Looking for new xanthine oxidase inhibitors: 3-Phenylcoumarins versus 2-phenylbenzofurans

Article abstract of DOI:10.1016/j.ijbiomac.2020.06.152

Overproduction of uric acid in the body leads to hyperuricemia, which is also closely related to gout. Uric acid production can be lowered by xanthine oxidase (XO) inhibitors. Inhibition of XO has also been proposed as a mechanism for improving cardiovascular health. Therefore, the search for new efficient XO inhibitors is an interesting topic in drug discovery. 3-Phenylcoumarins and 2-phenylbenzofurans are privileged scaffolds in medicinal chemistry. Their structural similarity makes them interesting molecules for a comparative study. Methoxy and nitro substituents were introduced in both scaffolds. The current study gives some insights into the synthesis and biological activity of these molecules against this important target. For the best compound of the series, the 3-(4-methoxyphenyl)-6-nitrocoumarin (4), the IC₅₀ value, type of inhibition, cytotoxicity on B16F10 cells and ADME theoretical properties, were determined. Docking studies were also performed in order to better understand the interactions of this molecule with the XO binding pocket. This work is a preliminary screening for further design and synthesis of new non-purinergic derivatives as potential compounds involved in the inflammatory suppression, specially related to gout.

Full text of DOI:10.1016/j.ijbiomac.2020.06.152

Journal Pre-proof
Looking for new xanthine oxidase inhibitors: 3-Phenylcoumarins
versus 2-phenylbenzofurans
Benedetta Era, Giovanna L. Delogu, Francesca Pintus, Antonella
Fais, Gianluca Gatto, Eugenio Uriarte, Fernanda Borges, Amit
Kumar, Maria J. Matos
PII:
S0141-8130(20)33593-5
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.06.152
BIOMAC 15909
Reference:
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
21 February 2020
2 April 2020
17 June 2020
Please cite this article as: B. Era, G.L. Delogu, F. Pintus, et al., Looking for new xanthine
oxidase inhibitors: 3-Phenylcoumarins versus 2-phenylbenzofurans, International Journal
of Biological Macromolecules (2020), https://doi.org/10.1016/j.ijbiomac.2020.06.152
This is a PDF file of an article that has undergone enhancements after acceptance, such
as the addition of a cover page and metadata, and formatting for readability, but it is
not yet the definitive version of record. This version will undergo additional copyediting,
typesetting and review before it is published in its final form, but we are providing this
version to give early visibility of the article. Please note that, during the production
process, errors may be discovered which could affect the content, and all legal disclaimers
that apply to the journal pertain.
© 2020 Published by Elsevier.

Journal Pre-proof
Looking for new xanthine oxidase inhibitors:
3-Phenylcoumarins versus 2-phenylbenzofurans
Benedetta Era,1,# Giovanna L. Delogu,1,# Francesca Pintus,1 Antonella Fais,1
Gianluca Gatto,2 Eugenio Uriarte,3,4 Fernanda Borges,5 Amit Kumar2 and
Maria J. Matos3,5,*
1
Department of Life and Environmental Sciences, University of Cagliari,
09042 Monserrato, Cagliari, Italy
2
Department of Electrical and Electronic Engineering, University of Cagliari,
09123, Via Marengo 2, Cagliari, Italy
3
Departamento de Química Orgánica, Facultade de Farmacia, Universidade
de Santiago de Compostela, 15782, Santiago de Compostela, Spain
4
Instituto de Ciencias Químicas Aplicadas, Universidad Autónoma de Chile,
7500912 Santiago, Chile
5
CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences,
University of Porto, 4169-007 Porto, Portugal
* Corresponding author: Maria J. Matos (maria.matos@fc.up.pt or
mariajoao.correiapinto@usc.es)
# These authors contributed equally to this work

