Synthesis, stereochemical separation, and biological evaluation of selective inhibitors of human MAO-B: 1-(4-Arylthiazol-2-yl)-2-(3- methylcyclohexylidene)hydrazines

Article abstract of DOI:10.1021/jm100120s

Novel 1-(4-arylthiazol-2-yl)-2-(3-methylcyclohexylidene)hydrazine derivatives have been investigated for their ability to inhibit selectively the activity of the human B isoform of monoamine oxidase. These compounds were obtained as racemates and (R)-enantiomers by a stereoconservative synthetic pattern in high yield and enantiomeric excess. The (S)-enantiomers of the most active derivatives have been separated by enantioselective HPLC. All compounds showed selective activity against hMAO-B with IC₅₀ ranging between 21.90 and 0.018 μM.

Full text of DOI:10.1021/jm100120s

6516 J. Med. Chem. 2010, 53, 6516–6520
DOI: 10.1021/jm100120s
Synthesis, Stereochemical Separation, and Biological Evaluation of Selective Inhibitors of
Human MAO-B: 1-(4-Arylthiazol-2-yl)-2-(3-methylcyclohexylidene)hydrazines
Franco Chimenti,† Daniela Secci,*,† Adriana Bolasco,† Paola Chimenti,† Arianna Granese,† Simone Carradori,†
‡
‡
§
§
§
ꢀ~
Matilde Yanez, Francisco Orallo, M. Luisa Sanna, Bruno Gallinella, and Roberto Cirilli
†Dipartimento di Chimica e Tecnologie del Farmaco, Universitaꢁ degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy,
‡
´
Departamento de Farmacologıa and Instituto de Farmacia Industrial, Facultad de Farmacia, Universidad de Santiago de Compostela,
Campus Universitario Sur, E-15782 Santiago de Compostela (La Corun~a), Spain, and §Dipartimento del Farmaco,
Istituto Superiore di Sanitaꢁ, V.le Regina Elena 299, 00161 Roma, Italy
Received December 11, 2009
Novel 1-(4-arylthiazol-2-yl)-2-(3-methylcyclohexylidene)hydrazine derivatives have been investigated
for their ability to inhibit selectively the activity of the human B isoform of monoamine oxidase. These
compounds were obtained as racemates and (R)-enantiomers by a stereoconservative synthetic pattern
in high yield and enantiomeric excess. The (S)-enantiomers of the most active derivatives have been
separated by enantioselective HPLC. All compounds showed selective activity against hMAO-B with
IC50 ranging between 21.90 and 0.018 μM.
Introduction
a substrate entrance cavity (∼290 A3) connected to a larger
substrate-binding cavity (∼ 420 A3). Ile199 was identified as
an important structural element because it acts as a gate that
allows the accessibility to the second cavity. In the closed
conformation, Ile199 physically separates the two cavities,
whereas in the presence of bulky ligands it adopts an open
conformation. Binding of clinical inhibitors such as rasagiline
and (R)-deprenyl induces a midspan type of cavity pushing
Ile199 into the open conformation. Furthermore, the obser-
vation that hMAO-B levels in humans increase 4- to 5-fold on
aging2,7 represents a rationale for the involvement of hMAO-B
in age-related neurological disorders such as Parkinson’s
and Alzheimer’s diseases.8-10 Increased hMAO-B activity
would be expected to diminish dopamine concentration and
to release catalytic reaction products (H2O2). Dopamine has
been involved in (R)-synuclein aggregation implicated in the
etiology of Parkinson’s disease, while high levels of H2O2
in the cell promote apoptotic signaling events and the deve-
lopment of Parkinson’sdisease. Inhibition of hMAO-B allows
a sort of neuroprotection against this bioactivation. In
