Exploration of chlorinated thienyl chalcones: A new class of monoamine oxidase-B inhibitors

Article abstract of DOI:10.1016/j.ijbiomac.2016.05.110

Chalcone has been reported to be a valid scaffold for the design of monoamine oxidase (MAO) inhibitors. This scenario has amplified the momentum for the discovery of heteroaryl based chalcone MAO inhibitors. In the present study, we have synthesized a ser

Full text of DOI:10.1016/j.ijbiomac.2016.05.110

Accepted Manuscript
Title: Exploration of Chlorinated Thienyl Chalcones: A New
Class of Monoamine Oxidase-B Inhibitors
Author: Bijo Mathew Abitha Haridas Gu¨lberk Uc¸ar Ipek
Baysal Adebayo A. Adeniyi Mahmoud E.S. Soliman Monu
Joy Githa Elizabeth Mathew Baskar Lakshmanan Venkatesan
Jayaprakash
PII:
S0141-8130(16)30523-2
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.05.110
BIOMAC 6171
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
13-3-2016
28-5-2016
30-5-2016
Please cite this article as: Bijo Mathew, Abitha Haridas, Gu¨lberk Uc¸ar, Ipek Baysal,
Adebayo A.Adeniyi, Mahmoud E.S.Soliman, Monu Joy, Githa Elizabeth Mathew,
Baskar Lakshmanan, Venkatesan Jayaprakash, Exploration of Chlorinated Thienyl
Chalcones: A New Class of Monoamine Oxidase-B Inhibitors, International Journal
of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.05.110
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Exploration of Chlorinated Thienyl Chalcones: A New Class of Monoamine Oxidase-B
Inhibitors
Bijo Mathewa*, Abitha Haridasb, Gülberk Uçarc*, Ipek Baysalc, Adebayo A. Adeniyid, Mahmoud E.S. Solimand,
Monu Joye, Githa Elizabeth Mathewf, Baskar Lakshmananb, Venkatesan Jayaprakashg
a) Division of Drug Design and Medicinal Chemistry Research Lab, Department of Pharmaceutical
Chemistry, Ahalia School of Pharmacy, Palakkad-678557, Kerala, India.
b) Department of Pharmaceutical Chemistry, Grace College of Pharmacy, Palakkad 678004, Kerala, India.
c) Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, 06100 Sıhhiye, Ankara,
Turkey.
d) School of Health Sciences, University of KwaZulu-Natal, Westville, Durban 4001, South Africa
e) The School of Pure & Applied Physics, M.G. University, Kottayam, India.
f) Department of Pharmacology, Grace College of Pharmacy, Palakkad 678004, Kerala, India.
g) Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi,
Jharkhand 835 215, India.
*Corresponding author: Bijo Mathew, Associate Professor, Division of Drug Design and
Medicinal Chemistry Research Lab, Department of Pharmaceutical Chemistry, Ahalia School of Pharmacy,
Palakkad-678557, Kerala, India
E-mail: bijovilaventgu@gmail.com, Phone: +919946700219.
Additional correspondence: Prof. Gülberk Uçar, Department of Biochemistry, Faculty of Pharmacy, Hacettepe
University, 06100 ,Sıhhiye, Ankara, Turkey
Email: gulberk@hacettepe.edu.tr
1These authors contributed equally.

Highlights
 Synthesized eleven chlorinated thienyl chalcones.
 Compounds showed reversible, selective and competitive MAO-B inhibition except
TC7.
 X-ray data of structure refinement of lead compound TC6.
 Structure–activity relationship has been established.
 Molecular docking and Molecular dynamics study of the lead compound.
Abstract
Chalcone has been reported to be a valid scaffold for the design of monoamine oxidase (MAO) inhibitors. This
scenario has amplified the momentum for the discovery of heteroaryl based chalcone MAO inhibitors. In the
present study, we have synthesized a series of eleven chlorinated thienyl chalcone derivatives substituted with a
different functional groups at the para- position on the ring B and investigated for their ability to inhibit human
MAO-A and -B. With the exception of compound (2E)-1-(4-chlorocyclopenta-1,3-dien-1-yl)-3-(4-
nitrophenyl)prop-2-en-1-one (TC7), which was a selective MAO-A inhibitor, all the other derivatives inhibited
hMAO-B potently and selectively with competitive mode of inhibition. The most potent compound (2E)-1-(4-
chlorocyclopenta-1,3-dien-1-yl)-3-(4-ethylphenyl)prop-2-en-1-one (TC6) was found to be the best activity and
higher selectivity towards hMAO-B with Ki and SI values of 0.31±0.02 µM and 16.84, respectively. All the
compounds presented in the current study are completely non-toxic with 74-88 % viable cells to hepatic cells at
100 µM concentration. Molecular docking and molecular dynamics simulation studies were carried out using
Autodock-4.2 and Amber 14 to understand the molecular level interaction and energy relation of MAO isoforms
with selective MAO-B inhibitor TC6.
Keywords: Thienyl chalcones, Human monoamine oxidase, Molecular docking, Molecular dynamics

1. Introduction
Monoamine oxidase (MAO) is located intracellularly in the mitochondrial outer membranes of neuronal, glial,
and other cells and catalyzes the oxidative deamination of monoamine neurotransmitters and xenobiotic amines
[1]. MAO consists of two isoforms, MAO-A and MAO-B, which are products of different genes [2]. Both
isoforms contain a non-covalent flavin adenine dinucleotide (FAD) co-factor and are involved in the oxidative
deamination of exogenous and endogenous amines, including neurotransmitters [3]. The reduced FAD is re-
oxidised by molecular oxygen to yield hydrogen peroxide, while the imine product is in most instances
hydrolysed to the corresponding aldehyde, thereby affecting the concentrations of neurotransmitter amines and
many xenobiotics [4].
Inhibitors of MAO-A are clinically used in the treatment of depression while MAO-B inhibitors are used in
the treatment of Parkinson’s disease (PD) in association with L-DOPA and/or Dopamine (DA) agonists and in
the management of symptoms associated with Alzheimer’s disease (AD) [5, 6]. MAO inhibitors that are
currently in practice produce side effects due to a lack of affinity and selectivity towards one of the isoforms.
Moreover, due to their irreversible nature they display typical drawbacks of long-lasting enzyme inhibition and
potential immunogenicity of enzyme-inhibitor adducts. In contrast, selective and reversible inhibitors have an
anticipated to have less toxicity since there is no requirement for de novo protein synthesis for the recovery of
enzymatic activity. So the development of a new class of potent, selective and reversible MAO inhibitors seems
to be the objective as they have safer therapeutic profiles [7].
Chalcones are broad family of compounds of both synthetic and natural sources. These derivatives have
received a great deal of attention due to their relatively easy synthesis, simple structures and wide range of
pharmacological activities. In the search for novel selective and reversible MAO-B inhibitors, chalcones have
emerged as a valid scaffold. Strategies by synthetic medicinal chemists includes the introductions of various
functional groups on ring A and B of chalcones as well as substitution with heterocycles such as furan,
thiophene, pyridine, piperidine, quinoline and pyrrole for developing more selective and potent MAO inhibitors.
Most of these studies revealed increase in potency due to the presence of lipophilic-electron withdrawing groups
such as chlorine, fluorine and trifluromethyl group at the para- position on the ring B of chalcones. These groups
could increase the lipophilic character of chalcones which favours the connection with the entrance cavity of the
active site of MAO-B [8-22]. In addition, naturally origin chalcones have been shown to inhibit hMAO-B [23].

