Monoamine oxidase inhibitory activity of 2-aryl-4H-chromen-4-ones

Article abstract of DOI:10.1016/j.bioorg.2014.11.008

A series of twenty 2-aryl-4H-chromen-4-one (flavones) derivatives (3a-3s) were synthesized and tested for hMAO inhibitory activity. Fifteen compounds (3a, 3c, 3e-3h, 3j-3p, 3r, 3s) were found to be selective towards MAO-B, while 3d was selective towards MAO-A, and 3b, 3i and 3q were non-selective. Experimental Selectivity Index for MAO-B ranges from 2.0 (3g, 3p) to 30.0 (3j). Compound 3j, which is carrying 3,4-di-OMeC₆H₃ groups at R position on the molecule, was found to be potent MAO-B inhibitor amongst the fifteen with K_i value for MAO-B of 0.16 ± 0.01 μM comparable to that of standard drug, Selegiline (K_i for MAO-B is 0.16 ± 0.01 μM). Compound 3j also appeared as the most selective MAO-B inhibitor according to its best selectivity index (30.0), which is comparable to that of Selegiline (SI_MAO-B = 35.0). Molecular docking and molecular dynamics simulation studies were carried out using Autodock-4.0 and Amber12 to understand the molecular level interaction and energy relation of MAO isoforms with selective inhibitors (3d and 3j). Simulation results are in good agreement with the experimental results. Leads identified may further be explored to develop potent isoform specific inhibitors of MAO.

Full text of DOI:10.1016/j.bioorg.2014.11.008

Bioorganic Chemistry 58 (2015) 72–80
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Monoamine oxidase inhibitory activity of 2-aryl-4H-chromen-4-ones
Badavath Vishnu Nayak a,1, S. Ciftci-Yabanoglu b,1, Soumendranath Bhakat c,1, Ajay Kumar Timiri a,
Barij N. Sinha a, G. Ucar b, Mahmoud E.S. Soliman c, Venkatesan Jayaprakash a,d,
⇑
a Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi, Jharkhand 835 215, India
b Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, Sıhhiye 06100, Ankara, Turkey
c School of Health Sciences, University of KwaZulu-Natal, Westville, Durban 4001, South Africa
d Valens Pharma Services, Regus Citi Centre, Level 6, Chennai Citi Centre, 10/11, Dr. Radhakrishnan Salai, Chennai, Tamil Nadu 600 004, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 September 2014
Available online 3 December 2014
A series of twenty 2-aryl-4H-chromen-4-one (flavones) derivatives (3a–3s) were synthesized and tested
for hMAO inhibitory activity. Fifteen compounds (3a, 3c, 3e–3h, 3j–3p, 3r, 3s) were found to be selective
towards MAO-B, while 3d was selective towards MAO-A, and 3b, 3i and 3q were non-selective. Experi-
mental Selectivity Index for MAO-B ranges from 2.0 (3g, 3p) to 30.0 (3j). Compound 3j, which is carrying
3,4-di-OMeC6H3 groups at R position on the molecule, was found to be potent MAO-B inhibitor amongst
Keywords:
Human monoamine oxidase
Flavones
Enzyme inhibitors
Molecular docking
Molecular dynamics
the fifteen with Ki value for MAO-B of 0.16 0.01
lM comparable to that of standard drug, Selegiline (Ki
for MAO-B is 0.16 0.01 M). Compound 3j also appeared as the most selective MAO-B inhibitor accord-
l
ing to its best selectivity index (30.0), which is comparable to that of Selegiline (SIMAO-B = 35.0). Molecular
docking and molecular dynamics simulation studies were carried out using Autodock-4.0 and Amber12
to understand the molecular level interaction and energy relation of MAO isoforms with selective inhib-
itors (3d and 3j). Simulation results are in good agreement with the experimental results. Leads identified
may further be explored to develop potent isoform specific inhibitors of MAO.
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
to the establishment of strict dietary restrictions by US-FDA [3].
Tricyclic antidepressants and Selective Serotonin Reuptake Inhibi-
Monoamine oxidases are responsible for the oxidative deamina-
tion of neurotransmitters and dietary amines. They contain flavin-
adenosine-dinucleotide (FAD) as co-enzyme and are located on the
outer mitochondrial membrane. There exists two different isoform,
MAO-A and MAO B, classified based on their specificity towards
their substrate and their selective inhibitors [1,2]. Serotonin,
adrenaline and noradrenaline are substrates for MAO-A, while
dopamine, phenylethylamine and benzylamine are substrates for
MAO-B [2]. Iproniazide was the first drug approved for the treat-
ment of depressive illness during 1950s and that was followed
by imipramine, phenelzine, isocarboxazid and tranylcypromine.
They were all found to be non-selective towards MAO isoforms
as well as not specific towards peripheral and brain MAO. Due to
which cheese-effect (Tyramine-induced hypertensive crisis) was
reported with use of tranylcypromine during mid 1960s, leading
tors emerged as an alternative to first generation antidepressants
as they can be administered without any dietary restrictions [4].
Research during the past two decades clearly established the ther-
apeutic utility of isoform selective and tissue specific MAO inhibi-
tors in the treatment of depressive illness (MAO-A inhibitors) and
neurodegenerative disorders (MAO-B inhibitors).
Chalcone derived pyrazolines were explored for their MAO
inhibitory and antidepressant activity by many research groups.
