Synthesis, molecular modeling, and selective inhibitory activity against human monoamine oxidases of 3-carboxamido-7-substituted coumarins

Article abstract of DOI:10.1021/jm801496u

A large series of 3-carboxamido-7-substituted coumarins have been synthesized and tested in vitro for their human monoamine oxidase A and B (hMAO-A and hMAO-B) inhibitory activity. Taking into account all the relevant structural information on MAOs reported in the literature, we made some changes in the coumarin nucleus and examined with particular attention the effect on activity and selectivity of substituting at position 3 with N-aryl or N-alkyl carboxamide and at position 7 with a benzyloxy or a 4'-F-benzyloxy group. Some of the assayed compounds proved to be potent, selective inhibitors of hMAO-B with IC50 values in the micromolar range. To better understand the enzyme-inhibitor interaction and to explain the selectivity of the most active compounds toward hMAOs, molecular modeling studies were carried out on new, high resolution, hMAO-A and hMAO-B crystallographic structures. ?2009 American Chemical Society.

Full text of DOI:10.1021/jm801496u

J. Med. Chem. 2009, 52, 1935–1942
1935
Synthesis, Molecular Modeling, and Selective Inhibitory Activity against Human Monoamine
Oxidases of 3-Carboxamido-7-Substituted Coumarins†
Franco Chimenti,‡ Daniela Secci,*,‡ Adriana Bolasco,‡ Paola Chimenti,‡ Bruna Bizzarri,‡ Arianna Granese,‡ Simone Carradori,‡
Matilde Ya´n˜ez,§ Francisco Orallo,§ Francesco Ortuso,| and Stefano Alcaro|
Dipartimento di Chimica e Tecnologie del Farmaco, UniVersita` degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy,
Departamento de Farmacolog´ıa, Facultad de Farmacia, UniVersidad de Santiago de Compostela, Campus UniVersitario Sur,
E-15782 Santiago de Compostela (La Corun˜a), Spain, Dipartimento di Scienze Farmacobiologiche, UniVersita` di Catanzaro “Magna
Graecia”, “Complesso Nin`ı Barbieri”, 88021 Roccelletta di Borgia (CZ), Italy
ReceiVed NoVember 27, 2008
A large series of 3-carboxamido-7-substituted coumarins have been synthesized and tested in vitro for their
human monoamine oxidase A and B (hMAO-A and hMAO-B) inhibitory activity. Taking into account all
the relevant structural information on MAOs reported in the literature, we made some changes in the coumarin
nucleus and examined with particular attention the effect on activity and selectivity of substituting at position
3 with N-aryl or N-alkyl carboxamide and at position 7 with a benzyloxy or a 4′-F-benzyloxy group. Some
of the assayed compounds proved to be potent, selective inhibitors of hMAO-B with IC50 values in the
micromolar range. To better understand the enzyme-inhibitor interaction and to explain the selectivity of
the most active compounds toward hMAOs, molecular modeling studies were carried out on new, high
resolution, hMAO-A and hMAO-B crystallographic structures.
Introduction
expression after the 60th year of life, especially in glial cells.8-10
MAO-B activity is believed to cause oxidative stress, during
catalytic turnover, because of the production of hydrogen
peroxide (H2O2) and other reactive oxygen species. Therefore,
a selective hMAO-B inhibitor, insofar as it decreases the rate
of oxidative deamination, could contribute to neuroprotection
and prevent neuronal degeneration.11
Monoamine oxidases (MAOs; EC 1.4.3.4)a are widespread
enzymes responsible for the regulation and metabolism of major
monoamine neurotransmitters (5-hydroxytryptamine (5-HT),
norepinephrine, dopamine), modulating their concentrations in
the brain and peripheral tissues. They are also involved in the
biodegradation of exogenous amines such as tyramine and
MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Accord-
ing to their substrate specificity, inhibitor sensitivity, and amino
acid sequence, the following two isoforms were fully character-
ized:1 hMAO-A and hMAO-B. Binda et al.2,3 described the
crystal structure of these two subtypes, highlighting the selective
interaction between them and their ligands. This led to renewed
interest in the rational design of potent, specific inhibitors with
therapeutic potential and no undesirable side effects.4 As
mentioned above, hMAOs play an important role in the
metabolism of several neurotransmitters and could be useful in
the treatment of a number of psychiatric and neurological
diseases. In fact, human MAO-B inhibitors are used alone or
in combination in the therapy of Alzheimer’s and Parkinson’s
diseases,5 while human MAO-A inhibitors are antidepressants
and antianxiety agents (Figure 1).6 Furthermore, interest in
selective inhibitors of hMAO-B has increased in recent years7
due to the discovery of an age-related increase in hMAO-B
In spite of such considerable progress in our understanding
of the interactions of the two enzyme forms with their preferred
substrates and inhibitors, no general rules are available for the
design of potent, selective inhibitors of hMAOs. At the same
time, a great deal of literature data on the importance of an
acyl group in position C3 on the coumarin nucleus considers it
crucial for a variety of pharmacological effects such as antico-
agulant,12 antineoplastic,13 antimicrobial,14,15 antidepressant,16
radical scavenger,17 anti-Helicobacter pylori,18 antiinflamma-
tory,19 protease, and ꢀ-secretase inhibitory effects20 and others.21
So far, among several MAO inhibitors, some (2H)-1-
benzopyran-2-one derivatives (coumarins) have been screened
for their good inhibitory activities (Figure 2).22-28 Starting from
these studies, our research group first investigated the MAO
inhibitory activity of N,N′-bis[2-oxo-2H-benzopyran]-3-car-
boxamides.29 The good results encouraged us to assay a large
array of 3-carboxamido coumarins in order to evaluate the
influence of a number of substitutions at this position as regards
size, length, and lipophilic or electronic characteristics
(H-bonding properties).
Chemistry. Most of the coumarin derivatives, (d) (Scheme
1), were synthesized starting from commercial substituted
salicylaldehydes according to the pathway reported in Scheme
1 and are listed in Table 1. After Knoevenagel cyclization to
ethyl esters of (2H)-1-benzopyran-2-one-3-carboxylic acids (a)
(Scheme 1), the compounds bearing an OH group at position 7
were subsequently functionalized by benzylation in the presence
of N,N′-dicyclohexyl-18-crown-6-ether, which by chelating
potassium ion facilitated the nucleophilic attack to improve the
yields of the related compounds (b) (Scheme 1). Hydrolysis with
10% sodium hydroxide and treatment of intermediates (a) and
†
PDB ID of hMAO-A: 2BXR and 2Z5X; PDB ID of hMAO-B: 1GOS
and 2BK3.
