Identification of Indole-Based Chalcones: Discovery of a Potent, Selective, and Reversible Class of MAO-B Inhibitors

Article abstract of DOI:10.1002/ardp.201600088

A series of 11 indole-based chalcones (IC1–11) with various electron donating and withdrawing groups at the para position of the phenyl ring B were synthesized. All the compounds were tested for their human monoamine oxidase (hMAO)-A and hMAO-B inhibitory

Full text of DOI:10.1002/ardp.201600088

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–11
Full Paper
Identification of Indole-Based Chalcones: Discovery of a Potent,
Selective, and Reversible Class of MAO-B Inhibitors
1
2
3
3
Rani Sasidharan , Sreedharannair Leelabaiamma Manju , Gulberk Uc¸ar Ã, Ipek Baysal ,
€
and Bijo Mathew4
Ã
1
College of Pharmaceutical Science, Government T.D. Medical College, Alappuzha, Kerala, India
Organic Chemistry Division, SAS, VIT University, Vellore, Tamil Nadu, India
Faculty of Pharmacy, Department of Biochemistry, Hacettepe University, Sıhhiye, Ankara, Turkey
Division of Drug Design and Medicinal Chemistry Research Lab, Department of Pharmaceutical
2
3
4
Chemistry, Ahalia School of Pharmacy, Palakkad, Kerala, India
A series of 11 indole-based chalcones (IC1–11) with various electron donating and withdrawing
groups at the para position of the phenyl ring B were synthesized. All the compounds were tested
for their human monoamine oxidase (hMAO)-A and hMAO-B inhibitory potencies. Most of the
synthesized candidates proved to be potent and selective inhibitors of MAO-B rather than MAO-A,
with a reversible and competitive mode. Among them, compound IC9 was found to be a potent
inhibitor of hMAO-B with Ki ¼ 0.01 Æ 0.005 mM and a selectivity index of 120. It was found to be
better than the standard drug, selegiline (hMAO-B with Ki ¼ 0.20 Æ 0.020 mM) with a selectivity index
of 30.55. PAMPA assays were carried out for all the compounds in order to evaluate the capacity of
the compounds to cross the blood–brain barrier. Moreover, the most potent MAO-B inhibitor, IC9,
was nontoxic at 5 and 25 mM, with 95.20 and 69.17% viable cells, respectively. The lead compound
IC9 has an antioxidant property of 1.18 Trolox equivalents by ABTS assay. Molecular modeling studies
were performed against hMAO-B to observe binding site interactions of the lead compound.
Keywords: 3-Acetyl indole / Chalcones / MAO / MAO-B / Molecular docking
Received: April 1, 2016; Revised: June 3, 2016; Accepted: June 3, 2016
DOI 10.1002/ardp.201600088
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
aldehyde [2, 3]. Particularly, MAO-A enzyme is responsible
for the deamination of the epinephrine, norepinephrine,
Introduction
Monoamine oxidases A and B (MAO-A and MAO-B) are flavin
adenine dinucleotide (FAD) dependent enzymes found in the
outer mitochondrial membrane in neuronal, glial, and other
mammalian cells [1]. Both these enzymes catalyze the two-
electron oxidation of biogenic amine substrates at the
a-carbon and the reduced FAD is re-oxidized by molecular
oxygen to yield hydrogen peroxide, while the imine product
is in most instances hydrolyzed to the corresponding
and serotonin, whereas the MAO-B enzyme metabolizes
b-phenethylamine [4]. Due to their fundamental role in
neurotransmitter metabolism, these isoforms are considered
as attractive drug targets for mood disorders and neurodegen-
erative diseases [5]. In fact, MAO-A inhibitors are frequently
used as antidepressants and antianxiety agents, while MAO-B
inhibitors are relevant tools in the therapy of Alzheimer’s and
Parkinson’s diseases as adjuvants to L-Dopa, the metabolic
precursor of dopamine [6].
Correspondence: Dr. S. L. Manju, Associate Professor, Department
of Chemistry, School of Advanced Sciences, VIT University, Vellore,
Tamil Nadu, India, 632014.
Ã
€
Additional correspondence: Prof. Dr. Gulberk Uc¸ar,
E-mail: slmanju@vit.ac.in
E-mail: gulberk@hacettepe.edu.tr;
Fax: 04162243092
Bijo Mathew, E-mail: bijovilaventgu@gmail.com.
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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–11
R. Sasidharan et al.
Currently, clinically used human MAO-B inhibitors behave as
selective and irreversible inhibitors. These candidates display
the typical drawbacks of long-lasting enzyme inhibition,
namely, de novo enzyme biosynthesis in the human brain and
potential immunogenicity of enzyme-inhibitor adducts, high
efficiency to target disruption, less sensitivity toward phar-
macokinetic parameters, and increased duration of action.
In general reversible inhibitors have safer profiles [7]. As
the general body of information grows, it is sensible to
continue to design new hMAO-B inhibitors to be used as
neuroprotectant therapeutics with higher selectivities and
fewer deleterious side effects.
Chalcones are open chain flavonoidal class of compounds
consisting of two rings A and B separated by a, b-unsaturated
carbonyl system. In this system, A ring is nearer to the carbonyl
group and B ring is nearer to the b-carbon [8]. The first time
MAO inhibitory activity of isolated isoliquiritigenin (natural
chalcone) from the roots of Glycyrrhiza uralensis was done
by Tanka et al. The inhibitory action of this chalcone was
investigated by using the mitochondrial MAO of rat liver as
the source of the enzyme [9, 10]. Their research set the stage
for the chalcone family toward MAO recognition. Based
upon the above observation, many of the research groups
have explored the importance of electron withdrawing and
electron donating motifs at various positions of A and B rings
of chalcones and evaluated their MAO inhibition. The most
active chalcones were characterized by the presence of
hydroxyl and methoxyl groups at ortho and para positions,
respectively, on the phenyl A ring and the lipophilic group
such as such as chlorine, fluorine, and trifluoromethyl at para
position on the B ring [11–15]. In addition, the incorporation
of heteronucleus such as furan, chromene, piperidine, pyrrole,
quinoline, and thiophene in the chalcone scaffold, also
validated as MAO-A and -B inhibitors [16–23].
residues. Moreover the substituted B ring of chalcones are
lined with the “entrance cavity” of MAO-B [13, 21–23].
