Development of fluorinated methoxylated chalcones as selective monoamine oxidase-B inhibitors: Synthesis, biochemistry and molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2015.07.001

A series of methoxylated chalcones with fluoro and trifluoromethyl derivatives were synthesized and investigated for their ability to inhibit human monoamine oxidase A and B. The chemical structures of the compounds have been characterized by means of the

Full text of DOI:10.1016/j.bioorg.2015.07.001

Bioorganic Chemistry 62 (2015) 22–29
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Short communication
Development of fluorinated methoxylated chalcones as selective
monoamine oxidase-B inhibitors: Synthesis, biochemistry and molecular
docking studies
Bijo Mathew a, , Githa Elizabeth Mathew b, Gülberk Uçar c, Ipek Baysal c, Jerad Suresh d,
⇑
Jobin Kunjumon Vilapurathu a, Aneesh Prakasan b, Jeethu Kuruppath Suresh b, Anjana Thomas b
a Division of Drug Design and Medicinal Chemistry Research Lab, Department of Pharmaceutical Chemistry, Grace College of Pharmacy, Palakkad 678004, Kerala, India
b Department of Pharmacology, Grace College of Pharmacy, Palakkad 678004, Kerala, India
c Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, 06100 Sıhhiye, Ankara, Turkey
d Department of Pharmaceutical Chemistry, Madras Medical College, Chennai 600004, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of methoxylated chalcones with fluoro and trifluoromethyl derivatives were synthesized and
investigated for their ability to inhibit human monoamine oxidase A and B. The chemical structures of
the compounds have been characterized by means of their 1H NMR, 13C NMR, Mass spectroscopic datas
and elemental analysis. The results demonstrate that these compounds are reversible and selective
Received 9 June 2015
Revised 6 July 2015
Accepted 7 July 2015
Available online 9 July 2015
MAO-B inhibitors with
a
competitive mode of inhibition. The most potent compound
(2E)-1-(4-methoxyphenyl)-3-[4-(trifluoromethyl)phenyl] prop-2-en-1-one showed the best activity
Keywords:
Methoxylated chalcone
MAO-A
MAO-B
Molecular docking
and higher selectivity towards hMAO-B with Ki and SI values of 0.22 0.01
that standard drug, Selegiline Ki and SI values were found as 0.33 0.03
l
l
M and 0.05 comparable to
M and 0.04, respectively.
Molecular docking studies were carried out to further explain the in vitro results of the new compounds,
and to identify the hypothetical binding mode for the compounds inside the inhibitor binding cavity of
hMAO-B.
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
brain seems to require about 40 days [3]. So, newly synthesized
reversible MAO-A and B inhibitors may cause a great healing value
Recognition of the catalytic site of monoamine oxidases (MAO)
represents as promising targets for intervention in many amine
oxidase-related disorders like depression and Parkinson’s disease
[1]. MAO inhibitors (MAOIs) are classified as reversible or
irreversible, according to their interaction with the isoform. MAO
irreversible inhibitors previously have been used as clinical drugs.
However, this type of inhibition has been shown to induce cardio-
vascular toxic effects, mainly provoked by the inhibition of the
peripheral MAO located in gut, liver and endothelium. On the other
hand, competitive, reversible inhibitors have less influence in the
enzyme recovery after withdrawal as the ingested tyramine is able
to displace the inhibitor from the MAO active site and be metabo-
lized in the normal way by a peripheral enzyme in the gut and liver
[2]. Besides, the slow and variable enzyme recovery following the
withdrawal of irreversible inhibitors is a disadvantage in clinical
use, since the turnover rate for MAO biosynthesis in the human
in this prospect.
In neurodegenerative disorders, including Parkinson’s and
Alzheimer’s diseases, MAO-B has been proposed to play a promi-
nent role, though generating reactive oxygen species (ROS) in the
oxidation of monoamine substrates. The oxidative pathway of
ROS can precipitate neuronal death in neurodegenerative disorders
[4]. Inhibitors of MAO with selectivity and specificity of MAO type
B prolong the activity of both endogenous and exogenously derived
dopamine, making them an option either as monotherapy in early
Parkinson’s disease or as adjuvant therapy in patients treated with
levodopa who are experiencing motor complications [5]. Thus the
objective of our research program is to develop some selective and
potent MAO-B inhibitors from therapeutically known scaffold.
Chalcones, three-carbon consists of
a, b-unsaturated carbonyl
system and are considered to be precursors of flavonoids and iso-
flavonoids [6]. The competence of chalcone scaffolds to work along
the central nervous system (CNS) is chiefly connected with its low
polar surface area that can aid to thwart the blood brain barrier
easily. This attribute is primarily made by the hydrophobic nature
⇑
Corresponding author.
E-mail address: bijovilaventgu@gmail.com (B. Mathew).
http://dx.doi.org/10.1016/j.bioorg.2015.07.001
0045-2068/Ó 2015 Elsevier Inc. All rights reserved.

B. Mathew et al. / Bioorganic Chemistry 62 (2015) 22–29
23
of two aromatic nucleus of A and B between the open chain of
a,
0.16 0.01 lM [18]. The present study aims to expand on the
b-unsaturated system [7]. The MAO inhibitory activities of chal-
cones have however, only been determined in a few instances for
naturally occurring chalcones using rat, bovine and hamster mono-
amine oxidases, while some synthetic chalcone derivatives were
screened against human MAO (hMAO) [8–16]. The gathered results
suggested that chalcone derivatives displayed a selective inhibi-
tory activity towards hMAO-B in the micro to nanomolar range.
