Adenosine N1-oxide analogues as inhibitors of orthopox virus replication

Article abstract of DOI:10.1135/cccc20061107

Several new types of adenosine N¹-oxide (ANO) derivatives including N¹-alkoxy and N⁶-alkyl as well as the analogues with a trihydroxycyclopentane ring in place of the ribose residue were synthesized and their antiviral properties were evaluated in Vero and LLC-MK2 cell cultures infected with vaccinia, mousepox, monkeypox, cowpox, and different isolates of smallpox viruses. The antiviral activity of ANO and its derivatives significantly depended on the virus type and cell cultures. Mousepox and monkeypox viruses were the most sensitive to these compounds, while vaccinia and cowpox viruses were inhibited at the concentrations 1-1.5 orders of magnitude higher. The toxicity of the synthesized compounds was much lower than that of ANO. Modifications of the ANO N⁶-position did not offer any advantages over the parent compound. The synthesized N¹-oxide derivatives of noraristeromycin retained the activity comparable with noraristeromycin and displayed a decreased toxicity. No direct correlation between antiviral activity and stability of the compounds was found.

Full text of DOI:10.1135/cccc20061107

Adenosine N-Oxide Analogues
1107
1
ADENOSINE N -OXIDE ANALOGUES AS INHIBITORS OF
ORTHOPOX VIRUS REPLICATION
a
a
a
Anastasiya L. KHANDAZHINSKAYA , Elena A. SHIROKOVA , Alexander V. SHIPITSIN ,
a
b
a
Inna L. KARPENKO , Evgenii F. BELANOV , Marina K. KUKHANOVA and
a,
Maksim V. YASKO *
a
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences,
3
2 Vavilov Str., Moscow 119991, Russian Federation
b
State Research Center of Virology and Biotechnology “Vektor”,
Koltsovo, Novosibirsk Region 633159, Russian Federation; e-mail: lhba-imb@mail.ru
Received March 1, 2006
Accepted April 27, 2006
Dedicated to Professor Antonín Holý on the occasion of his 70th birthday in recognition of his
outstanding contributions to the area of organic and medicinal chemistry.
1
1
6
Several n ew types of aden osin e N -oxide (ANO) derivatives in cludin g N -alkoxy an d N -alkyl
as well as th e an alogues with a trih ydroxycyclopen tan e rin g in place of th e ribose residue
were syn th esized an d th eir an tiviral properties were evaluated in Vero an d LLC-MK2 cell cul-
tures in fected with vaccin ia, m ousepox, m on keypox, cowpox, an d differen t isolates of sm all-
pox viruses. Th e an tiviral activity of ANO an d its derivatives sign ifican tly depen ded on th e
virus type an d cell cultures. Mousepox an d m on keypox viruses were th e m ost sen sitive to
th ese com poun ds, wh ile vaccin ia an d cowpox viruses were in h ibited at th e con cen tration s
1
–1.5 orders of m agn itude h igh er. Th e toxicity of th e syn th esized com poun ds was m uch
6
lower th an th at of ANO. Modification s of th e ANO N -position did n ot offer an y advan tages
over th e paren t com poun d. Th e syn th esized N -oxide derivatives of n oraristerom ycin re-
1
tain ed th e activity com parable with n oraristerom ycin an d displayed a decreased toxicity. No
direct correlation between an tiviral activity an d stability of th e com poun ds was foun d.
Keyw ord s: Nucleosides; Purin es; N-Oxides; Carbocyclic n ucleosides; Noraristerom ycin ;
An tivirals; Pox viruses.
Th e proph ylaxis an d treatm en t of th e disease in duced by sm allpox virus are
am on g th e m ost topical problem s of m odern m edicin e an d scien ce. Sin ce
th e vaccin ation was discon tin ued m ore th an 25 years ago, on ly few in divid-
uals are n ow protected again st th is in fection . Th e con cern about th e use of
sm allpox virus as a weapon for bioterrorism or biowarfare h as greatly in -
creased th e in terest in com poun ds capable of in h ibitin g th e orth opox virus
replication . Th e data on th e an tipox virus activity of com poun ds of various
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1107–1121
©
2006 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20061107

1
108
Khandazhinskaya et al.:
1
2
structures were recen tly sum m arized in reviews by De Clercq , Baker et al. ,
3
1
an d Sm ee et al. Of th e tested com poun ds, aden osin e N -oxide (1) an d
som e of its N -ben zyloxy derivatives were sh own to exh ibit a sign ifican t ac-
1
tivity again st vaccin ia virus in a cell culture assay an d in a m ouse m odel of
4
–6
7
vaccin ia-virus-in duced tailpox lesion . Kan e an d Sh um an sh owed th at
1
aden osin e N -oxide in h ibits vaccin ia virus replication in vitro by selectively
blockin g th e tran slation of viral m RNA, alth ough th e m ech an ism was un de-
term in ed. We describe in th is work th e syn th esis of four n ew types of ANO
1
6
an alogues, n am ely, N -alkoxy (2a–2e) an d N -alkyl (3a–3e) of ANO as well
as ANO carbocyclic an alogues 4 an d 5a, 5b (Ch art 1) an d th eir an tipox vi-
rus properties evaluated in cell cultures in fected with vaccin ia, m on keypox,
cowpox, an d sm allpox viruses.
Hal
NH2
N
NHR
N
O
R
O
N
N
N
N
N
N
HO
HO
O
O
HO
OH
2a-2e
HO
OH
3a-3e
NH2
N
2
2
2
2
2
a, R = CH2OC(O)CMe3
3a, R = Me
3b, R = cyclohexyl
3c, R = (CH ) OH
3d, R = (CH ) OH
3e, R = Bn
O
N
N
b, R = CH2OC(O)CHMe2
c, R = CH2COOEt
d, R = CH2COOH
2
2
N
HO
O
2 4
e, R = CH2CONH2
HO
OH
1 (ANO)
NH2
Hal
NH2
N
O
O R
N
N
N
N
N
N
N
HO
HO
5a, R = CH2COOEt
HO
OH
4
5b, R = CH2CONH2
HO
OH
5a,b
CHART 1
RESULTS AND DISCUSSION
Esters 2a an d 2b were obtain ed by alkylation of ANO (1) with (pivaloyl-
oxy)m eth yl ch loride or (butyryloxy)m eth yl ch loride, respectively, in DMF
5
sim ilarly to th e m eth od described in a previously publish ed procedure . As
com poun d 2b was partially degraded in th e process of reversed-ph ase ch ro-
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1107–1121

