New 5-unsubstituted dihydropyridines with improved CaV1.3 selectivity as potential neuroprotective agents against ischemic injury

Article abstract of DOI:10.1021/jm500263v

C₅-unsubstituted-C₆-aryl-1,4-dihydropyridines were prepared by a CAN-catalyzed multicomponent reaction from chalcones, β-dicarbonyl compounds, and ammonium acetate. These compounds were able to block Ca²⁺ entry after a dep

Full text of DOI:10.1021/jm500263v

Article
pubs.acs.org/jmc
New 5‑Unsubstituted Dihydropyridines with Improved CaV1.3
Selectivity as Potential Neuroprotective Agents against Ischemic
Injury
Giammarco Tenti,†,# Esther Parada,‡,# Rafael Leon,*,‡,§ Javier Egea,‡ Sonia Martínez-Revelles,∥
́
Ana María Briones,∥ Vellaisamy Sridharan,†,⊥ Manuela G. Lopez,‡,§,∥ María Teresa Ramos,†
́
and J. Carlos Menendez*,†
́
†Departamento de Química Organ
́
ica y Farmaceu
lo Hernando y Departamento de Farmacología y Terapeu
Madrid, 28029 Madrid, Spain
§Instituto de Investigacion
Sanitaria, Servicio de Farmacología Clínica, Hospital Universitario de la Princesa, 28006 Madrid, Spain
∥Departamento de Farmacología y Terapeu
tica, Facultad de Medicina, Universidad Autonoma de Madrid, Instituto de Investigacion
́
tica, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
‡Instituto Teofi
́
́
tica, Facultad de Medicina, Universidad Autonoma de
́
́
́
́
́
del Hospital Universitario La Paz (IdiPAZ), 28029 Madrid, Spain
⊥Department of Chemistry, School of Chemical and Biotechnology, SASTRA University, Thanjavur 613401, Tamil Nadu, India
S
* Supporting Information
ABSTRACT: C5-unsubstituted-C6-aryl-1,4-dihydropyridines
were prepared by a CAN-catalyzed multicomponent reaction
from chalcones, β-dicarbonyl compounds, and ammonium
acetate. These compounds were able to block Ca2+ entry after
a depolarizing stimulus and showed an improved Cav1.3/
Cav1.2 selectivity in comparison with nifedipine. Furthermore,
they were able to protect neuroblastoma cells against Ca2+
overload and oxidative stress models. Their selectivity ratio
makes them highly interesting for the treatment of neuro-
logical disorders where Ca2+ dyshomeostasis and high levels of
oxidative stress have been demonstrated. Furthermore, their
low potency toward the cardiovascular channel subtype makes them safer by reducing their probable side effects, in comparison
to classical 1,4-dihydropyridines. Some compounds afforded good protective profile in a postincubation model that simulates the
real clinical situation of ictus patients, offering a therapeutic window of opportunity of great interest for patient recovery after a
brain ischemic episode. Good activities were also found in acute ischemia/reperfusion models of oxygen and glucose deprivation.
homeostasis by blocking neuronal voltage-gated calcium
channels (VGCCs) could represent one important approach
for postischemic neuroprotection in humans.4
INTRODUCTION
■
Neurodegenerative disorders constitute a significant public
health problem worldwide, and among these, cerebrovascular
accidents represent one of the leading causes of death,
neurological disability, and cognitive impairment.1 In the past
years, significant progress has been made in the understanding
of the physiopathology of stroke, leading to the description of
several pathological features that are common with other
neurodegenerative disorders. These features include protein
misfolding and aggregation, excitotoxicity, oxidative stress, and
an ionic imbalance (especially with reference to Ca2+), which is
associated with mitochondrial dysfunction.2 These concepts are
the basis for the development of molecules that could be
employed as neuroprotective agents, a long-standing objective
of modern medicine.3 During cerebral ischemia, oxygen and
glucose deprivation induces a metabolic cascade that leads to
neuronal death. One of the most significant consequences of
these changes is the dysregulation of Ca2+ homeostasis, leading
to brain damage. In this context, the stabilization of Ca2+
VGCCs are multimeric membrane proteins that play a critical
role in many physiological functions in a variety of tissues.5
These proteins are usually classified into three broad groups
(CaV1, CaV2, and CaV3) and different subtypes depending on
the pore-forming subunit. The members of the CaV1 family
represent the most intensely studied channels and are classified
in four diverse isoforms (CaV1.1−CaV1.4) that differ in their
tissue distributions and physiological functions.6 CaV1.2
VGCCs are the major and most targeted isoform,7 found
mainly in the cardiovascular system, where they control
vascular tone, although they are also found in neurons.
CaV1.3 VGCCs have a tissue distribution similar to that of
Received: February 18, 2014
Published: April 22, 2014
© 2014 American Chemical Society
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Article
CaV1.2 VGCCs, but they are more neurospecific and are
believed to have an important role in neuronal excitability.8
Recent studies9 have demonstrated the implication of Ca2+
entry through CaV1.3 VGCCs in the generation of oxidative
stress underlying the pathogenesis of Parkinson’s disease.
Therefore, selective antagonism of CaV1.3 VGCCs is
considered a potential neuroprotective strategy that could
slow neuronal loss in the early stages of Parkinson’s disease.10
Among VGCCs blockers, 1,4-dihydropyridines (DHPs) can
certainly be considered the most extensively studied family
because of their pharmacological versatility.11 It is known that
high doses of DHPs, such as nifedipine, induce some beneficial
brain effects such as amelioration of age-related working
memory deficits among others.12 Indeed, nifedipine binds both
CaV1.2 and CaV1.3 isoforms, although it shows high selectivity
toward CaV1.2, a characteristic that is a key requirement for its
use in cardiovascular therapy, but that, coupled to its difficulty
in crossing the blood−brain barrier, prevents its use as a
neuroprotective agent.12 Therefore, a major drawback that
needs to be addressed in this area is the prevalence of vascular
side effects of currently used DHPs. Thus, a renewed interest
exists on the search and study of selective CaV1.3 L-type
VGCCs in the context of efforts to find CaV1.3 L-type VGCC-
selective protective agents for neurodegenerative diseases
(NDDs).10,13
Figure 1. Comparison between traditional 1,4-dihydropyridines with
cardiovascular activity (a) and the compounds studied in this paper
(b).
Scheme 1
Against this background, we have recently reported, in
preliminary fashion,14 the synthesis and initial pharmacological
evaluation of a small library of DHP derivatives able to prevent
neuronal [Ca2+]c overload by blockade of L-type VGCCs and
neuroprotective effect against oxidative stress in vitro. In this
work, we now report the extension of the initial library of
compounds and a full pharmacological characterization in order
to achieve a better understanding of their structure−activity
relationships (SARs). We have evaluated the ability of our
DHPs to antagonize selectively CaV1.3 VGCCs, with a view to
contribute to the search for new neuroprotective agents for the
treatment of NDDs and cerebral ischemia. We have also
extended the investigation of their biological activity by using
pharmacological assays that reproduce the pathological
conditions of an ischemic episode. In all cases, the substitution
pattern of these derivatives, especially the absence of a
substituent at C-5 and the presence of an aryl group at C-6,
was predicted to lead to a reduced vascular activity. It is
pertinent to note here that the C-5 substituent is known to
have an influence on the tissue selectivity of 1,4-dihydropyr-
idines and also on the antagonistic/agonistic character of the
DHPs (Figure 1).15 Additional neuroprotective mechanisms
have also been investigated.
which afforded compound 3a under the same conditions
employed for the multicomponent reaction.
This method allowed the preparation of good to excellent
yields of the compounds summarized in Table 1, bearing small
alkyl groups at C-2, ester, thioester, or ketone groups at C-3, a
variety of aryl, heteroaryl, or styryl substituents at C-4, no
substitution at C-5, and aryl or heteroaryl groups at C-6.