Few reports on MAO inhibitory activity [5–7] and antidepressant
activity of chalcones [8–10] were also available. Flavones another
oxygen containing heterocyclics derived from chalcones were also
reported to exhibit antidepressant [11–13] and MAO-inhibitory
activity [14–17]. Guided by these facts our group has synthesized
a series of 2-aryl-4H-chromen-4-one (Flavones) and tested them
for hMAO inhibitory activity.
Abbreviations: MAO, monoamine oxidase; hMAO, human MAO; rMAO, rat MAO;
SAR, structure–activity-relationship; bb, backbone; sc, side-chain.
2. Experimental
⇑
Corresponding author at: Department of Pharmaceutical Sciences and Tech-
2.1. Materials and methods
nology, Birla Institute of Technology, Mesra, Ranchi, Jharkhand 835 215, India.
E-mail addresses: gulberk@hacettepe.edu.tr (G. Ucar), venkatesanj@bitmesra.ac.
in (V. Jayaprakash).
Chemicals and solvents were of reagent grade and purchased
from Sigma–Aldrich/Merck/CDH/Rankem. Completion of reaction
1
These authors contributed equally.
http://dx.doi.org/10.1016/j.bioorg.2014.11.008
0045-2068/Ó 2014 Elsevier Inc. All rights reserved.

V.N. Badavath et al. / Bioorganic Chemistry 58 (2015) 72–80
73
was monitored on TLC plates (Merck). Melting points were deter-
mined using OPTIMELT (Stanford Research System) and are uncor-
rected. Intermediates were characterized by their FT-IR spectra
(FTIR-8400S-Schimadzu). Final compounds were characterized by
their 1H NMR (300 MHz, ECX-500, JEOL and 400 MHz, VNMRS400),
13C NMR (400 MHz, VNMRS400), in either CDCl3 or DMSO-d6 as a
solvent. The coupling constants (J) are expressed in hertz (Hz)
and chemical shifts (d) are reported in parts per million (ppm)
units relative to TMS. Mass spectra of the final compounds were
recorded by WATERS-Q-T of Premier-HAB213 using the ESI-MS
Electro spray Ionization technique. Molecular modelling studies
were carried using Maestro-8.5 (Schrodinger LLC) [18], AutoDock4
[19], Chimera [20] and Amber12 [21]. Microsoft Excel and Origin-
6.0 were used for generating graphs. Molecular Docking studies
were carried out on RHEL-5.0 Operating system installed on Dell
Precision workstation with Intel core 2 quad processor and 8 GB
RAM. While, Molecular dynamics (MD) simulations were run on
a super computer taking in account 5 gpus. Molecules were pre-
pared for MD simulation through PRODRG [22] and REDUCE [23]
web servers.
d (ppm) 7.05 (m, 1H), 7.09 (s, 1H, @CH), 7.2 (t, 1H), 7.25 (t, 1H),
7.65 (m, 1H), 7.9 (m, 1H), 7.95 (d, 1H), 8.05 (d, 1H), 8.2 (d, 1H),
10.65 (s, 1H, -OH).
2.2.2.5. 2-(3-Hydroxyphenyl)-4H-chromen-4-one (3e). Yield: 40%;
mp: 210 °C (Lit. 209–211 °C) [27]; 1H NMR (300 MHz, DMSO-d6)
d (ppm) 6.89 (s, 1H, @CHA), 7.28 (m, 1H), 7.4 (d,1H), 7.6 (t, 1H),
7.72 (t,1H), 7.83 (d, 1H), 8.16 (d, 1H), 8.24 (d,1H), 8.82 (s, 1H),
12.5 (s,1H, OH).
2.2.2.6. 2-(4-Hydroxyphenyl)-4H-chromen-4-one (3f). Yield: 50%;
mp: 190 °C (Lit. 186–187 °C) [28]; 1H NMR (400 MHz, DMSO-d6)
d (ppm) 6.85 (s, 1H, @CHA), 6.94 (d, 2H), 7.47 (t, 1H), 7.73–7.80
(m, 1H), 7.95(d, 2H), 8.02(d, 1H), 8.4(d, 1H),10.38 (s, 1H, OH); 13C
NMR (400 MHz, DMSO-d6)
d (ppm): 177.40, 163.59, 161.43,
156.0, 137.43, 134.51, 128.82, 125.79, 125.17, 123.73, 122.01,
118.81, 116.43, 105.24.
2.2.2.7. 2-(2-Methoxyphenyl)-4H-chromen-4-one (3g). Yield: 50%;
mp: 136 °C (Lit. 129–130 °C) [26]; 1H NMR (400 MHz, DMSO-d6):
d (ppm) 3.9 (s, 3H, -OCH3) 7.15 (d, 1H), 7.2 (m, 1H), 7.25 (m, 1H),
7.6 (m, 1H), 8.02 (d, 1H), 8.15 (m, 1H), 8.02 (s, 1H), 8.25 (d, 1H),
8.58 (d, 1H).
2.2. Chemistry
2.2.1. General procedure for synthesis of Chalcones (2a–2s)
To a solution of 20-hydroxy acetophenone (0.01 M) in ethanol
(10 mL) an equimolar quantity of appropriate benzaldehydes was
added and the mixture was maintained at <10 °C. Sodium hydrox-
ide solution (60% in water) was added to the mixture drop wise
with continuous stirring for a period of 30–45 min. The reaction
mixture was then left at room temperature for about 48 h with
occasional shaking. After 48 h, it was poured into ice-cold water
and the pH was then adjusted to pH-2 using 6 N hydrochloric acid.
The yellow precipitate obtained was filtered, washed with water
and dried. The crude product was then recrystallized from metha-
nol to give yellow product [24].
