Selective inhibitory activity against MAO and molecular modeling studies of 2-thiazolylhydrazone derivatives

Article abstract of DOI:10.1021/jm060869d

A series of 2-thiazolylhydrazone derivatives have been investigated for the ability to inhibit the activity of the A and B isoforms of monoamine oxidase (MAO) selectively. All of the compounds showed high activity against both the MAO-A and the MAO-B isoforms with pK_i values ranging between 5.92 and 8.14 for the MAO-A and between 4.69 and 9.09 for the MAO-B isoforms. Both the MAO-A and the MAO-B isoforms, deposited in the Protein Data Bank as model 2BXR and 1GOS, respectively, were considered in a computational study performed with docking techniques on the most active and MAO-B-selective inhibitor, 18.

Full text of DOI:10.1021/jm060869d

J. Med. Chem. 2007, 50, 707-712
707
Selective Inhibitory Activity against MAO and Molecular Modeling Studies of
2-Thiazolylhydrazone Derivatives
Franco Chimenti,† Elias Maccioni,| Daniela Secci,*,† Adriana Bolasco,† Paola Chimenti,† Arianna Granese,† Olivia Befani,‡
Paola Turini,‡ Stefano Alcaro,§ Francesco Ortuso,§ Maria C. Cardia,| and Simona Distinto|
Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente AttiVe and Dipartimento di Scienze Biochimiche “A. Rossi
Fanelli” and Centro di Biologia Molecolare del CNR, UniVersita` degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy,
Dipartimento di Scienze Farmaco Biologiche “Complesso Nin`ı Barbieri”, UniVersita` degli Studi di Catanzaro “Magna Graecia”, 88021
Roccelletta di Borgia (CZ), Italy, and Dipartimento Farmaco Chimico Tecnologico, UniVersita` degli Studi di Cagliari,
Via Ospedale 72, 09124 Cagliari, Italy
ReceiVed July 24, 2006
A series of 2-thiazolylhydrazone derivatives have been investigated for the ability to inhibit the activity of
the A and B isoforms of monoamine oxidase (MAO) selectively. All of the compounds showed high activity
against both the MAO-A and the MAO-B isoforms with pKi values ranging between 5.92 and 8.14 for the
MAO-A and between 4.69 and 9.09 for the MAO-B isoforms. Both the MAO-A and the MAO-B isoforms,
deposited in the Protein Data Bank as model 2BXR and 1GOS, respectively, were considered in a
computational study performed with docking techniques on the most active and MAO-B-selective inhibitor,
18.
Introduction
zymes.10,12 Recently, Parini et al. have reported that the activity
of the MAO-A isoform increases with age in rat hearts.13
Furthermore, it has been shown that MAO is responsible for
the biotransformation of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-
pyridine (MPTP) into 1-methyl-4-phenylpyridinium, a Parkin-
son’s-producing neurotoxin14-16 and may also contribute to the
apoptotic process because inhibition of MAO activity suppresses
cell death.17
MAO is a flavoprotein present in the outer mitochondrial
membranes of neuronal, glial, and other cells. It catalyzes the
oxidative deamination of biogenic amines in the brain and the
peripheral tissues, regulating their level. MAO exists in two
forms, namely, MAO-A and MAO-B.1 MAO-A is sensitive to
the selective inhibitor, clorgyline, whereas MAO-B is irrevers-
ibly inhibited by selegiline (Figure 1).
For all of these reasons, 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”.18
In humans MAO-B inhibitors (MAO-B-Is) are useful as
coadjuvants in the treatment of Parkinson’s disease and perhaps
Alzheimer’s disease,2,19,20 whereas MAO-A inhibitors (MAO-
A-Is) are antidepressant and antianxiety agents (Figure 1).21-23
A recent description of the crystal structure of the two
isoforms of human MAO by Binda et al. provides relevant
information of the mechanism underlying the selective interac-
tions between these proteins and their ligands. They are helpful
in probing the catalytic mechanism and in gaining a better
understanding of the pharmacophoric requirements needed for
the rational design of potent and selective enzyme inhibitors
with a therapeutic potential.24-29
Numerous compounds among the great variety of substituted
hydrazines behave as MAO inhibitors.30-32 A common structural
feature of substrates and inhibitors is an amino or imino group
that is assumed to play an essential role in orientation and
complex formation at the active site of the enzyme.33
In earlier papers by Cambria and others,34-37 it was shown
that numerous hydrazinothiazole derivatives inhibit MAO-B
activity in the range of micromolar concentration. Moreover,
benzylidene-hydrazinothiazole derivatives were reported to be
the most active, with their activity being further enhanced by
the presence of a methyl group at position 4 of the thiazole
nucleus. Starting from these studies, we assayed a series of
2-thiazolylhydrazone derivatives (1-18) for their ability to
inhibit MAO activity selectively. Though, except for 15 and
16, the compounds were already known, their potential inhibi-
tory activity against MAO isoforms had never been de-
MAO-A and MAO-B metabolize the principal biogenic
amines, dopamine (DA), noradrenalin (NA), adrenalin, serotonin
(5HT), and 2-phenylethylamine (PEA). MAO plays an important
role in neurotransmitters and is a scavenger of various other
amines.2 The typical substrate of MAO-A is 5HT, whereas the
substrates of MAO-B are â-phenylethylamine and benzylamine.
