Potent and Selective Monoamine Oxidase-B Inhibitory Activity: Fluoro- vs. Trifluoromethyl-4-hydroxylated Chalcone Derivatives

Article abstract of DOI:10.1002/cbdv.201500367

For various neurodegenerative disorders like Alzheimer's and Parkinson’s diseases, selective and reversible MAO-B inhibitors have a great therapeutic value. In our previous study, we have shown that a series of methoxylated chalcones with F functional gro

Full text of DOI:10.1002/cbdv.201500367

Article Type: Full Paper
Potent and Selective Monoamine Oxidase-B Inhibitory Activity: Fluoro- vs.
Trifluoromethyl-4-hydroxylated Chalcones Derivatives
1
2
3
3
by Bijo Mathew* , Githa Elizabeth Mathew , Gülberk Uçar* , Ipek Baysal , Jerad
4
2
2
5
Suresh , Sincy Mathew , Abitha Haridas , and Venkatesan Jayaprakash
1
Division of Drug Design and Medicinal Chemistry Research Lab, Department of
Pharmaceutical Chemistry, Ahalia School of Pharmacy, Palakkad -678557, Kerala, India
(phone: +919946700219; fax: 0491 2508537; e-mail: bijovilaventgu@gmail.com)
2
Department of Pharmacology, Grace College of Pharmacy, Palakkad 678004, Kerala,
India
3
Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, 06100
Sıhhiye, Ankara, Turkey
4
Department of Pharmaceutical Chemistry, Madras Medical College, Chennai-600004,
India
5
Department of Pharmaceutical Sciences and Technology, Birla Institute of
Technology, Mesra, Ranchi, Jharkhand 835 215, India
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/cbdv.201500367
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For various neurodegenerative disorders like Alzheimer’s and Parkinsonism diseases,
selective and reversible MAO B inhibitors have a great therapeutic value. In our previous
study, we have shown that a series of methoxylated chalcones with F functional group
exhibited high binding affinity towards for human monoamine oxidase-B (hMAO-B). In
continuation with our earlier study and to extend the structure–activity relationships, a series
of new five chalcones were studied for their inhibition of hMAO. The results demonstrated
that these compounds are reversible and selective hMAO-B inhibitors with a competitive
mode of inhibition. The most active compound, (2E)-1-(4-hydroxyphenyl)-3-[4-
(
trifluoromethyl)phenyl]prop-2-en-1-one exhibited K value 0.33 ± 0.01 µM towards hMAO-
i
B with a selectivity index of 26.36. Molecular docking study revealed that the presence of a
H-bond network in hydroxylated chalcone with the N5 atom of FAD is crucial for MAO-B
selectivity and potency.
Keywords: Chalcone, MAO-A, MAO-B, Molecular docking.
1
. Introduction
The Monoamine oxidase (MAO) enzyme consists of two isoforms, MAO-A and
MAO-B, which are widely distributed in central and peripheral nervous systems. These
isoforms are encoded by separate genes and share approximately 70% sequence identity at
the amino acid level and play crucial roles in the oxidative catabolism of neurotransmitter
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amines in central and peripheral tissues [1,2]. Selective inhibition of MAO-A results in the
elevated level of serotonin and noradrenaline and can be used for improving the symptoms of
depression [3]. While selective MAO-B inhibitors are used with L-DOPA and/or dopamine
agonists in the symptomatic treatment of Parkinson’s disease (PD) [4]. Side effects of most
current monoamine oxidase inhibitors (MAOi) are due to lack of affinity and selectivity
toward one of the isoforms. So, there is an urgent need to design new more potent, selective
and reversible inhibitors of MAO-A and MAO-B have great therapeutic value.
Considering the pharmacological importance of MAO inhibitors, the design of new
selective MAO inhibitors is pursued by several research groups. Chalcones are open-chain
flavonoids in which the two phenyl rings are linked by a three-carbon consists of α,β-
unsaturated carbonyl system. In this, aromatic ring A is generally nearer to the C=O group
and the B ring to the β C-atom of three-carbon unit. Chalcones are reported for their potential
MAO inhibition by different groups [5-11]. The most active chalcones were characterized by
the presence of OH/MeO substituents either ortho positions with Cl-atom at para position of
ring B. These compounds exhibited inhibitory activity in the nanomolar range with good
selectivity [12].
