New N4-hydroxycytidine derivatives: Synthesis and antiviral activity

Article abstract of DOI:10.1135/cccc20061099

Two series of N⁴-hydroxycytidine derivatives were synthesized and evaluated as potential antipox virus agents. Acylation of N ⁴-hydroxycytidine (1) with an excess of acyl chloride or imidazolide yielded the corresponding N⁴

Full text of DOI:10.1135/cccc20061099

New N4-Hydroxycytidine Derivatives
1099
NEW N4-HYDROXYCYTIDINE DERIVATIVES: SYNTHESIS AND
ANTIVIRAL ACTIVITY
a
a
a
Maksim A. IVANOV , Elena V. ANTONOVA , Aleksey V. MAKSIMOV ,
a
b
Lyudmila K. PIGUSOVA , Evgenii F. BELANOV and
Lyudmila A. ALEKSANDROVA
a1,
*
a
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences,
32 Vavilov Str., Moscow 119991, Russian Federation; e-mail: ala2004@zmail.ru
State Research Center of Virology and Biotechnology “Vektor”,
1
b
Koltsovo, Novosibirsk Region 633159, Russian Federation; e-mail: belanov@vector.nsc.ru
Received March 1, 2006
Accepted May 25, 2006
Dedicated to Professor Antonín Holý on the occasion of his 70th birthday in recognition of his
invaluable contributions to the area of medicinal chemistry.
Two series of N4-h ydroxycytidin e derivatives were syn th esized an d evaluated as poten tial
an tipox virus agen ts. Acylation of N4-h ydroxycytidin e (1) with an excess of acyl ch loride or
im idazolide yielded th e correspon din g N4-(acyloxy) derivatives. 5′-Ph osph on ates of 1 were
prepared by th e reaction of cytidin e 5′-ph osph on ates with aqueous h ydroxylam in e h ydro-
ch loride (pH 6.0). Nucleoside 1 an d its N4-(acyloxy) derivatives in h ibited replication of
pox viruses in cell cultures, N4-(pivaloyloxy)- an d N4-(ben zoyloxy)cytidin es bein g th e m ost
poten t. Th e syn th esized 5′-ph osph on ates were m ore cytotoxic th an th e paren t n ucleoside.
Keyw ord s: Nucleosides; Nucleoside an alogs; N4-Hydroxycytidin e; Nucleoside ph osph on ates;
Hydroxylam in e; Pyrim idin es; An tiviral agen ts; Pox viruses.
Nucleoside an alogues sh ow a h igh an tiviral poten tial. Nucleoside-derived
drugs are approved for th erapy of AIDS an d in fection s in duced by h erpex
virus, varicella zoster virus, an d h epatitis B virus. Of th eir sh ortcom in gs,
on e can n ote th eir cytotoxicity an d in sufficien t bioavailability, wh ich
could be im proved by proper m odification s of th eir structures. N4-Hydroxy-
cytidin e (1) was sh own to h ave a broad spectrum of an tiviral activity. In
particular, 1 in h ibited in cell culture assays som e clin ically im portan t in fec-
tion s, in duced by viruses of severe acute respiratory syn drom e, in fluen za A
an d B 1, m easles1, h epatitis C 2 an d som e oth ers. In th is paper we describe
th e syn th esis of n ew derivatives of 1 an d evaluation of th eir an tiviral prop-
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1099–1106
© 2006 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20061099

1100
Ivanov et al.:
erties in Vero an d LLC-MK2 cell cultures in fected with viruses of th e pox
fam ily.
We prepared a series of N4-(acyloxy) derivatives of 1 based on th e data on
th e positive effect of h ydroph obic substituen ts in th e m olecules of an ti-
virals3. Th e syn th esis of N4-(acyloxy) derivatives (2a–2d ) was perform ed ac-
cordin g to Sch em e 1. Acylation of 1 in pyridin e with a sm all excess of th e
acyl ch loride led to ben zoyl (2a) or pivaloyl (2b) derivatives of 1. In th e
case of N4-[(adam an tan e-1-carbon yl)oxy]cytidin e (2c) an d N4-(triph en yl-
acetoxy)cytidin e (2d ), im idazolides of adam an tan e-1-carboxylic or tri-
ph en ylacetic acid were used as acylatin g agen ts. Th e products were isolated
on a silica gel colum n in a gradien t of con cen tration s of MeOH in CHCl3
in 54–79% yields. We did n ot observe th e form ation of th e correspon din g
N-acyl derivatives, wh ich correlates with Mertes’s an d Sm rt’s data4 on th e
exclusive form ation of O-derivatives wh en m odified th e N4-(h ydroxy-
am in o) group of N4-h ydroxy-6-azacytidin e.