Two neurotransmitters that are fundamental in human depres-
sion, NA and 5HT, are primarily deaminated by MAO-A.
Inhibition of MAO-B increases DA and 5HT levels in mammals
and also exhibits neuroprotective effects.3 MAO-B activity has
been shown to increase with age and is particularly high around
the senile plaques (SP) in Alzheimer’s disease (AD) patients.3,4
The oxidative deamination of biogenic amines catalyzed by
MAO-B produces hydrogen peroxide and other reactive oxygen
species (ROS).3,5-8 Direct evidence for free radical involvement
in the etiology of Parkinson’s disease (PD) has been reported
with increased lipid peroxidation and superoxide dismutase
(SOD) activity in the substantia nigra of PD patients.5-9 CNS
degenerative disorders such as AD and PD have been associated
with oxidative stress, and increased MAO-B activity, with
reduced free radical scavenging capacity.3,5-12 On the contrary,
MAO-A activity does not increase with age, suggesting
independent regulation of the expression of these two isoen-
* Corresponding author. Phone, Fax: +39 06 4991 3763. E-mail,
daniela.secci@uniroma1.it.
† Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Bio-
logicamente Attive, Universita` degli Studi di Roma “La Sapienza”.
‡ Dipartimento di Scienze Biochimiche “A. Rossi Fanelli” and Centro
di Biologia Molecolare del CNR, Universita` degli Studi di Roma “La
Sapienza”.
§ Universita` degli Studi di Catanzaro “Magna Graecia”.
| Universita` degli Studi di Cagliari.
10.1021/jm060869d CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/25/2007

708 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Chimenti et al.
Figure 1. Irreversible (I), reversible (R), and selective MAO-A and/or MAO-B (A or A/B or B) inhibitors.
Table 1. Structures and Inhibitory Activities of Derivatives 1-18a
scribed.38,39 Furthermore, we considered both the MAO-A and
the MAO-B isoforms deposited in the Protein Data Bank as
models 1GOS24 and 2BXR,26 respectively, in a computational
study performed with docking techniques on the most active
inhibitor benzaldehyde-4-methyl-(4-phenyl-2-thiazolyl)hydra-
zone 18.
Chemistry
2-Thiazolylhydrazone derivatives (1-18) were synthesized
as reported in our previous communications.38,39
Cyclic ketones or aryl aldehydes reacted directly with
thiosemicarbazide, and the obtained thiosemicarbazones sub-
sequently reacted with R-halogenoketones to yield the 4-sub-
stituted thiazole ring derivatives. In the synthesis of all of the
compounds, isopropyl alcohol proved to be the best solvent for
our purpose. As a matter of fact, the reaction products precipitate
upon cooling, and they can be filtered and purified by crystal-
lization from ethanol or ethanol/isopropanol. All of the synthe-
sized products were characterized by analytical methods as
reported in the literature.38,39
Results and Discussion
All of the compounds were first evaluated for their ability to
inhibit MAO in the presence of kynuramine as a substrate. After
the first assessment of their inhibitory effect on the MAO, we
tested the compounds to determine their activity toward MAO-A
and MAO-B selectively in the presence of the specific substrates,
serotonin and benzylamine, respectively (Table 1).
From Table 1, which shows the structures and the MAO
inhibition data along with the MAO inhibitory selectivity (pSI:
log selectivity index ) pKi(MAO-A) - pKi(MAO-B)), it can be seen
that all of the synthesized compounds show high activity against
both the MAO-A and the MAO-B isoforms, with pKi values in
the range of 5.92 to 8.14 and 4.69 to 9.09, respectively.