In order to explore the influence of a higher rigidity on the
activity and selectivity of our compounds, we also prepared
Table 1. Scope and Yields of the DHP Synthesis
compd
R1
R2
R3
R4
C6H5
yield, %
3a17
3b14
3c
3d14
3e18
3f17
3g
3h17
3i14
3j
3k14
3l14
3m14
3n17
3o
3p19
3q
CH3
C2H5
C3H7
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OEt
OEt
OEt
StBu
CH3
OEt
OEt
OEt
StBu
OEt
StBu
StBu
StBu
OEt
OAllyl
OEt
OEt
OEt
OEt
OEt
StBu
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
95
92
81
99
56
71
86
73
69
75
67
99
97
94
78
96
78
62
57
49
52
C6H5
C6H5
C6H5
C6H5
4-ClC6H4
4-ClC6H4
C6H5
4-ClC6H4
4-MeOC6H4
4-MeOC6H4
3-MeOC6H4
4-BrC6H4
RESULTS AND DISCUSSION
C6H5
■
4-ClC6H4
4-MeC6H4
C6H5
Chemistry. For the preparation of the dihydropyridine
library, we employed a three-component reaction between β-
dicarbonyl compounds 1, chalcones 2, and ammonium acetate
as a source of ammonia in refluxing ethanol and in the presence
of ceric ammonium nitrate (CAN) as a Lewis acid catalyst
(Scheme 1).16 This formal [3 + 3] aza-annulation took place
presumably by a Hantzsch-like mechanism involving the initial
formation of a β-enaminone from compound 1 and ammonia,
followed by its Michael addition onto 2 and a final
cyclocondensation with loss of a molecule of water. This
mechanism was supported by performing the reaction between
commercially available ethyl 3-aminocrotonate and chalcone,
4-MeC6H4
4-MeC6H4
4-MeC6H4
C6H5
4-MeC6H4
C6H5
4-ClC6H4
C6H5
4-NO2C6H4
2-NO2C6H4
2-NO2C6H4
2-Thienyl
4-MeC6H4
4-ClC6H4
4-MeC6H4
2-Furyl
C6H5
3r
3s14
3t
4-MeC6H4
C6H5-CHC(CH3)
3u14
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hexahydroquinolines 5 by application of the CAN-catalyzed
multicomponent protocol to 1,3-cyclohexanedione and dime-
done (Scheme 2).20
(thioester and C4 p-Me-phenyl) and more than 4.8 times better
than 3m (thioester and C-6, C-4 p-Me-phenyl). The lower Ca2+
channel blocking properties of thioester derivatives might be
related to an increased steric hindrance originated by the high
volume of the t-Bu thioester group. As an exception, compound
3i, a thioester derivative bearing a C-4 p-OMe-phenyl
substituent, was slightly more potent than its parent ethyl
ester derivative 3h. On the other hand, compound 3m, showing
two p-Me-phenyl groups at C-4 and C-6, showed the lowest
potency, with an IC50 value above 100 μM. Among ethyl ester
derivatives, compound 3c was the most potent with an IC50
value of 9.9 μM.
Scheme 2
Regarding the effect of the substituents present at the
dihydropyridine ring, replacement of the methyl group at C-2
(3a) by an ethyl (3b) or propyl group (3c) improved their
blocking properties from 18.7 μM for compound 3a to 12.8 μM
for 3b and 9.9 μM for 3c. Bicyclic derivatives 5a and 5b, which
can also be viewed as C-2 modified analogues of 3a, showed
good potencies, especially compound 5b, which blocked the
Ca2+ signal induced by high potassium levels with an IC50 of 2.8
μM, being the most potent VGCCs blocker of the series and
almost as potent as nifedipine. Its analogue 5a, which shares the
same tetrahydroquinolin-5-(1H)-one core but lacks the two
methyl substituents at position 7, was 8.6 times less potent than
5b.
In terms of the substituents present at C-4 of the
dihydropyridine ring, compounds bearing unsubstituted phenyl
rings were generally better blockers than those having rings
possessing electron-donating or electron-withdrawing groups.
Finally, comparing different substituents at position C-6,
electron-withdrawing substituents derivatives were in general
more potent VGCC blockers than the corresponding phenyl
derivatives. Also generally speaking, derivatives bearing a p-Me-
phenyl moiety were poorer blockers than phenyl derivatives.
Functional Assay: CaV1.2 and CaV1.3 IC50 Determination
and Selectivity Comparison. As stated above, CaV1.2 L-type
VGCCs are the major isoform (∼90%),21 expressed in cardiac
myocytes, smooth muscle, and pancreas while CaV1.3 channels
are more neuron-specific (cerebral cortex, hippocampus, basal
ganglia, habenula, and thalamus) and are thought to serve
predominantly as modulators in the neuronal system. CaV1.3 is
connected to mitochondrial oxidative stress and increased
vulnerability in SNc dopaminergic neurons.9a,22
Because of our goal of reducing cardiovascular effects in
neuroprotective DHPs, it was crucial to study the CaV1.3/
CaV1.2 L-type VGCCs selectivity of these C-5 unsubstituted
DHPs. SH-SY5Y neuroblastoma cells were used to study the
blockade of CaV1.3 L-type VGCCs.23 Compounds 3n, 3u, and
5b were selected to calculate their IC50 values for CaV1.3 L-type
VGCCs because of their Ca2+ blockade properties and their
overall neuroprotective profile (see Tables 4 and 5).
Compound 5b showed the best IC50 to block Ca2+ entry
through neuronal L-type VGCCs (IC50 = 2.81 μM) displaying
only half potency of that calculated for nifedipine (IC50 = 1.35
μM), a classical, highly potent L-type VGCCs blocker.
Compounds 3n and 3u were less potent, with IC50 values of
20.6 and 63.1 μM, respectively (Table 3).
To calculate the CaV1.3/CaV1.2 selectivity ratio, we used the
relaxation induced by each compound in 70 mM K+
precontracted rat mesenteric resistance arteries. All DHPs
induced concentration-dependent relaxation responses. The
relaxation induced by nifedipine was higher than that induced
by 3n, 3u, and 5b (Table 3). Nifedipine blocked the CaV1.2
Pharmacology. Neuronal VGCC Blockade in SH-SY5Y
Neuroblastoma Cells. As previously mentioned, DHPs are
specific L-type VGCC blockers. Their activity and vascular
selectivity require an ester functionality at C-5 and a small alkyl
or aminoalkyl substituent at C-6.15 According to our
preliminary data, some compounds 3 blocked the [Ca2+]c
elevation elicited by 70 mM K+ in the neuronal type cells.14
IC50 values for Ca2+ signal blockade data of newly obtained
DHPs are collected in Table 2, using nifedipine as the reference
Table 2. IC50 Values of Compounds 3a−u and 5a,b
Calculated as Blockade of [Ca2+]c Increase Elicited by 70
mM K+ in SH-SY5Y Cells
a
compd
IC50 (μM)
nifedipine
3a
1.35 0.4
18.7 6.6
12.8 2.1
9.9 2.6
95.8 5.7
27.1 3.4
9.3 1.1
28.4 3.2
29.8 4.8
26.8 3.7
15.1 2.8
59.6 9.1
56.2 6.4
>100
3b14
3c
3d
3e
3f
3g
3h
3i14
3j
3k
3l
3m
3n
3o
20.6 2.1
18.5 1.1
12.2 1.4
44.1 8.2
57.1 6.6
14.9 3.8
55.5 5.4
63.1 3.0
24.0 6.4
2.8 0.8
3p
3q
3r
3s
3t
3u
5a
5b
a
IC50 values were determined from dose−response curves (1−100
μM). Data are expressed as the mean SEM of three to six different
cultures in triplicate.
drug. Most DHPs showed moderate to high potency to block
neuronal L-type VGCCs, with IC50 values ranging from more
than 100 μM (compound 3m) to 2.8 μM (compound 5b).
Regarding SAR studies related to the ester functionality, ethyl
ester derivatives were, in general, better VGCCs blockers than
tert-butyl thioester derivatives. Thus, compound 3n (ethyl ester
and C-4 p-Me-phenyl) was 2.7 times more potent than 3l
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bond donors at the stern, elimination of the port side ester
group does not abolish the channel-blocking effect of DHPs.27
We performed our docking calculations with nifedipine as a
reference compound. As shown in Figure 2A, nifedipine shares
a similar position as that found for (S)-nimodipine, in which
the NH group (stern) H-bonds to Y3i10. Nifedipine also has a
polar o-NO2 group at the bowsprit that H-bonds to Y4i11 and
this residue, in turn, H-bonds to the carboxymethyl ester
moiety present at port side.