Recently, our group has reported some potent, selective and reversible inhibitors of
hMAO-B having methoxylated and thiophene based chalcones with F and CF substitution.
3
Amongst them, (2E)-1-(4-methoxyphenyl)-3-[4-(trifluoromethyl)phenyl]prop-2-en-1-one and
(
2E)-1-(thiophen-2-yl)-3-[4-(trifluoromethyl)phenyl]prop-2-en-1-one exhibited K value 0.22
i
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±
0.01 and 0.90 ± 0.05 µM, respectively, towards hMAO-B. The study mainly highlighted the
effect of orientation of F and CF groups at the ring B of chalcone on the inhibition of hMAO
3
[
13, 14]. The ability of chalcone scaffolds to act on the central nervous system (CNS) mainly
associated with its low polar surface area that can help to cross the blood brain barrier easily.
This property, principally earned by the hydrophobic nature of two aromatic ring of A and B
at the terminals of open chain of α,β-unsaturated system.
The present study aimed at expanding on the structure–activity relationships (SARs)
of hMAO inhibition of chalcone derivatives by synthesizing a series of fluorinated chalcones
with OH substituted phenyl system. The information regarding the orientation of F and CF3
groups in the B ring system of hydroxylated chalcone and their effect on MAO inhibition is
not explored so far. Earlier we reported fluorochalcones from 2-acetylthiophene and 4-
methoxyacetophenone and observed that position of F (ortho, meta, para) on ring B had a
negligible impact on inhibition of both hMAO-A and hMAO-B. A similar pattern is observed
for CF group, but with slight improvement in potency for 4-CF derivative. This clearly
3
3
■showcasing■ the effect of electronegativity on biological activity. When thiophene is
replaced with para-methoxyphenyl ring, the position of F and CF has shown a great
3
difference in potency in inhibiting both hMAO-A and hMAO-B [13, 14]. Chalcones having F
substitution at ortho- and para-positions were found to inhibit hMAB-B potently in
comparison with their meta-counterpart, whereas the reverse has been observed for hMAO-
A. Similar trend was observed for CF derivatives, but here they are found to have potency
3
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equivalent to their F counterpart. From these past two experiences, we learnt that change in
biological activity due to nature and position of F groups on ring B has also been greatly
influenced by the nature and position of substituents on ring A. With this background, in the
presented research we evaluated effect of para-hydroxyphenyl as ring A that is a better H-
bond acceptor/donor when compared with para-methoxyphenyl. para-Hydroxyacetophenone
moiety was selected as starting reagent for the synthesis of various hydroxylated chalcones.
For this purpose, chalcones were obtained by a Claisen–Schmidt condensation of p-
hydroxyacetophenone with various F and CF substituted benzaldehydes under basic
3
conditions as outlined in the Scheme.
2
. Results and Discussion
2
.1. Chemistry
The synthesis of the fluorinated hydroxylated chalcones was achieved by following
Claisen–Schmidt condensation of 4-hydroxyacetophenone with various fluorinated aromatic
1
aldehydes under 40% KOH/EtOH medium (Scheme). H-NMR spectrum showed the peaks of
eleven H-atoms compromising of signals of the α,β-unsaturated unit, the substituted two
phenyl system and OH group. It has been noted that, the up-field H-atom of α C-atom and
down-field H-atom of β C-atom coupled with CH H-atom with coupling constants of (15.6 –
16.0 Hz). This large coupling constant shows the presence of (E)-configuration in the
1
3
fluorinated hydroxylated chalcones. In C-NMR spectra, the peaks for C=O C-atom are in
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the range of δ(C) 188.92 – 188.43. Mass spectra of the compounds postulated their
corresponding molecular weights.
2
.2. MAO inhibitory potencies of fluorinated hydroxylated chalcones
The potential inhibitory activities of the fluorinated hydroxylated chalcones on hMAO
isozymes were determined by measuring their effects on the production of H O from the
2
2
substrate (p-tyramine), using the Amplex Red MAO assay kit and enzymes (recombinant
MAO isoforms). All the newly synthesized chalcones were screened for their potential
hMAO inhibitory activity using recombinant hMAO-A and hMAO-B. The results of the
inhibitory potencies and selectivities of the chalcones under study towards MAO isoforms,
and reference compounds, are depicted in Table 1. The presented study documents that all the
fluorinated chalcones inhibited hMAO-B selectively and reversibly in a competitive mode.
