Synthesis, human monoamine oxidase inhibitory activity and molecular docking studies of 3-heteroarylcoumarin derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.01.033

Monoamine oxidase (MAO) is an important drug target for the treatment of neurological disorders. Series of 3-indolyl and 3-thiophenylcoumarins were synthesized and evaluated as inhibitors of the two human MAO isoforms, hMAO-A and hMAO-B. In general, the d

Full text of DOI:10.1016/j.ejmech.2011.01.033

European Journal of Medicinal Chemistry 46 (2011) 1147e1152
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, human monoamine oxidase inhibitory activity and molecular docking
studies of 3-heteroarylcoumarin derivatives
Giovanna Delogu a, Carmen Picciau a, Giulio Ferino b, Elías Quezada c, Gianni Podda a, Eugenio Uriarte b,
,
Dolores Viña d
*
a Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
b Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
c Departamento de Química, Facudade de Ciências, Universidade de Porto, 4169-007 Porto, Portugal
d Departamento de Farmacología, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Vida, 15782 Santiago de Compostela, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Monoamine oxidase (MAO) is an important drug target for the treatment of neurological disorders. Series
of 3-indolyl and 3-thiophenylcoumarins were synthesized and evaluated as inhibitors of the two human
MAO isoforms, hMAO-A and hMAO-B. In general, the derivatives were found to be selective hMAO-B
Received 30 September 2010
Received in revised form
12 January 2011
Accepted 18 January 2011
Available online 28 January 2011
inhibitors with IC50 values in the nanoMolar (nM) to microMolar (mM) range. Docking experiments were
carried out in order to compare the theoretical and experimental affinity of these compounds to the
hMAO-B protein. According to our results, docking experiments could be an interesting approach to try
to predict the activity of this class of coumarins against MAO-B receptors.
This article is dedicated to the memory of
Prof. Francisco Orallo.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
3-Heteroarylcoumarins
Perkin reaction
Monoamino oxidase (MAO)
Molecular docking
1. Introduction
mediators [4]. These properties determine the clinical importance
of MAO inhibitors. In fact, interest in selective MAO-B inhibitors has
MAO is a FAD-containing enzyme with two known isoforms
(MAO-A and MAO-B) and it is present in the outer membrane of
mitochondria in glial, neuronal and many other cells [1]. Deami-
nation of adrenaline, noradrenaline and serotonin is catalyzed by
increased in recent years due to their therapeutic potential in aging
related neurodegenerative diseases such as Alzheimer’s disease
(AD) and Parkinson’s disease (PD) [5]. Selective MAO-A inhibitors
have been used because of their therapeutic potential as anti-
depresants [6].
MAO-A whilst deamination of
b-phenylethylamine and benzyl-
amine is catalyzed by MAO-B [2]. The MAO metabolic reaction
involves the oxidation of the amine functional group via oxidative
The recent description of the crystal structure of the two iso-
forms of hMAO helps us to elucidate the underlying mechanisms. It
allows us to investigate the selective interactions between these
proteins and their ligands. It also allows us to probe the catalytic
mechanism, helping us to gain a complete understanding of the
pharmacophoric requirements necessary for the rational design of
new inhibitors [7,8].
Among the different existing inhibitors, those with a (1H)-ben-
zopyran structure have been deeply studied [9]. Substitution of the
coumarin nucleus at position 3 has been carried out with phenyl,
methyl, carboxylic acid, ethyl esther or acyl chloride groups [10]. Our
research group has reported in severalstudies, the importance tothe
MAO inhibitory activity of different substituents on the phenyl ring,
in the position 3 of the coumarin [11e13]. In this work we synthe-
sized and tested for MAO inhibitory activity some coumarin
cleavage of the
a-CH bond of the substrate with the ensuing
generation of an imine intermediate. This pathway is accomplished
by the reduction of the flavin cofactor that is reoxidized by molec-
ular oxygen, with simultaneous hydrogen peroxide release. Subse-
quently, the imine intermediate is hydrolyzed by a non-enzymatic
pathway yielding ammonia and the corresponding aldehyde [3].
This enzymatic function decreases the synaptic concentration of the
neurotransmitters mentioned above and controls a great extent the
neurone’s excitement in those possessing receptors for these
* Corresponding author. Tel.: þ34 981563100; fax: þ34 981594595.
