New hydroxypyridinone iron-chelators as potential anti-neurodegenerative drugs

Abstract

The neuroprotective action of a set of new hydroxypyridinone-based (3,4-HP) compounds (A, B and C), which are iron chelators extra-functionalized with a propargylamino group for potential MAO-B inhibition, was evaluated after cell treatment with MPP+ (an in vivo inducer of parkinsonism) and Abeta(1-40) and/or Abeta(1-42) peptides. Our results show that all these compounds improved cell viability in cells treated with MPP+ and Abeta(1-40) peptide or Abeta(1-42) peptide. In order to evaluate the cellular mechanisms underlying the activity of these compounds, we studied their protective role in caspase activation. All compounds tested were able to prevent MPP+ and Brefeldin A induced caspase-2 activation. They also showed quite effective in the inhibition of caspase-4 and caspase-3 activity, an effector caspase in the apoptotic process. Finally, detection of apoptotic-like cell death after cell exposure to MPP+ was also performed by TUNEL assay. Our results demonstrated that all tested compounds prevented DNA fragmentation by decreasing TUNEL positive cells. A, B and C were more effective than DFP (a 3,4-HP iron-chelating agent in clinical use) in MPP+ induced cell death. Therefore, these results evidenced a neuroprotective and antiapoptotic role for the compounds studied.

Full text

[Frontiers in Bioscience 13, 6763-6774, May 1, 2008]
6763
New hydroxypyridinone iron-chelators as potential anti-neurodegenerative drugs
Daniela Arduino1, Daniel Silva2, Sandra M. Cardoso,1,3 Silvia Chaves2 Catarina R. Oliveira1,4, M. Amelia Santos2
1Centro de Neurociencias e Biologia Celular, Universidade de Coimbra, 3030 Coimbra, Portugal, 2Centro Quimica Estrutural,
Instituto Superior Tecnico-UTL, Av. Rovisco Pais, 1049-001 Lisboa, Portugal, 3Instituto de Biologia, Faculdade de Medicina,
Universidade de Coimbra, 3030 Coimbra, Portugal, 4Instituto de Bioquimica, Faculdade de Medicina, Universidade de
Coimbra, 3030 Coimbra, Portugal
TABLE OF CONTENTS
1. Abstract
2. Introduction
3. Materials and methods
3.1. Materials
3.2. Synthesis of the compounds A-C
3.3. Partition coefficients
3.4. Cell culture and treatments
4. Results
4.1. Design and Chemistry
4.2. Hydroxypyridinone based compounds prevent cell death in PD and AD cellular models
4.2.1. In vitro neuroprotective activity
4.2.2. Hydroxypyridinone based compounds prevent caspase activation
5. Discussion
6. Acknowledgments
7. References
1. ABSTRACT
The neuroprotective action of a set of new
hydroxypyridinone-based (3,4-HP) compounds (A, B and
C), which are iron chelators extra-functionalized with a
propargylamino group for potential MAO-B inhibition, was
evaluated after cell treatment with MPP+ (an in vivo
inducer of parkinsonism) and Abeta1-40 and/or Abeta1-42
peptides. Our results show that all these compounds
improved cell viability in cells treated with MPP+ and
Abeta1-40 peptide or Abeta1-42 peptide. In order to evaluate
the cellular mechanisms underlying the activity of these
compounds, we studied their protective role in caspase
activation All compounds tested were able to prevent MPP+
and Brefeldin A induced caspase-2 activation. They also
sowed quite effective in the inhibition of caspase-4 and
caspase-3 activity, an effector caspase in the apoptotic
process. Finally, detection of apoptotic-like cell death after
cell exposure to MPP+ was also performed by TUNEL
assay. Our results demonstrated that all tested compounds
prevented DNA fragmentation by decreasing TUNEL
positive cells. A, B and C were more effective than DFP (a
3,4-HP iron-chelating agent in clinical use) in MPP+
induced cell death. Therefore, these results evidenced a
neuroprotective and antiapoptotic role for the compounds
studied.
2. INTRODUCTION
One third of the diseases in Europe correspond to
brain disorders and most of them are of neurodegenerative
nature, like Alzheimer`s disease (AD) and Parkinson`s
disease (PD). AD is the leading cause of dementia in
western countries and the most prevalent neurodegenerative
disease. The clinical hallmarks of AD are progressive
impairment in memory, cognition, orientation to physical
surroundings and language. AD is a progressive
neurological disease that results in irreversible loss of
neurons, particularly in the hippocampus and cortex. Other
pathological hallmarks are extracellular senile plaques
containing the beta-amyloid peptide (Abeta), and
intracellular neurofibrillary tangles (NFT) (1).
Parkinson’s disease is the most common
neurodegenerative movement disorder affecting more than
1% of the population above 60 years of age and for which
there is no effective treatment (2). Clinically, PD is
characterized by rigidity, tremor, slowness and balance
problems. PD pathology is characterized by selective
neuronal loss of dopaminergic neurons in the substantia
nigra pars compacta, and formation of Lewy bodies (LB),
which are intracytoplasmic inclusions, mainly composed of
α-synuclein and other cytoskeletal proteins (3).

