Monoamine oxidase inhibitory activities of heterocyclic chalcones

Article abstract of DOI:10.1016/j.bmcl.2015.09.049

Studies have shown that natural and synthetic chalcones (1,3-diphenyl-2-propen-1-ones) possess monoamine oxidase (MAO) inhibition activities. Of particular importance to the present study is a report that a series of furanochalcones acts as MAO-B selectiv

Full text of DOI:10.1016/j.bmcl.2015.09.049

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Monoamine oxidase inhibitory activities of heterocyclic chalcones
Corné Minders a,b, Jacobus P. Petzer a,b, Anél Petzer b, Anna C. U. Lourens a,b,
⇑
a Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
b Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Studies have shown that natural and synthetic chalcones (1,3-diphenyl-2-propen-1-ones) possess mono-
amine oxidase (MAO) inhibition activities. Of particular importance to the present study is a report that a
series of furanochalcones acts as MAO-B selective inhibitors. Since the effect of heterocyclic substitution,
other than furan (and more recently thiophene, piperidine and quinoline) on the MAO inhibitory proper-
ties of the chalcone scaffold remains unexplored, the aim of this study was to synthesise and evaluate
further heterocyclic chalcone analogues as inhibitors of the human MAOs. For this purpose, heterocyclic
chalcone analogues that incorporate pyrrole, 5-methylthiophene, 5-chlorothiophene and 6-methoxypyr-
Received 20 August 2015
Revised 17 September 2015
Accepted 21 September 2015
Available online xxxx
Keywords:
Chalcone
Monoamine oxidase (MAO)
Inhibition
Competitive
Reversible
idine substitution were examined. Seven of the nine synthesised compounds exhibited IC50 values <1
for the inhibition of MAO-B, with all compounds exhibiting higher affinities for MAO-B compared to the
MAO-A isoform. The most potent MAO-B inhibitor (4h) displays an IC50 value of 0.067 M while the most
potent MAO-A inhibitor (4e) exhibits an IC50 value of 3.81 M. It was further established that selected
lM
l
l
heterocyclic chalcones are reversible and competitive MAO inhibitors. 4h, however, may exhibit tight-
binding to MAO-B, a property linked to its thiophene moiety. We conclude that high potency chalcones
such as 4h represent suitable leads for the development of MAO-B inhibitors for the treatment of
Parkinson’s disease and possibly other neurodegenerative disorders.
Ó 2015 Elsevier Ltd. All rights reserved.
Neuropsychiatric and neurodegenerative disorders are often clo-
sely linked to reduced levels of monoamine neurotransmitters in
the central nervous system. For example, reduced levels of sero-
tonin, and to a lesser extent norepinephrine,1,2 have been impli-
cated in depressive illness, while depletion of central dopamine is
responsible for the characteristic motor symptoms of Parkinson’s
disease.3 Replacement therapy with metabolic precursors or agonist
drugs, and inhibition of neurotransmitter reuptake are approaches
frequently used to restore synaptic neurotransmission. Blocking
the metabolic degradation of the affected monoamines and thereby
increasing their levels in the central nervous system represent
another useful approach. The flavin adenine dinucleotide (FAD)-
containing enzyme monoamine oxidase (MAO) is a key metabolis-
ing enzyme of monoamine neurotransmitters in peripheral tissues
and in the central nervous system.4 MAO consists of two isoforms
of which the MAO-A form catalyses the degradation of serotonin,
as well as epinephrine and norepinephrine.5 MAO-A inhibitors are
thus an established class of antidepressant drugs. Inhibitors of the
MAO-B isoform, in turn, are used in the treatment of Parkinson’s dis-
ease where these drugs block the MAO-B-catalysed metabolism of
the direct metabolic precursor of dopamine. This combination fur-
ther enhances central dopamine levels and allows for the effective
L
-Dopa dose to be reduced.7–9
MAO inhibitors may also reduce potentially injurious by-
products of MAO-catalysed reactions. These are hydrogen peroxide,
which is generated from the re-oxidation of the FAD cofactor by
molecular oxygen, and aldehyde species, which form when the
imine product of most amine substrates are hydrolysed. Since these
by-products may contribute to the neurodegenerative processes in
Parkinson’s disease, MAO inhibitors may be neuroprotective by
reducing their central concentrations.4,10 In this respect, MAO-B
inhibitors may be of particular relevance to Parkinson’s disease
since MAO-B activity increases with age while MAO-A activity
remains unchanged.11 MAO-A inhibitors may have a similar role
in cardiovascular pathophysiology. Experimental evidence suggests
that MAO-A is a major source of hydrogen peroxide in the heart,
which may be responsible for increased oxidative stress and
age-related impairment of cardiac function.12 The by-products of
MAO-catalysis may target mitochondrial function and in this way
affect the function and viability of the myocardium.13
dopamine.5,6 MAO-B inhibitors are often combined with
L-Dopa,
Based on these considerations, MAO inhibitors are of impor-
tance in medicine and numerous research groups are involved in
the discovery of novel MAO inhibitors of both natural and
synthetic origin. Chalcones (1,3-diphenyl-2-propen-1-ones) (1) is
⇑
Corresponding author. Tel.: +27 18 2992266; fax: +27 18 2994243.
