.44 Additionally, NO may reduce inflammation
connected to oxidative stress by scavenging ROS, which can
adversely increase mucosal permeability and kill cells.44
Furthermore, it is found that NO as well as NO-derived
ROS, interact with peroxidases45 and lipoxygenases,46 altering
the generation of prostaglandins and leukotrienes, which are
signaling molecules involved in inflammation.47 As a result,
nitrones capable of acting as NO donors were introduced and
new therapeutic possibilities have been emerged.6−12
The nitrones were tested in vitro for their ability to release
NO by the action of a thiol cofactor. We tried to delineate, if
possible, the influence of nitrone functionality and structure on
NO release. NO donation with simultaneous production of
cyanide occurs by action of reducing agents like thiols. L-
Cysteine was used as a cofactor in this in vitro biological test.
The rate of NO release is affected by pH, and the mechanism
does not involve the intermediacy of S-nitrosothiols. In all
experiments, sodium nitroprusside (SNP), a source of NO, as
demonstrated by the ability to nitrosate amines and ketones,
was used as the reference drug. The screened compounds (final
concentration 100 μM) were tested for their ability to release
NO when dissolved in a phosphate buffer containing L-cysteine
(pH 7.4). Released NO was oxidized in the presence of air,
transforming it to nitrites, which were detected by the Griess
reaction.48
Virtually no formation of NO2− was detectable in the absence
of L-cysteine for nitrones 1−11. Very low if any formation of
NO2− was observed from nitrones 19 and 20 (Table 4). The
present study demonstrates that under the reported exper-
imental conditions the NO release from the tested compounds
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is very limited and low and seems to be independent of the
time.
4.1. Nitrones Reduce the Loss of Neuronal Viability
Induced by OGD. Cell viability assay by 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) determi-
nation was performed to evaluate the potential neuroprotective
effect on nitrones 1−11 against OGD. Exposure of neuronal
cultures to 4 h OGD (OGD 4 h) induced a significant decrease
in cell viability (65.2%, p < 0.0001 versus 100% control, by one-
sample t test), which was partially reversed after 24 h recovery
(reperfusion) (R24h, 77.0%; p < 0.01 versus OGD 4 h,
Student’s t test) but without reaching the 24 h control value (p
< 0.0001 versus 100% control, one-sample t test) (Figure 1).
Nitrones 1−11 (ranging from 0.1 μM to 1 mM) were added
at the beginning of reperfusion period to evaluate their
potential neuroprotective effect. Cytidine-5′-diphosphocholine
(citicoline or CDP-choline), a well-known neuroprotective
agent,52 and PBN, described as protective against free
radicals,17,18 were used as reference compounds to evaluate
the neuroprotection on neuronal cultures. Citicoline was
assayed from 1 μM to 1 mM, and we found a neuroprotective
effect at 10 and 100 μM (87.4 and 91.5%, respectively,
compared to 100% control) (Figure 2). PBN did not induce
neuroprotective effect in the range of assayed concentration
(0.1−10 mM; Figure 2).
The addition of 100−250 μM nitrone 1, 1 μM nitrone 2, or
10−100 μM nitrone 3 and 10, significantly increased cell
viability during reperfusion and returned near control values
(100.3−95.7%, 93.4%, 94.4−101.2%, and 94.9−96.1%, respec-
tively, compared to 100% control) (Figure 1). The neuro-
protection induced by nitrones 1−3 and 10 was compared with
citicoline and PBN, these nitrones providing significantly higher
neuroprotection than citicoline (Table 6). Conversely, and very
interestingly, selected compounds from Table 4, such as
nitrones 12, 13, 16, 19, and 20, showed no neuroprotective
activity (Figure 2).
Oxidation of NO by air can also yield nitrate, which is not
detectable by the Griess assay. Therefore, to absolutely
conclude that none of the nitrones released NO, we employed
nitrate reductase/NADPH system to see if any residual nitrate
can be reduced back to nitrite before using the Griess assay
again. Very small changes were observed (values in parentheses
in Table 4).
3.7. In Vivo Inhibitory Activity of the Carragenan Induced
Rat Paw Edema. Inflammation is a key defense mechanism,
mediated by a complex cascade of mediators. The inflammatory
response in stroke involves not only leukocytes, endothelial and
glial cells, but also neurons. After stroke, leukocytes home
toward the lesion, and brain parenchymal cells (microglia,
astrocytes, endothelia, even neurons) transform to an
inflammatory phenotype.49 Available literature links stroke-
induced inflammation to the progression of damage. Thus,
inhibition of inflammation would seem to be an obvious
therapeutic strategy.
For the in vivo screening, the selection of the compounds
was generally based on their good inhibitory activity on LOX,
hydroxyl radical scavenging activity, and antilipid peroxidation.
Some selected nitrones (12−14, 22) were examined by using
the functional model of carrageenan-induced rat paw edema.
Results were expressed as percentage of weight increase at the
right hind paw in comparison to the uninjected left hind paw.
The induced edema is a nonspecific inflammation highly
sensitive to NSAIDs. Thus, it has been accepted as a useful tool
to study new anti-inflammatory agents.50 It reliably predicts the
anti-inflammatory potency of the NSAIDs and detects during
the second phase which anti-inflammatory agents may play an
inhibitory role in prostaglandin amplification.51 As shown in
Table 5, nitrones 12 and 22 exhibited a comparable effect (26.5
4.2. Nitrones Attenuate Necrotic Neuronal Death Induced
by OGD. To confirm the neuroprotective effects of nitrones 1−
11 on cell viability, the effect of these compounds on necrotic
cellular death was measured by monitoring LDH release from
the cultured neuronal cells. Results from Figure 3 show that
exposure of neuronal cultures to 4 h OGD (OGD 4 h) followed
by 24 h reperfusion (R24h) induced a significant increase in
LDH release (3.23 0.26 times the control values; p < 0.001,
Student’s t test) (Figure 3). These increasing LDH release
values are in keeping with the loss in cell viability observed
under this condition (about 35%, Figure 1). Exposure of
neuronal cultures to just 4 h OGD (OGD 4 h) did not induce a
statistically significant increase in LDH release (38.05 8.69%)
when compared with control of 24 h reperfusion (C) (30.96
5.31%). However, this difference was statistically significant (p
< 0.05, Student’s t test) when compared with control of 4 h by
itself (19.77 4.10%) (not shown). This means that, during
the OGD treatment, neurons suffered a cellular membrane
damage, which not only cannot be repaired but is enhanced
during reperfusion. However, the presence of some nitrones
during this period reversed the necrotic death to a great extent
(Figure 3).
Table 5. In Vivo Anti-inflammatory Activities for Nitrones
12−14 and 22
a
b
nitrone
ICPE %
SD
ClogP
12
13
14
22
26.5* 0.8
no
1.32
2.56
3.09
2.01
4.18
no
23.2** 0.6
47** 1.0
indomethacin
a
Percentage of inhibition of carraggenan-induced rat paw edema
(ICPE %). Each value represents the mean DS obtained from six
animals in two independent experiments (n = 6 × 2). In all cases,
statistical significance against controls were performed by the Student’s
b
t test (*p < 0.1, **p < 0.01). Dose of the administered compounds:
0.01 mmol/kg.
and 23.2%), yet weaker compared to indomethacin (47%).
Lower lipophilicity is correlated with a biological effect in vivo
(nitrones 12 and 22). No biological response has been
produced by nitrones 13 and 14.