fact, agents that specifically limit the activity of hMAO-B
are likely to act as neuroprotectants.11 Indeed, treatment of
pre-Parkinson’s patients with selective hMAO-B inhibitors
has been shown to be effective in reducing the development of
this neurodegeneration. A disadvantage of the first selective
hMAO-B inhibitor, (R)-deprenyl, was its sympathomimetic
effect related toitschemical structure becauseitismetabolized
in vivo to methamphetamine compounds with sympatho-
mimetic activity. One advantage of rasagiline, therefore,
was that it was not an amphetamine derivative and showed
no sympathomimetic activity.12 Rasagiline [N-propargyl-1(R)-
aminoindan] is currently under development for the treat-
ment of Parkinson’s disease. It has been shown to have
neuroprotective properties and to be a potent, selective
inhibitor of hMAO-B. The (S)-enantiomer is a much less
potent (3 orders of magnitude) inhibitor of hMAO-B than
Monoamine oxidases A and B (EC 1.4.3.4) are outer
mitochondrial membrane bound enzymes involved in the
oxidative degradation of monoamine neurotransmitters and
xenobiotic arylalkylamines to aldehydes by using O2 as the
electron acceptor. To complete the catalytic cycle, the reduced
FADa cofactor reacts with O2 to generate oxidized flavin and
H2O2.1 All mammals contain MAO-A and MAO-B, which
are localized in different tissues. Human MAO-A (hMAO-A)
and human MAO-B (hMAO-B) are encoded by separate
genes and consist of different amino acid sequences that are
∼70% identical.2 The A isoform preferentially metabolizes
norepineprhine and 5-hydroxytryptamine and is inhibited by
clorgyline, whereas the B isoform prefers benzylamine as
substrate and is inhibited by (R)-deprenyl and rasagiline.
Tyramine and dopamine are equally metabolized by both
forms of the enzyme.3,4 The recent three-dimensional struc-
tures of hMAO-A5 and hMAO-B6 with their corresponding
inhibitors show overall similarity in their crystalline forms but
differ in oligomeric states and active site structures. In fact,
hMAO-B crystallizes as the dimer whereas hMAO-A crystal-
lizes as the monomer. In detail, the active site of hMAO-B is
characterized by a generally hydrophobic dipartite cavity with
*To whom correspondence should be addressed. Phone: þ39 06 4991
3763. Fax: þ39 06 49913772. E-mail: daniela.secci@uniroma1.it.
a Abbreviations: MAO, monoamine oxidase; FAD, flavin adenine dinu-
cleotide; IC50, 50% inhibitory concentration; HPLC, high-performance
liquid chromatography; CPSs, polysaccharide-based chiral stationary
phases; 3-MCE, 3-methylcyclohexanone; 3-MCET, 3-methylcyclohexyli-
dene thiosemicarbazone; Km, Michaelis constant; Vmax, maximum reaction
velocity; BTI-TN-5B1-4, insect cells infected with recombinant baculovirus
containing cDNA; PDB, Protein Data Bank; 2BXR, PDB code of hMAO-
A; 1GOS, PDB code of hMAO-B; SEM, standard error of the mean; P,
probability value; pIC50 = -log IC50. Abbreviations used for amino acids
follow the rules of the IUPACIUB Commission of Biochemical Nomen-
clature in J. Biol. Chem. 1972, 247, 977-983. Amino acid symbols denote
L-configuration unless otherwise indicated.
r
pubs.acs.org/jmc
Published on Web 08/17/2010
2010 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6517
Scheme 1. Synthesis of Racemic Compounds (1a-9a) and Their Corresponding (R)-Enantiomers
the (R)-enantiomer, but it retains its neuroprotective properties.
