Synthesis, stereochemical identification, and selective inhibitory activity against human monoamine oxidase-B of 2-methylcyclohexylidene-(4-arylthiazol-2- yl)hydrazones

Article abstract of DOI:10.1021/jm800132g

A series of 2-methylcyclohexylidene-(4-arylthiazol-2-yl)hydrazones have been investigated for their ability to inhibit selectively the activity of the human A and B isoforms of monoamine oxidase (MAO). The target compounds, which present a stereogenic center on the cyclohexane ring, were obtained as pure (R) and (S) enantiomers by enantioselective HPLC. The absolute configuration of homochiral forms isolated on a semipreparative scale was obtained by a combined strategy based on chemical correlation and single-crystal X-ray diffraction. All compounds showed higher activity against the human MAO-B isoform with IC ₅₀ values ranging between 26.81 ± 2.74 μM and 14.20 ± 0.26 nM, and the assays carried out on the pure enantiomers showed higher activity for the (R) form. A computational study was performed by molecular mechanics, DFT-based quantomechanics, and docking techniques on the most active and human MAO-B selective inhibitor 8.

Full text of DOI:10.1021/jm800132g

4874
J. Med. Chem. 2008, 51, 4874–4880
Articles
Synthesis, Stereochemical Identification, and Selective Inhibitory Activity against Human
Monoamine Oxidase-B of 2-Methylcyclohexylidene-(4-arylthiazol-2-yl)hydrazones
Franco Chimenti,† Elias Maccioni,‡ Daniela Secci,*,† Adriana Bolasco,† Paola Chimenti,† Arianna Granese,† Simone Carradori,†
Stefano Alcaro,§ Francesco Ortuso,§ Matilde Ya´n˜ez,| Francisco Orallo,| Roberto Cirilli, Rosella Ferretti, and
Francesco La Torre
Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente AttiVe, UniVersita` degli Studi di Roma “La Sapienza”, P.le A.
Moro 5, 00185 Roma, Italy, Dipartimento Farmaco Chimico Tecnologico, UniVersita` degli Studi di Cagliari, Via Ospedale 72, 09124 Cagliari,
Italy, Dipartimento di Scienze Farmaco Biologiche “Complesso Nin`ı Barbieri”, UniVersita` degli Studi di Catanzaro “Magna Graecia”,
88021 Roccelletta di Borgia (CZ), Italy, Departamento de Farmacolog´ıa, Facultad de Farmacia, UniVersidad de Santiago de Compostela,
Campus UniVersitario Sur, E-15782 Santiago de Compostela (La Corun˜a), Spain, and Dipartimento del Farmaco, Istituto Superiore di Sanita`,
V.le Regina Elena 299, 00161 Roma, Italy
ReceiVed February 7, 2008
A series of 2-methylcyclohexylidene-(4-arylthiazol-2-yl)hydrazones have been investigated for their ability
to inhibit selectively the activity of the human A and B isoforms of monoamine oxidase (MAO). The target
compounds, which present a stereogenic center on the cyclohexane ring, were obtained as pure (R) and (S)
enantiomers by enantioselective HPLC. The absolute configuration of homochiral forms isolated on a
semipreparative scale was obtained by a combined strategy based on chemical correlation and single-crystal
X-ray diffraction. All compounds showed higher activity against the human MAO-B isoform with IC50
values ranging between 26.81 ( 2.74 µM and 14.20 ( 0.26 nM, and the assays carried out on the pure
enantiomers showed higher activity for the (R) form. A computational study was performed by molecular
mechanics, DFT-based quantomechanics, and docking techniques on the most active and human MAO-B
selective inhibitor 8.
Introduction
while 2-phenylethylamine (PEA) and benzylamine are MAO-B
substrates.4 Clorgyline and moclobemide are two selective
reference inhibitors for MAO-A, while R-(s)-deprenyl (sel-
egiline), rasagiline, and lazabemide are well-known selective
inhibitors of MAO-B (Figure 1).5,6
Since they metabolize the principal biogenic amines, MAO-A
and MAO-B play an important role in the regulation of their
concentrations mainly in the central nervous system, where
abnormal values have been involved in psychiatric and neuro-
degenerative disorders such as Alzheimer’s disease (AD) and
Parkinson’s disease (PD).7,8 Therefore, selective and reversible
inhibitors of MAO-A or MAO-B may be useful therapeutic
agents devoid of unwanted side effects such as the so-called
“cheese effect”.9
Monoamine oxidases (MAOsa) are flavin adenine dinucleotide
(FAD) containing enzymes present in the outer mitochondrial
membranes of neuronal, glial, and other cells. They catalyze
the oxidation of endogenous and exogenous amines to the
corresponding aldehyde while releasing H2O2 and ammonia.1
MAOs exist in two forms, namely, MAO-A and MAO-B,
encoded by separate genes sharing a common intron/exon
organization.1–3 The two MAO isoforms have been identified
according to their substrate specificity and selective inhibitors.
The typical MAO-A substrate is 5-hydroxytryptamine (5-HT),
* To whom correspondence should be addressed. Phone: +39 06 4991
3763. Fax: +39 06 49913772. E-mail: daniela.secci@uniroma1.it.
†
Universita` degli Studi di Roma “La Sapienza”.
