Synthesis and study of a series of 3-arylcoumarins as potent and selective monoamine oxidase B inhibitors

Article abstract of DOI:10.1021/jm200716y

New series of 6-substituted-3-arylcoumarins displaying several alkyl, hydroxyl, halogen, and alkoxy groups in the two benzene rings have been designed, synthesized, and evaluated in vitro as human monoamine oxidase A and B (hMAO-A and hMAO-B) inhibitors.

Full text of DOI:10.1021/jm200716y

ARTICLE
pubs.acs.org/jmc
Synthesis and Study of a Series of 3-Arylcoumarins as Potent and
Selective Monoamine Oxidase B Inhibitors
Maria J. Matos,*,† Carmen Terꢀan,‡ Yunierkis Pꢀerez-Castillo,‡ Eugenio Uriarte,† Lourdes Santana,† and
Dolores Vi~na*,§
†Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain
‡Department of Organic Chemistry, Faculty of Chemistry, University of Vigo, 36310 Vigo, Spain
§Department of Pharmacology, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain
ABSTRACT: New series of 6-substituted-3-arylcoumarins dis-
playing several alkyl, hydroxyl, halogen, and alkoxy groups in the
two benzene rings have been designed, synthesized, and eval-
uated in vitro as human monoamine oxidase A and B (hMAO-A
and hMAO-B) inhibitors. Most of the studied compounds
showed a high affinity and selectivity to the hMAO-B isoen-
zyme, with IC50 values on nanomolar and picomolar range. Ten
of the 22 described compounds displayed higher MAO-B
inhibitory activity and selectivity than selegiline. Coumarin 7
is the most active compound of this series, being 64 times more
active than selegiline and also showing the highest hMAO-B
specificity. In addition, docking experiments were carried out on
hMAO-A and h-MAO-B structures. This study provided new information about the enzymeꢀinhibitor interaction and the potential
therapeutic application of this 3-arylcoumarin scaffold.
’ INTRODUCTION
inhibitors, such as clorgyline (irreversible) and moclobemide
(reversible), are used for the treatment of neurological disorders
like depression and anxiety,28 while selective and irreversible
MAO-B inhibitors, such as selegiline and rasagiline, are used in
the therapy of Parkinson’s disease.29,30 All of these aspects have
led to an intensive search for novel MAO inhibitors. Additionally,
the description of the crystal structure of the two MAO isoforms
by Binda et al. has provided relevant information about the
selective interactions and the pharmacophoric requirements
needed for the design of potent and selective inhibitors.31ꢀ34
In recent years, interest in selective hMAO-B inhibitors has
significantly intensified due to the discovery that expression
levels of this isoenzyme in neuronal tissue increase 4-fold with
age, resulting in an increment of dopamine metabolism, as well as
of production of hydrogen peroxide, which cause oxidative stress
and may play a relevant role in the etiology of neurodegenerative
diseases.35 As a result, MAO-B inhibitors would be useful as
adjuvant for the treatment of Parkinson’s and Alzheimer’s
diseases. For instance, it has been reported that selegiline
protects neuronal cells from the consequences of the oxidative
stress in these patients.36
Coumarins are a wide family of compounds present in
remarkable amounts in the nature.1,2 Representative compounds
occur for instance in the vegetable kingdom, either in free or
combined state.1,2 Because of their structural variability, these
heterocyclic compounds occupy an important role not only in
organic chemistry but also in medicinal chemistry.3,4 Therefore,
coumarins have been attracting considerable interest because of
their numerous biological activities. In the literature, coumarin
derivatives have been described as anticancer,5 antioxidant, or
anti-inflammatory,6 vasorelaxant,7 antimicrobial,8,9 antiviral,10,11
and enzymatic inhibitors.12ꢀ14 In particular, some coumarins had
been previously described as monoamine oxidase inhibitors.15ꢀ17
Monoamine oxidases (MAOs) are FAD-depending enzymes
found in the outer mitochondrial membrane of neuronal, glial,
and other mammalian cells.18 These enzymes are responsible for
catalyzing the oxidative deamination of neurotransmitters and
dietary amines, regulating intracellular levels of biogenic amines
in the brain and the peripheral tissues.19,20 Two enzymatic
isoforms, named MAO-A and MAO-B, have been identified on
the basis of their amino acid sequences,21 three-dimensional
structure,22 tissue distribution,23 inhibitor selectivity,24 and sub-
strate preferences.25 The hMAO-A isoform has a higher affinity for
serotonin and noradrenaline, whereas hMAO-B isoform prefer-
entially deaminates β-phenylethylamines and benzylamine.26,27
On the other hand, dopamine and tyramine are common
substrates for both isoforms. These physiologic properties de-
termine the clinical interest of MAO inhibitors. Selective MAO-A
In the past few years, a broad consensus has been reached
concerning the necessity for the research of new, more potent,
and less toxic MAO-B selective inhibitors. This research has
resulted in a significant number of compounds with different
Received: June 6, 2011
Published: September 18, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
the 3-arylcoumarins 1, 2, and 6ꢀ14. The treatment of the
3-(methoxyphenyl)-6-methylcoumarins 9,47 10,48 12, and 14 with
N-bromosuccinimide (NBS), in carbon tetrachloride (CCl4), under
reflux, using 2,20-azo-bis-iso-butyronitrile (AIBN) as catalyst,49
afforded the bromomethoxy derivatives 15ꢀ18. In addition, com-
pounds 3, 19ꢀ21 were obtained by acidic hydrolysis of the respec-
tive methoxy derivatives 2, 9, 10, and 11, using hydriodic acid
57% in the presence of acetic acid and acetic anhydride.48
Finally, the Williamson reaction45 of the hydroxycoumarin 3
with chloroacetone or cyclopentyl bromide, gave the corre-
sponding ethers 4 and 5, respectively. The Williamson reaction
of the hydroxycoumarin 20 with a chloroacetone gave the
corresponding derivative 22.
Figure 1. Coumarin-based MAO-A and MAO-B inhibitors.
’ BIOCHEMISTRY
Figure 2. Planed modifications and newly synthesized coumarins.
