Monoamine oxidase inhibition by selected anilide derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.08.036

A series of anilide derivatives were synthesized and evaluated as inhibitors of recombinant human monoamine oxidase (MAO) A and B. The most potent inhibitors among the derivatives that were initially evaluated were (2E)-N-(3-chlorophenyl)-3-phenylprop-2-enamide (2c) and (2E)-N-(3-bromophenyl)- 3-phenylprop-2-enamide (2d) with IC₅₀ values of 0.53 μM and 0.45 μM, respectively. These derivatives exhibited reversible and selective inhibition of MAO-B with binding affinities 37 fold higher for MAO-B than for MAO-A. Analysis of the possible binding interactions of these inhibitors with active site models of human MAO-A and -B led to the design of phenolic and benzonitrile derivatives of 2c and 2d. Among these were (2E)-N-(3-chlorophenyl)- 3-(4-hydroxyphenyl)prop-2-enamide (7c) and (2E)-N-(3-bromophenyl)-3-(4- hydroxyphenyl)prop-2-enamide (7d) which inhibited MAO-B selectively and reversibly with IC₅₀ values of 0.032 μM and 0.026 μM, respectively. These inhibitors were at least 14 fold more potent than 2c and 2d. This study concludes that N,3-diphenylprop-2-enamide is a suitable scaffold for the design of selective MAO-B inhibitors and structural modifications to enhance the binding affinities of the inhibitors for the MAO-B active site include substitution with halogens on the N-phenyl ring and substitution with hydroxyl and nitrile functional groups on the para and meta positions, respectively, of the C3 phenyl ring. Possible binding modes of these structures within the MAO-B active site are proposed with the emphasis on the interactions of the inhibitor halogens and the hydroxyl and nitrile functional groups with active site residues and water molecules.

Full text of DOI:10.1016/j.ejmech.2011.08.036

European Journal of Medicinal Chemistry 46 (2011) 5162e5174
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Monoamine oxidase inhibition by selected anilide derivatives
*
Lesetja Legoabe, Johann Kruger, Anél Petzer, Jacobus J. Bergh, Jacobus P. Petzer
Pharmaceutical Chemistry, School of Pharmacy, 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:
A series of anilide derivatives were synthesized and evaluated as inhibitors of recombinant human
monoamine oxidase (MAO) A and B. The most potent inhibitors among the derivatives that were initially
evaluated were (2E)-N-(3-chlorophenyl)-3-phenylprop-2-enamide (2c) and (2E)-N-(3-bromophenyl)-3-
Received 17 June 2011
Received in revised form
24 August 2011
Accepted 25 August 2011
Available online 30 August 2011
phenylprop-2-enamide (2d) with IC50 values of 0.53 mM and 0.45 mM, respectively. These derivatives
exhibited reversible and selective inhibition of MAO-B with binding affinities 37 fold higher for MAO-B
than for MAO-A. Analysis of the possible binding interactions of these inhibitors with active site models
of human MAO-A and eB led to the design of phenolic and benzonitrile derivatives of 2c and 2d. Among
these were (2E)-N-(3-chlorophenyl)-3-(4-hydroxyphenyl)prop-2-enamide (7c) and (2E)-N-(3-
bromophenyl)-3-(4-hydroxyphenyl)prop-2-enamide (7d) which inhibited MAO-B selectively and
Keywords:
Monoamine oxidase B
Reversible inhibitor
Selectivity
reversibly with IC50 values of 0.032 mM and 0.026 mM, respectively. These inhibitors were at least 14 fold
more potent than 2c and 2d. This study concludes that N,3-diphenylprop-2-enamide is a suitable scaffold
for the design of selective MAO-B inhibitors and structural modifications to enhance the binding affin-
ities of the inhibitors for the MAO-B active site include substitution with halogens on the N-phenyl ring
and substitution with hydroxyl and nitrile functional groups on the para and meta positions, respectively,
of the C3 phenyl ring. Possible binding modes of these structures within the MAO-B active site are
proposed with the emphasis on the interactions of the inhibitor halogens and the hydroxyl and nitrile
functional groups with active site residues and water molecules.
Anilide
Benzimidazole
Molecular docking
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
this region [4,5]. Considering that MAO-B exhibits an age-related
increase in most brain regions including the basal ganglia, inhibi-
Monoamine oxidase A and B (MAO-A and eB) are flavin adenine
dinucleotide (FAD)-containing enzymes that are attached to the
outer membrane of the mitochondrion. MAO-A and eB catalyze the
tion of this enzyme is particularly relevant in Parkinson’s disease
[5e7]. Two irreversible MAO-B inhibitors, (R)-deprenyl and rasa-
giline, are employed as adjunctive therapy to levodopa or as
monotherapy for Parkinson’s disease while the reversible inhibi-
tors, lazabemide and safinamide, have entered the clinical devel-
opment phases [8]. It should be noted that MAO-A may also
contribute to the metabolism of dopamine in the basal ganglia since
MAO-A inhibitors potentiate the elevation of central dopamine
levels in primates treated with levodopa [1,3]. Accordingly,
a-carbon oxidation of a variety of monoamine neurotransmitters
and are therefore considered to be drug targets for the treatment of
psychiatric and neurological disorders [1]. Since serotonin is
metabolized by MAO-A in the brain, MAO-A inhibitors have been
used in the treatment of depressive illness [2]. Both irreversible and
reversible MAO-A inhibitors have been established as effective in
this regard. Inhibitors of MAO-B are considered useful in the
treatment of Parkinson’s disease since these drugs may conserve
the depleted levels of dopamine in the basal ganglia of the
parkinsonian brain and may enhance the levels of dopamine
derived from its metabolic precursor, levodopa [1,3]. Although
dopamine is equally well metabolized by both MAO-A and eB,
MAO-B is the prevalent isoform in the basal ganglia and is therefore
considered to be primarily responsible for dopamine metabolism in
moclobemide,
a reversible MAO-A inhibitor, possesses mild
symptomatic benefits when combined with levodopa in the treat-
ment of Parkinson’s disease [9,10].
While irreversible inhibitors of MAO have been used extensively
as antiparkinsonian and antidepressant drugs, irreversible inhibi-
tion has a number of potential disadvantages [11]. These include
the loss of isoform selectivity at high drug concentrations or
following repeated drug administration. Also, following drug
withdrawal, the rate by which enzyme activity is recovered may be
variable and may require several weeks [12]. The turnover rate for
the biosynthesis of MAO-B in the human brain, for example, may be
as much as 40 days [13]. In contrast, following the administration of
* Corresponding author. Tel.: þ27 18 2992206; fax: þ27 18 2994243.
E-mail address: jacques.petzer@nwu.ac.za (J.P. Petzer).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.036

L. Legoabe et al. / European Journal of Medicinal Chemistry 46 (2011) 5162e5174
5163
H
N
O
2a
1,4-Diphenyl-2-butene (3)
O
H
N
N
Chalcone (4)
(E)-2-Styryl-1H-benzimidazole (1)
Fig. 2. The structures of 1,4-diphenyl-2-butene (3) and chalcone (4).
aim of this study was to evaluate the possibility that substituted
anilides may serve as a useful scaffold for the design of selective
inhibitors of MAO-B. In accordance with the expectation that
these anilides may be applied in the design of new MAO-B
inhibitors, the anilide derivatives reported here are structural
analogues of 1,4-diphenyl-2-butene (3) [16] and a synthetic series
of chalcones (4) [17] (Fig. 2) which have been reported to act as
inhibitors of MAO-B. Initially, 12 anilide derivatives (2ael) were
synthesized with the aim of determining the most suitable scaf-
fold size for MAO inhibition and four meta N-phenyl ring
Fig. 1. The structures of anilide derivative 2a and (E)-2-styryl-1H-benzimidazole (1).
reversible inhibitors, enzyme activity is recovered when the
inhibitor is cleared from the tissues and since reversible inhibitors
has a shorter duration of action the risk of loss of isoform selectivity
is reduced. In addition, when the inhibitors act in a competitive
manner, the inhibition may be relieved when the substrate
concentration is increased. This is especially relevant with the use
of irreversible MAO-A inhibitors. Irreversible MAO-A inhibitors
may potentiate the cardiovascular effects of indirectly acting
sympathomimetic amines such as tyramine since inactivation of
MAO-A in the gut wall greatly increases the quantity of tyramine
entering the systemic circulation [1,9]. In contrast, with reversible
inhibitors, the inhibitors may be displaced by tyramine which is
subsequently normally metabolized by the enzyme. For these
reasons several research groups are interested in the discovery of
novel MAO inhibitors which interact reversibly with the enzymes.
From these observations it is apparent that MAO-A and eB are
of significant pharmacological interest and inhibitors of these
isozymes are useful therapeutic agents. Based on reports that (E)-
2-styrylbenzimidazoles (1) (Fig. 1) act as reversible inhibitors of
MAO-B, in the present study, a series of structurally related ani-
lide derivatives (2ael) (Scheme 1) were synthesized and evalu-
ated as inhibitors of recombinant human MAO-B [14,15].
Depending on the nature of the C-2 substituent, the anilide
derivatives that were examined may be divided into three classes:
N,3-diphenylpropenamide (2aed), N,5-diphenylpentadienamide
(2eeh) and N,4-diphenylbutenamide (2iel) homologues. The
substituents were considered (H, CH3
, Cl, Br). By employing
molecular docking, this study also examined the molecular
interactions between selected anilide inhibitors and the active
sites of human MAO-A and eB. Based on these studies additional
structural modifications (compounds 7aeh) are made to the most
potent anilide inhibitors that were initially designed to produce
exceptionally potent MAO-B inhibitors with IC50 values in the low
nM range (Schemes 2 and 3).