Universita` degli Studi di Cagliari.
Universita` degli Studi di Catanzaro “Magna Graecia”.
Universidad de Santiago de Compostela.
In humans MAO-B inhibitors are useful in the treatment of
PD and AD,10–13 while MAO-A inhibitors are antidepressant
and antianxiety agents (Figure 1).14,15
‡
§
|
Istituto Superiore di Sanita`.
The recent description of the crystal structure of the two
isoforms of human MAO (hMAO) by Binda et al. has provided
relevant information about the recognition mechanism underly-
ing the selective interactions between these proteins and their
ligands, in order to probe the catalytic mechanism and gain a
better understanding of the pharmacophoric requirements needed
for the rational design of potent and selective enzyme inhibitors
with therapeutic potential.16–20
In the course of our research we have previously reported
the synthesis and inhibitory activity of a series of selective
2-thiazolylhydrazone MAO-B inhibitors,21 and as part of our
plan in this work, we study the inhibitory activity of 2-meth-
ylcyclohexylidene-(4-arylthiazol-2-yl)hydrazones on hMAO-A
a Abbreviations: MAO, monoamine oxidase; FAD, flavin adenine
dinucleotide; 5-HT (5-hydroxytryptamine); PEA, 2-phenylethylamine; AD,
Alzheimer’s disease; PD, Parkinson’s disease; IC50, 50% inhibition constant;
HPLC, high-performance liquid chromatography; CPSs, polysaccharide-
based chiral stationary phases; DFT-based, density functional theory; MC,
Monte Carlo; MM, molecular mechanics; MMFF, molecular mechanics
force field; QM, quantum mechanic; GB/SA, Gibbs Born/surface area;
2-MCE, 2-methylcyclohexanone; 2-MCET, 2-methylcyclohexylidene-thi-
osemicarbazone; Km, Michaelis constant; Vmax, maximum reaction velocity;
BTI-TN-5B1-4, insect cells infected with recombinant baculovirus contain-
ing 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 Nomenclature in
J. Biol. Chem. 1972, 247, 977-983. Amino acid symbols denote L-
configuration unless otherwise indicated.
10.1021/jm800132g CCC: $40.75
2008 American Chemical Society
Published on Web 07/31/2008

(4-Arylthiazol-2-yl)hydrazones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4875
Figure 1. Irreversible (I), reversible (R), and selective MAO-A and/or MAO-B (A or A/B or B) inhibitors.
Scheme 1. Synthesis of Compounds 1-9a
Figure 2. (R)- and (S)-Enantiomers of the 2-methylcyclohexylidene-
(4-arylthiazol-2-yl)hydrazones derivatives (1-9).
a Reagents and conditions: (i) thiosemicarbazide, 2-propanol, acetic acid;
(ii) R-halogenoketone, 2-propanol, room temp.
and hMAO-B. Owing to the presence of a stereogenic carbon
on the cyclohexyl ring, these compounds exist as (R)- and (S)-
enantiomers (Figure 2).
The first step toward the investigation of the impact of
absolute stereochemistry on the activity and selectivity was to
isolate sufficient quantities of the enantiomers of four of the
most active compounds to use in a parallel test in vitro. Our
strategy was to synthesize the racemic forms and successively
resolve them by HPLC on polysaccharide-based chiral stationary
phases (CPSs) on a semipreparative scale. A combined method
based on chemical correlation, single-crystal X-ray diffraction,
and enantioselective HPLC allowed us to assign the absolute
configuration of the isolated enantiomers.
Knowledge of the absolute configuration and in vitro biologi-
cal activity of homochiral forms was needed in the in silico
study of the interactions involving the receptor site.
In this paper we present the synthesis, the enantioseparation
of the 2-methylcyclohexylidene-(4-arylthiazol-2-yl)hydrazones
(1-9), the stereoselective synthesis of the pure enantiomers,
and the absolute configuration. We also report their inhibitory
activity against the A- and B-isoforms of hMAO, a computa-
tional approach for the study of the conformational properties
of the molecules, and the docking of the most active and
selective compound 8, using the crystal structure data and
models of the hMAO-A and hMAO-B, deposited in the Protein
Data Bank.
Figure 3. X-ray structure of (+)-(S)-2-MCET.
products were characterized by spectroscopic methods (see
Supporting Information).
For the stereocontrolled synthesis of the pure enantiomers
of compounds 3, 5, 8, and 9, we use the enantiopure forms of
2-MCET as chiral synthon on the basis of a previous study by
us on the enantioseparation and configurational stability of this
compound.24 The racemic 2-MCET obtained in the first step of
the reaction was separated by enantioselective HPLC on a
semipreparative scale, and the isolated pure enantiomers were
used for the second step.
The absolute configuration of the chiral synthon was estab-
lished by a single crystal X-ray diffraction analysis. The
molecular structure is illustrated in Figure 3. From the findings
of the X-ray structure analysis, we can assign unambiguously
the (S)-configuration to the C2 stereogenic center located on
the cyclohexyl ring (labeled C(1) in Figure 3) of the (+)-2-
MCET enantiomer.
