Synthesis and Comparative Study of Anti-Adenoviral Activity of 6-Azacytidine and Its Analogues

Article abstract of DOI:10.1080/15257770.2015.1034363

This paper presents the results of synthesis and study of cytotoxicity and the anti-adenoviral activity of new N4-derivatives of 6-azacytidine and its α-L-glycopyranosyl analogues obtained by the simplified one-pot version of the silyl condensation method. The resulting acylated 4-methylmercapto-1,2,4-triazin-3(2H)-one glycosides then underwent the amination and/or ammonolysis to provide 6-azacytidine glycoside analogues (2-6, 12, 15, 17) and compounds with modifications at both base and sugar fragments (11, 15). The evaluation of cytotoxicity and antiviral activity of new compounds against AdV5 showed high selectivity indexes for N4-methyl-6-azacytidine (2) and N,O-tetraacetyl-6-azacytidine (8). High anti-adenoviral activity of N4-methyl-6-azacytidine as well as very low cytotoxicity may suggest its further investigation as potential compound for the therapy of AdV infection.

Full text of DOI:10.1080/15257770.2015.1034363

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Synthesis and Comparative Study of Anti-
Adenoviral Activity of 6-Azacytidine and
Its Analogues
Inna Alexeevaa, Lydia Nosachb, Larisa Palchykovskaa, Lyubov Usenkoa
& Olga Povnitsab
a Institute of Molecular Biology and Genetics, National Academy of
Sciences, Kyiv, Ukraine
b D.K. Zabolotny Institute of Microbiology and Virology, National
Academy of Sciences, Kyiv, Ukraine
Published online: 13 Jul 2015.
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To cite this article: Inna Alexeeva, Lydia Nosach, Larisa Palchykovska, Lyubov Usenko &
Olga Povnitsa (2015) Synthesis and Comparative Study of Anti-Adenoviral Activity of 6-
Azacytidine and Its Analogues, Nucleosides, Nucleotides and Nucleic Acids, 34:8, 565-578, DOI:
10.1080/15257770.2015.1034363
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Nucleosides, Nucleotides and Nucleic Acids, 34:565–578, 2015
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2015.1034363
SYNTHESIS AND COMPARATIVE STUDY OF ANTI-ADENOVIRAL
ACTIVITY OF 6-AZACYTIDINE AND ITS ANALOGUES
Inna Alexeeva,1 Lydia Nosach,2 Larisa Palchykovska,1 Lyubov Usenko,1 and
Olga Povnitsa2
1Institute of Molecular Biology and Genetics, National Academy of Sciences, Kyiv, Ukraine
2D.K. Zabolotny Institute of Microbiology and Virology, National Academy of Sciences, Kyiv,
Ukraine
This paper presents the results of synthesis and study of cytotoxicity and the anti-adenoviral
2
activity of new N4-derivatives of 6-azacytidine and its α-L-glycopyranosyl analogues obtained by the
simplified one-pot version of the silyl condensation method. The resulting acylated 4-methylmercapto-
1,2,4-triazin-3(2Н)-one glycosides then underwent the amination and/or ammonolysis to provide
6-azacytidine glycoside analogues (2–6, 12, 15, 17) and compounds with modifications at both
base and sugar fragments (11, 15). The evaluation of cytotoxicity and antiviral activity of new
compounds against AdV5 showed high selectivity indexes for N4-methyl-6-azacytidine (2) and N,O-
tetraacetyl-6-azacytidine (8). High anti-adenoviral activity of N4-methyl-6-azacytidine as well as
very low cytotoxicity may suggest its further investigation as potential compound for the therapy of
AdV infection.
Keywords 6-azacytidine; nucleoside analogues; human adenovirus; antiviral agents
INTRODUCTION
Human adenoviruses (АdV) are ubiquitous infectious DNA viruses pos-
sessing a broad spectrum of pathogenicity. More than 50 human АdV
serotypes have been identified that are responsible for respiratory, gastroin-
testinal, and ocular diseases. АdV are able to persist in humans for a long
time in the latent state and can be reactivated by various factors, especially
grave problems they cause in immunocompromised hosts by the develop-
ment of generalized AdV infection. Disseminated AdV infections frequently
lead to a lethal outcome.
Currently known efficient inhibitors of АdV reproduction are repre-
sented by various classes of synthetic and natural compounds. In particu-
lar, compounds inhibiting АdV adsorption, virus DNA replication, synthesis
Received 23 March 2014; accepted 23 March 2015.
Address correspondence to Dr. Inna Alexeeva, Institute of Molecular Biology and Genetics, National
Academy of Sciences, 150 Zabolotnoho Str. 03680, Kyiv, Ukraine. E-mail: i.v.alexeeva@imbg.org.ua
565

