Polycyclic maleimide-based derivatives as first dual modulators of neuronal calcium channels and GSK-3β for Alzheimer's disease treatment

Article abstract of DOI:10.1016/j.ejmech.2018.12.003

Current healthcare has significantly increased the average life expectancy, leading to a consequently greater incidence of age-related diseases, such as Alzheimer's disease. Following a multitarget approach, in this paper a series of polycyclic maleimide-based derivatives were designed and synthesized aimed at simultaneously modulate neuronal calcium channels and glycogen synthase kinase 3-beta (GSK-3β), validated targets to combat Alzheimer’ disease. Different structural modifications were performed on the polycyclic scaffold in order to investigate the structure-activity relationships and compound 10 emerged as a promising non-toxic lead compound, endowed with calcium modulating brain-addressed properties and significant GSK-3β inhibitory activity. Moreover, the easily affordable polycyclic core appears as a new appealing privileged structure in medicinal chemistry.

Full text of DOI:10.1016/j.ejmech.2018.12.003

European Journal of Medicinal Chemistry 163 (2019) 394e402
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Polycyclic maleimide-based derivatives as first dual modulators of
neuronal calcium channels and GSK-3
treatment
b for Alzheimer's disease
Alessandra Bisi a, , Raquel L. Arribas , Matteo Micucci , Roberta Budriesi ,
**
b
a
a
c
c
a
a
Alessandra Feoli , Sabrina Castellano , Federica Belluti , Silvia Gobbi ,
b
a, *
Cristobal de los Rios , Angela Rampa
Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Via Belmeloro 6, 40126, Bologna, Italy
a
b
Instituto Teofilo Hernando and Departamento de Farmacología y Terapeutica, Facultad de Medicina, Universidad Autonoma de Madrid, C/Arzobispo
Morcillo, 4, 28029, Madrid, Spain
c
Epigenetic Med Chem Lab, Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, I-84084, Fisciano, Salerno, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 September 2018
Received in revised form
Current healthcare has significantly increased the average life expectancy, leading to a consequently
greater incidence of age-related diseases, such as Alzheimer's disease. Following a multitarget approach,
in this paper a series of polycyclic maleimide-based derivatives were designed and synthesized aimed at
30 November 2018
simultaneously modulate neuronal calcium channels and glycogen synthase kinase 3-beta (GSK-3b),
Accepted 2 December 2018
Available online 3 December 2018
validated targets to combat Alzheimer’ disease. Different structural modifications were performed on the
polycyclic scaffold in order to investigate the structure-activity relationships and compound 10 emerged
as a promising non-toxic lead compound, endowed with calcium modulating brain-addressed properties
Keywords:
Alzheimer's disease
and significant GSK-3
new appealing privileged structure in medicinal chemistry.
© 2018 Elsevier Masson SAS. All rights reserved.
b inhibitory activity. Moreover, the easily affordable polycyclic core appears as a
Maleimide-based polycyclic scaffold
2þ
Ca channels
GSK-3
Dual inhibitors
b
1
. Introduction
In this scenario, multiple lines of evidence have implicated
calcium dysregulation in brain aging and dementia [3]. Calcium
ions are critical mediators of cell signaling both for their electro-
genic functions (calcium-induced changes in membrane potential)
and for their roles as intracellular messengers (activation of
calcium-dependent enzymes). Accordingly, processes that result in
compromised calcium signaling can lead to a range of pathophys-
iological conditions, such as hypertension, cardiac hypertrophy and
a vast array of neurological problems [4]. Cellular calcium influx is
mediated by specific channels, membrane-spanning proteins
among which voltage-gated calcium channels (VGCCs) play a
pivotal role in regulating calcium current in response to membrane
depolarization. Their activity is essential to couple electrical signals
in the cell surface to physiological events in cells. The family of
VGCCs consists of ten members, which have been characterized in
mammals, and serve distinct roles in cellular signal transduction.
Alzheimer's disease (AD), the leading cause of dementia, is a
multifactorial pathology resulting from a complex network of
interrelated and not fully deciphered factors, and a long-term
effective therapy remains at the top of the list for unmet needs
[
1]. A promising advanced drug discovery approach to treat disor-
ders with such intricate pathological mechanisms consists in
designing multi-target-directed ligands (MTDLs), able to simulta-
neously interfere with different biochemical and physiological
processes that often concurrently occur [2].
Abbreviations: AD, Alzheimer's disease; GSK-3b, glycogen synthase kinase 3-
beta; MTDLs, multi-target-directed ligands; VGCCs, voltage-gated calcium chan-
nels; LTCCs, L-type Ca2 channels.
þ
According to the nomenclature adopted in 2000, the Ca
V
1 sub-
*
Corresponding author.
2
þ
family (Ca
responsible for long-lasting (L)-type Ca currents and activated
V V
1.1 to Ca 1.4) comprises L-type Ca channels (LTCCs),
*
* Corresponding author.
E-mail addresses: alessandra.bisi@unibo.it (A. Bisi), angela.rampa@unibo.it
2
þ
(
A. Rampa).
https://doi.org/10.1016/j.ejmech.2018.12.003
223-5234/© 2018 Elsevier Masson SAS. All rights reserved.
0

A. Bisi et al. / European Journal of Medicinal Chemistry 163 (2019) 394e402
395
after strong depolarization [5]. LTCCs play a key role in the regu-
lation of vascular smooth muscle contraction, and substances that
Table 1
2
þ
interfere with their function (Ca channel blockers, whose pro-
totypes include 1,4-dihydropyridines, DHPs) are widely used in the
therapy of hypertension.
2
þ
Considering the central role of Ca in brain signaling functions,
its imbalance is likely to be one of the main triggering causes of
neurodegeneration. Moreover, b-amyloid peptide (Ab), responsible
Compound
R
1
R
2
R
3
R
4
for the formation of senile plaques, one of the main AD hallmarks,
can induce neuronal membrane-associated oxidative stress,
resulting in disruption of intracellular calcium homeostasis and
making neurons vulnerable to calcium overload [6]. Accordingly,
the neuronal death elicited by the imbalance of this ion, observed
during disease evolution, suggests that drugs restoring the proper
1
2
3
4
5
6
7
8
9
H
H
Cl
H
H
H
H
H
H
Cl
H
Cl
H
H
H
H
H
H
Cl
H
H
H
H
H
H
NH
NHCOCH
H
H
H
H
2
3
NH
2
NHCOCH
3
2
þ
Ca levels in neurons might be a viable therapeutic option for AD
2þ
COCH3
[
7]. Furthermore, Ca
channel blockade reduces Ab-induced
H
neuronal decline in vitro, leading to neuroprotective effects in an-
imal models [8].
Some years ago, we reported a series of new anthracene-
maleimide-based LTCC blockers, among which 1 (Fig. 1) emerged
as the most promising compound [9]. Functional studies showed
that 1 (lacking intracellular effects in vascular smooth muscle)
would preferentially bind to inactivated LTCCs, directly interacting
with the pore-forming subunit of the channel, suggesting a
competitive interaction with the DHPs binding domain [9].
The maleimide-based polycyclic scaffold of compound 1 is
recognized as a valuable template in different fields of medicinal
chemistry, when decorated with appropriate substituents [9,10].
