Design, synthesis and biological evaluation of new antioxidant and neuroprotective multitarget directed ligands able to block calcium channels

Article abstract of DOI:10.3390/molecules25061329

We report herein the design, synthesis and biological evaluation of new antioxidant and neuroprotective multitarget directed ligands (MTDLs) able to block Ca²⁺ channels. New dialkyl 2,6-dimethyl-4-(4-(prop-2-yn-1-yloxy)phenyl)-1,4-dihydropyridi

Full text of DOI:10.3390/molecules25061329

molecules
Communication
Design, Synthesis and Biological Evaluation of New
Antioxidant and Neuroprotective Multitarget
Directed Ligands Able to Block Calcium Channels
Irene Pachòn Angona 1, Solene Daniel 1, Helene Martin 2, Alexandre Bonet 2, Artur Wnorowski 3,
Maciej Maj 3, Krzysztof Józ´wiak 3, Tiago Barros Silva 4, Bernard Refouvelet 1,
Fernanda Borges 4 , José Marco-Contelles 5,* and Lhassane Ismaili 1,
*
1
Neurosciences Intégratives et Cliniques EA 481, Pôle de Chimie Organique et Thérapeutique,
Univ. Bourgogne Franche-Comté, UFR Santé, 19, rue Ambroise Paré, F-25000 Besançon, France;
pachon.angona.irene@gmail.com (I.P.A.); solene.du70@hotmail.fr (S.D.);
bernard.refouvelet@univ-fcomte.fr (B.R.)
2
3
4
PEPITE EA4267, Laboratoire de Toxicologie Cellulaire, Univ. Bourgogne Franche-Comt
France; helene.martin@univ-fcomte.fr (H.M.); alexandre.bonet@univ-fcomte.fr (A.B.)
é, F-25000 Besançon,
Department of Biopharmacy, Medical University of Lublin, ul. W. Chodzki 4a, 20-093 Lublin, Poland;
artur.wnorowski@gmail.com (A.W.); maciejmaj@umlub.pl (M.M.); krzysztof.jozwiak@umlub.pl (K.J.)
CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto,
R. Campo Alegre 1021/1055, 4169-007 Porto, Portugal; ntb.silva@gmail.com (T.B.S.);
mfernandamborges@gmail.com (F.B.)
5
Laboratory of Medicinal Chemistry (IQOG, CSIC), Juan de la Cierva, 3, 28006 Madrid, Spain
Correspondence: iqoc21@iqog.csic.es (J.M.-C.); lhassane.ismaili@univ-fcomte.fr (L.I.)
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Maria Novella Romanelli and Silvia Dei
Received: 28 February 2020; Accepted: 12 March 2020; Published: 14 March 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: We report herein the design, synthesis and biological evaluation of new antioxidant and
neuroprotective multitarget directed ligands (MTDLs) able to block Ca2+ channels. New dialkyl
2,6-dimethyl-4-(4-(prop-2-yn-1-yloxy)phenyl)-1,4-dihydropyridine-3,5-dicarboxylate MTDLs 3a–t,
resulting from the juxtaposition of nimodipine, a Ca2+ channel antagonist, and rasagiline,
a known MAO inhibitor, have been obtained from appropriate and commercially available
precursors using a Hantzsch reaction. Pertinent biological analysis has prompted us to identify
the MTDL 3,5-dimethyl-2,6–dimethyl–4-[4-(prop–2–yn–1-yloxy)phenyl]-1,4-dihydro- pyridine-
3,5-dicarboxylate (3a), as an attractive antioxidant (1.75 TE), Ca2+ channel antagonist (46.95%
at 10
µM), showing significant neuroprotection (38%) against H2O2 at 10 µM, being considered thus a
hit-compound for further investigation in our search for anti-Alzheimer’s disease agents.
Keywords:
Alzheimer’s disease;
Ca2+ channel antagonists;
Hantzsch reaction;
multitarget directed ligands; neuroprotection; oxidative stress
1. Introduction
Alzheimer’s disease (AD) is a neurodegenerative pathology characterized by a highly
interconnected biological processes leading to neuronal death, accumulation and aggregation of
abnormal extracellular deposits of beta-amyloid peptide (A
hyperphosphorylated tau protein [ ], and low level of neurotransmitter acetylcholine. Therefore,
new strategies, based on the multitarget directed ligand (MTDL) approach [ ], have been developed for
β) and neurofibrillary tangles, composed of
1
2,3
the design of new drugs able to bind simultaneously at diverse enzymatic systems or receptors involved
in the progress of AD [4–8]. Accordingly, and following this paradigm a number of MTDLs has been
described by many research groups [9–11]. Our contributions in this area have used multicomponent
Molecules 2020, 25, 1329; doi:10.3390/molecules25061329
www.mdpi.com/journal/molecules

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reactions (MCRs) [12
–
16] as the method of choice for introducing rapidly and efficiently chemical
diversity in the search for new MTDLs.
In this context, we report herein the design, synthesis and biological evaluation of new MTDLs
able to lower the oxidative stress (OS), inhibit monoamine oxidase enzymes (MAOs), and block Ca2+
channels. OS plays a crucial role in the pathogeny of AD [17,18]. Diverse molecular processes converge
to generate high concentrations of radical oxygenated species (ROS) that oxidize proteins, nucleic acids,
polysaccharides and lipids, whose modification may affect their structure and biological functions
resulting in the neuronal death [19]. OS is caused by various underlying factors, such as mitochondrial
dysfunction [20
aggregation [18
,
21], and disruption of metal homeostasis (Cu, Fe, Zn), being also implicated in A
β
,22] and neuroinflammation [23 24]. Therefore, the antioxidant strategy is one of the
,
most promising pathways for the development of new drugs for aging-related diseases, such as AD [25].
MAO is an interesting pharmacological target for the development of new drugs for AD and other
neurodegenerative diseases, such as Parkinson’s disease (PD). In fact, MAO catalyze the deamination
of biogenic amines such adrenaline, dopamine, serotonin, thus leading to the production of H2O2,
which is a source of ROS, responsible of OS [26,
27]. Ca2+ channel blockade is also a well-established
pathway in the development of new drugs for AD, as evidenced by the example of nivaldipine,
currently under clinical development [28]. Ca2+ entry through voltage-gated L-type Ca2+ channels
(Cav 1.1–1.4) causes both Ca2+ overload and mitochondrial disruption, which leads to the activation of
the apoptotic cascade and cell death [29]. In addition, increased cytosolic calcium levels, involved in
the pathogenesis of AD, regulate glycogen synthase kinase, protein kinase C, and other kinases that
hyperphosphorylate tau, potentiate NFT formation [30] and also facilitate the formation of Aβ peptides
through calcium-mediated β-secretase activity [31–35].
The new MTDLs 3a–t reported here are dialkyl 2,6-dimethyl-4-(4-(prop-2-yn-1-yloxy)-
phenyl)-1,4-dihydropyridine-3,5-dicarboxylate derivatives (Figure 1), that result from the juxtaposition
of nimodipine, a Ca2+ channel blocker, and rasagiline, a known MAO inhibitor. Thus, the designed
MTDLs bear a 1,4-DHP, the core fragment of well-known Ca2+ channel antagonists, attached to a
propargylalkoxy motif, a well-known MAO pharmacophore, able to promote also neuronal survival
via neuroprotective/ neurorescue pathways [36,37].
Figure 1.
Structure of nimodipine, rasagiline, and the new MTDL dialkyl
2,6-dimethyl-4-(4-(prop-2-yn-1-yloxy)phenyl)-1,4-dihydropyridine-3,5-dicarboxylate derivatives 3a–t
reported here.

Molecules 2020, 25, 1329
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2. Results and Discussion
2.1. Synthesis
The synthesis of the new MTDL 3a
–
t
has been carried via a one-pot Hantzsch reaction of
aldehydes 1a
(Scheme 1). Aldehydes 1a
and propargyl bromide, under typical Williamson reaction conditions (Scheme 1). Aldehydes 2a
–
e
/
2a
–
e
with ethyl or methyl acetoacetate and ammonium carbonate in EtOH/water
were prepared from the appropriate substituted 4-hydroxybenzaldehydes
were
–e
–e
synthesized by a Mitsunobu reaction under the conditions described by Mertens [38], from but-3-yn-1-ol
and 3-substituted 4-hydroxybenzaldehydes, in the presence of Ph3P and diisopropyl azodicarboxylate
(DIAD), in THF, at room temperature (rt) (Scheme 1). All new compounds showed excellent analytical
and spectroscopic data, in good agreement with the expected values (see Material and Methods,
and Supplementary Material).
Scheme 1. Synthesis of dialkyl 2,6-dimethyl-4-(4-(alk-2-yn-1-yloxy)phenyl)-1,4-dihydropyridine-3,5-
dicarboxylates 3a–t.
To verify, the effectiveness of our design, compounds 3a–t
were submitted to Ca2+ channel
blockade, antioxidant and MAO inhibition evaluation assays, followed by the neuroprotection analysis
of selected compounds.
2.2. Biological Evaluation
2.2.1. Ca2+ Channel Blockade
The Ca2+ channel blockade capacity of compounds 3a-t, and nimodipine as standard, at 10
µM
concentration, has been carried out following the usual methodology [39]. As shown in Table 1,
the observed % values ranged from 20.2 (3k) to 47.0 (3a). The most potent DHP corresponded,
in decreasing order, to 3a (47.0
favorably witH-Nimodipine (52.8
(SAR), compounds bearing n = 1 length as linker showed better results than those bearing n = 2
linkers. In fact, only one adduct with n = 1 presented an inhibition value under 25% (3f, 22.9
±
6.6%), 3h (42.8
±
14.0%), and 3j (39.0
±
4.8%), comparing thus very
±
5.5%). From the point of view of the structure activity relationship
±
6.7%
), the rest surpassed 30% Ca2+ influx blockage, which is 2/3-fold the value of nimodipine (52.8%).
Concerning the influence of R1 over the blockade activity, no conclusions can be drawn. However, it is
worth mentioning that compounds 3a and 3h with R1 = H and Cl, respectively, are the most active
compounds, as they presented values of inhibition that almost doubled the rest (47% and 43%).

