Chasing ChEs-MAO B Multi-Targeting 4-Aminomethyl-7-Benzyloxy-2H-Chromen-2-ones

Article abstract of DOI:10.3390/molecules24244507

A series of 4-aminomethyl-7-benzyloxy-2H-chromen-2-ones was investigated with the aim of identifying multiple inhibitors of cholinesterases (acetyl- and butyryl-, AChE and BChE) and monoamine oxidase B (MAO B) as potential anti-Alzheimer molecules. Starting from a previously reported potent MAO B inhibitor (3), we studied single-point modifications at the benzyloxy or at the basic moiety. The in vitro screening highlighted triple-acting compounds (6, 8, 9, 16, 20) showing nanomolar and selective MAO B inhibition along with IC₅₀ against ChEs at the low micromolar level. Enzyme kinetics analysis toward AChE and docking simulations on the target enzymes were run in order to get insight into the mechanism of action and plausible binding modes.

Full text of DOI:10.3390/molecules24244507

Article
Chasing ChEs-MAO B Multi-Targeting
4-Aminomethyl-7-Benzyloxy-2H-Chromen-2-ones
Mariagrazia Rullo, Marco Catto, Antonio Carrieri, Modesto de Candia,
Cosimo Damiano Altomare and Leonardo Pisani *
Department of Pharmacy – Drug Sciences, University of Bari “Aldo Moro”, via E. Orabona 4, 70125 Bari, Italy;
mariagrazia.rullo@uniba.it (M.R.); marco.catto@uniba.it (M.C.); antonio.carrieri@uniba.it (A.C.);
modesto.decandia@uniba.it (M.d.C.); cosimodamiano.altomare@uniba.it (C.D.A.)
* Correspondence: leonardo.pisani@uniba.it; Tel.: +39-080-544-2803
Received: 14 November 2019; Accepted: 05 December 2019; Published: 9 December 2019
Abstract: A series of 4-aminomethyl-7-benzyloxy-2H-chromen-2-ones was investigated with the aim of
identifying multiple inhibitors of cholinesterases (acetyl- and butyryl-, AChE and BChE) and
monoamine oxidase B (MAO B) as potential anti-Alzheimer molecules. Starting from a previously
reported potent MAO B inhibitor (3), we studied single-point modifications at the benzyloxy or at the
basic moiety. The in vitro screening highlighted triple-acting compounds (6, 8, 9, 16, 20) showing
nanomolar and selective MAO B inhibition along with IC50 against ChEs at the low micromolar level.
Enzyme kinetics analysis toward AChE and docking simulations on the target enzymes were run in
order to get insight into the mechanism of action and plausible binding modes.
Keywords: acetylcholinesterase inhibitors; butyrylcholinesterase inhibitors; monoamine oxidase B
inhibitors; multi-target-directed ligands; coumarin derivatives; structure–activity relationships;
docking simulations
1. Introduction
Alzheimer’s disease (AD), the main cause of dementia, represents the sixth leading cause of
death in US, killing more patients than breast and prostate cancer combined [1]. Nowadays, most
AD cases are distributed to higher-income countries, but projections indicate that a prevalence
increase will take place in both low- and middle-income countries in the next two decades [2]. Even
if more than one century has passed from the seminal observation made by Alois Alzheimer, the
great efforts devoted to the understanding of AD aetiology are still not comprehensive [3]. As a
matter of fact, all the approved drugs exert palliative benefits by attenuating the cognitive symptoms
(e.g., memory loss) in mild cases, but they lose efficacy in the late stages of the disease when the
progressive loss of neurons results ultimately fatal [4]. Since the neuronal impairment is initially
restricted to cholinergic areas, with the exception of memantine, three out of the four marketed
drugs, as seen in Chart 1, aim at restoring basal neurotransmitter levels, namely acetylcholine (ACh),
by means of inhibiting acetylcholinesterase (AChE) [5]. More recently, for mild-to-severe AD cases,
the FDA approved the first drug combination that includes two active ingredients with different
mechanisms of action, that is donepezil (AChE inhibitor) and memantine (glutamate-receptor
blocker) [6]. This polypharmacological approach strengthens the idea that multifactorial
neurodegenerative disorders (NDs), including AD, might be more efficiently handled with
therapeutic protocols able to modulate two or more etiopathological mechanisms. In fact, NDs share
the multifactorial nature of cardiovascular and tumor diseases, which are commonly treated with
drug cocktails or combinations. The challenging goal of disclosing a single molecular tool endowed
Molecules 2019, 24, 4507; doi:10.3390/molecules24244507
www.mdpi.com/journal/molecules

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with multiple disease-related pharmacological activities represents the spirit of the so-called
multi-target strategy [7].
Chart 1. Drugs and combinations approved for the treatment of Alzheimer’s disease (AD).
In this study, we aimed at discovering novel multipotent anti-AD pharmacological tools
through the identification of multimodal compounds capable of counteracting cholinergic depletion
and oxidative stress conditions, by inhibiting ChEs and monoamine oxidase B (MAO B), as part of
our long-standing project. Neuroinflammation, a typical condition of AD brains, is, among others,
sustained by MAO B, whose upregulation in reactive astrocytes results in aberrant GABA levels [8]
and reactive oxygen species (ROS) production through the catalytic deamination of the starting
amine substrate into an aldehyde metabolite and hydrogen peroxide as the byproducts [9]. While
searching for new anti-Parkinson agents, we previously reported compound 3 (alias NW-1772,
compound 22b in ref. [10]) that behaved as a highly selective (SI, IC50 MAO A/IC50 MAO B = 457) and
in vivo potent rat MAO B inhibitor, with affinity in the nanomolar range (IC50 rat MAO B = 13 nM),
rapid blood–brain barrier penetration, and poor CYP450s liability. The X-ray crystal structure of 3 in
complex with hMAO B showed a binding mode in the aromatic cavity superimposed to that of
safinamide (Chart 1) [11], recently marketed as an anti-Parkinson drug in several EU countries. The
crystallographic pose within the enzymatic cleft showed the protonatable substituent placed close to
FAD coenzyme in the catalytic site whereas the benzyloxy group is anchored to the entrance cavity.
As a follow-up of our research on small-molecule agents for treating AD [12], we have recently
focused on single- [13] and multi-targeting coumarin derivatives [14]. The coumarin
(2H-chromen-2-one) nucleus represents a nature-friendly, easy-to-handle scaffold that has been
widely explored to hit several targets depending on the chemical decoration around the heterocyclic
skeleton, including antimicrobial [15], anticoagulant [16], antiproliferative [17], antiangiogenic [18],
and anti-HIV agents [19], among others. We found that compound 3 retained single-digit nanomolar
inhibitory activity and great selectivity toward human MAO B (IC50 human MAO B = 3.9 nM, SI =
167) and it was endowed with moderate affinity toward AChE (IC50 = 6.2 µM) and lower inhibitory
potency toward BChE (43% inhibition at 10 µM). Encouraged from in vitro data toward ChEs,
herein, the approach to get novel multipotent molecules started from hit 3 by introducing head
(substituent at position 4 of coumarin, coloured in red in Figure 1) or tail modifications (benzyloxy
group, blue-coloured in Figure 1) at the 4-aminomethyl-7-benzyloxycoumarin core, rooted on X-ray
complex analysis. In the first case, differently sized basic moieties were attached at C4 of the
coumarin core while maintaining the 3′-chlorobenzyloxy group at C7. In the second subset, the
unhindered C4-substituent (namely the methylaminomethyl group) was kept untouched and a
number of different substituents were introduced around the phenyl ring belonging to the