Focusing on therapeutic targets for AD, literature reports on in-
10
H
H
H
positions of the scaffold was investigated. Moreover, in a multi-
target perspective and considering the presence of the maleimide
moiety in different GSK-3b inhibitors, the new compounds were
also evaluated for their activity on this key kinase.
hibitors of glycogen synthase kinase-3
b
(GSK-3
b) bearing the
maleimide moiety [11e14]. Indeed, GSK-3
b
plays a pivotal role in
2. Results
the brain, where it regulates many crucial cellular processes, and its
dysregulation proved to be involved in the development of many
2.1. Chemistry
diseases, including AD. In this context, GSK-3b is the main kinase
responsible for tau phosphorylation, and intracellular aggregates of
hyperphosphorylated tau lead to the formation of neurofibrillary
tangles, another major hallmark for AD [15]. Thus, it is increasingly
evident that the role of hyperphosphorylation of tau in destabili-
zation of microtubule assembly and disturbance of axonal transport
According to Scheme 1, compounds 2e6 and 8e10 were pre-
pared via Diels-Alder cycloaddition, starting from maleimide and
the appropriately substituted anthracenes 11e18. Compound 7 was
obtained by acetylation of compound 6 with acetyl chloride in the
presence of K CO .
2
3
is detrimental in the neurodegenerative process, whereby GSK-3
b
Not commercially available anthracenes 14, 17 and 18 were
synthesized according to Scheme 2. 1-Aminoanthracene was acet-
ylated with acetyl chloride in the presence of K CO to obtain 14.
is widely considered a therapeutic target of interest, both in phar-
maceutical and academic laboratories [16].
2
3
Pursuing our efforts toward the development of small-
molecules as putative drug candidates for the treatment of AD,
we turned our attention to calcium channels as a new therapeutic
target to be exploited. Taking advantage of our previously reported
LTCC blockers, here we report a new series of maleimide-
anthracene-based compounds 2e10 (Fig. 1 and Table 1), in which
different chemical modifications on the polycyclic scaffold were
performed to deeply define structureeactivity relationship (SAR)
studies against LTCCs. In particular, the effect elicited by various
electron-withdrawing and electron-donating groups in different
The imine 17 resulted from the Schiff reaction of anthracene-9-
carbaldehyde with benzylamine. The amide 18 was synthesized
by condensation of the chloride of anthracene-9-carboxylic acid
with heptan-1-amine.
2
2
.2. Biological evaluation
.2.1. Functional study on cardiac system
The new compounds (2e10, Table 1) were tested for their car-
diovascular profile on isolated guinea pig left atrium driven at 1 Hz
Fig. 1. Design of the new compounds.

396
A. Bisi et al. / European Journal of Medicinal Chemistry 163 (2019) 394e402
3 2 3
Scheme 1. Preparation of compounds 2̶ 10. Reagents and conditions: i) Maleimide, toluene, reflux 5 h; ii) CH COCl, K CO , acetone, reflux 3 h.
for the new derivatives, since nifedipine showed inotropic effects as
well as negative chronotropic activity in the same experimental
conditions. For sake of clarity, the results reported on Table 2 were
also depicted in Fig. 2.
To assess the calcium antagonist activity of compounds 1e10,
2
þ
þ
the inhibition of Ca -induced contraction on K -depolarized
(
(
80 mM) guinea-pig vascular (aortic strips) and non-vascular
ileum) smooth muscle was evaluated. As reported in Table 3 and
Fig. 3, opposite to nifedipine, the compounds did not display any
vasorelaxant activity on aorta. On the contrary, all compounds,
except derivative 5, showed a calcium antagonist action on non-
vascular smooth muscle, with EC50 values ranging from 0.49 to
14.66 mM. This feature could be related to a selective modulation of
calcium influx on non-vascular smooth muscle, making these de-
rivatives devoid of relevant effects on blood pressure.
.2.2. Effect on Ca2 entry through neuronal VGCC
þ
2
To evaluate the blocking activity of the new derivatives, we
stimulated SH-SY5Y neuroblastoma cells with an extracellular so-
lution containing KCl 70 mM, leading to membrane depolarization
and VGCC opening, what induced [Ca ] elevation in the SH-SY5Y
Scheme 2. Preparation of anthracene derivatives 14, 17e18. Reagents and conditions:
i) CH COCl, K CO , acetone, reflux 3 h; ii) toluene, reflux 24 h; iii) SOCl , reflux 4 h; iv)
heptan-1-amine, triethylamine, CH Cl , r.t. 3 h.
2þ
3
2
3
2
cells cytosol. The presence of compounds 1e3 and 10 at the con-
2
2
centration of 10 mM mitigated such elevation better than the well-
2
þ
known Ca -antagonist nimodipine [18], used as standard in these
experiments (Table 4, center column). The ability of these four
and on a spontaneously beating right atrium, to evaluate their
inotropic and chronotropic effects, respectively. For compounds
devoid of negative chronotropic activity, the inotropy was also
checked in spontaneously beating right atria. Compound 1 and
nifedipine were also tested as reference compounds. Indeed due to
the peculiar structure of the compounds, this study was performed
in order to exclude a possible relevant binding to cardiac calcium
channels, which could be responsible for not negligible side effects.
From a first glance at the results (Table 2), all the tested mole-
cules (1e10) showed interesting cardiovascular activity profile.
Indeed, all derivatives displayed negative inotropic properties, with
þ
2þ
active compounds to reduce the 70 mM K -exerted neuronal Ca
increase was further assessed at different concentrations, with the
aim of calculating, by sigmoidal regression, their inhibitory con-
centration 50 (IC50), defined as the concentration of compound
þ
2þ
capable of halving the K -elicited Ca elevation (Table 4, right
column). These compounds had IC50s around 10 M, being com-
pound 10, with an IC50 of 9 M, the best VGCC blocker.
m
m
Gathering all the results, some SAR can be extracted. The pres-
ence of chlorine atoms in 1e3 is well accepted to feature a proper
blocking activity, unlike the presence of more polar groups, such as
amines, imines or amides. Indeed, it is widely known that chlorine
atoms are nicely accommodated in hydrophobic pockets. For this
reason, the long-chain, highly hydrophobic substituents found in
10 provide positive outcomes, despite the presence of a polar group
(amide) in an inner position. This hypothesis is confirmed by
comparing the blocking effect of 7 with that of 8, where the
replacement of the polar amide by the medium polar ketone
increased the blocking activity from 8 to 20% (Table 4). Otherwise,
the exception of compound 8, bearing
a strong electron-
withdrawing group in R3. This effect was also observed on the
right atrium in spontaneous activity, except for 5. In this respect,
chlorine atoms gave the best results (compounds 1e3) and are
better accommodated at C9eC10 positions, being 3 the most active
derivative.
On the contrary, 1e10 were all devoid of chronotropic proper-
ties. Therefore, an appreciable selectivity of action can be noticed

A. Bisi et al. / European Journal of Medicinal Chemistry 163 (2019) 394e402
397
Table 2
Activity of tested compounds on cardiac parameters.