2. Results and discussion
2.1. Chemistry
The target anilide derivatives (2ael) were prepared in relatively
high yields by reacting the appropriately substituted aniline (5)
with (E)-cinnamic acid (6a), (E,E)-5-phenyl-2,4-pentadienoic acid
(6b) or (E)-styrylacetic acid (6c) in the presence of the carbodiimide
dehydrating agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodii-
mide (EDAC) (Scheme 1) [15]. While (E)-cinnamic acid (6a) and (E)-
2a:
R = H
H
N
2b:
2c:
2d:
R = CH3
R
R = Cl
i
R = Br
O
O
HO2C
6a
2e:
R = H
H
N
2f:
R = CH3
2g:
2h:
R
R = Cl
i
i
NH2
R
R = Br
HO2C
HO2C
+
6b
5
H
N
R
2i:
R = H
2j: R = CH3
2k:
6c
O
R = Cl
2l: R = Br
Scheme 1. Synthetic pathway to anilide derivatives 2ael. Reagents and conditions: (i) EDAC, DMF, DMAP, imidazole, rt, 4 h.

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L. Legoabe et al. / European Journal of Medicinal Chemistry 46 (2011) 5162e5174
2.2. Enzymology
The MAO-B inhibitory properties of the anilide derivatives
(2ael) were investigated using commercially available (Sigma-
eAldrich) microsomes from insect cells containing recombinant
human MAO-B with kynuramine as the substrate [20]. The IC50
values for the inhibition of MAO-B by 2ael are presented in Table 1.
While all of the derivatives were found to be MAO-B inhibitors, 2c
and 2d were the only inhibitors with IC50 values in the sub-
micromolar range (0.53 mM and 0.45 mM, respectively). For the N,3-
diphenylpropenamide homologues (2aed), the unsubstituted (2a)
and methyl substituted (2b) anilides were approximately 10 fold
less potent as MAO-B inhibitors than the chlorine (2c) and bromine
(2d) substituted anilides. From this observation it can be concluded
that substitution at C-3 of the phenyl ring of the anilide moiety with
chlorine and bromine results in improved binding of the inhibitors
to the active site of MAO-B. This trend was also observed for the
N,5-diphenylpentadienamide (2eeh) and N,4-diphenylbutenamide
(2iel) derivatives with the chlorine and bromine substituted
homologues exhibiting better inhibition than the corresponding
unsubstituted and methyl substituted anilides. In general, the styryl
substituted anilides (2aed) were better inhibitors than the corre-
sponding phenylbutadienyl substituted anilides (2eeh) which
indicates that shorter side chain length is more optimal for MAO-B
inhibition. Since the cinnamyl substituted anilides (2iel) were
relatively weak MAO-B inhibitors it can be concluded that the
introduction of a sp3 hybridized carbon in the side chain reduces
binding affinity for the enzyme. To determine if the inhibition of
MAO-B by the test compounds is time-dependent, 2a at a concen-
Scheme 2. Synthetic pathway to anilide derivatives 7aed. Reagents and conditions: (i)
THF, CH2Cl2, triethylamine, acetic anhydride, DMAP, reflux, 30 min; (ii) 6 N HCl; (iii)
DMF, 2 M oxalyl chloride in DCM, rt; (iv) CH3OH/H2O, NaOH, rt; (v) 1 M HCl.
styrylacetic acid (6c) were commercially available, (E,E)-5-phenyl-
2,4-pentadienoic acid (6b) was synthesized from cynnamylidene-
malonic acid according to the literature procedure [18]. The trans
geometry about the ethenyl
p-bonds of 2ael was confirmed by
protoneproton coupling constants of 14.7e15.8 Hz.
Anilide derivatives 7aed in turn were prepared from (E)-3-
hydroxycinnamic acid (8a) or (E)-4-hydroxycinnamic acid (8b)
(Scheme 2). The phenolic hydroxyl groups of the hydroxycinnamic
acids were firstly protected as the acetic acid esters by reaction with
acetic anhydride in tetrahydrofurane (THF). The resulting acetox-
ycinnamic acids, 9a and 9b, were converted to the corresponding
acyl chlorides with oxalyl chloride and were subsequently reacted
with the appropriately substituted aniline (5) in CH2Cl2 The
resulting phenylcarbamoyl-ethenylphenyl acetate esters 10aed
were treated with NaOH in CH3OH/H2O to give the target anilide
derivatives 7aed [19]. Protoneproton coupling constants of
tration approximately two fold its IC50 value (9.72 mM) was pre-
incubated with recombinant human MAO-B (0.03 mg protein/mL)
for 0, 15, 30 and 60 min and the residual enzyme activity was
determined. As shown in Fig. 3, the enzyme activity is not reduced
with increased preincubation time which indicates that 2a is not
a time-dependent inhibitor of MAO-B over the period evaluated.
The anilide derivatives (2ael) were also evaluated as potential
inhibitors of recombinant human MAO-A. Commercially available
(SigmaeAldrich) insect cell microsomes containing recombinant
human MAO-A were employed as enzyme source and kynuramine
served as the substrate. The IC50 values for the inhibition of MAO-A
by the test compounds are presented in Table 1. The derivatives
were found to be relatively weak MAO-A inhibitors with IC50 values
15.4e15.8 Hz indicated that the geometries about the
7aed were trans.
p-bonds of
Anilide derivatives 7eeh were synthesized according to a similar
procedure described for the preparation of 2ael (Scheme 3). (E)-3-
Cyanocinnamic acid (12a) or (E)-4-cyanocinnamic acid (12b) were
reacted with the appropriately substituted aniline (5) in the pres-
ence of the dehydrating agent, EDAC. 4-Dimethylaminopyridine
(DMAP) and imidazole served as catalysts for the condensation
reaction. The starting cyanocinnamic acids 12a and 12b were
synthesized, in turn, by reaction of 3-cyanobenzaldehyde (11a) or 4-
cyanobenzaldehyde (11b), respectively, with malonic acid in pyri-
ranging from 16 to 335
mM. All of the compounds displayed
selective inhibition of the MAO-B isoform with the most potent
Table 1
The IC50 values for the inhibition of recombinant human MAO-A and eB by anilide
derivatives 2ael.
dine. The trans geometries about the ethenyl
p-bonds of 7eeh
were also confirmed by protoneproton coupling constants of
15.4e15.8 Hz.
MAO-B
MAO-A
Selectivityb
IC50 valuea
(m
M)
IC50 valuea
(mM)
2a
2b
2c
2d
2e
2f
2g
2h
2i
5.65 ꢁ 0.02
4.50 ꢁ 0.16
0.532 ꢁ 0.16
0.447 ꢁ 0.04
5.16 ꢁ 0.96
6.65 ꢁ 5.64
2.20 ꢁ 0.97
2.73 ꢁ 1.19
33.2 ꢁ 1.18
48.1 ꢁ 8.03
23.5 ꢁ 0.78
30.9 ꢁ 0.07
82.8 ꢁ 29.4
48.1 ꢁ 3.15
19.8 ꢁ 0.68
16.7 ꢁ 5.09
14.7
10.7
37.2
37.4
e
No inhibitionc
335 ꢁ 44.8
98.2 ꢁ 31.2
105 ꢁ 5.06
297 ꢁ 34.9
74.8 ꢁ 1.81
58.4 ꢁ 44.7
34.5 ꢁ 2.55
50.4
44.6
38.5
8.95
1.56
2.49
1.12
2j
2k
2l
a
The IC50 values are expressed as the mean ꢁ SD of duplicate determinations.
b
The selectivity for the inhibition of MAO-B is given as the ratio: IC50(MAO-A)/
Scheme 3. Synthetic pathway to anilide derivatives 7eeh. Reagents and conditions: (i)
malonic acid, pyridine, piperidine, 100 ꢀC, 4 h; (ii) 5 N HCl; (iii) EDAC, DMF, DMAP,
imidazole, rt, 4 h.
IC50(MAO-B).
c
No inhibition observed at a maximum concentration of 100
inhibitor.
mM of the test

L. Legoabe et al. / European Journal of Medicinal Chemistry 46 (2011) 5162e5174
5165
Table 2
The IC50 values for the inhibition of recombinant human MAO-A and eB by anilide
derivatives 7aeh.
8
6
4
2
0
MAO-B
MAO-A
Selectivityb
IC50 valuea
(
mM)
IC50 valuea
(mM)
7a
7b
7c
7d
7e
7f
1.33 ꢁ 0.23
1.01 ꢁ 0.08
No inhibitionc
No inhibitionc
47.3 ꢁ 8.43
35.6 ꢁ 3.47
13.5 ꢁ 1.88
27.2 ꢁ 7.71
184 ꢁ 13.1
36.2 ꢁ 4.92
e
e
0.032 ꢁ 0.005
0.026 ꢁ 0.005
0.090 ꢁ 0.003
0.059 ꢁ 0.005
0.407 ꢁ 0.014
0.289 ꢁ 0.023
1478
1369
150
461
452
125
7g
7h
0
15
30
60
a
The IC50 values are expressed as the mean ꢁ SD of triplicate determinations.
Preincubation time (min)
b
The selectivity for the inhibition of MAO-B is given as the ratio: IC50(MAO-A)/
IC50(MAO-B).
