Design, synthesis and biological evaluation of oxygenated chalcones as potent and selective MAO-B inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103335

The present study documents the synthesis of oxygenated chalcone (O1–O26) derivatives and their abilities to inhibit monoamine oxidases. All 26 derivatives examined showed potent inhibitory activity against MAO-B. Compound O23 showed the greatest inhibitory activity against MAO-B with an IC₅₀ value of 0.0021 μM, followed by compounds O10 and O17 (IC₅₀ = 0.0030 and 0.0034 μM, respectively). In addition, most of the derivatives potently inhibited MAO-A and O6 was the most potent inhibitor with an IC₅₀ value of 0.029 μM, followed by O3, O4, O9, and O2 (IC₅₀ = 0.035, 0.053, 0.072, and 0.082 μM, respectively). O23 had a high selectivity index (SI) value for MAO-B of 138.1, and O20 (IC₅₀ value for MAO-B = 0.010 μM) had an extremely high SI of >4000. In dialysis experiments, inhibitions of MAO-A and MAO-B by O6 and O23, respectively, were recovered to their respective reversible reference levels, demonstrating both are reversible inhibitors. Kinetic studies revealed that O6 and O23 competitively inhibited MAO-A and MAO-B, respectively, with respective K_i values of 0.016 ± 0.0007 and 0.00050 ± 0.00003 μM. Lead compound are also non-toxic at 200 μg/mL in normal rat spleen cells. Molecular docking simulations and subsequent Molecular Mechanics/Generalized Born Surface Area calculations provided a rationale that explained experimental data.

Full text of DOI:10.1016/j.bioorg.2019.103335

Journal Pre-proofs
Design, synthesis and biological evaluation of oxygenated chalcones as potent
and selective MAO-B inhibitors
Della Grace Thomas Parambi, Jong Min Oh, Seung Cheol Baek, Jae Pil Lee,
Anna Rita Tondo, Orazio Nicolotti, Hoon Kim, Bijo Mathew
PII:
S0045-2068(19)31268-4
DOI:
https://doi.org/10.1016/j.bioorg.2019.103335
YBIOO 103335
Reference:
Bioorganic Chemistry
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30 September 2019
2 October 2019
Please cite this article as: D. Grace Thomas Parambi, J. Min Oh, S. Cheol Baek, J. Pil Lee, A. Rita Tondo, O.
Nicolotti, H. Kim, B. Mathew, Design, synthesis and biological evaluation of oxygenated chalcones as potent and
selective MAO-B inhibitors, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103335
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Design, synthesis and biological evaluation of oxygenated chalcones as
potent and selective MAO-B inhibitors
Della Grace Thomas Parambi1#, Jong Min Oh2#, Seung Cheol Baek2, Jae Pil Lee2, Anna Rita
Tondo3, Orazio Nicolotti4, Hoon Kim2*, Bijo Mathew5*
1
Department of Pharmaceutical Chemistry, Jouf University, Sakaka, Al Jouf-2014, Saudi
Arabia.
2 Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon
National University, Suncheon 57922, Republic of Korea.
3
Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Via la Masa 19, 20156, Milano,
Italy.
4
Dipartimento di Farmacia—Scienze del Farmaco, Università degli Studi di Bari “Aldo
Moro”, via E. Orabona, 4, I-70125 Bari, Italy.
5Division of Drug Design and Medicinal Chemistry Research Lab, Department of
Pharmaceutical Chemistry, Ahalia School of Pharmacy, Palakkad-678557, Kerala, India.
#Authors contributed equally.
*Corresponding Authors:
Hoon Kim (H. Kim) (hoon@sunchon.ac.kr)
Bijo Mathew (B. Mathew) (bijovilaventgu@gmail.com) (bijo.mathew@ahalia.ac.in)
1

Abstract
The present study documents the synthesis of oxygenated chalcone (O1-O26) derivatives
and their abilities to inhibit monoamine oxidases. All 26 derivatives examined showed potent
inhibitory activity against MAO-B. Compound O23 showed the greatest inhibitory activity
against MAO-B with an IC50 value of 0.0021 µM, followed by compounds O10 and O17
(IC50 = 0.0030 and 0.0034 µM, respectively). In addition, most of the derivatives potently
inhibited MAO-A and O6 was the most potent inhibitor with an IC50 value of 0.029 µM,
followed by O3, O4, O9, and O2 (IC50 = 0.035, 0.053, 0.072, and 0.082 µM, respectively).
O23 had a high selectivity index (SI) value for MAO-B of 138.1, and O20 (IC50 value for
MAO-B = 0.010 µM) had an extremely high SI of > 4,000. In dialysis experiments,
inhibitions of MAO-A and MAO-B by O6 and O23, respectively, were recovered to their
respective reversible reference levels, demonstrating both are reversible inhibitors. Kinetic
studies revealed that O6 and O23 competitively inhibited MAO-A and MAO-B, respectively,
with respective Ki values of 0.016 ± 0.0007 and 0.00050 ± 0.00003 µM. Lead compound are
also non-toxic at 200µg/mL in normal rat spleen cells. Molecular docking simulations and
subsequent Molecular Mechanics/Generalized Born Surface Area calculations provided a
rationale that explained experimental data.
Keywords: Chalcone, MAO inhibition, Kinetics, Reversibility, Molecular docking.
2

1. Introduction
Monoamine oxidases (MAOs) are major metabolizing enzymes and attractive target for
the treatment of neurodegenerative disorders. MAOs are composed of a flavin adenine
dinucleotide (FAD) covalently bound to a cysteine residue, and therapeutics that inhibit these
enzymes are viewed as serious contenders for future therapy [1]. MAOs are vital for the
deamination, and thus, for regulations of the levels of biogenic amines like neuroamines,
xenobiotic amines (e.g., monoamine neurotransmitters), hormones, and exogenous amines
present in peripheral tissues and brain [2]. MAOs have been studied extensively and are
composed of two isoenzymes, that is, MAO-A and MAO-B, which are both associated with
the mitochondrial outer-membrane and exhibit different substrate specificities, inhibitor
sensitivities, and tissue localizations. Both isoenzymes are encoded on the X chromosome by
different genes [3], and are present in variable quantities in human tissues [4]. An elaborate
study on the kinetics of MAOs illustrated that the reaction pathways of MAO-A and B differ
and that this difference may depend upon the substrate used and their sources [5].
MAOs crucially modulate the functions of neurotransmitters, and thus, are of interest for
the treatment of depression and various neurodegenerative disorders [6]. Oxidative
deamination by MAOs can diminish levels of neurotransmitters in nerve terminals, but this
process generates free radicals, hydrogen peroxide, and reactive oxygen species (ROS) [7],
which can cause protein disruption, mitochondrial dysfunction, neuronal apoptosis, lipid
peroxidation, and eventually neuron death. Accordingly, the design and development of
specific drug candidates that inhibit the two isoforms of MAO have considerable therapeutic
potential [8]. Selective inhibitors of MAO-A are considered to be members of the third line
treatment arsenal for anxiety and depression, whereas MAO-B inhibitors have been
demonstrated to be effective treatments for neurodegenerative diseases such as Alzheimer’s
disease (AD) and Parkinson’s disease [9,10]. Many oxygen-containing scaffolds like
3