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Table 1. Calcium blockade percentages for compounds 3a
10 µM, and their ORAC (TE) values.a.
–t, expressed in percentage of inhibition at
Ca2+ Channel
inhib. at 10 µM (%)
ORAC b
Compounds
n
R1
R2
3a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
H
CH3
CH2CH3
CH3
47.0 ± 6.6
1.75 ± 0.07
1.44 ± 0.07
1.30 ± 0.02
1.60 ± 0.16
0.502 ± 0.01
0.98 ± 0.04
1.05 ± 0.05
1.35 ± 0.05
0.84 ± 0.03
1.41 ± 0.07
1.85 ± 0.17
2.45 ± 0.25
1.57 ± 0.14
2.78 ± 0.17
2.06 ± 0.03
2.77 ± 0.03
1.06 ± 0.11
0.83 ± 0.02
0.62 ± 0.03
1.43 ± 0.01
nd d
3b
H
33.2 ± 3.9
c
3c
OCH3
-
3d
OCH3
CH2CH3
CH3
33.3 ± 9.5
c
3e
OCH2CH3
-
3f
OCH2CH3
CH2CH3
CH3
22.9 ± 6.7
28.7 ± 4.3
42.8 ± 14.0
31.3 ± 5.8
39.0 ± 4.8
20.2 ± 3.5
22.5 ± 9.8
22.4 ± 6.8
3g
Cl
3h
Cl
CH2CH3
CH3
3i
Br
3j
Br
CH2CH3
CH3
3k
H
3l
H
CH2CH3
CH3
3m
OCH3
c
3n
OCH3
CH2CH3
CH3
-
3o
OCH2CH3
24.6 ± 2.2
26.9 ± 1.1
3p
OCH2CH3
CH2CH3
CH3
c
3q
Cl
-
3r
3s
Cl
CH2CH3
CH3
27.0 ± 5.8
c
Br
-
3t
Br
CH2CH3
24.2 ± 3.7
52.8 ± 5.5
nd d
Nimodipine
Melatonin
-
2.45 ± 0.09
a
b
Every percentage value is the mean of a triple of at least two different experiments. Data are expressed as
Trolox equivalents and are the mean (n = 3) ± SEM. c not active. nd: not determined.
d
2.2.2. Antioxidant Assay
The antioxidant activity of compounds 3a–t, compared to melatonin, used as positive control,
showing an ORAC value of 2.45 [12], was determined by the ORAC-FL method [40]. The antioxidant
activities are expressed as Trolox equivalents (TE) units. As shown in Table 1, the values for the
antioxidant capacity range from 0.52 (3e) to 2.78 (3n). Three compounds, 3l (2.45 TE), 3n (2.78 TE) and
3p (2.78 TE), showed antioxidant activities equal or higher than melatonin (2.45 TE). Concerning the
SAR, for the same R1 substituent, compounds with a linker length of n = 2 showed better ORAC values
than those with n = 1, except for the pairs 3g, 3q and 3h, 3r. For the same linker length the best results
for n = 1 were obtained for compounds bearing R1 = H, whereas for compounds with n = 2, the best
results corresponded to molecules bearing R1 = OMe or R1 = OEt.
2.2.3. hMAOs Inhibition
The effect of the compounds 3a
was evaluated by measuring the production of 4-hydroxyquinoline (4-HQ,
–
t
on the activity of both human MAO (hMAO) isoforms
max = 316 nm) from
λ
kynuramine, using microsomal recombinant hMAO isoforms. Unexpectedly and unfortunately these
compounds showed a very low inhibition.
Based on the previously described biological results, the three most balanced compounds (3a
3h and 3j) against calcium channel blockade and antioxidant activity were evaluated for their capacity
to protect human neuronal cells (SH-SY5Y cell line) from cell death.
,

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2.2.4. Neuroprotective Activity
Several in vitro approaches have been performed to mimic human neuronal features, based on
neuronal-like cells such as the neuroblastoma line SH-SY5Y, a human cell line that divides quickly
and has the ability to differentiate in post-mitotiC-Neurons, thus it is considered a convenient and
popular model to study neuroprotective activity for PD and AD [41]. For this purpose, cytotoxicity
was induced by mitochondrial respiratory chain blockers oligomycin rotenone (O/R) and by H2O2,
a well-known toxic responsible for the generation of ROS. Prior to the neuroprotective assay, the effect
of the compounds on the cell viability was evaluated at 1 and 10 µM, showing no cytotoxicity against
SH-SY5Y cells. As shown in Table 2, compounds 3a and 3j showed a modest neuroprotective effect
against O/R. However, and very interestingly, the two compounds showed an interesting effect against
H2O2, particularly at 10 µM where they showed a percentage of neuroprotection equal to 38 and 39 for
3a and 3j, respectively.
Table 2. Neuroprotective activity of compounds 3a
, 3h and 3j on H2O2 (200 µM) or oligomycin (O at
10 µM) /rotenone (R at 30 µM)-induced cell death in SH-SY5Y cells a.
Compounds
3a
Concentration
0.3 µM
H2O2 (%)
O/R (%)
21.66 +/− 0.02 *
11.21 +/− 0.04
np b
22.49 +/− 0.03 *
38.15 +/− 0.04 *
12.80 +/− 0.01
7.58 +/− 0.03
19.34 +/− 0.03 *
39.78 +/− 0.08 *
10 µM
0.3 µM
3h
3j
np b
10 µM
0.3 µM
14.59 +/− 0.03
np b
10 µM
a
Data are expressed as % neuroprotection
compared to the control cultures (one-way ANOVA); np: not protective.
±
SEM of quadruplicates from three different cultures; * p < 0.05, as
3. Materials and Methods
3.1. General Information
All reagents were purchased from Sigma Aldrich (Saint-Quentin Fallavier France) or TCI
1
(Zwijndrecht, Belgium). H- and 13C-NMR spectra were recorded on a Bruker (Wissembourg France)
spectrometer, operating at 400 and 100 MHz, respectively, in solution in dimethylsulfoxide (DMSO-d6)
at rt. Chemical shift values are given in
δ (ppm) relatively to TMS as internal reference. Coupling
constants are given in Hz. The following abbreviations were used: s= singlet, d= doublet, t= triplet,
q= quartet, m= multiplet. Elemental analyses were obtained by a Carlo Erba EA 1108 analyzer and
the analytical results were within
mass spectra were obtained at Centre Commun de Spectrom
±
0.2% of the theoretical values for all compounds. High resolution
trie de Masse, Lyon, France on a Bruker
é
micrOTOF-Q II spectrometer (Bruker Daltonics, Champs sur Marne France) in positive ESI-TOF
(electrospray ionization-time of flight).
3.2. Synthesis of Propargylic Aldehydes 1a–e
A suspension of the corresponding 4-hydroxybenzaldehyde (1 equiv) and K2CO3 (1.3 equiv) in
acetone (1.6 mmol/mL) was stirred at reflux for 30 min. The mixture was cooled to rt and propargyl
bromide (1.6 equiv) was added dropwise. The resulting suspension was stirred at reflux for 2 h 30 min.
After that time, the solvent was removed under pressure conditions. The residue was dissolved in
water and extracted with ethyl acetate three times. Organic layers were joined and dried over Na2SO4.
Activated carbon was added to the solution and the mixture is stirred over 15 min at 40 ◦C. The crude
was finally filtered over Celite®, the filtrate was evaporated and the obtained residue was recrystallized
from EtOAct/hexane (1:2 v/v) to afford the desired products in yields ranging from 34% to 98%.
4-(Prop-2-yn-1-yloxy)benzaldehyde (1a). The crude was prepared according to the general procedure
starting from commercially available 4-hydroxybenzaldehyde (1 equiv, 8.19 mmol, 1 g), K2CO3

Molecules 2020, 25, 1329
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(1.3 equiv, 10.65 mmol, 1.47 g) and propargyl bromide (1.6 equiv, 13.10 mmol, 0.992 mL) in acetone
(20 mL) to afford compound 1a (1.00 g, 77%). The crude was used without further purification.
1H-NMR (CDCl3)
2.4 Hz, 1H).
δ 9.91 (s, 1H), 7.93–7.80 (m, 2H), 7.14–7.07 (m, 2H), 4.78 (d, J = 2.4 Hz, 2H), 2.57 (t, J =
3-Methoxy-4-(prop-2-yn-1-yloxy)benzaldehyde (1b). The crude was prepared according to the general
procedure starting from commercially available 4-hydroxy-3-methoxybenzaldehyde (1 equiv, 6.58 mmol,
1 g), K2CO3 (1.3 equiv, 8.55 mmol, 1.181 g) and propargyl bromide (1.6 equiv, 10.52 mmol, 0.800 mL) in
acetone (16 mL) to afford compound 1b (1.16 g, 93%). The crude was used without further purification.
1H-NMR (CDCl3)
δ 9.87 (s, 1H), 7.52–7.39 (m, 2H), 7.14 (d, J = 8.1 Hz, 1H), 4.86 (d, J = 2.3 Hz, 2H), 3.94
(s, 3H), 2.56 (t, J = 2.4 Hz, 1H).
3-Ethoxy-4-(prop-2-yn-1-yloxy)benzaldehyde (1c). The crude was prepared according to the general
procedure starting from commercially available 3-ethoxy-4-hydroxybenzaldehyde (1 equiv, 6.02 mmol,
1.00 g), K2CO3 (1.3 equiv, 7.82 mmol, 1.081 g) and propargyl bromide (1.6 equiv, 9.63 mmol,
0.730 mL) in acetone (15 mL) to afford compound 1c (428.32 mg, 35%). The crude was used without
further purification.
3-Chloro-4-(prop-2-yn-1-yloxy)benzaldehyde (1d). The crude was prepared according to the general
procedure starting from commercially available 3-chloro-4-hydroxybenzaldehyde (1 equiv, 6.39 mmol,
1.00 g), K2CO3 (1.3 equiv, 8.31 mmol, 1.148 g) and propargyl bromide (1.6 equiv, 10.22 mmol, 0.78 mL)
in acetone (10 mL) to afford compound 1d (852.80 mg, 69%). The crude was used without further
1
purification. H-NMR (CDCl3)
δ 9.87 (s, 1H), 7.93 (d, J = 2.0 Hz, 1H), 7.79 (dd, J = 8.5, 2.0 Hz, 1H),
7.21 (d, J = 8.5 Hz, 1H), 4.88 (d, J = 2.4 Hz, 2H), 2.60 (t, J = 2.4 Hz, 1H).
3-Bromo-4-(prop-2-yn-1-yloxy)benzaldehyde (1e). The crude was prepared according to the general
procedure starting from commercially available 3-bromo-4-hydroxybenzaldehyde (1 equiv, 4.98 mmol,
1.00 g), K2CO3 (1.3 equiv, 6.47 mmol, 893.81 mg) and propargyl bromide (1.6 equiv, 7.96 mmol,
0.60 mL) in acetone (12 mL) to afford compound 1e (1.163 g 98%). The crude was used without further
1
purification. H-NMR (CDCl3)
δ 9.86 (s, 1H), 8.09 (d, J = 1.0 Hz, 1H), 7.82 (dd, J = 8.5, 1.1 Hz, 1H),
7.17 (d, J = 8.5 Hz, 1H), 4.89–4.81 (m, 2H), 2.60 (t, J = 2.4 Hz, 1H).
3.3. Synthesis of Propargylic Aldehydes 2a–e
A solution of 4-hydroxybenzaldehyde (1 equiv), triphenylphosphine (2 equiv) and 3-butyn-1-ol
(1.5 equiv) in THF (0.8 mmol/mL) was cooled to 0 ◦C. DIAD (1.5 equiv) is then added dropwise and
the resulting mixture is stirred overnight at rt. The solvent was evaporated, the residue solubilized in
ethyl acetate and washed 3 times with 1M NaOH solution and brine. The organic layers were dried
over Na2SO4, filtered and evaporated under pressure conditions. The residue was triturated with ethyl
ether and filtered. The filtrate was purified by flash chromatography with hexane/EtOAc (9:1 v/v) to
afford the desired products in yields ranging from 45% to 97%.
4-(But-3-yn-1-yloxy)benzaldehyde (2a). The crude was prepared according to the general
procedure starting from commercially available 4-hydroxybenzaldehyde (1 equiv, 16.38 mmol, 2.00 g),
triphenylphosphine (2 equiv, 32.76 mmol, 8.59 g), DIAD (1.5 equiv, 24.57 mmol, 4.84 mL) and
3-butyn-1-ol (1.5 equiv, 27.57 mmol, 1.860 mL) in THF (120 mL) to afford compound 2a (1.30 g, 46%).
1
The crude was used without further purification. H-NMR (400 MHz, CDCl3):
δ 2.06 (br s, 1 H, CH),
2.73 (dt, J = 2.4, 7.1 Hz, 2 H, CH2), 4.18 (t, J = 7.1 Hz, 2 H, CH2O), 7.02 (d, J = 8.5 Hz, 2 H, Harom),
7.84 (d, J = 8.5 Hz, 2 H, Harom), 9.90 (s, 1 H, CHO).
4-(But-3-yn-1-yloxy)-3-methoxybenzaldehyde (2b). The crude was prepared according to the general
procedure starting from commercially available 4-hydroxy-3-methoxybenzaldehyde (1 equiv, 6.57 mmol,
1.00 g), triphenylphosphine (2 equiv, 13.15 mmol, 3.45 g), DIAD (1.5 equiv, 9.86 mmol, 1.94 mL) and
3-butyn-1-ol (1.5 equiv, 9.86 mmol, 0.746 mL) in THF (60 mL) to afford compound 2b (674.9 mg, 50%).
1
The crude was used without further purification. H-NMR (CDCl3)
δ 9.85 (s, J = 4.2 Hz, 1H), 7.48–7.38
(m, 2H), 6.99 (dd, J = 8.1, 3.5 Hz, 1H), 4.28–4.18 (m, 2H), 3.97–3.89 (m, 3H), 2.77 (td, J = 7.3, 2.7 Hz, 2H),
2.09–2.00 (m, 1H).