In search of novel chalcone based MAO-inhibitors, a survey of chalcones reported for their MAO-inhibitory
activity was performed and a thorough SAR study has been reported by our group [24]. Guided by the facts in
the SAR study, we attempted to design a selective and reversible inhibitor of hMAO-B. Chalcones of 2-acetyl-5-
chloro thiophene were studied in the presented research with different electron donating and withdrawing groups
at the para- position on the ring B. In the present study, series of chlorinated thienyl chalcone derivatives were
prepared by the Claisen–Schmidt condensation of 2-acetyl 5-chloro-thiophene with substituted benzaldehydes
under basic conditions. Synthesized compounds were tested for their inhibitory activity on hMAO isoforms and
cytotoxic effects. Molecular docking and dynamics studies were carried out to get an insight of molecular
recognition process.
2. Experimental
2.1. Chemistry
2-Acetyl 5-chloro-thiophene and all the substituted benzaldehydes were procured from Sigma–Aldrich USA.
Melting points of all the synthesized derivatives were determined by open-capillary tube method and values 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, respectively (Bruker, Karlsruhe,
Germany). All NMR measurements were recorded in CDCl3, and the chemical shifts are reported in parts per
million (δ) downfield from the signal of tetramethylsilane (TMS) 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.
2.2. General procedure of chlorinated thienyl chalcones (TC1-TC11)
A mixture of 2-acetyl 5-chloro thiophene (0.01mol), para substituted benzaldehyde (0.01mol) and 40%
aqueous potassium hydroxide was added to 30 mL of ethanol and was stirred at room temperature for about 2-6
h. The resulting product was kept overnight in refrigerator. The solid separated was filtered, washed with water
and recrystallized from [Ethanol: Acetone: Chloroform- 1:1:1] yielded pure crystals.
2.2.1. (2E)-1-(4-chlorocyclopenta-1,3-dien-1-yl)-3-phenylprop-2-en-1-one (TC1): White powder. Yield: 89 %,
m.p. 208-210 oC. 1H NMR (400 MHz, CDCl3) δ ppm: 6.872-6.833 (d, 1H, J= 15.6 Hz, CHα), 7.004-6.994 (d, 1H,
J= 4.0 Hz, H4ʹ), 7.135-7.120 (t, 1H, J= 8.0 Hz, H4), 7.198-.7.178 (d, 2H, J= 8.0 Hz, H3&H5), 7.234-7.214 (d, 2H,
J= 8.0 Hz, H2&H6 ),7.425-7.415 (d, 1H, J= 4.0 Hz, H3ʹ), 7.628-7.690 (d, 1H, J= 15.2 Hz, CHβ). 13C NMR (100
MHz, CDCl3) δ ppm: 181.05 (C=O), 146.54 (C2ʹ), 144.23 (Cβ), 138.16 (C5ʹ), 133.74 (C3ʹ), 130.14 (C1), 128.13

(C4), 127.50 (C3&C5), 127.02 (C4ʹ), 125.34 (C2&C6), 121.03 (Cα). ESI-MS (m/z): calculated 248.72, observed
249.16 (M+)+, (M+2). Anal. calcd. for C13H9ClOS: C, 62.78; H, 3.65; S, 12.89. Found: C, 62.82; H, 3.74; S,
12.92.
2.2.2. (2E)-1-(4-chlorocyclopenta-1,3-dien-1-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one (TC2): Pale green
powder. Yield: 63 %, m.p. 158-160 oC. 1H NMR (400 MHz, CDCl3) δ ppm: 5.234 (s, 1H, OH), 6.470-6.531 (d,
1H, J= 15.6 Hz, CHα), 6.694-6.674 (d, 2H, J= 8.0 Hz, H3&H5), 6.894-6.884 (d, 1H, J= 4.0 Hz, H4ʹ), 7.136-7.116
(d, 2H, J= 8.0 Hz, H2&H6 ),7.326-7.316 (d, 1H, J= 4.0 Hz, H3ʹ), 7.534-7.496 (d, 1H, J= 15.2 Hz, CHβ). 13C NMR
(100 MHz, CDCl3) δ ppm: 181.82 (C=O), 160.21 (C4), 146.54 (C2ʹ), 145.34 (Cβ), 138.31 (C5ʹ), 134.34 (C3ʹ),
129.65 (C4ʹ), 128.76 (C1), 127.61 (C2&C6), 119.67 (Cα), 115.32 (C3&C5).ESI-MS (m/z): calculated 264.72,
observed 265.15 (M+)+, (M+2). Anal. calcd. for C13H9ClO2S: C, 58.98; H, 3.43; S, 12.11. Found: C, 58.99; H,
3.83; S, 12.12.
2.2.3. (2E)-1-(4-chlorocyclopenta-1,3-dien-1-yl)-3-(4-methoxyphenyl)prop-2-en-1-one (TC3): Cream powder.
Yield: 94 %, m.p. 115-117 oC. 1H NMR (400 MHz, CDCl3) δ ppm: 3.857 (s, 3H, OCH3), 6.908-6.886 (d, 2H, J=
8.4 Hz, H3&H5), 7.000-6.990 (d, 1H, J= 4.0 Hz, H4ʹ), 7.219-7.181 (d, 1H, J= 15.2 Hz, CHα), 7.599-7.578 (d, 2H,
J= 8.4 Hz, H2&H6 ), 7.625-7.615 (d, 1H, J= 4.0 Hz, H3ʹ), 7.827-7.789 (d, 1H, J= 15.2 Hz, CHβ). 13C NMR (100
MHz, CDCl3) δ ppm: 181.07 (C=O), 161.92 (C4), 144.54 (C2ʹ), 141.77 (Cβ), 139.56 (C5ʹ), 130.79 (C3ʹ), 129.02
(C4ʹ), 128.54 (C1), 127.62 (C2&C6), 118.04 (Cα), 114.50 (C3&C5), 55.16 (O-CH3). ESI-MS (m/z): calculated
278.75, observed 279.10 (M+)+, (M+2). Anal. calcd. for C14H11ClO2S: C, 60.32; H, 3.98; S, 11.50. Found: C,
60.88; H, 3.94; S, 11.62.
2.2.4. (2E)-1-(4-chlorocyclopenta-1,3-dien-1-yl)-3-(4-methylphenyl)prop-2-en-1-one (TC4): White powder.
Yield: 97 %, m.p. 138-140 oC. 1H NMR (400 MHz, CDCl3) δ ppm: 2.388 (s, 3H, CH3), 6.996-6.987 (d, 1H, J=
3.6 Hz, H3ʹ), 7.229-7.209 (d, 2H, J= 8.0 Hz, H3&H5), 7.294-7.256 (d, 1H, J= 15.2 Hz, CHα), 7.530-7.510 (d, 2H,
J= 8.0 Hz, H2&H6 ), 7.630-7.620 (d, 1H, J= 4.0 Hz, H4ʹ), 7.830-7.791 (d, 1H, J= 15.6 Hz, CHβ). 13C NMR (100
MHz, CDCl3) δ ppm: 181.12 (C=O), 144.59 (C2ʹ), 144.40 (C4), 141.40 (Cβ), 139.81 (C5ʹ), 131.00 (C3ʹ), 129.75
(C2&C6), 129.05 (C3&C5), 128.57 (C4ʹ), 127.66(C1), 119.35 (Cα), 21.55 (-CH3). ESI-MS (m/z): calculated
262.75, observed 263.10 (M+)+, (M+2). Anal. calcd. for C14H11ClOS: C, 64.00; H, 4.22; S, 12.20. Found: C,
64.08; H, 4.88; S, 12.22.
2.2.5. (2E)-1-(4-chlorocyclopenta-1,3-dien-1-yl)-3-[4-(dimethylamino)phenyl]prop-2-en-1-one (TC5): Orange
powder. Yield: 98 %, m.p. 154-156 oC. 1H NMR (400 MHz, CDCl3) δ ppm: 3.045 (s, 6H, N(CH3)2), 6.691-6.670