The most potent and selective hMAO-B inhibitor in this series was compound B5 that
inhibited hMAO-B isoform with a K value of 0.30 ± 0.05 µM and selective index of 26.36.
i
This was having a CF substitution at the para-position on the B ring of chalcone. The
3
following order of hMAO inhibitory activity was observed for the fluorinated hydroxylated
chalcones:
hMAO-A: 2-CF > 3-F > 2-F > 4-F > 4-CF3
3
hMAO-B: 4-CF > 2-F > 4-F > 2-CF > 3-F
3
3
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The study clearly indicated that all the chalcones under the present study exhibited
good activity in the submicromolar range towards the inhibition of hMAO-B. Chalcones
having F substitution at ortho- and para-position were found to be better than their meta-
counterpart in inhibiting hMAO-B, while a reverse trend has been observed for hMAO-A.
Chalcone having CF at ortho-position was found to inhibit hMAO-B at a concentration
3
equal to the one shown by the chalcones having F at ortho- and para-position. Chalcone
having CF at para-position was found to be better than their F counterparts (ortho, meta,
3
para) in inhibiting hMAO-B. The trend was quite similar to the one observed with the
fluorinated chalcones having thiophene as ring A, reported by our group [14], these
hydroxylated chalcones were found to be slightly better than their thienyl chalcones. But the
hydroxylated chalcones were not better than their methoxylated counterparts reported by our
group [13].
From the analysis of the inhibition data, it is clear that the presence of electron
withdrawing lipophilic group on the para-position of ring B of chalcone resulted the enhance
MAO-B inhibition, while the same substitution is associated with the decreased MAO-A
inhibition activity. Shifting the CF group from para- to ortho-position exhibiting three fold
3
less inhibition activity of MAO-B. Based on high inhibition potency and hMAO-B selectivity
as selection criteria, B5 was considered a promising lead compound for the development of
reversible hMAO-B inhibitors.
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The presence of CF substituent at para-position on ring B play a pivotal role in
3
determining a productive binding of chalcone-based hMAO-B inhibitors. The enhancement
of MAO-B inhibition potencies by CF substitution has been previously described [15-17].
3
Compounds B1, B3, and B4 were also found to be a potent MAO-B inhibitor with a K value
i
of 0.74, 0.83, and 0.90 µM, respectively.
2
.3. Kinetic analysis
Kinetic analyses were carried out for most potent hMAO-B inhibitor B5 from this
series. A set of Lineweaver–Burk plots were constructed in the absence and presence of
various concentrations of compound B5. The observation that the lines were linear and
intersects on the y-axis suggests that B5 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 vs. inhibitor concentration is shown in Fig. 2 and the K was estimated as 0.33 µM for
i
B5 competitively. This compound also showed the highest selectivity for hMAO-B (SI value
is calculated as 26.36).
2
.4. Reversibility studies
The reversibility of MAO inhibition with the newly synthesized compounds were
investigated, since it has been previously shown that reversibility of MAO inhibition is highly
important for drug safety over the irreversible inhibitors for the management of Parkinson’s
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disease. Reversibility of the inhibition of hMAO-B with the new compounds is shown in
Table 2. The results indicated that both MAO-A and MAO-B activities are recovered
following dialysis. The reversiblity of compound B5 (1.5 µM) was calculated as 24.77 ± 1.66
and 97.11 ± 2.33 before and after dialysis, respectively, whereas the percentage inhibition of
hMAO-B inhibition (%) of lazabemide (0.15 µM) was calculated as 7.85 ± 0.23 and 89.00 ±
5.20 before and after dialysis, respectively.
2
.5. Molecular docking
This preliminary molecular docking study points out that the presence of a H-bond
network in hydroxylated chalcone with the N5 atom of Flavin Adenine Dinucleotide (FAD) is
crucial for hMAO-B selectivity for compound B5. This stabilizing interaction makes the OH
group of ring A of fluorinated chalcones showed good proximity towards the isoalloxazine of
FAD unit (1.932 Å). This shorter distance between the phenolic OH and N5 atom of FAD
argues its enhanced binding affinities to the inhibitor binding cavity of MAO-B (Fig. 3).
