Homoisoflavonoids: Natural scaffolds with potent and selective monoamine oxidase-B inhibition properties

Article abstract of DOI:10.1021/jm1013709

A series of homoisoflavonoids [(E)-3-benzylidenechroman-4-ones 1a-w, 3-benzyl-4H-chromen-4-ones 2a-g, and 3-benzylchroman-4-ones 3a-e] have been synthesized and tested in vitro as inhibitors of human monoamine oxidase isoforms A and B (hMAO-A and hMAO-B).

Full text of DOI:10.1021/jm1013709

ARTICLE
pubs.acs.org/jmc
Homoisoflavonoids: Natural Scaffolds with Potent and Selective
Monoamine Oxidase-B Inhibition Properties
Nicoletta Desideri,*,† Adriana Bolasco,† Rossella Fioravanti,† Luca Proietti Monaco,† Francisco Orallo,‡
Matilde Yꢀa~nez,‡ Francesco Ortuso,§ and Stefano Alcaro§
†Dipartimento di Chimica e Tecnologie del Farmaco, Universitꢁa “La Sapienza” di Roma, P.le Aldo Moro, 5, 00185 Rome, Italy
‡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 Coru~na), Spain
§Dipartimento di Scienze Farmacobiologiche, Universitꢁa di Catanzaro “Magna Græcia”, Complesso Nini Barbieri, 88021 Catanzaro, Italy
S Supporting Information
b
ABSTRACT:
A series of homoisoflavonoids [(E)-3-benzylidenechroman-4-ones 1aꢀw, 3-benzyl-4H-chromen-4-ones 2aꢀg, and 3-benzylchro-
man-4-ones 3aꢀe] have been synthesized and tested in vitro as inhibitors of human monoamine oxidase isoforms A and B (hMAO-
A and hMAO-B). Most of the compounds were found to be potent and selective MAO-B inhibitors. In general, the (E)-3-
benzylidenechroman-4-ones 1aꢀw showed activities in the nano- or micromolar range coupled with high selectivity against hMAO-
B. The reduction of the exocyclic double bond results in compounds 3aꢀe selective against isoform B and active in the micromolar
range. In contrast, the endocyclic migration of the double bond (compounds 2aꢀg) generally produces the loss of the inhibitory
activity or a marked reduction in potency. (E)-3-(4-(Dimethylamino)benzylidene)chroman-4-one (1l) and (E)-5,7-dihydroxy-
3-(4-hydroxybenzylidene)chroman-4-one (1h) were the most interesting compounds of the entire series of inhibitors, showing
hMAO-B affinity better than the selective inhibitor selegiline. Molecular modeling studies have been carried out to explain the
selectivity of the most active homoisoflavonoids 1h and 1l.
’ INTRODUCTION
of clorgyline, whereas MAO-B preferentially metabolizes β-
phenylethylamine and benzylamine and is inhibited by
selegiline.1 Both isoenzymes deaminate dopamine, tyramine,
and tryptamine.1
Because of the important function played by MAO in the
metabolism of neurotransmitters, MAO inhibitors can be useful
in the treatment of many psychiatric and neurological diseases. In
fact, MAO-A inhibitors act as antidepressant and antianxiety
agents, whereas MAO-B inhibitors are used alone or in combina-
tion to treat Alzheimer’s and Parkinson’s diseases.13
Recognition of the importance of MAO as a drug target for
treatment of neurological disorders has produced an enormous
interest in the development of inhibitors of these enzymes.
Various structural classes of potent MAO inhibitors, including
Monoamine oxidase (MAO) is an ubiquitous membrane-
bound, flavin-containing enzyme, which is particularly abundant
in the liver and brain.1 MAO is located intracellularly in the
mitochondrial outer membranes of neuronal, glial, and other
cells and catalyzes the oxidative deamination of monoamine
neurotransmitters and xenobiotic amines to the corresponding
aldehydes with consumption of oxygen and production of
hydrogen peroxide, thereby affecting the concentrations of
neurotransmitter amines and many xenobiotic ones.2ꢀ4
Currently, two MAO isoforms, encoded by separate genes
sharing a common intron/exon organization,4ꢀ6 have been
identified based on their differential substrate specificity and
inhibitor sensitivity,7ꢀ9 tissue and cell distribution,10 and gene
expression characteristics.11,12
MAO-A preferentially deaminates serotonin, epinephrine, and
norepinephrine and is selectively inhibited by low concentrations
Received: October 21, 2010
Published: March 15, 2011
r
2011 American Chemical Society
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of (E)-3-benzylidenechroman-4-ones to further investigate the
structureꢀactivity relationships. The MAO recognition of the
most active compounds, 1h and 1l, was investigated by docking
experiments performed using available Protein Data Bank (PDB)
structures as receptor models.
’ CHEMISTRY
The (E)-3-benzylidenechroman-4-ones 1aꢀw were synthe-
sized by acid-catalyzed condensation of the appropriate chro-
man-4-one with substituted benzaldehydes (Scheme 1).
In general, the target compounds were prepared in good yield
using 85% phosphoric acid as the acid catalyst and heating the
mixture at 80 °C for 6 h. Under these conditions, the reaction of
chroman-4-one with N-(4-formylphenyl)acetamide gave a mix-
ture of amide 1k and the corresponding amine 1j, which were
easily separated by column chromatography. Starting from
substituted chroman-4-ones or 2-substituted benzaldehydes,
treatment with 85% phosphoric acid provided the corresponding
chromanones (1d, 1e, 1g, 1i, and 1sꢀv) in very low yields. To
obtain these compounds more efficiently, the condensations
were carried out in dry ethyl alcohol saturated with dry hydrogen
chloride. A single stereoisomer was obtained with both proce-
Figure 1. Some representative structures of nonselective and selective
MAO inhibitors.
hydrazide, amide, thiazole, imidazole, oxazolidinone, oxadiazo-
lone, diacylurea derivatives, etc., have been identified.14ꢀ27 In
spite of considerable progress in understanding the interactions
of the two enzyme forms with their corresponding substrates, no
general rules are yet available for the rational design of potent and
selective MAO inhibitors. There are many different structures of
MAO inhibitors partly because the active sites of the MAO
enzymes are ambiguous, which limits the design of potent
selective MAO inhibitors. Figure 1 shows the chemical structures
of some representative MAO inhibitors used in research or
clinical practice.
1
dures. The H NMR spectra allow the E configuration of the
double bound to be assigned on the basis of the chemical shift of
the olefinic proton, ranging from 7.6 to 7.9 ppm.
