Seleno-functionalization of quercetin improves the non-covalent inhibition of mpro and its antiviral activity in cells against sars-cov-2

Article abstract of DOI:10.3390/ijms22137048

The development of new antiviral drugs against SARS-CoV-2 is a valuable long-term strategy to protect the global population from the COVID-19 pandemic complementary to the vaccination. Considering this, the viral main protease (M^pro ) is among

Full text of DOI:10.3390/ijms22137048

International Journal of
Molecular Sciences
Article
Seleno-Functionalization of Quercetin Improves the
Non-Covalent Inhibition of Mpro and Its Antiviral Activity in
Cells against SARS-CoV-2
Francesca Mangiavacchi 1, Pawel Botwina 2,3 , Elena Menichetti 1,4, Luana Bagnoli 1 , Ornelio Rosati 1,
Francesca Marini 1 , Sérgio F. Fonseca 5, Laura Abenante 5, Diego Alves 5 , Agnieszka Dabrowska 2,3
,
Anna Kula-Pacurar 2 , David Ortega-Alarcon 6,7, Ana Jimenez-Alesanco 6,7, Laura Ceballos-Laita 6,8
,
Sonia Vega 6, Bruno Rizzuti 6,9 , Olga Abian 6,7,8,10,11, Eder J. Lenardão 5 , Adrian Velazquez-Campoy 6,7,8,11,12
,
Krzysztof Pyrc 2 , Luca Sancineto 1,
*
and Claudio Santi 1,
*
1
Group of Catalysis, Synthesis and Organic Green Chemistry, Department of Pharmaceutical Sciences,
University of Perugia Via del Liceo 1, 06100 Perugia, Italy; francesca.mangiavacchi@studenti.unipg.it (F.M.);
elena.menichetti1@studenti.unipg.it (E.M.); luana.bagnoli@unipg.it (L.B.); ornelio.rosati@unipg.it (O.R.);
francesca.marini@unipg.it (F.M.)
Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University,
Gronostajowa 7a, 30-387 Krakow, Poland; pawel.botwina@doctoral.uj.edu.pl (P.B.);
agnieszka.dabrowska@doctoral.uj.edu.pl (A.D.); anna.kula-pacurar@uj.edu.pl (A.K.-P.);
k.a.pyrc@uj.edu.pl (K.P.)
2
3
4
5
Microbiology Department, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University,
Gronostajowa 7, 30-387 Krakow, Poland
School of Science and Technology, Chemistry Division, University of Camerino, Via S. Agostino,
62032 Camerino, Italy
Laboratório de Síntese Orgânica Limpa—LASOL, CCQFA, Universidade Federal de Pelotas—UFPel,
P.O. Box 354, 96010-900 Pelotas, Brazil; tec.sergio_fonseca@yahoo.com.br (S.F.F.);
laura.abenante2018@gmail.com (L.A.); diego.alves@ufpel.edu.br (D.A.); lenardao@ufpel.edu.br (E.J.L.)
Institute for Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, and
GBsC-CSIC-BIFI, Universidad de Zaragoza, 50018 Zaragoza, Spain; dortega@bifi.es (D.O.-A.);
ajimenez@bifi.es (A.J.-A.); ceballos.laita@gmail.com (L.C.-L.); svega@bifi.es (S.V.) bruno.rizzuti@cnr.it (B.R.);
oabifra@unizar.es (O.A.); adrianvc@unizar.es (A.V.-C.)
Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza,
50009 Zaragoza, Spain
Instituto de Investigación Sanitaria de Aragón (IIS Aragon), 50009 Zaragoza, Spain
CNR-NANOTEC, SS Rende (CS), Licryl-UOS Cosenza and CEMIF.Cal, Department of Physics, University of
Calabria, 87036 Rende, Italy
Instituto Aragonés de Ciencias de la Salud (IACS), 50009 Zaragoza, Spain
Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas
Digestivas (CIBERehd), 28029 Madrid, Spain
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Mangiavacchi, F.; Botwina,
P.; Menichetti, E.; Bagnoli, L.; Rosati,
O.; Marini, F.; Fonseca, S.F.; Abenante,
L.; Alves, D.; Dabrowska, A.; et al.
Seleno-Functionalization of Quercetin
Improves the Non-Covalent
Inhibition of Mpro and Its Antiviral
Activity in Cells against SARS-CoV-2.
Int. J. Mol. Sci. 2021, 22, 7048.
6
7
8
9
https://doi.org/10.3390/ijms22137048
Academic Editor: Claudiu T. Supuran
10
11
Received: 5 June 2021
Accepted: 27 June 2021
Published: 30 June 2021
12
Fundación ARAID, Gobierno de Aragón, 50018 Zaragoza, Spain
Correspondence: luca.sancineto@unipg.it (L.S.); claudio.santi@unipg.it (C.S.)
*
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Abstract: The development of new antiviral drugs against SARS-CoV-2 is a valuable long-term
strategy to protect the global population from the COVID-19 pandemic complementary to the
vaccination. Considering this, the viral main protease (Mpro) is among the most promising molecular
targets in light of its importance during the viral replication cycle. The natural flavonoid quercetin
1
has been recently reported to be a potent Mpro inhibitor in vitro, and we explored the effect produced
by the introduction of organoselenium functionalities in this scaffold. In particular, we report here
a new synthetic method to prepare previously inaccessible C-8 seleno-quercetin derivatives. By
screening a small library of flavonols and flavone derivatives, we observed that some compounds
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
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Attribution (CC BY) license (https://
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4.0/).
inhibit the protease activity in vitro. For the first time, we demonstrate that quercetin (
tolylselenyl)quercetin (2d) block SARS-CoV-2 replication in infected cells at non-toxic concentrations,
with an IC50 of 192 M and 8 M, respectively. Based on docking experiments driven by experimental
1) and 8-(p-
µ
µ
evidence, we propose a non-covalent mechanism for Mpro inhibition in which a hydrogen bond
between the selenium atom and Gln189 residue in the catalytic pocket could explain the higher Mpro
activity of 2d and, as a result, its better antiviral profile.
Int. J. Mol. Sci. 2021, 22, 7048. https://doi.org/10.3390/ijms22137048
https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 7048
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Keywords: selenium; flavanols; main protease; SARS-CoV-2
1. Introduction
Flavonoids are valuable natural polyphenolic compounds endowed with a variety of
biological properties [ ] and are considered privileged scaffolds for rational drug design.
This group of compounds has been extensively studied, primarily because of their pro-
nounced antioxidant activity, which can proceed by several functioning mechanisms [ ].
1
1–5
Glutathione peroxidases (GPx) are a family of phylogenetically related redox enzymes
developed by living systems as protection against reactive oxygen species. Most of them
are characterized by the presence of a selenocysteine in which the selenium atom is the
actual catalyst for the glutathione-mediated reduction of peroxides [6], accounting for the
essential role of selenium as a micronutrient in mammals [ ]. A synergistic interaction
7
between quercetin and its homologue catechin with GPx was proved by Watanabe and
co-workers. The reversal of the hydrogen peroxide-induced cell damage mediated by such
flavonoids could be observed only in selenium-supplemented, GPx-overexpressing cells,
suggesting a direct interplay between flavonoids and the selenium-centered antioxidant
system [8].
In 2020, a global pandemic caused by the SARS-CoV-2 spread worldwide [9], and
while efforts were made to develop effective therapeutic strategies, none of the drugs
included in clinical trials offered a significant benefit. Even if vaccination is the method of
choice for viral infection containment, one must consider that some people will not develop
appropriate immunity, and the disease will remain a cause of severe infections and fatal
cases, especially in the high-risk groups. Furthermore, the emergence of escape variants
will likely increase the number of affected individuals. Consequently, the development of
small molecules able to block key steps of the viral replicative cycle is in high demand.
In this scenario, the seminal paper by Haitao Yang and co-workers, in which was
reported the isolation of the SARS-CoV-2 main protease (Mpro or 3CLpro) and the identi-
fication of some of its inhibitors, is worth mentioning [10]. In their study, among other
compounds, ebselen, one of the most investigated organoselenium compounds [11,12], was
identified as the most potent Mpro inhibitor, with half-maximal inhibitory concentration
(IC50) in the nanomolar range. Antiviral activity observed in cell models was noted at low
micromolar levels. A few months later, some of us reported a fast in vitro screening meant
to discover Mpro inhibitors and identified the flavonoid quercetin as a lead compound
binding to the active site with an inhibition constant of 7.4 µM [13]. More recently, it was
also demonstrated that rutin, a glycosylated conjugate of quercetin that commonly occurs
as a natural flavonoid, shares the same properties as the parent compound, although with
a slightly lower potency [14].
Considering our interest in the synthesis of organoselenium compounds with antiox-
idant activity, in light of the above-reported evidence and in the frame of our efforts to
develop antimicrobial compounds [15–18], we sought to prepare novel quercetin deriva-
tives decorated with organochalcogen moieties. These new hybrid compounds could
present improved Mpro inhibition and be endowed with the ability to hamper SARS-CoV-2
replication in a cellular context, thus being suitable to be considered for preclinical studies
required for the development of direct antiviral agents.
Some examples of flavonoids functionalized with selenium moieties have been previ-
ously reported in the literature and are collected in Figure 1. Double-selenenylated chrysin
derivatives (at C-6 and C-8 positions) having an improved antioxidant and anticancer
activity with respect to the parent natural compound were prepared by some of us through
a CuI-catalyzed C–Se coupling reaction [19].