Fig. 3. The time dependency of MAO-B inhibition by derivative 2a. The test inhibitor
was preincubated with recombinant human MAO-B for various periods of time
(0e60 min) and the residual enzyme catalytic rates (nmol 4-hydroxyquinoline formed/
min/mg protein) were measured. The catalytic rate recorded in the absence of the
inhibitor is 20.3 ꢁ 1.14 nmol/min/mg.
c
No inhibition observed at a maximum concentration of 100
inhibitor.
mM of the test
values 1.33 mM and 1.01 mM, respectively. The inhibition potencies of
7a and 7b are therefore approximately 2 fold weaker that those of
the lead compounds, 2c and 2d. It can therefore be concluded that
substitutionwith a hydroxyl functional group on the para position of
the C3 phenyl ring enhances the binding affinity of the anilide
derivatives for the MAO-B active site to a large degree while
substitution with a hydroxyl functional group on the meta position
reduces binding affinity. Using molecular docking studies and the
subsequent analysis of possible binding interactions between 7c and
7d and an active site model of MAO-B, possible reasons for this
behavior will be explored in more detail below. To determine if the
mode of MAO-B inhibition by the phenolic inhibitors are reversible
or irreversible, the time dependency of inhibition was evaluated for
one representative inhibitor, compound 7c. Compound 7c at
MAO-B inhibitors identified in the present study, 2c and 2d,
exhibiting binding affinities that are 37 fold higher for MAO-B than
for MAO-A. Based on their weak affinities for MAO-A, the interac-
tions of anilide derivatives 2ael with MAO-A were not further
characterized.
Based on the molecular docking studies detailed below (Section
2.3), and the analysis of the possible binding interactions of the
anilide derivatives 2c and 2d with activesite models of human MAO-
A and eB, a series of phenolic (7aed) and benzonitrile (7eeh)
derivatives of 2c and 2d were designed, synthesized and evaluated
as recombinant human MAO-A and eB inhibitors (Fig. 4). Anilide
derivatives 2c and 2d were selected as lead compounds since they
exhibited the most potent inhibitoryactivity towards MAO-B among
the anilides evaluated in the first part of this study. The IC50 values
that were recorded for these inhibitors are presented in Table 2. The
most potent MAO-B inhibitors among the phenolic derivatives
(7aed) were (2E)-N-(3-chlorophenyl)-3-(4-hydroxyphenyl)prop-
2-enamide (7c) and (2E)-N-(3-bromophenyl)-3-(4-hydroxyphenyl)
prop-2-enamide (7d) which were found to have IC50 values for the
aconcentration approximatelytwofold its IC50 value (0.064 mM) was
preincubated with recombinant human MAO-B (0.03 mg protein/
mL) for 0, 15, 30 and 60 min. The residual enzyme activity, the
capacity of MAO-B to oxidize kunuramine, was determined and as
shown in Fig. 5. Increased preincubation time of the enzyme with 7c
does not reduce the residual enzyme activity. This indicates that 7c is
not a time-dependent inhibitor of MAO-B over the period evaluated,
and therefore acts reversibly. To further support the finding that 7c
acts reversibly, a set of LineweavereBurk plots were constructed for
the inhibition of MAO-B by 7c (Fig. 6). The results show that the plots
arelinearand intersect at the y-axis which indicates thatthe mode of
inhibition is competitive and therefore reversible.
inhibition of MAO-B of 0.032 mM and 0.026 mM, respectively. These
inhibitors are therefore at least 14 fold more potent than the lead
compounds, 2c and 2d. Interestingly, both 7c and 7d contain the
phenolic hydroxyl functional group on the para position of the C3
phenyl ring. The homologues containing the phenolic hydroxyl
group in the meta position, compounds 7a and 7b, in contrast were
found to be only moderately potent inhibitors of MAO-B with IC50
2.8
2.4
2.0
1.6
1.2
0.8
0.4
0.0
8
6
4
2
-3
-2
-1
0
1
2
3
0
15
Preincubation time (min)
30
60
Log[7b]
Fig. 4. The sigmoidal doseeresponse curves of the initial rates of oxidation of
kynuramine by recombinant human MAO-B vs. the logarithm of concentration of
Fig. 5. The time dependency of MAO-B inhibition by derivative 7c. The test inhibitor
was preincubated with recombinant human MAO-B for various periods of time
(0e60 min) and the residual enzyme catalytic rates (nmol 4-hydroxyquinoline formed/
min/mg protein) were measured. The catalytic rate recorded in the absence of the
inhibitor is 2.51 ꢁ 0.03 nmol/min/mg.
inhibitor 7b (expressed in
concentration of kynuramine used was 30
nmoles 4-hydroxyquinoline formed/min/mg protein.
mM). The determinations were carried out in triplicate. The
mM and the rate data are expressed as

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L. Legoabe et al. / European Journal of Medicinal Chemistry 46 (2011) 5162e5174
FAD cofactor and the co-crystallised ligands were firstly corrected,
1.0
0.8
0.6
0.4
0.2
0.0
hydrogen atoms were added to the MAO-A and eB models and
a three-step energy minimization procedure of the models was
carried out. During the minimization procedure, the backbones of
the protein models were constrained. The co-crystallized ligands
were removed from the models and the binding sites were iden-
tified by the flood-filling algorithm. All crystal water molecules
were deleted from the models except for those in the MAO-A and
eB binding cavities that are considered non-displaceable by
binding ligands (see Experimental). After the structures of the
anilide derivatives that were investigated were constructed and
their geometries optimized within Discovery Studio, the LigandFit
application was used to dock the ligands into the active sites of
the MAO-A and eB models. This protocol has been shown to be
suitable for examining the binding of inhibitors to MAO-A and eB
since redocking of harmine (RMSD ¼ 0.64 Å) and safinamide
(RMSD ¼ 1.54 Å) into the active sites of the two enzymes, respec-
tively, yielded binding orientations that exhibited relatively small
RMSD values from the position of the co-crystallized ligands
[25,26].
-0.02
0.00
0.02
1/[S]
0.04
0.06
Fig. 6. Lineweaver-Burk plots of the oxidation of kynuramine by recombinant human
MAO-B in the absence (filled squares) and presence of various concentrations of 7c:
0.008 mM (open squares), 0.016 mM (filled circles) and 0.032 mM (open circles). The
rates (V) are expressed as nmol product formed/min/mg protein.
The active site of human MAO-B is reported to consist of two
cavities which consist of an entrance cavity which leads to the
substrate cavity [27]. The cavities are separated by the side chains
of Ile-199 and Tyr-326 [28]. Depending on the structure of the
bound inhibitor the two cavities may be either separate or fused.
For relatively large inhibitors such as 1,4-diphenyl-2-butene (3)
[16] the side chain of Ile-199 is rotated into an alternative confor-
mation which results in the fusion of the two cavities and allows
the inhibitor to traverse both active site cavities. Based on the
structural resemblance between 2c and 1,4-diphenyl-2-butene (3),
it may be expected that 2c and 3 should display similar binding
orientations in the MAO-B active site. In accordance with this
expectation, the best-ranked docking solution (Fig. 7) show that 2c
The recombinant human MAO-A inhibitory potential of the
phenolic type anilide derivatives (7aed) were also evaluated. As
shown in Table 2, the derivatives did not inhibit MAO-A catalytic
activity (even at a maximal tested concentration of 100
mM), or
were weak MAO-A inhibitors. It can therefore be concluded that
7aed are MAO-B selective inhibitors with
a reversible and
competitive mode of binding.
The results in Table 2 also shows that substitution of anilide
derivatives 2c and 2d with a nitrile functional group yields
compounds with improved MAO-B inhibition potencies. Among the
benzonitrile derivatives (7eeh), compounds 7e and 7f, which were
substituted with a nitrile functional group on the meta position of
the C3 phenyl ring, were the most potent MAO-B inhibitors with
IC50 values of 0.090 mM and 0.059 mM, respectively. These inhibitors
are therefore at least 5 fold more potent than the lead compounds,
2c and 2d. The homologues containing the nitrile functional group
on the para position of the C3 phenyl ring (7g and 7h) were less
potent MAO-B inhibitors than 7e and 7f. This finding is in contrast
to what has been observed for the anilides containing a phenolic
hydroxyl functional group. As discussed above, for this series
(7aed), the homologues containing the para phenolic hydroxyl
group were better MAO-B inhibitors than the meta substituted
homologues. It can therefore be concluded that substitution with
a nitrile functional group on the meta position of the C3 phenyl ring
enhances the MAO-B inhibition potency of the anilide derivatives to
a larger degree than substitution on the para position. Also of note,
all the benzonitrile derivatives (7eeh) were weak inhibitors of
MAO-A, and they may therefore be considered as MAO-B selective
inhibitors.
2.3. Molecular docking
To determine possible binding orientations of the inhibitors in
the active sites of MAO-A and eB, one of the potent MAO-B
inhibitors identified in the first part of this study, 2c was docked
into active site models of recombinant human MAO-A and eB [21].
For this purpose, the structures of human MAO-A co-crystallized
with harmine (PDB entry: 2Z5X) [22] and human MAO-B co-crys-
tallized with safinamide (PDB entry: 2V5Z) [23] were selected and
molecular docking was carried out according to a modification of
a previously reported protocol with the LigandFit application of the
Discovery Studio 1.7 modeling software (Accelrys) [21]. The protein
models were prepared within Discovery Studio 1.7 as previously
reported and involved the following steps [24]: The valences of the
Fig. 7. The best ranked docking solution for the binding of 2c (magenta colored) in the
active site of MAO-B (2V5Z.pdb) [23].

