Drug design, synthesis, in vitro and in silico evaluation of selective monoaminoxidase B inhibitors based on 3-acetyl-2-dichlorophenyl-5-aryl-2,3-dihydro-1,3,4-oxadiazole chemical scaffold

Article abstract of DOI:10.1016/j.ejmech.2015.12.026

With the aim to identify new, potent and selective monoamine oxidase B (MAO-B) inhibitors, molecular interaction field analysis has been applied to a MAO-B complex with 3-acetyl-2,5-diaryl-2,3-dihydro-1,3,4-oxadiazole chemical structure, known as a privileged scaffold for this target. Several compounds displayed potent in vitro activity, exhibiting IC₅₀ values in the medium to low nanomolar range. The enantiomers of most promising derivatives were separated by enantioselective HPLC and in vitro evaluated. Experimental results, according to theoretical drug design, clearly indicated a key role of the ligand stereochemistry in the target recognition/inhibition. In particular the (R)- enantiomers showed the best activity with respect to the (S)- stereoisomer. Finally, docking experiments coupled to molecular dynamics (MD) simulations, were applied for understanding the putative MAO -B binding modes of the new compounds providing detailed information for further structural optimization.

Full text of DOI:10.1016/j.ejmech.2015.12.026

European Journal of Medicinal Chemistry 108 (2016) 542e552
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Drug design, synthesis, in vitro and in silico evaluation of selective
monoaminoxidase B inhibitors based on 3-acetyl-2-dichlorophenyl-5-
aryl-2,3-dihydro-1,3,4-oxadiazole chemical scaffold
Simona Distinto a, Rita Meleddu a, Matilde Yanez b, Roberto Cirilli c, Giulia Bianco a,
Maria Luisa Sanna a, Antonella Arridu a, Pietro Cossu a, Filippo Cottiglia a, Cristina Faggi d,
,
Francesco Ortuso e *, Stefano Alcaro e, Elias Maccioni a
a
ꢀ
Dipartimento di Scienze della Vita e dell’Ambiente, Universita degli Studi di Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
b
~
Facultad de Farmacia, Departamento de Farmacología, Universidad de Santiago de Compostela, Campus Vida, La Coruna, 15782 Santiago de Compostela,
Spain
c
ꢀ
Dipartimento del Farmaco, Istituto Superiore di Sanita, V.le Regina Elena 299, 00161 Rome, Italy
d Dipartimento di Chimica “Ugo Schiff”, Via della Lastruccia, 3-13 - 50019 Sesto Fiorentino, Italy
e
ꢀ
Dipartimento di Scienze della Salute, Universita; “Magna Græcia” di Catanzaro, Campus “S. Venuta”, Viale Europa, 88100 Catanzaro, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
With the aim to identify new, potent and selective monoamine oxidase B (MAO-B) inhibitors, molecular
interaction field analysis has been applied to a MAO-B complex with 3-acetyl-2,5-diaryl-2,3-dihydro-
1,3,4-oxadiazole chemical structure, known as a privileged scaffold for this target. Several compounds
displayed potent in vitro activity, exhibiting IC50 values in the medium to low nanomolar range. The
enantiomers of most promising derivatives were separated by enantioselective HPLC and in vitro eval-
uated. Experimental results, according to theoretical drug design, clearly indicated a key role of the
ligand stereochemistry in the target recognition/inhibition. In particular the (R)- enantiomers showed
the best activity with respect to the (S)- stereoisomer. Finally, docking experiments coupled to molecular
dynamics (MD) simulations, were applied for understanding the putative MAO -B binding modes of the
new compounds providing detailed information for further structural optimization.
Received 10 March 2015
Received in revised form
10 December 2015
Accepted 12 December 2015
Available online 17 December 2015
Keywords:
2,3-Dihydro-1,3,4-oxadiazoles
Neurodegenerative disorders
MAO-B
© 2015 Elsevier Masson SAS. All rights reserved.
Docking
Molecular dynamics
1. Introduction
high sequence similarity, they differ in shape and volume of the
catalytic site allowing to discover selective binders. In fact, struc-
Rational design, synthesis, enantioseparation and enzymatic
characterization of new 3-acetyl-2,5-diaryl-2,3-dihydro-1,3,4-
oxadiazoles led to a series of novel monoamine oxidase (MAO)
inhibitors [1]. MAO are ubiquitous enzymes that play a key role in
the degradation of exogenous and endogenous amines. Dopamine
(DA), norepinephrine (NE), epinephrine, serotonin (5HT), and 2-
phenylethylamine (PEA) [2] are among the most common sub-
strates. The mammalian family of MAOs consists of two isozymes,
namely MAO-A and MAO-B, differing in their selectivity versus
substrates and inhibitors, 5HT and NE are preferentially deami-
nated by the A isoform, whereas 2-phenylethylamine and benzyl-
amine are MAO-B substrates [3]. Although the two isoforms share a
tural studies reported the presence of a single cavity of about
550 Å3 for MAO-A, whereas MAO-B has a tight and longer dipartite
cleft, named entrance and substrate cavities. These two pockets can
merge into a single cavity of ~700 Å3 [4]. Therefore, selective,
potent cavity-spanning, MAO-B inhibitors have been identified
such as safinamide [5,6], coumarin derivatives [7], farnesol [8],
caffeine derivatives [9,10] and the anti-diabetes drug pioglitazone
[11]. An increase of the DA levels as well as a neuroprotective effect
can be observed following the inhibition of MAO-B [12e16] As a
consequence, selective irreversible human MAO-B inhibitors
(hMAO-Bi) are used alone or in combination with other drugs, in
the treatment of both Parkinson's (PD) [17,18] and Alzheimer's (AD)
diseases [19e22]. In fact, the increased levels of MAO-B in both PD
patients and elderly population [23,24], may leads to an increased
production of hydrogen peroxide and other reactive oxygen spe-
cies, responsible for neuron degeneration [25e28]. Moreover, it has
* Corresponding author.
E-mail address: ortuso@unicz.it (F. Ortuso).
http://dx.doi.org/10.1016/j.ejmech.2015.12.026
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

S. Distinto et al. / European Journal of Medicinal Chemistry 108 (2016) 542e552
543
been proposed that the degeneration of the dopaminergic neurons,
observed in PD patients could be related to the radical species over-
production deriving from an accelerated dopamine metabolism
[29e31] Thus, neurological degeneration in the central nervous
system (CNS) and in particular in “substantia nigra”, could be
associated to the oxidative stress, with an increased MAO-B activity,
and with a decreased elimination rate of free radical species. MAO-
A expression, differently from MAO-B, does not increase with age,
suggesting that a totally independent mechanism regulates the
expression of the two enzymatic isoforms [32,33].
biological activity of the introduction of a 3,4-dichlorophenyl
moiety in the position of the dihydrooxadiazole scaffold.