2.2.2.8. 2-(3-Methoxyphenyl)-4H-chromen-4-one (3h). Yield: 65%;
mp: 125 °C (Lit. 130–131 °C) [29]; 1H NMR (400 MHz, DMSO-d6):
d (ppm) 3.8 (s, 3H, -OCH3) 7.1 (s, 1H, @CHA), 7 (d, 1H), 7.5 (m,
2H), 7.62 (s, 1H), 7.7 (d, 1H), 7.82 (m, 2H), 8.03 (d, 1H).
2.2.2.9. 2-(4-Methoxyphenyl)-4H-chromen-4-one (3i). Yield: 60%;
mp: 150 °C (Lit. 158–159 °C) [26]; 1H NMR (400 MHz, CDCl3): d
(ppm) 3.8 (s, 3H, OCH3), 6.75 (s, 1H, @CHA), 7 (d, 2H), 7.4(t, 1H),
7.55 (d,1H), 7.6 (t,1H), 7.8 (d,2H), 8.25 (d,1H).
2.2.2.10. 2-(3,4-Dimethoxyphenyl)-4H-chromen-4-one (3j). Yield:
65%; mp: 167.5 °C (Lit. 175–176 °C) [26]; 1H NMR (400 MHz,
CDCl3): d (ppm) 3.8 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 6.7 (s, 1H,
@CHA), 6.8 (s, 1H), 7 (d,1H), 7.39(t,1H), 7.4 (d,1H), 7.5 (d, 2H),
7.7 (m,2H), 8.2 (d,1H).
2.2.2. General procedure for the synthesis of flavones (3a–3s)
To a solution of Chalcones (2a–2s, 0.015 M) in 50 mL of
dimethyl sulfoxide an equimolar quantity of iodine (0.015 M)
was added. The reaction mixture was then stirred for about 30–
60 min at 140 °C. The reaction mixture was then treated with
aqueous sodium thiosulfate solution (20%) and extracted with
chloroform (3 ꢀ 30 mL). The combined organic phase was then
washed with brine, dried over anhydrous sodium sulphate and
concentrated by in rotary evaporator. The residue was purified
by recrystallization from suitable solvent to obtain desired final
compounds (3a–3t) [25].
2.2.2.11. 2-(4-Methylphenyl)-4H-chromen-4-one (3k). Yield: 70%;
mp: 106 °C (Lit. 110–111 °C) [26]; 1H NMR (400 MHz, DMSO-d6):
d (ppm) 2.2 (s, 1H, -CH3), 7.0 (s, 1H, @CH), 7.25 (t, 1H), 7.4 (m,
2H), 7.45(t, 1H), 7.6 (m, 1H), 8–8.08 (m, 2H), 8.15 (d, 1H, -OH).
2.2.2.12. 2-(4-Nitrophenyl)-4H-chromen-4-one (3l). Yield: 60%; mp:
232 °C (Lit. 234–236 °C) [25]; 1H NMR (300 MHz, CDCl3): d (ppm)
6.71 (s, 1H, @CHA), 7.1 (t, 1H), 7.38 (t, 1H), 7.49(d, 1H), 7.5 (d,
1H), 7.68 (m, 3H), 8.1 (dd, 1H); ESI-MS (m/z): Calculated 267.05,
obsrevd 267.60 (M+1)+.
2.2.2.1. 2-Phenyl-4H-chromen-4-one (3a). Yield: 60%; mp: 95 °C
(Lit. 96–97 °C) [26]; 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.0
(s, 1H, @CHA), 7.15 (d, 1H), 7.3 (t, 1H), 7.5 (t, 1H), 7.61 (m, 1H),
7.82 (m, 1H), 8.1 (m, 1H), 8.15 (d, 1H), 8.2 (m, 1H), 8.25 (d, 1H).
2.2.2.13. 4-(4-Oxo-4H-chromen-2-yl)benzonitrile (3m). Yield: 60%;
mp: 126 °C (Lit. 220–221 °C) [15]; 1H NMR (300 MHz, CDCl3): d
(ppm) 6.85 (s, 1H, @CHA), 7.42 (t, 1H), 7.55 (d, 1H), 7.71 (t, 1H),
7.8 (d, 2H), 8 (d, 2H), 8.2 (d, 1H); ESI-MS (m/z): Calculated,
247.06, observed 248.07 (M+1)+.
2.2.2.2. 2-(2-Chlorophenyl)-4H-1-Benzopyran-4-one (3b). Yield:
70%; mp: 195 °C (Lit. 192–193 °C) [25]; 1H NMR (300 MHz, CDCl3):
d (ppm) 6.63 (s, 1H, @CHA), 7.16 (t, 1H), 7.36–7.45 (m, 1H), 7.47–
7.54 (m, 1H), 7.6 (d, 1H), 7.68 (t, 1H), 7.77 (d, 1H), 7.12 (d, 1H),
8.17–8.23 (m, 1H).
2.2.2.14. 2-(4-Bromophenyl)-4H-chromen-4-one (3n). Yield: 60%;
mp: 173 °C (Lit. 173–176 °C) [30]; 1H NMR (300 MHz, CDCl3) d
(ppm) 6.77 (s, 1H, @CHA), 7.4 (t, 1H), 7.55 (d, 1H), 7.64 (m, 3H),
7.77 (d, 2H), 8.2 (d, 2H); ESI-MS (m/z): Calculated 299.98, observed
300.50 (M+1)+.