The presence of hydroxyl and methoxyl groups or two hydroxyl
substituents in the ortho and para position of the A aromatic moi-
ety as well as a chlorine atom in the para position of the B aromatic
moiety is found to be a fundamental requisite for activity. On the
other hand chalcones substituted with isoprenyloxyl substructures
have shown a lower activity [17].
structure–activity relationships (SARs) of MAO inhibition by chal-
cone derivatives by synthesizing a series of fluorinated chalcones
with methoxy substituted phenyl system. The information regard-
ing the orientation of fluoro and trifluoromethyl groups in the B
ring system of methoxylated chalcone and its effect of MAO inhibi-
tion is not explored so far. Para-methoxy acetophenone moiety
was selected as starting reagent for the synthesis of various
methoxylated chalcones. For this purpose, chalcones were
obtained by a Claisen–Schmidt condensation between p-methoxy
acetophenone and various substituted fluorine and trifluoromethyl
benzaldehydes under basic conditions – Scheme I (Fig. 2).
So far many of fluorine and trifluromethyl group
containing CNS drugs has been applied in clinical practice,
such as antidepressant-Fluoxetine, antipshychotic-Melperone,
anti-anxiety agent-Halazepam, benzodiazepine antagonist-
Flumazenil etc. (Fig. 1). Recently the cyclized product of
dimethoxyl chalcone (2-aryl-4H-chromen-4-one) was found to be
potent MAO-B inhibitor with Ki value for MAO-B of
2. Experimental
2.1. Materials and methods
All the fluorinated benzaldehydes were procured from
Sigma–Aldrich USA. Melting points of all the synthesized deriva-
tives were determined by open-capillary tube method and values
CH3
NH
O
O
F3C
CH3
N
F
Fluxetine
Melperone
CF3
O
N
Cl
N
COOC2H5
N
N
N
F
CH3
O
Halazepam
Flumazenil
Fig. 1. Fluorinated CNS drugs.
O
CH3
O
CHO
i
R
+
H3CO
R
OCH3
M1: R= 2-F
M2: R= 3-F
M3: R= 4-F
M4: R= 2-CF3
M5: R= 4-CF3
Fig. 2. Scheme 1 – Synthesis of fluorinated methoxylated chalcones; (i): Reagents and condition:C2H5OH, 40% KOH, stir. 4 h.

24
B. Mathew et al. / Bioorganic Chemistry 62 (2015) 22–29
were uncorrected. Proton (1H) and carbon (13C) nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker Avance III
600 spectrometer at frequencies of 400 MHz and 100 MHz, respec-
tively (Bruker, Karlsruhe, Germany). All NMR measurements were
conducted in CDCl3, and the chemical shifts are reported in parts
per million (d) downfield from the signal of tetramethylsilane
added to the deuterated solvent. Mass spectra were recorded on
a JEOL GCmate mass spectrometer. Elemental analyses (C, H, N)
were performed on a Leco CHNS 932 analyzer.
7.533–7.514 (t, 1H, J = 7.6 Hz, H5), 7.621–7.602 (t, 1H, J = 7.6 Hz,
H4), 7.754–7.734 (d, 1H, J = 8.0 Hz, H3), 7.853–7.834 (d, 1H,
0
0
J = 7.6 Hz, H6), 8.061–8.039 (d, 2H, J = 8.8 Hz, H4
& H6 ).
8.157–8.118 (d, 1H, J = 15.6 Hz, CHb). 13C NMR (100 MHz, CDCl3)
d ppm: 188.47, 163.65, 139.31, 134.30, 132.06, 131.02, 130.60,
129.60, 128.99, 128.08, 127.96, 126.52, 125.15, 122.64, 119.91,
113.93, 55.50. ESI-MS (m/z): calculated 306.279, observed
306.240 (M + 1)+. Anal. calcd. for C17H13F3O2: C, 66.67; H, 4.28.
Found: C, 66.78; H, 4.20.
2.2. Chemistry
2.2.5. (2E)-1-(4-methoxyphenyl)-3-[4-(trifluoromethyl) phenyl] prop-
2-en-1-one (M5)
A mixture of 4-methoxy acetophenone (0.01 mol), fluorinated
aldehyde (0.01 mol) and 40% aqueous potassium hydroxide
(15 mL) in ethanol (30 mL) was stirred at room temperature for
about 2–6 h. The resulting product was kept overnight in refriger-
ator. The solid separated was filtered, washed with water and
recrystallized from ethanol.
Pale yellow solid, Yield: 86%, M.P: 118–120 °C. 3.921 (s, 3H,
40-OCH3), 7.030–7.008 (d, 2H, J = 8.8 Hz, H3 & H5 ), 7.648–7.609
0
0
(d, 1H, J = 15.6 Hz, CH ), 7.699–7.679 (d, 2H, J = 8.0 Hz, H3 & H5,
a
7.768–7.748 (d, 2H, J = 8.0 Hz, H2 & H5), 7.837–7.798 (d, 1H,
J = 15.6 Hz, CHb), 8.083–8.061 (d, 2H, J = 8.8 Hz, H4 & H6 ). 13C
NMR (100 MHz, CDCl3) d ppm: 188.18, 163.72, 141.87, 138.52,
131.87, 131.54, 130.92, 130.74, 128.41, 127.93, 125.88, 125.81,
125.23, 124.16, 122.52, 113.98, 55.52. ESI-MS (m/z): calculated
306.279, observed 306.270 (M + 1)+. Anal. calcd. for C17H13F3O2:
C, 66.67; H, 4.28. Found: C, 66.56; H, 4.32.
