Unsaturated dideoxy fluoro-ketopyranosyl nucleosides as new cytostatic agents: A convenient synthesis of 2,6-dideoxy-3-fluoro-4-keto-β-d-glucopyranosyl analogues of uracil, 5-fluorouracil, thymine, N4-benzoyl cytosine and N6-benzoyl

Article abstract of DOI:10.1016/j.ejmech.2009.06.013

The β-protected nucleosides of uracil (2a), 5-fluorouracil (2b), thymine (2c), N⁴-benzoyl cytosine (2d) and N⁶-benzoyl adenine (2e) were synthesized by condensation of the peracetylated 3-deoxy-3-fluoro-d-glucopyranose (1) with the c

Full text of DOI:10.1016/j.ejmech.2009.06.013

European Journal of Medicinal Chemistry 44 (2009) 4764–4771
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Unsaturated dideoxy fluoro-ketopyranosyl nucleosides as new cytostatic agents:
A convenient synthesis of 2,6-dideoxy-3-fluoro-4-keto-
b- -glucopyranosyl
D
analogues of uracil, 5-fluorouracil, thymine, N4-benzoyl cytosine
and N6-benzoyl adenine
Stella Manta a, Niki Tzioumaki a, Evangelia Tsoukala a, Aggeliki Panagiotopoulou b,
Maria Pelecanou b, Jan Balzarini c, Dimitri Komiotis a
*
a Department of Biochemistry and Biotechnology, Laboratory of Organic Chemistry, University of Thessaly, 26 Ploutonos Street, 41221 Larissa, Greece
b Institute of Biology, National Centre for Scientific Research ‘‘Demokritos’’, 15310 Ag. Paraskevi, Athens, Greece
c 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
and N6-benzoyl adenine (2e) were synthesized by condensation of the peracetylated 3-deoxy-3-fluoro-
b
-protected nucleosides of uracil (2a), 5-fluorouracil (2b), thymine (2c), N4-benzoyl cytosine (2d)
Received 25 August 2008
Received in revised form
13 April 2009
Accepted 11 June 2009
Available online 18 June 2009
D-
glucopyranose (1) with the corresponding silylated bases. The nucleosides were deacetylated and several
subsequent protection and deprotection steps afforded the partially acetylated analogues 6a–e. Selective
iodination followed by hydrogenation gave the acetylated dideoxy analogues of uracil (8a), 5-fluorouracil
(8b), thymine (8c), N4-benzoyl cytosine (8d) and N6-benzoyl adenine (8e), respectively. Finally, direct
oxidation of the free hydroxyl group at the 40-position of 8a–e, and simultaneous elimination reaction of
Keywords:
the b-acetoxyl group, afforded the desired unsaturated 2,6-dideoxy-3-fluoro-4-keto-b-D-glucopyranosyl
Unsaturated dideoxy fluoro ketonucleosides
derivatives 9a–e. The new analogues were evaluated for antiviral and cytostatic activity. Compounds 9a–
e were not active against a broad panel of DNA and RNA viruses at subtoxic concentrations. However,
they were markedly cytostatic against a variety of tumor cell lines. The compounds should be regarded as
potential new lead compounds to be further investigated for anticancer therapy.
Ó 2009 Elsevier Masson SAS. All rights reserved.
b
-Elimination reaction
Antiviral
Antitumor activity
1. Introduction
antioxidant [22] and antibiotic [23] properties and as building
blocks in nucleic acid synthesis [24,25]. Among them, the unsatu-
Nucleoside analogues constitute an important class of antime-
tabolites used in the treatment of hematological malignancies and
in solid tumors [1,2]. These therapeutic compounds are frequently
used to inhibit DNA biosynthesis, a process that is essential for cell
growth and viral replication [3,4]. Substitution of hydrogen atoms
or hydroxyl groups for fluorine has been extensively employed in
the design of such analogues, as this can dramatically affect the
electronic structure of a nucleoside without significantly altering its
size and shape [5]. Thus, 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 anti-
viral agents [6–17].
rated ketonucleosides are a series of cytostatic drugs, which were
found to be highly cytotoxic in vitro [26–28] and to exert powerful
inhibitory action against L1210 leukemia in vivo [29,30]. These
agents are known to inhibit DNA, RNA, and protein synthesis [31]
and to interact with sulfhydryl groups of cellular proteins and
enzymes [32]. Moreover, the absence of a genotoxic effect [33]
makes some of these compounds particularly interesting and indi-
cates that they act by a mechanism that is probably different from
that associated with alkylating or intercalating antitumor drugs.
Based on the above observations, we have previously reported
the synthesis and biological evaluation of a series of unsaturated
fluoro ketopyranonucleoside analogues, which proved to be effi-
cient as tumor cell growth inhibitors and showed to have a prom-
ising potential in combating rotaviral infections. Our studies
indicated, inter alia, that the biological activity appeared to be
independent of the presence of a primary hydroxyl group since the
60-protected analogues exhibited the most promising cytotoxic and
antiviral properties [34–36].
Lately, nucleosides containing pyranosyl rings instead of fur-
anosyl ones have been evaluated for their potential antiviral [18–21],
* Corresponding author. Tel.: þ30 2410 565285; fax: þ30 2410 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.2009.06.013

S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 4764–4771
4765
In view of these facts, it was envisaged that a new class of
unsaturated 30-fluoro-40-ketopyranonucleoside analogues of uracil,
5-fluorouracil, thymine, N4-benzoyl cytosine and N6-benzoyl
adenine, respectively, bearing a methyl group in the 50-position of
the sugar moiety, could be of significant biological interest. In the
present study we attempt to describe the synthesis and biological
importance of these novel molecules.
which were reduced to form the deoxy nucleosides 8a–e by
hydrogen in the presence of palladium-on-carbon. In the final step,
oxidation of the fluoro acetylated dideoxy precursors 8a–e per-
formed by the acetic anhydride/dimethyl sulfoxide system [45]
afforded, after a
b-elimination reaction, the desired unsaturated 2,6-
dideoxy-3-fluoro-b-D-glycero-hex-2-enopyranosyl-4-ulose deriva-
tives of uracil (9a), 5-fluorouracil (9b), thymine (9c), N4-benzoyl
cytosine (9d) and N6-benzoyl adenine (9e), respectively. It should
therefore be mentioned that all attempts to remove the benzoyl
group of the target nucleoside analogues 9d and 9e were unsuc-
cessful and only degradation products were obtained [46–49].
The synthesized compounds were well characterized by NMR,
UV and IR spectroscopies, mass spectrometry and elemental anal-
2. Results and discussion
2.1. Chemistry
ysis. 1H NMR data obtained for the starting nucleosides 2a–e (J1 ,2
,
Our synthetic route to the target compounds 9a–e is shown in
0
0
Scheme 1. The protected 1-(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-
-glucopyranosyl) nucleosides of uracil (2a), 5-fluorouracil (2b) [37],
thymine (2c), N4-benzoyl cytosine (2d) [34] and 9-(2,4,6-tri-O-
acetyl-3-deoxy-3-fluoro-
-glucopyranosyl) nucleoside of N6-
b-
0
0
0
0
J2 ,3 , J3 ,4 ꢀ 9.0 Hz) revealed that, as expected, these compounds
had the
b
configuration and that it existed in the 4C1 conformation.
D
Finally, in compounds 9a–e, the presence of the C(O)CF]CH-
system was ascertained by the disappearance of the signal for H-40
in the 1H NMR spectra and the deshielding of other protons,
especially the olefinic proton H-20 as previously observed for other
b-D
benzoyl adenine (2e) [35], were obtained upon condensation of
1,2,4,6-tetra-O-acetyl-3-deoxy-3-fluoro-glucopyranose (1) [38,39]
with silylated uracil, 5-fluorouracil, thymine, N4-benzoyl cytosine
and N6-benzoyl adenine, respectively, in the presence of trime-
thylsilyl trifluoromethane-sulfonate or tin chloride, in refluxing
(2-deoxy-b-D-hex-2-enopyranosyl-4-ulose) nucleosides [50–52].