coumarin and chromone have attracted considerable attention because of their abilities to
inhibit both MAO isoforms [11-18].
Recently chalcone scaffolds have been shown to provide a basis for inhibiting MAOs [19-
27]. The relevance of the chalcone scaffold as a privileged structure in medicinal chemistry
has been highlighted in a very recent review [28, 29]. Despite of the disadvantages of
chalcone scaffold for capable of forming irreversible bonds with other molecules, resulting in
toxic effects, such as allergenic reactions, mutagenicity, and carcinogenicity, many promising
candidates from these family have been extensively researched and patented till now [30, 31].
Moreover it is further proved when browsing ChEMBLdb, a large collection of 611333 small
molecules provided with high quality experimental bioactivity data [32]. By using the
chalcone scaffold as a query in target prediction program such as the recent MuSSel, a
publicly available platform upon the request of a free license [33-35]. Results are enclosed as
supporting information
Decorating heterocycles with diverse functional groups around the α, β-unsaturated
linker of chalcone has revealed some fascinating chemistry [36]. Reports issued on the topic
show many α, β-unsaturated scaffolds selectively inhibit MAO-B rather than MAO-A [37-
39], and that this selectivity difference depends upon the nature and orientation of various
electron donating and withdrawing motifs on the phenyl system of chalcone scaffolds. By
introducing various electron donating and withdrawing groups on the phenyl ring or the
hetero cycle participation around the three carbon enone system, a new class of reversible and
selective chalcone-based MAO-B inhibitors has been identified. The presence of
dimethylamino, ethyl, bromo, or chloro groups at the para position of the phenyl A ring of
chalcone are associated with pronounced MAO-B inhibition, and it has been hypothesized
that these lipophilic groups are efficiently accommodated by the hydrophobic region of the
entrance cavity of MAO-B [41-47]. Recently our group also examined the bio-distributions
4

of potent chalcone based MAO-B inhibitors and found that molecules that bind more strongly
with human serum albumin (HAS) have better inhibitory characteristics [48,49]. In the
present study, we prepared 26 oxygenated chalcones and investigated their MAO inhibitory
profiles and structure activity relationships (SARs), especially the importance of the number
of alkyl groups between the two oxygen atoms in the A ring and the effect of various groups
on the B ring. The study mainly highlighted the importance of metheynedioxy (MDO) and
ethylenedioxy (EDO) ring in the chalcone scaffold towards MAO inhibition.
2. Result and discussion
2.1. Chemistry
Chalcones (O1-O26) were prepared by the Claisen-Schmidt condensation of 1-(2H-1,3-
benzodioxol-5-yl)ethan-1-one and 1-(2,3-dihydro-1,4-benzodioxin-6-yl)ethan-1-one in the
presence of various aryl substituted benzaldehydes in basic alcoholic medium (refer to
Scheme 1). All synthesized compounds were characterized by 1H-NMR, 13C-NMR, and mass
spectrometry. Large coupling constants (15Hz) showed that the double bonds of chalcones
were in the trans configuration [50]. Spectra are available in Supplementary materials.
5

Scheme 1: Synthetic route used to produce the 26 oxygenated chalcones
2.2. Biological evaluation
2.2.1. MAO inhibition studies
All 26 derivatives showed potent inhibitory activity against MAO-B at 10 µM with
residual activities of < 30% and most derivatives showed little residual activity (Table 1).
Compound O23 inhibited MAO-B most with an IC50 value of 0.0021 µM, followed by O10
and O17 (IC50 = 0.0030 and 0.0034 µM, respectively). Interestingly compound O23 inhibited
MAO-B 21 times more potently than the selective, reversible MAO-B inhibitor lazabemide
(IC50 = 0.046 μM) and 11 times more potently than the irreversible MAO-B inhibitor
6

pargyline (0.023 μM), which were used as reference compounds in the current study.
Compounds O20, O21, O22, O2, O3, O4, O7, O14, O24, and O26 also potently inhibited
MAO-B with the IC50 values ranging from 0.013 to 0.018 µM and were more potent than the
references. The other compounds inhibited MAO-B with IC50 values ranging from 0.021 to
0.068 µM. Regarding selectivity indices (SIs), O23 showed a high value for MAO-B at
138.1, while O10 and O17 had lower values (43.3 and 102.9, respectively) (Table 1).
Compound O20 (IC50 value for MAO-B = 0.010 µM) had an extremely high SI of > 4,000.
In addition, most of the compounds potently inhibited MAO-A at 10 µM by > 50%,
except compounds O15 and O20 (Table 1). Compound O6 inhibited MAO-A most with an
IC50 value of 0.029 µM, followed by O3, O4, O9, and O2 (IC50 = 0.035, 0.053, 0.072, and
0.082 µM, respectively). Compound O6 inhibited MAO-A 32 times more potently than
toloxatone (a MAO-A inhibitor; IC50 = 0.93 μM) in a non-selective manner, which offers an
advantage as its low SI can be utilized to treat the depressive symptoms associated with PD
[51]. Compounds O1, O5, O7, O8, O10, O17, and O23 also effectively inhibited MAO-A
with IC50 values ranging from 0.13 to 0.35 µM.
7

Table 1
Inhibition of recombinant human MAO enzymes by oxygenated chalconesa
Residual activity at 10 µM (%)
IC50 (µM)
Compounds
SIb
MAO-A
4.30 ± 2.35
13.1 ± 1.14
-3.97 ± 2.82
0.33 ± 1.40
2.64 ± 0.01
MAO-B
MAO-A
MAO-B
O1
O2
O3
O4
O5
-1.47 ± 3.46
1.47 ± 0.52
-6.62 ± 2.71
-6.25 ± 0.65
6.62 ± 8.12
0.31 ± 0.0026
0.082 ± 0.036
0.035 ± 0.0057
0.053 ± 0.0052
0.31 ± 0.031
0.026 ± 0.0030
0.015 ± 0.0025
0.015 ± 0.0037
0.013 ± 0.0037
0.045 ± 0.0014
11.9
5.47
2.33
4.08
0.69
O6
-2.32 ± 0.50
16.4 ± 5.59
5.30 ± 2.34
0.36 ± 0.52
4.70 ± 3.55
37.3 ± 1.26
3.52 ± 0.19
6.24 ± 3.77
41.1 ± 3.94
59.6 ± 1.60
17.5 ± 2.38
-9.31 ± 5.23
38.0 ± 1.12
17.1 ± 2.44
74.0 ± 7.23
-1.34 ± 7.33
7.38 ± 3.54
22.9 ± 4.46
46.8 ± 2.47
42.9 ± 0.81
43.4 ± 0.81
-
-4.04 ± 2.9
-4.59 ± 1.48
-1.42 ± 2.01
-2.82 ± 0.99
-3.18 ± 1.48
-6.72 ± 0.53
-8.49 ± 2.04
-11.0 ± 1.55
-3.7 ± 2.05
27.8 ± 3.25
-3.7 ± 3.09
-29.3 ± 1.92
7.3 ± 2.03
-4.4 ± 2.05
-6.37 ± 2.95
-12.8 ± 1.88
-14.2 ± 1.86
-8.86 ± 1.42
2.47 ± 2.48
-7.08 ± 1.93
-8.85 ± 3.42
-
0.029 ± 0.010
0.18 ± 0.0022
0.15 ± 0.0070
0.072 ± 0.021
0.13 ± 0.020
1.93 ± 0.046
1.25 ± 0.0049
1.43 ± 0.014
5.88 ± 1.40
24.9 ± 4.18
1.54 ± 0.22
0.35 ± 0.030
6.10 ± 0.059
0.74 ± 0.068
> 40
0.027 ± 0.0006
0.014 ± 0.00001
0.024 ± 0.00036
0.021 ± 0.0012
0.0030 ± 0.0009
0.029 ± 0.00031
0.027 ± 0.0035
0.024 ± 0.0047
0.018 ± 0.0022
0.068 ± 0.017
0.025 ± 0.0020
0.0034 ± 0.0011
0.032 ± 0.0061
0.025 ± 0.0032
0.010 ± 0.0040
0.010 ± 0.0013
0.010 ± 0.0052
0.0021 ± 0.00012
0.013 ± 0.0023
0.036 ± 0.00053
0.015 ± 0.0017
> 80
1.07
12.9
O7
O8
6.25
O9
3.43
O10
43.3
O11
66.6
O12
46.3
O13
59.6
O14
326.7
366.2
61.6
O15
O16
O17
102.9
190.6
29.6
O18
O19
O20
> 4,000
131.0
94.0
O21
1.31 ± 0.013
0.94 ± 0.069
0.29 ± 0.0069
10.18 ± 1.07
12.58 ± 1.39
8.39 ± 0.37
0.93 ± 0.027
> 80
O22
O23
138.1
783.1
349.4
559.3
O24
O25
O26
Toloxatonec
Lazabemide
Clorgyline
Pargyline
-
-
0.046 ± 0.0048
2.23 ± 0.21
-
-
0.0057 ± 0.00045
3.07 ± 0.17
-
-
0.023 ± 0.0041
a Results are expressed as the means ± standard errors of duplicate experiments.
b SI values are expressed for MAO-B versus MAO-A.
c
For reference compounds, inhibitory activities were determined after preincubating them
with enzymes for 30 min.
8