E-mail address: mdolores.vina@usc.es (D. Viña).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.033

1148
G. Delogu et al. / European Journal of Medicinal Chemistry 46 (2011) 1147e1152
Table 1
derivatives substituted at position 3 with different heterocyclic
rings. Additionally, we explored the importance of the number and
position of methoxy groups on (1H)-benzopyran structure.
IC50 values and hMAO-B selectivity index for the new compounds and reference
inhibitors on the enzymatic activity of human recombinant MAO isoforms expressed
in baculovirus infected BTI insect cells.
Compound
MAO-A (IC50
)
MAO-B (IC50
)
SI MAO-B
2. Results and discussion
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
**
**
**
**
6.37 ꢀ 0.43
13.3 ꢀ 0.89
1.92 ꢀ 0.13
m
m
m
M
M
M
>15b
>7.5b
>52b
>380b
>75
>210
>158b
>428b
e
2.1. Chemistry
262.95 ꢀ 17.61 nM
4.16 ꢀ 0.28
m
m
Ma
Ma
55.63 ꢀ 3.73 nM
The 3-heteroarylcoumarin derivatives were synthesized in
moderate yield (25e47%) via the classical Perkin reaction [14e17]
by condensation of the ortho-hydroxybenzaldehydes conveniently
substituted and the appropriately substituted acetic acids, using
N,N0-dicyclohexylcarbodiimide (DCC) as dehydrating agent (Fig. 1).
9.67 ꢀ 0.65
45.95 ꢀ 3.08 nM
**
**
**
**
**
**
633.55 ꢀ 42.43 nM
233.73 ꢀ 15.65 nM
**
**
**
**
e
The structures of these compounds were confirmed by 1H NMR, 13
C
e
e
NMR, mass spectrometry and elemental analyses.
R-(ꢂ)-Deprenyl
67.25 ꢀ 1.02
m
m
Ma
M
14.80 ꢀ 0.99 nM
4544
0.87
Iproniazide
6.56 ꢀ 0.76
7.54 ꢀ 0.36
mM
2.2. Enzyme inhibition studies
Each IC50 value is the mean ꢀ S.E.M. from five experiments (n ¼ 5). **Inactive at
100 M (highest concentration tested). At higher concentrations the compounds
m
precipitate. SI: hMAO-B selectivity index ¼ IC50(hMAO-A)/IC50(hMAO-B).
The potential effects of the new synthesized compounds on
hMAO activity were investigated by measuring their effects on the
production of hydrogen peroxide (H2O2) from p-tyramine, using
the AmplexÒ Red MAO assay kit (Molecular Probes, Inc., Eugene,
Oregon, USA) and MAO isoforms in microsomes 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). The inhibition of hMAO activity
was evaluated using the above method following the general
procedure described previously by us [18]. The test compounds did
not show any interference with the reagents used for biochemical
assay.
a
P < 0.01 vs the corresponding IC50 values obtained against MAO-B, as deter-
mined by ANOVA/Dunnett’s.
b
Values obtained under the assumption that the corresponding IC50 against
MAO-A is the highest concentration tested (100 mM).
the (1H)-benzopyran structure are inactive as MAO inhibitors.
Introduction of a methoxy group at either position 6 or 7 on the
(1H)-benzopyran structure, increases the MAO-B activity between
10 and 100 fold (compare 1a vs 2a and 3a; 1b vs 2b and 3b; 1c vs 2c)
with the only exception being compound 3c. Methoxy groups at
position 7 (compounds 2aec) improved the MAO-B inhibitory
activity compared to the corresponding compounds with the
methoxy group at position 6, however compounds 2b and 2c
showed a weak MAO-A inhibitory activity leading to a small
decrease in the B-selectivity.
The control activity of hMAO-A and hMAO-B using p-tyramine
as the common substrate was 165 ꢀ 2 pmol of p-tyramine oxidized
to p-hydroxyphenylacetaldehyde/min (n ¼ 20).
The results of the hMAO-A and hMAO-B inhibition studies with
compounds 1aec, 2aec, 3aec and 4aec are reported in Table 1
together with the selectivity index (SI hMAO-B ¼ [IC50(hMAO-A)]/
[IC50(hMAO-B)]). Enzymatic assays revealed that most of test
compounds were moderate to potent hMAO inhibitors at either low
micromolar to nanomolar concentrations, showing selectivity
toward hMAO-B.