New hydroxypyridinone iron-chelators as potential anti-neurodegenerative drugs
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The fact that age is a key risk factor in AD and
PD sporadic forms provides considerable support for the
free radical hypothesis, because effects of free radicals can
accumulate over the years (4, 5). Furthermore, a
considerable number of oxidative stress markers are found
in the brain of AD patients associated with senile plaques
and NFT (6, 7). Increased concentrations of copper, iron
and zinc are detected in affected brain regions within
amyloid plaques (8). Metal ions were shown are able to
bind to Abeta and to accelerate its aggregation and enhance
metal-catalysed oxidative stress associated with amyloid
plaque formation (9). The accumulation of the redox-active
metals, iron and copper, may be a major source of ROS,
which are in turn responsible for oxidative stress observed
in AD.
Moreover, there are several potential sources of
oxidative damage in PD and a number of substantial data
from post-mortem studies support an increased oxidative
damage in this disease. A consistent increase in lipid
peroxidation and an increase in protein carbonyl groups
have been reported in PD substantia nigra (10, 11). In
addition, a decrease in catalase and glutathione peroxidase
activities was reported, although superoxide dismutase
activity in substantia nigra of PD patients was shown to be
increased (12, 13) Also this is relevant to the fact that iron
levels, which in a non-pathological situation are
significantly higher in substantia nigra than in the other
brain regions, further increase in substantia nigra of PD
patients (13). Nigral dopaminergic neurons are particularly
exposed to oxidative stress, since dopamine can easily
auto-oxidize into toxic dopamine-quinone species,
superoxide radicals and hydrogen peroxide (14). Moreover,
dopamine can be metabolized via enzymatic deamination
by monoamine oxidase (MAO), with the production of
hydrogen peroxide (15), leading to oxidative damage and
subsequently to neuronal cell death.
Increasing evidences propose that apoptosis may
be one of the mechanisms leading to neuronal death in AD
and PD disorders. For instance, lymphocytes bearing
genetic or sporadic risk factors of AD show an increased
susceptibility to apoptotic cell death, suggesting that
apoptosis is a common feature shared by both sporadic and
familiar forms of AD (16). Apoptotic features have been
also shown in AD and PD patients by the demonstration of
DNA fragmentation and caspase activation (16-19).
Moreover, it has been described that pro-apoptotic genes
are up-regulated in substantia nigra of PD patients, and five
fold higher caspase-3 positive neurons were found in
patients compared to controls (18). These evidences,
together with other findings from different groups suggest
that, in pathological states of the brain associated with
induction of apoptosis, endoplasmic reticulum (ER)
dysfunction may be in an upstream process (5). A growing
number of reports recognize ER as key player in the
sensing and execution of apoptotic signals in familiar and
sporadic forms of AD and PD (20-23).
Thus, neuroprotective strategies employing
antioxidant, iron-chelating or free radical-scavenging
abilities is one approach to protect neurons and to restrict
the progression of these disorders. In fact, iron chelators,
antioxidants or monoamine oxidase B (MAO-B) inhibitors
have been shown to possess neuroprotective activity.
MAO-B inhibitors are amongst the drug candidates which
are claimed to have protective effects on both vascular and
neuronal tissues. There is, thus, an imperative need to
search for neuroprotective compounds with novel
mechanisms to assess their potential benefit in such
disorders.
In the present study, a number of novel
bitargeting ligands, based on 3-hydroxy-4-pyridinones (3,4-
HP), were developed and investigated in their protective
action, as potential anti-neurodegenerative drugs. In
particular, three hydroxypyridinone agents A, B and C,
which are iron chelators similar to deferriprone (DFP), a
chelator clinically used for iron removal form iron-overload
patients (24, 25), have been bifunctionalized with an
propargylamine group to account for their potential role on
the MAO-B inhibition (26, 27). After preparation and
characterization of the lipophilic character, we investigated
the neuroprotective efficacy of these novel compounds in
MPP+ (an in vivo inducer of parkinsonism) and Abeta1-40
and/or Abeta1-42 induced cell death.
3. MATERIALS AND METHODS
3.1. Materials
Optimum medium and fetal calf serum were
purchased from Gibco BRL, Life Technologies (Scotland,
UK). 1-methyl-4-phenylpyridinium (MPP+), dantrolene,
Brefeldin A, 3- (4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) and the substrate for
caspase-2 (N-acetyl-Val-Asp-Val-Ala-Asp-p-nitroanilide
(Ac-VDVAD-pNA)) were obtained from Sigma (St. Louis,
MO, USA). The synthetic Abeta1-40 and Abeta1-42 peptides
were from Bachem (Bubendorf, Switzerland). The
colorimetric substrate for caspase-3 (N-acetyl-Asp-Glu-
Val-Asp-p-nitroanilide (Ac-DEVD-pNA)) was from
Calbiochem, Merck KGaA (Darmstadt, Germany) whereas
the caspase-4 substrate (N-acetyl -Leu-Glu-Val-Asp-p-
nitroanilide (Ac-LEVD-pNA)) was obtained from MBL
International Corporation (Woburn, MA, USA). In situ cell
death detection kit was purchased from Roche Diagnostics
GmbH (Penzberg, Germany). Bio - Rad protein dye assay
was from Bio-Rad (Hercules, CA, USA). All other bench
reagents were of the highest grade of purity and were
commercially available.
3.2. Synthesis of the compounds A-C
Analytical grade reagents were purchased from
Aldrich, Sigma and Fluka and they were used as supplied.
Solvents were dried according to standard methods (28).
The chemical reactions were monitored by TLC using
alumina plates coated with silica gel 60 F254 (Merck).
Column flash chromatography separations were performed
on silica gel Merck 230-400 mesh ASTM. Melting points
were measured with a Leica Galen III hot stage apparatus
and are uncorrected. The 1H NMR spectra were recorded
on a Varian Unity 300 spectrometer at 25 oC. Chemical
shifts are reported in ppm (
δ
) with tetramethylsilane (TMS)
as internal reference, in organic solvents and sodium 3-