The inverted chirality leads to a different binding of the indan
ring, probably resulting from steric hindrance.13
In the course of our research,14 we have previously reported
the synthesis and MAO inhibitory activity of different series of
1-(4-arylthiazol-2-yl)-2-(cyclohexylidene)hydrazines.15-18 We
also pointed out that the (R)-enantiomers of reported chiral
derivatives (eutomers) were generally the most selective hMAO-
B inhibitors. For this reason it was important to have the pure
enantiomers of the most active compounds to evaluate the
influence of stereochemistry on the biological activity. In this
study, we present the synthesis of racemic 1-(4-arylthiazol-2-yl)-
2-(3-methylcyclohexylidene)hydrazines (1a-9a) and their cor-
responding (R)-enantiomers and the enantioseparation of the
(S)-homochiral forms. Indeed, owing to the presence of a
stereogenic carbon on the cyclohexyl ring, these compounds
exist as (R)- and (S)-enantiomers. The first step of the investiga-
tion of the impact of absolute stereochemistry on the activity
and selectivity was to isolate sufficient quantities of the enantio-
mers of four of the most active compounds to use in a parallel
test in vitro. Our strategy was to synthesize the racemic forms
Figure 1. Chromatograms of rac-5 and (R)-5a obtained by simul-
and successively resolve them by HPLC on polysaccharide-
based chiral stationary phases (CPSs) on a semipreparative
scale. Knowledge of the in vitro biological activity of homo-
chiral forms could be useful for the in silico study of the
interactions involving the receptor site.
taneous UV (black) and CD (gray) detection at 310 nm: column,
Chiralpak AS-H (250 mm ꢀ 4.6 mm i.d.); eluent, n-hexane-
2-propanol 90/10 (v/v); flow-rate, 1 mL min-1; temperature, 25 °C.
chromatographic run. The enantiomeric and diastereomeric
relationships between the chromatographic peaks were
established by spiking the racemic sample with the synthetic
(R)-enantiomer and monitoring the CD signal during the
HPLC stereoseparation as reported in our previous work.19
Chemistry and HPLC Enantioseparation
All derivatives were obtained by direct reaction between
3-methylcyclohexanone or (R)-3-methylcyclohexanone and
thiosemicarbazide in 2-propanol with catalytic amounts
of acetic acid (Scheme 1). The desired thiosemicarbazones (3-
MCET and (R)-3-MCET) were condensed with different and
freshly synthesized R-halo-acetophenone to give the corres-
ponding thiazoles (Hantzsch reaction) in high yields (84-
99%) and enantiomeric excess (g99%). All compounds were
fully characterized by 1H NMR and elemental analysis. The
synthetic protocol used for the preparation of (R)-enantio-
mers of 1a-9a starts from the commercially available (R)-3-
methylcyclohexanone and does not involve the stereogenic
center. Consequently, the absolute configuration of the thia-
zole derivatives in homochiral form was assigned by chemical
correlation. The HPLC enantioseparation of the most active
and selective compounds (3a, 5a, 8a, and 9a) was carried out
on the amylose-based Chiralpak AS-H CSP using binary
mixtures n-hexane-ethanol as mobile phases. As shown in
Figure 1, these conditions permitted a simultaneous diastereo-
and enantioseparation and, scaled up atsemipreparative level,
isolation of milligram amounts of each enantiomer for single
Biochemistry
The biological evaluation of the test drugs on hMAO activity
was investigated by measuring their effects on the production of
hydrogen peroxide (H2O2) from p-tyramine (a common sub-
strate for hMAO-A and hMAO-B), using the Amplex Red
MAO assay kit and microsomal MAO isoforms prepared from
insect cells (BTI-TN-5B1-4) infected with recombinant baculo-
virus containing cDNA inserts for hMAO-A or hMAO-B.20
The production of H2O2 catalyzed by MAO isoforms can be
detected using 10-acetyl-3,7-dihydroxyphenoxazine, a non-
fluorescent and highly sensitive probe that reacts with H2O2
in the presence of horseradish peroxidase to produce a fluore-
scent product, resorufin. New compounds and reference inhi-
bitors were unable to react directly with the Amplex Red
reagent, which indicates that these drugs do not interfere with
the measurements. On the other hand, in our experiments and
under our experimental conditions, hMAO-A displayed a
Michaelis constant (Km) equal to 457.17 ( 38.62 μM and a

6518 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Chimenti et al.