(E)-5,7-Dihydroxy-3-(4-hydroxybenzylidene)chroman-4-one
(1h) was synthesized by saponification of the corresponding
tribenzoate following published procedures.42
The reduction of (E)-3-(4-hydroxy-3-nitrobenzylidene)chro-
man-4-one (1q) with tin chloride in ethyl alcohol and concen-
trated hydrogen chloride provided the corresponding amino
derivative (1r).
The aim of the present study was the identification of novel
potent MAO inhibitors that could serve as potential lead
molecules for drug discovery.
The (E)-3-benzylidenechroman-4-ones 1aꢀg were converted
into the corresponding 3-benzyl-4-chromones 2aꢀg or 3-ben-
zylchroman-4-ones 3aꢀe according to procedures described
previously.29
Homoisoflavonoids constitute a small class of natural products
prevalently isolated from the bulbs, rhizomes, or roots of several
genera of Hyacinthaceae and Caesalpinioideae. These compounds
are structurally related to the more widespread flavonoids and,
according to their structural features, can be classified into three
types: 3-benzylidenechroman-4-ones, 3-benzyl-4H-chromen-4-
ones, and 3-benzylchroman-4-ones. Several natural and synthetic
homoisoflavonoids, like the related flavonoids, were found to
possess various biological properties such as antifungal,28
antiviral,29ꢀ31 antimutagenic,32,33 antiproliferative,34 antioxidant,35,36
antiallergic and antihistaminic,37 anti-inflammatory,38 and pro-
tein tyrosine kinase (PTK) inhibitor activities.39 However, no
data are available on the inhibitory activity of homoisoflavonoids
on MAOs.
Recently, several substituted chalcones and flavanones were
reported as potent and selective inhibitors of the B-isoform of
human MAO (hMAO).40,41 The structural relationship of
homoisoflavonoids to chalcones and flavanones prompted us
to investigate their inhibitory activity against the A and B
isoforms of hMAO. Initially, we tested the synthetic (E)-3-
benzylidenechroman-4-ones 1aꢀg, 3-benzyl-4-chromones 2aꢀg,
and 3-benzylchroman-4-ones 3aꢀe, previously studied as anti-
picornavirals.29ꢀ31 Because all the (E)-3-benzylidenechroman-4-
ones 1aꢀg, which may be regarded as rigid analogues
of chalcones, were potent and selective inhibitors of the B
isoform of hMAO, we planned the synthesis of a larger panel
’ BIOCHEMISTRY
The potential effects of the tested compounds 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,
Oregon, U.S.) and microsomal MAO isoforms prepared from
insect cells (BTI-TN-5B1-4) infected with recombinant baculo-
virus containing cDNA inserts for hMAO-A or hMAO-B (Sigma-
Aldrich Química S.A., Alcobendas, Spain). In this study hMAO
activity was evaluated using the above method following the
general procedure described previously by us.43 The tested drugs
(new compounds and reference inhibitors) themselves were
unable to react directly with the Amplex Red reagent, which
indicates that these compounds do not interfere with the
measurements. On the other hand, in our experiments and under
our experimental conditions, the control activity of hMAO-A and
hMAO-B (using p-tyramine as a common substrate for both
isoforms) was 165 ( 2 pmol of p-tyramine oxidized to
p-hydroxyphenylacetaldehyde/min (n = 20).
The results of hMAO-A and hMAO-B inhibition studies
are expressed as IC50 and reported in Tables 1 and 2 together
with the hMAO-B selectivity indexes (SI = IC50 MAO-A/IC50
MAO-B).
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ARTICLE
Scheme 1a
a Reagents and conditions: (i) 85% H3PO4, 80° C, 6 h; (ii) dry EtOH sat. HCl, room temp, 48 h.
In Table 1 are presented the data obtained for the (E)-3-
benzylidenechroman-4-ones 1aꢀw. Most of these homoisofla-
vonoids selectively inhibited the enzymatic activity of hMAO-B in
the nanomolar or micromolar range. Derivatives 1h, 1k, 1o, and
1r were also able to inhibit hMAO-A in the micromolar range.
Among these analogues, 1k and 1r were essentially nonselective
(SI = 2 and 3, respectively) whereas 1h and 1o exhibited hMAO-
B selectivity to different extents (SI = 1331 and 34, respectively).
Only the acid 1p was found to be completely inactive toward
both isoforms up to the highest concentration tested
(100 μM). (E)-3-[4-(Dimethylamino)benzylidene]chroman-4-
one (1l) was the most potent and selective hMAO-B inhibitor
identified in this study, exhibiting an IC50 of 8.51 nM and SI of
>11751. In comparison with the reference inhibitor selegiline,
compound 1l showed higher hMAO-B affinity and selectivity.
The replacement of the dimethylamino group with the amino
group resulted in compound 1j, about 100-fold less potent and
selective as a hMAO-B inhibitor (IC50 = 879.18 nM, SI > 114)
than 1l. Interestingly, acetylation of the amino group (compound
1k) produced a further decrease of hMAO-B inhibitory activity
and a dramatic reduction of selectivity (IC50 = 1.06 μM, SI = 2).
(E)-5,7-Dihydroxy-3-(4-hydroxybenzylidene)chroman-4-one
(1h) showed an affinity for hMAO-B (IC50 = 8.61 nM)
comparable to that for 1l. Although 1h was able to inhibit both
hMAO isoforms, it still exhibited an excellent hMAO-B selec-
tivity (SI = 1331). Although less active than 1h and 1l, (E)-5,7-
dichloro-3-(4-chlorobenzylidene)chroman-4-one (1e) showed
hMAO-B affinity and selectivity (IC50 = 13.03 nM, SI > 7675)
better than those of the reference compound, selegiline.
Several of these compounds (2e, 2f, 3a, and 3cꢀe) showed
selective hMAO-B inhibitory activities, whereas the 3-benzyl-4-
chromones 2aꢀd and 2g and the 3-benzylchroman-4-one 3b
were essentially inactive toward both isoforms up to the highest
concentration tested (100 μM). Disappointly, the endocyclic
migration of the double bond produces a loss of the inhibitory
activity (compounds 2aꢀd, 2g) or a marked reduction in
potency (compounds 2e and 2f) with respect to the correspond-
ing (E)-3-benzylidenechroman-4-ones 1aꢀg. The reduction of
the double bond results in compounds 3aꢀe, generally more
potent and selective than the corresponding 3-benzyl-4H-chro-
men-4-ones 2aꢀg. The only exception was 5,7-dichloro-3-(4-
chlorobenzyl)-4H-chromen-4-one (2e) that exhibited submicro-
molar potency toward hMAO-B, whereas the corresponding
3-benzylchroman-4-one (3e) was about 10-fold less active and
less selective. However, all the 3-benzylchroman-4-ones 3aꢀe
were less potent hMAO-B inhibitors than the corresponding
(E)-3-benzylidenechroman-4-ones 1aꢀe.
