An efficient synthesis of 3-fluoro-5-thio-xylofuranosyl nucleosides of thymine, uracil, and 5-fluorouracil as potential antitumor or/and antiviral agents

Article abstract of DOI:10.1016/j.bmc.2007.02.031

1,2:5,6-Di-O-isopropylidene-α-d-glucofuranose by the sequence of mild oxidation, reduction, fluorination, periodate oxidation, borohydride reduction, and sulfonylation gave 3-deoxy-3-fluoro-1,2-O-isopropylidene-5-O-p-toluenesulfonyl-α-d-xy lofuranose (5).

Full text of DOI:10.1016/j.bmc.2007.02.031

Bioorganic & Medicinal Chemistry 15 (2007) 3241–3247
An efficient synthesis of 3-fluoro-5-thio-xylofuranosyl nucleosides
of thymine, uracil, and 5-fluorouracil as potential antitumor
or/and antiviral agents
a
a
b
b
´
Evangelia Tsoukala, George Agelis, Jan Dolinsˇek, Tanja Botic,
Avrelija Cencicˇb,c and Dimitri Komiotisa,*
aDepartment of Biochemistry and Biotechnology, Laboratory of Organic Chemistry, University of Thessaly, 41221 Larissa, Greece
bDepartment of Microbiology, Biochemistry and Biotechnology, Faculty of Agriculture, University of Maribor,
Vrbanska c.30, 2000 Maribor, Slovenia
cDepartment of Biochemistry, Medical Faculty, University of Maribor, Slovenia
Received 4 December 2006; revised 12 February 2007; accepted 16 February 2007
Available online 20 February 2007
Abstract—1,2:5,6-Di-O-isopropylidene-a-D-glucofuranose by the sequence of mild oxidation, reduction, fluorination, periodate
oxidation, borohydride reduction, and sulfonylation gave 3-deoxy-3-fluoro-1,2-O-isopropylidene-5-O-p-toluenesulfonyl-a-D-xylof-
uranose (5). Tosylate 5 was converted to thioacetate derivative 6, which after acetolysis gave 1,2-di-O-acetyl-5-S-acetyl-3-deoxy-
3-fluoro-5-thio-D-xylofuranose (7). Condensation of 7 with silylated thymine, uracil, and 5-fluorouracil afforded nucleosides
1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-b-D-xylofuranosyl) thymine (8), 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-b-D-xylofuranosyl) ura-
cil (9), and 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-b-D-xylofuranosyl) 5-fluorouracil (10). Compounds 8, 9, and 10 are biologically
active against rotavirus infection and the growth of tumor cells.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
are identified in mammalian tissues, the mitochondrial
thymidine kinase (M-TK) and cytoplasmic thymidine
kinase (C-TK).2–7 The C-TK is described as the essential
precursor in human tumor cell lines and is believed to
play a significant role in the biosynthesis of TMP.8–12
These data suggest that the use of a drug, which blocks
de novo TMP biosynthesis and then selectively inhibits
the C-TK, might offer a possibility for effective antineo-
plastic chemotherapy.6,7 Among several nucleosides that
have been synthesized, 5-alkylthio-5-deoxythymidine
derivatives were found to be non-competitive inhibitors
of C-TK.5–7 Some other 5-alkylthionucleosides have
also been found to be potent antitumor or antiviral
agents.13–16
Although an upsurge in the search for new antitumor
agents has recently been observed, the survival rate of
patients still remains low and one of the reasons is the
poor sensitivity of tumors to drugs. Therefore, new
agents capable of suppressing tumor growth would con-
tribute greatly to a better prognosis and current therapy.
Thymidine 50-monophosphate (TMP), which is essential
for cell proliferation, can be furnished either from
deoxyuridine 5-phosphate (dUMP) via the de novo
pathway of biosynthesis or from exogenous thymidine.
Today, several drugs are effective in blocking this de
novo pathway.1 An alternate route to TMP involves
transfer of phosphate from a nucleoside 5-triphosphate
to thymidine, a reaction which is catalyzed by thymidine
kinase (TK).1–5 Two are the major forms of TK, which
On the other hand, a number of fluorine-substituted fur-
anosyl nucleoside analogues have demonstrated a sub-
stantial antiviral and anticancer potency.17–29 This has
been partly attributed to the small size and high electro-
negativity of fluorine, which is also capable of partici-
pating in hydrogen bonding.30,31 It appears that the
high strength of the C–F bond may hinder metabolic
pathways and may increase the effective lifetime of the
active molecule. Moreover, the presence of fluorine
Keywords: 3-Deoxy-3-fluoro-5-thio-xylofuranose; Nucleoside; Thy-
mine; Uracil; 5-Fluorouracil; Antiviral; Antitumor agent.