The biological evaluation of the test drugs on hMAO activity
was investigated by measuring their effects on the production of
hydrogen peroxide (H2O2) from p-tyramine (a common sub-
strate for h-MAO-A and hMAO-B), using the Amplex Red MAO
assay kit (Molecular Probes, Inc., Eugene, Oregon, USA) and
microsomal MAO isoforms prepared from insect cells (BTI-TN-
5B1ꢀ4) infected with recombinant baculovirus containing
cDNA inserts for hMAO-A or hMAO-B (Sigma-Aldrich Química
SA, Alcobendas, Spain) were used as source for the two micro-
somal MAO isoforms.53 The production of H2O2 catalyzed by
the two MAO isoforms can be detected using 10-acetyl-3,
7-dihydroxyphenoxazine (Amplex Red reagent), a nonfluores-
cent and highly sensitive probe that reacts with H2O2 in the
presence of horseradish peroxidase to produce a fluorescent
product, resorufin. New compounds and reference inhibitors
were unable to react directly with the Amplex Red reagent, which
indicates that these drugs do not interfere with the measure-
ments. On the other hand, in our experiments and under our
experimental conditions, hMAO-A displayed a Michaelis con-
stant (Km) equal to 457.17 ( 38.62 μM and a maximum reac-
tion velocity (Vmax) in the control group of 185.67 ( 12.06
(nmol p-tyramine/min)/mg protein, whereas hMAO-B showed
structural scaffolds.37ꢀ42 Therefore, the coumarin nucleus has
emerged in the 1990s as a promising scaffold for MAO
inhibitors.43 Structureꢀselectivity relationship studies about
coumarin derivatives suggested that selectivity was mainly de-
termined by the nature of the linkage between the coumarin and the
lipophilic aryl groups in the position 7.44 Therefore, the alkyl-
sulfonyloxy group provided MAO-A inhibitors like esuprone,
while a benzyloxy substituent led to potent and selective MAO-B
inhibitors, such as 7-benzyloxy-3,4-dimethylcoumarin (Figure 1).45
In relation to this research, we have previously reported
interesting MAO inhibitory properties for several 7-substituted
coumarins containing a benzene ring fused at 3,4 positions
(Figure 2).46 This condensation, in general, increased the
MAO-A and MAO-B inhibitory activities, and when an addi-
tional acetonyloxy group was present in the 7 position, this
resulted in compounds with very high MAO-B selectivity. These
results led us to analyze the importance of the aryl substituent
under the pyrone ring and the above-mentioned C7 substituent,
synthesizing a series of 3-arylcoumarin derivatives (Figure 2).
The substitution at C7 was removed and a smaller substituent
was introduced at C6 and the 3,4-condensed benzene ring has
moved at C3, including different substituent groups on it.47ꢀ49
The high MAO-B selectivity found for this new 3-arylcoumarin
scaffold encouraged us to synthesize and study the MAO
inhibitory activity of new analogues, in which a variety of groups
with different size, electronic, and lipophilic properties were
introduced in both aromatic rings, in order to clarify the influence
of the substitution pattern in the MAO inhibitory activity and
selectivity of the 3-arylcoumarin skeleton.
a Km of 220.33 ( 32.80 μM and V
of 24.32 ( 1.97
max
(nmol p-tyramine/min)/mg protein (n = 5). Most tested
compounds, concentration-dependently inhibited this enzy-
matic control activity (Table 1).
’ DOCKING STUDIES
To obtain information about enzymeꢀinhibitor interactions
that help us to explain the structural requirements for MAO
activity and selectivity of 3-arylcoumarin scaffold, a docking study
was performed. The crystallographic structure of MAO-B in
complex with a coumarin inhibitor (pdb code 2V61)52 and of
MAO-A in complex with harmine (pdb code 2ZX5)51 were used
to dock the derivates under study. The docking simulations were
carried out using the DOCK v. 6.3 package.54
Finally, on the basis of the significant structural information
currently available on MAOs,50ꢀ52 and to analyze the structural
requirements for MAO activity and selectivity, docking experi-
ments were carried out on hMAO-A and hMAO-B crystallo-
graphic structures.
To validate our docking protocol, we used the 7-(3-chloro-
benzyloxy)-4-[(methylamino)methyl]coumarin cocrystallized
with the MAO-B enzyme,52 as well as two other MAO inhibitors
(3-[(4-fluorophenyl)carbamoyl]coumarin and 3-[(4-methane-
sulfonylphenyl)carbamoyl]coumarin), all of them previously
subjected to molecular modeling studies and having bulky
substituents at the 3-position as is the case for the new coumarin
derivates reported in our manuscript (Figure 3).15
’ CHEMISTRY
The coumarin derivatives 1ꢀ22 were efficiently synthesized
according to the protocol outlined in Scheme 1. The chemical
structure of the compounds is organized in Table 1. The general
reaction conditions and the compounds characterization are
described in the Experimental Section.
Perkin condensation of different ortho-hydroxybenzaldehydes
with the adequate arylacetic acid, using N,N0-dicyclohexyl-
carbodiimide (DCC) as dehydrating agent,47,48 afforded
Although the proposed docking methodology is able to
reproduce the crystallographic orientation of the MAO-B
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Scheme 1a
a Reagents and conditions: (i) substituted phenylacetic acid, DCC, DMSO, 110 °C, 24 h; (ii) HI, AcOH, Ac2O, reflux, 3 h; (iii) chloroacetone, K2CO3,
acetone, reflux, 16 h; (iv) cyclopentyl bromide, K2CO3, acetone, reflux, 24 h; (v) NBS, AIBN, CCl4, reflux, 18 h.
coumarinic inhibitor (pdb code 2V61), the successful redocking
of the crystallographic inhibitor does not warrant a realistic
binding prediction of the new inhibitors proposed. To study
whether our methodology is able or not to reproduce the
previously described binding mode of noncrystallographic in-
hibitors, we redocked the last two above-mentioned compounds.
This yielded that the obtained binding modes are in agreement
with the results previously reported by Chimenti et al.15
During the validation of the docking protocol, we also
included 3, 5, and 6 structural water molecules in the receptor
structure, yielding similar scoring and orientations for the
compounds as in the water depleted receptor structure. This is
the main reason we did not use any structural water molecule in
the modeling of the compounds included in the paper. Further-
more, the use of the water depleted receptor would allow the
ligands to explore the lower region of the binding pocket. In this
way, the docking algorithm is able to explore orientations of the
inhibitors with the 3-substituent oriented to the upper subpocket
and to the FAD cofactor. Despite the fact that water molecules
were not considered during the ligands orientation step, the
predicted poses were rescored using the DOCK Amber-based
scoring function, which takes into account the solvent effect
using a continuous solvent model and account for small structur-
al rearrangements on the predicted complexes structures.55,56
These results support the use of our protocol for the prediction
and analysis of the binding mode of the coumarin derivatives
described in this study.
