Morpholine-based chalcones as dual-acting monoamine oxidase-B and acetylcholinesterase inhibitors: synthesis and biochemical investigations

Article abstract of DOI:10.1080/14756366.2020.1842390

Nine compounds (MO1–MO9) containing the morpholine moiety were assessed for their inhibitory activities against monoamine oxidases (MAOs) and acetylcholinesterase (AChE). Most of the compounds potently inhibited MAO-B; MO1 most potently inhibited with an IC₅₀ value of 0.030 μM, followed by MO7 (0.25 μM). MO5 most potently inhibited AChE (IC₅₀ = 6.1 μM), followed by MO9 (IC₅₀ = 12.01 μM) and MO7 most potently inhibited MAO-A (IC₅₀ = 7.1 μM). MO1 was a reversible mixed-type inhibitor of MAO-B (K_i = 0.018 μM); MO5 reversibly competitively inhibited AChE (K_i = 2.52 μM); and MO9 reversibly noncompetitively inhibited AChE (K_i = 7.04 μM). MO1, MO5 and MO9 crossed the blood–brain barrier, and were non-toxic to normal VERO cells. These results show that MO1 is a selective inhibitor of MAO-B and that MO5 is a dual-acting inhibitor of AChE and MAO-B, and that both should be considered candidates for the treatment of Alzheimer’s disease.

Full text of DOI:10.1080/14756366.2020.1842390

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Morpholine-based chalcones as dual-acting
monoamine oxidase-B and acetylcholinesterase
inhibitors: synthesis and biochemical
investigations
Rani Sasidharan, Bo Hyun Eom, Jeong Hyun Heo, Jong Eun Park, Mohamed
A. Abdelgawad, Arafa Musa, Nicola Gambacorta, Orazio Nicolotti,
Sreedharannair Leelabaiamma Manju, Bijo Mathew & Hoon Kim
To cite this article: Rani Sasidharan, Bo Hyun Eom, Jeong Hyun Heo, Jong Eun Park, Mohamed
A. Abdelgawad, Arafa Musa, Nicola Gambacorta, Orazio Nicolotti, Sreedharannair Leelabaiamma
Manju, Bijo Mathew & Hoon Kim (2021) Morpholine-based chalcones as dual-acting monoamine
oxidase-B and acetylcholinesterase inhibitors: synthesis and biochemical investigations, Journal of
Enzyme Inhibition and Medicinal Chemistry, 36:1, 188-197, DOI: 10.1080/14756366.2020.1842390
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 188–197
https://doi.org/10.1080/14756366.2020.1842390
SHORT COMMUNICATION
Morpholine-based chalcones as dual-acting monoamine oxidase-B and
acetylcholinesterase inhibitors: synthesis and biochemical investigations
Rani Sasidharana,b#, Bo Hyun Eomc#, Jeong Hyun Heoc#, Jong Eun Parkc, Mohamed A. Abdelgawadd,e, Arafa
f,g
h
h
b
i
ꢀ
Musa , Nicola Gambacorta , Orazio Nicolotti , Sreedharannair Leelabaiamma Manju , Bijo Mathew and
Hoon Kimc
b
aCollege of Pharmaceutical Science, Government T.D. Medical College, Alappuzha, India; Organic Chemistry Division, SAS, VIT University,
c
Vellore, India; Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University, Suncheon,
d
e
Republic of Korea; Pharmaceutical Chemistry Department, College of Pharmacy, Jouf University, Sakaka, Saudi Arabia; Pharmaceutical Organic
f
Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Beni Suef, Egypt; Department of Pharmacogonosy, College of Pharmacy, Jouf
g
h
University, Sakaka, Saudi Arabia; Department of Pharmacogonosy, Al-Azhar University, Cairo, Egypt; Dipartimento di Farmacia—Scienze del
i
ꢀ
Farmaco, Universita degli Studi di Bari “Aldo Moro”, Bari, Italy; Division of Drug Design and Medicinal Chemistry Research Lab, Department of
Pharmaceutical Chemistry, Ahalia School of Pharmacy, Palakkad, India
ABSTRACT
ARTICLE HISTORY
Received 16 September 2020
Revised 21 October 2020
Accepted 22 October 2020
Nine compounds (MO1–MO9) containing the morpholine moiety were assessed for their inhibitory activ-
ities against monoamine oxidases (MAOs) and acetylcholinesterase (AChE). Most of the compounds
potently inhibited MAO-B; MO1 most potently inhibited with an IC50 value of 0.030mM, followed by MO7
(0.25 mM). MO5 most potently inhibited AChE (IC50 ¼ 6.1 mM), followed by MO9 (IC50 ¼ 12.01 mM) and
MO7 most potently inhibited MAO-A (IC50 ¼ 7.1 mM). MO1 was a reversible mixed-type inhibitor of MAO-
B (Ki ¼ 0.018 mM); MO5 reversibly competitively inhibited AChE (Ki ¼ 2.52 mM); and MO9 reversibly non-
competitively inhibited AChE (Ki ¼ 7.04 mM). MO1, MO5 and MO9 crossed the blood–brain barrier, and
were non-toxic to normal VERO cells. These results show that MO1 is a selective inhibitor of MAO-B and
that MO5 is a dual-acting inhibitor of AChE and MAO-B, and that both should be considered candidates
for the treatment of Alzheimer’s disease.
