(Thiazol-2-yl)hydrazone derivatives from acetylpyridines as dual inhibitors of MAO and AChE: Synthesis, biological evaluation and molecular modeling studies

Article abstract of DOI:10.3109/14756366.2014.987138

Several (thiazol-2-yl)hydrazone derivatives from 2-, 3- and 4-acetylpyridine were synthesized and tested against human monoamine oxidase (hMAO) A and B enzymes. Most of them had an inhibitory effect in the low micromolar/high nanomolar range, being derivatives of 4-acetylpyridine selective hMAO-B inhibitors also at low nanomolar concentrations. The structure-activity relationship, as confirmed by molecular modeling studies, proved that the pyridine ring linked to the hydrazonic nitrogen and the substituted aryl moiety at C4 of the thiazole conferred the inhibitory effects on hMAO enzymes. Successively, the strongest hMAO-B inhibitors were tested toward acetylcholinesterase (AChE) and the most interesting compound showed activity in the low micromolar range. Our results suggest that this scaffold could be further investigated for its potential multi-targeted role in the discovery of new drugs against the neurodegenerative diseases.

Full text of DOI:10.3109/14756366.2014.987138

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–12
2015 Informa UK Ltd. DOI: 10.3109/14756366.2014.987138
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RESEARCH ARTICLE
(Thiazol-2-yl)hydrazone derivatives from acetylpyridines as dual
inhibitors of MAO and AChE: synthesis, biological evaluation and
molecular modeling studies
Melissa D’Ascenzio1, Paola Chimenti1, Maria Concetta Gidaro2, Celeste De Monte1, Daniela De Vita1, Arianna
1
1
1
2
2
3
Granese , Luigi Scipione , Roberto Di Santo , Giosue Costa , Stefano Alcaro , Matilde Yan˜ez , and Simone Carradori
4
`
´
2
1Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome, Italy, Dipartimento di Scienze della Salute,
3
4
`
Universita di Catanzaro, Catanzaro, Italy, University of Santiago de Compostela, Santiago de Compostela, Spain, and Department of Pharmacy,
‘‘G. D’Annunzio‘‘ University of Chieti-Pescara, Chieti Scalo (CH), Italy
Abstract
Keywords
Several (thiazol-2-yl)hydrazone derivatives from 2-, 3- and 4-acetylpyridine were synthesized
and tested against human monoamine oxidase (hMAO) A and B enzymes. Most of them had
an inhibitory effect in the low micromolar/high nanomolar range, being derivatives of
4-acetylpyridine selective hMAO-B inhibitors also at low nanomolar concentrations. The
structure–activity relationship, as confirmed by molecular modeling studies, proved that
the pyridine ring linked to the hydrazonic nitrogen and the substituted aryl moiety at C4 of
the thiazole conferred the inhibitory effects on hMAO enzymes. Successively, the strongest
hMAO-B inhibitors were tested toward acetylcholinesterase (AChE) and the most interesting
compound showed activity in the low micromolar range. Our results suggest that this scaffold
could be further investigated for its potential multi-targeted role in the discovery of new drugs
against the neurodegenerative diseases.
Acetylpyridine, AChE inhibitors, hMAO
inhibitors, molecular modeling,
neurodegenerative diseases,
(thiazol-2-yl)hydrazone derivatives
History
Received 14 October 2014
Revised 10 November 2014
Accepted 10 November 2014
Published online 25 March 2015
Introduction
neurodegeneration2,3. Furthermore, it has been demonstrated
that an over-expression of MAO-B isoform is involved in the
onset of neurodegenerative diseases such as Alzheimer’s (AD)
and Parkinson’s disease (PD), whereas the deregulation of
MAO-A activity in the cortex could be the cause of mental
disorders like depression, due to the reduction of catecholami-
nergic neurotransmitters4. Specifically, in patients affected by AD
there are structural alterations of numerous cerebral neuronal
circuits especially in the hippocampus and cortical areas,
characterized by the formation of senile plaques and neurofibril-
lary tangles. The degeneration of subcortical cholinergic and
monoaminergic systems also occurs, playing an important role in
the onset of AD due to the fact that these degenerated neurons,
with long and poorly myelinated axons, innervate the cerebral
areas involved in attention and memory acquisition and retrieval5.
On the other side, PD is the second most common neurodegen-
erative disease caused by the depletion of dopamine in
nigrostriatal neurons which are involved in the control of motor
function in the basal ganglia: the loss of dopaminergic neurons in
the substantia nigra provokes bradykinesia, rigidity, hypokinesia
and akinesia6,7. Considering all these features and the involvement
of a deregulated metabolism of monoamine transmitters in the
onset of neurodegenerative and mental disorders, the development
of new specific MAO inhibitors endowed with a selective activity
Monoamine oxidases A and B (MAO-A and -B) are flavin
adenine dinucleotide (FAD)-depending enzymes present in the
outer side of mitochondrial membrane. They are encoded by
different genes which are located on the X chromosome and could
derive from a common gene, because of their identical exon–
intron organization. MAO enzymes can be found in brain, liver,
blood platelets, placenta and gastrointestinal tract, but their action
and concentration in these tissues are different: MAO-A activity
predominates in the placenta and the gut, whereas MAO-B in the
blood platelets, the liver and in the human basal ganglia1. In
the brain, MAO-A is expressed in catecholaminergic neurons of
the cortex while MAO-B is present in serotonergic neurons. Both
enzymes catalyze the oxidative deamination of endogenous
neurotransmitters: even if they both degrade dopamine and
p-tyramine, MAO-A mostly oxidizes serotonin, noradrenaline
and adrenaline, whereas MAO-B generally metabolizes the small
amines like benzylamine and phenethylamine. This metabolic
pathway of endogenous amines is responsible for the production
of highly reactive peroxides causing oxidative damage and
and low toxicity is an open challenge8–14
.
Address for correspondence: Dr. Simone Carradori, Department of
Pharmacy, ‘‘G. D’Annunzio’’ University of Chieti-Pescara, Via dei
Vestini 31, 66100 Chieti Scalo (CH), Italy. E-mail: simone.carradori@
unich.it
Currently, there are three FDA-approved drugs based on
the ‘‘one drug, one target’’ paradigm (donepezil, galantamine
and rivastigmine) that improve symptoms by inhibiting

2
M. D’Ascenzio et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
acetylcholinesterase evidencing the limitations of this strategy for hydrogen peroxide (H2O2) from p-tyramine (a common substrate
the complex nature of AD. Conversely, the most current for both hMAO-A and hMAO-B), using the AmplexÕ Red MAO
innovative therapeutic approach is based on the ‘‘one drug, assay kit and microsomal MAO isoforms prepared from insect
multiple targets’’ paradigm, which suggests the use of compounds cells (BTI-TN-5B1-4) infected with recombinant baculovirus
with multiple activities at different target sites15,16. MTDL (multi- containing cDNA inserts for hMAO-A or hMAO-B. The produc-
target directed ligand) strategy targeting MAO-B represents a tion of H2O2 catalyzed by MAO isoforms can be detected using
promising approach to treat complex and multifaceted neurode- 10-acetyl-3,7-dihydroxyphenoxazine (AmplexÕ Red reagent), a
generative diseases17. This approach mainly includes, in AD, dual non-fluorescent and highly sensitive probe that reacts with H2O2
inhibitors of monoamine oxidase and acetylcholinesterase in the presence of horseradish peroxidase to produce a fluorescent
(AChE).
product, resorufin. In this study, hMAO activity was evaluated
The therapeutic potential of MAO-B inhibitors in AD has using the above method following the general procedure previ-
been also proposed due to their validated neuroprotective/ ously described by us33. The test drugs (new compounds and
neurorestorative and cognitive enhancing abilities besides their reference inhibitors) themselves were unable to react directly with
positive effect on monoaminergic transmission. In detail, scaf- the AmplexÕ Red reagent, which indicates that these drugs do not
folds containing a propargylamine moiety are of particular interfere with the measurements.
interest due to their reported beneficial actions18,19
.