E-mail address: arina.lourens@nwu.ac.za (A.C.U. Lourens).
http://dx.doi.org/10.1016/j.bmcl.2015.09.049
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
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2
C. Minders et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 1
a class of compounds that have been shown to inhibit the MAOs
(Fig. 1).14–20 Chalcones are intermediates in the biosynthesis of fla-
vonoids and are reported to possess a wide range of biological
activities.21,22 Since chalcones are relatively facile to prepare in
the laboratory, they often serve as reagents and intermediates in
organic chemistry. In fact, chalcones have been used in the synthe-
sis of pyrazoline derivatives as part of an effort to discover novel
MAO inhibitors.23–30 Literature reports that, with the appropriate
substitution, chalcones themselves are, however, high potency
MAO-B inhibitors. For example, synthetic chalcone 2 inhibits
The reported IC50 values for the inhibition of human MAO-B by selected chalcone
analogues31
MAO-B
IC50 (lM)
O
Cl
O
3a
3b
3c
3d
0.174
0.529
2.10
Cl
O
Cl
human MAO-B with an IC50 value of 0.0044
by 2 for which no inhibition of MAO-A is observed (up to
50 M), chalcones are in general selective MAO-B inhibitors. A
l
M.14 As exemplified
O
l
Cl
O
related study has recently investigated the human MAO inhibition
properties of a series of furanochalcones in an attempt, for the first
time, to determine the effect of heterocyclic substitution on inhibi-
tion potency.31 The resulting furanochalcones also proved to be
potent MAO-B-selective inhibitors with the most active compound
O
O
Cl
O
O
0.490
(3a) exhibiting an IC50 value of 0.174 lM (Table 1). Since the effect
of heterocyclic substitution other than furan, and more recently
thiophene, piperidine and quinoline,32,33 on the MAO inhibitory
properties of chalcones remains unexplored, the present study
3e
3f
0.616
0.200
0.275
Cl
Br
F
examines
a series of heterocyclic chalcone analogues that
O
O
incorporates pyrrole, 5-methylthiophene, 5-chlorothiophene and
6-methoxypyridine substitution (Table 2). This study is part of a
systematic investigation of the effect of heterocyclic substitution
of chalcones on MAO inhibition.
O
O
A further point of interest is that, in the reported study, two
representative substituted furanochalcones were found to inhibit
MAO-B reversibly.31 Reversibility of MAO inhibition is an impor-
tant consideration since the irreversible inhibition of MAO-A in
the periphery is associated with potentially fatal changes in
blood-pressure when these drugs are taken with certain foods.34,35
For this reason MAO-A inhibitors are used with caution in the clinic
and dietary restrictions are imposed. Reversible MAO-A inhibitors
are less likely to cause blood-pressure changes and are considered
to possess better safety profiles in this regard.36,37 Since MAO-B
inhibition is not associated with serious adverse effects, selective
(reversible or irreversible) MAO-B inhibitors are considered safer
drugs.38,39 It should, however, be kept in mind that long-lasting
enzyme inhibition by irreversible MAO-B inhibitors may in theory
lead to immunogenicity, and de novo synthesis of the MAO protein
is required for activity to recover.
