Synthesis and in vitro study of nitro- and methoxy-2-phenylbenzofurans as human monoamine oxidase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104616

A new series of 2-phenylbenzofuran derivatives were designed and synthesized to determine relevant structural features for the MAO inhibitory activity and selectivity. Methoxy substituents were introduced in the 2-phenyl ring, whereas the benzofuran moiety was not substituted or substituted at the positions 5 or 7 with a nitro group. Substitution patterns on both the phenyl ring and the benzofuran moiety determine the affinity for MAO-A or MAO-B. The 2-(3-methoxyphenyl)-5-nitrobenzofuran 9 was the most potent MAO-B inhibitor (IC₅₀ = 0.024 μM) identified in this series, whereas 7-nitro-2-phenylbenzofuran 7 was the most potent MAO-A inhibitor (IC₅₀ = 0.168 μM), both acting as reversible inhibitors. The number and position of the methoxyl groups on the 2-phenyl ring, have an important influence on the inhibitory activity. Molecular docking studies confirmed the experimental results and highlighted the importance of key residues in enzyme inhibition.

Full text of DOI:10.1016/j.bioorg.2020.104616

Bioorganic Chemistry 107 (2021) 104616
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and in vitro study of nitro- and methoxy-2-phenylbenzofurans as
human monoamine oxidase inhibitors
,
1
,
,
,d
Giovanna L. Delogua *, Amit Kumarb 1, Gianluca Gattob, Fernando Busteloc
,
e
e
c
c,d,*
˜
Lucía M. Saavedra , Maria Isabel Rodríguez-Franco , Reyes Laguna , Dolores Vina
a Department of Life and Environmental Sciences, University of Cagliari, 09042 Monserrato, Cagliari, Italy
b Department of Electrical and Electronic Engineering, University of Cagliari 09123 Cagliari, Italy
c Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Avda Barcelona s/n, Campus Vida 15782, Santiago
de Compostela, Spain
d Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela,
Spain
e
´
Instituto de Química Medica, Consejo Superior de Investigaciones Científicas (IQM-CSIC), c/ Juan de la Cierva 3, E-28006 Madrid, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new series of 2-phenylbenzofuran derivatives were designed and synthesized to determine relevant structural
features for the MAO inhibitory activity and selectivity. Methoxy substituents were introduced in the 2-phenyl
ring, whereas the benzofuran moiety was not substituted or substituted at the positions 5 or 7 with a nitro
group. Substitution patterns on both the phenyl ring and the benzofuran moiety determine the affinity for MAO-
A or MAO-B. The 2-(3-methoxyphenyl)-5-nitrobenzofuran 9 was the most potent MAO-B inhibitor (IC50 = 0.024
µM) identified in this series, whereas 7-nitro-2-phenylbenzofuran 7 was the most potent MAO-A inhibitor (IC50
= 0.168 µM), both acting as reversible inhibitors. The number and position of the methoxyl groups on the 2-
phenyl ring, have an important influence on the inhibitory activity. Molecular docking studies confirmed the
experimental results and highlighted the importance of key residues in enzyme inhibition.
2-Phenylbenzofurans
Monoamine oxidase inhibitors
Docking studies
1. Introduction
MAO isoforms in most species [4,5]. Because of the role of MAO in the
homeostasis of neurotransmitters in the brain, this enzyme has been
Monoamine oxidase (MAO) is a flavin adenine dinucleotide (FAD)
dependent enzyme responsible for the metabolism of monoamine neu-
rotransmitters, which plays an important role in brain development and
function [1].
recognized as an important target for neurological diseases, such as
depression, Parkinson’s disease (PD) or even Alzheimer’s disease (AD)
[6].
MAO-A inhibitors are well known as powerful anti-depressants, as
well as effective therapeutic agents for social phobia and panic disorder.
They are particularly effective in treatment-resistant depression and
atypical depression. However, only moclobemide is currently in clinical
use. Therefore, the search for novel MAO-A inhibitors certainly remains
an attractive topic [7].
Two isoforms of the enzymes, namely MAO-A and MAO-B are
encoded by distinct genes located on the X chromosome, and are
expressed in different amounts in a wide variety of tissues. Moreover,
MAO-A and MAO-B have been characterized by their amino acid se-
quences, three-dimensional structures, inhibitor selectivity, and sub-
strate preferences [2,3].
MAO-B inhibitors with a propargylamine molecular scaffolding such
as selegiline and rasagiline are used in the therapy for neurodegenera-
tive disorders, including PD. They enhance both endogenous dopamine
and the one produced from exogenously administrated levodopa [8] .
Serotonin, noradrenaline, and adrenaline are preferential substrates
for MAO-A and benzylamine and β-phenylethylamine for MAO-B.
Dopamine, tyramine, and tryptamine are common substrates for both
* Corresponding authors at: Department of Life and Environmental Sciences, University of Cagliari, 09042 Monserrato, Cagliari, Italy (G.L. Delogu); Center for
Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Avda Barcelona s/n, Campus Vida 15782, Santiago de
˜
Compostela, Spain (D. Vina).
˜
E-mail addresses: delogug@unica.it (G.L. Delogu), mdolores.vina@usc.es (D. Vina).
1
These authors should be considered joint first authors.
https://doi.org/10.1016/j.bioorg.2020.104616
Received 16 June 2020; Received in revised form 16 December 2020; Accepted 28 December 2020
Available online 5 January 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

G.L. Delogu et al.
Bioorganic Chemistry 107 (2021) 104616
While selegiline and rasagiline derivatives are irreversible inhibitors, a
new reversible MAO-B inhibitor, safinamide, has been approved by the
Food and Drug Administration in 2017 for PD therapy [9].
2-hydroxybenzaldehyde (6.60 mmol), in ethanol (20 mL), in an ice bath.
The reaction mixture was stirred at room temperature for 1 h. After that,
the solvent was removed, 1 N aqueous HCl solution (40 mL) was added
to the residue and extracted with diethyl ether. The solvent was evap-
orated under vacuum to give the desired compounds Ia-Ic [20–23].
The reaction of monoamines with MAO results in the generation of
H2O2 which, when accumulated, mediates neurotoxicity. As far as MAO-
B activity is increased in the older brain, MAO-B inhibitors could prevent
neuronal damage and secondary injuries by controlling oxidative stress.
Therefore, the neuroprotective role of MAO-B inhibitors justifies their
potential not only to manage PD but also to AD [10,11].
2.1.3. General procedure for the preparation of 2-hydroxybenzyltriphe-
nylphosphonium bromide (IIa-IIc).
A mixture of 2-hydroxybenzylalcohol (16.27 mmol) and PPh3⋅HBr
(16.27 mmol) in CH3CN (40 mL) was stirred under reflux for 2 h. The
solid formed was filtered and washed with CH3CN to give the desired
compounds IIa–IIc [20,24,25].
A benzofuran ring as a core of heterocyclic compounds is an essential
structural unit of various biologically active natural medicines and
synthetic chemical raw materials. Numerous studies have shown that 2-
phenylbenzofurans have strong biological activities such as anti-cancer,
anti-bacterial, anti-viral, anti-oxidative, anti-fungal, and anti-microbial.
Such a wide range of biological properties, inherent in benzofuran
scaffold, justifies the extensive interest in using benzofuran as a building
block of pharmacological agents [12].