Int. J. Mol. Sci. 2021, 22, 7048
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Previousworks
OH
O
O
ArSe)
CuI, DM2SO
ArSe
OH
O
O
Lenardao etal.
HO
R1
SeAr
R2
R2
HO
OH Se
R1
Quercetin (1) R1= R2= OH
Chrysin (5) R1= R2= H
Woollins’ reag.
MW, MeCN
R2
R2
HO
O
Antunesetal.
Thiswork
OH
O
O
OH
O
OH
OH
Et3N
Dioxane/H2O
,
OH
OH
OH
OH
HO
R-SeX
HO
O
Se
R
C-8 selectivity
Figure 1. Examples of Se-functionalized flavonoids.
A different seleno-functionalization was proposed by Antunes et al. with the treat-
ment of either quercetin or chrysin derivatives with Woollins’ reagent under microwave
irradiation, and the consequent introduction of a selone functionality at the C-4 position.
Moreover, in this case the introduction of a selenium atom in the flavonol scaffold resulted
in increased radical scavenging and anticancer activities, the latter demonstrated in a panel
of different cancer cell lines [20].
In this work, we report unprecedented reaction conditions for the selective function-
alization of the quercetin scaffold at the C-8 position with an alkyl/aryl organoselenium
moiety through a base-activated aromatic electrophilic substitution. All the new com-
pounds were fully characterized and assayed for the ability to inhibit SARS-CoV-2 Mpro
along with some previously prepared chrysin derivatives. The most potent derivatives were
tested for their antiviral properties in a cellular context, identifying derivatives suitable for
further preclinical evaluation.
2. Results and Discussion
2.1. Synthesis
In the frame of flavonoid modifications, some of us recently implemented a green
protocol for the deuteration of quercetin 3-O-rutinoside (rutin) at the aromatic positions
under basic conditions [21].
Using a similar strategy, we envisioned the possibility to exploit the reactivity of
flavonols with electrophilic selenium reagents for the C–Se bond formation at the aromatic
position of the resorcinol ring. Preliminary investigations (collected in Table 1) started
from the screening of the base and the electrophile as the best partners in the electrophilic
aromatic substitution. Based on the previously collected evidence [21], quercetin (1) and
tris(hydroxymethyl)aminomethane (Tris), in a molar ratio of 1:5, were mixed and dissolved
in the solvent (dioxane/water 4:1). Then, 2 molar equivalents of the electrophile were
added, and the mixture was stirred at room temperature until the complete consumption
of the starting material, monitored by TLC analysis. Among different selenium-centered
electrophiles, only PhSeCl, PhSeOTf [22], and N-PhSe-phthalimide (NPSeP) afforded, at
room temperature, the corresponding mono- and bis-selenenylated derivatives (2a and 3,
respectively), even if in poor to moderate yields (entries 1–3). Other electrophiles, such
as PhSeBr or PhSeI (not shown in Table 1), afforded a complex mixture of unidentified
compounds. A large amount of diphenyl diselenide (detected by TLC and in some cases
isolated by chromatography) probably indicated that the nature of the anion has a role

Int. J. Mol. Sci. 2021, 22, 7048
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in the stability of the electrophile under the applied basic conditions and bromide and
iodide derivatives are more prone to decomposition. Likely for the same reason, when the
reactions were conducted at 80 ◦C (entries 4 and 5), slightly reduced yields resulted. To
prevent the decomposition of the electrophile, the reactions with PhSeCl and NPSeP were
repeated under an argon atmosphere, reaching moderate to good yields of compounds 2a
and
different chemoselectivity; PhSeCl gave the prevalent formation of the C-8 functionalized
quercetin 2a in the presence of traces of the double-functionalized compound , both using
5 and 10 equivalents of base (entries 6 and 8). On the contrary, using NPSeP, 2a and were
3 after chromatographic purification. Notably, the two different reagents showed a
3
3
obtained in an overall yield of 81% but in a ratio of 37:63 (entry 7). Considering that the
selective C-8 selenenylation of flavonoids is an unprecedented synthetic opportunity, other
bases were investigated according to the conditions reported in Table 1 (entries 8–9), and
triethylamine afforded the best results in the synthesis of 2a, which was isolated in 55%
yield (entry 9).
Table 1. Optimization of the reaction conditions.
◦
Entry
Electrophile (E)
Base (B)
1:E:B
Time (h)
T ( C)
Yield % (2a:3)
1
2
3
4
5
PhSeCl
PhSeOTf a
NPSeP
PhSeCl
NPSeP
PhSeCl
NPSeP
PhSeCl
PhSeCl
PhSeCl
PhSeCl
Tris
Tris
Tris
Tris
Tris
Tris
Tris
Tris
1:2:5
1:2:5
1:2:5
1:2:5
1:2:5
1:2:5
1:2:5
1:2:10
1:2:5
1:2:5
1:2:2
72
24
4
4
4
2
1
2
2
rt
rt
rt
80
80
rt
rt
rt
rt
rt
rt
18 (18:traces)
11 (8:3)
47 (22:25)
11 (11:trace)
35 (10:25)
33 (33:trace)
81 (30:51)
22 (22:trace)
66 (55:11)
44 (44:trace)
9 (9:0)
6 b
7 b
8 b
9 b
10 b
11
Et3N
Arginine c
NaOH
2
6
a
b
Phenylselenyl trifluoromethanesulfonate was freshly prepared from phenylselenyl bromide and silver trifluoromethanesulfonate. The
c
reactions were performed under an argon atmosphere. The reaction mixture was not completely solubilized.
The structure of 2a was assigned comparing the 13C NMR spectra of compounds
acquired using a standard APT sequence (attached proton test; Figure 2). Starting
from the selected 13C NMR resonances unambiguously assigned in the literature [23
(spectrum in red color), the analysis clearly indicates that in 2a C-8 is a quaternary carbon
1, 2a,
and
3
]
(spectrum in blue color) and that in
black color).
3 both C-6 and C-8 are quaternary (spectrum in

Int. J. Mol. Sci. 2021, 22, 7048
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Figure 2. 13C spectra of 1, 2a, and 3 recorded using the APT pulse sequence.
Once the best conditions for the synthesis were identified, the scope of the reaction
was explored using different alkyl and ary selenyl chlorides 5a , which are commercially
available (5a) or generated in situ (5b ) by the treatment of the corresponding diselenides
4b ) with a stoichiometric amount of SO2Cl2. Diselenides were prepared according to
previously reported procedures [24 26], and the freshly prepared selenyl chlorides were
–e
–e
(
–e
–
immediately used after evaporation of the solvent and the unreacted sulfuryl chloride
under reduced pressure.
The data referring to the synthesis of the monoselenenylated compounds (2a–e) are
summarized in Table 2, and with the only exception of 3, in all the other cases the double-
functionalized derivatives were formed in traces and thus not isolated and characterized.
Even if yields ranged from acceptable, when using PhSeCl (5a) and alkylselenyl
chlorides 5b–c (entries 1–3), to poor, when using less electrophilic aryl (5d–e) selenenylating
reagents (entries 4–5), the protocol reported herein is interesting because, to the best
of our knowledge, it provides for the first time the possibility to access this class of
monoselenenylated flavonoids. To confirm the regioselectivity of the reaction, a NOESY
experiment was carried out on the butyl derivative 2c. An Overhauser effect was observed
between the hydrogen atoms of the alkyl chain and the H2’ and H6’ of the catechol (see
Supplementary Materials), in agreement with the structure assigned based on the 13C
NMR analysis.

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Table 2. Reaction scope.
Entry
Product
Yield (%)
1
58
2
3
4
48
46
32
5
21
2.2. Evaluation of Mpro Inhibition and Antiviral Assay
The small compound library herein synthesized, together with a series of selenium-
(6, 7a–g) and tellurium-containing (8, 9) chrysin analogues [27], reported in Figure 3,
was assayed through in vitro screening based on Mpro hydrolytic activity using a Förster
resonance energy transfer (FRET) substrate, very recently implemented by some of us [13].

