New pyrrole inhibitors of monoamine oxidase: Synthesis, biological evaluation, and structural determinants of MAO-A and MAO-B selectivity

Article abstract of DOI:10.1021/jm060882y

A series of new pyrrole derivatives have been synthesized and evaluated for their monoamine oxidase (MAO) A and B inhibitory activity and selectivity. N-Methyl,N-(benzyl),N-(pyrrol-2-ylmethyl)amine (7) and N-(2-benzyl),N-(1- methylpyrrol-2-ylmethyl)amine

Full text of DOI:10.1021/jm060882y

922
J. Med. Chem. 2007, 50, 922-931
New Pyrrole Inhibitors of Monoamine Oxidase: Synthesis, Biological Evaluation, and
Structural Determinants of MAO-A and MAO-B Selectivity
Giuseppe La Regina,† Romano Silvestri,*,† Marino Artico,† Antonio Lavecchia,*,‡ Ettore Novellino,‡ Olivia Befani,§
Paola Turini,§ and Enzo Agostinelli§
Dipartimento di Studi Farmaceutici, UniVersita` di Roma “La Sapienza”, Piazzale Aldo Moro 5, I-00185 Roma, Italy, Dipartimento di Chimica
Farmaceutica e Tossicologica, UniVersita` degli Studi di Napoli “Federico II”, Via Domenico Montesano 49, I-80131 Napoli, Italy, and
Dipartimento di Scienze Biochimiche “Rossi Fanelli” and Istituti di Biologia e Patologia Molecolare del CNR, UniVersita` di Roma
“La Sapienza”, Piazzale Aldo Moro 5, I-00185 Roma, Italy
ReceiVed July 26, 2006
A series of new pyrrole derivatives have been synthesized and evaluated for their monoamine oxidase (MAO)
A and B inhibitory activity and selectivity. N-Methyl,N-(benzyl),N-(pyrrol-2-ylmethyl)amine (7) and N-(2-
benzyl),N-(1-methylpyrrol-2-ylmethyl)amine (18) were the most selective MAO-B (7, SI ) 0.0057) and
MAO-A (18, SI ) 12500) inhibitors, respectively. Docking and molecular dynamics simulations gave
structural insights into the MAO-A and MAO-B selectivity. Compound 18 forms an H-bond with Gln215
through its protonated amino group into the MAO-A binding site. This H-bond is absent in the 7/MAO-A
complex. In contrast, compound 7 places its phenyl ring into an aromatic cage of the MAO-B binding
pocket, where it forms charge-transfer interactions. The slightly different binding pose of 18 into the MAO-B
active site seems to be forced by a bulkier Tyr residue, which replaces a smaller Ile residue present in
MAO-A.
Introduction
in Parkinson’s disease (PD), a neurodegenerative syndrome for
which the main therapy is the amelioration of symptoms with
L-DOPA and/or DA agonists.7 MAO-B is also involved in the
apoptotic process. At high concentrations, selegiline combined
with L-DOPA induces neuronal apoptosis, whereas at lower
concentration is a neuroprotector agent that prevents from the
apoptotic event.8 PF 9601N was recently discovered as an
irreversible MAO-B inhibitor that attenuates MPTP-induced
depletion of striatal dopamine levels in C57/BL6 mice.9 The
anti-MAO-B activity of the adenosine A2A receptor antagonist
KW-6002 may ameliorate its neuroprotective activity in anti-
PD therapy.10
Amine oxidases (amine: oxygen oxidoreductases, AOs) are
a heterogeneous superfamily of enzymes that catalyze the
oxidative deamination of mono-, di-, and polyamines. AOs differ
because of their molecular architecture, catalytic mechanisms,
and subcellular localizations. On the basis of the chemical nature
of the cofactor, AOs fall into two classes: FAD-AOs (EC
1.4.3.4) and Cu/TPQ-AOs (1.4.3.6)1. Both classes have been
isolated and characterized from micro-organisms, plants, and
mammals. FAD-AOs are mainly intracellular enzymes and are
often associated with the outer mitochondrial membrane.1
The isoforms MAO-A and MAO-B have been described on
the basis of their substrate and inhibitor specificity.2,3 MAO-A
catalyzes the oxidative deamination of serotonin (5-HT),
adrenaline (A), and noradrenaline (NA) and is selectively
inhibited by clorgyline (1) and moclobemide (2) (Chart 1).
MAO-B catalyzes the oxidative deamination of â-phenetylamine
and benzylamine and is selectively inhibited by selegiline (3).
Both isoenzymes deaminate dopamine (DA) in vitro and
tyramine, but human DA is preferentially metabolized by MAO-
B. Compounds 1 and 3 are irreversible inhibitors, whereas 2 is
a reversible MAO inhibitor. The kinetic aspects as well as the
possible functions of MAOs have been recently reviewed by
Tipton.4
We previously reported the synthesis of simple and highly
selective pyrrole MAO-A and MAO-B inhibitors. This new class
of MAO inhibitors was designed using a reference model that
we developed starting from the structures 2 and 3 (Chart 1).11
N-Methyl,N-propargyl,N-(pyrrol-2-ylmethyl)amine (4) was a
potent, although not selective, MAO-A inhibitor (Ki ) 0.0054
µM). The pyrrole-2-carboxyamides 5 an 6 showed high selectiv-
ity for the MAO-A (SIs ) 2025 and >2500, respectively; the
selectivity index (SI) was calculated as Ki(MAO-B)/Ki(MAO-
A) ratio, and N-methyl,N-benzyl,N-(pyrrol-2-ylmethyl)amine (7)
was highly selective for the B isoenzyme (SI ) 0.0057).11 These
results prompted us to synthesize new pyrrole analogues (8-
36) in order to extend the structure-activity relationship (SAR)
study. To investigate the structural determinants of MAO-A/B
selectivity, we carried out docking studies and molecular
dynamics (MD) simulations of the most selective inhibitors 7
and 18.
MAO-A and MAO-B have essential roles in vital physiologi-
cal processes and are involved in the pathogenesis of various
human diseases. The MAO inhibitors are used for the treatment
of psychiatric and neurological disorders.5,6 MAO-A inhibitors
are prescribed for mental depression. MAO-B inhibitors are used
Chemistry. The pyrrole-2-carboxamides 8, 11-16, 24, 27,
28, 32, and 33 were obtained in good yield by heating
2-trichloroacetyl-1H-pyrrole12 at 60 °C with appropriate amines
in the presence of triethylamine (Scheme 1). However, by use
of either N-methyl-R-phenylethylamine or N-methyl-R-cyclo-
hexylamine, this reaction produced a poor yield. To improve
the yield, we synthesized the amides 25, 29, and 30 using
pyrrole-2-carboxylic acid in the presence of (benzotriazol-1-
* To whom correspondence should be addressed. Phone: +39 06 4991
3800 (R.S.); +39 081 678 613 (A.L.). Fax: +39 06 491 491 (R.S.); +39
081 678 613 (A.L.). E-mail: romano.silvestri@uniroma1.it (R.S.);
lavecchi@unina.it (A.L.).
† Dipartimento di Studi Farmaceutici.
‡ Dipartimento di Chimica Farmaceutica e Tossicologica.
§ Dipartimento di Scienze Biochimiche “Rossi Fanelli” and Istituti di
Biologia e Patologia Molecolare del CNR.
10.1021/jm060882y CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/26/2007

New Pyrrole Inhibitors of Monoamine Oxidase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 923
Chart 1. Structures of Reference and New Pyrrole Inhibitors of Monoamine Oxidase
Scheme 1. Synthesis of Pyrrole-2-carboxamides 8, 11-16, 24, 25, 27-30, and 32-34a
a Reagents and Reaction Conditions. (a-d) XdCCl3, amine, Et3N, 60 °C, overnight. Amine: (a) Benzylamine or N-methylbenzylamine, (b) (R,S)-R-
phenylethylamine, (c) (R)-R-cyclohexylethylamine or (S)-R-cyclohexylethylamine, (d) Propargylamine or N-methylpropargylamine. (e, f) XdOH, amine,
BOP, Et3N, DMF, room temperature, overnight; Amine: (e) (R,S)-N-methyl-R-phenylethylamine, (f) (R)-N-methyl-R-cyclohexylethylamine or (S)-N-methyl-
R-cyclohexylethylamine. (g) CH3I, TBAHS, 50% NaOH/CH2Cl2, room temperature, overnight.
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) reagent and trietylamine in anhydrous dimethyl forma-
mide (DMF) at room temperature overnight. Compound 33 was
methylated at position 1 with iodomethane via a phase-transfer
reaction in the presence of tetrabutylammoniun hydrogen sulfate
in 50% NaOH/dichloromethame to furnish 34.