* To whom correspondence should be addressed. Phone: +39 06
49913149. Fax: +39 06 49913772. E-mail: daniela.secci@uniroma1.it.
‡
Dipartimento di Chimica e Tecnologie del Farmaco, Universita` degli
Studi di Roma “La Sapienza”.
§
Departamento de Farmacolog´ıa, Facultad de Farmacia, Universidad de
Santiago de Compostela.
|
Dipartimento di Scienze Farmacobiologiche, Universita` di Catanzaro
“Magna Graecia”.
a Abbreviations: MAO, monoamine oxidase; 5-HT (5-hydroxytryptamine);
MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MC, Monte Carlo;
PDB, Protein Data Bank. Abbreviations used for amino acids follow the
rules of the IUPACIUB Commission of Biochemical Nomenclature in
J. Biol. Chem. 1972, 247, 977-983. Amino acid symbols denote
L-configuration unless indicated.
10.1021/jm801496u CCC: $40.75
2009 American Chemical Society
Published on Web 03/06/2009

1936 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Chimenti et al.
Figure 1. Irreversible (I), reversible (R), and selective hMAO-A and hMAO-B inhibitors.
Scheme 1a
a Reagents and conditions: (i) Diethyl malonate, piperidine (cat.). EtOH
(ii) only when R1 ) OH, potassium carbonate, suitable benzyl bromide,
cis-dicyclohexyl-18-crown-6, anhydrous acetone. (iii) (1) NaOH 10%, (2)
HCl 3N, (3) SOCl2, reflux. (iv) HNR2R3, suitable solvent.
Figure 2. Structures of known coumarin-based MAO inhibitors.
(b) with thionyl chloride at reflux led to the desired derivatives
(c) (Scheme 1). These are particularly reactive toward nucleo-
philic substitution with different amines, which were used in a
double molar ratio in order to neutralize the excess of HCl.
Compounds 19 and 20 were prepared by alkaline hydrolysis
and chlorination of compound 15 as reported in our previous
article.29
Oxidation of compound 17 with oxone (2KHSO5 ·KHSO4 ·
K2SO4) led to compound 21 in quantitative yield. Hydrolysis
of arylmethyl ether 16 with hydroiodic acid 57% at reflux gave
the desired compound 22.
Biochemistry. The potential effects of the test drugs on
hMAO activity were investigated by measuring their effects on
the production of H2O2 from p-tyramine, using the Amplex Red
MAO assay kit (Molecular Probes, Inc., Eugene, OR) 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
Qu´ımica S.A., Alcobendas, Spain).
Red reagent), a nonfluorescent and highly sensitive probe that
reacts with H2O2 in the presence of horseradish peroxidase to
produce a fluorescent product, resorufin. In this study, hMAO
activity was evaluated using the above method following the
general procedure previously described by us.30 The test drugs
(new compounds and reference inhibitors) themselves 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, the control activity of hMAO-A and
hMAO-B (using p-tyramine as a common substrate for both
isoforms) was 165 ( 2 pmol of p-tyramine oxidized to
p-hydroxyphenylacetaldehyde/min (n ) 20).
Most tested drugs inhibited this enzymatic control activity
in a concentration-dependent way (see Table 1).
Results and Discussion
The production of H2O2 catalyzed by MAO isoforms can be
detected using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex
All synthesized compounds 1-59 were assayed for hMAO-A
and hMAO-B inhibitory activity. By analyzing the data reported

Human MAOs of 3-Carboxamido-7-Substituted Coumarins
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1937
Table 1. Structures and Biological Activity of Derivatives 1-59
compd
R
R1
R2
R3
IC50hMAO-A µM
IC50hMAO-B µM
SIf
>200
lit.
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C6H11
d
0.50 ( 0.03
25.36 ( 1.88
3.27 ( 0.26
d
31
32
33
2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
i-C3H7
i-C4H9
CH2CCH
d
>3.9
3
d
>31
4
d
5
CH2CH2NH2
Ph
d
7.68 ( 0.38
0.76 ( 0.04
0.91 ( 0.03
0.60 ( 0.01
0.64 ( 0.01
0.25 ( 0.01
0.25 ( 0.01
0.71 ( 0.01
0.89 ( 0.02
0.65 ( 0.02
d
0.82 ( 0.01
2.84 ( 0.21
0.067 ( 0.002
1.14 ( 0.09
d
>13
17
>110
24
>156
>400
>400
27
18
23
34
31
6
13.12 ( 0.91a
d
7
(2′-C6H5CH2-Ph)
(3′-CH3-Ph)
8
14.50 ( 1.13a
d
35
36
9
(3′-OCH3-Ph)
(3′-F-Ph)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
Ce
De
Ie
d
d
(3′-CF3-Ph)
(4′-CH3-Ph)
19.45 ( 1.53a
15.67 ( 1.15a
15.14 ( 1.47a
15.98 ( 0.98
d
31
(4′-C2H5-Ph)
(4′-i-C3H7-Ph)
(4′-COOEt-Ph)
(4′-OCH3-Ph)
(4′- SCH3-Ph)
(4′-F-Ph)
(4′-COOH-Ph)
(4′-COCl-Ph)
(4′-SO2CH3-Ph)
(4′-OH-Ph)
(2′-Cl-6′-CH3-Ph)
(2′,5′-CH3-Ph)
(2′,4′-CH3-Ph)
(2′,3′-CH3-Ph)
(2′,6′-CH3-Ph)
