Unsaturated fluoro-ketopyranosyl nucleosides: Synthesis and biological evaluation of 3-fluoro-4-keto-β-d-glucopyranosyl derivatives of N4-benzoyl cytosine and N6-benzoyl adenine

Article abstract of DOI:10.1016/j.ejmech.2007.04.001

The protected β-nucleosides 1-(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-β-d-glucopyranosyl)-N⁴-benzoyl cytosine (2a) and 9-(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-β-d-glucopyranosyl)-N⁶-benzoyl adenine (2b), were synthesized by the coupling

Full text of DOI:10.1016/j.ejmech.2007.04.001

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 420e428
http://www.elsevier.com/locate/ejmech
Short communication
Unsaturated fluoro-ketopyranosyl nucleosides: Synthesis and biological
evaluation of 3-fluoro-4-keto-b-D-glucopyranosyl derivatives of
N4-benzoyl cytosine and N6-benzoyl adenine
a
Stella Manta , George Agelis , Tanja Botic , Avrelija Cencic , Dimitri Komiotis
a
b
a,
ꢀ b,c
*
´
a Department of Biochemistry and Biotechnology, Laboratory of Organic Chemistry, University of Thessaly, 26 Ploutonos Street, 41221 Larissa, Greece
b Department of Microbiology, Biochemistry and Biotechnology, Faculty of Agriculture, University of Maribor, Vrbanska c.30, 2000 Maribor, Slovenia
c Department of Biochemistry, Medical Faculty, University of Maribor, Slovenia
Received 16 January 2007; received in revised form 4 April 2007; accepted 5 April 2007
Available online 25 April 2007
Abstract
The protected b-nucleosides 1-(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-b-D-glucopyranosyl)-N4-benzoyl cytosine (2a) and 9-(2,4,6-tri-O-acetyl-
3-deoxy-3-fluoro-b-D-glucopyranosyl)-N6-benzoyl adenine (2b), were synthesized by the coupling of peracetylated 3-deoxy-3-fluoro-D-gluco-
pyranose (1) with silylated N4-benzoyl cytosine and N6-benzoyl adenine, respectively. The nucleosides were deacetylated and several subsequent
protection and deprotection steps afforded the partially acetylated nucleosides of cytosine 7a and adenine 7b, respectively. Finally, direct
oxidation of the free hydroxyl group at 40-position of 7a and 7b, and simultaneous elimination reaction of the b-acetoxyl group, afforded
the desired unsaturated 3-fluoro-4-keto-b-D-glucopyranosyl derivatives. These newly synthesized compounds were evaluated for their potential
antitumor and antiviral activities. Compared to 5FU, the newly synthesized derivatives showed to be more efficient as antitumor growth inhib-
itors and they exhibited direct antiviral effect toward rotavirus.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Unsaturated fluoro ketonucleosides; b-Elimination reaction; Antiviral; Antitumor activity
1. Introduction
[9e12] and antibiotic [13] properties and as building blocks in
nucleic acid synthesis [14,15]. Removal of the hydroxyl
groups in the 20- and 30-positions of the aforementioned nucle-
osides has generated drugs of choice for treatment of certain
viral infections, including human immunodeficiency virus
(HIV) infection [16]. Furthermore, replacement of the glycane
moiety with a pseudo sugar, a substituted cyclohexene ring,
furnished enzymatically stable nucleic acid analogues [17]
which can be considered as biosteres of natural nucleosides
and nucleotides, and can play a major role in different domains
like therapy, diagnosis, and biotechnology. One series of un-
common six-membered nucleoside analogues, the unsaturated
ketonucleosides, are well established for their antineoplastic
activity and immunosuppressive effects [18e20]. It appeared
that these nucleosides not only exhibit growth inhibitory
activity against a variety of tumor cells [21,22] in vitro and
L1210 leukemia [23,24] in vivo, but they also may constitute
Nucleoside analogues have been widely explored as poten-
tial antiviral and antitumoral chemotherapeutic agents [1e4].
Consequently, a large number of modifications have been
made to both base and sugar moieties of natural nucleosides,
with the objective of increasing the therapeutic index of estab-
lished antiviral agents [5]. Over the past two decades, nucleo-
side chemistry has evolved to facilitate efficient routes to
effective agents for the treatment of AIDS [6], herpes [7]
and viral hepatitis [8].
The last decades, nucleosides with a six-membered carbo-
hydrate moiety have been evaluated for their potential antiviral
* Corresponding author. Tel.: þ302410 565285; fax: þ302410 565290.
E-mail address: dkom@bio.uth.gr (D. Komiotis).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.04.001

S. Manta et al. / European Journal of Medicinal Chemistry 43 (2008) 420e428
421
important synthetic intermediates in the nucleoside field owing
to their chemical reactivities in various media [25,26].
On the other hand, fluorine substitution has been exten-
sively investigated as a means of enhancing biological activity
and increasing chemical or metabolic stability [27e36]. It was
proved that the introduction of a fluorine atom in the sugar
moiety of the unsaturated nucleosides increases the activity
[37], raises the lipophilicity, and makes the penetration of
the drug through the cell membrane easier [38e41]. Specific
fluorination at the 20- and/or 30-position of the sugar moiety
of the nucleoside analogues has been studied in the pursuit
of safe, effective and chemically stable antiviral agents
[38,42e50].
In recent years a number of cytosine and adenine nucleo-
side analogues have been widely used not only as antileukemic
agents, but also as antitumor agents against solid tumors
[51e63]. Moreover, cytosine unsaturated nucleoside ana-
logues, such as D-20,30-didehydro-20,30-dideoxy-5-fluorocyti-
dine (D-d4FC) and its L-enantiomer (L-Fd4C), have been
identified as anti-HIVagents [64,65] and structureeactivity re-
lationships of 30-fluoro-20,30-unsaturated nucleosides, showed
potent activity in the cytosine derivatives [66].
