Synthesis of hantzsch adducts as cholinesterases and calcium flux inhibitors, antioxidants and neuroprotectives

Article abstract of DOI:10.3390/ijms21207652

We report herein the design, synthesis, biological evaluation, and molecular modelling of new inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), able to block Ca⁺² channels also showing antioxidant and neuroprotective activities. The new MTDL, dialkyl 2,6-dimethyl-4-(4-((5-aminoalkyl)oxy)phenyl)-1,4-dihydropyridine-3,5-dicarboxylate 3a-p, have been obtained via Hantzsch reaction from appropriate and commercially available precursors. Pertinent biological analysis has prompted us to identify MTDL 3h [dimethyl-4-(4-((5-(4-benzylpiperidin-1-yl)pentyl)oxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate] as an attractive inhibitor of AChE (1.8 μM) and BuChE (2 μM), Ca⁺² channel antagonist (47.72% at 10 μM), and antioxidant (2.54 TE) agent, showing significant neuroprotection 28.68% and 38.29% against H₂ O_2, and O/R, respectively, at 0.3 μM, thus being considered a hit-compound for further investigation in our search for anti-Alzheimer’s disease agents.

Full text of DOI:10.3390/ijms21207652

International Journal of
Molecular Sciences
Communication
Synthesis of Hantzsch Adducts as
Cholinesterases and Calcium Flux inhibitors,
Antioxidants and Neuroprotectives
Irene Pachón Angona 1, Helene Martin 2, Solene Daniel 1, Ignacio Moraleda 3, Alexandre Bonet 2,
Artur Wnorowski 4 , Maciej Maj 4, Krzysztof Jozwiak 4, Isabel Iriepa 3,
* ,
Bernard Refouvelet 1,*, 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.)
2
3
4
5
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 Organic Chemistry and Inorganic Chemistry, School Sciences, University of Alcalá,
Ctra. Barcelona, Km. 33.6, 28871 Alcalá de Henares, Spain; ignacio.moraleda@uah.es
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.)
Laboratory of Medicinal Chemistry (IQOG, CSIC), Juan de la Cierva, 3, 28006-Madrid, Spain;
iqoc21@iqog.csic.es
*
Correspondence: isabel.iriepa@uah.es (I.I.); bernard.refouvelet@univ-fcomte.fr (B.R.);
lhassane.ismaili@univ-fcomte.fr (L.I.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 11 September 2020; Accepted: 14 October 2020; Published: 16 October 2020
Abstract: We report herein the design, synthesis, biological evaluation, and molecular modelling
of new inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), able to
block Ca+2 channels also showing antioxidant and neuroprotective activities. The new MTDL,
dialkyl 2,6-dimethyl-4-(4-((5-aminoalkyl)oxy)phenyl)-1,4-dihydropyridine-3,5-dicarboxylate 3a-p
have been obtained via Hantzsch reaction from appropriate and commercially available
precursors. Pertinent biological analysis has prompted us to identify MTDL 3h
[dimethyl-4-(4-((5-(4-benzylpiperidin-1-yl)pentyl)oxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-
3,5-dicarboxylate] as an attractive inhibitor of AChE (1.8 M) and BuChE (2
M), Ca+2 channel
antagonist (47.72% at 10 M), and antioxidant (2.54 TE) agent, showing significant neuroprotection
28.68% and 38.29% against H2O2, and O/R, respectively, at 0.3 M, thus being considered
,
µ
µ
µ
µ
a hit-compound for further investigation in our search for anti-Alzheimer’s disease agents.
Keywords: Alzheimer’s disease; Ca+2 channel antagonists; Hantzsch reaction; multitarget directed
ligands; neuroprotection; oxidative stress
1. Introduction
Alzheimer’s disease (AD) is the most common dementia, mainly affecting the elderly population.
It causes the progressive loss of neurons, leading to an impairment of the cognitive function and a
loss behavioral abilities [1]. Although its etiopathology remains unclear, many risk factors appear to
increase the susceptibility and progression of this disorder, such as diet, environment and or genetics.
AD is histologically characterized by the presence of amyloid plaques and neurofibrillary
tangles (NFTs), mainly consisting of accumulations and depositions of β-amyloid (Aβ) peptide and
hyperphosphorylated tau protein (pTau) [
2
]. Other physiopathological features include synaptic
Int. J. Mol. Sci. 2020, 21, 7652; doi:10.3390/ijms21207652
www.mdpi.com/journal/ijms

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deterioration and disbalances of Ca+2 ions and biometals, which in turn cause oxidative stress. They are
all interconnected, resulting in a progressive and irreversible neurodegeneration leading to cell damage
and neuronal death [2].
To date, the only available therapy for this disorder is symptomatic and is far from stopping,
slowing or reversing the progression of AD. Hence, there is a need for developing new and more efficient
therapies [3,4]. The traditional mono-target approach is not adapted to such a multifactorial disease.
In response to this, a more recent method called multi-targeted directed ligands (MTDLs) has emerged.
These ligands are molecules bearing different pharmacophores in just one molecule, that are capable to
act simultaneously on several etiopathological AD features. Accordingly, several MTDLs have been
developed by many research groups [
multicomponent reactions (MCR) [
5
–
8
]. In the last decade, we have contributed to this area by using
9–
13] as a very suitable and efficient synthetic tool, with its easy
performance and its time-saving procedure leading a higher structural diversity.
In this context, we report herein the design, synthesis and biological evaluation of new MTDL as
inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), able to lower the oxidative
stress (OS), and block Ca+2 channels. In fact, it is well known that AChE inhibitors (AChEIs) not only
facilitate cholinergic transmission, but also interfere with the synthesis, deposition and aggregation of
toxic A
β
. [14
,15] In addition, other studies suggested that BuChE may also influence the aggregation
of A into neuritic plaques and formation of the NFT deposit. [16
β
,
17]. Otherwise, oxidative stress
(OS) caused by various underlying factors, such as mitochondrial dysfunction, and disruption of
metal homeostasis (Cu, Fe, Zn), plays a crucial role in the pathogeny of AD. Therefore, it is evident
that the antioxidant strategy constitutes one of the most promising pathways for the development
of new drugs for aging-related diseases [18]. Ca+2 channel blockade is also one of the promising
pathways in the development of new drugs for AD, as evidenced by the example of nivaldipine,
currently under clinical development [19]. The increase in cytosolic calcium levels causes mitochondrial
disruption, which leads to the activation of the apoptotic cascade and cell death [20], and potentiates
NFT formation [21].
2. Results
2.1. Synthesis
The synthesis of the new MTDL 3a-p has been carried out in one-pot Hantzsch reaction
of aldehydes 2-h with ethyl or methyl acetoacetate and ammonium carbonate, in EtOH/water
(Scheme 1). Aldehydes 2-h were synthesized in two steps. In the first step, the alkyl chain (n= 5
and n= 6) was incorporated by nucleophilic substitution on the hydroxyl group at the C4 of the
4-hydroxybenzaldehyde in the presence of K2CO3 in acetone to afford intermediates 1a-b. Then, in the
second step, compounds 1a-b were reacted with different amines, such as morpholine, diethylamine
N-methylpiperazine and 4-benzylpiperidine. All new compounds showed excellent analytical and
spectroscopic data, in good agreement with the expected values (see Material and Methods).
Scheme 1. Synthesis of dialkyl 2,6-dimethyl-4-(4-((5-aminoalkyl)oxy)phenyl)-1,4-dihydropyridine-
3,5-dicarboxylate 3a-p. Reagents and conditions: (
acetonitrile, 24 h, reflux; (c) EtOH/H2O, O.N., 75 ◦C.
a) K2CO3, acetonitrile, 18 h, reflux; (b) K2CO3,

