Dideoxy fluoro-ketopyranosyl nucleosides as potent antiviral agents: Synthesis and biological evaluation of 2,3- and 3,4-dideoxy-3-fluoro-4- and -2-keto-β-d-glucopyranosyl derivatives of N4-benzoyl cytosine

Article abstract of DOI:10.1016/j.ejmech.2009.01.020

The synthesis of the dideoxy fluoro ketopyranonucleoside analogues, 1-(2,3-dideoxy-3-fluoro-6-O-trityl-β-d-glycero-hexopyranosyl-4-ulos e)-N⁴-benzoyl cytosine (7a), 1-(3,4-dideoxy-3-fluoro-6-O-trityl-β-d-glycero-hexopyranosyl-2-ulos e)-N^4<

Full text of DOI:10.1016/j.ejmech.2009.01.020

European Journal of Medicinal Chemistry 44 (2009) 2696–2704
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Dideoxy fluoro-ketopyranosyl nucleosides as potent antiviral agents:
Synthesis and biological evaluation of 2,3- and 3,4-dideoxy-3-fluoro-4-
and -2-keto-
b-
-glucopyranosyl derivatives of N4-benzoyl cytosine
D
a
a
a
c
b
ˇ
ˇ
Stella Manta , Evangelia Tsoukala , Niki Tzioumaki , Ales Goropevsek , Ravi Teja Pamulapati ,
ˇ b
Avrelija Cencic c, Jan Balzarini d, Dimitri Komiotis a
,
,
*
a Department of Biochemistry and Biotechnology, Laboratory of Organic Chemistry, University of Thessaly, 26 Ploutonos Str., 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
d Rega Institute for Medical Research, Katholieke Universtiteit Leuven, 3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of the dideoxy fluoro ketopyranonucleoside analogues, 1-(2,3-dideoxy-3-fluoro-6-O-trityl-
b-D b-D-
-glycero-hexopyranosyl-4-ulose)-N4-benzoyl cytosine (7a), 1-(3,4-dideoxy-3-fluoro-6-O-trityl-
Received 20 November 2008
Received in revised form
16 January 2009
Accepted 20 January 2009
Available online 29 January 2009
glycero-hexopyranosyl-2-ulose)-N4-benzoyl cytosine (13a) and their detritylated analogues 8a and 14a,
respectively, is described. Condensation of peracetylated 3-deoxy-3-fluoro- -glucopyranose (1) with
D
silylated N4-benzoyl cytosine, followed by selective deprotection and isopropylidenation afforded
compound 2. Routine deoxygenation at position 20, followed by a deprotection-selective reprotection
sequence afforded the partially tritylated dideoxy nucleoside of cytosine 6, which upon oxidation of the
free hydroxyl group at the 40-position, furnished the desired tritylated 2,3-dideoxy-3-fluoro ketonu-
cleoside 7a in equilibrium with its hydrated form 7b. Compound 2 was the starting material for the
synthesis of the dideoxy fluoro ketopyranonucleoside 13a. Similarly, several subsequent protection and
deprotection steps as well as routine deoxygenation at position 40, followed by oxidation of the free
hydroxyl group at the 20-position of the partially tritylated dideoxy nucleoside 12, yielded the desired
carbonyl compound 13a in equilibrium with its hydrated form 13b. Finally, trityl removal from 7a/b and
13a/b provided the unprotected 2,3-dideoxy-3-fluoro-4-keto and 3,4-dideoxy-3-fluoro-2-ketopyr-
anonucleoside analogues 8a and 14a, in equilibrium with their gem-diol forms 8b and 14b. None of the
compounds showed inhibitory activity against a wide variety of DNA and RNA viruses at subtoxic
concentrations, except 7a/b that was highly efficient against rotavirus infection. Nucleoside 7a/b also
exhibited cytostatic activity against cells of various cancers. BrdU-cell cycle analysis revealed that the
mechanism of cytostatic activity may be related to a delay in G1/S phase and initiation of programmed
cell death.
Keywords:
Dideoxy fluoro ketonucleosides
Gem-diol
Antiviral
Antitumor activity
BrdU-cell cycle
Flow cytometry
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
important physiological and biogenetic properties. Among them,
nucleosides bearing cytosine as a base have been developed as
Nucleosides are structural modules of nucleic acids and there-
fore of fundamental importance in all living systems [1,2]. They
have been playing a major role in treating tumor and virus either as
selective inhibitors of certain obligatory enzymes for cancer or viral
replication [3], or as nucleic acid chain terminators, which interrupt
the replication of cancer cells or a virus [4–6]. Pyrimidine nucleo-
sides have attracted increased attention as a result of their
potent antitumor agents, which are effective not only on leukemias
and lymphomas, but also on a wide variety of solid tumors in vitro
as well as in vivo [7–13].
The last decades, nucleosides with a six-membered carbohy-
drate moiety have been evaluated for their potential antiviral
[14–18], antibiotic [19] and antioxidant [20] properties and as
building blocks in nucleic acid synthesis [21,22]. A series of six-
membered nucleoside analogues, the ketonucleosides, proved to
be key intermediates in synthetic and biosynthetic processes and
exhibited interesting antitumor and antiviral properties [23–27].
These nucleosides are known to inhibit DNA, RNA, and protein
* Corresponding author. Tel.: þ302410 565285; fax: þ302410 565290.
E-mail address: dkom@bio.uth.gr (D. Komiotis).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.01.020

S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 2696–2704
2697
synthesis [28], to interact with sulfhydryl groups of cellular
proteins and enzymes [29], while they were found to be highly
cytotoxic in vitro [24,30,31] and to exert powerful inhibitory
action against L1210 leukemia in vivo [32,33].
In view of these facts and taking into account that introduction
of fluorine into the sugar moiety of nucleosides enhances activity
[34–39], we have previously synthesized various unsaturated flu-
oro ketopyranonucleoside analogues, which proved to be efficient
as tumor growth inhibitors and showed to have a promising
potential in combating the rotaviral infections [40,41].
Thus, in continuation of our studies on the synthesis of sugar-
modified pyranonucleosides and based on the interesting biological
properties of deoxyhexopyranose nucleosides [14–16,42–45], we
decided to design and synthesize a new class of fluoro ketopyr-
anonucleoside analogues, the fluorinated 2,3-dideoxy-4-keto and
3,4-dideoxy-2-keto nucleosides of N4-benzoyl cytosine, in order to
assess their biological activity. Chemical synthesis and biological
activity of these compounds are presented herein.
amount of 4-dimethylaminopyridine (DMAP) in acetonitrile [48–
50], afforded the 20-O-phenoxythiocarbonyl derivative 3, in 70%
yield. The deoxygenation of compound 3 was carried out with tri-n-
butyltin hydride (Bu3SnH) to afford the 20-deoxynucleoside 4, in 60%
yield [50,51]. The 40,60-O-isopropylidene group of 4 was removed
upon treatment with aqueous trifluoroacetic acid (TFA) and selec-
tive protection of the primary 60-hydroxyl group with a trityl group
yielded the partially protected derivative 6, in 65% yield. Oxidation
of the fluoro tritylated dideoxy precursor 6 was performed with
pyridinium dichromate (PDC)/acetic anhydride (Ac2O) and afforded
40-keto form, 1-(2,3-dideoxy-3-fluoro-6-O-trityl-
b-D-glycero-hex-
opyranosyl-4-ulose)-N4-benzoyl cytosine (7a), in equilibrium with
its hydrated analogue 7b in a 3:7 ratio, as it was clearly determined
by integration of the related 1H NMR signals. It appears that the
presence of the electron-withdrawing fluorine atom
a to the
carbonyl, causes the easy hydration and gem-diol formation [52].
Finally, detritylation of 7a/b in CH2Cl2/formic acid, 1:1, led to the
corresponding dideoxy carbonyl compound, 1-(2,3-dideoxy-3-flu-
oro-
in equilibrium with its gem-diol 8b.
Compound [40,41] was the starting material for the
synthesis of 1-(3,4-dideoxy-3-fluoro- -glycero-hexopyranosyl-
b-D
-glycero-hexopyranosyl-4-ulose)-N4-benzoyl cytosine (8a)
2. Results and discussion
2
b-D
2.1. Chemistry
2-ulose)-N4-benzoyl cytosine (14a) (Scheme 2). Acetylation of the
nucleoside 2, followed by deisopropylidenation and finally by
selective tritylation of the primary hydroxyl group gave
compound 9 [41], which was then converted to the 40-deoxy
nucleoside analogue 11 via 40-O-phenoxythiocarbonyl derivative
10 by the similar methodology as described above. Deacetylation
of 11 was performed using NaOH/ethanol (EtOH)/pyridine [53] to
afford compound 12, in good yield (88%). Oxidation of the free
hydroxyl group in 20-position of the sugar moiety of nucleoside 12
with PDC/Ac2O afforded the desired 20-keto form, 1-(3,4-dideoxy-
Two different synthetic routes were investigated for the synthesis
of the title compounds, 1-(2,3-dideoxy-3-fluoro-
opyranosyl-4-ulose)-N4-benzoyl cytosine (8a) and 1-(3,4-dideoxy-
3-fluoro-
-glycero-hexopyranosyl-2-ulose)-N4-benzoyl cytosine
b-D-glycero-hex-
b-D
(14a). Inthe firstreactionscheme, condensation of3-deoxy-3-fluoro-
1,2,4,6-tetra-O-acetyl-glucopyranose (1) [46,47] (Scheme 1) with
silylated N4-benzoyl cytosine followed by selective deprotection and
specific acetalation using 2,2-dimethoxypropane in N,N-dime-
thylformamide (DMF), gave the 40,60-isopropylidene derivative 2
[40,41].
