A new series of flavones, thioflavones, and flavanones as selective monoamine oxidase-B inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.12.029

A new series of synthetic flavones, thioflavones, and flavanones has been synthesized and evaluated as potential inhibitors of monoamine oxidase isoforms (MAO-A and -B). The most active series is the flavanone one with higher selective inhibitory activity against MAO-B. Some of these flavanones (mainly the most effective) have been separated and tested as single enantiomers. In order to investigate the MAOs recognition of the most active and selective compounds, a molecular modeling study has been performed using available Protein Data Bank (PDB) structures as receptor models for docking experiments.

Full text of DOI:10.1016/j.bmc.2009.12.029

Bioorganic & Medicinal Chemistry 18 (2010) 1273–1279
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A new series of flavones, thioflavones, and flavanones as selective
monoamine oxidase-B inhibitors
a
a
a
Franco Chimenti a, Rossella Fioravanti a, , Adriana Bolasco , Paola Chimenti , Daniela Secci ,
Francesca Rossi a, Matilde Yáñez b, Francisco Orallo b, Francesco Ortuso c, Stefano Alcaro c,
Roberto Cirilli d, Rosella Ferretti d, M. Luisa Sanna d
*
a Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma ‘‘La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy
b Departamento de Farmacología and Instituto de Farmacia Industrial, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Universitario Sur,
E-15782 Santiago de Compostela (La Coruña), Spain
c Dipartimento di Scienze Farmacobiologiche, Università di Catanzaro ‘‘Magna Graecia”, ‘‘Complesso Ninì Barbieri”, 88021 Roccelletta di Borgia (CZ), Italy
d Istituto Superiore di Sanità, Dipartimento del Farmaco, Viale Regina Elena 299, I-00161 Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of synthetic flavones, thioflavones, and flavanones has been synthesized and evaluated as
potential inhibitors of monoamine oxidase isoforms (MAO-A and -B). The most active series is the flava-
none one with higher selective inhibitory activity against MAO-B. Some of these flavanones (mainly the
most effective) have been separated and tested as single enantiomers. In order to investigate the MAOs
recognition of the most active and selective compounds, a molecular modeling study has been performed
using available Protein Data Bank (PDB) structures as receptor models for docking experiments.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 2 November 2009
Revised 2 December 2009
Accepted 8 December 2009
Available online 4 January 2010
Keywords:
Flavones
MAO inhibitors
Molecular modeling
Enantiomeric separation
1. Introduction
Further in our recent paper16 we have reported the activity of
the flavone quercetin, which showed a selective MAO-A inhibitory
Flavonoids are an extensive group of polyphenolic compounds
present in plants, regularly consumed foods (e.g. vegetables and
fruits), olive oil, and beverages like tea and wine.1–3 They are usu-
ally subdivided, according to their chemical structure, into several
subclasses including anthocyanidins, flavanones, flavones, flavo-
nols, flavanonols (or dihydroflavonols), chalcones, isoflavones,
and flavanols (flavan-3-ols) (also called catechins). Besides their
physiological role in plants, these different flavonoids have been
reported to possess a wide range of biological activities, including
modulatory properties of several enzymes [e.g. activation of sirtu-
ins,4 inhibition of monoamine oxidase (MAO),5 and a number of
other biological activities such as anxiolytic,6 anti-inflammatory,3
antiviral,7 antiprotozoal,8 antioxidant,3 cardiovascular,3,9 and anti
carcinogenic properties.10–12
activity in the nanomolar range (IC50 = 10 nM).
Luteolin = R1= R2= R3= H, R4= OH.
R4
OR3
(MAO-A IC50 4.9 µM; MAO-B IC50
HO
R2
O
O
59.7 µM)5
Quercetin = R1=R4= OH, R2=R3=H
(MAO-A IC50 2.8 µM; MAO-B IC50
90.0 µM)5
R1
OH
Apigenin = R1=R2= R3=R4=H.
(MAO-A IC50 1.7 µM; MAO-B IC50 12.8 µM)5
Jaceosidine = R1=R3=H, R2=R4=OCH3.
(MAO IC50 19.0 µM)14,15
Concerning the effects of flavonoids on the enzymatic activity of
MAO (EC 1.4.3.4), several studies5,13–15 have previously described
the MAOIs (MAO Inhibitors) properties of jaceosidine, eupafolin,
luteolin, quercetin, and apigenin (Chart 1).
Eupafolin = R1= OH, R2= OCH3, R3= R4= H.
(MAO IC50 25.0 µM)14,15
R1
OH
Taxifolin = R1= OH
(MAO IC50 154.7 µM)5
HO
O
Aromadendrin = R1= H
OH
(MAO IC50 153.1 µM)5
OH
O
* Corresponding author. Tel.: +39 6 49693259; fax: +39 6 4462731.
E-mail address: rossella.fioravanti@uniroma1.it (R. Fioravanti).
Chart 1. Natural flavonoids having inhibitory activity against MAO.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.029

1274
F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 1273–1279
MAO is a flavoprotein located in the outer membrane of the
hydrogen peroxide (H2O2) from p-tyramine (a common substrate
for both hMAO-A and -B), using the Amplex Red MAO assay kit
(Molecular Probes, Inc., Eugene, Oregon, USA) and microsomal
MAO isoforms prepared from insect cells (BTI-TN-5B1-4) infected
with recombinant baculovirus containing cDNA inserts for
hMAO-A or -B (Sigma–Aldrich Química S.A., Alcobendas, Spain).