Table 3. IC50 Values for VGCC Blockade by Nifedipine and
Compounds 3n, 3u, and 5b
exptl IC50 (μM)
neuronal
(SH-SY5Y)
cardiovascular
(70 mM K+)
compd
CaV1.3/CaV1.2
nifedipine
1.35
20.6
63.1
2.81
0.00142
10.6
950.7
1.9
3n
3u
5b
20.3
3.1
0.34
8.3
Then we docked compound 3n using the previously
optimized settings, constraining the calculations to the DHP
binding region where, presumably, these new 5-unsubstituted
DHPs may interact. We docked the (S)-3n enantiomer because
it leaves the unsubstituted configuration at the port side
position, and as we mentioned before, this deletion does not
abolish the antagonistic activity by itself. The (S)-3n
enantiomer presents a 2-methyl and ethyl 3-carboxylate groups
at the starboard side and a bulky phenyl moiety at the port side,
and more importantly, it lacks the second carboxylic ester
functionality at the port side. Docking results of (S)-3n (Figure
2B) predict an orientation of the DHP core similar to that
found for nifedipine but show a noticeably different interaction
pattern. The inclusion of a bulky Ph group increases the steric
hindrance with F3p49, and therefore (S)-3n appears displaced
cardiovascular subtype more potently than neuronal channels
by a factor of 950, showing a high selectivity for the CaV1.2
VGCC subtype. On the contrary, our modifications in the DHP
structure have resulted in a drastic decrease in their selectivity
toward CaV1.2 cardiovascular subtype (Table 3). Compound
5b, the most potent VGCCs blocker of the new family, showed
the highest selectivity toward CaV1.2 subtype, since it was 8.3
times more potent toward this subtype than for neuronal
CaV1.3. Nevertheless, this result represents a CaV1.3/CaV1.2
selectivity more than 100 times lower than that of nifedipine.
This reduction on selectivity was even higher for compounds
3u and 3n, which were 306- and 500-fold less selective toward
CaV1.2 subtype than the reference compound. Similar results
were obtained when U46619 was used as precontraction
stimulus (data not shown). Thus, 3n and 3u have drastically
improved their selectivity toward the CaV1.3 by lowering their
potency toward the cardiovascular subtype while maintaining
their activity in the neuronal subtype. The CaV1.3/CaV1.2
achieved by these compounds represents a substantial improve-
ment even over isradipine, which so far was the DHP with the
highest relative affinity for CaV1.3 L-type VGCCs but which is
still CaV1.2-selective.22
Molecular Modeling. We performed docking calculations to
acquire deeper insights into the molecular basis for the
interaction of these new C5-unsubstituted DHPs with CaV1.2
L-type channels. Although the X-ray structure of the L-type
VGCCs is not available, several models have been described in
recent years, among which we have used the one developed by
Zhorov et al.24 We have maintained the nomenclature used by
these authors for a better understanding. Experimental
mutagenesis results25 and predicted interactions13b,24,26 have
located the DHP binding site in the interface of domains III
and IV of the pore-forming α1 subunit, in transmembrane
segments IIS5, IIIIS6, IVS6, and IIIP.
toward the side entry of the DHP binding site formed by M3i18
,
M4i12, T3o16, and S4i9 (not shown). Thus, the phenyl moiety at
the port side is partially accommodated in a hydrophobic
pocked formed by M3i19 and F3p49 and the core of DHP is
slightly displaced away from the selectivity pore of the channel.
The NH at the stern H-binds to Y3i10 and the carbonyl group at
the starboard establishes an H-bond to Q3o18
.
Comparing both complexes, we can conclude that the
absence of several H-bond interactions and the inclusion of a
bulky group at port side are destabilizing the interaction
between our 5-unsubstituted DHPs and the CaV1.2 L-type
VGCCs. These results, together with our experimental data,
give some hints of the reason for the observed decrease in
selectivity toward CaV1.2.
Neuroprotection: [Ca2+]c Overload Model. A tight control
of Ca2+ homeostasis by neurons is crucial for cell survival. In
several NDDs, Ca2+ dysregulation is thought to be one of the
main causes of neurodegeneration.28 Thus, drugs restoring the
Ca2+ balance should indeed be a therapeutic alternative for the
disease.29
In line with these findings, our preliminary study14 showed
an interesting neuroprotective profile of the C5-unsubstituted
DHPs against [Ca2+]c overload and oxidative stress. This study
It is known that although potent DHP antagonists have H-
bond acceptors at both the port and starboard sides and H-
Figure 2. Docked structures of nifedipine (orange sticks, panel A) and compound 3n (blue sticks, panel B) in the DHP binding site of the CaV1.2 L-
type VGCC model. H-bond interactions are represented dashed blue lines. Key interacting residues are displayed in colors selected by chain.
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was completed by investigating the potential neuroprotective
activity of the novel compounds 3a−u and 5a,b on SH-SY5Y
cells exposed for 24 h to a depolarizing concentration of K+,
which induced [Ca2+]c overload and consequently cell death.
Cells were co-incubated with drugs at 5 μM and high K+ (70
mM, hypertonic). Thereafter, MTT reduction was measured as
a parameter of cell death.30 As shown in Table 4, newly
showed the poorest profile. Generally, compounds bearing an
electron-donating group were better neuroprotectants than
those bearing an electron-withdrawing group.
VGCCs blockers have been previously studied as neuro-
protectant agents, and as a common feature, it has been widely
reported that their potency as [Ca2+]c elevation blockers and
the neuroprotective effect against Ca2+ overload are usually not
correlated.31 This characteristic is also relevant in our family of
DHPs; i.e., compound 5b showed the best Ca2+ signal blockade
(79% blockade), but it showed a moderate activity as
neuroprotectant in the [Ca2+]c overload model. However, 3s
blocked 36% of the Ca2+ signal and protected 40% of cells.
Similar results were obtained for compounds 3n and 3j, with
39% and 40% protection, respectively, which decreased only
43% and 48% the calcium increase elicited by 70 mM K+
(Table 2). These results led us to focus our attention on the
possibility of an additional mechanism of action for our
compounds, possibly related to their antioxidant action, as
previously described.14 The correlation between Ca2+ influx
through L-type VGCCs and increased mitochondrial oxidative
stress has been recently reported.32 Furthermore, L-type
antagonists reverted the oxidative stress measured in the
cells.33 Therefore, the neuroprotective effect of our compounds
against Ca2+ overload might be composed of VGCC blocker
potency and antioxidant effect. The protective capabilities of
these DHPs in an oxidative stress model are summarized in
Table 5.
Neuroprotection: Oxidative Stress Model. Oxidative stress
has been widely regarded as a key mechanism of neuronal death
in several NDDs. As a common feature, high concentrations of
reactive oxygen species (ROS) are produced by abnormal
pathological mitochondria.34 ROS oxidize lipids, causing
membrane impairment and inducing cell death.35 Oxidation
would also affect ribosomal functioning and reduce protein
synthesis.36 Free radicals may also oxidize DNA and RNA, as
shown by the detection of high levels of oxidized products in
vulnerable neurons.
Rotenone and oligomycin A block complexes I and V,
respectively, of the mitochondrial electron transport chain, thus
disrupting ATP synthesis.34 The mixture of rotenone plus
oligomycin A (rot/olig) constitutes a good model of oxidative
stress having its origin in mitochondria. Exposure to this
combination induces neurotoxicity, and it has been widely used
to evaluate potential protective drugs for neurodegenerative
diseases.37
First, we were interested in assessing the potential
antioxidant effect of compounds 3a−u and 5a,b with a co-
incubation protocol. C5-unsubstituted DHPs were co-incu-
bated for 8 h at a concentration of 5 μM with the stressor,
followed by a 16 h postincubation of drugs at the same
concentration without the toxic stimuli. These experimental
conditions were designed to study the potential neuro-
protective effect of the tested compounds based on their
antioxidant capabilities. Besides, as described in the Introduc-
tion, oxidative stress may be increased by [Ca2+]c overload;22
thus, in the co-incubation model of oxidative stress, neuro-
protection afforded by 5-unsubstituted DHPs could be
dependent on their VGCC antagonism properties. Results are
summarized in Table 5, compared with data for melatonin (0.3
μM) and nifedipine (5 μM), which were used as positive
control and reference compound, respectively. Tested com-
pounds showed, in general, interesting neuroprotective effects,
ranging from 32.5% of derivative 3a to 61.9% for 3e, bearing an
Table 4. Neuroprotection Exerted by Compounds 3a−u and
5a,b against Ca2+ Overload Induced by 70 mM K+ in SH-
a
SY5Y Human Neuroblastoma Cells
K+ (70 mM co-incubation)
compd
% survival
100
52.1 3.5###
% protection
basal
70 mM K+
nifedipine (5 μM)
62.9 3.4*
58.7 2.2*
67.6 3.1***
67.3 2.3***
65.4 3.3*
20.0
15.8
34.6
31.4
23.3
34.1
32.5
36.6
24.8
34.1
31.3
40.1
34.5
30.4
36.2
39.8
29.8
23.5
22.1
13.7
50.7
32.0
35.3
28.2
21.4
nifedipine (0.3 μM)
3a14
3b14
3c
3d14
3e14
3f14
3g
68.0 2.2***
67.4 1.4***
69.4 2.4***
65.1 4.2 *
69.4 3.6**
66.7 2.9***
72.7 2.9***
67.2 3.3***
67.4 1.9***
69.9 2.4***
72.3 3.6***
68.7 2.5***
64.6 4.3*
3h
3i14
3j
3k14
3l14
3m14
3n
3o
3p
3q
62.9 5.7*
59.3 5.5ns
3r
3s14
3t
75.6 2.1***
68.5 3.7**
69.0 2.6***
66.4 1.3***
64.0 4.1*
3u14
5a14
5b
a
Data are the mean
SEM of at least five different cultures in
triplicate. % protection was calculated considering MTT reduction by
nontreated cells (basal) as 100% survival. % of toxicity was normalized
for 70 mM K+ seen for each treatment and subtracted to 100. (∗∗∗) p
< 0.001. (∗∗) p < 0.01. (∗) p < 0.05. ns = not significant with respect
to 70 mM K+ treated cells. (###) p < 0.001 compared to basal
conditions. All compounds were assayed at 5 μM.