(2′,6′-F-Ph)
(3′,4′-CH3-Ph)
(3′,4′-OCH3-Ph)
(3′,5′-CH3-Ph)
(3′,5′-OCH3-Ph)
(2′,3′,4′,5′,6′-F-Ph)
(2′,3′,5′,6′-F-4′-CN-Ph)
(2′,3′,5′,6′-F-pyridin-4′-yl)
CH2Ph
<0.16
19
31
19
>122
5.2
14.76 ( 1.34a
13.06 ( 0.12a
9.97 ( 0.87a
9.67 ( 0.29
9.33 ( 0.73a
d
195
8.7
19
19
<0.10
0.0014 ( 0.00012
0.80 ( 0.01
19.07 ( 1.49
22.45 ( 1.52
13.74 ( 1.32
18.27 ( 1.57
1.29 ( 0.07
9.23 ( 0.02
0.38 ( 0.02
0.86 ( 0.04
0.70 ( 0.01
4.18 ( 0.12
0.46 ( 0.04
0.86 ( 0.02
0.58 ( 0.01
1.96 ( 0.15
d
6,664
>125
>5.2
0.71
33
37
d
15.95 ( 1.48a
12.32 ( 1.35
20.63 ( 1.07
9.79 ( 0.88a
12.76 ( 1.05b
18.77 ( 1.47a
9.45 ( 0.53a
17.56 ( 1.60a
61.46 ( 6.13a
d
0.89
1.1
7.6
1.4
49
38
11
25
15
>217
19
19
19
19
31
39
16.04 ( 0.95a
11.43 ( 0.74a
d
20
>51
CH2CH2Ph
d
Ph
d
8.89 ( 0.56
>11
CH2Ph
d
d
d
Ph
d
40
OCH2Ph
C6H11
d
64.45 ( 5.98
>1.5
OCH2Ph
i-C3H7
63.52 ( 5.34
58.64 ( 5.09a
60.93 ( 5.19a
61.38 ( 4.93a
51.89 ( 5.32
49.75 ( 2.36b
d
d
<0.63
6.1
OCH2Ph
i-C4H9
9.59 ( 0.45
3.69 ( 0.26
16.54 ( 0.87
63.52 ( 4.64
61.49 ( 3.47
4.39 ( 0.38
3.36 ( 0.09
0.99 ( 0.08
4.52 ( 0.33
3.45 ( 0.28
d
OCH2Ph
(4′-i-C3H7-Ph)
(2′,3′,5′,6′-F-4′-CN-Ph)
(2′-C6H5CH2-Ph)
(2′-CH3-6′-Cl-Ph)
(3′,4′-OCH3-Ph)
(3′,5′-OCH3-Ph)
CH2Ph
16
3.7
OCH2Ph
OCH2Ph
0.82
0.81
OCH2Ph
OCH2Ph
>23
OCH2Ph
52.89 ( 5.12
16
OCH2Ph
d
>101
OCH2Ph
NHPh
41.40 ( 2.45a
58.49 ( 4.33a
55.83 ( 3.68
59.56 ( 5.29b
49.20 ( 2.35a
52.62 ( 3.83a
d
9.2
OCH2(4′-F-Ph)
OCH2(4′-F-Ph)
OCH2(4′-F-Ph)
OCH2(4′-F-Ph)
OCH2(4′-F-Ph)
OCH2(4′-F-Ph)
OCH2Ph
(2′,3′,5′,6′-F-4′-CN-Ph)
(2′-C6H5CH2-Ph)
(2′-CH3-6′-Cl-Ph)
(3′,4′-OCH3-Ph)
(3′,5′-OCH3-Ph)
(4′-i-C3H7-Ph)
Ph
17
<0.56
1.4
17
41.64 ( 2.95
2.87 ( 0.25
21.56 ( 2.18
d
2.37 ( 0.08
4.98 ( 0.35
61.35 ( 1.13
0.020 ( 0.001
7.54 ( 0.36
c
2.4
d
>42
9.8
0.000065
3,362
0.87
<0.36
OCH2Ph
(3′,4′-OCH3-Ph)
48.95 ( 5.18a
0.004 ( 0.0002a
67.25 ( 1.02
6.56 ( 0.76
361.38 ( 19.37
Me
Each IC50 value is the mean ( SEM from five experiments. Level of statistical significance: a P < 0.01. Each IC50 value is the mean ( SEM from five
experiments. Level of statistical significance: b P < 0.05 versus the corresponding IC50 values obtained against hMAO-B, as determined by ANOVA/Dunnett’s.
c Inactive at 1 mM (highest concentration tested). d Inactive at 100 µM (highest concentration tested). e C ) Clorgyline, D ) R-(-)-deprenyl, I ) Iproniazid,
M ) Moclobemide. f SI: MAO-B selectivity index ) IC50 (MAO-A)/IC50
.
(MAO-B)
in Table 1, it can be seen that most compounds show a selective
inhibitory activity toward hMAO-B. Substitution at position 7
of the coumarin ring leads to compounds 41-58 with minor
activity toward the two isoforms, and substitution of both amidic
nitrogens (compounds 38-40 and 58) appears to be unfavorable.
Inhibitory activity increases when the nitrogen is substituted
with a phenyl group, and as regards compound 6 (IC50 against
hMAO-B ) 0.76 ( 0.04 µM), it can be seen that the presence
of an electrondrawing group such as fluorine (compound 10,
IC50 against hMAO-B ) 0.25 ( 0.01 µM) or trifluoromethyl

1938 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Chimenti et al.
Figure 3. Most stable binding poses of compounds 18 (top) and21 (bottom) with respect to hMAO-A (left side) and hMAO-B (right side). Ligands
are shown in CPK colored sticks, interacting residues are colored in green (hMAO-A) or orange (hMAO-B), carbons in stick rendering. FAD is
reported in spacefill with green (hMAO-A) or orange (hMAO-B) carbon atoms. Noninteracting aminoacids are in transparent green (hMAO-A) or
orange (hMAO-B) cartoon notation. Inhibitor-enzyme hydrogen bonds are reported in cyan dashed lines.
(compound 11, IC50 against hMAO-B ) 0.25 ( 0.01 µM) is
preferable for activity, whereas di- or poly substitution leads to
poorly active compounds.
A fluorine atom present in 4′-position of the N-phenyl moiety
improves activity compared to a fluorine atom present at the
cases the coumarin ring was involved in stacking contacts to
Tyr407 and Tyr444 of hMAO-A and to Tyr435 and Tyr398 of
hMAO-B. As regards the number of interactions with other
residues, 21 showed the most, followed by 18. Remarkably 18
showed no hydrogen bonds with the targets, while 21 in
hMAO-A did. This could partially explain the lower affinity
shown by 18 for hMAO-A compared to the other modeled
ligand, isoform selectivity could be due to the exclusive
recognition in hMAO-B of an additional cleft (Phe103, Pro104,
Trp119, Leu164, Leu167, Phe168) (Table 2).
