Coumarin-chalcone hybrids as inhibitors of MAO-B: Biological activity and in silico studies

Article abstract of DOI:10.3390/molecules26092430

Fourteen coumarin-derived compounds modified at the C3 carbon of coumarin with an α,β-unsaturated ketone were synthesized. These compounds may be designated as chalcocoumarins (3-cinnamoyl-2H-chromen-2-ones). Both chalcones and coumarins are recognized scaffolds in medicinal chemistry, showing diverse biological and pharmacological properties among which neuro-protective activities and multiple enzyme inhibition, including mitochondrial enzyme systems, stand out. The evaluation of monoamine oxidase B (MAO-B) inhibitors has aroused considerable interest as therapeutic agents for neurodegenerative diseases such as Parkinson’s. Of the fourteen chalcocumarins evaluated here against MAO-B, ChC4 showed the strongest activity in vitro, with IC₅₀ = 0.76 ± 0.08 μM. Computational docking, molecular dynamics and MM/GBSA studies, confirm that ChC4 binds very stably to the active rMAO-B site, explaining the experimental inhibition data.

Full text of DOI:10.3390/molecules26092430

molecules
Article
Coumarin-Chalcone Hybrids as Inhibitors of MAO-B:
Biological Activity and In Silico Studies
Guillermo Moya-Alvarado 1,†, Osvaldo Yañez 2,3,† , Nicole Morales 4, Angélica González-González 5,
Carlos Areche 6 , Marco Tulio Núñez 7 , Angélica Fierro 8,
*
and Olimpo García-Beltrán 9,10,
*
1
Biology Department, Johns Hopkins University, Baltimore, MD 21218, USA; gmoya@bio.puc.cl
Center of New Drugs for Hypertension (CENDHY), Santiago 8330015, Chile; osvyanezosses@gmail.com
Computational and Theoretical Chemistry Group, Departamento de Ciencias Químicas,
Facultad de Ciencias Exactas, Universidad Andres Bello, República 498, Santiago 7550196, Chile
Department of Physiology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile,
Santiago 8331150, Chile; nlmorales@uc.cl
Laboratorio de Interacciones Insecto-Planta, Instituto de Ciencias Biológicas, Universidad de Talca,
Casilla 747, Talca 3460000, Chile; angelica.gonzalez@utalca.cl
Department of Chemistry, Faculty of Sciences, Universidad de Chile, Las Palmeras 3425, Nuñoa,
Santiago 7800024, Chile; areche@uchile.cl
Biology Department, Faculty of Sciences, Universidad de Chile, Santiago 7800024, Chile; mnunez@uchile.cl
Department of Organic Chemistry, Faculty of Chemistry, Pontificia Universidad Católica de Chile, Casilla 306,
Santiago 6094411, Chile
Centro Integrativo de Biología y Química Aplicada (CIBQA), Universidad Bernardo O’Higgins,
General Gana 1702, Santiago 8370854, Chile
2
3
4
5
6
7
8
9
10
Facultad de Ciencias Naturales y Matemáticas, Universidad de Ibagué, Carrera 22 Calle 67,
Ibagué 730002, Colombia
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
*
†
Correspondence: afierroh@uc.cl (A.F.); jose.garcia@unibague.edu.co (O.G.-B.)
These authors contributed equally to this work.
Citation: Moya-Alvarado, G.; Yañez,
O.; Morales, N.; González-González,
A.; Areche, C.; Núñez, M.T.; Fierro,
A.; García-Beltrán, O.
Abstract: Fourteen coumarin-derived compounds modified at the C3 carbon of coumarin with an
-unsaturated ketone were synthesized. These compounds may be designated as chalcocoumarins
α,β
Coumarin-Chalcone Hybrids as
Inhibitors of MAO-B: Biological
Activity and In Silico Studies.
(3-cinnamoyl-2H-chromen-2-ones). Both chalcones and coumarins are recognized scaffolds in medic-
inal chemistry, showing diverse biological and pharmacological properties among which neuro-
protective activities and multiple enzyme inhibition, including mitochondrial enzyme systems,
stand out. The evaluation of monoamine oxidase B (MAO-B) inhibitors has aroused considerable
interest as therapeutic agents for neurodegenerative diseases such as Parkinson’s. Of the fourteen
chalcocumarins evaluated here against MAO-B, ChC4 showed the strongest activity in vitro, with
IC50 = 0.76 ± 0.08 µM. Computational docking, molecular dynamics and MM/GBSA studies, confirm that
ChC4 binds very stably to the active rMAO-B site, explaining the experimental inhibition data.
Molecules 2021, 26, 2430. https://
doi.org/10.3390/molecules26092430
Academic Editor: Maria João Matos
Received: 1 April 2021
Accepted: 18 April 2021
Published: 22 April 2021
Keywords: chalcocoumarin; MAO-B; molecular dynamics; in silico studies; neurodegenerative diseases
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Coumarins (
of natural and synthetic origin, that show numerous biological [
istry activities, such as anticoagulant, anticancer, antioxidant, antiviral, anti-diabetic, anti-
inflammatory, antibacterial, antifungal and anti-neurodegerative properties [ ], among
α
-benzopyrones, 2H-chromen-2-ones) are a large family of compounds,
1–6] and medicinal chem-
7–9
Copyright:
© 2021 by the authors.
which recent studies have paid special attention to enzyme inhibition. With regard to
monoamine oxidase (MAO) inhibition, recent findings have revealed that MAO affinity
and selectivity can be efficiently modulated by appropriate substitutions on the coumarin
ring system [1,10–13].
MAOs (EC 1.4.3.4) are flavoproteins located in the outer mitochondrial membrane
and involved in the oxidative deamination of endogenous and exogenous monoamines
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Attribution (CC BY) license (https://
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Molecules 2021, 26, 2430. https://doi.org/10.3390/molecules26092430
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Molecules 2021, 26, 2430
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using oxygen (O2) as electron acceptor. In humans they exist in two isoforms called MAO-
A and MAO-B. The high resolution crystal structures of both human isoforms A and B
(hMAO) rat MAO-A (rMAO-A) have made it possible to analyze binding modes of ligands
inside these macromolecules [14]. While the active site is formed by the common FAD
cofactor and similar amino acid residues in the different forms, these are distinguished by
their selectivity for substrates and inhibitors [15]. Thus, serotonin and noradrenaline are
substrates of MAO-A which is selectively inhibited by clorgyline, while MAO-B oxidizes
β-phenylethylamines and benzylamines and is selectively inhibited by l-deprenyl. MAO
genes are expressed in various tissues. However, in the brain, although both isoforms
are widely distributed, MAO-B is expressed in high concentrations in the hypothalamus,
striatum, globus pallidus and thalamus, and mainly in serotonergic cells while the A iso-
form is rather evenly distributed, mainly in the cortex, and in nuclei containing preferably
catecholaminergic and glial cells [16–21].
Although knowledge about MAO inhibition by compounds containing coumarin scaf-
folds is scarce, publications of articles describing new inhibitors of this class of compounds
are increasing. The variety of substitutions on the coumarin ring provide insight into the
influence on the activity-structure relationship. Among the most reported modifications
of the coumarin ring with MAO activity are on C3 and the steric effect of the substituent
appears to be important in modulating MAO-B inhibitory activity [11]. In addition, it
has been reported that the introduction of various substituents at the para position of the
3-phenyl ring is a good strategy for improving the desired MAO-B inhibitory activity [22
]
and when the 3-phenyl skeleton is replaced by a 3-benzoyl group, the activity is strongly
diminished [20]. It has also been observed that coumarins substituted with 3-indolyl and
3-thiophenyl shows greatest selective inhibition was against MAO-B [11,23,24].
In this work a merger of the coumarin scaffold and a 3-cinnamyl group led to new
hybrid (chalcocoumarin) derivatives (Scheme 1) that preserve structural characteristics
of compounds with the ability to interact with MAO. The synthetic strategy chosen al-
lowed a large variety of substituents on the cinnamyl benzene ring to be accessed using
different readily accessible benzaldehydes. Thus, the quantity and/or type of interactions
with the enzyme were explored involving some bulky groups to determine their possible
contribution to the biological activity as MAOIs. Our new compounds were screened
versus both MAO isoforms, and in silico studies were carried out to rationalize their main
interactions in the MAO active cavity. The computational biochemistry tools were used
considering the geometrical restrictions and most probable positions in the formation
of the ligand-receptor complex. The chalcocoumarin molecules were subjected to theo-
retical studies in which binding energies were estimated using docking and MM/GBSA
analysis. In addition, physicochemical parameters that are responsible for governing the
pharmacokinetic properties of drug molecules were determined.
Scheme 1. Hybrids of chalcocoumarin.