Molecules 2020, 25, 1329
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4-(But-3-yn-1-yloxy)-3-ethoxybenzaldehyde (2c). The crude was prepared according to the general
procedure starting from commercially available 3-ethoxy-4-hydroxybenzaldehyde (1 equiv, 6.02 mmol,
1.00 g), triphenylphosphine (2 equiv, 12.04 mmol, 3.16 g), DIAD (1.5 equiv, 9.03 mmol, 1.78 mL) and
3-butyn-1-ol (1.5 equiv, 9.03 mmol, 0.683 mL) in THF (60 mL) to afford compound 2c (1.423 g, 70%).
1
The crude was used without further purification. H-NMR (CDCl3)
δ 9.85 (s, 1H), 7.42 (dt, J = 5.5,
1.8 Hz, 2H), 6.99 (d, J = 8.0 Hz, 1H), 4.23 (t, J = 7.3 Hz, 2H), 4.15 (q, J = 7.0 Hz, 2H), 2.77 (td, J = 7.3,
2.7 Hz, 2H), 2.05 (t, J = 2.7 Hz, 1H), 1.47 (t, J = 7.0 Hz, 3H), 1.26 (d, J = 6.3 Hz, 3H).
4-(But-3-yn-1-yloxy)-3-chlorobenzaldehyde (2d). The crude was prepared according to the general
procedure starting from commercially available 3-ethoxy-4-hydroxybenzaldehyde (1 equiv, 6.39 mmol,
1.00 g), triphenylphosphine (2 equiv, 12.78 mmol, 3.35 g), DIAD (1.5 equiv, 9.58 mmol, 1.89 mL) and
3-butyn-1-ol (1.5 equiv, 9.58 mmol, 0.725 mL) in THF (60 mL) to afford compound 2d (1.30 g, 97%).
1
The crude was used without further purification. H-NMR (CDCl3)
δ
9.84 (s, 1H), 7.90 (d, J = 2.0 Hz,
1H), 7.76 (dd, J = 8.5, 2.0 Hz, 1H), 7.04 (d, J = 8.5 Hz, 1H), 4.26 (t, J = 7.1 Hz, 2H), 2.78 (td, J = 7.1, 2.7 Hz,
2H), 2.06 (t, J = 2.7 Hz, 1H).
3-Bromo-4-(but-3-yn-1-yloxy)benzaldehyde (2e). The crude was prepared according to the general
procedure starting from commercially available 3-bromo-4-hydroxybenzaldehyde (1 equiv, 4.97 mmol,
1.00 g), triphenylphosphine (2 equiv, 9.94 mmol, 2.61 g), DIAD (1.5 equiv, 7.46 mmol, 1.47 mL) and
3-butyn-1-ol (1.5 equiv, 7.46 mmol, 0.565 mL) in THF (60 mL) to afford compound 2e (631.20 mg, 50%).
1
The crude was used without further purification. H-NMR (CDCl3)
δ
9.85 (s, 1H), 8.09 (d, J = 2.0 Hz,
1H), 7.81 (dd, J = 8.5, 2.0 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 4.25 (t, J = 7.1 Hz, 2H), 2.80 (td, J = 7.1, 2.7 Hz,
2H), 2.07 (t, J = 2.7 Hz, 1H).
3.4. General procedure of compounds 3a–t
3-Substitued-4-alkynyloxy-benzaldehydes 1a–e and 2a–e (1 equiv) and the corresponding
acetoacetate (3.5 equiv) were dissolved in a mixture of EtOH (4 mmol/mL) and the same volume of H2O.
The resulting mixture is stirred and heated at 75 ◦C for 1 h. Next, ammonium carbonate (2.5 equiv)
was added to the mixture, and the reaction stirred and heated at 75 ◦C overnight. The desired product
precipitated once the crude reaction reached rt, or was triturated with diethyl ether. The solid was then
filtered and washed with diethyl ether again to finally afford compounds 3a–t in yields ranging from
12% to 76%.
3,5-Dimethyl-2,6–dimethyl–4-[4-(prop–2–yn–1-yloxy)phenyl]-1,4-dihydropyridine-3,5-dicarboxylate (3a,
Figure 2). Following the general procedure starting from 4-(prop-2-yn-1-yloxy)benzaldehyde (1a
,
2.25 mmol, 360.02 mg), methyl acetoacetate (3.5 equiv, 7.88 mmol, 0.849 mL) and ammonium carbonate
(2.5 equiv, 5.63 mmol, 540.51 mg) at 75 ◦C over 15 h, compound 3a (509.4 mg, 64%) was isolated as
1
white powder: H-NMR (CDCl3)
δ 7.18 (d, J = 8.7 Hz, 2H, 2HAr), 6.82 (d, J = 8.7 Hz, 2H, 2HAr), 5.67 (s,
1H, NH), 4.95 (s, 1H, H4), 4.62 (d, J = 2.4 Hz, 2H, 2H1’), 3.64 (s, 6H, 2CO2CH3), 2.50 (t, J = 2.4 Hz, 1H,
H3’), 2.32 (s, 6H, 2CH3). 13C-NMR (CDCl3)
δ
168.20, 156.19, 144.14, 140.94, 128.98, 128.79, 114.45, 114.37,
104.17, 79.03, 75.42, 55.91, 51.13, 38.58, 19.74. Anal. Calcd. for C20H21NO5: C, 67.59; H, 5.96; N, 3.94.
Found: C, 67.33; H, 6.04; N, 3.89.
Figure 2. Chemical stucture of 3a.

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3,5-Diethyl-2,6-dimethyl-4-[4-(prop-2-yn-1-yloxy)phenyl]-1,4-dihydropyridine-3,5-dicarboxylate (3b
Figure 3). Following the general procedure starting from 4-(prop-2-yn-1-yloxy)benzaldehyde (1a
,
,
1 equiv, 2.25 mmol, 360.02 mg), ethyl acetoacetate (3.5 equiv, 7.88 mmol, 1.00 mL) and ammonium
carbonate (2.5 equiv, 5.63 mmol, 540.51 mg) at 75 ◦C over 15 h, the solvent was reduced under pressure
conditions and the residue was triturated with diethyl ether. The resulting solid was stirred in diethyl
ether overnight and filtered to afford compound 3b (564.10 mg, 47%) as white powder: 1H-NMR
(CDCl3)
δ 7.23 – 7.12 (m, 2H, 2HAr), 6.86 – 6.75 (m, 2H, 2HAr), 5.58 (bs, 1H, NH), 4.94 (s, 1H, H4), 4.63 (d,
J = 2.4 Hz, 2H, 2H1’), 4.17 – 3.99 (m, 4H, 2OCH2CH3), 2.49 (t, J = 2.4 Hz, 1H, H3’), 2.32 (s, 6H, 2CH3),
1.22 (t, J = 7.1 Hz, 6H, 2CO2CH2CH3). 13C-NMR (101 MHz, CDCl3)
δ 167.79, 156.12, 143.72, 141.36,
129.17, 114.29, 104.49, 79.04, 75.38, 59.85, 56.00, 55.94, 38.94, 19.76, 14.41. HRMS ESI-TOF [M]+ m/z
Calcd. for C22H25NO5: 383,1726. Found: 383,1733.
Figure 3. Chemical stucture of 3b.
3,5-Dimethyl-4- [3-methoxy-4-(prop-2-yn-1-yloxy) phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (3c, Figure 4).
Following the general procedure starting from 3-methoxy-4-
(prop-2-yn-1-yloxy)benzaldehyde (1b, 1 equiv, 1.84 mmol, 350.00 mg), methyl acetoacetate (3.5 equiv,
6.44 mmol, 0.695 mL) and ammonium carbonate (2.5 equiv, 4.60 mmol, 442.01 mg) at 75 ◦C over 15 h,
1
compound 3c (324.10 mg, 46%) precipitated as beige crystals: H-NMR (CDCl3)
δ 6.88 (dd, J = 5.0,
3.1 Hz, 2H, 2HAr), 6.75 (dd, J = 8.3, 1.9 Hz, 1H, HAr), 5.70 (d, J = 32.2 Hz, 1H, NH), 4.97 (s, 1H, H4), 4.69
(d, J = 2.3 Hz, 2H, 2H1’), 3.82 (s, 3H, OCH3), 3.66 (s, 6H, 2CO2CH3), 2.48 (t, J = 2.3 Hz, 1H, H3’), 2.33 (s,
6H, 2CH3). 13C-NMR (CDCl3)
δ 168.19, 149.09, 145.43, 144.19, 141.75, 119.52, 114.06, 112.02, 104.00,
79.10, 75.63, 56.87, 55.95, 51.15, 38.94, 19.75. HRMS ESI-TOF [M]+ m/z Calcd. for C21H23NO6: 385,1513.
Found: 385,1525.
Figure 4. Chemical stucture of 3c.
3,5-Diethyl-4-[3-methoxy-4-(prop-2-yn-1-yloxy)phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(3d, Figure 5). Following the general procedure starting from 3-methoxy-4-(prop-2-yn-1-yloxy)
benzaldehyde (1b, 1 equiv, 1.84 mmol, 350.00 mg), ethyl acetoacetate (3.5 equiv, 6.44 mmol, 0.822 mL)
◦
and ammonium carbonate (2.5 equiv, 4.60 mmol, 442.01 mg) at 75 C over 15 h, compound 3d
1
(581.40 mg, 76%) precipitated as a yellow powder: H-NMR (CDCl3)
δ
6.89 (dd, J = 6.3, 5.3 Hz, 2H,

