Design, synthesis and biochemical evaluation of novel multi-target inhibitors as potential anti-Parkinson agents

Article abstract of DOI:10.1016/j.ejmech.2017.10.050

New 4-(3-nitrophenyl)thiazol-2-ylhydrazone derivatives are proposed as dual-target-directed monoamine oxidase B (MAO-B) and acetylcholinesterase (AChE) inhibitors, as well as antioxidant agents, for the treatment of neurodegenerative disorders such as Parkinson's disease. Rational molecular design, target recognition and predicted pharmacokinetic properties have been evaluated by means of molecular modelling. Based on these properties, compounds were synthesized and evaluated in vitro as MAO-B and AChE inhibitors, and compared to the activities at their corresponding isozymes, monoamine oxidase A (MAO-A) and butyrylcholinesterase (BuChE), respectively. Anti-oxidant properties, potentially useful in the treatment of neurodegenerative disorders, have been also investigated in vitro. Among the evaluated compounds, three inhibitors may be considered as promising dual inhibitors of MAO-B and AChE, in vitro. MAO-B inhibition was also shown to be competitive and reversible for compound 19.

Full text of DOI:10.1016/j.ejmech.2017.10.050

Accepted Manuscript
Design, synthesis and biochemical evaluation of novel multi-target inhibitors as
potential anti-Parkinson agents
Simone Carradori, Francesco Ortuso, Anél Petzer, Donatella Bagetta, Celeste
De Monte, Daniela Secci, Daniela De Vita, Paolo Guglielmi, Gokhan Zengin,
Abdurrahman Aktumsek, Stefano Alcaro, Jacobus P. Petzer
PII:
S0223-5234(17)30852-8
10.1016/j.ejmech.2017.10.050
EJMECH 9842
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 September 2017
Revised Date: 11 October 2017
Accepted Date: 16 October 2017
Please cite this article as: S. Carradori, F. Ortuso, Ané. Petzer, D. Bagetta, C. De Monte, D. Secci,
D. De Vita, P. Guglielmi, G. Zengin, A. Aktumsek, S. Alcaro, J.P. Petzer, Design, synthesis and
biochemical evaluation of novel multi-target inhibitors as potential anti-Parkinson agents, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.050.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and biochemical evaluation of novel multi-target
inhibitors as potential anti-Parkinson agents
a
Simone Carradori , Francesco Ortuso b,*, Anél Petzer c, Donatella Bagetta a, Celeste De Monte
d
d
e
d, Daniela Secci d,*, Daniela De Vita , Paolo Guglielmi , Gokhan Zengin , Abdurrahman
Aktumsek e, Stefano Alcaro b, Jacobus P. Petzer c
aDepartment of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, Via dei Vestini 31,
66100 Chieti, Italy
bDipartimento di Scienze della Salute, University “Magna Graecia” of Catanzaro, Campus “S.
Venuta”, Viale Europa, 88100 Catanzaro, Italy
cPharmaceutical Chemistry and Centre of Excellence for Pharmaceutical Sciences, North-West
University, Potchefstroom 2520, South Africa
dDipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” University of Rome, P.le A.
Moro 5, 00185 Rome, Italy
eSelcuk University, Science Faculty, Department of Biology, Konya, Turkey
*Corresponding authors
Email addresses: ortuso@unicz.it (F. Ortuso), daniela.secci@uniroma1.it (D. Secci)
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ABSTRACT
New 4-(3-nitrophenyl)thiazol-2-ylhydrazone derivatives are proposed as dual-target-directed
monoamine oxidase B (MAO-B) and acetylcholinesterase (AChE) inhibitors, as well as
antioxidant agents, for the treatment of neurodegenerative disorders such as Parkinson’s disease.
Rational molecular design, target recognition and predicted pharmacokinetic properties have
been evaluated by means of molecular modelling. Based on these properties, compounds were
synthesized and evaluated in vitro as MAO-B and AChE inhibitors, and compared to the
activities at their corresponding isozymes, monoamine oxidase A (MAO-A) and
butyrylcholinesterase (BuChE), respectively. Anti-oxidant properties, potentially useful in the
treatment of neurodegenerative disorders, have been also investigated in vitro. Among the
evaluated compounds, three inhibitors may be considered as promising dual inhibitors of MAO-
B and AChE, in vitro. MAO-B inhibition was also shown to be competitive and reversible for
compound 19.
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1. Introduction
Parkinson’s disease (PD) is a multifactorial, chronic neurodegenerative disorder with a high
social impact, particularly in Western countries [1, 2]. The physiopathology of PD, even if not
yet completely clear, reveals the deregulation of several genes, such as parkin, α-synuclein, DJ-
1, leucine-rich repeat kinase 2, and PTEN-induced putative kinase 1, that may induce protein
misfolding, phosphorylation and aggregation, mitochondrial dysfunction and oxidative stress [3].
In the early stages of PD these biochemical events result in the progressive loss of dopaminergic
neurons in the central nervous system (CNS), particularly in the pars compacta of the substantia
nigra. This pathology is associated with the appearance of intraneuronal protein aggregates
known as Lewy bodies. In later phases of PD noradrenergic, serotonergic and cholinergic
systems are also affected [3]. PD symptoms consisting in bradykinesia, resting tremor, muscle
rigidity, and postural instability can be attributed to the loss of dopaminergic neurons while, in
later phases, cognitive decline, sleep abnormalities, and depression occur due to the involvement
of the other neurotransmitter systems [4].
Currently, all available PD therapies are palliative and symptomatic, and their efficacy is
strictly related to the stage of the pathology: early pharmacological treatments achieve the best
results and may slow the disease development. Anti-Parkinson drugs, for the most part, act by
increasing dopaminergic tone via the biosynthesis of the dopamine (i.e., L-DOPA), by
stimulating dopamine receptors (i.e., Bromocriptine, Ropinirole), by limiting dopamine
metabolism with the inhibition of COMT (i.e., Tolcapone) or MAO-B (i.e., Selegiline,
Rasagiline, Safinamide) [5].
Moreover, considering that the MAO catalytic cycle produces several potentially neurotoxic
species such as hydrogen peroxide, ammonia, and aldehydes, MAO-B inhibitors may reduce
oxidative stress and neurotoxicity by reducing the formation of those metabolites [6]. While
providing effective in vivo MAO inhibition, early MAO inhibitors (i.e., Phenelzine,
Tranylcypromine and Isocarboxazide) possessed some safety problems. In contrast to more
recent compounds, these drugs are not selective for a particular MAO isoform and their
mechanism of inhibition is irreversible. Such nonselective irreversible MAO inhibitors prevent
the peripheral metabolism of tyramine, which may lead to a potentially lethal hypertensive crisis
known as “cheese effect” [7, 8]. Tyramine is an amine mostly present in cheese, red wine, meat
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and fish, which dictates that dietary restrictions should be imposed when treatment with
nonselective irreversible MAO inhibitors is initiated.
In the initial phase of PD, anti-muscarinic compounds can be administered to reduce
extrapyramidal symptoms [3]. In drug resistant patients, surgical deep brain stimulation can be
combined with pharmacological treatment [9]. To explain cognitive dysfunction in PD, a
convergent versus parallel model, according to dopamine-acetylcholine dependent alterations in
synaptic plasticity, has been proposed [10]. It has been suggested that AChE inhibitors, such as
Rivastigmine and Donepezil, may be effective for the treatment of the psychotic and cognitive
disorders associated with PD, without exacerbating motor symptoms [11, 12].
Due to its multifactorial physiopathology, the available PD treatments often require the co-
administration of several drugs. With the aim to improve therapeutic efficacy and patient
compliance, scientific research should be focused on a more promising multi-target-directed
strategy with a single compound, appropriately designed, and addressing different pathological
events [13, 14].