2.2.2. Structure activity relationships (SARs) of chalcones with respect to MAO inhibition
Considering the inhibitory profile of diverse oxygenated chalcones used in the study,
most of the compounds were selective for MAO-B inhibition, but the compounds with potent
MAO-A inhibitory activity were non-selective. We mainly focused on the effects of (a) the
number of alkyl groups between the two oxygen atoms in the A ring, and (b) of the various
electron donating withdrawing groups and their orientations in the B ring. Increasing the
number of alkyl groups between the two oxygen atoms improved MAO-B inhibition and
selectivity. Analogs with an ethylenedioxy ring on A ring tended to enhance selectivity for
MAO-B. The presence of fluorine atom at para position of phenyl ring B resulted in the
greatest MAO-B inhibition (O23 and O10), and shifting fluorine from para to meta or ortho
(O24 and O25) of ethylenedioxy chalcones reduced MAO-B inhibition but increased
selectivity. Introduction of a nitro group (electron withdrawing) at the para position of ring B
(O20) resulted in the highest selectivity (SI> 4,000) with a low IC50 value (0.010 μM) for
MAO-B. Introduction of an ethyl group in methylenedioxy chalcone (O6) resulted in a high
level of non-selective MAO-A inhibition. Introduction of methyl group on the para position
of ring B on EDO containing chalcone (O17) leads to an increasing MAO-B potency,
suggesting that methyl group is as good as -F atom. The SAR concluded that presence of
EDO unit have greater impact than the MDO in the chalcone scaffold for both potency and
selectivity on MAO-B inhibition. An overview of the SARs of oxygenated chalcones with
respect to MAO inhibition is provided in Fig.1.
9

O
MAO-B
inhibtion
4-F>Br>Cl
O
MAO-B
inhibition
n
SI
R
3F>2F>4F
when n=2
O
MAO-A
inhibition
R= C2H5 when n=1
Fig. 1: SARs of oxygenated chalcones towards MAO inhibition
2.2.3. Kinetics
Kinetic studies were conducted on MAO-A inhibition by O6 and MAO-B inhibition by
O23. Lineweaver-Burk plots and secondary plots showed O6 competitively inhibited MAO-
A with a Ki value of 0.016 ± 0.0007 µM (Figs. 2A&B), indicating O6 potently, reversibly,
and competitively inhibited MAO-A. MAO-B inhibition by O23 was competitive with a Ki
value of 0.00050 ± 0.00003 µM (Fig. 2C&D), which suggests O23 bound to the active site of
free MAO-B and that it acted as a potent, selective, and competitive inhibitor.
10

(A)
(B)
1800
1600
1400
1200
1000
800
10
8
0.04 uM
0.02 uM
0.01 uM
Control
6
4
600
400
2
200
0
0
-50
0
50
100
150
200
-0.02
-0.01
0
0.01
0.02
0.03
0.04
0.05
1/[S], mM-1
[O6], uM
(C)
(D)
11

7000
6000
5000
4000
3000
2000
1000
0
200
150
100
50
0.004 uM
0.002 uM
0.001 uM
Control
0
-10
0
10
20
30
40
-0.001
0
0.001
0.002
0.003
0.004
0.005
1/[S], mM-1
[O23], uM
Fig. 2. Lineweaver-Burk plots for MAO-A and MAO-B inhibitions by O6 (A) and O23 (C), respectively, and their respective secondary plots
(B) and (D) of the slopes vs. inhibitor concentrations.
12

2.2.4. Reversibility
Reversibility studies on the inhibitions of MAO-A and MAO-B were conducted using O6
and O23, respectively. Their relative activities of un-dialyzed (AU) and dialyzed (AD)
samples were calculated. In these experiments, inhibition of MAO-A by O6 was recovered
from 46.9% (value of AU) to 88.2% (value of AD), and this recovery was similar to that
observed for toloxatone (the reversible reference) (from 33.6 to 89.7%) (Fig. 3A). Inhibition
of MAO-B by O23 recovered from 26.9% (value of AU) to 90.9% (value of AD), and the
recovery was similar to that of lazabemide (the reversible reference) (from 39.8 to 91.9%)
(Fig. 3B). Inhibitions of MAO-A and MAO-B by the irreversible inhibitors, clorgyline and
pargyline, respectively, were not recovered. These experiments showed inhibitions of MAO-
A and MAO-B by O6 and O23, respectively, were recovered to respective reversible
reference levels, showing both acted as reversible inhibitors.
(A)
Undialyzed
Dialyzed
100
80
60
40
20
0
13

(B)
Undialyzed
Dialyzed
100
80
60
40
20
0
Fig. 3. Recoveries of MAO-A and MAO-B inhibitions by O6 (A) and O23 (B), respectively,
as determined by dialysis.
2.2.5. Cytotoxicity
Short term in vitro cytotoxicity studies were performed by Trypan blue dye exclusion test in
the rat spleen cells. The percentage of cell death was found to be 4 and 11 % at 200µg/mL for
the most potent MAO-B inhibitors O23 and O10 respectively. The details of the cytotoxicity
studies were shown in the Table 2.
14

Table 2: Trypan blue dye exclusion in vitro cytotoxicity test
Drug concentration (µg/mL) Percentage of cell death (%)
O10
O23
200
100
50
11
5
-
4
-
-
20
-
-
10
-
-
2.3. Computational studies
O10 and O23, which exhibited outstanding inhibitory activity against MAO-B, were
docked into the X-ray identified binding sites of MAO-A (PDB code: 2Z5X) and MAO-B
(PDB code: 2V5Z) as previously described [52]. In addition, docking scores and binding free
energies were calculated (refer to Table 3). The docking score values of the two compounds
were close to that of the X-ray cognate ligand (i.e., Safinamide) of MAO-B (-11.087 kcal/mol)
[53]. Interestingly, a significant energy gap between the docking scores of O10 and O23 with
MAO-A and that of the X-ray cognate ligand (i.e., Harmine) (-10.032 kcal/mol) of MAO-A
was found.
15