Molecules 2021, 26, 2430
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In the present study, a series of coumarin-chalcone hybrid compounds were synthe-
sized and tested on the 2 MAO isoforms. The activity shown was selective for MAO-B
and in particular, compound ChC4 showed the highest inhibitory activity on rMAO-B at
submicromolar concentrations. The results obtained will be useful to understand the mode
of inhibition of chalcocoumarins against rMAO-B, and to help predict the activities of these
new inhibitors that could be promising as therapeutics to treat neurodegenerative diseases
such as Parkinson’s disease.
2. Results and Discussion
2.1. Chemistry
The route employed to synthesize the compounds is summarized in Scheme 1. The
compounds were obtained starting from resorcinol (
Vilsmaier-Haack reaction [25]. Knoevenagel condensation of the aldehyde intermediate
with ethyl acetoacetate afforded hydroxycoumarin . The 7-hydroxycoumarin obtained
was methylated using a Williamson reaction using methyl sulfate as methylating agent,
obtaining the compound , finally the compounds derived the 3-cinnamoyl-2H-chromen-
2-one (ChC1 ChC14) (Table 1) were prepared in moderate yields (25–47%, unoptimized)
1), which was formylated using the
2
3
–
by Claisen-Schmidt condensation with the respective aldehyde (Supplementary Materials;
Scheme S1) [26]. Coumarin-chalcone hybrids have been studied and are currently still being
synthesized for various uses and their spectroscopy is well known, however, we will detail
some signals that are key to their identification. The 1H-NMR spectra of the compounds
ChC1
identifies this type of molecules, the neighboring vinyl protons of the
ketone appear at very close low field from the aromatic proton region. These protons
present signals corresponding to two doublets with variable between 8.5 and 7.0 and
with Jab = 16 Hz on average. this high constant corresponds to a trans isomer [27 29]. As
for the 13C-NMR spectrum, we will mention typical signals such as carbonyl shifts. first of
all, we will detail that the carbon of the -unsaturated ketone has a 190–180 ppm and
29], the compounds were
–
ChC14 present very similar chemical shift patterns with a particular signal that
α,β-unsaturated
δ
–
α
,β
δ
carbonyl carbons of α-pyrone δ 165–155 pmm on average [27–
characterized by 1H and 13C NMR (Supplementary Materials; Figures S1–S15).
Table 1. Structures of the synthetized chalcocoumarin hybrids.
Compounds
R1
R2
R3
R4
ChC1
ChC2
ChC3
ChC4
ChC5
ChC6
ChC7
ChC8
ChC10
ChC11
ChC12
H
OH
OCH3
H
H
H
OCH3
OCH3
H
H
H
H
H
H
OH
OCH3
H
OCH3
H
H
H
H
H
H
OCH3
H
H
OH
SCH3
OCH3
H
H
H
H
H
H
H
OCH3
OCH3
H
H
H
Br
Br

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Table 1. Cont.
Compounds
R1
R2
R3
R4
ChC13
ChC14
H
H
H
H
N(CH3)2
Br
H
H
2.2. Biological Analysis in Rat MAO
Fourteen derivatives differing in the substitution pattern of the cinnamyl benzene
ring were studied, these compounds were tested on rat MAO-A and B to determine their
inhibitory activity MAO. A general screening was carried out at 10 µM finding moderate
activity for some of the compounds against rMAO-B but none against rMAO-A. Thus, five
molecules were identified as possibly selective IMAO-B.
ChC4
cromolar in vitro potencies, all below 10
with a hydroxyl group on the meta position of the variable ring, displayed the highest
rMAO-B inhibitory activity (IC50 = 0.76 M). Interestingly, changing the position of the
,
ChC5
,
ChC6
,
ChC9 and ChC11 in MAO-B exhibited micromolar or submi-
µM (Table 2). Out of these ChC4, substituted
µ
hydroxyl group from meta to ortho or para (ChC2 and ChC10 respectively) led to loss of
the inhibitory activity. An approximately 12-fold lower IMAO activity was observed when
the hydroxyl group (in ChC4) was methylated (ChC5). This might be attributed to steric
hindrance and/or to the loss of hydrogen bonding donor quality which could be crucial
for some interaction in the binding site. Moving the methoxyl group from the meta to the
para position (ChC6 vs. ChC5) slightly increased potency.
Table 2. IC50 of the compounds in rMAO-A and rMAO-B.
Compounds
IC50 (µM) rMAO A
IC50 (µM) rMAO B
ChC1
ChC2
ChC3
ChC4
ChC5
ChC6
ChC7
ChC8
ChC9
ChC10
ChC11
ChC12
ChC13
ChC14
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.76 ± 0.08
9.63 ± 0.90
6.96 ± 0.07
>10
>10
3.71 ± 0.68
>10
5.88 ± 0.57
>10
>10
>10
Each IRC5e0pvlaalucienwgasthoebtmaineetdhforoxmy agnroavuepraogefoCf hthCre6e ewvaitluhaatiobnus l(kni=er3,).less electronegative and more
polarisable methylthio group (ChC11) only produced ChC6 is less potent than ChC11
The second most potent molecule was ChC9, with a methylenedioxy group bridging the
meta and para carbons. The methylenedioxy group increases the rigidity of the molecule,
possibly stabilizing the complex protein-ligand interaction. The same effect, extending the
rigidity, has been observed, on other derivatives as IMAO [30,31].
.

