(±)- BIGI-3h: Pentatarget-Directed Ligand combining Cholinesterase, Monoamine Oxidase, and Glycogen Synthase Kinase 3β Inhibition with Calcium Channel Antagonism and Antiaggregating Properties for Alzheimer's Disease

Article abstract of DOI:10.1021/acschemneuro.0c00803

Multitarget-directed ligands (MTDLs) are considered a promising therapeutic strategy to address the multifactorial nature of Alzheimer's disease (AD). Novel MTDLs have been designed as inhibitors of human acetylcholinesterases/butyrylcholinesterases, monoamine oxidase A/B, and glycogen synthase kinase 3β and as calcium channel antagonists via the Biginelli multicomponent reaction. Among these MTDLs, (±)-BIGI-3h was identified as a promising new hit compound showing in vitro balanced activities toward the aforementioned recognized AD targets. Additional in vitro studies demonstrated antioxidant effects and brain penetration, along with the ability to inhibit the aggregation of both τ protein and β-amyloid peptide. The in vivo studies have shown that (±)-BIGI-3h (10 mg/kg intraperitoneally) significantly reduces scopolamine-induced cognitive deficits.

Full text of DOI:10.1021/acschemneuro.0c00803

pubs.acs.org/chemneuro
Research Article
( )-BIGI-3h: Pentatarget-Directed Ligand combining Cholinesterase,
Monoamine Oxidase, and Glycogen Synthase Kinase 3β Inhibition
with Calcium Channel Antagonism and Antiaggregating Properties
for Alzheimer’s Disease
Lhassane Ismaili,* Julie Monnin, Adeline Etievant, Raquel L. Arribas, Lucía Viejo, Bernard Refouvelet,
Ondrej Soukup, Jana Janockova, Vendula Hepnarova, Jan Korabecny, Tomas Kucera, Daniel Jun,
Rudolf Andrys, Kamil Musilek, Aurelie Baguet, Eva M. García-Frutos, Angela De Simone,
Vincenza Andrisano, Manuela Bartolini, Cristóbal de los Ríos, José Marco-Contelles,
and Emmanuel Haffen
Cite This: ACS Chem. Neurosci. 2021, 12, 1328−1342
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Multitarget-directed ligands (MTDLs) are considered a promising therapeutic strategy to address the multifactorial
nature of Alzheimer’s disease (AD). Novel MTDLs have been designed as inhibitors of human acetylcholinesterases/
butyrylcholinesterases, monoamine oxidase A/B, and glycogen synthase kinase 3β and as calcium channel antagonists via the
Biginelli multicomponent reaction. Among these MTDLs, ( )-BIGI-3h was identified as a promising new hit compound showing in
vitro balanced activities toward the aforementioned recognized AD targets. Additional in vitro studies demonstrated antioxidant
effects and brain penetration, along with the ability to inhibit the aggregation of both τ protein and β-amyloid peptide. The in vivo
studies have shown that ( )-BIGI-3h (10 mg/kg intraperitoneally) significantly reduces scopolamine-induced cognitive deficits.
KEYWORDS: Alzheimer’s disease, calcium channel, GSK 3β, MAO, cholinesterases, Biginelli reaction
The formation of Aβ deposits in the brain is a critical step,
and it was associated with the progression and development of
AD. It is indeed now widely accepted that Aβ toxicity is
mainly related to Aβ prefibrillar and brain permeable species,
because Aβ oligomers have been shown to accelerate neuronal
cell death and to adversely affect synaptic function.4
INTRODUCTION
■
Alzheimer’s Disease (AD) is now considered an extensively
complex and multifactorial neurodegenerative disorder with
no available cure at the moment. Despite unprecedented
amounts of scientific publications (more than 100 K in
Pubmed), current offered treatments are limited to a
temporary relief of symptoms.1 AD is the most common
cause of memory impairment and dementia in elderly people.2
It is characterized by a series of highly interconnected
pathological processes that are hallmarked by (i) the
accumulation of abnormal levels of soluble aggregates and
extracellular deposits of β-amyloid peptide (Aβ) and (ii) the
formation of neurofibrillary tangles, which are composed of
hyperphosphorylated τ protein.3
Received: December 19, 2020
Accepted: March 22, 2021
Published: April 2, 2021
https://doi.org/10.1021/acschemneuro.0c00803
© 2021 American Chemical Society
ACS Chem. Neurosci. 2021, 12, 1328−1342
1328

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Hyperphosphorylation renders τ prone to aggregate and to
impair cell viability.5 This aggregation, once initiated, is self-
replicating, depleting the physiological τ pool in the neuron
and converting it to toxic neurofibrillary tangles which
continue to accumulate, leading to neuronal toxicity.6
These pathological structures ultimately lead to a
progressive loss of cholinergic neurons, memory impairment,
and cognitive dysfunction.7
The disease is also characterized by low levels of
acetylcholine, which is enhanced by the administration of
cholinesterase (ChE) inhibitors, one of the primary
therapeutic options available.8
Regarding mechanisms underlying neurodegeneration, the
increase in cytosolic calcium levels has been identified as a
crucial factor. Indeed, increased levels of calcium have been
shown to facilitate the formation of Aβ by increasing the
activity of β-secretase9,10 and regulating glycogen synthase
kinase 3 (GSK-3β).11 In addition, the entry of calcium
through L-type voltage-gated calcium channels (VGCC)
results in calcium overload, mitochondrial disruption, and
cell apoptosis activation.12
Furthermore, GSK-3β, which is a major kinase involved in
the phosphorylation of τ, has been implicated in AD and AD-
related τ pathology13,14 and has an important role in other AD
key features such as inflammation and apoptosis, and its levels
have been shown to be abnormally increased in the brain of
patients with AD.15
Figure 1. Design of BIGI compounds with potential inhibitory
activity against cholinesterase (ChE), MAO, and GSK-3β and with
calcium channel blockade activity.
Other significant factors such oxidative stress (OS) have
been identified as crucial elements in AD pathogenesis. OS is
caused by various underlying factors such as mitochondrial
dysfunction,16 disturbance in the homeostasis of biometals
(Cu, Fe, Zn) linked to their involvement in peptide Aβ
aggregation,17 neuroinflammation,18 or hydrogen peroxide
(H2O2) production in the MAO-catalyzed deamination
reaction of biogenic amines.19 In fact, it is well-known that
MAO-B plays a pivotal role in the progression of AD,20 and its
expression in neuronal tissues increases with aging, resulting in
increased dopamine metabolism and higher levels of hydrogen
peroxide that can cause further oxidative free radicals.
Given the above-mentioned evidence, the development of
multitarget-directed ligands (MTDLs), i.e., small molecules
able to hit multiple targets responsible for AD pathogenesis,
has been pursued, in recent years, as an innovative method for
AD medications and a therapeutic opportunity to face to this
complex scenario. Several MTDLs were developed with
promising profiles; however, none have been approved so
far for the treatment of AD.21−24
Multicomponent reactions (MCRs) are atom economic
where most atoms of starting materials, if not all, are found in
the product. Thus, MCRs are a useful alternative to sequential
multistep syntheses, allowing scaffold diversity and a rapid and
easy access to biologically relevant compounds.25,26 Therefore,
MCRs seem well-suited for the search of new MTDL able to
simultaneously interact with various biological targets
concerning AD.27
is an anti-AD drug that acts as a selective AChE inhibitor, and
chromone is a privileged scaffold in medicinal chemistry,
exhibiting a wide range of pharmacological activities such as
antioxidant properties and inhibitory activities against both
MAO and Aβ self-aggregation.31 The Biginelli one-pot
reaction, one of the very known multicomponent reactions,
was used not only for its interest as MCR but also to generate
the central dihydropyridinone core, which has a potential
calcium channel modulation activity similar to that of classical
dihydropyridines,32 such as SQ32926.32,33 (Figure 1). This
scaffold might also modulate GSK-3β, as it has the cyclic urea
group present in several GSK-3β inhibitors such as phenyl-
methylenehydantoins,15 SB-415286,34 or the triazinones
reported by Prati et al.35
From these studies, we have identified BIGI-3h, the first
small-molecule MTDL that can simultaneously modulate
ChEs, GSK3β, and monoamine oxidase (MAO); counteract
calcium overload through the inhibition of VGCC; exert
antioxidant activity; and inhibit Aβ and τ aggregation. This
multitarget profile might contribute to an original and
innovative therapeutic approach for the effective treatment
of AD.
RESULTS AND DISCUSSION
■
Chemistry. The syntheses of the BIGI compounds 3a−n
(Scheme 2) have been carried out using new one-pot Biginelli
reaction conditions of compounds 2a−c, ethyl acetoacetate,
different commercial 3-formyl chromones, and sodium
bisulfate as a catalyst in acetic acid at room temperature for
60 h, and they were refluxed for an additional 4 h.
Compounds 2a−c were obtained from commercial benzylpi-
peridines 1a,b (n = 0,1) or 1c36 (n = 2) with the benzoyl
isocyanate (BI), in CH2Cl2 at 0 °C for 1 h, followed by
In previous works, we have developed a number of new
MTDLs using MCR such Ugi and Hantsch.24,27−30 Herein,
we report the design; synthesis, via Biginelli MCR; and
biological evaluation of the new family of BIGI-3a−n named
ethyl 1-(2-(1-benzylpiperidin-4-yl)ethyl)-6-methyl-2-oxo-4-(4-
oxo-4H-chromen-3-yl)-1,2,3,4-tetrahydropyrimidine-5-carbox-
ylate (Figure 1). These compounds possess a selected
benzylpiperidine motif of donepezil and chromone. Donepezil
1329
https://doi.org/10.1021/acschemneuro.0c00803
ACS Chem. Neurosci. 2021, 12, 1328−1342

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Scheme 1. Synthetic Procedure for the Synthesis of Ureas 2a−c
Scheme 2. Synthesis of BIGI-3a−n Using One-Pot Biginelli Reaction
a
Table 1. Calcium Channel Blockade, hChE Inhibition, and ORAC Analysis of BIGI-3a−n and Reference Compounds
c
compound
calcium channel blockade (IC50 μM)
hAChE inhibition (IC50 μM)
hBuChE inhibition (IC50 μM)
ORAC
BIGI-3a
BIGI-3b
BIGI-3c
BIGI-3d
BIGI-3e
BIGI-3f
BIGI-3g
BIGI-3h
BIGI-3i
BIGI-3j
BIGI-3k
BIGI-3l
BIGI-3m
BIGI-3n
nifedipine
donepezil39
tacrine40
melatonin
ferulic acid
14
6
3.0 0.9
3
3
n.a.
n.a.
14.8 0.7
n.a.
3.29 0.07
2.06 0.17
3.51 0.09
2.53 0.17
3.26 0.12
2.20 0.05
2.56 0.13
3.65 0.07
3.09 0.07
3.24 0.02
2.75 0.16
1.68 0.18
3.29 0.10
6.08 0.38
0.23 0.01
0.06 0.00
0.30 0.01
0.29 0.01
0.06 0.00
0.15 0.00
0.18 0.01
0.50 0.02
0.17 0.01
0.34 0.02
0.58 0.02
b
36.1 3.1
6.58 0.34
8.40 0.34
5.96 0.22
15.7 0.9
17.3 0.9
4.29 0.09
6.51 0.45
2.95 0.14
6.26 0.31
14.4 1.2
4.35 0.23
b
9
2
1.9 0.6
2.2 0.6
1.7 0.6
1.4 0.4
8
1
3.8 0.1
1.4 0.5
3.2 0.2
5
2
1.7 0.4
6.2 0.2
b
b
b
b
2.45 0.04
b
b
b
0.02 0.01
1.33 0.04
b
7.40 0.39
0.049 0.002
b
2.5 0.1
3.7 0.2
b
b
a
Each IC50 is the mean standard error of mean of at least three different experiments performed in quadruplicate; n.a. = not active, % inhibition
b
c
under 50%. Not evaluated. Data are expressed as micromoles of trolox equivalents/μmol tested compound and are shown as means standard
error of means.
hydrolysis of the resulting compounds with NaOH for 48 h37
(Scheme 1). All new compounds were characterized using
analytical (HPLC) and spectroscopic data (1H and 13C NMR,
ESI-MS, or elemental analysis) showing data in good
agreement with their structures which are collected in the
chemical synthesis part and the Supporting Information
Biological Evaluation. To verify the effectiveness of our
compounds to simultaneously hit the selected targets and to
make a judicious use of resources, blockade of VGCC and
inhibition of human ChE (hChE), which are rational targets
for BIGIs, as well as antioxidant activity were selected as the
first filters for the selection of the promising compounds.
Then, the latter were evaluated as Aβ aggregation inhibitors
1330
https://doi.org/10.1021/acschemneuro.0c00803
ACS Chem. Neurosci. 2021, 12, 1328−1342