(d, 2H, J= 8.4 Hz, H3&H5), 7.134-7.096 (d, 1H, J= 15.2 Hz, CHα), 6.979-6.969 (d, 2H, J= 4.0 Hz, H4ʹ), 7.539-
7.517 (d, 2H, J= 8.8 Hz, H2&H6 ), 7.597-7.588 (d, 1H, J= 4.4 Hz, H3ʹ), 7.828-7.790 (d, 1H, J= 15.2 Hz, CHβ).
13C NMR (100 MHz, CDCl3) δ ppm: 181.11 (C=O), 152.37 (C4), 145.42 (C2ʹ), 145.20 (Cβ), 138.40 (C5ʹ), 130.56
(C2&C6), 130.10(C3ʹ), 127.47 (C4ʹ), 122.51(C1), 115.37 (Cα), 111.97 (C3&C5), 40.08 (N-(CH3)2). ESI-MS (m/z):
calculated 291.79, observed 292.00 (M+)+, (M+2). C15H14ClNOS: C, 61.74; H, 4.84; N, 4.80; S, 10.99. Found:
C, 61.78; H, 4.86; N, 4.88; S, 11.02.
2.2.6. (2E)-1-(4-chlorocyclopenta-1,3-dien-1-yl)-3-(4-ethylphenyl)prop-2-en-1-one (TC6): Cream powder.
Yield: 98 %, m.p. 108-110 oC. 1H NMR (400 MHz, CDCl3) δ ppm: 1.276-1.257 (t, 3H, J= 7.6 Hz, CH3), 2.679-
2.660 (q, 2H, J= 7.6 Hz, CH2), 7.004-6.995 (d, 1H, J= 3.6 Hz, H4ʹ), 7.138-7.100 (d, 1H, J= 15.2 Hz, CHα),
7.263-7.242 (d, 2H, J= 8.4 Hz, H3&H5), 7.546-7.566-7.546 (d, 2H, J= 8.0 Hz, H2&H6 ), 7.638-7.628 (d, 1H, J=
4.0 Hz, H3ʹ), 7.847-7.809 (d, 1H, J= 15.2 Hz, CHβ). 13C NMR (100 MHz, CDCl3) δ ppm: 181.15 (C=O), 147.70
(C2ʹ), 144.65 (Cβ), 139.45 (C5ʹ), 132.25(C4), 130.93 (C3ʹ), 128.69 (C2&C6), 128.60 (C3&C5), 127.89 (C4ʹ),
127.64(C1), 119.380 (Cα), 28.82 (CH2), 15.19 (CH3). ESI-MS (m/z): calculated 276.78, observed 277.10 (M+)+,
(M+2). C15H13ClOS: C, 65.09; H, 4.73; S, 11.58. Found: C, 65.08; H, 4.79; S, 11.60.
2.2.7. (2E)-1-(4-chlorocyclopenta-1,3-dien-1-yl)-3-(4-nitrophenyl)prop-2-en-1-one (TC7):Brown powder. Yield:
59 %, m.p. 200-202 oC. 1H NMR (400 MHz, CDCl3) δ ppm: 6.948-6.938(d, 1H, J= 4 Hz, H4ʹ), 7.342-7.303 (d,
1H, J= 15.6 Hz, CHα), 7.425-7.415 (d, 1H, J= 4 Hz, H3ʹ), 7.578-7.557 (d, 2H, J= 8.4 Hz, H2&H6 ), 7.595-7.585
(d, 1H, J= 4 Hz, H3ʹ), 7.639-7.600 (d, 1H, J= 15.6 Hz, CHβ). 8.210-8.190 (d, 2H, J= 8.0 Hz, H3&H5), 13C NMR
(100 MHz, CDCl3) δ ppm: 181.02 (C=O), 147.32(C4), 146.32 (C2ʹ), 145.53 (Cβ), 143.26 (C1), 139.13 (C5ʹ),
135.35 (C3ʹ), 130.78 (C4ʹ), 128.15 (C2&C6), 123.45 (Cα), 124.28 (C3&C5). ESI-MS (m/z): calculated 293.72,
observed 294.16 (M+)+, (M+2). C13H8ClO3S: C, 53.16; H, 2.75; N, 4.77; S, 10.92. Found: C, 53.18; H, 2.80; N,
4.84, S, 11.04.
2.2.8. (2E)-1-(4-chlorocyclopenta-1,3-dien-1-yl)-3-(4-chlorophenyl)prop-2-en-1-one (TC8): White powder.
Yield: 98 %, m.p. 156-158 oC. 1H NMR (400 MHz, CDCl3) δ ppm: 7.023-7.013 (d, 1H, J= 4 Hz, H3ʹ), 7.302-
7.263 (d, 1H, J= 15.6 Hz, CHα), 7.410-7.390 (d, 2H, J= 8.0 Hz, H3&H5), 7.577-7.556 (d, 2H, J= 8.4 Hz, H2&H6
),7.648-7.638(d, 1H, J= 4 Hz, H4ʹ), 7.803-7.764 (d, 1H, J= 15.6 Hz, CHβ). 13C NMR (100 MHz, CDCl3) δ ppm:
180.78 (C=O), 144.08 (C2ʹ), 143.02 (Cβ), 140.03 (C5ʹ), 136.75 (C3ʹ), 133.05 (C2&C6), 131.88 (C3&C5), 129.67
(C4ʹ), 129.33(C1), 127.75(C4), 120.85 (Cα). ESI-MS (m/z): calculated 283.17, observed 283.10 (M+)+, (M+2),
(M+4). C13H8Cl2OS: C, 55.14; H, 2.85; S, 11.32. Found: C, 55.17; H, 2.82; S, 11.36.

2.2.9. (2E)-3-(4-bromophenyl)-1-(4-chlorocyclopenta-1,3-dien-1-yl)prop-2-en-1-one (TC9): Pale white powder.
Yield: 98 %, m.p. 162-164 oC. 1H NMR (400 MHz, CDCl3) δ ppm: 7.022-7.012 (d, 1H, J= 4 Hz, H3ʹ), 7.324-
7.285 (d, 1H, J= 15.2 Hz, CHα), 7.503-7.482 (d, 2H, J= 8.4 Hz, H2&H6 ), 7.571-7.550 (d, 2H, J= 8.4 Hz,
H3&H5), 7.647-7.637 (d, 1H, J= 4 Hz, H4ʹ), 7.784-7.745 (d, 1H, J= 15.6 Hz, CHβ). 13C NMR (100 MHz, CDCl3)
δ ppm: 181.19 (C=O), 146.12 (C2ʹ), 143.25 (Cβ), 138.32 (C5ʹ), 132.13 (C3ʹ), 127.52 (C2&C6), 127.18 (C3&C5),
126.13 (C4ʹ), 125.21(C1), 122.15(C4), 120.13 (Cα). ESI-MS (m/z): calculated 327.62, observed 328.10 (M+)+,
(M+2). C13H8BrOS: C, 47.66; H, 2.46; S, 9.79. Found: C, 47.68; H, 2.52; S, 9.82.
2.2.10. (2E)-1-(4-chlorocyclopenta-1,3-dien-1-yl)-3-(4-fluorophenyl)prop-2-en-1-one (TC10): White powder.
Yield: 76 %, m.p. 114-116 oC. 1H NMR (400 MHz, CDCl3) δ ppm: 6.903-6.864 (d, 1H, J= 15.6 Hz, CHα), 7.246-
7.236 (d, 1H, J= 4 Hz, H4ʹ), 7.312-7.292 (d, 2H, J= 8.0 Hz, H3&H5), 7.461-7.441 (d, 2H, J= 8.0 Hz, H2&H6 ),
7.621-7.611 (d, 1H, J= 4 Hz, H3ʹ), 7.869-7.830 (d, 1H, J= 15.6 Hz, CHβ). 13C NMR (100 MHz, CDCl3) δ ppm:
180.18 (C=O), 160.35 (C4), 145.17 (C2ʹ), 144.44 (Cβ), 140.56 (C5ʹ), 138.35 (C3ʹ), 130.56(C1), 128.17 (C4ʹ), 127.45
(C2&C6), 121.32 (Cα), 115.62 (C3&C5). ESI-MS (m/z): calculated 266.71, observed 267.10 (M+)+, (M+2).
C13H8FOS: C, 58.54; H, 3.02; S, 12.02. Found: C, 58.68; H, 3.04; S, 12.08.
2.2.11. (2E)-1-(4-chlorocyclopenta-1,3-dien-1-yl)-3-[4-(trifluoromethyl)phenyl]prop-2-en-1-one (TC11): Brown
powder. Yield: 98%, m.p. 163-165 oC. 1H NMR (400 MHz, CDCl3) δ ppm: 6.749-6.710 (d, 1H, J= 15.6 Hz,
CHα), 6.951-6.941(d, 1H, J= 4 Hz, H4ʹ), 7.221-7.201 (d, 2H, J= 8.0 Hz, H2&H6 ), 7.327-7.317 (d, 1H, J= 4 Hz,
H3ʹ), 7.482-7.462 (d, 2H, J= 8.0 Hz, H3&H5), 7.672-7.633 (d, 1H, J= 15.6 Hz, CHβ). 13C NMR (100 MHz,
CDCl3) δ ppm: 180.53 (C=O), 146.54 (C2ʹ), 145.14 (Cβ), 138.23(C1), 137.45 (C5ʹ), 136.77 (C3ʹ), 129.88 (C4ʹ),
130.15(C4), 128.56 (C2&C6), 126.45 (C3&C5), 124.56(CF3)123.22 (Cα). ESI-MS (m/z): calculated 316.72,
observed 317.18 (M+)+, (M+2). C14H8ClF3OS: C, 53.09; H, 2.55; S, 10.12. Found: C, 53.12; H, 2.59; S, 10.16.
2.3. X-ray data collection & structure refinement of compound TC6
The cream crystals of TC6 were prismatic. The dimensions of the crystal used for data collection was 0.21 ×
0.24 × 0.41 mm3. The crystals belonged to Monoclinic system having space group P21/n with unit cell
dimensions, a = 7.5798Å, b = 17.4471Å, c = 10.9315Å, α = 90˚, β = 102.3˚ and γ = 90˚. Data are collected using
Bruker AXS Kappa Apex II CMOS detector diffractometer using graphite monochromatic Mo-K radiation
(λ=0.71073Å) at 293K. The data reduced using the program SAINT and empirical absorption corrections are
done using the SADABS. The structure is solved by direct method using SHELXS-2013. The top 18 peaks
formed the complete structure. The structure was refined using full-matrix least-squares method based on F2