2.1.3.1. 2-hydroxy-3-nitrophenylphosphonium bromide (IIa. It was ob-
tained with a yield of 95%. m.p. 115–117 ◦C; 1H NMR (500 MHz,
CDCl3), δ (ppm), J (Hz) = 4.90 (d, 2H, J = 14.8, CH2P), 6.80 (dd, 1H, J =
7.8 and 7.6, H-5), 7.20–7.32 (m, 1H, H-6), 7.50 (dd, 1H, J = 7.8 and 1.2,
H-4), 7.60–7.82 (m, 12H, ArH), 7.85–7.90 (m, 3H, ArH), 10.26 (s, 1H,
ArOH) ppm; 13C NMR (125 MHz, CDCl3), δ (ppm), J (Hz) = 36.58,
117.20, 120.12, 125.10, 131.98, 132.34, 133.46, 133.37, 136.65,
138.20, 152.05 ppm. MS (EI, 70 eV): m/z (%): 496 (98) [M + 2]+, 494
(100) [M]+, 448. Anal. calcd for C25H21BrO3P: C, 60.74%; H, 4.28%.
Found: C, 60.79%; H 4.35.
Recent studies have demonstrated enzymatic inhibition properties of
2-phenylbenzofuran derivatives, for example, against butyr-
ylcholinesterase (BchE) [13–15], and monoamine oxidase (MAO)
[16–18]. In general, benzofurans described as MAO inhibitors have a
higher selectivity for MAO-B isoform [17,18].
In our efforts to contribute to the development of novel compounds
that may be useful in the treatment of disorders associated with MAO,
we chose to focus on evaluating the relevance of structural features in
the MAOs inhibitory activity. In the present work, we describe the
synthesis and biological evaluation and molecular docking of a new
series of 2-phenylbenzofuran derivatives with methoxy and/or nitro
substitutions.
2.1.4. General procedure for the preparation of 2-phenylbenzofuran
(1–18).
A mixture of 2-hydroxybenzyltriphenylphosphonium bromide (1.11
mmol) and benzoyl chloride (1.11 mmol) in a mixed solvent (toluene 20
mL and Et3N 0.5 mL) was stirred under reflux for 2 h. The precipitate
was removed by filtration. The filtrate was concentrated, and the residue
was purified by silica gel chromatography (hexane/EtOAc 9:1) to give
the desired compounds.
In our previously studied benzofuran series, 2-(4-methoxyphenyl)-5-
nitrobenzofuran was found to be the most active compound, presenting
MAO-B selectivity and reversible inhibition (IC50 = 140 nM) [18,19]. In
the present work, we investigated the influence on the enzyme inhibi-
tion activity based on the position and number of methoxyl groups’
substitutions in the 2-phenyl ring. Besides, we examined the importance
of the presence and position of the nitro group on the benzofuran
scaffold.
2.1.4.1. 2-(2-methoxyphenyl)-7-nitrobenzofuran (1). It was obtained
1
◦
with a yield of 47%. m.p. 167–170 C. H NMR (500 MHz, CDCl3), δ
(ppm), J (Hz) = 4.03 (s, 3H, OCH3), 7.04 (d, 1H, J = 8.3, H-3′), 7.15 (t,
1H, J = 7.6, H-5′), 7.33 (t, 1H, J = 7.9, H-4′), 7.40 (t, 1H, H-5, J = 7.9),
7.45 (s, 1H, H-3), 7.88 (d, 1H, J = 7.8, H-6′), 8.24 (d, 1H, J = 7.8, H-6),
8.10 (d, 1H, J = 8.1, H-4) ppm; 13C NMR (125 MHz, CDCl3), δ (ppm), J
(Hz) = 56.81, 104.63, 113.84, 119.25, 119.62, 122.88, 123.47, 129.85,
129.87, 130.64, 131.26, 134.50, 157.40, 148.51, 153.20 ppm. MS (EI,
70 eV): m/z (%): 269 (100) [M]+, 238, 223, 193, 165. Anal. calcd for
2. Experimental
2.1. Chemistry
2.1.1. General information
C
15H11NO4: C, 66.91%; H, 4.12%; N, 5.20%. Found: C, 66.95%; H,
Starting materials and reagents were obtained from commercial
suppliers and were used without further purification. Melting points
(mp) are uncorrected and were determined with a Reichert Kofler
thermopan or in capillary tubes in a Buchi 510 apparatus. 1H NMR and
13C NMR spectra were recorded with a Varian INOVA 500 spectrometer
using DMSO‑d6 or CDCl3 as solvent. Chemical shifts (δ) are expressed in
parts per million (ppm) using TMS as an internal standard. Coupling
constants J are expressed in hertz (Hz). Spin multiplicities are given as s
(singlet), d (doublet), t (triplet) and m (multiplet). Mass spectrometry
was carried out with a Saturn 2000 ion-trap coupled with a Varian 3800
gas chromatograph operating under EI conditions. Elemental analyses
were performed by a Perkin-Elmer 240B microanalyzer and were within
± 0.4% of calculated values in all cases. The analytical results were ≥
95% purity for all compounds. Flash Chromatography (FC) was per-
formed on silica gel (Merck 60, 230–400 mesh); analytical TLC was
performed on precoated silica gel plates (Merck 60 F254). Organic so-
lutions were dried over anhydrous sodium sulfate. Concentration and
evaporation of the solvent after reaction or extraction were carried out
on a rotary evaporator (Büchi Rotavapor) operating at reduced pressure.
4.16%; N, 5.24%.
2.1.4.2. 2-(3-methoxyphenyl)-7-nitrobenzofuran (2). It was obtained
1
◦
with a yield of 45%. m.p. 115–117 C. H NMR (500 MHz, CDCl3), δ
(ppm), J (Hz) = 3.95 (s, 3H, OCH3), 6.98 (d, 1H, J = 8.2, H-4′), 7.12 (s,
1H, H-3), 7.38 (m, 2H, H-5′ and H-6′), 7.49 (s, 1H, H-2′), 7.55 (dd, 1H, J
= 7.7 and 1.9, H-5), 7.88 (d, 1H, J = 7.7, H-4), 8.12 (d, 1H, J = 8.0, H-6)
ppm; 13C NMR (125 MHz, CDCl3), δ (ppm), J (Hz) = 56.10, 104.18,
112.70, 115.98, 118.43, 122.92, 123.48, 128.98, 129.89, 130.68,
131.56, 134.46, 153.22, 158.15, 159.47 ppm. MS (EI, 70 eV): m/z (%):
269 (100) [M]+, 238, 223, 195, 126. Anal. calcd for C15H11NO4: C,
66.91%; H, 4.12%; N, 5.20%. Found: C, 66.96%; H, 4.17%; N, 5.23%.
2.1.4.3. 2-(4-methoxyphenyl)-7-nitrobenzofuran (3). It was obtained
1
◦
with a yield of 51%. m.p. 104–106 C; H NMR (500 MHz, CDCl3), δ
(ppm), J (Hz) = 3.87 (s, 3H, OCH3), 6.96 (s, 1H, H-3), 7.00 (d, 2H, J =
8.9, H-3′ and H-5′,), 7.31 (t, 1H, J = 7.9, H-5), 7.82 (d, 1H, J = 6.8, H-4),
7.88 (d, 2H, J = 8.9, H-2′ and H-6′), 8.06 (d, 1H, J = 8.0, H-6) ppm; 13C
NMR (125 MHz, CDCl3), δ (ppm), J (Hz) = 56.08, 102.93, 114.25,
122.93, 123.32, 123.52, 127.76, 129.94, 130.74, 134.56, 153.25,
158.33, 161.54 ppm. MS (EI, 70 eV): m/z (%): 269 (100) [M]+, 238,
223, 195, 152, 129. Anal. calcd for C15H11NO4: C, 66.91%; H, 4.12%; N,
2.1.2. General procedure for the preparation of 2-hydroxybenzylalcohols
(Ia-Ic).
Sodium borohydride (6.60 mmol) was added to a stirring solution of
2

G.L. Delogu et al.
Bioorganic Chemistry 107 (2021) 104616
5.20%. Found: C, 66.95%; H, 4.15%; N, 5.25%.
129.63, 130.84, 130.55, 144.43, 157.63, 159.12, 160.21 ppm. MS (EI,
70 eV): m/z (%): 269 (100) [M]+, 223, 195, 169, 152, 126. Anal. calcd
for C15H11NO4: C, 66.91%; H, 4.12%; N, 5.20%. Found: C, 66.92%; H,
4.15%; N, 5.26%.