2.2.2.3. 2-(4-Chlorophenyl)-4H-chromen-4-one (3c). Yield: 65%; mp:
184 °C (Lit. 186–188 °C) [25]; 1H NMR (300 MHz, CDCl3): d (ppm)
6.78 (s, 1H, @CH), 7.42 (t, 1H), 7.49 (d, 2H), 7.55 (d,1H), 7.69 (t,
1H), 7.82 (d. 2H), 7.19 (d,1H).
2.2.2.15. 2-(Naphthalen-1-yl)-4H-chromen-4-one (3o). Yield: 65%;
mp: 105 °C; (Lit. 107–109 °C) [31]; 1H NMR (300 MHz, CDCl3): d
(ppm) 6.6 (s, 1H, @CHA), 4.5 (t, 1H), 7.56 (m, 2H), 7.68 (t, 1H),
2.2.2.4. 2-(2-Hydroxyphenyl)-4H-chromen-4-one (3d). Yield: 55%;
mp: 203 °C (Lit. 209–210 °C) [26]; 1H NMR (400 MHz, DMSO-d6):

74
V.N. Badavath et al. / Bioorganic Chemistry 58 (2015) 72–80
7.2 (d, 1H), 7.9 (dd, 2H), 8 (d, 1H), 8.1 (dd, 2H), 8.2 (d, 1H); ESI-MS
B were incubated with a single concentration of the synthesized
compounds or the reference inhibitors in a sodium phosphate buf-
fer (0.05 M, pH 7.4) for 1 h at 37 °C. After this incubation period, an
aliquot was stored at 4 °C and used for the measurement of MAO-A
and -B activity. The remaining incubated sample was placed in an
Ultrafree-0.5 centrifugal tube with a 30 kDa Biomax membrane
and centrifuged at 9000ꢀ for 20 min at 4 °C. The enzyme retained
in the 30 kDa membrane was resuspended in a sodium phosphate
buffer at 4 °C and centrifuged again two successive times. After the
third centrifugation, the enzyme retained in the membrane was
resuspended in sodium phosphate buffer (300 mL) and an aliquot
of this suspension was used for MAO-A and -B activity determina-
tion. Control experiments were performed simultaneously (to
define 100% MAO activity) by replacing the inhibitors with appro-
priate dilutions of the vehicles. The corresponding values of per-
cent (%) MAO isoform inhibition was separately calculated for
samples with and without repeated washing.
(m/z): 273.09 (M+1)+.
2.2.2.16. 2-(Naphthalen-2-yl)-4H-chromen-4-one (3p). Yield: 63%;
mp: 155.6 °C (Lit. 148–150 °C) [31]; 1H NMR (300 MHz, CDCl3): d
(ppm) 6.93 (s, 1H, @CHA), 8.4 (s, 1H), 7.22 (d, 1H), 7.9–7.8 (m,
4H), 7.69 (t, 1H), 7.61 (d, 1H), 7.59–7.52 (m, 2H), 7.4 (t, 1H); ESI-
MS (m/z): Calculated 272.08, observed 273.09 (M+1)+.
2.2.2.17. 2-(Anthracen-9-yl)-4H-chromen-4-one (3q). Yield: 70%;
mp: 214 °C; 1H NMR (300 MHz, CDCl3): d (ppm) 6.67 (s, 1H,
@CHA), 7.47–7.54 (m, 6H), 7.7 (t, 1H), 7.95 (dd, 1H), 8.1 (dd, 2H),
8.4 (d, 1H), 8.6 (s, 1H); 13C NMR (400 MHz, CDCl3) d (ppm) 116.2,
118.4, 124.2, 124.9, 125.5, 125.6, 125.9, 126.3, 127.3, 128.7,
129.8, 139.2, 131, 134, 157.3, 16.7, 178.05 ESI-MS (m/z): Calculated
322.10, observed 323(M+1)+.
2.2.2.18. 2-(Thiophen-2-yl)-4H-chromen-4-one (3r). Yield: 56%; mp:
98 °C (Lit. 97–99 °C) [30]; 1H NMR (300 MHz, CDCl3): d (ppm) 6.9
(s, 1H, @CHA), 7.48 (t, 1H), 7.6 (d, 1H), 7.75 (t, 1H), 8.1 (d, 1H),
8.24 (d, 1H), 8.37 (d, 2H); ESI-MS (m/z): Calculated 228.02,
observed 228.60 (M+1)+.
2.4. Molecular docking simulation
In order to understand the molecular level interaction of the
potent molecule 3d and 3j towards MAO-A and MAO-B, molecular
docking studies were carried out using AutoDock4.0 [19]. Docking
protocol reported earlier by our group has been followed with
X-ray crystal structure of hMAO-A (2BXR) and hMAO-B (2BYB)
downloaded from Protein Data Bank (www.rcsb.org) [36]. Protein
preparation was carried out using Protein Preparation Wizard of
Maestro-8.5 (Schrodinger LLC) [37]. Water and covalently linked
ligands were deleted from the proteins and bond order was cor-
rected for FAD. After assigning charge and protonation state,
energy minimization was done using OPLS2005 force-field [38].
Ligands were prepared through PRODRG webserver (http://
davapc1.bioch.dundee.ac.uk/cgi-bin/prodrg) [22]. For docking, grid
parameter file (.gpf) and docking parameter files (.dpf) were writ-
ten using MGLTools-1.4.6 [19]. Receptor grids were generated
using 60 ꢀ 60 ꢀ 60 Grid points in xyz with grid spacing of
0.375 Å. Grid box was centered on N5 atom of FAD. Map types were
generated using autogrid-4.0. Docking was carried out with follow-
ing parameters: number of runs: 50, population size: 150, number
of evaluations: 2,500,000 and number of generations: 27,000, using
AutoDock4.0. Analysis of docking results was done using MGL-
Tools-1.4.6. Top scoring molecule in the largest cluster was ana-
lyzed for its interaction with the protein.
