6-Azacytidine - Compound with wide spectrum of antiviral activity

Article abstract of DOI:10.1081/NCN-100002508

6-Acytidine demonstrates activity against adenoviruses types 1, 2, 5. It inhibit synthesis of viral DNA and proteins. 6-AC shows antiherpetic and antiinfluenza action during experimental infection in mice. 6-AC is prospective for drug development as an antiviral substance with a wide spectrum of activity.

Full text of DOI:10.1081/NCN-100002508

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6-AZACYTIDINE - COMPOUND WITH WIDE SPECTRUM
OF ANTIVIRAL ACTIVITY
I. Alexeeva a , N. Dyachenko b , L. Nosach b , V. Zhovnovataya b , S. Rybalko c
, R. Lozitskaya d , A. Fedchuk e , V. Lozitsky e , T. Gridina e , A. Shalamay a , L.
Palchikovskaja a & O. Povnitsa b
a Institute of Molecular Biology and Genetics, Kyiv, Ukraine
b Institute of Microbiology and Virology, Kyiv-143, 03143, Ukraine
c Institute of Epidemiology and Infectious Diseases, Kyiv, Ukraine
d Physical-Chemical Institute, Odesa, Ukraine
e Institute of Emerging Infectious Diseases, Odesa, Ukraine
Version of record first published: 07 Feb 2007.
To cite this article: I. Alexeeva , N. Dyachenko , L. Nosach , V. Zhovnovataya , S. Rybalko , R. Lozitskaya , A. Fedchuk ,
V. Lozitsky , T. Gridina , A. Shalamay , L. Palchikovskaja & O. Povnitsa (2001): 6-AZACYTIDINE - COMPOUND WITH WIDE
SPECTRUM OF ANTIVIRAL ACTIVITY, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 1147-1152
To link to this article: http://dx.doi.org/10.1081/NCN-100002508
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1147–1152 (2001)
6-AZACYTIDINE - COMPOUND WITH WIDE
SPECTRUM OF ANTIVIRAL ACTIVITY
I. Alexeeva,1 N. Dyachenko,2,∗ L. Nosach,2 V. Zhovnovataya,2
S. Rybalko,3 R. Lozitskaya,4 A. Fedchuk,5 V. Lozitsky,5 T. Gridina,5
A. Shalamay,1 L. Palchikovskaja,1 and O. Povnitsa2
1Institute of Molecular Biology and Genetics, Kyiv, Ukraine
2Institute of Microbiology and Virology, Kyiv-143, Ukraine 03143
3Institute of Epidemiology and Infectious Diseases, Kyiv, Ukraine
4Physical-Chemical Institute, Odesa, Ukraine
5Institute of Emerging Infectious Diseases, Odesa, Ukraine
ABSTRACT
6-azacytidine demonstrates activity against adenoviruses types 1, 2, 5. It inhibit
synthesis of viral DNA and proteins. 6-AC shows antiherpetic and antiinfluenza
action during experimental infection in mice. 6-AC is prospective for drug
development as an antiviral substance with a wide spectrum of activity.
INTRODUCTION
The chemotherapy of virus infections is developing rather intensively now. It
is very important for medicine owing to the wide spread of influenza virus, herpes
simplex virus and adenoviruses and their important role in pathology. Modified
nucleosides have proven to be the useful substances for antiviral and anticancer
drug design. 6-Azacytidine (2-β-D-ribofuranosyl-5-amino-1,2,4-triazin-3(2H)-on;
6-AC) is an original structural cytidine analogue (Fig. 1). The objective of the
present investigation is to study of the antiviral activity 6-AC in different in vitro
and in vivo models.
∗Corresponding author. Fax at +38 044 266 23 79; E-mail: dyachenko@srv.imv.kiev.ua
1147
ꢀ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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ALEXEEVA ET AL.
Figure 1. 6-Azacytidine.
MATERIALS AND METHODS
The method of synthesis for 6-azacytidine, based on a simplified one-step
variant of a ꢁsilyl condensationꢂ, was developed in our laboratory. Selective N2-
glycosylation of 5-amino-1,2,4-triazine-3(2H)-one (6-azacytosine) by peracylri-
bofuranose in the presence of hexamethyldisilazane, trimethylchlorosilane, and tin
tetrachloride (catalytic) gives the tri-o-acyl derivative of 6-azacytidine in 68% yield.
The subsequent unblocking of the glycon moiety in alkaline medium led to unpro-
tected 6-AC (1). The structure of 6-AC was confirmed by their 1H-NMR spectral
data.
After crystallization from 80% ethanol 6-AC was obtained as colorless crys-
1
tals, m.p. 223–225◦C. UV(H2O ): λmax264 nm (lg ε 3.9). H-NMR (DMSO-d6)
δ: 8.018 (s, 1H, Ha-N); 7.901 (s, 1H, Hb-N); 7.503 (s, 1H, H-C5); 5.970 (d, 1H,
H-1ꢃ); 5.191 (d, 1H, OH-2ꢃ); 4.978 (d, 1H, OH-3ꢃ); 4.680 (t, 1H, OH-5ꢃ); 4.200
(dd, 1H, H-2ꢃ); 3.950 (dd, 1H, H-3ꢃ); 3.486 (m, 1H, Ha-5ꢃ); 3.359 (m, 1H,
Hb-5ꢃ).
Hep-2 or HeLa cells were grown in tubes with the strips of the cover glasses.
After 48 h, cells were infected with adenoviruses type 1, 2, 5 (Ad-1, Ad-2 or
Ad-5), further, after 60#min adsorption of virus, cells were washed with Hanks’
Table 1. Influence of 6-AC on the Ad 2 Reproduction in Hep-2 Cell Culture
Concentration
µg/ml
Inhibition of Number of Cells
with Intranuclear Inclusions, %
Virus Titres lg CTD50
125
62
16
8
0.5
0 (Control)
100
100
82
86
62
0
0
2.7
—
2.7
4.45
—