using SHELXL-2013. Five cycles of Isotropic refinement is followed by anisotropic refinement for the non-
hydrogen atoms. The position of all hydrogen atoms are geometrically fixed and treated with riding atoms, with
C-H distances of 0.93 or 0.96 Å. Anisotropic refinements of the non-hydrogen atoms along with isotropic
refinement of hydrogen atoms using 4714 reflections [25-27]. Materials for publications are prepared using,
ORTEP program (fig. 1).
2.4. Biochemistry
2.4.1. Chemicals
Recombinant hMAO-A and hMAO-B (expressed in baculovirus-infected BTI insect cells), R-(–)-deprenyl
hydrochloride (selegiline), moclobemide, lazabemide hydrate, resorufin, dimethyl sulfoxide (DMSO), and other
chemicals were purchased from Sigma-Aldrich (Munich, Germany). The Amplex®-Red MAO assay kit
(Mybiosource, USA) contained benzylamine, p-tyramine, clorgyline, pargyline and horseradish peroxidase.
2.4.2. Determination of inhibitory activities of the chlorinated thienylchalcones on human MAO-A and B
Human MAO isoform activities were determined by a fluorimetric method described and modified previously
using p-tyramine (0.05-0.50 mM) as common substrate. Study medium contained 0.1 mL of sodium phosphate
buffer (0.05 M, 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 37 oC in microplates, placed in the dark fluorimeter chamber. Reaction was started by
adding 200 µM Amplex Red reagent, 1 U/mL horseradish peroxidase (HRP), and p-tyramine. The production of
H2O2 catalyzed by MAO isoforms was detected using Amplex®-Red reagent, in the presence of HRP to produce
the fluorescent product resorufin. Resorufin was quantified at 37 oC in a multi detection microplate fluorescence
reader with excitation at 545 nm, and emission at 590 nm, over a 15 min period, in which the fluorescence
increased linearly. The specific fluorescence emission was calculated after subtraction of the background
activity, which was determined from wells containing all components except the hMAO isoforms, which were
replaced by a sodium phosphate buffer solution. In our experimental conditions, this background activity was
negligible [28-30].
Control experiments were carried out by replacing the compound and known 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 compounds to solutions containing only the Amplex Red reagent in a sodium
phosphate buffer. The new compounds and reference inhibitors themselves did not react directly with Amplex

Red reagent. Newly synthesized compounds did not cause any inhibition on the activity of HRP in the test
medium.
2.4.3. Kinetic experiments
Synthesized compounds were dissolved in dimethyl sulfoxide, with a maximum concentration of 1% and
used in a wide concentration range of 0.01-10.00 µM. The mode of MAO inhibition was examined using
Lineveawer-Burk plotting. The slopes of the Lineweaver-Burk plots were plotted versus the inhibitor
concentration and the Ki values were determined from the x-axis intercept as -Ki. Each Ki value is the
representative 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 [31].
2.4.4. Reversibility experiments
Reversibility of the MAO inhibition with the compounds was determined by dialysis method previously
described [32]. Dialysis tubing 16×25 mm (SIGMA, Germany) with a molecular weight cut-off of 12000 and a
sample capacity of 0.5-10 mL was used. Adequate amounts of the recombinant enzymes (hMAO-A or B) (0.05
mg/mL) were incubated with a concentration equal to five-fold of the IC50 values for the inhibition of hMAO-A
and -B, respectively in potassium phosphate buffer (0.05 M, pH 7.4, 5% sucrose containing 1% DMSO) for 15
min. at 37 oC. Another sets were prepared by preincubation of same amount of hMAO-A and –B with the
reference inhibitors (Moclobemide, Selegiline and Lazabemide). Enzyme-inhibitor mixtures were subsequently
dialyzed at 4°C in 80 ml of dialysis buffer (100 mM potassium phosphate, pH 7.4, 5% sucrose). The dialysis
buffer was replaced with fresh buffer at 3 h and 7 h after the start of dialysis. 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 mean ± SEM. For comparison, undialyzed mixtures of the MAOs and the inhibitors were
kept at 4 °C over the same time period.
2.5. Cytotoxicity studies
Cell viability was measured by quantitative colorimetric assay with 3-[4, 5 dimethylthiazol-2-yl]-2, 5-
diphenyl-tetrazolium bromide (MTT) (Sigma Aldrich) [33]. Human hepatoma cell line HepG2 (Invitrogen) was
cultured in Eagle's minimum essential medium (EMEM) supplemented with non-essential amino acids, 1 mM
sodium pyruvate, 10 % heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL
streptomycin. Cells were seeded in supplemented medium and maintained at 37 °C in a humidified atmosphere
of 5% CO2 and 95 % air. Exponentially growing HepG2 cells were subcultured in 96-well plates. The cells were

treated with the TC series compounds (1 µM, 10 µM and 100 µM) or 0.1% dimethylsulfoxide (DMSO) as a
vehicle control for 24 h. 50 μL of the MTT labelling reagent, at a final concentration of 0.5 mg/mL, was added to
each well at the end of the incubation time and the plate placed in a humidified incubator at 37 °C with 5 % CO2
and 95 % air (v/v) for 4 h until the appearance of purple formazan crystals formed. Then, the insoluble formazan
was dissolved with 100 µL of DMSO by shaking 1 h in darkness. MTT reduction was measured at 540 nm.
Control cells treated with 0.1% DMSO were used as 100% viability [34]. The concentration of DMSO in
compounds was kept below 1.25 %, which was found to be non-toxic to the cells. Significance was determined
using one-way analysis of variance (ANOVA). Results were expressed as mean±SEM. Differences are
considered statistically significant at p<0.05.
2. 6. Molecular docking studies
In the current molecular simulation study, AutoDock4.2 software was used to establish a ligand-based
computer modelling program for the prediction of binding energy of the selected compounds with MAO
isoenzymes [35].
2.6.1. Preparation of enzymes structure
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 [36]. 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 [37].
2.6.2. Preparation ligands structure
Ligands were prepared for docking through PRODRG webserver (http://davapc1.bioch.dundee.ac.uk/cgi-
bin/prodrg). Java Molecular Editor (JME, Novertis) available in the server being used for sketching the molecule
(input) and the output (pdb with poar hydrogens only) has been copied and saved in text file with *.pdb
extension [38].
2.7.3. Docking protocol
The receptor grids were generated with following parameters: Grid box dimension (xyz) of 60 × 60 × 60; grid
spacing of 0.375Å and centre of the grid box positioned on N5 atom of FAD (cofactor). The .gpf (grid
parameter file) file generated through MGLTools-1.5.6 was then used to generate map types through autogrid4
execution file. Similarly for each ligand, a docking parameter file (.dpf) has been written using MGLTools-1.5.6
with default parameters except: No. of runs: 50; population size: 300 and No. of evaluations set at Medium. The