Int. J. Mol. Sci. 2021, 22, 7048
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Figure 3. Chalcogen-containing chrysin derivatives.
As a first line of screening, all the compounds were assayed at 100
µ
M. Hits consisted
of compounds diminishing the Mpro enzymatic activity and were then investigated in depth
to determine their inhibition constant (Ki) and their half-maximal inhibitory concentration
(IC50). The obtained values are collected in Table 3 and Figure 4 and compared to those
reported in the literature for quercetin (1), which was selected as the reference compound.
Table 3. Ki and IC50 values of the tested compounds a.
Ki (µM)
CI95,Ki
IC50
CI95,IC50
Quercetin 1
7.4
1.8
ND
8.6
3.8
1.1
[5.8, 9.5]
[1.4, 2.2]
21
5.1
ND
24
11
3.0
13
[17, 28]
[4.1, 6.3]
2a
2b
2c
2d
2e
3
[6.9, 11]
[3.0, 5.0]
[0.85, 1,3]
[3.7, 5.8]
[20, 31]
[8.5, 14.2]
[2.4, 3.9]
[11, 16]
4.6
6
7a
8
9
ND
ND
1.1
ND
ND
3.3
2.2
[0.80, 1.6]
[0.57, 1.0]
[2.3, 4.8]
[1.7, 3.2]
0.77
a
Ki and IC50 were estimated from enzymatic assays as explained in the Materials and Methods section. ND = not determined (little or no
M). Uncertainties of the estimated values have been reported as 95% asymmetric confidence intervals (CI95).
significant inhibition at 100
µ

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100
80
60
40
20
0
2.5
2.0
1.5
1.0
0.5
0.0
2a
0
20
40
60
80
100 120 140
0
20
40
60
80
100
120
Concentration (
∝M)
time (min)
100
80
60
40
20
0
2.5
2.0
1.5
1.0
0.5
0.0
3
0
20
40
60
80 100 120 140
0
20
40
60
80
100
120
Concentration (
∝M)
time (min)
100
80
60
40
20
0
2.5
2.0
1.5
1.0
0.5
0.0
2d
0
20
40
60
80 100 120 140
0
20
40
60
80
100
120
Concentration (
∝M)
time (min)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
100
80
60
40
20
0
2e
0
20
40
60
80 100 120 140
0
20
40
60
80
100
120
Concentration (
∝M)
time (min)
Figure 4. Inhibition curves (left) and time-course monitoring of Mpro activity at increasing concentra-
tions of compounds (right). Increasing the concentration of compounds results in diminished Mpro
activity. Nonlinear least-square regression data analysis was employed to determine the inhibition
constant (Ki) and the half-maximal inhibitory concentration (IC50).
As a general comment, it is possible to underline that the introduction of an organose-
lenyl moiety in the quercetin structure endowed the molecule with a good ability to inhibit
Mpro. Indeed, quercetin has a Ki of 7.4
µM, whereas the vast majority of the tested com-
pounds displayed a higher potency, with the sole exception of 6 and 2b, which were devoid
of any noticeable anti-Mpro inhibitory activity. The selective introduction of the phenylse-
lenyl moiety at the C-8 position was highly effective, while the double substitution resulted
in a drop in the activity (2a vs.
to quercetin . The quercetin scaffold seems essential, as was demonstrated by the fact that
the 5,7-dihydroxy analogue chrysin derivative ( ) was completely inactive, indicating that
3), even if 3
is still a better Mpro inhibitor when compared
1
6
the higher degree of hydroxylation improves the target recognition. The best-in-class com-
pound, which surely deserves further investigation, is 2e, with a p-methoxyphenylselenyl
substituent at the C-8 position. Aliphatic substituents at the selenium atom are not well
tolerated, since the butyl (2c) and decyl (2b) derivatives yielded molecules with a poorer
propensity to block the enzymatic activity of Mpro
.
Many of the inhibitors identified and under development interact covalently with
Mpro. We assessed the reversibility of the interaction of compounds
and 2d by per-
forming experiments aimed at evaluating whether or not the enzyme–inhibitor complex
1

Int. J. Mol. Sci. 2021, 22, 7048
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dissociates under equilibrium: dialysis and size exclusion chromatography followed by
enzymatic activity quantification and near-UV circular dichroism spectra (inhibitor-bound
and inhibitor-free ones display markedly different near-UV circular dichroism spectra).
In the case of Mpro, the far-UV circular dichroism spectrum is quite insensitive to ligand
binding, but the near-UV circular dichroism spectrum changes considerably [13]; there-
fore, enzymatic activity and spectral properties can be employed as specific signature for
ligand-free Mpro. Both compound
1 and compound 2d exhibited a reversible interaction
with SARS-CoV-2 Mpro, provided that dilution is enough to dissociate both compounds
from Mpro (see Supplementary Materials), confirming the non-covalent interaction of the
selenium-derived compound.
Surprisingly, tellurium derivatives displayed a potent anti-Mpro activity (
9 and 8),
better than that displayed by the close selenium analogue, 7a, and by the whole set of
compounds. Of course, there are several concerns about the toxicity of organotellurium
compounds [28], which were indirectly confirmed by the in cellulo tests (reported below).
The most promising Mpro inhibitors (
1, 2a, 2d, 2e, 3, 8, and 9) were successively as-
sayed in an in vitro model of SARS-CoV-2 infection. The ability of each compound to
inhibit the virus replication was evaluated by infecting confluent Vero monolayers with
SARS-CoV-2 in the presence of a given concentration of each compound or DMSO as a
control. Mock-infected cells were also used as a negative control. In the initial experiments
all compounds were tested at five concentrations (100, 75, 50, 25, and 10 µM). After in-
cubation, the cells were scored under the inverted light microscope for the presence or
absence of the SARS-CoV-2-related cytopathic effect (CPE) and cytotoxicity (Table S1). Two
compounds inhibited the development of the CPE (2a and 2d). No CPE reduction was
recorded for other compounds (2e
and precipitated and were toxic, making it impossible to interpret the CPE reduction
for compound . The two most promising compounds (2a and 2d) and compound
tested at higher concentrations (500, 400, 300, 250, and 200 M), were further evaluated
, 3, 8, and 9) at non-toxic concentrations. Compounds
8
9
8
1,
µ
for the inhibition of the SARS-CoV-2 replication by means of RT-qPCR analysis. Anal-
ysis of 2a did not indicate any inhibition of SARS-CoV-2 (Figure S1). The cytotoxicity
of quercetin (500–100 µM) and 2d compound (100–10 µM) was also evaluated on Vero
cells and none of the tested concentrations exhibited cytotoxicity (Figure S2). The same
dose ranges of compounds were also tested in the in vitro cellular SARS-CoV-2 infection
model (Tables S1 and S2) and morphological changes were observed for 2d at 100 µM
concentration (Table S1). Both compounds exhibited CPE reduction as observed under
the inverted light microscope (Figure S3). Importantly, our RT-qPCR analysis, shown in
Figure 5, revealed that quercetin significantly inhibited SARS-CoV-2 infection at relatively
high concentrations, with IC50 = 192 µM (Figure S4). Quercetin derivative 2d inhibited the
virus replication at noticeably lower concentration (IC50 = 8 µM, Figure S4).
2d
Figure 5. Inhibition of SARS-CoV-2 replication by quercetin (compound
replication was evaluated at the given compound concentrations using RT-qPCR and the data are
presented as SARS-CoV-2 RNA copies per mL of the original sample. Bars show mean value SEM
1) and compound 2d. Virus
±
from three independent experiments. The significance level (* p < 0.05) is denoted with an asterisk in
the graph.

Int. J. Mol. Sci. 2021, 22, 7048
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This finding is in line with the Mpro inhibition, where 2d is among the most potent
compounds. No inhibition was observed for compounds 2e and 3, at non-toxic concentra-
tions, although these compounds are able to hamper the Mpro activity. The reason behind
the lack of activity for these compounds could be their poor bioavailability/penetration
into the cellular compartment, where the virus replicates, or their toxicity.
2.3. Molecular Docking of the Representative Compound 2d to SARS-CoV-2 Mpro
The binding to Mpro of the most promising compound found in the experiment,
compound 2d, was modeled in silico by using molecular docking simulations, largely
following a protocol that some of us have used to study the binding of quercetin and
its analogue rutin [13,14]. A blind search on the entire protein surface was performed at
very high exhaustiveness, and using two reference crystallographic structures [29]. The
results shown in Figure 6 indicate that compound 2d was predicted to bind in the protein
active site, with the double ring of the quercetin moiety in direct interaction with the
catalytic dyad, including residues His41 and Cys145. The best docking poses, assessed
in terms of both the most favorable affinity score and the number of binding modes in
the same docking cluster, shared a similar conformation. In particular, (i) the quercetin
scaffold occupied a single well-defined position, in contact with the two catalytic residues
(minimum distance: 3.5 and 3.7 Å with Cys145 and His41, respectively); (ii) the selenium
atom was an acceptor of a hydrogen bond with the side chain of Gln189 (donor–acceptor
distance: 3.8 Å); (iii) the adduct of quercetin had a larger variability in its position, having
the possibility of forming a number of weak hydrophobic contacts with various chemical
groups of the protein. The binding energy of the best three poses was –8.0
indicating a very favorable binding affinity.
±
0.2 kcal/mol,
Figure 6. Molecular docking of compound 2d to SARS-CoV-2 Mpro. (
poses bound in the protein active site. ( ) Detail of the docking poses highlighting the quercetin
scaffold approximately superimposed, while the chemical adduct (circled in green) shows a large
variability. ( ) The most favorable docking pose (binding energy 8.2 kcal/mol), with the side chains
A) The best three docking
B
C
−
of selected protein residues (the catalytic dyad His41/Cys145 and the hydrogen bonded residue
Gln189) explicitly shown.

