Investigations on the 2-thiazolylhydrazyne scaffold: Synthesis and molecular modeling of selective human monoamine oxidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.06.007

A new series of [4-(3-methoxyphenyl)-thiazol-2-yl]hydrazyne derivatives were synthesized in good yield (71-99%) and characterized by elemental analysis and ¹H NMR studies. The compounds were assayed for their in vitro human monoamine oxidase (hMAO) inhibitory activity and selectivity and most of them showed IC₅₀ values in the nanomolar range, thus demonstrating our interest in this privileged scaffold. The most active and selective derivative (20), bearing a pyridine moiety on the CN, displayed IC₅₀ = 3.81 ± 0.12 nM and selectivity ratio = 119 toward hMAO-B. Molecular modeling studies were carried out on recent and high resolution hMAO-A and hMAO-B crystallographic structures to better justify the enzyme-inhibitor interaction toward hMAO isoforms and to explain the structure-activity relationship of this kind of inhibitors.

Full text of DOI:10.1016/j.bmc.2010.06.007

Bioorganic & Medicinal Chemistry 18 (2010) 5715–5723
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Investigations on the 2-thiazolylhydrazyne scaffold: Synthesis and
molecular modeling of selective human monoamine oxidase inhibitors
a
a
a
Franco Chimenti a, Adriana Bolasco a, , Daniela Secci , Paola Chimenti , Arianna Granese ,
*
Simone Carradori a, Matilde Yáñez b, Francisco Orallo b, Francesco Ortuso c, Stefano Alcaro c
a Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma ‘La Sapienza’, P.le A. Moro 5, 00185 Rome, Italy
b Departamento de Farmacología and Instituto de Farmacia Industrial, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Universitario Sur,
E-15782 Santiago de Compostela (La Coruña), Spain
c Dipartimento di Scienze Farmacobiologiche, Università di Catanzaro ‘Magna Graecia’, ‘Complesso Ninì Barbieri’, 88021 Roccelletta di Borgia (CZ), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of [4-(3-methoxyphenyl)-thiazol-2-yl]hydrazyne derivatives were synthesized in good yield
(71–99%) and characterized by elemental analysis and 1H NMR studies. The compounds were assayed for
their in vitro human monoamine oxidase (hMAO) inhibitory activity and selectivity and most of them
showed IC50 values in the nanomolar range, thus demonstrating our interest in this privileged scaffold.
The most active and selective derivative (20), bearing a pyridine moiety on the C@N, displayed
IC50 = 3.81 0.12 nM and selectivity ratio = 119 toward hMAO-B. Molecular modeling studies were car-
ried out on recent and high resolution hMAO-A and hMAO-B crystallographic structures to better justify
the enzyme–inhibitor interaction toward hMAO isoforms and to explain the structure–activity relation-
ship of this kind of inhibitors.
Received 27 January 2010
Revised 3 June 2010
Accepted 4 June 2010
Available online 9 June 2010
Keywords:
hMAO inhibitors
2-Thiazolylhydrazyne
Molecular modeling
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
disturbances. In keeping with these premises, the development of
hMAO inhibitors has led to important breakthroughs in the ther-
It is well established that monoamine oxidases (MAOs; EC
1.4.3.4) play a significant role in the control of intracellular concen-
tration of monoaminergic neurotransmitters or neuromodulators
such as 5-hydroxytryptamine (5-HT), dopamine (DA), norepineph-
rine, and dietary amines. The rapid degradation of these molecules
ensures the proper functioning of synaptic neurotransmission and
is critically important for the regulation of emotional and other
brain functions. These flavoenzymes are located predominantly in
the outer mitochondrial membrane of neurons, glial, and other
mammalian cells. The enzyme exists in two isoforms, MAO-A and -
B, sharing about 70% amino acid identity, which are identified by
separated encoding genes, tissue distribution, their inhibitor sensi-
tivity and substrate selectivity.1 Monoamine oxidase-A and -B pref-
erentially deaminate 5-HT and phenylethylamines, respectively. In
the case of inhibitor sensitivity, MAO-A and -B forms are inhibited
irreversibly by low concentrations of clorgyline and R-(ꢀ)-deprenyl,
respectively. In Figure 1 we collected known clinical MAO inhibitors.
The by-products of MAO-mediated reactions include several
chemical species with neurotoxic potential, such as hydrogen per-
oxide, ammonia (or amine), and aldehydes. It is widely speculated
that prolonged or excessive activity of these enzymes may be
conducive to mitochondrial damages and neurodegenerative
apy of several neuropsychiatric disorders. Currently, the therapeu-
tic interest in human MAO inhibitors falls into two major
categories.2 hMAO-A inhibitors are used for the treatment of men-
tal disorders, particularly depression, whereas hMAO-B inhibitors
are used as co-adjuvants for the treatment of Parkinson’s and
Alzheimer’s disease. For these reasons, the active site of both iso-
forms has been recently crystallized with many selective reversible
inhibitors to highlight the structural information for the design of
new drugs.3,4
After our studies on the pyrazole5 and coumarin6 derivatives as
hMAO inhibitors, we focused our attention on an interesting scaf-
fold of molecules: (4-arylthiazol-2-yl)hydrazynes.
In fact, the same development of hMAO inhibitors started with
the serendipitous finding of antidepressant effects in patients trea-
ted with iproniazid, a hydrazine-based ant tubercular agent. An
almost common structural feature of substrate and inhibitors usu-
ally is an amino or imino group, which seems to play an essential
role in orientation and complex formation at the active site of the
enzyme. Furthermore, numerous substituted hydrazines have been
studied as MAO inhibitors and their specific mechanism of action
was fully investigated.7 Substituents on the aromatic ring at C4
position of thiazole ring also influenced the activity as demon-
strated by the introduction of several groups (NO2, CN, CH3, and
OCH3) or halogens (F and Cl) in the ortho and para positions in
our previous works,8,9 while the introduction of more steric
* Corresponding author. Tel.: +39 6 49913923; fax: +39 6 4462731.
