1,5-Diphenylpenta-2,4-dien-1-ones as potent and selective monoamine oxidase-B inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.11.006

A series of (2E,4E)-1-(2-hydroxyphenyl)-5-phenylpenta-2,4-dien-1-ones (3a-r) and (2Z,4E)-3-hydroxy-1-(2-hydroxyphenyl)-5-phenylpenta-2,4-dien-1-ones (6a-l) were synthesized and evaluated in vitro as inhibitors of the two human Monoamine oxidase (hMAO) iso

Full text of DOI:10.1016/j.ejmech.2012.11.006

European Journal of Medicinal Chemistry 59 (2013) 91e100
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
1,5-Diphenylpenta-2,4-dien-1-ones as potent and selective monoamine oxidase-B
inhibitors
,
a
a
a
b
Nicoletta Desideri a , Rossella Fioravanti , Luca Proietti Monaco , Mariangela Biava , Matilde Yáñez ,
*
Francesco Ortuso c, Stefano Alcaro c
a Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza e Università di Roma, P.le Aldo Moro 5, 00185 Rome, 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 della Salute, Università “Magna Græcia” di Catanzaro, Campus Universitario “S. Venuta”, Viale Europa, 88100 Catanzaro, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2012
Received in revised form
6 November 2012
Accepted 7 November 2012
Available online 15 November 2012
A series of (2E,4E)-1-(2-hydroxyphenyl)-5-phenylpenta-2,4-dien-1-ones (3aer) and (2Z,4E)-3-hydroxy-
1-(2-hydroxyphenyl)-5-phenylpenta-2,4-dien-1-ones (6ael) were synthesized and evaluated in vitro as
inhibitors of the two human Monoamine oxidase (hMAO) isoforms, MAO-A and MAO-B. Most of the
compounds showed a selective MAO-B inhibitory activity in the nanomolar or low micromolar range.
(2E,4E)-5-(4-Chlorophenyl)-1-(2-hydroxy-4-methoxyphenyl)penta-2,4-dien-1-one (3g) and (2E,4E)-5-
(4-chlorophenyl)-1-(2,4-dihydroxyphenyl)penta-2,4-dien-1-one (3h) were the most potent hMAO-B
inhibitors exhibiting IC50 of 4.51 nM and 11.35 nM, respectively, coupled with high selectivity. More-
over, partial recovery of MAO-B activity was observed after repeated washing in the presence of isatin
(reversible inhibitor) and compounds 3g and 3h suggesting a reversible inhibition of the enzyme.
Molecular mechanics and quantum chemistry methods were used to elucidate the MAO recognition of
the most active inhibitors 3g and 3h.
Keywords:
1,5-Diphenylpenta-2,4-dien-1-ones
Monoamine oxidase
hMAO-B inhibitors
Structureeactivity relationship
Docking
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
preponderant in placenta, lung and small intestine, while MAO B is
the only isoform in platelets and lymphocytes. In brain MAO B
Monoamine oxidase (MAO, EC 1.4.3.4) is a flavoenzyme that
catalyzes the deamination of a broad spectrum of biogenic and
xenobiotic amines. Two isoforms of MAOs have been identified and
designated as MAO-A and MAO-B. The MAO isozymes are products
of separate genes and they share approximately 70% amino acid
sequence identity [1]. Studies on X-ray crystallographic structures
of human MAO-A and MAO-B indicate that 6 of the 16 amino acid
residues comprising the active sites differ between the two
enzymes [2,3]. For these reasons, MAO-A and MAO-B have different
substrate and inhibitor specificities. MAO-A preferentially metab-
olizes the neurotransmitters, serotonin, epinephrine and norepi-
nephrine, and it is selectively inhibited by clorgyline. MAO-B are
(present in glial cells) predominates over MAO A [1]. These prop-
erties determine the therapeutic potential of MAO inhibitors. MAO-
A inhibitors have therapeutic utility for the treatment of depression
[5] whereas MAO-B inhibitors are used with L-DOPA and/or dopa-
mine agonists in the symptomatic treatment of Parkinson’s disease
[6,7], and are potential drugs in the therapy of Alzheimer’s disease
(AD) [8,9]. However, the first generation of non selective and irre-
versible MAO inhibitors was characterized by adverse side-effects
such as serious hypertensive crisis following the ingestion of die-
tary tyramine. The efforts devoted to the discovery of safer inhib-
itors led to the new generation of MAO inhibitors characterized by
the selectivity against MAO isoforms and in some cases by the
reversibility of action. As the ideal drug candidate has not been
achieved, researchers continued to explore this field.
Recently, we reported new series of MAO inhibitors related to
natural compounds as flavones, thioflavones and flavanones [10],
homoisoflavones [11], and chalcones [12], the biogenetic precur-
sors of flavonoids. The potent and selective hMAO-B inhibitory
activity of several synthetic chalcones [12] and of 1,4-diphenyl-2-
butene [13], prompted us to designed and tested against hMAO-A
and hMAO-B isoforms a series of structurally related (2E,4E)-1-(2-
predominantly involved in the metabolism of benzylamine and b-
phenylethylamine, and it is selectively inhibited by selegiline and
rasagiline. Dopamine and tyramine are considered to be a substrate
for both isozymes [4]. Although in humans both isozymes are
expressed in most peripheral tissues and organs, MAO-A is
* Corresponding author. Tel.: þ39 06 49913892; fax: þ39 06 49693268.
E-mail address: nicoletta.desideri@uniroma1.it (N. Desideri).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.006

92
N. Desideri et al. / European Journal of Medicinal Chemistry 59 (2013) 91e100
hydroxyphenyl)-5-phenylpenta-2,4-dien-1-ones
(2Z,4E)-3-hydroxy-1-(2-hydroxyphenyl)-5-phenylpenta-2,4-dien-
1-one (6aej).
(3aer)
and
hydroxyl groups to obtain the compounds 3h, 3n, 3o, 3p and 3q
was achieved during the work up of the reaction mixture.
(2Z,4E)-3-Hydroxy-1-(2-hydroxyphenyl)-5-phenylpenta-2,4-
dien-1-ones (6aej) were prepared according to the two step
procedure shown in Scheme 2. The appropriate 2-
hydroxyacetophenone (1aee) was first converted into the corre-
sponding 2-acetylphenyl (E)-cinnamate (5aej) by treatment with
substituted cinnamic acid (4aeg) in the presence of phosphoryl
chloride, in dry pyridine. In the second step, the Bakere
Venkataraman rearrangement of the formed ester (5aej) into
desired 6aej was achieved in the presence of potassium carbonate,
in dry acetone.
2. Results and discussion
2.1. Chemistry
(2E,4E)-1-(2-Hydroxyphenyl)-5-phenylpenta-2,4-dien-1-ones
(3aer) were synthesized by base-catalyzed reaction of the appro-
priate 2-hydroxyacetophenone (1aeg) and substituted cinna-
maldehyde (2aef). The condensation was performed in hydro-
alcoholic sodium hydroxide or in methanol using barium
hydroxide octahydrate as base (Scheme 1). The deprotection of
The hydroxy-analogs 6k and 6l were achieved by treatment
with boron tribromide of a solution of the corresponding methoxy
derivatives 6e and 6f in dry dichloromethane (Scheme 3).
R
OH
O
R3
R4
R1
R2
(i)
3a, 3b, 3c, 3f, 3j, 3k, 3l, 3m, 3n, 3o, 3p, 3q
R
OH
O
R3
R4
+
O
R1
R
OH
R3
R4
R2
H
(ii)
1a-g
2a-f
R1
R2
O
1a R = R1 = R2 = H
3d, 3e, 3g, 3h, 3i, 3r
1b R = R2 = H, R1 = Cl
1c R = R2= H, R1 = F
3a R = R1 = R2 = R3 = R4
= H
1d R = R1 = H, R2 = OCH3
1e R = OCH3, R1 = R2 = H
3b R = R2 = R3 = R4 H, R1 = Cl
=
3c R = R2 = R4
3d R = R2 = R4
3e R = R2 = R4
=
=
=
H, R1 = Cl, R3 = OCH3
H, R1 = Cl, R3 = NO2
H, R1 = R3 = Cl
1f R = (tetrahydro-2H-pyran-2-yl)oxy, R1 = R2 = H
1g R = R1 = H, R2 = (tetrahydro-2H-pyran-2-yl)oxy
3f R = R1 = R2 = R4
=
H, R3 = Cl
3g R = OCH3, R1 = R2 = R4
3h R = OH, R1 = R2 = R4 H, R3 = Cl
3i R = R2 = R3 = R4 H, R1 = F
3j R = R2 = R4 H, R1 = F, R3 = OCH3
3k R = R1 = R2 = R4 H, R3 = OCH3
3l R = R1 = R2 H, R3 = R4 = OCH3
3m R = R1 = H, R2 R3 = R4 = OCH3
=
H, R3 = Cl
2a R3 = R4 = H
=
2b R3 = Cl, R4 = H
=
2c R3 = OCH3, R4 = H
2d R3 = NO2, R4 = H
2e R3 = R4 = OCH3
2f R3 = OCOCH3, R4 = OCH3
=
=
=
=
3n R = R1 = R2 H, R3 = OH, R4 = OCH3
=
3o R = R1 = H, R3 = OH, R2
3p R = R1 = H, R2 = OH, R3
=
R4 = OCH3
R4 = OCH3
=
3q R = R1 = H, R2 = R3
=
OH, R4 = OCH3
3r R = R1 = R2 = R4 H, R3 = NO2
=
aReagents and conditions: (i) 60% NaOH, MeOH, r.t. 48 h; for 3n and 3o 60 °C 5 h; (ii) Ba(OH)2 . 8H2O, MeOH,
50 °C, 5 h.