2.1.4.4. 2-(3,4-dimethoxyphenyl)-7-nitrobenzofuran (4). It was obtained
1
◦
with a yield of 43%. m.p. 175–177 C; H NMR (500 MHz, CDCl3), δ
(ppm), J (Hz) = 3.96 (s, 3H, OCH3), 4.01 (s, 3H, OCH3), 7.04–6.94 (m,
2H, H-3 and H-5′), 7.33 (d, 1H, J = 7.9, H-6′), 7.44 (s, 1H, H-2′), 7.56
(dd, 1H, J = 8.4 and 1.9, H-5), 7.85 (d, 1H, J = 7.61, H-4), 8.08 (d, 1H, J
= 7.9, H-6) ppm; 13C NMR (125 MHz, CDCl3), δ (ppm), J (Hz) = 56.80,
104.16, 111.85, 112.72, 119.84, 122.90, 123.52, 123.98, 129.86,
130.65, 134.52, 149.96, 152.85, 153.24, 158.10 ppm. MS (EI, 70 eV):
m/z (%): 299 (100) [M]+, 268, 253, 237, 220, 210, 181. Anal. calcd for
C16H13NO5: C, 64.21%; H, 4.38%; N, 4.68%. Found: C, 64.25%; H,
4.42%; N, 4.70%.
2.1.4.10. 2-(3,4-dimethoxyphenyl)-5-nitrobenzofuran (10). It was ob-
tained with a yield of 76%. m.p. 207–208 ◦C; 1H NMR (500 MHz,
CDCl3), δ (ppm), J (Hz) = 3.96 (s, 3H, OCH3), 4.01 (s, 3H, OCH3), 6.97
(d, 1H, J = 8.4, H-5′), 7.01 (s, 1H, H-3), 7.37 (d, 1H, J = 1.8, H-2′), 7.47
(dd, 1H, J = 1.9 and 8.3H-6′), 7.57 (d, 1H, J = 9.0, H-7), 8.19 (dd, 1H, J
= 2.3 and 8.9, H-6), 8.47 (d, 1H, J = 2.3, H-4) ppm; 13C NMR (125 MHz,
CDCl3), δ (ppm), J (Hz) = 56.10, 56.12, 100.39, 108.10, 111.22, 111.49,
116.92, 118.64, 119.86, 122.19, 129.93, 144.38, 149.32, 150.42,
157.22, 159.33 ppm. MS (EI, 70 eV): m/z (%): 299 (100) [M]+, 268,
253, 237, 222. Anal. calcd for C16H13NO5: C, 64.21%; H, 4.38%; N,
4.68%. Found: C, 64.24%; H, 4.45%; N, 4.70%.
2.1.4.5. 2-(3,5-dimethoxyphenyl)-7-nitrobenzofuran (5). It was obtained
1
◦
with a yield of 46%. m.p. 202–204 C; H NMR (500 MHz, CDCl3), δ
(ppm), J (Hz) = 3.91 (s, 6H, OCH3), 6.56 (s, 1H, H-3), 7.09 (m, 3H, H-
2′and H-4′, H-6′), 7.37 (m, 1H, H-5), 7.89 (d, 1H, J = 7.7, H-4), 8.14 (d,
1H, J = 8.0, H-6) ppm; 13C NMR (125 MHz, CDCl3), δ (ppm), J (Hz) =
56.12, 102.84, 104.99, 105.72, 122.93, 123.54, 129.96, 130.77, 133.12,
134.68, 153.26, 157.20, 160.92 ppm. MS (EI, 70 eV): m/z (%): 299
(100) [M]+, 298, 283, 252, 267, 236, 221, 190. Anal. calcd for
2.1.4.11. 2-(3,5-dimethoxyphenyl)-5-nitrobenzofuran (11). It was ob-
tained with a yield of 79%. m.p. 178–180 ◦C; 1H NMR (500 MHz,
CDCl3), δ (ppm), J (Hz) = 3.91 (s, 6H, OCH3), 6.55 (s, 1H, H-3), 7.04 (s,
2H, H-2′ and H6′), 7.14 (s, 1H, H-4′), 7.62 (d, 1H, J = 9.0, H-7), 8.24 (dd,
1H, J = 2.4 and 8.9, H-6), 8.56 (d, 1H, J = 2.4, H-4) ppm; 13C NMR (125
MHz, CDCl3), δ (ppm), J (Hz) = 55.51, 101.29, 102.11, 103.42, 111.48,
117.34, 120.24, 129.56, 130.89, 144.33, 157.52, 159.02, 161.24 ppm.
MS (EI, 70 eV): m/z (%): 299 (100) [M]+, 283, 269, 253. Anal. calcd for
C16H13NO5: C, 64.21%; H, 4.38%; N, 4.68%. Found: C, 64.18%; H,
4.44%; N, 4.72%.
C
16H13NO5: C, 64.21%; H, 4.38%; N, 4.68%. Found: C, 64.26%; H,
4.44%; N, 4.69%.
2.1.4.6. 2-(3,4,5-trimethoxyphenyl)-7-nitrobenzofuran (6). It was ob-
tained with a yield of 54%. m.p. 145–147 ◦C; 1H NMR (500 MHz,
CDCl3), δ (ppm), J (Hz) = 3.93 (s, 3H, OCH3), 3.99 (s, 6H, OCH3), 7.07
(s, 1H, H-3), 7.18 (s, 2H, H-2′ and H-6′), 7.36 (t, 1H, J = 8.0, H-5), 7.87
(d, 1H, J = 7.7, H-4), 8.12 (d, 1H, J = 8.1, H-6) ppm; 13C NMR (125 MHz,
CDCl3), δ (ppm), J (Hz) = 56.81, 60.67, 103.72, 122.88, 123.47, 126.86,
129.85, 130.63, 134.50, 142.98, 153.20, 154.55, 157.20 ppm. MS (EI,
70 eV): m/z (%): 329 (100) [M]+, 268, 253, 225, 182. Anal. calcd for
2.1.4.12. 2-(2,4-dimethoxyphenyl)-5-nitrobenzofuran (12). It was ob-
tained with a yield of 55%. m.p. 186–188 ◦C; 1H NMR (500 MHz,
CDCl3), δ (ppm), J (Hz) = 3.78 (s, 3H, OCH3), 3.99 (s, 3H, OCH3),
6.64–6.59 (m, 2H, H-3′ and H-5′), 7.29 (s, 1H, H-3), 7.55 (d, 1H, J =
13.7, H-6′), 7.97 (d, 1H, J = 8.7, H-7), 8.17 (d, 1H, J = 9.0, H-6), 8.47 (s,
1H, H-4) ppm; 13C NMR (125 MHz, CDCl3), δ (ppm), J (Hz) = 56.08,
56.82, 100.69, 104.10, 106.32, 113.19, 115.62, 121.32, 121.56, 125.89,
129.53, 140.48, 147.79, 158.12, 160.22, 161.42 ppm. MS (EI, 70 eV):
m/z (%): 299 (100) [M]+, 268, 253, 237, 222. Anal. calcd for
C
17H15NO6: C, 62.00%; H, 4.59%; N, 4.25%. Found: C, 62.08%; H,
4.63%; N, 4.27%.