Molecules 2019, 24, 4507
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benzyloxy tail. To widen the structure–activity relationships, small groups (e.g., Me, Cl) were
inserted at positions 6 and 8 of the parent skeleton of 3. All compounds were screened toward
human MAOs and ChEs (AChE from electric eel and BChE from equine serum). In this regard, MAO
B selectivity was pursued to avoid safety issues related to the so-called “cheese-effect” whereas, on
the other side, AChE/BChE selectivity was not deemed critical since increasing lines of evidence
supported the involvement of BChE in chronic phases of AD thus claiming for BChE as a promising
therapeutic target [20].
Figure 1. Structural modifications of starting hit NW-1772 (3).
2. Results
2.1. Chemistry
Scheme 1 illustrates the synthetic route to obtain the final coumarin derivatives 3–27. Von
Pechmann cyclization on substituted 1,3-dihydroxybenzenes afforded the appropriate
4-chloromethyl-7-hydroxy coumarin 1a–d, which was alkylated with the suitable benzyl bromides
before reacting with primary or secondary amines to yield desired final compounds 3, 6–27. A
different route was followed to prepare coumarin 4–5 through intermediates 2e and 2g, requiring
the alkylation of 1a through a Mitsunobu etherification with commercially available piperonyl
alcohol or 1e, which was in turn obtained from borane-promoted reduction of
3-(dimethylamino)benzoic acid.
Scheme 1. Reagents and conditions: a) ethyl 4-chloroacetoacetate, conc. H
2
SO , 0 °C for 1–2 h (for 1a,
4
1c) or room temperature for 24 h (for 1b, 1d); b) suitable benzyl bromide, N,N-diisopropylethylamine
(DIEA), ethanol, 1.5–5 h, Δ; c) appropriate amine, dry tetrahydrofuran (THF) or dimethylformamide
(DMF),
DIEA
or
K2CO
3
,
4–72
h,
room
temperature
or
80
°C;
d)
4-(chloromethyl)-7-hydroxy-2H-chromen-2-one (1a), diisopropyl azodicarboxylate (DIAD), PPh ,
3
dry THF, 18 h, room temperature.

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2.2. In Vitro Screening
To identify novel multipotent inhibitors, the in vitro screening program started from evaluating
some congeners of 3 recruited from an in-house library of coumarin derivatives (6–9, 13–15, 17–21),
previously tested as rat MAOs inhibitors [10,21]. Moreover, newly synthesized compounds (4–5, 10–
12, 16, 22–27) were screened toward the target enzymes (MAOs and ChEs) in order to derive
structure–activity
relationships
focused
on
the
multi-targeting
features
of
4-aminomethyl-7-benzyloxy-2H-chromen-2-ones. In vitro enzymatic screening toward ChEs was
carried out according to the known Ellman’s protocol [22], by using eeAChE and esBChE as highly
homologous and cheaper surrogates for human enzymes. The enzyme inhibition assays on human
MAOs were performed by applying already reported methods [14]. The biological data for all
derivatives are collected in Table 1.
Table 1. Biological data of coumarin derivatives 3–27.
General
entry
R1
R2
R3
MAO A a,b
MAO B a,b
AChE a,c
BChE a,d
structure
3
4
5
6
7
8
9
Cl
H
H
H
H
H
0.65 ± 0.02
49 ± 1%
0.0039 ± 0.0008
0.57 ± 0.05
6.2  0.7
6.0  1.5
11  2
43 ± 5%
34  5%
20  4%
3.4  2.1
17  1%
2.9 ± 0.4
4.8 ± 0.3
NMe
2
OMe
–OCH
OMe
H
1.0 ± 0.1
0.067 ± 0.005
A
2
O–
1.2 ± 0.1
0.0075 ± 0.0001
0.0021 ± 0.0002
0.0022 ± 0.0001
0.0013 ± 0.0001
8.5  1.8
26  5%
2.00 ± 0.04
2.7 ± 1.0
H
Br
F
H
H
H
H
0.53 ± 0.01
2.31 ± 0.04
1.74 ± 0.04
H
H
General
entry
R1
R2
MAO A
MAO B
AChE
BChE
structure
10
11
12
H
Me
H
1.0 ± 0.1
0.58 ± 0.03
48 ± 1%
0.17 ± 0.01
0.068 ± 0.002
1.0 ± 0.1
7.2  0.6
47.1 ± 0.3%
7.9  0.4
46 ± 2%
48.1 ± 0.6%
27 ± 2%
B
Me
Cl
H
General
entry
R1
R2
MAO A
MAO B
AChE
BChE
structure
13
Bn
Bn
H
24 ± 3%
28 ± 1%
1.12 ± 0.01
32 ± 5%
32 ± 3%
46 ± 3%
9.2 ± 0.7
48 ± 1%
41 ± 3%
1.1 ± 0.1
1.7 ± 0.1
0.077 ± 0.014
2.6 ± 0.1
2.5 ± 0.5
5.9 ± 0.5
18 ± 3%
0.103 ± 0.001
0.332 ± 0.026
0.152 ± 0.020
0.273 ± 0.011
0.172 ± 0.001
0.066 ± 0.015
0.213 ± 0.002
0.0094 ± 0.0035
0.164 ± 0.002
0.0041 ± 0.0011
0.084 ± 0.002
0.061 ± 0.013
0.0011 ± 0.0003
0.030 ± 0.001
0.013 ± 0.001
0.031 ± 0.001
17  1
16  1
3.4 ± 1.3
6.3 ± 0.3
14  2%
2.3  0.9
46  2%
20  7%
7.0 ± 1.1
3.6  0.8
4.8  0.3
33  6%
28  1%
13 ± 2%
<5%
14
Me
15
pyrrolidin-1-yl
6.6  0.9
3.1  0.6
6.6  1.1
17  4
8.3  2.0
4.9  1.4
39 ± 1%
2.1  0.2
5.6  0.7
2.2  0.7
5.3  1.4
4.3  0.6
4.4  0.8
16
4-methylpiperazin-1-yl
morpholin-1-yl
17
18
H
H
Me
H
19
Me
nPr
nBu
C
20
21
H
22
CH
CH
CH
CH CONHMe
CH CONMe
CH CONH
2
C≡CH
H
23
2
C≡CH
Me
H
24
2
CONH
2
25
2
H
26
2
2
H
34 ± 2%
41 ± 2%
D
27
2
2
OMe
safinamide
donepezil
0.021 ± 0.002
2.3 ± 0.2

