New tools in nucleoside toolbox of tick-borne encephalitis virus reproduction inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.01.040

Design and development of nucleoside analogs is an established strategy in the antiviral drug discovery field. Nevertheless, for many viruses the coverage of structure-activity relationships (SAR) in the nucleoside chemical space is not sufficient. Here we present the nucleoside SAR exploration for tick-borne encephalitis virus (TBEV), a member of Flavivirus genus. Promising antiviral activity may be achieved by introduction of large hydrophobic substituents in the position 6 of adenosine or bulky silyl groups to the position 5′. Introduction of methyls to the ribose moiety does not lead to inhibition of TBEV reproduction. Possible mechanisms of action of these nucleosides include the inhibition of viral entry or interaction with TBEV non-structural protein 5 methyltransferase or RNA-dependent RNA polymerase domains.

Full text of DOI:10.1016/j.bmcl.2017.01.040

Accepted Manuscript
New Tools in Nucleoside Toolbox of Tick-Borne Encephalitis Virus Repro-
duction Inhibitors
Alexey A. Orlov, Mikhail S. Drenichev, Vladimir E. Oslovsky, Nikolay N.
Kurochkin, Pavel N. Solyev, Liubov I. Kozlovskaya, Vladimir A. Palyulin,
Galina G. Karganova, Sergey N. Mikhailov, Dmitry I. Osolodkin
PII:
S0960-894X(17)30052-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.01.040
BMCL 24616
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 December 2016
10 January 2017
12 January 2017
Please cite this article as: Orlov, A.A., Drenichev, M.S., Oslovsky, V.E., Kurochkin, N.N., Solyev, P.N.,
Kozlovskaya, L.I., Palyulin, V.A., Karganova, G.G., Mikhailov, S.N., Osolodkin, D.I., New Tools in Nucleoside
Toolbox of Tick-Borne Encephalitis Virus Reproduction Inhibitors, Bioorganic & Medicinal Chemistry Letters
(2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.01.040
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New Tools in Nucleoside Toolbox of Tick-Borne Encephalitis
Virus Reproduction Inhibitors
Alexey A. Orlova,b,#, Mikhail S. Drenichevc,#, Vladimir E. Oslovskyc, Nikolay N. Kurochkinc,
Pavel N. Solyevc, Liubov I. Kozlovskayaa, Vladimir A. Palyulinb, Galina G. Karganovaa,
Sergey N. Mikhailovc, Dmitry I. Osolodkin*,a,b
a
Chumakov Institute of Poliomyelitis and Viral Encephalitides, Chumakov FSC R&D IBP
RAS, 8 bd. 1 Poselok Instituta Poliomielita, Poselenie Moskovsky, Moscow 108819,
Russia
b Department of Chemistry, Lomonosov Moscow State University, 1 bd. 3 Leninskie Gory,
Moscow 119991, Russia
c
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova
Str., Moscow 119991, Russia
# Both authors contributed equally
* Corresponding Author. E-mail: dmitry_o@qsar.chem.msu.ru. Phone: +7-495-841-93-16.
Address: Chumakov Institute of Poliomyelitis and Viral Encephalitides, Chumakov FSC
R&D IBP RAS, 8 bd. 1 Poselok Instituta Poliomielita, Poselenie Moskovsky, Moscow
142782, Russia.
Abstract
Design and development of nucleoside analogs is an established strategy in the
antiviral drug discovery field. Nevertheless, for many viruses the coverage of
structure-activity relationships (SAR) in the nucleoside chemical space is not
sufficient. Here we present the nucleoside SAR exploration for tick-borne
encephalitis virus (TBEV), a member of Flavivirus genus. Promising antiviral activity
may be achieved by introduction of large hydrophobic substituents in the position 6
of adenosine or bulky silyl groups to the position 5'. Introduction of methyls to the
ribose moiety does not lead to inhibition of TBEV reproduction. Possible
mechanisms of action of these nucleosides include the inhibition of viral entry or
interaction with TBEV non-structural protein 5 methyltransferase or RNA dependent
RNA polymerase domains.
Keywords
antivirals; nucleosides; Flavivirus; tick-borne encephalitis.
Abbreviations
7DCMA, 7-deaza-2'-C-methyladenosine; DENV, dengue virus; EV-A71, enterovirus
A71; MTase, methyltransferase; NS5, non-structural protein 5; RAFI, rigid