obtained DHPs showed a similar profile of neuroprotection to
that of the previously reported analogues. Compounds 3c, 3g,
3h, 3o, 3p, 3r, and 5b exhibited comparable neuroprotective
activity of SH-SY5Y cells against [Ca2+]c overload. Protective
activities of new DHPs ranged from 22.1% (3q) to 40.1% (3j),
and the best neuroprotection result was obtained with
compound 3s, which was able to prevent the death of 51% of
cells. In general, compounds bearing an ester functionality
showed a higher neuroprotective profile compared to those
bearing a thioester moiety or polycyclic derivatives 5a and 5b,
the latter of which had a low neuroprotective activity.
Taking into account compounds of type 3 studied, 3s,
bearing a 2-thienyl substituent at C-4, showed the best
neuroprotective capability against [Ca2+]c overload and 3q
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Table 5. Neuroprotective Effect of C5-Unsubstituted DHPs 3a−u and 5a,b (5 μM) on SH-SY5Y Cell Viability against Toxicity
Induced by (a) 8 h Co-Incubation of rot/olig Mixture (30/10 μM) in the Presence of Drugs, Followed by 16 h Postincubation
a
of Drugs, or (b) 8 h Incubation of rot/olig Mixture without Any Drug, Followed by 16 h Postincubation of Drugs
8 h rot/olig co-incubation + 16 h postincubation
8 h rot/olig + 16 h postincubation
compd
basal
% survival
100
% protection
% survival
100
70.6 1.8###
87.8 3.9**
73.8 2.4ns
% protection
RO
65.6 1.3###
82.5 2.7***
78.4 2.9**
78.4 3.4**
77.4 5.9**
80.5 2.9**
79.5 6.0**
87.3 3.1***
82.7 3.2***
69.9 3.9ns
melatonin
44.7
29.3
32.5
32.8
42.9
39.6
61.9
46.2
11.9
46.2
45.7
47.8
57.9
49.4
36.2
53.3
41.5
58.3
11.7
nifedipine
3a
3b
3c
80.6 3.3ns
76.7 4.1ns
34.8
21.7
3d
3e
3f
3g
3h
3i
81.3 7.0**
82.3 3.9***
81.9 3.4**
85.7 4.3***
83.3 4.2***
79.9 2.9**
83.5 5.3**
79.7 3.2*
83.5 2.2*
81.8 3.1ns
76.6 3.9ns
74.2 5.1ns
83.4 3.1*
42.0
38.6
20.5
12.3
43.3
3j
3k
3l
3m
3n
3o
3p
3q
3r
88.0 2.6**
84.3 3.1*
60.1
44.1
64.6 1.5ns
69.9 2.2ns
11.7
60.0 3.1ns
3s
81.9 4.0***
78.0 3.6*
83.7 1.7***
80.3 3.9**
74.6 4.3ns
45.3
36.6
52.4
39.8
26.9
77.5 4.7ns
79.9 3.8ns
86.2 1.2**
25.6
33.2
52.2
3t
3u
5a
5b
a
Data are the mean SEM of five to seven different cultures in triplicate. % protection was calculated considering MTT reduction by nontreated
cells (basal) as 100% survival. % of toxicity was normalized for rot/olig seen for each treatment and subtracted to 100. (∗∗∗) p < 0.001. (∗∗) p <
0.01. (∗) p < 0.05. ns = not significant with respect to rot/olig treated cells. (###) p < 0.001 compared to basal conditions. All compounds were
assayed at 5 μM.
acetyl group instead of an ester or thioester substitution. In
general, in a comparison of DHPs possessing the same
substituents at positions 2, 3, and 6, ethyl ester and ketone
derivatives showed similar protection profiles indicating that
both groups have similar participation on their antioxidant
effect. For the ethyl ester family, derivatives bearing electron-
withdrawing groups were less potent neuroprotectants than
those with electron-donating groups. Thus, compounds 3p and
3r−q, having respectively 4-NO2 or 2-NO2 groups, were the
less active neuroprotectants in this model. On the other hand,
methyl- and methoxyphenyl derivatives showed a protective
effect over 40% in most cases, compound 3n being the best
with a 53.3% protection. Compounds bearing a p-chlorophenyl
moiety at the C-6 position showed similar protection values as
those compounds lacking this substituent, i.e., compounds 3h
(p-MeO-phenyl at C-4 and Ph at C-6) and 3j (m-MeO-phenyl
at C-4 and p-Cl-phenyl at C-6) with protection values of 46.2%
and 47.8%, respectively. Regarding the C2-substituent, an
increase of its steric volume was associated with an improve-
ment of their neuroprotective profile, with protection values of
32.5% (Me, 3a), 32.8% (Et, 3b), and 42.9% (Pr, 3c). A similar
correlation was also observed for their antioxidant effect.
Regarding thioester derivatives, all tested compounds showed
statistically significant neuroprotection with values ranging from
39.6% (3d) to 57.9% (3k). Compounds 3u and 3k afforded
neuroprotective effects over 50%. Finally, polycyclic derivatives
5b and 5a were also able to protect SH-SY5Y cells in a
statistically significant manner.
These encouraging results prompted us to study the
compounds with the best overall neuroprotective profile on a
second model of oxidative stress. A neuroprotective compound
with potential therapeutic interest will be used clinically after
neurons already have become vulnerable; for instance, diseases
are diagnosed after neuronal damage has already been
established.38 Because we wanted to simulate experimental
conditions closer to the clinical situation, in search for
compounds able to protect cells already exposed to oxidative
stress, we employed a protocol that consisted of an 8 h
incubation period with the stressor, followed by a 16 h
incubation period with the drug alone. We selected 11 DHPs to
be tested in this postincubation oxidative stress model, and data
are collected in Table 5. Melatonin (0.3 μM) and nifedipine (5
μM) were also tested as positive and reference compounds,
respectively. Among all tested compounds, derivatives 3h, 3l,
3n, 3o, and 3u demonstrated a neuroprotective effect after a 16
h postincubation in a statistically significant manner. Protection
values were, in all cases, over 40%, ranging from 43.3% of
derivative 3l to the 60.1% protection afforded by compound 3n.
It is interesting to emphasize that nifedipine, which showed
significant neuroprotection in the co-incubation stress model,
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lost its ability to protect in the postincubation model. This
result may indicate that neuroprotection is not fully dependent
on the Ca2+ signal blockade properties. Finally, compound 3u,
being the second best antioxidant found in this model, also
showed a good neuroprotective profile in the OGD model used
previously.14
Compounds 3n and 3u afforded maximum protection at 10 μM
(45%, Figure 3B). Nifedipine (10 μM) treatment produced no
significant protection, corroborating the results obtained in SH-
SY5Y cells subjected to rot/oligo stress postincubation.
