Synthesis, Biochemistry, and Computational Studies of Brominated Thienyl Chalcones: A New Class of Reversible MAO-B Inhibitors

Article abstract of DOI:10.1002/cmdc.201600122

A series of (2E)-1-(5-bromothiophen-2-yl)-3-(para-substituted phenyl)prop-2-en-1-ones (TB1–TB11) was synthesized and tested for inhibitory activity toward human monoamine oxidase (hMAO). All compounds were found to be competitive, selective, and reversibl

Full text of DOI:10.1002/cmdc.201600122

DOI: 10.1002/cmdc.201600122
Full Papers
Synthesis, Biochemistry, and Computational Studies of
Brominated Thienyl Chalcones: A New Class of Reversible
MAO-B Inhibitors
Bijo Mathew+,*[a] Abitha Haridas,[b] Gülberk UÅar+,*[c] Ipek Baysal,[c] Monu Joy,[d]
Githa E. Mathew,[e] Baskar Lakshmanan,[b] and Venkatesan Jayaprakash[f]
A series of (2E)-1-(5-bromothiophen-2-yl)-3-(para-substituted
phenyl)prop-2-en-1-ones (TB1–TB11) was synthesized and
tested for inhibitory activity toward human monoamine ox-
idase (hMAO). All compounds were found to be competitive,
selective, and reversible toward hMAO-B except (2E)-1-(5-bro-
mothiophen-2-yl)-3-(4-nitrophenyl)prop-2-en-1-one (TB7) and
(2E)-1-(5-bromothiophen-2-yl)-3-(4-chlorophenyl)prop-2-en-1-
one (TB8), which were selective inhibitors of hMAO-A. The
most potent compound, (2E)-1-(5-bromothiophen-2-yl)-3-[4-(di-
methylamino)phenyl]prop-2-en-1-one (TB5), showed the best
inhibitory activity and higher selectivity toward hMAO-B, with
Ki and SI values of 0.11Æ0.01 mm and 13.18, respectively.
PAMPA assays for all compounds were carried out in order to
evaluate the capacity of the compounds to cross the blood–
brain barrier. Moreover, the most potent MAO-B inhibitor, TB5,
was found to be nontoxic at 5 and 25 mm, with 95.75 and
84.59% viability among cells, respectively. Molecular docking
simulations were carried out to understand the crucial interac-
tions responsible for selectivity and potency.
Introduction
Monoamine oxidase A and B (MAO-A and MAO-B) are the two
isoenzymes responsible for catalyzing oxidative deamination of
neurotransmitters and dietary amines.[1] Increased MAO-A and
B activity is associated with decreased quantities of endoge-
nous and exogenous monoamines.[2] Inhibition of these en-
zymes has been proposed for the treatment of various patho-
geneses in the central nervous system (CNS) precipitated by in-
creased metabolism of biogenic amines.[3] Administration of
MAO-A inhibitors results in benefits for the treatment of anxi-
ety and depression.[4] MAO-B inhibitors are frequently used in
combination with levodopa (l-Dopa) and may safeguard the
depleted supply of dopamine in the Parkinsonian brain and
prolong the activity of dopamine derived from its metabolic
precursor, l-Dopa.[5,6] This combination therapy allows for the
effective l-Dopa dose to be decreased.[7] It should be noted
that activity and density of MAO-B in the CNS increase with
age, while MAO-A activity remains unchanged. The inhibition
of MAO-B-catalyzed oxidation of dopamine is therefore of en-
hanced relevance in the aged Parkinsonian brain.[8]
Depending on their interactions with MAO isoforms, bound
inhibitors are categorized as reversible or irreversible. Many
older generations of MAO inhibitors are nonselective in nature
but are commonly recognized as antidepressant agents.[9]
However, this type of inhibition has been shown to induce sig-
nificant toxic effects, mainly provoked by inhibition of the pe-
ripheral MAOs located in the gut, liver, and endothelium. Irre-
versible inhibition of intestinal MAO-A permits the production
of excessive amounts of tyramine in the systemic circulation,
which can induce the release of norepinephrine from peripher-
al neurons. This state leads to a potentially fatal increase in
blood pressure (cheese effect).[10] Currently used hMAO-B in-
hibitors include selective and irreversible types. These mole-
cules display the typical drawbacks of long-lasting enzyme in-
hibition, target disruption, poor pharmacokinetic parameters,
and potential immunogenicity of enzyme–inhibitor adducts.
However, a recently developed and novel class of reversible in-
hibitors has safer profiles.[11] As the general body of informa-
tion grows, it is prudent to continue to design new selective
and reversible MAO-B inhibitors to be used as various neuro-
degenerative diseases with higher specificities and fewer dele-
terious side effects.
[a] B. Mathew+
Division of Drug Design and Medicinal Chemistry Research Laboratory,
Department of Pharmaceutical Chemistry, Ahalia School of Pharmacy,
Palakkad 678557, Kerala (India)
E-mail: bijovilaventgu@gmail.com
[b] A. Haridas, Dr. B. Lakshmanan
Department of Pharmaceutical Chemistry,
Grace College of Pharmacy, Palakkad 678004, Kerala (India)
[c] Prof. Dr. G. UÅar,+ I. Baysal
Department of Biochemistry, Faculty of Pharmacy,
Hacettepe University, 06100 Sıhhiye, Ankara (Turkey)
E-mail: gulberk@hacettepe.edu.tr
[d] M. Joy
School of Pure & Applied Physics,
Mahatma Gandhi University, Kottayam 686560, (India)
[e] G. E. Mathew
Department of Pharmacology,
Grace College of Pharmacy, Palakkad 678004, Kerala (India)
[f] Dr. V. Jayaprakash
Department of Pharmaceutical Sciences and Technology,
Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand (India)
[+] These authors contributed equally to this work.
ChemMedChem 2016, 11, 1161 – 1171
1161
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Chalcones (1,3-diaryl-2-propen-1-ones) and their heterocyclic
analogues are considered to be open chain flavonoids, which
display a number of interesting biological properties. Chal-
cones may exist in two geometric isomeric forms, cis and trans,
with the trans isomer being the most thermodynamically
stable.[12–16] As chalcones are relatively easy to prepare, large
numbers of derivatives can be synthesized by substituting
with various aromatic and heteroaromatic nuclei. In fact, chal-
cones have been used as promising intermediates in the syn-
thesis of pyrazoline derivatives as part of an effort to discover
novel MAO inhibitors.[17,18] The MAO inhibitory activities of
chalcones reported by different groups revealed that these
compounds show a higher degree of MAO-B inhibition than
MAO-A inhibition. Most studies show that the presence of lipo-
philic electron-withdrawing groups such as chlorine, fluorine,
and trifluoromethyl groups at the para position on ring B of
chalcones increases MAO-B inhibitory activity. These groups
may increase the lipophilic character of chalcones, which
favors interaction with the entrance cavity of the MAO-B active
site.[19–27]
eight protons comprising signals from the a,b unsaturated
unit, the bromine-substituted thiophene, and the mono-substi-
tuted phenyl system. It has been noted that the upfield proton
of the a-carbon (a-C) and the downfield proton of the b-
carbon (b-C) coupled with the methine proton with a coupling
constant of 16.0 Hz. This large coupling constant value con-
firms the presence of trans configuration in the brominated
thienyl chalcones. Protons belonging to the to the aromatic
ring and thiophene ring were observed, with expected chemi-
cal shifts and integral values. 13C NMR spectra displayed car-
bonyl carbons for TB1–TB11 between d 180.0–181.0. The a-C
and b-C of chalcones were assigned as 119.5–121.4 and 144.4–
147.0, respectively. Characteristic peaks were observed in the
mass spectra of the synthesized compounds. The presence of
a bromine atom in all compounds showed characteristic [M+
2] isotope peaks. All compounds showed satisfactory results by
elemental analysis. The structure of TB5 was solved using
single crystal X-ray diffractometry (Figure 1).
Recently, our research group has reported a number of
potent, selective, and reversible inhibitors of hMAO-B, includ-
ing methoxylated and thiophene-based chalcones with fluoro
and trifluoromethyl substitutions. Among these, (2E)-1-(4-me-
thoxyphenyl)-3-[4-(trifluoromethyl)phenyl]prop-2-en-1-one and
(2E)-1-(thiophen-2-yl)-3-[4-(trifluoromethyl)phenyl]prop-2-en-1-
one exhibited Ki values of 0.22Æ0.01 and 0.90Æ0.05 mm, re-
spectively, toward hMAO-B. The study mainly highlighted the
effect of orientation of the fluorine and trifluoromethyl groups
on ring B of chalcone on hMAO inhibition.[28,29] Based on the
above consideration of the importance of selective and reversi-
ble MAO-B inhibitors, this attempt mainly focused on bromo-
substituted thienyl chalcones with a variety of electron-donat-
ing and -withdrawing groups at the para position on phenyl
ring B. Information regarding the effect of various groups at
the para position on ring B of brominated thienyl chalcones
and their effect on MAO inhibition has not been explored so
far.