2.1.4.7. 7-nitro-2-phenylbenzofuran (7). It was obtained with a yield of
45%. m.p. 118–119 ◦C; 1H NMR (500 MHz, CDCl3), δ (ppm), J (Hz) =
7.13 (s, 1H, H-3), 7.35 (t, 1H, H-4′), 7.53–7.40 (m- 2H, H-4 and H-5),
7.88 (d, 2H, J = 8.5, H-3′ and H-5′), 7.96 (d, 2H, J = 8.0, H-2′ and H-6′),
8.12 (dd, 1H, J = 8.2, H-6) ppm; 13C NMR (125 MHz, CDCl3), δ (ppm), J
(Hz) = 100.82, 120.48, 122.87, 125.46, 127.45, 128.93, 129.60,
133.28, 133.80, 146.85, 158.61 ppm. MS (EI, 70 eV): m/z (%): 239
(100) [M]+, 203, 193, 165. Anal. calcd for C14H9NO3: C, 70.29%; H,
3.79%; N, 5.86%. Found: C, 70.34%; H, 3.85%; N, 6.10%.
C
16H13NO5: C, 64.21%; H, 4.38%; N, 4.68%. Found: C, 64.26%; H,
4.46%; N, 4.69%.
2.1.4.13. 2-(3,4,5-trimethoxyphenyl)-5-nitrobenzofuran (13). It was ob-
tained with a yield of 58%. m.p. 250–252 ◦C; 1H NMR (500 MHz,
CDCl3), δ (ppm), J (Hz) = 3.93 (s, 3H, OCH3), 3.99 (s, 6H, OCH3), 7.11
(s, 1H, H-3), 7.27 (s, 2H, H-2′ and H-6′), 7.61 (d, 1H, J = 9.0, H-7), 8.23
(d, 1H, J = 9.0, H-6), 8.51 (s, 1H, H-4) ppm; 13C NMR (125 MHz, CDCl3),
δ (ppm), J (Hz) = 56.83, 60.74, 103.32, 103.75, 113.25, 121.20, 121.53,
125.82, 126.96, 140.52, 142.99, 154.56, 158.10, 158.61 ppm. MS (EI,
70 eV): m/z (%): 329 (100) [M]+, 283, 252, 221, 190. Anal. calcd for
C17H15NO6: C, 62.00%; H, 4.59%; N, 4.25%. found: C, 62.07%; H,
4.60%; N, 4.29%.
2.1.4.8. 2-(2-methoxyphenyl)-5-nitrobenzofuran (8). It was obtained
1
◦
with a yield of 47%. m.p. 174–176 C; H NMR (500 MHz, CDCl3), δ
(ppm), J (Hz) = 4.04 (s, 3H, OCH3), 7.05 (d, 1H, J = 8.3, H-3′) 7.11 (t,
1H, J = 7.6, H-5′), 7.40 (t, 1H, J = 7.8, H-4′), 7.45 (s, 1H, H-3), 7.57 (d,
1H, J = 9.0, H-6′), 8.06 (d, 1H, J = 7.8, H-7), 8.21(d, 1H, J = 9.0, H-6),
8.51 (s, 1H, H-4) ppm; 13C NMR (125 MHz, CDCl3), δ (ppm), J (Hz) =
56.82, 104.12, 113.10, 113.90, 119.30, 119.62, 121.32, 121.59, 125.62,
129.84, 131.25, 140.53, 147.83, 157.52, 158.12 ppm. MS (EI, 70 eV):
m/z (%): 269 (100) [M]+, 222, 195, 152, 131. Anal. calcd for
C15H11NO4: C, 66.91%; H, 4.12%; N, 5.20%. Found: C, 66.94%; H,
4.13%; N, 5.24%.
2.1.4.14. 5-nitro-2-phenylbenzofuran (14). It was obtained with a yield
of 40%. m.p. 158–159 ◦C; 1H NMR (500 MHz, CDCl3), δ (ppm), J (Hz) =
7.13 (s, 1H, H-3), 7.44–7.46 (m, 1H, H-4′), 7.48–7.52 (m, 2H, H-3′ and
H-5′), 7.59 (d, 1H, J = 9.2, H-7), 7.88 (d, 2H, J = 7.6, H-2′ and H-6′),
8.22 (dd, 1H, J = 2.0 and 8.8, H-6), 8.52 (d, 1H, J = 2.4, H-4) ppm; 13C
NMR (125 MHz, CDCl3), δ (ppm), J (Hz) = 101.58, 111.42, 117.25,
120.12, 125.28, 128.96, 129.25, 129.62, 129.66, 144.32, 157.58,
159.25 ppm. MS (EI, 70 eV): m/z (%):239 (100) [M]+, 203, 193, 165.
Anal. calcd for C14H9NO3: C, 70.29%; H, 3.79%; N, 5.86%. Found: C,
70.35%; H, 3.87%; N, 6.20%.
2.1.4.9. 2-(3-methoxyphenyl)-5-nitrobenzofuran (9). It was obtained
1
◦
with a yield of 62%. m.p. 188–189 C; H NMR (500 MHz, CDCl3), δ
(ppm), J (Hz) = 3.91 (s, 3H, OCH3), 6.97 (dd, 1H, J = 72.5 and 8.0, H-
4′), 7.12 (s, 1H, H-3), 7.36–7.43 (m, 2H, H-2′ and H5′), 7.47 (d, 1H, J =
7.6, H-6′), 7.60 (d, 1H, J = 9.0, H-7), 8.22 (dd, 1H, J = 2.4 and 9.0, H-6),
8.51 (d, 1H, J = 2.3, H-4) ppm; 13C NMR (125 MHz, CDCl3), δ (ppm), J
(Hz) = 55.52, 102.02, 110.70, 111.52, 115.51, 117.32, 117.94, 120.29,
2.1.4.15. 2-(2-methoxypheny1l)benzofuran (15). It was obtained with a
yield of 80%. m.p. 78–79 ◦C; H NMR (500 MHz, CDCl3), δ (ppm), J (Hz)
= 4.02 (s, 3H, OCH3), 7.02 (d, 1H, J = 8.2, H-3), 7.08–7.10 (m, 1H, H-
3

G.L. Delogu et al.
Bioorganic Chemistry 107 (2021) 104616
3′), 7.21–7.36 (m, 4H, H-5, H-6, H4′ and H5′), 7.52 (d, 1H, J = 8.2, H-7);
7.60 (d, 1H, J = 7.5, H-4); 8.08 (dd, 1H, J = 1.7, 7.7H-6′) ppm; 13C NMR
(125 MHz, CDCl3), δ (ppm), J (Hz) = 55.43, 106.32, 110.81, 111.05,
119.36, 120.79, 121.07, 122.61, 124.13, 127.05, 129.24, 129.88,
152.16, 153.94, 156.52 ppm. MS (EI, 70 eV): m/z (%): 224 (100) [M]+,
209, 193. Anal. calcd for C15H12O2: C, 80.34%; H, 5.39%; Found: C,
80.38%; H, 5.45%.
(excitation, 545 nm, emission, 590 nm) over a 15 min period, in which
the fluorescence increased linearly.
Control experiments were carried out simultaneously by replacing
the tested drugs with appropriate dilutions of the vehicles. In addition,
the possible capacity of the above tested drugs for modifying the fluo-
rescence generated in the reaction mixture due to non-enzymatic inhi-
bition (e.g., for directly reacting with Amplex Red reagent) was
determined by adding these drugs to solutions containing only the
Amplex Red reagent in a sodium phosphate buffer. The specific fluo-
rescence emission (used to obtain the final results) was calculated after
subtraction of the background activity, which was determined from
wells containing all components except the hMAO isoforms, which were
replaced by a sodium phosphate buffer solution [26].