Chemistry
Racemic 2-thiazolylhydrazone derivatives (1-9) were syn-
thesized as reported in previous communications by us22,23
(Scheme 1). 2-Methylcyclohexanone (2-MCE) reacted directly
with thiosemicarbazide, and the obtained 2-methylcyclohexy-
lidene-thiosemicarbazone (2-MCET) subsequently reacted with
R-halogenoketones to yield the 4-substituted thiazole derivatives.
In the synthesis of all compounds, 2-propanol proved to be the
best solvent for our purpose. As a matter of fact, the reaction
products precipitate and can be filtered and purified by crystal-
lization from ethanol or ethanol/isopropanol. All the synthesized
Starting from the enantiopure forms of 2-MCET of known
stereochemistry, we obtained enantioenriched forms of the target

4876 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Chimenti et al.
Table 1. Structures and Inhibitory Activities of Derivatives 1-9 and
Reference Compounds
a
a
compd
R
IC50 hMAO-A
IC50 hMAO-B
ratio
1
2
3
4
5
6
7
8
9
H
4-Cl
4-F
2,4-Cl
2,4-F
4-CH3
4-OCH3
4-NO2
4-CN
41.23 ( 3.96 µMb 711.23 ( 36.95 nM 58
35.22 ( 1.81 µMb 13.12 ( 0.51 µM 2.7
43.55 ( 3.61 µMb 203.83 ( 8.52 nM 214
44.70 ( 5.23 µMb 26.81 ( 2.74 µM 1.7
37.95 ( 3.41 µMb 14.20 ( 0.26 nM 2,673
d
142.64 ( 8.65 nM >701e
2.37 ( 0.14 µM 1.2
2.76 ( 0.17 µMb
d
32.33 ( 2.22 nM >3093e
31.03 ( 2.44 µMb 26.23 ( 1.02 nM 1,183
4.46 ( 0.32 nMb 61.35 ( 1.13 µM 0.000073
67.25 ( 1.02 µMb 19.60 ( 0.86 nM 3431
clorgyline
R-(-)-
deprenyl
iproniazide
moclobemide
6.56 ( 0.76 µM
361.38 ( 19.37 µM
7.54 ( 0.36 µM 0.87
<0.36f
c
a Each IC50 value is the mean ( SEM from five experiments (n ) 5).
b Level of statistical significance: P < 0.01 versus the corresponding IC50
values obtained against MAO-B, as determined by ANOVA/Dunnett’s test.
c Inactive at 1 mM (highest concentration tested). d Inactive at 100 µM
(highest concentration tested). At higher concentrations the compounds
precipitate. e Values obtained under the assumption that the corresponding
IC50 against hMAO-A is the highest concentration tested (100 µM). f Value
obtained under the assumption that the corresponding IC50 against hMAO-B
is the highest concentration tested (1 mM).
Figure 4. Check of the stereochemical course of the synthesis of (S)-3
starting from (S)-2-MCET: column, Chiralpak AS-H (250 mm × 4.6
mm i.d.); eluent, methanol; flow rate, 1 mL/min; temperature, 25 °C;
detection, UV at 310 nm.
molecules with correlated chirality. The stereochemical course
of the reaction was monitored by the same enantioselective
HPLC system used for the semipreparative enantioseparation.
The cyclization of the chiral synthon is stereoconservative as
demonstrated by the high ee values (95-98%) of the obtained
thiazoles.
Table 2. Structures and Inhibitory Activities of Separated Enantiomers
of Compounds 3, 5, 8, and 9
a
a
IC50 hMAO-A IC50 hMAO-B
ee
compd
R
(µM)
(nM)
ratio
(%)
As an example, in Figure 4 we report the chromatograms
relating to the control of the enantiomeric purity of (S)-3 and
the enantiomeric elution order assignment of its racemic form.
The assignment of the absolute configuration of the enanti-
omers of compounds 1-9 was determined by chemical cor-
relation using as reference compound the chiral synthon
2-MCET.24
(S)-3
(R)-3
(S)-5
(R)-5
(S)-8
(R)-8
(S)-9
(R)-9
4-F
4.94 ( 0.09b
3.53 ( 0.12b
4.81 ( 0.03b
6.83 ( 0.14b
42.31 ( 2.81b
43.95 ( 1.08b
7.22 ( 0.56b
5.50 ( 0.26b
47.24 ( 2.49
22.01 ( 1.32
36.06 ( 3.45
30.08 ( 2.71
16.58 ( 0.86
9.80 ( 0.67
63.02 ( 3.62
32.48 ( 2.25
105 >99
160 >95
133 >99
2,4-F
4-NO2
4-CN
227
95
2552 >99
4485 >99
115 >99
169 >99
a Each IC50 value is the mean ( SEM 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.
Results and Discussion
Thiazoles 1-9 were synthesized at the beginning as racemates
and evaluated for their ability to inhibit the A and B isoforms.