3′-position, as observed in compounds 18 and 10 with IC50
)
0.067 ( 0.002 and 0.25 ( 0.01 µM, respectively. The most
active and selective compound 21 has a methanesulfonyl group
at the 4′-position of the N-phenyl substituent (IC50 ) 0.0014 (
0.00012 µM).
With the aim of carrying out a detailed analysis of the hMAOs
binding modes of 18 and 21, we graphically inspected the most
stable configurations of all complexes of these compounds with
both isoforms (Figure 3). Both ligands showed productive
recognition of both hMAO-Aand hMAO-B. The coumarin
moiety was always located in the known hMAOs binding clefts,
leaving the lactone function close to the FAD cofactor. In all
Experimental Section
Chemistry. Unless otherwise noted, starting materials and
reagents were obtained from commercial suppliers and were used
without purification. Melting points (mp) were determined by the
capillary method on an FP62 apparatus (Mettler-Toledo) and are
1
uncorrected. H NMR spectra were recorded at 400 MHz on a

Human MAOs of 3-Carboxamido-7-Substituted Coumarins
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1939
Table 2. List of hMAO-A and hMAO-B Corresponding Residues
Recognized by 18 and 21
ArH), 8.96 (s, 1H, ArCHd), 9.03 (bs, 1H, NH, D2O exch). MS
m/z [MH+] ) 228.
N-(2-Benzylphenyl)-2-oxo-2H-chromene-3-carboxamide (7).
corresponding residues
compd
1
Yield 60%; mp 191-193 °C. H NMR (DMSO-d6) 4.04 (s, 2H,
hMAO-A
hMAO-B
18
21
ArCH2Ar), 7.19-7.22 (m, 5H, ArH), 7.46-7.54 (m, 3H, ArH),
7.77 (m, 2H, ArH), 8.03-8.05 (m, 3H, ArH), 8.97 (s, 1H, ArCHd),
10.64 (s, 1H, NH, D2O exch). MS m/z [MH+] ) 342.
Leu97
Leu88
a
a
Ala111
Phe112
Pro113
Trp128
Phe173
Leu176
Phe177
Ile180
Asn181
Tyr197
Cys201
Ile207
Phe208
Ser209
Val210
Gln215
Cys323
Ile325
Pro102
Phe103
Pro104
Trp119
Leu164
Leu167
Phe168
Leu171
Cys172
Tyr188
Cys192
Ile198
ab
b
b
ab
b
b
b
b
N-(3-Fluorophenyl)-2-oxo-2H-chromene-3-carboxamide (10).
Yield 73%; mp 242-243 °C. 1H NMR (DMSO-d6) 6.98-7.00 (m,
2H, ArH), 7.43-7.49 (m, 2H, ArH), 7.76-7.79 (m, 2H, ArH),
8.00-8.01 (m, 2H, ArH), 8.91 (s, 1H, ArCHd), 10.78 (s, 1H, NH,
D2O exch). MS m/z [MH+] ) 284.
b
b
b
ab
ab
ab
b
b
N-(3-Trifluoromethylphenyl)-2-oxo-2H-chromene-3-carboxa-
ab
ab+
ab
a
ab
ab
a
1
mide (11). Yield 60%; mp 210-211 °C. H NMR (DMSO-d6)
7.51-7.64 (m, 3H, ArH), 7.84-7.85 (m, 2H, ArH), 8.03-8.05 (m,
1H, ArH), 8.37-8.38 (m, 2H, ArH), 9.07 (s, 1H, ArCHd), 11.12
(bs, 1H, NH, D2O exch). MS m/z [MH+] ) 334.
ab
ab
a
a
ab
a
b
ab
a
a
ab
Ile199
N-(4-Ethylphenyl)-2-oxo-2H-chromene-3-carboxamide (13).
Ser200
Thr201
Gln206
Thr314
Ile316
Tyr326
Leu328
Phe343
Tyr398
Ser433
Gly434
Tyr435
FAD
1
Yield 58%; mp 176-179 °C. H NMR (DMSO-d6) 1.16 (s, 3H,
a
CH2CH3), 2.58 (s, 2H, ArCH2CH3), 7.23 (s, 2H, ArH), 7.46 (s,
1H, ArH), 7.55 (s, 1H, ArH), 7.63 (s, 2H, ArH), 7.77 (s, 1H, ArH),
8.00 (s, 1H, ArH), 8.91 (s, 1H, ArCHd), 10.59 (s, 1H, NH, D2O
exch). MS m/z [MH+] ) 294.
ab
a+
ab
ab
a
ab
ab
a
Ile335
Leu337
Phe352
Tyr407
Ser442
Gly443
Tyr444
FAD
N-(4-Isopropylphenyl)-2-oxo-2H-chromene-3-carboxamide)-
1
amide (14). Yield 68%; mp 160-165 °C. H NMR (DMSO-d6)
1.17-1.20 (d, J ) 6.1 Hz, 6H, CH(CH3)2), 2.85-2.86 (m, 1H,
ArCH(CH3)2), 7.23 (s, 2H, ArH), 7.44 (s, 1H, ArH), 7.55 (s, 1H, ArH),
7.61 (s, 2H, ArH), 7.76 (s, 1H, ArH), 7.99 (s, 1H, ArH), 8.90 (s, 1H,
ArCHd), 10.59 (s, 1H, NH, D2O exch). MS m/z [MH+] ) 308.
N-(4-Fluorophenyl)-2-oxo-2H-chromene-3-carboxamide (18).
ab
ab
ab
ab
ab
ab
1
a ) hMAO-A interaction. b ) hMAO-B interaction. + ) Hydrogen
bond interaction.
Yield 59%; mp 251-252 °C. H NMR (CDCl3) 7.07-7.11 (t, 2H,
ArH), 7.43-7.48 (t, 2H, ArH), 7.70-7.77 (m, 4H, ArH), 9.03 (s, 1H,
ArCHd), 10.83 (bs, 1H, NH, D2O exch). MS m/z [MH+] ) 284.
