Design, synthesis, and evaluation of 1, 4-benzodioxan-substituted chalcones as selective and reversible inhibitors of human monoamine oxidase B

Article abstract of DOI:10.1080/14756366.2020.1797711

The inhibition of monoamine oxidase B (MAO-B) could be an effective approach for the treatment of various neurological disorders. In this study, a series of 1, 4-benzodioxan-substituted chalcone derivatives were designed, synthesised and evaluated for the

Full text of DOI:10.1080/14756366.2020.1797711

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis, and evaluation of 1, 4-
benzodioxan-substituted chalcones as selective
and reversible inhibitors of human monoamine
oxidase B
Zhuo Kong , Demeng Sun , Yanmei Jiang & Yun Hu
To cite this article: Zhuo Kong , Demeng Sun , Yanmei Jiang & Yun Hu (2020) Design, synthesis,
and evaluation of 1, 4-benzodioxan-substituted chalcones as selective and reversible inhibitors
of human monoamine oxidase B, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1,
1513-1523, DOI: 10.1080/14756366.2020.1797711
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1513–1523
https://doi.org/10.1080/14756366.2020.1797711
RESEARCH PAPER
Design, synthesis, and evaluation of 1, 4-benzodioxan-substituted chalcones as
selective and reversible inhibitors of human monoamine oxidase B
ꢀ
ꢀ
Zhuo Kong , Demeng Sun , Yanmei Jiang and Yun Hu
Department of Bioengineering, Zunyi Medical University, Zhuhai, China
ABSTRACT
ARTICLE HISTORY
Received 17 May 2020
Revised 7 July 2020
Accepted 14 July 2020
The inhibition of monoamine oxidase B (MAO-B) could be an effective approach for the treatment of vari-
ous neurological disorders. In this study, a series of 1, 4-benzodioxan-substituted chalcone derivatives
were designed, synthesised and evaluated for their inhibitory activity against human MAO-B (hMAO-B).
The majority of these compounds showed inhibitory activity and high selectivity. The most potent com-
pound, (E)-1-(3-bromo-4-fluorophenyl)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)prop-2-en-1-one (22), exhib-
ited an IC50 of 0.026 mM with a selectivity index greater than 1538. Kinetics and reversibility studies
confirmed that the representative active compounds acted as competitive and reversible inhibitors of
hMAO-B. The enzyme-inhibitor interactions were investigated by molecular docking studies and the ration-
ale was provided. As these potent hMAO-B inhibitors exhibited low neurotoxicity and possessed promising
drug-like properties, we believe that these active compounds could be further investigated as potential
drug candidates for future in vivo studies.
KEYWORDS
Monoamine oxidase B
selective inhibitors;
reversible inhibitors;
heterocyclic chalcones
Introduction
inhibition, namely, de novo enzyme biosynthesis in the human
brain and potential immunogenicity of enzyme ꢁ inhibitor
adducts10. Reversible MAO-B inhibitors are believed to avoid these
side effects and have safer profiles, as the enzyme activity can be
recovered by simply terminating drug treatment. Recently, a
reversible MAO-B inhibitor, safinamide (IC50 ¼ 0.023 mM, Figure
1(A))11, has been approved for the treatment of PD. More efforts
will be devoted to the development of new reversible inhibitors
of MAO-B in the future.
In the search for new MAO-B inhibitors, chalcones have been
used as a valid scaffold (Figure 1(B)). Chimenti et al.12 have syn-
thesised plenty of chalcone derivatives based on the typical chal-
cone skeleton (1, 3-diphenyl-2-propen-1-one). They showed that,
with the appropriate substitution, these synthetic chalcone deriva-
tives exhibit high inhibitory potency and selectivity against human
MAO-B (hMAO-B). The most active chalcone compound, 5i, exhib-
its irreversible inhibition of hMAO-B with an IC50 value of
0.0044 mM. Interestingly, several groups13–16 have reported that
heterocyclic chalcone derivatives, in which the phenyl rings are
replaced with certain heterocyclic rings, such as furan and thio-
phene, are potential reversible hMAO-B inhibitors. The furanochal-
cone derivative 3a and thienylchalcone derivative 4h exhibit IC50
values of 0.174 mM and 0.067 mM13, respectively, for the inhibition
of hMAO-B.
Monoamine oxidases (MAO, EC 1.4.3.4) are flavin-dependent enzy-
mesthat play important roles in the metabolism of monoamine
neurotransmitters, such as dopamine, noradrenaline, serotonin. In
most mammalian tissues, there are two isoforms of MAO, MAO-A
and MAO-B, which share approximately 70% sequence identity. In
the human body, MAO-A predominates in sympathetic nerve ter-
minals and intestinal mucosa, whereas MAO-B is the predominant
isoform expressed in the brain1. Although both are active towards
dopamine and tyramine, the two isoforms of MAO show different
substrate specificities. MAO-A preferentially catalyses the oxidative
deamination of serotonin and noradrenaline, whereas the pre-
ferred
substrates
of
MAO-B
are
benzylamine
and
b-phenylethylamine2,3
.
Monoamines are usually degraded to aldehydes during cata-
lytic reactions and hydrogen peroxide (H2O2) is concomitantly
generated4. High levels of H2O2 result in the formation of reactive
oxygen species (ROS), which are neurotoxic and associated with
various neurodegenerative processes. The expression and activity
of MAO-B significantly increase in the brain of AD and PD
patients5. As the inhibition of MAO-B can enhance the level of
dopamine in the brain and reduce the formation of ROS, MAO-B
inhibitors are considered promising therapeutic agents for PD and
AD6. Currently, two types of MAO-B inhibitors are available for the
clinical treatment of PD. The first type is the irreversible MAO-B
inhibitors (Figure 1(A)), such as R-(–)-deprenyl (IC50 ¼ 0.0196 mM)7
and rasagiline (IC50 ¼ 0.069 mM)8. The propargyl group of this class
of inhibitors can form a covalent interaction with the flavin ring of
MAO-B, disabling it for further catalysis9. These so called “suicide
The 1, 4-benzodioxan derivatives also exhibit inhibition against
MAO-B (Figure 1(B)). For example, compound 7c inhibits the
hMAO-B with an IC50 value of 0.045 mM17. It is suggested that the
1, 4-benzodioxan moiety could be useful for the design of new
MAO-B inhibitors
Inspired by these results, we report the design, synthesis, and
inhibitors” display the typical drawbacks of long-lasting enzyme evaluation of a series of 1, 4-benzodioxan-substituted chalcone
CONTACT Yun Hu
These authors contributed equally to this work.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
ce04hy@mail2.sysu.edu.cn
Department of Bioengineering, Zunyi Medical University, Zhuhai Campus, Zhuhai 519041, China
ꢀ
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1514
Z. KONG ET AL.
(A)
(B)
Figure 1. (A) Structures of irreversible and reversible MAO inhibitors in clinical use; (B) Structures, potencies and inhibition modes of previously described MAO-B inhib-
itors. Middle, design strategy of 1, 4-benzodioxan-substituted chalcone compounds.
derivatives as new hMAO-B inhibitors. Additionally, the structure- Synthesis of chalcone compounds
activity relationship (SAR) is discussed and summarised. Hit com-
The chalcone compounds 1–28 were synthesised according to the
pounds from the screen assay exhibit high inhibitory activity and
synthetic protocol outlined in Scheme 1.
selectivity, which are new reversible inhibitors of hMAO-B.
General procedure for the synthesis of compound 1–9, 11–28
Acetophenones (1 mmol) and corresponding aromatic aldehyde
(1 mmol) were dissolved in 2 mL methanol. Then 2 mL 30% NaOH
Materials and methods
General methods
was added. The mixture was kept stirred at room temperature
and the reaction was monitored by TLC until completed. Under
ice bath, the mixture was diluted with water and adjusted to pH
¼ 2–3 with 5% HCl. The precipitate formed was filtered off and
then purified by chromatography column with ethyl acetate/pet-
roleum ether or recrystallization with ethanol.
The acetophenones and 1, 4-benzodioxan-6-carboxaldehydes were
purchased from Energy Chemical Company (Shanghai, China).