acetonitrile. The
b
configuration of these nucleosides was clearly
2.2. Antiviral, cytostatic and cytotoxic activity
established by their 1H NMR spectra. Removal of all O-acetyl pro-
tecting groups of the aforementioned compounds gave 3a–e, in
excellent yields [34,35,40]. Specific acetalation of the unprotected
analogues using 2,2-dimethoxypropane in N,N-dimethylformamide
(DMF) led to the 40,60-O-isopropylidene derivatives 4a–e, respec-
tively. Acetylation of the free hydroxyl group and finally deisopro-
pylidenation, afforded the partially acetylated analogues 6a–e
[34,35]. Replacement of the primary hydroxyl group of compounds
6a–e by an iodine atom using triphenylphosphine together with
iodine and imidazole [41–44], led to the desired iodo derivatives 7a–e
The compounds 9a–e were evaluated against a broad variety of
DNA and RNA viruses but found to be inactive at subtoxic concen-
trations. The cytostatic/cytotoxic activity measurements were based
on (i) the inhibition of tumor cell proliferation of murine leukemia
L1210, murine mammary carcinoma FM3A, human lymphocyte
Molt4/C8andCEMcells,or(ii)microscopicallydetectablealterationof
human embryonic lung (HEL), monkey kidney (Vero), and human
cervix carcinoma (HeLa) cell morphology or (iii) viability staining of
Madin–Darbycanine kidneycells by the colorimetric formasan-based
AcO
O
RO
B
H3C
B
O
O
O
i
iii
F
F
F
OAc
H3C
OAc
O
OR
OAc
OR
4a,b,c,d,e R = H
OR
2a,b,c,d,e R = Ac
1
iv
ii
5a,b,c,d,e R = Ac
3a,b,c,d,e R = H
v
R
HO
CH3
B
B
O
B
O
O
viii
vi
F
F
O
OH
OH
F
OAc
OAc
6a,b,c,d,e
9a,b,c,d,e
7a,b,c,d,e R = I
vii
8a,b,c,d,e R = H
4
6
B = a: Uracil, b: 5-Fluorouracil, c: Thymine, d: N -Benzoyl cytosine, e: N -Benzoyl adenine
Scheme 1. Reagents and conditions: i) silylated base, CH3CN, trimethylsilyl trifluomethane-sulfonate or tin chloride; ii) methanolic ammonia or ethanol, pyridine, NaOH, 0 ꢁC,
30 min, Amberlite IR-120 (Hþ) resin; iii) 2,2-dimethoxypropane, p-toluenesulfonic acid, DMF; iv) pyridine, acetic anhydride; v) 90% trifluoroacetic acid in methanol, 20 ꢁC, 10 min;
vi) iodine, triphenylphosphine, imidazole, tetrahydrofuran, 80 ꢁC, 1 h; vii) H2, 10% Pd/C, triethylamine, ethyl acetate, ethanol, 20 ꢁC, 24 h; viii) acetic anhydride/dimethyl sulfoxide,
100 ꢁC, 10 min.

4766
S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 4764–4771
Table 2
MTS assay. Compounds 9a–e showed a similar cytotoxic activity
against several monolayer cell types (MIC: w20 M). However, the
compoundsmarkedlyinhibitedcellproliferationofsuspensiontumor
cells at IC50 values that ranged between 0.49 and 16 M (Table 1).
Inhibitory activity of 9d and 9e against the incorporation of [3H]dThd, [3H]Urd and
m
[3H]Leu into cellular macromolecules.
a
Compound
IC50
(m
M) for incorporation into cellular macromolecules
m
[3H]dThd
L1210
[3H]Urd
[3H]Leu
There was no significant difference between the compounds in terms
of potency of inhibition, except for 9a–c that were more cytostatic for
MDCK cells when evaluated with the MTS dye staining method. Also,
when compounds such as 9d and 9e were evaluated on their inhibi-
tory potential against macromolecule (DNA, RNA, protein) synthesis
in L1210 and CEM cells, they remarkably inhibited radiolabeled
thymidine (DNA synthesis), uridine (RNA synthesis) and leucine
(protein synthesis) incorporation into methanol-insoluble cell
CEM
L1210
CEM
L1210
CEM
9d
9e
12
12
11
13
10
9.8
12
13
12
13
7.9
14
a
50% inhibitory concentration required to inhibit incorporation of radiolabeled
precursor in methanol-insoluble material.
The chemical shifts are expressed in parts per million (d) and
material to a similar extent (EC50: 7.9–14 mM) (Table 2) upon incu-
bation of the cell suspensions with the radiolabeled precursors and
the test compounds for 20 h.
following abbreviations were used: s ¼ singlet, br s ¼ broad singlet,
d ¼ doublet, dd ¼ doublet doublet, ddd ¼ doublet doublet doublet,
dtr ¼ doublet triplet and m ¼ multiplet. Mass spectra were obtained
with a Micromass Platform LC (ESI-MS). Infrared spectra were
obtained with a Nicolet 6700 FT-IR spectrometer. Optical rotations
were measured using Autopol I polarimeter. All reactions sensitive
to oxygen or moisture were carried out under nitrogen atmosphere
with dry solvents. Acetonitrile was distilled from calcium hydride
and stored over 3 Å molecular sieves. DMF and dimethyl sulfoxide
were also stored over 3 Å molecular sieves. Pyridine was stored over
pellets of potassium hydroxide. Tetrahydrofuran was freshly
distilled under nitrogen from sodium/benzophenone before use.
3. Conclusion
In conclusion, the synthesis of the unsaturated dideoxy fluoro-
ketopyranosyl nucleoside analogues bearing uracil, 5-fluorouracil,
thymine, N4-benzoyl cytosine and N6-benzoyl adenine, respectively
was undertaken, and the target nucleosides were evaluated for
their antiviral and cytostatic/cytotoxic activity. Compounds 9a–e
were not active against a broad panel of DNA and RNA viruses at
subtoxic concentrations. However, they were markedly inhibitory
against the proliferation of a variety of tumor cell lines. The
mechanism of antiproliferative activity is thus far unclear. It would
therefore be interesting to further explore the structure–activity
relationship by modifying the base part as well as replacing the
fluorine or methyl group by different chemical entities. We believe
that unsaturated dideoxy fluoro-ketopyranosyl nucleoside
analogues should be considered as novel lead compounds that have
to be further explored for antiproliferative (i.e. antitumor) activity.
4.2. Synthesis of 1-(2,6-dideoxy-3-fluoro-b-D-glycero-hex-2-
enopyranosyl-4-ulose)uracil (9a)
4.2.1. 1-(2,4,6-Tri-O-acetyl-3-deoxy-3-fluoro-b-D-
glucopyranosyl)uracil (2a)
A mixture of uracil (1.79 g, 15.99 mmol), hexamethyldisilazane
(HMDS) (4.18 mL,19.83 mmol) and saccharine (0.14 g, 0.74 mmol) in
anhydrous CH3CN (56 mL) was refluxed for 30 min under nitrogen.
Tetraacetylated 3-deoxy-3-fluoro-D-glucose (1) [38,39] (4.00 g,
11.42 mmol) and trimethylsilyl
trifluoromethane-sulfonate
4. Experimental part
(2.89 mL, 15.99 mmol) were then added and the reaction mixture
was refluxed for 3 h, cooled, neutralized with aqueous sodium
bicarbonate, and extracted with CH2Cl2 (1000 mL). The organic layer
was washed with water (3 ꢂ 20 mL) and dried over anhydrous
sodium sulfate, evaporated to dryness, finally purified by flash
column chromatography using ethyl acetate–n-hexane (7:3) as
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 spectrometer at
400, 376 and 100 MHz, respectively using CDCl3 and dimethyl
sulfoxide-d6 (DMSO-d6) with internal tetramethylsilane (TMS) for
1H and 13C and internal trifluorotoluene (TFT) for 19F.
eluant to give compound 2a as a solid. Yield: 3.40 g (74%); Rf ¼ 0.3 in
22
ethyl acetate–n-hexane (7:3); m.p. 115–117 ꢁC; [
a]
D
ꢃ5.3 (c 0.10,
CHCl3); UV (CHCl3): lmax 260 nm (3 d 8.50 (br
7068); 1H NMR (CDCl3):
s, NH), 7.35 (d, 1H, J5,6 ¼ 8.2 Hz, H-6), 5.840(d, 1H, H-5), 5.79 (d, 1H,
0
0
0
0
J1 ,2 ¼ 9.6 Hz, H-1 ), 5.30–5.22 (m, 2H, H-2 and H-4 ), 4.74 (dtr, 1H,
0
0
0
0
0
0
JF,3 ¼ 51.7 Hz, J2 ,3 ¼ 9.1 Hz, J3 ,4 ¼ 9.0 Hz, H-3 ), 4.30–4.11 (m, 2H, H-
6a0,6b0), 3.83 (m, 1H, H-50), 2.14 and 2.10 and 2.08 (3s, 9H, 3OAc); 19F
NMR:
d
ꢃ65.0; Anal. Calcd for C16H19FN2O9: C, 47.76; H, 4.76; N, 6.96.
Found: C, 47.65; H, 4.87; N, 6.84; ESI-MS (m/z): 403.34 (M þ Hþ).
Table 1
Cytostatic and cytotoxic activity of 9a–e against a panel of tumor cell lines.
4.2.2. 1-(3,4-Dideoxy-3-fluoro-b-D-glucopyranosyl)uracil (3a)
Compound IC50
L1210
2.9 ꢄ 1.1
(
m
M)
CC50
MDCK
2.5 ꢄ 1.2 2.2 ꢄ 0.9 0.6
(
m
M) MICc
HEL Vero HeLa
(mM)
a
b
A mixture of methanolic ammonia and compound 2a (3.40 g,
8.45 mmol) stirred for 4 h at room temperature. The reaction
mixture was concentrated and crude 3a was obtained as a colorless
FM3A
16 ꢄ 2
Molt4/C8 CEM
9a
9b
9c
9d
9e
20 20
20 20
20 20
20 20
20
20
20
20
oil and it was used without further purification. Yield: 2.05 g (88%);
0.82 ꢄ 0.47 0.49 ꢄ 0.45 3.3 ꢄ 0.2 3.7 ꢄ 0.6 0.6
2.8 ꢄ 1.0 10 ꢄ 8 2.5 ꢄ 1.3 2.6 ꢄ 1.1 0.7
1.4 ꢄ 0.0 11.9 ꢄ 5.2 1.6 ꢄ 0.2 1.7 ꢄ 0.5 8.2
2.4 ꢄ 0.7 6 ꢄ 1 5.0 ꢄ 3.9 4.3 ꢄ 3.4 12
22
[a
]
þ 5.3 (c 0.10, MeOH); UV (MeOH): lmax 260 nm (
3 6235); Anal.