amphipathic fusion inhibitor; RdRp, RNA dependent RNA polymerase; SAH, S-
adenosyl-L-homocysteine; TBEV, tick-borne encephalitis virus.

Nucleoside and nucleotide analogs and derivatives often show significant
antiviral activity. Numerous successful antiviral drugs are based on these
molecules.1 Whereas a large body of information is available for such viruses as
human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza A virus,
efficiency of nucleosides against other viruses remains poorly studied. For rational
design of antivirals it is required to know which structural features are important for
the inhibition of reproduction of these viruses.
Tick-borne encephalitis virus (TBEV) is a member of Flavivirus genus, which
includes enveloped viruses borne by blood-sucking arthropods, namely ticks and
mosquitoes. These viruses pose a threat for public health all over the world.
Mosquito-borne flaviviruses (dengue virus (DENV), West Nile virus, Japanese
encephalitis virus, yellow fever virus, Zika virus, etc.) are mostly spread in tropical
regions, while tick-borne flaviviruses, including TBEV, are common in temperate
climates of Europe, Russia, and northern Asia. Up to 10,000 clinical cases of tick-
borne encephalitis are registered annually,2 and about one-third of them lead to
prolonged sequela with different degree of severity. Genome of flaviviruses is
represented by 11 kb (+)RNA, and its replication is performed by NS5 protein
containing RNA dependent RNA polymerase (RdRp) and methyltransferase
(MTase) domains.3 These enzymes are common targets of nucleoside-based
antivirals.4 Moreover, RdRp sequences are rather conserved among RNA viruses,
and flavivirus RdRp is similar to HCV one, which gives a possibility to expand the
knowledge from one virus to another.3
Until 2015, there was no information on nucleoside inhibitors of TBEV
replication, and limited data were available for other classes.5,6 Notably, well-known
broad-spectrum antiviral drug ribavirin is not efficient as a TBEV reproduction
inhibitor.7-9 Then it was found that close analogs of nucleosides, e.g., 7-deaza-2'-C-
methyladenosine (7DCMA), inhibit replication of TBEV at micromolar
concentrations in an NS5-dependent manner,7 and nucleosides with large
hydrophobic substituents in the position 5 of nucleobase (RAFIs) show TBEV entry
inhibition at two-digit nanomolar concentrations, presumably due to interaction with
the viral membrane.10 In a follow-up study, SAR was explored around 7DCMA to
show that 2'-C-methylation of common nucleosides was the most efficient
approach, whereas 2'-O-methylation, 3'-O-methylation, and 3'-dehydroxylation did
not lead to nucleosides with improved anti-TBEV effect.8 Another adenosine analog
NITD008 (7-deaza-2'-C-ethynyladenosine) was shown to inhibit TBEV reproduction
along with three less common tick-borne flaviviruses on a micromolar level.11
Despite numerous nucleoside analogs tested on mosquito-borne flaviviruses,
there have been no systematic structure-activity studies, and only islands of
structure-activity relationship data for particular compound series are available from
different laboratories (Fig. 1). Tritylation12-15 or silylation16,17 of sugar moiety were