O
O
HNOCC6H5
HNOCC(CH3)3
N
N
O
N
HNOH
O
N
HO
(i)
HO
O
O
N
O
N
(ii)
HO OH
HO OH
HO
O
2a
2b
HO OH
1
O
O
(iv)
(iii)
HNOC
HNOCC(C6H5)3
N
N
O
N
O
N
HO
HO
O
O
HO OH
HO OH
2c
2d
(i) benzoyl chloride, Py, 4 oC, 20 h; (ii) pivaloyl chloride, Py, 4 oC, 20 h; (iii) 1-(adamantane-
-1-carbonyl)imidazolide, DMF, 20 oC, 20 h; (iv) 1-(triphenylacetyl)imidazolide, DMF, 20 oC, 20 h
SCHEME 1
An oth er approach of design in g n ew an tivirals in volves th e in troduct-
ion of 5′-ph osph on ate fragm en ts in to m olecules of n ucleoside an alogues.
For exam ple, 5′-ph osph on ate of 3′-deoxy-3′-azidoth ym idin e is used in m ed-
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New N4-Hydroxycytidine Derivatives
1101
ical practice as an an ti-HIV drug (Nicavir )5. Ph osph on ate derivatives of
acyclic n ucleosides h ave a broad spectrum of an an tiviral activity an d th ree
of th em are already m arketed – Hepsera
(aden ovir, PMEA), Viread
(ten ofovir, PMPA) an d Vistide (cidofovir, HPMPC)6.
5′-O-Ph osph on ate derivatives of cytidin e 3a–3c were prepared accordin g
to th e reported procedures7. Th e reaction of 3a–3c with aqueous h ydroxyl-
am in e h ydroch loride (pH 6) un der th e con dition s described in ref.8
(Sch em e 2) afforded derivatives 4a–4c, wh ich were purified by reversed-
ph ase silica gel ch rom atograph y an d isolated in 56–83% yields.
NH2
HNOH
O O
N
N
3, 4: a,
R = C2H5O
C
P
OH
O
N
O
N
O
R = H P
OH
RO
(i)
RO
O
O
b,
c,
O
HO OH
HO OH
R = HO
P
CH2
3a-3c
4a-4c
OH
(i) NH2OH·HCl/H2O, pH 6.0, 36 oC, 24 h
SCHEME 2
1
Th e structures of th e syn th esized com poun ds were con firm ed by UV, H,
13C an d 31P NMR, an d m ass spectra. It is n oteworth y th at substitution of
th e 4-am in o group in th e cytosin e base by a h ydroxylam in e group resulted
1
in a sign ifican t upfield sh ift of H-6, H-5 an d H-1′ reson an ces in th e H NMR
spectra com pared with th e paren t cytidin e ph osph on ates.
Stability of com poun ds 2a, 2b was evaluated in PBS an d n orm al h um an
blood serum at 37 °C accordin g to th e reported procedures9. Com poun ds
2a, 2b were stable in PBS to give on ly traces of 1 after 10 days. Half-life peri-
ods of com poun ds 2a, 2b in h um an blood serum were sh own to be 40 ± 5
an d 45 ± 5 m in , respectively, to give on ly 1.
An tiviral activity of th e syn th esized com poun ds was studied in Vero an d
LLC-MK2 kidn ey cell cultures in fected with various pox viruses: vaccin ia
(VV), m on keypox (MPV) an d cowpox (CPV) viruses. An tiviral efficien cy
was determ in ed usin g a Neutral Red uptake assay accordin g to ref.10 Th e
data were graph ed as sh own in Fig. 1 for 2a an d sum m arized in th e Table I.
Com poun d 1 effectively suppressed replication of pox viruses (VV, MPV
CPV) an d was m oderately toxic. An tiviral activities of 1 were h igh er th an
th ose of cidofovir, used as referen ce preparation in th e an tipox virus study-
in g10, alth ough th e latter dem on strated a con siderably lower toxicity. As a
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1102
Ivanov et al.:
result, selectivity in dexes (SI) of 1 were com parable with th ose of cidofovir
(3.6–15.3 versus 1.2–9.5 for differen t viruses an d cell cultures). An tiviral
properties of com poun ds 2a an d 2b were h igh er th an th ose of paren t 1 due
to lower cytotoxicity an d h igh er activity in both cell cultures. In LLC-MK-2
cells SI values of com poun ds 2a an d 2b ach ieved 50–100, wh ereas SI values
of 1 were 5–15 for differen t viruses. Th e sim ilar results were obtain ed in
Vero cells (IS 10–67 for com poun ds 2a an d 2b versus 3.6–5.6 for paren t 1).