New hydroxypyridinone iron-chelators as potential anti-neurodegenerative drugs
6765
(trimethylsilyl)- (2,2,3,3-d4)-propionate (DSS) in D2O
solutions. The following abbreviations are used: s = singlet;
d = duplet; t = triplet; q = quintuplet; m = multiplet; br =
broad. Mass spectra (FAB) were performed in a VG TRIO-
2000 GC/MS instrument. The High Resolution Mass
Spectra (HRMS) were obtained with a High Resolution
Fourier transform ion cyclotron resonance (FTICR)
instrument, Finnigan FT/MS 2001-DT, equipped with a
3.0-T superconducting magnet, by electron impact (EI),
typically with 15-eV electron beam energies, 5-micro
emission currents and 150°C sample temperatures.
Elemental analyses were performed on a Fisons EA1108
CHNF/O instrument.
1- (3´- (di (prop-2-ynyl)amino)propyl)-3-
hydroxy-2-methyl.pyridin-4 (1H)-one (A). To a solution of
1- (3´-aminopropyl)-3-hydroxy-2-methyl-pyridin-4 (1H)-
one (29) (1.50 g; 8.23 mmol) in anhydrous DMF (30 mL),
potassium carbonate (2.28 g; 16.5 mmol) was added and
the mixture was stirred for ca. 15 min. A solution of
propargyl bromide (0.71 mL; 8.23 mmol) in anhydrous
DMF (15 mL) was added dropwise to the reaction mixture
which was under nitrogen atmosphere and at 0ºC. The
reaction mixture was stirred for 24 h. After the end of the
reaction the solvent was evaporated to dryness and worked
up by adding water (70 mL) which was extracted with ethyl
acetate (8 × 30 mL). The organic layer was dried with
anhydrous sodium sulphate. The solvent was evaporated to
dryness and 1- (3- (di (prop-2-ynyl)amino)propyl)-3-
hydroxy-2-methylpyridin-4 (1H)-one was recrystallized
from ethanol/acetonitrile affording a dark yellow solid
(0.75 g) with 35% yield. M.p.: 138-140ºC. 1H-NMR
(CD3OD; δ/ppm): 7.65 (1 H, d, J = 7.5 Hz, 6-H
pyridinone), 6.39 (1 H, d, J = 6.6 Hz, 5-H pyridinone), 4.12
(2 H, t, J = 7.5 Hz, CH2 Pi), 3.45 (4 H, s, CH2-C≡C), 2.59
(2 H, t, J = 7.5 Hz, CH2NH2), 2.45 (3 H, s, CH3), 1.93 (2 H,
t, J = 7.2 Hz, CH2CH2CH2). 13C-NMR (CD3OD; δ/ppm):
170.6 (C=O), 139.1 (C-6), 132.9 (C-3), 112.6 (C-5), 112.0
(C-2), 79.2 (CH2C≡CH), 74.8 (CH2C≡CH), 52.8 (CH2N-
CH2C≡CH), 48.1 (N-CH2CH2CH2), 42.8 (N-CH2C≡CH),
28.8 (CH2CH2CH2), 11.9 (CH3 Pi). FAB-MS (m/z): 297
(M+K) +
, 259 (M+1)+, 219 (M- CH2C≡CH)+. Elemental
analysis: calculated for C15H18N2O2: C, 69.74; H, 7.02; N,
10.84; found: C, 69.76; H, 7.30; N, 11.01%.
3-Benzyloxy-2-methyl-1- (2- (piperazin-1’-
yl)ethyl)pyridin-4-one. To a solution of 1- (2-
aminoethyl)piperazine (3.83 mL; 29 mmol) in water (40
mL) at pH 13 was added 3-benzyloxy-2-methylpiran-4-one
(29) (6,26 g; 29 mmol) dissolved in methanol (40 mL). The
mixture was refluxed overnight. The reaction mixture was
concentrated to a ¼ of initial volume, the pH adjusted to 4
and the aqueous solution was washed with diethyl ether
(3×70 mL). The aqueous layer was basified to pH 8 and
extracted with dichloromethane (3×70 mL). The organic
layer was dried with anhydrous sodium sulphate. The
solvent was evaporated to dryness and the pure compound
(2.45 g; 7.5 mmol) was obtained (26% yield). 1H-NMR
(D2O; δ/ppm): 7.71 (1 H, d, J = 7.2 Hz, 6-H Pi), 6.56 (1 H,
d, J = 7.2 Hz, 5-H Pi), 5.06 (2 H, s, CH2Ph), 4.09 (2 H, t, J
= 6.9 Hz, CH2NH2), 3.21 (4 H, t, J =5.1 Hz, 3,4-H
piperazine), 2.6 (6 H, m, 2,6-H piperazine + CH2-N
piperazine), 2.05 (3 H, s, CH3). FAB-MS (m/z): 328
(M+1)+.
3-Hydroxy-2-methyl-1- (2- (piperazin-1-
yl)ethyl)pyridin-4-one. 3-benzyloxy-2-methyl-1- (2’-
(piperazin-1’-yl)ethyl)-pyridin-4-one (2.0 g; 5.53 mmol)
and Pd/C 10% (150 mg) were dissolved in methanol and
the solution was stirred at a pressure of 2 bar for 4 h at
room temperature. The inorganic material was filtered
off, the solvent evaporated to dryness and the remaining
solid was recrystallized from ethanol, affording a yellow
solid (1.83 g; 4.35 mmol) with a yield of 79%. 1H-NMR
(D2O; δ/ppm): 8.04 (1 H, d, J = 7.2 Hz, 6-H Pi), 7.09 (1
H, d, J = 7.2 Hz, 5-H Pi), 4.52 (2 H, t, J = 6.9 Hz, N-
CH2), 3.34 (4 H, t, J =5.1 Hz, 3,5-H piperazine), 3.08 (2
H, t, J = 6.9 Hz, CH2-N piperazine), 2.99 (4 H, t, J = 5.1
Hz, 2,6-H piperazine), 2.60 (3 H, s, CH3). FAB-MS
(m/z): 238 (M+1)+.
3-Hydroxy-2-methyl-1- (2- (4- (prop-2-
inyl)piperazin-1-yl)ethyl)pyridin-4-one (B). To a solution
of 3-hydroxy-2-methyl-1- (2- (piperazin-1-yl)ethyl)pyridin-
4-one (0,79 g; 3,33 mmol) in anhydrous DMF (40 mL) was
added potassium carbonate (0.92 g; 6.67 mmol) and the
mixture was stirred for ca. 10 min. A solution of propargyl
bromide (0.29 mL; 3.33 mmol) in anhydrous DMF (5 mL)
was added dropwise to the reaction mixture which was
under nitrogen atmosphere at 0ºC. The reaction mixture
was stirred for 24 h. After the end of the reaction the
solvent was evaporated to dryness and worked up by
adding water (50 mL) and extracting with ethyl acetate (4 ×
30 mL). The organic layer was dried with anhydrous
sodium sulphate. The solvent was evaporated to dryness
and the pure compound was recrystallized from ethanol,
affording a light beige solid (0.24 g) with 26% yield. M.p.:
213-215ºC. 1H-NMR (D2O; δ/ppm): 7.68 (1 H, d, J = 7.2
Hz, 6-H pyridinone), 6.52 (1 H, d, J = 7.2 Hz, 5-H
pyridinone), 4.24 (2 H, t, J = 7.8 Hz, N-CH2), 3.29 (2 H, s,
CH2-C≡C), 2.78 (2 H, t, J = 7.5 Hz, CH2-N piperazine
ring), 2.68 (8 H, m, H piperazine ring), 2.45 (3 H, s, CH3).
FAB-MS (m/z): 314 (M+K)+, 276 (M+H)+. HRMS m/z
calculated for C15H21N3O2: 275.163377; found:
275.163328.
3-Hydroxy-2-methyl-1- (3-
(methylamino)propyl)pyridin-4-one. To a solution of 1- (3-
aminopropyl)-3-hydroxy-2-methylpiridin-4-one (2.0 g;
11.0 mmol) in anhydrous ethanol (50 cm3) was added
paraformaldehyde (1.65 g; 5.5 mmol) and sodium
cyanoborohydride (0.83 g; 13.2 mmol). The mixture was
refluxed overnight. After the end of reaction the solvent
was evaporated to dryness. The residue was redissolved
in anhydrous methanol and the inorganic material was
filtered off. The solvent was evaporated to dryness and
the residue was recrystallized from ethanol/acetonitrile
affording a light yellow solid (1.05 g; 5.35 mmol) with
49% yield. M.p. 197-200ºC. 1
H-NMR (D2O; δ/ppm):
8.07 (1 H, d, J = 6.6 Hz, 6-H Pi), 7.07 (1 H, d, J = 6.6
Hz, 5-H Pi), 4.42 (2 H, t, J = 6.6 Hz, CH2N), 3.31 (2 H,
t, J = 6.6 Hz, CH2NH2), 2.91 (3 H, s, NHCH3), 2.57 (3
H, s, CH3), 2.30 (2 H, t, CH2CH2CH2). FAB-MS (m/z):
197 (M+1)+.