3-fluoro-6-O-trityl-b-D
-glycero-hexopyranosyl-2-ulose)-N4-benzoyl
cytosine (13a) in equilibrium with its hydrated analogue 13b in
Phenoxythiocarbonylation of 2 under a commonly used condi-
tion, phenyl chlorothionoformate (PhOC(S)Cl) and an excess
a 6:4 ratio, clearly determined by integration of the related 1H
AcO
O
O
O
CyBz
CyBz
CyBz
O
O
O
O
H3C
H3C
H3C
H3C
H3C
Ref. 40, 41
OAc
ii
i
F
F
F
F
H3C
OAc
O
O
O
OAc
OC(S)OPh
OH
1
2
3
4
iii
HO
TrO
TrO
HO
CyBz
CyBz
CyBz
CyBz
O
O
O
O
v
iv
vi
O
O
F
F
F
OH
F
OH
8a
7a
6
5
HO
TrO
HO
CyBz
CyBz
O
O
HO
F
F
HO
HO
8b
7b
Scheme 1. Reagents and conditions: (i) PhOC(S)Cl, DMAP, CH3CN, 20 ꢀC, 90 min; (ii) Bu3SnH, 1,1-azobis(cyclohexane carbonitrile), toluene, 80 ꢀC, 3 h; (iii) TFA, THF/H2O, 20 ꢀC,
90 min; (iv) pyridine, triphenylmethyl chloride, DMAP, 100 ꢀC, 1 h; (v) PDC, Ac2O, CH2Cl2, 55 ꢀC, 90 min; (vi) formic acid/CH2Cl2 (1:1), 20 ꢀC, 20 min.

2698
S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 2696–2704
TrO
O
TrO
TrO
CyBz
CyBz
CyBz
CyBz
H3C
H3C
O
O
O
O
Ref. 41
i
ii
F
F
F
F
HO
O
PhO(S)CO
OAc
OH
OAc
OAc
2
9
10
11
iii
TrO
HO
TrO
CyBz
CyBz
CyBz
O
O
O
v
iv
F
F
F
OH
O
14a
O
13a
12
TrO
HO
CyBz
OH
CyBz
OH
O
O
F
F
HO
HO
14b
13b
Scheme 2. Reagents and conditions: (i) PhOC(S)Cl, DMAP, CH3CN, 20 ꢀC, 90 min; (ii) Bu3SnH, 1,1-azobis(cyclohexane carbonitrile), toluene, 80 ꢀC, 3 h; (iii) EtOH, pyridine, NaOH,
ꢀC, 1 h, Amberlite IR-120 (Hþ) resin; (iv) PDC, Ac2O, CH2Cl2, 55 ꢀC, 90 min; (v) formic acid/CH2Cl2 (1:1), 20 ꢀC, 20 min.
0
NMR signals. Finally, deprotection of the primary hydroxyl group
of 13a/b was performed using CH2Cl2/formic acid, 1:1, and fur-
nished ketone 14a in equilibrium with its gem-diol 14b. It should
be mentioned that all attempts to remove the benzoyl group of
the target nucleoside analogues, were unsuccessful and only
degradation products were obtained [23,54,55].
2.2. Antiviral activity
Compounds 7a/b, 8a/b, 13a/b and 14a/b have been evaluated
against a broad variety of DNA and RNA viruses, including herpes
simplex virus type 1 [HSV-1 (KOS)], HSV-2 (G), vaccinia virus and
vesicular stomatitis virus (VSV) in HEL cell cultures; VSV, Cox-
sackie virus B4 and respiratory syncytial virus (RSV) in HeLa cell
cultures; parainfluenza-3 virus, reovirus-1, Sindbis virus, Cox-
sackie virus b4 and Punta Toro virus in Vero cell cultures; influ-
enza virus A (H1N1, H3N2) and influenza virus B in MDCK cell
cultures, feline corona virus (FIPV) and feline herpes virus in CRFK
cell cultures and human immunodeficiency virus (HIV-1(IIIB)) and
HIV-2(ROD) in CEM cell cultures. None of the compounds showed
inhibitory activity against any of these viruses at subtoxic
concentrations [i.e. MCC (minimal cytotoxic concentration): 100,
All compounds were well characterized by NMR and UV
spectroscopies, mass spectrometry and elemental analysis. The 1H
NMR spectrum of 7a/b showed
a
doublet at 6.35 ppm
0
0
(J1 ,2 ¼ 10.1 Hz) and a doublet of doublet of doublets at 5.22 ppm
0
0
0
0
0
0
(J3 ,F ¼ 47.0 Hz, J2 a,3 ¼ J2 b,3 ¼ 6.9 Hz), which are assigned to H-1
and H-30 protons, respectively, of keto compound 7a, as well as
0
0
a doublet at 5.94 ppm (J1 ,2 ¼ 10.6 Hz) and a doublet of doublet of
0
0
0
0
0
doublets at 4.72 ppm (J3 ,F ¼ 48.1 Hz, J2 a,3 ¼ 5.0 Hz, J2 b,3 ¼ 5.1 Hz),
assigned to H-10 and H-30 protons, respectively, of gem-diol 7b.
Similarly, in the H NMR spectrum of 13a/b, H-10 signals of keto
1
>100, 20 and >100
HeLa and Vero cell cultures]; CC50 (50%-cytostatic concentration):
11, 40, 0.6 and 45 M in MDCK cell cultures and 9.9, >100, 2.3 and
>100 M in CRFK cell cultures].
mM for 7a/b, 8a/b, 13a/b and 14a/b in HEL,
13a and gem-diol form 13b appeared as two sharp singlets at 6.41
and 5.86 ppm, respectively and H-30 signals of 13a and 13b
appeared as two doublet of doublet of doublets at 5.41 ppm
m
m
0
0
0
0
0
0
(J3 ,F ¼ 47.5 Hz, J3 ,4 a ¼ J3 ,4 b ¼ 7.4 Hz) and 4.82 ppm (J3 ,F ¼ 48.3 Hz,
The results of the antiviral assays against rotavirus on the newly
synthesized dideoxy fluoro-pyranonucleoside analogues 7a/b, 13a/
b and their detritylated analogues 8a/b and 14a/b, respectively, are
summarized and compared with AZT in Table 1.
In the neutralization assay, 8a/b was found to neutralize rota-
virus before its attachment to Caco-2 cells at the concentration of
J3 ,4 a ¼ 5.5 Hz, J3 ,4 b ¼ 5.3 Hz), respectively. Thus, in both cases, 1H
NMR spectra confirmed the equilibrium between keto and gem-
diol forms. However, 1H NMR spectra of 8a/b as well as 14a/b in
methanol-d4 (CD3OD) solution, showed a doublet at 5.94 ppm
0
0
0
0
0
0
0
(J1 ,2 ¼ 11.0 Hz) assigned to H-1 proton for 8b and a sharp singlet
at 5.85 ppm, assigned to H-10 proton for 14b, while H-30 signals
appeared as a doublet of doublet of doublets, for both compounds,
2.1 mM (vide IC50) as compared to AZT (75 mM). Similar results
were observed for 7a/b in inhibiting infectivity, following virus
attachment (Table 1); analogue 7a/b was actually more potent
than its congener 8a/b as it produced the same effect at a much
0
0
0
0
0
at 4.72 ppm (J3 ,F ¼ 48.4 Hz, J2 a,3 ¼ 4.8 Hz, J2 b,3 ¼ 4.9 Hz) and
0
0
0
0
0
4.94 ppm (J3 ,F ¼ 48.5 Hz, J3 ,4 a ¼ 5.5 Hz, J3 ,4 b ¼ 5.6 Hz), respec-
tively, indicating the presence of gem-diol forms exclusively.
Finally, the target compounds 7a/b, 8a/b and 13a/b, 14a/b were
characterized by the absence of the H-40 proton and H-20 proton
respectively, in their 1H NMR spectra.
lower concentration (0.63 mM) (treatment B). Interestingly, both
7a/b and its detritylated analogue 8a/b, although active in the
same antiviral assay, seem to exert their action by a different
mechanism.

S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 2696–2704
2699
Table 1
All of the tested compounds were cytotoxic on malignant cells of
different origin and had a substantial tumor selectivity index
(Table 2).
Antiviral activity of compounds 7a/b, 8a/b, 13a/b, 14a/b and AZT against rotavirus RF
strain on Caco-2 cells (IC50). CC50/IC50 ratios were calculated from CC50 values given
in Table 2.