CONCLUSION
■
Neuroprotection: Oxygen−Glucose Deprivation of Hippo-
campal Slices. Acute Model of Ischemia/Reperfusion. Oxygen
and glucose deprivation (OGD) is an acute model of the lesion
produced by [Ca2+]c overload during the OGD period followed
by free radical generation during the reoxygenation phase.39 In
neurons, the OGD period depolarizes the membrane after
mitochondrial failure. Depolarization induces substantial
[Ca2+]c elevation and a massive glutamate liberation, leading
to increased cytotoxicity. Among all the experimental models of
neurotoxicity elicited by Ca2+ overload, glutamate-induced Ca2+
overload seems to be the most relevant from a pathogenic point
of view and has been related to several neurodegenerative
diseases and stroke.40 Additionally, recent observations have
confirmed the influence of mitochondria-mediated cell Ca2+
regulation on glutamate-induced excitotoxicity.41 On the other
hand, the functional impairment of mitochondria, promoted by
the lack of oxygen, is increased when reoxygenation triggers a
massive production of reactive oxygen species raised by the
OGD-induced overwork of the NADPH oxidase (NOX)
enzyme.42
To further characterize the neuroprotective profile of
compounds 3u and 3n, we used this model, where toxicity
depends on [Ca2+]c overload and oxidative stress. Rat
hippocampal slices were subjected to 15 min OGD followed
by 120 min reoxygenation (see protocol in Figure 3A), and cell
viability was assessed by MTT reduction. Under these
experimental conditions, slices were treated with 3n or 3u at
increasing concentrations (1, 3, and 10 μM) and with
nifedipine (10 μM) as a control. Considering cell viability in
basal slices as 100%, OGD reduced cell viability by 40%.
In recent years, the different functions of CaV1.2 and CaV1.3 L-
type VGCCs have attracted a great deal of attention because of
their implication on different pathological conditions. In this
line, we have prepared a new class of C5-unsubstituted-C6-aryl-
1,4-dihydropyridines by a CAN-catalyzed multicomponent
reaction from chalcones, β-dicarbonyl compounds, and
ammonium acetate. These compounds were able to block
Ca2+ entry after a depolarizing stimulus and showed an
improved CaV1.3/CaV1.2 selectivity, when compared to
classical dihydropyridines. Docking studies have led to some
interesting conclusions on the interactions with the CaV1.2
channel that will be of interest for the future development of a
new generation of C5-unsubstituted 1,4-dihydropyridines. Our
DHPs protected neuroblastoma cells against [Ca2+]c overload
and oxidative stress-induced toxicity. Their selectivity ratio
makes them highly interesting for the treatment of neurological
disorders where Ca2+ dyshomeostasis and high levels of
oxidative stress have been demonstrated, since their low
potency toward the cardiovascular channel subtype makes
them potentially safer than classical 1,4-dihydropyridines as far
as cardiovascular side effects are concerned. Some compounds
afforded good protection in a postincubation model, which
better simulates the clinical condition, offering a therapeutic
window of opportunity of great interest for patient recovery
after a brain ischemic episode. Good activities were also found
in acute ischemia/reperfusion (oxygen and glucose deprivation)
models. Taken together, these compounds deserve further
investigation on neurological disease animal models to confirm
their good in vitro neuroprotective profile described here.
EXPERIMENTAL SECTION
Chemistry. The purity of new compounds was determined by
CHNS elemental analysis, and all values were verified to be within
0.4% of theoretical data.
■
General Procedure for the Synthesis of 1,4-Dihydropyridine
Derivatives (3) and 4,6,7,8-Tetrahydroquinolin-5(1H)-ones (5).
To a stirred solution of 1,3-diphenyl-2-propen-1-one derivatives (1
equiv, 2 mmol), 1,3-dicarbonyl compounds (1.1 equiv, 2.2 mmol), and
ammonium acetate (3 equiv, 6 mmol) in ethanol (3 mL) was added
ceric ammonium nitrate (CAN, 10% mol), and the resulting mixture
was refluxed for 4 h. After this time, ammonium acetate (1.5 equiv, 3
mmol) was again added and stirring was continued at the same
conditions for additional 4 h. After completion of the reaction
(checked by TLC), the mixture was allowed to cool to room
temperature, diluted with CH2Cl2 (20 mL), and washed with water to
remove CAN and the excess of ammonium acetate. The organic layer
was then washed with brine and dried over anhydrous Na2SO4, and
the solvent was evaporated under reduced pressure. The crude residue
was crystallized from EtOH or purified by silica gel column
chromatography using a petroleum ether−ethyl acetate mixture
(12:1 v/v) as eluent to give pure compounds (3a−u or 5a,b).
Characterization data for representative compounds follow. For full
characterization data, see the Supporting Information.
Figure 3. Post-OGD treatment with 3n and 3u protects hippocampal
slices against oxygen and glucose deprivation followed by reoxygena-
tion. (A) Protocol used to elicit toxicity. Hippocampal slices were
exposed for 15 min to OGD followed by 2 h in control solution
(Reox). 3n, 3u and nifedipine, when used, were present during the 2 h
reox period. (B) Cell viability was measured by the MTT reduction
activity. Values are expressed as the mean SEM of five independent
experiments: (∗∗∗) p < 0.001, compared to the basal; (##) p < 0.01
with respect to OGD-treated slices.
Ethyl 4,6-Diphenyl-2-propyl-1,4-dihydropyridine-3-carbox-
1
ylate (3c). Yellow paste; H NMR (CDCl3, 250 MHz) δ 1.08 (t, J
= 7.3 Hz, 3H, CH2CH2CH3), 1.17 (t, J = 7.1 Hz, 3H, OCH2CH3),
1.69−1.84 (m, 2H, CH2CH2CH3), 2.69−2.90 (m, 2H, CH2CH2CH3),
4.06 (q, J = 7.1 Hz, 2H, OCH2CH3), 4.73 (d, J = 5.6 Hz, 1H, C-4H),
5.20−5.23 (dd, J = 1.9, 5.6 Hz, 1H, C-5H), 5.68 (bs, 1H, NH), 7.15−
7.43 (m, 10H, ArH); 13C NMR (CDCl3, 63 MHz) δ 14.60
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(CH2CH2CH3), 14.64 (OCH2CH3), 22.7 (CH2CH2CH3), 36.1
(CH2CH2CH3), 41.4 (C-4), 59.7 (OCH2CH3), 99.0 (C-3), 105.4(C-
5), 125.5 (2xCHAr), 126.4 (CHAr), 128.1 (2 × CHAr), 128.6 (2 ×
CHAr), 128.9 (CHAr), 129.2 (2 × CHAr), 134.6, 136.4 (C-6CAr, C-
6), 149.4, 151.6 (C-4CAr, C-2), 168.5 (COOR); IR (NaCl) ν 3363,
2963, 2933, 2872, 1724, 1602, 1576, 1494, 1448, 1411, 1273, 1216,
1098, 698 cm−1; elemental analysis calcd (%) for C23H25NO2, C 79.51,
H 7.25, N 4.03; found, C 79.21, H 6.93, N 3.94.
Ethyl 4,6-Bis(4-chlorophenyl)-2-methyl-1,4-dihydropyri-
dine-3-carboxylate (3g). Yellow solid, mp >250 °C; 1H NMR
(CDCl3, 250 MHz) δ 1.17 (t, J = 7.1 Hz, 3H, OCH2CH3), 2.44 (s, 3H,
C-2CH3), 4.05 (q, J = 7.1 Hz, 2H, OCH2CH3), 4.69 (d, J = 5.5 Hz,
1H, C-4H), 5.15 (dd, J = 1.88, 5.5 Hz, 1H, C-5H), 5.57 (bs, 1H, NH),
7.26 (s, 4H, ArH), 7.35 (s, 4H, ArH); 13C NMR (CDCl3, 63 MHz) δ
14.7 (OCH2CH3), 21.1 (C-2CH3), 40.8 (C-4), 59.8 (OCH2CH3), 99.4
(C-3), 105.5 (C-5), 126.8 (2 × CHAr), 128.8 (2 × CHAr), 129.4 (2 ×
CHAr), 129.5 (2 × CHAr), 132.1, 134.0, 134.6, 134.8 (C-6CAr, C-6, 2
× ArCCl), 147.3, 147.5 (C-4CAr, C-2), 168.5 (COOR); IR (NaCl) ν
3348, 2982, 1724, 1597, 1491, 1270, 1231, 1091, 1015, 757 cm−1;
elemental analysis calcd (%) for C21H19Cl2NO2, C 64.96, H 4.93, N
3.61; found, C 64.78, H 4.84, N 3.43.