Table 3 shows the results of the reversibility and irreversibility
tests for the most effective compounds 1h and 1l. hMAO-A and
hMAO-B inhibition was irreversible in the presence of 1h as
shown by the lack of enzyme activity restoration after repeated
washing. Similar results were obtained for compound 1l and
selegiline against hMAO-B. On the contrary, significant recovery
of hMAO-A activity was observed after repeated washing of
moclobemide, indicating that this drug is a reversible inhibitor of
hMAO-A.
’ DOCKING STUDIES
The results for inhibitory effects and selectivity of the 3-benzyl-
4H-chromen-4-ones 2aꢀg and 3-benzylchroman-4-ones 3aꢀe on
hMAO isoforms are reported in Table 2.
Docking studies were carried out to evaluate the binding
modes of this class of homoisoflavonoids with respect to both
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Table 1. IC50 and SI for the Inhibitory Effects of (E)-3-Benzylidenechroman-4-ones 1aꢀw and Reference Inhibitors on the
Enzymatic Activity of Human Recombinant MAO Isoforms Expressed in Baculovirus Infected BTI Insect Cellsa
IC50
compd
R
R1
R2
R3
R4
R5
MAO-A
MAO-B
SId
1a
1b
1c
1d
1e
1f
H
H
H
H
Cl
H
H
H
H
Cl
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
f
f
f
f
f
f
f
479.70 ( 19.37 nM
55.37 ( 2.18 nM
154.23 ( 6.93 nM
31.82 ( 1.19 nM
13.03 ( 0.79 nM
58.90 ( 2.09 nM
1.57 ( 0.03 μM
8.61 ( 0.12 nM
331.11 ( 9.63 nM
879.18 ( 45.31 nM
1.06 ( 0.04 μM
8.51 ( 0.32 nM
104.34 ( 2.61 nM
476.51 ( 21.60 nM
140.52 ( 4.71 nM
f
>208h
>1806h
>648h
>3143h
>7675h
>1698h
>64h
OH
Cl
Cl
Cl
OCH3
OCH3
OH
1g
1h
1i
OCH3
OH
H
11.46 ( 0.43 μMb
1331
H
f
>302h
>114h
2.0
>11751h
>958h
>210h
34
1j
H
NH2
f
1k
1l
H
NHCOCH3
N(CH3)2
OH
2.10 ( 0.11 μMc
H
f
1m
1n
1o
1p
1q
1r
H
OCH3
OH
g
H
OCH3
OH
f
H
OH
4.74 ( 0.21 μMb
H
COOH
NO2
NH2
H
OH
f
H
OH
f
490.84 ( 12.73 nM
483.88 ( 16.21 nM
1.33 ( 0.07 μM
247.51 ( 6.31 nM
1.92 ( 0.08 μM
1.31 ( 0.04 μM
20.69 ( 0.37 μM
61.35 ( 1.13 μM
19.60 ( 0.86 nM
7.54 ( 0.36 μM
e
>204h
3.0
>75h
>404h
>52h
>76h
>4.8h
0.000073
3431
0.87
H
OH
1.44 ( 0.51 μMb
1s
1t
H
OH
OCH3
OCH3
H
g
H
OCH3
OCH3
OCH3
H
H
f
1u
1v
1w
H
OCH3
OCH3
OCH3
f
H
OCH3
OCH3
f
H
OCH3
f
clorgyline
selegiline
4.46 ( 0.32 nMb
67.25 ( 1.02 μMb
6.56 ( 0.76 μM
361 ( 19.37 μM
iproniazid
moclobemide
<0.36
a All IC50 values shown in this table are the mean ( SEM from five experiments. b Level of statistical significance: P < 0.01 versus the corresponding IC50
values obtained against MAO-B, as determined by ANOVA/Dunnett's. c Level of statistical significance: P < 0.05 versus the corresponding IC50 values
obtained against MAO-B, as determined by ANOVA/Dunnett's. d SI: hMAO-B selectivity index = IC50(hMAO-A)/IC50(hMAO-B). e Inactive at 1 mM
(highest concentration tested). f Inactive at 100 μM (highest concentration tested). g 100 μM inhibits the corresponding MAO activity by approximately
40ꢀ50%. At higher concentration the compounds precipitate. h Values obtained under the assumption that the corresponding IC50 against MAO-B is
the highest concentration tested (100 μM).
isoforms of human MAO. Crystallographic structures were
selected from the PDB to build our theoretical receptors models
(Experimental Section). Taking into account the experimental
hMAO inhibition data, compounds 1h and 1l were chosen for
our molecular modeling investigation. Although the irreversible
inhibitors usually form a covalent bond with a residue on the
enzyme, they may also act by other mechanisms.44 The so-called
tight-binding inhibitors, owing to the very low dissociation
constant, may show kinetics similar to the kinetics of covalent
irreversible inhibitors. In some cases, the inhibitors may rapidly
bind to the enzyme in a low-affinity enzymeꢀinhibitor (EI)
complex and then undergoes a slower rearrangement to a very
tightly bound EI* complex. For instance, R-(ꢀ)-deprenyl, an
irreversible MAO inhibitor, forms a noncovalent complex with
MAO as an initial and reversible step. Afterward, the interaction
of R-(ꢀ)-deprenyl with MAO leads to a reduction of the
enzyme-bound FAD and concomitant oxidation of the inhibitor.
This oxidized inhibitor is able to form a covalent bond at the N-5
position of FAD.45 The initial noncovalent binding to MAO has
been also described for other MAO inhibitors (e.g., clorgyline
derivatives).46 Moreover, we have already docked noncovalent
ligands40,41,47 showing an irreversible profile similar to 1h and 1l.
Both homoisoflavonoids were submitted to a conformational
search revealing, not surprisingly, only two conformers within 10
kcal/mol from the global minimum energy. Using the AutoDock
Vina method,48 the most populated structures of 1h and 1l were
submitted to flexible docking simulation with respect to both
hMAO-A and hMAO-B receptor models (Experimental
Section). The theoretical complexes were evaluated taking into
account two interaction energy descriptors. Best interaction
energy (IE) is the AutoDock Vina lowest ligand-target interac-
tion energy. ΔGbind is the Boltzmann averaged binding free
energy computed with the entire complex ensemble generated by
the docking program. In both cases, good qualitative agreements
with experimental inhibition data have been obtained (Table 4).
The most stable binding modes of both homoisoflavonoids in
the hMAO-A and -B active sites were graphically inspected
(Figure 2).