E-mail address: adriana.bolasco@uniroma1.it (A. Bolasco).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.007

5716
F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5715–5723
N
CH3
N
N
H
H
CH3
N
CH3
CH3
CH3
O
N
H
R-(-)-Deprenyl (I) (B)
Pargyline (I) (A/B)
O
Iproniazide (I) (A/B)
O
N
O
H
Cl
CH3
N
N
O
N
O
H
N
H
Cl
H3C
Isatin (R) (B)
Clorgyline (I) (A)
Isocarboxazide (I) (A/B)
Cl
NH
O
O
H
H3CO
N
OH
H3C
N
N
O
O
O
Br
Moclobemide (R) (A)
Brofaromine (R) (A)
O
Toloxatone (R) (A)
NH2
N
H
NH
N
Cl
Rasagiline (I) (B)
Lazabemide (R) (B)
Figure 1. Irreversible (I), reversible (R), and selective hMAO-A (A) and/or hMAO-B (B) inhibitors.
hindered coumarin and naphthalene nucleus at C4 position of thi-
azole led to a decreased hMAO inhibitory activity.10 That prompted
us to evaluate the presence of an electron-donating group such –
OCH3 in the meta-position of the aryl ring. The choice of different
aliphatic, cycloaliphatic, heterocyclic, and bicyclic moieties on
the C@N group allowed a better comprehension of the steric and
electronic influence on the enzyme–inhibitor interaction and a
modulation of the chemical structure of these derivatives.
In the past other authors have investigated some hydrazothiaz-
ole derivatives as regards their potency as rat MAO inhibitors, and
their thermotropic behavior in relation to the partition coefficient
in lipid membrane models, correlating a different binding of these
inhibitors to the enzyme surface according to their electronic or
lipophilic characteristics.11
As our major goal was to obtain a novel lead compound, in this
paper we report on the synthesis and the biological activity of a
large array of [4-aryl-2-thiazolyl]hydrazynes (1–27) as hMAO
inhibitors, substituted with a methoxy group in the meta-position
of the aromatic ring, in which the basic function, that supports the
primary interaction at the catalytic site, was simulated by the new
pharmacophoric structure. Furthermore, we performed a computa-
tional study on the most potent inhibitor (20) in order to rational-
ize the enzyme recognition with respect to hMAO-A and hMAO-B.
The new, high resolution, Protein Data Bank crystallographic struc-
tures were considered as receptor models of hMAO-A (PDB code
2Z5X) and hMAO-B (PDB code 2BK3), respectively.3,4
of acetic acid, and the obtained thiosemicarbazones were subse-
quently converted into (4-aryl-2-thiazolyl)hydrazynes by reaction
with
(Hantzsch reaction).
a
-bromo-30-methoxyacetophenone in the same solvent
a
-Bromo-30-methoxyacetophenone has been
synthesized by direct halogenation of 30-methoxyacetophenone
with bromine in chloroform. All synthesized products were puri-
fied with petroleum ether and diethyl ether and, if requested, by
chromatography before characterization by 1H NMR and elemental
analysis (see Section 5 and Supplementary data).
3. Biochemistry
The potential effects of the test drugs on hMAO activity were
investigated by measuring their effects on the production of hydro-
gen peroxide (H2O2) from p-tyramine (a common substrate for
both hMAO-A and hMAO-B), using the Amplex Red MAO assay
S
H
NH2
N
HN
N
i
H2N
O
NH2
S
N
S
OCH3
HN
N
iii
O
O
1-27
Br
CH3
ii
2. Chemistry
[4-Aryl-2-thiazolyl]hydrazyne derivatives (1–27) were synthe-
sized in high yields (71–99%) as reported in our previous commu-
nications8–10 (Scheme 1). Different carbonyl compounds reacted
directly with thiosemicarbazide in ethanol with catalytic amounts
OCH3
OCH3
Scheme 1. Reagents and conditions: (i) CH3COOH (cat.), EtOH, rt; (ii) CHCl3, Br2, rt;
(iii) EtOH, rt.

F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5715–5723
5717
kit (Molecular Probes, Inc., Eugene, Oregon, USA) and mitochon-
drial 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 Química S.A., Alcobendas,
Spain).
is characterized by a single large hydrophobic cavity which adapt
itself to the bulkiest moieties better than the two small hydropho-
bic cavities of hMAO-B. Accordingly, the anti-hMAO activity and
selectivity were influenced by the steric hindrance of the substitu-
ent on the hydrazonic nitrogen as well. In general, the bulkiest
compounds displayed both low inhibitory activity and the best
selectivity toward hMAO-A. In the reversibility and irreversibility
tests, hMAO-A and hMAO-B inhibition was irreversible in presence
of the compound 20, selected for docking experiments, as shown
by the lack enzyme activity restoration after repeated washing.
Similar results were obtained for R-(ꢀ)-deprenyl (Table 2). How-
ever, significant recovery of hMAO-A and hMAO-B activity was
observed after repeated washing of isatin, indicating that this drug
is a reversible inhibitor of hMAO isoforms. Similar results were
obtained for moclobemide on hMAO-A.
In principle, our results of reversibility with the compound 20
do not seem to correlate well with the conformational analysis
made in the docking studies, but a number of arguments may ex-
plain, at least in part, this discrepancy. In most cases, the irrevers-
ible inhibitors establish a covalent interaction with the active
center of the enzyme.13 However, not all irreversible inhibitors
form covalent adducts with their enzyme targets but they may also
act by other mechanisms. In fact, some reversible inhibitors bind so
tightly to their target enzyme that they are essentially irreversible.
These tight-binding inhibitors may show kinetic properties similar
to covalent irreversible inhibitors.
The production of H2O2 catalyzed by MAO isoforms can be
detected using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red
reagent), a non-fluorescent and highly sensitive probe that reacts
with H2O2 in the presence of horseradish peroxidase to produce
a fluorescent product, resorufin. In this study, hMAO activity was
evaluated using the above method following the general procedure
previously described by us.12 The test drugs (new compounds and
reference inhibitors) themselves were unable to react directly with
the Amplex Red reagent, which indicates that these drugs do not
interfere with the measurements. On the other hand, in our exper-
iments and under our experimental conditions, the control activity
of hMAO-A and hMAO-B (using p-tyramine as a common substrate
for both isoforms) was 165 2 pmol of p-tyramine oxidized to
p-hydroxyphenylacetaldehyde/min (n = 20). Most tested drugs
concentration-dependently inhibited this enzymatic control
activity.