Figure 1. ORTEP diagram of lead molecule TB5.
Pharmacological evaluation
MAO inhibition studies
Newly synthesized brominated thiophene chalcones were
screened for their inhibitory activities toward human MAO iso-
forms using recombinant enzymes. Enzymatic activities were
determined according to a previous method using the Amplex
Red MAO assay kit.[40–42] Compounds and reference inhibitors
(moclobemide, selegiline, and lazabemide) were independently
treated with Amplex Red reagent before the experiment. None
of the compounds interfered with the measurements. Test
compounds also did not interact with resorufin, as the fluores-
cence signal did not change when the test and reference com-
pounds were treated with various concentrations of resorufin.
Specific enzymatic activities were calculated as 155.12Æ
9.41 pmolmgÀ1 minÀ1 (n=3) for hMAO-A and 136.90Æ
8.85 pmolmgÀ1 minÀ1 (n=3) for hMAO-B. The experimental re-
sults are shown in Table 1. Selectivity indexes (SI) were ex-
pressed as Ki(MAO-A)/Ki(MAO-B). Selectivity toward MAO-A in-
creases as the corresponding SI decreases, while selectivity
toward MAO-B increases as the corresponding SI increases.
With the exception of TB7 and TB8, which were selective
MAO-A inhibitors, all other derivatives inhibited hMAO-B po-
tently and selectively with a competitive mode of inhibition.
Some structure–activity relationships (SARs) were inferred from
the data from enzymatic experiments. All synthesized com-
pounds contained a para-substituted phenyl moiety attached
Results and Discussion
Chemistry
Brominated thienyl chalcone derivatives (TB1–TB11) were effi-
ciently synthesized according to the pathway shown in
Scheme 1. The chemical structures of the compounds were
characterized by means of their 1H NMR, 13C NMR, and mass
1
spectroscopic (MS) data. H NMR spectra showed peaks from
Scheme 1. Synthesis of brominated thienyl chalcones. Reagents and condi-
tions: a) C2H5OH, 40% KOH, 4 h, room temperature.
ChemMedChem 2016, 11, 1161 – 1171
www.chemmedchem.org
1162
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Table 1. hMAO inhibitory activities of brominated thiophene-based chalcones.
Compound
R
Ki [mm][a]
SI[b]
Inhibition type
Reversibility
MAO selectivity
MAO-A
MAO-B
TB1
TB2
TB3
TB4
TB5
TB6
TB7
TB8
TB9
TB10
TB11
moclobemide
selegiline
lazabemide
H
4.55Æ0.27
2.85Æ0.11
1.40Æ0.10
2.54Æ0.17
1.45Æ0.09
1.25Æ0.09
0.54Æ0.03
0.50Æ0.03
1.05Æ0.09
4.06Æ0.19
3.85Æ0.20
0.15Æ0.01
6.55Æ0.40
547.22Æ21.30
1.22Æ0.10
1.02Æ0.09
0.55Æ0.11
0.31Æ0.01
0.11Æ0.01
0.19Æ0.01
3.05Æ0.15
9.30Æ0.62
0.25Æ0.01
1.71Æ0.12
0.96Æ0.07
1.75Æ0.14
0.14Æ0.01
0.007Æ0.001
3.73
2.79
2.54
8.20
13.18
6.57
0.18
0.05
4.20
2.37
4.01
0.09
46.79
78.17
competitive
competitive
competitive
competitive
competitive
competitive
competitive
competitive
competitive
competitive
competitive
competitive
suicide
reversible
reversible
reversible
reversible
reversible
reversible
reversible
reversible
reversible
reversible
reversible
reversible
irreversible
reversible
MAO-B
MAO-B
MAO-B
MAO-B
MAO-B
MAO-B
MAO-A
MAO-A
MAO-B
MAO-B
MAO-B
MAO-A
MAO-B
MAO-B
OH
OCH3
CH3
N(CH3)2
CH2CH3
NO2
Cl
Br
F
CF3
–
–
–
competitive
[a] Values are the meanÆSEM of three independent experiments. [b] Selectivity index, calculated as Ki(MAO-A)/Ki(MAO-B); selectivity toward MAO-A in-
creases as the corresponding SI decreases, while selectivity toward MAO-B increases as the corresponding SI increases.
to the 5-bromo thiophene by an enone linker. Among the syn-
thesized derivatives, nine compounds showed good inhibition
of MAO-B in the sub-micromolar range. The most potent com-
pound, TB5, with a dimethylamino group at the para position
of the phenyl system showed the best activity and higher se-
lectivity toward hMAO-B, with Ki and SI values of 0.11Æ
0.01 mm and 13.18, respectively. Its inhibitory potency was
found to be higher than that of selegiline but less than that of
lazabemide. According to the selectivity index, compound TB5
also appeared to be the most selective hMAO-B inhibitor in
this series. However, the selectivity of TB5 toward hMAO-B was
less than that of lazabemide and selegiline (SI values for selegi-
line and lazabemide were calculated as 46.79 and 78.17, re-
spectively). Among the synthesized brominated thienyl chal-
cones, the presence of electron-donating groups such as dime-
thylamino, ethyl, methyl, and methoxy groups at the para posi-
tion on the phenyl ring were found to be selective toward
MAO-B. The presence of a halogen, such as a chlorine or bro-
mine atom, on the phenyl ring showed mixed results for MAO
inhibition. Introduction of bromine at the para position on the
phenyl ring was more beneficial than a fluorine atom for MAO-
B inhibition. Chlorine substitution on the phenyl ring showed
potent and selective inhibition toward MAO-A, with a Ki value
of 0.50Æ0.03 mm (TB8). The following order of MAO inhibitory
activity was observed for the brominated thienyl chalcones:
ence of a lipophilic electron-donating dimethyl group on the
phenyl system. Introduction of an electron-withdrawing group,
such as a chlorine or nitro group, on the phenyl system dra-
matically abated anti-MAO-B activity and selectivity but im-
proved the anti-MAO-A activity. Using the high inhibition po-
tency and MAO-B selectivity from enzymatic studies as selec-
tion criteria, TB5 was considered a promising lead compound
for the development and optimization of selective MAO-B in-
hibitors for various neurodegenerative diseases.
Kinetics studies
Kinetic analyses were carried out for the most potent MAO-B
inhibitor (TB5) and MAO-A inhibitor (TB8) from this series to
gain further insight into the mode of MAO inhibition. A set of
Lineweaver–Burk plots were constructed in the absence and
presence of various concentrations of compounds TB5 and
TC8. The set consisted of five graphs, each constructed by
measuring MAO-B and MAO-A catalytic rates at different sub-
strate concentrations (0.1–1 mm). The first Lineweaver–Burk
plot was constructed in the absence of inhibitor, while the re-
maining four graphs were constructed in the presence of dif-
ferent concentrations of TB5 and TB8. The observation that
the lines were linear and intersected the y-axis suggested that
TB5 and TB8 interacts with the catalytic site of hMAO-B and
hMAO-A with a competitive mode of inhibition (Figures 2 and
3). Replots of the slopes of the Lineweaver–Burk plots versus
inhibitor concentrations are shown in Figures 4 and 5, and the
Ki values were estimated as 0.11 and 0.50 mm for TC5 (hMAO-B)
and TB8 (hMAO-A), respectively.
hMAO-A:
4-Cl>4-NO2 >4-Br>4-C2H5 >4-OCH3 >4-N-(CH3)2 >4-
CH3 >4OH>4-CF3 >4-F>4-H
hMAO-B:
4-N-(CH3)2 >4-C2H5 >4-Br>4-CH3 >4-OCH3 >4-CF3 >4-
OH>4-H>4-F>4-NO2 >4-Cl
As a MAO-B inhibitor, compound TB5 was 11-fold more
potent than parent compound TB1. This clearly suggests that
the enhanced anti-MAO-B activity of TB5 is due to the pres-
ChemMedChem 2016, 11, 1161 – 1171
www.chemmedchem.org
1163
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Figure 2. Lineweaver–Burk plots of hMAO-B activity in the absence and presence of various concentrations of compound TB5.
Figure 3. Lineweaver–Burk plots of hMAO-A activity in the absence and presence of various concentrations of compound TB8.