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To verify the effectiveness of our design, compounds 3a-p were submitted to AChE,
BuChE inhibition assay, Ca+2 channel blockade, antioxidant evaluation, followed by the neuroprotection
analysis of selected compounds.
2.2. Biological Evaluation
2.2.1. Inhibition of EeAChE/eqBuChE
For the ChE inhibition experiments, we used the Ellman protocol [22], the cheap and easily
available EeAChE and eqBuChE and tacrine as reference. As shown in Table 1, only compound 3h had
a percentage of inhibition against AChE at 10 mM higher than 50% and showed an IC50 equal to 1.8
µM, only 60-fold less active than tacrine (IC50 = 0.03
µM) [23
,
24]. However, five compounds 3c, 3h and
3k-l were active against BuChE with IC50 ranging from 2.0
µ
M for 3h to 9.6 M for 3k. Concerning the
µ
structure activity relationship (SAR), the diethylamine moeity seems to be favorable, regardless of the
length of the linker, but no additional clear SAR could be established.
Table 1. ChE inhibition and calcium blockade percentages for compounds 2a-p, and their ORAC
(TE) values.
AChE a
IC50 (µM)
BuChE a
IC50 (µM)
(%) Ca
Cpd
Amine
n
R
ORAC c
Flux Inhibition b
d
d
3a
3b
3c
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
OEt
OMe
OEt
18.58
31.89
58.21
39.55
40.51
38.4
1.31 ± 0.01
2.59 ± 0.04
2.64 ± 0.04
1.93 ± 0.68
1.78 ± 0.00
1.38 ± 0.02
2.24 ± 0.04
2.54 ± 0.04
1.22 ± 0.02
0.99 ± 0.09
1.21 ± 0.12
0.91 ± 0.01
1.10 ± 0.08
1.39 ± 0.12
1.36 ± 0.01
2.28 ± 0.01
nd
-
-
Morpholine
d
d
-
-
d
4.6 ± 0.1
-
Diethylamine
N-Me Piperazine
4-Bn Piperidine
Morpholine
d
d
3d
3e
OMe
OEt
-
-
d
d
-
-
d
d
3f
OMe
OEt
-
-
d
d
3g
3h
3i
53.6
-
-
OMe
OEt
1.8 ± 0.1
2.0 ± 0.1
47.72
47.55
52.57
31.66
39.74
50.6
d
d
-
-
d
d
3j
OMe
OEt
-
-
d
3k
3l
9.6 ± 0.3
8.7 ± 0.1
5.5 ± 0.1
-
Diethylamine
d
OMe
OEt
-
d
3m
3n
3o
3p
TA
NIM
-
N-Me Piperazine
4-N-Bn Piperidine
d
d
OMe
OEt
28.9
-
-
d
d
80.19
78.13
nd
-
-
d
d
OMe
-
-
—
—
—
0.03 ± 0.01
5.1 ± 0.1 nM
nd
nd
47.91
nd
MEL
nd
nd
nd
2.45 ± 0.09
a
Each IC50 value is the mean
±
SEM of quadruplicate of at least three different experiments; b Every percentage
c
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)
MEL = melatonin, TA = tacrine.
±
SEM. d % inhibition under 50% at 10
µM. nd: not determined. NIM = nimodipine,
2.2.2. Ca+2 Channel Blockade
Ca+2 channel blockade capacity of compounds 3a-p, compared to nimodipine as standard,
has been carried out following the usual methodology [12 13 25]. The new compounds showed a
,
,
good inhibition percentage of calcium flux ranging from 18.58 (3a) to 80.19 (3o). The most potent
compounds corresponded, in increasing order, to 3i (47.55), 3h (47.72), 3m (50.6), 3j (52.57), 3c (58.21),
3g (53.6), 3p (78.13) and 3o (80.19) comparing thus very favorably with nimodipine (47.91). It is
worth mentioning that compounds 3c, 3h and 3m are also the potent ChEI and are therefore the

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most balanced compounds in the two assays. From the point of view of SAR, compounds with
N-methylpiperazine and N-benzylpiperidine moieties showed better results with compounds bearing
ethylester than methylester groups, whatever the length of the linker. Except for the compounds
bearing the methylamine group, all compounds with the ethyester group showed better inhibition
when the length of the linker is equal to six than those with a linker with five carbons.
2.2.3. Antioxidant Analysis
The antioxidant activity expressed as Trolox equivalents (TE) units of compounds 3a-g, compared
to melatonin (MEL), used as positive control, with ORAC value 2.45 [9], was determined by the
ORAC-FL method [26]. As shown in Table 1, the values for the antioxidant capacity range from 0.91
(3l) to 2.64 (3c). Three compounds, 3b (2.59) and two of the most balanced compounds, 3c (2.64 TE)
and 3h (2.54 TE), showed antioxidant activities higher than melatonin (2.45 TE). The antioxidant power
of the third balanced compound 2m displayed only 1.10 TE, thus representing half of the activity of
melatonin. No evident SAR could be drawn for these results
2.2.4. Neuroprotective Activity
The most balanced compounds were then submitted to evaluation of their neuroprotection
activity. For this purpose, cytotoxicity was induced by H2O2, a well-known toxic responsible for
the generation of ROS and by mitochondrial respiratory chain blockers oligomycin rotenone (O/R).
Prior to the neuroprotective assay, the effect of the compounds on cell viability was evaluated at 1 and
10
displayed a modest neuroprotective effect against H2O2 and O/R. Nevertheless, and very interestingly,
compound 3h showed an interesting effect against H2O2, particularly at 0.3 M, where it showed a
percentage of neuroprotection equal to 28%. This neuroprotective positive effect is more pronounced
µM, showing no cytotoxicity against SH-SY5Y cells. As shown in Table 2, compounds 3c and 3m
µ
against O/R with a percentage equal to 38.29 and 33.90 at 0.3 and 1
melatonin with a percentage equal to 66.6 at 0.1 µM.
µM, respectively, compared to
Table 2. Neuroprotective activity of compounds 3c
, 3h and 3m on H2O2 (200 µM) or oligomycin O
at 10 µM)/rotenone (R at 30 µM)-induced cell death in SH-SY5Y cells a.
Compounds
3c
Concentration
0.3 µM
1 µM
H2O2 (%)
12.37 ± 0.08
24.17 ± 0.12
28.68 ± 0.02 *
23.32 ± 0.02 *
9.55 ± 0.00
O/R (%)
22.27 ± 0.09
25.87 ± 0.04
38.29 ± 0.04 *
33.90 ± 0.05 *
8.95 ± 0.00
0.3 µM
1 µM
0.3 µM
1 µM
3h
3m
4.63 ± 0.02
16.65 ± 0.00
66.6 ± 3.01 ***[10]
Melatonin
Data are expressed as % neuroprotection
*** p< 0.001 as compared to the control cultures (one-way ANOVA).
0.1 µM
55.4 ± 7.73 *[10]
a
±
SEM of quadruplicates from at least three different cultures; * p < 0.0,
2.3. Molecular Docking Studies
The potential binding mode of the most active compound, 3h, with hAChE (PDB: 1B41) and hBChE
(PDB: 4BDS) was investigated by molecular docking, which was performed by using Autodock
Vina [27] with structure images created by Discovery Studio. As we previously described [25],
conformational flexibility of AChE was considered by allowing eight side chains to be flexible during
the docking process.
The results show that 3h was quite strongly bound with the optimal conformation of AChE,
and its binding energy reached 10.3 kcal/mol. The whole molecule of 3h entered the binding pocket
−
of AChE in a linear mode, and the benzyl piperidinium moiety was inserted into the interior of the
pocket (Figures 1 and 2a). Due to the large molecular size, it could completely occupy the entire
cavity (Figure 2a) and the structure of one of the methoxycarbonyl groups on the dihydropyridine ring

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extends more outwardly, forming four hydrogen bonds with Ser293 and Glu292 (Figure 1). At the
bottom of the gorge, the phenyl ring interacted with Trp86 (anionic subsite, AS) and Tyr337 via
π
–
π
stacking interactions, and this ring was positioned to favor van der Waals interactions with the
catalytic triad (CT) residues His447 and Ser203. The protonated piperidine moiety interacted with
Asp74 and Tyr341, in the peripheral anionic site (PAS), via attractive charge and –cation interactions,
π
respectively. The pentamethylene chain interacted halfway down the gorge, with the acyl binding
pocket (ABP, Phe295, Phe297 and Phe338). The phenoxy ring interacted with Trp286 and Tyr341, in the
PAS, via π–π T-shaped interactions. At the mouth of the gorge, the NH dihydropyridine group also
formed a hydrogen bond with Trp286 (Figure 2b).
Docking experiments of a non-active compound, 3d, was then performed to predict why the other
dihydropiridines do not bind well to AChE. It showed that compound 3d has much lower affinity for
hAChE (binding energy, 6.8 kcal/mol) than compound 3h. This compound is accommodated in PAS
−
and no interaction with the catalytic triad aminoacids (His447, Ser203 and Glu334) has been established.
Contrary to compound 3h, the dihydropyridine moiety is oriented to the interior of the pocket and it
forms two hydrogen bonds between the NH group and two aminoacids of the PAS (Tyr124 and Asp74).
Additionally, the phenyl ring forms π–π T-shaped interaction with Trp286 (PAS). The diethylamonium
group extends outside the gorge interacting with His287 and Gln291.
Analysis of the binding mode of the compound 3h inside the active site of BChE revealed
that the compound stabilized itself inside the pocket through entangling several interactions
(docking score = −9.7 kcal/mol), adopting an U-shaped conformation (Figure 3a).
Figure 1. Binding mode of 3h in the active site of hAChE. Compound 3h is rendered as ball and stick
(carbon atoms in pink) and the side chain conformations of the mobile residues are illustrated in light
pink. The catalytic triad (CT) is colored in green, oxyanion hole (OH) in magenta, anionic subsite
(AS) in orange, except Trp86, acyl binding pocket (ABP) in yellow and peripheral anionic site (PAS)
in light pink. Hydrophobic contact,
H-bonds were in dark green dot lines, attractive charge and
bond in light green dot lines.
π
–
π
stacking and
π
–
π
T-shaped were depicted in purple dot lines,
–cation in orange and carbon hydrogen
π

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(b)
(a)
Figure 2.
(a) Three-dimensional surface representation of hAChE with compounds 3h (pink) and 3d
(yellow). (b) Two-dimensional schematic view of the interactions between 3h and hAChE.
(a)
(b)
Figure 3. Docking pose of compound 3h and tacrine inside gorge cavity of hBuChE. (
the accessible surface of the active site gorge (white), compound 3h is rendered in pink ball and stick
model. ( ) Compound 3h was shown in pink, tacrine in brown, catalytic anionic site (CAS) in green,
a) Top view of
b
OH in red, choline binding site (CBS) in violet, ABP in yellow, and PAS in blue.
The protein complex with the best-docked pose of compound 3h showed that both carboxylate
groups of the docked ligand were superimposed on the native co-crystallized tacrine (Figure 3b).
In this position, the dihydropyridine moiety is oriented toward the bottom of the active site, as with
tacrine, and it binds in the catalytic anionic site (CAS) region of the enzyme, establishing π–alkyl,
alkyl and carbon hydrogen interactions with many amino acids, including two key amino acids of
the choline binding site (CBS) pocket (Trp82 and Ala328). The phenoxy–alkyl linker chain went from
the bottom to the mouth of the gorge, allowing the benzyl–piperidinium moiety to enter the cavity
again. In this situation, π–anion interaction is established between Asp70 (peripheral anionic site, PAS)
and the phenoxy ring. In addition, the alkyl chain also interacts with Asn68 (PAS) via van der Waals
contacts (Figure 4).

