Anti-tick-borne encephalitis virus activity of novel uridine glycoconjugates containing amide or/and 1,2,3-triazole moiety in the linker structure

Article abstract of DOI:10.3390/ph13120460

Tick-borne encephalitis virus (TBEV) transmitted by ticks is a pathogen of great medical importance. As still no effective antiviral treatment is available, in the present study, a series of uridine glycoconjugates containing amide or/and 1,2,3-triazole moiety in the linker structure was synthesized and evaluated for the antiviral activity against two strains of TBEV: a highly virulent Hypr strain and less virulent Neudoerfl strain, using standardized previously in vitro assays. Our data have shown that four compounds from the series (18–21) possess strong activity against both TBEV strains. The half maximal inhibitory concentration (IC₅₀) values of compounds 18–21 were between 15.1 and 3.7 μM depending on the virus strain, which along with low cytotoxicity resulted in high values of the selectivity index (SI). The obtained results suggest that these compounds may be promising candidates for further development of new therapies against flaviviruses.

Full text of DOI:10.3390/ph13120460

pharmaceuticals
Article
Anti-Tick-Borne Encephalitis Virus Activity of Novel
Uridine Glycoconjugates Containing Amide or/and
1,2,3-Triazole Moiety in the Linker Structure
Gabriela Brzuska 1 , Gabriela Pastuch-Gawolek 2,3 , Monika Krawczyk 2,3
,
Boguslaw Szewczyk 1 and Ewelina Krol 1,
*
1
Department of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology, University of Gdansk and
Medical University of Gdansk, Abrahama 58, 80-307 Gdansk, Poland;
gabriela.brzuska@phdstud.ug.edu.pl (G.B.); boguslaw.szewczyk@biotech.ug.edu.pl (B.S.)
Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Faculty of Chemistry,
Silesian University of Technology, Krzywoustego 4, 44-100 Gliwice, Poland;
2
gabriela.pastuch@polsl.pl (G.P.-G.); monika.krawczyk@polsl.pl (M.K.)
Biotechnology Centre, Silesian University of Technology, Krzywoustego 8, 44-100 Gliwice, Poland
Correspondence: ewelina.krol@biotech.ug.edu.pl; Tel.: +48-58-523-63-83
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 30 November 2020; Accepted: 11 December 2020; Published: 13 December 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Tick-borne encephalitis virus (TBEV) transmitted by ticks is a pathogen of great medical
importance. As still no effective antiviral treatment is available, in the present study, a series of
uridine glycoconjugates containing amide or/and 1,2,3-triazole moiety in the linker structure was
synthesized and evaluated for the antiviral activity against two strains of TBEV: a highly virulent Hypr
strain and less virulent Neudoerfl strain, using standardized previously in vitro assays. Our data
have shown that four compounds from the series (18–21) possess strong activity against both TBEV
strains. The half maximal inhibitory concentration (IC50) values of compounds 18–21 were between
15.1 and 3.7 µM depending on the virus strain, which along with low cytotoxicity resulted in high
values of the selectivity index (SI). The obtained results suggest that these compounds may be
promising candidates for further development of new therapies against flaviviruses.
Keywords: uridine glycoconjugates; antiviral activity; glycosyltransferase inhibitors; flavivirus;
tick-borne encephalitis virus
1. Introduction
As the present pandemic situation shows, emerging viral infections cause many adverse effects,
not only directly on human health, but also on all aspects of our lives. One of such potential dangers of
viral origin are flaviviruses possessing positive-sense RNA genomes. Many viruses of the Flaviviridae
family are associated with human diseases of great medical importance. In the past few decades
flaviviruses, due to the climate change, have tremendously widened their range and have longer
periods of activity, resulting in over 400 million infections per year, due to which they are a constant
threat to public health [1]. Flaviviruses infect not only humans, but also many other animal hosts,
like birds, rodents, non-human primates and many other mammals. Their transmission is highly
correlated with the expansion of the geographical range of their insect vectors—ticks and mosquitos.
Recently, many epidemics and outbreaks of emerging and re-emerging flaviviruses (such as Zika virus
(ZIKV), dengue virus (DENV), yellow fever virus (YFV) or West Nile virus (WNV)) have been observed,
which has raised the importance of the research concerning treatment and prevention methods [2–4].
Pharmaceuticals 2020, 13, 460; doi:10.3390/ph13120460
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Within the group of tick-transmitted viruses, tick-borne encephalitis virus (TBEV) is one of the
most pathogenic flaviviruses affecting the human central nervous system. It causes a seasonal disorder
called tick-borne encephalitis (TBE), which may lead to severe and long-lasting complications including
meningitis, meningoencephalitis, partial paralysis, coma or even death [
30 countries of Europe and Asia and new endemic centers are constantly reported [
severalTBEVsubtypesarecirculating, withthreemainsubtypes: European(TBEV-Eu), Siberian(TBEV-Sib),
Far Eastern (TBEV-FE) and two newly discovered: Baikalian (TBEV-Bkl) and Himalayan (TBEV-Him) [ ].
5]. TBEV is endemic in over
6]. In these regions
7,8
These subtypes differ not only in the geographical distribution, which depends on their tick vector
(Ixodes ricinus—TBEV-Eu, Ixodes persulcatus—TBEV-FE and TBEV-Sib), but most importantly in the
severity of the TBE course and fatality rate. A bite of an TBEV infected tick is the most common route
of human infection; however, other routes like consumption of unpasteurized milk or milk products
from infected ruminants are also possible [
vaccination is not mandatory in most of the affected countries resulting in around 13,000 annual cases
worldwide [ 10]. Moreover, up to this date no effective and licensed treatment for TBEV infection is
available, although different antiviral compounds are being tested in the laboratories [11 14]. For the
9]. Although few vaccines against TBEV are on the market,
6,
–
treatment of infected patients, the only strategy being implemented in hospitals is to alleviate the symptoms.
In light of these facts, the research on the new antiviral drugs is highly justified.
From the structural point of view, members of the Flavivirus genus share several morphological
similarities. TBEV is a small, enveloped virus with a mean diameter of 50 nm. The genome, surrounded
by the capsid (C) is in the form of a ssRNA (+), which encodes a single polyprotein cleaved to three
structural: C, prM/M, E and five non-structural proteins (NS1–5) [15]. The prM/M and E proteins are
anchored in the envelope of the virus. The role of the E protein in the TBEV cycle is well characterized.
It mediates entry via receptor mediated endocytosis, and contains the major antigenic epitopes used
for generation of protective immune response [16]. Three of the flaviviral proteins usually possess at
least one N-glycosylation site: prM, E and NS1 and these proteins play a crucial role during the virion
assembly and maturation, entry to the cells, immune evasion and replication. The role of glycans in
the flavivirus replication cycle is not fully understood. For TBEV, it has been shown that removal
of the glycosylation site in the E protein leads to the reduction in infectivity of secreted virions [17].
Therefore, it is important to examine the role of glycans in each flavivirus infection separately and to
test the activity of glycosylation inhibitors as potential antiviral drugs.
Various glycosyltransferases (GT) may be utilized by flaviviruses to attach specific glycans onto
their proteins. Inhibition of one of the GT with synthetic compounds may be a promising strategy for
antiviral treatment for different flaviviruses. Previously, we have shown the antiviral activity of different
uridine derivatives targeting GT against members of the Flaviviridae family—classical swine fever
virus (CSFV), hepatitis C virus (HCV), and also against TBEV [18–20]. Moreover, the activity of these
compounds was tested against influenza virus (Orthomyxoviridae family member) [21]. The inhibitory
effect of these compounds was associated with the impaired maturation and secretion of infectious
viruses. These studies showed that GT inhibitors could be used as broad-spectrum antiviral drugs
against different human and animal viruses.
In this study, we present the design, synthesis and antiviral activity of a novel type of
uridine glycoconjugates, containing amide or/and 1,2,3-triazole moiety in the linker structure,
against two strains of tick-borne encephalitis virus, a main cause of human neuroinfections.
2. Results
2.1. Synthesis of Glycoconjugates 13–23
We designed compounds that can compete with natural glycosyltransferase substrates for sites in
their active centers. As previously published, natural donor-type substrates contain in their structure a
sugar unit linked via a diphosphate moiety to uridine [22,23]. Conventional modification methods of
glycosyltransferase inhibitors may include the replacement of the diphosphate moiety and connection of the