Figure 4. Replots of the slopes of the Lineweaver–Burk plots versus inhibitor TB5 concentration (hMAO-B).
the management of Parkinson’s disease.[30] All tested com-
Reversibility studies
pounds in the present study inhibited the hMAO isoforms re-
versibly. Table 2 presents the reversibility of hMAO-B inhibition
for the novel compounds. The reversibility of MAO inhibition
by brominated thiophene chalcones was investigated by deter-
mination of the recovery of MAO activity after dialysis of
enzyme–inhibitor mixtures. MAO isoforms were incubated in
the presence of the inhibitors at concentrations fivefold higher
Competitive reversible inhibitors have less influence in the
MAO enzyme recovery after withdrawal, as the ingested tyra-
mine is able to displace the inhibitor from the MAO active site
and is metabolized in the normal way by the peripheral
enzyme in the gut and liver. Moreover, reversible MAO-B inhib-
itors have significant advantages over irreversible inhibitors for
ChemMedChem 2016, 11, 1161 – 1171
www.chemmedchem.org
1164
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Figure 5. Replots of the slopes of the Lineweaver–Burk plots versus inhibitor TB8 concentration (hMAO-A).
allel, with the two best-described barriers being the
vascular BBB, consisting primarily of the capillary bed,
and the blood–cerebrospinal fluid (blood–CSF) barri-
er, consisting primarily of the choroid plexus.[33] The
BBB is nonselective toward drug uptake by diffusion
or active transport and creates major hurdles for suc-
cessful CNS drug development. Molecules like glu-
cose and fat-/lipid-soluble drugs can rapidly cross
into the brain, while delivery of many drugs into the
brain is difficult because of their poorly fat-soluble
nature.[34]
Table 2. Reversibility of hMAO activity by brominated thienyl chalcones.
Compound
hMAO-A activity [%][a]
hMAO-B activity [%][a]
Reversibility
–
before
after
before
after
washing
washing
washing
washing
no inhibitor
100Æ0.00
100Æ0.00
100Æ0.00
100Æ0.00
moclobemide 38.02Æ2.00 88.00Æ4.96 99.50Æ5.22 95.01Æ4.55 reversible
selegiline
lazabemide
TB1
TB2
TB3
TB4
TB5
TB6
TB7
TB8
TB9
TB10
TB11
90.16Æ4.82 88.33Æ4.30 50.99Æ2.05 53.44Æ2.75 irreversible
98.00Æ3.90 99.00Æ4.05 20.55Æ1.96 88.22Æ4.09 reversible
80.22Æ4.96 89.87Æ3.00 59.00Æ2.26 94.22Æ5.00 reversible
77.60Æ3.46 89.29Æ5.74 58.25Æ2.19 95.00Æ5.36 reversible
79.90Æ3.11 88.25Æ3.96 61.22Æ3.45 86.27Æ4.00 reversible
80.33Æ4.12 90.01Æ4.60 48.00Æ2.66 89.90Æ4.77 reversible
97.31Æ3.80 88.26Æ5.31 37.23Æ1.74 97.57Æ4.61 reversible
81.22Æ3.60 91.99Æ4.11 57.00Æ2.05 96.25Æ2.00 reversible
67.20Æ2.90 87.90Æ4.00 81.29Æ3.64 95.00Æ3.87 reversible
40.22Æ2.47 97.85Æ4.00 87.25Æ2.90 93.55Æ4.22 reversible
80.22Æ4.13 91.00Æ4.68 54.33Æ2.90 92.66Æ4.10 reversible
81.96Æ4.21 92.00Æ3.77 69.16Æ3.22 93.56Æ4.32 reversible
85.63Æ3.75 97.22Æ230 66.55Æ3.54 94.10Æ4.36 reversible
To evaluate the potential for the newly synthesized
chalcone derivatives to cross the BBB, a parallel artifi-
cial membrane permeation assay (PAMPA) for the
BBB was performed. Assay validation was performed
by comparing experimental permeabilities of nine
commercial drugs with reported values (Table 3). A
plot of experimental data versus literature values
a
good linear correlation of R2 =0.9934
[a] Values are the meanÆSEM of three independent experiments.
gave
(Figure 6). According to the limits established by Di
et al. for BBB permeation, test compounds were clas-
sified as follows:[35]
than the IC50 values for a period of 15 min and subsequently
dialyzed for 24 h. hMAO-B inhibition by compound TB5 and
hMAO-A inhibition by compound TB8 were completely re-
versed after 24 h of dialysis.
CNS+ (high predicted BBB permeation): Pe (10À6 cmsÀ1) >4.00
CNSÀ (low predicted BBB permeation): Pe (10À6 cmsÀ1) <2.00
CNSÆ (uncertain BBB permeation): Pe (10À6 cmsÀ1) from 4.00 to
Results suggested that brominated thienyl chalcones are re-
versible inhibitors of hMAO isoforms and have considerable
advantages over irreversible inhibitors, which may possess seri-
ous pharmacological side effects. The potent and selective
MAO-B inhibitor selegiline has been reported to have severe
side effects due to its amphetamine metabolites, and both se-
legiline and rasagiline are irreversible MAO-B inhibitors, trigger-
ing pharmacological side effects in long-term treatment of Par-
kinson’s disease.[31,32] Compound TB5 exhibited good reversibil-
ity of hMAO-B inhibition in the present series. The reversibility
values for compound TB5 was calculated as 37.23Æ1.74 and
97.57Æ4.61 before and after dialysis, respectively.
2.00.
In vitro blood–brain barrier (BBB) permeation assay
The blood–brain barrier (BBB) has great value for the delivery
of drugs to the CNS. The BBB consists of several barriers in par-
Figure 6. Linear correlation between experimental and reported permeabili-
ty of commercial drugs using the PAMPA BBB assay.
ChemMedChem 2016, 11, 1161 – 1171
www.chemmedchem.org
1165
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Table 3. Experimental permeability in the PAMPA BBB assay of brominat-
Table 4. In vitro cytotoxicity of brominated thiophene chalcones in
ed thiophene chalcones and commercial drugs used for assay validation.
HepG2 cells.
Compound[a]
Literature[b]
Pe [10À6 cmsÀ1
Experimental
Prediction
Compound
Viability [%]
[c]
[c]
]
Pe [10À6 cmsÀ1
]
1 mm
5 mm
25 mm
testosterone
verapamil
b-estradiol
progesterone
corticosterone
piroxicam
hydrocortisone
lomefloxacin
dopamine
TB1
TB2
TB3
TB4
TB5
TB6
TB7
TB8
TB9
17.0
16.0
12.0
9.3
5.1
2.5
1.8
1.1
0.2
–
–
–
–
–
–
–
–
–
16.72Æ1.52
14.97Æ1.65
10.72Æ1.69
7.88Æ0.33
4.08Æ1.03
2.23Æ0.53
1.53Æ0.48
1.09Æ0.23
0.36Æ0.14
6.45Æ0.96
6.67Æ2.06
8.41Æ2.25
8.22Æ1.79
15.08Æ1.60
9.72Æ1.58
5.72Æ1.23
10.25Æ1.67
9.96Æ2.68
8.02Æ1.02
5.19Æ0.92
CNS+
CNS+
CNS+
CNS+
CNS+
CNSÆ
CNSÀ
CNSÀ
CNSÀ
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
moclobemide
selegiline
lazabemide
TB1
TB2
TB3
TB4
TB5
TB6
TB7
95.70Æ4.26
95.88Æ2.05
96.00Æ3.85
97.21Æ3.05
84.51Æ2.75[a]
85.62Æ0.87[a]
96.01Æ2.31
97.50Æ2.02
94.21Æ2.59
96.34Æ4.10
97.00Æ1.45
95.54Æ3.36
97.67Æ1.45
98.01Æ1.53
77.22Æ2.90[a]
89.78Æ2.60[a]
90.25Æ4.12[a]
92.00Æ4.12[a]
60.70Æ2.16[b]
60.86Æ2.07[b]
75.79Æ2.23[b]
95.75Æ3.56
70.97Æ2.11[b]
91.17Æ2.24
94.82Æ2.21
93.29Æ1.64
93.46Æ3.55
92.43Æ1.44[a]
56.20Æ2.34[b]
60.05Æ2.05[b]
58.00Æ2.91[b]
42.69Æ1.18[c]
50.16Æ2.45[b]
57.67Æ2.18[b]
66.99Æ1.53[b]
84.59Æ2.54[a]
55.83Æ2.21[b]
76.81Æ1.59[b]
44.43Æ2.24[b]
92.86Æ1.49[a]
59.98Æ1.17[b]
83.30Æ1.65[a]
TB8
TB9
TB10
TB11
Data are the meanÆSEM of n=3 experiments. Cell viability is expressed
as a percentage of the control value; p<0.05 was considered statistically
significant ([a] p<0.05; [b] p<0.01; [c] p<0.001 vs. control).