Int. J. Mol. Sci. 2020, 21, 7652
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Figure 4. Two-dimensional schematic view of the interactions between 3h and hBChE.
The piperidinium ring, inside the cavity, pointed toward the catalytic triad (CT) amino acid
residues (Glu325, His438, and Ser198), causing several methylene groups to establish van der Waals
interactions with the last two amino acids. On the other hand, methylene groups can also interact with
Gly116 and Gly116 (oxyanion hole, OH) and Pro285 (located near the acyl pocket, ABP) (Figure 4).
Finally, the terminal phenyl group of the compound, going down, anchored inside the active
site gorge by establishing two
(Figure 4).
π–π interactions with Trp231 (located near the ABP) and Phe329 (ABP)
The docking results also reveal that BuChE could effectively accommodate compound 3c inside
the active site gorge. The ligand is placed into the binding pocket in a similar way to compound
3h (docking score =
−
8.7 kcal/mol) (Figure 5a). In this situation, one carboxylate group and the
dihydropyridine moiety of the docked ligand were superimposed onto the native co-crystallized
tacrine. The NH group, oriented toward the bottom of the active site, forms a hydrogen bond and
van der Waals interaction with the catalytic triad residues His438 and Ser198, respectively. Moreover,
methyl of the carboxylate groups interacts with Trp82 and Ala328 (CBS pocket), with Phe329 (ABP)
and with Tyr332 (PAS). The carboxylate group also interacts with Gly115 and Gly116 (OH) (Figure 6a).
The estimated binding energies for BuChE-3l and BuChE–3m complexes are
−
7.8 and 7.9 kcal/mol.
−
Both compounds showed a similar disposition inside the cavity, especially with regard to the
dihydropyridine moiety (Figure 5b). Contrary to compounds 3h and 3c, the NH group points
to the mouth of the gorge, placing the phenoxy–alkyl linker deep inside the cavity. Hydrogen bond was
observed between the oxygen of phenoxy group of 3l and 3m with Ser198 (catalytic triad). Apart from
this, His438 established π–π T-shaped interaction with the phenyl ring, π–alkyl and carbon hydrogen
interactions with the alkyl group of one carboxylate moiety. Residues such as Trp82 (CBS pocket),
Tyr332 and Thr120 (PAS) were also involved in other non-polar interactions (Figure 6b,c).

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The less active compound againts BuChE, 3k, was fitted on the enzyme in a mode similar to that
of the non-active compound (3d) (Figure 5c but there was difference between the energy of interaction
of the selected poses for both compounds (3k 7.5 kcal/mol, 3d 6,8 kcal/mol). The NH group is
)
:
−
:
−
pointed toward the entrance of the cavity, which placed the phenoxy–alkyl linker and one carboxylate
group in the bottom of the gorge. The alkyl group of the carboxylate moiety formed carbon hydrogen
bonds with His438 and a network of alkyl and π–alkyl interactions with Trp82, Met437 and Tyr440
(Figure 6d,e). Both compounds showed an unfavorable acceptor-acceptor interaction with Thr120 but
the non-active compound (3d) exhibited another such interaction with one amino acid of the catalytic
triad (Ser198) (Figure 6c).
(a)
(b)
(c)
Figure 5. Docking pose of compounds 3c
,
3d
,
3h and 3k-3m inside gorge cavity of hBuChE.
(
a) Compound 3h is shown in pink, compound 3c in orange. (b) Compound 3l is shown in green,
compound 3m in light violet. ( ) Compound 3d is shown in orange, compound 4k in light pink.
c
All compounds are represented in ball and stick models, CAS in green, OH in red, CBS in violet, ABP in
yellow, and PAS in blue.

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(a)
(b)
(c)
(d)
(e)
Figure 6. Two-dimensional schematic view of the interactions between 3c
3d (e) and hBChE.
(a), 3l (b), 3m (c), 3k (d),
3. Materials and Methods
3.1. Chemistry
3.1.1. General Methods
All reagents were purchased from Sigma Aldrich or TCI. Progress of the reactions was monitored
with TLC using aluminum sheets with silica gel 60 F254 from Merck (Kenilworth, NJ, USA).

Int. J. Mol. Sci. 2020, 21, 7652
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1H and 13C NMR spectra were recorded on a Bruker spectrometer, operating at 400 and 100 MHz,
respectively, using CDCl3 as a solvent. The chemical shifts are reported in parts per million (ppm),
using tetramethylsilane (TMS) as internal reference. The multiplicities of the signals are indicated
by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quadruplet; and m, multiplet
coupling constants, and they are expressed in Hz. High resolution mass spectra were obtained at
Centre Commun de Spectrométrie de Masse, Lyon, France on a Bruker micrOTOF-Q II spectrometer
(Bruker Daltonics) in positive ESI-TOF (electrospray ionization-time of flight). Elemental analyses
were obtained by a Carlo Erba EA 1108 analyzer, and the analytical results are within
theoretical values for all compounds.
±
0.2% of the
3.1.2. General Procedures for the Synthesis
General procedure
A
.
4-Hydroxybenzaldehyde (1 equiv) and the corresponding
bromo-chloroalkane (1.5 equiv) were introduced with K2CO3 (3 equiv) in acetonitrile (1.7 mmol/mL).
The reaction was stirred for 18 h at reflux. The crude oil was filtered over Celite® and the
filtrate was evaporated. The residue was filtered over silica with pentane to remove the excess
of bromo-chloroalkane and finally with DCM to get the desired compound as an oil. The crude was
then used without further purifications.
General procedure B. The corresponding halogenoalkoxy-benzaldehyde (1a
-b) was introduced
with the corresponding amine ( ) (1.5 or 2 equiv.) and K2CO3 (3e quiv.) in acetonitrile (0.6 mmol/mL).
A-D
The reaction was stirred for 24 h at reflux. The crude was filtered over Celite® and the filtrate was
evaporated. DCM was added to the filtrate and washed twice with water. The organic layers were
dried with brine and over Na2SO4 to finally be purified by flash column chromatography.
General procedure C. 4-alkyloxy-benzaldehyde (2a-h) (1 equiv.) and the corresponding
acetoacetate (3.5 equiv.) were dissolved in EtOH (4 mmol/mL). Successively, the same volume
of H2O was added dropwise while sonicating the crude. The resulting mixture was stirred and heated
◦
at 75 C for one hour. Finally, 2.5 equivalents of ammonium carbonate were added to the crude.
The reaction was then stirred and heated at 75 ◦C overnight. The desired product precipitated once the
crude reaction reached RT, or triturated in diethyl ether. The solid was then filtered and washed with
diethyl ether again to finally afford the desired final adducts from 12 to 76% yields.
Synthesis of Compounds 1a-b
4-((5-Bromopentyl)oxy)benzaldehyde (1a). The crude was prepared according to general
procedure A starting from commercially available 4-hydroxybenzaldehyde (1 equiv, 8.19 mmol,
1.00 g), 1-bromo-4-chloropentane (1.5 equiv, 12.28 mmol, 1.62 mL) and K2CO3 (3 equiv, 24.57 mmol,
3.40 g) in acetonitrile (25 mL) to afford 1.84 g of the desired compound as a transparent oil. The crude
was used without further purification.
4-((6-Bromohexyl)oxy)benzaldehyde (1b). The crude was prepared according to general
procedure A starting from commercially available 4-hydroxybenzaldehyde (1 equiv., 8.19 mmol,
1.00 g), 1-bromo-6-chlorohexane (1.5 equiv., 12.28 mmol, 1.83 mL) and K2CO3 (3 equiv., 24.57 mmol,
3.40 g) in acetonitrile (25 mL) to afford 1.57 g of the desired compound as a transparent oil. The crude
was used without further purification.
Synthesis of Compounds 2a-h
4-((5-Morpholinopentyl)oxy)benzaldehyde (2a). The crude was prepared according to general
procedure B starting from 4-((5-bromopentyl)oxy)benzaldehyde 1a (1 equiv., 6.78 mmol, 1.84 g),
commercially available morpholine (2 equiv., 13.56 mmol, 1.19 mL) and K2CO3 (3 equiv., 20.34 mmol,
2.81 g) in acetonitrile (25 mL) to afford 599.6 mg of the desired compound (32%) as a transparent oil.
1
The crude was used without further purifications. H NMR (400 MHz, CDCl3)
δ 9.88 (s, 1H), 7.89–7.75
(m, 2H), 7.05–6.94 (m, 2H), 4.05 (t, J = 6.4 Hz, 2H), 3.76 (s, 4H), 2.47 (d, J = 33.1 Hz, 6H), 1.94–1.77 (m, 2H),

