Chromone, a privileged scaffold for the development of monoamine oxidase inhibitors

Article abstract of DOI:10.1021/jm2004267

Two series of novel chromone derivatives were synthesized and investigated for their ability to inhibit the activity of monoamine oxidase. The SAR data indicate that chromone derivatives with substituents in position 3 of γ-pyrone nucleus act preferably a

Full text of DOI:10.1021/jm2004267

ARTICLE
pubs.acs.org/jmc
Chromone, a Privileged Scaffold for the Development of Monoamine
Oxidase Inhibitors†
Alexandra Gaspar,‡,§ Tiago Silva,‡ Matilde Yꢀa~nez,|| Dolores Vina,|| Franscisco Orallo,|| Francesco Ortuso,^
Eugenio Uriarte,§ Stefano Alcaro,*,^ and Fernanda Borges*,‡
‡CIQUP/Departamento de Química e Bioquímica, Faculdade de Ci^encias, Universidade do Porto, 4169-007 Porto, Portugal
§Departamento de Química Orgꢀanica and Departamento de Farmacología, Facultad de Farmacia, Universidad de Santiago de
Compostela, 15782 Santiago de Compostela, Spain
^Dipartimento di Scienze Farmacobiologiche, Facoltꢁa di Farmacia, Universitꢁa “Magna Græcia” di Catanzaro, Campus Universitario
“S. Venuta”, Viale Europa, 88100 Catanzaro, Italy
ABSTRACT: Two series of novel chromone derivatives were synthesized and investigated for
their ability to inhibit the activity of monoamine oxidase. The SAR data indicate that chromone
derivatives with substituents in position 3 of γ-pyrone nucleus act preferably as MAO-B
inhibitors, with IC50 values in the nanomolar to micromolar range. Almost all chromone
3-carboxamides display selectivity toward MAO-B. Identical substitutions on position 2 of γ-
pyrone nucleus result in complete loss of activity in both isoforms (chromones 2ꢀ12 except 3
and 5). Notably, chromone (19) exhibits an MAO-B IC50 of 63 nM, greater than 1000-fold
selectivity over MAO-A, and behaves as a quasi-reversible inhibitor. Docking experiments onto
the MAO binding of the most active compound highlight different interaction patterns among the isoforms A and B. The differential
analysis of the solvation effects among the chromone isomers gave additional insight about the superior outline of the 3-substituted
chromone derivatives.
’ INTRODUCTION
level of dopamine metabolism and in the production of higher
levels of hydrogen peroxide, which are thought to play a major
role in the etiology of neurodegenerative diseases such as
Parkinson’s and Alzheimer’s. MAO-B inhibitors are also currently
in clinical trials for the treatment of Alzheimer’s disease because an
increased level of MAO-B has been detected in the plaque-
associated astrocytes of brains from Alzheimer’s patients.4,5
Today efforts toward the development of monoamine oxidase
inhibitors are focused on selective MAO-A or MAO-B inhibitors.
Selective MAO-B inhibitors, alone or combined with L-Dopa, are
being examined in the treatment of, for example, schizophrenia,
Alzheimer’s disease, and Parkinson’s disease. The MAO-A inhibitors
are effective in the treatment of depression.
MAO-A is more sensitive to inhibition by clorgyline and moclo-
bemide, and MAO-B is selectively inhibited by low concentrations
of selegiline (R-(ꢀ)-deprenyl) and rasagiline (Scheme 2).1,4
All of these aspects have led to an intensive search for novel
MAO inhibitors, and this effort has increased considerably in
recent years. However, a large number of MAOs inhibitors
introduced into clinical practice were discarded because of
adverse effects, such as hepatotoxicity, orthostatic hypotension,
and the so-called “cheese effect”, which is characterized by
hypertensive crises.1 Currently, the MAO inhibitors of the new
generation are usually characterized by their relative specificities
for the MAO subtypes and in some cases by the reversibility of
their actions.
Monoamine oxidases (MAOs, EC 1.4.3.4) are widely distrib-
uted enzymes that contain a flavin adenine dinucleotide (FAD)
covalently bounded to a cysteine residue. Several living organ-
isms possess MAOs that are responsible for the major neuro-
transmitter degrading in the central nervous system (CNS) and
peripheral tissues.1,2
In mammals two isoforms of MAOs are present: MAO-A and
MAO-B. Both isoforms are involved in the oxidative deamination
of exogenous and endogenous amines, including neurotransmit-
ters, thus modulating their concentrations in the brain and
peripheral tissues.1 The MAO-A enzyme is responsible for the
deamination of the epinephrine, norepinephrine, and serotonin,
whereas the MAO-B enzyme metabolizes β-phenethylamine.1,3
The enzyme also plays an important role in the expression of
toxicity of the Parkinsonism-producing neurotoxin 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine by bioactivation into the toxic
metabolite, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium
ion.3
The MAO metabolic reaction involves the oxidation of the
amine function via oxidative cleavage of the R-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 molecular oxygen, with simulta-
neous hydrogen peroxide release. Subsequently, the imine inter-
mediate is hydrolyzed by a nonenzymatic pathway yielding
ammonia and the corresponding aldehyde (Scheme 1).2
Expression levels of MAO-B in neuronal tissue are enhanced
4-fold with aging, especially in glial cells, resulting in an increased
Received: April 10, 2011
Published: June 22, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
ARTICLE
Scheme 1
relationships were obtained by synthetic strategies and screened
toward human MAOs isoforms (hMAOs) to evaluate their
potency/selectivity ratio. The Protein Data Bank (PDB)12 avail-
ability of experimentally determined cocrystals of hMAO-A and -B
with different kinds of ligand allows us to also perform structure-
based molecular modeling studies with the aim to propose preferred
binding modes and to explain the reasons of the isoform selectivity
helping in the rational design of new inhibitors.