TB10
TB11
–
–
Molecular docking studies
[a] Compounds were dissolved in DMSO (5 mgmLÀ1) and diluted with
PBS/EtOH (70:30); final compound concentration: 100 mgmLÀ1. [b] Taken
from ref. [35]. [c] Values are expressed as the meanÆSD of three inde-
pendent experiments; –: no published details available.
Binding interaction analysis of MAO-B inhibitor TB5
To carry out a detailed study of the hypothetical MAO-B bind-
ing modes of the brominated thienyl chalcones, molecular
docking studies were performed. The active site of MAO-B is
characterized by two large pockets. Pocket 1 involves a large
aromatic cage made up of FAD, Phe343, and four tyrosine resi-
dues: Tyr188, 189, 435, and 398. Pocket 2 is a hydrophobic
pocket characterized by Leu171, Tyr326, Phe168, Ile198, and
Ile199. The smaller entrance cavity with Ile199 effectively
serves as a gate between these cavities.[36] Depending on the
structural nature of the bound inhibitor, the two cavities may
be either separate or fused.[37]
The data listed in Table 3 indicated that all of the brominat-
ed thiophene chalcones could cross the BBB to target the
enzyme in the CNS. Compounds TB5, TB8, TB9, and TB6
showed the highest permeability, suggesting that they may be
able to cross the BBB and reach biological targets located in
the CNS, consistent with our design strategy. These results also
encouraged us to carry out more detailed studies with these
new prodrugs.
The docking poses of MAO-B inhibitors suggest that the ter-
minal lipophilic groups such as dimethylamino, ethyl, bromo,
methyl, and methoxy at the para position on the chalcone
phenyl rings project into the entrance cavity of MAO-B, where
van der Waals interactions are predominant. The presence of
a bulky lipophilic dimethylamino group at the para position on
ring B of brominated thienyl chalcone TB5 can efficiently navi-
gate Ile199 and span both entrance and substrate cavities.
Docking studies of lead compound TB5 led to the proposed
binding mode in which a 5-bromo-substituted thiophene
binds pocket 1 and is anchored in the substrate cavity close to
the FAD cofactor, with a lipophilic dimethylamino positioned
in the entrance cavity of MAO-B. The interaction is stabilized
by the strong p–p stacking interaction between the phenyl
system of the chalcone and the FAD unit (Figure 7). The rotata-
ble bonds present in the compound result in a non-coplanar
confirmation between the phenyl system and the halogenated
thiophene and can make tight interactions in the inhibitor
binding cavity of MAO-B without any hydrogen bonding inter-
actions. These data provide evidence that MAO-B inhibitory
potency results from having a structure that binds to both the
entrance and substrate cavities.
Cytotoxicity studies
In vitro cytotoxicity of brominated thiophene chalcones and
standard MAO inhibitors were tested in human HepG2 hepatic
cancer cells at three different concentrations (1–25 mm)
(Table 4). The results showed that most of the novel com-
pounds were not toxic to hepatic cells at 1 mm concentration.
At 5 mm, compounds TB5, TB7, TB8, TB9, and TB10 were com-
pletely nontoxic to the cells. The most potent MAO-B inhibitor
of the present series, TB5, was nontoxic at 5 and 25 mm, result-
ing in cell viabilities of 95.75% and 84.59%, respectively. It was
noted that the presence of a nitrogen substituent, like a dime-
thylamino or nitro group, was responsible for the lower de-
grees of cytotoxicity of TB5 and TB7 at 25 mm, with 84.59%
and 76.81% cell viability, respectively. The presence of a dibro-
mo atom, both on thiophene and the phenyl ring of com-
pound TB9 showed the lowest cytotoxicity at 25 mm, with
92.86% cell viability in this series. Interestingly, the toxicity for
unsubstituted compound TB1 was greater than that of other
substituted heteroaryl chalcones at 25 mm in HepG2 cells.
ChemMedChem 2016, 11, 1161 – 1171
www.chemmedchem.org
1166
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
study documented that substitution at the para position on
the phenyl ring with electron-donating groups yields struc-
tures with potent MAO-B inhibitory activities. This behavior
was exemplified by TB5 (Ki =0.11 mm), TB6 (Ki =0.19 mm), TB4
(Ki =0.31 mm), and TB3 (Ki =0.55 mm), which possess dimethyla-
mino, ethyl, methyl, and methoxy groups, respectively, at the
para position of phenyl ring. PAMPA assays of all compounds
experimentally corroborated the results from MAO inhibition
studies. Previously, we reported fluorochalcones from 2-acetyl
thiophene, observing that the position of fluorine and trifluor-
omethyl groups (ortho, meta, para) on ring B had a negligible
impact on inhibition of both hMAO-A and hMAO-B. The most
potent hMAO-B inhibitor in this series, compound (2E)-1-(thio-
phen-2-yl)-3-[4-(trifluoromethyl)phenyl]prop-2-en-1-one, inhib-
ited the hMAO-B isoform potently and selectively, with a Ki
value of 0.90Æ0.05 mm. This compound has a trifluoromethyl
substituent at the para position of the B ring and is eightfold
less potent than TB5. From the above observation, we learned
that a change in hMAO-B inhibitory activity is due to the
nature and position of groups on phenyl ring B and is also
greatly influenced by the nature and position of substituents
on thiophene ring A. The SAR comparison is outlined in
Figure 9.
Figure 7. Docking pose of TB5 in the MAO-B active site. Yellow mesh indi-
cates p–p stacking interaction.
Binding interaction analysis of MAO-A inhibitor TB8
The inhibitor binding site of MAO-A is a single cavity that ex-
tends from the flavin ring to the cavity-shaping loop.[38] TB8
oriented the active site of MAO-A in such a way that the a,b-
unsaturated carbonyl linker oriented toward pocket 1 of the ar-
omatic cage lined by (FAD, Tyr407, and Tyr444). This places
the 5-bromo-substituted thiophene ring in pocket 2 (defined
by Ile180, Ile335, Leu337, and Phe352) and the para-chloro
phenyl ring in pocket 3 (defined by Gly74, Ile207, Phe208,
Glu216, and Trp441). TB8 is mainly stabilized by the phenyl
B ring and bromo-substituted thiophene A ring, making
a strong p–p stacking interaction with Phe352 in pocket 2 of
MAO-B (Figure 8). This stabilizing interaction results in the
enone linker of thienyl chalcone being within good proximity
(2.922 Š) of the isoalloxazine of the FAD unit. This shorter dis-
tance between the electron-rich carbonyl oxygen of chalcone
and the electron-deficient sp2 N5 atom of FAD supports its en-
hanced binding affinities to the inhibitor binding cavity of
MAO-A. This allows the formation of a charge-transfer bonding
interaction between the drug and the FAD unit, which may be
particularly important for MAO-A inhibition.[39]
Figure 9. SAR of thienyl chalcones for hMAO-B inhibition.
According to its significantly high hMAO-A inhibitory poten-
cy and selectivity, as well as its low toxicity, compound TB8 ap-
pears to be a promising candidate for the management of
major depression. Additionally, the reversibility of this com-
pound’s inhibitory activity appears to be an advantage for the
design and development of new antidepressants. The in vitro
BBB prediction of lead MAO-B inhibitor TB5 showed excellent
results and encouraged us to further explore the potential of
this family of derivatives as potential lead candidates for the
treatment of Parkinson’s disease. Recent studies suggest that
reversible MAO-B inhibitors might also be effective in treat-
ment of Alzheimer’s disease by selective inhibition of astrocytic
gamma-aminobutyric acid production, as well as in Parkinson’s
disease. The development of selective reversible MAO-B inhibi-
tors may reduce the adverse effects of irreversible inhibitors
and may be promising compounds for the treatment of other
Figure 8. Docking pose of TB8 in the MAO-A active site. Yellow mesh indi-
cates p-p stacking interaction.
