Synthesis and pharmacological assessment of diversely substituted pyrazolo[3,4-b]quinoline, and benzo[b]pyrazolo[4,3-g][1,8]naphthyridine derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.05.068

The synthesis and pharmacological analyses of a number of pyrazolo[3,4-b]quinoline and benzo[b]pyrazolo[4,3-g][1,8]naphthyridine derivatives are reported. We have synthesized the diversely substituted tacrine analogues 1-6, by Friedlaender-type reaction o

Full text of DOI:10.1016/j.ejmech.2011.05.068

European Journal of Medicinal Chemistry 46 (2011) 4676e4681
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Short communication
Synthesis and pharmacological assessment of diversely substituted
pyrazolo[3,4-b]quinoline, and benzo[b]pyrazolo[4,3-g][1,8]naphthyridine
derivatives
,
b
,
,
,
c
Daniel Silva a 1, Mourad Chioua b 1, Abdelouahid Samadi b, M. Carmo Carreiras a , María-Luisa Jimeno ,
*
Eduarda Mendes a, Cristóbal de los Ríos d e, Alejandro Romero f h, Mercedes Villarroya d
,
,
,
,f
Manuela G. López f g, José Marco-Contelles b
,
,
**
a Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal
b Laboratorio de Radicales Libres y Química Computacional (IQOG, CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain
c Centro de Química Orgánica “Lora Tamayo” (CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain
d Instituto de Investigación Sanitaria, Fundación de Investigación Biomédica, Hospital Universitario de la Princesa, C/Diego de León 62, 28006 Madrid, Spain
e Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Alcalá, Ctra. Barcelona, Km. 33.5, 28817 Alcalá de Henares, Spain
f Instituto Teófilo Hernando and Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo 4, 28029 Madrid, Spain
g Hospital La Paz Health Research Institute-IdiPAZ, Madrid, Spain
h Departamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and pharmacological analyses of a number of pyrazolo[3,4-b]quinoline and benzo[b]py-
razolo[4,3-g][1,8]naphthyridine derivatives are reported. We have synthesized the diversely substituted
tacrine analogues 1e6, by Friedländer-type reaction of readily available o-amino-1-methyl-pyrazole-
dicarbonitriles with cyclohexanone. The biological evaluation showed that pyrazolotacrines 1e6 are
inhibitors of Electrophorus electricus acetylcholinesterase (EeAChE), in the micromolar range, and quite
selective in respect to serum horse butyrylcholinesterase (eqBuChE) inhibition; the most interesting
inhibitor is N-(5-amino-1-methyl-6,7,8,9-tetrahydro-1H-benzo[b]pyrazolo[4,3-g][1,8]naphthyridin-3-yl)
Received 15 March 2011
Received in revised form
20 May 2011
Accepted 26 May 2011
Available online 7 June 2011
Keywords:
acetamide (5) [IC50 (EeAChE) ¼ 0.069 ꢀ 0.006
m
M; IC50 (eqBuChE) ¼ 6.3 ꢀ 0.6
mM]. Kinetic studies
Pyrazolotacrines
AChE/BuChE inhibitors
Oxidative stress
Neuroprotection
Alzheimer’s disease
showed that compound 5 is a mixed-type inhibitor of EeAChE (Ki ¼ 155 nM). Inhibitor 5 showed a 45%
neuroprotection value against rotenone/oligomycin A-induced neuronal death.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
described [2]. Classical pathological hallmarks are senile plaques,
consisting principally of amyloid-
b (Ab), and neurofibrillary
Alzheimer’s disease (AD) is the most prevalent neurodegener-
ative disorder, and one of the most common of all diseases in the
industrialized world [1]. AD is characterized by the gradual devel-
opment of forgetfulness, progressing to disturbances in language,
dyscalculia/acalculia, visuospatial disorientation, ideational and
ideomotor apraxia, akinesia, and mutism. The symptomatic course
of disease is generally 5 or more years, although a 7-year preclinical
period of stepwise decline in memory and attention span has been
tangles, which are composed of phosphorylated tau. Furthermore,
the basal nucleus of Meynert undergoes profound neuron loss, and
the neocortex exhibits a loss of cholinergic fibres and receptors and
a decrease of both choline acetyltransferase and acetylcholines-
terase (AChE) enzyme activity [2,3]. Since the symptoms of AD were
associated with an altered cholinergic function, research has
focused on the basal forebrain cholinergic system [3]. As a result,
the so-called “cholinergic hypothesis” was developed, which
postulated that a loss of cholinergic function in the central nervous
system contributed significantly to cognitive decline associated
with advanced age and AD [4]. Thus, drugs capable of inhibiting
AChE might potentiate central cholinergic function, therefore
improving cognition and perhaps even some of the behavioural
problems experienced by AD patients [5]. AChE inhibitors (AChEI)
* Corresponding author. Tel.: þ351 21 7946475; fax: þ351 21 7946470.
