Antiviral activity spectrum of phenoxazine nucleoside derivatives

Article abstract of DOI:10.1016/j.antiviral.2019.01.010

The phenoxazine scaffold is widely used to stabilize nucleic acid duplexes, as a part of fluorescent probes for the study of nucleic acid structure, recognition, and metabolism, etc. Here we present the synthesis of phenoxazine-based nucleoside derivatives and their antiviral activity against a panel of structurally diverse viruses: enveloped DNA herpesviruses varicella zoster virus (VZV) and human cytomegalovirus, enveloped RNA tick-borne encephalitis virus (TBEV), and non-enveloped RNA enteroviruses. Studied compounds were effective against DNA and RNA viruses reproduction in cell culture. 3-(2′-Deoxy-β-D-ribofuranosyl)-1,3-diaza-2-oxophenoxazine proved to be a potent inhibitor of VZV replication with superior activity against wild type than thymidine kinase deficient strains (EC₅₀ 0.06 and 10 μM, respectively). This compound did not show cytotoxicity on all the studied cell lines. Several compounds showed promising activity against TBEV (EC₅₀ 0.35–0.91 μM), but the activity was accompanied by pronounced cytotoxicity. These compounds may be considered as a good starting point for further structure optimization as antiherpesviral or antiflaviviral compounds.

Full text of DOI:10.1016/j.antiviral.2019.01.010

Accepted Manuscript
Antiviral activity spectrum of phenoxazine nucleoside derivatives
Liubov I. Kozlovskaya, Graciela Andrei, Alexey A. Orlov, Evgeny V. Khvatov,
Alexander A. Koruchekov, Evgeny S. Belyaev, Evgeny N. Nikolaev, Vladimir A.
Korshun, Robert Snoeck, Dmitry I. Osolodkin, Elena S. Matyugina, Andrey V. Aralov
PII:
S0166-3542(18)30717-4
https://doi.org/10.1016/j.antiviral.2019.01.010
AVR 4455
DOI:
Reference:
To appear in: Antiviral Research
Received Date: 28 November 2018
Revised Date: 14 January 2019
Accepted Date: 17 January 2019
Please cite this article as: Kozlovskaya, L.I., Andrei, G., Orlov, A.A., Khvatov, E.V., Koruchekov, A.A.,
Belyaev, E.S., Nikolaev, E.N., Korshun, V.A., Snoeck, R., Osolodkin, D.I., Matyugina, E.S., Aralov, A.V.,
Antiviral activity spectrum of phenoxazine nucleoside derivatives, Antiviral Research (2019), doi: https://
doi.org/10.1016/j.antiviral.2019.01.010.
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ACCEPTED MANUSCRIPT
Antiviral Activity Spectrum of Phenoxazine Nucleoside Derivatives
Liubov I. Kozlovskayaa,b,#, Graciela Andreic,#, Alexey A. Orlova,d,#, Evgeny V. Khvatova,
Alexander A. Koruchekova, Evgeny S. Belyaeve, Evgeny N. Nikolaevf, Vladimir A. Korshung,
Robert Snoeckc, Dmitry I. Osolodkina,b,d,*, Elena S. Matyuginah, Andrey V. Aralovg,*
a Institute of Poliomyelitis and Viral Encephalitides, FSBSI “Chumakov FSC R&D IBP RAS”,
Moscow 108819, Russia
b Institute of Translational Medicine and Biotechnology, Sechenov Moscow State Medical
University, Moscow 119991, Russia
c Rega Institute for Medical Research, KU Leuven, Leuven, Belgium
d Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia
e Frumkin Institute of Physical Chemistry and Electrochemistry of the Russian Academy of
Science, Moscow 119071 Russia
f Skolkovo Institute of Science and Technology, Moscow 143025, Russia
g Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
Moscow 117997, Russia
h Engelhardt Institute of Molecular Biology, Moscow 119991, Russia
# - equal contribution
Corresponding authors: AVA (Baruh238@mail.ru), DIO (dmitry_o@qsar.chem.msu.ru)
Abstract
The phenoxazine scaffold is widely used to stabilize nucleic acid duplexes, as a part of
fluorescent probes for the study of nucleic acid structure, recognition, and metabolism etc. Here
we present the synthesis of phenoxazine-based nucleoside derivatives and their antiviral activity