From the tested dideoxy fluoro-pyranonucleoside analogues,
compounds 7a/b and 13a/b exhibited a noteworthy cytotoxicity,
with good selectivity indices, particularly on AGS cells.
In comparison to the control compound, 5FU, compounds 7a/b,
13a/b and 14a/b were more effective on MCF-7, AGS and Caco-2
cells; compound 8a/b was more effective than 5FU only on AGS
cells.
From the results obtained, it is clear that some of the new
compounds show selectivity toward specific tumor cell lines. This is
not unprecedented as there are examples in the literature, which
demonstrate that the mode of inhibitory action especially on the
target enzymes in carcinogenic cells is not always similar even
among nucleoside antimetabolites which have the same nucleoside
base [56].
Compound
Treatment Aa
IC50
Treatment Ba
IC50
b
b
CC50/IC50
CC50/IC50
mg/mL
m
M
mg/mL
mM
7a/b
8a/b
13a/b
14a/b
AZT
n.e.
0.0008
n.e.
n.e.
0.02
n.e.
2.1
n.e.
n.e.
75
–
320
–
0.0004
n.e.
n.e.
n.e.
0.006
0.63
n.e.
n.e.
n.e.
22
32
–
–
–
–
0.75c
2.5c
n.e. ¼ no effect.
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.
CC50 value for AZT on Caco-2 cells ¼ 56.1
c
mM.
In the growth inhibition assay compounds 7a/b and 13a/b were
capable of inhibiting the growth of malignant cells in a range
comparable to 5FU (IC50, Table 2). In spite of the fact that differences
were observed among the tested compounds in the growth inhi-
bition activity in colon carcinoma cells, all analogues were strong
inhibitors of malignant cell growth of other cancer types.
Conversely to the previously reported unsaturated fluoro keto-
pyranonucleoside analogues [40,41], the new dideoxy fluoro-pyr-
anonucleosides, especially compounds 7a/b and 13a/b, showed
satisfactory, but similar, cytotoxicity against Caco-2 cells and MCF-7
cells. It appears that the existence of a carbon–carbon double bond
in the sugar residue does not seem to be a structural prerequisite
for cytotoxic and growth inhibition activity. On the contrary, the
presence of the keto-fluoro system is indispensable.
In contrast to compounds 7a/b and 8a/b, analogues 13a/b and
14a/b did not show any antiviral potency. We speculate that the
position of the keto group in the ring skeletons of compounds 7a/
b and 8a/b is critical in inhibiting the rotavirus replication in the
cell as well as for neutralizing rotavirus before its attachment.
According to the antiviral assay used, it can be suggested that the
tested compounds 7a/b and 8a/b showed specific activity against
rotavirus, and were capable of inhibiting the rotavirus infection in
cells at significantly lower concentrations than AZT.
Compared to the recently reported unsaturated fluoro keto-
pyranonucleoside analogues [40,41], the dideoxy fluoro ketopyr-
anonucleosides presented herein, were found to be more potent
and thus more suitable candidates in anti-rotavirus therapy. Thus, it
can be suggested that the incorporation of the carbon–carbon
double bond into the ring system of the dideoxy fluoro ketopyr-
anonucleosides has a marked reducing effect on their antiviral
activity. It seems that this structural feature of the carbohydrate
moiety influences the ability of the nucleoside analogue to partic-
ipate in the antiviral mechanism.
2.4. BrdU-cell cycle analysis
Many mechanisms have been implicated in 5FU resistance and/
or sensitivity, such as pharmacokinetic resistance, decreased
accumulation of activated metabolites, and altered effects on
thymidylate synthase (TS) [57,58]. Because fluoropyrimidine
treatment leads to cell cycle arrest [59,60], other parameters, such
as cell cycle distribution can also be used to predict ketonucleosides
sensitivity. In order to find out, whether the mechanism of anti-
tumor activity and/or resistance to dideoxy fluoro ketopyr-
anonucleoside analogues was similar to the mechanism of action of
5FU, the influences on cell cycle distribution and induction of
apoptosis were studied by BrdU-cell cycle analysis. Our results
clearly showed that in Caco-2 cell line, 5FU equitoxic treatment
resulted in an increase of the percentage of cells in G0/G1 and in %
decrease of cells in the G2/M phase (Table 3, compounds 7a/b and
13a/b, Fig. 1). However, BrdU analysis showed that a cell cycle delay
took place specifically at the G1/S interface as previously reported
for 5FU (Table 3, Fig. 1) [59,60]. Exposure of cancer cells to
compounds 7a/b, 13a/b and 5FU led to a % BrdU-positive cells or
labeling index (LI) increase. However, LI modifications were only
2.3. Cytotoxic and growth inhibition activity
The cytotoxic potential of compounds 7a/b, 8a/b, 13a/b and 14a/
b was studied on H4 normal human intestinal cells and HEL human
lung fibroblast cells, and on a series of various other human tumor
cells, such as human colonic adenocarcinoma derived Caco-2 cells,
gastric cancer derived AGS cells, and epithelial breast cancer
derived MCF-7 cells, Vero African green monkey cells and HeLa
human cervix carcinoma cells. The growth inhibition of Caco-2
cells, caused by the new compounds, was measured by determining
the 50% growth inhibitory concentration (IC50). The results are
summarized in Table 2 and compared with the values obtained for
5-fluorouracil (5FU).
Table 2
Cytotoxic effect (CC50, mM) of compounds 7a/b, 8a/b, 13a/b, 14a/b and 5-fluorouracil
(5FU) on Caco-2, H4, AGS and MCF-7 cells, and growth inhibition (IC50
2 cells.
, mM) on Caco-
Table 3
Cell cycle distribution of Caco-2 cells treated with compounds 7a/b, 13a/b and 5-
fluorouracil (5FU) as compared to untreated cells.
Compound Cytotoxic effect (CC50
,
m
M) TSI*
Growth inhibition
(IC50 M)
,
m
Compound
Cell cycle distribution (%)
%Apoptotic cells
(sub G1)
H4
Caco-2 AGS MCF-7 Caco-2 AGS MCF-7 Caco-2
7a/b
8a/b
13a/b
14a/b
5FU
100 20
659 659
50 20
659 264
3844 390
10
132 659
10 20
40
5.0
1.0
2.5
2.5
9.8
10.0 2.5
5.0 1.0
5.0 2.5
5.0 2.5
5.0 6.0
5.1
237
5.1
33
1.5
G0/G1
S (LI)
G2/M
G1/S
7a/b
13a/b
5FU
62.0
62.0
65.0
21.2
19.8
17.5
16.0
17.1
16.4
13.8
14.0
12.4
28.4
25.6
29.2
132 264
769 641
Untreated
63.1
13.7
22.3
9.9
25.6
*TSI: Tumor selectivity index (CC50 on H4 cells/CC50 on the specified host cells).

2700
S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 2696–2704
concentrations as compared to AZT or/and previously synthesized
corresponding unsaturated fluoro ketopyranonucleoside analogues
[40,41]. We speculate that modification in sugar component has
a very important role in this case. Furthermore, all newly synthe-
sized compounds showed antitumor activity against tested cell
lines, especially gastric cancer derived AGS cells. Compounds 7a/b,
13a/b and 14a/b were even more cytotoxic to all cancer cell lines
tested than control compound 5FU. Compounds 7a/b and 13a/
b also showed to be as effective in their antitumor activity and in
growth suppression of colon carcinoma cells as previously
synthesized corresponding unsaturated fluoro ketopyranonucleo-
side analogues [40,41].
We have also shown, using flow cytometric BrdU-cell cycle
analysis, that the mechanism of antitumor activity of the newly
synthesized compounds may be related to delay in G1/S and pro-
grammed cell death initiation. Moreover, our results indicated that
compound 7a/b may have strong implication in clinical settings,
especially for treatment of those tumors poorly responsive to the
existing chemotherapy agents.
In view of these considerations, there has been increasing
evidence suggesting a close correlation between the biological
activity and the presence of the a-fluorocarbonyl moiety. However,
Fig. 1. Flow cytometry results of cell cycle analysis using bivariate BrdU versus 7-AAD
labeling. Caco-2 cells were cultured as described in experimental procedures. Cells
were treated with analyzed compounds at concentrations yielding 50% growth inhi-
bition (IC50) or left untreated. During the last hour of incubation, cells were pulsed
the mechanism of action of the newly synthesized nucleosides is
thus far unclear. It would therefore be interesting to further explore
the structure–activity relationship by modifying the base part
(keeping or removing the benzyl part) as well as replacing the
fluorine group by different chemical entities.
with 10 mM BrdU solution. Samples for flow cytometry were processed using FITC-
BrdU flow kit including 7-AAD labeling. Representative dot-plot of treated cells, gated
according to their (FSC versus SSC) scatter properties is shown. Bivariate distributions
of BrdU content (FITC) versus DNA content (7-AAD) were analyzed. Cell cycle distri-
bution in G0/G1, S, G2/M phases and % of apoptotic (sub G1) cells was determined. The
G1/S subpopulation, corresponding to BrdU-positive cells containing G1 DNA, was
determined. Labeling index (LI) corresponds to S phase or percentage of BrdU-positive
cells.