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sugar unit via a non-charged linker to the uridine part. Such a linker should be capable of coordinating the
divalent metal ion (usually magnesium or manganese) bound in the active site of the enzyme. In the tested
glycoconjugates, the diphosphate moiety was replaced with an amide linker between carbohydrate and
uridine moiety (glycoconjugate structure type I, Figure 1) or with a methylene 1,2,3-triazolyl part linked to
a sugar or uridine moiety via an amide or ether linkage (glycoconjugate structures types II-IV, Figure 1).
Recently, the results of studies showing that a completely deprotected type IV glycoconjugate is able to
inhibit the activity of β-1,4-galactosyltransferase I (β4GalT) were reported [24]. There are also reports of
the synthesis of a (triazolyl) methyl amide-linked disaccharide nucleosides as analogues of nucleoside
diphosphate sugars. Some of the obtained 60-isonucleosides and triazole-containing glycoderivatives
displayed acetylcholinesterase inhibitory activities [25]. Nucleoside triphosphate mimetic, where the
phosphate residues-containing chain was replaced by an uncharged methylene-triazole moiety, are also
known to have occurred. These compounds have been evaluated as competitive inhibitors of Bacillus anthracis
pantothenate kinase [26]. Therefore, it can be assumed that the 1,2,3-triazole ring in the linker structure
is an important element for glycoconjugate activity. The aim of the present work was to investigate the
influence of the linker structure containing the 1,2,3-triazole system on the biological activity of the obtained
connections. Glycoconjugates structure type III have the reverse direction of linking reactive moieties
(as a result of the reaction between sugar azides and 50-propargyl uridine derivative) compared to the
glycoconjugate structure type IV. In turn, in the glycoconjugate structure of type II, an additional amide
moiety was introduced between the methylene 1,2,3-triazolyl part and uridine compared to the structure
type III. It was presumed that insertion into a linker of an additional amide bond, the presence of which
is observed in many biologically active compounds should improve glycoconjugates’ chelating ability,
and thus their binding ability at the metal-dependent enzyme active center [27].
Figure 1. The general structures of glycoconjugates, analogues of the natural β4GalT substrate—UDP-Gal.
The key reactions to obtain the planned glycoconjugate structures are the condensations
between the amine and carboxyl derivatives leading to the formation of an amide bond as well
as copper-catalyzed 1,3-dipolar azido-alkyne cycloaddition to form a 1,2,3-triazole system. Therefore,

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it was necessary to prepare appropriate sugar and uridine derivatives, which were then be used in the
synthesis of glycoconjugates.
The first part of the synthesis was devoted to the sugar building blocks necessary for glycoconjugates
synthesis. The synthetic pathways leading to the formation of sugar derivatives are presented in Scheme 1.
All substrates were prepared according to the previously published procedures involving the acetylation
of free sugars 1a or 2a and conversion of per-O-acetylated derivatives 1b or 2b into the corresponding
glycosyl bromides 1c or 2c
leading to obtain 2,3,4,6-tetra-O-acetyl-
benzyl protecting groups, it was necessary to remove the acetyls from compound
conditions [29] which allowed the attainment of deprotected -glycosyl azide which was benzylated with
benzyl bromide in the presence of sodium hydride in anhydrous DMF [30]. -D-Glycopyranosyl amines
3–5 were obtained from the corresponding glycosyl azides or 5a through a hydrogenation reaction in
[
22
,
28]. The glycosyl bromides were used immediately for further reactions
-glycosyl azides and 23]. To obtain glucosyl azide 5a with
under Zemplén
β
1
2 [
1
β
β
1, 2
a Parr apparatus using palladium hydroxide deposited on activated carbon [31].
Scheme 1. Synthesis of sugar derivatives. Reagents and conditions: (i) CH3COONa, Ac2O, b.p.,
1 h; (ii) CH3COOH, 33% HBr/AcOH, r.t. 1 h; (iii) NaN3, TBASH, CHCl3/NaHCO3, r.t. 2 h; (iv) 20%
Pd(OH)2/C, THF:EtOH (2:1, v/v), H2, 1.5 bar, 2 h; (v) 1. NaOMe, MeOH, r.t. 20 min; 2. Amberlyst-15, 3.
BnBr, NaH, DMF, r.t., 24 h; (vi) propargyl alcohol, BF3·Et2O, CH2Cl2, r.t., 1 h.
Sugar derivatives
6 and 7 in which the alkynyl moiety was introduced were prepared by reacting
per-O-acetylated D-glucose 1a or D-galactose 1b with propargyl alcohol in the presence of a Lewis acid as a
catalyst[32]. Theacetylneighboring-groupparticipationattheC-2positionofthesugarensuredtheformation
of only products of the
β-configuration. As a result, propargyl 2,3,4,6-tetra-O-acetyl-β-D-glycopyranosides
6 and 7 were obtained with good yields (89% and 83%, respectively).
The synthesis of uridine derivatives 8–10, a second component necessary for the construction
of glycoconjugates, as well as compounds 11a and 12a were described in the earlier works of our
group [18,24]. The counterpart of uridine derivative 10 with TBDMS-protecting groups was obtained by
adding propargyl amine to the compound 11a using DCC as an amide coupling agent (Scheme 2) [33].
The product 11 was obtained in the form of a colorless oil with a yield of 75%.
Scheme 2. Synthesis of uridine derivative 11. Reagents and conditions: (i) propargylamine, DCC,
CH2Cl2, r.t, 24 h.

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The last uridine derivative was obtained by reacting compound 12a with propargyl bromide in
anhydrous tetrahydrofuran using sodium anhydride as a base (Scheme 3). Carrying out the reaction
in an ultrasonic bath increases the contact surface of the reactants, and thus accelerates the reaction.
Thanks to the procedure used, the product 12 was obtained with a yield of 59%.
Scheme 3. Synthesis of uridine derivative 12. Reagents and Conditions: (i) propargyl bromide, NaH,
THF, r.t., ultrasonic bath, 4 h.
The synthesis of glycoconjugates was started with glycoconjugates 13–15 which were obtained in the
condensation reaction between 1-aminosugars 3–5 and uridine derivative 8 as an acid donor, using generated
in situ an amide coupling agent (4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methylmorpholinium chloride)
(DMTMM). The procedure worked well for glycoconjugates 13 and 14 obtained from acetyl protected
sugar derivatives (80% and 86% yield respectively, Scheme 4), however in the case of 1-aminosugar
with benzyl protection, it was possible to obtain glycoconjugate 15 only with a moderate yield
(42%). A possible explanation for this may be the excessively high reactivity of the amino sugar
with electron-donating protecting groups, which leads to the formation, in addition to the desired
glycoconjugate, also of by-products. Indeed, the spots, not only from the formed glycoconjugate,
but also from other sugar-containing products were observed on the thin layer chromatography (TLC)
plates used to follow the reaction progress.
Scheme 4. Synthesis of glycoconjugates type I. Reagents and conditions: (i) CDMT, NMM, MeOH, r.t., 5 h.
Other glycoconjugates, which were assumed to have a 1,2,3-triazole system in the linker structure
between the sugar fragment and uridine, were obtained using copper-catalyzed azide-alkyne 1,3-dipolar
cycloaddition (CuAAC) [34,35]. For the preparation of glycoconjugates 16–19 CuAAC reactions between
1-azido sugars or and uridine derivatives 10 or 11 were performed (Scheme 5). As the result of the
1
2
reactions, glycoconjugates 16–19 were obtained with good yields. In order to be able to assess whether
the amide bond introduced into the linker structure has a significant influence on the biological activity
of the final compounds, glycoconjugates 20 and 21 were prepared in parallel in which the amide bond
in the uridine fragment has been replaced with a linkage via an oxygen atom.

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Scheme 5. Synthesis of glycoconjugates type II and type III. Reagents and Conditions: (i) CuSO4
NaASC, THF/i-PrOH, H2O, r.t.
·5H2O,
The last group of type IV glycoconjugates was obtained in order to check whether the reverse
orientation of the 1,2,3-triazole system in the linker structure has a significant influence on the biological
activity of these connections. In case of the synthesis of glycoconjugates 22 and 23, propargyl glycosides
6
and 7 and the uridine derivative 9
containing an azide moiety in the C-50 position were used as
substrates for the CuAAC reaction (Scheme 6).
Scheme 6. Synthesis of glycoconjugates type IV. Reagents and Conditions: (i) CuSO4
THF/i-PrOH, H2O, r.t.
·
5H2O, NaASC,
All the carried out CuAAC reactions proceeded with good or very good yields and led to the formation
of only derivatives containing 1,4-disubstituted 1,2,3-triazole. The structures of all glycoconjugates were
confirmed by means of nuclear magnetic resonance (NMR) and high resolution mass spectroscopy (HRMS.)
2.2. Biological Evaluation
2.2.1. Antiviral Activity of Uridine Glycoconjugates Against Tick-Borne Encephalitis Virus
As the initial steps of the biological evaluation of synthesized compounds we performed
cytotoxicity studies according to the established method [19]. Cytotoxicity was analyzed using
MTS-based cell proliferation assay in the permissive for TBEV—A549 cell line. Cytotoxic concentration
(CC50) values for each compound were estimated in order to choose non-toxic doses for further antiviral
activity studies. CC50 values for the compounds 13–23 were 129, 114, 153, 136, 137, 69, 77, 184, 116,
173 and 157 µM, respectively. All of the uridine glycoconjugates showed dose-dependent cytotoxic

