Synthesis and biological evaluation of some novel 4′-thio-L-ribonucleosides with modified nucleobase moieties

Article abstract of DOI:10.1016/S0960-894X(03)00289-0

1,2,3,5-Tetra-O-acetyl-4-thio-β-L-ribofuranose (13) was synthesized by an improved five-step sequence starting from methyl α-D-lyxopyranoside. Compound 13 was then converted to the corresponding L-4′-thionucleosides 4-6 and 19 by a modified Vorbrueggen procedure. All of these nucleoside analogues were tested for their antitumour activity in vitro.

Full text of DOI:10.1016/S0960-894X(03)00289-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1849–1852
Synthesis and Biological Evaluation of Some Novel
-Thio-L-ribonucleosides with Modified Nucleobase Moieties
0
4
a,
a
a
a
Vjera Pejanovic
´
, * Zdenka Stokic, Biljana Stojanovic
b
´
´
, Vesna Piperski,
b
Mirjana Popsavin and Velimir Popsavin
aGalenika AD, Batajnicki drum bb, 11000 Belgrade, Yugoslavia
bDepartment of Chemistry, Faculty of Sciences, University of Novi Sad, Trg D. Obradovica 3, 21000 Novi Sad, Yugoslavia
Received 7 February 2003; revised 18 March 2003; accepted 19 March 2003
Abstract—1,2,3,5-Tetra-O-acetyl-4-thio-b-l-ribofuranose (13) was synthesized by an improved five-step sequence starting from
0
methyl a-d-lyxopyranoside. Compound 13 was then converted to the corresponding l-4 -thionucleosides 4–6 and 19 by a modified
Vorbru
#
¨
ggen procedure. All of these nucleoside analogues were tested for their antitumour activity in vitro.
2003 Elsevier Science Ltd. All rights reserved.
The discovery that replacement of the furanose ring-
oxygen with a sulphur atom leads to promising antiviral
or antitumour nucleosides has stimulated the synthesis
1
of this class of compounds. A number of syntheses of
0
2
b-d-4 -thionucleosides have been reported, along with
3
a limited number of syntheses of their l-enantiomers.
0
Some of these representatives, as for example d-4 -thio-
2
b,d
0
5
-fluorouridine
and 9-(4 -thio-b-d-ribofuranosyl)-6-
2
g
mercaptopurine showed marked cytotoxicity against
the leukemia L-1210 and certain human tumours. In
0
cleoside analogues have been synthesized, including the
contrast to l-4 -thionucleosides, a number of l-ribonu-
4
4
a
b-l-ribofuranosyl-5-fluorouracil
(1), b-l-ribofuran-
osyl-5-fluorocytosine (2), as well as the 9-(b-l-ribo-
furanosyl)-6-mercaptoguanine (3). However, there has
4
a
Chemistry
5
been no report in literature concerning either the synth-
esis or the biological data of 4 -thio-l-ribonucleosides
The 1,2,3,5-tetra-O-acetyl-4-thio-b-l-ribofuranose (13),
a key intermediate in the synthesis of the corresponding
0
bearing modified nucleobase moieties. In this paper we
0
4 -thio-l-nucleosides, has been synthesized previously
0
ribonucleosides of 5-fluorouracil (4), 5-fluorocytosine
6
wish to report on the synthesis of three novel 4 -thio-l-
by Reist et al. starting from d-lyxose. This approach
afforded the product 13 as a mixture of the corre-
(
5), and 6-mercaptoguanine (6), along with their effects
sponding a- and b-anomers in 13.6% overall yield from
six synthetic steps. By modifying the original procedure
6
on the proliferation of some malignant and normal
cells.
we have prepared pure b-anomer 13 starting from
methyl a-d-lyxopyranoside (7, Scheme 1), which is
6
7
readily available from d-lyxose, or d-xylose. The first
step of the sequence (7!8) was carried out as earlier
*Corresponding author at present address: Hemofarm Group, Hemo-
farm Institute, Beogradski put bb, 26300 Vrsac, Yugoslavia.