Conclusions
In the present study, a series of brominated thienyl chalcones
(TB1–TB11) were synthesized and evaluated as inhibitors of
MAO-A and MAO-B, and inhibitory activity was strongly affect-
ed by the type, electron density, and bulkiness of the substitu-
ents present at the para position on the phenyl ring. This
ChemMedChem 2016, 11, 1161 – 1171
www.chemmedchem.org
1167
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
1H, J=4.0 Hz, H3’), 7.60–7.58 (d, 2H, J=8.0 Hz, H2 and H6), 7.83–
7.79 ppm (d, 1H, J=16.0 Hz, CHb); 13C NMR (100 MHz, CDCl3): d=
181.0 (C=O), 161.9 (C4), 147.3 (C2’), 144.4 (Cb), 131.4 (C3’), 131.2 (C4’),
130.3 (C2 and C6), 127.3 (C1), 122.3 (Ca), 118.2 (C5’), 114.5 (C3 and
C5), 55.4 ppm (O-CH3); ESI-MS (m/z): calculated 323.20, observed
323.83 [M]+, [M+2]; Anal. calcd for C14H11BrO2S: C 52.03, H 3.43, S
9.92, found: C 52.17, H 3.42, S 10.01.
neurodegenerative diseases. Compound TB5 may have value
as a promising potent and reversible MAO-B inhibitor in this
regard. The systematic and finely tuned exploration of the vari-
ous electron-donating and -withdrawing groups at the para
position on the phenyl ring of the brominated thienyl chalcone
enabled precise development of a new MAO-B inhibitor. Over-
all, the study provided meaningful information for further de-
velopment of multifunctional drugs for neurodegenerative dis-
orders such as Parkinson’s and Alzheimer’s diseases.
(2E)-1-(5-Bromothiophen-2-yl)-3-(4-methylphenyl)prop-2-en-1-
one (TB4): Compound TB4 was isolated as an off-white powder
1
(yield: 86%): mp: 108–1108C; H NMR (400 MHz, CDCl3): d=2.39 (s,
3H, CH3), 7.14–7.13 (d, 1H, J=4.0 Hz, H3’), 7.23–7.21 (d, 2H, J=
8.0 Hz, H2 and H6), 7.29–7.25 (d, 1H, J=16.0 Hz, CHa), 7.53–7.51 (d,
2H, J=8.0 Hz, H3 and H5), 7.59–7.58 (d, 1H, J=4.0 Hz, H4’), 7.84–
7.80 ppm (d, 1H, J=16.0 Hz, CHb); 13C NMR (100 MHz, CDCl3): d=
180.9 (C=O), 147.2 (C2’), 144.7 (Cb), 141.4 (C3’), 131.8 (C4), 131.8 (C4’),
131.6 (C3 and C5), 129.7 (C2 and C6), 128.5 (C1), 122.5 (C5’), 119.5
(Ca), 21.5 ppm (CH3); ESI-MS (m/z): calculated 307.20, observed
307.79 [M]+, [M+2]; Anal. calcd for C14H11BrOS: C 54.74, H 3.61, S
10.44, found: C 54.70, H 3.74, S 10.23.
Experimental Section
General: 2-Acetyl 5-bromo-thiophene and all substituted benzalde-
hydes were procured from Sigma–Aldrich USA. Melting points of
all synthesized derivatives were determined by the open capillary
tube method, and values were uncorrected. H NMR and 13C NMR
spectra were recorded on a Bruker Avance III 600 spectrometer at
frequencies of 400 MHz and 100 MHz, respectively (Bruker, Karls-
ruhe, Germany). All NMR measurements were conducted in CDCl3,
and chemical shifts are reported in parts per million (d) downfield
from the signal of tetramethylsilane added to the deuterated sol-
vent. Mass spectra were recorded on a JEOL GCmate mass spec-
trometer. Elemental analyses (C, H, N) were performed on a Leco
CHNS 932 analyzer.
1
(2E)-1-(5-Bromothiophen-2-yl)-3-[4-(dimethylamino)
phenyl]-
prop-2-en-1-one (TB5): Compound TB5 was isolated as an orange
powder (yield: 96%): mp: 132–1348C; 1H NMR (400 MHz, CDCl3):
d=3.04 (s, 6H, N(CH3)2), 6.68–6.66 (d, 2H, J=8.0 Hz, H3 and H5),
7.10–7.08 (d, 2H, J=8.0 Hz, H2 and H6), 7.47–7.46 (d, 1H, J=4.0 Hz,
H3’), 7.55–7.51 (d, 1H, J=16.0 Hz, CHa), 7.55–7.54 (d, 1H, J=4.0 Hz,
H4’), 7.82–7.78 ppm (d, 1H, J=16.0 Hz, CHb); 13C NMR (100 MHz,
CDCl3): d=180.9 (C=O), 152.2 (C4), 147.9 (C2’), 145.8 (Cb), 131.2 (C3’),
130.7 (C2 and C6), 127.8 (C4’), 122.78 (C1), 121.4 (Ca), 115.1 (C5’),
111.8 (C3&C5), 40.0 ppm (N-(CH3)2); ESI-MS (m/z): calculated 336.24,
observed 336.89 [M]+, [M+2]; Anal. calcd for C15H14BrNOS: C
53.58, H 4.20, S 9.54, found: C 53.56, H 4.32, S 9.43.
Synthesis: A mixture of 2-acetyl 5-bromo thiophene (0.01 mol),
para-substituted benzaldehyde (0.01 mol), and 40% aqueous KOH
was added to EtOH (30 mL) and was stirred at room temperature
for 2–6 h. The resulting product was stored overnight in the refrig-
erator. The solid precipitate was filtered, washed with water, and
recrystallized from EtOH/acetone/chloroform (1:1:1) to yield pure
crystals.
(2E)-1-(5-Bromothiophen-2-yl)-3-(4-ethylphenyl)prop-2-en-1-one
(TB6): Compound TB6 was isolated as an off-white powder (yield:
95%): mp: 110–1128C; 1H NMR (400 MHz, CDCl3): d=1.27–1.25 (t,
3H, J=8.0 Hz, CH3), 2.71–2.69 (q, 2H, J=8.0 Hz, CH2), 7.14–7.13 (d,
2H, J=4.0 Hz, H4’), 7.25–7.23 (d, 2H, J=8.0 Hz, H3 and H5), 7.29–
7.25 (d, 1H, J=16.0 Hz, CHa), 7.55–7.53 (d, 2H, J=8.0 Hz, H2 and
H6), 7.58–7.57 (d, 2H, J=4.0 Hz, H3’), 7.84–7.80 ppm (d, 1H, J=
16.0 Hz, CHb); 13C NMR (100 MHz, CDCl3): d=180.9 (C=O), 147.7
(C2’), 147.2 (Cb), 144.7 (C4 and C3’), 132.4 (C1), 131.3 (C4’),128.6 (C2
and C6), 127.4 (C3 and C5), 122.5 (C5’), 119.5 (Ca), 28.3 (CH2),
15.6 ppm (CH3); ESI-MS (m/z): calculated 321.23, observed 321.84
[M]+, [M+2]; C15H13BrOS: C 56.08, H 4.08, S 9.98, found: C 56.12, H
4.32, S 9.92.
(2E)-1-(5-Bromothiophen-2-yl)-3-phenyl prop-2-en-1-one (TB1):
Compound TB1 was isolated as an off-white solid (yield: 94%):
mp: 106–1088C; 1H NMR (400 MHz, CDCl3): d=6.77–6.73 (d, 1H,
J=16.0 Hz, CHa), 7.00–6.99 (d, 1H, J=4.0 Hz, H4’), 7.14–7.12 (t, 1H,
J=8.0 Hz, H4), 7.20-.7.18 (d, 2H, J=8.0 Hz, H3 and H5), 7.24–7.22 (d,
2H, J=8.0 Hz, H2&H6), 7.43–7.42 (d, 1H, J=4.0 Hz, H3’), 7.63–
7.59 ppm (d, 1H, J=16.0 Hz, CHb); 13C NMR (100 MHz, CDCl3): d=
180.1 (C=O), 146.9 (C2’), 145.4 (Cb), 138.7 (C3’), 135.2 (C1), 132.2 (C4’),
128.5 (C3 and C5), 128.1 (C4), 126.2 (C2 and C6), 122.1 (C5’),
121.1 ppm (Ca); ESI-MS (m/z): calculated 248.72, observed 249.16
[M]+, [M+2]; Anal. calcd for C13H9BrOS: C 53.26, H 3.09, S 10.89,
found: C 53.18, H 3.14, S 10.96.
(2E)-1-(5-Bromothiophen-2-yl)-3-(4-nitrophenyl)prop-2-en-1-one
(TB7): Compound TB7 was isolated as a brown powder (yield:
74%): mp: 160–1628C; H NMR (400 MHz, CDCl3): d=6.96–6.92 (d,
(2E)-1-(5-Bromothiophen-2-yl)-3-(4-hydroxyphenyl)prop-2-en-1-
one (TB2): Compound TB2 was isolated as an off-white solid
(yield: 96%): mp: 80–828C; 1H NMR (400 MHz, CDCl3): d=5.28 (s,
1H, OH), 6. 54–6.50 (d, 1H, J=16.0 Hz, CHa), 6.67–6.65 (d, 2H, J=
8.0 Hz, H3 and H5), 6.89–6.88 (d, 1H, J=4.0 Hz, H4’), 7.13–7.11 (d,
2H, J=8.0 Hz, H2 and H6), 7.32–7.31 (d, 1H, J=4.0 Hz, H3’), 7.54–
7.50 ppm (d, 1H, J=16 Hz, CHb); 13C NMR (100 MHz, CDCl3): d=
181.0 (C=O), 159.3 (C4), 147.2 (C2’), 145.9 (Cb), 138.2 (C3’), 132.3 (C4’),
128.1 (C1), 127.4 (C2 and C6), 122.3 (C5’), 121.3 (Ca), 115.8 ppm (C3
and C5); ESI-MS (m/z): calculated 309.37, observed 310.01 [M]+,
[M+2]; Anal. calcd for C13H9BrO2S: C 50.50, H 2.93, S 10.37, found:
C 50.87, H 2.82, S 10.25.