We observed that the number and position of methoxy groups
on the (1H)-benzopyran structure is more important for hMAO
activity than the nature of the heterocycle ring in position 3. In fact,
compounds 4aec showing methoxy groups at positions 5 and 7 on
2.3. Molecular docking study
Docking experiments were carried out in order to compare the
theoretical and experimental affinity. A graphical inspection was
also made to propose the possible binding mode for the two most
active MAO-B inhibitor 2b and 2c. The Protein Data Bank [19] (PDB)
crystallographic structure of human MAO-B (PDB code 2V60) [8]
was considered as the receptor for docking simulations.
The scoring function estimates the affinity between ligands and
receptor [20,21]. In order to verify the usefulness of the scoring
R3
S
HO
O
R2
R1
a)
a)
+
O
O
S
R3
H
1a- 4a
R2
R1
O
S
HO
R3
OH
O
R2
R1
+
+
S
1
2
3
4
R1 = R2 = R3 = H
R1 =OCH3; R2 = R3 = H
R2 = OCH3; R1 = R3 = H
R1 = R3 = OCH3; R2 = H
O
O
1b- 4b
NH
HO
R3
O
R2
R1
a)
HN
O
O
1c- 4c
Fig. 1. Reagents and conditions: a) DCC/DMSO 110 ꢁC, 24e48 h.

G. Delogu et al. / European Journal of Medicinal Chemistry 46 (2011) 1147e1152
1149
Table 2
(ROC) curve. This representation is particularly suited to illustrating
the tendency of a binary model to either correctly classify the
compounds or predict false positives [26e28] (see Fig. 2). The area
under the curve is 0.93, indicating high predictive power for model.
By using docking calculations it is possible to differentiate between
true ligands and decoys (see Fig. 2).
Finally, we visually investigated the most stable configurations
of our active synthesized structures. In order to obtain a more
accurate model of inhibitor-enzyme interactions, the most stable
binding poses of our coumarins were subjected to the MacroModel-
eMBrAcE minimization module of Maestro [29]. In the eMBrAcE
calculation the ligands, pre-positioned with Glide, are minimized,
in turn, with the receptor.
We found two different binding patterns for 3-hetero-
arylcoumarins; all the 3-thiophen substituted coumarins show the
same orientation. For this reason, we focused our attention only on
the binding modes of the most active compounds 2b and 2c with
the MAO-B isoform (see Fig. 3). The MAO-B active site is known; it
extends from the substrate cleft, close to the FAD cofactor, to the
entrance cavity situated near the protein surface [30,31].
Comparison between experimental activity and predicted scoring function against
MAO-B.
Docking ranka
Drug
IC50
**
Docking scorec
b
1
2
3
4
5
6
7
8
4c
2a
2c
3a
2b
3b
1c
1b
3c
1a
4b
4a
ꢂ8.4274
ꢂ8.1976
ꢂ7.9367
ꢂ7.9025
ꢂ7.8267
ꢂ7.5740
ꢂ7.5170
ꢂ7.4479
ꢂ7.2776
ꢂ6.4593
ꢂ5.3446
ꢂ5.1865
0.2630
0.0459
0.6335
0.0556
0.2337
1.9200
13.3000
**
9
10
11
12
6.3700
**
**
**Inactive at 100 mM (highest concentration tested). At higher concentrations the
compounds precipitate.
a
Docking rank taking into account the study coumarins compounds.
Expressed in
Expressed in kJ/mol.
b
m
M.
c
function to recognize new molecules potentially active as MAO-B
inhibitors, different ligands were docked to the MAO-B protein.
The experimental IC50 values were compared with those for the
predicted activity according to the scoring function (see Table 2).
The square of the correlation coefficient (r2), between the scoring
function and the experimental MAO-B inhibitory activity pKi, was
calculated as 0.44. This result is reasonable considering that scoring
functions are not able to provide a good correlation between the
predicted and the experimental affinity [22e24]. In our case,
a certain tendency of the model to rank the compounds based on
their respective activity can be appreciated. In fact, when a cut-off
For the 3-thiophen structures the coumarin moiety is situated
up in the cavity, leaving the methoxy group directed toward FAD.