Scheme 1.

N. Desideri et al. / European Journal of Medicinal Chemistry 59 (2013) 91e100
93
R3
R4
R
OH
O
R3
R4
O
(i)
+
O
R
O
R1
R2
OH
R1
5a-j
R2
O
1a-e
4a-g
(ii)
1a R = R1 = R2 = H
1b R = R2 = H, R1 = Cl
1c R = R2= H, R1 = F
R
OH
R3
R4
1d R = R1 = H, R2 = OCH3
1e R = OCH3, R1 = R2 = H
R1
R2
O
OH
6a-k
5a, 6a R = R1 = R2 = R3 = R4
4a R3 = R4 = H
4b R3 = Cl, R4 = H
4c R3 = OCH3, R4 = H
4d R3 = CH3, R4 = H
4e R3 = NO2, R4 = H
4f R3 = R4 = OCH3
=
H
5b, 6b R = R2 = R4 H, R1 = R3 = Cl
=
5c, 6c R = R1 = R2 = R4
5d, 6d R = R2 = R3 = R4
=
H, R3 = Cl
H, R1 = F
=
5e, 6e R = R1 = R2 = R4
=
H, R3 = OCH3
4g R3 = OCOCH3, R4 = OCH3
5f, 6f R = R1 = R2
=
H, R3 = R4 = OCH3
R3 = R4 = OCH3
R = R1 = H, R3 = OCOCH3, R2
5g, 6g R = R1 = H, R2
=
5h
=
R4 = OCH3
=
R = R1 = H, R3 = OH, R2 R4 = OCH3
6h
5i, 6i
5j, 6j
R = R1 = R2 = R4
R = R1 = R2 = R4
=
=
H, R3 = NO2
H, R3 = CH3
aReagents and conditions: (i) POCl3, Py, r.t., 4 h; (ii) K2CO3, dry acetone, reflux, 24 h.
Scheme 2.
2.2. Biochemistry
hydrogen peroxide from p-tyramine using the Amplex Red MAO
assay kit 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. The hMAO activity was
The inhibitory potencies of the compounds on hMAO activity
were investigated by measuring their effects on the production of
Scheme 3.

94
N. Desideri et al. / European Journal of Medicinal Chemistry 59 (2013) 91e100
evaluated by measuring the fluorescence generated by resorufin
using the general procedure described previously by us [14].
The hMAO-A and hMAO-B inhibition data and the selectivity
indexes (SI ¼ IC50 hMAO-A/IC50 hMAO-B) of the (2E,4E)-1-(2-
the methoxyl group in the potent hMAO-B inhibitor 3g with the
hydroxyl group in the same position resulted in compound 3h, able
to inhibit both hMAO isoforms. Although about 2-fold less potent
than 3g as hMAO-B inhibitor, 3h remain potent in the nanomolar
range and endowed with an excellent selectivity (IC50 ¼ 11.35 nM,
SI ¼ 1354). In this series of penta-2,4-dien-1-ones (3aer), only the
trimethoxy analog 3m was essentially inactive against both iso-
hydroxyphenyl)-5-phenylpenta-2,4-dien-1-ones
(3aer)
and
(2Z,4E)-3-hydroxy-1-(2-hydroxyphenyl)-5-phenylpenta-2,4-dien-
1-ones (6ael) are reported in Tables 1 and 2, respectively, together
with the results obtained for the reference inhibitors (clorgyline,
selegiline, isatin, iproniazid and moclobemide).
forms up to the highest concentration tested (100
mM). The
replacement of one or two methoxyl groups in 3m with hydroxyl
groups results in compounds (3o, 3p and 3q) with affinity toward
both hMAO isoforms. On the contrary, the removal of one or two
methoxyl groups (compounds 3l and 3k, respectively) restored
only the MAO-B affinity in the micromolar range. The replacement
of the 3-methoxyl group in 3l with the hydroxyl group to obtain
compound 3n, confirmed the ability of poly-hydroxy substituted
analogs to inhibit both isoforms.
The introduction of a hydroxyl group in position 3 of the
chain results in the (2Z,4E)-3-hydroxy-1-(2-hydroxyphenyl)-5-
phenylpenta-2,4-dien-1-ones (6ael). This structural modification
produced a loss of the inhibitory activity or a marked reduc-
tion in potency with respect to unsubstituted (2E,4E)-1-(2-
hydroxyphenyl)-5-phenylpenta-2,4-dien-1-ones (3aer) (Table 2).
The only exception was represented by (2Z,4E)-1-(5-fluoro-
2-hydroxyphenyl)-3-hydroxy-5-phenylpenta-2,4-dien-1-one (6d)
that showed a potent hMAO-B inhibitory activity slightly higher
Generally, the new tested compounds selectively inhibited
the enzymatic activity of hMAO-B in the nanomolar or low
micromolar range. The most interesting compound of the entire
series of inhibitors was (2E,4E)-5-(4-chlorophenyl)-1-(2-hydroxy-
4-methoxyphenyl)penta-2,4-dien-1-one 3g, showing potent
hMAO-B activity and high selectivity (IC50 ¼ 4.51 nM, SI > 22,173).
Within the series of (2E,4E)-1-(2-hydroxyphenyl)-5-phenylpenta-
2,4-dien-1-ones (3aer), in addition to the affinity for hMAO-B,
some derivatives (3f, 3h, 3n, 3o, 3p and 3q) are also able to
inhibit hMAO-A in the micromolar range. These compounds
demonstrate a different extent of hMAO-B selectivity, showing SIs
ranging from 1.5 to 1354. With the exception of 3f, these analogs
bear at least another hydroxyl group in the aromatic rings in
addition to the hydroxyl group in position 2. The same behavior
was observed for related chalcones and homoisoflavones previ-
ously studied by us [11,12]. It is noteworthy that the replacement of
Table 1
hMAO inhibitory activities and selectivity indexes of (2E,4E)-1-(2-hydroxyphenyl)-5-phenylpenta-2,4-dien-1-ones (3aer), (E)-1-(5-fluoro-2-hydroxyphenyl)-3-phenylprop-
2-en-1-one (7) and reference inhibitors.
R
OH
R3
OH
R1
R4
F
R2
O
O
3a-r
7
a
a
Compd
R
R1
R2
R3
R4
MAO-A (IC50
)
MAO-B (IC50
)
SId
e
e
e
e
e
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
H
H
H
H
H
H
OCH3
OH
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
OCH3
OH
OH
H
H
H
OCH3
NO2
Cl
Cl
Cl
Cl
H
OCH3
OCH3
OCH3
OCH3
OH
OH
OCH3
OH
NO2
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
105.51 ꢀ 5.68 nM
54.77 ꢀ 2.63 nM
153.14 ꢀ 6.21 nM
>948h
>1826h
>653h
>93h
1.08 ꢀ 0.08
1.46 ꢀ 0.13
m
m
M
M
>68h
37.73 ꢀ 1.57
15.37 ꢀ 1.18
m
m
Mb
Mb
860.46 ꢀ 43.18 nM
4.51 ꢀ 0.28 nM
44
e
>22,173h
1354
11.35 ꢀ 0.73 nM
75.84 ꢀ 7.66 nM
725.92 ꢀ 31.56 nM
e
e
f
>1319h
>138h
>55h
F
H
H
H
H
H
H
H
H
F
1.81 ꢀ 0.15
m
m
M
M
14.61 ꢀ 2.18
>6.8h
f
f
e
1.56 ꢀ 0.19
1.03 ꢀ 0.25
25.16 ꢀ 1.96
1.43 ꢀ 0.22
m
m
m
m
Mb
Mb
Mb
Mc
501.66 ꢀ 49.19 nM
486.35 ꢀ 43.36 nM
3.1
2.1
17
1.5
1.45 ꢀ 0.16
m
M
3q
3r
7
969.44 ꢀ 87.45 nM
160.32 ꢀ 12.48 nM
667.89 ꢀ 59.32 nM
e
f
H
H
>624h
>150h
0.000073
3431
>4.7h
0.87
H
H
Clorgyline
Selegiline
Isatin
Iproniazid
Moclobemide
4.46 ꢀ 0.32 nMb
61.35 ꢀ 1.13
mM
67.25 ꢀ 1.02
m
Mb
19.60 ꢀ 0.86 nM
f
21.07 ꢀ 1.47
7.54 ꢀ 0.36
m
m
M
M
6.56 ꢀ 0.76
m
m
M
M
g
361.38 ꢀ 19.3
<0.36i
a
All IC50 values shown in this table are the mean ꢀ S.E.M. from five experiments. Level of statistical significance.
b
c
d
e
f
P < 0.01 or.