Journal Pre-proof
Abstract
Overproduction of uric acid in the body leads to hyperuricemia, which is also
closely related to gout. Uric acid production can be lowered by xanthine
oxidase (XO) inhibitors. Inhibition of XO has also been proposed as a
mechanism for improving cardiovascular health. Therefore, the search for new
efficient XO inhibitors is an interesting topic in drug discovery. 3-
Phenylcoumarins and 2-phenylbenzofurans are privileged scaffolds in
medicinal chemistry. Their structural similarity makes them interesting
molecules for a comparative study. Methoxy and nitro substituents were
introduced in both scaffolds. The current study gives some insights into the
synthesis and biological activity of these molecules against this important
target. For the best compound of the series, the 3-(4-methoxyphenyl)-6-
nitrocoumarin (4), the IC50 value, type of inhibition, cytotoxicity on B16F10
cells and ADME theoretical properties, were determined. Docking studies
were also performed in order to better understand the interactions of this
molecule with the XO binding pocket. This work is a preliminary screening for
further design and synthesis of new non-purinergic derivatives as potential
compounds involved in the inflammatory suppression, specially related to gout.
Keywords: 3-Phenylcoumarins; 2-Phenylbenzofurans; Xanthine oxidase
inhibitors

Journal Pre-proof
1. Introduction
Xanthine oxidase (XO, EC 1.2.3.2) is a form of molybdoflavin protein that
plays an important role at the end of the catabolic sequence of the purine
nucleotide metabolism in humans and a few other uricotelic species [1]. It
firstly catalyzes the oxidation of hypoxanthine to xanthine, and then
transforms it into uric acid, which is excreted in the urine [2]. This process
−
⋅
reduces molecular oxygen to O2 . Hydroxyl free radicals and hydrogen
peroxide, both of which are byproducts of XO activity, can cause oxidative
stress in human cells.
Overproduction or under excretion of uric acid in humans can lead to
hyperuricemia and gout, which is caused by crystallization and deposition of
uric acid in joints and surrounding tissue [3]. Gout is a metabolic disorder
associated with abnormal amounts of uric acid in the body, which causes
inflammation, gouty arthritis, and uric acid nephrolithiasis. Recent studies
have indicated that asymptomatic hyperuricemia is associated or may have a
causal relationship with cardiovascular diseases [4].
Allopurinol (1H-pyrazol[3,4-d]pyrimidin-4-ol, Fig. 1) is a substrate and specific
potent inhibitor of XO, and has been used for gout treatment for several years
[ 5 ]. Different studies have indicated that allopurinol may induce
hypersensitivity syndrome and Stevens-Johnson syndrome in patients [6,7,8].
More recently, febuxostat [2-(3-cyano-4-isobutoxyphenyl)-4-methyl-1,3-
thiazole-5-carboxylic acid, Fig. 1], a new non-purine XO inhibitor, has been
approved for the treatment of gout in the European Union and USA [5,9].
Many side effects of febuxostat have been reported [10].
H
O
N
O
N
N
O
S
HN
N
N
OH
Allopurinol
Febuxostat
Fig. 1. Chemical structure of allopurinol (reference compound) and febuxostat.
In this study, we intended to develop XO inhibitors with potent activity and low
toxicity, using compounds with a chemical structure distinct from allopurinol

Journal Pre-proof
and febuxostat, in order to increase the chemical spectrum of molecules
acting on this target. Therefore, XO inhibitory activity of two different scaffolds
was evaluated and compared with allopurinol.
2. Materials and methods
2.1. Synthesis
2.1.1. General
All reagents were purchased from Sigma-Aldrich and used without further
purification. All solvents were commercially available grade. Organic solutions
were dried over anhydrous Na2SO4. All reactions were carried out under
argon atmosphere, unless otherwise mentioned. Concentration and
Reaction mixtures were purified by flash column chromatography using Silica
Gel high purity grade (Merck grade 9385 pore size 60Å, 230-400 mesh
particle size). Reaction mixtures were analyzed by analytical thin-layer
chromatography (TLC) using plates precoated with silica gel (Merck 60 F254,
0.25 mm). Visualization was accomplished with UV light (254 nm) or
potassium permanganate (KMnO4).
2.1.2. General procedure for the preparation of 3-arylcoumarins 1-4. In a 20
mL dry Schlenk tube, to a solution of the conveniently substituted
salicylaldehyde (2.46 mmol) and the arylacetic acid (2.46 mmol), in acetic
anhydride (6 mL), NaH (2.46 mmol) was added in small portions, and the
reaction mixture was stirred for 3 hours, at room temperature. The obtained
crude was filtered and washed with diethyl ether. The obtained solid was then
purified by flash chromatography (hexane/ethyl acetate 9:1) to give the
desired coumarins 1-4.
2.1.3. General procedure for the preparation of 3-arylcoumarins 5 and 6. To a
20 mL two neck round bottomed flask was added a solution of 3-
chlorocoumarin (0.83 mmol), arylboronic acid (1.04 mmol), Na2CO3 (1.66
mmol), and Pd-salen complex (0.5 mol %), in DMF/H2O (1:1). The reaction
mixture was heated at 110 C for 120–180 minutes. The reaction was