4. Results and discussion
Structure–activity relationships (SARs) were inferred from data
of enzymatic experiments reported in Table 1.
With the only exceptions of derivatives 18 and 24, all tested
compounds (assayed as mixture of E and Z conformers) inhibited
hMAOs at micromolar or sub-micromolar concentration with ratio
values ranging from <0.0087 (25) to 119 (20). Aliphatic moieties
(1–5) showed that hMAO inhibitory activity and selectivity are lar-
gely influenced by the chain length. Elongation of the alkyl chain
produced slight improvements of hMAO-B inhibition (1–3), while
the biggest derivatives (4 and 5) were endowed with discrete
hMAO-A selectivity. Cyclization of the lateral chain of compound
3 to give 6 induced selectivity inversion, but the introduction of
a methyl group (7 and 8) or further elongation of the aliphatic cycle
(9–13) improved anti-hMAO-B activity and selectivity (i.e., com-
pound 10, IC50 hMAO-B = 12.22 0.43 nM and selectivity ratio =
60). It was clearly evident that the anti-hMAO activity and selectiv-
ity were strongly associated with the presence of heterocyclic or
bicyclic moiety (13–27) on the hydrazonic nitrogen. The bulkiest
compounds (23–27) displayed both low inhibitory activity and
the best selectivity toward hMAO-A (compound 25, IC50 hMAO-
A = 874.69 41.43 nM and ratio <0.0087).
Conversely, compounds 14, 15, 19, and 21, bearing a smaller
heterocycle in that position, were quite potent but less selective
hMAO-A inhibitors. Great improvement of the hMAO-B inhibitory
activity was obtained by replacing the hydrogen atom with a
methyl group at the C@N bond (compare compounds 14, 16, 19,
and 21–15, 17, 20, and 22). Among them, compound 20 was the
most selective hMAO-B inhibitor and almost five times more
potent than R-(ꢀ)-deprenyl (IC50 hMAO-B = 3.81 0.12 nM and
selectivity ratio = 119). The presence and the position of a methyl
group linked to the pyridine ring deeply affect the hMAO inhibitory
activity (18, 20, and 22) showing the lowest IC50 when it was intro-
duced in the meta-position. From the results obtained in this and in
our previous works,8–10 it is possible to state that moieties on the
C4 of the thiazole ring must be carefully evaluated. Molecular
modeling studies proved that this region was involved in the inter-
action between inhibitor and FAD cofactor which was placed inside
the active site of the enzyme and was responsible of its oxidative
activity. Therefore little changes of substituent dimension in this
position were scarcely tolerated. This evidence could be also justi-
fied on the basis of the detailed information that PDB provides
about the active site of both isoforms: the active site of hMAO-A
In these cases, some of these inhibitors rapidly bind to the en-
zyme in a low-affinity enzyme–inhibitor (EI) complex and this
then undergoes a slower rearrangement to a very tightly bound
*
EI complex. This kinetic behavior is called slow-binding. This slow
rearrangement after binding often involves conformational
a
change as the enzyme ‘clamps down’ around the inhibitor mole-
cule.14 Also some MAO irreversible inhibitors (the so-called suicide
inhibitors) act as a substrate for the target enzyme, which finally
generates a new compound that irreversibly inhibits MAO activity.
Therefore, the initial interaction of these inhibitors with MAO may
be different to the interaction obtained after several minutes of the
enzyme–inhibitor complex formation. R-(ꢀ)-deprenyl, for exam-
ple, first of all form a non-covalent complex with MAO as an initial,
reversible step. The subsequent interaction of R-(ꢀ)-deprenyl with
MAO leads to a reduction of the enzyme-bound flavin adenine
dinucleotide, and concomitant oxidation of the inhibitor. This oxi-
dized inhibitor then reacts with FAD at the N5-position in a cova-
lent manner.15 The initial non-covalent binding to MAO has been
also described for other MAO inhibitors.16 Finally, it is possible
that, in some cases of irreversible inhibition, a steric hindrance
might not allow the removal of the inhibitor from the enzymatic
active center (although its interaction with this binding site is
either very weak or reversible).
Bearing in mind all the above considerations and taking into
account that the docking studies make only a theoretical predic-
tion of the initial possible interaction inhibitor–enzyme, the results
obtained in these docking studies and in the reversibility experi-
ments may be different. Compound 20, the most potent and selec-
tive hMAO-B inhibitor, was submitted to a molecular modeling
study with the aim to evaluate its recognition with respect to both
A- and B-hMAO isoforms. Two new Protein Data Bank crystal struc-
tures were considered, after a preliminary pre-treatment, as recep-
tor models of hMAO-A and hMAO-B (see Section 5). The ligand was
built taking into account both E and Z isomers and submitted to
Monte Carlo conformational search followed by Boltzmann popu-
lation analysis at 300 K. Such an investigation revealed the much
higher probability of existence for E isomer (99.6%) with respect
to the Z one (0.4%) but, in order to analyze the binding modes of
20, we decided to consider both global minimum energy structures
for the next docking simulations. These calculations were

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F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5715–5723
Table 1
Structures and biological activity of derivatives 1–27 and reference inhibitors
Compound
X
IC50 hMAO-A
4.43 0.22
824.33 40.05 nM
IC50 hMAO-B
5.07 0.13 lM
Ratio
0.87
1.1
1
2
3
lM
771.82 30.05 nM
871.47 38.61 nM
1.31 0.03
1.15 0.01
1.38 0.04
l
l
l
Mb
Ma
M
1.5
4
5
6
6.72 0.32
***
lM
0.17
