ITH14001, a CGP37157-Nimodipine Hybrid Designed to Regulate Calcium Homeostasis and Oxidative Stress, Exerts Neuroprotection in Cerebral Ischemia

Article abstract of DOI:10.1021/acschemneuro.6b00181

During brain ischemia, oxygen and glucose deprivation induces calcium overload, extensive oxidative stress, neuroinflammation, and, finally, massive neuronal loss. In the search of a neuroprotective compound to mitigate this neuronal loss, we have designed and synthesized a new multitarget hybrid (ITH14001) directed at the reduction of calcium overload by acting on two regulators of calcium homeostasis; the mitochondrial Na⁺/Ca²⁺ exchanger (mNCX) and L-type voltage dependent calcium channels (VDCCs). This compound is a hybrid of CGP37157 (mNCX inhibitor) and nimodipine (L-type VDCCs blocker), and its pharmacological evaluation revealed a moderate ability to selectively inhibit both targets. These activities conferred concentration-dependent neuroprotection in two models of Ca²⁺ overload, such as toxicity induced by high K⁺ in the SH-SY5Y cell line (60% protection at 30 μM) and veratridine in hippocampal slices (26% protection at 10 μM). It also showed neuroprotective effect against oxidative stress, an activity related to its nitrogen radical scavenger effect and moderate induction of the Nrf2-ARE pathway. Its Nrf2 induction capability was confirmed by the increase of the expression of the antioxidant and anti-inflammatory enzyme heme-oxygenase I (3-fold increase). In addition, the multitarget profile of ITH14001 led to anti-inflammatory properties, shown by the reduction of nitrites production induced by lipopolysaccharide in glial cultures. Finally, it showed protective effect in two acute models of cerebral ischemia in hippocampal slices, excitotoxicity induced by glutamate (31% protection at 10 μM) and oxygen and glucose deprivation (76% protection at 10 μM), reducing oxidative stress and iNOS deleterious induction. In conclusion, our hybrid derivative showed improved neuroprotective properties when compared to its parent compounds CGP37157 and nimodipine.

Full text of DOI:10.1021/acschemneuro.6b00181

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Article
ITH14001, a CGP37157-nimodipine hybrid designed to regulate calcium
homeostasis and oxidative stress, exerts neuroprotection in cerebral ischemia
Izaskun Buendia, Giammarco Tenti, Patrycja Michalska, Iago Méndez-López, Enrique
Luengo, Michele Satriani, Juan Fernando Padin Nogueira, Manuela G. López,
María Teresa Ramos, Antonio G García, J. Carlos Menéndez, and Rafael León
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00181 • Publication Date (Web): 12 Oct 2016
Downloaded from http://pubs.acs.org on October 13, 2016
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ITH14001, a CGP37157ꢀnimodipine hybrid
designed to regulate calcium homeostasis and
oxidative stress, exerts neuroprotection in
cerebral ischemia
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#,‡
†,‡
#,§
#,§
Izaskun Buendia, Giammarco Tenti, Patrycja Michalska, Iago Méndez-López,
#
,§
#,†
#,§
Enrique Luengo, Michele Satriani, Fernando Padín-Nogueira, Manuela G.
#
,§
†
#,§
,†
López, Mª. Teresa Ramos, Antonio G. García, J. Carlos Menéndez* and Rafael
,
#,§
León*
#
Instituto Teófilo Hernando y Departamento de Farmacología y Terapéutica, Facultad
de Medicina. Universidad Autónoma de Madrid, 28029 Madrid, Spain.
†
Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia,
Universidad Complutense, 28040 Madrid, Spain.
§
Instituto de Investigación Sanitaria, Servicio de Farmacología Clínica, Hospital
Universitario de la Princesa, 28006 Madrid, Spain.
RECEIVED DATE (to be automatically inserted after your manuscript is accepted
if required according to the journal that you are submitting your paper to)
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ABSTRACT: During brain ischemia, oxygen and glucose deprivation induces calcium
overload, extensive oxidative stress, neuroinflammation and, finally, massive neuronal
loss. In the search of a neuroprotective compound to mitigate this neuronal loss, we
have designed and synthesized a new multitarget hybrid (ITH14001) directed at the
reduction of calcium overload by acting on two regulators of calcium homeostasis; the
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mitochondrial Na /Ca exchanger (mNCX) and Lꢀtype voltage dependent calcium
channels (VDCCs). This compound is a hybrid of CGP37157 (mNCX inhibitor) and
nimodipine (Lꢀtype VDCCs blocker), and its pharmacological evaluation revealed a
moderate ability to selectively inhibit both targets. These activities conferred
2+
concentrationꢀdependent neuroprotection in two models of Ca overload, such as
+
toxicity induced by high K in the SHꢀSY5Y cell line (60 % protection at 30 ꢀM) and
veratridine in hippocampal slices (26 % protection at 10 ꢀM). It also showed
neuroprotective effect against oxidative stress, an activity related to its nitrogen radical
scavenger effect and moderate induction of the Nrf2ꢀARE pathway. Its Nrf2 induction
capability was confirmed by the increase of the expression of the antioxidant and antiꢀ
inflammatory enzyme hemeꢀoxygenase I (3ꢀfold increase). In addition, the multitarget
profile of ITH14001 led to antiꢀinflammatory properties, shown by the reduction of
nitrites production induced by lipopolysaccharide in glial cultures. Finally, it showed
protective effect in two acute models of cerebral ischemia in hippocampal slices,
excitotoxicity induced by glutamate (31 % protection at 10 ꢀM) and oxygen and
glucose deprivation (76 % protection at 10 ꢀM), reducing oxidative stress and iNOS
deleterious induction. In conclusion, our hybrid derivative showed improved
neuroprotective properties when compared to its parent compounds CGP37157 and
nimodipine.
KEYWORDS. Calcium dyshomeostasis, cerebral ischemia, multitarget drugs,
mitochondrial NCX, voltage dependent calcium channels, Nrf2 inducers.
INTRODUCTION
2+
Calcium ion (Ca ) is the most important second messenger implicated in
neurotransmission as well as other cellular processes, such as the production of reactive
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oxygen or nitrogen species (ROS, RNS), mitochondrial bioenergetics, neurogenesis,
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autophagy and cell life/death signalling. The pleiotropic activity of this ion makes
necessary a tight control of its concentration fluctuations in all cellular compartments
and thus cells have developed a plethora of systems to strongly control the amplitude
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2+
and the temporal extension of its signals. However, these Ca buffering systems fail
2+
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under certain pathological conditions leading to dysregulation of Ca homeostasis.
2
+
Futile Ca cycling affects many cellular processes, principally the life/death
equilibrium leading to apoptosis and cell death. Neurodegenerative diseases are a
2+
prominent example where Ca dyshomeostasis has been related to disease onset and
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2+
development. In fact, neuronal death induced by Ca dysregulation has been widely
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reported in several neurological disorders such as Alzheimer´s disease, Parkinson´s
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disease or cerebral ischemia. Among these, cerebrovascular accidents are the second
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leading cause of death and the first cause of disability in the world.