Th e in troduction of m ore bulky substituen ts in to th e N4-position of th e
h eterocyclic base (com poun ds 2c an d 2d ) dram atically in creased cyto-
toxicity an d did n ot effect an tiviral activity.
As a rule, n ucleoside ph osph on ates exh ibit a lower activity in cell assays
if com pared with paren t n ucleosides, but due to a m ore poten t reduction of
cytotoxicity, th e SI of th ese com poun ds are h igh er5,11. Cytotoxicities of
N4-h ydroxycytidin e 5′-ph osph on ate derivatives 4 in Vero cell culture were
h igh er th en th at of th e paren t n ucleoside (CC50 5.4–38 µM) an d SI values of
com poun ds 4a an d 4b were low (3 an d 4.4 correspon din gly) Com poun d 4c
was in active in th ese experim en ts. A sim ilar result was obtain ed for 4′-th io-
5-eth yl-2′-deoxyuridin e, wh ose ph osph on ates were n eith er m ore active as
an ti h erpes sim plex virus agen ts n or less toxic9.
FIG. 1
Represen tative data from a sin gle experim en t for 2a again st various pox viruses on LLC-MK2
cells. Data are sh own for a sin gle replicate set of assays. IC50 values (µg/m l) for each strain are
sh own below th e graph , as th e R2 fit of th e poin ts to th e curve
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New N4-Hydroxycytidine Derivatives
1103
TABLE I
An tiviral activity an d cytotoxicity of N4-alkyloxycytidin es 2a–2d an d 5′-ph osph on ate deriva-
tives of N4-h ydroxycytidin e 4a–4c in Vero an d LLC-MK2 cell cultures
VV
MPV
CPV
Cell
CC50
Com pd
culture
µM
IC50, µM
IS
IC50, µM
IS
IC50, µM
IS
50
8.9
5.6
10.1
5.0
14.0
3.6
1
Vero
MK-2
Vero
MK-2
Vero
MK-2
Vero
MK-2
Vero
MK-2
Vero
Vero
Vero
Vero
MK-2
77
165
275
175
480
31
15.1
4.1
2.7
2.6
4.7
6.7
7.3
1.3
1.4
1.5
1.8
38
5.1
40.2
102
67
5.0
7.9
15.3
20.7
118
21
13.9
19.7
4.8
5.76
8.4
2a
2b
2c
2d
2.3
57.5
9.7
8.4
18.0
6.7
103
4.7
13.1
17.7
34
2.6
186
6.7
77
4.6
5.0
6.2
102
23
11.4
2.9
8.9
31.6
6.4
3.2
8.2
3.6
47
4.0
11.7
n .d.
n .d.
n .d.
9.5
22.5
n .d.
n .d.
n .d.
47.7
298.7
2.1
4a
4b
4c
6.6
5.4
38
4.4
3.0
1
n .d.
n .d.
n .d.
37.6
112.2
n .d.
n .d.
n .d.
7.5
Cido-
fovir
360
360
44.1
83.5
8.1
4.3
3.0
1.2
VV, vaccin ia virus; MPV, m on keypox virus; CPV, cowpox virus.
CC50 is th e m in im al con cen tration of th e com poun d, at wh ich 50% cell death occurs.
IC50 is th e con cen tration of com poun ds at wh ich proliferation of virus is in h ibited by 50%.
IS = CC50/IC50; n .d., n ot determ in ed.
To sum m arize, m odification of N4-h ydroxycytidin e 1 by acylation of th e
N4-position m ay h ave som e h opeful prospects, wh ereas its 5′-ph osph on yl-
ation seem s to be a dead lock.
EXPERIMENTAL
UV spectra (λ, n m ; ε) were m easured on a UV-2401 spectroph otom eter (Sh im adzu, Japan ).