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1.62 (bs, 2H), 1.56–1.45 (m, 2H). 13C NMR (101 MHz, CDCl3)
δ
190.77, 164.15, 131.19, 129.82, 114.74,
68.97, 44.97, 32.48, 28.91, 26.61, 26.59, 25.34
4-((5-(Diethylamino)pentyl)oxy)benzaldehyde (2b). The crude was prepared according to
general procedure B starting from 4-((5-bromopentyl)oxy)benzaldehyde 1a (1 equiv., 6.78 mmol,
1.84 g), commercially available diethylamine (2 equiv., 13.56 mmol, 1.404 mL) and K2CO3 (3 equiv.,
20.34 mmol, 2.81 g) in acetonitrile (25 mL) to afford 510.50 mg of the desired compound (29%) as a
1
transparent oil. The crude was used without further purifications. H NMR (400 MHz, CDCl3)
δ 9.87
(s, 1H), 7.85–7.78 (m, 2H), 7.03–6.92 (m, 2H), 4.03 (t, J = 6.5 Hz, 2H), 2.54 (q, J = 7.2 Hz, 5H), 2.45–2.36
(m, 2H), 1.82 (td, J = 13.3, 6.6 Hz, 3H), 1.56–1.43 (m, 4H), 1.39–1.31 (m, 2H), 1.03 (t, J = 7.2 Hz, 7H).
13C NMR (101 MHz, CDCl3)
24.10, 11.61.
δ 190.91, 164.21, 131.99, 129.78, 114.75, 68.30, 52.80, 46.86, 29.09, 26.83,
4-((5-(4-Methylpiperazin-1-yl)pentyl)oxy)benzaldehyde (2c). The crude was prepared according
to general procedure B starting from 4-((5-bromopentyl)oxy)benzaldehyde 1a (1 equiv., 5.08 mmol,
1.38 g), commercially available N-methylpiperazine (2 equiv., 10.16 mmol, 1.13 mL) and K2CO3
(3 equiv., 15.23 mmol, 2.11 g) in acetonitrile (25 mL) to afford 415.6 mg of the desired compound
1
(28%) as a transparent oil. The crude was used without further purifications. H NMR (400 MHz,
CDCl3)
δ 9.88 (s, 1H), 7.87–7.75 (m, 2H), 7.01–6.90 (m, 2H), 4.04 (t, J = 6.4 Hz, 2H), 2.52 (bs, 8H),
2.39 (dd, J = 17.5, 9.7 Hz, 2H), 2.31 (d, J = 9.9 Hz, 3H), 1.92–1.76 (m, 2H), 1.62–1.54 (m, 2H), 1.53–1.43
(m, 2H). 13C NMR (101 MHz, CDCl3)
46.01, 28.97, 26.58, 24.00.
δ 190.81, 164.17, 132.00, 129.80, 114.97, 68.19, 58.49, 55.07, 53.18,
4-((5-(4-Benzylpiperidin-1-yl)pentyl)oxy)benzaldehyde (2d). The crude was prepared according
to general procedure B starting from 4-((5-bromopentyl)oxy)benzaldehyde 1a (1 equiv., 1.48 mmol,
403.00 mg), commercially available 4-benzylpiperidine (2 equiv., 2.97 mmol, 0.521 mL) and K2CO3
(3 equiv., 4.45 mmol, 651.00 mg) in acetonitrile (8 mL) to afford 257.00 mg of the desired compound
1
(43%) as a transparent oil. The crude was used without further purifications. H NMR (400 MHz,
CDCl3)
δ 9.88 (s, 1H), 7.85–7.73 (m, 2H), 7.28 (d, J = 7.1 Hz, 2H), 7.19 (dd, J = 8.4, 6.3 Hz, 1H), 7.15–7.10
(m, 2H), 7.02–6.92 (m, 2H), 4.04 (t, J = 6.4 Hz, 2H), 3.04 (bs, 2H), 2.55 (d, J = 6.6 Hz, 2H), 2.45 (s, 2H),
1.99 (s, 2H), 1.88–1.79 (m, 3H), 1.68 (d, J = 11.4 Hz, 4H), 1.55–1.42 (m, 4H). 13C NMR (101 MHz, CDCl3)
δ
190.82, 164.02, 140.70, 132.00, 129.79, 129.13, 128.16, 125.78,114.75, 68.22, 58.92, 54.02„ 43.22, 37.97,
43.22, 37.97, 32.13, 28.98, 26.75.
4-((6-Morpholinohexyl)oxy)benzaldehyde (2e). The crude was prepared according to general
procedure B starting from 4-((6-bromohexyl)oxy)benzaldehyde 1b (1 equiv., 2.02 mmol, 500 mg),
commercially available morpholine (2 equiv., 4.04 mmol, 0.352 mL) and K2CO3 (3 Equiv., 6.06 mmol,
837.55 mg) in acetonitrile (20 mL) to afford 600 mg of the desired compound (48%) as a transparent oil.
1
The crude was used without further purifications. H NMR (400 MHz, CDCl3)
δ 9.88 (s, 1H), 7.86–7.80
(m, 2H), 7.06–6.95 (m, 2H), 4.04 (t, J = 6.4 Hz, 2H), 3.77 (s, 3H), 2.46 (d, J = 37.6 Hz, 6H), 1.82 (dd, J = 14.7,
6.7 Hz, 2H), 1.55–1.45 (m, 3H), 1.41 (dt, J = 8.7, 2.7 Hz, 2H), 1.25 (d, J = 4.9 Hz, 2H). 13C NMR (101 MHz,
CDCl3) δ 190.80, 164.20, 131.99, 129.79, 114.74, 66.94, 59.03, 44.97, 32.48, 29.01, 26.78, 26.43, 25.94
4-((6-(Diethylamino)hexyl)oxy)benzaldehyde (2f). The crude was prepared according to general
procedure B starting from 4-((6-bromohexyl)oxy)benzaldehyde 1b (1 equiv., 2.02 mmol, 500 mg),
commercially available diethylamine (2 equiv., 4.04 mmol, 0.416 mL) and K2CO3 (3 Equiv., 6.06 mmol,
837.55 mg) in acetonitrile (20 mL) to afford 237.4 mg of the desired compound (42%) as a transparent
oil. The crude was used without further purifications. 1H NMR (400 MHz, CDCl3)
δ 9.87 (s, 1H),
7.85–7.78 (m, 2H), 7.03–6.92 (m, 2H), 4.03 (t, J = 6.5 Hz, 2H), 2.54 (q, J = 7.2 Hz, 5H), 2.45–2.36 (m, 2H),
1.82 (td, J = 13.3, 6.6 Hz, 3H), 1.56–1.43 (m, 4H), 1.39–1.31 (m, 2H), 1.03 (t, J = 7.2 Hz, 7H). 13C NMR
(101 MHz, CDCl3) δ 190.82, 164.24, 132.00, 129.77, 114.76, 68.33, 52.82, 29.06, 27.44, 26.90, 25.98, 11.58
4-((6-(4-Methylpiperazin-1-yl)hexyl)oxy)benzaldehyde (2g). The crude was prepared according
to general procedure B starting from 4-((6-bromohexyl)oxy)benzaldehyde 1b (1 equiv., 4.03 mmol,
1 g), commercially available N-methylpiperazine (2 equiv., 8.06 mmol, 0.894 mL) and K2CO3 (3 Equiv.,
12.09 mmol, 1.67 g) in acetonitrile (40 mL) to afford 632 mg of the desired compound (49%) as a