L. Legoabe et al. / European Journal of Medicinal Chemistry 46 (2011) 5162e5174
5167
traverse both active site cavities with the 3-chloroanilide moiety
located within the entrance cavity while the styryl ring binds
towards the FAD cofactor in the substrate cavity. This orientation is
supported by a previous modeling study which suggests that the
chlorophenyl ring of chalcone derivatives also binds within the
the enhancement of productive hydrophobic and dipole interac-
tions between the benzyloxy side chain and the MAO-B entrance
cavity [25]. Reasons why alkyl substitution (for example compound
2b) on the anilide phenyl ring does not similarly enhance the
binding affinity of the inhibitors for the MAO-B active site is
unclear.
entrance cavity where it may be stabilized by He
p interactions
with the phenolic hydrogen of Tyr-326 [17]. In addition, hydrogen
bonding may occur between the phenolic hydrogen of Tyr-326 and
the anilide carbonyl oxygen of 2c. These findings support the
proposal that the side chain of Tyr-326 may plays an important role
in the recognition of inhibitors in the entrance cavity of MAO-B and
may, in certain instances, contribute to the observed selectivity of
MAO-B selective inhibitors [17]. In MAO-A, the residue that corre-
sponds to Tyr-326 (in MAO-B) is Ile-335. The resulting absence of
these stabilizing interactions may, in part, explain the lower MAO-A
inhibition potencies of these inhibitors. Consistent with this anal-
ysis, the best ranked docking solution of 2c within the active site of
MAO-A indicates that the inhibitor is not stabilized by hydrogen
bonding (Fig. 8). As mentioned above, the chloroanilide moiety of
2c located within the entrance cavity of MAO-B. Since the entrance
cavity is reported to be a hydrophobic environment [28], the
chloroanilide moiety is most likely stabilized to a large degree by
Van der Waals interactions within the entrance cavity. Therefore,
substitution with halogens on the anilide phenyl ring is expected to
enhance the productive interactions of the inhibitor with the
entrance cavity of MAO-B via hydrophobic burial and dipole
interactions. The enhancement of MAO-B inhibition potencies by
halogen substitution has been previously reported. For example,
chlorine and bromine substitution on the benzyloxy phenyl ring of
a series of 8-benzyloxycaffeines enhances the MAO-B inhibition
potencies 20 and 22 fold, respectively [25]. Modeling studies
suggest that this effect of halogen substitution is also dependent on
An important finding of the docking study is the observation
that the C3 phenyl ring (the styryl ring) of 2c is located in the polar
region of the MAO-B active site, the space near the FAD cofactor.
Since the C3 phenyl ring is unsubstituted it may be hypothesized
that the addition of polar functional groups may enhance the
binding affinity of the C3 phenyl ring via polar interactions with
amino acid residues and water molecules in the substrate cavity of
the enzyme. For example, X-ray crystal structures of both safina-
mide and 7-(3-chlorobenzyloxy)-4-formylcoumarin [23] in
complex with human MAO-B have shown that polar functional
groups of these inhibitors are involved in hydrogen bond interac-
tions with the amino acid residues in the substrate cavity of
the enzyme (Fig. 9). In the safinamideeMAO-B complex, the NH2 of
the propanamidyl moiety of safinamide is hydrogen bonded to the
carbonyl oxygen of side chain amidyl of Gln-206 [23] while
in the complex between MAO-B and 7-(3-chlorobenzyloxy)-4-
formylcoumarin, the formyl carbonyl oxygen is hydrogen bonded
to the phenolic hydrogen of Tyr-435 and an active site water
molecule [23]. Based on these observations, the addition of polar
substituents to the C3 phenyl ring of the most potent MAO-B
inhibitors identified in the first part of this study, 2c and 2d, may
further enhance their affinities for the enzyme’s active site. As
a potential hydrogen bond acceptor and donor, the phenolic
hydroxyl group was added to the meta and para positions of the C3
phenyl rings of 2c and 2d to yield structures 7aed. The hydroxyl
group was selected since it was previously shown to enhance the
MAO-B inhibition potencies of a series of chalcones, possibly by
undergoing hydrogen bonding within the enzymes substrate cavity
[17]. For example, trans-chalcone inhibits recombinant human
MAO-B with an IC50 value of 1.41
chalcone derivatives inhibits the enzyme with IC50 values ranging
from 0.0051 M to 0.16 M [17]. For the present study, the nitrile
mM while hydroxyl-substituted
m
m
functional group was also selected an as potential hydrogen bond
acceptor and was added to the meta and para positions of the C3
phenyl rings of 2c and 2d to yield structures 7eeh. The nitrile group
was selected based on as yet unpublished observations in our
laboratory that certain substituted benzonitriles and phthaloni-
triles are exceptionally potent inhibitors of human MAO-B.
Preliminary modeling studies suggest that this property may
most likely be attributed to the ability of the nitrile functional
H
CH3
O
NH2
N
F
H
O
Safinamide
CHO
O
Cl
O
O
7-(3-Chlorobenzyloxy)-4-formylcoumarin
Fig. 8. The best ranked docking solution for the binding of 2c (orange colored) in the
active site of MAO-A (2Z5X.pdb) [22]. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
Fig. 9. The structures of safinamide and 7-(3-chlorobenzyloxy)-4-formylcoumarin.

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L. Legoabe et al. / European Journal of Medicinal Chemistry 46 (2011) 5162e5174
Fig. 12. The best ranked docking solution for the binding of 7a (magenta colored) in
the active site of MAO-B (2V5Z.pdb) [23]. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
Fig. 10. The best ranked docking solution for the binding of 7c (magenta colored) in
the active site of MAO-B (2V5Z.pdb) [23]. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
MAO-B active site, near the FAD cofactor, where they may undergo
hydrogen bonding with a water molecule and the side chain of Tyr-
435. The absence of hydrogen bond interactions between the anilide
carbonyl oxygens of 7a and 7b and the phenolic hydrogen of Tyr-326
may explain, at least in part, their relatively weak MAO-B inhibition
potencies compared to 7c and 7d which is suggested (by the docking
study) to undergo this interaction. This analysis suggests that the
side chain of Tyr-326 may play an important role in the recognition
and stabilization of inhibitors in the entrance cavity of MAO-B.
Furthermore, while the polar interactions of the para and meta
hydroxyl groups and the MAO-B substrate cavity may be important
for enhancing the binding affinities of these anilide derivatives to
MAO-B, the loss of an interaction with Tyr-326 may, to a large
degree, negate the gain in these binding affinities. The improved
MAO-B inhibition potencies of the derivatives containing the para
phenolic hydroxyl groups (7c and 7d) compared to the corre-
sponding unsubstituted derivatives 2c and 2d may be explained by
the presence of the polar interactions between the phenolic
hydroxyl groups and the polar region of MAO-B. Even though
compounds 7a and 7b also possess these polar contacts the loss of
a potential hydrogen bond with Tyr-326 may explain the reduction
of MAO-B inhibition potencies compared to the corresponding
unsubstituted derivatives 2c and 2d.
As discussed above, substitution of anilide derivatives 2c and 2d
with a nitrile functional group on the C3 phenyl ring also yields
compounds with improved MAO-B inhibition potencies. The results
document that nitrile substitution on the meta position
(compounds 7e and 7f) of the C3 phenyl ring was more favorable
for MAO-B inhibition than substitution on the para position
(compounds 7g and 7h). To investigate this observation, 7e (meta
substituted nitrile) was docked into the MAO-B active site model as
described above. Compound 7e adopts a similar binding mode to
that observed for 2c with the C3 phenyl ring located in the
substrate cavity and the nitrile functional group positioned in the
polar region of the MAO-B active site, near the FAD cofactor
(Fig.13). Here, the nitrile nitrogen is within hydrogen bond distance
group to undergo hydrogen bond interactions with amino acid
residues in the substrate cavity of MAO-B.
Based on this analysis it was therefore expected that the
hydroxyl and nitrile substituted derivatives of 2c and 2d may exhibit
enhanced binding affinities to MAO-B. As discussed above, the para
substituted phenolic derivatives 7c and 7d were found to be
exceptionally potent MAO-B inhibitors, at least 14 fold more potent
than 2c and 2d. In contrast, meta substituted phenolic derivatives,
7a and 7b, were only moderately potent inhibitors of MAO-B,
approximately 2 fold weaker than 2c and 2d. When docked into
the MAO-B active site model according to the protocol described
above, the binding orientation that is adopted by 7c places the para
phenolic hydroxyl group in hydrogen bond contact distance to N5 of
the FAD cofactor as well as to a water molecule (Fig.10). Importantly,
7c exhibits a similar binding orientation to 2c (Fig. 11) and also
places the anilide carbonyl oxygen within hydrogen bond distance
to the phenolic hydrogen of Tyr-326. This binding mode is also
adopted by compound 7d (results not shown). In the docked
binding orientations of 7a (Fig. 12) and 7b (results not shown), the
anilide carbonyl oxygen is directed away from Tyr-326 with the
resulting loss of the potential important hydrogen bond interaction
with the phenolic hydrogen of Tyr-326. The meta phenolic hydroxyl
groups of 7a and 7b are however placed in the polar region of the
Fig. 11. The docked orientations of 2c and 7c within the active site of MAO-B.