2.2.2.19. 2-(Pyridin-3-yl)-4H-chromen-4-one (3s). Yield: 50%; mp:
153 °C (Lit. 128–130 °C) [30]; 1H NMR (300 MHz, CDCl3): d (ppm)
6.79 (s, 1H, @CHA), 7.35–7.49 (m, 2H), 7.5 (d, 1H), 7.6 (t, 1H),
8.12 (m, 2H), 8.7 (d, 1H), 9.1 (s, 1H); ESI-MS (m/z): Calculated
223.06, observed 223.60 (M+1)+.
2.3. Biochemistry
2.3.1. Determination of hMAO-A and -B activities
The activities of recombinant hMAO-A and hMAO-B were deter-
mined using p-tyramine as common substrate and calculated as
0.18 0.01 nmol/mg/min (n = 3) and 0.12 0.02 nmol/mg/min
(n = 3), respectively. The interactions of the synthesized com-
pounds with hMAO isoforms were determined by a fluorimetric
method described previously [28,32,33]. The production of H2O2
catalyzed by MAO isoforms was detected using a non-fluorescent
AmplexÒ-Red reagent which reacts with H2O2 in the presence of
horseradish peroxidase to produce the fluorescent product resoru-
fin. The reaction was started by the addition of 200 lM Amplex Red
reagent, 1 U/mL horseradish peroxidase, and p-tyramine (concen-
tration range 0.1–1 mM). Control experiments were carried out
using reference inhibitors (Selegiline and Moclobemide). The pos-
sible capacity of compounds to modify the fluorescence generated
in the reaction mixture due to non-enzymatic inhibition was deter-
mined by adding these compounds to solutions containing only the
Amplex Red reagent in a sodium phosphate buffer.
2.5. Molecular dynamics simulation
In order to understand the stability and energetics of the docked
complexes (compound 3d complexed with MAO-A (PDB: 2BXR)
and compound 3j complexed with MAO-B (PDB: 2BYB), we
prompted for a molecular dynamics simulation using GPU version
of Amber12. The water molecules and all non-standard compo-
nents were removed, making no changes in original co-ordinates.
Missing hydrogens were further optimized using REDUCE server
to generate a correct hydrogen bond network [23]. Chimera molec-
ular modelling and visualization suite (https://www.cgl.ucsf.edu/
chimera/) was used for structure preparation and visualization
purpose [20]. All graphs were generated using Origin 6.0 data anal-
ysis tool [39].
The prepared structures were then subjected to an all-atom
unrestrained molecular dynamics simulation in explicit solvent
using GPU based PMEMD programme provided with Amber 12
[21,40]. Protein structures were parameterized and represented
by ff03 variant of AMBER force field [41]. The tLeap module inte-
grated with Amber 12 was used to add hydrogen atoms and coun-
ter ions for neutralization. The geometry and charges of the ligands
were optimized with Gaussian 09 at HF/6-31G⁄ level [42]. Finally
2.3.2. Kinetic experiments
Synthesized compounds were dissolved in dimethylsulfoxide
(DMSO), with a maximum concentration of 1%; in the concentra-
tion range of 1–100 lM. Kinetic data were determined using the
Microsoft Excel package program. The slopes of the Lineweaver–
Burk plots were replotted 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. The protein content
was determined according to the Bradford method [34].
2.3.3. Reversibility experiments
Reversibility of the MAO inhibition with synthesized com-
pounds was evaluated by a centrifugation-ultrafiltration method
[35]. In brief, adequate amounts of the recombinant hMAO-A or

V.N. Badavath et al. / Bioorganic Chemistry 58 (2015) 72–80
75
Scheme 1. Reagents and conditions: (a) R-CHO, aq. NaOH (60%), EtOH, 48 h, rt; (b) DMSO, I2, reflux, 2 h.
entropy, respectively. Understanding of the binding free energy will
help in understanding the selectivity of compound 3d and 3j
towards MAO-A or MAO-B respectively.
ANTECHAMBER module was used to add partial atomic charges for
the ligands using restrained electrostatic potential procedure
(RESP) [43]. All systems were immersed in a orthorhombic box of
TIP3P water molecules such that no atom of either protein or
ligand was within 8 Å from any side of the box [44]. The partial
mesh Ewald (PME) method was employed to calculate long range
electrostatic potentials with a direct space and vdW cut-off of
12 Å [45]. All the systems were initially minimized for 1000 steps
of steepest descent followed by 1000 steps of conjugate gradient
with a restrained potential of 500 kcal/mol Å2 applied to the solute.
A further 100 steps unrestrained minimization of the all the sys-
tems were carried out for 1000 steps using conjugate gradient
algorithm. Prior to minimization, a canonical ensemble MD simu-
lation were carried out for 50 ps, with gradual heating from 0 K
to 300 K with a harmonic restrained of 5 kcal/mol Å2 and a Lange-
vin thermostat with a random collision frequency of 1/ps. The pre-
pared systems were subsequently equilibrated at 300 K using a
NPT ensemble for 500 ps with no restrained imposed and the pres-
sure was maintained at 1 bar. The SHAKE algorithm [46] was
applied to constrain all bond lengths involving hydrogen at their
equilibrium distance, a 2 fs time step and SPFP precision model
[47] was used for all MD runs. Finally a 10 ns production MD run
was performed for all the systems in a NPT ensemble [48] with a
pressure of 1 bar and a coupling constant of 2 ps. Resultant co-
ordinates were saved and analyzed in every 1 ps. Further analysis
of trajectories (RMSD, potential energy) was performed using
PTRAJ module implemented in Amber 12.