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1
transparent oil. The crude was used without further purifications. H NMR (400 MHz, CDCl3)
δ 9.85
(s, 1H), 7.87–7.68 (m, 2H), 6.99–6.91 (m, 2H), 4.01 (t, J = 6.5 Hz, 2H), 2.44 (bs, 8H), 2.36–2.30 (m, 2H),
2.26 (s, 3H), 1.94–1.71 (m, 2H), 1.55–1.41 (m, 4H), 1.36 (td, J = 8.6, 1.4 Hz, 2H). 13C NMR (101 MHz,
CDCl3) δ 190.84, 164.21, 132.00, 129.76, 114.7, 68.26, 58.59, 55.02, 53.14, 45.98, 28.99, 27.30, 26.75, 25.92.
4-((6-(4-Benzylpiperidin-1-yl)hexyl)oxy)benzaldehyde (2h). The crude was prepared according
to general procedure B. starting 4-((6-bromohexyl)oxy)benzaldehyde 1b (1 equiv., 2.84 mmol,
705.47 mg), 4-benzylpiperidine (2 equiv., 5.69 mmol, 1.00 mL) and K2CO3 (3 Equiv., 8.52 mmol,
1177.55 mg) in acetonitrile (28.2 mL) to afford 752 mg of the desired compound as a transparent oil.
The crude was used without further purifications.
Synthesis of Compounds 3a-p
Dimethyl-4-(4-((5-(diethylamino)pentyl)oxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (3d).
3d was prepared according to general procedure C starting from
4-((5-(diethylamino)pentyl)oxy)benzaldehyde 2b (1 equiv., 0.81 mmol, 214.00 mg), methyl acetoacetate
(3.5 equiv., 2.84 mmol, 306.7 mL) and ammonium carbonate (2.5 equiv., 2.03 mmol, 195.00 mg) at 75 ◦C
over 15h. After being worked up, the crude was finally purified by flash column chromatography
1
using DCM/MeOH 90/10 + 1% Et3N to afford 104.00 mg (28%) of 3d as yellow oil. H NMR (400 MHz,
CDCl3)
3.64 (d, J = 2.5 Hz, 6H), 2.59 (q, J = 7.1 Hz, 4H), 2.51–2.40 (m, 2H), 2.33 (s, 6H), 1.82–1.70 (m, 2H),
1.59–1.50 (m, 2H), 1.49–1.40 (m, 2H), 1.06 (t, J = 7.2 Hz, 7H). 13C NMR (101 MHz, CDCl3)
168.25,
δ 7.19–7.10 (m, 2H), 6.77–6.68 (m, 2H), 5.66 (s, 1H), 4.93 (s, 1H), 3.90 (t, J = 6.4 Hz, 2H),
δ
157.60, 144.08, 139.90, 128.69, 114.05, 104.23, 67.79, 52.84, 51.09, 46.97, 38.51, 29.82, 29.41, 26.66, 24.32,
19.70, 11.54. HRMS ESI-TOF [M]+ m/z calcd. for C26H38N2O5: 458.2770, found: 458.2781.
Diethyl-4-(4-((5-(diethylamino)pentyl)oxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (3c).
3c was prepared according to general procedure C starting from
4-((5-(diethylamino)pentyl)oxy)benzaldehyde 2b (1 equiv., 1.52 mmol, 400.00 mg), ethyl acetoacetate
(3.5 Equiv., 5.32 mmol, 520.00 mL) and ammonium carbonate (2.5 equiv., 3.80 mmol, 365.14 mg) at 75 ◦C
over 15h. After being worked up, the crude was finally purified by flash column chromatography using
1
DCM/MeOH 90/10 + 1% Et3N to afford 54.00 mg (7%) of 3c as colorless oil. H NMR (400 MHz, CDCl3)
δ
7.19–7.14 (m, 2H), 6.76–6.69 (m, 2H), 5.60 (s, 1H), 4.91 (s, 1H), 4.14–4.02 (m, 4H), 3.90 (t, J = 6.4 Hz,
2H), 2.61 (dd, J = 14.2, 7.1 Hz, 4H), 2.55–2.45 (m, 2H), 2.32 (s, 6H), 1.85–1.71 (m, 2H), 1.56 (dt, J = 10.1,
7.4 Hz, 2H), 1.45 (dd, J = 15.1, 8.0 Hz, 2H), 1.22 (t, J = 7.1 Hz, 6H), 1.07 (t, J = 7.1 Hz, 6H). 13C NMR
(101 MHz, CDCl3)
δ 167.83, 157.51, 143.64, 140.33, 129.08, 113.88, 104.56, 67.76, 59.81, 52.71, 46.95, 38.86,
29.36, 26.34, 24.28, 19.74, 14.41, 11.28. HRMS ESI-TOF [M]+ m/z calcd. for C28H42N2O5: 486,3071,
found: 486,3094.
Dimethyl-2,6-dimethyl-4-(4-((5-morpholinopentyl)oxy)phenyl)-1,4-dihydropyridine-3,5-
dicarboxylate (3b).
3b was prepared according to general procedure C starting from
4-((5-morpholinopentyl)oxy)benzaldehyde 2a (1 equiv., 1.49 mmol, 413.00 mg), methyl acetoacetate
(3.5 equiv., 5.96 mmol, 643.00 mL) and ammonium carbonate (2.5 equiv., 4.47 mmol, 429.00 mg) at 75 ◦C
over 15h. After being worked up, the crude was finally purified by flash column chromatography
1
using DCM/MeOH 97/3 + 1% Et3N to afford 147.00 mg (21%) of 3b as yellow oil. H NMR (400 MHz,
CDCl3) δ 7.19–7.10 (m, 2H), 6.78–6.66 (m, 2H), 5.72 (s, 1H), 4.93 (s, 1H), 3.89 (t, J = 6.4 Hz, 2H), 3.76–3.67
(m, 4H), 3.63 (s, 6H), 2.46 (bs, 4H), 2.40–2.33 (m, 2H), 2.32 (s, 6H), 1.81–1.72 (m, 2H), 1.61–1.51 (m, 2H),
1.50–1.41 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 168.22, 157.32, 143.99, 140.24, 128.83, 114.05, 104.31,
67.08, 63.73, 58.08, 52.04, 51.14, 38.60, 28.72, 23.71, 19.79. Anal. Calcd. for C26H36N2O6: C, 66.08; H,
7.68; N, 5.93; found: C, 64.78; H, 7.81; N, 5.98.
Diethyl-2,6-dimethyl-4-(4-((5-morpholinopentyl)oxy)phenyl)-1,4-dihydropyridine-3,5-
dicarboxylate (3a).
3a was prepared according to general procedure C starting from
4-((5-morpholinopentyl)oxy)benzaldehyde 2a (1 equiv., 0.927 mmol, 257.00 mg), ethyl acetoacetate
(3.5 equiv., 3.71 mmol, 473.00 mL) and ammonium carbonate (2.5 equiv., 2.78 mmol, 267.00 mg) at 75 ◦C
over 15h. After being worked up, the crude was finally purified by flash column chromatography using

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1
DCM/MeOH 95/5 + 1% Et3N to afford 223.00 mg (48%) of 3a as a yellow oil. H NMR (400 MHz, CDCl3)
7.21–7.08 (m, 2H), 6.78–6.65 (m, 2H), 5.57 (s, 1H), 4.92 (s, 1H), 4.21–4.00 (m, 4H), 3.90 (t, J = 6.4 Hz,
2H), 3.77 (bs, 4H), 2.51 (bs, 4H), 2.42 (bs, 2H), 2.32 (s, 6H), 1.88–1.68 (m, 2H), 1.60 (bs, 2H), 1.55–1.40
(m, 2H), 1.22 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3)
167.83, 157.48, 143.71, 140.34, 129.04,
δ
δ
113.84, 104.46, 67.69, 66.99, 59.78, 59.07, 53.83, 38.84, 29.33, 26.31, 24.13, 19.65, 14.39. Anal. Calcd.
for C28H40N2O6: C, 67.18; H, 8.05; N, 5.60; found: C, 66.01; H, 8.15; N, 5.79.
Dimethyl-2,6-dimethyl-4-(4-((5-(4-methylpiperazin-1-yl)pentyl)oxy)phenyl)-1,4-dihydropyridine-
3,5-dicarboxylate (3f).
3f was prepared according to general procedure C starting from
4-((5-(4-methylpiperazin-1-yl)pentyl)oxy) benzaldehyde 2c (1 equiv., 0.837 mmol, 243.00 mg),
methyl acetoacetate (3.5 equiv., 3.35 mmol, 362.00 mL) and ammonium carbonate (2.5 equiv., 2.51 mmol,
241.00 mg) at 75 ◦C over 15h. After being worked up, the crude was finally purified by flash column
chromatography using DCM/MeOH 92/8 + 1% Et3N to afford 46.05 mg (11%) of 3f as a yellow oil.
1H NMR (400 MHz, CDCl3)
δ 7.17–7.10 (m, 2H), 6.72 (d, J = 8.6 Hz, 2H), 5.89 (s, 1H), 4.92 (s, 1H),
4.08 (t, J = 6.5 Hz, 2H), 3.89 (t, J = 6.3 Hz, 2H), 3.63 (s, 6H), 3.53–3.47 (m, 4H), 2.38 (t, J = 2.4 Hz, 4H),
2.31 (d, J = 1.9 Hz, 9H), 1.81–1.72 (m, 2H), 1.72–1.63 (m, 2H), 1.56–1.46 (m, 2H). 13C NMR (101 MHz,
CDCl3) δ 168.26, 157.52, 155.58, 144.17, 140.00, 128.68, 114.02, 104.12, 67.57, 65.52, 54.74, 51.08, 46.13,
43.55, 38.49, 29.09, 28.85, 22.75, 19.63. HRMS ESI-TOF [M]+ m/z calcd. for C27H39N3O5: 485,2869,
found: 485,2890.
Diethyl-2,6-dimethyl-4-(4-((5-(4-methylpiperazin-1-yl)pentyl)oxy)phenyl)-1,4-dihydropyridine-
3,5-dicarboxylate (3e).
3e was prepared according to general procedure C starting from
4-((5-(4-methylpiperazin-1-yl)pentyl)oxy) benzaldehyde 2c (1 equiv., 0.689 mmol, 200.00 mg),
ethyl acetoacetate (3.5 equiv., 2.40 mmol, 308.12 mL) and ammonium carbonate (2.5 equiv., 1.72 mmol,
165.00 mg) at 75 ◦C over 15h. After being worked up, the crude was finally purified by flash column
chromatography using DCM/MeOH 98/2 + 1% Et3N to afford 114.7 mg (32%) of 3e as a light brown
1
solid. H NMR (400 MHz, CDCl3)
δ 7.22–7.09 (m, 2H), 6.79–6.62 (m, 2H), 5.54 (s, 1H), 4.92 (s, 1H),
4.18–4.00 (m, 4H), 3.89 (t, J = 6.4 Hz, 2H), 2.59 (bs, 8H), 2.45–2.39 (m, 2H), 2.36 (s, 3H), 2.32 (s, 6H),
1.81–1.71 (m, 2H), 1.63–1.53 (m, 2H), 1.50–1.40 (m, 2H), 1.22 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz,
CDCl3)
δ 167.82, 157.51, 143.58, 140.33, 129.09, 113.88, 104.61, 77.48, 77.16, 76.84, 67.68, 59.83, 58.43,
52.73, 45.78, 38.88, 29.32, 24.18, 19.78, 14.43. HRMS ESI-TOF [M]+ m/z calcd. for C29H43N3O5: 513,3197,
found: 513,3203.
Dimethyl-4-(4-((5-(4-benzylpiperidin-1-yl)pentyl)oxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-
3,5-dicarboxylate (3h).
3h was prepared according to general procedure C starting from
4-((5-(4-benzylpiperidin-1-yl)pentyl)oxy)benzaldehyde 2d (1 equiv., 0.783 mmol, 286.00 mg),
methyl acetoacetate (3.5 equiv., 3.13 mmol, 338.10 mL) and ammonium carbonate (2.5 equiv., 2.35 mmol,
◦
226.00 mg) at 75 C over 15h. After being worked up, the crude was finally purified by flash
column chromatography using DCM/MeOH 98/2 + 1% Et3N to afford 64.32 mg (15%) of 3h as
ayellow oil. 1H NMR (400 MHz, CDCl3)
δ 7.30–7.23 (m, 4H), 7.20–7.06 (m, 5H), 6.75–6.65 (m, 2H),
5.76–5.67 (m, 1H), 4.93 (s, 1H), 3.88 (t, J = 6.5 Hz, 2H), 3.63 (d, J = 2.5 Hz, 6H), 2.92 (d, J = 11.4 Hz, 2H),
2.53 (d, J = 7.0 Hz, 2H), 2.36–2.27 (m, 6H), 1.87 (t, J = 11.2 Hz, 2H), 1.79–1.70 (m, 2H), 1.63 (d, J = 12.1 Hz,
2H), 1.58–1.48 (m, 4H), 1.46–1.39 (m, 3H), 1.37–1.30 (m, 2H). 13C NMR (101 MHz, CDCl3)
δ 168.23,
157.59, 144.06, 140.80, 139.88, 129.23, 128.68, 128.27, 125.89, 114.03, 104.21, 67.73, 59.06, 54.07, 51.08,
43.29, 38.50, 38.03, 32.14, 29.36, 26.81, 24.34, 19.68. HRMS ESI-TOF [M]+ m/z calcd. for C34H44N2O5:
560,3238, found: 560,3250.
Diethyl-4-(4-((5-(4-benzylpiperidin-1-yl)pentyl)oxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-
3,5-dicarboxylate (3g).
3g was prepared according to general procedure C starting from
4-((5-(4-benzylpiperidin-1-yl)pentyl)oxy)-benzaldehyde 2d (1 equiv., 1.88 mmol, 687.00 mg),
ethyl acetoacetate (3.5 equiv., 7.52 mmol, 1.03 mL) and ammonium carbonate (2.5 Equiv., 5.64 mmol,
542.14 mg) at 75 ◦C over 15h. After being worked up, the crude was finally purified by flash column
chromatography using DCM/MeOH 97/3 + 1% Et3N to afford 227.00 mg (21%) of 3g as yellow oil.
1H NMR (400 MHz, CDCl3)
δ 7.27 (d, J = 7.8 Hz, 1H), 7.24 (s, 1H), 7.15 (dt, J = 8.1, 6.6 Hz, 5H), 6.74–6.67