Fig. 4. Top-scored docking poses of O10 (A) and O23 (B) in MAO B. Proteins are rendered
as cartoons while ligands and important residues are rendered as sticks. For the clarity of
description, only polar hydrogen atoms are shown. Black and red lines indicate possible
hydrophobic and hydrogen bond interactions.
Table 3. Docking scores and binding free energy values of O10 and O23 for MAO-A and
MAO-B.
MAO-A
Binding free energy
MAO-B
Docking score
Code
Docking score
(kcal/mol)
Binding free energy
(kcal/mol)
(kcal/mol)
(kcal/mol)
O10
O23
-8.598
-8.593
-27.13
-34.16
-10.092
-10.220
-47.70
-55.48
Of the two lead compounds, that are O10 and O23, the latter was nearest the FAD unit in
the inhibitor binding cavity of MAO-B and showed stronger  stacking interactions
between its benzoxolane moiety and Y435, Y398, and I199 of MAO-B. Noteworthy, the
Y326 to I335 mutation observed when comparing active site residues of MAO-B and MAO-
A is very much relevant for molecular selectivity. As shown in Fig. 4, Y326 plays a key role
for selectivity by establishing a potential hydrogen bond with hydrogen bond acceptor groups
of partner ligands O10 and O23. Notably, the relevance of Y326 in modulating selectivity
was further observed when docking O6, which is provided with moderate inhibition but very
16

low selectivity (IC50, MAO-A = 0.029 mM; IC50, MAO-B = 0.027 M). Further details are
reported as Supporting Information. In addition, binding free energy analyses indicated that
binding affinities of O23 and O10 with MAO-B were greater than that observed for MAO-A.
In-line with experimentally determined inhibition and SI values, MM-GBSA calculations
returned a higher binding free energy for O23 than O10.
Rotation through 180° allows O23 to adopt two binding conformations (Fig. 5), which
might explain the higher experimental SI of O23 for MAO-A. As shown on the right-hand
side of Figure 5, O23 shows the expected binding topology (that is the benzodioxane ring
facing the FAD) only paying a higher energetic cost (-8.061 kcal/mol versus -8.593 kcal/mol)
because of the steric hindrance of Y407 and Y444.
Fig. 5 Left-hand and right-hand poses produced the binding energies of -8.593 kcal/mol and -
8.061 kcal/mol, respectively, for the docking of O23 with MAO-A. Proteins are rendered as
cartoons while ligands and important residues are rendered as sticks. Orange bows indicate
steric hindrance.
2.3.1 In silico prediction of ADME/Tox properties
Finally, we carried out further in silico investigations on compounds O10 and O23. First,
we calculated a long list of pharmaceutically relevant properties by using QikProp Base a
quick, accurate, easy-to-use absorption, distribution, metabolism, and excretion (ADME)
prediction program designed by Professor William L. Jorgensen [54]. Based on this analysis,
17

we observed that that both O10 and O23 explored physicochemical properties experienced by
the 95% of known drugs. For instance, the predicted brain/blood partition coefficient is equal
to 0.051 and 0.030 for O10 and O23, respectively, being the recommended range for orally
delivered drugs to cross the blood-brain barrier between -3.00 and l.2. For the sake of
completeness, comprehensive spectra of all the predicted properties are enclosed in a file
available as Supplementary Materials.
3. Conclusion
The current study documents the synthesis of and the results of MAO inhibition studies on
a diverse class of chalcones (O1-O26) containing methylenedioxy and ethylenedioxy rings.
Most of the chalcones containing an ethylenedioxy ring exhibited potent and highly selective
MAO-B inhibition. The representative compounds O23, O10, and O17 potently inhibited
MAO-B with IC50 values of 0.0021, 0.0030, and 0.0034 μM, respectively, and did so more
effectively than the reference inhibitors, lazabemide and pargyline (IC50 values of 0.046 and
0.023 μM, respectively). Compound O6 inhibited MAO-A 32 times more than toloxatone
(the reference inhibitor; IC50 = 0.93 μM) but in a non-selective manner. In the SAR study, we
focused on the effects of the number of alkyl groups between the two oxygen atoms of the
chalcone A ring and of different groups on the phenyl B ring. Interestingly, the in vitro
cytotoxic studies results show that O23 and O10 is nontoxic at 200µg/ml with 4% and 11%
of percentage of death cells. Based on the inhibitory data obtained, we conclude chalcone
derivatives offer a means of discovering novel MAO inhibitors. Furthermore, our results raise
the possibility of developing reversible MAO inhibitors that might provide safer MAO-
inhibitor based therapies. We will study further in vivo analysis about the levels of biogenic
amines in near future by using these compounds.
18

4. Materials and Methods
4.1. Synthesis
Equimolar quantities of substituted benzaldehyde and 1-(2H-1,3-benzodioxol-5-yl)ethan-1-
one or 1-(2,3-dihydro-1,4-benzodioxin-6-yl)ethan-1-one were dissolved in 40% KOH and
ethanol. The reaction mixture was then stirred for 10 h and poured into ice-cold water. The
precipitated product was washed with water, dried, and recrystallized from ethanol.
4.1.1. (2E)-1-(2H-1,3-benzodioxol-5-yl)-3-phenylprop-2-en-1-one (O1): Yield:70%. 1H
NMR (500 MHz, CDCl3)δ: 6.07 (s, 2H, O-CH2-O), 6.91-6.89 (d, 1H, J= 10.0 Hz, -ArH),
7.43-7.41, (d, 3H, J= 10.0 Hz, -ArH), 7.48 (s, 1H, -ArH), 7.51-7.48 (1H, d, J= 15.0 Hz, -
13
CHα), 7.65-7.63 (d, 3H, J= 10.0 Hz, -ArH), 7.81-7.78 (d, 1H, J= 15.0 Hz, -CHβ). C-NMR
(125 MHz, CDCl3) δ: 101.81, 101.88, 107.78, 107.79, 108.44, 121.46, 124.69, 128.39,
128.94, 130.42, 144.27, 148.30, 151.71, 188.27. ESI-MS (m/z): Calculated- 252.2646,
Observed-252.2599.
4.1.2.
(2E)-1-(2H-1,3-benzodioxol-5-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one
(O2):
1
Yield:51%. H NMR (500 MHz, CDCl3)δ: 5.81 (s, 1H, OH), 6.07 (s, 2H, O-CH2-O), 6.91-
6.89 (d, 2H, J= 10.0 Hz, -ArH), 6.96-6.94 (d, 1H, J= 10.0 Hz, -ArH), 7.40-7.36 (1H, d, J=
16.0 Hz, -CHα), 7.62-7.60 (d, 2H, J= 10.0 Hz, -ArH), 7.64-7.63 (d, 1H, J= 10.0 Hz, -ArH),
7.80-7.77 (d, 1H, J= 15.0 Hz, -CHβ). 13C-NMR (125 MHz, CDCl3) δ: 101.80, 101.79, 107.66,
107.77, 108.14, 121.26, 124.62, 128.19, 128.44, 130.44, 144.26, 148.35, 151.51, 188.37. ESI-
MS (m/z): Calculated- 268.2640, Observed-268.2789.
4.1.3.
(2E)-1-(2H-1,3-benzodioxol-5-yl)-3-(4-methoxyphenyl)prop-2-en-1-one
(O3):
Yield:79%. 1H NMR (500 MHz, CDCl3)δ: 3.85 (s, 3H, OCH3), 6.06 (s, 2H, O-CH2-O), 6.90-
6.88 (d, 2H, J= 10.0 Hz, -ArH), 6.95-6.93 (d, 1H, J= 10.0 Hz, -ArH), 7.39-7.36 (1H, d, J=
19