The heterocyclic chalcone analogues, 4a–h, were synthesised by
the Claisen–Schmidt condensation between an aromatic aldehyde
and heteroaromatic methyl ketone in yields of 11–60%
(Scheme 1).40 For the synthesis of 4i, a substituted acetophenone
was reacted with a heteroaromatic aldehyde (yield 1.8%). For all
reactions, sodium hydroxide served as base. The structures and
purities of the chalcone analogues were verified by 1H NMR, 13C
NMR, mass spectrometry and HPLC analysis as cited in Supplemen-
tary material. The most characteristic signals observed in the 1H
NMR spectra, were those of the double bond protons, that were
3g
CF3
present as two doublets with coupling constants of 15.1–15.8 Hz,
indicating the trans geometry. In the 13C NMR spectra, the most
downfield signal, at 177.5–190.8 ppm, signified the presence of
the carbonyl group. For all compounds, NMR (chemical shifts, inte-
gration, multiplicities and coupling constants) and mass spectra
corresponded with the proposed structures. Purities, as deter-
mined by HPLC were acceptable (94–100%).
To determine the inhibitory activities of the heterocyclic chal-
cone analogues, the recombinant human MAO-A and MAO-B
enzymes were used according to a previously published proto-
col.41,42 For both MAO isoforms, kynuramine served as enzyme
substrate. Kynuramine is metabolised by the MAOs to yield 4-hy-
droxyquinoline, a metabolite which fluoresces in alkaline media.
MAO catalytic rates were thus determined by measuring the for-
mation of 4-hydroxyquinoline with fluorescence spectrophotome-
try. By carrying out these experiments in the presence of various
concentrations of the test inhibitors (0.003–100 lM), sigmoidal
plots of catalytic rate versus logarithm of inhibitor concentration
were constructed. From these plots IC50 values were calculated.
Examples of sigmoidal plots for the inhibition of MAO-A and
MAO-B are given in Figure 2.
The IC50 values for the inhibition of the human MAOs are given
in Table 2. From the results it is clear that all the heterocyclic chal-
cone analogues possess higher affinities for the MAO-B isoform
with selectivity index (SI) values of >4.6. The most selective
MAO-B inhibitor, compound 4h (SI = 240), also is the most potent
MAO-B inhibitor of the current study with an IC50 value of
O
A
B
1
0.067
range as that of the reference MAO-B inhibitor, lazabemide
(IC50 = 0.091
M).43 With the exception of 4b and 4f, all chalcones
display IC50 <1 M for the inhibition of MAO-B. The most potent
MAO-A inhibitor is compound 4e (IC50 = 3.81 M). Although not
considered highly potent, this value is similar to that of toloxatone,
lM. The inhibition potency of this compound is in the same
OH
O
l
l
H3CO
Cl
2
l
Figure 1. The structures of chalcones discussed in the text.
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C. Minders et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 2
The IC50 values for the inhibition of human MAO-A and MAO-B by heterocyclic chalcone analogues, 4a–i, and reference inhibitors
a
IC50
(l
M)
SIb
MAO-A
MAO-B
O
S
4a
4b
4c
7.56 1.36
0.133 0.048
57
44
46
Cl
H
CF3
O
Cl
N
143 24.1
15.3 6.16
3.27 0.419
0.330 0.053
O
Cl
H3CO
N
O
O
O
H
N
Br
F
4d
8.98 1.52
0.803 0.037
11
H
N
4e
4f
3.81 0.387
267 80.5
10.5 1.20
0.830 0.100
1.40 0.140
0.116 0.030
4.6
191
91
CF3
CF3
H
N
O
Br
F
S
4g
Cl
O
Br
S
4h
16.1 2.14
0.067 0.016
0.185 0.050
240
54
F
OH
O
S
4i
9.99 2.64
Cl
H3CO
Toloxatone
Lazabemide
3.92c
—
—
0.091c
a
b
c
All values are expressed as the mean standard deviation (SD) of triplicate determinations.
The selectivity index is the selectivity for the MAO-B isoform and is given as the ratio of IC50(MAO-A)/IC50(MAO-B).
Value obtained from Ref. 43.
O
O
O
100
75
50
25
0
a
Ar1
Ar1
Ar2
H
Ar2
Scheme 1. The general synthetic route for heterocyclic chalcone analogues, 4a–i.