’ CHEMISTRY
The functionalization of the chromone nucleous leads to the
obtention of two series of novel chromone carboxamides placed
at positions C2 and C3 of the γ-pyrone ring. The compounds
and the synthetic strategy used for their obtention have been
patented11 and are briefly depicted in Scheme 3. Chromone
carboxamide derivatives were synthesized straightforwardly, in
moderate/high yields, by a one-pot condensation reaction that
occurs, in equimolar amounts, between the corresponding
chromone carboxylic acid (1 or 13) and aniline (phenylamine)
or its ring-substituted derivatives. A similar condensation reac-
tion was performed with propylamine and cyclohexylamine. The
coupling reagents selected for carboxylic acid activation were
organophosphoric compounds, namely, (benzotriazol-1-yloxy)-
tris(dimethylamino)phosphonium hexafluorophosphate (BOP)
and (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluo-
rophosphate (PyBOP).13 In all the reactions, N,N-diisopropyl-
ethylamine (DIPEA) was used instead of the classic triethyl-
amine, since a significant increase of yield was observed due
mainly to an improvement in the purification steps.11
It must be highlighted that the synthetic strategies commonly
adopted for the synthesis of these types of amide are usually
based on a different approach: first, an acyl halide is obtained
through the reaction of a carboxylic acid with thionyl chloride,
followed by the reaction with a nucleofilic agent.14 In fact, the
method herein presented is particularly advantageous, since it
avoids the step of acyl halide obtention and the drawbacks related
with this type of reagent. Furthermore, the use of BOP or PyBOP
has several advantages, related with other coupling agents such as
dicyclohexylcarbodiimide (DCC), in the purification steps and in
the removal of side products. The syntheses of the dihydroxy-
lated compounds 5 and 17 were performed by a demethylation
reaction, using boron tribromide (BBr3), of the monomethoxy-
lated chromones 4 and 16, respectively. The selected strategy
avoids the prior protection of the hydroxyl groups of the starting
materials (Scheme 3).
Scheme 2
Despite considerable progress in understanding the interac-
tions of the two enzyme forms with their preferred substrates and
inhibitors, no general rules are yet available for the rational design
of potent and selective inhibitors of MAO possibly because the
mechanism of interaction of the new drugs with MAO isoforms
has not been fully characterized.
Privileged structures such as indoles, arylpiperazines, biphe-
nyls, and benzopyranes are currently ascribed as supportive
approaches in drug discovery. Different families of nitrogen
and oxygen heterocycles such as xanthones, coumarins, and their
precursors (chalcones) have also been extensively used as scaf-
folds in medicinal chemistry programs for searching novel MAO-
B inhibitors.6ꢀ8
Chromone scaffold [(4H)-1-benzopyran-4-one] has been
recognized as a pharmacophore of a large number of bioactive
molecules of either natural or synthetic origin. Until now, numerous
biological effects, especially in popular medicine, have been ascribed
to this benzo-γ-pyrone nucleus such as anti-inflammatory, anti-
tumoral, and antimicrobial activities.9 Enzymatic inhibition proper-
ties toward different systems such as oxidoreductases, kinases,
tyrosinases, cyclooxygenases have also been recognized.10 Recently,
our group has reported preliminary studies that point out the
relevance of these types of heterocyclic compound as monoamino
oxidase inhibitors.10,11
’ PHARMACOLOGY
Accordingly, our project has focused on the discovery of new
chemical entities (NCEs) for MAO inhibition incorporating
privileged structures with benzo-γ-pyrone substructure (Scheme 3).
Preliminary studies performed with chromones (1) and (13)
allow disclosure of the importance of the location of a carboxylic
moiety in the γ-pyrone nucleus. In fact, when the ꢀCOOH
substituent is in position 3 of the heterocyclic scaffold (13), it
binds the hMAO-B, exerting a selective inhibition with respect to
the A isoform (IC50(hMAO-B) = 0.048 ( 0.0026 nM; MAO-B
selective index (SI) of >2083).10,11 As the inhibition is of
irreversible type and in an attempt to develop novel reversible
and selective MAO-B inhibitors, the synthesis of 2- and 3-car-
boxamide chromone derivatives capable of establishing hydrogen
interactions with the enzyme was performed. Therefore, func-
tionalized chromone scaffolds suitable to establish structureꢀactivity
The potential effects of the test drugs on hMAO activity were
investigated by measuring their effects on the production of
hydrogen peroxide (H2O2) from p-tyramine (a common sub-
strate for both hMAO-A and hMAO-B), using the Amplex Red
MAO assay kit (Molecular Probes, Inc., Eugene, OR, U.S.) 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 (Sigma-Aldrich Química S.A.,
Alcobendas, Spain).
The production of H2O2 catalyzed by MAO isoforms can be
detected using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex
Red reagent), a nonfluorescent and highly sensitive probe that
reacts with H2O2 in the presence of horseradish peroxidase to
produce a fluorescent product, resorufin.
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Journal of Medicinal Chemistry
ARTICLE
Scheme 3a
a Reagents: (a) RNH2, BOP or PyBOP, DIPEA in DCM/DMF; (b) BBr3 in DCM.
In this study, hMAO activity was evaluated using the above-
mentioned method following the general procedure previously
described elsewhere.8,15 The drugs (novel compounds and
reference inhibitors) were unable to react directly with the
Amplex Red reagent, which indicates that they do not interfere
with the measurements.
In our experiments and under our experimental conditions,
hMAO-A displayed a Michaelis constant (Km) of 457.17 ( 38.62
μM and a maximum reaction velocity (Vmax) of 185.67 ( 12.06
(nmol/min)/mg protein whereas hMAO-B showed a Km of
220.33 ( 32.80 μM and a Vmax of 24.32 ( 1.97 (nmol/min)/mg
protein (n = 5).
The hMAO-A and hMAO-B inhibition and SI ([IC50 (MAO-
A)]/[IC50 (MAO-B)]) data are reported in Table 1.
Reversibility and irreversibility experiments were performed
to evaluate the type of the inhibitor, using R-(ꢀ)deprenyl
(irreversible inhibitor) and isatin (reversible inhibitor) as
standards.16
inhibitory potencies toward hMAO isoforms and selectivity of
the chromones under study, and reference compounds, are
shown in Table 1. By analyzing such data, one can conclude that
chromones bearing substituents in position 3 of γ-pyrone
nucleus act preferably as MAO-B inhibitors (iMAO-B) with
IC50 values in the nanomolar to micromolar range. The same
tendency was found with the chromone carboxylic acids.10 The
most promising iMAO-B chromones that are substituted in the
para position, with hydroxyl or methoxyl groups or a chloro
atom, of the side chain aromatic nucleus (compounds 15, 16 and
19, respectively) present IC50 values between 63 and 76 nM. The
most selective compounds possess electron withdrawing groups
(a Cl atom (compound 19) and a CF3 group (compound 21)) in
the para position of the exocyclic aromatic nucleus (SI of >1.585
and >909, respectively). The chromone derivative with a hydro-
xyl group in the para position (compound 15) is more active and
selective as iMAO-B than the compound with two hydroxyl
groups located in the para and meta positions (compound 17).