In our experiments and under our experimental conditions,
In a previous work we have demonstrated that the concurrent hMAO-A displayed a Michaelis constant (Km) equal to 457.17
inhibition of this enzyme and the binding to the 5-HT3 receptor 38.62 mM and a maximum reaction velocity (Vmax) in the control
can lead to interesting compounds20. AChE is a serine hydrolase group of 185.67 12.06 nmol p-tyramine/min/mg protein whereas
mainly found at neuromuscular junctions and cholinergic brain hMAO-B showed a Km of 220.33 32.80 mM and a Vmax equal to
synapses21. AD is associated with a loss of cholinergic neurons in 24.32 1.97 nmol p-tyramine/min/mg protein (n ¼ 5). Most tested
the brain and a decreased level of AChE, thus the major drugs concentration-dependently inhibited this enzymatic control
therapeutic goal in the AD treatment strategies is the inhibition activity (Tables 1–3).
of brain AChE22.
Taking into account our recent studies for the development of In vitro inhibition studies of AChE
new MAO inhibitors in which we explained that a pyridine ring
Electric eel AChE (EC 3.1.1.7), acetylthiocholine iodide, 5,50-
linked at N1 of the hydrazine moiety, together with the
dithio-bis-(2-nitrobenzoic acid) (DTNB) and donepezil, used as
simultaneous presence of an aromatic substituent at C4 of the
reference standard, were purchased from Sigma-Aldrich (Milan,
thiazole nucleus, could confer an inhibitory activity on hMAO
Italy). All other chemical and biological reagents and solvents
enzymes23,24, we designed 36 (thiazol-2-yl)hydrazone derivatives
used were of the highest analytical, commercially available grade.
of acetylpyridine regioisomers. Supported by molecular recog-
We determined the inhibitory activity by the spectrophotometric
nition studies we evaluated: the influence of 2-, 3- and
method of Ellman34 based on the reaction of released thiocholine
4-acetylpyridines bound to the hydrazonic nitrogen; the effect
to give a colored product with a chromogenic reagent DTNB, to
of several electron-withdrawing groups (chloride, bromide and
evaluate the inhibition of AChE. The study was carried out as
iodide), electron-donating groups (methoxy and methyl) and the
previously described35 using a double beam UV–Vis lambda 40
non-substituted phenyl ring as substituents of the aryl moiety
Perkin Elmer spectrophotometer and optical polystyrene cuvettes
linked at C4 of the pharmacophoric nucleus. Furthermore,
(10 ꢀ 10 ꢀ 45 mm, 340–800 nm optical transparency). AChE was
keeping in mind our study on coumarin derivatives as hMAO
daily tested to evaluate the effective enzymatic activity. Tested
inhibitors25,26, we also evaluated the effect on hMAO inhibition
compounds were dissolved in DMSO or H2O/DMSO. The final
due to the presence of this ring. In addition, considering the
DMSO content of the assay mixture (50.05%) had no effect on
structural analogy among our compounds and some cholinester-
enzyme activity. Each measure was repeated at least in triplicate.
ase inhibitors described in the literature27, we have selected the
In order to calculate the percent inhibition, each compound (final
most potent and selective hMAO-B inhibitors to define their
concentration 3 mM) was mixed in a polystyrene cuvette with
activity also towards acetylcholinesterase.
3.0 mL of 0.1 M (pH 7.4) phosphate buffer containing DTNB
(0.25 mM) and AChE (0.083 U mLꢁ1).
Materials and methods
The reaction was initiated by the addition of substrate,
Chemistry
acetylthiocholine iodide (final concentration 100 mM), then
changes in the absorbance at 412 nm at 25 ꢂC were recorded
between 0.5 and 1.5 min after substrate addition. The percent
inhibition was calculated using the following equation:
(4-Substituted-thiazol-2-yl)hydrazone compounds (1–36) were
synthesized in high yields as reported in our previous commu-
nications28–32. Acetylpyridine regioisomers reacted directly with
thiosemicarbazide in ethanol in presence of catalytic amounts of
acetic acid at room temperature. Then, the resulting thiosemi-
carbazones were reacted in ethanol with the appropriate
a-bromoacetophenone through Hantzsch cyclization to give
(4-substituted-thiazol-2-yl)hydrazone derivatives. All the synthe-
sized products were washed with n-hexane, petroleum ether
and diethyl ether and purified by chromatography (ethyl acetate/
petroleum ether) before characterization by spectroscopic
methods and elemental analysis.
AcꢁAi
inhibition ð%Þ ¼
ꢀ100
Ac
where Ai and Ac represent the change in the absorbance in the
presence of inhibitor and without inhibitor, respectively.
To calculate the Ki value for compound 26, the hydrolysis rates
(v) were measured at various acetylthiocholine (75–150 mM) and
inhibitor (0.5–2.5 mM) concentrations. The data were analyzed
with the enzyme kinetic module of SigmaPlot, version 8.02 a
(Sigma-Aldrich), in order to find the best fitting model of
inhibition, as indicated by calculated linear regression coeffi-
cient r2. Its Ki value was obtained according to Dixon’s method,
reporting the reciprocal of the hydrolysis rate versus the inhibitor
concentrations.
Biochemistry
In vitro inhibition studies of hMAO-A and hMAO-B
The biological evaluation of the test drugs on hMAO activity was
investigated by measuring their effects on the production of

DOI: 10.3109/14756366.2014.987138
(Thiazol-2-yl)hydrazone derivatives
3
Table 1. Inhibitory activity of 2-acetylpyridine derivatives 1–12 towards hMAO enzymes.
Compound
R
IC50 hMAO-A
**
IC50 hMAO-B
**
Ratio
1
2
3
9.43 0.72 mMb
6.18 0.42 mM
3.73 0.22 mM
1.53
1.27
4.74 0.32 mMb
4
2.39 0.16 mMa
880.30 49.16 nM
2.72
5
6
7
8
2.39 0.16 mMa
4.93 0.35 mM
3.38 0.15 mMb
6.18 0.12 mMa
1860.45 95.71 nM
1.28
0.92
1.39
4.44
5.36 0.43 mM
2436.75 101.94 nM
1393.10 96.73 nM
9
4.38 0.15 mMa
995.88 96.73 nM
4.40
10
11
5.00 0.21 mMb
2049.73 94.78 nM
4480.13 271.01 nM
2.44
1.04
4.64 0.13 mM
12
4.76 0.28 mM
***
50.05#
Refere notes of Table 3.

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M. D’Ascenzio et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Table 2. Inhibitory activity of 3-acetylpyridine derivatives 13–24 towards hMAO enzymes.
Compound
R
IC50 hMAO-A
IC50 hMAO-B
Ratio
0.99
13
5.02 0.16 mM
5.09 0.12 mM
3.89 0.06 mM
1.51 0.11 mM
14
15
4.35 0.38 mM
1.12
7.02
10.60 0.84 mMa
16
3.42 0.24 mMa
985.47 49.70 nM
3.47
17
18
19
20
***
***
6.74 0.36 mM
***
3.47 0.28 mMa
25.59 1.96 mM
5.15 0.27 mMa
0.51
50.26#
12.05
427.54 29.46 nM
21
10.08 0.64 mMa
1550.23 70.21 nM
6.50
22
23
***
***
3.36 0.18 mM
3570.11 120.06 nM
0.94
24
6.03 0.19 mM
***
50.06#
Refere notes of Table 3.