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against a panel of structurally diverse viruses: enveloped DNA herpesviruses varicella zoster
virus (VZV) and human cytomegalovirus, enveloped RNA tick-borne encephalitis virus (TBEV),
and non-enveloped RNA enteroviruses. Studied compounds were effective against DNA and
RNA viruses reproduction in cell culture. 3-(2’-Deoxy-β-D-ribofuranosyl)-1,3-diaza-2-
oxophenoxazine proved to be a potent inhibitor of VZV replication with superior activity against
wild type than thymidine kinase deficient strains (EC50 0.06 and 10 µM, respectively). This
compound did not show cytotoxicity on all the studied cell lines. Several compounds showed
promising activity against TBEV (EC50 0.35-0.91 µM), but the activity was accompanied with
pronounced cytotoxicity. These compounds may be considered as a good starting point for
further structure optimization as antiherpesviral or antiflaviviral compounds.
1. Introduction
Natural nucleosides are unique starting points for design and development of new therapeutic
agents because they are involved in all biological processes and are necessary building blocks of
DNA and RNA. Modified nucleosides form an important class of compounds that are widely
used for specific modulation of processes involving nucleosides. Currently more than 30
nucleoside/nucleotide analogues are approved for the treatment of cancer, viral, parasitic and
bacterial infections (De Clercq et al., 2016; Lapponi et al., 2016; Maffioli et al., 2017).
Nucleoside and acyclic analogues with expanded aromatic systems attract great attention,
because of their unique physico-chemical properties and wide range of biological activity. There
are several groups of such compounds (Figure 1): (1) hetero-expanded purine nucleosides 1 and
2 with increased aromaticity and polarizability, inhibiting the replication of hepatitis C virus
(Seley-Radtke et al., 2008; Zhang et al., 2008; Wauchope et al., 2012; Chen et al., 2015); (2)
etheno compounds, among which the tricyclic derivative of acyclovir 3 (TACV) shows an
inhibitory effect on herpes simplex viruses type 1 and 2 (HSV-1 & HSV-2) in vitro at

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concentrations of 2.7-7.6 µM, being less potent than acyclovir, but more selective (Boryski et al.,
1988). Another representative of this series, N2-etheno compound 4 with 3-(1,3-dihydroxy-2-
propoxy)-methyl chain of the ganciclovir type, showed antiviral activity in the range of 0.05-0.7
µM for HSV-1 and HSV-2 and also inhibited other herpesviruses, e.g., varicella zoster virus
(VZV) and cytomegalovirus (CMV) (Boryski et al., 1991; Golankiewicz et al., 1994); (3)
furano[2,3-d]pyrimidine and pyrrolo[2,3-d]pyrimidine nucleosides, of which furano[2,3-
d]pyrimidine 2’-deoxyribonucleosides 5a-b with C8 or C10 alkyl substituent in the 6-position
were the most potent and selective inhibitors of VZV to date (McGuigan et al., 1999; De Clercq,
2004; Andrei et al., 2005).
2-Oxo-1,3-diazaphenoxazine is a well known analog of cytosine with expanded heterocyclic ring
system that have already been used to stabilize nucleic acid duplexes (Lin and Matteucci, 1998;
Holmes et al., 2003; Ortega et al., 2007; Brown et al., 2014; Varizhuk et al., 2017; Kishimoto et
al., 2017) and i-motif (Tsvetkov et al., 2018), to recognize or detect 8-oxo-dG or a damaged
nucleotide within oligonucleotides (Nakagawa et al., 2007; Nasr et al. 2009; Li et al., 2010;
Taniguchi et al., 2014), and covalently capture 8-nitroguanosine (Fuchi and Sasaki, 2014; Fuchi
and Sasaki, 2015) and 8-thioguanosine (Fuchi et al., 2015). In addition, these derivatives are
considered as fluorescent probes for the study of nucleic acid structure, recognition, and
metabolism (Gardarsson et al., 2011; Rodgers et al., 2014). Given the ready incorporation of
phenoxazine nucleosides into a nucleic acid chain (Edwards et al., 2011), they may impair the
replication and transcription of cells and viruses, affecting the efficacy of DNA and RNA
synthesis by cellular or viral polymerases. Nevertheless, despite a large number of applications
in nucleic acid chemistry and molecular biology, antiviral properties of phenoxazine nucleosides
have not been properly characterized yet. In the same time, phenoxazines without a sugar moiety
have been reported to possess strong antiviral activity against CMV and moderate inhibition of