1
1H, J=16.0 Hz, CHa), 7.14–7.13 (d, 1H, J=4.0 Hz, H4’), 7.32–7.31 (d,
1H, J=4.0 Hz, H3’), 7.56–7.54 (d, 2H, J=8.0 Hz, H2 and H6), 7.66–
7.62 (d, 1H, J=16.0 Hz, CHb), 8.23–8.21 ppm (d, 2H, J=8.0 Hz, H3
and H5); 13C NMR (100 MHz, CDCl3): d=180.3 (C=O), 147.1 (C2’),
146.1 (C4), 145.6 (Cb), 142.4 (C1), 138.1 (C3’), 132.0 (C4’), 127.3 (C2
and C6), 122.6 (C5’), 121.3 (Ca), 120.0 ppm (C3 and C5); ESI-MS (m/z):
calculated 338.17, observed 338.80 [M]+, [M+2]; C13H8BrNO3S: C
46.17, H 2.38; N, 4.14, S 9.48, found: C 46.19, H 2.32; N, 4.24, S,
9.44.
(2E)-1-(5-Bromothiophen-2-yl)-3-(4-chlorophenyl)prop-2-en-1-
one (TB8): Compound TB8 was isolated as a pale white powder
(yield: 98%): mp: 140–1428C; H NMR (400 MHz, CDCl3): d=6.96–
6.95 (d, 1H, J=4.8 Hz, H4’), 7.12–7.11 (d, 1H, J=4.0 Hz, H3’), 7.34–
7.30 (d, 1H, J=15.6 Hz, CHa), 7.57–7.55 (d, 2H, J=8.0 Hz, H3 and
(2E)-1-(5-Bromothiophen-2-yl)-3-(4-methoxyphenyl)prop-2-en-1-
one (TB3): Compound TB3 was isolated as an off-white powder
(yield: 68%): mp: 130–1328C; H NMR (400 MHz, CDCl3): d=3.86 (s,
3H, OCH3), 6.95–6.93 (d, 2H, J=8.0 Hz, H3 and H5), 7.14–7.13 (d,
1H, J=4.0 Hz, H4’), 7.21–7.17 (d, 1H, J=16.0 Hz, CHa), 7.58–7.57 (d,
1
1
ChemMedChem 2016, 11, 1161 – 1171
www.chemmedchem.org
1168
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
H5), 7.67–7.65 (d, 2H, J=8.0 Hz, H2 and H6), 7.80–7.76 ppm (d, 1H,
J=16.0 Hz, CHb); 13C NMR (100 MHz, CDCl3): d=180.7 (C=O), 145.2
(Cb), 140.1 (C2’), 139.7 (C3’), 131.3 (C4), 130.2 (C1), 129.1 (C4’), 128.1
(C3 and C5), 127.1 (C2 and C6), 122.4 (C5’), 121.3 ppm (Ca); ESI-MS
(m/z): calculated 327.62, observed 328.81 [M]+, [M+2], [M+4];
C13H8ClBrOS: C 47.66, H 2.46, S 9.79, found: C 47.65, H 2.42, S 9.66.
378C in a multidetection microplate fluorescence reader with exci-
tation at 545 nm and emission at 590 nm over a 15 min period,
during which the fluorescence increased linearly. Specific fluores-
cence emission was calculated after subtraction of the background
activity, which was determined from wells containing all compo-
nents except the hMAO isoforms, which were replaced by
a sodium phosphate buffer solution. Under our experimental con-
ditions, this background activity was negligible. Control experi-
ments were carried out by replacing the compound and known in-
hibitors. The possible capacity of compounds to modify the fluo-
rescence generated in the reaction mixture due to non enzymatic
inhibition was determined by adding these compounds to solu-
tions containing only the Amplex Red reagent in a sodium phos-
phate buffer. The new compounds and reference inhibitors them-
selves did not react directly with Amplex Red reagent. Newly syn-
thesized compounds did not cause any inhibitory activity of HRP in
the test medium.
(2E)-3-(4-Bromophenyl)-1-(5-bromothiophen-2-yl)prop-2-en-1-
one (TB9): Compound TB9 was isolated as an off-white powder
1
(yield: 55%): mp: 138–1408C; H NMR (400 MHz, CDCl3): d=6.97–
6.93 (d, 1H, J=16.0 Hz, CHa), 7.14–7.13 (d, 1H, J=4.0 Hz, H4’),
7.22–7.21 (d, 1H, J=4.0 Hz, H3’), 7.29–7.27 (d, 2H, J=8.0 Hz, H2
and H6), 7.37–7.35 (d, 2H, J=8.0 Hz, H3 and H5), 7.57–7.53 ppm (d,
1H, J=16.0 Hz, CHb); 13C NMR (100 MHz, CDCl3): d=181.0 (C=O),
147.3 (C2’), 145.2 (Cb), 138.4 (C3’), 135.1 (C1), 132.3 (C4’), 131.2 (C3
and C5), 128.5 (C2 and C6), 122.4 (C4), 121.1 (C5’), 121.1 ppm (Ca);
ESI-MS (m/z): calculated 372.07, observed 372.91 [M]+, [M+2],
[M+4]; C13H8BrOS: C 41.96, H 2.17, S 8.62, found: C 41.66, H 2.22,
S 8.83.
Kinetics studies: Synthesized compounds were dissolved in
DMSO, at a maximum concentration of 1%, and used in a wide
compound concentration range of 0.10–1.00 mm. The mode of
MAO inhibition was examined using Lineweaver–Burk plotting. The
slopes of the Lineweaver–Burk plots were plotted versus the inhibi-
tor concentration, and the Ki values were determined from the x-
axis intercept as ÀKi. Each Ki value is the representative of a single
determination in which the correlation coefficient (R2) of the replot
of the slopes versus the inhibitor concentrations was at least 0.98.
SI values were calculated as Ki(hMAO-A)/Ki(hMAO-B). The protein
concentration was determined according to the Bradford
method.[43]
(2E)-1-(5-Bromothiophen-2-yl)-3-(4-fluorophenyl)prop-2-en-1-
one (TB10): Compound TB10 was isolated as an off-white powder
1
(yield: 92%): mp: 128–1308C; H NMR (400 MHz, CDCl3): d=6.60–
6.56 (d, 1H, J=16.0 Hz, CHa), 6.66–6.64 (d, 2H, J=8.0 Hz, H2 and
H6),7.14–7.13 (d, 1H, J=4.0 Hz, H4’), 7.32–7.30 (d, 2H, J=8.0 Hz, H3
and H5), 7.52–7.51 (d, 1H, J=4.0 Hz, H3’), 7.78–7.74 ppm (d, 1H, J=
16.0 Hz, CHb); 13C NMR (100 MHz, CDCl3): d=180.1 (C=O), 162.3
(C4), 147.19 (C2’), 145.4 (Cb), 138.5 (C3’), 132.4 (C4’), 130.9 (C1), 128.6
(C2 and C6), 122.5 (C5’), 121.4 (Ca), 115.6 ppm (C3 and C5); ESI-MS
(m/z): calculated 311.16, observed 311.93 [M]+, [M+2]; C13H8BrFOS:
C 50.18, H 2.59, S 10.30, found: C 50.38, H 2.44, S 10.18.
Reversibility of inhibition: Reversibility of MAO inhibition with the
compounds was determined by the dialysis method previously de-
scribed.[44] Dialysis tubing (16”25 mm, Sigma–Aldrich) with a mo-
lecular weight cutoff of 12000 Da and a sample capacity of 0.5–
10 mL was used. Adequate amounts of the recombinant enzymes
(hMAO-A or B, 0.05 mgmLÀ1) were incubated with a concentration
equal to fourfold of the IC50 values for the inhibition of hMAO-A
and -B, respectively, in potassium phosphate buffer (0.05m, pH 7.4)
containing 5% sucrose and 1% DMSO for 15 min at 378C. Another
set of enzymes was prepared by pre-incubation of same amount
of hMAO-A and -B with the reference inhibitors moclobemide, sele-
giline, and lazabemide. Enzyme–inhibitor mixtures were subse-
quently dialyzed at 48C in 80 mL of dialysis buffer (100 mm potas-
sium phosphate, pH 7.4) containing 5% sucrose. The dialysis buffer
was replaced with fresh buffer at 3 h and 7 h after the start of dial-
ysis. At 24 h after dialysis, residual MAO activities were measured.