Furthermore, given the presence of a chiral centre at the position 2
of the dihydrooxadiazole ring, the biological activity of the pure
enantiomers was investigated.
2
2. Results and discussion
2.1. Design of compounds 4a-j
Due to the relevance of this target many classes of compounds
were recently synthesized and tested for the inhibitory activity
towards MAO-B [34e37]. Previously, we identified highly potent
and selective MAO inhibitors 1-thiocarbamoyl-3,5-diaryl-4,5-
dihydro-(1H)-pyrazole [38] and 2-thiazolylhydrazones [39,40].
Moreover, in a recent study we demonstrated that the 2,3-dihydro-
1,3,4-oxadiazoles can be considered as a new interesting scaffold
for MAO-B selective inhibitors [1]. In this work, extending our
previous studies [36e39], we introduced a second chlorine atom on
the phenyl ring. According to our binding mode models, the second
chlorine atom points toward the Flavin Adenin Dinucleotide (FAD)
co-factor. Docking studies highlighted the preference of the chiral
carbon (R)- configuration for the binding to the MAO-B active site
(Fig. 1). Hence, according to the structural indication, acquired by
molecular interaction field analyses, we synthesised and tested a
series of 3-acetyl-2-(3,4-dichlorophenyl)-5-aryl-2,3-dihydro-1,3,4-
oxadiazoles 4a-j, in order to evaluate the influence on the
Starting from previously reported theoretical complexes be-
tween MAO-B and the 2-(4-chlorophenyl)-3-acetyl-5-(4-
chlorophenyl)-2,3-dihydro-1,3,4-oxadiazole [1] an extended mo-
lecular interaction fields (MIFs) analysis was carried out. After
removing the inhibitor, the interaction capabilities of the MAO-B
active site were investigated by means of appropriate probes
(Experimental section) as implemented in the program GRID [41].
The resulting isocontour maps were inspected taking into account
the two most energy stabilized previously reported poses (Pa and
Pb) of the inhibitor into the hMAO-B binding cleft [1]. MIFs indi-
cated strong interaction between the target and organic chlorine
suggesting the possibility to include a second chlorine atom on the
p-chlorophenyl ring substituent located at the position 2 of the
dihydrooxadiazole ring (Fig. 1).
Bromine contribution was, successively, evaluated by means of
BR probe. The visual inspection of the computed isocontour maps
revealed the possibility to replace the p-chlrophenyl at 5 position
moiety with the corresponding bromine analogues. In fact, taking
into account the same energy threshold (Experimental section), BR
map demonstrated wider surface than CL one but it did not
discriminated between the two aromatic rings located at oxadia-
zole ring positions 2 and 5 respectively (Fig. S1). The role of other
substituents was also investigated by computing the MIFs using C3
(methyl group), AR.COO (aromatic carboxylic moiety) and ON (nitro
group) probes. The C3 map graphical inspection has suggested the
methyl group as a possible substituent for both aromatic rings
(Fig. S2). The energy threshold required to overlap both Pa and Pb
poses was about 2 kcal/mol disfavoured with respect to halogen
mimicking probes, so we considered the introduction of the methyl
on the aromatic ring at position 5 only. A similar scenario was
observed for AR.COO, even if its recognition of the aromatic ring
located at oxadiazole position 5 was stronger (Fig. S3). On the other
hand, considering that our target is located in the CNS, a carboxylic
group was considered much too hydrophilic. As a consequence,
another electron withdrawing electron withdrawing substituent,
such as the nitro group, was theoretically investigated by means of
the ON GRID probe (Fig. S4). Nitro group MIFs overlapped both
aromatic rings. Finally, MIFs analyses interestingly highlighted a
key role of the oxadiazole ring chiral centre that allowed, in (R)-
configuration, the best superimposition between GRID maps and
the ligand aromatic rings.
A requirement important for selectivity seem to be the ability of
the inhibitors to fit within the elongated bipartite cavity of MAO-B,
which is bigger (700 Å3) and narrower than the single substrate
cavity of MAO-A (550 Å3) [4]. Therefore the shape of binding site
was considered and the skeleton of the series was maintained
because of its ability to occupy both cavities. In fact previous series
showed good selectivity toward MAO-B [1,38e40,42].
2.2. Synthesis
Fig. 1. Chlorine molecular interaction field (yellow wireframe grids) displayed on
previous reported [1] a) Pa and b) Pb theoretical complexes. The FAD cofactor is re-
ported in spacefill notation and the ligand in sticks cyan carbon and brown chlorine
atoms coloured. The rest of the enzyme is depicted in transparent violet cartoon (For
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
The 4a-j series was synthesized according to a previously
described procedure [1] as illustrated in Scheme 1.
Briefly, the suspension in acetic anhydride of the appropriate
benzoylhydrazide was heated under reflux until the formation of a

544
S. Distinto et al. / European Journal of Medicinal Chemistry 108 (2016) 542e552
Cl
H
Ar
O
Ar
Ar
O
N
ii
i
+
NH
NH2
NH
N
Cl
O
N
O
Cl
Cl
Cl
O
Cl
H3C
1
2
3a-j
4a-j
i: CH3COOH (CAT)/ EtOH, reflux; ii: (CH3CO)2O, reflux
Scheme 1. Synthetic pathway to compounds 4a-j.
Table 1
Inhibitory activities towards hMAO
dichlorophenyl)-5-aryl-2,3-dihydro-1,3,4-oxadiazoles derivatives 4a-j. Data are re-
yelloweorange solution was observed. The reaction mixture was
then poured onto ice water and vigorously stirred. The obtained
precipitate was then washed with 10% aqueous NaHCO3 solution,
water, and purified by crystallization. All of the products were
characterised by means of both analytical and spectroscopic
methods (Experimental section and Supplementary data) and
tested towards MAO-A and MAO-B.
A
and hMAO-B of 3-acetyl-2-(3,4-
ported in nM.