Adenosine N-Oxide Analogues
1109
m atograph y to give ANO, we purified it by dissolution in eth an ol an d sub-
sequen t precipitation with ch loroform . Esters 2c–2e were syn th esized in a
sim ilar m an n er usin g th e correspon din g derivatives of h aloacetic acids
(
Sch em e 1). Esters 2 were isolated as h alides (ch lorides in th e case of 2a, 2b,
brom ide 2c an d iodide 2e). Com poun d 2d was isolated in th e form of in n er
salt. It is n oteworth y th at am ide 2e was un stable durin g isolation ; th e prod-
uct of degradation was aden osin e.
Hal
NH2
N
NH2
N
O
O R
N
N
N
N
(i)
N
N
HO
HO
O
O
HO
OH
1 (ANO)
HO
OH
2a-2e
2
a
b
c
d
e
R
reagent (i)
CH2OC(O)CMe3
CH2OC(O)CHMe2
CH2COOEt
ClCH2OC(O)CMe3
ClCH2OC(O)CHMe2
BrCH2COOEt
CH2COOH
ICH2COOH
CH2CONH2
ICH2CONH2
SCHEME 1
1
6
6
-Alkyl N -oxides 3a–3e were obtain ed by oxidation of th e correspon din g
N -alkylpurin es 6a–6e with 3-ch loroperben zoic acid in aqueous m eth an ol
Sch em e 2). Nucleosides 6b–6d were prepared in h igh yields by treatin g
-ch loropurin e with th e correspon din g am in es in aqueous dioxan e. Com -
(
6
poun ds 6a, 6e were purch ased from Fluka. Th e yields of target 3a–3e
ran ged from 45 to 65%.
NHR
N
NHR
N
O
N
N
N
N
(i)
N
N
HO
HO
O
O
HO
OH
6a-6e
HO
OH
3a-3e
3
a
b
c
d
e
R
Me
cyclohexyl
(CH2)2OH
(CH2)4OH
Bn
(i) 3-chloroperbenzoic acid, H2O/MeOH
SCHEME 2
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1107–1121

1
110
Khandazhinskaya et al.:
6
A sim ilar oxidation of N -disubstituted purin es (6-m orph olin opurin e
6
6
riboside an d N ,N -dim eth yladen osin e) gave in osin e as a m ajor product
1
an d N -h ydroxyin osin e (7) as a m in or com pon en t. Th e structure of 7 was
con firm ed by ph ysicoch em ical m eth ods (UV, NMR an d elem en tal an alysis)
as well as by oxidation of in osin e with 3-ClC H CO H an d by deam in ation
6
4
3
8
of ANO with sodium n itrite in acid con dition s sim ilarly to ref.
Oxidation of a cyclopen ten e aden osin e an alogue (±)-5′-n oraristerom ycin
1
(
8) sm ooth ly afforded N -oxide 4 (Sch em e 3). As expected, alkylation of
1
N -oxide 4 with pivaloyloxym eth yl ch loride yielded th e correspon din g
N -ester. However, un like ester 2a, its carbocyclic an alogue was very un sta-
1
ble durin g ch rom atograph y, an d we failed to isolate it in pure state. Al-
1
th ough th e reaction of N -oxide 4 with brom oacetic ester or (iodom eth yl)-
acetam ide proceeded effectively, th e overall yield of esters 5a, 5b was rath er
low because of purification losses.
NH2
N
NH2
N
Hal
NH2
N
O
O R
N
N
N
N
N
N
N
N
N
HO
HO
HO
(i)
(ii)
HO
OH
8
HO
OH
4
HO
OH
5a,b
3
a
b
R
CH2COOEt
CH2CONH2
2
(i) 3-chloroperbenzoic acid, H O/MeOH
(ii) BrCH COOEt for 5a or ICH CONH for 5b
2
2
2
SCHEME 3
Th e syn th esized com poun ds were tested in Vero an d LLC-MK2 cell cul-
tures in fected with vaccin ia, m ousepox virus, m on keypox virus, cowpox vi-
rus, an d differen t isolates of sm allpox virus. Th e results are sum m arized in
Tables I–III. Th e represen tative data from a sin gle experim en t for com -
poun ds 2a an d 5a are sh own in Figs 1 an d 2. As on e can see from th e ta-
bles, th e an tiviral activity of paren t ANO an d its derivatives varied
sign ifican tly with th e virus type an d cell culture. Mousepox virus an d
m on keypox virus were m ost susceptible to ANO (IC50 0.001–0.05 µg/m l),
wh ereas th e in h ibition of vaccin ia an d cowpox virus replication required a
1
00–150-fold in crease in th e ANO con cen tration s (IC50 0.4–0.7 µg/m l). Th e
data for vaccin ia virus agree well with ANO in h ibitory con cen tration s
(
>0.32 µg/m l) reported earlier by Kwon g et al.5
In th e assay with vaccin ia virus, com poun ds 2a, 2c were about 35–80
tim es m ore active th an ANO. Th e activities of com poun ds 2a–2c were at
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1107–1121

Adenosine N-Oxide Analogues
1111
least on e order of m agn itude h igh er again st m on keypox virus com pared
with ANO in Vero cell culture. Except 2c, m odification s of th is type did n ot
h ave an y advan tages over ANO again st cowpox virus. Th e oth er com -
poun ds of th is type sh owed sim ilar or lower activity wh en com pared with
ANO. Th e toxicity assays sh owed essen tial advan tages of th e syn th esized
com poun ds, as th eir toxicities were 10–20-fold lower in n on -in fected Vero
cells com pared with th e paren t com poun d.
Com poun ds of type 3 presen t an oth er type of ANO m odification s. It was
6
sh own earlier th at proper m odification s of th e N -position of purin e-based
9
,10
agen ts led to h igh ly active in h ibitors of vaccin ia an d cowpox viruses
.
6
However, th e poten cy of com poun ds 3, in wh ich th e N -am in o group is
TABLE I
An tipox virus activity of th e syn th esized com poun ds in assay with Vero cells
IC50, µg/m l
CD50
Com pd
µg/m l
vaccin ia
m ousepox
m on keypox
cowpox
1
2
2
2
2
2
3
3
3
3
3
4
5
5
7
8
10
100
0.7
0.01
1.1
0.001
0.007
0.001
0.013
0.1
0.05
0.006
0.003
0.003
0.04
0.3
0.4
14.4
4.0
a
b
c
d
e
a
b
c
d
e
70
>200
>200
30
0.02
0.13
0.4
0.012
0.3
0.4
1.3
>200
>100
>200
>200
>200
100
0.7
0.6
0.28
24.7
1.7
0.8
23.2
1.8
35
>100
>100
49.0
1.9
1.5
0.6
1.9
2.2
2.4
2.2
6.3
21.0
1.6
0.75
0.1
0.6
0.57
0.03
0.16
23
a
>200
100
0.2
b
0.4
0.4
0.3
>200
10
16
6.4
>100
0.4
0.05
0.01
0.14
IC50, th e con cen tration th at causes a 50% reduction in virus replication ; CD50, th e con cen -
tration th at causes a 50% in h ibition of cell growth . Averages of th e data from th ree in de-
pen den t experim en ts. Experim en tal error 40–60%.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1107–1121