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HSV-1 and HSV-2 reproduction (Hayashi et al., 2008), thus showing possibility for further
analysis.
Here we report the synthesis of new and known phenoxazine nucleoside derivatives bearing a 2’-
deoxyribose or ribose moiety and their antiviral activity against a panel of structurally diverse
DNA and RNA viruses, comprising VZV (Human alphaherpesvirus 3) wild-type and thymidine
kinase deficient (TK-) strains, CMV (Human betaherpesvirus 5), TBEV (Tick-borne encephalitis
virus, a member of the Flavivirus genus), enterovirus A71 (EVA71, Enterovirus A),
coxsackievirus B1 (CVB1, Enterovirus B), and poliovirus 1 (PV1, Enterovirus C). Herpesviruses
are enveloped dsDNA viruses, while TBEV and enteroviruses represent (+)ssRNA viruses,
enveloped and non-enveloped, respectively. Flavi- and herpesviruses were suppressed by the
compounds of the series, and in most cases antiviral activity was accompanied by a pronounced
cytotoxicity, thus limiting direct applicability of the compounds. None of the compounds
inhibited the reproduction of enteroviruses. Phenoxazine 2’-deoxyriboside 7a potently inhibited
VZV without signs of cytotoxicity on all the studied cell lines and may be considered as a good
starting point for the further structure optimization.
2. Materials and methods
2.1. Chemical synthesis
Detailed chemical procedures are reported in the Supplemental information file.
2.2. Biological assays
2.2.1. Cells and viruses
Human embryonic lung (HEL) fibroblasts were purchased from ATCC [HEL 299 (ATCC®
CCL-137™).

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Porcine embryo kidney (PEK) and rhabdomyosarcoma (RD) cell lines were from FSBSI
“Chumakov FSC R&D IBP RAS”. The latter originated from NIBSC (UK).
Herpes simplex virus type 1 (HSV-1) strain KOS (ATCC® VR-1493™), thymidine kinase-
deficient (TK–) HSV-1 KOS strain resistant to ACV (ACVr) was selected in vitro under
increasing concentrations of ACV, herpes simplex virus type 2 (HSV-2) strain G (ATCC® VR-
734™); varicella-zoster virus (VZV) strain OKA (ATCC VR-795), TK–VZV strain 07-1 (kindly
provided by Shiro Shigeta, Fukushima Medical Center, Japan; human cytomegalovirus (CMV)
strains AD-169 (ATCC® VR-538™) and Davis (ATCC® VR-807™) were used at the Rega
Institute for Medical Research.
Tick-borne encephalitis virus strain Absettarov (GenBank accession no. KU885457) was isolated
from blood of the patient with acute TBE in 1951 in Russia.
Enterovirus A71 isolate 46973 (GenBank accession no. KJ645808) was isolated from patient
with acute flaccid paralysis in 2013 in Russia. Enterovirus B Coxscakievirus B1 isolate 48461
was isolated from patient with enteroviral meningitis in 2013 in Russia. Enterovirus C reference
vaccine strain Sabin 1 of poliovirus type 1 (GenBank access no. V01150) originated from
NIBSC (UK).
2.2.2. Cell toxicity evaluation and cytostatic assay in HEL cells
Cytotoxicity of the tested compounds was expressed as the minimum cytotoxic concentration
(MCC) or the compound concentration that caused a microscopically detectable alteration of cell
morphology. Alternatively, the cytostatic activity of the test compounds was measured based on
inhibition of cell growth. HEL cells were seeded at a rate of 5 x 103 cells/well into 96-well plates
and allowed to proliferate for 24 h. Then, medium containing different concentrations of the test
compounds starting at 100 µM was added. After 3 days of incubation at 37 °C, the cell number
was determined with a Coulter counter. The cytostatic concentration was calculated as the CC50,
(compound concentration required reducing cell proliferation by 50%).

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2.2.3. Cell toxicity assay in PEK cells
A cytotoxicity test in PEK cells was performed as described previously (Orlov et al. 2016). Stock
solutions of the compounds in DMSO (concentration of approximately 5-20 mM) were serially
diluted two-fold in medium 199 in Earle solution (FSBSI “Chumakov FSC R&D IBP RAS”,
Russia) to obtain final concentrations (stock/100 ꢀM). Equal volumes of compound dilutions
were added in two replicates to the cell monolayers. Control cells were treated with
corresponding sequential concentrations of DMSO in two replicates. After incubation at 37 °C
on days 1 and 7, cells morphology and vitality was assessed via microscope. CC50 was calculated
according to the Karber method (Lorenz and Bogel, 1973).
2.2.4. Cell toxicity assay in RD cells
Two-fold dilutions of compounds (concentration 5-20 mM) were prepared in Eagle minimal
essential medium with doubled amino acids and vitamins (2×EMEM, FSBSI “Chumakov FSC
R&D IBP RAS”, Russia) to obtain final concentrations starting from approx. stock/24 ꢀM. Equal
volumes of compound dilutions were added in four replicates to the 96-well plates and covered
with RD cell suspension (approx. 105 cells per well) in 2×EMEM with 5% FBS (Invitrogen,
South America). Control cells were treated with the same sequential concentrations of DMSO, as
in compound dilutions, in four replicates. After incubation at 37 °C on days 1 and 7, cells
morphology and vitality was assessed via microscope. CC50 values were calculated according to
the Karber method (Lorenz and Bogel, 1973).
2.2.5. Herpesviruses cytopathicity or plaque reduction test
Confluent HEL cell cultures in 96-well plates were inoculated with 100 CCID50 of virus (CCID50
is a virus dose to infect 50% of the cell cultures) (CMV) or with 20 plaque forming units (PFU)
(VZV). Following a 2 h adsorption period, viral inoculum was removed and the cell cultures
were incubated in the presence of varying concentrations of the test compounds starting at 100
µM. Viral cytopathicity (CMV) or plaque formation (VZV) was recorded as soon as it reached