Acetylation of the free hydroxyl group in the 20-position of
the sugar moiety of both the nucleosides with acetic anhy-
dride/pyridine afforded the desired acetylated derivatives 5a
and 5b in very good yield (89 and 80%). The 40,60-O-isopro-
pylidene group of both the derivatives was removed upon short
treatment with 90% trifluoroacetic acid in methanol. Selective
protection of the primary 60-hydroxyl group with a trityl group
yielded 7a and 7b (60 and 62%, respectively). Oxidation of the
fluoro acetylated precursors 7a and 7b, was performed with
pyridinium dichromate (PDC)/acetic anhydride and afforded,
after a b-elimination reaction, the desired, unsaturated
carbonyl compounds 1-(3-deoxy-3-fluoro-6-O-trityl-b-D-
glycero-hex-2-enopyranosyl-4-ulose)-N4-benzoyl cytosine (8a)
in 75% yield and 9-(3-deoxy-3-fluoro-6-O-trityl-b-D-glycero-
hex-2-enopyranosyl-4-ulose)-N6-benzoyl adenine (8b) in 50%
yield.
Finally, detritylation of 8a using a mixture of formic acid
and diethyl ether [74], afforded the corresponding deprotected
unsaturated 30-fluoro-40-keto nucleoside of N4-benzoyl cyto-
sine 9a in 50% yield.
Several approaches to obtain the desired detritylated deriv-
ative of N6-benzoyl adenine 9b were attempted. Treatment of
8b with a mixture of formic acid and diethyl ether [74], or
methanolic hydrogen chloride [75], or 70% acetic acid at
60 ꢀC [76], or sodiumeammonia [77], or zinc bromide in
dichloromethane [78], unfortunately, did not afford the corre-
sponding deprotected derivative of adenine 9b and only ade-
nine was isolated from the reaction mixture.
As a part of our continuing effort to develop new tumor-in-
hibitory agents, unsaturated 30-fluoro-20-ketonucleosides of
N4-benzoyl cytosine have been prepared and showed to have
a promising potential in combating the rotaviral infections
and in the treatment of colon cancer [67]. In extending these
studies, we decided to design and synthesize a new class of
unsaturated 30-fluoro-40-ketonucleosides, that of N4-benzoyl
cytosine and N6-benzoyl adenine, respectively.
All compounds were characterized by their elemental
1
analyses, H NMR and ESI-MS. H NMR data for 1-(2,4,6-
1
tri-O-acetyl-3-deoxy-3-fluoro-b-D-glucopyranosyl)-N4-benzoyl
0
0
0
0
0 0
2. Results and discussion
cytosine (2a) (J1 2 ¼ 9.4 Hz, J2 3 ¼ 9.1 Hz, J3 4 ¼ 9.0 Hz) and
9-(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-b-D-glucopyranosyl)-N6-
0
0
0
0
0 0
2.1. Chemistry
benzoyl adenine (2b) (J1 2 ¼ 9.6 Hz, J2 3 ¼ 9.1 Hz, J3 4 ¼
9.0 Hz) showed that, as expected, these compounds had the
b configuration and that it existed in the 4C1 conformation. Fur-
thermore, in compounds 8a, 8b and 9a, the presence of the
C(O)eCH]CH-system was ascertained by the disappearance,
in the 1H NMR spectrum, of the signal for H-40 and the deshield-
ing of other protons, especially the allylic proton H-20 as
previously observed for other (2-deoxy-b-D-hex-2-enopyrano-
syl-4-ulose) nucleosides [79e81]. Moreover, the small value
of J1 2 (1.6 Hz for 8a and 1.4 Hz for 8b) suggests that these
compounds adopt the sofa conformation [80] with H-10 perpen-
dicular to the ring. Such a conformation should be favoured,
because the bulky substituents at C-10 and C-50 are equatorial.
The starting materials, 1-(2,4,6-tri-O-acetyl-3-deoxy-
3-fluoro-b-D-glucopyranosyl)-N4-benzoyl cytosine (2a) and
9-(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-b-D-glucopyranosyl)-N6-
benzoyl adenine (2b), were obtained from condensation
of 1,2,4,6-tetra-O-acetyl-3-deoxy-3-fluoro-glucopyranose (1)
[68,69] with silylated N4-benzoyl cytosine and N6-benzoyl ade-
nine, respectively, in the presence of trimethylsilyl trifluome-
thane-sulfonate and tin chloride, in refluxing acetonitrile. The
coupling of the peracetylated sugar 1 with the silylated N4-ben-
zoyl cytosine and N6-benzoyl adenine, yielded only the b-nucle-
oside derivatives 2a and 2b (68 and 60%) after standard workup
and flash chromatography. The b configuration of these nucleo-
sides was clearly established by their 1H NMR spectra. Selective
deprotection of 2a and 2b using NaOHeethanolepyridine [70]
gave the base protected benzoylated derivatives 3a and 3b in ex-
cellent yield (90 and 88%, respectively). It is noteworthy that the
fullyunprotectedderivativeswereobtainedwhenthebprotected
nucleosides were treated by methanolic ammonia [71], or potas-
sium carbonateemethanol [72], or guanidine [73]. Treatment of
3a and 3b with 2,2-dimethoxypropane in dry N,N-dimethylfor-
mamide gave the 40,60-isopropylidene derivatives 4a and 4b, in
80 and 76% yield, respectively (Scheme 1).