Table 1. Structures and hMAO Inhibitory Activity of Derivatives and Reference Compoundsa
3-methylcyclohexylidene
2-methylcyclohexylidene
IC50 (μM)
IC50 (μM)
compd
R
hMAO-A
hMAO-B
ratio
17
48
compd
R
hMAO-A
41.23 ( 3.96c
hMAO-B
ratio
58
1a
(R)-1a
2a
H
H
37.84 ( 2.94b
38.61 ( 3.24b
23.12 ( 1.85
22.76 ( 1.64b
37.52 ( 3.21b
40.32 ( 3.31b
16.02 ( 0.78b
43.78 ( 4.17b
40.57 ( 3.45b
42.66 ( 3.09b
38.84 ( 3.04b
2.23 ( 0.19
1b
H
0.71 ( 0.036
13.12 ( 0.51
0.81 ( 0.075
21.90 ( 1.22
9.55 ( 0.76
0.41 ( 0.023
0.81 ( 0.068
0.38 ( 0.021
9.65 ( 0.0.78
3.68 ( 0.35
0.056 ( 0.0015
0.018 ( 0.00068
0.26 ( 0.017
2.86 ( 0.25
0.15 ( 0.0088
4.51 ( 0.28
4-Cl
4-Cl
1.1
2.4
2b
4-Cl
35.22 ( 1.81c
2.7
(R)-2a
3a
4-F
4-F
92
50
3b
4-F
4-F
43.55 ( 3.61c
3.53 ( 0.12c
4.94 ( 0.09c
44.70 ( 5.23c
0.20 ( 0.008
0.02 ( 0.001
0.04 ( 0.002
26.81 ( 2.74
214
160
105
1.7
(R)-3a
(RS)-3
4a
(R)-3b
(S)-3b
4b
4-F
42
4.5
4-F
2,4-Cl
2,4-Cl
2,4-Cl
2,4-F
2,4-F
2,4-F
4-CH3
4-CH3
4-OCH3
4-OCH3
4-NO2
4-NO2
4-NO2
4-CN
4-CN
4-CN
(R)-4a
5a
11
762
5b
2,4-F
2,4-F
2,4-F
4-CH3
37.95 ( 3.41c
6.83 ( 0.14c
0.014 ( 0.0002
0.03 ( 0.002
0.036 ( 0.003
0.142 ( 0.008
2,673
227
133
>701g
(R)-5a
(S)-5
6a
2158
39
>35g
>667g
1.3
(R)-5b
(S)-5b
6b
10.09 ( 0.65b
4.81 ( 0.03c
e
e
e
(R)-6a
7a
5.77 ( 0.13b
7b
4-OCH3
2.76 ( 0.17c
2.37 ( 0.14
1.2
(R)-7a
8a
4.26 ( 0.24c
3.12 ( 0.15
1.4
0.41 ( 0.013
0.086 ( 0.00041
0.21 ( 0.019
0.12 ( 0.0023
0.23 ( 0.0019
0.42 ( 0.029
61.35 ( 1.13
0.020 ( 0.00086
>244g
>1163g
476g
171
8b
4-NO2
4-NO2
4-NO2
4-CN
4-CN
4-CN
0.032 ( 0.002
0.009 ( 0.0006
0.016 ( 0.0008
0.026 ( 0.001
0.032 ( 0.002
0.063 ( 0.003
>3,093g
4,485
2,552
1,183
169
e
e
43.95 ( 1.08c
42.31 ( 2.81c
31.03 ( 2.44c
5.50 ( 0.26c
7.22 ( 0.56c
e
e
(R)-8a
(S)-8
9a
(R)-8b
(S)-8b
9b
20.57 ( 1.08b
21.90 ( 0.86b
17.14 ( 0.97b
0.00446 ( 0.00032
67.25 ( 1.02
6.56 ( 0.76
(R)-9a
(S)-9
C
95
41
(R)-9b
(S)-9b
115
0.000073
3362
0.87
<0.36h
>5.3g
D
I
M
7.54 ( 0.36
d
361.38 ( 19.37
f
Is
18.75 ( 1.24
a C=clorgyline. D=R-(-)-deprenyl.I=iproniazid. M=moclobemide. Is = Isatin. Ratio: hMAO-B selectivity index = IC50(hMAO-A) /IC50(hMAO-B) .