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
and for their abilities to cross the blood−brain barrier (BBB)
leading to a promising hit compound for AD treatment which
was further investigated as MAO, GSK3β, and τ aggregation
inhibitor.
Table 2. Efficacy of BIGI-3e, 3k, and 3h to Inhibit Aβ1‑42
Aggregation and Their Ability to Cross Blood−Brain
a
Barrier
b
compound
% inhibition Aβ1−42 aggregation
CNS
Calcium Channel Antagonism. We investigated the
blocking effect of the depolarization-induced Ca2+ uptake in
SH-SY5Y neuroblastoma cells stimulated with an extracellular
concentration of K+ 70 mM, which induces the cell Ca2+ level
elevation by opening VGCC, monitored with the Ca2+-
sensitive fluorescent dye Fluo-4. The results are listed in Table
1 showing that, among the 14 BIGIs assessed, 11 displayed
better VGCC inhibitory activity than that of nifedipine, which
is a calcium channel blocker medication used as positive
standard, with IC50 ranging from 1.4 to 6 μM. The three
compounds that possessed an IC50 higher than nifedipine
lacked substitution on the chromone moiety, regardless of the
length of the linker (Table 1).
Inhibition of Human AChE/Human BuChE. Cholines-
terase inhibition of compounds BIGI-3a−n was evaluated
following the Ellman protocol.38 As shown in Table 1, the
synthesized BIGIs generally exhibited good inhibition against
human acetylcholinesterase (hAChE) in the submicromolar
range and against human butyrylcholinesterase (hBuChE) in
the micromolar range, thus behaving as selective AChEI.
Particularly, compounds BIGI-3e and BIGI-3h, with a linker
length of n = 1, had an IC50 of 0.06 μM for hAChE and,
therefore, only 3-fold less actives than those of Donepezil with
IC50 of 0.02 μM, while the most potent inhibitor of hBuChE
was BIGI-3k (IC50 = 2.95 μM). It is worth noting that, BIGI-
3e, BIGI-3h, and BIGI-3k were also the best VGCC blockers
Concerning the structure activity relationship, a linker
length of n = 0 seemed detrimental for AChE inhibitory
activity. Except for compound BIGI-3a, all compounds
showed the same trend for hBuChE inhibition. For the
same linker length (n = 1 or 2), the best results were obtained
with a chromone bearing an ethyl group (R2 = Et) or having
no substituents.
Antioxidant Activity. The capacity of BIGI-3a−n to
reduce the amount of peroxyl radicals was determined by the
Oxygen Radical Absorbance Capacity by Fluorescence
(ORAC-FL) method,41,42 using Trolox as the standard
compound and fluorescein (FL) as the fluorescent probe.
The results were expressed as Trolox equivalents (μmol of
trolox/μmol of tested compound, TE). All compounds
exhibited strong antioxidant activity with Trolox equivalent
values ranging from 1.68 TE (BIGI-3l) to 3.65 TE (BIGI-
3h), similar to that of standards such as melatonin (2.5 TE)
and ferulic acid (3.7 TE).
Interestingly, the most balanced compounds considering
VGCC and hChE inhibition also showed strong antioxidant
activity; BIGI-3h was the most active compound, while BIGI-
3e and BIGI-3k showed, respectively, 3.26 and 2.75 TE.
Therefore, we examined whether these selected BIGI
compounds inhibited Aβ aggregation31 and crossed the
blood−brain barrier (BBB).
BIGI-3e
BIGI-3k
BIGI-3h
tacrine
63.0 2.1
63.8 0.8
66.0 0.7
6.3 2.1
CNS ( )
CNS (−)
CNS (+)
CNS (+)
n.d.
curcumin
74.5 0.5
a
Curcumin and tacrine were used as positive and negative reference
b
compounds, respectively. The % inhibition of Aβ1−42 aggregation by
the tested compounds. [Aβ] = [Inhibitor] = 50 μM. Data are the
means standard error of means of two experiments each performed
in duplicate. n.d. = not determined. CNS = central nervous system.
percentages in the narrow range 63−66% when tested at
equimolar concentrations with Aβ1−42. Their inhibitory
potencies were only slightly lower than that of curcumin, a
well-known antiaggregating agent against amyloid peptides,
tested under the same experimental conditions.
In Vitro Blood−Brain Barrier Permeability. A major
problem to overcome in CNS drug discovery is the ability of
the compound in question to cross the BBB at therapeutic
concentrations. Therefore, the three compounds were
evaluated using parallel artificial membrane permeability
assay (PAMPA)44 which is a high throughput technique
developed to predict BBB passive permeability. Despite all
three compounds having similar activity profiles, only
compound BIGI-3h could cross the BBB via passive diffusion
(Table 2). Consequently, BIGI-3h was selected as a
promising hit compound for AD treatment and was further
investigated.
IN-DEPTH EXPLORATION OF BIGI-3H
■
First, as BIGI-3h contains a stereocenter and chirality may
play a role in the biological activity toward selected targets, its
enantiomers were separated by chiral high performance liquid
chromatography, and each enantiomerically pure forms were
evaluated as calcium channel antagonists, ChE and Aβ
inhibitors. ( )-BIGI-3h, (+)-BIGI-3h, and (−)-BIGI-3h
were also evaluated to assess their inhibitory activity toward
MAO and GSK-3β, as well as against τ (306−336)
aggregation.
Results showed only slight activity variations in these pure
compounds compared to the racemate BIGI-3h (Table 3),
regarding the inhibition of hAChE (as confirmed by molecular
modeling, see below), the blockade of VGCC, and the
aggregation of Aβ as well as their ability to cross the BBB
(Table 4).
In Silico Modeling of the Two Enantiomers of BIGI-
3h. To disclose the proposed dual binding mode and to
assign the structural requirements determining the affinities of
each enantiomer, we have performed molecular modeling
studies for (R)-BIGI-3h and (S)-BIGI-3h in the hAChE
active site obtained from RCSB Protein Data Bank (PDB ID:
4EY7; Figure 2). The validity of our study is further
corroborated by the fact that the enzyme of the same origin
was used for in vitro evaluation.45 Moreover, the consistency is
also supported by the resemblance of BIGI-3h to donepezil
by possessing the N-benzylpiperidine fragment.
Inhibition of Aβ1−42 Aggregation. The inhibitory
activity of selected BIGI-3e, BIGI-3h, and BIGI-3k against
spontaneous aggregation of Aβ1−42 was determined in vitro
using a thioflavin T-based fluorometric assay.43 Curcumin was
used as positive control, while tacrine, the first approved anti-
AD drug, was used as negative control. The three BIGI
compounds, screened at 50 μM concentration, showed good
Aβ1−42 antiaggregation activity (Table 2), with inhibition
Indeed, both enantiomers share a high degree of homology
in their lodging and affinity to hAChE as predicted by our
1331
https://doi.org/10.1021/acschemneuro.0c00803
ACS Chem. Neurosci. 2021, 12, 1328−1342

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Table 3. Efficacy of BIGI-3h and Its Enantiomers to Inhibit hAChE, Calcium Channels, Monoamine Oxidase, and Glycogen
a
Synthase Kinase 3β and Aβ1−42 and τ(306−336) Aggregation and Predicted BBB Permeability of These Compounds
calcium
hAChE
(IC50 μM)
channel
MAO-A
(IC50 μM)
MAO-B
(IC50 μM)
Aβ1−42 aggregation
GSK-3β
(IC50 μM)
τ(306−336) aggregation
b
c
compound
)-BIGI-3h
(+)-BIGI-3h [αD] = 162°
(c 0.14, MeOH)
(IC50 μM)
(% inhibition)
(% inhibition)
(
0.06 0.00
0.09 0.00
1.4 0.4
63.6 1.3
66.5 0.9
29.1 0.5
10.6 1.5
73.7 5.4
78.3 3.0
5
2
75.6 35.7
195. 505
11.2 1.09
12.3 1.39
(−)-BIGI-3h [αD] =
0.07 0.00
1.6 0.4
63.5 0.2
24.3 1.2
75.3 1.6
−178° (c 0.13, MeOH)
clorgylin
0.045 0.003
pargyline
0.227 0.029
SB-415286
doxycycline
0.055
61.5 0.8
a
b
Each IC50 and % values are the mean standard error of mean of quadruplicate of at least three different experiments. [Aβ] = [inhibitor] = 50
c
μM. [τ(306−336) peptide] = [inhibitor] = 50 μM.
Table 4. Prediction of Blood−Brain Barrier Penetration of
catalytic triad showed van der Waals contacts with the ligand
further improving ligand stabilization.
a
Drugs Expressed as Pe
SEM
On the contrary, (S)-BIGI-3h revealed slightly different
orientation in the hAChE active site (Figure 2C,D). In a
similar fashion, Trp286 is implicated in π−π and cation−π
interactions with N-benzyl moiety (4.3 Å) and nitrogen from
piperidine (4.6 Å), respectively. Also, methyl and ethyl-
carboxylate appendages from ethyl 6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate displayed π−alkyl contacts
to Tyr341, Tyr337, Trp86, and Phe338. 6,8-Dichloro-4H-
chromen-4-one demonstrated only the displaced π−π
interaction with Trp86 (4.3 Å). His447 and Ser203 were
found to exert van der Waals forces, and Tyr124 formed a
hydrogen contact carbonyl from this fragment. The other
prominent interactions regarding the 6,8-dichloro-4H-chro-
men-4-one core observed in the (R)-BIGI-3h-hAChE
complex were omitted in the (S)-BIGI-3h-hAChE complex.
In our opinion, all these observations slightly favor the
accommodation of the (R)-BIGI-3h enantiomer over the (S)-
BIGI-3h. The crucial element in this process seems to be the
orientation of 6,8-dichloro-4H-chromen-4-one moiety in the
CAS of hAChE. These findings are also supported by the
calculated energies, used as scoring function where the
binding affinities were estimated −13.4 kcal/mol and −12.3
kcal/mol for (R)-BIGI-3h and (S)-BIGI-3h enantiomers,
respectively. Generally, weaker hAChE inhibition potency of
(R)-BIGI-3h and (S)-BIGI-3h compared to donepezil might
be attributed to inverted ligand anchoring in the gorge of the
enzyme (Figure 3).
MAO and GSK-3β Inhibition Assays. MAO inhibitory
activity was evaluated by the detection of the metabolites of a
deamination of a MAO substrate by the MAO-A or MAO-B.52
The method used kynuramine as the common substrate for
both MAO-A and MAO-B. The enzymes oxidize the substrate,
which leading to 4-hydroxyquinoline (4-HQ), were easily
identified in the reaction medium by HPLC-MS (m/z 146
ion, (M + H)+). Clorogyline, an irreversible and selective
MAOAI, and pargyline, an irreversible and selective MAOBI,
were used as references. As shown in Table 3, although
(+)-BIGI-3h and (−)-BIGI-3h showed weak MAO-A
inhibitory activity, both compounds could effectively inhibit
MAO-B in the micromolar range, with IC50 values of 11.2 and
12.34 μM, respectively, compared with that of standard
pargyline (IC50 = 0.227 μM).
BBB penetration estimation
compound
Pe SEM (*10−6 cm s−1
)
CNS ( )
(+)-BIGI-3h
(−)-BIGI-3h
( )-BIGI-3e
( )-BIGI-3k
tacrine
8.20 0.37
9.7 1.6
3.10 0.31
1.50 0.12
6.0 0.6
21.9 2.1
20.0 2.0
22.7 4.3
9.6 1.6
0.63 0.16
0.70 0.18
0.19 0.07
CNS+
CNS+
CNS
CNS−
CNS+
CNS+
CNS+
CNS+
CNS+
CNS−
CNS−
CNS−
donepezil
rivastigmine
testosterone
chlorpromazine
cefuroxim
obidoxime
furosemide
a
CNS(+) (high BBB permeation predicted); Pe (10−6 cm s−1) > 4.0,
CNS(−) (low BBB permeation predicted); Pe (10−6 cm s−1) < 2.0,
CNS( ) (BBB permeation uncertain); Pe (10−6 cm s−1) from 4.0 to
2.0.
experimental protocol exploiting AutoDock Tools.46−49 (R)-
BIGI-3h (Figure 2A,B) revealed dual binding site pattern with
N-benzylpiperidine being oriented outward to the cavity
gorge, while 6,8-dichloro-4H-chromen-4-one was placed
centrally. This is somewhat contradictory to donepezil-
hAChE anchoring in crystal structure, because N-benzylpiper-
idine was found to be stacked against Trp86 in the catalytic
anionic site (CAS; Figure 3).45,50 More in detail, the N-benzyl
ring of (R)-BIGI-3h is stacked against Trp286 (4.1 Å), a key
amino acid residue of the hAChE peripheral anionic site
(PAS).51 Additionally, the ligand is also engaged in cation−π
interaction between the nitrogen of piperidine and Trp286
(4.6 Å). In the midgorge AChE region, the ligand is
implicated in several π−alkyl interactions between Tyr124,
Phe338, and Phe297 and methyl together with ethyl-
carboxylate appendages of ethyl 6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate moiety. At the bottom of
the gorge, 6,8-dichloro-4H-chromen-4-one displayed putative
face-to-face π−π interaction with Trp86 (3.6 Å) and displaced
π−π interaction with Tyr337 (3.4 Å). Hydrogen situated at
the nitrogen from heterocyclic indole of Trp86 (2.2 Å)
formed favorable hydrogen contact to the carbonyl from the
ethyl-carboxylate appendage. Ser203 as well His447 from the
GSK-3β biochemical inhibition for ( )-BIGI-3h, (+)-BIGI-
3h,, and (−)-BIGI-3h was assessed using the Kinase-Glo
1332
https://doi.org/10.1021/acschemneuro.0c00803
ACS Chem. Neurosci. 2021, 12, 1328−1342

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Figure 2. Top-scored docking poses of (R)-BIGI-3h (A, B) and (S)-BIGI-3h (C, D) complexed with hAChE (PDB ID: 4EY7). Generally, for
panels A and C, (R)-BIGI-3h and (S)-BIGI-3h are shown in green and purple carbon atoms, respectively, and important amino acid residues
responsible for ligand anchoring are shown in blue; the catalytic triad is shown in yellow. Distances are measured in angstroms (Å). 3D poses
were generated using PyMOL viewer 2.4.1. Generally, for 2D representation rendered in panels B and D, all the plausible interactions are dashed
between appropriate amino acids and respective ligand parts. The 2D diagrams were created with Discovery Studio Visualizer v. 20.1.0.19295
̀
(BIOVIA, Dassault Systemes, Release 2019, San Diego, USA).
Figure 3. Spatial overlap between (R)-BIGI-3h (green) and (S)-BIGI-3h (purple) and crystal structure of donepezil (orange) embedded within
hAChE (PDB ID: 4EY7). For the sake of clarity, only Trp286 and Trp86 are displayed defining PAS and CAS regions of the enzyme,
respectively.
luminescent assay, which quantifies the decrease in ATP levels
following the kinase reaction.53 SB-41528634, a well-known
GSK3β inhibitor, was used as a positive control. As shown in
Table 3, ( )-BIGI-3h and (−)-BIGI-3h showed similar IC50
values (29.1 and 24.3 μM, respectively), whereas (+)-BIGI-3h
was slightly more potent (IC50 = 10.6 μM). These activities
within the micromolar range are in line with those of other
multitarget-based GSK-3β compounds.35
τ Aggregation and Inhibition Study. As hyper-
phosphorylated τ aggregation seems to proceed in a manner
similar to that of Aβ aggregation (conformational shift →
nucleation → elongation), we evaluated the ability of
( )-BIGI-3h and its enantiomers to inhibit the aggregation
of the tau(306−336) peptide comprising the R3 domain of τ
protein, in vitro. The importance of the τ R3 domain in τ
aggregation has been highlighted by a mutagenesis study.54
Furthermore, it has been recently shown that this fragment
1333
https://doi.org/10.1021/acschemneuro.0c00803
ACS Chem. Neurosci. 2021, 12, 1328−1342