0
0
2.2. Antiviral activity
To examine the potential antiviral properties of the nucleo-
sides, a colon adenocarcinoma Caco-2 cell line infected with
gastrointestinal rotavirus as a model virus was used. The re-
sults obtained by performing antiviral assay with the newly
synthesized compounds are summarized in Table 1 and com-
pared to AZT. Although compounds did not show antiviral ac-
tivity in neutralization assay they all showed the ability to
inhibit rotavirus infectivity following virus attachment. In

422
S. Manta et al. / European Journal of Medicinal Chemistry 43 (2008) 420e428
AcO
HO
AcO
B
O
B
O
O
ii
i
OAc
F
F
F
OAc
OH
OAc
OAc
OH
iii
OAc
3 a, b
1
2 a, b
O
H3C
H3C
HO
O
H3C
H3C
TrO
B
B
B
B
O
O
O
O
vi
v
iv
F
F
F
F
O
O
OH
OH
OAc
OAc
OH
OAc
7 a, b
5 a, b
6 a, b
4 a, b
vii
B
HO
TrO
O
B
a: B = N4- BzCyt
b: B = N6- BzAde
O
O
viii
O
F
F
9 a, b
8 a, b
Scheme 1. (i) Silylated base, CH3CN, trimethylsilyl trifluomethane-sulfonate or tin chloride; (ii) ethanol, pyridine, NaOH, 0 ꢀC, 30 min, Amberlite IR-120 (Hþ)
resin; (iii) 2,2-dimethoxypropane, p-toluenesulfonic acid, DMF, 60 ꢀC, 1 h; (iv) pyridine, acetic anhydride, 4 ꢀC, 12 h; (v) 90% trifluoroacetic acid in methanol,
20 ꢀC, 10 min; (vi) pyridine, triphenylmethyl chloride, 4-dimethylaminopyridine, 60 ꢀC, 12 h; (vii) PDC, acetic anhydride, CH2Cl2eDMF, 90 ꢀC, 100 min; (viii)
formic acid, diethyl ether, 20 ꢀC, 10 min.
comparison to AZT higher concentrations were needed for the
new compounds to obtain the same effect against virus and
CC50/IC50 values were lesser for the new compounds than
for AZT.
carcinoma (Caco-2), breast carcinoma (MCF-7), skin mela-
noma and normal human intestinal cells (H4, control cell
line). Results are summarized in Table 2. From the tested
unsaturated fluoro-ketopyranosyl nucleoside analogues, 1-(3-
deoxy-3-fluoro-6-O-trityl-b-D-glycero-hex-2-enopyranosyl-4-
ulose)-N4-benzoyl cytosine (8a) exhibited the best cytotoxic
2.3. Cytotoxic and growth inhibition activity
effect, particularly against skin melanoma cells (CC50
¼
3.3 mM). The same analogue of adenine 8b showed about
3-fold less cytotoxicity toward skin melanoma cells than 8a,
and toward other malignant cell lines their activity and selec-
tivity were comparable. In comparison to 5FU, compound 8a
showed to be 16-fold more selective in MCF-7 cells, 3-fold
more selective in skin melanoma cells and 2.5-fold more se-
lective in Caco-2 cells (see TSI values).
Compounds 8a, 8b and 9a were evaluated for their cyto-
toxic activity against several malignant tumor cell lines: colon
Table 1
Antiviral activity of nucleosides 8a, 8b, 9a and AZT against rotavirus RF
strain on Caco-2 cells (IC50
Compound Treatment Aa
IC50
)
Treatment Ba
IC50
b
CC50/IC50
CC50/IC50
Generally, all tested compounds exhibit higher cytotoxicity
in tumor cells than in the normal H4 cell line. Furthermore in
comparison to 5FU, compounds showed to be more cytotoxic
against all tumorgenic cells and more selective toward Caco-2
and MCF-7 cells. From the results obtained it can be observed
that some compounds have selective effect toward specific tu-
mor cell line used and it has been reported before that mode of
inhibitory action especially on the target enzymes in carcino-
genic cells is not always similar even among nucleoside anti-
metabolites which have the same nucleoside base [82].
mg/mL mM
mg/mL mM
8a
8b
n.e.
n.e.
n.e.
0.02
n.e.
n.e.
n.e.
74.84 0.75c
e
e
e
0.2
0.5
332.43 0.10
799.17 0.04
9a
AZT
0.5
0.006
1391.56 0.04
22.45 2.50
n.e. ¼ No effect.
a
Treatment A: neutralization of the virus in the solution before its attach-
ment; treatment B: inhibition of infectivity following virus attachment.
b
CC50/IC50 values were calculated using CC50 values in Table 2.
CC50 for AZT on Caco-2 cells ¼ 56.1 mM.
c

S. Manta et al. / European Journal of Medicinal Chemistry 43 (2008) 420e428
423
Table 2
reactions were carried out in dry solvents. Dichloromethane
was distilled from phosphorus pentoxide and stored over 4E
molecular sieves. Acetonitrile was distilled from calcium hy-
dride and stored over 3E molecular sieves. Dimethylforma-
mide (DMF) was also stored over 3E molecular sieves.
Pyridine was stored over pellets of potassium hydroxide.
Cytotoxic effect (CC50, mM) of compounds 8a, 8b, 9a and 5-fluorouracil
(5FU) on Caco-2, H4, MCF-7, and skin melanoma cells, and growth inhibition
(IC50, mM) on Caco-2 cells
Compound Cytotoxic effect (CC50, mM) TSIa
Growth
inhibition
(IC50, mM)
H4
Caco-2 Mela- MCF-7 Caco-2 Mela- MCF-7 Caco-2
noma
4.2. Synthesis of 1-(3-deoxy-3-fluoro-b-D-glycero-hex-2-
enopyranosyl-4-ulose)-N4-benzoyl cytosine (9a)
noma
8a
8b
9a
5FU
a
831.1 33.2
799.1 31.9
3.3
9.5
9.9 25.0
9.5 25.0
55.6 25.0
251.8 83.9
84.1 84.1
83.8 25.0
1.1
1.1
4.2.1. Synthesis of 1-(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-b-
D-glucopyranosyl)-N4-benzoyl cytosine (2a)
1391.5 55.6 16.6
16.6
1.5
3843.8 384.4 46.1 768.8 10
83.4
5
A mixture of N4-benzoyl cytosine (3.99 g, 18.55 mmol),
hexamethyldisilazane (HMDS) (4.8 mL, 23 mmol) and sac-
charine (0.16 g, 0.85 mmol) in anhydrous CH3CN (69 mL)
was refluxed for 30 min at 120 ꢀC. To this were added tetraa-
cetylated 3-deoxy-3-fluoro-D-glucose [68,69] (1) (5.00 g,
14.27 mmol) 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, and extracted with CH2Cl2 (1000 mL). The or-
ganic layer was washed with water (3 ꢁ 20 mL) and dried
over anhydrous sodium sulfate, evaporated to dryness, finally
purified with flash chromatography using ethyl acetateen-hex-
ane (8:2) as eluent to give compound 2a as a solid. Yield:
4.90 g (68%), Rf ¼ 0.35, m.p. 277e279 ꢀC; 1H NMR
TSI: tumor selectivity index (CC50 on H4 cells/CC50 on the specified host
cells).