< 0.014#
0.56
591.80 23.13 nMb
836.21 36.58 nMa
1.06 0.07
lM
7
26.64 0.81 nM
31
8
9
1.45 0.04
1.91 0.04
l
Ma
Ma
231.02 9.61 nM
31.87 1.26 nM
6.3
60
l
10
11
737.40 21.42 nMa
12.22 0.43 nM
72.70 2.31 nM
60
678.75 32.15 nMa
749.15 31.62 nMa
9.3
12
13
35.49 1.27 nM
29.54 1.07 nM
21
60
1.78 0.05 l
Ma
O
O
14
15
16
17
342.88 15.62 nMa
333.05 16.08 nMb
457.73 20.35 nM
636.08 27.65 nM
6.78 0.25
1.68 0.06
l
M
M
0.051
0.20
0.93
1.15
H
l
CH3
H
S
S
493.83 16.30 nM
551.38 12.35 nM
CH3
18
19
***
***
CH3
N
537.66 27.35 nMb
2.91 0.13
l
M
0.18
119
H
N
N
CH3
20
455.16 20.43 nMa
697.53 19.73 nMb
3.81 0.12 nM
21
22
H
1.14 0.03
l
M
0.61
32
N
N
CH3
3.71 0.08
3.64 0.06
l
Ma
116.38 4.25 nM
H
23
lM
***
<0.036#

F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5715–5723
5719
Table 1 (continued)
Compound
X
IC50 hMAO-A
***
IC50 hMAO-B
Ratio
H3C
24
25
**
H
874.69 41.43 nM
413.81 21.17 nMb
***
<0.0087#
O
O
H
26
27
799.69 27.61 nM
0.52
N
H
CH3
5.56 0.17
lM
6.97 0.23
l
M
0.80
O
O
a
C
D
I
M
Is
4.46 0.22 nM
67.25 1.02
6.56 0.26
61.35 1.13
19.60 0.86 nM
l
M
0.000073
3431
lM
l
M
7.54 0.36
*
l
M
0.87
361.38 19.37
***
l
M
<0.36§
>5.3Ê
18.75 1.24 lM
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 SEM from five experiments. Level of statistical significance: aP < 0.01 or bP < 0.05 versus the corresponding IC50 values obtained against hMAO-B,
as determined by ANOVA/Dunnett’s.
*
Inactive at 1 mM (highest concentration tested).
**
Inactive at 100
l
M (highest concentration tested).
One hundred micromolar inhibits the corresponding hMAO activity by approximately 40–45%. At higher concentration the compounds precipitate.
Values obtained under the assumption that the corresponding IC50 against hMAO-B is the highest concentration tested (100 M).
Value obtained under the assumption that the corresponding IC50 against hMAO-B is the highest concentration tested (1 mM).
Value obtained under the assumption that the corresponding IC50 against hMAO-A is the highest concentration tested (100 M).
***
#
l
§
Ê
l
Table 2
Reversibility and irreversibility of hMAO inhibition of derivative 20 and reference inhibitors
Compound
% hMAO-A inhibition
After repeated washing
% hMAO-B inhibition
After repeated washing
Before washing
Before washing
51.45 2.16
R-(ꢀ)-Deprenyl (20 nM)
52.05 2.08
Moclobemide (500
lM)
86.75 4.34
40.32 1.83
10.26 0.65a
12.10 0.52a
Isatin (100
Isatin (50
l
M)
M)
l
84.36 3.24
63.73 3.12
16.62 0.71a
67.18 3.21
20 (500 nM)
20 (5 nM)
59.14 2.02
65.13 2.94
Each value is the mean SEM from five experiments (n = 5).
a
Level of statistical significance: P < 0.01 versus the corresponding % hMAO-A or hMAO-B inhibition before washing, as determined by ANOVA/Dunnett’s.
Table 3
performed onto hMAO-A and hMAO-B receptor models and the
Interaction energies (kcal/mol) of compound 20 within hMAO-A and hMAO-B binding
pockets
recognition of 20 was evaluated using the interaction energy
parameter (Table 3).
D
Eint
vdW
ꢀ7.20
ꢀ36.30
El
4.50
ꢀ0.40
As reported in Table 3, in agreement with the experimental
data, compound 20 highlighted a better interaction to hMAO-B
than hMAO-A. The interaction energy components analysis indi-
cated a complete preference of compound 20 with respect to the
B isoform in terms of both vdW and electrostatic contributions.
These results were submitted to graphical inspection and the most
stable configurations of 20 in both enzymes were displayed in Fig-
ure 2. The analysis of compound 20 binding modes revealed that
the E isomer was more favoured than the Z on both receptor mod-
els. The configuration of compound 20 in hMAO-A and hMAO-B
was similar showing, in both case, the m-methoxyphenyl moiety
directed toward the FAD cofactor and the pyridine ring toward
the catalytic cleft outer side. Even if the number of interacting res-
idues was slightly larger with hMAO-A, the hMAO-B recognition
reported much more productive contributions: the ligand showed
a deeper position that allowed it to perform stacking contacts to
hMAO-A
hMAO-B
ꢀ2.70
ꢀ36.70
D
Eint, total interaction energy; vdW, van der Waals contribution; El, electrostatic
contribution.
Tyr398 and one hydrogen bond was established to the Ile198
backbone.
Moreover, into the preferred isoform, the m-methoxyphenyl
group was located close to Tyr188, Phe343, and Tyr435 and the
pyridine moiety was less exposed toward the solvent than into
the hMAO-A. The graphical inspection allowed a better apprecia-
tion of the details of the isoform recognition taking into account
the known differences in the binding pockets of MAO-A and -B.
In particular the corresponding residues Ile335 (hMAO-A) and

5720
F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5715–5723
were determined by the capillary method on an FP62 apparatus
(Mettler-Toledo) and are uncorrected. 1H NMR spectra were re-
corded at 400 MHz on a Bruker spectrometer using DMSO-d6 or
CDCl3 as solvent. Chemical shifts are expressed as d units (ppm)
relative to TMS. Coupling constants J are expressed in hertz (Hz).
Elemental analyses for C, H, and N were determined with a Per-
kin-Elmer 240 B microanalyzer and the analytical results were
P95% 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). 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 (Büchi Rotavapor) operating at reduced pressure. Com-
pound 17 is also commercially available.
5.2. General procedure for the synthesis of
acetophenone
a
-bromo-30-metoxy-
Equimolar quantities of 30-methoxyacetophenone and bromine,
both dissolved in chloroform, were stirred for 4 h at room temper-
ature until the presence of HBr disappeared. The solution was
evaporated under vacuum and the pale yellow solid was washed
with petroleum ether to give the product in good yield (97%).
