Acetylcholinesterase inhibition of diversely functionalized quinolinones for alzheimer’s disease therapy

Article abstract of DOI:10.3390/ijms21113913

In this communication, we report the synthesis and cholinesterase (ChE)/monoamine oxidase (MAO) inhibition of 19 quinolinones (QN1-19) and 13 dihydroquinolinones (DQN1-13) designed as potential multitarget small molecules (MSM) for Alzheimer’s disease therapy. Contrary to our expectations, none of them showed significant human recombinant MAO inhibition, but compounds QN8, QN9, and DQN7 displayed promising human recombinant acetylcholinesterase (hrAChE) and butyrylcholinesterase (hrBuChE) inhibition. In particular, molecule QN8 was found to be a potent and quite selective non-competitive inhibitor of hrAChE (IC₅₀ = 0.29 μM), with K_i value in nanomolar range (79 nM). Pertinent docking analysis confirmed this result, suggesting that this ligand is an interesting hit for further investigation.

Full text of DOI:10.3390/ijms21113913

International Journal of
Molecular Sciences
Communication
Acetylcholinesterase Inhibition of Diversely
Functionalized Quinolinones for Alzheimer’s
Disease Therapy
Óscar M. Bautista-Aguilera 1 , Lhassane Ismaili 2 , Mourad Chioua , Rudolf Andrys ,
3
4
4
, Petr Bzonek 4 , María Ángeles Martínez-Grau , Christopher D. Beadle ,
5
6
Monika Schmidt
7
8,9
1,10
2
Tatiana Vetman , Francisco López-Muñoz
, Isabel Iriepa
, Bernard Refouvelet ,
4
,
3,
Kamil Musilek * and José Marco-Contelles *
1
Department of Organic Chemistry and Inorganic Chemistry, Alcalá University, Alcalá de Henares,
8805 Madrid, Spain; oscar.bautista@uah.es (Ó.M.B.-A.); isabel.iriepa@uah.es (I.I.)
2
2
Laboratoire de Chimie Organique et Thérapeutique, Neurosciences Intégratives et Cliniques EA 481,
University Bourgogne Franche-Comté, UFR Santé, 19, rue Ambroise Paré, F-25000 Besançon, France;
lhassane.ismaili@univ-fcomte.fr (L.I.); bernard.refouvelet@univ-fcomte.fr (B.R.)
Laboratory of Medicinal Chemistry (IQOG, CSIC), C/ Juan de la Cierva 3, 28006 Madrid, Spain;
mchioua@gmail.com
Department of Chemistry, Faculty of Science, University of Hradec Kralove, Rokitanskeho 62, 500 03 Hradec
Kralove, Czech Republic; rudolf.andrys@uhk.cz (R.A.); monika.schmidt@uhk.cz (M.S.);
bzonepe@gmail.com (P.B.)
Lilly Research Laboratories, Eli Lilly & Company, Avda. de la Industria 30, Alcobendas, 28108 Madrid,
Spain; mamgrau@gmail.com
Lilly Research Centre, Eli Lilly & Company, Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK;
topher.beadle@gmail.com
3
4
5
6
7
8
Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, IN 46285, USA; vetman_tatiana@lilly.com
Faculty of Health, Camilo José Cela University of Madrid (UCJC), 28692 Villafranca del Castillo, Spain;
flopez@ucjc.edu
9
Neuropsychopharmacology Unit, “Hospital 12 de Octubre” Research Institute, 28041 Madrid, Spain
1
0
Institute of Chemical Research Andr
és M. del Río, Alcalá University, Alcalá de Henares, 28805 Madrid, Spain
*
Correspondence: kamil.musilek@uhk.cz (K.M.); iqoc21@iqog.csic.es (J.M.-C.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 10 April 2020; Accepted: 26 May 2020; Published: 30 May 2020
Abstract: In this communication, we report the synthesis and cholinesterase (ChE)/monoamine oxidase
MAO) inhibition of 19 quinolinones (QN1 19) and 13 dihydroquinolinones (DQN1 13) designed
(
-
-
as potential multitarget small molecules (MSM) for Alzheimer’s disease therapy. Contrary to our
expectations, none of them showed significant human recombinant MAO inhibition, but compounds
QN8
butyrylcholinesterase (hrBuChE) inhibition. In particular, molecule QN8 was found to be a potent
and quite selective non-competitive inhibitor of hrAChE (IC50 = 0.29 M), with K value in nanomolar
, QN9, and DQN7 displayed promising human recombinant acetylcholinesterase (hrAChE) and
µ
i
range (79 nM). Pertinent docking analysis confirmed this result, suggesting that this ligand is an
interesting hit for further investigation.
Keywords: Alzheimer’s disease; ChE/MAO inhibition; contilisant; dihydroquinolinones; docking;
quinolinones; synthesis
1
. Introduction
Alzheimer’s disease (AD), a pathology of ageing affecting mainly the elderly [
1], is a multifactorial,
neurodegenerative disease characterized by a progressive decline of memory and cognitive faculties [
2
].
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www.mdpi.com/journal/ijms

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Although the aetiology of AD is unknown, oxidative stress, deficit of neurotransmitters, and neuronal
death seem to play a critical role [ ]. To date, all the efforts directed to find the origin of the disease
have failed [ ], but as the number of suffering people is dramatically increasing, there is a strong and
3
4,5
urgent social need for therapeutic solutions. Acetylcholinesterase inhibitors (AChEI) such as donepezil,
rivastigmine, and galantamine, able to increase the level of biogenic amines in the brain, or memantine,
a non-competitive antagonist of the N-Methyl-D-Aspartate (NMDA) receptor, are the only drugs
currently administered to AD patients [
should be the starting point for designing more efficient clinical candidates for AD therapy. To do
this, the concept and application of multitarget small molecules (MSM) [ ] design strategy seem to us
the best way to cope with it. The ability of MSM to simultaneously modulate receptors or inhibit the
enzymatic systems involved in the progress of AD are the key factors of this approach [ ]. However,
6]. In spite of their limitations, we think that the use of AChEI
7
8,9
the proof that this approach is possible for complex diseases such as AD still has to be confirmed [10].
In a recent communication [11] we expanded the pharmacological profile of contilisant (Figure 1),
our most advanced lead compound for AD. We showed that contilisant is an antioxidant, permeable,
strong neuroprotective agent, able to increase the level of acetylcholine by inhibiting the cholinesterases
(
ChE) and monoamine oxidases (MAO), and modulating very specifically the histamine-3 (H3R) and
sigma-1 (S1R) receptors. In addition, contilisant overcomes the efficiency of donepezil in the in vivo
tests on suitable AD animal models [11].
Figure 1. Structure of contilisant, brexpiprazole, “quinolinones” (I), and “dihydroquinolinones” (II),
the new MSM designed in this work.
However, in our current research project, one of the main concerns was the irreversible MAO
inhibition shown by contilisant (Figure 1). Although this could be a point of debate [12], we decided
to design new ligands behaving pharmacologically like contilisant, but acting as MAO-reversible
inhibitors. In order to do this, we planned to substitute the N-methyl propargyl MAO-irreversible
inhibition motif by a typical MAO-reversible pharmacophore, using it at the same time as the
heterocyclic core instead of the indole ring. A number of options were possible to achieve these
goals, as coumarins [13], chromones [14], and chalcones [15] are well known reversible and selective
MAO-B inhibitors.
Thus, this simple design and recent communication on related chromenones [16] led us to select
“
quinolinones” (
basis of the availability of the starting materials, as well as the simple synthetic schemes to synthesize
them. Quinolinones have been previously described, with the antipsychotic drug “brexpiprazole” [17
Figure 1) being one of the best known examples. Dihydroquinolinones (Figure 1) bearing different
I, QN) and “dihydroquinolinones” (II, DQN) (Figure 1) as the MSM of choice, on the
]
(

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terminal amines have been reported as S1R antagonists for the potential use as analgesics [18], and as
new MSM showing ChE/MAO inhibition [19,20] and histamine 3 receptor antagonism [21] for the
treatment of AD.
In this work we describe the synthesis and biological evaluation of all these compounds designed
as MSM for AD therapy, an effort that has allowed us to identify compound QN8 (Figure 2) as a potent,
selective and non-competitive human recombinant acetylcholinesterase (hrAChE) inhibitor.
Me
O
N
N
N
N
N
(
)n
O
N
H
O
( )n
O
N
H
O
( )n
O
N
H
O
QN1 (n = 2)
QN2 (n = 3)
QN3 (n = 4)
QN4 (n = 3)
QN5 (n = 4)
QN6 (n = 1)
QN7 (n = 2)
QN8 (n = 3)
QN9 (n = 4)
N
N
N
(
)n
O
N
H
O
( )n
O
N
H
O
O
N
H
O
QN10 (n = 1)
QN11 (n = 2)
QN12 (n = 3)
QN13 (n = 4)
QN14
QN15 (n = 2)
QN16 (n = 4)
Me
N
N
(
)n
O
N
H
O
O
N
O
H
QN17 (n = 3)
QN18 (n = 4)
QN19
Figure 2. Structures of the “quinolinones” (QN1-19) described in this work.
2
. Results and Discussion
2
.1. Chemistry
Prompted by our experience in the area, we submitted this project to the ASL (Automated
Synthesis Lab) as part of the Eli Lilly’s Open Innovation Drug Discovery program [22], as this was an
opportunity to synthesize compounds remotely, explore novel synthetic approaches, improve reaction
efficiency, and test the feasibility of automatic synthetic processes to maximize the yield of targeted
compounds. The proposal was accepted, and we started the remote synthesis of the designed ligands
(Figures 2 and 3).
In this structure–activity relationship (SAR) analysis we had the opportunity to evaluate the
biological activity of different amines at variable distances from the quinolinone and dihydroquinolinone
cores. In Figure 2, we show the structures of the “quinolinones” QN1-19 that we prepared under
Lilly’s ASL program. These 19 ligands contain a basic amine (morpholine, N-methylpiperazine,
N-isopropylpiperazine, diethylamine, azepine, (R)-2-methylpyrrolidine, (S)-2-methylpyrrolidine, or
N-methylpiperidine) linked at the quinolinone core by an alkoxy group of different sizes. Note that
compounds QN7 and QN15-18 were prepared as formate salts. All these compounds are new (see the
Materials and Methods section).
Similarly, in Figure 3, we show the structures of the “dihydroquinolinones” DQN1-13 that we
synthesized remotely at Lilly’s ASL. These 13 compounds bear morpholine, N-methylpiperazine,
N-isopropylpiperazine, diethylamine, or N-methylpiperidine as the amino motif, with the
corresponding linkers to the 3,4-dihydroquinolin-2(1H)-one core (see the Materials and Methods section).