KEYWORDS
Morpholine-containing chal-
cone; monoamine oxidase;
acetylcholinesterase; dual-
acting inhibitor;
Docking analysis
1. Introduction
therapeutic targets in various neuropsychiatric illnesses and neuro-
degenerative disorders like Alzheimer’s disease (AD) and
Parkinson’s disease (PD)5. During the degradations of various bio-
genic neurotransmitters catalysed by MAO-A and MAO-B, hydro-
gen peroxide and reactive oxygen species (ROS) are produced as
major by-products, which might cause oxidative damage in brain
tissues. MAO inhibitors are considered potential neuroprotective
agents and up-regulating agents for neurotransmitter amines6.
Numerous studies have documented that selective and reversible/
irreversible MAO-B inhibitors are likely to play pivotal roles in AD-
related therapeutic strategies. Acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) inhibitors also play significant roles in
the maintenance of cholinergic functions and are used to provide
symptomatic relief in AD. Over the past two decades, many
researchers have tried to develop multi-acting MAO-B and AChE/
Due to the extreme complexities of brain systems and their
diverse dysfunctions, research focus is being directed towards the
design of multi-target directed ligands (MTDLs)1. In fact, it has
been suggested that functional equilibrium of brain after a neuro-
logic disorder is unlikely to be achieved by focussing on a single
molecular target2. The major challenge presented by the develop-
ment of MTDLs is to preserve balance between the effects drugs
have by acting at their individual molecular targets3. On the other
hand, some ligands can target two or more specific entities within
numerous biological networks. The likelihood of successful MTDL
design can be enhanced by considering the design of molecular
scaffolds via suitable molecular hybridisation and by understand-
ing the pathophysiologies of multifaceted diseases. The design
process can be accelerated by selecting pharmacophores based
on pre-clinical studies, emphasising structure–activity relationships
(SARs) and by performing in silico-based virtual screening4.
BChE inhibitors7–10
.
Morpholine is a tetrahydro-1,4-oxazine with a saturated hetero-
cyclic ring and provides a promising developmental starting point
due to its biological profile with metabolic stability. The presence
of a heteroatom like oxygen or nitrogen facilitates hydrogen
Monoamine oxidases (MAOs) play prominent roles in the inacti-
vations of various biogenic amines in central and peripheral tis-
sues. The isoenzymes of MAO-A and MAO-B are considered major bonding, and thus, interactions with enzymes, and the presence
CONTACT Hoon Kim
Suncheon 57922, Republic of Korea; Bijo Mathew
hoon@sunchon.ac.kr
Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University,
bijomathew@aims.amrita.edu Department of Pharmaceutical Chemistry, Amrita School of Pharmacy, Amrita
Vishwa Vidyapeetham, AIMS Health Sciences Campus, Kochi-682 041, India; Sreedharannair Leelabaiamma Manju
slmanju@vit.ac.in
Organic Chemistry
Division, SAS, VIT University, Vellore, India
#These authors contributed equally to this work.
ꢀ
Department of Pharmaceutical Chemistry, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, AIMS Health Sciences Campus, Kochi-682 041, India.