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Table 2. IC50 and SI for the Inhibitory Effects of 3-Benzyl-4H-chromen-4-ones 2aꢀg and 3-Benzylchroman-4-ones 3aꢀe and
Reference Inhibitors on the Enzymatic Activity of Human Recombinant MAO Isoforms Expressed in Baculovirus Infected BTI
Insect Cellsa
IC50
compd
R
R1
R2
R3
R4
R5
MAO-A
MAO-B
SIc
2a
2b
2c
2d
2e
2f
H
H
H
H
Cl
H
H
H
H
Cl
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
e
e
e
e
f
e
f
OH
Cl
e
f
Cl
Cl
438.60 ( 27.48 nM
21.59 ( 1.06 μM
f
>228g
>4.6g
OCH3
OCH3
H
e
e
e
e
e
e
e
2g
3a
3b
3c
3d
3e
OCH3
H
58.53 ( 2.24 μM
f
>1.7g
H
OH
Cl
H
7.37 ( 0.31 μM
3.07 ( 0.11 μM
4.59 ( 0.19 μM
61.35 ( 1.13 μM
19.60 ( 0.86 nM
7.54 ( 0.36 μM
d
>14g
>33g
>22g
H
Cl
Cl
Cl
clorgyline
selegiline
4.46 ( 0.32 nMb
67.25 ( 1.02 μMb
6.56 ( 0.76 μM
361 ( 19.37 μM
0.000073
3431
0.87
iproniazid
moclobemide
<0.36
a All IC50 values shown in this table are the mean ( SEM from five experiments. b Level of statistical significance: P < 0.01 versus the corresponding IC50
values obtained against MAO-B, as determined by ANOVA/Dunnett's. c SI: hMAO-B selectivity index = IC50(hMAO-A)/IC50(hMAO-B). d Inactive at
e
f
1 mM (highest concentration tested). Inactive at 100 μM (highest concentration tested). 100 μM inhibits the corresponding MAO activity by
approximately 40ꢀ50%. At higher concentration the compounds precipitate. g Values obtained under the assumption that the corresponding IC50
against MAO-B is the highest concentration tested (100 μM).
Table 3. Reversibility and Irreversibility of hMAO-B Inhibi-
tion of Derivatives 1h and 1la
Table 4. Comparison between Experimental IC50 Data and
Theoretical Binding Affinities of 1h and 1l with Respect to
hMAO-A and -B
% hMAO-A inhibition
hMAO-A
hMAO-B
compd
1h (50 μM)
before washing
after repeated washing
parameter
1h
1l
1h
1l
68.26 ( 4.34
85.98 ( 4.03
61.76 ( 4.98
11.45 ( 0.58b
a
IC50
11460.00
ꢀ6.30
ꢀ7.00
>100000.00
ꢀ1.88
8.61
8.51
moclobemide (500 μM)
b
ΔGbind
ꢀ9.10
ꢀ9.50
ꢀ9.35
ꢀ10.40
best IE c
ꢀ3.00
% hMAO-B inhibition
a Experimental inhibition data (nM). b Free energy of binding (kcal/
compd
before washing
after repeated washing
mol). c Best interaction energy (kcal/mol).
1h (10 nM)
48.66 ( 6.39
56.34 ( 5.37
53.28 ( 2.59
36.22 ( 4.26
58.67 ( 6.60
56.34 ( 2.01
1l (10 nM)
exclusive contact with Tyr69; conversely 1l was involved in
attractive hydrophobic contacts with Asn181 and Tyr444. The
effects of these different interactions on binding energy are
highlighted in Table 4.
selegiline (20 nM)
a Each value is the mean ( SEM from five experiments (n = 5). b Level of
statistical significance: P < 0.01 versus the corresponding % MAO-A or
MAO-B inhibition before washing, as determined by ANOVA/
Dunnett's.
The best poses of these compounds in the hMAO-B binding
site were different from the A isoform. These configurations
displayed the chromone ring far from the FAD cofactor. In both
1h and 1l hMAO-B complexes the interaction pattern was
similar: one hydrogen bond between the carbonyl oxygen of
the ligands and Tyr326-OH and several almost identical hydro-
phobic contacts. The slightly better interaction of 1l can be
attributed to its more hydrophobic nature compared with 1h.
Actually the chromone ring was surrounded by a large lipophilic
cage (Phe103, Pro104, Trp119, Leu164, Leu167, Phe168,
Leu171, Ile199, and Ile316) where 1h, because of its hydrophilic
substituents, was disfavored. Finally, the hMAO-B selectivity of
both compounds can be attributed to (a) the larger set of residues
interacting with hMAO-B and (b) the different hydrogen bond
Docking simulations generated several low energy binding
modes of 1h and 1l within both hMAO isoforms. These poses
revealed the ability of these ligands to alternatively recognize the
FAD with both benzyl and chromone rings (see Supporting
Information). Interestingly, the hMAO-A best poses of the 1h
and 1l chromone rings were located toward the enzymatic
cofactor (Figure 2a). Most of the ligandꢀenzyme interactions
appeared to be identical. The main difference between 1h and 1l
hMAO-A recognition was due to the presence of a phenol OH in
the former compound that established a hydrogen bond with
FAD N5 atom. Moreover, 1h, attracted by the FAD, resulted in
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Figure 2. Most representative binding modes of 1h (green) and 1l (white) into (a) hMAO-A and (b) hMAO-B catalytic sites. Common 1h and 1l
interacting residues are reported in magenta carbon atoms sticks. Exclusive 1h or 1l interacting residues are displayed according to the corresponding
ligands. The FAD cofactor is depicted using the spacefill representation. The rest of the enzyme is shown in yellow, transparent cartoon. Yellow dotted
lines indicate hydrogen bonds.
contributions, stronger in hMAO-B (Tyr326-OH O=1h/1l)
preparation of compound 1l). The solid was filtered off, washed with
water, and crystallized.
(E)-3-(4-(Dimethylamino)benzylidene)chroman-4-one (1l).
Yield: 77%. Mp = 151ꢀ153 °C (lit. 150ꢀ153 °C)34 from EtOH. The
compound exhibited spectroscopic data identical to those previously
reported.34
3 3 3
than in hMAO-A (FAD-N5 HO-1h).
3 3 3
’ CONCLUSION
In the current study we have synthesized and evaluated a series
of (E)-3-benzylidenechroman-4-ones 1aꢀw, 3-benzyl-4H-chro-
men-4-ones 2aꢀg, and 3-benzylchroman-4-ones 3aꢀe as inhi-
bitors of h-MAO isoforms A and B. In general, the active
compounds showed potent and selective activity toward the B
isoform. In particular, several (E)-3-benzylidenechroman-4-ones
exhibited potencies in the nanomolar range. Two derivatives, 1h
and 1l, were the most potent and selective hMAO-B inhibitors
with higher potency than the reference inhibitor selegiline and, in
the latter case, even higher selectivity. Molecular docking studies
suggest that stronger enzymeꢀinhibitor hydrogen bonds and
hydrophobic contacts in the hMAO-B active site can explain the
selectivity of both inhibitors for this isoform.