Journal Pre-proof
monitored by chromatography. After the completion of the reaction, the
mixture was extracted with ethyl acetate (3x20 mL). The organic extracts were
dried over anhydrous sodium sulphate, filtrated and evaporated under
vacuum. The obtained solid was purified by column chromatography
(hexane/ethyl acetate 9:1) to give the coumarins 5 and 6.
2.1.4. General procedure for the preparation of 2-hydroxybenzylalcohols Ia
and Ib. Sodium borohydride (6.60 mmol) was added to a stirred solution of 2-
hydroxybenzaldehyde (6.60 mmol), in ethanol (20 mL), in an ice bath. The
reaction mixture was stirred at room temperature for 1 hour. 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 evaporated under
vacuum to give the desired compounds Ia and Ib.
2.1.5.
General
procedure
for
the
preparation
of
2-
hydroxybenzyltriphenylphosphonium bromide IIa and IIb. A mixture of 2-
hydroxybenzylalcohol (16.27 mmol) and PPh3·HBr (16.27 mmol) in CH3CN
(40 mL) was stirred under reflux for 2 hours. The obtained solid was filtered
and washed with CH3CN to give the desired compounds IIa and IIb.
2.1.6. General procedure for the preparation of 2-phenylbenzofuran 7-12. 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 hours. 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 7-12.
2.2. Biological assays
2.2.1. XO inhibition
The biological assays were carried out following the protocol described below
[11]. The reaction mixture contained 0.1 M of a phosphate buffer solution (pH
7.5), an aqueous solution of XO (0.5 U/mL, Sigma Chemical Co) and 20 μL
DMSO, with or without the compound, at different doses. After mixing, a 0.82
mM of xanthine solution was added and the activity of the XO was determined

Journal Pre-proof
spectrophotometrically (Varian Cary 50) by measuring the formation of uric
acid at 295 nm, for 5 minutes, at 37 C. The percent of XO activity inhibition
was calculated as: inhibition (%) = (A–B)/A x 100%, where A represents the
difference in the absorbance of control sample between 0.5 and 1.0 minute,
and B represents the difference in absorbance of the test sample between 0.5
and 1.0 minute. The IC50 value, a concentration giving 50% inhibition of XO
activity, was determined by interpolation of dose-response curves. Allopurinol
was used as reference. All experiments were carried out three times.
Continuous variables with normal distribution were presented as mean ± SD.
T fi 2% DMSO / z m
activity. The mode of inhibition of the enzyme was performed using the
Lineweaver-Burk plot. Enzyme kinetics were determined in the absence and
presence of different concentrations of the tested samples (2.5, 5.0 and 7.5
µM), as well as different xanthine concentrations (16.6, 20, 25 and 50 µM) as
substrate.
2.2.2. Cytotoxicity
B16F10 mouse melanoma cells (CRL-6475) were purchased from the
American Type Culture Collection (ATCC, Manassas, VA, USA). The cells
D b ’ M E M m DMEM m
with 10% fetal bovine serum (FBS, Gibco, NY, USA), and 1%
penicillin/streptomycin at 37 C in a humidified atmosphere with 5% CO2. The
cytotoxic effect of compound 4 was evaluated on B16F10 cells using the 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [12].
Briefly, cells were seeded in a 96-well plate (5x103 cells/well) and incubated
for 48 hours, at 37 C, with the samples at different concentrations from 10 to
150 µM. As DMSO was used to dissolve the compounds, all the activities
were measured in the presence of DMSO (0.5%), as solvent control. After the
incubation time, cells were labelled with MTT solution for 3 hours at 37 C.
The resulting violet formazan precipitates were dissolved in DMSO and color
development was measured at 570 nm, using a microplate reader (Infinite 200,
Tecan, Austria). The absorbance is proportional to the number of viable cells.