The (pyrrol-2-ylmethyl)amines 19, 21-23, 26, 31, and 36
were synthesized by a reaction of pyrrole with appropriate
amines in the presence of 37% formaldehyde at 0 °C for 30
min (Scheme 2). Compounds 17 and 20 were prepared by
NaBH3CN reduction of the corresponding (pyrrol-2-ylmethyl-
en)amines in THF/isopropanol at room temperature for 10 min
in acidic medium. The intermediates methylenamines were
obtained by heating pyrrole-2-carboxyaldehyde with the proper
amine at 50 °C for 1 h. Similarly, compound 35 was obtained
by reduction of the corresponding methylenamine; the NaBH4
reduction in methanol at 0 °C for 30 min produced a good yield.
Compound 18 was prepared by a two-step procedure without
isolation of the intermediate methylenamine. Accordingly,
benzylamine was added to a solution of 1-methylpyrrole-2-
carboxaldehyde, and then this mixture was treated with NaBH3-
CN in 6 N HCl/ methanol at room temperature overnight.
Biology. Bovine brain mitochondria isolated according to
Basford13 were used as a source of the two MAO isoforms.
The new pyrrole analogues were tested in comparison with
moclobemide (MCL), clorgyline (CLG), and selegiline (SLG)
as reference drugs. MAO-A and MAO-B activity was deter-
mined by a fluorometric assay, using kinuramine as a substrate,
in the presence of their specific inhibitors (L-deprenyl 1 µM
for MAO-A and clorgyline 1 µM for MAO-B).14 The four final
concentrations ranged from 5 µM to 0.1 mM. Dixon plots
showed that the inhibition was not competitive. The inhibitory
activity (Ki) and A selectivity (SI) of compounds 4 and 7-36
are summarized in Table 1. All compounds were reversible
inhibitors. In fact, 95-100% of enzyme activity was restored
only by dialysis after 24 h (the dialysis was performed in a

924 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
La Regina et al.
Scheme 2. Synthesis of Pyrrole-2-ylmethylamines 17-23, 26, 31, 35, and 36a
a Reagents and Reaction Conditions. (a-d) XdH, R1dH, CH3, amine, 37% HCHO, CH3CN, 0 °C, 30 min. Amine: (a) Benzylamine or
N-methylphenylalkylamines, (b) (R,S)-N-methyl-R-phenylethylamine, (c) (R)-N-methyl-R-cyclohexylethylamine, (d) N-methylpropargylamine, (e) XdCHO,
R1dH, (i) Amine, 50 °C, 1 h, (ii) NaBH3CN, 1N HCl, THF, i-PrOH, room temperature, 10 min; (f) XdCHO, R1dCH3, Benzylamine, NaBH3CN, 6 N HCl,
THF, MeOH, room temperature, overnight; (g) XdCHO, R1dH, (i) Propargylamine, THF, room temperature, 24 h, molecular sieves, (ii) NaBH4, MeOH,
0 °C, 30 min.
cold room in the presence of 0.1 M potassium phosphate buffer
at pH 7.2). Because of the high affinity of the inhibitors, no
dissociation of the enzyme-inhibitor complex was detected
during the activity assay, and the inhibition was apparently
irreversible. In these experimental conditions the substrate did
not compete with the inhibitor. Accordingly, a decrease of Vmax
was observed, while the Km value was unchanged.
compounds 9, 11, 13, and 15 with 10, 12, 13, and 16). Great
improvement of the MAO-B inhibitory activity was obtained
by replacing the methylene (n ) 1) of 10 with an ethylene group
(n ) 2) (compound 12 that was 2500 times more active than
10). Further elongation of the linker group (n ) 3 or 4) caused
abatement of activity (compare 12 with 13-16).
Reduction of unmethylated carboxamides to the correspond-
ing amines did not affect the MAO-A inhibitory activity; the
only exception were those compounds bearing the ethylene
linker group (20 was 21 times more potent than 11; compare
9-12, 14, and 16 with 7, 17, and 20-23). Reduction of 10
produced 7, a compound endowed with high MAO-B inhibitory
activity and selectivity.11 Derivatives 17-23 were surprisingly
weak MAO-B inhibitors, with the only exception being 20 (Ki-
(MAOB) ) 0.7 µM). It was clearly evident that the MAO-B
selectivity was strongly associated with the presence of the
N-benzyl,N-methylamino group.
As an MAO-A inhibitor, 18 was 140 times more potent than
the parent compound 17 and displayed high selectivity (SI )
12 500). Introduction of a methyl group at 18’s NH (19)
dramatically abated anti-MAO-A activity and selectivity.
The anti-MAO potency displayed by some cyclohexyl deriva-
tives in a preliminary screening (data not shown) and led us to
synthesize compounds 27-31 as pure enantiomers. The anti-
MAO-A activity of 27-30 was dependent on the methylcar-
boxamide rather than the chiral center (compare 28 with 27 and
30 with 29). On the contrary, as MAO-B inhibitor (R)-derivative
29 was 136 times more potent than the corresponding enantio-
mer (S)-30, while (R)-27 and (S)-28 were almost equipotent.
Replacing the benzyl of 9 with a propargyl group gave
compound 32. As an MAO-A inhibitor, 32 was as active as 9.
The corresponding N-methylcarboxamide 33 was 8 times more
potent than 32. Reduction of 32 to 35 resulted in a significant
improvement of the anti-MAO-B activity (Ki ) 0.062 µM). The
methylated analogue 4 greatly showed improved anti-MAO-A
(Ki ) 0.0054 µM) and anti-MAO-B (Ki ) 0.02 µM) activity.
However, this compound was poorly selective.
Results and Discussion
With the only exception of 7 and 24, tested derivatives
inhibited the MAO-A at sub-micromolar concentration. Six
compounds (7, 12, 20, 29, 35, and 36) inhibited the B isoform
in the sub-micromolar range of concentration (several derivatives
inhibited the MAO-B at micromolar concentration). SI values
ranged from 12 500 (18) to 0.0057 (7). The amides 12 and 29
showed the greatest MAO-A inhibitory activity (Ki(MAO-A)
) 0.007 and 0.0017 µM, respectively). Compound 18 was the
most selective MAO-A inhibitor (Ki(MAO-A) ) 0.024 µM, Ki-
(MAO-B) ) 300 µM, SI ) 12 500). Compound 29 was almost
31 times more potent than clorgyline; however, because of its
potent MAO-B inhibitory activity (Ki(MAO-B) ) 0.03 µM), it
was not selective. Compounds 12, 20, 29, 35, and 36 were potent
but poor selective MAO-B inhibitors.
Structure-activity relationships (SARs) were inferred from
data of enzymatic experiments reported in Table 1. First we
analyzed how the length of the linker group affected the anti-
MAO activity (Chart 2). N-Phenyl-1H-pyrrole-2-carboxamide
(8, n ) 0, Ki ) 0.4 µM) inhibited the MAO-A at a concentration
1.6 times higher than that obtained with 9 (n ) 1, Ki ) 0.25
µM).11 Elongation of the alkyl chain (n ) 2, 3) produced slight
decreases of MAO-A inhibition. In contrast, 15 (n ) 4) was
4.5 times more potent than 9 (compare 9 with 8, 11, 13, and
15). N-Methylcarboxamides 10 (n ) 1) and 16 (n ) 4) were
2.4 and 1.6 times less active than the parent compounds 9 and
15, respectively. Conversely, compounds 12 (n ) 2) and 14 (n
) 3) were almost 60 and 17 times more active than 11 and 13
(compare compounds 9, 11, 13, and 15 with 10, 12, 13, and
16). As MAO-B inhibitors, the N-methylcarboxamides were
more potent than the unmethylated counterparts (compare
Molecular Modeling. We carried out docking experiments
and molecular dynamics (MD) simulations of compounds 7 and

New Pyrrole Inhibitors of Monoamine Oxidase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 925
Table 1. Structures and Monoamine Oxidase Inhibitory Activities of
Derivatives 4 and 7-36a
Table 2. Result of 50 Independent Autodock and GOLD Docking Runs
for Each Liganda
ligand MAO isoform Ntot focc ∆Gbind GOLD fitness scoreb
7
7
18
18
A
B
A
B
19
8
10
16
20
20
17
13
-8.75
-9.13
-8.47
-8.45
47.6
51.9
52.4
40.2
a Ntot is the total number of clusters; the number of results in the top
cluster is given by the frequency of occurrence, focc; ∆Gbind(kcal/mol) is
the estimated free energy of binding for the top cluster results. b Higher
scores indicate more favorable binding (kJ/mol).
Ki (µM)
Ki (µM)
compd
R1
R2
n
*
MAO-A MAO-B
SIb
interactions, all complexes obtained from docking were submit-
ted to MD simulations for 400 ps at 300 K (constant temper-
ature). The dynamic stability of each docked conformation was
monitored by computing the root-mean-squared deviations
(rmsd) of the ligands, relative to their initial docked orientation.
The enzyme-ligand hydrogen bond distances were monitored
during the complete MD trajectory.
