Targeting sars-cov-2 polymerase with new nucleoside analogues

Article abstract of DOI:10.3390/molecules26113461

Despite the fact that COVID-19 vaccines are already available on the market, there have not been any effective FDA-approved drugs to treat this disease. There are several already known drugs that through drug repositioning have shown an inhibitory activity against SARS-CoV-2 RNA-dependent RNA polymerase. These drugs are included in the family of nucleoside analogues. In our efforts, we synthesized a group of new nucleoside analogues, which are modified at the sugar moiety that is replaced by a quinazoline entity. Different nucleobase derivatives are used in order to increase the inhibition. Five new nucleoside analogues were evaluated with in vitro assays for targeting polymerase of SARS-CoV-2.

Full text of DOI:10.3390/molecules26113461

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molecules
Article
Targeting SARS-CoV-2 Polymerase with New
Nucleoside Analogues
Vasiliki Daikopoulou 1, Panagiotis Apostolou 1 , Sofia Mourati 1, Ioanna Vlachou 1, Maria Gougousi 1
and Ioannis Papasotiriou 2,
*
1
Research Genetic Cancer Centre S.A. Industrial Area of Florina, GR53100 Florina, Greece;
daikopoulou.vasiliki@rgcc-genlab.com (V.D.); apostolou.panagiotis@rgcc-genlab.com (P.A.);
mourati.sofia@rgcc-genlab.com (S.M.); vlachou.ioanna@rgcc-genlab.com (I.V.);
gougousi.maria@rgcc-genlab.com (M.G.)
Research Genetic Cancer Centre International GmbH, Baarerstrasse, 95, 6301 Zug, Switzerland
Correspondence: papasotiriou.ioannis@rgcc-international.com
2
*
Abstract: Despite the fact that COVID-19 vaccines are already available on the market, there have
not been any effective FDA-approved drugs to treat this disease. There are several already known
drugs that through drug repositioning have shown an inhibitory activity against SARS-CoV-2 RNA-
dependent RNA polymerase. These drugs are included in the family of nucleoside analogues. In
our efforts, we synthesized a group of new nucleoside analogues, which are modified at the sugar
moiety that is replaced by a quinazoline entity. Different nucleobase derivatives are used in order
to increase the inhibition. Five new nucleoside analogues were evaluated with in vitro assays for
targeting polymerase of SARS-CoV-2.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Keywords: nucleoside derivatives; COVID-19; quinazoline moiety
Citation: Daikopoulou, V.;
Apostolou, P.; Mourati, S.; Vlachou, I.;
Gougousi, M.; Papasotiriou, I.
1. Introduction
Targeting SARS-CoV-2 Polymerase
with New Nucleoside Analogues.
Molecules 2021, 26, 3461. https://
doi.org/10.3390/molecules26113461
The ongoing pandemic COVID-19 has already infected more than 142 million people
worldwide resulting in over 3 million reported deaths, with significant social and eco-
nomic repercussions [1]. This disease is caused by severe acute respiratory syndrome
coronavirus (SARS-CoV-2), there is no specific drug for inhibiting the replication of this
virus. Coronaviruses are a large family of single-stranded RNA viruses (+ssRNA) that
Academic Editor: Diego Muñoz-Torrero
infect different animal species [2,3]. The SARS-CoV-2 RNA-dependent RNA polymerase
Received: 5 May 2021
Accepted: 2 June 2021
Published: 7 June 2021
(RdRp) is responsible for the replication of the virus. It can cause sickness with symptoms
ranging from a common cold to severe symptoms and death. Their positive sense RNA
acts like a messenger RNA (mRNA) and can be directly translated. However, these viruses
encode for RNA-dependent RNA polymerases in order to accomplish their replication, by
synthesizing a negative sense RNA strand which then acts as the template for the positive
sense strand synthesis. Therefore, RdRp could be used as a potential drug target [4].
There are several ways to inhibit the replication process of the virus. The first is
through the inhibition of the polymerase itself; therefore, no replication is taking place.
In another way, specific nucleoside analogues are used, which in turn does not allow the
polymerization of the viral RNA. Nucleoside analogues are modified, and upon being incor-
porated to the nucleotide chain, they cannot form bonds with the next-added nucleotides,
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terminating the reaction [
group, avoiding the formation of the 30-50 phosphodiester bond. Such inhibitors have
been widely used both for DNA as well as for RNA polymerases [ ]. The coronavirus
RdRp could be targeted by the biggest class of small molecule-based antivirals which is
the nucleoside analogues [ 10]. In recent studies many nucleoside analogues have been
reported as candidate inhibitors of the SARS-CoV-2 RdRp [11 12]. Remdesivir ( ) [13 15],
5
]. In general, nucleoside analogue inhibitors lack the 30-OH
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Attribution (CC BY) license (https://
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4.0/).
6,7
8
–
,
1
–
Sofosbuvir (2) [16] and Ribavirin (3) [17
,18] (Figure 1) have been evaluated against RdRp.
Molecules 2021, 26, 3461. https://doi.org/10.3390/molecules26113461
https://www.mdpi.com/journal/molecules