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effects with relatively moderate level of toxicity; below a concentration of 25
µM all of the compounds
did not significantly influence the viability of the cells.
Preliminary screening of antiviral effects of eleven tested compounds was performed using
two TBEV strains (Hypr and Neudoerfl) by assessing cytopathic effect (CPE) inhibition assay and
plaque reduction assay as previously reported [19]. The compounds were tested at the non-toxic
concentration of 25 µM. TBEV Hypr strain is a highly cytopathic virus causing A549 cell death 96 h
post-infection. To analyze the antiviral activity of the tested compounds, their protective effect on
cell survival was measured using a colorimetric assay. In A549-TBEV infected DMSO-treated cells
the rate of cell death was about 50% (Figure 2). In A549-TBEV, infected cells treated with 25
compounds 18 19 20 and 21, the rate of cell death was significantly decreased. Two compounds
20 and 21) were the most active from the series. TBEV-induced CPE effect was nearly completely
inhibited after treatment with 25 M of both compounds. The cell death was also strongly reduced after
µM of
,
,
(
µ
treatment with two other compounds (18 and 19). The inhibition of cell death was not observed for
other tested compounds. A strong CPE was still observed in A549 cells treated with these compounds,
indicating that these compounds have almost no antiviral activity against TBEV.
Figure 2. The effect of uridine glycoconjugates on tick-borne encephalitis virus (TBEV)-induced
cytopathic effect. A549-infected cells (TBEV Hypr strain (multiplicity of infection (MOI) of 0.1)) were
treated with 25 µM of tested compounds or DMSO. After 48 h, the cell death was measured and
calculated as the percentage of the cell viability. Error bars represent standard deviations from 3 separate
experiments. ns: not significant; ***: p < 0.001 compared to infected DMSO-treated cells by one-way
ANOVA with Dunnett’s post-test.
These results were further confirmed using plaque reduction assay where the dose of 25 µM of
all synthesized compounds was tested for the inhibition of TBEV propagation in A549 cells using the
immunoperoxidase monolayer assay (IPMA). A low multiplicity of infection (0.001) of low cytopathic
Neudoerfl strain was used to detect the single areas of infected cells by the immunostaining of E protein.
The infected DMSO-treated cells were used as positive control. Viral plaques were counted and expressed
as percentage in comparison to the number of plaques detected in the positive control set as 100%.
As shown in Figure 3 compounds 18, 19, 20 and 21 effectively inhibited virus propagation in comparison
to infected DMSO-treated cells. The reduction in size and number of plaques after 48 h compounds
treatment was observed. As in previous experiment compounds 20 and 21 were the most active causing
nearly a complete inhibition of TBEV propagation. Treatment with compounds 13–17 and 22–23 had no
effect on TBEV infection; therefore, these compounds were not used in further analysis.

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Figure 3. The effect of uridine glycoconjugates on TBEV infection in A549 cells. A549 cells were infected
with the TBEV Neudoerfl strain at an MOI of 0.001. At 2 h post-infection (p.i.) the virus was removed,
cells were washed and overlaid with carboxymethylcellulose in Dulbecco’s Modified Eagle’s Medium
with DMSO or various doses of tested compounds. At 48 h p.i., cells were fixed, and infected foci
were visualized by immunostaining with the monoclonal anti-Flavivirus group antigen antibody (4G2).
Viral plaques were counted and expressed as percentage in comparison to the number of plaques
detected in DMSO-treated infected cells set as 100%. Error bars represent standard deviations from
3 separate experiments. ns: not significant; ***: p < 0.001 compared to infected DMSO-treated cells by
one-way ANOVA with Dunnett’s post-test.
2.2.2. Dose-Response Activity of Uridine Glycoconjugates Against Tick-Borne Encephalitis Virus
Four compounds that showed strong TBEV antiviral activity in the initial experiments were further
evaluated to determine the dose-response inhibitory effect. Neudoerfl TBEV-infected monolayers of
A549 cells (multiplicity of infection (MOI) = 0.001) were incubated with increasing concentrations of
the most active compounds: 18, 19, 20, 21 (0–25 µM) diluted in the overlay medium for 48 h and the
plaque reduction assay described above was performed. Plaques were immunostained and counted.
All four tested compounds showed a dose-dependent reduction in the number of positive infected
foci (Figure 4). Compounds 20 and 21 were the most active because the highest inhibition of plaque
formation was observed in the case of all tested doses of these compounds. At the dose of 25
plaques were detected. The dose of 12.5 M of both compounds significantly reduced the number of
plaques by 96% and 92%, respectively, when compared to infected DMSO-treated cells. Moreover, the
dose of 6.25 M caused 75% and 62% reductions in the number of plaques for compounds 20 and 21
respectively. For less active compounds 18 and 19, the decrease in the number of plaques at the highest
doses of 25 and 12.5 M was also observed. An amount of 25 M of both compounds reduced the
number of plaques by 98% and 81%, respectively.
Next, compounds 18–21 have been tested for their effect on the infectious virus titers in a dose-response
assay. A549 cells were infected with low MOI of the virus (0.001, Neudoerfl strain) and treated with 0–25
µM no
µ
µ
,
µ
µ
µM
concentrations of compounds. The culture supernatants collected 72 h p.i. were used to determine the viral
titers using the plaque assay. The results were used to determine half-maximal inhibitory concentration
(IC50) values and to calculate selectivity index (SI) values (CC50/IC50 ratio) shown in Table 1.
The titer for the positive control (TBEV collected from non-treated cells) was 1
unit/mL (PFU/mL). Four tested compounds (18 19 20 and 21) significantly reduced TBEV titers in a
dose-dependent manner confirming their anti-TBEV activity (Figure 5). Compounds 20 and 21 were
×
107 plaque-forming
,
,
the most active. After treatment with 25
virus was detected. Treatment with 6.25
µ
M of both compounds and 12.5
µM of compound 20 no
µM of compound 20 and 12.5 M of compound 21 caused
µ
103–104-fold reduction in viral titer, respectively. Compounds 18 and 19 caused nearly 103–106-fold
reduction in viral titer after treatment with 25 and 12.5
µM. The highest IC50 value—3.7 µM was
obtained for compound 20 and due to its low cytotoxicity it reached a high SI of 49.7.

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Figure 4. The effect of uridine glycoconjugates on TBEV infection in A549 cells. A549 cells were infected
with the TBEV Neudoerfl strain at an MOI of 0.001. At 2 h p.i. the virus was removed, cells were
washed and overlaid with carboxymethylcellulose in Dulbecco’s Modified Eagle’s Medium with DMSO
or various doses of tested compounds (0–25 µM). At 48 h p.i., cells were fixed, and infected foci were
visualized by immunostaining with the monoclonal anti-Flavivirus group antigen antibody (4G2).
Viral plaques were counted and expressed as percentage in comparison to the number of plaques
detected in DMSO-treated infected cells set as 100%. Error bars represent standard deviations from
3 separate experiments.
Table 1. Antiviral activity of uridine glycoconjugates 18–21 against TBEV Neudoerfl strain.
Type of Uridine Glycoconjugate Structure
II
Compound
CC50 (µM) a
SI c
7.4
IC50 (µM) b
18
19
20
21
69
77
9.3
15.1
3.7
5.1
184
116
49.7
24.7
III
4.7
a
Concentration of the compound causing reduction in cell viability by 50%; b Concentration to reduce infectious
viral titers by 50%; c Selectivity index (SI): CC50/IC50
.
Figure 5. The effect of uridine glycoconjugates 18
with the TBEV Neudoerfl strain at an MOI of 0.1 and treated with various doses of tested compounds
(0–25 M). At 72 h p.i., culture supernatants were collected to determine TBEV titers by plaque assay.
Error bars represent standard deviations from 3 separate experiments.
, 19, 20 and 21 on TBEV titers. A549 cells were infected
µ