In the present study, taking into account our previous works [15-21], novel 4-(3-
nitrophenyl)thiazol-2-ylhydrazones have been designed, synthesized and evaluated as dual-
target-directed MAO-B and AChE inhibitors with reversible modes of inhibition according to the
SAR studies proposed for this scaffold [22]. To investigate the abilities of the synthesized
compounds to access the brain, their permeation across the blood-brain barrier (BBB) has been
theoretically investigated and predicted in silico. Finally, the anti-oxidant properties of the
compounds were investigated by five different assays in vitro. Based on the results of this study,
three thiazolylhydrazones, 8, 19 and 20, were identified as promising lead compounds for the
development of novel treatments for neurodegenerative disorders such as PD.
2. Results and discussion
2.1. Rational drug design
The molecular design workflow consisted of four steps: (a) first the shape and volume
complementarity of the common scaffold were evaluated with respect to the targets, (b) then
analyses of molecular interaction fields (MIFs) and quantum mechanics simulation suggested
type and position of substituents, (c) pharmacokinetic properties of the designed compounds
were theoretically predicted, and (d) finally the interaction of the MAOs and cholinesterases
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(ChEs) with the proposed derivatives were investigated by means of docking experiments
(Experimental section).
Because we have already demonstrated the MAO inhibitory properties of thiazolylhydrazone
derivatives [23, 24], our goal was to rationally design new analogues that retain MAO-B
inhibition, but also possessing inhibitory activity against ChEs. Via molecular modelling, the
target recognition of the common scaffold, 4-(phenyl)thiazol-2-ylhydrazone (1) was firstly
evaluated (Figure 1). In both MAO isoforms, 1 exhibited several binding modes, but in the most
stable orientations the unsubstituted hydrazone moiety was directed towards the FAD cofactor,
while the phenyl ring was located at the catalytic site entrance (Supplementary material, Figure
S1a-b). The ChEs interacted with 1 principally by means of stacking contacts with Trp residues
(Supplementary material, Figure S1c-d). In AChE, the external Trp286 was better recognized
than the internal Trp86 residue, while in BuChE, where Ala277 replaces the AChE Trp286, 1
interacted with the internal Trp82 residue (corresponding to Trp86 in AChE). The binding
orientation of 1 to the MAOs could allow for covalent bond formation between N5 of the FAD
and the hydrazone group of 1, which consequently could lead to irreversible target inhibition. In
order to prevent such a possibility, two methyl groups were introduced to replace the hydrazone
terminal hydrogen atoms, thus yielding compound 2 (Figure 1).
Fig. 1. Chemical structures of 1 and 2.
The binding mode of 2 to MAO-A was completely opposite compared to 1. In this instance,
the phenyl ring was directed towards the FAD, thus excluding the possibility of covalent bond
formation by means of the hydrazone moiety. Conversely, in MAO-B and the ChEs little
difference was observed between the recognition of 1 and 2 (Supplementary material, Figure
S2). In AChE, 2 slightly altered its position to interact with the inner Trp86 instead of the
external Trp286, assuming a binding mode and position similar to that reported in the crystal
structure 4EY7 for the interaction of AChE with the known selective inhibitor, Donepezil
(Supplementary material, Figure S3) [25]. For the recognition of both 1 and 2, the known key
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role of stacking interactions was confirmed by graphical analysis. In order to improve such a
favourable contribution, ortho-, meta- and para-nitrophenyl derivatives of 2 were designed
according to the findings of molecular interaction fields (MIFs) analyses. Theoretical models,
excluding ligands, for the MAOs and ChEs were mapped using ON GRID probe. The resulting
MIFs were superimposed with the highest scored docking pose of 2 (Supplementary material,
Figures S4 and S5). It was not surprising to observe a good correlation between the MAOs
computed MIFs and all positions of the ligand phenyl moiety. In fact, the nitro group was already
included at the para position, leading to improved interaction with the MAOs [21]. In contrast,
the analyses of the MIFs of the ChEs suggested suitable substitution on the ortho and meta
positions only. In view of the key role of stacking interactions in target recognition, we have
investigated the effect of the position of nitro group on the coplanarity of the phenyl and thiazole
rings. In fact, coplanar conformations allow for electron delocalization between the two aromatic
rings, potentially improving stacking contacts to the targets. The relative energy profile as a
function of the phenyl-thiazole dihedral angle (Figure 2) was computed using quantum
mechanics methods (Experimental section).
Fig. 2. The effect of the position of nitro group (ortho-, meta- and para-) on the relative energy profile of 4-
(phenyl)thiazol-2-ylhydrazone scaffold as a function of the phenyl-thiazole dihedral angle.
The dihedral energy profiles clearly indicate that the nitro group at the ortho position prevents
coplanarity of the phenyl and thiazolyl rings, while meta- and para-nitro derivatives allow for
coplanarity, to the same degree. Considering the MIFs and quantum mechanics analysis, the
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subsequent compounds were designed as meta-nitro derivatives only. In order to evaluate the
effect of the nitro group on target recognition, compound 2 was substituted with the nitro group
in the meta position to yield compound 3. The new derivative was submitted to docking
simulation as previously reported for 1 and 2. The highest scored docking poses of 3
(Supplementary material, Figure S6) were similar to those of 2, with some differences in
theoretical target affinity (XP-GScore). The m-nitro derivative 3 exhibited similar interaction
energies compared to precursor 2 at all targets except MAO-A. In this respect, the binding of
compound 3 to MAO-A was weaker by 1.6 kcal/mol compared to 2, thus providing better MAO-
B selectivity, at least theoretically. In order to suggest novel and more potent dual-target-directed
MAO-B and AChE inhibitors, known building blocks [20] were used to design a library of
alkyl/cycloalkyl-substituted derivatives of 3 and the resulting structures were virtually screened
against the MAO and ChE targets. The pharmacokinetics properties of the new compounds, such
as oral absorption and blood-brain barrier permeation (Experimental section), and their
interaction with the targets were computationally investigated (Table 1).
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Table 1. Chemical structures of the 4-(3-nitrophenyl)thiazol-2-ylhydrazones (3-20) and their theoretical properties
and docking scores for binding to the MAOs and ChEs.
Docking scorec
Compound
R
%OAa QPlogBBb
MAO-A MAO-B AChE BuChE
3
4
89.36
92.59
-0.92
-0.95
-1.05
-0.95
-1.00
-1.14
-1.15
-1.05
-1.25
-1.15
-1.35
-0.95
-0.92
-0.90
-0.90
-0.83
-5.93
-5.92
-5.85
-2.59
0.56
-7.15
-7.22
-8.06
-7.30
-7.83
-7.94
-8.14
-7.80
-8.87
-7.53
-9.05
-7.84
-7.60
-9.05
-8.45
-7.89
-6.51
-6.59
-7.03
-6.58
-7.68
-7.65
-7.68
-7.39
-8.51
-9.28
-7.93
-6.45
-8.23
-9.88
-8.98
-8.29
-7.31
-5.34
-6.79
-5.36
-8.16
-5.68
-5.73
-5.61
-8.37
-6.75
-8.60
-6.21
-7.13
-6.62
-6.79
-5.89
5
94.83
6
96.41
7
100.00
96.77
0.50
8
100.00
100.00
100.00
100.00
100.00
92.84
-2.41
0.28
9
10
11
12
13
14
15
16
17
18
-2.71
0.01
-2.95
-6.71
-0.85
-1.26
0.61
94.58
100.00
96.81
100.00
1.79
92.39
-0.97
-0.97
-2.76
0.76
-7.98
-9.14
-7.68
-7.26
-6.68
-6.33
19
20
100.00
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aPercentage of oral dose absorbed; bBlood-brain barrier permeation as pLog(IN/OUT); cBest pose XP-GScore in
kcal/mol.