3. Results and discussion
3.1. Chemistry
All the compounds were synthesized according to the reactions
outlined in Scheme 1. Chalcones (2a–2s) were prepared through
Claisen–Schmidt condensation of appropriate aromatic/heteroaro-
matic aldehydes with 20-hydroxyacetophenone in 60% aqueous
ethanolic sodium hydroxide solution. The final products (3a–3s)
were obtained by refluxing chalcones (2a–2s) with iodine, dimeth-
ylsulfoxide (DMSO). Chalcones were identified by comparing their
melting point with that of melting point reported in the literature
and by IR spectra. IR peaks due to a,b-carbonyl group and phenolic
hydroxyl group were displayed in the ranges 1610–1699 cmꢁ1 and
3025–3396 cmꢁ1; respectively. Final compounds (3a–3s) were
characterized by 1H NMR, 13C NMR and ESI-MS spectroscopy. Pro-
ton in 3rd position of chromenone appeared as a singlet between d
6.6 and d 7.10, methyl protons (3k) appeared as a singlet at d 2.2,
methoxy protons (3g–3j) appeared as singlet between d 3.80–
3.99 and hydroxyl proton (3d–3f) appeared as singlet between d
8.15–12.5. 13C NMR displayed carbonyl carbon (3f, 3q) between d
177–178. Mass spectra of compounds 3l-3s displayed (M+1)+ peak.
MM-GBSA based end-point energy calculation was performed
to understand free energy differences between two different states
[49–52]. Understanding of binding free energy profile provides an
energetic insight into the process of binding. In this study the bind-
ing free energies of compound 3d and 3j bound with both MAO-
isoforms were calculated using 1000 snapshots taken from 10 ns
MD trajectory at 10 ps intervals. The binding free energy for anal-
ysis was carried out by the following set of equations:
3.2. Biochemistry
All the compounds were screened for their hMAO inhibitory
activity using recombinant hMAO-A and hMAO-B (Table 1). Sixteen
compounds (3a, 3c, 3e–3i, 3j–3p, 3r, 3s) were found to be selective
towards hMAO-B, while one compound (3d) was selective towards
hMAO-A and other two (3b and 3q) were non-selective. Amongst
the hMAO-B inhibitors, the most potent compound was 3j (Ki for
D
D
Gbind ¼ Gcomplex ꢁ ðGreceptor þ Gligand
Gbind ¼ Egas þ Gsol ꢁ T
Þ
ð1Þ
ð2Þ
ð3Þ
ð4Þ
ð5Þ
hMAO-B is 0.16 0.01 lM). Compound 3j also appeared as the
most selective MAO-B inhibitor in this series with SIMAO-B of 30.0,
which is comparable to that of Selegiline, the well-known selective
MAO-B inhibitor (SIMAO-B = 35.0). Compound 3d, having 2-OH-C6H4
group at R position of the molecule, inhibited hMAO-A potently
D
S
Egas ¼ Eint þ Evdw þ Eele
Gsol ¼ GGB þ GSA
and selectively (Ki hMAO-A is 0.52 0.03 lM; SIMAO-A is 11.5).
Fig. 1 displays the SI for MAO-A and MAO-B of the compounds
3a-3s. All tested compounds inhibited the hMAO reversibly. Table 2
indicated the reversibility of hMAO-B inhibition with the com-
pounds 3a–3s. Compound 3d, which inhibited hMAO-A reversibly
is not included in the Table 2. The percentage inhibition (%) of
hMAO-A of compound 3d (200 nM) was calculated as
89.05 5.68 and 8.44 0.61 before and after washing, respectively
whereas the percentage inhibition of hMAO-A inhibition (%) of
moclobemide (200 nM) was calculated as 86.95 4.77 and
9.02 0.55 before and after washing, respectively.
GSA
¼
cSASA
where
DGbind is the total binding free energy, Greceptor and Gligand
correspond to the energies of protein and ligand respectively. Egas
is the gas-phase energy; Eint is the internal energy; and Eele and EvdW
are the Coulomb and Van der Waals energies, respectively. The sol-
vation free energy, denoted by Gsol, can be decomposed into polar
and nonpolar contribution states. The polar solvation contribution,
GGB, is determined by solving the GB equation, whereas, GSA, the
nonpolar solvation contribution is estimated from the solvent
accessible surface area (SASA) determined using a water probe
radius of 1.4 Å. T and S correspond to temperature and total solute
A brief discussion of SAR with respect to MAO-B inhibition has
been presented below. Amongst the fifteen selective MAO-B inhib-
itors, compounds having deactivating functional groups at para
position of the phenyl ring (3c, 3l, 3m, 3n) were found to be

76
V.N. Badavath et al. / Bioorganic Chemistry 58 (2015) 72–80
Table 1
Human MAO inhibitory activity of compounds (3a–3s).