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(m, 2H), 5.88 (bs, 1H), 4.91 (s, 1H), 4.14–4.00 (m, 4H), 3.88 (t, J = 6.4 Hz, 2H), 2.95 (d, J = 11.3 Hz, 2H),
2.53 (d, J = 7.0 Hz, 2H), 2.38–2.32 (m, 2H), 2.31 (s, 6H), 1.91 (t, J = 11.2 Hz, 2H), 1.79–1.70 (m, 2H),
1.64 (d, J = 13.0 Hz, 2H), 1.60–1.48 (m, 3H), 1.47–1.34 (m, 4H), 1.21 (t, J = 7.1 Hz, 6H). 13C NMR
(101 MHz, CDCl3)
δ 167.83, 157.42, 143.82, 140.63, 140.32, 129.17, 128.98, 128.23, 125.87, 113.79, 104.30,
77.48, 77.16, 76.84, 67.66, 59.72, 58.90, 53.94, 43.16, 38.78, 37.88, 31.88, 29.27, 26.56, 24.25, 19.54, 14.35.
HRMS ESI-TOF [M]+ m/z calcd. for C36H48N2O5: 588,3535, found: 588,3563.
3,5-Dimethyl-4-(4-{[6-(diethylamino)hexyl]oxy}phenyl)-2,6–dimethyl-1,4–dihydropyridine-3,5-
dicarboxylate (3l).
3l was prepared according to general procedure C starting from
4-((6-(diethylamino)hexyl)oxy)benzaldehyde 2f (1 equiv., 0.55 mmol, 160.7 mg), methyl acetoacetate
(3.5 equiv., 1.93 mmol, 0.279 mL) and ammonium carbonate (2.5 equiv., 1.38 mmol, 132.60 mg) at 75 ◦C
over 15h. After being worked up, the crude was finally purified by flash column chromatography
using AE/MeOH 96/4 + 1% Et3N to afford 25.10 mg (3%) of 3l as yellow oil. 1H NMR (400 MHz,
CDCl3) δ 7.19–7.11 (m, 2H), 6.72 (d, J = 8.6 Hz, 2H), 5.79 (s, 1H), 4.92 (s, 1H), 3.88 (t, J = 6.4 Hz, 2H),
2.69 (dd, J = 14.0, 6.9 Hz, 4H), 2.63–2.50 (m, 2H), 2.33 (s, 6H), 1.75 (dd, J = 14.0, 7.2 Hz, 2H), 1.62–1.53
(m, 2H), 1.50–1.41 (m, 2H), 1.36 (dd, J = 14.9, 7.8 Hz, 2H), 1.12 (t, J = 7.2 Hz, 6H). 13C NMR (101 MHz,
CDCl3)
δ 168.24, 157.59, 144.04, 139.94, 128.73, 114.05, 104.28, 67.67, 51.11, 46.84, 38.54, 29.35, 27.28,
26.06, 19.76. HRMS ESI-TOF [M]+ m/z calcd. for C27H40N2O5: 472,2927, found: 472,2937.
3,5-Diethyl-4-(4-{[6-(diethylamino)hexyl]oxy}phenyl)–2,6–dimethyl-1,4–dihydropyridine-3,5-
dicarboxylate (3k).
3k was prepared according to general procedure C starting from
4-((6-(diethylamino)hexyl)oxy)benzaldehyde 2f (1 equiv., 0.90 mmol, 249 mg), ethyl acetoacetate
(3 equiv., 2.69 mmol, 0.263 mL) and ammonium carbonate (2 equiv., 1.80 mmol, 172.58 mg) at 70 ◦C over
15h. After being worked up, the crude was finally purified by flash column chromatography using
1
AE/MeOH 98/2 + 1% Et3N to afford 129.2 mg (25%) of 3k as yellow oil. H NMR (400 MHz, CDCl3)
δ
7.20–7.10 (m, 2H), 6.77–6.67 (m, 2H), 5.87 (s, 1H), 4.91 (s, 1H), 4.13–3.98 (m, 4H), 3.88 (t, J = 6.4 Hz, 2H),
2.67 (q, J = 7.2 Hz, 4H), 2.55 (dd, J = 9.3, 6.7 Hz, 2H), 2.31 (s, 6H), 1.78–1.68 (m, 2H), 1.50 (dddd, J = 21.2,
15.5, 10.0, 7.3 Hz, 5H), 1.38–1.29 (m, 2H), 1.21 (t, J = 7.1 Hz, 6H), 1.08 (t, J = 7.2 Hz, 6H). 13C NMR
(101 MHz, CDCl3)
δ 167.81, 157.55, 143.58, 140.28, 129.08, 113.89, 104.61, 67.78, 59.83, 46.93, 38.88, 29.43,
27.46, 26.15, 19.79, 14.43. HRMS ESI-TOF [M]+ m/z calcd. for C29H44N2O5: 500,3236, found: 500,3250.
3,5-Dimethyl-2,6-dimethyl-4-(4-{[6-(morpholin-4-yl)hexyl]oxy}phenyl)-1,4-dihydropyridine-
3,5-dicarboxylate (3j).
3j was prepared according to general procedure C starting from
4-((6-morpholinohexyl)oxy)benzaldehyde 2e (1 equiv., 0.69 mmol, 200 mg), methyl acetoacetate
(3.5 equiv., 2.40 mmol, 0.259 mL) and ammonium carbonate (2.5 equiv., 1.72 mmol, 165.03 mg) at 60 ◦C
over 15h. After being worked up, the crude was finally purified by flash column chromatography
1
using DCM/MeOH 95/5 + 1% NH3 to afford 53 mg (7%) of 3j as birght orange oil. H NMR (400 MHz,
CDCl3) δ 7.15 (d, J = 8.7 Hz, 2H), 6.73 (d, J = 8.7 Hz, 2H), 5.63 (s, J = 14.2 Hz, 1H), 4.93 (s, J = 4.1 Hz,
1H), 3.89 (t, J = 6.4 Hz, 2H), 3.80 (s, 4H), 3.64 (d, J = 2.4 Hz, 6H), 2.66–2.51 (m, J = 9.6, 7.8 Hz, 4H),
2.47 (bs, J = 15.5 Hz, 2H), 2.33 (s, 6H), 1.80–1.68 (m, 2H), 1.60 (bs, 2H), 1.52–1.42 (m, 2H), 1.41–1.31
(m, 2H). 13C NMR (101 MHz, CDCl3)
δ 168.23, 157.61, 143.99, 139.92, 128.91, 128.73, 114.06, 113.97,
104.31, 67.70, 58.99, 53.52, 51.12, 38.55, 29.35, 27.23, 26.09, 19.77. HRMS ESI-TOF [M]+ m/z calcd.
for C27H38N2O6: 486,2711, found: 486,2730.
3,5-Ethyl-2,6-dimethyl-4-(4-{[6-(morpholin-4-yl)hexyl]oxy}phenyl)-1,4-dihydropyridine-3,5-
dicarboxylate (3i).
3i was prepared according to general procedure C starting from
4-((6-morpholinohexyl)oxy)benzaldehyde 2e (1 equiv., 0.412 mmol, 120 mg), ethyl acetoacetate
(2.5 equiv., 1.03 mmol, 0.089 mL) and ammonium carbonate (1.5 equiv., 0.62 mmol, 59.38 mg) at 70 ◦C
over 15h. After being worked up, the crude was finally purified by flash column chromatography
using DCM/MeOH 96/4 + 1% NH3 to afford 37.00 mg (19%) of 3i as a colorless oil. 1H NMR
(400 MHz, CDCl3)
3.89 (t, J = 6.4 Hz, 2H), 3.76 (bs, 4H), 2.49 (bs, 4H), 2.38 (bs, 2H), 2.32 (s, 6H), 1.80–1.68 (m, 2H), 1.50–1.41
(m, 3H), 1.40–1.31 (m, 3H), 1.22 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3)
167.82, 157.55,
δ 7.21–7.12 (m, 2H), 6.77–6.69 (m, 2H), 5.54 (s, 1H), 4.92 (s, 1H), 4.16–4.02 (m, 4H),
δ