2.1.4.16. 2-(3-methoxyphenyl)benzofuran (16). It was obtained with a
yield of 88%. m.p. 49–50 ◦C; 1H NMR (500 MHz, CDCl3), δ (ppm), J (Hz)
= 3.90 (s, 3H, OCH3), 6.91 (ddd, 1H, J = 0.7, 2.6 and 8.2, H-4′), 7.03 (s,
1H, H-3); 7.22–7.25 (m, 1H, H-5), 7.28–7.31 (m, 1H, H-2′), 7.36 (t, 1H,
J = 8.2, H-5′), 7.41–7.42 (m, 1H, H-6), 7.46–7.47 (m, 1H, H-6′), 7.53 (d,
1H, J = 8.4, H-7), 7.58 7.59 (m, 1H, H-4) ppm; 13C NMR (125 MHz,
CDCl3), δ (ppm), J (Hz) = 55.3, 101.62, 110.14, 111.16, 114.43 ,
117.55, 120.97, 122.96, 124.36, 129.14, 129.83, 131.72, 154.84,
155.72, 159.86. ppm. MS (EI, 70 eV): m/z (%): 224(100) [M]+, 293,
195, 169, 152, 126. Anal. calcd for C15H12O2: C, 80.34%; H, 5.39%;
Found: C, 80.33%; H, 5.41%.
2.3. Reversibility
To evaluate whether compounds 7 and 9 are reversible or irrevers-
ible hMAO-A and hMAO-B inhibitors respectively, a dilution method
was used [27]. A 100X concentration of the enzyme used in the exper-
iments described above was incubated with a concentration of inhibitor
equivalent to 10-fold its IC50 value. After 30 min, the mixture was
diluted 100-fold and MAO inhibition was determined as above.
Reversible inhibitors show linear progress with a slope equal to ≈ 91%
of the slope of the control sample, whereas irreversible inhibition rea-
ches only ≈ 9% of this slope. Control tests were performed by pre-
incubating and diluting the enzyme in the absence of inhibitor.
2.1.4.17. 2-(3,4-dimethoxyphenyl)benzofuran (17). It was obtained
1
◦
with a yield of 84%. m.p. 120–121 C; H NMR (500 MHz, CDCl3), δ
(ppm), J (Hz) = 3.96 (s, 3H, OCH3), 4.02 (s, 3H, OCH3), 6.94 (s, 1H, H-
3), 6.97 (d, 1H, J = 8.4, H-5′), 7.24 (td, 1H, J = 1.14 and 7.30, H-5), 7.28
(td, 1H, J = 1.40 and 7.30, H-2′), 7.41 (d, 1H, J = 1.98, H-6′), 7.47 (dd,
1H, J = 2.00 and 8.35, H-6), 7.52–7.55 (m, 1H, H-7), 7.57–7.60 (m, 1H,
H-4) ppm; 13C NMR (125 MHz, CDCl3), δ (ppm), J (Hz) = 55.97, 56.01,
100.12, 108.21, 111.04, 11.45, 118.23, 120.76, 122.93, 123.6, 123.97,
129.56, 149.32, 149.61, 154.83, 156.04 ppm. MS (EI, 70 eV): m/z (%):
254 (100) [M]+, 239, 223, 192. Anal. calcd for C16H14O3: C, 75.57%; H,
5.55%; Found: C, 75.60%; H, 5.60%.
2.4. Cytotoxicity
The SH-SY5Y cells grew in a culture medium containing Nutrient
Mixture F-12 Ham (Ham’s F12) and Minimum Essential Medium Eagle
(EMEM) (mixture 1:1) and supplemented with 15% Fetal Bovine Serum
(FBS), 1% L-Glutamine, 1% non-essential amino acids (all of them from
Sigma-Aldrich S.A.) and 1% of penicillin G/streptomycin sulfate (Gibco,
Invitrogen). The cells grew in 75 cm2 flasks in an incubator (Forma
Direct Heat CO2, Thermo Electron Corporation), under conditions of
saturated humidity with a partial pressure of 5% CO2 in the air, at 37 ◦C,
until reaching the confluence, 90–95% of the flask surface. To carry out
the cytotoxicity assays, the cells were seeded in sterile 96-well plates,
with a density of 2x105 cells/mL and grown distributed in aliquots of
2.1.4.18. 2-(2,4-dimethoxyphenyl)benzofuran (18). It was obtained
with a yield of 65%. m.p. 49–52 ◦C; 1H NMR (500 MHz, CDCl3), δ (ppm),
J (Hz) = 3.84 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 6.55 (d, 1H, J = 2.0, H-
3), 6.60 (dd, 1H, J = 2.5 and 8.5, H-6), 7.17–7.24 (m, 3H, H-43, H-5 and
H-6), 7.48 (d, 1H, J = 7.5, H-7), 7.56 (d, 1H, J = 7.5, H-4), 7.97 (d, 1H, J
= 8.5, H-6′) ppm; 13C NMR (125 MHz, CDCl3), δ (ppm), J (Hz) = 55.45,
0.98.72, 104.23, 104.82, 110.43, 112.74, 120.24, 122.65, 123.59,
127.93, 130.02, 152.42, 153.64, 157.76, 160.88 ppm. MS (EI, 70 eV):
m/z (%): 254 (100) [M]+, 239, 223, 192. Anal. calcd for C16H14O3: C,
75.57%; H, 5.55%; Found: C, 75.39%; H, 5.41%.
100
Subsequently, the cultures were treated with the compounds dis-
solved in DMSO, at 50 or 30 M concentration (0.5% or 0.3% DMSO/
100 L well) and incubated for 24 h. After this time, cell viability was
determined using MTT (5 mg/mL in Hank’s). 10 L of MTT solution
were added to each well containing 100 L of culture medium and the
cells were incubated for 2 h as above described. Then, culture medium
μL for 24 h under the conditions above described [28].
μ
μ
μ
2.2. Determination of human MAO (hMAO) isoforms activity
μ
The effects of the tested compounds on the enzymatic activity of
hMAO isoforms were evaluated by a fluorimetric method using an
Amplex Red MAO assay kit (InvitrogenTM) and MAO isoforms prepared
from insect cells (BTI-TN-5B1-4) infected with recombinant baculovirus
containing cDNA inserts from MAO-A or MAO-B (Sigma-Aldrich).
Briefly, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4) containing
the tested drugs in several concentrations and adequate amounts of re-
combinant hMAO-A or hMAO-B required and adjusted to obtain in our
experimental conditions the same reaction velocity [165 pmol of p-
tyramine/min (hMAO-A: 1.1 µg protein; specific activity: 150 nmol of p-
tyramine oxidized to p-hydroxyphenylacetaldehyde/min/mg protein;
hMAOB: 7.5 µg protein; specific activity: 22 nmol of p-tyramine trans-
formed/min/mg protein)] were placed in each well of a 96-well black
plate (MicrotestTM 96 well assay plate, BD Biosciences) and incubated
in the dark fluorimeter chamber for 15 min at 37 ◦C. The reaction was
started by adding (final concentrations) 200 µM Amplex Red reagent, 1
U/mL horseradish peroxidase and 1 mM p-tyramine. The production of
H2O2 and, consequently, of resorufin was quantified at 37 ◦C in a mul-
tidetection microplate fluorescence reader (FLX800TM, Bio-Tek In-
struments, Inc., Winooski, VT, USA) based on the fluorescence generated
was removed, 100 μL DMSO/well was added to solve the formazan
crystals formed by the viable cells and the absorbance (λ 540 nm) was
quantified in a plate reader. The viability (percentage) was calculated as
[Absorbance (treatment)/Absorbance (negative control)] 100% [29].
2.5. In silico prediction of ADME properties and metabolic stability
The Molinspiration Cheminformatics software (https://www.
molinspiration.com/) was used to calculate the physicochemical prop-
erties of the 2-phenylbenzofuran derivatives. The octanol/water parti-
tion coefficient (LogP), the polar surface area (TPSA), the number of
atoms and molecular weight (MW), the number of H-bond acceptors
(ON) and H-bond donors (OHNH), the volume (V) and the number of
rotatable links (rotb) were calculated, as well as the prediction of the
violations of Lipinski rules (n viol) [30]. These results together give an
idea of the potential bioavailability of these compounds.
To assess metabolism we performed an in silico prediction using the
MaxEfficience mode of GLORY system (https://acm.zbh.uni-hamburg.
de/glory/) which combines sites of metabolism prediction (SoM)
4

G.L. Delogu et al.