The theoretical pharmacokinetic data correlated well with those of known CNS active drugs
[26], and suggested that the 4-(3-nitrophenyl)thiazol-2-ylhydrazones could exhibit good
absorption after oral administration, and could also penetrate the blood-brain. Analyses of the
docking poses (Figures S7-S22) and scores suggested that these compounds could possess good
MAO selectivity with a strong preference for the MAO-B isoform. This may be attributed to the
productive hydrogen bond interaction between the thiazole ring nitrogen and the Cys172 in
MAO-B, an interaction that is not possible in MAO-A since Asn181 is the corresponding residue
in this position. Moreover, for compounds 7, 8, 10, 12, 17, 18 and 20, these results highlighted a
disfavoured recognition of MAO-A as demonstrated by their target interaction energies greater
than 0 kcal/mol. This finding suggests that the addition of steric hindrance at the hydrazone may
be used to obtain MAO isoform selectivity. For the ChEs, the docking simulations did not
suggest that the newly synthesized compounds will exhibit notable selectivity even if 4, 8-10, 12
and 16-20, preferred AChE compared to BuChE. On the basis of the above theoretical analysis,
derivatives 3-20 were selected for synthesis and in vitro biological evaluation.
2.2. Synthesis
4-(3-Nitrophenyl)thiazol-2-ylhydrazone derivatives (3-20) were synthesized in high yields as
reported in our previous communications (Table 1) [27]. A carbonyl compound was reacted with
thiosemicarbazide in ethanol at room temperature, with acetic acid serving as the catalyst
(Scheme 1). Then, the resulting thiosemicarbazones were reacted in ethanol with 2-bromo-3’-
nitroacetophenone to furnish the proposed derivatives by Hantzsch cyclization. All the
synthesized products were washed with petroleum ether and diethyl ether and purified by column
chromatography (ethyl acetate/petroleum ether) before characterization by spectroscopic
methods (IR, 1H and 13C NMR) and elemental analysis to ensure high purity (≥96%).
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Scheme 1. Synthesis of 4-(3-nitrophenyl)thiazol-2-ylhydrazone derivatives 3-20. Reagents and conditions: (i)
thiosemicarbazide, EtOH, acetic acid (cat.), rt; (ii) 2-bromo-3’-nitroacetophenone, EtOH, rt.
2.3. In vitro MAO inhibition study
All the synthesized compounds were found to inhibit the human MAO enzymes, particularly
the MAO-B isoform, with promising IC50 values. Several cycloaliphatic derivatives displayed
inhibitory action against MAO-B at low nanomolar concentrations (Table 2). Conversely, these
compounds inhibited MAO-A in the high micromolar range. Derivatives possessing linear
aliphatic substituents (e.g. 1, 2, 8-11) were particularly active MAO-B inhibitors (IC50 values
ranging from 0.101 to 0.716 µM). Compounds substituted with a five-membered linear chain (5,
6) or branched chain (7) exhibited very good MAO-B inhibition (IC50 values of 0.055, 0.053 and
0.012 µM, respectively). Among the cycloaliphatic substituents, the remarkable MAO-B
inhibition was exhibited by cycloheptylidene 17 (IC50 = 0.0086 µM) and cyclooctylidene 18
(IC50 = 0.010 µM). The introduction of an exocyclic aliphatic ring improved the MAO-B
inhibition potency and selectivity within this library with the cyclopropylethylidene 19 and
cyclohexylethylidene 20 possessing IC50 values of 0.0053 µM and 0.0072 µM for MAO-B,
respectively. These in vitro biological data thus supported the findings of the molecular
modelling study, and showed that the 4-(3-nitrophenyl)thiazol-2-ylhydrazone derivatives were
indeed potent MAO inhibitors with manifest selectivity for the MAO-B isoform.
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Table 2. Inhibitory activities of compounds 3-20 towards MAO and ChE targets.
MAO-Aa
1.07 ± 0.981
20.5 ± 2.29
38.0 ± 1.31
24.3 ± 2.91
4.41 ± 0.554
3.99 ± 0.218
6.33 ± 0.116
4.86 ± 0.254
7.15 ± 0.076
6.03 ± 0.230
10.9 ± 1.67
3.46 ± 0.263
2.56 ± 0.358
4.25 ± 0.237
1.59 ± 0.201
1.82 ± 0.061
2.66 ± 0.051
29.1 ± 2.72
-
MAO-Ba
0.716 ± 0.521
0.351 ± 0.043
0.055 ± 0.0079
0.053 ± 0.0074
0.012 ± 0.0015
0.101 ± 0.0089
0.118 ± 0.021
0.189 ± 0.017
0.355 ± 0.035
0.182 ± 0.030
0.626 ± 0.053
0.095 ± 0.014
0.047 ± 0.0061
0.237 ± 0.051
0.0086 ± 0.0017
0.010 ± 0.0061
0.0053 ± 0.00080
0.0072 ± 0.0018
-
AChEb BuChEb
Compound
3.00
14.00
31.00
9.00
2.30
47.00
24.00
15.00
5.28
10.00
3.00
14.15
6.92
18.00
1.44
0.80
44.00
41.00
13.44
-
12.85
12.13
5.16
7.45
8.98
6.35
11.34
11.02
8.68
10.01
9.54
12.44
8.41
4.87
7.11
7.64
12.77
6.22
13.62
-
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Galantamine
Clorgyline
R-(-)-Deprenyl
0.00446 ± 0.00032
67.25 ± 1.02
61.35 ± 1.13
0.01960 ± 0.00086
-
-
a IC50 values in µM; b percentage of target inhibition at 3 µM
The most potent MAO-B inhibitor of the series, compound 19, was further investigated to
determine if it acts as a reversible or irreversible MAO-B inhibitor. For this purpose, dialysis was
used. MAO-B and compound 19 (at a concentration equal to 4 × IC50) were combined and
incubated for 15 min. The mixture was subsequently dialyzed for 24 h and diluted twofold, to
yield a concentration of 19 equal to 2 × IC50, and the residual enzyme activity was measured. As
positive control, incubation and dialysis of MAO-B with the irreversible MAO-B inhibitor, (R)-(-
)-deprenyl, were carried out while as negative control a similar experiment was carried out in
absence of inhibitor. For comparison, the residual MAO activities of undialyzed mixtures of
MAO-B and 19 were also measured after twofold dilution as above. The results of the dialysis
study show that, after incubation with 19, dialysis restores MAO-B activity to 91% of the
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negative control value (Figure 3). This demonstrated that 19 is a reversible MAO-B inhibitor
since, for reversible inhibition, dialysis is expected to restore enzyme activity to 100%. In
contrast, after incubation with (R)-(-)-deprenyl, dialysis does not restore MAO-B activity with
the residual activity at only 1.9%. In undialyzed mixtures, MAO-B inhibition persists with the
activity at 46%.
Subsequently, Lineweaver-Burk plots for the inhibition of MAO-B by 19 were constructed to
demonstrate that this compound is a competitive MAO-B inhibitor (Figure 3). Six Lineweaver-
Burk plots were thus constructed at the following concentrations of 19: 0 µM, ¼ × IC50, ½ ×
IC50, ¾ × IC50, 1 × IC50 and 1¼ × IC50. The Lineweaver-Burk plots were found to intersect on
the y-axis, providing evidence of competitive inhibition and thus further supporting for a
reversible interaction. From the replot of the slopes of the Lineweaver-Burk plots versus
inhibitor concentration, a Ki value of 0.0073 µM for the inhibition of MAO-B by 19 was
estimated.
1200
A
B
100
75
800
400
100
75
50
25
0
-0.006 -0.003 0.000 0.003 0.006
[I], µM
50
25
0
-0.02
0.00
0.02
0.04
0.06
1/[S]
Fig. 3. A: Reversibility of the inhibition of MAO-B by 19. MAO-B and 19 (at 4 × IC50) were preincubated for 15
min, dialyzed for 24 h and the residual enzyme activity was measured (19-dialyzed). MAO-B was similarly
preincubated in the absence (No inhibitor–dialyzed) and presence of the irreversible inhibitor, (R)-(-)-deprenyl
(Depr-dialyzed), and dialyzed. For comparison, the residual MAO activity of undialyzed mixtures of MAO-B with
19 is also shown (19-undialyzed). B: Lineweaver-Burk plots of human MAO-B activities in the absence (filled
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squares) and presence of various concentrations of compound 19. The inset is a graph of the slopes of the
Lineweaver-Burk plots versus inhibitor concentration.