Compound
Ar
hMAO-A (IC50
)
hMAO-B (IC50)
4a
**
69360 3510
4b
4c
4d
4e
4f
**
**
**
**
**
**
***
2.3. Biological activity
The inhibitory activities of compounds 4a-j on human recom-
binant MAO eA and eB isoforms, expressed in baculovirus infected
BTI infected cells, as IC50, are reported in Table 1. Tested compounds
demonstrated no interference with the measurements, since they
were unable to react directly with the Amplex Red reagent. The
kinetic parameters of hMAO-A and hMAO-B were evaluated in the
presence of different tyramine concentrations. In our experiments,
hMAO-A displayed a Michaelis constant (Km) of 514 46.8
maximum reaction velocity (Vmax) of 301.4 27.9 nmol/min/mg
protein, whereas hMAO-B showed a Km of 104.7 16.3 M and a
217.99 10.62
19.35 0.68
626.82 36.52
9.46 0.57
**
mM and a
m
V
max of 28.9 6.3 nmol/min/mg protein (n ¼ 5). Active compounds
4g
showed reversible behaviour according to the method proposed by
Cer et al. [43]. Therefore, reported IC50 is a useful tool to determine
the relative activity of the compounds within the series as well as to
determine substituent and key positions in the scaffold.
4h
4i
**
**
**
67.69 4.27
Half of the tested compounds exhibited inhibitory activity to-
wards hMAO-B at nM concentration, with the exception of com-
pound 4b which inhibited the corresponding MAO activity by
approximately 40e50% at the concentration of 100
pounds 4g and 4i that were inactive at 100 M. Compounds 4a and
4j were effective in the M range. Interestingly, none of the tested
compounds exhibited activity towards the A isoform of the enzyme
**
mM and com-
4j
2460 170
m
m
at the tested concentration of 100 mM.
Clorgyline
Deprenyl
Iproniazide
Moclobemide
4.60 0.30
67250 1020
6560 760
61350 1130
19.00 0.86
7540 360
*
2.4. Structure activity relationships
3613.80 193.70
Biological tests confirmed our hypothesis, that the introduction
of a 3,4-dichlorophenyl moiety in the position 5 of the dihy-
drooxadiazole ring could lead to more effective and selective
compounds with respect to the previously reported mono-chloro
derivatives (i.e. the mono-chloro analogue of 4f was inactive on
All IC50 values shown in this table are the mean S.E.M. from five experiments.
* Inactive at 1 ꢀ 106 nM (highest concentration tested).
** Inactive at 100000 nM (highest concentration tested). At higher concentration the
compounds precipitate.
*** 100000 nM inhibits the corresponding MAO activity by approximately 40e45%.
At higher concentration the compounds precipitate.
MAO-A whereas demonstrated
a
MAO-B IC50 equal to
121.62 9.63 nM) [1]. Although an excellent remote Hammett
correlation (sp or sþp ) has been found for para substitution in the
two aryl rings at the positions 2 and 5 of the dihydrooxadiazole
[44], the electronic effect of the substituent in the para position of
the phenyl moiety at the position 5 of the dihydrooxadiazole did
not seem to play a relevant role in determining the potency of the
inhibitors. On the contrary, just the presence of a substituent in this
position seemed to be essential for the activity, probably due to the
need of bulky substituent in this position. This hypothesis was
corroborated by the low activity exhibited by compound 4a and 4b
bearing a pyridil and a phenyl substituent respectively. However
either the introduction of a dimethylamino moiety or of a cyano as
in compound 4g and 4i, led to a decrease of the inhibitory activity. It
is worth to note that the introduction of a methyl substituent in the
ortho position, as in compound 4j, led to decrease of the inhibitory
potency more than 100 fold higher than the corresponding para-
substituted compound 4d. Hence a complex blend of electronic
and steric effects should be considered to determine the best
substitutions for the biological activity.

S. Distinto et al. / European Journal of Medicinal Chemistry 108 (2016) 542e552
545
Table 2
2.5. Enantiomeric mixture resolution
Inhibitory activities towards hMAO-B of 3-acetyl-2-(3,4-dichlorophenyl)-5-aryl-2,3-
dihydro-1,3,4-oxadiazoles 4e and 4f as pure enantiomers and racemates. Data re-
ported in nM.
All the synthesized compounds have a stereogenic centre at the
position 2 of the heterocyclic ring. 4d, 4e and 4f were chosen to
perform the separation of the single enantiomers and to evaluate
the influence of stereochemistry on their biological properties.
Repetitive semipreparative HPLC resolutions carried out on the
Chiralpak IA chiral stationary phase (CSP) using the mixture
dichloromethane/DEA 100/0.1 (v/v) as eluent enabled us to easily
collect tens of mg of enantiopure samples (Table S1). The enan-
tiomeric nature of the samples obtained on mg-scale was demon-
strated by polarimetric (Table S1) and circular dichroism (CD)
analysis (Fig. S5).
The chiroptical properties of the enantiopure antipodes were
perfectly specular (Table S1 and Fig. S2). The absolute configuration
of the enantiomers of 4e and 4f was empirically established by CD
correlation method using the enantiomers of 4d as reference
samples. Crystallization of the second eluted enantiomer of (-)-4d
(in the chromatographic conditions reported in Table S1) from
ethanol/water afforded single crystals which were suitable for X-
ray diffraction analysis. An Oak Ridge Thermal Ellipsoid Plot Pro-
gram (ORTEP) view of (S)-(-)- 4d is showed in Fig. 2. The replace-
ment of the methyl group on the phenyl ring (compound 4d) by a
chlorine atom (compound 4e) or a nitro group (compound 4f) did
not produce significantly alteration in the spectral location of the
maximum and minimum of the representative CD bands recorded
in ethanol solution (Fig. S5).
Compound
) 4e
Structure
hMAO-B (IC50)
(
626.82 36.52
203.59 18.61
**
(R)-(þ) 4e
(S)-(-) 4e
(
) 4f
9.46 0.57
7.61 0.64
523.46 41.30
(R)-(þ) 4f
(S)-(-) 4f
Each IC50 values is the mean S.E.M. from five experiments.
** Inactive at 100000 nM (highest concentration tested).
varies with a Ki ranging from 35 to 0.10 mM. Among the docking
settings implemented in Glide [46] software (see the Experimental
section), Quantum Mechanics-polarized ligand docking (QMPL)
workflow [47,48] successfully reproduced the crystallographic
poses of 5 out of 7 compounds within 1.6 Å root-mean-square
deviation (RMSD). However, also the docking pose of farnesol, the
co-crystallized ligand reported in 2BK3 [8] PDB model, in absence
of electrondensity maps could be considered good. In fact, farnesol
has a plausible pose rotated of almost of 180ꢁ and the second best
pose, is slightly shifted compared to experimental data even if with
an RMSD value > 2 Å (Fig. 3). (Table S1).
QMPL docking workflow combines docking with ab initio for
ligand charges calculation within the protein environment. QMPL
workflow consists of three steps: in the first one proteinꢂligand
complexes are generated with Glide (Grid Based Ligand Docking
with Energetics).