Accordingly, it can be also anticipated that the para-position of the CF substituent on the
3
aromatic nucleus of B ring of chalcone can influence the strength of the H-bond and π-π
stacking interaction and consequently affecting the potency of this type of hMAO-B inhibitor.
Another possible explanation of MAO-B selectivity of B5 may be that CF group, by virtue
3
of its higher Hammett constant, forms more productive interactions with the hMAO-B active
site.
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Depending on the bound substrate/inhibitor, MAO-B active site can exhibit a bipartite
cavity so that the ligand has to negotiate a smaller entrance room before entering the substrate
cavity where FAD is accommodated [18]. The presence of CF group on the para-position of
3
ring B can contribute significant lipophilic nature to the molecule. Since the entrance cavity is
reported to be a highly hydrophobic environment, the CF substituent is stabilized principally
3
by Van der Waals interactions within the entrance cavity. The strong electron withdrawing
CF group in the para-position of the B ring of the B5 significantly reduces the electron
3
density and is capable of forming more π-π stacking interaction with FAD unit of the
inhibitor binding cavity of MAO-B [19].
3
. Conclusion
In the present work, evidence was acquired to demonstrate that hydroxylated chalcone
is a privileged scaffold for the design of potent, selective, and reversible MAO-B inhibitors.
It may thus be concluded that B5 is a promising lead for the development of reversible and
selective MAO-B inhibitors, which may be used in the treatment of Parkinson’s disease. It is
also mentioned that these lead chalcones are the versatile intermediates for the preparation of
2-pyrzolines compounds for the development of remarkable MAO inhibition properties.
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We sincerely thank the Department of Biochemistry, Faculty of Pharmacy, Hacettepe
University, Ankara, Turkey, for carrying out the enzyme inhibition studies of hMAO
isoforms.The authors are thankful to IIRBS, Mahatma Gandhi University, Kottayam, India
for carrying out the spectral analysis.
4
4
4
. Experimental Part
.1. Chemistry
.2. General procedure for the synthesis of chalcones
A mixture of 4-hydroxyacetophenone (0.01 mol, 1 equiv.), fluorinated aldehyde (0.01
mol, 1 equiv.) and 40% aq. KOH (15 ml) in EtOH (30 ml) was stirred at r.t. for about 2 – 6 h.
The resulting product was kept overnight in refrigerator. The separated solid was filtered,
washed with H O and recrystallized from EtOH. If the solid was not formed, dil. HCl is
2
slightly added to the mixture was to afford products.
(2E)-3-(2-Fluorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (B1). Yield: 53%.
1
Brown powder. M.p. 138 – 140°. H-NMR: 5.910 (s, 1 H); 7.696 – 7.657 (dd, J = 15.6, 1 H);
1
3
7
1
2
.933 – 7.894 (dd, J = 15.6, 1 H); 6.955 – 8.043 (m, 8 H). C-NMR: 188.92; 163.01; 160.17;
36.99; 131.69; 131.60; 131.26; 131.05; 129.88; 124.50; 116.40; 116.18; 115.52. ESI-MS:
+
42.256 ([M + H] ).
(2E)-3-(3-Fluorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (B2). Yield: 50%.
1
Cream powder. M.p: 114 – 116°. H-NMR: 5.932 (s, 1 H); 7.698 – 7.659 (dd, J = 15.6, 1 H);
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1
3
7
1
.931 – 7.896 (dd, J = 15.6, 1 H); 6.952 – 8.034 (m, 8 H). C-NMR: 188.43; 163.35; 161.14;
37.19; 131.33; 131.43; 131.25; 129.62; 124.48; 116.12; 115.87. ESI-MS: 242.243 ([M +
+
H] ).
(2E)-3-(4-Fluorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (B3). Yield: 56%.
1
Pale-yellow powder. M.p: 158 – 160°. H-NMR: 6.10 (s, 1 H); 7.508 – 7.469 (dd, J = 15.6, 1
1
3
H); 7.814 – 7.775 (dd, J = 15.6, 1 H); 6.955 – 8.043 (m, 8 H). C-NMR: 188.67; 162.34;
1
60.67; 136.64; 131.19; 131.87; 131.75; 128.68; 124.92; 116.72; 115.12. ESI-MS: 242.244
+
(
[M + H] ).