N-(4-Methanesulfonylphenyl)-2-oxo-2H-chromene-3-carboxa-
mide (21). Compound 17 (1 mmol) was heated at 200 °C in
methanol (50 mL). A solution of oxone (3 mmol) in H2O (10 mL)
was added dropwise followed by 24 h of stirring at room
temperature. The suspension was filtered and the crude product was
Bruker spectrometer using DMSO-d6 or CDCl3 as solvent. Chemical
shifts are expressed as δ units (ppm) relative to TMS. Coupling
constants J are expressed in hertz (Hz). Elemental analyses for C,
H, and N were determined with a Perkin-Elmer 240 B microana-
lyzer, and the analytical results were g95% purity for all com-
pounds. All reactions were monitored by TLC performed on 0.2
mm thick silica gel plates (60 F254 Merck). Preparative flash column
chromatography was carried out on silica gel (230-400 mesh, G60
Merck). Mass spectra (EI) were obtained with a Fisons QMD 1000
mass spectrometer (70 eV, 200 µA, ion source temperature 200
°C). The samples were introduced directly into the ion source. In
the mass spectra, the fragment ion at m/z ) 173, corresponding to
the 3-acyl coumarin structure was always the most abundant
observed.
1
purified by crystallization (EtOH). Yield 99%; mp >280 °C. H
NMR (CDCl3) 3.07 (s, 3H, SO2CH3), 7.42-7.49 (t, 2H, ArH),
7.70-7.75 (t, 2H, ArH), 7.93-7.96 (m, 4H, ArH), 9.05 (s, 1H,
ArCHd), 11.12 (bs, 1H, NH, D2O exch). MS m/z [MH+] ) 344.
N-(2-Chloro-6-methylphenyl)-2-oxo-2H-chromene-3-carboxa-
mide (23). Yield 60%; mp 202-206 °C. 1H NMR (DMSO-d6) 2.22
(s, 3H, ArCH3), 7.15-7.26 (m, 3H, ArH), 7.34-7.38 (m, 2H, ArH),
7.77 (s, 1H, ArH), 8.01 (s, 1H, ArH), 8.93 (s, 1H, ArCHd), 10.23
(bs, 1H, NH, D2O exch). MS m/z [MH+] ) 314.
N-(2,4-Dimethylphenyl)-2-oxo-2H-chromene-3-carboxam-
ide (25). Yield 58%; mp 243-246 °C. 1H NMR (DMSO-d6) 2.22
(s, 3H, ArCH3), 2.29 (s, 3H, ArCH3), 7.05 (s, 1H, ArH), 7.55-7.80
(m, 3H, ArH), 8.01-8.04 (m, 3H, ArH), 9.00 (s, 1H, ArCHd),
10.60 (bs, 1H, NH, D2O exch). MS m/z [MH+] ) 294.
The synthesis of some of these compounds has been described
in previous references and was performed with slight changes. Their
analytical and spectral data were in full agreement with those
reported in the literature.
General Procedure for the Synthesis of Coumarin Deriva-
tives 1-59. The starting coumarin-3-carboxylic acid ethyl ester (a)
(Scheme 1) was prepared by Knoevenagel reaction between diethyl
malonate (1 mmol) and the appropriate salicylaldehyde (1 mmol)
with catalytic amounts of piperidine in ethanol. Then, if there was
an OH group at position 7, etherification was performed by adding
a suitable benzyl bromide (1 mmol) and potassium carbonate (1
mmol) in dry acetone, using N,N′-dicyclohexyl-18-crown-6-ether
(1 mmol) as a chelating agent. All derivatives (a) and (b) (Scheme
1) were dissolved in 10% NaOH (50 mL) and 3 N HCl (50 mL)
was added. The suspension was filtered and the solid refluxed under
magnetic stirring with thionyl chloride (30 mL) to give the desired
compound (c) (Scheme 1). Finally, a solution of the appropriate
amine (2 mmol) in toluene or ethyl ether was added dropwise and
stirred for 96 h at reflux. The organic layer was concentrated under
vacuum, cooled to 4 °C, and filtered. The solid was purified by
crystallization from ethanol or chromatography.
N-(2,3-Dimethylphenyl)-2-oxo-2H-chromene-3-carboxam-
ide (26). Yield 57%; mp 227-229 °C. 1H NMR (DMSO-d6) 2.21
(s, 3H, ArCH3), 2.29 (s, 3H, ArCH3), 7.00-7.03 (m, 2H, ArH),
7.46-7.49 (m, 2H, ArH), 7.79 (m, 1H, ArH), 8.02-8.05 (m, 2H,
ArH), 9.03 (s, 1H, ArCHd), 10.56 (bs, 1H, NH, D2O exch). MS
m/z [MH+] ) 294.
N-(2,6-Dimethylphenyl)-2-oxo-2H-chromene-3-carboxam-
1
ide (27). Yield 53%; mp 223-225 °C. H NMR (CDCl3) 2.31 (s,
6H, 2ArCH3), 7.26-7.27 (d, J0 ) 8.1 Hz, 1H, ArH), 7.42-7.48
(m, 3H, ArH), 7.71-7.75 (m, 3H, ArH), 9.04 (s, 1H, ArCHd),
10.18 (bs, 1H, NH, D2O exch). MS m/z [MH+] ) 294.
N-(2,6-Difluorophenyl)-2-oxo-2H-chromene-3-carboxamide (28).
Yield 54%; mp 169-171 °C. 1H NMR (CDCl3) 6.98 (s, 1H, ArH),
7.26-7.33 (m, 3H, ArH), 7.43 (s, 1H, ArH), 7.52-7.57 (m, 3H,
ArH), 9.05 (s, 1H, ArCHd), 10.30 (bs, 1H, NH, D2O exch). MS
m/z [MH+] ) 302.