Left atrium
Right atrium
Comp.
negative inotropy
negative inotropy
negative chronotropy
Activitya (M ± SEM)
EC50b
(m
M)
95% conf lim (ꢀ10ꢁ6
)
Activityc (M ± SEM)
EC50b
(
m
M)
95% conf lim (ꢀ10ꢁ6
)
Activityd (M ± SEM)
e
87 ± 1.9e
94 ± 2.7e
97 ± 2.3
92 ± 2.1
34 ± 1.2
79 ± 0.4
93 ± 1.1
4 ± 0.1e
85 ± 1.6
85 ± 1.7e
1
2
3
4
5
6
7
8
9
1
80 ± 1.7
93 ± 1.1
0.43
0.87
0.22
1.91
0.32
1.49
3.65
0.31e0.55
0.74e1.02
0.13e0.35
1.64e2.21
0.21e0.47
1.06e1.88
3.27e3.85
2.48
1.08
0.47
1.87
2.04e3.01
0.80e1.19
0.30e0.72
1.49e2.03
47 ± 0.9
37 ± 0.9
#
e
97 ± 1.9
82 ± 2.5
65 ± 1.4
42 ± 2.6
e
f
8 ± 0.6
g
74 ± 2.2
79 ± 1.2
11 ± 0.3
75 ± 2.1
76 ± 1.4
97 ± 2.0
2.20
2.18
1.83e2.43
1.77e2.43
15 ± 0.3
37 ± 1.7f
33 ± 2.1
26 ± 1.8f
45 ± 1.4
0.85
1.91
0.26
0.57e1.26
0.93e3.10
0.19e0.36
2.87
0.51
2.43e3.03
0.37e0.71
e
0
e
h,i
Nif.
85 ± 4.2
a
ꢁ5
Decrease in developed tension on isolated guinea pig left atrium at 5 ꢀ 10 M, expressed as percent changes from the control (n ¼ 5e6). The left atria were driven at 1 Hz.
ꢁ
5
The 5 ꢀ 10 M concentration gave the maximum effect for most compounds.
b
Calculated from log concentrationꢁresponse curves (Probit analysis by Litchfield and Wilcoxon [17] with n ¼ 6e7). When the maximum effect was <50%, the EC50 values
were not calculated.
c
ꢁ5
Decrease in developed tension on guinea pig spontaneously beating isolated right atrium at 5 ꢀ 10 M, expressed as percent changes from the control (n ¼ 7e8). The
ꢁ
5
5
ꢀ 10 M concentration gave the maximum effect for most compounds.
d
Decrease in atrial rate on guinea pig spontaneously beating isolated right atrium at 10 M, expressed as percent changes from the control (n ¼ 7e8). The 10ꢁ5 M con-
ꢁ5
centration gave the maximum effect for most compounds. Pretreatment heart rate ranged from 165 to 190 beats/min.
e
f
ꢁ5
At 10 M.
At 5 ꢀ 10 M g At 10 M.
ꢁ5
ꢁ6
h
i
ꢁ7
At 10 M.
EC50 ¼ 0.039
mM (c.l. 0.031e0.051). # Inactive up to the concentration indicated above.
3b, we employed the LANCE Ultra TR-FRET assay, one of the most
well established methods to determine kinase activity [19,20]. An
initial screening was performed using a single concentration
(10 mM) of each compound and the percentage of enzyme residual
activity was calculated with respect to vehicle (DMSO). For com-
parison purpose, the reference standard compound SB216763 was
also tested (IC50 ¼ 11.4 ± 2.1 nM in our assay conditions).
Among the tested compounds, derivatives 3, 9 and 10 exhibited
a weak but distinct inhibition of GSK-3b. In particular, it is note-
worthy that this activity is related to the presence of a substituent
in R1.
To ascertain whether the observed effect was dose-dependent,
different concentrations were investigated and the results are dis-
played in Fig. 5. All the three tested compounds demonstrated an
unambiguous dose-dependent effect in this assay, even with
different biochemical potency.
Compound 10, bearing an N-heptylamide substituent, was the
most potent of series (55% and 10% of GSK-3
.12 M and at 50 M, respectively). In contrast, the elimination of
the long alkyl chain reduced the inhibitor activity (48 and 51% of
GSK-3 residual activity at 50 M for 3 and 9, respectively).
b residual activity at
Fig. 2. Potency of compounds (pEC50) on guinea pig cardiac parameters: negative
inotropy (light grey) and negative chronotropy (dark grey). The dashed line shows the
reference values of negative inotropic potency for NIF (pEC50). The dotted line shows
the reference values of negative chronotropic potency for NIF (pEC50). When error bar
are not shown, these are covered by the point itself.
3
m
m
b
m
3
. Discussion
the change of the substituted position in the aromatic rings did not
play a key role in the blocking activity of the compounds. Overall,
these results underline that non-polar substitution on these poly-
cyclic derivatives enhances their VGCC blocking activity.
It is well known that calcium imbalance in specific brain areas is
involved in triggering neurodegeneration, and calcium channels
modulators could be useful in diseases like AD [8]. Indeed, voltage-
dependent currents, due to an increase in Ca influx via LTCC, are
enhanced in ageing hippocampal neurons [21], and LTCC antago-
nists could counteract this effect in aging-related diseases. More-
over, recent in vivo calcium imaging studies in a mouse model of AD
have demonstrated aberrant increases in neuronal Ca concen-
tration close to A deposits [22]. A possible explanation could be
that A peptides form Ca - permeable pore in the plasma mem-
2þ
2.2.3. Cytotoxicity
We investigated the effects of the best VGCC blockers (1e3, 10)
on human neuronal SH-SY5Y cells viability by means of the color-
imetric MTT assay. As depicted in Fig. 4, the loss of cell viability,
after 48 h of exposure with compounds, was irrelevant and statis-
tically non-significant.
2
þ
b
2þ
b
brane, thereby rendering neurons vulnerable to excitotoxicity and
apoptosis [7].
2.2.4. GSK-3b activity
In order to test whether compounds 1e10 can also inhibit GSK-
In a previous work, we have developed a series of LTCC blockers

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A. Bisi et al. / European Journal of Medicinal Chemistry 163 (2019) 394e402
Table 3
Activity of tested compounds on smooth muscle.
Aorta
Ileum
Compound
Activitya (M ± SEM)
Activitya (M ± SEM)
90 ± 1.7c
EC50b
(
m
M)
95% conf lim (ꢀ10ꢁ6
)
1
2
3
4
5
6
7
8
9
1
21 ± 1.6
3.33
1.18
0.49
14.66
2.77e3.02
0.89e1.32
0.31e0.66
12.19e17.38
e
9 ± 0.8
7 ± 0.4
8 ± 0.7
6 ± 0.2
#
10 ± 0.7
18 ± 0.7
#
90 ± 1.4
68 ± 2.4
c
97 ± 1.6
12 ± 0.
90 ± 1.5e
67 ± 1.8e
85 ± 1.8
85 ± 2.4
94 ± 3.8
1.27
2.51
0.77
12.06
3.67
0.86e1.90
2.07e2.99
0.53e1.01
11.09e12.73
2.74e4.91
0
6 ± 0.3
82 ± 1.3
c,d
f
Nif.
70 ± 0.36
0.0015
0.0011e0.0022
a
Percent inhibition of calcium-induced contraction on K -depolarized (80 mM) guinea pig aortic strips and longitudinal smooth muscle (at 5 ꢀ 10 M). The 5 ꢀ 10ꢁ5
þ
ꢁ5
M
concentration gave the maximum effect for most compounds.
b
Calculated from log concentration-response curves (Probit analysis by Litchfield and Wilcoxon [17] with n ¼ 6e7). When the maximum effect was <50%, the IC50 values
were not calculated.
c
d
e
ꢁ6
At 10 M.