The present results allow us to point out that the inclusion of this
type of substituent in the meta position of the 3-aryl substituent
could not be beneficial. The derivative 22 with the para position
substituted with a strong electron polar and bulky withdrawing
group (nitro group) shows a loss of selectivity with an IC50
’ RESULTS AND DISCUSSION
The chemical structures of the examined compounds are
depicted in Scheme 3. The results of the in vitro evaluation of
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Table 1. MAO Inhibitory Activities of Chromone Carboxamides and Reference Inhibitorsa
compd
R
IC50(hMAO-A) (μM)
IC50(hMAO-B) (μM)
SI
2
Ph
c
c
3
40-OH-Ph
30-OH-40-OCH3-Ph
30,40-OH-Ph
30,40-OCH3-Ph
40-Cl-Ph
65.23 ( 5.82
41.90 ( 2.79
1.6
4
c
c
5
0.19 ( 0.016
2.66 ( 0.13
c
0.071
6
c
7
c
c
8
40-OCF3-Ph
40-CF3-Ph
40-NO2-Ph
C6H11
c
c
9
c
c
10
11
12
14
15
16
17
18
19
20
21
22
23
24
c
c
c
d
C3H7
c
d
Ph
c
0.40 ( 0.022
0.064 ( 0.0054
0.076 ( 0.0032
0.16 ( 0.013
2.33 ( 0.07
0.063 ( 0.0042
1.08 ( 0.072
0.11 ( 0.0074
11.78 ( 0.79
0.93 ( 0.062
37.69 ( 1.68
0.017 ( 0.0019
7.54 ( 0.36
>250e
74
40-OH-Ph
30-OH-40-OCH3-Ph
30,40-OH-Ph
30,40-OCH3-Ph
40-Cl-Ph
40-OCF3-Ph
40-CF3-Ph
40-NO2-Ph
C6H11
4.76 ( 0.39b
8.34 ( 0.27b
110
0.43 ( 0.0035b
2.7
c
>43e
>1585e
>93e
>909 e
0.94
>107e
>2.7e
4043
0.87
c
c
c
11.11 ( 0.74
c
C3H7
c
R-(ꢀ)-deprenyl
iproniazid
68.73 ( 4.21b
6.56 ( 0.76
a All IC50 values shown in this table are the mean ( SEM from five experiments. SI: hMAO-B selectivity index = IC50(hMAO-A)/IC50(hMAO-B).
b Level of statistical significance: P < 0.01 versus the corresponding IC50 values obtained against MAO-B, as determined by Student's t test. The IC50
values of compounds 2 and 14 were taken from the literature.11c c Inactive at 100 μM (highest concentration tested). d Inactive at 1 mM (highest
concentration tested). e Values obtained under the assumption that the corresponding IC50 against MAO-A or MAO-B is the highest concentration
tested (100 μM or 1 mM).
of 11 μM for both MAO isoforms. It seems that the electron
withdrawing nature, in particular the resonance effect (weak
donating), and the spatial volume of the substituents located in
the aromatic ring have a particular effect on the potency of the
3-carboxamides.
Table 2. Reversibility and Irreversibility of hMAO
Inhibitiona
% hMAO-B inhibition
compd
before washing
after repeated washing
The chromone carboxamide derivative with an unsubstituted
exocyclic aromatic moiety (compound 14) is twice more active
than compound 23, a carboxamide with a cyclohexyl instead of
the aromatic ring (IC50 of 400 and 930 nM, respectively). The
absence of a ring led to a dramatic loss of activity (see linear
alkylcarboxamide 24; IC50 of 37 μM). All of the previously
mentioned chromone carboxamides, except chromone 22, are
inactive toward MAO-A.
R-(ꢀ)-deprenyl (25 nM)
isatin (20 μM)
53.62 ( 5.15
47.55 ( 5.42
60.31 ( 7.74
59.60 ( 6.96
47.72 ( 6.21
0b
23.50 ( 3.71b
15 (100 nM)
19 (100 nM)
29.14 ( 4.16
a Each value is the mean ( SEM from five experiments (n = 5). b Level of
statistical significance: P < 0.05 versus the corresponding % hMAO-B
inhibition before washing, as determined by Student's t test.
Of note is that the chromones bearing the same type of
substituents in position 2 of γ-pyrone nucleus (chromones
2ꢀ12) present a total loss of MAO inhibition except with
compounds 3 and 5. Compound 3, with a hydroxyl group in the
para position of the exocyclic aromatic ring, has a comparable
inhibitory activity for both isoforms. The introduction of
another hydroxyl group in the meta position (compound 5)
considerably increases the potency and selectivity toward
hMAO-A.
The reversibility studies performed with the chromones 15
and 19 revealed that the carboxamides behave as quasi-reversible
MAO-B inhibitors. In fact, the percent of enzymatic inhibition
was much lower after repeated washing with respect to R-(ꢀ)-
deprenyl (irreversible inhibitor) (Table 2).
The data so far obtained are in accordance with preliminary
results,11 since it supports the assumption that the positive
hydrophobicity (+π) of the substituent, besides inductive and
mesomeric effects, located on the phenyl exocyclic moiety
markedly influences the potency and selectivity of the chromone
carboxamides. The tendency is in perfect agreement with the
parameter space of the descriptors defined in the so-called
Craig’s plot.
In order to highlight the reasons of MAO selectivity, we have
carried out a structure-based molecular modeling study using
hMAOs cocrystals deposited into the PDB.12 Similar to our previous
work,17we have focused our attention onto the most active and
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Figure 2. Difference (in kJ/mol) between water solvation energies of
3ꢀ10 with respect to their isomers 15ꢀ22.
moiety was situated close to the FAD and the p-cloroanilide side
chain occupied the entrance cavity at the loop formed by residues
99ꢀ112. Two intermolecular hydrogen bonds were observed
between the carboxyamide and the Cys172 and between the
chromone sp2 oxygen and the Tyr326. The hydrophobic side
chain of 19 productively interacts with Pro102, Phe103, Leu167,
and Ile199.
