Fluoro-ketopyranosyl nucleosides: Synthesis and biological evaluation of 3-fluoro-2-keto-β-d-glucopyranosyl derivatives of N4-benzoyl cytosine

Article abstract of DOI:10.1016/j.bmc.2006.10.033

1,2:5,6-Di-O-isopropylidene-α-d-glucofuranose on mild oxidation, reduction, fluorination, and deisopropylidenation followed by acetylation gave peracetylated 3-deoxy-3-fluoro-d-glucopyranose. This was coupled with silylated N⁴-benzoyl cytosine.

Full text of DOI:10.1016/j.bmc.2006.10.033

Bioorganic & Medicinal Chemistry 15 (2007) 980–987
Fluoro-ketopyranosyl nucleosides: Synthesis and biological
evaluation of 3-fluoro-2-keto-b-D-glucopyranosyl
derivatives of N4-benzoyl cytosine
a
a
b
Stella Manta, George Agelis, Tanja Botic, Avrelija Cencic and Dimitri Komiotisa,
ˇb,c
*
´
aDepartment of Biochemistry and Biotechnology, Laboratory of Organic Chemistry, University of Thessaly, 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 20 April 2006; revised 9 October 2006; accepted 17 October 2006
Available online 19 October 2006
Abstract—1,2:5,6-Di-O-isopropylidene-a-D-glucofuranose on mild oxidation, reduction, fluorination, and deisopropylidenation fol-
lowed by acetylation gave peracetylated 3-deoxy-3-fluoro-D-glucopyranose. This was coupled with silylated N4-benzoyl cytosine.
The nucleoside was deacetylated and after several subsequent protection and deprotection steps afforded the desired 3-fluoro-2-ke-
to-b-D-glucopyranosyl derivatives. These novel synthesized compounds were evaluated for antiviral and cytotoxic activities against
rotavirus, vesicular stomatitis virus, and the human colon adenocarcinoma cell line Caco-2, and have a promising potential in com-
bating the rotaviral infections and in the treatment of colon cancer. As compared to AZT, a nucleoside analogue of reverse trans-
criptase inhibitor, the novel synthesized 1-(3,4-dideoxy-3-fluoro-b-D-glycero-hex-3-enopyranosyl-2-ulose)-N4-benzoyl cytosine
showed to be more effective at lower concentrations in inhibition of rotavirus infection as well as in the same range of antitumor
activity.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
In recent years a number of five-membered cytosine
nucleoside analogues, such as 1-(2-deoxy-2-methylene-
b-D-erythro-pentofuranosyl) cytosine (DMDC),12
20-deoxy-20,20-difluorocytidine (gemcitabine),13 and
1-(2-C-cyano-2-deoxy-b-D-arabino-pentofuranosyl) cy-
tosine (CNDAC),14 have been developed as potent anti-
tumor agents, which are effective not only on leukemias
and lymphomas, but also on a wide variety of solid tu-
mors in vitro as well as in vivo. Moreover, cytosine
unsaturated nucleoside analogues, such as D-20,30-dide-
hydro-20,30-dideoxy-5-fluorocytidine (D-d4FC)15 and
its L-enantiomer (L-Fd4C),16 have been identified as
antiHIV agents. Structure–activity relationships of L-
30-fluoro-20,30-unsaturated nucleosides showed potent
activity in the cytosine and 5-fluorocytosine derivatives
(EC50 = 0.089 and 0.018 lM, respectively).17
Nucleoside analogues display a wide range of biological
activities as antitumor, antiviral, and chemotherapeutic
agents.1–4 A number of fluorine-substituted nucleoside
analogues have demonstrated their potent antiviral
activity as well as favorable chemical and pharmacolog-
ical properties by virtue of the small size and highly elec-
tronegative nature of the fluorine atom, which is also
capable of participating in hydrogen bonding.5–7 It ap-
pears that the high strength of the C–F bond may hinder
metabolism pathways and may increase the effective life-
time of the active molecule. Furthermore, introduction
of a fluorine atom in the sugar moiety raises the lipophil-
icity and makes the penetration of the drug through the
cell membrane easier.8–11
The last decades, nucleosides with a six-membered car-
bohydrate moiety have been evaluated for their potential
antiviral18–21 and antibiotic22 properties and as building
blocks in nucleic acid synthesis.23,24 Among them, the
unsaturated ketonucleosides are well-established for
their antineoplastic activity and immunosuppressive
Keywords: Unsaturated fluoro ketonucleosides; b-elimination reaction;
Gem-diol; Antiviral; Antitumor activity.