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2HAr), 6.78 (dd, J = 8.3, 2.0 Hz, 1H, HAr), 5.61 (s, 1H, NH), 4.95 (s, 1H, H4), 4.69 (d, J = 2.4 Hz, 1H, 2H1’),
4.18 – 4.04 (m, 4H, 2CO2CH2CH3), 3.83 (s, 3H, OCH3), 2.47 (t, J = 2.4 Hz, 1H, H3’), 2.33 (s, 6H, 2CH3),
1.23 (t, J = 7.1 Hz, 6H, 2CO2CH2CH3). 13C-NMR (CDCl3)
δ 167.79, 148.96, 145.35, 143.79, 142.23, 119.91,
114.07, 112.37, 104.34, 79.11, 75.59, 59.87, 56.92, 55.92, 39.28, 19.78, 14.48. HRMS ESI-TOF [M]+ m/z
Calcd. for C23H27NO6: 413,1829. Found: 413,1838.
Figure 5. Chemical stucture of 3d.
3,5-Dimethyl-4-[3-ethoxy-4-(prop-2-yn-1-yloxy)phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(3e, Figure 6). Following the general procedure starting from 3-ethoxy-4-(prop-2-yn-1-yloxy)
benzaldehyde (1c, 1 equiv, 1.22 mmol, 191.30 mg), methyl acetoacetate (3.5 equiv, 4.30 mmol, 0.462 mL)
and ammonium carbonate (2.5 equiv, 3.06 mmol, 294.04 mg) at 75◦ C over 15 h, a precipitated was
isolated that was filtered and washed in diethyl ether:pentane (1:2 v/v) to afford compound 3a
1
(192.70 mg, 40%) as a white powder: H-NMR (CDCl3)
δ 6.88 (dd, J = 7.7, 5.1 Hz, 2H, 2HAr), 6.75 (dd, J
= 8.3, 1.9 Hz, 1H, HAr), 5.61 (s, 1H, NH), 4.95 (s, 1H, H4), 4.69 (d, J = 2.2 Hz, 1H, 2H1’), 4.06 (q, J =
7.0 Hz, 2H, OCH2CH3), 3.65 (s, 6H, 2CO2CH3), 2.47 (t, J = 2.3 Hz, 1H, H3’), 2.33 (s, 6H, 2CH3), 1.42 (t, J
= 7.0 Hz, 3H, OCH2CH3). 13C-NMR (CDCl3)
δ 168.21, 148.45, 145.76, 144.17, 141.89, 119.62, 114.91,
113.68, 104.02, 79.32, 75.50, 64.44, 57.10, 51.15, 38.90, 19.74, 14.96. HRMS ESI-TOF [M]+ m/z Calcd. for
C22H25NO6: 399,1673. Found: 399,1682.
Figure 6. Chemical stucture of 3e.
3,5-Diethyl-4-[3-ethoxy-4-(prop-2-yn-1-yloxy)phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (3f
,
Figure 7). Following the general procedure starting from 3-ethoxy-4-(prop-2-yn-1-yloxy)benzaldehyde
(
1c, 1 equiv, 1.01 mmol, 205.00 mg), ethyl acetoacetate (3.5 equiv, 3.54 mmol, 0.452 mL) and ammonium
carbonate (2.5 equiv, 2.53 mmol, 242.63 mg) at 75 ◦C over 15 h, the solvent was removed under pressure
conditions and the residue was triturated with diethyl ether. The resulting solid was stirred in diethyl
1
ether overnight and filtered to afford compound 3f (272.90 mg, 52%) as abrown powder: H-NMR
(CDCl3)
δ 6.89 (dd, J = 5.0, 3.2 Hz, 2H, 2HAr), 6.77 (dd, J = 8.3, 1.8 Hz, 1H, HAr), 5.54 (s, 1H, NH),
4.94 (s, 1H, H4), 4.69 (d, J = 2.3 Hz, 2H, 2H1’), 4.16 – 3.98 (m, 6H, OCH2CH3 and 2CO2CH2CH3), 2.46

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(t, J = 2.3 Hz, 1H, H3’), 2.33 (s, 6H, 2CH3), 1.42 (t, J = 7.0 Hz, 3H, OCH2CH3), 1.23 (t, J = 7.1 Hz,
6H, 2CO2CH2CH3). 13C-NMR (CDCl3)
δ 167.80, 148.45, 145.71, 143.72, 142.29, 120.02, 114.93, 114.01,
104.38, 79.37, 75.45, 64.46, 59.88, 57.18, 39.22, 19.81, 15.04, 14.48. HRMS ESI-TOF [M]+ m/z Calcd. for
C24H29NO6: 427,1977. Found: 427,1995.
Figure 7. Chemical stucture of 3f.
3,5-Dimethyl-4-[3-chloro-4-(prop-2-yn-1-yloxy)phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(3g, Figure 8). Following the general procedure starting from 3-chloro-4-(prop-2-yn-1-yloxy)
benzaldehyde (1d, 1 equiv, 1.54 mmol, 300.00 mg), methyl acetoacetate (3.5 equiv, 5.39 mmol, 0.578 mL)
and ammonium carbonate (2.5 equiv, 3.85 mmol, 369.95 mg) at 75 ◦C over 15 h, the observed precipitate
was filtered and washed in diethyl ether to afford compound 3g (405.71 mg, 68%) as a white solid:
1H-NMR (400 MHz, CDCl3)
δ
7.23 (d, J = 2.1 Hz, 1H, HAr), 7.13 (dd, J = 8.5, 2.1 Hz, 1H, HAr), 6.94
(d, J = 8.5 Hz, 1H, HAr), 5.64 (s, 1H, NH), 4.94 (s, 1H, H4), 4.71 (d, J = 2.3 Hz, 2H, 2H1’), 3.66 (s, 6H,
2CO2CH3), 2.52 (t, J = 2.3 Hz, 1H, H3’), 2.34 (s, 6H, 2CH3). 13C-NMR (101 MHz, CDCl3)
167.96, 151.68,
δ
144.38, 142.15, 129.74, 126.98, 122.80, 114.02, 103.76, 78.46, 76.09, 57.02, 51.22, 38.67, 19.83. HRMS
ESI-TOF [M]+ m/z Calcd. for C20H20ClNO5: 389,1024. Found: 389,1030.
Figure 8. Chemical stucture of 3g.
3,5-Diethyl-4-[3-chloro-4-(prop-2-yn-1-yloxy)phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (3h
,
Figure 9 . Following the general procedure starting from 3-chloro-4-(prop-2-yn-1-yloxy)benzaldehyde
)
(
1d, 1 equiv, 1.54 mmol, 300.00 mg), ethyl acetoacetate (3.5 equiv, 5.39 mmol, 0.688 mL) and ammonium
carbonate (2.5 equiv, 3.85 mmol, 369.95 mg) at 75 ◦C over 15 h, the precipitate was filtered and washed
1
with diethyl ether to afford compound 3h (416.9 mg, 68%) as a white solid: H-NMR (CDCl3)
δ 7.29 (s,
1H, HAr), 7.16 (dd, J = 8.5, 2.2 Hz, 1H, HAr), 6.95 (d, J = 8.5 Hz, 1H, HAr), 5.61 (s, 1H, NH), 4.94 (s, 1H,
H4), 4.74 (d, J = 2.4 Hz, 2H, 2H1’), 4.21 – 4.03 (m, 4H, 2CO2CH2CH3), 2.54 (t, J = 2.4 Hz, 1H, H3’), 2.36 (s,
6H, 2CH3), 1.25 (t, J = 7.1 Hz, 6H, 2CO2CH2CH3). 13C-NMR (CDCl3)
δ 296.64, 280.65, 273.12, 271.69,
259.32, 256.41, 251.67, 243.07, 233.13, 207.55, 205.15, 189.07, 186.15, 168.17, 148.91, 143.51. Anal. Calcd.
for C22H24ClNO5: C, 63.23; H, 5.79; N, 3.35. Found: C, 62.93; H, 5.88; N, 3.39.