Ischemia, commonly known as stroke, occurs after the interruption of the cerebral
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blood flow, causing a decreased delivery of oxygen and nutrients into the brain.
Oxygen and glucose deprivation (OGD) during ischemia leads to cytosolic calcium
2+
(
[Ca ] ) overload prompted by a massive release of the excitatory amino acid
c
14
glutamate. Thereafter, mitochondrial failure induces the production of free radical
species that is exacerbated during the reoxygenation (reox) phase. At the same time, the
elevated levels of oxidative stress and the initial neuronal death induce glial activation
and neuroinflammation. Reactive glia increases the production of free radicals and
15
16
releases proꢀinflammatory cytokines, contributing to a massive neuronal death.
Currently, the only approved treatment for ischemic stroke is the tissue plasminogen
activator (tPA), a drug that accelerates thrombus removal, although its use is highly
restricted due to its small therapeutic window. In search for alternative approaches,
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neuroprotection represents a useful instrument to prevent the onset and to combat the
progression of this pathological condition. In this context, the most widely accepted
targets to develop neuroprotective agents against ischemic stroke are: the reduction of
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[
c
Ca ] overload, the decrease of ROS production during the reox phase and the
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reduction of glial activation. However, clinical trials of singleꢀtarget drugs aimed at one
of these pathological processes have failed so far, probably because the complexity of
the pathological cascade of events involved in the pathogenesis of cerebral ischemia. It
has thus become apparent during the last years that multitarget drugs should be
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potentially more effective for the treatment of this condition.
2
+
c
In order to reduce the initial [Ca ] overload, several targets have been proposed,
+
2+
including the mitochondrial Na /Ca exchanger (mNCX) and the voltage dependent
calcium channels (VDCCs). The mNCX protein has been widely used as a
2
+
18
neuroprotective target as it regulates mitochondrial Ca cycling (mCC). It is
2
+
c
considered responsible for about 50% of the [Ca ] overload occurring during
+
19
ischemia, due to increased levels of intracellular Na . Along with a reduction of
2+
2+
[
Ca ] overload, the mNCX blockade strategy induces mitochondrial calcium ([Ca ] )
c m
2+
elevation increasing the activity of Ca dependent dehydrogenases and augmenting
ATP synthesis and thus favouring neuronal viability. The mNCX antagonist that has
received most attention is 7ꢀchloroꢀ5ꢀ(2ꢀchlorophenyl)ꢀ1,5ꢀdihydroꢀ4,1ꢀbenzothiazepinꢀ
2(3H)ꢀone (CGP37157, figure 1) which acts as a neuroprotective drug against
18, 20
ischemia.
Furthermore, this compound reduced neuronal damage in in vivo models
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of cerebral ischemia.
On the other hand, VDCC and specifically the Lꢀtype VDCCs are considered the most
2+
2+
c
permeable channels to Ca and the most important contributors to [Ca ] overload in
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pathological conditions. Focusing on ischemic stroke, nimodipine reduces infarct size
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after stroke and improves the clinical outcome in acute ischemic stroke in animal
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models. However, clinical trials have recurrently failed to show efficacy, which has
been related to the hypotension caused by the potent blockade of vascular smooth
2
5
muscle cells associated to the high selectivity of 1,4ꢀdihydropyridines as blockers of
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the cardiovascular Lꢀtype VDCCs (Ca
V V
1.2) relative to the neuronal subtype (Ca 1.3).
The relevance of the Lꢀtype VDCCs as a therapeutic target has been reinforced by the
2
+
recent finding of an important link between Ca entry through Lꢀtype VDCCs and the
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production of free radical species. Thus, Lꢀsubtype VDCCs blockade simultaneously
2
+
reduces Ca overload and oxidative stress, two of the main pathological features in
2
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ischemic stroke.
Regarding oxidative damage, the phase II antioxidant and antiꢀinflammatory response
is a highly promising target against oxidative stress and neuroinflammation occurring in
ischemic stroke. Several inducers of the nuclear factor E2ꢀrelated factor 2 (Nrf2)
transcriptional factor, the master regulator of phase II antioxidant response, have
2
8
demonstrated high neuroprotection in different animal models.
Furthermore,
3
ꢀbutylphthalide (NBP), a component of celery oil that acts as an Nrf2 inducer, has
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found clinical use in China to treat ischemic stroke.
Taking into account these precedents, we have recently proposed the multitarget
combination of mNCX antagonism and Lꢀtype VDCC blockade, as an interesting
2
+
strategy to reꢀestablish the mCC reducing [Ca ]
c
overload, and antioxidant effect as
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complementary mechanisms of action to provide neuronal survival.
Under these premises, we report here the design, synthesis, and pharmacological
evaluation of a new CGP37157ꢀnimodipine hybrid, named ITH14001, endowed with
VDCC blocker and mNCX antagonist properties. Furthermore, calcium influx through
Lꢀtype VDCCs induces the production of free radicals and thus a moderate blockade of
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this channel is expected to mitigate oxidative stress occurring during the reoxygenation
phase in ischemia. The study of this compound as a calcium cycle regulator and
2+
neuroprotective agent in several models of [Ca ]
c
overload, oxidative stress, cerebral
ischemia and neuroinflammation is reported.
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RESULTS AND DISCUSSION
Rational design of a multitarget neuronal calcium stabilizer. Against the
aforementioned background, we undertook a rational pharmacophoreꢀdirected design
towards a multitarget calcium stabilizer. Our initial concept involved linking two
structurally active fragments, namely the benzothiazepine derivative CGP37157 to
interact with mNCX, and a 1,4ꢀdihydropyridine derivative to target the Lꢀtype VDCCs,
in order to intervene directly in the modulation of both the cellular and mCC. As the
CGPꢀ37157 structure did not offer many options to establish a link with another
molecule without affecting its activity, we selected as the mNCXꢀblocking partner a
benzodiazepinone analogue of the compound. This choice emerged from previous
structureꢀactivityꢀrelationships of mNCX inhibitors, which showed that simple
replacement of the sulphur atom at the 4ꢀposition by a nitrogen atom bearing a 2ꢀ
hydroxyethyl substituent afforded a compound with a comparable activity to the parent
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CGP37157. We envisaged that this dimethylene chain could act as the linker between
the benzodiazepine moiety and the dihydropyridine group, whereas the OH could be
used for the formation of an ester with one of the carbonyl functions located at Cꢀ3 or
Cꢀ5 of the dihydropyridine. On the other hand, we decided to use nimodipine as the
dihydropyridine component of our hybrid due to its wellꢀknown behaviour as a calcium
channel blocker, and also because one of its ester groups has a good structural similarity
with the planned linker (figure 1).
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Figure 1. Rational design of the multitarget neuronal calcium stabilizer ITH14001.