4. Experimental
4.1. Chemistry
Melting points were recorded in a Mel-Temp apparatus and are
uncorrected. Thin-layer chromatography (TLC) was performed 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 column chromatography was performed
using silica gel (240–400 mesh, Merck). 1H, 19F and 13C NMR spectra
were obtained at room temperature with a Bruker 400 spectrom-
eter at 400, 376 and 100 MHz, respectively using CDCl3 and CD3OD
with internal tetramethylsilane (TMS) for 1H and 13C and internal
trifluorotoluene (TFT) for 19F.
related to the G1/S phase accumulation, and not to a real enhanced
S-phase fraction. In fact, BrdU analysis showed that G1 cells, as
determined by 7-AAD analysis, were composed of BrdU-positive
and BrdU-negative cell subpopulations (Fig. 1). These subpopula-
tions cannot be distinguished using 7-AAD analysis alone.
This is viewed as a consequence of the cell fraction that was able
to incorporate BrdU and maintain its G1 DNA content. This fraction
was considered to be in phase G1, by 7-AAD analysis, whereas it
was included in the labeled subpopulation by BrdU analysis. Thus,
cells performing repair synthesis of DNA, with no net DNA increase,
would be included in the LI score, but could still have a DNA content
that was indistinguishable from G1 cells.
Synchronization of cells, caused by a delay in G1/S, may be
related to the programmed cell death initiation. Induced delay in
G1/S was representative of apoptosis involvement and of 5FU
treatment sensibility. Actually, sub-G1 peak, representative of
apoptotic cells was detected using flow cytometry already in
untreated cells but was enhanced in cells treated with compounds
7a/b and 5FU.
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, ddd ¼ doublet doublet doublet, dtr ¼ doublet triplet,
dq ¼ doublet quartet and m ¼ multiplet. Mass spectra were
obtained with a Micromass Platform LC (ESI-MS). Optical rotations
were measured using Autopol I polarimeter. All reactions were
carried out in dry solvents. CH2Cl2 was distilled from phosphorous
pentoxide and stored over 4 Å molecular sieves. CH3CN and toluene
were distilled from calcium hydride and stored over 3 Å molecular
sieves. Pyridine was stored over pellets of potassium hydroxide. All
reactions sensitive to oxygen or moisture were carried out under
nitrogen atmosphere.
The apoptotic activity of the tested compounds was observed in
the p53-mutated (Caco-2) cell line, suggesting that ketonucleoside-
induced apoptosis is mediated by a p53-independent mechanism.
This finding could have important implications in a clinical setting,
since many tumors have p53 alterations and mutated p53 has been
shown to be associated with a poor response to chemotherapy.
4.2. Synthesis of 1-(2,3-dideoxy-3-fluoro-b-D-glycero-
hexopyranosyl-4-ulose)-N4-benzoyl cytosine (8a) and
hydrated analogue 8b
4.2.1. 1-(3-Deoxy-3-fluoro-4,6-O-isopropylidene-2-O-
3. Conclusion
phenoxythiocarbonyl-b-D
-glucopyranosyl)-N4-benzoyl cytosine (3)
Compound 2 [40,41] (2.93 g, 6.99 mmol) was dissolved in
anhydrous CH3CN (46 mL). DMAP (1.71 g, 13.98 mmol) was added
and the mixture was stirred for 15 min. Then, PhOC(S)Cl (1.04 mL,
7.69 mmol) was added dropwise, and the resulting solution was
In summary, the newly synthesized fluorinated 2,3-dideoxy-4-
keto and 3,4-dideoxy-2-keto nucleosides of N4-benzoyl cytosine
showed good antiviral activity toward rotavirus in very low

S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 2696–2704
2701
stirred for 90 min at room temperature. The reaction was quenched
by adding water (1.1 mL). The solvent was removed under reduced
pressure, the obtained residue was dissolved in EtOAc (500 mL),
washed with water (1 ꢁ30 mL), dried over anhydrous magnesium
sulfate (MgSO4), evaporated under reduced pressure, and purified
by flash column chromatography using ethyl acetate–n-hexane
(m, 1H, H-50), 3.53 (m, 2H, H-6a0,6b0), 2.80 and 1.74 (2 m, 2H, H-20);
19F NMR:
d
ꢂ65.5; Anal. Calcd for C36H32FN3O5: C, 71.39; H, 5.33; N,
6.94. Found: C, 71.11; H, 5.17; N, 6.65; ESI-MS (m/z): 606.67 (M þ Hþ).
4.2.5. 1-(2,3-Dideoxy-3-fluoro-6-O-trityl-b-D-glycero-
hexopyranosyl-4-ulose)-N4-benzoyl cytosine (7a) and
(4:6) as eluant to afford 3 as a yellow syrup. Yield: 2.72 g (70%);
hydrated analogue 7b
22
Rf ¼ 0.52 in ethyl acetate–n-hexane (6:4); [
UV (CHCl3): lmax 260 nm (
1H, NH), 7.90–7.54 (m, 7H, Bz, H-5 and H-6), 7.41–7.09 (m, 5H,
a
]
D
þ 76.0 (c 0.5, CHCl3);
A mixture of 6 (0.83 g, 1.38 mmol; dried by co-evaporation with
toluene), PDC (0.78 g, 2.07 mmol) and Ac2O (0.65 mL, 6.9 mmol)
was refluxed at 55 ꢀC in dry CH2Cl2 (12 mL) for 90 min. After cooling,
ethyl acetate (1.6 mL) was added and the resulting slurry was
transferred on the top of a silica-gel column packed in ethyl acetate.
The solutionwas filtered through the column and washed with ethyl
acetate (30 mL) until the product was eluted completely. The
solvent was evaporated and the residue was rendered free of Ac2O
and pyridine by co-evaporation with dry toluene (3ꢁ). Finally,
purification by flash column chromatography using ethyl acetate–n-
hexane (6:4) as eluant afforded 7a/b as a white foam. Yield: 0.42 g
(50%), Rf ¼ 0.4 in ethyl acetate–n-hexane (8:2); 1H NMR for 7a
3
29,207); 1H NMR (CDCl3):
d 8.73 (br s,
0
0
0
0
C6H5), 6.26 (d, 1H, J1 ,2 ¼ 8.0 Hz, H-1 ), 5.90 (m, 1H, H-2 ), 4.95 (dtr,
0
0
0
0
0
0
1H, J3 ,F ¼ 53.0 Hz, J2 ,3 ¼ J3 ,4 ¼ 8.8 Hz, H-3 ), 4.06 (m, 2H, H-
6a0,6b0), 3.89 (m, 1H, H-40), 3.62 (m, 1H, H-50), 1.61 and 1.52 (2s, 6H,
2 ꢁ CH3); Anal. Calcd for C27H26FN3O7S: C, 58.37; H, 4.72; N, 7.56.
Found: C, 58.19; H, 4.97; N, 7.45; ESI-MS (m/z): 556.59 (M þ Hþ).
4.2.2. 1-(2,3-Dideoxy-3-fluoro-4,6-O-isopropylidene-
hexopyranosyl)-N4-benzoyl cytosine (4)
b-D-glycero-
Compound 3 (2.72 g, 4.9 mmol) was coevaporated three times
with anhydrous toluene, dissolved in toluene (106 mL), and
degassed with nitrogen for 30 min. To this solution were added
Bu3SnH (3.95 mL, 14.7 mmol) and 1,1-azobis(cyclohexane carbon-
itrile) (0.36 g, 1.47 mmol) and the mixture was heated to 80 ꢀC for
3 h. After cooling to room temperature, the mixture was evapo-
rated, the residue was purified by flash column chromatography
using ethyl acetate–n-hexane (6:4) as eluant and compound 4 was
(CDCl3):
d 9.17 (br s, 1H, NH), 7.99–7.28 (m, 22H, 3C6H5, Bz, H-5 and
0
0
0
0
H-6), 6.35 (d, 1H, J1 ,2 ¼ 10.1 Hz, H-1 ), 5.22 (ddd, 1H, J3 ,F ¼ 47.0 Hz,
0
0
0
0
0
0
0
0
J2 a,3 ¼ J2 b,3 ¼ 6.9 Hz, H-3 ), 3.82–3.72 (m, 3H, H-5 and H-6a ,6b ),
3.20 and 2.25 (2 m, 2H, H-20); 1H NMR for 7b (CDCl3):
9.17 (br s,1H,
NH), 7.99–7.28 (m, 22H, 3C6H5, Bz, H-5 and H-6), 5.94 (d, 1H,
d
0
0
0
0
0
0
J1 ,2 ¼ 10.6 Hz, H-1 ), 4.72 (ddd, 1H, J3 ,F ¼ 48.1 Hz, 0J2 a,3 ¼ 5.0 Hz,
0
0
0
0
0
J2 b,3 ¼ 5.1 Hz, H-3 ), 3.73–3.58 (m, 3H, H-5 and H-6a ,6b ), 2.60 and
2.07 (2 m, 2H, H-20); Anal. Calcd for C36H30FN3O5$0.5H2O: C, 70.59;
H, 5.07; N, 6.86. Found: C, 70.71; H, 4.93; N, 7.04; ESI-MS (m/z):
604.64 (M þ Hþ) for 7a; ESI-MS (m/z): 622.64 (M þ Hþ) for 7b.