*
Corresponding author. Tel.: +302410 565285; fax: +302410
565290; e-mail: dkom@bio.uth.gr
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.02.031

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E. Tsoukala et al. / Bioorg. Med. Chem. 15 (2007) 3241–3247
enhances lipophilicity and makes the penetration of the
drug through the cell membrane easier.32–35 The intro-
duction of fluorine into the sugar moiety, especially in
the C-2 and/or C-3 positions, induces antiviral selectiv-
ity, as in the case of 2-fluoroarabinothymidine, which
demonstrates selective inhibition of Herpes Simplex
viruses (HSV-1 and HSV-2).21–23 Moreover, 3-fluoro-3-
deoxy-thymidine was proven to be very active against
human immunodeficiency viruses (HIV) and also a bet-
ter inhibitor of HIV replication than 3-azido-3-deoxy-
thymidine (AZT).23–25
followed by stereoselective reduction with sodium boro-
hydride in methanol and tosylation with p-toluenesulfonyl
chloride in pyridine, gave the corresponding 1,2:5,6-di-
O-isopropylidene-3-O-toluenesulfonyl-a-D-allofuranose
(2).26–28 (Scheme 1) Displacement of the tosyloxy group
at C-3 of 2 by a fluorine atom was effected by reacting 2
with potassium fluoride in acetamide. Selective removal
of the 5,6-O-isopropylidene group afforded the 3-deoxy-
3-fluoro-1,2-O-isopropylidene-a-D-glucofuranose (3),29
which upon periodate oxidation, followed by borohy-
dride reduction of the resulting aldehyde (one pot), gave
3-deoxy-3-fluoro-1,2-O-isopropylidene-a-D-xylofura-
nose (4). Sulfonylation of 4 with p-toluenesulfonyl
chloride in pyridine proceeded readily, giving the tosyl
derivative 5 as a white solid. Tosylate 5 was in turn trea-
ted with potassium thioacetate in hot N,N-dimethyl-
formamide36 to give the corresponding thioacetate 6,
the infrared spectrum of which showed a characteristic
absorption at 1697 cmꢀ1 (S-acetyl group); the NMR
spectrum and chemical analysis were also consistent
with structure 6. Acetolysis37 of 6 in the presence of ace-
tic acid, acetic anhydride, and sulfuric acid afforded a
mixture of the anomeric diacetates 7 (ratio of a to b ano-
mer, 1:2). Compound 7 showed strong IR absorptions at
The above observations and the continuous demand for
new antitumor and antiviral agents prompted us to
design and synthesize a series of 1-(5-S-acetyl-3-deoxy-
3-fluoro-5-thio-b-D-xylofuranosyl) nucleosides of thy-
mine, uracil, and 5-fluorouracil (5FU), which we report
herein.
2. Results and discussion
2.1. Synthesis
1
Oxidation of the commercially available 1,2:5,6-di-O-
isopropylidene-a-D-glucofuranose (1) with pyridinium
dichromate and acetic anhydride in dichloromethane,
1750 cmꢀ1 (OAc) and 1697 cmꢀ1 (SAc), while in its H
NMR spectrum appeared prominent 3-proton methyl
signals, belonging to the acetylthio moiety (a:d 2.36,
H3C
H3C
H3C
H3C
O
O
O
O
HO
HO
O
O
O
OH
F
i
ii
O
O
O
CH3
CH3
CH3
CH3
CH3
CH3
O
O
O
TsO
2
3
1
iii
(92%)
RO
AcS
AcS
O
O
O
OAc
F
F
F
v
vi
(88%)
(82%)
O
O
CH3
CH3
CH3
CH3
AcO
O
O
7
6
4 R = H
iv
(91%)
5 R = Ts
vii
O
N
R
NH
O
AcS
8 R = CH3
O
F
9 R = H
10 R = F
AcO
Scheme 1. Reagents and Conditions: (i) a—PDC/Ac2O/CH2Cl2; b—NaBH4/MeOH; c—TsCl/pyridine; (ii) a—KF/acetamide; b—70% AcOH; (iii)
NaIO4/MeOH/NaBH4; (iv) TsCl/pyridine; (v) KSAc/DMF/100 ꢁC; (vi) Ac2O/CH3COOH/H2SO4; (vii) silylated base/Me3SiOSO2CF3.