** Corresponding author. Fax: þ34 91 5644853.
E-mail addresses: iqoc21@iqog.csic.es (M. Carmo Carreiras), mcdamaso@ff.ul.pt
(J. Marco-Contelles).
1
Those authors have contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.068

D. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4676e4681
4677
Table 1
Inhibition of AChE from Electrophorus electricus (EeAChE) and horseserum butyrylcholinesterase (eqBuChE) by the pyrazolo-THA analogues 1e6 and THA.
Compound
Structure
IC50
(
m
M) EeAChE
IC50
(
m
M) eqBuChE
Selectivity BuChE/AChE
NH2
NC
N
1
0.57 ꢀ 0.04
1.70 ꢀ 0.02
0.6 ꢀ 0.1
100
>175
Me
N
N
NH2
N
NC
N
2
3
4
5
6
6.3 ꢀ 0.6
5.4 ꢀ 0.6
>100
3.7
N
Me
O
NH2
N
H2
N
C
9
> 10.7
91.3
39
Me
N
N
O
O
H2N
HN
N
C
9.3 ꢀ 1.5
N
N
Me
NH2
N
AcHN
N
0.069 ꢀ 0.006
6.3 ꢀ 0.6
9.0 ꢀ 0.9
N
N
Me
Ph
NH2
AcHN
N
0.23 ꢀ 0.05
N
N
N
Me
NH2
Tacrine
0.027 ꢀ 0.002
0.0052 ꢀ 0.0002
0.193
N
aData are expressed as the mean ꢀ SEM of at least three different experiments in quadruplicate.
may inhibit AChE via a competitive mechanism, by interacting with
the catalytic centre (CC) of the enzyme, via a non-competitive
mechanism, by binding with the peripheral anionic site (PAS), or
via both mechanisms, by exerting a dual binding AChE inhibition
[5]. For a while the treatment with AChEI was reported to produce
only symptomatic improvement, having no effect in the course of
the disease [6]. However, other studies indicated that AChE inter-
agents can attenuate neuronal damage and death from cytotoxic
insults, and therefore might affect AD pathogenesis [12].
Considering the non-cholinergic aspects of AChE related to its
PAS [7e9] in associating with Ab, an attractive target for the design
of new anti-dementia drugs emerged. Peripheral or dual site
inhibitors of AChE may simultaneously alleviate the cognitive
deficit in AD patients and prevent the assembly of Ab, which will
acts with A
promoting A
increase A -dependent neurotoxicity [9]. These reports arose a new
b
by a hydrophobic environment close to the PAS, thus
b
fibril formation [7,8]. Besides, AChEeA complexes
b
b
enthusiasm in AChEI. Interestingly, early reports demonstrated the
ability of cholinesterase inhibitors (ChEI) to enhance the release of
non-amyloidogenic soluble derivatives of amyloid precursor
protein (APP) in vitro and in vivo, and possibly to slow down
formation of amyloidogenic compounds in the brain [10]. The
increase of soluble APP (APPs) was also consistent with AChE
inhibition [11]. Numerous clinical trials have demonstrated the
safety and efficacy of ChEI in the treatment of AD. Besides, there is
growing evidence from preclinical studies indicating that these
Scheme 1. Synthesis of 4-amino-2-methyl-5,6,7,8-tetrahydro-2H-pyrazolo[3,4-b]
quinoline-3-carbonitrile (1).

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D. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4676e4681
Scheme 2. Synthesis of 4-amino-2-methyl-5,6,7,8-tetrahydro-2H-pyrazolo[3,4-b]quinoline-3-carbonitrile (2), and 1-methyl-4-((6-phenoxyhexyl)amino)-5,6,7,8-tetrahydro-2H-
pyrazolo[3,4-b]quinoline-3-carboxamide (4).
delay the neurodegenerative process [13]. This strategy was
pursued for several medicinal chemists who have been developing
new compounds with dual AChE inhibitory activity [14e17].