15.0 Hz, -CHα), 7.53 (s, 1H, -ArH), 7.60-7.58, (d, 2H, J= 10.0 Hz, -ArH), 7.65-7.63 (d, 1H,
13
J= 10.0 Hz, -ArH), 7.79-7.76 (d, 1H, J= 15.0 Hz, -CHβ). C-NMR (125 MHz, CDCl3) δ:
54.41, 101.82, 107.88, 108.41, 114.37, 119.32, 123.45, 124.48, 127.68, 130.09, 130.22,
144.12, 148.22, 151.51, 161.55, 188.31. ESI-MS (m/z): Calculated- 282.2906, Observed-
282.2879.
4.1.4.
(2E)-1-(2H-1,3-benzodioxol-5-yl)-3-(4-methylphenyl)prop-2-en-1-one
(O4):
1
Yield:75%. H NMR (500 MHz, CDCl3)δ: 2.39 (s, 3H, CH3), 6.07 (s, 2H, O-CH2-O), 6.91-
6.88 (d, 1H, J= 10.0 Hz, -ArH), 7.23-7.21 (d, 2H, J= 10.0 Hz, -ArH), 7.47-7.42 (1H, d, J=
15.0 Hz, -CHα), 7.55-7.53, (d, 2H, J= 10.0 Hz, -ArH), 7.64 (s, 1H, -ArH), 7.67-7.65 (d, 1H,
13
J= 10.0 Hz, -ArH), 7.80-7.77 (d, 1H, J= 15.0 Hz, -CHβ). C-NMR (125 MHz, CDCl3) δ:
21.53, 101.84, 107.89, 108.43, 120.65, 124.60, 127.23, 128.41, 129.68, 132.21, 133.08,
140.94, 144.36, 148.25, 151.61, 188.37. ESI-MS (m/z): Calculated- 266.2912, Observed-
266.2899.
4.1.5. (2E)-1-(2H-1,3-benzodioxol-5-yl)-3-[4-(dimethylamino)phenyl]prop-2-en-1-one (O5):
1
Yield:74%. H NMR (500 MHz, CDCl3)δ: 3.04 (s, 6H, N(CH3)2), 6.05 (s, 2H, O-CH2-O),
6.91-6.89 (d, 1H, J= 10.0 Hz, -ArH), 6.68-6.66 (d, 2H, J= 10.0 Hz, -ArH), 6.89-6.87 (d, 1H, -
ArH), 7.31-7.28 (1H, d, J= 15.0 Hz, -CHα), 7.55-7.53, (d, 3H, J= 10.0 Hz, -ArH), 7.65-7.63
13
(d, 1H, J= 10.0 Hz, -ArH), 7.79-7.76 (d, 1H, J= 15.0 Hz, -CHβ). C-NMR (125 MHz,
CDCl3) δ: 40.15, 101.71, 107.83, 108.41, 111.79, 112.00, 116.41, 122.70, 124.17, 130.32,
133.74, 142.80, 145.26, 148.07, 151.15, 151.19, 188.43. ESI-MS (m/z): Calculated-
295.3324, Observed-295.3298.
4.1.6. (2E)-1-(2H-1,3-benzodioxol-5-yl)-3-(4-ethylphenyl)prop-2-en-1-one (O6): Yield:77%.
20

1H NMR (500 MHz, CDCl3)δ: 1.27-1.25 (t, 3H , J= 10.0 Hz, CH3), 2.71-2.69 (q, 2H , J= 10.0
Hz, CH2), 6.04 (s, 2H, O-CH2-O), 6.90-6.88 (d, 1H, J= 10.0 Hz, -ArH), 7.25-7.23 (d, 1H, J=
10.0 Hz, -ArH), 7.47-7.43 (1H, d, J= 15.0 Hz, -CHα), 7.55-7.53, (d, 3H, J= 10.0 Hz, -ArH),
7.57 (s, 1H, -ArH), 7.65-7.63 (d, 1H, J= 10.0 Hz, -ArH), 7.80-7.77 (d, 1H, J= 15.0 Hz, -CHβ).
13C-NMR (125 MHz, CDCl3) δ: 15.35, 28.84, 101.84, 107.88, 107.96 108.42, 120.68, 124.59,
128.50, 132.44, 144.37, 147.23, 148.24, 151.60, 188.34, 196.77. ESI-MS (m/z): Calculated-
280.3178, Observed-280.3091.
4.1.7. (2E)-1-(2H-1,3-benzodioxol-5-yl)-3-(4-nitrophenyl)prop-2-en-1-one (O7): Yield:80%.
1H NMR (500 MHz, CDCl3)δ: 6.07 (s, 2H, O-CH2-O), 6.93-6.91 (d, 1H, J= 10.0 Hz, -ArH),
7.26-7.24 (d, 2H, J= 10.0 Hz, -ArH), 7.62-7.59 (1H, d, J= 15.0 Hz, -CHα), 7.68 (s, 1H, -
ArH), 7.77-7.79, (d, 2H, J= 10.0 Hz, -ArH), 7.81-7.78 (d, 1H, J= 15.0 Hz, -CHβ), 8.28-8.26
13
(d, 2H, J= 10.0 Hz, -ArH). C-NMR (125 MHz, CDCl3) δ: 101.95, 102.06, 107.95, 108.04,
124.22, 125.05, 125.44, 127.43, 128.50, 132.33, 140.98, 141.17, 152.24, 187.35. ESI-MS
(m/z): Calculated- 297.2622, Observed-297.2598.
4.1.8.
(2E)-1-(2H-1,3-benzodioxol-5-yl)-3-(4-chlorophenyl)prop-2-en-1-one
(O8):
1
Yield:73%. H NMR (500 MHz, CDCl3)δ: 6.06 (s, 2H, O-CH2-O), 6.91-6.89 (d, 1H, J= 10.0
Hz, -ArH), 7.40-7.38 (d, 2H, J= 10.0 Hz, -ArH), 7.40-7.38 (d, 2H, -ArH), 7.48-7.45 (1H, d,
J= 15.0 Hz, -CHα), 7.53 (s, 1H, J= 10.0 Hz, -ArH),7.58-7.56, (d, 1H, J= 10.0 Hz, -ArH),
7.73-7.73 (d, 1H, J= 15.0 Hz, -CHβ). 13C-NMR (125 MHz, CDCl3) δ: 101.92, 107.95, 108.41,
122.08, 124.74, 129.22, 129.53, 132.78, 133.47, 136.28, 142. 76, 151.85, 187.96. ESI-MS
(m/z): Calculated- 286.7097, Observed-286.6999.
4.1.9.
(2E)-1-(2H-1,3-benzodioxol-5-yl)-3-(4-bromophenyl)prop-2-en-1-one
(O9):
21