IC50 ¼ 0.009
m
M (c.l. 0.003e0.020).
ꢁ5
f
ꢁ9
At 10 M At 5 ꢀ 10 M # Inactive up to the concentration indicated above.
Fig. 4. Effect of compounds 1, 2, 3, and 10 on the viability of SH-SY5Y cells. SH-SY5Y
cells were subjected to compounds for 48 h at 10 and 30 M. Cell viability was
m
measured by the method of the MTT reduction. Data are expressed as the mean ± SEM
of cell viability compared with that of the basal group, i.e. cells only treated with
vehicle (Veh. DMSO, white bar), of three independent experiments. Differences be-
tween basal and test groups were statistically not significant.
Fig. 3. Potency of compounds (pEC50) on guinea pig smooth muscle parameters:
vascular (aorta) (light grey) and non-vascular (ileum) (dark grey). The dashed line
shows the reference potency values for NIF (pEC50) on vascular tissues. The dotted line
shows the reference potency values for NIF (pEC50) on non-vascular tissues. When
error bar are not shown, these are covered by the point itself.
bearing a new maleimide-related polycyclic scaffold [9], which
could also remind of the maleimide moiety found in recently re-
ported GSK-3b inhibitors [11e14]. Given the long lasting experience
Table 4
Blockade of the Ca2 entry elicited by K 70 mM-evoked depolarization in SH-SY5Y
þ
þ
of our research group in designing molecules for AD, we decided to
exploit this new versatile structure to design a new series of
compounds to be assessed for their potential therapeutic in AD.
First, the activity on the heart was evaluated to rule out any
possible side effects. The compounds showed moderate negative
inotropic properties and, unlike nifedipine, were all devoid of
chronotropic activity, highlighting a remarkable selectivity. These
2
þ
a
cells by derivatives 1e10, measured by the Ca -sensitive probe Fluo-4 .
Compound
% Blockadeb
IC50 mM
Nimodipine
25 ± 5
39 ± 14
37 ± 11
38 ± 12
11 ± 14
0 ± 11
6 ± 3
8 ± 13
20 ± 15
4 ± 9
14 ± 5
15 ± 4
11 ± 1
15 ± 5
1
2
3
4
5
6
7
8
9
2
þ
results could reflect a different expression of Ca channel subtypes
in tissues used for functional assays. In particular, the negative
inotropic effect is due to interaction with the Ca
the negative chronotropic effect seems to be related to interaction
with the Ca 1.3 isoform [23]. Furthermore, all compounds were
v
1.2 isoform, while
v
10
56 ± 9
9 ± 3
devoid of vasorelaxant activity on aorta, being thus unable to affect
the blood pressure.
a
Data are mean of at least three independent experiments ± SEM in triplicates,
þ
2þ
comparing with the K -induced Ca increase in the absence of compounds.
b
Then, we evaluated the blocking activity of the new derivatives
Compounds evaluated at 10 mM.
2
þ
on SH-SY5Y neuroblastoma cells Ca channels. This cell line ex-
2
þ
presses VGCC of neuronal type [24], and similar intracellular Ca
-

A. Bisi et al. / European Journal of Medicinal Chemistry 163 (2019) 394e402
399
Fig. 5. Effect of compounds 3, 9 and 10 on GSK-3
b enzymatic activity. The activity of the compounds on GSK-3b was determined using a TR-FRET assay. Each compound inhibited
the enzyme in a dose-dependent manner. Values obtained for each compound are the means ± SD determined for three separate experiments.
buffering machinery to primary neural cultures, so it has been
widely used to study the physiopathology of neurodegeneration
and the effect of neuroprotective compounds directed to counteract
motif in medicinal chemistry.
In summary, a series of polycyclic maleimide-based derivatives
were designed and synthesized as dual calcium channels and GSK-
2
þ
Ca
depolarization-stimulated calcium uptake into SH-SY5Y cells. In
particular compound 10, with an IC50 of 9 M, turned out to be the
most potent VGCC blocker. It is well known that, like the heart,
brain also expresses two sub-types of neuronal LTCCs, Ca 1.2 (90%)
and Ca 1.3 (10%) isoforms, which regulate neuronal excitability and
synaptic plasticity. Notably, in most cases, a single neuron ex-
overload. Compounds 1e3 and 10 inhibited the
3b modulators for AD treatment. The new compounds showed
interesting properties on the selected targets, highlighting the
potential of this easily affordable polycyclic scaffold as a new
privileged structure in medicinal chemistry. From a multitarget
point of view, 10 represents a promising non-toxic lead compound,
bypassing the cardiovascular side effects resulting from peripheral
L-type channel blockade and embracing in a new atypical scaffold
m
v
v
2
þ
presses both of them [25,26]. Therefore, mitigation of Ca entry
excess elicited by 10 may restore the altered synaptic transmission
and synaptic plasticity to normal parameters, ending the enhanced
neuronal vulnerability.
calcium modulating brain-addressed properties and GSK-3
erly adjusted inhibitory activity.
b prop-
4. Experimental
4.1. Chemistry
Previous studies showed that 1 preferentially binds to inacti-
vated LTCCs [9], therefore the same behavior can be speculated for
10, and this might also increase its therapeutic value by targeting
only excitotoxic or pathologically active cells, thus sparing normal
synapses [22,27].
4.1.1. General methods
Melting points were measured in glass capillary tubes on a
Büchi SMP-20 apparatus and are uncorrected. NMR spectra were
Furthermore, 10 exhibited a slight but definitive inhibition of
GSK-3
marks of AD. GSK-3
functions by reducing many inflammatory responses by both as-
trocytes and microglia in the CNS. Our organism is not prepared to
restore the overexpression of this enzyme in different pathological
conditions such as AD, thus a smooth inhibition of GSK-3b, able to
decrease levels of activity to physiological ones, would be enough
to produce an important therapeutic effect [28]. In this way, a
b
, the link between A
b
and tau protein, the two major hall-
3
recorded in CDCl , unless otherwise indicated, on a Varian Gemini
1
13
b
inhibitors may improve deficient cognitive
spectrometer at 400 MHz ( H) and 100 MHz ( C). Chemical shifts
are reported in parts per million (ppm) relative to tetramethylsilane
(TMS), and spin multiplicities are given as s (singlet), d (doublet), t
(triplet), m (multiplet) or br (broad). Direct infusion ES-MS spectra
were recorded on a Waters Micromass ZQ 4000 apparatus. High
resolution mass spectra (HRMS) were recorded on a Waters Xevo
Q-Tof. Chromatographic separations were performed by flash
chromatography on silica gel columns (Kieselgel 40,
0.040e0.063 mm; Merck). Organic solutions were dried over
anhydrous sodium sulfate. Elemental analysis was used to deter-
mine purity of the described compounds. All chemicals were
moderate inhibition restores GSK-3b activity to normal levels in a
disease-affected tissue, with negligible effect on healthy tissues.
Notably, compound 10 contains an amide bond, widespread in drug
candidates, that, due to its intrinsic stability, represents a privileged

400
A. Bisi et al. / European Journal of Medicinal Chemistry 163 (2019) 394e402
purchased from Aldrich Chemistry, Milan (Italy), or from Alfa Aesar,
Milan (Italy), and were of the highest purity. Compounds were
named relying on the naming algorithm developed by Cam-
bridgeSoft Corporation and used in ChemDraw Professional 15.0.