VDCC-L; hence they may prevent the Ca2þ overload of cells from is
chemically compromised tissue, as well as the alteration of ener-
getic metabolism of these cells by the Ca2þ overload of its mito-
chondria [31]. Nimodipine, also a neuronal VDCC-L blocker protects
neurons from death evoked by focal cerebral ischemia [32].
In this context, some years ago we undertook a research project
directed to the synthesis of multipotent compounds designed to
target both AChE and neuronal Ca2þ modulation, capable of
showing neuroprotection and antioxidant properties. Hence, we
have synthesized and evaluated several families of hybrid
compounds such as IeVIII [33e44], where the benzene ring in
tacrine (THA) was substituted by a 4H-pyran ring (I) [33e38],
a pyridine ring (II) [34,36,37,39], a DHP ring (III) [40], a furan ring
(IV) [35,41], a thiophene ring (V) [41], an oxazole ring (VI) [36,42],
The mechanism of A
cellular calcium homeostasis resulting in elevated rest intracell-
ular free calcium concentration ([Ca2þ)i] and increased [Ca2þ
b toxicity also involves destabilization of
]
i
responses to excitatory aminoacids and membrane depolarization.
Thus, A renders neurons more vulnerable to environmental insults
[18e20].
b
Due to the multiple events that take place in the cascade of the
neurodegenerative disease new therapeutic strategies were
devised and the new drug candidates were designed to act on
multiple neural and biochemical targets for the treatment of
cognition impairment, motor dysfunction, depression and neuro-
degeneration [21]. Examples include the development of single
molecular entities that combine two or more of the following
properties: cholinesterase inhibition, activation or inhibition of
a
1H-chromeno[2,3-b]quinolin-1-one ring (VII) [43], and
3,4,5,7,8,9,10,12-octahydrodibenzo[b,g][1,8]naphthyridin-1(2H)-
one ring (VIII) [44], respectively (Chart 1). Other THA analogues
have also been reported from other research groups [45e47].
With these models in mind, and attempting to get more equi-
potent AChE versus butyrylcholinesterase inhibitors (BuChEI),
endowed with an additional neuroprotective profile, we have now
designed a series of new THA analogues showing pyrazole as the
privileged heterocyclic ring system [48e50]. Therefore, we have
carried out the synthesis and biological evaluation of the diversely
substituted pyrazolotacrines (pyrazolo-THA) 1e6 (Table 1) bearing
either the pyrazolo[3,4-b]quinoline (IX) or benzo[b]pyrazolo[4,3-g]
[1,8]naphthyridine (X) (Chart 1) heterocyclic ring systems.
specific subtypes of acetylcholine receptors or
a-adrenoceptors,
anti-inflammatory activity, monoamine oxidase inhibition, neuro-
protection, and anti-apoptotic activity among others [21]. There-
fore, in order to address the different targets that contribute to AD
pathogenesis a new generation of multipotent compounds for AD
emerged [22e27]. The strategy of our group has been devoted to
the development of AChE inhibitors endowed with a neuronal
Ca2þchannel antagonist effect. Ca2þ modulates many neural
processes, including synaptic plasticity and apoptosis. Dysregula-
tion of intracellular Ca2þ signalling has been implicated in the
pathogenesis of AD. Increased intracellular Ca2þ elicits the char-
Barreiro and co-workers [51] designed
a few positional
acteristic lesions of this disorder, including the accumulation of A
the hyperphosphorylation of tau and neuronal death. Conversely,
neurodegeneration that is induced by A or tau is probably medi-
b
,
pyrazolo-THA analogues that were planned to investigate the
contribution of different N-phenyl orientations around the azahe-
terocyclic system in the AChE activity. By performing molecular
modelling (MM) studies they have observed interaction of the
azaheterocyclic ring of some pyrazolo derivatives in the crystallo-
graphic structure of the complex THAeAChE. With the exception of
the tetracyclic and the linear extended pyrazolo-THA derivatives,
the AChE inhibitory activity of the synthesized compounds was, in
general, modest [51]. According to MM results the poor activity
might be ascribed to the angular pattern adopted by the N-phenyl
group attached to the pyrazolopyridine system, which introduced
severe steric constraints into the molecular fit with Ile439/His440
and Tyr334/Trp432 on the active site of AChE. Interestingly,
a possible neuroprotective effect was observed in two pairs of the
positional pyrazolo-THA isomers investigated; the results showed
they were not significantly toxic to neurons and revealed
b
ated by changes in Ca2þ homeostasis. Disruption of Ca2þ regulation
in the endoplasmic reticulum mediates the most significant signal-
transduction cascades that are associated with AD. Moreover,
mutations that cause familial AD have been linked to intracellular
Ca2þ signalling pathways. Destabilization of Ca2þ signalling seems
to be central to the pathogenesis of AD, and targeting this process
might be therapeutically beneficial [18,28,29]. In addition, Ca2þ
entry through L-type voltage-dependent Ca2þ channels (VDCC-L,
Cav 1.1e1.4) causes both Ca2þ overload and mitochondrial disrup-
tion, which leads to the activation of the apoptotic cascade and cell
death [30]. These data justify the research for a possible therapeutic
effect in brain ischemia of drugs that block the entry of Ca2þ into
the cell. The dihydropyridines (DHP) are primary modulators of the
Scheme 3. Synthesis of 4-amino-2-methyl-5,6,7,8-tetrahydro-2H-pyrazolo[3,4-b]quinoline-3-carboxamide (3).

D. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4676e4681
4679
a tendency to reduce lactate dehydrogenase (LDH) release below
control levels in a concentration-dependent manner [51].
horseserum (eqBuChE), according to Ellman’s protocol [58]. The
corresponding IC50 values are shown in Table 1. Pyrazolo[3,4-b]
quinolines 1e4 were less potent AChEI than benzo[b]pyrazolo[4,3-
g][1,8]naphthyridines 5 and 6. Nevertheless, compounds 5/6 and
2/3 revealed similar BuChE inhibitory activity. Among the pyrazolo
[3,4-b]quinolines 1e4, the most potent AChEI 1 and 3 bear a methyl
group at N2. The presence of a methyl at N1 in compounds 2 and 4,
as well as the N-alkylation in compound 4 are structural factors that
decrease the AChE inhibitory potency. Interestingly, unlike the
results found with AChE, moving the methyl group from N2 to N1,
2. Chemistry
2.1. Pyrazolo [3,4-b]quinolines
The precursors for the synthesis of 4-amino-1-pyrazolo[3,4-b]
quinoline derivatives (1e4) (Schemes 1e3) were the readily available
3-amino-1-methyl-pyrazole-4,5-dicarbonitrile (7) and 5-amino-1-
methyl-pyrazole-3,4-dicarbonitrile (8) [52]. The Friedländer reac-
tion (FR) [53] of 3-amino-1-methyl-pyrazole-4,5-dicarbonitrile (7)
with cyclohexanone, under the standard experimental conditions
[33], gave a complex reaction mixture, from which we were unable to
isolate the desired compound. However, the use of trimethylsilyl
triflate (TMSOTf)-EtOAc [54] system to promote the reaction gave the
expected 4-amino-2-methyl-5,6,7,8-tetrahydro-2H-pyrazolo[3,4-b]
quinoline-3-carbonitrile (1), albeit in poor yield (Scheme 1).
Conversely, the same reaction between the isomer 5-amino-1-
methyl-pyrazole-3,4-dicarbonitrile (8) and cyclohexanone afforded
the required pyrazolo-THA 4-amino-1-methyl-5,6,7,8-tetrahydro-1H-
pyrazolo[3,4-b]quinoline-3-carbonitrile (2) in 60% yield (Scheme 2).
The reaction of compound 7 with N-benzylpiperidin-4-one
(9), using the TMSOTf/EtOAc reaction conditions [54], did not
yield the expected product, but amide 10 (Scheme 3), as the
result of the regioselective hydrolysis of the nitrile at C5 in
compound 7. The position of the carboxamido function was also
supported by selective NOE experiments between the two singlet
amido protons and the NeMe group, although the unequivocal
final confirmation was obtained from the FR between 10 and
cyclohexanone using AlCl3 as promotor to give pyrazolo-THA 3
(Scheme 3) in 28% yield (7% when using TMSOTf/EtOAc [54])
(Supplementary material).
BuChE inhibition was improved from more than 100 to 6
mM.
Transformation of the nitrile into an amide group at C3 also
increased the BuChE inhibitory activity. Focussing on benzo[b]
pyrazolo[4,3-g][1,8]naphthyridines
5 and 6, the presence of
a phenyl ring in compound 6 impaired AChE inhibition, as
compared to compound 5, which lacks this phenyl ring, while
BuChE inhibitory activity was quite similar. Overall, the most potent
inhibitor for both enzymes was N-(5-amino-1-methyl-6,7,8,9-
tetrahydro-1H-benzo[b]pyrazolo[4,3-g][1,8]naphthyridin-3-yl)
acetamide (5) [IC50 (EeAChE)
¼
69
ꢀ
6 nM, and IC50
(eqBuChE) ¼ 6.3 ꢀ 0.6
mM], 2.5-fold less active than THA, but much
less potent BuChEI.