Supplemental data for this article can be accessed here.
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
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.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
189
Figure 1. Morpholine-containing, CNS active, FDA approved drugs. 1, moclobemide; 2, reboxetine.
of electron-deficient atoms may also impart hydrophobic interac-
tions with morpholine. From the synthetic perspective, various
molecular scaffolds have been added to morpholine by replacing
its secondary nitrogen11. Moclobemide (1) and reboxetine (2)
(both antidepressants) provide examples of FDA-approved drugs
containing the morpholine moiety (Figure 1). These drugs revers-
ibly inhibit MAO-A and selectively inhibit norepinephrine reuptake
in the central nervous system (CNS), and thus, block the human
a4b2 nicotinic acetylcholine receptor. In addition, more than 20
drugs containing the morpholine moiety have been FDA
approved; they include mycophenolate mofetil (an immunosup-
pressant), linezolid and finafloxacin (antibiotics), geftinib (an anti-
neoplastic and epidermal growth factor inhibitor), rivaroxaban (an
anticoagulant and factor Xa inhibitor), and eteplirsen, which is
used to treat Duchenne muscular dystrophy12. Considering the
importance of morpholine nucleus, it is worthwhile to design mor-
pholine derived compounds of medicinal chemistry interest.
The chalcones framework is considered an excellent starting
point for the design for MAO-B and AChE enzyme inhibitors13–15
.
The presence of an a-b unsaturated ketone and three rotatable
bonds in chalcones can provide different bonding orientations16.
The literature amply demonstrates that most chalcones are potent,
reversible and selective MAO-B inhibitors17. In addition, the pres- Figure 2. MTDL design of MAO-B and AChE inhibitors.
ence and orientation of various electron-donating and withdraw-
ing groups on the phenyl/heteroaryl A and B rings of chalcones
can impart electrophilic character to the Michael acceptor present.
aldehydes and 4-morpholine acetophenone in the presence of an
alcoholic basic medium33. The H1 NMR showed that the ring pro-
tons N-CH2 and O-CH2 of morpholine ring resonated at 3.32–3.31
and 3.75–3.88 ppm as triplets, respectively. The large coupling
constant (J ¼ 15 Hz) value of Ha and Hb protons confirmed the
trans configuration of the morpholine chalcones. The deshielded
values between 186.88–186.07 in C13-NMR clearly evidenced the
formation of sp2 carbonyl functional group of a,b-unsaturated
The introductions of electron-donating/lipophilic halogens onto
the phenyl B ring of chalcones have resulted in the syntheses of
highly selective MAO-B inhibitors18–27. Many studies have reported
that the presence of various alkylamino groups on the A ring pro-
vides AChE inhibitory activity28–31. The FDA-approved drugs like
flurbiprofen and rivastigmine linked with chalcone moiety were
developed as selective ChE/MAO-B inhibitors for the prophylactic
agents for AD32. The design strategy of the current study is
depicted in Figure 2 and the synthetic route is shown in
Scheme 1. A recent study showed that the presence of a pendant
morpholine ring on the chalcone A ring favoured hMAO-B inhibi-
tory activity33,34. The difference in MAO-B inhibition profiles
became more obvious when chalcone B ring was substituted
using a fluoro or trifluoromethyl group33.
1
ketones. The identities of the compounds were determined by H-
NMR, 13 C-NMR and Mass spectrometry (Supplementary materials).
2.2. Analysis of enzyme inhibitory activities
2.2.1. Inhibitory activities
In this study, nine morpholine-based compounds (MO1–MO9)
were analysed with respect to their abilities to inhibit MAO-A,
MAO-B and AChE. Most of the compounds inhibited MAO-B by ꢁ
50% at 1.0 mM, except MO3 and MO5 (Table 1). Compound MO1
most potently inhibited MAO-B (IC50 ¼ 0.030 mM), followed by
2. Results and discussion
2.1. Chemistry
Morpholine-containing a,b-unsaturated ketones were prepared by MO7, MO8, MO4, MO9, MO6, MO2, MO3 and MO5 (IC50 ¼ 0.25,
condensation between various aromatic/para substituted 0.32, 0.33, 0.36, 0.64, 0.70, 1.01 and 1.31 mM, respectively). In

190
R. SASIDHARAN ET AL.
Scheme 1. The synthetic route used to produce the target compounds (MO1–MO9).