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2.3. Molecular Docking and Ligand Efficiency Analysis
To analyze the changes in potency of the coumarin-chalcone hybrids, docking studies
were carried out using the crystal structure of rMAO-A and the homology model of rMAO-
B (Supplementary Materials; Figures S16 and S17), analyzing the possibility that each one of
them has to form a stable complex with each of the 14 molecules synthesized by us. Table 3
shows that most of these molecules present better interactions with the rMAO-B binding
site, since the corresponding energies are at least 2.5 kcal mol more negative in all but one
of the cases. This difference could be due to the substitutes present in the molecules. The
results of the molecular docking experiments showed more favorable interactions (more
negative
∆
E
binding) for the complexes in rMAO-B than in rMAO-A, with average values
around
−
9.26 kcal 6.57 kcal
·
mol−1 vs. mol−1 respectively) which are in accord with the
−
·
experimental data for the whole series. In the rMAO-B, although no major differences were
observed in the binding modes of the active compounds, subtle energy differences were
identified. The results of this molecular docking study point to strong interactions of the
chalcocoumarins in the binding pocket of rMAO-B, but considerably weaker in rMAO-A.
Table 3. Molecular docking results for ChC1–ChC14 in the rMAO-A/rMAO-B models. Intermolecular docking energy
values (∆Ebinding), Kd values and calculated Ligand Efficiency (LE) for the rMAO-A and rMAO-B complexes.
Docking Results a
Ligand Efficiency
rMAO-A
rMAO-B
rMAO-A
rMAO-B
Compound
∆Ebinding (kcal·mol−1
)
∆Ebinding (kcal·mol−1
)
Kd
LE (kcal·mol−1
)
Kd
LE (kcal·mol−1
)
2.03×10−5
1.22×10−5
1.22×10−5
2.41×10−5
1.72×10−5
1.45×10−5
2.85×10−5
1.22×10−5
1.03×10−5
2.03×10−5
2.03×10−5
1.22×10−5
7.41×10−6
1.45×10−5
1.52×10−7
6.57×10−8
2.14×10−7
6.57×10−8
9.21×10−8
1.52×10−7
4.20×10−7
9.21×10−8
5.55×10−8
1.80×10−7
3.00×10−7
5.28×10−6
5.55×10−8
1.29×10−7
ChC1
ChC2
ChC3
ChC4
ChC5
ChC6
ChC7
ChC8
ChC9
ChC10
ChC11
ChC12
ChC13
ChC14
−6.4
−6.7
−6.7
−6.3
−6.5
−6.6
−6.2
−6.7
−6.8
−6.4
−6.4
−6.7
−7.0
−6.6
−9.3
−9.8
−9.1
−9.8
−9.6
−9.3
−8.7
−9.6
−9.9
−9.2
−8.9
−7.2
−9.9
−9.4
0.278
0.279
0.268
0.262
0.260
0.264
0.229
0.248
0.261
0.246
0.256
0.248
0.269
0.275
0.404
0.408
0.364
0.408
0.384
0.372
0.322
0.355
0.380
0.353
0.356
0.266
0.380
0.391
a
In each site, the energy was calculated to see which site bound more strongly to the ligand. In bold ChC4 displayed the highest rMAO-B
inhibitory activity.
When analyzing the docking results for rMAO-B from the conformational viewpoint,
it is necessary to consider the residues that constitute the substrate-binding site of rMAO-B,
which is composed of the FAD cofactor, two flanking residues, Tyr398 and Tyr435, that
form an “aromatic box”, and a number of others, particularly Cys172, Tyr326, Met341,
Ser200 Gln206 and Thr314 [32,33]. The results show that all the chalcocoumarins settle
in the active site of rMAO-B (Supplementary Materials; Figure S18), with the benzene
ring of the coumarin moiety close to the FAD, more specifically the central N-5, at a
distance of about 4.0 Å. The benzene ring of the cinnamyl moiety extends into the generally
hydrophobic entrance cavity adjoining the substrate-binding site. The mere length of the
ChC molecules indicates that to bind in the active site of MAO-A the latter must undergo a
rearrangement of the residues separating the entrance and the substrate cavities, which
may explain their general preference for MAO-B.
ChC4 was located inside the cavity interacting with Tyr435, Tyr398, Tyr60, Phe343,
Asn83, Arg307, Thr314, and Leu328. Two hydrogen bonds where generated with Asn83 and,
via its C-30 hydroxyl group, Thr316. ChC2 actually when interact with the amino acids
into the pocket adopt a planar conformation because the hydrogen bond confirms our
discussion that could be responsible for none activity of ChC2 in rMAO-B. A quantum
geometric optimization of ChC2 and ChC4 at the M05-2X-D3/6-31G(d,p) level of theory,
showed their C-20 and C-30 hydroxyl groups pointing in opposite directions, suggesting

Molecules 2021, 26, 2430
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different preferred intramolecular interactions (Figure 1). Both the different electronic
potential distribution and the resulting preferred intermolecular interaction might be
responsible for the difference in in IC50 values.
The best three ligands obtained from the docking exhibit low Kd values, these lig-
ands are ChC4, ChC9 and ChC13, which means that these ligand/rMAO-B complexes
are the most stable in the series. These results are consistent with those obtained in
the docking experiments in which these complexes were the most stable according to
their
LE > 0.3 kcal
for development as an rMAO-B inhibitor with a LE value of 0.408. Although ChC2 and
ChC4 have similar Kd, LE and Ebinding values, the ChC2 molecule does not show in vitro
∆
Ebinding values. The proposed tolerable values of LE for inhibitor candidates are
·
mol−1
[34–36]. According to this reference value, ChC4 is a good prospect
∆
activity against rMAO-B, on the other hand, ChC4 has a good inhibitory activity against
rMAO-B, since micromolar concentrations are needed to inhibit it, which is consistent with
the values obtained for the Kd. ChC1 would appear to be almost as good, with LE = 0.404,
but again its activity, if any, is worse than our cutoff value. The low micromolar-active
ChC5, ChC6, ChC9 and ChC11 have LE values of 0.384, 0.372, 0.380 and 0.356, respectively,
and the less (or in-) active ChC13 and ChC14 have LE values of 0.380 and 0.391.
Figure 1.
(right). (
(
A
) NCIplot of the non-covalent interaction regions with isosurface gradient (0.6 au) for ChC2 (left) and ChC4
B) Electrostatic potential (in a.u.) of ChC2 (left) and ChC4 (right) mapped on the 0.001 a.u. isodensity surface for
the selected structure computed at the M05-2X-D3/6-31G(d,p) level of theory.
2.4. Analysis of Molecular Dynamics Simulations
Molecular dynamics simulations were performed for 100 ns to analyze the confor-
mational stability of the rMAO-B/ChC2 and rMAO-B/ChC4 complexes. The RMSD, a
quantitative parameter, was used to estimate the stability of the protein-ligand systems
and the apoprotein. The RMSD in Figure 2A shows that the rMAO-B/ChC2 and rMAO-
B/ChC4 complexes remain highly stable throughout the simulation time. We can see that
the structures of the complexes does not change significantly. The RMSD values for the
ChC4 complex are remarkably constant about 1.5 Å, with a very slight instability and
increase near the end of the simulation. The ChC2 complex shows similar, somewhat less
stable behavior for almost 40 ns, and then its RMSD value falls abruptly to about 1.0 Å and
rises slowly with appreciable fluctuations to about 1.2 Å at 100 ns, indicative of weaker

Molecules 2021, 26, 2430
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binding in the rMAO-B site. However, a maximum difference of 3.0 Å in the RMSD is
taken to indicate that a system is in equilibrium [37], so this condition is fulfilled by both
compounds. To complement the analysis carried out calculating the RMSD, the Radius of
Gyration (RGyr) was analyzed for the same runs. From this analysis (Figure 2B), we can
conclude that the RGyr of ChC2 and ChC4 oscillate in a narrow interval between 4.3–4.8 Å.
These stable values during the 100 ns simulation indicate again that ligand binding does
not induce major conformational changes in the protein structure.
Figure 2. (A) Root Mean Square Deviation (RMSD) and (B) Radius of gyration (RGyr) as a function of
simulation times for the complexes formed between rMAO-B and ChC2 and ChC4.
Structural studies in MAO have shown that two residues Tyr398 and Tyr435 in MAO-B
located in the active site approximately perpendicular to the FAD play a functional role in
this enzyme, acting as a cofactor stabilizing the active site, forming an aromatic box whose
function is to stabilize the ligand [13]. Molecular simulation results show a difference in
the interaction of the compounds ChC2 and ChC4 with the FAD cofactor, see Figure 3A.
Compound ChC2 shows a spacing that fluctuates between 17.0 Å and 20.0 Å from its
original position, signifying a null interaction with the FAD cofactor. On the other hand,
the compound ChC4 is within the range of interaction with the FAD cofactor. This distance
was measured between the nitrogen atom of the alloxazine planar ring of FAD and the
center of the benzaldehyde aromatic ring of compounds, Figure 3B.
Molecular dynamics simulations showed of rMAO-B that residues that interact with
the ligands ChC2 and ChC4, see Figure 4. The most frequent residues in rMAO-B/ChC2
were Ile164, Ile199, Leu167, Leu171, Phe168, Pro104, Trp119, Val316, Phe103, Pro102,
Tyr115 and Thr196. In contrast, the most frequent residues in rMAO-B/ChC4 were Ile164,
Ile199, Leu171, Phe168, Pro104, Trp119, Tyr326, Val316, Cys172 and Tyr115 with van der
Waals and hydrogen bonds interactions. Highlighting residues Cys172 and Tyr326, which