The production of H2O2 catalyzed by MAO isoforms, can be de-
tected using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red re-
agent), a non-fluorescent compound and highly sensitive probe
that reacts with H2O2 in the presence of horseradish peroxidase
to produce a fluorescent product, resorufin. In this study hMAO
activity was evaluated using the above method following the gen-
eral previously procedure described by.28
The test drugs (new compounds and reference inhibitors) them-
selves were unable to react directly with the Amplex Red reagent,
which indicates that these drugs do not interfere with the mea-
surements. On the other hand, in our experiments and under our
experimental conditions, the control activity of hMAO-A and -B
(using p-tyramine as a common substrate for both isoforms) was
165 2 pmol of p-tyramine oxidized to p-hydroxyphenylacetalde-
hyde/min (n = 20).
mitochondria that contains a covalently bound flavin adenine
dinucleotide (FAD) as a coenzyme and that has considerable phys-
iological and pharmacological interest due to its central role in the
metabolism of monoamine neurotransmitters. MAO exists in two
isoforms, MAO-A and MAO-B, which share approximately 70%
sequence identity on the amino acid levels and differ in their
substrate specificity, susceptibility to specific inhibitors, and
three-dimensional structure.17,18 As indicated above, these MAO
isoforms catalyze the biotransformation by oxidation of endoge-
nous neurotransmitter monoamines and also the metabolization
of various exogenous primary, secondary, and tertiary amines in
the central nervous system as well as in peripheral tissues.19
MAO-A preferentially deaminates 5-hydroxytryptamine (5-HT),
norepinephrine, and epinephrine and is irreversibly inhibited by
low concentrations of clorgyline whereas MAO-B preferentially
deaminates dopamine, b-phenylethylamine, and benzylamine
and is irreversibly inhibited by R-(ꢀ)-deprenyl.
It is well known that major depression is related to the deficit of
monoamines (basically norepinephrine and 5-HT) at critical syn-
apses in the central nervous system20 whereas Parkinson’s disease
is mainly due to a deficit of dopamine.21
Most tested flavonoids concentration-dependently and selec-
tively inhibited the enzymatic control activity of MAO-B (see Ta-
bles 1–3).
All these findings support the clinical importance of MAO inhib-
itors in the treatment of several neurological and psychiatric disor-
ders. Thus, selective MAO-A inhibitors are used as anti-depressant
and anti-anxiety drugs whereas selective MAO-B inhibitors can be
mainly used to treat Parkinson’s disease either alone or in combi-
4. Results and discussion
nation with L
-DOPA.22,23
All synthesized compounds 2a–p, 3a–p, and 4a–p were assayed
for hMAO-A and -B inhibitory activity. By analyzing the data re-
ported in Tables 1–3 it can be seen that the most active and
hMAO-B selective compounds belong to flavanones 2a–p deriva-
tives and among them the best results (in the low micro molar
range) were observed with compounds 2f, 2g, 2h, 2j, 2k, and 2n,
substituted with the fluorine atom and the methyl or the methoxy
group. It seems that the substitution pattern of these groups do not
Bearing in mind the above considerations, we consider that nat-
ural products as flavonoids (e.g. flavones and flavanones) are
promising lead compounds for developing effective agents to com-
bat neurological diseases. Therefore, as part of our continuous
search for potential selective MAO inhibitors, we have synthesized
flavones, thioflavones, and flavanones to investigate their inhibi-
tory activity on human MAO isoforms (hMAO-A and -B). Due to
the presence of a stereogenic carbon on the flavanone ring (flava-
nones exist as (R)- and (S)-enantiomers), we performed on the
most active compounds the enantioseparation by chiral HPLC
and tested the single enantiomers. Furthermore, the most promis-
ing compound binding mode to both hMAO-A and B catalytic clefts
has been investigated by means of molecular modeling carried out
using PDB crystallographic structures as receptor models for dock-
ing simulations.
OHC
+
R
OH
CH3
R1
O
i
2. Chemistry
R1
R1
The syntheses of the flavones and flavanones derivatives were
carried out using chalcones as starting products24,25; (Scheme 1)
whereas the thioflavones derivatives were prepared from the pre-
viously synthesized flavones. The synthesis of chalcone derivatives
was performed according to procedures previously described.26
Briefly, we used the aldolic condensation with Ba(OH)2ꢁ8H2O as
base. In this process, 20-hydroxyacetophenone reacted with an
aldehyde. Chalcones 1a–p and sodium acetate were heated in
refluxing ethanol to obtain derivatives 2a–p. To obtain derivatives
3a–p and 4a–p, suitable chalcones 1a–p were dissolved in DMSO
(dimethyl sulfoxide) and iodine was added. After oxidative cycliza-
tion of chalcones, the derivatives 3a–p were treated with Lawes-
son’s reagent,27 in refluxing benzene for 2 h to obtain 4a–p
derivatives.
R
O
ii
R
OH
O
2 a-p
O
1 a-p
iii
R1
R1
R
O
S
R
O
iv
O
3 a-p
4 a-p
3. Biochemistry
Scheme 1. Reagents and conditions: (i) Ba(OH)2ꢁ8H2O, ethanol, 30 °C; (ii)
CH3COONa, ethanol, 24–48 h, reflux; (iii) I2/DMSO, reflux; (iv) Lawesson’s reagent,
benzene, reflux, 2 h.