With the aim to further explore the structure–activity
relationships (SARs) of MAO inhibition by chalcone deriva-
tives, we planned the introduction of indole ring system in the
A ring with a variety of electron donating and withdrawing
groups at the para position of phenyl ring B. The information
regarding the exploration of various groups at the para
position of ring B system of indole-based chalcone and
its effect of MAO inhibition is not explored so far. 3-Acetyl
indole moiety was selected as starting reagent for the
synthesis of various indole-based chalcones, since indole
is often present in compounds with remarkable MAO
inhibitory activities [25–29]. Accordingly to these results, in
this work we describe the synthesis, biological evaluation, and
molecular modeling of indole-based chalcone derivatives as
selective MAO-B inhibitors. The pharmacological profiles of
these compounds including MAO-A and MAO-B inhibition,
the kinetics and reversibility of enzyme inhibition, blood–
brain barrier permeation, and cytotoxicity and antioxidant
effects were evaluated.
Results and discussion
Chemistry
The synthetic strategy of indolyl chalcones is outlined in
Scheme 1. The commercially available 3-acetyl indole and
various para-substituted benzaldehydes underwent Claisen–
Schmidt condensation in the presence of a base followed by
acidification with dil. HCl to afford corresponding indolyl
chalcones (IC1–11). The chemical structures of the compounds
have been ascertained by means of their 1H NMR, mass
spectroscopic data, and elemental analysis. 1H NMR spectrum
showed different type of protons corresponding to the signals
of the a,b- unsaturated unit, indole, and the mono-
substituted phenyl system. It has been noted that the upfield
proton of a-carbon and downfield proton of b-carbon
coupled with methine proton with coupling constants 15.2–
15.8 Hz. The large coupling constants suggest the presence of
trans configuration in the indolyl chalcones. The protons
belonging to the aromatic ring and indole ring are observed
with the expected chemical shift and integral value. The
Many of the studies clearly revealed that chalcone
compounds showed selective, reversible/irreversible, and
potent MAO-B inhibition than MAO-A [24]. Recently, our
group reported potent, selective, and reversible hMAO-B
inhibitory properties of methoxylated and thienyl chalcones.
Molecular modeling studies of these chalcones in the hMAO-B
active site showed that the A ring of the ligand oriented
toward the FAD co-factor and is stabilized by hydrogen
bonds and hydrophobic interactions with Tyr398 and Tyr435
Scheme 1. Synthesis of indolyl chalcones. Reagents and conditions: (a) C2H5OH, 10% NaOH, reflux, 12–15 h.
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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–11
MAO Inhibitory Activity of Indolylchalcones
characteristic peaks were observed in the mass spectra of
the synthesized compounds. The presence of chlorine and
bromine atom in the compounds of IC8 and IC9 showed
characteristic [Mþ2] isotope peaks.
calculated and experimental results are shown in Table 1.
Selectivity indexes (SI) were expressed as Ki(MAO-A)/Ki(MAO-B).
Selectivity toward MAO-A increases as the corresponding SI
decreases.
It can be seen from Table 1 that all the tested compounds
showed much better inhibitory activities against hMAO-B
than hMAO-A except compound IC7 which inhibited hMAO
isoforms non-selectively, appeared as moderate to good
inhibitors of hMAO-B with Ki values in the submicromolar
range. Among these derivatives, compound IC9 exhibited
potent activity and higher selectivity toward hMAO-B with Ki
and SI values of 0.01 Æ 0.005 mM and 120. It was found to be
20-folds more efficient than the standard drug, selegiline
(hMAO-B with Ki ¼ 0.20 Æ 0.020 mM) with selectivity index of
30.55. This inhibitory activity in nanomolar range indicated
that the crucial role of substituent at the para position
of the phenyl ring B might be important for the hMAO-B
activity and selectivity.
In general, variation of substituents in the para position of
phenyl B ring system attempted in the heteroaryl chalcone
scaffold resulted in improved inhibitory activity. In particular,
the enhancement of lipophilicity (compounds IC1–IC11)
by incorporating an electron donating lipophilic groups or
bulky halogen at the para position on phenyl B ring system of
Pharmacological evaluation
MAO inhibition studies
To determine the inhibitory activities of the indole-based
chalcones, the recombinant human MAO-A and MAO-B
enzymes were used as enzyme sources and the activity was
determined according to a previously published protocol
using the Amplex Red MAO assay kit [30–32]. A reversible
and selective hMAO-A inhibitor, moclobemide, and an
irreversible (suicide) and selective hMAO-B inhibitor, selegi-
line were employed as the positive control compounds
in this test. Compounds and reference inhibitors were
independently treated with Amplex Red reagent before the
experiment. None of them interfered 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 concen-
trations of resorufin. Specific enzyme activities were calcu-
lated as 164.99 Æ 11.00 pmol/mg/min (n ¼ 3) for hMAO-A
and 141.20 Æ 6.89 pmol/mg/min (n ¼ 3) for hMAO-B. The
Table 1. hMAO inhibitory activities of indole-based chalcones.