566
Inna Alexeeva et al.
of structural viral proteins, and proteolytic processing of some structural
polypeptides were found. Some of the discussed compounds that demon-
strated anti-adenoviral activity in vitro and in vivo could be potent antiviral
drugs.[1–4] Nevertheless, only two nucleoside analogues, namely cydofovir
and ribavirin, are currently used in clinical practice for the treatment of
patients with life-threatening AdV infections.[3] Some side effects of these
drugs have limited their application in the treatment of AdV diseases.
6-Azacytidine (2-β-D-ribofuranosyl-5-amino-1,2,4-triazin-3(2H)-one, 6-
azaCyd), a structural analogue of natural cytidine, was synthesized by the
Czech–Ukrainian team,[5] and studied first as a potential cancerostatic.[6,7]
More recent research on antiviral properties of 6-azaСyd has demonstrated
its high activity relative to human AdV in vitro and in vivo systems.[8–13] In
cell cultures, the anti-adenoviral effect of 6-azaСyd was expressed by inhibit-
ing the formation of virus-specific intranuclear DNA-containing inclusion
bodies, the synthesis of viral proteins and infectious virus.[12,14]
The application of 6-azaСyd on a model of disseminated AdV infection
in newborn Syrian hamsters led to significant decrease of virus titer and
shortening of the time of virus clearance in organs.[11,12]
This work describes the synthesis and comparative results of the studies
of anti-adenoviral activity of novel and already known 6-azaСyd structural
analogues (N -α-L-glycosides 6-azacytosine, its N 4-derivatives, and seco-6-aza-
nucleosides). The goal of the present study was to find the most effective
inhibitor against AdV in its parental nucleoside form and to determine
separate molecular fragment that enhances the anti-AdV activity.
RESULTS AND DISCUSSION
Chemistry
Chemical strategy for the synthesis of 6-azaСyd analogues was based
on the bioisosteric substitution of individual atoms, functional groups, and
molecular fragments. Efficient methods that have been developed allowed
us to obtain compounds in sufficiently high yields.
The synthesis of 6-azaCyd glycoside analogues was performed by a simpli-
fied one-pot version of the “silyl condensation” method.[10,14–16] This one-pot
procedure involves the formation of silyl ether of heterocyclic aglycone with
excess trimethylchlorosilane (TMSCl) and hexamethyldisilasane (HDMS)
and its interaction with protected carbohydrate activated by tin(IV) chlo-
ride (Schemes 1, 2).[17] As expected, β-anomers of nucleosides were formed
in all cases according to NMR data (J1’–2’ coupling constants in the range
3.6–4.0 Hz). Glycosylation position was confirmed by a bathochromic shift
of UV absorbance maxima of nucleosides in acidic medium (0.1 M HCl),
which is a characteristic for N2-glycosides of 5-amino-1,2,4-triazune.[15] The

Anti-Adenoviral Activity of 6-Azacytidine and Analogues
567
resulting acylated N2-glycosides of 5-metylmercapto-1,2,4-triazin-3(2Н)-one
(MMT) (compounds 1, 10, 13) and 3,5-dithio-(2,3,4,5)-tetrahydro-1,2,4-
triazine (DTT) N2-riboside (16) then underwent amination and/or am-
monolyzis for obtaining the corresponding N -glycosides of 1,2,4-triazine
base (2–7, 12, 15, 17) and compounds with modifications at both nucle-
oside fragments (11, 14). The glycosidic bond of N2-triazinyl nucleosides
(in contrast to that in N4-derivatives) is known to be stable to alkaline hy-
drolysis.[11] Deblocking of protecting groups in triacetyl glycosides 1, 10, 13
by treatment with aqueous ammonia/ethanol (4:1, v/v, RT) resulted in si-
multaneous substitution of 5-methylmercapto group for amino group and
formation of corresponding N-glycosides of 6-azacytosine (Scheme 1). This
general procedure previously reported by our laboratory was easily applied
for 6-azaCyd analogues.[11] Also, the selective amination of triazinebase and
ammonolyzis acetoxy-groups ribofuranoside (16) into the target 6-azaCyd
thio-analogue (17) was achieved in fairly good yield by action of methanolic
ammonia in a sealed vessel at room temperature (Scheme 2). The treatment
of acetylated nucleosides 1, 10, 13 with an excess (1.5–2.0 eq.) of appropriate
alkylamine in absolute ethanol afforded amino-derivatives. Next, the hydrol-
ysis of acetyl groups of carbohydrate fragment with ammonium hydroxide
gave glycosides 2–6, 11, 14, respectively. The N,O-tetraacetyl derivative 8 was
prepared according to the procedure given in literature.[18]
Acyclic 6-azaCyd and 5-methyl-6-azaCyd 2’,3’- seco-nucleosides (9, 9a)
were obtained via the periodate oxidation of carbohydrate residue of cor-
responding ribofuranosides (NaIO4, 1.1 eq.) followed by the reduction of
thus generated aldehyde groups by sodium borohydride as reported previ-
ously.[18,19]
The structures of the obtained compounds were confirmed by spectral
data (1H NMR, UV, and MS) and their comparison with the spectra of 6-
azacytidine and its derivatives with known structure.[9,11] Thus, the position
of anomeric proton of exoN-monosubstituted β-D-ribofuranosyl derivatives
2–6 (δ 5.94–5.96 ppm with a coupling constant value of 3.6–4.0 Hz) and
their λmax (266–272 nm) is in agreement with reported data.[9] Chemical
shifts of anomeric protons and J1’–2’ coupling constants of rhamno- and ara-
binopyranosyl derivatives 11–15 are close to those of previously reported
glucopyranosyl analogues.[11] Note that the NMR spectra of free nucleosides
12, 15, as well as seco-nucleoside 9, show the doublet splitting of exo-NH2
group signal characteristic for 6-azacytidine (ꢀδ 0.11–0.12 ppm), which is
[19]
most pronounced in compounds 9a
and 17 (ꢀδ 0.55 and 0.39 ppm,
respectively). The non-equivalence of amino group protons results from
its restricted rotation and the asymmetry of 1,2,4-triazinyl heterocycle elec-
tronic structure, which is enhanced by the introduction of substituents at
C-3 and/or C-6 positions of the ring. It is assumed that one of the NH2-
group protons is involved in intramolecular hydrogen binding to the nitro-
gen atom N -4.[20] This interpretation is consistent with quantum-chemical