On the basis of the interesting antioxidant profile obtained
by most of the tested nitrones, we next carried out the
neuroprotection analyses.
4. Neuroprotection Evaluation against Experimental
Ischemia. The neuroprotective effect of nitrones was evaluated
on primary neuronal cultures from cerebral cortex, cultured for
6−8 days and subjected to oxygen−glucose deprivation
(OGD).
Nitrones neuroprotective effects (ranging from 1 to 250 μM)
on necrotic cell death were assayed by adding them at the
beginning of the reperfusion period, and their effect was
compared with the neuroprotective effect of citicoline, as a
known neuroprotective agent,52 and PBN, as a neuroprotective
agent against free radicals.17,18
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Figure 1. Effect of nitrones on primary neuronal cultures exposed to oxygen−glucose deprivation (OGD). Bar chart showing the percentage of cell
viability at 24 h of recovery after 4 h OGD, either untreated (R24h) or treated with different concentrations (μM) of nitrones 1−11, or citicoline.
The value induced by OGD at 4 h without recovery period (OGD 4 h) is also indicated. Cell viability corresponding to control cells (1.534 0.09
AU) was considered as 100%. The values represent the average of three to four independent experiments; error bars representing the SEM *P <
0.05, and **P < 0.01 versus R24h by one-way analysis of variance following Dunnett’s post test, when analysis of variance was significant. +P < 0.05
versus R24h by Student’s t test. Statistical significances below R24h value were not shown.
Table 6. Neuroprotective Effect for Nitrones 1−3 and 10 in
Neuronal Cultures Exposed to Oxygen−Glucose
a
Deprivation (OGD)
molecule
nitrone
concentration
neuroprotection (%)
1
100 μM
250 μM
1 μM
101.3 3.3***
81.2 5.0***
71.2 4.0**
75.7 4.9**
105.2 4.3***
77.6 3.1***
83.0 2.7***
2
3
10 μM
100 μM
10 μM
100 μM
10
Figure 2. Effect of nitrones on primary neuronal cultures exposed to
oxygen−glucose deprivation (OGD). Bar chart showing the
percentage of cell viability at 24 h of recovery after 4 h OGD, either
untreated (R24h) or treated with different concentrations (μM) of
nitrones 12, 13 (PBN), 16, 19, 20, or citicoline. The value induced by
OGD at 4 h is also indicated. Cell viability corresponding to control
cells (1.497 0.05 AU) was considered as 100%. The values represent
the average of three to five independent experiments; error bars
representing the SEM *P < 0.05 versus R24h by Student’s t test.
Statistical significances below R24h value were not shown.
PBN
citicoline
5 mM
−13.4 1.9***
53.2 1.8
100 μM
a
Neuroprotection was defined as the percentage to reach the control
value, defined as 100%, from R24h value, defined as 0%. Data were
obtained from Figure 1 (Figure 2 for PBN). **P < 0.01, and ***P <
0.001 compared to citicoline, by Student’s t test.
concentrations (Table 7). As shown in the table, a smaller
efficiency (near to 50% neuroprotection) was also obtained at
100−250 μM nitrone 4, 100 μM nitrone 8, 250 μM nitrone 9,
and 1−10 μM nitrones 10 and 11.
Citicoline, tested from 1 to 250 μM, decreased LDH release
in a dose-dependent manner, having significant neuroprotective
effect at concentrations between 10 and 250 μM (35, 43 and
59%, respectively) (Figure 3). PBN also induced neuro-
protective effects at the same concentrations, from 30 to
50%. The addition of 1−250 μM of nitrone 1, 1−10 μM of
nitrone 2 or 1−250 μM of nitrone 3, 100−250 μM of nitrone 4
or 250 μM of nitrone 5, 1−10 μM of nitrone 6 or 250 μM of
nitrone 7, 100 μM or 250 μM of nitrones 8 and 9, respectively,
and 1 μM nitrones of 10 and 11, significantly decreased the
LDH release during recovery.
Maximal neuroprotective effects (statistically significant
when higher than 50% neuroprotection) were obtained at
250 μM nitrone 1, 1−10 μM nitrone 2, 10−100 μM nitrone 3,
1−10 μM nitrone 6, and 250 μM nitrone 7, an effect
comparable with citicoline or PBN at 100−250 μM
5. In Vitro Blood−Brain Barrier Permeation Assay. In
today’s drug discovery research, screening for the BBB
penetration is of great importance. One major problem for
successful CNS drugs is to cross the BBB and reach their
therapeutic targets. On the other hand, for drugs acting in
peripheral tissues, penetration into the CNS might cause
unwanted side effects. In the last years, several in vitro methods
have been performed to predict the BBB permeation of
investigational drugs. Among them, the parallel artificial
membrane permeation assays (PAMPA) have the advantage
of predicting passive blood−brain barrier permeation with high
success, high throughput, and reproducibility.
To evaluate brain penetration of compounds here described,
we used the PAMPA-BBB method described by Di et al.,53 and
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Figure 3. Effect of nitrones on LDH release in primary cortical neuronal cultures exposed to oxygen−glucose deprivation (OGD). Bar chart shows
the percentage of LDH release at 24 h of recovery after 4 h OGD, either untreated (R24h) or treated with different concentrations (μM) of nitrones
1−11, or citicoline or PBN. The value induced by OGD at 4 h without recovery period (OGD 4 h) is also indicated. LDH release corresponding to
R24h cells exposed to 4 h OGD and 24 h of reperfusion (R24h) (35.42 3.49%) was considered as 100%. The values represent the average of four
independent experiments; error bars representing the SEM *P < 0.05, **P < 0.01, and ***P < 0.001 versus R24h by one-way analysis of variance
followed by Holm−Sidak’s post test, when analysis of variance was significant. Statistical significances above R24h value were not shown.
a
Table 7. Neuroprotective Effect for Nitrones 1−11 against
Table 8. Experimental Permeability (Pe 10−6 cm s−1) in the
PAMPA-BBB Assay for Nitrones 1, 2, 10, and 11 with Their
Predictive Penetration in the CNS
Necrotic Cell Death in Neuronal Cultures Exposed to
a
Oxygen−Glucose Deprivation (OGD)
concentration
neuroprotection against necrotic cell death
(%)
nitrone
Pe
prediction
nitrone
(μM)
1
27.0 0.4
15.0 0.1
22.0 1.3
16.8 0.3
cns+
cns+
cns+
cns+
1
250
1
70.08 7.52*
70.17 3.95**
62.22 3.36**
63.01 3.26**
66.82 4.57**
53.80 6.01
2
2
3
4
6
10
11
10
10
a
PBS:EtOH (70:30). Data are the mean SD of three independent
experiments.
100
100
250
1
standard.53 Thus, it is expected that they could penetrate into
the CNS and reach their biological targets located in the CNS.
56.95 10.6
61.87 2.81**
114.17 15.69**
96.26 6.62***
55.57 12.51
79.21 19.55
45.37 2.41
10
7
250
100
250
10
CONCLUSIONS
■
8
Continuing with our nitrones for stroke project development,19
we have now investigated and report here the in vitro
antioxidant activity of nitrones 1−25. From data in Tables 3
and 4, and regarding their ability to interact with AAPH, the
highest activity was observed for nitrones 19 and 20 followed
by 21, which incorporated moieties 3-MeO and 4-OH on the
phenyl ring, as well as nitrones 2 and 10 with a 3-(nitrooxy)
propoxy) benzylidene group. The N-substituent does not seem
to influence either the antilipid peroxidation activity or the
overall lipophilicity. From the results, it is obvious that
inhibition of lipid peroxidation activity is not always
accompanied by radical scavenging ability and vice versa. This
can be attributed to the different chemical reactions involved in
the various assays. Particularly effective antioxidants are the
phenoxide anions from phenolic compounds. As shown, and as
expected, nitrones 4 and 5 bearing free phenol groups provided
the best RSA (%) values, in the 79−82% range, in DPPH test.