E. Tsoukala et al. / Bioorg. Med. Chem. 15 (2007) 3241–3247
3243
b:d 2.37) and 6-proton methyl peaks assigned to the acet-
oxyl group (d: 2.06–2.16). Condensation of the anomeric
mixture 7 with the appropriate silylated nucleic acid
bases in the presence of trimethylsilyl trifluoromethane-
sulfonate afforded solely the protected b-nucleosides,
that is the title 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-b-
D-xylofuranosyl) nucleosides of thymine (8), uracil (9),
and 5FU (10), respectively.
inhibition of Caco-2 cells induced by the new com-
pounds was measured by determining the minimal
inhibitory concentration (IC50). The results are sum-
marized in Table 2 and compared with the values ob-
tained for 5FU.
The tested compounds exhibited higher cytotoxicity in
tumor cells than in the normal H4 cell line, with the
exception of compound 8, which was 2-fold more toxic
(vide TSI values) in skin melanoma and MCF-7 cells
than in normal cells. Compounds 9 and 10 were highly
selective for malignant cells. Comparison with 5FU re-
vealed that these molecules were 2.5-fold Caco-2 cells
selective and as selective as 5FU in skin melanoma
and MCF-7 cells; compound 10 did not show any
MCF-7 cells selectivity. This selective activity of the
new compounds is noteworthy and merits further
investigation.
2.2. Antiviral activity
The results from the antiviral assays on the newly syn-
thesized compounds are summarized in Table 1 and
compared to AZT. In the neutralization assay all three
compounds exhibited much higher efficacy (IC50) than
AZT. Compound 8 showed the highest direct antiviral
effect on rotavirus as the virus vanished at a very low
concentration (0.002 mg/mL), while slightly higher con-
centrations were necessary in the case for compounds 9
and 10 (0.006 mg/mL), in order to achieve the same re-
sult. Compound 8 exhibited the highest selectivity as
the corresponding CC50/IC50 value reached an optimum
level. All compounds were also capable of inhibiting the
rotavirus infection in Caco-2 cells, as strong inhibition
of infectivity was observed after rotavirus attachment
to the cells. Inhibition of rotavirus infectivity following
virus attachment of all tested compounds was compara-
ble to that of AZT or even slightly better, while their
CC50/IC50 values were almost of the same magnitude.
The effect of compounds 8–10 on cell growth was deter-
mined using Caco-2 cells. As determined by colony
numbers after 10 days of incubation, it was found that
the new compounds are capable of inhibiting the growth
of these cells in a concentration-dependent manner.
Compound 8 exhibited growth inhibitory activity (IC50
1.9 lM) similar to 5FU (IC50 1.5 lM), but 4-fold less tu-
mor selectivity (TSI 2.5 vs 10). Compounds 9 and 10
were also found to be strong inhibitors of cell growth,
but although their effect was somewhat less pronounced
than that of 5FU, they were highly selective in malig-
nant cells, as they exhibited a 2.5-fold higher TSI values
than 5FU.
2.3. Cytotoxic and growth inhibition activity
The cytotoxicity of compounds 8–10 was measured on
H4 normal human intestinal cells and on a series of
other human tumor cells, such as human colonic ade-
nocarcinoma derived Caco-2 cells, skin melanoma
cells, and epithelial breast cancer derived MCF-7 cell
line, and is expressed by the CC50 values. The growth
3. Conclusion
In conclusion, the newly synthesized 1-(3-deoxy-3-flu-
oro-5-S-acetyl-5-thio-b-D-xylofuranosyl)
nucleosides
Table 1. Antiviral activity of nucleosides 8–10 and AZT against rotavirus RF strain on Caco-2 cells (IC50
)
Compound
Treatment Aa
Treatment Ba
b
IC50
CC50/IC50
IC50
CC50/IC50
(mg/mL)
(lM)
(mg/mL)
(lM)
8
0.002
0.006
0.006
0.020
5.55
17.32
16.47
74.84
10
0.006
0.006
0.006
0.006
16.65
17.32
16.47
22.45
3.33
3.33
3.33
2.5
9
10
3.33
3.33
0.75c
AZT
CC50/IC50 ratios were calculated from CC50 values given in Table 2.
a Treatment A, Neutralization of the virus in the solution before its attachment. Treatment B, Inhibition of infectivity following virus attachment.
b CC50/IC50 values were calculated using CC50 values in Table 2.
c CC50 for AZT on Caco-2 cells = 56.1 lM.