Chemistry. We started by synthesizing initially the azaꢀanalogue of CGP37157 via a
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modification of the literature method. As illustrated in Scheme 1, 2ꢀaminoꢀ2’,5ꢀ
dichlorobenzophenone 2 was condensed with 2ꢀaminoethanol to afford the imine 3 that
was subsequently reduced to the βꢀaminoalcohol 4. With this compound in hand, we
proceeded to the formation of the diazepine ring by its treatment with bromoacetyl
bromide in a oneꢀpot, multi step fashion, obtaining compound 5 as the result of the
reaction of the βꢀhydroxy group with a second equivalent of bromoacetyl bromide.
After subsequent hydrolysis of 5 by classical baseꢀpromoted saponification we obtained
the expected azaꢀanalogue of CGP37157 6 in an overall 47% yield.
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Scheme 1. Synthesis of the azaꢀanalogue of CGP37157 6. Reagents and conditions: (i)
2ꢀaminoethanol (neat), 140 ºC, 20 h; (ii) NaBH (CN), MeOH/AcOH, 0 ºC to room
3
temperature, 18 h; (iii) (a) bromoacetyl bromide, 0 ºC to room temperature, 17 h; (b)
DIEA, 50 ºC, 5 h; (iv) 2 M aqueous KOH, MeOH, room temperature, 2 h.
Based on our previous experience on the synthesis of bioactive dihydropyridine
26
derivatives we decided to synthesize the target compound in a multicomponent
fashion, employing a modified threeꢀcomponent Hantzsch dihydropyridine synthesis to
build the nimodipine fragment. Thus, we prepared on one hand, the βꢀketoester 7
employing a transesterification of methyl acetoacetate with compound 6 in the presence
®
32
of the acidic ion exchange resin Amberlyst 15 and, on the other hand, the conjugated
3
3
enone 8 by using the classical conditions for the Knoevenagel condensation. The final
threeꢀcomponent reaction between compounds 7, 8 and ammonium acetate in refluxing
ethanol afforded the desired hybrid ITH14001 in good yield (72%) (Scheme 2).
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Scheme 2. Multicomponent synthesis of the hybrid compound ITH14001. Reagents
and conditions: (i) Amberlistꢀ15, toluene, reflux, 15 h; (ii) Piperidine, AcOH, benzene,
reflux, 16 h; (iii) EtOH, reflux, 15 h.
Pharmacology.
Compound ITH14001 modulates both the L-type VDCCs and the mNCX
transporter. In order to determine the potential activity of our compound over the mCC
and Lꢀtype VDCCs we used primary cultures of bovine chromaffin cells (BCCs), a
wellꢀaccepted model of paraneuron where we established the percentage of VDCCsꢀ
34
subtypes expressed by these cells. As shown in figure 2, ITH14001 did not change the
2+
2+
[
Ca ]
c c
peak in BCCs (figure 2A). Nevertheless, it altered the kinetics of [Ca ]
clearance at 10 µM, augmenting the time constant (τ) of the slow component of the
2+
[
Ca ] clearance from 32.9 ± 2.8 s to 52.6 ± 5.1 s (p < 0.005) (figure 2B). This result
c
2+
shows that although ITH14001 does not block the Ca entry through VDCCs in BCCs,
2+
c
it mitigates the clearance of [Ca ] by mitochondria, suggesting that it might block the
3
5
mNCX. Furthermore, ITH14001 does not alter the VDCCs currents in BCCs as shown
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in figure 2C; this correlates with the observation that the compound did not alter the
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c
peak [Ca ] transient generated by K in furaꢀ2ꢀloaded BCCs (figure 2A).
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Figure 2. A) Representative traces of [Ca ]
c
evoked by K pulses (5 s, 70 mM) in
2
+
BCCs. [Ca ]
c
measurements were done with Furaꢀ2 AM dye (1 h incubation, 10 µM).
P2 and P3 represent the second and the third pulse under compound (ITH14001, 10 µM)
incubation. B) Quantification of both (fast and slow) clearance time constant of the
2+
[
c
Ca ] peaks represented in A. C) Intensityꢀvoltage (IꢀV) calcium current curve elicited
by 50ꢀms depolarizing pulses in BCCs under the perforated patch configuration. IꢀV
curves were performed perfusing the cells with tyrode medium (Control) or tyrode with
ITH14001 (10 µM). D) Quantification of the calcium current peak (ICa) in BCCs or in
embryo chromaffin cells (ECCs) without (control) or with ITH14001 treatment in BCCs
(3 µM) and in ECCs (10 µM). Data are mean ± SEM of triplicates of four independent
experiments: *p < 0.05 compared with control; **p < 0.01, compared with control
conditions.
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To further analyze whether the effect of ITH14001 is specific over mNCX, the
voltageꢀdependent calcium currents in chromaffin cells from rat embryo (ECC) were
measured. The rat ECCs is an excellent model to study the Lꢀtype VDCC due to its high
34b
expression in this type of cells. In ECCs current through Lꢀtype channel corresponds
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to 60 % of the total, differently from BCCs where it only accounts for 20 %. In this
case, the electrophysiological experiments showed a significant blockade of the peak ICa
(
186.1 ± 27.6 pA) in the control vs. under ITH14001 preꢀincubation (105.0 ± 6.0 pA,
figure 2D) (p < 0.05). Therefore, we can conclude that ITH14001 modulates calcium
signalling by blocking specifically the mNCX and also the Lꢀtype VDCC. Given its
relatively low potency to target Lꢀtype VDCCs, it is expected that compound ITH14001
would have less hypotensive effects compared classical dihydropyridines which are
known to have a potent vasodilatory effect.
2
+
As described, the ischemic cascade starts with the mitochondrial failure and [Ca ]
c
overload followed by intensive oxidative stress damage and glial activation during the
reoxygenation phase. The dual mechanism of action of ITH14001 reduces the initial
2+
[
Ca ] overload acting over the mNCX and Lꢀtype VDCCs. This target combination
c
2+
helps to mitigate futile mCC and [Ca ] overload favouring mitochondria stabilization
c
and energy recovery. The moderate activity over Lꢀtype VDCCs might also be of
interest since it can reduce secondary effects over the cardiovascular system, an effect
2
6
related to the failure of nimodipine in clinical trials for cerebral ischemia.
ITH14001 exerts neuroprotective effects against the toxicity elicited by [Ca ]
overload. We next evaluated the potential neuroprotective effect of compound
2+
c
2
+
+
ITH14001 in two different cytotoxic models related to [Ca ]
c
overload; a) high [K ]
and b) veratridine. First we used the SHꢀSY5Y neuroblastoma cell line exposed to a
+
2+
c
depolarizing concentration of K during 24 h, which causes [Ca ] overload and
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mitochondrial dysfunction inducing a 45 % cell death. Cells were coꢀincubated with
increasing concentrations of ITH14001 or reference compounds (nimodipine and
+
CGP37157) and high K (70 mM) during 24 h. Thereafter, cell viability was assayed by
37
the MTT method. Our hybrid compound exerted neuroprotection at 10 ꢀM (25 %
protection) and 30 ꢀM (60 % protection) (p < 0.001). Compared to reference
compounds, nimodipine and CGP37157, with neuroprotective effects of 47 and 40 %,
respectively, ITH14001 showed a higher neuroprotective effect at 30 ꢀM (figure 3),
although the differences were not significant.