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Most of the compounds in Figure 3 are new, but the morpholine DQN1
[18], the N-isopropylpiperazines
DQN6-8
[19], and the diethylamines DQN9 [21] and DQN10-12 [19] have been previously described
in the literature.
Figure 3. Structures of the “dihydroquinolinones” (DQN1-13) described in this work.
A number of compounds in these families were prepared by Mitsunobu reaction of commercially
available 7-hydroxyquinolin-2(1H)-one (
Scheme 2) with the appropriate alcohol. Another set of compounds were synthesized by O-alkylation
of compounds and with the corresponding halides in two steps (Schemes 1 and 2). The present
1) (Scheme 1) and 7-hydroxy-3,4-dihydroquinolin-2(1H)-one (2)
(
1
2
design is very versatile, allowing us also to change the length of the linker connecting the heterocyclic
core with the cyclic (or acyclic) amine.
Scheme 1. Synthetic methods for the preparation of the “quinolinones” (QN1-19).

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Scheme 2. Synthetic methods for the preparation of the “dihydroquinolinones” (DQN1-13).
As described in the Materials and Methods section, starting from commercial 7-hydroxyquinolin-
(1H)-one ( ) and the appropriate aminoalcohol, compounds QN6 (10%), QN10 (60%), QN11
60%), QN14 (30%), and QN19 (30%) (Figure 2) were obtained by Mitsunobu reaction using
2
(
1
cyanomethylenetributylphosphorane, in toluene at reflux (Scheme 1) [23]. Compounds QN1 (20%),
QN2 (20%), QN3 (10%), QN4 (20%), QN5 (20%), QN7 (30%), QN8 (30%), QN9 (10%), QN12 (20%),
QN13 (50%), QN15 (10%), QN16 (10%), QN17 (30%), and QN18 (10%) (Figure 2) were synthesized
by reacting K CO , 7-hydroxyquinolin-2(1H)-one (
1
), and the corresponding 1,n-dibromoalkane in
2
◦
3
CH CN at 50 C, with the subsequent addition of the appropriate amine (Scheme 1) [24].
3
Starting from commercially available 7-hydroxy-3,4-dihydroquinolin-2(1H)-one (2) and
the appropriate aminoalcohol, compounds DQN1 (80%), DQN2 (80%), DQN5 (30%),
DQN9 (70%), and DQN13 (70%) (Figure 3) were obtained by Mitsunobu reaction using
cyanomethylenetributylphosphorane in toluene at reflux (Scheme 2) [23]. Molecules DQN3 (30%),
DQN4 (30%), DQN6 (20%), DQN7 (30%), DQN8 (30%), DQN10 (50%), DQN11 (60%), and DQN12
(
50%) (Figure 3) were synthesized by reacting K CO , 7-hydroxy-3,4-dihydroquinolin-2(1H)-one (2),
2 3
◦
and the corresponding 1,n-dibromoalkane in CH CN at 50 C, with the subsequent addition of the
3
appropriate amine (Scheme 2) [24].
All compounds showed proper analytical and spectroscopic data in good agreement with
their structures.
2
.2. Biological Evaluation
The compounds were screened for inhibition at human recombinant monoamine oxidases
(
hrMAO-A and hrMAO-B), human recombinant acetylcholinesterase (hrAChE) and human recombinant
butyrylcholinesterase (hrBuChE), at two concentrations (1 and 10 M). Clorgylin and pargylin were
the standards used for hrMAO-A and hrMAO-B inhibition, respectively, at low concentrations (0.05
and 0.5 M) due to their high ability to decrease MAO activity. Tacrine (1 and 10 M) was chosen as
the standard for hrAChE and hrBuChE inhibition.
µ
µ
µ

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The inhibition of hrMAO-A/B by novel compounds was rather weak compared with the standards
used (data not shown).
These compounds were then tested on hrAChE/hrBuChE [25
QN9, and DQN7 showed high hrAChE inhibition (88–95% at 10
hrBuChE inhibition (45–65% at 10 µM concentration).
,
26]. As a result, compounds QN8
,
µM concentration) and moderate
On the basis of these data, three compounds (QN8
determination in hrAChE and hrBuChE (Table 1, Figures 4 and 5). Compound QN8 demonstrated the
highest inhibition for hrAChE (IC50 = 0.29 0.02 M) with the highest selectivity for hrAChE (hrBuChE
IC50 = 12.73 0.45 M), and was further tested in kinetic experiments (Figure 6) [27]. Compound
QN8 resulted as a non-competitive inhibitor of hrAChE, with K value in nanomolar range (79 7 nM).
, QN9, and DQN7) were chosen for IC50
±
µ
±
µ
±
i
In comparison with donepezil, QN8 is six-fold less potent as hrAChEI (Table 1), and shows a Ki value
two-fold higher than donepezil (Ki = 39 nM) [28].
Table 1.
Determination of IC50 for human recombinant acetylcholinesterase (hrAChE) and
butyrylcholinesterase (hrBuChE) inhibition by ligands QN8, QN9, DQN7, and donepezil.
N
N
N
N
O
N
H
O
(
)n
O
N
H
O
QN8 (n = 3)
QN9 (n = 4)
DQN7
IC5 (µM) a
0
Selectivity Index IC50
hrBuChE)/IC50 (hrAChE)
Compound
(
hrAChE
hrBuChE
QN8
QN9
DQN7
0.29 ± 0.02
0.96 ± 0.04
1.58 ± 0.15
0.048 ± 0.003
12.73 ± 0.45
6.70 ± 0.32
16.73 ± 0.46
3.17 ± 0.1
44
7
11
66
Donepezil [28]
a
IC50 values are expressed as the mean ± standard error of the mean for triplicate determinations.
Figure 4. IC50 determination for hrAChE inhibition by QN8, QN9, and DQN7.
Figure 5. IC50 determination for hrBuChE inhibition by QN8, QN9, and DQN7.

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Figure 6. Inhibition kinetics of hrAChE by compound QN8.
Compounds DQN6-8 and DQN10-12 have been previously described and tested as MAO and
ChE inhibitors [19]. We observed low hrMAO inhibition at 1 and 10 M concentration (data not shown)
and did not determine their IC . In a formerly published study [19], hrMAO inhibition was reported
µ
50
in the double digit micromolar range for most of the compounds, but the source of MAOs was not
mentioned or referenced.
Regarding the inhibition of the hChEs, the previous data [19] were different from ours, but used
eel, equine, or rat AChE and BuChE enzymes. Because of the higher relevance, we think that only data
determined for human enzymes should be considered for further investigation.
Our SAR design was based on two similar cores, a variable linker and a limited number of
differently functionalized basic centres (Figures 2 and 3). On the basis of the comparison between
compounds QN8 (0.29 µM) and DQN7 (1.58 µM), the quinolinone core looked more potent than
the semi-unsaturated dihydroquinolinone as hrAChE inhibitor. The isopropylpiperazine seemed
also essential for the hrAChE activity with the butoxy group as the optimal linker between the core
and basic centre. The branched isopropyl substituent boosted the hrAChE inhibition as observed
when comparing QN8 (0.29
assessment is also valid for the compounds QN9 (0.96
low inhibition percentage). These SAR findings open new directions to develop further modified
hrAChE inhibitors with hrBuChE selectivity.
µM) with the methylpiperazine QN4 (low inhibition percentage). This
µ
M) and DQN7 (1.58 M) vs. QN5 and DQN3
µ
(
2
.3. Computational Chemistry: Docking of Compound QN8
To justify the observed in vitro enzymatic activity and shed light into the hrAChE active site and
binding mode of compound QN8, we performed molecular docking studies using software AutoDock
Vina 1.1.2 [29].
Compound QN8 showed quite high binding affinity ( 10.3 kcal/mol), indicating a tight binding to
−
the enzyme. The docking analysis revealed that compound QN8 spanned the narrow AChE active site
interacting with the anionic subsite (AS) halfway down the gorge, and in the mouth of the gorge at the
peripheral anionic site (PAS), but not with the catalytic triad (CT: residues His447, Glu334, and Ser203)
of hAChE (Figure 7). In this binding mode, the quinolinone ring interacted with Trp86 (at the AS) and
with Tyr337 via
and Tyr341, at the PAS, via attractive charge and
π
–
π
stacking interactions. The diprotonated piperazine moiety interacted with Asp74
-cation interactions, respectively. The oxygen ether
π
formed a hydrogen bond with Gly121 in the oxyanion hole (OH), while Tyr124 formed a hydrogen bond
with the hydrogen of one of the quaternized nitrogen of the piperazine moiety. In addition, some carbon
hydrogen interactions were observed between the ligand and Thr83, Tyr124, and Ty337 (Figure 8).

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Figure 7. Binding mode of QN8 in the active site of hrAChE. Compound QN8 is rendered as sticks
carbon atoms in blue) and the side chain conformations of the mobile residues are illustrated in light
(
blue. Catalytic triad (CT) is coloured in green, oxyanion hole (OH) in magenta, anionic subsite (AS)
in orange except Trp86, the acyl binding pocket (ABP) in yellow, and peripheral anionic site (PAS) in
light blue.
Figure 8. 2D schematic view of the interactions between QN8 and hrAChE.
In conclusion, docking results based on the interactions of compound QN8 at the AS, PAS and OH
are in good agreement with the experimental kinetic data, indicating and confirming a non-competitive
type of inhibition.
2
.4. Predicted Physico-Chemical Properties Analysis for Ligand QN8
The physico-chemical properties of ligand QN8 were evaluated with QikProp module of
Schrodinger (QikProp, version 5.1, Schrodinger, LLC, New York, NY, 2017-1). The selected properties
are known to influence metabolism, cell permeation, and bioavailability (Table 2).