Molecules 2019, 24, 4507
a IC50 (µM) or % inhibition at 10 µM. Values are the mean ±SEM of three independent experiments.
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b Human recombinant monoamine oxidase (MAO). c acetylcholinesterase (AChE) from electric eel.
d butyrylcholinesterase (BChE) from equine serum.
3. Discussion
3.1. Structure–Activity Relationships
Structural modifications around the benzyloxy-tail region markedly affected MAO B affinity.
Indeed, the presence of hydrogen-bonding acceptor (HBA) groups at position 3 (4) or 3,5 (5) reduced
MAO B affinity and selectivity. These substitution patterns were scarcely tolerated from both ChEs
as well. Interestingly, the presence of the piperonyl group (6) or meta-halogens (e.g., Cl in 3, Br in 8,
and F in 9) produced MAO B selective compounds almost equipotent to the unsubstituted
benzyloxy-derivative 7. This compound was the least active ChEs inhibitor (26% and 17% of
inhibition at 10 µM toward AChE and BChE, respectively). Conversely, halogens at the meta position
improved the inhibitory activity against both AChE and BChE, particularly Br and F.
When little modifications were applied around the heterocyclic backbone, the inhibition of
ChEs was moderate-to-poor and MAO B/A isoform selectivity fell down drastically. A methyl group
at position 6 or 8 produced an affinity drop toward MAO B compared to 3. A more dramatic
decrease was returned by Cl at position 6.
Then, the investigation was shifted to the modification of the basic protonatable head. As
expected from crystallographic poses, the steric hindrance of the basic head played a detrimental
role in the binding interactions with hMAO B. Confirming the prediction of a 3D-QSAR model
previously developed on rat enzymes [23], bulky groups (benzyl in 13–14, aliphatic 5–6 membered
nitrogen-containing cycles in 15–17) led to a significant reduction of MAO B inhibitory potency
compared to 3. Both molecular simplification of the basic head into primary amine 18 and the
modification of the basic head into a tertiary amine, albeit unhindered, did not ameliorate the
multi-targeting activity (compare 14 with 13, 23 with 22, 18–19 with 3). In addition, tertiary amines
(14, 19, and 23) displayed a lower B/A selectivity. The homologation of the alkyl chain on the basic
nitrogen of 3 to linear n-propyl (20) and n-butyl groups (21) progressively reduced MAO B
inhibitory potencies (3 < 20 < 21), too. The introduction of a terminal propargyl group led to a potent
and selective MAO B inhibitor (22) endowed with a three-fold improved AChE affinity compared to
3 (IC50 values equal to 2.1 and 6.2 µM, respectively) and a slightly lower BChE inhibition. Finally,
glycine-inspired terminal chains were introduced (24–26). These coumarins were potent MAO B
inhibitors, with compound 25 being a more potent MAO B inhibitor than 3 (IC50 = 1.1 nM). Within
this subset, it is worth noting that derivative 25 represented the most selective MAO B inhibitor of
the whole series and 24 showed no selectivity at all, providing nanomolar inhibition of both MAO
isoforms (IC50 = 77 and 61 nM toward MAO A and B, respectively). The increasing hindrance on the
terminal amide and/or the reduced HB networking did not clearly correlate with dual AChE/MAO B
affinity. Moreover, these polar chains were not tolerated by BChE.
In order to probe potential additive effects, we selected and combined one head and tail
modification among the previous ones by joining a 3,5-dimethoxybenzyl group (from 5) to a
Gly-NH fragment (from 24) in the same molecule. Interestingly, the resulting derivative 27 showed
2
a higher MAO B affinity and selectivity than 5 and 24, whereas ChEs inhibition was from moderate
(AChE) to low (BChE).
Concerning trends in structure–activity relationships, as seen in Figure 2, MAO B affinity is
much more sensitive than AChE to modifications at the protonatable moiety, where bulkier groups
and unhindered tertiary amines led to less active MAO B inhibitors. Halogens (particularly F and Br)
in the meta position of the benzyloxy tail represent the most effective substituents for good triple
targeting activity and high B/A selectivity.

Molecules 2019, 24, 4507
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Figure 2. Pictorial representation of structure–activity relationships.
Upon looking for MTDLs, an appropriately balanced bioactivity ratio is a challenging, yet
desirable, goal [24]. Most of the inhibitors described herein missed this outcome, since their
inhibitory readouts were biased against MAO B, and their IC50 values differ by more than one order
of magnitude, showing nanomolar MAO B affinity along with moderate ChEs inhibition in the
micromolar range (6, 8, 9, 20). Instead, compound 16 was endowed with a potent and well-balanced
bioactivity profile (submicromolar inhibition of MAO B, low micromolar inhibition of AChE and
BChE), showing AChE/MAO B and BChE/MAO B IC50 ratio equal to 11 and 9, respectively.
As reported in Table 2, these multimodal hit compounds were endowed with encouraging
drug-like features, showed no violation of Lipinsky’s RO5, and they were predicted to be CNS
permeant.
Table 2. Computed drug-like properties for the most active ChEs-MAO B inhibitors.
Entry
MW a
TPSA (Å2) a
HBA a
HBD a cLogP b BBB c RO5 violation a
6
8
339.34
374.23
313.32
398.88
357.83
69.93
51.47
51.47
45.92
51.47
6
4
5
5
4
1
1
1
0
1
2.89
3.80
3.08
3.53
4.69
+
+
+
+
+
0
0
0
0
0
9
16
20
a MW: molecular weight; TPSA: topological polar surface area; HBA: number of hydrogen bond
acceptors; HBD: number of hydrogen bond donors; RO5: violation of Lipinsky’s rule of 5. Properties
c
computed with SwissADME web tool. b Estimated with ACD/Labs 9.04. BBB penetration predicted
with the BOILED-Egg method through the free web-tools available at http://www.swissadme.ch.
3.2. AChE Kinetics
Enzyme kinetics were used as tool for investigating the inhibition mechanism toward AChE.
Compounds 8, bearing a meta-bromobenzyloxy substituent at coumarin C7, and 24, branched with a
glycinamide chain as the variation at position 4, were chosen as prototypes bearing different
head-or-tail modification schemes. Both compounds showed among the highest AChE inhibitory
potencies (IC50 in the low micromolar range). Similarly, with donepezil [25], Lineweaver–Burk plots
in Figure 3 indicated a mixed-type mechanism of inhibition toward eeAChE, which suggested the
possibility of occupying the peripheral anionic subsite (PAS) region, at least in part. Moreover,
compound 8 showed a K
i
equal to 2.91 µM and derivative 24 returned a K = 3.63 µM.
i

Molecules 2019, 24, 4507
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Figure 3. Electric eel AChE enzyme kinetics for compounds 8 (left) and 24 (right).
The chance of occupying the PAS of AChE acquires relevance in relation to AD, where the
chaperone activity of this region enhancing the fibrillization of neurotoxic amyloid monomers has
been well-documented [26].
3.3. Docking Studies
In order to get clues regarding a plausible binding mode within target enzymes, docking
simulations were run for compounds 8 and 24. As it could have been expected from the high
similarity with compound 3, the binding modes within hMAO B enzymatic cleft were close to those
of the cocrystallized ligand (PDB entry 2V61, data not shown) [11].
Aiming at studying binding interactions with AChE, the hAChE/donepezil X-ray complex
(PDB entry 6O4W) [27] was enrolled to this end, given that hAChE and eeAChE, used for in vitro
screening, share 88% identity and 93% overall sequence homology [28]. As shown in Figure 4, in
both instances, the heterocyclic cores were accommodated at the PAS in a way that most likely
resembled the pose of the native ligand (e.g., donepezil), as seen in Figure S1, even if the coumarin
ring twisting did not accomplish a proper geometry matching Trp286 for an optimal face-to-face π–
π interaction. Nonetheless, the whole molecular architecture was further stabilized by a network of
polar contacts (hydrogen and π-cation bonds) anchoring the basic heads to residues lining the
entrance of the active-site gorge (e.g., Ser293, Trp286). Additional hydrophobic interactions were
captured by 8 and 24 with the side chains of Phe338, Tyr337, and Tyr341. Interestingly, the same
outcomes were obtained when applying a different X-ray complex (PDB entry 6EY6) [29], where
galantamine is totally embedded within the catalytic region and a different rotamer was described
for Tyr337 (Phe330 in eeAChE), pointing toward the mid-gorge, as seen in Figure S2. In these runs,
compounds 8 and 24 were unable to occupy either the catalytic region, or to fit CAS (data not
shown), showing similar binding epitopes pointing to the outer mid-gorge region and Trp286 at the
PAS.