1
Yield:76%. H NMR (500 MHz, CDCl3)δ: 6.06 (s, 2H, O-CH2-O), 6.91-6.89 (d, 1H, J= 10.0
Hz, -ArH), 7.49-7.46 (1H, d, J= 15.0 Hz, -CHα), 7.55-7.50 (d, 5H, -ArH), 7.65-7.63 (d, 1H,
J= 10.0 Hz, -ArH),7.40-7.38 (d, 2H, J= 10.0 Hz, -ArH), 7.40-7.38 (d, 2H, -ArH), 7.73-7.70
13
(d, 1H, J= 15.0 Hz, -CHβ). C-NMR (125 MHz, CDCl3) δ: 101.86, 102.02, 107.93, 108.39,
122.15, 124.63, 129.72, 131.61, 132.75, 133.87, 142.79, 148.35, 151.85, 187.92. ESI-MS
(m/z): Calculated- 331.1607, Observed-331.1599.
4.1.10.
(2E)-1-(2H-1,3-benzodioxol-5-yl)-3-(4-fluorophenyl)prop-2-en-1-one
(O10):
1
Yield:76%. H NMR (500 MHz, CDCl3)δ: 6.03 (s, 2H, O-CH2-O), 6.91-6.89 (d, 1H, J= 10.0
Hz, -ArH), 7.11-7.09 (d, 2H, J= 10.0 Hz, -ArH), 7.43-7.40 (d, 1H, J= 15.0 Hz, -CHα), 7.52 (s,
13
1H, -ArH), 7.63-7.61 (d, 3H, J= 10.0 Hz, -ArH), 7.77-7.74 (d, 1H, J= 15.0 Hz, -CHβ). C-
NMR (125 MHz, CDCl3) δ: 101.99, 107.97, 108.40, 116.01, 121.35, 124.68, 130.23, 142.94,
148.33, 151.80, 162.97, 164.97, 188.03, 196.45. ESI-MS (m/z): Calculated- 270.2551,
Observed-270.2489.
4.1.11.
(2E)-1-(2H-1,3-benzodioxol-5-yl)-3-(3-fluorophenyl)prop-2-en-1-one
(O11):
1
Yield:74%. H NMR (500 MHz, CDCl3)δ: 6.03 (s, 2H, O-CH2-O), 6.91-6.89 (d, 1H, J= 10.0
Hz, -ArH), 7.11-7.09 ( d, 2H, J= 10.0 Hz, -ArH), 7.40-7.39 (d, 3H, , J= 5.0 Hz, -ArH), 7.49-
7.46 (d, 1H, J= 15.0 Hz, -CHα), 7.53 (s, 1H, -ArH), 7.66-7.64 (d, 1H, J= 10.0 Hz, -ArH),
7.75-7.72 (d, 1H, J= 15.0 Hz, -CHβ). 13C-NMR (125 MHz, CDCl3) δ: 101.85, 101.93, 107.95,
122.82, 124.47, 124.81, 130.44, 130.51, 142.71, 142.73, 151.89, 162.04, 164.00, 187.88,
196.22. ESI-MS (m/z): Calculated- 270.2551, Observed-270.00.
4.1.12.
(2E)-1-(2H-1,3-benzodioxol-5-yl)-3-(2-fluorophenyl)prop-2-en-1-one
(O12):
1
Yield:68%. H NMR (500 MHz, CDCl3)δ: 6.06 (s, 2H, O-CH2-O), 6.91-6.89 (d, 1H, J= 10.0
22

Hz, -ArH), 7.15-7.13 ( d, 1H, J= 10.0 Hz, -ArH), 7.54 (s, 1H, -ArH), 7.62-7.59 (d, 1H, J=
15.0 Hz, -CHα), 7.66-7.64 (d, 4H, J= 10.0 Hz, -ArH), 7.89-7.86 (d, 1H, J= 15.0 Hz, -CHβ).
13C-NMR (125 MHz, CDCl3) δ: 101.81, 101.90, 107.93, 124.46, 124.85, 129.82, 131.65,
137.01, 148.32, 151.81, 160.69, 161.95, 162.71, 188.24, 196.47. ESI-MS (m/z): Calculated-
270.2551, Observed-270.2488.
4.1.13.
(2E)-1-(2H-1,3-benzodioxol-5-yl)-3-[4-(trifluoromethyl)phenyl]prop-2-en-1-one
1
(O13): Yield:69%. H NMR (500 MHz, CDCl3)δ: 6.08 (s, 2H, O-CH2-O), 6.91-6.89 (d, 1H,
J= 10.0 Hz, -ArH), 7.40-7.37 (d, 1H, J= 15.0 Hz, -CHα), 7.49 (s, 1H, -ArH), 7.52-7.50 (d,
2H, J= 10.0 Hz, -ArH), 763-7.61 (d, 2H, J= 10.0 Hz, -ArH), 7.74-7.72 ( d, 1H, J= 10.0 Hz, -
ArH), 7.83-7.81 (d, 4H, J= 10.0 Hz, -ArH), 8.12-8.09 (d, 1H, J= 15.0 Hz, -CHβ). ESI-MS
(m/z): 13C-NMR (125 MHz, CDCl3) δ: 101.95, 107.51, 108.51, 125.06, 126.23, 126.31,
127.92, 129.57, 132.07, 132.42, 134.42, 139.58, 151.96, 188.03. Calculated- 320.2626,
Observed-320.2599.
4.1.14.
(2E)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-phenylprop-2-en-1-one
(O14):
Yield:72%. 1H NMR (500 MHz, CDCl3)δ: 4.31-4.29 (t, 4H, J= 10.0 Hz, O-CH2-CH2-O),
6.95-6.93 (d, 1H, J= 10.0 Hz, -ArH), 7.42-7.40, (d, 3H, J= 10.0 Hz, -ArH), 7.52-7.49 (1H, d,
J= 15.0 Hz, -CHα),7.63 (s, 1H, -ArH), 7.65-7.63 (d, 3H, J= 10.0 Hz, -ArH), 7.81-7.78 (d, 1H,
13
J= 15.0 Hz, -CHβ). C-NMR (125 MHz, CDCl3) δ: 64.70, 117.17, 118.05, 121.70, 122.67,
127.97, 128.07, 128.93, 130.37, 131.92, 135.01, 143.43, 144.11, 147.94, 188.55. ESI-MS
(m/z): Calculated- 266.2912, Observed-266.2921.
4.1.15.
(2E)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one
1
(O15): Yield:47%. H NMR (500 MHz, CDCl3)δ: 4.30-4.28 (t, 4H, J= 10.0 Hz, O-CH2-CH2-
23

