Selected C7-substituted chromone derivatives as monoamine oxidase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2012.08.003

A series of C7-substituted chromone (1-benzopyran-4-one) derivatives were synthesized and evaluated as inhibitors of recombinant human monoamine oxidase (MAO) A and B. The chromones are structurally related to a series of C7-functionalized coumarin (1-benzopyran-2-one) derivatives which has been reported to act as potent MAO inhibitors. The results of the current study document that the chromones are highly potent reversible inhibitors of MAO-B with IC₅₀ values ranging from 0.008 to 0.370 μM. While the chromone derivatives also exhibit affinities for MAO-A, with IC₅₀ values ranging from 0.495 to 8.03 μM, they are selective for the MAO-B isoform. Structure-activity relationships (SAR) show that 7-benzyloxy substitution of chromone is suitable for MAO-B inhibition with tolerance for a variety of substituents and substitution patterns on the benzyloxy ring. It may be concluded that 7-benzyloxychromones are appropriate lead compounds for the design of reversible and selective MAO-B inhibitors. With the aid of modeling studies, potential binding orientations and interactions of selected chromone derivatives in the MAO-A and -B active sites are examined.

Full text of DOI:10.1016/j.bioorg.2012.08.003

Bioorganic Chemistry 45 (2012) 1–11
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Bioorganic Chemistry
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Outstanding Paper
Selected C7-substituted chromone derivatives as monoamine oxidase inhibitors
Lesetja J. Legoabe a, Anél Petzer a, Jacobus P. Petzer b,
⇑
a Unit for Drug Research and Development, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
b 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:
Received 29 March 2012
Available online 31 August 2012
A series of C7-substituted chromone (1-benzopyran-4-one) derivatives were synthesized and evaluated
as inhibitors of recombinant human monoamine oxidase (MAO) A and B. The chromones are structurally
related to a series of C7-functionalized coumarin (1-benzopyran-2-one) derivatives which has been
reported to act as potent MAO inhibitors. The results of the current study document that the chromones
Keywords:
Monoamine oxidase
Inhibition
Reversible
Chromone
are highly potent reversible inhibitors of MAO-B with IC50 values ranging from 0.008 to 0.370
the chromone derivatives also exhibit affinities for MAO-A, with IC50 values ranging from 0.495 to
8.03 M, they are selective for the MAO-B isoform. Structure–activity relationships (SAR) show that
lM. While
l
7-benzyloxy substitution of chromone is suitable for MAO-B inhibition with tolerance for a variety of
substituents and substitution patterns on the benzyloxy ring. It may be concluded that 7-benzyloxychr-
omones are appropriate lead compounds for the design of reversible and selective MAO-B inhibitors.
With the aid of modeling studies, potential binding orientations and interactions of selected chromone
derivatives in the MAO-A and -B active sites are examined.
Coumarin
Structure–activity relationship
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
MAO-A active site is a single cavity, the MAO-B active site consists
of an entrance cavity (300 Å3) which leads, from the surface of the
The monoamine oxidases (MAO) are flavoenzymes which play
an important role in the oxidative catabolism of amine neurotrans-
mitters such as dopamine, serotonin and epinephrine [1]. All
mammals contain two distinctive MAO enzymes, MAO-A and
MAO-B, which are localized in the outer membrane of the mito-
chondria [2,3]. These enzymes are encoded by separate genes and
share approximately 70% sequence identity at the amino acid level
[3]. Based on the observation that the genes exhibit identical exon–
intron organizations, it is thought that the two MAO genes evolved
by duplication of a single ancestral gene [4]. Although the struc-
tures of the MAOs are highly similar, they exhibit different sub-
strate and inhibitor specificities. Most notably, MAO-A utilizes
serotonin as substrate while the dietary amines, b-phenylethyl-
amine and benzylamine, are MAO-B selective substrates [5,6].
Dopamine, epinephrine and norepinephrine are considered to be
substrates for both isoforms [1,5]. Relatively minor differences in
the architectures of their respective active sites are thought to
determine the observed differences in specificities of MAO-A and
-B. The volume of the MAO-A active site is ꢀ400 Å3 while the
MAO-B active site cavity has a volume of ꢀ700 Å3 [7,8]. While the
enzyme, to the substrate cavity (ꢀ400 Å3) [4]. Since the side chain
of residue Ile-199 may adopt two different conformations, the
active site of MAO-B may exist as two separate cavities or fuse into
a large single cavity. The rotamer conformation of the Ile-199 side
chain is determined by the nature of the bound ligand. With rela-
tively large ligands bound to MAO-B, the side chain of Ile-199 ro-
tates from the normally ‘‘closed’’ conformation into the ‘‘open’’
conformation. This allows for the fusion of the active site cavities,
and is necessary for the accommodation of larger ligands [9]. The
residue in MAO-A which corresponds to Ile-199 in MAO-B, is Phe-
208. The increased bulk of the phenyl side chain of Phe-208 may
impede the binding of larger MAO-B selective inhibitors to MAO-
A. In contrast to the Ile-199 side chain, the side chain of Phe-208
residue in MAO-A does not adopt an alternate conformation. Based
on these observations, the differences in size and conformational
flexibility of Ile-199 and Phe-208 are determinants of the differen-
tial MAO-A and -B substrate and inhibitor specificities [4]. Another
notable difference between the MAO-A and -B active sites involves
a restriction imposed on the MAO-B active site by the phenolic ring
of the Tyr-326 side chain. Residue Tyr-326 is found at the boundary
between the substrate and entrance cavities of MAO-B and may ob-
struct certain ligands from adopting similar orientations to those
observed in the MAO-A active site [7]. The corresponding residue
to Tyr-326 in MAO-A, is Ile-335. The smaller size of the Ile-335 side
chain allows for specific binding orientations in the MAO-A active
Abbreviations: DMSO, dimethyl sulfoxide; FAD, flavin adenine dinucleotide;
MAO, monoamine oxidase; PDB, protein data bank; RMSD, root mean square
deviation; SAR, structure–activity relationship; SI, selectivity index.
⇑
Corresponding author. Fax: +27 18 2994243.
E-mail address: jacques.petzer@nwu.ac.za (J.P. Petzer).
0045-2068/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2012.08.003

2
L.J. Legoabe et al. / Bioorganic Chemistry 45 (2012) 1–11
Parkinson’s disease by reducing the generation of hydrogen perox-
O
O
ide and dopanal [19]. These species are metabolic by-products of
the MAO-B catalyzed oxidation of dopamine and may lead to
neuronal damage when produced in excessive quantities in the
brain [20–23]. Increased levels of hydrogen peroxide may promote
7
7
O
O
1
2
apoptotic signaling events while dopanal has been implicated in a-
synuclein aggregation [24]. Both these processes are thought to be
involved in the development of Parkinson’s disease.
Fig. 1. The structures of chromone (1) and coumarin (2).
Based on the pharmacological importance of MAO inhibitors,
the design of new MAO inhibitors is pursued by several research
groups. The present study aims to discover novel highly potent hu-
man MAO inhibitors by employing the chromone (1-benzopyran-
4-one) structure as scaffold. Chromone (1) is a member of the
benzopyrone class of compounds and a structural isomer of cou-
marin (1-benzopyran-2-one, 2) (Fig. 1). Coumarins, in particular
C7-functionalized coumarin derivatives, have been shown to act
as potent reversible inhibitors of the MAO enzymes [25]. Substitu-
tion of coumarin and coumarin derivatives at C7 of the benzopy-
rone ring with the benzyloxy moiety yields compounds which
are particularly potent and selective MAO-B inhibitors [25]. The
present study investigated the possibility that C7 substitution of
chromone may similarly lead to structures endowed with potent
MAO inhibitory properties. For this purpose alkyloxy substituents
– benzyloxy, phenylethoxy and phenylpropoxy – were selected
for substitution at C7 of the chromone ring. To further examine
the structure–activity relationships (SAR) for MAO inhibition, the
first member of the present series, the 7-benzyloxychromone
derivative (3a), was substituted on the benzyloxy phenyl ring with
alkyl groups (CH3, CN, CF3) and halogens (Cl, Br, F) (Table 1). The
view that the chromone ring may act as a scaffold for the design
of MAO inhibitors is supported by recent reports of a subset of
C2- and C3-substituted chromone derivatives which act as inhibi-
tors of MAO-B [26,27]. A small series of C6- and C7-substituted
chromones have also been found to be inhibitors of both MAO iso-
zymes [25]. This research forms part of a recent study by us, which
examined the MAO inhibitory properties of a series of C6-substi-
tuted chromones [28].
site which would not be possible in MAO-B. These structural differ-
ences may also be considered as determinants of the differential
MAO-A and -B substrate and inhibitor specificities [4].
The MAO enzymes are of medical and pharmacological impor-
tance. Since MAO-A catalyzes the oxidation of central and periphe-
ral serotonin, MAO-A inhibitors are used in the management of
anxiety disorder and depression [5,10]. The finding that MAO-A
levels exhibit an age-dependent increase in the hearts of rats,
suggests that MAO-A may also play a role in cardiac cellular degen-
eration [11]. Since hydrogen peroxide is generated by the MAO cat-
alytic cycle, increased levels of reactive oxygen species, derived
from hydrogen peroxide, may lead to apoptosis and necrosis of
cardiac cells. MAO-B inhibitors, in turn, are used in the therapy of
Parkinson’s disease [12]. MAO-B levels and activity exhibits an
age-dependent increase in most brain regions, including the basal
ganglia [13–15]. This increased MAO-B activity is expected to
diminish dopamine levels and contribute to dopamine depletion
in the parkinsonian brain. MAO-B inhibitors may therefore exert a
dopamine sparing effect and are used as adjuvant therapy to levo-
dopa, the metabolic precursor of dopamine. In accordance with this
analysis, it has been demonstrated that, in primates, MAO-B inhib-
itors enhance dopamine levels derived from levodopa [16,17].
MAO-B inhibitors may also indirectly increase striatal extracellular
dopamine levels by inhibiting the catabolism b-phenylethylamine.
This increases the central levels of b-phenylethylamine which is
both a releaser of dopamine as well as an inhibitor of active
dopamine uptake [18]. Interestingly, MAO-B inhibitors may also
protect against the neurodegenerative processes associated with
Table 1
The structures of the C7-substituted chromone derivatives (3a–o) that were examined in this study.
O
7
R
O
O
3
R
R
R
R
CH2
CH2
CH2
CH2
(CH2)2
(CH2)3
CH2
CH2
CH2
CH2
c
d
h
l
a
e
i
b
Cl
F
Cl
F
CH2
CH2
CH2
Br
f
g
k
Br
CH2
j
CN
CN
CF3
(CH2)2
Br
o
m
n

L.J. Legoabe et al. / Bioorganic Chemistry 45 (2012) 1–11
3
Table 2
O
O
The IC50 values for the inhibition of recombinant human MAO-A and -B by the C7-
substituted chromomes 3a–o.
R
Br
O
+
7
HO
4
O
7
R
O
O
3
K2CO3
7
R
O
O
R
IC50
(
l
M)a
SIb
3
MAO-A
MAO-B
Scheme 1. Synthetic route to C7-substituted chromone derivatives (3a–o).
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
C6H5–CH2–
C6H5–(CH2)2–
C6H5–(CH2)3–
5.33 0.105
2.19 0.057
3.08 0.096
1.52 0.027
0.495 0.019
1.73 0.044
0.592 0.060
2.10 0.153
1.17 0.032
8.03 0.497
7.12 0.353
0.784 0.019
1.57 0.043
1.97 0.098
0.718 0.027
0.085 0.005
0.115 0.003
0.121 0.018
0.017 0.002
0.029 0.005
0.014 0.001
0.018 0.005
0.044 0.004
0.025 0.002
0.062 0.008
0.075 0.002
0.370 0.033
0.117 0.002
0.008 0.002
0.317 0.028
63
19
25
89
17
124
33
48
47
130
95
2
2. Results
3-ClC6H4–CH2–
4-ClC6H4–CH2–
3-BrC6H4–CH2–
4-BrC6H4–CH2–
3-FC6H4–CH2–
4-FC6H4–CH2–
3-CH3C6H4–CH2–
4-CH3C6H4–CH2–
3-CNC6H4–CH2–
4-CNC6H4–CH2–
4-CF3C6H4–CH2–
4-BrC6H4–(CH2)2–
2.1. Chemistry
The route followed for the synthesis of the target C7-substituted
chromone derivatives (3a–o) is shown in Scheme 1 [28]. Com-
pounds 3a–o were synthesized in relatively good yields (25–84%)
by reacting 7-hydroxy-4-chromone (4) with the appropriate alkyl
bromides. These reactions were conducted in the presence of
K2CO3, and acetone served as solvent. In most instances, the
products were purified by recrystallization from ethanol. Com-
pounds 3b and 3o, however, required purification by column chro-
matography as cited in Section 4. The structures and purities of the
new compounds were verified by 1H NMR, 13C NMR, mass spec-
trometry and HPLC (see Section 4).
13
246
2
a
All values are expressed as the mean 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).
b
the test inhibitors, sigmoidal dose–response curves were con-
structed and the corresponding IC50 values were calculated
(Fig. 2). All determinations were carried out in triplicate.
2.2. MAO inhibition studies
To examine the MAO-A and -B inhibitor properties of the C7-
substituted chromone derivatives (3a–o), recombinant human
MAO-A and -B were employed as enzyme sources. The mixed
MAO-A/B substrate, kynuramine, was used for the enzyme activity
measurements of both isozymes [29,30]. Kynuramine is oxidized
by the MAO isozymes to yield 4-hydroxyquinoline which fluo-
resces in the alkaline medium used to terminate the enzymatic
reactions. Using fluorescence spectrophotometry, the concentra-
tions of 4-hydroxyquinoline in the reactions were measured
without interference from kynuramine or the test inhibitors. From
the rate data obtained in the presence of various concentrations of
2.2.1. IC50 values for the inhibition of MAO-B
The IC50 values for the inhibition of MAO-B by the C7-substituted
chromone derivatives 3a–o are presented in Table 2. The results
show that the chromones are highly potent inhibitors of human
MAO-B with all of the recorded IC50 values in the nanomolar range.
In accordance with literature reports that the benzyloxy side chain
enhances the MAO inhibition potencies of coumarin derivatives
[25], the present study finds that benzyloxy substitution of chro-
mone at C7 also yields compounds with potent MAO-B inhibitory
properties. For example, the first member of the series, the 7-ben-
zyloxychromone derivative 3a, exhibited an IC50 value of
0.085
ylene and ethylene units to yield the phenylethoxy (3b,
IC50 = 0.115 M) and phenylpropoxy (3c, IC50 = 0.121 M) substi-
lM. Extending the length of the C7 side chain of 3a with meth-
100
75
l
l
tuted homologues, respectively, resulted in a slight loss of MAO-B
inhibition potency. Although not statistically significant (p > 0.05),
from these results it may be concluded that benzyloxy substitution
at C7 of chromone is more favorable for potent MAO-B inhibition
than substitution with the phenylethoxy and phenylpropoxy moie-
ties. Substitution on the benzyloxy phenyl ring of derivative 3a with
halogens leads to a relatively large enhancement of MAO-B inhibi-
tion potency. For example, meta and para substitution of 3a with
chlorine, bromine and fluorine yielded structures (3d–i) with IC50
MAO-A
MAO-B
50
25
values ranging from 0.014 to 0.044 lM. These derivatives are
approximately two–sixfold more potent as MAO-B inhibitors than
3a (p < 0.05 in all instances). Alkyl substitution on the benzyloxy
phenyl ring of derivative 3a also yielded structures with potent
MAO-B inhibitory properties. For example, structures 3j–k, the
methyl substituted homologues, were more potent MAO-B
inhibitors than the unsubstituted homologue 3a, although by a
small degree and not statistically significant (p > 0.05). Similarly,
-4
-3
-2
-1
0
1
2
3
Log [3g]
Fig. 2. The sigmoidal dose–response curves of the human MAO-A (circles) and
MAO-B (squares) catalytic rates as a function of the logarithm of the concentration
of inhibitor 3g (expressed in
and the values are given as mean SD.
lM). The determinations were carried out in triplicate

4
L.J. Legoabe et al. / Bioorganic Chemistry 45 (2012) 1–11
the trifluoromethyl substituted homologue 3n was also found to be
more potent than 3a (p < 0.05). In fact 3n proved to be the most
potent inhibitor of the present series with an IC50 value of
0.008 lM. Interestingly, the cyano substituted derivatives 3l–m
exhibited weaker MAO-B inhibitory potencies than the correspond-
ing unsubstituted homologue 3a (3l, p < 0.05; 3m, p > 0.05). Among
the chromone derivatives substituted with a benzyloxy moiety at
C7, those containing cyano groups on the benzyloxy ring were the
weakest MAO-B inhibitors. In spite of this, compounds 3l and 3m
may still be viewed as potent MAO-B inhibitors with IC50 values of
0.370 lM and 0.117 lM, respectively. In accordance with the view
that benzyloxy substitution at C7 of chromone is more favorable
for MAO-B inhibition than phenylethoxy substitution (see above),
para bromine substitution of the 7-phenylethoxychromone deriva-
tive 3b, yielded structure 3o (IC50 = 0.317 lM) which is approxi-
mately 17-fold weaker (p < 0.05) as an MAO-B inhibitor than the
para bromine substituted 7-benzyloxychromone derivative 3g
(IC50 = 0.018 lM). Interestingly, meta and para substitution of a
particular substituent (Cl, Br, F, CH3) on 3a yielded similar MAO-B
inhibition activities for both positions (p > 0.05). The only exception
is cyano substitution with the para substituted derivative, 3m being
3-fold more potent than the corresponding meta substituted deriv-
ative 3l (p < 0.05). This result shows that, for the most part, the
substitution pattern has little effect on the MAO-B inhibitory
potencies of 7-benzyloxychromone derivatives.
2.2.2. IC50 values for the inhibition of MAO-A
As shown in Table 2 the C7-substituted chromone derivatives
3a–o are also inhibitors of human MAO-A. The IC50 values that
were recorded for the series ranged from 0.495 to 8.03 lM. The
selectivity index (SI) values indicated that the chromones are
selective inhibitors of the MAO-B isozyme. The most selective com-
pound, derivative 3n, is also the most potent MAO-B inhibitor of
the present series. In contrast to the results obtained with MAO-
B, the phenylethoxy (3b, IC50 = 2.19
IC50 = 3.08 M) substituted homologues were more potent MAO-
inhibitors than the 7-benzyloxychromone derivative 3a
(IC50 = 5.33 M) (p < 0.05 in both instances). Meta and para substi-
tution on the benzyloxy phenyl ring of derivative 3a with halogen
containing functional groups (Cl, Br, F, CF3) and the cyano group led
to an enhancement of MAO-A inhibition potency (p < 0.05 in all in-
stances), with the homologue containing a para chlorine substitu-
ent, compound 3e, exhibiting the most potent MAO-A inhibition of
lM) and phenylpropoxy (3c,
l
Fig. 3. Recovery of enzyme activity after dilution. MAO-A (top panel) and MAO-B
(bottom panel) were preincubated with compound 3g at concentrations equal to
A
l
10 Â IC50 and 100 Â IC50 for 30 min and then diluted to 0.1 Â IC50 and 1 Â IC50
,
respectively. The residual enzyme activities were subsequently measured.
tives are also reversible inhibitors of MAO-A and -B, the reversibil-
ity of inhibition of a selected inhibitor of the present series,
compound 3g, was evaluated. Compound 3g was selected as repre-
sentative inhibitor since it displayed potent inhibition towards
both MAO-A and -B. For the purpose of the reversibility study,
the recoveries of the enzymatic activities after dilution of
enzyme-inhibitor complexes were evaluated. The human MAO en-
zymes were firstly incubated with compound 3g at concentrations
of 10 Â IC50 and 100 Â IC50 for 30 min. These incubations were
subsequently diluted 100-fold to yield concentrations of the inhib-
itor of 0.1 Â IC50 and 1 Â IC50, respectively, and the residual en-
zyme catalytic rates were measured. As shown in Fig. 3, after
dilution of 3g to a concentration equal to 0.1 Â IC50, the MAO-A
and -B activities were recovered to levels of 94% and 96% of the
control values, respectively. This behavior is consistent with a
reversible interaction of 3g with MAO-A and -B. In contrast, after
treatment of MAO-A and -B with the irreversible inhibitors, pargy-
line and (R)-deprenyl, respectively, enzyme activity is not
regained.
the series (IC50 = 0.495 lM). In contrast to the results obtained
with MAO-B, methyl substitution on the benzyloxy ring of deriva-
tive 3a was associated with reduced MAO-A inhibitory potency
(p < 0.05 in both instances). For example, the derivatives substi-
tuted at the meta (3j) and para (3k) positions with the methyl
group exhibited IC50 values of 8.03
Interestingly, para bromine substitution of the 7-phenylethoxychr-
omone derivative 3b, to yield structure 3o (IC50 = 0.718 M), re-
lM and 7.12 lM, respectively.
l
sulted in enhanced MAO-A inhibition compared to 3b (p < 0.05).
This result is dissimilar to that obtained with MAO-B since bro-
mine substitution of 3b leads to a decrease of MAO-B inhibition po-
tency. Interestingly, for most 7-benzyloxychromone derivatives,
para substitution of a particular substituent (Cl, Br, F, CH3) yielded
improved MAO-A inhibition (p < 0.05) compared to meta substitu-
tion. The only exception is cyano substitution with the meta substi-
tuted derivative, 3l exhibiting 2-fold higher potency than the
corresponding para substituted derivative 3m (p < 0.05).
The possibility that 3g acts as a competitive inhibitor of human
MAO-A and -B was also investigated. Lineweaver–Burk double re-
ciprocal plots for the catalytic activities of MAO-A and -B were con-
structed in the absence and presence of three different
concentrations of 3g. The results, shown in Fig. 4, document that
the double reciprocal plots constructed with both MAO-A and -B
2.3. Reversibility of MAO inhibition
Literature reports that 7-benzyloxycoumarin derivatives are
competitive inhibitors, and therefore reversible inhibitors, of rat
MAO-A and -B [25]. To verify that 7-benzyloxychromone deriva-

L.J. Legoabe et al. / Bioorganic Chemistry 45 (2012) 1–11
5
100
75
50
25
0
-0.02
0.00
0.02
0.04
0.06
1/[S]
100
75
50
25
0
Fig. 5. The predicted binding orientations of 3g (yellow) and 3n (cyan) within the
MAO-B active site. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)
-0.02
0.00
0.02
0.04
0.06
1/[S]
Fig. 4. A set of four Lineweaver–Burk plots were constructed for the oxidation of
kynuramine by human MAO-A (top) and MAO-B (bottom). The data were collected
in the absence (filled squares) and presence of three different concentrations of 3g.
The inhibitor concentrations selected for the MAO-A studies were 0.148
squares), 0.296 M (filled circles) and 0.592 M (open circles) while the inhibitor
concentrations selected for the MAO-B studies were 0.0045 M (open squares),
0.009 M (filled circles), 0.018 M (open circles).
lM (open
l
l
l
l
l
are linear and each set has a common y-intercept. This behavior
indicates that 3g acts as a competitive inhibitor of human MAO-
A and -B, and provides additional support for the finding that 3g
is a reversible MAO inhibitor.
2.4. Molecular modeling
As discussed in Section 1, the availability of the crystallographic
structures of MAO-A and -B greatly assists in the design of inhibi-
tors of these enzymes and may provide a structural rationale for
the observed inhibitory properties of certain classes of compounds.
To provide additional insight into the MAO inhibition modes of C7-
substituted chromone derivatives, possible binding orientations
and interactions of selected inhibitors, compounds 3g and 3n, were
explored via molecular docking studies. For this purpose, the X-ray
crystal structures of human MAO-A cocrystallized with harmine
(PDB entry: 2Z5X) [7] and human MAO-B cocrystallized with 7-
(3-chlorobenzyloxy)-4-formylcoumarin (PDB entry: 2V60) [31]
served as protein models. The protein model of MAO-B is particu-
larly suitable for the docking studies since this model would enable
the comparison of the binding orientation of the coumarin moiety
of the cocrystallized ligand with the chromone moieties of 3g and
3n. Furthermore, in this model the side chain of Ile-199 is rotated
into the ‘‘open’’ conformation which allows for larger ligands, such
as the chromone derivatives examined here, to fit into the MAO-B
Fig. 6. The reported three-dimensional structure of 7-(3-chlorobenzyloxy)-4-
formylcoumarin in complex with the human MAO-B active site (2V60.pdb) [31].
active site. The preparation of the models and the docking calcula-
tions were carried out with the Windows based Discovery Studio
3.1 modeling software (Accelrys) [32] according to the literature
procedure [28,33]. The CDOCKER module of Discovery Studio 3.1
was utilized for the docking calculations. This docking protocol
has been shown to be appropriate for predicting binding orienta-
tions of ligands in the active sites of MAO-A and -B [28].
The highest ranked docking orientation of compound 3g in the
active site of the MAO-B model is illustrated in Fig. 5. The 10 high-
est ranked solutions display similar binding orientations with

6
L.J. Legoabe et al. / Bioorganic Chemistry 45 (2012) 1–11
with the active site residues compared to the folded conformation,
the ability of the C7-substituted chromone derivatives to bind in an
extended conformation to MAO-B may explain their selective
inhibition of the MAO-B isozyme. The assumption that the ex-
tended conformation is more favorable for MAO-B inhibition is in
accordance to the observation that inhibitors, which span both
active site cavities, are frequently more potent inhibitors than
those binding only in the substrate cavity [9].
It is noteworthy that in both MAO-A and -B, compound 3n
exhibits virtually identical binding modes to 3g. This suggests
that not many differences in the binding orientations of the
7-benzyloxychromone class of compounds exist and explain
the similar MAO-A and -B inhibitory activities among these
derivatives.
3. Discussion and conclusion
The present study examined the MAO inhibitory properties of a
series of C7-substituted chromones and finds that these com-
pounds are highly potent inhibitors of human MAO-B. For compar-
ison, the reversible MAO-B inhibitor safinamide exhibits an IC50
value of 0.08–0.5
lM, and 6-benzyloxychromone inhibits MAO-B
with an IC50 value of 0.053
l
M (SI = 62) under similar conditions
Fig. 7. The predicted binding orientations of 3g (purple) and 3n (cyan) within the
MAO-A active site. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)
[28,31]. The results further document that a representative mem-
ber of the series acts reversibly in a competitive manner. Among
the compounds studied, benzyloxy substitution of chromone is
the most favorable for MAO-B inhibition. The effects on MAO-B
inhibition by different substituents and substitution patterns on
the benzyloxy ring of 7-benzyloxychromone were also examined.
With the exception of cyano substitution, all substitution and sub-
stitution patterns enhanced MAO-B inhibition potency. The model-
ing studies propose that the C7-substituted chromones bind to
both active site cavities of MAO-B with the C7 side chain occupying
the entrance cavity while the chromone moiety binds within the in
substrate cavity. Since the entrance cavity is reported to be a highly
hydrophobic space, it may be expected that an enhancement of the
lipophilicity of the C7 side chain will result in more productive Van
der Waals interactions with the entrance cavity, and thus an
enhancement in binding affinity [34]. Substitution on the benzyl-
oxy phenyl ring with halogens and alkyl groups will increase the
lipophilicity of the C7 side chain, and may explain the observed
enhancements of MAO-B inhibition potencies compared to the
unsubstituted homologue 3a.
RMSD values of less than 0.21 Å from the position of the best
ranked solution. The chromone moiety of 3g is located within the
substrate cavity of the enzyme, in close proximity of the flavin ade-
nine dinucleotide (FAD) cofactor. The orientation and binding posi-
tion of the chromone are similar to that of the coumarin ring of the
cocrystallized ligand (Fig. 6) [31]. This binding position of 3g may
allow for the formation of a
p–p interaction (5.2 Å) between the
chromone ring and the aromatic ring of Tyr-398. In addition, the
benzopyrone carbonyl oxygen of 3g is within hydrogen bond dis-
tance to an active site water molecule. The formyl carbonyl oxygen
of the cocrystallized ligand, which binds in the same region as the
benzopyrone carbonyl oxygen of 3g, is reported to also undergo a
hydrogen bond interaction with this water molecule. Also similar
to the cocrystallized ligand, the C7 side chain of 3g extends into
the entrance cavity of the MAO-B active site. Since the entrance
cavity is reported to be a highly hydrophobic environment, the
C7 substituents are stabilized principally by Van der Waals interac-
tions within the entrance cavity [34].
The highest ranked docking orientation of compound 3g in the
active site of the MAO-A model is illustrated in Fig. 7. Compound
3g exhibits a similar binding orientation in the MAO-A active site
to that observed in MAO-B. The chromone moiety of 3g binds in
the proximity of the FAD cofactor with the C7 benzyloxy side chain
extending towards the entrance of the active site. A hydrogen on
C7-substituted chromones are also inhibitors of human MAO-A.
All compounds, however, display selective inhibition of MAO-B,
with the most potent MAO-B inhibitor, compound 3n, exhibiting a
246-fold selectivity for MAO-B. Analyses of the predicted binding
orientations and interactions of a representative chromone, com-
pound 3g, in the MAO-A and -B active sites show that the chromone
moiety of 3g may undergo p–p interactions and hydrogen bonding
within the MAO-B active site while, in MAO-A, these interactions
are predicted not to occur. The absence of these potentially stabiliz-
ing interactions in MAO-A may explain the lower MAO-A inhibition
potencies of the chromones compared to their potencies for the inhi-
bition of MAO-B. The modeling studies also suggest that, while 3g
binds in an extended conformation in the MAO-B active site, it
adopts a folded conformation in MAO-A. Compared to the folded
conformation, the extended conformation may place the benzyloxy
phenyl ring in a more favorable position/orientation to undergo
productive interactions in the hydrophobic entrance cavity space.
This may contribute to the selective inhibition of the MAO-B
isozyme by the C7-substituted chromones. Another dissimilarity
between the binding orientations of 3g within MAO-A and -B is
the orientation of the chromone moiety within the substrate cavity.
Compared to its orientation in MAO-B, the chromone ring of 3g is
the benzyloxy ring of 3g may undergo a potential p–r interaction
with the phenyl ring of Phe-208. Interestingly, 3g binds in a folded
conformation in the MAO-A active site, while exhibiting an ex-
tended conformation in the MAO-B active site. As discussed in Sec-
tion 1, the increased bulk of the phenyl side chain of Phe-208 may
impede the binding of relatively large inhibitors in the MAO-A ac-
tive site. To avoid structural overlap with Phe-208 in the MAO-A
active site, larger inhibitors bind in a folded conformation. The
ability of the side chain of Ile-199 to rotate from the MAO-B active
site cavity enables larger inhibitors to bind in an extended confor-
mation in MAO-B. The differences in size and conformational flex-
ibility of Ile-199 and Phe-208 may therefore explain the different
binding modes of 3g to MAO-A and -B [4]. Assuming that the
extended conformation results in more productive interactions

L.J. Legoabe et al. / Bioorganic Chemistry 45 (2012) 1–11
7
O
O
5
O
N
H
5
O
N
N
N
O
N
O
6
O
O
N
H
6
O
7
Fig. 8. The structures of 5-benzyloxyisatin (5), 8-benzyloxycaffeine (6) and 6-
benzyloxyisatin (7).
rotated through ꢀ180° in MAO-A. This places the benzopyrone
carbonyl oxygen in a region in the MAO-A substrate cavity where
the oxygen is predicted not to undergo hydrogen bonding. In
contrast, in MAO-B, the benzopyrone carbonyl oxygen is directed to-
wards a water molecule with which hydrogen bonding is predicted
to occur. The potential formation of this stabilizing hydrogen bond
interaction may explain, at least in part, the higher inhibition poten-
cies of the chromones for MAO-B. The finding that fused aromatic
moieties exhibit differing orientations in the MAO-A and -B active
sites is in agreement with literature which reports that, in the
MAO-B substrate cavity, the isatin ring of the reversible MAO inhib-
itor, 5-benzyloxyisatin (5), may also be rotated through ꢀ180° com-
pared to its position in MAO-A (Fig. 8) [35]. Similarly modeling
studies have shown that in MAO-B, the caffeine ring of 8-benzyloxy-
caffeine (6) may also be rotated through ꢀ180° compared to its po-
sition in MAO-A [30]. Interestingly, the modeling studies indicate
that inhibitors 3g and 3n exhibits highly similar binding orienta-
tions to both MAO-A and -B. In spite of this, compound 3n displays
a much larger degree of MAO-B inhibition and selectivity than 3g.
While the molecular reasons for this are unclear, a possible explana-
Fig. 9. A comparison of the predicted binding orientations of 3g and its C6-
substituted homologue within the MAO-B active site.
ꢀ180° compared to the orientation of the isatin moiety of the 6-
benzyloxyisatin isomer (7) [35]. These data suggest that the binding
orientation adopted by the chromone ring in the MAO-B substrate
cavity is determined by the position of the substituent. For optimal
placement of a C7 substituent in the entrance cavity, the chromone
ring adopts a different binding orientation in the substrate cavity
compared to the orientation of the chromone rings of C6-substi-
tuted homologues. Since the C6-substituted chromones are more
potent MAO-B inhibitors than the C7-substituted chromones, it
may be concluded that the binding mode of the C6-substituted
homologues allows for more productive interactions with the
MAO-B active site. Among these is a potential hydrogen bond with
Tyr-398 which is predicted to occur with the C6-substituted chro-
mones, but not the C7-substituted chromones [28].
tion may be that the CF3 group of 3n (
er Hammett constant ( p), forms more productive interactions with
the MAO-B active site than the 3g bromine ( p = 0.23) [30]. It has
previously been demonstrated that the MAO-B inhibitory activities
of a series of 8-benzyloxycaffeines correlates with increasing val-
rp = 0.54), by virtue of its high-
4. Experimental section
r
r
4.1. Chemicals and instrumentation
r
Unless otherwise noted, the reagents required for the chemical
synthesis were acquired from Sigma–Aldrich and were used with-
out purification. Proton (1H) and carbon (13C) NMR spectra were
recorded on a Bruker Avance III 600 spectrometer at frequencies
of 600 MHz and 150 MHz, respectively. All NMR measurements
were conducted in CDCl3 containing tetramethylsilane. Chemical
shifts are given in parts per million (d) downfield from the signal
of tetramethylsilane. Spin multiplicities are reported as s (singlet),
d (doublet), dd (doublet of doublets), t (triplet), q (quartet) or m
(multiplet). Direct insertion high resolution mass spectra (HRMS)
were obtained on a DFS high resolution magnetic sector mass spec-
trometer (Thermo Electron Corporation) in electron ionization (EI)
mode. Melting points (mp) were measured with a Buchi M-545
melting point apparatus and are uncorrected. The purities of the fi-
nal synthesized products were evaluated by HPLC analyses which
were conducted with an Agilent 1100 HPLC system equipped with
a quaternary pump and an Agilent 1100 series diode array detector
(see Supplementary material). HPLC grade acetonitrile (Merck) and
Milli-Q water (Millipore) was used for the chromatography.
ues [30]. In contrast, MAO-A inhibition does not correlate with
r
[30].
As mentioned in Section 1, we have recently reported the MAO
inhibitory properties of a series of C6-substituted chromones [28].
Compared to the C6-substituted chromones, which exhibited IC50
values for the inhibition of human MAO-B of 0.002–0.076
lM, the
C7-substituted homologues (IC50 = 0.008–0.370 M) may be con-
l
sidered as less potent inhibitors. For example, the C6-substituted
homologue of 3n was found to be a fourfold more potent inhibitor
than 3n (IC50 = 0.008 lM) with an IC50 value of 0.002 lM. Interest-
ingly, modeling studies indicate that C6-substituted chromones ex-
hibit similar binding modes in the MAO-B active site compared to
the C7-substituted chromones [28]. The principal difference is that,
the chromone rings of the C6-substituted chromones are rotated by
ꢀ180° compared to the orientations of the C7-substituted homo-
logues (Fig. 9). This finding is similar to a previous report that, in
the MAO-B substrate cavity, the isatin moiety of the reversible
MAO inhibitor, 5-benzyloxyisatin (5), may also be rotated through

8
L.J. Legoabe et al. / Bioorganic Chemistry 45 (2012) 1–11
Fluorescence spectrophotometry was carried out with a Varian
Cary Eclipse fluorescence spectrophotometer. Microsomes from in-
sect cells containing recombinant human MAO-A and -B (5 mg/mL)
and kynuramine.2HBr were from Sigma–Aldrich.
176.88, 162.69, 158.05, 154.84, 137.64, 134.67, 130.02, 128.51,
127.40, 127.38, 125.34, 119.12, 114.81, 112.96, 101.49, 69.56.
HRMS m/z: calcd for C16H11ClO3, 286.0397, found 286.0395. Purity
(HPLC): 99%.
4.2. Synthesis of the C7 substituted chromone derivatives (3a–o)
4.2.5. 7-[(4-Chlorobenzyl)oxy]-4H-chromen-4-one (3e)
The title compound (brown powder) was prepared by reacting
7-hydroxychromone and 4-chlorobenzyl bromide in acetone, with
a yield of 73%: mp 126.1–127.3 °C (ethanol). 1H NMR (Bruker
Avance III, 600 MHz, CDCl3) d 8.09 (d, J = 8.8 Hz, 1H), 7.74 (d,
J = 5.9 Hz, 1H), 7.35 (s, 4H), 7.00 (d, J = 8.9 Hz, 1H), 6.86 (s, 1H),
6.25 (d, J = 5.9 Hz, 1H), 5.09 (s, 2H). 13C NMR (Bruker Avance III,
7-Hydroxy-4-chromone (3 mmol) was dissolved in 20 ml of
acetone. To this solution, anhydrous potassium carbonate
(6 mmol) and the appropriate alkyl bromide (6 mmol) were added,
and the reaction mixture was heated at reflux and stirred for 24 h.
The reaction was filtered through a pad of Celite and the solvent
was subsequently removed in vacuo. The crude residue was tritu-
rated with 50 mL diethyl ether, the resulting solid material was
collected by filtration and dried overnight at 60 °C. Compounds
3b and 3o required column chromatographic purification. For
these compounds, the residue obtained after removal of the reac-
tion solvent was chromatographed using neutral alumina as sta-
tionary phase and dichloromethane-ethylacetate (4:1) as an
eluent. The chromone derivatives were recrystallized from ethanol.
150 MHz, CDCl3)
d 176.87, 162.76, 158.04, 154.83, 134.22,
134.09, 128.91, 128.77, 127.33, 119.04, 114.84, 112.94, 101.45,
69.66. HRMS m/z: calcd for
C16H11ClO3, 286.0397, found
286.0395. Purity (HPLC): 99%.
4.2.6. 7-[(3-Bromobenzyl)oxy]-4H-chromen-4-one (3f)
The title compound (white powder) was prepared by reacting
7-hydroxychromone and 3-bromobenzyl bromide in acetone, with
a yield of 82%: mp 145.4–145.5 °C (ethanol). 1H NMR (Bruker
Avance III, 600 MHz, CDCl3) d 8.11 (d, J = 8.9 Hz, 1H), 7.75 (d,
J = 6.1 Hz, 1H), 7.58 (t, J = 1.8 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.34
(d, J = 7.8 Hz, 1H), 7.29–7.22 (m, 1H), 7.02 (dd, J = 8.9, 2.4 Hz, 1H),
6.87 (d, J = 2.4 Hz, 1H), 6.26 (d, J = 6.0 Hz, 1H), 5.08 (s, 2H). 13C
4.2.1. 7-(Benzyloxy)-4H-chromen-4-one (3a)
The title compound (light orange powder) was prepared by
reacting 7-hydroxychromone and benzyl bromide in acetone, with
a yield of 49%: mp 129.2–130.3 °C (ethanol). 1H NMR (Bruker
Avance III, 600 MHz, CDCl3) d 8.10 (d, J = 8.9 Hz, 1H), 7.74 (d,
J = 6.0 Hz, 1H), 7.45–7.30 (m, 5H), 7.02 (dd, J = 8.9, 2.4 Hz, 1H),
6.89 (d, J = 2.4 Hz, 1H), 6.25 (d, J = 6.0 Hz, 1H), 5.13 (s, 2H). 13C
NMR (Bruker Avance III, 150 MHz, CDCl3)
d 176.90, 162.69,
158.06, 154.86, 137.90, 131.46, 130.33, 130.30, 127.41, 125.84,
122.82, 119.14, 114.82, 112.98, 101.50, 69.51. HRMS m/z: calcd
for C16H11BrO3, 329.9892, found 329.9889. Purity (HPLC): 99%.
NMR (Bruker Avance III, 150 MHz, CDCl3)
d 176.93, 163.06,
158.09, 154.80, 135.59, 128.73, 128.38, 127.47, 127.23, 118.91,
114.95, 112.91, 101.43, 70.48. HRMS m/z: calcd for C16H12O3,
252.0786, found 252.0793. Purity (HPLC): 99%.
4.2.7. 7-[(4-Bromobenzyl)oxy]-4H-chromen-4-one (3g)
The title compound (light brown powder) was prepared by
reacting 7-hydroxychromone and 4-bromobenzyl bromide in ace-
tone, with a yield of 84%: mp 139.7–139.8 °C (ethanol). 1H NMR
(Bruker Avance III, 600 MHz, CDCl3) d 8.21 (d, J = 6.0 Hz, 1H), 7.94
(d, J = 8.8 Hz, 1H), 7.61 (d, J = 8.3 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H),
7.21 (d, J = 2.4 Hz, 1H), 7.12 (dd, J = 8.8, 2.4 Hz, 1H), 6.27 (d,
J = 6.0 Hz, 1H), 5.23 (s, 2H). 13C NMR (Bruker Avance III, 150 MHz,
CDCl3) d 175.64, 162.51, 157.64, 156.52, 135.55, 131.48, 130.04,
126.48, 121.32, 118.28, 115.12, 112.19, 101.87, 69.19. HRMS m/z:
calcd for C16H11BrO3, 329.9892, found 329.9885. Purity (HPLC):
99%.
4.2.2. 7-(2-Phenylethoxy)-4H-chromen-4-one (3b)
The title compound (white powder) was prepared by reacting
7-hydroxychromone and 2-phenylethyl bromide in acetone, with
a yield of 34%: mp 86.8–89.7 °C (ethanol). 1H NMR (Bruker Avance
III, 600 MHz, CDCl3) d 8.07 (d, J = 8.9 Hz, 1H), 7.73 (d, J = 5.9 Hz,
1H), 7.36–7.21 (m, 5H), 6.94 (dd, J = 8.9, 2.3 Hz, 1H), 6.79 (d,
J = 2.3 Hz, 1H), 6.24 (d, J = 6.0 Hz, 1H), 4.23 (t, J = 7.0 Hz, 2H), 3.12
(t, J = 7.0 Hz, 2H). 13C NMR (Bruker Avance III, 150 MHz, CDCl3) d
176.95, 163.21, 158.13, 154.77, 137.52, 128.93, 128.56, 127.13,
126.71, 118.72, 114.75, 112.88, 100.90, 69.20, 35.43. HRMS m/z:
calcd for C17H14O3, 266.0942, found 266.0938. Purity (HPLC): 99%.
4.2.8. 7-[(3-Fluorobenzyl)oxy]-4H-chromen-4-one (3h)
The title compound (brown powder) was prepared by reacting
7-hydroxychromone and 3-fluorobenzyl bromide in acetone, with
a yield of 70%: mp 180.6–181.8 °C (ethanol). 1H NMR (Bruker
Avance III, 600 MHz, CDCl3) d 8.11 (d, J = 8.8 Hz, 1H), 7.75 (d,
J = 5.8 Hz, 1H), 7.35 (q, J = 7.2 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H),
7.14 (d, J = 9.4 Hz, 1H), 7.03 (m, 2H), 6.87 (s, 1H), 6.26 (d,
J = 5.9 Hz, 1H), 5.13 (s, 2H). 13C NMR (Bruker Avance III,
4.2.3. 7-(3-Phenylpropoxy)-4H-chromen-4-one (3c)
The title compound (cream colored powder) was prepared by
reacting 7-hydroxychromone and 3-phenylpropyl bromide in ace-
tone, with a yield of 68%: mp 93.1–94.0 °C (ethanol). 1H NMR (Bru-
ker Avance III, 600 MHz, CDCl3) d 8.09 (d, J = 8.9 Hz, 1H), 7.75 (d,
J = 6.0 Hz, 1H), 7.28 (m, 2H), 7.19 (m, 3H), 6.95 (dd, J = 8.9,
2.4 Hz, 1H), 6.78 (d, J = 2.4 Hz, 1H), 6.25 (d, J = 6.0 Hz, 1H), 4.01
(t, J = 6.2 Hz, 2H), 2.81 (t, J = 7.5 Hz, 2H), 2.19–2.10 (m, 2H). 13C
150 MHz, CDCl3)
d 176.90, 163.79, 162.74, 162.15, 158.07,
154.85, 138.20, 130.37, 130.32, 127.39, 122.72, 119.12, 115.35,
115.21, 114.84, 114.32, 114.17, 112.97, 101.52, 69.62. HRMS m/z:
calcd for C16H11FO3, 270.0692, found 270.0681. Purity (HPLC): 99%.
NMR (Bruker Avance III, 150 MHz, CDCl3)
158.20, 154.79, 140.97, 128.48, 128.45, 127.14, 126.08, 118.66,
114.80, 112.89, 100.84, 67.46, 31.96, 30.43. HRMS m/z: calcd for
d 177.03, 163.49,
C18H16O3, 280.1099, found 280.1099. Purity (HPLC): 99%.
4.2.9. 7-[(4-Fluorobenzyl)oxy]-4H-chromen-4-one (3i)
4.2.4. 7-[(3-Chlorobenzyl)oxy]-4H-chromen-4-one (3d)
The title compound (brown powder) was prepared by reacting
7-hydroxychromone and 4-fluorobenzyl bromide in acetone, with
a yield of 67%: mp 120.6–121.3 °C (ethanol). 1H NMR (Bruker
Avance III, 600 MHz, CDCl3) d 8.09 (d, J = 8.9 Hz, 1H), 7.74 (d,
J = 5.8 Hz, 1H), 7.42–7.37 (m, 2H), 7.07 (t, J = 8.2 Hz, 2H), 7.00 (d,
J = 8.9 Hz, 1H), 6.87 (s, 1H), 6.25 (d, J = 5.9 Hz, 1H), 5.08 (s, 2H).
13C NMR (Bruker Avance III, 150 MHz, CDCl3) d 176.89, 162.85,
161.83, 158.06, 154.83, 131.38, 131.36, 129.43, 129.38, 127.29,
The title compound (brown powder) was prepared by reacting
7-hydroxychromone and 3-chlorobenzyl bromide in acetone, with
a yield of 77%: mp 136.8–137.0 °C (ethanol). 1H NMR (Bruker
Avance III, 600 MHz, CDCl3) d 8.11 (d, J = 9.0 Hz, 1H), 7.75 (d,
J = 6.0 Hz, 1H), 7.42 (d, J = 1.9 Hz, 1H), 7.35–7.26 (m, 3H), 7.02
(dd, J = 8.9, 2.4 Hz, 1H), 6.87 (d, J = 2.4 Hz, 1H), 6.25 (d, J = 6.0 Hz,
1H), 5.10 (s, 2H). 13C NMR (Bruker Avance III, 150 MHz, CDCl3) d

L.J. Legoabe et al. / Bioorganic Chemistry 45 (2012) 1–11
9
118.99, 115.75, 115.61, 114.86, 112.92, 101.42, 69.79. HRMS m/z:
4.2.15. 7-[2-(4-Bromophenyl)ethoxy]-4H-chromen-4-one (3o)
calcd for C16H11FO3, 270.0692, found 270.0694. Purity (HPLC): 99%.
The title compound (white powder) was prepared by reacting
7-hydroxychromone and 4-bromophenethyl bromide in acetone,
with a yield of 25%: mp 93.1–93.8 °C (ethanol). 1H NMR (Bruker
Avance III, 600 MHz, CDCl3) d 8.07 (d, J = 8.9 Hz, 1H), 7.74 (d,
J = 6.0 Hz, 1H), 7.43 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 8.3 Hz, 2H),
6.92 (dd, J = 8.9, 2.4 Hz, 1H), 6.78 (d, J = 2.4 Hz, 1H), 6.24 (d,
J = 6.0 Hz, 1H), 4.20 (t, J = 6.7 Hz, 2H), 3.07 (t, J = 6.7 Hz, 2H). 13C
4.2.10. 7-[(3-Methylbenzyl)oxy]-4H-chromen-4-one (3j)
The title compound (brown powder) was prepared by reacting
7-hydroxychromone and 3-methylbenzyl bromide in acetone, with
a yield of 73%: mp 89.0–91.8 °C (ethanol). 1H NMR (Bruker Avance
III, 600 MHz, CDCl3) d 8.14 (d, J = 9.2 Hz, 1H), 7.79 (d, J = 6.0 Hz,
1H), 7.35–7.25 (m, 3H), 7.20 (d, J = 7.5 Hz, 1H), 7.07 (dd, J = 8.9,
2.3 Hz, 1H), 6.93 (s, 1H), 6.29 (d, J = 6.0 Hz, 1H), 5.13 (s, 2H), 2.41
(s, 3H). 13C NMR (Bruker Avance III, 150 MHz, CDCl3) d 176.94,
163.13, 158.10, 154.79, 138.48, 135.49, 129.14, 128.62, 128.21,
127.20, 124.58, 118.87, 114.96, 112.90, 101.40, 70.56, 21.38. HRMS
m/z: calcd for C17H14O3, 266.0943, found 266.0937. Purity (HPLC):
99%.
NMR (Bruker Avance III, 150 MHz, CDCl3)
d 176.92, 163.04,
158.11, 154.80, 136.63, 131.63, 130.67, 127.22, 120.60, 118.84,
114.66, 112.92, 100.94, 68.77, 34.85. HRMS m/z: calcd for C17H13-
BrO3, 344.0048, found 344.0033. Purity (HPLC): 99%.
4.3. Human MAO inhibition studies
Microsomes from insect cells containing recombinant human
MAO-A and -B were obtained from commercial sources (Sigma–Al-
drich). The enzymatic reactions were conducted in potassium
phosphate buffer (100 mM, pH 7.4, made isotonic with KCl
4.2.11. 7-[(4-Methylbenzyl)oxy]-4H-chromen-4-one (3k)
The title compound (light pink powder) was prepared by react-
ing 7-hydroxychromone and 4-methylbenzyl bromide in acetone,
with a yield of 67%: mp 118.2–119.3 °C (ethanol). 1H NMR (Bruker
Avance III, 600 MHz, CDCl3) d 8.09 (d, J = 8.9 Hz, 1H), 7.74 (d,
J = 5.8 Hz, 1H), 7.31 (d, J = 7.5 Hz, 2H), 7.19 (d, J = 7.6 Hz, 2H),
7.01 (d, J = 8.9 Hz, 1H), 6.88 (s, 1H), 6.25 (d, J = 5.9 Hz, 1H), 5.08
(s, 2H), 2.35 (s, 3H). 13C NMR (Bruker Avance III, 150 MHz, CDCl3)
d 176.94, 163.14, 158.09, 154.78, 138.26, 132.53, 129.39, 127.62,
127.17, 118.83, 114.99, 112.88, 101.39, 70.45, 21.18. HRMS m/z:
calcd for C17H14O3, 266.0943, found 299.0929. Purity (HPLC): 99%.
20.2 mM) to a final volume of 500
(45 M for MAO-A and 30 M for MAO-B), various concentrations
of the test inhibitors (0–100 M) and 4% DMSO as cosolvent. The
lL, and contained kynuramine
l
l
l
reactions were initiated by the addition of MAO-A or -B
(0.0075 mg protein/mL) and were subsequently incubated for
20 min at 37 °C. The reactions were terminated by the addition
of 400 lL NaOH (2 N) and 1000 lL water, centrifuged for 10 min
at 16,000g and the concentrations of 4-hydroxyquinoline in the
supernatants were measured spectrofluorometrically (kex = 310 -
nm; kem = 400 nm) [29,33,36]. A linear calibration curve was con-
structed by preparing samples containing 4-hydroxyquinoline
4.2.12. 3-{[(4-Oxo-4H-chromen-7-yl)oxy]methyl}benzonitrile (3l)
The title compound (brown crystals) was prepared by reacting
7-hydroxychromone and 3-cyanobenzyl bromide in acetone, with
a yield of 66%: mp 156.8–157.0 °C (ethanol). 1H NMR (Bruker
Avance III, 600 MHz, CDCl3) d 8.11 (d, J = 8.5 Hz, 1H), 7.76 (d,
J = 5.6 Hz, 1H), 7.23 (s, 1H), 7.66 (d, J = 7.5 Hz, 1H), 7.62 (d,
J = 7.5 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.01 (d, J = 9.0 Hz, 1H), 6.87
(s, 1H), 6.25 (d, J = 6.0 Hz, 1H), 5.15 (s, 2H). 13C NMR (Bruker
Avance III, 150 MHz, CDCl3) d 176.79, 162.35, 157.99, 154.90,
137.26, 131.92, 131.46, 130.66, 129.56, 127.49, 119.28, 118.36,
114.63, 112.97, 112.91, 101.53, 69.04. HRMS m/z: calcd for
(0.047–1.56
To each calibration standard, volumes of 400
1000 L water were added. The appropriate control samples were
l
M) dissolved in 500
lL potassium phosphate buffer.
l
L NaOH (2 N) and
l
included to confirm that the test inhibitors do not fluoresce or
quench the fluorescence of 4-hydroxyquinoline under these assay
conditions. IC50 values were estimated from sigmoidal dose–re-
sponse curves (graphs of the initial rate of kynuramine oxidation
versus the logarithm of inhibitor concentration). Each sigmoidal
curve was constructed from six different inhibitor concentrations
spanning at least three orders of magnitude. Data analyses were
carried out with the Prism 5 software package (GraphPad) employ-
ing the one site competition model. IC50 values were determined in
triplicate and are expressed as mean standard deviation (SD).
One-way analyses of variances (ANOVA) with Tukey’s post hoc test
was used to determine if statistical differences exist between the
means of the IC50 values among the test compounds.
C17H11NO3, 277.0739, found 277.0733. Purity (HPLC): 99%.
4.2.13. 4-{[(4-Oxo-4H-chromen-7-yl)oxy]methyl}benzonitrile (3m)
The title compound (orange powder) was prepared by reacting
7-hydroxychromone and 4-cyanobenzyl bromide in acetone, with
a yield of 68%: mp 213.7–213.8 °C (ethanol). 1H NMR (Bruker
Avance III, 600 MHz, CDCl3) d 8.11 (d, J = 8.9 Hz, 1H), 7.75 (d,
J = 6.1 Hz, 1H), 7.68 (d, J = 7.8 Hz, 2H), 7.54 (d, J = 7.9 Hz, 2H),
7.02 (d, J = 8.9 Hz, 1H), 6.86 (s, 1H), 6.26 (d, J = 5.9 Hz, 1H), 5.19
(s, 2H). 13C NMR (Bruker Avance III, 150 MHz, CDCl3) d 176.78,
162.36, 158.00, 154.89, 140.97, 132.52, 127.56, 127.52, 119.30,
118.43, 114.66, 113.00, 112.16, 101.54, 69.27. HRMS m/z: calcd
for C17H11NO3, 277.0739, found 277.0733. Purity (HPLC): 99%.
4.4. Recovery of enzyme activity after dilution
Compound 3g at concentrations equal to 10 Â IC50 and
100 Â IC50 for the inhibition of the respective MAO isozymes was
preincubated with recombinant human MAO-A or -B (0.75 mg/
ml) for 30 min at 37 °C in potassium phosphate buffer (100 mM,
pH 7.4, made isotonic with KCl). Control incubations were con-
ducted in the absence of inhibitor, and DMSO (4%) was added as
co-solvent to all preincubations. The reactions were subsequently
diluted 100-fold with the addition of a solution of kynuramine to
yield final concentrations of compound 3g equal to 0.1 Â IC50 and
4.2.14. 7-{[4-(Trifluoromethyl)benzyl]oxy}-4H-chromen-4-one (3n)
The title compound (brown powder) was prepared by reacting
7-hydroxychromone and 4-(trifluoromethyl)benzyl bromide in
acetone, with a yield of 69%: mp 159.3–159.4 °C (ethanol). 1H
NMR (Bruker Avance III, 600 MHz, CDCl3) d 8.11 (d, J = 8.9 Hz, 1H),
7.75 (d, J = 5.9 Hz, 1H), 7.65 (d, J = 7.8 Hz, 2H), 7.54 (d, J = 7.8 Hz,
2H), 7.02 (d, J = 8.9 Hz, 1H), 6.87 (s, 1H), 6.25 (d, J = 5.9 Hz, 1H),
5.19 (s, 2H). 13C NMR (Bruker Avance III, 150 MHz, CDCl3) d
176.86, 162.61, 158.05, 154.87, 139.66, 130.63, 130.42, 127.44,
127.40, 125.71 (q), 124.83, 123.02, 119.19, 114.78, 112.98,
101.51, 69.52. HRMS m/z: calcd for C17H11F3O3, 320.0660, found
320.0659. Purity (HPLC): 99%.
1 Â IC50. The final concentrations of kynuramine were 45
lM and
30 M for MAO-A and -B, respectively, and the final concentrations
l
of MAO-A and -B were 0.0075 mg/mL. The reactions were incubated
for a further 20 min at 37 °C, terminated and the residual rates of 4-
hydroxyquinoline formation were measured as described above.
The residual enzyme catalytic rates were expressed as mean SD.
As control experiments, pargyline (IC50 = 12.97
lM) and (R)-
deprenyl (IC50 = 0.079 M) were similarly preincubated with
l

10
L.J. Legoabe et al. / Bioorganic Chemistry 45 (2012) 1–11
recombinant human MAO-A and -B (0.75 mg/ml) at concentrations
equal to 100 Â IC50 and diluted 100-fold with the addition of kynur-
amine to yield final concentrations of the inhibitors equal to
1 Â IC50 [37].
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2012.08.
003.
4.5. Lineweaver–Burk plots
A set consisting of four Lineweaver–Burk plots were con-
structed for the inhibition of recombinant human MAO-A and -B
by the selected inhibitor 3g. For this purpose, the rates of kynur-
amine oxidation by MAO-A and -B were measured in the absence
and presence of three different concentrations of 3g. The enzy-
matic reactions were conducted as described above and contained
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