Tel.:+381-13-821-068; fax:+381-11-2351-994; e-mail: vpejanovic@
hemofarm.co.yu
6
described. However, 4-S-acetyl derivative 10 was syn-
thesized from alcohol 8 via the triflic ester 9. This
enabled the usage of milder reaction conditions in the
ˇ
0
960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00289-0

1850
V. Pejanovi c´ et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1849–1852
ꢀ
Scheme 1. Conditions: (a) Me
from 8; (d) 9:1 TFA–H
2
C(OMe) , TsOH, Me
O, rt, 2h; (e) 15:15:1 AcOH–Ac
2
2
CO, rt, 24 h, 78%; (b) Tf
ꢀ
2
O, DMAP, Py, CH Cl
, 0 C, 48 h, 58% from 10 (21% overall from 7).
2
2
, ꢁ15 C, 0.5 h; (c) KSAc, DMF, rt, 2h, 47%
2
2
O–H SO
2
4
Scheme 2. Conditions: for 14, (a) (i) 5-fluorouracil, TMSCl, (NH
cytosine, TMSCl, (NH SO
4
)
2
SO
4
, HMDS"# , 4 h; (ii) 13, TMSOTf, MeCN, rt, 6 h; for 15, (a) (i) 5-fluoro-
ꢀ
4
) , HMDS"# , 3 h; (ii) 13, TMSOTf, MeCN, 40 C, 2h; for 16; (a) (i) 6-mercaptoguanine, TMSCl, (NH
h; (ii) 13, TMSOTf, MeCN, rt, 2h; for 17; (a) (i) Thymine, TMSCl, (NH
2
4
4
)
2
SO
4
, HMDS"# ,
5 )
1.5% of 18; (b) NaOMe, MeOH, rt, 2h for 14, 1 h for 15, 20 min for 17, 49% of 4, 67% of 5, 90% of 19; (c) NH
4
2
SO
4
, HMDS"# , 5 h; (ii) 13, TMSOTf, MeCN, rt, 6 h, 48% of 17 and
ꢀ
3
/MeOH, 0 C, 24 h, 46%.
subsequent nucleophilic displacement process with
thioacetate anion. Thus, the reaction of 9 with potas-
sium thioacetate in N,N-dimethylformamide was com-
pleted in 2h at room temperature, in contrast to the
reaction of the corresponding tosyl derivative with
The chemical yields of the obtained protected nucleo-
sides 14–16 were fair, and the ratio of the b- and
a-anomers was variable. As expected, the b-anomers
were the major components in the mixtures owing to
2-O-acetyl participation. The presence of a considerable
amount of a-anomer 16 is not entirely unexpected since
6
ꢀ
potassium thiobenzoate, which required 24 h at 105 C
to be completed. Hydrolytic removal of the iso-
propylidene protective group in 10 gave a 2:1 mixture of
1
1
¨
it is well known that the Vorbruggen method shows a
lower selectivity with purine nucleobases. The structural
elucidation and assignment of a- and b-anomers of 14
was achieved by NMR spectroscopy, in particular by
virtue of the observed NOE after irradiation of multi-
8
the expected diol 11 and the 4-O-acetyl derivative 12.
As the compounds 11 and 12 could not be separated by
column chromatography, their mixture was subjected to
9
0
0
acetolysis under the Whistler reaction conditions, to
plets at 3.69 (H-4 , major isomer) and 3.96 ppm (H-4 ,
minor isomer). Only the minor isomer clearly exhibited
afford the crystalline b-anomer 13 in 58% yield with
respect to 10. Thus obtained sample 13, was for the first
time fully characterized by the corresponding physical
0
NOE between H-6 of the nucleobase and H-4 , thus
implying a spatial vicinity of these protons. Such an
arrangement is only possible if the minor isomer 14
represents an a-anomer, as confirmed by molecular
1
0
and spectral data.
