Benzo[b]tiophen-3-ol derivatives as effective inhibitors of human monoamine oxidase: design, synthesis, and biological activity

Article abstract of DOI:10.1080/14756366.2019.1653864

A series of benzo[b]thiophen-3-ols were synthesised and investigated as potential human monoamine oxidase (hMAO) inhibitors in vitro as well as ex vivo in rat cortex synaptosomes by means of evaluation of 3,4-dihydroxyphenylacetic acid/dopamine (DOPAC/DA)

Full text of DOI:10.1080/14756366.2019.1653864

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Benzo[b]tiophen-3-ol derivatives as effective
inhibitors of human monoamine oxidase: design,
synthesis, and biological activity
Paolo Guglielmi, Daniela Secci, Anél Petzer, Donatella Bagetta, Paola
Chimenti, Giulia Rotondi, Claudio Ferrante, Lucia Recinella, Sheila Leone,
Stefano Alcaro, Gokhan Zengin, Jacobus P. Petzer, Francesco Ortuso &
Simone Carradori
To cite this article: Paolo Guglielmi, Daniela Secci, Anél Petzer, Donatella Bagetta, Paola
Chimenti, Giulia Rotondi, Claudio Ferrante, Lucia Recinella, Sheila Leone, Stefano Alcaro, Gokhan
Zengin, Jacobus P. Petzer, Francesco Ortuso & Simone Carradori (2019) Benzo[b]tiophen-3-
ol derivatives as effective inhibitors of human monoamine oxidase: design, synthesis, and
biological activity, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 1511-1525, DOI:
10.1080/14756366.2019.1653864
To link to this article: https://doi.org/10.1080/14756366.2019.1653864
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 31, NO. 01, 1511–1525
https://doi.org/10.1080/14756366.2019.1653864
RESEARCH PAPER
Benzo[b]tiophen-3-ol derivatives as effective inhibitors of human monoamine
oxidase: design, synthesis, and biological activity
a
a
b
Paolo Guglielmi , Daniela Secci , Anel Petzer , Donatella Bagettac,d, Paola Chimentia, Giulia Rotondia,
ꢀ
Claudio Ferrantee, Lucia Recinellae, Sheila Leonee, Stefano Alcaroc,d , Gokhan Zenginf , Jacobus P. Petzerb,
Francesco Ortusoc,d
and Simone Carradorie
b
aDipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, Rome, Italy; Pharmaceutical Chemistry, School of Pharmacy,
c
Centre of Excellence for Pharmaceutical Sciences, North-West University, Potchefstroom, South Africa; Dipartimento di Scienze della Salute,
d
“Magna Graecia” University of Catanzaro, Campus Universitario “S. Venuta”, Viale Europa Loc. Germaneto, Catanzaro, Italy; Net4Science
Academic Spin-Off, Campus Universitario “S. Venuta”, Viale Europa Loc. Germaneto, “Magna Graecia” University of Catanzaro, Catanzaro, Italy;
f
eDepartment of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, Chieti, Italy; Department of Biology, Science Faculty, Selcuk University,
Konya, Turkey
ABSTRACT
ARTICLE HISTORY
Received 4 July 2019
Revised 2 August 2019
Accepted 2 August 2019
A series of benzo[b]thiophen-3-ols were synthesised and investigated as potential human monoamine oxi-
dase (hMAO) inhibitors in vitro as well as ex vivo in rat cortex synaptosomes by means of evaluation of
3,4-dihydroxyphenylacetic acid/dopamine (DOPAC/DA) ratio and lactate dehydrogenase (LDH) activity.
Most of these compounds possessed high selectivity for the MAO-B isoform and a discrete antioxidant
and chelating potential. Molecular docking studies of all the compounds underscored potential binding
site interactions suitable for MAO inhibition activity, and suggested structural requirements to further
improve the activity of this scaffold by chemical modification of the aryl substituents. Starting from this
heterocyclic nucleus, novel lead compounds for the treatment of neurodegenerative disease could
be developed.
KEYWORDS
MAO-B inhibitors;
benzothiophene; molecular
modelling; rat cortex
synaptosomes; antioxidant
activity; Parkinson’s disease
1. Introduction
participates in the Fenton reaction6 and reacts with certain cations
such as Cuþ and/or Fe2þ which leads to reactive the formation of
oxygen species (ROS). ROS are less stable than H2O2 and immedi-
ately react with the surrounding protein systems, which leads to
structural/functional damages and cells death7. MAO-mediated
oxidative stress has been associated with different neurodegenera-
tive pathologies as well as cardiomyopathies. This suggests that
reducing the activity of the hMAOs could be useful in order to
Monoamine oxidases (MAOs; EC 1.4.3.4) are mitochondrial bound
flavoenzymes, which catalyse the oxidative degradation of amines.
The two human isoforms (hMAO-A and B) are quite similar sharing
ꢀ70% sequence identity1,2 and are co-expressed, to almost the
same extent, in the majority of human tissues. However, some dif-
ferences in the distribution exist with hMAO-A being the isoform
predominantly expressed in placenta and intestinal tract, while
hMAO-B is more abundant in the brain and liver. In the central
nervous system (CNS), they modulate monoamine levels and thus
participate in the complex system that controls the physiological
and functional concentrations of these neurotransmitters. The two
MAO enzymes share similar affinity for dopamine, epinephrine,
norepinephrine, and tyramine; serotonin is the preferred substrate
for hMAO-A, while hMAO-B has high affinity for benzylamine3.
The products deriving from hMAO enzymatic activity are alde-
hydes and the ammonium ion, while the by-product hydrogen
peroxide (H2O2) is formed in order to regenerate the catalytically
active form of the FAD cofactor4. Although aldehydes do not
appear to accumulate in the healthy brain, some studies have
shown that elevated concentration of these products may exert
cytotoxic effects5. Moreover, the abnormal expression or increased
activity of hMAOs may lead to the excessive production of H2O2
protect cells8–10
.
An extensive number of natural and synthetic compounds
have shown effective inhibition of the hMAOs4,11,12. Keeping in
mind the structure and properties of endogenous substrates and
reversible inhibitors that bind to hMAOs, Wouters et al.13 pro-
posed that ideal MAO inhibitors should be flat molecules (Figure
1(a)) with specific dimensions depending on the isoform
(11.5 ꢁ 5.5 ꢁ 1.8 Å for hMAO-A and 8.5 ꢁ 5.1 ꢁ 1.8 Å for hMAO-B).
The pharmacophore reported in Figure 1(a) was inspired by
isatin and indole analogues which possess different enzyme spe-
cificities, inhibiting hMAO-B and hMAO-A, respectively14,15
.
Although the dimensions of compounds may explain the different
affinities for the MAO isoforms (hMAO-A can accommodate larger
molecules than hMAO-B), the differences of the electron density
in the molecules play an important role in enzyme selectivity. As a
matter of the fact, follow-up studies focussed on the isosteric sub-
that can expose cells to oxidative damage. In this respect, H2O2 stitution of the nitrogen of the indole system with an oxygen
CONTACT Simone Carradori
simone.carradori@unich.it
Department of Pharmacy, “G. D’Annunzio” University of Chieti-Pescara, Via dei Vestini 31, Chieti
66100, Italy
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1512
P. GUGLIELMI ET AL.
(a)
(b)
(c)
Figure 1. (a) Molecular properties of the pharmacophore for the inhibition of the hMAOs. The part of the structure shown in red corresponds to the chalcone moiety
(adapted from reference13); (b) Structure of aurone (I) and indanone (II) derivatives; (c) Benzo[b]thiophen-3-ol scaffold: similarities and differences with previously
reported compounds.
atom or methylene group, to obtain respectively aurone16 and hydroxyl radicals possess a very short half-life, estimated at 1 ns,
indanone derivatives17,18 (Figure 1(b)).
Aurone derivatives (Figure 1(b,I)) showed selective inhibition
towards rat MAO-B (rMAO-B) with IC50 values ranging from 11.6 to
26.3 mM, without activity against the A isoform. On the other
they can be highly toxic to biomolecules and the use of chelating
agents should repair this metal dyshomeostasis leading to
reduced damage derived from oxidative stress.
The structure of benzo[b]thiophen-3-ol has been previously
hand, indanone derivatives (Figure 1(b,II)) showed good inhibitory studied from a chemical point of view and several research
profile in the low micromolar range especially for hMAO-B groups have proposed synthetic strategies to obtain this class of
(0.0052 < IC50 hMAO-B (mM) <2.74). All these structures (indole compounds23–25. Here, we propose a new one-step, very simple
analogues, aurone, and indanone derivatives) share a common synthetic procedure which allowed us to obtain the desired com-
structural feature, and is thus similar to the chalcone moiety pounds PM1-PM20 (Scheme 1). Methyl 2-mercaptobenzoate and
(highlighted in red), whose ability to bind to hMAO enzymes have a-bromo acetophenone in equimolar amount, were reacted in
been reported in the past by our group19. With the aim to explore
methanol in the presence of potassium hydroxide. The reaction
new structures for the inhibition of hMAO, we proposed a new
scaffold based on benzo[b]thiophen-3-ol structure (Figure 1(c)).
This scaffold retained some similarity with the compounds dis-
cussed above, for example the presence of bicyclic system con-
nected with a “bridge” to an aromatic/heteroaromatic ring.
Other important differences among these scaffolds are the isos-
teric replacement of the oxygen atom of aurones with sulphur
and the presence of 1,3-diketonic system that, via keto-enol tauto-
merism, generates the corresponding chalcone while also possess-
ing the potential for metal chelation20. This is a very interesting
aspect and provides the possibility to obtain multi-target-directed
drugs in the light of the evidence that some cations may contrib-
ute to neurodegeneration in CNS tissues. As mentioned above,
these ions are implicated in the Fenton reaction6 which catalyses
was performed in a nitrogen atmosphere (to avoid sulphur oxida-
tion) and at room temperature for 1–2 h. After this time, an
excess of potassium hydroxide was added, and the temperature
was raised to 60 ꢂC. The completion of reactions was usually
reached in 4–5 h producing all the compounds PM1-PM20 in
high yields.