(E)-3-(4-Hydroxy-3-methoxybenzylidene)chroman-4-one
(1m). Yield: 71%. Mp = 130ꢀ131 °C (lit. 126ꢀ129 °C)49 from ethyl
alcohol. IR (KBr): 3200, 1655 cmꢀ1. 1H NMR: δ (ppm) 9.75 (bs, 1H,
OH), 7.88 (dd, 1H, H5, J5ꢀ6 = 8.0 Hz, J5ꢀ7 = 1.6 Hz), 7.71 (t, 1H, dCH,
Jall = 1.8 Hz), 7.58 (ddd, 1H, H7, J6ꢀ7 = 7.2 Hz, J7ꢀ8 = 8.4 Hz, J5ꢀ7 = 1.6
Hz), 7.12 (ddd, 1H, H6, J6ꢀ7 = 7.2 Hz, J5ꢀ6 = 8.0 Hz, J6ꢀ8 = 1.0 Hz),
7.07 (d, 1H, H20, J2 -6 = 1.7 Hz), 7.05 (dd, 1H, H8, J7ꢀ8 = 8.4 Hz, J6ꢀ8
=
0
0
1.0 Hz), 6.93 (dd, 1H, H60, J2 -6 = 1.7 Hz), 6.90 (d, 1H, H5 , J5 -6 = 8.4
Hz), 5.47 (d, 2H, H2, Jall= 1.7 Hz), 3.84 (s, 3H, OCH3). 13C NMR: δ
(ppm) 180.9, 160.4, 148.8, 147.6, 137.2, 135.8, 127.7, 127.1, 125.2,
124.4, 121.8, 121.6, 117.7, 115.6, 114.8, 67.5, 55.6.
0
0
0
0
0
(E)-3-(3-Hydroxy-4-methoxybenzylidene)chroman-4-one
(1n). Yield: 78%. Mp = 192ꢀ193 °C from EtOH. IR (KBr): 3150,
1650 cmꢀ1. 1H NMR: δ (ppm) 9.34 (bs, 1H, OH), 7.88 (dd, 1H, H5,
J5ꢀ6 = 7.9 Hz, J5ꢀ7 = 1.8 Hz), 7.64 (t, 1H, =CH, Jall = 1.8 Hz), 7.58 (ddd,
1H, H7, J6ꢀ7 = 7.2 Hz, J7ꢀ8 = 8.4 Hz, J5ꢀ7 = 1.8 Hz), 7.13 (ddd, 1H, H6,
J6ꢀ7 = 7.2 Hz, J5ꢀ6 = 7.9 Hz, J6ꢀ8 = 1.0 Hz), 7.07ꢀ7.03 (m, 2H, H8,
H50), 6.94ꢀ6.91 (m, 2H, H20, H60), 5.43 (d, 2H, H2, Jall = 1.8 Hz), 3.85
(s, 3H, OCH3). 13C NMR: δ (ppm) 180.9, 160.4, 149.4, 146.5, 136.8,
135.9, 128.3, 127.1, 126.5, 122.9, 121.8, 121.5, 117.7, 117.2, 112.0,
67.4, 55.6.
(E)-3-(3,4-Dihydroxybenzylidene)chroman-4-one (1o). Yield:
54%. Mp = 227ꢀ230 °C (lit. 224ꢀ225 °C)49 from EtOH. IR (KBr): 3400,
1640 cmꢀ1. 1H NMR: δ (ppm) 9.50 (bs, 2H, 2OH), 7.87 (dd, 1H, H5,
J5ꢀ6 = 7.8 Hz, J5ꢀ7 = 1.7 Hz), 7.61 (t, 1H, =CH, Jall= 1.8 Hz), 7.58 (ddd,
1H, H7, J6ꢀ7 = 7.4 Hz, J7ꢀ8 = 8.4 Hz, J5ꢀ7 = 1.7 Hz), 7.12 (ddd, 1H, H6,
J6ꢀ7 = 7.4 Hz, J5ꢀ6 = 7.8 Hz, J6ꢀ8 = 1.0 Hz), 7.05 (dd, 1H, H8, J7ꢀ8 = 8.4
Hz, J6ꢀ8 = 1.0 Hz), 6.89ꢀ6.80 (m, 3H, H20, H50, H60), 5.43 (d, 2H, H2,
Jall = 1.8 Hz). 13C NMR: δ (ppm) 180.9, 160.3, 147.8, 145.3, 137.2, 135.8,
127.3, 127.1, 125.2, 123.4, 121.7, 121.6, 117.7, 115.8, 67.5.
’ EXPERIMENTAL SECTION
Chemistry. Chemicals were purchased from Sigma-Aldrich and
used without further purification. Melting points were determined on a
Stenford Research Systems OptiMelt (MPA-100) apparatus and are
uncorrected. 1H NMR and 13C NMR spectra were recorded in DMSO-
d6, unless indicated otherwise, on a Bruker AM-400 spectrometer, using
TMS as internal standard. IR spectra were recorded in KBr disks on an
FT-IR PerkinElmer Spectrum 1000. All compounds were routinely
checked by thin-layer chromatography (TLC), 1H NMR, and 13C NMR.
TLC was performed on silica gel or aluminum oxide fluorescent coated
plates (Merck, Kieselgel, or aluminum oxide 60 F254). Components
were visualized by UV light. Compound purity was determined by
elemental analysis and was confirmed to be g95% for all compounds.
(E)-3-Benzylidenechroman-4-ones (1aꢀg), 3-benzyl-4-chromones
(2aꢀg), and 3-benzylchroman-4-ones (3aꢀe) were prepared as pre-
viously described by us.29 (E)-5,7-Dihydroxy-3-(4-hydroxybenzylide-
ne)chroman-4-one (1h) was obtained by saponification of the
corresponding tribenzoate as previously reported.42
(E)-2-Hydroxy-5-[(4-oxochroman-3-ylidene)methyl]benzoic
Acid (1p). Yield: 74%. Mp = 234ꢀ238 °C from EtOH. IR (KBr):
General Procedure for the Synthesis of (E)-3-Benzylide-
nechroman-4-ones 1lꢀq and 1w. A mixture of the appropriate
chroman-4-one (0.01 mol) and substituted benzaldehyde (0.01 mol) in
85% phosphoric acid (20 mL) was heated at 80 °C for 6 h. After cooling,
the mixture was diluted with iceꢀwater (and made alkaline for the
3500ꢀ2300, 1650, 1600 cmꢀ1
.