1H NMR spectra were recorded on an AMX III-400 (Bruker, U.S.A.) with a workin g frequen cy
of 400 MHz for 1H NMR (Me4Si an d sodium 3-(trim eth ylsilyl)propan e-1-sulfon ate (DSS) were
in tern al stan dards for CDCl3 or DMSO-d6 an d D2O solution s, respectively), 101 MHz for
13C NMR an d 162 MHz for 31P NMR. 31P NMR spectra were recorded with 1H-decouplin g
an d 85% H3PO4 as an extern al stan dard. Ch em ical sh ifts (δ-scale) are given in ppm ; cou-
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1104
Ivanov et al.:
plin g con stan ts (J) in Hz. Mass spectra were registered on a Com pact MALDI 4 spectrom eter
(Kratos An alytical, U.S.A.). Meltin g poin ts were determ in ed on a Kofler block an d are un cor-
rected.
Startin g Materials an d Solven ts
Adam an tan e-1-carboxylic acid, Et3N, ben zoyl ch loride an d pivaloyl ch loride were purch ased
from Fluka; pyridin e, dioxan e, DMF, triph en ylacetic acid, 1,1′-carbon yldiim idazole were
from Aldrich ; Silica gel 60 (40–63 µm ) an d LiCh roprep RP-18 (25–40 µm ) were from Merck,
h ydroxylam in e h ydroch loride was from Reakh im (Russia). Cytidin e 5′-(eth oxycarbon yl)-
ph osph on ate (3a), cytidin e 5′-ph osph on ate (3b) an d 5′-O-(ph osph on om eth yl)cytidin e (3c)
were prepared accordin g to publish ed procedures7. Cidofovir was a kin d gift of Dr Rosen berg
(In stitute of Organ ic Ch em istry an d Bioch em istry, Academ y of Scien ces of th e Czech Republic).
N4-(Ben zoyloxy)cytidin e (2a) an d N4-(Pivaloyloxy)cytidin e (2b)
Ben zoyl ch loride or pivaloyl ch loride (0.34 m m ol) was added to a precooled (–20 °C) solu-
tion of 1 (80 m g, 0.31 m m ol) in dry pyridin e (2 m l). Th e m ixture was kept at 4 °C for 20 h ,
MeOH (1 m l) was added an d th e solven ts were evaporated. Th e residue was dissolved in
ch loroform (0.5 m l), applied on to a silica gel colum n (2 × 22 cm ), an d eluted with a gradi-
en t of m eth an ol in ch loroform (0→20%, 750 m l). Th e target fraction s were evaporated, th e
residue was crystallized from MeOH to give 2a (76 m g, 72%) an d 2b (89 m g, 79%).
N4-(Benzoyloxy)cytidine (2a): For C16H17N3O7 MS: m/z 362.3 (M+). M.p. 208–209 °C. UV
(MeOH): λm ax 276 (7200), 231 (7300). 1H NMR (CD3OD): 7.54 m , 6 H (C6H5, H-6); 5.91 d,
1 H, J1′,2′ = 4.7 (H-1′); 5.82 d, 1 H, J5,6 = 8.1 (H-5); 4.18 m , 2 H (H-2′, H-3′); 3.99 t, 1 H,
J4′,3′ ~ J4′,5′ = 2.8 (H-4′); 3.82 dd, 2 H, Jgem = 12.7, J5′a,4′ = 3.9 (H-5′a); 3.76 dd, 2 H Jgem
=
12.7, J5′b,4′ = 3.7 (H-5′). 13C NMR (CD3OD): 61.09 (C-5′); 69.99 (C-2′); 72.93 (C-3′); 84.78
(C-4′); 87.31 (C-1′); 96.32 (C-5); 128.34 (CH-m-Bz); 129.86 (CH-i-Bz); 130.01 (CH-o-Bz);
133.03 (CH-p-Bz); 133.30 (C-6); 134.35 (C-2); 149.07 (C-4); 179.07 (C=O).
N4-(Pivaloyloxy)cytidine (2b): For C14H21N3O7 MS: m/z 342.3 (M+). M.p. 119–120 °C. UV
(MeOH): λm ax 277 (7100), 227 (7150). 1H NMR (CD3OD): 7.52 d, 1 H, J5,6 = 8.1 (H-6);
6.07 d, 1 H (H-5); 5.88 d, 1 H, J1′,2′ = 4.9 (H-1′); 5.87 d, 1 H, J1′,2′ = 4.4 (H-2′a); 4.18 m , 2 H
(H-2′b, H-3′); 4.02 m , 1 H (H-4′); 3.8 dd, 2 H, Jgem = 12.7, J5′a,4′ = 3.86 (H-5′a); 3.76 dd, 2 H,
J5′b,4′ = 3.7 (H-5′b); 1.22 m , 9 H (3 CH3). 13C NMR (CD3OD): 26.83 ((CH3)3); 39.75
(C(CH3)3); 61.09 (C-5′); 70.01 (C-2′); 72.99 (C-3′); 87.38 (C-4′); 91.27 (C-1′); 96.51 (C-5);
133.92 (C-6); 136.35 (C-2); 149.05 (C-4); 187.12 (C=O).