P < 0.05 versus the corresponding IC50 values obtained against MAO-B, as determined by ANOVA/Dunnett’s.
SI: hMAO-B selectivity index ¼ IC50(hMAO-A)/IC50(hMAO-B).
Inactive at 100
100
Inactive at 1 mM (highest concentration tested).
mM (highest concentration tested).
m
M inhibits the corresponding MAO activity by approximately 40e50%. At higher concentration the compounds precipitate.
g
h
i
Values obtained under the assumption that the corresponding IC50 against MAO-A is the highest concentration tested (100 mM).
Values obtained under the assumption that the corresponding IC50 against MAO-B is the highest concentration tested (1 mM).

N. Desideri et al. / European Journal of Medicinal Chemistry 59 (2013) 91e100
95
Table 2
hMAO inhibitory activities and selectivity indexes of (2Z,4E)-3-hydroxy-1-(2-hydroxyphenyl)-5-phenylpenta-2,4-dien-1-ones (6ael) and reference inhibitors.
6a-l
R4
a
a
Compd
R
R1
R2
R3
MAO-A (IC50
)
MAO-B (IC50
)
SId
e
e
e
e
e
e
f
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
H
H
Cl
Cl
H
OCH3
OCH3
OCH3
OH
NO2
CH3
OH
OH
H
H
H
H
1.16 ꢀ 0.03
m
M
>86h
e
f
50.44 ꢀ 4.61 nM
>1983h
f
H
e
e
OCH3
OCH3
OCH3
H
H
H
3.76 ꢀ 0.42
1.52 ꢀ 0.04
m
m
Mb
Mb
1.68 ꢀ 0.21
2.67 ꢀ 0.28
3.77 ꢀ 0.19
m
m
m
M
M
M
2.2
e
e
>37h
>27h
57
H
H
H
26.46 ꢀ 1.97 nM
OH
431.07 ꢀ 52.45 nMc
4.46 ꢀ 0.32 nMb
654.32 ꢀ 61.05 nMb
0.66
Clorgyline
Selegiline
Isatin
Iproniazid
Moclobemide
61.35 ꢀ 1.13
m
M
0.000073
3431
67.25 ꢀ 1.02
m
Mb
19.60 ꢀ 0.86 nM
f
21.07 ꢀ 1.47
7.54 ꢀ 0.36
m
m
M
M
>4.7h
0.87
6.56 ꢀ 0.76
m
m
M
M
g
361.38 ꢀ 19.3
<0.36i
a
All IC50 values shown in this table are the mean ꢀ S.E.M. from five experiments. Level of statistical significance.
b
c
d
e
f
P < 0.01 or.
P < 0.05 versus the corresponding IC50 values obtained against MAO-B, as determined by ANOVA/Dunnett’s.
SI: hMAO-B selectivity index ¼ IC50(hMAO-A)/IC50(hMAO-B).
Inactive at 100
100
Inactive at 1 mM (highest concentration tested).
mM (highest concentration tested).
m
M inhibits the corresponding MAO activity by approximately 40e50%. At higher concentration the compounds precipitate.
g
h
i
Values obtained under the assumption that the corresponding IC50 against MAO-A is the highest concentration tested (100 mM).
Values obtained under the assumption that the corresponding IC50 against MAO-B is the highest concentration tested (1 mM).
than the corresponding (2E,4E)-1-(5-fluoro-2-hydroxyphenyl)-5-
phenylpenta-2,4-dien-1-one (3i) (IC50 ¼ 50.44 nM and 75.84 nM,
respectively) and better selectivity (SI > 1983 and >1319, respec-
tively). Also in this series of inhibitors, the compounds substituted
with more than one hydroxyl group in the aromatic rings (6h, 6k
and 6l) are able to inhibit both hMAO isoforms. In particular, 6l was
the only hMAO-A inhibitor of both series active in the sub-
micromolar range. Moreover, its potency toward B isoform was
slightly higher than the potency toward A isoform, resulting in an
essentially nonselective compound.
The reversibility tests revealed the lack of enzyme activity
restoration after repeated washing for compounds 3b, 6d and 6k.
Similar results were obtained for selegiline a well-known irre-
versible MAO-B inhibitor. On the contrary, significant recovery of
MAO-B activity was observed after repeated washing of isatin
(reversible inhibitor) and compound 3g. The enzymatic inhibition
lowered in a minor extent after repeated washing in the presence of
3h (Table 3). These results suggest that the novel potent hMAO-B
inhibitors 3g and 3h are reversible inhibitors.
To evaluate whether the most potent hMAO-B inhibitors (3b, 3g,
3h, 6d, 6k) are reversible or irreversible hMAO-B inhibitors, the so-
called repeated washing method was used [12]. The results ob-
tained using selegiline (irreversible inhibitor) and isatin (reversible
inhibitor) as reference compounds are reported in Table 3.
2.3. Molecular modeling studies
With the aim to rationalize the different hMAO inhibition of the
analogs 3g and 3h, molecular modeling studies were carried out.
Following our previous experience [15] we have adopted both
molecular mechanics and quantum chemistry methods to elucidate
the mechanism of inhibition of previously reported compounds. In
particular, the recognition of hMAO-A and -B and the role of the p-
methoxy substituent, available into the 3g in replacement of the 3h
hydroxy group, were investigated.
In order to obtain reasonable configuration of both inhibitors
into the hMAO-A and -B binding clefts, docking experiment were
performed. The top ranked theoretical complex revealed remark-
able similarities, actually, in all cases the ligands showed the
phenolic ring and the p-chlorophenyl moieties respectively posi-
tioned toward the FAD cofactor and the entrance gorge surrounded
by hydrophobic residues. With the aim to improve the analysis of
3g and 3h hMAO recognition, new complexes between our inhib-
itors and both target isoform models were manually built by means
of a 180ꢁ rotation of the corresponding top ranked poses. Resulting
Table 3
Reversibility and irreversibility of hMAO-B inhibition of derivatives 3b, 3g, 3h, 6d
and 6k.
Comp
% MAO-B inhibition
Before washing
After repeated washinga
3b (100 nM)
3g (10 nM)
3h (10 nM)
6d (100 nM)
6k (100 nM)
58.10 ꢀ 0.10
61.34 ꢀ 2.38
51. 52 ꢀ 3.18
62.35 ꢀ 0.45
66.70 ꢀ 1.52
64.31 ꢀ 2.08
53.08 ꢀ 1.64
60.30 ꢀ 2.53
37.51 ꢀ 1.13
42.02 ꢀ 0.70
68.80 ꢀ 3.33
68.55 ꢀ 4.39
31.22 ꢀ 0.91
54.63 ꢀ 1.72
Isatin (30
mM)
Selegiline (20 nM)
Each value is the mean ꢀ S.E.M. from five experiments (n ¼ 5).
a
Level of statistical significance: P < 0.01 versus the corresponding MAO-B
inhibition before washing, as determined by ANOVA/Dunnett’s.

96
N. Desideri et al. / European Journal of Medicinal Chemistry 59 (2013) 91e100
structures, after energy optimization and re-scoring procedure,
revealed interaction energy remarkably weaker than the original
top ranked poses (see Table 1 in Supplementary content). Such
a scenario and the docking score (see Figure S1 in Supplementary
content) were not in agreement to the experimental IC50 data, as
a consequence, each top ranked docking complex was submitted to
12 ns of molecular dynamics (MD). The resulting four trajectories
were analyzed in terms of inhibitor target interaction energy and
geometry (Section Experimental section) reporting a good quali-
tative agreement to the experimental inhibition data. Actually,
hMAO-B$3g resulted as the most energy favored complex with an
advantage of about 10 kcal/mol with respect to the hMAO-A$3g,
conversely predicted as the worst one. The hMAO-B binding ener-
gies of 3h indicated a disadvantage of 3.4 kcal/mol with respect to
3g on the contrary, into the hMAO-A, 1.2 kcal/mol demonstrated its
better recognition than 3g (see Figure S2 in Supplementary
content). The hMAO-B selectivity of the selected compounds was
also remarked by computing, during the molecular dynamics runs,
the target backbone root mean square deviation (RMSd) with
respect to the corresponding starting structures (see Figure S3 in
Supplementary content). As reported by the average RMSd values,
the target isoform B accommodate both inhibitors with lower
residues interacting to 3g and 3h, all MD trajectory frames were
investigated. The contribution of each residue to the inhibitor
binding was expressed as percentage of frames reporting its
interaction to the ligand (Fig. 1).