(2E)-1-(4-Hydroxyphenyl)-3-[2-(trifluoromethyl)phenyl]prop-2-en-1-one (B4).
1
Yield: 44%. Brown powder. M.p: 173 – 175°. H-NMR: 5.693 (s, 1 H); 8.154 – 8.114 (dd, J =
1
3
1
1
2
6, 1 H); 7.834 – 7.795 (dd, J = 15.6, 1 H); 6.952 – 8.028 (m, 8 H). C-NMR: 188.90;
63.01; 160.17; 136.99; 131.69; 131.26; 131.05; 129.88; 124.50; 116.40; 115.52. ESI-MS:
+
92.252 ([M + H] ).
(2E)-1-(4-Hydroxyphenyl)-3-[4-(trifluoromethyl)phenyl]prop-2-en-1-one (B5).
1
Yield: 62%. Pale-yellow powder. M.p: 161 – 163°. H-NMR: 5.827 (s, 1 H); 7.847 – 7.808
1
3
(
dd, J = 15.6, 1 H); 7.641 – 7.602 (dd, J = 15.6, 1 H); 6.868 – 8.259 (m, 8 H). C-NMR:
1
88.92; 163.71; 161.37; 136.87; 131.39; 131.12; 131.01; 129.18; 124.58; 116.67; 115.63.
+
ESI-MS: 292.252 ([M + H] ).
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4
4
.3. Biochemistry
.3.1. Determination of inhibitory activities of the chalcones on human MAO-A and B
hMAO isoform activities were determined by a fluorimetric method described and
modified previously by using recombinant hMAO-A and hMAO-B (expressed in baculovirus-
infected BTI insect cells, Sigma, Germany) as enzyme sources and p-tyramine (0.05 – 1.00
mM) as common substrate. Specific MAO activities were calculated as 168.22 ± 9.55 pmol
–
1
–1
–1
–1
mg min (n = 3) for hMAO-A and 148.25 ± 8.60 pmol mg min (n = 3) for hMAO-B [20
–
22].
Study medium contained 0.1 ml of sodium phosphate buffer (0.05M, pH 7.4), various
concentrations of the synthesized compounds or known inhibitors (moclobemide, selegiline
and lazabemide), and recombinant hMAO-A or hMAO-B. This mixture was incubated for 15
min at 37° 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 H O catalyzed by MAO isoforms was detected using Amplex -Red
2
2
reagent, in the presence of HRP to produce the fluorescent product resorufin. Resorufin was
quantified at 37° 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
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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 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.
4
.3.2. Kinetic experiments
Synthesized compounds were dissolved in DMSO, with a maximum concentration of
% and used in a wide concentration range of 0.01 µM – 10.00 mM. The mode of MAO
1
inhibition was examined using Lineveaver–Burk plotting. The slopes of the Lineweaver–Burk
plots were plotted vs. the inhibitor concentration, and the K values were determined from the
i
x-axis intercept as -K . Each K value is the representative of single determination where the
i
i
2
correlation coefficient (R ) of the replot of the slopes vs. the inhibitor concentrations was at
least 0.98. SI was calculated as K (hMAO-A)/K (hMAO-B. The protein was determined
i
i
according to the Bradford method [23].
4
.3.3. Reversibility experiments
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Reversibility of the MAO inhibition with the compounds was determined by dialysis
method previously described [24]. Dialysis tubing 16 × 25 mm (Sigma, Germany) with a
molecular weight cut-off of 12,000 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 IC values for the inhibition of hMAO-A and -B,
5
0
respectively, in potassium phosphate buffer (0.05M, pH 7.4, 5% sucrose containing 1%
DMSO) for 15 min at 37°. Another sets were prepared by preincubation of same amount of
hMAO-A and B with the reference inhibitors (moclobemide, selegiline and lazabemide)
considering their IC values reported for the inhibition of hMAO-A and -B previously
5
0
reported. IC value for the inhibition of hMAO-A with moclobemide was taken as 4 µM and
5
0
IC values for the inhibition of hMAO-B with selegiline and lazabemide were taken as 0.079
5
0
µM and 0.03 µM, respectively [25, 26]. Enzyme-inhibitor mixtures were subsequently
dialyzed at 4° 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
2
4 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° over the
same time period [27].