The pure enantiomers of 4e and 4f, respectively the less active
and the most active compounds in the nM range, were then eval-
uated for their ability to inhibit the two different MAO isoforms.
Also in this case no activity towards MAO-A at the concentration of
100 mM was observed. The results for MAO-B isoform are reported
in Table 2. In agreement with molecular modelling suggestion the
activity is exclusively (4e) or mostly (4f) associated with the (R)-
enantiomer.
2.6. Molecular modelling
In the second step, a mixed quantum mechanical/molecular
mechanics (QM/MM) method is used to compute the ligand charge
distribution. The protein is defined as the MM region, and the
ligand is defined as the QM region. In the third step, the ligands are
submitted to another Glide docking run where the ligand charges
are substituted with the new charge sets calculated in the second
step [47,48].
In order to rationalize at molecular level the enzyme inhibition
of most promising compounds, suggesting their putative binding
mode, docking and molecular dynamics (MD) simulations were
carried out. Thus, we validated our docking protocol performing
self and cross-docking of seven crystallized inhibitors, (Tables S1
and S2), into the hMAO-B crystal structure deposited into the
Protein Data Bank [45] with code 2V5Z [7]. The purpose of this task
was to estimate the efficiency of our docking protocol in predicting
the correct orientation of original inhibitors into the substrate
binding cavity. The biological activity of reference compounds
The same protocol was applied to dock the separated enantio-
mers (R)-4e and (R)-4f. Due to lack of accuracy of docking scores,
the simple docking experiment alone is not often sufficient to rank
binding scores with a linear correlation with experimentally
measured binding affinities of known complexes [49]. Therefore we
decided to use a combination of docking and MD. Hence, to better
estimate the ligandereceptor interaction energies, the poses,
reporting an RMSD >2 Å within the top five ranked binding poses,
1
2
were submitted to 10 ns of MD simulations (pose and Table 3).
Actually, explicit solvent MD, coupled with efficient free-energy
sampling algorithms, can potentially offers accurate prediction of
ligands to proteins binding free energies. Firstly, the molecular
dynamic simulation protocol was validated considering the 7
crystal taken into account for docking validation in three possible
system setting: monomer, dimer and dimer in membrane (Fig. 4).
We decided to consider the enzyme embedded into the mito-
chondrial membrane since in literature has been reported its key
role in compound recognition. In particular, it was reported that the
loop 85e112 is involved in the substrate catalytic site entrance
Fig. 2. An ORTEP view of the molecular structure of (S)-(-)-4d.

546
S. Distinto et al. / European Journal of Medicinal Chemistry 108 (2016) 542e552
Fig. 3. Results of seven self and cross-docking experiments performed using 2V5Z as receptor model. Below each docking superimposition is reported the RMSD between co-
crystallized (in ball and sticks) and first pose (in sticks). For 2BK3 ligand is reported also the second docking pose. The inhibitor ID correspondence is SAG: Safinamide; C17: 7-
[(3-chlorobenzyl)oxy]-2-oxo-2H-chromene- 4-carbaldehyde; RM1: N-methyl-1(R)-aminoindan;
chlorobenzyl)oxy]-4-[(methylamino)methyl]- 2H-chromen-2-one; ISN: isatin; FOH: farnesol.
1 PB: 1,4-diphenyl-2-butene[(1E)-4-phenylbut-1-enyl]benzene; C18:7-[(3-
Table 3
correct prediction of the interaction energy.
Averaged energies of interaction between new compounds and hMAO-B. Tot-E,
Coul-E and vdW-E are respectively the total interaction energy, its electrostatic and
van der Waals terms expressed in kcal/mol. The different starting poses are indi-
cated by1 and2.
Thus the monomer MD setting was used to run further simu-
lation taking into account 2 starting poses per compound. The best
pose results are illustrated in Fig. 6. Compound 4e was able to enter
deeply in the binding cavity and was stabilized by hydrophobic and
aromatic interactions (Fig. 6aec). Although the dichlorophenyl
Tot-E
Coul-E
vdW-E
(R)-4e1
(R)-4e2
(R)-4f1
(R)-4f2
ꢂ56.76
ꢂ56.19
ꢂ64.15
ꢂ63.75
ꢂ5.38
ꢂ2.98
ꢂ9.26
ꢂ6.89
ꢂ45.78
ꢂ51.79
ꢂ50.39
ꢂ48.12
moiety was able to rotate, still the
pep interaction with Phe343
was conserved. On the contrary, compound 4f was kept between
the entrance and the catalytic cavity separated by the Ile199 due to
Fig. 4. Overview of MAO B a) monomer; b) homodimer; c) homodimer embedded in a lipid bilayer.
modulation [50]. We observed that the estimated interaction en-
ergies well correlate with G of dissociation data with all settings
(Fig. 5). However, the monomer shows the best correlation with a
r2 ¼ 0.89 (0.98 if we leave the crystal 1OJA out of the evaluation).
The inhibitor ID correspondence is SAG: Safinamide; C17: 7-[(3-
chlorobenzyl)oxy]-2-oxo-2H-chromene- 4-carbaldehyde; RM1: N-
the interaction of the bulky nitro moiety with residues and waters
in this region. More in details, compound 4f resulted stabilized
within the binding site through an array of aromatic (Tyr326,
Phe168), hydrophobic (Leu171, Ile199, Leu167, Ile198), and H-bond
interactions with binding site residues (Fig. 6def and Fig. S6).
The H-bond between the 4f carbonyl and the hydroxyl group of
the important residue Tyr435, which, together FAD and Tyr398,
delimits the aromatic cage pivotal for MAO activity, was monitored
and found to be stable along the whole MD simulation (Fig. S7). In
addition, the number of good contacts was higher for 4f compared
to 4e (Fig. S6). Furthermore, the averaged energy of interaction
(Table 3) was in accordance with the difference of activity and
underlines the higher weight of van der Waals interactions
D
methyl-1(R)-aminoindan;
phenylbut-1-enyl]benzene;
1
PB: 1,4-diphenyl-2-butene[(1E)-4-
C18:7-[(3-chlorobenzyl)oxy]-4-
[(methylamino)methyl]- 2H-chromen-2-one; ISN: isatin; FOH:
farnesol.