The coupling reactions of 13 with 5-fluorouracil,
-fluorocytosine and 6-mercaptoguanine were performed
by applying a modified Vorbruggen method. Thus, the
¨
nucleobases were refluxed in hexamethyldisilazane, and
the resulting silylated bases were reacted with 13, in the
presence of trimethylsilyl triflate as a catalyst in dry ace-
tonitrile as a solvent at room temperature, to afford the
corresponding protected nucleosides 14–16 (Scheme 2).
The results are summarized in Table 1.
5
Table 1. Coupling reactions of modified nucleobases with 13
1
1
a
b
Silylated base
5-Fluorouracil
Product
Yield (%)
b/a Ratio
14
15
16
79
59
54
5.5:1
10:1
2.5:1
5
6
-Fluorocytosine
-Mercaptoguanine
a
Isolated yields.
The ratios of the a- and b-anomers were determined by H NMR.
b
1

V. Pejanovi c´ et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1849–1852
1851
modeling (HyperChem Molecular Modeling Package,
Table 2. In vitro cytotoxicity of synthesized compounds
release 6.03; Hypercube Inc., Gainesville, FL). The
conformational search was performed by varying sugar
endocyclic torsion angles in a-14 by the usage directed
method. The conformations obtained were geometry
optimized by AMBER 94 force field to reach rms gra-
a
Compd
IC50 mM
C6
HTB14
HeLa
NB4
T47D
NHDF
4
5
6
19
>100
>100
>100
>100
41.5
83.3
62.1
67.8
>100
95.9
>100
100
>100
b
>100
b
>100
>100
>100
>100
GSA
>100
>100
GSA
GSA
>100
˚
dients of <0.01 kcal/(A mol). The structures having
b
energies of 6 kcal/mol higher than the lowest energy
conformation, as well as those with relative energy dif-
ferences within 0.05 kcal/mol were discarded in the
post-optimization runs. At least four low-energy con-
aIC50 is a compound concentration required to inhibit the cell growth
by 50% compared to an untreated control.
b
GSA – Growth-stimulatory activity.
0
formations were found that have the H-4 /H-6 distance
˚
in the range of 2.70–3.18 A. These findings are con-
sistent with the NOE results.
All tested compounds showed a moderate growth
inhibitory activity only against HTB14 human glioma
cells, while on C6 rat glioma and HeLa cells the highest
compound concentration tested (100 mM) caused inhib-
ition of cell proliferation from 15 to 50%. These com-
pounds had no growth inhibitory effect on NHDF cell
line at any concentration tested. Such a result is con-
sistent with earlier findings that most of l-nucleoside
analogues are not recognized by normal cellular
enzymes and are less toxic to normal cells. However,
compounds 5 and 6 exhibited a significant growth sti-
mulatory activity towards NB4 and T47D cells at con-
centrations 0.78–1.56 mM. Compound 5 enhanced the
growth of both NB4 and T47D cell lines by 63 and
86%, respectively, while compound 6 stimulated the cell
proliferation of T47D by 89% with respect to an
untreated control. To the best of our knowledge com-
pounds 5 and 6 represent the first nucleoside analogues
with growth stimulatory activity towards NB4 and
T47D cells.