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Figure 3. Neuroprotective effect of ITH14001 against [Ca ]
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overload: A)
+
neuroprotective effect of ITH14001 against the cytotoxic effect induced by high K (70
mM). Cells were coꢀincubated with increasing concentrations of ITH14001 or control
+
compounds (nimodipine and CGP37157, 30 ꢀM) and high K during 24 h. B)
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Cytoprotective effect against the toxicity elicited in rat hippocampal slices by
veratridine (30 ꢀM) during 3.5 h alone or coꢀincubated with the indicated treatments.
Data are expressed as mean ± SEM of four different experiments in triplicate. ***p <
###
#
0
.001 compared with basal conditions; p < 0.001 and p < 0.05 compared with the
corresponding toxic stimulus.
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The study of the neuroprotective profile of hybrid ITH14001 against [Ca ] overload
c
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was completed using veratridine as toxic stimulus, in rat hippocampal slices. This
+
2+
+
c c
alkaloid induces [Na ] /[Ca ] overload by blocking Na channels inactivation, leading
+
2+
to mitochondria being unable to modulate Na and Ca and thus implicating the
mNCX. Using a protocol previously described by our group, we employed 30 ꢀM of
1
8a
veratridine during 3.5 h that induced 38 % of cell death (figure 3B). In this model,
1
8a
CGP37157 achieved maximum protection at 30 ꢀM and thus we used CGP37157 and
nimodipine at this concentration for comparative purposes. ITH14001 was used at 10
ꢀ
M as it was able to protect almost at the same extent of that achieved with 30 ꢀM in
the previous model. As shown in figure 3B, our hybrid derivative was able to induce a
6 % of neuroprotection, a value very close of that achieved by CGP37157 (31 %) at 30
M with no significant differences. Nimodipine showed a very potent neuroprotective
2
ꢀ
effect being able to rescue 76 % of viability at 30 ꢀM.
Compound ITH14001 exerts antioxidant properties. Among the different
alterations observed in brain ischemia, oxidative stress and neuroinflammation are two
2
+
key elements that trigger neuronal death. The uncontrolled entry of Ca through the Lꢀ
10
type VDCCs has been recently related directly to an increase of ROS, and thus the
moderate blockade of Lꢀtype VDCCs by hybrid derivative ITH14001 might improve its
neuroprotective effect against oxidative stress. Therefore, we used the rotenone and
oligomycin A combination (rote/olig) in the SHꢀSY5Y neuroblastoma cell line as
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oxidative stress model to test this hypothesis. The toxic combination increases ROS
production, damaging mitochondria, reducing ATP production and causing cell death
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simulating the pathological events occurring in cerebral ischemia. As shown in figure
4A, rote/olig caused 38 % of cell death, which was prevented by ITH14001 in a
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concentration dependent manner from 0.3 ꢀM to 30 ꢀM. In this case, melatonin and
nimodipine were included as positive control and reference compound. CGP37157 was
not included since it is toxic at the concentration of 30 ꢀM in SHꢀSY5Y neuroblastoma
cells (see supporting information) and it was unable to protect in this oxidative stress
1
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model.
protection at 0.3 and 1 ꢀM, and higher neuroprotective effect was achieved at 10 ꢀM (p
0.05) and 30 ꢀM (p < 0.01). Compared to nimodipine, hybrid derivative ITH14001
Compared to positive control melatonin, ITH14001 showed similar
<
showed an improved neuroprotective effect (figure 4A).
In view of the protective effect against oxidative stress we envisaged the possibility of
an additional scavenger effect in our hybrid. We first used the oxygen radical
absorbance capacity (ORAC) assay to test the ROS scavenging effect, but our
compound was inactive in this assay (data not shown). We then submitted the
compound to the α,αꢀdiphenylꢀβꢀpicrylhydrazyl (DPPH) assay, in which the radicals
generated are RNS. Interestingly, ITH14001 showed a good activity in this assay, being
two times more potent than melatonin at the same concentration as a RNS scavenger (p
< 0.05) (figure 4B).
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Figure 4. Neuroprotective effect against oxidative stress and DPPH reduction effect of
ITH14001: A) protective effect of increasing concentrations of ITH14001 against the
toxicity induced by rotenone/oligomycin A (30/10 ꢀM) with a coꢀincubation protocol
during 24 h. Data are expressed as means ± SEM of five different experiments in
###
#
triplicate. ***p < 0.001 compared to basal conditions; p < 0.001 and p < 0.05
&&
&
compared to toxic stimulus. p < 0.01 and p < 0.05 compared to melatonin. . B)
Scavenger effect of ITH14001 in the DPPH assay compared to the antioxidant
neurohormone melatonin. Values are expressed as percentage of DPPH radical
reduction as mean ± SEM of four different experiments in triplicate. *p < 0.05
compared with melatonin 1 mM.
ITH14001 reduces the toxicity elicited by glutamate and oxygen and glucose
deprivation in brain hippocampal slices. As summarized above, glutamateꢀinduced
2+
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excitotoxicity is an important process causing neuronal death through [Ca ] overload
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and mitochondrial failure in cerebral ischemia. In fact, one of the most widely used in
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vitro models of stroke is the glutamateꢀinduced toxicity in rat hippocampal slices. As
2+
we have demonstrated, ITH14001 exhibits Ca regulatory activity and antioxidant
properties and thus we considered of interest to explore its potential protective effect in
this neurotoxicity model. The experimental protocol used is described in the upper part
of figure 5A; after the stabilization period (45 min, 34 ºC), rat hippocampal slices were
incubated with glutamate alone (1 mM), or coꢀincubated with increasing concentrations
of ITH14001 (1, 10 and 30 ꢀM) or control compounds, nimodipine (30 ꢀM) and
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CGP37157 (30 ꢀM), during 4 h at 37 ºC. Under these conditions glutamate induced
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5 % reduction of cellꢀviability (measured by MTT reduction) (figure 5A), a value
43
similar to that previously reported. Interestingly, ITH14001 reduced this neuronal
lesion at all concentrations assayed, achieving a 25%, 76% and 60% neuroprotection at
1, 10 and 30 ꢀM, respectively. The parent compounds nimodipine (68 % protection)
and CGP37157 (67 % protection), used at 30 ꢀM exhibited an effect similar to that
achieved by ITH14001 at 3ꢀfold lower concentration (10 ꢀM, 76 % protection).