DOI: 10.3109/14756366.2014.987138
(Thiazol-2-yl)hydrazone derivatives
5
Table 3. Inhibitory activity of 4-acetylpyridine derivatives 25–36 and reference drugs towards hMAO enzymes.
Compound
R
IC50 hMAO-A
***
IC50 hMAO-B
Ratio
25
14.14 0.80 mM
47.07#
26
27
2.53 0.06 mMa
4.24 0.09 nM
5.02 0.23 nM
597.00
910.36
4.57 0.16 mMa
28
26.93 1.23 mMa
12.79 0.98 nM
2105.55
29
30
31
32
24.01 1.62 mMa
13.00 0.57 mMa
0.49 0.03 mMb
8.06 0.64 mMa
22.59 1.06 nM
73.52 6.98 nM
842.30 36.19 nM
260.38 11.61 nM
1062.42
176.82
0.58
30.95
33
14.91 0.96 mMa
1662.28 31.45 nM
8.97
34
35
***
62.59 3.91 nM
41597.70#
7.65 0.54 mMa
3311.26 114.23 nM
2.31
(continued )

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M. D’Ascenzio et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Table 3. Continued
IC50 hMAO-A
Compound
R
IC50 hMAO-B
Ratio
36
4.87 0.39 mMa
1280.65 40.95 nM
3.80
Clorgyline
R-(-)-Deprenyl
Iproniazid
Moclobemide
Isatin
4.46 0.32 nMa
67.25 1.02 mM
6.56 0.76 mM
361.38 19.37 mM
***
61.35 1.13 mM
19.60 0.86 nM
7.54 0.36 mM
*
0.000073
3431.12
0.87
50.36#
45.33#
18.75 1.24 mM
Ratio: hMAO-B selectivity index ¼ IC50 (hMAO-A)/IC50 (hMAO-B).
Each IC50 value is the mean S.E.M. from five experiments (n ¼ 5).
Level of statistical significance: ap50.01 or bp50.05 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 mM (highest concentration tested).
***100 mM inhibits the corresponding MAO activity by approximately 40–50%. At higher concentration the
compounds precipitate.
#Values obtained under the assumption that the corresponding IC50 against MAO is the highest concentration tested
(100 mM for compounds and Isatin or 1 mM for Moclobemide).
Results and discussion
IC50 of 19.60 nM). Finally, unlike its 2- and 3-acetylpyridine
analogues (selective MAO-A inhibitors with ratio 50.06), the
coumarin derivative of 4-acetylpiridine (36) possessed a reversed
selectivity (ratio ¼ 3.80). These biological results demonstrated
the importance of the pyridine ring linked at the hydrazonic N1
and of a substituted aryl moiety at C4 of the thiazole nucleus
for the development of new hMAO inhibitors. Moreover,
4-acetylpyridine derivatives could be further investigated for
their potential role in the management of neurodegenerative
diseases caused by hMAO-B over-expression.
To better understand the molecular mechanism of hMAO-A
and hMAO-B inhibition, docking simulations were performed for
the most active compound 26 and its 2- and 3-acetylpyridine
analogues, compounds 14 and 2, respectively. Both E and Z
isomers of the 3D molecules were docked into the binding pocket
of the enzymes in order to estimate the ligand binding affinity by
means of Glide SP scoring function (Gscore) in kcal/mol36. For
each compound the corresponding final score has been considered
as the average of E and Z isomers Gscore and, in according to
the biological data, confirmed the better accommodation into the
hMAO-B active site with respect the hMAO-A one (Table 4). On
the basis of not discriminant Gscore values, for the best pose
of docked compounds the binding energy (dG Bind) by Prime
MM-GBSA utility37 were also calculated and, in agreement
with the experimental IC50 data, compound 26 showed the best
dG Bind value for the hMAO-B isoform, corresponding to
ꢁ66.94 kcal/mol.
We designed 36 (thiazol-2-yl)hydrazone derivatives of 2-, 3- and
4-acetylpyridine as potential human MAO inhibitors. According
to the biological results reported in Tables 1–3, with the only
exception of products 1, 17 and 22 that were not active, all the
compounds have shown to be hMAO inhibitors in low micro-
molar/high nanomolar range and most of the 4-acetylpyridine
derivatives displayed a selective inhibitory activity at low
nanomolar concentrations. As regards 2-acetylpyridine deriva-
tives (Table 1), the presence of a methyl group (compound 4),
a methoxy group (8) and a further phenyl moiety (9) at the para
position of the aromatic ring conferred a discrete selectivity
towards B isoform. In detail, compound 4 had an IC50 of 2.39 mM
against hMAO-A and 880.30 nM against hMAO-B, compound 8
possessed an IC50 of 6.18 mM against hMAO-A and 1393.10 nM
against hMAO-B, while the IC50 values of compound 9 towards
hMAO-A and -B were 4.38 mM and 995.88 nM, respectively.
Regarding 3-acetylpyridine derivatives (Table 2), the selectivity
towards hMAO-B of compounds containing a methyl group (16),
a methoxy group (20) and a further phenyl moiety (21) at C4
position of the aryl moiety increased. Moreover, for compound 20
the concentration required for the inhibitory effect on hMAO-B
diminished (IC50 of 427.54 nM). On the contrary, the presence of
the coumarin ring in 2- and 3-acetylpyridines 12 and 24 conferred
a high hMAO-A selectivity with an IC50 of 4.76 and 6.03 mM
respectively, which were better than that of the reference drug
moclobemide (361.38 mM). Finally, 4-acetylpyridine derivatives
reported in Table 3 have proven to be the most active and selective
inhibitors. In this series, 4-chlorophenyl (29) and 2,4-dichlor-
ophenyl (34) derivatives showed a great selectivity of action
towards B isoform at very low concentrations (22.59 and
62.59 nM, respectively). Also derivatives 26, 27 and 30, which
contained 2-, 3- and 4-bromophenyl substituents respectively,
were selectively active against hMAO-B (4.24 nM for compound
26, 5.02 nM for 27 and 73.52 nM for 30). Compound 27 was more
selective (ratio ¼ 910.36) than the other two bromophenyl
regioisomers as well as compounds 32 and 33 with respect
to their corresponding 2- and 3-acetylpyridine derivatives. The
4-methylphenyl substituted compound 28 was not only one of the
most efficacious products (12.79 nM), but also the most selective
human MAO-B inhibitor of this scaffold (ratio ¼ 2105.55, which
is similar to that of the reference drug R-(ꢁ)-deprenyl, that had an
Despite the different inhibitory activity of compounds 26, 14
and 2, in terms of IC50 values, the configuration of the double
bond and the substitution to the pyridine ring did not remarkably
influence the E and Z isomers recognition into the hMAO-B
catalytic site: in all cases the bromophenyl substituent was
oriented toward the FAD cofactor and the pyridine was located at
the entrance site (Figure S1). This binding mode is common to the
E, but not to the Z isomers, docked into the hMAO-A lipophilic
cage: the configuration of the double bond caused the orientation
of pyridine ring toward the FAD with the bromophenyl substituent
located at the gate of the catalytic site (Figure S2).
As reported in our previous work, amino acid residues in the
area opposite to the flavin: hMAO-A ILE180, ASN181, PHE208,
SER209 and ILE335 with the corresponding hMAO-B LEU171,
CYS172, ILE199, SER200 and TYR326, respectively, strongly

DOI: 10.3109/14756366.2014.987138
(Thiazol-2-yl)hydrazone derivatives
7
modify shape and size of the active site and, as a consequence, of the compounds displayed a strong alignment of pyridine and
the different substrate recognition30. The hMAO-A binding pocket bromophenyl rings into the catalytic site and no different
is shorter and wider than the longer and narrower pocket of hydrophobic ligand–target interactions (Figure S1).
hMAO-B38 and its smaller cavity volume could explain the better
In order to further investigate the stereochemical requirement
biological data of most of the derivatives 1–36 (Tables 1–3) for for optimal hMAO-B inhibition, both E and Z isomers of
the hMAO-B isoform, as corroborated by molecular modeling compounds 26 and 2 were submitted to a molecular dynamic
studies.