1-4, 2(3H)-thiazolone-4-phenyl-cycloalkylidenehydrazone
derivatives, showed selective inhibitory activity against MAO-A
with pKi’s in the range of 6.85 to 7.69 and the highest pSI’s of
2.7 for 3 and 1.48 for 4. 4-Phenyl-2-thiazolylhydrazone deriva-
tives showed inhibitory activity against MAO-A with pKi’s in
the range of 5.92 to 7.67 and against MAO-B with pKi’s from
7.00 to 9.09. 18 was the most potent of the series, which reached
the very interesting inhibitory activity value of pKi 9.09. 6 and
18 showed significant MAO-B selectivity with higher pSI values
of -1.12 and -1.42, respectively. 4-Methyl-2-thiazolylhydra-
zone derivatives showed interesting inhibitory activity against
both MAO-A and MAO-B, without any remarkable selectivity.
The computational work was carried out with the purpose of
rationalizing by molecular modeling the selectivity toward MAO
a The data represent mean values of at least of three separate experiments.
b pSI: log selectivity index ) pKi
- pKi
(MAO-A)
(MAO-B).
isoforms of the most active and selective MAO-B inhibitor 18.
We applied the same docking approach as in other recent
communications of ours,40 that is, performing a flexible docking

SelectiVe Inhibitory ActiVity against MAO
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 709
Figure 2. Most stable complexes of 18 and MAO isoforms: A (a, b), B (c, d), and B with Ile199 mutated in Phe199 (e, f). Interacting residues
of the active site are shown in labeled polytubes, FAD in the CPK rendering, 18 in the blue carbon polytube, and other aminoacids in the ribbon
in MAO-A (a), MAO-B (c), and mutated MAO-B (e) isoforms. LigPlot representation of the same MAO-A (b), MAO-B (d), and mutated MAO-B
(f) complexes reported without hydrogens. Hydrogen bonds are reported as dashed lines between heavy atoms.
search with the Monte Carlo (MC) method implemented in the
Macromodel software41 as described in the Experimental
Section.
mutated in Phe, the resulting ∆∆G was equal to -44.87 kcal/
mol, still in agreement with the isoform selectivity inhibition.
The analysis of the stereoisomer contributions to the MAO
binding showed that in both isoforms the E form plays a more
important role than the Z form, with ∆∆GE-Z differences on
the order of 2 to 3 kcal/mol. Moreover, the greater importance
of the E form has also been highlighted by a Boltzmann
population analysis, which was carried out on enantiomer
ensembles, and in all simulations for both MAO-A and MAO-B
complexes it reported a probability of existence of about 100%.
With the aim of understanding the reason for the selectivity
of 18 at the molecular level, using the graphical tools cited in
the Experimental Section we inspected the most stable geom-
etries of this compound within the enzymatic clefts of MAO-A
and MAO-B. Figure 2 shows pictures of these geometries. In
MAO-A, (Figure 2a,b) recognition of 18 is relatively close to
the FAD. The phenyl ring of the R1 substituent interacted with
hydrophobic residues such as Phe177, Leu176, Phe173, and
Ile325, establishing about 20 contacts computed by the LigPlot43
analysis. The strongest interaction was found with another
hydrophobic residue (Phe208) with 22 contacts. Minor interac-
tions were established with other residues, in particular, by the
R substituent. In this energy-minimum configuration, we found
no intermolecular hydrogen bonds.
We built and energy-minimized a preliminary model of 18.
We then created a starting configuration of the 18 complex with
each enzyme, searching the most stable GLIDE42 pose with the
pretreated enzyme models as reported in the Experimental
Section. In agreement with the selectivity data for 18, in both
cases GLIDE energies (GE) and scores (GS) were lower in
MAO-B (GE ) -43.2 and GS ) -8.14) than in MAO-A (GE
) -16.5 and GS ) -6.36).
These energy-minimum configurations were used for a more
accurate and complete docking search with the MC method.