Int. J. Mol. Sci. 2021, 22, 7048
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A number of conclusions can be drawn from these observations. First, the quercetin
scaffold is crucial to bind Mpro, and has a distinct association mode to the protein active
site. However, compared to the binding of (unmodified) quercetin [13], for compound 2d
the presence of a chemical adduct breaks the pseudo-symmetry in the molecular structure,
reducing the possible anchoring modes of the parent scaffold to a single conformation.
This is also at variance with rutin, whose chemical adduct still allows two possible binding
modes for the sugar moiety [14]. The reason for this behavior is the presence of the selenium
atom, which contributes to the binding of the compound by forming a hydrogen bond. We
note that the presence of this conserved bond, together with the specificities of the nature
of selenium as a hydrogen bond acceptor compared to other elements [30], would also
explain in a rather straightforward way the strong potency of our quercetin derivatives
with respect to the parent compound. In our simulations, compared to quercetin [13],
not only compound 2d showed a higher selectivity in the binding conformation, but
the predicted affinity was also more favorable by 1 kcal/mol. The chemical adduct of
compound 2d had a higher variability in the conformation compared to the quercetin
scaffold and contributed to a lesser extent to the binding. Therefore, we suggest that the
binding of the other selenium-derivative compounds is modulated by subtle differences in
the interactions of their adducts with the entrance region of the Mpro active site, and likely
also by entropic contributions. These effects are difficult to capture by molecular docking
due to the limitation of this technique, which does not take into account the dynamics of
the protein–ligand complex.
3. Materials and Methods
3.1. Synthesis: General Remarks
Solvents and reagents were used as received unless otherwise noted. The starting
materials are commercially available; diselenides 4b
7a–g , and belong to the same batch described in ref. [27] and were synthesized as
reported in the literature; the physical and spectral data of 7a , and are reported
–f [25,26] and chrysin derivatives 6,
,
8
9
6,
–
g,
8
9
below. Reactions were conducted in round-bottom flasks and were stirred with Teflon-
coated magnetic stirring bars.
The analytical thin layer chromatography (TLC) was performed on silica gel 60 F254-
precoated aluminum foil sheets (Merck, Darmstadt, Germany) and visualized by UV
irradiation (Spectroline® UV light, Sigma-Aldrich, St. Louis, MI, USA) or by using I2 or a
KMnO4 stain (Merck, Darmstadt, Germany). Silica gel Kiesinger 60 (70–230 mesh) was used
for flash column chromatography and a 240–400 mesh for normal column chromatography.
◦
NMR spectroscopic (Bruker, Fällanden, Switzerland) experiments were performed at 25 C
on a Bruker DRX 400 MHz (UniPG-Italy) and a Varian INOVA 300 MHz (UFRGS-Brazil).
1H and 13C NMR chemical shifts (
δ
) are reported in parts per million (ppm), and they
are relative to TMS (0.0 ppm), and the residual solvent peak (DMSO-d6, 2.50 ppm or CDCl3,
7.27 for 1H NMR, and 39.52 ppm or 77.0 ppm for 13C NMR). 77Se chemical shifts (
) are
δ
reported in parts per million (ppm), and they are relative to diphenyl diselenide (447 ppm)
in DMSO-d6. Data are reported as follows: chemical shift (multiplicity, coupling constants,
where applicable, and the number of hydrogen atoms). Abbreviations are as follows:
s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublet), dt (doublet of triplet),
tt (triplet of triplet), m (multiplet), br.s. (broad signal). Coupling constant (J) is quoted
in Hz to the nearest 0.1 Hz. High-resolution mass spectrometry (HRMS) measurements
were performed using a Synapt G2-Si mass spectrometer (Waters, Milford, USA) equipped
with an APCI source and quadrupole time-of-flight mass analyzer. The mass spectrometer
was operated in the positive and negative ion detection modes with discharge current set
at 4.0
were processed using MassLynx 4.1 software (Waters, Milford, USA) incorporated into the
instrument. Melting points (m.p.) were determined on Mel-Temp apparatus or a Marte
PFD III instrument (Brazil) with a 0.1 ◦C precision, and are uncorrected.
µ
A. The heated capillary temperature was 350 ◦C. The results of the measurements
®

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3.1.1. General Procedure for the In Situ Formation of RSeCl (5b–f)
Diselenide 4b (0.22 mmol) was placed in a round-bottom flask under an argon
–
f
atmosphere, and dichloromethane (1 mL) was added. The resulting solution was stirred at
0 ◦C, and sulfuryl chloride (0.22 mmol) was added dropwise. The reaction mixture was
stirred at room temperature for 1 h, while protected from light. After 1 h, the solvent was
removed under reduced pressure, and the corresponding organoselenyl chloride was used
without further purification.
3.1.2. General Procedure for the Seleno-Functionalization of 1
Quercetin
1 (0.2 mmol) and triethylamine (1.0 mmol) were placed in a round-bottom
flask and sonicated until homogeneity. The flask was connected to a vacuum line, and
several evacuations and purges with argon were made. Then, a mixture of 1,4-dioxane
(1.5 mL) and water (0.5 mL) was added to solubilize the reagents, followed by the addition
of RSeX (0.44 mmol) in 1,4-dioxane (0.5 mL). The reaction mixture was stirred for the time
indicated in Table 2. The reactions were monitored by TLC (eluent CH2Cl2/MeOH 9:1).
The reaction was quenched with 10% aqueous HCl, extracted with EtOAc (3
dried over Na2SO4 and concentrated under reduced pressure. Products were purified by
column chromatography using a mixture CH2Cl2/MeOH as eluent.
×
20 mL),
3.1.3. Physical Data
2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-8-(phenylselanyl)-4H-chromen-4-one (2a)
◦
Yellow solid (m.p. 211–213 C; 53 mg, 0.12 mmol, isolated yield 58% purified by sil-
1
ica gel chromatography; eluent DCM/MeOH 98:2). H NMR (400 MHz, DMSO-d6):
δ
12.88 (s, 1H), 11.3 (br.s., 1H), 9.59 (s, 2H), 9.25 (s, 1H), 7.80–7.77 (m, 1H), 7.45–7.40 (m,
1H), 7.23–7.10 (m, 5H), 6.78 (d, J = 8.4 Hz, 1H), 6.43 (s, 1H) ppm. 13C NMR (100.62 MHz,
DMSO-d6):
δ 176.0, 165.5, 162.0, 156.4, 147.9, 147.3, 145.1, 136.0, 132.7, 129.4 (2C), 128.6 (2C),
126.0, 122.1, 120.0, 115.6, 115.5, 103.7, 98.2, 92.0 ppm. 77Se NMR (76.27 MHz, DMSO-d6):
222.0 ppm. HRMS (ESI-TOF): calc. for C21H15O7Se [M+H]+: 458.9983; found 458.9985.
δ
2-(3,4-Dihydroxyphenyl◦)-3,5,7-trihydroxy-8-(decylselenyl)-4H-chromen-4-one (2b)
Yellow solid (m.p. 155–159 C; 50 mg, 0.10 mmol, isolated yield 48% purified by sil-
1
ica gel chromatography; eluent DCM/MeOH 98:2). H NMR (400 MHz, DMSO-d6):
δ 12.73
(s, 1H), 9.54 (br.s, 3H), 7.90 (s, 1H), 7.74 (d, J = 7.5 Hz, 1H), 6.89 (d, J = 7.8 Hz, 1H), 6.35 (s,
1H), 2.80–2.77 (m, 2H), 1.44–1.03 (m, 16H), 0.82–0.79 (m, 3H) ppm. 13C NMR (100.62 MHz,
DMSO-d6):
δ 176.0, 165.0, 160.9, 156.3, 147.9, 147.2, 145.2, 135.8, 122.4, 120.0, 115.6, 115.5,
103.5, 97.9, 91.8, 31.3, 29.6, 29.0, 29.0, 28.8, 28.7, 28.5, 26.8, 22.2, 14.0 ppm. 77Se NMR
(76.27 MHz, DMSO-d6):
523.1235, found 523.1233.
δ
115.7 ppm. HRMS (ESI-TOF): calc. for C25H31O7Se [M+H]+:
2-(3,4-Dihydroxyphenyl◦)-3,5,7-trihydroxy-8-(butylselenyl)-4H-chromen-4-one (2c)
Yellow solid (m.p. 159–163 C; 40 mg, 0.09 mmol, isolated yield 46% purified by sil-
1
ica gel chromatography; eluent DCM/MeOH 98:2). H NMR (400 MHz, DMSO-d6):
δ 12.72
(s, 1H), 9.73–9.32 (m, 3H), 7.88 (d, J = 2.1 Hz, 1H), 7.73 (dd, J = 2.0 and J = 8.5 Hz, 1H), 6.90
(d, J = 8.5 Hz, 1H), 6.35 (s, 1H), 2.79 (t, J = 7.2 Hz, 2H), 1.47–1.42 (m, 2H), 1.34–1.28 (m, 2H),
0.74 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (100.62 MHz, DMSO-d6):
δ 176.1, 165.1, 160.9, 156.3,
148.0, 147.2, 145.2, 135.8, 122.4, 120.1, 115.7, 115.5, 103.5, 97.9, 92.0, 32.0, 26.7, 22.1, 13.5 ppm.
77Se NMR (76.27 MHz, DMSO-d6):
δ 115.4 ppm. HRMS (ESI-TOF): calc. for C19H19O7Se
[M+H]+: 439.0296, found 439.0299.
2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-8-(p-tolylselanyl)-4H-chromen-4-one (2d)
◦
Yellow solid (m.p. 203–206 C; 40 mg, 0.06 mmol, isolated yield 32% purified by sil-
1
ica gel chromatography; eluent DCM/MeOH 98:2). H NMR (400 MHz, DMSO-d6):
δ 12.85
(s, 1H), 9.67–9.59 (m, 2H), 9.27 (br.s, 1H), 7.80 (d, J = 2.1 Hz, 1H), 7.46 (dd, J = 2.1 Hz and
J = 8.5 Hz, 1H), 7.13 (d, J = 8.1 Hz, 2H), 6.99 (d, J = 8.0 Hz, 2H), 6.79 (d, J = 8.5 Hz, 1H), 6.41
(s, 1H), 2.16 (s, 3H) ppm. 13C NMR (100.62 MHz, DMSO-d6):
δ
176.0, 165.3, 161.9, 156.3,
147.9, 147.3, 145.1, 136.0, 135.4, 130.0 (2C), 129.1 (2C), 128.8, 122.2, 120.0, 115.6, 103.7, 98.1,