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Table 2. Physico-chemical properties of compound QN8 calculated using QikProp. a.
Molecule
QN8
MW
343.468
QPPCaco
87.998
SASA
708.219
PSA
Volume
1221.055
QPlogBB
−0.038
DonorHB AccptHB
QPlogPo/w
QPlogS
1.000
metab
3
7.250
2.449
−1.920
Molecule
QPlogKhsa % HOA
0.185 76.087
ROF
ROT
QN8
60.557
0
0
a
MW: molecular weight of the molecule (130–725). SASA: total solvent accessible surface area, in square angstroms,
using a probe with a 1.4 Å radius (limits 300–1000). Volume: total solvent-accessible volume, in cubic angstroms,
using a probe with a 1.4 Å radius (limits 500–2000). donorHB: estimated number of hydrogen bonds that would be
accepted by the solute (limits: 0–6). accptHB: estimated number of hydrogen bonds that would be donated by the
solute (limits: 2–20). QPlogPo/w: predicted octanol/water partition coefficient (limits
−
2.0–6.5). QPlogS: predicted
aqueous solubility. S, in mol/dm , is the concentration of the solute’s saturated solution that is in equilibrium
with crystalline solid (limits 6.5–0.5). QPPCaco: predicted apparent Caco-2 cell permeability in nanometres
per second. Caco-2 cells is a model for the gut–blood barrier. QikProp predictions are for non-active transport.
<25 poor, >500 great). PSA: Van der Waals surface area of polar nitrogen and oxygen atoms (limits 7–200). QPlog
BB: predicted brain/blood partition coefficient (limits 3.0–1.2). metab: number of likely metabolic reactions (limits
–8). QPlogKhsa: prediction of binding to human serum albumin (limits 1.5–1.5). HOA: predicted qualitative
human oral absorption on 0% to 100% scale. ROF: number of violations of Lipinski’s Rule Of Five [30] (molecular
weight <500, QPlogPo/w <5, number of hydrogen bond donor 5, number of hydrogen bond acceptors HB 10).
32] (QPlogS> 5.7, QPCaco >22 nm/s, number of primary
3
−
(
−
1
−
≤
≤
ROT: number of violations of Jorgensen’s rule of three [31
metabolites <7).
,
−
Central Nervous System (CNS) drugs tend to be more lipophilic, less polar, less flexible, and have
lower molecular weight and molecular volume than drugs used for other therapeutics. Usually, CNS
drugs show values of molecular weight (MW) < 450, number of hydrogen bond donors < 3, number
of hydrogen bond acceptors < 7, partition coefficient (QPlogPo/w) < 5, polar surface area (PSA) < 90
square Å, number of rotatable bonds < 8, and hydrogen bonds < 8.
The partition coefficient (QPlogPo/w), which is critical for estimation of absorption within the
body, is 2.449 for ligand QN8. A value that ranges 1 to 3 is most favourable for blood–brain barrier
(
BBB) penetration and CNS activity. The gut–blood barrier permeability was predicted using Caco-2
cell permeability (QPPCaco) as model and it showed good value at 87.998 (<25 poor, >500 great).
Further, the prediction for human serum albumin binding using QPlogKhsa showed that the value for
QN8 lay within the expected range (from
−
1.5 to 1.5). Likewise, the brain/blood partition coefficient
(
QPlogBB) showed a satisfactory value (Table 2). The percentage of oral drug absorption predicted for
the test compound was adequate, with a high percentage (>70%) of human oral absorption, indicating
their possibilities in oral drug formulation.
The predicted properties of compound QN8 (Table 2) were in the ranges defined by QikProp for
95% of known oral drugs and also satisfied the Lipinski’s rule of five [30]. In conclusion, on the basis
of the physico-chemical properties shown in Table 2, compound QN8 shows characteristics that are
typical of a drug-like molecule with potential BBB permeability.
3
3
3
. Materials and Methods
.1. Chemistry
.1.1. General Methods
1H-NMR (400 MHz) spectra were recorded on a Bruker Avance III 400 NMR spectrometer (Bruker,
Billerica, MA, USA). Chemical shifts
δ are given in parts per million referring to the signal centre
using the solvent peaks for reference: DMSO-d 2.49 ppm. LC/MS were recorded in an Agilent
6
HP1100 liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA). Electrospray
mass spectrometry measurements (acquired in positive mode) were performed on a mass selective
detector quadrupole spectrometer interfaced to the HP1100 HPLC. The conditions followed were as
follows: column: HPH Phenomenex Kinetix EVO 2.6u, 2.1
×
30 mm; flow rate: 0.85 mL/min; gradient:
5
–100% B; run time: 2 min; solvent A: 10 mM ammonium bicarbonate (pH = 10); solvent B: acetonitrile.

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The purity of all products, as checked with LC/MS using a diode array detector coupled to a mass
spectrometer, was higher than 95%.
3
.1.2. General Procedures for the Synthesis of Quinolinones (QNs)
a) General Procedure A via Mitsunobu reaction: A mixture of 7-hydroxyquinolin-2(1H)-one
(
(
(
0.5 mmol), the appropriate aminoalcohol (0.5 mmol), and cyanomethylenetributylphosphorane
0.7 mmol) in toluene (5 mL) was heated at 100 C for 16 h. The reaction mixture was diluted with
◦
methanol and loaded into 10 g Strong Cation Exchange (SCX) column. Resin was washed with
methanol, and crude product was eluted in 2N ammonia in methanol before evaporation of the solvent.
The residue was purified by column chromatography (CH Cl /MeOH (from 1% to 5%)).
2
2
(
b) General Procedure B via O-Alkylation: Step 1. A mixture of potassium carbonate (2.5 mmol),
7
-hydroxyquinolin-2(1H)-one (0.5 mmol), and the corresponding 1,n-dibromoalkane (1.0 mmol) in
◦
acetonitrile (5 mL) was heated at 50 C for 20 h. After cooling, acetonitrile (10 mL) was added,
the inorganic salts were filtered off, and the solvent was evaporated. Step 2. To a stirred solution of the
crude intermediate in acetonitrile (5 mL), we added the appropriate amine (2.0 mmol). After stirring
◦
at 50 C for 15 h, we diluted the reaction mixture with methanol and loaded it into 10 g SCX resin.
Resin was washed with methanol, and crude product was eluted with 2N ammonia in methanol before
evaporation of the solvent. The residue was purified by column chromatography (CH Cl /MeOH
2
2
(from 1% to 5%)).
(
c) General Procedure C via O-Alkylation: Step 1. A mixture of potassium carbonate (2.5 mmol),
7
-hydroxyquinolin-2(1H)-one (0.5 mmol), and the corresponding 1,n-dibromoalkane (1.0 mmol) in
◦
acetonitrile (5 mL) was heated at 50 C for 20 h. After cooling, acetonitrile (10 mL) was added,
the inorganic salts were filtered off, and the solvent was evaporated. Step 2. To a stirred solution of the
crude intermediate in acetonitrile (5 mL), we added the appropriate amine (0.5 mmol). After stirring
◦
at 50 C for 15 h, we diluted the reaction mixture with methanol and loaded it into 10 g SCX resin.
Resin was washed with methanol, and crude product was eluted with 2N ammonia in methanol before
evaporation of the solvent. The residue was purified by column chromatography (CH Cl /MeOH
2
2
(
from 1% to 5%)).
-(3-Morpholinopropoxy)quinolin-2(1H)-one (QN1). Following General Procedure B, the reaction
of a mixture of potassium carbonate (345.5 mg, 2.5 mmol), 7-hydroxyquinolin-2(1H)-one (80.6 mg,
.5 mmol), and 1,3-dibromopropane (202 mg, 1.0 mmol) in acetonitrile (5 mL) afforded a crude
7
0
intermediate that was suspended in acetonitrile (5 mL), and treated with morpholine (174.3 mg,
1
2
.0 mmol) to give compound QN1 (22.73 mg, 20% yield). H-NMR (400 MHz, DMSO-d )
δ: 11.55
6
(
1
(
br s, 1H), 7.81 (d, J = 9.5 Hz, 1H), 7.59 (dd, J = 8.3, 1.3 Hz, 1H), 6.79–6.75 (m, 2H), 6.30 (d, J = 9.5 Hz,
H), 4.05 (t, J = 6.5 Hz, 2H), 3.58–3.54 (m, 4H), 2.40–2.37 (m, 6H), 1.91–1.86 (m, 2H). LC/MS m/z 289.2
M + H), t = 0.65 min.
R
7-(4-Morpholinobutoxy)quinolin-2(1H)-one (QN2). Following General Procedure B, the reaction of a
mixture of potassium carbonate (345.5 mg, 2.5 mmol), 7-hydroxyquinolin-2(1H)-one (80.6 mg, 0.5 mmol),
and 1,4-dibromobutane (216 mg, 1.0 mmol) in acetonitrile (5 mL) afforded a crude intermediate that was
suspended in acetonitrile (5 mL), and treated with morpholine (174.3 mg, 2.0 mmol) to give compound
1
QN2 (20.53 mg, 20% yield). H-NMR (400 MHz, DMSO-d )
δ: 11.55 (br s, 1H), 7.80 (d, J = 9.5 Hz,
6
1H), 7.55 (dd, J = 8.4, 1.3 Hz, 1H), 6.79–6.76 (m, 2H), 6.29 (d, J = 9.5 Hz, 1H), 4.03 (t, J = 6.4 Hz, 2H),
3.56-3.52 (m, 4H), 2.33–2.30 (m, 6H), 1.76–1.73 (m, 2H), 1.58–1.55 (m, 2H). LC/MS m/z 303.2 (M + H),
tR = 0.71 min.
7-(5-Morpholinopentyloxy)quinolin-2(1H)-one (QN3). Following General Procedure C, the reaction
of a mixture of potassium carbonate (345.5 mg, 2.5 mmol, 7-hydroxyquinolin-2(1H)-one (80.6 mg,
0.5 mmol), and 1,5-dibromopentane (230 mg, 1.0 mmol) in acetonitrile (5 mL) afforded a crude
intermediate that was suspended in acetonitrile (5 mL), and treated with morpholine (43.6 mg,
1
0
.5 mmol) to give compound QN3 (19.87 mg, 10% yield). H-NMR (400 MHz, DMSO-d )
δ: 11.56 (br s,
6
1
H), 7.80 (d, J = 9.5 Hz, 1H), 7.55 (dd, J = 8.3, 1.3 Hz, 1H), 6.79–6.76 (m, 2H), 6.30 (d, J = 9.5 Hz, 1H), 4.01