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Figure 9. Chemical stucture of 3h.
3,5-Dimethyl-4-[3-bromo-4-(prop-2-yn-1-yloxy)phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate(3i
Figure 10). Following the general procedure starting from 3-bromo-4-(prop-2-yn-1-yloxy)benzaldehyde
1e, 1 equiv, 1.05 mmol, 250.00 mg), methyl acetoacetate (3.5 equiv, 3.66 mmol, 0.395 mL) and ammonium
,
(
carbonate (2.5 equiv, 2.63 mmol, 252.24 mg) at 75 ◦C over 15 h, the precipitate was filtered and washed
with diethyl diethyl ether:pentane (1:1 v/v) to afford compound 3i (266.20 mg, 58%) as a white solid:
1H-NMR (CDCl3) δ 7.40 (d, J = 2.2 Hz, 1H, HAr), 7.17 (dd, J = 8.5, 2.2 Hz, 1H, HAr), 6.92 (d, J = 8.5 Hz,
1H, HAr), 5.63 (s, 1H, NH), 4.94 (s, 1H, H4), 4.71 (d, J = 2.4 Hz, 2H, 2H1’), 3.66 (s, J = 6.2 Hz, 6H,
2CO2CH3), 2.52 (t, J = 2.4 Hz, 1H, H3’), 2.34 (s, 6H, 2CH3). 13C-NMR (CDCl3)
δ 167.95, 152.59, 144.38,
142.54, 132.78, 127.78, 113.81, 112.07, 103.77, 78.45, 76.10, 57.07, 51.22, 38.62, 19.84. HRMS ESI-TOF [M]+
m/z Calcd. for C20H20BrNO5: 433,0516. Found: 433,0525.
Figure 10. Chemical stucture of 3i.
3,5-Diethyl-4-[3-bromo-4-(prop-2-yn-1-yloxy)phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (3j
,
Figure 11). Following the general procedure starting from 3-bromo-4-(prop-2-yn-1-yloxy)benzaldehyde
(
1e, 1 equiv, 1.05 mmol, 205.00 mg), ethyl acetoacetate (3.5 equiv, 3.66 mmol, 0.467 mL) and ammonium
carbonate (2.5 equiv, 2.63 mmol, 252.24 mg) at 70 ◦C over 15 h, the solvent was removed under pressure
conditions and the residue was triturated with diethyl ether. The resulting solid was stirred in diethyl
ether:pentane (1:1 v/v) overnight and filtered to afford compound 3j (202.71 mg, 42%) as white powder:
1H-NMR (CDCl3)
δ 7.43 (d, J = 2.1 Hz, 1H, HAr), 7.18 (dd, J = 8.5, 2.1 Hz, 1H, HAr), 6.91 (d, J = 8.5 Hz, 1H,
HAr), 5.56 (s, 1H, NH), 4.91 (s, 1H, H4), 4.71 (d, J = 2.4 Hz, 2H, 2H1’), 4.22 – 4.01 (m, 4H, 2CO2CH2CH3),
2.51 (t, J = 2.4 Hz, 1H, H3’), 2.34 (s, 6H, 2CH3), 1.23 (t, J = 7.1 Hz, 6H, 2CO2CH2CH3). 13C-NMR (CDCl3)
δ
167.51, 152.46, 143.99, 142.98, 133.29, 128.09, 113.76, 111.78, 104.06, 78.44, 76.06, 59.97, 57.10, 39.03,
19.83, 14.42. HRMS ESI-TOF [M]+ m/z Calcd. for C22H24BrNO5: 461,0829. Found: 461,0838.

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Figure 11. Chemical stucture of 3j.
3,5-Dimethyl-4-[4-(but-3-yn-1-yloxy)phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (3k
,
Figure 12). Following the general procedure starting from 4-(but-3-yn-1-yloxy)benzaldehyde (2a
)
(1 equiv, 1.43 mmol, 250.00 mg), methyl acetoacetate (3.5 equiv, 5.02 mmol, 0.640 mL) and ammonium
◦
carbonate (2.5 equiv, 3.58 mmol, 343.52 mg) at 75 C over 15 h, the solvent was removed under
pressure conditions and the resulting residue was purified by flash column chromatography using
1
hexane/EtOAc (7/
(CDCl3)
7.0 Hz, 2H, 2H1’), 3.64 (s, 6H, 2CO2CH3), 2.64 (td, J = 7.0, 2.7 Hz, 2H, 2H2’), 2.33 (s, 6H, 2CH3), 2.01 (t, J
·
3) + 1% Et3N, to afford compound 3k (144.2 mg, 27%) as white crystals: H-NMR
7.21–7.10 (m, 2H, 2HAr), 6.79–6.72 (m, 2H, 2HAr), 5.61 (s, 1H, NH), 4.94 (s, 1H, H4), 4.04 (t, J =
δ
= 2.7 Hz, 1H, H4’). 13C-NMR (CDCl3)
δ 168.20, 156.93, 144.03, 140.51, 129.01, 128.82, 114.28, 114.19,
104.27, 80.72, 69.90, 66.01, 51.13, 38.60, 19.77, 19.70. HRMS ESI-TOF [M]+ m/z Calcd. for C21H23NO5:
397,1879. Found: 397,1889.
Figure 12. Chemical stucture of 3k.
3,5-Diethyl-4-[4-(but-3-yn-1-yloxy)phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
Figure 13). Following the general procedure starting from 4-(but-3-yn-1-yloxy)benzaldehyde (2a
(
3l
,
,
1 equiv, 1.43 mmol, 250.00 mg), ethyl acetoacetate (3.5 equiv, 5.02 mmol, 0.540 mL) and ammonium
carbonate (2.5 equiv, 3.58 mmol, 343.52 mg) at 75 ◦C over 15 h, the solvent was removed under pressure
conditions and the residue was triturated with diethyl ether. The resulting residue was recrystallized
in DCM/diethyl ether (1:1 v/v) to afford compound 3l (70.10 mg, 12%) as a white solid: 1H-NMR
(CDCl3)
δ 7.23–7.11 (m, 2H, 2HAr), 6.78–6.72 (m, 2H, 2HAr), 5.57 (s, 1H, NH), 4.92 (s, 1H, H4), 4.15–3.99
(m, 6H, 2CO2CH2CH3 and 2H1’), 2.64 (td, J = 7.0, 2.7 Hz, 2H, 2H2’), 2.32 (s, 6H, 2CH3), 2.01 (t, J =
2.7 Hz, 1H, H4’), 1.22 (t, J = 7.1 Hz, 6H, 2CO2CH2CH3). 13C-NMR (CDCl3)
δ 167.79, 156.86, 143.65,
140.92, 129.18, 114.11, 104.54, 80.73, 69.90, 66.03, 59.84, 38.93, 19.77, 19.70, 14.42. HRMS ESI-TOF [M]+
m/z Calcd. for C23H27NO5: 369,1570. Found: 369,1576.

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Figure 13. Chemical stucture of 3l.
3,5-Dimethyl-4-[4-(but-3-yn-1-yloxy)-3-methoxyphenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
3m, Figure 14). Following the general procedure starting from 4-(but-3-yn-1-yloxy)-
(
3-methoxybenzaldehyde (2b, 1 equiv, 1.47 mmol, 300.00 mg), methyl acetoacetate (3.5 equiv,
5.14 mmol, 0.594 mL) and ammonium carbonate (2.5 equiv, 3.68 mmol, 353.13 mg) at 75 ◦C over 15 h,
the precipitate was filtered and washed in diethyl ether:pentane (1:1 v/v) to afford compound 3m
1
(420.5 mg, 72%) as a white solid: H-NMR (CDCl3)
δ
6.87 (s, 1H, HAr), 6.74 (d, J = 0.8 Hz, 2H, 2HAr),
5.63 (s, 1H, NH), 4.95 (s, 1H, H4), 4.10 (t, J = 7.4 Hz, 2H, 2H1’), 3.82 (s, 3H, OCH3), 3.66 (s, 6H, 2CO2CH3),
2.68 (td, J = 7.4, 2.7 Hz, 2H, 2H2’), 2.34 (s, 6H, 2CH3), 2.01 (t, J = 2.7 Hz, 1H, H4’). 13C-NMR (CDCl3) δ
168.19, 149.13, 146.29, 144.09, 141.35, 119.75, 113.79, 112.39, 104.09, 80.54, 70.03, 67.23, 56.14, 51.14, 38.94,
19.78, 19.61. Anal. Calcd. for C22H25NO5: C, 66.15; H, 6.31; N, 3.51. Found: C, 66.23; H, 6.36; N, 3.59.
Figure 14. Chemical stucture of 3m.
3,5-Diethyl-4-[4-(but-3-yn-1-yloxy)-3-methoxyphenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (3n
,
Figure 15). Following the general procedure starting from 4-(but-3-yn-1-yloxy)-3-methoxybenzaldehyde
(
2b, 1 equiv, 1.47 mmol, 300.00 mg), ethyl acetoacetate (3.5 equiv, 5.14 mmol, 0.656 mL) and ammonium
carbonate (2.5 equiv, 3.68 mmol, 353.13 mg) at 75 ◦C over 15 h, the solvent was removed under pressure
conditions and the residue was triturated with diethyl ether. The resulting solid was stirred in diethyl
ether:pentane (1:1 v/v) overnight and filtered to afford compound 3n (520.92 mg, 83%) as a light
1
yellowish solid: H-NMR (CDCl3)
δ 6.88 (d, J = 1.7 Hz, 1H, HAr), 6.79–6.70 (m, 2H, 2HAr), 5.55 (s,
1H, NH), 4.94 (s, 1H, H4), 4.10 (pd, J = 8.2, 3.7 Hz, 6H, 2CO2CH2CH3 and 2H1’), 3.82 (s, 3H, OCH3),
2.68 (td, J = 7.5, 2.7 Hz, 2H, 2H2’), 2.33 (s, 6H, 2CH3), 2.01 (t, J = 2.7 Hz, 1H, H4’), 1.23 (t, J = 7.1 Hz,
6H, 2CO2CH2CH3). 13C-NMR (CDCl3)
δ 167.78, 148.99, 146.21, 143.70, 141.81, 120.14, 113.78, 112.73,
104.42, 80.56, 70.01, 67.27, 59.88, 56.12, 39.26, 19.81, 19.62, 14.49. HRMS ESI-TOF [M]+ m/z Calcd. for
C24H29NO6: 427,1985. Found: 427,1995.