With the aim to more deeply investigate the reasons for the
different recognition of 19 in hMAO-A with respect to the
hMAO-B, the best docking poses, reported in Figure 1b, were
superposed using the FAD and the active site backbone
residues as geometry references. Such a procedure indicated
that hMAO-B configuration of 19 in hMAO-A loses the
hydrogen bond contribution because Cys172 and Tyr326,
available into the former enzyme, are replaced by Asn181 and
Ile335, respectively. Moreover, in the hMAO-A cleft the 19
p-cloroanilide moiety bumps against the side chain of Phe208
corresponding to the sterically smaller hMAO-B Ile199. The
same analysis has been carried out on the 19 hMAO-A best
pose with respect to hMAO-B. In this case we can report
clashes between the chromone moiety and the Tyr326 and no
hydrogen bond. In order to reduce clashes, both new models
have been refined with Glide software, but after the optimiza-
tion the scenario was not so different: the clashes were
reduced, but with respect to the docking best poses, the
inhibitor was located more distant from the FAD and no
intermolecular hydrogen bonds were established between the
ligand and the targets.
After the docking experiments with the most active and
selective compound we have repeated the same procedure trying
to reproduce by interaction energies the biological trend re-
ported in Table 1, at least for the quantitatively measured values.
Since no good correlation was found for the entire set, we have
focused our attention onto the conformational and solvation
properties of our derivatives. In particular a comparison of the
isomers bearing the side chain at the 2- or 3-position onto the
chromone ring has been carried out. The conformational proper-
ties, investigated by means of Monte Carlo (MC) search,
indicated few possible local minimum energy structures in
particular for compounds 15ꢀ22. Such data could be considered
as an entropy benefit of these ligands with respect to their
isomers 3ꢀ10. The comparative solvation analysis in water
revealed a remarkable energy advantage of compounds 3ꢀ10
with respect to their isomers 15ꢀ22 (Figure 2). Such evidence
could contribute to explain the better activity of chromone
derivatives reporting the carboxyamide side chain at position 3
than at position 2. In fact, the interaction with the target of 3ꢀ10
could be penalized into the aqueous environment.
Figure 1. Most stable docking poses of 19 into (a) hMAO-A and
(b) hMAO-B binding sites. Ligand is reported in green carbon sticks.
Interacting residues are in gray carbon sticks, and FAD is in space-fill.
Yellow dotted lines indicate ligandꢀenzyme intermolecular hydrogen
bonds: /, ligand interaction to side chain; //, ligand interaction to
backbone; ///, ligand interaction to both backbone and side chain.
selective compound. So in this series, we have selected 19 as a
case study for our theoretical investigation. Also in the present
study we have adopted the docking program Glide18 and the
PDB models 2Z5X19 and 2V5Z20 for mimicking the hMAO-A
and -B targets, respectively. Details of the molecular modeling
procedures are reported in the Experimental Section.
Compound 19 target interaction energies, ꢀ7.12 and
ꢀ207.68 kJ/mol for hMAO-A and -B, respectively, were in
good agreement with the experimental biological data. The
docking proposed configuration indicated a strong difference
depending on the target. Actually, the only [hMAO-A 19]
3
Glide proposed configuration showed the chromone moieties
in the entrance gorge while the p-cloroanilide side chain
recognized the FAD cofactor suggesting a πꢀπ interaction to
Tyr407 and Tyr444. Several other residues were involved in
such a binding mode but only van der Waals contacts can be
designated (Figure 1a).
The interaction of 19 to hMAO-B was found to be much more
productive with respect to the previous one. First of all, the
interaction energy was remarkably lower than in hMAO-A and a
number of binding modes were proposed by Glide. Moreover,
the most stable configuration was in agreement with literature
data as concerns the intramolecular hydrogen bond and with the
positioning of the chromone ring.17Actually, the chromone
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’ CONCLUSIONS
constants (J) are given in Hz. Electron impact mass spectrometry (EI-MS)
was carried out on a VG AutoSpec instrument. The data are reported as m/z
(% of relative intensity of the most important fragments). Melting points
were obtained on a Stuart Scientific SMP1 apparatus and are uncorrected.
Synthesis. General Procedure for Amide Obtention. The
synthetic procedure corresponds to a one-pot condensation reaction
that occurs, in equimolar amounts, between the carboxylic acid and the
respective amine according to the following general procedure. 2-Car-
boxychromone (1) or 3-carboxychromone (13) (0.50 g, 2.63 mmol)
was dissolved in 6 mL of DMF and 0.37 mL of diisopropylethylamine
(DIPEA). The solution was then cooled at 0 ꢀC in an iceꢀwater bath,
and a BOP (1.16 g, 2.63 mmol) or PyBOP (1.37 g, 2.63 mmol) solution
in CH2Cl2 (6 mL) was added. The mixture was stirred for 30 min.
Afterward, the corresponding amine (phenylamine, 4-hydroxyphenyla-
mine, 3-hydroxy-4-methoxyphenylamine, 3,4-dimethoxyphenylamine,
cyclohexylamine, or propylamine) was added in equimolar amount.
The temperature was gradually increased to room temperature. The
mixture was stirred for additional 4 h.
General Procedure for Demethylation Reactions. The
monomethoxylated chromone carboxamide 4 or 16 (0.25 g, 0.80 mmol)
was suspended in anhydrous dichloromethane and under argon atmo-
sphere. The resulting suspension was stirred, cooled at ꢀ80 ꢀC, and
BBr3 (6 mL of a 1 M solution in dicloromethane) was added dropwise.
Once the addition was completed, the reaction mixture was allowed to
warm to room temperature with continuous stirring. After BBr3 destruction
with methanol, the purification process was carried out straightforwardly.
The structural elucidation of the other carboxamides was described
elsewhere.