D
Calcd for C10H13FN2O6: C, 43.48; H, 4.74; N, 10.14. Found: C, 43.39;
20 20 100
H, 4.86; N, 9.97; ESI-MS (m/z): 277.23 (M þ Hþ).
a
50% inhibitory concentration, or compound concentration required to inhibit
cell proliferation by 50%.
4.2.3. 1-(3-Deoxy-3-fluoro-4,6-O-isopropylidene-b-D-
glucopyranosyl)uracil (4a)
b
50% cytotoxic concentration, or compound concentration required to reduce cell
viability by 50% as measured by MTS dye staining.
c
Compound 3a (2.05 g, 7.44 mmol) was dissolved in a mixture of
Minimal inhibitory concentration, or compound concentration required to
induce a microscopically visible alteration of cell morphology.
29.8 mL of 2,2-dimethoxypropane and 94.1 mL of dry DMF. To this

S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 4764–4771
4767
was added p-toluenesulfonic acid (1.70 g, 8.93 mmol) and the
4.2.7. 1-(2-O-Acetyl-3,6-dideoxy-3-fluoro-
glucopyranosyl)uracil (8a)
b-D-
mixture stirred at room temperature for 2 h. The reaction mixture
was neutralized with triethylamine so that pH did not exceed 7. The
mixture was concentrated under high vacuum to eliminate the
DMF. Purification by flash column chromatography using ethyl
After two vacuum/H2 cycles to remove air from the reaction tube,
the stirred mixture of 7a (1.13 g, 2.64 mmol), 10% Pd/C (0.36 g) and
triethylamine (0.73 mL, 5.28 mmol) in ethyl acetate (80.8 mL) and
ethanol (80.8 mL) was hydrogenated at ambient pressure (balloon)
and temperature (ca. 20 ꢁC) for 24 h. The reaction mixture was
filtered and the filtrate was concentrated in vacuum. The residue
was purified by flash column chromatography using ethyl acetate–
acetate–n-hexane (8:2) as eluant gave 4a as a yellowish oil.
22
Yield: 1.53 g (65%); Rf ¼ 0.25 in ethyl acetate–n-hexane (8:2); [
a]
D
ꢃ7.0 (c 0.33, CHCl3); UV (CHCl3): lmax 257 nm (
3
5704); 1H NMR
(CDCl3):
d
9.71 (br s, NH), 7.31 (d, 1H0, J5,6 ¼ 8.1 Hz, H-6), 5.82 (d, 1H,
0
0
0
H-5), 5.71 (d, 1H, J1 ,2 ¼ 9.2 Hz, H-1 ), 4.66 (dtr, 1H, JF,3 ¼ 53.2 Hz,
n-hexane (7:3) as eluant to give 8a as a white foam. Yield: 0.60 g
22
0
0
0
0
0
0
0
J2 ,3 ¼ 8.5 Hz, J3 ,4 ¼ 8.8 Hz, H-3 ), 4.02–3.88 (m, 2H, H-2 and H-4 ),
3.86–3.73 (m, 2H, H-6a0,6b0), 3.53 (m, 1H, H-50), 1.55 and 1.48 (2s,
6H, 2 ꢂ CH3); Anal. Calcd for C13H17FN2O6: C, 49.37; H, 5.42; N, 8.86.
Found: C, 49.15; H, 5.55; N, 9.03; ESI-MS (m/z): 317.27 (M þ Hþ).
(75%); Rf ¼ 0.38 in ethyl acetate–n-hexane (7:3); [
a
]
þ7.0 (c 0.50,
D
CHCl3); UV (CHCl3): lmax 256 nm (
3
6812); 1H NMR (CDCl3):
d 8.34
(br s, NH), 7.33 (d, 1H, J5,6 ¼ 8.2 Hz, H-6), 5.80 (d, 1H, H-5), 5.73 (d,
1H, J1 ,2 ¼ 9.4 Hz, H-10), 5.16 (m, 1H, H-20), 4.59 (dtr, 1H,
0
0
0
0
0
0
0
0
JF,3 ¼ 52.4 Hz, J2 ,3 ¼ 8.6 Hz, J3 ,4 ¼ 8.9 Hz, H-3 ), 3.68–3.50 (m, 2H,
H-40 and H-50), 2.08 (s, 3H, OAc), 1.39 (d, 3H, J5 ,6 ¼ 5.7 Hz, H-6 ); 19F
0
0
0
4.2.4. 1-(2-O-Acetyl-3-deoxy-3-fluoro-4,6-O-isopropylidene-b-D-
glucopyranosyl)uracil (5a)
NMR:
d
ꢃ65.5; Anal. Calcd for C12H15FN2O6: C, 47.68; H, 5.00; N, 9.27.
Found: C, 47.97; H, 5.18; N, 8.91; ESI-MS (m/z): 303.24 (M þ Hþ).
To a solution of 4a (1.53 g, 4.84 mmol) in dry pyridine (24.2 mL)
was added acetic anhydride (0.91 mL, 9.68 mmol) and the resultant
mixture was stirred at room temperature for 1 h. Methanol
(0.45 mL) was added to quench the reaction and the mixture was
concentrated under high vacuum to remove the solvents. Purifi-
cation by flash column chromatography using ethyl acetate–n-
4.2.8. 1-(2,6-Dideoxy-3-fluoro-b-D-glycero-hex-2-enopyranosyl-4-
ulose)uracil (9a)
To a solution of 8a (0.60 g, 1.98 mmol) in dimethyl sulfoxide
(9.7 mL) was added acetic anhydride (4.84 mL, 51.28 mmol). The
mixture was heated at 100 ꢁC for 10 min, then cooled to room
temperature, diluted with ethyl acetate and washed with water.
The organic layer was dried over anhydrous sodium sulfate, filtered
and evaporated to dryness. The residue was purified by flash
column chromatography using ethyl acetate–n-hexane (6:4) as
hexane (1:1) as eluant gave 5a as a white powder. Yield: 1.54 g
22
(89%); Rf ¼ 0.25 in ethyl acetate–n-hexane (1:1); [
CHCl3); UV (CHCl3): lmax 255 nm (
(br s, NH), 7.32 (d, 1H, J5,6 ¼ 8.2 Hz, H-6), 5.82-5.77 (m, 2H,
a
]
ꢃ2.0 (c 0.39,
D
3
8165); 1H NMR (CDCl3):
d 8.50
0
0
0
0
0
0
J1 ,2 ¼ 9.2 Hz, H-5 and H-1 ), 5.79 (d, 1H, J1 ,2 ¼ 9.0 Hz, H-1 ), 5.23
(m, 1H, H-20), 4.70 (dtr, 1H, JF,3 ¼053.0 Hz, J2 ,3 ¼ 8.8 Hz,
eluant to afford pure 9a as a white solid. Yield: 0.31 g (65%); Rf ¼ 0.4
0
0
0
22
0
J3 ,4 ¼ 9.0 Hz, H-3 ), 4.03–3.94 (m, 1H, H-4 ), 3.91–3.75 (m, 2H, H-
6a0,6b0), 3.50 (m, 1H, H-50), 2.09 (s, 3H, OAc), 1.55 and 1.48 (2s, 6H,
2 ꢂ CH3); Anal. Calcd for C15H19FN2O7: C, 50.28; H, 5.34; N, 7.82.
Found: C, 50.12; H, 5.56; N, 7.50; ESI-MS (m/z): 359.34 (M þ Hþ).
in ethyl acetate–n-hexane (6:4); m.p. 247–250 ꢁC; [
a
]
ꢃ2.0 (c
0
0
D
0.50, CHCl3); UV (CHCl3): lmax 255 nm (
3
10814); 1H NMR (DMSO-
0
d6):
d
7.69 (d, 1H, J5,6 ¼ 8.0 Hz, H-6), 7.01 (dd, 1H, JF,1 ¼12.5 Hz,
0
0
0
0
0
J1 ,2 ¼ 1.3 Hz, H-1 ), 6.81 (dd, 1H, JF,2 ¼ 6.4 Hz, H-2 ), 5.67 (d, 1H, H-
5), 4.69 (m, 1H, H-50), 1.30 (d, 3H, J5 ,6 ¼ 6.5 Hz, H-6 ); 13C NMR
0
0
0
(DMSO-d6):
d
189.05 (C-40); 163.00 (C-4); 153.54 (C-30); 150.06 (C-
4.2.5. 1-(2-O-Acetyl-3-deoxy-3-fluoro-b-D-glucopyranosyl)uracil
(6a)
2); 141.71 (C-6); 123.36 (C-20); 102.79 (C-5); 77.32 (C-10); 75.85 (C-
50); 14.81 (C-60); 19F NMR: –64.3; IR (Neat, cmꢃ1): 1711.10 (keto
d
Product 5a (1.54 g, 4.31 mmol), obtained from the previous
procedure, was dissolved in 21.6 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. Purification by flash column
group); Anal. Calcd for C10H9FN2O4: C, 50.01; H, 3.78; N, 11.66.