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Figure 5. ITH14001 protected rat hippocampal slices against the excitotoxicity elicited
by glutamate and the toxicity induced by OGD/reox. A) After the stabilization period
(45 min), slices were incubated with glutamate (1 mM) alone or coꢀincubated with the
compounds during 4 h. Thereafter, viability was determined by the MTT method. B)
After 45 min stabilization slices were subjected to OGD (15 min) followed by
reoxygenation (2 h). Treatments were incubated after the OGD period, during the
reoxygenation phase. Thereafter, viability was determined by the MTT method. Data
are expressed as mean ± SEM of five different experiments in triplicate. ***p < 0.001
##
#
compared with basal conditions; p < 0.01 and p < 0.05 compared with the
&
correspondent toxic stimulus. p < 0.05 compared with CGP37157.
The pharmacological profile and neuroprotective properties of the ITH14001
prompted us to use a more physiological brain ischemia model. We selected the oxygen
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and glucose deprivation (OGD) followed by reoxygenation (reox) model in rat
hippocampal slices. In this model, the OGD period (15 min) induces mitochondrial
2+
depolarization, glutamate release and [Ca ]
c
overload. During the reoxygenation phase,
44
there is a massive release of ROS and RNS. We have selected a postꢀOGD incubation
protocol to better reproduce the clinical condition where patients are treated after the
brain ischemic episode has occurred. Rat hippocampal slices subjected to 15 min of
OGD followed by 2 h of reox caused 40 % cell death (p < 0.001), whereas the postꢀ
OGD treatment with increasing concentrations of ITH14001 afforded 31 % (p < 0.05)
and 34 % (p < 0.05) neuroprotection at 10 and 30 ꢀM, respectively. Interestingly, the
neuroprotective effect exerted by ITH14001 was not observed for the parent
compounds, CGP37157 and nimodipine, at the concentration of 10 ꢀM, showing a
statistically significant (p < 0.05) better neuroprotection than both at this concentration.
The neuroprotection values for both parent compounds are in line with results
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previously described in our group.
ITH14001 reduces ROS production and iNOS over-expression induced in the
OGD/reox ischemia model. As previously described, increased ROS production and
neuroinflammation are two of the bestꢀdescribed alterations occurring after brain
45
ischemic episode. OGD/reox causes an increase of 95 % of ROS production in
hippocampal slices, measured as H DCFDA increase of fluorescence (figure 6A and
C). In slices treated with ITH14001 (10 ꢀM), ROS levels were significantly reduced
almost to basal levels (p< 0.001). This reduction might be related to its ability to
2
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2+
2+
modulate Ca currents (figure 1), reducing Ca overload, which stabilize the
mitochondria. Additionally, the partial blockade of Lꢀtype VDCC (figure 1) might help
to reduce oxidative stress. Furthermore, our compound can trap RNS thereby reducing
46
nitrosative damage (figure 4B).
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Figure 6. ITH14001 reduces ROS production and iNOS expression in rat hippocampal
2
slices after OGD/reox. ROS production measured as H DCFDA fluorescence intensity
increase after OGDꢀreox period. Slices were subjected to the same OGD/reox protocol
loaded with the fluorescent dye during the stabilization period. Compound ITH14001
was incubated at 10 ꢀM after the 15 min OGD period, during the reox phase. A)
Representative microphotographs of ROS production and C) ROS production
quantification. Westernꢀblot analysis of iNOS expression in hippocampal slices
subjected to OGD/reox alone or incubated postꢀOGD with ITH14001 (10 ꢀM). B)
Representative western blot traces and D) iNOS expression quantification. Data are
expressed as mean ± SEM of five different experiments in quadruplicate. ***p < 0.001
##
#
compared with basal conditions; p < 0.005 and p < 0.05 compared with the
corresponding toxic stimulus.
In an effort to study the neuroinflammatory component of the OGD/reox model in
hippocampal slices, we measured the induction of inducible nitric oxide synthase
(iNOS) enzyme occurring during the OGD/reox. As shown in figure 6B and 6D,
OGD/reox doubled the expression of iNOS in rat hippocampal slices (p < 0.05). Postꢀ
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OGD treatment with ITH14001 (10 ꢀM) completely prevented the deleterious overꢀ
expression of this proꢀinflammatory enzyme (p < 0.005). Thus, ITH14001 was able to
reduce neuroinflammation occurring during ischemia, a complementary activity to its
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+
Ca regulation and antioxidant properties. In the aggregate, these properties are an
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interesting multitarget combination to prevent brain ischemia damage.
ITH14001 is able to induce the phase II antioxidant and anti-inflammatory
2+
response in primary glial cultures. Together with the Ca cycling regulation and the
antioxidant activity, we were interested in further demonstrating the potential antiꢀ
inflammatory activity of compound ITH14001 as part of its mechanism of action.
ITH14001 was able to reduce OGD/reox damage by reducing ROS production (figure
5B and 6A) and iNOS expression (figure 6B), the latter result indicating a potential
direct effect over the activation of glial cells. In this line, the protection afforded by our
compound against oxidative stress might be related not only to its calcium regulation
and antioxidant activities, but also to an increase in the antioxidant capability of the cell.
The Nrf2 factor is the master regulator of the phase II antioxidant and antiꢀinflammatory
response, and small compounds can induce its activity to trigger a neuroprotective
28
response against oxidative stress. Among others, Nrf2 induces the expression of the
antioxidant and antiꢀinflammatory enzyme hemeꢀoxygenaseꢀ1 (HOꢀ1) and the catalytic
subunit (GCLc) of glutathioneꢀCꢀligase, the enzyme that catalyses the limiting step of
the de novo synthesis of glutathione. On the other hand, it has been recently
demonstrated that nimodipine is able to protect against oxidative stress by moderately
47
inducing the Nrf2 response. Thus, we tested the possibility of Nrf2 induction by
ITH14001 compared with CGP37157 and nimodipine at the same concentration (30
ꢀM) (Nrf2 induction doseꢀresponse curves of all compounds are included in the
Supporting Information). Figure 7A shows that compound ITH14001 was able to
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significantly (p < 0.001) increase luciferase expression by 2.1 fold. This Nrf2ꢀinduction
ability was significantly (p < 0.05) higher than that observed for nimodipine (1.9 fold
induction). In this model, GGP37157 was completely unable to induce the Nrf2ꢀARE
response at this concentration, being significantly less potent than our hybrid derivative
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(
p < 0.001) (figure 7A).
ROS and RNS overꢀproduction are related to the increase in inflammatory cytokines
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observed after brain ischemia. After demonstrating the antioxidant effect and Nrf2
induction capability of hybrid ITH14001, we were interested in elucidating its potential
antiꢀinflammatory properties. For this purpose, primary glial cell cultures were exposed
during 18 h to 1 ꢀg/ml of lipopolysaccharide (LPS), which is widely used to cause an
inflammatory response. Under these experimental conditions, the production of nitrites
increased four times compared to basal conditions (figure 7B) (p < 0.005). Treatment
with 1 and 10 ꢀM of ITH14001 did not offer any antiꢀinflammatory effect; however, at
3
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This antiꢀinflammatory activity complements the multitarget profile of our hybrid
derivative, making it particularly attractive against brain ischemia.
