Potent and highly selective dual-targeting monoamine oxidase-B inhibitors: Fluorinated chalcones of morpholine versus imidazole

Article abstract of DOI:10.1002/ardp.201800309

Two series of fluorinated chalcones containing morpholine and imidazole-based compounds (f1–f8) were synthesized and evaluated for recombinant human monoamine oxidase (MAO)-A and -B as well as acetylcholinesterase inhibitory activities. Our results indica

Full text of DOI:10.1002/ardp.201800309

Received: 24 October 2018
Revised: 5 December 2018
Accepted: 14 December 2018
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DOI: 10.1002/ardp.201800309
FULL PAPER
Potent and highly selective dual-targeting monoamine
oxidase-B inhibitors: Fluorinated chalcones of morpholine
versus imidazole
Bijo Mathew1
Seung C. Baek2
Della G. Thomas Parambi3
Jae P. Lee2
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Githa E. Mathew4
Sivaraman Jayanthi5
Vinod Devaraji5
Clariya Rapheal1
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Devikrishna Vinod1
Shahin Shad Kondarath1
Md. S. Uddin6
Hoon Kim2
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1 Division of Drug Design and Medicinal Chemistry Research Lab, Department of Pharmaceutical Chemistry, Ahalia School of Pharmacy, Palakkad, India
2 Department of Pharmacy and Research Institute of Life Pharmaceutical Sciences, Sunchon National University, Suncheon, Republic of Korea
3 Department of Pharmaceutical Chemistry, Jouf University, Sakaka, Saudi Arabia
4 Department of Pharmacology, Grace College of Pharmacy, Palakkad, India
5 Computational Drug Design Lab, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore, India
6 Department of Pharmacy, Southeast University, Dhaka, Bangladesh
Correspondence
Abstract
Bijo Mathew, Division of Drug Design and
Medicinal Chemistry Research Lab, Department
of Pharmaceutical Chemistry, Ahalia School of
Pharmacy, Palakkad-678557, Kerala, India.
Email: bijovilaventgu@gmail.com
Two series of fluorinated chalcones containing morpholine and imidazole-based
compounds (f1–f8) were synthesized and evaluated for recombinant human
monoamine oxidase (MAO)-A and -B as well as acetylcholinesterase inhibitory activities.
Our results indicate that morpholine containing chalcones are highly selective MAO-B
inhibitors having reversibility properties. All the imidazole-based fluorinated chalcones
showed weak MAO inhibitions in both isoforms. Among the tested compounds, (2E)-3-
(3-fluorophenyl)-1-[4-(morpholin-4-yl)phenyl]prop-2-en-1-one (f2) showed potent
inhibitory activity for recombinant human MAO-B (IC50 = 0.087 μM) with a high
selectivity index (SI) of 517.2. In the recovery experiments using dialysis, the residual
activity of MAO-B inhibited by f2 was close to that with the reversible reference
inhibitor. Inhibition assays revealed that the Ki values of f1 and f2 for MAO-B were 0.027
and 0.020 μM, respectively, with competitive patterns. All the morpholine-based
compounds (f1–f4) showed moderate inhibition toward acetylcholinesterase with IC50
values ranging between 24 and 54 μM. All morpholine-containing compounds exhibit
good blood–brain barrier permeation in the PAMPA method. The rational approach
regarding the highly selective MAO-B inhibitor f2 was further ascertained by induced fit
docking and molecular dynamics simulation studies.
Hoon Kim, Department of Pharmacy and
Research Institute of Life Pharmaceutical
Sciences, Sunchon National University,
Suncheon 57922, Republic of Korea.
Email: hoon@sunchon.ac.kr
Funding information
Basic Science Research Program through the
National Research Foundation (NRF) of Korea,
which is funded by the Ministry of Education,
Grant number: 2017R1D1A3B03028559
K E Y W O R D S
acetylcholinesterase, chalcone, entrance cavity, imidazole, MAO-B selective inhibitor,
molecular dynamics, morpholine
Bijo Mathew and Seung C. Baek contributed equally to this work.
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Arch Pharm Chem Life Sci. 2019;e1800309.
wileyonlinelibrary.com/journal/ardp
© 2019 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201800309

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MATHEW ET AL.