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are important for the active site of the rMAO-B flavoprotein. Tyr326 and Cys172 are
key residues that determines substrate and inhibitor specificity, also exhibits conforma-
tional changes on the inhibitor binding and restricts the binding of certain inhibitors
(e.g., harmine) to human MAO-B [38]. These results documents that ChC4 is a reversible
inhibitor of rMAO-B.
Figure 3.
(A
) Last frame of the molecular simulation showing the positions between the FAD molecule
and ChC2
–
ChC4 compounds interacting with rMAO-B. ( ) Distance as a function of simulation time,
B
between the nitrogen atom of the aloxazine planar ring of FAD and the center of the benzaldehyde
aromatic ring, for compounds ChC2 and ChC4. Dashed lines represent the position of the nitrogen
atom of the aloxazine planar ring.
Figure 4. Frequency of the appearance of residues at a distance of 3.0 Å or closer from ligands (
calculated using MD procedures.
A) ChC2 and (B) ChC4
The analyses of trajectories indicate that during most of the simulation the ligand
ChC4 maintain hydrogen bonds with residues of the active site of rMAO-B. However, the
number of hydrogen bonds formed was different for ChC2 and ChC4 (Figure 5). ChC2
formed two hydrogen bonds between the residues Glu483 and Tyr115, highlighting the
participation of the residues Val316, Ala325, Ile164 and Leu167. Finally, ChC4 formed
two hydrogen bonds with the Phe168, Cys172, Ile164 and Tyr115, highlighting the par-
ticipation of the residues Ile199, Trp119 and Tyr326. These residues, see Figure 6, are

Molecules 2021, 26, 2430
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consistent with previous theoretical-experimental studies carried out [39
,
40] where they
detail the interaction that some of the synthesized compounds have with the active site
of the rMAO-B. This difference in the formation of hydrogen bonds with key residues in
rMAO-B could be explained the difference in experimental activity between the ChC2 and
ChC4 compounds.
Figure 5. Fraction (in %) of intermolecular hydrogen bonds for rMAO-B interacting with (A) ChC2 and (B) ChC4. The
graph bar shows the most common hydrogen bonds formed between the residues on the pocket and the inhibitors.
Figure 6. Schematic representations at the end (100 ns) of their respective production runs for ligands (A)
ChC2 and (B) ChC4 bound to rMAO-B. (I) The surrounding amino acid residues in the binding pocket of
rMAO-B within 4Å from ligands. (II) Two-dimensional interaction map of ChC2 and ChC4 and rMAO-B.
The arrows indicate potential interactions between amino acid residues and the ligands.
Finally, the binding free energy (MM/GBSA) was computed after the MD simulation;
the last 70 ns for all the complexes and the results are given in Table 4. The compound
ChC2 has a binding free energy of -29.06 kcal
mol−1 with rMAO-B enzyme, while the
·
compound ChC4 showed relatively binding free energy of –25.87 kcal·mol−1. The results

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obtained from MM/GBSA show a slight difference in their binding free energy between
ChC2 and ChC4 compounds bound to rMAO-B. This slight difference is due to the R1 to
R2 position of the hydroxyl group in benzaldehyde aromatic ring. In particular, the ChC4
compound has a better activity at the experimental and in silico level.
Table 4. Predicted binding free energies (kcal
mol−1) and individual energy terms calculated from
·
molecular dynamics simulation through the MM-GBSA protocol for rMAO-B complexes.
−1
Calculated Free Energy Descomposition (kcal·mol
)
Compounds
∆Gbinding
∆EvdW
∆Eelect
∆Epot
ChC2
ChC4
−29.06 ± 0.10
−25.87 ± 0.09
−39.69 ± 0.13
−44.94 ± 0.09
15.03 ± 0.17
24.90 ± 0.04
−24.65 ± 0.10
−20.03 ± 0.09
2.5. In Silico Pharmacokinetic Prediction
A good drug candidate is absorbed in required time and well distributed throughout
the system for its effective metabolism and action. Toxicity is another very important factor
that often overshadows the ADME behaviour. SwissADME explorer online was used for
in silico prediction of drug likeness of the synthesized compounds (ChC1
–ChC14) based
on various molecular descriptors and the results are presented in Table 5.
Table 5. In silico predicted physicochemical properties of all compounds ChC1–ChC14.
MW
(g/mol)
TPSA
(Å2)
Log Kp
(cm/s)
Nº
Violations
Compounds Log P
HBA
HBD
RB
Log S
ChC1
ChC2
ChC3
ChC4
ChC5
ChC6
ChC7
ChC8
ChC9
ChC10
ChC11
ChC12
ChC13
ChC14
3.38
2.9
307.32
323.32
337.35
323.32
337.35
337.35
367.37
367.37
351.33
353.35
353.41
495.14
351.42
386.22
63.6
83.83
72.83
83.83
72.83
72.83
82.06
82.06
82.06
93.06
88.9
4
5
5
5
5
5
6
6
6
6
4
5
4
4
1
2
1
2
1
1
1
1
1
2
1
1
1
1
4
4
5
4
5
5
6
6
4
5
5
5
6
4
−4.89
−4.94
−5.06
−4.94
−5.06
−5.06
−5.22
−5.22
−5.08
−5.11
−5.95
−6.49
−5.86
−5.61
−5.43
−5.79
−5.64
−5.79
−5.64
−5.64
−5.84
−5.84
−5.84
−5.99
−5.35
−5.62
−5.04
−5.43
0
0
0
0
0
0
0
0
0
0
0
0,1 *
0
0
3.36
2.97
3.27
3.27
3.28
3.27
3.23
2.92
3.82
4.48
4.24
3.91
72.83
63.6
63.6
MW = 150–500 g/mol; TPSA = 20 Å2–130 Å2; HBA = N
Insoluble < 10 < Poorly < 6 < Moderately < 4 < Soluble <
Lipinski, Ghose, Veber, Egan and Muegge rules. * Violation of Ghose and Muegge rules.
º
of H-bond acceptors
2; Log P
≤
10; HBD = N
º
of H-bond donor
≤
5; RB = 0–9; Log S =
Violations of
−
−
−
−
≤
5; log Kp ≥ −2.5 considered to be permeable; N
º
The most potent compound ChC4 in biological experiment data having logP value of
2.97, it’s clear that it doesn’t violate of five Lipinski rules, while the other molecules have
logP values in the range of 2.90–4.48 and are expected to be orally active. In addition, the
logS values for ChC4 have a value of
−
4.94 indicating proper solubility, which is an indica-
tion of favorable drug like property, makes compound ChC4 promising drug candidate
for further research and development. Thirteen of fourteen synthesized molecules do not
break the rules of Lipinski, Ghose, Veber, Egan and Muegge, since the molecule ChC12
breaks the rules of Ghose and Muegge.
The Boiled-egg model is proposed as an accurate predictive model that works by
computing the lipophilicity and polarity of small molecules. The Boiled-egg analysis
of the fourteen molecules (Figure 7) has shown that compounds ChC1
ChC6 ChC12 ChC13 and ChC14 are highly absorbable at the brain barrier, whereas
compounds ChC2 ChC4 ChC7 ChC8 ChC9 ChC10 and ChC11 are highly absorbable
in the gastrointestinal tract.
, ChC3, ChC5,
,
,
,
,
,
,
,