Int. J. Mol. Sci. 2020, 21, 3913
11 of 20
(
t, J = 6.5 Hz, 2H), 3.56–3.52 (m, 4H), 2.30–2.26 (m, 6H), 1.76–1.73 (m, 2H), 1.54–1.37 (m, 4H). LC/MS
m/z 317.2 (M + H), t = 0.77 min.
R
7
-[4-(4-Methylpiperazin-1-yl)butoxy]quinolin-2(1H)-one (QN4). Following General Procedure B,
the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol), 7-hydroxyquinolin-2(1H)-one
80.6 mg, 0.5 mmol), and 1,4-dibromobutane (216 mg, 1.0 mmol) in acetonitrile (5 mL) afforded a crude
(
intermediate that was suspended in acetonitrile (5 mL), and treated with N-methylpiperazine (200.3 mg,
1
2
.0 mmol) to give compound QN4 (29.38 mg, 20% yield). H-NMR (400 MHz, DMSO-d )
δ: 11.56 (br s,
6
1H), 7.80 (d, J = 9.5 Hz, 1H), 7.55 (dd, J = 8.4, 1.3 Hz, 1H), 6.83–6.76 (m, 2H), 6.29 (d, J = 9.5 Hz, 1H),
4
3
.02 (t, J = 6.4 Hz, 2H), 2.37–2.27 (m, 7H), 2.14 (s, 3H), 1.75–1.73 (m, 2H), 1.57–1.55 (m, 2H) (the missing
H must be hidden under the DMSO-d signal). LC/MS m/z 316.2 (M + H), t = 0.68 min.
6
R
7
-[5-(4-Methylpiperazin-1-yl)pentyloxy]quinolin-2(1H)-one (QN5). Following General Procedure C,
the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol), 7-hydroxyquinolin-2(1H)-one
80.6 mg, 0.5 mmol), and 1,5-dibromopentane (230 mg, 1.0 mmol) in acetonitrile (5 mL) afforded a
(
crude intermediate that was suspended in acetonitrile (5 mL), and treated with N-methylpiperazine
1
(
50.08 mg, 0.5 mmol) to give compound QN5 (27.16 mg, 20% yield). H-NMR (400 MHz, DMSO-d )
6
δ
: 11.48 (br s, 1H), 7.80 (d, J = 9.4 Hz, 1H), 7.59 (dd, J = 8.4, 1.3 Hz, 1H), 6.78–6.76 (m, 2H), 6.29 (d,
J = 9.4 Hz, 1H), 4.00 (t, J = 6.4 Hz, 2H), 2.32–2.26 (m, 10H), 2.14 (s, 3H), 1.75–1.72 (m, 2H), 1.44–1.41 (m,
4
H). LC/MS m/z 330.2 (M + H), t = 0.73 min.
R
7
-[2-(4-Isopropylpiperazin-1-yl)ethoxy]quinolin-2(1H)-one (QN6). Following General Procedure A,
7
-hydroxyquinolin-2(1H)-one (80.6 mg, 0.5 mmol) and 2-(4-isopropylpiperazin-1-yl)ethan-1-ol (86.1 mg,
0
.5 mmol) were reacted with cyanomethylenetributylphosphorane (169 mg, 0.7 mmol) in toluene
1
(5 mL) to give compound QN6 (20.91 mg, 10% yield). H-NMR (400 MHz, DMSO-d )
δ: 11.53 (br s,
6
1
4
3
H), 7.81 (d, J = 9.4 Hz, 1H), 7.56 (dd, J = 8.4, 1.4 Hz, 1H), 6.82–6.79 (m, 2H), 6.30 (d, J = 9.4 Hz, 1H),
.10 (t, J = 5.8 Hz, 2H), 2.70 (t, J = 5.8 Hz, 2H), 2.59–2.27 (m, 9H), 0.96 (d, J = 6.3 Hz, 6H). LC/MS m/z
16.2 (M + H), t = 0.69 min.
R
7
-[3-(4-Isopropylpiperazin-1-yl)propoxy]quinolin-2(1H)-one formate salt (QN7). Following General
Procedure B, the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol), 7-hydroxyquinolin-
(1H)-one (80.6 mg, 0.5 mmol), and 1,3-dibromopropane (202 mg, 1.0 mmol) in acetonitrile
5 mL) afforded a crude intermediate that was suspended in acetonitrile (5 mL), and treated with
2
(
1
-isopropylpiperazine (256.4 mg, 2.0 mmol) to give compound QN7 as free base. Treatment with 0.1%
1
formic acid/H O afforded compound QN7 (46.98 mg, 30% yield). H-NMR (400 MHz, DMSO-d )
δ:
2
6
1
1
1.56 (s, 1H), 7.81 (d, J = 9.5 Hz, 1H), 7.55 (dd, J = 8.5, 1.3 Hz, 1H), 6.81–6.79 (m, 2H), 6.30 (d, J = 9.5 Hz,
H), 4.06 (t, J = 5.9 Hz, 2H), 2.59–2.38 (m, 11H), 1.90–1.87 (m, 2H), 0.96 (d, J = 6.5 Hz, 6H). LC/MS m/z
3
30.2 (M + H), t = 0.27 min.
R
7
-[4-(4-Isopropylpiperazin-1-yl)butoxy]quinolin-2(1H)-one (QN8). Following General Procedure C,
the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol), 7-hydroxyquinolin-2(1H)-one
80.6 mg, 0.5 mmol), and 1,4-dibromobutane (216 mg, 1.0 mmol) in acetonitrile (5 mL) afforded a
(
crude intermediate that was suspended in acetonitrile (5 mL), and treated with 1-isopropylpiperazine
1
(
64.1 mg, 0.5 mmol) to give compound QN8 (49.56 mg, 30% yield). H-NMR (400 MHz, DMSO-d )
6
δ
: 11.56 (br s, 1H), 7.80 (d, J = 9.5 Hz, 1H), 7.55 (dd, J = 8.3, 1.3 Hz, 1H), 6.79–6.77 (m, 2H), 6.29 (d,
J = 9.5 Hz, 1H), 4.02 (t, J = 6.4 Hz, 2H), 2.58 (t, J = 6.5 Hz, 2H), 2.40–2.30 (m, 9H), 1.81–1.69 (m, 2H),
.63–1.50 (m, 2H), 0.95 (d, J = 6.5 Hz, 6H). LC/MS m/z 344.2 (M + H), t = 0.80 min.
1
R
7
-[5-(4-Isopropylpiperazin-1-yl)pentyloxy]quinolin-2(1H)-one (QN9). Following General Procedure C,
the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol), 7-hydroxyquinolin-2(1H)-one
80.6 mg, 0.5 mmol), and 1,5-dibromopentane (230 mg, 1.0 mmol) in acetonitrile (5 mL) afforded a
(
crude intermediate that was suspended in acetonitrile (5 mL) and treated with 1-isopropylpiperazine
1
(
64.1 mg, 0.5 mmol) to give compound QN9 (22.19 mg, 10% yield). H-NMR (400 MHz, DMSO-d )
6
δ
: 11.55 (br s, 1H), 7.80 (d, J = 9.5 Hz, 1H), 7.55 (dd, J = 8.3, 1,3 Hz, 1H), 6.80–6.77 (m, 2H), 6.29 (dd,
J = 9.5 Hz, 1H), 4.00 (t, J = 6.4 Hz, 2H), 2.57-1.73 (m, 11H), 1.75–1.65 (m, 2H), 1.45-1.42 (m, 4H), 0.95 (d,
J = 6.5 Hz, 6H). LC/MS m/z 358.2 (M + H), t = 0.86 min.
R