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1
INTRODUCTION
discovery of novel selective MAO-B and AChE inhibitors.[16–20]
Furthermore, dual and multi-targeted approaches toward MAO-B
and AChE inhibitions for the development of potential therapeutic
agents for AD and PD have recently been reported.[21]
Parkinson's disease (PD) encompasses a multicentric progressive loss
of specific neuronal cell populations resulting in the development of
the movement disorder. PD is the second most prevalent age-related
neurodegenerative disease that results from the loss of nigrostriatal
dopaminergic neurons.[1] The neurodegeneration is initiated decades
before it affects the motor function, and the phenotype is usually
characterized by resting tremors, rigidity, and bradykinesia.[2] Mono-
amine oxidase (MAO; EC 1.4.3.4) has increased expression levels in
neuronal tissues as well as gastro and hepatic tissues.[3] MAO-A and
MAO-B inhibitors are in clinical use for the treatment of neurological
and psychiatric disorders, respectively.[4] Biochemically, these two
isoenzymes are differentiated by their substrate and inhibitor
specificities.[5] Inhibition of isoform B, which is mainly localized in
the raphe nucleus of serotonergic neuronal cell bodies, leads to
elevated levels of dopamine (DA) in Parkinsonism patients. The
phenomenal advances in neurochemistry have greatly helped in
unfolding the pathophysiology of this disorder and provided the basis
for the introduction of levodopa. The new understanding and
disclosure of the mechanisms of 1-methyl-4-phenyl-1,2,5,6-tetrahy-
dropyridine (MPTP) in primate models has triggered a resurgence
concerning the etiological factors that enhance the inhibition of MAO-
B with deprenyl, the first MAO-B inhibitor which potentiates the
effects of levodopa and prolongs the life of PD patients.[6]
Chalcone chemistry is a fascinating area in the field of medicinal
chemistry due to its wide target-based mechanism, easy synthesis, and
structural versatility.[22] Chemically, it consists of two aryl or heteroaryl
rings separated by an α–β unsaturated carbonyl linker; ring A is nearer
to the carbonyl system and ring B is nearer to the β-carbon of the
unsaturated unit[23] (Figure 1). Numerous structural manipulations in
chalcone motifs have been endeavored to develop highly selective
reversible MAO inhibitors. These efforts are mainly focused for the
development of MAO-B inhibitors rather than MAO-A.[24] It has
largely been reported that the presence of electron donating or
lipophilic groups such as methyl, dimethylamino, ethyl, bromine,
fluorine, and trifluoromethyl groups at ring B of chalcones are
favorable for MAO-B inhibitory activity.[25–29] Simultaneously, the
effect of heterocyclic systems such as thiophene, indole, furan, and
imidazole in ring A also produced promising MAO-B inhibitory
potencies by maintaining the above-mentioned pharmacophore on
ring B.[30–40] These two structural manipulations have resulted in the
development of a diverse class of MAO-B inhibitors encompassing
competitive mode of inhibition. The current study focuses on the
effect of the morpholine and imidazole heterocyclic systems at the
para position of the ring A of phenyl system and the fine tuning of
fluorine on the phenyl ring B in the chalcones. Our group has
previously reported a new class of fluorinated chalcones as hMAO-B
inhibitors (Figure 2). Accordingly, the current study describes the
synthesis of morpholine/imidazole-based fluorinated chalcones, and
investigates their potential for MAO inhibition, kinetics, reversibility
mode, and blood–brain barrier (BBB) permeation property. In addition,
AChE inhibitory actions are also investigated. Finally, we identify the
lead molecule from the in vitro results of MAO-B inhibitor by
performing a detailed molecular dynamics simulation.