Experimental
Ki value (mM)a)
Experimental
Inhibition
type
MAO
selectivity
Compounds
R
MAO-A
MAO-B
SIb)
Reversibility
IC1
IC2
IC3
IC4
IC5
IC6
IC7
IC8
4–H
4–OH
4–OCH3
4–CH3
4–N(CH3)2
4–CH2CH3
4–NO2
4–Cl
4–Br
4–F
4–CF3
–
–
1.50 Æ 0.09
1.98 Æ 0.11
1.20 Æ 0.09
1.40 Æ 0.10
0.80 Æ 0.04
0.74 Æ 0.03
0.32 Æ 0.01
1.00 Æ 0.07
1.20 Æ 0.11
2.90 Æ 0.15
1.50 Æ 0.11
0.17 Æ 0.020
6.11 Æ 0.331
0.14 Æ 0.010
0.35 Æ 0.021
0.11 Æ 0.009
0.09 Æ 0.004
0.15 Æ 0.009
0.11 Æ 0.007
0.30 Æ 0.010
0.28 Æ 0.019
0.01 Æ 0.005
0.30 Æ 0.020
0.19 Æ 0.009
1.68 Æ 0.110
0.20 Æ 0.020
10.71
5.66
10.91
15.55
5.33
6.73
1.07
3.57
120
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Suicide
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Irreversible
MAO-B
MAO-B
MAO-B
MAO-B
MAO-B
MAO-B
Non-selective
MAO-B
MAO-B
MAO-B
MAO-B
MAO-A
MAO-B
IC9
IC10
IC11
Moclobemide
Selegiline
9.66
7.90
0.10
30.55
inhibitor
a) Value represents the mean Æ SEM of three independent experiments.
b) Selectivity index. It was calculated as Ki(MAO-A)/Ki(MAO-B). Selectivity toward MAO-A increases as the corresponding SI
decreases while selectivity toward MAO-B isoform increases as the corresponding SI increases.
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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–11
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indolylchalcolones resulted in improved inhibitory activity
against the MAO-B isozyme. The potential hMAO-B inhibitory
activity and selectivity can be attributed to the presence of a
4-bromo substituent on the phenyl system (IC9). Presence of
halogen such as chlorine or fluorine atom of phenyl ring
showed almost similar results of MAO-B inhibition with Ki
value of 0.28 and 0.30 mM, respectively. The presence of
hydrophilic groups, like hydroxyl on the phenyl B ring led to a
35-fold decrease in potency (Ki ¼ 0.35 mM) in IC2 compared to
the corresponding bromo derivative IC9 against hMAO-B. The
following ranking order of MAO inhibitory activity was
observed for the indole-based chalcones as per experimental
inhibitory data:
series have been used as promising intermediates in the
synthesis of 2-pyrazoline derivatives as part of an effort to
discover novel MAO inhibitors [33]. Another motivating
aspect is the inhibitory capacity IC7 compound which showed
Ki value of 0.30 and 0.32 mM toward both MAO-B and -A,
respectively. It is to be noted that dual-target-directed MAO-B
and MAO-A inhibitors may have enhanced therapeutic value,
making such compounds ideal adjuvants to L-DOPA treatment
in Parkinson’s disease, particularly when associated with
depressive state of mood disorders [34].
Kinetic studies
Kinetic analyses were carried out for most potent MAO-B
inhibitor IC9 from this series to gain the further insight into the
mode of MAO inhibition. A set of Lineweaver–Burk plots was
constructed in the absence and presence of various concen-
trations of compound IC9. The set consisted of five graphs,
each constructed by measuring MAO-B and MAO-A catalytic
rate at different substrate concentrations (0.01–0.15 mM).
The observation that the lines were linear and intersect on
the y-axis suggests that IC9 interacts with the catalytic site
of hMAO-B, with a competitive mode of inhibition (Fig. 1). The
replots of the slopes of the Lineweaver–Burk plots versus
inhibitor concentration are shown in Fig. 2 and the Ki was
estimated as 0.012 mM for IC9 (hMAO-B).
hMAO-A: 4-NO2 > 4-C2H5 > 4-N-ðCH3Þ2 > 4-Cl > 4-OCH3
> 4-Br > 4-CH3 > 4-CF3 > 4-H > 4-OH > 4-F
hMAO-B: 4-Br > 4-CH3 > 4-C2H5 > 4-OCH3 > 4-H
> 4-N-ðCH3Þ2 > 4-CF3 > 4-Cl > 4-NO2 > 4-F > 4-OH
Thus, substitution with lipophilic motifs such as bromo,
methyl, ethyl, and methoxyl groups at the para position on
the phenyl ring B increased the potency toward hMAO-B
compared to standard selegiline. This behavior is exemplified
by IC9 (Ki ¼ 0.01 mM), IC4 (Ki ¼ 0.09 mM), IC6 (Ki ¼ 0.11 mM),
and IC3 (Ki ¼ 0.11 mM). Among the non-functionalized phenyl
ring compound IC1 showed Ki value of 0.14 mM against
hMAO-B, which further supports the above observation
claiming the crucial role of the nature of the lipophilic para
substitution on phenyl ring for the development of potent
hMAO-B inhibitory activity. The potent chalcones from this
Reversibility studies
Newly synthesized indole-based chalcone derivatives inhib-
ited 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 compounds
was investigated by determination of the recoveries of MAO
Figure 1. Lineweaver–Burk plots of hMAO-B activity in the absence and presence of various concentrations of compound IC9.
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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–11
MAO Inhibitory Activity of Indolylchalcones
Figure 2. Replots of the slopes of the Lineweaver–Burk plots versus inhibitor IC9 concentration (hMAO-B).
activity after dialysis of enzyme-inhibitor mixtures. MAO
isoforms were incubated in the presence of the inhibitors, at
concentrations equal to 4 Â IC50, for a period of 15 min and
subsequently dialyzed for 24 h. hMAO-B inhibition by
compound IC9 was completely reversed after 24 h of dialysis.