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Molecules 2021, 26, 3461
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Emtricitabine combined with Tenofovir, that are also known nucleoside analogues were
assessed for their antiviral activity [19]. In fact, clinical trials have been designed to start
using this combination of drugs in patients [20,21]. Furthermore, it is important to highlight
that besides targeting RdRp, several recent proposals have been published for the treatment
of SARS-CoV-2 targeting the SARS-CoV-2 Main Protease [22,23].
Figure 1. Remdesivir (1), Sofosbuvir (2) and Ribavirin (3).
Nucleoside analogue inhibitors are chemically synthesized derivatives of a nucleobase
conjugated with a sugar moiety in which either the heterocyclic ring or the sugar moiety has
been modified (Figure 2). Nucleoside analogues could be used as anticancer agents [24
–26],
as antibacterial agents [27,28] and as antiviral agents [29,30].
Aromatic heterocyclic base
Base
N
O
-O P O
O-
Phosphate
O
OHOH
Sugar
Figure 2. Areas for modification on nucleoside analogues.
Previously, we reported the conjugation of an adenine with a quinazoline moiety [31].
Quinazolines are heterocyclic compounds and they have been investigated for their ther-
apeutic potential. They have been evaluated as antimicrobial, anti-inflammatory and as
anticancer agents and it has been proved that they play a significant role in medicinal
chemistry [32–34]. In this study we synthesized nucleoside analogues which consist of
purine and pyrimidine derivatives and a quinazoline moiety. Next, we aimed to identify
whether the different analogues that have been designed and synthesized in our laboratory,
could affect the SARS-CoV-2 polymerase, and particularly the catalytic subunit NSP12.
The evaluation of the nucleosides we usedon in vitro assays, based on synthesis of DNA
or RNA, using RNA as a template, and NSP12, as well as commercial RNA-dependent
RNA polymerases.

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2. Results
2.1. Synthesis of Modified Nucleoside Analogues
Our approach to synthesize a new group of nucleoside analogues was based on
the modification of the sugar moiety into a quinazoline moiety (Figure 3). Nucleoside
derivatives constitute a large class of antiviral agents and quinazoline analogues are
molecules that have shown antiviral activity as well [35,36].
Aromatic heterocyclic base
Base
N
N
R
Quinazoline
moiety
N
O
P
O
O-
Phosphate
O-
Figure 3. Replacement of sugar moiety with a quinazoline moiety for synthesis of new nucleo-
side analogues.
The selective hydrolysis of the commercially available material
according to the literature [37]. The conjugation of quinazoline with different nucleobase
derivatives was performed via nucleophilic aromatic substitution (SNAr). The compound
4, was performed
5
6
was synthesized by the coupling of an adenine with the quinazoline 5 under nitrogen
atmosphere, adding material silica gel as support. The desired product was formed in
41% yield. In order to synthesize compounds
7 and 8, N-acetyl guanine [38] and N-acetyl
cytosine [39] were synthesized accordingly, with already known procedures. The protection
of these two molecules was performed to be directed to the formation of the specific N-C
bond. The synthesis of compound
7 was performed with a yield of 37% and that of the
compound 8 with a yield of 43% (Figure 4).
Figure 4. Synthesis of nucleoside analogues
6
–
8
. Reagents and conditions: (a) sodium hydroxide solution (2% NaOH), r.t.
◦
4 h, (b) for compound
6, adenine, cesium carbonate (Cs2CO3), silica gel, inert atmosphere, 130 C, 3.5 h, (c) for compound
7
,
◦
N-acetyl guanine, cesium carbonate (Cs2CO3), silica gel, inert atmosphere, 130 C, 24 h, (d) for compound
cytosine, cesium carbonate (Cs2CO3), silica gel, inert atmosphere, 130 C, 24 h.
8
, N-acetyl
◦

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A deprotection step was performed in compound
8
in order to remove the acetyl
group and receive the compound 9 in 82% yield (Figure 5).
Figure 5. Deprotection of compound
65 C, 24 h.
8. Reagents and conditions: (a) methylamine solution (40%),
◦
As we have already mentioned, the conjugation is achieved by regioselective N-
arylation via nucleophilic aromatic substitution (SNAr). In order to evaluate whether a
potential group on the quinazoline moiety will affect this step, we decided to insert a
fluorine substituent to check if there is an increase of the yield. Since fluorine is considered
an activated substrate for nucleophilic aromatic substitution, it was deliberately chosen. For
this purpose, the compounds 11 and 12 were synthesized according to the literature. [40,41]
Then, a selective hydrolysis of the quinazoline moiety was achieved and the compound 13
was formed in 55% yield. The conjugation of the adenine with compound 13 took place
overnight and was formed in 53% yield. It is worth mentioning that in this experiment, we
did receive higher yield than the yield in the conjugation without the fluorine substituent
(Figure 6).
◦
Figure 6. Synthesis of compound 14. Reagents and conditions: (a) urea, 150 C, 7 h, (b) POCl3, N, N-dimethylaniline,
◦
◦
110 C, 7 h, (c) sodium hydroxide solution (NaOH 2%), 60 C, 6 h, (d) adenine, cesium carbonate (Cs2CO3), silica gel, inert
◦
atmosphere, 130 C, 24 h.
According to the mechanism of action of a molecule during the inhibition of a poly-
merase, it is crucial that the molecule contains a hydroxyl or a phosphate group. Compounds
6, 7, 8 and 14 contain a hydroxyl group in the quinazoline moiety for this purpose. In order
to evaluate a molecule which contains a phosphate group, we synthesized compound 15
.
The phosphorylation was performed by using phosphorus pentoxide (Figure 7).