2.4. In vitro cholinesterases inhibition study
To determine if the 4-(3-nitrophenyl)thiazol-2-ylhydrazones (3-20) may act as dual-target-
directed inhibitors, they were also evaluated as potential AChE and BuChE inhibitors. For this
purpose, Galantamine served as reference inhibitor, a compound which exhibits 13.44% and
13.62% inhibition of AChE and BuChE, respectively, at 3 µM. As reported in Table 2, all the
compounds are moderate inhibitors of both ChE isoenzymes. Three derivatives, 8, 19 and 20,
display the best inhibitory activity (˃41%) of AChE at 3 µM, and also possess good selectivity
for AChE over BuChE. These compounds may be considered as promising dual-target-directed
inhibitors (MAO-B and AChE) for the treatment of neurodegenerative diseases as also
previously reported [28].
2.5. Anti-oxidant in vitro assays
The in vitro anti-oxidant activity of this library (compounds 3-20) has been evaluated by five
experimental approaches, using trolox as a reference compound. Among free radical scavenging
methods, DPPH assay is highly sensitive, simple and not costly. In addition, ABTS test could be
applicable to both hydrophilic and lipophilic compounds. The free radical scavenging potential
of the 4-(3-nitrophenyl)thiazol-2-ylhydrazones was slightly lower than trolox, whereas in the
reducing power (important for the inhibition of the Fenton reaction between metal ions and the
byproducts of MAO-mediated reaction) and total anti-oxidant capacity in the
phosphomolybdenum (PhosphoMo) assay, the test and reference compounds display almost
comparable potencies. These ancillary biological activities could be useful for the treatment of
multifactorial neurodegenerative diseases where oxidative stress enhances cognitive impairment
and inflammatory processes.
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Table 3. Anti-oxidant activities of compounds 3-20 compared to the reference drug trolox.
Anti-oxidant assays
Compound
PhosphoMoa
1.75 ± 0.08
1.65 ± 0.13
1.20 ± 0.08
1.10 ± 0.02
1.13 ± 0.03
1.15 ± 0.05
1.46 ± 0.06
1.25 ± 0.02
1.47 ± 0.08
1.56 ± 0.04
1.24 ± 0.08
1.34 ± 0.01
1.43 ± 0.07
1.13 ± 0.08
1.23 ± 0.03
1.22 ± 0.09
1.47 ± 0.07
1.19 ± 0.03
1.13 ± 0.05
FRAPa
CUPRACa
DPPHb
ABTSb
0.34 ± 0.01 0.51 ± 0.02 1.16 ± 0.03 1.07 ± 0.02
0.42 ± 0.01 0.58 ± 0.01 1.18 ± 0.02 1.04 ± 0.01
0.45 ± 0.01 0.55 ± 0.05 1.20 ± 0.02 1.04 ± 0.01
0.34 ± 0.01 0.46 ± 0.02 1.13 ± 0.01 1.04 ± 0.01
0.42 ± 0.01 0.57 ± 0.01 1.35 ± 0.01 1.05 ± 0.02
0.33 ± 0.01 0.46 ± 0.02 1.21 ± 0.01 1.04 ± 0.01
0.37 ± 0.01 0.52 ± 0.02 1.18 ± 0.02 1.06 ± 0.02
0.39 ± 0.02 0.53 ± 0.02 1.23 ± 0.02 1.04 ± 0.01
0.37 ± 0.05 0.59 ± 0.09 1.17 ± 0.09 1.05 ± 0.07
0.41 ± 0.02 0.55 ± 0.01 1.26 ± 0.02 1.04 ± 0.01
0.39 ± 0.01 0.53 ± 0.01 1.15 ± 0.01 1.11 ± 0.10
0.40 ± 0.03 0.51 ± 0.03 1.18 ± 0.04 1.10 ± 0.09
0.33 ± 0.01 0.32 ± 0.01 1.20 ± 0.02 1.10 ± 0.08
0.39 ± 0.01 0.39 ± 0.02 1.18 ± 0.01 1.09 ± 0.07
0.42 ± 0.03 0.41 ± 0.02 1.22 ± 0.01 1.07 ± 0.05
0.39 ± 0.02 0.44 ± 0.03 1.28 ± 0.02 1.09 ± 0.08
0.47 ± 0.02 0.53 ± 0.02 1.26 ± 0.02 1.06 ± 0.03
0.67 ± 0.03 0.61 ± 0.01 1.35 ± 0.01 1.05 ± 0.02
0.16 ± 0.01 0.24 ± 0.02 0.40 ± 0.01 0.66 ± 0.02
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Trolox
aEC50 values in mM; bIC50 values in mM.
2.6. Pan Assay INterference compoundS (PAINS) evaluation
Compounds 3-20, due to their chemical scaffold, dual inhibition and modest anti-oxidant
properties could be suspected of artificial activity. In view of this, and according to a recently
published editorial [29], all designed inhibitors have been evaluated by means of three different
theoretical tools such as ZINC PAINS Pattern Identifier [30], False Positive Remover [31] and
FAF-Drug4 [32]. None of the considered algorithms reported our compounds as potential PAINS
or covalent inhibitors. ZINC PAINS Pattern Identifier suggested 3, 4 and 6 to be further
investigated by means of aggregation tests only. Because 3, 4 and 6 have not demonstrated
interesting activities in vitro, the evaluation of their aggregative properties has been not carried
out. A further indication that our compounds are not PAINS can be taken from their
experimental activities: 3-20, in general, discriminated between target isoforms. Finally, the
14

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inhibition of MAO-B, affected by compound 19, has been demonstrated as a competitive and
reversible process. Taking into account the above reported results and considerations, we are
confident that our designed inhibitors, in particular the most promising 8, 19 and 20, did not
show artificial activities.
3. Conclusions
A multidisciplinary approach allowed the identification of three new promising dual inhibitors
that could be useful in Parkinson’s disease therapy. A molecular modelling-based rational drug
design approach has driven the synthesis of a small library of 4-(3-nitrophenyl)thiazol-2-
ylhydrazone derivatives. Experimental evaluations have confirmed, in vitro, the inhibition of
both MAO-B and AChE, and the selectivity with respect to the corresponding MAO-A and
BuChE isozymes. Anti-oxidant properties of these new inhibitors have been also demonstrated,
thus highlighting a useful synergistic effect for the treatment of neurodegenerative disorders.
Further study, based on these results, will be carried out to investigate the biological properties
of the identified inhibitors on cell lines and in vivo models.
4. Experimental protocols
4.1. Molecular modelling
Theoretical studies were performed using the Schrödinger Suite version 2015-4 [33]. The three-
dimensional models of the designed compounds were built using the Maestro GUI [34] and
optimized by means of the OPLS-2005 force field [35] Solvent effects were taken into account
using the GB/SA water implicit solvation model [36] Target theoretical structures were obtained
from Protein Data Bank (PDB) [37]: crystallographic entries 2Z5X [38], 2V5Z [39], 4M0E [40],
and 1P0I [41] for MAO-A, MAO-B, AChE and BuChE, respectively. In order to prepare the
models for simulation, all PDB entries were submitted to a preliminary pre-treatment: ligands
and co-crystallized water molecules were removed, missing hydrogen atoms were added and, in
the MAO targets, FAD connectivity was fixed. Torsion energy profiles of ortho-, meta- or para-
substituted nitrophenyl ring with respect to the thiazole ring were investigated at DTF level of
theory using the B3LYP functional and the 6-31G** basis set, as implemented in Jaguar software
[42]. For each isomer, the torsion scan was carried out from 0° to 360° degree at regular intervals
of 6°. The energy of the corresponding 61 conformers, for each derivative, was computed after
15

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optimisation in vacuo. Molecular interaction fields (MIFs) analysis was performed by applying
the program GRID [43, 44] to the structure models of the MAOs and ChEs. ON and C3 probes
were used to highlight target favourable interaction areas for nitro and methyl substituents,
respectively. The resolution of the computed MIFs was fixed at 0.33 Å (NPLA=3). Other GRID
directives were left to default values. Oral bioavailability (as %) and BBB permeation [26] were
computed using the QikProp tool [45]. Docking experiments were carried out with the Glide
software [46]. Target binding sites were defined by means of a regular grid box of about 64,000
Å3 centred in the MAOs, on the FAD N5 atom and in the ChEs, on the catalytic serine residue.