N-(Prop-2-ynyl)-2-oxo-2H-chromene-3-carboxamide (4). Yield
58%; mp 182-183 °C. 1H NMR (CDCl3) 2.27 (s, 1H, CCH), 4.26
(s, 2H, NHCH2), 7.38-7.43 (m, 2H, ArH); 7.67-7.71 (m, 2H,
N-(3,4-Dimethoxyphenyl)-2-oxo-2H-chromene-3-carboxam-
ide (30). Yield 77%; mp 215-217 °C. 1H NMR (DMSO-d6) 3.69
(s, 3H, ArOCH3), 3.77 (s, 3H, ArOCH3), 6.99 (m, 1H, ArH),

1940 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Chimenti et al.
7.45-7.59 (m, 3H, ArH), 7.67-7.90 (m, 3H, ArH), 8.97 (s, 1H,
ArCHd), 10.25 (bs, 1H, NH, D2O exch). MS m/z [MH+] ) 326.
N-(3,5-Dimethylphenyl)-2-oxo-2H-chromene-3-carboxam-
ide (31). Yield 52%; mp 205-207 °C. 1H NMR (DMSO-d6) 2.25
(s, 6H, 2 ArCH3), 6.78 (s, 1H, ArH), 7.29-7.36 (m, 2H, ArH),
7.42-7.57 (m, 2H, ArH), 7.75-7.99 (m, 2H, ArH), 8.88 (s, 1H,
ArCHd), 10.56 (bs, 1H, NH, D2O exch). MS m/z [MH+] ) 294.
N-(3,5-Dimethoxyphenyl)-2-oxo-2H-chromene-3-carboxam-
N-(3,5-Dimethoxyphenyl)-7-benzyloxy-2-oxo-2H-chromene-
3-carboxamide (49). Yield 65%; mp 242-243 °C. 1H NMR
(DMSO-d6) 3.74 (s, 6H, 2ArOCH3), 5.28 (s, 2H, ArCH2), 6.29 (s,
1H, ArH), 6.93-6.95 (m, 2H, ArH), 7.24-7.30 (m, 3H, ArH),
7.42-7.48 (m, 4H, ArH), 7.98-8.00 (m, 1H, ArH), 8.95 (s, 1H,
ArCHd), 10.53 (s, 1H, NH, D2O exch). MS m/z [MH+] ) 432.
N-Benzyl-7-benzyloxy-2-oxo-2H-chromene-3-carboxamide (50).
1
Yield 58%; mp 183-184 °C. H NMR (CDCl3) 4.66-4,68 (m,
1
ide (32). Yield 55%; mp 207-209 °C. H NMR (CDCl3) 3.84 (s,
2H, NHCH2), 5.17 (s, 2H, ArCH2), 6.93 (s, 1H, ArH), 7.01-7.04
(m, 1H, ArH), 7.35-7.36 (m, 5H, ArH), 7.42-7.44 (m, 5H, ArH),
7.67-7.68 (m, 1H, ArH), 8.88 (s, 1H, ArCHd), 9.29 (bs, 1H, NH,
D2O exch). MS m/z [MH+] ) 386.
6H, 2ArOCH3), 6.42 (s, 1H, ArH), 6.99-7.00 (m, 2H, ArH),
7.45-7.49 (m, 2H, ArH), 7.79-7.81 (m, 2H, ArH), 9.01 (s, 1H,
ArCHd), 10.87 (bs, 1H, NH, D2O exch). MS m/z [MH+] ) 326.
N-Methyl-N-phenyl-2-oxo-2H-chromene-3-carboxamide (38).
Yield 65%; mp 138-140 °C. 1H NMR (CDCl3) 3.49 (s, 3H, NCH3),
7.23-7.24 (m, 5H, ArH), 7.41-7.42 (m, 2H, ArH), 7.47-7.49 (m,
2H, ArH), 7.72 (s, 1H, ArCHd); MS m/z [MH+] ) 280.
N′-Phenyl-7-benzyloxy-2-oxo-2H-chromene-3-carbohy-
1
drazide (51). Yield 61%; mp 230-232 °C. H NMR (DMSO-d6)
5.25 (s, 2H, ArCH2), 6.70-6.75 (m, 3H, ArH), 6.83-6.85 (m, 2H,
ArH), 7.20-7.29 (m, 5H, ArH), 7.32-7.37 (m, 3H, ArH), 7.44 (s,
1H, ArCHd), 7.80 (bs, 1H, NH, D2O exch), 8.55 (bs, 1H, NH,
D2O exch). MS m/z [MH+] ) 387.
N-Methyl-N-benzyl-2-oxo-2H-chromene-3-carboxamide (39).
1
Yield 99%; mp 173-177 °C. H NMR (DMSO-d6) 2.50 (s, 3H,
NCH3), 4.08 (s, 2H, ArCH2), 7.41-7.43 (m, 5H, ArH), 7.53-7.55
(m, 4H, ArH), 9.30 (s, 1H, ArCHd); MS m/z [MH+] ) 294.
N-Cyclohexyl-7-benzyloxy-2-oxo-2H-chromene-3-carboxam-
ide (41). Yield 99%; mp 198-200 °C. 1H NMR (DMSO-d6)
1.27-1.44 (m, 5H, cyclohexyl), 1.60-1.74 (m, 3H, cyclohexyl),
1.97-2.00 (m, 2H, cyclohexyl), 3.96-3.99 (m, 1H, cyclohexyl),
5.16 (s, 2H, ArCH2), 6.93-7.01 (m, 2H, ArH), 7.37-7.43 (m, 5H,
ArH), 7.58-7.60 (m, 1H, ArH), 8.70 (s, 1H, ArCHd), 8.83 (s,
1H, NH, D2O exch). MS m/z [MH+] ) 378.
N-Isopropyl-7-benzyloxy-2-oxo-2H-chromene-3-carboxam-
ide (42). Yield 84%; mp 186-188 °C. 1H NMR (DMSO-d6)
1.26-1.28 (d, J ) 6.2 Hz, 6H, CH(CH3)2), 4.24-4.26 (m, 1H,
NHCH(CH3)2), 5.17 (s, 2H, ArCH2), 6.93-7.01 (m, 2H, ArH),
7.26-7.43 (m, 5H, ArH), 7.60-7.62 (m, 1H, ArH), 8.67 (s, 1H,
ArCHd), 8.83 (s, 1H, NH, D2O exch). MS m/z [MH+] ) 338.