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Figure 4. Predicted binding mode of compounds 8 (left) and 24 (right) within hAChE (PDB entry
6O4W). For a data summary, see Table S1.
Regardless of its large structural homology (about 70%) with AChE, BChE features a larger
cavity (almost 200 Å3), making it accessible to bulkier binders. Docking studies involving hBChE
(PDB entry 6F7Q) retrieved plausible binding poses for coumarin 8, as seen in Figure 5, suggesting
the ability of this derivative to suitably interact with aromatic residues at CAS (Trp82) and PAS
(Tyr332) of BChE. On the other side, compound 24 adopted an unsuitable coumarin-ring twist for
interacting with Trp82, thus showing lower anti-BChE activity, as can be seen for compounds 24–27
bearing differently substituted Gly-NH side chains.
2
Figure 5. Predicted binding mode of compounds 8 (left) and 24 (right) within human BChE (PDB
entry 6F7Q).
4. Materials and Methods
4.1. Enzyme Inhibition Assays
All enzymes and reagents were purchased from Sigma Aldrich, Milan, Italy. Inhibition of
human recombinant MAO
A and B was assayed by monitoring the fluorescence of
4-hydroxyquinoline produced from the oxidation of substrate kynuramine, following a previously
published method [14]. Acetylcholinesterase from electric eel and butyrylcholinesterase from equine
serum were used in the Ellman’s spectrophotometric method as already described [13]. In all cases, a
96-well plate procedure was used with an Infinite M1000 Pro plate reader (Tecan, Cernusco sul
Naviglio, Italy). Inhibition values were determined as IC50 values by statistical analysis using Prism
(GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, USA). For less
active compounds, percent inhibition was measured at 10 µM. Inhibition kinetics of eeAChE were

Molecules 2019, 24, 4507
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determined as described [13], using four concentrations of inhibitor (0 to 5 µM) and six
concentrations of substrate acetylthiocholine (33 to 200 µM).
4.2. Molecular Modeling
Compounds 8 and 24, with standard values of bond lengths and valence angles, were built
within the Maestro software package [30] as protonated skeletons, and passed to Open Babel for a
10,000 steps of Steepest Descent minimization using the Universal Force Field. Chain A of hAChE
(PDB 6O4W) and hBChE (PDB entry 6F7Q) [31] enzyme structures were passed to the Protein
Preparation Wizard interface of Maestro, where water molecules were removed from the protein
target and hydrogen atoms were added, their positions optimized, and the protonation states of
residues determined according to PROPKA prediction at pH 7.0. AMBER UNITED force field
electrostatic charges [32] were loaded on the protein structure, while the molcharge complement of
QUACPAC [33] was used in order to achieve Marsili–Gasteiger charges for the inhibitors. Affinity
maps were first calculated on a 0.375 Å spaced 85 × 85 × 85 Å3 cubic box, centered on the inhibitors’
crystallized structures, and the accessibility of the binding site was exploited throughout 250 runs of
the Lamarckian Genetic Algorithm (LGA) implemented in AUTODOCK 4.2.6 [34].
Bridging water molecules were taken into account by using the automated hydration procedure
in AUTODOCK [35], and the population size and the number of energy evaluations figures were set
to 300 and 10,000,000, respectively. Among all the plausible binding poses, either the best
AUTODOCK free energy scoring function pose and/or the most populated cluster was finally
selected as representative of the ChEs/inhibitor complex.
4.3. Chemistry
Starting materials, reagents, and analytical grade solvents were purchased from Sigma-Aldrich,
Milan, Italy. The purity of all the intermediates, checked by HPLC, was always better than 95%. All
the newly prepared and tested compounds showed purity higher than 95% (elemental analysis).
Elemental analyses were performed on the EuroEA 3000 analyzer for the final compounds tested as
MAOs and ChEs inhibitors. The measured values for C, H, and N agreed to within 0.40% of the
theoretical values. Column chromatography was performed using Merck silica gel 60 (0.063–0.200
mm, 70–230 mesh). Flash chromatographic separations were performed on a Biotage SP1
purification system using flash cartridges prepacked with KP-Sil 32–63 µm, 60 Å silica. All reactions
were routinely checked by TLC using Merck Kieselgel 60 F254 aluminum plates and visualized by UV
light or iodine. Regarding the reaction requiring the use of dry solvents, the glassware was
flame-dried and then cooled under a stream of dry argon before the use. Nuclear magnetic
resonance spectra were recorded on a Varian Mercury 300 instrument (at 300 MHz) or on an Agilent
Technologies 500 apparatus (at 500 MHz) at ambient temperature in the specified deuterated
solvent. Chemical shifts (δ) are quoted in parts per million (ppm) and are referenced to the residual
solvent peak. The coupling constants J are given in Hertz (Hz). The following abbreviations were
used: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), m (multiplet), br (broad signal);
signals due to OH and NH protons were located by deuterium exchange with D O. The attribution
2
1
of H NMR signals was supported by comparison with literature data of close analogues. Melting
points for solid final compounds were determined by the capillary method on a Stuart Scientific
SMP3 electrothermal apparatus and are uncorrected. HRMS experiments were performed with a
dual electrospray interface (ESI) and a quadrupole time-of-flight mass spectrometer (Q-TOF, Agilent
6530 Series Accurate-Mass Quadrupole Time-of-Flight LC/MS, Agilent Technologies Italia S.p.A.,
Cernusco sul Naviglio, Italy). Full-scan mass spectra were recorded in the mass/charge (m/z) range
50–3000 Da. The following derivatives (intermediates or tested final compounds) have been already
described in the literature: 4-(chloromethyl)-7-hydroxy-2H-chromen-2-one (1a) [13],
4-(chloromethyl)-7-hydroxy-8-methyl-2H-chromen-2-one
7-[(3-chlorobenzyl)oxy]-4-(chloromethyl)-2H-chromen-2-one
4-(chloromethyl)-7-[(3-fluorobenzyl)oxy]-2H-chromen-2-one
7-[(3-bromobenzyl)oxy]-4-(chloromethyl)-2H-chromen-2-one
(1d)
(2a)
[36],
[36],
[10],
[21],
(2b)
(2c)