The growth inhibition of Caco-2 cells induced by the new
compounds was additionally measured by determining the
minimal inhibitory concentration, IC50. The results are sum-
marized at the end of Table 2 and compared with the values
obtained with 5FU. Compounds 8a and 8b exhibit slightly bet-
ter growth inhibitory activity (IC50 1.1 mM) than 5FU (IC50
1.5 mM) but with higher tumor selectivity (see TSI values).
Compound 9a was also able to inhibit cell growth although
the effect was somewhat less pronounced than that of 5FU
but with higher selectivity for Caco-2 cells.
(CDCl3): d 7.88 (d, 1H, J6,5 ¼ 7.2 Hz, H-6), 7.84e7.48 (m,
3. Conclusion
0
0
0
6H, Bz and H-5), 6.10 (d, 1H, J1 ,2 ¼ 9.4 Hz, H-1 ),
0
0
0
0
0
5.39e5.22 (m, 2H, J2 ,1 ¼ 9.4 Hz, J4 ,3 ¼ 9.0 Hz, H-2 and
In conclusion to biological assays, compounds 8a, 8b
and 9a were evaluated for their antiviral and antitumor cell
activities. Among them 1-(3-deoxy-3-fluoro-6-O-trityl-b-
D-glycero-hex-2-enopyranosyl-4-ulose)-N4-benzoyl cytosine
(8a) exhibited the best cytotoxic effect, particularly against
skin melanoma cells (CC50 ¼ 3.3mM). In general, all tested
compounds had better cytotoxic activities than the corre-
sponding standard 5FU. The compounds also showed rather
modest but direct activity against rotavirus.
H-40), 4.80 (dtr, 1H, JF,3 ¼ 51.7 Hz, J3 ,2 ¼ 9.1 Hz,
0
0
0
0
0 0
0
0
J3 ,4 ¼ 9.0 Hz, H-3 ), 4.34e4.11 (m, 2H, H-6a ,6b ), 3.86 (m,
1H, H-50), 2.16 and 2.11 and 2.07 (3s, 9H, 3OAc); 19F
NMR: d ꢂ65.0. Anal. calcd. for C23H24FN3O9: C, 54.65, H,
4.79, F, 3.76, N, 8.31; found: C, 54.70, H, 4.80, F, 3.75, N,
8.42; ESI-MS (m/z): found 506.43 (M þ Hþ).
4.2.2. Synthesis of 1-(3,4-dideoxy-3-fluoro-b-
D-glucopyranosyl)-N4-benzoyl cytosine (3a)
Compound 2a (4.90 g, 9.7 mmol) was dissolved in etha-
nolepyridine (97 mL þ 29.1 mL), 2 M NaOH (19.4 mL) was
added and the mixture was stirred for 30 min at 0 ꢀC. Amber-
lite IR-120 (Hþ) was added to neutralize the base. The suspen-
sion was filtered, the resin was washed with ethanol and
pyridine (100 mL þ 100 mL) and the filtrate was evaporated.
The solid residue was triturated with diethyl ether
(2 ꢁ 30 mL) and CH2Cl2 (2 ꢁ 30 mL) and filtered. Crude 3a
was obtained as a yellow foam and it was used without further
purification. Yield: 3.31 g (90%); ESI-MS (m/z): found 380.37
(M þ Hþ).
4. Experimental part
4.1. Chemistry
Melting points were recorded in a Mel-Temp apparatus and
are uncorrected. Thin-layer chromatography (TLC) was per-
formed on Merck precoated 60F254 plates. Reactions were
monitored by TLC on silica gel, with detection by UV light
(254 nm) or by charring with sulfuric acid. Flash chromatog-
raphy was performed using silica gel (240e400 mesh, Merck).
NMR spectra were recorded at room temperature with
a Brucker 400 MHz spectrometer using CDCl3, CD3OD,
4.2.3. Synthesis of 1-(3-deoxy-3-fluoro-4,6-
1
with internal tetramethylsilane (TMS) for H and internal tri-
O-isopropylidene-b-D-glucopyranosyl)-N4-benzoyl
cytosine (4a)
fluorotoluene (TFT) for 19F. The chemical shifts are expressed
in parts per million (d) and following abbreviations were used:
s ¼ singlet, br s ¼ broad singlet, d ¼ doublet, dd ¼ doublet
doublet, dtr ¼ doublet triplet and m ¼ multiplet. Mass spectra
were obtained with a Micromass Platform LC (ESI-MS). All
Compound 3a (3.31 g, 8.73 mmol) was dissolved in a mix-
ture of 34.92 mL of 2,2-dimethoxypropane and 110.4 mL of
dry DMF. To this was added p-toluenesulfonic acid (2.65 g,
13.97 mmol) and the mixture was stirred at 60 ꢀC for 1 h.