0
0
2.2.1. (2E)-3-(2-fluorophenyl)-1-(4-methoxyphenyl) prop-2-en-1-one
(M1)
Pale white solid, Yield: 88%, M.P: 89–91 °C. 1H NMR (400 MHz,
CDCl3) d ppm: 3.920 (s, 3H, 40-OCH3), 7.026–7.004 (d, 2H, J = 8.8 Hz,
0
0
H3 & H5 ), 7.128–7.110 (t, 1H, J = 7.2 Hz, H5), 7.150–7.143 (d, 1H,
J = 2.8 Hz, H3), 7.398–7.379 (t, 1H, J = 7.6 Hz, H4), 7.418–7.398
2.3. Biochemistry
(d, 1H, J = 8.0 Hz, H6), 7.577–7.538 (d, 1H, J = 15.6 Hz, CH ),
a
7.792–7.753 (d, 1H, J = 15.6 Hz, CHb), 8.077–8.055 (d, 2H,
2.3.1. Chemicals
J = 8.8 Hz, H4 & H6 ). 13C NMR (100 MHz, CDCl3) d ppm: 188.33,
164.30, 163.61, 161.84, 142.43, 142.41, 137.45, 137.37, 130.87,
124.47, 124.44, 123.10, 117.23, 117.01, 113.93, 55.52. ESI-MS
(m/z): calculated 256.271, observed 256.270 (M + 1)+. Anal. calcd.
for C16H13FO2: C, 74.99; H, 5.11. Found: C, 74.88; H, 5.21.
0
0
hMAO-A and hMAO-B (both recombinant, expressed in
baculovirus-infected BTI insect cells), R-(–)-deprenyl hydrochlo-
ride (selegiline), moclobemide, resorufin, dimethyl sulfoxide
(DMSO), and other chemicals were purchased from Sigma–
Aldrich (Munich, Germany). The AmplexÒ-Red MAO assay kit
(Cell Technology Inc., Mountain View, CA, USA) contained benzy-
lamine, p-tyramine, clorgyline (MAO-A inhibitor), pargyline
(MAO-B inhibitor), and horseradish peroxidase.
2.2.2. (2E)-3-(3-fluorophenyl)-1-(4-methoxyphenyl) prop-2-en-1-one
(M2)
1H NMR (400 MHz, CDCl3) d ppm: Pale white solid, Yield: 82%,
M.P: 94–96 °C. 3.913 (s, 3H, 40-OCH3), 7.020–6.998 (d, 2H,
0
0
J = 8.8 Hz, H3
&
H5 ), 7.149–7.127 (d, 1H, J = 8.8 Hz, H4),
2.3.2. Determination of inhibitory activities of the fluorinated
methoxylated chalcones on human MAO-A and B
7.234–7.215 (t, 1H, J = 7.6 Hz, H6), 7.386–7.369 (t, 1H, J = 6.8 Hz,
H5), 7.681 (s, 1H, H2), 7.702–7.662 (d, 1H, J = 16.0 Hz, CH ),
The activities of hMAO-A and hMAO-B were determined using
p-tyramine as common substrate and calculated as 161.50
8.33 pmol/mg/min (n = 3) and 140.00 7.99 pmol/mg/min (n = 3),
respectively. The interactions of the synthesized fluorinated
methoxylated chalcones with hMAO isoforms were determined by
a fluorimetric method described and modified previously [19–21].
Study medium contained 0.1 mL of sodium phosphate buffer
(0.05 M, pH 7.4), various concentrations of the newly synthesized
compounds or reference compounds, and adequate amounts of
recombinant hMAO-A or hMAO-B. This mixture was incubated for
15 min at 37 °C in a 96-well microplates, placed in the dark
fluorimeter chamber. After this incubation period, the reaction
a
7.930–7.890 (d, 1H, J = 16.0 Hz, CHb), 8.079–8.057 (d, 2H,
J = 8.8 Hz, H4 & H6 ).13C NMR (100 MHz, CDCl3) d ppm: 188.69,
163.54, 162.99, 160.48, 136.67, 131.61, 131.53, 130.96, 129.80,
124.62, 124.45, 123.28, 116.37, 116.16, 113.89, 55.49. ESI-MS
(m/z): calculated 256.271, observed 256.260 (M + 1)+. Anal. calcd.
for C16H13FO2: C, 74.99; H, 5.11. Found: C, 74.91; H, 5.17.
0
0
2.2.3. (2E)-3-(4-fluorophenyl)-1-(4-methoxyphenyl) prop-2-en-1-one
(M3)
Pale white solid, Yield: 90%, M.P: 106–108 °C. 1H NMR
(400 MHz, CDCl3) d ppm: 3.908 (s, 3H, 40-OCH3), 7.013–6.992
0
0
(d, 2H, J = 8.4 Hz, H3 & H5 ), 7.143–7.722 (d, 2H, J = 8.4 Hz, H3
&
was started by adding 200 lM Amplex Red reagent, 1 U/mL horse-
H5, 7.508–7.469 (d, 1H, J = 15.6 Hz, CH ), 7.664–7.650 (d, 2H,
radish peroxidase (HRP), and p-tyramine (concentration range
a
J = 5.6 Hz, H2
& H5), 7.801–7.862 (d, 1H, J = 15.6 Hz, CHb),
0.05–1.00 mm).