Journal Pre-proof
2.3. Molecular Docking
The crystal structure (PDB ID: 1FIQ) of XO [13] and the three-dimensional
structure of the ligands, obtained using open babel software [14], were used
for the computational study. The structure of the ligands was geometry
optimized using quantum chemistry calculations [ 15 ]. The protein-ligand
docking was performed using the COACH-D server [16], which predicts ligand
poses by using a consensus of five methods. The final step in the protocol
[17] consisted of docking the ligand into the predicted binding pockets to build
their complex structures, employing the molecular docking algorithm
AutoDock Vina [18]. For each predicted binding pocket, the best match to the
consensus prediction of the binding residues was then selected.
3. Results and discussion
3.1. Synthesis
Based on previous findings in the field [19], and in our experience with
substituted 3-phenylcoumarins and 2-phenylbenzofurans, in the present work
we propose the synthesis (Scheme 1), XO inhibition evaluation (Table 1 and
Fig. 2), cytotoxicity evaluation (Fig. 3) and a comparative study of a series of
different substituted compounds from both families. With the aim of finding the
structural features for the biological activity, we decided to explore the
importance of the nature and position of small groups (methoxy and nitro
substituents) into both scaffolds (Scheme 1).
The derivatives 1-4 [20,21,22] were efficiently synthesized by a condensation
reaction in a dry Schlenk tube, in presence of sodium hydride and with acetic
anhydride as solvent, at room temperature, for three hours. The reaction
mixture was purified by flash chromatography, using hexane/ethyl acetate as
eluent, in a proportion of 9:1. Starting from the commercially available
salicylaldehydes and the respectively substituted arylacetic acids, we
obtained four derivates in good yields (60–75%).
The derivatives 5 [23] and 6 [23] were efficiently synthesized by a direct
cross-coupling reaction, in presence of sodium carbonate (Na₂CO₃), N,N’-
bis(salicylidene)-ethylenediamino-palladium (salen-Pd), in DMF/H2O (1:1), at
110 C, for 120-180 minutes. The reaction mixture was purified by flash

Journal Pre-proof
chromatography, using hexane/ethyl acetate as eluent, in a proportion of 9:1.
Starting from the 3-chlorocoumarin and the respectively substituted
arylboronic acids, we obtained two derivates in good yields (56 and 64%).
Compounds 7-12 [24,25,26,27,28,29] were prepared via a Wittig reaction,
according to the protocol outlined in Scheme 1. The desired Wittig reagents
were readily prepared from the conveniently substituted 2-hydroxybenzyl
alcohols Ia and Ib and PPh3·HBr, in acetonitrile, at 82 C, for two hours. The
key step for the formation of the benzofuran moiety was achieved by an
intramolecular reaction between the 2-hydroxybenzyltriphosphonium salt IIa
and IIb and the appropriate benzoyl chloride, in toluene, at 110 C, for 2 hours,
to give the six desired derivates in good yields (40-96%).

Journal Pre-proof
Scheme 1. Synthetic methodology to prepare compounds 1-12. Reagents
and conditions: a) NaH (1.0 equiv), acetic anhydride, r.t., 3 h; b) Na₂CO₃ (2.0
equiv), palladium complex (0.5 mol %), DMF/H2O (1:1), 110 C, 120-180 min;
c) NaBH4, EtOH, 0 C to RT, ~2 h; d) PPh3·HBr, CH3CN, 82 C, 2 h; e)
toluene, Et3N, 110 C, 2 h.
3.2. Enzyme inhibitory studies
The inhibitory effect of compounds 1-12 on XO activity was determined
spectrophotometrically by monitoring the formation of uric acid at 295 nm.
Bovine milk xanthine oxidase (1332 residues), used in our enzymatic assay, is
90% homologous to human liver enzyme (1333 residues). Six 3-
phenylcoumarins and six 2-phenylbenzofurans were evaluated for their ability
to inhibit XO, and an overview of the effects is summarized in Table 1.
Table 1. XO inhibitory activity % I b 100 μM IC50) of 3-
phenylcoumarins, 2-phenylbenzofurans and allopurinol. Type of inhibition for
compound 4 and allopurinol, the reference compound.
1-6
Inhibitory
activity
(Inhibition % ±
SD)
Type of
inhibition
Compound
R1
R2
R3
IC50 (μM) ± SD
1
2
3
4
5
H
H
H
H
H
75.4 ± 0.2
19.2 ± 0.7
76.6 ± 1.6
85.1± 3.2
40.4 ± 4.0
-
NO2
-
NO2 OMe
H
-
NO2
H
H
OMe
H
8.4 ± 1.7
-
uncompetitive
OMe