*
Corresponding author. Tel.: +30 2410 565285; fax: +30 2410
565290; e-mail: dkom@bio.uth.gr
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.033

S. Manta et al. / Bioorg. Med. Chem. 15 (2007) 980–987
981
effects.25–27 It appeared that these nucleosides not only
exhibit growth inhibitory activity against a variety of tu-
mor cells28,29 in vitro and L1210 leukemia30,31 in vivo,
but they also may constitute important synthetic inter-
mediates in the nucleoside field owing to their chemical
reactivity in various media.32,33 Furthermore, fluorine
introduction in 30-position of an unsaturated 40-ketonu-
cleoside leads to an important difference in the toxicity
toward neoplastic and normal cells.34
without purification and then treated with acetic anhy-
dride–pyridine.46
Finally, deacetylation of 9, whichis very unstable under ba-
sic conditions, was performed with methanolic hydrogen
chloride47 to afford 1-(3,4-dideoxy-3-fluoro-b-D-glycero-
hex-3-enopyranosyl-2-ulose)-N4-benzoyl cytosine (10).
2.2. Biological activity
The above observations and the continuous demand for
new antiviral and anticancer agents prompted us to
design and synthesize a new class of unsaturated fluo-
ro-ketonucleosides, that of N4-benzoyl cytosine. This
conjugation reported for the first time appeared to us
as a challenge and the first biological results confirm
our hypothesis.
To investigate the biological activity of ketonucleosides,
we used in vitro tests mimicking the intestinal tract. To
examine the potential antiviral properties of the ketonu-
cleosides, two cell models were used. First one, a small
intestinal pig IPEC-J2 cell line infected with VSV and
second one, embryonic monkey kidney MA 104 cell line
and gastrointestinal rotavirus were used as model virus.
Three different approaches were followed to test the pos-
sible inhibitory activity on VSV and rotavirus infectivity
as described in experimental part. Experiments demon-
strated that all compounds may have the antiviral activ-
ity, but the effect is virus selective. While no significant
protection was observed in the case of VSV for com-
pounds 8 and 9 when compounds were added before,
during or after infection of IPEC-J2 cells, compound
10 was able partially to inhibit virus infectivity following
virus attachment at the concentration of 7 lg/mL
(Fig. 1). It could well be that more concentrated com-
pounds would elicit the inhibitory activity but the con-
centrations over the 0.02 mg/mL caused cytotoxicity of
the IPEC-J2 cell line. Those findings are in accordance
with previously published results showing that nitro-de-
rivatives but not amino-derivatives of ketonucleosides
inhibit replication of murine leukemia virus but at cyto-
toxic doses.48,49 In spite of the fact that higher concen-
trations of all tested compounds were toxic to the MA
104 cells, the concentrations below 0.02 mg/mL inhibit-
ed the rotavirus infectivity (Table 1 and Fig. 2).
Although the compound 8 was less toxic to the MA
104 cells even at higher concentrations (Table 1 and
Fig. 2), the IC50 value was 0.07 mg/mL. This value
was similar to the one of AZT, which inhibited rotavirus
infection of the cells at the concentration of 0.06 mg/mL
(Table 1). This was significantly higher than for the com-
pounds 9 and 10 against rotavirus infection either in
inhibiting infectivity following virus attachment or neu-
tralizing the virus in solution before its attachment to
the cell surface (Table 1 and Fig. 2). The best IC50 value
was obtained for the compounds 9 and 10 (0.02 mg/mL)
in inhibiting infectivity following virus attachment by
60% and 75%, respectively (Fig. 2). On the contrary,
only the compound 9 did not elicit inhibitory activity
in neutralization of the virus in solution before the rota-
virus attachment (Table 1). None of the tested com-
pounds was able to trigger the antiviral state in the
IPEC-J2 and MA 104 cells significantly, whereas AZT
showed positive results at the concentrations as low as
0.004 mg/mL (Fig. 3 and Table 1). Interestingly, all
three compounds analyzed, at the concentration of
30 ng/mL, stimulated the proliferation of MA 104 cells
increasing the cell number for 40% (compound 8), 55%
(compound 9), and 70% (compound 10) as compared
to the control, non-treated cells (Fig. 4), respectively.