We proposed a reaction mechanism for the synthesis of the ben-
zo[b]thiophen-3-ol derivatives as reported in Scheme 2. The first
phase of the reaction is the nucleophilic attack of the deprotonated
thiol group on a-position of the ketone to obtain the intermediate
W. After an intramolecular crossed aldolic reaction between the
methylene y and methyl ester functional group with consequent
methanol elimination, the benzo[b]thiophen-3-ol is obtained26.
The synthesised compounds PM1-PM20 were then evaluated
the production of hydroxyl radicals from hydrogen peroxide, a as hMAO inhibitors using the recombinant hMAOs as enzyme
well-known by-product of MAO enzymatic activity21,22. Since sources. The inhibitory activities of compounds PM1-PM20 are

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1513
Scheme 1. Synthesis of compounds PM1-PM20.
Scheme 2. Proposed mechanism of reaction.
summarised in Table 1 along with selectivity index (SI) values oven-dried glassware. Solvents and reagents were used as sup-
given as the ratio (IC50 hMAO-A)/(IC50 hMAO-B).
Compounds that showed the best IC50 values towards hMAO-B
(PM4, PM5, PM6, PM9, PM10, PM12, and PM13) were also tested
plied without further purification. All melting points were meas-
R
ured on
a
StuartV melting point apparatus SMP1 and are
ꢂ
uncorrected (temperatures are reported in C). Fluorescence spec-
in cortex synaptosomes in both basal and LPS-induced inflamma- trophotometry was carried out with a Varian Cary Eclipse fluores-
1
tory conditions, to estimate the capability of reducing 3,4-dihy- cence spectrophotometer. H and 13C NMR spectra were recorded
droxyphenylacetic acid/ dopamine (DOPAC/DA) ratio and lactate at 400 and 101 MHz, respectively, on a Bruker spectrometer using
dehydrogenase (LDH) activity27–29. The most active compounds
CDCl3 and DMSO-d6 as the solvents at room temperature. The
samples were analysed with a final concentration of ꢀ30 mg/ml.
(PM4, PM5, PM6, PM9, PM10, PM12, and PM13) were further
Chemical shifts are expressed as d units (parts per million) relative
to the solvent signal. H spectra are reported as follows: dH (spec-
tested for their antioxidant and metal chelating activity with the
aim to demonstrate the possibility of ancillary effects in neurode-
generative disorders. Finally, all the compounds were analysed by
molecular modelling to better corroborate the biological data and
to further determine which tautomer was responsible for the
observed biological activity.
1
trometer frequency, solvent): chemical shift/ppm (multiplicity,
J-coupling constant(s), number of protons, assignment). 13C spec-
tra are reported as follows: dC (spectrometer frequency, solvent):
chemical shift/ppm (J-coupling constant C-F, assignment).
Multiplicity is abbreviated as follows: br – broad; s – singlet; d –
doublet; t – triplet; q – quartette; and m – multiplet. Coupling
constants J are given in Hertz (Hz). The processing and analyses of
the NMR data were carried out with MestreNova. Column chroma-
2. Experimental protocols
2.1. General
R
tography was carried out using Sigma-AldrichV (St. Louis, MO), sil-
Unless otherwise indicated, all reactions were carried out under ica gel (high purity grade, pore size 60 Å, 230–400 mesh particle
a positive nitrogen pressure (balloon pressure) in washed and size). All the purifications and reactions were monitored by TLC

1514
P. GUGLIELMI ET AL.
which was performed on 0.2 mm thick silica gel-aluminium backed benzothiophene), 7.87–7.89 (m, 2H, Ar), 8.09 (d, J¼ 8.0 Hz, 1H, ben-
zothiophene), 13.49 (brs, 1H, OH, D2O exch.). 13 C NMR (101 MHz,
plates (60 F254, Merck, Kenilworth, NJ)Visualisation was carried out
CDCl3): d 21.5 (CH3), 109.7 (benzothiophene), 123.0 (benzothio-
phene), 124.0 (benzothiophene), 124.7 (benzothiophene), 125.6
(Ar), 128.6 (Ar), 128.9 (Ar), 130.1 (benzothiophene), 130.3 (benzo-
thiophene), 133.4 (Ar), 138.3 (Ar), 138.7 (Ar), 140.8 (benzothio-
phene), 165.3 (Cbenzothiophene-OH), 192.1 (C═ O). Anal. Calcd for
C16H12O2S: C, 71.62; H, 4.51. Found: C, 71.64; H, 4.50.
under ultra-violet irradiation (254 nm). Elemental analyses for C, H,
and N were recorded on a Perkin-Elmer 240 B microanalyzer
obtaining analytical results within 0.4% of the theoretical values
for all compounds. Where given, systematic compound names are
those generated by ChemBioDraw Ultra 12.0 following IUPAC con-
ventions. Microsomes from insect cells containing recombinant
hMAO-A and hMAO-B (5 mg protein/ml) and kynuramine dihydro-
bromide were obtained from Sigma-Aldrich (St. Louis, MO).
2.2.4. (3-Hydroxybenzo[b]thiophen-2-yl)(p-tolyl)methanone (PM4)
Yellow powder, mp 99–101 ꢂC, 77% yield; 1H NMR (400 MHz,
CDCl3): d 2.49 (s, 3H, CH3), 7.35–7.37 (m, 2H, Ar), 7.43–7.47 (m, 1H,
benzothiophene), 7.55–7.59 (m, 1H, benzothiophene), 7.75 (d,
J¼ 8.2 Hz, 1H, benzothiophene), 7.99–8.01 (m, 2H, Ar), 8.09 (d,
J¼ 8.0 Hz, 1H, benzothiophene), 13.6 (brs, 1H, OH, D2O exch.). 13 C
NMR (101 MHz, CDCl3): d 21.7 (CH3), 109.5 (benzothiophene), 122.9
(benzothiophene), 123.9 (benzothiophene), 124.7 (benzothio-
phene), 128.6 (Ar), 129.4 (Ar), 130.0 (benzothiophene), 130.3 (ben-
zothiophene), 135.5 (Ar), 140.7 (benzothiophene), 143.5 (Ar), 165.3
(Cbenzothiophene-OH), 191.4 (C═ O). Anal. Calcd for C16H12O2S: C,
71.62; H, 4.51. Found: C, 71.59; H, 4.47.
2.2. Chemistry
In an oven dried flask containing a stirring solution of methyl 2-
mercaptobenzoate (1.0 equiv.) in methanol (10 ml), freshly ground
potassium hydroxide (1.5 equiv.) and the appropriate a-bromoace-
tophenone (1.0 equiv.) were added. The reaction was performed
at room temperature for 1–2 h. After this time, an excess of potas-
sium hydroxide was added (1.5 equiv.) and the temperature was
raised to 60 ꢂC. The progression of reaction was monitored by TLC
and completion was usually reached in 4–5 h. The reaction was
poured into ice-cold water (30 ml) and the pH was adjusted to the
value of ꢀ7 with a 2 N HCl solution to induce the complete pre-
cipitation of the desired compound. The resulting benzo[b]thio-
phen-3-ol was collected by vacuum filtration and washed with hot
methanol (20 ml). This procedure was used in order to obtain all
the compounds PM1-PM20 in high yields and a good level
of purity.
2.2.5. (3-Hydroxybenzo[b]thiophen-2-yl)(3-methoxyphenyl)me-
thanone (PM5)
1
Yellow powder, mp 77–79 ꢂC, 83% yield; H NMR (400 MHz, CDCl3):
d 3.93 (s, 3H, OCH3), 7.16–7.19 (m, 1H, Ar), 7.44–7.49 (m, 1H Ar þ
1H benzothiophene), 7.56–7.60 (m, 1H Ar þ 1H benzothiophene),
7.66–7.69 (m, 1H, Ar), 7.76 (d, J¼ 8.2 Hz, 1H, benzothiophene),
7.87–7.89 (m, 2H, Ar), 8.09 (d, J¼ 8.1 Hz, 1H, benzothiophene),
13.45 (brs, 1H, OH, D2O exch.). 13C NMR (101 MHz, CDCl3): d 55.5
(OCH3), 109.7 (benzothiophene), 119.2 (Ar), 120.8 (Ar), 123.0 (ben-
zothiophene), 124.0 (benzothiophene), 124.8 (benzothiophene),
129.8 (Ar), 130.2 (benzothiophene), 130.3 (benzothiophene), 139.5
(Ar), 140.8 (benzothiophene), 159.8 (Ar), 165.4 (Cbenzothiophene-OH),
191.6 (C═O). Anal. Calcd for C16H12O3S: C, 67.59; H, 4.25. Found: C,
67.61; H, 4.29.
2.2.1. (3-Hydroxybenzo[b]thiophen-2-yl)(phenyl)methanone (PM1)
Yellow powder, mp 118–120 ꢂC, 80% yield; 1H NMR (400 MHz,
DMSO-d6): d 7.48–7.52 (m, 1H, benzothiophene), 7.58–7.72 (m, 3H
Ar þ 1H benzothiophene), 7.92–7.95 (m, 2H, Ar), 7.98 (d, J¼ 8.2 Hz,
1H, benzothiophene), 8.05 (d, J¼ 8.0 Hz, 1H, benzothiophene),
12.02 (brs, 1H, OH, D2O exch.). 13C NMR (101 MHz, DMSO-d6): d
112.2 (benzothiophene), 123.9 (benzothiophene), 124.0 (benzo-
thiophene), 125.5 (benzothiophene), 128.8 (Ar), 129.1 (Ar), 130.4
(benzothiophene), 131.1 (Ar), 133.1 (Ar), 138.6 (benzothiophene),
140.0 (benzothiophene), 160.3 (Cbenzothiophene-OH), 190.9 (C═ O).