THA is usually associated with hepatotoxicity. Though this work
reports the in vitro investigation of a number of THA analogues as
new multi-target-directed-ligands, hepatotoxicity studies have not
been included in this paper. In fact, THA leads to elevated amino-
transferase levels in 50% of patients. In spite of these high enzyme
levels, severe hepatoxicity is rarely seen, and enzyme values return
to normal following withdrawal of the drug [59]. THA undergoes
extensive oxidative metabolism to a variety of mono- and dihy-
droxylated metabolites in animals and humans [60e63]. THA bio-
activation by cytochrome (CYP) P450 enzymes, mainly CYP1A2
isoform is responsible for the protein-reactive metabolites (PRM)
observed in microsomes of all animal species studied. The
production of PRM is supported by a two-step mechanism in the
bioactivation of THA involving an initial 7-hydroxylation followed
by a postulated 2-electron oxidation to yield a reactive quinone
methide [62,63]. However, due to the nature of the pyrazole
derivatives structure a quinone methide is not feasible to be
formed. Nevertheless drug metabolism studies are meant to be
carried out after activity in vivo is confirmed. Besides, the fact that
THA was shown to alter intracellular glutathione concentration in
cultured hepatocytes might suggest the involvement of generation
In order to prepare dual AChEI [55], pyrazolo-THA 2 was reacted
with (6-bromohexyloxy)benzene (11) (Supplementary material), in
the presence of t-BuOH/t-BuOK. Under these conditions, the amino
at C4 was alkylated, and the nitrile group was transformed into
a carboxamide, to provide pyrazolo-THA 4 (Scheme 2) [56].
2.2. Benzo[b]pyrazolo[4,3-g][1,8]naphthyridines
Next, the N-(6-amino-5-cyano-1-methyl-1H-pyrazolo[3,4-b]
pyridin-3-yl)acetamides 17 and 18 [57] were used for the synthesis
of the pyrazolo-THA 5 and 6 under the standard FR conditions
(Scheme 4) (Supplementary material).
Control
70
10nM
3. Results and discussion
30nM
60
100nM
3.1. Pharmacology
300nM
50
1uM
3.1.1. AChE/BuChE inhibitory activity
40
Pyrazolo-THA 1e6 have been evaluated as inhibitors of
AChE from Electrophorus electricus (EeAChE), and BuChE from
30
20
10
0
-4
-2
0
2
4
6
8
10
-1 -1
[ATCh] (mM )
Fig. 1. Steady-state inhibition of AChE hydrolysis of acetylthiocholine (ATCh) by
compound 5. LineweavereBurk reciprocal plots of initial velocity and substrate
concentrations (0.1e1 mM) are presented. Lines were derived from a weighted least-
squares analysis of data.
Scheme 4. Friedländer reaction of pyrazolo[3,4-b]pyridines 17 and 18.

4680
D. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4676e4681
NO2
NH2
O
O
O
O
O
N
Tacrine
N
H
Nimodipine
NH2
NH2
Ar NH2
Ar
Ar NH2
EtO2C
N
O
EtO2C
Ar
Ar
X
( )n
( )n
N
( )n
N
X
N
( )n
N
N
II
IV X= O
V X= S
I X= O
III X= NH
VI
Ar NH2
O
NH2
N
R1
N
R1
NH2
N
N
N
( )n
N
N
X
N
R2
R2
VII X= O
VIII X= NH
(X)
(IX)
Chart 1. General structure and heterocyclic ring systems for the tacrine analogues previously described by our group (IeVIII) (Refs. [13e24]), and reported in the work (IX, X).
of reactive oxygen species (ROS) and lipid peroxidation, because
intracellular glutathione represents an important protective factor
against oxidative damage. Regarding this hypothesis, it has been
previously reported that THA, at high concentrations induced lipid
peroxidation in isolated rat hepatocytes, which was prevented by
preincubating the cells with vitamin E, a naturally occurring anti-
oxidant [64e66]. Thus, some considerations can be done; if ROS
generation was involved in pyrazolo-THAs toxicity, it could be
speculated that pyrazolo-THA 5 might not trigger this adverse
effect since this compound was shown to have antioxidant prop-
erties on SH-SY5Y neuroblastoma, showing a protection of 48%.