Results suggest that newly synthesized indolylchalcones are
reversible inhibitors of hMAO isoforms and have considerable
advantages compared to irreversible inhibitors which may
possess serious pharmacological side effects. The potent and
selective MAO-B inhibitor selegiline has been reported to
have severe side effects due to its amphetamine metabolites
and both selegiline and rasagiline are irreversible MAO-B
inhibitors triggering pharmacological side effects in long term
treatment of Parkinson’s disease [35]. Reversibility of hMAO-B
inhibition with compound IC9 was found to be good in the
present series. The reversiblity of compound IC9 was
calculated as 28.10 Æ 1.56 and 96.00 Æ 3.13 before and after
dialysis, respectively.
In vitro blood–brain barrier permeation assay
Newly synthesized derivatives were screened for their ability
to cross the blood–brain barrier (BBB) since this is the first
requirement for the design of successful CNS drugs. To
investigate whether the present compounds could pass this
barrier, a widely known parallel artificial membrane perme-
ation assay of blood–brain barrier (PAMPA-BBB) was used.
Assay validation was made by comparing experimental
permeabilities of nine commercial drugs with reported values
(Table 3). A plot of experimental data versus bibliographic
values gave a good linear correlation (R2 ¼ 0.9965, Fig. 3).
Table 2. Reversibility of the inhibition of hMAOs by indole-based chalcones.
Test compounds
incubated with
hMAO
hMAO-A activity
before washing
(%)
hMAO-A activity
after washing
(%)
hMAO-B activity
before washing
(%)
hMAO-B activity
after washing
(%)
Reversibility
IC1
IC2
IC3
IC4
IC5
IC6
IC7
IC8
90.25 Æ 3.77
87.20 Æ 3.60
89.55 Æ 4.75
90.66 Æ 4.05
90.01 Æ 3.55
87.44 Æ 4.00
50.68 Æ 2.66
88.21 Æ 4.00
95.20 Æ 5.00
94.78 Æ 4.20
89.99 Æ 2.60
100 Æ 0.00
94.00 Æ 4.61
91.25 Æ 3.05
88.20 Æ 3.78
88.22 Æ 4.00
92.78 Æ 4.00
92.26 Æ 4.50
88.21 Æ 4.00
93.06 Æ 3.74
94.60 Æ 3.55
95.55 Æ 4.30
92.40 Æ 3.22
100 Æ 0.00
40.55 Æ 1.96
51.20 Æ 2.30
42.20 Æ 2.10
40.00 Æ 2.01
55.00 Æ 2.16
52.98 Æ 2.44
49.88 Æ 2.02
53.20 Æ 4.05
28.10 Æ 1.56
51.28 Æ 3.16
50.22 Æ 2.16
100 Æ 0.00
90.44 Æ 3.27
88.27 Æ 2.37
94.00 Æ 3.25
87.55 Æ 4.09
92.39 Æ 4.23
94.00 Æ 4.01
90.32 Æ 4.03
90.47 Æ 3.33
96.00 Æ 3.13
95.00 Æ 2.85
95.80 Æ 2.56
100 Æ 0.00
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
–
IC9
IC10
IC11
With no inhibitor
Moclobemide
Selegiline
45.56 Æ 3.02
92.21 Æ 3.90
89.25 Æ 3.77
90.05 Æ 5.552
99.00 Æ 4.87
51.00 Æ 3.41
94.89 Æ 5.22
52.09 Æ 3.00
Reversible
Irreversible
Each value represents the mean Æ SEM (n ¼ 3).
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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–11
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Table 3. Permeability (Pe 10À 6 cm sÀ 1) in the PAMPA-BBB assay for commercial drugs used in the validation of the assay
and for indole-based chalcones.
Bibliography Pe
Experimental Pe
Compoundsa)
(Â10À 6 cm sÀ 1 b)
)
(Â10À 6 cm sÀ 1 c)
)
Prediction
IC1
IC2
IC3
IC4
IC5
IC6
IC7
IC8
10.55 Æ 1.05
9.04 Æ 0.54
12.87 Æ 1.05
14.02 Æ 1.10
8.88 Æ 0.39
9.00 Æ 0.45
4.11 Æ 0.16
6.22 Æ 0.51
15.27 Æ 1.09
10.02 Æ 0.98
9.57 Æ 0.52
16.72 Æ 1.52
14.97 Æ1.65
10.72 Æ 1.69
7.88 Æ 0.33
4.08 Æ 1.03
2.23 Æ 0.53
1.53 Æ 0.48
1.09 Æ 0.23
0.36 Æ 0.14
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ/À
CNSÀ
CNSÀ
CNSÀ
IC9
IC10
IC11
Testosterone
Verapamil
b-Estradiol
Progesterone
Corticosterone
Piroxicam
Hydrocortisone
Lomefloxacin
Dopamine
17.0
16.0
12.0
9.3
5.1
2.5
1.8
1.1
0.2
a) Compounds were dissolved in DMSO at 5 mg/mL and diluted with PBS/EtOH (70:30). The final concentration of compounds was
100 mg/mL.
b) Taken from [36].
c) Values were expressed as the mean Æ SEM of three independent experiments.
According to the limits established by Di et al. for blood–brain
barrier permeation [36], test compounds were classified as
follows:
concentrations (1–25 mM) (Table 4). The results showed that
almost all of the novel compounds were not toxic to hepatic
cells at 1 mM concentration. At 5 mM, compounds IC3, IC4, IC9,
and IC10 were found to be completely nontoxic to the cells.