2.2.6. (3-Hydroxybenzo[b]thiophen-2-yl)(4-methoxyphenyl)metha-
Anal. Calcd for C15H10O2S: C, 70.80; H, 3.96. Found: C, 70.85; none (PM6)
Yellow powder, mp 181–183 ꢂC, 73% yield; 1H NMR (400 MHz,
H, 3.99.
DMSO-d6): d 3.88 (s, 3H, OCH3), 7.13–7.15 (m, 2H, Ar), 7.49–7.53
(m, 1H, benzothiophene), 7.62–7.66 (m, 1H, benzothiophene),
7.98–8.01 (m, 2H Ar þ 1H benzothiophene), 8.03 (d, J¼ 8.0 Hz, 1H,
benzothiophene), 12.76 (brs, 1H, OH, D2O exch.). 13C NMR
(101 MHz, DMSO-d6): d 56.1 (OCH3), 111.4 (benzothiophene), 114.6
(Ar), 123.7 (benzothiophene), 123.9 (benzothiophene), 125.5 (ben-
zothiophene), 130.3 (benzothiophene), 130.7 (benzothiophene),
130.9 (Ar), 131.3 (Ar), 139.7 (benzothiophene), 160.9 (Ar), 163.5
(Cbenzothiophene-OH), 189.5 (C═ O). Anal. Calcd for C16H12O3S: C,
67.59; H, 4.25. Found: C, 67.63; H, 4.24.
2.2.2. (3-Hydroxybenzo[b]thiophen-2-yl)(o-tolyl)methanone (PM2)
Yellow powder, mp 131–133 ꢂC, 86% yield; 1H NMR (400 MHz,
CDCl3): d 2.50 (s, 3H, CH3), 7.32–7.36 (m, 2H, Ar), 7.43–7.48 (m, 1H
Ar þ 1H benzothiophene), 7.54–7.58 (m, 1H, benzothiophene),
7.63–7.65 (m, 1H, Ar), 7.71 (d, J ¼ 8.2 Hz, 1H, benzothiophene), 8.09
(d, J¼ 8.0 Hz, 1H, benzothiophene), 12.84 (brs, 1H, OH, D2O exch.).
13 C NMR (101 MHz, CDCl3): d 19.7 (CH3), 112.0 (benzothiophene),
123.1 (benzothiophene), 124.0 (benzothiophene), 124.7 (benzo-
thiophene), 125.5 (Ar), 127.4 (Ar), 130.0 (benzothiophene), 130.5
(benzothiophene), 130.9 (Ar), 131.3 (Ar), 136.3 (Ar), 138.3 (Ar),
141.0 (benzothiophene), 160.3 (Cbenzothiophene -OH), 190.9 (C═ O).
Anal. Calcd for C16H12O2S: C, 71.62; H, 4.51. Found: C, 71.68;
H, 4.52.
2.2.7. (3-Fluorophenyl)(3-hydroxybenzo[b]thiophen-2-yl)methanone
(PM7)
Yellow powder, mp 133–135 ꢂC, 82% yield; 1H NMR (400 MHz,
CDCl3): d 7.31–7.36 (m, 1H, Ar), 7.45–7.49 (m, 1H, benzothiophene),
7.52–7.62 (m, 1H Ar þ 1H benzothiophene), 7.74–7.77 (m, 1H Ar þ
1H benzothiophene), 7.88 (d, J¼ 7.7 Hz, 1H, Ar), 8.09 (d, J¼ 8.1 Hz,
1H, benzothiophene), 13.49 (brs, 1H, OH, D2O exch.). 13 C NMR
2.2.3. (3-Hydroxybenzo[b]thiophen-2-yl)(m-tolyl)methanone (PM3)
Yellow powder, mp 122–124 ꢂC, 76% yield; 1H NMR (400 MHz,
CDCl3): d 2.49 (s, 3H, CH3), 7.44–7.48 (m, 2H Ar þ 1H benzothio-
(101 MHz, CDCl3):
d
109.4 (benzothiophene), 115.4 (d, JC-F
¼
phene), 7.56–7.60 (m, 1H, benzothiophene), 7.76 (d, J¼ 8.2 Hz, 1H, 23.1 Hz, Ar), 119.5 (d, JC-F ¼ 21.3 Hz, Ar), 123.0 (benzothiophene),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1515
124.1 (benzothiophene), 124.2 (d, JC-F ¼ 3.2 Hz, Ar), 124.9 (benzo- (benzothiophene), 131.2 (Ar), 132.2 (benzothiophene), 135.1 (Ar),
thiophene), 130.2 (benzothiophene), 130.4 (benzothiophene),
130.4 (d, JC-F ¼7.8 Hz, Ar), 140.2 (d, JC-F ¼6.8 Hz, Ar), 140.8 (benzo-
thiophene), 162.7 (d, JC-F ¼248.5 Hz, Ar), 165.8 (Cbenzothiophene-OH),
190.2 (C═O). Anal. Calcd for C15H9FO2S: C, 66.17; H, 3.33. Found: C,
66.18; H, 3.37.
139.5
(benzothiophene),
140.0
(benzothiophene),
157.3
(Cbenzothiophene-OH), 186.7 (C═ O). Anal. Calcd for C15H8Cl2O2S: C,
55.75; H, 2.50. Found: C, 55.80; H, 2.53.
2.2.12.
(3-Bromophenyl)(3-hydroxybenzo[b]thiophen-2-yl)metha-
none (PM12)
Yellow powder, mp 169–171 ꢂC, 90% yield; 1H NMR (400 MHz,
CDCl3): d 7.42–7.49 (m, 1H Ar þ 1H benzothiophene), 7.58–7.62
(m, 1H, benzothiophene), 7.74–7.77 (m, 1H Ar þ 1H benzothio-
phene), 7.99 (d, J¼ 7.8 Hz, 1H, Ar), 8.09 (d, J¼ 8.0 Hz, 1H, benzo-
thiophene), 8.18–8.19 (m, 1H, Ar), 13.27 (brs, 1H, OH, D2O exch.).
13 C NMR (101 MHz, CDCl3): d 109.4 (benzothiophene), 123.0 (Ar),
123.1 (benzothiophene), 124.1 (benzothiophene), 124.9 (benzo-
thiophene), 126.9 (Ar), 130.1 (benzothiophene), 130.3 (Ar), 130.5
(benzothiophene), 131.4 (Ar), 135.5 (Ar), 140.0 (Ar), 140.8 (benzo-
thiophene), 165.7 (Cbenzothiophene-OH), 190.1 (C═O). Anal. Calcd for
C15H9BrO2S: C, 54.07; H, 2.72. Found: C, 54.10; H, 2.76.
2.2.8. (4-Fluorophenyl)(3-hydroxybenzo[b]thiophen-2-yl)methanone
(PM8)
Yellow powder, mp 127–129 ꢂC, 87% yield; 1H NMR (400 MHz,
CDCl3): d 7.12 (t, J¼ 8.6 Hz, 2H, Ar), 7.34 (t, J¼ 7.6 Hz, 1H, benzo-
thiophene), 7.46 (t, J¼ 7.6 Hz, 1H, benzothiophene), 7.63 (d,
J ¼ 8.2 Hz, 1H, benzothiophene), 7.95–8.01 (m, 2H Ar þ 1H benzo-
thiophene), 13.32 (brs, 1H, OH, D2O exch.). 13C NMR (101 MHz,
CDCl3): d 109.2 (benzothiophene), 115.9 (d, JC-F ¼21.9 Hz, Ar), 123.0
(benzothiophene), 124.0 (benzothiophene), 124.8 (benzothio-
phene), 130.2 (benzothiophene), 130.3 (benzothiophene), 131.0 (d,
JC-F ¼ 9.2 Hz, Ar), 134.4 (d, JC-F ¼3.0 Hz, Ar), 140.6 (benzothio-
phene), 165.4 (d, JC-F ¼254.7 Hz, Ar), 165.6 (Cbenzothiophene-OH),
190.2 (C═O). Anal. Calcd for C15H9FO2S: C, 66.17; H, 3.33. Found: C,
66.20; H, 3.39.
2.2.13. (4-Bromophenyl)(3-hydroxybenzo[b]thiophen-2-yl)methanone
(PM13)
Yellow powder, mp 168–170 ꢂC, 84% yield; 1H NMR (400 MHz,
CDCl3): d 7.46 (t, J¼ 7.6 Hz, 1H, benzothiophene), 7.59 (t, J¼ 7.6 Hz,
1H, benzothiophene), 7.70 (d, J¼ 8.5 Hz, 2H, Ar), 7.75 (d, J¼ 8.2 Hz,
1H, benzothiophene), 7.94 (d, J¼ 8.5 Hz, 2H, Ar), 8.08 (d, J¼ 8.0 Hz,
1H, benzothiophene), 13.39 (brs, 1H, OH, D2O exch.). 13 C NMR
(101 MHz, CDCl3): d 109.3 (benzothiophene), 123.0 (benzothio-
phene), 124.1 (benzothiophene), 124.9 (benzothiophene), 127.7
(Ar), 130.0 Ar), 130.2 (benzothiophene), 130.4 (benzothiophene),
132.0 (Ar), 136.9 (Ar), 140.7 (benzothiophene), 165.7
(Cbenzothiophene-OH), 190.4 (C═ O). Anal. Calcd for C15H9BrO2S: C,
54.07; H, 2.72. Found: C, 54.08; H, 2.72.