2. Results and discussion
2.1. Synthesis
Commercially available 1,2:5,6-di-O-isopropylidene-a-
D-glucofuranose (1) was readily transformed in five steps
into the corresponding 3-deoxy-3-fluoro-1,2,4,6-tetra-O-
acetyl-glucopyranose (2)35,36 (Scheme 1). Condensation
of 2 with silylated N4-benzoyl cytosine in presence of
trimethylsilyl trifluomethane-sulfonate yielded the
desired b-protected nucleoside 3. Selective deprotection
of 3 using sodium hydroxide (NaOH)/ethanol (EtOH)/
pyridine37 gave benzoylated derivative 4 in excellent
yield. However, when compound 3 was treated by
methanolic ammonia,38 or potassium carbonate
(K2CO3)/methanol (MeOH),39 or guanidine,40 the fully
unprotected nucleoside was obtained. Treatment of 4
with 2,2-dimethoxypropane in dry N,N-dimethylform-
amide (DMF) gave the 40,60-isopropylidene derivative
5 as a bright yellow oil.
The oxidation of nucleoside 5 was a crucial step because
of the instability of the molecule possessing the keto–
fluoro system. The presence of an electron-withdrawing
fluorine atom a to the carbonyl causes its easy hydra-
tion and gem-diol formation.34 Different oxidation
methods have been studied. The use of pyridinium
dichromate (PDC)/3E molecular sieves,41 dimethylsulf-
oxide (DMSO)/acetic anhydride,42 and DMSO/oxalyl
chloride43 gave invariably a mixture of the desired 20-
ketonucleoside,
1-(3-deoxy-3-fluoro-4,6-O-isopropyl-
idene-b-D-glucopyranosyl-2-ulose)-N4-benzoyl cytosine
(6) and its gem-diol 7 in a 7:3 ratio.44,45 The two deriv-
atives were separated in low yields for characterization
of compound 6 by NMR spectroscopy and chemical
analysis. Deisopropylidenation of 6 with trifluoroacetic
acid afforded the unprotected ketonucleoside 8, which,
upon treatment with acetic anhydride–pyridine gave
the unsaturated ketonucleoside 9 after a b-elimination
reaction.
However, compound 9 was obtained in a 66% overall
yield when 5 was oxidized with PDC–acetic anhydride
and the crude reaction mixture was deisopropylidinated

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S. Manta et al. / Bioorg. Med. Chem. 15 (2007) 980–987
H3C
H3C
O
O
AcO
AcO
CyBz
O
O
O
a
F
OAc
F
OH
OAc
O
OAc
CH3
CH3
OAc
OAc
O
1
2
d
3
b
O
O
O
HO
CyBz
OH
H3C
H3C
O
CyBz
O
CyBz
O
H3C
H3C
H3C
H3C
O
CyBz
O
c
F
F
F
F
O
O
O
OH
OH
OH
OH
7
6
4
5
d, e, f
e
HO
HO
AcO
f
g
CyBz
O
CyBz
O
O
O
CyBz
O
F
OH
F
O
F
8
9
10
Scheme 1. Reagents and conditions: (a) N4-benzoyl cytosine, trimethylsilyl trifluoromethane-sulfonate, HMDS, saccharine, CH3CN, 120 ꢁC, 5 h,
reflux; (b) EtOH, pyridine, 0 ꢁC, 30 min, Amberlite IR-120 (H+) resin; (c) 2,2-dimethoxypropane, p-toluenesulfonic acid, DMF, 60 ꢁC, 1 h; (d) PDC,
acetic anhydride, CH2Cl2, 20 ꢁC, 3 h; (e) 90% trifluoroacetic acid in MeOH, 20 ꢁC, 10 min; (f) pyridine–acetic anhydride, 0 ꢁC, 1 h; (g) dry methanolic
hydrogen chloride, CH2Cl2, 20 ꢁC, 24 h.
Furthermore, we have examined the antiproliferative
and cytotoxic potency of ketonucleosides, using human
100
colon adenocarcinoma cell line Caco-2. All compounds
analyzed, diminished the growth of Caco-2 cells as
90
determined by colony numbers after 10 days of incuba-
tion (Table 2) in a concentration-dependent manner
with the IC50 value being 2.5 mg/mL for the compound
8, 1.0 mg/mL for the compound 9, and 0.2 mg/mL for
the compound 10, respectively. As compared to AZT,
compounds 9 and 10 showed similar antiproliferative
activity to AZT, while compound 8 was active only at
significantly higher concentrations (2.5 mg/mL) (Table
2). Moreover, the compounds 9 and 10 showed the high-
est cytotoxic potency with the IC50 value being 0.2 mg/
mL equally, whereas significantly higher concentration
(1.5 mg/mL) was needed for the compound 8 to elicit
the cytotoxic effect on Caco-2 cells after 72 h of co-incu-
bation (Table 2). Our results confirmed previously pub-
lished data where it was reported that keto-C-glycosides
and methylene nucleoside analogues inhibit the growth
of some tumor cell lines.49,25
80
70
60
50
40
30
20
10
0
4
5
6
7
8
9
dilution (log3)
Figure 1. Compound 10 inhibition of the VSV infectivity following
virus attachment to IPEC-J2 cells. Cell monolayer was first co-
incubated with VSV for 1 h, after the remaining virus was washed off
the monolayers, threefold serial dilutions of the compound 10 were
performed in culture medium without serum. Following 24 h incuba-
tion at 37 ꢁC in a humidified atmosphere of 5% CO2, the plates were
stained with Crystal violet in EtOH, rinsed with water, and destained
with 10% (v/v) acetic acid. The A590 was measured, and the results were
expressed, for each dilution, by mean ratios (%, SD) of absorbances
in treated wells (n = 6) to those in control wells (n = 6).