According to these data, the presence of the membrane into the
theoretical models, although relevant in the stage of compound
entrance and recognition, was found to be not essential for the

S. Distinto et al. / European Journal of Medicinal Chemistry 108 (2016) 542e552
547
Fig. 5. MD energy analysis. Inhibitors are arranged from left to right by increasing magnitude of DG of dissociation. Sum of binding energy components for each inhibitor and setting
(a) monomer, (c) dimer, (e) membrane. The Total energy (Tot_E) of interaction is shown with a line. Besides each histogram: figures (b), (d) and (f), the related linear regression plot
and r2 values for the sum of all interaction energies between each inhibitor and MAO B.
contribution.
with favourable score. In conclusion, 4d revealed to have slightly
more favorable physicochemical properties and therefore seems to
be a good candidate for future development.
With the aim to estimate the drug-likeness of the compounds, in
silico ADME-tox prediction has been carried out [51]. Since 4f was
identified as the best inhibitor of our series, we investigated its
absorption, distribution, metabolism, excretion and toxicity
(ADMEtox) properties using the Qikprop [52] software. Such an
approach provides molecular properties by means of a comparison
of the novel compound with respect to the 95% of known drugs. The
computed molecular descriptors are shown in Table S4. All prop-
erties were within the ranges specified. The predicted IC50 for hu-
3. Conclusion
Given our interest in the development of MAO-B inhibitors as a
potential therapeutic strategy for neurodegenerative diseases, we
developed molecules with optimized activity toward B isoform.
Therefore, we synthesised a new series of 2-(3,4-dichlorophenyl)-
3-acetyl-5- aryl-2,3-dihydro-1,3,4-oxadiazoles. The introduction of
a second chlorine atom in the 5-phenyl substituent improved po-
tency. Several compounds showed inhibition properties in the nM
range and a high selectivity toward MAO-B. As previously observed
for similar compounds, the (R)-enantiomer exhibits a higher
inhibitory activity compared to both the racemic mixture and the
(S)-enantiomer. We have validated a computational protocol with
good qualitative accord between experimental and theoretical ac-
tivities. Therefore, the devised workflow will be applied for future
studies.
ꢀ
man Ether-a-go-go-Related Gene (HERG) K þ Channel Blockage was
borderline in its value. However, considering the possibility of a
limited error of prediction in discriminating a safe or not-safe
compound, biological experiments are needed. Tissue distribution
is an important element of the pharmacokinetic (PK) profile of a
drug united with knowledge of the in vitro activity. The activity of
this compound should occur in the brain, therefore we paid great
attention on analysing in silico molecular descriptors that are
required for a CNS active agent like the polar surface area (PSA) and
the logarithm of the bloodebrain barrier partition (LogBB). The
values indicate that newly synthesized molecule would cross
bloodebrain barrier (BBB) with a low rate. Hence, we checked if
this was an issue of other interesting compounds of the series and
in particular of 4d, which also has good inhibitory activity
(19.35 0.68 nM). In this case the prediction of CNS activity was
positive. Therefore, this compound appears more interesting in this
respect. Finally, qualitative human oral absorption was predicted
4. Experimental
4.1. Chemistry and compounds characterization
4.1.1. General methods
Melting points were uncorrected and were determined on a

548
S. Distinto et al. / European Journal of Medicinal Chemistry 108 (2016) 542e552
Fig. 6. a) d) Superimposed structures of 10 ns MD simulations frames of [4e 4f- MAO-B] complex coloured by time-step: initial (red), final (blue) along with intermediate structures
snapshots. b) e) close-up of the binding cavity; c) f) 2D representing compound and interacting residues(For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.).
Reichert Kofler thermopan apparatus. 1H NMR spectra were
recorded on a Varian Unity 500 and on a Bruker 400, using tetra-
methylsilane (TMS) as internal standard (chemical shifts in
C, 53.59; H, 3.30; N, 12.50, Found C, 53.55; H, 3.29; N, 12.46.
1H-NMR (500 MHz, DMSO):
2.39 (s, 3H), 7.24 (s, 1H), 7.50 (d,
1H, J ¼ 8 Hz), 7.73 (m, 3H, J ¼ 8, and 4 Hz), 7.82 (s, 1H), 8.75 (s, 2H).
13C-NMR (100 MHz, DMSO):
21.38, 91.36, 120.39, 127.23,
129.26, 131.39, 131.71, 132.94, 137.15, 150.80 (2C), 153.15, 157.01,
166.44, 166.55.
d
d
values). NMR spectra are reported in Supplementary data. Elec-
tron ionisation (EI) mass spectra were obtained by a Fisons QMD
1000 mass spectrometer (70 eV, 200 A, ion source temperature
d
m
200 ꢁC). The samples were introduced directly into the ion source.
TLC analyses were performed on silica gel 60 F254 plates. All syn-
thesized compounds were purified by crystallization from an
appropriate solvent. Elemental analyses were obtained on a Perkin
Elmer 240 B microanalyzer. Analytical data of the synthesised
compounds are in agreement within 0.4% of the theoretical
values.
4.1.2.2. 4b: 2-(3,4-dichlorophenyl)-3-acetyl-5- phenyl-2,3-dihydro-
1,3,4-oxadiazole. C16H12Cl2N2O2. White crystals, M.p. 99e100 ꢁC
(ethanol), MS (m/z) 335, yield 43%. CHN %: Calc. C, 57.33; H, 3.61; N,
8.36, Found C, 57.36; H, 3.63; N, 8.37.
1H-NMR (400 MHz, DMSO):
d 2.27 (s, 3H), 7.22 (s, 1H), 7.48 (dd,
1H, J ¼ 8.3 and 2.0 Hz), 7.54 (t, 2H, J ¼ 7.6 Hz), 7.61 (t, 1H, J ¼ 7.2 Hz),
7.73 (d, 1H, J ¼ 8.4 Hz), 7.95 (d, 1H, J ¼ 2 Hz), 7.84 (m, 2H).
13C-NMR (100 MHz, DMSO):
d 21.39, 90.56, 123.89, 126.83 (2C),
4.1.2. Synthesis of compounds 4a-j [1]
127.03, 129.12, 129.32 (2C), 131.46, 131.68, 132.24, 132.79, 137.57,
154.80, 167.27.
Equal amounts of aromatic aldehyde (0.018 mol) and the
appropriate arylhydrazide (0.018 mol) were refluxed in ethanol
(60 mL) under vigorous stirring from 1 to 3 h in presence of acetic
acid as catalyst (0.5 mL). With respect to the different aromatic
substituents in positions 2 and 5 of the oxadiazole ring, a differently
coloured solution is obtained. The reaction solution is monitored by
TLC (eluent dichloromethane: methanol 20:1). After the mixture is
cooled at room temperature, the precipitated product is filtered off
and then washed with isopropyl ether. Obtained arylidenearylhy-
drazides (0.003 mol) are refluxed in 6 mL of acetic anhydride under
vigorous stirring from 15 min to 2 h. The suspension is monitored
by TLC (eluent chloroform: methanol 20:1). The solution is then
poured onto ice-water (100 g) and vigorously stirred. A precipitate
is formed which is washed with NaHCO3 (10% aqueous solution) to
remove the acetic acid. The obtained solid is further purified by
crystallization.