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Figure 7. Predictive human intestinal absorption (HIA) model and blood-brain barrier permeation
(BBB) method (boiled-egg plot) of the 14 compounds.
The ADMET properties showed much similarity among the thirteen molecules that
can be used for advanced clinical trials.
3. Materials and Methods
3.1. Solvents and Reagents
Solvents and reagents (analytical grade and spectroscopic grade) were obtained from
Sigma-Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany). Melting points
were determined on a Galen III hot-plate microscope (Reichert-Jung, St. Louis, MO, USA)
1
equipped with a thermocouple. H- and 13C-NMR spectra were recorded on a 400 MHz
multidimensional spectrometer (Bruker Corporation, Billerica, MA, USA) using the solvent
or the TMS signal as an internal standard.
3.2. Synthesis
3-Cinnamoyl-7-methoxy-2H-chromen-2-one (ChC1). 3-Acetyl-7-methoxy-2H-chromen-
2-one (0.44 g, 2.0 mmol) and benzaldehyde (0.21 g, 2.0 mmol) were dissolved in 25 mL of
DCM and to this solution 0.5 mL of piperidine were added. The mixture was kept at reflux
temperature, monitoring the reaction by TLC for 10 h. The solution was concentrated under
reduced pressure and dissolved in a small aliquot of DCM and then MeOH was added
in excess to induce precipitation. This procedure was performed twice. The precipitate
was finally purified by column chromatography on silica gel eluting with DCM: 0.25 g
yellow solid, 40.8%, m.p. 190–192 º δ 8.59 (s, 1H, Ar-H), 8.01 (d, 1H,
C; 1H NMR (CDCl3):
J = 15.8 Hz, Ar-CH), 7.85 (d, 1H, J = 15.8 Hz, CO-CH=), 7.67 (s, 2H, Ar-H), 7.56 (d, 1H,
J = 8.6 Hz, Ar-H), 7.40 (s, 3H, Ar-H,), 6.90 (dd, 1H, J = 8.6, 1.0 Hz, Ar-H), 6.85 (s, 1H, Ar-H),
3.91 (s, 3H, OCH3). 13C-NMR (CDCl3):
δ
56.1, 100.2, 112.4, 113.8, 124.0, 129.0, 130.6, 131.5,
135.3, 144.3, 148.5, 157.8, 160.0, 165.0, 186.3.
(E)-3-(3-(2-Hydroxyphenyl)acryloyl)-7-methoxy-2H-chromen-2-one (ChC2). 3-Acetyl-7-
methoxy-2H-chromen-2-one (0.44 g, 2.0 mmol) and o-hydroxybenzaldehyde (0.24 g, 2.0 mmol)
were reacted and worked up according to the previous procedure: 1.75 g, pale white solid,
95.6%, m.p.; 188–190 ◦C. 1H-NMR (DMSO-d6),
δ 10.43 (s, 1H, OH), 8.71 (s, 1H, Ar-H), 8.04
(d, 1H, J = 15.9 Hz, Ar-CH=), 7.92 (d, 1H, J = 8.8 Hz, Ar-H), 7.89 (d, 1H, J = 15.9 Hz), 7.69
(dd, 1H, J = 7.7, 1.0 Hz, Ar-H), 7.35 (t, 1H, J = 7.0 Hz, Ar-H), 7.13 (d, 1H, J = 2.2 Hz, Ar-H),

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7.08 (dd, 1H, J = 8.6, 2.2 Hz, Ar-H), 7.00 (d, 1H, J = 8.0 Hz, Ar-H), 6.94 (t, 1H, J = 7.5 Hz,
Ar-H), 3.96 (s, 3H, OCH3), 13C-NMR (DMSO-d6):
δ 56.7, 100.8, 112.5, 113.9, 116.8, 120.0,
121.8, 121.9, 124.3, 129.2, 132.3, 132.6, 139.5, 148.2, 157.4, 157.8, 159.4, 165.1, 187.1.
(E)-7-methoxy-3-(3-(2-methoxyphenyl)acryloyl)-2H-chromen-2-one (ChC3). 3-Acetyl-7-methoxy-
2H-chromen-2-one (0.44 g, 2.0 mmol) and o-methoxybenzaldehide (0.27 g, 2.0 mmol), were
reacted and worked ◦up according to the previous procedure: 0.330 g, pale white solid,
1
49%, m.p.. 184–186 C; H-NMR (CDCl3):
δ
8.55 (s, 1H, =C-H), 8.21 (d, 1H, Ar-CH=,
J = 15.8 Hz), 8.04 (d, 1H, CO-CH=, J = 15.8 Hz), 7.71 (d, 1H, Ar-H, J = 7.6 Hz), 7.56 (d, 1H,
Ar-H, J = 8.4 Hz), 7.37 (t, 1H, Ar-H, J = 7.9), 6.98 (t, 1H, Ar-H, J = 7.7 Hz), 6.92 (d, 1H, Ar-H,
J = 8.2 Hz), 6.90 (d, 1H, Ar-H, J = 8.5 Hz), 6.85 (s, 1H, Ar-H), 3.91 (s, 6H, 2 OCH3); 13C-NMR
(DMSO-d6):
δ 55.6, 56.0, 100.4, 111.2, 112.5, 113.7, 120.8, 121.9, 124.1, 124.6, 129.3, 131.3,
132.0, 139.9, 148.2, 157.6, 159.0, 159.8, 165.0, 186.8.
(E)-3-(3-(3-hydroxyphenyl)acryloyl)-7-methoxy-2H-chromen-2-one (ChC4). 3-Acetyl-7-
methoxy-2H-chromen-2-one (0.44 g, 2.0 mmol) and m-hydroxybenzaldehyde (0.24 g,
2.0 mmol) were reacted and worked up according to the previous procedure: 0.195 g,
◦
white solid, 30%, m.p. 184–186 C; 1H NMR (DMSO-d6):
δ 9.77 (sbr, 1H), 8.76 (s, 1H, =C-H),
7.94 (d, 1H, Ar-H, J = 8.0 Hz), 7.82 (d, 1H, Ar-CH=, J = 15.9 Hz), 7.71 (d, 1H, CO-CH=,
J = 15.9 Hz), 7.62 (d, 1H, Ar-H, J = 8.6 Hz), 7.33 (m, 1H, Ar-H), 7.25-7.17 (m, 2H, Ar-H), 7.15
(s, 1H, Ar-H), 7.09 (dd, 1H, Ar-H, J = 8.0, 1.0 Hz), 6.94 (dd, 1H, Ar-H, J = 8.0, 1.0 Hz), 3.97
(s, 3H, OCH3); 13C NMR (DMSO-d6): 56.7, 100.9, 112.5, 114.0, 114.8, 118.5, 120.5, 121.6,
124.9, 130.6, 132.5, 136.3, 143.9, 148.6, 157.5, 158.2, 159.4, 165.3, 186.7.
(E)-7-methoxy-3-(3-(3-methoxyphenyl)acryloyl)-2H-chromen-2-one (ChC5). 3-Acetyl-7-methoxy-
2H-chromen-2-one (0.44 g, 2.0 mmol) and m-methoxybenzaldehide (0,27 g, 2.0 mmol), were
reacted and worked up according to the previous procedure: 0,280 g, pale white solid, 42%,
◦
m.p. 164–166 C; 1H-NMR (CDCl3):
δ 8.58 (s, 1H, =C-H), 7.99 (d, 1H, Ar-CH=, J = 15.9 Hz),
7.82 (d, 1H, CO-CH=, J = 15.9 Hz), 7.57 (d, 1H, Ar-H, J = 8.0 Hz), 7.35-7.25 (m, 2H, Ar-H),
7.18 (s, 1H, Ar-H) 6.96 (dd, 1H, Ar-H, J = 8.0, 2.0 Hz), 6.91 (dd, 1H, Ar-H, J = 8.8, 2.0 Hz),
6.85 (d, 1H, Ar-H, J = 2.0 Hz), 3.83 (s, 3H, OCH3), 3.76 (s, 3H, OCH3); 13C-NMR (CDCl3):
δ
55.8, 56.5, 100.8, 112.8, 113.9, 114.3, 117.1, 121.7, 122.0, 124.9, 130.3, 131.8, 136.8, 144.8, 149.0,
158.1, 160.2, 160.3, 165.8, 186.8.
(E)-7-methoxy-3-(3-(4-methoxyphenyl)acryloyl)-2H-chromen-2-one (ChC6). 3-Acetyl-7-methoxy-
2H-chromen-2-one (0.44 g, 2.0 mmol) and p-methoxybenzaldehide (0.27 g, 2.0 mmol), were
reacted and worked◦up according to the previous procedure: 0.26 g, pale white solid,
1
39%, m.p. 158–160 C; H-NMR (CDCl3):
δ
8.58 (s, 1H, =C-H), 7.91 (d, 1H, Ar-CH=,
J = 15.9 Hz), 7.84 (d, 1H, CO-CH=, J = 15.9 Hz), 7.64 (d, 2H, Ar-H, J = 8.0 Hz), 7.56 (d, 1H,
Ar-H, J = 8.8 Hz), 6.93 (d, 2H, Ar-H, J = 8.0 Hz), 6.91 (dd, 1H, Ar-H, J = 8.8, 2.0 Hz), 6.85
(d, 1H, Ar-H, J = 2.0 Hz), 3.92 (s, 3H, OCH3), 3.86 (s, 3H, OCH3); 13C-NMR (CDCl3):
δ
57.2, 57.8, 102.0, 114.2, 115.5, 116.1, 123.4, 123.6, 129.6, 132.5, 133.0, 146.3, 150.0, 159.4, 161.6,
163.5, 166.8, 188.0.
(E)-3-(3-(3,4-dimethoxyphenyl)acryloyl)-7-methoxy-2H-chromen-2-one (ChC7). 3-Acetyl-
7-methoxy-2H-chromen-2-one (0.44 g, 2.0 mmol) and 3,4-dimethoxybenzaldehide (0.33 g,
2.0 mmol) were reacted and worked up according to the previous procedure: 0.42 g, bright
yellow solid, 57%, m.p. 182–184 ◦C; 1H-NMR (CDCl3):
δ 8.55 (s, 1H, =C-H), 8.16 (d, 1H,
Ar-CH=, J = 15.9 Hz), 8.00 (d, 1H, CO-CH=, J = 15.9 Hz), 7.55 (d, 1H, Ar-H, J = 8.6 Hz), 7.34
(d, 1H, Ar-H, J = 1.0 Hz), 7.19 (dd, 1H, AR-H, J = 8.0, 8.0 Hz) 7.07 (d, 1H, Ar-H, J = 8.0 Hz),
7.00 (dd, 1H, Ar-H, J = 8.0, 2.0 Hz), 6.94(d, 1H, Ar-H, J = 2.0 Hz), 3.90 (s, 3H, OCH3), 3.89 (s,
3H, OCH3), 3.87 (s, 3H, OCH3); 13C-NMR (CDCl3):
δ 55.9, 56.0, 61.5, 100.4, 112.4, 113.8, 114.4,
119.9, 121.6, 124.2, 125.5, 129.2, 131.3, 139.1, 148.4, 149.2, 153.2, 157.7, 159.7, 165.1, 186.7.
(E)-3-(3-(2,5-dimethoxyphenyl)acryloyl)-7-methoxy-2H-chromen-2-one (ChC8). 3-Acetyl-
7-methoxy-2H-chromen-2-one (0.44 g, 2.0 mmol) and 2,5-dimethoxybenzaldehide (0.33 g,
2.0 mmol), were reacted and worked up according to the previous procedure: 0,45 g, bright
yellow solid, 62%, m.p. 174–176 ◦C; 1H-NMR (CDCl3):
δ 8.55 (s, 1H, =C-H), 8.17 (d, 1H,
Ar-CH=, J = 15.6 Hz), 8.0 (d, 1H, CO-CH=, J = 15.6 Hz), 7.6 (dd, 1H, Ar-H, J = 8.0, 9.0 Hz),
7.2 (d, 1H, Ar-H, J = 2.0 Hz), 6.92-6.97 (m, 4H, Ar-H), 3.91 (s, 3H, OCH3), 3.87 (s, 3H, OCH3),