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2.2.3. The Activity of Uridine Glycoconjugates on Protein Synthesis
Previously, we reported that the synthesis of viral glycoproteins of CSFV and influenza virus is
impaired after treatment with uridine derivatives [36 37]. The uridine glycoconjugates described in
,
this report were also designed to target glycosyltransferases, therefore we assessed the influence of
tested compounds on TBEV glycoprotein production using Western blot analysis of TBEV Neudoerfl
strain infected and treated cells lysates. A dose-dependent reduction in the production of E protein was
observed after treatment with compounds 18
shown in Figure 6. To our surprise, all tested doses of compound 20 (25–1.5625
,
19 and 21. The representative results for compound 21 are
M) caused the complete
µ
inhibition of viral protein synthesis as E protein was not detected in Western blot analysis (Figure 6).
After treatment with each of tested compounds, the non-glycosylated and under-glycosylated form of
E protein were not detected probably due to their degradation.
Figure 6. Analysis of TBEV E glycoprotein synthesis after treatment with uridine glycoconjugates.
A549 cells were infected with TBEV Neudoerfl strain, MOI of 0,1. At 2 h p.i., cells were treated with
various doses of compound 20, 21 or DMSO diluted in the medium for 48 h. Cell lysates were prepared
and separated by SDS-PAGE (gradient 10–20% polyacrylamide). The E protein was detected in Western
blot analysis using monoclonal anti-Flavivirus group antigen antibody (4G2).
3. Discussion
Due to the continuous changes of the global climate, which influences the geographical distribution of
ticks and mosquitoes, flaviviruses are an expanding threat to the human population. New endemic centers
and outbreaks of emerging and re-emerging flaviviruses are often reported. Although, vaccines against some
flaviviruses are commercially available e.g., anti-TBEV and anti-YFV, vaccination is not mandatory, or it is
not 100% effective. Moreover, in spite of much effort put in the development of the effective antiviral therapy
against flaviviruses, no drugs were approved for clinical use. Tick-borne encephalitis virus causing the most
important and serious neurological infections is one of the most pathogenic flaviviruses constituting a public
health problem in Europe and Asia. Several thousand cases of TBE are registered annually, however due to
the large number of asymptomatic infections the scale is much wider.
N-glycans attached to flavivirus proteins play multiple roles in the replication cycle including
attachment to the host cell, assembly and secretion of virions. Glycosylated E protein of flaviviruses may
interact with cell surface lectins during the attachment step. The interaction of C-type lectin dendritic
cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and DC-SIGN-related
protein (DC-SIGNR) with E glycoprotein of many flaviviruses: ZIKV, WNV, DENV and Japanese
encephalitis virus (JEV) was reported [38–42]. The interaction with DC-SIGN and DC-SIGNR depends on
the virus, presence of the glycan as well as on the complexity of the glycan chain. However, these type of
lectins are expressed only on the macrophages and dendritic cells (DCs) or on microvascular endothelial
cells [43]. Another study showed that glycosylation of ZIKV E protein plays a role during attachment
to other cell types like A549 cell line, but is dispensable for infection in Vero cells [44]. Both cell lines
lack DC-SIGN or DC-SIGNR, which may suggest that other lectins may be involved in the attachment
of virus to some cell types. Moreover, glycosylation of both flavivirus envelope proteins prM and E
could be also a crucial factor for proper folding, assembly and egress through the secretory pathway,
although involvement of glycans in these processes has not been fully understood. The ablation of
the N-glycosylation site in the prM or E protein of TBEV, ZIKV or WNV influences the expression and

Pharmaceuticals 2020, 13, 460
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secretion of virus-like particles or subviral particles [45–47]. These reports suggest that glycan chains may
stabilize formation of E protein dimers or may interact with some chaperone-like proteins during the
transport of virions through the secretory compartments. For TBEV it has been shown that inhibition of
N-glycosylation of E protein or glucose trimming of the carbohydrate side chain of this protein results in
the decrease in VLPs secretion, suggesting the role of N-glycans in assembly and/or secretion step [48].
In another study, it has been proven that the glycan associated with TBEV E-154 glycosylation site
plays an important role in the secretion of VLPs from mammalian cells, however the glycosylation of
the prM protein plays a less important role in this process [45]. Moreover, the impairment of TBEV E
protein glycosylation did not affect the total level of secreted E protein in mammalian cells, however the
conformation structure of the protein was affected resulting in the reduction in infectivity of secreted
virions also in a mice model. In contrast, the deletion of the glycosylation site of E protein did not affect
TBEV growth in tick cells [17]. The significant difference in the carbohydrate profile of TBEV E protein
when grown in human and tick cells can be the explanation of this phenomenon [49]. As glycosylation
plays highly crucial, yet multifaceted roles during the flavivirus replication cycle, we anticipate that
targeting of this process may be a promising approach to antiviral therapy.
As the first step of our research, we evaluated the antiviral activity of 11 novel uridine
glycoconjugates containing amide or/and 1,2,3-triazole moiety in the linker structure against TBEV,
a member of the Flavivirus genus. At the initial screening, four compounds (18
, 19, 20, 21) at
the non-toxic dose (25 M) significantly increased cell survival after infection of A549 cells with
µ
a pathogenic strain of TBEV (Hypr strain) (Figure 2). The antiviral activity of all glycoconjugates
was further evaluated in the virus plaque formation assay, using a low pathogenic strain of TBEV
(Neudoerfl strain) (Figure 3). Again, only these four compounds showed strong antiviral activity at
the same dose, reducing the formation of viral plaques. Compounds 18 and 19 belong to the type II
glycoconjugates, while compounds 20 and 21 to type III (Figure 1). It is worth noting that both active
types of glycoconjugates contain a 1,2,3-triazole ring in the linker structure which results from the
CuAAC reaction of glycosyl azides with propargyl derivatives of uridine. The presence of TBDMS
groups in the uridine fragment turned out to be significant factor for the activity. This is evidenced by
the significantly lower activity of glycoconjugates 16 and 17 containing isopropylidene protection in
the uridine fragment, which represent the analogues of active compounds 18 and 19. The presence
of an amide moiety in glycoconjugates 18 and 19 also significantly affects the cytotoxicity of these
compounds, which translates into a reduced selectivity index. When the amide bond in the uridine
fragment was replaced with a linkage by an oxygen atom (compounds 20 and 21), the cytotoxicity
of the glycoconjugates thus obtained decreased two-fold, while their activity increased significantly.
On the other hand, the type of sugar unit present in the glycoconjugates has a little effect on the
observed activity and cytotoxicity.
Dose-dependent studies of the influence of four the most active compounds were performed using
a plaque reduction assay (Figure 4). Moreover, the dose-dependent activity on TBEV titer reduction
were used for the estimation of IC50 values (Figure 5, Table 1). Compound 20 showed the most potent
antiviral activity, reaching 3.7
of compound 21 was also high—4.7
level of TBEV propagation inhibition (IC50 = 9.3 and 15.1
µ
M as IC50 value, which resulted in the highest SI of 49,7. The IC50 value
M. Two other compounds (18 and 19) demonstrated moderate
M, respectively). Our observations are
µ
µ
similar to previously published data, that the lack of N-glycosylation site in the E protein of TBEV
reduces virion secretion out of mammalian cells [17].
To further characterize the activity of tested compounds, we investigated their influence on the
production of E protein using Western blot analysis, as tested compounds were designed to inhibit the
activity of β-1,4-galactosyltransferase I during the late steps of N-glycosylation process. The production of
E protein was affected by compounds studied by us in a dose-dependent manner. The production of viral
proteins was greatly decreased at all tested concentrations as shown for the representative compound
21 (Figure 6). For the most active compound 20, the complete inhibition of viral protein synthesis was
observed at all tested doses of this compound. It is worthwhile to note that neither less glycosylated nor

Pharmaceuticals 2020, 13, 460
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non-glycosylated forms of E protein were detected probably due to the quick degradation process of
non-matured forms. The reduction in the glycoprotein E production after inhibitory treatment can be the
reason for the decrease in TBEV viral production observed in previous experiments.
Summarizing, the four tested compounds (18, 19, 20 and 21) showed significant antiviral activity
against two strains of tick-borne encephalitis virus. On the basis of these results, we conclude that
compounds targeting N-glycosylation process of viral proteins may constitute a new promising
approach in antiviral strategy.
4. Materials and Methods
4.1. General Information
Nuclear magnetic resonance (1H-NMR and 13C-NMR) spectra were determined in CDCl3 or
DMSO-d6 using tetramethyl silane (TMS) as an internal standard and recorded with an Agilent
spectrometer at a frequency of 400 MHz or with a Varian spectrometer at a frequency of 600 MHz. NMR
solvents were purchased from ACROS Organics (Geel, Belgium). Chemical shifts (δ) are given in parts
per million (ppm) and coupling constants (J) are given in Hz. Splitting patterns are designated as follows:
s, singlet; d, doublet; dd, doublet of doublets; t, triplet; dd~t, doublet of doublets resembling a triplet;
ddd, doublet of doublet of doublets; m, multiplet; bs, broad singlet. Optical rotations were measured
with a JASCO P-2000 polarimeter using a sodium lamp (589.3 nm) at room temperature. Melting point
measurements were performed on OptiMelt (MPA 100) Stanford Research Systems. Mass spectra were
recorded with a WATERS LCT Premier XE LC/MS system (high-resolution mass spectrometer equipped
with an electron spray ionization source and a high-resolution orthogonal TOF analyzer). Reactions were
monitored by Thin-Layer Chromatography (TLC) on precoated plates of silica gel 60 F254 (Merck KGaA,
Darmstadt, Germany). TLC plates were inspected under UV light (λ = 254 nm) or charring after spraying
with 10% sulfuric acid in ethanol. Products were purified using column chromatography performed
on Silica Gel 60 (70–230 mesh, Merck KGaA, Darmstadt, Germany) developed with toluene/EtOAc or
CHCl3/MeOH solvent systems in various volume ratios. All evaporations were performed on a rotary
evaporator under reduced pressure at 45 ◦C.
2,3,4,6-Tetra-O-acetyl-
azide ( ) [23], 2,3,4,6-tetra-O-acetyl-
D-galactopyranosyl amine ( ), 2,3,4,6-tetra-O-benzyl-
propargyl 2,3,4,6-tetra-O-acetyl- -D-glucopyranoside
D-galactopyranoside
32], 20,30-O-isopropylidene-uridine-50-carboxylic acid
50-azido-50-deoxy-20,30-O-isopropylidene-uridine ( ) [51], N-propargyl-20,30-O-isopropylideneuridine-
50-amide 24], 20,30-di-O-tert-butyldimethylsilyl-uridine-50-carboxylic acid
10 11a 18],
20,30-di-O-tert-butyldimethylsilyl-uridine (12a) [52] as well as glycoconjugates (13, 14, 16 and 17) [29
β
-D-glucopyranosyl azide (
-D-glucopyranosyl amine (
-D-galactopyranosyl amine (
), propargyl 2,3,4,6-tetra-O-acetyl-
50],
1
), 2,3,4,6-tetra-O-acetyl-
), 2,3,4,6-tetra-O-acetyl-
) [31],
β-D-galactopyranosyl
2
β
3
β-
4
β
5
β
(6
β-
(7
)
[
(8)
[
9
(
)
[
(
) [
]
were prepared according to the respective published procedures. Sugars, uridine and other
used chemicals were purchased from Sigma-Aldrich (Steinheim, Germany), ACROS Organics
(Geel, Belgium) or Avantor (Gliwice, Poland) and were used without purification.
4.2. Chemistry
4.2.1. Procedure for Synthesis of N-Propargyl-20,30-O-Tert-Butyldimethylsilyluridine-50-Amide 11
20,30-Di-O-tert-butyldimethylsilyl-uridine-50-carboxylic acid 11a (224 mg, 0.46 mmol) was
dissolved in dichloromethane (6 mL) followed by addition of DCC (96 mg, 0.46 mmol) and propargyl
amine (32 µL, 0.50 mmol) and mixed for 24 h at r.t. The reaction progress was monitored on TLC
in an eluents system toluene:ethyl acetate (1:1). After the reaction was completed, the reaction
mixture was concentrated under diminished pressure and the crude product was purified by column
chromatography (toluene:ethyl acetate; gradient: 10:1 to 6:1) to give 11 (180 mg, 75%) as a colorless oil.