The potential effects of the test drugs on hMAO activity were
investigated by measuring their effects on the production of

F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 1273–1279
1275
Table 1
Table 2
Structures and inhibitory activity of derivatives 2a–p
Structures and inhibitory activity of derivatives 3a–p
R1
R1
O
R
O
R
O
O
SI–
Compd
R
R1
IC50
lM hMAO-A
IC50
l
M hMAO-B
SI#
Compd
R
R1
IC50
lM hMAO-A
IC50
lM hMAO-B
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
***
***
**
**
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
H
H
H
H
F
F
F
F
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
H
F
CH3
OCH3
H
2.87 0.09
4.41 0.13
2.07 0.11
2.20 0.10
3.77 0.16
0.67 0.04
0.16 0.006
0.17 0.005
3.62 0.10
0.13 0007
0.50 0.02
3.35 0.07
1.47 0.03
0.17 0.009
0.55 0.02
7.18 0.06
>35b
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
H
H
H
H
F
F
F
F
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
H
F
CH3
OCH3
H
>23b
***
2.38 0.07
27.87 1.04
>42b
>3.6b
>7.7b
>48b
>45b
***
***
**
>27b
12.94 0.47
***
F
>149b
>625b
>588b
>28b
F
CH3
OCH3
H
4.33 0.39
>23b
>75b
**
CH3
OCH3
H
***
**
1.34 0.02
**
**
***
***
***
***
F
>769b
>200b
>30b
F
***
***
**
CH3
OCH3
H
CH3
OCH3
H
>68b
>588b
>182b
>14b
F
5.14 0.18
11.34 0.26
49.72 1.83
>19b
>8.8b
>2b
**
F
**
CH3
OCH3
CH3
OCH3
**
M hMAO-B 61.35 1.13; SI#
hMAO-B
Clorgyline (IC50
0.000065); R-(ꢀ)-deprenyl (IC50
0.019 0.0009; SI# 3.539); Iproniazid (IC50
hMAO-B 7.54 0.36; SI# 0.87); Moclobemide (IC50
M hMAO-B *; SI# <0.36c); –SI: hMAO-B selectivity index = IC50 (hMAO-A)/IC50 (hMAO-
l
M hMAO-A 0.004 0.0003a; IC50
hMAO-A 67.25 1.02a; IC50
hMAO-A 6.56 0.76; IC50
M hMAO-A 361.38 19.37; IC50
l
M hMAO-B 61.35 1.13; SI#
Clorgyline (IC50
l
M hMAO-A 0.004 0.0003a; IC50
hMAO-A 67.25 1.02a; IC50
hMAO-A 6.56 0.76; IC50
M hMAO-A 361.38 19.37; IC50
M hMAO-B*; SI# <0.36c); –SI: hMAO-B selectivity index = IC50 (hMAO-A)/IC50 (hMAO-B)
l
lM
lM
hMAO-B
0.000065); R-(ꢀ)-deprenyl (IC50
0.019 0.0009; SI# 3.539); iproniazid (IC50
hMAO-B 7.54 0.36; SI# 0.87); Moclobemide (IC50
lM
lM
l
M
l
lM
l
M
l
lM
l
l
.
B). Each IC50 value is the mean S.E.M. from five experiments.
Each IC50 value is the mean S.E.M. from five experiments. Level of statistical sig-
nificance: aP < 0.01 versus the corresponding IC50 values obtained against hMAO-B,
as determined by ANOVA/Dunnett’s. bValues obtained under the assumption that
the corresponding IC50 against hMAO-A is the highest concentration tested
Level of statistical significance: aP < 0.01 versus the corresponding IC50 values
obtained against hMAO-B, as determined by ANOVA/Dunnett’s. bValues obtained
under the assumption that the corresponding IC50 against hMAO-A is the highest
concentration tested (100 l
M). cValue obtained under the assumption that the
(100 l
M). cValue obtained under the assumption that the corresponding IC50
corresponding IC50 against hMAO-B is the highest concentration tested (1 mM).
against hMAO-B is the highest concentration tested (1 mM).
*Inactive at 1 mM (highest concentration tested).
Inactive at 100 lM (highest concentration tested).
*Inactive at 1 mM (highest concentration tested).
**
**
Inactive at 100
l
M (highest concentration tested).
***
100
lM inhibits by approximately 40%.
affect the activity. The introduction of a double bond in the
flavones 3a–p and of the sulfur in the thioflavones 4a–p leads to
a relevant decrease of the inhibitory activity although remain a
hMAO-B selectivity. The most active compounds 2f, 2g, 2h, 2j,
2k, and 2n, first assayed as racemates, were resolved by enantiose-
lective HPLC and tested again as single enantiomers. The direct
HPLC enantiomeric separation was accomplished on the immobi-
lised polysaccharide-based Chiralpak IA chiral stationary phases
(CSP) using pure methanol as a mobile phase. The absolute config-
uration of the enantiopure forms isolated at semipreparative scale
was empirically assigned by comparing the circular dichroism (CD)
spectra of the enantiomers of flavanone29 with those of the struc-
turally analogues 2f, 2g, 2h, 2j, 2k, and 2n (see Supplementary
data).
In the reversibility and irreversibility tests, hMAO-B inhibition
was irreversible in presence of the compounds selected for docking
experiments [2j, (R)-2j and (S)-2j] (see below) as shown by the lack
enzyme activity restoration after repeated washing. Similar results
were obtained for R-(ꢀ)-deprenyl (Table 4). However, significant
recovery of hMAO-A activity was observed after repeated washing
of moclobemide, indicating that this drug is a reversible inhibitor
of hMAO-A.
structures, after a preliminary treatment, were adopted as targets
(see Experimental section). The molecular modeling investigation,
in qualitative agreement to the experimental data, reported the
capability of both 2j enantiomers to fit within hMAO-A and -B cat-
alytic sites with some differences in terms of affinity. Actually both
isomers recognized preferentially the hMAO-B binding cleft and,
between them, the (S)-2j highlighted most favorable interactions.