New hydroxypyridinone iron-chelators as potential anti-neurodegenerative drugs
6766
3-Hydroxy-2-methyl-1- (3-methyl-3- (prop-2’-
inylamino))pyridin-4-one (C). To a solution of 3-hydroxy-
2-methyl-1- (3´- (methylamino)propyl)piridin-4-one (1.0 g;
5.10 mmol) in anhydrous DMF (50 mL) was added
potassium carbonate (1.41 g; 10.2 mmol) and the mixture
was stirred for ca. 10 min. A solution of propargyl bromide
(0.66 mL; 6.12 mmol) in anhydrous acetonitrile (20 mL)
was added dropwise to the reaction mixture which was
under nitrogen atmosphere and at 0ºC. The reaction mixture
was stirred for 80 h. After the end of the reaction the
solvent was evaporated to dryness and redissolved in
anhydrous methanol to remove any inorganic material by
precipitation. The solvent was evaporated to dryness and
the compound was recrystallized from methanol/ethyl
acetate, affording a highly hygroscopic yellow solid (0.57
g) with 48% yield. 1H-NMR (D2O; δ/ppm): 8.18 (1 H, d, J
= 7.2 Hz, 6-H pyridinone), 7.09 (1 H, d, J = 7.2 Hz, 5-H
pyridinone), 4.42 (2 H, t, J = 7.8 Hz, CH2N pyridinone),
3.67 (2 H, t, J = 6.9 Hz, CH2N), 4.21 (2 H, s, CH2-C≡C),
3.60 (3 H, s, CH2NHCH3), 3.20 (3 H, s, N-CH3), 2.60 (3 H,
s, CH3), 2.41 (2 H, t, J = 7.5 Hz, CH2CH2CH2). 13C-NMR
(CD3OD; /ppm): 153.0 (C-6), 145.0 (C-3), 144.7 (C-5),
115.2 (C-2), 84.3 (CH2C≡CH), 79.8 (CH2C≡CH), 56.7
(CH2N-CH2C≡CH), 54.9 (N-CH2CH2CH2), 52.3 (N-
CH2C≡CH), 25.8 (CH2CH2CH2), 15.3 (CH3 Pi). FAB-MS
(m/z): 235 (M+1)+. HRMS m/z calculated for C13H18N2O2:
234.1368279; found: 234.136796.
3.3. Partition coefficients
The octanol-water partition coefficients (log
P) were calculated at 25ºC, based on the
concentration ratio of each compound between 1-
octanol and a buffered aqueous phase (Tris, pH =
7.4). The two phases were previously pre-saturated
with respect to each other. The “shakeflask” method
was used, as previously described (29, 30), and the
species concentrations were evaluated by UV-Vis
spectrophotometry, using the hydroxypyridinone
benzenoid bands at ca 280-290 nm.
3.4. Cell culture and treatments
Human teratocarcinoma NT2 cells were
purchased from Stratagene (La Jolla, CA, USA) and were
cultured as described previously (31). Briefly, the cells
were grown in Optimen medium with 10% heat inactivated
fetal calf serum, containing 50 U/mL penicillin, and 50
µg/mL streptomycin, under a humidified atmosphere of
95% air- 5% CO2 at 37ºC. Cells were plated at 0.1 × 106
/mL for cell viability assays, 0.5 × 106 /mL for
measurement of caspase activity and 0.75 × 106 /mL for in
situ cell death assay. Medium was changed after 24 h and
immediately before treatments. MPP+ was prepared as 10
mM stocks (in DMSO) immediately before use and added
to the medium to 1 mM final concentration. Abeta1-40 (5
µM) and Abeta1-42 (1 µM) peptides were added from a
231.5 µM and 221.5 µM stock PBS solutions, respectively.
Previously, the peptides were dissolved in sterile distilled
water at a concentration of 6 mg/mL and diluted to 1
mg/mL (231.5 M or 221.5 µM, respectively) with PBS and
then incubated for 5-7 days to induce fibril formation.
Brefeldin A (2 µM) was added from 5 mM stock ethanol
solution.
The tested compounds (A, B, C, DFP) were
dissolved in DMSO at a concentration of 2 mM and
aliquots were stored at -20ºC. These compounds were
added to the medium to 5 µM final concentration. In
parallel experiments and before the addition of stress
inducers, cells were also preincubated for 1 h with
dantrolene (10 µM), an inhibitor of ryanodine receptors of
ER, which was used as a control for neuroprotective action.
For combinatorial treatments, both simultaneous and
sequential treatment approaches were tested and the
selected compounds concentrations shown to exert the
maximal protective action and had no toxic effects to cells.
The final concentration of ethanol/DMSO in culture media
did not exceed 0.05% (v/v) and no alterations on cells were
observed. For all conditions tested, control experiments
were performed in which the compounds tested or the
stress agents was not added.
Cell reduction ability as a surrogate of cell
viability was measured by using a quantitative colorimetric
assay with MTT according to the method of Mosmann
(1983) (32). In brief, cells were incubated with MTT
solution (in sodium medium) for 3 h at 37ºC. The medium
was then discarded, the stained cells were dissolved with
isopropanol/HCl, and thereafter the absorbance at 570 nm
was measured using a Spectramax Plus 384
spectrophotometer (Molecular Devices). MTT reduction
ability was expressed as a percentage of the control value
obtained for untreated cells.
Caspase activation assays were performed by
using the method described by Cregan et al. (1999) (33),
with slight modifications. After described treatments, cells
were washed once in PBS and harvested on ice with a lysis
buffer containing 25 mM HEPES, 1 mM EDTA, 1 mM
EGTA, 2 mM MgCl2, and protease inhibitors (0.1 M
PMSF, 2 mM DTT, and a 1:1000 dilution of a protease
inhibitor cocktail). The cellular suspension was
frozen/defrosted three times on liquid nitrogen and
sonicated twice for 30 sec. Then, the lysate was centrifuged
for 10 min at 20,200 × g and at 4ºC. The resulting
supernatant was collected and assayed for protein
concentration using Bio-Rad protein dye assay reagent. To
evaluate caspase activation, cell extracts containing 50 µg
or 100 µg of protein were incubated at 37ºC for 2 h in 25
mM HEPES, pH 7.5 containing 0.1% (w/v) CHAPS, 10%
(w/v) sucrose, 2 mM DTT with 100 M Ac-VDVAD-
pNA, 100 µM Ac-DEVD-pNA and 50 µM Ac-LEVD-
pNA, colorimetric substrates for caspase-2, 3 and 4,
respectively. Substrate cleavage was detected at 405 nm
using a Spectramax Plus 384 spectrophotometer (Molecular
Devices) and the results were expressed as the increase
above control value obtained for untreated cells.
Detection of apoptotic cell death after cell
exposure to MPP+ was performed by using an in situ cell
death detection kit, based on labelling of DNA strand
breaks (TUNEL technology), according to the procedure
provided by the manufacturer. Briefly, cells in coverslips
were fixed in 4% paraformaldehyde in PBS, pH 7.4, for 1
h. Then, cells were washed twice with PBS and immersed
in a permeabilisation solution (0.1% Triton X-100 in 0.1%