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completion in the control virus-infected cell cultures that were not treated with the test
compounds. Antiviral activity was expressed as the EC50 [compound concentration required
reducing virus-induced cytopathicity (CMV) or viral plaque formation (VZV) by 50%].
2.2.6. TBEV plaque reduction test
Plaque reduction test was performed as previously described32. In brief, stock solutions of the
compounds in DMSO were diluted in medium 199 in Earle solution (FSBSI “Chumakov FSC
R&D IBP RAS”, Russia) and added to the cells simultaneously with the virus (20-40 PFU/well)
at final concentration of approximately 50-200ꢀM in each well in 8 replicates. Virus with
corresponding DMSO dilution was used as a control. Cells were incubated at 37 °C for 1 h for
infectious virus adsorption. Then, each well was overlaid with 1 mL of 1.26% methylcellulose
(Sigma) containing 2% FBS (Invitrogen, South America). After incubation at 37 °C for 6 days,
the cells were fixed with 96% ethanol. Plaques were stained with 0.4% gentian violet and
counted. EC50 were calculated according to the Reed-and-Meunch method (Reed and Muench,
1938).
2.2.7. Enteroviruses cytopathic effect inhibition test
Cytopathic effect inhibition test against representatives of Enterovirus genus was performed as
described previously (Kozlovskaya et al. 2018). In brief, eight 2-fold dilutions of stock solutions
of the compounds in 4 replicates were prepared in 2×EMEM (FSBSI “Chumakov FSC R&D IBP
RAS”, Russia) to obtain a final concentration series starting from approx. stock/24 ꢀM.
Compound dilutions were mixed with equal volumes of the enterovirus suspension containing
100 TCID50 (50% tissue culture infectious dose). Control cells were treated with the same
sequential concentrations of DMSO as in compound dilutions. After 1 h incubation at 36.5 °C
the RD cell suspension (approx.105 cells per well) in 2×EMEM containing 5% FBS (Invitrogen,
South America) was added to experimental mixtures. Each experiment contained virus dose
titration in the inoculate to assure the acceptable dose-range. After a 5-day incubation at 37 °C,

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cytopathic effect (CPE) was visually accessed via microscope. EC50 values were calculated
according to the Karber method (Lorenz and Bogel, 1973).
3. Results
3.1. Chemistry
Twelve of 18 tested compounds were synthesized earlier and described in scientific or patent
literature. Of these known derivatives, compounds 7a, 8a, and 11a were prepared by a method
developed by Matteucci group (Lin et al., 1995; Lin and Matteucci, 1998). The structures of 7b,
11b, and 12b were covered by patents without synthesis details (Manoharan and Rajeev, 2009a;
Manoharan and Rajeev, 2009b; Manoharan, 2009; Manoharan et al., 2010). The synthesis of 7c,
8b-c, and 13b was described in patents (Manoharan and Rajeev, 2009a; Manoharan and Rajeev,
2009b; Manoharan, 2009; Manoharan et al., 2010; Lin and Matteucci, 1997). Compound 12a
was an intermediate in the synthesis of G-clamp H-phosphonate/phosphoramidite, used without
isolation and characterization (Lin and Matteucci, 1998; Ortega et al., 2007), and was also
described without synthetic details in patents (Lin and Matteucci, 1997; Bodepudi et al, 2016).
Finally, the preparation of 1,3-diaza-2-oxophenoxazine 17 was described earlier (Edwards et al.,
2011). Compounds 10b, 13a, 14a-b, 15, and 16 were newly synthesized.
In general, a well-developed methodology with small changes was followed for the synthesis
(Lin et al., 1995; Lin and Matteucci, 1998; Ortega et al., 2007). The preparation of the 5’-OH and
5’-O-DMTr phenoxazine nucleosides 7a-c and 8a-c starting from corresponding 5-bromouracil
derivatives 6a-c (Scheme 1) is given in full detail in the Supplementary Material.
G-clamp-based derivatives 12a-b and 14a-b was prepared as described (Scheme 2, see also
Supplementary Material for details).
To prepare new N10-alkylated derivatives, 8a was alkylated with iodoacetamide in the presence
of DBU, yielding 15, which, in turn, was 5΄-O-deprotected for the preparation of 16 (Scheme 3).