(MD) of 5 ns carried out by Desmond package41. The
However, the docking simulation and the best pose of ligands ‘‘Simulation Event Analysis’’ panel was used to calculate the
in complex with the hMAO-B crystallographic model, down- fluctuation of energy during the dynamic simulation, considering
loaded from the Protein Data Bank (PDB) site39 with the code as final score (MDE) the average of E and Z isomers energy
2V5Z40, was unable to explain the molecular basis of the low values: the derivative 26 with the MDE value of ꢁ59.88 kcal/mol
nanomolar IC50 value for the 4-acetylpyridine 26 better than the showed the best theoretical result (Table 4). Therefore, to display
micromolar values of its analogues 14 and 2: the superimposition interactions of ligands during the MD with the amino acid
residues lining the catalytic site of the target, ‘‘Simulation
Table 4. Scoring values (Gscore) and binding energy values (dG Bind)
after GlideSP docking and Prime-MMGBSA simulations, for the best
submitted to graphical inspection and the last frames are reported
pose of ligands 2, 14 and 26 within the hMAO-A and hMAO-B active
Interaction Diagram’’ panel was used. The MD results were
in Figures 1 and 2.
sites.
The MD analysis of compound 26 revealed that both the
isomers, during the simulation, are able to establish not only
hMAO-A
hMAO-B
dG Bind
hydrophobic contacts with critical amino acid residues of the
binding site, but polar contacts too. Moreover, the E isomer
hydrazonic moiety was involved in two H-bonds with SER200
residue, while the same molecule portion in the Z isomer
established two H-bonds with TYR326 (Figure 1). On the other
hand, the MD analysis of compound 2 showed for the E isomer
Compound
Gscore
dG Bind
Gscore
MDE
2
14
26
ꢁ6.25
ꢁ6.08
ꢁ6.00
ꢁ51.94
ꢁ21.52
ꢁ8.58
ꢁ8.98
ꢁ8.97
ꢁ8.95
ꢁ63.32
ꢁ65.48
ꢁ66.94
ꢁ58.14
ꢁ59.88
Fluctuation of energy values during a molecular dynamic simulation
(MDE) were also calculated for compounds 2 and 26 in complex with
the hMAO-B crystal model. All theoretical data are reported in kcal/mol
considering the average of E and Z isomers values.
thiazole ring (tautomeric form)
a
polar contact with
TYR326,while the hydrazonic portion and the pyridine of the Z
isomer were involved in several H-bond contacts with SER200
and TYR326 amino acid residues (Figure 2). On the basis of these
Figure 1. The dynamic simulation last frame of both E (A) and Z (B) isomers of compound 26 and their H-bond contacts into the hMAO-B binding
site. The inhibitor E and Z isomers are rendered as carbon-pink sticks, the pocket amino acids are labeled as carbon-blue sticks and FAD co-factor as
carbon-cyan sticks. Target is described as blue cartoon and all non-carbon atoms are colored according to atom types. For the colored version of this
figure, please refer to the online version of the article.
Figure 2. The dynamic simulation last frame of both E (A) and Z (B) isomers of compound 2 and their H-bond contacts into the hMAO-B binding site.
The inhibitor E and Z isomers are rendered as carbon-yellow sticks, the pocket amino acids are labeled as carbon-blue sticks and FAD co-factor as
carbon-cyan sticks. Target is described as blue cartoon and all non-carbon atoms are colored according to atom types. For the colored version of this
figure, please refer to the online version of the article.

8
M. D’Ascenzio et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
results, we concluded that the pyridine ring linked to the stabilized by PHE331 (Figure 3). Therefore, the 4-pyridine ring
hydrazonic nitrogen and the substituted aryl moiety at C4 of the linked to the hydrazonic nitrogen also conferred the inhibitory
pharmacophoric thiazole conferred a great inhibitory effect on activity on the AChE enzyme in addition to selective hMAO-B
hMAO-B enzyme.
Finally, the most potent and selective hMAO-B inhibitors
inhibition and could be considered for future investigations.
(26–29 and 34) were chosen to perform the enzyme inhibition Experimental
tests of AChE (Table 5). Among them, compound 26 was found as
Chemistry
the most active compound with inhibition values of 50% at 3 mM.
The inhibition percentages of 27–29 and 34 showed these Starting materials and reagents used in the synthetic procedures
compounds as inactive at the tested concentration, with values were obtained from commercial suppliers and were used without
ranging from 2% (compound 34) to 7% (compound 28). The type further purification. Melting points (mp) were determined by the
of inhibition and Ki value were determined only for compound 26. capillary method on an FP62 apparatus (Mettler-Toledo) and are
1
Our data show that 26 is
a
competitive inhibitor with uncorrected. Temperatures are reported in ꢂC. H spectra were
Ki ¼ 1.82 0.55 mM (r2 ¼ 0.96) as also corroborated by molecular recorded on 400 MHz Bruker spectrometer using DMSO-d6 as the
docking study.
solvent. Chemical shifts are reported in parts per million (ꢀ)
The X-ray model of AChE with the PDB code 1EVE42, downfield from the signal of tetramethylsilane (TMS) which was
co-crystallized with Donepezil, was used to generate docking added to the deuterated solvent. The assignment of exchangeable
ligand poses of all compounds tested in vitro. Scoring values protons (NH) was confirmed by the addition of D2O. Coupling
Gscore and Embrace Free Energy (EFE)43 calculated as the constants J are expressed in hertz (Hz). Elemental analyses for C,
average of E and Z isomers values are reported in Table 5. H and N were determined with a Perkin-Elmer 240 B microana-
According to the biological results, in silico data showed lyzer and the analytical results were ꢃ95% purity for all
compound 26 with the acceptable Gscore value of ꢁ7.81 kcal/ compounds. All reactions were monitored by TLC performed
mol and the best EFE value of ꢁ63.98 kcal/mol but in all cases on 0.2 mm thick silica gel plates (60 F254 Merck). Visualization
less than the positive control Donepezil values (Gscore ¼ was carried out under ultra-violet irradiation (254 nm).
ꢁ9.89 kcal/mol; EFE ¼ ꢁ253.67 kcal/mol). Both the isomers of Preparative flash column chromatography was carried out on
26 were stabilized, into the binding pocket, through a hydrophobic silica gel (230–400 mesh, G60 Merck). Organic solutions were
interaction between the thiazole and PHE288 amino acid residue, dried over anhydrous sodium sulfate. Concentration and evapor-
involved in the H-bond contact with the pyridine ring of the ation of the solvent after reaction or extraction were carried out on
molecule, too. The E isomer established an additional non-polar a rotary evaporator (Bu¨chi Rotavapor); where given, systematic
contact with the THP84, common binding residue within the compound names are those generated by ChemDraw ultra
AChE–Donepezil complex, while the Z isomer best pose was following IUPAC conventions.
General procedure for the synthesis of derivatives 1–36
Table 5. Experimental and theoretical inhibition data of derivatives
26–29, 34 and reference drug Donepezil towards AChE target.