In order to analyze the binding modes of 2j enantiomers within
the MAO isoforms, the most stable theoretical complexes were
graphically inspected reporting similar interaction and binding
modes.
Furthermore in Table 6 was reported the average G score values
for the modeled compounds. As displayed inFigure 1 and according
to interaction energy data, the (R)-2j showed a more productive
recognition with respect to the (S)-2j into the hMAO-A. In the
hMAO-A the (R)-2j was able to deeply penetrate into the active
sites highlighting hydrophobic contacts to the FAD. Such an obser-
vation cannot be addressed to the (S)-2j located at a larger distance
from the cofactor. Taking into account the orientation of both
enantiomers into the hMAO-A binding cleft, (S)-2j appeared
slightly translated toward the outer side. So, the most part of inter-
acting residues was shared between the enantiomers but (R)-2j
performed stronger interaction to Tyr407 and Tyr444 with respect
to (S)-2j who, vice versa, was able to hydrophobically contact
Ala111, Ile325, and Leu97.
Compound 2j, that evidenced the best inhibitory activity as
racemate, was the best compound also in the two enantiomeric
forms (Table 5). Because of this result we selected this compound
for the subsequent molecular modeling investigation.
The recognition of both (R)-2j and (S)-2j enantiomers was eval-
uated by means of docking studies with respect to hMAO-A and -B
receptor models. Two new, high resolution, PDB crystallographic
A similar scenario was observed into the hMAO-B case but the
binding mode differences were less remarkable (Fig. 2).
Although (R)-2j bound relatively deeper in the active site cavity,
the (S)-enantiomer also was recognized by the FAD cofactor. The

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F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 1273–1279
Table 3
Table 5
Structures and inhibitory activity of derivatives 4a–p
Inhibitory activity of single enantiomer of the most active flavanones 2
SIa
R1
Comp.
IC50
lM hMAO-A
IC50 lM hMAO-B
(R)(+)2f
(S)(-)2f
**
**
**
**
**
**
**
**
**
**
**
**
0.65 0.03
0.62 0.02
0.15 0.01
0.17 0.01
0.15 0.01
0.15 0.01
0.14 0.01
0.11 0.01
0.53 0.03
0.59 0.03
0.20 0.01
0.16 0.01
>154b
>161b
>667b
>588b
>667b
>667b
>714b
>909b
>189b
>169b
>500b
>625b
O
R
(R)(+)2g
(S)(ꢀ)2g
(R)(+)2h
(S)(ꢀ)2h
(R)(+)2j
(S)(ꢀ)2j
(R)(+)2k
(S)(ꢀ)2k
(R)(+)2n
(S)(ꢀ)2n
S
Compd
R
R1
IC50
l
M hMAO-A
IC50
l
M hMAO-B
SI–
**
***
4a
4b
4c
4d
4e
4f
H
H
H
H
F
F
H
F
**
**
CH3
OCH3
H
6.69 0.13
>15b
**
0.48 0.024
>208b
***
***
**
***
F
6.89 0.19
>15b
>62b
Each IC50 value is the means SEM from five experiments.
a
***
**
SI: hMAO-B selectivity index = IC50 (hMAO-A)/IC50
Values obtained under the assumption that the corresponding IC50 against
MAO-A is the highest concentration tested (100 lM).
.
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
F
F
CH3
OCH3
H
1.61 0.04
(hMAO-B)
b
***
**
***
***
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
**
F
CH3
OCH3
H
6.72 0.08
>15b
**
**
***
**
***
***
**
F
CH3
OCH3
7.61 0.43
3.34 0.16
>13b
>30b
***
**
Table 6
Theoretical affinity of 2j, (R)-2j, and (S)-2j with respect to MAO isoforms
Clorgyline (IC50
l
M hMAO-A 0.004 0.0003a; IC50
hMAO-A 67.25 1.02a; IC50
hMAO-A 6.56 0.76; IC50
M hMAO-A 361.38 19.37; IC50
M hMAO-B*; SI# <0.36c) SI: hMAO-B selectivity index = IC50 (hMAO-A)/IC50
l
M hMAO-B 61.35 1.13; SI#
hMAO-B
Comp
hMAO-A*
hMAO-B*
0.000065); R-(ꢀ)-deprenyl (IC50
0.019 0.0009; SI# 3.539); Iproniazid (IC50
hMAO-B 7.54 0.36; SI# 0.87); Moclobemide (IC50
lM
lM
( )-2j
(R)-2j
(S)-2j
ꢀ6.75
ꢀ6.93
ꢀ6.58
ꢀ7.45
ꢀ7.44
ꢀ7.47
l
M
l
lM
l
.
(hMAO-B)
*
Each IC50 value is the means SEM from five experiments.
Average G score values.
Level of statistical significance: aP < 0.01 versus the corresponding IC50 values
obtained against hMAO-B, as determined by ANOVA/Dunnetts. bValues obtained
´
under the assumption that the corresponding IC50 against hMAO-A is the highest
concentration tested (100 l
M). cValue obtained under the assumption that the
corresponding IC50 against hMAO-B is the highest concentration tested (1 mM).
*Inactive at 1 mM (highest concentration tested).
**
Inactive at 100
l
M (highest concentration tested).