5.3. General procedure for the synthesis of derivatives 1–27
The appropriate carbonyl compound (50 mmol) was dissolved in
100 mL of ethanol and stirred with an equimolar quantity of thio-
semicarbazide for 24 h at room temperature with catalytic amounts
of acetic acid. The desired thiosemicarbazone precipitated from
reaction mixture was filtered, washed with suitable solvent (petro-
leum ether, hexane, and diethyl ether), and dried. Equimolar
amounts of the prepared thiosemicarbazone (50 mmol) and freshly
synthesized
a
-bromo-30-methoxyacetophenone (50 mmol) were
reacted in ethanol at room temperature under magnetic stirring
for 4 h. The suspension was filtered and dried to give compounds
1–27 in high yield.
Figure 2. Compound 20 recognition of hMAO-A (a) and hMAO-B (b). Ligand is
reported in sticks, interacting residues in green carbon colored sticks, FAD cofactor
is displayed as
a spacefill structure. The rest of the enzyme is displayed in
transparent green cartoon. Yellow dotted line indicates hydrogen bond interaction.
5.3.1. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(propan-2-yli-
dene)hydrazine (1)
Tyr326 (hMAO-B) appeared relevant to explain the molecular rec-
ognition of the compound 20. In the B isoform Tyr326, due to its
consistent steric effects, induced the inhibitor to interact with
the FAD. Conversely, the hMAO-A Ile335, characterized by a lower
hindrance with respect to the corresponding hMAO-B residue, al-
lowed the ligand to assume multiple poses into the binding cleft
with higher enthalpic and entropic penalties.
We found a molecular rationale for the hMAO-A and hMAO-B
selectivity of this new class of 2-thiazolylhydrazyne inhibitors.
These findings increase our confidence in our model and stimulate
us to continue our investigations in designing more potent and
selective analogs. The results obtained in this study indicate that
some of the compounds tested may have interesting therapeutic
potential as original chemical models (templates) for the design
and subsequent development of new drugs (potent and selective
hMAO-A or hMAO-B inhibitors) useful for improving the pharma-
cological treatment of major depressive disorders and neurodegen-
erative diseases (e.g., Parkinson’s disease), respectively.
Yield 99%; mp 213–214 °C; 1H NMR (CDCl3) 2.12 (s, 3H, CH3),
2.21 (s, 3H, CH3), 3.90 (s, 3H, OCH3), 6.70 (s, 1H, C5H-thiaz.),
6.98–7.01 (m, 1H, Ar), 7.26–7.29 (m, 2H, Ar), 7.37–7.39 (m, 1H,
Ar), 12.45 (br s, 1H, NH, D2O exch.).
5.3.2. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(pentan-3-ylidene)-
hydrazine (2)
Yield 89%; mp 158–159 °C; 1H NMR (CDCl3) 1.14–1.18 (m, 3H,
CH3), 1.23–1.27 (m, 3H, CH3), 2.41–2.43 (m, 2H, CH2), 2.55–2.57
(m, 2H, CH2), 3.91 (s, 3H, OCH3), 6.69 (s, 1H, C5H-thiaz.), 7.01 (s,
1H, Ar), 7.27–7.28 (m, 2H, Ar), 7.37–7.39 (m, 1H, Ar), 12.35 (br s,
1H, NH, D2O exch.).
5.3.3.1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(pentan-2-ylidene)-
hydrazine (3)
Yield 84%; mp 150–151 °C; 1H NMR (CDCl3) 0.96–1.00 (m, 3H,
CH3), 1.65–1.68 (m, 2H, CH2), 2.15 (s, 3H, CH3), 2.36–2.38 (m, 2H,
CH2), 3.93 (s, 3H, OCH3), 6.70 (s, 1H, C5H-thiaz.), 6.98–7.00 (m,
1H, Ar), 7.37–7.40 (m, 3H, Ar), 12.29 (br s, 1H, NH, D2O exch.).
5. Experimental
5.1. Chemistry
5.3.4. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(heptan-2-
ylidene)-hydrazine (4)
Yield 89%; mp 134–135 °C; 1H NMR (CDCl3) 0.90–0.93 (m, 3H,
CH3), 1.34–1.35 (m, 2H, CH2), 1.56–1.63 (m, 4H, 2ꢁ CH2), 2.19
(s, 3H, CH3), 2.35–2.39 (m, 2H, CH2), 3.93 (s, 3H, OCH3), 6.69
Starting materials and reagents were obtained from commercial
suppliers and were used without purification. Melting points (mp)

F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5715–5723
5721
(s, 1H, C5H-thiaz.), 7.01 (s, 1H, Ar), 7.27–7.30 (m, 2H, Ar), 7.37–7.39
(m, 1H, Ar), 12.30 (br s, 1H, NH, D2O exch.).
C5H-thiaz.), 7.01 (s, 1H, Ar), 7.26–7.29 (m, 2H, Ar), 7.30–7.41 (m,
1H, Ar), 12.24 (br s, 1H, NH, D2O exch.).
5.3.5. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(octan-2-ylidene)-
hydrazine (5)
5.3.13. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-cycloheptylid-
ene-hydrazine (13)
Yield 99%; mp 109–110 °C; 1H NMR (DMSO-d6) 0.86 (s, 3H,
CH3), 1.27–2.49 (m, 10H, CH2), 2.49 (s, 3H, CH3), 3.78 (s, 3H,
OCH3), 6.65 (s, 1H, C5H-thiaz.), 6.96–6.99 (m, 2H, Ar), 7.09 (s, 1H,
Ar), 7.72–7.75 (m, 2H, Ar), 11.20 (br s, 1H, NH, D2O exch.).
Yield 71%; mp 194–195 °C; 1H NMR (CDCl3) 1.57–1.60 (m, 4H,
cycloheptyl), 1.65–1.67 (m, 4H, cycloheptyl), 2.80–2.86 (m, 2H,
cycloheptyl), 2.87–2.90 (m, 2H, cycloheptyl), 3.92 (s, 3H, OCH3),
7.26 (s, 1H, C5H-thiaz.), 7.27–7.29 (m, 1H, Ar), 7.30–7.33 (m, 3H,
Ar), 12.30 (br s, 1H, NH, D2O exch.).