The most potent MAO-B inhibitor IC9 in the current study was
nontoxic at 5 and 25 mM, with 95.20 and 69.17% viable cells,
respectively.
À
Á
CNS þ ðhigh BBB permeation predictedÞ: Pe 10À 6 cmsÀ 1
> 4:00
À
Á
CNS À ðlow BBB permeation predictedÞ: Pe 10À 6 cmsÀ 1
< 2:00
À
Á
CNS Æ ðBBB permeation uncertainÞ: Pe 10À 6 cmsÀ 1
from 4:00 to 2:00
The data showed in Table 3 indicated that all of the indole-
based chalcones can cross the BBB to target the enzyme
in the central nervous system. Compounds IC9, IC4, and
IC3 showed the highest permeability suggesting that they
may cross the BBB easily and reach the biological targets
located in the CNS, which was consistent with our design
strategy.
Cytotoxicity studies
The toxicities of the newly synthesized derivatives were
determined in order to obtain data for the assessment of their
possible drug-like properties. In vitro cytotoxicity of the novel
compounds was tested in HepG2 cells at three different
Figure 3. Linear correlation between experimental and
reported permeability of commercial drugs using the PAMPA-
BBB assay.
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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–11
MAO Inhibitory Activity of Indolylchalcones
Table 4. In vitro cytotoxicity test and ABTS assay for the indole-based chalcones.
Viability (%)
Compounds
1 mM
5 mM
25 mM
ABST assaya)
IC1
IC2
IC3
IC4
IC5
IC6
IC7
IC8
IC9
IC10
IC11
88.22 Æ 3.05Ã
97.55 Æ 5.14
98.15 Æ 1.86
97.00 Æ 2.98
97.84 Æ 5.53
96.80 Æ 3.87
96.00 Æ 2.85
85.21 Æ 2.56Ã
97.86 Æ 4.45
96.88 Æ 2.74
96.54 Æ 3.78
62.49 Æ 1.44ÃÃ
89.65 Æ 2.75Ã
95.77 Æ 3.01
94.89 Æ 3.55
82.58 Æ 1.26ÃÃ
56.58 Æ 1.71ÃÃ
56.31 Æ 1.87ÃÃ
63.64 Æ 0.68ÃÃ
95.20 Æ 3.11
95.22 Æ 2.15
84.33 Æ 2.55Ã
58.23 Æ 3.00ÃÃ
57.54 Æ 1.44ÃÃ
62.21 Æ 1.47ÃÃ
60.87 Æ 2.17ÃÃ
73.38 Æ 1.22ÃÃ
56.58 Æ 1.71ÃÃ
53.16 Æ 0.93ÃÃÃ
52.28 Æ 1.46ÃÃÃ
69.17 Æ 0.60ÃÃ
74.57 Æ 0.97ÃÃ
65.80 Æ 1.99ÃÃ
0.48
0.54
0.67
0.73
0.68
0.58
0.32
0.80
1.18
1.32
0.35
Data were expressed as mean Æ SEM (n ¼ 3). Cell viability was expressed as a percentage of the control value. p < 0.05 was
considered as statistically significant. (Ãp < 0.05, ÃÃp < 0.01, ÃÃÃp < 0.001 vs. control).
a)Data were expressed as Trolox equivalent (mmol Trolox)/(mmol tested compound).
ABTS radical cation scavenging assay
hMAO-B, either substrates or competitive inhibitors bind to
hydrophobic cavity which includes: an “entrance cavity” lined
with Ile199 and the “substrate cavity”. Here smaller entrance
cavity with Ile199 effectively serves as a gate between these
cavities. Depending on the structure of the bound inhibitor,
the two cavities may be either separate or fused [39, 40]. The
bromo-substituted phenyl ring of IC9 is oriented horizontally
sandwiched between the phenolic side chains of Tyr435
and Tyr398 residues, and it approached from the re face of
FAD making one p–p stacking interaction with Tyr435 in
the active site of hMAO-B. The interaction of IC9 is also
stabilized by one more p–p stacking interaction of carbocyclic
ring of indole with the FAD along with one intermolecular
hydrogen bond was observed between the NH group of
indole and the Tyr325 (Fig. 4).
Compounds IC1–11 were tested for their antioxidant activities
by using the 2,20-azino-bis-2-ethybenz-thiazoline-6-sulfonic
acid (ABTS) method. Trolox (a water-soluble vitamin E analog)
was used as a reference standard [37, 38]. Their antioxidant
activities were provided as a Trolox equivalent, with their
relative potency at 25 mM compared with Trolox. As shown
in Table 4, compounds IC8, IC9, and IC10 had the ability
to scavenge the ABTS radical with 0.80, 1.18, 1.32 Trolox
equivalents, respectively. The most active compounds were
observed for the compounds with halogen substitution at the
para position on ring B of indolyl chalcones. However,
electron withdrawing group such as nitro and trifluoromethyl
substitution on the phenyl ring were unfavorable to the
ABTS radicals scavenging activity.
Computational studies
Molecular docking studies
Conclusion
To investigate the hypothetical molecular mechanism of
hMAO-B inhibition by the lead compound IC9, molecular
docking studies were carried out using AutoDock4.2. In
From inspection of the chemical structures, it can be
concluded that among the synthesized compounds, the
Figure 4. Docking pose of IC9 in the MAO-B
active site: Yellow mesh indicates p–p stack-
ing interaction and blue line indicates
H-bonding interaction.