Found: C, 50.19; H, 3.56; N, 11.39; ESI-MS (m/z): 241.17 (M þ Hþ).
chromatography using ethyl acetate as eluant gave 6a as a foam.
4.3. Synthesis of 1-(2,6-dideoxy-3-fluoro-b-D-glycero-hex-2-
enopyranosyl-4-ulose)5-fluorouracil (9b)
22
Yield: 1.23 g (90%); Rf ¼ 0.23 in ethyl acetate; [
a]
þ 7.0 (c 0.50,
D
MeOH); UV (MeOH): lmax 257 nm (
3
3530); Anal. Calcd for
C12H15FN2O7: C, 45.29; H, 4.75; N, 8.80. Found: C, 45.64; H, 4.57; N,
The isopropylidene 5-fluorouracil derivative 4b was synthesized
by condensation of peracetylated 3-deoxy-3-fluoro- -glucopyr-
anose (1) [38,39] with silylated 5-fluorouracil, followed by depro-
tection of the -nucleoside formed 2b and finally by specific
9.19; ESI-MS (m/z): 319.27 (M þ Hþ).
D
4.2.6. 1-(2-O-Acetyl-3-deoxy-3-fluoro-6-iodo-b-D-
b
glucopyranosyl)uracil (7a)
acetalation of the fully unprotected analogue 3b, as previously
A
mixture of 6a (1.23 g, 3.88 mmol), imidazole (0.53 g,
described [37].
7.76 mmol), triphenylphosphine (1.53 g, 5.82 mmol) and iodine
(1.48 g, 5.82 mmol) in tetrahydrofuran (38 mL) was stirred under
reflux at a bath temperature of 80 ꢁC for 1 h. The reaction mixture
was then cooled to room temperature and concentrated in vacuum.
Purification by flash column chromatography using ethyl acetate–
4.3.1. 1-(2-O-Acetyl-3-deoxy-3-fluoro-4,6-O-isopropylidene-
glucopyranosyl)5-fluorouracil (5b)
b-D-
5-Fluorouracil derivative 5b was synthesized from 1-(3-deoxy-
3-fluoro-4,6-O-isopropylidene- -glucopyranosyl)5-fluorouracil
b-D
n-hexane (6:4) as eluant gave 7a as a syrup. Yield: 1.13 g (68%);
(4b) [37] by the same methodology as described for the synthesis of
5a. Compound 5b was obtained as a white powder following
purification by flash column chromatography using ethyl acetate–
22
Rf ¼ 0.24 in ethyl acetate–n-hexane (6:4); [
a
]
D
ꢃ3.0 (c 0.50, CHCl3);
UV (CHCl3): lmax 256 nm (
3
5621); 1H NMR (CDCl3):
d
8.03 (br s,
0
0
NH), 7.36 (d, 1H, J5,6 ¼ 8.4 Hz, H-6), 5.87–5.81 (m, 2H, J1 ,2 ¼ 9.2 Hz,
n-hexane (1:1) as eluant. Yield: 1.80 g (80%); Rf ¼ 0.29 in ethyl
22
H-5 and H-10), 5.18 (m, 1H, H-20), 4.70 (dtr, 1H, JF,3 ¼ 52.3 Hz,
0
acetate–n-hexane (1:1); [
a]
þ 2.0 (c 0.27, CHCl3); UV (CHCl3): lmax
D
261 nm (
3
2556); 1H NMR (CDCl3):
d 8.30 (br s, NH), 7.38 (d, 1H,
0
0
0
0
0
0
J2 ,3 ¼ 8.5 Hz, J3 ,4 ¼ 8.9 Hz, H-3 ), 3.89 (m, 1H, H-4 ), 3.56 (m, 2H, H-
6a0,6b0), 3.17 (m, 1H, H-50), 2.09 (s, 3H, OAc); Anal. Calcd for
C12H14FIN2O6: C, 33.66; H, 3.30; N, 6.54. Found: C, 33.93; H, 3.52; N,
6.30; ESI-MS (m/z): 429.17 (M þ Hþ).
0
0
0
0
J6,F5 ¼ 5.6 Hz, H-6), 5.75 (dd, 1H, J1 ,F5 ¼1.3 Hz, J1 ,2 ¼ 9.3 Hz, H-1 ),
5.16 (m, 1H, H-20), 4.70 (dtr, 1H, JF,3 ¼ 52.9 Hz, J2 ,3 ¼ J3 ,4 ¼ 9.0 Hz,
0
0
0
0
0
H-30), 3.98 (m, 1H, H-40), 3.93–3.78 (m, 2H, H-6a0,6b0), 3.50 (m, 1H,

4768
S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 4764–4771
H-50), 2.10 (s, 3H, OAc), 1.56 and 1.48 (2s, 6H, 2 ꢂ CH3); Anal. Calcd
for C15H18F2N2O7: C, 47.88; H, 4.82; N, 7.44. Found: C, 47.75; H, 5.15;
N, 7.67; ESI-MS (m/z): 377.32 (M þ Hþ).
4.4. Synthesis of 1-(2,6-dideoxy-3-fluoro-b-D-glycero-hex-2-
enopyranosyl-4-ulose)thymine (9c)
4.4.1. 1-(2,4,6-Tri-O-acetyl-3-deoxy-3-fluoro-b-D-
glucopyranosyl)thymine (2c)
4.3.2. 1-(2-O-Acetyl-3-deoxy-3-fluoro-b-D-glucopyranosyl)5-
fluorouracil (6b)
5-Fluorouracil derivative 6b was synthesized from 5b by the
same methodology as described for the synthesis of 6a. Compound
6b was obtained as a foam following purification by flash column
A mixture of thymine (2.02 g, 15.99 mmol), HMDS (4.18 mL,
19.83 mmol) and saccharine (0.13 g, 0.74 mmol) in anhydrous
CH3CN (56 mL) was refluxed for 30 min, under nitrogen. Tetraace-
tylated 3-deoxy-3-fluoro-D-glucose (1) [38,39] (4.00 g, 11.42 mmol)
and tin chloride (1.87 mL, 15.99 mmol) were then added and the
reaction mixture was refluxed for 2 h, cooled, neutralized with
aqueous sodium bicarbonate, and extracted with CH2Cl2 (1000 mL).
The organic layer was washed with water (3 ꢂ 20 mL) and dried
over anhydrous sodium sulfate, evaporated to dryness, finally
purified by flash column chromatography using ethyl acetate–n-
hexane (8:2) as eluant to give compound 2c as a solid. Yield: 3.42 g
chromatography using ethyl acetate–n-hexane (8:2) as eluant.
22
Yield: 1.39 g (87%); Rf ¼ 0.26 in ethyl acetate–n-hexane (8:2); [
a]
D
þ14.0 (c 0.50, MeOH); UV (MeOH): lmax 262 nm (
3 6394); Anal.
Calcd for C12H14F2N2O7: C, 42.86; H, 4.20; N, 8.33. Found: C, 43.04;
H, 4.09; N, 8.10; ESI-MS (m/z): 337.23 (M þ Hþ).
(72%); Rf ¼ 0.35 in ethyl acetate–n-hexane (8:2); m.p. 180–182 ꢁC;
4.3.3. 1-(2-O-Acetyl-3-deoxy-3-fluoro-6-iodo-b-D-
glucopyranosyl)5-fluorouracil (7b)
5-Fluorouracil derivative 7b was synthesized from 6b by the
same methodology as described for the synthesis of 7a. Compound
7b was obtained as an oil following purification by flash column
chromatography using dichloromethane–methanol (9.5:0.5) as
22
[
a
]
ꢃ10.5 (c 0.10, CHCl3); UV (CHCl3): lmax 260 nm (
3
7894); 1H
D
NMR (CDCl3):
d
8.20 (br s, NH), 7.16 (s0, 1H, H-6), 5.78 (d, 1H,
0
0
0
0
J1 ,2 ¼ 9.6 Hz, H-1 ), 5.33–5.22 (m, 2H, H-2 and H-4 ), 4.73 (dtr, 1H,
0
0
0
0
0
0
JF,3 ¼ 51.8 Hz, J2 ,3 ¼ 9.1 Hz, J3 ,4 ¼ 9.0 Hz, H-3 ), 4.30–4.12 (m, 2H,
H-6a0,6b0), 3.85–3.80 (m, 1H, H-50), 2.15 and 2.10 and 2.08 (3s, 9H,
3OAc), 1.97 (s, 3H, 5-CH3); 19F NMR:
d
ꢃ65.0; Anal. Calcd for
eluant. Yield: 1.04 g (56%); Rf ¼ 0.20 in dichloromethane–methanol
C17H21FN2O9: C, 49.04; H, 5.08; N, 6.73. Found: C, 49.17; H, 4.92; N,
22
(9.5:0.5); [
(
a]
ꢃ2.0 (c 0.50, CHCl3); UV (CHCl3): lmax 263 nm
D
6.58; ESI-MS (m/z): 417.35 (M þ Hþ).