The incorporation of N-tert-butyl or benzyl groups, as well as 3-
(nitrooxy) propoxy) benzylidene and quinoline moieties,
provide nitrones with increased antilipid peroxidation activity,
hydroxyl radical scavenging activity, and LOX inhibition.
Overall, and among the different nitrones investigated, 2, 4,
10, 11, 13, and 22 showed interesting antioxidant activities.
We assayed the nitrones for neuroprotection activity by
MTT determination on neuronal cultures. In these experi-
ments, well-known neuroprotective agents, such as nitrone
PBN17,18 and citicoline,52 were included. Citicoline had
9
10
11
1
47.48 3.33
citicoline
100
250
100
250
61.26 8.26
84.64 3.48***
67.82 4.70**
73.20 5.83**
PBN
a
Neuroprotection against necrotic cell death was defined as the
percentage to reach the control C value (defining as 100%
neuroprotection the difference between R24h and C percentages).
Data were obtained from Figure 3. *P < 0.05, **P <0.01, and **P <
0.001 compared to 50% neuroprotection, by one-sample Student’s t
test.
́
subsequently optimized by Rodriguez-Franco and colleagues
for molecules with limited water solubility.54−56 In the last
years, this method was successfully applied by some of us to
different classes of compounds.57−64 The in vitro permeability
(Pe) values of derivatives 1, 2, 10, and 11 through a lipid extract
of porcine brain were determined by using PBS/ethanol
(70:30) (Table 8). In the same assay, 15 commercial drugs of
known CNS penetration were also tested and their
experimental values were compared to reported values, giving
a good lineal correlation. All tested nitrones showed good
permeability values, close to the reported for verapamil (Pe = 16
× 10−6 cm s−1), which is generally used as a high permeability
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rotary evaporator. Flash column chromatography was performed 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. The purity of compounds was checked by
elemental analyses, conducted on a Carlo Erba EA 1108 apparatus, and
confirmed to be ≥95%. 2,2-Diphenyl-1-picrylhyrazyl (DPPH) radical,
sodium benzoate, FeSO4·7H2O, EDTA, 30% H2O2, nitro blue
tetrazolium chloride, β-nicotinamide adenine dinucleotide 2′-phos-
phate reduced tetrasodium salt hydrate (NADPH), and nitrate
reductase from Arabidopsis thaliana, were purchased from Sigma-
Aldrich. Phosphate buffer (0.1 M and pH 7.4) was prepared mixing an
aqueous KH2PO4 solution (50 mL, 0.2 M) and an aqueous of NaOH
solution (78 mL, 0.1 M), and the pH (7.4) was adjusted by adding a
solution of KH2PO4 or NaOH. Nordihydroguaiaretic acid (NDGA),
trolox, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), so-
dium nitroprusside (SNP), and caffeic acid (CA) were purchased from
the Aldrich Chemical Co. Milwaukee, WI, (USA). Soybean LOX,
linoleic acid sodium salt, xanthine, xanthine oxidase, and indomethacin
were obtained from Sigma Chemical, Co. (St. Louis, MO, USA), and
carrageenan, type K, was commercially available. For the in vivo
experiments, male and female Fischer-344 rats (180−240 g) were
used. For the in vitro tests, a Lambda 20 (Perkin−Elmer) UV−vis
double beam spectrophotometer was used.
General Procedure for Nitrone Synthesis. Method A: In a 20
mL glass tube equipped with septa, the aldehyde, dry Na2SO4 (3 mol
equiv), and triethylamine (2 mol equiv) were suspended in dry THF.
Then, the hydroxylamine hydrochloride (2 equiv) was added. The
mixture was stirred for 30 s and then exposed to MWI (250 W) at 90
°C during the time indicated for each compound. When the reaction
was over (TLC analysis), the reaction mixture was diluted with water,
extracted with AcOEt, dried over anhydrous sodium sulfate, filtered,
and the solvent was evaporated. The resultant solid was purified by
column chromatography to give pure compounds. Method B: Methyl-
2-nitropropane (2 mol equiv) and zinc (3 mol equiv) were added to a
solution of the aldehyde in EtOH (0.14M). The reaction mixture was
cooled to 0 °C, and glacial acetic acid (6 equiv) was added dropwise.
The reaction mixture was stirred at room temperature for 14 h and
then was kept at 0 °C for 2 h. The precipitate was filtered and washed
with EtOH. The solvent was evaporated and the resultant solid was
purified by column chromatography to give pure compounds.
(Z)-2-Methyl-N-(3-(trifluoromethyl)benzylidene)propan-2-
amine Oxide (1).21 Following the general procedure (method B),
reaction of commercial 3-(trifluoromethyl)benzaldehyde (100 mg,
0.58 mmol), 2-methyl-2-nitropropane (0.125 mL, 1.15 mmol), Zn
(115 mg, 1.73 mmol), and acetic acid (0.20 mL, 3.45 mmol) in EtOH
(3 mL), after the specified reaction time, and column chromatography
(hexane/EtOAc, 8:2, v/v), gave nitrone 1 (54 mg, 39%) as a solid: mp
52−4 °C. IR (KBr) νmax 3436, 2985, 1567, 1341, 1173, 1116 cm−1. 1H
NMR (400 MHz, CDCl3) δ 8.69 (s, 1H, H-2′, Ph), 8.38 (d, 1H, J = 8.0
Hz, H-4′, Ph), 7.64−7.62 (m, 2H, NCH, H-6′, Ph), 7.52 (t, 1H, J =
8.0 Hz, H-5′, Ph), 1.62 [s, 9H, C(CH3)3]. 13C NMR (101 MHz,
CDCl3) δ 131.6 (C-6′, Ph), 130.8 (q, J = 32.9 Hz, CF3), 128.8 (C-5′,
Ph), 128.6 (CN), 127.9 (C-3′ Ph), 126.4 (C-4′ Ph), 125.2 (C-2′
Ph), 122.5 (C-1′, Ph), 71.5 [C(CH3)3], 28.3 [C(CH3)3]. MS (EI) m/z:
245 (M)+, 205, 163, 121, 79, 57. MS (ESI): 246.0 (M + H)+. Anal.