L. Legoabe et al. / European Journal of Medicinal Chemistry 46 (2011) 5162e5174
5169
to an active site water molecule. This polar interaction may, at least
in part, explain the improved MAO-B inhibition potencies of the
benzonitrile derivatives (7ee7h) compared to the corresponding
unsubstituted derivatives 2c and 2d. Of importance is the obser-
vation that the anilide carbonyl oxygen of 7e may also undergo
a potential hydrogen bond interaction with the phenolic hydrogen
of Tyr-326. Compound 7f exhibits a similar binding mode and
interactions (results not shown). In the docked binding orientations
of 7g (Fig. 14) and 7h (results not shown) within the MAO-B active
site, the anilide carbonyl oxygens of these inhibitors may also
undergo a potential hydrogen bond with the phenolic side chain of
Tyr-326. The observation that all of the nitrile substituted anilide
derivatives (7eeg) may be involved in hydrogen bonding with Tyr-
326 may explain the finding that all these derivatives are potent
MAO-B inhibitors, with IC50 values in the nM range. This further
supports the suggestion that Tyr-326 may play a significant role in
inhibitor stabilization within the MAO-B active site.
3. Conclusion
In the first part of this study a series of anilide derivatives (2ael)
were examined in order to determine if they may act as useful
scaffolds for the design of selective reversible inhibitors of MAO-B.
Of the three types of anilide derivatives investigated, the N,3-
diphenylpropenamides (2aed) were found to be the most prom-
ising class of MAO-B inhibitors, with those homologues substituted
with chlorine (2c) and bromine (2d) on the C-3 of the phenyl ring of
the anilide moiety being the most potent inhibitors (IC50 values of
0.53 mM and 0.45 mM, respectively). These inhibitors were selective
Fig. 13. The best ranked docking solution for the binding of 7e (magenta colored) in
the active site of MAO-B (2V5Z.pdb) [23]. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
for the MAO-B isoform, and as shown by example with 2c, these
inhibitors bind reversibly to the enzyme. It can therefore be
concluded that N,3-diphenylprop-2-enamide is a suitable scaffold
for the design of potent MAO-B inhibitors and that substitution of
the anilide C-3 phenyl ring with halogens is a general strategy to
enhance the binding affinities for the MAO-B active site.
In the second part of this study, a series of phenolic (7aed) and
benzonitrile (7eeh) derivatives of 2c and 2d were examined as
potential inhibitors of MAO-A and eB. As discussed above, the
design of these derivatives were based on the docking study which
suggests that the C3 phenyl ring of 2c is located in the polar region
of the MAO-B active site, the space near the FAD cofactor. Substi-
tution on the C3 phenyl ring with polar functional groups may
enhance the binding affinity of the C3 phenyl ring within the polar
substrate cavity of the enzyme. The results of the enzymatic studies
indicated that the phenolic derivatives 7aed are selective MAO-B
inhibitors and that the homologues containing phenolic hydroxyl
functional groups on the para position of the C3 phenyl ring (7c and
7d) enhanced the MAO-B inhibition potencies of the lead
compounds, 2c and 2d, by at least 14 fold. The homologues con-
taining phenolic hydroxyl groups in the meta position (7a and 7b),
in contrast were only moderately potent MAO-B inhibitors with
potencies approximately 2 fold weaker than those of the lead
compounds. It can therefore be concluded that substitution with
a hydroxyl functional group on the para position (and not the meta
position) of the C3 phenyl ring enhances the MAO-B inhibition
potencies of the N,3-diphenylpropenamide type anilide derivatives.
The benzonitrile derivatives were also found to potent inhibitors
with the homologues containing the nitrile functional group on the
meta position of the C3 phenyl ring (7e and 7f) exhibiting at least 5
fold more potent MAO-B inhibition potencies (IC50 values of
0.090
mM and 0.059
mM, respectively) than the lead compounds, 2c
and 2d (IC50 values of 0.532
m
M and 0.447 M, respectively). The
m
homologues containing the nitrile functional group on the para
position (7g and 7h) were also potent MAO-B inhibitors with IC50
values (0.407 mM and 0.289 mM, respectively) comparable to that of
Fig. 14. The best ranked docking solution for the binding of 7g (magenta colored) in
the active site of MAO-B (2V5Z.pdb) [23]. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)

5170
L. Legoabe et al. / European Journal of Medicinal Chemistry 46 (2011) 5162e5174
compounds 2c and 2d. It may therefore be concluded that substi-
tution with a nitrile functional group on the meta position (and to
a lesser degree, on the para position) of the C3 phenyl ring
enhances the MAO-B inhibition potencies of the N,3-
diphenylpropenamide anilide derivatives.
2H, J ¼ 7.5 Hz), 7.63 (d, 1H, J ¼ 7.5 Hz), 7.76 (d, 1H, J ¼ 15.8 Hz), 7.94
(s,1H), 8.52 (s, 1H); 13C NMR (Bruker Avance III 600, CDCl3)
118.75,
120.48, 122.59, 123.16, 127.38, 127.95, 128.79, 130.05, 130.27, 134.27,
139.34, 142.85, 164.69; EIMS m/z 301 (Mꢃþ); HRMS calcd.
301.01023, found 301.00943; Purity (HPLC): 97.3%.
d
4. Experimental
4.2.2. (2E,4E)-N-(3-Methylphenyl)-5-phenylpenta-2,4-dienamide (2f)
The title compound was prepared from 3-methylaniline and
4.1. Chemicals and instrumentation
(E,E)-5-phenyl-2,4-pentadienoic acid in
150e152 ꢀC (ethanol); UV (CH3CN) lmax 320 nm (ε 54130); 1H NMR
(Bruker Avance III 600, CDCl3)
2.31 (s, 3H), 6.12 (d, 1H, J ¼ 14.7 Hz),
6.84e6.92 (m, 3H), 7.20 (t, 1H, J ¼ 7.9 Hz), 7.26e7.33 (m, 3H),
7.38e7.55 (m, 6H); 13C NMR (Bruker Avance III 600, CDCl3)
21.47,
117.03, 120.57, 124.12, 125.15, 126.13, 127.07, 128.74, 128.83, 136.14,
137.98, 138.92, 139.93, 142.22, 164.21; EIMS m/z 263 (Mꢃþ); HRMS
calcd. 263.13101, found 263.13064; Purity (HPLC): 97.5%.
a yield of 22%; mp
All reagents used in the synthetic procedures were obtained
from SigmaeAldrich and were used without purification. 1H NMR
and 13C NMR spectra were recorded on a Bruker Avance III 600
spectrometer in CDCl3 or DMSO-d6. Chemical shifts are reported in
d
d
parts per million (d) downfield from the signal of tetramethylsilane
which was added to the deuterated solvent. Spin multiplicities are
given as s (singlet), brs (broad singlet), d (doublet), dd (doublet of
doublets), t (triplet) or m (multiplet). Direct insertion electron
impact ionization (EIMS) and high resolution mass spectra (HRMS)
were obtained on a DFS high resolution magnetic sector mass
spectrometer (Thermo Electron Corporation). Melting points (mp)
were determined on a Stuart SMP10 melting point apparatus and
are uncorrected. Fluorescence spectrophotometry was conducted
4.2.3. (2E,4E)-N-(3-Chlorophenyl)-5-phenylpenta-2,4-dienamide (2g)
The title compound was prepared from 3-chloroaniline and
(E,E)-5-phenyl-2,4-pentadienoic acid in a yield of 42%; mp 174 ꢀC
(ethanol/ethyl acetate, 3:1); UV (CH3CN) lmax 321 nm (ε 53850); 1H
NMR (Bruker Avance III 600, DMSO-d6)
d
6.33 (d, 1H, J ¼ 14.9 Hz),
7.04e7.17 (m, 3H), 7.30e7.41 (m, 5H), 7.51 (d, 1H, J ¼ 8.3 Hz), 7.59
with
a
Varian Cary Eclipse fluorescence spectrophotometer.
(m, 2H), 7.92 (s, 1H), 10.33 (s, 1H); 13C NMR (Bruker Avance III 600,
Microsomes from insect cells containing recombinant human
MAO-A and eB (5 mg/mL) and kynuramine.2HBr were obtained
from SigmaeAldrich. UVeVis spectra of the test compounds were
DMSO-d6) d 117.56, 118.63, 122.93, 124.82, 126.71, 127.16, 128.81,
130.45, 133.09, 136.13, 139.44, 140.79, 141.41, 164.04; EIMS m/z 283
(Mꢃþ); HRMS calcd. 283.07639, found 283.07614; Purity (HPLC):
99.1%.
recorded at a concentration of 10 mM in acetonitrile with a Shi-
madzu Multispec-1501 photodiode array spectrophotometer.
Column chromatography was carried out with Silica gel 60 (Fluka;
0.063e0.2 mm) while thin layer chromatography (TLC) was carried
out using silica gel 60 (Merck) with UV254 fluorescent indicator. To
determine the purities of the synthesized compounds, HPLC anal-
yses were performed with an Agilent 1100 HPLC system equipped
with a quaternary pump, an Agilent 1100 series diode array
4.2.4. (2E,4E)-N-(3-Bromophenyl)-5-phenylpenta-2,4-dienamide (2h)
The title compound was prepared from 3-bromoaniline and
(E,E)-5-phenyl-2,4-pentadienoic acid in a yield of 40%; mp 172 ꢀC
(ethanol); UV (CH3CN) lmax 321 nm (ε 55580); 1H NMR (Bruker
Avance III 600, DMSO-d6)
d
6.33 (d, 1H, J ¼ 15.1 Hz), 7.06 (d. 1H,
J ¼ 15.4 Hz), 7.12e7.17 (m, 1H), 7.22 (d, 1H, J ¼ 7.9 Hz), 7.26e7.33 (m,
detector and a Venusil XBP C18 column (4.60 ꢂ 150 mm, 5
mm) (see
2H), 7.36e7.40 (m, 3H), 7.56e7.59 (m, 3H), 8.06 (s,1H),10.32 (s,1H);
Supplementary Material). HPLC grade acetonitrile (Merck) and
Milli-Q water (Millipore) was used for the chromatography.