Reaction conditions: (a) ketone (1 equiv), aldehyde (1 equiv), NaOH (40%;
0.5 equiv), EtOH, rt.
4h
4e
a MAO-A inhibitor in clinical use, which is reported to inhibit
MAO-A with an IC50 value of 3.92 l
M.43
From the inhibition data, the following structure–activity rela-
tionships (SARs) and comparisons may be made: (1) by comparing
the MAO-A and MAO-B inhibition potencies of the pyrrole
derivatives, it is clear that trifluoromethyl substitution on the para
position of the phenyl ring (4e) is preferable over substitution in
the meta position (4f) for both isoforms. In fact the para substituted
compound is the most potent MAO-A inhibitor of the current
series. (2) A comparison of the MAO-B inhibition potencies of the
-3
-2
-1
0
1
2
3
Log[I]
Figure 2. The sigmoidal plots for the inhibition of MAO-A by 4e (filled circles) and
MAO-B by 4h (open circles).
5-chlorothiophene derivatives, 4a (IC50 = 0.133
(IC50 = 0.116 M), indicate that a 3-bromo-4-fluorophenyl and
4-trifluoromethylphenyl substituents yields similar MAO-B
l
M) and 4g
inhibition potencies. (3) Previously reported compound 2, the most
active analogue among a series of 1,3-diphenylprop-2-en-1-ones,
was resynthesised for comparative purposes.14 In the present
l
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4
C. Minders et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
study this compound exhibits an IC50 value of 0.093 0.022
the inhibition of human MAO-B, which is slightly less potent than
the most active analogue, 4h (IC50 = 0.067 M). This indicates that
heterocyclic substitution of the chalcone scaffold (with a methylth-
iophene ring in particular) is a viable design strategy. It should be
noted that the difference in activities of these compounds are
within experimental error, and their activities may thus be viewed
as similar. The difference in the IC50 values of 2 in the present and
the reported study (IC50 = 0.0044 lM) may be due to the use of dif-
ferent substrates and experimental conditions. (4) Comparison of
the MAO-B inhibition potencies of 3-bromo-4-fluorophenyl
derivatives 4h (IC50 = 0.067 lM) and 4g (IC50 = 0.116 lM) reveals
l
M for
Since reversibility of MAO inhibition is frequently a considera-
tion in inhibitor design and development, the reversibility of
MAO inhibition by the most potent MAO-A and MAO-B inhibitors,
4e, and 4h, respectively, were examined. This study also investi-
gated the reversibility of MAO-B inhibition by compound 4d. To
examine the reversibility of inhibition, the recoveries of enzyme
activity after dilution of enzyme–inhibitor mixtures were evalu-
ated. MAO-A and MAO-B were preincubated (for 30 min) with
the test compounds at concentrations of 10 Â IC50 and 100 Â IC50
for the inhibition of the respective enzymes. These mixtures were
subsequently diluted 100-fold to yield inhibitor concentrations of
0.1 Â IC50 and 1 Â IC50, and the residual MAO activities were mea-
sured. For reversible inhibition, enzyme activity is expected to
recover to 90% after dilution of the enzyme–inhibitor mixtures to
an inhibitor concentration of 0.1 Â IC50, while enzyme activity is
expected to recover to 50% after dilution to 1 Â IC50. As positive
controls, the irreversible MAO-A and MAO-B inhibitors, pargyline
and (R)-deprenyl, respectively, were also evaluated. For irre-
versible inhibition, enzyme activity is not expected to recover after
dilution of enzyme–inhibitor mixtures.