Table 1. Inhibitions of MAO-A, MAO-B, and AChE by MO1–MO9a
Residual activities (%)
IC50 (mM)
MAO-A
(10 mM)
MAO-B
(1.0 mM)
AChE
(10 mM)
BChE
(10 mM)
Compounds
MAO-A
MAO-B
AChE
BChE
SIb
MO1
MO2
MO3
MO4
MO5
MO6
MO7
MO8
94.2 0.62
93.8 1.34
91.0 3.17
70.2 1.24
88.1 3.43
12.3 1.58
ꢂ16.5 4.53
83.7 4.02
79.8 1.54
38.1 9.50
54.5 5.28
79.9 5.28
49.0 1.45
58.6 3.39
64.1 1.37
26.1 5.44
34.6 5.44
51.3 2.42
53.4 0.99
72.3 1.34
79.3 2.82
66.0 8.79
34.5 9.51
76.9 3.54
65.3 1.55
50.6 3.61
50.4 2.42
62.85 2.97
63.55 3.30
56.78 2.97
80.30 6.43
60.86 1.07
70.96 0.36
70.21 4.76
74.61 0.73
70.21 5.50
>40
>40
>40
25.8 1.58
>40
8.7 1.32
7.1 0.41
>40
>40
1.08 0.025
—
0.030 0.062
0.70 0.23
1.01 0.08
0.33 0.03
1.31 0.26
0.64 0.04
0.25 0.05
0.32 0.16
0.36 0.16
—
16.1 2.24
30.2 3.24
>40
28.42 0.02
6.1 0.0048
>40
20.48 1.10
12.07 1.18
12.01 2.13
>40
>40
>40
>1333.3
> 57.1
>39.6
78.2
>30.5
13.6
28.4
>125.0
>111.1
>40
18.09 0.38
>40
24.83 0.34
>40
MO9
>40
Toloxatone
Lazabemide
Clorgyline
Pargyline
Tacrine
0.063 0.015
—
0.028 0.0043
0.007 0.00070
—
0.27 0.019
0.060 0.0022
aValues above are the means SEs of duplicate or triplicate experiments.
bSI ¼ IC50 of MAO-A/IC50 of MAO-B.
AChE values were determined after pre-incubation compounds and enzymes for 15 min.
addition, MO5 most potently inhibited AChE (IC50 ¼ 6.1 mM), fol- reported that shifting of morpholine ring to the phenyl B ring of
chalcones showed moderate AChE inhibition35. Results of SAR
analysis of MAO-B/AChE inhibitions by the nine compounds are
lowed by MO9 and MO8 (IC50 ¼ 12.01 and 12.07 mM, respect-
ively). MO7 most potently inhibited MAO-A (IC50 ¼ 7.1 mM),
followed by MO6 (IC50 ¼ 8.7 mM). MO5 most potently inhibited
provided in Figure 3.
BChE (IC50 ¼ 18.09 mM), with weaker inhibitory activity compared
to AChE, followed by MO7 (IC50 ¼ 24.83 mM) (Table 1).
Morpholine-containing compounds in this study shared a 1–(4-
2.2.2. Kinetics of MAO-B inhibition
Lineweaver–Burk plots and secondary plots of MAO-B inhibition by
morpholinophenyl) prop-2-en-1-one structure. Introduction of
various electron-donating and withdrawing groups onto the para
MO1 showed that MO1 is a mixed inhibitor of MAO-B (Ki
0.018 0.002) (Figure 4(A,B)). These results suggested that MO1 binds
¼
position of the phenyl B ring of the basic chalcone scaffold
afforded different derivatives. Surprisingly, unsubstituted MO1
inhibited MAO-B more potently than the other eight derivatives.