All simulations were computed using Glide ligand flexible algorithm at extra-precision (XP)
level. Authors will release the atomic coordinates and experimental data upon article publication.
4.2. Chemistry
Starting materials and reagents used in the synthetic procedures were obtained from
commercial suppliers and were used without purification. Where mixtures of solvents are
specified, the stated ratios are volume:volume. All melting points were measured on a Stuart®
melting point apparatus SMP1, and are uncorrected. Temperatures are reported in °C. 1H and 13C
NMR spectra were recorded at 400 MHz and 101 MHz, respectively, on a Bruker spectrometer
using CDCl3 and DMSO-d6 as the solvents. Chemical shifts are expressed as δ units (parts per
million) relative to the solvent signal. Coupling constants J are given in Hertz (Hz). IR (neat)
spectra were recorded on a FT-IR Perkin-Elmer SpectrumOne instrument with the ATR system.
Absorption bands (νmax) are reported as the wavenumbers (cm-1). Elemental analyses for C, H,
and N were recorded on a Perkin-Elmer 240 B microanalyzer and the analytical results were
within ± 0.4% of the theoretical values for all compounds. All reactions were monitored by TLC
performed on 0.2 mm thick silica gel plates (60 F254, Merck). Visualization was carried out under
UV irradiation (254 nm). Preparative flash column chromatography was carried out on silica gel
(high purity grade, pore size 60 Å, 200-425 mesh particle size). Organic solutions were dried
over anhydrous sodium sulfate. Evaporation of the solvent after reaction was carried out on a
rotary evaporator (Büchi Rotavapor). Where given, systematic compound names are those
generated by ChemBioDraw Ultra® 12.0 following IUPAC conventions.
4.3. General synthetic procedure
The appropriate carbonyl compound (1.0 equiv.) was dissolved in 50 mL of ethanol and
magnetically stirred with an equimolar amount of thiosemicarbazide for 24-72 h at room
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temperature, using acetic acid as the catalyst. The obtained suspension was filtered, and the solid
washed consecutively with petroleum ether and diethyl ether. The thiosemicarbazone (1.0 equiv.)
thus obtained was reacted with 2-bromo-3’-nitroacetophenone dissolved in ethanol (50 mL)
under magnetic stirring at room temperature for 24-72 h. The reaction was monitored by TLC
until completion. The resulting 4-(3-nitrophenyl)thiazol-2-ylhydrazone derivative was collected
by filtration, washed with petroleum ether and diethyl ether, and purified by column
chromatography using ethyl acetate:petroleum ether as mobile phase, to give compounds 3-20 in
good yields. Chemical-physical data are characteristic for the most predominant E geometric
isomer because of the upshielding observed in the 1H NMR spectra for the NH signal and for the
absence of any Z-stabilizing functional groups [27].
In general, the IR spectrum (neat) for derivatives 3-20 showed stretching absorption bands at
approximately 3300 cm-1 for NH, at 3030 cm-1 due to the stretching of Csp2-H, at 1630 cm-1 for
the C=N and at 1580 and 1440 cm-1 for C=C.
4.4. Characterization data for compounds 3-20
4.4.1. 1-(4-(3-Nitrophenyl)thiazol-2-yl)-2-(propan-2-ylidene)hydrazine (3) [47]
4.4.2. 1-(Butan-2-ylidene)-2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazine (4)
1
Yellow powder, mp 165-170 °C, 73% yield; H NMR (400 MHz, CDCl3): δ 1.16-1.19 (t, 3H,
CH3), 2.20 (s, 3H, CH3), 2.43-2.45 (q, 2H, CH2), 6.95 (s, 1H, C5H-thiazole), 7.74-7.77 (m, 1H,
Ar), 8.18-8.20 (m, 1H, Ar), 8.24-8.27 (m, 1H, Ar), 8.52 (s, 1H, Ar), 12.31 (bs, 1H, NH, D2O
13
exch.). C NMR (101 MHz, DMSO-d6): δ 11.0 (CH3), 16.9 (CH3), 31.6 (CH2), 106.6 (Ar-C5,
thiazole), 120.4 (Ar), 122.4 (Ar), 130.6 (Ar), 132.0 (Ar), 136.6 (Ar), 147.9 (Ar-NO2), 148.7 (Ar-
C4, thiazole), 154.7 (C=N), 170.9 (C=N, thiazole).
4.4.3. 1-(4-(3-Nitrophenyl)thiazol-2-yl)-2-(pentan-2-ylidene)hydrazine (5)
Green powder, mp 175-179 °C, 64% yield; 1H NMR (400 MHz, CDCl3): δ 0.97-1.00 (m, 3H,
CH3), 1.64-1.68 (m, 2H, CH2), 2.18 (s, 3H, CH3), 2.36-2.40 (m, 2H, CH2), 6.94 (s, 1H, C5H-
thiazole), 7.54-7.56 (m, 1H, Ar), 8.29-8.32 (m, 2H, Ar), 8.53 (s, 1H, Ar), 12.23 (s, 1H, NH, D2O
exch.). 13C NMR (101 MHz, DMSO-d6): δ 14.0 (CH3), 16.9 (CH3), 19.6 (CH2), 32.8 (CH2),
106.0 (Ar-C5, thiazole), 120.4 (Ar), 122.4 (Ar), 130.3 (Ar), 132.0 (Ar), 136.6 (Ar), 147.9 (Ar-
NO2), 148.8 (Ar-C4, thiazole), 153.6 (C=N), 166.0 (C=N, thiazole).
4.4.4. 1-(4-(3-Nitrophenyl)thiazol-2-yl)-2-(pentan-3-ylidene)hydrazine (6)
17

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Green powder, mp 177-179 °C, 76% yield; 1H NMR (400 MHz, CDCl3): δ 1.15-1.28 (t, 6H, 2
x CH3 ), 2.43-2.45 (q, 2H, CH2), 2.54-2.56 (q, 2H, CH2 ), 6.95 (s, 1H, C5H-thiazole), 7.74-7.76
(m, 1H, Ar), 8.26-8.30 (m, 1H, Ar), 8.35-8.37 (m, 1H, Ar) , 8.52 (s, 1H, Ar), 12.32 (s, 1H, NH,
D2O exch.). 13C NMR (101 MHz, DMSO-d6): δ 10.2 (CH3), 11.0 (CH3), 23.2 (CH2), 29.0 (CH2),
106.6 (Ar-C5, thiazole), 120.4 (Ar), 122.3 (Ar), 130.6 (Ar), 132.0 (Ar), 136.6 (Ar), 148.2 (Ar-
NO2), 148.7 (Ar-C4, thiazole), 155.6 (C=N), 171.1 (C=N, thiazole).
4.4.5. 1-(4-Methylpentan-2-ylidene)-2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazine (7)
Brown oil, 64% yield; 1H NMR (400 MHz, CDCl3): δ 0.97-0.99 (d, J = 6.8 Hz, 6H, 2 x CH3),
1.90 (s, 3H, CH3), 2.01-2.02 (m, 1H, CH), 2.21-2.23 (d, J = 7.2 Hz, 2H, CH2), 7.02 (s, 1H, C5H-
thiazole), 7.53-7.57 (t, 1H, Ar), 8.11-8.15 (t, 2H, Ar), 8.40 (bs, 1H, NH, D2O exch.), 8.66 (s, 1H,
13
Ar). C NMR (101 MHz, DMSO-d6): δ 17.1 (CH3), 22.8 (2 x CH3), 25.8 (CH), 47.5 (CH2),
106.6 (Ar-C5, thiazole), 120.4 (Ar), 122.4 (Ar), 130.6 (Ar), 132.0 (Ar), 136.8 (Ar), 148.3 (Ar-
NO2), 148.7 (Ar-C4, thiazole), 153.0 (C=N), 170.9 (C=N, thiazole).