Due to wide movements into the hMAO-A cleft, both
compounds showed a larger number of interacting residues in this
isozyme (see Figures S4 and S5 in Supplementary content). The
percentage of frames reporting 3g or 3h interacting to the FAD
remarked that our ligands were located into the hMAO-B binding
cleft (3g ¼ 93.78%; 3h ¼ 92.12%) deeper than into the hMAO-A
(3g ¼ 82.16%; 3h ¼ 82.99%). Such an information suggested to
investigate the stacking interaction among the phenolic ring of our
compounds and the hMAO-A Tyr407 and 444 and the corre-
sponding hMAO-B Tyr398 and 435. The compound 3g into the
hMAO-B showed stacking with both residues for more than 90% of
the simulation time, while in all other cases a preference for one of
the two tyrosines was observed. The stacking stability could be
attributed to the 3g p-methoxy phenyl group. In order to deep
investigate the role of such a moiety, quantum mechanics calcula-
tions of both ligands molecular orbital were performed (Section
Experimental section).
As expected, even if molecular orbital distribution of our
compounds was similar, 3g revealed HOMO/LUMO areas
surrounding the p-methoxy phenyl ring larger than 3h (see
Figure S5 in Supplementary content) suggesting stronger interac-
tion to the tyrosine residues. Moreover, such a substituent high-
lighted a different role depending on the target. In the hMAO-A p-
ꢀ
ꢀ
ꢀ
perturbation (3g ¼ 1.86 A; 3h ¼ 2.44 A) than hMAO-A (3g ¼ 3.79 A;
ꢀ
3h ¼ 4.12 A). Such data were not a surprise because it is known [16]
3
ꢀ
that hMAO-B active site (w700 A ) is larger than hMAO-A one
3
ꢀ
(w400 A ), and contributed to rationalize the isoform selectivity of
our compounds. With the aim to identify the most relevant
Fig. 1. Representative molecular dynamics binding modes of 3g (left side) and 3h (right side) into the hMAO-A (top side) and hMAO-B (bottom side) active sites. Residues
interacting to the inhibitors with a frequency higher than 60% are reported in green carbons sticks or space fill, ligands are depicted in gray carbons balls and sticks. Yellow dotted
lines indicate hydrogen bonds. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

N. Desideri et al. / European Journal of Medicinal Chemistry 59 (2013) 91e100
97
methoxy phenyl ring cannot interact to Tyr197 and allowed the
hydrogen bonding between 3g o-phenolic group and both Asn181
side chain and Ile207 backbone. Unlike 3h, the interaction network
has forced the removal of the 3g aromatic ring from the FAD and
consequently from the Tyr407-444 stacking. Into the hMAO-B, the
substitution of the Asn181 with the Cys172 was mainly responsible
of our compounds isoform selectivity. In fact, the flexibility of the
Cys side chain was lower than Asn and the hydrogen bond to
Cys172 was unable to withdraw from the FAD both our inhibitors
that, therefore, established stacking contacts to Tyr398 and 435.
The hMAO-B active site, larger than hMAO-A, allowed the entrance
of solvent water molecules that contributed to the complexes
stabilization performing hydrogen bond bridge between the keto-
group of our compounds and the Tyr326 side chain.
hydroxyl group (synthesis of compounds 3neq), the mixture was
stirred at room temperature for 1 h. The resulting suspension was
filtered (3j) or extracted with AcOEt (3leq). In the latter case, the
combined organic phases were washed with saturated NaHCO3
solution and brine, then dried over Na2SO4, filtered and evaporated
to dryness. The crude product was purified by crystallization from
AcOEt (3j) or chromatographed on a silica gel column eluting with
AcOEt/petroleum ether 1:1 (3l and 3m), AcOEt/petroleum ether 1:3
(3p), AcOEt/petroleum ether 1:4 (3q) or CH2Cl2 (3n and 3o).
4.1.1.1. (2E,4E)-1-(5-Fluoro-2-hydroxyphenyl)-5-(4-methoxyphenyl)
penta-2,4-dien-1-one (3j). Yield: 78%; mp 191e193 ꢁC from AcOEt.
IR (KBr) 3450, 1620 cmꢂ1 1H NMR (CDCl3):
. d (ppm) 12.70 (s, 1H,
OH), 7.75 (dd, 1H, H
b
, Ja ¼ 14.5 Hz, Jb ¼ 10.3 Hz), 7.55e7.47 (m,
-
b
-g
3H, H6, H20, H60), 7.23 (dt, 1H, H4, J4-F ¼ 8.8 Hz, J3-4 ¼ 8.8 Hz, J4-
3. Conclusion
¼ 2.8 Hz), 7.12e6.86 (m, 6H, H
a, Hg, Hd
, H3, H30, H50), 3.87 (s, 3H,
6
OCH3). 13C NMR (CDCl3):
d
(ppm) 192.8 (d, JC-F ¼ 2.3 Hz),161.1, 159.7,
A new class of highly potent and selective hMAO-B inhibitors
was identified. The most potent derivatives 3g and 3h showed
inhibitory activity in the low nanomolar range coupled with high
selectivity. Moreover, the results of reversibility tests suggest that
these two compounds are reversible inhibitors of MAO-B. Based on
their potent and selective MAO-B inhibitory properties, these
compounds could be useful for the development of promising drug
candidate for the therapy of neurodegenerative diseases.
154.9 (d, JC-F ¼ 236.1 Hz),147.0,143.6,129.2, 128.7,124.4,123.5 (d, JC-
¼ 23.7 Hz), 121.7, 119.7 (d, JC-F ¼ 7.3 Hz), 119.6 (d, JC-F ¼ 6.1 Hz),
F
114.5, 114.4 (d, JC-F ¼ 21.3 Hz), 55.4.
4.1.1.2. (2E,4E)-5-(3,4-Dimethoxyphenyl)-1-(2-hydroxyphenyl)
penta-2,4-dien-1-one (3l). Yield: 48%; mp 193e196 ꢁC from
acetone. IR (KBr) 1632 cmꢂ1. 1H NMR (CDCl3):
d
(ppm) 12.96 (s, 1H,
2OH), 7.84 (dd, 1H, H6, J5-6 ¼ 8.1 Hz, J4-6 ¼ 1.6 Hz), 7.72 (dd, 1H, H
Jaꢂ ¼ 14.6 Hz, Jbꢂ ¼ 10.6 Hz), 7.47 (ddd, 1H, H4, J4-5 ¼ 7.2 Hz, J3-
b
,
b
g
4. Experimental section
¼ 8.5 Hz, J4-6 ¼ 1.6 Hz), 7.19 (d,1H, H , Jaꢂ ¼ 14.6 Hz), 7.08 (dd,1H,
a
4
b
H60, J5 -6 ¼ 8.3 Hz, J2 -6 ¼ 1.9 Hz), 7.05e6.86 (m, 6H, H
g, Hd, H3, H5,
0
0
0
0
4.1. Chemistry
H20, H50), 3.95 (s, 3H, OCH3), 3.93 (s, 3H, OCH3). 13C NMR (CDCl3):
d
(ppm) 193.7, 163.6, 150.7, 149.4, 145.9, 143.0, 136.1, 129.4, 129.2,
Chemicals were purchased from SigmaeAldrich 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 detected with
a Bruker AM-400 spectrometer, using TMS as internal standard. IR
spectra were recorded on a FT-IR PerkinElmer Spectrum 1000. All
compounds were routinely checked by thin-layer chromatography
(TLC) and 1H NMR. TLC was performed on silica gel or aluminum
oxide fluorescent coated plates (Fluka, DC-Alufolien Kieselgel or
aluminum oxide F254). Compound purity was determined by
elemental analysis and was confirmed to be >95% for all the tested
compounds. Analytical results are within ꢀ0.40% of the theoretical
values (Table 2 in Supplementary content). The synthetic inter-
mediates 2-hydroxy-4-[(tetrahydro-2H-pyran-2-yl)oxy]acetophe-
none (1f) [12], 2-hydroxy-6-[(tetrahydro-2H-pyran-2-yl)oxy]
acetophenone (1g) [17], (E)-3,4-dimethoxycinnamaldehyde (2e)
[18], and (E)-3-(4-acetoxy-3-methoxy)cinnamic acid (2f) [19] were
synthesized following the procedure previously described. (2E,4E)-
1-(2-Hydroxyphenyl)-5-phenylpenta-2,4-dien-1-ones (3aef, 3i,
3k, 3r) [20,21], 20-cinnamoyloxyacetophenones (5aee, 5i, 5j)
[21,22], (2Z,4E)-3-hydroxy-1-(2-hydroxyphenyl)-5-phenylpenta-
2,4-dien-1-ones (6aee, 6i, 6j, 6k) [21,22], and (E)-1-(5-fluoro-2-
hydroxyphenyl)-3-phenylprop-2-en-1-one (7) [21] were prepared
as previously reported by us.