Journal Pre-proof
6
H
H
OMe
22.5 ± 1.2
-
7-12
Inhibitory
activity
(Inhibition % ±
SD)
Type of
inhibition
Compound
R1
R2
R3
IC50 (μM) ± SD
7
H
H
H
H
H
H
26.3 ± 3.6
-
8
NO2
NO2
*
-
9
OMe
H
36.5 ± 0.9
-
-
10
NO2 OMe
NI
*
11
12
H
H
H
OMe
H
-
OMe
NI
-
Allopurinol [30]
0.1
competitive
* At 100 µM (highest concentration tested) the compounds precipitated.
NI = No inhibition observed at 100 µM.
3-(4-Methoxyphenyl)-6-nitrocoumarin (4) proved to be the best candidate of
the series. Therefore, the IC50 of this compound was calculated (IC50 = 8.4 μM,
Table 1). A kinetic study of this compound was also performed to understand
the mechanism of enzymatic inhibition. The inhibitory binding mode was
studied by using Lineweaver-Burk double reciprocal plots and the results are
presented in Fig. 2. Analyzing the chart, plots of 1/V versus 1/[S] gave a
family of parallel lines for increasing concentration. This behavior indicates
that compound 4 interacts with the enzyme in an uncompetitive way. The type
of inhibition is different to the reference compound, allopurinol (Table 1).

Journal Pre-proof
12
10
8
6
4
2
0
-0.08 -0.04
0
0.04
0.08
1/Xanthine
Fig. 2. Lineweaver-Burk plots for the XO inhibition caused by compound 4.
Concentrations of compound 4 were 0 ● , 2.5 ○ , 5.0 □ and 7.5 ■ μM.
3.3. Cytotoxicity assay analysis
After obtaining encouraging results from the previous experiments, biosafety
effectiveness of the promising compound 4 was further evaluated, and the
results represented in Fig. 3.
Fig. 3. The effect of compound 4 on B16F10 cell viability. Cells were treated
with different concentrations of compound 4 (10-150 μM) and their viability
was evaluated by MTT assay.

Journal Pre-proof
The results obtained support the fact that compound 4 is non-cytotoxic on
B16F10 cells at the concentration in which the XO activity is inhibited (IC50 =
8.4 µM).
3.4. Molecular and physicochemical properties of compounds
To better correlate the drug-like properties of the best compound (4),
theoretical calculations were performed. For comparison, calculations were
also performed for the 2-(4-methoxyphenyl)-5-nitrobenzofuran (10, 2-
phenylbenzofuran with same substitution pattern of 4) and allopurinol, the
reference compound. Physicochemical properties, such as lipophilicity,
expressed as the octanol/water partition coefficient, topological polar surface
area (TPSA), molar refractivity (steric factor), number of hydrogen acceptors,
number of hydrogen bond donors, volume, rotatable bonds and water
solubility were calculated using Swiss ADME web tool [31] and Molinspiration
program [32]. TPSA is commonly used as medicinal chemistry metric for the
m z ’ b m the desired
target. The obtained in silico results are summarized in Table 2.
Table 2. Physicochemical properties of compounds 4 and 10, and allopurinol,
used as reference.
Compound 4
Compound 10
Allopurinol
Volume (Å3)
248.88
229.89
108.68
Molar Refractivity
(m3mol-1)
83.23
76.96
34.51
Rotatable bonds
H-bond acceptors
H-bond donors
3
5
0
3
3
0
0
3
2

Journal Pre-proof
Polar surface
area (Å2)
85.26
68.19
4.02
74.44
0.06
Lipophilicity
(WLogP)
3.38
Moderate
Yes
Water solubility
Moderate
Yes
Very soluble
Yes
Drug likeness
(Lipinski)
Subsequently, utilizing the lipophilicity and TPSA descriptors, two crucial
pharmacokinetic parameters namely gastrointestinal absorption (GA) and
blood-brain barrier (BBB) were predicted using the novel Brain Or
IntestinaLEstimateD permeation (BOILED-Egg) method (Fig. 4).
Fig. 4. BOILED-Egg construction plot for compounds 4 and 10, and
allopurinol, using WLogP and TPSA parameters. The white region represents
the physicochemical space of GA and the yellow region of BBB.
It is interesting to note that the promising compound 4 and allopurinol share
the physiochemical space that represents high probability of absorption in the
gastrointestinal tract. Since the highest concentration of XO in humans is