Calcd for C12H14F3NO: C, 58.77; H, 5.75; N, 5.71. Found: C, 58.83;
H, 6.02; N, 5.99.
significant neuroprotective effect at 100 μM (89.3%), in
accordance with other studies.65 However, PBN, ranging
from 100 μM to 10 mM, had no neuroprotective effect, even
at the concentrations described as protective (1−10 mM) in
other studies.66,67 However, nitrones 1−3 and 10 exhibited the
highest neuroprotective effect. In neuronal cultures treated with
these nitrones, the cell viability was restored during reperfusion
period against OGD-induced damage. The neuroprotection
induced by nitrones 1−3 and 10 was compared with citicoline
and PBN and shown in Table 6. From these data, some simple
structure−activity relationships can be inferred. First of all, it
seems that both the presence of a strong electron-withdrawing
group (CF3) in the benzene ring, or the quinoline heterocyclic
ring system conjugated with the nitrone moiety, are key
structural motifs for the remarkable neuroprotective capacity
observed in compounds 1−3. Conversely, the presence of a
highly hindered phenol moiety in nitrones 4 and 5, or the
bromofuran ring system in nitrones 6 and 7, are associated with
strong toxicity along with no activity, or toxicity at high
concentrations, respectively. Finally, in nitrones 8−11, the type
of the N-alkyl residue on the nitrogen bearing the nitrone
motif, determined their neuroprotective profile: nitrones 8 and
9, bearing the tert-butyl chain, are more toxic at high
concentrations than nitrones 10 and 11 bearing the benzyl
group. This is similar to what we observed in the antioxidant
activity for the same nitrones determined by DPPH (see
above).
In general, the effect of these nitrones on necrotic cellular
death, measured by the LDH release method, is in good
agreement with data from neuroprotective activity on cell
viability shown above. Thus, the trifluoromethyl group in the
benzene ring of nitrone 1 and the quinoline ring in nitrones 2
and 3 seem to provide nitrones with maximal neuroprotective
capacity. For nitrones 8−11, only nitrone 9, bearing a tert-butyl
substituent on the nitrone motif, was significantly neuro-
protective. However, and at least as far as necrotic cell death is
concerned, small concentrations of nitrones 10 and 11, bearing
a benzyl group, could result in a good neuroprotective capacity.
On the contrary, at high concentrations, these nitrones become
toxic.
It is also worth pointing out that the theoretical calculations
for the neuroprotection are in good agreement with the
experimental data for nitrones 2, 3, 6−9, and 11, unlike
nitrones 1, 4, 5, and 10. Similarly, for the BBB prediction, the
theoretical calculations are in agreement with the experimental
data for nitrones 1−7, but not for nitrones 8−11. The
theoretical analyses were utterly successful in identifying the
quinolinyl nitrones 2 and 3 as potent neuroprotective agents
able to cross the BBB.
Overall, and among the different molecules investigated,
permeable quinolinyl nitrones 2 and 3 show potent combined
antioxidant and neuroprotective properties and consequently
can be considered as new lead compounds for further
development in specific tests for potential stroke treatment.
EXPERIMENTAL SECTION
■
(Z)-N-[(2-Chloro-6-methylquinolin-3-yl)methylene]-2-meth-
ylpropan-2-amine Oxide (2). Following the general procedure
(method B), reaction of commercial 2-chloro-6-methylquinoline-3-
carbaldehyde (300 mg, 1.46 mmol), 2-methyl-2-nitropropane (0.32
mL, 2.92 mmol), Zn (290 mg, 4.38 mmol), and acetic acid (0.50 mL,
8.75 mmol) in EtOH (8 mL), after the specified reaction time, and
column chromatography (hexane/EtOAc, 85:15, v/v) gave nitrone 2
(112 mg, 28%) as a solid: mp 120−3 °C. IR (KBr) νmax 3436, 2967,
General Methods. Reactions were monitored by TLC using
precoated silica gel aluminum plates containing a fluorescent indicator
(Merck, 5539). Detection was done by UV (254 nm), followed by
charring with sulfuric−acetic acid spray, 1% aqueous potassium
permanganate solution, 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
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1
1548, 1364, 1333, 1185, 1131 cm−1. H NMR (400 MHz, CDCl3) δ
(m, 6H, NCH, 5Ph), 6.46 (d, 1H, J = 3.0 Hz, H-4′, Ar), 5.01 (s, 2H,
CH2Ph). 13C NMR (101 MHz, CDCl3) δ 148.8 (C-2′, Ar), 132.4,
129.5, 129.3, 129.1 (Ph), 124.4, 124.2 (CN, C-5′, Ar), 117.5 (C-3′,
Ar), 114.2 (C-4′, Ar), 69.6 (CH2Ph); MS (EI) m/z: 281, 279 (M)+, 91.
MS (ESI) m/z: 282.0, 280.0 (M + H)+. Anal. Calcd for C12H10BrNO2:
C, 51.45; H, 3.60; N, 5.00; Br, 28.53. Found: C, 51.74; H, 3.69; N,
5.16; Br, 28.08.
10.29 (s, 1H, H-4′, Ar), 8.27 (s, 1H, NCH), 7.85 (d, 1H, J = 8.8 Hz,
H-8′, Ar), 7.63 (s, 1H, H-5′, Ar), 7.56 (d, 1H, J = 8.8 Hz, H-7′, Ar),
2.52 (s, 3H, CH3), 1.67 [s, 9H, C(CH3)3]. 13C NMR (101 MHz,
CDCl3) δ 148.0 (Ar), 145.5 (Ar), 137.6 (Ar), 136.5 (C-4′, Ar), 133.7
(C-7′, Ar), 127.8, 127.7 (C-5′, C-8′, Ar), 127.2 (Ar), 125.4 (CN),
122.7 (Ar), 72.3 [C(CH3)3], 28.3 [C(CH3)3)], 21.6 (CH3). MS (EI)
m/z: 276 (M)+, 241, 185, 167, 57. MS (ESI) m/z: 277.0 (M + H)+.
Anal. Calcd for C15H17ClN2O: C, 65.10; H, 6.19; N, 10.12; Cl, 12.81.
Found: C, 65.37; H, 5.91; N, 9.93; Cl, 13.07.
(Z)-N-[(2-Chloro-6-methylquinolin-3-yl)methylene]-1-phe-
nylmethanamine Oxide (3). Following the general procedure
(method A), reaction of commercial 2-chloro-6-methylquinoline-3-
carbaldehyde (200 mg, 0.97 mmol), Na2SO4 (410 mg, 2.92 mmol),
Et3N (0.30 mL, 1.95 mmol), and N-benzylhydroxylamine hydro-
chloride (310 mg, 1.95 mmol) in THF (4 mL), after 30 min, and
column chromatography (hexane/EtOAc, 7:3, v/v), gave nitrone 3
(192 mg, 64%) as a solid: mp 154−156 °C. IR (KBr) νmax 3436, 2918,
1569, 1555, 1429, 1342, 1180, 1146, 1048 cm−1. 1H NMR (400 MHz,
CDCl3) δ 10.20 (s, 1H, H-4′, Ar), 8.10 (s, 1H, NCH), 7.84 (d, 1H, J
= 8.3 Hz, H-8′, Ar), 7.62 (s, 1H, H-5′, Ar), 7.57 (d, 1H, J = 8.3 Hz, H-
7′, Ar), 7.54−7.51 (m, 2H, Ph), 7.47−7.41 (m, 3H, Ph), 5.16 (s, 2H,
CH2Ph), 2.52 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ 147.4,
145.6, 137.7 (3Ar), 136.8 (C-4′, Ar), 133.9 (C-7′, Ar), 132.7 (Ph),
129.5, 129.4, 129.3, 129.1 (CN, 3Ph), 127.8 (2Ar), 127.0 (C-8′, Ar),
122.2 (Ar), 72.2 (CH2Ph), 21.6 (CH3). MS (EI) m/z: 310 (M)+, 275,
91. MS (ESI) m/z: 311.0 (M + H)+. Anal. Calcd for C18H15ClN2O: C,
69.57; H, 4.86; N, 9.01; Cl, 11.41. Found: C, 69.52; H, 5.14; N, 9.30;
Cl, 11.63.