124.9, 122.5, 121.7, 120.2, 118.7, 118.6, 111.3, 109.5, 56.0, 55.9.
4.1.1.3. (2E,4E)-5-(3,4-Dimethoxyphenyl)-1-(2-hydroxy-6-methoxy-
phenyl)penta-2,4-dien-1-one (3m). Yield: 48%; mp 136e139 ꢁC
from AcOEt/petroleum ether. IR (KBr) 1621 cmꢂ1 1H NMR
.
(CDCl3):
d
(ppm) 13.28 (s, 1H, OH), 7.66 (dd,1H, H
b
, Ja ¼ 14.8 Hz, Jb
-
b
-
¼ 10.4 Hz), 7.40 (d, 1H, H , Ja ¼ 14.8 Hz), 7.34 (t, 1H, H4, J3-4 ¼ J4-
a
g
-b
¼ 8.3 Hz), 7.08 (dd, 1H, H60, J5 -6 ¼ 8.3 Hz, J2 -6 ¼ 1.9 Hz), 7.05 (d,
0
0
0
0
5
1H, H20, J2 -6 ¼ 1.9 Hz), 6.97 (d, 1H, H
d
, Jg ¼ 15.3 Hz), 6.89 (dd, 1H,
0
0
-d
0
0
H
g
, Jb ¼ 10.4 Hz, Jg ¼ 15.3 Hz), 6.86 (d, 1H, H50, J5 -6 ¼ 8.3 Hz),
-g
-d
6.60 (d, 1H, H5, J4-5 ¼ 8.3 Hz), 6.41 (d, 1H, H3, J3-4 ¼ 8.3 Hz), 3.95 (s,
3H, OCH3), 3.94 (s, 3H, OCH3), 3.92 (s, 3H, OCH3). 13C NMR (CDCl3):
d
(ppm) 194.2, 164.9, 160.9, 150.4, 149.3, 144.2, 141.8, 135.6, 129.9,
129.5, 125.6, 121.5, 112.0, 111.3, 111.0, 109.5, 101.5, 55.99, 55.96,
55.85.
4.1.1.4. (2E,4E)-5-(4-Hydroxy-3-methoxyphenyl)-1-(2-hydroxyphenyl)
penta-2,4-dien-1-one (3n). Yield: 80%; mp 124e126 ꢁC from AcOEt/
petroleum ether. IR (KBr) 3455, 1630 cmꢂ1. 1H NMR (CDCl3):
d
(ppm)
12.96 (s, 1H, 2OH), 7.84 (dd, 1H, H6, J4-6 ¼ 1.5 Hz, J5-6 ¼ 8.1 Hz), 7.71
(dd, 1H, H
, Ja ¼ 14.6 Hz, Jb ¼ 10.7 Hz), 7.47 (ddd, 1H, H4, J4-
b
-b
-g
¼ 7.3 Hz, J3-4 ¼ 8.5 Hz, J4-6 ¼ 1.5 Hz), 7.18 (d, 1H, H , Ja ¼ 14.6 Hz),
a
5
-b
7.07 (dd, 1H, H60, J5 -6 ¼ 8.2 Hz, J2 -6 ¼ 1.8 Hz), 7.03e6.87 (m, 6H, H
g,
0
0
0
0
Hd
, H3, H5, H20, H50), 5.84 (s, 1H, 40OH), 3.95 (s, 3H, OCH3). 13C NMR
(CDCl3):
d (ppm) 193.7, 163.6, 147.5, 146.9, 146.0, 143.2, 136.1, 129.4,
4.1.1. General procedure for the synthesis of (2E,4E)-1-(2-
128.7, 124.6, 122.2, 122.1, 120.1, 118.7, 113.6, 114.9, 109.1, 56.0.
hydroxyphenyl)-5-phenylpenta-2,4-dien-1-ones (3j, 3leq)
An aqueous solution of 60% sodium hydroxide (50 mL) was
added dropwise to a stirred suspension of the appropriate 20-
hydroxyacetophenone (1a, 1c, 1d, 1g) (10 mmol) and substituted
cinnamaldehyde (2c, 2e, 2f) (11 mmol) in methanol (50 mL), cooled
in ice bath. The mixture was stirred at room temperature for 48 h
(3j, 3l, 3m, 3p, 3q) or heated at 60 ꢁC for 5 h (3n, 3o). After that
period, the suspension was diluted with ice and water and acidified
with 2 N hydrochloric acid. For the eventual deprotection of the
4.1.1.5. (2E,4E)-5-(4-Hydroxy-3-methoxyphenyl)-1-(2-hydroxy-6-
methoxyphenyl)penta-2,4-dien-1-one (3o). Yield: 58%; mp 113e
115 ꢁC from AcOEt/petroleum ether. IR (KBr) 3443, 1620 cmꢂ1. 1H
NMR (CDCl3):
d
(ppm) 13.29 (s, 1H, 2OH), 7.65 (dd, 1H, H
b
, Ja
-
¼ 14.8 Hz, Jb ¼ 10.3 Hz), 7.39 (d,1H, H
a
, Ja ¼ 14.8 Hz), 7.33 (t,1H,
b
-
g
-b
H4, J4-5 ¼ J3-4 ¼ 8.3 Hz), 7.06 (dd, 1H, H60, J5 -6 ¼ 8.2 Hz, J2 -
0
0
0
¼ 1.9 Hz), 7.00 (d, 1H, H20, J2 -6 ¼ 1.9 Hz), 6.95e6.83 (m, 3H, H
g
,
0
0
60
Hd
, H50), 6.60 (dd, 1H, H5, J4-5 ¼ 8.3 Hz, J3-5 ¼ 0.8 Hz), 6.40 (dd, 1H,

98
N. Desideri et al. / European Journal of Medicinal Chemistry 59 (2013) 91e100
H3, J3-4 ¼ 8.3 Hz, J3-5 ¼ 0.8 Hz), 5.83 (s, 1H, 40OH), 3.95 (s, 3H, OCH3),
165.1,143.5, 140.1,134.9,133.6,132.3,128.9,128.8,127.9,125.1,112.9,
108.3, 102.6.
3.93 (s, 3H, OCH3). 13C NMR (CDCl3):
d (ppm) 194.2, 164.9, 160.9,
147.1,146.8, 144.3,142.0,135.6,129.7,129.0, 125.3,121.8,114.8,112.0,
110.9, 109.1, 101.5, 56.0, 55.8.
4.1.4. General procedure for the synthesis of 2-acetylphenyl (E)-
cinnamates (5feh)
4.1.1.6. (2E,4E)-1-(2,6-Dihydroxyphenyl)-5-(3,4-dimethoxyphenyl)
Phosphorus oxychloride (30 mmol) were added to a solution of
the appropriate 2-hydroxyacetophenone (1a, 1d) (10 mmol) and
substituted cinnamic acid (4f, 4g) (12 mmol) in dry pyridine
(20 mL) cooled in ice bath. The solution was stirred at room
temperature for 4 h, than it was poured into ice and water and
acidified with 2 N HCl. The precipitate was removed by filtration,
washed with water and purified by silica gel column chromatog-
raphy eluting with AcOEt/light petroleum (1:3).
penta-2,4-dien-1-one (3p). Yield: 38%; mp 185e189 ꢁC from EtOH.
IR (KBr) 3405, 1625 cmꢂ1 1H NMR (CD3COCD3):
. d (ppm) 11.58 (s,
0
0
2H, OH), 7.75e7.64 (m, 2H, Ha, Hb
), 7.31 (d, 1H, H20, J2 -6 ¼ 2.0 Hz),
7.26 (t, 1H, H4, J3-4 ¼ 8.2 Hz), 7.17e7.10 (m, 3H, H
g, Hd
, H60), 6.97 (d,
1H, H50, J5 -6 ¼ 8.3 Hz), 6.43 (d, 2H, H3, H5, J3-4 ¼ 8.2 Hz), 3.87 (s, 3H,
0
0
OCH3), 3.85 (s, 3H, OCH3). 13C NMR (DMSO D6):
d (ppm) 194.0,
160.6, 150.2, 149.0, 144.7, 142.2, 134.8, 129.5, 129.0, 125.4, 121.8,
111.7, 111.6, 109.8, 107.1, 55.56, 55.57.
4.1.4.1. 2-Acetylphenyl (E)-3,4-dimethoxycinnamate (5f). Yield:
73%; mp 102e104 ꢁC from EtOH. The compound exhibited spec-
troscopic data identical to those previously reported [23].