1
112
Khandazhinskaya et al.:
substituted with an alkyl group, was lower th an th at of ANO towards all
th e tested viruses, alth ough th ey were n on -toxic up to th e con cen tration of
2
00 µm ol/l.
Som e in terestin g results were obtain ed for n oraristerom ycin derivatives 4
an d 5a, 5b. 5′-Noraristerom ycin (8) is kn own to be active again st a wide
1
1–13
spectrum of viruses in cludin g th e pox virus fam ily
an d is targeted at
cellular S-aden osyl-L-h om ocystein e (SAH) h ydrolase, wh ich is crucial for
1
4
RNA m aturation . SAH h ydrolase in h ibitors m ay serve as prom isin g an tivi-
ral agen ts. Som e kn own SAH h ydrolase in h ibitors were active in vitro
again st a broad spectrum of viruses in cludin g vaccin ia virus but n ot again st
6
cowpox virus . Herein , we sh owed th at com poun ds 5a, 5b obtain ed by
1
m odifyin g th e N -atom of n oraristerom ycin , exh ibit th e sim ilar activity in
Vero cell culture again st all th e tested viruses in cludin g cowpox virus com -
TABLE II
An tipox virus activity of th e syn th esized com poun ds in th e assay in LLC-MK2 cells
IC50, µg/m l
CD50
Com pd
µg/m l
vaccin ia
m ousepox
m on keypox
cowpox
1
2
2
2
2
2
3
3
3
3
3
4
5
5
7
8
14
3.6
6.0
0.03
0.1
3.7
0.6
4.2
9.7
3.4
23
a
b
c
d
e
a
b
c
d
e
32
24
32
0.06
0.624
0.2
0.2
94.5
10.5
105
3.6
>200
17
24
100
23
70
11
8.5
21
20
0.5
3.0
5.7
>100
>200
>200
>200
>100
>200
>200
>200
>100
>100
>100
>100
>100
85
>100
>100
18.6
>100
9.0
>100
>100
41
>100
>100
16
>100
>100
1.0
>100
>100
>100
>100
22
a
>100
0.4
2.7
b
0.2
0.1
>100
>100
>100
0.5
16.52
0.8
18
For sym bols, see Table I.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1107–1121

Adenosine N-Oxide Analogues
1113
TABLE III
Activities an d cytotoxicities of th e selected com poun ds again st sm allpox virus strain s in
Vero cell culture
In dia 3a
6-58
Con go-9
Butler
CD50
µg/m l
Com pd
IC50
µg/m l
IC50
µg/m l
IC50
µg/m l
IC50
µg/m l
IS
IS
IS
IS
1
~10
~200
70
0.01
1110
0.004
2270
0.001 10000
0.007 28570
0.003 10000
0.007
1538
2
2
4
8
a
0.008 25000
0.004 17500
0.008 25000
0.003 10000
0.005 40000
b
0.006
1.3
5000
>80
>100
10
0.13
0.01
>770
1000
0.47
>215
227
0.8
>120
227
0.044
0.044
0.04
250
For sym bols, see Table I. IS = CD50/IC50
FIG. 1
1
Represen tative data from a sin gle experim en t on th e efficacy of N -[(pivaloyloxy)m eth oxy]-
aden osin e ch loride (2a) in Vero cells in fected with various pox viruses. IC values (µg/m l) for
each virus are sh own below th e graph . R are poin t deviation s to th e curve
5
0
2
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1107–1121

1
114
Khandazhinskaya et al.:
pared with paren t 8 (Table I). It sh ould be also n oted th at com poun ds 5a,
5
b sh owed decreased toxicity towards n on -in fected Vero cell culture.
Th e syn th esized com poun ds were also tested in LLC-MK2 cell culture
(
Table II). A com parison of Tables I an d II dem on strates th at drug efficacies
an d cellular toxicities varied con siderably an d depen ded both on th e virus
type an d cell lin e. As a rule, th e syn th esized com poun ds sh owed a m uch
lower efficacy in LLC-MK2 cell lin e com pared with th e results for Vero cells.
Com poun ds 2a, 2b, wh ich in h ibited m on keypox virus in LLC-MK2 cells
with th e activity h igh er th an th e paren t com poun d, were an exception .
Th ese data partially correlate with th e earlier publish ed data, wh ere it was
sh own th at 1-[(2,4-difluoroben zyl)oxy]aden osin e perch lorate (DFBA) m an i-
fested a sign ifican t activity again st poxvirus fam ily in Vero cells, but n ot in
2
LLC-MK2 cells .
Th e selected com poun ds were also tested in Vero cell culture again st a
n um ber of sm allpox virus strain s (Table III). Th e efficacy of esters 2a, 2b in
th ese experim en ts was h igh er th an th at of th e referen ce ANO due to both
lower toxicity an d sligh tly h igh er activity. At th e sam e tim e, carbocyclic de-
rivative 4 sh owed a decreased activity com pared with th e referen ce 5′-n or-
aristerom ycin for th e tested virus strain s, alth ough it was ten fold less toxic.
FIG. 2
1
Represen tative data from a sin gle experim en t on th e efficacy of N -[(eth oxycarbon yl)-
m eth oxy]-9-[(1β,2α,3α,4β)-2,3,4-trih ydroxycyclopen tyl]aden in e brom ide (5a) in Vero cells in -
fected with various pox viruses. For oth er sym bols, see legen d to Fig. 1
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1107–1121

