Synthesis of 4,6-dideoxy-3-fluoro-2-keto-β-d-glucopyranosyl analogues of 5-fluorouracil, N6-benzoyl adenine, uracil, thymine, N4-benzoyl cytosine and evaluation of their antitumor activities

Article abstract of DOI:10.1016/j.bioorg.2009.11.001

The synthesis of the unsaturated 4,6-dideoxy-3-fluoro-2-keto-β-d-glucopyranosyl nucleosides of 5-fluorouracil (6a), N⁶-benzoyl adenine (6b), uracil (6c), thymine (6d) and N⁴-benzoyl cytosine (6e), is described. Monoiodination of compounds 1a,b, followed by acetylation, catalytic hydrogenation and finally regioselective 2′-O-deacylation afforded the partially acetylated dideoxynucleoside analogues of 5-fluorouracil (5a) and N⁶-benzoyl adenine (5b), respectively. Direct oxidation of the free hydroxyl group at the 2′-position of 5a,b, with simultaneous elimination reaction of the β-acetoxyl group, afforded the desired unsaturated 4,6-dideoxy-3-fluoro-2-keto-β-d-glucopyranosyl derivatives 6a,b. Compounds 1c-e were used as starting materials for the synthesis of the dideoxy unsaturated carbonyl nucleosides of uracil (6c), thymine (6d) and N⁴-benzoyl cytosine (6e). Similarly a protection-selective deprotection sequence followed by oxidation of the free hydroxyl group at the 2′-position of the dideoxy benzoylated analogues 9c-e with simultaneous elimination reaction of the β-benzoyl group, gave the desired nucleosides 6c-e. None of the compounds was inhibitory to a broad spectrum of DNA and RNA viruses at subtoxic concentrations. The 5-fluorouracil derivative 6a was more cytostatic (50% inhibitory concentration ranging between 0.2 and 12 μM) than the other compounds.

Full text of DOI:10.1016/j.bioorg.2009.11.001

Bioorganic Chemistry 38 (2010) 48–55
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of 4,6-dideoxy-3-fluoro-2-keto-b-D-glucopyranosyl analogues of
5-fluorouracil, N6-benzoyl adenine, uracil, thymine, N4-benzoyl cytosine and
evaluation of their antitumor activities
Stella Manta a, Evangelia Tsoukala a, Niki Tzioumaki a, Christos Kiritsis a, Jan Balzarini b, Dimitri Komiotis a,
*
a Department of Biochemistry and Biotechnology, Laboratory of Organic Chemistry, University of Thessaly, 41221 Larissa, Greece
b 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:
Received 7 October 2009
Available online 20 November 2009
The synthesis of the unsaturated 4,6-dideoxy-3-fluoro-2-keto-b-
D
-glucopyranosyl nucleosides of 5-fluoro-
uracil (6a), N6-benzoyl adenine (6b), uracil (6c), thymine (6d) and N4-benzoyl cytosine (6e), is described.
Monoiodination of compounds 1a,b, followed by acetylation, catalytic hydrogenation and finally regiose-
lective 20-O-deacylation afforded the partially acetylated dideoxynucleoside analogues of 5-fluorouracil
(5a) and N6-benzoyl adenine (5b), respectively. Direct oxidation of the free hydroxyl group at the 20-position
of 5a,b, with simultaneous elimination reaction of the b-acetoxyl group, afforded the desired unsaturated
Keywords:
Unsaturated dideoxy fluoro ketonucleosides
b-Elimination reaction
Antitumor activity
4,6-dideoxy-3-fluoro-2-keto-b-D-glucopyranosyl derivatives 6a,b. Compounds 1c–e were used as starting
materials for the synthesis of the dideoxy unsaturated carbonyl nucleosides of uracil (6c), thymine (6d) and
N4-benzoyl cytosine (6e). Similarly a protection-selective deprotection sequence followed by oxidation of
the free hydroxyl group at the 20-position of the dideoxy benzoylated analogues 9c–e with simultaneous
elimination reaction of the b-benzoyl group, gave the desired nucleosides 6c–e. None of the compounds
was inhibitory to a broad spectrum of DNA and RNA viruses at subtoxic concentrations. The 5-fluorouracil
derivative 6a was more cytostatic (50% inhibitory concentration ranging between 0.2 and 12 lM) than the
other compounds.
Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction
anonucleosides, which not only were found to have significant
in vitro and in vivo inhibitory activity against various types of can-
cer cells [27,28] but they also appeared to be highly reactive sulf-
hydryl blocking agents [29]. In this regard, we have been actively
engaged in the study of various unsaturated ketopyranonucleo-
sides [22,30–34] and have developed stereoselective synthetic
methods for these biologically important targets in medicinal
chemistry.
In the course of our studies, we have prepared the unsaturated
30-fluoro-40- and 20-ketonucleosides [30,31], which proved to be effi-
cient as tumor cell growth inhibitors and had a promising potential
in combating rotaviral infections. Our recent research also disclosed
thatthe60-protectedanalogueswereendowedwithhighercytotoxic
and antiviral potency, indicating that the presence of a primary hy-
droxyl group might not be critical for biological activity. This obser-
vation encouraged us to develop the synthesis of the dideoxy
unsaturated 30-fluoro-40-ketopyranonucleoside analogues bearing
a methyl group in the 50-position of the sugar moiety [32]. As
expected, these 60-OH-lacking analogues have emerged as more
effective inhibitory agents against a variety of tumor cell lines.
With the above applications in mind and in order to elucidate
the structural requirements for biological activity of unsaturated
ketonucleosides, we decided to design and synthesize the dideoxy
Nucleoside analogues have emerged as important therapeutic
agents for the development of antiviral and antitumor drugs [1–
4]. Antiviral nucleoside analogues inhibit replication of the viral
genome, whereas anticancer nucleoside analogues inhibit cellular
DNA replication and repair [5]. Attachment of fluorine atoms on
the sugar and heterocyclic moieties of such analogues is known
to be of advantage both for an improved activity, higher bioavail-
ability as well as for causing a retarded catabolism of several drugs
[6–9]. Therefore, specific fluorination at the 20- and/or 30-position
of the sugar moiety of the nucleosides has been studied in the pur-
suit of safe, effective and chemically stable antiviral agents [9–15].
The last decades, nucleosides containing pyranosyl rings in-
stead of furanosyl ones have been evaluated for their potential
antiviral [16–22], antioxidant [23] and antibiotic [24] properties
and as building blocks in nucleic acid synthesis [25,26]. The keto-
hexose unsaturated nucleosides are a series of sugar modified pyr-
* Corresponding author. Address: University of Thessaly, Department of Bio-
chemistry and Biotechnology, Laboratory of Organic Chemistry, 26 Ploutonos Str.,
41221 Larissa, Greece. Fax: +30 2410 565290.
E-mail address: dkom@bio.uth.gr (D. Komiotis).
0045-2068/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2009.11.001

S. Manta et al. / Bioorganic Chemistry 38 (2010) 48–55
49
unsaturated 30-fluoro-20-ketopyranonucleosides of 5-fluorouracil,
N6-benzoyl adenine, uracil, thymine, and N4-benzoyl cytosine
respectively, bearing a methyl group in the 50-position of the sugar
moiety. Chemical synthesis and biological activity of these com-
pounds are presented herein.
Calcd for C14H15F2IN2O7: C, 34.44; H, 3.10; N, 5.74. Found: C,
34.69; H, 2.92; N, 5.85. ESI-MS (m/z): 489.19 (M+H+).