8
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
0
1
1
2
2
3
3
4
4
1
1
1
1
2
2
3
4
0.4
0.25
0.6
>100
150
150
>250.0
600.0
250.0
>238.1
17.1
9c
10c
11
0.42
0.007
0.70
0.04
0.055
0.09
0.35
3.5
0.024
0.15
0.02
0.05
0.05
0.1
>100
12
13
14
15
0.12
25
8.9
57
2.5
35.7
222.5
1036.4
27.7
20.5
0.0057
12500.0
566.6
35.0
96.0
40.0
25.0
50.6
450.0
15.0
13.8
10.6
17.6
The use of the crystal structure of rat MAO-A and human
MAO-B for docking studies is justified by the following facts:
(i) the crystal structures of bovine MAO-A and MAO-B
isoforms are unknown; (ii) the rat and bovine sequences of
MAO-A are characterized by 85.3% of identity and 95.4% of
homology at the binding site, while the human and bovine
sequences of MAO-B have 91.3% of identity and 97.1% of
homology,22 and (iii) all active-site residues are largely con-
served across the MAO isoforms sequenced so far. Only one
mutation is found at the catalytic site of the human and bovine
sequence of MAO-B: Ile199 is replaced by Phe199.23
AutoDock provided well-clustered docking results for com-
pounds 7 and 18. The 50 independent docking runs, carried out
for each ligand, converged to a small number of different
positions (“clusters” of results differing by less than 1.5 Å rmsd).
Generally, the top ranking clusters (i.e., those with the most
favorable ∆Gbind) were also associated with the highest fre-
quency of occurrence, which suggested a good convergence of
the search algorithm. The best results in terms of free energy
of binding were all located in a similar position into the active
site.
16
17
7.2
0.02
300
85
0.7
4.8
2.0
2.5
61.8
4.5
1.5
3.6
3.5
7c
18
19c
20
CH3 CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
21
22
23
24
25
26
27
28
CH3
CH3
CH3
H
CH3
CH3
H
R,S
R,S
R,S
R
S
R
1.22
0.01
0.1
0.26
0.33
0.0017
0.02
0.02
0.23
0.075
0.83
0.3
0.0054
0.44
11.5
0.054
3.8
H
29
30
31
CH3
CH3
CH3
H
0.03
4.1
7.0
70
50
S
R
205.0
350.0
304.3
666.6
>120.5
2.1
32
33c
34
CH3
CH3 CH3
H
H
>100
35
H
CH3
0.62
0.02
4c
3.7
36
CH3 CH3
>100
>100
>227.3
MCLd
CLGe
SLGf
>87
58
0.97
1074.1
0.25
Docking studies with GOLD showed that 7 and 18 occupied
the same binding locations suggested by AutoDock into the
MAO-A and MAO-B. The GOLD run recognized a series of
variable conformations of the ligand docked into the binding
site, together with an associated scoring function and other
measures of the corresponding protein-ligand interaction
energy. The GOLD score consisted of hydrogen-bonding,
complex energy, and ligand internal-energy terms.
a Data represent mean values for at least three separate experiments each
performed in duplicate. Standard errors were within 2%. b SI ) Ki(MAO-
B)/Ki(MAO-A) ratio. c Reference 11. d MCL, moclobemide. e CLG, clor-
gyline. f SLG, selegiline.
Chart 2. Structure-activity Relationships Remarksa
Docking results (the total number of clusters, the number of
results in the most populated cluster, the relative estimated free
energy of binding, and the GOLD fitness score) are summarized
in Table 2.
Binding Mode of 7 and 18 into MAO-A. Docking studies
of compound 7 into the active site of MAO-A provided well-
clustered solutions. The top result ranked with a clearly better
score than all other results (∆Gbind ) -8.75 kcal/mol, found
20 times out of 50). Similarly, the top-ranked binding mode
obtained for 18 (∆Gbind ) -8.47 kcal/mol, found 17 times out
of 50) was located in a comparable position into the active site.
Surprisingly, docked conformations of 7 and 18 proposed by
GOLD assumed binding poses that strongly resembled the top-
ranking ones found by AutoDock, with fitness scores of 47.6
and 52.4 kJ/mol, respectively.
a (+) Positive or (-) negative effect of the chemical modifications on
the inhibition of the indicated MAO isoform.
18 using the 3-D X-ray crystal structures of rat MAO-A (PDB
code 1O5W)15 and human MAO-B (PDB code 1GOS).16
Docking calculations were performed using the automated
docking tools AutoDock 3.0.517,18 and GOLD 2.2,19,20 which
well reproduced experimentally found binding modes of several
ligands.17,18,21
In order to take into account the protein flexibility and
evaluate the dynamic stability of the predicted ligand/enzyme
It is worth noting that the scoring functions found by
Autodock were not well correlated with the experimental data,

926 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
La Regina et al.
while GOLD found interesting correlations with the experi-
mental data from an energetic point of view. The reason way
Autodock gave unsatisfactory results might be a result of some
simplifications residing in this software: (A) no explicit water
molecules were considered in docking studies; (B) solvation
and entropic effects were not taken into account. Accordingly,
Binda et al. emphasized the role of structural water molecules
into the catalytic region close to the FAD cofactor, also in the
presence of noncovalent ligands complexed to the MAO-B
isoform.24 Structural water molecules were neglected in our
docking simulations, but we aim to include them in our
forthcoming studies.
Visual inspection of the poses of 7 and 18 into the MAO-A
binding site revealed that the phenyl rings are placed in the
“aromatic cage” and are oriented to establish π-π stacking
interactions with Tyr407 and Tyr444 side chains as well as a
T-shaped π-π interactions with the FAD aromatic ring.
Moreover, a hydrogen bond between the protonated amino group
of 18 and the carbonyl oxygen of Gln215 side chain is also
observed. This H-bond is absent in the 7/MAO-A complex. The
methyl group on the positively charged nitrogen atom of 7
caused an increase of the steric hindrance into the binding cavity,
thus preventing from forming a reinforced H bond between the
proton on the amino group and the Gln215 residue. In both
solutions, the pyrrole ring is embedded in a large hydrophobic
pocket formed by Ile180, Phe208, Val210, Ile325, Ile335,
Ile337, and Met350.
MD trajectories (data not shown) suggest that MAO-A
produces stable complexes with the inhibitors 7 and 18.
Compound 7, after small amplitude fluctuations in the binding
site for 100 ps, rapidly achieves stable interactions with the
enzyme key residues throughout the trajectory. The low rmsd
(1.45 Å) of compound 18 indicates no significant deviation from
the initial docked conformation during the MD simulation for
400 ps. Figure 1 shows the binding mode of 7 (a) and 18 (b)
into the MAO-A active site as the average structure calculated
on the whole 300 ps of the production step. Visual inspection
of the complex models indicates that the higher anti-MAO-A
potency of 18 in comparison with 7 (Ki ) 0.024 and 3.5 µM,
respectively) may be ascribable to the H-bond between the
protonated amino group of 18 and the carbonyl oxygen of
Gln215 side chain that is absent in the 7/MAO-A complex.
Figure 1. Binding modes of compound 7 (a) and 18 (b) into the
MAO-A binding cavity. For clarity, only interacting residues are
displayed. Ligand (cyan), FAD cofactor (yellow), and interacting key
residues (white) are represented as stick models, while the proteins
(purple) are represented as ribbons. The van der Waals volume of Ile335
is displayed as a transparent yellow surface.
Binding Mode of 7 and 18 into MAO-B. AutoDock found
different binding poses for compounds 7 and 18, with highly
populated clusters (20/50 and 13/50, respectively) and estimated
binding free energy of -9.13 and -8.45 kcal/mol, respectively.
In contrast, GOLD successfully calculated a single binding pose
of 7 and 18 into the MAO-B active site. However, the associated
binding orientations strongly resembled the top-ranked ones
found by AutoDock, with fitness scores of 51.9 kJ/mol for 7
and 40.2 kJ/mol for 18.
Analysis of the MD trajectories of compounds 7 and 18
complexed with MAO-B reveals that both ligands adopt a stable
binding pose during the simulation time, as confirmed by their
low rmsd fluctuations (1.01 Å for 7 and 2.01 Å for 18).
Moreover, the H bonds between 7 and both Tyr435 and Gln206
side chains were also stable throughout the MD simulation, thus
explaining the higher anti-MAO-B potency of 7 (7, Ki ) 0.02
µM; 18, 300 µM). Figure 2 displays the binding mode of 7 (a)
and 18 (b) into the MAO-B active site as the average structure
calculated on the whole 300 ps of the production step. These
results provide a molecular rationale for the MAO-A selectivity
of 18. In fact, the binding pose of 18 into the MAO-B active
site seems to be forced by the bulkier Tyr326 residue (displayed
as a transparent yellow surface in Figure 2). The Tyr326 residue,
which is specific of the MAO-B, is replaced by the smaller Ile
residue (Ile335, displayed as a transparent yellow surface in
Figure 1) in the MAO-A isoform. This amino acid forces the
ligand to adopt a different pose into MAO-B, thus preventing
from recurring charge-transfer interactions of the phenyl ring
with the “aromatic cage”. Ile335 and Tyr326, the major
The phenyl ring of 7 is hosted into the “aromatic cage” framed
by Tyr188, Tyr398, Tyr435, and the FAD aromatic ring, where
it forms a number of charge-transfer interactions. Unexpectedly,
the phenyl ring of 18 is positioned just underneath the enzymatic
“aromatic cage” and seems unable to form any charge-transfer
interaction with the enzyme. Two H-bonds are also observable
for 7 between the protonated aminomethyl group and the
phenolic oxygen of Tyr435 and the pyrrole NH and the Gln206
carbonyl oxygen. Compound 18 forms only one H-bond between
its protonated amino group and the Gln206 side chain. In both
cases, the binding is further stabilized by hydrophobic interac-
tions between the pyrrole ring and a large lipophilic cleft made
up by Phe168, Leu171, Ile198, Ile199, and Tyr326 side chains.