N-Isobutyl-7-benzyloxy-2-oxo-2H-chromene-3-carboxamide
(43). Yield 64%; mp 160-161 °C. 1H NMR (DMSO-d6) 0.98-1.00
(d, J ) 6.0 Hz, 6H, CH(CH3)2), 1.93-1.95 (m, 1H, CH(CH3)2),
3.32-3.33 (m, NHCH2CH), 5.17 (s, 2H, ArCH2), 6.96-7.03 (m,
2H, ArH), 7.27-7.43 (m, 5H, ArH), 7.60-7.63 (m, 1H, ArH), 8.84
(s, 1H, ArCHd), 8.99 (bs, 1H, NH, D2O exch). MS m/z [MH+] )
352.
N-(4-Cyano-2,3,5,6-tetrafluorophenyl)-7-(4-fluorobenzyloxy)-
2-oxo-2H-chromene-3-carboxamide (52). Yield 56%; mp 237-238
°C. 1H NMR (DMSO-d6) 5.27 (s, 2H, ArCH2), 7.12-7.15 (m, 1H,
ArH), 7.24-7.30 (m, 3H, ArH), 7.52-7.53 (m, 2H, ArH),
7.98-8.01 (m, 1H, ArH), 8.99 (s, 1H, ArCHd), 10.72 (bs, 1H,
NH, D2O exch). MS m/z [MH+] ) 487.
N-(2-Benzylphenyl)-7-(4-fluorobenzyloxy)-2-oxo-2H-chromene-
3-carboxamide (53). Yield 57%; mp 205-208 °C. 1H NMR
(DMSO-d6) 4.04 (s, 2H, ArCH2Ar), 5.26 (s, 2H, ArCH2), 7.13-7.24
(m, 11H, ArH), 7.26-7.28 (m, 1H, ArH), 7.54-7.55 (m, 2H, ArH),
7.98-8.02 (m, 2H, ArH), 8.98 (s, 1H, ArCHd), 10.70 (bs, 1H,
NH, D2O exch). MS m/z [MH+] ) 480.
N-(2-Methyl-6-chlorophenyl)-7-(4-fluorobenzyloxy)-2-oxo-2H-
chromene-3-carboxamide (54). Yield 59%; mp 236-238 °C. 1H
NMR (DMSO-d6) 2.24 (s, 3H, ArCH3), 5.27 (s, 2H, ArCH2),
7.12-7.13 (m, 1H, ArH), 7.20-7.25 (m, 5H, ArH), 7.40-7.42 (m,
1H, ArH), 7.53-7.55 (m, 2H, ArH), 7.97-7.99 (m, 1H, ArH), 8.99
(s, 1H, ArCHd), 10.19 (s, 1H, NH, D2O exch). MS m/z [MH+] )
438.
N-(3,4-Dimethoxyphenyl)-7-(4-fluorobenzyloxy)-2-oxo-2H-
chromene-3-carboxamide (55). Yield 58%; mp 182-183 °C. 1H
NMR (DMSO-d6) 3.74 (s, 3H, ArOCH3), 3.76 (s, 3H, ArOCH3),
5.24 (s, 2H, ArCH2), 6.97-6.99 (m, 1H, ArH), 7.12-7.20 (m, 5H,
ArH), 7.40-7.42 (m, 1H, ArH), 7.52-7.54 (m, 2H, ArH),
7.97-7.99 (m, 1H, ArH), 8.96 (s, 1H, ArCHd), 10.49 (s, 1H, NH,
D2O exch). MS m/z [MH+] ) 450.
N-(4-Isopropylphenyl)-7-benzyloxy-2-oxo-2H-chromene-3-
carboxamide (44). Yield 69%; mp 275-278 °C. 1H NMR (DMSO-
d6) 1.25-1.27 (d, J ) 6.1 Hz, 6H, CH(CH3)2), 2.96-2.99 (m, 1H,
ArCH(CH3)2), 5.19 (s, 2H, ArCH2), 6.99-7.07 (m, 2H, ArH),
7.25-7.27 (m, 2H, ArH), 7.43-7.45 (m, 5H, ArH), 7.63-7.65 (m,
3H, ArH), 8.98 (s, 1H, ArCHd), 10.77 (s, 1H, NH, D2O exch).
MS m/z [MH+] ) 414.
N-(3,5-Dimethoxyphenyl)-7-(4-fluorobenzyloxy)-2-oxo-2H-
chromene-3-carboxamide (56). Yield 52%; mp 210-214 °C. 1H
NMR (DMSO-d6) 3.74 (s, 6H, 2ArOCH3), 5.25 (s, 2H, ArCH2),
6.32 (s, 1H, ArH), 6.94 (s, 2H, ArH), 7.19-7.20 (m, 1H, ArH),
7.26-7.27 (m, 3H, ArH), 7.52-7.53 (m, 2H, ArH), 7.97-7.99 (m,
1H, ArH), 8.93 (s, 1H, ArCHd), 10.53 (bs, 1H, NH, D2O exch).
MS m/z [MH+] ) 450.
N-(4-Cyano-2,3,5,6-tetrafluorophenyl)-7-benzyloxy-2-oxo-2H-
chromene-3-carboxamide (45). Yield 72%; mp 242-245 °C. 1H
NMR (DMSO-d6) 5.28 (s, 2H, ArCH2), 7.12-7.24 (m, 2H, ArH),
7.30-7.49 (m, 5H, ArH), 7.98-8.00 (m, 1H, ArH), 9.00 (s, 1H,
ArCHd), 10.75 (bs, 1H, NH, D2O exch). MS m/z [MH+] ) 469.
N-(2-Benzylphenyl)-7-benzyloxy-2-oxo-2H-chromene-3-car-
N-(4-Isopropylphenyl)-7-(4-fluorobenzyloxy)-2-oxo-2H-
chromene-3-carboxamide (57). Yield 56%; mp 235-237 °C. 1H
NMR (DMSO-d6) 1.25-1.27 (d, J ) 6.1 Hz, 6H, CH(CH3)2),
2.96-2.99 (m, 1H, ArCH(CH3)2), 5.19 (s, 2H, ArCH2), 6.99-7.07
(m, 2H, ArH), 7.27-7.29 (m, 2H, ArH), 7.43-7.45 (m, 4H, ArH),
7.67-7.69 (m, 3H, ArH), 8.98 (s, 1H, ArCHd), 10.77 (s, 1H, NH,
D2O exch). MS m/z [MH+] ) 432.