***
100
lM inhibits by approximately 40%.
number of interacting residues was equivalent and a large part of
them was shared. So the slightly advantage of (S)-2j interaction en-
ergy with respect to (R)-2j could be addressed to the better accom-
modation of its fluorophenyl moiety into the lipophilic cage
delimited by Trp119, Phe168, Leu171, Ile199, and Tyr326. The
analysis of the residues involved in the recognition of 2j enantio-
mers in both MAO isoforms revealed that the selectivity was not
addressed to canonical differences between the catalytic gorges
such as Ile199 and Tyr326 (hMAO-B), respectively, versus Phe208
and Ile335 (hMAO-A).
Actually all these crucial amino acids interacted with both 2j
enantiomers as shown in Figures 1 and 2. Since the average G score
values (see Table 6) take into account other effects (solvation, pen-
alty clashes, loss of ligand internal degrees of freedom, etc) the
hMAO-B selectivity can be addressed to those terms. Finally in or-
der to clarify the role of R1 we have compared the hMAO-B
Figure 1. Superimposition of best fully energy minimized hMAO-A poses of (R)-2j
and (S)-2j. The ligand is reported as white and green carbon polytube, respectively.
The interacting residues with (R)-2j and (S)-2j are colored in white and green,
respectively. The FAD cofactor is displayed as a spacefill structure. Non-polar
hydrogen atoms are omitted for clarity.
Table 4
Reversibility and irreversibility of hMAO inhibition of compound 2j and of its separated enantiomers
Compd
% hMAO-A inhibition
After repeated washing
% hMAO-B inhibition
After repeated washing
Before washing
84.75 4.34
Before washing
51.45 2.69
(R)(ꢀ)Da (20 nM)
52.05 2.88
Ma (500
lM)
10.26 0.65b
2j (100 nM)
38.36 3.15
35.61 2.90
45.29 3.92
36.51 2.56
37.23 3.14
42.58 3.37
(R)(+)2j (100 nM)
(S)(ꢀ)2j (100 nM)
a
D, (R)(ꢀ)deprenyl; M, moclobemide. Each value is the means SEM from five experiments (n = 5).
b
´
Level of statistical significance: P < 0.01 versus the corresponding % hMAO-A inhibition before washing, as determined by ANOVA/Dunnetts.

F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 1273–1279
1277
5. Experimental
5.1. General procedure for the synthesis of 20-hydroxy
chalcones 1a–p
A solution of suitable 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 the pH of the reaction mixture was brought
back to 7.0 by the careful addition of HCl 1 N solution. The aqueous
layer was extracted with ethyl acetate, washed with brine, dried
(Na2SO4), and then concentrated in vacuo. The residue was purified
by crystallization from ethanol, to afford chalcones 1a–p.
5.2. General procedure for the synthesis of flavanones 2a–p
Figure 2. Superimposition of best fully energy minimized hMAO-B poses of (R)-2j
and (S)-2j. The ligand is reported as white and green carbon polytube, respectively.
The interacting residues with (R)-2j and (S)-2j are colored in white and green,
respectively. The FAD cofactor is displayed as a spacefill structure. Non-polar
hydrogen atoms are omitted for clarity.
Chalcones 1a–p (0.2 mmol) and sodium acetate (2.0 mmol)
were heated in refluxing ethanol (2 mL) for 24–48 h. The mixture
was then allowed to cool to r.t. and poured into ice water
(10 mL) and extracted with CH2Cl2 (3 ꢂ 10 mL). The combined or-
ganic phase was washed with brine, dried over Na2SO4, and then
concentrated in vacuo. The residue was column chromatographed
on silica gel, eluting with mixture of ethyl acetate/hexane 1:4, to
afford flavanones 2a–p which were crystallized from suitable sol-
vent (see Supplementary data).
interaction energies of 2j, 2i, 2k, and 2l averaging the values of
both enantiomers. The results (not shown) indicated the fluoro-
phenyl as the best moiety with the optimal compromise of lipo-
philicity and steric hindrance. In principle, our results of
irreversibility obtained with the compounds 2j, (R)-2j, and (S)-2j,
do not seem to correlate well with the conformational analysis
made in the docking studies but a number of arguments may ex-
plain, at least in part, this discrepancy. In most cases, the irrevers-
ible inhibitors establish a covalent interaction with the active
center of the enzyme.30
5.3. General procedure for the synthesis of flavones 3a–p
To a solution of chalcones 1a–p (0.015 moL) in 50 ml of DMSO
(dimethyl sulfoxide) were added iodine (0.015 mol). The reaction
mixture was stirred for 30 min at 130 °C. Then the mixture was
treated with sodium thiosulfate (solution 20%) and extracted with
CHCl3 (3 ꢂ 10 mL). The combined organic phase were washed with
brine, dried over Na2SO4, and then concentrated in vacuo. The res-
idue was purified by crystallization from suitable solvent (see Sup-
plementary data).
However, not all irreversible inhibitors form covalent adducts
with their enzyme targets but they may also act by other mecha-
nisms. In fact, some reversible inhibitors bind so tightly to their
target enzyme that they are essentially irreversible. These tight-
binding inhibitors may show kinetics similar to covalent irrevers-
ible inhibitors. In these cases, some of these inhibitors rapidly bind
to the enzyme in a low-affinity enzyme-inhibitor (EI) complex and
this then undergoes a slower rearrangement to a very tightly
bound EI* complex. This kinetic behavior is called slow-binding.