Adenosine N-Oxide Analogues
1115
We assum ed th at th e syn th esized com poun ds can be depot form s of ANO
(
2a–2e) or n oraristerom ycin (5a, 5b), wh ich un derwen t in tracellular h ydro-
lysis to release paren t ANO or n oraristerom ycin , respectively. Th e HPLC
an alysis of product com position after ch em ical or en zym atic h ydrolysis of
2
a an d 5a, 5b con firm ed th at th e m ajor h ydrolysis products were ANO
an d n oraristerom ysin . Th e h alf-life (t0.5) of ester 2a in th e ph osph ate buffer
(
1
pH 7.0, 37 °C) was about 2.5 h , wh ereas for 2c it fell to 20 m in (TLC an d
H NMR m on itorin g). In 100% h um an blood serum , t0.5 of 2a was on ly
1
0 m in , with ANO as a m ajor h ydrolysis product. Carbocyclic ester 5a was
stable in PBS, but its h alf-life in h um an blood serum did n ot exceed 15 m in ,
an d th e m ajor h ydrolysis products were N-oxide 4 an d n oraristerom ycin .
Th ese data allow a suggestion th at th ese com poun ds are depot form s of
ANO an d n oraristerom ycin , respectively. A h igh er activity of 2a m igh t be
accoun ted for by a better cellular uptake due to its h igh er h ydroph obicity
an d in tracellular degradation to ANO, wh ich m an ifests sign ifican t an tipox
5
virus activity . At th e sam e tim e, an tiviral activity of com poun d 2c again st
differen t pox viruses in Vero cell culture is rath er h igh , alth ough it is h y-
drolyzed to give a m ixture of products, in wh ich a portion of ANO is con -
siderably lower com pared with 2a. A com parison of an tiviral activity an d
stability of th e com poun ds in differen t m edia did n ot en able a direct
activity–stability correlation . Th e data obtain ed im ply th at th e m ech an ism
1
of action of differen t N -oxide derivatives m ay vary accordin g to th eir struc-
tures. On e can n ot exclude th at som e ANO derivatives are ph osph orylated
to give th e correspon din g triph osph ates th at m igh t be in corporated in to
viral RNA an d block its tran slation sim ilar to th e m ech an ism described
7
earlier for ANO . It is also n oteworth y th at m ost of th e syn th esized com -
poun ds are m arkedly less toxic th an th e paren t com poun d.
EXPERIMENTAL
6
6
N -Meth yladen osin e (6a), N -ben zyladen osin e (6e), in osin e an d 3-ch loroperben zoic acid
were from Fluka; pyridin e an d DMF were from Aldrich . Colum n ch rom atograph y was
perform ed on Silica gel 60 (40–63 µm ); reversed-ph ase ch rom atograph y was carried out on
LiCh roprep RP-8 an d LiCh roprep RP-18 (25–40 µm ) (Merck); HPLC was perform ed on a
Gilson ch rom atograph (Fran ce) supplied with an LKB 2220 in tegrator. Nucleosil 100 C-18
(
5 µm ) colum n (4 × 150 m m ) an d two elutin g system s were used: (a) Buffer A: 5 m M
NaH PO buffer, pH 5.4; buffer B: 70% aceton itrile; gradien t: 0–5 m in , 0% B; 5–7 m in ,
2
4
0
–10% B; 7–20 m in , 10–20% B; 20–35 m in , 20–70%; 35–40 m in , 70–100% B; th e flow
rate was 0.5 m l/m in . (b) Buffer A: H O; buffer B: 100% eth an ol; gradien t: 0–5 m in , 0% B;
2
5
0
–30 m in , 0–30% B; 30–40 m in , 30–32% B; 40–45 m in , 32–100% B; th e flow rate was
.5 m l/m in .
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1107–1121

1
116
Khandazhinskaya et al.:
NMR spectra were recorded on an AMX III-400 spectrom eter (Bruker) with th e workin g
1
frequen cy 400 MHz for H NMR (Me Si as an in tern al stan dard for organ ic solven ts an d so-
4
1
3
dium 3-(trim eth ylsilyl)propan e-1-sulfon ate for D O) an d 101 MHz for C NMR. Ch em ical
2
sh ifts (δ-scale) are given in ppm , couplin g con stan ts (J) in Hz. UV spectra (λ in n m ) were re-
corded on a Sh im adzu UV-1201 spectroph otom eter (Japan ) in m eth an ol or H O at pH 7.0.
2
Elem en tal an alysis was perform ed on a Th erm o Fin igan (CHNS) an alyzer, m odel EA1112
(
Italy).
1
N -[(Pivaloyloxy)m eth oxy]aden osin e Ch loride (2a)
(
(
Pivaloyloxy)m eth yl ch loride (200 µl, 1.39 m m ol) was added to a suspen sion of ANO
130 m g, 0.46 m m ol) in DMF (2 m l), an d th e m ixture was stirred at 20 °C for 18 h . Th e so-
lution was quen ch ed with water (10 m l), evaporated in vacuum , diluted with water (2 m l)
an d applied on to a colum n LiCh roprep RP-18 eluted with water. Th e target fraction s were
1
evaporated an d lyoph ilized to give 155 m g (78%). UV (MeOH): λm ax 258. H NMR
(
(
DMSO-d ): 10.56 s an d 9.94 s, 2 H (NH ); 8.97 s, 1 H (H-8); 8.82 s, 1 H (H-2); 5.95 m , 3 H
6 2
H-1′ an d OCH O); 5.62 br s, 1 H (2′-OH); 5.34 br s, 1 H (3′-OH); 5.09 br s, 1 H (5′-OH);
2
4
.49 m , 1 H (H-2′); 4.15 m , 1 H (H-3′); 3.99 m , 1 H (H-4′); 3.63 m , 2 H (H-5′); 1.19 s, 9 H
1
3
(
(CH ) ). C NMR (D O): 180.1 (C=O); 149.7 (C-6); 146.1 (C-4); 144.8 (C-8 an d C-2); 120.0
3 3 2
(
C-5); 93.5 (OCH O); 89.4 (C-1′); 86.0 (C-4′); 74.9 (C-2′); 70.6 (C-3′); 61.6 (C-5′); 39.2
2
(
CMe ); 26.5 (Me ). For C16H24ClN O ·0.5H O (442.9) calculated: 43.42% C, 5.69% H,
3
3
5
7
2
1
5.82% N; foun d: 43.53% C, 5.81% H, 15.75% N.
1
N -[(Isobutyryloxy)m eth oxy]aden osin e Ch loride (2b)
(
(
Isobutyryloxy)m eth yl ch loride (200 µl, 1.50 m m ol) was added to a suspen sion of ANO
130 m g, 0.46 m m ol) in DMF (2 m l), an d th e m ixture was stirred at 20 °C for 18 h . Th e so-
lution was diluted with CCl4 (5 m l) an d cooled to 0 °C. After 18 h , th e oil-like residue was
separated an d dissolved in eth an ol (1 m l). Th e solution was quen ch ed with ch loroform
(
10 m l), an d th e m ixture was stirred at 0 °C for 18 h . Th e precipitate was filtered off, dried
in vacuum , dissolved in water (5 m l), an d lyoph ilized to give 38 m g (20%) of th e product.
1
UV (MeOH): λm ax 258. H NMR (DMSO-d ): 10.54 s an d 9.96 s, 2 H (NH ); 9.04 s, 1 H (H-8);
6
2
8
5
1
.83 s, 1 H (H-2); 5.95 d, 1 H, J = 5.3 (H-1′); 5.93 s, 2 H (OCH O); 5.65 br s, 1 H (2′-OH);
.38 br s, 1 H (3′-OH); 5.12 br s, 1 H (5′-OH); 4.50 m , 1 H (H-2′); 4.16 m , 1 H (H-3′); 4.00 m ,
2
H (H-4′); 3.63 m , 2 H (H-5′); 2.72 m , 1 H (CH (i-Pr)); 1.19 d, 6 H, J = 6.9 (Me ). For
2
C15H22ClN O ·0.25H O (424.3) calculated: 42.47% C, 5.34% H, 16.51% N; foun d: 42.56% C,
5
7
2
5
.41% H, 16.74% N.
1
N -[(Eth oxycarbon yl)m eth oxy]aden osin e Brom ide (2c)
(
(
Eth oxycarbon yl)m eth yl brom ide (165 µl, 1 m m ol) was added to a suspen sion of ANO
300 m g, 0.9 m m ol) in DMF (5 m l), an d th e m ixture was stirred at 20 °C for 18 h . Th e solu-
tion was diluted with CCl4 (5 m l) an d cooled to 0 °C. After 18 h , th e oil-like residue was
separated an d dissolved in eth an ol (1 m l). Th e solution was quen ch ed with ch loroform
(
10 m l), an d th e m ixture was stirred at 0 °C for 18 h . Th e precipitate was filtered off, dried
in vacuum , dissolved in water (5 m l), an d lyoph ilized to give 78 m g (21%) of ester 2c.
1
UV (H O): λ
258 (ε 14500). H NMR (DMSO-d ): 10.48 s an d 9.79 s, 2 H (NH ); 9.09 s,
6 2
2
m ax
1
H (H-2); 8.81 s, 1 H (H-8); 5.94 d, 1 H, J = 5.6 (H-1′); 5.62 m , 1 H (2′-OH); 5.35 m , 1 H
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1107–1121