All reactions were carried out in triplicate, and the residual enzyme
catalytic rates were expressed as means ÆSEM. For comparison,
undialyzed mixtures of the MAOs and the inhibitors were kept at
48C over the same time period.
(2E)-1-(5-Bromothiophen-2-yl)-3-[4-(trifluoromethyl)phenyl]prop-
2-en-1-one (TB11): Compound TB11 was isolated as a pale pink
powder (yield: 97%): mp: 136–1388C; 1H NMR (400 MHz, CDCl3):
d=6.66–6.62 (d, 1H, J=15.6 Hz, CHa), 6.95–6.94 (d, 1H, J=4.0 Hz,
H4’), 7.22–7.20 (d, 2H, J=8.0 Hz, H2 and H6), 7.33–7.32 (d, 1H, J=
4.0 Hz, H3’), 7.46–7.44 (d, 2H, J=8.0 Hz, H3 and H5), 7.58–7.54 ppm
(d, 1H, J=16.0 Hz, CHb); 13C NMR (100 MHz, CDCl3): d=180.0 (C=
O), 147.4 (C2’), 145.6 (Cb), 138.3 (C1), 137.79 (C3’), 132.7 (C4’), 130.3
(C4), 126.7 (C2 and C6), 125.1 (C3 and C5), 124.5 (CF3), 122.4 (C5’),
121.2 ppm (Ca); ESI-MS (m/z): calculated 361.17, observed 361.88
[M]+, [M+2]; C14H8BrF3OS: C 46.56, H 2.23, S 8.88, found: C 46.43,
H 2.43, S 8.86.
Reagents: Recombinant hMAO-A and hMAO-B (expressed in bacu-
lovirus-infected BTI insect cells), R-(À)-deprenyl hydrochloride (sele-
giline), moclobemide, lazabemide hydrate, resorufin, dimethyl sulf-
oxide (DMSO), and other chemicals were purchased from Sigma–
Aldrich (Munich, Germany). The Amplex Red MAO assay kit (Mybio-
source, USA) contained benzylamine, p-tyramine, clorgyline, pargy-
line, and horseradish peroxidase.
In vitro BBB permeation assay: The ability of newly synthesized
compounds that penetrate the BBB was determined using
a PAMPA for the BBB, according to a previous method.[35] Briefly,
compounds and commercial drugs were dissolved in DMSO at
a concentration of 5 mgmLÀ1. They were diluted with a mixture of
phosphate-buffered saline solution and EtOH (PBS/EtOH, 70:30) to
give a final concentration of 25 mgmLÀ1. The filter membrane in
the donor microplate was coated with porcine polar brain lipid
(PBL) dissolved in dodecane (4 mL, 20 mgmLÀ1). Diluted solution
(200 mL) and PBS/EtOH (70:30, 300 mL) were added to the donor
and acceptor wells, respectively. The donor filter plate was carefully
placed on the acceptor plate. This sandwich system was stored at
258C for 16 h. The donor plate was then carefully removed, and
Determination of hMAO activity: hMAO activities were deter-
mined by a fluorimetric method described and modified previously
using p-tyramine (0.05–0.50 mm) as a common substrate.[40–42] The
study medium contained sodium phosphate buffer (0.1 mL, 0.05m,
pH 7.4), various concentrations of the synthesized compounds or
known inhibitors (moclobemide, selegiline and lazabemide), and
recombinant hMAO-A or hMAO-B. This mixture was incubated for
15 min at 378C in microplates in a dark fluorimeter chamber. The
reaction was started by adding Amplex Red reagent (200 mm),
horseradish peroxidase (HRP, 1 UmLÀ1), and p-tyramine (0.05–
0.50 mm). The production of H2O2 catalyzed by MAO isoforms was
detected using Amplex Red reagent in the presence of HRP to pro-
duce the fluorescent product resorufin. Resorufin was quantified at
ChemMedChem 2016, 11, 1161 – 1171
www.chemmedchem.org
1169
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
the concentrations of the compounds and the commercial drugs
in the acceptor, donor, and reference wells were measured with
a UV plate reader.
Keywords: chalcones · cytotoxicity · molecular docking ·
monoamine oxidases
Cytotoxic activity: Cell viability was measured by a quantitative
colorimetric assay with 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenylte-
trazolium bromide (MTT, Sigma–Aldrich). The HepG2 human hepa-
toma cell line (Invitrogen) was cultured in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with l-glutamine (2 mm),
heat-inactivated fetal bovine serum (FBS, 10%), penicillin
(100 UmLÀ1), and streptomycin (100 mgmLÀ1). Cells were seeded in
supplemented medium and maintained at 378C in a humidified at-
mosphere of 5% CO2 and 95% air. Exponentially growing HepG2
cells were subcultured in 96-well plates. The cells were treated
with the reference MAO inhibitors and novel brominated chalcone
compounds (1 mm, 5 mm, and 25 mm) or in 0.1% DMSO as a vehicle
control for 24 h. MTT labeling reagent (10 mL), at a final concentra-
tion of 5 mgmLÀ1, was added to each well at the end of the incu-
bation time, and the plate was placed in a humidified incubator at
378C with 5% CO2 and 95% air (v/v) for 4 h, until purple formazan
crystals appeared. Then, the insoluble formazan was dissolved in
DMSO (100 mL) by shaking for1 h in the dark. MTT reduction was
measured at 590 nm. Control cells treated with 0.1% DMSO were
used as 100% viability.[45,46] Significance was determined using Stu-
dent’s t-test. Results were expressed as meanÆSEM. Differences
were considered statistically significant at p<0.05.
[1] K. F. Tipton, Cell Biochem. Funct. 1986, 4, 79–87.
[2] D. ViÇa, M. J. Matos, G. Ferino, E. Cadoni, R. Laguna, F. Borges, E. Uriarte,
L. Santana, ChemMedChem 2012, 7, 464–470.
[3] T. Rahman, M. Rahmatullah, Bioorg. Med. Chem. Lett. 2010, 20, 537–540.
[4] J. Shih, K. Chen, M. J. Ridd, Annu. Rev. Neurosci. 1999, 22, 197–217.
[5] B. Drukarch, F. L. van Muiswinkel, Biochem. Pharmacol. 2000, 59, 1023–
1031.
[6] H. H. Fernandez, J. J. Chen, Pharmacotherapy 2007, 27, 174S–185S.
[7] A. Petzer, P. Grobler, J. J. Bergh, J. P. Petzer, J. Pharm. Pharmacol. 2014,
66, 677–687.
[8] C. J. Fowler, A. Wiberg, L. Oreland, J. Marcusson, B. Winblad, J. Neural
Transm. 1980, 49, 1–20.
[9] S. Carradori, J. P. Petzer, Expert Opin. Ther. Pat. 2015, 25, 91–110.
[10] M. Da Prada, G. Zürcher, I. Wüthrich, W. E. Haefely, J. Neural Transm.
Suppl. 1988, 26, 31–56.
[11] S. Carradori, R. Silvestri, J. Med. Chem. 2015, 58, 6717–6732.
[12] Z. A. Nowakowska, Eur. J. Med. Chem. 2007, 42, 125–137.
[13] N. K. Sahu, S. S. Balbhadra, J. Choudhary, D. V. Kohli, Curr. Med. Chem.
2012, 19, 209–225.
[14] B. Mathew, J. Suresh, S. Anbazghagan, J. Paulraj, G. K. Krishnan, Biomed.
Prev. Nutr. 2014, 4, 451–458.
[15] P. Singh, A. Anand, V. Kumar, Eur. J. Med. Chem. 2014, 85, 758–777.
[16] D. K. Mahapatra, V. Asati, S. K. Bharti, Eur. J. Med. Chem. 2015, 92, 839–
865.