Most MAO-B inhibitors are devoid of the “cheese effect,” a
property that attributes these drugs as exciting prospects for further
investigation as neurodegenerative disease therapeutics.[7] Based on
their kinetics of inhibition, there are two classes of MAO-B inhibitors:
reversible and irreversible.[8] Reversible inhibitors are usually struc-
turally similar to MAO substrates and actively bind to the site but are
metabolized slower. Irreversible or suicide inhibitors first bind in a
reversible competitive manner and are then oxidized by a FAD
cofactor, which then makes it unavailable for amine metabolism.[9]
Usually, the MAO-B inhibitors are designed to elevate the reduced DA
concentrations responsible for PD, wherein at least 80% of the enzyme
needs to be inhibited to achieve pharmacological action.[10]
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2
RESULTS AND DISCUSSION
Alzheimer's disease (AD) is another common neurodegenerative
disease characterized by dementia, behavioral disturbances, and
difficulties in daily living activities.[11] The deficiency in cholinergic
neurotransmission results in memory and cognitive deficits in AD
patients. Inhibition of acetylcholinesterase (AChE), which catalyzes the
degradation of acetylcholine, improves the cholinergic function, and is
a target which represents an innovative therapeutic weapon for the
treatment of AD.[12] Studies reveal that selective MAO-B inhibitors
such as selegiline, rasagiline, and safinamide retard the further
neurodegeneration and have a positive effect on memory modalities
in AD.[13] Recent in vivo studies in mice reported the correlation
between the progress of AD and MAO activity.[14] Based on the drug
design concept of “one molecule, multiple-targets,”[15] we aim to
synthesize a multi-target chalcone-based molecular frame work
possessing inhibitory potential to both MAO-B and AChE. Chalcone
motifs are extensively recognized by many groups addressing the
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2.1 Chemistry
The synthesis of fluorinated heteroaryl chalcones was
accomplished
by
Claisen–Schmidt
condensation
between
FIGURE 1 Chalcone motif

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FIGURE 2 Potent fluorinated chalcones as MAO-B inhibitors reported by our group[26–28]
morpholine/imidazole-based heteroaryl methyl ketones and various
fluorinated benzaldehydes in the presence of an alkaline alcoholic
medium (Scheme 1). The 1H NMR spectra of f1–f4 showed that H1 and
focuses on (i) the effect of morpholine and imidazole heterocyclic
system at the para position of ring A of the chalcones and (ii) effect and
orientation of the fluorine unit at B ring. All the imidazole-based
fluorinated chalcones showed lesser MAO inhibition in both isoforms,
except f8 (hMAO-B IC50 = 9.07 μM), which was previously reported
along with f7.[40] Two of the most active compounds from these series
were morpholine-containing compounds f1 and f2 with IC50 values of
0.14 and 0.087 μM, respectively, against hMAO-B. These compounds
differed in the position of the fluorine atom on ring B of the chalcone
core. It is noticeable that shifting of fluorine to ortho and para positions
of ring B of chalcones decreases the MAO-B inhibitory potency when
compared to meta substitution. This fluorine orientation pattern has no
impact on imidazole-based chalcones. This suggests a clear structure–
activity relation principle that the fluorine orientation pattern in
morpholine containing compounds renders effectiveness against
hMAO-B inhibition.
H
2 protons of the morpholine ring resonated at 3.33–3.36 and 3.84–
3.88 ppm as triplets, respectively. Hα and Hβ protons of fluorinated
heteroaryl chalcones appeared as sharp doublets at 7.33–7.58 and
7.68–8.15 ppm, respectively. The large coupling constant (J) of these
doublets (16 Hz) confirmed the trans configuration of the chalcones.
Mass spectra of all fluorinated chalcones showed intensive molecular
ions, secondary to the structure of the targeted compounds.
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2.2 Biology
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2.2.1 MAO inhibition studies
The preliminary investigation suggests that morpholine containing
fluorinated chalcone compounds are highly selective hMAO-B
inhibitors as compared to imidazole (Table 1). The study mainly
In morpholine containing compounds, shifting of fluorine to para
(f3) decreased the potency toward hMAO-B (IC50 = 0.21 μM).
SCHEME 1 Synthesis of fluorinated chalcones

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TABLE 1 Inhibition of MAO-A, MAO-B, and AChE by fluorinated heteroaryl chalconesa
Residual activity at 10 µM (%)
IC50 (µM)
Compounds
MAO-A
MAO-B
AChE
MAO-A
18.69 0.34
45.0 2.84
>50.0
MAO-B
AChE
SIb
f1
62.0 1.4
78.5 0.7
88.5 2.1
87.5 2.1
87.5 0.7
84.0 2.8
80.5 2.1
84.0 1.4
−2.6 0.1
−3.4 0.4
1.06 1.4
34.0 5.7
78.3 7.1
59.4 5.7
69.2 7.1
42.5 7.8
84.4 0.39
80.1 0.27
80.5 0.05
77.0 0.96
93.9 0.42
95.8 0.67
95.4 0.32
98.0 0.75
0.14 0.005
0.087 0.008
0.21 0.006
2.30 0.20
>10.0
48.06 0.55
>50.0
133.5
517.2
>47.6
>4.3
f2
f3
>50.0
f4
>50.0
24.52 0.27
>50.0
f5
>50.0
f6
>50.0
>10.0
>50.0
f7
>50.0
>10.0
>50.0
f8
>50.0
9.07 0.52
–
>50.0
>1.1
Toloxatone
Lazabemide
Clorgyline
Pargyline
Tacrine
0.96 0.036
–
0.032 0.0056
>2.0
0.0046 0.0002
>2.0
0.097 0.0047
0.24 0.015
aResults are expressed as means SD of duplicate experiments. The value for tacrine was obtained after 15 min pre-incubation with enzyme.
bSI values for MAO-B were obtained by dividing IC50 values of MAO-A by those of MAO-B.