This slow rearrangement after binding often involves a conforma-
tional change as the enzyme ‘‘clamps down” around the inhibitor
molecule.31 In contrast, some irreversible MAO inhibitors (the so-
called suicide inhibitors) act as a substrate for the target enzyme,
which finally generates a new compound that irreversibly inhibits
MAO activity. Therefore, the initial interaction of these inhibitors
with MAO may be different to the interaction obtained after sev-
eral minutes of the enzyme–inhibitor complex formation (see
above). R-(ꢀ)-deprenyl, for example, first of all form a non-
covalent complex with MAO as an initial, reversible step. The sub-
sequent interaction of R-(ꢀ)-deprenyl with MAO leads to a reduc-
tion of the enzyme-bound FAD, and concomitant oxidation of the
inhibitor. This oxidized inhibitor then reacts with FAD at the
N-5-position in a covalent manner.32 The initial non-covalent bind-
ing to MAO has been also described for other MAO inhibitors (e.g.
clorgyline derivatives).33 Finally, it is possible that, in some cases
of irreversible inhibition, a steric hindrance may prevent the
release of the inhibitor from the enzymatic active center (although
its interaction with this binding site is either very weak or
reversible).
5.4. General procedure for the synthesis of thioflavones 4a–p
A 100 mL flask was charged with flavones 3a–p (0.05 mol) and
Lawesson’s reagent (0.025 mol), whereupon the temperature of the
reaction mixture increases to 78–80 °C. After 5 min, 35 mL of ben-
zene was added, the mixture was heated at refluxing for 2 h, then
was cooled to r.t., and the solvent removed under vacuum. The
crude solid was purified by suitable method (see Supplementary
data).
5.5. Determination of hMAO isoform activity
The effects of the test compounds on hMAO isoform enzymatic
activity were evaluated by a fluorimetric method following the
experimental protocol previously described.28
Briefly, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4) con-
taining various concentrations of the test drugs (new compounds
or reference inhibitors) and adequate amounts of recombinant
hMAO-A or -B required and adjusted to obtain in our experimental
conditions the same reaction velocity, i.e., to oxidize (in the control
group) 165 pmol of p-tyramine/min (hMAO-A: 1.1
cific activity: 150 nmol of p-tyramine oxidized to p-hydroxyphe-
nylacetaldehyde/min/mg protein; hMAO-B: 7.5 protein;
lg protein; spe-
l
g
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, USA)
placed in the dark multimode microplate reader chamber. After
this incubation period, the reaction was started by adding (final
Bearing in mind all the above considerations and taking into ac-
count that the docking studies make only a theoretical prediction
of the initial possible interaction inhibitor–enzyme, the results ob-
tained in these docking studies and in the reversibility experiments
may be different.

1278
F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 1273–1279
concentrations) 200
l
M Amplex Red reagent, 1 U/ml horseradish
The mobile phases were filtered and degassed by sonication imme-
diately before using. In analytical enantioseparations, standard
solutions were prepared by dissolving about 1 mg of racemic sam-
peroxidase and 1 mM p-tyramine. The production of H2O2 and,
consequently, of resorufin was quantified at 37 °C in a multimode
microplate reader (Fluostar Optima, BMG Labtech GmbH, Offen-
burg, Germany), based on the fluorescence generated (excitation,
545 nm, emission, 590 nm) over a 15 min period, in which the fluo-
rescence increased linearly.
Control experiments were carried out simultaneously by replac-
ing the test drugs (new compounds and reference inhibitors) with
appropriate dilutions of the vehicles. In addition, the possible
capacity of the above test drugs to modify the fluorescence gener-
ated in the reaction mixture due to non-enzymatic inhibition (e.g.
for directly reacting with Amplex Red reagent) was determined by
adding these drugs to solutions containing only the Amplex Red re-
agent in a sodium phosphate buffer.
ple, into 10 ml of methanol. The injection volume was 10–20 ll. In
a semipreparative enantioseparation 1 mL sample loop was used.
After semipreparative separation, the collected fractions were ana-
lyzed by chiral analytical columns to determine their enantiomeric
excess (ee).
The column hold-up time (t0 = 3.0 min for the 250 ꢂ 4.6 mm i.d.
column) was determined from the elution of an unretained marker
(toluene), using methanol as eluent, delivered at a flow-rate of
1.0 mL/min.
The chromatographic and polarimetric data of the enantiomers
separated at semipreparative scale are summarized in Supplemen-
tary data, Table 4. Specific rotations were measured at 589 nm by a
Perkin-Elmer polarimeter model 241 equipped with a Na lamp. The
volume of the cell was 1 mL and the optical path was 10 cm. The
system was at a temperature of 20 °C by a Neslab RTE 740 cryostat.
The circular dichroism (CD) spectra of the enantiomers of 2f, 2g,
2h, 2j, 2k, and 2n, dissolved in ethanol (concentration about
0.2 mg/mL), in a quartz cell (0.1 cm-path length) at 25 °C, were
measured by using a Jasco Model J-700 spectropolarimeter (see
Supplementarydata, Fig. 1). The spectra are average computed over
three instrumental scans and the intensities are presented in terms
of ellipticity values (mdeg).
The specific fluorescence emission (used to obtain the final re-
sults) was calculated after subtraction of the background activity,
which was determined from vials containing all components ex-
cept the MAO isoforms, which were replaced by a sodium phos-
phate buffer solution.
5.6. Reversibility and irreversibility experiments
To evaluate whether some of the tested compounds, 2j, (R)-2j,
and (S)-2j, are reversible or irreversible hMAO-B inhibitors, an
effective centrifugation–ultrafiltration method (so-called repeated
washing) previously described was used.26
7. Molecular modeling
Briefly, adequate amounts of the recombinant hMAO-B were
incubated together with a single concentration (see Table 4) of
the test drugs or the reference inhibitor R-(ꢀ)-deprenyl in a so-
dium phosphate buffer (0.05 M, pH 7.4) for 15 min at 37 °C.