Molecules 2021, 26, 2430
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3.82 (s, 3H, OCH3); 13C-NMR (CDCl3):
δ
56.2, 56.4, 56.6, 100.7, 100.7, 112.8, 112.9, 113.7,
114.1, 114.3, 118.3, 125.1, 131.7, 131.9, 139.9, 148.6, 153.9, 154.0, 158.0, 165.4, 187.1.
(E)-3-(3-(benzo[d][1,3]dioxol-5-yl)acryloyl)-7-methoxy-2H-chromen-2-one (ChC9). 3-Acetyl-
7-methoxy-2H-chromen-2-one (0.44 g, 2.0 mmol) and piperonal (0.30 g, 2.0 mmol) were
reacted and worked up according to the previous procedure: 0.29 g, yellow solid, 41%, m.p.
◦
178–180 C; 1H-NMR (DMSO-d6):
δ 8.62 (s, 1H, =C-H), 7.84 (d, 1H, J = 12.0 Hz), 7.63 (d, 1H,
Ar-CH=, J = 15.7 Hz), 7.56 (d, 1H, CO-CH= J = 15.7 Hz), 7.68 (d, 1H, Ar-H, J = 8.0 Hz), 7.33
(s, 1H, Ar-H), 7.25 (d, 1H, Ar-H, J = 8.0 Hz), 7.06 (s, 1H, Ar-H), 7.03-6.94 (m, 3H, Ar-H), 6.07
(s, 2H, OCH2O), 3.87 (s, 3H, OCH3); 13C-NMR (CDCl3):
δ 55.8, 100.6, 101.2, 106.7, 108.4,
111.5, 122.8, 125.7, 128.1, 130.8, 134.6, 142.2, 147.3, 147.9, 153.6, 160.9, 163.5, 187.1.
(E)-3-(3-(4-hydroxy-3-methoxyphenylacryloyl)-7-methoxy-2H-chromen-2-one (ChC10). 3-
Acetyl-7-methoxy-2H-chromen-2-one (0.44 g, 2.0 mmol) and vainillin (0.30 g, 2.0 mmol),
were reacted and wor◦ked up according to the previous procedure: 0,340 g, yellow solid,
1
48.3%, m.p. 210–212 C; H-NMR (CDCl3):
δ
8.65 (s, 1H, =C-H), 8.27 (d, 1H, Ar-CH=,
J = 16 Hz), 8.10 (d, 1H, CO-CH=, J = 16 Hz), 7.65 (d, 1H, Ar-H, J = 8.0 Hz), 7.32 (d, 1H, Ar-H,
J = 2.0 Hz), 7.45 (dd, 1H, Ar-H, J = 8.8, 2.0 Hz) 7.00 (dd, 1H, Ar-H, J = 8.8, 2.0 Hz), 6.91 (d,
1H, Ar-H, J = 8.8 Hz), 6.93 (d, 1H, Ar-H, J = 2.0 Hz), 3.97 (s, 3H, OCH3), 3.92 (s, 3H, OCH3);
13C-NMR (CDCl3):
δ 55.9, 56.0, 100.4, 112.4, 112.5, 113.3, 113.7, 117.9, 121.7, 124.6, 124.8,
131.3, 139.6, 148.2, 153.5, 153.6, 157.6, 159.7, 165.7, 186.7.
(E)-3-(3-(4-mercaptophenyl)acryloyl)-7-methoxy-2H-chromen-2-one (ChC11). 3-Acetyl-7-
methoxy-2H-chromen-2-one (0.44 g, 2.0 mmol) and methyl(4-vinylphenyl)sulfane (0.27g,
2.0 mmol), were reacted and worked up according to the previous procedure: 0,305 g,
yellow solid, 43%.m.p. 196–198 ◦C; 1H-NMR (CDCl3):
δ 8.58 (s, 1H, =C-H), 7.99 (d, 1H,
Ar-CH=, J = 15.7 Hz), 7.82 (d, 1H, CO-CH=, J = 15.7 Hz), 7.58 (m, 3H, Ar-H), 7.26 (m, 3H,
Ar-H 6.91 (dd, 1H, Ar-H, J = (8.0, 2.0), 6.86 (d, 1H, Ar-H, J = 2.0 Hz), 3.91 (s, 3H, OCH3),
2.50 (s, 3H, SCH3); 13C-NMR (CDCl3):
δ 15.6, 56.4, 100.8, 112.9, 114.3, 121.9, 123.6, 126.3,
129.6, 131.7, 131.9, 142.9, 144.4, 148.8, 158.1, 160.2, 165.5, 165.6, 186.7.
(E)-3-(3-(3,5-dibromo-4-methoxyphenyl)acryloyl)-7-methoxy-2H-chromen-2-one (ChC12). 3-Acetyl-
7-methoxy-2H-chromen-2-one (0,44 g, 2.0 mmol) and 3,5-dibromo-4-methoxybenzaldehyde
(0.58 g, 2.0 mmol), were reacted and worked up according to the previous procedure:
0.21 g, yellow solid, 21.4%, m.p. 228–230 ◦C; 1H-NMR (CDCl3):
δ 8.57 (s, 1H, =C-H), 8.18.
(s, 2H, Ar-H), 7.95 (d, 1H, Ar-CH=, J = 16 Hz), 7.81 (d, 1H, CO-CH=, J = 16 Hz), 7.63 (d, 1H,
Ar-H, J = 8.0 Hz), 7.22 (dd, 1H, Ar-H, J = 8.0, 2.0 Hz), 6.98 (d, 1H, Ar-H, J = 2.0 Hz) 4.01 (s,
3H, OCH3), 3.95 (s, 3H, OCH3); 13C-NMR (CDCl3):
δ 60.9, 56.5, 100.5, 110.8, 111.7, 117.4,
123.7, 126.9, 132.3, 133.6, 136.1, 141.9, 147.7, 154.0, 154.5, 157.6, 159.4, 163.2, 188.1.
(E)-3-(3-(4-(dimethylamino)phenyl)acryloyl)-7-methoxy-2H-chromen-2-one (ChC13). 3-Acetyl-
7-methoxy-2H-chromen-2-one (0.44 g, 2.0 mmol) and 4-(dimethylamino)benzaldehyde (0.27 g,
2.0 mmol), were reacted and worked up according to the previous procedure: 0.15 g, red
solid, 22%, m.p. 220–222 ◦C; 1H-NMR (CDCl3): δ 8.56 (s, 1H, =C-H), 7.97 (d, 1H, Ar-CH=,
J = 15.7 Hz), 7.81 (d, 1H, CO-CH=, J = 15.7 Hz), 7.58 (d, 1H, Ar-H, J = 8.8 Hz), 7.54 (d, 2H,
Ar-H, J = 8.6 Hz), 6.88 (d, 1H, Ar-H, J = 8.0, 2.0 Hz), 6.85 (d, 1H, Ar-H, J = 2.0 Hz), 6.68 (d,
2H, Ar-H, J = 8.8 Hz), 3.90 (s, 3H, OCH3), 3.04 (s, 6H, N(CH3)2); 13C-NMR (CDCl3):
δ 40.5,
56.4, 64.1, 100.7, 112.2, 113.0, 114.0, 119.3, 122.6, 123.3, 131.4, 131.5, 146.4, 148.2, 152.6, 158.0,
160.3, 165.2, 186.4.
(E)-3-(3-(4-bromophenyl)acryloyl)-7-methoxy-2H-chromen-2-one (ChC14). 3-Acetyl-7-methoxy-
2H-chromen-2-one (0.44 g, 2.0 mmol) and p-methoxybenzaldehide (0.27 g, 2.0 mmol), were
reacted and worked up according to the previous procedure: 0.27 g, pale white solid, 35%,
◦
m.p. 158–160 C; 1H-NMR (CDCl3):
δ 8.76 (s, 1H, =C-H), 7.94 (d, 1H, Ar-H, J = 8.0, 8.0 Hz),
7.82 (d, 1H, Ar-CH=, J = 16 Hz), 7.71 (d, 1H, Ar-CH=, J = 16 Hz), 7.33 (d, 1, Ar-H, J = 8.0,
8.0 Hz), 7.25-7.18 (m, 2H, Ar-H), 7.15 (s, 1H, Ar-H), 7.09 (dd, 1H Ar-H, J = 8.0, 2.0 Hz), 6.94
(dd, 1H Ar-H, J = 8.0, 2.0 Hz), 3.97 (s, 3H, OCH3); 13C-NMR (CDCl3):
δ
57.2, 60.8, 110.2,
112.4, 118.5, 126.1, 129.6, 133.0, 134.2, 136.5, 142.0, 147.3, 154.1, 154.4, 159.6, 160.5, 186.7.