4.1.2.7. 2-acetyl-9,10-dihydro-9,10- [3,4]epipyrroloanthracene-12,14-
dione (8). Using the previous procedure and starting from 1-
(anthracen-2-yl)ethan-1-one (16), 8 was obtained. Yield 35%, mp
ꢂ
1
244e245 C. H NMR
d
: 2.58 (d, J ¼ 10.4 Hz, 3H), 3.26e3.32 (m, 2H),
4.85 (t, J ¼ 3.2 Hz, 2H), 7.18e7.24 (m, 2H Ar), 7.32e7.49 (m, 3H Ar.),
13
4.1.2. General procedure for the synthesis of compounds 1e10 via
3
7.79e7.97 (m, 2H Ar). C NMR d: 26.63 (CH ), 45.30 (CH), 45.32
Diels-Alder cycloaddition
(CH), 47.59 (CH), 47.85 (CH), 124.38, 124.75, 125.25, 125.26, 125.31,
127.41, 127.54, 136.01, 139.24, 140.87, 141.94, 144.00, 176.20 (CO),
A solution of maleimide (1.0 mmol) and the selected anthracene
derivative (1.0 mmol) in toluene was refluxed for 3 h and allowed to
stand overnight. The reaction mixture was then heated under
reflux for another 90 min. After cooling, the precipitate was
collected by filtration and purified by recrystallization from ethyl
acetate, unless otherwise indicated.
176.25 (CO), 197.57 (COCH
ESI þ [M þ 23]: calcd for C20
3
). ESI-MS (m/z): 340 (M þ Na). HRMS
H15NO
3
Na, 340.0950. Found: 340.0949.
4.1.2.8. 9-((benzylimino)methyl)-9,10-dihydro-9,10-
[3,4]epi-
pyrroloanthracene-12,14-dione (9). Using the previous procedure
ꢂ
1
and starting from 17, 9 was obtained. Yield 87%, mp 243e245 C. H
NMR
: 3.35 (d, J ¼ 3.1 Hz, 1H), 3.81 (d, J ¼ 3.1 Hz, 1H), 4.82 (s, 1H),
5.11e5.22 (m, 2H), 7.08e7.61 (m, CH þ 13H Ar). C NMR
(C), 48.96 (CH), 50.12 (CH), 52.43 (CH), 65.90 (CH ), 123.55, 123.89,
24.09, 125.48, 126.50, 127.00, 127.13, 127.17 (2C), 127.23, 128.31,
4.1.2.1. 2-chloro-9,10-dihydro-9,10- [3,4]epipyrroloanthracene-12,14-
d
13
dione (1). Using the previous procedure and starting from 2-
chloroanthracene, 1 was obtained [9].
d: 45.91
2
1
4
1
.1.2.2. 1-chloro-9,10-dihydro-9,10-
2,14-dione (2). Using the previous procedure and starting from 1-
chloroanthracene, 2 was obtained [9].
[3,4]epipyrroloanthracene-
128.68 (2C), 138.37, 138.79, 138.96, 141.41, 141.86, 162.17 (C¼N),
þ
175.48 (CO), 176.14 (CO). ESI-MS (m/z): 393 (M þ H ), 415 (M þ Na).
20 2 2
HRMS ESI þ [M þ 23]: calcd for C26H N O Na, 415.1422. Found:
415.1424.
4
1
9
.1.2.3. 9,10-dichloro-9,10-dihydro-9,10- [3,4]epipyrroloanthracene-
2,14-dione (3). Using the previous procedure and starting from
,10-dichloroanthracene 12, was obtained. Yield 36%,
: 3.50 (s, 2H), 7.26e7.40 (m, 4H Ar),
4.1.2.9. N-heptyl-12,14-dioxo-9,10-
[3,4]epipyrroloanthracene-
3
9(10H)-carboxamide (10). Using the previous procedure and
ꢂ
1
ꢂ
1
mp > 250 C. H NMR
d
starting from 18, 10 was obtained. Yield 45%, mp 180e182 C. H
NMR
13
7.74e7.76 (m, 2H Ar), 7.85e7.87 (m, 2H Ar), 9.38 (br, 1H, NH).
C
d
: 0.91 (t, J ¼ 6.8 Hz, 3H), 1.26e1.35 (m, 4H), 1.42e1.51 (m, 4H),
NMR
d: 55.56 (2CH), 62.92 (2C), 123.09, 123.65, 128.56, 128.96,
1.72e1.81 (m, 2H), 3.28 (dd, J ¼ 8.8 and 3.2 Hz, 1H), 3.55e3.73 (m,
3H), 4.77 (d, J ¼ 2.8 Hz, 1H), 7.21e7.24 (m, 3H Ar), 7.32e7.40 (m, 4H
1
36.79, 139.93, 171.50 (2CO). ESI-MS (m/z): 366 (M þ Na). HRMS
13
ESI þ [M þ 23]: calcd for C18
H11Cl
2
NO Na, 366.0064. Found:
2
Ar), 7.49e7.51 (m, 1H Ar). C NMR
d
: 14.22 (CH
), 31.92 (CH ), 40.24 (CH
CH), 48.87 (CH), 50.45 (C), 59.09 (CH), 123.77, 124.71, 124.89,
3
), 22.75 (CH
2
), 27.24
3
66.0066.
(CH
(
2
), 29.14 (CH
2
), 29.70 (CH
2
2
2
N), 45.83
4.1.2.4. 1-amino-9,10-dihydro-9,10-
[3,4]epipyrroloanthracene-
125.62, 127.07, 127.50, 127.57, 127.72, 137.64, 137.92, 140.82, 140.90,
1
2,14-dione (4). Using the previous procedure and starting from
168.36 (CO), 175.50 (CO), 176.31 (CO). ESI-MS (m/z): 439 (M þ Na).
anthracen-1-amine (13), 4 was obtained. The product was then
converted in the hydrochloride salt affording 4 HCl. Yield 6%,
HRMS ESI þ [M þ 23]: calcd for C26
28 2 3
H N O Na, 439.1998. Found:
.
439.2000.
ꢂ
1
mp > 250 C (MeOH/ethyl ether). H NMR
.67 (d, J ¼ 9.6 Hz, 2H), 6.59e6.76 (m, 1H Ar), 6.78e6.81 (m, 1H Ar),
.05e7.18 (m, 1H Ar), 7.19e7.21 (m, 1H Ar), 7.25e7.29 (m, 2H Ar),
d: 3.24e3.28 (m, 2H),
4
7
4.1.3. General procedure for the synthesis of amide compounds 7, 14
A stirred mixture of the selected amine (1 eq.), acetylchloride
2 3
(1.1 eq.) and K CO in acetone (40 mL) was refluxed for 3 h. The hot
1
3
7.32e7.34 (m, 2H Ar), 7.53 (br, 2H, NH
2
). C NMR
d: 44.83 (CH),
4
7.12 (CH), 47.65 (CH), 47.77 (CH),120.98,125.15,126.01 (2C),126.85
reaction mixture was filtered and evaporated to dryness. The crude
residue was purified via flash column chromatography (toluene/
acetone 4:1).