Each IC50 value is the mean ( S.E.M. from five experiments (n = 5). b Level of statistical significance: P < 0.01 versus the corresponding IC50 values obtained
against hMAO-B, as determined by ANOVA/Dunnett’s test. c Level of statistical significance: P < 0.05 versus the corresponding IC50 values obtained against
hMAO-B, as determined by ANOVA/Dunnett’s test. d Inactive at 1 mM (highest concentration tested). e Inactive at 100 μM (highest concentration tested). At
higher concentrations the compounds precipitate. f 100 μM inhibits the corresponding hMAO activity by approximately 40-45%. At higher concentration the
compound precipitates. g Values obtained under the assumption that the corresponding IC50 against hMAO-A is the highest concentration tested (100 μM).
h Value obtained under the assumption that the corresponding IC50 against hMAO-B is the highest concentration tested (1 mM).
maximum reaction velocity (Vmax) in the control group of
185.67 ( 12.06 (nmol p-tyramine/min)/mg protein, whereas
hMAO-B showed a Km of 220.33 ( 32.80 μM and a Vmax of
24.32 ( 1.97 (nmol p-tyramine/min)/mg protein (n = 5). Most
tested drugs concentration-dependently inhibited this enzymatic
control activity (Table 1).
In Table 1 we compared the biological results for the two
series of derivatives obtained by shifting the chiral methyl group
on the cycloaliphatic portion. As regards the racemic com-
pounds (1a-9a and 1b-9b), with the exception of derivatives
4a, they all showed a better inhibitory activity in the 2-methyl-
cyclohexylidene series, and the 3-methylcyclohexylidene series
maintained the same trend and hMAO-B selectivity.
Results and Discussion
Structure-activity relationship within this small library
revealed that racemic compounds with a 4-Cl, 4-CH3, and
4-OCH3 substituted phenyl group (2a, 6a, and 7a) or an
unsubstituted (1a) one at the C4 position of thiazole ring
displayed hMAO inhibitory activity in the micromolar range,
with selectivity toward B isoform. The main difference in the
hMAO inhibition of these derivatives originated from the
replacement of the chlorine, methyl, and methoxy on the
aromatic moiety by a fluorine atom (3a and 5a), a nitro
(8a), and a cyano (9a) group (inhibitory activity in the
nanomolar range). The number and the position of fluorine
Racemic 1-(4-arylthiazol-2-yl)-2-(3-methylcyclohexylide-
ne)hydrazines (1a-9a) and their corresponding (R)-enantio-
mers were synthesized and evaluated for their ability to inhibit
the A and B isoforms of hMAO. All compounds wereselective
hMAO-B inhibitors in the micromolar or submicromolar
range, for which it was important to assay the pure enantio-
mers of the most active ones to evaluate the influence of
stereochemistry on their inhibitory activity. With this in mind,
we also performed a direct HPLC separation of the (S)-forms
from the racemic mixtures.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6519
on the phenyl ring of the molecule seemed to be responsible
for the best activity and selectivity toward hMAO-B, since
5a (2,4-fluoro disubstituted derivative) had IC50 = 0.056 (
0.0015 μM and selectivity ratio was 762. Conversely, the
introduction of two more sterically hindered chlorine atoms
in ortho and para positions (4a) might reduce the compound
binding in the activesitecavityof the hMAO-B. A preliminary
molecular modeling approach was carried out to justify the
importance of this substituted aromatic ring in the interaction
with both isoforms (see Supporting Information). Superim-
positions of the best fully energy minimized hMAO-A and -B
poses of the most selective derivative (5a) indicated that the
aromatic and the thiazole moieties were located close to the
FAD coenzyme, suggesting a functional role in catalysis as
also demonstrated by the presence of a structural “aromatic
cage” in hMAOs and in several flavin-dependent amine
oxidizing enzymes described by others.21 The sum of these
interactions, in particular the hydrogen bond between Tyr326
and hydrazone NH, could also explain the better hMAO-B
recognition of this scaffold.