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3.3. Biological Assessment
The effect of coumarin derivatives on MAO-A and MAO-B were measured using a sus-
pension of crude rat brain mitochondria as enzyme source. 4-Dimethylaminophenethylamine
(4-DMAPEA, 2.5 µM) and 5-hydroxytryptamine (5-HT, 100 mM) were used as substrates selec-
tive of MAO-B or MAO-A, respectively. Evaluation of the test compounds on rMAO activity
was executed by measuring their effects on the production of 4-dimethylaminophenylacetic
acid (DMAPAA) by rMAO-B and 5-hydroxyindoleacetic acid (5-HIAA) by rMAO-A with
O2 using HPLC-ED (L-7110 LaChrom and amperometric detector L-3500 LaChrom Recipe,
Hitachi, (Tokyo, Japan) (for more detail see methodological references [41
,42]). The IC50
values (average SD was measured in two independent experiments each in triplicate)
±
were assessed representing percentage of inhibition in function of the negative logarithm of
different inhibitor concentrations (10−4 to 10−8) using the GraphPad Prism software [43].
3.4. Computational Analysis
3.4.1. Homology Modeling
Human monoamine oxidase B (hMAO-B) at 1.6Å resolution was used as template
(PDB code 1OJ9) to obtain a 3D structure of rat MAO-B (rMAO-B) using homology model-
ing. The amino acid sequence and crystal structure of the protein was extracted from NCBI
and PDB databases [44,45] considering the high level of amino acid identity (around 90%)
the target protein and template were aligned through a single alignment using MultAlin
interface [46]. MODELLER9v6 program [47] was used and 100 structures were prepared
using standard parameters and the outcomes were ranked on the basis of the internal
scoring function of the program (DOPE score). The best model was chosen as the target
model. The cofactor FAD was placed inside of MAO using the corresponding crystal
coordinates. To analyze the rMAO-B model, VMD program [48] was used to evaluate
the 3D distribution and general physical chemistry characteristics. Then, stereochemical
and energetic quality of the homology models was evaluated using PROSAII server [49],
ANOLEA server [50] and Procheck program [51]. The crystal structure of rMAO-A (PDB
code 1O5W [52]) and model of rMAO-B isoform were submited to H++ server [53,54] to
computes pK values of ionizable groups and adds missing hydrogen atoms according to
the specified pH of the environment as is described in H++ server.
3.4.2. Molecular Docking
Coumarin-chalcone hybrids were docked in the binding cavity of rMAO-A (PDB code
1O5W) and homology model for rMAO-B using AutoDock 4.012 suite. In general, the grid
maps were calculated using the AutoGrid 4.0 option and were centered on the sites described
before. The volume chosen for the grid maps were made up of 60
×
60
×
60 points, with a
grid-point spacing of 0.375 Å. The author’s option was used to define the rotating bond
in the ligand. In the Lamarckian genetic algorithm (LGA) dockings, an initial population
of random individuals with a population size of 150 individuals, a maximum number of
2.5
×
107 energy evaluations, a maximum number generation of 27,000, a mutation rate of
0.02 and crossover rate of 0.80 were employed. Each complex was built using the lowest
docked-energy binding positions. Van der Waals interaction cutoff distances were set at
12 Å and dielectric constant was 10. The partial charges of each ligand were determined
with PM6-D3H4 semi-empirical method [55,56] implemented in the MOPAC2016 [57]
software. PM6-D3H4 [56] introduces dispersion and hydrogen-bonded corrections to the
PM6 method.
3.4.3. Ligand Efficiency Approach
Ligand efficiency (LE) calculations were performed using one parameter Kd. The
Kd parameter corresponds to the dissociation constant between a ligand/protein, and
their value indicates the bond strength between the ligand/protein [34–36]. Low values