The appropriate 2-, 3- or 4-acetylpyridine (50 mmol) was
dissolved in 50 mL of ethanol and magnetically stirred with
equimolar amounts of thiosemicarbazide for 24 h at room
temperature with acetic acid as the catalyst. The resulting
thiosemicarbazone precipitating from reaction mixture was
filtered and washed with solvents (n-hexane, petroleum ether
and diethyl ether). The obtained thiosemicarbazone (50 mmol)
reacted with an equimolar quantity of the corresponding
a-bromoacetophenone dissolved in ethanol (50 mL) under mag-
netic stirring at room temperature. The reaction was monitored
until completion by TLC. The resulting (thiazol-2-yl)hydrazone
was filtered, washed with petroleum ether and diethyl ether, and,
if necessary, purified by chromatography on silica gel (ethyl
acetate: n-hexane, 3:1), to give all compounds in high yields
Compound
Inhibition (%)
Gscore
EFE
26
27
28
29
50
3
7
4
2
98
ꢁ7.81
ꢁ7.62
ꢁ7.66
ꢁ7.42
ꢁ7.89
ꢁ9.89
ꢁ63.98
ꢁ55.81
ꢁ55.85
ꢁ57.55
ꢁ54.99
ꢁ253.67
34
Donepezil
The inhibition activity of studied compounds at 3 mM, after in vitro assay,
was expressed as percentages. For the best pose of ligands within the
AChE active site, in silico, GlideSP score (Gscore) and Embrace Free
Energy (EFE) after docking simulation and Embrace minimization were
reported in kcal/mol considering the average of E and Z isomers values.
Figure 3. The docking best pose of both E (A) and Z (B) isomers of compound 26 and their H-bond contact into the AChE binding site, superimposed
to Donepezil co-crystallized inhibitor. The E and Z isomers are rendered as carbon-pink sticks, Donepezil as carbon-green sticks and the pocket amino
acids are labeled as carbon-blue sticks. Target is described as blue cartoon and all non-carbon atoms are colored according to atom types. For the
colored version of this figure, please refer to the online version of the article.

DOI: 10.3109/14756366.2014.987138
(Thiazol-2-yl)hydrazone derivatives
9
Scheme 1. Synthesis of (thiazol-2-yl)hydrazone derivatives 1–36. Reagents and conditions: (i) EtOH, acetic acid (cat.), rt; (ii) EtOH, rt. For
substituents (R) see Tables 1–3.
(78–99%). In preceding works, products 4, 8, 16, 20, 28, 3244, 7, (400 MHz, DMSO-d6): ꢀ 2.45 (s, 3H, CH3), 7.22–7.25 (m, 1H,
19, 3145, 12, 24, 3646, 18 and 2147, 5, 17, 2948 have already been Ar), 7.32 (s, 1H, thiazole), 7.39–7.43 (m, 1H, Ar), 7.53–7.55 (m,
synthesized and characterized for other purposes and were tested 1H, Ar), 8.10–8.17 (m, 3H, Ar), 8.73–8.75 (m, 1H, Ar), 11.87 (bs,
in this paper to ensure a complete structure–activity relationship 1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-d6): ꢀ 13.95,
(Scheme 1).
100.21, 123.33, 124.45, 125.02, 126.22, 127.98, 130.43, 130.78,
135.89, 136.22, 142.34, 146.67, 148.22, 152.09, 170.11. Anal.
Calcd for C16H12Cl2N4S: C, 52.90; H, 3.33; N, 15.42. Found: C,
53.11; H, 3.50; N, 15.28.
Characterization data for new compounds
1-(4-(2-Chlorophenyl)thiazol-2-yl)-2-(1-(pyridin-2-yl)ethylidene)
1
1-(4-(2,4-Dimethoxyphenyl)thiazol-2-yl)-2-(1-(pyridin-2-
yl)ethylidene)hydrazine (11): m.p. 218–220 ꢂC; 81% yield; 1H
NMR (400 MHz, DMSO-d6): ꢀ 2.40 (s, 3H, CH3), 3.79 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3), 7.15–7.17 (m, 1H, Ar), 7.23 (s, 1H,
thiazole), 7.37–7.40 (m, 1H, Ar), 7.48–7.50 (m, 1H, Ar), 8.00–
8.10 (m, 3H, Ar), 8.64–8.66 (m, 1H, Ar), 11.58 (bs, 1H, NH, D2O
exch.); 13C NMR (101 MHz, DMSO-d6): ꢀ 13.87, 57.23, 57.44,
101.01, 121.89, 123.34, 124.02, 125.46, 127.21, 130.97, 131.33,
135.61, 135.89, 142.63, 146.32, 148.44, 151.11, 170.74. Anal.
Calcd for C18H18N4O2S: C, 61.00; H, 5.12; N, 15.81. Found: C,
60.82; H, 4.97; N, 15.64.
hydrazine (1): m.p. 235–237 ꢂC; 78% yield; H NMR (400 MHz,
DMSO-d6): ꢀ 2.49 (s, 3H, CH3), 7.40–7.53 (m, 3H, Ar), 7.55 (s,
1H, thiazole), 7.60–7.63 (m, 1H, Ar), 8.00–8.10 (m, 2H, Ar),
8.67–8.69 (m, 2H, Ar), 12.73 (bs, 1H, NH, D2O exch.); 13C NMR
(101 MHz, DMSO-d6): ꢀ 13.98, 102.34, 123.98, 125.12, 125.78,
126.15, 127.19, 129.99, 130.67, 132.68, 135.67, 142.24, 146.43,
148.34, 152.89, 170.80. Anal. Calcd for C16H13ClN4S: C, 58.44;
H, 3.98; N, 17.04. Found: C, 58.22; H, 4.12; N, 16.89.
1-(4-(2-Bromophenyl)thiazol-2-yl)-2-(1-(pyridin-2-yl)ethylidene)
1
hydrazine (2): m.p. 210–211 ꢂC; 80% yield; H NMR (400 MHz,
DMSO-d6): ꢀ 2.41 (s, 3H, CH3), 7.44–7.53 (m, 2H, Ar), 7.55 (s,
1H, thiazole), 7.59–7.63 (m, 2H, Ar), 8.00–8.10 (m, 2H, Ar),
8.67–8.69 (m, 2H, Ar), 12.17 (bs, 1H, NH, D2O exch.); 13C NMR
(101 MHz, DMSO-d6): ꢀ 13.78, 101.67, 123.56, 124.68, 125.01,
125.95, 127.44, 130.67, 130.99, 133.13, 135.02, 144.56, 146.97,
148.32, 154.01, 171.03. Anal. Calcd for C16H13BrN4S: C, 51.48;
H, 3.51; N, 15.01. Found: C, 51.60; H, 3.73; N, 15.20.
1-(4-(2-Chlorophenyl)thiazol-2-yl)-2-(1-(pyridin-3-
yl)ethylidene)hydrazine (13): m.p. 230–232 ꢂC; 97% yield; 1H
NMR (400 MHz, DMSO-d6): ꢀ 2.43 (s, 3H, CH3), 7.38–7.40 (d,
J ¼ 8.0 Hz, 2H, Ar), 7.44 (s, 1H, thiazole), 7.66–7.74 (m, 2H, Ar),
8.14–8.16 (d, J ¼ 8.0 Hz, 2H, Ar), 8.94–9.13 (m, 1H, Ar), 9.31 (s,
1H, Ar), 11.95 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
DMSO-d6): ꢀ 13.91, 100.22, 123.18, 123.97, 124.44, 125.56,
127.12, 129.78, 130.21, 132.44, 135.34, 143.01, 146.36, 147.53,
152.35, 170.20. Anal. Calcd for C16H13ClN4S: C, 58.44; H, 3.98;
N, 17.04. Found: C, 58.71; H, 4.09; N, 16.90.