8.065–8.043 (d, 2H, J = 8.8 Hz, H4 & H6 ). 13C NMR (100 MHz,
CDCl3) d ppm: 188.46, 165.19, 163.50, 162.69, 142.60, 131.39,
131.36, 131.05, 130.79, 130.25, 121.66, 121.16, 116.16, 115.95,
113.98, 55.49. ESI-MS (m/z): calculated 256.271, observed
256.250 (M + 1)+. Anal. calcd. for C16H13FO2: C, 74.99; H, 5.11.
Found: C, 74.83; H, 5.16.
The production of H2O2 catalyzed by MAO isoforms was
detected using AmplexÒ-Red reagent, a non-fluorescent probe that
reacts with H2O2 in the presence of horse radish peroxidase to pro-
duce the fluorescent product resorufin. The reaction was started by
0
0
adding 200 lM Amplex Red reagent, 1 U/mL horseradish peroxi-
dase, and p-tyramine (concentration range 0.1–1 mM). Control
experiments were carried out by replacing the compound and ref-
erence inhibitors. The possible capacity of compounds to modify
the fluorescence generated in the reaction mixture due to
non-enzymatic inhibition was determined by adding these com-
pounds to solutions containing only the Amplex Red reagent in a
sodium phosphate buffer.
2.2.4. (2E)-1-(4-methoxyphenyl)-3-[2-(trifluoromethyl) phenyl]
prop-2-en-1-one (M4)
Yellow solid, Yield: 87%, M.P: 78–80 °C. 1H NMR (400 MHz,
CDCl3)
d
ppm: 3.911 (s, 3H, 40-OCH3), 7.017–6.995 (d, 2H,
0
0
J = 8.4 Hz, H3
&
H5 ), 7.472–7.433 (d, 1H, J = 15.6 Hz, CH ),
a

B. Mathew et al. / Bioorganic Chemistry 62 (2015) 22–29
25
2.3.3. Kinetic experiments
2.4.3. Docking protocol
All the synthesized fluorinated methoxylated chalcones were
dissolved in dimethyl sulfoxide, with a maximum concentration
of 1% and used in the concentration range of 0.01–2.00 lM. The
The receptor grid of both enzymes was developed by using
60 ꢁ 60 ꢁ 60 grid points in xyz with grid spacing of 0.375 Å and
the grid box was centred on N5 atom of FAD (cofactor). The
Lamarckian genetic algorithm was applied for all molecular dock-
ing simulations. Population size of 300, mutation rate of 0.02 and
crossover rate of 0.8 were set as the parameters. Simulations were
performed using up to 2.5 million energy evaluations with a utter-
most of 27,000 multiplications. Each simulation was performed 50
times, yielding 50 docked conformations. In the automated dock-
ing procedure, AutoDock output record the result of docking stud-
ies with an extension file ‘dlg’. The output file involves many
details about the docking regarding the binding energy and inhibi-
tion constant of ten conformers used in the respective ligand. From
the docking results, the lowest docking energy conformations were
also the largest conformational cluster, which was chosen to repre-
sent the most favourable binding mode between the ligands and
enzymes [28].
mode of MAO inhibition was examined using Lineveawer-Burk
plotting. The slopes of the Lineweavere-Burk plots were plotted
versus the inhibitor concentration and the Ki values were deter-
mined from the x-axis intercept as ꢀKi. Each Ki value is the repre-
sentative of single determination where the correlation coefficient
(R2) of the replot of the slopes versus the inhibitor concentrations
was at least 0.98. SI was calculated as Ki (hMAO-A)/Ki (hMAO-B).
The protein was determined according to the Bradford method
[22].
2.3.4. Reversibility experiments
Reversibility of the MAO inhibition with synthesized com-
pounds was determined by centrifugation-ultrafiltration method
previously described [23]. Briefly, adequate amounts of the
recombinant enzymes (hMAO-A or B) were incubated with a single
concentration of the synthesized compounds or the reference inhi-
bitors in a sodium phosphate buffer (0.05 M, pH 7.4) for one hour
at 37 °C. An aliquot was stored at 4 °C and used for the measure-
ment of MAO-A and -B activity. The remaining incubated sample
was placed in an Ultrafree-0.5 centrifugal tube with a 30 kDa
Biomax membrane and centrifuged at 9000g for 20 min at 4 °C.
The enzyme retained in the 30 kDa membrane was resuspended
in a sodium phosphate buffer at 4 °C and centrifuged two more
times. After the third centrifugation, the enzyme retained in the
membrane was resuspended in sodium phosphate buffer
(300 mL) and an aliquot of this suspension was used for MAO-A
and -B activity determination. Control experiments were per-
formed simultaneously (to determine the 100% MAO activity) by
replacing the compounds and standards with appropriate dilutions
of the vehicles. The corresponding values of percent (%) MAO iso-
form inhibition was separately calculated for samples with and
without repeated washing.