Fig. 3a illustrates the most stable configuration of 2b. As show in
the figure, 2b occupies the entrance hydrophobic cleft formed by
Phe103, Pro104, Leu164, Phe168, Leu171, Cys172, Ile199, Ile316 and
Tyr326. No hydrogen bonds (H-bonds) were detected for 2b.
Conversely, good van der Waals interactions with Ile199, Phe168,
Gln206 and Tyr326 were observed. Besides this, electrostatic
interactions with Tyr326 and Leu164 were also evident. In the
binding mode of 2c a deep recognition of the ligand into the MAO-B
active site was observed (see Fig. 3b). The indole system was placed
facing the FAD cofactor in the middle between the aromatic resi-
value of IC50 ¼ 1.0
mM is used in order to differentiate the
compounds in two groups (high and low affinity), the model is able
to recognize clearly both groups. The most active compounds
occupy the first positions according to the scoring function.
dues Tyr398 and Tyr435. Tyr398 is involved in a double
pep
stacking interaction, with phenyl and pyrrol rings respectively. A H-
bond concerning the indole NH of 2c and the N5 nitrogen of the
FAD cofactor, helps to stabilize the indole moiety of the coumarin.
Furthermore 2c has strong van der Waals interactions with Gln206,
Tyr326, Leu171 and Cys172.
However, the compounds with IC50 > 1.0 mM, are ranked in the last
positions. The only exception is the inactive coumarin 4c which is
ranked in the first positions (see Table 2).
A second study was carried out in order to assess whether the
model discriminates between ligands and decoys. The synthesized
coumarins were dispersed in a pool of 120 decoys extracted from
the ZINC database [25]. After completing docking calculations, the
data was plotted to produce a receiver operative characteristics
Even, though we cannot observe
a significant difference
between the experimental and theoretical activity of compounds
2c and 2b; in this study we have tried to establish how these
compounds interact differently in the MAO-B binding pocket.
Docking of compound 2c, showed that stability of this coumarin
Fig. 2. ROC curve relative to synthesized coumarins and 120 ZINC decoys.

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G. Delogu et al. / European Journal of Medicinal Chemistry 46 (2011) 1147e1152
and -B. In general, the derivatives were found to be selective for the
MAO-B isoform with 7-methoxy-3-heteroarylcoumarins exhibiting
inhibition potencies in the low nanoMolar range. Docking experi-
ments showed that the affinity of the studied compounds to MAO-B
is similar to that obtained experimentally.
According to our results, docking experiments could be an
interesting approach to try to predict the activity for this class of
coumarins in MAO-B receptor. Moreover, the two different binding
patterns for 2b and 2c proposed in this work, could help us to better
understand the modes of interaction of new 3-heteroarylcoumarins.
4. Experimental
4.1. General methods
Melting points (mp) are uncorrected and were determined with
a Reichert Kofler thermopan or in capillary tubes in a Buchi 510
apparatus. 1H NMR (300 MHz) and 13C NMR (75.4 MHz) spectra
were recorder with a Bruker AMX spectrometer. Chemical shifts (d)
are expressed in parts per million (ppm) using TMS as an internal
standard. Spin multiplicities are given as s (singlet), d (doublet), dd
(doublet of doublets) and m (multiplet). Mass spectrometry was
carried out with a Kratos MS-50 or a Varian MAT-711 spectrometer.
Elemental analyses were performed by a PerkineElmer 240B
microanalyzer and were within ꢀ0.4% of calculated values in all
cases. Flash Chromatography (FC) was performed on silica gel
(Merck 60, 230e400 mesh); analytical TLC was performed on
precoated silica gel plates (Merck 60 F254).
4.2. Synthesis
4.2.1. General synthetic method for the coumarin skeleton
A
solution of ortho-hydroxybenzaldehyde (1e4, 8 mmol),
substituted acetic acid (aec, 10 mmol) and DCC (12 mmol) in
dimethylsulfoxide (DMSO, 10 mL), was heated (oil bath) at 110 ꢁC
for 24e48 h. On completion of the reaction, cold water (100 mL)
and acetic acid (15 mL) were added. The reaction mixture was
stirred at room temperature for 4 h and extracted with diethyl ether
(4 ꢃ100 mL). The precipitated dicyclohexylurea was filtered off. The
filtrate was extracted with 5% aqueous NaHCO3 (200 mL). The
organic phase was stirred for 1 h with 5% aqueous sodium meta-
bisulfite in order to remove the unreacted hydroxybenzaldehyde.