The analysis of the influence of the stereochemistry on the
biological behavior confirmed the results previously obtained.16
Except for derivatives 3a and 9a (which, for both the racemic and
the (R)-homochiral forms, displayed inhibitory activity in the
same concentration range), the 3-methylcyclohexylidene series
showed a stronger interaction with hMAO-B when assayed as
pure enantiomer, the (R)-homochiral form being more active
and selective than the corresponding (S)-enantiomers. This
behavior could be also highlighted in the 2-methylcyclohexyli-
dene series (compare 9a to 9b and (R)-9a to (R)-9b).
Moreover, as in the 2-methylcyclohexylidene series, we also
decided to separate and evaluate the (S)-forms of the same
derivatives (4-F, 2,4-F, 4-NO2, and 4-CN) because in the
3-methylcyclohexylidene series they displayed high inhibitory
activities as well. For 5a, 8a, and 9a, it was possible to state
that the (S)-homochiral form represented the distomer while
the (R)-homochiral one was the eutomer. Instead the hMAO-
B selectivity was only implemented for derivatives (R)-8a and
(R)-5a with selectivity ratios of >1163 and 2158, respectively.
In terms of anti-hMAO-A activity, the same substitutents
(4-CH3 and 4-NO2) on the aromatic ring proved to be inactive
in both series.
As a consequence, the shifting of the chiral methyl group on
the cyclohexylidene moiety confirmed the choice of this phar-
macophore and revealed that the CH3 group on the asymmetric
carbon of the six-membered ring may represent only the main
(R)- and (S)-recognition difference allowing additional hydro-
phobic contacts (see Supporting Information). These findings
increase our confidence in this model and stimulate us to
continue investigations in designing more potent and selective
analogues that may have interesting therapeutic potential as
original chemical models (templates) for the design and sub-
sequent development of new drugs useful for improving the
pharmacological treatment of major depressive disorders and
neurodegenerative diseases.
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 microanalyzer, and the analytical results
indicated g95% purity for all compounds. 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). The
lipophilic parameter ClogP was calculated for each molecule
by using ChemDraw Ultra 8.0. The synthesis of some com-
pounds has been described in previously22 and was performed
with slight changes.
General Procedure for the Synthesis of Racemic Derivatives
1a-9a and (R)-Enantiomers. The appropriate carbonyl com-
pound (50 mmol) was dissolved in 100 mL of 2-propanol and
stirred with an equimolar quantity of thiosemicarbazide for 24 h
at room temperature with catalytic amounts of acetic acid. The
desired thiosemicarbazone precipitated from the mixture and
was filtered, washed with suitable solvent, and dried under
vacuum. Equimolar amounts of the prepared thiosemicarba-
zone (50 mmol) and freshly synthesized, according to classical
methods, substituted R-halo-acetophenone (50 mmol), both
stirred in 2-propanol, were reacted at room temperature for
2 h. The precipitate was filtered, washed with petroleum ether and
diethyl ether, and dried under vacuum to give all compounds in
high yields (84-99%). All the yields are on isolated basis.
Determination of hMAO Isoform Activity. Briefly, 0.1 mL of
sodium phosphate buffer (0.05 M, pH 7.4) containing different
concentrations of the test drugs (new compounds or reference
inhibitors) 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) 165 pmol of p-tyramine/min (hMAO-A, 1.1 μg of
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 trans-
formed/min)/mg protein) was incubated for 15 min at 37 °C in a
flat-black-bottom 96-well microtest plate (BD Biosciences,
Franklin Lakes, NJ) placed in the dark multimode microplate
reader chamber. After this incubation period, the reaction was
started by adding (final concentrations) 200 μM Amplex Red
reagent, 1 U/mL horseradish peroxidase, and 1 mM p-tyramine
(final saturating concentration). The production of H2O2 and
consequently of resorufin was quantified at 37 °C in a multi-
detection microplate fluorescence reader (Fluostar Optima,
BMG Labtech GmbH, Offenburg, Germany) based on the
fluorescence generated (excitation, 545 nm; emission, 590 nm)
over 15 min, in which the fluorescence increased linearly.