Table 2. Cytotoxic effect (CC50, lM) of compounds 8–10 and 5-fluorouracil (5FU) on Caco-2, H4, MCF 7, and skin melanoma cells, and growth
inhibition (IC50 lM) on Caco-2 cells
Compound
Cytotoxic effect (CC50 lM)
TSIa
Growth inhibition (IC50 lM)
H4
138.7
Caco-2
Skin melanoma
MCF-7
Caco-2
Skin melanoma
MCF-7
Caco-2
8
9
55.5
57.8
277.5
17.3
16.5
46.1
277.5
288.7
2.5
25
0.5
83.5
83.2
83.4
0.5
5
1.9
5.8
1443.7
1372.4
3843.8
10
5FU
54.9
384.4
1372.4
768.8
25
10
1
16.5
1.5
5
a TSI, tumor selectivity index (CC50 on H4 cells/CC50 on the specified host cells).

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E. Tsoukala et al. / Bioorg. Med. Chem. 15 (2007) 3241–3247
8–10 are good candidates for the development of poten-
tial antiviral agents, as significantly lower concentra-
tions of these agents with respect to AZT were needed
to neutralize rotavirus infectivity. Further investigation
will enable us to elucidate potential mechanisms of their
activity and application. The most promising antitumor
activity was observed in the case of colon carcinoma
treatment, where growth inhibition and cytotoxic effect
were achieved at low concentration in comparison to
5FU, although the antitumor activity of the new com-
pounds was in most cases cell type depended.
4.1.2. 3-Deoxy-3-fluoro-1,2-O-isopropylidene-5-O-p-tolu-
enesulfonyl-a-D-xylofuranose (5). To a solution of com-
pound 4 (4.0 g, 20.7 mmol) in dry pyridine (53.1 mL)
was added p-toluenesulfonyl chloride (5.2 g, 27.6 mmol)
and kept overnight at room temperature. After neutral-
ization (NaHCO3) and extraction with EtOAc (4·
500 mL), the combined extracts were dried over anhy-
drous sodium sulfate and evaporated to dryness. Purifi-
cation of the residue by flash chromatography with
EtOAc/hexane (1:9) yielded the title compound (6.5 g,
91%, Rf = 0.2 in EtOAc/hexane, 1:9) as a white solid.
mp 61 ꢁC.
4. Experimental
1H NMR (CDCl3): d 7.4–7.8 (m, 4H, tosyl group), 5.93
(d, J1,2 = 3.7 Hz, 1H, H-1), 4.96 (dd, J3,F = 50.1 Hz,
J3,4 = 1.8 Hz, 1H, H-3), 4.67 (dd, J2,F = 10.5 Hz,
J2,1 = 3.7 Hz, 1H, H-2), 4,35-4,49 (m, J4,F = 28.1 Hz,
J4,3 = 1.8 Hz, 1H, H-4), 4.18–4.29 (m, 2 H, H-5a and
H-5b), 2.47 (s, 3H, ArCH3), 1.48 (s, 3H, CH3), 1.33 (s,
3H, CH3).
4.1. General procedure
Solutions were removed in vacuo below 40 ꢁC under re-
duced pressure. Melting points were determined on a
Mel-Temp apparatus and are uncorrected. Flash chro-
matography was performed with Silica Gel 60 (220–
440 mesh, Merck). TLC was carried out on Silica gel
(240–400 mesh, Merck), and the developing solvents
were as specified. NMR spectra were recorded at room
temperature with a Brucker 400 MHz spectrometer
using CDCl3 as solvent and TMS as internal standard.
Chemical shifts (d) were given in ppm measured down-
field from TMS, and spin–spin coupling constants are
in Hz. Infrared spectra were obtained with a Perkin–El-
mer Model 1600 FT-IR spectrophotometer. Optical
rotations were measured using a Schmidt and Haensch
polarimeter. All reactions were carried out in dry sol-
vents. CH3CN was distilled from calcium hydride and
stored over 3E molecular sieves. N,N-dimethylformam-
ide (DMF) was also stored over 3E molecular sieves,
and pyridine over potassium hydroxide pellets.
Found: C, 52.23; H, 5.69; F, 5.72. Calcd for
C15H19FO6S: C, 52.01; H, 5.53; F, 5.48.
ESI-MS m/z (relative intensity, %): 347.4 [(M+H+), 100].