New Pyrrole Inhibitors of Monoamine Oxidase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 927
methyl group at position 1 of the pyrrole resulted in a reduction
of anti-MAO-B activity. It is intriguing that the two closely
related structures 7 and 18 showed the highest selectivity against
the MAO-B (7, SI ) 0.0057) and the MAO-A (18, SI ) 12 500).
Docking studies and MD simulations indicated that the high
MAO-A inhibitory potency of 18 (Ki ) 0.024 µM) may be
ascribable to the H bond between its protonated amino group
and the carbonyl oxygen of Gln215 side-chain. This H-bond is
absent in the 7/MAO-A complex. Conversely, into the MAO-B
binding site, 7 forms two H bonds, whereas 18 seems to form
only one H bond between the protonated amino group and
Gln206 side chain. In the B isoform, the phenyl ring of 7 is
hosted into an aromatic cage, where it forms a number of charge-
transfer interactions. On the contrary, the phenyl ring of 18 is
unable to establish any charge-transfer interaction with the
enzyme. This compound seems to be forced by a bulkier Tyr
residue, specific of this isoform (into MAO-A this aminoacid
is replaced by a smaller Ile residue). We found a molecular
rationale for the MAO-A and MAO-B selectivity of this new
class of pyrrole inhibitors. These findings increase our confi-
dence in our model and stimulate us to continue our investiga-
tions in designing more potent and selective analogues.
Experimental Section
Chemistry. Melting points (mp) were determined on a Bu¨chi
510 apparatus and are uncorrected. Infrared spectra (IR) were run
on Perkin-Elmer 1310 and SpectrumOne spectrophotometers. Band
position and absorption ranges are given in cm-1. Proton nuclear
magnetic resonance (1H NMR) spectra were recorded on Bruker
AM-200 (200 MHz) and Bruker Avance 400 MHz FT spectrometers
in the indicated solvent. Chemical shifts are expressed in δ units
(ppm) from tetramethylsilane. Column chromatographies were
packed with alumina (Merck, 70-230 mesh) and silica gel (Merck,
70-230 mesh). Aluminum oxide thin-layer chromatography (TLC)
cards (Fluka, aluminum oxide precoated aluminum cards with
fluorescent indicator at 254 nm) and silica gel TLC cards (Fluka,
silica gel precoated aluminum cards with fluorescent indicator at
254 nm) were used for TLC. Developed plates were visualized with
a Spectroline ENF 260C/F UV apparatus. Organic solutions were
dried over anhydrous sodium sulfate. Concentration and evaporation
of the solvent after reaction or extraction was carried out on a rotary
evaporator (Bu¨chi Rotavapor) operating at reduced pressure.
Elemental analyses were found within (0.4% of the theoretical
values.
Figure 2. Binding modes of compound 7 (a) and 18 (b) into the
MAO-B binding cavity. For clarity, only interacting residues are
displayed. Ligand (orange), FAD cofactor (yellow), and interacting key
residues (white) are represented as stick models, while the proteins
(green) are represented as ribbons. The van der Waals volume of Tyr326
is displayed as a transparent yellow surface. H-bonds are shown as
dashed yellow lines.
General Procedure for the Synthesis of Compounds 8-16,
24, 27, 28, 32, and 33. Example: N-Phenyl-1H-pyrrole-2-
carboxamide (8). A mixture of 2-trichloroacetyl-1H-pyrrole (2.76
g, 0.013 mol), aniline (1.49 g, 1.46 mL, 0.016 mol), and triethyl-
amine (1.62 g, 2.23 mL, 0.016 mol) was heated overnight at 60
°C. The mixture was evaporated in vacuo to give a crude residue
which was triturated with n-hexane. The solid was filtered, washed
with n-hexane, and then purified by alumina column chroma-
tography (chloroform as eluent) to afford 8 (1.27 g, 52%) as white
structural differences between the two MAO isoforms,25 seem
to play a significant role in the recognition of these derivatives.
Conclusions
SAR analysis clarified structural requirements for the good
activity and selectivity of this new class of anti-MAO agents.
Essential structural features for an active agent have included
(Chart 2): (i) a methyl group at the carboxamide function
increasead the anti-MAO-A and -B activity; (ii) the length of
the linker (n ) 1-4) had no effect on the anti-MAO-A activity
of amides 8-16; (iii) an ethylene linker (n ) 2, 11 and 12)
yielded compounds with increased anti-MAO-B activity; (iv)
reduction of the carbonyl to a methylene resulted in potent
MAO-B inhibitors; (v) introduction of a N-benzyl,N-methyl-
amino group (7) resulted in compounds with greater anti-
MAO-B activity; (vi) a N-methyl,N-(R)-R-(1-cyclohexyl)ethyl-
amino group (29) increased both MAO-A and MAO-B inhibi-
tory activity, with concomitant loss of selectivity; (vii) the
propargyl group was a valid bioisostere of the benzyl for the
anti-MAO-A activity of the amides; (viii) introduction of a
1
crystals, mp 152-155 °C (from benzene). H NMR (DMSO-d6):
δ 6.17 (m, 1H), 6.96-7.08 (m, 3H), 7.32 (t, J ) 7.89 Hz, 2H),
7.74 (d, J ) 7.82 Hz, 2H), 9.72 (br s, 1H, disappeared on treatment
with D2O), 11.62 ppm (br s, 1H disappeared on treatment with
D2O). IR: ν 1635, 2924, 3319 cm-1. Anal. Calcd (C11H10N2O
(186.21)) C, H, N.26
N-Benzyl-1H-pyrrole-2-carboxamide (9). This compound was
synthesized as we previously reported.11
N-Benzyl,N-methyl-1H-pyrrole-2-carboxamide (10). This com-
pound was synthesized as we previously reported.11
N-2-Phenylethyl-1H-pyrrole-2-carboxamide (11). It was syn-
thesized as 8 using 2-phenylethylamine. Yield 87%, mp 125 °C
(from toluene). 1H NMR (DMSO-d6): δ 2.81 (t, J ) 7.45 Hz, 2H),
3.38-3.49 (m, 2H), 6.04-6.07 (m, 1H), 6.74 (s, 1H), 6.80-6.83
(m, 1H), 7.16-7.37 (m, 5H), 8.09 (br s, disappeared on treatment

928 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
La Regina et al.
with D2O, 1H), 11.42 ppm (br s, disappeared on treatment with
D2O, 1H). IR: ν 1612, 3271, 3381 cm-1. Anal. Calcd (C13H14N2O
(214.27)) C, H, N.
(m, 1H), 8.00 (br s, disappeared on treatment with D2O, 1H), 11.00
ppm (br s, disappeared on treatment with D2O, 1H). IR: ν 1622,
3264, 3283, 3360 cm-1. Anal. Calcd (C8H8N2O (148.16)) C, H, N.
N-Methyl,N-propargyl-1H-pyrrole-2-carboxamide (33). This
compound was synthesized as we previously reported.11
N-(2-Phenylethyl),N-methyl-1H-pyrrole-2-carboxamide (12).
It was synthesized as 8 using N-methyl-N-(2-phenylethyl)amine.
1
Yield 39%, oil. H NMR (CDCl3): δ 2.97 (t, J ) 7.43 Hz, 2H),
General Procedure for the Synthesis of Compounds 25, 29,
and 30. Example: (R,S)-N-methyl,N-(R-phenylethyl)-1H-pyr-
role-2-carboxamide (25). BOP reagent (3.32 g, 0.0075 mol) was
added to a solution of pyrrole-2-carboxylic acid (0.83 g, 0.0075
mol), (R,S)-N-methyl-R-phenylethylamine (2.03 g, 0.015 mol), and
triethylamine (2.28 g, 3.17 mL, 0.0225 mol) in anhydrous DMF (5
mL). The reaction mixture was stirred at room temperature
overnight. Water was added while stirring, and the mixture was
extracted with ethyl acetate. The organic layer was separated,
washed with brine, and dried. The solvent was evaporated to afford
a residue which was purified by silica gel column chromatography
3.17 (s, 3H), 3.82 (t, J ) 7.43 Hz, 2H), 6.25-6.27 (m, 1H), 6.56-
6.57 (m, 1H), 6.93 (s, 1H), 7.21-7.25 (m, 3H), 7.29-7.33 (m,
2H), 9.89 ppm (br s, disappeared on treatment with D2O, 1H). IR:
ν 1586, 3240 cm-1. Anal. Calcd (C14H16N2O (228.29)) C, H, N.