Replacing fluorine with trifluoromethyl group at the same position (f4)
also resulted in decreased inhibition (IC50 = 2.30 μM). Interestingly, f2
showed an extremely high selectivity index (SI) for MAO-B (517.2),
suggesting that f2 is a highly selective inhibitor for MAO-B. The
potency of f2 for MAO-B (Ki = 0.020) was higher than the synthesized
furanochalcone derivatives (Ki > 0.072 μM), except for one
compound (2E,4E)-1-(furan-2-yl)-5-phenylpenta-2,4-dien-1-one (F1)
(Ki = 0.0041 μM) previously reported by our group.[39] However, the SI
value of f2 (517.2) in this study was three times higher than that of the
lead furanochalone (F1) of the previous study (172.4).[39] However,
although the potency (IC50 = 0.087 μM) of f2 for MAO-B was 2.7 times
lower than the marketed drug for MAO-B lazabemide
(IC50 = 0.032 μM), the values were within a comparable range in
nanomolar concentration. The potency of f2 (0.087 μM) was also more
than to the standard hMAO-B irreversible type inhibitor pargyline
(0.097 μM) (Table 1). Furthermore, our studies revealed that the f2
molecule has some structural similarity with the FDA approved
selective hMAO-A inhibitor moclobemide (Figure 3). The morpholine
containing tail, electron rich linker and halogenated phenyl head are
common structural features of both motifs, which may contribute to
the similarity in targeting MAO.
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2.2.2 AChEe inhibition
As seen in Table 1, all compounds are less potent than the reference
compound tacrine for AChE inhibition. However, the morpholine-
based compounds (f1–f4) showed moderate inhibition toward AChE,
with IC50 values ranging between 24 and 54 μM, based on the reported
classification.[41] The data showed that the imidazole nucleus may not
be a crucial factor for the inhibitory potency against AChE in
fluorinated chalcones. Effect of fluorine orientation also shows
moderate inhibition in morpholine containing compounds for AChE
FIGURE 3 Similar structural features of f2 and moclobemide

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inhibition. The ortho- and para-substituted analogues f1 and f3 show
slightly higher AChE inhibition compared to the meta-substituted
analogue f3, unlike observed for MAO-B inhibition. Interestingly, the
presence of trifluoromethyl group at the para position of ring B of
morpholine chalcones shows two times higher potency than the
fluorinated compounds.
was nearly instantaneous. In reversibility experiments, AD and AU
values obtained for f2 were 70.1% and 26.7%, respectively (Figure 5).
For the reference experiments, AD and AU values for lazabemide were
73.4% and 35.0%, and for pargyline were 28.5% and 27.5%,
respectively. After dialysis, inhibition by pargyline remained constant.
However, inhibition by lazabemide was greatly recovered, similar to
the inhibition by f2. These results indicate that f2 is a reversible
inhibitor of MAO-B.
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2.2.3 Kinetics
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2.3 BBB prediction
The inhibition modes of f1 and f2 for MAO-B were analyzed by
Lineweaver–Burk plots. Plots for both f1 and f2 were linear
intersecting the y-axis (Figure 4A and C). The secondary plot of slopes
versus inhibitor concentrations provided the Ki values of f1 and f2 for
MAO-B inhibitions, determined to be 0.027 0.002 and
0.020 0.002 μM, respectively (Figure 4B and D). These results
indicate that f1 and f2 are selective and reversible competitive
inhibitors of MAO-B. Comparing the inhibition constants, the
inhibition constant of f2 was much lower than the irreversible
reference hMAO-B inhibitor selegiline (0.14 μM), as previously
reported by our research group.[32–36]
The parallel artificial membrane permeation assay (PAMPA) is used to
determine the BBB permeation potential of a compound. According to
the limits established by Di et al.,[42] BBB permeation test compounds
are classified as follows:
ꢀ
ꢁ
CNSþ ðhigh BBB permeation predictedÞ : Pe ꢀ10ꢁ6 cmsꢁ1
> 4:00
ꢀ
ꢁ
CNSꢁ ðlow BBB permeation predictedÞ : Pe ꢀ10ꢁ6 cmsꢁ1
< 2:00
Reversibility studies
No changes in activity were observed for f2 with MAO-B up to 30 min
of preincubation, suggesting the interaction between MAO-B and f2
Table 2 indicates the permeability of commercial drugs obtained
by PAMPA-BBB assay and the top ranked four morpholine containing
FIGURE 4 Lineweaver–Burk plots for inhibition of MAO-B by f1 (A) and f2 (C), and their respective secondary plots of slopes versus
inhibitor concentrations for f1 (B) and f2 (D)

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generate least energy with best pose. Results indicate that it holds
π–π interactions with Tyr326 and Tyr398 surrounded by hydrophobic
pocket environment Ile326, Trp119, Pro104, Phe103, Leu164,
Leu167, Ile199, Cys172, Tyr188, Tyr326, Leu328, Tyr60, and
Tyr435. The fluorinated A ring of morpholine chalcone was directed
toward the FAD unit within close proximity of the N5 atom (Figure 6).