(Z)-N-[(5-Bromofuran-2-yl)methylene]-2-methylpropan-2-
amine Oxide (7). Following the general procedure (method A),
reaction of commercial 5-bromofuran-2-carbaldehyde (150 mg, 0.86
mmol), Na2SO4 (730 mg, 5.14 mmol), Et3N (0.52 mL, 3.43 mmol),
and N-t-butylhydroxylamine hydrochloride (430 mg, 3.42 mmol) in
THF (5 mL), after 1 h, and column chromatography (hexane/EtOAc,
6:4, v/v), gave nitrone 7 (150 mg, 71%) as a solid: mp 93−5 °C. IR
1
(KBr) νmax 3436, 2978, 1476, 1357, 1202, 1116, 1013 cm−1. H NMR
(400 MHz, CDCl3) δ 7.71 (d, 1H, J = 3.3 Hz, H-3′, Ar), 7.69 (s, 1H,
NCH), 6.46 (d, 1H, J = 3.3 Hz, H-4′, Ar), 1.57 (s, 9H, CH3). 13C
NMR (101 MHz, CDCl3) δ 149.6 (C-2′, Ar), 123.9 (CN), 120.6
(C-3′, Ar), 117.0 (C-4′, Ar), 114.0 (C-5′, Ar), 69.9 [C(CH3)3], 28.0
[C(CH3)3]. MS (EI) m/z: 247, 245 (M)+, 191, 189, 79, 57. MS (ESI)
m/z: 248.0, 246.0 (M + H)+. Anal. Calcd for C9H12BrNO2: C, 43.92;
H, 4.91; N, 5.69; Br, 32.47. Found: C, 43.80; H, 4.75; N, 5.81; Br,
31.93.
(Z)-2-Methyl-N-(4-(3-(nitrooxy)propoxy)benzylidene)-
propan-2-amine Oxide (8). Following the general procedure
(method A), reaction of 3-(4-formylphenoxy)propyl nitrate20 (67
mg, 0.30 mmol), Na2SO4 (128 mg, 0.90 mmol), Et3N (0.09 mL, 0.60
mmol), and N-t-butylhydroxylamine hydrochloride (75 mg, 0.60
mmol) in THF (4 mL), after 18 h, and column chromatography
(hexane/EtOAc, 2:8, v/v), gave nitrone 8 (56 mg, 63%) as a solid: mp
99−101 °C. IR (KBr) νmax 3436, 2982, 1623, 1609, 1513, 1281, 1269,
1177, 1109 cm−1. 1H NMR (400 MHz, CDCl3) δ 8.28 (d, 2H, J = 8.5
Hz, H-2′, Ph), 7.48 (s, 1H, NCH), 6.92 (d, 2H, J = 8.5 Hz, H-3′,
Ph), 4.67 (t, 2H, J = 6.0 Hz, CH2ONO2), 4.12 (t, 2H, J = 6.0 Hz,
CH2OAr), 2.23 (q, 2H, J = 6.0 Hz, CH2CH2CH2), 1.61 [s, 9H,
C(CH3)3]. 13C NMR (101 MHz, CDCl3) δ 159.6 (C-4′, Ph), 130.8
(C-2′ Ph), 129.6 (CN), 124.4 (C-1′, Ph), 114.2 (C-3′ Ph), 70.2
[C(CH3)3], 69.8 (CH2ONO2), 63.6 (CH2OAr), 28.3 [C(CH3)3], 26.9
(CH2CH2CH2). MS (EI) m/z: 296 (M)+, 240, 194, 177, 136, 57. MS
(ESI) m/z: 297.0 (M + H)+. Anal. Calcd for C14H20N2O5: C, 56.75; H,
6.80; N, 9.45. Found: C, 56.76; H, 6.50; N, 9.24.
(Z)-N-(3,5-Di-tert-butyl-4-hydroxybenzylidene)-1-phenylme-
thanamine Oxide (4).22 Following the general procedure (method
A), reaction of commercial 3,5-di-t-butyl-4-hydroxybenzaldehyde (200
mg, 0.82 mmol), Na2SO4 (470 mg, 3.29 mmol), Et3N (0.25 mL, 1.64
mmol), and N-benzylhydroxylamine hydrochloride (260 mg, 1.64
mmol) in THF (4 mL), after 40 min, and column chromatography
(hexane/EtOAc, 6:4, v/v), gave nitrone 4 (264 mg, 95%) as a solid:
mp 225−6 °C. IR (KBr) νmax 3623, 3430, 2956, 1593, 1438, 1412,
1239, 1146, 1119 cm−1. H NMR (400 MHz, CDCl3) δ 8.16 (s, 2H,
1
H-2′, Ar), 7.50−7.34 (m, 6H, NCH, 5Ph), 5.61 (s, 1H, OH), 5.03
(s, 2H, CH2Ph), 1.45 (s, 18H, CH3). 13C NMR (101 MHz, CDCl3) δ
155.9 (C-4′, Ar), 135.8 (C-3′, Ar), 135.1 (CN), 133.5, 129.1, 128.8,
128.7 (CH2Ph), 126.4 (C-2′, Ar), 122.2 (C-1′, Ar), 70.6 (CH2Ph), 34.3
[C(CH3)3], 30.1 [C(CH3)3]. MS (EI) m/z: 339 (M)+, 323, 266, 91.
MS (ESI) m/z: 340.3 (M + H)+. Anal. Calcd for C22H29NO2: C,
77.84; H, 8.61; N, 4.13. Found: C, 77.51; H, 8.43; N, 4.30.
(Z)-2-Methyl-N-(4-(2-(nitrooxy)ethoxy)benzylidene)propan-
2-amine Oxide (9). Following the general procedure (method A),
reaction of 2-(4-formylphenoxy)ethyl nitrate20 (120 mg, 0.57 mmol),
Na2SO4 (480 mg, 3.41 mmol), Et3N (0.35 mL, 2.27 mmol), and N-t-
butylhydroxylamine hydrochloride (290 mg, 2.27 mmol) in THF (4
mL), after 20 h, and column chromatography (hexane/EtOAc, 2:8, v/
v), gave nitrone 9 (72 mg, 45%) as a solid: mp 66−9 °C. IR (KBr)
(Z)-N-(3,5-Di-tert-butyl-4-hydroxybenzylidene)-2-methyl-
propan-2-amine Oxide (5).22 Following the general procedure
(method A), reaction of commercial 3,5-di-t-butyl-4-hydroxybenzalde-
hyde (120 mg, 0.49 mmol), Na2SO4 (280 mg, 1.95 mmol), Et3N (0.15
mL, 0.98 mmol), and N-t-butylhydroxylamine hydrochloride (123 mg,
0.98 mmol) in THF (4 mL), after 16 h, and column chromatography
(hexane/EtOAc, 6:4, v/v), gave nitrone 5 (105 mg, 70%) as a solid:
mp 230−3 °C. IR (KBr) νmax 3436, 2969, 1438, 1360, 1195, 1093
1
νmax 3436, 2981, 1636, 1604, 1285, 1259, 1111 cm−1. H NMR (400
MHz, CDCl3) δ 8.29 (d, 2H, J = 8.6 Hz, H-2′, Ph), 7.47 (s, 1H, N
CH), 6.91 (d, 2H, J = 8.6 Hz, H-3′, Ph), 4.82 (t, 2H, J = 4.4 Hz,
CH2ONO2), 4.29 (t, 2H, J = 4.4 Hz, CH2OAr), 1.60 [s, 9H,
C(CH3)3]. 13C NMR (101 MHz, CDCl3) δ 159.2 (C-4′, Ph), 131.0
(C-2′ Ph), 129.6 (CN), 125.1 (C-1′, Ph), 114.4 (C-3′ Ph), 71.0
(CH2ONO2), 70.5 [C(CH3)3], 64.2 (CH2OAr), 28.5 [C(CH3)3]. MS
(EI) m/z: 282 (M)+, 180, 163, 57. MS (ESI) m/z: 283.0 (M + H)+.