5.3.6. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-cyclopentylidene-
hydrazine (6)
5.3.14. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(furan-2-ylmeth-
ylene)-hydrazine (14)
Yield 73%; mp 185–186 °C; 1H NMR (CDCl3) 1.85–1.87 (m, 2H,
cyclopentyl), 1.93–1.97 (m, 2H, cyclopentyl), 2.53–2.55 (m, 2H,
cyclopentyl), 2.65–2.67 (m, 2H, cyclopentyl), 3.91 (s, 3H, OCH3),
6.62 (s, 1H, C5H-thiaz.), 7.00 (m, 1H, Ar), 7.26–7.29 (m, 2H, Ar),
7.30–7.38 (m, 1H, Ar), 12.28 (br s, 1H, NH, D2O exch.).
Yield 99%; mp 139–140 °C; 1H NMR (DMSO-d6) 3.98 (s, 3H,
OCH3), 7.72–7.91 (m, 3H, furan), 7.90 (s, 1H, C5H-thiaz.), 8.33–
8.72 (m, 4H, Ar), 9.08 (s, 1H, CH@), 11.95 (br s, 1H, NH, D2O exch.).
5.3.15. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(1-(furan-2-yl)
ethylidene)-hydrazine (15)
5.3.7. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(2-methylcyclop-
entylidene)hydrazine (7)
Yield 80%; mp 224–225 °C; 1H NMR (DMSO-d6) 2.23 (s, 3H,
CH3), 3.78 (s, 3H, OCH3), 6.60 (s, 1H, C5H-thiaz.), 6.75–6.98 (m,
2H, furan), 7.33–7.42 (m, 4H, Ar), 7.58 (s, 1H, furan), 11.28 (br s,
1H, NH, D2O exch.).
Yield 79%; mp 172–173 °C; 1H NMR (CDCl3) 1.20 (s, 3H, CH3),
1.21 (s, 1H, cyclopentyl), 1.60–1.61 (m, 2H, cyclopentyl), 2.75–
2.90 (m, 2H, cyclopentyl), 3.92 (s, 3H, OCH3), 6.72 (s, 1H, C5H-thi-
az.), 7.00 (s, 1H, Ar), 7.26–7.29 (m, 2H, Ar), 7.30–7.38 (m, 1H, Ar),
12.28 (br s, 1H, NH, D2O exch.).
5.3.16. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(thiophen-2-
ylmethylene)hydrazine (16)
5.3.8. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(3-methylcyclop-
entylidene)hydrazine (8)
Yield 82%; mp 185–186 °C; 1H NMR (DMSO-d6) 3.78 (s, 3H,
OCH3), 6.80 (s, 1H, C5H-thiaz.), 7.08–7.10 (m, 1H, thiophene),
7.30–7.32 (m, 1H, thiophene), 7.33–7.40 (m, 4H, Ar), 7.56–7.58
(m, 1H, thiophene), 8.22 (s, 1H, CH@), 12.22 (br s, 1H, NH, D2O
exch.).
Yield 99%; mp 189–190 °C; 1H NMR (DMSO-d6) 1.00–1.03 (m,
3H, CH3), 1.12–1.15 (m, 1H, cyclopentyl), 1.89–2.43 (m, 5H, cyclo-
pentyl), 2.5–2.67 (m, 1H, cyclopentyl), 3.90 (s, 3H, OCH3), 6.61 (s,
1H, C5H-thiaz.), 7.24–7.38 (m, 4H, Ar), 10.70 (br s, 1H, NH, D2O
exch.).
5.3.17.1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(1-(thiophen-2-yl)-
ethylidene)hydrazine (17)
5.3.9. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-cyclohexylidene-
hydrazine (9)
Yield 75%; mp 220–221 °C; 1H NMR (DMSO-d6) 2.33 (s, 3H,
CH3), 3.79 (s, 3H, OCH3), 6.80 (s, 1H, C5H-thiaz.), 7.05–7.07 (m,
1H, thiophene), 7.30–7.38 (m, 2H, thiophene), 7.42–7.52 (m, 4H,
Ar), 11.40 (br s, 1H, NH, D2O exch.).
Yield 76%; mp 181–182 °C; 1H NMR (CDCl3) 1.57–1.60 (m, 4H,
cyclohexyl), 1.62–1.66 (m, 2H, cyclohexyl), 1.68–179 (m, 2H, cyclo-
hexyl), 2.66–2.72 (m, 2H, cyclohexyl), 3.91 (s, 3H, OCH3), 6.69 (s,
1H, C5H-thiaz.), 7.00–7.01 (m, 1H, Ar), 7.26–7.29 (m, 1H, Ar),
7.30–7.39 (m, 2H, Ar), 12.28 (br s, 1H, NH, D2O exch.).
5.3.18. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(1-(pyridin-2-yl)-
ethylidene)hydrazine (18)
Yield 75%; mp 236–237 °C; 1H NMR (DMSO-d6) 2.45 (s, 3H,
CH3), 3.79 (s, 3H, OCH3), 7.90 (s, 1H, C5H-thiaz.), 8.28–8.35 (m,
4H, Ar), 8.40–8.78 (m, 4H, pyridine), 10.90 (br s, 1H, NH, D2O
exch.).
5.3.10. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(2-methylcyclo-
hexylidene)hydrazine (10)
Yield 99%; mp 156–157 °C; 1H NMR (CDCl3) 1.15–1.16 (m, 3H,
CH3), 1.58–1.59 (m, 2H, cyclohexyl), 1.60–1.62 (m, 1H, cyclohexyl),
1.80–1.90 (m, 1H, cyclohexyl), 2.00–2.07 (m, 2H, cyclohexyl), 2.19–
2.89 (m, 3H, cyclohexyl), 3.91 (s, 3H, OCH3), 6.60 (s, 1H, C5H-thiaz.),
7.00 (s, 1H, Ar), 7.26–7.29 (m, 2H, Ar), 7.30–7.38 (m, 1H, Ar), 12.20
(br s, 1H, NH, D2O exch.).