The test drugs (new compounds and reference inhibitors)
themselves do not directly react with the Amplex Red reagent,
which indicates that these drugs do not interfere with the
measurements. In our experiments and our experimental condi-
tions, hMAO-A displayed a Michaelis constant (Km) of 457.17
( 38.62 µM and a maximum reaction velocity (Vmax) of 185.67
( 12.06 (nanomol/min)/mg protein whereas hMAO-B showed
a Km of 220.33 ( 32.80 µM and a Vmax of 24.32 ( 1.97
(nanomol/min)/mg protein (n ) 5). The tested drugs (new
compounds and reference inhibitors) inhibited these control
enzymatic MAO activities, and the inhibition was concentration
dependent.
As shown in Table 1, the new compounds were significantly
selective hMAO-B inhibitors. For this reason it was important
to have the pure enantiomers of the most active compounds in
order to evaluate the influence of stereochemistry on the
inhibitory activity. With this in mind we performed a chro-
matographic separation of the enantiomers.
The HPLC enantioseparation was carried out on the amylose-
based Chiralpak AD and Chiralpak AS-H CSPs using normal-
phase and polar organic conditions (for details, see the
Supporting Information). The absolute configuration of the
isolated homochiral forms on semipreparative level was assigned
by comparing their elution order with that of the enantiomers
of known stereochemistry obtained by stereoselective synthesis.
The choice to use 2-MCET instead of 2-MCE as a chiral
synthon was based on the high configurational stability and
better availability of the pure enantiomers of the former, as
previously reported in a recent work by us.24
In Table 1 we report the inhibitory activity of racemic
compounds 1-9 against hMAO-A and hMAO-B, while Table
2 shows the activities of the pure enantiomers of compounds 3,
5 (the most active), 8 (the most selective), and 9, selected among
all the racemic compounds for the next experiments.
From the results reported in Table 2 we can point out that
the (R)-enantiomers are the most active and B-selective with
hMAO-B IC50 values ranging between 32.48 ( 2.25 and 9.80
( 0.67 nM and hMAO-B selectivity ratios ranging between 160
and 4485. Just to clarify, the results obtained in this work for
the racemic mixture of compound 1, 2-methylcyclohexylidene-
(4-arylthiazol-2-yl)hydrazone partially disagree with the results
previously published for the same compound.21 This discrepancy
may be due, at least in part, to the different methods and
experimental conditions used in both studies. Compound (R)-
8, with the best hMAO-B activity and B-selectivity, was
therefore selected for the subsequent computational study.
In the reversibility and irreversibility tests, hMAO-A and
hMAO-B inhibition was irreversible in presence of the racemic
mixture and the enantiomers of compound 8 as shown by the
lack enzyme activity restoration after repeated washing (Table
3). Similar results were obtained for the racemic mixture and
the enantiomers of compound 5 against hMAO-A and hMAO-B
(data not shown) and for R-(s)-deprenyl against hMAO-B
(Table 3). However, significant recovery of hMAO-A activity

(4-Arylthiazol-2-yl)hydrazones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4877
Table 3. Reversibility and Irreversibility of MAO Inhibitiona
hMAO-A inhibition (%)
after repeated
compd
before washing
washing
moclobemide (500 µM)
8 (100 µM)
(S)-8 (100 µM)
(R)-8 (100 µM)
86.75 ( 4.34
23.41 ( 1.62
69.47 ( 3.76
60.56 ( 2.98
10.26 ( 0.65b
26.61 ( 1.54
71.47 ( 3.47
58.43 ( 2.76
hMAO-B inhibition (%)
after repeated
washing
compd
before washing
R-(-)-deprenyl (20 nM)
8 (50 nM)
(S)-8 (50 nM)
48.67 ( 2.34
58.65 ( 2.97
74.32 ( 4.14
77.83 ( 3.89
49.14 ( 2.80
60.19 ( 3.26
79.26 ( 4.32
81.63 ( 4.25
(R)-8 (50 nM)
a Each value is the mean ( SEM from five experiments (n ) 5). b Level
of statistical significance: P < 0.01 versus the corresponding % hMAO-A
inhibition before washing as determined by ANOVA/Dunnett’s test.
Figure 5. Superimposition of best fully energy minimized hMAO-A
poses of (R)-8 and (S)-8 on protein CR atoms. The ligand is depicted
as orange carbon and light-blue carbon polytube models, respectively.
The FAD cofactor is displayed with surface color coded carbons. The
interacting residues with (R)-8 and (S)-8 are reported as orange and
light-blue carbon wires, respectively. Hydrogen atoms are omitted for
clarity, with the exception of the amminic one of 8. Hydrogen bonds
are reported as dotted lines. MAO residues establishing hydrogen bonds
with 8 are rendered as polytubes.
Table 4. Thermodynamic Docking Results of 8·hMAO Complexes and
Comparison with Experimental Inhibition Data
a
c
enzyme
C*
CdN
∆∆Eint
pIC50
4.36
hMAO-A
R
E
Z
E
Z
E
Z
E
Z
-40.58b
-33.53
-41.03b
-36.77
-42.32
-46.84b
-43.78b
-42.25
S
R
S
4.37
8.00
7.78
hMAO-B
correlation between theoretical and experimental data was
observed. The trend can be highlighted, especially considering
the lowest value of the E and Z configuration interaction energies
for each of the eight complex ensembles. In three of the four
complexes, that is, [(R)-8 · hMAO-A], [(S)-8 · hMAO-A], and
[(S)-8·hMAO-B], the E configuration was ranked as the most
stable of 8 for recognition within the enzymes. Conversely, the
[(R)-8·MAO-B] complex was consistently stable when the Z
configuration is adopted. In order to understand the thermody-
namic results at the molecular level, we analyzed the four most
stable complexes. In this analysis we considered the number of
contacts and intermolecular hydrogen bonds established by
compound 8 and the hMAO residues as described in the
Experimental Section.