13C NMR (Bruker Avance III 600, DMSO-d6)
d 117.93, 121.47, 121.59,
124.81, 125.81, 126.70, 127.15, 128.80, 130.75, 136.12, 139.44, 140.93,
141.39, 164.00; EIMS m/z 327 (Mꢃþ); HRMS calcd. 327.02588, found
327.02491; Purity (HPLC): 97.0%.
4.2. Synthesis of the anilide derivatives 2ael
The appropriately substituted aniline (5) (10 mmol), the
carboxylic acid (6aec) (10 mmol) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDAC, 15 mmol) were dis-
solved in a minimum amount of N,N-dimethylformamide (DMF). 4-
Dimethylaminopyridine (DMAP, 0.5 mmol) and imidazole
(0.5 mmol) were added as catalysts. The reaction was stirred for 4 h
at room temperature and an excess amount of water was added to
the mixture to precipitate the product. Except for 2f, which was
isolated via extraction to ethyl acetate (50 mL), the products were
collected by filtration and purified by recrystallization as indicated
below. Compounds 2bed were purified by silica gel column chro-
matography using ethyl acetate/petroleum ether (1:1) as mobile
phase. For previously described compounds the recorded and
literature melting points are as follows: 2a: mp 150 ꢀC (ethanol), lit.
150e153 ꢀC [29]; 2b: mp 112 ꢀC, lit. 112 ꢀC [30]; 2c: mp 115 ꢀC, lit.
125e126 ꢀC [31]; 2e: mp 189 ꢀC (ethanol/ethyl acetate, 3:1), lit.
189e190 ꢀC [32]; 2i: mp 95 ꢀC (ethanol), lit. 94.5e95 ꢀC [33].
4.2.5. (3E)-N-(3-Methylphenyl)-4-phenylbut-3-enamide (2j)
The title compound was prepared from 3-methylaniline and (E)-
styrylacetic acid in a yield of 21%; mp 92 ꢀC (ethyl acetate/petro-
leum ether, 1.5:1); UV (CH3CN) lmax 254 nm (ε 34840); 1H NMR
(Bruker Avance III 600, CDCl3)
d
2.30 (s, 3H), 3.30 (d, 2H, J ¼ 7.5 Hz),
6.33e6.38 (m, 1H), 6.59 (d, 1H, J ¼ 15.8 Hz), 6.91 (d, 1H, J ¼ 7.5 Hz),
7.17 (t, 1H, J ¼ 7.9 Hz), 7.24e7.29 (m, 2H), 7.31e7.34 (m, 3H), 7.39 (m,
2H), 7.41 (s, 1H); 13C NMR (Bruker Avance III 600, CDCl3)
d 21.42,
41.90, 116.97, 120.53, 121.89, 125.26, 126.36, 127.94, 128.65, 128.78,
135.30, 136.38, 137.54, 138.90, 168.80; EIMS m/z 251 (Mꢃþ); HRMS
calcd. 251.13101, found 251.13094; Purity (HPLC): 98.4%.
4.2.6. (3E)-N-(3-Chlorophenyl)-4-phenylbut-3-enamide (2k)
The title compound was prepared from 3-chlorolaniline and
(E)-styrylacetic acid in a yield of 18%; mp 91 ꢀC (ethyl acetate/
petroleum ether, 1:1); UV (CH3CN) lmax 254 nm (ε 35520); 1H
NMR (Bruker Avance III 600, CDCl3)
d
3.31 (d, 2H, J ¼ 6.4 Hz),
6.31e6.36 (m, 1H), 6.60 (d, 1H, J ¼ 15.8 Hz), 6.06 (d, 1H,
J ¼ 7.15 Hz), 7.20 (t, 1H, J ¼ 8.0 Hz), 7.24e7.28 (m, 1H), 7.31e7.35
(m, 3H), 7.38 (d, 2H, J ¼ 7.5 Hz), 7.49 (s, 1H), 7.61 (s, 1H); 13C NMR
4.2.1. (2E)-N-(3-Bromophenyl)-3-phenylprop-2-enamide (2d)
The title compound was prepared from 3-bromoaniline and
cinnamic acid in a yield of 53%: mp 136e137 ꢀC (ethyl acetate/n-
hexane, 1:1); UV (CH3CN) lmax 294 nm (ε 27710); 1H NMR
(Bruker Avance III 600, CDCl3)
d 41.85, 117.79, 119.94, 121.37,
124.49, 126.39, 128.09, 128.70, 129.96, 134.63, 135.69, 136.21,
138.74, 168.91; EIMS m/z 271 (Mꢃþ); HRMS calcd. 271.07639, found
271.07630; Purity (HPLC): 98.7%.
(Bruker Avance III 600, CDCl3)
J ¼ 7.9 Hz), 7.24 (d, 1H, J ¼ 7.9 Hz), 7.30 (m, 2H), 7.34 (m, 1H), 7.42 (d,
d
6.69 (d, 1H, J ¼ 15.4 Hz), 7.16 (t, 1H,

L. Legoabe et al. / European Journal of Medicinal Chemistry 46 (2011) 5162e5174
5171
4.2.7. (3E)-N-(3-Bromophenyl)-4-phenylbut-3-enamide (2l)
The title compound was prepared from 3-bromolaniline and (E)-
styrylacetic acid in a yield of 41%; mp 98 ꢀC (ethanol); UV (CH3CN)
lmax 254 nm (ε 34700); 1H NMR (Bruker Avance III 600, CDCl3)
(m, 2H), 7.29 (t, 1H, J ¼ 7.91 Hz), 7.50 (d, 1H, J ¼ 15.43 Hz), 7.56 (d,
1H, J ¼ 8.28 Hz), 8.06 (s, 1H), 9.66 (brs, 1H), 10.36 (s, 1H); 13C NMR
(Bruker Avance III 600, DMSO-d6)
d 113.93, 117.23, 117.98, 119.04,
121.52, 121.64, 125.95, 130.07, 130.83, 135.80, 140.86, 141.03, 157.78,
163.84; EIMS m/z 317 (Mꢃþ); HRMS calcd. 317.00514, found
317.0048; Purity (HPLC): 99.4%.
d
3.31 (d 2H, J ¼ 7.2 Hz), 6.31e6.36 (m, 1H), 6.60 (d, 1H, J ¼ 15.8 Hz),
7.14 (t, 1H, J ¼ 8.0 Hz), 7.21 (d, 1H, J ¼ 8.3 Hz), 7.26 (m, 1H), 7.43 (t,
2H, J ¼ 7.53 Hz), 7.38 (d, 2H, J ¼ 7.53 Hz), 7.41 (d, 1H, J ¼ 7.91 Hz),
7.48 (s, 1H), 7.74 (s, 1H); 13C NMR (Bruker Avance III 600, CDCl3)
4.3.3. (2E)-N-(3-Chlorophenyl)-3-(4-hydroxyphenyl)prop-2-
enamide (7c)
The title compound was prepared from 3-chlorolaniline and (E)-
4-hydroxycinnamic acid (8b) in a yield of 73%; mp 200e201 ꢀC
(ethyl acetate); UV (CH3CN) lmax 315 nm (ε 40600); 1H NMR
d
41.82, 118.31, 121.36, 122.57, 122.76, 126.38, 127.42, 128.08, 128.69,
130.26, 135.66, 136.21, 138.87, 168.95; EIMS m/z 315 (Mꢃþ); HRMS
calcd. 315.02588, found 315.02447; Purity (HPLC): 97.9%.
4.3. Synthesis of the anilide derivatives 7aed
(Bruker Avance III 600, DMSO-d6)
d
6.57 (d, 1H, J ¼ 15.43 Hz), 6.82
(d, 2H, J ¼ 8.66 Hz), 6.09 (dd, 1H, J ¼ 1.51, 7.91 Hz), 7.33 (t, 1H,
J ¼ 7.91 Hz), 7.46 (d, 2H, J ¼ 8.66 Hz), 7.51 (m, 2H), 7.93 (s, 1H), 9.97
(brs, 1H), 10.27 (s, 1H); 13C NMR (Bruker Avance III 600, DMSO-d6)
Protection: (E)-3-Hydroxycinnamic acid (8a) or (E)-4-
hydroxycinnamic acid (8b) (30.46 mmol) was suspended in
a solution of tetrahydrofurane (THF, 30 mL) and CH2Cl2 (30 mL) and
triethylamine (TEA, 73.1 mmol), DMAP (5.2 mmol) and acetic
anhydride (73.1 mmol) were added. The reaction mixture was
heated under reflux for 60 min at 90 ꢀC. The THF and CH2Cl2 were
removed under reduced pressure and the residue was treated with
6 M HCl (100 mL). The resulting light yellow precipitate was
collected by filtration, washed with water and dried to give the
acetoxycinnamic acid intermediates 9a and 9b in yields of 81% and
93%, respectively [19].
d
115.89,117.49,118.04,118.56,122.79,125.55,129.68,130.46,133.11,
140.93, 141.04, 159.40, 164.33; EIMS m/z 273 (Mꢃþ); HRMS calcd.
273.05566, found 273.0571; Purity (HPLC): 99.1%.