4.1.3. 3-Deoxy-3-fluoro-1,2-O-isopropylidene-5-S-acetyl-
5 (6.5 g,
5-thio-a-D-xylofuranose (6). The tosylate
18.8 mmol) was heated with potassium thioacetate
(2.9 g, 25.5 mmol) in DMF (71 mL) at 100 ꢁC for 1 h.
The reaction mixture was neutralized with aqueous
NaHCO3. After that, the mixture was concentrated un-
der high vacuum pump to eliminate the DMF. The res-
idue was partitioned between water and EtOAc, the
organic extract was dried over anhydrous sodium sul-
fate, filtered, and evaporated to dryness. The residue
was purified by flash chromatography using EtOAc/hex-
ane (1:9) as eluent to give compound 7 (4.1 g, 87%,
Rf = 0.3 in EtOAc/hexane, 1:9) as a yellow syrup.
4.1.1. 3-Deoxy-3-fluoro-1,2-O-isopropylidene-a-D-xylo-
furanose (4). The diol 326–29 (5.0 g, 22.5 mmol) was
added to a stirred solution of NaIO4 (5.09 g, 23.8 mmol)
in H2O (79 mL) and MeOH (79 mL) leading to immedi-
ate precipitation of NaIO3. After 1 h at room tempera-
ture, any residual periodate was destroyed with a drop
of ethylene glycol. The reaction mixture was stirred for
1 h at room temperature with NaBH4 (2.0 g,
52.3 mmol), neutralized with aqueous NaHCO3, and
then extracted with ethyl acetate (EtOAc) (4· 500 mL).
The organic layer was washed with NaHSO4, dried over
anhydrous sodium sulfate, evaporated to dryness, and
purified by column chromatography with EtOAc/hexane
(2:8) to give compound 4 (4.0 g, 92%, Rf = 0.3 in EtOAc/
hexane, 2:8).
IR (Nujol): 1697 (SAc) cmꢀ1
.
1H NMR (CDCl3): d 5.96 (d, J1,2 = 3.8 Hz, 1H, H-1),
4.88 (dd, J3,F = 50.1 Hz, J3,4 = 2.1 Hz, 1H, H-3), 4.68
(dd, J2,F = 10.8 Hz, J2,1 = 3.8 Hz 1H, H-2), 4,19-4,34
(m, J4,F = 28.4 Hz, J4,3 = 2.1 Hz, 1H, H-4), 3.12–3.26
(m, 2H, H-5a and H-5b), 2.36 (s, 3H, SAc), 1.47 (s,
3H, CH3), 1.31 (s, 3H, CH3).
Found: C, 48.27; H, 5.89; F, 7.74. Calcd for
C10H15FO4S: C, 47.99; H, 6.04; F, 7.59.
ESI-MS m/z (relative intensity, %): 251.4 [(M+H+), 100].
1H NMR (CDCl3): d 5.91 (d, J1,2 = 3.8 Hz, 1 H, H-1),
4.92 (dd, J3,F = 50.4 Hz, J3,4 = 2.3 Hz, 1H, H-3), 4.62
(dd, J2,F = 11.1 Hz, J2,1 = 3.8 Hz, 1 H, H-2), 4.28 (m,
1H, H-4), 3.82 (m, 2H, H-5a and H-5b), 1.41 (s, 3H,
CH3), 1.25 (s, 3H, CH3).
4.1.4.
1,2-Di-O-acetyl-5-S-acetyl-3-deoxy-3-fluoro-5-
thio-D- xylofuranose (7). Compound 6 (4.1 g, 16.5 mmol)
was acetolyzed with 350 mL of a mixture of acetic anhy-
dride–acetic acid–sulfuric acid (70:30:1, v/v). After 3
days, anhydrous ether (600 mL) was added, followed
by sodium acetate (25 g). The mixture was filtered and
the residue was washed with ether (2· 500 mL). The
combined solutions were co-evaporated with toluene
and the residue was purified by flash chromatography
Found: C, 49.67; H, 7.00; F, 9.63. Calcd for C8H13FO4:
C, 50.00; H, 6.82; F, 9.89.
ESI-MS m/z (relative intensity, %): 193.3 [(M+H+), 100].

E. Tsoukala et al. / Bioorg. Med. Chem. 15 (2007) 3241–3247
3245
on a silica gel column using EtOAc/hexane (2:8) as eluent.
Pure compound 7 was collected as a thick syrup (4.0 g,
82%, Rf = 0.3 in EtOAc/hexane, 2:8).
IR (Nujol): 1697 (SAc), 1750 (OAc) cmꢀ1
.