3H, C-2CH3), 3.91 (q, J = 7.1 Hz, 2H, OCH2CH3), 5.21 (d, J = 5.1
Hz, 1H, C-4H), 5.40 (dd, J = 1.7, 5.1 Hz, 1H, C-5H), 5.57 (bs, 1H,
NH), 7.27−7.34 (m, 1H, ArH), 7.36 (s, 4H, ArH), 7.53−7.66 (m, 2H,
ArH), 7.76 (dd, J = 1, 8.2 Hz, 1H, C-4ArH); 13C NMR (CDCl3, 63
MHz) δ 14.3 (OCH2CH3), 20.8 (C-2CH3), 37.2 (C-4), 59.8
(OCH2CH3), 98.7 (C-3), 104.7 (C-5), 123.6 (CHAr), 126.8 (2 ×
CHAr), 127.1 (CHAr), 129.4 (2 × CHAr), 131.8 (CHAr), 133.6
(CHAr), 134.3, 134.4, 135.0 (C-6CAr, C-6, ArCCl), 143.7, 148.2,
148.7 (C-4CAr, C-2, ArCNO2), 167.8 (COOR); IR (NaCl) ν 3188,
1721, 1651, 1610, 1528, 1482, 1439, 1269, 1094, 826, 747 cm−1;
elemental analysis calcd (%) for C21H19ClN2O4, C 69.22, H 5.53, N
7.69; found, C 69.11, H 5.37, N 7.66.
Ethyl 6-(Furan-2-yl)-2-methyl-4-(4-tolyl)-1,4-dihydropyri-
dine-3-carboxylate (3t). Yellow syrup; 1H NMR (CDCl3, 250
MHz) δ 1.18 (t, J = 7.1 Hz, 3H, OCH2CH3), 2.32 (s, 3H, CH3), 2.44
(s, 3H, CH3), 4.05 (q, J = 7.1 Hz, 2H, OCH2CH3), 4.64 (d, J = 5.65
Hz, 1H, C-4H), 5.37 (dd, J = 1.58, 5.65 Hz, 1H, C-5H), 5.90 (bs, 1H,
NH), 6.40−6.44 (m, 2H, furylH), 7.10 (d, J = 8.02 Hz, 2H, C-4ArH),
7.23 (d, J = 8.02 Hz, 2H, C-4ArH), 7.39−7.40 (m, 1H, furylH); 13C
NMR (CDCl3, 63 MHz) δ 14.7 (OCH2CH3), 21.1 (CH3), 21.5
(CH3), 40.1 (C-4), 59.7 (OCH2CH3), 99.7, 103.4 (C-3, C-5), 105.1
(CH-furyl), 112.0 (CH-furyl), 126.3 (C-6), 128.1 (2 × CHAr), 129.4
(2 × CHAr), 136.0 (ArCCH3), 141.9 (CH-furyl), 146.0, 146.6, 149.1
(C-4CAr, C-2, C-furyl), 168.7 (COOR); IR (NaCl) ν 3348, 2980,
1724, 1662, 1604, 1268, 1096, 751 cm−1; elemental analysis calcd (%)
for C20H21NO3, C 74.28, H 6.55, N 4.33; found, C 73.99, H 6.37, N
4.22.
Docking Calculations. The crystal structure of the CaV1.2 L-
subtype VGCC has not been described, and therefore, for molecular
modeling studies we used the CaV1.2 L-subtype VGCC model
developed by D. Tikhonov and B. S. Zhorov and kindly shared with us
by Prof. Zhorov.24 Docking was performed with the program Molegro
Virtual Docker43 using the molecular docking algorithm Moldock
score. Prior to docking, the structures of nifedipine and (S)-3n were
built and their energies were minimized using Gaussian software
(Frisch, M. J.; et al. Gaussian 03, revision B.04; Gaussian, Inc.:
Pittsbusrg, PA, 2003). Calculations were run on an iMac with a 3.4
GHz i7 processor and 16 GB DDR3. Ligand binding cavities were
identified using the Molegro expanded van der Waals molecular
surface prediction algorithm with a grid resolution of 0.5 Å. A total of
100 docking runs with a population size of 100 were calculated over a
16 Å radius surrounding the predicted DHP binding site cavity with a
grid resolution of 0.3, a maximum of 15 000 iterations per position, a
scaling factor of 0.50, and crossover rate of 0.90 using the Moldock
score algorithm. Moldock optimizer function was used to more
precisely optimize H-bond geometries by calculating the position of
the hydrogen atoms for any hydrogen donors (both in the ligand and
in the proteins). Similar positions were clustered using a root mean
squared deviation (rmsd) of 1.5 Å. Prepositioned ligands were
randomized in the predicted cavity prior to each docking run, and
docking was constrained to the predicted DHPs binding site cavity. In
order to verify that positions resulting from in silico docking represent
correctly bound conformations, each position was visually inspected
and compared. Positions were also inspected and compared with the
rerank score algorithm, protein interaction, hydrogen bonding, and
affinity interaction energies and ordered by the energy of interaction
protein−ligand. Complexes were optimized using Moloc software44
(www.moloc.ch) with standard force field and optimization parame-
ters. During energy minimization the position of amino acid side
chains were fixed while allowing all ligand atoms to move.
Ethyl 6-(4-Chlorophenyl)-4-(3-methoxyphenyl)-2-methyl-
1
1,4-dihydropyridine-3-carboxylate (3j). Yellow syrup; H NMR
(CDCl3, 250 MHz) δ 1.17 (t, J = 7.12 Hz, 3H, OCH2CH3), 2.44 (s,
3H, C-2CH3), 3.81 (s, 3H, OCH3), 4.08 (q, J = 7.12 Hz, 2H,
OCH2CH3), 4.69 (d, J = 5.5 Hz, 1H, C-4H), 5.19 (dd, J = 1.2, 5.5 Hz,
1H, C-5H), 5.54 (bs, 1H, NH), 6.72−6.77 (dd, J = 2.5, 8.1 Hz, 1H, C-
4ArH), 6.89−6.96 (m, 2H, C-4ArH), 7.23 (t, J = 7.8 Hz, 1H, C-4ArH),
7.35 (s, 4H, C-6ArH); 13C NMR (CDCl3, 63 MHz) δ 14.7
(OCH2CH3), 21.1 (C-2CH3), 41.3 (C-4), 55.5 (OCH3), 59.7
(OCH2CH3), 99.6 (C-3), 105.9 (C-5), 111.4 (CHAr), 114.2
(CHAr), 120.6 (CHAr), 126.8 (2 × CHAr), 129.3(2 × CHAr),
129.6 (CHAr), 133.8, 134.7, 134.8 (C-6CAr, C-6, ArCCl), 147.1, 150.7
(C-4CAr, C-2), 160.0 (ArC-OCH3), 168.6 (COOR); IR (NaCl) ν
3356, 2980, 1724, 1676, 1596, 1486, 1286, 1266, 1224, 1093, 754
cm−1; elemental analysis calcd (%) for C22H22ClNO3, C 68.83, H 5.78,
N 3.65; found, C 68.59, H 5.67, N 3.72.
Allyl 6-(4-Chlorophenyl)-2-methyl-4-phenyl-1,4-dihydropyr-
idine-3-carboxylate (3o). Orange syrup; 1H NMR (CDCl3, 250
MHz) δ 2.45 (s, 3H, C-2CH3), 4.52 (dd, J = 0.85, 5.3 Hz 2H,
OCH2CHCH2), 4.74 (d, J = 5.58 Hz, 1H, C-4H), 5.10−5.23 (m,
3H, C-5H, OCH2CHCH2), 5.60 (bs, 1H, NH) 5.76−5.93 (m, 1H,
OCH2CHCH2), 7.17−7.24 (m, 1H, ArH), 7.28−7.38 (m, 8H,
ArH); 13C NMR (CDCl3, 63 MHz) δ 18.8 (C-2CH3), 38.8 (C-4), 62.1
(OCH2CHCH2), 96.8 (C-3), 103.7 (C-5), 115.0 (OCH2CH
CH2), 124.2 (CHAr), 124.4 (2 × CHAr), 125.6 (2 × CHAr), 126.3 (2
× CHAr), 130.9 (OCH2CHCH2), 131.2, 132.2, 132.3 (C-6CAr, C-
6, ArCCl), 145.2, 146.4 (C-4CAr, C-2), 165.8 (COOR); IR (NaCl) ν
3342, 2926, 1678, 1606, 1484, 1221, 1095 cm−1; elemental analysis
calcd (%) for C22H20ClNO2, C 72.22, H 5.51, N 3.83; found, C 71.97,
H 5.43, N 4.00.
