Monoamine oxidase isoform-dependent tautomeric influence in the recognition of 3,5-diaryl pyrazole inhibitors

Article abstract of DOI:10.1021/jm060868l

A series of 3,5-diaryl pyrazoles were prepared and assayed for their ability to inhibit reversibly monoamine oxidase-A (MAO-A) and monoamine oxidase B (MAO-B). Several compounds show inhibitory activity with concentration values in the nanomolar range. A computational work was carried out on the two most selective inhibitors that have tautomeric pyrazole forms. The binding free energies of these compounds for each MAO isoform were influenced by the tautomeric equilibria.

Full text of DOI:10.1021/jm060868l

J. Med. Chem. 2007, 50, 425-428
425
Letters
Table 1. Chemical and Physical Data and Anti-MAO Activity of
Pyrazole 3a-ka
Monoamine Oxidase Isoform-Dependent
Tautomeric Influence in the Recognition of
3,5-Diaryl Pyrazole Inhibitors
mp
(°C)
yield
pIC50
pIC50
pSIb
cmpd
R
R1
(%) MAO-A MAO-B selectivity
3a
3b
3c
3d
3e
3f
3g
3h
H
H
H
4-Cl
4-Cl
4-F
4-CH3
4-CH3 4-Cl
4-CH3 4-F
H
193-5 24
217-9 35
196-8 25
240-2 35
220-2 35
182-4 29
193-5 43
211-3 39
215-7 27
9.00
6.52
5.39
8.69
8.52
5.20
5.82
5.52
5.64
7.85
4.94
6.42
4.42
8.00
8.22
7.52
5.22
7.39
4.00
9.00
8.05
7.00
6.35
2.00
4.82
6.00
1.00
-1.70
-2.13
3.47
1.13
1.20
-3.18
-2.53
-1.36
1.50
2.94
1.60
Franco Chimenti,† Rossella Fioravanti,*,† Adriana Bolasco,*,†
Fedele Manna,† Paola Chimenti,† Daniela Secci,*,†
Olivia Befani,‡ Paola Turini,‡ Francesco Ortuso,§ and
Stefano Alcaro§
4-Cl
4-F
4-Cl
4-F
4-F
H
Dip. di Studi di Chimica e Tecnologia delle Sostanze
Biologicamente AttiVe, UniVersita` di Roma “La Sapienza”, P.le
Aldo Moro, 5, 00185 Rome, Italy, Dip. di Scienze Biochimiche “A.
Rossi Fanelli” and Centro di Biologia Molecolare del CNR,
UniVersita` di Roma “La Sapienza”, P.le Aldo Moro, 5,
00185 Rome, Italy, and Dip. di Scienze Farmacobiologiche,
UniVersita` di Catanzaro “Magna Græcia”, Complesso Nin`ı
Barbieri, 88021 Catanzaro, Italy
3j
3k
4-CH3 4-CH3 171-4 35
MCLc
TOLd
SELe
-1.58
a The data represent mean values of at least three separate experiments.
b pSI ) log selectivity index ) pIC50 (MAO-A) - pIC50 (MAO-B).
c Moclobemide. d Toloxatone. e Selegiline.
ReceiVed July 24, 2006
Abstract: A series of 3,5-diaryl pyrazoles were prepared and assayed
for their ability to inhibit reversibly monoamine oxidase-A (MAO-A)
and monoamine oxidase B (MAO-B). Several compounds show
inhibitory activity with concentration values in the nanomolar range.
A computational work was carried out on the two most selective
inhibitors that have tautomeric pyrazole forms. The binding free energies
of these compounds for each MAO isoform were influenced by the
tautomeric equilibria.
Figure 1. General formula of 3,5-diaryl pyrazole.