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
can induce the aggregation of the τ microtubule-binding
region in cells;55 thus, it represents a good in vitro model to
monitor τ aggregation and screen for aggregation inhibitors.
Therefore, aggregation inhibition experiments were per-
formed by incubating the τ(306−336) peptide in the absence or
presence of the tested compounds at a concentration ratio of
[inhib]/[τ] = 1/1, equal to 50 μM. Thioflavin T was used as a
fluorescent dye to detect fibril formation. ( )-BIGI-3h
showed an interesting inhibitory pattern: It delayed the lag
phase (t1/2 from 4.5 to 6.3 h) (Figure 4) and inhibited
proof-of-concept regarding the brain activity of (
)-BIGI-
3h, using the novel object recognition (NOR) test, a widely
used test for cognition assessment.56 As expected, control
mice (vehicle/DMSO group) spent more time exploring the
novel object than the familiar one (RI = 0.67, Figure 5).
Compared with that of the control treatment, scopolamine (1
mg/kg) treatment resulted in a decrease in the recognition
index in mice, suggesting a working memory impairment. In
fact, scopolamine produced a memory acquisition deficit, thus
constituting an appropriate model to evaluate short-term
memory. Importantly, mice treated with 10 mg/kg of (
)-BIGI-3h after scopolamine treatment showed significantly
improved recognition index. These results showed that
recognition memory deficits caused by scopolamine (1 mg/
kg) injection are restored by 10 mg/kg of ( )-BIGI-3h
(Figure 5). This activity can likely be mainly attributed to the
anticholinesterase properties of this compound. It will
nevertheless be interesting to identify the target engagement
of ( )-BIGI-3h, through a comprehensive study on an
appropriate animal model of Alzheimer’s disease.
CONCLUSION
■
Based on our confidence that MCRs represent a useful tool in
medicinal chemistry and symbolize the most appropriate
method for accessing many different types of pharmacophores,
thus expressing particular relevance for the discovery of
bioactive compounds, we have designed and synthesized, via
the Biginelli multicomponent reaction, 14 new compounds.
From all the biological and physicochemical results gathered
in this study, we have identified ( )-ethyl 1-(2-(1-
benzylpiperidin-4-yl)ethyl)-4-(6,8-dichloro-4-oxo-4H-chro-
men-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-car-
boxylate. ( )-BIGI-3h is the first MTDL that simultaneously
addressed several key targets involved in the AD pathological
cascade.
This newly designed and well-balanced compound showed
potent and well-balanced in vitro inhibitory activities against
neurotransmitter-catabolizing enzymes (ChE and MAO) and
GSK3β kinase, along with VGCC blockade. ( )-BIGI-3h also
showed potent inhibition of τ and Aβ aggregation, antioxidant
activity, and good brain permeation. Moreover, BBB
permeation was also confirmed by in vivo studies, which
Figure 4. Inhibitory effects of compounds ( )-BIGI-3h, (+)-BIGI-
3h, and (−)-BIGI-3h against the aggregation of τ(306−336) [τ(306−336)
peptide] = [inhibitor] = 50 μM. Aggregation is monitored by the
increase in fluorescence emission in the presence of thioflavin T dye.
τ(306−336) aggregation by 73.7%, while the anti-ChE drug
tacrine did not significantly interfere with aggregation of the
τ(306−336) peptide (Table 3). Doxycycline, which was used as
positive control, gave a percentage of inhibition of 61.5
0.8%, when assayed in the same experimental conditions
(Table 3). A delay in the lag phase is an interesting feature for
a potential inhibitor, as it implies potent inhibition in the early
phase of τ aggregation and higher efficacy in preventing fibril
formation. Pure enantiomers were tested under the same
experimental conditions, but no significant difference in anti-τ
aggregation activity was noted compared with that of the
racemic mixture.
In Vivo Study of BIGI-3h. (+)-BIGI-3h and (−)-BIGI-3h
showed biological activity similar to that of the racemic
mixture. Hence, an in vivo study was performed to obtain
Figure 5. Effect of combined administration of scopolamine/dimethyl sulfoxide (scop/DMSO), donepezil, and ( )-BIGI-3h on recognition
index. BIGI-3h at 1 mg/kg (bigi1); BIGI-3h at 10 mg/kg (bigi10). ** p < corrected significance threshold.
1334
https://doi.org/10.1021/acschemneuro.0c00803
ACS Chem. Neurosci. 2021, 12, 1328−1342

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Chemical Synthesis. General Procedure for the Synthesis
of Ureas 2a−c.37 To a solution of the corresponding 4-amino-
benzylpiperidine (1 g) in dichloromethane (10 mL) at 0 °C, a 20 mL
solution of benzoyl isocyanate (1.2 equiv) in dichloromethane was
added. The reaction was stirred for 1 h at room temperature. The
solvent was evaporated, and the crude oil was recrystallized in diethyl
ether to give a white solid. Yield: 72%. The product was reacted
without further purification.
also showed that ( )-BIGI-3h (10 mg/kg) could significantly
restore the cognitive deficit of scopolamine-treated mice in the
NOR test. This beneficial activity can likely be mainly
attributed to the anticholinesterase properties of this
compound. Notwithstanding, specific AD murine models
would be required to further in vivo assess the multifunctional
profile of ( )-BIGI-3h; its unique pharmacological profile
makes this compound eligible for further studies for a possible
effective treatment of AD.
The corresponding benzoyl compound was hydrolyzed for 48 h at
room temperature in a 1:1 solution of methanol in 1 M aqueous
NaOH (30 mL). The solvents were concentrated under vacuum and
extracted three times with ethyl acetate. The combined organic layers
were washed with brine, dried over Na2SO4, and evaporated. The
obtained solid was washed with diethyl ether to give a white solid.
Yields: 41−55%.
METHODS
■
Chemistry. General Methods. All the commercially available
reagents and solvents were purchased from Sigma-Aldrich, VWR, and
TCI. Reactions were followed by analytical thin layer chromatog-
raphy (TLC) on precoated TLC plates (layer 0.20 mm silica gel 60
1-((1-Benzylpiperidin-4-yl)methyl)urea (2a). Yield: 41% 1H NMR
(400 MHz, CDCl3): δ 7.36−7.30 (m, 4H), 7.28−7.23 (m, 1H), 5.03
(bs, 1H), 4.60 (s, 2H), 3.50 (s, 2H), 3.10−3.00 (m, 2H), 2.95−2.85
(m, 2H), 2.02−1.89 (m, 2H), 1.74−1.62 (m, 2H), 1.57−1.41 (m,
1H), 1.37−1.21 (m, 2H).
with a fluorescent indicator UV254. H and 13C NMR spectra were
1
recorded on a Bruker spectrometer; operating at (1H NMR at 400
MHz, 13C NMR at 100 MHz), in solution in dimethyl sulfoxide
(DMSO-d6) or chloroform (CDCl3) at 20 °C; chemical shift values
are given in δ (ppm) relative to TMS as internal reference. Coupling
constants are given in hertz (Hz). The following abbreviations were
used: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet.
High resolution mass spectra were obtained at Centre Commun de
1-(2-(1-Benzylpiperidin-4-yl)ethyl)urea (2b). Yield: 43%. 1H
NMR (400 MHz, CDCl3): δ 7.36−7.30 (m, 4H), 7.28−7.23 (m,
1H), 4.67 (bs, 1H), 4.46 (bs, 2H), 3.50 (s, 2H), 3.25−3.13 (m, 2H),
2.94−2.82 (m, 2H), 2.03−1.87 (m, 2H), 1.73−1.59 (m, 2H), 1.50−
1.40 (m, 2H), 1.37−1.22 (m, 3H).
́
Spectrometrie de Masse, Lyon, France, on a Bruker micrOTOF-Q II
1-(3-(1-Benzylpiperidin-4-yl)propyl)urea (2c). Yield: 35%. 1H
NMR (400 MHz, CDCl3): δ 7.29−7.26 (m, 5H), 4.45 (bs, 1H),
4.35 (bs, 2H), 3.47 (s, 2H), 3.16−3.10 (m, 2H), 2.88−2.84 (m, 2H),
1.95−1.87 (m, 2H), 1.65−1.53 (m, 2H), 1.48−1.46 (m, 2H). 1.27−
1.19 (m, 5H).
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 were within
0.2% of the theoretical values for all compounds. The purity of the
new compounds was checked by elemental analyses, conducted on a
Carlo Erba EA 1108 apparatus, and confirmed to be >95%. The
BIGI were found also to be ≥95% pure by HPLC analysis using a
Hitachi Lachrom Elite series instrument equipped with a L2400
Lachrom Elite DAD detector and a Uptisphere ODB column (4.6
mm × 100 mm, Ø= 3 μm). Peaks were detected at 210 nm, and the
system was operated at 25 °C with a flow rate of 2 mL/min. The
mobile phase was an isocratic mixture of acetonitrile and water (1:1,
v/v) containing 0.1% (w/v) of monopotassium phosphate.
General Procedure for the Synthesis of Biginelli Com-
pounds BIGI-3a−n. To a solution of 150 mg of the corresponding
commercially chromones in 10 mL of acetic acid, 1.2 equiv of the
corresponding urea 2a−c was added. The reaction was stirred for 2
h. Ethyl acetoacetate (1.25 equiv) and 1 equiv of sodium bisulfate
were added. The reaction was stirred for 60 h at room temperature
and then refluxed for 4 h. The catalyst was filtered off, and the
solvent was evaporated under vacuum. The crude oil was purified by
flash chromatography eluted with dichloromethane/MeOH/
NH3(aq) 96:4:0.4 to give the pure product. Yield 45−78%.
( )-Ethyl 1-((1-Benzylpiperidin-4-yl)methyl)-6-methyl-2-oxo-4-
(4-oxo-4H-chromen-3-yl)-1,2,3,4-tetrahydro pyrimidine-5-carboxy-
late BIGI-3a. Following the general procedure, compound BIGI-3a
was obtained as a brown powder with a yield of 52%. 1H NMR (400
MHz, CDCl3): δ 8.24−8.21 (dd, 1H), 7.65−7.72 (m, 1H), 7.63 (s,
1H), 7.45−7.42 (m, 2H), 7.29−7.26 (m, 5H), 6.02−6.01 (dd, 1H),
5.61−5.60 (dd, 1H), 4.18−4.08 (m, 2H), 4.07−3.99 (m, 1H), 3.47
(s, 2H), 3.36−3.31 (dd, 1H), 2.88−2.77 (dd, 2H), 2.59 (s, 3H),
1.95−1.86 (m, 3H), 1.61−1.47 (m, 2H), 1.33−1.26 (m, 2H), 1.19−
1.16 (t, 3H). 13C NMR (101 MHz, CDCl3): δ 176.55, 164.65,
162.42, 155.45, 152.67, 151.65, 150.90, 133.01, 128.28, 127.27,
124.83, 124.44, 122.90, 122.27, 117.15, 98.88, 59.44, 51.88, 45.81,
28.67, 15.54, 13.24. Anal. Calcd for C30H33N3O5: C, 69.88; H, 6.45;
N, 8.15; found, C, 70.05; H, 6.51; N, 7.99.
( )-Ethyl 1-((1-Benzylpiperidin-4-yl)methyl)-4-(6-bromo-4-oxo-
4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate BIGI-3b. Following the general procedure, compound
BIGI-3b was obtained as a brown yellow powder with a yield of 63%.
1H NMR (400 MHz, CDCl3): δ 8.27−8.26 (d, 1H), 7.69−7.66 (dd,
1H), 7.55 (s, 1H), 7.26−7.16 (m, 6H), 5.97−5.96 (d, 1H), 5.48−
5.47 (d, 1H), 4.10−3.99 (m, 2H), 3.52−3.89 (m, 1H), 3.49−3.37
(m, 3H), 2.79−2.75 (m, 2H), 2.51 (s, 3H), 1.79−1.75 (m, 2H),
1.59−1.51 (m, 3H), 1.22−1.15 (m, 2H), 1.19−1.15 (m, 3H). 13C
NMR (101 MHz, CDCl3): δ 176.17, 165.57, 155.21, 153.48, 153.03,
151.85, 137.83, 136.97, 129.31, 128.47, 128.18, 127.08, 125.25,
123.67, 120.15, 118.88, 99.53, 63.15, 60.43, 53.47, 46.60, 40.58,
36.52, 33.64, 32.07, 31.73, 16.11, 14.30. Anal. Calcd for
C30H32BrN3O5: C, 60.61; H, 5.43; N, 7.07; Br, 13.44; found, C,
60.52; H, 5.48; N, 7.12; Br, 13.37.
Analyses by chiral SFC were run on Waters SFC/MS equipped
with a UV-DAD (220 and 254 nm) detector and MS (APCI:
modes). Analyses were performed isocratic on a Chiralpak AS-H (4.6
mm × 100 mm, particle size 5 μm) with flow rate of 5 mL/min.
Mobile phase was 0.2% isopropylamine CO2/EtOH (55:45).
Separations by preparative chiral SFC were run on a Sepiatec Prep
100 SFC equipped with UV-DAD and a Chiralpak AS-H column (21
mm × 250 mm, particle size 5 μm). Peaks were detected at 220 and
254 nm, and the system was operated at 40 °C with a flow rate of 65
mL/min with the same mobile phase.
PAINS Analysis. The undetected pan assay interference
compounds (PAINS) with false-positive activities seriously com-
promise their outcomes in medicinal chemistry programs. Thus,
although our designed and synthesized BIGIs are not known as
classes of PAINS,57−60 BIGI-3h was submitted to Free ADME-Tox
filtering tool (FAFDrugs4) program (http://fafdrugs4.mti.univ-paris-
diderot.fr/) and to the “False Positive Remover” software (http://
www.cbligand.org/PAINS/). The results indicate that it is not
classified as PAINS. BIGI-3h was also not recognized as PAINS
according swissadme.ch (see Figure S27 in Supporting Information).
Finally, compound BIGI-3h showed 71% of similarity with a
previously reported aggregator61,62 according to the software
“Aggregator Advisor” (http://advisor.bkslab.org/). Then, because
the aggregators are nonspecific inhibitors, BIGI-3h was evaluated
against an unrelated enzyme (the alkaline phosphatase, see
Supporting Information). The results indicated no inhibition, proving
that BIGI-3h is not an aggregator. In addition, the previously
reported aggregator showed another chemical structure compared to
our compounds, exhibiting higher molecular weight equal to 840 g
mol−1 and many conjugated double bonds (2 biphenyl groups with a
total of five benzene rings in its structure).
1335
https://doi.org/10.1021/acschemneuro.0c00803
ACS Chem. Neurosci. 2021, 12, 1328−1342