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in AREc32 and glial cells. A) Nrf2 induction in AREc32 cells by ITH14001,
nimodipine and CGP37157 at 30 ꢀM. Luciferase relative activity was normalized to
control conditions assigning the value of 1. Data are expressed as mean ± SEM of
duplicates of five different experiments. ***p < 0.001 compared to basal conditions;
###
#
p < 0.001 and p < 0.05 compared to ITH14001. B) Reduction of nitrite production
by compound ITH14001 upon stimulation of primary glial cultures with LPS (1
ꢀg/mL). Data were normalized to basal conditions (100 %). Data are expressed as mean
±
SEM of five different experiments in triplicate. **p < 0.005 compared with basal
#
conditions; p < 0.05 compared with LPSꢀinduced nitrite production. C) Westernꢀblot
analysis of HOꢀ1 and GCLc enzymes induced by ITH14001 (30 ꢀM) in glial cells
primary cultures. D) Quantification of HOꢀ1 and E) GCLc overꢀexpression normalized
to basal conditions induced by ITH14001 (30 ꢀM). Data are expressed as mean ± SEM
of four different cultures. **p < 0.01 and *p < 0.05 compared with basal conditions.
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Additionally, we investigated the Nrf2ꢀinduction properties in glial cells as a potential
mechanism of action to reduce oxidative stress and neuroinflammation. Thus, we
measured the expression of HOꢀ1 and GCLc in primary glial cultures, which would be
involved in the antioxidant and antiꢀinflammatory effects of ITH14001. Mixed glial
cells primary cultures were preꢀtreated with ITH14001 (30 ꢀM) during 24 h. After this
period, glial cells were processed to measure protein expression by westernꢀblot (figure
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7C). ITH14001 induced by 3ꢀfold HOꢀ1 enzyme (p< 0.01) (figure 7D) and by 40 %
GCLc (p< 0.05) (figure 7E), compared to nonꢀtreated cells. Therefore, the extra
antioxidant and antiꢀinflammatory effects observed with ITH14001 can be related, at
least in part, to the induction of Nrf2 and the increased expression HOꢀ1 and GCLc
enzymes.
Finally, the new compound has shown no hepatotoxicity or neurotoxicity up to 30 ꢀM
(figure 8C and 7D) revealing a better safety profile compared to CGP37157, which was
significantly toxic at 30 ꢀM (figure 8A).
A
B
SH-SY5Y
00
HepG2
1
100
50
0
50
***
0
Basal
1
3
10
30
Basal
1
3
10
30
CGP37157 (
ꢀ
M)
CGP37157 (ꢀM)
C
D
SH-SY5Y
00
HepG2
1
100
5
0
50
0
0
Basal
1
3
10
M)
30
Basal
1
3
10
M)
30
ITH14001 (
ꢀ
ITH14001 (
ꢀ
Figure 8: Cytotoxicity elicited by compound ITH14001 and CGP37157 in the
neuroblastoma cell line SHꢀSY5Y and hepatotoxicity in the hepatocarcinoma HepG2
2
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cells. Viability was measured as MTT reduction in presence of increasing
concentrations of compounds. Data are expressed as mean ± SEM of four different
experiments in triplicate. ***p < 0.001 compared to basal conditions.
0
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CONCLUSIONS
In conclusion, compound ITH14001, designed as a hybrid of CGP37157 and
nimodipine, caused neuroprotection on in vitro cellular and tissue slices models related
to cerebral ischemia. In this report we demonstrate its activity over mNCX (figure 1)
and VDCC being a moderate blocker. Both activities are related to the neuroprotection
2+
c
exerted by compound ITH14001 against [Ca ] overload, being of interest in the first
phase of ischemic damage. We also describe its antioxidant properties including RNS
scavenger and Nrf2 induction capability that might be related to its neuroprotective
activity against oxidative stress. Finally, we have also demonstrated its antiꢀ
inflammatory profile in primary glial cultures, which represent and improvement
compared to its parent compounds. The plethora of activities demonstrated by hybrid
ITH14001 are of high interest for the treatment of cerebral ischemia since this
pathological condition exhibit a complex combination of pathological pathways related
2
+
to Ca dyshomeostasis, oxidative stress and neuroinflammation. Taken together,
ITH14001 is a multitarget compound of potential interest for the treatment of brain
ischemia conditions.
METHODS
Chemistry. All reagents (Aldrich, Fluka, SDS, Probus) and solvents (SDS), were of
commercial quality and were used as received. Reactions were monitored by thin layer
chromatography, on aluminium plates coated with silica gel with fluorescent indicator
(SDS CCM221254). Separations by flash chromatography were performed on silica gel
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(SDS 60 ACC 40ꢀ63 ꢁm) or neutral alumina (Merck S22). Melting points were
measured on a Reichert 723 hot stage microscope, and are uncorrected. Infrared spectra
were recorded on an Agilent Cary630 FTIR spectrometer with a diamond ATR
accessory for solid and liquid samples, requiring no sample preparation. NMR spectra
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1
were obtained on a Bruker Avance 250 spectrometer operating at 250 MHz for H and
13
63 MHz for C (CAI de Resonancia Magnética Nuclear, Universidad Complutense).
NMR chemical shifts marked as * show that the assignment can be interchanged
between two or more magnetic nuclei with similar chemical shifts. Elemental analyses
were determined by CAI de Microanalisis Elemental, Universidad Complutense, using a
Leco 932 CHNS combustion microanalyzer. Experimental procedures and
characterization data for new compounds follow. For full experimental procedures and
characterization data, see the Supporting Information.