Other reagents and solvents were commercially available and
were used without further purification. Reactions were monitored
by TLC on a glass plate coated with silica gel with fluorescent
indicator (GF254). Column chromatography was performed on sil-
ica gel (200 ꢁ 300 mesh). All melting points were measured on
the OptiMelt automated melting point system. 1H NMR and 13C
NMR spectra were recorded using TMS as an internal standard
with a Bruker BioSpin Ultrashield 400 NMR system. The Purities of
compounds used for biological evaluation (>95%) were deter-
mined on a Waters Ultimate HPLC system using UV monitor at
254 and 365 nm for detection. The compounds were eluted with
Acetonitrile/water (0.1% TFA, w/v) in ratios of 20:80–75:25 at a
flow rate of 0.3 mL/min. The Compounds purities were calculated
as the percentage peak area of the analysed compound, and
retention times (tR) were presented in minutes. High resolution
mass spectra (HRMS) were recorded on Agilent Technologies 6530
Q-TOF. The human recombinant MAO-A and -B enzymes, Amplex
Red reagent, horseradish peroxidase, R-(-)-deprenyl, rasagiline and
safinamide were obtained from Sigma Aldrich.
Synthesis of compound 10
4 mmol 2, 4-dihydroxyacetophenone and 2 mmol Pyridinium 4-tol-
uenesulfonate were dissolved in 25 mL CH2Cl2. 6 mmol 3,4-dihy-
dro-2H-pyran was slowly added to the solution and the mixture
was kept stirred at room temperature. The reaction was monitored
by TLC until completed. The organic solvent was removed under
vacuum and the crude product was used without further
purification.
6 mmol 1, 4-benzodioxan-6-carboxaldehyde was dissolved in
ethanol and mixed with the above product. Then 6 mL 30% NaOH
aqueous solution was added. The mixture was kept stirred at
room temperature and the reaction was monitored by TLC until
completed. Under ice bath, the mixture was diluted with water
and adjusted to pH ¼ 2–3 with 5% HCl. The intermediate formed
were filtered off and then purified by column chromatography.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1515
Scheme 1. Synthesis of Compounds 1–28.
The intermediate was then dissolved in 10 mL methanol and was NMR (101 MHz, DMSO-d6) d 189.42, 150.06, 148.58, 144.52, 138.24,
added with 172 mg p-toluenesulfonic acid. The mixture was kept 133.41, 129.67, 129.17, 128.91, 126.42, 120.47, 108.99, 107.45,
stirred at room temperature and the reaction was monitored by 102.13. HRMS: m/z [M þ H]þ calcd for C16H12O3: 253.0800,
TLC until completed. Under ice bath, the mixture was diluted with found: 253.0883.
excessive water. The yellow crude product formed were then fil-
tered off and purified by recrystallization with ethanol.
(E)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-phenylprop-2-en-1-one
(4) Yield 63.52%; m.p.: 129.7–130.7 ꢂC; UPLC purity 99.77%, tR
1
(E)-3-(naphthalen-2-yl)-1-phenylprop-2-en-1-one (2) Yield 38.8%; ¼1.704 min. H NMR (400 MHz, DMSO-d6) d 8.15 (d, J ¼ 7.6 Hz, 2H),
m.p.: 157.9–159.4 ꢂC; UPLC purity 98.85%, tR ¼8.950 min. 1H NMR 7.81 (d, J ¼ 15.5 Hz, 1H), 7.68 ꢁ 7.64 (m, 2H), 7.59 ꢁ 7.55 (m, 2H),
(400 MHz, DMSO-d6) d 8.35 (s, 1H), 8.21 (d, J ¼ 7.5 Hz, 2H), 8.14 (d, 7.51 (s, 1H), 7.38 (d, J ¼ 8.1 Hz, 1H), 6.94 (d, J ¼ 8.3 Hz, 1H), 4.30 (q,
J ¼ 8.6 Hz, 1H), 8.08 (d, J ¼ 15.6 Hz, 1H), 8.01 ꢁ 7.90 (m, 2H), J ¼ 5.1 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) d 189.45, 146.35,
7.72 ꢁ 7.57 (m, 5H). 13C NMR (101 MHz, DMSO-d6) d 189.64, 144.51, 144.39, 144.09, 138.25, 133.40, 129.18, 128.91, 128.64, 123.52,
138.10, 134.42, 133.62, 133.43, 132.80, 131.17, 129.28, 129.02, 120.59, 117.90, 117.69, 64.89, 64.43. HRMS: m/z [M þ H]þ calcd for
128.95, 128.19, 127.92, 127.26, 124.91, 122.85. HRMS: m/z [M þ H]þ C17H14O3: 267.1016, found: 267.1085.
calcd for C19H14O: 259.1117, found: 259.1117.
(E)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-(2-hydroxyphenyl)-
(E)-3-(benzo[d][1,3]dioxol-5-yl)-1-phenylprop-2-en-1-one (3) yield prop-2-en-1-one (5) Yield 49.25%; m.p.: 120.8–122.3 ꢂC; UPLC pur-
30.84%; m.p.: 117.2–118.3 ꢂC; UPLC purity 99.18%, tR ¼1.697 min. ity 97.49%, tR ¼ 2.237 min, 1H NMR (400 MHz, DMSO-d6) d 12.73
1H NMR (400 MHz, DMSO-d6) d 8.18 ꢁ 8.14 (m, 1H), 7.84 (d, (s, 1H), 8.30 (d, J ¼ 7.2 Hz, 1H), 7.92 (d, J ¼ 15.4 Hz, 1H), 7.77 (d,
J ¼ 15.5 Hz, 0H), 7.75 ꢁ 7.63 (m, 1H), 7.57 (t, J ¼ 7.5 Hz, 1H), 7.34 J ¼ 15.4 Hz, 1H), 7.59–7.55 (m, 2H), 7.41 (dd, J ¼ 8.4, 1.7 Hz, 1H),
(dd, J ¼ 8.1, 1.7 Hz, 0H), 7.00 (d, J ¼ 8.0 Hz, 0H), 6.12 (s, 1H). 13C 7.02- 6.98 (m, 2H), 6.95 (d, J ¼ 8.4 Hz, 1H), 4.31 (dd, J ¼ 9.8, 5.0 Hz,

1516
Z. KONG ET AL.
4H). 13C NMR (101 MHz, DMSO-d6) d 194.07, 162.57, 146.76, 145.50, 64.43, 56.23, 56.08. HRMS: m/z [M þ H]þ calcd for C19H18O5:
144.14, 136.73, 131.33, 128.42, 124.05, 120.99, 119.92, 119.55, 327.1227, found: 327.1251.
118.19, 118.01, 117.98, 64.94, 64.42. HRMS: m/z [M-H]þ calcd for
C17H14O4: 281.0819, found: 281.0827.
(E)-3–(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-(2-fluorophenyl)prop-
2-en-1-one (12) Yield 14.1%; m.p.: 93.7–95.4 ꢂC; UPLC purity
98.88%, tR ¼7.654 min. 1H NMR (400 MHz, DMSO-d6) d 7.76 (t,
(E)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1–(3-methoxyphenyl)-
prop-2-en-1-one (6) Yield 75.1%; m.p.: 94.4–95.6 ꢂC; UPLC purity J ¼ 7.4 Hz, 1H), 7.65 (q, J ¼ 6.8, 6.4 Hz, 1H), 7.54 (d, J ¼ 15.7 Hz, 1H),
97.46%, tR ¼7.624 min. 1H NMR (400 MHz, DMSO-d6) d 7.84 ꢁ 7.71 7.42 ꢁ 7.23 (m, 5H), 6.92 (d, J ¼ 8.4 Hz, 1H), 4.29 (q, J ¼ 4.9 Hz, 4H).
(m, 2H), 7.65 (d, J ¼ 15.5 Hz, 1H), 7.61 (s, 1H), 7.56 ꢁ 7.42 (m, 2H), 13C NMR (101 MHz, DMSO-d6) d 189.23, 161.81, 159.32, 146.58,
7.37 (d, J ¼ 8.4 Hz, 1H), 7.22 (d, J ¼ 8.2 Hz, 1H), 6.93 (d, J ¼ 8.4 Hz, 145.08, 144.10, 134.47, 134.38, 130.88, 130.86, 128.16, 127.68,
1H), 4.29 (q, J ¼ 5.2 Hz, 4H), 3.85 (s, 3H). 13C NMR (101 MHz, DMSO- 127.55, 125.25, 125.23, 124.24, 124.21, 123.27, 118.04, 117.75,
d6) d 189.20, 159.99, 146.35, 144.47, 144.07, 139.71, 130.31, 128.62, 117.14, 116.92, 64.89, 64.40. HRMS: m/z [M þ H]þ calcd for
123.59, 121.43, 120.62, 119.45, 117.88, 117.73, 113.43, 64.88, 64.42, C17H13FO3:285.0921, found: 285.0809.