Molecules 2019, 24, 4507
10 of 16
7-(benzyloxy)-4-(chloromethyl)-2H-chromen-2-one
(2d)
[10],
[10],
[10],
[10],
[10],
[21],
[10],
[10],
[10],
[21],
[21],
[10],
[10],
[21],
7-(1,3-benzodioxol-5-ylmethoxy)-4-(chloromethyl)-2H-chromen-2-one
7-[(3-chlorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-2-one
(2g)
(3)
7-(1,3-benzodioxol-5-ylmethoxy)-4-[(methylamino)methyl]-2H-chromen-2-one
(6)
7-(benzyloxy)-4-[(methylamino)methyl]-2H-chromen-2-one
(7)
7-[(3-bromobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-2-one
7-[(3-fluorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-2-one
4-[(benzylamino)methyl]-7-[(3-chlorobenzyl)oxy]-2H-chromen-2-one
(8)
(9)
(13)
4-{[benzyl(methyl)amino]methyl}-7-[(3-chlorobenzyl)oxy]-2H-chromen-2-one
7-[(3-chlorobenzyl)oxy]-4-(pyrrolidin-1-ylmethyl)-2H-chromen-2-one
7-[(3-chlorobenzyl)oxy]-4-(morpholin-4-ylmethyl)-2H-chromen-2-one
(14)
(15)
(17)
4-(aminomethyl)-7-[(3-chlorobenzyl)oxy]-2H-chromen-2-one
(18)
7-[(3-chlorobenzyl)oxy]-4-[(dimethylamino)methyl]-2H-chromen-2-one
7-[(3-chlorobenzyl)oxy]-4-[(propylamino)methyl]-2H-chromen-2-one
4-[(butylamino)methyl]-7-[(3-chlorobenzyl)oxy]-2H-chromen-2-one (21) [21].
(19)
(20)
General
procedure
for
the
preparation
of
starting
7-hydroxy-4-chloromethyl-2H-chromen-2-ones 1b–c: Prepared according to procedure described in
ref. [10]or [36]. The appropriate resorcinol (55 mmol) was dissolved in conc. sulfuric acid (60 mL) at
0 °C. Then ethyl 4-chloroacetoacetate (8.2 mL, 50 mmol) was added dropwise while cooling. After
stirring for 1 h at 0 °C (for 1c) or for 24 h at room temperature (for 1b), the reaction mixture was
poured onto ice. The precipitate was collected, filtered, washed with abundant water, and purified
as detailed below, when necessary.
4-(chloromethyl)-7-hydroxy-6-methyl-2H-chromen-2-one (1b): Prepared from 2,4-dihydroxytoluene (6.8
1
g, 55 mmol). Used without further purification. Brown solid; yield: 8.2 g, 73%. H NMR (300 MHz,
Acetone-d
6
) δ: 2.27 (s, 3H, CH
3
), 4.91 (s, 2H, CH Cl), 6.38 (s, 1H, H-3coum), 6.75 (s, 1H, H-8coum), 7.61 (s,
2
1H, H-5coum), OH not detectable.
6-Chloro-4-(chloromethyl)-7-hydroxy-2H-chromen-2-one (1c): Prepared from 4-chlororesorcinol (8.0 g, 55
mmol). Purified through flash chromatography (elution gradient: methanol in chloroform 0%→5%).
1
White solid; yield: 5.8 g, 47%. H NMR (300 MHz, DMSO-d
6) δ: 4.97 (s, 2H, CH
2
Cl), 6.47 (s, 1H,
H-3coum), 6.92 (s, 1H, H-8coum), 7.84 (s, 1H, H-5coum), 11.48 (s, 1H, dis. with D
2
O, OH).
[3-(Dimethylamino)phenyl]methanol (1e): 3-(Dimethylamino)benzoic acid (3.3 g, 20 mmol) was
dissolved in dry THF (100 mL) and then 1.0 M BH solution in THF (60 mL, 60 mmol) was added
3
dropwise while cooling at 0 °C. The reaction mixture was slowly warmed at ambient temperature
and kept under magnetic stirring for 24 h. After cooling through an external ice bath, the excess
borane was quenched by slowly adding methanol. The solvents were removed under reduced
pressure. The resulting mixture was partitioned with ethyl acetate (200 mL) and 2 N aq. NaOH (3 ×
50 mL), and washed with brine (1 × 50 mL). The organic layer was dried over Na
2
SO , concentrated
4
under vacuum and purified through flash chromatography (elution gradient: methanol in
1
chloroform 0% → 5%). Yellow oil; yield: 2.7 g, 89%. H NMR (300 MHz, CDCl
3
) δ: 1.67 (br, 1H, dis.
with D
2
O, OH), 2.96 (s, 6H, N(CH
3
)2
), 4.65 (s, 2H, CH
2
OH), 6.66–6.75 (m, 3H, Ar), 7.21–7.23 (m, 1H,
Ar).
4-(Chloromethyl)-7-{[3-(dimethylamino)benzyl]oxy}-2H-chromen-2-one (2e): Phenol 1a (1.6 g, 7.5 mmol)
was dissolved in dry THF (60 mL) followed by the addition of 1e (2.3 g, 15 mmol), DIAD (3.0 g, 15
mmol) and a solution of PPh (3.9 g, 15 mmol) in THF (15 mL), via syringe at 0 °C. The mixture was
3
stirred at room temperature overnight and then concentrated to dryness under rotary evaporation.
The resulting oil residue was purified through flash chromatography (elution gradient: EtOAc in
1
n-hexane 0% → 80%). Brown solid; yield: 0.80 g, 31%. H NMR (300 MHz, CDCl
3
) δ: 2.97 (s, 6H,
N(CH
3
)2
), 4.61 (s, 2H, CH
2
Cl), 5.10 (s, 2H, CH O), 6.39 (s, 1H, H-3coum), 6.71–6.76 (m, 3H, Ar), 6.93 (d, J
2
= 2.5 Hz, 1H, H-8coum), 6.97 (dd, J = 8.8, 2.5 Hz, 1H, H-6coum), 7.20–7.23 (m, 1H, Ar), 7.56 (d, J = 8.8 Hz,
1H, H-5coum).