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
( )-Ethyl 1-((1-Benzylpiperidin-4-yl)methyl)-4-(7-ethyl-4-oxo-
4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate BIGI-3c. Following the general procedure, compound
40.49, 36.42, 33.47, 31.74, 31.38, 20.87, 16.09, 14.26. Anal. Calcd for
C32H36ClN3O5: C, 66.48; H, 6.28; N, 7.27; Cl, 6.13; found, C, 66.34;
H, 6.35; N, 7.18; Cl, 6.24.
1
BIGI-3c was obtained as a purple powder with a yield of 78%. H
( )-Ethyl 1-(2-(1-Benzylpiperidin-4-yl)ethyl)-4-(6,8-dichloro-4-
oxo-4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimi-
dine-5-carboxylate BIGI-3h. Following the general procedure,
compound BIGI-3h was obtained as a brown yellow powder with
NMR (400 MHz, CDCl3): δ 7.96−7.95 (d, 1H), 7.53 (s, 1H), 7.45−
7.43 (dd, 1H), 7.29−7.27 (m, 1H), 7.21−7.15 (m, 5H), 5.97−5.95
(d, 1H), 5.54−5.53 (d, 1H), 4.10−3.92 (m, 3H), 3.37 (s, 2H), 3.28−
3.23 (dd, 1H), 2.78−2.66 (m, 4H), 2.51 (s, 3H), 1.84−1.75 (m,
2H), 1.48−1.37 (m, 3H), 1.27−1.17 (m, 5H), 1.11−1.08 (t, 3H).
13C NMR (101 MHz, CDCl3): δ 177.64, 165.73, 154.94, 153.78,
1
a yield of 75%. H NMR (400 MHz, CDCl3): δ 8.10−8.09 (d, J =
4.0 Hz, 1H), 7.75−7.74 (d, 2H), 7.37−7.34 (m, 5H), 6.16−6,15 (d,
1H), 5.54−5,53 (d, 1H) 4.20−4.10 (m, 2H), 3.88−3.86 (m, 1H),
3.76 (s, 2H), 3.66−3.57 (m, 1H), 3.19−3.16 (m, 2H), 2.59 (s, 3H),
2.23−2.18 (m, 2H), 1.79−1.72 (m, 2H), 1.62−1.59 (m, 3H), 1.57−
1.55 (m, 1H), 1.20 (m, 1H), 1.18 (t, J = 8.0 Hz, 3H). 13C NMR
(101 MHz, CDCl3): δ 176.00, 175.75, 165.49, 153.70, 153.20,
151.69, 150.88, 134.24, 131.29, 130.58, 128.77, 125.82,
124.58,124.10, 123.90, 99.51, 61.56, 60.67, 52.32, 46.85, 40.53,
36.04, 32.81, 30.28, 29.95, 22.12,16.26, 14.38. HRMS ESI-TOF [M +
H]+ m/z meas. (measured) for C31H34Cl2N3O5: 598.1862; found,
598.1870. Anal. Calcd for C31H33Cl2N3O5: C, 62.21; H, 5.56; N,
7.02; Cl, 11.85; found, C, 62.38; H, 5.47; N, 7.13; Cl, 11.74.
(+)-Ethyl 1-(2-(1-Benzylpiperidin-4-yl)ethyl)-4-(6,8-dichloro-4-
oxo-4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimi-
dine-5-carboxylate (+) BIGI-3h. Retention time on analytical chiral
HPLC: 0.48 min. [α]D = +162° (c 0.14, MeOH), HRMS ESI-TOF
[M + H]+ m/z meas. for C31H34Cl2N3O5: 598.1872; found,
598.1870.
152.55, 151.95, 141.83, 134.30, 129.19, 128.20, 127.11, 123.91,
123.69, 123.06, 118.02, 100.04, 62.91, 60.43, 52.85, 47.08, 46.77,
36.71, 29.72, 28.39, 16.51, 15.54, 14.28. Anal. Calcd for C32H37N3O5:
C, 70.70; H, 6.86; N, 7.73; found, C, 70.85; H, 6.81; N, 7.69.
( )-Ethyl 1-(2-(1-Benzylpiperidin-4-yl)ethyl)-6-methyl-2-oxo-4-
(4-oxo-4H-chromen-3-yl)-1,2,3,4-tetrahydro pyrimidine-5-carboxy-
late BIGI-3d. Following the general procedure, compound BIGI-3d
1
was obtained as a red powder with a yield of 45%. H NMR (400
MHz, CDCl3): δ 8.25−8.22 (d, 1H), 7.72−7.67 (t, 1H), 7.61 (s,
1H), 7.46−7.43 (m, 2H), 7.28−7.26 (m, 5H), 5.97−5.96 (d, 1H),
5.59−5.58 (d, 1H), 4.19−4.06 (m, 2H), 3.98−3.92 (m, 1H), 3.60−
3.5 (m, 1H), 3.42 (s, 2H), 2.85−2.81 (d, 2H), 2.6 (s, 3H), 1.85−
1.75 (m, 2H), 1.70−1.67 (m, 1H), 1.62−1.53 (m, 2H), 1.42−1.33
(m, 1H), 1.29−1.27 (m, 3H), 1.25−1.15 (m, 3H). 13C NMR (101
MHz, CDCl3): δ 177.52, 165.64, 156.48, 153.58, 152.75, 151.80,
134.00, 129.27, 128.17, 127.06, 125.85, 125.43, 123.95, 123.31,
118.20, 99.70, 63.23, 60.38, 53.56, 53.52, 46.56, 40.46, 36.51, 33.59,
32.11, 31.72, 31.64, 29.70, 16.07, 14.27. Anal. Calcd for C31H35N3O5:
C, 70.30; H, 6.66; N, 7.93; found, C, 70.11; H, 6.72; N, 7.99.
( )-Ethyl 1-(2-(1-Benzylpiperidin-4-yl)ethyl)-4-(6-bromo-4-oxo-
4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate BIGI-3e. Following the general procedure, compound
(−)-Ethyl 1-(2-(1-Benzylpiperidin-4-yl)ethyl)-4-(6,8-dichloro-4-
oxo-4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimi-
dine-5-carboxylate (−) BIGI-3h. Retention time on analytical chiral
HPLC: 0.72 min. [α]D = −178° (c 0.13, MeOH), HRMS ESI-TOF
[M + H]+ m/z meas. for C31H34Cl2N3O5: 598.1874; found,
598.1870.
( )-Ethyl 1-(3-(1-Benzylpiperidin-4-yl)propyl)-6-methyl-2-oxo-4-
(4-oxo-4H-chromen-3-yl)-1,2,3,4-tetrahydro pyrimidine-5-carboxy-
late BIGI-3i. Following the general procedure, compound BIGI-3i
was obtained as a brown powder with a yield of 55%. 1H NMR (400
MHz, CDCl3): δ 8.16−8.14 (dd, 1H), 7.62−7.55 (m, 2H), 7.37−
7.33 (m, 2H), 7.24−7.16 (m, 5H), 5.93−5.92 (dd, 1H), 5.52−5.51
(dd, 1H), 4.11−3.99 (m, 2H), 3.75−3.80 (m, 1H), 3.47−3.43 (m,
3H), 2.81−2.78 (m, 2H), 2.53 (s, 3H), 1.89−1.83 (t, 2H), 1.53−
1.49 (m, 3H), 1.35−1.45 (m, 1H), 1.18−1.08 (m, 8H). 13C NMR
(101 MHz, CDCl3): δ 177.54, 165.69, 156.48, 153.62, 152.75,
151.87, 134.01, 129.39, 127.11, 125.85, 125.43, 123.95, 123.33,
118.18, 99.55, 63.17, 60.39, 53.62, 53.57, 46.59, 42.75, 35.28, 33.42,
31.99, 37.29, 16.17, 14.28. Anal. Calcd for C32H37N3O5: C, 70.70; H,
6.86; N, 7.73; found, C, 70.58; H, 6.92; N, 7.70.
1
BIGI-3e was obtained as a brown powder with a yield of 68%. H
NMR (400 MHz, CDCl3): δ 8.33 (s, 1H), 7.75−7.73 (d, 1H), 7.61
(s, 1H), 7.33−7.26 (m, 6H), 60.3−6.02 (d, 1H), 5.54−5.53 (d, 1H),
4.14−4.06 (m, 2H), 3.94−3.91 (m, 1H), 3.59−3.50 (m, 1H), 3.42
(s, 2H), 2.84−2.80 (d, 2H), 2.58 (s, 3H), 1.83−1.80 (m, 2H), 1.68−
1.57 (m, 3H), 1.24−1.18 (m, 4H), 1.16−1.14 (m, 3H). 13C NMR
(101 MHz, CDCl3): δ 176.17, 165.55, 155.19, 153.46, 153.02,
151.80, 137.40, 136.97, 129.39, 128.44, 128.20, 127.18, 125.22,
123.62, 120.14, 118.88, 99.51, 63.04, 60.43, 53.38, 53.34, 46.58,
40.55, 36.45, 33.55, 31.90, 31.56, 16.11, 14.27. Anal. Calcd for
C31H34BrN3O5: C, 61.19; H, 5.63; N, 6.91; Br, 13.13; found, C,
61.42; H, 5.49; N, 6.84; Br, 13.05.
( )-Ethyl 1-(2-(1-Benzylpiperidin-4-yl)ethyl)-4-(7-ethyl-4-oxo-
4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate BIGI-3f. Following the general procedure, compound
( )-Ethyl 1-(3-(1-Benzylpiperidin-4-yl)propyl)-4-(6-bromo-4-oxo-
4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate BIGI-3j. Following the general procedure, compound
1
BIGI-3f was obtained as a brown powder with a yield of 57%. H
NMR (400 MHz, CDCl3): δ 7.96−7.95 (d, 1H), 7.53 (s, 1H), 7.46−
7.43 (dd, 1H), 7.29−7.15 (m, 6H), 5.96−5.95 (d, 1H), 5.51−5.50
(d, 1H), 4.10−3.99 (m, 2H), 3.85−3.95 (m, 1H), 3.50−3.45 (m,
1H), 3.34 (s, 2H), 2.77−2.66 (m, 4H), 2.52 (s, 3H), 1.77−1.74 (m,
2H), 1.62−1.59 (m, 1H), 1.54−1.51 (m, 2H), 1.30−1.35 (m, 2H),
1.17−1.23 (m, 5H), 1.11−1.08 (t, 3H). 13C NMR (101 MHz,
CDCl3): δ 177.64, 165.68, 154.93, 153.64, 152.66, 151.76, 141.79,
138.00, 134.26, 129.27, 128.17, 127.04, 123.91, 123.74, 123.11,
118.05, 99.83, 63.24, 60.35, 53.57, 53.53, 46.62, 40.45, 36.53, 33.60,
32.15, 31.74, 28.39, 16.07, 15.57, 14.28. Anal. Calcd for C33H39N3O5:
C, 71.07; H, 7.05; N, 7.53; found, C, 70.95; H, 7.11; N, 7.62.
( )-Ethyl 1-(2-(1-Benzylpiperidin-4-yl)ethyl)-4-(6-chloro-7-meth-
yl-4-oxo-4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyri-
midine-5-carboxylate BIGI-3g. Following the general procedure,
compound BIGI-3g was obtained as a purple powder with a yield of
1
BIGI-3j was obtained as a brown powder with a yield of 49%. H
NMR (400 MHz, CDCl3): δ 8.28−8.27 (d, 1H), 7.68−7.66 (dd,
1H), 7.55 (s, 1H), 7.27−7.18 (m, 6H), 5.86−5.85 (d, 1H), 5.50−
5.49 (d, 1H), 4.11−3.99 (m, 2H), 3.78 (m, 1H), 3.45−3.41 (m, 2H),
3.48 (s, 1H), 2.81−2.76 (m, 2H), 2.53 (s, 3H), 1.87−1.81 (t, 2H),
1.53−1.49 (m, 3H), 1.35−1.40 (m, 1H), 1.12−1.09 (m, 8H). 13C
NMR (101 MHz, CDCl3): δ 176.25, 165.60, 155.23, 153.46, 152.96,
151.98, 137.01, 129.29, 128.47, 128.16, 127.01, 125.23, 123.62,
120.14, 118.91, 99.32, 63.35, 60.44, 53.76, 46.57, 42.83, 35.46, 33.49,
32.18, 27.28, 16.20, 14.28. Anal. Calcd for C32H36BrN3O5: C, 61.74;
H, 5.83; N, 6.75; Br, 12.83; found, C, 61.82; H, 5.77; N, 6.81; Br,
12.74.
( )-Ethyl 1-(3-(1-Benzylpiperidin-4-yl)propyl)-4-(7-ethyl-4-oxo-
4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetr hydropyrimidine-5-
carboxylate BIGI-3k. Following the general procedure, compound
1
66%. H NMR (400 MHz, CDCl3): δ 8.15 (s, 1H), 7.5 (s, 1H),
1
BIGI-3k was obtained as a brown powder with a yield of 63%. H
7.31−7.27 (m, 6H), 6.03−6.02 (d, 1H), 5.54−5.53 (d, 1H), 4.16−
4.08 (m, 2H), 3.9 (m, 1H), 3.58−3.48 (m, 3H), 2.90−2.87 (d, 2H),
2.57 (s, 3H), 2.48 (s, 3H), 2.01 (s, 1H), 1.90−1.86 (d, 2H), 1.67−
1.58 (m, 3H), 1.27−1.24 (m, 3H), 1.18−1.13 (m, 3H). 13C NMR
(101 MHz, CDCl3): δ 176.28, 165.58, 154.71, 153.53, 152.78,
151.70, 143.38, 137.04, 132.17, 129.49, 128.22, 127.26, 125.43,
123.29, 122.98, 119.93, 99.64, 62.82, 60.39, 53.20, 53.15, 46.57,
NMR (400 MHz, CDCl3): δ 7.96−7.95 (d, 1H), 7.53 (s, 1H), 7.45−
7.42 (dd, 1H), 7.29−7.19 (m, 6H), 5.91−5.90 (d, 1H), 5.52−5.51
(d, 1H), 4.10−3.99 (m, 2H), 3.77−3.81 (m, 1H), 3.45−3.50 (m,
3H), 2.88−2.81 (d, 2H), 2.72−2.66 (dd, 2H), 2.53 (s, 3H), 1.82−
1.90 (m, 1H), 1.52−1.50 (m, 3H), 1.42−1.45 (m, 1H), 1.23−1.08
(m, 12H). 13C NMR (101 MHz, CDCl3): δ 177.68, 165.71, 154.93,
1336
https://doi.org/10.1021/acschemneuro.0c00803
ACS Chem. Neurosci. 2021, 12, 1328−1342