Bioorganic Chemistry 107 (2021) 104616
Scheme 1. Synthesis of 2-phenylbenzofuran derivatives via Wittig reaction. Reagents and conditions: a) NaBH4, EtOH, 0 ◦C to rt, 2 h; b) PPh3 HBr, CH3CN, 82 ◦C, 2
h; c) toluene, Et3N, 110 ◦C, 2 h.
performed with FAME2 and a collection of rules for metabolic reactions
mediated by the cytochrome P450 enzyme family. A priority score for
each predicted metabolite based on the SoM probability of the atoms
involved in the transformation and whether the reaction type is common
or not let to rank the predicted metabolites for a particular molecule
[31,32].
2.6. In vitro blood–brain barrier permeation assay (PAMPA-BBB)
Prediction of the brain penetration was evaluated using the PAMPA-
BBB assay, as previously described [33–37]. Solutions were added to 96-
well plates using a semi-automatic electronic pipette (CyBi®-SELMA)
and UV reading with a microplate spectrophotometer (Multiskan Spec-
trum, Thermo Electron Co.). Commercial drugs, phosphate buffered
saline solution at pH 7.4 (PBS), and dodecane were purchased from
Sigma, Aldrich, Acros, and Fluka. Millex filter units (PVDF membrane,
5

G.L. Delogu et al.
Bioorganic Chemistry 107 (2021) 104616
the active site was selected based on binding energy values that were
obtained from docking experiments.
Table 1
IC50 values and MAO-B selectivity index (SI) [IC50 (MAO-A)]/[IC50 (MAO-B)] for
the inhibitory effects of test drugs (new compounds and reference inhibitors) on
the enzymatic activity of human recombinant MAO isoforms expressed in
baculovirus infected BTI insect cells.
3. Results and discussion
Compounds
IC50 hMAO-B (
μ
M)
IC50 hMAO-A (
μ
M)
S.I.
3.1. Chemistry
1
> 100
> 100
—
2
0.534 ± 0.037
0.342 ± 0.018
15.64 ± 2.06
28.00 ± 3.18
> 100
1.69 ± 0.11
0.538 ± 0.11
> 100
3.16
Compounds 1–18 were efficiently synthesized by an intramolecular
Wittig reaction (Scheme 1). The desired Wittig reagent was readily
prepared from the conveniently substituted 2-hydroxybenzyl alcohol Ia-
c and triphenylphosphine hydrobromide (PPh3⋅HBr) [20–23]. The key
step for the formation of the benzofuran moiety was achieved by an
intramolecular reaction between 2-hydroxybenzyltriphosphonium salts
IIa-c [20,24,25], and the appropriate benzoylchloride.
3
1.57
4
> 6.39a
> 3.57a
—
5
> 100
6
> 100
7
9.72 ± 0.65
29.99 ± 3.68
0.168 0.011
> 100
0.02
8
> 3.33a
> 4,212a
—
9
0.024 0.003
> 100
> 100
10
> 100
The benzofuran structures were then confirmed by employing 1H
NMR, 13C NMR, mass spectrometry, and elemental analyses.
11
> 100
> 100
—
12
1.72 ± 0.27
> 100
0.184 0.028
> 100
0.11
13
—
14
0.551 ± 0.072
15.80 ± 0.38
0.620 ± 0.064
4.48 ± 0.20
4.29 ± 0.29
0.017 ± 0.002
61.35 ± 1.13
> 100
> 100
> 181.5a
6.33
3.2. Pharmacology
15
**
16
**
161.3
> 22.3a
4.23
3.2.1. MAO in vitro inhibition
17
> 100
The results of the hMAO-A and hMAO-B inhibition studies for com-
pounds 1–18 (IC50) and MAO-B selectivity index are reported in Table 1.
The test compounds did not show any interference with the reagents
used for the biochemical assay. Enzymatic assays revealed that most of
the test compounds were moderate to potent hMAO inhibitors at either
low micromolar to nanomolar concentrations.
18
18.12 ± 1.45
68.73 ± 4.20
0.001 ± 0.0003
361.38 ± 19.37
90 ± 2.47
selegiline
clorgyline
moclobemide
safinamide#
4,043
0.000016
< 3.62a
1,500
0.060 ± 0.005
Each IC50 value is the mean ± SEM of three experiments (n = 3). [a] Values
obtained under the assumption that the corresponding IC50 against MAO-A or
2-(n-Methoxyphenyl)benzofurans 15–18 resulted as potent and se-
lective MAO-B inhibitors. For these derivatives, a methoxyl substituent
at the meta position of the 2-phenyl ring is most favorable for the activity
than ortho substitution. Therefore, compound 16 is approximately 25
times more potent than 15. However, a double substitution at meta and
para positions (compound 17) leads to similar results than ortho and para
substitutions (compound 18) but major MAO-B selectivity.
MAO-B is the highest concentration tested (100 µM). ** 100 μM concentration
inhibits enzymatic activity by approximately 45–50%. Compounds precipitate at
higher concentrations. #Safinamide data from ref. [45]x
diameter 25 mm, pore size 0.45 μm) were acquired from Millipore. The
porcine brain lipid (PBL) was obtained from Avanti Polar Lipids. The
donor microplate was a 96-well filter plate (PVDF membrane, pore size
An additional substitution with a nitro group at 7 position on the
benzofuran moiety leads, in general, to decreased MAO-B activity. Thus,
compounds 15 and 17 resulted as more potent MAO-B inhibitors than 1
and 4, respectively. For these 2-(n-methoxyphenyl)-7-nitrobenzofurans,
the most active compound 3 links the methoxyl group at the para po-
sition. However, it shows a low selectivity. Besides, it is noteworthy that
the absence of methoxyl substituents on 2-phenyl of 7-nitrobenzofuran
leads to a potent and quite selective inhibitor of the MAO-A isoform,
compound 7, which is 2,151 times more potent than moclobemide.
Conversely, 2-phenyl-5-nitrobenzofuran (14) without methoxyl
substituents on the phenyl ring resulted in a potent and selective MAO-B
inhibitor. In general, 2-phenyl-5-nitrobenzofuran with only one
methoxyl substituent on the phenyl ring resulted as more potent MAO-B
inhibitors than the corresponding 2-phenyl-7-nitrobenzofuran. 2-(3-
Methoxylphenyl)-5-nitrobenzofuran (9) resulted as the most potent
MAO-B inhibitor in the studied series with a potency similar to
selegiline.
0.45 μm) and the acceptor microplate was an indented 96-well plate,
both from Millipore. The acceptor 96-well microplate was filled with
200
μL of PBS: EtOH (70:30) and the filter surface of the donor plate was
impregnated with 4
μ
L of PBL in dodecane (20 mg mLꢀ 1). Compounds
were dissolved in PBS: EtOH (70:30), filtered through a Millex filter, and
then added to the donor wells (200 μL). The donor filter plate was
carefully put on the acceptor plate to form a sandwich, which was left
undisturbed for 240 min at room temperature. After incubation, the
donor plate is carefully removed and the concentration of compounds in
the acceptor wells was determined by UV–Vis spectroscopy. Every
sample is analyzed at five wavelengths, in four wells and at least in two
independent runs, and the results are given as the mean ± SD. In each
experiment, 11 quality control standards of known BBB permeability
were included to validate the analysis set.
2.7. Molecular modeling studies
Increasing the number of methoxyl groups on the phenyl ring, we
noticed a decrease in the activity, which is most effective for 2-phenyl-5-
or 7-nitrobenzofurans than for 2-phenylbenzofurans. Substitution with
three methoxyl groups in para and in both meta positions on the 2-
phenyl ring in compounds 6 and 13, leads to loss of activity.