Int. J. Mol. Sci. 2021, 22, 7048
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92.5, 20.6 ppm. 77Se NMR (76.27 MHz, DMSO-d6):
δ
215.4 ppm. HRMS (ESI-TOF): calc. for
C22H17O7Se [M+H]+ 473.0140, found 473.0140.
2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-8-(◦(4-methoxyphenyl)selanyl)-4H-
chromen-4-one (2e) Yellow solid (m.p. 194–197 C; 20 mg, 0.04 mmol, isolated yield
1
21% purified by silica gel chromatography; eluent DCM/MeOH 98:2). H NMR (400 MHz,
DMSO-d6):
(d, J = 8.1 Hz, 2H), 6.85 (d, J = 8.2 Hz, 1H) 6.78 (d, J = 8.1 Hz, 2H), 6.40 (s, 1H), 3.64 (s,
3H) ppm. 13C NMR (100.62 MHz, DMSO-d6):
176.0, 165.3, 161.8, 158.3, 156.2, 148.0, 147.3,
δ 12.84 (s, 1H), 9.60–9.33 (br.s, 3H), 7.85 (s, 1H), 7.54 (d, J = 7.7 Hz, 1H), 7.25
δ
145.2, 136.0, 131.6 (2C), 122.2 (2C), 120.1, 115.7, 115.6, 115.1 (2C), 103.6, 98.1, 93.5, 55.2 ppm.
77Se NMR (76.27 MHz, DMSO-d6):
δ 208.1 ppm. HRMS (ESI-TOF): calc. for C22H17O8Se
[M+H]+: 489.0089 found 489.0092.
2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-6,8-bis(phenylselanyl)-4H-chromen-4-
one (3) Yellow solid (m.p. 206–210 ◦C; 62.5 mg, 0.10 mmol, isolated yield 51% purified by
1
silica gel chromatography; eluent DCM/MeOH 98:2). H NMR (400 MHz, DMSO-d6):
δ
13.93 (s, 1H), 10.34 (s, 1H), 9.81 (s, 1H), 9.71 (s, 1H), 9.31 (s, 1H), 7.83 (s, 1H), 7.51–7.49 (m,
1H), 7.27–7.16 (m, 10H), 6.81 (d, J = 8.5 Hz, 1H) ppm. 13C NMR (100.62 MHz, DMSO-d6):
δ
175.8, 165.1, 163.9, 156.9, 148.1, 147.7, 145.1, 136.1, 134.4, 132.2, 132.0, 129.4 (2C), 129.3
(2C), 128.7 (3C), 126.2, 126.0, 123.0, 121.9, 120.1, 115.6, 104.0, 98.0, 92.7 ppm. 77Se NMR
(76.27 MHz, DMSO-d6):
δ 228.6, 226.0 ppm. HRMS (ESI-TOF): calc. for C27H19O7Se2
[M+H]+: 614.9461, found 614.9456.
5,7-Dihydroxy-2-phenyl-6,8-bis(phenylselanyl)-4H-chromen-4-one (6) [19] Yellow
solid (m.p. 188.7–190.6 ◦C; 251 mg, 0.44 mmol, isolated yield 89% purified by silica gel
1
chromatography; eluent hexane/AcOEt 80:20). H NMR (400 MHz, CDCl3/DMSO-d6):
δ
14.08 (s, 1H); 7.93 (s, 1H); 7.84 (d, J = 7.0 Hz, 2H), 7.52–7.38 (m, 2H), 7.33–7.31 (m, 1H);
7.20–7.15 (m, 3H); 6.72 9s, 1H) ppm. 13C NMR (100 MHz, CDCl3/DMSO-d6):
δ
180.6, 163.8,
163.2, 162.3, 187.1, 130.5, 130.3, 130.0, 128.9, 127.7, 127.5, 127.4, 127.3, 124.9, 124.7, 124.6,
103.8, 103.7, 97.8, 91.7 ppm.
5-Hydroxy-2-phenyl-7-(2-(phenylselanyl)ethoxy)-4H-chromen-4-one (7a) [27] White
solid (m.p. 155–156 ◦C; 377 mg, 0.86 mmol, isolated yield 86% purified by silica gel chro-
1
matography; eluent hexane/AcOEt 20:80). H NMR (300 MHz, CDCl3):
δ 12.69 (s, 1H);
7.86–7.83 (m, 2H); 7.59–7.50 (m, 5H); 7.31–7.29 (m, 3H); 6.63 (s, 1H); 6.38 (d, J = 7.2 Hz, 1H);
6.26 (d, J = 2.1 Hz, 1H); 4.24 (t, J = 7.2 Hz, 2H); 3.23 (t, J = 7.2 Hz, 2H) ppm. 13C NMR
(75 MHz, CDCl3):
δ 182.3, 164.2, 163.8, 162.0, 157.6, 133.2, 131.8, 131.1, 129.2, 129.0, 127.5,
126.2, 105.7, 98.5, 92.9, 25.5 ppm.
5-Hydroxy-7-[(2-mesitylselanyl)ethoxy]-2-phenyl-4H-chromen-4-one (7b)
[27]
White/pink solid (m.p. 135.7–138.4◦C; 289 mg, 0.60 mmol, isolated yield 60% purified by
1
silica gel chromatography; eluent hexane/AcOEt 20:80). H NMR (300 MHz, CDCl3):
δ
12,70 (s, 1H); 7.86–7.84 (m, 2H); 7.53–7.52 (m, 3H); 6.95 (s, 2H); 6.63 (s, 1H); 6.35 (d, J = 2.2 Hz,
1H); 6.23 (d, J = 2.2. Hz, 1H); 4.12 (t, J = 7.0 Hz, 2H); 2.99 9t, J = 7.0 Hz, 2H); 2.55 (s, 6H);
2.26 (s, 3H) ppm. 13C NMR (75 MHz, CDCl3):
δ 182.3, 164.9, 164.2, 163.9, 162.1, 157.6, 143.1,
138.6, 131.8, 131.2, 129.0, 128.6, 126.4, 126.2, 105.8, 105.7, 98.4, 93.0, 77.4, 76.6, 67.9, 25.2, 24.5,
20.9 ppm.
5-Hydroxy-2-phenyl-7-[2-(o-tolylselanyl)ethoxy]-4H-chromen-4-one (7c) [27] Light
yellow solid (m.p. 123.9–127.4 ◦C; 321 mg, 0.73 mmol, isolated yield 71% purified by silica
1
gel chromatography; eluent hexane/AcOEt 20:80). H NMR (300 MHz, CDCl3):
δ 12.70 (s,
1H); 7.87–7.84 (m, 2H); 7.53–7.50 (m, 4H); 7.26–7.10 (m, 3H); 6.64 (s, 1H); 6.40 (d, J = 2.2 Hz,
1H); 6.28 40 (d, J = 2.2 Hz, 1H); 4.23 (t, J = 7.2 Hz, 2H); 3.21 (t, J = 7.2 Hz, 2H); 2.45 (s,
3H) ppm. 13C NMR (75 MHz, CDCl3):
δ 182.4, 164.2, 163.9, 162.1, 157.6, 139.9, 132.3, 131.8,
131.2, 130.2, 129.8, 129.0, 127.4, 126.7, 126.2, 105.8, 98.6, 93.0, 67.7, 24.4, 22.5 ppm.
7-{2-[(4-Fluorophenyl)selanyl]ethoxy}-5-hydroxy-2-phenyl-4H-chromen-4-one
◦
(7d)
[
27] White solid (m.p. 171.1–173.7 C; 282 mg, 0.62 mmol, isolated yield 62% pu-
rified by silica gel chromatography; eluent hexane/AcOEt 20:80). 1H NMR (300 MHz,
CDCl3):
δ 12.70 (s, 1H); 7.87–7.84 (m, 2H); 7.59–7.53 (m, 5H); 7.07–6.97 (m, 2H); 6.64 (s, 1H);
6.36 (d, J = 2.2 Hz, 1H); 6.26 (d, J = 2.2 Hz, 1H); 4.23 (t, J = 7.1 Hz, 2H); 3.18 (t, J = 7.1 Hz,