Then, an aliquot of this incubated was stored at 4 °C and used
for subsequent measurement of hMAO-B activity under the exper-
imental conditions indicated above (see the subsection determina-
The Protein Data Bank34 (PDB) crystallographic structures
2Z5X35 and 2BK336 were considered as receptor model of hMAO-
A and -B, respectively.
Both (R) and (S) enantiomers of 2j were built by means of the
Maestro GUI37 and energy minimized using the OPLS-AA38 force
field as implemented in Macromodel ver. 7.2.39 Water solvent ef-
fects were taken into account using the implicit solvation model
GB/SA.40 The optimized structures were submitted to docking sim-
ulations with respect to hMAO-A and -B PDB crystallographic
structures. Both receptor models required graphical manipulation:
the co-crystallized ligands, harmine and farnesol, respectively, for
2Z5X and 2BK3, were removed, FAD double bonds were corrected,
and hydrogen atoms were added onto both proteins and cofactors.
According to the Glide41 methodology, a regular box, of about
110,000 Å3, centered onto the cofactor N5 atom, was considered
as the enzyme active site for both hMAO-A and -B models. In order
to take into account the induced fit phenomena, 2j enantiomers
were docked using the Glide ‘‘flexible” algorithm. The binding
affinity has been evaluated using the G Score average value com-
puted from the ten best generated poses for each enantiomer into
the hMAO-A and –B, respectively (see Supplementary data). The
most stable complexes were submitted to energy minimization
using the same force field and aqueous environment previously re-
ported. The resulting optimized structures were considered for the
binding modes graphical analysis. PyMol ver. 0.9842 was used to
create Figs. 1 and 2.
tion of MAO activity). The remaining incubated sample (300 lL)
was placed in a Ultrafree-0.5 centrifugal tube (Millipore, Billerica,
USA) with a 30 kDa Biomax membrane in the middle of the tube
and centrifuged (9000g, 20 min, 4 °C) in a centrifuge (J2-MI, Beck-
man Instruments, Inc., Palo Alto, California, USA). The enzyme re-
tained 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 mem-
brane was resuspended in sodium phosphate buffer (300 lL) and
an aliquot of this suspension was used for subsequent hMAO-B
activity determination.
Similar studies were carried out on hMAO-A activity in presence
of the reference inhibitor moclobemide under the experimental
conditions described above.
Control experiments were performed simultaneously (to define
100% hMAO activity) by replacing the test drugs with appropriate
dilutions of the vehicles. The corresponding values of percent (%)
hMAO inhibition were separately calculated for samples with and
without repeated washing.
The results obtained in this study indicate that flavonoids may
have interesting therapeutic potential as original chemical models
(templates) for the design and subsequent development of new
drugs (selective and efficient MAO-B inhibitors) useful for improv-
ing the pharmacological treatment of neurodegenerative diseases
(e.g. Parkinson’s disease).
6. Enantioseparation studies
HPLC enantioseparations were performed by using stainless-
steel Chiralpak IA (250 ꢂ 4.6 mm I.D. and 250 ꢂ 10 mm I.D.)
(Daicel, Chemical Industries, Tokyo, Japan) columns. HPLC-grade
solvents were supplied by Carlo Erba (Milan, Italy). HPLC apparatus
consisted in a Perkin–Elmer (Norwalk, CT, USA) 200 lc pump
equipped with a Rheodyne (Cotati, CA, USA) injector, a HPLC Dio-
nex (CA, USA) Model TCC-100 oven and a Jasco (Jasco, Ishikawa-
cho, Hachioji City, Tokyo, Japan) Model 2095 Plus UV/CD detector.
Acknowledgments
This work was supported by Grants from MURST (Italy). Minis-
terio de Sanidad y Consumo (Spain; FISS PI061537) and Consellería

F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 1273–1279
1279
19. Kopin, I. J. Pharmacol. Rev. 1985, 37, 338.
20. Schildkraut, J. J. Am. J. Psychiatry 1965, 122, 509.
21. Grimbergen, Y. A.; Langston, J. W.; Roos, R. A.; Bloem, B. R. Expert Rev.
Neurother. 2009, 9(2), 279.
de Innovación e Industria de la Xunta de Galicia (Spain; INCI-
TE07PXI203039ES, INCITE08E1R203054ES and 08CSA019203PR).
22. Henchcliffe, C.; Schumacher, H. C.; Burgut, F. T. Expert. Rev. Neurother. 2005, 5,
811.
Supplementary data
23. Youdim, M. B.; Edmondson, D.; Tipton, K. F. Nat. Rev. Neurosci. 2006, 7,
295.
24. Lim, S. S.; Jung, S. H.; Ji, J.; Shin, K. H.; Keum, S. R. J. J. Pharm. Pharmacol. 2001,
53, 653.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.12.029.
25. Fukai, T.; Nomura, T. Heterocycles 1991, 32(3), 499.
26. Chimenti, F.; Fioravanti, R.; Bolasco, A.; Chimenti, P.; Secci, D.; Rossi, F.; Yanez,
M.; Orallo, F.; Ortuso, F.; Alcaro, S. J. Med. Chem. 2009, 52(9), 2818.
27. Levai, A. Heterocyclic Commun. 1999, 5, 419.
28. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.; Bizzarri, B.; Granese, A.;
Carradori, S.; Yanez, M.; Orallo, F.; Ortuso, F.; Alcaro, S. J. Med. Chem. 2009,
52(9), 1935.