568
Inna Alexeeva et al.
SCHEME 1 Synthesis of novel compounds 2–6, 11, 12, 14, 15. Reagents and conditions: I – peracetyl-
D-ribofuranose, II – peracetyl-L-arabinopyranose, III – peracetyl-L-rhamnopyranose. (a) Peracetyl-sugar,
TMSCl, HMDS, SnCl4, CH3CN, RT, 4–5 hours; (b) amine/ethanol, RT, 0.5 hour; (c) aq. NH3/ethanol,
4:1 v/v, RT, 16–18 hours; (d) Ac2O/Py;[18] (e) NaIO4, then NaBH4.[19]
characteristics of 6-azacytidine. This nucleoside exhibits a high dipole
moment and can actively participate in the formation of hydrogen bonds
and dipole–dipole and π-stacking interactions.[21,22] Formation of similar H-
bonds and other interaction can be suggested for its glycoside analogues as
well.
SCHEME 2 Synthesis of compounds 16, 17. Reagents and conditions: (a) TMSCl, HMDS, SnCl4, CH3CN,
RT, 6 hours; (b) methanolic ammonia, sealed flask, RT, 24 hours.

Anti-Adenoviral Activity of 6-Azacytidine and Analogues
569
TABLE 1 The effect of 6-azacytidine derivatives and analogues on the reproduction of AdV5
adenovirus in vitro
Compound
Name (short name)
Cell line Serotype ЕС50 (μМ)∗ СС50 (μМ)∗
SI
2
3
4
N4-methyl-6-azacytidine
N4-allyl-6-azacytidine
N4-
Hep-2
Hep-2
Hep-2
Ad-5
Ad-5
Аd-5
0.4
56
25.4
1210
1700
920
>3000
30
36
(dimethylaminoethyl)-
6-azacytidine
5
6
N4-(pyridine-3-ylmethyl)-
6-azacytidine
N4-(carbamoylmethyl)-6-
azacytidine
Hep-2
Hep-2
Ad-5
Ad-5
24.0
26.0
560
23
3320
125
7
8
9
9a
11
6-azacytidine (6-azaCyd)
N,O-tetraacetyl-6-azaCyd
6-azaCyd (seco)
5-metyl-6-azaCyd (seco)
2-α-L-arabinopyranosyl-
N 4–methyl-6-
Hep-2
HeLa
Hep-2
Hep-2
Hep-2
Ad-5
Ad-2
Ad-2
Ad-2
Ad-5
2.0
0.3
>100
30
760
380
—
500
1000
380
>1200
—
16
33
31
azacytosine
12
14
2-α-L-arabinopyranosyl-6-
azacytosine
Hep-2
Hep-2
Ad-2
Ad-5
>100
>100
> 400
4
2-α-L-rhamnopyranosyl-
—
—
N 4–methyl-6-
azacytosine
15
2-α-L-rhamnopyranosyl-6-
azacytosine
Hep-2
Ad-5
>100
—
—
17
18
19
2-thio-6-аzаСyd
Ribavirin
Сidofovir
HeLa
Hep-2
Hep-2
Ad-2
Ad-5
Ad-5
0.8
33
10.7
24.0
2000
3560
30.0
60
334
∗The average from three independent experiments, standard deviation below 15%.
ANTIVIRAL ACTIVITY
Anti-adenoviral activity of 6-azaСyd derivatives was evaluated using the
cytomorphological test developed previously by us that allows to precisely
calculate the number of infected cells by the presence of AdV-induced
DNA-containing intranuclear inclusion bodies. The correctness of this
method was confirmed by the fluorescent antibodies method (MFA) ac-
cording to the presence of major structural protein hexon in nucleus.
Both methods demonstrated correlation between anti-adenoviral activity
index values for 6-azaCyd, 6-azauridine, and ribavirin.[12,14,23] This direct
quantitative virus-specific method allows correct determination of anti-
adenoviral effect of tested compounds and has several advantages over
the indirect assays, such as cytopathic effect (СРЕ), plaque formation,
or viability of infected cells (МТТ test) in the primary screening of
compounds.
As can be seen in Table 1, 6-azaCyd (7) is an efficient and selective
inhibitor of AdV with EC50 of 2.0 μM and a selectivity index (SI) of 380.