Journal Pre-proof
present in the intestinal and liver tracts, GA is an important parameter that
should be considered for the identification of lead molecules.
To better understand the uncompetitive interaction displayed by compound 4
and XO binding pocket residues, molecular docking calculations were
performed for the protein-ligand complexes (Fig. 5). Since, compound 10
presents the same substitution pattern (Fig. 5C) as compound 4 (Fig. 5B), for
comparison, even if compound 10 proved to be experimentally non-active,
calculations were performed for both molecules.
Fig. 5. Modeling XO–compound complexes. (A) XO protein structure shown
using surface and cartoon, ligand (as licorice) and binding region highlighted
with dashed circle representation. (B) Zoomed view of predicted ligand
binding site for compound 4. (C) Zoomed view of predicted ligand binding site
for compound 10.
The docking results indicated a better binding energy for compound 4 (-8.8
kcal/mol), while compound 10 displayed a lower value of -7.5 kcal/mol. The
binding sites for the compounds 4 and 10, and the amino acid residues
responsible for the interactions, are shown in detail in Fig. 6.

Journal Pre-proof
Fig. 6. Predicted docked conformation of compound 4 and 10 bound on XO
binding pocket. Hydrophobic residues are in green, positive charged residues
in indigo, while polar residues are represented in light blue.
The binding site for the two compounds is quite similar. However, they differ in
the orientation/positioning of the methoxy group. Indeed, the
orientation/positioning of methoxy group of compound 4 in the binding site
, π-stacking interaction (T-type) with
residue Phe798, cation–π -bridge interaction involving the
Arg912 residue. Therefore, resulting in a better docking energy with respect to
compound 10. Moreover, the residues involved in the interaction with the
compounds are different from the catalytic important residues (Glu802,
Arg880, Glu1261) present in the active site of XO protein, supporting the
uncompetitive inhibition characteristic of compound 4.
4. Conclusions
In summary, we investigated the XO inhibitory activity of twelve compounds,
and found eight active molecules, displaying three of them inhibitory activity
above 75%, at 100 µM. All the 3-phenylcoumarins proved to be active on the
studied target. The most active molecule –3-(4-methoxyphenyl)-6-
nitrocoumarin (4)– presented an IC50 of 8.4 μM XO, b
enzyme in an uncompetitive way. Our results highlighted a relationship
between the scaffold, the nature of the substituents and their position within

Journal Pre-proof
the scaffolds, and XO inhibitory activity. Besides being the most promising
compound against the enzyme, compound 4 proved to be non-cytotoxic on
B16F10 cells at the concentration in which XO activity is inhibited (~10 μM).
Also, this molecule presents pharmacokinetics and drug-likeness
characteristics that support it as a promising candidate for designing effective
non-purinergic XO inhibitors.
CRediT authorship contribution statement
Benedetta Era: Conceptualization, Methodology, Investigation, Writing -
review & editing, Visualization Giovanna L. Delogu: Methodology,
Investigation, Writing - review & editing, Visualization Francesca Pintus:
Methodology, Writing - review & editing Antonella Fais: Conceptualization,
Investigation, Visualization, Writing - review & editing, Resources Gianluca
Gatto: Funding acquisition, Resources Eugenio Uriarte: Funding acquisition,
Resources Fernanda Borges: Funding acquisition, Supervision Amit Kumar:
Methodology, Investigation, Writing - review & editing, Data Curation Maria J.
Matos: Conceptualization, Methodology, Investigation, Writing - Original Draft,
Visualization, Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work
reported in this paper.
Acknowledgements
This project was partially supported by the University of Porto, University of
Santiago de Compostela, Xunta de Galicia (Galician Plan of Research,
Innovation and Growth 2011–2015, Plan I2C, ED481B 2014/086–0 and
ED481B 2018/007) and Fundação para a Ciência e Tecnologia (FCT,
CEECIND/02423/2018). The present work was partially supported by FIR
(Fondo Integrativo per la Ricerca – annualità 2018), University of Cagliari.
This project was partially supported by Foundation for Science and
Technology (FCT), and FEDER/COMPETE (POCI-01-0145-FEDER-006980).
This article is based upon work from COST Action CA15135.