3. Result and discussion
3.1. Chemistry
The synthesis of the fluorinated methoxylated chalcones was
conducted by the Claisen–Schmidt condensation between
4-methoxyacetophenone with various fluorinated aromatic alde-
hydes under 40% KOH ethanolic medium. 1H NMR spectrum
showed the peaks of 13 protons compromising of signals of the
a-b unsaturated unit, the substituted two phenyl system and
methoxyl group. It has been noted that, the up field proton of
a
carbon and down field proton of b carbon coupled with methine
proton with coupling constants (15.6–16.0 Hz). This indicates the
presence of trans (E) configuration in the fluorinated methoxylated
chalcones. A sharp singlet shielded protons in the rage of d 3.908–
3.9021 of chalcones strongly recommended the presence of meth-
oxyl group. 13C NMR spectra, the peaks for carbonyl carbon of chal-
cone and methoxyl group are in the range of d 188.69–188.18 and
55.49–55.52 respectively. All compounds provided satisfactory ele-
mental analyses.
2.4. Molecular docking methodology
In the current molecular simulation study, AUTODOCK4.2 soft-
ware was used to establish a ligand-based computer modelling
program for the prediction of binding energy of the selected
compounds with MAO isoenzymes [24].
3.2. Biochemistry
The potential inhibitory activities of the fluorinated methoxy-
lated chalcones on hMAO isozymes were determined by measuring
their effects on the production of H2O2 from the substrate
(p-tyramine), using the Amplex Red MAO assay kit and enzymes
(recombinant MAO isoforms). The synthesized compounds did
not exhibit any interference with the reagents in the assay med-
ium. The results concerning to hMAO-A and hMAO-B inhibitory
activities with the synthesized compounds are presented in
Table 1 together with their MAO selectivity indices. The MAO inhi-
bitory activity of chalcone scaffolds has been described previously
by different groups and appear to travel along the same style as in
the fluorinated methoxylated chalcones. The results demonstrate
that these compounds exhibited moderate to good inhibitory activ-
ities towards MAO-B than MAO-A. In agreement with the experi-
mental data, all the fluorinated methoxylated chalcones showed
productive recognition and selective inhibitory activity towards
hMAO-B in the submicromolar range. The following order of
MAO inhibitory activity was observed for the fluorinated methoxy-
lated chalcones:
2.4.1. Preparation of enzymes
Preparation Wizard of Maestro-8.4 (Schrodinger LLC) has been
used to prepare protein. Crystallographic models 2BXR (hMAO-A)
and 2BYB (hMAO-B) were downloaded from www.rcsb.org [25].
Initially the PDB files of the enzymes were refined by removing
the B-chain and keeping the cofactor FAD. Water and covalently
linked ligands were deleted and bond order was corrected for
FAD. After assigning charge and protonation state finally energy
minimization was done using OPLS2005 force field. PDB written
by Maestro for prepared proteins were rewritten by pdb format
for AutoDock compatible atom type [26].
2.4.2. Preparation ligands
Ligands were prepared through PRODRG webserver (http://
davapc1.bioch.dundee.ac.uk/cgi-bin/prodrg). The structures of the
ligands were constructed by using ChemDraw (Cambridge Soft,
Massachusetts, USA) and saved in MDL Mol format. This format
can be uploaded in the PRODRUG web server and the energy min-
imized ligand structures were saved in the pdb format for further
docking studies [27].
hMAO-A : 2-CF3 > 3-F > 2-F > 4-F > 4-F3
hMAO-B : 4-CF3 > 4-F > 2-F > 3-F > 2-CF3

26
B. Mathew et al. / Bioorganic Chemistry 62 (2015) 22–29
Table 1
Experimentally determined Ki values for the inhibition of hMAO-A and -B with synthesized fluorinated methoxylated chalcones.
Compounds
R
Ki (MAO-A) (
l
M)a
Ki (MAO-B) (
l
M)a
SIb
Inhibition type
Reversibility
Selectivity
M1
M2
M3
M4
2-F
3-F
4-F
2-CF3
4-CF3
–
2.34 0.19
1.46 0.11
2.39 0.20
0.90 0.22
4.32 0.25
8.15 1.33
0.28 0.02
0.35 0.02
0.27 0.01
0.36 0.02
0.22 0.01
0.35 0.02
1.45 0.04
0.12
0.24
0.12
0.40
0.05
0.04
140.0
Competitive
Competitive
Competitive
Competitive
Competitive
Suicide
Reversible
Reversible
Reversible
Reversible
Reversible
Irreversible
Reversible
MAO-B
MAO-B
MAO-B
MAO-B
MAO-B
MAO-B
MAO-A
M5
Selegiline
Moclobemide
–
0.010 0.002
Competitive
a
Each value represents the mean SEM of three independent experiments.
The selectivity index (SI) was calculated as Ki (MAO-B)/Ki (MAO-A).
b
All the fluorinated chalcones exhibited good activity in the submi-
cromolar range towards MAO-B, but among them the highest activ-
ity was observed for the methoxylated chalcones that were
substituted with a trifluromethyl group in the 4-position of the B
ring. Our data revealed that the newly synthesized compounds
exhibited strong and selective inhibitory activity against hMAO-B.