Int. J. Mol. Sci. 2020, 21, 3913
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7-[2-(Diethylamino)ethoxy]quinolin-2(1H)-one (QN10)
.
Following General Procedure A,
7-hydroxyquinolin-2(1H)-one (80.6 mg, 0.5 mmol) and 2-(diethylamino)ethan-1-ol (58.6 mg, 0.5 mmol)
were reacted with cyanomethylenetributylphosphorane (169 mg, 0.7 mmol), in toluene (5 mL, 47.3 mmol)
1
to give compound QN10 (77.06 mg, 60% yield). H-NMR (400 MHz, DMSO-d )
δ: 11.56 (s, 1H), 7.81
6
(d, J = 9.4 Hz, 1H), 7.56 (dd, J = 8.4, 1.3 Hz, 1H), 6.79–6.77 (m, 2H), 6.30 (d, J = 9.4 Hz, 1H), 4.05 (t,
J = 6.2 Hz, 2H), 2.80 (t, J = 6.2 Hz, 2H), 2.57 (q, J = 7.1 Hz, 4H), 0.99 (t, J = 7.1 Hz, 6H). LC/MS m/z 261.2
(
M + H), t = 0.78 min.
R
7-[3-(Diethylamino)propoxy]quinolin-2(1H)-one (QN11). Following General Procedure A,
7
-hydroxyquinolin-2(1H)-one (80.6 mg, 0.5 mmol) and 3-(diethylamino)propan-1-ol (65.6 mg, 0.5 mmol)
were reacted with cyanomethylenetributylphosphorane (169 mg, 0.7 mmol) in toluene (5 mL) to give
1
compound QN11 (77.0 mg, 60% yield). H-NMR (400 MHz, DMSO-d )
δ: 11.51 (br s, 1H), 7.80 (d,
6
J = 9.5 Hz, 1H), 7.55 (dd, J = 8.5, 1.3 Hz, 1H), 6.80–6.77 (m, 2H), 6.29 (d, J = 9.5 Hz, 1H), 4.04 (t, J = 6.3 Hz,
H), 2.46 (q, J = 7.1 Hz, 4H), 1.84–1.81 (m, 2H), 0.95 (t, J = 7.1 Hz, 6H) (the missing 2H must be hidden
under the DMSO-d signal). LC/MS m/z 275.2 (M + H), t = 0.81 min.
2
6
R
7
-[4-(Diethylamino)butoxy]quinolin-2(1H)-one (QN12). Following General Procedure B, the reaction
of a mixture of potassium carbonate (345.5 mg, 2.5 mmol), 7-hydroxyquinolin-2(1H)-one (80.6 mg,
.5 mmol), and 1,4-dibromobutane (216 mg, 1.0 mmol) in acetonitrile (5 mL) afforded a crude
0
intermediate that was suspended in acetonitrile (5 mL), and treated with diethylamine (146.3 mg,
1
2
.0 mmol) to give compound QN12 (27.81 mg, 20% yield). H-NMR (400 MHz, DMSO-d )
δ: 11.56 (s,
6
1
H), 7.80 (d, J = 9.4 Hz, 1H), 7.56 (dd, J = 8.4, 1.3 Hz, 1H), 6.82–6.78 (m, 2H), 6.29 (d, J = 9.4 Hz, 1H), 4.02
(
(
t, J = 6.6 Hz, 2H), 2.43 (q, J = 7.1 Hz, 4H), 1.77–1.71 (m, 2H), 1.57–1.51 (m, 2H), 0.95 (t, J = 7.1 Hz, 6H)
the missing 2H must be hidden under the DMSO-d signal). LC/MS m/z 289.2 (M + H), t = 0.84 min.
6
R
7
-[5-(Diethylamino)pentyloxy]quinolin-2(1H)-one (QN13). Following General Procedure B, the
reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol), 7-hydroxyquinolin-2(1H)-one
80.6 mg, 0.5 mmol), and 1,5-dibromopentane (230 mg, 1.0 mmol) in acetonitrile (5 mL) afforded a
(
crude intermediate that was suspended in acetonitrile (5 mL), and treated with diethylamine (146.3 mg,
1
2
.0 mmol) to give compound QN13 (82.72 mg, 50% yield). H-NMR (400 MHz, DMSO-d )
δ: 11.56 (br
6
s, 1H), 7.80 (d, J = 9.5 Hz, 1H), 7.55 (dd, J = 8.3, 1.3 Hz, 1H), 6.80–6.77 (m, 2H), 6.29 (d, J = 9.5 Hz, 1H),
.00 (t, J = 6.5 Hz, 2H), 2.49–2.35 (m, 6H), 1.79–1.73 (m, 2H), 1.46–1.38 (m, 4H), 0.94 (t, J = 7.1 Hz, 6H).
LC/MS m/z 303.2 (M + H), t = 0.90 min.
4
R
7
-[2-(Azepan-1-yl)ethoxy]quinolin-2(1H)-one (QN14)
.
Following General Procedure A,
7
0
-hydroxyquinolin-2(1H)-one (80.6 mg, 0.5 mmol) and 2-(azepan-1-yl)ethan-1-ol (71.6 mg,
.5 mmol) were reacted with cyanomethylenetributylphosphorane (169 mg, 0.7 mmol) in toluene
1
(5 mL, 47.3 mmol) to give compound QN14 (46.52 mg, 30% yield). H-NMR (400 MHz, DMSO-d )
δ:
6
8
.28 (d, J = 9.0 Hz, 1H), 7.62 (dd, J = 8.8, 0.7 Hz, 1H), 7.44–7.40 (m, 2H), 6.33 (d, J = 9.0 Hz, 1H), 4.19 (t,
J = 5.7 Hz, 2H), 3.62–3.55 (m, 4H), 2.73 (t, J = 5.7 Hz, 2H), 2.49–2.43 (m, 8H). LC/MS m/z 287.2 (M + H),
tR = 0.90 min.
(
R)-7-[3-(2-Methylpyrrolidin-1-yl)propoxy]quinolin-2(1H)-one formate salt (QN15). Following General
Procedure B, the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol),
-hydroxyquinolin-2(1H)-one (80.6 mg, 0.5 mmol), and 1,3-dibromopropane (202 mg, 1.0 mmol)
7
in acetonitrile (5 mL) afforded a crude intermediate that was suspended in acetonitrile (5 mL) and
treated with (R)-2-methylpyrrolidine (170.3 mg, 2.0 mmol) to give compound QN15 as a free base.
1
Treatment with 0.1% formic acid/H O afforded compound QN15 (18.05 mg, 10% yield). H-NMR
2
(
(
(
400 MHz, DMSO-d6) δ: 11.55 (s, 1H), 7.80 (d, J = 9.4 Hz, 1H), 7.55 (dd, J = 8.4, 1.3 Hz, 1H), 6.86–6.75
m, 2H), 6.29 (d, J = 9.4 Hz, 1H), 4.05 (t, J = 6.4 Hz, 2H), 3.10–3.06 (m, 1H), 2.92–2.89 (m, 1H), 2.26–2.23
m, 1H), 2.15–2.10 (m, 1H), 2.05–2.00 (m, 1H), 1.93–1.84 (m, 3H), 1.73–1.58 (m, 2H), 1.35–1.21 (m, 1H),
1
.00 (d, J = 6.0 Hz, 3H). LC/MS m/z 287.2 (M + H), t = 0.39 min.
R
(
R)-7-[5-(2-Methylpyrrolidin-1-yl)pentyloxy]quinolin-2(1H)-one formate salt (QN16). Following
General Procedure B, the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol),
-hydroxyquinolin-2(1H)-one (80.6 mg, 0.5 mmol), and 1,5-dibromopentane (230 mg, 1.0 mmol)
7

Int. J. Mol. Sci. 2020, 21, 3913
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in acetonitrile (5 mL) afforded a crude intermediate that was suspended in acetonitrile (5 mL) and
treated with (R)-2-methylpyrrolidine (170.3 mg, 2.0 mmol) to give compound QN16 as free base.
1
Treatment with 0.1% formic acid/H O afforded compound QN16 (31.92 mg, 10% yield). H-NMR
2
(
(
(
400 MHz, DMSO-d6) δ: 11.53 (br s, 1H), 7.80 (d, J = 9.5 Hz, 1H), 7.55 (dd, J = 8.4, 1.2 Hz, 1H), 6.80–6.75
m, 2H), 6.29 (d, J = 9.5 Hz, 1H), 4.01 (t, J = 6.5 Hz, 2H), 3.05–3.01 (m, 1H), 2.74–2.65 (m, 1H), 2.21–2.16
m, 1H), 1.99–1.94 (m, 2H), 1.86–1.71 (m, 3H), 1.64–1.57 (m, 2H), 1.55–1.36 (m, 4H), 1.30–1.23 (m, 1H),
1
.00 (d, J = 6.0 Hz, 3H). LC/MS m/z 315.2 (M + H), t = 0.49 min.
R
(
S)-7-[4-(2-Methylpyrrolidin-1-yl)butoxy]quinolin-2(1H)-one formate salt (QN17). Following General
Procedure B, the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol),
-hydroxyquinolin-2(1H)-one (80.6 mg, 0.5 mmol), and 1,4-dibromobutane (216 mg, 1.0 mmol) in
7
acetonitrile (5 mL) afforded a crude intermediate that was suspended in acetonitrile (5 mL), and treated
with (S)-2-methylpyrrolidine (170.3 mg, 2.0 mmol) to give compound QN17 as free base. Treatment
1
with 0.1% formic acid/H O afforded compound QN17 (39.42 mg, 30% yield). H-NMR (400 MHz,
2
DMSO-d6)
δ
: 11.57 (s, 1H), 8.18 (s, 1H), 7.80 (d, J = 9.5 Hz, 1H), 7.55 (dd, J = 8.3, 1.4 Hz, 1H), 6.80–6.78
(m, 2H), 6.29 (dd, J = 8.5, 1.8 Hz, 1H), 4.03 (t, J = 6.4 Hz, 2H), 3.17–3.08 (m, 1H), 2.84–2.78 (m, 1H),
2.32–2.28 (m, 1H), 2.20–2.06 (m, 2H), 1.92–1.57 (m, 7H), 1.34–1.25 (m, 1H), 1.05 (d, J = 6.1 Hz, 3H).
LC/MS m/z 301.2 (M + H), t = 0.42 min.
R
(
S)-7-[5-(2-Methylpyrrolidin-1-yl)pentyloxy]quinolin-2(1H)-one formate salt (QN18)
General Procedure B, the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol),
-hydroxyquinolin-2(1H)-one (80.6 mg, 0.5 mmol), and 1,5-dibromopentane (230 mg, 1.0 mmol)
.
Following
7
in acetonitrile (5 mL) afforded a crude intermediate that was suspended in acetonitrile (5 mL) and
treated with (S)-2-methylpyrrolidine (170.3 mg, 2.0 mmol) to give compound QN18 as free base.
1
Treatment with 0.1% formic acid/H O afforded compound QN18 (27.01 mg, 10% yield). H-NMR
2
(
(
(
400 MHz, DMSO-d6) δ: 11.50 (br s, 1H), 7.80 (d, J = 9.5 Hz, 1H), 7.55 (dd, J = 8.3, 1,2 Hz 1H), 6.80–6.77
m, 2H), 6.29 (d, J = 9.5 Hz, 1H), 4.00 (t, J = 6.5 Hz, 2H), 3.05–2.98 (m, 1H), 2.75–2.65 (m, 1H), 2.26–2.12
m, 1H), 2.03–1.94 (m, 2H), 1.87–1.74 (m, 3H), 1.64–1.58 (m, 2H), 1.51–1.40 (m, 4H), 1.29–1.25 (m, 1H),
1
.00 (d, J = 6.0 Hz, 3H). LC/MS m/z 315.2 (M + H), t = 0.49 min.
R
7-[(1-Methylpiperidin-4-yl)oxy]quinolin-2(1H)-one (QN19)
.
A mixture of 7-hydroxyquinolin-
2(1H)-one (80.6 mg, 0.5 mmol), 1-methylpiperidin-4-ol (172.8 mg, 1.5 mmol), and
cyanomethylenetributylphosphorane (362.0 mg, 1.5 mmol) in toluene (5 mL, 47.3 mmol) was heated
◦
at 100 C for 16 h. The reaction mixture was diluted with methanol and loaded into 10 g SCX resin.
Resin was washed with methanol, and crude product was eluted with 2N ammonia in methanol before
evaporation of the solvent. The residue was purified by column chromatography (CH Cl /MeOH
2
2
1
(
from 1% to 5%)) to give compound QN19 (46.1 mg, 30% yield). H-NMR (400 MHz, DMSO-d ) δ:
6
1
1
1
1.49 (s, 1H), 7.80 (d, J = 9.5 Hz, 1H), 7.55 (dd, J= 8.3, 1,3 Hz, 1H), 6.82–6.79 (m, 2H), 6.30 (d, J = 9.5 Hz,
H), 4.38 (tt, J = 8.2, 4.0 Hz, 1H), 3.17 (d, J = 5.2 Hz, 1H), 2.62–2.58 (m, 2H), 2.19 (s, 3H), 2.16–2.12 (m,
H), 1.98–1.95 (m, 2H), 1.71–1.62 (m, 2H). LC/MS m/z 259.2 (M + H), t = 0.67 min.
R
3
.1.3. General Procedures for the Synthesis of DihydroQuinolinones (DQNs)
a) General Procedure via Mitsunobu reaction: mixture of 7-hydroxy-3,4-
(
A
A
dihydroquinolin-2(1H)-one (0.5 mmol), the appropriate aminoalcohol (0.5 mmol), and
◦
cyanomethylenetributylphosphorane (0.7 mmol) in toluene (5 mL) was heated at 100 C for 16 h.
The reaction mixture was diluted with methanol and loaded into 10 g SCX resin. Resin was washed
with methanol, and crude product was eluted in 2N ammonia in methanol before evaporation of the
solvent. The residue was purified by column chromatography (CH Cl /MeOH (from 1% to 5%)).
2
2
(
b) General Procedure B via O-Alkylation: Step 1. A mixture of potassium carbonate (2.5 mmol),
7
-hydroxy-3,4-dihydroquinolin-2(1H)-one (0.5 mmol), and the corresponding 1,n-dibromoalkane
◦
(1.0 mmol) in acetonitrile (5 mL) was heated at 50 C for 20 h. After cooling, acetonitrile (10 mL)
was added, the inorganic salts were filtered off, and the solvent was evaporated. Step 2. To a stirred
solution of the crude intermediate in acetonitrile (5 mL), we added the appropriate amine (2.0 mmol).