l
that an electron donating methyl substituent in the thiophene ring
(4h) results in slightly improved MAO-B inhibition compared to
substitution with an electron withdrawing chlorine substituent
(4g). (5) When the MAO-B inhibition potencies of the 3-bromo-
4-fluorophenyl derivatives 4g (IC50 = 0.116
(IC50 = 0.803 M), and the MAO-B inhibition potencies of the
4-trifluoromethylphenyl derivatives 4a (IC50 = 0.133 M) and 4e
(IC50 = 0.830 M) are compared, the results indicate that
lM) and 4d
l
l
l
5-chlorothiophene substitution is preferable over pyrrole
substitution. Similarly, when the MAO-B inhibition potencies of
The results show that the pyrrole derivative, 4e, behaves as a
reversible inhibitor of both MAO-A and MAO-B (Fig. 3). After dilu-
the 3-chlorophenyl derivatives, 4c (IC50 = 0.330
l
M) and 4b
tion, of mixtures containing MAO and 4e to 0.1 Â IC50 and 1 Â IC50
,
(IC50 = 3.27 M) are compared, it is evident that 6-methoxypyri-
l
the MAO-A activity is recovered to levels 90% and 67%, respec-
tively, of the negative control value (experiment conducted in
absence of inhibitor). After dilution, MAO-B activity is recovered
to levels of 85% and 32%, respectively, of the negative control value.
In contrast, after similar treatment of MAO-A and MAO-B with the
irreversible inhibitors pargyline and (R)-deprenyl, respectively, the
MAO activities are not recovered as dilution to concentrations of
0.1 Â IC50 resulted in the recovery of only 1.2% and 3.4% enzyme
activity. These results were as expected, since a reversible mode
of binding to MAO-B was also illustrated for a related series of
furanochalcones.31
dine substitution is also preferable over pyrrole substitution.
As mentioned above, in a previous study a series of furanochal-
cones has been shown to inhibit the MAOs.31 The inhibition poten-
cies of the heterocyclic chalcones of the current study compounds
may thus be compared to the results obtained with the furanochal-
cones (Table 1). This direct comparison is possible, since these
derivatives were evaluated under the same experimental condi-
tions. In order to identify the heterocyclic substituent that confers
the most potent MAO-B inhibition activity, the potencies of the
following derivatives were compared: (1) the 3-chlorophenyl
derivatives 3b–d (IC50 = 0.529, 2.10, 0.490
l
M, respectively), 4b
Unexpected results were, however, obtained when the
reversibility of binding of the most potent MAO-B inhibitor, 4h,
was examined. As shown in Figure 4, after dilution of mixtures
containing MAO-B and 4h to concentrations equal to 0.1 Â IC50
and 1 Â IC50, the MAO-B activities are recovered to levels of only
26% and 5%, respectively, of the control value. This behaviour is
not fully consistent with a reversible interaction of 4h with
MAO-B. As mentioned above, for reversible inhibition, after dilu-
tion of enzyme–inhibitor mixtures to inhibitor concentrations of
0.1 Â IC50 and 1 Â IC50, enzyme activity is expected to recover to
(IC50 = 3.27 M) and 4c (IC50 = 0.330 M). An analysis of the MAO-B
l
l
inhibition activities of these compounds indicate that the effect of
the heteroaromatic/aromatic substituent on activity, in decreasing
order is: 6-methoxypyridine > 5-methylfuran > phenyl > furan >
pyrrole. It should be noted that the activities of 3b, 3d and 4c are
similar in range. (2) The 3-bromo-4-fluorophenyl derivatives, 3f
(IC50 = 0.200
lM), 4d (IC50 = 0.803
lM), 4g (IC50 = 0.116 lM) and
4h (IC50 = 0.067
l
M). An analysis of the MAO-B inhibition activities
indicates that the effect of the heteroaromatic substituent for these
compounds, on activity, in decreasing order is: 5-methylthio-
phene > 5-chlorothiophene > 5-methylfuran > pyrrole. It should be
noted that the activities of 3f and 4g are, however, similar. (3) The
MAO-A
MAO-B
100
75
50
25
0
100
75
50
25
0
4-trifluoromethylphenyl derivatives, 3g (IC50 = 0.275
lM), 4a
(IC50 = 0.133 M) and 4e (IC50 = 0.830 M). An analysis of the
l
l
MAO-B inhibition activities indicate that the effect of the
heteroaromatic substituent for these compounds, on activity, in
decreasing order is: 5-chlorothiophene > 5-methylfuran > pyrrole.
These results show that substitution with a 5-methylthiophene
group is an improvement on furan or methylfuran substitution,
and results in optimal MAO-B inhibition activity, while pyrrole sub-
stitution is associated with decreased MAO-B inhibition activity.