The molecular docking studies were done for different com-
pounds of the studied series. Although we mainly focused the
present results on two representative compounds, the coumarin
derivatives 9 and 10, with substituents in para and meta positions
(Figure 4). They interact with the isoenzyme in the well-known
binding pocket,32,33,50,52 with the substituent at C6 pointing to
the FAD cofactor, Phe343, and Tyr60. The coumarin ring is
oriented toward the bottom of the substrate cavity, interacting
with the FAD cofactor as well as with Tyr398, Tyr435, and
Gln206 through van der Waals and hydrophobic interactions,
including πꢀπ interactions with these tyrosine residues.
In addition, the predicted orientation of the coumarin moiety
allows the interaction of its oxygen atoms of the coumarin moiety
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Table 1. Structure and hMAO Inhibitory Activity in Vitro of the 3-Arylcoumarin Derivatives 1ꢀ22 and Reference Compoundsa
compd
R
R20
R30
CH3
R40
R50
IC50 hMAO-A (μM)
IC50 hMAO-B
SId
1
2
3
4
5
6
7
8
9
OCH3
OCH3
OH
H
H
H
b
17.05 ( 0.88 nM
1.52 ( 0.08 nM
67.10 ( 2.99 nM
5.52 ( 0.29 μM
14.47 ( 0.78 μM
283.75 ( 19.90 nM
0.31 ( 0.02 nM
15.01 ( 0.83 nM
13.05 ( 0.90 nM
>5882e
>6789e
372
>18e
>6.9e
H
H
CH3
CH3
CH3
CH3
H
H
b
H
H
H
24.90 ( 1.33
C3H5O2
C5H9O
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
b
H
H
H
b
H
H
H
b
>352e
H
H
CH3
H
H
b
>333333e
>6667e
>7663
>125000
H
CH3
H
H
c
H
OCH3
H
H
b
10
H
OCH3
H
H
b
0.80 ( 0.05 nM
b
11
OCH3
H
H
H
b
12
OCH3
OCH3
OCH3
Br
H
OCH3
H
b
8.98 ( 1.42 nM
2.73 ( 0.12 nM
160.64 ( 1.01 nM
0.74 ( 0.02 nM
3.25 ( 0.17 nM
54.03 ( 3.91 μM
b
>11136e
9209
>623e
>135870e
>31250e
>1.9e
13
H
OCH3
OCH3
OCH3
Br
25.14 ( 1.68
14
H
OCH3
H
b
15
H
b
16
H
OCH3
OCH3
OCH3
H
H
b
17
Br
Br
H
H
OCH3
OCH3
H
b
18
OCH3
OH
H
b
19
b
155.59 ( 17.09 nM
650.03 ( 34.82 nM
120.02 ( 6.43 nM
180.04 ( 9.65 nM
19.60 ( 0.86 nM
7.54 ( 0.36 μM
>643e
54
20
H
OH
H
H
35.04 ( 1.88
21.58 ( 1.16
c
21
OH
H
H
H
180
22
C3H5O2
H
H
>555e
3431
0.87
Selegiline
67.25 ( 1.02
6.56 ( 0.76
Iproniazide
a Each IC50 value is the mean ( SEM from five experiments (n = 5). b Inactive at 100 μM (highest concentration tested), at higher concentrations the
c
compounds precipitate. 100 μM inhibits the corresponding hMAO activity by approximately 40ꢀ45%, at higher concentrations the compounds
precipitate. d Selectivity index: MAO-B selectivity ratios [IC50 (MAO-A)]/[IC50 (MAO-B)] for inhibitory effects of both new compounds and reference
inhibitors. e Values obtained under the assumption that the corresponding IC50 against MAO-A is the highest concentration tested (100 μM).
Figure 3. Compounds used to validate the docking protocol.
with Cys172, showing a favorable geometry for the formation
of hydrogen bonds between the ligand and the receptor.
Moreover, the coumarin core also interacts with Ile198,
Ile199, and Leu171. Finally, the substituted 3-phenyl ring is
oriented to an entrance cavity, an hydrophobic subpocket
existing only in the MAO-B isoform, which is defined by
Leu171, Ile199, Tyr326, Phe168, Ile316, Trp119, Pro102, and
Pro104. This is probably one explanation to the MAO-B
selectivity of the described compounds.
’ RESULTS AND DISCUSSION
All described coumarins in this report (compounds 1ꢀ22)
were efficiently synthesized and evaluated for their ability to
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Figure 4. Molecular docking studies of coumarin derivatives 9 and 10. Most stable binding poses of compound 10 (a) and 9 (b) into MAO-B binding
site, and compound 10 (c) into MAO-A binding site. Compounds are colored by atom type, and predicted H-bonds are represented as green
pseudobonds. Superposition of binding poses of compound 10 (d) to both isoenzymes, MAO-A (ligand and Phe208 in magenta), and MAO-B.
inhibit the A and B isoforms of hMAO. The corresponding IC50
values and MAO-B selectivity ratios [IC50 (MAO-A)]/[IC50
(MAO-B)] are shown in Table 1. The chemical structures of the
newly designed compounds, as well as the biological and docking
results, can help us with an interesting SAR study. From the
experimental results, it can be observed that most of the tested
compounds are selective inhibitors toward MAO-B, with IC50
values in the low micro, nano, and picomolar range. Ten of the 22
tested compounds have similar or better IC50 than the selegiline
(IC50 = 19.60 nM, reference MAO-B inhibitor). Two of the most
active compounds are the coumarin derivatives 7 and 10, with
IC50 against hMAO-B of 0.31 and 0.80 nM, respectively. Both
compounds 7 and 10 are para or meta substituted in the 3-phenyl
ring with a methyl or a methoxy group, respectively. With the aim
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of introducing a group with different physicochemical properties
under the 3-phenyl ring of the coumarin moiety, we have
brominated the different 3-methoxyphenyl derivatives. There-
fore, compound 15 has the para and meta positions substituted
with a methoxy and a bromo groups, respectively. This com-
pound proved to be one of the three most active compounds,
with IC50 against hMAO-B of 0.74 nM. This new substitution
makes this compound a better MAO-B inhibitor than the
corresponding derivative without the bromo atom, compound
9, with an IC50 of 13.05 nM. On the other hand, compound 16,
with the para and meta positions substituted with a bromo and a
methoxy groups, respectively, seems to lose MAO-B inhibitory
activity. So, we can infer that the presence of an electron donor
at the para position is an important modification to improve
the activity. From the biological data, the coumarin 7, with a
p-methyl substituent in the 3-phenyl ring and a 6-methyl sub-
stituent in the coumarin aromatic ring, was the most potent and
selective one against MAO-B isoenzyme. This compound was 63
times more active than the selegiline, resulting also much more
selective than it (selectivity MAO-B index >333333).