1H NMR: δ (ppm) 11.87 (bs, 1H,
COOH), 7.87ꢀ7.80 (m, 2H, H5, H20), 7.69 (bs, 1H, dCH), 7.63ꢀ7.54
(m, 2H, H60, H7), 7.12ꢀ7.01 (m, 3H, H50, H6, H8), 5.40 (d, 2H, H2, Jall =
1.4 Hz). 13C NMR: δ (ppm) 180.9, 171.2, 162.0, 160.5, 137.4, 136.0, 135.6,
132.7, 129.4, 127.1, 125.0, 121.8, 121.4, 117.8, 117.7, 113.5, 67.2.
2160
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ARTICLE
(E)-3-(4-Hydroxy-3-nitrobenzylidene)chroman-4-one (1q).
Yield: 77%. Mp = 203ꢀ205 °C from EtOAc. IR (KBr): 3240, 1655,
1530, 1300 cmꢀ1. 1H NMR: δ (ppm) 11.65 (bs, 1H, OH), 7.94 (d, 1H,
(E)-3-(2-Hydroxy-4-methoxybenzylidene)chroman-4-one
(1s). Purified by column chromatography, eluting with AcOEt/petro-
leum ether (1:5). Yield: 56%. Mp = 158ꢀ160 °C from EtOH. IR (KBr):
3124, 1655 cmꢀ1. 1H NMR: δ (ppm) 10.34 (bs, 1H, OH), 7.89ꢀ7.86
(m, 2H, dCH, H5), 7.57 (ddd, 1H, H7, J6ꢀ7 = 7.2 Hz, J7ꢀ8 = 8.4 Hz,
H20, J2 -6 = 2.2 Hz), 7.88 (dd, 1H, H5, J5ꢀ6 = 7.9 Hz, J5ꢀ7 = 1.8 Hz), 7.70
0
0
(bs, 1H, =CH), 7.64 (dd, 1H, H60, J2 -6 = 2.2 HzJ5 -6 =8.7 Hz), 7.60 (ddd,
0
0
0
0
1H, H7, J6ꢀ7 = 7.3 Hz, J7ꢀ8 =8.4 Hz, J5ꢀ7 = 1.8Hz), 7.23 (d, 1H, H50, J5 -6
= 8.7 Hz), 7.13 (ddd, 1H, H6, J6ꢀ7 = 7.3 Hz, J5ꢀ6 = 7.9 Hz, J6ꢀ8 = 0.9 Hz),
7.05 (dd, 1H, H8, J7ꢀ8 = 8.4 Hz, J6ꢀ8 = 0.9 Hz), 5.44 (d, 2H, H2, Jall = 1.8
Hz). 13C NMR: δ (ppm) 180.5, 160.5, 152.8, 137.2, 136.5, 135.1, 134.4,
130.2, 127.2, 127.1, 124.9, 121.9, 121.3, 119.3, 117.8, 67.2.
J5ꢀ7 = 1.8 Hz), 7.12 (ddd, 1H, H6, J6ꢀ7 = 7.2 Hz, J5ꢀ6 = 7.9 Hz, J6ꢀ8
=
0
0
1.0 Hz), 7.08 (d, 1H, H60, J5 -6 = 8.2 Hz), 7.04 (dd, 1H, H8, J7ꢀ8 = 8.4
0
0
Hz, J6ꢀ8 = 1.0 Hz), 6.53ꢀ6.49 (m, 2H, H30, H50), 5.33 (d, 2H, H2, Jall
=
1.8 Hz), 3.77 (s, 3H, OCH3). 13C NMR: δ (ppm) 181.2, 162.2, 160.5,
158.6, 135.8, 132.8, 131.7, 127.3, 127.1, 121.8, 121.7, 117.8, 114.0, 105.4,
101.1, 67.9, 55.2.
(E)-3-(3,4,5-Trimethoxybenzylidene)chroman-4-one (1w).
Yield: 63%. Mp = 105ꢀ106 °C (lit. 108ꢀ109 °C)50 from EtOAc/
(E)-3-(2,4-Dimethoxybenzylidene)chroman-4-one (1t). Yield:
64%. Mp = 123ꢀ125 °C (lit. 133ꢀ135 °C)34 from MeOH. The
compound exhibited spectroscopic data identical to those previously
reported.34
1
petroleum ether. IR (KBr): 1660 cmꢀ1. H NMR: δ (ppm) 7.90 (dd,
1H, H5, J5ꢀ6 = 7.8 Hz, J5ꢀ7 = 1.8 Hz), 7.73 (bs, 1H, dCH), 7.60 (ddd,
1H, H7, J7ꢀ8 = 8.4 Hz, J6ꢀ7 = 7.2 Hz, J5ꢀ7 = 1.8 Hz), 7.14 (ddd,1H, H6,
J6ꢀ7 = 7.2 Hz, J5ꢀ6 = 7.8 Hz, J6ꢀ8 = 1.0 Hz), 7.06 (dd, 1H, H8, J7ꢀ8 = 8.4
Hz, J6ꢀ8 = 1.0 Hz), 6.77 (s, 2H, H20, H60), 5.50 (d, 2H, H2 Jall = 1.8 Hz),
3.84 (s, 6H, 2OCH3), 3.74 (s, 3H, OCH3). 13C NMR: δ (ppm) 181.0,
160.6, 152.8, 138.9, 136.9, 136.2, 130.0, 129.2, 127.2, 121.9, 121.5, 117.8,
108.0, 67.4, 60.1, 56.0.
Synthesis of (E)-3-(4-Aminobenzylidene)chroman-4-one
(1j) and (E)-N-(4-[(4-Oxochroman-3-ylidene)methyl]phenyl)
acetamide (1k). A mixture of chroman-4-one (0.01 mol) and
4-aminobenzaldehyde (0.01 mol) in 85% H3PO4 (20 mL) was heated
at 80 °C for 6 h. After cooling, the mixture was diluted with iceꢀwater
and alkalinized with 2 N NaOH. The solid was filtered off and washed
with water. The obtained mixture of (E)-3-(4-aminobenzylidene)chroman-
4-one (1j) and (E)-N-(4-[(4-oxochroman-3-ylidene)methyl]phenyl)-
acetamide (1k) was separated by column chromatography on silica gel,
eluting with AcOEt/petroleum ether (1:1).
(E)-3-(2,3-Dimethoxybenzylidene)chroman-4-one (1u).