Pharmaceuticals 2020, 13, 460
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N-propargyl-20,30-di-O-tert-butyldimethylsilyl-uridine-50-amide(11): [α]2D5
=
−
27.7 (c = 2.4, CHCl3),
1H-NMR (CDCl3, 400 MHz)
δ
:
−
0.02, 0.04, 0.12, 0.18 (4s, 12H, CH3Si), 0.85, 0.93 (2s, 6H, (CH3)3C),
2.23 (dd~t, 1H, J = 2.4 Hz, J = 2.4 Hz, CH2CCH), 4.02–4.15 (m, 2H, CH2CCH), 4.26 (dd, 1H, J = 1.2 Hz,
J = 4.3 Hz, H-30), 4.39 (d, 1H, J = 1.2 Hz, H-40), 4.68 (dd, 1H, J = 4.3 Hz, J = 7.2 Hz, H-20), 5.39 (d, 1H,
J = 7.2 Hz, H-10), 5.78 (d, 1H, J = 8.0 Hz, H-5ur), 7.44 (d, 1H, J = 8.0 Hz, H-6ur), 7.77 (t, 1H, J = 5.1 Hz,
NH
CH2). 13C-NMR (CDCl3, 100 MHz)
δ
:
−5.13,
−4.74,
−4.61 (CH3Si), 17.85, 17.99 ((CH3)3
C), 25.67,
25.76 ((
C
H3)3C), 28.80 (NH
C
H2CCH), 70.65, 71.44, 74.73 (NHCH2C
85.24(C-40), 95.34 (C-10), 102.77 (C-5ur), 144.41 (C-6ur), 150.38 (C-2ur), 162.08 (C-4ur), 169.09 (CONH).
C
H, C-20, C-30), 79.03 (NHCH2
CCH),
ESI-HRMS: calcd for C24H41N3O6Si2Na [M + Na]+: m/z 546.2432. Found: m/z 546.2435.
4.2.2. Procedure for Synthesis Of 20,30-Di-O-Tert-Butyldimethylsilyl-50-O-Propargyl-Uridine 12
20,30-Di-O-tert-butyldimethylsilyluridine 12a (482 mg, 1.03 mmol) was dissolved in THF (10 mL)
followed by addition of sodium hydride (130 mg, 5.42 mmol). The flask with the reaction mixture was
placed in an ice bath and stirred for 30 min. Next propargyl bromide (102 µL, 1.18 mmol) was added
and the whole mixture was placed in an ultrasonic bath for 4 h. The reaction progress was monitored
on TLC in an eluents system toluene:ethyl acetate (1:1). After the reaction was completed, methanol
(1 mL) and water (5 mL) were added to the reaction mixture. The product was extracted with ethyl
acetate (3
×
10 mL). The combined organic phases were dried over anhydrous MgSO4, filtered and
concentrated under reduced pressure. The crude product was purified by column chromatography
(toluene:ethyl acetate; gradient: 20:1 to 8:1) to give 12 (309 mg, 59%) as a white solid.
20,30-Di-O-tert-butyldimethylsilyl-50-O-propargyl-uridine (12): [α]D25 = 37.6 (c = 1.0, CHCl3),
1
m.p. = 66–68 ◦C, H-NMR (CDCl3, 400 MHz)
δ
: 0.08, 0.09, 0.12, 0.13 (4s, 12H, CH3Si), 0.90, 0.91
), 3.68 (dd, 1H, J = 2.0 Hz,
(2s, 18H, (CH3)3C), 2.49 (dd~t, 1H, J = 2.4 Hz, J = 2.4 Hz, CH2CC
H
J = 10.6 Hz, H-50b), 3.87 (dd, 1H, J = 2.2 Hz, J = 10.6 Hz, H-50a), 4.08–4.18 (m, 3H, H-20, H-30, H-40),
3.96–4.27 (m, 2H, CH2CCH), 4.16 (ddd, 1H, J = 2.6 Hz, J = 6.3 Hz, J = 17.6 Hz, NHCH2CCH),
5.70 (dd, 1H, J = 2.2 Hz, J = 8.0 Hz, H-5ur), 5.79 (d, 1H, J = 3.1 Hz, H-10), 8.00 (d, 1H, J = 8.2 Hz,
H-6ur), 8.62 (s, 1H, NH). 13C-NMR (CDCl3, 100 MHz)
18.07 ((CH3)3 ), 25.79, 25.83 (( H3)3C), 58.58 (O
CH2C H), 78.68 (CH2
CH), 82.59 (C-40), 89.60 (C-1)’, 101.69 (C-5ur), 140.48 (C-6ur), 150.19 (C-2ur),
δ
:
−
5.01,
−
4.86,
−
4.57, 4.30 (CH3Si), 18.01,
−
C
C
C
H2CCH), 67.96 (C-50), 70.70, 75.15, 75.91 (C-20, C-30,
C
C
163.20 (C-4ur). ESI-HRMS: calcd for C24H42N2O6Si2Na [M + Na]+: m/z 533.2479. Found: m/z 533.2478.
4.2.3. General Procedure for Synthesis of Glycoconjugates 13–15 By Amide Bond Formation
20,30-O-Isopropylidene-uridine-50-carboxylic acid
(3 mL) followed by addition of acetylated glycosyl amine
glycosyl amine (156 mg, 0.29 mmol) and mixed for 5 min at r.t. Then, 2-chloro-4,6-dimethoxy-1,3,5-
triazine (56 mg, 0.32 mmol) and 4-methylmorpholine (35 L, 0.32 mmol) were added and the reaction
8
(86 mg, 0.29 mmol) was dissolved in MeOH
or (100 mg, 0.29 mmol) or benzylated
3
4
5
µ
was continued for 5 h at r.t. The reaction mixture was concentrated under reduced pressure, dissolved
in CH2Cl2 (30 mL) and washed with brine (5 mL), saturated NaHCO3 (5 mL) and once again with
brine (5 mL). The organic layer was dried over anhydrous MgSO4 and concentrated under reduced
pressure. Crude products were purified by column chromatography (chloroform:methanol; gradient:
100:1 to 75:1 in case of products 13 and 14 or toluene:ethyl acetate; gradient 4:1 to 2:1 in case of product
15) to give 13 (144 mg, 80%) or 14 (155 mg, 86%) or 15 (100 mg, 42%) as white solids.
N-(2”,3”,4”,6”-tetra-O-benzyl-
β
-D-galactopyranosyl)-20,30-O-isopropylidene-uridine-50-amide
: 1.21, 1.53 (2s, 6H,
(
Glycoconjugate 15): [α]D20 17.3 (c = 1.0, CHCl3), 1H-NMR (CDCl3, 400 MHz)
=
−
δ
(CH3)2C), 3.52–3.59 (m, 2H, H-6agal, H-6bgal), 3.68 (dd, 1H, J = 2.4 Hz, J = 9.4 Hz, H-3gal), 3.69 (m, 1H,
H-5gal), 3.82 (dd~t, 1H, J = 9.0 Hz, J = 9.4 Hz, H-2gal), 3.99 (d, 1H, J = 2.4 Hz, H-4gal), 4.40 and 4.45
(qAB, 2H, J = 12.0 Hz, CH2Ph), 4.43 (d, 1H, J = 3.2 Hz, H-40), 4.59 and 4.91 (qAB, 2H, J = 11.3 Hz,
C
H2Ph), 4.66 and 4.73 (qAB, 2H, J = 11.6 Hz, CH2Ph), 4.75 and 4.92 (qAB, 2H, J = 11.7 Hz, CH2Ph),
4.77 (dd, 1H, J = 3.1 Hz, J = 6.6 Hz, H-20), 4.82 (dd, 1H, J = 3.2 Hz, J = 6.6 Hz, H-30), 5.20 (dd~t, 1H,
J = 9.0 Hz, J = 9.4 Hz, H-1gal), 5.43 (dd, 1H, J = 3.1 Hz, H-10), 5.62 (dd, 1H, J = 1.9 Hz, J = 7.8 Hz,