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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–11
R. Sasidharan et al.
hMAO-B inhibitory activity was strongly affected by the size
and nature of the substituent groups at the para position on
the phenyl ring of indolyl chalcones. This study highlighted
the importance of bromine atom at the para position on
phenyl ring that could significantly enhance the hMAO-B
inhibitory activities of indolyl chalcones. The Ki value of
hMAO-B inhibition of the compounds ranges from 0.01 to
0.35 mM, with compound IC9 being the most potent and more
efficient than the standard selegiline. Besides, this compound
is good antioxidant with brain penetration capacity for CNS
activity. In the cell viability, IC9 was shown to have very low
toxicity in HepG2 cells and showed good hepato protective
action. Altogether, the multifunctional effects of this indolyl
derivative qualify them as potential selective and reversible
type of hMAO-B inhibitor and might be promising lead
candidates for further research for the development of
anti-Parkinson’s agents.
(2E)-3-(4-Hydroxyphenyl)-1-(1H-indol-3-yl)prop-2-en-1-
one (IC2)
Pale brown, yield: 45%, m.p. 210–212°C. 1H NMR (400 MHz,
DMSO) d ppm: 5.22 (s, 1H, OH), 7.19 (d, 1H, J ¼ 14.4 Hz, Ha),
7.19–7.31 (m, 3H, Ar-H), 7.47 (d, 1H, J ¼ 15.6 Hz, Hb), 8.17–8.30
(m, 4H, Ar-H), 11.91 (s, 1H, NH): ESI-MS (m/z): calculated
263.290, observed 263.222 [Mþ]þ. Anal. calcd. for C17H13NO2:
C, 77.55; H, 4.98; N, 5.32. Found: C, 77.63; H, 4.86; N, 5.46.
(2E)-1-(1H-Indol-3-yl)-3-(4-methoxyphenyl)prop-2-en-1-
one (IC3)
Pale yellow, yield: 52%, m.p. 202–204°C. 1H NMR (400 MHz,
DMSO) d ppm: 3.86 (s, 3H, OCH3), 6.92–7.79 (m, 9, Ar-H), 7.21
(d, 1H, J ¼ 15.6 Hz, CHa), 7.83(d, 1H, J ¼ 15.6 Hz, CHb), 10.80
(s, 1H, NH): ESI-MS (m/z): calculated 277.317, observed 277.270
[Mþ]þ. Anal. calcd. for C18H15NO2: C, 77.96; H, 5.45; N, 5.05.
Found: C, 77.93; H, 5.46; N, 5.02.
(2E)-1-(1H-Indol-3-yl)-3-(4-methylphenyl)prop-2-en-1-one
(IC4)
Experimental
Pale yellow, yield: 51%, m.p. 208–210°C. 1H NMR (400 MHz,
DMSO) d ppm: 3.41 (s, 3H, CH3), 6.81–7.76 (m, 9, Ar-H), 7.36 (d,
1H, J ¼ 15.6 Hz, CHa), 7.95 (d, 1H, J ¼ 15.6 Hz, CHb), 11.08 (s, 1H,
NH): ESI-MS (m/z): calculated 261.317, observed 261.278
[Mþ]þ. Anal. calcd. for C18H15NO: C, 82.73; H, 5.79; N, 5.36.
Found: C, 82.83; H, 5.81; N, 5.22.
Chemistry
General
3-Acetyl indole and all the aromatic 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)
nuclear magnetic resonance (NMR) spectra were recorded on
a Bruker Avance III 600 spectrometer at frequency of 400 MHz
(Bruker, Karlsruhe, Germany). All NMR measurements were
conducted in CDCl3, and the chemical shifts are reported in
parts per million (d) downfield from the signal of tetra-
methylsilane added to the deuterated solvent. Mass spectra
(2E)-3-[4-(Dimethylamino)phenyl]-1-(1H-indol-3-yl)prop-
2-en-1-one (IC5)
Orange, yield: 61%, m.p. 195–197°C. 1H NMR (400 MHz,
DMSO) d ppm: 3.13 (s, 6H, N(CH3)2), 6.89–7.92 (m, 9, Ar-H),
6.92. (d, 1H, J ¼ 15.2 Hz, CHa), 7.85 (d, 1H, J ¼ 15.6 Hz, CHb),
11.08 (s, 1H, NH): ESI-MS (m/z): calculated 290.359, observed
290.996 [Mþ]þ. Anal. calcd. for C18H18N2O: C, 78.59; H, 6.25;
N, 9.65. Found: C, 78.39; H, 6.80; N, 9.72.
were recorded on
Elemental analyses (C, H, N) were performed on a Leco CHNS
932 analyzer.
a JEOL GCmate mass spectrometer.
The InChI codes of the investigated compounds are
provided as Supporting Information.
(2E)-3-(4-Ethylphenyl)-1-(1H-indol-3-yl)prop-2-en-1-one
(IC6)
Brown, yield: 51%, m.p. 198–200°C. 1H NMR (400 MHz, DMSO)
d ppm: 1.43 (t, 3H, CH3), 2.91 (q, 2H, CH2), 6.82 (d, 1H,
J ¼ 16.0 Hz, CHa), 6.93–7.97 (m, 9, Ar-H), 7.84 (d, 1H, J ¼ 15.6
Hz, CHb), 11.18 (s, 1H, NH): ESI-MS (m/z): calculated 275.344,
observed 275.029 [Mþ]þ. Anal. calcd. for C19H17NO: C, 82.88;
H, 6.22; N, 5.09. Found: C, 82.83; H, 6.24; N, 5.12.
General procedure for the synthesis of IC1–11
To a solution of 3-acetylindole (1 mmol) and appropriate
aldehyde (1 mmol) in ethanol (20 mL) was added 10% sodium
hydroxide (2 mL) and refluxed the reaction mixture for 15 h.