3. Conclusion
In conclusion, fluoro-ketopyranosyl nucleosides, in par-
ticular compound 10, are potential antiadenocarcinoma
agents and moreover, compound 10 is a promising can-

S. Manta et al. / Bioorg. Med. Chem. 15 (2007) 980–987
983
Table 1. IC50 (mg/mL) of compounds 8, 9, 10, and AZT for the antiviral activity against rotavirus RF strain
IC50 (mg/mL)
Compound
8
9
10
AZT
Antiviral state
Neutralization of the virus in the solution before its attachment
Inhibition of infectivity following virus attachment
0
0
0
0.004
0.06
0.015
0.07
0.07
0.02
0
0.02
0.0008
3,5
3,0
2,5
2,0
,5
,0
0,5
0,0
2
3
4
5
6
7
8
dilution (log 3)
compound 8 compound 9 compound
0
cells rotavirus
Figure 2. Inhibition of the rotavirus infectivity following virus attachment to MA 104 cells. Cell monolayer was first co-incubated with rotavirus RF
strain for 1 h, after, the remaining virus was washed off the monolayers, threefold serial dilutions of each compound were performed in culture
medium without serum. Following further 72 h incubation at 37 ꢁC in a humidified atmosphere of 5% CO2, the plates were stained with Crystal violet
in EtOH, rinsed with water, and destained with 10% (v/v) acetic acid. The A590 was measured, and the results were expressed, for each dilution, by
mean ratios (%, SD) of absorbances in treated wells (n = 6) to those in control wells (n = 6).
3,5
3,0
2,5
2,0
1,5
1,0
0,5
0,0
1
2
3
4
5
6
7
8
dilution (log 3)
compound 8 compound 9 compound 10 cells rotavirus
Figure 3. Ability of antiviral state induction in rotavirus-infected MA 104 cells. Washed monolayer of MA104 cells was first incubated with threefold
serial dilutions of the compounds for 10 h at 37 ꢁC in the atmosphere of 5% CO2. After incubation the compounds were washed off with DMEM
without supplements and monolayer was challenged immediately with rotavirus (100 lL per well). After 72 h of incubation for rotavirus, the plates
were stained with Crystal violet in EtOH, rinsed with water, and destained with 10% (v/v) acetic acid. The A590 was measured, and the results were
expressed, for each dilution, by mean ratios (%, SD) of absorbances in virus-infected wells (n = 6) to those in control (only virus-infected) wells
(n = 6).
didate in development of antirotavirus drugs as (i) inhib-
itor of infectivity following virus attachment and (ii)
neutralizing agent of the virus in solution before attach-
ment to the cell surface. Moreover, as compared to
AZT, a nucleoside analogue of reverse transcriptase
inhibitor,
1-(3,4-dideoxy-3-fluoro-b-D-glycero-hex-3-

984
S. Manta et al. / Bioorg. Med. Chem. 15 (2007) 980–987
180,0
160,0
140,0
120,0
100,0
80,0
60,0
40,0
20,0
0,0
1
2 3 4 5 6 7 8
1
2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
dilutions (log 3)
Figure 4. Cytotoxicity of compound 8 (black box), compound 9 (dashed box), and compound 10 (gray box) to MA 104 cells. Each of the compounds
at the initial concentration of 0.2 mg/mL was serially diluted in cell culture medium and left on MA104 cell monolayer seeded in 96-well plates.
Following 72 h incubation at 37 ꢁC in a humidified atmosphere of 5% CO2, the plates were stained with Crystal violet in EtOH, rinsed with water,
and destained with 10% (v/v) acetic acid. The A590 was measured, and the results were expressed, for each dilution, by mean ratios (%, SD) of
absorbances in treated wells (n = 4) to those in control wells (n = 8).