570
Inna Alexeeva et al.
It should be noted that ribavirin (18) and cidofovir (19) reference com-
pounds studied under the same experimental conditions appeared to be
much weaker AdV inhibitors (EC50 33 and 10.7 μM, respectively) than 6-
azaCyd.
The modified 6-azaСyd derivatives 2–6, 17 containing various sub-
stituents at С-3 and С-5 positions of 1,2,4-triazinyl aglycone demonstrated
significant structure-dependent anti-adenoviral activity. They inhibited AdV
reproduction with inhibitory concentration ranging from 0.4 to 56 μМ and
low cytotoxicity. Compounds 4, 5, and 6 showed decrease in antiviral ef-
fect (EC50 24–26 μM) in comparison with the parent compound, but these
values are close to those obtained for ribavirin. The lowest cytotoxicity was
demonstrated by compound 6. The most efficient compounds in this se-
ries of 6-azaCyd derivatives were N4-methyl-6-azaCyd (2) аnd 2-thio-6-azaCyd
(17), with ЕС50 values being 0.4 μМ аnd 0.8 μМ, respectively. However,
these compounds demonstrated very different cytotoxicity levels that have
an effect on selectivity index. The most active compound 2 has high SI value
(>3000), whereas it was low for compound 17 (SI ∼ 30). As shown in Ta-
ble 1, compound 8 with substituents located at both moieties of nucleoside
molecule also appeared to be an efficient AdV inhibitor (ЕС50 0.3 μМ, SI
value = 1200).
Seco-nucleoside 9 with unsubstituted aglycones, α-L-arabinopyranosyl-6-
аzаСуt (12) and α-L-rhamnopyranosyl-6-аzаСуt (15), as well as other gly-
cosides containing carbohydrate fragments other than ribofuranose, have
lost their ability to inhibit АdV replication (EC50 > 100 μM). Presence of
a methyl group at C-5 or C-6 position of aglycone of seco-nucleoside 9a and
α-L-arabinopyranoside 11, respectively, enhanced their anti-adenoviral ac-
tivity (ЕС50 ∼ 30 μМ) but they showed no increase in antiviral effect in
comparison with the parent compound. However, the same modification of
α-L-rhamnopyranoside 14 was inefficient (EC50 > 100 μM).
Previous studies on anti-adenoviral activity of various 6-azaСyd ana-
logues, including 2ꢁ-deoxy- and 2ꢁ,3ꢁ-dideoxynucleosides,[9] did not reveal
structural elements enhancing the antiviral effect. In this work, we stud-
ied a wide range of compounds. N-glycopyranosides of 6-azacytosine and
seco-nucleoside 9 were inactive against AdV even at high concentrations
(>100 μM), which may be due to their structural features, especially the
conformation state of carbohydrate fragment in these compounds result-
ing in the loss of their affinity to viral targets. Similar results concerning
the decrease or loss of activity after the substitution of ribofuranose residue
for other sugars were obtained with ribavirin[24] and 6-azacytidine glycoside
analogues.[11] At the same time, significant increase in anti-adenoviral ac-
tivity observed for compounds 2, 9a, and 11 in comparison with 7, 9, and
12 was probably caused by the presence of СН3-group at C-5 or C-6 posi-
tion of triazinyl aglycone. It is known that the introduction of a lipophilic
methyl group into the compound structure may enhance its interaction with

Anti-Adenoviral Activity of 6-Azacytidine and Analogues
571
appropriate target area. In addition, electron-donor properties of methyl
group can affect molecule ionization that is frequently reflected in biolog-
ical characteristics of compounds as well. Thus, the presence of N4-methyl
group in compound 2 led to almost five-fold increase of its anti-adenoviral
activity as compared with the parent 6-azaCyd. However, in the case of α-
L-rhamnopyranoside 14, the presence of СН3 groups at both N4-positions
of aglycone did not lead to expected result. It can be supposed that the
C-5ꢁ methyl group of a carbohydrate moiety found in compounds 14 and
15 may significantly restrict the conformational possibilities of nucleosides
and their interaction with the target.[25] Elongation of alkyl chain of N4-
substituent (compounds 3, 4, and 6) or the introduction of pyridyl group
(5) resulted in the weakening of biological effect (Table 1). Thus, methyl
substituent is an important pharmacophoric element for enhancing the
anti-adenoviral activity for 9a and 11 glycoside analogues and N4-methyl
derivative 2.
Compound 17 is one of the best АdV inhibitors, but at the same time, it
has the highest cytotoxicity. This effect may be possibly associated with the
easiness of oxidation of С-3 thiol group in its imine tautomer of triazinyl
ring with a formation of disulfide bond with biopolymers.
Compound 8 acylated at amino and hydroxyl groups (depot form of 6-
azaCyd) exhibited high anti-adenoviral activity, perhaps due to the increased
lipophilicity.
The inhibitory effect of 6-azaCyd was analyzed in our previous stud-
ies also at the level of viral proteins synthesis. Electrophoretic analysis of
14C-labeled viral proteins synthesized in 48 hours after infecting and cell
treatment with 6-azaCyd showed the absence of some early and late proteins
being synthesized prior and after DNA replication, respectively. These pro-
teins required for DNA replication include the DNA-binding protein (DBP,
72 kDa), the precursor of terminal protein (pTP, 75 kDa), which may form
durable heterodimer with AdV-polymerase and serves as a primer for AdV
DNA replication and AdV DNA polymerase, and 140 kDa protein. Blocking
of the synthesis of major structural protein (hexon) was also demonstrated
by the fluorescent antibodies technique.[8,23] It should be noted that the
effect of 6-azaСyd on the synthesis of viral proteins is comparable with that
of ribavirin reported by us previously,[14]
The 6-azaCyd inhibition of the formation of intranuclear DNA-
containing inclusion bodies and the synthesis of early virus-induced and
structural viral proteins allow us to suppose that 6-azaCyd is able to com-
pletely block the expression of adenoviral genome.
CONCLUSIONS
A series of new 6-azaCyd analogues and derivatives were synthesized
and their antiviral activity against АdV reproduction in cell cultures was