O
O
R
Compound
R-
Experimental
Exp MAO-B SIa Exp MAO-A SIa Inhibition type Reversibility MAO-Selectivity
Ki value MAO-A (
l
M)b Ki value MAO-B ( M)b
l
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
C6H5
2.50 0.12
1.90 0.15
3.90 0.20
0.52 0.03
3.02 0.21
5.06 0.21
3.22 0.25
1.99 0.13
2.64 0.18
1.06 0.09
1.20 0.10
0.60 0.04
5.98 0.26
0.80 0.04
1.05 0.09
1.65 0.11
0.86 0.07
1.80 0.15
0.16 0.01
0.99 0.05
0.20 0.01
0.90 0.05
0.52 0.04
0.39 0.02
1.30 0.11
1.15 0.12
2.91 0.20
1.90 0.16
0.16 0.01
1.40 0.11
2.4
1.6
6.5
0.1
3.8
4.8
2.0
2.3
1.5
30.0
3.4
4.2
9.1
3.8
2.1
2.0
1.6
2.2
3.1
35.63
0.02
0.4
0.6
0.2
11.5
0.3
0.2
0.5
0.4
0.7
0.0
0.3
0.2
0.1
0.3
0.5
0.5
0.6
0.5
0.3
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
MAO-B selective
Non-selective
2-Cl-C6H4
4-Cl-C6H4
2-OH-C6H4
3-OH-C6H4
4-OH-C6H4
2-OMe-C6H4
3-OMe-C6H4
4-OMe-C6H4
MAO-B selective
MAO-A selective
MAO-B selective
MAO-B selective
MAO-B selective
MAO-B selective
MAO-B selective
MAO-B selective
MAO-B selective
MAO-B selective
MAO-B selective
MAO-B selective
MAO-B selective
MAO-B selective
Non-selective
3,4-OMe-C6H3 4.80 0.10
4- Me-C6H4
4-NO2-C6H4
4-CN-C6H4
4-Br-C6H4
Naphth-1-yl
Naphth-2-yl
Anthracen-9-yl 1.88 0.09
Thiophen-2-yl 6.44 0.51
Pyridin-3-yl
3.32 0.20
0.84 0.05
8.21 0.55
2.00 0.16
0.80 0.05
2.55 0.20
3q
3r
3s
MAO-B selective
MAO-B selective
5.93 0.37
5.70 0.05
0.03 0.01
Selegiline
Moclobemide
–
–
0.03
46.66
Irreversible MAO-B selective
Reversible MAO-A selective
a
Selectivity index calculated with Experimental Ki values: It was calculated as Ki (MAO-A)/Ki (MAO-B) for MAO-B and Ki (MAO-B)/Ki (MAO-A) for MAO-A. Selectivity
towards MAO isoforms increases as the corresponding SI decreases.
b
Each value represents the mean SEM of three independent experiments.
Table 2
Reversibility of hMAO-B inhibition with the compounds 3a–3s.
Compounds (200 nM)
hMAO-B inhibitiona (%)
Before washing
After washing
Reversibility
3a
3b
3c
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
84.50 4.91
80.06 4.28
90.76 5.88
87.00 5.22
87.65 4.30
80.02 4.39
85.81 5.06
80.12 5.33
92.19 5.06
86.90 5.54
88.22 5.13
89.00 4.12
87.35 5.28
85.90 5.16
84.13 5.22
78.90 4.10
82.44 5.02
85.66 4.90
50.05 3.11
9.68 0.65
8.68 0.55
7.89 0.41
8.18 0.47
8.00 0.69
8.05 0.70
9.89 0.80
8.56 0.58
6.94 0.42
8.88 0.55
7.95 0.48
6.99 0.39
7.75 0.55
8.96 0.80
8.90 0.71
8.10 0.60
9.00 0.54
8.95 0.57
48.97 2.86
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Irreversible
3s
Selegiline
a
Each value represents the mean S.E.M of three experiments.
selective and potent inhibitors of hMAO-B compared with com-
pound having unsubstituted phenyl ring (3a). Strength of deacti-
vating group was designated as (NO2 > Br > Cl > CN) and well
correlates with potency. Cyano group, being stronger than halo-
gens, exhibited lesser potency due to difference in interaction with
the protein, MAO-B (see Section 3.3). But the selectivity towards
MAO-B (SIMAO-B = KiMAO-A/KiMAO-B) was found to be in reverse
order. Similarly compounds with activating functional group at
meta position of the phenyl ring (3e, 3h) were also found to be bet-
ter than 3a but definitely not better than compounds with deacti-
vating functional groups at para position (3c, 3l, 3m, 3n). These
two compounds (3e, 3h) did not displayed significant effect on

V.N. Badavath et al. / Bioorganic Chemistry 58 (2015) 72–80
77
Fig. 1. Predicted Ki vs Experimental Ki (A and B) and SI (C) Plot against MAO-isoforms.
Table 3
Total binding free energy profile of compound 3d and 3k with MAO-isoforms.
Complex
Eelec
EvdW
D
Ggas
D
Gsolv
DGbind
3d-MAO-A
3d-MAO-B
3j-MAO-A
3j-MAO-B
ꢁ12.3188 0.1017
ꢁ9.3756 0.1097
ꢁ10.1897 0.1586
ꢁ15.4189 0.1009
ꢁ67.8562 0.1285
ꢁ80.3953 0.2019
ꢁ65.8738 0.1996
ꢁ80.1658 0.2002
ꢁ95.3066 0.2864
20.6595 0.1063
16.6729 0.09689
20.2647 0.1048
24.1065 0.1151
ꢁ59.5155 0.2069
ꢁ49.3863 0.2095
ꢁ60.9011 0.2695
ꢁ70.1468 0.2182
ꢁ56.6836 0.2185
ꢁ70.9590 0.2246
ꢁ79.53674 0.1586
selectivity towards MAO-B. Compound 3j (R = 3,4-di-OCH3) was
found to be most potent MAO-B inhibitor with best selectivity
index of all the twenty compounds studied. These differences
may be due to difference in binding mode rather than simply the
strength of activating functional groups and additive effects due
to their combination. Replacing phenyl ring with naphth-2-yl
(3p), anthracen-9-yl (3q), thiophen-2-yl (3r) and pyridin-3-yl
(3s) did not improved potency as well as selectivity towards
MAO-B. Compound 3p with naphth-1-yl replacing phenyl (3a)
was found to be 2.7-fold better than 3a, but displayed no great dif-
ference in Selectivity towards MAO-B. While Compound 3r with
antracen-9-yl replacing phenyl (3a) was found to be non-selective
towards MAO-isoforms. Increasing bulkiness with addition of more
rings gradually shifts MAO-B selective ? Non-selective and may
further be towards MAO-A. Further Molecular docking analysis
was carried out to understand the interactions at molecular level
that are responsible for potency and selectivity.