Ethyl 2-Methyl-4-(2-nitrophenyl)-6-(4-tolyl)-1,4-dihydropyr-
idine-3-carboxylate (3q). Yellow syrup; 1H NMR (CDCl3, 250
MHz) δ 0.98 (t, J = 7.1 Hz, 3H, OCH2CH3), 2.38 (s, 3H, CH3), 2.49
(s, 3H, CH3), 3.92 (q, J = 7.1 Hz, 2H, OCH2CH3), 5.21 (d, J = 5.05
Hz, 1H, C-4H), 5.38 (dd, J = 1.8, 5.05 Hz, 1H, C-5H), 5.67 (bs, 1H,
NH), 7.19 (d, J = 7.95 Hz, 2H, C-6ArH), 7.25−7.33 (m, 3H, ArH),
7.55 (dt, J = 1.0, 7.42 Hz, 1H, C-4ArH), 7.66 (dd, J = 1.22, 7.87 Hz,
1H, C-4ArH), 7.74 (dd, J = 1.0, 8.08 Hz, 1H, C-4ArH); 13C NMR
(CDCl3, 63 MHz) δ 14.3 (OCH2CH3), 20.8 (CH3), 21.6 (CH3), 31.1
(C-4), 59.7 (OCH2CH3), 98.5 (C-3), 103.6 (C-5), 123.5 (CHAr),
125.3 (2 × CHAr), 126.7 (CHAr), 129.9 (2 × CHAr), 131.9 (CHAr),
133.0, 133.5, 135.1 (C-6CAr, CHAr, C-6), 144.1, 148.2, 148.9 (C-
4CAr, C-2, ArCNO2), 168.0 (COOR); IR (NaCl) ν 3332, 2976, 1674,
1608, 1524, 1486, 1355, 1221, 1084, 750 cm−1; elemental analysis
calcd (%) for C22H22N2O4, C 69.83, H 5.86, N 7.40; found, C 69.86, H
5.69, N 7.64.
Pharmacology. SH-SY5Y Neuroblastoma Cells Culture. SH-
SY5Y cells were cultured following supplier instructions in a 1:1
mixture of F12 (Ham 12) and Eagle’s MEM supplemented with 15
nonessential amino acids, 10% heat-inactivated fetal bovine serum
(FBS), 1 mM sodium pyruvate, 100 μg/mL streptomycin, and 100
units/mL penicillin. Cells were kept at 37 °C in a humidified
atmosphere of 95% air and 5% CO2. For experimental procedures,
cells were cultured in 48-well plates at a density of 1 × 105 cells/well.
Treatments were performed in 1% FBS medium unless other
concentration is stated. Cells were used up to 13 passages.
Ethyl 6-(4-Chlorophenyl)-2-methyl-4-(2-nitrophenyl)-1,4-di-
hydropyridine-3-carboxylate (3r). Yellow syrup; 1H NMR
(CDCl3, 250 MHz) δ 0.97 (t, J = 7.1 Hz, 3H, OCH2CH3), 2.49 (s,
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Ca2+ Signal Measurements. Neuroblastoma cells were cultured
in bottom-transparent black 96-well plates at a density of 5 × 104 cells/
well. After 2 days in culture, cells were loaded with 10 μM Fluo-4/AM
for 1 h at 37 °C in culture medium without FBS. After loading period,
cells were washed twice with Krebs-HEPES solution and kept at room
temperature for 15 min before starting the experiment. Compounds at
the desired concentration were incubated for 15 min before injecting
the stimulus (70 mM KCl). Fluorescence intensity was measured for
12 s at 485 and 520 nm wavelengths of excitation and emission,
respectively, in a microplate reader (FLUOstar Optima, BMG,
Offenburg, Germany). Once the experiment was finish, 50 μL of
Triton (5%) was added to measure maximum fluorescence (Fmax),
followed by 50 μL of MnCl2 to obtain the minimum fluorescence
(Fmin). Potassium evoked responses were expressed as % of
fluorescence at each time point (F) minus minimum fluorescence
values divided by Fmax − Fmin. The maximum value of F520 obtained for
each well was considered as the peak F520 value.
incubating the hippocampal slices in a glucose-free Krebs solution
equilibrated with 95% N2/5% CO2 at 37 °C over 15 min. Glucose was
replaced by 2-deoxyglucose. After a 15 min OGD period, slices were
reincubated in oxygenated normal Krebs solution for 120 min
(reoxygenation period) at 37 °C.
Viability Quantification by MTT Reduction in SH-SY5Y
Neuroblastoma Cells and Hippocampal Slices. Cell viability
was measured by MTT reduction, which measures the mitochondrial
activity of living cells, by quantitative colorimetric assay.45 Briefly,
MTT was added to each well at a final concentration of 5 mg/mL and
incubated at 37 °C in the dark for 2 h. Then the culture medium was
eliminated and the precipitate was dissolved by adding 300 μL/well
(48-well plate) of DMSO. An amount of 100 μL of the resulting
colored solution from each well was transferred to a transparent 96-
well plate, and optical density was measured in an ELISA reader at 540
nm. Control cells treated with DMSO were taken as 100% viability.
Hippocampal cell viability was determined using the same colorimetric
probe.
Hippocampal slices were immediately transferred to a 96-well plate
after reoxygenation and incubated with MTT at a final concentration
of 0.5 mg/mL in Krebs bicarbonate solution for 30 min at 37 °C.
Finally, the precipitate was dissolved in 200 μL of DMSO and optical
density was measured. Absorbance of control slices was taken as 100%
viability.
Neuroprotection against Ca2+ Overload Induced by High K+.
Stock solutions of the compounds under assay were prepared in
DMSO at 10−2 M concentration and kept at −20 °C. Compounds
were diluted with neuroblastoma cells culture medium to the desired
concentration (5 μM), and cells were co-incubated for 24 h with the
toxic stimulus. The control group was treated with the same amount of
DMSO alone.
Vascular Reactivity. Third order branches of mesenteric artery
from 6-month-old Wistar Kyoto rats (2 mm length) were mounted in
a small-vessel dual chamber myograph to monitor isometric tension.
Two steel wires (40 μm diameter) were introduced through the lumen
of the segments and mounted as previously described.46 After a 30 min
equilibration period in oxygenated Krebs−Henseleit solution (KHS)
at 37 °C (pH 7.4), segments were stretched to their optimal lumen
diameter for active tension development.46 Then segments were
washed with KHS and equilibrated for 30 min. Contractility of
segments was then tested by an initial exposure to a high-K+ solution
(120 mM KCl−KHS). The presence of endothelium was determined
by the ability of 10 μM acetylcholine to induce relaxation in arteries
precontracted with phenylephrine at a concentration that produces
approximately 50% of the contraction induced by KCl−KHS. In
different segments, concentration−response curves (0.01 nM to 0.1
μM) to nifedipine, 3n, 3u, and 5b were performed in arteries
precontracted with a 70 mM KCl−KHS solution.
Statistical Analysis. All values are expressed as the mean SEM,
and “n” represents the number of different cultures or animals used.
The IC50 or EC50 values were calculated by nonlinear regression
analysis of each individual concentration−response curve using
GraphPad Prism software (San Diego, CA, USA). Results were
analyzed using comparisons between experimental and control groups
performed by one-way ANOVA followed by Newman-Keuls post hoc
test. Differences were considered to be statistically significant when p
≤ 0.05.
Neuroprotection against Oxidative Stress Induced by
Rotenone−Oligomycin A Cocktail. (a) Co-Incubation Protocol.
Compounds were diluted from stock solutions with neuroblastoma
cells culture media (1% FBS) containing the rotenone−oligomycin A
mixture (30 μM/10 μM, respectively) to the desired concentration (5
μM). Then cells were co-incubated for 24 h with each treatment and
toxic stimulus rot/olig. Control cells were treated with the same
concentration of DMSO without any drug. Melatonin (0.3 μM) and
nifedipine (5 μM) were used as positive and reference controls,
respectively.
(b) Postincubation Protocol. Cells were cultured for 24 h. Culture
medium was replaced by media containing toxic stimulus cocktail
(rot/olig 30/10 μM), and cells were incubated for 8 h at 37 °C.