572
Inna Alexeeva et al.
evaluated. The results of the present study showed the increase of antiade-
noviral effect of 2, 8, and 17 in comparison with 6-azaCyd and reference
compounds (ribavirin and cydofovir). Two compounds were found to ex-
hibit the activity exceeding five to seven times that of 6-azaCyd, namely 2
(N4-methyl-6-azaCyd) and 8 (N,O-tetraacetyl-6-azacytidine). Anti-adenoviral
action of compounds 2, 9a, and 11 is determined by the presence of СН3
group, which may be a key pharmacophore in antiviral activity of the stud-
ied modified nucleosides. High antiviral effect of N4-methyl-6-azacytidine
against AdV (EC50 0.4 μM, SI > 3000) suggests its further investigation as a
potential treatment for AdV infection.
EXPERIMENTAL
Chemistry
All reagents and solvents for synthesis were from UkrOrgSyntez
(Ukraine), Fluka (Switzerland), and Reachim (Russia); they were purified
and dried by standard methods. Reference compounds 1-β-D-ribofuranosyl-
1,2,4-triazole-3-carboxamide (Ribavirin, Virazole) and 1-(S)-[3-hydroxy-2-
(phosphonomethoxy)-propyl]cytosine (Cydofovir, Vistide) were obtained
from Gilead Sciences (USA).
Thin-layer chromatography (TLC) was performed on Silica gel 60F254
plates (Merck, Germany) in the solvent system chloroform/methanol (9:1
or 14:1, v/v). The glycosides of 1,2,4-triazines were visualized on TLC plates
using a specific color reaction with Dische’s reagent. Column chromatog-
raphy was carried out on silica gel (63–200 mesh, Merck) in the gradient
of methanol in chloroform. The absorption spectra were recorded with
1
a Shimadzu UV-3100 spectrophotometer (Japan). H NMR spectra were
recorded on a Varian Mercury 400 spectrometer (400 MHz) in DMSO-d6
with tetramethylsilane as an internal standard; chemical shifts are given in
ppm. Chromato-mass spectra were obtained with an Agilent 1100 LC/MSD
SL instrument (Germany) in a positive mode. Melting points of new com-
pounds were determined using a Boetius PNMK 05 apparatus (Nagema,
Germany).
General procedure for the preparation of nucleoside intermediates 1,
10, 13. To a solution of 4-methylmercapto-1,2,4-triazin-3(2H)-one (MMT
base, 0.429 g, 3 mmol) and corresponding peracylated sugar (3.3 mmol)
in absolute acetonitrile (25 mL) was added hexamethyldisilazane (HMDS,
0.52 mL, 2.4 mmol), trimethylchlorosilane (TMSCl, 0.6 mL, 4.8 mmol), and
SnCl4 (0.72 mL, 6.0 mmol), and the mixture was stirred under anhydrous
conditions for 4–6 hours at room temperature. The reaction was monitored
by TLC. The solvent was removed under reduced pressure and the crude
product was purified by silica gel column chromatography in a gradient