All nine derivatives exhibited MAO-B selectivity. In particular, MO1
(IC50 ¼ 0.030 mM) was ꢁ2 times more potent than the reversible
MAO-B inhibitor lazabemide (IC50 ¼ 0.063 mM), and had a high
selectivity index (SI ¼ >1333.3), which meant that it was the best
inhibitor in the series. Regarding MAO-B inhibition, MO1 was
slightly less potent than the reference irreversible MAO-B inhibitor
pargyline (IC50 ¼ 0.028 mM). The presence of dimethylamino,
chloro and bromo substituents at the para position of the chal-
to an allosteric site other than the substrate-binding site of MAO-B.
2.2.3. Kinetics of AChE inhibitions
Lineweaver–Burk plots and secondary plots showed that MO5 and
MO9 competitively and non-competitively, respectively, inhibited
AChE (Figure 5(A,C)) with Ki values of 2.52 0.17 and 7.04
0.32 mM, respectively (Figure 5(B,D)), which suggest that MO5 is a
potent, selective and competitive inhibitor of AChE that binds to
cone B ring conferred moderate AChE inhibition. Recently, it was the active site of AChE, and that MO9 binds to a site other than

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
191
Figure 3. SAR analysis of MAO-B and AChE inhibitors.
(A)
(B)
120
100
80
60
40
20
0
Control
3000
2500
2000
1500
1000
500
0.0125 uM
0.025 uM
0.05 uM
0
-10
-5
0
5
10
15
20
25
30
-0.02
0
0.02
0.04
0.06
0.08
1/[S], mM-1
[MO1], uM
Figure 4. Lineweaver–Burk plots of MAO-B inhibition by MO1 (A), and respective secondary plots (B) of slopes vs. inhibitor concentrations.
the active site of AChE and changes the 3D structure of the or depression. The PAMPA used was validated by comparing the
enzyme, thus inhibiting enzyme activity.
experimentally determined permeabilities of eight commercial
drugs with reported values (Table 2). According to reported BBB
permeation limits, compounds were classified as follows:36 CNSþ
(high): Pe (10ꢂ6 cm sꢂ1) > 4.00, CNS- (low): Pe (10ꢂ6 cm sꢂ1
)
2.2.4. Analysis of the reversibilities of MAO-B and AChE inhibitions
Inhibition assays were carried out after preincubating MAO-B or
AChE with the inhibitors for 15 min. The reversibilities of MAO-B
inhibition by MO1 and of AChE inhibitions by MO5 and MO9
were investigated by dialysis34. Dialysis recovered the inhibition of
MAO-B by MO1 from 21.6% (AU) to 77.1% (AD), which was similar
to that shown by lazabemide (from 38.8 to 90.0%), a reversible
MAO-B inhibitor (Figure 6(A)). On the other hand, inhibition of
MAO-B by pargyline (an irreversible inhibitor) was recovered from
10.7 to 16.5%. In addition, inhibitions of AChE by MO5 and MO9
were recovered by dialysis from 35.8% (AU) to 79.2% (AD) and
from 33.8 to 80.7%, respectively, which were similar to that
observed for tacrine (from 32.8 to 91.7%), a reversible AChE inhibi-
tor (Figure 6(B)). These results indicate that MO1 is a reversible
inhibitor of MAO-B, and that MO5 and MO9 are reversible inhibi-
tors of AChE.
<2.00 and CNS (uncertain): Pe (10ꢂ6 cm sꢂ1) from 4.00 to 2.00.
Our results suggest that all nine synthesised derivatives could
cross the BBB, and that MO1, MO5 and MO9 would be the
most effective.
2.4. Cytotoxic studies
The biocompatibilities of MO1, MO5 and MO9 were investigated
using an MTT (3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay and normal VERO cells37,38. Results showed that
MO1, MO5 and MO9 were non-toxic to normal VERO cells with
IC50 values of 195.14, 185.44 and 188.34 mg/mL, respectively, indi-
cating that the therapeutic potentials of these compounds would
be suitable.