Adenosine N-Oxide Analogues
1117
(
3′-OH); 5.12 s, 2 H (OCH ); 5.11 m , 1 H (5′-OH); 4.46 m , 1 H (H-2′); 4.25 q, 2 H, J = 7.2
2
(
OCH CH ); 4.55 m , 1 H (H-3′); 3.99 m , 1 H (H-4′); 3.66–3.58 m , 2 H (H-5′); 1.25 t, 3 H, J =
2
3
7
.2 (OCH CH ). For C14H20BrN O ·0.5H O (459.3) calculated: 36.63% C, 4.61% H, 15.26% N;
2 3 5 7 2
foun d: 36.86% C, 4.72% H, 15.05% N.
1
N -(Carboxym eth oxy)aden osin e (2d )
Iodoacetic acid (70 m g, 0.45 m m ol) was added to a suspen sion of ANO (100 m g, 0.3 m m ol)
in DMF (3 m l), th e m ixture was stirred at 20 °C for 18 h an d evaporated. Th e residue was
dissolved in water applied on to a LiCh roprep RP-18 colum n an d eluted with water to give
1
3
1% (30.6 m g) of product 2d . UV (H O): λ
258 (ε 14500). H NMR (D O): 8.97 s, 1 H
2
m ax
2
(
H-2); 8.51 s, 1 H (H-8); 6.08 d, 1 H, J = 5 (H-1′); 4.92 s, 2 H (OCH ); 4.66 m , 1 H (H-2′);
2
4
.40 m , 1 H (H-3′); 4.22 m , 1 H (H-4′); 3.66–3.88 m , 2 H (H-5′). For C12H15N O ·1.5H O
5 7 2
(
368.3) calculated: 39.16% C, 4.92% H, 19.02% N; foun d: 39.23% C, 5.04% H, 18.85% N.
1
N -(Carbam oylm eth oxy)aden osin e Iodide (2e)
Carbam oym eth yl iodide (80 m g, 0.5 m m ol) was added to a suspen sion of ANO (130 m g,
.4 m m ol) in DMF (2 m l), an d th e m ixture was stirred at 20 °C for 18 h an d evaporated. Th e
residue was dissolved in water, applied on to a LiCh roprep RP-18 colum n an d eluted with 4%
0
1
MeOH to give 44% (66.2 m g) of th e product. UV (H O): λ
258 (ε 14500). H NMR
m ax
2
(
CD OD): 9.10 s, 1 H (H-2); 8.71 s, 1 H (H-8); 6.10 d, 1 H, J = 5 (H-1′); 5.15 s, 2 H (OCH );
3 2
4
.62 m , 1 H (H-2′); 4.36 m , 1 H (H-3′); 4.16 m , 1 H (H-4′); 3.77–3.81 m , 2 H (H-5′). For
C12H17IN O ·0.75H O (481.7) calculated: 29.92% C, 3.87% H, 17.45% N; foun d: 29.73% C,
6
6
2
3
.83% H, 17.59% N.
9
-[(1β,2α,3α,4β)-2,3,4-Trih ydroxycyclopen tyl]aden in e-1-oxide (4)
A suspen sion of (±)-5′-n oraristerom ycin (8) (50 m g, 0.2 m m ol), DMF (5 m l), MeOH (5 m l)
an d H O (5 m l) was h eated un der stirrin g un til com plete dissolution . 3-Ch loroperben zoic
2
acid (250 m g, 1 m m ol) was added to th e solution , an d th e m ixture was stirred at 20 °C for
1
8 h . Th e solution was evaporated up to th e volum e of 2 m l, diluted with water (20 m l),
evaporated to th e volum e 2 m l, an d diluted with water (20 m l). Th e filtrate was evaporated
an d ch rom atograph ed on a LiCh roprep RP-18 colum n eluted with water. Th e yield of th e
1
product was 84% (44.7 m g). UV (H O): λ
262 (ε 14500). H NMR (DMSO-d ): 8.59 s, 1 H
2
m ax
6
(
(
H-8); 8.35 s, 2 H (H-2); 5.17 m , 5.03 m an d 4.91 m , 3 H (3 × OH); 4.69 m an d 4.48 m , 2 H
H-2′ an d H-3′); 3.90 m an d 3.76 m , 2 H (H-1′ an d H-4′); 2.63 m an d 1.82 m , 2 H (H-5′). For
C11H15N O ·0.5H O (290.3) calculated: 45.56% C, 5.56% H, 24.15% N; foun d: 45.63% C,
5
4
2
5
.68% H, 24.22% N.
1
N -[(Eth oxycarbon yl)m eth oxy]-9-[(1β,2α,3α,4β)-2,3,4-trih ydroxycyclopen tyl]aden in e
Brom ide (5a)
(
0
Eth oxycarbon yl)m eth yl brom ide (15 µl, 0.13 m m ol) was added to a suspen sion of 4 (32 m g,
.12 m m ol) in DMF (1.5 m l), an d th e m ixture was stirred at 20 °C for 18 h , evaporated in
vacuum of oil pum p with out h eatin g, an d diluted with eth yl acetate. Th e resultin g precipi-
tate was wash ed with eth yl acetate, dissolved in water, an d lyoph ilized to give 76% of com -
1
poun d 5a. UV (H O): λ
262 (ε 14500). H NMR (DMSO-d ): 10.40 br s an d 9.71 br s, 2 H
2
m ax
6
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1107–1121