New hydroxypyridinone iron-chelators as potential anti-neurodegenerative drugs
6767
sodium citrate) for 2 min on ice. After washing twice with
PBS, cells were incubated with the nucleotide mixture and
terminal deoxynucleotidyl-transferase in 50 µL of
equilibration buffer, in a humidified atmosphere at 37°C for
1 h in the dark, to allow that the end labelling reactions
occur. The reaction was stopped by washing three times in
PBS. Ultimately, the cells were treated with
DakoCytomation Fluorescent mounting solution on a
microscope slide and analyzed in an Axiovert 200
fluorescence microscope (Zeiss, Germany). The
photographs were taken at 40× magnification.
All data were expressed as mean ±S.E.M. of at
least three independent experiments performed in
duplicates. Statistical analyses were performed using one-
way ANOVA followed by Bonferroni Multiple-
Comparisons Procedure as post -hoc test. A P - value< 0.05
was considered statistically significant.
4. RESULTS
4.1 Design and Chemistry
The option for the hydroxypyridinone-based
derivatives (3,4-HPs) in this study was based on the fact
that, besides their recognized excellent iron-chelating
properties, other biologically relevant properties can be
combined by the easy introduction of a wide variety of
functional groups, according to the desired roles of the final
molecular entity. 3,4-HPs are heterocyclic O,O-chelators
with high affinity for iron (compare to deferriprone, which
is clinically used as iron scavenger (25)); they are very
stable, non-toxic and bio-available at any physiological pH
conditions; they are easily made and ring-substituted to
tune a diversity of physico-chemical or biological
properties, including lipo-hydrophilicity and permeability
to the BBB and the interaction with specific relevant bio-
sites or proteins (34, 35); they can form neutral metal-
complexes which can easily efflux from brain (36).
This set of new compounds includes a free 3-
hydroxy-4-pyridinone group (3,4-HP) to chelate the iron
(III) and at least one propargylamine group to interact and
inhibit the MAO-B activity. These two pharmacophore
units are separated by spacers of slightly different size and
type to account for some differentiation in the lipophilicity
and membrane crossing abilities and also for interaction
with biological targets. The piperazine segment may also
be expected to have some interaction with Cu (II), which is
involved in the binding of amyloid plaque precursor
proteins. An additional benefit of application of these
compounds is that they are cheap and easily made. The
synthetic route for the preparation of the new A-C
compounds involves always a previous preparation of the
corresponding 3-hydroxy-2-methyl-4-pyridinone backbone,
possessing an alkylamine spacer as the ring N-substituent.
This terminal amine group can be either primary or
secondary amine group, and it works as the actual point of
attachment between the propargylic and the 3,4-HP units.
The preparation of the amino derivatives of 3,4-HP follows
standard strategies previously described (28, 34). That
coupling reaction involves a nucleophilic substitution of
propargyl bromide, under anhydrous DMF conditions and
in presence of base (potassium carbonate). Noteworthy to
mention that for this last reaction, the phenolic group of
hydroxyyridinone cannot be protected with benzyl group,
because its final deprotection via hydrogenolysis would
lead back to the disconnection of the propargyl group. In
case of compound B, the O-benzylation was used to
simplify the working up of the products, but the O-
deprotection was required before the amine propargylation.
The results of the lipophilicity studies (log P,
octanol/water) evidenced that A and C are more lipophilic
than DFP, a benefit for their bioavailability and BBB
requirements as potential drugs, although the lipophilicity
charater of B and DFP are quite similar. Therefore, while in
A and C the hydrophilic character of the neutral amine
group is compensated by the lipophilic N-substituent alkyl
groups, in case of B one the amine groups of the piperazine
unit may be protonated at neutral pH (26-a), thus
contributing to some extra-hydrophilic character. Although
no solution studies were performed to assess iron-chelating
efficacy of this set compounds, it is anticipated that it
should be close to the reported for DFP and other
bifunctional hydroxypyridonate derivatives (37), because
the spacer group is long enough to diminish the effect of
different substituent. In case of compound B, the amine
groups of the piperazine moiety (boat conformation) have
some affinity for Cu (II) and so, besides its high affinity for
Fe (III) due to the hydroxypyridinone moiety, some extra-
interaction with soft double-charged metal ions can also
occur.
4.2. Hydroxypyridinone based compounds prevent cell
death in PD and AD cellular models
4.2.1. In vitro neuroprotective activity
To evaluate the potential therapeutic action of
hydroxypyridinone- based compounds, NT2 cells were
treated with MPP+ and Abeta1-40 or Abeta1-42 peptides,
which constitute good ex vivo cellular models for studying
neurodegeneration associated to PD and AD disorders.
Figure 1 shows a significant decrease in cell viability after
a 24 h treatment with 1 mM MPP+ and 1 µM Abeta1-42
peptides. Hydroxypyridinone- based compounds by itself
did not affect cell viability at the same end point (data not
shown). Furthermore, all these compounds (A, B, C and
DFP) improved cell viability in cells treated either with
MPP+, Abeta1-40 and Abeta1-42 peptides.
4.2.2. Hydroxypyridinone based compounds prevent
caspase activation
Apoptosis is mediated by a family of cysteinyl-
containing, aspartate - specific proteases (caspases) that,
in turn, activated the caspase cascade resulting in cell
death (38). Different caspases mediate cell death in
response to different apoptotic stimuli. Caspase-2 plays
an important function in initiation of ER stress apoptosis
and nuclear stress signalling. Moreover, the role of this
caspase in neuronal cells has been of particular interest
because of its essential role in neuronal death elicited by
growth factor deprivation and amyloid-beta toxicity
(39). Recent studies show that caspase-4 in humans and
caspase-12 in mouse mediate ER stress induced
apoptosis (40, 41).

New hydroxypyridinone iron-chelators as potential anti-neurodegenerative drugs
6768
Figure 1. Effect of A, B, C and DFP on MPP+ and Abeta1-40 or Abeta1-42 peptides toxicity in NT2 cells. NT2 cells were treated
with MPP+ (1 mM) and Abeta1-40 (5 µM) or Abeta1-42 (1 µM) peptides, for 24h, in the absence or the presence of either 5 µM of
compounds A, B, C or DFP. Evaluation of cell viability was performed by using MTT reduction test. Results are expressed as the
percentage of NT2 untreated cells, with the mean ± S.E.M. derived from 6 different experiments. *p < 0.05, significantly
different when compared with NT2 untreated cells; §p < 0,05; §§p < 0,01; §§§p < 0,001, significantly different when compared
with Abeta1-42 treated cells; #p < 0,05; ##p < 0,01, significantly different when compared with MPP+
treated cells.
We observed that an increase in VDVAD (N-
acetyl-Val-Asp-Val-Ala-Asp-p-nitroanilide) cleavage
(caspase-2 activation) occurred after cells treatment with
MPP+ or Brefeldin A (a classical ER stress agent, acting by
ER - Golgi protein trafficking inhibition) for 24 h.
Nevertheless, it was also evident that the co-treatment with
either of these hydroxypyridinone compounds restrained
this increase. So, it means that all these compounds are able
to protect cells against MPP+ and Brefeldin A induced
caspase-2 activation. Similar results were observed
relatively to caspase-4 activation (Figure 2B). Cell
exposure to MPP+, but not to Abeta1-42 peptide, for 24 h,
resulted in a significant increase in LEVD cleavage, which
was remarkably reduced in combinatorial treatments with
A or DFP, most effective compounds in prevention of
caspase-4 activation. In previous work from our laboratory,
it was demonstrated that MPP+ and Abeta1-40 induced
mitochondrial dependent apoptosis via mitochondrial
cytochrome c release and caspase activation in these
cellular models (unpublished work and 42). Additionally,
since caspase-3 is an effector caspase in the apoptotic
process, and to better characterize the neuroprotection
mechanisms mediated by these compounds, we analysed
the protective effect of these compounds against ER and
mitochondria stressors (Brefeldin A, and MPP+,
respectively) on caspase-3 activation. As shown in Figure
2C, when cells were co-treated with A and C compounds,
the caspase-3 activation was decreased.
Since caspases were shown to be activated, we
intended to further analyse DNA fragmentation in cells
upon MPP+ exposure and to evaluate the effectiveness of
these compounds on rescue cells from MPP+ mediated cell
death. After 24 h exposure to MPP+, apoptotic cells were
observed both by phase contrast and fluorescence
microscopy (Figure 3). All tested compounds prevented
DNA fragmentation by decreasing TUNEL positive cells,
A, B and C being the most successful compounds in
prevention of MPP+ induced cell death.
5. DISCUSSION
The major mechanisms underlying the
neurodegenerative processes in Parkinson’s and
Alzheimer’s diseases are not yet known. Nonetheless,
many biochemical evidences provided support to a cascade
of events, including oxidative stress, iron deposition at the
site of neuronal lesion, inflammatory processes,
excitotoxicity, and apoptosis. Taking into account that
these events involve multifactorial conditions, the
development of drugs that could modulate multiple targets
simultaneously with eventual synergistic side effects seems
the most wised strategy for improving drug efficacy. In
fact, the design of compounds as dual target drugs has been
object of recent research but mostly based on the task of
extra-functionalization of high cost mono-target drugs
(43,44). Recently, 8-hydroxyoxyquinoline-bearing
derivatives have also been developed to combine the iron-
chelating and MAO-B inhibitory properties (45).
In the present study a set of bitargeting ligands,
based on 3-hydroxy-4-pyridinones were designed and their
protective action, as potential anti-neurodegenerative drugs,
was investigated. Firstly, our results demonstrated that all
the selected compounds provided substantial protection
from cell death induced by MPP+, Abeta1-40 and Abeta1-42
treatment of cultured human teratocarcinoma NT2 cells, a
cell line that has been shown to exhibit a neuronal
phenotype and has several neurochemical markers (46).
MPP+, Abeta1-40 and Abeta1-42 treatments in NT2 cells
constitute a reliable model for screening potential
neuroprotective compounds because they mimic some