In hMAO-A (Figure 5) 8 enantiomers showed a similar
recognition pattern. In both cases the ligand established two
hydrogen bonds. One was between the nitro group as acceptor
and the phenolic hydroxyl moiety of Tyr444 as donor, the other
was between the amine hydrogen as donor and the hydroxyl
oxygen of Thr336 as acceptor. In the superimposition of these
two complexes, which were carried out on the CR atoms of the
enzyme, the aromatic and the thiazole moieties of the enanti-
omers fit each other almost perfectly Conversely, the methyl
moiety on the asymmetric carbon of the six-membered ring takes
a different position within the cleft. The main consequence of
this was that an additional hydrophobic contact of (S)-8 was
established with Phe208. This justifies the slightly better
recognition of this enantiomer compared to (R)-8, as demon-
strated by the interaction energies in agreement with the
experimental inhibition data (Table 3). (R)-8 and (S)-8 respec-
tively established 28 and 32 contacts with 13 and 14 residues
of hMAO-A, as shown in Figure 5.
a In kcal/mol. b ∆∆ interaction energy minimum. c pIC50 ) -log IC50.
was observed after repeated washing of moclobemide, indicating
that this drug is a reversible inhibitor of hMAO-A.
This selection of compound 8 was carried out following a
similar approach reported in our previous communication.21 For
the presence in 8 of the asymmetric methylcyclohexylidene
moiety and the thiosemicarbazone imine NdC double bond,
we have modeled the E and Z isomers separately and kept the
R configuration fixed by Monte Carlo (MC) simulations and
the molecular mechanics force field (MMFF). The conforma-
tional distribution in the energies window within 12.5 kcal/mol
above the global minimum structure was larger for the (R)-E
than for the (R)-Z stereoisomer, with 59 and 40 unique
conformers, respectively. The most stable conformers were then
compared by a quantum mechanic (QM) method, as described
in the Experimental Section. The MM and QM energy evalu-
ation of the global (R)-E and (R)-Z minimum conformers of 8
revealed differences in stability, which were lower than 0.25
and 0.35 kcal/mol when measured in different solvating
environments. Therefore, for compound 8, in agreement with
previous observations by us on other thiazole derivatives,21 both
stereoisomers can exist in physiological conditions and can
contribute almost equally to the binding properties within the
hMAO isoforms.
After converting the conformers of the R into the S enantiomer
in both E and Z configurations, we used the four 8 ensembles
to perform docking experiments on the hMAO pretreated
enzyme models as described in the Experimental Section. First,
in the rigid docking we included all the conformers obtained in
the MC search. Then we fully energy-minimized the configura-
tions of the eight possible combinations of hMAO isoforms and
ligand stereoisomer. The thermodynamic interaction results are
reported in Table 4.
In MAO-B (Figure 6) the molecular recognition of (R)-8
and (S)-8 was significantly different. Even when the aromatic
and the thiazole moieties were located approximately in the
same area, i.e., close to the FAD, the fit between them was
not perfect. The reason for this could be the different
configuration of the CdN double bond, Z in (R)-8 and E in
Assuming that the enzyme inhibition is a function of the
interaction energy, from a comparison with pIC50 a good

4878 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Chimenti et al.
geometry revealed fluctuations lower than 1.5 and 0.6 Å within
the MAO A and B isoforms, respectively (see Supporting
Information). This result suggests that our ligands are more
favored to assume the bioactive conformations found within the
MAO-B isoform than that within the A one. In agreement with
the experimental selectivity inhibition our ligands appear to be
conformationally preorganized to fit into the MAO-B gorge with
a low entopic cost.
In conclusion, the molecular modeling study of the most
active and selective hMAO inhibitor 8 was qualitatively in
agreement with the selectivity for isoform B as well as the
different enantioselective inhibition of the enantiomers for the
same enzyme. Moreover, both MM and QM studies indicated
that both E and Z configurations are thermodynamically possible
for the inhibitor 8. The docking results show that the Z
configuration has the best recognition for hMAO-B. This
information should be taken into account in the rational design
of new more potent and selective inhibitors.
Figure 6. Superimposition of best fully energy minimized hMAO-B
poses of (R)-8 and (S)-8 on protein CR atoms. The ligand is depicted
as orange carbon and light-blue carbon polytube models, respectively.
The FAD cofactor is displayed with surface color coded carbons. The
interacting residues with (R)-8 and (S)-8 are reported as orange and
light-blue carbon wires, respectively, and common interacting residues
are labeled yellow. Hydrogen atoms are omitted for clarity, with the
exception of the amminic one of 8. Common hydrogen bonds are
reported as yellow dotted lines, the one specific for the (R)-8, orange.