4.4.6. 1-(Hex-5-en-2-ylidene)-2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazine (8)
Yellow powder, mp 180-182 °C, 71% yield; 1H NMR (400 MHz, CDCl3): δ 2.19 (s, 3H, CH3),
2.37-2.39 (m, 2H, CH2), 2.49-2.51 (m, 2H, CH2), 5.04-5.06 (m, 2H, =CH2), 5.90-5.94 (m, 1H,
=CH), 6.96 (s, 1H, C5H-thiazole), 7.54-7.56 (m, 1H, Ar), 8.19-8.21 (d, J = 7.4 Hz, 1H, Ar), 8.30-
13
8.31 (d, J = 7.4 Hz, 1H, Ar), 8.52 (s, 1H, Ar), 12.27 (bs, 1H, NH, D2O exch.). C NMR (101
MHz, DMSO-d6): δ 17.1 (CH3), 30.3 (CH2), 37.6 (CH2), 106.6 (Ar-C5, thiazole), 115.6 (=CH2),
120.4 (Ar), 122.4 (Ar), 130.6 (Ar), 132.0 (Ar), 136.6 (Ar), 138.3 (=CH-), 148.0 (Ar-NO2), 149.0
(Ar-C4, thiazole), 152.9 (C=N), 170.8 (C=N, thiazole).
4.4.7. 1-(Hexan-2-ylidene)-2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazine (9)
1
Green powder, mp 156-160 °C, 82% yield; H NMR (400 MHz, DMSO-d6): δ 0.89-0.93 (m,
3H, CH3), 1.32-1.35 (m, 2H, CH2), 1.49-1.53 (m, 2H, CH2), 1.93 (s, 3H, CH3), 2.23-2.27 (m, 2H,
CH2), 7.56 (s, 1H, C5H-thiazole), 7.68-7.73 (m, 1H, Ar), 8.13-8.15 (m, 1H, Ar), 8.28-8.31 (m,
13
1H, Ar), 8.70 (s, 1H, Ar), 10.80 (bs, 1H, NH, D2O exch.). C NMR (101 MHz, DMSO-d6): δ
14.3 (CH3), 17.0 (CH3), 22.2 (CH2), 28.3 (CH2), 38.0 (CH2), 106.6 (Ar-C5, thiazole), 120.4 (Ar),
122.6 (Ar), 130.6 (Ar), 132.0 (Ar), 137.0 (Ar), 148.2 (Ar-NO2), 148.7 (Ar-C4, thiazole), 156.6
(C=N), 170.9 (C=N, thiazole).
4.4.8. 1-(Hexan-3-ylidene)-2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazine (10)
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Yellow powder, mp 153-155 °C, 84% yield; 1H NMR (400 MHz, DMSO-d6): δ 0.92-0.96 (m,
3H, CH3), 1.03-1.08 (m, 3H, CH3), 1.48-1.50 (m, 2H, CH2), 2.23-2.30 (m, 2H, CH2), 2.33-2.37
(m, 2H, CH2), 7.56 (s, 1H, C5H-thiazole), 7.68-7.77 (m, 1H, Ar), 8.13-8.15 (m, 1H, Ar), 8.28-
8.30 (m, 1H, Ar), 8.70 (s, 1H, Ar), 11.00 (bs, 1H, NH, D2O exch.). 13C NMR (101 MHz, DMSO-
d6): δ 11.0 (CH3), 14.4 (CH3), 19.5 (CH2), 29.5 (CH2), 37.8 (CH2), 106.6 (Ar-C5, thiazole), 120.4
(Ar), 122.4 (Ar), 130.6 (Ar), 132.0 (Ar), 136.6 (Ar), 148.0 (Ar-NO2), 148.7 (Ar-C4, thiazole),
157.3 (C=N), 171.0 (C=N, thiazole).
4.4.9. 1-(Heptan-2-ylidene)-2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazine (11)
Green powder, mp 79-81 °C, 73% yield; 1H NMR (400 MHz, slightly soluble in DMSO-d6): δ
0.85-0.89 (m, 3H, CH3), 1.25-1.30 (m, 4H, 2 x CH2), 1.49-1.51 (m, 2H, CH2), 1.91 (s, 3H, CH3),
2.20-2.24 (t, 2H, CH2), 7.55 (s, 1H, C5H-thiazole), 7.69-7.71 (m, 1H, Ar), 8.11-8.14 (m, 1H, Ar),
8.27-8.29 (m, 1H, Ar), 8.67 (s, 1H, Ar), 10.83 (bs, 1H, NH, D2O exch.).
4.4.10. 1-(Heptan-3-ylidene)-2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazine (12)
Brown oil, 69% yield; 1H NMR (400 MHz, CDCl3): δ 0.97-1.00 (m, 3H, CH3), 1.17-1.20 (t,
3H, CH3), 1.39-1.47 (m, 2H, CH2), 1.53-1.68 (m, 2H, CH2), 2.25-2.40 (m, 4H, CH2), 7.02 (s, 1H,
C5H-thiazole), 7.54-7.58 (t, 1H, Ar), 8.11-8.15 (m, 2H, Ar), 8.44 (bs, 1H, NH, D2O exch.), 8.67
(s, 1H, Ar). 13C NMR (101 MHz, DMSO-d6): δ 10.2 (CH3), 14.3 (CH3), 22.7 (CH2), 25.8 (CH2),
27.6 (CH2), 35.4 (CH2), 106.6 (Ar-C5, thiazole), 120.4 (Ar), 122.7 (Ar), 130.6 (Ar), 132.0 (Ar),
136.6 (Ar), 147.8 (Ar-NO2), 148.6 (Ar-C4, thiazole), 158.2 (C=N), 171.0 (C=N, thiazole).
4.4.11. 1-(Octan-2-ylidene)-2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazine (13)
Yellow powder, mp 135-137 °C, 81% yield; 1H NMR (400 MHz, DMSO-d6): δ 0.85-0.88 (m,
3H, CH3), 1.28-1.29 (m, 6H, 3 x CH2), 1.49-1.53 (m, 2H, CH2), 1.92 (s, 3H, CH3), 2.22-2.25 (m,
2H, CH2), 7.57 (s, 1H, C5H-thiazole), 7.68-7.74 (m, 1H, Ar), 8.12-8.15 (m, 1H, Ar), 8.28-8.30
(m, 1H, Ar), 8.68-8.69 (m, 1H, Ar), 10.84 (bs, 1H, NH, D2O exch). 13C NMR (101 MHz,
DMSO-d6): δ 14.4 (CH3), 17.0 (CH3), 22.5 (CH2), 26.1 (CH2), 28.7 (CH2), 31.6 (CH2), 38.3
(CH2), 106.6 (Ar-C5, thiazole), 120.4 (Ar), 122.4 (Ar), 130.6 (Ar), 132.0 (Ar), 136.6 (Ar), 145.3
(Ar-NO2), 148.7 (Ar-C4, thiazole), 153.8 (C=N), 170.9 (C=N, thiazole).