obtained as a white solid. Yield: 1.19 g (60%); Rf ¼ 0.26 in ethyl
22
acetate–n-hexane (6:4); m.p. 188–190 ꢀC;
CHCl3); UV (CHCl3): lmax 260 nm (
[
a
]
þ 43.0 (c 0.5,
D
3 d 8.68
19,352); 1H NMR (CDCl3):
(br s, 1H, NH), 7.89–7.46 (m, 7H, Bz, H-5 and H-6), 5.95 (d, 1H,
0
0
0
0
0
J1 ,2 ¼ 10.7 Hz, H-1 ), 4.78 (m, 1H, H-3 ), 3.98 (m, 1H, H-4 ), 3.87 (m,
4.2.6. 1-(2,3-Dideoxy-3-fluoro-b-D-glycero-hexopyranosyl-4-
2H, H-6a0,6b0), 3.47 (m, 1H, H-50), 2.83 and 1.77 (2 m, 2H, H-20), 1.58
ulose)-N4-benzoyl cytosine (8a) and hydrated analogue 8b
and 1.48 (2s, 6H, 2 ꢁ CH3); 19F NMR:
d
ꢂ65.0; Anal. Calcd for
Compound 7a/b (0.42 g, 0.69 mmol) was dissolved in a mixture
of CH2Cl2 (2.45 mL) and formic acid (2.45 mL, 90%). The solution
was stirred for 20 min at room temperature, diluted with toluene,
and co-distilled several times with the same solvent to avoid ester
formation [61]. The concentrated residue was purified by flash
column chromatography using ethyl acetate–methanol (9.8:0.2) as
eluant and compound 8a/b was obtained as a white powder. Yield:
C20H22FN3O5: C, 59.55; H, 5.50; N,10.42. Found: C, 59.33; H, 5.61; N,
10.56; ESI-MS (m/z): 404.41 (M þ Hþ).
4.2.3. 1-(2,3-Dideoxy-3-fluoro-b-D
-glycero-hexopyranosyl)-N4-
benzoyl cytosine (5)
To a stirred solution of compound 4 (1.19 g, 2.94 mmol) in 4:1
tetrahydrofuran (THF)/H2O (13.6 mL) at 0 ꢀC was added TFA
(1.1 mL). The resulting solution was allowed to warm to room
temperature, stirred for 90 min and then was concentrated at 40 ꢀC
under high vacuum in order to remove traces of TFA. Purification by
flash column chromatography using ethyl acetate–methanol
(9.7:0.3) as eluant gave compound 5 as yellowish foam. Yield:
0.14 g (52%); Rf ¼ 0.15 in ethyl acetate–methanol (9.8:0.2);
22
[
a
]
þ 48.0 (c 0.5, MeOH); UV (MeOH): lmax 258 nm (
3
16,169); 1H
D
NMR (CD3OD):
d 8.29–7.53 (m, 7H, Bz, H-5 and H-6), 5.94 (d, 1H,
0
0
0
0
0
0
J1 ,2 ¼ 11.0 Hz, H-1 ), 4.72 (ddd, 1H, J3 ,F ¼ 48.4 Hz, 0J2 a,3 ¼ 4.8 Hz,
0
0
0
0
J2 b,3 ¼ 4.9 Hz, 1H, H-3 ), 4.06–3.94 (m, 2H, H-6a ,6b ), 3.57 (m, 1H,
H-50), 2.50 and 2.09 (2 m, 2H, H-20); 13C NMR (CD3OD):
d 169.23
0.77 g (72%); Rf ¼ 0.2 in ethyl acetate–methanol (9.7:0.3);
(C]O); 165.05 (C-4); 157.18 (C-2); 146.41 (C-6); 134.71 (Carom);
134.18 (CHarom); 129.88 (2CHarom); 129.24 (2CHarom); 99.08 (C-40);
94.05 (C-5); 92.54 (C-30); 80.51 (C-50); 78.46 (C-10); 61.44 (C-60);
22
[
a]
þ 30.0 (c 0.5, MeOH); UV (MeOH): lmax 258 nm (
3
17,223); 1H
D
NMR (CD3OD):
d 7.91–7.30 (m, 7H, Bz, H-5 and H-6), 5.81 (d, 1H,
J1 ,2 ¼ 10.8 Hz, H-1 ), 4.65 (m, 1H, H-3 ), 3.89–3.75 (m, 2H, H-
6a0,6b0), 3.63 (m, 1H, H-40), 3.43 (m, 1H, H-50), 2.43 and 1.88 (2 m,
2H, H-20); Anal. Calcd for C17H18FN3O5: C, 56.20; H, 4.99; N, 11.56.
Found: C, 56.38; H, 4.86; N, 11.67; ESI-MS (m/z): 364.32 (M þ Hþ).
30.79 (C-20); 19F NMR:
d
ꢂ63.2; Anal. Calcd for C17H18FN3O6: C,
0
0
0
0
53.83; H, 4.78; N, 11.08. Found: C, 53.98; H, 4.66; N, 11.19; ESI-MS
(m/z): 380.36 (M þ Hþ).
4.3. Synthesis of 1-(3,4-dideoxy-3-fluoro-b-D-glycero-
4.2.4. 1-(2,3-Dideoxy-3-fluoro-6-O-trityl-
hexopyranosyl)-N4-benzoyl cytosine (6)
b
-D-glycero-
hexopyranosyl-2-ulose)-N4-benzoyl cytosine (14a) and hydrated
analogue 14b
To a solution of compound 5 (0.77 g, 2.12 mmol) in dry pyridine
(10.6 mL) was added triphenylmethyl chloride (0.89 g, 3.18 mmol)
and a catalytic amount of DMAP. The mixture was stirred at 100 ꢀC
for 1 h and then concentrated. Purification by flash column chro-
matography using ethyl acetate–n-hexane (7:3) as eluant gave pure
4.3.1. 1-(2-O-Acetyl-3-deoxy-3-fluoro-4-O-phenoxythiocarbonyl-
6-O-trityl-b-D
-glucopyranosyl)-N4-benzoyl cytosine (10)
Cytosine derivative 10 was synthesized from 9 [41] by the same
methodology as described for the synthesis of 3. Compound 10 was
obtained as a white solid following purification by flash column
chromatography using ethyl acetate–n-hexane (1:1) as eluant.
6 as a white solid. Yield: 0.83 g (65%); Rf ¼ 0.52 in ethyl acetate–
22
methanol (9.8:0.2); m.p. 150–153 ꢀC; [
a
]
þ 63.0 (c 0.5, CHCl3); UV
D
(CHCl3): lmax 258 nm (
3
16,111); 1H NMR (CDCl3):
d
8.91 (br s, 1H,
Yield: 1.51 g (55%); Rf ¼ 0.2 in ethyl acetate–n-hexane (1:1); m.p.
22
NH), 7.95–7.29 (m, 22H, 3C6H5, Bz, H-5 and H-6), 5.86 (d, 1H,
153–156 ꢀC; [
a
]
þ 21.0 (c 0.5, CHCl3); UV (CHCl3): lmax 260 nm
D
J1 ,2 ¼ 10.5 Hz, H-1 ), 4.74 (m, 1H, H-3 ), 3.90 (m, 1H, H-4 ), 3.62
(3 d 8.66 (br s, 1H, NH), 7.90–6.81 (m, 27H,
21,501); 1H NMR (CDCl3):
0
0
0
0
0

2702
S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 2696–2704
0
0
0
Bz, H-5, H-6 and 4C6H5), 6.15 (d,1H, J1 ,2 ¼ 9.1 Hz, H-1 ), 5.98 (m,1H,
Compound 14a/b was obtained as a white powder following puri-
fication by flash column chromatography using ethyl acetate–
H-40), 5.32 (m, 1H, H-20), 4.97 (dtr, 1H, J3 ,F ¼ 51.9 Hz, J2 ,3 ¼ 9.0 Hz,
0
0
0
0
0
0
0
0
0
J3 ,4 ¼ 9.1 Hz, H-3 ), 4.00 (m, 1H, H-5 ), 3.42–3.30 (m, 2H, H-6a ,6b ),
2.09 (1s, 3H, OAc); Anal. Calcd for C45H38FN3O8S: C, 67.57; H, 4.79;
N, 5.25. Found: C, 67.69; H, 4.64; N, 5.53; ESI-MS (m/z): Found
800.88 (M þ Hþ).