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Figure 15. Chemical stucture of 3n.
3,5-Dimethyl-4-[4-(but-3-yn-1-yloxy)-3-ethoxyphenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (3o
,
Figure 16). Following the general procedure starting from 4-(but-3-yn-1-yloxy)-3-ethoxybenzaldehyde
(
2c, 1 equiv, 1.60 mmol, 350.00 mg), methyl acetoacetate (3.5 equiv, 5.61 mmol, 0.679 mL) and ammonium
carbonate (2.5 equiv, 4.00 mmol, 384.36 mg) at 75 ◦C over 15 h, the precipitate was filtered and washed
1
in diethyl ether:pentane (1:1 v/v) to afford compound 3o (288.40 mg, 44%) as a white solid: H-NMR
(CDCl3)
4H, OC
2.02 (t, J = 2.6 Hz, 1H, H4’), 1.42 (t, J = 7.0 Hz, 3H, OCH2CH3). 13C-NMR (CDCl3)
δ
6.89 (s, 1H, HAr), 6.77 (s, 2H, 2HAr), 5.67 (s, 1H, NH), 4.95 (s, 1H, H4), 4.09 (dt, J = 2.3, 7.1 Hz,
2CH3 and 2H1’), 3.67 (s, 6H, 2CO2CH3), 2.69 (td, J = 7.4, 2.6 Hz, 2H, 2H2’), 2.35 (s, 6H, 2CH3),
168.20, 148.54,
H
δ
146.73, 144.08, 141.58, 120.21, 120.01, 114.84, 114.43, 104.10, 80.72, 69.88, 67.61, 64.76, 51.13, 38.91, 19.75,
19.68, 15.03. HRMS ESI-TOF [M]+ m/z Calcd. for C23H27NO6: 413,1831. Found: 413,1838.
Figure 16. Chemical stucture of 3o.
3,5-Diethyl-4-[4-(but-3-yn-1-yloxy)-3-ethoxyphenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (3p
,
Figure 17). Following the general procedure starting from 4-(but-3-yn-1-yloxy)-3-ethoxybenzaldehyde
(
2c, 1 equiv, 1.60 mmol, 350.00 mg), ethyl acetoacetate (3.5 equiv, 5.61 mmol, 0.716 mL) and ammonium
carbonate (2.5 equiv, 4.00 mmol, 384.36 mg) at 75 ◦C over 15 h, the precipitate was filtered and washed
in diethyl ether:pentane (1:1 v/v) to afford compound 3p (340.31 mg, 48%) as a white solid: H-NMR
(CDCl3)
δ 6.88 (d, J = 1.3 Hz, 1H, HAr), 6.81–6.70 (m, 2H, 2HAr), 5.52 (s, 1H, NH), 4.93 (s, 1H, H4),
4.17 – 3.99 (m, 8H, OCH2CH3 and 2H1’ and 2CO2CH2CH3), 2.67 (td, J = 7.4, 2.7 Hz, 2H, 2H2’), 2.33
(s, 6H, 2CH3), 2.00 (t, J = 2.7 Hz, 1H, H4’), 1.40 (t, J = 7.0 Hz, 3H, OCH2CH3), 1.23 (t, J = 7.1 Hz, 6H,
2CO2CH2CH3). 13C-NMR (CDCl3)
δ 167.79, 148.52, 146.68, 143.64, 141.97, 120.41, 114.81, 114.76, 104.44,
80.71, 69.86, 67.66, 64.80, 59.86, 39.20, 19.81, 19.70, 15.10, 14.48. Anal. Calcd. for C25H31NO6: C, 68.01;
H, 7.08; N, 3.17. Found: C, 67.87; H, 7.15; N, 3.25. HRMS ESI-TOF [M]+ m/z Calcd. for C25H31NO6:
441,2137. Found: 441,2151.

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Figure 17. Chemical stucture of 3p.
3,5-Dimethyl-4-[4-(but-3-yn-1-yloxy)-3-chlorophenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (3q
,
Figure 18). Following the general procedure starting from 4-(but-3-yn-1-yloxy)-3-chlorobenzaldehyde
(
2d, 1 equiv, 1.44 mmol, 300.00 mg), methyl acetoacetate (3.5 equiv, 5.03 mmol, 0.543 mL) and
ammonium carbonate (2.5 equiv, 3.60 mmol, 345.92 mg) at 78 ◦C over 15 h, the solvent was removed
under pressure conditions and the resulting residue was purified by flash column chromatography
1
using hexane/EtOAc (6/4) + 1% Et3N, to afford compound 3q (76.05 mg, 13%) as a white solid: H-NMR
(CDCl3)
5.64 (s, 1H, NH), 4.92 (s, 1H, H4), 4.10 (t, J = 7.2 Hz, 2H, 2H1’), 3.65 (s, J = 4.3 Hz, 6H, 2CO2CH3), 2.70
(td, J = 7.2, 2.7 Hz, 2H, 2H2’), 2.34 (s, 6H, 2CH3), 2.02 (t, J = 2.7 Hz, 1H, H4’). 13C-NMR (CDCl3)
167.84,
δ 7.22 (d, J = 2.2 Hz, 1H, HAr), 7.11 (dd, J = 8.5, 2.2 Hz, 1H, HAr), 6.78 (d, J = 8.5 Hz, 1H, HAr),
δ
152.29, 144.19, 141.61, 129.55, 126.95, 122.72, 113.59, 103.67, 80.11, 70.01, 67.27, 51.06, 38.55, 19.68, 19.48.
HRMS ESI-TOF [M]+ m/z Calcd. for C21H22ClNO5: 403,1180. Found: 403,1187.
Figure 18. Chemical stucture of 3q.
3,5-Diethyl-4-[4-(but-3-yn-1-yloxy)-3-chlorophenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (3r
,
Figure 19). Following the general procedure starting from 4-(but-3-yn-1-yloxy)-3-chlorobenzaldehyde
(
2d) (1 equiv, 1.44 mmol, 300.00 mg), ethyl acetoacetate (3.5 equiv, 5.03 mmol, 0.643 mL) and ammonium
◦
carbonate (2.5 equiv, 3.60 mmol, 345.92 mg) at 79 C over 15 h, the solvent was removed under
pressure conditions and the resulting residue was purified by flash column chromatography using
hexane/EtOAc (6/4) + 1% Et3N, to afford compound 3r (80.23 mg, 13%) as a white solid: H-NMR
1
(CDCl3)
δ 7.25 (d, J = 2.2 Hz, 1H, HAr), 7.12 (dd, J = 8.4, 2.2 Hz, 1H, HAr), 6.78 (d, J = 8.5 Hz, 1H,
HAr), 5.57 (s, 1H, NH), 4.90 (s, 1H, H4), 4.20–4.01 (m, 6H, 2H1’ and 2CO2CH2CH3), 2.70 (td, J = 7.3,
2.7 Hz, 2H, 2H2’), 2.33 (s, 6H, 2CH3), 2.02 (t, J = 2.7 Hz, 1H, H4’), 1.23 (t, J = 7.1 Hz, 6H, 2CO2CH2CH3).
13C-NMR (CDCl3)
δ 167.55, 152.33, 143.95, 142.16, 130.16, 127.41, 122.61, 113.64, 104.08, 80.26, 70.14,
67.44, 59.95, 39.06, 22.09, 19.81, 19.62, 14.41. HRMS ESI-TOF [M]+ m/z Calcd. for C23H26ClNO5:
431,1491. Found: 431,1500.

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Figure 19. Chemical stucture of 3r.
3,5-Dimethyl-4-[3-bromo-4-(but-3-yn-1-yloxy)phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (3s
,
Figure 20). Following the general procedure starting from 3-bromo-4-(but-3-yn-1-yloxy)benzaldehyde
(
2e, 1 equiv, 0.77 mmol, 195.00 mg), methyl acetoacetate (3.5 equiv, 2.69 mmol, 0.291 mL) and ammonium
carbonate (2.5 equiv, 1.93 mmol, 189.97 mg) at 75 ◦C over 15 h, the precipitated was filtered and washed
1
in diethyl ether:pentane (1:3 v/v) to afford compound 3s (142.40 mg, 23%) as a white solid: H-NMR
(CDCl3)
δ 7.38 (d, J = 1.8 Hz, 1H, HAr), 7.16 (d, J = 8.4 Hz, 1H, HAr), 6.76 (d, J = 8.4 Hz, 1H, HAr), 5.62 (s,
1H, NH), 4.92 (s, 1H, H4), 4.10 (t, J = 7.2 Hz, 2H, 2H1’), 3.65 (s, 6H, 2CO2CH3), 2.70 (td, J = 7.1, 2.5 Hz,
2H, 2H2’), 2.34 (s, 6H, 2CH3), 2.03 (d, J = 2.4 Hz, 1H, H4’). 13C-NMR (CDCl3)
δ 167.95, 153.32, 144.30,
142.15, 132.73, 127.89, 113.49, 103.82, 80.27, 70.15, 67.46, 51.20, 38.65, 19.83, 19.62. HRMS ESI-TOF [M]+
m/z Calcd. for C21H22BrNO5: 447,0666. Found: 447,0681.
Figure 20. Chemical stucture of 3s.
3,5-Diethyl-4-[3-bromo-4-(but-3-yn-1-yloxy)phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (3t
Figure 21). Following the general procedure starting from 3-bromo-4-(but-3-yn-1-yloxy)benzaldehyde
2e, 1 equiv, 0.77 mmol, 195.00 mg), ethyl acetoacetate (3.5 equiv, 1.69 mmol, 0.343 mL) and ammonium
,
(
carbonate (2.5 equiv, 1.93 mmol, 184.97 mg) at 85 ◦C over 15 h, the solvent was removed under pressure
conditions and the residue was triturated with diethyl ether. The resulting solid was stirred in diethyl
ether:pentane (1:1 v/v) overnight and filtered to afford compound 3t (280.92 mg, 49%) as a white solid:
1H-NMR (CDCl3) δ 7.42 (d, J = 2.1 Hz, 1H, HAr), 7.16 (dd, J = 8.4, 2.2 Hz, 1H, HAr), 6.88 – 6.66 (m, 1H,
HAr), 5.55 (s, 1H, NH), 4.90 (s, 1H, H4), 4.31 – 4.02 (m, 6H, 2H1’ and 2CO2CH2CH3), 2.70 (td, J = 7.3,
2.7 Hz, 2H, 2H2’), 2.33 (s, 6H, 2CH3), 2.03 (t, J = 2.7 Hz, 1H, H4’), 1.23 (t, J = 7.1 Hz, 6H, 2CO2CH2CH3).
13C-NMR (CDCl3)
δ 167.56, 153.21, 144.02, 142.58, 133.20, 128.18, 113.42, 111.93, 104.03, 80.27, 70.14,
67.48, 59.95, 39.01, 19.77, 19.61, 14.41. HRMS ESI-TOF [M]+ m/z Calcd. for C23H26BrNO5: 475,0983.
Found: 475,0994.