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1,3-Diaza-2-oxophenoxazine 17 was prepared earlier starting from 5-bromouracil (Edwards et
al., 2011). For the synthesis of 17, an alternative method was developed using glycosidic bond
cleavage of 7a in the presence of HClaq. (Scheme 3).
3.2. Biology
All synthesized final compounds together with some intermediates (Figure 2) were tested for
their antiviral activities against the human pathogenic VZV, CMV, TBEV and enteroviruses of
A, B, and C species.
Inhibition of dsDNA herpesviruses was measured by viral cytopathic effect (CPE) reduction
assay (CMV) or plaque reduction assay (VZV) in human embryonic lung (HEL) cell
monolayers. Wild type [OKA (TK+)] and thymidine kinase deficient [07-1 (TK-)] VZV strains,
and AD-169 and Davis CMV strains were used. Antiviral activity is expressed as the 50%
effective concentration (EC50) or concentration required to reduce viral CPE (CMV) or viral
plaque formation (VZV) by 50%. Cytotoxicity in the same cell line was assessed by two
parameters: minimal cytotoxic concentration (MCC) that caused a microscopically detectable
alteration of cell morphology in confluent monolayers and by cytostatic activity, i.e. 50%
cytostatic concentration (CC50) or concentration required to reduce cell growth by 50% on
replicating growing cells. The results are given in Table 1.
Compounds 7a, 8a, 8b, 12a, 14a, 15 showed activity against VZV. Compound 7a emerged as
the most active one against VZV [OKA (TK+)] strain (EC50 = 0.06 µM), but it was about 170
times less active against the [07-1 (TK-)] strain. Compared to the reference anti-VZV drugs, 7a
was 42-fold more active than acyclovir and about 3-fold less active than brivudine against the
OKA strain and similar to the reference drug, compound 7a had decreased activity against TK
deficient VZV. Interestingly, 7a was not cytotoxic or cytostatic for HEL cells at the maximum

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concentration tested (i.e. 100µM), resulting in selectivity indices (ratio CC50/EC50) of 1667
(OKA strain) and 10 (07-1 strain).
Compounds 8a, 8b, 12a, 14a, 15 showed anti-VZV activity in the micromolar range and proved
equally active against VZV TK+ and TK- strains, being their EC50’s comparable to the ones of
the reference compound acyclovir against the OKA strain. In contrast to 7a, compounds 8a, 8b,
12a, 14a, 15 proved cytostatic for HEL cells (CC50 9.7-37 µM) and thus poorly selective. These
compounds were not only cytostatic, but they were also able to alter cell morphology, when
studied on confluent HEL cells.
8a, 8c, 12b, 13b, 14a, and 15 showed only minor activity against CMV strain Davis with EC50’s
in the micromolar range, comparable to ganciclovir, but less effective than cidofovir. However,
none of the compounds (except 8a) showed inhibitory effect against CMV strain AD-169 in the
studied range of concentrations. Considering their cytostatic and cytotoxic effects on HEL cells,
and the lack of activity for the AD-169 CMV strain, 8a, 8c, 12b, 13b, 14a, 15 cannot be
considered as CMV leads. Additionally, none of the compounds showed activity when tested
against HSV-1 and HSV-2 by CPE reduction assay in HEL cells.
Inhibition of reproduction of (+)ssRNA viruses was measured using 50% plaque reduction assay
for TBEV in porcine embryo kidney (PEK) cells’ monolayer and 50% cytopathic effect
inhibition assay for enteroviruses in growing human rhabdomyosarcoma (RD) cells. Acute and
chronic cytotoxicity in the same cell lines were also assessed as cytotoxic concentration (CC50)
required to cause a microscopically detectable alteration of cell morphology in 50% cells in
confluent PEK cells and cytotoxic concentration (CC50) required to reduce cell growth by 50%
on replicating growing RD cells. The results are given in Table 2. None of the tested compounds
inhibited the reproduction of enteroviruses, while most of them were substantially toxic for
growing RD cells, especially when the cumulative effect was estimated as chronic cytotoxicity.
On the other hand, the compounds did not show signs of acute toxicity on PEK cells, but chronic