4.1.2.3. 4c: 2-(3,4-dichlorophenyl)-3-acetyl-5-(4-methoxyphenyl)-
2,3-dihydro-1,3,4-oxadiazole. C17H14Cl2N2O3. Pale yellow crystals,
M.p. 118e120 ꢁC (ethanol), MS (m/z) 364, yield 45%. CHN %: Calc. C,
55.91; H, 3.86; N, 7.67, Found C, 55.89; H, 3.86; N, 7.65.
1H-NMR (500 MHz, DMSO):
d 2.25 (s, 3H), 3.82 (s, 3H), 7.07 (d,
2H, J ¼ 9 Hz), 7.17 (1H, s), 7.45 (dd, 1H, J ¼ 8.5 and 2.0 Hz), 7.71 (d,
1H, J ¼ 8.5 Hz), 7.75 (d, 1H, J ¼ 2 Hz), 7.77 (d, 2H, J ¼ 8.5 Hz).
13C-NMR (100 MHz, DMSO):
d 21.32, 55.66, 90.16, 114.75 (2C),
115.93, 126.89, 128.69 (2C), 128.99, 131.40, 131.61, 132.67, 137.67,
154.81, 162.29, 167.01.
4.1.2.4. 4d: 2-(3,4-dichlorophenyl)-3-acetyl-5-(4-methylphenyl)-2,3-
dihydro-1,3,4-oxadiazole. C17H14Cl2N2O2. Pale yellow crystals, M.p.
120e122 ꢁC (isopropyl ether), MS (m/z) 348, yield 75%. CHN %: Calc.
C, 58.47; H, 4.04; N, 8.02, Found C, 58.50; H, 4.03; N, 8.05.
4.1.2.1. 4a: 2-(3,4-dichlorophenyl)-3-acetyl-5- pyridin-2,3-dihydro-
1,3,4-oxadiazole. C15H11Cl2N3O2. Pink powdery solid, M.p.
117e118 ꢁC (isopropyl ether), MS (m/z) 294, yield 92%. CHN %: Calc.
1H-NMR (400 MHz, DMSO):
d
2.26 (s, 3H), 2.38 (s, 3H), 7.2 (s,
1H), 7.34 (d, 2H, J ¼ 8 Hz), 7.46 (dd, 1H, J ¼ 8 and 2 Hz), 7.72 (m, 3H,

S. Distinto et al. / European Journal of Medicinal Chemistry 108 (2016) 542e552
549
J ¼ 8, and 2 Hz), 7.77 (d, 1H, J ¼ 2 Hz).
13C-NMR (100 MHz, DMSO):
21.32, 21.38, 90.37, 121.06, 126.83
58.47; H, 4.04; N, 8.02 Found C,58.49; H, 4.06; N, 8.04.
1H-NMR (400 MHz, DMSO):
2.43 (3H, s), 2.70 (3H, s), 7.45 (1H,
d
d
(2C), 126.98, 129.07, 129.87 (2C), 130.17, 131.47, 132.76, 137.63,
142.44, 154.94, 167.17.
s), 7.54 (1H, t, CH, J ¼ 7.33 Hz), 7.78 (1H, d, J ¼ 7.6 Hz), 7.88 (1H, dd,
J ¼ 8.2 and 2.2), 8.09 (1H, td, J ¼ 7.6 and 1.5 Hz), 8.14 (3H, m).
13C-NMR (100 MHz, DMSO):
d 20.18, 23.11, 91.37, 126.39, 128.28,
4.1.2.5. 4e: 2-(3,4-dichlorophenyl)-3-acetyl-5-(4-chlorophenyl)-2,3-
dihydro-1,3,4-oxadiazole. C16H11Cl3N2O2. Pale yellow crystals, M.p.
119e120 ꢁC (ethanol); MS (m/z) 367, yield 92%. CHN %: Calc. C,
51.99; H, 3.00; N, 7.58 Found C, 52.02; H, 3.02; N, 7.60.
128.39, 128.56, 128.95, 129.71, 130.58, 131.34, 132.12, 132.98, 133.35,
136.50, 154.70, 157.99.
4.1.3. HPLC resolution of 4d-f
1H-NMR (400 MHz, DMSO):
d
2.27 (3H, s), 7.22 (1H, s), 7.48 (1H,
The enantioseparations of 4d-f were performed by using the
dd, J ¼ 8.4 and 2.0 Hz), 7.61 (2H, d, J ¼ 8.8 Hz), 7.72 (1H, d,
stainless-steel Chiralpak IA (250 mm
ꢀ
4.6 mm i.d. and
J ¼ 8.4 Hz), 7.80 (1H, d, J ¼ 2.0 Hz), 7.84 (2H, d, J ¼ 8.8 Hz).
250 ꢀ 10 mm i.d.) (Chiral Technologies Europe, Illkirch, France)
columns. All chemicals and solvents for HPLC were purchased from
Aldrich (Italy) and used without further purification. The analytical
HPLC apparatus consisted of a PerkineElmer (Norwalk, CT, USA)
200 lc pump equipped with a Rheodyne (Cotati, CA, USA) injector, a
13C-NMR (100 MHz, DMSO):
d 21.39, 90.88, 122.83, 127.10,
128.63 (2C), 129.17, 129.49 (2C), 131.44, 131.70, 132.85, 136.88,
137.41, 154.02, 167.34.
4.1.2.6. 4f: 2-(3,4-dichlorophenyl)-3-acetyl-5-(4-nitrophenyl)-2,3-
dihydro-1,3,4-oxadiazole. C16H11Cl2N3O4. Yellow crystals, M.p.
165e166 ꢁC (ethanol); MS (m/z) 348, yield 47%. CHN %: Calc. C,
50.55; H, 2.92; N, 11.05 Found C, 50.53; H, 2.91; N, 11.03.
20-ml sample loop, a HPLC Dionex CC-100 oven (Sunnyvale, CA,
USA) and a Jasco (Jasco, Tokyo, Japan) Model CD 2095 Plus UV/CD
detector. For semipreparative separations a PerkineElmer 200 LC
pump equipped with a Rheodyne injector, a 1 mL sample loop, a
PerkineElmer LC 101 oven and Waters 484 detector (Waters Cor-
poration, Milford, MA, USA) were used. The signal was acquired and
processed by Clarity software (DataApex, Prague, The Czech
Republic).