Molecules 2019, 24, 4507
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General procedure for the preparation of 7-benzyloxy-4-chloromethyl-2H-chromen-2-ones 2f,
2h–j: The appropriate 7-OH-coumarin 1a–d (20 mmol) was suspended in ethanol (100 mL), before
adding DIEA (7.0 mL, 40 mmol) and the suitable benzyl bromide (40 mmol). The mixture was
refluxed until reaction completion (TLC control, 1.5–5 h) and the solvent was removed under rotary
evaporation. The resulting crude was purified through flash chromatography as indicated below.
4-(Chloromethyl)-7-[(3,5-dimethoxybenzyl)oxy]-2H-chromen-2-one (2f): Prepared from 1a (4.2 g, 20
mmol) and 3,5-dimethoxybenzyl bromide (9.2 g, 40 mmol). Purified through flash chromatography
(elution gradient: chloroform in n-hexane 60% → 100%). White solid; yield: 4.4 g, 61%. 1H NMR (300
MHz, DMSO-d
6
) δ: 3.73 (s, 6H, OCH
3
), 4.97 (s, 2H, CH
2
Cl), 5.16 (s, 2H, CH O), 6.43–6.45 (m, 1H, Ar),
2
6.53 (s, 1H, H-3coum), 6.59-6.63 (m, 2H, Ar), 7.03–7.12 (m, 2H, H-6coum + H-8coum), 7.75 (d, J = 8.4 Hz, 1H,
H-5coum).
7-[(3-Chlorobenzyl)oxy]-4-(chloromethyl)-6-methyl-2H-chromen-2-one (2h): Prepared from 1b (4.5 g, 20
mmol) and 3-chlorobenzyl bromide (5.3 mL, 40 mmol). Purified through flash chromatography
1
(elution gradient: EtOAc in n-hexane 10% → 60%). Yellow solid; yield: 4.0 g, 57%. H NMR (300
MHz, DMSO-d
6
) δ: 2.12 (s, 3H, CH
3
), 4.96 (s, 2H, CH
2
Cl), 5.26 (s, 2H, CH O), 6.47 (s, 1H, H-3coum), 7.11
2
(s, 1H, H-8coum), 7.39–7.46 (m, 3H, Ar), 7.53 (s, 1H, Ar), 7.65 (s, 1H, H-5coum).
6-Chloro-7-[(3-chlorobenzyl)oxy]-4-(chloromethyl)-2H-chromen-2-one (2i): Prepared from 1c (4.9 g, 20
mmol) and 3-chlorobenzyl bromide (5.3 mL, 40 mmol). Purified through flash chromatography
1
(elution gradient: chloroform in n-hexane 50% → 80%). White solid; yield: 6.6 g, 89%. H NMR (300
MHz, DMSO-d
6
) δ: 5.01 (s, 2H, CH
2
Cl), 5.35 (s, 2H, CH O), 6.54 (s, 1H, H-3coum), 7.36 (s, 1H, H-8coum),
2
7.38–7.45 (m, 3H, Ar), 7.55 (s, 1H, Ar), 7.95 (s, 1H, H-5coum).
7-[(3-Chlorobenzyl)oxy]-4-(chloromethyl)-8-methyl-2H-chromen-2-one (2j): Prepared from 1d (4.5 g, 20
mmol) and 3-chlorobenzyl bromide (5.3 mL, 40 mmol). Purified through flash chromatography
1
(elution gradient: chloroform in n-hexane 10% → 80%). Off-white solid; yield: 5.7 g, 81%. H NMR
(300 MHz, Acetone-d
6
) δ: 2.24 (s, 3H, CH
3
), 4.98 (s, 2H, CH
2
Cl), 5.30 (s, 2H, CH O), 6.50 (s, 1H,
2
H-3coum), 7.15 (d, J = 8.8 Hz, 1H, H-6coum), 7.41–7.46 (m, 3H, Ar), 7.54 (s, 1H, Ar), 7.67 (d, J = 8.8 Hz, 1H,
H-5coum).
General procedure for the preparation of final compounds 4–5, 10–12. To a stirred 2.0 N
methylamine solution in THF (10 mL), the suitable chloride 2e–f, 2h–j was added portionwise (1.0
mmol, six aliquots, 1 h time interval) under an Ar atmosphere. The mixture was stirred at room
temperature for 6–24 h until disappearance of the starting material (TLC control). After the
evaporation of excess amine and THF under reduced pressure, the resulting crude was purified as
indicated below.
7-{[3-(Dimethylamino)benzyl]oxy}-4-[(methylamino)methyl]-2H-chromen-2-one (4): Prepared from 2e
(0.34 g, 1.0 mmol). Purified through flash chromatography (eluent: EtOAc). Yellow solid;
1
low-melting; yield: 0.27 g, 80%. H-NMR (300 MHz, DMSO-d
6
) δ: 2.33 (s, 3H, CH
O), 6.29 (s, 1H, H-3coum), 6.66–6.75 (m, 3H, Ar),
7.00 (dd, J = 8.8, 2.5 Hz, 1H, H-6coum), 7.04 (d, J = 2.5 Hz, 1H, H-8coum), 7.21–7.23 (m, 1H, Ar), 7.73 (d, J =
8.8 Hz, 1H, H-5coum), NH not detected. Anal. (C20 ) calcd. % C, 70.99; H, 6.55; N, 8.28. Found %
[M + Na]+ m/z 361.1523, found
2
NHCH
3
), 2.86 (s,
6H, N(CH
3
)
2
), 3.80 (s, 2H, CH
2
NHCH
3
), 5.17 (s, 2H, CH
2
H22
N
2O3
C, 71.12; H, 6.46; N, 8.18. HRMS (Q-TOF) calcd. for C20
361.1524.
H22
N
2
O3
7-[(3,5-Dimethoxybenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-2-one (5): Prepared from 2f (0.36 g,
1.0 mmol). Purified through flash chromatography (eluent: EtOAc). Yield: 0.22 g, 61%. 1H-NMR (300
MHz, DMSO-d
CH O), 6.28 (s, 1H, H-3coum), 6.44 (t, J = 2.2 Hz, 1H, Ar), 6.60 (d, J = 2.2 Hz, 2H, Ar), 6.99 (dd, J = 8.8 Hz,
2.5 Hz, 1H, H-6coum), 7.03 (d, J = 2.5 Hz, 1H, H-8coum), 7.73 (d, J = 8.8 Hz, 1H, H-5coum), NH not detected.
Anal. (C20 21NO ) calcd. % C, 67.59; H, 5.96; N, 3.94. Found % C, 67.68; H, 6.01; N, 3.87. HRMS
(Q-TOF) calcd. for C20
[M + Na]+ m/z 378.1312, found 378.1312.
6
) δ: 2.32 (s, 3H, CH
2
NHCH
3
), 3.72 (s, 6H, OCH
3
), 3.81 (s, 2H, CH
2
NHCH
3
), 5.14 (s, 2H,
2
H
5
H21NO
5