Journal Pre-proof
References

Journal Pre-proof
CRediT authorship contribution statement
Benedetta Era: Conceptualization, Methodology, Investigation, Writing -
review & editing, Visualization Giovanna L. Delogu: Methodology,
Investigation, Writing - review & editing, Visualization Francesca Pintus:
Methodology, Writing - review & editing Antonella Fais: Conceptualization,
Investigation, Visualization, Writing - review & editing, Resources Gianluca
Gatto: Funding acquisition, Resources Eugenio Uriarte: Funding acquisition,
Resources Fernanda Borges: Funding acquisition, Supervision Amit Kumar:
Methodology, Investigation, Writing - review & editing, Data Curation Maria J.
Matos: Conceptualization, Methodology, Investigation, Writing - Original Draft,
Visualization, Supervision.
[1] R. Hille, Molybdenum-containing hydroxylases, Arch. Biochem. Biophys.
433(1) (2005) 107-116.
[2] K. Tomovic, B.S. Ilic, Z. Smelcerovic, M. Miljkovic, D. Yancheva, M. Kojic,
A.T. Mavrova, G. Kocic, A. Smelcerovic, Benzimidazole-based dual dipeptidyl
peptidase-4 and xanthine oxidase inhibitors, Chem. Biol. Interact. 315 (2020)
1088733.
[3] P. Richette, T. Bardin, Gout, Lancet 375(9711) (2010) 318-328.
[4] E. Agabiti-Rosei, G. Grassi, Beyond gout: uric acid and cardiovascular
diseases, Curr. Med. Res. Opin. 3 (2013) 33-39.
[5] J.A. Singh, K.S. Akhras, A. Shiozawa, Comparative effectiveness of urate
lowering with febuxostat versus allopurinol in gout: analyses from large U.S.
managed care cohort, Arthritis Res. Ther. 17(1) (2015) 120.
[6] B. Hammer, A. Link, A. Wagner, M. Böhm, Hypersensitivity syndrome
during therapy with allopurinol in asymptomatic hyperuricemia with a fatal
outcome, Dtsch. Med. Wochenschr. 126(47) (2001) 1331-1334.
[7] S. Bashir, S.M. Shah, I. Babar, Allopurinol induced Stevens-Johnson
syndrome: a case report, J. Pak. Med. Assoc. 50(6) (2000) 207-209.

Journal Pre-proof
[8] G.B. Elion, A. Kovensky, G.H. Hitchings, Metabolic studies of allopurinol,
an inhibitor of xanthine oxidase, Biochem. Pharmacol. 15(7) (1966) 863-880.
[9] C.L. Gray, N.E. Walters-Smith, Febuxostat for treatment of chronic gout,
Am. J. Health Syst. Pharm. 68(5) (2011) 389-398.
[10] B.L. Love, R. Barrons, A. Veverka, K.M. Snider, Urate-lowering therapy
for gout: focus on febuxostat, Pharmacotherapy 30(6) (2010) 594-608.
[11] S. Floris, A. Fais, A. Rosa, A. Piras, H. Marzouki, R. Medda, A.M.
González-Paramás, A. Kumar, C. Santos-Buelga, B. Era, Phytochemical
composition and the cholinesterase and xanthine oxidase inhibitory properties
of seed extracts from the Washingtonia filifera palm fruit, RSC Adv. 9 (2019)
21278.
[12] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays, J. Immunol. Methods. 65
(1983) 55-63.
[13] E. Cristofer, T.E. Bryan, K. Okamoto, T. Nishino, T. Nishino, E.F. Pai,
PNAS 97(20) (2000) 10723-10728.
[14] N.M. O'Boyle, M. Banck, C.A. James, C. Morley, T. Vandermeersch, G.R.
Hutchison, Open Babel: An open chemical toolbox, J. Cheminform. 3 (2011)
33.
[15] A. Kumar, G. Cappellini, F. Delogu, Electronic and optical properties of
chromophores from hexeneuronic acids, Cellulose 26 (2019) 1489-1501.
[16] Q. Wu, Z. Peng, Y. Zhang, J. Yang, COACH-D: improved protein-ligand
binding site prediction with refined ligand-binding poses through molecular
docking, Nucleic Acids Res. 46 (2018) W438-W442.
[17] J. Yang, A. Roy, Y. Zhang, Protein-ligand binding site recognition using
complementary binding-specific substructure comparison and sequence
profile alignment, Bioinformatics 29(20) (2013) 2588-2595.
[18] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of
docking with a new scoring function, efficient optimization, and multithreading,
J. Comput. Chem. 31 (2010) 455-461.
[19] A. Fais, B. Era, S. Asthana, V. Sogos, R. Medda, L. Santana, E. Uriarte,
M.J. Matos, F. Delogu, A. Kumar, Coumarin derivatives as promising xanthine
oxidase inhibitors, Inter. J. Biolog. Macromol. 120 (2018) 1286-1293.