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
153.65, 152.62, 151.82, 141.79, 134.28, 129.43, 129.37, 128.23,
127.20, 127.19, 123.89, 123.72, 123.09, 118.03, 99.64, 63.17, 63.15,
60.36, 53.63, 53.59, 46.62, 42.71, 35.31, 33.36, 31.94, 31.93, 31.91,
31.88, 29.72, 28.39, 27.28, 16.16, 15.54, 14.28. Anal. Calcd for
C34H41N3O5: C, 71.43; H, 7.23; N, 7.35; found, C, 71.35; H, 7.28;
N, 7.44.
In Vitro Anticholinesterase Assay. The AChE and BuChE
inhibitory activity of the tested compounds was determined using a
38
́
modified Ellmans method. Human erythrocytes acetylcholinester-
ase (hAChE; EC 3.1.1.7), human recombinant butyrylcholinesterase
́
(hBuChE; EC 3.1.1.8), 5,5′-dithiobis(2-nitrobenzoic acid) (Ellmans
reagent, DTNB), phosphate buffer (PB, pH 7.4), acetylthiocholine
(ATC), and butyrylthiocholine (BTC), were purchased from Sigma-
Aldrich (Prague, Czech Republic). Nunc polystyrene 96-well
microplates with a flat-bottom shape (ThermoFisher Scientific,
USA) were used for the measurement, and all the assays were
carried out in 0.1 M KH2PO4/K2HPO4 buffer, pH 7.4. Enzyme
solution was prepared at activity 2.0 units/mL in 2 mL of aliquots.
The assay mixture (100 μL) consisted of 40 μL of 0.1 M phosphate
buffer (pH 7.4), 20 μL of 0.01 M DTNB, 10 μL of enzyme, and 20
μL of 0.01 M substrate (ATC iodide solution). Tested compounds
were dissolved first in methanol or in DMSO and then further
diluted by assay buffer to the final concentration. The highest
concentration tested was 31.6 μM, which contained 0.5% of
cosolvent (methanol or DMSO) in the assay mixture. Such level of
cosolvents was proved to have no effect on the activity of the
enzyme. Enzyme with the inhibitor was preincubated for 5 min in the
assay mixture and then the reaction was started by addition of 20 μL
of substrate (ATC for AChE, BTC for BChE). The enzyme activity
was determined by measuring the increase in absorbance at 412 nm
at 37 °C in 2 min intervals using a Multimode microplate reader
Synergy 2 (Vermont, USA). Each concentration was assayed in
triplicate. The obtained data were used to compute the percentage
inhibition (I; eq 1):
( )-Ethyl 1-(3-(1-Benzylpiperidin-4-yl)propyl)-4-(6-chloro-7-
methyl-4-oxo-4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahy-
dropyrimidine-5-carboxylate BIGI-3l. Following the general proce-
dure, compound BIGI-3l was obtained as a brown yellow powder
1
with a yield of 62%. H NMR (400 MHz, CDCl3): δ 8.33 (s, 1H),
7.75 (s, 1H), 7.24−7.16 (m, 6H), 5.88−5.87 (d, 1H), 5.50−5.49 (d,
1H), 4.10−3.99 (m, 2H), 3.78−3.58 (m, 1H), 3.47−3.42 (m, 3H),
2.79−2.77 (d, 2H), 2.52 (s, 3H), 2.41 (s, 3H), 1.87−1.82 (t, 2H),
1.54−1.49 (m, 3H), 1.35−1.45 (m, 1H), 1.18−1.08 (m, 8H). 13C
NMR (101 MHz, CDCl3): δ 176.36, 165.64, 154.75, 153.52, 152.76,
151.89, 143.41, 132.20, 129.29, 128.17, 127.02, 125.45, 123.31,
122.99, 119.93, 99.44, 63.33, 60.40, 53.71, 46.54, 42.80, 35.46, 33.46,
32.15, 27.28, 20.87, 16.18, 14.28. Anal. Calcd for C33H38ClN3O5: C,
66.94; H, 6.47; N, 7.10; Cl, 5.99; found, C, 67.90; H, 6.43; N, 7.19;
Cl, 5.98.
( )-Ethyl 1-(3-(1-Benzylpiperidin-4-yl)propyl)-4-(6,8-dichloro-4-
oxo-4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimi-
dine-5-carboxylate BIGI-3m. Following the general procedure,
compound BIGI-3m was obtained as a brown powder with a yield
1
of 49%. H NMR (101 MHz, CDCl3): δ 8.03−8.02 (d, 1H), 7.66−
7.64 (dd, 2H), 7.25−7.18 (m, 5H), 5.83−5.82 (d, 1H), 5.49−5.48
(d, 1H), 4.12−4.00 (m, 2H), 3.70−3.80 (m, 1H), 3.47−3.45 (m,
3H), 2.87−2.81 (m, 2H), 2.53 (s, 3H), 1.80−1.95 (m, 2H), 1.50−
1.65 (m, 3H), 1.45−1.48 (m, 1H), 1.18−1.35 (m, 8H). 13C NMR
(101 MHz, CDCl3): δ 175.82, 165.46, 153.37, 152.99, 152.12,
150.80, 134.17, 131.21, 129.43, 128.24, 127.19, 125.69, 124.48,
124.00, 123.81, 99.05, 63.20, 60.52, 53.59, 46.56, 42.94, 35.35, 33.42,
31.94, 29.71, 27.28, 16.22, 14.30. Anal. Calcd for C32H35Cl2N3O5: C,
62.75; H, 5.76; N, 6.86; Cl, 11.58; found, C, 62.98; H, 5.72; N, 6.74;
Cl, 11.41.
ΔA
ΔA0
I = 1 −
i [%]
(1)
where ΔAi indicates the absorbance change provided by cholinester-
ase exposed to AChE inhibitors and ΔA0 indicates absorbance
change caused by intact cholinesterase (phosphate buffer was used
instead of AChE inhibitor solution). The inhibition potency of the
tested compounds was expressed as the IC50 value (the concentration
of inhibitor which causes 50% cholinesterase inhibition). Calculations
were performed using software Microsoft Excel (Redmont, WA,
USA) and GraphPad Prism version 5.02 for Windows, GraphPad
Software, San Diego, CA, USA.
( )-Ethyl 1-(3-(1-Benzylpiperidin-4-yl)propyl)-4-(6-isopropyl-4-
oxo-4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4 -tetrahydropyrimi-
dine-5-carboxylate BIGI-3n. Following the general procedure,
compound BIGI-3n was obtained as a brown powder with a yield
of 45%. 1H NMR (400 MHz, CDCl3): δ 7.98−7.97 (d, 1H), 7.54 (d,
2H), 4.46−7.50 (dd, 1H), 7.32−7.20 (m, 5H), 6.18−6.17 (d, 1H),
5.49−5.48 (d, 1H), 4.10−3.99 (m, 2H), 3.9 (s, 2H), 3.70−3.80 (m,
1H), 3.45−3.50 (m, 1H), 3.23−3.20 (m, 2H), 2.98−2.94 (m, 1H),
2.5 (s, 3H), 1.60−1.65 (m, 2H), 1.48−1.08 (m, 18H). 13C NMR
(101 MHz, CDCl3): δ 177.63, 165.65, 154.93, 153.87, 152.73,
151.41, 146.44, 133.04, 131.10, 130.91, 129.03, 129.82, 123.70,
123.05, 122.46, 118.06, 99.72, 80.62, 80.40, 51.80, 46.82, 42.58,
33.86, 32.47, 30.32, 29.70, 29.51, 29.40, 26.93, 23.94, 23.91, 16.16,
14.26. Anal. Calcd for C35H43N3O5: C, 71.77; H, 7.40; N, 7.17;
found, C, 72.03; H, 7.39; N, 7.09.
Oxygen Radical Absorbance Capacity Assay (ORAC). The
radical scavenging capacity of the TFAHs was determined by the
ORAC-FL method using fluorescein as a fluorescent probe.41,42
A
Varioskan Flash plate reader with built-in injectors (Thermo
Scientific) was used. The reaction was carried out at 37 °C in 75
mM phosphate buffer (pH = 7.4), and the final volume reaction
mixture was 200 μL. In a black 96-well microplate (Nunc),
antioxidant (20 μL) and fluorescein (FL, 120 μL, final concentration
of 70 nM) were incubated for 15 min at 37 °C. Then, 2,2′-
azobis(amidinopropane) dihydrochloride (AAPH, 60 μL, final
concentration of 40 mM) solution was added rapidly using the
built-in injector. The fluorescence was measured every minute for 60
min at 485 nm (excitation wavelength) and 535 nm (emission
wavelength). All the reactions were made in triplicate and at least
three different assays were performed for each sample. Antioxidant
curves (fluorescence versus time) were first normalized to the curve
of the blank (without antioxidant); then, the area under the
fluorescence decay curve (AUC) was calculated as AUC = 1 +
sum(fi/f 0), where f 0 is the initial fluorescence reading at 0 min and fi
is the fluorescence value at time i.
Biology. Measurement of Cytosolic Ca2+ in SH-SY5Y Cells.
SH-SY5Y cells were maintained similarly to what was previously
described.63 Free cytosolic Ca2+ was measured using the fluorescent
Ca2+ indicator Fluo-4/AM (Fluo-4 acetoxymethylyester). SH-SY5Y
cells were seeded onto 96-well black plates at a density of 105 cells
per well, achieving confluence after 48 h. Cells were washed with
Krebs-HEPES (KH) solution (KH, in mM: 144 NaCl, 5.9 KCl, 1.2
MgCl2, 2 CaCl2, 11 D-glucose, 10 HEPES, pH 7.4). Cells were
loaded with 10 mM Fluo-4/AM and 0.2% pluronic acid in KH and
incubated for 45 min at 37 °C in the dark. Then, cells were washed
twice with KH to remove the excess of probe. Tested compounds
were incubated 10 min before K+ 70 mM was applied to evoke the
increment of cytosolic Ca2+. To normalize Fluo-4 signals, Triton X-
100 (5%) and 1 M MnCl2 were applied to register both maximal and
minimal fluorescence, respectively. The experiments were analyzed at
excitation and emission wavelengths of 485 and 520 nm, respectively,
and fluorescence was measured in a fluorescence microplate reader
(FLUOstar Optima, BMG, Germany). Data were calculated as a
The net AUC corresponding to a sample was calculated as follows:
NetAUC = AUCantioxidant − AUCblank
Regression equations were calculated by plotting the net AUC
against the antioxidant concentration. The ORAC value was obtained
by dividing the slope of the latter curve between the slope of the
Trolox curve obtained in the same assay. Final ORAC values were
expressed as micromoles (μmol) of Trolox equivalent/μmol.
percentage of Fmax − Fmin
.
1337
https://doi.org/10.1021/acschemneuro.0c00803
ACS Chem. Neurosci. 2021, 12, 1328−1342