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143.57, 140.31, 129.09, 113.89, 104.61, 67.77, 59.83, 59.11, 53.69, 38.88, 29.40, 27.32, 26.14, 19.78, 14.42.
HRMS ESI-TOF [M]+ m/z calcd. for C29H42N2O6: 514,3028, found: 514,3043.
3,5-Dimethyl-2,6-dimethyl-4-(4-{[6-(4-methylpiperazin-1-yl)hexyl]oxy}phenyl)-1,4-dihydro-
pyridine-3,5-dicarboxylate (3n). 3n was prepared according to general procedure C starting
from 4-((6-(4-methylpiperazin-1-yl)hexyl)oxy)benzaldehyde 2g (1 equiv., 0.82 mmol, 250 mg),
methyl acetoacetate (4 equiv., 3.29 mmol, 0.355 mL) and ammonium carbonate (3 equiv., 2.46 mmol,
236.38 mg) at 70 ◦C over 15h. After beingworked up, the crude was finally purified by flash column
chromatography using EA/MeOH 95/5 + 1% Et3N to afford 61.6 mg of 3n as white powder (15%).
1H NMR (400 MHz, CDCl3)
δ 7.15 (d, J = 8.6 Hz, 2H), 6.72 (d, J = 8.6 Hz, 2H), 5.66 (s, 1H), 4.93 (s, 1H),
3.88 (t, J = 6.4 Hz, 2H), 3.64 (sJ, 6H), 2.54 (bs, 8H), 2.43–2.36 (m, 2H), 2.33 (s, 9H), 1.80–1.66 (m, 2H),
1.59–1.50 (m, 2H), 1.48–1.39 (m, 2H), 1.35 (dd, J = 14.0, 7.0 Hz, 2H). 13C NMR (101 MHz, CDCl3)
δ
168.23, 157.63, 144.01, 139.88, 128.71, 114.06, 104.29, 67.76, 58.60, 54.86, 52.95, 51.11, 45.92, 38.52, 29.83,
29.39, 27.40, 26.70, 26.14, 19.76. Anal. Calcd. for C28H41N3O5: C, 64.48; H, 8.27; N, 8.41; found: C,
65.48; H, 8.31; N, 8.53. HRMS ESI-TOF [M]+ m/z calcd. for C28H41N3O5: 499,3039, found: 499,3046.
3,5-Diethyl-2,6-dimethyl-4-(4-{[6-(4-methylpiperazin-1-yl)hexyl]oxy}phenyl)-1,4-dihydro-
pyridine-3,5-dicarboxylate (3m). 3m was prepared according to general procedure C starting
from 4-((6-(4-methylpiperazin-1-yl)hexyl) oxy)benzaldehyde 2g (1 equiv., 0.70 mmol, 212 mg),
ethyl acetoacetate (3 equiv., 2.09 mmol, 0.204 mL) and ammonium carbonate (2 equiv., 1.39 mmol,
133.95 mg) at 70 ◦C over 15h. After was worked up, the crude was finally purified by flash column
chromatography using EA/MeOH 97/3 + 1% Et3N to afford 144.4 mg (20%) of 3m as a yellow oil.
1H NMR (400 MHz, CDCl3)
δ 7.16 (d, J = 8.7 Hz, 2H), 6.71 (d, J = 8.7 Hz, 2H), 5.62 (s, 1H), 4.91 (s, 1H),
4.14–3.99 (m, 4H), 3.88 (t, J = 6.4 Hz, 2H), 2.59 (bs, 8H), 2.49–2.39 (m, 2H), 2.36 (s, 3H), 2.32 (s, 6H),
1.78–1.68 (m, 2H), 1.60–1.50 (m, 2H), 1.50–1.41 (m, 2H), 1.40–1.31 (m, 2H), 1.22 (t, J = 7.1 Hz, 6H).
13C NMR (101 MHz, CDCl3)
δ 167.82, 157.53, 143.63, 140.31, 129.07, 113.88, 104.56, 67.74, 59.81,
58.41, 54.51, 52.60, 45.69, 38.86, 29.37, 27.31, 26.09, 19.76, 14.42. HRMS ESI-TOF [M]+ m/z calcd.
for C30H45N3O5: 527,3341, found: 527,3359.
3,5-Dimethyl-4-(4-{[6-(4-benzylpiperidin-1-yl)hexyl]oxy}phenyl)-2,6-dimethyl-1,4-dihydro-
pyridine-3,5-dicarboxylate (3p). 3p was prepared according to general procedure C starting
from 4-((6-(4-benzylpiperidin-1-yl)hexyl)oxy)benzaldehyde 2h (1 equiv., 0.79 mmol, 300 mg),
methyl acetoacetate (3 equiv., 2.37 mmol, 0.256 mL) and ammonium carbonate (2 equiv., 1.58 mmol,
152.02 mg) at 70 ◦C over 24h. After being worked up, the crude was finally purified by flash column
chromatography using DCM/MeOH 92/8 + 1% NH3 to afford 29.91 mg (7%) of 3p as light yellow
oil. 1H NMR (400 MHz, CDCl3)
δ 7.30–7.26 (m, 1H), 7.25 (s, 1H), 7.22–7.11 (m, 5H), 6.79–6.66
(m, 2H), 5.72 (d, J = 13.5 Hz, 1H), 4.93 (s, 1H), 3.87 (t, J = 6.4 Hz, 2H), 3.64 (s, 6H), 3.03 (bs, 2H),
2.55 (d, J = 6.3 Hz, 2H), 2.42 (bs, 2H), 2.33 (s, 6H), 2.02 (bs, 2H), 1.77–1.63 (m, 5H), 1.63–1.52 (m, 4H),
1.49–1.40 (m, 3H), 1.34 (dd, J = 13.7, 6.2 Hz, 2H). 13C NMR (101 MHz, CDCl3)
δ 168.25, 157.47, 144.12,
140.06, 129.14, 128.93, 128.75, 128.66, 126.53, 114.03, 113.94, 104.21, 67.39, 59.84, 57.72, 53.12, 51.10, 42.12,
38.54, 36.84, 29.12, 29.03, 26.72, 25.71, 23.63, 19.73. HRMS ESI-TOF [M]+ m/z calcd. for C35H46N2O5:
574,3391, found: 574,3407.
3,5-Dimethyl-4-(4-{[6-(4-benzylpiperidin-1-yl)hexyl]oxy}phenyl)-2,6-dimethyl-1,4-dihydro-
pyridine-3,5-dicarboxylate (3o). 3o was prepared according to general procedure C starting
from 4-((6-(4-benzylpiperidin-1-yl)hexyl)oxy)benzaldehyde 2h (1 equiv., 0.79 mmol, 300 mg),
ethyl acetoacetate (2.5 equiv., 1.97 mmol, 0.251 mL) and ammonium carbonate (1.5 equiv., 1.19 mmol,
114.01 mg) at 75 ◦C over 20h. The crudewas dissolved in MeOH and solvent was evaporated in vacuo.
The residue was dissolved in DCM and washed with water and brine. The organic layer was then
dried over Na2SO4 and finally purified by flash column chromatography using DCM/MeOH 96/4
1
+ 1% NH3 to afford 144.4 mg (31%) of 3o as dark yellow oil. H NMR (400 MHz, CDCl3)
δ
7.30–7.26
(m, 1H), 7.25 (s, 1H), 7.21–7.10 (m, 5H), 6.76–6.64 (m, 2H), 5.68 (s, 1H), 4.91 (s, 1H), 4.14–4.03 (m, 4H),
3.88 (t, J = 6.4 Hz, 2H), 2.95 (d, J = 11.2 Hz, 2H), 2.53 (d, J = 7.0 Hz, 2H), 2.32 (s, 7H), 1.90 (t, J = 11.2 Hz,
2H), 1.78–1.69 (m, 2H), 1.64 (d, J = 12.8 Hz, 2H), 1.58–1.49 (m, 4H), 1.47–1.29 (m, 7H), 1.22 (t, J = 7.1 Hz,