Polar residue Gln206 collectively gives the protein-ligand complex
total energy of −20834 kcal/mol, docking score of −11.520, and
contains ligand Epik penalty which gives a glide XP score of −10.381
and subsequently an IFD score of −20941.70, which are good affinity
scores for ligand binding. Stable protein-ligand pose are subjected to
explicit molecular dynamics to check the stability, interactions, and
consistency of protein ligand complex for 50 ns time period. As per
RMSD (root mean square deviations) analysis, the protein C-alpha and
ligand were stable in the range 1.7–2.2 Å for a long duration of
simulation time without any major fluctuations; we therefore conclude
that the complexes were energetically compatible (Figure 7). The
interaction patterns were strongly observed with π–π interactions
with Tyr326 and other hydrophobic networks, H-bonds with amino
acids such as His90, Phe99, Pro104, Trp119, Leu167, Phe168, Leu171,
Cys172, Ile198, Ile199, Ile316, and Tyr326, and H bonds with Thr201,
Gln205, Gln206, Tyr206, Tyr326, Phe343, Tyr398, and Tyr435. Major
water bridges between the protein and ligand formed were Ile199,
Ser200, Thr201, Gly205, Gly206, Thr316, Tyr326, and Phe343,
whereas minor bridges were with formed Gln65, Tyr60, Gly101, and
Cys172 (Figures 8 and 9). The overall impression of the ligand–protein
interaction analysis and C-alpha protein and ligand RMSD plots and
nonbonding interactions indicate that f2 is reasonably stable in the
MAO-B protein, and we hypothesize that f2 is an active molecule with
major Tyr326 π–π and hydrophobic interaction that could be the
critical cause evoking the biological response.
FIGURE 5 Reversibility mode of f2
fluorinated chalcones. The results show that all the tested chalcones
can efficiently cross the BBB to target the MAO-A and MAO-B
enzymes in the central nervous system (CNS), which is consistent with
our design strategy. All the morpholine containing chalcones show
higher permeability than the standard testosterones. We particularly
highlight that the highest permeability value achieved of f4 in the series
may be due to the presence of a lipophilic trifluoromethyl group.
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2.4 Computational studies
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2.4.1 Molecular docking and dynamics
Docking software performance was validated by calculating the crystal
pose RMSD (root mean square deviation) in place against the pose
generated by the Glide software for ligands. When two crystal protein
PDBs such as 2V61 (ligand id C18) and 2V5Z (ligand id HRM) were
used, RMSD values were 0.862 and 1.121 by Glide XP docking
(Figure 6). Therefore, we used same settings for this study.
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3
CONCLUSION
The study mainly corroborated the MAO and AChE inhibitory effect of
morpholine and imidazole heterocyclic nucleus on the para position of
ring A of fluorinated chalcones. Fine tuning of the fluorine atom on the
The compound, (2E)-3-(3-fluorophenyl)-1-[4-(morpholin-4-yl)-
phenyl]prop-2-en-1-one (f2) was docked against MAO-B (2V61) to
TABLE 2 PAMPA-BBB of morpholine-based fluorinated chalcones and commercial drugs
Compoundsa
Bibliography Pe (×10−6 cms−1 b
)
Experimental Pe (×10−6 cms−1 c
)
Prediction
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS−
CNS−
f1
–
18.31 0.24
18.14 0.26
18.58 0.65
19.43 0.45
17.33 0.12
08.13 0.42
0.21 0.01
1.71 0.02
f2
–
f3
–
f4
–
Testosterone
Progesterone
Dopamine
Hydrocortisone
17.0
9.3
0.2
1.8
aCompounds were dissolved in DMSO (5 mg/mL) and diluted to be 100 μg/mL with PBS/EtOH (70:30).