The B isoform is known to be important, especially after the
discovery that MAO-B oxidizes a Parkinsonism-producing
neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, to
form an active neurotoxin, 1-methyl-4-phenylpyridinium ion.7
Because L-deprenyl, a representative MAO-B inhibitor, may
have neuroprotective activity especially for dopamine neurons,
it is used in treatment of Parkinson’s disease. MAO-B inhibitors
are also currently used in clinical trials in the treatment of
Alzheimer’s disease because an increased level of MAO-B has
been detected in the plaque-associated astrocytes of brains from
Alzheimer’s patients.8
Monoamine oxidases (MAO, EC 1.4.3.4), widely distributed
in all living organisms, are FAD-containing enzymes, tightly
bound to the outer mitochondrial membrane. In mammals, two
different isoforms of MAOs are present, namely, MAO-A and
MAO-B. MAO-A is located predominantly in catecholaminergic
neurons, while MAO-B is present in serotonergic neurons and
glia.1,2 Both isoforms are characterized by specific substrates
and inhibitors. MAO-A has a higher affinity for serotonin and
norepinephrine and is more sensitive to inhibition by clorgyline,
while MAO-B preferentially deaminates â-phenylethylamines
and benzylamine and is sensitive to low concentrations of
L-deprenyl.3,4 Dopamine, tyramine, and tryptamine are common
substrates for both MAOs.
Efforts have been addressed to the design, synthesis, and study
of new reversible and selective MAO inhibitors, leading to
compounds such as toloxatone5 and moclobemide.6 These
compounds selectively block MAO-A and do not form a
covalent bond with the MAO enzyme, acting via a reversible
inhibitory mechanism. These MAO-A inhibitors retain anti-
depressant effects in animal models and are devoid of the severe
food and drug incompatibilities induced by the first generation
of MAO inhibitors.
Different families of heterocycles containing two or four
nitrogen atoms have been used as scaffolds for synthesizing
reversible and selective monoamino oxidase inhibitors (MAO-
Is-A and MAO-Is-B). In particular, Wouters et al.9 studied
oxadiazolone, tetrazole, oxadiazinone, and indenopyridazine
derivatives as potent, reversible, and selective MAO-B inhibi-
tors. Altomare et al. studied the MAO inhibitory activity of
isoquinoline derivatives and condensed pyridazines.10,11 From
the activities of diazine derivatives as MAO inhibitors, it
emerges that most of the condensed pyridazines are reversible
MAO-B inhibitors, with none or little effect on MAO-A, while
the condensed pyrimidines are reversible inhibitors endowed
with an appreciable selectivity toward MAO-A. Substituents on
the diazine nucleus modulate the inhibitory activity. In contrast,
the nature of the ring(s) condensed with diazine appears to affect
selectivity toward MAO-A and MAO-B significantly.
* To whom correspondence should be addressed. Tel.: +39-06-
49913975. Fax: +39-06-49913763. E.mail: rossella.fioravanti@uniroma1.it.
(R.F.); adriana.bolasco@uniroma1.it (A.B.); daniela.secci@uniroma1.it
(D.S.).
† Dip. di Studi di Chimica e Tecnologia delle Sostanze Biologicamente
Attiv, Universita` di Roma “La Sapienza”.
With this in mind, and continuing in our study of reversible
and selective pyrazole derivatives as inhibitors of MAO-A and
MAO-B isoforms,12 we here report the synthesis of a new series
of compounds featuring the pyrazole ring, to verify if the planar
conformation affects the inhibitory activity. The 3,5-diphenyl
‡ Dip. di Scienze Biochimiche “A. Rossi Fanelli” and Centro di Biologia
Molecolare del CNR, Universita` di Roma “La Sapienza”.
§ Dip. di Scienze Farmacobiologiche, Universita` di Catanzaro “Magna
Græcia”.