Also, it is important to point out that the presence of a nitro group at
5 position of the 2-(2,4-dimethoxyphenyl)benzofuran increases its
inhibitory activity against both MAO-B and MAO-A. However, a more
significant increase is noted against MAO-A. Furthermore, compound 12
is almost 100 times more potent as MAO-A inhibitor than compound 18.
The three-dimensional (3D) protein structures of human MAO-A
(PDB id: 2Z5X) [38], and human MAO-B (PDB id: 2 V61) [39] were
accessed from the protein data bank. The two-dimensional (2D) sketch
of the ligands (6, 7, 9, 12, and 13) was created using the Marvin JS tool
(www.chemaxon.com), and subsequently, the 3D-coordinates were
generated using Open Babel software [40]. Molecular docking studies
were performed using the COACH-D web-server [41], which employs
five individual methods to predict the position and conformation of the
ligands within the protein-binding site [42,43]. The refinement of the
predicted ligand-binding poses was then done using AutoDock Vina
[44].
3.3. Reversibility
The co-factor FAD in the X-ray structures of the protein was not
removed, and the active site was defined by the centroid of co-
crystalllized ligands that were already present respectively in the pro-
tein X-ray structures (2Z5X, 2 V61). The best pose of each compound in
Reversibility experiments were performed to evaluate the type of
inhibition exerted by derivatives 7, the most potent MAO-A inhibitor,
and 9, the most potent MAO-B inhibitor in this series. An effective
6

G.L. Delogu et al.
Table 2
Bioorganic Chemistry 107 (2021) 104616
hMAO-B inhibitors, respectively (Table 2). Although there is some
controversy about the most appropriate type of inhibition for MAO in-
hibitors, reversible inhibitors appear to have a better safety profile, and
they represent an alternative to the irreversible inhibitors currently in
therapeutic use.
Reversibility results of hMAO-A inhibition for compound 7
and hMAO-B inhibition for compound 9 and reference
inhibitors.
Compounds
Slope (ΔUF/t) [%][a]
7
48.40 ± 3.50
67.95 ± 2.75
9.50 ± 0.20
8.70 ± 0.58
9
3.4. Cytotoxicity
selegiline
clorgyline
[a]
The cytotoxic effects of compounds 1–18 were evaluated by using
Values represent the mean ± SEM of n = 2 experiments
relative to control; data show recovery after dilution of
hMAO-A activity incubated with compound 7 or clorgyline
and hMAO-B activity incubated with compound 9 or
selegiline.
the human neuroblastoma cell line SH-SY5Y. Cells were treated with the
compounds at 50 μM concentration and incubated for 24 h. Afterward,
the percentage of cell viability was measured as MTT reduction [29].
As depicted in Fig. 1, most of the 2-phenylbenzofuran derivatives
evaluated lack cellular toxicity at 50 μM concentration. Only the com-
pounds 14 and 18 appreciably reduced the viability of the SH-SY5Y cells
at this concentration. This cytotoxicity disappears with decreasing
concentration. Thus, at 30 μM concentration, which is higher than that
inhibiting MAO-B in vitro, the viability of the cells after treatment with
compounds 14 and 18 was 99.0 ± 6.6 and 84.3 ± 5.6%. At this con-
centration, no statistically significant differences with the viability of
the control were observed (P < 0.05).
3.5. In silico and in vitro prediction of ADME properties
In silico calculations using the Molinspiration Cheminformatics
software (https://www.molinspiration.com/) showed that all the 2-phe-
nylbenzofurans studied have adequate physicochemical properties to
display good bioavailability, and none of them violate the Lipinski rule
of five (Table 3). The main differences between our derivatives and the
reference inhibitors are noted in the LogP and TPSA values. In general,
LogP is much higher concerning the reference compounds moclobemide,
selegiline and safinamide but comparable to clorgyline. For compounds
linking a nitro group, the value of TPSA is comparable to that of the
reference inhibitors moclobemide or safinamide.
Fig. 1. Cytotoxic activity of compounds 1–18 after 24 h incubation in SH-SY5Y
cells. Cell viability was measured as MTT reduction and data were normalized
as % of control. Results are expressed as mean ± SEM from at least 3 different
cultures. *P ≤ 0.05 versus the cells treated with DMSO (0.5%). Comparisons
were performed by one-way ANOVA followed by the Dunnett’s test.
dilution method was used [27], and selegiline (irreversible MAO-B in-
hibitor) and clorgyline (irreversible MAO-A inhibitor) were taken as
standards. Compounds 7 and 9 resulted as reversible hMAO-A and
Moreover, the ability of new 2-phenylbenzofuran derivatives to cross
the blood–brain barrier (BBB) was predicted by the in vitro parallel
artificial membrane permeability assay for the BBB (PAMPA-BBB),
Table 3
Structural properties of 2-phenylbenzofuran derivatives 1–18, and reference compounds.
Compounds
Log Pa
TPSA (Å2)b
MW (Da)c
nOHd
nOHNHd
Volumee
Lipinskif
1
4.10
4.12
4.15
3.74
4.13
3.72
4.09
4.12
4.05
3.76
4.16
4.16
3.75
4.12
4.19
4.21
3.83
4.22
3.74
1.69
2.64
2.91
68.28
68.20
68.20
77.43
77.43
86.67
58.96
68.20
104.79
77.43
77.43
77.43
86.67
58.96
22.37
22.37
31.61
31.61
12.47
41.57
3.24
269.26
269.26
269.26
299.28
299.28
329.31
239.23
269.26
269.26
299.28
299.28
299.28
329.31
239.23
224.26
224.26
254.28
254.28
272.18
268.74
187.29
320.35
5
5
5
6
6
7
4
5
5
6
6
6
7
4
2
2
3
3
2
4
1
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
3
229.89
229.89
229.89
255.44
255.44
280.98
204.35
229.89
229.89
255.44
255.44
255.44
280.98
204.35
206.56
206.56
232.10
232.10
238.91
240.70
202.64
279.04
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
clorgyline
moclobemide
selegiline
safinamide
64.36
a
Log P – expressed as the logarithm of octanol/water partition coefficient.
TPSA – topological polar surface area.
b
c
d
e
f
MW – molecular weight.
Number of hydrogen bond acceptors (nOH) and donors (nOHNH).
Molecular volume.
Number of violations of Lipinski’s rules.
7

G.L. Delogu et al.
Bioorganic Chemistry 107 (2021) 104616
little solubility and/or too low UV-response, and therefore, they could
not be tested. In each experiment, 11 commercial drugs of known brain
permeability were also tested and their permeability values normalized
to the reported PAMPA-BBB data. As previously established by Di et al.
[33], compounds with Pe > 4.0⋅10-6 cm⋅sꢀ 1 would cross the BBB (cns + ),
whereas those displaying Pe < 2.0⋅10-6 cm⋅sꢀ 1 would not reach the CNS
(cns-). Between these values, the predicted CNS permeability was un-
certain (cns +/-) [33]. As shown in table 4, the 2-phenylbenzofuran
derivatives 3–7 and 17 were predicted to be CNS-permeable, whereas
16 and 18 could experience difficulties crossing the BBB.
Table 4
CNS-Permeability prediction based on the PAMPA-BBB assay (Pe, 10-6 cm sꢀ 1).
Compounds
Pe (10-6 cm sꢀ 1
)
CNS Prediction
3
8.8 ± 0.16
9.3 ± 0.27
10.2 ± 0.05
7.1 ± 0.71
8.6 ± 0.02
0.7 ± 0.05
9.4 ± 0.01
2.1 ± 0.08
CNS +
CNS +
CNS +
CNS +
CNS +
CNS -
4
5
6
7
16
17
18
CNS +
CNS + / -
aResults are the mean ± SD of at least two independent experiments, where
every sample was analyzed at five wavelengths in four different wells.