Int. J. Mol. Sci. 2020, 21, 3913
14 of 20
◦
After stirring at 50 C for 15 h, we diluted the reaction mixture with methanol and loaded it into 10 g
SCX resin. Resin was washed with methanol, and crude product was eluted with 2N ammonia in
methanol before evaporation of the solvent. The residue was purified by column chromatography
[
CH Cl /MeOH (from 1% to 5%)].
2
2
(
c) General Procedure C via O-Alkylation: Step 1. A mixture of potassium carbonate (2.5 mmol),
7
-hydroxy-3,4-dihydroquinolin-2(1H)-one (0.5 mmol), and the corresponding 1,n-dibromoalkane
◦
(1.0 mmol) in acetonitrile (5 mL) was heated at 50 C for 20 h. After cooling, acetonitrile (10 mL)
was added, the inorganic salts were filtered off, and the solvent was evaporated. Step 2. To a stirred
solution of the crude intermediate in acetonitrile (5 mL), we added the appropriate amine (0.5 mmol).
◦
After stirring at 50 C for 15 h, we diluted the reaction mixture with methanol and loaded it into 10 g
SCX resin. Resin was washed with methanol, and crude product was eluted with 2N ammonia in
methanol before evaporation of the solvent. The residue was purified by column chromatography
(
CH Cl /MeOH (from 1% to 5%)).
2
2
7-(2-Morpholinoethoxy)-3,4-dihydroquinolin-2(1H)-one (DQN1). Following General Procedure A,
7
0
4
-hydroxy-3,4-dihydroquinolin-2(1H)-one (81.6 mg, 0.5 mmol) and 2-morpholinoethan-1-ol (65.6 mg,
.5 mmol) were reacted with cyanomethylenetributylphosphorane (169 mg, 0.7 mmol) in toluene (5 mL,
1
7.3 mmol) to give compound DQN1 (110.47 mg, 80% yield). H-NMR (400 MHz, DMSO-d )
δ: 9.97 (s,
6
1
3
H), 7.05 (d, J = 8.2 Hz, 1H), 6.50 (dd, J = 8.2, 2.6 Hz, 1H), 6.43 (d, J = 2.6 Hz, 1H), 4.00 (t, J = 5.7 Hz, 2H),
.59–3.57 (m, 4H), 2.80–2.77 (m, 2H), 2.68–2.65 (m, 2H), 2.47–2.40 (m, 6H). LC/MS m/z 277.2 (M + H),
tR = 0.63 min.
7-[2-(4-Methylpiperazin-1-yl)ethoxy]-3,4-dihydroquinolin-2(1H)-one (DQN2). Following General
Procedure A, 7-hydroxy-3,4-dihydroquinolin-2(1H)-one (81.6 mg, 0.5 mmol) and 2-(4-methylpiperazin-
1
0
-yl)ethan-1-ol (72.1 mg, 0.5 mmol) were reacted with cyanomethylenetributylphosphorane (169 mg,
.7 mmol) in toluene (5 mL, 47.3 mmol) to give compound DQN2 (119.38 mg, 80% yield). H-NMR
1
(
400 MHz, DMSO-d6)
J = 2.6 Hz, 1H), 3.99 (t, J = 5.8 Hz, 2H), 2.78 (t, J = 7.5 Hz, 2H), 2.65 (t, J = 5.8 Hz, 2H), 2.52–2.48 (m, 4H),
.43–2.40 (m, 4H), 2.31–2.25 (m, 2H), 2.15 (s, 3H). LC/MS m/z 290.2 (M + H), t = 0.60 min.
δ: 9.97 (s, 1H), 7.05 (d, J = 8.3 Hz, 1H), 6.50 (dd, J = 8.3, 2.6 Hz, 1H), 6.43 (d,
2
R
7
-[4-(4-Methylpiperazin-1-yl)butoxy]-3,4-dihydroquinolin-2(1H)-one (DQN3). Following General
Procedure C, the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol), 7-hydroxy-
,4-dihydroquinolin-2(1H)-one (81.6 mg, 0.5 mmol), and 1,4-dibromobutane (216 mg, 1.0 mmol) in
3
acetonitrile (5 mL) afforded a crude intermediate that was suspended in acetonitrile (5 mL), and
treated with N-methylpiperazine (50.08 mg, 0.5 mmol) to give compound DQN3 (41.70 mg, 30% yield).
1
H-NMR (400 MHz, DMSO-d6) δ: 9.97 (s, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.48 (dd, J = 8.3, 2.6 Hz, 1H), 6.43
(
8
d, J = 2.6 Hz, 1H), 3.90 (t, J = 6.4 Hz, 2H), 2.78 (dd, J = 8.5, 6.5 Hz, 2H), 2.43–2.39 (m, 4H), 2.34–2.24 (m,
H), 2.14 (s, 3H), 1.75–1.63 (m, 2H), 1.59–1.47 (m, 2H). LC/MS m/z 318.2 (M + H), t = 0.72 min.
R
7
-[5-(4-Methylpiperazin-1-yl)pentyloxy]-3,4-dihydroquinolin-2(1H)-one (DQN4). Following General
Procedure C, the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol), 7-hydroxy-
,4-dihydroquinolin-2(1H)-one (81.6 mg, 0.5 mmol), and 1,5-dibromopentane (230 mg, 1.0 mmol)
3
in acetonitrile (5 mL) afforded a crude intermediate that was suspended in acetonitrile (5 mL), and
treated with N-methylpiperazine (50.08 mg, 0.5 mmol) to give compound DQN4 (43.22 mg, 30% yield).
1
H-NMR (400 MHz, DMSO-d6) δ: 9.97 (s, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.48 (dd, J = 8.3, 2.6 Hz, 1H),
6
.43 (d, J = 2.6 Hz, 1H), 3.88 (t, J = 6.5 Hz, 2H), 2.80–2.76 (m, 2H), 2.43–2.39 (m, 5H), 2.38–2.19 (m, 7H),
2
.14 (s, 3H), 1.69–1.65 (m, 2H), 1.52–1.31 (m, 4H). LC/MS m/z 332.2 (M + H), t = 0.79 min.
R
7-[2-(4-Isopropylpiperazin-1-yl)ethoxy]-3,4-dihydroquinolin-2(1H)-one
(DQN5)
.
Following
General Procedure A, 7-hydroxy-3,4-dihydroquinolin-2(1H)-one (81.6 mg, 0.5 mmol)
and 2-(4-isopropylpiperazin-1-yl)ethan-1-ol (63.6 mg, 0.5 mmol) were reacted with
cyanomethylenetributylphosphorane (169 mg, 0.7 mmol) in toluene (5 mL) to give compound DQN5
1
(
46.62 mg, 30% yield). H-NMR (400 MHz, DMSO-d )
δ
: 9.97 (s, 1H), 7.05 (d, J = 8.3 Hz, 1H), 6.50 (dd,
6
J = 8.3, 2.6 Hz, 1H), 6.43 (d, J = 2.6 Hz, 1H), 3.98 (t, J = 5.8 Hz, 2H), 2.78 (t, J = 7.5 Hz, 2H), 2.65–2.59 (m,
H), 2.47–2.37 (m, 9H), 0.96 (d, J = 6.5 Hz, 6H). LC/MS m/z 318.2 (M + H), t = 0.72 min.
4
R