55.82. HRMS: m/z [M þ H]þ calcd for C18H16O4: 297.1121,
(E)-1-(2-chlorophenyl)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)prop-
found: 297.1134.
2-en-1-one (13) Yield 36%; m.p.: 67.5–69.4 ꢂC; UPLC purity 99.39%,
1
(E)-3–(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-(4-hydroxyphenyl)- tR ¼7.686 min. H NMR (400 MHz, DMSO-d6) d 7.59 ꢁ 7.51 (m, 3H),
prop-2-en-1-one (7) Yield 29%; m.p.: 206.3–207.7 ꢂC; UPLC purity 7.50 ꢁ 7.47 (m, 1H), 7.35 ꢁ 7.29 (m, 2H), 7.24 (dd, J ¼ 8.4, 2.0 Hz,
99.79%, tR ¼2.327 min. 1H NMR (400 MHz, DMSO-d6) d 10.41 (s, 1H), 7.12 (d, J ¼ 16.0 Hz, 1H), 6.90 (d, J ¼ 8.4 Hz, 1H), 4.33 ꢁ 4.24 (m,
1H), 8.08 (d, J ¼ 8.7 Hz, 2H), 7.77 (d, J ¼ 15.5 Hz, 1H), 7.59 (d, 4H). 13C NMR (101 MHz, DMSO-d6) d 193.44, 146.69, 146.58, 144.12,
J ¼ 15.5 Hz, 1H), 7.47 (d, J ¼ 1.7 Hz, 1H), 7.35 (dd, J ¼ 8.4, 1.8 Hz, 139.35, 132.12, 130.51, 130.34, 129.67, 127.98, 127.81, 124.92,
1H), 6.93 (d, J ¼ 8.6 Hz, 2H), 6.89(s, 1H), 4.30 (q, J ¼ 4.7 Hz, 4H). 13C- 123.45, 118.03, 117.77, 64.90, 64.38. HRMS: m/z [M þ H]þ calcd for
NMR (101 MHz, DMSO-d6) d 187.49, 162.48, 146.03, 144.05, 143.05, C17H13ClO3: 301.0626, found: 301.0645.
131.54, 129.77, 128.86, 123.21, 120.65, 117.86, 117.47, 115.77,
(E)-1-(2-bromophenyl)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)prop-
64.85, 64.43. HRMS: m/z [M-H]þ calcd for C17H14O4: 281.0819, 2-en-1-one (14) Yield 39.3%; m.p.: 70.3–72.2 ꢂC; UPLC purity
found: 281.0831.
99.68%, tR ¼7.864 min. 1H NMR (400 MHz, DMSO-d6) d 7.73 (d,
(E)-3–(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-(4-methoxyphenyl)- J ¼ 7.8 Hz, 1H), 7.54 ꢁ 7.49 (m, 2H), 7.47 ꢁ 7.43 (m, 1H), 7.34 ꢁ 7.23
prop-2-en-1-one (8) Yield 43.9%; m.p.: 93.8–94.9 ꢂC; UPLC purity (m, 3H), 7.09 (d, J ¼ 16.0 Hz, 1H), 6.90 (d, J ¼ 8.4 Hz, 1H), 4.34 ꢁ 4.23
98.82%, tR ¼7.483 min. 1H NMR (400 MHz, DMSO-d6) d 8.17 (d, (m, 4H). 13C NMR (101 MHz, DMSO-d6) d 194.36, 146.78, 146.70,
J ¼ 8.8 Hz, 2H), 7.80 (d, J ¼ 15.5 Hz, 1H), 7.62 (d, J ¼ 15.5 Hz, 1H), 144.13, 141.41, 133.59, 132.12, 129.54, 128.26, 127.98, 124.75,
7.49 (d, J ¼ 2.0 Hz, 1H), 7.36 (dd, J ¼ 8.4, 2.0 Hz, 1H), 7.08 (d, 123.46, 119.05, 118.03, 117.76, 64.90, 64.38. HRMS: m/z [M þ H]þ
J ¼ 8.7 Hz, 2H), 6.93 (d, J ¼ 8.4 Hz, 1H), 4.30 (q, J ¼ 5.0 Hz, 4H), 3.87 calcd for C17H13BrO3: 345.0121, found: 345.0138.
(s, 3H). 13C NMR (101 MHz, DMSO-d6) d 187.68, 163.56, 146.14,
(E)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-(3-fluorophenyl)prop-
144.07, 143.49, 131.29, 131.11, 128.80, 123.37, 120.56, 117.86, 2-en-1-one (15) Yield 70.4%; m.p.: 128.3–12.4 ꢂC; UPLC purity
117.56, 114.41, 64.86, 64.43, 56.01. HRMS: m/z [M þ H]þ calcd for 93.14%, tR ¼7.813 min. 1H NMR (400 MHz, DMSO-d6) d 8.00 (d,
C18H16O4: 297.1121, found: 297.1041.
J ¼ 7.7 Hz, 1H), 7.96 (d, J ¼ 9.8 Hz, 1H), 7.81 (d, J ¼ 15.5 Hz, 1H), 7.68
(E)-3–(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-(3-hydroxyphenyl)- (d, J ¼ 15.5 Hz, 1H), 7.61 (td, J ¼ 8.0, 5.8 Hz, 1H), 7.54 ꢁ 7.48 (m,
prop-2-en-1-one (9) Yield 63.76%; m.p.: 196.8–198.5 ꢂC; UPLC pur- 2H), 7.39 (dd, J ¼ 8.5, 2.1 Hz, 1H), 6.93 (d, J ¼ 8.4 Hz, 1H), 4.30 (q,
ity 99.26%, tR ¼6.407 min. 1H NMR (400 MHz, DMSO-d6) d 9.77 (s, J ¼ 5.4 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) d 188.18, 164.02,
1H), 7.71 (d, J ¼ 15.6 Hz, 1H), 7.67 ꢁ 7.56 (m, 2H), 7.48 ꢁ 7.46 (m, 161.59, 146.52, 145.13, 144.09, 140.52, 140.46, 131.36, 131.29,
2H), 7.38 ꢁ 7.34 (m, 2H), 7.05 (dd, J ¼ 8.1, 2.5 Hz, 1H), 6.93 (d, 128.52, 125.06, 125.03, 123.82, 120.38, 120.17, 117.89, 117.79,
J ¼ 8.4 Hz, 1H), 4.30 (q, J ¼ 4.8 Hz, 4H). 13C NMR (101 MHz, DMSO- 115.55, 115.33, 64.90, 64.42. HRMS: m/z [M þ H]þ calcd for
d6) d 189.39, 158.14, 146.29, 144.16, 144.08, 139.70, 130.22, 128.65, C17H13FO3: 285.0921, found: 285.0929.
123.40, 120.78, 120.55, 119.97, 117.90, 117.66, 115.05, 64.88, 64.42.
(E)-1-(3-bromophenyl)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)prop-
HRMS:
m/z
[M þ H]þ
calcd
for
C17H14O4:
283.0965, 2-en-1-one (16) Yield 25.53%; m.p.: 119.3–120.4 ꢂC; UPLC purity
99.08%, tR ¼2.557 min. 1H NMR (400 MHz, DMSO-d6) d 8.32 (t,
found: 283.0982.
(E)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1–(2,4-dihydroxyphenyl)- J ¼ 1.8 Hz, 1H), 8.15 (d, J ¼ 7.8 Hz, 1H), 7.90 ꢁ 7.77 (m, 2H), 7.68 (d,
prop-2-en-1-one (10) Yield 16.45%; m.p.: 169.5–170.8 ꢂC; UPLC pur- J ¼ 15.5 Hz, 1H), 7.61 ꢁ 7.47 (m, 2H), 7.40 (dd, J ¼ 8.4, 2.1 Hz, 1H),
1
ity 93.36%, tR ¼1.582 min. H NMR (400 MHz, DMSO-d6) d 13.54 (s, 6.94 (d, J ¼ 8.3 Hz, 1H), 4.30 (q, J ¼ 5.3 Hz, 4H). 13C NMR (101 MHz,
1H), 10.76 (s, br, 1H), 8.21 (d, J ¼ 9.0 Hz, 1H), 7.83 (d, J ¼ 15.4 Hz, DMSO-d6) d 188.06, 146.56, 145.26, 144.10, 140.27, 136.04, 131.41,
1H), 7.71 (d, J ¼ 15.3 Hz, 1H), 7.53 (d, J ¼ 1.5 Hz, 1H), 7.39 ꢁ 7.37 (m, 128.53, 127.90, 123.90, 122.76, 120.10, 117.89, 117.85, 64.91, 64.42.