2.2.9. (3-Chlorophenyl)(3-hydroxybenzo[b]thiophen-2-yl)methanone
(PM9)
Yellow powder, mp 166–168 ꢂC, 89% yield; 1H NMR (400 MHz,
CDCl3): d 7.45–7.52 (m, 1H Ar þ 1H benzothiophene), 7.58–7.62
(m, 1H Ar þ 1H benzothiophene), 7.76 (d, J¼ 8.2 Hz, 1H, benzo-
thiophene), 7.94–7.96 (m, 1H, Ar), 8.03–8.04 (m, 1H, Ar), 8.09 (d,
J ¼ 8.0 Hz, 1H, benzothiophene), 13.29 (brs, 1H, OH, D2O exch.).
13C NMR (101 MHz, CDCl3): d 109.4 (benzothiophene), 123.0 (ben-
zothiophene), 124.1 (benzothiophene), 124.9 (benzothiophene),
126.5 (Ar), 128.5 (Ar), 130.0 (Ar), 130.2 (benzothiophene), 130.5
(benzothiophene), 132.6 (Ar), 135.0 (Ar), 139.8 (Ar), 140.8 (benzo-
thiophene), 165.7 (Cbenzothiophene-OH), 190.2 (C═O). Anal. Calcd for
C15H9ClO2S: C, 62.40; H, 3.14. Found: C, 62.42; H, 3.11.
2.2.14. 4–(3-Hydroxybenzo[b]thiophene-2-carbonyl)benzonitrile
(PM14)
Orange powder, mp 176–178 ꢂC, 81% yield; 1H NMR (400 MHz,
DMSO-d6): d 7.48 (t, J¼ 7.6 Hz, 1H, benzothiophene), 7.62 (t,
J¼ 7.6 Hz, 1H, benzothiophene), 7.96–8.04 (m, 4H Ar þ 1H benzo-
thiophene), 8.08 (d, J¼ 8.1 Hz, 1H, benzothiophene), 11.95 (brs, 1H,
OH, D2O exch.). 13C NMR (101 MHz, DMSO-d6): d 114.2 (Ar), 114.5
(benzothiophene), 118.8 (CN), 124.1 (benzothiophene), 124.2 (ben-
zothiophene), 125.4 (benzothiophene), 129.4 (Ar), 130.2 (benzo-
2.2.10. (4-Chlorophenyl)(3-hydroxybenzo[b]thiophen-2-yl)metha-
none (PM10)
Yellow powder, mp 164–166 ꢂC, 88% yield; 1H NMR (400 MHz,
CDCl3): d 7.44–7.48 (m, 1H, benzothiophene), 7.52–7.55 (m, 2H,
Ar), 7.57–7.61 (m, 1H, benzothiophene), 7.75 (d, J¼ 8.2 Hz, 1H, ben-
zothiophene), 8.00–8.04 (m, 2H, Ar), 8.08 (d, J¼ 8.1 Hz, 1H, benzo-
thiophene), 13.40 (brs, 1H, OH, D2O exch.). 13 C NMR (101 MHz,
CDCl3): d 109.3 (benzothiophene), 123.0 (benzothiophene), 124.1
(benzothiophene), 124.9 (benzothiophene), 129.1 (Ar), 129.9 (Ar),
130.2 (benzothiophene), 130.4 (benzothiophene), 136.5 (Ar), 139.1
(Ar), 140.7 (benzothiophene), 165.7 (Cbenzothiophene-OH), 190.3 (C═
O). Anal. Calcd for C15H9ClO2S: C, 62.40; H, 3.14. Found: C, 62.35;
H, 3.17.
thiophene),
131.8
(benzothiophene),
132.8
(Ar),
140.0
(benzothiophene), 143.1 (Ar), 158.3 (Cbenzothiophene-OH), 188.9 (C═
O). Anal. Calcd for C16H9NO2S: C, 68.80; H, 3.25; N, 5.01. Found: C,
68.83; H, 3.29; N, 4.97.
2.2.15. (3-Hydroxybenzo[b]thiophen-2-yl)(4-nitrophenyl)methanone
(PM15)
Orange powder, mp 205–207 ꢂC, 79% yield; 1H NMR (400 MHz,
DMSO-d6): d 7.49 (t, J¼ 7.6 Hz, 1H, benzothiophene), 7.58–7.71 (m,
1H, benzothiophene), 7.99 (d, J¼ 8.1 Hz, 1H, benzothiophene),
8.06–8.11 (m, 2H Ar þ 1H benzothiophene), 8.38 (d, J¼ 8.1 Hz, 2H,
Ar), 11.94 (brs, 1H, OH, D2O exch.). 13C NMR (101 MHz, DMSO-d6):
2.2.11. (2,4-Dichlorophenyl)(3-hydroxybenzo[b]thiophen-2-yl)me-
thanone (PM11)
White powder, mp 149–151 ꢂC, 88% yield; 1H NMR (400 MHz,
DMSO-d6): d 7.44 (t, J¼ 7.6 Hz, 1H, benzothiophene), 7.58–7.60 (m,
2H Ar þ 1H benzothiophene), 7.78 (s, 1H, Ar), 7.94 (d, J¼ 8.1 Hz,
1H, benzothiophene), 8.06 (d, J¼ 8.1 Hz, 1H, benzothiophene),
11.74 (brs, 1H, OH, D2O exch.). 13 C NMR (101 MHz, DMSO-d6): d
116.5 (benzothiophene), 124.2 (benzothiophene), 125.2 (benzo-
d
114.5 (benzothiophene), 123.9 (Ar), 124.1(benzothiophene),
124.2 (benzothiophene), 125.4 (benzothiophene), 130.1 (Ar), 130.2
(benzothiophene), 131.9 (benzothiophene), 140.0 (benzothio-
thiophene), 128.0 (Ar), 129.4 (Ar), 130.0 (Ar), 130.1 phene), 144.8 (Ar), 149.6 (Ar-NO2), 158.1 (Cbenzothiophene-OH), 188.5

1516
P. GUGLIELMI ET AL.
Table 1. Inhibitory activity (IC50) and selectivity index (SI) of compounds PM1-PM20 towards hMAO-A and
hMAO-B.
IC50 SD (µM)a
SIb
Compound
Substituent
hMAO-A
hMAO-B
13.3 0.29
18.7 2.99
12.6 1.58
2.71 0.14
33.0 0.88
4.18 0.77
25.1 2.17
13.4 0.92
51.0 1.72
13.8 0.51
7.39 0.15
23.4 3.03
1.81 0.13
0.47 0.02
0.78 0.07
0.28 0.03
1.44 0.40
2.28 0.18
0.55 0.09
0.89 0.07
1.8
0.8
PM1
PM2
PM3
PM4
PM5
PM6
PM7
PM8
PM9
PM10
6.9
5.8
42.3
14.9
17.4
5.8
92.7
15.5
43.4 2.81
37.3 1.01
1.2
PM11
63.2 3.48
21.1 1.98
21.2 3.27
0.35 0.08
0.88 0.08
4.51 0.13
180
23.9
4.7
PM12
PM13
PM14
18.8 1.53
50.1 2.56
53.8 2.90
6.44 0.68
2.75 0.02
49.7 1.90
56.9 5.62
7.56 0.70
6.8
1.0
0.9
0.8
PM15
PM16
PM17
PM18
36.5 1.07
42.5 1.88
5.59 0.74
6.5
PM19
PM20
1.08 0.097
39.4
aValues are the mean SD of triplicate determinations. bSelectivity index for the MAO-B isoform, given as the
ratio: (IC50 hMAO-A)/(IC50 hMAO-B).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1517
(C═O). Anal. Calcd for C15H9NO4S: C, 60.20; H, 3.03; N, 4.68. Found: (C═ O) Anal. Calcd for C21H14O2S: C, 76.34; H, 4.27. Found: C,
C, 60.21; H, 3.05; N, 4.69.
76.38; H, 4.31.
2.2.16. (3-Hydroxybenzo[b]thiophen-2-yl)(pyridin-3-yl)methanone
(PM16)
2.2.20. (3-Hydroxybenzo[b]thiophen-2-yl)(naphthalen-2-yl)metha-
none (PM20)
Yellow powder, mp 126–128 ꢂC, 90% yield; 1H NMR (400 MHz,
CDCl3): d 7.44–7.49 (m, 1H, benzothiophene), 7.57–7.68 (m, 2H Ar
þ 1H benzothiophene), 7.78 (d, 2H, J¼ 8.2 Hz, 1H, benzothio-
phene), 7.93–7.95 (m, 1H, Ar), 7.99–8.05 (m, 2H, Ar), 8.09–8.12 (m,
1H Ar þ 1H benzothiophene), 8.65 (s, 1H, Ar), 13.58 (brs, 1H, OH,
D2O exch.). 13C NMR (101 MHz, CDCl3): d 109.8 (benzothiophene),
123.0 (benzothiophene), 124.0 (benzothiophene), 124.4 (Ar), 124.8
(benzothiophene), 127.0 (Ar), 127.9 (Ar), 128.5 (Ar), 128.7 (Ar),
129.5 (Ar), 129.9 (Ar), 130.2 (benzothiophene), 130.4 (benzothio-
phene), 132.4 (Ar), 135.3 (Ar), 135.3 (Ar), 139.8 (Ar), 140.8 (benzo-
thiophene), 145.5 (Ar), 165.6 (Cbenzothiophene-OH), 191.14 (C═ O).
Anal. Calcd for C19H12O2S: C, 74.98; H, 3.97. Found: C, 74.99;
H, 4.02.