Due to their similar chromatographic properties, the
a- and b-anomers 14–16 could not be separated by silica
gel column chromatography, but were in the mixtures
further converted to the corresponding deprotected
nucleosides 4–6. As shown in Scheme 2, deacetylation of
0
1
4, 15 and 16 gave the corresponding 4 -thio-l-ribonu-
cleosides 4 (49%), 5 (67%) and 6 (46%), in b/a ratios of
:1, 10:1 and 2.5:1, respectively. Similarly to their syn-
1
8
6
thetic precursors, the a- and b-anomers of 4–6 could not
be separated by column chromatography. However,
even in the mixture their structure, including their ste-
reochemistry at the anomeric positions was successfully
1
12ꢁ14
resolved by H NMR spectroscopy. The most reli-
able method for assignment of anomeric configuration
of free N- and C-nucleosides is the usage of differences
0
0
in H-1 chemical shifts. Namely, the H-1 proton of
b-anomers appears at higher field than those of
a-anomers due to the shielding effects of a cis hydroxyl
0
16
group at C-2 . It appears that this rule is also valid for
0
revealed a high-field chemical shift for H-1 of the
the 4 -thioribonucleosides, since NMR peak analysis
0
In summary we have developed an improved five-step
route to 1,2,3,5-tetra-O-acetyl-4-thio-b-l-ribofuranose
(13), a key intermediate in the synthesis of several new
0
b-anomer with respect to H-1 of the corresponding
0
a-anomer for each of the synthesized compounds 4–6.
l-4 -thioribonucleosides (4–6, and 19). The products
obtained were evaluated for their cytotoxicity profile to
certain malignant and normal cell lines. Further work
directed to syntheses of novel l-4 -thioribonucleosides
0
moderate in vitro cytotoxicity against certain human
tumours, its l-configuration counterpart 19 was also
prepared for comparison (Scheme 2). Condensation of
Since 4 -thio-b-d-ribofuranosylthymine demonstrated a
0
with modified base moieties is currently underway.
2
b
1
3 with silylated thymine, in presence of trimethylsilyl
triflate, gave the corresponding b- 17 and a-anomer 18
in an approximate ratio of 30:1. After chromato-
graphic purification on a column of silica gel (1:1
hexane–EtOAc), pure b-anomer 17 was isolated in
Acknowledgements
This work was supported by a research grant from the
Ministry of Science, Technologies and Development of
the Republic of Serbia (Grant No. 1896). The authors
are grateful to Mrs. T. Marinko-Covell (University of
Leeds, UK) for recording the mass spectra. The authors
also thank Dr. Z. Pietrzkowsky (ICN Pharmaceuticals,
Inc., Costa Mesa, CA) for MTS assay.
4
8% yield, along with a small amount of 18 (1.5%).
The major product 17 was deprotected with sodium
methoxide in methanol to afford the corresponding
1
5
nucleoside 19 (90%) after silica gel column chroma-
tography.
References and Notes
Biological Evaluation
Compounds 4–6 and 19 were evaluated for their in vitro
1
1
. (a) Dyson, M. R.; Coe, P. L.; Walker, R. T. J. Med. Chem.
991, 34, 2782. (b) Secrist, III, J. A.; Tiwari, K. N.; Riordan,
1
7
cytotoxicity to C6 rat glioma, HTB14 human glioma,
HeLa human cervical carcinoma, NB4 leukemia, T47D
breast cancer, as well as to normal human dermal
fibroblast (NHDF) cell lines. The results are shown in
Table 2.
J. M.; Montgomery, J. A. J. Med. Chem. 1991, 34, 2361.
. (a) Naka, T.; Minakawa, N.; Abe, H.; Kaga, D.; Matsuda,
2
A. J. Am. Chem. Soc. 2000, 122, 7233. (b) Clement, M. A.;
Berger, S. H. Med. Chem. Res. 1992, 2, 154. (c) Bellon, L.;

1852
V. Pejanovi c´ et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1849–1852
1
Barascut, J.-L.; Imbach, J.-L. Nucleosides Nucleotides 1992,
1, 1467. (d) Bobek, M.; Bloch, A.; Parthasarathy, R.; Whis-
tler, R. L. J. Med. Chem. 1975, 18, 784. (e) Ototani, N.;
Whistler, R. L. J. Med. Chem. 1974, 17, 535. (f) Bobek, M.;
Whistler, R. L.; Bloch, A. J. Med. Chem. 1970, 13, 411.