2.2.3. 1-(2,4-Di-O-acetyl-3,6-dideoxy-3-fluoro-b-D-glucopyranosyl)5-
fluorouracil (4a)
After two vacuum/H2 cycles to remove air from the reaction
tube, the stirred mixture of 3a (2.51 g, 5.14 mmol), 10% Pd/C
(0.70 g) and triethylamine (1.42 mL, 10.28 mmol) in AcOEt
(157.3 mL) and EtOH (157.3 mL) was hydrogenated at ambient
pressure (balloon) and temperature (ca. 20 °C) for 24 h. The reac-
tion mixture was filtered and the filtrate was concentrated in vac-
uum. The residue was purified by flash chromatography (hexane/
AcOEt, 6:4) and compound 4a (1.21 g, 65%, Rf = 0.31 in hexane/
2. Experimental
2.1. General methods
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). UV–Vis spectra were
recorded on a PG T70 UV–VIS spectrometer and mass spectra were
obtained with a Micromass Platform LC (ESI-MS). Optical rotations
were measured using Autopol I polarimeter. 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 methanol-d4 (CD3OD) with internal tetramethylsilane (TMS)
for 1H and 13C and internal trifluorotoluene (TFT) for 19F.
The chemical shifts are expressed in parts per million (d) and
following abbreviations were used: s = singlet; br s = broad singlet;
d = doublet; dd = doublet doublet; dtr = doublet triplet; m = multi-
plet. All reactions sensitive to oxygen or moisture were carried out
under nitrogen atmosphere with dry solvents. Dimethyl sulfoxide
was stored over 3 Å molecular sieves. Pyridine was stored over pel-
lets of potassium hydroxide. Tetrahydrofuran was freshly distilled
under nitrogen from sodium/benzophenone before use.
AcOEt, 6:4) was obtained as a white foam. ½a D22
þ 12:0 (c 0.50,
ꢀ
CHCl3); kmax 265 nm (e
9574); 1H NMR (CDCl3): d 8.76 (br s, 1H,
0
0
NH), 7.44 (d, 1H, J6,F5 = 5.7 Hz, H-6), 5.73 (d, 1H, J1 ,2 = 9.4 Hz, H-
10), 5.17 (m, 1H, H-20), 5.04 (m, 1H, H-40), 4.69 (dtr, 1H,
JF,3 = 51.8 Hz, J2 ,3 = J3 ,4 = 9.1 Hz, H-30), 3.71 (m, 1H, Y-50), 2.16
0
0
0
0
0
and 2.08 (2s, 6Y, 2JAc), 1.28 (d, 3Y, J5 ,6 = 6.1 Hz, H-60); Anal. Calcd
for C14H16F2N2O7: C, 46.41; H, 4.45; N, 7.73. Found: C, 46.64; H,
4.34; N, 7.92. ESI-MS (m/z): 363.27 (M+H+).
0
0
2.2.4. 1-(4-O-Acetyl-3,6-dideoxy-3-fluoro-b-
fluorouracil (5a)
D-glucopyranosyl)5-
To a solution of 4a (1.21 g, 3.34 mmol) in pyridine (19 mL) was
added hydroxylamine hydrochloride (0.93 g, 13.4 mmol) and anhy-
drous sodium acetate (1.1 g, 13.4 mmol) with vigorous stirring at
20 °C. After 12 h acetone (0.95 mL) was added and the solvents were
removed under high vacuum. Purification by flash chromatography
(hexane/AcOEt, 6:4) gave 5a (0.53 g, 50%, Rf = 0.17 in hexane/AcOEt,
6:4) as a colourless oil. ½a D22
ꢀ
þ 6:0 (c 0.50, CHCl3); kmax 266 nm (
e
5701); 1H NMR (CDCl3): d 9.85 (br s, 1H, NH), 7.43 (d, 1H,
J6,F5 = 5.7 Hz, H-6), 5.66 (d, 1H, J1 ,2 = 9.0 Hz, H-10), 4.95 (m, 1H, H-
0
0
40), 4.63 (dtr, 1H, JF,3 = 52.0 Hz, J2 ,3 = 8.8 Hz, J3 ,4 = 9.0 Hz, H-30),
0
0
0
0
0
2.2. Synthesis of 1-(4,6-dideoxy-3-fluoro-b-
enopyranosyl-2-ulose)5-fluorouracil (6a)
D-glycero-hex-3-
3.85 (m, 1H, Y-20), 3.74 (m, 1H, Y-50), 2.15 (s, 3Y, JAc), 1.25 (d, 3Y,
J5 ,6 = 6.1 Hz, H-60); Anal. Calcd for C12H14F2N2O6: C, 45.01; H, 4.41;
N, 8.75. Found: C, 45.19; H, 4.29; N, 8.57. ESI-MS (m/z): 321.24
(M+H+).