4.1.1.7. (2E,4E)-1-(2,6-Dihydroxyphenyl)-5-(4-hydroxy-3-methoxy-
phenyl)penta-2,4-dien-1-one (3q). Yield: 45%; mp 176e179 ꢁC from
AcOEt/petroleum ether. IR (KBr) 3374, 1628 cmꢂ1 1H NMR
.
(CD3COCD3):
(m, 2H, Ha, Hb
d
(ppm) 11.5 (s, 2H, OH), 8.05 (s, 1H, OH), 7.73e7.64
4.1.4.2. 2-Acetyl-3-methoxyphenyl (E)-3,4-dimethoxycinnamate
(5g). Yield: 90%; mp 96e98 ꢁC from EtOH. The compound exhibi-
ted spectroscopic data identical to those previously reported [23].
), 7.31 (d, 1H, H20, J2 -6 ¼ 1.9 Hz), 7.26 (t, 1H, H4, J3-
0
0
, H60), 6.85 (d, 1H, H50, J5 -
0
¼ 8.2 Hz), 7.12e7.09 (m, 3H, H
g, Hd
4
60
¼ 8.2 Hz), 6.43 (d, 2H, H3, H5, J3-4 ¼ 8.2 Hz), 3.91 (s, 3H, OCH3). 13
C
NMR (DMSO D6):
d
(ppm) 194.0, 160.6, 148.5, 148.0, 145.0, 142.8,
4.1.4.3. 2-Acetyl-3-methoxyphenyl (E)-4-acetoxy-3-methoxycinna-
134.8, 128.9, 127.8, 124.5, 122.1, 115.6, 111.6, 110.5, 107.1, 55.7.
mate (5h). Yield: 48%; mp 143e146 ꢁC from AcOEt/petroleum
ether. IR (KBr) 1767, 1715, 1633 cmꢂ1. 1H NMR (CDCl3):
d (ppm) 7.78
4.1.2. Synthesis of (2E,4E)-5-(4-chlorophenyl)-1-(2-hydroxy-4-
methoxyphenyl)penta-2,4-dien-1-one (3g)
(d, 1H, H
b
, Ja ¼ 15.9 Hz), 7.38 (t, 1H, H5, J4-5 ¼ J5-6 ¼ 8.3 Hz), 7.18e
-
b
7.14 (m, 2H, H60, H20), 7.07 (d,1H, H50, J5 -6 ¼ 8.4 Hz), 6.85 (d,1H, H4,
0
0
Barium hydroxide octahydrate (15 mmol) was added to
a mixture of 2-hydroxy-4-methoxyacetophenone (1e) (10 mmol)
and 4-chlorocinnamaldehyde (2b) (10 mmol) in EtOH (200 mL).
The mixture was stirred at 50 ꢁC for 5 h and at room temperature
overnight. After removal of ethanol in vacuum, water was added to
the residue and the pH was adjusted to 2 with 2 N HCl. The mixture
was extracted with AcOEt. The combined organic phases were
washed with saturated NaHCO3 solution and brine, than dried over
Na2SO4, filtered and evaporated to dryness. The residue was puri-
fied by silica gel column chromatography eluting with AcOEt/
petroleum ether 1:4. Yield: 40%; mp 182e183 ꢁC from AcOEt. IR
J4-5 ¼ 8.3 Hz), 6.81 (d, 1H, H6, J3-4 ¼ 8.3 Hz), 6.52 (d, 1H, H
a, Ja
-
¼ 15.9 Hz), 3.89 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 2.51 (s, 3H,
b
COCH3), 2.32 (s, 3H, OCOCH3).
4.1.5. General procedure for the synthesis of (2Z,4E)-3-hydroxy-1-
(2-hydroxyphenyl)-5-phenylpenta-2,4-dien-1-ones (6f, 6g)
Anhydrous K2CO3 (50 mmol) was added to a solution of the
appropriate 2-acetylphenyl (E)-cinnamate (5f, 5g) (10 mmol) in dry
acetone (100 mL), and the mixture was refluxed for 24 h with
stirring. After cooling, the mixture was poured into ice and the pH
was adjusted to 4 with 2 N HCl. The solid was removed by filtration,
dissolved in chloroform and washed with brine. The organic phase
was dried over Na2SO4, filtered and evaporated to dryness. The
residue was purified by crystallization.
(KBr) 3454, 1621 cmꢂ1. 1H NMR (DMSO D6):
d (ppm) 13.46 (s, 1H,
OH), 7.92 (d, 1H, H6, J5-6 ¼ 9.4 Hz), 7.65 (dd,1H, H , Ja ¼ 14.8 Hz, Jb
b
-
b
-
¼ 8.4 Hz), 7.43 (d, 2H, H20, H60, J2 -3 ¼ 8.4 Hz), 7.34 (d, 2H, H3 , H5 ,
0
0
0
0
g
0
0
J2 -3 ¼ 8.4 Hz), 7.14 (d, 1H, H
a
, Ja ¼ 14.8 Hz), 7.03e6.94 (m, 2H, H
g,
-b
Hd
), 6.48e6.45 (m, 2H, H3, H5), 3.85 (s, 3H, OCH3). 13C NMR (DMSO
4.1.5.1. (2Z,4E)-5-(3,4-Dimethoxyphenyl)-3-hydroxy-1-(2-hydroxy-
phenyl)penta-2,4-dien-1-one (6f). Yield: 40%; mp 126e128 ꢁC from
EtOH. The compound exhibited spectroscopic data identical to
those previously reported [23].
D6): (ppm) 191.7, 166.7, 166.2, 144.0, 140.5, 135.1, 134.6, 131.1, 129.1,
d
128.5, 127.4, 124.3, 114.1, 107.7, 101.1, 55.6.
4.1.3. Synthesis of (2E,4E)-5-(4-chlorophenyl)-1-(2,4-dihydroxy-
phenyl)penta-2,4-dien-1-one (3h)
4.1.5.2. (2Z,4E)-5-(3,4-Dimethoxyphenyl)-3-hydroxy-1-(2-hydroxy-
6-methoxyphenyl)penta-2,4-dien-1-one (6g). Yield: 40%; mp 131e
132 ꢁC from AcOEt. The compound exhibited spectroscopic data
identical to those previously reported [23].
Barium hydroxide octahydrate (15 mmol) was added to
a mixture of 2-hydroxy-4-[(tetrahydro-2H-pyran-2-yl)oxy]aceto-
phenone (1g) [12] (10 mmol) and 4-chlorocinnamaldehyde (2b)
(10 mmol) in EtOH (200 mL). The mixture was stirred at 50 ꢁC for
5 h and at room temperature overnight. After removal of ethanol in
vacuum, water was added to the residue, the pH was adjusted to 2
with 2 N HCl, and the mixture was stirred at room temperature for
1 h. After this period, the solid was removed by filtration, washed
with water and purified by silica gel column chromatography
eluting with AcOEt/petroleum ether 1:4. Yield: 61%; mp 217e220 ꢁC
4.1.6. (2Z,4E)-3-Hydroxy-5-(4-hydroxy-3-methoxyphenyl)-1-(2-
hydroxy-6-methoxyphenyl)penta-2,4-dien-1-one (6h)
Anhydrous K2CO3 (50 mmol) was added to a solution of 2-
acetyl-3-methoxyphenyl (E)-3-(4-acetoxy-3-methoxyphenyl)cin-
namate (5h) (10 mmol) in dry acetone (100 mL), and the mixture
was refluxed for 24 h with stirring. After cooling, the mixture was
poured into ice (50 g) and water (50 mL), and stirred at room
temperature for 1 h. After that period, the pH was adjusted to 4
with 2 N HCl, the solid was removed by filtration and purified by
silica gel column chromatography eluting with CH2Cl2. Yield: 25%;
mp 162e164 ꢁC from AcOEt/petroleum ether. IR (KBr) 3406,
from AcOEt/petroleum ether. IR (KBr) 3440, 3241, 1636 cmꢂ1 1H
.
NMR (DMSO D6): d (ppm) 13.36 (s, 1H, OH), 10.80 (bs, 1H, OH), 7.92
(d, 1H, H6, J5-6 ¼ 8.9 Hz), 7.62 (d, 2H, H20, H60, J2 -3 ¼ 8.4 Hz), 7.58
0
0
(dd, 1H, H
b
, Ja ¼ 14.8 Hz, Jb ¼ 9.6 Hz), 7.50e7.46 (m, 3H, H
a,
-
b
-g
H30, H50), 7.27 (dd, 1H, H
g
, Jg ¼ 15.5 Hz, Jb ¼ 9.6 Hz), 7.22 (d, 1H,
-d
-g
Hd
, Jg ¼ 15.5 Hz), 6.42 (dd, 1H, H5, J5-6 ¼ 8.9 Hz, J3-5 ¼ 2.3 Hz), 6.30
1626 cmꢂ1
.