This is exemplified by the finding that 4h is 2.6-fold more potent
as a MAO-B inhibitor than the most potent furanochalcone (3a)
e
IC50
IC 50
y
or
n
i
it
nhib
No I
l
1 x
Parg
=
]
= 0.1 x
]
(IC50 = 0.174 l
M) investigated by Robinson et al.,31 and has similar
[4e
[4e
activity to 2, previously investigated by Chimenti and co-workers.14
It should be kept in mind that this is a preliminary study and future
work will include the expansion of this series, to investigate the
effect of further variations in the substitution of the phenyl ring in
particular, and also to investigate the effect of heteroaromatic
substitution other than those considered in this study.
Figure 3. Dilution reverses MAO-A and MAO-B inhibition by 4e. The MAOs and 4e
were incubated at inhibitor concentrations of 10 Â IC50 and 100 Â IC50 for 30 min
and subsequently diluted 100-fold to 0.1 Â IC50 and 1 Â IC50. For comparison, MAO-
A
and MAO-B were similarly preincubated with pargyline and (R)-deprenyl,
respectively, at concentrations equal to 10 Â IC50 and diluted to 0.1 Â IC50. The
residual enzyme activities after dilution were measured and are shown.
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C. Minders et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
MAO-B
MAO-B
MAO-B
100
75
50
25
0
100
75
50
25
0
100
75
50
25
0
l
r
IC50
IC50
ito
reny
nhib
No I
1 x
=
]
= 0.1 x
NI
4h
dialysed
depr
4h
]
(R)-Dep
[4d
[4d
undialysed
Figure 4. Dilution reverses MAO-B inhibition by 4d but not inhibition by 4h. MAO-
B and the test inhibitors were incubated at inhibitor concentrations of 10 Â IC50 and
Figure 5. Dialysis reverses MAO-B inhibition by 4h. MAO-B and 4h (at
a
concentration of 4 Â IC50) were incubated for 15 min, dialysed for 24 h and the
residual enzyme activity was measured (4h dialysed). Similar dialysis of MAO-B in
the absence (NI dialysed) and presence of the irreversible inhibitor, (R)-deprenyl
(depr dialysed) were also carried out. The residual MAO-B activity of undialysed
mixtures of MAO-B with 4h was also recorded (4h undialysed).
100 Â IC50 for 30 min and subsequently diluted 100-fold to 0.1 Â IC50 and 1 Â IC50
.
For comparison, MAO-B was similarly preincubated with (R)-deprenyl at
a
concentration equal to 10 Â IC50 and diluted to 0.1 Â IC50. The residual enzyme
activities after dilution were measured and are shown.
90% and 50%, respectively. A possible explanation for this finding is
that 4h may exhibit tight-binding to the MAO-B enzyme, and the
inhibition caused by this compound is not readily reversed by
dilution. This possibility is further explored below where the
reversibility of MAO-B inhibition by 4h is examined by dialysis.
To investigate whether potential tight-binding of 4h was due to
the presence of the phenyl or thiophene moieties, the reversibility
of MAO-B inhibition of 4d was also examined. These results, given
in Figure 4, show that after dilution of mixtures containing MAO-B
2400
MAO-B
1800
1200
600
100
−0.03 0.00 0.03 0.06 0.09
75
[I], µ
M
50
25
0
and 4d to inhibitor concentrations equal to 0.1 Â IC50 and 1 Â IC50
,
the MAO-B activities are recovered to levels of 81% and 60%,
respectively, of the control value. This behaviour is consistent with
a reversible interaction of 4d with MAO-B. From these results it
could thus be derived that the tight-binding observed for 4h is
due to the presence of the thiophene moiety, as the pyrrole
derivative 4d, with a similar phenyl substituent, binds reversibly
to MAO-B. Potential tight-binding of inhibitors to the MAOs has
been previously reported for a variety of different classes of
compounds.44–47
-0.02
0.00
0.02
1/[S]
0.04
Figure 6. Lineweaver–Burk plots of human MAO-B activities in the absence (filled
squares) and presence of various concentrations of 4h [IC50(MAO-B) = 0.067 M].