Int. J. Mol. Sci. 2021, 22, 7048
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2H) ppm. 13C NMR (75 MHz, CDCl3):
δ
182.4, 164.3, 163.9, 162.6 (d, 1JC-F = 247.9 Hz), 162.1,
157.6, 136.0 (d, 3JC-F = 8.0 Hz), 131.8, 131.1 129.0, 126.2, 123.2 (d, 4JC-F = 3.4 Hz), 116.4 (d,
2JC-F = 21.5 Hz), 105.8, 98.5, 92.9, 67.8, 26.4 ppm.
5-Hydroxy-2-phenyl-7-{2-[(3-(trifluoromethyl)phenyl]selanyl)ethoxy}-4H-chromen-
4-one (7e)
[
27] White solid (m.p. 154.1–157.7 ◦C; 289 mg, 0.57 mmol, isolated yield 57%
1
purified by silica gel chromatography; eluent hexane/AcOEt 20:80). H NMR (300 MHz,
CDCl3):
δ 12.70 (s, 1H); 7.87–7.44 (m, 3H); 7.74 (d, J = 7.7 Hz, 1H); 7.55–7.50 (m, 4H); 7.41
(t, J = 7.7 Hz, 1H); 6.64 (s, 1H); 6.39 (d, J = 2.2 Hz, 1H); 6.27 (d, J = 2.2. Hz, 1H); 4.29 (t,
J = 6.8 Hz, 2H); 3.30 (t, J = 6.8 Hz, 2H) ppm13C NMR (75 MHz, CDCl3):
δ 182.4, 164.9, 164.0,
163.9, 162.1, 157.6, 136.0 (q, 4JC-F = 1.26 Hz), 131.8, 131.4 (q, 2JC-F = 32.5 Hz), 131.1, 130.4,
129.5 (q, 3JC-F = 3.80 Hz), 129.0, 126.2, 124.1 (q, 3JC-F = 3.77 Hz), 123.5 (q, 1JC-F = 272.7 Hz),
105.8, 105.7, 98.4, 93.0, 67.8, 25.9 ppm.
5-Hydroxy-7-{2-[(2-methoxyphenyl)selanyl]ethoxy}-2-phenyl-4H-chromen-4-one
◦
(7f)
[
27] Grey solid (m.p. 132.9–135.6 C; 378 mg, 0.84 mmol, isolated yield 81% puri-
fied by silica gel chromatography; eluent hexane/AcOEt 20:80). 1H NMR (300 MHz,
CDCl3):
δ 12.65 (s, 1H); 7.85–7.82 (m, 2H); 7.51–7.44 (m, 4H); 7.25 (ddd, J = 8.1, 7.4 and
1.6 Hz, 1H); 6.91–6.85 (m, 2H); 6.61 (s, 1H); 6.40 (d, J = 2.2 Hz, 1H); 6.27 (d, J = 2.2 Hz, 1H);
4.26 (t, J = 7.3 Hz, 2H); 3.88 (s, 3H); 3.23 (t, J = 7.3 Hz, 2H) ppm. 13C NMR (75 MHz, CDCl3):
δ
182.4, 164.9, 164.4, 163.9, 162.2, 158.3, 157.7, 132.4, 131.7, 131.3, 129.0, 128.6, 126.2, 121.5,
117.9, 110.8, 105.8, 105.8, 98.6, 93.1, 68.2, 55.8, 23.0 ppm.
7-[2-(Benzylselanyl)ethoxy]-5-hydroxy-2-phenyl-4H-chromen-4-one (7g) [27] Green
solid (m.p. 115.4- 118.2 ◦C; 336 mg, 0.74 mmol, isolated yield 74% purified by silica gel
1
chromatography; eluent hexane/AcOEt 20:80). H NMR (300 MHz, CDCl3):
δ 12.71 (s,
1H); 7.87–7.84 (m, 2H); 7.52–7.50 (m, 3H); 7.32–7.25 (m, 5H); 6.63 (s, 1H); 6.41 (d, J = 6.7 Hz,
1H); 6.28 (d, J = 2.2 Hz, 1H); 4.13 (t, J = 7.0 Hz, 2H); 3.89 (t, J = 6.7 Hz, 2H) ppm. 13C NMR
(75 MHz, CDCl3):
δ 182.3, 164.2, 163.8, 162.1, 157.6, 138.8, 131.8, 131.1, 129.0, 128.8, 128.6,
126.9, 126.2, 105.7, 105.7, 98.5, 93.0, 68.7, 27.7, 21.4 ppm.
5-Hydroxy-2-phenyl-7-[2-(phenyltellanyl)ethoxy]-4H-chromen-4-one (8) [27] Yellow
solid (m.p. 128–129 ◦C; 407 mg, 0.82 mmol, isolated yield 87% purified by silica gel chro-
1
matography; eluent hexane/AcOEt 20:80). H NMR (300 MHz, CDCl3):
δ 12.69 (s, 1H);
7.86–7.79 (m, 4H); 7.53–7.50 (m, 3H); 7.33–7.21 (m, 3H); 6.63 (s, 1H); 6.37 (d, J = 2.2 Hz, 1H);
6.26 (d, J = 2.2 Hz, 1H); 4.34 (t, J = 7.6 Hz, 2H); 3.19 (t, J = 7.6 Hz, 2H) ppm. 13C NMR
(75 MHz, CDCl3):
δ 182.3, 164.1, 163.8, 162.0, 157.7, 138.8, 131.8, 131.1, 129.3, 129.0, 128.1,
126.2, 105.7, 105.6, 98.6, 93.0, 69.9, 5.9 ppm.
7-{2-[(4-Chlorophenyl)tellanyl]ethoxy}-5-hydroxy-2-phenyl-4H-chromen-4-one
◦
(9)
[
27] Yellow solid (m.p. 140.3–143 C; 297 mg, 0.57 mmol, isolated yield 57% puri-
fied by silica gel chromatography; eluent hexane/AcOEt 20:80). 1H NMR (300 MHz,
CDCl3):
J = 8.4 Hz, 2H); 6.64 (s, 1H); 6.38–6.37 (d, J = 2.4 Hz, 2H); 6.27–6.26 (d, J = 2.1 Hz, 2H); 4.34
(t, J = 7.5 Hz, 2H); 3.19 (t, J = 7.5 Hz, 2H) ppm. 13C NMR (75 MHz, CDCl3):
182.3, 164.9,
δ 12.70 (s, 1H); 7.87–7.84 (m, 2H); 7.71 (d, J = 8.4 Hz, 2H); 7.53–7.51 (m, 3H); 7.20 (d,
δ
164.0, 163.9, 162.1, 157.6, 140.2, 134.8, 131.8, 131.1, 129.6, 129.0, 126.2, 108.1, 105.8, 98.5, 93.1,
69.7, 6.5 ppm.
3.2. SARS-CoV-2 Mpro Expression and Purification
Mpro was expressed in a pET22b plasmid transformed into BL21 (DE3) Gold E. coli
◦
strain. Small-scale cultures grown in LB/ampicillin (100
µ
g/mL) at 37 C overnight
were employed for inoculating 4 L large-scale cultures of LB/ampicillin (100
incubated at 37 C until reaching OD close to 0.6 at 600 nm. Protein expression was
µ
g/mL)
◦
induced with 1 mM isopropyl 1-thio-◦
β
-D-galactopyranoside (IPTG) at 18 ◦C for 5 h. Cells
were harvested by centrifugation at 4 C for 10 min at 10,000 rpm (Beckman Coulter Avanti
J-26 XP Centrifuge) and resuspended in lysis buffer (sodium phosphate 50 mM, pH 7,
sodium chloride 500 mM). Cells were lysed by sonication (Sonics Vibra-Cell Ultrasonic
Liquid Processor) in ice, adding benzonase 20 U/mL (Merck-Millipore, Burlington, MA,
USA) and lysozyme 0.5 mg/mL (Carbosynth, Berkshire, UK). Cell debris was removed