13. Selected data for 5 (10:1 mixture of b- and a-anomer): H
NMR (methanol-d ): b-anomer: d 3.42(m, 1H, =5.2,
1
J
0
0
6
3 ,4
a,5
,3
0
J
4
0
,5
0
a
=4.3, J
0
4
0
,5
0
b
=4.4 Hz, H-4 ), 3.79 (dd, 1H, J
5
0
0
b
=11.8
=3.8 Hz,
=4.9 Hz, H-2 ), 5.98 (dd, 1H,
0
Hz, H-5 a), 3.87 (dd, 1H, H-5 b), 4.12(dd, 1H, J
H-3 ), 4.22 (dd, 1H, J
2
0
0
0
0
0
0
1
,2
0
J1 =1.8 Hz, H-1 ), 8.51 (d, 1H, J =7.1 Hz, H-6,);
0
,F 6,F
3
. Satoh, H.; Yoshimura, Y.; Sakata, S.; Miura, S.; Machida,
H. Bioorg. Med. Chem. Lett. 1998, 8, 989 and references therein.
. (a) For examples, see: Griffon, J.-F.; Mathe, C.; Faraj, A.;
0
a-anomer: d 6.29 (dd, 1H, J
1
0
,F=1.9 Hz, J
1
0
,2
0
=4.8 Hz, H-1 ),
):
b-anomer: d 53.83 (C-4 ), 62.97 (C-5 ), 66.70 (C-1 ), 74.66
1
3
4
´
8.29 (d, 1H, J6,F=7.3 Hz, H-6); C NMR (methanol-d
6
0
0
0
Aubertin, A.-M.; De Clercq, E.; Balzarini, J.; Sommadossi, J.-
P.; Gosselin, G. Eur. J. Med. Chem. 2001, 36, 447. (b) Lin, T.-
S.; Luo, M.-Z.; Liu, M. C.; Pai, S. B.; Dutschman, G. E.;
Cheng, Y.-C. J. Med. Chem. 1994, 37, 798. (c) Mansuri,
M. M.; Farina, V.; Starrett, J. E., Jr.; Benigni, D. A.; Bran-
kovan, V.; Martin, J. C. Bioorg. Med. Chem. Lett. 1991, 1, 65.
0
0
(C-3 ), 79.74 (C-2 ), 128.13 (d, J6,F=33.9 Hz, C-6), 138.28 (d,
5,F=243.2 Hz, C-5), 157.30 (C-2), 159.30 (d, J4,F=14.3 Hz,
C-4); a-anomer: d 54.41 (C-4 ), 62.19 (C-1 ), 64.56 (C-5 ), 75.37
J
0
0
0
0
0
+
(C-2 ), 76.8 (C-3 ); HR MS: m/z 278.0611 (M +H); calcd for
S: 278.0611.
14. Selected data for 6 (2.5:1 mixture of b- and a-anomer): H
=11,
9 3 4
C H13FN O
1
5
. Heitner, H. I.; Lippard, S. J.; Sunshine, H. R. J. Am. Chem.
Soc. 1972, 94, 8936.
. Reist, E. J.; Gueffroy, D. E.; Goodman, L. J. Am. Chem.
Soc. 1964, 86, 5658.
. Popsavin, V.; Grabez, S.; Stojanovic
Pejanovic, V.; Miljkovic, D. Carbohydr. Res. 1999, 321, 110.