Int. J. Mol. Sci. 2020, 21, 3913
15 of 20
7
-[3-(4-Isopropylpiperazin-1-yl)propoxy]-3,4-dihydroquinolin-2(1H)-one (DQN6). Following General
Procedure C, the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol),
-hydroxy-3,4-dihydroquinolin-2(1H)-one (81.6 mg, 0.5 mmol), and 1,3-dibromopropane (202 mg,
.0 mmol) in acetonitrile (5 mL) afforded a crude intermediate that was suspended in acetonitrile (5 mL),
7
1
and treated with 1-isopropylpiperazine (64.1 mg, 0.5 mmol) to give compound DQN6 (31.79 mg, 20%
1
yield). H-NMR (400 MHz, DMSO-d )
δ: 9.96 (br s, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.48 (dd, J = 8.2,
6
2
2
.6 Hz, 1H), 6.43 (d, J = 2.6 Hz, 1H), 3.91 (t, J = 6.4 Hz, 2H), 2.78 (dd, J = 8.5, 6.5 Hz, 2H), 2.62–2.55 (m,
H), 2.49–2.40 (m, 6H), 2.40–2.31 (m, 5H), 1.86–1.80 (m, 2H), 0.96 (d, J = 6.5 Hz, 6H). LC/MS m/z 332.2
(
M + H), t = 0.78 min.
R
7
-[4-(4-Isopropylpiperazin-1-yl)butoxy]-3,4-dihydroquinolin-2(1H)-one (DQN7). Following General
Procedure C, the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol),
-hydroxy-3,4-dihydroquinolin-2(1H)-one (81.6 mg, 0.5 mmol), and 1,4-dibromobutane (216 mg,
.0 mmol) in acetonitrile (5 mL) afforded a crude intermediate that was suspended in acetonitrile (5 mL),
7
1
and treated with 1-isopropylpiperazine (64.1 mg, 0.5 mmol) to give compound DQN7 (58.01 mg, 30%
1
yield). H-NMR (400 MHz, DMSO-d )
δ
: 9.97 (s, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.48 (dd, J = 8.2, 2.6 Hz,
6
1
(
3
H), 6.43 (d, J = 2.6 Hz, 1H), 3.90 (t, J = 6.4 Hz, 2H), 2.78 (t, J = 7.5 Hz, 2H), 2.59–2.54 (m, 3H), 2.46–2.37
m, 6H), 2.36–2.23 (m, 4H), 1.71–1.65 (m, 2H), 1.55–1.51 (m, 2H), 0.95 (d, J = 6.5 Hz, 6H). LC/MS m/z
46.2 (M + H), t = 0.83 min.
R
7
-[5-(4-Isopropylpiperazin-1-yl]pentyloxy)-3,4-dihydroquinolin-2(1H)-one (DQN8). Following General
Procedure C, the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol),
-hydroxy-3,4-dihydroquinolin-2(1H)-one (81.6 mg, 0.5 mmol), and 1,4-dibromopentane (230 mg,
.0 mmol) in acetonitrile (5 mL) afforded a crude intermediate that was suspended in acetonitrile (5 mL),
7
1
and treated with 1-isopropylpiperazine (64.1 mg, 0.5 mmol) to give compound DQN8 (50.70 mg, 30%
1
yield). H-NMR (400 MHz, DMSO-d )
δ
: 9.97 (s, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.48 (dd, J = 8.2, 2.6 Hz,
6
1
(
6
H), 6.43 (d, J = 2.6 Hz, 1H), 3.88 (t, J = 6.5 Hz, 2H), 2.78 (t, J = 7.5 Hz, 2H), 2.60–2.54 (m, 4H), 2.47–2.40
m, 5H), 2.34–2.32 (m, 2H), 2.26–2.22 (m, 2H), 1.73–1.66 (m, 2H), 1.50–1.34 (m, 4H), 0.95 (d, J = 6.5 Hz,
H). LC/MS m/z 360.2 (M + H), t = 0.90 min.
R
7
-[2-(Diethylamino)ethoxy]-3,4-dihydroquinolin-2(1H)-one (DQN9). Following General Procedure
A, 7-hydroxy-3,4-dihydroquinolin-2(1H)-one (81.6 mg, 0.5 mmol) and 2-(diethylamino)ethan-1-ol
65.6 mg, 0.5 mmol) were reacted with cyanomethylenetributylphosphorane (169 mg, 0.7 mmol) in
toluene (5 mL, 47.3 mmol) to give compound DQN9 (97.32 mg, 70% yield). 1H-NMR (400 MHz,
DMSO-d6) : 9.96 (s, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.48 (dd, J = 8.2, 2.6 Hz, 1H), 6.43 (d, J = 2.6 Hz,
H), 3.93 (t, J = 6.2 Hz, 2H), 2.76 (q, J = 7.1 Hz, 4H), 2.59–2.50 (m, 4H), 2.42 (t, J = 6.2 Hz, 2H), 0.97 (t,
J = 7.1 Hz, 6H). LC/MS m/z 263.2 (M + H), t = 0.82 min.
(
δ
1
R
7-[3-(Diethylamino)propoxy]-3,4-dihydroquinolin-2(1H)-one (DQN10). Following General Procedure
B, the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol), 7-hydroxy-3,4-
dihydroquinolin-2(1H)-one (81.6 mg, 0.5 mmol), and 1,3-dibromopropane (202 mg, 1.0 mmol) in
acetonitrile (5 mL) afforded a crude intermediate that was suspended in acetonitrile (5 mL), and treated
1
with diethylamine (146.28 mg, 2.0 mmol) to give compound DQN10 (63.15 mg, 50% yield). H-NMR
(
400 MHz, DMSO-d6)
J = 2.6 Hz, 1H), 3.92 (t, J = 6.3 Hz, 2H), 2.78 (dd, J = 8.4, 6.5 Hz, 2H), 2.50–2.37 (m, 8H), 1.81–1.74 (m,
H), 0.94 (t, J = 7.1 Hz, 6H). LC/MS m/z 277.2 (M + H), t = 0.84 min.
δ: 9.96 (s, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.48 (dd, J = 8.2, 2.6 Hz, 1H), 6.43 (d,
2
R
7-[4-(Diethylamino)butoxy]-3,4-dihydroquinolin-2(1H)-one (DQN11). Following General Procedure
B, the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol), 7-hydroxy-3,4-
dihydroquinolin-2(1H)-one (81.6 mg, 0.5 mmol), and 1,3-dibromobutane (216 mg, 1.0 mmol) in
acetonitrile (5 mL) afforded a crude intermediate that was suspended in acetonitrile (5 mL), and treated
1
with diethylamine (146.28 mg, 2.0 mmol) to give compound DQN11 (80.66 mg, 60% yield). H-NMR
(
400 MHz, DMSO-d6)
J = 2.6 Hz, 1H), 3.90 (t, J = 6.5 Hz, 2H), 2.78 (t, J = 7.5 Hz, 2H), 2.47–2.37 (m, 8H), 1.72–1.65 (m, 2H),
.53–1.46 (m, 2H), 0.95 (t, J = 7.1 Hz, 6H). LC/MS m/z 291.2 (M + H), t = 0.87 min.
δ: 9.97 (s, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.48 (dd, J = 8.2, 2.6 Hz, 1H), 6.43 (d,
1
R