1
118
Khandazhinskaya et al.:
(
NH ); 9.05 s an d 8.64 s, 2 H (H-2 an d H-8); 5.25 m , 1 H (OH); 5.11 s, 2 H (O-CH -COO);
2
2
5
7
1
1
.09 m an d 5.06 m , 2 H (2 × OH); 4.77 m an d 4.49 m , 2 H (H-2′ an d H-3′); 4.21 q, 2 H, J =
.2 (CH CH ); 3.93 m an d 3.77 m , 2 H (H-1′ an d H-4′); 2.63 m an d 1.76 m , 2 H (H-5′);
2
3
.24 t, 3 H (CH CH ). For C15H22BrN O ·0.75H O (461.8) calculated: 39.03% C, 5.13% H,
2
3
5
6
2
5.17% N; foun d: 39.28% C, 5.21% H, 14.96% N.
1
N -(Carbam oylm eth oxy)-9-[(1β,2α,3α,4β)-2,3,4-trih ydroxycyclopen tyl]aden in e
Iodide (5b)
Carbam oylm eth yl iodide (25 m g, 0.13 m m ol) was added to a suspen sion of 4 (32 m g,
.12 m m ol) in DMF (2 m l), an d th e m ixture was stirred at 20 °C for 18 h , evaporated, dis-
solved in water, an d ch rom atograph ed on a LiCh roprep RP-18 colum n eluted with water.
0
1
Yield 57%. UV (H O): λ
262 (ε 14500). H NMR (DMSO-d ): 9.85 br s, 2 H (NH ); 9.03 s
6 2
2
m ax
an d 8.45 s, 2 H (H-2 an d H-8); 7.65 s an d 7.09 s, 2 H (CONH ); 5.22 m , 5.09 m an d 5.02 m ,
2
3
H (3 × OH); 4.65 s, 2 H (O-CH -COO); 4.48 m , 3.92 m , 3.83 m an d 3.76 m , 4 H (H-1′,
2
H-2′, H-3′ an d H-4′); 2.62 m an d 1.74 m , 2 H (H-5′). For C13H19IN O ·0.75H O (479.8) calcu-
6
5
2
lated: 32.55% C, 4.30% H, 17.52% N; foun d: 32.71% C, 4.36% H, 17.41% N.
6
Syn th esis of N -Substituted Aden osin es 6. Gen eral Procedure
Alkylam in e (1.0 m m ol) an d water (0.1 m l) were added to a suspen sion of 6-ch loropurin e
riboside (57.5 g, 0.2 m m ol) in dioxan e (3 m l) an d th e suspen sion was stirred at room tem -
perature. After 3 h , th e clear solution was evaporated, an d th e residue was diluted with
m eth an ol–eth er (1:1 for 6b, 1:5 for 6c, an d 1:3 for 6d ). After 18 h at +4 °C, th e supern atan t
was decan ted, evaporated, an d repeatedly treated with th e m eth an ol–eth er solution . Com -
bin ed precipitates were purified by reverse-ph ase ch rom atograph y an d lyoph ilized.
6
6
N ,N -Dim eth yladen osin e an d 6-m orph olin opurin e riboside were prepared in a sim ilar
m an n er from 6-ch loropurin e riboside an d dim eth ylam in e or m orph olin e, respectively.
6
N -Cyclohexyladenosine chloride (6b ): Yield ca. 90%. UV (MeOH): λm ax 269 (ε 15400).
1
H NMR (CD OD): 8.23 s an d 8.14 s, 2 H (H-2 an d H-8); 5.98 d, 1 H, J = 5.8 (H-1′); 4.70 t,
3
1
1
H, J = 5.5 (H-2′); 4.26 dd, 1 H, J = 5.3 an d 3.9 (H-3′); 4.21 q, 1 H, J = 3.5 (H-4′); 3.80 dd,
H, J = 3.1 an d 12.6 (H-5′a); 3.72 dd, 1 H, J = 3.8 an d 12.6 (H-5′b); 1.96 br s, 1 H (cyclo-
h exyl); 1.65–1.49 m , 4 H (cycloh exyl); 1.42–1.19 m , 6 H (cycloh exyl). For C16H24ClN O ·
5
4
0
1
.25H O (300.4) calculated: 49.25% C, 6.32% H, 17.95% N; foun d: 49.16% C, 6.21% H,
2
8.04% N.
6
N -(2-Hydroxyethyl)adenosine chloride (6c): Yield ca. 90%. UV (MeOH): λ
267 (ε 16200).
m ax
1
H NMR (CD OD/D O): 8.25 s an d 8.23 s, 2 H (H-2 an d H-8); 5.96 d, 1 H, J = 6.2 (H-1′);
3
2
4
3
.74 t, 1 H, J = 5.1 (H-2′); 4.32 dd, 1 H, J = 5.4 an d 2.5 (H-3′); 4.17 q, 1 H, J = 2.7 (H-4′);
.88 dd, 1 H, J = 2.5 an d 12.5 (H-5′a); 3.74 dd, 1 H, J = 2.8 an d 12.5 (H-5′b); 3.77 m , 4 H
(
CH O an d CH N (h ydroxyeth yl)). For C12H17N O ·0.75HCl·0.5H O (347.6) calculated:
2 2 5 5 2
4
1.55% C, 5.44% H, 20.19% N; foun d: 41.76% C, 5.53% H, 20.44% N.
N -(4-Hydroxybutyl)adenosine chloride (6d ): Yield ca. 90%. UV (MeOH): λm ax 269 (ε 16000).
6
1
H NMR (CD OD/D O): 8.26 s an d 8.22 s, 2 H (H-2 an d H-8); 5.98 d, 1 H, J = 6.5 (H-1′);
3
2
4
3
.73 t, 1 H, J = 6.1 (H-2′); 4.33 dd, 1 H, J = 5.1 an d 2.6 (H-3′); 4.22 q, 1 H, J = 2.5 (H-4′);
.88 dd, 1 H, J = 2.8 an d 12.8 (H-5′a); 3.74 dd, 1 H, J = 2.9 an d 12.8 (H-5′b); 3.62 m , 4 H
(
CH O an d CH N (h ydroxybutyl)); 1.75 m an d 1.67 m , 4 H (CH CH (h ydroxybutyl)). For
2 2 2 2
C14H22ClN O ·0.25H O (380.3) calculated: 44.23% C, 5.96% H, 18.42% N; foun d: 44.11% C,
5
5
2
5
.88% H, 18.54% N.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1107–1121