Orange powder, mp 120–122 ꢂC, 79% yield; 1H NMR (400 MHz,
DMSO-d6): d 7.47–7.51 (m, 1H, benzothiophene), 7.58–7.64 (m, 1H
Pyr þ 1H benzothiophene), 7.99 (d, J¼ 8.1 Hz, 1H, benzothio-
phene), 8.10 (d, J¼ 8.1 Hz, 1H, benzothiophene), 8.21–8.24 (m, 1H,
Pyr), 8.79–8.80 (m, 1H, Pyr), 9.00–9.01 (m, 1H, Pyr), 11.92 (brs, 1H,
OH, D2O exch.). 13C NMR (101 MHz, DMSO-d6): d 109.5 (benzothio-
phene), 123.1 (benzothiophene), 123.8 (Pyr), 124.2 (benzothio-
phene), 125.1 (benzothiophene), 130.0 (benzothiophene), 130.7
(benzothiophene), 136.3 (Pyr), 136.4 (Pyr), 140.8 (benzothiophene),
148.8 (Pyr), 152.4 (Pyr), 165.9 (Cbenzothiophene-OH), 189.3 (C═ O).
Anal. Calcd for C14H9NO2S: C, 65.87; H, 3.55; N, 5.49. Found: C,
65.89; H, 3.57; N, 5.53.
2.2.17. (3-Hydroxybenzo[b]thiophen-2-yl)(pyridin-4-yl)methanone
(PM17)
2.3. Biological assays
Orange powder, mp 203–205 ꢂC, 85% yield; 1H NMR (400 MHz,
CDCl3): d 7.46–7.50 (m, 1H, benzothiophene), 7.60–7.64 (m, 1H,
benzothiophene), 7.76 (d, J¼ 8.2 Hz, 1H, benzothiophene),
7.85–7.86 (m, 2H, Pyr), 8.10 (d, J¼ 8.1 Hz, 1H, benzothiophene),
8.87–8.89 (m, 2H, Pyr), 13.12 (brs, 1H, OH, D2O exch.). 13C NMR
(101 MHz, CDCl3): d 109.4 (benzothiophene), 121.6 (Pyr), 123.1
(benzothiophene), 124.3 (benzothiophene), 125.1 (benzothio-
phene), 130.0 (benzothiophene), 130.9 (benzothiophene), 141.0
2.3.1. hMAO-a and hMAO-B inhibition studies
IC50 values for the inhibition of hMAO-A and hMAO-B were meas-
ured according to the literature protocol30,31 with the commer-
cially available recombinant enzymes (Sigma-Aldrich, St. Louis,
MO) serving as enzyme sources. Kynuramine was used as sub-
strate for both hMAO isoforms. The oxidation of kynuramine by
the hMAOs yields 4-hydroxyquinoline which was measured by
fluorescence spectrophotometry. By assessing the hMAO activities
in the presence of a range of different inhibitor concentrations
(0.003–100 mM), the IC50 values were measured. The enzyme reac-
tions (200 ml) were carried out in white 96-well microtiter plates
(Eppendorf, Hamburg, Germany) in potassium phosphate buffer
(pH 7.4, 100 mM) and contained kynuramine (50 mM), the test
inhibitors spanning at least three order of magnitude
(0.003–100 mM) and hMAO-A (0.0075 mg protein/ml) or hMAO-B
(0.015 mg protein/ml). The reactions were initiated with the add-
ition of enzyme, incubated for 20 min at 37 ꢂC, and at endpoint
were treated with 2 N NaOH (80 ml) to terminate the enzyme reac-
tions. The fluorescence intensity of 4-hydroxyquinoline, the prod-
uct formed by the MAO-catalysed oxidation of kynuramine, was
measured (kex¼310 nm; kem¼400 nm). Sigmoidal plots of 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 measurements.
(benzothiophene), 144.7 (Pyr), 150.6 (Pyr), 165.4 (Cbenzothiophene
-
OH), 189.7 (C═ O). Anal. Calcd for C14H9NO2S: C, 65.87; H, 3.55; N,
5.49. Found: C, 65.85; H, 3.50; N, 5.55.
2.2.18. (3-Hydroxybenzo[b]thiophen-2-yl)(thiophen-3-yl)methanone
(PM18)
Yellow powder, mp 182–184 ꢂC, 73% yield; 1H NMR (400 MHz,
CDCl3): d 7.44–7.48 (m, 1H thiophene þ 1H benzothiophene),
7.57–7.61 (m, 1H, benzothiophene), 7.78 (d, J¼ 8.2 Hz, 1H, benzo-
thiophene), 7.80–7.81 (m, 1H, thiophene), 8.07 (d, J¼ 8.0 Hz, 1H,
benzothiophene), 8.39–8.40 (m, 1H, thiophene), 13.55 (brs, 1H, OH,
D2O exch.). 13C NMR (101 MHz, CDCl3): d 109.4 (benzothiophene),
123.0 (benzothiophene), 123.9 (benzothiophene), 124.8 (benzo-
thiophene), 126.6 (thiophene), 127.6 (thiophene), 130.2 (benzo-
thiophene), 130.4 (benzothiophene), 132.2 (thiophene), 140.2
(thiophene), 141.0 (benzothiophene), 165.7 (Cbenzothiophene-OH),
184.6 (C═O). Anal. Calcd for C13H8O2S: C, 59.98; H, 3.10. Found: C,
60.02; H, 3.12.
2.3.2. Rat cortex synaptosomes
Male adult Sprague-Dawley rats (200–250 g) were housed in
Plexiglass cages (40 cm ꢁ 25 cm ꢁ 15 cm), two rats per cage, in
climatised colony rooms (22 1 ꢂC; 60% humidity), on a 12 h/12 h
light/dark cycle (light phase: 07:00–19:00 h), with free access to
tap water and food, 24 h/d throughout the study, with no fasting
periods. Rats were fed a standard laboratory diet (3.5% fat, 63%
carbohydrate, 14% protein, 19.5% other components without cal-
oric value; 3.20 kcal/g). Housing conditions and experimentation
procedures were strictly in accordance with the European Union
ethical regulations on the care of animals for scientific research.
According to the recognised ethical principles of “Replacement,
Refinement and Reduction of Animals in Research”, specimens
2.2.19. [1,1’-biphenyl]-4-yl(3-hydroxybenzo[b]thiophen-2-yl)metha-
none (PM19)
Yellow powder, mp 162–164 ꢂC, 89% yield; 1H NMR (400 MHz,
CDCl3): d 7.44–7.54 (m, 3H Ar þ 1H benzothiophene), 7.57–7.61
(m, 1H, benzothiophene), 7.69–7.71 (m, 2H, Ar), 7.77–7.80 (m, 2H
Ar þ 1H benzothiophene), 8.11 (d, J¼ 8.1 Hz, 1H, benzothiophene),
8.17–8.19 (m, 2H, Ar), 13.61 (brs, 1H, OH, D2O exch.). 13C NMR
(101 MHz, CDCl3): d 109.6 (benzothiophene), 123.0 (benzothio-
phene), 124.0 (benzothiophene), 124.8 (benzothiophene), 127.3
(Ar), 127.4 (Ar), 128.3 (Ar), 129.0 (Ar), 129.1 (Ar), 130.2 (benzothio-
phene), 130.3 (benzothiophene), 136.9 (Ar), 139.8 (benzothio- were obtained as residual material from vehicle-treated rats rando-
phene), 140.8 (Ar), 145.5 (Ar), 165.6 (Cbenzothiophene-OH), 191.14 mised in our previous experiments approved by Local Ethical

1518
P. GUGLIELMI ET AL.
Committee (University “G. d’Annunzio” of Chieti-Pescara) and (1.25–20.00 ng/ml) were obtained daily progressively diluting stock
Italian Health Ministry (Project N. 880 definitely approved by
Italian Health Ministry on 24th August 2015).
solutions in mobile phase.
Synaptosomes were prepared from a pool of frontal and par-
ietal cortex, which are more sensitive to oxidative stress compared
to other areas such as occipital and dorsal cortex32. Briefly, the
frontal and parietal cortex was quickly dissected, homogenised in
0.32 M saccharose solution and centrifuged, first at 4000ꢁg for
10 min, and then at 12000ꢁg for 20 min, to isolate neuronal end-
ings from cell nuclei and glia. The purified synaptosomes were
suspended at 37 ꢂC, under O2/CO2 95%/5%, pH 7.35–7.45, in
Krebs-Ringer buffer (mM: NaCl 125, KCl 3, MgSO4 1.2, CaCl2 1.2,
Tris–HCl 10, glucose 10). Then, the synaptosome suspension was
divided into fractions (each containing 100 mg of tissue in 3 ml
medium) that were incubated at 37 ꢂC, under agitation for 30 min
(incubation period), and treated with a pharmacological stimulus
as follows: i) Krebs-Ringer buffer (vehicle); ii) vehicle plus oxidant
stimulus [LPS 10 lg/ml]; iii) vehicle plus oxidant stimulus and MAO
inhibitors (20 nM–1 lM). After the incubation period, synaptosome
suspension was centrifuged (12,000ꢁg for 20 min) and the super-
natant assayed for LDH, DA, and DOPAC determination.
2.3.5. Statistical analysis
GraphPad Prism version 5.01 for Windows (GraphPad Software,
San Diego, CA) was used as statistical analysis software.
Experiments were performed at least in triplicate and results are
presented as mean standard deviation (SD). One-way analysis of
variance (ANOVA) followed by Newman–Keuls post-hoc test was
employed to assess significant differences (p<.05). As regards the
animals randomised for each experimental group, the number
was calculated on the basis of the “Resource Equation”
N¼(E þ T)/T35.
2.4. Molecular modelling
All molecular modelling simulations were carried out by means of
36
€
€
the Schrodinger Suite version 2018–1 (Schrodinger LLC, NY) .