(
(
2C), 126.98, 127.13, 127.66, 138.83, 139.42, 144.97 (CNH
CO), 178.32 (CO). ESI-MS (m/z): 291 (M þ H ). HRMS ESI þ [M þ 1]:
2
), 177.98
þ
15 2 2
calcd for C18H N O , 291.1133. Found: 291.1131.
4.1.3.1. N-(12,14-dioxo-9,10-dihydro-9,10- [3,4]epipyrroloanthracen-
4
1
.1.2.5. N-(12,14-dioxo-9,10-dihydro-9,10- [3,4]epipyrroloanthracen-
-yl)acetamide (5). Using the previous procedure and starting from
2-yl)acetamide (7). Using the previous procedure and starting from
ꢂ
1
6, 7 was obtained (23% yield), mp 174e175 C. H NMR
d: 2.16 (d,
ꢂ
1
14, 5 was obtained. Yield 25%, mp 182e183 C. H NMR
d: 2.16 (s,
J ¼ 12.8 Hz, 3H), 3.25e3.27 (m, 2H), 4.76 (d, J ¼ 4.4 Hz, 2H),
13
3
H), 3.25e3.27 (m, 2H), 4.81 (d, J ¼ 4.4 Hz, 2H), 7.21e7.58 (m, 7H
3
7.15e7.45 (m, 7H Ar). C NMR d: 23.99 (CH ), 40.65 (CH), 46.58
13
Ar). C NMR
8.91 (CH), 122.21, 122.39, 122.49, 124.79, 125.56, 127.09, 127.25,
32.58, 135.82, 141.27, 142.48, 143.15, 168.92 (COCH ), 177.80 (CO),
78.13 (CO). ESI-MS (m/z): 355 (M þ Na). HRMS ESI þ [M þ 23]:
calcd for C20 Na, 355.1059. Found: 355.1058.
d
: 23.98 (CH
3
), 40.61 (CH), 46.47 (CH), 48.65 (CH),
(CH), 48.75 (CH), 48.96 (CH), 122.25, 122.41, 122.52, 124.67, 125.77,
4
1
1
127.19, 127.83, 132.59, 136.02, 141.29, 142.53, 143.65, 168.96
þ
3
(COCH
3
), 177.85 (CO), 178.38 (CO). ESI-MS (m/z): 333 (M þ H ), 355
(M þ Na). HRMS ESI þ [M þ 23]: calcd for C20
16 2 3
H N O Na, 355.1059.
H
16
N
2
O
3
Found: 355.1057.
4
.1.2.6. 2-amino-9,10-dihydro-9,10-
[3,4]epipyrroloanthracene-
4.1.3.2. N-(anthracen-1-yl)acetamide (14). Using the previous pro-
cedure and starting from anthracen-1-amine (13), 14 was obtained.
12,14-dione (6). Using the previous procedure and starting from
ꢂ
1
ꢂ
1
anthracen-2-amine (15), 6 was obtained. Yield 93%. mp > 250 C. H
NMR : 3.22e3.26 (m, 2H), 3.64 (br, 2H, NH
), 4.65 (d, J ¼ 9.6 Hz,
H), 6.44e6.75 (m, 1H Ar.), 6.66e6.75 (m, 1H Ar), 7.05e7.24 (m, 3H
Yield 56%, mp 202e204 C. H NMR
7.2e8.6 (m, 9H Ar). C NMR d: 24.12, 108.83, 118.84, 119.25, 121.22,
d
: 2.42 (s, 3H), 3.98 (br, 1H, NH),
13
d
2
2
125.63, 125.77, 125.96, 126.88, 128.18, 128.75, 130.47, 131.49, 132.38,
142.24, 168.91.
13
Ar), 7.32e7.34 (m, 2H Ar). C NMR
CH), 48.61 (CH), 106.83, 121.44, 124.61, 125.66, 126.31, 127.09,
27.48, 128.82, 138.29, 139.08, 140.36, 144.62 (CNH ), 177.38 (CO),
78.22 (CO). ESI-MS (m/z): 291 (M þ H ), 313 (M þ Na). HRMS
ESI þ [M þ 23]: calcd for C18 Na, 313.0953. Found: 313.0955.
d: 44.95 (CH), 46.18 (CH), 47.83
(
1
1
2
4.1.4. 1-(anthracen-9-yl)eN-benzylmethanimine (17)
A mixture of anthracene-9-carbaldehyde (0.5 g, 2.43 mmol) and
benzylamine (0.29 g, 2.67 mmol) in toluene (40 mL) was refluxed
þ
14 2 2
H N O

A. Bisi et al. / European Journal of Medicinal Chemistry 163 (2019) 394e402
401
for 24 h with Dean-Stark apparatus. The mixture was washed with
water (3 ꢀ 25 mL), and the organic layer was evaporated under
reduced pressure. The residue was purified by flash chromatog-
Spain) in its pro-drug form (acetoxymethyl ester) at 10
m
M in
Krebs-HEPES buffer (composition in mM: 145 NaCl, 5.6 KCl, 1.2
MgCl , 2 CaCl , 11 glucose, and 10 HEPES; pH 7.4) for 45 min, along
2
2
ꢂ
1
raphy affording 13 (0.18 g, 26% yield), mp 70e72 C (ligroin).
NMR
: 5.21 (s, 2H), 7.23e8.61 (m, 12H Ar), 8.79 (s, 1H, CH ¼ ), 9.01
d, 1H Ar), 9.59 (s, 1H Ar). C NMR
25.77, 127.94, 128.16, 128.47, 128.35, 128.42, 130.99, 131.78, 135.38,
H
with pluronic acid 0.2%, used to facilitate its entry through the
plasma membrane. Then, cells were gently washed out with Krebs-
HEPES and incubated with compounds at the desired concentration
or vehicle (DMSO) for 10 min. Afterwards, SH-SY5Y cells were
d
13
(
d: 64.51, 123.92, 125.31, 125.43,
1
þ
138.92, 160.86.
exposed to K 70 mM by injecting a solution with high KCl con-
2
þ
centration, inducing cell depolarization and subsequent Ca in-
crease, which was monitored by reading the Fluo-4-derived
fluorescence in a multi-well fluorescence reader (FluoStar Optima,
4.1.5. N-heptylanthracene-9-carboxamide (18)
Anthracene-9-carboxylic acid (1 g, 4.4 mmol) was treated with
2
þ
SOCl
2
(0.66 mL, 8.8 mmol) and the mixture was heated on a steam
was removed under reduced
BMG Labtech, Ortenberg, Germany). To calculate the Ca increase
in presence of compounds compared with the control situation,
fluorescence in each well was obtained according to the following
bath for 4 h, the excess of SOCl
2
pressure and the resulting acyl chloride was added to a solution of
n-heptylamine (0.33 mL, 2.2 mmol) and TEA (0.61 mL, 4.4 mmol) in
2
þ
c
formula:
maximal fluorescence reached, F
before injecting the solution of high K , Fmax is the fluorescence
obtained after treating cells with 5% Triton X (100 L), used to lysate
cells and recover the maximal fluorescence of the well, and Fmin is
the fluorescence obtained when 100 L of MnCl 1M were applied
to the cell lysate, which fully quenched the fluorescent dye, thus
D
[Ca
]
¼ (F
x
e F
0
)/(Fmax e Fmin), whereby F
x
is the
CH
water. The mixture was extracted with ethyl acetate, washed with
water, dried over Na SO and evaporated to dryness, to obtain the
2
Cl
2
(3 mL). The mixture was stirred 3 h at rt and poured in ice/
0
is the average fluorescence
þ
2
4
m
desired compound, which was used for the following step without
further purification.
m
2
2
þ
4
4
.2. Biology
obtaining the minimum fluorescence. After monitoring the Ca
oscillations for 2 min, Fmax then Fmin were obtained by successive
.2.1. Functional studies
addition of the Triton, then the MnCl
evaluated in a blind fashion.