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Figure 21. Chemical stucture of 3t.
3.5. Biological Evaluation
3.5.1. Calcium Channel Blockade
Human neuroblastoma cell line SH-SY5Y (CRL-2266) obtained from the American Type Culture
Collection (Manassas, VA, USA) was maintained at 37◦C in humidified atmosphere of 5% CO2/95%
air. Mixture of Dulbecco’s Modified Eagle Medium and Nutrient Mixture F-12 (1:1) containing 10% of
fetal bovine serum, was used as a culture medium (Thermo Fisher Scientific). The cells were seeded
out in 96-well dark-walled plates at a density of 1
×
105 cells per well. The cells were between second
and eighth passage number at the time of the experiment. After 24 h, the cells were loaded with
FLIPR Calcium 6 indicator (Molecular Devices, San Jose, CA, USA) for 2 h at 37 ◦C, according to the
manufacturers protocol. Compounds of interest were dissolved in appropriate amount of DMSO,
in order to prepare stock solutions at 10 mM concentration. They were then subsequently diluted
to a final concentration of 10 µ
M in Hanks0 Balanced Salt Solution (HBSS, Thermo Fisher Scientific,
Waltham, MA, USA) buffered with HEPES (Sigma-Aldrich) at pH = 7.4 and as such used for treatment
of indicator loaded cells (10 min at 37 ◦C). Fluorescence of indicator loaded cells was measured with
Synergy H1 (Biotek Instruments, Winooski, VT, USA) multilabel plate reader at excitation and emission
wavelengths of 485 and 525 nm, respectively. The baseline fluorescence was recorded for 5 sec. Then,
the cells were stimulated with KCl/CaCl2 solution (in HBSS, final concentration of KCl and CaCl2
was 90 mM and 5 mM, respectively) and the fluorescence was recorded for further 30 s. Dimethyl
sulfoxide 1% solution in HBSS was used as a vehicle control. Nimodipine (10 µM) was used as
a reference inhibitor. Compounds were assessed at the same final concentration as nimodipine in
triplicates in three independent experiments. Fluorescent intensity values were normalized to the
baseline. Outlier detection by Grubbs’ test was performed and outlying values were excluded from the
further analysis.
3.5.2. Oxygen Radical Absorbance Capacity Assay
The antioxidant activity of hybrids 3a-tI was carried out by the ORAC-FL using fluorescein as a
fluorescent probe. Briefly, fluorescein and antioxidant were incubated in a black 96-well microplate
(Nunc, Thermo Scientific, 67403 Illkirch France) for 15 min at 37 ◦C. 2,20-Azobis(amidinopropane)
dihydrochloride was then added quickly using the built-in injector of a Varioskan Flash plate reader
(Thermo Scientific). The fluorescence was measured at 485 nm (excitation wavelength) and 535 nm
(emission wavelength) each min for 1h. All the reactions were made in triplicate and at least three
different assays were performed for each sample.
3.5.3. hMAOs Inhibition Screening
The effect of the test compounds on the activity of both hMAO isoforms was evaluated by measuring
the production of 4-hydroxyquinoline (4-HQ, λmax= 316 nm) from kynuramine, using microsomal

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recombinant hMAO isoforms. Prior to the each experiment, stock solutions were prepared in the
following conditions: test compounds/standard inhibitors (2 mM in DMSO), kynuramine (400 µM in
dH2O), hMAO-A (36.7
in sodium phosphate buffer 0.05 M pH 7.4,). The amount of hMAO-A (specific activity = 116 pmol
kynuramine/min/ g protein) and hMAO-B (specific activity = 16.4 pmol kynuramine/min/ µg
µg/mL in sodium phosphate buffer 0.05 M pH 7.4,) and hMAO-B (147.8 µg/mL
µ
protein) was adjusted to obtain the same reaction rate (50 pmol kynuramine/min) under our
experimental conditions. The assays were run in flat bottom 96-well microplates (BRANDplates,
Pure GradeTM, BRAND GMBH, Wertheim, Germany) using a multimode plate reader (Biotek Synergy
HT). Each experiment included control wells (vehicle) and standard inhibitors (clorgyline for hMAO-A
and deprenyl for hMAO-B). In a typical experiment, 1
(test wells) or DMSO (control wells), 20 L of kynuramine and 160
(0.05 M, pH 7.4) were incubated at 37 ◦C for 10 min and the absorbance at 316 nm was read to correct
the background signal. After this incubation, 20 L of hMAO-A or hMAO-B were added and the
µL the test compounds/standard inhibitors
µ
µL of sodium phosphate buffer
µ
absorbance at 316 nm was read each minute over 45 min. Using a previously determined calibration
curve for 4-HQ, the amount of 4-HQ formed was calculated and the n (4-HQ)= f (t) plots were obtained
for each treatment. The reaction rates (ν0, indicted by the slopes) were used to calculate the % of
inhibition, according to the following formula: % inhibition = [ν0 (control) - ν0 (test)]/ ν0 (control) *
100. The IC50 of the standard inhibitors were determined using the same protocol, using increasing
concentrations of chlorgyline and deprenyl (0–100 nM) and tracing the dose-response curves. All values
(% inhibition and IC50) are the mean ± SD from three experiments.
3.5.4. Effect of Compounds 3a, 3h and 3j on H2O2 (200
R at 30 µM)-Induced Cell Death in SH-SY5Y Cells
µM) and Oligomycin/Rotenone (O at 10µM and
SH-SY5Y cells were seeded in 96-well culture plates at a density of 12
DMEM/F12 (1:1) medium supplemented with 10% fetal bovine serum, 1
×
104 cells per well in
non-essential amino
×
acids, 100 units/mL penicillin and 10 mg/mL streptomycin (Dutscher, 67170 Brumath, France).
After 48 h of incubation, the cultures were treated with 100 L of the test compounds or DMSO
(0.1%) in the same medium. Following 24 h, the cells were co-incubated with H2O2 (200 M)
or a mixture of oligomycin/rotenone (10 M/ 30 M) with or without the tested compounds at
µ
µ
µ
µ
noncytotoxic concentrations for an additional period of 24 h. The percent of cell viability was
measured using CellTiter 96 AQueous Non-Radioactive Cell Proliferation (MTS) Assay (Promega,
Charbonnières-les-Bains, France).
4. Conclusions
Compounds 3a–t have been successfully synthesized in modest to high yields by Hantzsch
multicomponent reactions, and their biological evaluation, as potential MAO inhibitors, Ca2+
channel blockers, antioxidant and neuroprotective agents has been assessed. Unfortunately,
our molecules displayed very low MAO inhibition power. However, concerning Ca2+ channel
blockade results, it is worth mentioning that compounds 3a and 3h showed Ca2+ influx blockage
values of 47%, and 42.8%, respectively, very close to the standard reference nimodipine (52.8%)
at 10
µ )
M. The most active compounds were those with n = 1 linker length, with R1= H (3a
and R1= Cl (3h). This suggests that the C3 position at the aryl core is involved in the Ca2+
channel blockade, and that this finding is a good strategy for further pharmacomodulation studies.
The antioxidant activity allowed us to identify molecules 3l (2.45 TE), 3n (2.78 TE) and 3p (2.78 TE)
as interesting antioxidants with ORAC values equal or higher than melatonin (2.45TE). Moreover,
and very interestingly, the most potent Ca2+ channel agonists, 3a and 3h, showed interesting ORAC
values equal to 1.75TE and 1.35TE. The neuroprotective activity results support the interest of
compounds 3a and 3h, particularly for their neuroprotective activity against H2O2 in SHSY5Y cells.
To sum up, the biological analyses have prompted us to identify the multifunctional compound
3,5-dimethyl-2,6–dimethyl–4-[4-(prop–2–yn–1-yloxy)phenyl]-1,4-dihydropyridine-3,5-dicarboxylate

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(
3a) as an attractive antioxidant (1.75 TE), Ca2+ channel antagonist (47.0% at 10
µM), showing significant
neuroprotection (38%) against H2O2 at 10 µM, being considered thus as a new hit-compound for
further investigation in our search for anti-Alzheimer’s disease agents. These preliminary results
confirmed in part the efficiency of our design. Thus, based on the poor MAO inhibition results,
we intend to design and prepare new compounds witn nitrogen instead of oxygen at the aromatic ring,
that we hope and expect to be more appropriate for efficient MAO inhibition. This work is now in
progress in our laboratories, and the results will be presented elsewhere in due course.
Supplementary Materials: The following are available online, Figures are NMR Spectra of compounds 3a–t.
Author Contributions: Conceptualization, L.I.; methodology, L.I., H.M., K.J. and F.B.; investigation, I.P.A., S.D.,
A.B., M.M., A.W., and T.B.S.; resources, B.R.; writing—original draft preparation, L.I. and K.J.; writing—review
and editing, J.M.-C.; supervision, L.I.; funding acquisition, L.I. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was supported by the Regional Council of Franche-Comté (2016YC-04540 and 04560) and
supported by Foundation for Science and Technology (FCT) and FEDER/COMPETE (Grants UID/QUI/00081/2020,
PTDC/MED-QUI/29164/2017, and SFRH/BPD/114945/2016).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Bartus, R.; Dean, R.; Beer, B.; Lippa, A. The cholinergic hypothesis of geriatric memory dysfunction. Science
1982, 217, 408–414. [CrossRef] [PubMed]
2.
Bond, M.; Rogers, G.; Peters, J.; Anderson, R.; Hoyle, M.; Miners, A.; Moxham, T.; Davis, S.; Thokala, P.;
Wailoo, A.; et al. The effectiveness and cost-effectiveness of donepezil, galantamine, rivastigmine and
memantine for the treatment of Alzheimer’s disease (review of Technology Appraisal No. 111): A systematic
review and economic model. Heal. Technol. Assess. 2012, 16, 1–470. [CrossRef] [PubMed]
Gasque, M.J.O.; Marco-Contelles, J. Alzheimer’s Disease, the “One-Molecule, One-Target” Paradigm, and the
Multitarget Directed Ligand Approach. ACS Chem. Neurosci. 2018, 9, 401–403. [CrossRef]
Morphy, R.; Rankovic, Z. Designing multiple ligands - medicinal chemistry strategies and challenges.
Curr. Pharm. Des. 2009, 15, 587–600. [CrossRef] [PubMed]
3.
4.
5.
Agis-Torres, A.; Sollhuber, M.; Fernández, M.; Sánchez-Montero, J.M. Multi-Target-Directed ligands and
other therapeutic strategies in the search of a real solution for Alzheimer’s disease. Curr. Neuropharmacol.
2014, 12, 2–36. [CrossRef] [PubMed]
6.
Cavalli, A.; Bolognesi, M.L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Recanatini, M.; Melchiorre, C.
Multi-target-Directed ligands to combat neurodegenerative diseases. J. Med. Chem. 2008, 51, 347–372.
[CrossRef]
7.
8.
León, R.; Garcia, A.G.; Marco-Contelles, J. Recent advances in the multitarget-directed ligands approach for
the treatment of Alzheimer’s disease. Med. Res. Rev. 2013, 33, 139–189. [CrossRef]
Ismaili, L.; Refouvelet, B.; Benchekroun, M.; Brogi, S.; Brindisi, M.; Gemma, S.; Campiani, G.; Filipic, S.;
Agbaba, D.; Esteban, G.; et al. Multitarget compounds bearing tacrine-and donepezil-like structural and
functional motifs for the potential treatment of Alzheimer’s disease. Prog. Neurobiol. 2017, 151, 4–34.
[CrossRef]
9.
Prati, F.; De Simone, A.; Bisignano, P.; Armirotti, A.; Summa, M.; Pizzirani, D.; Scarpelli, R.; Perez, D.I.;
Andrisano, V.; Perez-Castillo, A.; et al. Multitarget Drug Discovery for Alzheimer’s Disease: Triazinones as
BACE-1 and GSK-3β Inhibitors. Angew. Chem. Int. Ed. 2014, 54, 1578–1582. [CrossRef]
10. Gandini, A.; Bartolini, M.; Tedesco, D.; Mart
ínez-González, L.; Roca, C.; Campillo, N.E.; Zaldivar-Diez, J.;
Perez, C.; Zuccheri, G.; Miti, A.; et al. Tau-Centric Multitarget Approach for Alzheimer’s Disease:
Development of First-in-Class Dual Glycogen Synthase Kinase 3
β and Tau-Aggregation Inhibitors.
J. Med. Chem. 2018, 61, 7640–7656. [CrossRef]
11. Nepovimova, E.; Korabecny, J.; Dolezal, R.; Babkova, K.; Ondrejicek, A.; Jun, D.; Hepnarova, V.; Horova, A.;
Hrabinova, M.; Soukup, O.; et al. Tacrine–Trolox Hybrids: A Novel Class of Centrally Active, Nonhepatotoxic
Multi-Target-Directed Ligands Exerting Anticholinesterase and Antioxidant Activities with Low In Vivo
Toxicity. J. Med. Chem. 2015, 58, 8985–9003. [CrossRef]