Molecules 2019, 24, 4507
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7-[(3-Chlorobenzyl)oxy]-8-methyl-4-[(methylamino)methyl]-2H-chromen-2-one (10): Prepared from 2j
(0.35 g, 1.0 mmol). Purified through flash chromatography (eluent: EtOAc). White solid; mp: 145–146
1
°C; yield: 0.11 g, 33%. H-NMR (300 MHz, DMSO-d
6
) δ: 2.23 (s, 3H, CH
O), 6.29 (s, 1H, H-3coum), 7.06 (d, J = 8.8 Hz, 1H, H-6coum),
7.39–7.44 (m, 3H, Ar), 7.52 (s, 1H, Ar), 7.62 (d, J = 8.8 Hz, 1H, H-5coum), NH not detected. Anal.
(C19 18ClNO ) calcd. % C, 66.38; H, 5.28; N, 4.07. Found % C, 66.45; H, 5.40; N, 4.02. HRMS (Q-TOF)
calcd. for C19 18ClNO
[M + Na]+ m/z 366.0867, found 366.0869.
3
), 2.32 (s, 3H, CH
2
NHCH
3
),
3.81 (s, 2H, CH
2
NHCH
3
), 5.27 (s, 2H, CH
2
H
3
H
3
7-[(3-Chlorobenzyl)oxy]-6-methyl-4-[(methylamino)methyl]-2H-chromen-2-one
hydrochloride
(11):
Prepared from 2h (0.35 g, 1.0 mmol). Purified through flash chromatography (eluent: EtOAc) and
transformed into the corresponding hydrochloride salt by treatment with 4.0 N HCl solution in
1
1,4-dioxane. White solid; mp > 230 °C; yield: 0.17 g, 44%. H-NMR (300 MHz, DMSO-d
6
) δ: 2.27 (s,
O), 6.42 (s, 1H,
H-3coum), 7.14 (s, 1H, H-8coum), 7.38–7.45 (m, 3H, Ar), 7.53 (s, 1H, Ar), 7.66 (s, 1H, H-5coum), 9.88 (br s,
2H, dis. with D ). Anal. (C19 19Cl NO ) calcd. % C, 60.01; H, 5.04; N, 3.68. Found % C, 60.12;
O, NH +
H, 5.03; N, 3.72. HRMS (Q-TOF) calcd. for C19 18ClNO
[M + Na]+ m/z 366.0867, found 366.0870.
3H, CH
3
), 2.68 (s, 3H, CH
2
NH +
2
CH
3
), 4.42 (s, 2H, CH
2
NH +
2
CH
3
), 5.28 (s, 2H, CH
2
2
2
H
2
3
H
3
6-Chloro-7-[(3-chlorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-2-one (12): Prepared from 2i (0.37
g, 1.0 mmol). Purified through flash chromatography (eluent: EtOAc). White solid; mp: 176–177 °C;
yield: 0.13 g, 37%. 1H-NMR (300 MHz, DMSO-d
2H, CH O), 6.34 (s, 1H, H-3coum), 7.31 (s, 1H, H-8coum), 7.42–7.48 (m, 3H, Ar), 7.54 (s, 1H, Ar), 7.93 (s,
1H, H-5coum), NH not detected. Anal. (C18 15Cl NO ) calcd. % C, 59.36; H, 4.15; N, 3.85. Found % C,
15Cl NO
[M + Na]+ m/z 386.0321, found
6
) δ: 2.31 (s, 3H, NHCH
3
), 3.81 (s, 2H, CH
2
NH), 5.33 (s,
2
H
2
3
59.48; H, 4.20; N, 3.83. HRMS (Q-TOF) calcd. for C18
386.0324.
H
2
3
General procedure for the preparation of final compounds 16, 22–27. After dissolving chloride
2a or 2f (1.0 mmol) in 8 mL of dry THF (dry acetone or DMF for the synthesis of 16 and 24,
respectively), an excess amine was added, followed by a proton scavenger (K
2
CO for 16, 22 and 23:
3
0.28 g, 2.0 mmol; DIEA for 24–27: 1.4 mL, 8.0 mmol). The reaction mixtures were kept under
magnetic stirring at room temperature for 24–72 h (for 16, 22, 23) or heated at 80 °C for 4–6 h (24–27).
After evaporation of the solvent under rotary evaporation, the resulting crude was purified as
indicated below.
7-[(3-Chlorobenzyl)oxy]-4-[(4-methylpiperazin-1-yl)methyl]-2H-chromen-2-one (16): Prepared from 2a
(0.34 g, 1.0 mmol) and 1-methylpiperazine (0.17 mL, 1.5 mmol). The crude was partitioned between
brine (80 mL) and diethyl ether (50 mL). The organic phase was dried over Na
to dryness. Crystallization from hot ethanol afforded the title compound 16. Pale yellow solid; mp:
133–134 °C; yield: 0.19 g, 47%. 1H-NMR (300 MHz, DMSO-d
) δ: 2.13 (s, 3H, NCH ), 2.23–2.36 (m, 4H,
N-cycle), 2.40–2.46 (m, 4H, N-cycle), 3.61 (s, 2H, CH N), 5.23 (s, 2H, CH O), 6.28 (s, 1H, H-3coum), 7.01
(dd, J = 8.8, 2.5 Hz, 1H, H-6coum), 7.06 (d, J = 2.5 Hz, 1H, H-8coum), 7.39–7.43 (m, 3H, Ar), 7.53 (s, 1H,
Ar), 7.84 (d, J = 8.8 Hz, 1H, H-5coum). Anal. (C22 23ClN ) calcd. % C, 66.24; H, 5.81; N, 7.02. Found %
23ClN
[M + Na]+ m/z 421.1289, found
2
SO
4
and concentrated
6
3
2
2
H
2O3
C, 66.41; H, 5.90; N, 6.93. HRMS (Q-TOF) calcd. for C22
421.1289.
H
2
O3
7-[(3-Chlorobenzyl)oxy]-4-[(prop-2-yn-1-ylamino)methyl]-2H-chromen-2-one (22): Prepared from 2a (0.34
g, 1.0 mmol) and propargylamine (0.64 mL, 10 mmol). Purified through flash chromatography (elution
gradient: EtOAc in n-hexane 20% → 100%). White solid; mp: 114–115 °C; yield: 0.24 g, 69%. 1H-NMR
(300 MHz, DMSO-d
CH NHCH C≡CH), 5.23 (s, 2H, CH
(d, J = 2.5 Hz, 1H, H-8coum), 7.37–7.43 (m, 3H, Ar), 7.53 (s, 1H, Ar), 7.74 (d, J = 8.8 Hz, 1H, H-5coum), NH
not detected. Anal. (C20 16ClNO ) calcd. % C, 67.90; H, 4.56; N, 3.96. Found % C, 68.02; H, 4.50; N, 3.99.
HRMS (Q-TOF) calcd. for C20 16ClNO
[M + Na]+ m/z 376.0711, found 376.0713.
6) δ: 2.71–2.76 (m, 1H, C≡CH), 3.37 (s, 2H, NHCH2C≡CH), 3.90 (s, 2H,
2
2
2
O), 6.28 (s, 1H, H-3coum), 7.01 (dd, J = 8.8, 2.5 Hz, 1H, H-6coum), 7.07
H
3
H
3
7-[(3-Chlorobenzyl)oxy]-4-{[methyl(prop-2-yn-1-yl)amino]methyl}-2H-chromen-2-one hydrochloride (23):
Prepared from 2a (0.34 g, 1.0 mmol) and N-methylpropargylamine (0.25 mL, 3.0 mmol). Purified
through flash chromatography (elution gradient: EtOAc in n-hexane 10% → 70%) and transformed