MAO residues establishing hydrogen bonds with 8 are rendered as
polytubes.
Experimental Section
Chemistry. General Procedure for the Preparation of 2-MCET
and 2-Thiazolylhydrazone Derivatives (1-9). 2-Methylcyclohex-
anone (50 mmol) was dissolved in 100 mL of 2-propanol and
refluxed under magnetic stirring with an equimolar amount of
thiosemicarbazide for 24 h. The thiosemicarbazone 2-MCET
precipitated from the reaction mixture, was filtered, and was
crystallized from EtOH/CH2Cl2. The chemical and physical proper-
ties are reported in the Supporting Information.
Equimolar amounts of 2-MCET and bromoacetophenone, both
dissolved in 2-propanol, were reacted at room temperature under
stirring for 2 h. The white precipitate was filtered and crystallized
from ethanol or ethanol/isopropanol to give compounds 1-9. For
compounds 1-9 we report the chemical and physical properties
(see Supporting Information).
Pharmacological Studies: Determination of hMAO Isoforms
Inhibitory Activity. The potential effects of the test drugs on hMAO
activity were investigated by measuring their effects on the
production of hydrogen peroxide from p-tyramine (a common
substrate for both hMAO-A and hMAO-B), using the Amplex Red
MAO assay kit (Molecular Probes, Inc., Eugene, OR) and microso-
mal MAO isoforms prepared from insect cells (BTI-TN-5B1-4)
infected with recombinant baculovirus containing cDNA inserts for
hMAO-A or hMAO-B (Sigma-Aldrich Qu´ımica S.A., Alcobendas,
Spain).
The production of H2O2 catalyzed by hMAO isoforms can be
detected using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red
reagent), a nonfluorescent, highly sensitive, and stable probe that
reacts with H2O2 in the presence of horseradish peroxidase to
produce a fluorescent product: resorufin. In this study hMAO
activity was evaluated using the above method following the general
procedure described previously by us.25
Briefly, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4)
containing various concentrations of the test drugs (new compounds
or reference inhibitors) and adequate amounts of the recombinant
hMAO-A or hMAO-B were adjusted to obtain in our experimental
conditions the same rection velocity, i.e., to oxidize (in the control
group) the same concentration of substrate: 165 pmol of p-tyramine/
min (hMAO-A, 1.1 µg; specific activity, 150 (nmol of p-tyramine
oxidized to p-hydroxyphenylacetaldehyde/min)/mg protein; hMAO-
B, 7.5 µg; specific activity, 22 (nmol of p-tyramine transformed/
min)/mg protein) was incubated for 15 min at 37 °C in a flat-black-
bottom 96-well microtiter (Microtest plate, BD, Franklin Lakes,
NJ) placed in the dark fluorimeter chamber. After this incubation
period, the reaction was started by adding (final concentrations)
200 µM Amplex Red reagent, 1 U/mL horseradish peroxidase, and
1 mM p-tyramine. The production of H2O2 and, consequently, of
resorufin was quantified at 37 °C in a multidetection microplate
fluorescence reader (FLX800, Bio-Tek Instruments, Inc., Winooski,
VT) based on the fluorescence generated (excitation, 545 nm;
(S)-8, best poses in the hMAO-B isoform. The interaction
pattern is actually different also in terms of intermolecular
hydrogen bonds. Two common hydrogen bonds were formed.
One was between the thiazole nitrogen as acceptor and the
amide hydrogen of the Gln206 side chain as donor, and the
other was between the amine hydrogen as donor and the
hydroxyl oxygen of Tyr326 as acceptor. For the (R)-8
hMAO-B complex an additional hydrogen bond was also
detected between the nitro group as donor and the hydroxyl
oxygen of Tyr188 as acceptor. This additional H-bond, found
in the (R)-8 compound Z configuration, can explain the larger
experimental inhibition difference of stereoisomers 8 onto
the hMAO-B isoform versus the hMAO-A (Table 4). In terms
of contacts and residues involved in hMAO-B recognition,
an analysis of the most stable complexes revealed consistent
differences. (R)-8 and (S)-8 respectively established 37 and
34 contacts with 13 and 11 residues of hMAO-B as shown
in Figure 6. In this case the common contact residues are
only eight, confirming a more consistent diversity in enan-
tioselective recognition in the hMAO-B isoform compared
to the hMAO-A. Another important consideration in the
analysis of the isoform binding poses can be a double stacking
of the nitrobenzene moiety with the Tyr398 and Tyr435 side
chains (Figure 6). This is supposed to be a crucial interaction
in the recognition of hMAO inhibitors. Since in hMAO-A
the corresponding stacking with Tyr407 and Tyr444 was
prevented by the hydrogen bond with the nitro group of 8
(Figure 5), the selectivity for the hMAO-B isoform can also
be explained by the fact that the partial double π-π
interaction is missing.
In order to better explore the flexibility of the optimized
docking configurations, the best poses were submitted to
molecular dynamics experiments. The analysis of the results
confirmed that the configurations obtained in both MAO
isoforms with the stereoisomes of 8 were stable. The root-mean-
square deviation (rmsd) with respect to the starting ligand

(4-Arylthiazol-2-yl)hydrazones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4879
emission, 590 nm) over a 15 min period in which the fluorescence
increased linearly.