[42]
bTaken from Ref.
.
cValues were shown as the mean SEM of three independent experiments.

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FIGURE 6 Docking validation-crystal ligand pose (gray) versus docked pose (green) for 2V61 and 2V5Z
various positions of ring B of chalcones markedly alters the activity.
The results document that morpholine bearing fluorinated chalcones
are potent and highly selective hMAO-B inhibitors with moderate
AChE inhibition. All the imidazole-based fluorinated chalcones showed
weak MAO inhibitions in both isoforms. We further postulate that f2 is
a potential candidate as a multi-targeting compound, being a potent
and selective MAO-B inhibitor (0.087 μM) and moderate AChE
inhibitor (53.41 μM). The Ki values of potent molecules f1 and f2 for
MAO-B were 0.027 and 0.020 μM, respectively, and showed
competitive inhibitions. Percentage of the relative activity of f2 on
dialyzed and undialyzed values is 70.1 and 26.7%, respectively,
indicating the formation of a reversible hMAO-B-inhibitor complex.
FIGURE 7 Induced fit best pose of f2 for 3D-dimensional (ligand in ball and stick and interacting residues with molecular surface with FAD
cofactor) (A) and 2D-interaction image (B) against MAO-B

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The original spectra of the investigated compounds are provided
as Supporting Information, as are their InChI codes together with some
biological activity data.
(2E)-3-(2-Fluorophenyl)-1-[4-(morpholin-4-yl)phenyl]prop-2-en-
1-one (f1)
Yellow, m.p. 118–120°C. 1H NMR (400 MHz, CDCl3) δ: 3.35–3.34 (4H,
t, morpholine -N-(CH2)2), 3.87–3.86 (4H, t, morpholine O-(CH2)2),
6.93–6.90 (2H, d,H3′ & H5′), 6.93–6.84 (2H, d, H3 & H5), 7.17–7.10
(2H, m, H4 & H6), 7.37–7.33 (1H, d, J = 16 Hz, -CHα), 7.89–7.85 (1H, d,
J = 16 Hz, -CHβ), 8.03–8.00 (2H, d, H2′ & H6′). ESI-MS (m/z): calculated
311.350, observed 311.349.
FIGURE 8 Protein-ligand RMSD
(2E)-3-(3-Fluorophenyl)-1-[4-(morpholin-4-yl)-phenyl]prop-2-
en-1-one (f2)
The BBB permeation assay method strongly indicates that all the
morpholine containing fluorinated chalcones have the ability to cross
the BBB which is a consistent requirement for the development of
multi-targeted MAO-B inhibitors for various neurodegenerative
disorders. Computational studies revealed that morpholine bearing
phenyl ring of f2 exhibits a π–π stacking interaction with Tyr326.
Highly selective inhibition of hMAO-B with moderate inhibition with
AChE of f2 shows the potential therapeutic application of this
compound for the treatment of AD and PD.
Yellow, m.p. 116–118°C. 1H NMR (400 MHz, CDCl3) δ: 3.33–3.32
(4H, t, morpholine -N-(CH2)2), 3.85–3.84 (4H, t, morpholine O-(CH2)2),
6.90–6.87 (2H, d, H3′ & H5′), 7.40–7.06 (4H, m, H2, H4, H5 & H6), 7.56–
7.52 (1H, d, J = 16 Hz, -CHα), 7.75–7.71 (1H, d, J = 16 Hz, -CHβ), 8.01–
7.98 (2H, d, H2′ & H6′). ESI-MS (m/z): calculated 311.350, observed
311.349.
(2E)-3-(4-Fluorophenyl)-1-[4-(morpholin-4-yl)phenyl]prop-2-en-
1-one (f3)
Pale yellow, m.p. 150–152°C. 1H NMR (400 MHz, CDCl3) δ: 3.35–3.34
(4H, t, morpholine -N-(CH2)2), 3.88–3.87 (4H, t, morpholine O-(CH2)2),
6.90–6.87 (2H, d, H3′ & H5′), 7.12–7.00 (2H, m, H3 & H5), 7.50–7.46
(1H, d, J = 16 Hz, -CHα), 7.64–7.61 (2H, m, H2 & H6), 7.77–7.73 (1H, d,
J = 16 Hz, -CHβ), 8.01–7.99 (2H, d, H2′ & H6′). ESI-MS (m/z): calculated
311.350, observed 311.350.