As a matter of fact, in the latter search the imine bond was also
set to rotate freely, exploring both E and Z configurations. The
generation of the binding modes in the enzyme proved to be
quite difficult in the MAO-A isoform, where more than 27
configurations were found with an average number of duplicates
of 1.33. Conversely, in MAO-B the MC search was more
successful, with 87 configurations and an average number of
duplicates of 2.54. These statistical data suggested a more
accessible MAO-B cleft for 18. After full minimization, all of
the MC-generated configurations were submitted to a statistical
thermodynamic analysis according to the MOLINE method.43
The free complexation energy (∆∆G) was once in better
agreement with the experimental selectivity trend, with values
of -41.91 (MAO-A) and -47.05 (MAO-B) kcal/mol. Repeating
the calculation with the MAO-B model with Ile199 virtually
Conversely, recognition of 18 within the MAO-B cleft proved
to be more productive (Figure 2c,d). In this case, two hydro-
phobic sites of the enzyme were implicated in the most stable
binding mode. One was based on hydrophobic amino acids

710 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Chimenti et al.
mL, 0.1 N). The 2-thiazolylhydrazone derivatives were dissolved
in dimethyl sulfoxide (DMSO) and added to the reaction mixture
at concentrations ranging from 0 to 10-3 mM. To study the effect
of the inhibition of 2-thiazolylhydrazone derivatives on the activities
of both MAO-A and MAO-B separately, the mitochondrial fractions
were preincubated at 38 °C for 30 min with the appropriate specific
irreversible inhibitor (0.5 µM L-deprenyl to eliminate MAO-B from
the assay of MAO-A activity and 0.05 µM clorgyline to eliminate
MAO-A from the assay for the B isoform). Fluorimetric measure-
ments were recorded with a Perkin-Elmer LS 50B spectrofluorim-
eter. The protein concentration was determined according to
Bradford.47
Dixon plots were used to estimate the inhibition constant (Ki)
of the inhibitors (Table 1). The data are the means of three
experiments performed in duplicate. It is interesting that all of the
compounds act through the reversible mode, as shown by dialysis
performed over 24 h in a cold room against a potassium phosphate
buffer (0.1 M, pH 7.2) capable of restoring 90-100% of the activity
of the enzyme.
Molecular Modeling. Following the computational work of
recent communications of ours,40 the Protein Data Bank48 crystal-
lographic models of human MAO-A and MAO-B, respectively
known by the pdb codes 2BXR26 and 1GOS,24 were considered
for the docking experiments knowing that the comparison of the
human and bovine sequences at the catalytic site revealed a high
level of identity.49 Both structures were obtained as adducts with
two similar compounds, clorgyline for 2BXR and pargyline for
1GOS, covalently linked to the FAD N5 nitrogen. To simulate the
active site of the bovine MAO-B, the 1GOS model was virtually
mutated, replacing the Ile199 with a Phe residue.
Phe168, Trp119, Leu167, Leu164, Pro104, Ile316, and Ile199
(about 20 contacts). The other was based on FAD cofactors
Tyr398 and Tyr435 (about 35 contacts). With these two aromatic
residues, a π-π stacking interaction is established with the R1
phenyl moiety. This type of interaction was found to be a
peculiarity of many potent MAO inhibitors.44 Moreover, an
intermolecular hydrogen bond interaction between the iminic
hydrogen and the Cys172 side chain stabilized this configuration.
The same simulation carried out with a MAO-B model
obtained; replacing Ile199 with Phe199 led to a similar binding
mode. The list of residues involved in the most stable complex
was very similar to that of the parent model. The difference in
this list was merely qualitative. Instead of Leu167 and Phe343,
this model included Gly434 and Ile198. The reason for this
different residue recognition should be attributed to an ap-
proximately 180° rotation of the binding mode around the
thiazole moiety as shown in Figure 2e,f. The most relevant
consequence in the interaction pattern was due to an additional
hydrophobic contact between Phe199 and the toluene moiety
of 18 and the lack of hydrogen bonding with Cys172. Therefore,
the ligand-enzyme interaction energy obtained in this case was
relatively higher than the binding pose computed with the
original 1GOS model. Despite the mutation of the Ile199
residue, which involved a partial destabilization of about 2.18
kcal/mol in the free energy of complexation of 18 within the
MAO-B structures, all of the docking models resulted in
agreement with the experimental selectivity.
Globally, the number of contacts and the different hydrogen
bond network of 18 docked into the two clefts explained the
higher selectivity for the MAO-B isoform. Finally, it is
interesting that the same extended conformation of compound
18 recognized the MAO-A and MAO-B isoforms in an opposite
configuration with respect to the R and R1 aromatic moieties
closer to the cofactor (Figure 2).