Maestro GUI37 was employed to build the 3D theoretical struc-
tures of our derivatives. Both tautomers/conformers were taken
into account. Quantum mechanics optimisation was performed by
means of Jaguar38 software using the DFT B3LYP method and
2.3.3. LDH activity determination
ꢄꢄ
6–311 G
as basis set. The resulting structure population was
LDH activity was measured by evaluating the consumption of
NADH in 20 mM HEPES-Kþ (pH 7.2), 0.05% bovine serum albumin,
20 mM NADH and 2 mM pyruvate using a microplate reader (exci-
tation 340 nm, emission 460 nm) according to manufacturer’s
protocol (Sigma-Aldrich, St. Louis, MO). Extracts were tested at
25 mg/ml. Data were obtained from triplicate test and expressed
as relative variations compared to vehicle-treated cells33.
estimated according to Boltzmann analysis at 300 K. Protein Data
Bank (PDB)39 crystallographic structures, deposited with the corre-
sponding codes 2Z5X40 and 6FW041, were used as receptor mod-
els of hMAO-A and hMAO-B, respectively. Both PDB structures
were prepared as follows before the docking simulation: missing
atoms and FAD bond order were fixed, hydrogen atoms were
added and co-crystallised water molecules and ligands, harmine
and chlorophenyl-chromone-carboxamide for 2Z5X and 6FW0,
respectively, were removed. After preparation, the Glide42 software
was used to generate ligand configurations in the enzymatic
clefts, which were defined by a regular box of about 64,000 Å3
centred on the FAD N5 atom. Ligand structural flexibility was
taken into account by the software by a maximum of 10 docking
configurations for ligand were generated. The binding affinity was
estimated by means of the standard precision (SP) scoring func-
tion and the top-ranked complexes, according to Glide Score,
were considered for the binding modes analyses. In order to
evaluate the non-bonded interaction energy contribution, the
Glide Energy and its components Glide Ecoul and EvdW
were examined.
2.3.4. Neurotransmitter extraction and high-performance liquid
chromatography (HPLC) determination
Extracellular DA, 5-HT, and NE levels were analysed through HPLC
apparatus consisting of a Jasco (Tokyo, Japan) PU-2080 chromato-
graphic pump and an ESA (Chelmsford, MA) Coulochem III coulo-
metric detector, equipped with microdialysis cell (ESA-5014b)
porous graphite working electrode and solid-state palladium refer-
ence electrode. The analytical conditions for biogenic amine iden-
tification and quantification were selected as previously
reported34. Briefly, the analytical cell was set at ꢃ0.150 V, for
detector 1 and at þ0.300 V, for detector 2, with a range of 100 nA.
The chromatograms were monitored at the analytical detector 2.
Integration was performed by Jasco Borwin Chromatography soft-
ware version 1.5 . The chromatographic separation was performed
by isocratic elution on Phenomenex Kinetex reversed-phase col-
2.5. Radical scavenging and chelating activities
umn (C18, 150 mm ꢁ 4.6 mm i.d., 2.6 mm). The mobile phase was 2.5.1. Free radical scavenging activity (DPPH)
(10:90, v/v) acetonitrile and 75 mM, pH 3.00 phosphate buffer con- Each test solution (1 ml) was added to 2,2-diphenyl-1-picrylhydra-
taining octanesulfonic acid 1.8 mM, EDTA 30 mM and triethylamine
0.015% v:v. Flow rate was 0.6 ml/min and the samples were manu-
ally injected through a 20 ml loop. Neurotransmitter peaks were
identified by comparison with the retention time of pure standard.
Neurotransmitter concentrations in the samples were calculated
by linear regression curve (y ¼ bx þ m) obtained with standard.
Neither internal nor external standard was necessary for neuro-
transmitter quantification and all tests performed for method val-
idation yielded results in accordance to limits indicated in official
guidelines for applicability in laboratory trials. The standard stock
solutions of DA and DOPAC at 2 mg/ml were prepared in bi-dis-
zyl (DPPH) solution (4 ml, 0.004% methanolic solution). The sample
absorbance was noted at 517 nm after 30 min incubation at room
temperature in the dark43.
2.5.2. 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)
radical cation scavenging activity
ABTS.þ radical cation was produced in situ by reacting a 7 mM
ABTS solution with 2.45 mM potassium persulfate and allowing
the mixture to incubate for 12–16 h in the dark at room tempera-
ture. The ABTS solution was first diluted with methanol to an
tilled water containing 0.004% EDTA and 0.010% sodium bisul- absorbance of 0.700 0.02 at 734 nm. Each test solution (1 ml) was
phite. The stock solutions were stored at 4 ꢂC. Work solutions mixed with ABTS solution (2 ml)44.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1519
2.5.3. Evaluation of total antioxidant capacity by phosphomolyb- hMAO-B, with the best efficacy recorded when the methoxy group
was placed on the para-position of the phenyl ring (PM6, IC50
hMAO-B ¼ 0.28 mM). Similar to the methyl-substituted derivatives,
the placement of the methoxy substituent on the meta-position
negatively affected inhibition activity against hMAO-A (PM5, IC50
hMAO-A ¼ 33.0 mM), thus improving the selectivity index (SI ¼
42.3). In the light of the above, it may be concluded that electron
donor groups improve inhibitory activity towards hMAO-B when
they are substituted on the meta- and para-positions.
Furthermore, when these groups are placed on the meta-position,
they also led to an increase in selectivity due to the reduction of
hMAO-A inhibition. A different trend was observed for halogen-
substituted derivatives (PM7-PM13). The data show that when the
substituent changed from fluoro to chloro and finally to bromo,
there was an increment of inhibitory activity and selectivity
towards hMAO-B, according to the increased size and reduced
electronegativity of the halogen, with the best inhibition and
selectivity shown by compound PM12 (IC50 hMAO-B ¼ 0.35 mM; SI
¼ 180). In the light of the above, we conclude that halogens,
which represent electron-withdrawing groups, positively affect
both inhibition activity and selectivity when they are placed on
the meta position (e.g. PM12, IC50 hMAO-B ¼ 0.35 mM vs. PM13,
IC50 hMAO-B ¼ 0.88 mM). This trend differs from that observed with
the electron donor groups (e.g. methyl or methoxy). Other com-
pounds such as PM14 and PM15, which are substituted on the
para position with CN and NO2, exhibited hMAO-B inhibition in
the low micromolar range (PM14, IC50 hMAO-B ¼ 4.51 mM; PM15,
IC50 hMAO-B ¼ 2.75 mM) with little selectivity between the two iso-
forms (PM14 SI ¼ 4.7; PM15 SI ¼ 6.8). The presence of a hetero-
cyclic ring for compounds PM16-PM19 negatively affected the
inhibition of hMAO-B. Finally, substitution of the phenyl ring with
the bulky naphthyl, but not biphenyl, improved both activity and
selectivity towards hMAO-B (PM20, IC50 hMAO-B ¼ 1.08 mM; SI
¼ 39.4).
denum assay
Each test solution (0.3 ml) was mixed with 3 ml of reagent solution
(0.6 M sulphuric acid, 28 mM sodium phosphate and 4z mM
ammonium molybdate). The sample absorbance was read at
695 nm after 90 min incubation at 95 ꢂC45.
2.5.4. Cupric ion reducing (CUPRAC) method
Each test solution (0.5 ml) was added to reaction mixture contain-
ing 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 as fol-
lows: sample solution (0.5 ml) and reaction mixture (3 ml) without
CuCl2. The absorbances were read at 450 nm after 30 min of incu-
bation at room temperature46.
2.5.5. Ferric reducing antioxidant power (FRAP) method
Each sample solution (0.1 ml) was added to the 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). Then, the absorbance was read at
593 nm after a 30 min incubation at room temperature47.
2.5.6. Metal chelating activity on ferrous ions
Each test solution (2 ml) was added to FeCl2 solution (0.05 ml,
2 mM). The reaction was initiated by the addition of 5 mM ferro-
zine (0.2 ml). Similarly, a blank was prepared as follows: test solu-
tion (2 ml), FeCl2 solution (0.05 ml, 2 mM) and water (0.2 ml). The
absorbances of sample and blank were subsequently noted at
562 nm after 10 min incubation at room temperature48.
3. Result and discussion
3.1. In vitro MAO inhibition study
3.2. Evaluation of DOPAC/DA ratio and LDH activity
The synthesised compounds PM1-PM20 were evaluated as poten-
tial inhibitors of hMAO-A and hMAO-B, and the activities are given
as the IC50 values in Table 1. Among these derivatives, only com-
pounds PM2, PM17, and PM18 were superior inhibitors of hMAO-
A compared to hMAO-B, although only to a small extent. All other
compounds were selective hMAO-B inhibitors with IC50 values in
the micromolar/low micromolar range. The simplest compound of
this series, PM1, containing an unsubstituted phenyl ring bound
to the carbonyl “bridge,” showed similar inhibition against both
the isoforms with poor selectivity (IC50 hMAO-A ¼ 13.3 mM; IC50
hMAO-B ¼ 7.39 mM; SI ¼ 1.8). When substitution on the phenyl
ring occurred, we observed different effects depending on the
position and chemical nature of the substituent. In fact, the pres-
ence of a weak electron donor such as the methyl group on phe-
nyl ring, improved inhibition activity towards hMAO-B if placed on
the meta (PM3, IC50 hMAO-B ¼ 1.81 mM) or para (PM4, IC50 hMAO-
B ¼ 0.47 mM) positions. PM4 also displayed improved inhibition
activity towards hMAO-A (PM4, IC50 hMAO-A ¼ 2.71 mM), account-
ing for the slightly reduced SI compared with PM3 (PM3, IC50
hMAO-A ¼ 12.6 mM). On the other hand, when this substituent was
Figures 2–5 show that deprenyl, an irreversible and selective
MAO-B inhibitor, and the new MAO inhibitors PM4, PM5, PM6,
PM9, PM10, PM12, and PM13 were able to exert modulatory
effects on cortex synaptosome DA/DOPAC ratio and LDH activity,
in both basal and LPS-induced inflammatory conditions.