Int. J. Mol. Sci. 2021, 22, 7048
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by centrifugation at 4 ◦C for 30 min at 20,000 rpm, and by subsequent filtration (0.45
µm
pore membrane). Affinity chromatography (ÄKTA FPLC System, GE Healthcare Life
Sciences, Pittsburgh, PA, USA) using a cobalt HiTrap TALON column (GE Healthcare
Life Sciences) allowed fast purification in a single chromatographic step, applying an
imidazole 10–250 mM gradient. Purity was assessed by SDS-PAGE, and pure protein
fractions were pooled and dialyzed to remove imidazole in a buffer (sodium phosphate
50 mM, pH 7, and sodium chloride 150 mM). Protein concentration was quantitated using
an extinction coefficient of 32890 M−1 cm−1 at 280 nm. Protein identity was assessed by
mass spectrometry (LC-ESI-MS/MS).
3.3. SARS-CoV-2 Mpro Proteolytic Activity Assay
A continuous assay based on Förster resonance energy transfer (FRET) to measure
in vitro the catalytic activity of Mpro was implemented by using the substrate (Dabcyl)
KTSAVLQSGFRKME(Edans)-NH2 (Biosyntan GmbH, Berlin, Germany) [31] The enzymatic
reaction was initiated by adding substrate at 20
µM (final concentration) to the enzyme at
0.2 M (final concentration) in a final volume of 100
µ
µL. The reaction buffer was sodium
phosphate 50 mM, pH 7, NaCl 150 mM, and DMSO 2.5%. Fluorescence emission was mea-
sured in a FluoDia T70 microplate reader (Photon Technology International, Birmingham,
NJ, USA) for 20 min (excitation wavelength, 380 nm; emission wavelength, 500 nm). The
initial slope of the time evolution curve of the fluorescence emission signal provided a
direct quantification of the enzymatic activity. The Michaelis–Menten constant, Km, and
the catalytic rate constant or turnover number, kcat, were previously estimated (Km = 11
µM
and kcat = 0.040 s−1) [13].
3.4. SARS-CoV-2 Mpro Inhibition Assay
The in vitro inhibition potency of the compounds against Mpro was assessed through
the estimation of the inhibition constant, Ki, and the half-maximal inhibitory concentration,
IC50, from experimental inhibition curves. Inhibition curves were obtained by measuring
the enzyme activity (at fixed 0.2
concentration) as a function of compound concentration (serial 2-fold dilution from 125
to 0 M), maintaining the percentage of DMSO (2.5%) constant. The enzymatic activity was
µM enzyme concentration and fixed 20 µM substrate
µM
µ
quantitated as the initial slope of the substrate fluorescence emission time evolution curve,
and was plotted as a function of compound concentration. The ratio between the activity
(slope) in the presence and absence of the compound provides the residual percentage of
activity at a given compound concentration. Nonlinear regression analysis employing a
simple inhibition model (considering inhibitor depletion due to enzyme binding) allowed
us to estimate the apparent inhibition constant, Kiapp, for each compound according to
Equation (1):
!
r
ꢀ
ꢁ
1
2
[EI] =
[I]T + [E]T + Kapp
−
[I]T + [E]T + Kapp 2 − 4[E]T[I]T
i
i
!
r
ꢀ
ꢁ
[I]T − [E]T − Kapp
+
[I]T + [E]T + Kapp 2 − 4[E]T[I]T
1
,
(1)
[I] = [I]T − [EI] =
2
i
i
v([I])
[EI]
[E]T
1
= 1 −
v([I] = 0)
=
[I]
1+ K
app
i
where [EI] is the concentration of the enzyme–inhibitor complex, [E]T and [I]T are the total
concentrations of the enzyme and the inhibitor, Kiapp is the apparent inhibition constant for
the inhibitors (quercetin and derivatives), [I] is the concentration of the free inhibitor, and v
is the initial slope of the enzymatic activity trace at a given (free) inhibitor concentration [I]
(or total inhibitor concentration [I]T). No approximation for the free inhibitor concentration
(e.g., assuming to be equal to the total inhibitor concentration) was made, thus having
general validity for any total enzyme and inhibitor concentration and any value of the
inhibition constant (even for tight-binding inhibitors). In addition, if the inhibitor acts

Int. J. Mol. Sci. 2021, 22, 7048
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through a purely competitive mechanism, the previous equation can be substituted by
Equation (2):
v([I])
v([I] = 0)
1
1
=
=
,
(2)
[I]
[I]
ꢀ
ꢁ
1 +
1 +
Kiapp
[S]
m
Ki 1+ K
where Ki is the intrinsic (i.e., substrate concentration-independent) inhibition constant,
m is the Michaelis–Menten constant for the enzyme–substrate interaction, and [S] is the
K
substrate concentration. Because Km and [S] are known, the intrinsic inhibition constant
can be determined. Furthermore, approximating the free compound concentration by the
total compound concentration and neglecting ligand depletion, the Kiapp in Equation (2) is
equivalent to the IC50, as reported in Table 3. However, it should be emphasized that the
IC50 is an assay-dependent inhibition potency index (among other parameters, it depends
on the enzyme and substrate concentrations, as well as on the Km); thus, the intrinsic
inhibition constant is a better inhibition potency index. Uncertainties have been reported
as 95% profile likelihood asymmetric confidence intervals, as especially recommended
for equilibrium constants (i.e., binding association/dissociation constants and inhibition
constants) [32].
3.5. Molecular Simulations
Molecular modeling of the binding between Mpro and compound 2d was carried
out by using an in-house version of the docking engine AutoDock Vina 1.1.2 [33]. The
protocol followed was analogous to the one that some of us already used to assess the
binding to the same protein of quercetin and its glycoside derivative rutin [13,14]. In brief,
Mpro was modeled on the basis of the crystallographic structures 6Y2E and 6Y2F [29],
deposited in the Protein Data Bank (PDB), which report the protein in an unliganded
form and complexed with an α-ketoamide inhibitor, respectively. Compound 2d was
modeled starting from quercetin and attaching in silico the chemical adduct. An energy
minimization of its structure was carried out by using the universal force field (UFF) [34].
The presence of the metal in compound 2d was taken into account by modifying
the docking protocol as detailed hereafter. The ordinary version of AutoDock Vina treats
selenium as sulfur; therefore an updated version was used in which the correct simulation
parameters were included. The well depths of the van der Waals interactions (εi = 0.291
kcal/mol) and the atomic solvation parameter (solpar
=
−
1.1
×
10–3) were imported from
the extended scoring function of AutoDock [35]; these terms in AutoDock Vina take part
also in the evaluation of the electrostatic interactions. Furthermore, the correct covalent
radius of selenium (rcov = 1.16 Å) was used [36]. A blind search on the whole protein
volume was performed, with an exhaustiveness 16 times larger than the default value [37].
3.6. Cells and Viruses
Vero cells (Cercopithecus aethiops; kidney epithelial; ATCC CCL-81) were cultured in
Dulbecco’s MEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5%
fetal bovine serum (heat-inactivated; Thermo Fisher Scientific, Waltham, MA, USA) and
antibiotics: penicillin (100 U/mL) and streptomycin (100 µg/mL).
Reference SARS-CoV-2 strain 026V-03883 was kindly granted by Christian Drosten,
Charité—Universitätsmedizin, Berlin, Germany, by the European Virus Archive—Global
(EVAg); https://www.european-virus-archive.com/, accessed on 15 April 2021).
All SARS-CoV-2 stocks were generated by infecting monolayers of Vero cells. The cells
◦
were incubated at 37 C under 5% CO2. The virus-containing medium was collected at day
◦
2 post-infection (p.i.), aliquoted, and stored at
cells were prepared in the same manner.
−
80 C. Control samples from mock-infected
Virus yields were assessed by titration on fully confluent cells in 96-well plates ac-
cording to the method of Reed and Muench [38]. Plates were incubated at 37 ◦C, and the
cytopathic effect (CPE) was scored by observation under an inverted microscope.