5.3.19. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(pyridin-3-
ylmethylene)hydrazine (19)
Yield 99%; mp 214–215 °C; 1H NMR (DMSO-d6) 3.80 (s, 3H,
OCH3), 6.88 (s, 1H, C5H-thiaz.), 7.31–7.35 (m, 1H, Ar), 7.41–7.45
(m, 3H, Ar), 8.14–8.15 (m, 1H, pyridine), 8.62–8.70 (m, 3H, pyri-
dine), 9.03 (s, 1H, CH@), 12.30 (br s, 1H, NH, D2O exch.).
5.3.11. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(3-methylcyclo-
hexylidene)hydrazine (11)
Yield 93%; mp 148–149 °C; 1H NMR (CDCl3) 1.02–1.03 (m, 3H,
CH3), 1.09–1.11 (m, 2H, cyclohexyl), 1.58–1.69 (m, 5H, cyclo-
hexyl), 2.38–2.40 (m, 1H, cyclohexyl), 3.18–3.20 (m, 1H, cyclo-
hexyl), 3.91 (s, 3H, OCH3), 6.67 (s, 1H, C5H-thiaz.), 7.01 (s, 1H,
Ar), 7.22–7.28 (m, 2H, Ar), 7.30–7.36 (m, 1H, Ar), 12.18 (br s,
1H, NH, D2O exch.).
5.3.20. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(1-(pyridin-3-yl)-
ethylidene)hydrazine (20)
Yield 89%; mp 249–250 °C; 1H NMR (DMSO-d6) 2.40 (s, 3H,
CH3), 3.79 (s, 3H, OCH3), 6.89 (s, 1H, C5H-thiaz.), 7.33–7.35 (m,
1H, Ar), 7.44–7.51 (m, 3H, Ar), 8.19–8.20 (m, 2H, pyridine), 8.83–
8.85 (m, 2H, pyridine), 12.25 (br s, 1H, NH, D2O exch.).
5.3.12. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(4-methylcy-
clohexylidene)hydrazine (12)
5.3.21. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(pyridin-4-
ylmethylene)hydrazine (21)
Yield 76%; mp 152–153 °C; 1H NMR (CDCl3) 0.98 (s, 3H, CH3),
1.24–1.25 (m, 2H, cyclohexyl), 1.34–1.45 (m, 2H, cyclohexyl),
1.57–1.66 (m, 2H, cyclohexyl), 1.90–2.01 (m, 1H, cyclohexyl),
2.20–3.20 (m, 2H, cyclohexyl), 3.92 (s, 3H, OCH3), 6.67 (s, 1H,
Yield 99%; mp 250–251 °C; 1H NMR (DMSO-d6) 3.79 (s, 3H,
OCH3), 6.88 (s, 1H, C5H-thiaz.), 7.37–7.45 (m, 3H, Ar), 7.55 (s, 1H,
Ar), 8.10–8.13 (m, 4H, pyridine), 8.81 (s, 2H, CH@), 13.10 (br s,
1H, NH, D2O exch.).

5722
F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5715–5723
5.3.22. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(1-(pyridin-4-yl)-
ethylidene)hydrazine (22)
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 non-
enzymatic inhibition (e.g., for directly reacting with Amplex Red
reagent) was determined by adding these drugs to solutions con-
taining only the Amplex Red reagent in a sodium phosphate buffer.
The specific fluorescence emission (used to obtain the final
results) was calculated after subtraction of the background activ-
ity, 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 practically negligible.
Yield 80%; mp 169–170 °C; 1H NMR (DMSO-d6) 2.40 (s, 3H,
CH3), 3.79 (s, 3H, OCH3), 6.81 (s, 1H, C5H-thiaz.), 7.33–7.35 (m,
1H, Ar), 7.40–7.45 (m, 3H, Ar), 8.15–8.16 (m, 2H, pyridine), 8.81–
8.83 (m, 2H, pyridine), 12.20 (br s, 1H, NH, D2O exch.).
5.3.23. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(naphthalen-1-
ylmethylene)hydrazine (23)
Yield 85%; mp 201–202 °C; 1H NMR (DMSO-d6) 3.80 (s, 3H,
OCH3), 6.92 (s, 1H, C5H-thiaz.), 7.38–7.46 (m, 3H, Ar), 7.59–7.67
(m, 3H, Ar), 7.96–7.99 (m, 3H, Ar), 8.25–8.30 (m, 2H, Ar), 8.91 (s,
1H, CH@), 11.45 (br s, 1H, NH, D2O exch.).
5.3.24. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(1-(naphthalen-
2-yl)ethylidene)hydrazine (24)
5.5. Reversibility and irreversibility experiments
Yield 77%; mp 235–236 °C; 1H NMR (DMSO-d6) 2.42 (s, 3H,
CH3), 3.79 (s, 3H, OCH3), 6.95 (s, 1H, C5H-thiaz.), 7.25–7.48 (m,
7H, Ar), 7.90–8.20 (m, 4H, Ar), 11.30 (br s, 1H, NH, D2O exch.).
To evaluate whether the compound 20 is reversible or irrevers-
ible hMAO inhibitor, an effective centrifugation–ultrafiltration
method (so-called repeated washing) previously described by us
was used.7 Briefly, adequate amounts of recombinant hMAO-A or
hMAO-B were incubated together with a single concentration
(see Table 2) of the compound 20 or the reference inhibitors
R-(ꢀ)-deprenyl, isatin, and moclobemide 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 sample was stored at 4 °C and used for
subsequent measurement of hMAO activity under the experimen-
tal conditions indicated above (see the Section 5.4). The remaining
5.3.25. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(benzodioxol-5-
ylmethylene)-hydrazine (25)
Yield 79%; mp 229–230 °C; 1H NMR (DMSO-d6) 3.62 (s, 3H,
OCH3), 6.06 (s, 2H, OCH2O), 6.84 (s, 1H, C5H-thiaz.), 7.01–7.40
(m, 7H, Ar), 7.99 (s, 1H, CH@), 12.19 (br s, 1H, NH, D2O exch.).