2.5. Total ROSs assay
2.3. Blood–brain barrier (BBB) permeation
ROS are considered to be the radicals primarily responsible for the
neuronal death observed in various neurodegenerative disor-
ders39. The lead compounds MO1, MO5 and MO9 were subjected
The synthesised derivatives were screened for their abilities to
cross the BBB using the parallel artificial membrane permeability
assay (PAMPA) because this ability is a critical developmental
requirement for any drug targeting neurodegenerative disorders to ROS assays using Hela cells. ROS levels significantly increased in

192
R. SASIDHARAN ET AL.
(A)
(B)
600
500
400
300
200
100
0
25
20
15
10
5
Control
3 uM
6 uM
12 uM
0
-15
-5
5
15
25
-5
0
5
10
15
1/[S], mM-1
[MO5], uM
(C)
(D)
1000
Control
35
30
25
20
15
10
5
900
800
700
600
500
400
300
200
100
0
2 uM
4 uM
8 uM
0
0
-10
-5
5
10
-15
-5
5
15
25
1/[S], mM-1
[MO9], uM
Figure 5. Lineweaver–Burk plots of AChE inhibition by MO5 (A) and MO9 (C), and respective secondary plots (B and D) of slopes vs. inhibitor concentrations.
Table 2. Experimental membrane permeabilities of the synthetic and refer-
ence inhibitors.
Undialyzed
Dialyzed
(A)
100
80
60
40
20
0
Bibliography
Experimental
Compounds
Pe (ꢃ10ꢂ6 cm sꢂ1
)
Pe (ꢃ10ꢂ6 cm sꢂ1
)
Prediction
Testosterone
Verapamil
b-estradiol
Progesterone
Piroxicam
Hydrocortisone
Lomefloxacin
Dopamine
MO1
17.0
16.0
12.0
9.3
2.5
1.8
1.1
0.2
—
16.86 0.67
15.69 0.44
11.88 0.53
9.10 0.11
2.35 0.30
1.71 0.05
1.26 0.01
0.21 0.01
16.34 0.16
14.44 0.81
14.61 0.08
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ/-
CNS-
CNS-
CNS-
CNSþ
CNSþ
CNSþ
Control
MO1 Lazabemide Pargyline
Control
MO1 Lazabemide Pargyline
MO5
MO9
—
—
Undialyzed
Dialyzed
(B)
100
80
60
40
20
0
H2O2-treated Hela cells as determined by fluorescent image analy-
ses (Figure 7(B)). MO1 and MO5 (Figure 7(C,D)) at 40 mg/mL
resulted in significantly lower ROS levels than MO9 (Figure 7(E)).
Interestingly, the colony formation induced by H2O2 was markedly
reduced by the lead MAO-B inhibitor MO1, which suggested that
the molecule can be highly recommended for the arrest of free
radicals in the neurodegenerative disorders.
control
MO5
MO9
Tacrine
conrtrol
MO5
MO9
Tacrine
Figure 6. Dialysis recoveries of MAO-B inhibition by MO1 (A) and AChE inhibition
by MO5 and MO9 (B). The concentrations of the inhibitors used were approxi-
mately 2 ꢃ IC50 values: MO1, 0.06 mM; MO5, 12.0 mM; MO9, 24.0mM; lazabemide,
0.12 mM; pargyline, 0.06 mM; and tacrine, 0.54 mM (tacrine was used as a reference
reversible inhibitor). For recovery experiments, preincubated enzyme mixtures
were dialysed as described in the text.
2.6. Computational studies
Initially, MO1 and MO5 were used as bases for profiling the
pharmacological spectra of putative protein targets using the free
web platform MuSSel40,41. Interestingly, MAO-B and AChE were

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
193
Figure 7. Inhibition of H2O2-induced ROS generation in human cervical cancer cells. (A) Treatment-naïve control cells; (B) Cells treated with 100lg/mL of H2O2; (C)
Cells treated with 40 lg/mL of MO1; (D) Cells treated with 40 lg/mL of MO5; (E) Cells- treated with 40lg/mL of MO9.
Table 3. Docking score values for bindings between MO1 and MO5 and MAO-B
predicted to be 13th and 14th targets for MO1 and 7th and 15th
or AChE.
targets for MO5, respectively. Details are provided in Supporting
Docking score (kcal/mol)
Information.
Docking studies were performed on MO1 and MO5 using the
Compounds
AChE
MAO-B
BChE
X-ray resolved structures of MAO-A, MAO-B and AChE in the
MO1
MO5
ꢂ10.31
ꢂ10.12
ꢂ8.67
ꢂ9.34
ꢂ7.42
ꢂ6.66
Protein Data Bank (PDB) as entries 2Z5X, 2VZ5 and 4EY742–45
.