This preliminary study, carried out with the most active
compound of this series, explained the nature of the selectivity
of this inhibitor and can aid the design of new compounds for
introducing appropriate substituents onto the 2-thiazolylhydra-
zone scaffold.
After the covalent bond between the FAD moiety and the
cocrystallized inhibitor was removed, the pretreatment of the
original pdb models consisted of a 48 kcal/mol constrained energy
minimization of those residues within a 15 Å radius of the N5 of
the isoalloxazine ring in order to restore the natural planarity of
the isoalloxazine FAD ring and relax the active-site amino acids.
In the resulting energy-minimum structures, ligands were removed
and used as receptor models. This was done with the force field
AMBER* united atom notation and the GB/SA water implicit model
of solvation as implemented in MacroModel ver 7.2.41
Starting geometries of 18 complexes with MAO-A and MAO-B
were generated by the GLIDE docking method.42 Both pretreated
enzyme models were submitted to map calculations using a box of
about 110 000 Å3 centered on the FAD N5 atom. Flexible docking
of the 18 compound was done by generating a maximum number
of 5000 configurations. The most stable GLIDE pose was analyzed
and used as a starting configuration for a more accurate docking
search performed by Monte Carlo using the MOLS directive for
rototranslational settings, that is, allowing each ligand to rotate a
full (180° and to translate with a tolerance of (10 Å. One thousand
MC iterations were carried out, and each configuration was energy-
minimized with the AMBER* united atom and the GB/SA water
implicit model of solvation. The same constrained energy minimi-
zation conditions were adopted during the MC search. The obtained
MC ensemble was then full energy-minimized, removing any
constant force. The same docking route was carried out with the
original crystallographic coordinates, that is, with the nonplanar
FAD ring conformation, obtaining no significant difference in the
results because of the final full energy-minimization procedure.
The interaction energy of all complexes before and after full
relaxation were computed according to the MOLINE method43 and
converted to average state equations.
Experimental Section
Chemistry. General Procedure for the Preparation of 2-Thia-
zolylhydrazone Derivatives (1-18). The appropriate cyclic ketones
or aryl aldehydes were dissolved in 2-propanol, which is the most
suitable solvent, and were refluxed under magnetic stirring with
thiosemicarbazide for 24 h. The obtained thiosemicarbazones
subsequently reacted with 2-bromo-acetophenone at room temper-
ature or with 2-chloro-acetone at reflux, with both dissolved in
2-propanol. After cooling to room temperature, the solution was
filtered, and the desired derivatives were crystallized from ethanol
or ethanol/isopropanol to give compounds 1-18, respectively. Only
for compounds 15 and 16 do we report the chemical and physical
properties (see Supporting Information).
Biochemistry. All of the chemicals were commercial reagents
of analytical grade and were used without purification. Bovine brain
mitochondria were used as the enzyme source and were isolated
according to the Basford method.45 In all of the experiments, the
AO activities of the beef brain mitochondria were determined by a
fluorimetric method according to Matsumoto et al.46 using kynuramine
as a substrate at four different final concentrations ranging from 5
µM to 0.1 mM.
All calculations were made with a Linux cluster of 8 Intel Xeon
dual processors at 3.2 GHz with 2 GB of RAM. Graphic
manipulations and analysis of the docking experiments were
performed by the Maestro Graphical User Interface version 4.1.012
for Linux operating systems.41 Ligplot version 4.050 and Pymol
version 0.9850 were used to create Figure 2.
Briefly, the incubation mixtures contained potassium phosphate
buffer (0.1 mL, 0.25 M, pH 7.4), mitochondria (6 mg/mL), and
drug solutions with final concentrations ranging from 0 to 10-3
µM. The solutions were incubated at 38 °C for 30 min. The addition
of perchloric acid ended the reaction. The samples were centrifuged
at 10 000g for 5 min, and the supernatant was added to NaOH (2.7
Acknowledgment. This work was supported by grants from
MURST. We also acknowledge Mr. Anton Gerada, a profes-

SelectiVe Inhibitory ActiVity against MAO
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 711
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(26) De Colibus, L.; Li, M.; Binda, C.; Lustig, A.; Edmondson, D. E.;
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sional translator, Fellow of the Institute of Translation and
Interpreting of London and Member of the AIIC (Association
Internationale des Interpreˆtes de Confe´rences-Geneva), for the
revision of the manuscript.
Supporting Information Available: Analytical data for new
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
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