1H NMR (CDCl3): d 7.37 (d, J6 ;5 ¼ 8:2 Hz, 1H, H-60),
0
0
0
0
6.03 (d, J1,2 = 2.0 Hz, 1H, H-1), 5.79 (d, J5 ;6 ¼ 8:2 Hz,
1H, H-50), 5.24 (dd, J2,F = 16.0 Hz, J2,1 = 2.0 Hz, 1H,
H-2), 4.97 (dd, J3,F = 50.0 Hz, J3,4 = 2.2 Hz, 1H, H-3),
4.16-4.27 (m, J4,F = 28.9 Hz, J4,3 = 2.2 Hz, 1H, H-4),
3.24-3.36 (m, 2H, 1H, H-5a and 1H, H-5b), 2.40 (s,
3H, SAc), 2.15 (s, 3H, OAc).
IR (Nujol): 1697 (SAc), 1750 (OAc) cmꢀ1.
1H NMR (CDCl3): d 6.45 and 6.11 (a: d, J1,2 = 4.8 Hz,
0.3H, H-1; b: s, J1,2 = 0.7 Hz, 0.7H, H-1), 5.29–5.33
(m, 1H, H-2), 4.97 (dd, J3,F = 50.3 Hz, J3,4 = 3.7 Hz
1H, H-3), 4.31–4.48 (m, J4,F = 25.8 Hz, J4,3 = 3.7 Hz,
1H, H-4), 3.14–3.34 (m, 2H, H-5a and H-5b), 2.36 and
2.37 (b and a, s, 3H, SAc), 2.06–2.16 (m, 6H, OAc).
Found: C, 45.33; H, 4.63; F, 5.26; N, 7.82. Calcd for
C13H15FN2O6S: C, 45.08; H, 4.37; F, 5.49; N, 8.09.
Found: C, 45.08; H, 5.29; F, 6.31. Calcd for
C11H15FO6S: C, 44.89; H, 5.14; F, 6.46.
ESI-MS m/z (relative intensity, %): 347.5 [(M+H+), 100].
4.1.7. 1-(5-S-Acetyl-3-deoxy-3-fluoro-5-thio-b-D- xylofur-
anosyl) 5-fluorouracil (10). A mixture of 5FU (2.5 g,
18.9 mmol), hexamethyldisilazane (5.0 mL, 23.4 mmol),
and saccharine (0.159 g, 0.87 mmol) in anhydrous
CH3CN (59 mL) was refluxed at 120 ꢁC for 20 min.
After cooling to rt, diacetylated compound 7 (4.0 g,
13.5 mmol) was added, followed by trimethylsilyl
trifluoromethane-sulfonate (3.4 mL, 19 mmol). The
reaction mixture was stirred at rt for 6 h, then diluted
with EtOAc (1000 mL), washed with aqueous
NaHCO3 and finally with water. The organic
layer was dried over sodium sulfate, filtered, and
evaporated to syrup. The resulting material was purified
by column chromatography using EtOAc/hexane
(5:5) to give pure 10 (2.7 g, 55%, Rf = 0.3 in EtOAc/
hexane, 5:5).
ESI-MS m/z (relative intensity, %): 295.4 [(M+H+), 100].
4.1.5. 1-(5-S-Acetyl-3-deoxy-3-fluoro-5-thio-b-D- xylofur-
anosyl) thymine (8). A mixture of thymine (2.4 g,
18.9 mmol), hexamethyldisilazane (4.9 mL, 23.3 mmol),
and saccharine (0.115 g, 0.63 mmol) in anhydrous
CH3CN (65 mL) was refluxed at 120 ꢁC for 20 min.
To this were added diacetylated 3-deoxy-3-fluoro-5-
S-acetyl-5-thio-D-xylofuranose
7 (4.0 g, 13.5 mmol)
and trimethylsilyl trifluoromethane-sulfonate (3.4 mL,
19 mmol). The reaction mixture was refluxed at 80 ꢁC
for 2 h. The mixture was neutralized with aqueous
NaHCO3, then diluted with water and extracted with
EtOAc (1000 mL). The extract was dried over sodium
sulfate, filtered, and evaporated to a syrup, which was
purified by column chromatography using EtOAc/
hexane (8:2) to afford the title compound (2.7 g, 56%,
Rf = 0.35 in EtOAc/hexane, 8:2).
22
½aꢁ þ 36:8 (c 0.25, CHCl3)
D
IR (Nujol): 1697 (SAc), 1750 (OAc) cmꢀ1
.