3.6. In silico metabolism
With the aim of advancing further on the potential therapeutic suc-
cess of these series of 2-phenylbenzofuran derivatives we perform an in
silico prediction of the metabolism for the most potent compounds 7, 9
and 16 corresponding to different series. GLORY combines SoM pre-
diction with FAME2 and a new collection of rules for metabolic reactions
mediated by the cytochrome P450 enzyme family. The importance of
CYPs to drug discovery is clear from the observation that many drugs are
described by Di et al. [33] and then adapted by us for testing molecules
with limited water-solubility [34–37]. To measure the passive CNS-
permeation of new compounds we selected a mixture of PBS:EtOH
(70:30) as solvent. In these conditions, the 2-phenylbenzofuran de-
rivatives 3–7 and 16–18 gave solutions with a suitable UV-signal to be
detected in the experiment, while the rest of the compounds showed
Table 5
The most possible metabolites for compounds 7,9 and 16.
Comp.
structure
score
Comp.
Structure
score
Comp.
structure
score
7
9
16
M7.1
M7.2
M7.3
2.43
2.04
1.92
M9.1
M9.2
M9.3
4.38
4.38
1.66
M16.1
M16.2
M16.3
4.34
4.34
2.92
OCH2OH
O2
N
O
M7.4
M7.5
1.87
1.84
M9.4
M9.5
1.61
1.43
M16.4
M16.5
2.51
2.32
Fig. 2. Structures of human MAO complexes with top ranked ligands. (A) hMAO-B in complex with compound 9 (C-9), and (B) hMAO-A complex with compound 7
(C-7). The binding site of the ligands is represented as the region enclosed within the pink oval. The residues forming aromatic cage are shown as green circle, and the
residue determining ligand specificity is shown as black-filled circle. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
8

G.L. Delogu et al.
Bioorganic Chemistry 107 (2021) 104616
inhibitory profile against the two investigated enzymes (hMAO-B,
hMAO-A). The most significant difference between the two enzymes can
be noted in the structure of their active site cavities; a single cavity (with
~ 500 Å3 volume) in hMAO-A, while a bipartite cavity (entrance ~ 300
Å3 volume, substrate cavity ~ 400 Å3 volume) in hMAO-B.
Table 6
Predicted binding free energy (ΔG) for the ligands under investigation. The
ligand with better binding energy and (IC50) value are highlighted in bold.
Ligand
IC50
(μ
M)
ΔG (kcal/mol)
MAO-B
MAO-A
MAO-B
MAO-A
The computational docking results for ligands studied revealed that
they all bind non-covalently to the enzyme. The docked conformation of
the top-ranked ligand complexes superimposed nicely with the X-ray
structure ligands (supplementary information Fig. S1). The binding free
energies of investigated ligands against the two enzymes are shown
Table 6.
6
> 100
9.72
> 100
0.168
>100
0.184
> 100
68.73
0.001
361
ꢀ 7.1
ꢀ 7.6
¡8.7
ꢀ 7.5
ꢀ 6.2
ꢀ 7.1
ꢀ 7.3
ꢀ 7.2
ꢀ 0.4
¡8.2
ꢀ 5.9
ꢀ 7.9
ꢀ 0.3
ꢀ 6.8
ꢀ 7.2
ꢀ 8.1
7
9
0.024
1.72
12
13
> 100
0.017
61.35
> 100
selegiline
clorgyline
moclobemide
Among the 2-phenylbenzofuran derivatives, compound 9 displayed
better binding affinity to hMAO-B, while compound 7 to hMAO-A. The
docking results were in good agreement with the experimental data
(Table 6). Furthermore, the binding free energy trend among the ligands
for both the proteins was consistent with the noted experimental trend
(IC50) values.
metabolized by CYPs. Results of GLORY suggest the most probably sites
of metabolism (SoM). The predicted metabolites for the most active
compounds in each series ranked by empirical probability scores are
described in table 5.
The top-ranked docked conformation of compound 9 bound to
hMAO-B protein (Fig. 3A, Fig. 3B), revealed hydrophobic interactions
between the nitro group (in position 5 of scaffold) and residues Tyr 398
and Tyr 435. These two residues, together with FAD, form an aromatic
cage in the substrate cavity region [49]. Further, we also found phenyl
ring of ligand interacting with residues Tyr 326 and Ile 199: the former
known to be involved in substrate specificity [4], and the latter acting as
gate [50] between the entrance and substrate-binding cavities of hMAO-
3.7. Molecular docking
Molecular docking is an important tool used in drug discovery, which
has been successfully applied to identify promising inhibitors against
protein complexes associated to various diseases [46–48]. Here, mo-
lecular docking studies for the different ligand–protein complexes
(Fig. 2) were performed to provide insights into ligand’s selective
Fig. 3. Zoomed view of predicted docked positions for the top ligands bound to the MAO protein. In (A), and (B) the docked position and interacting picture of
compound 9 bound to hMAO-B protein is shown. In (C), and (D) is shown the docked position and interacting picture of compound 7 bound to hMAO-A. In (B), and
(D) the polar residues are shown in parrot green, and polar residues in cyan. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
9

G.L. Delogu et al.
Bioorganic Chemistry 107 (2021) 104616
´
B protein. The methoxy substituent of the ligand was found to be
involved in hydrophobic interactions with residues Leu 164, Leu 167,
and Phe 168. Compound 12, which shares the same nitro group position
as compound 9, but has additional methoxy substituent, resulted in a
similar docked orientation but was more distant from the aromatic cage
residues (supplementary information Fig. S2).
(Centro singular de investigacion de Galicia accreditation 2019-2022)
and the European Union (European Regional Development Fund -
ERDF), is gratefully acknowledged. The present work was partially
`
supported by FIR (Fondo Integrativo per la Ricerca – annualita 2018)
University of Cagliari. G. L. Delogu is grateful to R. Mascia (University of
Cagliari) for his technical assistance. M.I.R.-F. thanks the financial
support from Spanish Ministry of Science, Innovation and Universities
(grant RTI2018-093955-B-C21) and General Council for Research and
Innovation of the Community of Madrid / European Structural Funds
(grant B2017/BMD-3827 - NRF24ADCM).
Concerning the hMAO-A protein and among the studied derivatives,
compound 7 displayed better binding energy (Table 4), and the top-
ranked docked conformation shown in Fig. 3C and 3D. The nitro
group of compound 7 was involved in hydrophobic interaction with Tyr
407 (aromatic cage), Asn 181, and Gln 215. Further, the phenyl-ring of
compound 7 formed strong hydrophobic interactions with residues Ile
180, Phe 208, Cys 323, Ile 335, and Leu 337. Indeed, the critical role of
residue Ile 335 in determining substrate and inhibitor specificities have
been reported previously [4].
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104616.
Compound 7 (Fig. 3D) differs from 9 (Fig. 3B) in the nitro group
position and also lacks methoxy substituent in the phenyl-ring. The
compounds 12 and 9 resulted in a docked orientation with the methoxy
group towards the aromatic cage, and nitro group at the other end
(supplementary information Fig. S3). The docking results suggest that
interaction of the nitro group with the aromatic cage residues can be the
reason for better affinity displayed by compound 7, concerning the other
ligands. Further the docking results of ligands with three methoxy
substituents in the 2-phenyl ring (compound 6, compound 13) indicated
unfavourable binding with the hMAO-A enzyme. The reason for
unfavourable binding could be related unfavorable contacts displayed
by methoxy substituents with key residues: Phe 352, Ile 335, and Gln
215. Indeed, this observation is consistent with a previous docking study
[51], wherein the addition of other methoxy groups in the ligand
resulted in unfavourable binding characteristics to hMAO-A enzyme. In
summary, docking results were consistent with the reported experi-
mental IC50 values for the 2-phenylbenzofuran derivatives. Further-
more, molecular insights into ligand interaction with key residues of the
protein facilitating the formation of stable protein–ligand complexes
have been illustrated.
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