Molecular docking studies: In the current molecular simulation
study, AutoDock 4.2 software was used to establish a ligand-based
computer modeling program for the prediction of binding energy
of the selected compounds with hMAO isoforms.[47] The docking
protocol used X-ray crystal structures of hMAO-A (PDB ID: 2BXR)
and hMAO-B (PDB ID: 2BYB) downloaded from the Protein Data
Bank (PDB; www.rcsb.org).[48] Protein preparation was carried out
using the Protein Preparation Wizard of Maestro 8.5 (Schrçdinger,
LLC).[49] Ligands were prepared through the PRODRG webserver
(davapc1.bioch.dundee.ac.uk/cgi-bin/prodrg).[50] The receptor grids
for both enzymes were generated with following parameters: grid
box dimension (xyz) of 60”60”60; grid spacing of 0.375 Š, and
center of the grid box positioned on the N5 atom of FAD (cofac-
tor). The grid parameter file (.gpf) generated through
MGLTools 1.5.6 was then used to generate map types through
anautogrid4 execution file. Similarly, for each ligand, a docking pa-
rameter file (.dpf) was written using MGLTools 1.5.6 with default pa-
rameters except: Number of runs=50; population size=300 and
number of evaluations=medium. The Lamarckian genetic algo-
rithm was selected for all molecular docking simulations. The .dpf
file generated for each ligand was then used for running molecular
docking simulations using an AutoDock 4 execution file, which
generated docking log files (.dlg) containing results of the simula-
tion. Using the Analyze module in MGLTools 1.5.6, the docking log
files were analyzed. The top scoring molecule from the largest clus-
ter was considered for analysis.[51,52]
[17] D. Secci, S. Carradori, A. Bolasco, B. Bizzarri, M. D’Ascenzio, E. Maccioni,
Curr. Top. Med. Chem. 2012, 12, 2240–2257.
[18] B. Mathew, J. Suresh, S. Anbazhagan, G. E. Mathew, Cent. Nerv. Syst.
Agents Med. Chem. 2013, 13, 195–206.
[19] S. Tanaka, Y. Kuwai, M. Tabata, Planta Med. 1987, 53, 5–8.
[20] F. Chimenti, R. Fioravanti, A. Bolasco, P. Chimenti, D. Secci, F. Rossi, M.
YµÇez, F. Orallo, F. S. Alcaro, J. Med. Chem. 2009, 52, 2818–2824.
[21] S. J. Robinson, J. P. Petzer, A. Petzer, J. J. Bergh, A.C.U. Lourens, Bioorg.
Med. Chem. Lett. 2013, 23, 4985–4989.
[22] G. Jo, S. Ahn, B. G. Kim, H. R. Park, Y. H. Kim, H. A. Choo, D. Koh, Y.
Chong, J. H. Ahn, Y. Lim, Bioorg. Med. Chem. 2013, 21, 7890–7897.
[23] N. Morales-Camilo, C. O. Salas, C. Sanhueza, C. Espinosa-Bustos, M.
Reyes-Parada, F. Gonzalez-Salas, C. Sanhauez, C. Espinosa-Bustos, S. Se-
pulveda-Boza, M. Reyes-Parada, F. Gonzalez-Nilo, M. Caroli-Rezende, A.
Fierro, Chem. Biol. Drug Des. 2015, 85, 685–695.
[24] J. W. Choi, B. K. Jang, N. Cho, J. H. Park, S. K. Yeon, E. J. Ju, Y. S. Lee, G.
Han, A. M. Pae, D. J. Kim, K. D. Park, Bioorg. Med. Chem. 2015, 23, 6486–
6496.
[25] S. Zaib, S. U. F. Rizvi, S. Aslam, M. Ahmad, S. M. A. Abid, M. al-Rashida, J.
Iqbal, Med. Chem. 2015, 11, 580–589.
[26] S. Zaib, S. U. F. Rizvi, S. Aslam, M. Ahmad, M. al-Rashida, J. Iqbal, Med.
Chem. 2015, 11, 497–505.
[27] C. Minders, J. P. Petzer, A. Petzer, A. C. U. Lourens, Bioorg. Med. Chem.
Lett. 2015, 25, 5270–5276.
[28] B. Mathew, G. E. Mathew, G. UÅar, I. Baysal, J. Suresh, J. K. Vilapurathu, A.
Prakasan, J. K. Suresh, A. Thomas, Bioorg. Chem. 2015, 62, 22–29.
[29] B. Mathew, G. UÅar, S. Y. Ciftci, I. Baysal, J. Suresh, G. E. Mathew, J. K. Vila-
purathu, N. A. Moosa, N. Pullarottil, L. Viswam, A. Haridas, F. Fathima,
Lett. Org. Chem. 2015, 12, 605–613.
[30] M. B. H. Youdim, Y. S. Bakhle, Br. J. Pharmacol. 2006, 147, S287–S296.
[31] R. Ramsay, Curr. Top. Med. Chem. 2012, 12, 2189–2209.
[32] C. Binda, E. M. Milczek, D. Bonivento, J. Wang, A. Mattevi, D. E. Edmond-
son, Curr. Top. Med. Chem. 2011, 11, 2788–2796.
Acknowledgements
[33] W. A. Banks, BMC Neurol. 2009, 9, S3.
[34] M. P. Schaddelee, H. L. Voorwinden, D. Groenendaal, A. Hersey, A. P. Ij-
zerman, M. Danhof, A. G. De Boera, Eur. J. Pharm. Sci. 2003, 20, 347–
356.
[35] L. Di, E. H. Kerns, K. Fan, O. J. McConnell, G. T. Carter, Eur. J. Med. Chem.
2003, 38, 223–232.
The authors thank the Institute for Intensive Research in Basic
Sciences (IIRBS), Mahatma Gandhi University (Kottayam, India)
and the Sophisticated Analytical Instrument Facility, Indian Insti-
tute of Technology (Chennai, India) for carrying out the spectral
analysis.
[36] C. Binda, M. Li, F. Hubalek, N. Restelli, D. E. Edmondson, Proc. Natl. Acad.
Sci. USA 2003, 100, 9750–9755.
ChemMedChem 2016, 11, 1161 – 1171
www.chemmedchem.org
1170
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
[37] L. Legoabe, J. Kruger, A. Petzer, J. J. Bergh, J. P. Petzer, Eur. J. Med. Chem.
2011, 46, 5162–5174.
[46] C. F. Wu, R. Bertorelli, M. Sacconi, G. Pepeu, S. Consolo, Neurobiol. Aging
1988, 9, 357–361.
[47] G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S. Good-
sell, A. J. Olson, J. Comput. Chem. 2009, 30, 2785–2791.
[48] L. De Colibus, M. Li, C. Binda, A. Lustig, D. E. Edmondson, A. Mattevi,
Proc. Natl. Acad. Sci. USA 2005, 102, 12684–12689.
[49] B. Vishnu Nayak, S. Ciftci-Yabanoglu, S. S. Jadav, M. Jagrat, B. N. Sinha,
G. Ucar, V. Jayaprakash, Eur. J. Med. Chem. 2013, 69, 762–767.
[50] A. W. Schüttelkopf, D. M. van Aalten, Acta Crystallogr. Sect. D 2004, 60,
1355–1363.
[51] B. V. Nayak, S. Ciftci-Yabanoglu, S. Bhakat, A. K. Timiri, B. N. Sinha, G.
Ucar, M. E. S. Soliman, V. Jayaprakash, Bioorg. Chem. 2015, 58, 72–80.
[52] V. N. Badavath, I. Baysal, G. Ucar, S. K. Mondal, B. N. Sinha, V. Jayapraksh,
Arch. Pharm. Chem. Sci. Ed. 2016, 349, 9–19
[38] R. K. Tripathi, S. Krishnamurty, S. R. Ayyannan, ChemMedChem 2016, 11,
119–132.
[39] F. Manna, F. Chimenti, F. A. Bolasco, D. Secci, B. Bizzarri, O. Befani, P.
Turini, B. Mondovi, S. Alcaroc, A. Tafi, Bioorg. Med. Chem. Lett. 2002, 12,
3629–3633.
[40] M. C. Anderson, F. Hasan, J. M. McCrodden, K. F. Tipton, Neurochem. Res.
1993, 18, 1145–1149.
[41] M. YµÇez, N. Fraiz, E. Cano, F. Orallo, Biochem. Biophys. Res. Commun.
2006, 344, 688–695.
[42] F. Chimenti, E. Maccioni, D. Secci, A. Bolasco, P. Chimenti, A. Granese, S.
Carradori, S. Alcaro, F. Ortuso, M. YµÇez, J. Med. Chem. 2008, 51, 4874–
4880.
[43] M. M. Bradford, Anal. Biochem. 1976, 72, 248–254.
[44] A. Petzer, A, Pienaar, J. P. Petzer, Life Sci. 2013, 93, 283–287.
[45] M. B. Hansen, S. E. Nielsen, K. Berg, J. Immunol. Methods 1989, 119,
203–210.
Received: March 1, 2016
Revised: March 30, 2016
Published online on May 9, 2016
ChemMedChem 2016, 11, 1161 – 1171
www.chemmedchem.org
1171
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim