Chalcones: A valid scaffold for monoamine oxidases inhibitors

Article abstract of DOI:10.1021/jm801590u

A large series of substituted chalcones have been synthesized and tested in vitro for their ability to inhibit human monoamine oxidases A and B (hMAO-A and hMAO-B). While all the compounds showed hMAO-B selective activity in the micro- and nanomolar ranges, the best results were obtained in the presence of chlorine and hydroxyl or methoxyl substituents. To better understand the enzyme-inhibitor interaction and to explain the selectivity of the most active compounds toward hMAO-B, molecular modeling studies were carried out on new, high resolution, hMAO-B crystallographic structures. For the only compound that also showed activity against hMAO-A as well as low selectivity, the molecular modeling study was also performed on the hMAO-A crystallographic structure. The docking technique provided new insight on the inhibition mechanism and the rational drug design of more potent/selective hMAO inhibitors based on the chalcone scaffold.

Full text of DOI:10.1021/jm801590u

2818
J. Med. Chem. 2009, 52, 2818–2824
Chalcones: A Valid Scaffold for Monoamine Oxidases Inhibitors
Franco Chimenti,† Rossella Fioravanti,*,† Adriana Bolasco,† Paola Chimenti,† Daniela Secci,† Francesca Rossi,† Matilde Ya´n˜ez,‡
Francisco Orallo,‡ Francesco Ortuso,§ and Stefano Alcaro§
Dipartimento di Chimica e Tecnologie del Farmaco, UniVersita` degli Studi di Roma “La Sapienza”, P. le A. Moro 5, 00185 Roma, Italy,
Departamento de Farmacolog´ıa and Instituto de Farmacia Industrial, Facultad de Farmacia, UniVersidad de Santiago de Compostela, Campus
UniVersitario Sur, E-15782 Santiago de Compostela (La Corun˜a), Spain, Dipartimento di Scienze Farmacobiologiche, UniVersita` di Catanzaro
“Magna Graecia”, “Complesso Nin`ı Barbieri”, 88021 Roccelletta di Borgia (CZ), Italy
ReceiVed December 17, 2008
A large series of substituted chalcones have been synthesized and tested in vitro for their ability to inhibit
human monoamine oxidases A and B (hMAO-A and hMAO-B). While all the compounds showed hMAO-B
selective activity in the micro- and nanomolar ranges, the best results were obtained in the presence of
chlorine and hydroxyl or methoxyl substituents. To better understand the enzyme-inhibitor interaction and
to explain the selectivity of the most active compounds toward hMAO-B, molecular modeling studies were
carried out on new, high resolution, hMAO-B crystallographic structures. For the only compound that also
showed activity against hMAO-A as well as low selectivity, the molecular modeling study was also performed
on the hMAO-A crystallographic structure. The docking technique provided new insight on the inhibition
mechanism and the rational drug design of more potent/selective hMAO inhibitors based on the chalcone
scaffold.
Introduction
Monoamine oxidases (MAOs; EC 1.4.3.4a) are widespread
enzymes responsible for the regulation and metabolism of major
monoamine neurotransmitters (5-hydroxytryptamine (5-HT),
norepinephrine, dopamine), modulating their concentrations in
the brain and peripheral tissues. Two isoforms of MAOs were
fully characterized:1 hMAO-A and hMAO-B, according to their
substrate specificity, inhibitor sensitivity, and amino acid
sequence. Binda et al.2,3 described the crystal structure of these
two subtypes, highlighting the selective interaction between them
and their ligands. The researcher’s interest in the rational design
of potent, specific inhibitors with therapeutic potential and no
undesirable side-effects has actually been renewed.4 As men-
tioned above, hMAOs play an important role in the metabolism
of several neurotransmitters, and their inhibitors could be useful
in the treatment of psychiatric and neurological diseases. Human
MAO-B inhibitors are used alone or in combination in the
therapy of Alzheimer’s and Parkinson’s diseases, and in 2006 a
new MAO B inhibitor, safinamide, is about to enter phase III
trials,5 while human MAO-A inhibitors are used as antidepres-
sants and antianxiety agents.6 Furthermore, interest in selective
inhibitors of hMAO-B has increased in recent years7 due to the
discovery of an age-related increase in hMAO-B expression after
Figure 1. General chemical structure of synthesized trans-chalcones.
the 60th year of life especially in glial cells.8-10 Therefore, a
selective hMAO-B inhibitor, could contribute to neuroprotection
and prevent neuronal degeneration.11
Because no general rules are available for the design of potent
and selective inhibitors of hMAOs, in pursuing our studies in
the field, we choose natural products as a significant source of
new drug candidates. An impressive number of modern drugs
have been developed from natural sources, especially from plants
used as traditional folk medicine.
Chalcones (trans-1,3-diphenyl-2-propen-1-ones) are the bio-
genetic precursors of all known flavonoids and are abundant in
edible plants.12 Chemically, they consist of open-chain fla-
vonoids in which the two aromatic rings are joined by a three-
carbon R,ꢀ-unsaturated carbonyl system (Figure 1). They present
a broad spectrum of biological activities12,13 such as anticancer,
anti-inflammatory, antimalarial, antifungal,14,15 antilipidemic,
and antiviral activities.16
For example, xanthoangelol has been reported to induce
apoptosis and to inhibit tumor promotion and metastasis in
several cancer cell lines.17,18 Broussochalcone A,19 dimethyl-
aminochalcones,20 and cardamonin21 possess anti-inflammatory
activity.22 Licochalcone A, a substance found in the roots of
the Chinese liquorice, showed antimalarial activity.23 4′,4-
Dichlorochalcone exhibits antilipidemic activity24 (Figure 2).
There are other chalcones with very interesting biological
activity such as butein25 and isoliquiritigenin.21 These chalcones,
like trans-resveratrol,26 remarkably activate sirtuin27 enzymatic
activity, mimic the beneficial effects of caloric restriction (CR),
retard the aging process, and increase longevity in the budding
yeast Saccharomyces cereVisiae. However, the information on
the inhibitory monoamine oxidase (MAO) activity of these
* To whom correspondence should be addressed. Phone. +39 06
49913975. Fax: +39 06 49913772. E-mail: rossella.fioravanti@uniroma1.it.
†
Dipartimento di Chimica e Tecnologie del Farmaco, Universita` degli
Studi di Roma “La Sapienza”.
‡
Departamento de Farmacolog´ıa and Instituto de Farmacia Industrial,
Facultad de Farmacia, Universidad de Santiago de Compostela.
§
Dipartimento di Scienze Farmacobiologiche, Universita` di Catanzaro
“Magna Graecia”.
a Abbreviations: MAO, monoamine oxidase; 5-HT (5-hydroxytryptamine);
CR, caloric restriction; IC50, 50% inhibitory concentration; Michaelis
constant (Km); maximum reaction velocity (Vmax); MC, Monte Carlo; PDB,
protein data bank; 2BXR and 2Z5X, PDB code of hMAO-A; 1GOS and
2BK3, PDB code of hMAO-B; FAD, flavin adenine dinucleotide; CVDW,
Coulomb van der Waals; SEM, standard error of the mean; P, probability
value; pIC50 ) -log IC50. Abbreviations used for amino acids follow the
rules of the IUPACIUB Commission of Biochemical Nomenclature in
J. Biol. Chem. 1972, 247, 977-983. Amino acid symbols denote L-
configuration unless indicated.
10.1021/jm801590u CCC: $40.75
2009 American Chemical Society
Published on Web 04/20/2009

Chalcones: A Valid Scaffold for MAO Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2819
Figure 2. Structures of known trans-chalcones with biological activities.
molecules reported in the literature is limited and directed to
behavioral tests and assays on rat mitochondrial MAOs.28
Therefore, considering that chalcones are a structurally simple
group of compounds that for our knowledge have not been
assayed on human MAOs and that we previously29,30 reported
a number of synthetic inhibitors of MAO-A and -B, in which
chalcones are intermediate compounds, in this investigation we
present the synthesis and the inhibitory activity against the A-and
B-isoforms of hMAO of a number of substituted chalcones.
We also report on a computational study using the crystal
structure data and models of hMAO-A and hMAO-B, deposited
in the Protein Data Bank, with the aim of rationalizing the
binding modes of these compounds with human MAO isoforms.
produce a fluorescent product, resorufin. In this study, hMAO
activity was evaluated using the above method following the
general procedure described previously by us.32 The test drugs
(new compounds and reference inhibitors) themselves were
unable to react directly with the Amplex Red reagent, which
indicates that these drugs do not interfere with the measure-
ments. In our experiments and under our experimental condi-
tions, hMAO-A displayed a Michaelis constant (Km) of 457.17
( 38.62 µM and a maximum reaction velocity (Vmax) of 185.67
( 12.06 nmol/min/mg protein, whereas hMAO-B showed a Km
of 220.33 ( 32.80 µM and a Vmax of 24.32 ( 1.97 nmol/min/
mg protein (n ) 5).
Most tested drugs inhibited this enzymatic control activity
in a concentration-dependent way (see Table 1).
Chemistry
Results and Discussion
The general synthetic strategy employed to prepare chalcones
in excellent yield (Table 1) was based on the Claisen-Schmidt
condensation reported previously.31 By condensing substituted
acetophenone with aromatic aldehydes, using solid barium
hydroxide octahydrate as catalyst in methanol at 40 °C, we
obtained derivatives 5 (Scheme 1). The starting materials were
commercially available. This method for the preparation of
chalcones is particularly attractive because it specifically gener-
ates the (E)-isomer.
The hMAO-A and hMAO-B inhibition data are reported in
Table 1 together with the selectivity index (selectivity index SI
) IC50 hMAO-A /IC50 hMAO-B). It can be observed that all
compounds show a selective inhibitory activity toward hMAO-B
in the micro- and nanomolar range. The most active compounds,
5i and 5m (IC50 ) 0.0044 ( 0.00027 µM and 0.0051 ( 0.00019
µM, respectively), are disubstituted in the 2′- and 4′-position
of the B aromatic moiety with two hydroxyls or hydroxyl and
methoxy groups and in 4-position of the A aromatic moiety
with a chlorine atom. Compound 5i also shows the highest
MAO-B selectivity (SI > 11364). The other compounds show
good activity in the nanomolar range and selectivity, but among
them the highest activity was detected for the compounds that
are substituted with a chloro atom in the 4-position and with
hydroxy or methoxy groups in the 4′-position. From this data,
we can indicate that the presence in 2′- and 4′- position of the
B aromatic moiety of the two previously indicated substituents
and of a chlorine atom in 4-position of the A aromatic moiety
is a fundamental requisite for inhibitory activity. The chalcones
5a-e, 4′-isoprenyloxy substituted, show lower activity.
Biochemistry
The potential effects of the test drugs on hMAO activity were
investigated by measuring their effects on the production of
hydrogen peroxide (H2O2) from p-tyramine using the Amplex
Red MAO assay kit (Molecular Probes, Inc., Eugene, OR) and
microsomal MAO isoforms prepared from insect cells (BTI-
TN-5B1-4) infected with recombinant baculovirus containing
cDNA inserts for hMAO-A or hMAO-B (Sigma-Aldrich
Qu´ımica SA, Alcobendas, Spain).
The production of H2O2 catalyzed by MAO isoforms can be
detected using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex
Red reagent), a nonfluorescent and highly sensitive probe that
reacts with H2O2 in the presence of horseradish peroxidase to
In the reversibility and irreversibility tests, h-MAO-B inhibi-
tion was irreversible in presence of the compounds 5a, 5i, and

2820 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Table 1. Structures and Inhibitory Activity of Derivatives 5a-p
Chimenti et al.
compd
R
R1
R2
hMAO-A (IC50 µM)
hMAO-B (IC50 µM)a
SI
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
A
(CH3)2CdCHCH2O-
OH
OH
OH
OH
OH
H
H
H
OH
H
OH
H
H
CH3
OCH3
OCH2Ph
Cl
Cl
Cl
OCH2Ph
Cl
H
Cl
Cl
Cl
Cl
OCH2Ph
Cl
**
**
**
**
**
**
**
**
***
**
***
***
18.76 ( 0.92
1.03 ( 0.09
**
>1.1b
(CH3)2CdCHCH2O-
>19b
(CH3)2CdCHCH2O-
(CH3)2CdCHCH2O-
**
(CH3)2CdCHCH2O-
1.25 ( 0.04
**
>16b
OCH2Ph
OCH3
OCH3
OCH3
H
H
OH
OH
F
0.054 ( 0.0033
0.45 ( 0.028
0.0044 ( 0.00027
1.41 ( 0.070
0.16 ( 0.0092
0.031 ( 0.00083
0.0051 ( 0.00019
0.071 ( 0.0022
0.50 ( 0.030
0.47 ( 0.017
61.35 ( 1.13
0.020 ( 0.00086
7.54 ( 0.36
*
>370b
>44b
>11364b
>14b
>312b
>1613b
971
OH
OH
H
4.95 ( 0.28a
***
>704b
>40b
F
**c
SO2CH3
H
**c
>43b
0.0045 ( 0.00032a
67.25 ( 1.02a
6.56 ( 0.76
361.38 ( 19.37
0.000073
3362
B
C
D
0.87
<0.36c
SI: hMAO-B selectivity index ) IC50
/IC50 (hMAO-B). Each IC50 value is the mean ( S.E.M. from five experiments. * Inactive at 1mM (highest
(hMAO-A)
concentration tested). ** Inactive at 20 µM (highest concentration tested). *** Inactive at 50 µM (highest concentration tested). A ) clorgyline; B )R-
(-)-deprenyl; C ) iproniazid; D ) moclobemide. Level of statistical significance: a P < 0.01 versus the corresponding IC50 values obtained against hMAO-
B, as determined by ANOVA/Dunnett’s. b Values obtained under the assumption that the corresponding IC50 against MAO-A is the highest concentration
tested (20 µM or 50 µM). c Value obtained under the assumption that the corresponding IC50 against MAO-B is the highest concentration tested (1 mM).
Scheme 1a
a Reagents: (i) 3,3-dimethylallyl bromide, anhydrous K2CO3, acetone, rt; (ii) benzyl chloride, KI, Na2CO3, dry ethanol, refluxed; (iii) 3,4-dihydro-R-
pyran, pyridinium p-toluenesulfonate, methylene chloride, rt; (iv) benzaldehyde, barium hydroxide octahydrate, methanol, 40° C; (v) p-toluenesulfonic acid,
methanol, rt.
5m (chosen for docking experiments; see below) as shown by
the lack of enzyme activity restoration after repeated washing.
Similar results were obtained for R-(-)-deprenyl (Table 2).
With the aim of analyzing representatively the 5a, 5i, and
5m binding modes, the most stable configurations of all
complexes were graphically inspected (Figure 3).

Chalcones: A Valid Scaffold for MAO Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2821
Table 2. Reversibility and Irreversibility of hMAO-B Inhibition of
Derivatives 5a, 5m, and 5ia
recorded on a Bruker AMX (300 MHz) using tetramethylsilane
(TMS) as internal standard (chemical shifts in δ values, J in Hz).
Elemental analyses for C, H, and N were determined with a Perkin-
Elmer 240 B microanalyzer, and the analytical results were g95%
purity for all compounds.
Synthesis of 4′-Isoprenyloxy Acetophenone 2. 3,3-Dimethyl-
allyl bromide (0.04 mol) was added dropwise to a mixture of 2,4-
hydroxy acetophenone (0.03 mol) and anhydrous K2CO3 (0.04 mol)
in acetone (100 mL). The mixture was stirred for 3 h at room
temperature and then filtered. Removal of acetone from the filtrate
left a residue, which was crystallized from petroleum ether to give
a solid compound; mp 46-47 °C.
Synthesis of 4′-Benzyloxy Acetophenone 3. 4-Hydroxy ac-
etophenone (0.022 mol), potassium iodide (300 mg), and sodium
carbonate (0.022 mol) were stirred in dry ethanol (80 mL), after
which benzyl chloride (0.022 mol) in dry ethanol (20 mL) was
added dropwise. The reaction mixture was stirred and refluxed for
4 h. The reaction mixture was poured onto ice and the solid obtained
was filtered, washed with water, and dried. The yielded crude
residue 4′-benzyloxy acetophenone 3, which was crystallized from
ethanol; mp 82-83 °C.
Synthesis of 4′-Pyranosyl-2′-hydroxy Acetophenone 4. 2,4-
Dihydroxyacetophenone (0.025 mol) and pyridinium p-toluene-
sulfonate (0.0006 mol) were stirred and reacted for 0.5 h in
dichloromethane (80 mL), after which 3,4-dihydro-R-pyran(0.14
mol), dissolved in 20 mL of dichloromethane, was added dropwise.
The reaction mixture was stirred at room temperature for 4 h and
then was washed twice with water, dried, and evaporated in vacuo.
The yielded crude residue, 4′-pyranosyl-2′-hydroxy acetophenone,
was crystallized from petroleum ether to give a solid compound;
mp 46-47 °C.
Synthesis of 4′-Isoprenyloxy Chalcones 5a-e. A solution of
isoprenyloxy acetophenone 2 (0.01 mol) and the suitable benzal-
dehyde (0.01 mol) dissolved in ethanol was treated with barium
hydroxide (0.01 mol). The solution was stirred for 24 h at 30 °C.
After removal of ethanol, water was added to the residue and the
solution was adjusted to pH 2 with diluted HCl and then extracted
with chloroform. The organic layer was washed with water and
then dried over anhydrous sodium sulfate. The removal of the
chloroform left a solid, which was purified by crystallization from
ethanol.
Synthesis of 4′-Benzyloxy Chalcones 5f. A solution of 4′-
benzyloxy acetophenone 3 (0.01 mol) and the suitable benzaldehyde
(0.01 mol) dissolved in ethanol was treated with barium hydroxide
(0.01 mol). The solution was stirred for 24 h at 30 °C. After removal
of ethanol, water was added to the residue and the solution was
adjusted to pH 2 with diluted HCl and then extracted with
chloroform. The organic layer was washed with water and then
dried over anhydrous sodium sulfate. The removal of the chloroform
left a solid, which was purified by crystallization from ethanol.
Synthesis of Chalcones 5g-k and 5n-p. A solution of
acetophenone (0.025 mol), benzaldehyde (0.025 mol), and barium
hydroxide octahydrate (0.025 mol) was dissolved in MeOH (100
mL). The reaction mixture was stirred for 12 h at 40 °C and then
evaporated in vacuo. Water (100 mL) was added and the mixture
was neutralized with HCl 2 M and extracted with EtOAc. The
organic layer was separated, washed with water, and dried. The
removal of the chloroform left a solid, which was purified by
crystallization from a suitable solvent.
% hMAO-B inhibition
compd
before washing
after repeated washing
5a (20 µM)
54.32 ( 2.21
55.12 ( 3.67
59.13 ( 3.17
50.78 ( 2.15
58.51 ( 2.98
61.38 ( 4.26
60.26 ( 3.23
51.75 ( 2.32
5m (5 nM)
5i (5 nM)
R-(-)-deprenyl (20 nM)
a Each value is the mean ( SEM from five experiments (n ) 5).
Chalcone derivatives 5a, 5i, and 5m, were selected on the
basis of their experimentally demonstrated activity. In particular,
5a was chosen because it was the least active compound, 5i
because it proved to be the most potent and selective, and 5m
because it was the only compound that showed affinity for both
enzyme isoforms. Consequently, the molecular recognition of
5a and 5i was evaluated only with respect to hMAO-B isoform,
while 5m was analyzed against both isoenzymes.
The new Protein Data Bank (PDB)33 high resolution crystal-
lographic structures were considered as receptor models: 2Z5X34
and 2BK335 for hMAO-A and hMAO-B, respectively.
The computational approach consisted of a preliminary
conformational search of the ligands, followed by the flexible
docking of global minimum energy conformers to the targets.
In agreement with the experimental data, all ligands showed
productive recognition of the target. The hydroxy-phenyl ring
was always placed toward the FAD cofactor. Its positioning, in
the hMAOs active site, was strictly related to the substituents.
The 5a prenyloxy moiety hindrance prevented a deeper recogni-
tion of the hMAO-B binding cleft, while the smaller methoxyl
and hydroxyl groups, shown by 5i and 5m, allowed a fruitful
interaction with the catalysis involved FAD and tyrosine amino
acidic residues. In all cases, hydrogen bonds stabilized the
recognition of the ligands. Such an observation was remarkable
for the 5m·hMAO-B complex, where two hydrogen bonds, with
FAD and Tyr435, respectively, contributed to explain the greater
activity of this inhibitor. The number of interacting residues
was quite similar in all complexes (Table 3) and, on analyzing
the different activities and selectivities, we attributed a pivotal
role to Tyr326 in the hMAOs binding modes.
Because of its prenyloxy group, the less potent inhibitor 5a
was placed far from Tyr326 in hMAO-B simulations. The 5i
compound recognized Tyr326 well, while the most active
inhibitor, 5m, showed an H-π interaction through its chlo-
rophenyl ring and two hydrogen bonds with other relevant
residues (Figure 3). In hMAO-A, 5m compound lost the
contribution of Tyr326, which is altered in Ile335, and also that
of other important amino acidic residues (Table 3). The resulting
effect implied an experimental inhibition and a theoretical
affinity that was quite similar to 5a in hMAO-B. This scenario
could be considered an explanation of the different behavior
shown by 5a, 5i, and 5m with respect to hMAOs inhibition.
Conclusion
Synthesis of 2′,4′-Dihydroxy Chalcones 5l-m. A solution of
4′-pyranosyl-2′-hydroxy acetophenone (0.01 mol) and the suitable
benzaldehyde (0.01 mol) dissolved in ethanol was treated with
barium hydroxide (0.01 mol). The solution was stirred for 24 h at
30 °C. After removal of ethanol, water was added to the residue
and the solution was adjusted to pH 2 with diluted HCl and then
extracted with chloroform. The organic layer was washed with water
and then dried over anhydrous sodium sulfate. The crude product
(0.025 mol) obtained after removal of the chloroform was dissolved
in methanol with p-toluenesulfonic acid (0.025 mol). The reaction
mixture was stirred for 4 h at room temperature and then evaporated
in vacuo. The mixture was diluted with water (100 mL), neutralized
In the present work, we have focused our attention onto the
inhibitory activity of chalcones on hMAOs and demonstrate that
they are more active and selective against the B-isoform; for
these reasons, we can indicate chalcones as a useful scaffolds
for hMAOs inhibitors and we plan to extend and report
structure-activity relationship studies, including other substit-
uents, in a further communication.
Experimental Section
Chemistry. Melting points are uncorrected and were determined
1
on a Reichert Kofler thermopan apparatus. H NMR spectra were

2822 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Chimenti et al.
Figure 3. Most stable configuration of 5a, 5i, and 5m into the hMAO-B (a-c) and 5m into the hMAO-A (d) active sites. Hydrogen atoms are
hidden for clarity, interacting residues are displayed in wireframe, ligand in sticks, FAD in sticks green carbon colored, and the rest of the enzymes
are depicted in transparent green (hMAO-B) or yellow (hMAO-A) cartoon.
Table 3. Relevant Interacting Residues List for 5a, 5i, and 5m hMAOs
reaction velocity in our experimental conditions, i.e., to oxidize
(in the control group) the same concentration of substrate, were
used: 165 pmol of p-tyramine/min (hMAO-A, 1.1 µg protein;
specific activity, 150 nmol of p-tyramine oxidized to p-hydrox-
yphenylacetaldehyde/min/mg protein; hMAO-B, 7.5 µg protein;
specific activity, 22 nmol of p-tyramine transformed/min/mg
protein) were incubated for 15 min at 37 °C in a flat black-bottom
96-well microtest plate (BD Biosciences, Franklin Lakes, NJ) placed
in a dark fluorimeter chamber. After this incubation period, the
reaction was started by adding (final concentrations) 200 µM
Amplex Red reagent, 1 U/mL horseradish peroxidase, and 1 mM
p-tyramine. The production of H2O2 and, consequently, of resorufin,
was quantified at 37 °C in a multidetection microplate fluorescence
reader (FLX800, Bio-Tek Instruments, Inc., Winooski, VT) based
on the fluorescence generated (excitation, 545 nm; emission, 590
nm) over a 15 min period, in which the fluorescence increased
linearly.
Control experiments were carried out simultaneously by replacing
the test drugs (new compounds and reference inhibitors) with
appropriate dilutions of the vehicles. In addition, the potential ability
of the test drugs to modify the fluorescence generated in the reaction
mixture due to nonenzymatic inhibition (e.g., for directly reacting
with Amplex Red reagent) was determined by adding these drugs
to solutions containing only the Amplex Red reagent in a sodium
phosphate buffer.
Recognitiona
corresponding
residues
corresponding
residues
ligands
ligands
hMAO-A hMAO-B 5a 5i 5m hMAO-A hMAO-B 5a 5i 5m
Tyr69
Tyr60
b
ab
a
b
b
b
ab
ab
a
ab
ab
ab
ab
Leu97
Phe112 Phe103
Trp128 Trp119
Leu176 Leu167
Leu88
a
b
Ala111
Pro113
Phe173
Phe177
Pro102
Pro104
Leu164
Phe168
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
ab
Ile180
Ile207
Ser209
Leu171
Ile198
Ser200
Asn181 Cys172
Phe208
Val210
Gln215
Ile325
Leu337 Leu328
Phe352
Tyr444
Ile199
Thr201
Gln206
Ile316
a
a
Gly214 Gly205
Cys323 Thr314
b
b
b
b
b
b
b
b
b
b
b
Ile335
Tyr326
b+ ab
Met350 Met341
a
ab
a+b+
Phe343
Tyr435 b+
Tyr407
Tyr398
FAD
b
b
b
b
ab+ FAD
a a: hMAO-A interaction; b: hMAO-B interaction; +: hydrogen bond.
with 5% NaHCO3 (50 mL), and extracted with EtOAc. The organic
layer was separated, washed with water, dried, and evaporated in
vacuo. The crude chalcones obtained were purified by crystallization
from ethanol.
Determination of hMAO Isoform Activity. The effects of the
test compounds on hMAO isoform enzymatic activity were evalu-
ated by a fluorimetric method following the experimental protocol
previously described by us.32
Briefly, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4)
containing the test drugs (new compounds or reference inhibitors)
in various concentrations and adequate amounts of recombinant
hMAO-A or hMAO-B required and adjusted to obtain the same
To determine the kinetic parameters of hMAO-A and hMAO-B
(Km and Vmax), the corresponding enzymatic activity of both
isoforms was evaluated (under the experimental conditions de-
scribed above) in the presence of a number (a wide range) of
p-tyramine concentrations.
The specific fluorescence emission (used to obtain the final
results) was calculated after subtraction of the background activity,

Chalcones: A Valid Scaffold for MAO Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2823
Table 4. Comparison between Experimental pIC50 and Theoretical
Interaction Energy Values for Compounds 5a, 5i, and 5ma
and Conseller´ıa de Innovacio´n e Industria de la Xunta de Galicia
(Spain; INCITE07PXI203039ES, INCITE08E1R203054ES, and
08CSA019203PR). Francisco Orallo is especially grateful to the
Conseller´ıa de Educacio´n y Ordenacio´n Universitaria de la
Xunta de Galicia (Spain) for financial support aimed to
intensifying his research activity and reducing his teaching
during the 2007-2008 academic year [Programa de promocio´n
de intensificacio´n de la actividad investigadora en el sistema
Universitario de Galicia (SUG)]. We also acknowledge Anton
Gerada, a professional translator, Fellow of the Institute of
Translation and Interpreting of London and Member of AIIC
(Association Internationale des Interpreˆtes de Confe´rences,
Geneva), for revision of the manuscript.
pIC50
Glide ECvdW
compd
hMAO-A
hMAO-B
hMAO-A
hMAO-B
5a
5i
5m
4.73
8.35
8.30
-38.48
-44.66
-43.25
5.31
-39.52
a ECvdW is reported in kcal/mol.
which was determined from vials containing all components except
the hMAO isoforms, which were replaced by a sodium phosphate
buffer solution.
Reversibility and Irreversibility Experiments. To evaluate
whether some of the tested compounds (5a, 5i, and 5m) are
reversible or irreversible hMAO-B inhibitors, an effective centrifu-
gation-ultrafiltration method (so-called repeated washing) was
used.36
Supporting Information Available: Analytical and spectral data
for new compounds 2-4 and 5a-p and a few details on
pharmacological studies. This material is available free of charge
via the Internet at http://pubs.acs.org.
Briefly, adequate amounts of the recombinant hMAO-B were
incubated together with a single concentration (see Table 2) of the
test drugs or the reference inhibitor R-(-)-deprenyl in a sodium
phosphate buffer (0.05 M, pH 7.4) for 15 min at 37 °C.
After this incubation period, an aliquot of this incubated was
stored at 4 °C and used for subsequent measurement of hMAO-B
activity under the experimental conditions indicated above (see the
section Determination of MAO Activity). The remaining incubated
sample (300 µL) was placed in a Ultrafree-0.5 centrifugal tube
(Millipore, Billerica, MA) with a 30 kDa Biomax membrane in
the middle of the tube and centrifuged (9000g, 20 min, 4 °C) in a
centrifuge (J2-MI, Beckman Instruments, Inc., Palo Alto, CA). The
enzyme retained in the 30 kDa membrane was resuspended in
sodium phosphate buffer at 4 °C and centrifuged again (under the
same experimental conditions described above) two successive
times. After the third centrifugation, the enzyme retained in the
membrane was resuspended in sodium phosphate buffer (300 µL)
and an aliquot of this suspension was used for subsequent hMAO-B
activity determination.
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