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toxicity was pronounced as well for series 8 and 12. Potent inhibition of TBEV reproduction was
shown by DMTr protected molecules (EC50 0.35-10.7 µM).
The compounds have also been evaluated against other RNA viruses: enveloped (-)ssRNA
vesicular stomatitis virus (family Rhabdoviridae) and respiratory syncytial virus (family
Pneumorviridae) (in HeLa cells), non-enveloped dsRNA reovirus-1 (family Reoviridae),
enveloped (+)ssRNA Sindbis virus (family Togaviridae) and yellow fever virus (family
Flaviviridae), enveloped (-)ssRNA Punta Toro virus (in Vero cells) and enveloped (-)ssRNA
influenza virus A and influenza virus B (in MDCK cell) by CPE reduction assay, but no antiviral
activity was found at the highest compound concentration (100 µM) studied (Głowacka et al.
2014).
4. Discussion
The phenoxazine scaffold is widely used to stabilize nucleic acid duplexes, as potential
fluorescent probes for the study of nucleic acid structure, recognition, and metabolism,etc. Here
we presented the synthesis of phenoxazine-based nucleoside derivatives and their antiviral
activity against a panel of structurally diverse viruses and human pathogenic VZV, CMV, TBEV
and enteroviruses of A, B, and C species. Exploration of structure-activity and structure-toxicity
relationships for phenoxazine derivatives employed three variation points: pattern of
phenoxazine moiety modification (7, 8 vs. 11, 13 vs. 12, 14 vs. 15, 16), substitution of pentose 2’
position (a vs. b vs. c), and 5’-O-dimethoxytritylation of pentose (7, 11, 12, 16 vs. 8, 13, 14, 15).
Different susceptibility patterns were observed for DNA and RNA viruses, depending on the
pentose hydroxylation state. Deoxyribose derivatives tend to inhibit multiplication of dsDNA
viruses VZV and CMV more consistently compared with ribose derivatives. Unsubstituted
compound 7a showed the most potent anti-VZV activity in the series, whereas its ribose analogs

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7b, c were much less active. Most of the active compounds showed higher inhibition rate and
range against VZV variants, than against CMV ones. The most potent VZV replication inhibitor
7a is the deoxyribose derivative of unsubstituted phenoxazine that showed inhibition in
micromolar range for both TK+ and TK- strains. This compound selectively inhibits the
replication of the VZV OKA strain, and does not cause toxic or cytostatic effects in any of the
studied cell lines under the studied conditions. Anti-VZV and anti-CMV activity of less specific
compounds 8a and 12a is accompanied by substantial toxicity against all the studied cell lines,
thus restricting their further development.
Substitution in the phenoxazine core was only partially sampled in our series. Introduction of 2-
aminoethoxy moiety in position 9 (series 12), that is responsible for additional hydrogen bonding
with complementary guanine within duplexes, slightly decreased anti-VZV activity and
introduced moderate anti-TBEV activity, but was associated with increased cytotoxicity.
Protection of 2-aminoethoxy with Cbz group eliminated the anti-herpes virus activity. N10-(2-
Amino-2-oxoethyl)ation of phenoxazine also eliminated the antiviral activity of unsubstituted
deoxyribosides. 2’-O-Methylation of ribose (7c) deteriorated the potency of the compound, but
did not induce the cytotoxicity.
5’-O-Dimethoxytritylation rendered all of the compounds consistently active against TBEV
replication in vitro, no matter the hydroxylation state of position 2’. This observation is in line
with previous findings (Angusti et al., 2008; De Burghgraeve et al., 2013; Chatelain et al., 2013;
Saudi et al., 2014; Vernekar et al., 2015; McGuigan et al., 2016;Orlov et al., 2017) and adds
DMTr groups to the arsenal of hydrophobic substituents leading to anti-flaviviral activity of 5’-
O-substituted nucleosides. Notably, VZV and CMV were also occasionally inhibited by these
derivatives, making this substitution pattern efficient not only for ssRNA flaviviruses, but also
for dsDNA herpesviruses. Although, activity was accompanied by pronounced toxicity.
Nevertheless, compounds from series 13-15 were not toxic for PEK cells, and thus could be
further pursued as flavivirus replication inhibitors.