N4-[(Adam an tan e-1-carbon yl)oxy]cytidin e (2c) an d N4-(Triph en ylacetoxy)cytidin e (2d )
1,1′-Carbon yldiim idazole (81 m g, 0.5 m m ol) was added to a solution of adam an tin e-
1-carboxylic or triph en ylacetic acid (0.33 m m ol) in DMF (5 m l) an d th e m ixture was kept
at 20 °C for 1 h , th en th e m ixture was added to a precooled (–20 °C) solution of 1 (78 m g,
0.3 m m ol) in DMF (3 m l). After 20 h at 4 °C, th e solven t was evaporated an d th e residue
was partition ed between ch loroform (20 m l) an d water (5 m l). Th e organ ic layer was wash ed
with water (5 m l), dried over an h ydrous Na2SO4, an d evaporated. Th e residue was dissolved
in ch loroform (0.5 m l), applied on to a silica gel colum n (2.5 × 9 cm ), an d eluted with a gra-
dien t of m eth an ol in ch loroform (0→7%, 300 m l). Th e target fraction s were evaporated in
vacuum to give 68 m g of 2c (54%) or 94 m g of 2d (59%) as a wh ite foam .
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New N4-Hydroxycytidine Derivatives
1105
N4-[(Adamantane-1-carbonyl)oxy]cytidine (2c): For C20H27N3O7 (421.2) MS: m/z 420 (M+).
UV (MeOH): λm ax 276 (7200), 230 (7250). 1H NMR: (CDCl3): 9.75 s, 1 H (3-NH); 7.05 d, 1 H,
J6,5 = 8.1 (H-6); 5.82 d, 1 H, J1′,2′ = 5.6 (H-1′); 5.8 d, 1 H (H-5); 4.27 t, 1 H, J2′,1′
(H-2′); 4.17 t, 1 H, J3′,2′
3J3′,4′ = 5.6 (H-3′); 4.04 m , 1 H (H-4′); 3.7 dd, 2 H, Jgem = 12.7,
3J5′a,4′ = 3.1 (H-5′a); 3.61 dd, 2 H, J5′b,4 = 4.4 (H-5′b); 1.94 m , 15 H (Ada).
~
3J2′,3′ = 5.6
~
N4-(Triphenylacetoxy)cytidine (2d ): For C29H27N3O7 (529.6) MS: m/z 528 (M+). UV (MeOH):
λm ax 270 (7400), 228 (7300). 1H NMR: (CDCl3): 9.45 s, 1 H (3-NH); 7.46 d, 1 H, J6,5 = 8.06
(H-6); 7.2 m , 15 H (3 C6H5); 6.81 d, 1 H (H-5); 5.86 d, 1 H, J1′,2′ = 5.0 (H-1′); 4.27 t, 1 H,
J2′,1′ ~ J
= 5.2 (H-2′); 4.16 t, 1 H, J3′,2′ ~ J3′,4′ = 5.1 (H-3′); 4.12 m , 1 H (H-4′); 3.82 dd, 2 H,
2′,3′
Jgem = 12.7, J5′a,4′ = 3.8 (H-5′a); 3.76 dd, 2 H, J5′b,4′ = 4.2 (H-5′b).
N4-Hydroxycytidin e 5′-(Eth oxycarbon yl)ph osph on ate (4a), N4-Hydroxycytidin e
5′-Ph osph on ate (4b) an d N4-Hydroxy-5′-O-(ph osph on om eth yl)cytidin e (4c)
A solution of h ydroxylam in e h ydroch loride (174 m g, 2.5 m m ol) in water (3 m l, pH 6.0) was
added to th e solution of correspon din g cytidin e derivative 3a–3c (0.5 m m ol) in water (3 m l).
Th e m ixture was kept at 36 °C for 24 h , th en con cen trated to 3 m l. Th e residue was applied
on to a LiCh roprep RP-18 colum n (2 × 17 cm ) an d eluted in a lin ear gradien t of MeOH
(0→5%, 600 m l) in aqueous 0.01 M NH4HCO3. Th e target fraction s were evaporated, co-
evaporated with water (2 × 5 m l), th e residue was dissolved in water (5 m l) an d freeze-dried
to yield 148 m g (72%) of 4a, 121 m g (71%) of 4b or 153 m g (83%) of 4c as am m on ium
salts.