1H), 6.94 (d, J ¼ 8.4 Hz, 1H), 6.43 ꢁ 6.40 (m, 1H), 6.30 (d, J ¼ 2.0 Hz, HRMS: m/z [M þ H]þ calcd for C17H13BrO3: 345.0121,
1H), 4.31 ꢁ 4.29 (m, 4H).13C-NMR (101 MHz, DMSO-d6) d 191.94,
found: 345.0124.
166.32, 165.61, 146.44, 144.14, 144.11, 133.55, 128.60, 123.77,
(E)-1-(3-chlorophenyl)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)prop-
119.65, 117.91, 117.79, 113.47, 108.63, 103.04, 64.90, 64.42. HRMS: 2-en-1-one (17) Yield 49.25%; m.p.: 125.5–127.0 ꢂC; UPLC purity
m/z [M þ H]þ calcd for C17H14O5: 299.0914, found: 299.0919.
95.22%, tR ¼2.366 min. 1H NMR (400 MHz, DMSO-d6) d 8.20 (t,
(E)-3–(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1–(3,4-dimethoxyphe- J ¼ 1.9 Hz, 1H), 8.11 (d, J ¼ 7.9 Hz, 1H), 7.83 (d, J ¼ 15.5 Hz, 1H),
nyl)prop-2-en-1-one (11) Yield 55.16%; m.p.: 110.0–111.1 ꢂC; 7.76 ꢁ 7.64 (m, 2H), 7.63 ꢁ 7.54 (m, 2H), 7.40 (dd, J ¼ 8.4, 2.1 Hz,
UPLC purity 94.77%, tR ¼6.966 min. 1H NMR (400 MHz, DMSO- 1H), 6.94 (d, J ¼ 8.4 Hz, 1H), 4.30 (q, J ¼ 5.3 Hz, 4H). 13C NMR
d6) d 7.91 (dd, J ¼ 8.4, 2.0 Hz, 1H), 7.81 (d, J ¼ 15.5 Hz, 1H), 7.62 (101 MHz, DMSO-d6) d 188.12, 146.55, 145.25, 144.10, 140.07,
(d, J ¼ 13.1 Hz, 2H), 7.50 (s, 1H), 7.37 (dd, J ¼ 8.4, 2.0 Hz, 1H),
134.26, 133.13, 131.15, 128.57, 128.53, 127.53, 123.89, 120.11,
7.09 (d, J ¼ 8.4 Hz, 1H), 6.93 (d, J ¼ 8.3 Hz, 1H), 4.30 (q, J ¼ 5.1 Hz, 117.89, 117.84, 64.91, 64.42. HRMS: m/z [M þ H]þ calcd for
4H), 3.87 (d, J ¼ 4.0 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) d C17H13ClO3: 301.0626, found: 301.0638.
187.67, 153.56, 149.23, 146.12, 144.06, 143.41, 131.17, 128.81,
(E)-1–(4-chlorophenyl)-3–(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)prop-
123.69, 123.39, 120.51, 117.84, 117.57, 111.34, 111.27, 64.87, 2-en-1-one (18) Yield 58.43%; m.p.: 102.2–103.3 ꢂC; UPLC purity

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1517
99.59%, tR ¼2.383 min. 1H NMR (400 MHz, DMSO-d6) d 8.18 (d, 128.35, 124.31, 123.13, 120.53, 120.12, 118.09, 117.92, 110.76,
J ¼ 8.5 Hz, 2H), 7.80 (d, J ¼ 15.5 Hz, 1H), 7.70 ꢁ 7.60 (m, 3H), 7.38 64.95, 64.40.
(E)-1-(5-chloro-2-hydroxyphenyl)-3-(2,3-dihydrobenzo[b][1,4]dioxin-
(dd, J ¼ 8.4, 2.0 Hz, 1H), 6.93 (d, J ¼ 8.3 Hz, 1H), 4.30 (d, J ¼ 4.1 Hz,
4H). 13C NMR (101 MHz, DMSO-d6) d 188.31, 146.47, 144.89, 144.09,
138.37, 136.89, 130.84, 129.28, 128.55, 123.69, 120.23, 117.91,
117.76, 64.90, 64.43. HRMS: m/z [M þ H]þ calcd for C17H13ClO3:
301.0626, found: 301.0632.
6-yl)prop-2-en-1-one (25) Yield 60.12%; m.p.: 136.2–137.3 ꢂC; UPLC
1
purity 98.79%, tR ¼3.216 min. H NMR (400 MHz, DMSO-d6) d 12.59
(s, 1H), 8.32 (s, 1H), 7.97 ꢁ 7.71 (m, 2H), 7.63 ꢁ 7.36 (m, 3H), 6.98
(dd, J ¼ 31.8, 8.5 Hz, 2H), 4.31 (s, 4H). 13C NMR (101 MHz, DMSO-d6)
d 192.95, 160.80, 146.89, 146.16, 144.13, 135.96, 130.12, 128.35,
124.33, 123.33, 122.48, 120.12, 120.06, 118.08, 117.92, 64.95, 64.41.
HRMS: m/z [M þ H]þ calcd for C17H13ClO4: 317.0575,
found: 317.0593.
(E)-1–(4-bromophenyl)-3–(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)
prop-2-en-1-one (19) Yield 50.48%; m.p.: 136.7–138.2 ꢂC; UPLC
purity 99.71%, tR ¼2.525 min. 1H NMR (400 MHz, DMSO-d6) d 8.09
(d, J ¼ 8.6 Hz, 2H), 7.84 ꢁ 7.73 (m, 3H), 7.67 (d, J ¼ 15.5 Hz, 1H), 7.51
(s, 1H), 7.38 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.93 (d, J ¼ 8.3 Hz, 1H), 4.30 (q,
J ¼ 4.8 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) d 188.52, 146.48,
144.93, 144.09, 137.21, 132.24, 130.96, 128.55, 127.56, 123.70,
120.20, 117.91, 117.77, 64.90, 64.43. HRMS: m/z [M þ H]þ calcd for
C17H13BrO3: 345.0121, found: 345.0132.
(E)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-(4-fluorophenyl)prop-
2-en-1-one (20) Yield 36.63%; m.p.: 135.5–136.5 ꢂC; UPLC purity
96.31%, tR ¼7.718 min. 1H NMR (400 MHz, DMSO-d6) d 8.25 (dd,
J ¼ 8.5, 5.5 Hz, 2H), 7.81 (d, J ¼ 15.5 Hz, 1H), 7.66 (d, J ¼ 15.5 Hz,
1H), 7.51 (s, 1H), 7.44 ꢁ 7.30 (m, 3H), 6.93 (d, J ¼ 8.4 Hz, 1H), 4.30
(q, J ¼ 5.1 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) d 187.94, 166.67,
164.17, 146.39, 144.55, 144.08, 134.91, 134.89, 131.93, 131.83,
128.59, 123.61, 120.31, 117.88, 117.69, 116.26, 116.05, 64.88, 64.42.
HRMS: m/z [M þ H]þ calcd for C17H13FO3: 285.0921,
found: 285.0940.
(E)-1-(4-chloro-2-hydroxyphenyl)-3-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)prop-2-en-1-one (26) Yield 37.89%; m.p.: 155.7–156.1 ꢂC; UPLC
1
purity 95.71%, tR ¼9.415 min. H NMR (400 MHz, DMSO-d6) d 12.89
(s, 1H), 8.29 (d, J ¼ 8.6 Hz, 1H), 7.85 (d, J ¼ 15.4 Hz, 1H), 7.76 (d,
J ¼ 15.4 Hz, 1H), 7.53 (d, J ¼ 2.1 Hz, 1H), 7.39 (dd, J ¼ 8.4, 2.1 Hz,
1H), 7.09 (d, J ¼ 2.1 Hz, 1H), 7.05 (dd, J ¼ 8.6, 2.1 Hz, 1H), 6.94 (d,
J ¼ 8.4 Hz, 1H), 4.31 (q, J ¼ 5.3 Hz, 4H). 13C NMR (101 MHz, DMSO-
d6) d 193.15, 163.01, 146.86, 145.83, 144.13, 140.54, 132.88, 128.33,
124.10, 120.30, 120.04, 119.81, 118.02, 117.97, 117.83, 64.94, 64.41.