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Inhibition of Aβ1−42 Self-Aggregation. 1,1,1,3,3,3-Hexafluoro-
2-propanol (HFIP) pretreated Aβ1−42 samples (Bachem AG,
Switzerland) were resolubilized with a MeCN/0.3 mM Na2CO3/
250 mM NaOH (48.4/48.4/3.2) mixture to have a stable stock
solution ([Aβ1−42] = 500 μM).43 Experiments were performed by
incubating the peptide in 10 mM phosphate buffer (pH 8.0)
containing 10 mM NaCl, at 30 °C for 24 h (final Aβ concentration =
50 μM with and without inhibitor (50 μM). The inhibitor was
dissolved in methanol and diluted in the assay buffer. Blanks
containing inhibitor and ThT were also prepared and evaluated to
account for quenching and fluorescence properties. To quantify
amyloid fibril formation, the ThT fluorescence method was used.64,65
After incubation, samples were diluted to a final volume of 2.0 mL
with 50 μM glycine−NaOH buffer (pH 8.5) containing 1.5 μM ThT.
A 300 s scan of fluorescence intensity was carried out (λexc = 446
nm; λem = 490 nm), and values at plateau were averaged after
subtracting the background fluorescence of 1.5 μM ThT solution.
The fluorescence intensities were compared, and the % inhibition
was calculated.
PAMPA Assay. To predict passive blood-brain penetration of
novel compounds, a modification of the parallel artificial membrane
permeation assay (PAMPA) has been used based on reported
protocol.44,66 The filter membrane of the donor plate was coated
with PBL (Polar Brain Lipid, Avanti, USA) in dodecane (4 μL of 20
mg/mL PBL in dodecane), and the acceptor well was filled with 300
μL of PBS pH 7.4 buffer (VA). Tested compounds were dissolved
first in DMSO (0.5% (V/V) final), and that was diluted with PBS pH
7.4 to reach the final concentration 50 μM in the donor well. Donor
solution (300 μL) was added to the donor wells (VD), and the donor
filter plate was carefully put on the acceptor plate so that coated
membrane was “in touch” with both donor solution and acceptor
buffer. Test compound diffused from the donor well through the
lipid membrane (area = 0.28 cm2) to the acceptor well. The
concentration of the drug in both donor and the acceptor wells was
assessed after 3, 4, 5, and 6 h of incubation in quadruplicate using the
UV plate reader Synergy HT (Biotek, USA) at the maximum
absorption wavelength and the standard curve for each compound.
The data are presented as the permeability (Pe) according eq 1:67,68
combines a Markov chain Monte Carlo like method for the global
search and a Broyden-Fletcher-Goldfarb-Shano gradient approach for
the local search.46 It is a type of memetic algorithm based on
interleaving stochastic and deterministic calculations.69 Each docking
task was repeated 30 times with the exhaustiveness parameter set to
16, employing 16 CPU in parallel multithreading. From the obtained
results, the solutions reaching the minimum predicted Gibbs binding
energy were taken as the top-scoring modes. The graphic
representations of the docked poses were rendered in PyMOL
2.4.1 (The PyMOL Molecular Graphics System, Version 2.4.1
̈
Schrodinger, LLC.). The 2D diagrams were generated using
Discovery Studio Visualizer v. 20.1.0.19295 (BIOVIA, Dassault
̀
Systemes, Release 2019, San Diego, USA).
MAO-A and MAO-B Inhibition. The inhibition assay for MAO-
A and MAO-B was conducted in a white opaque 96-well microplate.
The reaction mixture contains 42 nM MAO-A or 200 nM MAO-B
and inhibitors (+)-BIGI-3h and (−)-BIGI-3h in final concentrations
ranging from 0.1 to 200 μM in 50 mM potassium phosphate buffer
with 20% (v/v) glycerol (pH 7.5). The mixture was preincubated at
37 °C for 15 min, and subsequently, substrate kynuramine was added
to the final concentration of 50 μM. The whole reaction mixture was
incubated at 37 °C for 30 min. The deamination product of
kynuramine formed during the enzymatic reaction (4-hydroxyquino-
line) was determined by fluorescence detection (ex 310/em 400 nm)
using microplate reader (Spark multimode microplate reader, Tecan
Group Ltd.). IC50 values were calculated by adjusting the
experimental data (enzyme activity versus concentration of inhibitor)
to nonlinear regression curves using GraphPad Prism 7.
The determination of Ki values was performed for MAO-B enzyme
with three different concentrations of each inhibitor: 5, 15, and 50
μM. The final concentrations of these inhibitors in the reaction
mixture were proposed to cover the entire inhibitory range.
GSK-3β Inhibition In Vitro Assay. Human recombinant GSK-
3β, prephosphorylated polypeptide substrate and white 96-well plates
were purchased from Millipore (Millipore Iberica S.A.U.). Kinase-
Glo Luminescent Kinase Assay was obtained from Promega
(Promega Biotech Iberica, SL). Adenosinetriphosphate (ATP) and
all other reagents were from Sigma-Aldrich. Assay buffer contained
50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
(pH 7.5), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM
ethylene glycol tetraacetic acid (EGTA), and 15 mM magnesium
acetate. The in solution experiments for GSK-3β inhibition analysis
were performed by following the method developed by Baki et al.53
In a typical assay, 10 μL of test compound (dissolved in DMSO at 1
mM concentration and diluted in advance in assay buffer to the
desired concentration) and 10 μL (20 ng) of enzyme were added to
each well in the presence of 20 μL of assay buffer containing GSM
substrate and ATP in order to reach 25 and 1 μM final
concentrations, respectively. The final DMSO percentage in the
reaction mixture did not exceed 5%. After a 30 min incubation time
at 30 °C, the enzymatic reaction was stopped by adding 40 μL of
Kinase-Glo reagent. Glow-type luminescence was recorded after 10
min using a VictorX 3 PerkinElmer plate reader. The activity was
found proportional to the difference of the total and consumed ATP.
The inhibitory activities were calculated on the basis of maximal
kinase (average positive), and luciferase activities (average negative)
were measured in the absence of inhibitor and in the presence of
i
y
[drug]acceptor
[drug]equilibrium
j
z
j
z
z
j
Pe = C × −ln 1 −
where C
j
j
j
z
z
z
k
{
i
j
y
VD × VA
(VD + VA) × area × time
z
j
j
z
z
=
j
j
z
z
(1a)
k
{
In Silico Experimental Section. From the online PDB database
(www.pdb.org), a model of hAChE (PDB ID: 4EY7, resolution: 2.35
Å) was downloaded and prepared for flexible molecular docking by
MGL Tools utilities.45 The preparation of this receptor involved
removal of the surplus copies of the enzyme chains, nonbonded
inhibitors, addition of polar hydrogens, and merging of nonpolar
ones. Default Gasteiger charges were assigned to all atoms. Flexible
parts of the enzymes were determined by a spherical selection of
residues (R = 11 Å) approximately around the center of the active
site. In the same points, the centers of the grid box of 33 × 33 × 33
Å3 were positioned. The rotatable bonds in the flexible residues were
detected automatically by AutoDock Tools 1.5.4 program. Given the
limitation of the program used for flexible molecular docking, water
molecules had to be removed from the system. The following xyz-
coordinate of the grid box center was applied: hAChE (10.698,
−58.115, −23.192). The studied ligands were first drawn in
HyperChem 8.0 and then manually protonated as suggested by
MarvinSketch 6.2.0. software (http://www.chemaxon.com), geo-
metrically optimized by semiempirical quantum-chemistry PM3
method and stored as pdb files. The structures of the ligands were
processed for docking in a similar way as above-mentioned flexible
parts of the receptor by AutoDock Tools 1.5.4 program. Molecular
docking was carried out in AutoDock Vina 1.1.2 program utilizing
computer resources of the Czech National Grid Infrastructure
MetaCentrum. The search algorithm of AutoDock Vina efficiently
reference compound inhibitor (SB-415286 Merck Millipore, IC50
=
55 nM) at total inhibition concentration (5 μM),70 respectively. The
linear regression parameters were determined, and the IC50 was
extrapolated (GraphPad Prism 4.0, GraphPad Software Inc.).
τ(306−336) Peptide Aggregation and Its Inhibition by ThT
Fluorimetric Assay. The τ(306−336) (1 mg) (Bachem AG, Germany)
was initially dissolved in 1,1,1,3,3,3,-hexafluoro-2-propanol (HFIP),
gently vortexed, sonicated, and kept overnight at room temperature.
Subsequently, the sample was aliquoted dried and stored at −20 °C.
Stock solutions of τ(306−336) peptide (500 μM) were prepared in
ultrapure water and immediately used. Stock solution of thioflavin T
(ThT, 500 μM) was prepared in 56.3 mM phosphate buffer (PB, pH
= 7.4), while stock solution of inhibitors and doxycycline (20 mM)
1338
https://doi.org/10.1021/acschemneuro.0c00803
ACS Chem. Neurosci. 2021, 12, 1328−1342

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
were prepared in DMSO/methanol 10/90. The τ(306−336) aggregation
was monitored at 30 °C in a black, clear bottom 96-well plate
(Greiner) by EnSpire multiplate reader (PerkinElmer) using the ThT
fluorimetric assay6 with some variations. The excitation and emission
wavelengths were set at 446 and 490 nm, respectively. Assay samples
were prepared by diluting τ(306−336) stock solution to 50 μM in the
assay mixture which consisted in 20 μM ThT, 48.1 mM PB (final
concentrations) in final 100 μL volume (final DMSO and MeOH
content: 0.05% and 0.45%, respectively). Inhibition experiments were
performed by incubating τ(306−336) peptide at the given conditions in
the presence of tested inhibitors at 50 μM. Fluorescence data were
recorded every 10 min overnight with 1 min shaking at 800 rpm
prior to each reading. Each inhibitor was assayed in triplicate in at
least two independent experiments. Estimation of the inhibitory
potency (%) was carried out by comparing fluorescence values at the
plateau (average fluorescence intensity value in the 12−16 h range).
nose to the object at a distance of less than 2 cm and/or touching it
with the vibrissae or nose and/or climbing on it. The recognition
index was calculated as follows: time spent exploring the novel
object/(time spent exploring the novel object + time exploring the
familiar object). Statistical analyses were performed using the Χ2 test.
We used the Bonferroni correction for multiple comparisons. We
considered differences to be significant at p < 0.05. Results are
presented as mean and standard error.
All animal experiments comply with the ARRIVE (Animal
Research: Reporting of In Vivo Experiments) guidelines and are
carried out in accordance with the EU Directive 2010/63/EU for the
care and the use of laboratory animals. The protocol were approved
́
by the Franche-Comte University’s Animal Care Committee (Protol
number: 2015-004-EH-12PR)
ASSOCIATED CONTENT
■
The % inhibition is expressed as the mean
SEM. Quenching of
sı
ThT fluorescence was evaluated by preparing blank solutions
containing inhibitor/reference compound and preformed fibrils of
τ(306−336) peptide.
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00803.
In Vivo Test. For behavioral studies, we used 50 swiss female 10-
week-old mice weighing 25−30 g. Memory impairment was induced
by the potent antimuscarinic drug scopolamine hydrobromide (1
mg/kg, Sigma) dissolved in saline buffer. To explore the potential of
( )-BIGI-3h as a cognitive enhancer, we administered the hybrid
drug at doses of 1 and 10 mg/kg. We tested the antiamnesic effect of
donepezil (Tocris Bioscience, R&D Systems Inc.) at a dose of 1 mg/
kg. Both products, ( )-BIGI-3h and donepezil, were dissolved in
saline buffer containing 3.6% DMSO. All drug solutions were
independently prepared immediately before their use and were
administered intraperitoneally in 10 mL/kg of vehicle volume.
Different batches of mice were used for each experiment. The
animals were distributed into 5 experimental groups (I−V): Control
group I mice were administered 3.6% DMSO in saline buffer, labeled
as “control vehicle”; experimental groups II−V consisted of (II) mice
administered scopolamine dissolved in 3.6% DMSO in saline buffer,
labeled as “scopo/DMSO”, (III) mice coadministered scopolamine
plus Donepezil, labeled as “Scopo/donep”, and ( IV and V) mice
coadministrated scopolamine plus ( )-BIGI-3h at 1 and 10 mg/kg
labeled, respectively, “Scopo/BIGI-3h 1” and “Scopo/BIGI-3h 10”.
Object Recognition Task. Spontaneous object recognition was
tested as described previously56 with slight modifications. All
behavioral experiments took place during the light phase of the 12
h light/dark cycle between 10 am and 3 pm to minimize the
influence of circadian rhythm. The ORT was performed in a round
plastic open field (diameter: 48 cm) with black walls (40 cm high).
The apparatus was placed in a poorly illuminated (60 lx, where lx
stands for lux) and noise-isolated room, and the mice were randomly
placed in the experimental room 1 h before beginning the
experimental task each day. The behavior of the animals (time
spent exploring the objects and overall locomotor activity) was
registered during precise time periods. On the first day, animals were
allowed to explore the apparatus individually for 5 min in the absence
of objects (habituation session). On the second day, 30 min before
the trial session, mice were administered treatments as indicated in
the previous section and then subjected to the acquisition trial (T1).
Mice were individually allowed, for 5 min, to freely explore 2
identical plastic objects placed in the arena, located in symmetric
positions at a distance of about 7 cm away from walls. Then, the
rodents were removed from the open field for an interval of 1 h.
After this delay, we performed the retention trial (T2). Animals were
returned for 5 min to the bin where 1 of the original objects had
been replaced by a novel object (neutral to naive mice) with a
different shape, color, and textures than the remaining object
previously presented in the acquisition trial (familiar object). To
score the exploration behavior of the mice, a camera was mounted
above the center of the arena. The total time spent in active
exploration of each object during T2 was determined through
analyzation of the video recording. As a criterion of active object
exploration, we measured the time spent by the animal directing its
1H NMR and 13C NMR spectra for key compounds
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
Lhassane Ismaili − Neurosciences intégratives et cliniques EA
481, Univ. Bourgogne Franche-Comté, F-25000 Besanco̧ n,
France; orcid.org/0000-0002-6013-3150;
Phone: +33381665543; Email: lhassane.ismaili@univ-
fcomte.fr
Authors
Julie Monnin − Neurosciences intégratives et cliniques EA
481, Univ. Bourgogne Franche-Comté, F-25000 Besancon,
France
Adeline Etievant − Neurosciences intégratives et cliniques EA
481, Univ. Bourgogne Franche-Comté, F-25000 Besancon,
̧
̧
France
Raquel L. Arribas − Servicio de Farmacología Clínica,
Instituto de Investigación Sanitaria, Hospital Universitario
de la Princesa, 28006 Madrid, Spain; Instituto Teofilo
Hernando, Universidad Autónoma de Madrid, 28029
Madrid, Spain
Lucía Viejo − Servicio de Farmacología Clínica, Instituto de
Investigación Sanitaria, Hospital Universitario de la
Princesa, 28006 Madrid, Spain; Instituto Teofilo Hernando,
Universidad Autónoma de Madrid, 28029 Madrid, Spain
Bernard Refouvelet − Neurosciences intégratives et cliniques
EA 481, Univ. Bourgogne Franche-Comté, F-25000
Besanco̧ n, France
Ondrej Soukup − Biomedical Research Center, University
Hospital Hradec Kralove, Czech Republic, University of
Defence, 50003 Hradec Kralove, Czech Republic;
orcid.org/0000-0001-6376-8701
Jana Janockova − Biomedical Research Center, University
Hospital Hradec Kralove, Czech Republic, University of
Defence, 50003 Hradec Kralove, Czech Republic
Vendula Hepnarova − Department of Toxicology and
Military Pharmacy, Faculty of Military Health Sciences,
66210 Brno, Czech Republic
Jan Korabecny − Biomedical Research Center, University
Hospital Hradec Kralove, Czech Republic, University of
Defence, 50003 Hradec Kralove, Czech Republic
1339
https://doi.org/10.1021/acschemneuro.0c00803
ACS Chem. Neurosci. 2021, 12, 1328−1342