The mobile phases were filtered and degassed by sonication
immediately before. After the semipreparative separation, the
collected fractions were analyzed on chiral analytical columns to
determine their enantiomeric excess.
Control experiments were carried out simultaneously by replacing
the test drugs (new compounds and reference inhibitors) with
appropriate dilutions of the vehicles. In addition, the possible
capacity of the above test drugs to modify the fluorescence
generated in the reaction mixture due to nonenzymatic inhibition
(e.g., for directly reacting with Amplex Red reagent) was deter-
mined by adding these drugs to solutions containing only the
Amplex Red reagent in a sodium phosphate buffer.
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 de-
scribed above) in the presence of a number (a wide range) of
p-tyramine concentrations.
The specific fluorescence emission (used to obtain the final
results) was calculated after subtraction of the background activity,
which was determined from vials containing all components except
the MAO isoforms, which were replaced by a sodium phosphate
buffer solution.
The corresponding IC50 values and MAO-B selectivity ratios
[IC50(MAO-A)]/[IC50(MAO-B)] for the inhibitory effects of test
drugs (new compounds and reference inhibitors) are shown in
Tables 1 and 2.
Reversibility and Irreversibility Experiments. To evaluate
whether the test compounds are reversible or irreversible MAO
inhibitors, an effective centrifugation-ultrafiltration method (so-
called repeated washing) was used. Briefly, adequate amounts of
the recombinant hMAO-A or hMAO-B were incubated together
with a single concentration (see Table 3) of the test drugs [racemic
mixture and enantiomers of compound 5 and 8 and the reference
inhibitors moclobemide and R-(s)-deprenyl in a sodium phosphate
buffer (0.05 M, pH 7.4)] for 15 min at 37 °C. After this incubation
period, an aliquot of this incubated sample was stored at 4 °C and
used for subsequent measurement of MAO inhibitory activity under
the experimental conditions indicated above (see the subsection on
determination of MAO inhibitory activity). The remaining incubated
sample (300 µL) was placed in a Ultrafree-0.5 centrifugal tube
(Millipore, Billerica, MA) with a 30 kDa Biomax membrane in
the middle of the tube and centrifuged (9000g, 20 min, 4 °C) in a
centrifuge (J2-MI, Beckman Instruments, Inc., Palo Alto, CA). The
enzyme retained in the 30 KDa membrane was resuspended in
sodium phosphate buffer at 4 °C and centrifuged again (under the
same experimental conditions described above) two successive
times. After the third centrifugation, the enzyme retained in the
membrane was resuspended in sodium phosphate buffer (300 µL)
and an aliquot of this suspension was used for subsequent MAO
activity determination.
Control experiments were performed simultaneously (to define
100% MAO activity) by replacing the test drugs with appropriate
dilutions of the vehicles. The corresponding values of percent (%)
MAO isoform inhibition were separately calculated for samples
with and without repeated washing.
X-Ray Crystal Structure Analysis. (+)-2-MCET crystallizes
from a solution of dichloromethane and ethanol as colorless needles.
Crystal data: C16H30N6S2, M ) 370.58, orthorhombic, space group
P212121, a ) 8.194(1) Å, b ) 13.053(1) Å, c ) 19.254(1) Å, V
) 2059.3(3) Å3, Z ) 4, Dc ) 1.195, µ ) 2.416 mm-1, F(000) )
800.
Molecular Modeling. The computational work was carried out
in three steps considering the most active and selective compound
8 with a similar approach reported in our previous communication.21
All calculations were carried out with a Linux cluster of 16 Intel
Xeon dual processors at 3.2 GHz with 2 Gb of RAM. Graphic
manipulations and analysis of the docking experiments were
performed with the Maestro Graphical User Interface, version
4.1.012, for Linux operating systems.26 Ligplot, version 4.0,27 and
Pymol, version 0.98,28 were used to create Figures 5 and 6.
The first step was the conformational analysis of 8, which is
complicated by the presence of the asymmetric methylcyclohexy-
lidene moiety and the configuration of the thiosemicarbazone imine
NdC double bond. Two Monte Carlo (MC) simulations were
carried out using the (R)-enantiomer built in the E and Z configura-
tions. Five-thousand conformations were randomly generated and
energy-minimized with the MMFF force field in GB/SA water as
implemented in the Macromodel package.29 The conformational
deduplication criterion was set as default, i.e., with a cutoff of 1
kcal/mol and an rms threshold of 0.25 Å.
The E and Z global minimum conformers were both submitted
to an ab initio single point DFT-B3LYP energy calculation with
Jaguar,26 at 6-311G**++ basis set in three different environments:
vacuum, water, and 1,2-dichloroethane.
The mirror images of the (S)-E and (S)-Z stereoisomers were
respectively obtained by inverting the sign of the Z atomic
coordinates of the (R)-E and (R)-Z conformers of 8 obtained in the
MC search using the MOLINE conversion module.30
The second step of the modeling work was the pretreatment of
the hMAO enzyme crystallographic models. Following computa-
tional work in our recent communications,31 the Protein Data
Bank32 crystallographic models of hMAO-A and hMAO-B, re-
spectively known by the PDB codes 2BXR17 and 1GOS,16 were
used for the docking experiments. Both structures were obtained
as adducts with two similar compounds, clorgyline for 2BXR and
pargyline for 1GOS, covalently linked to the FAD N5 nitrogen.