424
S. Manta et al. / European Journal of Medicinal Chemistry 43 (2008) 420e428
The reaction mixture was neutralized with triethylamine so pH
did not exceed 7. The mixture was concentrated under high
vacuum to eliminate the DMF. Purification with flash chroma-
tography using ethyl acetateen-hexane (9:1) gave 4a as
d 7.90 (d, 1H, J6,5 ¼ 7.5 Hz, H-6), 7.85e7.26 (m, 21H, Bz and
0
0
0
H-5 and 3C6H5), 6.03 (d, 1H, J1 ,2 ¼ 9.4 Hz, H-1 ), 5.20 (m,
1H, H-20), 4.70 (dtr, 1H, JF,3 ¼ 52.0 Hz, J3 ,2 ¼ 8.9 Hz,
0
0
0
0
0
0
0
J3 ,4 ¼ 8.9 Hz, H-3 ), 4.13 (m, 1H, H-4 ), 4.09e3.64 (m, 2H,
H-6a0,6b0), 3.48 (m, 1H, H-50), 2.05 (1s, 3H, OAc); 19F NMR:
d ꢂ64.9. Anal. calcd. for C38H34FN3O7: C, 68.77, H, 5.16, F,
2.86, N 6.33; found: C, 68.56, H, 5.27, F, 2.72, N, 6.40; ESI-
MS (m/z): found 664.67 (M þ Hþ).
1
a bright yellow oil. Yield: 2.93 g (80%), Rf ¼ 0.3; H NMR
(CDCl3): d 7.88 (d, 1H, J6,5 ¼ 7.9 Hz, H-6), 7.84e7.43 (m,
0
0
0
6H, Bz and H-5), 5.86 (d, 1H, J1 ,2 ¼ 9.2 Hz, H-1 ), 4.68
0
0
0
0
0
0
(dtr, 1H, JF,3 ¼ 53.2 Hz, J3 ,2 ¼ 8.7 Hz, J3 ,4 ¼ 8.7 Hz, H-3 ),
0
0
0
0
0
4.05e3.87 (m, 2H, J2 ,1 ¼ 9.2 Hz, J4 ,3 ¼ 8.7 Hz, H-2 and
H-40), 3.86e3.73 (m, 2H, H-6a0,6b0), 3.51 (m, 1H, H-50),
1.53 and 1.45 (2s, 6H, 2 ꢁ CH3); 19F NMR: d ꢂ64.9. Anal.
calcd. for C20H22FN3O6: C, 57.28, H, 5.29, F, 4.53, N,
10.02; found: C, 57.16, H, 5.31, F, 4.24, N, 10.03; ESI-MS
(m/z): found 420.41 (M þ Hþ).
4.2.7. Synthesis of 1-(3-deoxy-3-fluoro-
6-O-trityl-b-D-glycero-hex-2-enopyranosyl-
4-ulose)-N4-benzoyl cytosine (8a)
A mixture of 7a (2.23 g, 3.36 mmol, dried by co-evapora-
tion with toluene), PDC (1.9 g, 5.04 mmol) and acetic anhy-
dride (3.17 mL, 33.60 mmol) was refluxed at 90 ꢀC in dry
CH2Cl2 (32 mL) and dry DMF (10 mL) for 100 min. The mix-
ture was then concentrated under high vacuum to remove the
solvents. Purification with flash chromatography using ethyl
acetateen-hexane (7:3) afforded pure 8a as a white foam.
4.2.4. Synthesis of 1-(2-O-acetyl-3-deoxy-3-fluoro-
4,6-O-isopropylidene-b-D-glucopyranosyl)-
N4-benzoyl cytosine (5a)
To a solution of 4a (2.93 g, 6.99 mmol) in dry pyridine
(35 mL) was added acetic anhydride (0.59 mL, 6.29 mmol)
at 0 ꢀC and the resulted mixture was stirred overnight at
4 ꢀC. Methanol (0.3 mL) was added to quench the reaction
and the mixture was concentrated under high vacuum to re-
move the solvents. Purification with flash chromatography us-
ing ethyl acetateen-hexane (7:3) gave 5a. Yield: 2.87 g
1
Yield: 1.51 g (75%), Rf ¼ 0.39; H NMR (CDCl3): d 7.95 (d,
1H, J6,5 ¼ 7.5 Hz, H-6), 7.90e7.24 (m, 21H, Bz and H-5
0
0
0
0
and 3C6H5), 7.00 (dtr, 1H, J2 ,1 ¼ 1.6 Hz, J2 ,5 ¼ 1.8 Hz,
JF,2 ¼ 7.3 Hz, H-20), 6.58 (dd, 1H, J1 ,2 ¼ 1.6 Hz,
0
0
0
0
0
0
JF,1 ¼ 10.5 Hz, H-1 ), 4.50 (m, 1H, H-5 ), 3.76e3.62 (m,
2H, H-6a0,6b0); 19F NMR: d ꢂ63.2. Anal. calcd. for
C36H28FN3O5: C, 71.87, H, 4.69, F, 3.16, N, 6.98; found: C,
71.75, H, 4.88, F, 3.05, N, 7.07; ESI-MS (m/z): found
602.63 (M þ Hþ).
(89%), Rf ¼ 0.4; 1H NMR (CDCl3):
d 7.94 (d, 1H,
J6,5 ¼ 6.5 Hz, H-6), 7.80e7.50 (m, 6H, Bz and H-5), 6.09
0
0
0
0
(d, 1H, J1 ,2 ¼ 9.3 Hz, H-1 ), 5.26 (m, 1H, H-2 ), 4.76 (dtr,
0
0
0
0
0
0
1H, JF,3 ¼ 53.0 Hz, J3 ,2 ¼ 8.9 Hz, J3 ,4 ¼ 8.8 Hz, H-3 ), 4.03
(m, 1H, H-40), 3.99e3.83 (m, 2H, H-6a0,6b0), 3.57 (m, 1H,
H-50), 2.09 (1s, 3H, OAc), 1.59 and 1.51 (2s, 6H, 2 ꢁ CH3);
19F NMR: d ꢂ65.0. Anal. calcd. for C22H24FN3O7: C, 57.26,
H, 5.24, F, 4.12, N, 9.11; found: C, 57.14, H, 5.37, F, 4.02,
N, 9.01; ESI-MS (m/z): found 462.42 (M þ Hþ).