O), 6.94-6.92 (d, 1H, J= 10.0 Hz, -ArH), 7.44-7.42, (d, 3H, J= 10.0 Hz, -ArH), 7.51-7.48
(1H, d, J= 15.0 Hz, -CHα), 7.65-7.63 (d, 3H, J= 10.0 Hz, -ArH), 7.80-7.77 (d, 1H, J= 15.0
13
Hz, -CHβ). C-NMR (125 MHz, CDCl3) δ: 64.99, 117.15, 118.35, 121.71, 122.17, 127.53,
128.17, 128.53, 130.35, 131.62, 135.80, 143.44, 144.71, 147.44, 188.35. ESI-MS (m/z):
Calculated- 282.2906, Observed-282.2899.
4.1.16.
(2E)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-(4-methoxyphenyl)prop-2-en-1-one
1
(O16): Yield:82%. H NMR (500 MHz, CDCl3)δ: 3.85 (s, 3H, OCH3), 4.32-4.30 (t, 4H, J=
10.0 Hz, O-CH2-CH2-O), 6.94-6.92 (d, 2H, J= 10.0 Hz, -ArH), 6.95 (s, 1H, -ArH), 7.40-7.37
(1H, d, J= 15.0 Hz, -CHα), 7.60-7.58, (d, 4H, J= 10.0 Hz, -ArH), 7.78-7.75 (d, 1H, J= 15.0
Hz, -CHβ). 13C-NMR (125 MHz, CDCl3) δ: 54.41, 64.69, 114.35, 117.22, 119.37, 122.54,
123.45, 127.73, 130.13, 132.20, 143.37, 143.96, 147.74, 161.51, 188.59. ESI-MS (m/z):
Calculated- 296.3172, Observed-296.3099.
4.1.17.(2E)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-(4-methylphenyl)prop-2-en-1-one (O17):
1
Yield:81%. H NMR (500 MHz, CDCl3)δ: 2.39 (s, 3H, OCH3), 4.33-4.31 (t, 4H, J= 10.0 Hz,
O-CH2-CH2-O), 6.96-6.94 (d, 2H, J= 10.0 Hz, -ArH), 7.23-7.21 (d, 2H, J= 10.0 Hz, -ArH),
7.48-7.45 (1H, d, J= 15.0 Hz, -CHα), 7.54-7.52, (d, 1H, J= 10.0 Hz, -ArH), 7.60-7.58, (d, 2H,
13
J= 10.0 Hz, -ArH), 7.79-7.76 (d, 1H, J= 15.0 Hz, -CHβ). C-NMR (125 MHz, CDCl3) δ:
21.53, 64.70, 117.26, 118.26, 120.69, 122.62, 124.56, 128.41, 129.66, 132.27, 140.88,
143.18, 143.40, 144.21, 147.83, 188.65. ESI-MS (m/z): Calculated- 280.3178, Observed-
280.3099.
4.1.18. (2E)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-[4-(dimethylamino)phenyl]prop-2-en-1-
1
one (O18): Yield:80%. H NMR (500 MHz, CDCl3)δ: 3.04 (s, 6H, N(CH3)2), 4.32-4.30 (t,
4H, J= 10.0 Hz, O-CH2-CH2-O), 6.95-6.93 (d, 1H, J= 10.0 Hz, -ArH), 7.62-7.60 (d, J= 10.0
Hz, 6H, ArH), 7.33-7.30 (1H, d, J= 15.0 Hz, -CHα), 7.79-7.76 (d, 1H, J= 15.0 Hz, -CHβ). 13C-
24

NMR (125 MHz, CDCl3) δ: 40.16, 64.67, 111.25, 116.49, 117.84, 120.31, 122.40, 130.30,
132.76, 141.45, 145.09, 151.89, 188.70. ESI-MS (m/z): Calculated- 309.3590, Observed-
309.3498.
4.1.19. (2E)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-(4-ethylphenyl)prop-2-en-1-one (O19):
1
Yield:75%. H NMR (500 MHz, CDCl3)δ: 1.28-1.26 (t, 3H , J= 10.0 Hz, CH3), 2.69-2.67 (q,
2H , J= 10.0 Hz, CH2), 4.30-4.28 (t, 4H, J= 10.0 Hz, O-CH2-CH2-O), 6.92-6.90 (d, 1H, J=
10.0 Hz, -ArH), 7.28-7.26 (d, 1H, J= 10.0 Hz, -ArH), 7.45-7.42 (1H, d, J= 15.0 Hz, -CHα),
7.57-7.55, (d, 3H, J= 10.0 Hz, -ArH), 7.58 (s, 1H, -ArH), 7.65-7.63 (d, 1H, J= 10.0 Hz, -
ArH), 7.81-7.78 (d, 1H, J= 15.0 Hz, -CHβ).13C-NMR (125 MHz, CDCl3) δ: 15.33, 28.84,
66.65, 101.14, 107.08, 107.926 108.44, 120.08, 124.53, 128.53, 132.44, 144.17, 147.53,
148.26, 151.62, 188.44, 196.17. ESI-MS (m/z): Calculated- 294.3444, Observed-294.3399.
4.1.20. (2E)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-(4-nitrophenyl)prop-2-en-1-one (O20):
1
Yield:72%. H NMR (500 MHz, CDCl3)δ: 4.31-4.29 (t, 4H, J= 10.0 Hz, O-CH2-CH2-O),
7.00-6.90 (d, 1H, J= 10.0 Hz, -ArH), 7.623-7.60 (1H, d, J= 15.0 Hz, -CHα), 7.78-7.78 (d,
4H, J= 10.0 Hz, -ArH), 7.81-7.78 (d, 1H, J= 15.0 Hz, -CHβ), 8.28-8.26 (d, 2H, J= 10.0 Hz, -
ArH). 13C-NMR (125 MHz, CDCl3) δ: 64.66, 117.17, 117.49, 118.13, 122.47, 122.84,
124.19, 125.46, 128.86, 131.12, 140.82, 141.23, 143.58, 144.71, 148.45, 187.65. ESI-MS
(m/z): Calculated- 311.2888, Observed-311.2798.
4.1.21. (2E)-3-(4-chlorophenyl)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)prop-2-en-1-one (O21):
1
Yield:81%. H NMR (500 MHz, CDCl3) δ: 4.34-4.32 (t, 4H, J= 10.0 Hz, O-CH2-CH2-O),
6.96-6.94(1H, d, J= 10.0 Hz, -ArH), 7.39-7.37 (2H, d, J= 10.0 Hz, -ArH), 7.48-7.45 (1H, d,
J= 15.0 Hz, -CHα), 7.59-7.57 (4H, d, J= 10.0 Hz, -ArH), 7.74-7.71 (d, 1H, J= 15.0 Hz, -CHβ).
25

13C-NMR (125 MHz, CDCl3) δ: 64.12, 117.17, 117.81, 118.03, 122.01, 122.68, 129.218,
129.52, 132.72, 133.50, 136.19, 142.56, 143.46, 148.06, 188.21, 196.70. ESI-MS (m/z):
Calculated- 300.7363, Observed-300.00.
4.1.22.
(2E)-3-(4-bromophenyl)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)prop-2-en-1-one
(O22): Yield:63%. 1H NMR (500 MHz, CDCl3) δ: 4.35-4.33 (t, 4H, J= 10.0 Hz, O-CH2-
CH2-O), 6.97-6.95(1H, d, J= 10.0 Hz, -ArH), 7.48-7.46 (2H, d, J= 10.0 Hz, -ArH), 7.53-7.50
(1H, d, J= 15.0 Hz, -CHα), 7.59-7.57 (4H, d, J= 10.0 Hz, -ArH), 7.73-7.70 (d, 1H, J= 15.0
13
Hz, -CHβ). C-NMR (125 MHz, CDCl3) δ: 64.13, 117.35, 118.04, 122.20, 122.69, 124.58,
129.73, 130.86, 131.75, 132.15, 133.94, 142.65, 143.47, 148.07, 188.23. ESI-MS (m/z):
Calculated- 345.1873, Observed-345.1799.
4.1.23. (2E)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-(4-fluorophenyl)prop-2-en-1-one (O23):
1
Yield:72%. H NMR (500 MHz, CDCl3) δ: 4.34-4.32 (t, 4H, J= 10.0 Hz, O-CH2-CH2-O),
6.96-6.94 (1H, d, J= 10.0 Hz, -ArH), 6.97-6.95(1H, d, J= 10.0 Hz, -ArH), 7.12-7.10 (d, 1H,
J= 15.0 Hz, -CHβ), 7.60-7.58 (4H, d, J= 10.0 Hz, -ArH), 7.74-7.71 (d, 1H, J= 15.0 Hz, -CHβ).
13C-NMR (125 MHz, CDCl3) δ: 64.14, 116.00, 117.33, 118.03, 121.42, 122.66, 130.22,
131.84, 142.80, 143.46, 148.002, 162.95, 164.95, 188.34. ESI-MS (m/z): Calculated-
284.2817, Observed-284.2793.
4.1.24. (2E)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-(3-fluorophenyl)prop-2-en-1-one (O24):
1
Yield:71%. H NMR (500 MHz, CDCl3)δ: : 4.32-4.30 (t, 4H, J= 10.0 Hz, O-CH2-CH2-O),
6.03 (s, 2H, O-CH2-O), 6.97-6.95 (d, 1H, J= 10.0 Hz, -ArH), 7.10-7.08 ( d, 1H, J= 10.0 Hz, -
ArH), 7.39-7.37 (d, 3H, J= 10.0 Hz, -ArH), 7.50-7.47 (d, 1H, J= 15.0 Hz, -CHα), 7.60-7.58
13
(d, 2H, J= 10.0 Hz, -ArH), 7.75-7.72 (d, 1H, J= 15.0 Hz, -CHβ). C-NMR (125 MHz,
CDCl3) δ: 64.72, 114.31, 117.36, 118.06, 122.87, 124.87, 130.42, 139.49, 137.26, 142.56,
143.49, 148.12, 162.04, 164.00, 188.19, 196.61. ESI-MS (m/z): Calculated- 284.2817,
Observed-284.2787.
26