Anal. Calcd for C13H18N2O5: C, 55.31; H, 6.43; N, 9.92. Found: C,
55.54; H, 6.18; N, 9.92.
cm−1. H NMR (400 MHz, CDCl3) δ 8.21 (s, 2H, H-2′, Ar), 7.44 (s,
1
1H, NCH), 5.56 (s, 1H, OH), 1.60 [s, 9H, NC(CH3)3], 1.46 [s,
18H, ArC(CH3)3]. 13C NMR (101 MHz, CDCl3) δ 155.6 (C-4′, Ar),
135.7 (C-3′, Ar), 130.6 (CN), 126.5 (C-2′, Ar), 122.9 (C-1′, Ar),
69.9 [NC(CH3)3], 34.4 [ArC(CH3)3], 30.2 [ArC(CH3)3], 28.5
[NC(CH3)3]. MS (EI) m/z: 305 (M)+, 249, 234, 57. MS (ESI) m/
z: 306.3 (M + H)+. Anal. Calcd for C19H31NO2: C, 74.71; H, 10.23; N,
4.59. Found: C, 74.83; H, 10.12; N, 4.81.
(Z)-N-(4-(3-(Nitrooxy)propoxy)benzylidene)-1-phenylme-
thanamine Oxide (10). Following the general procedure (method
A), reaction of 3-(4-formylphenoxy)propyl nitrate20 (80 mg, 0.36
mmol), Na2SO4 (200 mg, 1.42 mmol), Et3N (0.11 mL, 0.71 mmol),
and N-benzylhydroxylamine hydrochloride (113 mg, 0.71 mmol) in
THF (3 mL), after 4 h, and column chromatography (hexane/EtOAc,
3:7, v/v), gave nitrone 10 (102 mg, 87%) as a solid: mp 96−98 °C. IR
(Z)-N-[(5-Bromofuran-2-yl)methylene]-1-phenylmethan-
amine Oxide (6). Following the general procedure (method A),
reaction of commercial 5-bromofuran-2-carbaldehyde (110 mg, 0.63
mmol), Na2SO4 (360 mg, 2.51 mmol), Et3N (0.19 mL, 1.25 mmol),
and N-benzylhydroxylamine hydrochloride (200 mg, 1.25 mmol) in
THF (4 mL), after 30 min, and column chromatography (hexane/
EtOAc, 6:4, v/v), gave nitrone 6 (142 mg, 81%) as a solid: mp 108−
1
(KBr) νmax 3436, 1624, 1605, 1282, 1256, 1174 cm−1. H NMR (400
MHz, CDCl3) δ 8.20 (d, 2H, J = 8.0 Hz, H-2′, Ph), 7.50−7.31 (m, 6H,
NCH, 5CH2Ph), 6.90 (d, 2H, J = 8.0 Hz, H-3′, Ph), 5.03 (s, 2H,
CH2Ph), 4.66 (t, 2H, J = 6.0 Hz, CH2ONO2), 4.10 (t, 2H, J = 5.6 Hz,
CH2OAr), 2.22 (t, 2H, J = 6.0, 5.6 Hz, CH2CH2CH2). 13C NMR (101
110 °C. IR (KBr) νmax 3436, 1477, 1222, 1134, 1010 cm−1. H NMR
1
(400 MHz, CDCl3) δ 7.71 (d, 1H, J = 3.0 Hz, H-3′, Ar), 7.44−7.43
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MHz, CDCl3) δ 159.8 (C-4′, Ph), 133.8 (CN), 133.3 (CH2Ph),
130.6 (C-2′ Ph), 129.2, 129.0, 128.9 (3CH2Ph), 123.8 (C-1′, Ph),
114.2 (C-3′ Ph), 70.8 (CH2Ph), 69.8 (CH2ONO2), 63.5 (CH2OAr),
26.9 (CH2CH2CH2). MS (EI) m/z: 330 (M)+, 153, 91;. MS (ESI) m/
z: 331.0 (M + H)+. Anal. Calcd for C17H18N2O5: C, 61.81; H, 5.49; N,
8.48. Found: C, 62.02; H, 5.38; N, 8.70.
in phosphate buffer (50 mM, pH 7.4), the tested nitrones
(concentration 0.1 mM), and ascorbic acid (10 mM). After 30 min
of incubation (37 °C), the reaction was stopped with CCl3COOH
(17% w/v) (Table 3). Trolox was used as an appropriate standard.
Scavenging Activity of Superoxide Anion Radical.34 The
superoxide anion was generated by the xanthine−xanthine oxidase
system and measured by the nitroblue tetrazolium (NBT) method.
NBT and tested nitrone (0.1 mM final concentration) and xanthine
oxidase 40 μL (1.4 mL/60 mg, 0.07U/mL) were added to the reaction
mixture in phosphate buffer pH 7.4 (0.1M) containing xanthine. After
incubating for 10 min at rt, the absorbance was recorded at 560 nm.
Caffeic acid was used as an appropriate standard.
(Z)-N-(4-(2-(Nitrooxy)ethoxy)benzylidene)-1-phenylmethan-
amine Oxide (11). Following the general procedure (method A),
reaction of 2-(4-formylphenoxy)ethyl nitrate20 (70 mg, 0.33 mmol),
Na2SO4 (190 mg, 1.33 mmol), Et3N (0.10 mL, 0.66 mmol), and N-
benzylhydroxylamine hydrochloride (106 mg, 0.66 mmol) in THF (4
mL), after 3 h, and column chromatography (hexane/EtOAc, 3:7, v/
v), gave nitrone 11 (102 mg, 87%) as a solid: mp 133−5 °C. IR (KBr)
Soybean LOX Inhibition Study in Vitro.69 The tested nitrones,
dissolved in DMSO, were incubated at rt with sodium linoleate (0.1
mL) and 0.2 mL of enzyme solution (0.1 mg/mL in saline). The
conversion of sodium linoleate to 13-hydroperoxylinoleic acid at 234
nm was recorded and compared with the appropriate standard
inhibitor (NDGA). The type of inhibition was studied. The nitrones
(in a standard concentration) were added to a series of different
substrate concentrations (sodium linoleate). The reactions are started
by adding the same amount of enzyme to each of the substrate
concentrations containing the tested nitrone. The results support
competitive inhibition.