Molecules 2021, 26, 2430
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indicate strong binding of the molecule to the protein. Kd calculations were done using the
following Equations (1) and (2):
∆G0 = −2.303RTlog(Kd)
(1)
(2)
0
∆G
2.303RT
Kd = 10
where
∆
G0 is the binding energy (kcal mol−1) obtained from docking experiments, R is
·
the gas constant, and T is the temperature in Kelvin. In standard conditions of aqueous
solution at 298.15 K, neutral pH and remaining concentrations of 1 M. The LE allows us to
compare molecules according to their average binding energy [36,58]. Thus, it determined
as the ratio of binding energy per non-hydrogen atom, as follows (Equation (3)) [34
–36
,
59]:
2.303RT
HAC
LLE = −
log(Kd)
(3)
where Kd is obtained from Equation (2) and HAC denotes the heavy atom count (i.e.,
number of non-hydrogen atoms) in a ligand.
3.4.4. Molecular Dynamic Simulations
Two complexes were built for each modeled ChC2/rMAO-B and ChC4/rMAO-B,
and each model was confined inside a periodic simulation box. Wa−ter model TIP3P [60
]
with 20.459 molecules was used as solvent. Furthermore, Na+ and Cl ions were added to
neutralize the systems and maintain an ionic concentration of 0.15 mol
try optimizations of the two molecules were carried out with the density functional theory
method by a M05-2X [61]-D3 [62] in conjunction with the 6-31G(d,p) basis set. ChC2 ChC4
and FAD compounds were parametrized using LigParGen web server and implementing
the OPLS-AA/1.14*CM1A(-LBCC) force field parameters for organic ligands [63 65]. The
·
L
−1.The full geome-
,
–
partial charges of each ligand were determined with generated by the restrained electro-
static potential (RESP) model [66]. MD simulations were carried out using the modeled
CHARMM22 and CHARMM36 force fields [67,68] within the NAMD software [69]. First,
each system included 20,000 steps of conjugate-gradient energy minimization followed
by 10 ns of simulation with the protein backbone atoms fixed and gradually releasing the
backbone over 50,000 ps with 10 to 0.001 kcal
mol−1Å−2 restraints. The total duration of
·
simulation was approximately 100 ns for each system. During the MD simulations, motion
equations were integrated with a 1 fs time step in the NPT ensemble at a pressure of 1 atm.
The SHAKE algorithm was applied to all hydrogen atoms, and the van der Waals cutoff
was set to 12 Å. The temperature was maintained at 310 K, employing the Nosée-Hoover
thermostat method with a relaxation time of 1 ps. The Nosée-Hoover-Langevin piston
was used to control the pressure at 1 atm. Long-range electrostatic forces were taken into
account by means of the particle-mesh Ewald approach. Data were collected every 1 ps
during the MD runs. Molecular visualization of the systems and MD trajectory analysis
were carried out with the VMD software package [48].
3.4.5. Free Energy Calculation
The molecular MM/GBSA method was employed to estimate the binding free energy
of the rMAO-B/ligand complexes. For calculations from a total of 100 ns of MD, the last
70 ns were extracted for analysis, and the explicit water molecules and ions were removed.
The MM/GBSA analysis was performed on three subsets of each system: the protein alone,
the ligand alone, and the complex (protein-ligand). For each of these subsets, the total free
energy (∆Gtot) was calculated as follows (Equation (4)):
∆Gtot = EMM + Gsolv − T∆Sconf
(4)
where EMM is the bonded and Lennard–Jones energy terms; Gsolv is the polar contribution of
solvation energy and non-polar contribution to the solvation energy; T is the temperature;

Molecules 2021, 26, 2430
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and
∆
Sconf corresponds to the conformational entropy [70]. Both EMM and Gsolv were
calculated using NAMD software with the generalized Born implicit solvent model [71
tot was calculated as a linear function of the solvent-accessible surface area, which was
calculated with a probe radius of 1.4 Å [73]. The binding free energy of rMAO-B and ligand
,72].
∆
G
complexes (∆Gbind) were calculated by the difference where ∆Sconf values are the averages
over the simulation (Equation (5)):
∆Gbind = ∆Gtot(complex) − ∆Gtot(protein) − ∆Gtot(ligand)
3.4.6. ADMET Prediction
(5)
The ADMET properties of a compound deal with its absorption, distribution, metabolism,
excretion, and toxicity in and through the human body. ADMET, which constitutes the
pharmacokinetic profile of a drug molecule, is very essential in evaluating its pharmaco-
dynamic activities. In this study for all molecules, we have used the SwissADME [74
]
prediction tool, for in silico physicochemical properties such as molecular hydrogen bond
acceptor (HBA), hydrogen bond donor (HBD), weight (MW), topological polar surface
area (TPSA), rotatable bond count (RB), octanol/water partition coefficient (LogP), wa-
ter solubility (LogS) and skin permeation (logKp). Further the ligands were analyzed for
Bioavailability property using Boiled Egg analysis [75].
4. Conclusions
Fourteen compounds derived from chalcocoumarins were synthesized and evaluated
against monoamine oxidase enzyme isoforms. The experimental results obtained against
MAO-A and MAO-B show that the compounds ChC4
exhibit MAO-B affinity at micro and sub-micromolar concentrations, in particular ChC4
which shows an IC50 value of 0 0.76 0.08 M. Where compound ChC4 is highlighted
, ChC5, ChC6, ChC9 and ChC11
±
µ
in molecular modeling, ADMET predictions, docking and MM/GBSA calculations, these
results suggest that compound ChC4 has the appropriate interactions with the active site
of rMAO-B. Furthermore, the ADMETox values obtained for the compound ChC4 indicate
adequate solubility in the gastrointestinal tract, which is a favourable indication for it to
be a promising drug candidate for further research and development. This compound
complies with the interactions described for the active site of rMAO-, fitting into a distance
close enough to the nitrogen atom of the aloxazine planar ring of FAD to form an interaction
necessary for the inhibition of rMAO-B. These analyses may be important initial steps
towards the development of new drugs in the fight against depressive disorder and
Parkinson’s disease.
Supplementary Materials: The following are available online. Scheme S1. Synthetic route to
compounds ChC1–ChC14; Figure S1–S15: –ChC14: Figure S16.
1H and 13C NMR spectrum of ChC1
Homology modeling analysis; Figure S17. Ramachandran plot generated via PROCHECK for the
rMAO-B model; Figure S18. Alignment of all the ChC ligands docked in complex with rMAO-B.
Author Contributions: O.Y., M.T.N., C.A., A.F. and O.G.-B. contributed to the conception and design
of the study; O.Y., A.F. and A.G.-G. preformed the theoretical calculations; O.Y., A.F., and O.G.-B.
organized the database; A.F., M.T.N., G.M.-A. and N.M. design and performance of biological assay;
O.Y., A.F. and O.G.-B. wrote the first draft of the manuscript. All authors contributed to manuscript
revision, read and approved the submitted version. All authors have read and agreed to the published
version of the manuscript.
Funding: Ministry of Science, Technology and Innovation, the Ministry of Education, the Ministry
of Industry, Commerce and Tourism, and ICETEX, Programme Ecosistema Científico-Colombia
Científica, from the Francisco José de Caldas Fund, Grand RC-FP44842-212-2018.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conflicts of Interest: The authors declare that there is no conflict of interest.

Molecules 2021, 26, 2430
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Sample Availability: Samples of compounds ChC1–ChC14 are available from the authors.
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