Yield: 53%. Mp = 108ꢀ109 °C EtOAc/petroleum ether. IR (KBr):
1673 cmꢀ1. 1H NMR: δ (ppm) 7.90 (dd, 1H, H5, J5ꢀ6 = 7.8 Hz, J5ꢀ7
1.7 Hz), 7.82 (bs, 1H, dCH), 7.61 (ddd, 1H, H7, J7ꢀ8 = 8.9 Hz, J6ꢀ7
=
=
7.2 Hz, J5ꢀ7 = 1.7 Hz), 7.22ꢀ7.12 (m, 3H, H6, H50, H60), 7.05 (d, 1H,
H8, J7ꢀ8 = 8.3 Hz), 6.83 (dd, 1H, H40, J4 -5 = 6.9 Hz, J4 -6 = 2.2 Hz),
5.29 (d, 2H, H2 Jall = 1.8 Hz), 3.86 (s, 3H, OCH3), 3.74 (s, 3H,
OCH3). 13C NMR: δ (ppm) 181.2, 160.8, 152.5, 147.6, 136.3, 132.2,
131.3, 127.6, 127.3, 124.1, 122.0, 121.8, 121.5, 118.0, 114.8, 67.6,
60.6, 55.8.
0
0
0
0
(E)-3-(2,3,4-Trimethoxybenzylidene)chroman-4-one (1v).
Yield: 43%. Mp = 116ꢀ118 °C (lit. viscous oil)50 from acetone. IR
(KBr): 1668 cmꢀ1. 1H NMR: δ (ppm) 7.89 (dd, 1H, H5, J5ꢀ6 = 7.8 Hz,
J5ꢀ7 = 1.8 Hz), 7.79 (bs, 1H, dCH), 7.60 (ddd, 1H, H7, J7ꢀ8 = 8.9 Hz,
J6ꢀ7 = 7.2 Hz, J5ꢀ7 = 1.8 Hz), 7.13 (ddd, 1H, H6, J6ꢀ7 = 7.2 Hz, J5ꢀ6
=
7.8 Hz, J6ꢀ8 = 1.0 Hz), 7.05 (dd, 1H, H8, J7ꢀ8 = 8.9 Hz, J6ꢀ8 = 1.0 Hz),
(E)-3-(4-Aminobenzylidene)chroman-4-one (1j). Yield: 30%.
Mp = 164ꢀ66 °C from EtOAc/petroleum ether. IR (KBr): 3430, 3340,
1650 cmꢀ1. 1H NMR: δ (ppm) 7.85 (dd, 1H, H5, J5ꢀ6 = 7.8 Hz, J5ꢀ7 = 1.8
Hz), 7.62 (bs, 1H, =CH), 7.55 (ddd, 1H, H7, J6ꢀ7 = 7.2 Hz, J7ꢀ8 = 8.4 Hz,
0
6.98 (s, 1H, H60, J2 -6 = 8.7 Hz), 6.92 (s, 1H, H5 , J2 -6 = 8.7 Hz), 5.33 (d,
2H, H2, Jall = 1.8 Hz), 3.88 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.80 (s,
3H, OCH3). 13C NMR: δ (ppm) 181.1, 160.6, 155.3, 152.7, 141.7,
136.0, 132.0, 129.5, 127.2, 125.6, 121.9, 121.6, 120.3, 117.8, 107.7, 67.7,
61.3, 60.5, 56.0.
0
0
0
0
J5ꢀ7 = 1.8 Hz), 7.21 (d, 2H, H20, H60, J2 -3 = 8.6 Hz), 7.10 (ddd, 1H, H6,
J6ꢀ7 =7.2Hz,J5ꢀ6 =8.2Hz,J6ꢀ8 = 1.0 Hz), 7.03 (dd, 1H, H8, J7ꢀ8 =8.4Hz,
0
0
J6ꢀ8 = 1.0 Hz), 6.66 (d, 2H, H30, H50, J2 -3 = 8.6 Hz), 5.95 (bs, 2H, NH2),
5.43 (d, 2H, H2, Jall = 1.8 Hz). 13C NMR: δ (ppm) 180.6, 160.2, 151.2,
137.8, 135.4, 133.0, 127.0, 124.6, 121.8, 121.6, 121.0, 117.6, 113.5, 67.8.
(E)-N-(4-[(4-Oxochroman-3-ylidene)methyl]phenyl)acetamide
(1k). Yield: 25%. Mp = 175ꢀ176 °C from EtOAc/petroleum ether. IR
(KBr): 3300, 1664, 1660 cmꢀ1. 1H NMR: δ (ppm) 10.23 (s, 1H, NH),
7.88 (dd, 1H, H5, J5ꢀ6 = 7.8 Hz, J5ꢀ7 = 1.6 Hz), 7.74ꢀ7.70 (m, 3H, H20,
H60, dCH), 7.59 (ddd, 1H, H7, J6ꢀ7 = 7.1 Hz, J7ꢀ8 = 8.4 Hz, J5ꢀ7 = 1.6
0
0
Synthesis of (E)-3-(3-Amino-4-hydroxybenzylidene)chro-
man-4-one (1r). Tin(II) chloride (0.03 mol) in EtOH (15 mL) was
added to a suspension of (E)-3-(4-hydroxy-3-nitrobenzylidene)chro-
man-4-one (1q) (0.005 mol) in EtOH(35 mL) and concentrated HCl
(75 mL). The mixture was refluxed for 2 h under stirring. After cooling,
the mixture was neutralized with 2 N NaOH and the solid was filtered
off. The product was purified by column chromatography on silica gel
(AcOEt/petroleum ether 1:1). Yield: 46%. Mp = 192ꢀ195 °C from
EtOAc/petroleum ether. IR (KBr): 3460, 3380, 1655 cmꢀ1. 1H NMR: δ
(ppm) 9.80 (bs, 1H, OH), 7.86 (d, 1H, H5, J5ꢀ6 = 7.6 Hz), 7.59ꢀ7.55
(m, 2H, dCH, H7), 7.12 (t, 1H, H6, J5ꢀ6 = J6ꢀ7 = 7.6 Hz), 7.04 (d, 1H,
Hz), 7.44 (d, 2H, H30, H50, J2 -3 = 8.6 Hz), 7.13 (ddd, 1H, H6, J6ꢀ7 = 7.1
0
0
Hz, J5ꢀ6 = 7.8 Hz, J6ꢀ8 = 0.7 Hz), 7.06 (dd, 1H, H8, J7ꢀ8 = 8.4 Hz, J6ꢀ8
=
0.7 Hz), 5.45 (d, 2H, H2, Jall = 1.6 Hz), 2.09 (s, 3H, CH3). 13C NMR: δ
(ppm) 181.0, 168.7, 160.5, 140.8, 136.3, 136.0, 131.5, 129.1, 128.3,
127.2, 121.9, 121.5, 118.7, 117.8, 67.5, 24.1.