The activity data of new reported compounds point out that
almost all the substitutions at ortho position of the 3-phenyl ring
seem to be unfavorable for the activity. Compounds 11 and 18,
with a methoxy or a bromo substituent, respectively, at the ortho
position are inactive at 100 μM (highest concentration tested).
The corresponding compound 14, compared with compound 18
but without the ortho-bromo atom, has an activity in the
nanomolar range. Compound 17, also with a bromo atom at
ortho position, has an MAO-B inhibitory activity in the high
micromolar range, while their correspondent nonbrominated
compound 12, with a “free” ortho position, has an activity against
MAO-B in the low nanomolar range. It seems clear that the
presence of different substituents in the ortho position annulated
or strongly decreased the inhibitory MAO-B activity. An excep-
tion occurs when the ortho position is substituted with a hydroxyl
group (compound 21). In this case, the methoxy derivative
(compound 11) has no activity against MAO-B and the hydroxyl
one has an IC50 in the nanomolar range. This is going to be
explained later in the discussion of the Docking Studies. More-
over, inhibitory MAO-B activity increases when the small
lipophilic groups (methoxy or methyl) are included at meta or
para positions. Nevertheless, the dimethoxy-substituted com-
pound 13 is less active than the meta-monosubstituted one
(compound 10). In addition, a hydroxyl group incorporated in
para- or meta-positions of the phenyl ring of the moiety seems to
be less favorable for the MAO-B inhibition than a methoxy or
methyl group at the same position, as it can be seen by comparing
the IC50 values of compounds 7ꢀ10 with those of compounds
19 and 20, respectively. Finally, a methyl group in the 6 position
of the coumarin moiety (compound 7) provides better MAO-B
inhibitory activity than a methoxy group (compound 2) or a
hydroxyl group (compound 3). Also, bulkier alkoxy substituents
at C6, such as an 2-oxopropoxy group (compound 4) or a
cyclopentyloxy group (compound 5), significantly reduced the
activity, being better tolerated in the 3-phenyl ring, in particular
in the meta position (compound 22). Almost all the studied
compounds with substituents in the 3-phenyl ring proved to be
most active against the MAO-B than the nonsubstituted 6-methyl-
3-phenylcoumarin 6 (IC50 = 283 nM). Compounds substituted in
the ortho position (excepting compound 21, already mentioned),
compound 20 and compounds bulky substituted (compounds 4
and 5) in position 6 were the only exception.
On the basis of the docking results, the proposed binding
mode for this series of compounds in the pocket of hMAO-B is
consistent with the observation that only small substituents are
tolerated at position 6, as can be seen analyzing the results of the
compounds 4 and 5. The increase in the size of substituents at C6
results in a disruption of the proposed binding mode because part
of the space occupied by the coumarin ring will be used by this
bulky substituent, resulting in the loss of the key interactions to
stabilize the ligandꢀenzyme complex.
Additionally, the poses predicted by the docking simulations
show a small angle between the planes defined by the coumarin
and the 3-phenyl ring. These conformations, which are sterically
prohibited when a methoxy group is included at 20 position of the
3-phenyl ring, are however permitted for a hydroxyl group at the
same position. These conformational changes can explain why
the ortho position substitutions of the 3-phenyl ring considerably
decreased or abolished the activity and why the compound 21
has activity against MAO-B, whereas the corresponding methoxy
derivative 11 is inactive at the highest tested concentration
against the same enzyme (Table 1). Moreover, the docking
results indicate that a substituent at para- or meta-position of the
3-phenyl ring makes the compound occupy the above-men-
tioned hydrophobic subpocket (Figure 4a and 4b).
Finally, the comparison between the predicted binding mode
of compound 10 to MAO-A (Figure 4c) with its binding mode to
MAO-B (Figure 4d) corroborates that entrance cavity only exits
in MAO-B because in MAO-A its formation is prevented by the
presence of the Phe208 residue instead of the Ile199 present in
the same position of MAO-B.
On the basis of the docking studies information, the high
MAO-B selectivity of these series of compounds could be
explained by taking into account two factors: the ability of the
compounds to recognize and exploit the entrance cavity in
MAO-B and the fact that, according to predicted binding mode,
in MAO-A these compounds should occupy a more distant
position into the binding pocket to fit in the cavity and interact
with the binding points (ligand in magenta ball and sticks,
Figure 4d). This movement to a deeper region in the cavity
results in the displacement of some water molecules conserved in
the X-ray structures of the MAOs, which is not necessary for the
binding to MAO-B, according to the proposed models. This
displacement of the water molecules is an energetically expensive
process that negatively influences the formation of stable com-
plexes between the inhibitors and MAO-A.
’ CONCLUSION
In this paper, we have used the Perkin reaction as a key step of
a good methodology for the efficient and general synthesis of a
selected series of 3-arylcoumarins. Most of the studied com-
pounds show a high affinity and selectivity for the MAO-B
isoenzyme. Several compounds of these series proved to have
much higher MAO-B inhibitory activity and selectivity than the
selegiline. The docking studies performed on these compounds
show that their interactions with the MAO-B binding pocket are
more intense than those with the MAO-A one. The docking
results are in accord with the biological data and allowed us to
support an interesting SAR study. Additionally, all the results
show that a small substitution at C6 (methyl or methoxy groups)
of the coumarin core seems to be important when a phenyl group
is located at C3. A bulkier group at C6 (compounds 4 and 5)
drastically decreases the MAO-B inhibitory activity. With a small
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Journal of Medicinal Chemistry
ARTICLE
substituent at C6, both kind and position of substituents included
in the 3-aryl group play a crucial role in activity and selectivity.
Meta and para substituents were the most favorable positions for
the desired activity. Currently, further research is going to be
conducted on the 3-arylcoumarin scaffold as potential agents for
the treatment of Parkinson’s disease.
128.3, 129.2, 129.5, 132.3, 134.1, 134.8, 138.0, 139.5, 139.8, 151.6, 160.8.