New hydroxypyridinone iron-chelators as potential anti-neurodegenerative drugs
6769
Figure 2. Caspase activation in NT2 cells induced by MPP+ and Abeta1-42 or Brefeldin A (BFA). (A) Effect of compounds A, B,
C, DFP and dantrolene (DNT) on caspase 2-like activity (Ac-VDVAD-pNA cleavage) increase; (B) Effect of compounds A, B, C
and DFP on caspase 4-like activity (Ac-LEVD-pNA cleavage) increase; (C) Effect of compounds A, B, C, DFP and dantrolene
on caspase 3-like activity (Ac-DEVD-pNA cleavage) increase. Caspase activation was evaluated spectrophotometrically at 405
nm as described in Materials and methods. Data are expressed relative to the basal activity observed in the untreated NT2 cells,
with the mean ± S.E.M. derived from 4 different experiments. . *p < 0,05, significantly different when compared with NT2
untreated cells; #p < 0,05; ###p < 0,001, significantly different when compared with MPP+
treated cells; +++p < 0,001,
significantly different when compared with BFA treated cells.

New hydroxypyridinone iron-chelators as potential anti-neurodegenerative drugs
6770
Figure 3. Effect of A, B, C and DFP on MPP+ induced apoptotic cell death in NT2 cells. NT2 cells were treated with MPP+ (1
mM) alone or in the presence of compound A, B, C or DFP (5 µM), for 24 h. Apoptotic cell death was determined by TUNEL
assay and was analysed by phase contrast and fluorescence microscopy. Merged images illustrate that all tested compounds
prevented DNA fragmentation by decreasing TUNEL positive cells, being A, B and C most effective compounds in prevention of
MPP+ induced cell death.

[Frontiers in Bioscience 13, 6763-6774, May 1, 2008]
6771
Table 1. Structural formula and log P of the compounds used in study
Compound name Molecular Structure Log P
A
N
ON
HO
-0.13
B NO
HO
NN
-0.94
C
N
ON
HO
-0.32
DFP
NO
HO
-1.03
aspects of neurodegeneration underlying the Parkinson’s
and Alzheimer’s diseases physiopathology, respectively. In
addition, it was also evident that, in both cellular models,
A, B and C compounds were more effective than DFP, a
chelator clinically used for iron removal (Figure 1).
This finding can be explained regarding the
hypothesis that neuroprotection in neurodegenerative
diseases requires a permeable drug combining antioxidant
and iron chelating properties. Several lines of evidence
propose that metal ion homeostasis is severely deregulated
and may be related to oxidative damage in AD brain (47).
Increased concentrations of copper, iron and zinc are
detected in affected brain regions within amyloid plaques
(48).These metals bind to Abeta and were shown to
accelerate its aggregation and enhance metal-catalysed
oxidative stress, associated with amyloid plaque formation
(9). In addition, copper, iron and zinc have been reported to
increase Abeta toxicity (8,49).
Thus, our findings demonstrating that compound A
markedly decreased Abeta toxicity (Figure 1) and suppressed
caspase-4 activation (Figure 2B) can be explained taking into
account its antioxidant ability and the presence of a piperazine
moiety which exhibits some affinity for Cu (II), preventing
further Abeta aggregation. Therefore, the probable copper
chelating action could prevent Abeta induced toxicity, by
reducing the possibility of generation of toxic-amyloid
peptides, which can disrupt intraneuronal Ca2+ levels, leading
to ER perturbated Ca2+ homeostasis and, subsequently, to ER
associated caspases activation.
In addition, accumulating evidences have shown
that iron-dependent oxidative stress, increased levels of
iron, increased MAO-B activity, and depletion of
antioxidant enzymes in the brain may be the main
pathogenic factors in PD (4). Besides, it is plausible that
stress responses from ER involves transcriptional
signalling, intracellular Ca2+ mobilization, activation of
apical caspases like, caspases-4 or -2, that may proceed
with or without the contribution of mitochondrial-
dependent apoptotic pathway (50). In this case, the results
of the present study show that compounds A and C
significantly reduced MPP+-induced caspases-2, -4 and -3
activation (Figure 2 A, B and C) in a similar way than
dantrolene, an inhibitor of ryanodine receptors of ER.
Dantrolene prevents Ca2+ release from ER lumen to cytosol,
and was used as a control for neuroprotection activity
(Figure 2 A). In addition, caspases-mediated apoptotic cell
death inhibition was corroborated by the efficiency of these
compounds on rescue cells from MPP+ mediated cell death
(Figure 3). Herein, modified compounds A, B and C were
more successful than DFP in preventing MPP+ induced cell
death. Thus, we suggest that compounds A and C could act
at the ER stress level that may proceed with mitochondrial-
dependent caspases activation, by preventing the successive
caspase-3 activation and, subsequently, DNA
fragmentation.