4.3.4. (2E)-N-(3-Bromophenyl)-3-(4-hydroxyphenyl)prop-2-
enamide (7d)
The title compound was prepared from 3-bromolaniline and
(E)-4-hydroxycinnamic acid (8b) in a yield of 82%; mp 204e205 ꢀC
(ethyl acetate); UV (CH3CN) lmax 316 nm (ε 39700); 1H NMR
Conjugation: (E)-3-Acetoxycinnamic acid (9a) or (E)-4-
acetoxycinnamic acid (9b) (7.27 mmol) was suspended in DMF
(10 mL) and a 2 M solution of oxalyl chloride in CH2Cl2 (7.5 mL) was
added slowly at room temperature. As oxalyl chloride was added the
white suspensionbecame clearand a gasevolved. After2 h of stirring
at room temperature the reaction was concentrated under reduced
pressure toyield aviscous oil. After the additionof another portion of
CH2Cl2 (40 mL), the appropriately substituted aniline (5) (22 mmol)
was added slowly over a period of 5 min. The resulting light yellow
precipitatewas removed byfiltration. Thefiltratewastriturated with
400 mL petroleum ether to give the phenylcarbamoyl-
ethenylphenyl acetate 10aed in yields of 61e98% [19].
Deprotection: Esters 10aed (3.17 mmol) was suspended in
CH3OH/H2O (1:1, 30 mL) and NaOH (6.2 mmol) was added. After the
reaction was stirred at room temperature for 2 h the pH was
adjusted to 6 with 1 M HCl. The resulting light yellow precipitate
was collected by filtration, washed with water and recrystallized as
indicated below to yield the target anilide derivatives 7aed [19].
(Bruker Avance III 600, DMSO-d6)
d
6.57 (d, 1H, J ¼ 15.43 Hz), 6.82
(d, 2H, J ¼ 8.66 Hz), 7.22 (d, 1H, J ¼ 7.91 Hz), 7.27 (t, 1H, J ¼ 7.91 Hz),
7.46 (d, 2H, J ¼ 8.66 Hz); 7.50 (d, 1H, J ¼ 15.81 Hz), 7.55 (d, 1H,
J ¼ 8.28 Hz), 8.06 (s, 1H), 9.98 (brs, 1H), 10.25 (s, 1H); 13C NMR
(Bruker Avance III 600, DMSO-d6)
d 115.89, 117.88, 118.04, 121.41,
121.62, 125.55, 125.69, 129.69, 130.77, 141.05, 141.08, 159.40, 164.32;
EIMS m/z 317 (Mꢃþ); HRMS calcd. 317.00514, found 317.0081; Purity
(HPLC): 99.4%.
4.4. Synthesis of the anilide derivatives 7eeh
3-Cyanobenzaldehyde (11a) or 4-cyanobenzaldehyde (11b)
(15 mmol) and malonic acid (30 mmol) were dissolved in pyridine
(30 mL). Piperidine (3.0 mmol) was added and the reaction was
heated for 4 h at 100 ꢀC. The reaction was cooled to room
temperature and 5 M HCl (25 mL) was added slowly. The resulting
white precipitate was collected by filtration, washed with water
and dried to give (E)-3-cyanocinnamic acid (12a) or (E)-4-
cyanocinnamic acid (12b) in yields of 81% and 78%, respectively.
(E)-3-Cyanocinnamic acid (12a) or (E)-4-cyanocinnamic acid
(12b) (4.04 mmol) and the appropriately substituted aniline (5)
(4.04 mmol) were dissolved in a minimum amount of DMF. EDAC
(6.06 mmol) was added to the reaction mixture followed by DMAP
(0.2 mmol) and imidazole (0.2 mmol) as catalysts. The reaction was
stirred for 4 h at room temperature and an excess amount of water
was added. The resulting precipitate, the anilide derivatives (7eeh),
was collected by filtration and purified by silica gel column chro-
matography using ethyl acetate/petroleum ether (1:1) as mobile
phase.
4.3.1. (2E)-N-(3-Chlorophenyl)-3-(3-hydroxyphenyl)prop-2-
enamide (7a)
The title compound was prepared from 3-chlorolaniline and (E)-
3-hydroxycinnamic acid (8a) in a yield of 84%; mp 125e127 ꢀC
(ethyl acetate); UV (CH3CN) lmax 290 nm (ε 26700); 1H NMR
(Bruker Avance III 600, DMSO-d6)
d
6.72 (d, 1H, J ¼ 15.81 Hz), 6.82
(d, 1H, J ¼ 7.91 Hz), 6.99 (s, 1H), 7.04 (d, 1H, J ¼ 7.53 Hz), 7.12 (d, 1H,
J ¼ 7.91 Hz), 7.23 (t, 1H, J ¼ 7.91 Hz), 7.35 (t, 1H, J ¼ 7.91 Hz), 7.51 (m,
2H), 7.93 (s, 1H), 9.66 (brs, 1H), 10.38 (s, 1H); 13C NMR (Bruker
Avance III 600, DMSO-d6)
d 113.93, 117.22, 117.60, 118.66, 119.03,
121.53, 123.04, 130.06, 130.51, 133.14, 135.80, 140.72, 141.03, 157.78,
163.86; EIMS m/z 273 (Mꢃþ); HRMS calcd. 273.05566, found
273.0602; Purity (HPLC): 94.3%.
4.4.1. (2E)-N-(3-Chlorophenyl)-3-(3-cyanophenyl)prop-2-enamide
(7e)
The title compound was prepared from 3-chlorolaniline and (E)-
3-cyanocinnamic acid (12a) in a yield of 46%; mp 200e203 ꢀC (ethyl
acetate); UV (CH3CN) lmax 281 nm (ε 25100); 1H NMR (Bruker
4.3.2. (2E)-N-(3-Bromophenyl)-3-(3-hydroxyphenyl)prop-2-
enamide (7b)
The title compound was prepared from 3-bromolaniline and
(E)-3-hydroxycinnamic acid (8a) in a yield of 72%; mp 192e193 ꢀC
(ethyl acetate); UV (CH3CN) lmax 291 nm (ε 29400); 1H NMR
Avance III 600, DMSO-d6)
d
6.88 (d, 1H, J ¼ 15.81 Hz), 7.13 (d, 1H,
J ¼ 8.66 Hz), 7.36 (t, 1H, J ¼ 8.28 Hz), 7.52 (d, 1H, J ¼ 8.66 Hz), 7.64
(m, 2H), 7.86 (d, 1H, 7.91 Hz), 7.92 (s, 1H), 7.96 (d, 1H, J ¼ 7.91 Hz),
8.09 (s, 1H), 10.46 (s, 1H); 13C NMR (Bruker Avance III 600, DMSO-
(Bruker Avance III 600, DMSO-d6)
d
6.71 (d, 1H, J ¼ 15.81 Hz), 6.82
(dd, 1H, J ¼ 1.88, 7.91 Hz), 6.99 (s, 1H), 7.04 (d, 1H, J ¼ 7.91 Hz), 7.23
d6)
d 112.18, 117.71, 118.42, 118.75, 123.26, 124.28, 130.23, 130.54,

5172
L. Legoabe et al. / European Journal of Medicinal Chemistry 46 (2011) 5162e5174
131.63, 131.85, 133.05, 133.15, 135.92, 138.52, 140.50, 163.35; EIMS
m/z 282 (Mꢃþ); HRMS calcd. 282.05600, found 282.0566; Purity
(HPLC): 98.7%.
constructed with known amounts (0.047e1.56
metabolite dissolved in 500 potassium phosphate buffer
(100 mM, pH 7.4, made isotonic with KCl 20.2 mM). To each cali-
bration standard, volumes of 400 L NaOH (2 N) and 1000 L water
mM) of the authentic
m
L
m
m
4.4.2. (2E)-N-(3-Bromophenyl)-3-(3-cyanophenyl)prop-2-enamide
(7f)
The title compound was prepared from 3-bromolaniline and
(E)-3-cyanocinnamic acid (12a) in a yield of 40%; mp 200e201 ꢀC
(ethyl acetate); UV (CH3CN) lmax 281 nm (ε 27200); 1H NMR
were added. The necessary control samples were included to
confirm that the test inhibitors do not fluoresce or quench the
fluorescence of 4-hydroxyquinoline under these assay conditions.
IC50 values were calculated by plotting the initial rate of kynuramine
oxidation versus the logarithm of the inhibitor concentration to
obtain a sigmoidal doseeresponse curve. Foreach sigmoidal curve, 6
different inhibitor concentrations spanning at least 3 orders of
a magnitude were used. These kinetic data were fitted to the one site
competition model incorporated into the Prism software package
(GraphPad)and the IC50 values weredetermined in triplicateand are
expressed as mean ꢁ standard deviation (SD).
(Bruker Avance III 600, DMSO-d6)
d
6.88 (d, 1H, J ¼ 15.81 Hz), 7.26
(d, 1H, J ¼ 8.28 Hz), 7.30 (t, 1H, J ¼ 7.91 Hz), 7.56 (d, 1H, J ¼ 8.28 Hz),
7.65 (m, 2H), 7.86 (d, 1H, J ¼ 7.91 Hz), 7.96 (d, 1H, J ¼ 7.91 Hz), 8.06
(s, 1H), 8.10 (s, 1H), 10.45 (s, 1H); 13C NMR (Bruker Avance III 600,
DMSO-d6)
d 112.17, 118.08, 118.41, 121.59, 121.63, 124.27, 126.15,
130.22, 130.84, 131.63, 131.84, 133.06, 135.90, 138.51, 140.64, 163.32;
EIMS m/z 326 (Mꢃþ); HRMS calcd. 326.00548, found 326.0074;
Purity (HPLC): 99.1%.