HRMS: m/z [M þ H]þ calcd for C17H13ClO4: 317.0575,
found: 317.0593.
(E)-3-(benzo[d][1,3]dioxol-5-yl)-1–(3-bromophenyl)prop-2-en-1-one
(27) Yield 22.14%; m.p.: 111.8–112.6 ꢂC; UPLC purity 98.78%, tR
¼2.539 min. 1H NMR (400 MHz, DMSO-d6) d 8.33 (s, 1H), 8.15 (d,
J ¼ 7.8 Hz, 1H), 7.88 ꢁ 7.81 (m, 2H), 7.75 ꢁ 7.69 (m, 2H), 7.54 (t,
J ¼ 7.9 Hz, 1H), 7.36 (dd, J ¼ 8.0, 0.9 Hz, 1H), 7.00 (d, J ¼ 8.0 Hz, 1H),
6.13 (s, 2H).13C NMR (101 MHz, DMSO-d6) d 188.01, 150.26, 148.60,
145.37, 140.25, 136.05, 131.42, 131.39, 129.57, 127.87, 126.85,
122.77, 119.93, 108.98, 107.54, 102.18. HRMS: m/z [M þ H]þ calcd
for C16H13BrO3: 330.9968, found: 330.9968.
(E)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-(4-(trifluoromethyl)-
phenyl)prop-2-en-1-one (21) Yield 41.9%; m.p.: 139.0–140.2 ꢂC;
1
UPLC purity 96.39%, tR ¼8.629 min. H NMR (400 MHz, DMSO-d6) d
8.32 (d, J ¼ 8.1 Hz, 2H), 7.92 (d, J ¼ 8.2 Hz, 2H), 7.82 (d, J ¼ 15.6 Hz,
1H), 7.71 (d, J ¼ 15.5 Hz, 1H), 7.53 (d, J ¼ 2.0 Hz, 1H), 7.40 (dd,
J ¼ 8.4, 2.1 Hz, 1H), 6.95 (d, J ¼ 8.4 Hz, 1H), 4.31 (q, J ¼ 4.9 Hz, 4H).
13C NMR (101 MHz, DMSO-d6) d 188.85, 146.64, 145.57, 144.11,
141.50, 132.88, 132.56, 129.67, 128.44, 126.13, 126.09, 125.67,
123.81, 122.97, 120.34, 117.94, 117.86, 64.92, 64.42. HRMS: m/z
[M þ H]þ calcd for C18H13F3O3: 335.0890, found: 335.0912.
(E)-3-(benzo[d][1,3]dioxol-5-yl)-1-(4-chlorophenyl)prop-2-en-1-one
(28) Yield 16.27%; m.p.: 125.5–127.1 ꢂC; UPLC purity 99.03%, tR
1
¼2.311 min. H NMR (400 MHz, DMSO-d6) d 8.18 (d, J ¼ 8.2 Hz, 2H),
7.82 (d, J ¼ 15.5 Hz, 1H), 7.74 ꢁ 7.66 (m, 2H), 7.63 (d, J ¼ 8.3 Hz, 2H),
7.34 (dd, J ¼ 8.0, 1.7 Hz, 1H), 7.00 (d, J ¼ 8.0 Hz, 1H), 6.12 (s, 2H).
13C NMR (101 MHz, DMSO-d6) d 188.26, 150.18, 148.58, 145.00,
138.40, 136.87, 130.81, 129.58, 129.27, 126.64, 120.08, 108.99,
107.46, 102.16. HRMS: m/z [M þ H]þ calcd for C16H11ClO3:
287.0469, found: 287.0488.
(E)-1–(3-bromo-4-fluorophenyl)-3–(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)prop-2-en-1-one (22) Yield 41.2%; m.p.: 160.1–161.6 ꢂC; UPLC
purity 97.93%, tR ¼8.688 min. 1H NMR (400 MHz, DMSO-d6) d 8.51
(dd, J ¼ 6.9, 2.1 Hz, 1H), 8.24 ꢁ 8.18 (m, 1H), 7.83 (d, J ¼ 15.4 Hz,
1H), 7.67 (d, J ¼ 15.4 Hz, 1H), 7.54 (d, J ¼ 2.4 Hz, 2H), 7.38 (dd,
J ¼ 8.5, 2.0 Hz, 1H), 6.92 (d, J ¼ 8.4 Hz, 1H), 4.30 (q, J ¼ 4.8 Hz, 4H).
13C NMR (101 MHz, DMSO-d6) d 186.79, 162.72, 160.21, 146.55,
145.28, 144.08, 136.18, 136.15, 134.39, 130.91, 130.83, 128.51,
123.94, 119.83, 117.85, 117.82, 117.67, 117.45, 109.36, 109.15,
64.91, 64.42. HRMS: m/z [M þ H]þ calcd for C17H12BrFO3: 363.0027,
found: 363.0040.
hMAO inhibition assay
The hMAO inhibition assay was performed as previously
described18. Adequate amounts of recombinant hMAO-A/B were
acquired and adjusted to 12.5 mg/mL or 75 mg/mL for hMAO-A or
hMAO-B, respectively, with sodium phosphate buffer (50 mM, pH
7.4). The test compounds were dissolved in DMSO as stock solu-
tions (10 mM) and diluted with sodium phosphate buffer (50 mM,
pH 7.4) to the corresponding concentrations. Then the test com-
pounds (20 mL) and hMAO (80 mL) were incubated for 15 min at
37 ꢂC in a flat, black-bottomed 96-well microtest plate in the dark.
The reaction was initiated by adding Amplex Red reagent
(200 mM, final concentration), horseradish peroxidase (2 U/mL, final
concentration), and 2 mM p-tyramine for hMAO-A or 2 mM benzyl-
amine (final concetration) for hMAO-B and incubated at 37 ꢂC for
20 min. Activity was quantified in a multi detection microplate
fluorescence reader (SpectraMax M5, Molecular Devices, USA)
based on the fluorescence generated (excitation, 545 nm; emis-
(E)-1-(3,4-dichlorophenyl)-3–(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)
prop-2-en-1-one (23) Yield 62.7%; m.p.: 156.6–157.9 ꢂC; UPLC pur-
ity 93.35%, tR ¼9.358 min. 1H NMR (400 MHz, DMSO-d6) d 8.40 (s,
1H), 8.11 (d, J ¼ 8.4 Hz, 1H), 7.85 ꢁ 7.81 (m, 2H), 7.69 (d, J ¼ 15.4 Hz,
1H), 7.55 (s, 1H), 7.40 (d, J ¼ 8.4 Hz, 1H), 6.93 (d, J ¼ 8.3 Hz, 1H),
4.31 (q, J ¼ 5.2 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) d 187.18,
146.64, 145.57, 144.09, 138.35, 136.26, 132.36, 131.50, 130.79,
128.90, 128.48, 124.00, 119.80, 117.87, 64.92, 64.42. HRMS: m/z
[M þ H]þ calcd for C17H12Cl2O3: 335.0236, found: 335.0252.