3 d 8.35 (br s, NH), 7.43 (d, 1H,
6140); 1H NMR (CDCl3):
0
0
0
0
J6,F5 ¼ 5.6 Hz, H-6), 5.82 (dd, 1H, J1 ,F5 ¼1.0 Hz, J1 ,2 ¼ 9.7 Hz, H-1 ),
4.4.2. 1-(3,4-Dideoxy-3-fluoro-b-D-glucopyranosyl)thymine (3c)
Thymine derivative 3c was synthesized from 2c by the same
methodology as described for the synthesis of 3a. Compound 3c
5.11 (m, 1H, H-020), 4.70 (dtr, 1H, JF,3 ¼ 52.0 Hz, J2 ,3 ¼ 8.9 Hz,
0
0
0
0
0
0
0
0
J3 ,4 ¼ 9.1 Hz, H-3 ), 3.89 (m, 1H, H-4 ), 3.56 (m, 2H, H-6a ,6b ), 3.19
(m, 1H, H-50), 2.10 (s, 3H, OAc); Anal. Calcd for C12H13F2IN2O6: C,
32.31; H, 2.94; N, 6.28. Found: C, 32.13; H, 3.25; N, 6.40; ESI-MS
(m/z): 447.13 (M þ Hþ).
was obtained as a colorless oil and it was used without further
22
purification. Yield: 2.14 g (90%); [
(MeOH): lmax 260 nm (
a
]
þ 5.3 (c 0.10, MeOH); UV
D
3
6794); Anal. Calcd for C11H15FN2O6: C,
45.52; H, 5.21; N, 9.65. Found: C, 45.35; H, 5.14; N, 9.53; ESI-MS
(m/z): 291.26 (M þ Hþ).
4.3.4. 1-(2-O-Acetyl-3,6-dideoxy-3-fluoro-b-D-glucopyranosyl)5-
fluorouracil (8b)
4.4.3. 1-(3-Deoxy-3-fluoro-4,6-O-isopropylidene-b-D-
glucopyranosyl)thymine (4c)
Thymine derivative 4c was synthesized from 3c by the same
methodology as described for the synthesis of 4a. Compound 4c was
obtained as a yellowish oil following purification by flash column
5-Fluorouracil derivative 8bwas synthesized from 7b by the same
methodology as described for the synthesis of 8a. Compound 8b was
obtained as a foam following purification by flash column chroma-
tography using ethyl acetate–n-hexane (2:8) as eluant. Yield: 0.51 g
22
(68%); Rf ¼ 0.10 in ethyl acetate–n-hexane (2:8); [
a
]
þ12.0 (c 0.50,
D
CHCl3); UV (CHCl3): lmax 263 nm (
3
7708); 1H NMR (CDCl3):
d
8.55 (br
chromatography using ethyl acetate–n-hexane (7:3) as eluant.
22
0
Yield: 1.76 g (72%); Rf ¼ 0.25 in ethyl acetate–n-hexane (7:3); [
ꢃ6.0 (c 0.50, CHCl3); UV (CHCl3): lmax 263 nm (
(CDCl3):
a]
D
0
0
s, NH), 7.40 (d,1H, J6,F5 ¼ 5.7 Hz, H-6), 5.70 (d,1H, J1 ,2 ¼ 9.6 Hz, H-1 ),
5156); 1H NMR
5.10 (m, 1H, H-20 0), 4.60 (dtr, 1H, JF,3 ¼ 52.0 Hz, 0 J2 ,3 ¼ 8.5 Hz,
3
0
0
0
0
0
0
d
8.00 (br s, NH), 7.11 (s, 1H, H-6), 5.76 (d, 1H, J1 ,2 ¼ 9.3 Hz,
0
0
J3 ,4 ¼ 8.9 Hz, H-3 ), 3.69–3.51 (m,0 2H, H-4 and H-5 ), 2.09 (s, 3H,
H-10), 4.71 (dtr, 1H, JF,3 ¼ 53.3 Hz, J2 ,3 ¼ 8.6 Hz, J3 ,4 ¼ 8.7 Hz, H-3 ),
4.00–3.87 (m, 2H, H-20 and H-40), 3.84–3.73 (m, 2H, H-6a0,6b0), 3.56
(m, 1H, H-50), 2.96 and 2.88 (2s, 6H, 2 ꢂ CH3), 1.88 (s, 3H, 5-CH3);
Anal. Calcd for C14H19FN2O6: C, 50.91; H, 5.80; N, 8.48. Found: C,
50.72; H, 6.08; N, 8.80; ESI-MS (m/z): 331.32 (M þ Hþ).
0
OAc),1.41 (d, 3H, J5 ,6 ¼ 5.6 Hz, H-6 ); 19F NMR:
d
ꢃ64.3, ꢃ63.2; Anal.
0
0
0
0
0
0
0
Calcd for C12H14F2N2O6: C, 45.01; H, 4.41; N, 8.75. Found: C, 44.90; H,
4.65; N, 8.89; ESI-MS (m/z): 321.26 (M þ Hþ).
4.3.5. 1-(2,6-Dideoxy-3-fluoro-b-D-glycero-hex-2-enopyranosyl-4-
ulose)5-fluorouracil (9b)
4.4.4. 1-(2-O-Acetyl-3-deoxy-3-fluoro-4,6-O-isopropylidene-b-D-
5-Fluorouracil derivative 9b was synthesized from 8b by the
same methodology as described for the synthesis of 9a. Compound
9b was obtained as a white powder following purification by flash
column chromatography using ethyl acetate–n-hexane (2:8) as
glucopyranosyl)thymine (5c)
Thymine derivative 5c was synthesized from 4c by the same
methodology as described for the synthesis of 5a. Compound 5c
was obtained as a white foam following purification by flash
column chromatography using ethyl acetate–n-hexane (7:3) as
eluant. Yield: 0.22 g (55%); Rf ¼ 0.16 in ethyl acetate–n-hexane
22
(2:8); [
a
]
þ6.0 (c 0.50, CHCl3); UV (CHCl3): lmax 259 nm (
3 9433);
eluant. Yield: 1.63 g (82%); Rf ¼ 0.37 in ethyl acetate–n-hexane
D
1H NMR (DMSO-d6):
d
8.15 (d, 1H, J6,F5 ¼ 6.7 Hz, 0H-6), 6.93 (d, 1H,
22
(7:3); [
a
]
ꢃ2.0 (c 0.28, CHCl3); UV (CHCl3): lmax 261 nm (
3 3756);
D
0
1H NMR (CDCl3):0 d 8.38 (br s, NH), 07.12 (s, 1H, H-6), 5.78 (d, 1H,
0
0
JF,1 ¼12.4 Hz, H-1 ), 6.81 (d, 1H, JF0,2 ¼ 5.2 Hz, H-2 ), 4.69 (m, 1H, H-
50), 1.31 (d, 3H, J5 ,6 ¼ 6.5 Hz, H-6 ); 13C NMR (DMSO-d6):
d
189.03
0
0
0
0
0
J1 ,2 ¼ 9.5 Hz, H-1 ), 5.25 (m, 1H, H-2 ), 4.69 (dtr, 1H, JF,3 ¼0 53.0 Hz,
(C-40); 153.66 (C-4); 151.41 (C-30); 148.79 (C-2); 139.41 (C-5);
0
0
0
0
0
J2 ,3 ¼ 8.8 Hz, J3 ,4 ¼ 8.9 Hz, H-3 ), 4.00–3.77 (m, 3H, H-4 and H-
6a0,6b0), 3.50 (m, 1H, H-50), 2.08 (s, 3H, OAc), 1.95 (s, 3H, 5-CH3), 1.56
and 1.48 (2s, 6H, 2 ꢂ CH3); Anal. Calcd for C16H21FN2O7: C, 51.61; H,
5.68; N, 7.52. Found: C, 51.75; H, 5.46; N, 7.63; ESI-MS (m/z): 373.33
(M þ Hþ).
125.96; (C-6); 122.69 (C-20); 77.52 (C-10); 75.79 (C-50); 14.82 (C-60);
19F NMR:
d
–65.0, –65.5; IR (Neat, cmꢃ1): 1714.45 (keto group);
Anal. Calcd for C10H8F2N2O4: C, 46.52; H, 3.12; N, 10.85. Found: C,
46.38; H, 3.34; N, 10.74; ESI-MS (m/z): 259.16 (M þ Hþ).

S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 4764–4771
4769
4.4.5. 1-(2-O-Acetyl-3-deoxy-3-fluoro-
b
-D-glucopyranosyl)thymine
by selective deprotection of the
b-nucleoside formed 2d, specific
(6c)
acetalation of the base protected analogue 3d, conventional acet-
ylation of the partially protected derivative 4d and finally by dei-
sopropylidenation of the acetylated analogue 5d, as previously
described [34,35].
Thymine derivative 6c was synthesized from 5c by the same
methodology as described for the synthesis of 6a. Compound 6c
was obtained as a powder following purification by flash column
chromatography using ethyl acetate as eluant. Yield: 1.31 g (90%);
22
Rf ¼ 0.19 in ethyl acetate; [
a]
þ12.0 (c 0.50, MeOH); UV (MeOH):
4.5.1. 1-(2-O-Acetyl-3-deoxy-3-fluoro-6-iodo-b-D-glucopyranosyl)-
D
lmax 262 nm (
3
7427); Anal. Calcd for C13H17FN2O7: C, 46.99; H, 5.16;
N4-benzoyl cytosine (7d)
N, 8.43. Found: C, 47.23; H, 4.98; N, 8.54; ESI-MS (m/z): 333.27
(M þ Hþ).