4.6. Time-dependent and competitive inhibition studies
4.4.3. (2E)-N-(3-Chlorophenyl)-3-(4-cyanophenyl)prop-2-enamide
(7g)
The title compound was prepared from 3-chlorolaniline and (E)-
4-cyanocinnamic acid (12b) in a yield of 51%; mp 193 ꢀC (ethyl
acetate); UV (CH3CN) lmax 294 nm (ε 37200); 1H NMR (Bruker
This study show that the test anilide derivatives act as moderate
to potent MAO-B inhibitors while possessing little or weak MAO-A
inhibition potential. To determine whether the inhibitors interact
reversibly or irreversibly with MAO-B, time-dependant inhibition
studies were carried with two selected inhibitors, 2a and 7c.
Avance III 600, DMSO-d6)
d
6.91 (d, 1H, J ¼ 15.43 Hz), 7.12 (d, 1H,
Compound 2a (9.72 mM) or 7c (0.064 mM) was preincubated with
J ¼ 8.66 Hz), 7.36 (t, 1H, J ¼ 7.91 Hz), 7.51 (d, 1H, J ¼ 8.28 Hz), 7.65 (d,
1H, J ¼ 15.43 Hz), 7.80 (d, 2H, J ¼ 8.28 Hz), 7.89 (d, 2H, J ¼ 8.28 Hz),
7.92 (s, 1H), 10.50 (s, 1H); 13C NMR (Bruker Avance III 600, DMSO-
recombinant human MAO-B (0.03 mg protein/mL) for periods of 0,
15, 30, 60 min at 37 ꢀC in potassium phosphate buffer (100 mM, pH
7.4, made isotonic with KCl 20.2 mM). These inhibitor concentra-
tions are approximately 2 fold the measured IC50 values for the
inhibition of human MAO-B (Tables 1 and 2). Following the addition
d6)
d 111.80, 117.71, 118.61, 118.76, 123.30, 125.27, 128.46, 130.54,
132.88, 133.15, 138.84, 139.16, 140.46, 163.23; EIMS m/z 282 (Mꢃþ);
HRMS calcd. 282.05600, found 282.0563; Purity (HPLC): 99.0%.
of kynuramine (30
20 min at 37 ꢀC. The final volumes of all incubations were 500
The reactions were terminated with 200 L NaOH (2 N) and
a volume of 1200 L distilled water and the rates of formation of 4-
m
M), the reactions were incubated for a further
mL.
4.4.4. (2E)-N-(3-Bromophenyl)-3-(4-cyanophenyl)prop-2-enamide
(7h)
m
m
The title compound was prepared from 3-bromolaniline and
(E)-4-cyanocinnamic acid (12b) in a yield of 60%; mp 199e202 ꢀC
(ethyl acetate); UV (CH3CN) lmax 292 nm (ε 33600); 1H NMR
hydroxyquinoline were measured and quantified as described
above. All measurements were carried out in triplicate and are
expressed as mean ꢁ SD. The final enzyme concentration in the
incubations was 0.015 mg protein/mL and the final concentration of
2a and 7c were 4.86 mM and 0.032 mM, respectively. These
concentrations are approximately equal to the IC50 values for the
inhibition of MAO-B by the respective inhibitors.
To determine the mode of inhibition, sets of LineweavereBurk
plots were constructed for the inhibition of the MAO-B catalyzed
kynuramine oxidation by a selected representative inhibitor,
compound 7c. The initial rates by which MAO-B catalyzes the
oxidation of kynuramine were determined at four different kynur-
(Bruker Avance III 600, DMSO-d6)
d
6.91 (d, 1H, J ¼ 15.43 Hz), 7.26
(d, 1H, J ¼ 7.91 Hz), 7.30 (t, 1H, J ¼ 7.91 Hz), 7.57 (d, 1H, J ¼ 7.91 Hz),
7.65 (d, 1H, J ¼ 15.81 Hz), 7.80 (d, 2H, J ¼ 8.28 Hz), 7.89 (d, 2H,
J ¼ 8.28 Hz), 8.06 (s, 1H), 10.45 (s, 1H); 13C NMR (Bruker Avance III
600, DMSO-d6)
d
111.80, 118.09, 118.61, 121.61, 121.64, 125.26,
126.20, 128.46, 130.84, 132.87, 138.83, 139.15, 140.61, 163.21; EIMS
m/z 326 (Mꢃþ); HRMS calcd. 326.00548, found 326.0062; Purity
(HPLC): 95.7%.
4.5. MAO-A and eB inhibition studies
amine concentrations (15e90 mM) in the absence of the test inhib-
itor and also in the presence of three different concentrations of the
As enzyme sources, commercially available (SigmaeAldrich)
microsomes from insect cells, containing recombinant human MAO-
A and eB, were employed. The enzyme activity measurements were
based on the MAO-A or MAO-B (0.0075 mg protein/mL) catalyzed
oxidation of kynuramine to 4-hydroxyquinoline [20]. The incuba-
inhibitor. The concentrations of 7c that were selected for this
purpose were 0.008 mM, 0.016 mM and 0.032 mM while the concen-
tration of MAO-B was 0.015 mg/mL. The enzymatic reactions and
measurements were carried out as described above. Linear regres-
sion analysis was performed using the Prism software package.
tions were conducted in 500
(100 mM, pH 7.4, made isotonic with KCl 20.2 mM) in the presence of
various concentrations of the test inhibitors (0e100 M) and 4%
DMSO as cosolvent. For MAO-A activity measurements the reactions
contained 45 M kynuramine while for the for MAO-B activity
measurements the kynuramine concentrationwas 30 M. Following
a 20 min incubation at 37 ꢀC, the reactions were terminated by the
addition of 400 L NaOH (2 N) and 1000 L water. The reactions were
centrifuged for 10 min at 16,000 g and concentration measurements
of 4-hydroxyquinoline in the supernatants of the samples were
carried out spectrofluorometrically at excitation and emission
wavelengths of 310 nm and 400 nm, respectively. The quantitative
estimations were made with the aid of a linear calibration curve
mL potassium phosphate buffer
4.7. Molecular docking studies
m
The molecular docking studies were carried out with the
Windows based Discovery Studio 1.7 molecular modeling software
[24]. The crystallographic structures of MAO-B co-crystallized with
safinamide (PDB code: 2V5Z) [23] and MAO-A co-crystallized with
harmine (PDB code: 2Z5X) [22] were retrieved from the Broo-
khaven Protein Data Bank (www.rcsb.org/pdb). Hydrogen atoms
were added to the receptor models according to the appropriate
protonation states of the ionizable amino acids at pH 7.4. The
valences of the FAD cofactors (oxidized state) and co-crystallized
ligands were also corrected and hydrogen atoms were added
m
m
m
m

L. Legoabe et al. / European Journal of Medicinal Chemistry 46 (2011) 5162e5174
5173
according to the appropriate protonation states at pH 7.4 and the
References
structure was typed automatically with the Momany and Rone
CHARMm forcefield. The protein backbone was fixed while the
structure was energy minimized. Firstly, a steepest descent mini-
mization was carried out followed by conjugate gradient minimi-
zation. For both protocols the termination criteria was set to
a maximum of 2500 steps or a minimum value of 0.1 for the root
mean square of the energy gradient. Finally an adopted basis
Newton-Rapheson minimization was carried out with the termi-
nation criteria set to a maximum of 5000 steps or a minimum value
for the root mean square of the energy gradient of 0.01. For these
minimization protocols the implicit generalized Born solvation
model with simple switching was used with the dielectric constant
set to 4. For both the MAO-A and eB models, the co-crystallized
water molecules were deleted with the exception of 4 active site
waters. The X-ray crystallographic structures of MAO-B have shown
that three active site water molecules (HOH 1155, 1170 and 1351; A-
chain of 2V5Z) are conserved, all in the vicinity of the FAD cofactor
[23]. Also, docking studies in our laboratory suggest that HOH 1346
(MAO-B, A-chain) may also frequently be involved in interaction
with reversible inhibitors and was therefore also retained. For the
docking studies with MAO-A, the crystal waters were also removed
with the exception of HOH 710, 739 and 725 (2Z5X) which occupies
the analogous positions in the MAO-A active site to those positions
that are occupied in the MAO-B active site by the waters cited
above. HOH 726 which is located between the aromatic residues
Tyr-407 and Tyr-444 was also retained since previous docking
studies in our laboratory suggested that reversible inhibitors do not
bind in close proximity to this polar space and thus do displace this
water molecule [25]. The models were subsequently deprived of
the co-crystallized ligand and the backbone constraint and the
binding sites were identified by a flood-filling algorithm. The
structures of compounds 2c and 7aed were constructed within
Discovery Studio and the hydrogen atoms were added according to
the appropriate protonation states at pH 7.4. Their geometries were
briefly optimized in Discovery Studio using a fast Dreiding-like
forcefield (1000 iterations) and atom potential types and partial
charges were subsequently automatically assigned with the
Momany and Rone CHARMm forcefield. Docking of the ligands
were subsequently carried out with the LigandFit application of
Discovery Studio which uses total ligand flexibility whereby the
final ligand conformations are determined by the Monte Carlo
conformation search method set to a variable number of trial runs.
The docking solutions were further refined using the Smart Mini-
mizer algorithm. The parameters for the docking runs were set to
their default values and ten possible binding solutions were
computed for each docked ligand. The best-ranked binding
conformation of each ligand was determined according to the
DockScore values. The illustrations were prepared in PyMOL [34].
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