Among these five compounds, M5 showed the best activity and
higher selectivity towards hMAO-B with Ki and SI values of
Kinetic analyses were carried out for most potent MAO-B inhi-
bitor M5 from this series. The aim of this experiment was to find
the mode of MAO-B inhibition by fluorinated methoxylated chal-
cones. A set of Lineweaver-Bruke plots were constructed in the
absence and presence of various concentrations of compound
M5. The observation that the lines were linear and intersects on
the y-axis suggests that M5 interacts with the catalytic site of
hMAO-B, with a competitive mode of inhibition. (Fig. 3). The
replots of the slopes of the Lineweaver–Burk plots versus inhibitor
concentration is shown in Fig. 4 and the Ki was estimated as
0.22 0.01
the reference selective and irreversible MAO-B inhibitor (Ki and SI
values were found as 0.33 0.03 M and0.04, respectively). Based
lM and 0.05, respectively, surpassing that of selegiline,
l
0.22 lM for M5. In the reversibility and irreversibility tests,
on high inhibition potency and MAO-B selectivity as selection crite-
ria, M5 was considered a promising lead compound for the develop-
ment of reversible MAO-B inhibitors. Compounds M3 and M1 were
also found to be a potent MAO-B inhibitor with a Ki value of 0.27
hMAO-B inhibition was found to be reversible in presence of the
compounds as shown by the recovery enzyme activity after
repeated washing. Table 2 presents the reversibility of hMAO-B
inhibition with the chalcone compounds. Reversibility of hMAO-B
inhibition with compound M5 was found to be good in this series.
The percentage inhibition (%) of hMAO-B of compound M5
and 0.28 lM respectively. Substitution of both trifluromethyl and
fluoro group in the para position of the B ring of methoxylated chal-
cone enhances the MAO-B inhibition potencies with greater extent.
The results also demonstrate that the increasing the bulkiness with
addition of trimethyl group in the para position of ring B in
methoxylated chalcones favours the activity ratio towards MAO-B.
A logical explanation for this observation is that the higher degree
of lipophilicity of the trifluromethyl group compared with the fluo-
rine atom facilitates more productive Van der Waals interactions
with the inhibitor binding cavity of MAO-B.
(0.5 lM) was calculated as 86.90 6.33 and 11.02 0.10 before
and after washing, respectively.
Another interesting aspect is the inhibitory capacity M4
compound which showed Ki value of 0.36 and 0.90
lM towards
both MAO-B and respectively. It is to be noted that
A
dual-target-directed MAO-A and B inhibitors may have enhanced
therapeutic value, making such compounds ideal adjuvants to
L-Dopa treatment in Parkinson’s disease, particularly when
Fig. 3. Lineweaver–Burk plots of the oxidation of p-tyramine by recombinant hMAO-B. The plots were constructed in the absence and presence of various concentrations of
compound (0.10–1.50 M) M5.
l

B. Mathew et al. / Bioorganic Chemistry 62 (2015) 22–29
27
Fig. 4. Replots of the slopes of the Lineweaver–Burk plots versus inhibitor (M5) concentration.
3.3. Molecular docking studies
Table 2
Reversibility of hMAO-B inhibition the newly synthesized fluorinated methoxylated
chalcones.
Molecular docking studies were carried out to further explain
the in vitro results of the new compounds, and to identify the hypo-
thetical binding mode for the compounds inside the inhibitor bind-
ing cavity of hMAO-A and B. Molecular docking scores and the
calculated inhibition constants towards both MAO-A and MAO-B
of titled derivatives were shown in Table 3. Experimental datas
concerning the hMAO inhibitory activities of fluorinated methoxy-
lated chalcones were found to be in a good agreement with the cal-
culated data obtained from the molecular docking studies (Figs. 5
and 6). The inhibitor binding cavity of MAO-B is characterized by
two large pockets. Pocket 1 involves, a large aromatic cage made
up of FAD, Phe 343 and four tyrosine residues Tyr 188, 189, 435
and 398. Pocket 2 a hydrophobic pocket characterized by Leu
171, Tyr 326, Phe 168, Ile 198 and 199. Here smaller entrance cav-
ity’ with Ile 199 effectively serves as a gate between these cavities
[30]. Depending on the structure of the bound inhibitor the two
cavities may be either separate or fused [31].
Compound (50
lM)
hMAO-B inhibition (%)
Before washing
After repeated washing
Selegiline
M1
M2
M3
M4
47.09 3.25
82.46 6.03
80.24 5.67
83.00 6.05
77.75 5.00
86.90 6.33
46.38 3.10
14.21 1.20
15.00 1.18
13.90 1.05
17.18 1.30
11.02 0.10
M5
Each value represents the mean SEM of three experiments.
associated with depression [29]. Thus, besides deriving additional
SARs for the inhibition of the chalcone type MAOs and potentially
discovering highly potent MAO-B inhibitors, this study will also
facilitate the comparison of the MAO-B inhibition properties of
fluorine and the trifluoromethyl group in the various positions of
the B ring of chalcones.
The docking studies of M5 reveals that the p-methoxyl group in
the A ring of chalcone is positioned between the phenolic side
Table 3
Theoretically determined binding energy and Ki values of the newly synthesized fluorinated thienylchalcones for the inhibition of hMAO-A and -B.
Si
No.
Code MW
Binding energy for MAO-A
(kcal/mol)
Binding energy for MAO-B
(kcal/mol)
Calculated Ki value for MAO- Calculated Ki value for MAO- Calculated
A (l
M)
B (l
M)
SI*
1.
2.
3.
4.
5.