Molecules 2019, 24, 4507
13 of 16
into the corresponding hydrochloride salt by treatment with 4.0 N HCl solution in 1,4-dioxane.
White solid; mp: 183–184 °C (dec.); yield: 0.36 g, 88%. 1H-NMR (300 MHz, D
O+DMSO-d ) δ: 2.65 (s,
1H, C≡CH), 2.70 (s, 3H, NH+(CH )), 3.95 (s, 2H, NH+(CH
)CH C≡CH), 4.22 (s, 2H,
CH )CH C≡CH), 5.25 (s, 2H, CH O), 6.53 (s, 1H, H-3coum), 7.05 (dd, J = 8.8, 2.2 Hz, 1H, H-6coum),
NH+(CH
7.12 (d, J = 2.2 Hz, 1H, H-8coum), 7.37–7.43 (m, 3H, Ar), 7.53 (s, 1H, Ar), 7.88 (d, J = 8.8 Hz, 1H, H-5coum).
Anal. (C21 19Cl NO ) calcd. % C, 62.39; H, 4.74; N, 3.46. Found % C, 62.51; H, 4.70; N, 3.31. HRMS
(Q-TOF) calcd. for C21 18ClNO
[M + Na]+ m/z 390.0867, found 390.0868.
2
6
3
3
2
2
3
2
2
H
2
3
H
3
N2-({7-[(3-chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}methyl)glycinamide (24): Prepared from 2a (0.34 g,
1.0 mmol) and glycinamide hydrochloride (0.55 g, 5.0 mmol). Purified through column
chromatography (eluent: 5% methanol in chloroform). Off-white solid; mp: 183–184 °C; yield: 0.24 g,
1
64%. H-NMR (300 MHz, DMSO-d
6
) δ: 3.12 (s, 2H, CH
2
CO), 3.87 (s, 2H, CH
2
NH), 5.22 (s, 2H, CH
), 7.31 (s, 1H,
), 7.38–7.43 (m, 3H, Ar), 7.53 (s, 1H, Ar), 7.73 (d, J = 8.8 Hz, 1H, H-5coum), aminic NH not
detected. Anal. (C19 17ClN ) calcd. % C, 61.21; H, 4.60; N, 7.51. Found % C, 61.33; H, 4.64; N, 7.45.
HRMS (Q-TOF) calcd. for C19 17ClN
[M + Na]+ m/z 395.0769, found 395.0769.
2
O),
6.35 (s, 1H, H-3coum), 7.00 (dd, J = 8.8, 2.5 Hz, 1H, H-6coum), 7.07 (m, 2H, H-8coum + CONH
a
CONH
b
H
2O4
H
O
2 4
N2-({7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}methyl)-N1-methylglycinamide (25): Prepared from
2a (0.34 g, 1.0 mmol) and 2-amino-N-methylacetamide hydrochloride (0.62 g, 5.0 mmol). Purified
through column chromatography (eluent: 10% methanol in chloroform). White solid; mp: 168–169
°C; yield: 0.24 g, 61%. 1H-NMR (300 MHz, DMSO-d
CH CO), 3.86 (s, 2H, CH NH), 5.22 (s, 2H, CH O), 6.32 (s, 1H, H-3coum), 7.00 (dd, J = 8.8, 2.5 Hz, 1H,
H-6coum), 7.07 (d, J = 2.5 Hz, 1H, H-8coum), 7.38-7.43 (m, 3H, Ar), 7.52 (s, 1H, Ar), 7.72 (d, J = 8.8 Hz, 1H,
H-5coum), 7.73–7.78 (m, 1H, CONH), aminic NH not detected. Anal. (C20 19ClN ) calcd. % C, 62.10; H,
19ClN
[M + Na]+ m/z
6
) δ: 2.58 (d, J = 4.7 Hz, 3H, CONHCH3), 3.14 (s, 2H,
2
2
2
H
2O4
4.95; N, 7.24. Found % C, 62.42; H, 4.79; N, 7.15. HRMS (Q-TOF) calcd. for C20
H
2
O4
409.0926, found 409.0942.
N2-({7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}methyl)-N1,N1-dimethylglycinamide (26): Prepared
from 2a (0.34 g, 1.0 mmol) and 2-amino-N,N-dimethylacetamide acetate (0.81 g, 5.0 mmol). Purified
through column chromatography (eluent: 5% methanol in chloroform). White solid; mp: 134–135 °C;
yield: 0.21 g, 53%. 1H-NMR (300 MHz, DMSO-d
CH CO), 3.84 (s, 2H, CH NH), 5.22 (s, 2H, CH
H-6coum), 7.06 (d, J = 2.5 Hz, 1H, H-8coum), 7.38–7.42 (m, 3H, Ar), 7.52 (s, 1H, Ar), 7.75 (d, J = 8.8 Hz, 1H,
H-5coum), aminic NH not detected. Anal. (C21 21ClN ) calcd. % C, 62.92; H, 5.28; N, 6.99. Found % C,
63.09; H, 5.20; N, 7.06. HRMS (Q-TOF) calcd. for C21 21ClN
[M + Na]+ m/z 423.1082, found 423.1089.
6
) δ: 2.82 (s, 3H, NCH
3
), 2.87 (s, 3H, NCH3), 3.40 (s, 2H,
2
2
2
O), 6.30 (s, 1H, H-3coum), 7.00 (dd, J = 8.8, 2.5 Hz, 1H,
H
2
O4
H
2O4
N2-({7-[(3,5-dimethoxybenzyl)oxy]-2-oxo-2H-chromen-4-yl}methyl)glycinamide (27): Prepared from 2f
(0.36 g, 1.0 mmol) and glycinamide hydrochloride (0.55 g, 5.0 mmol). Purified through column
chromatography (eluent: 10% methanol in chloroform). White solid; mp: 130–131 °C; yield: 0.29 g,
1
74%. H-NMR (500 MHz, DMSO-d
6
) δ: 3.32 (s, 2H, CH
O), 6.36 (s, 1H, H-3coum), 6.45 (t, J = 2.4 Hz, 1H, Ar), 6.62 (d, J = 2.4 Hz, 2H, Ar), 7.00 (dd, J
= 8.8, 2.5 Hz, 1H, H-6coum), 7.05 (m, 2H, H-8coum + CONH ), 7.31 (s, 1H, CONH ), 7.73 (d, J = 8.8 Hz, 1H,
) calcd. % C, 63.31; H, 5.57; N, 7.03. Found % C,
[M + Na]+ m/z 421.1370, found 421.1371.
2
CO), 3.73 (s, 6H, OCH
3
), 3.88 (s, 2H, CH NH),
2
5.15 (s, 2H, CH
2
a
b
H-5coum), aminic NH not detected. Anal. (C21
H22N2
O
6
63.33; H, 5.51; N, 6.98. HRMS (Q-TOF) calcd. for C21
H
22
N
2
O6
5. Conclusions
In the present work, we aimed at finding novel multipotent compounds capable of inhibiting
AD-related enzymatic targets, namely MAO B and ChEs, by exploring structural modifications
around the 4-aminomethyl-7-benzyloxy-2H-chromen-2-one core at the basic protonatable head or at
the benzyloxy tail. A larger activity window was observed in the case of MAO B, where structural
requirements, basically the influence of steric bulk within the protonatable group, were confirmed.
Unfortunately, the structural modifications explored herein did not succeed in tightening the
binding with ChEs and in improving inhibition to a higher extent. The kinetic analysis indicated a
mixed-type inhibition for AChE, which suggests the possibility for ligands to bind peripheral

Molecules 2019, 24, 4507
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subsites. Docking simulations corroborated these results, highlighting productive binding
interactions at the PAS region of both ChEs and suggesting the inability of these molecules to deeply
lock into the catalytic region of AChE. Irrespective of the basic moiety, the binding contacts were
limited to PAS residues and the coumarin derivatives were unable to reach and interact with the
catalytic Ser203 and the CAS (e.g., Trp86).
We succeeded in identifying different coumarins with an interesting multiple activity showing
nanomolar activity toward MAO B along with AChE and/or BChE inhibition in the low micromolar
range (e.g., 3, 6, 8, 9, 20, 22–27). A more balanced multi-targeting compound was identified in
derivative 16 (IC50 = 0.27 µM, 3.1 µM and 2.3 µM toward MAO B, AChE, and BChE, respectively). In
light of these data, further hit optimization programs along with deeper in vitro and computational
studies will be fostered to achieve balanced profiles with higher ChEs affinity and to rule out
off-target activities (e.g., through off-target battery docking).
Abbreviations:
The following abbreviations are used in this manuscript:
ACh: acetylcholine
AChE: acetylcholinesterase
AD: Alzheimer’s disease
EtOAc: ethyl acetate
EU: European Union
BBB: blood-brain barrier
BChE: butyrylcholinesterase
CAS: catalytic anionic subsite
ChE: cholinesterase
DIAD: diisopropyl azodicarboxylate
DIEA: N,N-diisopropylethylamine
DMF: N,N-dimethylformamide
DMSO: dimethylsulfoxide
FAD: flavin adenine dinucleotide
FDA: Food and Drug Administration
GABA: γ-amminobutirric acid
HBA: hydrogen bond acceptor
HBD: hydrogen bond donor
HIV: human immunodeficiency virus
HRMS: high-resolution mass
MAO A: monoamine oxidase A
MAO B: monoamine oxidase B
MTDL: multi-target-directed ligand
PAS: peripheral anionic subsite
PD: Parkinson’s disease
PDB: protein data bank
Q-TOF: quadrupole-time of flight
THF: tetrahydrofuran
TPSA: topological polar surface area
Supplementary Materials: The following are available online, Figure S1: Michaelis-Menten curves toward
eeAChE for compounds 8 and 24, Figure S2: Superimposition of donepezil / 8 binding poses within human
AChE, Figure S3: Different rotamers for mid-gorge Tyr337 in hAChE complexes, Table S1: hAChE docking data
for compounds 8, 24, donepezil.
Author Contributions: Funding acquisition, C.D.A. and L.P.; Investigation, M.R., M.C., A.C. and L.P.;
Supervision, L.P.; Writing—original draft, L.P.; Writing—review & editing, M.C., M.d.C., C.D.A. and L.P.
Funding: L.P. and M.C. acknowledge financial support from Italian Ministry for Education and Research
(Fondo di Finanziamento per le Attività Base di Ricerca, FFABR 2017). Authors are also grateful for the financial
support from the University of Bari (Fondi di Ateneo 2012).

Molecules 2019, 24, 4507
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Conflicts of Interest: The authors declare no conflict of interest.
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