4.4.12. 1-Cyclopentylidene-2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazine (14) [47]
4.4.13. 1-Cyclohexylidene-2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazine (15)
1
Grey powder, mp 178-185 °C, 69% yield; H NMR (400 MHz, CDCl3): δ 1.68-1.70 (m, 2H,
cyclohexane), 1.78-1.81 (m, 4H, cyclohexane), 2.41-2.44 (m, 2H, cyclohexane), 2.61-2.64 (m,
19

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2H, cyclohexane), 6.94 (s, 1H, C5H-thiazole), 7.74-7.75 (m, 1H, Ar), 8.20-8.25 (m, 2H, Ar), 8.52
(s, 1H, Ar), 12.28 (bs, 1H, NH, D2O exch.). 13C NMR (101 MHz, DMSO-d6): δ 25.5 (CH2), 26.0
(CH2), 27.3 (CH2), 27.7 (CH2), 35.2 (CH2), 106.5 (Ar-C5, thiazole), 120.5 (Ar), 122.4 (Ar), 130.6
(Ar), 132.1 (Ar), 136.4 (Ar), 147.7 (Ar-NO2), 148.7 (Ar-C4, thiazole), 156.8 (C=N), 170.8 (C=N,
thiazole).
4.4.14. 2-(2-(4-methylcyclohexylidene)hydrazinyl)-4-(3-nitrophenyl)thiazole (16)
Grey powder, mp 174-176 °C, 78% yield; 1H NMR (400 MHz, CDCl3): δ 0.98-0.99 (d, 3H, J =
6.5 Hz, CH3), 1.24-1.25 (m, 2H, cyclohexane), 1.34-1.45 (m, 2H, cyclohexane), 1.57-1.66 (m,
2H, cyclohexane), 1.90-2.01 (m, 1H, cyclohexane), 2.20-3.20 (m, 2H, cyclohexane), 6.67 (s, 1H,
C5H-thiazole), 7.01 (s, 1H, Ar), 7.26-7.29 (m, 2H, Ar), 7.30-7.41 (m, 1H, Ar), 12.24 (bs, 1H,
NH, D2O exch.). 13C NMR (400 MHz, DMSO-d6): 13C NMR (101 MHz, DMSO-d6): δ 21.8
(CH3), 26.9 (CH2), 31.5 (CH), 34.0 (CH2), 34.4 (CH2), 35.3 (CH2), 106.5 (Ar-C5, thiazole), 120.4
(Ar), 122.4 (Ar), 130.6 (Ar), 132.1 (Ar), 136.6 (Ar), 147.8 (Ar-NO2), 148.7 (Ar-C4, thiazole),
156.5 (C=N), 170.8 (C=N, thiazole).
4.4.15. 1-Cycloheptylidene-2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazine (17)
1
White powder, mp 194-195 °C, 83% yield; H NMR (400 MHz, CDCl3): δ 1.64-1.71 (m, 6H,
cycloheptane), 1.86-1.87 (m, 2H, cycloheptane), 2.57-2.59 (m, 2H, cycloheptane), 2.69-2.71 (m,
2H, cycloheptane), 6.96 (s, 1H, C5H-thiazole), 7.75-7.77 (m, 1H, Ar), 8.19-8.31 (m, 2H, Ar),
8.52 (s, 1H, Ar), 12.11 (bs, 1H, NH, D2O exch.). 13C NMR (101 MHz, DMSO-d6): δ 24.5 (CH2),
27.7 (CH2), 30.3 (CH2), 30.3 (CH2), 31.4 (CH2), 36.8 (CH2), 106.7 (Ar-C5, thiazole), 120.5 (Ar),
122.4 (Ar), 130.6 (Ar), 132.1 (Ar), 136.5 (Ar), 147.7 (Ar-NO2), 148.7 (Ar-C4, thiazole), 159.4
(C=N), 170.7 (C=N, thiazole).
4.4.16. 1-Cyclooctylidene-2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazine (18)
Yellow powder, mp 180-183 °C, 81% yield; 1H NMR (400 MHz, CDCl3): δ 1.43-1.44 (m, 2H,
cyclooctane), 1.57-1.59 (m, 4H, cyclooctane), 1.85-1.86 (m, 2H, cyclooctane), 1.96-1.98 (m, 2H,
cyclooctane), 2.48-2.51 (m, 2H, cyclooctane), 2.62-2.65 (m, 2H, cyclooctane), 6.94 (s, 1H, C5H-
thiazole), 7.76-7.78 (m, 1H, Ar), 8.21-8.22 (m, 1H, Ar), 8.29-8.32 (m, 1H, Ar), 8.52 (s, 1H, Ar),
12.33 (bs, 1H, NH, D2O exch.). 13C NMR (101 MHz, DMSO-d6): δ 24.1 (CH2), 25.3 (CH2), 25.5
(CH2), 26.7 (CH2), 27.3 (CH2), 28.5 (CH2), 36.1 (CH2), 106.6 (Ar-C5, thiazole), 120.5 (Ar),
122.5 (Ar), 130.6 (Ar), 132.1 (Ar), 136.4 (Ar), 147.6 (Ar-NO2), 148.7 (Ar-C4, thiazole), 160.1
(C=N), 170.6 (C=N, thiazole).
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4.4.17. 1-(1-Cyclopropylethylidene)-2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazine (19)
Yellow powder, mp 150-155 °C, 89% yield; 1H NMR (400 MHz, DMSO-d6): δ 0.72-0.75 (m,
4H, cyclopropane), 1.64-1.67 (m, 1H, cyclopropane), 1.79 (bs, 3H, CH3), 7.55 (s, 1H, C5H-
thiazole), 7.69 (t, J= 8.0 Hz, 1H, Ar), 8.13 (d, J= 8.0 Hz, 1H, Ar), 8.27 (d, J= 7.6 Hz, 1H, Ar),
8.66 (s, 1H, Ar), 10.90 (bs, 1H, NH, D2O exch.). 13C NMR (101 MHz, DMSO-d6): δ 6.2 (2 x
CH2, cyclopropane), 14.7 (CH, cyclopropane), 18.0 (CH2), 106.5 (Ar-C5, thiazole), 120.4 (Ar),
122.4 (Ar), 130.6 (Ar), 132.0 (Ar), 136.5 (Ar), 147.8 (Ar-NO2), 148.7 (Ar-C4, thiazole), 155.4
(C=N), 170.7 (C=N, thiazole).
4.4.18. 1-(1-Cyclohexylethylidene)-2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazine (20)
1
White powder, mp 177-179 °C, 78% yield; H NMR (400 MHz, CDCl3): δ 1.31-1.36 (m, 5H,
cyclohexane), 1.84-1.87 (m, 5H, cyclohexane), 2.13 (s, 3H, CH3), 2.16-2.19 (m, 1H,
cyclohexane), 6.95 (s, 1H, C5H-thiazole), 7.71-7.74 (t, 1H, Ar), 8.17-8.19 (d, J= 8.0 Hz, 1H, Ar),
13
8.27-8.29 (d, J = 8.0 Hz, 1H, Ar), 8.53 (s, 1H, Ar), 11.91 (bs, 1H, NH, D2O exch.). C NMR
(101 MHz, DMSO-d6): δ 15.5 (CH2), 26.0 (2 x CH2, cyclohexane), 30.3 (2 x CH2, cyclohexane),
46.3 (CH3), 106.7 (Ar-C5, thiazole), 120.4 (Ar), 122.3 (Ar), 130.6 (Ar), 132.0 (Ar), 136.8 (Ar),
148.2 (Ar-NO2), 148.7 (Ar-C4, thiazole), 156.6 (C=N), 171.1 (C=N, thiazole).
4.5. MAO-A and MAO-B inhibition studies
IC50 values for the inhibition of MAO-A and MAO-B were measured according to the literature
protocol [48, 49]. Recombinant human MAO-A and MAO-B (Sigma-Aldrich) were used as
enzyme sources, while kynuramine served as substrate for both enzyme isoforms. The enzyme
reactions were carried out in potassium phosphate buffer (pH 7.4, 100 mM) and contained
kynuramine (50 µM), the test inhibitors (0.003–100 µM) and MAO-A (0.0075 mg/mL) or MAO-
B (0.015 mg/mL). After initiation with the addition of enzyme, the reactions were incubated for
20 min and at endpoint were treated with NaOH to terminate the enzyme reactions. The
fluorescence intensity of 4-hydroxyquinoline, the product formed by MAO-catalyzed oxidation
of kynuramine, was measured (λex = 310 nm; λem = 400 nm). Sigmoidal plots of MAO catalytic
rate versus logarithm of inhibitor concentration were constructed and the IC50 values were
estimated and reported as the mean ± standard deviation (SD) of triplicate experiments.