Molecules 2020, 25, 1329
20 of 21
12. Benchekroun, M.; Ismaili, L.; Pudlo, M.; Luzet, V.; Gharbi, T.; Refouvelet, B.; Marco-Contelles, J.
Donepezil–ferulic acid hybrids as anti-Alzheimer drugs. Futur. Med. Chem. 2015, 7, 15–21. [CrossRef]
13. Benchekroun, M.; Romero, A.; Egea, J.; Leon, R.; Michalska, P.; Buendía, I.; Jimeno, M.L.; Jun, D.; Janockova, J.;
Hepnarova, V.; et al. The Antioxidant Additive Approach for Alzheimer’s Disease Therapy: New Ferulic
(Lipoic) Acid Plus Melatonin Modified Tacrines as Cholinesterases Inhibitors, Direct Antioxidants, and
Nuclear Factor (Erythroid-Derived 2)-Like 2 Activators. J. Med. Chem. 2016, 59, 9967–9973. [CrossRef]
14. Ismaili, L.; Carreiras, M.D.C. Multicomponent Reactions for Multitargeted Compounds for Alzheimer’s
Disease. Curr. Top. Med. Chem. 2018, 17, 3319–3327. [CrossRef]
15. Malek, R.; Arribas, R.L.; Palomino-Antolín, A.; Totoson, P.; Demougeot, C.; Kobrlova, T.; Soukup, O.; Iriepa, I.;
Moraleda, I.; Diez-Iriepa, D.; et al. New Dual Small Molecules for Alzheimer’s Disease Therapy Combining
Histamine H3 Receptor (H3R) Antagonism and Calcium Channels Blockade with Additional Cholinesterase
Inhibition. J. Med. Chem. 2019, 62, 11416–11422. [CrossRef]
16. Malek, R.; Maj, M.; Wnorowski, A.; Józ´wiak, K.; Martin, H.; Iriepa, I.; Moraleda, I.; Chabchoub, F.;
Marco-Contelles, J.; Ismaili, L. Multi-target 1,4-dihydropyridines showing calcium channel blockade and
antioxidant capacity for Alzheimer’s disease therapy. Bioorganic Chem. 2019, 91, 103205. [CrossRef]
17. Chen, Z.; Zhong, C. Oxidative stress in Alzheimer’s disease. Neurosci. Bull. 2014, 30, 271–281. [CrossRef]
18. von Bernhardi, R.; Eugen
Diverse Pathogenic Mechanisms. Antioxid. Redox Signal. 2011, 16, 974–1031. [CrossRef]
19. Pratic , D. Oxidative stress hypothesis in Alzheimer’s disease: A reappraisal. Trends Pharmacol. Sci. 2008
29, 609–615. [CrossRef]
ín, J. Alzheimer’s Disease: Redox Dysregulation As a Common Denominator for
ò
,
20. Federico, A.; Cardaioli, E.; Da Pozzo, P.; Formichi, P.; Gallus, G.N.; Radi, E. Mitochondria, oxidative stress
and neurodegeneration. J. Neurol. Sci. 2012, 322, 254–262. [CrossRef]
21. Yan, M.H.; Wang, X.; Zhu, X. Mitochondrial defects and oxidative stress in Alzheimer disease and Parkinson
disease. Free. Radic. Boil. Med. 2012, 62, 90–101. [CrossRef]
22. Greenough, M.A.; Camakaris, J.; Bush, A. Metal dyshomeostasis and oxidative stress in Alzheimer’s disease.
Neurochem. Int. 2013, 62, 540–555. [CrossRef]
23. Candore, G.; Bulati, M.; Caruso, C.; Castiglia, L.; Romano, G.C.; Di Bona, D.; Duro, G.; Lio, M.; Matranga, D.;
Pellicanò, M.; et al. Inflammation, Cytokines, Immune Response, Apolipoprotein E, Cholesterol, and
Oxidative Stress in Alzheimer Disease: Therapeutic Implications. Rejuvenation Res. 2010, 13, 301–313.
[CrossRef]
24. Lee, Y.-J.; Han, S.-B.; Nam, S.-Y.; Oh, K.-W.; Hong, J.T. Inflammation and Alzheimer’s disease.
Arch. Pharmacal Res. 2010, 33, 1539–1556. [CrossRef]
25. Rosini, M.; Simoni, E.; Milelli, A.; Minarini, A.; Melchiorre, C. Oxidative Stress in Alzheimer’s Disease:
Are We Connecting the Dots? J. Med. Chem. 2013, 57, 2821–2831. [CrossRef]
26. Alper, G.; Girgin, F.K.; Ozgönül, M.; Mentes¸, G.; Ersöz, B. MAO inhibitors and oxidant stress in aging brain
tissue. Eur. Neuropsychopharmacol. 1999, 9, 247–252. [CrossRef]
27. Marin, D.B.; Bierer, L.M.; Lawlor, B.A.; Ryan, T.M.; Jacobson, R.; Schmeidler, J.; Mohs, R.C.; Davis, K.L.
l-Deprenyl and physostigmine for the treatment of Alzheimer’s disease. Psychiatry Res. 1995, 58, 181–189.
[CrossRef]
28. Godyn´, J.; Jon´czyk, J.; Panek, D.; Malawska, B. Therapeutic strategies for Alzheimer’s disease in clinical
trials. Pharmacol. Rep. 2016, 68, 127–138. [CrossRef]
29. Chakroborty, S.; Stutzmann, G.E. Calcium channelopathies and Alzheimer’s disease: Insight into therapeutic
success and failures. Eur. J. Pharmacol. 2014, 739, 83–95. [CrossRef]
30.
Á
vila, J.; P
Tau phosphorylation and assembly. Neurotox. Res. 2004, 6, 477–482. [CrossRef]
31. Chen, C.-H.; Zhou, W.; Liu, S.; Deng, Y.; Cai, F.; Tone, M.; Tone, Y.; Tong, Y.; Song, W. Increased
NF- B signalling up-regulates BACE1 expression and its therapeutic potential in Alzheimer’s disease.
érez, M.; Lim, F.; Gómez-Ramos, A.; Hernández, F.; Lucas, J.J. Tau in neurodegenerative diseases:
κ
Int. J. Neuropsychopharmacol. 2012, 15, 77–90. [CrossRef] [PubMed]
32. Cho, H.J.; Jin, S.M.; Youn, H.D.; Huh, K.; Mook-Jung, I. Disrupted intracellular calcium regulates BACE1
gene expression via nuclear factor of activated T cells 1 (NFAT 1) signaling. Aging Cell 2008, 7, 137–147.
[CrossRef]

Molecules 2020, 25, 1329
21 of 21
33. Hayley, M.; Perspicace, S.; Schulthess, T.; Seelig, J. Calcium enhances the proteolytic activity of BACE1:
An in vitro biophysical and biochemical characterization of the BACE1–calcium interaction. Biochim. et
Biophys. Acta (BBA) Biomembr. 2009, 1788, 1933–1938. [CrossRef]
34. Oules, B.; Del Prete, D.; Greco, B.; Zhang, X.; Lauritzen, I.; Sevalle, J.; Moreno, S.; Paterlini-Br
Trebak, M.; Checler, F.; et al. Ryanodine receptor blockade reduces amyloid- load and memory impairments
in Tg2576 mouse model of Alzheimer disease. J. Neurosci. 2012, 32, 11820–11834. [CrossRef]
35. Small, D.H.; Gasperini, R.; Vincent, A.J.; Hung, A.C.; Foa, L. The Role of A -Induced Calcium Dysregulation
échot, P.;
β
β
in the Pathogenesis of Alzheimer’s Disease. J. Alzheimers Dis. 2009, 16, 225–233. [CrossRef]
36. Weinreb, O.; Mandel, S.; Youdim, M.B.H.; Amit, T. Targeting dysregulation of brain iron homeostasis in
Parkinson’s disease by iron chelators. Free Radic. Biol. Med. 2013, 62, 52–64. [CrossRef]
37. Kumar, B.; Kumar, M.; Dwivedi, A.R.; Kumar, V. Synthesis, Biological Evaluation and Molecular Modeling
Studies of Propargyl-Containing 2,4,6-Trisubstituted Pyrimidine Derivatives as Potential Anti-Parkinson
Agents. ChemMedChem 2018, 13, 705–712. [CrossRef]
38. Mertens, M.D.; Hinz, S.; Müller, C.E.; Gütschow, M. Alkynyl–coumarinyl ethers as MAO-B inhibitors.
Bioorganic Med. Chem. 2014, 22, 1916–1928. [CrossRef]
39. Chioua, M.; Buzzi, E.; Moraleda, I.; Iriepa, I.; Maj, M.; Wnorowski, A.; Giovannini, C.; Tramarin, A.; Portali, F.;
Ismaili, L.; et al. Tacripyrimidines, the first tacrine-dihydropyrimidine hybrids, as multi-target-directed
ligands for Alzheimer’s disease. Eur. J. Med. Chem. 2018, 155, 839–846. [CrossRef]
40. Davalos, A.; Gómez-Cordovés, C.; Bartolomé, B. Extending Applicability of the Oxygen Radical Absorbance
Capacity (ORAC−Fluorescein) Assay. J. Agric. Food Chem. 2004, 52, 48–54. [CrossRef]
41. Marrazzo, P.; Angeloni, C.; Hrelia, S. Combined Treatment with Three Natural Antioxidants Enhances
Neuroprotection in a SH-SY5Y 3D Culture Model. Antioxidants 2019, 8, 420. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
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