Lamarckian genetic algorithm was selected for all molecular docking simulations. The .dpf file generated for
each ligand was then used for running molecular docking simulation using autodock4 execution file, which will
generate docking log file (*.dlg) containing results of the simulate+on. Through analyze module in MGLTool-
1.5.6, the docking log files were analysed. Top scoring molecule from the largest cluster was considered for
analysis. [39].
2.7. Molecular dynamics study
All atoms unrestrained molecular dynamics (MD) of the two complexes (compound TC6 complexed with
MAO-A (PDB: 2BXR) and MAO-B (PDB: 2BYB), were carried out in Amber 14 using GPU based PMEMD
programme [40].The protein structures were parametrized with amber force field (leaprc.ff12SB) [41]. The force
field of the ligand TC6 was derived using the restrained electrostatic potential (RESP) method. The ligand was
first optimized using Gaussian 09 at HF/6-31G* level of theory and the partial atomic charges were fitted using
the R.E.D package [42]. The simulation was carried out in explicit solvent of an orthorhombic box of TIP3P
water molecules such that no solute atoms was within 8 Å from any side of the box [43]. The tLeap module of
Amber 15 was used to add hydrogen atoms and counter ions Cl- (three atoms) and Na+ (two atoms) were added
for neutralization of MAO-A and MAO-B respectively. The long range electrostatic potential was calculated by
using Partial Mesh Ewald (PME) method with a direct space and vdW cut-off of 12 Å [44].
Each of the systems was minimized twice, first with initial minimization for 1000 steps (500 steepest descent
followed by 500 steps of conjugate gradient) with strong constrained on both ligands and the protein while the
second minimization was for 100 steps with no constrain. This was followed by a canonical ensemble MD
simulation where the systems were gradually heated up to temperature of 300.00 K for a simulation steps of
25,000 using Langevin thermostat with a collision frequency of 1.0 ps-1 and a harmonic restrained of 5 kcal/mol
Å was applied on the solutes. This was followed by another 25,000 steps of NPT simulation to control the
density of the systems and 250,000 steps of equilibration at 300.00 K temperature, 1 bar pressure and a coupling
constant of 2 ps. Thereafter, the production MD was carried out and the frame was recorded at every 500 steps of
simulation and a time step of 2 fs was used all through the MD. All bond lengths involving hydrogen atoms were
restrained using SHAKE algorithm [45].
The package BIO3D was used for the analysis of the conformational changes in the trajectory of the protein
while the secondary structures were predicted using DSSP [46]. The visualization of the results was done using
VMD, Chimera, Pymol and Ligplus [47-50]. The change in the binding free energy of the systems from their

unbound state to bound state was calculated using Poisson Boltzman (MM-PBSA) and Generalized Born (MM-
GBSA) methods [51-54]. Both the GBSA and PBSA binding energy of the two systems were computed using a
total of 1000 snapshots taken from 10000 ps of MD trajectory at 10 ps intervals. The binding free energy can be
represented with the equation (1):
∆G0bind =∆G0Gas, complex +∆G0solv, complex – [∆G0receptor +∆G0ligand] (1)
Solvation free energies were calculated as contribution from electrostatic by either solving the linearised Poisson
Boltzman (GPB) or Generalized Born equation (GGB) with addition of empirical term for hydrophobic
contributions (GSA). The GSA can be calculated from the solvent accessible surface area (SASA) equation (2).
∆G0solv = ∆G0elect, ε =80 - ∆G0elect, ε =1 +∆G0
(2)
hydrophobic
The free energy in vacuum can be approximated as the average interaction energy between receptor and ligand,
and putting the entropy change upon binding into consideration if desired equation (3).
∆G0Gas =∆E0MD -T. ∆S0NMA (3)
3. Results and Discussion
3.1. Chemistry
The chlorinated chalcone (TC1-TC11) derivatives were efficiently synthesized according to the pathway
shown in Scheme 1. The chemical structures of the compounds have been characterized by means of their 1H
NMR, 13C NMR, mass spectroscopic datas and elemental analysis. 1H-NMR spectrum showed different types of
protons corresponding to the signals of α-β unsaturated unit, chlorine substituted thiophene and the mono
substituted phenyl system. It has been noted that, the up field proton of α-carbon and down field proton of β-
carbon coupled with methine proton with coupling constants (15.2-15.8 Hz). This suggests the presence of trans
configuration in the chlorinated thienyl chalcones. The protons belonging to the to the aromatic ring and
thiophene ring are observed with the expected chemical shift and integral value. 13C NMR displayed carbonyl
carbon of (TC1-TC11) between δ 180.53–181.82. The characteristic peaks were observed in the mass spectra of
the synthesized compounds. The presence of chlorine atom in all the compounds showed a characteristic [M+2]
isotope peaks. The presence of [M+4] isotope peak in compound TC8 clearly showed the presence of two
chlorine atoms.
3.2. Biochemistry

3.2.1. MAO inhibition studies
Taking into the account of previous studies of chalcones on MAO inhibition, our current work is focused on
modifying the molecular structure of very potent MAO-B inhibitors by introducing new functional groups at the
para position on the B ring of chlorinated thienyl chalcones to improve their activities. All the newly synthesized
compounds were investigated for their potential hMAO inhibitory activity using recombinant hMAO-A and
hMAO-B. Enzyme activities were determined by a fluorimetric method using the Amplex Red MAO assay kit.
Novel compounds and reference inhibitors (moclobemide, selegiline and lazabemide) were treated with Amplex
Red reagent, and it was shown that the test compounds did not interfere with the measurements. Test compounds
either did not interact with resorufin since the fluorescence signal did not change when the test and reference
compounds were treated with various concentrations of resorufin. The inhibition constant (Ki) and selectivity
indexes (SI) of the titled compounds are depicted in the Table 1. Specific enzyme activities were calculated as
169.33±8.98 pmol/mg/min (n=3) for hMAO-A and 140.22±6.05 pmol/mg/min (n=3) for hMAO-B.
With the exception of TC7, which was a selective MAO-A inhibitor, all derivatives inhibited hMAO-B
potently and selectively with competitive mode of inhibition and some structure-activity relationships (SARs)
were inferred from the data of enzymatic experiments. The most potent compound TC6 with an ethyl group at
the para position on the phenyl system showed the best activity and higher selectivity towards hMAO-B with Ki
and SI values of 0.31±0.02 µM and 16.84. Among the synthesized chlorinated thiophene chalcones, presence of
lipophilic electron donating groups such as ethyl, methyl, methoxyl and dimethylamino groups at the para
position on the phenyl ring were found to be selective towards the inhibition of MAO-B. Presence of halogen
such as chlorine and bromine atoms were also showed favourable activity towards MAO-B. Introduction of
deactivating nitro group shifted potent and selective towards MAO-A with a Ki value of 0.49±0.02 µM. By
analysing hMAO-B inhibition potencies of the chlorinated thiophene chalcones derivatives, it is clear that
electron donating substitutions at the para position on the phenyl ring is preferable over substitutions with
electron withdrawing nitro and trifluoromethyl groups. The following order of MAO inhibitory activity was
observed for the chlorinated thienyl chalcones:
hMAO-A:4-NO2>4-Br>4-Cl>4-N-(CH3)2>4-CH3>>4-F>4-OCH3>4-H>4-CF3>4-C2H5>4OH
hMAO-B: 4-C2H5 > 4-N-(CH3)2>4-CH3>4-OCH3>4-Br>4-Cl>4-H>4-OH>4-CF3>4-F>4-NO2
3.2.2. Kinetic studies
Kinetic analyses were carried out for most potent MAO-B inhibitor TC6 from this series. The purpose of this
experimentation was to ascertain the mode of MAO-B inhibition by chlorinated thienyl chalcones. A set of