1-(4-(3-Bromophenyl)thiazol-2-yl)-2-(1-(pyridin-2-yl)ethylidene)
1
hydrazine (3): m.p. 229–231 ꢂC; 82% yield; H NMR (400 MHz,
DMSO-d6): ꢀ 2.42 (s, 3H, CH3), 7.36–7.39 (m, 2H, Ar), 7.50–7.51
(m, 2H, Ar), 7.60 (s, 1H, thiazole), 7.87–7.89 (d, J ¼ 8.0 Hz, 1H,
Ar), 8.09–8.16 (m, 2H, Ar), 8.67–8.68 (m, 1H, Ar), 11.73 (bs, 1H,
NH, D2O exch.); 13C NMR (101 MHz, DMSO-d6): ꢀ 13.99,
101.24, 124.01, 124.12, 124.99, 125.14, 127.96, 132.02, 133.13,
133.68, 134.80, 144.36, 146.90, 147.98, 155.10, 170.50. Anal.
Calcd for C16H13BrN4S: C, 51.48; H, 3.51; N, 15.01. Found: C,
51.21; H, 3.32; N, 14.83.
1-(4-(2-Bromophenyl)thiazol-2-yl)-2-(1-(pyridin-3-
yl)ethylidene)hydrazine (14): m.p. 220–221 ꢂC; 91% yield; 1H
NMR (400 MHz, DMSO-d6): ꢀ 2.39 (s, 3H, CH3), 7.08 (s, 1H,
thiazole), 7.26–7.28 (d, J ¼ 8.0 Hz, 1H, Ar), 7.29–7.30 (m, 1H,
Ar), 7.36–7.37 (m, 1H, Ar), 7.64–7.75 (m, 1H, Ar), 8.38–8.40 (d,
J ¼ 8.0 Hz, 2H, Ar), 8.74–8.75 (m, 1H, Ar), 9.16 (s, 1H, Ar),
12.00 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-d6):
ꢀ 13.66, 100.77, 122.18, 124.02, 125.11, 126.88, 128.77, 130.43,
130.99, 131.17, 135.21, 144.76, 146.41, 147.22, 152.44, 170.11.
Anal. Calcd for C16H13BrN4S: C, 51.48; H, 3.51; N, 15.01.
Found: C, 51.25; H, 3.20; N, 15.17.
1-(4-(4-Bromophenyl)thiazol-2-yl)-2-(1-(pyridin-2-yl)ethylidene)
1
hydrazine (6): mp 239–241 ꢂC; 80% yield; H NMR (400 MHz,
DMSO-d6): ꢀ 2.48 (s, 3H, CH3), 7.51 (m, 1H, Ar), 7.60–7.62 (m,
2H, Ar), 7.63 (s, 1H, thiazole), 7.82–7.84 (m, 1H, Ar), 7.85 (m,
1H, Ar), 8.14 (m, 2H, Ar), 8.53 (m, 1H, Ar), 11.77 (bs, 1H, NH,
D2O exch.); 13C NMR (101 MHz, DMSO-d6): ꢀ 13.89, 100.78,
122.12, 122.98, 124.34, 125.46, 126.79, 130.98, 131.54, 144.67,
147.32, 148.29, 153.66, 170.19. Anal. Calcd for C16H13BrN4S: C,
51.48; H, 3.51; N, 15.01. Found: C, 51.70; H, 3.73; N, 15.19.
1-(4-(4-Biphenyl)thiazol-2-yl)-2-(1-(pyridin-2-yl)ethylidene)
1-(4-(3-Bromophenyl)thiazol-2-yl)-2-(1-(pyridin-3-
yl)ethylidene)hydrazine (15): m.p. 250–252 ꢂC; 86% yield; 1H
NMR (400 MHz, DMSO-d6): ꢀ 2.40 (s, 3H, CH3), 7.38–7.42 (m,
1H, Ar), 7.50–7.53 (m, 1H, Ar), 7.58 (s, 1H, thiazole), 7.89–7.91
(m, 2H, Ar), 8.10–8.11 (m, 1H, Ar), 8.74 (m, 2H, Ar), 9.06 (s, 1H,
Ar), 11.68 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
DMSO-d6): ꢀ 13.87, 100.20, 123.00, 124.78, 126.34, 126.97,
127.13, 131.78, 132.45, 133.66, 136.79, 145.45, 145.32, 148.22,
154.87, 171.56. Anal. Calcd for C16H13BrN4S: C, 51.48; H, 3.51;
N, 15.01. Found: C, 51.59; H, 3.29; N, 14.77.
1
hydrazine (9): m.p. 208–210 ꢂC; 79% yield; H NMR (400 MHz,
DMSO-d6): ꢀ 2.44 (s, 3H, CH3), 7.38–7.50 (m, 5H, Ar), 7.53 (s,
1H, thiazole), 7.72–7.79 (m, 4H, Ar), 7.98–8.14 (m, 3H, Ar),
8.65–8.67 (m, 1H, Ar), 11.84 (bs, 1H, NH, D2O exch.); 13C NMR
(101 MHz, DMSO-d6): ꢀ 13.77, 101.23, 124.16, 124.97, 125.01,
125.55, 126.51, 127.66, 128.23, 130.98, 131.54, 133.80, 135.33,
147.23, 148.44, 148.97, 154.33, 171.44. Anal. Calcd for
C22H18N4S: C, 71.32; H, 4.90; N, 15.12. Found: C, 71.10; H,
5.07; N, 15.29.
1-(4-(2,4-Dichlorophenyl)thiazol-2-yl)-2-(1-(pyridin-3-
yl)ethylidene)hydrazine (22): m.p. 225–227 ꢂC; 90% yield; 1H
NMR (400 MHz, DMSO-d6): ꢀ 2.35 (s, 3H, CH3), 7.43–7.46 (m,
3H, Ar), 7.52 (s, 1H, thiazole), 7.69–7.90 (m, 2H, Ar), 8.55–8.56
(m, 1H, Ar), 8.9 (s, 1H, Ar), 11.46 (bs, 1H, NH, D2O exch.); 13
C
NMR (101 MHz, DMSO-d6): ꢀ 13.44, 100.89, 124.28, 124.98,
125.37, 127.67, 127.89, 130.34, 130.89, 134.65, 135.72, 144.01,
1-(4-(2,4-Dichlorophenyl)thiazol-2-yl)-2-(1-(pyridin-2-yl)ethy-
lidene)hydrazine (10): m.p. 198–200 ꢂC; 85% yield; 1H NMR

10 M. D’Ascenzio et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
146.42, 148.00, 152.67, 171.01. Anal. Calcd for C16H12Cl2N4S: Anal. Calcd for C16H12Cl2N4S: C, 52.90; H, 3.33; N, 15.42.
C, 52.90; H, 3.33; N, 15.42. Found: C, 53.05; H, 3.14; N, 15.69. Found: C, 52.69; H, 3.57; N, 15.20.