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Table 2. Influence of 6-AC on the Ad 5 Reproduction in Hep-2 Cell Culture
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Concentration
of 6-AC, µg/ml
Percent of Cells with
Viral Inclusion Bodies
Inhibition of
Infection, %
125
62
16
8
0.5
0 (Control)
0
0
0
0
24
62
100
100
100
100
61
solution and incubated in Eagle medium carrying 6-AC of varying concentrations.
48 h following infection the cells were fixed, stained with 0.01% acridine orange
solution and investigated by luminescent microscopy. The infected cells were re-
vealed by presence of the virus-specific intranuclear inclusions. An inhibition of
Figure 2. The inhibitory action of 6-AC on quantity of ad-1 infected cells with intranuclear inclusion
(1) and hexon antigen (2) in cell culture HeLa.

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ALEXEEVA ET AL.
Table 3. Prevention Action of 6-AC During Experimental Herpetic Infection
Lethal Cases
Doses
mg/kg
Quantity of Mice
in Experiment
Preparation
6-AC
Total
%
MP∗∗
IE∗∗
0.01
0.1
0.5
1.0
1.0
14
14
14
12
14
14
8
12
10
2
4
14
57.1
85.7
71.4
16.6
28.5
1.73
1.16
1.40
6.02
3.50
42.19
13.79
28.57
83.38
71.14
Acyclovir
Placebo
100
∗-Multiple of protection.
∗∗-Index of efficiency.
virus reproduction in cell cultures by 6-AC was determined by a reduction in the
number (percent) of cells with inclusions (1).
Mice infected intracerebrally with herpes simplex virus (HSV) type 1 were
injected intramusculary with 6-AC in 24 hrs before or after infection.
Influenza infection in mice (12–14 g) was modeled by intranasal challenge
of A/PR/8/34 (H1N1) strain using 4 animals per each logarithmic dilution of virus
containing material. 6-AC was instillated intranasally in dose 25 mg/kg per day
during 5 days beginning from 24 hours before infection. The death of animals was
registered during 14 days after infection.
RESULTS
6-AC over a wide range of concentrations (0,5–125 µg/ml) inhibits the syn-
thesis of infective Ad, formation of intranuclear inclusion bodies and hexon antigen
(Tables 1 and 2, Fig. 2). It also inhibits the synthesis of viral DNA and viral polypep-
tides (2, 3). 6-AC in some high concentrations completely caused the switch-off of
adenovirus genome expression.
The selectivity index of 6-AC was 62–125 towards Ad 1 and Ad 2 and 250
towards Ad 5. 6-AC was effective on the model of herpetic meningoencephalitis
Table 4. Therapeutic Action of 6-AC During Experimental Herpetic Infection
Lethal Cases
Doses
mg/kg
Quantity of Mice
in Experiment
Preparation
6-AC
Total
%
MP
IE
0.01
0.1
0.5
1.0
14
14
14
14
14
10
10
4
6
14
71.4
71.4
28.5
42.8
100
1.40
1.40
3.50
2.30
28.57
28.57
71.40
56.59
Acyclovir
Placebo

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Figure 3. Efficacy of 6-AC during experimental influenza infection in mice.

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ALEXEEVA ET AL.
in mice. It was more active than reference compound acyclovir (“KRKA”, Slovenia)
if preparations were used on preventive or therapeutic schemes (Tables 3 and 4).
The intranasal instillations of 6-AC decreased lethality reliably during exper-
imental influenza infection in mice (Fig. 3). The difference between control and
experimental groups was 1,0 lg LD50.
So, 6-AC has a wide spectrum of antiviral activity. It is a prospective substance
for drug development and for synthesis of its derivatives.
6-AC could be used as the reference antiviral preparation to be compared
with its derivatives containing macrocyclic pyridinophanes [4], which are under
investigation in our group through INTAS-grant 97-31528.
ACKNOWLEDGEMENTS
This study is partially supported by INTAS (Grant 97-31528, which dedicate
to study of anticancer and antiviral properties of macrocyclic pyridinophanes). We
are indebted to Organizing Committee of XIV International Roundtable on Nucle-
osides, Nucleotides and their Biological Applications for waiving of Registration
Fee. Danyla Kryzhanovsky and Andrew Fedchuk are acknowledged for their tech-
nical assistance during preparation of this report.
REFERENCES
1. Alexeeva I.; Palchikovskaya L.; Shalamay A.; Nosach L.; Zhovnovataya V.; Povnitsa
O.; Dyachenko N. Acta Biochim. Polonica, 2000, 47, 95–101.
2. Dyachenko N.; Nosach L.; Alexeeva I.; Zhovnovataya V.; Povnitsa O.; Chernomaz A.;
Shalamay A. Acta Biochim. Polonica, 2000, in press.
3. Nosach L.; Butenko S.; Timofeeva M.; Dyachenko N.; Tikhomirova T.; Alekseeva I.;
Chernetsky V. Mikrobiol. Zhurnal, 1989, 1, 73–75 (In Ukrainian).
4. Kuzmin V.E.; Lozitsky V.P.; Kamalov R.N.; Zheltvay A.I.; Fedtchouk A.S.;
Kryzhanovsky D.N., Acta Biochimica Polonica, Vol. 47, No. 3, 2000, 867–875.

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