1H NMR (CDCl3):
d
(ppm) (CDCl3): 14.85 (s, 1H, OH),
-
d
(d, 1H, H3, J3-5 ¼ 2.3 Hz). 13C NMR (DMSO D6):
d
(ppm) 191.1, 165.6,
12.77 (s, 1H, OH), 7.58 (d, 1H, H
d
, Jg ¼ 15.8 Hz), 7.32 (t, 1H, H4, J3-
-d

N. Desideri et al. / European Journal of Medicinal Chemistry 59 (2013) 91e100
99
¼ J4-5 ¼ 8.3 Hz), 7.15 (dd, 1H, H60, J5 -6 ¼ 8.2 Hz, J2 -6 ¼ 1.9 Hz), 7.06
4.2.2. Reversibility and irreversibility experiments
0
0
0
0
4
(d,1H, H20, J2 -6 ¼ 1.9 Hz), 6.95 (d,1H, H5 , J5 -6 ¼ 8.2 Hz), 6.77 (s,1H,
To evaluate whether some of the tested compounds (3b, 3g, 3h,
6d and 6k) are reversible or irreversible hMAO-B inhibitors, an
effective centrifugationeultrafiltration method (so-called repeated
washing) was used [12].
0
0
0
0
0
Ha
), 6.60 (d, 1H, H3, J3-4 ¼ 8.3 Hz), 6.45 (d, 1H, H , Jg ¼ 15.8 Hz),
g
-d
6.43 (d, 1H, H5, J4-5 ¼ 8.3 Hz), 5.99 (s, 1H, OH), 3.97 (s, 3H, OCH3),
3.96 (s, 3H, OCH3). 13C NMR (CDCl3):
d
(ppm) 194.7, 175.2, 164.0,
160.3, 147.8, 146.8, 139.6, 134.9, 127.9, 122.7, 120.4, 114.9, 111.1, 110.7,
109.7, 103.2, 101.7, 56.0, 55.9.
4.3. Molecular modeling
4.1.7. (2Z,4E)-5-(3,4-Dihydroxyphenyl)-3-hydroxy-1-(2-
hydroxyphenyl)penta-2,4-dien-1-one (6m)
Theoretical 3D models of the most active and hMAO-B selective
compounds, 3g and 3h were built by means of Maestro [25] GUI
version 9.1. The inhibitor structures were optimized by means of
energy minimization carried out with the OPLS-2005 force field [26]
and the GB/SA [27] water implicit solvation model. All these
preliminary calculation were computed by the version 9.8 of the
MacroModel [28] software. Docking simulation was performed
using the ligand flexible algorithm of Glide [29] at XP precision level.
Thetargetmodels ofhMAO-A andhMAO-Bweretwohighresolution
crystal structures corresponding to the Protein Data Bank codes
2Z5X [2] and 2V5Z [3] respectively. Co-crystallized ligands, harmine
for 2Z5X and safinamide for 2V5Z, and water molecules were
removed from both target models and their binding clefts were
defined by means of a regular box of about 1000 A , centered onto
the N5 FAD cofactor. Molecular dynamics (MD) simulations were
carried out, up to 12 ns at 300 K, applying the version 2.4 of the
DESMOND [30] package and the OPLS-AA force field onto the most
stable complex of both inhibitors in hMAO-A and -B. The explicit
solvation model TIP3 was adopted for taking into account water
solvent effects. The four MD trajectories, each consisting of 240
structures sampled at regular interval equal to 5 ps, were graphical
inspected and analyzed using VMD version 1.8.7 [31]. The molecular
orbital (MO) analysis of compounds 3g and 3h was carried out at DFT
level of theory using the hybrid functional B3LYP and the 6-31G**
basis set. For mimicking the enzyme environment, the implicit
solvation model PBF chloroform was adopted. The structure of both
inhibitors, selected for the MO study, were obtained from the last
molecular dynamics sampled frame and energy minimized during
the DFT calculation. Quantum mechanics calculations were per-
formed by means of the Jaguar [32]. Molecular modeling figures
were depicted by PyMol version 1.3 [33].
A
solution 1 M of boron tribromide in dichloromethane
(100 mL) was added dropwise to a stirred solution of 6f (10 mmol)
in dry dichloromethane (200 mL) cooled in ice bath. The reaction
mixture was stirred in ice bath for 1 h and then at room temper-
ature for 20 h. After this period, the mixture was poured into ice
and water and vigorously stirred. The obtained precipitate was
filtered, washed with water, and purified by silica gel column
chromatography eluting with AcOEt/petroleum ether 1:1. Yield:
40%; mp 200e201 ꢁC from AcOEt. IR (KBr) 3385,1635 cmꢂ1. 1H NMR
(acetone d6):
d (ppm) 14.73 (s, 1H, OH), 12.21 (s, 1H, OH), 8.45 (bs,
2H, 2OH), 7.92 (dd, 1H, H6, J5-6 ¼ 8.3 Hz, J4-6 ¼ 1.5 Hz), 7.60 (d, 1H,
H
d
, Jg ¼ 15.8 Hz), 7.51 (ddd, 1H, H4, J3-4 ¼ 8.4 Hz, J4-5 ¼ 7.2 Hz, J4-
3
ꢀ
-
d
¼ 1.5 Hz), 7.21 (d, 1H, H20, J2 -6 ¼ 2.0 Hz), 7.09 (dd, 1H, H60, J5 -
0
0
0
6
60
0
0
¼ 8.2 Hz, J2 -6 ¼ 2.0 Hz), 6.97e6.94 (m, 2H, H3, H5), 6.90 (d, 1H,
H50, J5 -6 ¼ 8.2 Hz), 6.69 (d, 1H, H
g
, Jg ¼ 15.8 Hz), 6.68 (s, 1H, H
a).
0
0
-
d
13C NMR (DMSO D6):
d (ppm) 191.4, 177.7, 159.9, 148.5, 145.7, 140.5,
134.9, 129.2, 126.3, 121.6, 120.1, 119.2, 117.7, 115.8, 114.7, 114.4, 98.5.
4.2. Biochemistry
4.2.1. Determination of hMAO isoform activity
The effects of the tested compounds on hMAO isoform enzymatic
activity were evaluated by measuring their effects on the production
of hydrogen peroxide from p-tyramine using the Amplex Red MAO
assay kit (Molecular Probes, Inc., Eugene, Oregon, USA) and micro-
somal MAO isoforms prepared from insect cells (BTI-TN-5B1-4)
infected with recombinant baculovirus containing cDNA inserts for
hMAO-A or hMAO-B (SigmaeAldrich Química S.A., Alcobendas,
Spain). Briefly, various concentrations of the test drugs or reference
inhibitors and adequate amounts of recombinant hMAO-A or
hMAO-B (required to obtain the same reaction velocity) were
incubated for 15 min at 37 ꢁC in a sodium phosphate buffer (0.05 M,
pH 7.4). The reaction was started by adding (final concentrations)
Acknowledgments
This work was supported by the Italian MIUR (Ministero del-
l’Istruzione, dell’Universita e della Ricerca). We thank dr Ivano
Pindinello for technical assistance.
200 mM Amplex Red reagent, 1 U/mL horseradish peroxidase and
1 mM p-tyramine which is totally sufficient to saturate both MAO
isoforms in these experimental conditions. The hMAO activity was
evaluated by measuring the fluorescence generated by resorufin
using the general procedure described previously by us [14]. The
possible capacity of the above test drugs to modify the fluorescence
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 reagent in a sodium
phosphate buffer. For fluorescent compounds, incubations with
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.11.006.
References
60
mM kynuramine (SigmaeAldrich Química S.A., Alcobendas,
Spain) were performed at 37 ꢁC and pH 7.4 in a sodium phosphate
buffer (Na2HPO4/KH2PO4 isotonized with KCl). The rate of oxidation
of the non selective substrate into 4-hydroxyquinolone was moni-
tored at 314 nm using a Pharmacia Biotech Ultrospec 4000 UV/
Visible Spectrophotometer [24]. IC50 values were estimated by non-
linear regression analysis using GraphPad Prism software (San
Diego, USA), with X ¼ log molar concentration of tested compound
and Y ¼ percentage of inhibition of control resorufin production.
This regression was performed using data obtained with 4e6
different concentrations of each tested compound. IC50 values are
the mean ꢀ S.E.M. from five experiments.
[1] J.C. Shih, K. Chen, M.J. Ridd, Monoamine oxidase: from genes to behaviour,
Annu. Rev. Neurosci. 22 (1999) 197e217.
[2] S.Y. Son, J. Ma, Y. Kondou, M. Yoshimura, E. Yamashita, T. Tsukihara, Structure
ꢀ
of human monoamine oxidase A at 2.2-A resolution: the control of opening
the entry for substrates/inhibitors, Proc. Natl. Acad. Sci. U. S. A. 105 (2008)
5739e5744.