1-(4-(2,4-Dimethoxyphenyl)thiazol-2-yl)-2-(1-(pyridin-3-
1-(4-(2,4-Dimethoxyphenyl)thiazol-2-yl)-2-(1-(pyridin-4-yl)
yl)ethylidene)hydrazine (23): m.p. 205–208 ꢂC; 95% yield; 1H ethylidene)hydrazine (35): m.p. 230–233 ꢂC; 93% yield; 1H
NMR (400 MHz, DMSO-d6): ꢀ 2.36 (s, 3H, CH3), 3.87 (s, 3H, NMR (400 MHz, DMSO-d6): ꢀ 2.56 (s, 3H, CH3), 3.83 (s, 3H,
OCH3), 4.00 (s, 3H, OCH3), 7.34 (s, 1H, thiazole), 7.74–7.76 (m, OCH3), 3.87 (s, 3H, OCH3), 7.17–7.277 (m, 1H, Ar), 7.32 (s, 1H,
3H, Ar), 8.24–8.28 (m, 2H, Ar) 8.74–8.79 (m, 1H, Ar), 9.03 (s, thiazole), 7.47–7.54 (m, 1H, Ar), 7.68–7.70 (m, 1H, Ar), 8.00–
1H, Ar), 10.95 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, 8.10 (m, 2H, Ar), 8.57–8.66 (m, 2H, Ar) 12.58 (bs, 1H, NH, D2O
DMSO-d6): ꢀ 13.59, 57.39, 57.97, 100.53, 122.76, 122.56, 125.87, exch.); 13C NMR (101 MHz, DMSO-d6): ꢀ 13.67, 57.88, 58.12,
126.84, 126.98, 129.54, 132.67, 134.64, 134.93, 141.63, 145.02, 100.11, 122.59, 126.17, 126.44, 126.94, 128.88, 133.12, 134.33,
149.43, 150.32, 170.24. Anal. Calcd for C18H18N4O2S: C, 61.00; 134.77, 145.13, 149.77, 150.78, 170.06. Anal. Calcd for
H, 5.12; N, 15.81. Found: C, 61.19; H, 5.31; N, 15.99.
1-(4-(2-Chlorophenyl)thiazol-2-yl)-2-(1-(pyridin-4-
C18H18N4O2S: C, 61.00; H, 5.12; N, 15.81. Found: C, 60.78; H,
4.96; N, 15.54.
yl)ethylidene)hydrazine (25): m.p. 222–225 ꢂC; 87% yield; 1H
NMR (400 MHz, DMSO-d6): ꢀ 2.36 (s, 3H, CH3), 7.41–7.50 (m, Determination of hMAO isoform activity
2H, Ar), 7.89 (s, 1H, thiazole), 7.56–7.57 (m, 2H, Ar), 8.38–8.40
The effects of the test compounds on the enzymatic activity of
(d, J ¼ 8.0 Hz, 2H, Ar), 8.66–8.67 (m, 2H, Ar), 10.43 (bs, 1H, NH,
D2O exch.); 13C NMR (101 MHz, DMSO-d6): ꢀ 13.89, 100.66,
123.88, 123.99, 124.78, 128.66, 130.74, 132.66, 132.89, 136.44,
147.45, 149.54, 157.67, 171.98. Anal. Calcd for C16H13ClN4S: C,
58.44; H, 3.98; N, 17.04. Found: C, 58.62; H, 3.79; N, 17.27.
1-(4-(2-Bromophenyl)thiazol-2-yl)-2-(1-(pyridin-4-
yl)ethylidene)hydrazine (26): m.p. 227–230ꢂC; 80% yield; 1H
NMR (400 MHz, DMSO-d6): ꢀ 2.42 (s, 3H, CH3), 7,32–7.34 (m,
1H, Ar), 7.39 (s, 1H, thiazole), 7.48 (m, 1H, Ar), 7.88–7.90 (d,
J ¼ 8.0 Hz, 2H, Ar), 8.27–8.29 (d, J ¼ 8.0 Hz, 2H, Ar), 8.90–8.91
(m, 2H, Ar), 12.50 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
DMSO-d6): ꢀ 13.90, 100.66, 125.12, 125.54, 125.89, 128.45,
130.48, 130.97, 133.78, 135.54, 143.54, 148.52, 157.33, 171.19.
Anal. Calcd for C16H13BrN4S: C, 51.48; H, 3.51; N, 15.01.
Found: C, 51.20; H, 3.24; N, 15.22.
hMAO isoform were evaluated by a fluorimetric method follow-
ing the experimental protocol previously described by us33.
0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4) containing
different concentrations of the test drugs and adequate amounts of
recombinant hMAO-A or hMAO-B required and adjusted to
obtain the same reaction velocity in our experimental conditions,
i.e. to oxidize in the control group 165 pmol of p-tyramine/min,
were incubated for 15 min at 37 ꢂC in a flat-black-bottom 96-well
microtestÔ plate (BD Biosciences, Franklin Lakes, NJ) placed in
a dark fluorimetric chamber. The reaction was started by adding
200 mM AmplexÕ Red reagent, 1 U/mL horseradish peroxidase
and 1 mM p-tyramine. The production of H2O2 and, consequently,
of resorufin was quantified at 37 ꢂC in a multidetection microplate
fluorescence reader (Fluostar Optima, BMG Labtech GmbH,
Offenburg, Germany) based on the fluorescence generated
(excitation, 545 nm, emission, 590 nm) over a 15 min period, in
which the fluorescence increased linearly. Control experiments
were carried out simultaneously by replacing the test compounds
and reference inhibitors with appropriate dilutions of the vehicles.
In addition, the potential ability 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 AmplexÕ Red
reagent) was determined by adding these drugs to solutions
containing only the AmplexÕ Red reagent in a sodium phosphate
buffer. The drugs, vehicle and chemicals used in the experiments
were the new compounds, moclobemide (F. Hoffmann-La Roche
Ltd., Basel, Switzerland), dimethyl sulfoxide (DMSO), R-(ꢁ)-
deprenyl hydrochloride, iproniazid phosphate, isatin (Sigma-
Aldrich, Spain), resorufin sodium salt, clorgyline hydrochloride,
p-tyramine hydrochloride, sodium phosphate and horseradish
peroxidase (supplied in the AmplexÕ Red MAO assay kit from
Molecular Probes). Unless otherwise specified, results shown in
the text and tables are expressed as mean standard error of
the mean (S.E.M.) from n experiments. Significant differences
between two means were determined by one-way analysis of
variance (ANOVA) followed by the Dunnett’s post-hoc test.
Graph Pad Prism Software (GraphPad Software, San Diego, CA)
was used to perform statistical analyses and to calculate IC50
values and kinetic parameters.
1-(4-(3-Bromophenyl)thiazol-2-yl)-2-(1-(pyridin-4-
yl)ethylidene)hydrazine (27): m.p. 260–262 ꢂC; 89% yield; 1H
NMR (400 MHz, DMSO-d6): ꢀ 2.40 (s, 3H, CH3), 7.37–7.41 (m,
1H, Ar), 7.53–758 (m,1H, Ar), 7.66 (s, 1H, thiazole), 7.88–7.90
(d, J ¼ 8.0 Hz, 1H, Ar), 8.09–8.10 (m, 1H, Ar), 8.21–8.22 (m, 2H,
Ar), 8.86–8.87 (m, 2H, Ar), 12.18 (bs, 1H, NH, D2O exch.); 13C
NMR (101 MHz, DMSO-d6): ꢀ 13.79, 101.11, 125.33, 125.46,
125.67, 128.43, 130.12, 130.34, 134.34, 135.44, 144.79, 148.12,
157.20, 171.11. Anal. Calcd for C16H13BrN4S: C, 51.48; H, 3.51;
N, 15.01. Found: C, 51.26; H, 3.36; N, 14.80.
1-(4-(4-Bromophenyl)thiazol-2-yl)-2-(1-(pyridin-4-yl)ethylidene)
hydrazine (30): m.p. 275–277 ꢂC; 97% yield; 1H NMR (400 MHz,
DMSO-d6): ꢀ 2.39 (s, 3H, CH3), 7.57–7.61 (m, 2H, Ar), 7.63 (s,
1H, thiazole), 7.82–7.84 (d, J ¼ 8.0 Hz, 2H, Ar), 8.19–8.21 (d,
J ¼ 8.0 Hz, 2H, Ar), 8.85–8.86 (m, 2H, Ar), 12.04 (bs, 1H, NH,
D2O exch.); 13C NMR (101 MHz, DMSO-d6): ꢀ 13.80, 100.45,
123.22, 124.56, 125.78, 126.88, 131.11, 131.34, 147.58, 149.89,
153.45, 171.15. Anal. Calcd for C16H13BrN4S: C, 51.48; H, 3.51;
N, 15.01. Found: C, 51.33; H, 3.25; N, 14.79.