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Tomas Kucera − Department of Toxicology and Military
Pharmacy, Faculty of Military Health Sciences, 66210 Brno,
Czech Republic
Daniel Jun − Department of Toxicology and Military
Pharmacy, Faculty of Military Health Sciences, 66210 Brno,
Czech Republic
Rudolf Andrys − Faculty of Science, Department of
Chemistry, University Hradec Kralove, 50003 Hradec
Kralove, Czech Republic
Kamil Musilek − Faculty of Science, Department of
Chemistry, University Hradec Kralove, 50003 Hradec
Kralove, Czech Republic; orcid.org/0000-0002-7504-
4062
related patent application. The other authors declare no
conflicts of interest.
ACKNOWLEDGMENTS
■
L.I. thanks the Regional Council of Franche-Comté (2016YC-
04540 and 04560) for financial support, Mrs. M.-J. Henriot
(PHV Pharma) for her support in the HPLC analyses, and
Vincent Luzet for preliminary results in synthesis. O.S., J.J.,
and J.K. acknowledge the support from the grant by Czech
Science Foundation no. 20-29633J. T.K., D.J., and V.H.
acknowledge support from the Long-term Development Plan
(Faculty of Military Health Sciences). R.A. and K.M. thank
the Ministry of Education, Youth and Sports of the Czech
Republic (ERDF no. CZ.02.1.01/0.0/0.0/16_025/0007444).
Aurelie Baguet − Université Bourgogne Franche Comté,
̂
INSERM, UMR1098, Interactions Hote-Greffon-Tumeur/
Ingénierie Cellulaire et Génique, F-25000 Besanco̧ n, France
ABBREVIATIONS
■
Eva M. García-Frutos − Instituto de Ciencia de Materiales de
Madrid, CSIC, Cantoblanco, 28049 Madrid, Spain;
orcid.org/0000-0001-6270-1126
MTDLs multitarget-directed ligands
MAO
monoamine oxidase
GSK-3β glycogen synthase kinase
VGCC L-type voltage-gated calcium channels
Angela De Simone − Department for Life Quality Studies,
Alma Mater Studiorum University of Bologna, 47921
Rimini, Italy
Vincenza Andrisano − Department for Life Quality Studies,
Alma Mater Studiorum University of Bologna, 47921
Rimini, Italy; orcid.org/0000-0003-4396-1904
Manuela Bartolini − Department of Pharmacy and
Biotechnology, Alma Mater Studiorum University of
Bologna, 40126 Bologna, Italy; orcid.org/0000-0002-
2890-3856
Cristóbal de los Ríos − Servicio de Farmacología Clínica,
Instituto de Investigación Sanitaria, Hospital Universitario
de la Princesa, 28006 Madrid, Spain; Instituto Teofilo
Hernando, Universidad Autónoma de Madrid, 28029
Madrid, Spain; orcid.org/0000-0002-6456-7589
José Marco-Contelles − Laboratory of Medicinal Chemistry,
Instituto de Química Orgánica General, Consejo Superior de
Investigaciones Científicas, 28006 Madrid, Spain;
orcid.org/0000-0003-0690-0328
ChE
cholinesterase, hChE human cholinesterase
hAChE human acetylcholinesterase
hBuChE human butyrylcholinesterase
REFERENCES
■
(1) Cacabelos, R. (2018) Have There Been Improvements in
Alzheimer’s Disease Drug Discovery over the Past 5 Years? Expert
Opin. Drug Discovery 13 (6), 523−538.
(2) Querfurth, H. W., and LaFerla, F. M. (2010) N. Engl. J. Med.
362 (4), 329−344.
(3) Bartus, R. T., Dean, R. L., Beer, B., and Lippa, A. S. (1982) The
Cholinergic Hypothesis of Geriatric Memory Dysfunction. Science
217 (4558), 408−414.
(4) Hamley, I. W. (2012) The Amyloid Beta Peptide: A Chemist’s
Perspective. Role in Alzheimer’s and Fibrillization. Chem. Rev. 112
(10), 5147−5192.
(5) Liu, M., Dexheimer, T., Sui, D., Hovde, S., Deng, X., Kwok, R.,
Bochar, D. A., and Kuo, M.-H. (2020) Hyperphosphorylated Tau
Aggregation and Cytotoxicity Modulators Screen Identified Pre-
scription Drugs Linked to Alzheimer’s Disease and Cognitive
Functions. Sci. Rep. 10 (1), 16551.
(6) Gandini, A., Bartolini, M., Tedesco, D., Martinez-Gonzalez, L.,
Roca, C., Campillo, N. E., Zaldivar-Diez, J., Perez, C., Zuccheri, G.,
Miti, A., et al. (2018) Tau-Centric Multitarget Approach for
Alzheimer’s Disease: Development of First-in-Class Dual Glycogen
Synthase Kinase 3β and Tau-Aggregation Inhibitors. J. Med. Chem. 61
(17), 7640−7656.
Emmanuel Haffen − Neurosciences intégratives et cliniques
EA 481, Univ. Bourgogne Franche-Comté, F-25000
Besanco̧ n, France
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.0c00803
Author Contributions
L.I. conceived, designed, synthesized compounds, determined
ORAC values and supervised the whole project. J.M. and A.E.
designed and carried out the in vivo NOR studies. E.H. (lead),
R.L.A., and L.V. carried out the calcium channel study.
C.d.l.R. (lead), J.J., V.H., and T.K. carried out the PAMPA.
O.S. and D.J. (leads) performed cholinesterase inhibition. J.J.
performed the molecular docking studies. R.A. did the MAO
inhibition with K.M. (lead). A.B. did the alkaline phosphatase
experiment. L.I., B.R., and E.M.G.-F. wrote the original draft.
A.d.S. and V.A. designed and performed GSK3β inhibition;
M.B. and J.J. designed and performed Aβ and τ(306-336) self-
aggregation experiments. J.M.-C. supervised biological evalua-
tion and chiral separation. The manuscript was revised and
approved by all authors.
(7) Gómez-Isla, T., Hollister, R., West, H., Mui, S., Growdon, J. H.,
Petersen, R. C., Parisi, J. E., and Hyman, B. T. (1997) Neuronal Loss
Correlates with but Exceeds Neurofibrillary Tangles in Alzheimer’s
Disease. Ann. Neurol. 41 (1), 17−24.
(8) Masters, C. L., Bateman, R., Blennow, K., Rowe, C. C., Sperling,
R. A., and Cummings, J. L. (2015) Alzheimer’s Disease. Nat. Rev. Dis.
Primers 1 (1), 1−18.
(9) Chen, C.-H., Zhou, W., Liu, S., Deng, Y., Cai, F., Tone, M.,
Tone, Y., Tong, Y., and Song, W. (2012) Increased NF-ΚB Signalling
up-Regulates BACE1 Expression and Its Therapeutic Potential in
Alzheimer’s Disease. Int. J. Neuropsychopharmacol. 15 (1), 77−90.
̀
(10) Oules, B., Del Prete, D., Greco, B., Zhang, X., Lauritzen, I.,
Sevalle, J., Moreno, S., Paterlini-Bréchot, P., Trebak, M., Checler,
et al. (2012) Ryanodine Receptor Blockade Reduces Amyloid-β Load
and Memory Impairments in Tg2576 Mouse Model of Alzheimer
Disease. J. Neurosci. 32 (34), 11820−11834.
Notes
(11) Avila, J., Pérez, M., Lim, F., Gómez-Ramos, A., Hernández, F.,
and Lucas, J. J. (2004) Tau in Neurodegenerative Diseases: Tau
Phosphorylation and Assembly. Neurotoxic. Res. 6 (6), 477−482.
The authors declare the following competing financial
interest(s): L.I. declares a conflict of interest based on a
1340
https://doi.org/10.1021/acschemneuro.0c00803
ACS Chem. Neurosci. 2021, 12, 1328−1342