methanol (9.8:0.2) as eluant. Yield: 0.11 g (50%), Rf ¼ 0.15 in ethyl
22
acetate–methanol (9.8:0.2);
[
a
]
þ 52.0 (c 0.5, MeOH); UV
(MeOH): lmax 258 nm (
3
21,395); 1DH NMR (CD3OD):
d
8.39–7.50 (m,
7H, Bz, H-5 and H-6), 5.85 (s, 1H, H-10), 4.94 (ddd, 1H, J3 ,F ¼ 48.5 Hz,
0
J3 ,4 a ¼ 5.5 Hz, J3 ,4 b ¼ 5.6 Hz, H-30), 3.87 (m, 1H, H-50), 3.67 (m, 2H,
0
0
0
0
4.3.2. 1-(2-O-Acetyl-3,4-dideoxy-3-fluoro-6-O-trityl-
b
-
D
-glycero-
H-6a0,6b0), 2.12–1.96 (m, 2H, H-40); 13C NMR (CD3OD):
d 169.27
hexopyranosyl)-N4-benzoyl cytosine (11)
(C]O); 164.88 (C-4); 150.06 (C-2); 141.63 (C-6); 134.23 (Carom);
134.16 (CHarom); 129.87 (2CHarom); 129.22 (2CHarom); 98.41 (C-20);
94.38 (C-5); 92.88 (C-30); 83.11 (C-10); 70.62 (C-50); 65.12 (C-60);
Cytosine derivative 11 was synthesized from 10 by the same
methodology as described for the synthesis of 4. Compound 11 was
obtained as a white solid following purification by flash column
chromatography using ethyl acetate–n-hexane (1:1) as eluant.
30.79 (C-40); 19F NMR:
d
ꢂ64.3; Anal. Calcd for C17H18FN3O6: C,
53.83; H, 4.78; N, 11.08. Found: C, 54.03; H, 4.89; N, 10.87; ESI-MS
Yield: 0.83 g (68%); Rf ¼ 0.18 in ethyl acetate–n-hexane (1:1); m.p.
(m/z): 380.34 (M þ Hþ).
22
138–140 ꢀC; [
a]
þ19.0 (c 0.5, CHCl3); UV (CHCl3): lmax 260 nm (
3
D
20,723); 1H NMR (CDCl3):
d
8.65 (br s, 1H, NH), 7.90–7.27 (m, 22H,
4.4. Methods for measurement of biological activity
0
0
0
3C6H5, Bz, H-5 and H-6), 5.95 (d, 1H, J1 ,2 ¼ 9.1 Hz, H-1 ), 5.04 (m,
1H, H-20), 4.85 (m, 1H, H-30), 3.85 (m, 1H, H-50), 3.26 (dq, 2H,
4.4.1. Cells and culture conditions
J5 ,6 a ¼ 4.9 Hz, J5 ,6 b ¼ 4.7 Hz, J6 a,6 b ¼ 10.0 Hz, H-6a0,6b0), 2.40 and
The human colon adenocarcinoma cell line Caco-2 (a generous
gift of Dr. Rene´ L’Harridon, INRA, VIM, Jouy-en-Josas, France), MCF-
7 cell line derived from breast carcinoma, gastric cancer derived
AGS cell line and non-tumorigenic human fetal small intestine cell
line H4 (control) were used for experiments. Cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM) (Sigma–Aldrich,
Grand Island, USA), supplemented with 5% fetal calf serum (Cam-
0
0
0
0
0
0
1.91 (2 m, 2H, H-40), 2.06 (1s, 3H, OAc); 19F NMR:
d
ꢂ65.5; Anal.
Calcd for C38H34FN3O6: C, 70.47; H, 5.29; N, 6.49. Found: C, 70.59; H,
5.11; N 6.74; ESI-MS (m/z): 648.70 (M þ Hþ).
4.3.3. 1-(3,4-Dideoxy-3-fluoro-6-O-trityl-
hexopyranosyl)-N4-benzoyl cytosine (12)
b-D-glycero-
Compound 11 (0.83 g,1.28 mmol) was dissolved in EtOH–pyridine
(12.8 þ 3.85 mL), 2 M NaOH (0.86 mL) was added and the mixture
stirred for 1 h at 0 ꢀC. Amberlite IR-120 (Hþ) was added to neutralize
the base. The suspension was filtered, the resin was washed with
EtOH and pyridine (10 þ 10 mL) and the filtrate was evaporated. The
solid residue was purified by flash column chromatography using
brex, Verviers, 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% CO2
atmosphere in tissue culture flasks until confluent. Cell culture
medium was regularly changed.
ethyl acetate aseluantand 12 wasobtainedasayellowishfoam. Yield:
4.4.2. Nucleoside solutions
22
0.682 g (88%); Rf ¼ 0.4 in ethyl acetate; [
a
]
þ14.0 (c 0.5, CHCl3); UV
Dideoxy fluoro ketonucleosides were freshly prepared in sterile
dimethyl sulfoxide (DMSO) at the concentration of 0.5 mg/mL. The
final concentration of DMSO was below 0.1% of cell culture medium.
All solutions were protected against light.
D
(CHCl3): lmax 260 nm(
3
21,804); 1H NMR (CDCl3):
d 9.01 (brs,1H, NH),
0
0
7.90–7.28 (m, 22H, 3C6H5, Bz, H-5 and H-6), 5.82 (d, 1H, J1 ,2 ¼ 9.1 Hz,
H-10), 4.78 (m, 1H, H-30), 3.89 (m, 1H, H-50), 3.70 (m, 1H, H-20), 3.25
(dq, 2H, J5 ,6 a ¼ 5.3 Hz, J5 ,6 b ¼ 4.6 Hz, J6 a,6 b ¼ 9.9 Hz, H-6a0,6b0), 2.33
AZT (RetrovirÒ) GlaxoSmithKline, USA, a drug used for anti-
retroviral therapy (ART) was used as a standard compound in bio-
logical experiments, prepared in the same way as dideoxy fluoro
ketonucleosides.
0
0
0
0
0
0
and 1.82 (2 m, 2H, H-40); 19F NMR:
d
ꢂ63.2; Anal. Calcd for
C36H32FN3O5: C, 71.39; H, 5.33; N, 6.94. Found: C, 71.64; H, 4.97; N,
6.72; ESI-MS (m/z): 606.64 (M þ Hþ).
5FU (fluorouracilum), Lederle Arzeinmittel GmbH, BRD, a drug
used forantitumor therapy was used as standard compound (control)
in growth inhibition and cytotoxicity assessment of compounds,
prepared in the same way as dideoxy fluoro ketonucleosides.
4.3.4. 1-(3,4-Dideoxy-3-fluoro-6-O-trityl-b-D-glycero-
hexopyranosyl-2-ulose)-N4-benzoyl cytosine (13a) and hydrated
analogue 13b
Cytosine derivative 13a/b was synthesized from 12 by the same
methodology as described for the synthesis of 7a/b. Compound
13a/b was obtained as a white foam following purification by flash
column chromatography using ethyl acetate as eluant. Yield: 0.35 g
4.4.3. Virus propagation
The rotavirus RF strain was propagated in Caco-2 cells in the
presence of trypsin (1 mg per mL of DMEM) as described previously
[62]. 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 centrifugation. Virus was stored at
ꢂ70 ꢀC until used.
(52%); Rf ¼ 0.39 in ethyl acetate; 1H NMR for 13a (CDCl3):
d
8.98 (br
s, 1H, NH), 8.15–7.27 (m, 22H, 3C6H5, Bz, H-5 and H-6), 6.41 (s, 1H,
H-10), 5.41 (ddd, 1H, J3 ,F ¼ 47.5 Hz, J3 ,4 a ¼ J3 ,4 b ¼ 7.4 Hz, H-30), 4.29
0
0
0
0
0
(m, 1H, H-50), 3.43 (dq, 2H, J5 ,6 a ¼ 5.1 Hz, J5 ,6 b ¼ 5.9 Hz,
0
0
0
0
J6 a,6 b ¼ 10.0 Hz, H-6a0,6b0), 2.82 and 2.29 (2 m, 2H, H-40); 1H NMR
0
0
for 13b (CDCl3):
d
8.98 (br s, 1H, NH), 8.15–7.27 (m, 22H, 3C6H5, Bz,
4.4.4. Antiviral assay
The potential antiviral activity of the newly synthesized
compounds was tested against rotavirus by investigating:
H-5 and H-6), 5.86 (s, 1H, H-10), 4.82 (ddd, 1H, J3 ,F ¼ 48.3 Hz,
0
J3 ,4 a ¼ 5.5 Hz, J3 ,4 b ¼ 5.3 Hz, H-30), 3.89 (m, 1H, H-50), 3.23 (dq, 2H,
0
0
0
0
J5 ,6 a ¼ 4.1 Hz, J5 ,6 b ¼ 4.7 Hz, J6 a,6 b ¼ 10.0 Hz, H-6a0,6b0), 2.16–1.96
(m, 2H, H-40); Anal. Calcd for C36H30FN3O5$0.5H2O: C, 70.59; H,
5.07; N, 6.86. Found: C, 70.33; H, 5.18; N, 7.07; ESI-MS (m/z): 604.66
(M þ Hþ) for 13a; ESI-MS (m/z): 622.67 (M þ Hþ) for 13b.