The organic phase was washed with water, dried (Na2SO4) and the
solvent removed under reduced pressure. The residue was purified
by column chromatography (Hexane/EtOAc, 9:1).
4.2.2. 3-(Thiophen-2-yl)coumarin (1a) [29]
Yield 34%; mp 166e167 ꢁC. 1H NMR (300 MHz) (CDCl3):
d
¼ 7.12
(m, 1H), 7.28 (d, J ¼ 9.4 Hz, 1H), 7.35 (d, J ¼ 9.4 Hz, 1H), 7.42 (d,
J ¼ 5.1 Hz, 1H), 7.52 (m, 2H), 7.80 (d, J ¼ 3.8 Hz, 1H), 7.99 (s, 1H). 13C
Fig. 3. Most stable binding poses of compounds 2b (a) and 2c (b) into the MAO-B
binding pocket (PDB code: 2V60). For clarity only interacting residues, labeled in white
wire, are displayed. Ligands and FAD cofactor are depicted in tube; carbon atoms are
colored in green and purple for ligand and FAD respectively. In (a) all hydrogens atoms
are omitted. In (b) only the hydrogen atom forming H-bond, displayed in yellow dot
line, is maintained. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
NMR (75.4 MHz) (CDCl3):
d
¼ 116.4, 119.3, 121.7, 124.6, 127.0, 127.5,
127.7, 129.6, 131.1, 135.5, 135.9, 152.6, 160.1. EI-MS (m/z): 229
[M þ 1]þ, 228 [Mþ]. Anal. Calcd. for C13H8O2S: C 68.40, H 3.53;
Found: C 68.52, H 3.59.
4.2.3. 3-(Thiophen-3-yl)coumarin (1b) [32]
Yield 37%; mp 175e176 ꢁC. 1H NMR (300 MHz) (CDCl3):
d
¼ 7.30
into the binding site, is due both to H-bonds formed with the FAD
cofactor, aromatic rings interactions, and van der Walls forces. For
2b instead, we can appreciate that the ligand-receptor complex is
stabilized not so deeply into the binding cleft through van der
Waals forces and electrostatic interactions.
(d, J ¼ 7.5 Hz, 1H), 7.35 (m, 2H), 7.53 (m, 3H), 7.93 (s, 1H), 8.18 (d,
J ¼ 3.0 Hz, 1H). 13C NMR (75.4 MHz) (CDCl3):
¼ 116.3, 119.4, 122.6,
d
124.5, 125.6, 126.0, 126.2, 127.7, 131.1, 134.3, 137.2, 152.8, 160.0. EI-
MS (m/z): 229 [M þ 1]þ, 228 [Mþ]. Anal. Calcd. for C13H8O2S: C
68.40, H 3.53; Found: C 68.50, H 3.6.
3. Conclusions
4.2.4. 3-(Indol-3-yl)coumarin (1c)
In the current study, three series of 3-heteroarylcoumarin
derivatives were synthesized and evaluated as inhibitors of MAO-A
Yield 33%; mp 190e191 ꢁC. 1H NMR (300 MHz) (CDCl3):
(m, 3H), 7.48 (m, 2H), 7.58 (dd, J ¼ 7.6; 1.3 Hz, 1H), 7.98 (m, 1H); 8.16
d
¼ 7.32

G. Delogu et al. / European Journal of Medicinal Chemistry 46 (2011) 1147e1152
1151
(s, 1H), 8.20 (d, J ¼ 2.6 Hz, 1H), 8.65 (br; 1H). 13C NMR (75.4 MHz)
d
¼ 55.6, 56.0, 92.4, 95.1, 126.5, 127.1, 127.3, 127.5, 131.5, 136.9, 153.2,
(CDCl3):
d
¼ 111.9,116.3,119.5,120.2,120.9,122.7,122.9,124.4,125.5,
155.3,156.9,160.0,163.4. EI-MS (m/z): 289 [M þ 1]þ, 288 [Mþ]. Anal.