0
0
2.2.1. 1-(3-Deoxy-3-fluoro-6-iodo-b-D-glucopyranosyl)5-fluorouracil (2a)
A mixture of 1-(3-deoxy-3-fluoro-b-
D-glucopyranosyl)5-fluoro-
uracil (1a) [32] (2.5 g, 8.5 mmol), imidazole (1.16 g, 17.0 mmol),
triphenylphosphine (3.34 g, 12.75 mmol) and iodine (3.24 g,
12.75 mmol) in tetrahydrofuran (83.2 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 under vacuum.
Purification by flash chromatography (hexane/AcOEt, 2:8), gave 2a
2.2.5. 1-(4,6-Dideoxy-3-fluoro-b-
ulose)5-fluorouracil (6a)
D-glycero-hex-3-enopyranosyl-2-
To a solution of 5a (0.53 g, 1.67 mmol) in dimethyl sulfoxide
(8.18 mL) was added acetic anhydride (4.08 mL, 43.25 mmol).
The mixture was heated at 100 °C for 10 min, then cooled to room
temperature, diluted with AcOEt and washed with water. The or-
ganic layer was dried over anhydrous sodium sulfate, filtered and
evaporated to dryness. The residue was purified by flash chroma-
tography (CH2Cl2/MeOH, 9.5:0.5) and compound 6a (0.24 g, 55%,
Rf = 0.43 in CH2Cl2/MeOH, 9.5:0.5) was obtained as a white pow-
(2.34 g, 68%, Rf = 0.36 in hexane/AcOEt, 2:8) as a syrup. ½a D22
ꢁ 2:0
ꢀ
(c 0.50, MeOH); kmax 262 nm (e
10801); 1H NMR (CD3OD): d 7.89
(d, 1H, J6,F5 = 6.3 Hz, H-6), 5.64 (d, 1H, J1 ,2 = 9.6 Hz, H-10), 4.42
0
0
(dtr, 1H, JF,3 = 52.5 Hz, J2 ,3 = 8.7 Hz, J3 ,4 = 9.0 Hz, H-30), 3.87 (m,
1H, H-20), 3.65–3.41 (m, 3H, H-40 and H-6a0,6b0), 3.23 (m, 1H, H-
50); Anal. Calcd for C10H11F2IN2O5: C, 29.72; H, 2.74; N, 6.93. Found:
C, 29.84; H, 2.63; N, 7.15. ESI-MS (m/z): 405.10 (M+H+).
0
0
0
0
0
der: mp. 197–199 °C; ½a D22
ꢀ
ꢁ 20:0 (c 0.50, CHCl3); kmax 263 nm (
e
1078); 1H NMR (CDCl3): d 10.47 (br s, 1H, NH), 7.48 (d, 1H,
J6,F5 = 5.6 Hz, H-6), 7.16 (d, 1H, JF,1 = 5.5 Hz, H-10), 6.59 (dd, 1H,
0
JF,4 = 11.3 Hz, J4 ,5 = 1.3 Hz, H-40), 4.94 (m, 1H, H-50), 1.51 (d, 3Y,
0
0
0
J5 ,6 = 6.8 Hz, H-60); 13C NMR (CDCl3): d 188.26, 159.32, 157.01,
149.18, 143.91, 125.88, 116.90, 85.37, 69.66, 21.08; 19F NMR: d
ꢁ65.0, ꢁ65.5. Anal. Calcd for C10H8F2N2O4: C, 46.52; H, 3.12; N,
10.85. Found: C, 46.35; H, 3.40; N, 10.74. ESI-MS (m/z): 259.16
(M+H+).