Control experiments were carried out simultaneously by repla-
cing 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 fluore-
scence generated in the mixture due to nonenzymatic inhibition
(e.g., for directly reacting with Amplex Red reagent) was
determined by adding these drugs to solutions containing only
the Amplex Red reagent in a sodium phosphate buffer. To
determine the kinetic parameters of hMAO-A and hMAO-B
(Km and Vmax), the corresponding enzymatic activity of both
isoforms was evaluated (under the experimental conditions
described above) in the presence of a number (a wide range) of
p-tyramine concentrations. The specific fluorescence emission
(used to obtain the final results) was calculated after subtraction
of the background activity, which was determined from vials
containing all components except the hMAO isoforms, which
were replaced by a sodium phosphate buffer solution. In our
experimental conditions, this background activity was practi-
cally negligible.
Experimental Section
Chemistry. Unless otherwise noted, starting materials and
reagents were obtained from commercial suppliers and were
used without further purification. Melting points (mp) were
determined by the capillary method on an FP62 apparatus
HPLC Stereoseparation. HPLC stereoseparations were per-
formed using stainless-steel Chiralpak AS-H (250 mm ꢀ 4.6 mm
i.d. and 250 mm ꢀ 10 mm i.d.) columns (Chiral Technologies
Europe, Illkirch, France). HPLC-grade solvents were supplied
1
(Mettler-Toledo) and are uncorrected. H NMR spectra were
recorded at 400 MHz on a Bruker spectrometer using CDCl3 as
solvent. Chemical shifts are expressed as δ units (ppm) relative

6520 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Chimenti et al.
by Carlo Erba (Milan, Italy). The HPLC apparatus consisted of
a Perkin-Elmer (Norwalk, CT) 200 lc pump equipped with a
Rheodyne (Cotati, CA) injector, a 1000 μL sample loop, and a
HPLC Dionex TCC-100 oven (Sunnyvale, CA). The detector
for HPLC was a Jasco (Jasco, Tokyo, Japan) model CD 2095
Plus UV/CD. The signal was acquired and processed by Clarity
software (DataApex, Prague, Czech Republic). The mobile
phases were filtered and degassed by sonication just before
use. After semipreparative separation, the collected fractions
were analyzed by a chiral analytical column to determine their
enantiomeric excess (ee).
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Acknowledgment. This work was supported by Grant FIRB
RBI067F9E (Italy), Spanish Ministerio de Sanidad y Consumo
ꢀ
´
(Grant FISS PI061537), Consellerıa de Innovacion y Industria
ꢀ
(13) Binda, C.; Hubalek, F.; Li, M.; Herzig, Y.; Sterling, J.; Edmond-
son, D. E.; Mattevi, A. Crystal structures of monoamine oxidase B
in complex with four inhibitors of the N-propargylaminoindan
class. J. Med. Chem. 2004, 47, 1767–1774.
de la Xunta de Galicia (Spain, Grants INCITE07PXI203039ES,
INCITE08E1R203054ES, and 08CSA019203PR), and Spanish
ꢀ
Ministerio de Ciencia y Innovacion (Grant FISS PS09/00618).
(14) Bolasco, A.; Fioravanti, R.; Carradori, S. Recent development of
monoamine oxidase inhibitors. Expert Opin. Ther. Pat. 2005, 15
(12), 1763–1782.
(15) Chimenti, F.; Maccioni, E.; Secci, D.; Bolasco, A.; Chimenti, P.;
Granese, A.; Befani, O.; Turini, P.; Alcaro, S.; Ortuso, F.; Cardia,
M. C.; Distinto, S. Selective inhibitory activity against MAO and
molecular modeling studies of 2-thiazolylhydrazone derivatives.
J. Med. Chem. 2007, 50, 707–712.
Supporting Information Available: Analytical data for new
compounds, chromatography conditions, computational data,
and details of pharmacological studies. This material is available
free of charge via the Internet at http://pubs.acs.org.
(16) Chimenti, F.; Maccioni, E.; Secci, D.; Bolasco, A.; Chimenti, P.;
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