The contents of reaction mixture were poured into ice-cold
water and neutralized with dilute hydrochloric acid. The
solid so obtained was filtered, dried, and recrystallized from
suitable solvent to afford (IC1–11) [41].
(2E)-1-(1H-Indol-3-yl)-3-(4-nitrophenyl)prop-2-en-1-one (IC7)
Yellow, yield: 58%, m.p. 212–214°C. 1H NMR (400 MHz, DMSO) d
ppm: 6.91 (d, 1H, J ¼ 16.0 Hz, CHa), 6.83–7.99 (m, 9, Ar-H), 7.73 (d,
1H, J ¼ 15.6 Hz, CHb), 11.21 (s, 1H, NH): ESI-MS (m/z): calculated
292.289, observed 292.230 [Mþ]þ. Anal. calcd. for C17H12N2O3:
C, 69.86; H, 4.14; N, 9.58. Found: C, 69.80; H, 4.24; N, 9.62.
(2E)-1-(1H-Indol-3-yl)-3-phenylprop-2-en-1-one (IC1)
Pale white, yield: 40%, m.p. 173–175°C. 1H NMR (400 MHz,
DMSO) d ppm: 1H NMR (400 MHz, DMSO) d ppm: 7.21 (d, 1H,
J ¼ 14.4 Hz, Ha), 7.13–7.45 (m, 4H, Ar-H), 7.49 (d, 1H, J ¼ 15.6
Hz, Hb), 8.19–8.45 (m, 4H, Ar-H), 11.98 (s, 1H, NH): ESI-MS
(m/z): calculated 247.267, observed 247.291 [Mþ]þ. Anal.
calcd. for C17H13NO: C, 82.57; H, 5.30; N, 5.66. Found: C, 82.68;
H, 5.34; N, 5.86.
(2E)-3-(4-Chlorophenyl)-1-(1H-indol-3-yl)prop-2-en-1-one
(IC8)
Pale white solid, yield: 61%, m.p. 200–202°C. 1H NMR
(400 MHz, CDCl3) d ppm: 7.17–8.30 (m, 9, Ar-H), 8.17 (d, 1H,
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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–11
MAO Inhibitory Activity of Indolylchalcones
J ¼ 15.6 Hz, CHa), 8.76 (d, 1H, J ¼ 15.6 Hz, CHb), 11.91 (s, 1H,
NH); ESI-MS (m/z): calculated 281.736, observed 282.213
[Mþ]þ, [Mþ2]. Anal. calcd. for C17H12ClNO: C, 72.47; H,
4.29; N, 4.97. Found: C, 72.45; H, 4.94; N, 4.92.
Resorufin was quantified at 370°C in a multidetection
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.
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 con-
taining only the Amplex Red reagent in a sodium phosphate
buffer. The new compounds and reference inhibitors them-
selves did not react directly with Amplex1 Red reagent. Newly
synthesized compounds did not cause any inhibition on the
activity of HRP in the test medium.
(2E)-3-(4-Bromophenyl)-1-(1H-indol-3-yl)prop-2-en-1-one
(IC9)
Dark yellow solid, yield: 73%, m.p. 180–182°C. 1H NMR
(400 MHz, CDCl3) d ppm: 6.66–7.78 (m, 9, Ar-H), 7.51 (d, 1H,
J ¼ 16.0 Hz, CHa), 7.82 (d, 1H, J ¼ 16.0 Hz, CHb), 11.35 (s, 1H,
NH); ESI-MS (m/z): calculated 326.187, observed 326.191
[Mþ]þ, [Mþ2]. Anal. calcd. for C17H12BrNO: C, 62.60; H,
3.71; N, 4.29. Found: C, 62.55; H, 3.84; N, 4.22.
(2E)-3-(4-Fluorophenyl)-1-(1H-indol-3-yl)prop-2-en-1-one
(IC10)
1
Yellow solid, yield: 53%, m.p. 218–220°C. H NMR (400 MHz,
CDCl3) d ppm: 6.75–7.98 (m, 9, Ar-H), 7.59 (d, 1H, J ¼ 16.0 Hz,
CHa), 7.92 (d, 1H, J ¼ 16.0 Hz, CHb), 11.29 (s, 1H, NH); ESI-MS
(m/z): calculated 265.281, observed 265.243 [Mþ]þ. Anal.
calcd. for C17H12FNO: C, 76.97; H, 4.56; N, 5.28. Found: C, 76.95;
H, 4.64; N, 5.26.
Kinetic studies
Synthesized compounds were dissolved in dimethyl sulfoxide,
with a maximum concentration of 1% and used in a wide
concentration range of 0.010–0.15 mM. The mode of MAO
inhibition was examined using Lineweaver–Burk plotting.
The slopes of the Lineweaver–Burk plots were plotted versus
the inhibitor concentration and the Ki values were deter-
mined from the x-axis intercept as À 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 [42].
(2E)-1-(1H-Indol-3-yl)-3-[4-(trifluoromethyl)phenyl]prop-
2-en-1-one (IC11)
Pale brown, yield: 93%, m.p. 192–194°C. 1H NMR (400 MHz,
CDCl3) d ppm: 6.85–7.94 (m, 9, Ar-H), 7.49 (d, 1H, J ¼ 16.0 Hz,
CHa), 7.86 (d, 1H, J ¼ 16.0 Hz, CHb), 11.41 (s, 1H, NH); ESI-MS
(m/z): calculated 315.289, observed 315.289 [Mþ]þ. Anal.
calcd. for C17H12FNO: C, 68.57; H, 3.84; N, 4.44. Found: C, 68.88;
H, 3.80; N, 4.36.