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
(12) Chakroborty, S., and Stutzmann, G. E. (2014) Calcium
Channelopathies and Alzheimer’s Disease: Insight into Therapeutic
Success and Failures. Eur. J. Pharmacol. 739, 83−95.
(13) L’Episcopo, F., Drouin-Ouellet, J., Tirolo, C., Pulvirenti, A.,
Giugno, R., Testa, N., Caniglia, S., Serapide, M. F., Cisbani, G.,
Barker, R. A., et al. (2016) GSK-3β-Induced Tau Pathology Drives
Hippocampal Neuronal Cell Death in Huntington’s Disease:
Involvement of Astrocyte-Neuron Interactions. Cell Death Dis. 7,
No. e2206.
Tacrines as Cholinesterases Inhibitors, Direct Antioxidants, and
Nuclear Factor (Erythroid-Derived 2)-Like 2 Activators. J. Med.
Chem. 59 (21), 9967−9973.
́
(30) Malek, R., Maj, M., Wnorowski, A., Józwiak, K., Martin, H.,
Iriepa, I., Moraleda, I., Chabchoub, F., Marco-Contelles, J., and
Ismaili, L. (2019) Multi-Target 1,4-Dihydropyridines Showing
Calcium Channel Blockade and Antioxidant Capacity for Alzheimer’s
Disease Therapy. Bioorg. Chem. 91, 103205.
(31) Jalili-Baleh, L., Babaei, E., Abdpour, S., Nasir Abbas Bukhari,
S., Foroumadi, A., Ramazani, A., Sharifzadeh, M., Abdollahi, M., and
Khoobi, M. (2018) A Review on Flavonoid-Based Scaffolds as Multi-
Target-Directed Ligands (MTDLs) for Alzheimer’s Disease. Eur. J.
Med. Chem. 152, 570−589.
(32) Matos, L. H. S., Masson, F. T., Simeoni, L. A., and Homem-
de-Mello, M. (2018) Biological Activity of Dihydropyrimidinone
(DHPM) Derivatives: A Systematic Review. Eur. J. Med. Chem. 143,
1779−1789.
(33) Grover, G. J., Dzwonczyk, S., McMullen, D. M., Normandin,
D. E., Parham, C. S., Sleph, P. G., and Moreland, S. (1995)
Pharmacologic Profile of the Dihydropyrimidine Calcium Channel
Blockers SQ 32,547 and SQ 32,926 [Correction of SQ 32,946]. J.
Cardiovasc. Pharmacol. 26 (2), 289−294.
(34) Cohen, P., and Goedert, M. (2004) GSK3 Inhibitors:
Development and Therapeutic Potential. Nat. Rev. Drug Discovery 3
(6), 479−487.
(35) 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. (2015) Multitarget Drug Discovery for Alzheimer’s
Disease: Triazinones as BACE-1 and GSK-3β Inhibitors. Angew.
Chem., Int. Ed. 54 (5), 1578−1582.
(36) Choi, J. Y., Calvet, C. M., Gunatilleke, S. S., Ruiz, C.,
Cameron, M. D., McKerrow, J. H., Podust, L. M., and Roush, W. R.
(2013) Rational Development of 4-Aminopyridyl-Based Inhibitors
Targeting Trypanosoma Cruzi CYP51 as Anti-Chagas Agents. J. Med.
Chem. 56 (19), 7651−7668.
(14) Beurel, E., Grieco, S. F., and Jope, R. S. (2015) Glycogen
Synthase Kinase-3 (GSK3): Regulation, Actions, and Diseases.
Pharmacol. Ther. 0, 114−131.
(15) Maqbool, M., Mobashir, M., and Hoda, N. (2016) Pivotal
Role of Glycogen Synthase Kinase-3: A Therapeutic Target for
Alzheimer’s Disease. Eur. J. Med. Chem. 107, 63−81.
(16) Yan, M. H., Wang, X., and Zhu, X. (2013) Mitochondrial
Defects and Oxidative Stress in Alzheimer Disease and Parkinson
Disease. Free Radical Biol. Med. 62, 90−101.
(17) Greenough, M. A., Camakaris, J., and Bush, A. I. (2013) Metal
Dyshomeostasis and Oxidative Stress in Alzheimer’s Disease.
Neurochem. Int. 62 (5), 540−555.
(18) Candore, G., Bulati, M., Caruso, C., Castiglia, L., Colonna-
̀
Romano, G., Di Bona, D., Duro, G., Lio, D., Matranga, D., Pellicano,
M., et al. (2010) Inflammation, Cytokines, Immune Response,
Apolipoprotein E, Cholesterol, and Oxidative Stress in Alzheimer
Disease: Therapeutic Implications. Rejuvenation Res. 13 (2−3), 301−
313.
(19) Rosini, M., Simoni, E., Milelli, A., Minarini, A., and Melchiorre,
C. (2014) Oxidative Stress in Alzheimer’s Disease: Are We
Connecting the Dots? J. Med. Chem. 57 (7), 2821−2831.
(20) Reis, J., Cagide, F., Valencia, M. E., Teixeira, J., Bagetta, D.,
Pérez, C., Uriarte, E., Oliveira, P. J., Ortuso, F., Alcaro, S., et al.
(2018) Multi-Target-Directed Ligands for Alzheimer’s Disease:
Discovery of Chromone-Based Monoamine Oxidase/Cholinesterase
Inhibitors. Eur. J. Med. Chem. 158, 781−800.
(21) Albertini, C., Salerno, A., Pinheiro, P. de S. M., and Bolognesi,
M. L. (2020) From Combinations to Multitarget-Directed Ligands:
A Continuum in Alzheimer’s Disease Polypharmacology. Med. Res.
Rev., 1.
(22) Prati, F., Cavalli, A., and Bolognesi, M. L. (2016) Navigating
the Chemical Space of Multitarget-Directed Ligands: From Hybrids
to Fragments in Alzheimer’s Disease. Molecules 21 (4), 466.
(23) Guzior, N., Wieckowska, A., Panek, D., and Malawska, B.
(2014) Recent Development of Multifunctional Agents as Potential
Drug Candidates for the Treatment of Alzheimer’s Disease. Curr.
Med. Chem. 22 (3), 373−404.
(37) Carling, W. R., and Moore, K. W. (1997) Imidazolone and
Oxazolone Derivatives as Dopamine Antagonists, US5698573A.
(38) Ellman, G. L., Courtney, K. D., Andres, V., jr., and
Featherstone, R. M. (1961) A New and Rapid Colorimetric
Determination of Acetylcholinesterase Activity. Biochem. Pharmacol.
7 (2), 88−95.
́
(39) Pachón-Angona, I., Refouvelet, B., Andrys, R., Martin, H.,
Luzet, V., Iriepa, I., Moraleda, I., Diez-Iriepa, D., Oset-Gasque, M.-J.,
Marco-Contelles, et al. (2019) Donepezil + Chromone + Melatonin
Hybrids as Promising Agents for Alzheimer’s Disease Therapy. J.
Enzyme Inhib. Med. Chem. 34 (1), 479−489.
(24) Malek, R., Arribas, R. L., Palomino-Antolin, A., Totoson, P.,
Demougeot, C., Kobrlova, T., Soukup, O., Iriepa, I., Moraleda, I.,
Diez-Iriepa, D., et al. (2019) 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. 62 (24), 11416−11422.
(25) Dömling, A., Wang, W., and Wang, K. (2012) Chemistry and
Biology Of Multicomponent Reactions. Chem. Rev. 112 (6), 3083−
3135.
(40) Benchekroun, M., Bartolini, M., Egea, J., Romero, A., Soriano,
E., Pudlo, M., Luzet, V., Andrisano, V., Jimeno, M. L., Lopez, M. G.,
et al. (2015) Novel Tacrine-Grafted Ugi Adducts as Multipotent
Anti-Alzheimer Drugs: A Synthetic Renewal in Tacrine-Ferulic Acid
Hybrids. ChemMedChem 10 (3), 523−539.
(41) Ou, B., Hampsch-Woodill, M., and Prior, R. L. (2001)
Development and Validation of an Improved Oxygen Radical
Absorbance Capacity Assay Using Fluorescein as the Fluorescent
Probe. J. Agric. Food Chem. 49 (10), 4619−4626.
(42) Dávalos, A., Gómez-Cordovés, C., and Bartolomé, B. (2004)
Extending Applicability of the Oxygen Radical Absorbance Capacity
(ORAC-Fluorescein) Assay. J. Agric. Food Chem. 52 (1), 48−54.
(43) Bartolini, M., Bertucci, C., Bolognesi, M. L., Cavalli, A.,
Melchiorre, C., and Andrisano, V. (2007) Insight into the Kinetic of
Amyloid Beta (1−42) Peptide Self-Aggregation: Elucidation of
Inhibitors’ Mechanism of Action. ChemBioChem 8 (17), 2152−2161.
(44) Di, L., Kerns, E. H., Fan, K., McConnell, O. J., and Carter, G.
T. (2003) High Throughput Artificial Membrane Permeability Assay
for Blood−Brain Barrier. Eur. J. Med. Chem. 38 (3), 223−232.
(45) Cheung, J., Rudolph, M. J., Burshteyn, F., Cassidy, M. S., Gary,
E. N., Love, J., Franklin, M. C., and Height, J. J. (2012) Structures of
Human Acetylcholinesterase in Complex with Pharmacologically
Important Ligands. J. Med. Chem. 55 (22), 10282−10286.
(26) Akritopoulou-Zanze, I. (2008) Isocyanide-Based Multicompo-
nent Reactions in Drug Discovery. Curr. Opin. Chem. Biol. 12 (3),
324−331.
(27) Ismaili, L., and do Carmo Carreiras, M. (2018) Multi-
̀
component Reactions for Multitargeted Compounds for Alzheimers
Disease. Curr. Top. Med. Chem. 17 (31), 3319−3327.
(28) Benchekroun, M., Ismaili, L., Pudlo, M., Luzet, V., Gharbi, T.,
Refouvelet, B., and Marco-Contelles, J. (2015) Donepezil−Ferulic
Acid Hybrids as Anti-Alzheimer Drugs. Future Med. Chem. 7 (1),
15−21.
(29) Benchekroun, M., Romero, A., Egea, J., León, R., Michalska, P.,
Buendía, I., Jimeno, M. L., Jun, D., Janockova, J., Sepsova, V., et al.
(2016) The Antioxidant Additive Approach for Alzheimer’s Disease
Therapy: New Ferulic (Lipoic) Acid Plus Melatonin Modified
1341
https://doi.org/10.1021/acschemneuro.0c00803
ACS Chem. Neurosci. 2021, 12, 1328−1342

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
(46) Trott, O., and Olson, A. J. (2009) AutoDock Vina: Improving
the Speed and Accuracy of Docking with a New Scoring Function,
Efficient Optimization, and Multithreading. J. Comput. Chem. 31 (2),
455−461.
(47) Spilovska, K., Korabecny, J., Sepsova, V., Jun, D., Hrabinova,
M., Jost, P., Muckova, L., Soukup, O., Janockova, J., Kucera, T., et al.
(2017) Novel Tacrine-Scutellarin Hybrids as Multipotent Anti-
Alzheimer’s Agents: Design, Synthesis and Biological Evaluation.
Molecules 22 (6), 1006.
(48) Korabecny, J., Andrs, M., Nepovimova, E., Dolezal, R.,
Babkova, K., Horova, A., Malinak, D., Mezeiova, E., Gorecki, L.,
Sepsova, V., et al. (2015) 7-Methoxytacrine-p-Anisidine Hybrids as
Novel Dual Binding Site Acetylcholinesterase Inhibitors for
Alzheimer’s Disease Treatment. Molecules 20 (12), 22084−22101.
(49) Nepovimova, E., Korabecny, J., Dolezal, R., Babkova, K.,
Ondrejicek, A., Jun, D., Sepsova, V., Horova, A., Hrabinova, M.,
Soukup, O., et al. (2015) 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. 58 (22), 8985−9003.
(50) Zemek, F., Drtinova, L., Nepovimova, E., Sepsova, V.,
Korabecny, J., Klimes, J., and Kuca, K. (2014) Outcomes of
Alzheimer’s Disease Therapy with Acetylcholinesterase Inhibitors and
Memantine. Expert Opin Drug Saf 13 (6), 759−774.
(51) Inestrosa, N. C., Alvarez, A., Pérez, C. A., Moreno, R. D.,
Vicente, M., Linker, C., Casanueva, O. I., Soto, C., and Garrido, J.
(1996) Acetylcholinesterase Accelerates Assembly of Amyloid-β-
Peptides into Alzheimer’s Fibrils: Possible Role of the Peripheral Site
of the Enzyme. Neuron 16 (4), 881−891.
(62) Werner, S., Turner, D. M., Lyon, M. A., Huryn, D. M., and
Wipf, P. (2006) A Focused Library of Tetrahydropyrimidinone
Amides via a Tandem Biginelli-Ugi Multi-Component Process.
Synlett 2006 (14), 2334−2338.
(63) Gonzalez, D., Arribas, R. L., Viejo, L., Lajarin-Cuesta, R., and
de los Rios, C. (2018) Substituent Effect of N-Benzylated Gramine
Derivatives That Prevent the PP2A Inhibition and Dissipate the
Neuronal Ca2+ Overload, as a Multitarget Strategy for the
Treatment of Alzheimer’s Disease. Bioorg. Med. Chem. 26 (9),
2551−2560.
(64) Naiki, H., Higuchi, K., Hosokawa, M., and Takeda, T. (1989)
Fluorometric Determination of Amyloid Fibrils in Vitro Using the
Fluorescent Dye, Thioflavin T1. Anal. Biochem. 177 (2), 244−249.
(65) LeVine, H., 3rd. (1993) Thioflavine T Interaction with
Synthetic Alzheimer’s Disease Beta-Amyloid Peptides: Detection of
Amyloid Aggregation in Solution. Protein Sci. 2 (3), 404−410.
(66) Lemes, L. F. N., de Andrade Ramos, G., de Oliveira, A. S., da
Silva, F. M. R., de Castro Couto, G., da Silva Boni, M., Guimaraes,
̃
M. J. R., Souza, I. N. O., Bartolini, M., Andrisano, V., et al. (2016)
Cardanol-Derived AChE Inhibitors: Towards the Development of
Dual Binding Derivatives for Alzheimer’s Disease. Eur. J. Med. Chem.
108, 687−700.
(67) Sugano, K., Hamada, H., Machida, M., and Ushio, H. (2001)
High Throughput Prediction of Oral Absorption: Improvement of
the Composition of the Lipid Solution Used in Parallel Artificial
Membrane Permeation Assay. J. Biomol. Screening 6 (3), 189−196.
(68) Wohnsland, F., and Faller, B. (2001) High-Throughput
Permeability PH Profile and High-Throughput Alkane/Water Log P
with Artificial Membranes. J. Med. Chem. 44 (6), 923−930.
(69) Liu, B., Wang, L., and Jin, Y.-H. (2007) An Effective PSO-
Based Memetic Algorithm for Flow Shop Scheduling. IEEE Trans
Syst. Man Cybern B Cybern 37 (1), 18−27.
(70) Coghlan, M. P., Culbert, A. A., Cross, D. A., Corcoran, S. L.,
Yates, J. W., Pearce, N. J., Rausch, O. L., Murphy, G. J., Carter, P. S.,
Roxbee Cox, L., et al. (2000) Selective Small Molecule Inhibitors of
Glycogen Synthase Kinase-3 Modulate Glycogen Metabolism and
Gene Transcription. Chem. Biol. 7 (10), 793−803.
(52) Yan, Z., Caldwell, G. W., Zhao, B., and Reitz, A. B. (2004) A
High-Throughput Monoamine Oxidase Inhibition Assay Using
Liquid Chromatography with Tandem Mass Spectrometry. Rapid
Commun. Mass Spectrom. 18 (8), 834−840.
(53) Baki, A., Bielik, A., Molnár, L., Szendrei, G., and Keseru, G. M.
̈
(2007) A High Throughput Luminescent Assay for Glycogen
Synthase Kinase-3beta Inhibitors. Assay Drug Dev. Technol. 5 (1),
75−83.
(54) von Bergen, M., Friedhoff, P., Biernat, J., Heberle, J.,
Mandelkow, E.-M., and Mandelkow, E. (2000) Assembly of τ
Protein into Alzheimer Paired Helical Filaments Depends on a Local
Sequence Motif (306VQIVYK311) Forming β Structure. Proc. Natl.
Acad. Sci. U. S. A. 97 (10), 5129−5134.
(55) Stöhr, J., Wu, H., Nick, M., Wu, Y., Bhate, M., Condello, C.,
Johnson, N., Rodgers, J., Lemmin, T., Achyraya, S., et al. (2017) 31-
Residue Peptide Induces Aggregation of Tau’s Microtubule-Binding
Region in Cells. Nat. Chem. 9 (9), 874−881.
(56) Antunes, M., and Biala, G. (2012) The Novel Object
Recognition Memory: Neurobiology, Test Procedure, and Its
Modifications. Cogn Process 13 (2), 93−110.
(57) Baell, J. B., and Holloway, G. A. (2010) New Substructure
Filters for Removal of Pan Assay Interference Compounds (PAINS)
from Screening Libraries and for Their Exclusion in Bioassays. J. Med.
Chem. 53 (7), 2719−2740.
(58) Capuzzi, S. J., Muratov, E. N., and Tropsha, A. (2017)
Phantom PAINS: Problems with the Utility of Alerts for Pan-Assay
INterference CompoundS. J. Chem. Inf. Model. 57 (3), 417−427.
(59) Jasial, S., Hu, Y., and Bajorath, J. (2017) How Frequently Are
Pan-Assay Interference Compounds Active? Large-Scale Analysis of
Screening Data Reveals Diverse Activity Profiles, Low Global Hit
Frequency, and Many Consistently Inactive Compounds. J. Med.
Chem. 60 (9), 3879−3886.
(60) Baell, J. B., and Nissink, J. W. M. (2018) Seven Year Itch: Pan-
Assay Interference Compounds (PAINS) in 2017Utility and
Limitations. ACS Chem. Biol. 13 (1), 36−44.
(61) Ferreira, R. S., Simeonov, A., Jadhav, A., Eidam, O., Mott, B.
T., Keiser, M. J., McKerrow, J. H., Maloney, D. J., Irwin, J. J., and
Shoichet, B. K. (2010) Complementarity between a Docking and a
High-Throughput Screen in Discovering New Cruzain Inhibitors. J.
Med. Chem. 53 (13), 4891−4905.
1342
https://doi.org/10.1021/acschemneuro.0c00803
ACS Chem. Neurosci. 2021, 12, 1328−1342