Cytosine derivative 7d was synthesized from 1-(2-O-acetyl-3-
deoxy-3-fluoro- cytosine (6d)
b-D
-glucopyranosyl)-N4-benzoyl
[34,35] by the same methodologyas described for the synthesis of 7a.
Compound 7d was obtained as a yellow syrup following purification
by flash column chromatography using dichloromethane–methanol
4.4.6. 1-(2-O-Acetyl-3-deoxy-3-fluoro-6-iodo-b-D-
glucopyranosyl)thymine (7c)
Thymine derivative 7c was synthesized from 6c by the same
methodology as described for the synthesis of 7a. Compound 7c
was obtained as a syrup following purification by flash column
chromatography using ethyl acetate–n-hexane (4:6) as eluant.
(9.5:0.5) as eluant. Yield: 1.56 g (62%); Rf ¼ 0.35 in dichloromethane–
22
methanol (9.5:0.5); [
a
]
þ6.0 (c 0.50, CHCl3); UV (CHCl3): lmax
D
260 nm (
3
18038); 1H NMR (CDCl3)
d
0
8.77 (br s,1H, NH), 7.90–7.50 (m,
0
0
7H, Bz, H-5 and H-6), 6.14 (d, 1H, J1 ,2 ¼ 9.2 Hz, H-1 ), 5.18 (m, 1H, H-
0
20), 4.80 (dtr, 1H, JF,3 ¼ 52.2 Hz, J2 ,3 ¼ J3 ,4 ¼ 8.9 Hz, H-3 ), 3.91 (m,
0
0
0
0
0
Yield: 1.13 g (65%); Rf ¼ 0.16 in ethyl acetate–n-hexane (4:6);
22
[
a
]
þ 3.0 (c 0.28, CHCl3); UV (CHCl3): lmax 261 nm (
3
5102); 1H
1H, H-40) 3.62-3.56 (m, 2H, H-6a0,6b0), 3.26 (m, 1H, H-50), 2.06 (s, 3H,
D
NMR (CDCl3):
d
8.53 (br s, NH), 7.19 (s, 1H, H-6), 5.78 (d, 1H,
OAc); 19F NMR:
d
ꢃ65.0. Anal. Calcd for C19H19FIN3O6: C, 42.95; H,
0
0
0
0
0
J1 ,2 ¼ 9.3 Hz, H-1 ), 5.53 (m, 1H, H-2 ), 4.98 (m, 1H, 0H-4 ), 4.27 (ddd,
3.60; N, 7.91. Found: C, 43.15; H, 3.47; N, 8.23; ESI-MS (m/z): 532.29
(M þ Hþ).
0
0
0
0
0
1H, JF,3 ¼ 48.9 Hz, J2 ,3 ¼ 8.8 Hz, J3 ,4 ¼ 8.9 Hz, H-3 ), 3.43-3.23 (m,
2H, H-6a0,6b0), 3.14 (m, 1H, H-50), 2.07 (s, 3H, OAc), 1.99 (s, 3H, 5-
CH3); Anal. Calcd for C13H16FIN2O6: C, 35.31; H, 3.65; N, 6.34.
Found: C, 34.99; H, 3.77; N, 6.48; ESI-MS (m/z): 443.19 (M þ Hþ).
4.5.2. 1-(2-O-Acetyl-3,6-dideoxy-3-fluoro-b-D
-glucopyranosyl)-N4-
benzoyl cytosine (8d)
Cytosine derivative 8d was synthesized from 7d by the same
methodology as described for the synthesis of 8a. Compound 8d
was obtained as a yellowish foam following purification by flash
4.4.7. 1-(2-O-Acetyl-3,6-dideoxy-3-fluoro-b-D-
glucopyranosyl)thymine (8c)
Thymine derivative 8c was synthesized from 7c by the same
methodology as described for the synthesis of 8a. Compound 8c
was obtained as a colorless oil following purification by flash
column chromatography using ethyl acetate–n-hexane (4:6) as
column chromatography using ethyl acetate as eluant. Yield: 0.66 g
22
(55%); Rf ¼ 0.24 in ethyl acetate; [
a
]
þ 26.0 (c 0.50, CHCl3); UV
D
(CHCl3): lmax 260 nm (
3
18145); 1H NMR (CDCl3)
d
7.91–7.45 (m, 7H,
0
0
0
0
Bz, H-5 and H-6), 6.00 (d, 1H, J1 ,2 ¼ 9.3 Hz, H-1 ), 5.18 (m, 1H, H-2 ),
0
0
0
0
0
0
eluant. Yield: 0.58 g (72%); Rf ¼ 0.12 in ethyl acetate–n-hexane
4.71 (dtr, 1H, JF,3 ¼ 52.2 Hz, J2 ,3 ¼ J3 ,4 ¼ 8.7 Hz, H-3 ), 3.70–3.57
22
(m, 2H, H-40 and H-50), 2.04 (s, 3H, OAc), 1.41 (d, 3H, J5 ,6 ¼ 5.8 Hz,
0
0
(4:6); [
a
]
D
þ 10.0 (c 0.50, CHCl3); UV (CHCl3): lmax 262 nm (
3 5106);
1H NMR (CDCl3):0 d 8.03 (br s, NH), 07.18 (s, 1H, H-6), 5.64 (d, 1H,
H-60); 19F NMR:
d
ꢃ65.5. Anal. Calcd for C19H20FN3O6: C, 56.29; H,
0
0
0
J1 ,2 ¼ 9.3 Hz, H-1 ), 5.05 (m, 1H, H-2 ), 4.77 (m, 1H, H-3 ), 3.80 (m,
4.97; N, 10.37. Found: C, 56.61; H, 4.85; N, 10.63; ESI-MS (m/z):
406.36 (M þ Hþ).
1H, H-40), 2.33 (m, 1H, H-50), 2.06 (s, 3H, OAc), 1.96 (s, 3H, 5-CH3),
1.34 (d, 3H, J5 ,6 ¼ 6.2 Hz, H-6 ); 19F NMR: –65.5; Anal. Calcd for
C13H17FN2O6: C, 49.37; H, 5.42; N, 8.86. Found: C, 49.65; H, 5.29; N,
8.75; ESI-MS (m/z): 317.26 (M þ Hþ).
0
0
0
4.5.3. 1-(2,6-Dideoxy-3-fluoro-
b-D-glycero-hex-2-enopyranosyl-4-
ulose)-N4-benzoyl cytosine (9d)
Cytosine derivative 9d was synthesized from 8d by the same
methodology as described for the synthesis of 9a. Compound 9d was
obtained as a white foam following purification by flash column
4.4.8. 1-(2,6-Dideoxy-3-fluoro-b-D-glycero-hex-2-enopyranosyl-4-
ulose)thymine (9c)
Thymine derivative 9c was synthesized from 8c by the same
methodology as described for the synthesis of 9a. Compound 9c
was obtained as a white solid following purification by flash
column chromatography using ethyl acetate–n-hexane (4:6) as
chromatography using ethyl acetate–n-hexane (9:1) as eluant. Yield:
22
0.28 g (50%); Rf ¼ 0.30 in ethyl acetate–n-hexane (9:1); [
(c 0.50, CHCl3); UV (CHCl3): lmax 260 nm (
d6): 8.19 (br s, 1H, NH), 8.00–7.39 (m, 7H, Bz, H-5 and H-6), 7.07 (d,
a]
D
þ 61.0
3
16459); 1H NMR (DMSO-
d
0
0
0
0
eluant. Yield: 0.27 g (58%); Rf ¼ 0.11 in ethyl acetate–n-hexane
1H, JF,1 ¼12.3 Hz, H-1 ), 7.00 (d, 1H, JF,2 ¼ 6.0 Hz, H-2 ), 4.75 (m, 1H,
22
(4:6); m.p. 112-115 ꢁC; [
a
]
þ 13.0 (c 0.50, CHCl3); UV (CHCl3): lmax
H-50), 1.33 (d, 3H, J5 ,6 ¼ 6.5 Hz, H-6 ); 13C NMR (DMSO-d6):
d 189.06
0
0
0
D
259 nm (
3
11,335); 1H0NMR (DMSO-d6):
d
7.56 (s, 1H, H-6), 6.98 (d,
(C-40); 167.52 (C]O); 163.99 (C-4); 153.48 (C-30); 151.32 (C-2);
146.87 (C-6); 135.60 (Carom); 135.05 (CHarom); 132.95 (2CHarom);
128.53 (2CHarom); 123.52 (C-20); 97.45 (C-5); 78.76 (C-10); 76.18 (C-
0
0
0
0
1H, JF,1 ¼12.5 Hz, H-1 ), 6.80 (dd, 1H, JF,2 ¼ 6.5 Hz, J1 ,2 ¼1.4 Hz, H-
20), 4.67 (m, 1H, H-50), 1.77 (s, 3H, 5-CH3), 1.29 (d, 3H, J5 ,6 ¼ 6.5 Hz,
0
0
H-60); 13C NMR (DMSO-d6):
d
198.07 (C-40); 163.83 (C-4); 151.42 (C-
50); 14.86 (C-60); 19F NMR:
d
ꢃ64.3; IR (Neat, cmꢃ1): 1719.29 (keto
30); 150.14 (C-2); 136.98 (C-6); 123.60 (C-5); 110.60 (C-20); 77.14 (C-
group); Anal. Calcd for C17H14FN3O4: C, 59.47; H, 4.11; N, 12.24.