found to be especially attractive strategies of achieving antiflaviviral effect for
nucleosides. On the contrary, acyclic nucleoside analogs were shown to be
ineffective against flavivirus reproduction in cells.15,18 Nucleobase was varied in a
rather conservative manner, mostly by changing of a hydrogen bonding or electron
density pattern by swapping nitrogen and carbon atoms.19,20
[Figure 1 should be placed here]
Figure 1. Antiflaviviral activity of nucleosides according to literature.7,8,10-15, 20, 36, 37
Modifications of the nucleoside scaffold leading to antiviral activity are colored
green, the ones not introducing activity – red. * 1’-C-analogs were assessed as 4-
aza-7,9-dideazaadenosine derivatives. † 4-substituted adenosines were highly toxic
in PS cells. 2’-C-ethynyl derivatives were active with moderate toxicity in another
assay. ‡ O-tritylated analogs were tested only for fludarabine and inosine. § O-
tritylated analogs with various substituents; compounds with trityl moieties in 3’-O
and 5’-O positions were the most active. # O-tritylated analogs were tested for 5-
aza-cytidine.
In this study we expand the anti-TBEV nucleoside SAR by variation of
substituents in the position N6 of adenosine along with examples of N2 substituted
guanosines, N4 substituted cytidine, polar nucleobase ribosides, and 3'-C-
methylated nucleosides. The study of N6 substituted adenosines was particularly
inspired by high potency of N6-benzyladenosine and N6-furfuryladenosine against
Lassa and Marburg viruses revealed in high-throughput screening campaigns21,22
and by activity of compounds from this series against enterovirus A71.23,24
The previously published procedures were used for the synthesis of N6-
substituted adenosines 1 and 2b-j,23,24 3’-C-methylnucleosides 325,26, 2a, 4a, 4b,27 5
and 628 (Table 1); synthesis and characterization data for compounds 1e-k are
provided in the Supplementary Information.
Anti-TBEV activity was assessed in the plaque reduction assay in PEK
(porcine embryo kidney) cells using the method described earlier.10 Samples of the
compounds were incubated with TBEV (strain Absettarov) for one hour and then
added to the cell monolayer, achieving the final 50 µM concentration for each
compound. Compounds that showed the decrease of plaque count at 50 µM
concentration were used for the subsequent cell toxicity (CC50) and antiviral
efficiency (EC50) determination. The results are given in Table 1.

Table 1.
Anti-TBEV activity and cytotoxicity of nucleosides.
N6-Substituted adenosines
Substituent
R1
CC50
CC50
EC50,
µM
No.
1a
Compound name
R2
(24h), µM (7d), µM
N6-Benzyladenosine
N6-Furfuryladenosine
H
>50
ND
>50
ND
>50
>50
1b
H
H
N6-(E)-(3-Phenyl-2-
propen-1-yl)adenosine
1c
1d
ND
ND
ND
ND
>50
>48
N6-(3-phenyl-2-propin-1-
H
H
yl)-adenosine
1e
N6-Trityladenosine
ND
ND
>50
N6-(β-Naphthylmethyl)-
1f
H
H
ND
ND
>50
adenosine
N6-(9-Anthranylmethyl)-
1g
>50
>50
15±2
adenosine
N6-(1-Pyrenylmethyl)-
1h
1i
H
>50
>50
9
9
6±1
2±1
adenosine
N6-Benzyl-5ʹ-O-
trityladenosine

N6-Benzyl-5′-O-tert-
butyldimethylsilyl-
adenosine
1j
>50
>50
>50
>50
20±10
5±4
N6-Benzyl-5′-O-
triisopropylsilyladenosine
1k
1l
N6-Benzyladenine
>50
>50
>50
2a
2b
N6-Methyladenosine
CH3
H
H
ND
ND
ND
ND
>50
>50
N6-Isopentenyladenosine
2c
2d
N6-Propargyladenosine
H
H
ND
ND
ND
ND
>50
>50
N6-(Z)-(4-Hydroxy-2-
butenyl)adenosine
2e
2f
N6-Neryladenosine
H
H
ND
ND
ND
ND
>50
>50
N6-Geranyladenosine
N6-(5-Hexene-2-yne-1-
2g
2h
H
H
ND
ND
ND
ND
>50
>50
yl)adenosine
N6-Cyclohexyladenosine
N6-(S)-(-)-
Perillyladenosine
2i
2j
H
H
ND
ND
ND
ND
>50
>50
N6-(1R)-(-)-
Myrtenyladenosine
3’-C-Methylnucleosides
CC50
CC50
EC50,
µM
No.
3a
Compound name
Structure
(24h), µM (7d), µM
3ʹ-C-Methyluridine
ND
ND
>50