H8, J7ꢀ8 = 8.3Hz), 6.78ꢀ6.75 (m, 2H, H20, H60), 6.61(d, 1H, H50, J5 -6
=
0
0
7.9 Hz), 5.43 (d, 2H, H2, Jall= 1.5 Hz), 4.76 (bs, 2H, NH2). 13C NMR: δ
(ppm) 181.0, 160.4, 146.5, 137.9, 137.0, 135.7, 127.1, 126.8, 125.3,
121.8, 121.7, 120.9, 117.7, 115.9, 114.3, 67.7.
General Procedure for the Synthesis of (E)-3-Benzylide-
nechroman-4-ones 1i and 1sꢀv. A mixture of the appropriate
chroman-4-one (0.01 mol) with substituted benzaldehyde (0.01 mol) in
dry EtOH saturated with HCl (40 mL) was stirred at room temperature
for 48 h. After this time, the mixture was diluted with iceꢀwater and the
solid filtered off and washed with water. The product was purified by
column chromatography on silica gel or by crystallization.
Pharmacological Studies. Determination of hMAO Iso-
form Activity. The effects of the test compounds on hMAO isoform
enzymatic activity were evaluated by a fluorimetric method following the
experimental protocol previously described.52
Reversibility and Irreversibility Experiments. To evaluate
whether some of the tested compounds (1h and 11) are reversible or
irreversible hMAO-B inhibitors, an effective centrifugationꢀultrafiltration
method (so-called repeated washing) was used.40
Molecular Modeling. Compounds 1h and 1l were built by means
of the Maestro GUI.53 Conformational properties of both molecules
have been investigated by means of 1000 steps of Monte Carlo (MC)
search as implemented in Macromodel, version 7.2.54 Each conformer
was energy-minimized using the AMBER*55 force field in united atoms
(E)-3-Benzylidene-6-chlorochroman-4-one (1i). Yield: 86%.
Mp
=
158ꢀ159 °C (lit. 148ꢀ150 °C)51 from EtOAc. IR
(KBr): 1660 cmꢀ1. H NMR (CDCl3): δ (ppm) 7.98 (d, 1H, H5,
J5ꢀ7 = 2.6 Hz), 7.90 (bs, 1H, dCH), 7.49ꢀ7.37 (m, 4H, H7, H30-H50),
7.33ꢀ7.30 (m, 2H, H20, H60), 6.93 (d, 1H, H8, J7ꢀ8 = 8.8 Hz), 5.35 (d,
2H, H2, Jall = 1.8 Hz). 13C NMR (DMSO d6): δ (ppm) 180.2, 159.2,
137.3, 135.7, 133.5, 130.3, 129.8, 128.8, 126.0, 125.9, 124.8, 122.4,
120.3, 67.5.
1
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Journal of Medicinal Chemistry
ARTICLE
notation. Water solvent effects have been taken into account by means of
the implicit model GB/SA.56 The global minimum energy structures of
1h and 1l were considered for the next docking simulations. Crystal-
lographic structures, deposited into the Protein Data Bank (PDB)57 with
codes 2Z5X58 and 2V60,59 were considered, after graphical manipula-
tion (fixing missing atoms and/or bond order, adding nonaliphatic H
atoms), as receptor models of hMAO-A and hMAO-B, respectively. The
cocrystallized ligands harmine and 7-(3-chlorobenzyloxy)-4-carboxalde-
hyde-coumarin, respectively, for 2Z5X and 2V60, were removed, FAD
double bonds were fixed, and hydrogen atoms were added onto both
proteins and cofactors.
(5) Shih, J. C.; Chen, K. Regulation of MAO-A and MAO-B gene
expression. Curr. Med. Chem. 2004, 11, 1995–2005.
(6) Shih, J. C.; Chen, K.; Ridd, M. J. Monoamine oxidase: from genes
to behavior. Annu. Rev. Neurosci. 1999, 22, 197–217.
(7) Youdim, M. B. H.; Finberg, J. P. M. New directions in mono-
amine oxidase A and B. Selective inhibitors and substrates. Biochem.
Pharmacol. 1991, 41 (2), 155–162.
(8) Gottowik, J.; Cesura, A. M.; Malherbe, P.; Lang, G.; Prada, M. D.
Characterisation of wild-type and mutant forms of human monoamine
oxidase A and B expressed in a mammalian cell line. FEBS Lett. 1993,
317, 152–156.
(9) Geha, R. M.; Rebrin, I.; Chen, K.; Shih, J. C. Substrate and
inhibitor specificities for human monoamine oxidase A and B are
influenced by a single amino acid. J. Biol. Chem. 2001, 276, 9877–9882.
(10) Westlund, K. N.; Denney, R. M.; Kochersperger, L. M.; Rose,
R. M. Distinct monoamine oxidase A and B populations in primate brain.
Science 1985, 230, 181–183.
(11) Bach, A. W. J.; Lan, N. C.; Johnson, D. L.; Abell, C. W.;
Bembenek, M. E.; Kwan, S. W.; Seeburg, P. H.; Shih, J. C. cDNA cloning
of human liver monoamine oxidase A and B: molecular basis of
differences in enzymic properties. Proc. Natl. Acad. Sci. U.S.A. 1988,
85, 4934–4938.
According to the AutoDock Vina48 docking methodology, a regular
box of 15 625 Å3, centered on the FAD N5, was considered as the active
site in both hMAO-A and -B receptor models. The global minimum
energy conformers of 1h and 1l were submitted to flexible docking
simulations. The estimation of the ΔGbind was carried out according to
the following equation:
n
ΔGbind ¼ ∑ ðIE ÞðP Þ
c
c
100
c¼1
(12) Grimsby, J.; Chen, K.; Wang, L. J.; Lan, N. C.; Shih, J. C.
Human monoamine oxidase A and B genes exhibit identical
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where ΔGbind is the Boltzmann averaged binding free energy, c is the
configuration of the ligandꢀtarget complex, n is the maximum number
of configurations, IEc is the AutoDock Vina interaction energy of the
configuration c, and Pc is the Boltzman population at 300 K for
configuration c.
’ ASSOCIATED CONTENT
S
Supporting Information. Elemental analysis results (C,
b
H, N, Cl) of tested compounds and molecular modeling details.
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ39-06-49913892. Fax: þ39-06-49693268. E-mail:
nicoletta.desideri@uniroma1.it.
’ ACKNOWLEDGMENT
We thank MURST (Italy) for financial support.
’ ABBREVIATIONS USED
MAO, monoamine oxidase; MAO-A, monoamine oxidase A; MAO-
B, monoamine oxidase B; PTK, protein tyrosine kinase; hMAO,
human MAO; PDB, Protein Data Bank
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