Docking protocols were performed as we described in a previous
study13. Molecular docking simulations were performed to investi-
gate interactions between MO1 and MO5 and MAO-A, MAO-B MO5 hydrogen-bonded with F295 and formed p ꢂ p interactions
and AChE. The docking scores resulting from in silico simulations with the side chain of W286 at the peripheral anionic subsite
are reported in Table 3. Results showed that the carbonyl and the (PAS) of AChE (Figure 8(A)). Also, the dimethylamino group of
4-morpholin-phenyl groups of the chalcone scaffolds of MO1 and MO5 established a weak hydrophobic interaction with W86 at the

194
R. SASIDHARAN ET AL.
Figure 8. Top scored poses of MO1 and MO5 for binding with AChE (A), MAO-B (B) and MAO-A (C) are shown on left and right sides, respectively. MO1, MO5 and
key enzyme residues are displayed as green, yellow and grey sticks, respectively. Green and red arrows indicate p–p interactions and hydrogen bonds, respectively.
catalytic anionic subsite (CAS). MO1 and MO5 formed p ꢂ p inter- BChE binding pocket; in particular MO1 engaged a p–p inter-
actions with the selective MAO-B residue Y326 (Figure 8(B)). In action with F329, but both compounds cannot make crucial inter-
addition, the styryl and dimethylaminophenyl groups of MO1 and actions with the catalytic W82 residue (Figure 8(D)).
MO5, respectively, established p–p interactions with Y398 and
Overall, computational analyses provided a sound explanation
were sandwiched within an aromatic cage formed by Y328, Y435 of the experimental data. Docking scores and the binding modes
and FAD. MO1 engaged in a p ꢂ p interaction with Y407 and was obtained demonstrated that MO1 and MO5 can meaningfully
entangled in a hydrophobic cage consisting of Y407, Y444 and interact with MAO-B as indicated by IC50 experimental values.
FAD of MAO-A (Figure 8(C)), whereas MO5 established p–p inter- Interestingly, even if MO1 had adopted a similar binding mode
actions with a selective MAO-A residue F208 using its 4-morpho- in MAO-A and MAO-B binding pockets, the two docking score
line-phenyl group. In order to point out the selectivity of MO1 values would have been quite different, due to a lack of p–p
and MO5, molecular docking analyses were performed on BChE interaction with the MAO-A selective residue F208 and lesser
crystal structure. Docking score values of MO1 and MO5 for BChE hydrophobic interactions. On the other hand, MO5 assumed a
were worse than those for AChE (Table 3). Interestingly, MO1 and switched pose within the MAO-A binding pocket, with its mor-
MO5 can predominantly establish hydrophobic interactions in the pholine ring facing FAD, presumably because the dimethylamino

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
195
substituent on the phenyl ring did not allow adequate access to and then dialysing solutions with stirring for 6 h with a buffer
the binding site. As regards AChE, molecular docking awarded a
better score for MO5 than MO1, which also agreed with experi-
mental data, despite the resemblance between their bind-
ing poses.
change at 3 h. Residual activities were calculated using the meas-
ured activities of undialyzed (AU) and dialysed (AD) solutions and
the activities of untreated controls (i.e. without inhibitor).
Disclosure statement
3. Conclusion
No potential conflict of interest was reported by the authors.
We describe the synthesis of morpholine-containing a,b-
unsaturated ketones, and the results of an investigation of their
MAOs and AChE inhibition profiles. Most of the nine compounds
synthesised exhibited potent MAO-B inhibition with moderate
AChE inhibition. Interestingly, MO1 (the lead compound) inhibited
MAO-B inhibition in the low nanomolar range and was more
potent than lazabemide (the reference compound). The low cyto-
toxicity and the ability of MO1 to transit the BBB support our
drug design strategy. In addition, the ROS scavenging efficacy of
MO1 suggests improved neuroprotective effects.
Funding
This research was supported by the National Research Foundation
of Korea (NRF) grant funded by the Republic of Korea government
(Grant No. NRF-2019R1A2C1088967 to H. Kim).
ORCID
Hoon Kim
http://orcid.org/0000-0002-7203-3712
4. Experimental
4.1. Enzyme assays
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