127.2, 127.6, 130.1, 134.9, 136.2, 143.5, 152.2, 160.8. EI-MS (m/z): 262
[M þ 1]þ, 261 [Mþ]. Anal. Calcd. for C17H11NO2: C 78.15, H 4.24;
Found: C 78.20, H 4.30.
Calcd. for C15H12O4S: C 62.49, H 4.20; Found: C 62.55, H 4.27.
4.2.12. 5,7-Dimethoxy-3-(thiophen-3-yl)coumarin (4b)
Yield 35%; mp 158e159 ꢁC. 1H NMR (300 MHz) (CDCl3):
d
¼ 3.85
4.2.5. 7-Methoxy-3-(thiophen-2-yl)coumarin (2a) [33]
(s, 3H), 3.90 (s, 3H), 6.28 (d, J ¼ 1.8 Hz, 1H), 6.42 (d, J ¼ 1.8 Hz, 1H),
7.35 (dd, J ¼ 4.8; 2.5 Hz, 1H), 7.52 (d, J ¼ 4.8 Hz, 1H), 8.10 (d,
Yield 27%; mp 155e156 ꢁC. 1H NMR (300 MHz) (CDCl3):
d
¼ 3.89
(s, 3H), 6.85 (s, 1H), 6.88 (d, J ¼ 8.4 Hz, 1H), 7.11 (m, 1H), 7.38 (dd,
J ¼ 4.0; 1.0 Hz, 1H), 7.45 (d, J ¼ 8.4 Hz, 1H), 7.74 (dd, J ¼ 4.0; 1.0 Hz,
J ¼ 2.5 Hz, 1H), 8.20 (s, 1H). 13C NMR (75.4 MHz) (CDCl3):
¼ 55.6,
d
55.8, 92.2, 94.8, 104.3, 117.3, 124.4, 125.2, 126.1, 132.7, 135.0, 155.3,
156.8, 160.4, 163.2. EI-MS (m/z): 289 [M þ 1]þ, 288 [Mþ]. Anal.
Calcd. for C15H12O4S: C 62.49, H 4.20; Found: C 62.53, H 4.24.
1H), 7.96 (s, 1H). 13C NMR (75.4 MHz) (CDCl3):
113.1, 126.1,126.5, 126.8, 127.4, 128.7,136.0, 136.4, 154.5, 161.0, 162.5.
EI-MS (m/z): 259 [M þ 1]þ, 258 [Mþ]. Anal. Calcd. for C14H10O3S: C
65.10, H 3.90; Found: C 65.17, H 3.96.
d
¼ 55.8, 100.4, 112.9,
4.2.13. 5,7-Dimethoxy-3-(indol-3-yl)coumarin (4c)
Yield 43%; mp 179e180 ꢁC. 1H NMR (300 MHz) (CDCl3):
d
¼ 3.88
4.2.6. 7-Methoxy-3-(thiophen-3-yl)coumarin (2b)
(s, 3H), 3.95 (s, 3H), 5.68 (d, J ¼ 7.2 Hz, 1H), 6.33 (d, J ¼ 2.0 Hz, 1H),
6.48 (d, J ¼ 2.0 Hz, 1H), 7.33 (m, 2H), 7.89 (dd, J ¼ 7.2; 1.2 Hz, 1H),
8.17 (s, 1H), 8.33 (dd, J ¼ 7.0; 1.2 Hz, 1H), 8.48 (br, 1H). 13C NMR
Yield 25%; mp 168e169 ꢁC. 1H NMR (300 MHz) (CDCl3):
d
¼ 3.87
(s, 3H), 6.85 (m, 2H), 7.36 (dd, J ¼ 5.0; 3.0 Hz, 1H), 7.42 (d, J ¼ 8.4 Hz,
1H), 7.50 (dd, J ¼ 5.0; 1.2 Hz, 1H), 7.87 (s, 1H), 8.11(dd, J ¼ 3.0; 1.2 Hz,
(75.4 MHz) (CDCl3):
d
¼ 55.8, 56.0, 92.3, 95.0, 104.6, 113.6, 115.3,
1H). 13C NMR (75.4 MHz) (CDCl3):
125.0, 125.5, 126.0, 128.6, 134.6, 137.5, 154.6, 160.8, 162.4. EI-MS (m/
z): 259 [M þ 1]þ, 258 [Mþ]. Anal. Calcd. for C14H10O3S: C 65.10, H
3.90; Found: C 65.15, H 3.97.
d
¼ 55.7, 100.3, 112.8, 113.0, 119.3,
115.8, 119.6, 122.6, 124.5, 127.6, 133.3, 136.0, 151.0, 155.0, 156.8, 161.1,
163.2. EI-MS (m/z): 322 [M þ 1]þ, 321 [Mþ]. Anal. Calcd. for
C19H15NO4: C 71.02, H 4.71; Found: C 71.10, H 4.77.