1
boxamide (46). Yield 61%; mp 207-209 °C. H NMR (DMSO-
d6) 4.05 (s, 2H, ArCH2Ar), 5.28 (s, 2H, ArCH2), 7.03-7.29 (m,
8H, ArH), 7.31-7.48 (m, 5H, ArH), 7.98-8.03 (m, 4H, ArH), 8.98
(s, 1H, ArCHd), 10.65 (s, 1H, NH, D2O exch). MS m/z [MH+] )
462.
N-(2-Methyl-6-chlorophenyl)-7-benzyloxy-2-oxo-2H-chromene-
3-carboxamide (47). Yield 55%; mp 203-205 °C. 1H NMR
(DMSO-d6) 2.23 (s, 3H, ArCH3), 5.28 (s, 2H, ArCH2), 7.05-7.07
(m, 1H, ArH), 7.18-7.21 (m, 3H, ArH), 7.39-7.46 (m, 3H, ArH),
7.49-7.50 (m, 3H, ArH), 7.98-8.00 (m, 1H, ArH), 8.93 (s, 1H,
ArCHd), 10.20 (s, 1H, NH, D2O exch). MS m/z [MH+] ) 420.
N-(3,4-Dimethoxyphenyl)-7-benzyloxy-2-oxo-2H-chromene-
3-carboxamide (48). Yield 62%; mp 183-185 °C. 1H NMR
(DMSO-d6) 3.74 (s, 3H, ArOCH3), 3.76 (s, 3H, ArOCH3), 5.27 (s,
2H, ArCH2), 6.90-6.93 (m, 2H, ArH), 7.25-7.27 (m, 2H, ArH),
7.30-7.39 (m, 3H, ArH), 7.48-7.50 (m, 2H, ArH), 7.98-8.01 (m,
2H, ArH), 8.96 (s, 1H, ArCHd), 10.50 (s, 1H, NH, D2O exch).
MS m/z [MH+] ) 432.
N-Methyl-N-phenyl-7-benzyloxy-2-oxo-2H-chromene-3-car-
1
boxamide (58). Yield 60%; mp 157-158 °C. H NMR (DMSO-
d6) 3.33 (s, 3H, NCH3), 5.17 (s, 2H, ArCH2), 6.99-7.01 (m, 4H,
ArH), 7.24-7.46 (m, 8H, ArH), 7.58-7.60 (m, 1H, ArH), 8.04 (s,
1H, ArCHd); MS m/z [MH+] ) 386.
N-(3,4-Dimethoxyphenyl)-7-benzyloxy-8-methyl-2-oxo-2H-
chromene-3-carboxamide (59). Yield 59%; mp 220-222 °C. 1H
NMR (DMSO-d6) 2.28 (s, 3H, ArCH3), 3.74 (s, 3H, ArOCH3), 3.76
(s, 3H, ArOCH3), 5.32 (s, 2H, ArCH2), 6.98-7.00 (m, 1H, ArH),
7.24-7.50 (m, 8H, ArH), 7.80-7.85 (m, 1H, ArH), 8.80 (s, 1H,
ArCHd), 10.50 (bs, 1H, NH, D2O exch). MS m/z [MH+] ) 445.
Determination of hMAO Isoform Activity. The effects of the
test compounds on the enzymatic activity of hMAO isoform were

Human MAOs of 3-Carboxamido-7-Substituted Coumarins
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1941
Table 3. Comparison between Theoretical Affinity and Experimental
evaluated by a fluorimetric method following the experimental
protocol previously described by us.30
Inhibition Dataa
Briefly, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4)
containing the test drugs (new compounds or reference inhibitors)
at 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 (BD
Biosciences, Franklin Lakes, NJ) 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
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 (FLX800, Bio-Tek
Instruments, Inc., Winooski, VT) based on the fluorescence
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
generated in the reaction mixture due to nonenzymatic inhibition
(e.g., for directly reacting with Amplex Red reagent) was deter-
mined by adding these drugs to solutions containing only the
Amplex Red reagent in a sodium phosphate buffer.
hMAO-A
hMAO-B
compd
pIC50
∆G
pIC50
∆G
18
21
4.89
5.03
-35.80
-37.70
7.18
8.85
-38.00
-39.10
a ∆G are reported in kcal/mol. pIC50 ) -log IC50.
To improve our binding models including enzyme flexibility,
all generated complex configurations (10 and 8 for 18 and 21,
respectively) were submitted to energy minimization using the same
force field and environment reported for the ligand Monte Carlo
search. After full relaxation, the interaction energy of all complexes
was computed according to the MOLINE method49 and converted
to average state equations (Table 3).
Acknowledgment. This work was supported by MURST (Italy),
Ministerio de Sanidad y Consumo (Spain; FISS PI061537) and Xunta
de Galicia (Spain; INCITE07PXI203039ES, INCITE08E1R203054ES,
and 08CSA019203PR). Francisco Orallo is especially grateful to
the Conseller´ıa de Educacio´n y Ordenacio´n Universitaria de la
Xunta de Galicia (Spain) for giving him financial support to
intensify his research activity and to reduce his teaching during
the academic year 2007-2008 (programa de promocio´n de
intensificacio´n de la actividad investigadora en el sistema
Universitario de Galicia (SUG)). We also thank Anton Gerada,
a professional translator and Fellow of the Institute of Transla-
tion and Interpreting of London and Member of AIIC (Associa-
tion Internationale des Interpreˆtes de Confe´rences, Geneva), for
the revision of the manuscript.
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.
Supporting Information Available: Chemistry and pharmaco-
logical studies. This material is available free of charge via the
Internet at http://pubs.acs.org.
MolecularModeling.Asreportedinourrecentcommunications,23,29
computational studies were performed in order to rationalize the
recognition of the most potent inhibitors 18 and 21 with respect to
human MAO-A and MAO-B. The new, high resolution, Protein
Data Bank41 (PDB) crystallographic structures were considered as
receptor models of hMAO-A (PDB code 2Z5X42) and hMAO-B
(PDB code 2BK343), respectively.
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The theoretical approach was carried out by following three main
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Three-dimensional models of compounds 18 and 21 were built
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