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The reason for the potent antiviral effect of 5’-O-substituted nucleosides is not completely
understood (Eyer et al., 2018). Nevertheless, certain hypotheses may be drawn based on
literature analysis. 5’-O-Tritylated nucleosides are efficient cell permeable inhibitors of host
enzyme thymidine phosphorylase (Liekens et al, 2006). The role of this enzyme in the
pathogenesis of flaviviruses and enteroviruses was never shown. Nevertheless, trytilated uridines
inhibit flavivirus RNA-dependent RNA polymerase (De Burghgraeve et al., 2013). Besides, the
thymidine phosphorylase plays an important role in the metabolism of nucleoside antivirals used
for treatment of herpesviral infections (Guenther et al, 2002). Thus, one may suggest
simultaneous action of the tritylated nucleosides onto the host and virus enzymes as the reason
for improved antiviral activity compared to unsubstituted nucleoside analogs, which may be also
less cell-permeable.
Another effect worth noticing is the increase in cytotoxicity of most of the compounds when
tested on growing cells. The effect is expected, as any stabilization of DNA or RNA-DNA
duplexes would affect most strikingly highly metabolically active growing and replicating cells.
Stabilization of nucleic acid duplexes would make it harder for cellular machinery to unwind or
separate the strands that would decrease DNA replication and transcription. During the
experiments we observed that cells grown in confluent monolayers, where cellular growth was
blocked (contact inhibition), were less affected and showed higher CC50 or MCC values in
comparison with cytostatic effect of the same compound studied in growing cells: MCC values
were substantially lower than CC50 values for HEL cells, and comparison CC50 values for
confluent PEK cells were higher for than the ones obtained on growing RD cells. Therefore, one
can propose that fast replicating viruses, like enteroviruses, would be affected by nucleic acid
stabilizing agents. Nevertheless, all studied compounds were inactive against three Enterovirus
species.

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5. Conclusion
The synthesis of phenoxazine-based nucleoside derivatives and their antiviral activity against a
panel of structurally and replicatively diverse viruses with DNA and RNA genomes, consisting
of varicella zoster virus (VZV), human cytomegalovirus (CMV), tick-borne encephalitis virus
(TBEV), and a selection of enteroviruses was presented. 3-(2’-Deoxy-β-D-ribofuranosyl)-1,3-
diaza-2-oxophenoxazine 7a proved to be a potent inhibitor of VZV replication with higher
activity against wild type than thymidine kinase deficient strains (EC50 0.06 and 10 µM,
respectively). This compound did not show cytotoxicity on all the studied cell lines and may be
considered as a good starting point for the further structure optimization. Several compounds
showed promising activity against TBEV (EC50 0.35-0.91 µM), but the activity was
accompanied with pronounced cytotoxicity. Nevertheless, compounds from similar series were
not toxic for PEK cells, and thus could be further pursued as flavivirus replication inhibitors.
Acknowledgements
This work was supported by the Russian Science Foundation (project no. 16-15-10307 –
determination of antiviral activity against TBEV and enteroviruses). The authors are also grateful
to Ellen De Waegenaeren and Leentje Persoons, for excellent technical assistance.
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Table 1. Anti-herpesvirus activity and corresponding cytotoxicity of phenoxazine nucleosides.
Human embryonic lung
VZV EC50 (µM)
CMV EC50 (µM)
(HEL) cells
Cytotoxicity (µM)
Cmpd #
07-1
Cell
morphology
(MCC)
OKA strain
(TK+)
AD-169
strain
Davis
strain
Cell growth
strain
(CC50)
(TK-)
0.06 ± 0.01 10 ± 5
n/aa
n/a
n/a
4
n/a
n/a
49
>100
>100
>100
20
>100
NDb
7a
n/a
n/a
7b
7c c
55
47
ND
1.5 ± 0.3
2.7 ± 1.3
n/a
1.2 ± 0.5
1.8
4
12.2 ± 0.8
9.7 ± 1.5
ND
8a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
2.9
n/a
n/a
n/a
2.19
10.9
1.27
n/a
4
9 ± 5
20
8b
8c c
10b
11b
12a c
12b c
13b c
14a
14b
15 c
16
n/a
n/a
n/a
>100
>100
4
ND
n/a
n/a
ND
2.23
n/a
1.46
3.57
n/a
ND
20
ND
n/a
60 ± 40
9 ± 5
4
ND
1.7 ± 0.1
n/a
n/a
14.4 ± 0.7
ND
n/a
8.94
n/a
4.63
n/a
47 ± 27
>100
>100
37 ± 5
ND
n/a
n/a
17 c
67
n/a
ND
acyclovir
2.5±0.4
acyclovir ganciclovir ganciclovir
>350
>300
>440
>300
39±9
brivudin cidofovir cidofovir
0.51 0.3
5.99
1.06
Reference
drugs
brivudin
0.022±0.008 1.9±0.5
EC50 – Effective concentration required to reduce virus plaque formation by 50% (20 PFU VZV)
or viral CPE (CMV)
MCC – Minimal cytotoxic concentration that causes a microscopically detectable alteration of
cell morphology
CC50 – Cytotoxic concentration required to reduce cell growth by 50%.
an/a – Not active against the respective virus at non-toxic concentrations
b ND – Not determined
c Experiments for low active compounds were performed once in 2 replicates