MS m/z 251 ([M + 1]+, 27), 250 (M+, 100), 222 (72), 221 (39), 178
(32), 150 (10), 136 (10), 124 (10). Anal. Calcd for C17H14O2: C, 81.58;
H, 5.64. Found: C, 81.56; H, 5.62.
General Procedure for the Preparation of 3-(Bromometh-
oxyphenyl)coumarins (15ꢀ18). A solution of 3-(methoxyphenyl)-
coumarins (3.76 mmol), NBS (4.51 mmol), and AIBN (cat.) in CCl4
(5 mL) was stirred under reflux for 18 h. The resulting solution was filtered
to remove the succinimide. The solvent was evaporated under vacuum
and purified by flash chromatography (hexane/ethyl acetate 95:5).
3-(3-Bromo-4-methoxyphenyl)-6-methylcoumarin (15). Yield: 41%;
mp 206ꢀ207 °C. 1H NMR (CDCl3) 2.42 (s, 3H, ꢀCH3), 3.94 (s, 3H,
ꢀOCH3), 6.97 (d, 1H, H-50 J = 8.7), 7.23ꢀ7.35 (m, 3H, H-20, H-60,
H-5), 7.69ꢀ7.74 (m, 2H, H-7, H-8), 7.89 (s, 1H, H-4). 13C NMR
(CDCl3) 20.8, 56.3, 111.5, 111.6, 116.1, 119.3, 126.3, 127.6, 128.5,
129.0, 132.5, 133.1, 134.2, 139.1, 151.5, 156.2, 160.6. MS m/z (%) 347
(18), 346 (98), 345 ([M + 1]+, 19), 344 (M+, 100), 303 (45), 301 (45),
275 (11), 250 (17), 222 (13), 194 (11), 178 (13), 165 (58), 163 (11),
139 (15), 132 (42), 82 (18), 76 (14), 63 (19), 50 (14). Anal. Calcd for
C17H13BrO3: C, 59.15; H, 3.80. Found: C, 59.10; H, 3.72.
3-(4-Bromo-3-methoxyphenyl)-6-methylcoumarin (16). Yield: 51%;
mp 139ꢀ140 °C. 1H NMR (CDCl3) 2.49 (s, 3H, ꢀCH3), 3.89 (s, 3H,
ꢀOCH3), 6.49 (dd, 1H, H-7, J = 7.4, J = 1.3), 7.08ꢀ7.12 (m, 3H, H-20,
H-60, H-8), 7.32ꢀ7.42 (m, 2H, H-5, H-50), 7.74 (s, 1H, H-4). 13C NMR
(CDCl3) 33.1, 55.4, 114.2, 114.7, 117.1, 119.7, 120.9, 126.8, 128.2,
128.7, 129.6, 132.2, 134.3, 135.7, 139.4, 153.2, 159.5. MS m/z (%) 346
(45), 345 ([M + 1]+, 10), 344 (M+, 100), 266 (49), 265 (45), 238 (24),
237 (67), 194 (42), 165 (29), 133 (28). Anal. Calcd for C17H14O2: C,
59.15; H, 3.80. Found: C, 59.17; H, 3.83.
3-(2-Bromo-3,5-dimethoxyphenyl)-6-methylcoumarin (17). Yield:
46%; mp 178ꢀ179 °C. 1H NMR (CDCl3) 2.40 (s, 3H, ꢀCH3), 3.79 (s,
3H, ꢀOCH3), 3.87 (s, 3H, ꢀOCH3), 6.51 (s, 2H, H-40, H-60),
7.26ꢀ7.33 (m, 3H, H-5, H-7, H-8), 7.60 (s, 1H, H-4). 13C NMR
(CDCl3) 20.8, 55.6, 56.4, 100.1, 104.3, 107.4, 116.4, 118.7, 127.9, 129.0,
132.8, 134.2, 137.6, 142.3, 152.1, 157.0, 159.7. MS m/z (%) 377 (42),
376 ([M + 1]+, 18), 375 (M+, 100), 282 (59), 279 (14), 239 (11) 208
(9), 152 (10), 118 (9), 58 (12). Anal. Calcd for C18H15BrO4: C, 57.62;
H, 4.03. Found: C, 57.61; H, 4.08.
3-(2-Bromo-3,4,5-trimethoxyphenyl)-6-methylcoumarin (18). Yield:
50%; mp 167ꢀ168 °C. 1H NMR (CDCl3) 2.40 (s, 3H, ꢀCH3), 3.83 (s,
3H, ꢀOCH3), 3.90 (s, 6H, ꢀ(OCH3)2), 6.72 (s, 1H, H-60), 7.25 (d, 1H,
H-8, J = 8.4), 7.29 (d, 1H, H-5, J = 1.9), 7.34 (dd, 1H, H-7, J = 8.4, J =
2.0), 7.63 (s, 1H, H-4). 13C NMR (CDCl3) 20.8, 55.6, 61.1, 61.1, 110.1,
110.4, 116.4, 118.7, 127.8, 128.4, 131.2, 132.9, 134.3, 142.7, 143.4, 151.2,
152.0, 152.7, 160.1. MS m/z (%) 408 (32), 407 ([M + 1]+, 12), 406 (M+,
68), 404 (5) 326 (22), 325 (59), 282 (15), 264 (15) 169 (9), 139 (10).
Anal. Calcd for C19H17BrO5: C, 56.31; H, 4.23. Found: C, 56.38;
H, 4.28.
’ EXPERIMENTAL SECTION
Chemistry. Melting points were determined using a Reichert Kofler
thermopan or in capillary tubes on a B€uchi 510 apparatus and are
uncorrected. IR spectra were recorded on a Perkin-Elmer 1640FT
spectrophotometer. 1H and 13C NMR spectra were recorded on a
Bruker AMX spectrometer at 300 and 75.47 MHz, respectively, using
TMS as internal standard (chemical shifts in δ values, J in Hz). Mass
spectra were obtained using a Hewlett-Packard 5988A spectrometer.
Elemental analyses were performed using a Perkin-Elmer 240B micro-
analyser and were within (0.4% of calculated values in all cases. Silica gel
(Merck 60, 230ꢀ00 mesh) was used for flash chromatography (FC).
Analytical thin layer chromatography (TLC) was performed on plates
precoated with silica gel (Merck 60 F254, 0.25 mm). The purity of
compounds 1ꢀ22 was assessed by HPLC and was found to be higher
than 95%.