Synthesis
benzo[ ][1,4]diazepin-4-yl)ethyl 2-bromoacetate (5). To a stirred solution of 4 (2.116
g, 6.8 mmol) in 35 mL of CH Cl at 0 °C was added bromoacetyl bromide (1.48 mL,
of
2-(7-chloro-5-(2-chlorophenyl)-2-oxo-2,3,4,5-tetrahydro-1H-
e
2
2
17.0 mmol), and stirring was continued at 0 °C for 1 hour and then at room temperature
for 16 h. Diethylamine (17.4 mL, 100.0 mmol) was added, and the mixture was stirred
at 50 °C for 5 h. The mixture was cooled to room temperature and diluted with CH
2 2
Cl ;
the CH Cl solution was washed with water and dried over anhydrous magnesium
2
2
sulphate. After filtration, the filtrate was concentrated to dryness, and the crude product
was purified using silica gel column chromatography (hexane:AcOEt 100/0 to 60/40) to
give 5 as a white solid (1.894 g, 59%). Mp: 148ꢀ149 °C. IR (neat) ν 2948, 1733, 1664,
ꢀ1 1
1
025 cm . H NMR (250 MHz, CDCl
3.00ꢀ3.15 (m, 1H, one H of CH ꢀβ), 3.47 (d, J = 15.5 Hz, 1H, one H of CH
J = 15.5 Hz, 1H, one H of CH ꢀ3), 3.85 (s, 2H, CH ꢀ2), 4.14ꢀ4.29 (m, 1H, one H of
3
) δ (ppm) 2.84ꢀ3.00 (m, 1H, one H of CH
2
ꢀβ),
2
2
ꢀ3), 3.58 (d,
2
2
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CH ꢀα), 4.33ꢀ4.49 (m, 1H, one H of CH ꢀα), 5.31 (s, 1H, CHꢀ5), 7.05 (d, J = 8.5 Hz,
2
2
1H, CHꢀ9), 7.25 (dd, J = 8.3, 2.2 Hz, 1H, CHꢀ8), 7.45ꢀ7.29 (m, 4H, CHꢀ6, CHꢀ4’, CHꢀ
13
5
’ and CHꢀ6’), 7.60 (dd, J = 7.3, 1.5 Hz, 1H, CHꢀ3’), 9.12 (s, 1H, CONH). C NMR
(63 MHz, CDCl
3
) δ (ppm) 172.39 (Cꢀ2=O), 167.45 (Cꢀ1”=O), 137.26 (Cꢀ1’*), 137.02
0
1
2
3
4
5
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1
2
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4
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(Cꢀ5a*), 134.90 (Cꢀ9a), 132.78 (Cꢀ6’*), 131.14 (CHAr), 130.93 (Cꢀ7*), 130.68 (CHAr),
130.50 (CHAr), 129.93 (CHAr), 129.34 (CHAr), 127.52 (CHAr), 122.56 (CHAr), 65.45
(CHꢀ5), 64.17 (CH ꢀα), 53.23 (CH ꢀβ), 52.30 (CH ꢀ3), 26.18 (CH ꢀ2”). Anal. Calcd.
2 2 2 2
(%) for C19
2 2 3
H17BrCl N O : C: 48.33, H: 3.63, N: 5.93; found: C: 47.96, H: 3.48, N: 6.06.
Synthesis
benzo[
of
2-(7-chloro-5-(2-chlorophenyl)-2-oxo-2,3,4,5-tetrahydro-1H-
e
][1,4] diazepin-4-yl)ethyl 3-oxobutanoate (7). A mixture of methyl
acetoacetate (0.332 g, 2.85 mmol), 6 (1.053 g, 3 mmol) and Amberlystꢀ15 (10% by
weight of βꢀketoester, 0.033 g) in toluene (10 mL) was heated to 110 °C in a flask
provided with a distillation condenser (DeanꢀStark) to remove MeOH. After completion
of the reaction, monitored by TLC, the catalyst was filtered off and the filtrate was
concentrated and purified using silica gel column chromatography (CH
2 2
Cl :AcOEt from
100:0 to 75:25) to give 7 as a pale brown solid (0.640 g, 49%). Mp 68ꢀ69 °C. IR (neat)
ꢀ1
1
ν 2918, 1712, 1665, 1034 cm . H NMR (250 MHz, CDCl
”), 2.80ꢀ2.96 (m, 1H, one H of CH ꢀβ), 2.97ꢀ3.11 (m, 1H, one H of CH
(m, 4H, CH ꢀ3 and CH ꢀ2”), 4.12ꢀ4.28 (m, 1H, one H of CH ꢀα), 4.28ꢀ4.42 (m, 1H, one
H of CH ꢀα), 5.29 (s, 1H, CHꢀ5), 6.61 (d, J = 2.3 Hz, 1H, CHꢀ6), 6.98 (d, J = 8.5 Hz,
H, CHꢀ8), 7.18ꢀ7.47 (m, 4H, CHꢀ9, CHꢀ4’, CHꢀ5’ and CHꢀ6’), 7.54 (dd, J = 7.2, 2.2
3
) δ (ppm) 2.27 (s, 3H, CH
3
ꢀ
4
2
2
ꢀβ), 3.36ꢀ3.67
2
2
2
2
1
13
Hz, 1H, CHꢀ3’), 8.38 (s, 1H, CONH). C NMR (63 MHz, CDCl
”=O), 172.62 (Cꢀ2=O), 167.38 (Cꢀ1”=O), 137.35 (Cꢀ1’*), 136.93 (Cꢀ5a*), 134.86 (Cꢀ
9a), 132.80 (Cꢀ2’*), 131.10 (CHAr), 130.86 (Cꢀ7*), 130.67 (CHAr), 130.50 (CHAr),
29.89 (CHAr), 129.29 (CHAr), 127.49 (CHAr), 122.55 (CHAr), 65.42 (CHꢀ5), 63.33
3
) δ (ppm) 200.88 (Cꢀ
3
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(CH ꢀα), 53.24 (CH ꢀβ), 52.34 (CH ꢀ3), 50.35 (CH ꢀ2”), 30.66 (CH ꢀ4”). Anal. calcd
2
2
2
2
3
(
%) for C H Cl N O : C: 57.94, H: 4.63, N: 6.44; found: C: 57.82, H: 4.78, N: 6.33.