2
solutions. Compounds were
The pharmacological profile of all compounds was derived on
guinea-pig isolated left and right atria to evaluate their inotropic
þ
and chronotropic effects, respectively, and on K -depolarized
4.2.4. Toxicity assays
(
(
80 mM) guinea-pig vascular (aortic strips) and non vascular
ileum) smooth muscle to assess calcium antagonist activity. All
SH-SY5Y cells were maintained similarly to what was previously
described [32]. Cells were seeded in 48-well plates at a seeding
density of 70 000 cells/well. Before cells confluence, compounds
dissolved in MEM/F12 media supplemented with 10% fetal bovine
serum (FBS) were preincubated for 24 h. Then, media was replaced
by fresh new media with 1% FBS and compounds and the culture
maintained for 24 h more. Then, the colorimetric dye 3-[4,5-
dimethylthiazole-2-il]-2,5-diphenyltetrazolium bromide (MTT)
was applied to cells at the concentration of 1.2 mM and incubated
for 2 h in dark. Media were removed and the precipitated, purple-
colored formazan, generated by the reduction of MTT, was dis-
solved with 0.3 mL of DMSO. Solutions were transferred to a 96-
well plate to measure the absorbance of each well at 540 nm in a
multi-well plate reader, whereby the more purple absorbance, the
more cell viability. Cells non-treated with compounds defined the
maximal cell viability, normalized to 100%, and SH-SY5Y cells
exposed to compounds showed a cell viability of the expressed
percentage compared with the basal group.
procedures followed the guidelines of animal care and approved by
the Ethics Committee of the University of Bologna (Bologna, Italy)
(
ID: 737/2017). The methods were previously described [29].
Briefly, compounds were checked at increasing doses (0.1, 0.5, 1, 5,
0, 50, 100 Mol/L) to evaluate the percent decrease of developed
1
m
tension on isolated left atrium driven at 1 Hz (negative inotropic
activity), the percent decrease in atrial rate on spontaneously
beating right atrium (negative chronotropic activity). For com-
pounds devoid of negative chronotropic activity, the inotropy was
also checked in spontaneously beating right atria. The percent in-
þ
hibition of calcium-induced contraction was evaluated on K -
depolarized smooth muscle strips.
Data were analyzed using Student's t-test and presented as
mean ± S.E.M [17]. Since the drugs were added in cumulative
manner, the differences between the control and the experimental
values at each concentration were tested for a P value < 0.05. The
potency of drugs defined as EC50 and IC50 was evaluated from log
concentration-response curves (Probit analysis using Litchfield and
4.2.5. GSK-3b inhibition
®
Wilcoxon [17] or GraphPad Prism software [30,31] in the appro-
priate pharmacological preparations).
The assays were performed in white Optiplate-384 at room
ꢂ
temperature (22 C) in a final volume of 25
mL, using the following
2
Kinase Buffer: 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl ,
4
.2.2. SH-SY5Y neuroblastoma cells culture
The SH-SY5Y neuroblastoma cell line was acquired from ATCC
2 mM DTT and 0.01% Tween-20. The compounds were dissolved in
DMSO and then diluted in kinase buffer, keeping constant the
concentration of DMSO (3%) in each well. In each assay, the com-
pound SB216763 was used as positive control (IC50 11.4 ± 2.1 nM),
ꢂ
(
Manassas, USA; cat#CRL-2266). Cells were kept at 37 C under
2
humidity and an atmosphere enriched with 5% CO . Cell batches of
low number of passages lacked mycoplasma, according to the
Mycoplasma detection kit QuickTest (Houston, USA) assay. Cells
maintenance and culture were performed according to what have
been described [18]. All the experiments were conducted in sterile
conditions.
while DMSO was used as reference. GSK-3
tration) was first incubated with the three compounds for 30 min,
then a mixture of ATP (10 M, final concentration) and ULight-GS
b (0.5 nM, final concen-
m
(Ser641/pSer657) Peptide (50 nM, final concentration) was added.
The reaction was incubated for 1 h, afterwards the kinase reaction
was stopped by adding 24 mM EDTA and 2 nM (final concentration)
of Eu-anti-phospho-GS (Ser641) antibody, both diluted in Detection
Buffer 1ꢀ. After an incubation of 1 h, the TR-FRET signal was read
with the EnSight Multimode Plate Reader (excitation at 320 nm and
emissions at 615 and 665 nm). Values obtained for each compound
are the means ± SD determined for three separate experiments. The
þ
4
.2.3. Fluorescence-based Ca2 increases measurements
Following a protocol recently described [18], SH-SY5Y neuro-
blastoma cells, seeded in clear-bottom, black 96-well plates, and
with 100% of confluence (36e72 h after seeding), were charged
with the Ca2 -sensitive Fluo-4 dye (Life Technologies, Alcobendas,
þ

402
A. Bisi et al. / European Journal of Medicinal Chemistry 163 (2019) 394e402
tested compounds are soluble in the assay conditions as deter-
mined at the maximum concentration tested (nephelometry
determination).
as potent, selective inhibitors of glycogen synthase kinase-3
2013) 5498e5516.
15] M. Llorens-Martín, J. Jurado, F. Hern ꢀa ndez, J. Avila, GSK-3
b, a pivotal kinase in
Alzheimer disease, Front. Mol. Neurosci. 7 (46) (2017) 1e11.
16] I. Khan, M.A. Tantray, M. Sarwar Alam, H. Hamid, Natural and synthetic
bioactive inhibitors of glycogen synthase kinase, Eur. J. Med. Chem. 125
b, Molecules 18
(
ꢀ
[
[
Appendix A. Supplementary data
(2017) 464e477.
[
[
17] R.J. Tallarida, R.B. Murray, Manual of Pharmacologic Calculations with Com-
puter Programs, second ed., Springer-Verlag, New York, 1987.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2018.12.003.
18] R. Lajarín-Cuesta, C. Nanclares, J.-A. Arranz-Tagarro, L. Gonz ꢀa lez-Lafuente,
R.L. Arribas, M. Araujo de Brito, L. Gandía, C. de los Ríos, Gramine derivatives
targeting Ca2 channels and ser/thr phosphatases: a new dual strategy for the
þ
References
treatment of neurodegenerative diseases, J. Med. Chem. 59 (2016)
6265e6280.
[
19] D.J. Moshinsky, L. Ruslim, R.A. Blake, F. Tang, A widely applicable, high-
throughput TR-FRET assay for the measurement of kinase autophosphor-
ylation: VEGFR-2 as a prototype, J. Biomol. Screen 8 (2003) 447e452.
20] D. Patnaik, X. Jun, M.A. Glicksman, G.D. Cuny, R.L. Stein, J.M.G. Higgins,
Identification of small molecule inhibitors of the mitotic kinase haspin by
high-throughput screening using a homogeneous time-resolved fluorescence
resonance energy transfer assay, J. Biomol. Screen 13 (2008) 1025e1034.