3ʹ-C-Methyl-2’-
hydroxythymidine
3b
ND
ND
>50
3c
3ʹ-C-Methylcytidine
ND
ND
>50
3d
3ʹ-C-Methylguanosine
ND
ND
>50
Polar hydrophilic nucleosides
Structure
CC50
CC50
EC50,
µM
No.
4a
Compound name
(24h), µM (7d), µM
1N-Methyladenosine
ND
ND
>50
2-Amino-9-ribosyl-7-
methyl-9H-purin-7-ium-6-
olate
4b
ND
ND
>50

N2-Substituted guanosines
CC50
CC50
EC50,
µM
No.
5a
Compound name
R
(24h), µM (7d), µM
N2-Benzoylguanosine
ND
ND
ND
ND
>50
>50
5b
N2-Isobutyrylguanosine
N4-Substituted cytidine
6
N4-Benzoylcytidine
ND
ND
>50
*ND — not determined (the compound was ineffective at 50 µM concentration and further
investigation was not performed)
Cell toxicity of all the active molecules in the series was not observed in 50
µM concentrations after 24 h incubation, but chronic (7 d) toxicity was observed for
N6-(2-pyrenylmethyl)adenosine 1h and N6-benzyl-5'-O-trityladenosine 1i. As these
molecules are very hydrophobic, further modifications are required to achieve
optimal pharmacokinetics.
Most compounds of the series did not show TBEV reproduction inhibition in
vitro. That suggests that the decoration of nucleobase amino groups as well as 3’-
C-methylation of ribose do not usually lead to the emergence of this kind of activity.
Nevertheless, the anti-TBEV activity gradually increased with the increase of N6
substituent size from methyl to 2-pyrenylmethyl: whereas methyladenosine 2a and
benzyladenosine 1a were not active, anthracenylmethyladenosine 1g was definitely
active, and N6-(2-pyrenylmethyl)adenosine 1h showed clear antiviral effect on a
micromolar level (on par with previously reported nucleosides7,8), though being
rather toxic after 7 days incubation. Removal of ribose from 1a, giving
benzyladenine 1l, did not lead to any activity. Due to clear dependence of the
efficiency on the size of aromatic substituent, the activity of 1h may be attributed at