Int. J. Mol. Sci. 2020, 21, 3913
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7
-[5-(Diethylamino)pentyloxy]-3,4-dihydroquinolin-2(1H)-one (DQN12). Following General Procedure
B, the reaction of a mixture of potassium carbonate (345.5 mg, 2.5 mmol), 7-hydroxy-3,4-
dihydroquinolin-2(1H)-one (81.6 mg, 0.5 mmol), and 1,3-dibromopentane (230 mg, 1.0 mmol) in
acetonitrile (5 mL) afforded a crude intermediate that was suspended in acetonitrile (5 mL) and treated
1
with diethylamine (146.28 mg, 2.0 mmol) to give compound DQN12 (76.63 mg, 50% yield). H-NMR
(400 MHz, DMSO-d6)
δ: 9.97 (s, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.48 (dd, J = 8.2, 2.6 Hz, 1H), 6.43 (d,
J = 2.6 Hz, 1H), 3.88 (t, J = 6.5 Hz, 2H), 2.78 (t, J = 7.5 Hz, 2H), 2.48–2.37 (m, 6H), 2.35 (t, J = 6.6 Hz, 2H),
1
.73–1.66 (m, 2H), 1.44–1.38 (m, 4H), 0.94 (t, J = 7.1 Hz, 6H). LC/MS m/z 305.2 (M + H), t = 0.95 min.
R
7-[(1-Methylpiperidin-4-yl)oxy]-3,4-dihydroquinolin-2(1H)-one (DQN13). A mixture of 7-hydroxy-
3
,4-dihydroquinolin-2(1H)-one (81.6 mg, 0.5 mmol), 1-methylpiperidin-4-ol (172.8 mg, 1.5 mmol), and
cyanomethylenetributylphosphorane (362.0 mg, 1.5 mmol) in toluene (5 mL, 47.3 mmol) was heated at
◦
1
00 C for 16 h. The reaction mixture was diluted with methanol, loaded into 10 g SCX resin. Resin was
washed with methanol, and crude product was eluted in 2N ammonia in methanol before evaporation
of the solvent. The residue was purified by column chromatography (CH Cl /MeOH (from 1% to 5%))
2
2
1
to give compound DQN13 (87.79 mg, 70% yield). H-NMR (400 MHz, DMSO-d ) δ: 9.93 (s, 1H), 7.04
6
(
d, J = 8.3 Hz, 1H), 6.50 (dd, J = 8.3, 2.6 Hz, 1H), 6.44 (d, J = 2.6 Hz, 1H), 4.24-4.21 (m, 1H), 2.78 (t,
J = 7.5 Hz, 2H), 2.61–2.57 (m, 2H), 2.46–2.40 (m, 2H), 2.20 (s, 3H), 2.17–2.09 (m, 2H), 1.92–1.87 (m, 2H),
.65–1.58 (m, 2H). LC/MS m/z 261.2 (M + H), t = 0.70 min.
1
R
3
.2. Biological Evaluation
3
.2.1. Human MAO Activity Assay
The hrMAO-A and hrMAO-B enzymes were purchased from Sigma-Aldrich (St. Louis, MO,
USA). The reaction mixture contained hrMAO-A (2.5
6.25 g/mL protein final concentration) enzyme and tested compound in final concentration of 1
and 10
µg/mL protein final concentration) or hrMAO-B
(
µ
µ
M in 50 mM potassium phosphate buffer with 20% (v/v) glycerol (pH 7.5). The mixture
◦
was pre-incubated at 37 C for 5 min and subsequently substrate kynuramine was added to the
final concentration of 60
µ
M in the case of hrMAO-A and 30
µ
M in the case of hrMAO-B. The final
◦
volume of reaction mixture was 0.1 mL. The whole reaction mixture was incubated at 37 C for 30 min.
The reaction was stopped by the addition of 200
down to 0 C. The sample was then centrifuged (16,500
µ
L acetonitrile/methanol mixture (ratio 1:1) and cooling
◦
×
g) for 10 min. The deamination product of
kynuramine formed during the enzymatic reaction 4-hydroxyquinoline (4-HQ) was determined by
HPLC–MS on a 2.1 mm 50 mm, 1.8 m Zorbax RRHD Eclipse plus C18 column (Agilent) by using
×
µ
a 6470 Series Triple Quadrupole mass spectrometer (Agilent) (electrospray ionisation—positive ion
mode). Three MRM transitions were followed for kynuramine (165.1 => 30.2, 165.1 => 118.0, 165.1 =>
136.0) and 4-HQ (146.1 => 51.1, 146.1 => 77.0, 146.1 => 91.0). Eluents: (A) 0.1% formic acid in water;
(B) 0.1% formic acid in acetonitrile.
3
.2.2. Human Esterase Activity Assay
Inhibition Efficiency Screening and Determination of IC50
The hrAChE and hrBuChE were prepared as recombinant proteins at the University of Hradec
Kralove. For their production, the mammalian expression system was used. Briefly, the DNA sequence
encoding human AChE and BuChE was obtained from UniProtKB Server (www.uniprot.org, accession
numbers: P22303 and P06276) and de novo synthesized as GeneArt Strings DNA fragments by
GeneArt Gene Synthesis Service (Thermo Fisher Scientific, Pardubice, Czech Republic). The DNA
fragments were PCR-amplified using gene-specific primers, adding the DNA sequence for C-terminal
6 His-tag. The amplicons were inserted into the mammalian pcDNA3.4 vector by TOPO cloning
×
technology, and the final DNA constructs were verified by Sanger sequencing (ABI PRISM 3130xl).
For protein expression, the DNA constructs were transiently transfected into Hek293 derivatives.

Int. J. Mol. Sci. 2020, 21, 3913
17 of 20
◦
Recombinant proteins were collected from culture supernatant 6 days later and stored at
further purification.
−
80 C for
The hrAChE and hrBuChE were purified using a NGC Medium-Pressure Chromatography System
(Bio-Rad, USA) [25]. The total volume of 6–8 mL of medium containing secreted protein was desalted
using 5 mL HiTrap Desalting column (GE Healthcare, Prague, Czech Republic) equilibrated with
buffer A (20 mM sodium phosphate buffer, 150 mM NaCl, 15 mM imidazole, and 20% glycerol;
pH 7.4). Acquired supernatant was loaded onto a 1 mL HisTrap FF column (GE Healthcare, Prague,
Czech Republic) equilibrated with buffer A. Captured proteins were eluted with buffer B (20 mM
sodium phosphate buffer, 150 mM NaCl, 500 mM imidazole, and 20% glycerol; pH 7.4). Imidazole
was subsequently removed by repeated centrifugation in Amicon Ultra-4 (Ultracel-10K) tube (Merck
Millipore). Protein concentration was determined by linearized Bradford method adapted for
9
6-well plate.
The catalytic activity of enzymes was determined by standard Ellman method [26] adapted for
6-well plates. The reaction mixture contained hrAChE (70 ng/mL protein final concentration) or
9
hrBuChE (220 ng/mL protein), tested compound at required concentration (varying from 0.1
µ
M up to
0
8
7
0
µ
M), and 500
µ
M 5,5 -dithiobis-2-nitrobenzoic acid (DTNB) in 20 mM sodium phosphate buffer (pH
◦
.4). The mixture was pre-incubated at 37 C for 15 min, and subsequently substrate acetylthiocholine
M.
L. The product formed during the reaction 5-thio-2-nitrobenzoic
iodide (ATChI) or butyrylthiocholine iodide (BuTChI) was added to the final concentration of 1000
The final volume of reaction was 100
µ
µ
acid (TNB) was determined by following its absorbance at 436 nm. The catalytic activity was evaluated
◦
as amount of product (%) formed by enzyme after 10 min of incubation at 37 C. IC50 values of
individual compounds were determined by non-linear regression using GraphPad Prism 7.
3
.2.3. Determination of Inhibition Kinetics
Compound with the highest inhibition potential against hrAChE was further analysed regarding
its inhibition kinetics parameters (inhibition mechanism and inhibitory constant). Thus, esterase
activity assay was carried out at various concentrations of substrate ATChI (ranging from 25 M to
000 M) and various concentrations of tested compound (0.1 M, 0.3 M, and 1 M). Inhibition
µ
2
µ
µ
µ
µ
mechanism and kinetic constant were determined by non-linear regression and double reciprocal
method by Lineweaver-Burk [27] using GraphPad Prism 7.
3
.3. Docking Analysis
Compound QN8 was prepared using “build” option within Discovery Studio 2.1 to create
three-dimensional geometry, and assign proper bond orders and ionization states prior to virtual
screening. Protein crystal structure of hAChE (PDB ID: 1B41) was prepared prior to docking, using
protein model tool in Discovery Studio in order to add hydrogen atoms and to assign proper bonds,
bond orders, hybridization, and charges. In addition, cocrystal ligands and water molecules were
removed. AutoDockTools (ADT; version 1.5.4) was used to add hydrogens and partial charges for
proteins and ligands using Gasteiger charges. To give flexibility to the binding site, residues lining the
AChE (Trp286, Tyr124, Tyr337, Tyr341, Tyr72, Asp74, Thr75, Trp86) were allowed to move during the
docking search as performed by software AutoDock Vina 1.1.2 [29]. The ADT program was used to
generate the docking input files. Docking calculations were performed with the program AutoDock
Vina 1.1.2 and default parameters were used except num modes, which was set to 40. The grid box
was built with a resolution of 1 Å and 60
protein (x = 116.546; y = 110.33; z = 134.181). The best Vina scored poses were considered and the
docked ligand output files were viewed and analysed using Discovery Studio.
×
60
×
72 points, and it was positioned at the middle of the
−

Int. J. Mol. Sci. 2020, 21, 3913
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4
. Conclusions
In this work, we described the synthesis of 19 quinolinones (QN1
DQN1
synthesized by Mitsunobu or O-alkylation protocols starting from 7-hydroxyquinolin-2(1H)-one
) (Scheme 1) and 7-hydroxy-3,4-dihydroquinolin-2(1H)-one ( ) (Scheme 2), respectively, and suitably
functionalized precursors.
-
19) and 13 dihydroquinolinones
(
-13) designed as potential MSM for AD therapy. QN and DQN ligands were easily
(1
2
The biological analysis of these compounds on selected targets (hrChEs/hrMAOs) involved in
the pathology of the disease produced interesting results. Thus, contrary to our expectations, none
of them showed significant hrMAO inhibition. However, molecules QN8
promising hrAChE and hrBuChE inhibition. In particular, molecule QN8 resulted as a potent and
selective non-competitive hrAChE inhibitor (IC50 = 0.29 M), with K value in the nanomolar range
79 nM).
Pertinent docking analysis of hit-compound QN8 confirmed the observed kinetic and hrChE
, QN9, and DQN7 showed
µ
i
(
inhibition results, suggesting that this ligand is an interesting hit for further investigation. In addition,
theoretical physico-chemical property analysis of QN8 confirmed that this is an attractive ligand for
deeper investigation in the search of more efficient molecules for AD therapy.
Author Contributions: J.M.-C. designed the project; K.M. supervised the biological analysis; C.D.B.,
M.C., R.A., M.S., and P.B. performed the synthetic and biological studies; I.I. carried out the computational analysis;
L.I., F.L.-M., and B.R. analysed the data; M. .M.-G. and T.V. supervised the project; K.M. and J.M.-C. organised
the draft manuscript; M.
Ó.M.B.-A.,
Á
Á
.M.-G. corrected it for its final version; and all the authors participated in reviewing and
editing the manuscript. All authors read and approved the final manuscript.
Funding: R.A., M.S., P.B., and K.M. were supported by European Regional Development Fund/European Social
Fund (ERDF/ESF, project PharmaBrain, no. CZ.02.1.01/0.0/0.0/16_025/0007444), University of Hradec Kralove
(
no. SV2113-2019, VT2019-2021), and EU COST action CA15135 MuTaLig. J.M.C. thanks Ministerio de Econom
ía
(
MINECO, SAF2015-65586-R) and Universidad Camilo Jos Cela (UCJC, grants UCJC 2020-03, and UCJC 2020-33)
é
for support.
Conflicts of Interest: The authors declare no conflict of interest.
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