21] M.J. Berridge, Calcium regulation of neural rhythms, memory and Alzheimer's
disease, J. Physiol. 592 (2014) 281e293.
[
1] M. Prince, A. Wimo, M. Guerchet, G.-C. Ali, Y.-T. Wu, M. Prina, World Alz-
heimer Disease Report, Alzheimer’s Disease International, London, UK, 2015,
pp. 25e46.
2] J.R. Morphy, Chapter 10 the challenges of multi-target lead optimization, in:
J.R. Morphy, C.J. Harris (Eds.), Designing Multi-target Drugs, The Royal Society
of Chemistry, London, 2012, pp. 141e154.
[
[
[
[
[
[
3] N.J. Ortner, J. Striessnig, L-type calcium channels as drug targets in CNS dis-
[
[
orders, Channels 10 (1) (2016) 7e13.
4] G.W. Zamponi, Targeting voltage-gated calcium channels in neurological and
psychiatric diseases, Nat. Rev. j Drug Discov. 15 (2016) 19e34.
5] D.J. Triggle, L-type calcium channels, Curr. Pharmaceut. Des. 12 (4) (2006)
22] J.-A. Arranz-Tagarro, C. de los Rios, A.G. Garcia, J.-F. Padin, Recent patents on
calcium channel blockers: emphasis on CNS diseases, Expert Opin. Ther. Pat.
24 (9) (2014) 959e977.
4
43e457.
6] R. Resende, C. Pereira, P. Agostinho, A.P. Vieira, J.O. Malva, C.R. Oliveira, Sus-
ceptibility of hippocampal neurons to A peptide toxicity is associated with
[
[
23] K.V. Kuchibhotla, S.T. Goldman, C.R. Lattarulo, H.Y. Wu, B.T. Hyman,
B.J. Bacskai, Aß plaques lead to aberrant regulation of calcium homeostasis
in vivo resulting in structural and functional disruption of neuronal networks,
Neuron 59 (2008) 214e225.
b
perturbation of Ca2þ homeostasis, Brain Res. 1143 (2007) 11e21.
7] G. Zundorf, G. Reiser, Calcium dysregulation and homeostasis of neural cal-
cium in the molecular mechanisms of neurodegenerative diseases provide
multiple targets for neuroprotection, Antioxidants Redox Signal. 14 (2011)
[
24] S.R. Sousa, I. Vetter, L. Ragnarsson, R.J. Lewis, Expression and pharmacology of
endogenous Ca
v
channels in SH-SY5Y human neuroblastoma cells, PloS One 8
(3) (2013) e59293.
1275e1288.
[
[
25] J. Striessnig, A. Pinggera, G. Kaur, G. Bock, P. Tuluc, L-type Ca2þ channels in
[
8] V. Nimmrich, A. Eckert, Calcium channel blockers and dementia, Br. J. Phar-
macol. 169 (2013) 1203e1210.
9] S. Bova, S. Saponara, A. Rampa, S. Gobbi, L. Cima, F. Fusi, G. Sgaragli, M. Cavalli,
C. de los Rios, J. Striessnig, A. Bisi, Anthracene based compounds as new L-type
Ca2þ channel blockers: design, synthesis, and full biological profile, J. Med.
Chem. 52 (5) (2009) 1259e1262.
heart and brain, WIREs Membr. Transp. Signal. 3 (2014) 15e38.
26] G.W. Zamponi, J. Striessnig, A. Koschak, A.C. Dolphin, The physiology, pa-
thology, and pharmacology of voltage-gated calcium channels and their
future therapeutic potential, Pharmacol. Rev. 67 (2015) 821e870.
[
[
[
[
27] S. Navakkodea, L. Chao, T.W. Soong, Altered function of neuronal L-type cal-
cium channels in ageing and neuroinflammation: implications in age-related
synaptic dysfunction and cognitive decline, Ageing Res. Rev. 42 (2018) 86e99.
28] A. Martinez, D.I. Perez, C. Gil, Lessons learnt from glycogen synthase kinase 3
inhibitors development for Alzheimer's disease, Curr. Top. Med. Chem. 13
[
[
10] A. Bisi, S. Gobbi, L. Merolle, G. Farruggia, F. Belluti, A. Rampa, J. Molnar,
E. Malucelli, C. Cappadone, Design, synthesis and biological profile of new
inhibitors of multidrug resistance associated proteins carrying a polycyclic
scaffold, Eur. J. Med. Chem. 92 (2015) 471e480.
11] D.G. Smith, M. Buffet, A.E. Fenwick, D. Haigh, R.J. Ife, M. Saunders,
B.P. Slingsby, R. Stacey, R.W. Ward, 3-Anilino-4-arylmaleimides: potent and
selective inhibitors of glycogen synthase kinase-3 (GSK-3), Bioorg. Med.
Chem. Lett 11 (2001) 635e639.
(2013) 1808e1819.
29] M. Micucci, A. Angeletti, M. Cont, I. Corazza, R. Aldini, E. Donadio, A. Chiarini,
R. Budriesi, Hibiscus Sabdariffa L. flowers and Olea Europea L. leaves extract-
based formulation for hypertension care: in vitro efficacy and toxicological
profile, J. Med. Food 19 (2016) 504e512.
[
12] Q. Ye, Y. Shen, Y. Zhou, D. Lv, J. Gao, J. Li, Y. Hu, Design, synthesis and eval-
[
[
[
30] H. Motulsky, A. Christopoulos, Fitting models to biological data using linear
uation of 7-azaindazolyl-indolyl-maleimides as glycogen synthase kinase-3
GSK-3 ) inhibitors, Eur. J. Med. Chem. 68 (2013) 361e371.
b
and non linear regression. www.graphpad.com, 2003.
(
b
31] H.J. Motulsky, Prism 5 Statistics Guide, GraphPad Software Inc., San Diego, CA,
[
13] Q. Ye, W. Mao, Y. Zhou, L. Xu, Q. Li, Y. Gao, J. Wang, C. Li, Y. Xu, Y. Xu, H. Liao,
L. Zhang, J. Gao, J. Li, T. Pang, Synthesis and biological evaluation of 3-([1,2,4]
triazolo[4,3-a]pyridin-3-yl)-4-(indol-3-yl)-maleimides as potent, selective
2007. www.graphpad.com.
32] L. Gonzalez-Lafuente, J. Egea, R. Leon, F.J. Martinez-Sanz, L. Monjas, C. Perez,
C. Merino, A.M. Garcia-De Diego, M.I. Rodriguez-Franco, A.G. Garcia,
M. Villarroya, M.G. Lopez, C. de Los Rios, Benzothiazepine CGP37157 and its
isosteric 2’-methyl analogue provide neuroprotection and block cell calcium
entry, ACS Chem. Neurosci. 3 (2012) 519e529.
GSK-3b inhibitors and neuroprotective agents, Bioorg. Med. Chem. 23 (2015)
1179e1188.
[
14] Q. Ye, M. Li, Y. Zhou, T. Pang, L. Xu, J. Cao, L. Han, Y. Li, W. Wang, J. Gao, J. Li,
Synthesis and biological evaluation of 3-Benzisoxazolyl-4-indolylmaleimides