Biological assays
Chemicals
Recombinant hMAO-A and hMAO-B (expressed in baculovirus-
infected BTI insect cells), R-(À )-deprenyl hydrochloride (selegi-
line), moclobemide, resorufin, dimethyl sulfoxide (DMSO),
and other chemicals were purchased from Sigma–Aldrich
(Munich, Germany). The Amplex1-Red MAO assay kit (Mybio-
source, USA) contained benzylamine, p-tyramine, clorgyline,
pargyline, and horseradish peroxidase.
Reversibility of inhibition
Reversibility of the MAO inhibition with the compounds was
determined by dialysis method previously described [43].
Dialysis tubing 16 Â 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 fourfold 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°C. Other sets were prepared by
preincubation of same amount of hMAO-A and -B with the
reference inhibitors (moclobemide and selegiline). 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 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 Æ SEM. For comparison, undialyzed
mixtures of the MAOs and the inhibitors were kept at 4°C
over the same time period.
Determination of hMAO activity
hMAO activites were determined by a fluorimetric method
described and modified previously using p-tyramine (0.05–
0.50 mM) as common substrate [30–32]. 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°C in microplates, placed in the dark
fluorimeter chamber. Reaction was started by adding 200 mM
Amplex Red reagent, 1 U/mL horseradish peroxidase (HRP),
and p-tyramine. The production of H2O2 catalyzed by MAO
isoforms was detected using Amplex1-Red reagent, in the
presence of HRP to produce the fluorescent product resorufin.
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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–11
R. Sasidharan et al.
In vitro blood–brain barrier permeation assay
ABTS solution, respectively. After the addition of either
Trolox or another antioxidant to the ABTS solution, complete
mixing of the reactants was achieved by bubbling three to
four times using plastic pipettes. The optical absorbance
of ABTS at 415 nm was measured at 6 min after addition
and equilibrated at 30°C. Each individual treatment was
repeated three times and the results of the experiments were
compared [37, 38].
The ability of newly synthesized compounds to penetrate into
brain was determined using a parallel artificial membrane
permeation assay (PAMPA) for blood–brain-barrier according
to a previous method [36]. Briefly, compounds and the
commercial drugs were dissolved in DMSO at a concentration
of 5 mg/mL. They were diluted with a mixture 70:30 of
phosphate buffered saline solution and ethanol (PBS/EtOH) to
give a final concentration of 25 mg/mL. The filter membrane
in donor microplate was coated with porcine polar brain
lipid (PBL) dissolved in dodecane (4 mL, 20 mg/mL). A total of
200 mL of diluted solution and 300 mL of PBS/EtOH (70:30)
were added to the donor and the acceptor wells, respectively.
The donor filter plate was carefully placed on the acceptor
plate. Sandwich system was kept at 25°C for 16 h. The donor
plate was carefully removed, and the concentrations of the
compounds and the commercial drugs in the acceptor, donor,
and reference wells were measured with a UV plate reader.
Molecular docking studies
In the current molecular simulation study, AUTODOCK4.2
software was used to establish a ligand-based computer
modeling program for the prediction of binding energy of the
selected compounds with hMAO isoforms [46]. Docking
protocol was done with X-ray crystal structure of hMAO-A
(2BXR) and hMAO-B (2BYB) downloaded from Protein
Data Bank (www.rcsb.org) [47]. Protein preparation was
carried out using Protein Preparation Wizard of Maestro-8.5
(Schrodinger LLC) [48]. Ligands were prepared through
PRODRG webserver (http://davapc1.bioch.dundee.ac.uk/cgi-
bin/prodrg) [49]. The receptor grids for both enzymes were
generated with following parameters: grid box dimension
Cytotoxic activity
Cell viability was measured by quantitative colorimetric assay
with
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium
̊
bromide (MTT) (Sigma–Aldrich). Human hepatoma cell line
HepG2 (Invitrogen) was cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM) supplemented with 2 mM L-Gluta-
mine, 10% heat-inactivated fetal bovine serum (FBS), 100
units/mL penicillin, and 100 mg/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 novel compounds (1, 5, and 25 mM)
or 0.1% DMSO as a vehicle control for 24 h. A total of 10 mL of
the MTT labeling reagent, at a final concentration of 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 appearence of purple
formazan crystals formed. Then, the insoluble formazan was
dissolved with 100 mL of dimethylsulfoxide (DMSO) by shaking
1 h in darkness. MTT reduction was measured at 590 nm.
Control cells treated with 0.1% DMSO were used as 100%
viability [44, 45]. Significance was determined using Student’s
t-test. Results were expressed as mean Æ SEM. Differences are
considered statistically significant at p < 0.05.
(xyz) of 60 Â 60 Â 60; grid spacing of 0.375 A and center
of the grid box positioned on N5 atom of FAD (co-factor). 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 number 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 (Ã.dlg) containing results of the simulation. Through
analyze module in MGLTool-1.5.6, the docking log files were
analyzed. Top scoring molecule from the largest cluster was
considered for analysis [50].
The authors are thankful to IIRBS, Mahatma Gandhi
University, Kottayam and SAIF, IIT, Chennai, India for carrying
out the spectral analysis.
The authors have declared no conflict of interest.
Antioxidant activity
The ABTS radical cation scavenging method can be described
as follows: 2,20-Azino-bis-2-ethylbenzthiazoline-6-sulfonic
acid (ABTS) was dissolved in purified water to a 7 mM
concentration. The ABTS radical cation (ABTS•+) was produced
by reacting ABTS stock solution with 2.45 mM potassium
persulfate (final concentration) and allowing the mixture to
stand in the dark at room temperature for at least 18 h before
use. The stock solution of ABTS was serially diluted with
sodium phosphate buffer (50 mM, pH 7.4) to 100 mM. Trolox
and all the target compounds at different concentrations
(total volume of 50 mL) were added to 150 mL of 100 mM
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