M1
M2
M3
M4
M5
256.27 ꢀ7.78
ꢀ8.74
ꢀ8.78
ꢀ8.81
ꢀ8.78
ꢀ8.71
2.00
1.43
2.06
0.75
2.66
0.39
0.37
0.35
0.37
0.42
0.19
0.25
0.16
0.49
0.15
256.27 ꢀ7.97
256.27 ꢀ7.76
306.28 ꢀ8.36
306.28 ꢀ7.61
*
Molecular weight.
5
4
3
2
1
0
MAO-A Calculated
MAO-A Experimental
MAO-B Calculated
MAO-B Experimental
M1
M2
M3
M4
M5
Fig. 5. Experimental and calculated Ki values corresponding to the inhibition of hMAO-A and -B with M series compounds.

28
B. Mathew et al. / Bioorganic Chemistry 62 (2015) 22–29
0.6
0.4
0.2
SI Calculated
SI Experimental
0
M1
M2
M3
M4
M5
Fig. 6. Experimental and calculated selectivity indices (calculated as Ki (MAO-B)/Ki (MAO-A) for the M series towards hMAO isoforms.
Fig. 7. Docking picture of M5 in the MAO-B active site.
Fig. 8. Docking picture of M3 in the MAO-B active site: Yellow mesh indicates p–p stacking interaction.
chains of Tyr398 and Tyr435 MAO-B and is anchoring from the re
face of FAD with a distance of 3.564 Å (Fig. 7). This interaction is
interaction of the fluorinated methoxylated chalcones showed
good proximity towards the isoalloxazine of FAD unit. The potent
inhibitor M3 also showed a similar type of interactions like M5
in the inhibitor binding cavity of MAO-B (Fig. 8). From the close
inspection of docking pose of M3, fluorine atom in the B ring of
chalcone lined with the entrance cavity of Ile199 and the methoxyl
group was positioned between the Tyr435 and Tyr 396 in the aro-
matic cage and is anchoring from the re face of FAD. M3 is mainly
mainly stabilized by the strong
p–p interaction between B ring
of the M5 and the FAD unit. The strong electron withdrawing triflu-
oromethyl group in the para position of the B ring of the methoxy-
lated chalcone significantly reduces the electron density and is
capable of forming more
p–p stacking interaction with aromatic
cages of the inhibitor binding cavity of MAO-B [32]. This stabilizing

B. Mathew et al. / Bioorganic Chemistry 62 (2015) 22–29
29
stabilized by the strong p–p interaction of FAD unit and Tyr398 of
References
B and A ring respectively. As mentioned above, the trifluromethyl
and the fluorine groups of the M5 and M3 of B ring are located
within the entrance cavity of MAO-B since it is a hydrophobic envi-
ronment [33].
[1] M. Yáñez, J.F. Padín, J.A. Arranz-Tagarro, M. Camiña, R. Laguna, Curr. Top. Med.
Chem. 12 (2012) 2275–2282.
[2] M.B.I. Youdim, Y.S. Bakhle, Br. J. Pharmacol. 147 (2006) S287–S296.
[3] L. Oreland, S.S. Jossan, P. Hartvig, S.M. Aquilonius, B. Langstrom, J. Neural.
Transm. Suppl. 32 (1990) 55–59.
[4] J. Reis, I. Encarnação, A. Gaspar, A. Morales, N. Milhazes, F. Borges, Curr. Top.
Med. Chem. 12 (2012). 2177–2118.
[5] H.H. Fernandez, J.J. Chen, Pharmacotherapy 27 (2007) 174S–185S.
[6] G. Di Carlo, N. Mascolo, A.A. Izzo, F. Capasso, Life Sci. 65 (1999) 337–353.
[7] O. Afzal, S. Bawa, S. Kumar, R. Kumar, Md.Q. Hassan, Lett. Drug Des. Discov. 10
(2013) 75–85.
[8] S. Tanaka, Y. Kuwai, M. Tabata, Planta Med. 53 (1987) 5–8.
[9] T. Hatano, T. Fukuda, T. Miyase, T. Noro, T. Okuda, Chem. Pharm. Bull. 39 (1991)
1238–1243.
[10] X. Pan, L.D. Kong, Y. Zhang, C.H. Cheng, R.X. Tan, Acta Pharmacol. Sinica 21
(2000) 949–953.
[11] H. Haraguchi, Y. Tanaka, A. Kabbash, T. Fujioka, T. Ishizu, A. Yagi,
Phytochemistry 65 (2004) 2255–2260.
[12] F. Chimenti, R. Fioravanti, A. Bolasco, P. Chimenti, D. Secci, F. Rossi, M. Yanez, F.
Orallo, F. Ortuso, S. Alcaro, J. Med. Chem. 52 (2009) 2818–2824.
[13] G. Jo, S. Ahn, B.G. Kim, H.R. Park, Y.H. Kim, A.H. Cho, D. Koh, Y. Chong, J.H. Ahn,
Y. Lim, Biorg. Med. Chem. 21 (2013) 7890–7897.
The presence trifluromethyl and the fluorine groups in the B
ring of chalcones are stabilized to a large degree by Van der
Waals interactions within the entrance cavity of Ile199.
Therefore, substitution with halogens on the chalcone system is
expected to enhance the productive interactions of the inhibitor
with the entrance cavity of MAO-B via hydrophobic burial and
dipole interactions. The enhancement of MAO-B inhibition poten-
cies by halogen substitution has been previously described. For
example, chlorine and bromine substitution on the benzyloxy phe-
nyl ring of a series of 8-benzyloxycaffeines enhances the MAO-B
inhibition potencies 20 and 22-fold, respectively [34].