0
0
2.2.2. 1-(2,4-Di-O-acetyl-3-deoxy-3-fluoro-6-iodo-b-
glucopyranosyl)5-fluorouracil (3a)
D-
To a solution of 2a (2.34 g, 5.78 mmol) in dry pyridine (28.9 mL)
was added acetic anhydride (1.36 mL, 14.45 mmol) and the re-
sulted mixture was stirred at room temperature for 2 h. MeOH
(0.7 mL) was added to quench the reaction and the mixture was
concentrated under high vacuum to remove the solvents. Purifica-
tion by flash chromatography (hexane/AcOEt, 1:1) afforded 3a
(2.51 g, 89%, Rf = 0.67 in hexane/AcOEt, 3:7) as a yellow oil.
2.3. Synthesis of 9-(4,6-dideoxy-3-fluoro-b-
D-glycero-hex-3-
enopyranosyl-2-ulose)-N6-benzoyl adenine (6b)
½
a 2D2
ꢀ
þ 2:0 (c 0.26, CHCl3); kmax 266 nm (
e
8727); 1H NMR (CDCl3):
d 8.94 (br s, 1H, NH), 7.46 (d, 1H, J6,F5 = 5.6 Hz, H-6), 5.85 (d, 1H,
2.3.1. 9-(3-Deoxy-3-fluoro-6-iodo-b-
adenine (2b)
D
-glucopyranosyl)-N6-benzoyl
J1 ,2 = 9.3 Hz, H-10), 5.25–5.17 (m, 2H, H-20 and H-40), 4.77 (dtr,
0
0
1H, JF,3 = 51.6 Hz, J2 ,3 = J3 ,4 = 9.1 Hz, H-30), 3.54 (m, 1H, Y-50),
3.40–3.19 (m, 2H, H-6a0,6b0), 2.19 and 2.09 (2s, 6Y, 2JAc); Anal.
Adenine derivative 2b was synthesized from 9-(3-deoxy-3-flu-
0
0
0
0
0
oro-b-
D
-glucopyranosyl)-N6-benzoyl adenine (1b) [31] by the same

50
S. Manta et al. / Bioorganic Chemistry 38 (2010) 48–55
methodology as described for the synthesis of 2a. Purified by flash
chromatography (CH2Cl2/MeOH, 9:1) and compound 2b (2.1 g,
66%, Rf = 0.39 in CH2Cl2/MeOH, 9:1) was obtained as a thick syrup.
2.4. Synthesis of 1-(4,6-dideoxy-3-fluoro-b-
enopyranosyl-2-ulose)uracil (6c)
D-glycero-hex-3-
½
a 2D2
ꢀ
ꢁ 4:0 (c 0.50, MeOH); kmax 279 nm (
e
6448); 1H NMR (CD3OD):
d 8.77 and 8.63 (2s, 2H, H-2,8), 8.12–7.58 (m, 5Y, Bz), 5.85 (d, 1H,
2.4.1. 1-(3-Deoxy-3-fluoro-6-iodo-b-
Uracil derivative 2c was synthesized from 1-(3-deoxy-3-fluoro-
-glucopyranosyl)uracil (1c) [32] by the same methodology as
D
-glucopyranosyl)uracil (2c)
J1 ,2 = 8.8 Hz, H-10), 4.64–4.50 (m, 2H, H-20 and H-30), 3.77 (m, 1H,
H-40), 3.65–3.45(m, 3H, H-50 and H-6a0,6b0); Anal. Calcd for C18H17FI-
N5O4: C, 42.12; H, 3.34; N, 13.64. Found: C, 42.30; H, 3.17; N, 13.86.