References and notes
1. Harborne, J. B.; Baxter, H. Ed.; The Handbook of Natural Flavonoids; 1999; Vol.
2, pp 879–889.
2. Aherne, S. A.; O’Brien, N. M. Nutrition 2002, 18, 75.
3. Middleton, E., Jr.; Kandaswami, C.; Theoharides, T. C. Pharmacol. Rev. 2000, 52,
673.
4. Orallo, F. Curr. Med. Chem. 2008, 15(19), 1887.
29. Cirilli, R.; Ferretti, R.; De Santis, E.; Gallinella, B.; Zanitti, L.; La Torre, F. J.
Chromat A 2008, 1190(1–2), 95.
30. Tipton, K. F.; Boyce, S.; O’Sullivan, J.; Davey, G. P.; Healy, J. Curr. Med. Chem.
2004, 11, 1965.
5. Hua Han, X.; Su Hong, S.; Hwang, J. S.; Koo Lee, M.; Hwang, B. Y.; Seup Ro, J.
Arch. Pharm. Res. 2007, 30, 13.
31. Szedlacsek, S. E.; Duggleby, R. G. Meth. Enzymol. 1995, 249, 144.
32. Gerlach, M.; Riederer, P.; Youdim, M. B. Eur. J. Pharmacol. 1992, 226, 97.
33. O’Brien, E. M.; Tipton, K. F.; Meroni, M.; Dostert, P. J. Neural. Transm. Suppl.
1994, 41, 295.
34. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235.
35. Son, S. Y.; Ma, J.; Kondou, Y.; Yoshimura, M.; Yamashita, E.; Tsukihara, T. Proc.
Natl. Acad. Sci. USA 2008, 105, 5739. Data deposition: www.pdb.org (PDB ID
code2Z5X).
36. Hubalek, F.; Binda, C.; Khalil, A.; Li, M.; Mattevi, A.; Castagnoli, N.; Edmondson,
D. E. J. Biol. Chem. 2005, 280, 15761. Data deposition: www.pdb.org (PDB ID
code 2BK3).
37. Maestro ver. 4.1 Schroedinger Inc.: Portland, OR, 1998–2001.
38. Kaminski, G.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. L. J. Phys. Chem. B
2001, 105, 6474.
39. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.; Caufield,
C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440.
40. Hasel, W.; Hendrickson, T. F.; Still, W. C. Tetrahedron Comput. Methodol. 1988, 1,
103.
41. [a] Glide ver. 4.1, Schroedinger Inc.: Portland, OR, 1998–2001.; (b) Eldridge, M.
D.; Murray, C. W.; Auton, T. R.; Paolini, G. V.; Mee, R. P. J. Comput. Aided Mol.
Des. 1997, 11, 425.
6. Marder, M.; Paladini, A. C. Curr. Top. Med. Chem. 2002, 2, 853.
7. Manthey, J. A.; Grohmann, K.; Guthrie, N. Curr. Med. Chem. 2001, 8, 135.
8. Fournet, A.; Munoz, V. Curr. Top. Med. Chem. 2002, 2, 1215.
9. Manach, C.; Mazur, A.; Scalbert, A. Curr. Opin. Lipidol. 2005, 16, 77.
10. (a) Cardenas, M.; Marder, M.; Blank, V. C.; Roguin, L. P. Bioorg. Med. Chem. 2006,
14, 2966. and references therein; (b) Akama, T.; Shida, Y.; Sugaya, T.; Ishida, H.;
Gomi, K.; Kasai, M. J. J. Med. Chem. 1996, 39, 3461; (c) Cushman, M.;
Nagarathnam, D. J. Nat. Prod. 1991, 54, 1656; (d) Hayashi, T.; Uchida, K.;
Hayashi, K.; Niwayama, S.; Morita, N. Chem. Pharm. Bull. 1988, 36, 4849; (e)
Beutler, J. A.; Cardellina, J. H., II; Lin, C. M.; Hamel, E.; Cragg, G. M.; Boyd, M. R.
Bioorg. Med. Chem. Lett. 1993, 3, 581.
11. Li, Y.; Fang, H.; Xu, W. Mini Rev. Med. Chem. 2007, 7(7), 663.
12. Shankar, S.; Ganapathy, S.; Srivastava, R. K. Front Biosci. 2007, 12, 4881.
13. Singh, A.; Naidu, P. S.; Kulkarni, S. K. Pharmacology 2003, 68, 81.
14. Lee, S. J.; Chung, H. Y.; Lee, I. K.; Oh, S. U.; Yoo, I. D. Food Sci. Biotechnol. 2000, 9,
179.
15. Brahmachari, G.; Gorai, D. Curr. Org. Chem. 2006, 10, 873.
16. Chimenti, F.; Cottiglia, F.; Bonsignore, L.; Casu, L.; Casu, M.; Floris, C.; Secci, D.;
Bolasco, A.; Chimenti, P.; Granese, A.; Befani, O.; Turini, P.; Alcaro, S.; Ortuso, F.;
Trombetta, G.; Loizzo, A.; Guarino, I. J. Nat. Prod. 2006, 69, 945.
17. Binda, C.; Hubálek, F.; Li, M.; Edmondson, D. E.; Mattevi, A. FEBS Lett. 2004,
564(3), 225.
42. DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific: San
Carlos, CA, 2002. http://www.pymol.org.
18. Edmondson, D. E.; Mattevi, A.; Binda, C.; Li, M.; Hubalek, F. Curr. Med. Chem.
2004, 11, 1983.