0
0
0
0
0
0
a) The inhibition of infectivity following virus attachment: Washed
monolayers of Caco-2 and H4 cells were first incubated with
rotavirus for 1 h at 37 ꢀC in the atmosphere 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 the monolayer was treated immediately with
the tested compounds or AZT added in 3-fold serial dilutions
(initial concentration of 0.5 mg/mL). After 72 h of incubation
4.3.5. 1-(3,4-Dideoxy-3-fluoro-b-D-glycero-hexopyranosyl-2-
ulose)-N4-benzoyl cytosine (14a) and hydrated analogue 14b
Cytosine derivative 14a/b was synthesized from 13a/b by the
same methodology as described for the synthesis of 8a/b.

S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 2696–2704
2703
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 the mean ratios (%, ꢃSD) of absorbances in
virus-infected wells (n ¼ 6) to those in control (only virus
versus DNA content (7-AAD) were analyzed. Cell cycle distribution
in G0/G1, S, G2/M phases and % of apoptotic (sub G1) cells was
determined according to gating represented in Fig. 1. Also the G1/S
subpopulation, corresponding to BrdU-positive cells containing G1
DNA, was determined. Labeling index (LI) corresponded to S phase
or percentage of BrdU-positive cells (Fig. 1).
infected) wells (n ¼ 6). The 50% inhibitory concentration (IC50
)
of the tested compounds was obtained from the concentra-
tion–effect curve.
4.4.8. Antiviral activity assays
b) The neutralization of the virus in solution before attachment: 3-
fold dilutions of each of the tested compounds or AZT (initial
concentration of 0.5 mg/mL) were first co-incubated with
rotavirus in DMEM supplemented with trypsin for 12 h prior
the infection of Caco-2 and H4 cell monolayer at 37 ꢀC and 5%
CO2. Residual viral infectivity was measured after 72 h post
infection. Rotavirus alone was treated in the same way as the
control. 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 measured, and the results
were expressed, for each dilution, by the mean ratios (%, ꢃSD)
of absorbencies in virus-infected wells (n ¼ 6) to those in
control (only virus infected) wells (n ¼ 6). The 50% inhibitory
concentration (IC50) of the tested compounds was obtained
from the concentration–effect curve.
The compounds were evaluated against the following viruses:
herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-
deficient (TKꢂ) HSV-1 KOS strain resistant to ACV (ACVr), herpes
simplex virus type 2 (HSV-2) strains Lyons and G, vaccinia virus
Lederle strain, respiratory syncytial virus (RSV) strain Long, vesic-
ular stomatitis virus (VSV), Coxsackie B4, Parainfluenza-3,
Reovirus-1, Sindbis, Punta Toro, influenza virus type A (H1N1,
H3N2) and type B and feline corona virus. The antiviral, other than
anti-HIV, assays were based on inhibition of virus-induced cyto-
pathicity in human embryonic lung (HEL) fibroblasts, African green
monkey cells (Vero), human epithelial cervix carcinoma cells
(HeLa), Crandel feline kidney cells (CFKC) and Madin-Darby canine
kidney cells (MDCK). Briefly, confluent cell cultures in microtiter
96-well plates were inoculated with 100 CCID50 of virus (1 CCID50
being the virus dose to infect 50% of the cell cultures). After a 1–2 h
adsorption period, residual virus was removed, and the cell cultures
were incubated in the presence of varying concentrations of the
test compounds. Viral cytopathicity was recorded as soon as it
reached completion in the control virus-infected cell cultures that
were not treated with the test compounds. Antiviral activity was
expressed as the EC50 or compound concentration required to
reduce virus-induced cytopathicity by 50%.
4.4.5. Cytotoxicity assay
Caco-2 cells, H4 cells, MCF-7 (breast carcinoma) and gastric
cancer derived AGS cells (6X106 cells per plate) were seeded in P96
plates and treated with the compounds or AZT 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 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 (%, ꢃSD) of absorbances in treated wells (n ¼ 2) to
those in control wells (n ¼ 24). The 50% cytotoxic concentration
(CC50) of the tested compounds was obtained from the concen-
tration–effect curve.
4.4.9. Inhibition of HIV-induced cytopathicity in CEM cells
HumanCEMcell cultures (w3 ꢁ 105 cells mLꢂ1)were infected with
w100 CCID50 HIV-1(IIIB) or HIV-2(ROD)per mL and seeded in 96-well
(200 mL/well) microtiter plates, containing appropriate dilutions of
the test compounds. After 4 days of incubation at 37 ꢀC, syncytia
formation was examined microscopically in the CEM cell cultures.
4.4.10. Cytostatic and cytotoxicity assays
4.4.6. Growth inhibition assay
The cytostatic concentration (for MDCK and CRFK cells) was
calculated as the CC50, or the compound concentration required to
reduce cell proliferation by 50% relative to the number of cells in
the untreated controls. CC50 values were estimated from graphic
plots of the number of cells (percentage of control) as a function of
the concentration of the test compounds. Alternatively, cytotoxicity
of the test compounds (for HEL, Vero and HeLa cells) was expressed
as the minimum cytotoxic concentration (MCC) or the compound
concentration that caused a microscopically detectable alteration of
cell morphology.
It was performed on Caco-2 cell line by modified method
described previously [63]. Briefly, in 96-well plates, six wells of 2-fold
dilutions of each compound or 5FU (initial concentration of 0.5 mg/
mL) were applied to 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 dilu-
tion, by the mean ratios (%, ꢃSD) of colony number in treated wells
(n ¼ 2) to those in control wells (n ¼ 24). The 50% growth inhibitory
concentration (IC50) of the tested compounds was obtained from the
concentration–effect curve.
Acknowledgements
4.4.7. Flow cytometric BrdU-cell cycle analysis
Caco-2 cells in Dulbecco’s modified Eagle’s medium (DMEM)/
10% fetal bovine serum were seeded in P96 plates (1 ꢁ106 cells per
well) and treated with the compounds at concentrations yielding
50% growth inhibition (IC50) or left untreated. Then, the cells were
incubated at 37 ꢀC in the humidified incubator for 24 h. During the
This work was supported in part by the Greek General Secre-
tariat for Research and Technology Program No. 3245C and bilateral
scientific-technology project between Greece and Slovenia:
‘‘Synthesis and biological evaluation as potential anticancerogenic
and antiviral agents of amino-fluoronucleosides of adenine and
cytosine’’. The authors are grateful to Dr. K. Antonakis (Ecole 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
last hour of incubation cells were pulsed with 10 mM BrdU solution.
Samples for flow cytometry were processed using FITC-BrdU
flow kit (BD Biosciences) according to the manufacturer protocol.
Flow cytometry was performed on BD LSR II (BD Biosciences)
instrument. The data were analyzed using FACSDiva software (BD
Biosciences) on cells gated according to their (FSC versus SSC)
scatter properties. Bivariate distributions of BrdU content (FITC)
ˇ
and fruitful aid, to Lidija Gadisnik, Leentje Persoons, Frieda
Demeyer, Vicky Broeckx, A. Camps, S. Carmans and L. Van den
Heurck for the excellent technical performance as well as to ELPEN
pharmaceuticals for financial support.

2704
S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 2696–2704
[28] C. Aujard, Y. Moule, E. Chany-Morel, K. Antonakis, Biochem. Pharmacol. 27
References
(1978) 1037–1042.
[29] T. Halmos, A. Cardon, K. Antonakis, Chem. Biol. Interact. 46 (1983) 11–29.
[30] K. Antonakis, I. Chouroulinkov, C.R. Acad. Sci. Paris 273D (1971) 2661–2663.
[31] K. Antonakis, I. Chouroulinkov, Biochem. Pharmacol. 23 (1974) 2095–2100.
[32] K. Antonakis, T. Halmos, J. Bach, I. Chouroulinkov, Eur. J. Med. Chem. 15 (1980)
237–240.
[33] I. Chouroulinkov, K. Antonakis, C.R. Acad. Sci. Hebd Seances Acad. Sci. D 285
(1977) 1021–1024.
[34] P. Liu, A. Sharon, C.K. Chu, J. Fluorine Chem. 129 (2008) 743–766.
[35] K. Lee, Y. Choi, G. Gumina, W. Zhou, R.F. Schinazi, C.K. Chu, J. Med. Chem. 45
(2002) 1313–1320.
[36] Y. Chong, H. Choo, S. Lee, Y. Choi, R.F. Schinazi, C.K. Chu, J. Med. Chem. 45
(2002) 4888–4898.
[37] K.W. Pankiewicz, Carbohydr. Res. 327 (2000) 87–105.
[38] J.L. Clark, J.C. Mason, L. Hollecker, L.J. Stuyver, P.M. Tharnish, T.R. McBrayer,
M.J. Otto, A.P. Furman, R.F. Schinazi, K.A. Watanabe, Bioorg. Med. Chem. Lett.
16 (2006) 1712–1715.
[1] D. Voet, J.G. Voet, Biochemistry, second ed. John Wiley & Sons, New York, 1995.