Journal Pre-proof
[20] M. Crawford, J.A.M. Shaw, Perkin coumarin synthesis, J. Chem. Soc.
(1953) 3435-3439.
[21] N.R. Krishnaswamy, T.R. Seshadri, B.R. Sharma, Nitro derivatives of 3-
phenylcoumarins, Ind. J. Chem. 7(1) (1969) 49-55.
[22] M.J. Matos, S. Vazquez-Rodriguez, L. Santana, E. Uriarte, C. Fuentes-
Edfuf, Y. Santos, A. Munoz-Crego, Synthesis and structure-activity
relationships of novel amino/nitro substituted 3-arylcoumarins as antibacterial
agents, Molecules 18 (2013) 1394-1404.
[23] J.F. Archer, J. Grimshaw, Electrochemical reactions. VI. Application of
the Hammett relation to the polarographic reduction of substituted 9-
benzylidenefluorenes and 3-phenylcoumarins, J. Chem. Soc. [Section] B:
Phys. Org. 3 (1969) 266-270.
[24] G. Ferino, E. Cadoni, M.J. Matos, E. Quezada, E. Uriarte, L. Santana, S.
Vilar, N.P. Tatonetti, M. Yañez, D. Viña, C. Picciau, S. Serra, G. Delogu, MAO
inhibitory activity of 2-arylbenzofurans versus 3-arylcoumarins : synthesis, in
vitro study, and docking calculations, ChemMedChem 8 (2013) 956-966.
[25] A. Bochicchio, L. Chiummiento, M. Funicello, M.T. Lopardo, P. Lupattelli,
Efficient synthesis of 5-nitro-benzo[b]furans via 2-bromo-4-nitro-phenyl
acetates, Tetrahedron Lett. 51 (2010) 2824-2827.
[26] M.J. Bosiak, A convenient synthesis of 2-arylbenzo[b]furans from aryl
halides and 2-halophenols by catalytic one-pot cascade method, ACS Catal. 6
(2016) 2429-2434.
[27] H. Hao, W. Chen, J. Zhu, C. Lu, Y. Shen, Design and synthesis of 2-
phenylnaphthale noids and 2-phenylbenzofuranoids as DNA topois omerase
inhibitors and antitumor agents, Eur. J. Med. Chem. 102 (2015) 277-287.
[28] W. Pu, Y. Yuan, D. Lu, X. Wang, H. Liu, C. Wang, F. Wang, G. Zhang, 2-
Phenylbenzo[b]furans: synthesis and promoting activity on estrogen
biosynthesis, Bioorg. Med. Chem. Lett. 26 (2016) 5497-5500.
[29] D.D. Qin, W. Chen, X. Tang, W. Yu, A.A. Wu, Y. Liao, H.B. Chen,
Accessing
2-arylbenzofurans
by
CuI2(pip)2catalyzed
tandem
couplingcyclization reaction: mechanistic studies and application to the
synthesis of stemofuran A and moracin M, Asian J. Org. Chem. 5 (2016)
1345-1352.

Journal Pre-proof
[ 30 ] R. Arimboor, M. Rangan, S.G. Aravind, C. Arumughan,
Tetrahydroamentoflavone (THA) from Semecarpus anacardium as a potent
inhibitor of xanthine oxidase, J. Ethnopharmacology 133 (2011) 1117-1120.
[31] A. Daina, O. Michielin, V. Zoete, SwissADME: a free web tool to evaluate
pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small
molecules, Sci. Rep. 7 (2017) 42717.
[
32
]
Molinspiration
Cheminformatics.
Available
online:
http://www.molinspiration.com/services/properties.html (accessed on January
2020).

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6