Particularly, in Figure 2, it is possible to observe that deprenyl
and PM9 stimulated DOPAC/DA ratio, while PM10, PM12, and
PM13 inhibited basal DOPAC/DA ratio. On the other hand, no sig-
nificant effect was exerted by PM4, PM5, and PM6. When the
synaptosomes were challenged with an inflammatory LPS stimulus
(Figure 3), we observed that deprenyl and all PM inhibitors were
able to reduce DOPAC/DA ratio. Additionally, the present mole-
cules were more effective than deprenyl, with the highest inhibi-
tory effects displayed by PM10, PM12, and PM13.
DOPAC/DA ratio has long been proposed as an index of MAO-
B activity27, while microdialysis studies demonstrated the ability of
LPS to increase monoamine degradation and extracellular DOPAC
levels, in mouse prefrontal cortex49. Additionally, we have recently
reported that the pro-oxidant stimulus induced by amyloid b-pep-
located at the ortho-position, we observed reduced inhibition tide could reduce monoamine levels, in rat cortex synapto-
somes50,51. Despite the agreement between the data reported in
activity against both the isoforms, with a slight preference for
hMAO-A (PM2, IC50 hMAO-A ¼ 18.7 mM; IC50 hMAO-B ¼ 23.4 mM; SI Figure 3 and the MAO-B inhibitory activity described in Table 1,
¼ 0.8). A similar trend was observed for compounds substituted the contrasting results obtained in basal condition (Figure 2), fol-
with the methoxy group, which is considered to be a stronger lowing PMs treatment, suggested the possible onset of pro-
electron donor than the methyl (PM5 and PM6). For these two inflammatory/pro-oxidant effects, which could have overcome the
derivatives, we also recorded different inhibition activities towards intrinsic MAO inhibitory activity of these molecules, thus leading

1520
P. GUGLIELMI ET AL.
ꢄꢄꢄ
p<.001 vs. vehicle group. All the com-
Figure 2. Effect of the PM series of inhibitors on DOPAC/DA ratio in rat cortex synaptosomes. ANOVA: p<.0001; post-hoc:
pounds and deprenyl were tested at the same concentrations: 20 nM, 0.1lM, and 1 lM.
ꢄ
ꢄꢄꢄ
p<.001 vs.
Figure 3. Effect of the PM series of inhibitors on DOPAC/DA ratio in rat cortex synaptosomes challenged with LPS. ANOVA: p<.0001; post-hoc: p<.05,
LPS group. All the compounds and deprenyl were tested at the same concentrations: 20 nM, 0.1lM and 1 lM.
to increased DA turnover in rat cortex synaptosomes. In order to stimulatory effect at the highest tested concentration (1 mM). We
test this hypothesis, we performed a second set of experiments to cannot exclude that the highest tested deprenyl concentration
evaluate the effect of deprenyl and the PM inhibitors on cortex (1 mM) could be toxic for cortex synaptosomes. Similarly, the PM
synaptosome LDH activity, in both basal and LPS-induced inflam- inhibitors displayed a significant LDH inhibitory activity, which is
matory condition (Figures 4 and 5).
more evident around their respective IC50 values, which are
included in the range 0.1–1 mM (Table 1). Unlike deprenyl, all the
PM molecules inhibited LDH activity in the concentration range
(0.1–1 lM). Our findings of reduced LPS-induced LDH activity by
both deprenyl (20 nM) and PMs (0.1–1 lM), in rat cortex synapto-
LDH has long been considered a valuable marker of tissue
damage28,29. Additionally, antioxidants are able to downregulate
their ex vivo activity52. In this study, we observed that, except for
compound PM13, most of the PM inhibitors upregulated basal
LDH activity (Figure 4). Conversely, when synaptosomes were per- somes, are consistent with the reported antioxidant activity of
fused with Krebs-Ringer buffer added with LPS, deprenyl inhibited MAO inhibitors in vivo54,55. On the other hand, we should consider
LDH activity at the lowest concentration (20 nM), which is very that the contrasting finding of stimulation of LDH activity (Figure
close to its MAO-B IC50 value (17 nM)53, despite exerting a 4), induced by deprenyl and PM inhibitors in basal condition,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1521
ꢄ
ꢄꢄ
ꢄꢄꢄ
p<.001 vs. vehicle group.
Figure 4. Effect of the PM series of inhibitors on LDH activity in rat cortex synaptosomes. ANOVA: p<.0001; post-hoc: p<.05, p<.01,
All the compounds and deprenyl were tested at the same concentrations: 20 nM, 0.1lM, and 1 lM.
ꢄ
ꢄꢄ
p<.01,
Figure 5. Effect of the PM series of inhibitors on LDH activity in rat cortex synaptosomes challenged with LPS. ANOVA: p<.0001; post-hoc: p<.05,
ꢄꢄꢄ
p<.001 vs. LPS group. All the compounds and deprenyl were tested at the same concentrations: 20 nM, 0.1lM, and 1 lM.
could be related to the employed ex vivo experimental model. inhibitors PM10, PM12, and PM13 which exerted the highest
Specifically, it is well known that antioxidants in the cell medium
could exert pro-oxidative effects, by generating hydrogen perox-
ide and thus activating adaptive responses of cells to mild oxida-
tive stress. In this context, it is rational to hypothesise that our
results of blunted LPS-induced DOPAC/DA ratio and LDH activity,
in cortex synaptosomes treated with both deprenyl and PMs,
could be related to both MAO-B inhibition activity and improved
neuron antioxidant defence system. Taken together, these findings
support further investigation of the inhibition efficacy of the PM
series in in vivo experimental models of neuroinflammation and
inhibitory efficacy on both LPS-induced DOPAC/DA ratio and LDH
activity (Figures 3 and 5).
3.3. Antioxidant and chelating activity
The most active compounds, investigated in the previous ex vivo
tests, were also studied as putative antioxidants and metal chelat-
ing agents in six in vitro spectrophotometric assays (Table 2). In
general, they were less potent with respect to the reference com-
oxidative stress. Particularly, future studies should involve pounds (Trolox as antioxidant and EDTA as chelating agent). Only

1522
P. GUGLIELMI ET AL.
Table 2. IC50 values in the antioxidant assays (mM).
Samples
DPPH
ABTS
>5
>5
3.18 0.40
2.49 0.30
3.28 0.44
3.42 0.66
2.68 0.75
0.15 0.01
–
FRAP
CUPRAC
Chelating ability
Phosphomolybdenum assay
PM4
PM5
PM6
PM9
PM10
PM12
PM13
Trolox
EDTA
1.13 0.01
1.20 0.02
1.13 0.01
1.31 0.04
1.10 0.01
1.11 0.01
1.11 0.01
0.09 0.01
–
3.04 0.03
3.27 0.08
3.11 0.11
>5
3.50 0.07
3.95 0.16
2.74 0.10
0.20 0.01
–
1.88 0.08
1.14 0.04
1.62 0.10
3.57 0.11
1.41 0.12
1.13 0.06
1.43 0.11
0.38 0.01
–
2.63 0.42
1.74 0.19
1.89 0.08
1.97 0.09
2.35 0.13
1.55 0.28
4.71 1.04
2.63 0.42
0.05 0.01
4.40 0.07
4.40 0.70
>5
>5
>5
>5
>5
2.17 0.10
–
Table 3. Theoretical interaction energy and its van der Waals (vdW) and
Coulomb (Coul) components are reported (in kcal/mol) for each ligand-tar-
get complex.
A1
A2
hMAO-A
Interaction Energy
Coul
hMAO-B
Interaction Energy
Coul
Compounds
VdW
Total
VdW
Total
PM1
PM2
PM3
PM4
PM5
PM6
PM7
PM8
ꢃ26.91
ꢃ30.73
ꢃ15.49
ꢃ15.12
ꢃ33.95
ꢃ36.02
ꢃ31.50
ꢃ16.59
ꢃ16.87
ꢃ18.41
ꢃ33.83
ꢃ19.74
ꢃ36.96
ꢃ20.86
ꢃ37.63
ꢃ35.75
ꢃ27.60
ꢃ38.05
ꢃ11.88
ꢃ26.78
ꢃ0.81
ꢃ0.24
ꢃ5.59
ꢃ4.88
ꢃ1.00
ꢃ1.23
ꢃ1.77
ꢃ2.93
ꢃ5.11
ꢃ3.10
ꢃ1.49
ꢃ3.46
ꢃ2.20
ꢃ2.78
ꢃ2.86
ꢃ1.91
ꢃ3.00
ꢃ0.61
ꢃ0.54
ꢃ0.69
ꢃ27.72
ꢃ30.97
ꢃ21.08
ꢃ20.00
ꢃ34.94
ꢃ37.25
ꢃ33.27
ꢃ19.51
ꢃ21.97
ꢃ21.50
ꢃ35.32
ꢃ23.20
ꢃ39.16
ꢃ23.63
ꢃ40.49
ꢃ37.66
ꢃ30.60
ꢃ38.66
ꢃ12.42
ꢃ27.47
ꢃ39.51
ꢃ37.60
ꢃ45.05
ꢃ40.88
ꢃ44.92
ꢃ43.11
ꢃ42.08
ꢃ35.20
ꢃ46.49
ꢃ43.35
ꢃ33.39
ꢃ47.43
ꢃ44.01
ꢃ41.96
ꢃ43.49
ꢃ42.30
ꢃ36.93
ꢃ41.52
ꢃ24.22
ꢃ37.34
0.51
ꢃ0.79
ꢃ0.47
0.02
ꢃ39.00
ꢃ38.39
ꢃ45.52
ꢃ40.87
ꢃ45.61
ꢃ42.84
ꢃ41.36
ꢃ36.64
ꢃ47.28
ꢃ43.57
ꢃ33.79
ꢃ48.29
ꢃ44.28
ꢃ46.51
ꢃ44.96
ꢃ43.14
ꢃ38.15
ꢃ42.59
ꢃ24.70
ꢃ38.50
B1
B2
ꢃ0.69
0.27
0.72
ꢃ1.44
ꢃ0.80
ꢃ0.22
ꢃ0.40
ꢃ0.86
ꢃ0.27
ꢃ4.55
ꢃ1.47
ꢃ0.84
ꢃ1.22
ꢃ1.08
ꢃ0.48
ꢃ1.16
PM9
PM10
PM11
PM12
PM13
PM14
PM15
PM16
PM17
PM18
PM19
PM20
Figure 6. 2 D structures of the conformers/tautomers investigated by quantum-
mechanics approach for each compound reported in Table 1.
in the phosphomolybdenum test, were PM4 and PM5 comparable
to the reference drug.