Pharmaceuticals 2020, 13, 460
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H-5ur), 7.09 (d, 1H, J = 7.8 Hz, H-6ur), 7.20 (d, 1H, J = 9.4 Hz, NHamide), 7.23–7.38 (m, 20H, H-Ph),
8.22 (bs, 1H, NH) : 27.14, 25.02 (( H3)2C), 68.10 (C-6gal), 72.54, 73.45,
13C-NMR (CDCl3, 100 MHz)
73.46, 74.16 (
H2Ph), 74.80, 75.01 (C-4gal, C-5gal), 78.04 (C-30), 78.98, 82.07 (C-2gal, C-3gal), 82.67
.
δ
C
C
(C-20), 83.29 (C-1gal), 85.31 (C-40), 96. 86 (C-10), 102.91 (C-5ur), 127.13, 127.41, 127.47, 127.63, 127.73,
127.76, 127.81, 127.86, 127.94, 127.99, 128.22, 128.28, 128.3, 128.35, 128.41, 128.44, 137.73, 138.01, 138.40,
138.78 (C-Ph), 142.78, 149.81 (C-6ur, C-2ur), 162.20 (C-4ur), 169,08 (CONH).ESI-HRMS: calcd for
C46H49N3O11Na [M + Na]+: m/z 842.3265. Found: m/z 842.3268.
4.2.4. General Procedure for Synthesis of Glycoconjugates 16–23 by CuAAC Reaction
Procedure
A: To a solution of O-acetylated glycosyl azides 5 or 6 (86 mg, 0.23 mmol)
and N-propargyl-20,30-O-isopropylidene-uridine-50-amide 10 (77 mg, 0.23 mmol) or N-propargyl-
20,30-di-O-tert-butyldimethylsilyl-uridine-50-amide 11 (120 mg, 0.23 mmol) or 20,30-di-O-tert-
butyldimethylsilyl-50-O-propargyl-uridine 12 (117 mg, 0.23 mmol) in THF (2 mL) and i-PrOH (2 mL)
mixture, CuSO4 5H2O (12 mg, 0.046 mmol) dissolved in H2O (1 mL) was added followed by addition of
·
sodium ascorbate (18 mg, 0.09 mmol) dissolved in H2O (1 mL). The reaction mixture was stirred for 5 h
(during synthesis of products 16 and 17) or for 24 h at r.t. (synthesis of products 18–23). Mixture was
concentrated in vacuo and purified by column chromatography to give products 16–23.
1-(2”,3”,4”,6”-tetra-O-acetyl-β
-D-glucopyranosyl)-4-(20,30-di-O-tert-butyldimethylsilyl-uridine-50-
carbonylaminomethyl)-[1,2,3]-triazole (Glycoconjugate 18): The crude product was purified by column
chromatography (toluene:ethyl acetate; gradient: 6:1 to 1:1) to give 18 (142 mg, 69%) as a white solid.
[α]2D5
= δ: -0.04, 0.04, 0.13, 0.19
32.6 (c = 1.0, CHCl3), m.p. = 117–120 ◦C, 1H-NMR (CDCl3, 400 MHz)
−
(4s, 12H, CH3Si), 0.84, 0.94 (2s, 18H, (CH3)3C), 1.90, 2.04, 2.07, 2.09 (4s, 12H, CH3CO), 4.01 (ddd, 1H,
J = 2.0 Hz, J = 4.4 Hz, J = 9.8 Hz, H-5glu), 4.17 (dd, 1H, J = 2.0 Hz, J = 12.6 Hz, H-6aglu), 4.31–4.36
(m, 2H, H-6bglu, H-30), 4.42 (s, 1H, H-40), 4.52–4.70 (m, 2H, CH2), 4.71 (dd, 1H, J = 4.3 Hz, J = 7.8 Hz,
H-20), 5.23 (m, 1H, H-4glu), 5.37–5.45 (m, 3H, H-10, H-2glu, H-3glu), 5.77 (dd, 1H, J = 2.0 Hz, J = 8.0 Hz,
H-5ur), 5.88 (m, 1H, H-1glu), 7.41 (d, 1H, J = 8.0 Hz, H-6ur), 7.78 (s, 1H, H-5triaz), 8.08 (t, 1H, J = 5.1
Hz, N
17.83, 17.98 ((CH3)3
H
CH2), 9.05 (s, 1H, NH). 13C-NMR (CDCl3, 100 MHz)
), 20.24, 20.48, 20.50, 20.65 ( H3CO), 25.66, 25.76 ((
δ:
−
5.17,
−
4.74,
−
4.71,
−
4.55 (CH3Si),
H2),
C
C
CH3)3C), 34.99 (NHC
61.42 (C-6glu), 67.61, 70.59, 70.61, 72.70, 74.76, 75.19 (C-4glu, C-3glu, C-2glu, C-5glu, C-20, C-30), 85.56,
85.73 (C-40, C-1glu), 95.02 (C-10), 102.89 (C-5ur), 120.11 (C-5triaz.), 144.28, 145.04 (C-6ur, C-4triaz.), 150.48
(C-2ur), 162.50 (C-4ur), 169.30, 169.36, 169.94, 170.52 (CO). ESI-LRMS: calcd for [M + Na]+: m/z 919.3553.
Found: m/z 919.3560.
1-(2”,3”,4”,6”-tetra-O-acetyl-β
-D-galactopyranosyl)-4-(20,30-di-O-tert-butyldimethylsilyl-uridine-50-
carbonylaminomethyl)-[1,2,3]-triazole (Glycoconjugate 19): The crude product was purified by column
chromatography (toluene:ethyl acetate; gradient: 8:1 to 1:1) to give 19 (148 mg, 72%) as a white solid.
[α]2D5
= δ: -0.05, 0.04, 0.13,
16.4 (c = 1.0, CHCl3), m.p. = 126–128 ◦C, 1H-NMR (CDCl3, 400 MHz)
−
0.20 (4s, 12H, CH3Si), 0.84, 0.94 (2s, 18H, (CH3)3C), 1.91, 2.02, 2.05, 2.22 (4s, 12H, CH3CO), 4.16–4.26
(m, 3H, H-5gal, H-6agal, H-6bgal), 4.31 (dd, 1H, J = 0.8 Hz, J = 4.3 Hz, H-30), 4.42 (d, 1H, J = 0.8 Hz,
H-40), 4.52 (m, 1H, CH), 4.66–4.74 (m, 2H, CH, H-20), 5.24 (dd, 1H, J = 3.5 Hz, J = 10.0 Hz, H-3gal),
5.42 (d, 1H, J = 7.8 Hz, H-10), 5.53 (dd~t, 1H, J = 9.4 Hz, J = 10.0 Hz, H-2gal), 5.55 (d, 1H, J = 3.5 Hz
,
H-4gal), 5.76 (dd, 1H, J = 2.3 Hz, J = 8.0 Hz, H-5ur), 5.84 (d, 1H, J = 9.4 Hz, H-1gal), 7.42 (d, 1H,
J = 8.0 Hz, H-6ur), 7.83 (s, 1H, H-5triaz), 8.04 (t, 1H, J = 5.3 Hz, N
CH2), 8.85 (s, 1H, NH). 13C-NMR
(CDCl3, 100 MHz) 5.14, 4.69, 4.67, 4.51 (CH3Si), 17.87, 18.04 ((CH3)3 ), 20.37, 20.50, 20.63,
20.65 ( H3CO), 25.69, 25.80 (( H3)3C), 35.00 (NH
H2), 61.21 (C-6gal), 66.88 (C-4gal), 68.16 (C-30), 70.69
H
δ:
−
−
−
−
C
C
C
C
(C-3gal), 70.85 (C-2gal), 74.21 (C-5gal), 74.81 (C-20), 85.62, 86.30 (C-40, C-1gal), 94.98 (C-10), 102.87 (C-5ur),
120.32 (C-5triaz.), 144.26, 144.75 (C-6ur, C-4triaz.), 150.42 (C-2ur), 162.38 (C-4ur), 169.36, 169.55, 169.94,
170.36 (CO). ESI-LRMS: calcd for [M + Na]+: m/z 919.3553. Found: m/z 919.3563.
1-(2”,3”,4”,6”-tetra-O-acetyl-
methyl)-[1,2,3]-triazole (Glycoconjugate 20): The crude product was purified by column chromatography
(toluene:ethyl acetate; gradient: 6:1 to 2:1) to give 20 (146 mg, 72%) as a white solid. [α]2D5 = 4.0 (c = 1.0
β
-D-glucopyranosyl)-4-(20,30-di-O-tert-butyldimethylsilyl-uridine-50-O-
,