After removal of the covalent bond between the FAD moiety
and the cocrystallized inhibitor, pretreatment of the original PDB
models consisted of a 48 kcal/mol constrained energy minimization
of those residues out of a radius of 15 Å from N5 of the
isoalloxazine ring in order to restore the natural planarity of the
isoalloxazine FAD ring and relax the active site amino acids. The
resulting energy minimum structure ligands were removed and used
as receptor models. This was done with the AMBER* force field
united atom notation and the GB/SA water model of solvation as
implemented in MacroModel.26
The third step was the docking into the hMAO receptor models.
All energy minimum conformers of the four 8 stereoisomers were
docked in the hMAO binding sites with the GLIDE docking
method.33 Both pretreated enzyme models were submitted to map
calculations using a box of about 110 000 Å3 centered on the FAD
N5 atom. The rigid docking of the 8 stereoisomers was done by
generating a maximum number of 5000 configurations, scaling the
ligand van der Waals radii by 50%. All complex configurations
were fully energy-minimized with the AMBER* united atom and
the GB/SA water model of solvation.26
After full relaxation, the interaction energy of all complexes was
computed according to the MOLINE method30 and compared to
the experimental inhibition data.
In order to highlight the hMAO residues involved in van der
Waals and hydrogen bonds in the recognition of 8 in the enzyme,
the most stable fully energy-minimized configurations (Table 4)
of four complexes [(R)-8·hMAO-A], [(S)-8·hMAO-A], [(R)-
8·hMAO-B], and [(S)-8·hMAO-B] were analyzed using the LigPlot
program.27
Chromatographic (HPLC) Resolution of Racemic Samples
1-9. HPLC enantioseparations were performed using stainless-
steel Chiralpak AD (250 mm × 4.6 mm i.d. and 250 mm × 10
mm i.d.) and Chiralpak AS-H (250 mm × 4.6 mm i.d. and 250
mm × 10 mm i.d.) (Daicel, Chemical Industries, Tokyo, Japan)
columns. HPLC-grade solvents were supplied 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 HPLC Dionex (CA) model TCC-100 oven, and a Jasco (Jasco,
Ishikawa-cho, Hachioji City, Tokyo, Japan) model 2095 Plus UV/
CD detector.
Acknowledgment. This work was supported by grants
from MURST (Italy), Ministerio de Sanidad y Consumo (Spain,

4880 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Chimenti et al.
deposition: www.pdb.org (PDB ID codes 2BXR, 2BXS, and 2BYB)
(18) Binda, C.; Huba´lek, F.; Li, M.; Edmondson, D. E.; Mattevi, A. Crystal
structure of human monoamine oxidase B, a drug target enzyme
monotopically inserted into the mitochondrial outer membrane. FEBS
Lett. 2004, 564 (3), 225–228.
(19) Binda, C.; Edmondson, D. E.; Mattevi, A. Structure-function relation-
ships in flavoenzyme-dependent amine oxidations. A comparison of
polyamine oxidase and monoamine oxidase. J. Biol. Chem. 2002, 277
(27), 23973–23976.
(20) Huba´lek, F.; Binda, C.; Khalil, A.; Li, M.; Mattevi, A.; Castagnoli,
N.; Edmondson, D. E. Demonstration of isoleucine 199 as a structural
determinant for the selective inhibition of human monoamine oxidase
B by specific reversible inhibitors. J. Biol. Chem. 2005, 280 (16),
15761–15766.
(21) 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.
Grant FISS PI061537), and Xunta de Galicia (Spain, Grant
PGIDIT05BTF20302PR). We acknowledge Anton Gerada, a
professional 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. We acknowledge Dott. Cristina Faggi
of the Department of Organic Chemistry of the University of
Florence for the X-ray structure analysis. F.O. especially grateful
to the Conseller´ıa de Educacio´n e Ordenacio´n Universitaria de
la Xunta de Galicia (Spain) for giving him financial support to
intensify his research activity and to reduce his teaching during
the academic year 2007-2008 [programa de promocio´n de
intensificacio´n de la actividad investigadora en el sistema
Universitario de Galicia (SUG)].
Supporting Information Available: Analytical and spectral data
for new compounds, chromatography conditions, X-ray data,
computational data, and a few details on pharmacological studies.
This material is available free of charge via the Internet at http://
pubs.acs.org.
(22) Maccioni, E.; Cardia, M. C.; Bonsignore, L.; Plumitallo, A.; Pellerano,
M. L.; DeLogu, A. Synthesis and anti-microbial activity of isothi-
osemicarbazones and cyclic analogues. Farmaco 2002, 57 (10), 809–
817.
(23) Maccioni, E.; Cardia, M. C.; Distinto, S.; Bonsignore, L.; De.Logu,
A. An investigation of the biological effect of structural modifications
of isothiosemicarbazones and their cyclic analogues. Farmaco 2003,
58 (9), 951–959.
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