22
½aꢁ þ 28:9 (c 0.25, CHCl3)
D
1H NMR (CDCl3): d 7.74 (d, J6,F5 = 5.8 Hz, 1H, H-60),
6.01 (dd, J1,2 = 1.9 Hz, J1,F5 = 1.7 Hz, 1H, H-1), 5.23
(dd, J2,F = 16.0 Hz, J2,1 = 1.9 Hz, 1H, H-2), 4.98 (dd,
J3,F = 50.0 Hz, J3,4 = 2.2 Hz, 1H, H-3), 4.16–4.28 (m,
J4,F = 28.8 Hz, J4,3 = 2.2 Hz, 1H, H-4), 3.25–3.37 (m,
2H, 1H, H-5a and 1H, H-5b), 2.40 (s, 3H, SAc), 2.15
(s, 3H, OAc).
IR (Nujol): 1697 (SAc), 1750 (OAc) cmꢀ1
.
1H NMR (CDCl3): d 8.67 (br s, NH), 7.26 (s, 1H, H-60),
6.06 (d, J1,2 = 2.2 Hz, 1H, H-1), 5.23 (d, J2,F = 16.9 Hz,
J2,1 = 2.2 Hz, 1H, H-2), 4.99 (dd, J3,F = 50.2 Hz,
J3,4 = 2.3 Hz, 1H, H-3), 4.11–4.26 (m, J4,F = 28.7 Hz,
J4,3 = 2.3 Hz, 1H, H-4), 3.29–3.33 (m, 2H, H-5a and
H-5b), 2.40 (s, 3H, SAc), 2.14 (s, 3H, OAc), 1.94 (s,
3H, 50-CH3).
Found: C, 43.11; H, 3.63; F, 10.66; N, 7.42. Calcd for
C13H14F2N2O6S: C, 42.86; H, 3.87; F,10.43; N, 7.69.
Found: C, 46.91; H, 5.13; F, 5.46; N, 7.52. Calcd for
C14H17FN2O6S: C, 46.66; H, 4.75; F, 5.27; N, 7.77.
ESI-MS m/z (relative intensity, %): 365.5 [(M+H+), 100].
4.2. Methods for measurement of biological activity
ESI-MS m/z (relative intensity, %): 361.4 [(M+H+), 100].
4.2.1. Cells and culture conditions. The human colonic
adenocarcinoma Caco-2 cells were a generous gift of
dr. Rene L’Harridon, INRA, VIM, Jouy-en-Josas,
France; human fetal small intestine cell line H4, breast
carcinoma cell line MCF 7, and skin melanoma cell line
were used. Cells were grown in Dulbecco’s modified Ea-
gle’s medium (DMEM, Sigma–Aldrich, Grand Island,
USA), supplemented with 5% fetal calf serum (Camb-
rex, Verviers, Belgium), L-glutamine (2 mmol/L, Sigma,
St. Louis, USA), penicillin (100 Us/mL, Sigma, St.
Louis, USA), and streptomycin (1 mg/mL, Fluka, Buchs,
Switzerland) at 37 ꢁC in 5% CO2 atmosphere in tissue
4.1.6. 1-(5-S-Acetyl-3-deoxy-3-fluoro-5-thio-b-D- xylofur-
anosyl) uracil (9). Compound 9 was obtained from
the 1,2-Di-O-acetyl-5-S-acetyl-3-deoxy-3-fluoro-5-thio-
D- xylofuranose (7) (4.0 g, 13.5 mmol), according to
the general procedure as described for 1-(5-S-acetyl-3-
deoxy-3-fluoro-5-thio-b-D-xylofuranosyl) thymine (8).
After purification on silica gel column using EtOAc/hex-
ane (8:2), the title nucleoside 9 was obtained (2.4 g, 51%,
Rf = 0.3 in EtOAc/hexane, 8:2).
22
½aꢁ þ 27:6 (c 0.25, CHCl3)
D

3246
E. Tsoukala et al. / Bioorg. Med. Chem. 15 (2007) 3241–3247
culture flasks until confluent. Cell culture medium was
regularly changed.
ratios (%, SD) of absorbances in virus-infected wells
(n = 6) in comparison to those in control (only virus-in-
fected) wells (n = 6). The minimal inhibitory concentra-
tion (IC50) of the tested compounds was obtained from
the concentration–effect curve.