10); 75.83 (C-50); 14.81 (C-60); 11.84 (CH3); 19F NMR:
d
–63.2; IR
Found: C, 59.63; H, 3.94; N, 12.56; ESI-MS (m/z): 344.32 (M þ Hþ).
(Neat, cmꢃ1): 1651.83 (keto group); Anal. Calcd for C11H11FN2O4: C,
51.97; H, 4.36; N,11.02. Found: C, 51.79; H, 4.25; N,11.21; ESI-MS (m/
z): 255.23 (M þ Hþ).
4.6. Synthesis of 9-(2,6-dideoxy-3-fluoro-b-D-glycero-hex-2-
enopyranosyl-4-ulose)-N6-benzoyl adenine (9e)
4.5. Synthesis of 1-(2,6-dideoxy-3-fluoro-
b
-
D
-glycero-hex-2-
The partially protected adenine derivative 6e was prepared by
enopyranosyl-4-ulose)-N4-benzoyl cytosine (9d)
the coupling reaction of peracetylated 3-deoxy-3-fluoro-
pyranose (1) [38,39] with silylated N6-benzoyl adenine followed by
selective deprotection of the -nucleoside formed 2e, specific
D-gluco-
The partially protected cytosine derivative 6d was prepared by
b
the coupling reaction of tetraacetylated 3-deoxy-3-fluoro-
D
-glu-
acetalation of the base protected analogue 3e, conventional acet-
ylation of the partially protected derivative 4e and finally by
copyranose (1) [38,39] with silylated N4-benzoyl cytosine followed

4770
S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 4764–4771
deisopropylidenation of the acetylated analogue 5e, as previously
described [35].
to infect 50% of the cell cultures). After a 1 h virus adsorption
period, residual virus was removed, and the cell cultures were
incubated in the presence of varying concentrations (200, 40, 8, .
4.6.1. 9-(2-O-Acetyl-3-deoxy-3-fluoro-6-iodo-
b
-
D
-glucopyranosyl)-
mM) of the test compounds. Viral cytopathicity was recorded as
N6-benzoyl adenine (7e)
soon as it reached completion in the control virus-infected cell
cultures that were not treated with the test compounds. The
methodology of the anti-HIV assays was as follows: human CEM
(w3 ꢂ105 cells/cm3) cells were infected with 100 CCID50 of
Adenine derivative 7e was synthesized from 9-(2-O-acetyl-3-
deoxy-3-fluoro-
b-D
-glucopyranosyl)-N6-benzoyl adenine (6e) [35]
by the same methodology as described for the synthesis of 7a.
Compound 7e was obtained as a white solid following purification by
flash column chromatography using dichloromethane–methanol
HIV(IIIB) or HIV-2(ROD)/ml and seeded in 200-mL wells of a micro-
titer plate containing appropriate dilutions of the test compounds.
After 4 days of incubation at 37 ꢁC, HIV-induced CEM giant cell
formation was examined microscopically.
(9:1) as eluant. Yield: 1.62 g (65%); Rf ¼ 0.48 in dichloromethane–
22
methanol (9:1), m.p. 95–98 ꢁC; [
a]
D
ꢃ6.0 (c 0.50, CHCl3); UV (CHCl3):
lmax 279 nm (3 d 9.25 (br s, 1H, NH), 8.84 and
7288); 1H NMR (CDCl3)
0
0
8.26 (2s, 2H, H-2,8), 8.05-7.45 (m, 5H, Bz), 5.98 (d,1H, J1 ,2 ¼ 9.3 Hz, H-
4.8. Cytostatic/toxic activity assays
10), 5.58 (m, 1H, 0H-20), 4.79 (dtr, 1H, JF,3 ¼ 52.1 Hz, J2 ,3 ¼ 8.4 Hz,
0
0
0
0
0
0
0
0
J3 ,4 ¼ 8.9 Hz, H-3 ), 4.02 (m, 1H, H-4 ), 3.57 (m, 2H, H-6a ,6b ), 3.37
Murine leukemia L1210, murine mammary carcinoma FM3A,
and human lymphocyte Molt4/C8 and CEM cells were seeded in 96-
well microtiter plates at 50,000 (L1210, FM3A) or 75,000 (Molt,
(m, 1H, H-50), 1.84 (s, 3H, OAc); 19F NMR:
d
ꢃ65.5. Anal. Calcd for
C20H19FIN5O5: C, 43.26; H, 3.45; N, 12.61. Found: C, 43.38; H, 3.67; N,
12.42; ESI-MS (m/z): 556.28 (M þ Hþ).
CEM) cells per 200-mL well in the presence of different concentra-
tions of the test compounds. After 2 (L1210, FM3A) or 3 (Molt, CEM)
days, the viable cell number was counted using a Coulter counter
apparatus. The 50% cytostatic concentration (CC50) was defined as
the compound concentration required to inhibit tumor cell prolif-
eration by 50%.
4.6.2. 9-(2-O-Acetyl-3,6-dideoxy-3-fluoro-b-D
-glucopyranosyl)-N6-
benzoyl adenine (8e)
Adenine derivative 8e was synthesized from 7e by the same
methodology as described for the synthesis of 8a. Compound 8e
was obtained as a clear viscous oil following purification by flash
column chromatography using ethyl acetate as eluant. Yield: 0.88 g
Acknowledgements
22
(70%); Rf ¼ 0.19 in ethyl acetate; [
a
]
þ4.0 (c 0.50, CHCl3); UV
D
(CHCl3): lmax 274 nm (
3
6699); 1H NMR (CDCl3)
d 9.08 (br s,1H, NH),
This work was supported in part by the Postgraduate Pro-
grammes ‘‘Biotechnology-Quality assessment in Nutrition and the
Enviroment’’, ‘‘Application of Molecular Biology-Molecular
Genetics-Molecular Markers’’, Department of Biochemistry and
Biotechnology, University of Thessaly. The financial support was
also provided by the ‘‘Geconcerteerde Onderzoeksacties’’ (GOA no.
05/19) of the K.U.Leuven. The authors are grateful to Dr. K. Anto-
nakis (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 aid, to Mrs. Lizette van
Berckelaer, Leentje Persoons, Frieda De Meyer, Vickie Broeckx and
Leen Ingels for dedicated technical help.
8.84 and 8.22 (2s, 2H, H-2,8), 8.03–07.45 (m, 5H, Bz), 5.85 (d, 1H,
0
0
0
0
J1 ,2 ¼ 9.5 Hz, H-1 ), 5.59 (m, 1H, H-2 ), 4.68 (dtr, 1H, JF,3 ¼ 52.1 Hz,
0
0
0
0
0
0
0
J2 ,3 ¼ J3 ,4 ¼ 8.4 Hz, H-3 ), 3.78–3.69 (m, 2H, H-4 and H-5 ), 1.83 (s,
3H, OAc), 1.43 (d, 3H, J5 ,6 ¼ 5.0 Hz, H-6 ); 19F NMR:
d
ꢃ65.0. Anal.
0
0
0
Calcd for C20H20FN5O5: C, 55.94; H, 4.69; N, 16.31. Found: C, 56.19;
H, 4.81; N, 16.16; ESI-MS (m/z): 430.42 (M þ Hþ).
4.6.3. 9-(2,6-Dideoxy-3-fluoro-b-D-glycero-hex-2-enopyranosyl-4-
ulose)-N6-benzoyl adenine (9e)
Adenine derivative 9e was synthesized from 8e by the same
methodology as described for the synthesis of 9a. Compound 9e
was obtained as a white solid following purification by flash
column chromatography using ethyl acetate–n-hexane (9:1) as
References
eluant. Yield: 0.41 g (55%); Rf ¼ 0.20 in ethyl acetate–n-hexane
22
(9:1), m.p. 205-207 ꢁC; [
a
]
þ 9.0 (c 0.50, CHCl3); UV (CHCl3): lmax
[1] C.M. Galmarini, J.R. Mackey, C. Dumontet, Leukemia 15 (2001) 875–890.
[2] B.D. Cheson, Semin. Oncol. 19 (1992) 695–706.
[3] D.M. Huryn, M. Okabe, Chem. Rev. 92 (1992) 1745–1768.
[4] G.A. Patani, E.J. LaVoie, Chem. Rev. 96 (1996) 3147–3176.
[5] L. Agrofoglio, E. Suhas, A. Farese, R. Condom, R. Guedj, Tetrahedron 50 (1994)
10611–10670.
[6] A. Van Aerschot, P. Herdewijn, J. Balzarini, R. Pawels, E. De Clercq, J. Med.
Chem. 32 (1989) 1743–1749.