In the present work evidence was acquired to demonstrate
that chromone is a valid scaffold for the design of potent,
selective, and reversible MAO inhibitors. Generally chromones
bearing substituents in position 3 of γ-pyrone nucleus act as
hMAO-B selective inhibitors and those bearing substituents in
position 2 of the same nucleus selectively inhibit the hMAO-A or
are inactive. The introduction of a chloro substituent in the para
position of the exocyclic aromatic ring of chromone 3-phenyl-
carboxamides was crucial for the obtention of a potent and
selective iMAO (IC50 = 63 nM; SI > 1585). The easy synthetic
accessibility and potentially low toxicity of chromones9 make
them “privileged” scaffolds. Docking experiments, carried out
with the most active and selective compound 19, indicated the
reasons for the better stabilization within the hMAO-B isoform
versus the -A one. A comparison of 3ꢀ10 and 15ꢀ22 conforma-
tional properties indicated an entropy advantage of the latter in
the interaction with both targets. The solvation energies comparison
reported a preference of the 2-substituted chromone derivatives for
the aqueous solvent. Both results suggested a penalization of 3ꢀ10
in target recognition with respect to 15ꢀ22.
Additional studies are warranted for a systematic lead optimi-
zation, modulated by appropriate modifications of length, size,
and chemical nature of the substituents, a process that can lead to
in the future to a drug candidate.
’ EXPERIMENTAL SECTION
N-(3,4-Dihydroxyphenyl)-4-oxo-4H-1-benzopyran-2-car-
boxamide (5). Yield: 65%. Mp: 267ꢀ271 ꢀC. 1H NMR: δ = 6.75 (1H,
d, J = 8.5, H(50)), 6.94 (1H, s, H(3)), 7.04 (1H, dd, J = 8.5, 2.4, H(60)),
7.32 (1H, d, J = 2.4, H(20)), 7.58 (1H, ddd, J = 8.0, 6.8, 1.2, H(6)), 7.85
(1H, d, J = 8.0, H(8)), 7.93 (1H, ddd, J = 8.5, 7.0, 1.5, H(7)), 8.08 (1H,
dd, J = 7.6, 1.4, H(5)), 8.94 (1H, s, 30-OH), 9.17 (1H, s, 40-OH), 10.49
(1H, s, CONH). EI-MS m/z: 298 (10), 297 (M+•, 52), 296 (19), 146
(100), 124 (92), 105 (57), 97 (16), 89 (74), 77 (15), 69 (19), 68 (15),
63 (16).
Chemistry. Chromone-2-carboxylic and chromone-3-carboxylic
acids, benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluoro-
phosphate (BOP), benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate (PyBOP), N,N-diisopropylethylamine (DIPEA),
dimethylformamide (DMF), boron tribromide (BBr3), and aniline and
its derivatives were purchased from Sigma-Aldrich Química S.A. (Sintra,
Portugal). All other reagents and solvents were pro analysis grade and
were acquired from Merck (Lisbon, Portugal) and used without addi-
tional purification.
N-(4-(Chlorophenyl)-4-oxo-4H-1-benzopyran-2-carboxa-
1
mide (7). Yield: 50%. Mp: 268ꢀ271 ꢀC. H NMR: δ = 6.97 (1H, s,
Thin-layer chromatography (TLC) was carried out on precoated
silica gel 60 F254 (Merck) with layer thickness of 0.2 mm. For analytical
control the following systems were used: ethyl acetate/petroleum ether,
ethyl acetate/methanol, and chloroform/methanol in several propor-
tions. The spots were visualized under UV detection (254 and 366 nm)
and iodine vapor. Normal-phase column chromatography was per-
formed using silica gel 60, 0.2ꢀ0.5 or 0.040ꢀ0.063 mm (Merck).
Following the workup and after extraction, the organic phases were
dried over Na2SO4. Solutions were decolorized with activated charcoal
when necessary. The recrystallization solvents were ethyl acetate or ethyl
ether/n-hexane. Solvents were evaporated in a Buchi Rotavapor.
The purity of the final products (>97% purity) was verified by high-
performance liquid chromatography (HPLC) equipped with a UV
detector. Chromatograms were obtained in an HPLC/DAD system, a
Jasco instrument (pump model 880-PU and solvent mixing model 880-
30, Tokyo, Japan) equipped with a commercially prepacked Nucleosil
RP-18 analytical column (250 mm ꢁ 4.6 mm, 5 μm, Macherey-Nagel,
Duren, Germany) and UV detection (Jasco model 875-UV) at the maximum
wavelength of 254 nm. The mobile phase consisted of a methanol/water
or acetonitrile/water (gradient mode, room temperature) at a flow rate
of 1 mL/min. The chromatographic data were processed in a Compaq
computer fitted with CSW 1.7 software (DataApex, Czech Republic).
Apparatus. 1H NMR data were acquired, at room temperature, on a
Br€uker AMX 300 spectrometer operating at 300.13 MHz. Dimethylsulf-
oxide-d6 was used as a solvent. Chemical shifts are expressed in δ (ppm)
values relative to tetramethylsilane (TMS) as internal reference. Coupling
H(3)), 7.48 (2H, d, J = 8.8, H(30), H(50)), 7.56 (1H, m, H(6)),
7.83ꢀ7.96 (4H, m, H(7), H(8), H(20), H(60)), 8.08 (1H, dd, J = 7.9,
1.6, H(5)), 10.87 (1H, s, CONH). EI-MS m/z: 301 (34), 300 (33), 299
(M+•, 100), 298 (50), 282 (15), 270 (24), 173 (14), 145 (28), 101 (18),
90 (10), 89 (89), 69 (16), 63 (14).
N-(4-(Trifluoromethoxy)phenyl)-4-oxo-4H-1-benzopyr-
an-2-carboxamide (8). Yield: 56%. Mp: 241ꢀ244 ꢀC. 1H NMR: δ =
7.00 (1H, s, H(3)), 7.45 (2H, d, J = 8.2, H(30), H(50)), 7.57 (1H, dd, J =
8.2, 7.4, H(6)), 7.90ꢀ7.97 (4H, m, H(7), H(8), H(20), H(60)), 8.08
(1H, d, J = 7.6, H(5)), 10.92 (1H, s, CONH). EI-MS m/z: 349 (M+•,
86), 348 (100), 332 (21), 320 (41), 264 (12), 176 (17), 173 (21), 145
(44), 117 (18), 101 (20), 89 (98), 69 (24).