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4
EXPERIMENTAL
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4.1 Chemistry
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4.1.1 Synthesis
Equimolar quantities of substituted fluorinated benzaldehyde and
4′-morpholinoacetophenone or 4′-imidazoleacetophenone were dis-
solved in 40% KOH. The reaction mixture was stirred for 12 h and was
poured into ice-cold water. The precipitated product was washed
using water, dried, and recrystallized from methanol.
(2E)-1-[4-(Morpholin-4-yl)phenyl]-3-[4-(trifluoromethyl)phenyl]-
prop-2-en-1-one (f4)
Yellow, m.p. 85–87°C. 1H NMR (400 MHz, CDCl3) δ: 3.36–3.35 (4H, t,
J = 4 hz, morpholine -N-(CH2)2), 3.88–3.87 (4H, t, J = 8 Hz, morpholine
FIGURE 9 Protein interaction fractions with f2

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O-(CH2)2), 6.90–6.87 (2H, d, H3′ & H5′), 7.12–7.00 (2H, m, H3 & H5),
7.51–7.47 (1H, d, J = 16 Hz, -CHα), 7.64–7.61 (2H, m, H2 & H6), 7.84–
7.80 (1H, d, J = 16 Hz, -CHβ), 8.01–7.99 (2H, d, H2′ & H6′). ESI-MS
(m/z): calculated 361.357, observed 361.350.
4.2.1 Analysis of inhibitory activities and kinetics
The inhibitory potential of the eight compounds for MAO-A or MAO-B
were primarily analyzed at 10 μM concentrations. Thereafter, we
determined the IC50 values for compounds possessing greater than
50% inhibitory activity. Reversible and irreversible reference inhibitors
were included as standards. The time-dependence of the inhibition by
the most potent compound f2 for MAO-B was further investigated, as
previously described.[45] Ki values and inhibition types of the inhibitors
were determined by kinetic studies, as described previously.[43] The
AChE inhibitory activities by the compounds were measured by pre-
incubating for 15 min with the enzyme prior to the measurement.
(2E)-3-(2-Fluorophenyl)-1-[4-(1H-imidazol-1-yl)phenyl]prop-2-
en-1-one (f5)
Orange, m.p. 135–137°C. 1H NMR (400 MHz, CDCl3) δ: 6.93–6.90
(2H, d, H3′ & H5′), 6.88–6.79 (2H, d, H3 & H5), 7.12–7.15 (2H, m, H4 &
H6), 7.20–7.25 (3H, d, ImH), 7.38–7.33 (1H, d, J = 16 Hz, -CHα), 8.15–
8.11 (1H, d, J = 16 Hz, -CHβ), 8.03–8.00 (2H, d, H2′ & H6′). ESI-MS
(m/z): calculated 292.307, observed 292.299.
(2E)-3-(3-Fluorophenyl)-1-[4-(1H-imidazol-1-yl)phenyl]prop-2-
en-1-one (f6)
|
4.2.2 Analysis of inhibitor reversibility
Pale red, m.p. 148–150°C. 1H NMR (400 MHz, CDCl3) δ: 6.88–6.82
(2H, d, H3′ & H5′), 7.26–7.21 (3H, d, ImH), 7.40–7.06 (4H, m, H2, H4, H5
& H6), 7.43–7.39 (1H, d, J = 16 Hz, -CHα), 7.75–7.71 (1H, d, J = 16 Hz,
-CHβ), 8.01–7.98 (2H, d, H2′ & H6′). ESI-MS (m/z): calculated 292.307,
observed 292.299.
The reversibility of the most potent inhibitor f2 for MAO-B was
investigated by the dialysis method using DiaEasy dialyzers (BioVision
Inc., Milpitas, CA, USA), as previously described.[45] Concentrations
used were ∼2 × IC50: 0.18 μM of f2, 0.08 μM of lazabemide, and
0.20 μM of pargyline. Residual activities for undialyzed and dialyzed
sets were separately measured, after preincubation with MAO-B for
30 min. The relative values for undialyzed (AU) and dialyzed (AD) assays
were calculated comparing with each control set without inhibitor. The
reversibility pattern was concluded by comparing AU and AD values
with the references.