Lineweaver-Bruke plots were constructed in the absence and presence of various concentrations of compound
TC6. The observation that the lines were linear and intersects on the y-axis suggests that TC6 interacts with the
catalytic site of hMAO-B, with a competitive mode of inhibition (fig. 2). The replot of the slopes versus
inhibitor concentration is shown in (fig. 3) and the Ki was estimated as 0.31 µM for TC6.
3.2.3. Reversibility studies
Reversible MAO-B inhibitors have significant advantages over the irreversible inhibitors for the management
of Parkinson’s disease [7]. Taking into the advantage of this drug design aspect, the goal of our study is to design
and development of some new class of reversible type of MAO-B inhibitors. All tested synthesised compounds
inhibited the hMAO isoforms reversibly. Table 2 presents the reversibility of hMAO-B inhibition with the novel
compounds. The reversibility of MAO inhibition by the new derivatives was investigated by measuring the
recoveries of MAO activities after dialysis of enzyme-inhibitor mixtures. MAO isoforms were incubated in the
presence of the representative inhibitors, at concentrations equal to 5 × IC50, for a period of 15 min and
subsequently dialysed for 24 h. As shown in Table 2, MAO-B inhibition by compound TC6 is completely
reversed after 24 h of dialysis. As shown in the table 2, after similar preincubation and dialysis of MAO-B with
the irreversible inhibitor selegiline, the enzyme action was not retrieved. Results suggest that the TC series are
reversible inhibitors of hMAO and have considerable advantages compared to irreversible inhibitors which may
possess serious pharmacological side effects.
3.3. Cytotoxicity studies
In vitro cytotoxicity of the synthesized compounds is tested in Hep2 cells at three different concentrations (1–
100 µM). The results showed that all the compounds presented in the current study are completely non-toxic
with 74-88 % viable cells to hepatic cells at 100 µM concentration. At 10 µM, only the difference between the %
viability values of the compounds TC5, TC7 and TC11 were found to be statistically significant at p>0.05
(Table 3).
3.4. Molecular docking studies
Several researchers preferred to use the PDB of MAO co-crystallized with noncovalent ligands (2Z5X,
2V60, 2BK3) [55, 56] for molecular docking studies. The recent report of Badavth et al., suggested that the
experimental and predicted (Estimated Ki value) values did not correlate well with these [57]. Hence, we
adopted the earlier protocol reported by our group with 2BXR and 2BYB [58].

The 5-chloro thiophene nucleus of TC6 is located within the substrate cavity of the MAO-B enzyme, in close
proximity of the FAD cofactor (Fig. 4). Since the entrance cavity is reported to be a highly hydrophobic space, it
may be expected that an enhancement of the lipophilicity of the ethyl substitution on ring B in chlorinated
thienyl chalcone will result in more productive Van der Waals interactions with the entrance cavity, and thus an
enhancement in binding affinity. This may as well explain the observed enhancements of MAO-B inhibition
potency of TC6 compared to the unsubstituted homologue TC1.
The details of the component energies of all the ligands towards the active site of MAO-A and B are
presented in table 4. This revealed a factor that an increase in the VdW energy of the ligands showed increase in
potency as well as selectivity toward hMAO-B [58]. Compound TC6 was found to be the most potent MAO-B
inhibitor among the series with VdW energy of -10.44. The binding interaction of TC6 is majorly stabilized by
the π-π stacking interaction between the ethyl substituted phenyl ring and the aromatic ring of Tyr-435.
3.5. Molecular dynamics study
3.5.1. Binding energy analysis
Molecular dynamics study is a very important tool to understand the stability and the binding mode of the
ligand-receptor interactions. It also helps to understand the dynamics of the protein, which represent the true
nature of protein better than ordinary docking, which does not completely consider the dynamic of protein. In
addition to normal molecular dynamics, we employed all-atom enhanced sampling method of accelerated
molecular dynamics (AMD) in order to understand the possible rare events that can take place in the interaction
of the ligand TC6 with the two receptors after a much longer molecular dynamics [59]. AMD is an important
method, because of the ability to accelerates and extends the time scale in molecular dynamics simulations
through addition of a bias potential to the true potential such that will enhance the escape rates from potential
wells. This method has been successfully applied to investigate conformational changes in proteins that typically
occur on the millisecond time scale [60].
The root means square displacement (RMSD) of the protein backbone residues during the course of
molecular dynamics (MD) and the changes in the potential (EPTOT) energy of the complexes MAO-A-TC6 and
MAO-B-TC6 are shown in Fig. 5. The RMSD of the two complexes shows that stable conformational changes
where obtained for both complexes during the normal MD and AMD methods. The total potential energy plots
for normal and AMD are within the same range and have similar feature which is an indication that the

accelerated molecular dynamics does not change the potential surface of the systems. The EPTOT plot of MAO-
A is characterized with higher energy level compare to MAO-B. The conformational changes in complex MAO-
B is more significant compare to complex MAO-A which are characterized with higher and lower RMSD
respectively.
The results from the MM-PBSA and its complementary MM-GBSA method for the binding analysis of ligand
TC6 with MAO-A and MAO-B are shown in Table 1. The two methods of binding studies show that TC6 have
stronger affinity for MAO-B than MAO-A during the normal MD which completely agrees with the
experimental result. However, during the AMD, a significant decrease in the interacting free energy was
obtained compare to normal MD and only MM-GBSA shows a better interaction of TC6 with MAO-B compare
to MAO-A (Table 1 and Fig. 6) while MM-PBSA completely disagree with experimental result. The favourable
interaction of TC6 with MAO-B is from the higher change ΔEelec and ΔGgas. As estimated from MM-GBSA, the
difference in the binding free energy of the interaction of TC6 with MAO-B compare to MAO-A is -5.474
kcal/mol in normal MD and -1.624 kcal/mol in AMD. Since AMD simulation method is able to account for the
possible rare events of the protein complex, the significant decrease in the activities of the TC6 interacting with
both MAO-A and MAO-B is an indication of possible reduction in the efficiency of TC6 as a limiting factors
that can arise from huge conformational changes of the receptor. The decomposition of changes in the binding
energy base on the residual contribution is shown in Table 2. As observed in residue decomposition for the
interaction of TC6-MAO-A, the first five residues which were ranked during the normal MD to have the highest
energy change (ILE 165, ASN 166, PHE 193, TYR 389, and TYR 426) are also ranked as top five in its AMD
but not in the same order. In TC6-MAO-B, only three residues GLN 205, TYR 397 and TYR 434 out of the first
five that were ranked during the normal MD to have the highest energy contribution to binding were also ranked
during the AMD.
3.5.2. Study of conformational changes
An insight into the structural distribution and the conformational changes during the MD was obtained using
the principal components analysis (PCA) which is orthogonal eigenvectors. The PCA is very significant because
it gives special insight into the dynamics and conformational changes in both MAO-A and MAO-B as shown in