Anti-Adenoviral Activity of 6-Azacytidine and Analogues
573
of MeOH (0–5%) in CHCl3. The acetylated nucleosides were obtained as
chromatographically homogeneous oils (yields 75–82%).
N2-(2ꢁ,3ꢁ,5ꢁ-tri-O-acetyl)-β-D-ribofuranosyl-5-methylmercapto-1,2,4-triaz-
in-3(2H)-one (1). Colorless foam (0.98 g, 82%). UV (CHCl3): λmax 232,
302 nm; λmin 257 nm. 1H NMR: δ 7.97 (s, 1H, C5H), 6.11 (d, 1H, 1ꢁ-H, J =
3.2 Hz), 5.38 (dd, 1H, 2ꢁ-H), 5.21 (dd, 1H, 3ꢁ-H), 4.30 (m, 2H, 4ꢁ-H, 5ꢁ-H),
4.07 (m, 1H, 5ꢁ-H), 2.56 (s, 3H, SCH3), 2.08–2.02 (m, 9H, 3Ac). LC–MS:
m/z 402.3 [M+H]+.
General procedure for the synthesis of N4-derivatives of 6-azacytidine
(2-6). To a suspension of 1.0 mmol intermediate 1 in 8 mL of ethanol was
added an appropriate amino component (1.5–2 mmol), and the reaction
mixture was stirred at room temperature for several hours (TLC control).
The reaction mixture was concentrated to dryness under reduced pressure
to afford clear oil. Deacylation of glycoside derivatives was carried out with
a mixture of 25% aqueous ammonia and ethanol (4:1, v/v) for 16 hours
at 18◦C. The solvent was removed in vacuo and the residue was crystallized
from ethanol.
2-(β-D-ribofuranosyl)-5-methylamino-1,2,4-triazin-3(2H)-one (2). Yield
from 1: 0.19 g (73%), mp: 219–221◦C. UV (H2О): λmax 272 nm (lg ε 3.96).
1H NMR: δ 8.45 (d, 1H, NH, J = 4.2 Hz), 7.46 (s, 1H, C5H), 5.94 (d, 1H,
1ꢁ-H, J = 3.6 Hz), 4.97 (d, 1H, 2ꢁ-OH, J = 4.2 Hz), 4.75 (d, 1H, 3ꢁ-OH, J =
5.6 Hz), 4.43 (t, 1H, 5ꢁ-OH, J = 5.6; 6.0 Hz), 4.18 (dd, 1H, 2ꢁ-H), 3.98 (dd,
1H, 3ꢁ-H), 3.76 (t, lH, 4ꢁ-H), 3.49 (m, 1H, 5ꢁ-Hβ), 3.38 (m, 1H, 5ꢁ- Hα), 2.84
(d, 3H, CH3, J = 4,8 Hz). LC–MS: m/z 259.2 [M+H]+.
2-(β-D-ribofuranosyl)-5-allylamino-1,2,4-triazin-3(2H)-one (3). Yield
from 1: 0.169 g (56%), mp: 147–149◦C. UV (H2О): λmax 273 nm (lg ε 3.84).
1H NMR: δ 8.55 (t, 1H, NH), 7.52 (s, 1H, C5H), 7.33 (s, 1H, HC = ), 5.95
(d, 1H, 1ꢁ-H, J = 4.0 Hz), 5.14–5.27 (dd, 2H, CH2), 4.95 (d, 1H, 2ꢁ-OH, J =
5.6 Hz), 4.72 (d, 1H, 3ꢁ-OH, J = 6.0 Hz), 4.39 (t, 1H, 5ꢁ-OH, J = 5.6; 6.0 Hz),
4.20 (dd, 1H, 2ꢁ-H), 3.95–4.02 (m, 3H, CH2-N, 3ꢁ-H), 3.78 (t, 1H, 4ꢁ-H), 3.52
(m, 1H, 5ꢁ-Hα), 3.40 (m, 1H, 5ꢁ- Hβ). LC–MS: m/z 285.0 [M+H]+.
2-(β-D-ribofuranosyl)-5-(2-dimethylaminoethyl)-amino-1,2,4-triazin-3
(2H)-one (4). Yield from 1: 0.22 g (70%), mp: 240-242◦C. UV (H2О): λmax
1
267.5 nm (lg ε 3.95). H NMR: δ 8.36 (br.s, 1H, NH), 7.55 (s, 1H, C5H),
2.21 (s, 6H, CH3), 5.94 (d, 1H, 1ꢁ-H, J = 3.6 Hz), 4.97 (d, 1H, 2ꢁ-OH, J =
4.2 Hz), 4.76 (d, 1H, 3ꢁ-OH, J = 7.0 Hz), 4.44 (t, 1H, 5ꢁ-OH, J = 5.6; 6.0 Hz),
4.18 (dd, 1H, 2ꢁ-H), 3.98 (dd, 1H, 3ꢁ-H), 3.76 (dd, 1H, 4ꢁ-H), 3.51 (m, 1H,
5ꢁ-Hα), 3.38–3.97 (m, 3H, CH2, 5ꢁ-Hβ), 2.43 (t, 2H, CH2). LC–MS: m/z
316.2 [M+H]+.
2-(β-D-ribofuranosyl)-5-(pyridin-3-ylmethyl)-amino-1,2,4-triazin-3(2H)-
one (5). Yield from 1: 0.20 g (62%), mp: 117–120◦C. UV (H2О): λmax
1
267 nm (lg ε 3.95). H NMR: δ 8.95 (br.s, 1H, NH), 8.54 (s, 1H, Py), 8.44
(d, 1H, Py), 7.74 (dd, 1H, Py), 7.54 (s, 1H, C5H), 6.61 (t, 1H, Py), 5.94 (d,
1H, 1ꢁ-H, J = 4.0 Hz), 4.97 (d, 1H, 2ꢁ-OH), 4.74 (d, 1H, 3ꢁ-OH), 4.55 (d, 2H,