1-(4-(4-Biphenyl)thiazol-2-yl)-2-(1-(pyridin-4-yl)ethylidene)
hydrazine (33): m.p. 245–247 ꢂC; 78% yield; 1H NMR (400 MHz,
DMSO-d6): ꢀ 2.24 (s, 3H, CH3), 7.41–7.49 (m, 5H, Ar), 7.53 (s,
1H, thiazole), 7.70–7.89 (m, 4H, Ar), 7.96–8.22 (m, 2H, Ar),
8.55–8.77 (m, 2H, Ar), 11.84 (bs, 1H, NH, D2O exch.); 13C NMR
(101 MHz, DMSO-d6): ꢀ 13.89, 100.55, 124.67, 125.07, 125.69,
127.41, 127.92, 128.66, 130.62, 131.50, 133.32, 135.64, 147.77,
149.06, 155.01, 171.22. Anal. Calcd for C22H18N4S: C, 71.32; H,
4.90; N, 15.12. Found: C, 71.60; H, 4.72; N, 14.98.
Study of AChE inhibition
Percent inhibition of AChE: The spectrophotometric method of
1-(4-(2,4-Dichlorophenyl)thiazol-2-yl)-2-(1-(pyridin-4-yl)ethy- Ellman34 was used to assess the inhibitory activity of the selected
lidene)hydrazine (34): m.p. 241–243 ꢂC; 92% yield; 1H NMR compounds 26–29 and 34 toward AChE. 3 mL of 0.1 M (pH 7.4)
(400 MHz, DMSO-d6): ꢀ 2.43 (s, 3H, CH3), 7.43–7.46 (m, 3H, phosphate buffer containing DTNB (0.25 mM) and AChE
Ar), 7.55 (s, 1H, thiazole), 7.79–7.81 (m, 2H, Ar), 8.55–8.56 (m, (0.083 U mLꢁ1) was mixed in a polystyrene cuvette of 1 cm
2H, Ar), 11.98 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, path length with 1 mL of the studied compounds, previously
DMSO-d6): ꢀ 13.67, 100.80, 121.12, 123.54, 125.88, 127.86, solubilized in DMSO in order to obtain a 9 mM solution. The
129.02, 130.44, 134.45, 135.16, 146.01, 148.55, 152.43, 171.05. reaction was started by adding 30 mL of an aqueous solution

DOI: 10.3109/14756366.2014.987138
(Thiazol-2-yl)hydrazone derivatives 11
(pH ¼ 7.4, phosphate buffer) of acetylthiocoline (10 mM) to the molecules, the results of the docking and dynamic experiments
enzyme-inhibitor mixture. The production of the yellow anion was and to generate all figures.
recorded at 412 nm and 25 ꢂC between 0.5 and 1.5 min. As a
control, an identical solution of the enzyme without the inhibitor
is processed following the same protocol to determine the 100%
of enzyme activity.
Conclusion
Starting from our recent discoveries in the field of new hMAO
inhibitors, we synthesized and tested 36 hydrazothiazole deriva-
tives of 2-, 3- and 4-acetylpyridine bearing different aromatic
substituents at C4 of the thiazole. Most of the products were
active in the low micromolar/high nanomolar range, and some of
them were effective at low nanomolar concentrations towards
MAO-B isoform. While the coumarin derivatives 12 and 24 were
more active and more selective on hMAO-A enzyme with respect
to the reference drug Moclobemide, compounds 26, 27 and 28
showed an inhibitory effect on hMAO-B at lower concentrations
than that required by R-(ꢁ)-Deprenyl. Moreover, compound 28
displayed a selectivity of inhibition that was very similar to that of
the above mentioned reference drug. As demonstrated by
molecular modeling studies, the presence of an acetylpyridine
linked at the hydrazonic nitrogen together with an aryl moiety at
C4 position of the pharmacophoric nucleus could be crucial for
the inhibitory effect of this scaffold of molecules on monoamine
oxidase enzymes. Furthermore, 4-acetylpyridine nucleus con-
ferred to the hydrazothiazole derivatives the best hMAO-B
inhibitory activity. We have also investigated the anticholinester-
ase activity of hydrazones 26–29 and 34 selected from the most
active and selective hMAO-B inhibitors. The percentage of
inhibition of compound 26 prompted us to determine its type of
inhibition and the Ki value. Our results show that 26 inhibits
AChE in the low micromolar range with a competitive mechan-
ism, in agreement with the molecular docking study; therefore, the
dual effect of derivate 26 against MAO-B and AChE makes it a
good candidate for further study in the effort at developing new
anti-neurodegenerative agents.
Kinetic analysis of AChE inhibition: 3 mL of 0.1 M (pH 7.4)
phosphate buffer containing DTNB (0.25 mM) and AChE
(0.083 U mLꢁ1) was mixed with the opportune volume of
compound 26, solubilized in H2O/DMSO (10:1) in order to
obtain the final solutions of 26 between 0.5 and 2.5 mM and the
final DMSO content of the assay mixture 50.05%. The reaction
was started by adding the proper volume of an aqueous solution
(pH 7.4, phosphate buffer) of acetylthiocoline (10 mM) to the
enzyme–inhibitor mixture. The production of the yellow anion
was recorded at 412 nm and 25 ꢂC between 0.5 and 1.5 min. To
obtain estimates of the mechanism of action of compound 26, the
plot of 1/v versus [I] was constructed at different concentrations of
the inhibitor (0.5, 1.0, 1.5, 2.0 and 2.5 mM) and three concentra-
tions of substrate (75, 100 and 150 mM). Each determination was
performed in triplicate. The recorded data were analyzed with the
enzyme kinetic module of SigmaPlot, version 8.02 a.
Molecular modeling studies
The hMAO-A and hMAO-B enzyme models 2Z5X49 and 2V5Z40
were downloaded from the Protein Data Bank (PDB)39 site, while
the crystallographic structure with PDB code 1EVE42 was used
for the AChE target. Both E and Z isomers of the 3D compounds
26, 14 and 2 were built by means Maestro GUI 9.7 in the
Scrodinger Suite 201450. Protonated and tautomeric forms at pH
7.4 for each molecule were calculated using the ‘‘LigPrep’’
module51 and the lowest energy conformations, generated in the
absence of the receptor with the OPLS_2005 force field, were
used as starting points for the docking simulations. To study all
interactions of each target with the ligands, water molecules were
removed and all hydrogen atoms were added during the prepar-
ation procedure of the enzymes carried out by ‘‘Protein
Preparation Wizard’’ function. Docking simulations were per-
formed with the software Glide 6.236 on the hMAO-A, hMAO-B
and AChE crystal structures. Semi-flexible approach was con-
sidered: the flexible ligands were sequentially docked in the
binding pocket of the rigid targets recording 10 poses per ligand.
Declaration of interest
This work was supported by ‘‘Progetto di Ateneo Ricerca 2013’’
(P. Chimenti) and Interregional Research Center for Food Safety and
Health at the Magna Græcia University of Catanzaro (MIUR PON
a3_00359).
The authors state no conflict of interest and have received no payment
in preparation of this manuscript.
˚
A cube of length 25 A was defined as binding region, using the
‘‘Receptor Grid Generation’’ panel with the default options,
centered on the former positions of the removed co-crystallized
inhibitors with the proteins, into each catalytic site. The accuracy
of GlideSP has been shown to successfully reproduce experimen-
tally observed binding modes of co-crystallized inhibitors, in
terms of root-mean squared deviation (RMSD) that were
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Supplementary material available online
Supplementary Figures S1 and S2.