understanding of compound 3d with MAO-A and compound 3j
with MAO-B. Binding mode analysis of compound 3d with MAO-
A (Fig. 2A) has revealed three H-bonding interaction viz., (i) hydro-
xyl hydrogen of 3d with side-chain hydroxyl oxygen of SER209, (ii)
hydroxyl oxygen of 3d with backbone amino hydrogen of SER209
and (iii) chromenone ring oxygen with hydroxyl hydrogen of
TYR444. The o-hydroxy phenyl group at 2nd position of chrome-
none also establishes pi-pi interaction with TRP441. These interac-
tions kept pocket1 (Aromatic cage: FAD, TYR407, TYR444)
unoccupied, pocket 3 (ILE180, ILE335, LEU337, MET350, PHE352)
partially occupied by chromenone benzene ring and pocket 2
(GLY71, GLN74, ARG206, ILE207, PHE208, GLU216, TRP441) par-
tially occupied by o-hydroxy phenyl ring at 2nd position of
chromenone. All the other compounds with activating functional
group 3e-3k that are showing selectivity towards MAO-B binds dif-
ferently with MAO-A. The chromenone benzene ring was accom-
modated in pocket 2 while phenyl ring at the 2nd position of
chromenone ring in pocket 3. This is mainly due to the position
and bulkiness of the substitution in the phenyl ring at the 2nd posi-
tion of chromenone and relatively polar nature of the pocket 2
(compared with pocket 1 and 3). Polar character of pocket 2 is
due to the amino acids: GLN74, ARG206, GLU216 and TRP441
3.3. Molecular docking study
Molecular docking study was performed with the above-men-
tioned protocol (see Section 2.4) to understand the molecular level

78
V.N. Badavath et al. / Bioorganic Chemistry 58 (2015) 72–80
Fig. 2. (A) Interaction of compound 3d with human MAO-A (PDB Code: 2BXR); (B) Interaction of compound 3j with human MAO-B (PDB Code: 2BYB).
and is slightly electropositive due to GLN74, ARG206 and TRP441.
This makes this pocket suitable for fragments with polar and elec-
tronegative in nature. These factors favored the establishment of (i)
H-bonding interaction between chromenone carbonyl oxygen and
backbone amino hydrogen of SER209 and (ii) pi–pi interaction
between chromenone benzene ring with PHE352. The VdW inter-
action of 3d is thus less than compounds 3e–3k, but the difference
in binding mode with favourable H-bonding and Electrostatic
interaction makes the molecule potent than their counterparts
(Table 2).
TYR398, TYR435), while phenyl ring at 2nd position of the chrome-
none having two-methoxy substitution is placed in the narrow
hydrophobic tunnel leading to solvent exposed entrance. Whereas
the analysis of compounds containing deactivating functional
groups at para position of phenyl ring (3c, 3l–3n) at 2nd position
of chromenone revealed difference in binding mode. In all the
cases the phenyl ring carrying deactivating functional group is con-
veniently accommodated in aromatic cage and orienting the ben-
zene ring of chromenone towards hydrophobic tunnel. The
potent one amongst the four, 3l, shows pi-pi interaction between
chromenone benzene ring and TYR435. Compounds 3c and 3m dis-
played two pi-pi interaction, one between benzene ring of chrome-
none and TRY435 and the other between phenyl ring at 2nd
position of chromenone and TYR435. While, the least potent one
Binding mode analysis of compound 3j with MAO-B (Fig. 2B)
has revealed pi-pi interaction between phenyl ring at 2nd position
of chromenone and TYR435 and FAD. The benzene ring of chrome-
none is comfortably accommodated in aromatic cage (FAD,

V.N. Badavath et al. / Bioorganic Chemistry 58 (2015) 72–80
79
Fig. 3. Potential energy and RMSD vs Time curves for compounds 3d and 3j with both isoforms of MAO.
shows pi-pi interaction between phenyl rings at 2nd position of
3.4. Molecular dynamics simulation
chromenone and TYR435.
The observations clearly indicated that the nature and position
of the substituents determines the orientation of the molecule
inside the active site of MAO-isoforms and in turn determines
the potency and selectivity.
Molecular docking sometimes leads to artifacts [53]. Molecular
dynamics (MD) simulation proven as a useful tool to understand
the stability of the docked complexes as well as the free energy
profile of ligand–protein complexes. It was observed from the

80
V.N. Badavath et al. / Bioorganic Chemistry 58 (2015) 72–80
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potential energy and RMSD plots that all the systems were well
stabilized after a 10 ns MD simulation (Fig. 3). Also the RMSD
and potential energy parameters were well converged over the
time which made all systems ideal for a MM-GBSA based binding
free energy analysis. MD based binding free energy analysis proven
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Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2014.
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