4.2.2. Fluoro-thiofuranosyl nucleosides. Stock drug solu-
tions were freshly prepared in sterile dimethylsulfoxide
(DMSO) at the concentration of 0.5 mg/mL. The final
concentration of DMSO in the cell culture medium
was less than 0.1%. All solutions were protected against
light.
4.2.5. Growth inhibition assay. It was performed on
Caco-2 cell line by modified method described previ-
ously.39 Briefly, in 96-well plates, six wells of 3-fold dilu-
tions of compound (initial concentration of 0.5 mg/mL)
were applied to monolayers of 10 cells/well in DMEM/
10% fetal bovine serum. Incubation was performed at
37 ꢁC in the humidified incubator for 10 days. The col-
onies were counted in each well and the results were ex-
pressed, for each dilution, by the mean ratios (%, SD)
of colony number in treated wells (n = 2) in contrast
with those in control wells (n = 24). The minimal inhib-
itory concentration (IC50) of the tested compounds was
obtained from the concentration–effect curve.
AZT (Retrovirꢂ) GlaxoSmithKline,USA, a drug used
for antiretroviral therapy (ART) was used as a standard
compound in antiviral experiments and 5FU as a stan-
dard compound in antitumor experiments. Solutions
were prepared in the same way as those of fluoro-thio-
furanosyl nucleosides.
4.2.3. Virus propagation. Rotavirus RF strain was prop-
agated on Caco-2 monolayers in the presence of trypsin
(1 lg per mL of DMEM) as described previously.38
Supernatant containing the virus was collected from
the flasks when cytopathic effect (CPE) was observed
(24–48 h at 37 ꢁC) by microscopy and clarified by centri-
fugation. Virus was stored at ꢀ70 ꢁC until used. For the
antiviral assay, virus with 1.5 tissue culture infective
dose 50% units per mL (TCID50/mL) was used
(100 lL per well).
4.2.6. Cytotoxicity assay. Caco-2, H4, MCF 7, and skin
melanoma cells (6· 106 cells per plate) were seeded in P
96 plate and treated with the compounds at 3-fold serial
dilutions of each compound (initial concentration of
0.5 mg/mL). Then, the cells were incubated at 37 ꢁC in
the humidified incubator for 72 h. The plates were
stained with Crystal Violet in ethanol, rinsed with water,
and destained with 10% (v/v) acetic acid. The A590 was
measured, and the results were expressed, for each dilu-
tion, by the mean ratios (%, SD) of absorbances in
treated wells (n = 2) compared to those in control wells
(n = 24). The minimal inhibitory concentration (CC50)
of the tested compounds was obtained from the concen-
tration–effect curve.
4.2.4. Antiviral assay. The potential antiviral activity of
the newly synthesized compounds was tested against
rotavirus by investigating:
(a) The inhibition of infectivity following virus attach-
ment: Washed monolayer Caco-2 cells were first incu-
bated with rotavirus for 1 h at 37 ꢁC in the presence of
5% CO2 (time for virus to attach to cell membrane
receptors). After incubation, the remaining virus was
washed off with DMEM without supplements and
monolayer was treated immediately with the nucleoside
added in 3-fold serial dilutions (initial concentration of
0.5 mg/mL). After 72 h of incubation for rotavirus, the
plates were stained with Crystal Violet in ethanol, rinsed
with water, and destained with 10% (v/v) acetic acid.
The A590 was measured, and the results were expressed,
for each dilution, by the mean ratios (%, SD) of absor-
bances in virus-infected wells (n = 6) compared to those
in control (only virus-infected) wells (n = 6). The mini-
mal inhibitory concentration (IC50) of the tested com-
pounds was obtained from the concentration–effect
curve.
Acknowledgments
The authors are grateful to Dr. K. Antonakis (Ec. Nat.
Sup. de Chimie de Paris; CNRS-FRE 2463) for encour-
aging this work, to Dr. T. Halmos (Ec. Nat. Sup. de
Chimie de Paris) and to Associate Professor K. Litinas
(Chem. Dpt. Arist. Univ. of Thessaloniki) for their un-
stinted and fruitful aid, as well as ELPEN pharmaceuti-
cals for financial support.
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(b) The neutralization of the virus in solution before
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(initial concentration of 0.5 mg/mL) were first pre-incu-
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sin for 12 h prior to the infection of cell monolayer at
37 ꢁC and 5% CO2. Residual viral infectivity was mea-
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alone was treated in the same way as the control. After
72-h of incubation, the plates were stained with Crystal
Violet in ethanol, rinsed with water, and destained with
10% (v/v) acetic acid. The A590 was measured, and the
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