[2] G. Gumina, Y. Chong, H. Choo, G. Song, C.K. Chu, Curr. Top. Med. Chem. 2
(2002) 1065–1086.
[3] D.C. Orr, H.T. Figueiredo, C.L. Mo, C.R. Penn, J.M. Cameron, J. Biol. Chem. 267
(1992) 4177–4182.
[4] Y. Kitade, C. Kozaki, T. Miwa, M. Nakanishi, Tetrahedron 58 (2002) 1271–1277.
[5] M.A. Turner, X. Yang, D. Yin, K. Kuczera, R.T. Borchardt, P.L. Howell, Cell Bio-
chem. Biophys. 33 (2000) 101–125.
[6] K.S. Anderson, Biochim. Biophys. Acta 1587 (2002) 296–299.
[7] T.S. Lin, M.Z. Luo, M.C. Liu, R.H. Clarkatzenburg, Y.C. Cheng, W.H. Prusoff,
W.R. Mancini, G.I. Birnbaum, E. Gabe, J. Giziewicz, J. Med. Chem. 34 (1991)
2607–2615.
[8] C.H. Baker, J. Banzon, J.M. Bollinger, J. Stubbe, V. Samano, M.J. Robins, J. Med.
Chem. 34 (1991) 1879–1884.
[9] A.H. Cory, V. Samano, M.J. Robins, J.G. Cory, Biochem. Pharmacol. 47 (1994)
365–371.
´
ˇ
[39] E. Tsoukala, G. Agelis, J. Dolinsek, T. Botic, A. Cencic, D. Komiotis, Bioorg. Med.
Chem. 15 (2007) 3241–3247.
[10] W. Plunkett, P. Huang, Y.Z. Xu, V. Heinemann, R. Gunewald, V. Gandhi, Semin.
Oncol. 22 (Suppl. 11) (1995) 3–10 and references cited therein.
[11] A. Azuma, Y. Nakajima, N. Nishizono, N. Minakawa, M. Suzuki, K. Hanaoka,
T. Kobayashi, M. Tanaka, T. Sasaki, A. Matsuda, J. Med. Chem. 36 (1993)
4183–4189.
[12] A. Azuma, K. Hanaoka, A. Kurihara, T. Kobayashi, S. Miyaushi, N. Kamo,
M. Tanaka, T. Sasaki, A. Matsuda, J. Med. Chem. 38 (1995) 3391–3397.
[13] A. Matsuda, A. Azuma, Nucleosides Nucleotides 14 (1995) 461–471.
[14] I. Verheggen, A. Van Aerschot, S. Toppet, R. Snoeck, G. Janssen, J. Balzarini,
E. De Clercq, P. Herdewijn, J. Med. Chem. 36 (1993) 2033–2040.
[15] I. Verheggen, A. Van Aerschot, L. Van Meervelt, J. Rozenski, L. Wiebe, R. Snoeck,
G. Andrei, J. Balzarini, P. Claes, E. De Clercq, P. Herdewijn, J. Med. Chem. 38
(1995) 826–835.
´
ˇ
[40] S. Manta, G. Agelis, T. Botic, A. Cencic, D. Komiotis, Bioorg. Med. Chem. 15
(2007) 980–987.
ˇ
[41] S. Manta, G. Agelis, T. Botic´, A. Cencic, D. Komiotis, Eur. J. Med. Chem. 43 (2008)
420–428.
[42] M.J. Pe´rez-Pe´rez, E. De Clercq, P. Herdewijn, Bioorg. Med. Chem. Lett. 6 (1996)
1457–1460.
[43] K. Augustyns, F. Vandendriessche, A. Van Aerschot, R. Busson, C. Urbanke,
P. Herdewijn, Nucleic Acids Res. 20 (1992) 4711–4716.
[44] K. Augustyns, J. Rozenski, A. Van Aerschot, R. Busson, P. Claes, P. Herdewijn,
Tetrahedron 50 (1994) 1189–1198 and references cited therein.
[45] K. Augustyns, J. Rozenski, A. Van Aerschot, G. Janssen, P. Herdewijn, J. Org.
Chem. 58 (1993) 2977–2982.
[46] W. Sowa, H.S. Thomas, Can. J. Chem. 44 (1966) 836–837.
[47] A.B. Foster, R. Hems, M.J. Webber, Carbohydr. Res. 5 (1967) 292–301.
[48] M. Dunkel, W. Pfleiderer, Nucleosides Nucleotides 11 (1992) 787–819.
[49] M.J. Robins, J.S. Wilson, F. Hansske, J. Am. Chem. Soc. 105 (1983) 4059–4065.
[50] T.-C. Chien, C.-S. Chen, J.-W. Chern, J. Chin. Chem. Soc. 52 (2005) 1237–1244.
[51] D.H.R. Barton, S.W. McCombie, J. Chem. Soc., Perkin Trans.116 (1975) 1574–1585.
[52] F. Leclercq, M.J. Egron, K. Antonakis, I. Bennani-Baiti, C. Frayssinet, Carbohydr.
Res. 228 (1992) 95–102.
[53] J. Milecki, A. Foldesi, A. Fischer, R.W. Adamiak, J. Chattopadhyaya, J. Labelled
Cpd Radiopharm. 44 (2001) 763–783.
[54] J. Gutzwiller, M. Uskokovic, J. Am. Chem. Soc. 92 (1970) 204–205.
[55] D.L. Boger, K. Machiya, J. Am. Chem. Soc. 114 (1992) 10056–10058.
[56] A. Matsuda, T. Sasaki, Cancer Sci. 95 (2004) 105–111.
[57] G.J. Peters, C.L. Van der Wilt, B. Van Triest, G. Codacci-Pisanelli, P.G. Johnston,
C.J. Van Groeningen, H.M. Pinedo, Eur. J. Cancer 31A (1995) 1299–1305.
[58] H.M. Pinedo, G.F. Peters, J. Clin. Oncol. 6 (1988) 1653–1664.
[59] M. Pickard, C. Dive, A.R. Kinsella, Br. J. Cancer 72 (1995) 1389–1396.
[60] G.A. Piazza, A.K. Rahm, T.S. Finn, B.H. Fryer, H. Li, A.L. Stoumen, R. Pamukcu,
D.J. Ahnen, Cancer Res. 57 (1997) 2452–2459.
[61] D. Komiotis, G. Agelis, S. Manta, N. Tzioumaki, E. Tsoukala, J. Carbohydr. Chem.
25 (2006) 441–450.
[62] M. Tsurusawa, M. Niwa, N. Katano, T. Fujimoto, Cancer Res. 48 (1988) 4288–4293.
[63] J.L. Tonkinson, P. Marder, S.L. Andis, R.M. Schultz, L.S. Gossett, C. Shih,
L.G. Mendelsohn, Cancer Chemother. Pharmacol. 39 (1997) 521–531.
[16] T. Ostrowski, B. Wroblowski, R. Busson, J. Rozenski, E. De Clercq, M.S. Bennet,
J.N. Champness, W.C. Summers, M.R. Sanderson, P. Herdewijn, J. Med. Chem.
41 (1998) 4343–4353.
ˇ
[17] G. Agelis, N. Tzioumaki, T. Tselios, T. Botic´, A. Cencic, D. Komiotis, Eur. J. Med.
Chem. 43 (2008) 1366–1375.
ˇ
[18] G. Agelis, N. Tzioumaki, T. Botic´, A. Cencic, D. Komiotis, Bioorg. Med. Chem. 15
(2007) 5448–5456.
[19] A. Haouz, V. Vanheusden, H. Munier-Lechman, M. Froeyen, P. Herdewijn,
S. Van Galenbergh, M. Delarue, J. Biol. Chem. 278 (2003) 4963–4971.
[20] C. Spanou, S. Manta, D. Komiotis, A. Dervishi, D. Kouretas, Int. J. Mol. Sci. 8
(2007) 695–704.
[21] K. Vastmans, S. Pochet, A. Peys, L. Kerremans, A. Van Aerschot, C. Hendrix,
P. Marliere, P. Herdewijn, Biochemistry 39 (2000) 12757–12765.
[22] K. Vastmans, M. Froeyen, L. Kerremans, S. Pochet, P. Herdewijn, Nucleic Acids
Res. 29 (2001) 3154–3163.
[23] M.J. Egron, F. Leclercq, K. Antonakis, I. Bennani-Baiti, C. Frayssinet, Carbohydr.
Res. 248 (1993) 143–150.
[24] J. Paterson, C. Uriel, M.J. Egron, J. Herscovici, K. Antonakis, M. Alaoui, Anti-
microb. Agents Chemother. 42 (1998) 779–784.
[25] M. Alaoui, C. Lasne, K. Antonakis, I. Chouroulinkov, Mutagenesis 6 (1986)
411–417.
[26] A.R. Khan, K.X. Mulligan, K.K. Redda, A.P. Ollapally, Synth. Commun. 32 (2002)
1023–1030.
[27] M. Alaoui, M.J. Egron, M. Bessodes, K. Antonakis, I. Chouroulinkov, Eur. J. Med.
Chem. 22 (1987) 305–310.