[3] C. Binda, J. Wang, L. Pisani, C. Caccia, A. Carotti, P. Salvati, D.E. Edmondson,
A. Mattevi, Structures of human monoamine oxidase
B complexes with
selective noncovalent inhibitors: safinamide and coumarin analogs, J. Med.
Chem. 50 (2007) 5848e5852.
[4] M.B. Youdim, D.E. Edmondson, K.F. Tipton, The therapeutic potential of
monoamine oxidase inhibitors, Nat. Rev. Neurosci. 7 (2006) 295e309.
[5] M. Yamada, H. Yasuhara, Clinical pharmacology of MAO inhibitors: safety and
future, Neurotoxicology 25 (2004) 215e221.
̀

100
N. Desideri et al. / European Journal of Medicinal Chemistry 59 (2013) 91e100
[6] B. Drukarch, F.L. Van Muiswinkel, Drug treatment of Parkinson’s disease. Time
[21] C. Conti, P. Mastromarino, P. Goldoni, G. Portalone, N. Desideri, Synthesis and
anti-rhinovirus properties of fluoro-substituted flavonoids, Antivir. Chem.
Chemother. 16 (2005) 267e276.
[22] N. Desideri, C. Conti, P. Mastromarino, F. Mastropaolo, Synthesis and anti-
rhinovirus activity of 2-styrylchromones, Antivir. Chem. Chemother. 11
(2000) 321e327.
for phase II, Biochem. Pharmacol. 59 (2000) 1023e1031.
[7] A.H. Schapira, Treatment options in the modern management of Parkinson
disease, Arch. Neurol. 64 (2007) 1083e1088.
[8] P. Riederer, W. Danielczyk, E. Grunblatt, Monoamine oxidase-B inhibition in
Alzheimer’s disease, Neurotoxicology 25 (2004) 271e277.
[9] T. Thomas, Monoamine oxidase-B inhibitors in the treatment of Alzheimer’s
disease, Neurobiol. Aging 21 (2000) 343e348.
[10] F. Chimenti, R. Fioravanti, A. Bolasco, P. Chimenti, D. Secci, F. Rossi, M. Yanez,
F. Orallo, F. Ortuso, S. Alcaro, A new series of flavones, thioflavones, and
flavanones as selective monoamine oxidase-B inhibitors, Bioorg. Med. Chem.
18 (2010) 1273e1279.
[23] C.M.M. Santos, A.M.S. Silva, J.A.S. Cavaliero, Efficient syntheses of new
polyhydroxylated 2,3-diaryl-9H-xanthen-9-ones, Eur. J. Org. Chem. (2009)
2642e2660.
[24] U. Thull, S. Kneubühler, B. Testa, M.F. Borges, M.M. Pinto, Substituted
xanthones as selective and reversible monoamine oxidase A (MAO-A) inhib-
itors, Pharm. Res. 10 (1993) 1187e1190.
[11] N. Desideri, A. Bolasco, R. Fioravanti, L. Proietti Monaco, F. Orallo, M. Yañez,
F. Ortuso, S. Alcaro, Homoisoflavonoids: natural scaffold with potent and
selective monoamine oxidase-B inhibition properties, J. Med. Chem. 54 (2011)
2155e2164.
[12] F. Chimenti, R. Fioravanti, A. Bolasco, P. Chimenti, D. Secci, F. Rossi, M. Yañez,
F. Orallo, F. Ortuso, S. Alcaro, Chalcones: a valid scaffold for monoamine
oxidases inhibitors, J. Med. Chem. 52 (2009) 2818e2824.
[13] C. Binda, M. Li, F. Hubalek, N. Restelli, D.E. Edmondson, A. Mattevi, Insights
into the mode of inhibition of human mitochondrial monoamine oxidase B
from high-resolution crystal structures, Proc. Natl. Acad. Sci. U. S. A. 100
(2003) 9750e9755.
[14] M. Yáñez, N. Fraiz, E. Cano, F. Orallo, Inhibitory effects of cis- and trans-
resveratrol on noradrenaline and 5-hydroxytryptamine uptake and on
monoamine oxidase activity, Biochem. Biophys. Res. Commun. 344 (2006)
688e695.
[15] F. Chimenti, E. Maccioni, D. Secci, A. Bolasco, P. Chimenti, A. Granese,
S. Carradori, S. Alcaro, F. Ortuso, M. Yáñez, F. Orallo, R. Cirilli, R. Ferretti, F. La
Torre, Synthesis, stereochemical identification, and selective inhibitory
activity against human monoamine oxidase-B of 2-methylcyclohexylidene-
(4-arylthiazol-2-yl)hydrazones, J. Med. Chem. 51 (2008) 4874e4880.
[16] D.E. Edmondson, C. Binda, J. Wang, A.K. Upadhyay, A. Mattevi, Molecular and
mechanistic properties of the membrane-bound mitochondrial monoamine
oxidases, Biochemistry 20 (2009) 4220e4230.
[17] M. Deodhar, D.StC. Black, N. Kumar, Acid catalyzed stereoselective rear-
rangement and dimerization of flavenes: synthesis of dependensin, Tetrahe-
dron 63 (2007) 5227e5235.
[18] A.I. Vedernikov, S.P. Gromov, Convenient method for the preparation of crown
ether cinnamaldehydes, Synthesis (2001) 889e892.
[25] Maestro, Version 9.1, Schrodinger, LLC, New York, NY, 2010.
[26] G. Kaminski, R.A. Friesner, J. Tirado-Rives, W.L. Jorgensen, Evaluation and
reparametrization of the OPLS-AA force field for proteins via comparison with
accurate quantum chemical calculations on peptides, J. Phys. Chem. B 105
(2001) 6474e6487.
[27] W. Hasel, T.F. Hendrickson, W.C. Still, A rapid approximation to the solvent
accessible surface areas of atoms, Tetrahedron Comput. Methodol. 1 (1988)
103e116.
[28] (a) MacroModel, Version 9.8, Schrodinger, LLC, New York, NY, 2010;
(b) F. Mohamadi, N.G.J. Richards, W.C. Guida, R. Liskamp, M. Lipton,
C. Caufield, G. Chang, T. Hendrickson, W.C. Still, MacroModel-an integrated
software system for modeling organic and bioorganic molecules using
molecular mechanics, J. Comput. Chem. 11 (1990) 440e467.
[29] (a) Glide, Version 5.6, Schrodinger, LLC, New York, NY, 2010;
(b) R.A. Friesner, J.L. Banks, R.B. Murphy, T.A. Halgren, J.J. Klicic, D.T. Mainz,
M.P. Repasky, E.H. Knoll, D.E. Shaw, M. Shelley, J.K. Perry, P. Francis, P.S. Shenkin,
Glide: a new approach for rapid, accurate docking and scoring. 1. Method and
assessment of docking accuracy, J. Med. Chem. 47 (2004) 1739e1749;
(c) T.A. Halgren, R.B. Murphy, R.A. Friesner, H.S. Beard, L.L. Frye, W.T. Pollard,
J.L. Banks, Glide: a new approach for rapid, accurate docking and scoring. 2.
Enrichment factors in database screening, J. Med. Chem. 47 (2004) 1750e1759;
(d) R.A. Friesner, R.B. Murphy, M.P. Repasky, L.L. Frye, J.R. Greenwood,
T.A. Halgren, P.C. Sanschagrin, D.T. Mainz, Extra precision Glide: docking and
scoring incorporating a model of hydrophobic enclosure for proteineligand
complexes, J. Med. Chem. 49 (2006) 6177e6196.
[30] K.J. Bowers, E. Chow, H. Xu, R.O. Dror, M.P. Eastwood, B.A. Gregersen, J.L.
Klepeis, I. Kolossváry, M.A. Moraes, F.D. Sacerdoti, J.K. Salmon, Y. Shan, D.E.
Shaw, Scalable algorithms for molecular dynamics simulations on commodity
clusters, in: Proceedings of the ACM/IEEE Conference on Supercomputing
(SC06), Tampa, Florida, November 2006, pp. 11e17.
[31] W. Humphrey, A. Dalke, K. Schulten, VMD e visual molecular dynamics, J. Mol.
Graph. 14 (1996) 33e38.
[32] Jaguar, Version 5.6, Schrodinger, LLC, New York, NY, 2010.
[33] The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC.
[19] A.M. Condo Jr., D.C. Baker, R.A. Moreau, K.B. Hicks, Improved method for the
synthesis of trans-feruloyl-b-sitostanol, J. Agric. Food Chem. 49 (2001) 4961e
4964.
[20] N. Desideri, P. Mastromarino, C. Conti, Synthesis and evaluation of anti-
rhinovirus activity of 3-hydroxy and 3-methoxy 2-styrylchromones, Antivir.
Chem. Chemother. 14 (2003) 195e203.