General Procedure for the Preparation of 3-Phenylcou-
marins (1, 2, 6ꢀ14). A solution of 2-hydroxy-6-methylbenzaldehyde/
2-hydroxy-6-methoxybenzaldehyde (7.34 mmol) and the corresponding
phenylacetic acid (9.18 mmol) in dimethyl sulfoxide (15 mL) was
prepared. N,N0-Dicyclohexylcarbodiimide (11.46 mmol) was added,
and the mixture was heated in an oil bath at 110 °C for 24 h. Ice
(100 mL) and acetic acid (10 mL) were added to the reaction mixture.
After keeping it at room temperature for 2 h, the mixture was extracted
with ether (3 ꢁ 25 mL). The organic layer was extracted with sodium
bicarbonate solution (50 mL, 5%) and then water (20 mL). The solvent
was evaporated under vacuum, and the dry residue was purified by FC
(hexane/ethyl acetate 9:1).
6-Methoxy-3-(3-methylphenyl)coumarin (1). Yield 79%; mp 98ꢀ
99 °C. 1H NMR (CDCl3) 2.46 (s, 3H, ꢀCH3), 3.90 (s, 3H, ꢀOCH3),
7.01 (d, 1H, H-5, J = 2.8), 7.14 (dd, 1H, H-7, J = 9.0, J = 2.9), 7.25ꢀ7.41
(m, 3H, H-50, H-40, H-8), 7.50ꢀ7.54 (m, 2H, H-20, H-60), 7.79 (s, 1H,
H-4). 13C NMR (CDCl3) 21.5, 55.8, 109.9, 117.4, 119.0, 120.0, 125.7,
128.3, 128.8, 129.2, 129.6, 134.7, 138.0, 139.6, 147.9, 156.1, 160.7. MS
m/z 267 ([M + 1]+, 30), 266 (M+, 100), 238 (38), 195 (25), 167 (14),
165 (17), 152 (24). Anal. Calcd for C17H14O3: C, 76.68; H, 5.30. Found:
C, 76.72; H, 5.38.
6-Methoxy-3-(4-methylphenyl)coumarin (2). Yield 76%; mp 130ꢀ
131 °C. 1H NMR (CDCl3) 2.40 (s, 3H, ꢀCH3), 3.86 (s, 3H, ꢀOCH3),
7.01 (d, 1H, H-7, J = 3.9), 7.13ꢀ7.17 (m, 2H, H-5, H-8), 7.30ꢀ7.35 (m,
2H, H-30, H-50), 7.65 (d, 2H, H-20, H-60, J = 8.1), 7.78 (s, 1H, H-4). 13C
NMR (CDCl3) 21.3, 55.8, 109.8, 117.4, 118.9, 120.1, 128.4, 128.6,
129.2, 131.9, 138.9, 139.0, 147.9, 156.1, 160.8. MS m/z 267 ([M + 1]+,
20), 266 (M+, 100), 238 (25), 195 (18), 165 (9), 152 (16). Anal. Calcd
for C17H14O3: C, 76.68; H, 5.30. Found: C, 76.67; H, 5.23.
General Procedure for the Preparation of Hydroxy-3-
phenylcoumarins (3, 20ꢀ21). A solution of substituted 6-meth-
oxy-3-phenylcoumarin (0.50 mmol) in acetic acid (5 mL) and acetic
anhydride (5 mL), at 0 °C, was prepared. Hydriodic acid 57% (10 mL)
was added dropwise. The mixture was stirred, under reflux temperature,
for 3 h. The solvent was evaporated under vacuum, and the dry residue
was purified by CH3CN crystallization.
6-Hydroxy-3-(4-methylphenyl)coumarin (3). Yield 61%; mp 214ꢀ
215 °C. 1H NMR (DMSO-d6) 2.32 (s, 3H, ꢀCH3), 7.01 (d, 1H, H-7, J =
8.8, J = 2.9), 7.07 (d, 1H, H-5, J = 2.8), 7.21ꢀ7.26 (m, 3H, H-30, H-50,
H-8), 7.59 (d, 2H, H-20, H-60, J = 8.1), 8.08 (s, 1H, H-4), 9.77 (s, 1H,
ꢀOH). 13C NMR (DMSO-d6) 21.3, 113.0, 117.1, 120.0, 120.5, 127.2,
128.8, 129.2, 132.3, 138.5, 140.4, 146.7, 154.2, 160.5. MS m/z (%) 253
([M + 1]+, 51), 252 (M+, 100), 225 (35), 224 (96), 223 (62) 195 (13),
181 (17), 165 (19), 152 (31), 139 (10), 125 (15), 115 (15). Anal. Calcd
for C16H12O3: C, 76.18; H, 4.79. Found: C, 76.11; H, 4.77.
6-Methyl-3-(4-methylphenyl)coumarin (7). Yield 72%; mp: 139ꢀ
140 °C. 1H NMR (CDCl3) 2.40 (s, 6H, (ꢀCH3)2), 7.21ꢀ7.30 (m, 5H,
H-5, H-7, H-8, H-30, H-50), 7.60 (d, 2H, H-20, H-60, J = 8.2), 7.72 (s, 1H,
H-4). 13C NMR (CDCl3) 21.1, 21.6, 116.4, 119.7, 127.9, 128.4, 128.7,
129.4, 132.2, 132.5, 134.3, 139.0, 139.4, 139.5, 151.8, 161.2. MS m/z 251
([M + 1]+, 32), 250 (M+, 100), 222 (56), 221 (24), 178 (27), 150 (12),
136 (12), 124 (19). Anal. Calcd for C17H14O2: C, 81.58; H, 5.64. Found:
C, 81.52; H, 5.60.