4.2.8. Synthesis of 1-(3-deoxy-3-fluoro-
b-D-glycero-hex-2-enopyranosyl-4-ulose)-
N4-benzoyl cytosine (9a)
Compound 8a (1.51 g, 2.51 mmol) was dissolved in a mix-
ture of formic acid and diethyl ether (10 mL þ 10 mL). The
mixture was stirred for 10 min at room temperature, diluted
with toluene and co-distilled several times with the same sol-
vent to avoid ester formation [83]. The mixture was concen-
trated under high vacuum and then purified with flash
chromatography using ethyl acetate to afford pure 9a. Yield:
4.2.5. Synthesis of 1-(2-O-acetyl-3-deoxy-
3-fluoro-b-D-glucopyranosyl)-
N4-benzoyl cytosine (6a)
Product 5a (2.87 g, 6.22 mmol), obtained from the previous
procedure, was dissolved in 31.1 mL of 90% trifluoroacetic
acid in methanol. The solution was stirred for 10 min at
room temperature and then concentrated at 40 ꢀC under high
vacuum in order to remove traces of trifluoroacetic acid. Puri-
fication with flash chromatography using ethyl acetate gave
6a. Yield: 2.36 g (90%), Rf ¼ 0.12; ESI-MS (m/z): found
422.39 (M þ Hþ).
1
0.45 g (50%), Rf ¼ 0.35; H NMR (CD3OD): d 8.23 (d, 1H,
J6,5 ¼ 7.5 Hz, H-6), 8.04e7.38 (m, 6H, Bz and H-5), 7.08
0
0
0
0
0
0
(dtr, 1H, J2 ,1 ¼ 1.4 Hz, J2 ,5 ¼ 1.9 Hz, JF,2 ¼ 7.0 Hz, H-2 ),
0
0
0
0
6.85 (dd, 1H, J1 ,2 ¼ 1.4 Hz, JF,1 ¼ 11.7 Hz, H-1 ), 4.64 (m,
1H, H-50), 4.14e3.97 (m, 2H, H-6a0,6b0); 19F NMR:
d ꢂ65.0. Anal. calcd. for C17H14FN3O5: C, 56.83, H, 3.93,
F, 5.29, N, 11.69; found: C, 56.74, H, 4.05, F, 5.14, N,
11.89; ESI-MS (m/z): found 360.29 (M þ Hþ).
4.2.6. Synthesis of 1-(2-O-acetyl-3-deoxy-
3-fluoro-6-O-trityl-b-D-glucopyranosyl)-
4.3. Synthesis of 9-(3-deoxy-3-fluoro-6-O-trityl-
b-D-glycero-hex-2-enopyranosyl-4-ulose)-
N6-benzoyl adenine (8b)
N4-benzoyl cytosine (7a)
To a solution of compound 6a (2.36 g, 5.6 mmol) in dry pyr-
idine (28 mL) was added triphenylmethyl chloride (1.87 g,
6.72 mmol) and a catalytic amount of 4-dimethylaminopyri-
dine. The mixture was stirred overnight at 60 ꢀC and then con-
centrated. Purification with flash chromatography using ethyl
acetateen-hexane (7:3), gave pure 7a as a white solid. Yield:
2.23 g (60%), Rf ¼ 0.37, m.p. 184e186 ꢀC; 1H NMR (CDCl3):
4.3.1. Synthesis of 9-(2,4,6-tri-O-acetyl-
3-deoxy-3-fluoro-b-D-glucopyranosyl)-
N6-benzoyl adenine (2b)
A mixture of N6-benzoyl adenine (4.44 g, 18.55 mmol),
HMDS (4.8 mL, 23 mmol) and saccharine (0.16 g, 0.85 mmol)

S. Manta et al. / European Journal of Medicinal Chemistry 43 (2008) 420e428
425
in anhydrous CH3CN (69 mL) was refluxed for 30 min at
120 ꢀC. To this were added tetraacetylated 3-deoxy-3-flu-
oro-D-glucose [68,69] (1) (5.00 g, 14.27 mmol) and tin chlo-
ride (2.35 mL, 19.98 mmol). The reaction mixture was
refluxed at 100 ꢀC for 5 h, then cooled, neutralized with
aqueous sodium bicarbonate, and extracted with CH2Cl2
(1000 mL). The organic layer was washed with water
(3 ꢁ 20 mL) and dried over anhydrous sodium sulfate, evap-
orated to dryness, finally purified with flash chromatography
using ethyl acetateen-hexane (8:2) as eluent to give com-
pound 2b as a solid. Yield: 4.53 g (60%), Rf ¼ 0.24, m.p.
118e120 ꢀC; 1H NMR (CDCl3): d 9.07 (1H, br s, eNH), 8.77
1.87 (1s, 3H, OAc), 1.61 and 1.52 (2s, 6H, 2 ꢁ CH3); 19F
NMR: d ꢂ65.0. Anal. calcd. for C23H24FN5O6: C, 56.90, H,
4.98, F, 3.91, N, 14.43; found: C, 57.03, H, 4.86, F, 4.04, N,
14.22; ESI-MS (m/z): found 486.47 (M þ Hþ).
4.3.5. Synthesis of 9-(2-O-acetyl-3-deoxy-
3-fluoro-b-D-glucopyranosyl)-
N6-benzoyl adenine (6b)
Adenine derivative 6b was synthesized from 5b by the sim-
ilar procedure as described for 6a. Purified with flash chroma-
tography using ethyl acetate as eluent and it was obtained as
a white foam. Yield: 1.83 g (90%), Rf ¼ 0.1; ESI-MS (m/z):
found 446.38 (M þ Hþ).
and 8.19 (2H, 2s, H-2,8), 7.98e7.42 (m, 5H, 0Bz), 5.84 (d, 1H,
0
0
0
J1 ,2 ¼ 9.6 Hz, H-1 ), 5.70e5.58 (m, 1H, H-2 ), 5.43e5.30 (m,
1H, H-40), 4.80 (dtr, 1H, JF,3 ¼ 51.6 Hz, J3 ,2 ¼ 9.1 Hz,
4.3.6. Synthesis of 9-(2-O-acetyl-3-deoxy-
3-fluoro-6-O-trityl-b-D-glucopyranosyl)-
N6-benzoyl adenine (7b)
Adenine derivative 7b was synthesized from 6b by the simi-
lar procedure as described for 7a. Purified with flash chromatog-
raphy using ethyl acetateen-hexane (7:3) as eluent and it was
obtained as a white solid. Yield: 1.75 g (62%), Rf ¼ 0.43, m.p.