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Inna Alexeeva et al.
CH2, J = 7.6 Hz), 4.44 (t, 1H, 5ꢁ-OH), 4.18 (dd, 1H, 2ꢁ-H), 3.98 (dd, 1H,
3ꢁ-H), 3.76 (dd, l H, 4ꢁ-H), 3.51 (m, 1H, 5ꢁ-Hα), 3.43 (m, 1H, 5ꢁ-Hβ), 1.07 (s,
1H, Py). LC–MS: m/z 336.0 [M+H]+.
2-(β-D-ribofuranosyl)-5-(carbamoylmethyl)-amino-1,2,4-triazin-3(2H)-
one (6). Yield from 1: 0.181 g (60%), mp: 110–112◦C. UV (H2О): λmax
271 nm (lg ε 3.96). 1H NMR: δ 8.95 (br.s, 1H, NH), 7.68 (s, 1H, C5H), 7.55
(s, 1H, NH2), 7.17 (s, 1H, NH2), 5.98 (d, 1H, 1ꢁ-H, J = 3.9 Hz), 5.20 (d, 1H,
2ꢁ-OH, J = 5.4 Hz), 4.96 (d, 1H, 3ꢁ-OH, J = 6.0 Hz), 4.67 (t, 1H, 5ꢁ-OH, J =
5.7; 6.0 Hz), 4.22 (dd, 1H, 2ꢁ-H, J = 4.0; 5.6 Hz), 3.97 (dd, 1H, 3’-H, J = 6.0;
6.0 Hz), 3.89 (d, 2H, CH2, J = 5.2 Hz), 3.76 (dd, 1H, 4ꢁ-H, J = 5.6; 6.0 Hz),
3.52–3.38 (m, 2H, 5ꢁ- Hα, 5ꢁ-Hβ). LC–MS: m/z 302.0 [M+H]+.
2-(β-D-ribofuranosyl)-5-amino-1,2,4-triazin-3(2H)-one (6-azaCyd, 7).
Yield from 1: 0.226 g (93%), mp: 223–225◦C. UV (H2О): λmax 264 nm (lg
1
ε 3.89), UV (0,1 M HCl): λmax 285 (lg ε 3.96). H NMR: δ 7.91, 7.77 (2s,
2H, NH2), 7.45 (s, 1H, C5H), 5.94 (d, 1H, 1ꢁ-H, J = 4.0 Hz), 4.95 (d, 1H,
2ꢁ-OH, J = 5.2 Hz), 4.72 (d, 1H, 3ꢁ-OH, J = 6.0 Hz), 4.12 (t, 1H, 5ꢁ-OH, J =
5.2; 6.0 Hz), 4.20 (dd, 1H, 2ꢁ-H, J = 4.0; 5.6 Hz), 3.98 (dd, 1H, 3’-H, J = 6.0;
6.0 Hz), 3.77 (dd, 1H, 4ꢁ-H, J = 5.6; 4.4 Hz), 3.52 (m, 1H, 5ꢁ- Hα), 3.40 (m,
1H, 5ꢁ-Hβ). LC–MS: m/z 245.1 [M+H]+.
2-β-D-(2’, 3’, 5’- tri-O-acetyl)-N5-acetylamino-1,2,4-triazin-3(2H)-one (8).
Compound 8 was obtained as reported previously.[18] Yield 94%, mp:
147–150◦C (EtОH). UV (H2О): λmax 265 nm (lg ε 4.06). 1H NMR: δ 11.50 (s,
1H, NH), 8.72 (s, 1H, C5H), 6.21 (d, 1H, 1ꢁ-H, J = 3.2 Hz), 5.58 (t, 1H, 2ꢁ-H,
J = 3.5 Hz), 5.40 (t, 1H, 3ꢁ-H, J = 5.6 Hz), 4.32 (m, 2H, 4’-H, 5’-H), 4.06 (dd,
1H, 5’-H, J = 4.4 Hz), 2.18 (s, 3H, Ac), 2.08 (2s, 6H, 2Ac), 2.02 (s, 3H, Ac).
5-Amino-2-[2ꢁ-hydroxy-1ꢁ-(2ꢁꢁ-hydroxy-1ꢁꢁ-hydroxymethyl-ethoxy)-ethyl]-
1,2,4-triazin-3(2H)-one (9) was synthesized as a white solid by the procedure
described in our paper.[19] Yield 92%, mp: 143–146◦C. UV (H2О): λmax
263.5 nm (lg ε 3.79).
2-(α-L-arabinopyranosyl)-5-methylamino-1,2,4-triazin-3(2H)-one
(11).
Yield from 10: 0.162 g (63%), mp: 225–228◦C. UV (H2О): λmax 275.5 nm
1
(lg ε 3.94). H NMR: δ 8.49 (d, 1H, NH, J = 4.6), 7.55 (s, 1H, C5-H), 5.92
(d, 1H, 1ꢁ-H, J = 3.6 Hz), 5.42 (d, 1H, 2ꢁ-OH), 5.25 (d, 1H, 3ꢁ-OH), 4.72
(t, 1H, 4ꢁ-OH), 4.41 (dd, 1H, 2ꢁ-H), 3.83 (s, 2H, 3ꢁ-H, 4ꢁ-H), 3.53 (m, 1H,
5ꢁ-Hα), 3.40 (m, 1H, 5ꢁ-Hβ), 2.80 (d, 3H, CH3, J = 4.6 Hz). LC–MS: m/z
259.2 [M+H]+.
2-(α-L-arabinopyranosyl)-5-amino-1,2,4-triazin-3(2H)-one (12). Yield
from 10: 0.157 g (61%), mp: 240–242◦C. UV (H2О): λmax 264 nm (lg ε
1
3.82), UV (0,1 M HCl): λmax 270 nm (lg ε 3.94). H NMR: δ 7.88, 7.76 (2s,
2H, NH2), 7.53 (s, 1H, C5-H), 5.91 (d, 1H, 1ꢁ-H, J = 5.6 Hz), 5.27 (d, 1H,
2ꢁ-OH, J = 6.4 Hz), 5.13 (d, 1H, 3ꢁ-OH, J = 6.0 Hz), 4.56 (t, 1H, 4ꢁ-OH), 4.40
(dd, 1H, 2ꢁ-H), 3.83 (m, 2H, 3ꢁ-H, 4ꢁ-H), 3.55 (m, 1H, 5ꢁ-Hα), 3.41 (m, 1H,
5ꢁ-Hβ). LC–MS: m/z 245.0 [M+H]+.