(2E)-3-(4-Fluorophenyl)-1-[4-(1H-imidazol-1-yl)phenyl]prop-2-
en-1-one (f7)
Brick red, m.p. 140–142°C. 1H NMR (400 MHz, CDCl3) δ: 6.90–6.86
(2H, d, H3′ & H5′), 7.10–7.02 (2H, m, H3 & H5), 7.24–7.20 (3H, d, ImH),
7.46–7.42 (1H, d, J = 16 Hz, -CHα), 7.68–7.63 (2H, m, H2 & H6), 7.79–
7.75 (1H, d, J = 16 Hz, -CHβ), 8.03–8.00 (2H, d, H2′ & H6′). ESI-MS
(m/z): calculated 292.307, observed 292.299.
|
4.3 BBB assay
The top-ranked four synthesized morpholine-based fluorinated
chalcones and the commercial drugs were dissolved in DMSO to a
final concentration of 5 mg/mL. Compounds were then diluted with a
mixture of phosphate-buffered saline solution and ethanol (PBS/
EtOH, 70:30) to a final concentration of 25 μg/mL. The filter
membrane in donor microplate was coated with polar brain lipid
(PBL) dissolved in dodecane (4 μg/mL, 20 mg/mL). A total of 200 μL of
diluted solution and 300 μL of PBS/EtOH (70:30) were added to the
donor and the acceptor wells, respectively. The donor plate was
carefully placed on the acceptor plate and the sandwich was kept at
25°C for 16 h. The donor plate was carefully removed, and then the
concentrations of the compounds in the acceptor, donor, and
reference wells were measured with a UV plate reader.[42]
(2E)-1-[4-(1H-Imidazol-1-yl)phenyl]-3-[4-(trifluoromethyl)-
phenyl]prop-2-en-1-one (f8)
Saffron red, m.p. 152–154°C. 1H NMR (400 MHz, CDCl3) δ: 6.91–6.88
(2H, d, H3′ & H5′), 7.14–7.06 (2H, m, H3 & H5), 7.25–7.21 (3H, d, ImH),
7.42–7.38 (1H, d, J = 16 Hz, -CHα), 7.65–7.62 (2H, m, H2 & H6), 7.72–
7.68 (1H, d, J = 16 Hz, -CHβ), 8.04–8.00 (2H, d, H2′ & H6′). ESI-MS
(m/z): calculated 342.314, observed 342.315.
|
4.2 Enzyme assays
MAO-A and MAO-B activities were measured by the continuous
method using kynuramine (0.06 mM) and benzylamine (0.3 mM) as
the substrates as described previously[40] and expressed as changes
in absorbance per min. Km values for kynuramine and benzylamine
were 0.035 and 0.15 mM, respectively; the concentrations for both
substrates were 1.7× and 2.0 × Km values, respectively. The
chemicals and enzymes used were as described previously.[43]
AChE activity was assayed using the method previously de-
scribed,[44] with slight modifications. The mixture was reacted at
25°C for 10 min with monitoring at 412 nm using AChE (0.2 U/mL)
from Electrophorus electricus (Type VI-S, Sigma). The mixture
contained 0.5 mM of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)
and 0.5 mM of acetylthiocholine iodide (ACTI) in 0.5 mL of 50 mM
sodium phosphate (pH 7.5).
|
4.4 Computational studies
All computational works were performed using Schrodinger suite
(Small-Molecule Drug Discovery Suite 2018-2, Schrödinger, LLC, New
York, NY, 2018).
|
4.4.1 Protein and ligand preparation
The protein (PDB: 2V61) used in the study was prepared using the
protein preparation wizard where it was processed for fixing the
structural issues arising from X-ray crystallography experimental

10 of 11
MATHEW ET AL.
|
limitations such as missing hydrogens, side chains or loops and bond
orders concerns.[46] In this process, protein is also completely reviewed
and modified to retain water and heteroatoms critical for calculations,
followed by optimization of hydrogen bond orientations and restrained
minimization. A convergence threshold of 0.30 Å with OPLS 2005
force field was used to generate least energy and a problem free
protein system to be used for the computational studies.[47] All the
organic ligands used in the manuscripts are prepared by Ligprep and
used 3D coordinates using OPLS 2005 to maintain molecule's integrity
and stereochemistry, ionization at biological pH with minimized 3D
coordinates using OPLS 2005 force field.[48]
ORCID
Bijo Mathew
http://orcid.org/0000-0002-6658-4497
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The authors have declared no conflict of interest.

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How to cite this article: Mathew B, Baek SC, Thomas
Parambi DG, et al. Potent and highly selective dual-targeting
monoamine oxidase-B inhibitors: Fluorinated chalcones of
morpholine versus imidazole. Arch Pharm Chem Life Sci.
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