5.3.26. 1-(4-(3-Methoxyphenyl)thiazol-2-yl)-2-((1H-indol-3-yl)-
methylene)-hydrazine (26)
incubated sample (300 lL) was placed in an Ultrafree-0.5 centrifu-
Yield 75%; mp 229–230 °C; 1H NMR (DMSO-d6) 3.79 (s, 3H,
OCH3), 6.96 (s, 1H, C5H-thiaz.), 7.15–7.23 (m, 4H, Ar), 7.30–7.44
(m, 4H, indole), 7.77 (s, 1H, CH@), 8.23 (s, 1H, NH-indole, D2O
exch.), 11.55 (br s, 1H, NH, D2O exch.).
gal tube (Millipore, Billerica, USA) with a 30 kDa Biomax mem-
brane in the middle of the tube and centrifuged (9000g, 20 min,
4 °C) in a centrifuge (J2-MI, Beckman Instruments, Inc., Palo Alto,
California, USA). The enzyme retained in the 30 kDa membrane
was resuspended in sodium phosphate buffer at 4 °C and centri-
fuged again (under the same experimental conditions described
above) two successive times. After the third centrifugation, the en-
zyme retained in the membrane was resuspended in sodium phos-
5.3.271. -(4-(3-Methoxyphenyl)thiazol-2-yl)-2-(1-(coumarin-3-yl)-
ethylidene)hydrazine (27)
Yield 95%; mp 242–243 °C; 1H NMR (DMSO-d6) 2.27 (s, 3H,
CH3), 3.79 (s, 3H, OCH3), 6.90 (s, 1H, C5H-thiaz.), 7.37–7.40 (m,
4H, Ar), 7.44–7.45 (m, 2H, coumarin), 7.50–7.70 (m, 2H, coumarin),
8.16 (s, 1H, ArCH@), 11.40 (br s, 1H, NH, D2O exch.).
phate buffer (300 lL) and an aliquot of this suspension was used
for subsequent hMAO isoform activity determination. Control
experiments were performed simultaneously (to define 100%
hMAO activity) by replacing the test drugs with appropriate dilu-
tions of the vehicles. The corresponding values of percent (%)
hMAO isoform inhibition were separately calculated for samples
with and without repeated washing. Furthermore, Ki values for
each compound have been calculated and reported in Supplemen-
tary data.
5.4. Determination of hMAO isoform activity
The effects of the test compounds on the enzymatic activity of
hMAO isoform were evaluated by a fluorimetric method following
the experimental protocol previously described by us.12 Briefly,
0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4) containing dif-
ferent concentrations of the test drugs (new compounds or refer-
ence inhibitors) and adequate amounts of recombinant hMAO-A
or hMAO-B required and adjusted to obtain in our experimental
conditions the same reaction velocity, that is, to oxidize (in the
5.6. Molecular modeling
The Protein Data Bank17 (PDB) crystallographic structures
2Z5X3 and 2BK34 were considered as receptor models of hMAO-A
and hMAO-B, respectively. Both E and Z isomers of compound 20
were built by means of the Maestro GUI.18 2000 steps of Monte
Carlo conformational search were applied to all rotatable bonds
of this inhibitor. The OPLS-AA19 force field, as implemented in
the version 7.2 of Macromodel software,20 was adopted to evaluate
the energy of the Monte Carlo structures. Water solvent effects
were taken into account using the implicit solvation model
GB/SA.21 Such an approach led to the identification of 66 and 72
unique conformations for the E and Z isomers, respectively. The
Boltzmann analysis was applied to both Monte Carlo structure
ensembles reporting a E:Z population ratio equal to 99.6:0.4. The
global minimum energy structures of E and Z isomers were sub-
mitted to docking simulations with respect to hMAO-A and -B
PDB crystallographic structures. Both receptor models required
graphical manipulation: the co-crystallized ligands, harmine and
farnesol, respectively, for 2Z5X and 2BK3, were removed, FAD
control group) 165 pmol of p-tyramine/min (hMAO-A: 1.1
protein; specific activity: 150 nmol of p-tyramine oxidized to
p-hydroxyphenylacetaldehyde/min/mg protein; hMAO-B: 7.5
lg
lg
protein; specific activity: 22 nmol of p-tyramine transformed/
min/mg protein) were incubated for 15 min at 37 °C in a flat-
black-bottomed 96-well microtest plate (BD Biosciences, Franklin
Lakes, NJ, USA) placed in the dark multimode microplate reader
chamber. After this incubation period, the reaction was started
by adding (final concentrations) 200 lM Amplex Red reagent,
1 U/mL horseradish peroxidase, and 1 mM p-tyramine (final satu-
rating concentration). The production of H2O2 and, consequently,
of resorufin was quantified at 37 °C in a multidetection microplate
fluorescence reader (Fluostar Optima, BMG Labtech GmbH, Offen-
burg, Germany) based on the fluorescence generated (excitation,
545 nm, emission, 590 nm) over a 15 min period, in which the fluo-
rescence increased linearly. Control experiments were carried out

F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5715–5723
5723
F.; Fioravanti, R.; Bolasco, A.; Manna, F.; Chimenti, P.; Secci, D.; Rossi, F.; Turini,
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4135; (c) Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.; Bizzarri, B.; Granese,
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double bonds were fixed, and hydrogen atoms were added onto
both proteins and cofactors. According to the Glide22 methodology,
a regular box, of about 110,000 Å3, centered onto the cofactor N5
atom, was considered as the enzyme active site for both hMAO-A
and -B models. In order to take into account the induced fit phe-
nomena, the ligand was docked using the Glide ‘flexible’ algorithm.
The binding affinity was evaluated using the free energy of com-
plexation (Ecvdw). The most stable complexes were submitted to
energy minimization using the same force field and aqueous envi-
ronment previously reported. The resulting optimized structures
were considered for the binding modes graphical analysis. PyMol
ver. 0.9823 was used to create Figure 2.
8. Chimenti, F.; Maccioni, E.; Secci, D.; Bolasco, A.; Chimenti, P.; Granese, A.;
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This work was supported by grants from MURST (Italy). Minis-
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