Fig. 7a. The average structures for the three predicted conformational changes based on the PCA analysis of the
normal MD are shown in Fig. 7b. The average geometries of the three groups of conformations clearly show that
the changes are predominantly the loop region of the receptors (7b). The region of the PCA that has the lowest
energy are represented are circled round in the plot of changes in the potential energy (7c). The first three PC
(PC-1, PC-2 and PC-3) captured 39% in MAO-A but 41.4% in MAO-B of the total variance in MD trajectory of
the complexes. During the MD, a periodic jump in the conformation was observed for the complexes as clearly
shown by the continuous colour scale of the PC from blue to red to green but there is no significant
conformational change of α-helices and β-helices two protein backbones. This is more significant changes in the
binding position of TC6 from the averages geometries of the three clusters of conformations for MAO-A
compare to MAO-B as suggested from PCA analysis (Fig. 7). The highest value for the residual fluctuations
from the PCA analysis for normal MD for MAO-A was a little above 0.12 while that of MAO-B was a little
above 0.15 which is a clear indication that that there is overall more residual conformational change in MAO-B
compare to MAO-A.
Using the results from the AMD, the conformational changes of complexes MAO-A and MAO-B are shown
in the Fig. 8 and 9 respectively. Both the root means square fluctuation (RMSF) and the PCA (PC1 and PC2)
show the high level of residual fluctuations in MAOA are in the region 200-250 residues while in MAOB are
found in the region 450-495residues. The three predicted conformational changes are show in black, red and
green colour (8c and 9c). The free energy surface was constructed using python script for reweighting of AMD
simulations (8e and 9e) and also from change in the potential energy (8f and 9f) [61]. The two free energy
surfaces clearly shown that the best conformation for MAO-A corresponds to black colour region of PCA while
that of MAO-B correspond to green region. The structural comparisons of the best conformation in MAO-A and
MAO-B during the AMD with that of normal M-D are shown in Fig. 10.
The observed conformational changes in MAO-A are mainly loop region while it involves both the α-helices
and loop in MAO-B. Also higher levels of hydrophobic interactions are observed in the ligand interaction with
MAO-B compare to MAO-A (10a and 10b). The results suggest that complex TC6-MAO-B is prone to higher
conformational changes as a possible rare event than complex TC6-MAO-A (Fig. 10) which is also supported
with more significant reduction in its interacting energy compare to MAO-A during the AMD (Table 5). This is
further supported from the observed higher values of RMSF (≈ 4.0, Fig. 9a), PC1 and PC2 residual fluctuations
(≈ 0.25, Fig. 9b) and the fluctuation of the three groups of predicted conformational changes (≈ 3.5, Fig. 9d) for

the TC6-MAO-B interaction compare to what was observed for the corresponding parameters in Fig. 8 for the
interaction of TC6-MAO-A.
The residue-residue correlation plots were obtained by computing dynamical cross-correlation matrix
(DCCM) according to the following equation (4) [62].
c i, j


C i, j 
(4)


c i,i 1/2 c j, j
1/2




The ‘C(i,j)’ represent the covariance matrix element of protein fluctuation between residues ‘i’ and ‘j’. The
plots of the dynamical cross-correlation motions (DCCM) for the TC6-MAO-A and TC6-MAO-B are shown in
Fig. 11 and their graphical representation in 3D showing only the most significant networks of correlation and
anticorrelation (|C(i,j)| ≥ 0.6) . In the two complexes, most of the observed DCCM especially for |C(i,j)| ≥ 0.7
were mainly from the neighbouring residues (intradomain correlation) that form a secondary structure while very
few were from distant amino acids belonging to different regions or domains (interdomain correlation). A higher
level of locally coupled motions was observed for the α-helices and β-helices residues with their surroundings
compare to the loop region. Both TC6-MAO-A and TC6-MAO-B show a significant level of correlation but a
higher networks of correlation was observed for TC6-MAO-B compare to TC6-MAO-A. Also, TC6-MAO-A
show a significant level of anticorrelation (blue lines) during the dynamics of its atoms which was found to be
between the β-sheet and the loop.
The molecular dynamic simulation shows a better interaction of TC6 with MAO-B compare to MAO-A. This
is confirmed from the results of the binding free energy using MM-PBSA and MM-GBSA during the normal
MD but only MM-GBSA confirms this observation during the AMD simulation. Using AMD to study the
possible rare events in the complexes, the results suggest that complex TC6-MAO-B is prone to a more
significant conformational changes than TC6-MAO-A which can significantly reduce the binding affinity of the
inhibitor. The molecular dynamics of the two complexes also give further information about the differences in
binding mode of TC6. Some of the observed differences in binding of TC6 to MAO-A compare to MAO-B are
reduction in the size of binding site, higher RMSD of the protein backbone residues, stronger fluctuation of the
potential energy, higher values of the RMSF and PCA of the residues though over lower range of residues. These
properties are clear indication that the binding of TC6 to MAO-A is weaker compare to its binding with MAO-
B.

4. Conclusion
The present study discovers a number of potent new MAO-B inhibitors among the heterocyclic chlorinated
thiophene based chalcone class of compounds. Experimental results further revealed that all the heterocyclic
chalcones under the present study are reversible type MAO inhibitor. The molecules here reported may be
considered promising hit compounds for the discovery of new neuroprotectants and for the development of
multi-target directed ligands having MAO-B inhibition as the core activity. The finely tuned exploration of the
various groups at the para position on the ring B of chlorinated chalcone allowed a precise development of a new
class of MAO-B inhibitors. The lead chalcones can be further optimized for the preparation of 2-pyrazolines, a
promising structural scaffold for the development of inhibitors of MAO-A and B.
Acknowledgements
The authors thankful to IIRBS, Mahatma Gandhi University, Kottayam, India and SAIF-IIT, Chennai for
carrying out the spectral analysis. The authors also thanks to the School of Pure & Applied Physics, M.G.
University, Kottayam, India for X-ray diffraction analysis.
References
[1] R.H. Haung, R, Faulkner, J. Biol. Chem. 256 (1981) 9211–9215.
[2] J.C. Shih, K. Chen, M.J. Ridd, Annu. Rev. Neurosci. 22 (1999) 197-217.
[3] M. Yamada, H. Yasuhara, Neurotoxicology 25 (2004) 215–221.
[4] D.E. Edmondson, Curr. Pharm. Des. 20 (2014) 155-160.
[5] J.M. Andrews, C.B. Nemeroff, Am. J. Med. Chem. 97 (1994) 24S-32S.
[6] B. Drukarch, F.L. van Muiswinkel, Biochem. Pharmacol. 59 (2000) 1023−1031.
[7] S. Carradori, R. Silvestri, J. Med. Chem. 58 (2015) 6717-6732.
[8] S. Tanaka, Y. Kuwai, M. Tabata, Planta Med. 53 (1987) 5-8.
[9] H. Haraguchi, Y. Tanaka, A. Kabbash, T. Fujioka, T. Ishizu, A. Yagi, Phytochemistry 65 (2004) 2255–2260.
[10] X. Pan, L. D. Kong, Y. Zhang, C. H. Cheng, R. X. Tan, Acta Pharmacol. Sinica 21 (2000) 949–953.
[11] 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.
[12] S.J. Robinson, J.P. Petzer, A. Petzer, J.J. Bergh, A.C.U. Lourens, Bioorg. Med. Chem. Lett. 23 (2013) 4985-
4989.