1H-NMR (500 MHz, DMSO):
d 2.29 (3H, s), 7.26 (1H, s), 7.51 (1H,
dd, J ¼ 8 and 1.5 Hz), 7.72 (1H, d, J ¼ 8.5 Hz), 7.83 (1H, d, J ¼ 2.0 Hz),
8.07 (2H, d, J ¼ 8 Hz), 8.35 (2H, d, J ¼ 9 Hz).
13C-NMR (100 MHz, DMSO):
d 21.38, 91.46, 122.51, 124.41 (2C),
127.20, 128.12 (2C), 129.23, 129.83, 131.38, 132.94, 137.14, 149.25,
153.26, 167.55.
4.1.4. Circular dichroism
The CD spectra were measured in ethanol solution by using a
Jasco Model J-700 spectropolarimeter. The concentration was
0.2 mg/mL. The optical path and temperature were set at 0.1 mm
and 20 ꢁC, respectively. The spectra were average computed over
three instrumental scans and the intensities were presented in
terms of ellipticity values (mdeg).
4.1.2.7. 4g: 2-(3,4-dichlorophenyl)-3-acetyl-5-(4-N,N-dimethylami-
nophenyl)-2,3-dihydro-1,3,4-oxadiazole. C18H17Cl2N3O2.
Yellow
crystals, M.p. 156e158 ꢁC (isopropyl ether); MS (m/z) 377, yield 76%.
CHN %: Calc. C, 57.16; H, 4.53; N, 11.11 Found C, 57.18; H, 4.55; N,
11.13.
1H-NMR (400 MHz, DMSO):
d 2.23 (3H, s), 2.99 (6H, s), 6.77 (2H,
d, J ¼ 8.8 Hz), 7.13 (1H, s), 7.43 (1H, dd, J ¼ 8.4, and 2 Hz), 7.63 (2H, d,
4.1.5. Polarimetry
J ¼ 9.2 Hz), 7.72 (2H, m, J ¼ 8.4 Hz).
Specific rotations were measured at 589 nm by a PerkinElmer
polarimeter model 241 equipped with a Na/Hg lamp. The volume of
the cell was 1 cm3 and the optical path was 10 cm. The system was
set at 20 ꢁC.
13C-NMR (100 MHz, DMSO):
d 21.36, 39.80 (2C), 89.55, 109.72,
111.68 (2C), 126.85, 128.20 (2C), 128.94, 131.45, 131.61, 132.58,
137.99, 152.58, 155.69, 166.68.
4.1.2.8. 4h: 2-(3,4-dichlorophenyl)-3-acetyl-5-(4-bromophenyl)-2,3-
dihydro-1,3,4-oxadiazole. C16H11BrCl2N2O2. White cottony solid,
M.p. 140e142 ꢁC (ethanol); MS (m/z) 411, yield 47%. CHN %: Calc. C,
46.41; H, 2.68; N, 6.77 Found C, 46.40; H, 2.69; N, 6.78.
4.1.6. Crystal structure determination for compound 4d
C17H14Cl2N2O2 M ¼ 349.21, Monoclinic, space group P 21,
a ¼ 9.095(1), b ¼ 6.552(1), c ¼ 13.870(1)Å,
b
¼ 95.982(4),
V ¼ 822.0(2)Å3, Z ¼ 2 Dc ¼ 1.415,
m
¼ 3.643 mm-1, F(000) ¼ 362.
1H-NMR (400 MHz, DMSO):
d
2.27 (3H, s), 7.22 (1H, s), 7.48 (1H,
3040 reflections were collected with a 4.89<q
< 69.78 range with a
dd, J ¼ 8.4 and 2.0 Hz), 7.72 (1H, d, J ¼ 8.4 Hz), 7.76 (4H, m,
completeness to theta 95.7%; 2307 were unique, the parameters
were 213 and the final R index was 0.0446 for reflections having
I > 2sI and 0.0689 for all data. A colourless prismatic shaped crystal
J ¼ 2.0 Hz), 7.79 (1H, d, J ¼ 2 Hz).
13C-NMR (100 MHz, DMSO):
d
21.39, 90.88,123.17,125.77,127.10,
128.74 (2C), 129.17, 131.44, 131.70, 132.41 (2C), 132.85, 137.40,
154.13, 167.34.
(0.08 ꢀ 0.06 ꢀ 0.02) was used for data collection. Hydrogen atoms
were assigned in calculated positions, except H2 (on asymmetric
C2) which was found in the F.D map. RX-analysis was carried out
with a Goniometer Oxford Diffraction KM4 Xcalibur2 at room
4.1.2.9. 4i: 2-(3,4-dichlorophenyl)-3-acetyl-5-(4-cyanophenyl)-2,3-
dihydro-1,3,4-oxadiazole. C17H11Cl2N3O2. Pale yellow crystals, M.p.
148e150 ꢁC (ethanol/isopropanol); MS (m/z) 359, yield 86%. CHN %:
Calc. C, 56.69; H, 3.08; N, 11.67; N, 6.77 Found C, 56.68; H, 3.09, N,
11.68.
temperature. Cu/Ka radiation (40 mA/-40 KV), monochromated by
an Oxford Diffraction Enhance ULTRA assembly, and an Oxford
Diffraction Excalibur PX Ultra CCD were used for cells parameters
determination and data collection. The integrated intensities,
1H-NMR (400 MHz, DMSO):
d
2.28 (3H, s), 7.25 (1H, s); 7.50 (1H,
measured using the u scan mode, were corrected for Lorentz and
dd, J ¼ 8.4, and 2.0 Hz); 7.72 (1H, d, J ¼ 8 Hz), 7.83 (1H, d, J ¼ 2.0 Hz),
polarization effects [53]. Direct methods of SIR2004 [54] were used
in solving the structures and they were refined using the full-
matrix least squares on F2 provided by SHELXL97 [55]. Multi-scan
symmetry-related measurement was used as experimental ab-
sorption correction type. The non-hydrogen atoms were refined
anisotropically whereas hydrogen atoms were refined as isotropic.
The X-ray CIF file for this structure has been deposited at the
Cambridge Crystallographic Data Centre and allocated with the
deposition number CCDC 1030508. Copies of the data can be
7.99 (4H, m, J ¼ 8.6 Hz).
13C-NMR (100 MHz, DMSO):
d 21.39, 91.34, 114.13,118.30, 127.22,
127.48 (2C), 128.23, 128.72, 129.25, 131.41, 131.73, 133.21 (2C),
137.22, 153.51, 167.54.