Table 2. IC50 (mg/mL) of compounds 8, 9, 10, and AZT for growth inhibition and cytotoxic activity on Caco-2 cells
IC50 (mg/mL)
Compound
8
9
10
AZT
Growth inhibition
Cytotoxicity
2.5
1.5
1.0
0.2
0.2
0.2
0.015
0.015
enopyranosyl-2-ulose)-N4-benzoyl cytosine (10), showed
to be more effective at lower concentrations in inhibition
of rotavirus infection as well as in the same range of
antitumor activity.
and trimethylsilyl trifluomethane-sulfonate (3.6 mL,
19.98 mmol). The reaction mixture was refluxed at
120 ꢁC for 5 h, then cooled, neutralized with aqueous
sodium bicarbonate (NaHCO3), and extracted with
CH2Cl2 (1000 mL). The organic layer was washed with
water (1· 20 mL) and dried over anhydrous sodium sul-
fate, evaporated to dryness, and finally purified by flash
chromatography (hexane/ethyl acetate (EtOAc), 2:8) to
give compound 3 (4.90 g, 68%, Rf = 0.35) as a solid.
Mp 277 ꢁC.
4. Experimental
4.1. General procedure
Melting points were recorded in a Mel-Temp apparatus
and are uncorrected. Thin-layer chromatography (TLC)
was performed on Silica gel (240–400 mesh, Merck).
NMR spectra were recorded at room temperature with
a Brucker 250 MHz spectrometer in chloroform-d
(CDCl3) with internal tetramethylsilane (TMS). All
reactions were carried out in dry solvents. Dichloro-
methane (CH2Cl2) was distilled from phosphorus pent-
oxide (P2O5) and stored over 4E molecular sieves.
Acetonitrile (CH3CN) was distilled from calcium hy-
dride (CaH2) and stored over 3E molecular sieves.
DMF was also stored over 3E molecular sieves. Pyridine
was stored over pellets of potassium hydroxide (KOH).
1HNMR (CDCl3): d 7.88 (d, 1H, J6,5 = 7.2 Hz, H-6),
0
0
7.84–7.48 (m, 6H, Bz and H-5), 6.10 (d, 1H, J1 ;2 ¼ 9:4
Hz, H-10), 5.39–5.22 (m, 2H, J2 ;1 ¼ 9:4 Hz, J4 ;3
¼
0
0
0
0
9:0 Hz, H-20 and H-40), 4.8 (dtr, 1H, JF;3 ¼ 51:7 Hz,
0
J3 ;2 ¼ 9:1 Hz, J3 ;4 ¼ 9:0 Hz, H-30), 4.34–4.11 (m, 2H,
H-6a0,6b0), 3.86 (m, 1H, H-50), 2.16–2.11–2.07 (3s, 9H,
3OAc).
0
0
0
0
Found: C, 54.70; H, 4.80; F, 3.75; N, 8.42. Calcd for
C23H24FN3O9: C, 54.65; H, 4.79; F, 3.76; N, 8.31.
4.1.2. 1-(3,4-Dideoxy-3-fluoro-b-D-glucopyranosyl)-N4-
benzoyl cytosine (4). Compound 3 (4.90 g, 9.7 mmol)
was dissolved in EtOH–pyridine (97 + 29.1 mL), 2 M
NaOH (19.4 mL) was added, and the mixture stirred
for 30 min at 0 ꢁC. Amberlite IR-120 (H+) was added
to neutralize the base. The suspension was filtered, the
resin was washed with EtOH and pyridine (100 +
100 mL), and the filtrate was evaporated. The solid res-
idue was triturated with diethyl ether (2· 30 mL) and
4.1.1. 1-(3-Deoxy-3-fluoro-2,4,6-tri-O-acetyl-b-D-gluco-
pyranosyl)-N4-benzoyl cytosine (3). A mixture of N4-ben-
zoyl cytosine (3.99 g, 18.55 mmol), hexamethyldisilazane
(HMDS) (4.8 mL, 23 mmol), and saccharine (0.16 g,
0.85 mmol) in anhydrous CH3CN (69 mL) was refluxed
for 30 min at 120 ꢁC. To this were added tetraacetylated
3-deoxy-3-fluoro-D-glucose35,36 (2) (5.00 g, 14.27 mmol)

S. Manta et al. / Bioorg. Med. Chem. 15 (2007) 980–987
985
CH2Cl2 (2· 30 mL), and filtered. Product 4 (3.31 g, 90%)
was used without further purification.
quench the reaction and the mixture concentrated under
high-vacuum pump to remove the solvents. Purification
by flash chromatography (hexane/EtOAc, 3:7) gave the
title compound 9 (1.85 g, 66% from 5, Rf = 0.27).