[7] K. Lee, Y. Choi, E. Gullen, S. Schlueter-Wirtz, R.F. Schinazi, Y.C. Cheng, C.K. Chu,
J. Med. Chem. 42 (1999) 1320–1328.
[8] K. Lee, Y. Choi, G. Gumina, W. Zhou, R.F. Schinazi, C.K. Chu, J. Med. Chem. 45
(2002) 1313–1320.
[9] Y. Chong, H. Choo, S. Lee, Y. Choi, R.F. Schinazi, C.K. Chu, J. Med. Chem. 45
(2002) 4888–4898.
[10] E. Matthes, C. Lehmann, M. Von Janta-Lipinski, D. Scholz, Biochem. Biophys.
Res. Commun. 165 (1989) 488–495.
[11] A. Van Aerschot, D. Everaert, J. Balzarini, K. Augustyns, L. Jie, G. Jannsen,
O. Peeters, N. Blaton, C. De Ranter, E. De Clercq, P. Herdewijn, J. Med. Chem. 33
(1990) 1833–1839.
D
279 nm (
2,8), 8.05-7.54 (m, 5H, Bz), 7.34 (d, 1H, JF,1 ¼12.3 Hz, H-1 ), 7.15 (d,
3 d 8.80 and 8.71 (2s, 2H, H-
9038); 1H NMR (DMSO-d6):
0
0
0
0
0
0
0
1H, JF,2 ¼ 6.2 Hz, H-2 ), 4.85 (m, 1H, H-5 ), 1.32 (d, 3H, J5 ,6 ¼ 6.5 Hz,
H-60); 13C NMR (DMSO-d6):
d
189.32 (C-40); 165.75 (C]O); 153.21
(C-30); 152.19 (C-2); 151.05 (C-6); 150.60 (C-4); 143.38 (C-8); 133.26
(Carom); 132.77 (CHarom); 132.58 (2CHarom); 128.54 (2CHarom);
125.12 (C-5); 123.10 (C-20); 76.45 (C-10); 76.19 (C-50); 14.85 (C-60);
19F NMR:
d
ꢃ63.2; IR (Neat, cmꢃ1): 1714.45 (keto group); Anal.
Calcd for C18H14FN5O3: C, 58.85; H, 3.84; N, 19.07. Found: C, 58.98;
H, 3.62; N, 19.18; ESI-MS (m/z): 368.35 (M þ Hþ).
4.7. Antiviral activity assays
The antiviral assays, other than the anti-HIV assays, were based
on inhibition of virus-induced cytopathicity in HEL [herpes simplex
virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccinia virus and vesicular
stomatitis virus], Vero (parainfluenza-3, reovirus-1, Sindbis virus
and Coxsackie B4), HeLa (vesicular stomatitis virus, Coxsackie virus
B4, and respiratory syncytial virus) or MDCK [influenza A (H1N1;
H3N2) and influenza B] cell cultures. Confluent cell cultures (or
nearly confluent for MDCK cells) in microtiter 96-well plates were
inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose
[12] P. Herdewijn, R. Pawels, M. Baba, J. Balzarini, E. De Clercq, J. Med. Chem. 30
(1987) 2131–2137.
[13] E. Matthes, D. Scholz, G. Sydow, M. Von Janta-Lipinski, H.A. Rosenthal,
P. Langen, Z. Klin. Med. 45 (1990) 1255–1258.
[14] S.M. Daluge, D.J. Purifoy, P.M. Savina, M.H. St. Clair, N.R. Parry, I.K. Dev,
P. Novak, K.M. Ayers, J.E. Reardon, G.B. Roberts, J.A. Fyfe, M.R. Blum,
D.R. Averett, R.E. Dornsife, B.A. Domin, R. Ferone, D.A. Lewis, T.A. Krenitsky,
Antimicrob. Agents Chemother. 38 (1994) 1590–1603.
[15] 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.

S. Manta et al. / European Journal of Medicinal Chemistry 44 (2009) 4764–4771
4771
[16] P. Liu, A. Sharon, C.K. Chu, J. Fluorine Chem. 129 (2008) 743–766.
[17] K.W. Pankiewicz, Carbohydr. Res. 327 (2000) 87–105.
[18] 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.
[19] 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.
[33] M.A. Alaoui-Jamali, C. Lasne, K. Antonakis, I. Chouroulinkov, Mutagenesis 6
(1986) 411–417.
[34] S. Manta, G. Agelis, T. Botic´, A. Cencicˇ, D. Komiotis, Bioorg. Med. Chem. 15
(2007) 980–987.
[35] S. Manta, G. Agelis, T. Botic´, A. Cencicˇ, D. Komiotis, Eur. J. Med. Chem. 43 (2008)
420–428.
ˇ
[36] G. Agelis, N. Tzioumaki, T. Tselios, T. Botic´, A. Cencic, D. Komiotis, Eur. J. Med.
[20] 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.
[21] Y. Maurinsh, J. SchramL, H. De Winter, N. Blaton, O. Peeters, E. Lescrinier,
J. Rozenski, A. Van Aerschot, E. De Clercq, R. Busson, P. Herdewijn, J. Org. Chem.
62 (1997) 2861–2871.
Chem. 43 (2008) 1366–1375.
[37] M.J. Egron, D. Komiotis, I. Dorange, J. Herscovici, A.P. Ollapally, K. Antonakis,
Nucleosides, Nucleotides Nucleic Acids 24 (2005) 243–246.
[38] W. Sowa, H.S. Thomas, Can. J. Chem. 44 (1966) 836–837.
[39] A.B. Foster, R. Hems, M.J. Webber, Carbohydr. Res. 5 (1967) 292–301.
[40] V. Vanheusden, R. Busson, P. Herdewijn, Van S. Calenbergh, J. Org. Chem. 69
(2004) 4446–4453.
[41] P.J. Garegg, B. Samuelsson, J. Chem. Soc., Perkin Trans. 1 (1980) 2866–
2869.
[42] B. Classon, P.J. Garegg, B. Samuelsson, Can. J. Chem. 59 (1981) 339–343.
[43] P.J. Garegg, R. Johansson, C. Ortega, B. Samuelsson, J. Chem. Soc., Perkin Trans.
1 (1982) 681–683.
[44] B. Classon, Z. Liu, B. Samuelsson, J. Org. Chem. 53 (1988) 6126–6130.
[45] J.D. Albright, L. Goldman, J. Org. Chem. 30 (1965) 1107–1110.
[46] S. Manta, E. Tsoukala, N. Tzioumaki, A. Goropevsˇek, R.T. Pamulapati, A. Cencicˇ,
J. Balzarini, D. Komiotis, Eur. J. Med. Chem. 44 (2009) 2696–2704.
[47] M.J. Egron, F. Leclercq, K. Antonakis, I. Bennani-Baiti, C. Frayssinet, Carbohydr.
Res. 248 (1993) 143–150.
[22] C. Spanou, S. Manta, D. Komiotis, A. Dervishi, D. Kouretas, Int. J. Mol. Sci. 8
(2007) 695–704.
[23] A. Haouz, V. Vanheusden, H. Munier-Lechman, M. Froeyen, P. Herdewijn,
S. Van Galenbergh, M. Delarue, J. Biol. Chem. 278 (2003) 4963–4971.
[24] K. Vastmans, S. Pochet, A. Peys, L. Kerremans, A. Van Aerschot, C. Hendrix,
P. Marliere, P. Herdewijn, Biochemistry 39 (2000) 12757–12765.
[25] K. Vastmans, M. Froeyen, L. Kerremans, S. Pochet, P. Herdewijn, Nucleic Acids
Res. 29 (2001) 3154–3163.
[26] K. Antonakis, I. Chouroulinkov, C.R. Acad. Sci. Paris 273D (1971) 2661–2663.
[27] K. Antonakis, I. Chouroulinkov, Biochem. Pharmacol. 23 (1974) 2095–2100.
[28] J. Paterson, C. Uriel, M.J. Egron, J. Herscovici, K. Antonakis, M. Alaoui, Anti-
microb. Agents Chemother. 42 (1998) 779–784.
[29] K. Antonakis, T. Halmos, J. Bach, I. Chouroulinkov, Eur. J. Med. Chem. 15 (1980)
237–240.
[30] I. Chouroulinkov, K. Antonakis, C.R. Acad. Sci. Hebd Seances Acad. Sci. D 285
(1977) 1021–1024.
[48] J. Gutzwiller, M. Uskokovic, J. Am. Chem. Soc. 92 (1970) 204–205.
[49] D.L. Boger, K. Machiya, J. Am. Chem. Soc. 114 (1992) 10056–10058.
[50] M. Bessodes, R. Lakaf, K. Antonakis, Carbohydr. Res. 148 (1986)
148–152.
[31] C. Aujard, Y. Moule, E. Chany-Morel, K. Antonakis, Biochem. Pharmacol. 27
(1978) 1037–1042.
[32] T. Halmos, A. Cardon, K. Antonakis, Chem. Biol. Interact. 46 (1983) 11–29.
[51] T. Halmos, K. Antonakis, Carbohydr. Res. 68 (1979) 61–69.
[52] F. Leclercq, K. Antonakis, Carbohydr. Res. 181 (1988) 217–222 and references
cited therein.