[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, Bioorg.
Med. Chem. 21 (2013) 7890-7897.
[14] N. Morales-Camilo, C.O. Salas, C. Sanhueza, C. Espinosa-Bustos, S. Sepúlveda-Boza, M. Reyes-Parada, F.
Gonzalez-Nilo, A. Fierro, Chem. Biol. Drug Des. 85 (2015) 685-695.
[15] S. Zaib, S.U.F. Rizvi, S. Aslam, M, Ahmad, S.M.A. Abid, M, al-Rashida, J. Iqbal, Med. Chem. 11 (2015)
580-589.
[16] S. Zaib, S.U.F. Rizvi, S. Aslam, M. Ahmad, M. al-Rashida, J. Iqbal, Med. Chem. 11 (2015), 497-505.
[17] B. Mathew, G.E. Mathew, G. Uçar, I. Baysal, J. Suresh, J. K. Vilapurathu, A. Prakasan, J. K. Suresh , A.
Thomas, Bioorg. Chem. 62 (2015) 22-29.
[18] B. Mathew, G. Uçar, S. Yabanoğlu-Çiftçi, I. Baysal, J. Suresh, G.E. Mathew, J.K. Vilapurathu, A.M.
Nadeena, P. Nabeela, V. Lakshmi, A. Haridas, F. Fathima, Lett. Org. Chem. 12 (2015) 605-613
[19] 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. 23 (2015) 6486-6496.
[20] C. Minders, J.P. Petzer, A. Petzer, A.C.U. Lourens, Bioorg. Med. Chem. Lett. 25 (2015) 5270-5276.
[21] A. Hammuda, R. Shalaby, S. Rovida, D. E. Edmondson, C. Binda, A. Khali, Eur. J. Med. Chem. 114 (2016)
162-169.
[22] B. Mathew, A. Haridas, G. Uçar, I. Baysal, M. Joy, G.E. Mathew, B. Lakshmanan, V. Jayaprakash,
ChemMedChem. (2016).doi:10.1002/cmdc.201600122.
[23] S. Carradori, M. D’Ascenzio, P. Chimenti, D. Secci, A. Bolasco, Mol. Divers. 18 (2014) 18:219-243.
[24] B. Mathew, A. Haridas, J. Suresh, G.E. Mathew, G. Ucar, V. Jayaprakash, Cent. Nerv. Syst. Agents Med.
Chem. (2016) doi:10.2174/1871524915666151002124443.
[25] G.M. Sheldrick, Acta Cryst. C71 (2015) 3-8.
[26] L.G. Farrugia, J. Appl. Cryst. 45 (2012) 849-854.
[27] A.L. Spek, Acta Cryst. D65 (2009) 148-155.

[28] M.C. Anderson, F. Hasan, J.M. McCrodden, K.F. Tipton, Neurochem. Res. 18 (1993) 1145–1149.
[29] 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.
[30] M. Yáñez, N. Fraiz, E. Cano, F. Orallo, Biochem. Biophys. Res. Commun. 344 (2006) 688–695.
[31] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254.
[32] 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.
[33] M.B. Hansen, S.E. Nielsen, K. Berg, J. Immunol. Meth. 119 (1989) 203-210.
[34] C.F. Wu, R. Bertorelli, M. Sacconi, G. Pepeu, S. Consolo, Neurobiol. Aging, 9 (1988) 357-361.
[35] 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.
[36] L. De Colibus, M. Li, C. Binda, A. Lustig, D.E. Edmondson, A. Mattevi, Proc. Natl. Acad. Sci. USA 102
(2005) 12684–12689.
[37] 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.
[38] A.W. Schuttelkopf, D.M. van Aalten, Acta Crystallogr. D Biol. Crystallogr. 60 (2004) 1355–1363.
[39] B. Mathew, J. Suresh, S. Anbazhagan, S. Dev. Biomed. Aging Pathol. 4 (2014) 327-333.
[40] A.W. Götz, M.J. Williamson, D. Xu, D. Poole, S. Le Grand, R.C. Walker, J. Chem. Theory Comput. 8
(2012) 1542–1555.
[41] K. Lindorff-Larsen, S. Piana, K. Palmo, P. Maragakis, J.L. Klepeis, R.O. Dror, D.E. Shaw, Proteins: Struct.,
Funct., Bioinf. 78 (2010) 1950–1958.
[42] F.V. Dupradeau, A. Pigache, T. Zaffran, C. Savineau, R. Lelong, N. Grivel, D. Lelong, W. Rosanski, P.
Cieplak, Phys. Chem. Chem. Phys. 12 (2010) 7821-7839.

[43] W.L. Jorgensen, J. Chandrasekhar, J.D. Madura, R.W. Impey, M.L. Klein, J. Chem. Phys. 79 (1983) 926–
935.
[44] U. Essmann, L. Perera, M.L. Berkowitz, T. Darden, H. Lee, L.G. Pedersen, J. Chem. Phys. 103 (1995)
8577–8593.
[45] J.P. Ryckaert, G. Ciccotti, H.J. Berendsen, J. Comput. Phys. 23 (1977) 327–341.
[46] B.J. Grant, A.P.C. Rodrigues, K.M. Elsawy, J.A. McCammon, L.S.D. Caves, Bioinformatics, 22 (2006),
2695-2696.
[47] W. Humphrey, A. Dalke, K. Schulten. J. Mol. Graph. 14 (1996) 33–38.
[48] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, T.E. Ferrin, J.
Comput. Chem. 25 (2004) 1605–1612.
[49] Schrodinger LLC. 2015. “The PyMOL Molecular Graphics System, Version 1.8.
[50] R.A. Laskowski, M.B. Swindells, J. Chem. Inf. Model. 51 (2011) 2778-2786.
[51] P.A. Kollman, I. Massova, C. Reyes, B. Kuhn, S. Huo, L. Chong, M. Lee, T. Lee, Y. Duan, W. Wang, Acc.
Chem. Res. 33 (2000) 889–897.
[52] I. Massova, P.A. Kollman, Perspect. Drug Discovery Des. 18 (2000) 113–135.
[53] V. Tsui, D.A. Case, Biopolymers, 56 (2000) 275–291.
[54] A. Onufriev, D. Bashford, D.A. Case, J. Phys. Chem. B, 104 (2000) 3712–3720.
[55] F. Chimenti, A. Bolasco, D. Secci, P. Chimenti, A. Granese, S. Carradori, M. Yáñez, F. Orallo, F. Ortuso,S.
Alcaro, . Bioorg. Med. Chem. 18 (2010) 5715−5723.
[56] N. Desideri, A. Bolasco, R. Fioravanti, L. Proietti Monaco, F. Orallo, M. Yáñez, F. Ortuso, S. Alcaro, J.
Med. Chem. 54 (2011) 2155−2164.
[57] V. N. Badavath, I. Baysal, G. Ucar, B. N. Sinha, V. Jayapraksh, ACS Med. Chem. Lett. 7 (2016) 56-61.
[58] V. N. Badavath, I. Baysal, G. Ucar, S. K. Mondal, B. N. Sinha, V. Jayapraksh, Arch. Pharm. Chem. Life
Sci. 349 (2016) 9–19.

[59] D. Hamelberg, J. Mongan, J.A. McCammon, J. Chem. Phys. 120 (2004) 11919-11929.
[60] L.C.T. Pierce, R.S. Ferrer, A.F. de Oliveria, J.A. McCammon, R.C. Walker. J. Chem. Theory Comput. 8
(2012) 2997-3002.
[61] Y. Miao, W. Sinko, L. Pierce, D. Bucher, R.C. Walker , J.A McCammon, J. Chem. Theory Comput. 10
(2014) 2677-2689.
[62] P.H. Huenberger, A.E. Mark, W.F. van Gunsteren, J. Mol. Biol. 252 (1995) 492-503.

Fig 1. ORTEP diagram of compound TC6 [lead molecule]

Fig.2. Lineweaver-Burk plots of hMAO-B activity in the absence and presence of various concentrations of
compound TC6

Fig. 3. The graph of the slopes of Lineweaver-Burk plots versus inhibitor concentrations. Ki was calculated as
0.31 µM

Fig. 4. Interaction of compound TC6 with the hMAO-B (PDB: 2BYB) active site: Yellow mesh indicates π-π
stacking interaction

Classical
AMD
Fig. 5. The potential energy (EPTOT) and the RMSD of all the mass except hydrogen atoms obtained from the
trajectories of the normal MD and AMD

-21.983
-21.194
-25.448
-20.157
-37.245
-35.05
-42.714
-36.674
Methods
Fig. 6. The changes in the total free energy of the TC6 interaction with MAO-A and MAO-B using the
MM-PBSA and MM-GBSA methods from the trajectory of the normal MD and AMD.