4.1.3.
1-(3-Deoxy-3-fluoro-4,6-O-isopropylidene-b-D-
glucopyranosyl)-N4-benzoyl cytosine (5). Compound 4
(3.31 g, 8.73 mmol) was dissolved in a mixture of
34.92 mL of 2,2-dimethoxypropane and 110.4 mL of
dry DMF. To this was added (2.65 g, 13.97 mmol) of p-
toluenesulfonic acid and the mixture stirred at 60 ꢁC for
1 h. The reaction mixture was neutralized with triethyl-
amine so pH did not exceed 7. After that, the mixture
was concentrated under high-vacuum pump to eliminate
the DMF. Purification with flash chromatography (hex-
ane/EtOAc, 1:9), gave 5 (2.93 g, 80%, Rf = 0.3) as a bright
yellow oil.
1HNMR (CDCl3): d 7.89 (d, 1H, J6,5 = 7.5 Hz, H-6)
7.58–7.43 (m, 6H, Bz and H-5), 6.56 (dd, 1H,
JF;4 ¼ 11:0 Hz, J4 ;5 ¼ 1:5 Hz, H-40), 5.88 (s, 1H, H-
10), 5.03 (m, 1H, H-50), 4.35–4.2 (m, 2H, H-6a0,6b0),
2.05 (s, 3H, OAc).
0
0
0
Found: C, 57.03; H, 4.00; F, 4.75; N, 10.45. Calcd for
C19H16FN3O6: C, 56.86; H, 4.02; F, 4.73; N, 10.47.
4.1.6. 1-(3,4-Dideoxy-3-fluoro-b-D-glycero-hex-3-enopyr-
anosyl-2-ulose)-N4-benzoyl cytosine (10). To a solution
of 9 (1.85 g, 4.61 mmol) in dry CH2Cl2 (9.2 mL) was
added, at room temperature, 9.2 mL of methanolic
hydrogen chloride prepared by addition at 0 ꢁC, of ace-
tyl chloride (0.4 mL) to dry MeOH (10 mL). After 24 h,
reaction was complete, and the mixture was diluted with
CH2Cl2 and treated with an excess of NaHCO3. After
washing twice with water, drying over anhydrous mag-
nesium sulfate (MgSO4) and concentration, the crude
mixture was purified by flash chromatography (EtOAc)
to give 10 (0.83 g, 50%, Rf = 0.29).
1HNMR (CDCl3): d 7.88 (d, 1H, J6,5 = 7.9 Hz, H-6),
0
0
7.84–7.43 (m, 6H, Bz and H-5), 5.86 (d, 1H, J1 ;2
¼
9:2 Hz, H-10), 4.68 (dtr, 1H, JF;3 ¼ 53:2 Hz, J3 ;2
¼
0
0
0
8:7 Hz, J3 ;4 ¼ 8:7 Hz, H-30), 4.05–3.87 (m, 2H,
0
0
J2 ;1 ¼ 9:2 Hz, J4 ;3 ¼ 8:7 Hz, H-20 and H-40), 3.86–
3.73 (m, 2H, H-6a0,6b0), 3.51 (m, 1H, H-50), 1.53–1.45
(2s, 6H, 2·CH3).
0
0
0
0
Found: C, 57.16; H, 5.31; F, 4.24; N, 10.03. Calcd for
C20H22FN3O6: C, 57.28; H, 5.29; F, 4.53; N, 10.02.
4.1.4.
1-(3-Deoxy-3-fluoro-4,6-O-isopropylidene-b-D-
1HNMR (CDCl3): d 7.89 (d, 1H, J6,5 = 7.5 Hz, H-6),
7.59–7.43 (m, 6H, Bz and H-5), 6.61 (dd, 1H,
glucopyranosyl-2-ulose)-N4-benzoyl cytosine (6). A mix-
ture of 5 (2.93 g, 6.98 mmol; dried by co-evaporation
with toluene), PDC (3.15 g, 8.38 mmol), and acetic
anhydride (1.98 mL, 20.94 mmol) was stirred in dry
CH2Cl2 (58.1 mL) for 3 h. After cooling, EtOAc
(10 mL) was added and the resulting slurry was trans-
ferred on the top of a silica-gel column packed in
EtOAc. The solution was filtered through the column
and washed with EtOAc (92 mL) until the product was
eluted completely. The solvent was evaporated and the
residue was rendered free of acetic anhydride and pyri-
dine by co-evaporation with dry toluene (3·) to afford
2.45 g of crude 6. Purification by flash chromatography
(hexane/EtOAc, 2:8) gave pure 6 in 22% yield.