Int. J. Mol. Sci. 2020, 21, 7652
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6H). 13C NMR (101 MHz, CDCl3)
δ
167.82, 157.55, 143.66, 140.77, 140.27, 129.24, 129.05, 128.28, 125.91,
113.87, 104.52, 67.81, 59.79, 59.11, 43.27, 38.84, 38.01, 32.05, 29.41, 27.57, 26.89, 26.15, 19.70, 14.40. Anal.
Calcd. for C37H50N2O5: C, 73.72; H, 8.36; N, 4.65; found: C, 71.09; H, 8.47; N, 4.58. HRMS ESI-TOF
[M]+ m/z calcd. for C37H50N2O5: 602,3705, found: 602,3720.
3.2. Biological Evaluation
3.2.1. Inhibition of EeAChE and eqBuChE
The evaluation of the activity of compounds against both EeAChE and eqBuChE was evaluated
following the Ellman [22] spectrophotometric method. Briefly, the reaction happens in a final volume
of 3 mL of a 0.1 M phosphate-buffered solution at pH = 8.0, containing 5,50-dithiobis-2-nitrobenzoic
acid, EeAChE or eqBuChE, tested compound and bovine albumin serum phosphate-buffered (pH = 8)
solution After this pre-incubation period, acetylthiocholine iodide or butyrylthiocholine iodide was
added, allowing 15 min of additional incubation time. For IC50, inhibition curves were built by
pre-incubating this blend at room temperature with nine concentrations of each compound for
10 min [28].
3.2.2. Calcium Channel Blockade
The calcium channel blockade assay was performed as described earlier [29]. Briefly, the SH-SY5Y
cells were seeded out in 96-well dark-walled plates at a density of 1
×
105 cells per well. After 24 h,
the cells were loaded with FLIPR Calcium 6 indicator (Molecular Devices) for 2 h at 37 ◦C, according to
the manufacturer’s protocol. Compounds were dissolved in an appropriate amount of DMSO at 10 mM
concentration and diluted to a final concentration of 10 µ
M in Hanks0 Balanced Salt Solution (HBSS,
Thermo Fisher Scientific) buffered with HEPES (Sigma-Aldrich) at pH = 7.4, and as such used were for
treatment of indicator loaded cells (10 min at 37 ◦C). The fluorescence of indicator-loaded cells was
measured with Synergy H1 (Biotek Instruments, USA) multilabel plate reader at excitation at 485 nm
and emission at 525 nm wavelengths. The baseline fluorescence was recorded for 5 s. Then, the cells
were stimulated with KCl/CaCl2 solution (in HBSS, final concentration of KCl and CaCl2 was 90 and
5 mM, respectively) and the fluorescence was recorded for a further 30 s. Dimethyl sulfoxide 1% solution
in HBSS was used as a vehicle control. Nimodipine (Cayman Chemical Company) was used as a
reference inhibitor at 10 µM and compounds were assessed at the same final concentration in triplicate
in three independent experiments. Fluorescent intensity values were normalized to the baseline.
3.2.3. Oxygen Radical Absorbance Capacity Assay
The antioxidant activity of compounds 3a-p was performed by the ORAC-FL using fluorescein as
a fluorescent probe. Briefly, fluorescein and antioxidant were incubated in a black 96-well microplate
(Nunc) for 15 min at 37 ◦C. 2,20-Azobis(amidinopropane) dihydrochloride was then added quickly
using the built-in injector of Varioskan Flash plate reader (Thermo Scientific). The fluorescence was
measured at 485 nm (excitation wavelength) and 535 nm (emission wavelength) each min for 1 h.
All the reactions were made in triplicate and at least three different assays were performed for each
sample [9,30].
3.2.4. Effect of Compounds 3c, 3h and 3m on H2O2 on a Mixture of Oligomycin A/Rotenone-Induced
Cell Death in SH-SY5Y Cells
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, 1X non-essential amino acids,
100 units/mL penicillin and 10 mg/mL streptomycin (Dutscher, France). After 48 h of incubation,
×
104 cells per well in
the cultures were treated with 100
After 24 h, the cells were co-incubated with H2O2 (200
with or without the tested compounds at noncytotoxic concentrations for an additional period of
µl of the test compounds or DMSO (0.1%) in the same medium.
µ
M) or O/R (O at 10 M/R at 30 M)
µ
µ

Int. J. Mol. Sci. 2020, 21, 7652
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24 h. The percentage of cell viability was measured using CellTiter 96 AQueous Non-Radioactive Cell
Proliferation (MTS) Assay (Promega, France).
3.3. Molecular Modelling
Compounds 3c, 3d, 3h and 3k-3m were built using Discovery Studio 2.1 (DS 2.1) and protonated
on a piperidine ring following the energy-minimization using the adopted-based Newton–Raphson
algorithm. The protein crystal structure of hAChE in complex with fasciculine (PDB ID: 1B41)
was prepared prior to docking, using the protein model tool in Discovery Studio. Cocrystal ligands and
water molecules were removed and AutoDockTools (ADT; version 1.5.6) was used to add hydrogens
and partial charges for proteins and ligands using Gasteiger charges.
To give flexibility to the binding site, residues lining the AChE (Trp286, Tyr124, Tyr337, Tyr341,
Tyr72, Asp74, Thr75, Trp86) were allowed to move during the docking search as performed by software
Autodock Vina [27]. The grid box was built with a resolution of 1 Å and 60
×
60
×
72 points and it was
positioned at the middle of the protein (x = 116.546; y = 110.33; z = −134.181).
For hBuChE, the three-dimensional structure in complex with tacrine has been used (PDB ID:
4BDS). The preparation of this protein was done following the same protocol described before for
hAChE. All dockings were performed where a box of 66
×
66
×
70 Åwith grid points separated 1 Å,
was positioned at the middle of the protein (x = 136.0; y = 123.59; z = 38.56).
The ADT program was used to generate the docking input files. Docking calculations were
performed as blind dockings with the program Autodock Vina [27] and default parameters were used,
except num_modes, which was set to 40.
The best Vina scored poses were considered and the docked ligand output files were viewed and
analyzed using DS.
4. Conclusions
AD is a neurodegenerative disorder and is the most common form of senile dementia. The four
current AD pharmacotherapies focus on the impairment of cholinergic and glutamatergic systems,
but with limited therapeutic success. So, it is urgent to develop new hits to combat AD. For this
purpose, compounds 3a-g have been successfully synthesized by Hantzsch multicomponent
reaction, and their biological evaluation, as potential cholinesterase inhibitors, Ca+2 channel blockers,
antioxidant and neuroprotective agents has been assessed. The inhibition of cholinesterase allowed
us to identify five compounds, 3c
3h as a micromolar inhibitor against AChEI. Concerning Ca+2 channel blockade results, it is worth
mentioning that compounds 3c 3g-j, 3m and 3o-p showed inhibitions higher than or comparable
with nimodipine. Compounds 3b and 3h showed antioxidant activities higher than melatonin
, 3h and 3k-l, as micromolar inhibitors against BuChE and only
,
-
c
used as standard. To sum up, these biological analyses have prompted us to identify compound 3h
(dimethyl-4-(4-((5-(4-benzylpiperidin-1-yl)pentyl)oxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate) as the most balanced MTDL, showing IC50 equal to 1.8 and 2
µ
M against AChE and
BuChE, respectively, and a percentage of Ca+2 channel blockade equal to 47.72% at 10
µM with
additional interesting antioxidant effect (2.54 TE). The neuroprotective activity results support the
interest in compound 3h, particularly for its neuroprotective activity against H2O2 (28.68%) and O/R
(38.29%) in SHSY5Y cells at 0.3
µM, thus being considered as a new hit-compound for further
investigation in our search for anti-AD agents. This work is now in progress in our laboratories,
to develop analogues with best pharmacological profile, and the results will be presented elsewhere in
due course.
Author Contributions: Conceptualization, L.I.; methodology, L.I., H.M., K.J., I.I., and F.B.; investigation, I.P.A.,
S.D., A.B., M.M., A.W., I.M., and T.B.S.; resources, B.R.; writing—original draft preparation, L.I. and K.J.;
writing—review and editing, I.I., J.M.-C.; review and editing. 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).

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Conflicts of Interest: The authors declare no conflict of interest.
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