N-(3-Phenylpropyl)-1H-pyrrole-2-carboxamide (13). It was
synthesized as 8 using 3-phenylpropylamine. Yield 57%, mp 80-
82 °C (from ethanol). 1H NMR (DMSO-d6): δ 1.76-1.79 (m, 2H),
2.59 (t, J ) 7.64 Hz, 2H), 3.18-3.23 (m, 2H), 6.04-6.06 (m, 1H),
6.73 (s, 1H), 6.81 (s, 1H), 7.13-7.21 (m, 5H), 7.97 (br s,
disappeared on treatment with D2O, 1H), 11.37 ppm (br s,
disappeared on treatment with D2O, 1H); IR: ν 1590, 3181, 3285
cm-1. Anal. Calcd (C14H16N2O (228.29)) C, H, N.
1
(ethyl acetate as eluent). H NMR (CDCl3): δ 1.61 (d, J ) 6.95
Hz, 3H), 2.94 (s, 3H), 6.19 (m, 1H), 6.24-6.25 (m, 1H), 6.57 (s,
1H), 6.95 (s, 1H), 7.26-7.38 (m, 5H), 9.94 ppm (br s, disappeared
on treatment with D2O, 1H). IR: ν 1583, 3251 cm-1. Anal. Calcd
(C14H16N2O (228.29)) C, H, N.
N-(3-Phenylpropyl),N-methyl-1H-pyrrole-2-carboxamide (14).
It was synthesized as 8 using N-methyl-N-(3-phenylpropyl)amine.
1
Yield 70%, mp 92 °C (from ethanol). H NMR (DMSO-d6): δ
1.99 (m, 2H), 2.68 (t, J ) 7.74 Hz, 2H), 3.22 (s, 3H), 3.61 (t, J )
7.44 Hz, 2H), 6.23 (s, 1H), 6.51 (s, 1H), 6.92 (s, 1H), 7.19-7.21
(m, 3H), 7.25-7.31 (m, 2H), 9.55 ppm (br s, disappeared on
treatment with D2O, 1H). IR: ν 1587, 3245 cm-1. Anal. Calcd
(C15H18N2O (242.32)) C, H, N.
(R)-N-(R-Cyclohexylethyl),N-methyl-1H-pyrrole-2-carbox-
amide (29). It was prepared as 25 using (R)-N-methyl-N-(R-
cyclohexylethyl)amine. Yield 61%, oil. 1H NMR (CDCl3): δ 0.8-
1.06 (m, 2H), 1.12-1.25 (m, 6H), 1.40-1.42 (m, 1H), 1.58-1.63
(m, 3H), 1.75-1.81 (m, 2H), 3.12 (s, 3H), 4.53-4.57 (m, 1H),
6.21 (s, 1H), 6.59 (s, 1H), 6.92 (s, 1H), 9.77 ppm (br s, disappeared
on treatment with D2O, 1H). IR: ν 1578, 3252 cm-1. Anal. Calcd
(C14H22N2O (234.34)) C, H, N.
N-(4-Phenylbutyl)-1H-pyrrole-2-carboxamide (15). It was
synthesized as 8 using 4-phenylbutylamine. Yield 57%, mp 98-
1
100 °C (from ethanol). H NMR (DMSO-d6): δ 1.45-1.51 (q, J
) 7.27 Hz, 2H), 1.53-1.59 (q, J ) 7.70 Hz, 2H), 2.58 (t, J ) 7.47
Hz, 2H), 3.21 (q, J ) 6.53 Hz, 2H), 6.02-6.04 (m, 1H), 6.70-
6.72 (m, 1H), 6.79-6.80 (s, 1H), 7.12-7.18 (m, 3H), 7.22-7.26
(m, 2H), 7.92 (br s, disappeared on treatment with D2O, 1H), 11.35
ppm (br s, disappeared on treatment with D2O, 1H). IR: ν 1602,
3178, 3279 cm-1. Anal. Calcd (C15H18N2O (242.32)) C, H, N.
N-(4-Phenylbutyl),N-methyl 1H-pyrrole-2-carboxamide (16).
It was synthesized as 8 using N-methyl-N-(4-phenylbutyl)amine.
Yield 40%, mp 75 °C (from ethanol). 1H NMR (CDCl3): δ 1.66-
1.68 (m, 4H), 2.66 (t, J ) 7.11 Hz, 2H), 3.22 (s, 3H), 3.59 (t, J )
7.11 Hz, 2H, 2H), 6.24-6.26 (m, 1H), 6.53 (s, 1H), 6.91 (s, 1H),
7.15-7.20 (m, 3H), 7.25-7.29 (m, 2H), 9.61 ppm (br s, disappeared
on treatment with D2O, 1H). IR: ν 1664, 3225 cm-1. Anal. Calcd
(C16H20N2O (256.35)) C, H, N.
(S)-N-(R-Cyclohexylethyl),N-methyl-1H-pyrrole-2-carbox-
amide (30). It was synthesized as 25 using (S)-N-methyl-N-(R-
cyclohexylethyl)amine. Yield 85%, oil. 1H NMR (CDCl3): δ 0.8-
1.06 (m, 2H), 1.12-1.25 (m, 6H), 1.40-1.42 (m, 1H), 1.58-1.63
(m, 3H), 1.75-1.81 (m, 2H), 3.12 (s, 3H), 4.53-4.57 (m, 1H),
6.21 (m, 1H), 6.59 (m, 1H), 6.92 (m, 1H), 9.77 ppm (br s,
disappeared on treatment with D2O, 1H). IR: ν 1578, 3252 cm-1
.
Anal. Calcd (C14H22N2O (234.34)) C, H, N.
N-Propargyl,N-methyl-1-methyl-1H-pyrrole-2-carboxamide
(34). Iodomethane (0.85 g, 0.37 mL, 0.006 mol) was added to an
ice-cooled mixture of 33 (0.32 g, 0.002 mol), tetrabutylammonium
hydrogen sulfate (0.68 g, 0.002 mol), dichlorometane (10 mL), and
50% NaOH solution (7 mL). The reaction was stirred at room
temperature overnight. Water was added while stirring and the
mixture extracted with dichlorometane. The organic layer was
separated, washed with brine, and dried. The solvent was evaporated
to afford a residue which was purified by silica gel column
(R,S)-N-(R-Phenylethyl)-1H-pyrrole-2-carboxamide (24). It
was synthesized as 8 using (R,S)-R-phenylethylamine. Yield 35%,
mp 144-147 °C (from ethanol). 1H NMR (DMSO-d6): δ 1.43 (d,
J ) 7.00 Hz, 3H), 5.10-5.14 (m, 1H), 6.06 (d, J ) 8.20 Hz, 1H),
6.82 (s, 1H), 6.87 (s, 1H), 7.17-7.36 (m, 5H), 8.26 (br s,
disappeared on treatment with D2O, 1H), 11.36 ppm (br s,
1
chromatography (chloroform as eluent). Yield 57%, oil. H NMR
DMSO-d6: δ 2.36 (t, J ) 4.87 Hz, 1H), 3.07 (s, 3H), 3.68 (s, 3H),
4.28 (d, J ) 4.87 Hz, 2H), 6.04-6.07 (m, 1H), 6.48-6.50 (m,
1H), 6.91-6.93 ppm (m, 1H). IR: ν1622, 2116, 3108, 3285, 3487
cm.1 Anal. Calcd (C10H12N2O (176.22)) C, H, N.
disappeared on treatment with D2O, 1H). IR: ν 1604, 3285 cm-1
Anal. Calcd (C13H14N2O (214.27)) C, H, N.
.
(R)-N-(R-Cyclohexylethyl)-1H-pyrrole-2-carboxamide (27). It
General Procedure for the Synthesis of Compounds 4, 7, 19,
21, 22, 23, 26, 31, and 36. Example: N-Methyl,N-(2-phenyleth-
yl),N-(pyrrol-2-ylmethyl)amine (21). Formaldehyde (37% water
solution, 0.41 mL, 0.016 mol) and N-methyl-2-phenylethylamine
(2.16 g, 0.016 mol) were added to an ice-cooled solution of pyrrole
(1.07 g, 0.016 mol) in acetonitrile (42 mL). The reaction was stirred
for 30 min at room temperature. After quenching on crushed ice,
the mixture was made basic with 50% NaOH and extracted with
ethyl acetate. The organic layer was separated, washed with brine,
and dried. The solvent was evaporated to give a residue that was
purified by alumina column chromatography (chloroform a eluent).