JF;4 ¼ 11:4 Hz, J4 ;5 ¼ 1:1 Hz, H-40), 5.22 (s, 1H, H-
0
0
0
10), 4.89 (m, 1H, H-50), 3.88–3.77 (m, 2H, H-6a0,6b0).
Found: C, 56.72; H, 3.91; F, 5.26; N, 11.73. Calcd for
C17H14FN3O5: C, 56.83; H, 3.93; F, 5.29; N, 11.69.
4.2. Methods for measurement of biological activity
4.2.1. Cells and culture conditions. The IPEC-J2 cell line
(Intestinal pig epithelial-cell jejenum) was a generous gift
of Prof. Anthony Blikslager (North Carolina State Uni-
versity, USA). Human colon adenocarcinoma Caco-2
and embryonic rhesus monkey kidney MA104 cells were
a generous gift of dr. Rene L’Harridon, INRA, VIM,
Jouy-en-Josas, France. Cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM) (Sigma–Aldrich,
Grand Island, USA), supplemented with 5% fetal calf
serum (Cambrex, Verviers, Belgium), L-glutamine
(2 mmol/L, Sigma, St. Louis, USA) penicillin (100 U/
mL, Sigma, St. Louis, USA) and streptomycin (1 mg/
mL, Fluka, Buchs, Switzerland) at 37 ꢁC in 5% carbon
dioxide (CO2) atmosphere in tissue culture flasks until
confluent. Cell culture medium was regularly changed.
1HNMR (CDCl3): d 8.05 (d, 1H, J6,5 = 7.6 Hz, H-6), 8.0–
7.44 (m, 6H, Bz and H-5), 5.92 (1s, 1H, H-10), 4.65 (dd,
1H, J3 ;4 ¼ 9:2 Hz, JF;3 ¼ 51:4 Hz, H-30), 4.13–4.03 (m,
0
0
0
1H, J4 ;3 ¼ 9:2 Hz, H-40), 3.93–3.77 (m, 2H, H-6a0,6b0),
0
0
3.53 (m, 1H, H-50), 1.54–1.44 (2s, 6H, 2·CH3).
Found: C, 57.26; H, 4.33; F, 4.92; N, 10.35. Calcd for
C20H20FN3O6: C, 57.55; H, 4.83; F, 4.55; N, 10.07.
4.1.5.
1-(6-O-Acetyl-3,4-dideoxy-3-fluoro-b-D-glycero-
hex-3-enopyranosyl-2-ulose)-N4-benzoyl cytosine (9).
Crude 6 (2.0 g, 4.79 mmol), obtained from the previous
procedure, was dissolved in 23.95 mL of 90% trifluoro-
acetic acid in MeOH. The solution was stirred for
10 min at room temperature and then concentrated at
40 ꢁC under high-vacuum pump to remove traces of tri-
fluoroacetic acid. Crude 8 (1.79 g, 99%) was dissolved in
dry pyridine (9.5 mL), acetic anhydride (1.79 mL) was
added, and the mixture stirred at 0 ꢁC for 1 h under
strictly dry conditions. MeOH (2.0 mL) was added to
4.2.2. Fluoro-ketopyranosyl nucleosides. Compounds
8–10 were freshly prepared in sterile DMSO at the con-
centration of 5 mg/mL. The final concentration of
DMSO was below 0.1% of cell culture medium. All solu-
tions were protected against light.
AZT (Retrovirꢂ) GlaxoSmithKline, USA, a drug used
for antiretroviral therapy (ART) was used as a standard
compound in biological experiments, prepared in the
same way as fluoro-ketopyranosyl nucleosides.

986
S. Manta et al. / Bioorg. Med. Chem. 15 (2007) 980–987
4.2.3. Virus propagation. Vesicular stomatitis virus
(VSV)—Indiana strain was propagated in IPEC-J2
monolayers. Supernatant containing the virus was col-
lected from the flasks when cytopathic effect (CPE)
was observed (24–48 h at 37 ꢁC) by microscopy and clar-
ified by centrifugation. Virus was stored at ꢀ70 ꢁC until
used. For the antiviral assay, virus with 1.436 tissue cul-
ture infective dose 50% units per mL (TCID50/mL) was
used (100 lL per well). The bovine RF strain was propa-
gated in MA104 cells in the presence of trypsin as
described previously.50 Virus titers present in the clari-
fied lysed-cell supernatants were determined as above.
sured, and the results were expressed, for each dilution,
by the mean ratios (%, SD) of absorbances in virus-in-
fected wells (n = 6) to those in control (only virus infect-
ed) wells (n = 6). The minimal inhibitory concentration
(IC50) of the tested compounds was obtained from the
concentration–effect curve.