. The by-product 12 was presumably formed from the
NMR (dMSO-d ): b-anomer: d 3.30–3.45 (m, 3H, J
0 0
6
5 a,5 b
0
0
6
J4
0
0
=4.4 and 7 Hz, 2ꢂH-5 and H-4 ), 3.96 (dd, 1H,
,5
0
0
J
2
0
,3
0
=3.4, J
3
0
,4
0
=7.1 Hz, H-3 ), 4.15 (t, 1H, J
1
0
,2
0
=3 Hz, H-2 ),
2
), 7.90 (s, 1H,
0
7
´
, B.; Popsavin, M.;
5.14 (d, 1H, H-1 ), 6.38 and 6.40 (2ꢂbs, 2H, NH
´
´
H-8), 12.60 (bs, 1H, NH); a-anomer: d 3.30–3.45 (overlapped
0
8
m, 2H, 2ꢂH-5 ), 3.49 (m, 1H, J
4
0
,5
0
=7.6 and 3.7, J
0
3
0
,4
0
=7.6
=3.4 Hz, H-3 ), 4.23 (dd, 1H,
0
thioacetate 11 by a competitive S-O acetyl migration process.
For a related example see: Heap, J. M.; Owen, L. N. J. Chem.
Soc. (C) 1970, 707.
Hz, H-4 ), 3.84 (dd, 1H, J
0
2
0
,3
0
0
0
13
J
1
0
,2
=4.5, H-2 ), 5.89 (d, 1H, H-1 ); C NMR (dMSO-d
0
6
):
0
0
b-anomer: d 50.23 (C-1 ), 52.34 (C-4 ), 64.03 (C-5 ), 74.28 (C-
3 ), 79.22 (C-2 ), 123.25, 151.97 and 158.28 (C-2, C-4 and C-5),
0
0
9
1
1
. Bobek, M.; Whistler, R. L. Methods Carbohydr. Chem.
972, 6, 292.
0
139.05 (C-8) 159.58 (C-6); a-anomer: d 49.06 (C-1 ), 52.86 (C-
4 ), 63.43 (C-5 ), 75.05 and 76.03 (C-2 and (C-3 ); FAB MS:
ꢀ
0
0
0
0
0. Selected data for 13: mp 64–65 C (from MeOH), [a]
D
+
+
+
106 (c 0.1 in CHCl ); ref 12 (for d-enantiomer): mp 64–
m/z 338 (M +Na), 316 (M +1).
15. Selected data for 19: mp 171–177 C; [a]
MeOH); H NMR (methanol-d
(m, 1H, J =3.6, J =5.1, J
3
ꢀ
1
ꢀ
6
2
J
5 C, [a]
D
ꢁ102( c 2in CHCl
3
); H NMR (CDCl
3
): d 2.07,
D
+47 (c 1 in
6
): d 1.90 (bs, 3H, 5-Me), 3.39
1
.08, 2.11 and 2.14 (4ꢂs, 3H each, 4ꢂMeCO), 3.80 (ddd, 1H,
0
0
=4.7 Hz, H-4 ), 3.77 (dd,
4 ,5 b
=6.8, J =5.5, J =8.5 Hz, H-4), 4.14 (dd, 1H,
4,5b
0
0
0
0
0
4
,5a
3,4
3 ,4
4 ,5 a
0 0
0
=11.7 Hz, H-5 a), 3.84 (dd, 1H, H-5 b), 4.19 (t, 1H,
5 a,5 b
J5a,5b=11.4 Hz, H-5a), 4.37 (dd, 1H, H-5b), 5.36 (dd, 1H,
2,3=3.7 Hz, H-3), 5.56 (dd, 1H, J1,2=2Hz, H- 2) , 5.81 (d, 1H,
H-1); 13C NMR (CDCl
): d 20.23, 20.25, 20.31 and 20.52
1H, J
0
0
0
J
J
2
0
,3
0
=3.7 Hz, H-3 ), 4.30 (dd, 1H, J
1
0
,2
0
=6.5 Hz, H-2 ), 6.06
(d, 1H, H-1 ), 8.04 (bs, 1H, H-6); C NMR (methanol-d ): d
12.50 (5-Me), 54.11 (C-4 ), 63.86 (C-5 ), 64.87 (C-1 ), 75.14 (C-
0
13
3
6
0
0
0
(
4ꢂMeCO), 45.23 (C-4), 64.36 (C-5), 73.55 (C-3), 75.50 (C-2),
0
0
7
MS: m/z 335 (M +H).