OA, a potent and non-selective inhibitor of serine/threonine phos-
phatases, induces tau hyperphosphorylation and cell death in both
primary cortical neurons and neuroblastoma cell lines, as well as
in vivo models [70e72]. In this model, measured with the method of
MTT reduction, pyrazolo-THA 5 was not able to improve neuronal
viability of cells damaged with OA (Supplementary material).
4. Conclusions
We have first reported the synthesis and pharmacological
evaluation of a series of novel fused pyrazolo-THA possessing the
pyrazolo[3,4-b]quinoline (IX), or benzo[b]pyrazolo[4,3-g][1,8]
naphthyridine (X) heterocyclic ring systems. Pyrazolo-THA 1e6,
designed as multipotent agents, since they combine ChE inhibition
with a neuroprotective profile, were prepared by FR-type reaction
of readily available precursors with cyclohexanone. The biological
evaluation showed that some of these molecules were potent and
selective AChEI; the most interesting inhibitor was N-(5-amino-1-
methyl-6,7,8,9-tetrahydro-1H-benzo[b]pyrazolo[4,3-g][1,8]naph-
thyridin-3-yl)acetamide (5), a mixed-type inhibitor for EeAChE,
showing a Ki of 155 nM. Interestingly, pyrazolo-THA 5 indicated
3.1.2. Kinetic study of the AChE inhibition by compound 5
The AChE inhibition mechanism accomplished by pyrazolo-THA
1e6 was investigated using pyrazolo-THA 5, the most potent AChEI.
The type of inhibition was elucidated from the analysis of Line-
weavereBurk reciprocal plots (Fig. 1) showing both increasing
slopes (lower Vmax) and intercepts (higher KM) with higher inhib-
itory concentration [67]. This suggests a mixed-type inhibition. A Ki
value of 155 nM was estimated from the slopes of double reciprocal
plots versus compound 5 concentrations (Supplementary material).
a
45% neuroprotection value against rotenone/oligomycin
3.1.3. Neuroprotective effect of compounds 1e6 against rotenone/
oligomycin A-induced cell death in SH-SY5Y neuroblastoma cells
The exposure of SH-SY5Y cells to a mixture of rotenone plus
oligomycin A (Rot/Olig) constitutes a good model of oxidative stress
having its origin in mitochondria [68]. Rotenone blocks complex I of
mitochondrial electron transport chain and oligomycin A inhibits
complex V, thus ATP synthesis was completely disrupted; they
elicit neurotoxicity and allow the evaluation of potential neuro-
protective AChEI drugs used to treat AD patients, such as galant-
amine, donepezil, and rivastigmine. Cells exposed to Rot/Olig in the
absence of the compound showed 35% cell death with respect to
control cells (13%), measured as % of LDH release [69]. Measured at
A-induced cell death, but was not able to improve cell viability of
SH-SY5Y cells damaged with OA. To sum up, among all these
compounds, pyrazolo-THA 5 can be considered as a promising hit
for further development of a new family of neuroprotective AChEI.
Acknowledgements
We thank V. Gómez and L. González-Lafuente for technical
support. D. Silva thanks Fundação para a Ciência e Tecnologia do
Ministério da Ciência, Tecnologia e Ensino Superior of Portugal
for his grant belonging to project PTDC/SAU-NEU/64151/2006.
M. Chioua thanks Instituto de Salud Carlos III (MICINN) for
1
mM, only pyrazolo-THA 5 was able to reduce the Rot/Olig-induced
a “Sara Borrell” post-doctoral contract. JMC thanks MICINN
neuronal death. In terms of protection, pyrazolo-THA 5 afforded
48% protection. Melatonin, used as reference, protected a 48% at the
concentration of 10 nM (Supplementary material).
Encouraged by the neuroprotective activity of pyrazolo-THA 5
against Rot/Olig neurotoxicity, its effect against a toxicity model well
related to AD, e.g. the exposure to okadaic acid (OA), was evaluated.
(SAF2006-08764-C02-01; SAF2009-07271) financial support. MGL
thanks MICINN Ref. SAF2009-12150. DS thanks COST Action D34.
This work was partly supported by grants RD06/0026/1002
(RENEVAS), ISCIII, MICINN, S/SAL-0275-2006, Comunidad de
Madrid, and Fundación CIEN, project 1004040042. JMC thanks Prof.
A. García for his continuous interest and support.

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4681
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