147e149 ꢀC; 1H NMR (CDCl3): d 9.20 (1H, br s, eNH), 8.87
and 8.28 (2H, 2s, H-2,8), 8.10e7.22 (m, 20H, Bz and 3C6H5),
0
0
0
0
0
0
0
J3 ,4 ¼ 9.0 Hz), 4.29e4.08 (m, 2H, H-6a ,6b ), 3.90 (m, 1H,
H-50), 2.11, 2.01 and 1.78 (3s, 9H, 3OAc); 19F NMR: d ꢂ65.0.
Anal. calcd. for C24H24FN5O8: C, 54.44, H, 4.57, F, 3.59, N,
13.23; found: C, 54.58, H, 4.38, F, 3.72, N, 13.39; ESI-MS (m/
z): found 530.46 (M þ Hþ).
4.3.2. Synthesis of 9-(3,4-dideoxy-
3-fluoro-b-D-glucopyranosyl)-
N6-benzoyl adenine (3b)
Adenine derivative 3b was synthesized from 2b by the sim-
ilar procedure as described for 3a. Yield: 3.04 g (88%),
obtained as a yellow foam and it was used without further pu-
rification. ESI-MS (m/z): found 404.38 (M þ Hþ).
0
0
0
0
5.88 (d, 1H, J1 ,2 ¼ 9.5 Hz, H-1 ), 5.61 (m, 1H, H-2 ), 4.73
0
0
0
0
0
0
(dtr, 1H, JF,3 ¼ 52.1 Hz, J3 ,2 ¼ 8.9 Hz, J3 ,4 ¼ 8.9 Hz, H-3 ),
4.21 (m, 1H, H-40), 3.75 (m, 1H, H-50), 3.60e3.48 (m, 2H, H-
6a0,6b0), 1.85 (1s, 3H, OAc); 19F NMR: d ꢂ65.5. Anal. calcd.
for C39H34FN5O6: C, 68.11, H, 4.98, F, 2.76, N, 10.18; found:
C, 67.99, H, 4.74, F, 2.88, N, 10.08; ESI-MS (m/z): found
688.74 (M þ Hþ).
4.3.3. Synthesis of 9-(3-deoxy-
3-fluoro-4,6-O-isopropylidene-b-D-glucopyranosyl)-
N6-benzoyl adenine (4b)
4.3.7. Synthesis of 9-(3-deoxy-3-fluoro-
Adenine derivative 4b was synthesized from 3b by the sim-
ilar procedure as described for 4a. Purified with flash chroma-
tography using ethyl acetate as eluent and it was obtained as
6-O-trityl-b-D-glycero-hex-2-enopyranosyl-4-ulose)-
N6-benzoyl adenine (8b)
Adenine derivative 8b was synthesized from 7b by the simi-
lar procedure as described for 8a. Purified with flash chromatog-
raphy using ethyl acetateen-hexane (7:3) as eluent and it was
1
a bright yellow oil. Yield: 2.54 g (76%), Rf ¼ 0.43; H NMR
(CDCl3): d 9.04 (1H, br s, eNH), 8.39 and 8.0 (2H, 2s,
0
1
0
H-2,8), 7.94e7.42 (m, 5H, Bz), 5.65 (d, 1H, J1 ,2 ¼ 8.9 Hz, H-
obtained as a white foam. Yield: 0.8 g (50%), Rf ¼ 0.38; H
10), 4.70 (dtr, 1H, JF,3 ¼ 39.5 Hz, J3 ,2 ¼ 8.9 Hz, J3 ,4 ¼ 8.7 Hz,
H-30), 4.56 (m, 1H, H-20), 4.10e3.94 (m, 1H, H-40), 3.93e3.70
(m, 2H, H-6a0,6b0), 3.51 (m, 1H, H-50), 1.52 and 1.43 (2s, 6H,
2 ꢁ CH3); 19F NMR: d ꢂ64.9. Anal. calcd. for C21H22FN5O5:
C, 56.88, H, 5.00, F, 4.28, N, 15.79; found: C, 56.76, H, 4.82,
F, 4.42, N, 15.90; ESI-MS (m/z): found 444.41 (M þ Hþ).
NMR (CDCl3): d 9.05 (1H, br s, eNH), 8.88 and 8.27 (2H, 2s,
H-2,8), 8.10e7.22 (m, 20H, Bz and 3C6H5), 7.07 (dtr, 1H,
0
0
0
0
0
0
0
0
0
0
0
J2 ,1 ¼ 1.4 Hz, J2 ,5 ¼ 1.9 Hz, JF,2 ¼ 6.7 Hz, H-2 ), 6.74 (dd,
0
0
0
0
0
1H, J1 ,2 ¼ 1.4 Hz, JF,1 ¼ 10.2 Hz, H-1 ), 4.57 (m, 1H, H-5 ),
3.73e3.66 (m, 2H, H-6a0,6b0); 19F NMR: d ꢂ64.3. Anal. calcd.
for C37H28FN5O4: C, 71.03, H, 4.51, F, 3.04, N, 11.19; found: C,
71.19, H, 4.34, F, 3.25, N, 10.98. ESI-MS (m/z): found 626.66
(M þ Hþ).