1
νmax 3436, 1618, 1606, 1284, 1255, 1176, 1147 cm−1. H NMR (400
MHz, CDCl3) δ 8.21 (d, 2H, J = 8.8 Hz, H-2′, Ph), 7.49−7.32 (m, 6H,
NCH, 5CH2Ph), 6.91 (d, 2H, J = 8.8 Hz, H-3′, Ph), 5.03 (s, 2H,
CH2Ph), 4.82 (t, 2H, J = 4.4 Hz, CH2ONO2), 4.28 (t, 2H, J = 4.4 Hz,
CH2OAr). 13C NMR (101 MHz, CDCl3) δ 159.5 (C-4′, Ph), 133.9
(CN), 133.5 (CH2Ph), 130.9 (C-2′ Ph), 129.5, 129.2 (3CH2Ph),
124.5 (C-1′, Ph), 114.5 (C-3′ Ph), 71.1 (CH2Ph), 71.0 (CH2ONO2),
64.2 (CH2OAr). MS (EI) m/z: 316 (M)+, 91. MS (ESI) m/z: 317.0
(M + H)+. Anal. Calcd for C16H16N2O5: C, 60.75; H, 5.10; N, 8.86.
Found: C, 60.92; H, 4.97; N, 8.96.
Chemical Determination of Antioxidant Activity. We used
different types of assays to measure in vitro antioxidant activity of
nitrones, such as the inhibition of lipid peroxidation (LP) induced by
AAPH, the DMSO method for the hydroxyl radical scavenging activity,
the superoxide anion radical activity, and the in vitro inhibition of
soybean lipoxygenase (LOX). All require a spectrophotometric
measurement and a certain reaction time in order to obtain
reproducible results. Each in vitro experiment was performed at least
in triplicate, and the standard deviation of absorbance was less than
10% of the mean.
Quantitative Nitrite Assays for NO Release.70 A solution of the
examined nitrones (final concentration 100 μM (Table 1) (20 μL),
dissolved in DMSO, was added to 2 mL of 50 mmol phosphate buffer
(pH = 7.4) containing the appropriate amount of thiol cofactor 1 mM
(L-cysteine). After 5 or 60 min at 37 °C, 1 mL of the reaction mixture
was treated with 250 μL of the Griess reagent (4 g sulfanilamide, 0.2 g
N-naphthyl-ethyldiamine dihydrochloride, 10 mL of 85% phosphoric
acid in distilled water, final volume 100 mL). After the mixture stood
for 10 min at rt, absorbance was recorded at 540 nm. Sodium
nitroprusside (SNP) was used as standard reference compound (100
μM). The yield in nitrite (NO2− release) for each nitrone, as function
of L-cysteine (mM concentration) is given in Table 4, expressed as %
NO2− (mol/mol). The nitrite determination was repeated, using the
nitrate reductase (0.1 U/mL)/NADPH (1 mM) system71,72 to see if
any residual nitrate could be reduced back to nitrite before using the
Inhibition of Linoleic Acid Lipid Peroxidation.27,68 Production
of conjugated diene hydroperoxide by oxidation of linoleic acid in an
aqueous dispersion was monitored at 234 nm. AAPH was used as a
free radical initiator. This assay can be used to follow oxidative changes
and to understand the contribution of each tested nitrone. Ten μL of
the 16 mM linoleic acid sodium salt solution was added to the UV
cuvette containing 0.93 mL of 0.05 M phosphate buffer, pH 7.4,
prethermostated at 37 °C. The oxidation reaction was initiated at 37
°C under air by the addition of 50 μL of 40 mM AAPH solution.
Oxidation was carried out in the presence of aliquots (10 μL). In the
assay without antioxidant, lipid oxidation was measured in the
presence of the same level of DMSO. The rate of oxidation at 37
°C was monitored by recording the increase in absorption at 234 nm
caused by conjugated diene hydroperoxides. Trolox was used as an
appropriate standard.
Antioxidant Activity of Nitrones Determined by DPPH
Radical Method.31 The assay was carried out in 96-well microplates,
and the final reaction mixture was 200 μL per well. A methanol DPPH
solution (1.35 × 10−4 M, final concentration) (2.7 mL) was mixed with
different concentrations of the nitrones (0, 100, 200, 300, 400, and 500
μM, final concentration) (0.3 mL) freshly prepared from a methanolic
stock solution (10−3 M). The resulting mixture was incubated for 2 h
at rt in the dark. After this time, 200 μL were placed in a 96-well
microplate and the absorbance of DPPH radical was measured at 517
nm in a spectrophotometric plate reader (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% (radical scavenging
activity), calculated as follows: RSA% = 100[(Ao − Ai)/Ao) × 100,
where Ao and Ai are the DPPH absorbance in absence and in presence
of added nitrone concentration i, respectively. Data are expressed as
means.
Griess assay. Values are given as means
SD of four to six
experiments. SD values were always lower than 10%. Calibration curve
10−100 nmol/mL sodium nitrite standard solutions were used for the
calibration curve.
In Vivo Assays. Acute Toxicity. In acute toxicity preliminary
experiments (data not given), in vivo the nitrones 12, 13, 14 and 22
were examined in doses from 0.05 up to 0.1 mmol/kg body weight
(lethal range 30−80% after 24 h).
Inhibition of the Carrageenan-Induced Edema..69 Edema was
induced in the right hind paw of Fisher 344 rats (150−200 g) by the
intradermal injection of 0.1 mL 2% carrageenan in water. Both sexes
were used. Females pregnant were excluded. Each group was
composed of six animals. For each nitrone, the experiment was
performed twice for validation. The animals, which had been bred in
our laboratory, were housed under standard conditions and received a
diet of commercial food pellets and water ad libitum during the
maintenance but were entirely fasted during the experiment period. In
our studies, we followed the recognized national and European ethical
guidelines on animal experimentation. The experiments conform to
the law for the protection of experimental animals (Republic of
Greece) and are registered at the Veterinary Administration of the
Republic of Greece. The tested nitrones, 0.01 mmol/kg body weight,
were diluted in water with a few drops of Tween 80 and ground in a
mortar before use, and they were intraperitoneally simultaneously
given with the carrageenan injection. The rats were euthanized 3.5 h
after carrageenan administration. The difference between the weight of
the injected and uninjected paws was calculated for each animal. The
change in paw weight was compared with that in control animals
(treated with water or Tween−water) and expressed as a percent
inhibition of the edema % ICPE values (Table 5). Indomethacin was
tested as a reference compound in 0.01 mmol//kg (47%). Values %
ICPE are the mean from two different experiments (n = 6 animals
Competition of the Tested Nitrones with DMSO for
Hydroxyl Radicals.31 The hydroxyl radicals generated by the Fe3+/
ascorbic acid system were detected by the determination of
formaldehyde produced from the oxidation of DMSO. The reaction
mixture contained EDTA (0.1 mM), Fe3+ (167 μM), DMSO (33 mM)
164
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Article
each time) with a standard error of the mean less than 10%.
(LDH in the culture medium plus LDH inside the cells). Increased
LDH release denotes increase in plasma membrane damage produced
by necrotic cell death.
%ICPE inhibition = (A − A
/A ) × 100
c
c
treat
In Vitro BBB Permeation Assay. Prediction of the brain
penetration was evaluated using a parallel artificial membrane
permeation assay (PAMPA), in a similar manner as previously
described.53−64 Commercial drugs, phosphate buffered saline solution
at pH 7.4 (PBS), and dodecane were purchased from Sigma, Aldrich,
Acros, and Fluka. Millex filter units (PVDF membrane, diameter 25
mm, pore size 0.45 μm) were acquired from Millipore. PBL was
obtained from Avanti Polar Lipids. The donor microplate was a 96-
well filter plate (PVDF membrane, pore size 0.45 μm) and the
acceptor microplate was an indented 96-well plate, both from
Millipore. The acceptor 96-well microplate was filled with 180 μL of
PBS/ethanol (70:30), and the filter surface of the donor microplate
was impregnated with 4 μL of PBL in dodecane (20 mg mL−1).