4
.4. Molecular docking methodology
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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 [28]. Docking protocol done with X-ray crystal
structure of hMAO-A (2BXR) and hMAO-B (2BYB) downloaded from Protein Data Bank
(
www.rcsb.org) [29]. Protein preparation was carried out using Protein Preparation Wizard of
Maestro-8.5 (Schrodinger LLC) [30]. Ligands were prepared through PRODRG webserver
http://davapc1.bioch.dundee.ac.uk/cgi-bin/prodrg) [31]. Grid preparation and the docking
parameters are prepared on the basis of reported method [32].
(
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Received December 1, 2015
Accepted March 7, 2016
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Table 1. Experimental K values corresponding to the inhibition of hMAO isoforms by the chalcones.
i
a
b
Compound
Experimental K values [µM] )
SI )
Inhibition type
Reversibility
MAO Selectivity
i
MAO-A
4.96 ± 0.23
MAO-B
B1
0.74 ± 0.04
1.03 ± 0.09
0.83 ± 0.04
0.90 ± 0.05
0.33 ± 0.01
5.50 ± 0.13
0.09 ± 0.04
0.006 ± 0.001
6.70
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Suicide inhibitor
Competitive
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Irreversible
Reversible
B
B
B
B
B
A
B
B
B2
4.22 ± 0.28
5.55 ± –0.36
3.61 ± 0.19
4.10
6.69
B3
B4
4.01
B5
8.70 ± 0.45
26.36
0.11
Moclobemide
Selegiline
0.61 ± 0.03
11.20 ± 1.08
1320.00 ± 302.50
124.44
Lazabemide
220.00
a
b
)
Values represents the mean ± SEM of three independent experiments. ) Selectivity index. It was calculated as K (MAO-A)/K (MAO-B).
i
i
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a
Table 2. Reversibility of hMAO inhibition by the chalcones )
Test compound
hMAO-AActivity
before dialysis [%]
100 ± 0.00
hMAO-AActivity
after dialysis [%]
99.74 ± 1.45
98.55 ± 2.88
96.44 ± 2.99
98.03 ± 4.31
90.34 ± 2.38
88.45 ± 3.00
85.25 ± 3.25
90.00 ± 2.21
97.00 ± 2.08
hMAO-B Activity
before dialysis [%]
100 ± 0.00
hMAO-B Activity
after dialysis [%]
100.59 ± 1.64
98.21 ± 4.00
11.77 ± 5.45
89.00 ± 5.20
90.26 ± 5.55
89.40 ± 4.67
87.16 ± 3.90
90.21 ± 5.03
97.11 ± 2.33
Reversibility
With no inhibitor
Moclobemide (16.00 µM)
Selegiline (0.35 µM)
Lazabemide (0.15 µM)
B1 (3.00 µM)
–
15.00 ± 1.04
77.90 ± 4.09
80.23 ± 3.87
68.65 ± 2.55
76.22 ± 3.21
80.00 ± 4.02
75.90 ± 3.00
86.24 ± 3.55
68.24 ± 3.69
9.88 ± 4.12
Reversible
Irreversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
7.85 ± 0.23
38.77 ± 1.98
57.90 ± 3.59
39.29 ± 2.35
52.88 ± 3.75
24.77 ± 1.16
B2 (4.00 µM)
B3 (3.50 µM)
B4 (4.00 µM)
B5 (1.50 µM)
a
)
hMAO Isoforms were preincubated with no inhibitor, with the newly synthesized compounds and also with the reference inhibitors for 15
min, and enzyme activities were measured. The mixtures were then dialyzed for 24 h and residual activities were determined. Each value
represents the mean ± SEM of three independent.
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Captions:
Scheme 1. Synthesis of fluorinated hydroxylated chalcones.
Fig. 1. Lineweaver–Burk plots of the oxidation of p-tyramine by recombinant hMAO-B. The
plots were constructed in the absence and presence of various concentration of compound B5.
Fig. 2. Replots of the slopes of the Lineweaver–Burk plots vs. inhibitor B5 concentration.
Fig. 3. Docking pose of B5 in the MAO-B active site. Yellow mesh indicates π-π stacking
interaction.
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This article is protected by copyright. All rights reserved.