N-(4-(Trifluoromethyl)phenyl)-4-oxo-4H-1-benzopyran-
1
2-carboxamide (9). Yield: 50%. Mp: 258ꢀ261 ꢀC. H NMR: δ =
7.02 (1H, s, H(3)), 7.58 (1H, ddd, J = 8.1, 7.0, 1.2, H(6)), 7.81 (2H, d,
J = 8.8, H(30), H(50)), 7.86 (1H, dd, J = 8.4, 1.0, H(8)), 7.94 (1H, ddd,
J = 8.6, 7.0, 1.6, H(7)), 8.06 (2H, d, J = 8.4, H(20), H(60)), 8.09 (1H, dd,
J = 7.6, 1.6, H(5)), 11.04 (1H, s, CONH). EI-MS m/z: 333 (M+•, 35),
332 (100), 303 (16), 173 (15), 145 (32), 117 (12), 101 (11), 89 (58), 69
(15), 57 (12).
N-(4-Nitrophenyl)-4-oxo-4H-1-benzopyran-2-carboxamide
(10). Yield: 15%. Mp: >280 ꢀC. 1H NMR: δ = 7.04 (1H, s, H(3)), 7.59
(1H, ddd, J = 8.2, 7.0, 1.2, H(6)), 7.86 (1H, d, J = 7.6, H(8)), 7.96 (1H,
ddd, J = 8.6, 7.0, 1.6, H(7)), 8.10 (3H, m, H(5), H(20), H(60)), 8.34 (2H,
m, H(30), H(50)), 11.22 (1H, s, CONH). EI-MS m/z: 310 (M+•, 6), 309
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Journal of Medicinal Chemistry
ARTICLE
(22), 123 (45), 111 (38), 101 (45), 99 (54), 97 (69), 95 (32), 87 (63),
85 (90), 83 (51), 73 (74), 71 (69), 69 (56), 58 (35), 57 (100), 55 (65).
N-Cyclohexyl-4-oxo-4H-1-benzopyran-2-carboxamide (11).
Yield: 60%. Mp: 183ꢀ184 ꢀC. 1H NMR: δ = 1.12ꢀ1.86 (10H, m, 2 ꢁ
H(20), 2 ꢁ H(30), 2 ꢁ H(40), 2 ꢁ H(50), 2 ꢁ H(60)), 3.78 (1H, m,
H(10)), 6.83 (1H, s, H(3)), 7.54 (1H, dd, J = 7.9, 7.2, H(6)), 7.79 (1H, d,
J = 8.4, H(8)), 7.90 (1H, ddd, J = 8.5, 7.0, 1.4, H(7)), 8.05 (1H, d, J = 7.9,
H(5)), 8.88 (1H, d, J =8.1, CONH). EI-MS m/z: 271 (M+•, 38), 228 (12),
191 (19), 190 (100), 173 (16), 145 (12), 89 (39).
(0.7H, d, J = 7.6, H(5)), 8.11 (0.3H, d, J = 7.6, H(5)), 8.44 (0.7H, d, J =
14.8, H(2)), 8.60 (0.3H, d, J = 14.8, H(2)), 10.31 (0.3H, brs, CONH),
11.85 (0.7H, brs, CONH). EI-MS m/z: 271 (M+•, 100), 228 (18), 188
(23), 175 (23), 173 (18), 121 (31), 97 (22), 57 (17).
N-Propyl-4-oxo-4H-1-benzopyran-3-carboxamide (24).
Yield: 18%. Mp: 139ꢀ142 ꢀC. 1H NMR: δ = 1.04 (3H, m, CH3), 1.77
(2H, m, CH3CH2), 3.52 (2H, m, NHCH2), 7.23ꢀ7.30 (2H, m, H(6),
H(8)), 7.57 (1H, ddd, J = 8.5, 7.0, 1.5, H(7)), 8.03 (0.7H, d, J = 7.6,
H(5)), 8.10 (0.3H, d, J = 7.7, H(5)), 8.40 (0.7H, d, J = 15.0, H(2)), 8.55
(0.3H, d, J = 15.0, H(2)), 10.25 (0.3H, brs, CONH), 11.90 (0.7H, brs,
CONH). EI-MS m/z: 232 (18), 231 (M+•, 100), 215 (56), 203 (17), 202
(19), 189 (10), 188 (20), 175 (50), 173 (10), 122 (19), 121 (16), 92
(15).
Determination of hMAO Isoform Activity. The effects of the
test compounds on hMAO isoform enzymatic activity were evaluated by
a fluorimetric method following the experimental protocol previously
described elsewhere.8 Briefly, 0.1 mL of sodium phosphate buffer
(0.05 M, pH 7.4) containing various concentrations of the test drugs
(new compounds or reference inhibitors) and adequate amounts of recom-
binant 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) 165 pmol of p-tyramine/min (hMAO-A, 1.1 μg protein;
specific activity, 150 nmol of p-tyramine oxidized to (p-hydroxyphenyl-
acetaldehyde/min)/mg protein; hMAO-B, 7.5 μg of protein; specific
activity, 22 nmol of (p-tyramine transformed/min)/mg protein), was
incubated for 15 min at 37 ꢀC in a flat-black-bottom 96-well microtest
plate (BD Biosciences, Franklin Lakes, NJ, U.S.) placed in a dark
multimode microplate reader chamber. After this incubation period,
the reaction was started by adding (final concentrations) 200 μM
Amplex 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 multimode microplate reader (Fluostar Optima,
BMG Labtech GmbH, Offenburg, Germany), based on the fluorescence
generated (excitation, 545 nm, emission, 590 nm) over a 15 min period,
in which the fluorescence increased linearly.
N-Propyl-4-oxo-4H-1-benzopyran-2-carboxamide (12).