6-Methyl-3-(3-methylphenyl)coumarin (8). Yield 75%; mp 84ꢀ
1
85 °C. H NMR (CDCl3) 2.39 (s, 6H, ꢀ(CH3)2), 6.98ꢀ7.02 (m,
3H, H-40, H-7, H-8), 7.13ꢀ7.24 (m, 4H, H-20, H-5, H-50, H-60), 7.72 (s,
1H, H-4). 13C NMR (CDCl3) 20.8, 21.5, 116.1, 119.4, 125.6, 127.6,
7133
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Journal of Medicinal Chemistry
ARTICLE
3-(3-Hydroxyphenyl)-6-methylcoumarin (20). Yield 53%; mp 160ꢀ
161 °C. 1H NMR (DMSO-d6) 2.32 (s, 3H, ꢀCH3), 6.77 (dd, 1H, H-40,
J = 7.1, J = 2.4), 7.05ꢀ7.07 (m, 2H, H-5, H-8), 7.21ꢀ7.25 (m, 2H, H-50,
H-60), 7.38 (dd, 1H, H-7, J = 8.5, J = 1.8), 7.50 (s, 1H, H-20), 8.08 (s, 1H,
H-4), 9.52 (s, 1H, ꢀOH). 13C NMR (DMSO-d6) 20.8, 115.9, 116.1,
119.6, 127.3, 128.7, 129.7, 133.0, 134.2, 136.4, 140.8, 151.5, 157.5, 160.2.
MS m/z 254 (12), 253 ([M + 1]+, 63), 252 (M+, 73), 251 (15), 225 (46),
223 (53), 195 (33), 194 (18), 181 (29), 178 (17), 166 (12), 165 (42),
152 (43), 139 (13), 126 (24), 111 (19), 63 (16), 51 (16). Anal. Calcd for
C16H12O3: C, 76.18; H, 4.79. Found: C, 76.20; H, 4.83.
3-(2-Hydroxyphenyl)-6-methylcoumarin (21). Yield 56%; mp 167ꢀ
168 °C. 1H NMR (DMSO-d6) 2.38 (s, 3H, ꢀCH3), 6.30ꢀ6.35 (m, 2H,
H-30, H-50), 7.22ꢀ7.24 (d, 1H, H-5, J = 1.5), 7.29ꢀ7.31 (m, 2H, H-40,
H-60), 7.34 (d, 1H, H-8, J = 8.4), 7.44 (dd, 1H, H-7, J = 8.4, J = 1.5), 7.97
(s, 1H, H-4), 9.60 (s, 1H, ꢀOH). 13C NMR (DMSO-d6) 20.3, 115.6,
118.7, 119.0, 122.3, 125.9, 128.0, 129.6, 130.8, 132.3, 133.6, 141.8, 151.2,
155.0, 159.5. MS m/z 253 ([M + 1]+, 18), 252 (M+, 100), 251 (10), 234
(12), 223 (58), 195 (35), 194 (5), 181 (18), 165 (17), 152 (18), 126 (5),
63 (6), 51 (6). Anal. Calcd for C16H12O3: C, 76.18; H, 4.79. Found: C,
76.20; H, 4.81.
Determination of hMAO Isoform Activity. Briefly, 0.1 mL of
sodium phosphate buffer (0.05 M, pH 7.4) containing different con-
centrations of 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, i.e., to oxidize (in the control
group) the same concentration of substrate: 165 pmol of p-tyramine/
min (hMAO-A, 1.1 μg protein; specific activity, 150 nmol of p-tyramine
oxidized to p-hydroxyphenylacetaldehyde/min/mg protein; hMAO-B,
7.5 μg protein; specific activity, 22 nmol of p-tyramine transformed/
min/mg protein) were incubated for 15 min at 37 °C in a flat-black-
bottom 96-well microtest plate and placed in the dark fluorimeter
chamber. After this incubation period, the reaction was started by adding
(final concentrations) 200 μM Amplex Red reagent, 1 U/mL horse-
radish peroxidase, and 1 mM p-tyramine. The production of H2O2 and,
consequently, of resorufin was quantified at 37 °C in a multidetection
microplate fluorescence reader (FLX800, Bio-Tek Instruments, Inc.,
Winooski, VT, USA) based on the fluorescence generated (excitation,
545 nm, emission, 590 nm) over a 15 min period, in which the
fluorescence increased linearly.
General Procedure for the Preparation of 2-Oxopropoxy-
6-phenylcoumarin (4, 22). Under a suspension of anhydrous
K2CO3 (0.25 mmol) and the corresponding hydroxycoumarin (0.13
mmol), in anhydrous acetone (3 mL), the chloroketone (0.25 mmol)
was added. The suspension was stirred, at reflux temperature, for 16 h.
The mixture was cooled, and the precipitate was recovered by filtration
and washed with anhydrous acetone (3 ꢁ 40 mL). The solvent was
evaporated under vacuum, and the dry residue was purified by FC
(hexane/ethyl acetate 85:15).
Control experiments were carried out simultaneously by replacing
the test drugs (new compounds and reference inhibitors) with appro-
priate dilutions of the vehicles. In addition, the possible capacity of the
above test drugs to modify the fluorescence generated in the reaction
mixture due to nonenzymatic inhibition (e.g., for directly reacting with
Amplex Red reagent) was determined by adding these drugs to solutions
containing only the Amplex Red reagent in a sodium phosphate buffer.
To determine the kinetic parameters of hMAO-A and hMAO-B (Km and
Vmax), the corresponding enzymatic activity of both isoforms was
evaluated (under the experimental conditions described above) in the
presence of a number (a wide range) of p-tyramine concentrations.
The specific fluorescence emission (used to obtain the final results)
was calculated after subtraction of the background activity, which was
determined from vials containing all components except the hMAO
isoforms, which were replaced by a sodium phosphate buffer solution. In
our experimental conditions, this background activity was practically
negligible.
Molecular Docking Simulations. The crystallographic structure
of MAO-B in complex with a coumarin inhibitor (pdb code 2V61)52 and
the MAO-A in complex with harmine (pdb code 2ZX5)51 were used to
dock the coumarins under study. For both isoenzymes, hydrogen atoms
and charges were added to the receptor structure using the UCSF
Chimera,57 and the atomic charges of the FAD cofactor were calculated
using GAMESS.58
Three-dimensional conformers for the compounds were generated
using the OMEGA software.59 A maximum of 50000 conformations per
molecule were generated using an energy window of 10 kcal. All
rotatable bonds were considered during the torsion search, using the
Merck Molecular Force Field (MMFF), and duplicate conformers were
discarded based on a root-mean-square (rms) value of 0.5 Å. A
maximum number of 300 conformers were saved for each compound.