4.1.2.10. 4j:
2-(3,4-dichlorophenyl)-3-acetyl-5-(2-methylphenyl)-
2,3-dihydro-1,3,4-oxadiazole. C17H14Cl2N2O2. White solid, M.p.
147e150 ꢁC (hexane); MS (m/z) 348, yield 32%. CHN %: Calc. C,

550
S. Distinto et al. / European Journal of Medicinal Chemistry 108 (2016) 542e552
obtained, free of charge, from CCDC,12 Union Road, Cambridge, CB2
1EZ UK (e-mail: deposit@ccdc.cam.ac.uk; http://www.ccdc.cam.ac.
uk).
ligand reported into the PDB entry 2V5Z [7].
4.3.3. Molecular dynamics
The docking resulting complexes were solvated with a box of
TIP3P (Transferable Intermolecular Potential 3-Point) water [65]
and counter ions were added creating an overall neutral system
simulating approximately 0.15 M NaCl. The ions were equally
distributed in a water box. The final system was subjected to a MD
simulation up to 10 ns using Desmond [66]. Restraint force of
50 kcal/(mol$Å) was added to monomer and dimer transmembrane
portion whereas the dimer in membrane was left without any re-
straint. The solvated models were optimized, and subsequently the
MTK_NPT (Martyna-Tobias-Klein with constant Number of parti-
cles, Pressure and Temperature) ensemble was employed [67]. The
default stages in the relaxation process for the NPT ensemble
included two energy minimizations and four simulation steps.
During the energy minimizations, two runs of 2000 iteration were
processed using the steepest descent method: during the first run,
the protein structure was fixed by a force restraint constant of
50 kcal/(mol$Å) and in the second all restraints were removed.
With the first simulation, at NVT (constant Number of particles,
Volume, and Temperature) ensemble, the system reached a tem-
perature of 10 K. In the following three simulations in the NPT
ensemble, the system was heated up to 325 K and the pressure was
kept constant at 1 bar using the Berendsen thermostatebarostat.
During the production phase, temperature and pressure were kept
4.2. Enzymatic assay
MAO inhibition measurements were evaluated following the
general procedure previously described by us [42]. Briefly, test
drugs and adequate amounts of recombinant hMAO-A or hMAO-B
(SigmaeAldrich Quıímica S.A., Alcobendas, Spain) required and
adjusted to oxidize 165 pmol of p-tyramine/min in the control
group, were incubated for 15 min at 37 ꢁC in a flat-black-bottom 96-
well microtest plate (BD Biosciences, Franklin Lakes, NJ) placed in
the dark fluorimeter chamber. The reaction was started by adding
200 mM Amplex Red reagent (Molecular Probes, Inc., Eugene, OR),
1 U/mL horseradish peroxidase, and 1 mM p-tyramine and the
production of resorufin, was quantified at 37 ꢁC in a multidetection
microplate fluorescence reader (FLX800, Bio-Tek Instruments, Inc.,
Winooski, VT) based on the fluorescence generated (excitation,
545 nm; emission, 590 nm). The specific fluorescence emission was
calculated after subtraction of the background activity, which was
determined from vials containing all components except the hMAO
isoforms, which were replaced by a sodium phosphate buffer
solution.
4.3. Molecular modelling
ꢀ
constant using the Nose-Hoover thermostatebarostat. The energy
4.3.1. MAO-B active site characterization
and trajectory were recorded every 1.2 ps and 4.8 ps, respectively.
For multiple time step integration, RESPA (REversible reference
System Propagator Algorithm) [68] was applied to integrate the
equation of motion with Fourier-space electrostatics computed
every 6 fs, and all remaining interactions computed every 2 fs. All
chemical bond lengths involving hydrogen atoms were fixed with
SHAKE [69]. Short range cut-off was set to 9 Å and the smooth
particle mesh Ewald method (PME) [70] were used for long range
electrostatic interaction. The MD trajectories were analyzed in
terms of interaction energies and geometries.
The previous reported1 theoretical complex between the PDB
MAO-B and the most active compound 2-(4-chlorophenyl)-3-
acetyl-5-(4-chlorophenyl)-2,3-dihydro-1,3,4-oxadiazole
was
considered for the active site characterization. The binding cleft
was defined by means of a regular box of 1000 Å3 centred onto the
ligand position. After removing the inhibitor, GRID [41] software
was applied for mapping the target previous defined using CL, BR,
AR.COOꢂ, C3 and ON probes for mimicking organic chlorine and
bromine atoms, aromatic carboxylic moiety, methyl and nitro
group oxygen respectively. Default software directives were taken
into account except for the number of planes per Angstrom (NPLA)
that was increased till 3 corresponding to a grid spacing equal to
0.333 Å. Computed isocontour maps were displayed using VMD ver.
1.9.1 [56] and graphical inspected considering an energy threshold
equal to ꢂ5.5 kcal/mol for all probes except C3 whose maps were
analyzed at ꢂ3.5 kcal/mol.
4.3.4. ADME-tox prediction
The energy minimized ligands structures were evaluated for
their drug-like properties using Qikprop [52] which predicts
physically significant descriptors and pharmaceutically relevant
properties of organic molecules [71,72]. Results are reported in
Table S3.
4.3.2. Docking protocol
Acknowledgment
Three-dimensional coordinates of the receptor [7] were ob-
tained from the Protein Data Bank (PDB). The protein was processed
and the internal hydrogen bonding network of the receptor was
optimized using the algorithm implemented in Protein Preparation
wizard [57]. In order to compare energetic analysis it has been
necessary complete the transmembrane portion considering the
The authors wish to acknowledge Fondazione Banco di Sarde-
gna for financial support.
Appendix A. Supplementary data
aminoacid sequence and
a
ꢂhelix packing reported in literature
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.12.026.
[58,59]. The mitochondrial bilayer membrane was simulated with
an Dipalmitoylphosphatidylcholine (DPPC) bilayer [60]. Three-
dimensional ligand structures for all docking experiments were
built using Maestro GUI. Structures were optimized by means of
energy minimization carried out using the Merck Molecular Force
Field (MMFFs) [61], the Generalized Born/Surface Area (GB/SA) [62]
water implicit solvation model and the PolakeRibier Coniugate
Gradient (PRCG) method, converging on gradient with a threshold
of 0.05 kJ (molÅ)ꢂ1 as implemented in Macromodel. Molecular
docking studies were performed using Glide SP [46], Glide XP [63],
QMPL [47,48] and Induced Fit workflow protocol [64]. Grids were
defined around the refined structure by centring on co-crystallized
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