(E)-1-(5-bromo-2-hydroxyphenyl)-3-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)prop-2-en-1-one (24) Yield 58.33%; m.p.: 141.8–143.7 ꢂC; UPLC
1
purity 98.33%, tR ¼9.201 min. H NMR (400 MHz, DMSO-d6) d 12.59
(s, 1H), 8.41 (s, 1H), 7.87 (d, J ¼ 15.3 Hz, 1H), 7.75 (d, J ¼ 15.3 Hz, sion, 590 nm). The specific fluorescence emission was calculated
1H), 7.67 (d, J ¼ 8.4 Hz, 1H), 7.57 (s, 1H), 7.41 (d, J ¼ 8.4 Hz, 1H), after subtraction of the background activity. The background
6.95 (t, J ¼ 9.7 Hz, 2H), 4.30 (q, J ¼ 4.8 Hz, 4H). 13C NMR (101 MHz, activity was determined from wells containing all components
DMSO-d6) d 192.88, 161.14, 146.88, 146.15, 144.13, 138.72, 132.94, except the hMAO isoforms, which were replaced by a sodium

1518
Z. KONG ET AL.
phosphate buffer solution. The percentage inhibition was calcu- MTT assay
lated by the following expression: (1 – IFi/IFc) ꢃ 100, in which IFi
The 3–(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT)
and IFc are the fluorescence intensities obtained for hMAO in the
presence and absence of inhibitors after subtracting the respective
background. The synthetic compounds were first screened for the
inhibition of hMAO-B at 1 mM, which showed more than 70%
enzyme inhibition were then subjected to IC50 determination
assay was performed as previously described22. BV2 Cells were
plated at 5000 cells per well and allowed to grow at 37 ꢂC over-
night. Then the cells were treated with various concentrations of
the tested compounds for 24 h at 37 ꢂC in a humidified atmos-
phere with 5% CO2, 0.5% DMSO acted as the vehicle control. The
cell culture medium was then removed, followed by the addition
of fresh cell culture medium containing 5.0 g/L MTT (20 mL/per
well) and incubation at 37 ꢂC in a humidified, 5% CO2 atmosphere
for 4 h. After this incubation period, the cell culture medium was
removed and the formed formazan crystals dissolved in 100%
DMSO. The absorbance was measured at 570 nm in a multi-well
plate reader. The results are expressed as % relative cell viability
compared to DMSO vehicle control.
Enzyme kinetic study
To evaluate the mode of MAO-B inhibition, the representative
compound was evaluated in substrate-dependent kinetic experi-
ments performed as previously described. Sets of Lineweaver–Burk
plots were generated. The reciprocal MAO-B activity was plotted
against the reciprocal substrate concentration. The initial catalytic
rates of human MAO-B were measured at four different concentra-
tions of the substrate benzylamine (1, 2, 4 and 8 mM) in the
absence (basal sample) and in the presence of four different con-
centrations (IC50, 3/2 IC50, 2 IC50 and 5/2 IC50) of the inhibitors.
The enzymatic reactions and measurements were performed using
human MAO-B assay conditions as described above for the deter-
mination of IC50 values.
Results
Inhibitory activity of monoamine oxidase
The inhibitory effects of 28 chalcone derivatives on human MAO-B
and MAO-A enzymes were screened using a fluorescence-based
assay. The tested concentration was 1 mM unless otherwise stated.
Irreversible inhibitors R-(–)-deprenyl and rasagiline were used as
positive controls for MAO-B inhibition at 0.1 mM. Clorgyline was
used as a positive control for MAO-A inhibition. The results are
summarised in Table 1.
Reversibility study of MAO-B inhibition
To investigate the reversibility of the MAO-B inhibition by the
active compounds, we performed time-dependent inhibition
experiments using human MAO-B as previously described11.
R-(–)-deprenyl, rasagiline and safinamide were used as the refer-
ence compounds. The hMAO-B enzyme was pre-incubated with
the representative compounds at the concentrations of their cor-
responding IC80 for 15 min and the reaction was initiated by add-
ing work solutions as described above. The activity of the enzyme
was monitored by fluorescence measurements over a period of
120 min. Control experiments without inhibitors were run simul-
taneously. The enzymatic activity was determined as described
above. The percentage of enzyme activity was plotted against the
incubation time to determine time-dependent enzyme inhibition.
Parent chalcone compound 1 exhibited a moderate inhibition
effect against hMAO-B (41.6% inhibition at 10 mM), with an IC50 of
Table 1. MAO inhibitory activities of 1, 4-benzodioxan-substituted chalcone
derivatives
hMAO-B
hMAO-A
IC50 (lM)
Selectivity
Index (SI)a
Compound
Inhibition (%)
IC50 (lM)
1
2
3
4
5
6
7
8
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
N.D.
41.6% 0.003b
18.1% 3.38
13.04 0.06
N.D.
>3
N.D.
50.8%
9
N.D.
N.D.
84.9% 2.5
75.0% 1.7
78.0% 0.27
26.0% 2.3
51.7% 6.9
86.3% 0.9
69.5% 0.4
22.4% 0.9
89.2% 0.46
92.4% 0.59
92.6% 0.38
0.22 0.005
0.3 0.02
0.34 0.01
N.D.
>186
>133
>118
N.D.
N.D.
0.15 0.007
N.D.
N.D.
>267
N.D.
9
10
11
12
13
14
15
16
17
18
19
20
Molecular docking study
N.D.
N.D.
0.12 0.01
0.054 0.006
0.081 0.004
0.13 0.007
0.055 0.002
0.057 0.004
N.D.
>333
>741
>494
>308
>727
>702
N.D.
A molecular docking study was performed to investigate the pos-
sible interaction between the active compounds and hMAOs. The
crystal structures of hMAO-B (PDB code 2V61)19 and hMAO-A
(2Z5X)20 were adopted and the compounds were docked into the
active sites following the standard dock protocol in the software
Molecular Operating Environment (MOE) 2019. For the docking
into hMAO-B, nine conserved water molecules were included in
89.2%
1
93.3% 0.68
93.4% 0.57
69.9% 7.2
68.1% 2.0
87.5% 0.29
76.8% 1.1
95.6% 0.21
88.8% 0.75
77.1% 3.65
78.4% 1.4
62.3% 3.2
80.4% 0.44
57.5% 3.2
92% 0.738
75% 0.012
N.D.
N.D.
N.D.
0.068 0.007
0.23 0.0005
0.026 0.001
0.17 0.02
0.25 0.03
0.32 0.05
N.D.
>588
>174
>1538
>235
>160
>125
N.D.
the docking process according to previous reports21. Default set- 21
tings were used, unless stated otherwise. The top-ranked poses of
22
23
24
25
26
27
28
the docked compounds were selected and analysed.
0.28 0.002
N.D.
>143
N.D.
N.D.
Theoretical calculation of physicochemical parameters
R-(-)-deprenyl
rasagiline
clorgyline
0.0196b
The relevant physicochemical parameters, including molecular
weight (M.W), hydrogen bond acceptor and donor (HBA, HBD),
number of rotatable bonds (NRB), topological surface area (tPSA)
and the octanol/water partition coefficient (LogP), were calculated
using the descriptors module in MOE 2019.
N.D.
0.069c
N.D.
86.6% 0.4d
N.D.
N.D.
Data are represented as means SEM from at least three independent experi-
ments. aSelectivity index (SI) ¼ IC50 (hMAO-A)/IC50 (hMAO-B); bsee Ref. 6; csee
Ref. 7; d% inhibition at 0.1 lM; N.D., no determined.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1519
Figure 2. The SAR of 1, 4-benzodioxan-substituted chalcones towards MAO-B inhibition.
13.04 mM. The replacement of the B ring of 1 with naphthyl (2) or 3-dioxaindane moiety, the corresponding compounds 27 and 28
the 1, 3-benzodioxole (3) moiety resulted in increased inhibitory exhibited lower inhibitory activities (27, IC50 ¼ 0.28 mM; 28, 57.5%
activities (18.1% and 50.8% inhibition, respectively), whereas the
replacement of the B ring with the 1, 4-benzodioxan moiety, hit
potent lead compound 4, which exhibited a 55-fold higher hMAO-
B inhibition (IC50 ¼ 0.22 mM) than 1. These results suggested that
the replacement of the B ring of the parent chalcone with fused-
ring moieties increases the inhibitory activity and that the 1,
4-benzodioxan substituent is preferable. Thus, we considered
inhibition at 1 mM) than their parent compounds, which further
suggested that the 1, 4-benzodioxan moiety is a crucial pharmaco-
phore for this class of inhibitors.
An overview of the SAR of 1, 4-benzodioxan-substituted chal-
cones with respect to MAO-B inhibition is provided in Figure 2.
To hMAO-A, the 1, 4-benzodioxan-substituted chalcone com-
pounds exhibited weak inhibitory activities, with less than 50%
inhibition even at 40 mM. A high selectivity index (SI) value was
observed regarding the active compounds. The most potent
hMAO-B inhibitor (22) exhibited an SI value of greater than 1538.
Thus, we concluded that the active compounds in the 1, 4-benzo-
dioxan-substituted chalcones are highly selective hMAO-
B inhibitors.
compound
4 as a new lead scaffold for further structural
modifications.