21
20
2
2
4
Synthesis
benzo[
of
3-(2-(7-chloro-5-(2-chlorophenyl)-2-oxo-2,3,4,5-tetrahydro-1H-
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e
][1,4]diazepin-4-yl)ethyl) 5-isopropyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-
dihydropyridine-3,5-dicarboxylate 1 (ITH14001). A solution of 7 (192 mg, 0.44
mmol) and ammonium acetate (68 mg, 0.88 mmol) in ethanol (2 mL) was stirred for 30
min at room temperature; 8 (122 mg, 0.44 mmol) was then added and the mixture was
heated under reflux and stirred for 15 hours. After this time, the mixture was diluted
2 2
with CH Cl (20 mL), washed with water followed by brine, and dried over anhydrous
magnesium sulfate. Evaporation of the solvent under reduced pressure left a crude that
was purified using silica gel column chromatography (dichlorometane:ethyl acetate
from 100:0 to 90:10) to give ITH14001 as a light yellow solid (218 mg, 72%). Mp: 149ꢀ
ꢀ1
1
1
50°C. IR (neat) ν 1675, 1526, 1348, 1101 cm . H NMR (250 MHz, CDCl
δ (ppm) 1.06 (d, J = 6.2 Hz, 3H, OCH(CH one isomer), 1.07 (d, J = 6.2 Hz, 3H,
OCH(CH one isomer), 1.22 (d, J = 6.0 Hz, 3H, OCH(CH one isomer), 1.24 (d, J =
.8 Hz, 3H, OCH(CH ) one isomer), 2.32 (s, 6H, Cꢀ6CH two isomers), 2.37 (s, 3H, Cꢀ
3
) 2 isomers
3 2
)
)
3 2
3 2
)
5
2
3
2
3
CH
3
one isomer) 2.38 (s, 3H, Cꢀ2CH
ꢀ3’ two isomers), 4.06ꢀ4.14 (m, 2H, CH
ꢀα one isomer), 4.89ꢀ5.00 (m, 2H, OCH(CH
3
one isomer), 2.77ꢀ3.04 (m, 4H, CH
2
ꢀβ, two
ꢀα one
two
isomers), 3.35ꢀ3.56 (m, 4H, CH
2
2
isomer), 4.23ꢀ4.39 (m, 2H, CH
2
3
)
2
isomers), 5.06 (s, 1H, CHꢀ4 one isomer), 5.12 (s, 1H, CHꢀ4 one isomer), 5.16 (s, 1H,
CHꢀ5’ one isomer), 5.28 (s, 1H, CHꢀ5’ one isomer), 5.73 (bs, 2H, NHꢀ1 two isomers),
6
.43 (d, J = 2.0 Hz, 1H, NHꢀ1’ one isomer), 6.62 (d, J = 2.1 Hz, 1H, NHꢀ1’ one isomer),
.94 (d, J = 5.5 Hz, 1H, ArH one isomer), 6.98 (d, J = 5.5 Hz, 1H, ArH one isomer),
6
7.16ꢀ7.39 (m, 11H, ArH two isomers), 7.52ꢀ7.55 (m, 1H, ArH one isomer), 7.64 (d, J =
.5 Hz, 2H, ArH two isomers), 7.92ꢀ7.97 (m, 2H, ArH two isomers), 8.23ꢀ8.04 (m, 4H,
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3
ArH two isomers). C NMR (63 MHz, CDCl ) 2 isomers δ (ppm) 172.35 (Cꢀ2’=O one
3
isomer), 171.83 (Cꢀ2’=O one isomer), 167.26 (Cꢀ5 C=O* one isomer), 167.22 (Cꢀ5
C=O* one isomer), 166.96 (Cꢀ3 C=O* one isomer), 166.91 (Cꢀ3 C=O* one isomer),
1
50.28 (Cꢀ6* one isomer), 150.22 (Cꢀ6* one isomer), 148.53 (Cꢀ2* one isomer), 148.47
Cꢀ2* one isomer), 145.64 (CNO * one isomer), 145.45 (CNO * one isomer), 144.84
(CAr* one isomer), 144.78 (CAr* one isomer), 137.46 (Cꢀ1”* one isomer), 137.30 (Cꢀ
”* one isomer), 137.02 (Cꢀ5a* one isomer), 136.82 (Cꢀ5a* one isomer), 135.01 (CHAr
0
1
2
3
4
5
6
7
8
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0
1
2
3
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(
2
2
1
one isomers), 134.95 (CHAr one isomer), 134.82 (Cꢀ9a* one isomer), 134.81 (Cꢀ9a*
one isomer), 133.01 (Cꢀ2”’* one isomer), 132.75 (Cꢀ2”* one isomer), 131.05 (CHAr
one isomer), 130.96 (Cꢀ7 one isomer), 130.77 (CHAr + Cꢀ7 one isomer), 130.64 (CHAr
one isomer), 130.55 (CHAr one isomer), 130.50 (CHAr one isomer), 130.33 (CHAr one
isomer), 129.75 (CHAr one isomer), 129.67 (CHAr one isomer), 129.22 (CHAr one
isomer), 128.99 (CHAr one isomer), 128.94 (CHAr one isomer), 127.50 (CHAr one
isomer), 127.30 (CHAr one isomer), 123.59 (CHAr one isomer), 123.53 (CHAr one
isomer), 122.46 (CHAr two isomers), 121.76 (CHAr two isomers), 104.24 (Cꢀ5 two
isomers), 103.47 (Cꢀ3 one isomer), 103.29 (Cꢀ3 one isomer), 67.84 (OCH(CH ) two
3
2
isomers), 65.35 (CHꢀ5’ one isomer), 65.07 (CHꢀ5’ one isomer), 61.74 (CH ꢀα one
2
isomer), 61.15 (CH
2
ꢀα one isomer), 53.30 (CH
ꢀ3’ two isomers), 40.41 (CHꢀ4 one isomer), 40.33 (CHꢀ4 one
two isomers OCH(CH one isomer), 22.19 (Cꢀ2 CH one
isomer), 20.37 (OCH(CH ) one isomer), 20.21 (OCH(CH ) one isomer), 20.04
2 2
ꢀβ one isomer), 52.68 (CH ꢀβ one
isomer), 52.48 (CH
2
isomer), 22.52 (Cꢀ6 CH
3
3
)
2
3
3
2
3 2
+
(
OCH(CH ) two isomer). MS (TOFꢀpos) m/z calcd for C H Cl N O [M+H]
3 2 35 34 2 4 7
6
2 4 7
93.187, found 693.042. Anal. calcd (%) for C35H34Cl N O : C: 60.61, H: 4.94, N: 8.08;
found: C: 60.23, H: 4.94, N: 8.10.
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Pharmacology. DMEM/F12, rotenone, oligomycin A, LPS (lipopolysaccharide),
NEDA (Nꢀ(1ꢀNaphthyl)ethylenediamine dihydrochloride), DAPSONE (4,4´ꢀdiaminoꢀ
diꢀphenylsulfone) were obtained from Sigma (Madrid, Spain). 2,7ꢀDichlorofluorescein
2
diacetate (H DCFDA) was purchased from Molecular Probes (Invitrogen, Madrid,
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Spain). Penicillin, pyruvate, and 5 % heatꢀinactivated fetal bovine serum (FBS),
DMEMꢀglutamax, were purchased from Invitrogen. Luciferase Assay System (E1500)
was purchased from Promega (Madison, WI, USA).
Animals. P3ꢀ10 or 3ꢀmonths (300 g) male SpragueꢀDawley (SD) rats were housed
under controlled temperature and lighting conditions. Food and water were provided ad
libitum. All the efforts were made to minimize number of animals and animal suffering.
All the performed procedures were carried out following the Guide for the Care and Use
of Laboratory Animals, which were preꢀapproved by the ethics committees for the care
and use of animals in research of the Universidad Autónoma de Madrid, Spain, and
according to the European Guidelines for the use and care of animals for research in
accordance with the European Union Directive of 22 September 2010 (2010/63/UE) and
with the Spanish Royal Decree of 1 February 2013 (53/2013).
Culture of Bovine Chromaffin cells. Bovine chromaffin cells (BCCs) were isolated
49
from calves adrenal glands, following standard methods. Cells were suspended in
culture medium (DMEM) supplemented with fetal calf serum (5 %), cytosine
arabinoside (10 µM), fluorodeoxyuridine (10 µM), streptomycin (50 µg/ml) and
penicillin (50 IU/ml). Cells were plated on 12ꢀmm diameter glass coverslips at low
5
density (1.0 x 10 cells/coverslip) for patchꢀclamp studies. To study the changes of
2
+
5
[
Ca ]
c
, cells density was 5 x 10 cells/coverslip (25ꢀmm diameter glass coverslips).
Primary cells cultures were kept in an incubator at 37°C, waterꢀsaturated and in a 5 %
CO
2
ꢀ95 % air atmosphere. Experiments were carried out 24 h thereafter.
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