4.5.1. Investigating reversibility of inhibition by dialysis
The reversibility of MAO-B inhibition by compound 19 was investigated by dialysis according
to the literature protocol [48]. MAO-B (0.03 mg protein/mL) and the test compound at an
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ACCEPTED MANUSCRIPT
inhibitor concentration equal to 4 × IC50 were incubated for 15 min at 37 °C. The buffer was
potassium phosphate buffer (100 mM, pH 7.4, 5% sucrose) and all incubations contained 4%
DMSO as co-solvent. As negative control, MAO-B was incubated in the absence of inhibitor,
while as positive control MAO-B was incubated with the irreversible inhibitor (R)-(-)-deprenyl
[IC50(MAO-B) = 0.019 µM], with the inhibitor concentration equal to 4 × IC50 [50]. The
mixtures were subsequently dialyzed at 4 °C with the dialysis buffer being replaced with fresh
buffer at 3 h and 7 h after the start of dialysis. After 24 h of dialysis, the mixtures were diluted
twofold with the addition of the enzyme substrate, kynuramine, to yield an inhibitor
concentration equal to 2 × IC50 and kynuramine concentration of 50 µM. These reactions were
incubated for 20 min at 37 ºC, terminated with the addition of NaOH and the concentrations of 4-
hydroxyquinoline were measured by fluorescence spectrophotometry as described above. For
comparison, undialyzed mixtures of MAO-B and 19 were maintained at 4 °C for 24 h and the
residual MAO-B activity was measured as for the dialyzed samples. All reactions were carried
out in triplicate and the residual enzyme catalytic rates were expressed as mean ± SD.
4.5.2. Lineweaver-Burk plots
A set consisting of six Lineweaver-Burk plots was constructed for the inhibition of MAO-B by
19. For this purpose, the following six inhibitor concentrations were used: 0 µM, ¼ × IC50, ½ ×
IC50, ¾ × IC50, 1 × IC50 and 1¼ × IC50. For each plot, kynuramine was used at 15–250 µM, and
the concentration of MAO-B was 0.015 mg protein/mL. All enzyme reactions and activity
measurements were carried out as described above for the dialysis experiments. The Ki value was
estimated from a plot of the slopes of the Lineweaver-Burk plots versus inhibitor concentration
(x-axis intercept equals –Ki).
4.6. Cholinesterases inhibition studies
To evaluate the capacity of the 4-(3-nitrophenyl)thiazol-2-ylhydrazones to inhibit ChE activity,
the protocol of Ellman was used [51]. Electric eel AChE (EC 3.1.1.7), acetylthiocholine iodide
(ASCh), 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB) and the reference drug, galantamine, were
purchased from Sigma-Aldrich (Milan, Italy). The assay was carried out with a double beam
UV–Vis lambda 40 Perkin Elmer spectrophotometer using optical polystyrene cuvettes (10 x 10
x 45 mm, 340–800 nm optical transparency). The tested compounds were dissolved in DMSO
and were added to the assay mixtures to yield a final DMSO content of <0.05%, a concentration
that does not affect enzyme activity. This method is based on the cleavage of DTNB by
22

ACCEPTED MANUSCRIPT
thiocholine (SCh), which is formed by the enzymatic hydrolysis of the ASCh. This cleavage
yields 5-mercapto-2-nitrobenzoic with a strong yellow color and a maximum wavelength of
absorbance at 412 nm. The changes in the absorbance at 25 °C were recorded between 0.5 and
1.5 min after substrate addition to the reaction mixture containing AChE, DTNB and the test
compound in phosphate buffer (pH = 7.4). For the inhibition of BuChE, the sample solutions (50
µL) contained the test inhibitor, DTNB [5,5-dithio-bis(2-nitrobenzoic) acid] (125 µL) and the
BuChE (horse serum butyrylcholinesterase, EC 3.1.1.8, Sigma) solution (25 µL) in Tris-HCl
buffer (pH 8.0). The reactions were carried out in a 96-well microplate and incubated for 15 min
at 25 °C. The reactions were initiated with the addition of butyrylthiocholine chloride (BTCl) (25
µL). The blank was prepared as above, but without enzyme (AChE or BuChE). The sample and
blank absorbances were recorded at 405 nm after 10 min incubation at 25 °C. The absorbance of
the blank was subtracted from that of the sample. The AChE/BuChE inhibition potency of each
compound was expressed as percent inhibition obtained by the following equation:
ꢇꢈ − ꢇꢉ
ꢅ ꢆ
ꢀꢁℎꢀꢂꢀꢃꢀꢄꢁ % =
∙ 100
ꢇꢈ
where Ai and Ac represent the change in the absorbance in the presence of inhibitor and absence
of inhibitor, respectively.
4.7. Anti-oxidant activity evaluation
4.7.1. Phosphomolybdenum assay
The total anti-oxidant activity of the compounds was evaluated by phosphomolybdenum
method according to Zengin et al. with slight modification [52]. The sample solution (0.3 mL)
was combined with 3 mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate
and 4 mM ammonium molybdate) and the absorbance was recorded at 695 nm after 90 min
incubation at 95 °C. The EC50, which is the effective concentration at which the absorbance was
0.5, was calculated for each compound and trolox.
4.7.2. Radical scavenging activity
The radical scavenging effect of the compounds on the 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical was estimated according to Zengin et al. [53]. The sample solution (1 mL) was added to 4
mL of a 0.004% solution of DPPH in methanol. The sample absorbance was recorded at 517 nm
after 30 min incubation at room temperature in dark. The scavenging activity of the compounds
on the ABTS radical cation [2,2’-azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid] was
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ACCEPTED MANUSCRIPT
measured according to the method of Zengin et al. with slight modification [54]. Briefly, ABTS.+
was produced directly by reacting a 7 mM ABTS solution with 2.45 mM potassium persulphate
and allowing the mixture to incubate for 12-16 h in the dark at room temperature. Prior to
initiating the assay, the ABTS solution was diluted with methanol to an absorbance of 0.700 ±
0.02 at 734 nm. The sample solution (1 mL) was added to ABTS.+ solution (2 mL) and mixed,
and the sample absorbance was recorded at 734 nm after 30 min incubation at room temperature.
The corresponding IC50 value, which is the effective concentration at which 50% of
DPPH/ABTS radicals are scavenged, was calculated for each compound and trolox.
4.7.3. Reducing power (CUPRAC and FRAP tests)
The cupric ion reducing activity (CUPRAC) was determined according to the method of
Zengin et al. [55]. The sample solution (0.5 mL) was added to a premixed reaction mixture
containing CuCl2 (1 mL, 10 mM), neocuproine (1 mL, 7.5 mM) and NH4Ac buffer (1 mL, 1 M,
pH 7.0). Similarly, a blank was prepared by adding sample solution (0.5 mL) to a premixed
reaction mixture (3 mL) without CuCl2. The absorbances of the sample and blank were
subsequently recorded at 450 nm after 30 min incubation at room temperature. The absorbance
of the blank was subtracted from that of the sample.
The FRAP (ferric reducing antioxidant power) assay was carried out as described by Zengin et
al. with slight modification [55]. The sample solution (0.1 mL) was added to the premixed FRAP
reagent (2 mL) containing acetate buffer (0.3 M, pH 3.6), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ)
(10 mM) in 40 mM HCl and ferric chloride (20 mM) in a ratio of 10:1:1 (v/v/v). The absorbance
of the sample was subsequently recorded at 593 nm after 30 min incubation at room temperature.
Both results were expressed as EC50 values.
Acknowledgments
This article is based upon work from COST Action CA15135, supported by COST.
Appendix A. Supplementary material
Top scored docking poses and elemental analysis results can be found at http://……….
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