least partially to the interaction with the viral membrane, analogously to RAFIs.10
Possible mechanism of action of the identified TBEV inhibitors could be
related to the interaction with NS5 protein, which consists of MTase and RdRp
domains, or cell entry inhibition.
Similar substituted nucleosides showed inhibition of flavivirus NS5 activities
in the enzymatic assays.12,16,17,29 We employed homology modelling of TBEV NS5
and molecular docking as the tools that could allow us to assess the relevance of
this mode of action to our compounds.
The model of full-length TBEV NS5 was built in Modeller 9.1530 based on the
Japanese encephalitis virus NS5 template (PDB ID 4K6M31). The SAH molecule in
MTase domain was retained, and RdRp domain was in the conformation similar to
other flavivirus RdRps in complex with inhibitors. Docking of the ligand
conformations generated with OMEGA32 was performed using FRED and
HYBRID.33
Modification of N6-benzyladenosine with bulky protective groups on 5'-
hydroxyl group is in line with the previous findings for mosquito-borne
flaviviruses12-17: compounds 1i-k efficiently inhibit reproduction of TBEV. This
modification is compatible with NS5 MTase domain interaction shown previously for
similarly substituted compounds.17,29 Docking results show the possibility of
realization of binding modes consistent with this hypothesis (Fig. 2A).
[Figure 2 should be placed here]
Figure 2. Docking of active molecules into binding sites of TBEV NS5 model.
A) Methyltransferase binding site. S-Adenosyl-L-homocysteine molecule is shown
with cyan carbons, 1j — with green carbons. B) RdRp binding site. TBEV NS5
model is red, X-ray structures of DENV3 RdRp with inhibitors are overlapped (5IQ6,
brown; 5K5M, violet). Example docking poses of 1h (gray carbons) and 1g (light
blue carbons) are shown. The figure was prepared in VIDA 4.3.038.
Inhibition of flaviviral NS5 RdRp by bulky hydrophobic nucleosides was also
previously shown.12,16 We used information available from X-ray structures of DENV
RdRp complexed with non-nucleoside inhibitors (PDB IDs: 5IQ634, 5K5M35) to
assess the possibility of interaction between our molecules and TBEV RdRp model.
The generated binding modes overlap with both known inhibitor binding sites,
suggesting that the interaction is theoretically possible. The large volume of the
binding site is consistent with the lack of activity of smaller nucleosides: only pyrene
and anthracene are large enough to occupy a significant portion of the binding site
and prevent RNA binding (Fig. 2B). Thus, both NS5 domains may contribute to the
antiviral activity of these compounds.

The role of identified compounds in entry inhibition needs to be evaluated in
additional studies, for example, time-of-addition test.
The molecules presented here provide important novel information for further
development of flavivirus reproduction inhibitors based on the nucleoside scaffold.
Large aromatic substituents are shown to be the most efficient when introduced into
the nucleobase of adenosine. Modification of 5’ hydroxyl with bulky hydrophobic
groups proved its feasibility for flavivirus reproduction inhibition. Docking simulation
suggests that the interaction with NS5 protein is quite possible for the studied
compounds, as well as the inhibition of TBEV reproduction on the entry phase.
Further mechanism of action studies for these N6-substituted adenosines should
provide reasonable details for the design of efficient TBEV antivirals.
Acknowledgments
The images of the screwdriver and the wrench (distributed by CC-GFDL license)
used in the graphical abstract were created by Joel Yoder. The authors thank Yulia
Rogova, Oleg Samsonov, Anastasia Eletskaya for technical assistance. Free academic
licenses was kindly provided by ChemAxon and OpenEye Scientific Software, Inc.
This study was supported by Russian Science Foundation (grant number 16-15-
10307). The authors declare no competing financial interest. The funding source had no
role in study design, in the collection, analysis and interpretation of data, in the writing of
the report, and in the decision to submit the article for publication.

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Figure 1
modi ed nucleobase
O
N
A
NH2
G
O
NH2
-CN
-CH3
-vinyl
-ethynyl
N
N
R
NH
N
N
N
1'C*
N
N
N
HN
HN
N
NH2
N
NH2
N
O
O
-CH3
2'C
2'O
-ethyl
-ethynyl
-vinyl
OH
OH
†
-CH3
-ethynyl
2'C
2'O
OH
OH
OH
OH
-methylethynyl
-OCH3
‡
-OTr
5'O
-OCH3
‡
-OTr
3'O
-OCH3
3'O
-OCH3
3'deoxy
3'deoxy
U
C
NH2
N
O
modi ed nucleobases
-perylen-3-yl ethynyl
HN
5-
-pyren-1-yl ethynyl
O
N
N
O
OH
O
O
-CH
-CH3, F
-CH
-CH3, F
-ethynyl
2'C
2'O
2'C
2'O
3
3
OH
OH
#
§
OH
OH
OH
-OTr
-OCH3
-OTr
-OCH3
#
-N
4'C
-OTr
5'O
3
§
#
-OTr
-OCH3
3'O
-OTr
3'O
-OCH3
3'deoxy

Figure 2

Graphical abstract