Anti-Adenoviral Activity of 6-Azacytidine and Analogues
575
2-(α-L-rhamnopyranosyl)-5-methylamino-1,2,4-triazin-3(2H)-one
(14).
Yield from 13: 0.176 g (56%), mp: 245–248◦C. UV (H2О): λmax 274 nm (lg
1
ε 3.95). H NMR: δ 8.58 (d, 1H, NH, J = 4.4 Hz), 7.44 (s, 1H, C5-H), 5.93
(d, 1H, 1ꢁ-H, J = 4.0 Hz), 4.672 (d, 1H, 2ꢁ-OH), 4.66 (d, 1H, 3ꢁ-OH), 4.45
(s, 1H, 4ꢁ-OH), 3.99 (d, 1H, 2ꢁ-H, J = 3.6 Hz), 3.93 (br s, 1H, 3ꢁ-H), 3.64 (t,
1H, 4ꢁ-H, J = 6.4; 6.8 Hz), 3.31 (d, 1H, 5ꢁ-H), 2.51 (d, 3H, CH3, J = 4,4 Hz),
1.183 (d, 3H, CH3, J = 6,4 Hz). LC–MS: m/z 273.0 [M+H]+.
2-(α-L-rhamnopyranosyl)-5-amino-1,2,4-triazin-3(2H)-one (15). Yield
from 13: 0.168 g (65%), mp: 262–264◦C. UV (H2О): λmax 271.3 nm (lg ε
1
3.65), UV (0,1 M HCl): λmax 279.7 (lg ε 4.01). H NMR: δ 7.79, 7.68 (2s,
2H, NH2), 7.43 (s, 1H, C5-H), 5.93 (d, 1H, 1ꢁ-H, J = 3.6 Hz), 4.66 (s, 1H,
2ꢁ-OH), 4.61 (s, 1H, 3ꢁ-OH), 4.45 (s, 1H, 4ꢁ-OH), 3.90–4.00 (dd, 2H, 3ꢁ-H;
2ꢁ-H), 3.65 (t, 1H, 4ꢁ-H, J = 6.4; 7.0 Hz), 3.30 (m, 1H, 5ꢁ-H), 1.18 (d, 3H,
CH3, J = 7.0 Hz). LC–MS: m/z 259.2 [M+H]+.
2-(β-D-Ribofuranosyl)-5-amino-1,2,4-triazin-3(2H)-thione (2S-6-azaCyd,
2-thio-6-azacytidine, 17). The acetylated 2-β-D-ribofuranosyl-1,2,4-triazin-
3,5(2H,4H)-dithione was prepared by the reaction of 2,4-dithio-6-
azauracil[25] (0.145 g, 1 mmol) with tetraacetylribofuranose (0.35 g, 1 mmol)
using the conventional methodology. Purification by silica gel flash chro-
matography (chloroform–methanol, 95:5 v/v) gave 0.31 g (77%) of the
product 16 as a red-brown solid. It was dissolved in methanolic ammonia
(15 mL), and gaseous ammonia was passed through this solution for 2 hours
at room temperature. The mixture was kept in a sealed flask for 24 hours. The
solvent was removed in vacuo and the residue was crystallized from ethanol.
Yield 0.17 g (73%), mp: 235-239◦C (lit. 235–238◦C[26]). UV (EtОH): λmax
242 nm (lg ε 4.09); 282 nm (lg ε 3.83). 1H NMR: δ 8.53, 8.14 (2s, 2H, NH2),
7.74 (s, 1H, C5H), 7.04 (d, 1H, 1ꢁ-H, J = 3.6 Hz), 5.07 (d, 1H, 2ꢁ-OH, J =
4.8 Hz), 4.76 (d, 1H, 3ꢁ-OH, J = 6.4 Hz), 4.43 (t, 1H, 5ꢁ-OH, J = 4.8; 6.0 Hz),
4.12 (dd, 1H, 2ꢁ-H, J = 4.0; 4,8 Hz), 4.00 (dd, 1H, 3ꢁ-H, J = 6.0; 5.6 Hz),
3.80 (dd, 1H, 4ꢁ-H, J = 6.0; 6.0 Hz), 3.54 (m, 1H, 5ꢁ-H), 3.45 (m, 1H, 5ꢁ-H).
LC–MS: m/z 261.0 [M+H]+.
BIOLOGY
Cells and Viruses
Reference strains of AdV (types 2 and 5) obtained from the Institute of
Microbiology, Semmelweis University Medical School (Budapest, Hungary)
and monolayer culture Hep-2 cells were used in the study. Cells were grown
in a medium containing equal volumes of medium 199 and Eagle medium,
supplemented with 10% of heat-inactivated fetal bovine serum (Sigma),
100 U/mL of penicillin and 100 mg/mL of streptomycin. Infected cells
were incubated in maintenance medium (Eagle medium without serum).

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Inna Alexeeva et al.
Antiviral and Cytotoxicity Assays
Anti-AdV activity was determined by a method developed in our labora-
tory, and is based on the calculation of infected cells with DNA-containing
intranuclear inclusion bodies induced by the virus.[14] In preliminary experi-
ments, we have found the virus infection dose that in 48 hours post-infection
induced intranuclear inclusion bodies in 70–80% of infected cells. Cells were
grown in tubes with strips of cover glasses. In 48 hours, cells were infected
with AdV and after 1 hour of the adsorption of virus at room temperature,
cells were washed with Hank’s solution and pre-incubated in maintenance
medium containing test compounds at varying concentrations. The infected
cells, which were not treated with the assayed compounds, were used as a
positive control of virus infection. After 48 hours of incubation at 37◦C, the
cells were fixed with 96% ethanol, washed with Hank’s solution, stained with
0.01% acridine orange solution, and the number of infected cells with DNA-
containing inclusion bodies was counted using a luminescent microscope
LM-2 (LOMO, Russia). On each of the three slides, 500 cells were counted
and the percentage of infected cells was determined. The 50% effective
concentration (EC50) was determined as the compound concentration de-
creasing the percentage of infected cells with intranuclear inclusion bodies
by 50% in comparison with the virus control.
The cytotoxicity of the compounds was determined in Hep-2 cells by
a standard MTT test, a colorimetric assay for cell viability.[27] Hep-2 cells
growing in 96-well plates were treated by serial dilutions of tested compounds
for four days. The compound concentration that reduced the absorbance
of mock-infected cells at 570 nm by 50% of that of the control was defined
as 50% cytotoxic concentration (CC50). Selectivity index was determined
as a ratio of minimal cytotoxic concentration (CC50) to minimal effective
concentration (EC50). Three independent experiments were performed to
determine the CC50 and EC50 values for each compound presented in the
Table.
ACKNOWLEDGMENT
The authors are grateful to Dr. Igor Dubey for valuable discussions and
support.
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