4.3. Determination of MAO isoforms activity
4.2.7. 3-(Indol-3-yl)-7-methoxycoumarin (2c)
Yield 29%; mp 185e186 ꢁC. 1H NMR (75.4 MHz) (CDCl3):
d
¼ 3.95
The effects of the new synthesized compounds on hMAO iso-
form enzymatic activity were evaluated by a fluorimetric method
following the experimental protocol previously described by
us [15].
Briefly, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4)
containing the test drugs (new compounds or reference inhibitors)
in various concentrations and adequate amounts of recombinant
hMAO-A or hMAO-B required and adjusted to obtain in our
experimental conditions the same reaction velocity, (hMAO-A:
(s, 3H), 6.89 (m, 2H), 7.28 (m, 2H), 7.47 (m, 2H), 7.95 (m, 1H), 8.10
(s, 1H), 8.13 (d, J ¼ 3.0 Hz, 1H), 8.57 (br, 1H). 13C NMR (300 MHz)
(CDCl3):
d
¼ 55.7, 100.3, 100.0, 111.7, 112.6, 113.6, 119.4, 119.5, 120.6,
122.5, 126.0, 126.8, 128.1, 135.5, 136.2, 153.8, 161.1, 161.6. EI-MS (m/
z): 292 [M þ 1]þ, 291 [Mþ]. Anal. Calcd. for C18H13NO3: C 74.22, H
4.50; Found: C 74.29, H 4.56.
4.2.8. 6-Methoxy-3-(thiophen-2-yl)coumarin (3a)
Yield 47%; mp 150e151 ꢁC. 1H NMR (300 MHz) (CDCl3):
d
¼ 3.85
1.1
mg protein; specific activity: 150 nmol of p-tyramine oxidized to
(s, 3H), 6.96 (d, J ¼ 2.8 Hz, 1H), 7.09 (m, 2H), 7.27 (d, J ¼ 9.0 Hz, 1H),
7.42 (dd, J ¼ 5.1; 1.0 Hz, 1H), 7.79 (dd, J ¼ 3.7; 1.0 Hz, 1H), 7.94
p-hydroxyphenylacetaldehyde/min/mg protein; hMAO-B: 7.5 mg
protein; specific activity: 22 nmol of p-tyramine transformed/min/
mg protein) were incubated for 15 min at 37 ꢁC in a flat-black-
bottom 96-well microtestÔ plate, placed in the dark fluorimeter
chamber. After this incubation period, the reaction was started by
(s, 1H). 13C NMR (75.4 MHz) (CDCl3):
119.6, 121.9, 127.0, 127.5, 127.7, 135.2, 135.9, 147.1, 156.2, 161.1. EI-MS
(m/z): 259 [M þ 1]þ, 258 [Mþ]. Anal. Calcd. for C14H10O3S: C 65.10, H
3.90; Found: C 65.15, H 3.94.
d
¼ 55.7, 109.4, 117.3, 119.0,
adding (final concentrations) 200 m
M AmplexÒ Red reagent,1 U/mL
horseradish peroxidase and 1 mM p-tyramine. The production of
H2O2 and, consequently, of resorufin was quantified at 37 ꢁC in
a multidetection microplate fluorescence reader (FLX800Ô, Bio-
TekÒ Instruments, Inc., Winooski, VT, USA) based on the fluores-
cence generated (excitation, 545 nm, emission, 590 nm) over
a 15 min period, in which the fluorescence increased linearly.
Control experiments were carried out simultaneously by
replacing the test drugs (new compounds and reference inhibitors)
with appropriate dilutions of the vehicles. In addition, the possible
capacity of the above test drugs to modify the fluorescence gener-
ated in the reaction mixture due to non-enzymatic inhibition (e.g.,
for directly reacting with AmplexÒ Red reagent) was determined by
adding these drugs to solutions containing only the AmplexÒ Red
reagent in a sodium phosphate buffer.