Yield 7%, oil. 1H NMR (CDCl3): δ 2.31 (s, 3H), 2.64 (t, J ) 7.52
Hz, 2H), 2.79 (t, J ) 7.52 Hz, 2H), 3.55 (s, 2H), 6.00 (s, 1H),
6.09-6.11 (m, 1H), 6.63-6.64 (m, 1H), 7.17-7.32 (m, 5H), 8.21
ppm (br s, disappeared on treatment with D2O, 1H). IR: ν 3428
cm-1. Anal. Calcd (C14H18N2 (214.31)) C, H, N.
was synthesized as 8 using (R)-R-cyclohexylethylamine. Yield 78%,
1
mp 163-166 °C (from ethanol). H NMR (DMSO-d6): δ 0.88-
0.92 (m, 2H), 1.03-1.14 (m, 6H), 1.32-1.34 (m, 1H), 1.56-1.71
(m, 5H), 3.76-3.78 (m, 1H), 6.02 (br s, disappeared on treatment
with D2O, 1H), 6.77-6.80 (m, 2H), 7.54-7.56 (m, 1H), 11.30 ppm
(br s, disappeared on treatment with D2O, 1H). IR: ν 1602, 3285
cm-1. Anal. Calcd (C13H20N2O (220.31)) C, H, N.
(S)-N-(R-Cyclohexylethyl)-1H-pyrrole-2-carboxamide (28). It
was synthesized as 8 using (S)-R-cyclohexylethylamine. Yield 75%,
1
mp 163-166 °C (from ethanol). H NMR (DMSO-d6): δ 0.88-
0.92 (m, 2H), 1.04-1.14 (m, 6H), 1.32-1.35 (m, 1H), 1.56-1.71
(m, 5H), 3.76-3.78 (m, 1H), 6.02 (br s, disappeared on treatment
with D2O, 1H), 6.76-6.80 (m, 2H), 7.54-7.56 (m, 1H), 11.30 ppm
(br s, disappeared on treatment with D2O, 1H). IR: ν 1602, 3285
cm-1. Anal. Calcd (C13H20N2O (220.31)) C, H, N.
N-Methyl,N-(propargyl),N-(pyrrol-2-ylmethyl)amine (4). This
N-Propargyl-1H-pyrrole-2-carboxamide (32). It was synthe-
sized as 8 using propargylamine. Yield 82%, mp 110-112 °C (from
ethanol). 1H NMR (DMSO-d6): δ 2.36 (t, J ) 4.99 Hz, 1H), 4.00-
4.02 (m, 2H), 6.01-6.03 (m, 1H), 6.71-6.73 (m, 1H), 6.74-6.76
compound was synthesized as we previously reported.11
N-Methyl,N-(benzyl),N-(pyrrol-2-ylmethyl)amine (7). This
compound was synthesized as we previously reported.11

New Pyrrole Inhibitors of Monoamine Oxidase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 929
N-Methyl,N-(benzy),N-(1-methylpyrrol-2-ylmethyl)amine (19).
This compound was synthesized as we previously reported.11
N-Methyl,N-(3-phenylpropyl),N-(pyrrol-2-ylmethyl)amine (22).
It was synthesized as 21 using N-methyl,N-(3-phenylpropyl)amine.
with D2O, 1H), 5.93-5.95 (m, 2H), 6.66-6.69 (m, 1H), 7.18-
7.22 (m, 3H), 77.25-7.33 (m, 2H), 10.71 ppm (br s, disappeared
on treatment with D2O, 1H); IR: ν 3314 cm-1. Anal. Calcd
(C13H16N2 (200.28)) C, H, N.
1
Yield 9%, oil. H NMR (CDCl3): δ 1.78-1.86 (q, J ) 7.55 Hz,
N-(Propargyl),N-(pyrrol-2-ylmethyl)amine (35). To a solution
of pyrrole-2-carboxaldehyde (1.0 g, 0.01 mol) in THF (62.5 mL)
was added propargylamine (2.75 g, 3.43 mL, 0.05 mol). The
reaction mixture was stirred at room temperature for 24 h over
molecular sieves 3 Å and filtered. Collected solvent was evaporated
to give N-(propargyl),N-(pyrrol-2-ylmethylene)amine (1.1 g, 85%)
as a yellow oil, which was used without further purification. Sodiun
borohydride (0.42 g, 0.0011 mol) was added to an ice-cooled
solution of the latter amine (1.32 g, 0.01 mol) in methanol (73 mL).
The reaction was stirred at room temperature for 45 min. The
solvent was distilled, water was added, and the mixture was
extracted with ethyl acetate. The organic layer was separated,
washed with brine, and dried. The solvent was evaporated to afford
a brownish oil that solidified on standing. The crude product was
purified by alumina column chromatography (ethyl acetate as
eluent) to afford 20, yield 52%, mp 50 °C (from cyclohexane). 1H
NMR (DMSO-d6): δ 3.06 (t, J ) 4.7 Hz, 1H), 3.24 (d, J ) 4.81
Hz, 2H), 3.65 (s, 2H), 4.12 (br s, disappeared with treatment with
D2O, 1H), 5.86-5.90 (m, 2H), 6.60-6.61 (m, 1H), 10.56 ppm (br
s, disappeared on treatment with D2O, 1H); IR: ν 2117, 3176, 3250,
3272 cm-1. Anal. Calcd (C8H10N2 (134.18)) C, H, N.
2H), 2.18 (s, 3H), 2.40 (t, J ) 7.36 Hz, 2H), 2.62 (t, J ) 7.71 Hz,
2H), 3.49 (s, 2H), 6.00 (s, 1H), 6.11-6.13 (m, 1H), 6.71-6.73
(m, 1H), 7.17-7.21 (m, 3H), 7.25-7.30 (m, 2H), 8.55 ppm (br s,
disappeared on treatment with D2O, 1H). IR: ν 3379 cm-1. Anal.
Calcd (C15H20N2 (228.34)) C, H, N.
N-Methyl,N-(3-phenylbutyl),N-(pyrrol-2-ylmethyl)amine (23).
It was synthesized as 21 using N-methyl,N-(3-phenylbutyl)amine.
1
Yield 6%, oil. H NMR (CDCl3): δ 1.52-1.60 (m, 4H), 2.21 (s,
3H), 2.42 (t, J ) 7.35 Hz, 2H), 2.59 (t, J ) 7.29 Hz, 2H), 3.54 (s,
2H), 6.02 (s, 1H), 6.10-6.11 (m, 1H), 6.73-6.74 (m, 1H), 7.13-
7.19 (m, 3H), 7.25-7.28 (m, 2H), 8.98 ppm (br s, disappeared on
treatment with D2O, 1H). IR: ν 3339 cm-1. Anal. Calcd (C16H22N2
(242.37)) C, H, N.
(R,S)-N-Methyl,N-(R-phenylethyl),N-(pyrrol-2-ylmethyl)-
amine (26). It was synthesized as 21 using (R,S)-N-methyl,N-(R-
1
phenylethyl)amine. Yield 14%, oil. H NMR (CDCl3): δ 1.40 (d,
J ) 6.80 Hz, 3H), 2.16 (s, 3H), 3.40 (d, J ) 13.84 Hz, 1H), 3.55
(d, J ) 13.84 Hz, 1H), 3.63 (q, J ) 6.79 Hz, 1H), 5.99 (s, 1H),
6.11-6.13 (m, 1H), 6.72-8.74 (m, 1H), 7.24-7.37 (m, 5H), 8.23
ppm (br s, disappeared on treatment with D2O, 1H). IR: ν 3436
cm-1. Anal. Calcd (C14H18N2 (214.31)) C, H, N.
N-(2-Benzyl),N-(1-methylpyrrol-2-ylmethyl)amine (18). A so-
lution of 6 N HCl in MeOH (1:1) (15 mL) and benzylamine (0.98
g, 0.0016 mol) was added to a mixture of 1-methylpyrrole-2-
carboxaldehyde (1.0 g, 0.0092 mol), THF (114 mL), and methanol
(114 mL). After stirring for 30 min at 0 °C, sodium cyanoboro-
hydride (0.678 g, 0.0108 mol) was added, then the reaction mixture
was stirred overnight while the reaction temperature was warmed
to room temperature. The solvent was evaporated, water was added,
and the mixture was extracted with ethyl acetate. The organic layer
was separated, washed with brine, and dried. The solvent was
evaporated to afford a brownish oil that solidified on standing. The
crude product was purified by alumina gel column chromatography
(chloroform as eluent). Yield 62%, mp 215-223 °C (from
cyclohexane). 1H NMR (CDCl3): δ 3.63 (s, 3H), 3.73 (s, 2H), 3.82
(s, 2H), 6.03-6.06 (m, 2H), 6.58 (t, J ) 4.21 Hz, 1H), 7.25-7.36
(m, 5H), 8.22 ppm (br s, disappeared on treatment with D2O, 1H).
IR: ν 3318, cm-1. Anal. Calcd (C13H16N2 (200.28)) C, H, N.
Biology. Mitochondria Preparation. Mitochondria were pre-
pared according to Basford.13 The following reagents were used.