New hydroxypyridinone iron-chelators as potential anti-neurodegenerative drugs
6772
Overall, we were able to prove that compound A
was the most efficient product acting on several cellular
targets. The high neuroprotective effect of this compound,
in these cellular models, may be due to the combined
potential actions of iron chelation, antioxidant, MAO B
inhibition and good permeability. Among these compounds
only DMHP was assessed for the iron (III) chelating and
other anti-oxidant properties, although all of them are
expected to present similar behaviours. In fact, the affinity
of the 3-hydroxy-4-pyridinones for iron (III) showed to be
quite high (pFe = 19.4 for DMHP) and with minimal
dependence on the type of N-substituent group (37). On the
other hand, preliminary studies indicated other anti-oxidant
properties for DMHP, namely those associated with the
inhibition of deoxyribose degradation and of lipoperoxidation
(IC50 ca 200 and 20 µM, respectively) (51).
The results in this study are consistent with the
lipophilicity studies, which evidenced that A and C are
more lipophilic than DFP, representing a benefit for their
bioavailability and Blood Brain Barrier crossing facility.
The particular properties of compound A show that it is a
superior product comparing with the others, exerting its
effects by inhibiting MAO dependent generation of
hydrogen peroxide, and chelating iron-dependent
generation of reactive hydroxyl radical (by Fenton reaction)
from hydrogen peroxide generated by other reactions.
Taking together, the several targets and various
biochemical properties of compound A make of it a
potential drug in PD and AD treatment.
6. ACKNOWLEDGMENTS
The authors are grateful to Dra. Isabel Nunes-
Correia (Centro de Neurociências e Biologia Celular,
Universidade de Coimbra, Portugal) for cell culture
support. The authors also thank the Portuguese Fundação
para a Ciência e Tecnologia (FCT) (project
PCDT/QUI/56985/04) and Instituto de Investigação
Interdisciplinar (III/39/2007) for financial support.
7. REFERENCES
1. Selkoe, D.J.: Clearing the brain's amyloid cobwebs.
Neuron 32, 177-80 (2001)
2. Roman, M.J., D.C. Delis, J.V. Filoteo, T.L. Demadura, J.
Paulsen, N.R. Swerdlow, M.R. Swenson, D. Salmon, N.
Butters & C. Shults: Is there a "subcortical" profile of
attentional dysfunction? A comparison of patients with
Huntington's and Parkinson's diseases on a global-local
focused attention task. J Clin Exp Neuropsychol 20, 873-84
(1998)
3. Forno, L.S.: Neuropathology of Parkinson's disease. J
Neuropathol Exp Neurol 55, 259-72 (1996)
4. Cardoso, S.M., P.I. Moreira, P. Agostinho, C. Pereira &
C.R. Oliveira: Neurodegenerative pathways in Parkinson's
disease: therapeutic strategies. Curr Drug Targets CNS
Neurol Disord 4, 405-19 (2005)
5. Pereira, C., P. Agostinho, P.I. Moreira, S.M. Cardoso &
C.R.Oliveira: Alzheimer's disease-associated neurotoxic
mechanisms and neuroprotective strategies. Curr Drug
Targets CNS Neurol Disord 4, 383-403 (2005)
6. Butterfield, D.A., J. Drake, C. Pocernich & A. Castegna:
Evidence of oxidative damage in Alzheimer's disease
brain: central role for amyloid beta-peptide. Trends Mol
Med. 7, 548-54 (2001)
7. Pratico, D., C.M. Clark, V.M. Lee, J.Q. Trojanowski,
J. Rokach & G.A. FitzGerald: Increased 8,12-iso-
iPF2alpha-VI in Alzheimer's disease: correlation of a
noninvasive index of lipid peroxidation with disease
severity. Ann Neurol 48, 809-12 (2000)
8. Crichton, R.R. & R.J. Ward: Metal-based
Neurodegeneration. John Wiley & Sons Lda, Chichester,
pp 62-87 (2006).
9. Smith, M.A., P.L. Harris, L.M. Sayre & G. Perry:
Iron accumulation in Alzheimer disease is a source of
redox-generated free radicals. Proc Natl Acad Sci USA
94, 9866-9868 (1997)
10. Dexter, D.T., C.J. Carter, F.R. Wells, F. Javoy-Agid,
Y. Agid, A. Lees, P. Jenner & C.D. Marsden: Basal lipid
peroxidation in substantia nigra is increased in
Parkinson's disease. J Neurochem 52, 381-9 (1989)
11. Floor, E., M.G. Wetzel: Increased protein oxidation
in human substantia nigra pars compacta in comparison
with basal ganglia and prefrontal cortex measured with
an improved dinitrophenylhydrazine assay. J
Neurochem 70, 268-75 (1998)
12. Saggu, H., J. Cooksey, D. Dexter, F.R. Wells, A.
Lees, P. Jenner & C.D. Marsden: A selective increase in
particulate superoxide dismutase activity in
parkinsonian substantia nigra. J Neurochem 53, 692-7
(1989)
13. Ben-Shachar, D., P. Riederer & M.B. Youdim: Iron-
melanin interaction and lipid peroxidation: implications
for Parkinson's disease. J Neurochem 57, 1609-14
(1991)
14. Graham, D.G.: Oxidative pathways for
catecholamines in the genesis of neuromelanin and
cytotoxic quinones. Mol Pharmacol 14, 633-43 (1978)
15. Maker, H.S., C. Weiss, D.J. Silides & G. Cohen:
Coupling of dopamine oxidation (monoamine oxidase
activity) to glutathione oxidation via the generation of
hydrogen peroxide in rat brain homogenates. J Neurochem
36, 589-93 (1981)
16. Schindowski, K., T. Kratzsch, J. Peters, B. Steiner, S.
Leutner, N. Touchet, K. Maurer, C. Czech, L. Pradier, L.
Frölich, W.E. Müller & A. Eckert: Impact of aging:
sporadic and genetic risk factors on vulnerability to
apoptosis in Alzheimer's disease. Neuromolecular Med 4,
161-78 (2003)
17. Tatton, N.A., A. Maclean-Fraser, W.G. Tatton, D.P.
Perl & C.W. Olanow: A fluorescent double-labelling
method to detect and confirm apoptotic nuclei in
Parkinson's disease. Ann Neurol 44, S142-8 (1998)
18. Hartmann, A., S. Hunot, P.P. Michel, M.P. Muriel,
S. Vyas, B.A. Faucheux, A. Mouatt-Prigent, H. Turmel,
A. Srinivasan, M. Ruberg, G.I. Evan, Y. Agid & E.C.
Hirsch: Caspase-3: A vulnerability factor and final effector
in apoptotic death of dopaminergic neurons in Parkinson's
disease. Proc Natl Acad Sci USA 14; 97, 2875-80 (2000)
19. Tatton, N.A.: Increased caspase 3 and Bax
immunoreactivity accompany nuclear GAPDH
translocation and neuronal apoptosis in Parkinson's
disease. Exp Neuro 166, 29-43 (2000)