Thereafter, compounds were diluted from stock solutions with
neuroblastoma cell culture medium (1% FBS) to the desired
concentration (5 μM). Then cells were incubated for 16 h with
each treatment and without toxic stimulus. Control cells were treated
with the same concentration of DMSO without any drug. Melatonin
and nifedipine were used as positive and reference controls,
respectively.
Animal Usage and Hippocampal Slice Preparation. The
experiments were performed after protocol approval by the institu-
tional Ethic Committee of the Universidad Autonoma de Madrid,
́
Spain, according to the European Guideline for the Use and Care of
Animals for Research. All efforts were made to minimize animal
suffering and to reduce the number of animals used in the experiments.
Experiments were completed in hippocampal slices from adult male
Sprague−Dawley rats (275−325 g). The protocol for hippocampal
slice preparation was similar to that used by Egea et al.39 with slight
modifications. After quick decapitation of rats (pentobarbital
anesthesia, 60 mg/kg ip), forebrains were rapidly removed and kept
in ice-cold Krebs bicarbonate dissection buffer (pH 7.4) (in mM):
KCl, 2; NaCl, 120; CaCl2, 0.5; NaHCO3, 26; MgSO4, 10; KH2PO4,
1.18; glucose, 11; sucrose, 200. Then dissected hippocampi were
quickly glued down leaning vertically against agar blocks in small
chamber, submerged in cold, oxygenated dissection buffer, and
sectioned in transverse slices of 200 μM thick using a vibratome
(Leica, Hidelberg, Germany).
ASSOCIATED CONTENT
* Supporting Information
■
S
General experimental methods in chemistry, reaction con-
ditions, compound characterization, NMR spectra, and
pharmacology protocols used. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*R.L.: fax, 34-91-4973543; phone, 34-91-4972766; e-mail,
rafael.leon@uam.es.
■
Neuroprotection against Oxidative Stress Induced by
Oxygen and Glucose Deprivation (OGD) in Hippocampal
Slices. We have used a previously described protocol.39 Solutions
were prebubbled with either 95% O2/5% CO2 or 95% N2/5% CO2 gas
mixtures for 45 min before slice immersion to ensure O2 or N2
saturation. After a stabilization period of 30 min, hippocampal slices of
the control group were incubated for 15 min in Krebs solution
equilibrated with 95% O2/5% CO2 at 37 °C. OGD was achieved by
*J.C.M.: fax, 34-91-3941822; phone, 34-91-3941840; e-mail,
josecm@farm.ucm.es.
Author Contributions
#G.T. and E.S. contributed equally.
G.T. participated in the design and synthesis of new DHPs
and drafting and critical revision of the manuscript. E.P.
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Article
(b) Triggle, D. J. L-type calcium channels. Curr. Pharm. Des. 2006,
12, 443−457.
participated in the design of pharmacological experiments, data
acquisition and analysis of neuroprotection experiments, and
critical revision of the manuscript. S.M.-R. and A.M.B.
contributed to data acquisition and data analysis/interpretation
and critical revision of the manuscript. J.E. and M.G.L.
contributed to pharmacological data analysis/interpretation
and critical revision of the manuscript. V.S. participated in the
design and synthesis of new DHPs. M.T.R. contributed to the
design and synthesis supervision of new DHPs, data analysis/
interpretation, and critical revision of the manuscript. R.L.
contributed to concept/design, acquisition of data and analysis
of VGCCs blockade IC50 calculation and molecular modeling,
drafting of the manuscript, critical revision of the manuscript,
and approval of the article. J.C.M. contributed to concept/
design, synthesis supervision, drafting of the manuscript, critical
revision of the manuscript, and approval of the article.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(11) (a) Edraki, N.; Mehdipour, A. R.; Khoshneviszadeh, M.; Miri, R.
Dihydropyridines: evaluation of their current and future pharmaco-
logical applications. Drug Discovery Today 2009, 14, 1058−1066.
(b) Ioan, P.; Carosati, E.; Micucci, M.; Cruciani, G.; Broccatelli, F.;
Zhorov, B. S.; Chiarini, A.; Budriesi, R. 1,4-Dihydropyridine scaffold in
medicinal chemistry, the story so far and perspectives (part 1): action
in ion channels and GPCRs. Curr. Med. Chem. 2011, 18, 4901−4922.
(12) Striessnig, J.; Koschak, A.; Sinnegger-Brauns, M. J.; Hetzenauer,
A.; Nguyen, N. K.; Busquet, P.; Pelster, G.; Singewald, N. Role of
voltage-gated L-type Ca2+ channel isoforms for brain function.
Biochem. Soc. Trans. 2006, 34, 903−909.
Prof. Boris Zhorov is gratefully acknowledged for providing the
CaV1.2 PDB model used in molecular modeling. R.L. thanks
Instituto de Salud Carlos III for a research contract under the
Miguel Servet Program (Grant CP11/00165). G.T. thanks
Universidad Complutense for a predoctoral fellowship. A.M.B.
thanks MINECO for a research contract under the Ramon
Cajal program (Grant RyC-2010-06473). We also thank the
continued support of Fundacion Teofilo Hernando, Madrid,
́
y
́
́
Spain. Financial support came from the following: Spanish
́
Ministerio de Economia y Competitividad, MINECO (Grants
(13) (a) Kang, S.; Cooper, G.; Dunne, S. F.; Luan, C. H.; Surmeier,
D. J.; Silverman, R. B. Structure−activity relationship of N,N′-
disubstituted pyrimidinetriones as Ca(V)1.3 calcium channel-selective
antagonists for Parkinson’s disease. J. Med. Chem. 2013, 56, 4786−
4797. (b) Locatelli, A.; Cosconati, S.; Micucci, M.; Leoni, A.; Marinelli,
L.; Bedini, A.; Ioan, P.; Spampinato, S. M.; Novellino, E.; Chiarini, A.;
Budriesi, R. Ligand based approach to L-type calcium channel by
imidazo[2,1-b]thiazole-1,4-dihydropyridines: from heart activity to
brain affinity. J. Med. Chem. 2013, 56, 3866−3877. (c) Chang, C. C.;
Cao, S.; Kang, S.; Kai, L.; Tian, X.; Pandey, P.; Dunne, S. F.; Luan, C.
H.; Surmeier, D. J.; Silverman, R. B. Antagonism of 4-substituted 1,4-
dihydropyridine-3,5-dicarboxylates toward voltage-dependent L-type
Ca2+ channels CaV 1.3 and CaV 1.2. Bioorg. Med. Chem. 2010, 18,
3147−3158.
CTQ-2012-33272 to J.C.M. and SAF-2012-32223 to M.G.L.);
People Programme (Marie Curie Actions) of the European
Union’s Seventh Framework Programme (FP7/2007−2013)
under REA Grant PCIG11-GA-2012-322156 to R.L.; IS Carlos
III, Programa Miguel Servet (Grant CP11/00165) to R.L.; IS
Carlos III (Grant PI13/01488 to A.M.B.), and the Spanish
Ministry of Health (Instituto de Salud Carlos III) Grant
RENEVAS-RETICS-RD06/0026 to M.G.L.
ABBREVIATIONS USED
■
CAN, ceric ammonium nitrate; [Ca2+]c, cytosolic calcium
concentration; CaV1.3, L-type voltage-gated calcium channel
1.3; CaV1.2, L-type voltage-gated calcium channel 1.2; DHP,
1,4-dihydropyridine; NDD, neurodegenerative disease; OGD,
oxygen and glucose deprivation; ROS, reactive oxygen species;
rot/olig, mixture of 30 μM rotenone and 10 μM oligomycin A;
SAR, structure−activity relationship; SNc, substantia nigra pars
compacta; VGCC, voltage-gated calcium channel.
(14) Tenti, G.; Egea, J.; Villarroya, M.; Leon
́
́
, R.; Fernandez, J. C.;
Padín, J. F.; Sridharan, V.; Ramos, M. T.; Menen
́
dez, J. C.
Identification of 4,6-diaryl-1,4-dihydropyridines as a new class of
neuroprotective agents. MedChemComm 2013, 4, 590−594.
(15) Triggle, D. J. 1,4-Dihydropyridines as calcium channel ligands
and privileged structures. Cell. Mol. Neurobiol. 2003, 23, 293−303.
́
(16) For a review, see the following: Sridharan, V.; Menendez, J. C.
Cerium(IV) ammonium nitrate as a catalyst in organic synthesis.
Chem. Rev. 2010, 110, 3805−3849.
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