Reagents: medium A contained 0.4 M sucrose, 0.001 EDTA, 0.02%
PES or heparin and pH value was adjusted to 6.8-7.0 by addition
of KOH; medium F made was made up of the medium A to which
Ficoll was added to a final concentration of 8%. Calf or beef brains
were removed from the animals within 5-10 min after their death.
The brains were immediately placed in cold medium A and then
stored on ice, to be transported to the laboratory. In a cold room,
at 5 °C, the cerebral hemispheres were removed from the brains
and the meninges were taken up with forceps. The gray matter was
scraped from the cortices using a dull spatula. Two brains yield
corresponded to about 100 g of wet tissue, which was homogenized
in 2 mL Medium A (2 mL/g of wet tissue). The homogenate was
kept at pH 7.0 by adding of some drops of Tris buffer 2 M and 1
mg of ꢀ-aminocaproic acid/g of tissue. Then the mixture was stirred
at 0-4 °C for 15 min. The suspension was diluted with medium A
(20 mL/g of the original tissue) and centrifuged twice, first at 184
g for 20 min and then without transferring of the supernatant at
1153 g for others for 20 min. The residue R1 was discarded, while
the supernatant S1 was centrifuged at 12 000 g for 15 min, to yield
a crude mitochondria pellet R2, the supernatant S2 was discarded.
The fraction R2 was dissolved in medium F (6 mL/g of original
tissue), gently homogenized, and centrifuged at 12 000 g for 30
min. The resulting mitochondria fraction R3 was washed using 4
mL of medium A/g of original tissue and again centrifuged at
12 000 g for 15 min to yield the final mitochondria fraction R4,
which was homogenized in potassium phosphate buffer pH 7.4,
(R)-N-(R-Cyclohexylethyl)-N-methyl,N-(pyrrol-2-ylmethyl)-
amine (31). It was synthesized as 21 using (R)-N-(R-cyclohexyl-
ethyl)-N-methylamine. Yield 15%, oil. 1H NMR (CDCl3): δ 0.85-
0.93 (m, 6H), 1.16-1.28 (m, 7H), 2.10 (s, 3H), 2.29-2.31 (m,
1H), 2.60-2.63 (m, 1H), 3.49 (d, J ) 12 Hz, 1H), 3.62 (d, J )
11.5 Hz, 1H), 5.97-5.99 (m, 1H), 6.12-6.14 (m, 1H), 6.73-6.74
(m, 1H), 8.47 ppm (br s, disappeared on treatment with D2O, 1H).
Anal. Calcd (C14H24N2 (220.36)) C, H, N.
N-Methyl,N-(propargyl),N-(1-methylpyrrol-2-ylmethyl)-
amine (36). It was synthesized as 21 using 1-methyl-1H-pyrrole
and N-methylpropargylamine. Yield 5%, mp 148-155 °C (from
1
benzene). H NMR (CDCl3): δ 2.25 (t, J ) 4.7 Hz, 1H), 2.27 (d,
J ) 4.7 Hz, 2H), 2.29 (s, 3H), 3.49 (s, 2H), 3.63 (s, 3H), 6.01-
6.04 (m, 2H), 6.57-6.60 ppm (m, 1H). IR: ν 3094, 2725, 2116
cm-1. Anal. Calcd (C10H14N2 (162.23)) C, H, N.
N-(Benzyl),N-(pyrrol-2-ylmethyl)amine (17). A mixture of
pyrrole-2-carboxaldehyde (1.0 g, 0.01 mol) and benzylamine (2.14
g, 2.18 mL, 0.02 mol) was heated at 50 °C for 1 h. After cooling,
the reaction mixture was evaporated and the crude product was
triturated with n-hexane to give N-benzyl-N-(1H-pyrrol-2-ylmeth-
ylene)amine as a white solid (1.7 g, 92%).27 A solution of the latter
compound (0.5 g, 0.0027 mol) in THF (4.4 mL) and isopropanol
(13.2 mL) was added to a mixture of sodium cyanoborohydride
(0.19 g, 0.003 mol) and 1 N HCl in anhydrous diethylether (4 mL)
at 0 °C. The reaction was stirred for 10 min at room temperature,
then made basic with potassium carbonate saturated solution, and
extracted with ethyl acetate. The organic layer was separated,
washed with brine, and dried. The solvent was evaporated to give
a residue that was purified by silica gel column chromatography
(chloroform as eluent). Yield 44%, mp 95-98 °C (toluene/
cyclohexane). 1H NMR (DMSO-d6): δ 3.80 (s, 2H), 3.81 (s, 2H),
4.37 (br s, disappeared on treatment with D2O, 1H), 5.94-6.00
(m, 2H), 6.71 (s, 1H), 7.26-7.42 (m, 5H), 10.81 ppm (br s,
disappeared on treatment with D2O, 1H). IR: ν 3375, 3297 cm-1
Anal. Calcd (C12H14N2 (186.26)) C, H, N.
.
N-(2-Phenylethyl),N-(pyrrol-2-ylmethyl)amine (20). It was
synthesized as 17 using 2-phenylethylamine. The intermediate
reaction gave N-(2-phenylethyl)-N-(1H-pyrrol-2-ylmethylene)amine,
1
yield 79%, mp 98-101 °C (cyclohexane). H NMR (DMSO-d6):
δ 2.70-2.87 (m, 4H), 2.77 (s, 2H), 3.99 (br s, disappeared on
treatment with D2O, 1H), 5.93-5.95 (m, 2H), 6.65-6.69 (m, 1H),
7.06-7.33 (m, 5H), 10.71 ppm (br s, disappeared on treatment with
D2O, 1H). IR: ν 2425, 3314 cm-1. Sodium cyanoborohydride
1
reduction afforded 20, yield 27%, oil. H NMR (DMSO-d6): δ
2.80 (m, 4H), 3.78 (s, 2H), 3.99 (br s, disappeared with treatment

930 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
La Regina et al.
0.25 M. The yield of mitochondria protein obtained was between
100 and 140 mg per 50 g wet weight of the original tissue.
Activity Assay. Mono amine oxidase activity was determined
using kinuramine as a substrate, at four different final concentrations
ranging from 5 µM to 0.1 mM, by a sensitive fluorometric assay
according to Matsumoto et al.14 In all assays the incubation mixtures
contained: potassium phosphate buffer pH 7.4, mitochondria (6
mg/mL), drug solutions in DMSO, added to the reaction mixture
at a final concentration ranging from 0 to 10-3 µM. Solutions were
preincubated for 30 min before adding the substrate and then
incubated for others 30 min. The inhibitory activities of both
MAO-A and MAO-B separately were determined after incubation
of the mitochondrial fractions for 30 min at 38 °C, in the presence
of the specific inhibitor (L-deprenyl 1 µM to estimate the MAO A
activity or clorgyline 1 µM to assay the isoform B). It was taken
into account that MAO-A is irreversibly inhibited by low concen-
tration of clorgyline but is unaffected by low concentration of
L-deprenyl, utilized contrary in the form MAO-B. The addition of
percloric acid ended the reaction. Then the samples were centrifuged
at 10 000 g for 5 min, and the supernatant was added to 2.7 mL of
1 N NaOH. Fluorometric measurements were recorded at λexc 317
nm and λem 393 nm, using a Perkin-Elmer LS 50B spectrofluo-
rometer. The protein concentration was determined according to
Goa.28 Dixon plot were used to estimate the inhibition constant
(Ki) of the inhibitors. Data are the means of three or more
experiments each performed in duplicate.
Computational Chemistry. Molecular modeling and graphics
manipulations were performed using the SYBYL software package
(Sybyl Molecular Modeling System, version 7.0, Tripos Inc., St.
Louis, MO), running it on a Silicon Graphics Tezro R16000
workstation. Model building of compounds 7 and 18 was ac-
complished with the TRIPOS force field29 available within SYBYL.
Point charges for the inhibitors were calculated using the Gasteiger-
Marsili method.30 Energy minimizations of the 7/MAO-A, 7/MAO-
B, 18/MAO-A, and 18/MAO-B complexes were realized by
employing the INSIGHT II/DISCOVER software packages (Insight
II Molecular Modeling Package and Discover 2.2000 Simulation
Package, Accelrys Inc., San Diego, CA), selecting the CVFF force
field.31
Docking Simulations. Docking was performed using AutoDock
3.0.517,18 and GOLD 2.219,20 software packages. AutoDock combines
a rapid energy evaluation through precalculated grids of affinity
potentials with a variety of search algorithms to find suitable binding
positions for a ligand on a given protein. While the protein is
required to be rigid, the Autodock allows torsional flexibility in
the ligand. GOLD is an automated ligand-docking program that
uses a genetic algorithm to explore the full range of ligand
conformational flexibility. Moreover, it permits some protein
conformational freedom in the sense that torsion angles of serine,
threonine, and tyrosine hydroxyl groups as well as lysine amine
groups are optimized by the search algorithm during the posing.