Compounds were dissolved in PBS/ethanol (70:30) at 100 μg mL−1,
filtered through a Millex filter, and then added to the donor wells (180
μL). The donor filter plate was carefully put on the acceptor plate to
form a sandwich, which was left undisturbed for 240 min at 25 °C.
After incubation, the donor plate is carefully removed and the
concentration of compounds in the acceptor wells was determined by
UV spectroscopy. Every sample is analyzed at five wavelengths, in four
wells and at least in three independent runs, and the results are given
Ac = mean weight of the injected and uninjected (carageenan
injection) paws of the group of the control animals, and Atreat = mean
weight of the injected and uninjected paws (carageenan injection) of
the group of the treated animals with each examined compound.
Primary Neuronal Cultures. Primary neuronal cultures from rat
cerebral cortex were prepared as previously described.73,74 All
procedures associated with animal experiments were approved by
́
the Ethics Committee of the Hospital Ramon y Cajal and Universidad
Complutense de Madrid (UCM, Madrid (Spain). Cell suspensions
from cerebral cortex were prepared from 16- to 17-day-old Sprague−
Dawley rat embryos. Living cells in cell suspension were counted by
trypan blue exclusion method. Cells were seeded on plastic multidishes
precoated with 0.05 mg/mL poly-D-lysine at a density of 2.5 × 105
cells/cm2 and were kept at 37 °C in a 6.5% CO2 atmosphere in high-
glucose Dulbecco’s medium supplemented with 15% heat-inactivated
(56 °C for 30 min) fetal calf serum. After 24 h, cultured cells were
placed in, and maintained in, serum-free medium (Dulbecco’s: Ham’s
F12, 1:1 [vol/vol], 5 mg/mL glucose, 2 mM L-glutamine, and 1 mM
sodium pyruvate, and supplemented with 100 μg/mL transferrin, 100
μM putrescine, 20 nM progesterone, 30 nM sodium selenite, and 5
μg/mL insulin), as described by Bottenstein and Sato.75 Six- to eight-
day cultures were used in the experiments and contained 90% β-III-
tubulin-positive mature neurons, as described previously.76
as the mean
standard deviation. In each experiment, 15 quality
control standards of known BBB permeability were included to
validate the analysis set.
Exposure of Cell Cultures to Oxygen−Glucose Deprivation
and Treatments. Primary neuronal cultures were exposed to OGD
so as to induce cellular damage. Cultured cells were washed and placed
in glucose-free Dulbecco’s medium (bubbled with 95% N2/5% CO2
for 30 min) and maintained in an anaerobic chamber containing a gas
mixture of 95% N2/5% CO2 and humidified at 37 °C at a constant
pressure of 0.15 bar. Cells were exposed to OGD for a period of 4 h
(OGD 4 h). At the end of the OGD period, culture medium was
replaced with oxygenated serum-free medium, and cells were placed
and maintained in the normoxic incubator for 24 h to recovery
(R24h). In the neuroprotection experiments, citicoline or nitrones (0.1
μM−1 mM) were added at the beginning of recovery period. Control
cultures in Dulbecco’s medium containing glucose were kept in the
normoxic incubator for the same period of time as the OGD, and then
culture medium was replaced with serum-free medium and cells were
returned to the normoxic incubator until the end of recovery period.
Control experiments included the same amounts of vehicle (final
concentration <0.1% dimethyl sulfoxide or ethanol). The experimental
procedures were blindly performed, assigning a random order to each
assayed nitrone. Nitrones were analyzed independently three−five
times with different batches of cultures, and each experiment was run
in quadruplicate.
Cell Viability Assay. Cell viability was evaluated by quantification
of living, metabolically active cells, as determined by a colorimetric
assay using the photometric reduction of MTT to a blue formazan
product. The assay of living, metabolically active cells was performed
by incubating with 0.2 mg/mL MTT in the culture medium for 1.5 h
in an incubator at 37 °C in a 6.5% CO2 atmosphere. After the
incubation period, cells were lysed with an equal volume of 10 mM
HCl and 10% SDS overnight. Values were quantified by absorbance
(test 595 nm, reference 690 nm). Decreased MTT activity denotes
impairment of mitochondrial function and is considered to be an index
of cell damage. Primary neuronal cultures were collected at times
indicated for MTT determination.
AUTHOR INFORMATION
■
Corresponding Author
*(D.H.‑L.) Phone: +30 2310997627. Fax: +30 2310997852.
E‑mail: hadjipav@pharm.auth.gr. (A.A.) Phone: +34
913368400. Fax: +34 91336-9016. E‑mail: alberto.alcazar@
hrc.es. (M.J.O.-G.) Phone: 34-1-3941788. Fax: 34-1-3941779.
E-mail: mjoset@farm.ucm.es. (J.M.‑C.) Phone: +34
915622900. Fax: +34 915644853. E‑mail: iqoc21@iqog.csic.es.
Author Contributions
▽These authors have contributed equally to this work.
ACKNOWLEDGMENTS
■
J. Marco-Contelles thanks MICINN (SAF2006-08764-C02-01
and SAF2009-07271), Comunidad de Madrid (S/SAL-0275-
2006), for support. E. Soriano thanks MICINN for grant
CTQ2009-10478. M. J. Oset-Gasque thanks MICINN
(SAF2009-11219, SAF2010-20337), ISCIII [Retic RENEVAS
(RD06/0026/012)], and UCM-Santander (GR35/10-B) for
financial support. A. Alcaz
III (ISCIII) grants FIS08/0761 and RETICS-RD06/0026/008
from the MICINN and to M. Gomez-Calcerrada for technical
́
ar thanks Institute of Health Carlos
́
́
assistance. M. I. Rodriguez-Franco thanks MICINN (SAF2006-
01249 and SAF2009-13015-C02-01) for support. We also wish
to thank Drs C. Hansch and A. Leo as well as Biobyte Corp.
201 West 4th Street, Suite 204, Claremont, California, 91711,
USA, for free access to the C-QSAR program. J.M.C. thanks
also Prof. Jose
and support. J.M.C. thanks Javier Moron
́
Castillo (USC, Spain) for his continuous interest
for his help in
́
Measurement of LDH Activity. For these assays, cultured
neurons grown in 24-well culture dishes at a density of 0.5 × 106
cells/well were used. LDH activity was measured as the rate of
decrease of the absorbance at 340 nm, resulting from the oxidation of
NADH to NAD+ as described.77,78 Briefly, the culture medium was
collected and the neurons were lysed with 0.1 M Tris-HCl (pH 7.4)
containing 0.1% Triton X-100, cell suspensions were centrifuged at
13000g for 5 min, and LDH activity was measured in both
preparations, culture medium and cell supernatants. Data are given
as percentage of LDH release with respect to the total LDH content
improving the manuscript.
ABBREVIATIONS USED
■
AAPH, 2,2′-azobis(2-amidinopropane) dihydrochloride; BBB,
blood−brain barrier; citicoline, cytidine-5′-diphosphocholine;
LDH, lactic acid dehydrogenase; LOX, lipoxygenase; LP, lipid
peroxydation; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide; NO, nitric oxide; OGD, oxygen−glucose
165
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Journal of Medicinal Chemistry
Article
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deprivation; PBN, phenyl-tert-butylnitrone; PBL, porcine brain
lipid; ROS, reactive oxygen species
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