Adenosine N-Oxide Analogues
1119
6
Syn th esis of N -Substituted Derivatives 3. Gen eral Procedure
3
-Ch loroperben zoic acid (100 m g, 40–63%, ~0.3 m m ol) was added to a suspen sion of
6
N -alkyladen osin e (0.1 m m ol) in aqueous m eth an ol (1 m l). Th e reaction m ixture was stirred
at 37 °C for 18 h , th en an excess of 3-ch loroperben zoic acid (50 m g, ~0.15 m m ol) was
added, an d th e reaction solution was stirred at 37 °C for an oth er 24 h . Th e reaction m ix-
ture was evaporated an d th e residue was partition ed between water an d eth yl acetate. Th e
aqueous layer was evaporated an d th e residue was ch rom atograph ed on a reverse-ph ase
LiCh roprep RP-18 colum n in a gradien t of aceton itrile in 0.05 M NH HCO . Th e target 3a–3e
4
3
were repeatedly purified by reverse-ph ase ch rom atograph y an d lyoph ilized.
6
6
Oxidation s of N ,N -dim eth yladen osin e, 6-m orph olin opurin e riboside an d in osin e were
carried out in a sim ilar fash ion . Isolation of h ydroxyin osin e 7 was perform ed sim ilarly to
th e isolation of N-oxides 3.
6
1
N -Methyladenosine N -oxide (3a): Yield 53%. UV (H O): λ
234 (ε 35000) an d 269 (ε 9000).
m ax
2
1
H NMR (D O): 8.56 s an d 8.44 s, 2 H (H-2 an d H-8); 6.10 d, 1 H, J = 5.3 (H-1′); 4.74 t, 1 H,
2
J = 5 (H-2′); 4.44 dd, 1 H, J = 4.4 an d 5.0 (H-3′); 4.27 q, 1 H, J = 3.6 (H-4′); 3.91 dd, 1 H,
J = 3.1 an d 12.8 (H-5′a); 3.83 dd, 1 H, J = 4.1 an d 12.8 (H-5′b); 3.54 s, 3 H (Me). For
C11H15N O ·0.5H O (306.3) calculated: 43.18% C, 5.26% H, 22.88% N; foun d: 43.36% C,
5
5
2
5
.30% H, 22.69% N.
6
1
N -Cyclohexyladenosine N -oxide (3b): Yield 56%. UV (MeOH): λ
237 (ε 32000) an d 273
m ax
1
(
(
(
ε 10500). H NMR (CD OD): 8.50 s an d 8.37 s, 2 H (H-2 an d H-8); 5.95 d, 1 H, J = 5.7
H-1′); 4.70 t, 1 H, J = 5.3 (H-2′); 4.27 dd, 1 H, J = 5.4 an d 3.9 (H-3′); 4.20 q, 1 H, J = 3.5
H-4′); 3.79 dd, 1 H, J = 3.1 an d 12.5 (H-5′a); 3.72 dd, 1 H, J = 3.9 an d 12.5 (H-5′b); 1.97 m ,
3
1
H (cycloh exyl); 1.70 m , 2 H (cycloh exyl); 1.57 m , 2 H (cycloh exyl); 1.41 m , 4 H (cyclo-
h exyl); 1.20 m , 2 H (cycloh exyl). For C16H23N O ·0.25H O (369.9) calculated: 51.97% C,
5
5
2
6
2
.40% H, 18.94% N; foun d: 52.18% C, 6.46% H, 18.82% N.
6
1
N -(2-Hydroxyethyl)adenosine N -oxide (3c): Yield 45%. UV (H O): λ
234 (ε 31500) an d
m ax
2
69 (ε 9800). 1H NMR (D O): 8.44 s an d 8.30 s, 2 H (H-2 an d H-8); 5.96 d, 1 H, J = 6.2
2
(
H-1′); 4.71 t, 1 H, J = 5.1 (H-2′); 4.32 t, 1 H, J = 4.4 (H-3′); 4.12 q, 1 H, J = 3.6 (H-4′); 4.06 t,
2
5
H, J = 5.5 (CH N (h ydroxyeth yl)); 3.76 dd, 1 H, J = 3.1 an d 12.8 (H-5′a); 3.73 t, 2 H, J =
2
1
3
.5 (CH O (h ydroxyeth yl)); 3.68 dd, 1 H, J = 3.9 an d 12.8 (H-5′b). C NMR (D O): 158.23
2
2
(
(
C-6); 150.72 (C-4); 145.00 (C-2); 144.06 (C-8); 122.66 (C-5); 89.29 (C-1′); 86.30 (C-4′); 74.68
C-2′); 71.31 (C-3′); 62.10 (C-5′); 60.61 (CH O (h ydroxyeth yl)); 40.11 (CH N (h ydroxy-
2
2
eth yl)). For C12H17N O ·0.5H O (336.3) calculated: 42.89% C, 5.40% H, 20.84% N; foun d:
5
6
2
4
3.15% C, 5.51% H, 20.75% N.
6
1
N -(4-Hydroxybutyl)adenosine N -oxide (3d ): Yield 62%. UV (H O): λ
235 (ε 31000) an d
m ax
2
2
5
2
71 (ε 9600). 1H NMR (CD OD/D O): 8.43 s an d 8.30 s, 2 H (H-2 an d H-8); 5.96 d, 1 H, J =
3 2
.3 (H-1′); 4.70 t, 1 H, J = 5 (H-2′); 4.28 t, 1 H, J = 5.7 (H-3′); 4.12 q, 1 H, J = 4 (H-4′); 3.91 t,
H, J = 6.9 (CH N (h ydroxybutyl); 3.77 dd, 1 H, J = 3.1 an d 12.8 (H-5′a); 3.74 dd, 1 H, J = 4
2
an d 12.8 (H-5′b); 3.49 t, 2 H, J = 6.7 (CH O (h ydroxybutyl)); 1.65 m an d 1.51 m , 4 H
2
1
3
(
CH CH (h ydroxybutyl)). C NMR (CD OD/D O): 158.56 (C-6); 150.86 (C-4); 145.93 (C-2);
2 2 3 2
1
5
44.81 (C-8); 122.56 (C-5); 89.41 (C-1′); 86.48 (C-4′); 74.82 (C-2′); 71.44 (C-3′); 62.39 (C-5′);
8.75 (CH O (h ydroxybutyl)); 39.14 (CH N (h ydroxybutyl)); 26.07 an d 29.33 (CH CH
2
2
2
2
(
h ydroxybutyl)). For C14H21N O ·0.5H O (364.4) calculated: 46.18% C, 6.08% H, 19.24% N;
5 6 2
foun d: 46.31% C, 6.13% H, 19.10% N.
6
1
N -Benzyladenosine N -oxide (3e): Yield 65%. UV (MeOH): λ
233 (ε 36500) and 271 (ε 8600).
m ax
1
H NMR (CD OD): 8.56 s an d 8.35 s, 2 H (H-2 an d H-8); 7.19–7.33 m , 5 H (Ph ); 5.97 d, 1 H,
3
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1107–1121