As for the compounds that possess electron-donating groups
-OH and -OCH3 at different positions on the A ring, only 9, which
bears a 30-OH substituent, exhibited higher MAO-B inhibitory
activity (IC50 ¼ 0.15 mM) than 4. The introduction of 20-OH, 40-OH,
30-OCH3 and 40-OCH3 at the A ring resulted in lower inhibitory
activity (5, IC50 ¼ 0.3 mM; 6, IC50 ¼ 0.34 mM; 7, 26.0%; 8, 51.7%
inhibition) than 4. The 20, 40-dihydroxy and 30, 40-dimethoxy substi-
tuted compounds 10 and 11 also showed lower inhibitory activity
than 4 (69.5% and 22.4% inhibition, respectively). These results
suggested that substitution with the hydroxyl group at the meta-
position of the A ring is favourable to increase the inhibitory activ-
ity of 1, 4-benzodioxan chalcones.
The 1, 4-benzodioxan chalcones that bear electron-withdrawing
groups at different positions of the A ring mostly exhibited inhibi-
tory activities against hMAO-B. Introduction of halogen atoms at
the ortho- and meta-position of the A ring resulted in a signifi-
cantly higher inhibitory potency than 4. The 20-F, 20-Cl, and 20-Br
substituted compounds 12, 13 and 14 showed a 1.8–4.1-fold
Kinetics study
Kinetic analyses were carried out with representative compounds
16, 17 and 22. The purpose of this experiment was to determine
the binding mode of these potent compounds with MAO-B.
Lineweaver–Burk graphs were constructed in the absence or pres-
ence of inhibitors at various concentrations. The lines are linear
and intersect at the y-axis (Figure 3), which suggests that com-
pounds 16, 17, and 22 are competitive inhibitors of hMAO-B.
Reversibility study
To investigate the reversibility of the enzymatic inhibition, we per-
formed time-dependent inhibition studies using representative
inhibitors 16, 17, and 22 at the concentration of their IC80. R-
(-)-Deprenyl, rasagiline and safinamide were used as reference
inhibitors and evaluated under the same experimental conditions.
The hMAO-B activity (% of control) was measured for 120 min in
the presence of the tested inhibitors.
When treated with irreversible inhibitors, the residual activity
of hMAO-B decayed continuously throughout the incubation
period (Figure 4(A)), which indicates that inhibiting hMAO-B by
these compounds is irreversible. In the case of the reversible
inhibitor safinamide, the residual activity of hMAO-B began to
increase after 15 min, indicating that the inhibitor can be replaced
by the competing substrate and the enzymatic activity is recov-
ered11. Similarly, after incubation with compounds 16, 17, and 22
for 20 min, the residual activities of hMAO-B began to increase
gradually (Figure 4(B)). These results clearly showed that these
active compounds 16, 17, and 22 act as reversible hMAO-
B inhibitors.
higher in inhibitory potency (12, IC50 ¼ 0.12 mM; 13, IC50
¼
0.054 mM; 14, IC50 ¼ 0.081 mM) and the 30-F, 30-Br, and 30-Cl substi-
tuted compounds 15, 16, and 17 showed 1.7–4-fold higher in
inhibitory potency (IC50 ¼ 0.13, 0.055, and 0.057 mM, respectively)
than 4. However, the introduction of 40–Cl and 40–Br (18, and 19)
at the A ring reduced the inhibitory activity compared to 16 and
17 and only 69.9% and 68.1% inhibition were observed at 1 mM,
respectably, suggesting that 40-Cl and 40-Br were not well toler-
ated. The introduction of 40-F maintained potent inhibitory activity
(20, IC50 ¼ 0.068 mM). As the 40-CF3 substituted compound (21)
only exhibited inhibition potency comparable to that of 4, we
concluded that substitution with halogen atoms at the ortho- and
meta-position of the A ring is favourable to increase the inhibitory
activity of 1, 4-benzodioxan chalcones. The introduction of both
40-F and 30-Br at the A ring led to the most potent compound
(22) with an IC50 value as low as 0.026 mM, which was comparable
to that of safinamide.
The introduction of 30, 40-dichloro substituents resulted in less
potent inhibitory activity (23, IC50 ¼ 0.17 mM) than 17, which
might be influenced by the 40–Cl substitution. The introduction of
20-OH to compounds 16–18 also resulted in lower inhibitory activ-
ities (24, IC50 ¼ 0.25 mM; 25, IC50 ¼ 0.32 mM; 26, 62.3% inhibition
Molecular docking studies
at 1 mM) compared to the corresponding compounds, which indi- To investigate the possible interaction mechanism between
cated that the 20-OH was not tolerated either. After the replace- the active compounds and hMAO-B, we performed a molecular
ment of the 1, 4-benzodioxan moiety of 16 and 18 with a 1, docking study using the docking module in the software MOE.

1520
Z. KONG ET AL.
(A)
(B)
(C)
2.5
4
3
2
1
4
3
2
1
control
control
control
0.054
0.081
0.108
0.135
µM
0.039
0.059
0.079
0.099
µ
M
M
M
0.057
0.086
0.114
0.143
µM
µ
M
µ
µ
2.0
1.5
1.0
0.5
µ
M
M
M
µ
M
µ
µ
µ
M
µ
M
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1/[S](mM-1) (compound 16)
1/[S](mM-1) (compound 17)
1/[S](mM-1) (compound 22)
Figure 3. Lineweaver–Burk plots for hMAO-B inhibition by compound 16, 17, and 22 (A–C).
90
80
70
60
50
(A)
(B)
Safinamide
Selegilin
Rasagiline
16
17
22
60
40
20
0
40
80
120
0
40
80
120
Time / min
Time / min
Figure 4. Time-dependent inhibition of hMAO-B by reference compounds R-(–)-deprenyl, rasagiline and safinamide (0.05 lM, 0. 20 lM and 0.06 lM, respectively, A)
and test compounds 16, 17, and 22 (0.28 lM, 0.28 lM and 0.19 lM, respectively, B). The remaining activity was expressed as % of activity.
The co-crystal structure of hMAO-B (PDB code 2V61) was single substrate cavity of approximately 550 Å3, which is shorter
employed and the compounds were docked into the active site.
in length than that of hMAO-B, which is approximately 700 Å3 9
.
The active site of MAO-B consists of two cavities, the “entrance Compound 22 adopted an extended molecular conformation
cavity” and the “substrate cavity”. The entrance cavity is separated located in the active site of hMAO-A (Figure 5(D)). In contrast to
the binding mode with hMAO-B, the 1, 4-benzodioxan ring of 22
located apart from FAD while the A ring and substituents 30-Br
and 40-F were pressed close to FAD, which indicated a strong
steric hindrance effect between them. Therefore, we hypothesise
that steric hindrance might be the key factor that prevents the
compounds from binding to hMAO-A.
from the outer surface by the cavity gating loop (residues 99–110)
and the substrate cavity extends from flavin adenine dinucleotide
(FAD) to the entrance cavity23. Parent chalcone compound 1
adopted an extended pose across both cavities of MAO-B (Figure
5(A)). The A ring occupied the hydrophobic pocket in the entrance
cavity and the B ring was directed towards the hydrophobic cavity
in front of FAD. The main interaction between them was thought
to be hydrophobic. As for compound 4, the A ring occupied a
similar position at the entrance cavity whereas the 1, 4-benzo-
dioxan moiety pointed towards the substrate cavity, the oxygen
atom of which established a hydrogen bond with Cys172 (3.3 Å,
Figure 5(A)). This may explain the higher inhibitory activity of
compound 4 than of compound 1. Compounds 16 and 22
adopted similar binding poses with hMAO-B, but their oxygen
atom at the 1, 4-benzodioxan ring could establish hydrogen
bonds with Tyr435 (3.4 Å, Figure 5(B)), instead of Cys172 (> 5.0 Å),
Evaluation of drug-likeness
As there are specific physicochemical requirements for drugs that
act on the central nervous system (CNS), we estimated the drug-
likeness of the active compounds using theoretical calculations
inMOE. Relevant physicochemical parameters were calculated,
including MW, HBA, HBD, NRB, tPSA, Log P and Log BB. The
results are summarised in Table 2. According to these data, all
selected active compounds were in agreement with Lipinski’s rule
of five for drug-likeness without any violation. The calculated val-
ues of these parameters were all within the suggested limits set
for the development of CNS drugs (MW < 400, HBA ꢄ 7, HBD ꢄ3,
NRB < 8, cLogP ¼ 2–5, and tPSA < 70 Å2)26. All these compounds
exhibited a log BB value greater than ꢁ1, which indicated that
these active compounds might possess good blood-brain barrier
(BBB) permeability27. We further employed the Cbligand-BBB pre-
dictor program28 to predict the ability of these compounds to
cross the BBB. All the active compounds were predicted to be BBB
(þ) (Table 2). Similar results were observed for the clinical drugs
R-(–)-deprenyl, rasagiline, safinamide and clorgyline.
which is in line with the binding mode of chromone inhibitors24
.