Pharmaceuticals 2020, 13, 460
15 of 20
CHCl3), m.p. = 105–108 ◦C, 1H-NMR (CDCl3, 400 MHz)
0.90 (2s, 18H, (CH3)3C), 1.87, 2.03, 2.04, 2.07 (4s, 12H, CH3CO), 3.68 (dd, 1H, J = 1.4 Hz, J = 10.6 Hz,
δ:
−
0.01, 0.06, 0.07, 0.08 (4s, 12H, CH3Si), 0.88,
H-50a), 4.03 (ddd, 1H, J = 1.8 Hz, J = 4.9 Hz, J = 10.1 Hz, H-5glu), 4.09–4.15 (m, 3H, H-20, H-30, H-40),
4.18 (dd, 1H, J = 1.8 Hz, J = 12.7 Hz, H-6aglu), 4.35 (dd, 1H, J = 4.9 Hz, J = 12.7 Hz, H-6bglu), 4.64 and
4.72 (qAB, 2H, J = 11.9 Hz, CH2), 5.25 (dd~t, 1H, J = 9.4 Hz, J = 10.1 Hz, H-4glu), 5.38 (dd~t, 1H,
J = 9.0 Hz, J = 9.8 Hz, H-2glu), 5.43 (dd~t, 1H, J = 9.4 Hz, J = 9.8 Hz, H-3glu), 5.65 (dd, 1H, J = 1.8 Hz,
J = 8.2 Hz, H-5ur), 5.78 (d, 1H, J = 3.1 Hz, H-10), 5.89 (d, 1H, J = 9.0 Hz, H-1glu), 7.75 (s, 1H, H-5triaz),
7.94 (s, 1H, NH), 7.95 (d, 1H, J = 8.2 Hz, H-6ur). 13C-NMR (CDCl3, 100 MHz)
4.32 (CH3Si), 18.01, 18.10 ((CH3)3 ), 20.13, 20.50, 20.54, 20.68 ( H3CO), 25.80, 25.84 ((C
δ
:
−
4.98,
−
4.82,
−
4.62,
−
C
C
H3)3C),
61.54 (C-6glu), 64.32 (CH2), 67.75 (C-50), 68.60, 70.39 (C-3glu, C-4glu), 70.94, 72.51, 75.41, 75.84 (C-2glu
,
C-5glu, C-20, C-30), 82.83, 85.91 (C-40, C-1glu), 89.46 (C-10), 101.86 (C-5ur), 120.60 (C-5triaz.), 140.75,
144.71 (C-6ur, C-4triaz.), 150.11 (C-2ur), 162.75 (C-4ur), 169.02, 169.37, 169.80, 170.39 (CO). ESI-LRMS:
calcd for [M + Na]+: m/z 906.3600. Found: m/z 906.3581.
1-(2”,3”,4”,6”-tetra-O-acetyl-β-D-galactopyranosyl)-4-(20,30-di-O-tert-butyldimethylsilyl-uridine-50-O-
methyl)-[1,2,3]-triazole (Glycoconjugate 21): The crude product was purified by column chromatography
(toluene:ethyl acetate; gradient: 6:1 to 2:1) to give 21 (150 mg, 74%) as a white solid. [α]2D5 = 17.2 (c = 1.0
,
CHCl3), m.p. = 104–107 ◦C, 1H-NMR (CDCl3, 400 MHz)
δ: 0.05, 0.06, 0.07, 0.08 (4s, 12H, CH3Si), 0.88,
0.90 (2s, 18H, (CH3)3C), 1.88, 2.02, 2.04, 2.21 (4s, 12H, CH3CO), 3.68 (dd, 1H, J = 2.2 Hz, J = 10.8 Hz
,
H-50a), 3.88 (dd, 1H, J = 2.2 Hz, J = 10.8 Hz, H-50b), 4.09–4.27 (m, 6H, H-5gal, H-6agal, H-6bgal, H-20,
H-30, H-40), 4.65 and 4.73 (qAB, 2H, J = 11.9 Hz, CH2), 5.27 (dd, 1H, J = 3.5 Hz, J = 10.2 Hz, H-3gal),
5.50 (dd~t, 1H, J = 9.4 Hz, J = 10.2 Hz, H-2gal), 5.57 (d, 1H, J = 3.5 Hz, H-4gal), 5.66 (dd, 1H, J = 2.4 Hz
J = 8.2 Hz, H-5ur), 5.79 (d, 1H, J = 3.1 Hz, H-10), 5.86 (d, 1H, J = 9.4 Hz, H-1gal), 7.80 (s, 1H, H-5triaz),
7.95 (d, 1H, J = 8.2 Hz, H-6ur), 8.27 (s, 1H, NH). 13C-NMR (CDCl3, 100 MHz)
4.84, 4.69, 4.47,
4.19 (CH3Si), 18.14, 18.24 ((CH3)3 ), 20.36, 20.61, 20.73, 20.76 ( H3CO), 25.93, 25.98 (( H3)3C),
,
δ:
−
−
−
−
C
C
C
61.14 (C-6gal), 64.51 (CH2), 66.91, 68.18, 68.75, 70.76, 71.10, 74.36, 76.00 (C-50, C-4gal, C-3gal, C-30, C-2gal
,
C-5gal, C-20), 82.92, 86.55 (C-40, C-1gal), 89.54 (C-10), 101.99 (C-5ur), 120.85 (C-5triaz.), 140.84, 144.78
(C-6ur, C-4triaz.), 150.32 (C-2ur), 163.07 (C-4ur), 169.36, 169.88, 170.03, 170.44 (CO). ESI-LRMS: calcd for
[M + Na]+: m/z 906.3600. Found: m/z 906.3604.
20,30-O-isopropylidene-50-deoxy-50-[4-(2”,3”,4”,6”-tetra-O-acetyl-
β-D-glucopyranosyl-oxymethyl)-1,2-3-
triazol-1-yl]uridine (Glycoconjugate 22): The crude product was purified by column chromatography
(toluene:ethyl acetate; gradient: 15:1 to 2:1 and next chloroform:methanol; gradient 100:1 to 40:1)
to give 22 (142 mg, 89%) as a white solid. [α]2D4 = 7.3 (c = 1.0, CHCl3), m.p. = 108–110 C,
◦
1H-NMR (CDCl3, 400 MHz
) δ: 1.33, 1.55 (2s, 6H, (CH3)2C), 1.99, 2.00, 2.03, 2.09 (4s, 12H, CH3CO),
3.73 (ddd, 1H, J = 2.3 Hz, J = 4.9 Hz, J = 9.8 Hz, H-5glu), 4.15 (dd, 1H, J = 2.3 Hz, J = 12.1 Hz, H-6aglu),
4.23 (dd, 1H, J = 4.9 Hz, J = 12.7 Hz, H-6bglu), 4.47 (ddd, 1H, J = 4.7 Hz, J = 6.6 Hz, J = 6.8 Hz, H-40),
4.67 (d, 1H, J = 8.2 Hz, H-1glu), 4.68 (dd, 1H, J = 6.8 Hz, J = 14.1 Hz, H-50a), 4.73 (dd, 1H, J = 4.3 Hz,
J = 14.1 Hz, H-50b), 4.81 and 4.93 (qAB, 2H, J = 12.5 Hz, CH2), 4.91 (dd, 1H, J = 4.7 Hz, J = 6.7 Hz, H-30),
5.10 (dd, 1H, J = 8.2 Hz, J = 9.4 Hz, H-2glu), 5.17 (dd, 1H, J = 2.0 Hz, J = 6.7 Hz, H-20), 5.19 (dd~t, 1H
J = 9.4 Hz J = 9.8 Hz, H-4glu), 5.21 (dd~t, 1H, J = 9.4 Hz, J = 9.4 Hz, H-3glu), 5.50 (d, 1H, J = 2.0 Hz
,
,
,
H-10), 5.75 (dd, 1H, J = 2.3 Hz, J = 7.8 Hz, H-5ur), 7.08 (d, 1H, J = 7.8 Hz, H-6ur), 7.56 (s, 1H, H-5triaz),
8.59 (s, 1H, NH). 13C-NMR (CDCl3, 100 MHz)
δ: 20.62, 20.70, 20.77 (CH3CO), 25.28, 27.13 (CH3)2C),
51.64 (C-50), 61.92, 63.16 (C-6glu, CH2), 68.42, 71.28, 71.97, 72.67 (C-2glu, C-3glu, C-4glu, C-5glu), 81.54,
84.03, 85.92 (C-20, C-30, C-40), 96.33 (C-10), 100.23 (C-1glu), 103.01 (C-5ur), 114.96 (CH3)2C), 124.29
(C-5triaz.), 143.22, 144.11 (C-6ur, C-4triaz.), 149.75 (C-2ur), 162.48 (C-4ur), 169.56, 170.24, 170.74 (CO).
ESI-LRMS: calcd for [M + H]+: m/z 696.2364. Found: m/z 696.2407.
20,30-O-isopropylidene-50-deoxy-50-[4-(2”,3”,4”,6”-tetra-O-acetyl-
β-D-galactopyranosyl-oxymethyl)-1,2-3-
triazol-1-yl]uridine (Glycoconjugate 23): The crude product was purified by column chromatography
(toluene:ethyl acetate; gradient: 15:1 to 2:1 and next chloroform:methanol; gradient 100:1 to 40:1) to
give 23 (104 mg, 65%) as a white solid. [α]2D4 = 1.2 (c = 1.0, CHCl3), m.p. = 101–105 C, H-NMR
◦
1
(CDCl3, 400 MHz) δ: 1.34, 1.55 (2s, 6H, (CH3)2C), 1.98, 2.00, 2.06, 2.15 (4s, 12H, CH3CO), 3.95 (ddd, 1H,