1
Yield: 84%. Mp: 167ꢀ171 ꢀC. H NMR (CDCl3): δ = 1.02 (3H, t,
J = 7.4, CH3), 1.70 (2H, m, CH3CH2), 3.47 (2H, m, NHCH2), 7.02 (1H,
bs, CONH), 7.17 (1H, s, H(3)), 7.46 (1H, ddd, J = 8.0, 7.0, 1.0, H(6)),
7.53 (1H, dd, J = 7.9, 1.0, H(8)), 7.74 (1H, ddd, J = 8.6, 7.0, 1.3, H(7)),
8.22 (1H, dd, J=8.0, 1.6, H(5)). EI-MSm/z: 232 (16), 231 (M+•, 100), 216
(26), 203 (12), 202 (22), 190 (10), 189 (30), 174 (19), 173 (93), 159
(20), 146 (12), 145 (43), 101 (15), 89 (83), 69 (17), 63 (11).
N-(3,4-Dihydroxyphenyl)-4-oxo-4H-1-benzopyran-3-car-
1
boxamide (17). Yield: 50%. Mp: 266ꢀ269 ꢀC. H NMR: δ = 6.82
(1H, d, J = 8.4, H(50)), 6.94 (1H, dd, J = 8.5, 2.4, H(60)), 6.99 (1H, d, J =
2.5, H(20)), 7.33ꢀ7.39 (2H, m, H(6), H(8)), 7.69 (1H, dd, J = 8.4, 7.1,
H(7)), 7.99 (1H, dd, J = 7.7, 1.3, H(5)), 8.64 (0.7H, s, H(2)), 8.68
(0.7H, s, H(2)), 8.72 (0.3H, s, H(2)), 9.43 (1H, s, OH), 9.46 (1H, s,
OH), 11.73 (0.3H, s, CONH), 11.80 (0.3H, s, CONH), 13.42 (0.7H, s,
CONH), 13.46 (0.7H, s, CONH). EI-MS m/z: 297 (M+•, 7), 281 (11),
208 (13), 207 (73), 173 (100), 149 (24), 121 (72), 97 (20), 95 (27), 83
(27), 81 (33), 77 (21), 73 (36), 71 (26), 69 (40), 55 (57).
N-(4-(Chlorophenyl)-4-oxo-4H-1-benzopyran-3-carboxa-
mide (19). Yield: 47%. Mp: 255ꢀ259 ꢀC. 1H NMR: δ = 7.34 (1H, d,
J = 7.8, H(8)), 7.38 (1H, dd, J = 7.6, 1.3, H(6)), 7.54 (2H, d, J =
8.8, H(30), H(50)), 7.70ꢀ7.73 (3H, m, H(20), H(60), H(7)), 7.99 (1H,
dd, J = 7.8, 1.5, H(5)), 8.85 (0.7H, d, J = 13.8, H(2)), 8.88 (0.3H, d, J =
14.8, H(2)), 8.88 (0.3H, d, J = 14.8, H(2)), 11.84 (0.3H, d, J = 14.8,
CONH), 11.84 (0.3H, d, J = 14.8, CONH), 13.39 (0.7H, d, J = 13.8,
CONH). EI-MS m/z: 301 (37), 300 (21), 299 (M+•, 88), 174 (16), 173
(100), 151 (18), 121 (37), 92 (11), 89 (10).
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 MAO
isoforms, which were replaced by a sodium phosphate buffer solution.
Reversibility and Irreversibility Experiments. To evaluate
whether compounds 15 and 19 are reversible or irreversible hMAO-B
inhibitors, an effective centrifugationꢀultrafiltration method (so-called
repeated washing) was used.8 Briefly, adequate amounts of the recom-
binant hMAO-B were incubated with a single concentration (see
Table 2) of the compounds 15 and 19 or the reference inhibitors
R-(ꢀ)-deprenyl and isatin in a sodium phosphate buffer (0.05 M, pH
7.4) for 15 min at 37 ꢀC. After this incubation period, an aliquot of this
incubated mixture was stored at 4 ꢀC and used for subsequent
measurement of hMAO-B activity under the experimental conditions
indicated above (see the section on determination of MAO activity).
The remaining incubated sample (300 μL) was placed in an Ultrafree-
0.5 centrifugal tube (Millipore, Billerica, MA, U.S.) with a 30 kDa
Biomax membrane in the middle of the tube and centrifuged (9000g,
N-(4-(Trifluoromethoxy)phenyl)-4-oxo-4H-1-benzopyran-
3-carboxamide (20). Yield: 55%. Mp: 223ꢀ226 ꢀC. 1H NMR: δ =
7.31ꢀ7.38 (2H, m, H(6), H(8)), 7.46 (2H, d, J = 8.7, H(30), H(50)),
7.71 (1H, ddd, J = 8.5, 7.0, 1.5, H(7)), 7.79 (2H, d, J = 8.7, H(20), H(60)),
8.00 (1H, dd, J = 7.8, 1.5, H(5)), 8.85 (0.7H, d, J = 13.5, H(2)), 8.89
(0.3H, d, J = 15.3, H(2)), 11.84 (0.3H, d, J = 15.2, CONH), 13.42 (0.7H,
d, J = 13.5, CONH). EI-MS m/z: 349 (M+•, 31), 201 (13), 174 (12), 173
(100), 121 (30), 92 (10).
N-(4-(Trifluoromethyl)phenyl)-4-oxo-4H-1-benzopyran-3-
carboxamide (21). Yield: 55%. Mp: 251ꢀ254 ꢀC. 1H NMR: δ = 7.36
(1H, d, J = 8.0, H(8)), 7.39 (1H, dd, J = 8.0, 1.7, H(6)), 7.73 (1H, ddd,
J = 8.3, 7.6, 1.6, H(7)), 7.83ꢀ7.92 (4H, m, H(20), H(30), H(50), H(60)),
8.00 (1H, dd, J = 7.8, 1.4, H(5)), 8.95 (0.7H, d, J = 13.7, H(2)), 8.98
(0.3H, d, J = 14.6, H(2)), 11.90 (0.3H, d, J = 14.6, CONH), 13.40 (0.7H,
d, J = 13.7, CONH). EI-MS m/z: 333 (M+•, 58), 212 (11), 185 (19), 173
(100), 145 (15), 121 (33), 92 (13).