10.1021/jm060868l CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/04/2007

426 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3
Letters
Scheme 1a
a Reagents and conditions: (i) Ba(OH)2, EtOH 96%, 30 °C; (ii) H2O2 35%, EtOH dry, K2CO3; (iii) N2H4, p-toluenesulfonic acid, xylenes, refluxed.
pyrazole derivatives 3a-k, listed in Table 1 and shown in Figure
1, were prepared according to the methods shown in Scheme
1.
Treatment of an appropriate acetophenone with benzaldehydes
gave R,â-unsaturated ketones (chalcones) 1a-k by a typical
Figure 2. Tautomeric forms of pyrazole.
base-catalyzed aldol condensation13 in 50-80% yields (Scheme
1). The reaction of 2-propen-1-ones 1a-k with an excess of
aqueous hydrogen peroxide in ethanol gave the corresponding
chalcone epoxide 2a-k; the reaction of suitable epoxide with
Taking into account that 1H-pyrazoles can exist in two
tautomeric forms a and b (Figure 2) and that when the
substituents at the 4-position of the 3- and 5-aryl moieties are
hydrazine monohydrate 98% and p-toluenesulfonic acid mono-
different (R * R1), the two tautomers are nonequivalent,15,16
hydrate in boiling ethanol gave the corresponding 3,5-diphenyl
we highlighted the effect of tautomerism of the most active and
pyrazoles 3a-k.14 The structures of compounds 3a-k were
selective derivative 3g on the interaction with the active site of
1
confirmed by IR, mass spectra, and H NMR analyses.
the enzyme. The same procedure was repeated also for
Pyrazoles 3a-k were evaluated for their ability to inhibit
MAO-A and MAO-B. The pIC50 values against the two MAO
isoforms and the A-selectivity are summarized in Table 1. The
A-selectivity has been expressed as pSI and represents the log
selectivity index calculated as pIC50 (MAO-A) - pIC50 (MAO-
B). The positive value means a MAOI-A and the negative one
means a MAOI-B.
compound 3h, which also exists in two tautomeric forms and
shows good inhibitory activity against MAO-B associated with
good selectivity.
Taking into account the large electronic delocalization of these
3,5-diaryl pyrazole derivatives, both a and b tautomeric isomers
of 3g and 3h were built in planar conformation and energy
evaluated by ab initio techniques.17
All the tested derivatives showed a reversible mode of action,
because dialysis for 24 h in a cold room against a 0.1 M
potassium phosphate buffer (pH 7.2) could restore 90 to 100%
of the enzyme activity.
The computational study was carried out following our
previously reported approaches,12 focusing on the most active
and selective inhibitors, 3g and 3h, which can undergo the
tautomeric equilibrium shown in Figure 2.
As reported in Table 1, some derivatives of 3 show inhibitory
activity with concentration values in the nanomolar range. The
best MAO-A inhibitors are 3a, 3d, and 3e, with pIC50 values
in the nanomolar range (8.52-9.00), while the best MAO-B
inhibitors are 3a, 3b, 3g, and 3h, with pIC50 values in the
nanomolar range (8.00-9.00). Compound 3a shows good
inhibitory activity against MAO-A and MAO-B but low
selectivity (pIC50 MAO-A ) 9.00, pIC50 MAO-B ) 8.00, and
pSI ) 1.00). Compounds 3b and 3c, which bear a halogen atom
on the 4-position of the 3,5-aryl moieties, show good inhibitory
activity against MAO-B associated with good inhibitory selec-
tivity. Compound 3d, which bears the chloro substituent in the
4-position of the aryl moieties, shows good inhibitory activity
against MAO-A associated with high selectivity.
Compound 3e, which bears fluorine and chlorine atoms on
the 4-position of the 3,5-aryl moieties, and compound 3f, which
bears the fluorine atoms on the 4-position of the aryl moieties,
show lower inhibitory activity on both isoforms.
Finally, compounds 3g (R ) 4-CH3, R1 ) H), 3h (R )
4-CH3, R1 ) 4-Cl), and 3j (R ) 4-CH3, R1 ) 4-F) show good
inhibitory activity against MAO-B associated with high selectiv-
ity.