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J = 1.0 Hz, J = 6.5 Hz, J = 6.8 Hz, H-5gal), 4.13 (dd, 1H, J = 6.5 Hz, J = 11.2 Hz, H-6agal), 4.17 (dd, 1H,
J = 6.8 Hz, J = 11.2 Hz, H-6bgal), 4.46 (m, 1H, H-40), 4.65 (d, 1H, J = 8.2 Hz, H-1gal), 4.80 and 4.97 (qAB,
2H, J = 12.5 Hz, CH2), 4.68–4.77 (m, 2H, H-50a, H-50b), 4.87 (dd, 1H, J = 4.7 Hz, J = 6.8 Hz, H-30),
5.02 (dd, 1H, J = 3.5 Hz, J = 10.2 Hz, H-3gal), 5.06 (dd, 1H, J = 2.0 Hz, J = 6.8 Hz, H-20), 5.21 (dd, 1H,
J = 8.2 Hz, J = 10.2 Hz, H-2gal), 5.90 (dd, 1H, J = 1.0 Hz, J = 3.5 Hz, H-4gal), 5.55 (d, 1H, J = 2.0 Hz,
H-10),5.79 (dd, 1H, J = 2.0 Hz, J = 7.8 Hz, H-5ur), 7.11 (d, 1H, J = 7.8 Hz, H-6ur), 7.58 (s, 1H, H-5triaz),
9.20 (s, 1H, NH). 13C-NMR (CDCl3, 100 MHz)
δ: 20.56, 20.64, 20.70, 20.80 (CH3CO), 25.24, 27.10
(C
H3)2C), 51.60(C-50), 61.31, 63.22 (CH2, C-6gal), 67.04, 68.88, 70.75, 70.86 (C-2gal, C-3gal, C-4gal, C-5gal),
81.39, 83.91, 85.74 (C-20, C-30, C-40), 95.86 (C-10), 100.82 (C-1gal), 103.05 (C-5ur), 115.00 (CH3)2C), 124.34
(C-5triaz.), 143.24, 144.10 (C-6ur, C-4triaz.), 150.01 (C-2ur), 162.92 (C-4ur), 169.73, 170.11, 170.29, 170.49
(CO). ESI-LRMS: calcd for [M + H]+: m/z 696.2364. Found: m/z 696.2468.
4.3. Biological Evaluation of Compounds Activity
4.3.1. Antiviral Compounds
The synthesized compounds were dissolved in DMSO and stored at −20◦C until future use.
4.3.2. Cells and Viruses
The A549 cell line (adenocarcinomic human alveolar basal epithelial cells, ATCC® CCL-185
™) was
cultured in Dulbecco’s Modified Eagle’s Medium (D-MEM) (Corning, New York, USA) supplemented
with 8% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 g/mL streptomycin at
37 ◦C under 5% CO2. The Neudoerfl TBEV strain, kindly provided by Dr. Karin Stiasny (Center for
Virology, Medical University of Vienna, Vienna, Austria) and the Hypr TBEV strain, kindly provided by
Dr. Daniel Ru˚žek (Department of Virology, Veterinary Research Institute, Brno, Czech Republic) were
cultured in A549 cells for 3–4 days. The plaque assay method was used to determine TBEV virus titers.
4.3.3. Cell Viability Assay
A549 cell viability and compound cytotoxicity were estimated by the CellTiter 96 AQueous
non-radioactive cell proliferation assay (MTS) (Promega, Madison, WI, USA) according to the
manufacturer’s instructions. The cytotoxicity measurements were done in three replicates.
The half-maximal cytotoxic concentration (CC50) values of compounds that are cytotoxic in 50% were
calculated using the GraphPad Prism software (version 5.01, GraphPad Software, San Diego, CA, USA
)
from the dose–response curves.
4.3.4. CPE Inhibition Assay
A549 cells were seeded together with different doses of tested compounds or DMSO
(positive control) and infected with TBEV (Hypr strain; multiplicity of infection (MOI) of 0.1) for
96 h. CPE was measured by the colorimetric CytoTox 96® Non-Radioactive Cytotoxicity Assay
(Promega, Madison, WI, USA) according to the manufacturer’s instructions. One-way ANOVA
with Dunnett’s post-test was performed for statistical analysis using the GraphPad Prism software
(version 5.01, GraphPad Software, San Diego, CA, USA).
4.3.5. Plaque Assay
Monolayers of A549 cells grown in 24-well culture plates were inoculated with 10-fold dilutions
of TBEV strains for 2 h at 37 ◦C. After the virus was removed, the cells were overlaid with
carboxymethylcellulose in Modified Eagle’s Medium (MEM) medium. After 5 days the medium was
removed, the cells were washed with PBS and stained with the solution of 0.005% amido black to count
the plaque-forming units (PFU). The virus titers were expressed in terms of PFU/mL.

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4.3.6. Plaque Reduction Assay
Monolayers of A549 cells seeded in 12-well plates were inoculated with TBEV at MOI of
0.001. Virus inoculum was removed after 2 h at 37 ◦C, the cells were washed and overlaid with 1%
carboxymethylcellulose in Modified Eagle’s Medium (MEM) medium with DMSO or tested compounds.
After three days, cells were washed with phosphate-buffered saline (PBS) and fixed with methanol.
The infected virus foci were visualized by immunostaining with the monoclonal anti-Flavivirus group
antigen antibody (4G2) (Absolute Antibody, Oxford, UK; diluted 1:1500 in PBS, 0,05% Tween 80, 5% FBS)
as a primary antibody and anti-mouse secondary antibody conjugated with horseradish peroxidase
(HRP) (diluted 1:2000) in PBS containing 0,05% Tween 80 and 5% FBS. Plaques were detected using a
Vector Nova Red kit (Vector Laboratories Ltd., Peterborough, UK) according to the manufacturer’s
protocol and counted. If necessary, one-way ANOVA with Dunnett’s post-test was performed in
some experiments for statistical analysis using the GraphPad Prism software (version 5.01, GraphPad
Software, San Diego, CA, USA).
4.3.7. Dose-Response Antiviral Activity of Tested Compounds
A549 cells were infected with TBEV Neudoerfl strain (MOI = 0.1) and treated with different
concentrations of the tested compounds (0 to 25 µM). Culture supernatants were collected two days
post-infection and used for the estimation of viral titers using the plaque assay. The dose–response
curves were prepared using GraphPad Prism software and used for calculations of the half-maximal
inhibitory concentration (IC50) values, indicating the concentration required to reduce the viral titer by
50% compared to the non-treated virus infected cells.
4.3.8. Western Blot Analysis
A monolayer of A549 cells in 12-well plates was inoculated with TBEV at an MOI of 0.1 for 2 h.
The virus was collected and fresh medium with different concentrations of compounds or DMSO
was added and cultured for further 48 h at 37 ◦C. Cells were lysed at 4 ◦C for 1 h with TNET buffer
(20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100). Total concentration of
proteins in lysates was measured using a Quick Start
™ Bradford Protein Assay (Bio-Rad) according
to the manufacturer’s protocol. An amount of 50 g of proteins of each lysate were separated on a
µ
10–20% SDS-PAGE gel under denaturing and reducing conditions, transferred to PVDF membranes,
and detected with a specific monoclonal anti-Flavivirus group antigen antibody (4G2) (1:1500 dilution)
followed by anti-mouse peroxidase (HRP)-conjugated secondary antibodies (diluted 1:2000). Blots were
developed using a Super Signal
™ West Pico Plus Substrate system (Pierce, Dallas, TX, USA) using
Chemidoc system Alliance™ Q9-Series (UVITEC, Cambrige, United Kingdom).
Author Contributions: Conceptualization, E.K. and G.P.-G.; methodology, E.K., G.B. and G.P.-G; investigation, E.K.,
G.B. and M.K.; writing—original draft preparation, E.K., G.B. and G.P.-G.; writing—review and editing, E.K.,
G.P.-G. and B.S.; visualization, G.B.; supervision, B.S.; project administration, E.K.; funding acquisition, E.K.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Science Centre, Poland, grant number UMO-2015/19/D/NZ6/01717.
Ewelina Krol was the recipient of the fellowship L’Oréal – UNESCO For Women in Science.
Acknowledgments: We thank Karin Stiasny from the Center for Virology, Medical University of Vienna,
Vienna, Austria for the TBEV Neudoerfl strain. We also thank Daniel Ru˚žek from Department of Virology,
Veterinary Research Institute, Brno, Czech Republic for the TBEV Hypr strain.
Conflicts of Interest: The authors declare no conflict of interest.
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