N-(4-Nitrophenyl)-4-oxo-4H-1-benzopyran-3-carboxamide
(22). Yield: 35%. Mp: >280 ꢀC. 1H NMR: δ = 7.35ꢀ7.41 (2H, m, H(6),
H(8)), 7.74 (1H, ddd, J = 8.2, 7.3, 1.7, H(7)), 7.93ꢀ7.96 (2H, m, H(20),
H(60)), 8.00 (1H, dd, J = 7.9, 1.6, H(5)), 8.32 (2H, d, J = 9.2, H(30),
H(50)), 8.97 (0.7H, d, J = 13.5, H(2)), 8.99 (0.3H, d, J = 14.3, H(2)),
11.94 (0.3H, d, J = 14.2, CONH), 13.39 (0.7H, d, J = 13.3, CONH). EI-
MS m/z: 310 (M+•, 34), 309 (18), 280 (15), 173 (100), 123 (20), 121
(45), 116 (16), 109 (16).
N-Cyclohexyl-4-oxo-4H-1-benzopyran-3-carboxamide(23).
Yield: 30%. Mp: 181ꢀ184 ꢀC. 1H NMR: δ = 1.32ꢀ1.94 (10H, m, 2 ꢁ
H(20), 2 ꢁ H(30), 2 ꢁ H(40), 2 ꢁ H(50), 2 ꢁ H(60)), 3.70 (1H, m,
H(10)), 7.32 (2H, m, H(6), H(8)), 7.66 (1H, dd, J = 8.0, 7.3, H(7)), 8.02
5171
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Journal of Medicinal Chemistry
ARTICLE
20 min, 4 ꢀC) in a centrifuge (J2-MI, Beckman Instruments, Inc., Palo
Alto, CA, U.S.). The enzyme retained in the 30 kDa membrane was
resuspended in sodium phosphate buffer at 4 ꢀC and centrifuged again
(under the same experimental conditions described above) two succes-
sive times. After the third centrifugation, the enzyme retained in the
membrane was resuspended in sodium phosphate buffer (300 μL) and
an aliquot of this suspension was used for subsequent hMAO-B activity
determination. Similar studies were carried out on MAO-A activity in the
presence of the reference inhibitor moclobemide under the experimental
conditions described above.
Control experiments were performed simultaneously (to define 100%
hMAO activity) by replacing the test drugs with appropriate dilutions of the
vehicles. The corresponding values of percent hMAO inhibition were
separately calculated for samples with and without repeated washing.
Statistical Analysis of Data. Data were expressed as the mean
((SEM) and were analyzed by ANOVA followed by Dunnett’s test.
Groups of test data (mean ( SD) were comparing using the Student’s t
test for paired observations. Values were considered to differ signifi-
cantly at the level of p < 0.05.
Molecular Modeling. The most active and selective compound 19
was built by means of the Maestro21 and energy minimized with 2000
steps of PolakꢀRibiere conjugated gradient algorithm applied to OPLS-
2005 force field22 as implemented in MacroModel.23Solvating effects
were simulated by means of the GB/SA24 water implicit model as
implemented in the same program. Docking simulations were carried
out using the ligand flexible algorithm of Glide.18The target models of
hMAO-A and hMAO-B were obtained from two Protein Data Bank high
resolution crystal structures 2Z5X19 and 2V5Z,20 respectively. After
removal of cocrystallized ligands, harmine for 2Z5X and safinamide for
2V5Z, the binding sites were defined by means of a regular box of about
1000 Å3 centered onto the N5 FAD cofactor. Glide XP scoring function
was adopted for stability evaluation of the complexes. The quality of our
protocols has been evaluated by submitting the X-ray cocrystallized
ligands to redocking. In both hMAO-A and -B cases the theoretical
complexes were almost identical to the experimental ones, as reported by
the root mean square deviation equal to 0.26 and 0.21 Å, respectively.
In order to take into account the solvation effects of the other
compounds 3ꢀ10 and 15ꢀ22, models of these derivatives were
submitted to 1000 iterations of Monte Carlo conformational search as
implemented in MacroModel23 and energy minimized with the same
protocol, force field, and implicit model of solvation considered for the
most selective compound 19.
’ ABBREVIATIONS USED
CNS, central nervous system; FAD, flavin adenine dinucleotide;
MAO, monoamine oxidase; MAO-A, monoamine oxidase A; MAO-
B, monoamine oxidase B; hMAO, human MAO isoform; PDB,
Protein Data Bank; SAR, structureꢀactivity relationship
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’ AUTHOR INFORMATION
Corresponding Author
*For S.A.: phone, +39 09613694197; fax, +39 0961391490;
e-mail, alcaro@unicz.it. For F.B.: phone, +351 220402560; fax,
+351 220402659; e-mail, fborges@fc.up.pt.
’ ACKNOWLEDGMENT
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Granese, A.; Carradori, S.; Yꢀa~nez, M.; Orallo, F.; Ortuso, F.; Alcaro, S.
Synthesis, molecular modeling, and selective inhibitory activity against
human monoamine oxidases of 3-carboxamido-7-substituted coumarins.
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Bolasco, A.; Chimenti, P.; Secci, D.; Rossi, F.; Yꢀa~nez, M.; Orallo, F.;
Ortuso, F.; Alcaro, S. Chalcones: a valid scaffold for monoamine oxidases
inhibitors. J. Med. Chem. 2009, 52, 2818–2824. (c) Chimenti, F.;
Fioravanti, R.; Bolasco, A.; Chimenti, P.; Secci, D.; Rossi, F.; Yꢀa~nez,
M.; Orallo, F.; Ortuso, F.; Alcaro, S.; Cirilli, R.; Ferretti, R.; Sanna, M. L.
A new series of flavones, thioflavones, and flavanones as selective
monoamine oxidase-B inhibitors. Bioorg. Med. Chem. 2010, 18, 1273–
1279.
This work was supported by the Foundation for Science and
Technology (FCT), Portugal (Projects PTDC/QUI/70359/
2006 and PTDC/QUI-QUI/113687/2009). A.G. (Grant
SFRH/BD/43531/2008) and F.B. (Grant SFRH/BSAB/1090/
2010) are thankful for the FCT grants. The authors are grateful to
Dr. Alfredo Mellace (Department of Chemistry, Nassau Com-
munity College NY, USA) for proofreading the revised manuscript.
’ DEDICATION
†This article is dedicated to the memory of Prof. Francisco
Orallo, the pharmacologist and friend who believed in us.
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