Pretreatment of the enzyme models is summarized in the
Supporting Information.
Thanks to the extreme simplicity of the compound in terms
of free rotatable bonds, we could skip the conformational search.
Energy optimization was carried out by the DFT B3LYP method
with the 6-311G**++ basis set. The internal energy difference
between the two optimized isomers proved to be lower than
0.08 kcal/mol, demonstrating that both are significantly popu-
lated at room temperature. Therefore, both a and b tautomers
were included in the docking experiments using crystallographic
MAO-A and MAO-B models,18,19 available in the Protein Data
Bank (PDB) as targets.20 After pretreatment of the isoform
models, the GLIDE approach21 was used to generate configura-
tions of the two tautomers in the enzymatic clefts using a box
of about 110 000 Å3 centered on the FAD N5 atom. The most
stable 10 GLIDE poses of the 5000 generated were fully energy
minimized with the AMBER* united atom force field in GB/
SA water22,23 and submitted to the MM-GBSA binding mode
analysis.24
Each energy minimum pose was submitted to a refinement
based on a 500 ps molecular dynamics run carried out at 300
°K with a 1.5 fs time step. Fifty conformations were regularly
collected each 10 ps and energy minimized, in the same force
field and solvation conditions, to obtain a final ensemble. The
thermodynamic evaluation was performed on these optimized
Among all, compound 3g is the most interesting inhibitor,
with a good inhibitory activity on the MAO-B isoform (pIC50
MAO-B ) 9.00) and a high selectivity (pSI ) -3.18).

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3 427
Table 2. Thermodynamic Details of 3g and 3h Tautomers (Tau) into
the Enzymatic Clefts: Relative Internal Energies (RIE) and Boltzmann
Population (Prob %)
MAO-A
prob %
MAO-B
prob %
cmpd
Tau
RIE
RIE
3g
a
b
a
b
74.5
0.0
0.0
0
100
100
0
84.6
0.0
33.0
0.0
0
100
0
3h
17.7
100
Table 3. Number of Optimized Configurations (nconf) and Binding
Free Energy (‚Gbind) in kcal/mol Computed at 300°K for Each 3g and
3h Tautomer (Tau) Complexed into the MAO-A and MAO-B Enzymatic
Clefts
MAO-A
‚Gbind
MAO-B
‚Gbind
cmpd
Tau
nconf
nconf
3g
a
b
a
b
39
41
43
44
-47.16
-43.69
-42.24
-35.21
45
44
43
49
-41.38
-46.60
-44.14
-44.34
Figure 3. Most probable and stable complex of 3g (tautomer b) and
MAO-B. Interacting residues of the active site are shown in labelled
polytubes, FAD in CPK rendering, the compound in blue carbon
polytube, and other aminoacids in ribbon. Hydrogen bonds are displayed
as dashed lines.
3h
configurations, considering the Boltzmann population at 300°K
of the enzyme-ligand complex computed unifying a and b
tautomer ensembles related to the same compound/isoform
complex. Such an approach allowed us to obtain a single
ensemble for each compound with respect to each enzyme. To
identify the role of each tautomer in the MAO recognition, the
probability was carried out using the all-energy contributions
of the 1:1 complexes (Table 2).
Further details of modeling protocol are reported in the
Supporting Information.
As shown in Table 2, a consistent effect in the tautomer
recognition was highlighted by the RIE and Boltzmann popula-
tion, with extreme values equal to 0 or 100%, derived from
global complex internal energies.
sional translator, Fellow of the Institute of Translation and
Interpreting of London and Member of AIIC (Association
Internationale des Interpreˆtes de Confe´rences - Geneva) for the
revision of the manuscript.
Supporting Information Available: 1H NMR spectral data and
elemental analyses of derivatives 3a-k. Molecular modeling details
about the computational protocol, one table, and seven figures. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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