Afterward, AM1-BCC charges were added to each conformer using the
MOLCHARGE program, which is part of the QUACPAC package.60
Rigid docking was carried out for each ligand conformer using the
DOCK 6.3 package.54 A maximum of 5000 orientations per ligand was
assayed. The energy grid-based scoring function was selected for poses
quality evaluation. The five lowest scored poses for each ligand
conformer were saved, allowing for a maximum of 1500 saved poses
for each compound. Afterward, the five lowest scored conformers of
each ligand were rescored, using the DOCK Amber-based scored
function, considering only the flexibility of the ligand. Finally, the pose
with the lowest Amber-based scoring value for each compound was
rescored, with the same scoring function, considering both the ligand
and the binding. We chose the Amber-based scoring function to take
3-(4-Methylphenyl)-6-(2-oxopropoxy)coumarin (4). Yield 74%; mp
1
128ꢀ129 °C. H NMR (CDCl3) 2.33 (s, 3H, ꢀCH3), 2.42 (s, 3H,
ꢀCH3), 4.64 (s, 2H, ꢀCH2), 6.96 (d, 1H, H-5, J = 2.9), 7.14 (dd, 1H,
H-7, J = 9.0, J = 2.9), 7.28ꢀ7.33 (m, 3H, H-30, H-50, H-8), 7.60ꢀ7.65
(m, 2H, H-20, H-60), 7.74 (s, 1H, H-4). 13C NMR (CDCl3) 21.3, 26.6,
73.5, 111.0, 117.7, 119.2, 120.2, 128.4, 129.0, 129.2, 131.7, 138.6, 139.1,
148.4, 154.2, 160.6, 204.7. MS m/z 309 ([M + 1]+, 31), 308 (M+, 100),
266 (12), 265 (50), 236 (15), 235 (26), 207 (31) 195 (12), 179 (16),
178 (16), 165 (11), 153 (12), 129 (20). Anal. Calcd for C19H16O4: C,
74.01; H, 5.23. Found: C, 73.90; H, 5.18.
6-Methyl-3-[3-(2-oxopropoxy)phenyl]coumarin (22). Yield 71%;
1
mp 107ꢀ108 °C. H NMR (CDCl3) 2.31 (s, 3H, ꢀCH3), 2.43 (s,
3H, ꢀCH3), 4.61 (s, 2H, ꢀCH2), 6.94 (d, 1H, H-5, J = 1.2), 7.24ꢀ7.42
(m, 6H, H-7, H-8, H-20, H-40, H-50, H-60), 7.77 (s, 1H, H-4). 13C NMR
(CDCl3) 20.8, 26.6, 73.1, 114.8, 115.0, 116.1, 119.2, 121.9, 127.7, 129.7,
132.6, 134.2, 136.4, 140.2, 151.6, 157.6, 160.6, 205.3. MS m/z 309 ([M +
1]+, 14), 308 (M+, 70), 266 (55), 265 (50), 237 (29), 236 (21), 235
(20), 178 (24), 165 (10). Anal. Calcd for C19H16O4: C, 74.01; H, 5.23.
Found: C, 74.08; H, 5.30.
Preparation of 6-(2-Cyclopentyloxy)-3-(4-methylphenyl)-
coumarin (5). Under a suspension of anhydrous K2CO3 (0.25 mmol)
and the 6-hydroxycoumarin 3 (0.13 mmol), in anhydrous acetone
(3.0 mL), the cyclopentyl bromide (0.25 mmol) was added. The
suspension was stirred, at reflux temperature, for 24 h. The mixture
was cooled, and the precipitate was recovered by filtration and washed
with anhydrous acetone (3 ꢁ 40.0 mL). The solvent was evaporated
under vacuum, and the dry residue was purified by FC (hexane/ethyl
acetate 9:1). Yield 58%; mp 147ꢀ148 °C. 1H NMR (CDCl3) 1.37ꢀ1.99
(m, 8H, (ꢀCH2)4), 2.52 (s, 3H, ꢀCH3), 4.89 (s, 1H, H-100), 7.05 (d,
1H, H-5, J = 2.7), 7.17 (dd, 1H, H-7, J = 9.0, J = 2.7), 7.36ꢀ7.40 (m, 3H,
H-30, H-50, H-8), 7.72 (d, 2H, H-20, H-60, J = 8.1), 7.84 (s, 1H, H-4). 13C
NMR (CDCl3) 21.3, 24.0, 32.8, 80.0, 111.8, 117.3, 120.1, 120.3, 128.4,
128.4, 129.1, 132.0, 138.8, 139.2, 147.6, 154.6, 160.9. MS m/z321([M+1]+,
7), 320 (M+, 28), 253 (19), 252 (100), 224 (38), 223 (13), 152 (11).
Anal. Calcd for C21H20O3: C, 78.75; H, 6.29. Found: C, 78.69; H, 6.24.
7134
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Journal of Medicinal Chemistry
ARTICLE
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California, San Francisco (supported by NIH P41 RR001081).
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: +34 981 563100. Fax: +34 981 544912. E-mail:
mariajoao.correiapinto@rai.usc.es (M.J.M.) and mdolores.vina@
usc.es (D.V.).
’ ACKNOWLEDGMENT
We are grateful to the Xunta de Galicia (09CSA030203PR,
PGIDIT07PXIB, 10PXIB203303PR) and Ministerio de Sanidad
y Consumo (FIS PI09/00501) for financial support. M. Jo~ao
Matos thanks Fundac-~ao de Ci^encia e Tecnologia for the fellow-
ship. Y. Pꢀerez-Castillo thanks OpenEye Scientific Software for
providing an Academic License for using their software at the
Laboratory for Medicinal Chemistry of the Rega Institute for
Medical Research at the Katholieke Universiteit Leuven.
’ DEDICATION
This article is dedicated to the memory of Prof. Francisco Orallo
’ ABBREVIATIONS USED
hMAO, human monoamine oxidase; FAD, flavin adenine dinu-
cleotide; IC50, half maximal inhibitory concentration; cDNA,
cDNA; SAR, structureꢀactivity relationship; PDB, Protein
Data Bank; HPLC, high-performance liquid chromatography
(16) Pisani, L.; Muncipinto, G.; Miscioscia, T. F.; Nicolotti, O.;
Leonetti, F.; Catto, M.; Caccia, C.; Salvati, P.; Soto-Otero, R.; Mendez-
Alvarez, E.; Passeleu, C.; Carotti, A. Discovery of a novel class of potent
coumarin monoamine oxidase B inhibitors: development and biophar-
macological profiling of 7-[(3-chlorobenzyl)oxy]-4-[(methylamino)-
methyl]-2H-chromen-2-one methanesulfonate (NW-1772) as a highly
potent, selective, reversible, and orally active monoamine oxidase B
inhibitor. J. Med. Chem. 2009, 52, 6685–6706.
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