9.08 (C-1), 168.95, 169.10, 169.37 and 169.93 (4ꢂMeCO); CI
3 ), 78.98 (C-2 ), 111.88 (C-5), 138.91 (C-6), 153.16 (C-2),
+
+
+
163.78 (C-4); FAB MS: m/z 297 (M +Na), 275 (M +1).
16. Chu, C. K.; El-Kabbani, F. M.; Thompson, B. B.
Nucleosides Nucleotides 1984, 3, 1.
17. Neutralred Bioassay (Clonetics) was used for evaluation
of all synthesised compounds for their in vitro cytotoxicity
against the C6, HTB14 and HeLa cell lines. The effect of syn-
thesized compounds to proliferation of NB4, T47D and
NHDF cells was established by MTS assay (Promega, G5430).
1
1
1
1. Vorbru
981, 114, 1234.
2. Selected data for 4 (6:1 mixture of b- and a-anomer): H
¨
ggen, H.; Krollikiewics, K.; Bennua, B. Chem. Ber.
1
NMR (DMSO-d
J3
6
): b-anomer: d 3.2(m, 1H, J
4
0
,5
0
=5.2and 5.5,
0
0
0
0
=2.1 Hz, H-4 ), 3.50–3.80 (several signals, 2H, 2ꢂH-5 ),
,4
0
4
2
8
3
.00 (dd, 1H, J
0
0
=3.4 Hz, H-3 ), 4.14 (dd, 1H, J
0
0
=6 Hz, H-
2
,3
1 ,2
0
0
), 5.00–5.75 (several signals, 3H, 3ꢂOH), 5.83 (d, 1H, H-1 ),
0
.32(d, 1H, J6,F=7 Hz, H-6); a-anomer: d 3.38 (m, 1H, H-4 ),
.50–3.80 (several signals, 4H, H-2 , H-3 and 2ꢂH-5 ), 5.00–5.75
The cells were grown in an appropriate culture medium
ꢀ
0
0
0
at 37 C in a humidified atmosphere with 5% CO . The
2
0
(
(
(
several signals, 3H, 3ꢂOH), 6.03 (d, J
1
0
,2
0
=5.2Hz, H-1 ), 8.26
tested compounds were added to the cells seeded in triplicates
in the concentration range of 0.78–100 mM and incubated for
72h.
1
3
6
d, J6,F=7.3 Hz, H-6); C NMR (DMSO-d ): b-anomer: d 53.13
0
0
0
0
0
C-4 ), 62.71 (C-5 ), 63.26 (C-1 ), 73.07 (C-3 ), 76.54 (C-2 ),
1
1
25.96 (d, J6,F=34.3 Hz, C-6), 139.97 (d, J5,F= 230.8 Hz, C-5),
49.92(C- 2) , 157.04 (d, 4,F=26.2 Hz, C-4); a-anomer: d 53.80
18. For recent reviews see: Chen, S. H. Curr. Med. Chem.
2002, 9, 899. Gumina, G.; Song, G. Y.; Chu, C. K. FEMS
Microbiol. Lett. 2001, 202, 9. Maury, G. Antivir. Chem. Che-
moth. 2000, 11, 165. Wang, P.; Hong, J. H.; Cooperwood, J. S.;
Chu, C. K. Antiviral Res. 1998, 40, 19.
J
0 0 0 0 0
(
C-4 ), 60.03 (C-5 ), 63.49 (C-1 ), 73.84 and 74.72(C-2 and C-3 ),
1
1
29.31 (d, J6,F=35.3 Hz, C-6), 138.14 (d, J5,F=230 Hz, C-5),
49.92(C- 2) , 160.13 (d, J4,F=33.9 Hz, C-4).