4.3.4. Synthesis of 9-(2-O-acetyl-3-deoxy-3-
fluoro-4,6-O-isopropylidene-b-D-glucopyranosyl)-
N6-benzoyl adenine (5b)
4.4. Methods for measurement of biological activity
Adenine derivative 5b was synthesized from 4b by the sim-
ilar procedure as described for 5a. Purified with flash chroma-
tography using ethyl acetateen-hexane (6:4) as eluent and it
was obtained as a yellow foam. Yield: 2.22 g (80%),
Rf ¼ 0.2; 1H NMR (CDCl3): d 9.00 (1H, br s, eNH), 8.85 and
4.4.1. 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 foetal 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% foetal calf serum (Cambrex, Verviers,
8.22 (2H, 2s, H-2,8), 8.06e7.52 (m, 5H, 0Bz), 5.91 (d, 1H,
0
0
0
J1 ,2 ¼ 9.5 Hz, H-1 ), 5.72 (m, 1H, H-2 ), 4.80 (dtr, 1H,
0
0
0
0
0
0
JF,3 ¼ 52.9 Hz, J3 ,2 ¼ 8.8 Hz, J3 ,4 ¼ 8.9 Hz, H-3 ), 4.12 (m,
1H, H-40), 4.06e3.84 (m, 2H, H-6a0,6b0), 3.64 (m, 1H, H-50),

426
S. Manta et al. / European Journal of Medicinal Chemistry 43 (2008) 420e428
Belgium), L-glutamine (2 mmol/L, Sigma, St. Louis, USA),
penicillin (100 units/mL, Sigma, St. Louis, USA) and strepto-
mycin (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.
each dilution, by the mean ratios (%, ꢃSD) of absorbances in
virus-infected wells (n ¼ 6) in comparison to those in control
(only virus-infected) wells (n ¼ 6). The minimal inhibitory con-
centration (IC50) of the tested compounds was obtained from the
concentrationeeffect curve.
4.4.2. Nucleoside solutions
4.4.5. Growth inhibition assay
Stock drug solutions were freshly prepared in sterile di-
methyl sulfoxide (DMSO) at the concentration of 0.5 mg/
mL. The final concentration of DMSO in the cell culture me-
dium was less than 0.1%. All solutions were protected against
light.
AZT (RetrovirÒ) GlaxoSmithKline, USA, a drug used for
antiretroviral therapy was used as a standard compound in an-
tiviral experiments and 5FU as a standard compound in antitu-
mor experiments.
It was performed on Caco-2 cell line by modified method de-
scribed previously [85]. Briefly, in 96-well plates, six wells of
3-fold dilutions of each compound (initial concentration of
0.5 mg/mL) were applied to monolayers of 10 cells/well in
DMEMe10% foetal 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 the mean ratios (%, ꢃSD) of colony number
in treated wells (n ¼ 2) in contrast to those in control wells
(n ¼ 24). The minimal inhibitory concentration (IC50) of the
tested compounds was obtained from the concentrationeeffect
curve.
4.4.3. Virus propagation
Rotavirus RF strain was propagated on Caco-2 monolayers
in the presence of trypsin (1 mg per mL of DMEM) as de-
scribed previously [84]. Supernatant containing the virus was
collected from the flasks when cytopathic effect (CPE) was
observed (24e48 h at 37 ꢀC) by microscopy and clarified by
centrifugation. Virus was stored at ꢂ70 ꢀC until used. For
the antiviral assay, virus with 1.5 tissue culture infective
dose 50% units per mL (TCID50/mL) was used (100 mL per
well).
4.4.6. 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 com-
pounds at 3-fold serial dilutions of each compound (initial con-
centration 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 de-
stained with 10% (v/v) acetic acid. The A590 was measured, and
the results were expressed, for each dilution, by the mean ratios
(%, ꢃSD) of absorbances in treated wells (n ¼ 2) compared to
those in control wells (n ¼ 24). The minimal inhibitory concen-
tration (CC50) of the tested compounds was obtained from the
concentrationeeffect curve.
4.4.4. Antiviral assay
The potential antiviral activity of the newly synthesized
compounds was tested against rotavirus by investigating:
a) The inhibition of infectivity following virus attachment:
Washed monolayer Caco-2 cells were first incubated with
rotavirus for 1 h at 37 ꢀC in the presence of 5% CO2 (time
for virus to attach to cell membrane receptors). After incuba-
tion, the remaining virus was washed off with DMEM without
supplements and monolayer was treated immediately with the
nucleosides added in 3-fold serial dilutions (initial concentra-
tion 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 dilu-
tion, by the mean ratios (%, ꢃSD) of absorbances in virus-
infected wells (n ¼ 6) compared to those in control (only
virus-infected) wells (n ¼ 6). The minimal inhibitory concen-
tration (IC50) of the tested compounds was obtained from the
concentrationeeffect curve.
b) The neutralization of the virus in solution before attach-
ment: Three-fold dilutions of each tested compound (initial con-
centration of 0.5 mg/mL) were first pre-incubated with rotavirus
in DMEM supplemented with trypsin for 12 h prior to the infec-
tion of cell monolayer at 37 ꢀC and 5% CO2. Residual viral in-
fectivity was measured after 72 h post-infection for rotavirus.
Rotavirus alonewas 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 results were expressed, for
Acknowledgements
This work was supported in part by the Greek General Sec-
retariat for Research and Technology Program No. 3245C and
bilateral scientific-technology project between Greece and
Slovenia: SYNTHESIS AND BIOLOGICAL EVALUATION
AS POTENTIAL ANTICANCEROGENIC AND ANTIVI-
RAL AGENTS OF AMINO-FLUORONULEOSIDES OF
ADENINE AND CYTOSINE. The authors are grateful to
Dr. K. Antonakis (Ec. Nat. Sup. de Chimie de Paris; CNRS-
FRE 2463) for encouraging this work, to Dr. T. Halmos (Ec.
Nat. Sup. de Chimie de Paris) for the unstinted and fruitful
ˇ
aid, to Lidija Gadisnik for the excellent technical performance
as well as to ELPEN pharmaceuticals for financial support.
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