N4-Hydroxycytidine 5′-(ethoxycarbonyl)phosphonate (4a): For C12H18N3O10P (395.3) MS: m/z
394 (M+). UV: (H2O, pH 2) λm ax 282 (10400), 221 (8990); (H2O, pH 7) λm ax 271 (8100), 235
(8400). 1H NMR (D2O): 6.88 d, 1 H, J6,5 = 8.4 (H-6); 5.64 d, 1 H, J1′,2′ = 6.5 (H-1′); 5.51 d,
1 H (H-5); 4.04–4.00 m , 2 H (H-2′, H-3′); 3.97–3.95 m , 2 H (CH2CH3); 3.93 m , 1 H (H-4′);
3.91–3.87 m , 2 H (H-5′); 1.18 t, 3 H, JCH2,CH3 = 7.1 (CH2CH3). 31P NMR (D2O): –4.51 s.
N4-Hydroxycytidine 5′-phosphonate (4b): For C9H14N3O8P (323.2) MS: m/z 322 (M+). UV:
(H2O, pH 2) λm ax 282 (10300), 222 (8950); (H2O, pH 7) λm ax 269 (8150), 235 (8600).
1H NMR (D2O): 6.85 d, 1 H, J5,6 = 8.1 (H-6); 6.49 d, 1 H, JH,P = 641 (H-P); 5.52 d, 1 H (H-5);
5.64 d, 1 H, J1′,2′ = 6.5 (H-1′); 4.05–3.99 m , 2 H (H-2′, H-3′); 3.92 m , 1 H (H-4′); 3.83–3.73 m ,
2 H (H-5′). 31P NMR (D2O): 7.12 s. 13C NMR (D2O): 60.48 d, JC,P = 2.28 (C-5′); 67.46 (C-2′);
69.85 (C-3′); 80.28 d, JC,P = 7.63 (C-4′); 85.69 (C-1′); 96.27 (C-5); 128.62 (C-6); 144.11 (C-2);
148.64 (C-4).
N4-Hydroxy-5′-O-(phosphonomethyl)cytidine (4c): For C10H16N3O9P (353.2) MS: m/z 352
(M+). UV: (H2O, pH 2) λm ax 281 (10500), 221 (9050); (H2O, pH 7) λm ax 271 (8200), 235
(8500). 1H NMR (D2O): 7.40 d, 1 H, J6,5 = 5.3 (H-6); 5.72 m , 2 H (H-5, H-1′); 4.17 t, 1 H,
J2′,1′ = 5.3, J2′,3′ = 5.3 (H-2′); 4.10 t, 1 H, J3′,2′ = 4.4, J3′,4′ = 5.0 (H-3′); 4.00 d, 1 H, J4′,3′ = 3.1
(H-4′); 3.75 dd, 1 H, Jgem = 11.2, J5′a,4′ = 2.5 (H-5′H); 3.65 dd, 1 H, J5′b,4′ = 3.4 (H-5′b); 3.55 d
an d 3.57 d, 2 H, JHa,P = JHb,P = 9.0 (PCH2O). 31P NMR (D2O): 15.94 s. 13C NMR (D2O):
68.72 d, JC,P = 156 (C-P); 70.46 (C-2′); 72.37 (C-3′); 73.82 d, JC,P = 9.8 (C-5′); 83.41 (C-4′);
86.89 (C-1′); 98.33 (C-5); 130.28 (C-6); 143.46 (C-2); 149.45 (C-4).
Th e in vitro cytotoxicity an d an tiviral effects of syn th esized com poun ds in Vero, an d
LLC-MK2 kidn ey cell cultures in fected with viruses of pox virus fam ily (vaccin ia,
m on keypox an d cowpox viruses) were determ in ed usin g a n eutral red uptake assay as de-
scribed previously10. Th e data were plotted an d an alyzed by usin g th e software program
(Molecular Devices, Men lo Park, U.S.A.).
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 1099–1106

1106
Ivanov et al.:
The work was supported by the Russian Foundation for Basic Research (projects 05-04-49492 and
05-04-49500), the program of the Presidium of Russian Academy of Sciences on molecular and
cellular biology, and ISTC grant 1989.
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