1
120
Khandazhinskaya et al.:
J = 6 (H-1′); 5.12 br s, 2 H (CH Ph ); 4.64 t, 1 H, J = 5.3 (H-2′); 4.30 t, 1 H, J = 5 (H-3′);
2
4
.14 q, 1 H, J = 4 (H-4′); 3.81 dd, 1 H, J = 3.2 an d 12.8 (H-5′a); 3.72 dd, 1 H, J = 3.8 an d 12.8
(
H-5′b). For C17H19N O ·0.25H O (377.9) calculated: 54.06% C, 5.20% H, 18.54% N; foun d:
5
5
2
5
3.97% C, 5.13% H, 18.76% N.
N -Hydroxyinosine (7): Yields < 5% (based on N ,N -dim eth yladen osin e), 10% (based on
1
6
6
6
-m orph olin opurin e riboside), 24% (oxidation of in osin e), an d ca. 60% (deam in ation of
1
ANO [6]). UV (H O): λ
229 (ε 35000) an d 257 (ε 8500). H NMR (D O): 8.35 s an d 8.15 s,
2
m ax
2
2
H (H-2 an d H-8); 5.94 d, 1 H, J = 5.9 (H-1′); 4.69 t, 1 H, J = 5 (H-2′); 4.34 dd, 1 H, J = 4.4
an d 5.2 (H-3′); 4.17 q, 1 H, J = 3.5 (H-4′); 3.81 dd, 1 H, J = 2.8 an d 12.9 (H-5′a); 3.83 dd,
H, J = 4 an d 12.9 (H-5′b). 13C NMR (D O): 163.03 (C-6); 151.58 (C-4); 144.96 (C-2); 144.04
1
2
(
C-8); 125.45 (C-5); 89.24 (C-1′); 86.26 (C-4′); 74.62 (C-2′); 71.07 (C-3′); 62.06 (C-5′). For
C10H12N O ·0.75NH ·0.25H O (301.5) calculated: 39.84% C, 4.93% H, 22.07% N; foun d:
4
6
3
2
3
9.75% C, 4.84% H, 22.21% N.
An tiviral Activity
All virus strain s an d cell cultures were obtain ed from th e collection of State Research Cen ter
of Virology an d Biotech n ology “Vektor”, Koltsovo, Novosibirsk Region . Th e syn th esized
com poun ds were screen ed again st cowpox (Grish ak strain ), m on keypox (Zair 599 strain ),
m ousepox virus (K-1 strain ), vaccin ia virus (strain LIVP) used for vaccin ation of world popu-
lation , an d variola m ajor (leth ality 30–40%) of differen t geograph ical origin : In dia 3a
(
In dia), 6–58 (Pakistan ), Con go-9 (Africa), an d Butler variola m in or (leth ality 1–2%).
An tiviral activity of th e com poun ds was tested sim ilarly to th e earlier described proce-
2
dure . Briefly, an tiviral com poun ds were dissolved in DMSO or water to a con cen tration of
2
0 m g/m l followed by serial dilution s with RPMI m edia an d added to 96-well m icrolitre
plates con tain in g Vero or LLC-MK2 cells. Fin al drug con cen tration s ran ged from 100 to
.0005 µg/m l. At each drug con cen tration , th ree wells were in fected with 105 pfu/well of
0
orth opox virus, wh ile th ree wells were left un in fected for toxicity tests. Th e plates were
in cubated in a CO2 in cubator at 37 °C for 5 days, th e m edium -drug m ixture was aspirated
2
an d cells were stain ed with Neutral Red as described earlier . Th e absorban ces were read at
4
5
90 n m on a Bio-tek plate reader. Th e drug con cen tration s th at reduced cell viability by
0% (CD50) an d th eir an tiviral efficacy (IC50) were calculated usin g th e software program
(
Molecular Devices, Men lo Park, U.S.A.).
The work was supported by the Russian Foundation for Basic Research, project 05-04-49500,
the program of Presidium of Russian Academy of Sciences on Molecular and Cellular Biology, and
the Ministry of Science of Russian Federation (project 11).
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