New hydroxypyridinone iron-chelators as potential anti-neurodegenerative drugs
6773
20. Paschen, W.: Endoplasmic reticulum: a primary target
in various acute disorders and degenerative diseases of the
brain. Cell Calcium 34, 365-83 (2003)
21. Ryu, E.J., H.P. Harding, J.M. Angelastro, O.V. Vitolo,
D. Ron & L.A. Greene: Endoplasmic reticulum stress and
the unfolded protein response in cellular models of
Parkinson's disease. J Neurosc 22, 10690-8 (2002)
22. Yamamuro, A., Y. Yoshioka, K. Ogita & S. Maeda:
Involvement of endoplasmic reticulum stress on the cell
death induced by 6-hydroxydopamine in human
neuroblastoma SH-SY5Y cells. Neurochem Res 31, 657-64
(2006)
23. Hoozemans, J.J., E.S. van Haastert, P. Eikelenboom,
R.A. de Vos, J. M. Rozemuller & W. Scheper: Activation
of the unfolded protein response in Parkinson's disease.
Biochem Biophys Res Commun 354, 707-11 (2007)
24. Hider, R.C., G.J. Kontoghiorghes & J. Silver:
Pharmaceutical compositions. UK PATENT gb2118176,
1983
25. Kontoghiorghes, G.J., M.A. Aldouri, L. Sheppard &
A.V. Hoffbrand: 1,2-dimethyl-3-hydroxypyrid-4-one, an
orally active chelator for treatment of iron overload. Lancet
1 (8545), 1294-1295 (1987)
26. Hubálek, F., C. Binda, M. Li, J. Sterling, D.E.
Edmondson & A. Mattevi: Inactivation of purified human
recombinant monoamine oxidases A and B by rasagiline
and its analogues. J Med Chem 47, 1767-1774 (2004)
27. Youdim, M.B.H. & Y.S. Bakhle: Monoamine oxidase:
isoforms and inhibitors in Parkinson's disease and
depressive illness. British J Pharmacol 147, 287-296
(2006)
28. Armarego, W.L.F. & D.D. Perring (eds) Purification of
Laboratory Chemicals 4rd ed., Butterworth-Heinemann
Press, Oxford (1999).
29. Santos, M.A., R. Grazina, A.Q. Neto, G. Cantinho, L.
Gano & L. Patricio: Synthesis, chelating properties towards
gallium and biological evaluation of two N-substituted 3-
Hydroxy-4-Pyridinones. J Inorg Biochem 78, 303-311
(2000)
30. Leo, A., C. Hansch & D. Elkins: Partition coefficients
and their uses. Chem Rev 71, 526-616 (1971).
31. Cardoso, S.M. & C.R. Oliveira: The role of calcineurin
in amyloid-beta-peptides-mediated cell death. Brain Res
1050, 1-7 (2005)
32. Mosmann, T.: Rapid colorimetric assay for cellular
growth and survival: application to proliferation and
cytotoxicity assays. J Immunol Methods 65, 55-63 (1983)
33. Cregan, S.P., J.G. MacLaurin, C.G. Craig, G.S.
Robertso, D.W. Nicholson, D.S. Park & R.S. Slack: Bax-
dependent caspase-3 activation is a key determinant in p53-
induced apoptosis in neurons. J Neurosc 19, 7860-9 (1999)
34. a) Santos, M.A.: Hydroxypyridinone complexes with
aluminium. In vitro studies and in vivo perspectives. Coord
Chem Rev 228, 187-203 (2002); b) Santos, M.A.: Recent
developments on 3-hydroxy-4-pyridinones in view of
clinical applications. Mono and combined ligand
approaches. Coord Chem Rev (2008)
35. Hider, R.C. & Z.D. Liu: Emerging understanding of the
advantage of small molecules such as hydroxypyridinones
in the treatment of iron overload. Curr Med Chem 10,
1051-1064 (2003)
36. Florence, A.L., A. Gauthier, R.J. Ward & R.R.
Crichton. Influence of hydroxypyridones and
desferrioxamine on the mobilization of aluminium from
tissues of aluminium-loaded rats. Neurodegeneration 4,
449-455 (1995)
37. Santos, M.A., M. Gil, L. Gano, S. Chaves: Bifunctional
3-hydroxy-4-pyridinone derivatives as potential
pharmaceuticals. Synthesis, complexation with Fe (III), Al
(III) and Ga (III), and in vivo evaluation with 67Ga. J Bio
Inorg Chem 10, 564-580 (2005)
38. Chang, H.Y. & X. Yang: Proteases for cell suicide:
functions and regulation of caspases. Microbiol Mol Biol
Rev 64, 821–846 (2000)
39. Troy, C.M. & M.L. Shelanski: Caspase-2 redux. Cell
Death Differ 10, 101-7 (2003)
40. Nakagawa, T., H. Zhu, N. Morishima, E. Li, J. Xu,
B.A. Yankner & J. Yuan: Caspase-12 mediates
endoplasmic-reticulum-specific apoptosis and cytotoxicity
by amyloid-beta. Nature 403, 98-103 (2000)
41. Hitomi, J., T. Katayama, Y. Eguchi, T. Kudo, M.
Taniguchi, Y. Koyama, T. Manabe, S. Yamagishi, Y.
Bando, K. Imaizumi, Y. Tsujimoto & M. Tohyama:
Involvement of caspase-4 in endoplasmic reticulum stress-
induced apoptosis and Abeta-induced cell death. J Cell Biol
165, 347-56 (2004)
42. Cardoso, S.M., R.H. Swerdlow & C.R. Oliveira:
Induction of cytochrome c-mediated apoptosis by amyloid
beta 25-35 requires functional mitochondria. Brain Res
931, 117-25 (2002)
43. Youdim, M.B., M. Fridkin & H. Zheng: Novel
bifunctional drugs targeting monoamine oxidase inhibition
and iron chelation as an approach to neuroprotection in
Parkinson's disease and other neurodegenerative diseases. J
Neural Transm 111, 1455-1471 (2004)
44. Sterling, J., Y. Herzig, T. Goren, N. Finkelstein, D.
Lerner, W. Goldenberg, I. Miskolczi, S. Molnar, F. Rantal,
T. Tamas, G. Toth, A. Zagyva, A. Zekany, J. Finberg, G.
Lavian, A. Gross, R. Friedman, M. Razin, W. Huang, B.
Krais, M. Chorev, M. B. Youdim & M. Weinstock,: Novel
dual inhibitors of AChE and MAO derived from hydroxy
aminoindan and phenethylamine as potential treatment for
Alzheimer's disease. J Med Chem 45, 5260-5279 (2002)
45. Zheng, H., L.M. Weiner, O. Bar-Am, S. Epsztejn, Z.I.
Cabantchik, A. Warshawsky, M.B. Youdim & M. Fridkin:
Design, synthesis, and evaluation of novel bifunctional
iron-chelators as potential agents for neuroprotection in
Alzheimer's, Parkinson's, and other neurodegenerative
diseases. Bioorg Med Chem 13, 773-783 (2005)
46. Cardoso, S.M., S. Santos, R. H. Swerdlow & C. R.
Oliveira: Functional mitochondria are required for amyloid
beta-mediated neurotoxicity. FASEB J 15, 1439-1441
(2001)
47. Rogers J.T. & D. K. Lahiri: Metal and inflammatory
targets for Alzheimer's disease. Curr Drug Targets 5, 535-
551 (2004).
48. Lovell, M.A., J. D. Robertson, W.J. Teesdale, J.L.
Campbell & W.R. Markesbery: Copper, iron and zinc in
Alzheimer's disease senile plaques. J Neurol Sci 158, 47-52
(1998)
49. Moreira, P., C. Pereira, M.S. Santos & C. Oliveira:
Effect of zinc ions on the cytotoxicity induced by the

New hydroxypyridinone iron-chelators as potential anti-neurodegenerative drugs
6774
amyloid beta-peptide. Antioxi Redox Signal 2, 317-325
(2000)
50. Hicks, S.W. & C.E. Machamer: Golgi structure in stress
sensing and apoptosis. Biochim Biophys Acta 1744, 406-14
(2005)
51. S. Canário, S. Chaves, M.L. Mira & M.A. Santos: Anti-
oxidant properties of O,O- and O,S-Donor Ligands with
pyrone and pyridinone molecular scaffolds. In Press
Abbreviations: AchE: acetylcholinesterase; AD:
Alzheimer`s disease; ER: endoplasmic reticulum; MAO:
Monoamine oxidase; MPP+: 1-methyl-4-phenylpyridinium;
MTT: 3- (4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; NFT: Neurofibrillary tangles;
PD: Parkinson´s disease; ROS: reactive oxygen species;
TUNEL: Terminal uridine deoxynucleotidyl transferase
dUTP nick end labeling
Key Words: Alzheimer`s disease, Parkinson`s Disease,
Neurodegeneration, Apoptosis; Mpp+, Hydroxypyridinone,
Chelation, Therapy; Iron Chelator
Send correspondence to: M. Amelia Santos, Centro de
Quimica Estrutural, Instituto Superior Tecnico (UTL), Av
Rovisco Pais, 1, 1049-001 Lisboa, Tel:00351-218419273,
Fax: 00351-218464455, E-mail: masantos@ist.utl.pt
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