4.1.25. (2E)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-(2-fluorophenyl)prop-2-en-1-one (O25):
1
Yield:65%. H NMR (500 MHz, CDCl3)δ: 4.35-4.35 (t, 4H, J= 10.0 Hz, O-CH2-CH2-O),
6.97-6.95 (d, 3H, J= 10.0 Hz, -ArH), 7.38-7.35 (d, 1H, J= 15.0 Hz, -CHα), 7.63-7.61 (d, 5H,
13
J= 10.0 Hz, -ArH), 7.89-7.86 (d, 1H, J= 15.0 Hz, -CHβ). C-NMR (125 MHz, CDCl3) δ:
64.74, 117.43, 118.12, 122.78, 123.92, 125.86, 128.89, 131.52, 138.42, 142.04, 143.52,
188.05. ESI-MS (m/z): Calculated- 284.2817, Observed-284.2794.
4.1.26. (2E)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-[4-(trifluoromethyl)phenyl]prop-2-en-1-
one (O26): Yield: 67%. 1H NMR (500 MHz, CDCl3)δ: 4.35-4.32 (t, 4H, J= 10.0 Hz, O-CH2-
CH2-O), 4.35-4.35 (t, 4H, J= 10.0 Hz, O-CH2-CH2-O), 6.98-6.96 (d, 1H, J= 10.0 Hz, -ArH),
7.58-7.55 (d, 1H, J= 15.0 Hz, -CHα), 7.61-7.59 (d, 2H, J= 10.0 Hz, -ArH), 7.44-7.42 (d, 4H,
13
J= 10.0 Hz, -ArH), 7.80-7.77 (d, 1H, J= 15.0 Hz, -CHβ). C-NMR (125 MHz, CDCl3) δ:
64.78, 117.13, 118.22, 122.08, 123.12, 125.83, 128.39, 131.62, 138.62, 142.44, 143.42,
188.35. ESI-MS (m/z): Calculated- 334.2892, Observed-334.2799.
4.2. Enzyme assays
4.2.1. Assays of MAO-A and MAO-B
MAO activities were assayed using recombinant human MAO-A and MAO-B in the presence
of 0.06 mM kynuramine (1.7 × Km) or 0.3 mM benzylamine (2.1 × Km) as substrates,
respectively, as described previously [55]. Km values of these substrates were 0.036 mM and
0.14 mM, respectively. Enzymes and chemicals were purchased from Sigma-Aldrich (St.
Louis, MO, USA).
4.2.2. Analysis of enzyme inhibition and kinetics
The inhibitory activities of the 26 compounds against MAO-A and MAO-B were first
examined at a concentration of 10 µM, and inhibitory potencies were then determined using
27

IC50 values. Time-dependent inhibitions, reversibilities, and kinetic studies were performed
on the most potent inhibitors, i.e., O6 for MAO-A and O23 for MAO-B, as previously
described [56]. Kinetic experiments were carried out at five substrate and three inhibitor
concentrations.
4.2.3. Analysis of inhibitor reversibilities
Reversibilities of compounds O6 and O23 were analyzed using a dialysis method after
preincubation with MAO-A and MAO-B, respectively, for 30 min, as previously described
[57]. The concentrations used were; 0.06 µM for O6, 0.0040 µM for O23, 2.0 µM for
toloxatone (a reversible MAO-A reference inhibitor), 0.014 µM for clorgyline (an irreversible
MAO-A reference inhibitor), 0.080 µM for lazabemide (a reversible MAO-B reference
inhibitor), and 0.20 µM for pargyline (an irreversible MAO-B reference inhibitor). The
relative activities of un-dialyzed (AU) and dialyzed (AD) samples were compared for
determination of reversibility patterns.
4.3. Computational studies
4.3.1. Docking methodology
Starting with the fetching from the Protein Data Bank [52, 53] of the 3D structures of MAO-
A (PDB ID: 2Z5X) and MAO-B (PDB ID: 2V5Z), a pre-treatment of the enzymes was
carried out by means of protein preparation module available from the Schrödinger suite [58]:
the latter exploits an optimization of a protein structure by adjusting the imprecisions that
may occur within a X-ray crystal structure; in doing that, 9 water molecules within MAO-A
and 8 water molecules within MAO-B were preserved and not eliminated. The prepared
proteins were subjected to docking simulations carried out by using the QM polarized ligand
docking available from Schrödinger Suite; such protocol allows a certain conformational
flexibility for ligand to be docked while rigidity of the protein structures is retained [59]. The
28

ligand center of mass of the X-ray cognate ligand of both PDB structures was taken as
reference for the cubic grid center.
The QM-polarized ligand docking protocol is arranged in three steps, that are: a) a standard
precision (SP) initial docking using Glide; b) the calculation of QM partial charges of the
docked ligand based on the field generated by the receptor; c) a standard precision (SP) re-
docking phase upon each ligand pose considering computed QM based charges.
During the analysis of docked poses, steric hindrance between ligands and protein residues
was also evaluated according to the following computation of bad contacts:
퐷12
퐶 =
(푅1 + 푅2)
where D12 is the distance between atoms 1 and 2, and R1 and R2 are the van der Waals radii
of atoms 1 and 2. A range of C values between 0.75 and 0.89 flags a contact between the
ligand and the receptor as “bad” [60].
4.3.2. MM-GBSA calculations
In order to estimate ligand-binding affinities, a Molecular Mechanics/Generalized Born
Surface Area (MM-GBSA) method was added to the workflow for the calculation of the
binding free energies (G) between protein and ligands [61]. Such method is implemented in
Prime available in the Schrodinger software 2018-2. Provided that ΔEMM is the term referred
to the minimized energy of the ligand-protein complex, ΔGsolv is the term referred to the
solvation energy and ΔGSA is the term referred to the surface area energy, the binding free
energies (G) of compounds with respect to MAO-A and MAO-B were calculated as follows:
∆퐺푏푖푛푑 = ∆퐸푀푀 + ∆퐺푠표푙푣 + ∆퐺푆퐴
Obtained docking poses were minimized using Prime.
4.3.3. ADME-Tox in silico prediction
A panel of pharmaceutically relevant properties (to name a few, the octanol/water and
29