4.2.9. 6-Methoxy-3-(thiophen-3-yl)coumarin (3b)
Yield 43%. mp 164e165 ꢁC. 1H NMR (300 MHz) (CDCl3):
d
¼ 3.84
(s, 3H), 6.95 (d, J ¼ 3.0 Hz, 1H), 7.06 (dd, J ¼ 9.0; 3.0 Hz, 1H), 7.25
(d, J ¼ 9.0 Hz,1H), 7.37 (dd, J ¼ 5.1;3.0 Hz,1H), 7.50(dd, J ¼ 5.1;1.3 Hz,
1H), 7.86 (s, 1H), 8.18 (dd, J ¼ 3.0; 1.3 Hz, 1H). 13C NMR (75.4 MHz)
(CDCl3):
d
¼ 55.7, 109.6, 117.2, 118.9, 119.7, 122.8, 125.6, 126.0, 126.1,
134.3, 136.9,147.2,156.0, 160.1. EI-MS (m/z): 259 [M þ 1]þ, 258 [Mþ].
Anal. Calcd. for C14H10O3S: C 65.10, H 3.90; Found: C 65.20, H 3.99.
4.2.10. 3-(Indol-3-yl)-6-methoxycoumarin (3c)
Yield 38%; mp 188e189 ꢁC. 1H NMR (300 MHz) (CDCl3):
d
¼ 3.84
(s, 3H), 5.87 (d, J ¼ 7.7 Hz,1H), 6.93 (d, J ¼ 2.8 Hz,1H), 7.04 (dd, J ¼ 9.0;
2.8 Hz, 1H), 7.30 (m, 3H), 7.79 (d, J ¼ 7.7 Hz, 1H), 8.01 (s, 1H), 8.27
(s, 1H), 8.30 (br, 1H). 13C NMR (75.4 MHz) (CDCl3):
d
¼ 55.7, 109.3,
The specific fluorescence emission (used to obtain the final
results) was calculated after subtraction of the background activity,
which was determined from vials containing all components except
the hMAO isoforms, which were replaced by a sodium phosphate
buffer solution.
112.6, 115.4, 117.2, 118.7, 119.3, 119.8, 122.7, 124.6, 125.7, 127.3, 135.9,
137.0, 146.7, 150.8, 156.1, 160.5. EI-MS (m/z): 292 [M þ 1]þ, 291 [Mþ].
Anal. Calcd. for C18H13NO3: C 74.22, H 4.50; Found: C 74.30, H 4.52.
4.2.11. 5,7-Dimethoxy-3-(thiophen-2-yl)coumarin (4a)
Yield 45%; mp 177e178 ꢁC. 1H NMR (300 MHz) (CDCl3):
d
¼ 3.86
4.4. Ligand docking
(s, 3H), 3.93 (s, 3H), 6.31 (d, J ¼ 2.1 Hz, 1H), 6.46 (d, J ¼ 2.1 Hz, 1H),
7.10 (dd, J ¼ 5.0; 3.6 Hz, 1H), 7.36 (d, J ¼ 5.0 Hz, 1H), 7.72
(d, J ¼ 3.6 Hz, 1H), 8.29 (s, 1H). 13C NMR (75.4 MHz) (CDCl3):
All docking calculations were performed with Maestro 9.0
package [34]. The crystal structure of hMAO-B was prepared for

1152
G. Delogu et al. / European Journal of Medicinal Chemistry 46 (2011) 1147e1152
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ꢀ
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Acknowledgments
This work was partially supported by grants from Xunta de
Galicia (Spain; PGIDIT09CSA030203PR and 10PXIB203303PR),
University of Cagliari (ex 60% 2009), Fondazione Banco Sardegna
2007 and MIUR 2008.
E. Quezada thanks for a postdoctoral grant from FCT (Portugal).
G. Ferino is grateful to Regione Autonoma della Sardegna for
a Master & Back scholarship. Authors thanks S. Vilar for helpful
suggestions on the docking experiments. Technical support from
Red Gallega de Boinformatica and Centro de Supercomputacion de
Galicia (CESGA) is gratefully acknowledged.
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