4. Conclusion
[14] S.J. Robinson, J.P. Petzer, A. Petzer, J.J. Bergh, A.C.U. Lourens, Bioorg. Med.
Chem. Lett. 23 (2013) 4985–4989.
[15] B. Evranos-Aksöz, S. Yabanog˘lu-Çiftçi, G. Uçar, K. Yelekçi, R. Ertan, Bioorg. Med.
Chem. Lett. 24 (2014) 3278–3284.
[16] N. Morales-Camilo, C.O. Salas, C. Sanhueza, C. Espinosa-Bustos, S. Sepúlveda-
Boza, M. Reyes-Parada, F. Gonzalez-Nilo, M. Caroli-Rezende, A. Fierro, Chem.
Biol. Drug Des. 85 (2015) 685–695.
[17] A.M. Helguera, G. Pérez-Machado, M.N.D.S. Cordeiro, F. Borges, Mini Rev. Med.
Chem. 12 (2012) 907–919.
[18] B.V. Nayak, S. Ciftci-Yabanoglu, S. Bhakat, A.K. Timiri, B.N. Sinha, G. Ucar, M.E.S.
Soliman, V. Jayaprakash, Bioorg. Chem. 58 (2015) 72–80.
[19] M.C. Anderson, F. Hasan, J.M. McCrodden, K.F. Tipton, Neurochem. Res. 18
(1993) 1145–1149.
[20] F. Chimenti, E. Maccioni, D. Secci, A. Bolasco, P. Chimenti, A. Granese, S.
Carradori, S. Alcaro, F. Ortuso, M. Yáñez, J. Med. Chem. 51 (2008) 4874–4880.
[21] M. Yáñez, N. Fraiz, E. Cano, F. Orallo, Biochem. Biophys. Res. Commun. 344 (2)
(2006) 688–695.
[22] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254.
[23] F. Chimenti, S. Carradori, D. Secci, A. Bolasco, B. Bizzarri, P. Chimenti, A.
Granese, M. Yáñez, F. Orallo, Eur. J. Med. Chem. 45 (2010) 800–804.
[24] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J.
Olson, J. Comput. Chem. 30 (2009) 2785–2791.
The study mainly highlights the effect and orientation of fluo-
rine and the trifluromethyl group in the B ring of a methoxylated
chalcones towards MAO inhibition. The results document that all
the chalcone derived compounds showed potent MAO-B inhibitory
activity in the submicromolar range. Compound M5 is not simply a
high potent MAO-B inhibitor, but also revealed relatively good
selectivity for MAO-B over the MAO-A isoform. The study revealed
the importance of lipophilic halogen groups in the para position of
the B ring of chalcone compounds and is promising for the devel-
opment of new selective and reversible type of MAO-B inhibitors. It
may thus be concluded that M5 is a promising lead for the devel-
opment of reversible and selective MAO-B inhibitors, which can be
used in the treatment of Parkinson’s disease. To our best knowl-
edge, this is the first report on the MAO inhibition activity of fluo-
rinated methoxylated chalcones. Further structural optimization
and the modification of this scaffold are well under way in our
laboratory.
[25] L. De Colibus, M. Li, C. Binda, A. Lustig, D.E. Edmondson, A. Mattevi, Proc. Natl.
Acad. Sci. USA 102 (36) (2005) 12684–12689.
[26] B.V. Nayak, S. Ciftci-Yabanoglu, S.S. Jadav, M. Jagrat, B.N. Sinha, G. Ucar, V.
Jayaprakash, Eur. J. Med. Chem. 69 (2013) 762–767.
[27] A.W. Schuttelkopf, D.M. van Aalten, Acta Crystallogr. D Biol. Crystallogr. 60
(2004) 1355–1363.
Acknowledgment
[28] B. Mathew, J. Suresh, S. Anbazhagan, S. Dev, Biomed. Aging Pathol. 4 (2014)
297–301.
[29] L.J. Legoabe, A. Petzer, J.P. Petzer, Chem. Biol. Drug Des. (2015), http://
dx.doi.org/10.1111/cbdd.12508.
[30] C. Binda, M. Li, F. Hubalek, N. Restelli, D.E. Edmondson, Proc. Natl. Acad. Sci.
USA 100 (2003) 9750–9755.
[31] L. Legoabe, J. Kruger, A. Petzer, J.J. Bergh, J.P. Petzer, Eur. J. Med. Chem. 46
(2011) 5162–5174.
The authors extend their appreciation to Kerala State Council
for Science, Technology & Environment, Kerala, India (application
no: 01/SPS-54/2015/CSTE) for the financial support. The authors
also thankful to IIRBS, Mahatma Gandhi University, Kottayam,
India for carrying out the spectral analysis.
[32] D.E. Edmondson, A. Mattevi, C. Binda, M. Li, F. Hubalek, Curr. Med. Chem. 11
(2004) 1983–1993.
[33] L. Novaroli, A. Daina, E. Favre, J. Bravo, A. Carotti, F. Leonetti, M. Catto, P.A.
Carrupt, M. Reist, J. Med. Chem. 49 (2006) 6264–6272.
[34] B. Strydom, S.F. Malan Jr., N. Castagnoli, J.J. Bergh, J.P. Petzer, Bioorg. Med.
Chem. 18 (2010) 1018–1028.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2015.07.
001.