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Table 2. Inhibition of (+)ssRNA viruses’ reproduction by phenoxazine nucleosides and
corresponding cytotoxicity.
PEK cells CC50
(µM)
RD cells CC50 (µM)
TBEV EC50 (µM) Enteroviruses EC50 (µM)
Absettarov strain EVA71 CVB1 PV1
Cmpd #
24 h
7 d
24 h
7 d
> 25
n/aa
n/a
n/a
n/a
n/a
n/a
n/a
ND
n/a
n/a
n/a
ND
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
ND
n/a
n/a
n/a
ND
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
ND
n/a
n/a
n/a
ND
n/a
n/a
n/a
n/a
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
7a
> 25
7b
47 ± 1
7c
3.4 ± 1.4
0.9 ± 0.3
1.44 ± 0.01
> 75
23 ± 14 <12.5
4.8 ± 0.9 4.8 ± 0.9
8a
>100
<12.5
28 ± 11 10 ± 2
10 ± 2
4.8 ± 0.9
4.8 ± 0.9
19± 4
8b
>50
8c
NDb
> 50
> 80
> 100
>100
> 50
>40
ND
77 ± 15
ND
10b
11a
11b
12a
12b
13a
13b
14a
14b
15
n/a
> 50
> 80
<12.5
<16
ND
n/a
> 100
> 100
12 ± 1
4.8 ± 0.9 1.2 ± 0.2
14.1±1.9
0.7± 0.5
0.4 ± 0.3
0.9 ± 0.3
2.40 ± 0.02
2.0 ± 0.5
10 ± 2
ND
3.4 ± 0.7
ND
> 50
>40
19 ± 4
16 ± 3
9.6 ± 1.9
> 50
>60
32 ± 11 10 ± 2
35 ± 12 6.8 ± 1.3 8.1 ± 1.6
> 50
> 50
>90
ND
10 ± 2
> 100
>100
10 ± 2
> 100
>100
121 ±
43
n/a
n/a
n/a
n/a
n/a
n/a
n/a
16
17
107.9 ± 0.4
N6-(9-
Reference Anthranylmethyl)-
compounds adenosine
7.8 ± 0.4
>180
N6-(9-
N6-Benzyladenosine
Anthranylmethyl)- N6-Benzyladenosine
adenosine
2.5 ± 0.5 8 ± 3 10 ± 1
> 50
> 50
10 ± 2
10 ± 2
EC50 – Effective concentration required to reduce virus plaque formation by 50% (20-40 PFU
TBEV) or to reduce cytopathic effect by 50% (enteroviruses).
CC50 – Cytotoxic concentration required to reduce cell growth by 50.
a n/a – Not active against respective virus in non-toxic concentrations
b ND – Not determined

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Figure 1. Nucleoside and acyclic analogues with an expanded aromatic system.
Figure 2. Phenoxazine derivatives tested for their antiviral activities against the human
pathogenic VZV, CMV, TBEV and enteroviruses of A, B, and C species.
Scheme 1. The preparation of the 5’-OH and 5’-O-DMTr phenoxazine derivatives. Reagents and
conditions: (a) PPh3, CCl4, CH2Cl2, reflux; then 2-aminophenol, DIPEA, rt; (b) DIPEA,
C2H5OH, reflux, then aq NH3, rt; (c) DMTr-Cl, Py, rt.
Scheme 2. The preparation of the 5’-OH and 5’-O-DMTr G-clamp-based derivatives. Reagents
and conditions: (a) PPh3, CCl4, CH2Cl2, reflux; then 2-aminoresorcinol, DBU, rt; (b) benzyl-N-
(2-hydroxyethyl)carbamate, DEAD, PPh3, CH2Cl2, rt; (c) DIPEA, C2H5OH, reflux, then aq NH3,
rt; (d) H2/Pd, CH3OH, rt; (e) DMTr-Cl, Py, rt.
Scheme 3. The preparation of the N10-alkylated derivatives and 1,3-diaza-2-oxophenoxazine.
Reagents and conditions: (a) iodoacetamide, DBU, CH2Cl2, rt; (b) acetic acid/H2O, 50°C; (c)
conc. HCl/H2O, 60°C.

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•
•
•
1,3-Diaza-2-oxophenoxazine nucleoside derivatives were synthesized.
These compounds were evaluated against DNA and RNA viruses in vitro.
3-(2’-Deoxy-β-D-ribofuranosyl)-1,3-diaza-2-oxophenoxazine proved to be a potent inhibitor of
VZV replication.
•
Several derivatives showed promising activity against TBEV, but the activity was accompanied
with pronounced cytotoxicity.