3.4. Molecular modelling studies
Before evaluating target recognition, the possibility that our com-
pounds could exist in different tautomeric and conformational
states have been taken into account. In particular, the keto-enol
tautomerism and formation of intramolecular hydrogen bonds
(HBs) were investigated. For each compound, four states (Figure
6) were optimised at quantum mechanics level and submitted to
the Boltzmann population analysis at 300 K (Table S1).
which is replaced by Ile199 in hMAO-B, and thus assumes a differ-
ent binding mode with the benzothiophene moiety involved in
p–p stacking with Tyr407. However, such geometry didn’t prevent
unfavourable steric contacts to hMAO-A residues, Asn181
and Gln215.
For all compounds, the A1 form described a population larger
than 95% disregarding the substitution pattern on the aryl ring.
Therefore, this form was submitted to molecular docking simula-
tions. Docking analysis was focussed on the binding modes which
showed the best theoretical affinity according to the Glide Score.
Even if all compounds recognised both hMAO isoforms, they dis-
played a better theoretical interaction energy towards the hMAO-
B, excluding PM11 (Table 3). By computing r2 between theoretical
interaction energies and experimental inhibition data, considered
a -log(IC50), a strong linear correlation was not found (r2 ¼0.53)
but a qualitative accord only. This was not a surprise because IC50
does not depend on the ligand target interaction only solvation
effect, ligand–-ligand, and ligand–other solutes interaction, not
considered in docking simulation, contribute. Analysing the theor-
etical complexes in terms of interaction energy contribution, we
found that, in general, the van der Waals (vdW) component
strongly favoured hMAO-B binding compared to hMAO-A
(Table 3).
Regarding the binding modes of the other derivatives, by vis-
ual inspections it was observed that, considering the orientation
of the benzothiophene portion, they could assume different bind-
ing modes in the hMAO-B active site, thus maintaining (PM2,
PM4, PM6, PM7, PM8, PM11, PM13, and PM17) (Figures S1–S8)
or losing (PM1, PM3, PM5, PM9, PM10, PM14, PM15, PM16,
PM18, PM19, and PM20) (Figures S9–S19) the intra-molecular HB.
In particular, derivatives PM1, PM4, PM6, PM7, PM11, PM13, and
PM19 directed the benzothiophene moiety towards the FAD
cofactor forming p–p interactions with active site aromatic amino
acids. Among these, only PM13 does not establish productive
contacts with the target. In contrast, the benzothiophene moiety
of other compounds was directed towards the entry gorge, which
resulted in the positioning of their hydroxyl group close to the
side chain of Tyr326 (PM2, PM3, PM5, PM8, PM9, PM10, PM15,
PM16, PM17, PM18, and PM20) or to the Ile199 backbone
(PM20), thus establishing HB to the target. Thus, the docking
results suggested that both p–p interactions and HBs with Tyr326
could play a key role in hMAO-B binding by most of the com-
We ascribe the hMAO-B preference of PM12, as reported in
Figure 7, to the intermolecular HB with Tyr326 side chain, which is
replaced in hMAO-A by the Ile335. Instead, in the hMAO-A pocket, pounds. Regarding hMAO-A, an intra-molecular HB was present
the binding of PM12 is hampered by the presence of the Phe208 only in the docking poses of compounds PM1, PM17, and PM19

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1523
(a)
(b)
Trp128
Trp119
Cys172
Asn181
Tyr407
Ile199
Phe208
Tyr435
Tyr398
Pro102
Thr314
Tyr444
Ala111
Tyr326
Ile335
Phe343
Phe352
Gln206
Gln215
Cys323
Tyr60
Tyr69
Figure 7. The most stable binding modes of PM12 in the (a) hMAO-A and (b) hMAO-B active sites, represented in grey and lilac colouring, respectively. The ligand is
depicted in polytube with the carbons coloured green, the FAD cofactor is displayed in space fill and the most relevant ligand interacting amino acids are shown as
thin tubes. Yellow, light blue, and orange dotted lines represent inter-molecular hydrogen bonds, p–p interactions and unfavourable contacts, respectively.
(Figures S20–S22). Although the docking complexes of PM1 and theoretical complexes, the intra-molecular HB was lost and
PM17 were quite similar, PM19 presented an opposite binding
orientation. In this respect, PM19 fitted in the hMAO-A cleft orien-
tated with the biphenyl moiety towards the outside. In addition,
for such compounds conflicts with both tyrosine residues close to
the cofactor and with the Ala111 backbone were suggested.
Similar to PM1 and PM17, compound PM11 (Figure S23) bound
with the benzothiophene core towards the entry of the active
site, but the presence of the 2,4-dichlorine substituted phenyl ring
led to the loss of the intra-molecular HB and to steric conflict with
Tyr69. This type of unfavourable interactions was present in most
of the hMAO-A docking solutions. For example, compounds PM2,
PM7, PM16, and PM18 (Figures S24–S27) displayed similar bind-
ing modes, but PM16 and PM18 formed HB and p–p interactions
with Phe208, whereas for PM2 and PM7, which possessed bulkier
substituents, unfavourable contacts with Phe208 and Tyr407 were
recorded. These contacts penalised the recognition of derivatives
PM3 (Figure S28) and PM9 (Figure S29) mainly due to interaction
with Tyr407 and Asn181 residues. Thus, the presence of a methyl
group or chlorine atom on the meta-position of the phenyl ring
resulted in similar unfavourable interactions with hMAO-A. For
compounds PM4, PM5, PM10, and PM20 (Figures S30–S33) simi-
lar docking poses were predicted. Favourable p–p contacts and
very similar recognition of derivatives PM6, PM8, PM13, PM14,
and PM15 (Figures S34–S38) by hMAO-A were predicted, although
these compounds are differently substituted on the para-position
of the phenyl ring. Particularly, the poses of PM6, PM13, and
PM15 were almost identical as well as those of PM8 and PM14.
Conversely, for compounds PM4 and PM10, substituted on the
para-position of the phenyl ring with a methyl group and chlorine
atom, respectively, the hydroxyl group was directed towards the
cofactor. In conclusion, molecular modelling proposed that ben-
zo[b]thiophen-3-ol tautomer A1 is the most populated state for
each compound of our series. In qualitative agreement to the bio-
logical assays, all derivatives were able to recognise both hMAOs
but preferred hMAO-B in terms of interaction energy and product-
ive contacts. Such a preference seemed to be driven by the vdW
replaced, in hMAO-B only, by a favourable inter-molecular inter-
action to Tyr326.
4. Conclusions
This library of benzothiophen-3-ol derivatives was demonstrated
to be a very interesting scaffold to design high potency hMAO-B
inhibitors, with IC50 values in the low micromolar to nanomolar
range. Moreover, the introduction of specific substituents on the
phenyl resulted in highly selective inhibition of this isozyme.
These compounds were also characterised by an effective ex vivo
hMAO-B inhibition as well as by limited antioxidant and chelating
properties. Furthermore, the binding modes and energies of the
designed molecules were predicted in silico, thus corroborating
the potential of this scaffold for the treatment of neurodegenera-
tive disorders.
Disclosure statement
The authors state no conflict of interest and they have received
no payment in preparation of this manuscript.
Funding
This work was supported by “Progetto di Ricerca Ateneo La
Sapienza 2014-C26A14AC5L” (Italy) and POR FESR LAZIO 2014/
2020 – REGIONE LAZIO – Avviso pubblico LIFE 2020 (Prof. Daniela
Secci). The monoamine oxidase inhibition experiments were sup-
ported by grants from the National Research Foundation of South
Africa (Grant specific unique reference numbers (UID) 85642,
96180). Opinions expressed and conclusions arrived at, are those
of the authors and therefore the NRF do not accept any liability in
interaction energy. Finally, in a large part of the most stable regard thereto.

1524
P. GUGLIELMI ET AL.
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ORCID
Stefano Alcaro
http://orcid.org/0000-0002-0437-358X
http://orcid.org/0000-0002-5165-6013
http://orcid.org/0000-0001-6235-8161
http://orcid.org/0000-0002-8698-9440
Gokhan Zengin
Francesco Ortuso
Simone Carradori
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scaffold for monoamine oxidases inhibitors. J Med Chem
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theses, UV-Vis, IR, 1H, 13C and 19F NMR spectral studies of
the complexes of U(VI), Fe(III), V(V) and Ca(II) with 2-thenoyl-
trifluoroacetone (HTTA). Inorg Chim Acta 1990;176:143–8.
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