4.2.5. Growth inhibition assay. It was performed on
Caco-2 cell line by modified method described previous-
ly.51 Briefly, in 96-well plates, six wells of threefold dilu-
tions of each compound or AZT (initial concentration
5 mg/mL) were applied to monolayers of 10 cells/well
in Dulbecco’s modified Eagle’s medium (DMEM)/10%
fetal bovine serum. Incubation was performed at 37 ꢁC
in the humidified incubator for 10 days. The colonies
were counted in each well and the results were expressed,
for each dilution, by mean ratios (%, SD) of colony
number in treated wells (n = 2) to those in control wells
(n = 24). The minimal inhibitory concentration (IC50) of
the tested compounds was obtained from the concentra-
tion–effect curve.
4.2.4. Antiviral assay. The potential antiviral activity of
the novel synthesized compounds was tested against
rotavirus or VSV by investigating:
(a) The ability of antiviral state induction in the infected
cells: washed monolayer of MA104 or IPEC-J2 cells was
first incubated with threefold serial dilutions of the com-
pounds or AZT for 10 h at 37 ꢁC in the atmosphere of
5% CO2. After incubation the compounds were washed
off with DMEM without supplements and monolayer
was challenged immediately with rotavirus or VSV virus
(100 lL per well). After 24 h post-infection for VSV and
72 h post-infection for rotavirus, the plates were stained
with Crystal violet in EtOH, rinsed with water, and
destained with 10% (v/v) acetic acid. The A590 was mea-
sured, and the results were expressed, for each dilution,
by mean ratios (%, SD) of absorbances in virus-infect-
ed wells (n = 6) to those in control (only virus infected)
wells (n = 6).
4.2.6. Cytotoxicity assay. Caco-2 cells (6 · 106 cells per
plate) were seeded in P 96 plate and treated with the
compounds at threefold serial dilutions of each com-
pound or AZT (initial concentration 5 mg/mL). Then,
the cells were incubated at 37 ꢁC in the humidified incu-
bator for 72 h. The plates were stained with Crystal vio-
let in EtOH, 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 absorbances in treated wells (n = 2) to those
in control wells (n = 24). The minimal inhibitory concen-
tration (IC50) of the tested compounds was obtained
from the concentration–effect curve.
(b) The inhibition of infectivity following virus attach-
ment: washed monolayer of MA104 or IPEC-J2 cells
was first incubated with rotavirus or VSV for 1 h at
37 ꢁC in the atmosphere of 5% CO2 (time for virus to at-
tach to cell membrane receptors). After incubation the
remaining virus was washed off with DMEM without
supplements and monolayer was treated immediately
with the F-ketonucleosides or AZT, added in threefold
serial dilutions. After 24 h post-infection for VSV and
72 h post-infection for rotavirus, the plates were stained
with Crystal violet in EtOH, rinsed with water, and
destained with 10% (v/v) acetic acid. The A590 was mea-
sured, and the results were expressed, for each dilution,
by the mean ratios (%, SD) of absorbances in virus-in-
fected wells (n = 6) to those in control (only virus infect-
ed) wells (n = 6). The minimal inhibitory concentration
(IC50) of the tested compounds was obtained from the
concentration–effect curve.
Acknowledgments
This work was supported in part by the Greek General
Secretariat for Research and Technology Program No.
3245C and bilateral scientific-technology project be-
tween Greece and Slovenia: Synthesis and biological
evaluation as potential anticancerogenic and antiviral
agents of amino-fluoronuleosides of adenine and cyto-
sine. The authors are grateful to Dr. K. Antonakis
(Ec. Nat. Sup. de Chimie de Paris; CNRS-FRE 2463)
for encouraging this work and to Dr. T. Halmos (Ec.
Nat. Sup. de Chimie de Paris) for the unstinted and
ˇ
fruitful aid and to Lidija Gradisnik for the excellent
technical performance.
(c) The neutralization of the virus in solution before
attachment: threefold dilutions of each of the tested
compounds or AZT were first co-incubated with rotavi-
rus or VSV in DMEM for 12 h prior to the infection of
cell monolayer at 37 ꢁC and 5% CO2. Residual viral
infectivity was measured after 24 h post-infection for
VSV and 72 h post-infection for rotavirus. Rotavirus
or VSV alone was treated in the same way as the con-
trol. After 72 h of incubation, the plates were stained
with Crystal violet in EtOH, rinsed with water, and
destained with 10% (v/v) acetic acid. The A590 was mea-
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