The inhibitor concentrations used were: ¼ Â IC50, ½ Â IC50, ¾ Â IC50, 1 Â IC50 and
1¼ Â IC50 and the inset is a graph of the slopes of the Lineweaver–Burk plots versus
inhibitor concentration.
l
Since the inhibition of MAO-B by compound 4h may not to be
readily reversible and 4h therefore may exhibit a degree of
tight-binding to the MAO-B active site, the reversibility of MAO-B
inhibition by 4h was further examined by dialysis. For this purpose
MAO-B and 4h, at a concentration of 4 Â IC50, were preincubated
for a period of 15 min and subsequently dialysed for 24 h. The
residual enzyme activity was measured and compared to similar
dialysis experiments performed in the absence of inhibitor and
presence of the irreversible inhibitor, (R)-deprenyl. For reversible
inhibition, the enzyme activity is expected to recover to 100% after
dialysis. The results, given in Figure 5, show that inhibition of
MAO-B by 4h is almost completely reversed after 24 h of dialysis,
with the MAO-B activity recovering to a level of 83% of the control
value (activity after dialysis in absence of inhibitor). In contrast,
MAO-B activity in undialysed mixtures of the enzyme and 4h is
only 18% of the control value. This behaviour is consistent with
reversible inhibition of MAO-B by 4h. As expected, enzyme activity
is not recovered after dialysis of mixtures containing MAO-B and
(R)-deprenyl, with activity at only 5% of the control value.
To further investigate MAO-B inhibition by 4h, the present
study examines the mode (e.g., competitive) of inhibition. For this
purpose Lineweaver–Burk plots were constructed. Six plots were
constructed employing the following inhibitor concentrations:
Table 3
The percentage viable HeLa cells remaining after treatment with 4d, 4e and 4h,
compared to untreated cells (100%)
% viable cellsa
1
l
M
10 lM
4d
4e
4h
99.4 3.4
98.0 8.6
4.66 6.9*
95.7 4.0
77.5 11.6*
104 4.7
*
p <0.05 compared to untreated cells.
Values are given as mean SD of triplicate determinations.
a
intersect on the y-axis (Fig. 6). This suggests that 4h is a competi-
tive inhibitor of human MAO-B. This is in agreement with the find-
ing that this compound is a reversible MAO-B inhibitor. Global
(shared) fitting of the inhibition data directly to the Michaelis–
Menten equation yields
a Ki value of 0.0064 0.0004 lM
(r2 = 0.999) for the inhibition of MAO-B.
To obtain an early assessment of the ‘drug-like’ properties of
some the heterocyclic chalcones, the toxicity of selected inhibitors,
4d, 4e and 4h, towards cultured cells was determined. For this
0
lM, ¼ Â IC50, ½ Â IC50, ¾ Â IC50, 1 Â IC50 and 1¼ Â IC50. The
results show that the Lineweaver–Burk plots are linear and
Please cite this article in press as: Minders, C.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.049

6
C. Minders et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
purpose, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) cell viability assay was employed.48,49 Cytotoxicity
was evaluated by exposing HeLa cells for 24 h to concentrations of
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.09.
049.
1 and 10 lM of the test compounds. To determine if statistical dif-
ferences exist between mean values of the drug-treated and drug-
naive cells, the viability data were analysed by One-way ANOVA
followed by Post-hoc Dunnett’s analysis. As shown in Table 3, the
most potent MAO-B inhibitor of the present series, 4h, was non-
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95.7% viable cells, respectively, remaining. Compound 4e, the most
potent MAO-A inhibitor, however, exhibited significant toxicity
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l
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Compared to 4h, compound 4e is thus toxic to cultured HeLa cells.
To determine whether the pyrrole or phenyl substituents were
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In conclusion, this study discovers a number of potent new
MAO-B inhibitors among the heterocyclic chalcone class of com-
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potent chalcone analogue. 4h is more potent as a MAO-B inhibitor
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The NMR and MS spectra were recorded by André Joubert and
Johan Jordaan of the SASOL Centre for Chemistry, North-West
University. This work is based on the research supported in part
by the Medical Research Council of South Africa and National
Research Foundation of South Africa (Grant specific unique
reference numbers (UID) 85642, 80647, 96180 and 76308). The
Grantholders acknowledge that opinions, findings and conclusions
or recommendations expressed in any publication generated by
the NRF supported research are that of the authors, and that the
NRF accepts no liability whatsoever in this regard.
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