These groups are allowed to rotate freely to favor intramolecular
(with other residues of the protein) and intermolecular (with the
ligand trial solution) H-bond formation. GOLD requires a user-
defined binding site. It searches for a cavity within the defined
area, and considers all the solvent-accessible atoms in that area as
active-site atoms. On the basis of the GOLD score, for each
molecule a bound conformation with high score was considered as
the best bound conformation. The score function that was imple-
mented in GOLD consisted basically of H-bonding, complex
energy, and ligand internal energy terms. A population of possible
docked orientations of the ligand is set up at random. Each member
of the population is encoded as a “chromosome”, which contains
information about the mapping of ligand H-bond atoms onto
(complementary) protein H-bond atoms, mapping of hydrophobic
points of the ligand onto protein hydrophobic points, and the
conformation around flexible ligand bonds and protein OH groups.
A number of parameters control the precise operation of the genetic
algorithm.
SYBYL fragment library. The ligands were modeled in their
protonated form. Geometry optimizations were carried out with the
SYBYL/MAXIMIN2 minimizer by applying the BFGS (Broyden,
Fletcher, Goldfarb, and Shannon) algorithm32 and setting a rms
gradient of the forces acting on each atom of 0.001 kcal mol-1
Å-1 as the convergence criterion. Partial atomic charges were
assigned by using the Gasteiger-Marsili formalism.
Protein Setup. The crystal structures of MAO-A (entry code:
1O5W)15 and of MAO-B (entry code: 1GOS)16 recovered from
Brookhaven Protein Database33 were used. The structures were set
up for docking as follows: polar hydrogens were added by using
the BIOPOLYMERS module within the SYBYL program (residues
Arg, Lys, Glu, and Asp were considered ionized, while all His were
considered to be neutral by default), Kollman united atom partial
charges were assigned, and all waters were removed.
Original PDB structures were subjected to a preliminary con-
strained energy minimization of those residues out of a radius of
15 Å from the N5 of the isoalloxazine ring in order to restore the
natural planarity of the isoalloxazine FAD ring and relax the active
site aminoacids. In the resulting energy minimized structures, the
covalent ligands (pargyline for 1GOS and clorogyline for 1O5W)
were removed and used as starting models for docking simulations.
AutoDock Docking. Docking of compounds 7 and 18 to both
MAO-A and MAO-B was carried out using the empirical freeenergy
function and the Lamarckian genetic algorithm, applying a standard
protocol with an initial population of 50 randomly placed individu-
als, a maximum number of 1.5 × 106 energy evaluations, a mutation
rate of 0.02, a crossover rate of 0.80, and an elitism value of 1.
Proportional selection was used, where the average of the worst
energy was calculated over a window of the previous 10 genera-
tions. For the local search, the so-called pseudo Solis and Wets
algorithm was applied by using a maximum of 300 iterations. The
probability of performing local search on an individual in the
population was 0.06, and the maximum number of consecutive
successes or failures before doubling or halving the local search
step size was 4.
Fifty independent docking runs were carried out for each ligand.
Results differing by less than 1 Å in positional rmsd were clustered
together and represented by the result with the most favorable free
energy of binding (∆Gbind). Finally, the compounds were set up
for docking with the help of AutoTors, the main purpose of which
is to define the torsional degrees of freedom to be considered during
the docking process. All torsion angles for each compound were
considered flexible. Solvation parameters were added to the final
protein file by using the ADDSOL utility of AutoDock. The grid
maps representing the proteins in the actual docking process were
calculated with AutoGrid. The grids (one for each atom type in
the ligand plus one for electrostatic interactions) were chosen to
be sufficiently large to include not only the active site but also
significant portions of the surrounding surface. The dimensions of
the grids were thus 60 Å × 60 Å × 60 Å, with a spacing of 0.375
Å between the grid points. The grid center was centered on the
FAD N5 atom using the original PDB models without their covalent
ligands.
GOLD Docking. An active site of radius 15 Å was defined
considering the phenolic oxygen atom of Tyr435 and Tyr444 as
the center of the MAO-B and MAO-A, respectively. Fifty inde-
pendent docking runs were performed for each docking experiment.
All docking runs were carried out using standard default settings
with a population size of 100, a maximum number of 100 000
operations, and a mutation and crossover rate of 95. The best
generated 10 solutions of each ligand were ranked according to
their fitness scores calculated by the GOLD Chem-Score function.
Molecular Dynamics Simulations. Refinement of the inhibitor/
enzyme complexes was achieved by energy minimization with the
CVFF force field, permitting only the ligand and the side chain
atoms of the protein within a radius of 10 Å around the ligand to
relax. Calculations were performed by 3000 steps of steepest
descents and 2000 steps of conjugate gradients (down to a maximal
atomic rmsd of 10.0 and 0.01 kcal/Å, respectively). The geometry-
optimized complexes were then used as the starting point for
Ligand Setup. The structures of the ligands 7 and 18 were
constructed using standard bond lengths and bond angles of the

New Pyrrole Inhibitors of Monoamine Oxidase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 931
Lamarckian Genetic Algorithm and an Empirical Binding Free Energy
Function. J. Comput. Chem. 1998, 19, 1639-1662.
subsequent 400 ps MD simulation, during which the protein
backbone atoms were constrained as done in the previous step. A
distance-dependent dielectric of 40 was applied. The cutoff radius
for nonbonded interactions was 12 Å, with a secondary cutoff radius
of 15 Å. The molecular system was allowed to equilibrate to 300
K for 100 ps and then kept at this temperature throughout the 300
ps of production run, with a step length of 1 fs. Coordinates were
saved every 1 ps and used to calculate the averaged structures from
the simulations. The averaged structures over the last 300 ps of
the simulations were energy minimized as previously described and
stored as the final conformation of the ligand-enzyme complexes.
(18) Goodsell, D. S.; Morris, G. M.; Olson, A. J. Automated docking of
flexible ligands: applications of AutoDock. J. Mol. Recognit. 1996,
9, 1-5.
(19) GOLD 2.2; CCDC Software Limited: Cambridge, UK, 2004.
(20) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
Development and validation of a genetic algorithm for flexible
docking. J. Mol. Biol. 1997, 267, 727-748.
(21) Wang, R.; Lu, Y.; Wang, S. Comparative evaluation of 11 scoring
functions for molecular docking. J. Med. Chem. 2003, 46, 2287-
2303.
(22) Database searching (SWISS-PROT), sequence alignment, and analysis
of rat, bovine and human MAO sequences were carried out using
FASTA (Pearson, W. R. Proc. Natl. Acad. Sci. U.S.A. 1988, 85,
2444-2448.) and BLAST programs (Wang, S.; Pak, Y. J. Phys.
Chem. B. 2000, 104, 354-359.).
(23) Crystallographic analysis of human MAO-B in complex with several
inhibitors (PDB codes: 1S2Q, 1S2Y, 1S3E, and 1S3B) revealed the
presence of two cavities inside the protein: the “substrate” cavity
(in front of the flavin ring) and the “entrance” cavity (a channel
towards the catalytic site). The side chain of Ile199 is as a key
structural element that opens and closes the connection between the
two cavities. In the closed-conformation, Ile199 physically separates
the two cavities. In the presence of bulky ligands this residue adopts
an open conformation that allows the ligand to reach the susbtrate
cavity. We used the crystal structure of the human MAO-B/pargyline
complex, which is characterized by an Ile199 open conformation.
Ile199 is conserved in all the known MAO-B sequences, with the
only exception of bovine MAO-B, where it is replaced with Phe.
Edmondson et al. (Huba`lek, F.; Binda, C.; Khalil, A.; Li, M.; Mattevi,
A.; Castagnoli, N.; Edmondson, D. E. Demonstration of isoleucine
199 as a strctural determinant for the selective inhibition of human
monoamine oxidase B specific reversible inhibitors. J. Biol. Chem.
2005, 280, 15761-15766.) demonstrated that with rasagiline and
isatin the side chain of Phe199 adopted almost identical conformations
(instead of the open/closed-conformations of Ile199), without chang-
ing in either kinetics or binding affinity.
(24) Binda, C.; Li, M.; Huba`lek, F.; Restelli, N.; Edmondson, D. E.;
Mattevi, A. Insights into the mode of inhibition of human mitochon-
drial monoamine oxidase B from high-resolution crystal structures.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9750-9755.
(25) De Colibus, L.; Li, M.; Binda, C.; Lustig, A.; Edmondson, D. E.;
Mattevi, A. Three-dimensional structure of human monoamine
oxidase A (MAO A): relation to the structures of rat MAO A and
human MAO B. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 12684-
12689.
Acknowledgment. This work was partially supported by the
Italian MIUR (Ministero dell’Istruzione, dell’Universita` e della
Ricerca) and by funds MIUR-PRIN 2005 (Cofin) (EA).
Supporting Information Available: Elemental analyses of new
derivatives 8, 11-18, 20-32, and 34-36. This material is available
free of charge via Internet at http://pubs.acs.org.
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