Int. J. Mol. Sci. 2021, 22, 7048
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3.7. Evaluation of Viral Infection
Vero cells were seeded in culture medium on 96-well plates (TPP, Trasadingen, Switzer-
land) at 2 days before infection. Subconfluent cells were infected with SARS-CoV-2 viruses
at 1600 50% tissue culture infectious dose (TCID50)/mL. Infection was performed in the
presence of test compounds dissolved in DMSO (100 mM stocks) or with DMSO as a
◦
control. After 2 h of incubation at 37 C, cells were rinsed twice in PBS, and a fresh medium
with the given inhibitor or solvent was added. The infection was carried out for 2 days,
and the cytopathic effect (CPE) was assessed using the inverted light microscope. Culture
supernatants were collected from wells where CPE reduction was observed.
3.8. Isolation of Nucleic Acids, Reverse Transcription, and Quantitative PCR
A viral DNA/RNA kit (A&A Biotechnology, Gdan´sk, Poland) was used for nucleic
acid isolation from cell culture supernatants. RNA was isolated according to the manufac-
turer’s instructions.
Viral RNA was quantified using quantitative PCR coupled with reverse transcription
(RT-qPCR) (GoTaq Probe 1-Step RT-qPCR System, Promega, Poland) using a CFX96 Touch
real-time PCR detection system (Bio-Rad, Munich, Germany). The reaction was carried
out in the presence of the probes and primers (Fwd: CAC ATT GGC ACC CGC AAT
C; Rev: GAG GAA CGA GAA GAG GCT TG; probe: 6FAM-ACT TCC TCA AGG AAC
AAC ATT GCC A-BHQ-1). The heating scheme was as follows: 15 min at 45 ◦C and 2 min
◦
◦
◦
◦
at 95 C, followed by 40 cycles of 15 s at 95 C and 1 min at 58 C or 60 C. In order
to assess the copy number of the N gene, standards were prepared. The PCR product
was amplified and cloned into pTZ57R/T plasmids using an InsTAclone PCR cloning
kit (Thermo Scientific). The resulting plasmid was linearized, and its concentration was
assessed using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Waltham,
MA, USA) and the number of copies was deducted based on the Avogadro constant. The
obtained standards were serially diluted and used as an input for real-time PCR.
3.9. Cell Viability Assay
Cell viability was evaluated using the XTT Cell Viability Assay kit (Biological Indus-
tries, Cromwell, CT, USA) according to the manufacturer’s protocol. Vero, A549ACE2+,
CRFK, HRT-18, LLC-MK2, and HSF cells were cultured on 96-well plates. Cells were incu-
◦
bated with ACF for 24 h at 37 C in an atmosphere containing 5% CO2. After incubation, the
medium was discarded and 100 µL of fresh medium was added to each well. Then, 25 µL
of the activated 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide
(XTT) solution was added and samples were incubated for 2 h at 37 ◦C. The absorbance
(λ = 450 nm) was measured using a Spectra MAX 250 spectrophotometer (Molecular De-
vices, San Jose, CA, USA). The obtained results were normalized to the control samples,
where cell viability was set to 100%.
3.10. Statistical Analyses
The results are expressed as mean
±
standard error of the mean (SEM). For the
determination of the half-maximal inhibitory concentration (IC50), a dose–response curve
fit using a nonlinear regression model was performed using GraphPad 8.0 software. To
determine the significance of the results obtained, the Kruskal–Wallis non-parametric test
was used and p-values < 0.05 were considered significant.
4. Conclusions
In this communication, we reported a new metal-free protocol for the synthesis of a
small library of selenoquercetin analogues. Then, the obtained compounds were screened
in vitro as SARS-CoV-2 main protease inhibitors through a recently implemented method.
The most significant derivatives are endowed with a low micromolar potency which is
better than that shown by quercetin, highlighting that the seleno-functionalization as well
as the presence of the catechol unity improves the Mpro inhibitory activity. The most

Int. J. Mol. Sci. 2021, 22, 7048
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effective Mpro inhibitors were further analyzed using an in cellulo model of SARS-CoV-2
infection, identifying compound 2d as a reliable preclinical candidate. In addition, we
demonstrated for the first time that quercetin 1, besides being a low micromolar Mpro
inhibitor, shows antiviral activity with IC50 = 194
µM. The introduction of the para-tolyl-
selenyl fragment at the quercetin C-8 position led to a 24-fold improvement of the antiviral
potency (IC50 = 8 µM), in good qualitative agreement with the indications deriving from
the Mpro inhibition assay. Finally, experimental and in silico investigation suggested that
the inhibition occurs according to a non-covalent interaction in which the selenium atom is
involved in a hydrogen bond with Gln189, helping to anchor the quercetin scaffold and
blocking the catalytic dyad His41/Cys145.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/ijms22137048/s1.
Author Contributions: Conceptualization: C.S., L.S., K.P., A.K.-P. and A.V.-C.; methodology:
F.M. (Francesca Marini), C.S., P.B., A.D., D.A., E.J.L., D.O.-A., A.J.-A., L.C.-L., B.R., O.A. and A.V.-C.;
software, B.R., O.A. and A.V.-C.; formal analysis, E.J.L., D.O.-A., A.J.-A., L.C.-L., S.V., B.R., O.A.
and A.V.-C.; investigation: F.M. (Francesca Mangiavacchi), E.M., O.R., L.B., K.P., A.K.-P., S.F.F., L.A.,
D.O.-A., A.J.-A., L.C.-L., S.V. and B.R.; resources: C.S., F.M., O.A., A.V.-C. and E.J.L.; data curation:
L.S., K.P., A.K.-P., A.V.-C. and E.J.L.; visualization: O.R.; writing—original draft preparation, F.M.
(Francesca Mangiavacchi), L.B., K.P., A.K.-P. and E.J.L.; writing—review and editing, K.P., A.K.-P.,
D.A., E.J.L., B.R., O.A., A.V.-C., L.S. and C.S.; supervision, C.S., L.S., K.P., A.K.-P., B.R., O.A. and
A.V.-C.; funding acquisition, C.S., K.P., O.A., A.V.-C. and E.J.L. All authors have read and agreed to
the published version of the manuscript.
Funding: This work was supported by the University of Perugia “Fondo per la Ricerca di Base
2019”(C.S.); Fundación hna (to A.V.C. and O.A.); Miguel Servet Program from Instituto de Salud Car-
los III (CPII13/00017 to O.A.); Fondo de Investigaciones Sanitarias from the Instituto de Salud Carlos
III, and the European Union (ERDF/ESF, “Investing in your future”) (PI18/00349 to O.A. and an FIS
Research Contract to L.C.L.); Spanish Ministry of Economy and Competitiveness (BFU2016-78232-P
to A.V.C.); Spanish Ministry of Science, Innovation and Universities (FPI Predoctoral Research Con-
tract BES-2017-080739 to D.O.A.); Diputación General de Aragón (Predoctoral Research Contract
2019 to A.J.A., “Protein Targets and Bioactive Compounds Group” E45_20R to A.V.C., “Digestive
Pathology Group” B25_20R to O.A.); and Centro de Investigación Biomédica en Red en Enfermedades
Hepáticas y Digestivas (CIBERehd). This study was financed in part by the Coordenação de Aper-
feiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001. FAPERGS, CNPq,
and FINEP are acknowledged for financial support. CNPq is also acknowledged for fellowships to
E.J.L. and D.A. This work was supported by the subsidy from the Polish Ministry of Science and
Higher Education for research on the SARS-CoV-2 and a grant from the National Science Center
UMO-2017/27/B/NZ6/02488 to K.P.
Data Availability Statement: All the chemical data are within the article and its supporting in-
formation. All the biological data on antiviral activity are within the article and its supporting
information.
Acknowledgments: The work described in this paper is part of the scientific activity of the interna-
tional multidisciplinary “SeS Redox and Catalysis” network. The proteomic analysis was performed
in the Proteomics Platform from Servicios Científico Técnicos (IACS—Universidad de Zaragoza) that
belongs to ProteoRed, PRB3-ISCIII.
Conflicts of Interest: The authors declare no conflict of interest.
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