Additionally, the 30-Br of compounds 16 and 22 form halogen
bonds with the carbonyl oxygen of Leu164 with a bond distance
of 3.3 Å, C ꢁ Br ꢁ O angle of 163ꢂ, and Br ꢁ OꢁC angle of 137ꢂ
with Leu164, which might play a key role in increasing the inhibi-
tory activity of both compounds25. Compound 22 exhibited an
approximately 2.6-fold greater inhibition activity than 16, which
might be attributed to the additional substitution with 40-F at the
A ring. The 40-F of 22 was embedded in the local cavity formed
by residues Phe103, Phe104, and Trp119 (Figure 5(B–C)), which
might strengthen the binding contact with the enzyme and con-
sequently lead to increased inhibitory activity.
We also attempted to explain why the active compounds
exhibited weak inhibitory activity against hMAO-A using a docking
Cell cytotoxicity assay
study. The most active compound (22), which showed the highest The cytotoxicity of the potent compounds 16, 17 and 22 was
SI, was docked into the active site of hMAO-A. hMAO-A has a evaluated in BV2 microglia cells using the MTT assay. For all the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1521
Figure 5. Docking poses of chalcone compounds 1, 4, 16, and 22 in the hMAO-B (PDB code: 2V61) (A–C) or hMAO-A (2Z5X) (D) active site. (A) docking poses of com-
pounds 1 (gray) and 4 (yellow); (B) docking poses of compounds 4 (yellow), 16 (cyan), and 22 (brown); (C) the docking pose of 22 in the active site of hMAO-B; (D)
the docking pose of 22 in the active site of hMAO-A. Hydrogen bonds are shown as yellow dotted lines, halogen bonds are shown as blue dotted lines. For clarity,
only the relevant residue side chains are shown. FAD is rendered as white sticks. Red bows indicate steric hindrance.
Table 2. Calculation of the drug-like properties of selected active compounds and MAO-B standard inhibitors
entries
M.W
HBA
HBD
NRB
clogP (o/w)
tPSA (Å2)
Log BBa
lip_violation
Predicted BBB (þ/–)b
13
16
17
20
300.7410
345.1920
300.7410
284.2860
363.1820
187.2860
171.2340
320.3490
272.1750
<500
3
3
3
3
3
1
1
3
2
0
0
0
0
0
0
1
2
0
3
3
3
3
3
5
3
7
7
4.2500
4.4950
4.2890
3.8130
4.6460
2.8110
2.4620
2.8540
3.7060
2–5
35.5300
35.5300
35.5300
35.5300
35.5300
3.2400
12.0300
64.3500
12.4700
<90
0.26
0.30
0.27
0.19
0.32
0.52
0.34
0.38
0.52
ꢅ ꢁ1
0
0
0
0
0
0
0
0
0
þ
þ
þ
þ
þ
þ
þ
þ
þ
22
R-(-)-deprenyl
rasagiline
safinamide
clorgyline
Suggested limits
a
<7
<3
<8
ꢀ
Log BB ¼ –0.0148 ꢃ tPSA þ 0.152 clogP þ 0.139, see Ref. 25; BBB( ): blood-brain barrier permeability.
16
17
22
100
50
0
M
M
m
m
SO
DM
Figure 6. Cell viability of BV2 cells after treatment with compounds 16, 17, and 22 at the concentrations of 5 and 25mM for 24 h.
5
25

1522
Z. KONG ET AL.
tested compounds, no considerable decrease in cellular viability
was observed, even at the highest tested concentration (25 mM)
(Figure 6). The results suggested that compounds 16, 17, and 22
exhibit low cytotoxicity and might have a wide safety window.
8. Matos MJ, Vilar S, Gonzalez-Franco RM, et al. Novel (couma-
rin-3-yl)carbamates as selective mao-b inhibitors: synthesis,
in vitro and in vivo assays, theoretical evaluation of adme
properties and docking study. Eur J Med Chem 2013;63:
151–61.
9. De Colibus L, Li M, Binda C, et al. Three-dimensional struc-
ture of human monoamine oxidase a (mao a): relation to
the structures of rat mao a and human mao b. Proc. Natl.
Acad. Sci. USA 2005;102:12684–9.
10. Carradori S, Silvestri R. New frontiers in selective human
mao-b inhibitors. J Med Chem 2015;58:6717–32.
Conclusion
In the current study, a series of 1, 4-benzodioxan-substituted chal-
cone compounds were designed, synthesised, and evaluated for
the inhibition of hMAO-B. Mostshowed potent inhibitory activity
and high selectivity. SAR analysis suggested that the 1, 4-benzo-
dioxan moiety is a key pharmacophore and substitution with halo- 11. Reis J, Cagide F, Chavarria D, et al. Discovery of new chem-
gen atoms at the ortho- or meta-position of the A ring would
significantly increase the inhibitory potency of this class of inhibi-
tor. The introduction of 40-F at the A ring would be favourable.
The most potent compound (22), bearing both 30-Br and 40-F at
the A ring, exhibited an IC50 value of 0.026 mM with an SI value
greater than 1538, which was comparable to that of safinamide.
Kinetics and reversibility studies confirmed that the representative
compounds 16, 17, and 22 acted as competitive and reversible
inhibitors of hMAO-B. Owing to their promising drug-like proper-
ties and low cytotoxicity, we believe that the active 1, 4-benzo-
dioxan-substituted chalcone compounds could serve as valuable
drug candidates for further development.
ical entities for old targets: insights on the lead optimization
of chromone-based monoamine oxidase b (mao-b) inhibi-
tors. J Med Chem 2016;59:5879–93.
12. Chimenti F, Fioravanti R, Bolasco A, et al. Chalcones: a valid
scaffold for monoamine oxidases inhibitors. J Med Chem
2009;52:2818–24.
13. Minders C, Petzer JP, Petzer A, Lourens A. Monoamine oxi-
dase inhibitory activities of heterocyclic chalcones. Bioorg
Med Chem Lett 2015;25:5270–6.
14. Jo G, Ahn S, Kim B-G, et al. Chromenylchalcones with inhibi-
tory effects on monoamine oxidase b. Bioorg Med Chem
2013;21:7890–7.
15. Zaib S, Umar Farooq Rizvi S, Aslam S, et al. Quinolinyl-
thienyl chalcones as monoamine oxidase inhibitors and their
in silico modeling studies. Med Chem 2015;11:580–9.
16. Sasidharan R, Manju SL, Uc¸ar G, et al. Identification of
indole-based chalcones: discovery of a potent, selective, and
reversible class of mao-b inhibitors. Arch. Pharm. 2016;349:
627–37.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
17. Engelbrecht I, Petzer JP, Petzer A. The synthesis and evalu-
ation of sesamol and benzodioxane derivatives as inhibitors
of monoamine oxidase. Bioorg Med Chem Lett 2015;25:
1896–900.
This work was supported by the National Natural Science
Foundation of China under Grant [No. 81560562 and 21762055];
the Science and Technology Cooperation Plan of Guizhou
Province under Grant [Qian Ke He LH Zi (2015) 7559].
18. Lu C, Guo Y, Yan J, et al. Design, synthesis, and evaluation
of multitarget-directed resveratrol derivatives for the treat-
ment of Alzheimer’s disease. J Med Chem 2013;56:5843–59.
19. Binda C, Wang J, Pisani L, et al. Structures of human mono-
amine oxidase b complexes with selective noncovalent
inhibitors: safinamide and coumarin analogs. J Med Chem
2007;50:5848–52.
20. Son S-Y, Ma J, Kondou Y, et al. Structure of human mono-
amine oxidase a at 2.2- resolution: the control of opening
the entry for substrates/inhibitors. Proc Natl Acad Sci 2008;
105:5739–44.
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