ESI-MS (m/z): 514.25 (M+H+).
0
0
b-
D
described for the synthesis of 2a. Purified by flash chromatography
(CH2Cl2/MeOH, 9:1) and compound 2c (1.26 g, 60%, Rf = 0.11 in
CH2Cl2/MeOH, 9:1) was obtained as a yellow foam. ½a D22
þ 2:0 (c
ꢀ
2.3.2. 9-(2,4-Di-O-acetyl-3-deoxy-3-fluoro-6-iodo-b-
glucopyranosyl)-N6-benzoyl adenine (3b)
D-
0.31, MeOH); kmax 257 nm (e
3220); 1H NMR (CD3OD): d 8.33 (br
s, 1H, NH), 7.67 (d, 1H, J5,6 = 8.1 Hz, H-6), 5.76 (d, 1H, H-5), 5.65
(d, 1H, J1 ,2 = 9.4 Hz, H-10), 4.44 (dtr, 1H, JF,3 = 52.4 Hz,
0
0
0
Adenine derivative 3b was synthesized from 2b by the same
methodology as described for the synthesis of 3a. Purified by flash
chromatography (hexane/AcOEt, 2:8) and compound 3b (2.0 g,
J2 ,3 = J3 ,4 = 8.7 Hz, H-30), 3.87 (m, 1H, H-20), 3.65–3.43 (m, 3H, H-
40 and H-6a0,6b0), 3.22 (m, 1H, H-50); Anal. Calcd for C10H12FIN2O5:
C, 31.11; H, 3.13; N, 7.26. Found: C, 30.89; H, 3.27; N, 7.07. ESI-MS
(m/z): 387.13 (M+H+).
0
0
0
0
82%, Rf = 0.4 in AcOEt) was obtained as
ꢁ 2:0 (c 0.50, CHCl3); kmax 280 nm (
12921); 1H NMR (CDCl3):
d 8.86 and 8.32 (2s, 2H, H-2,8), 8.08–7.55 (m, 5Y, Bz), 5.99 (d, 1H,
a yellowish syrup.
½
a 2D2
ꢀ
e
J1 ,2 = 9.3 Hz, H-10), 5.71 (m, 1H, H-20), 5.35 (m, 1H, H-40), 4.88 (dtr,
0
0
2.4.2. 1-(2,4-Di-O-acetyl-3-deoxy-3-fluoro-6-iodo-b-
glucopyranosyl)uracil (3c)
D-
1H, JF,3 = 51.6 Hz, J2 ,3 = J3 ,4 = 8.9 Hz, H-30), 3.71 (m, 1H, H-50),
3.42–3.23 (m, 2H, H-6a0,6b0), 2.23 and 1.87 (2s, 6Y, 2JAc); Anal.
Calcd for C22H21FIN5O6: C, 44.24; H, 3.54; N, 11.72. Found: C,
44.09; H, 3.24; N, 11.91. ESI-MS (m/z): 598.35 (M+H+).
0
0
0
0
0
Uracil derivative 3c was synthesized from 2c by the same meth-
odology as described for the synthesis of 3a. Purified by flash chro-
matography (hexane/AcOEt, 4:6) and compound 3c (1.32 g, 86%,
Rf = 0.62 in AcOEt) was obtained as a white solid: mp. 138–
2.3.3. 9-(2,4-Di-O-acetyl-3,6-dideoxy-3-fluoro-b-
N6-benzoyl adenine (4b)
D-glucopyranosyl)-
140 °C; ½a 2D2
ꢀ
ꢁ 1:0 (c 0.16, CHCl3); kmax 256 nm (
e
4882);1H NMR
(CDCl3): d 8.32 (br s, 1H, NH), 7.34 (d, 1H, J5,6 = 8.2 Hz, H-6), 5.85
Adenine derivative 4b was synthesized from 3b by the same
methodology as described for the synthesis of 4a. Purified by flash
chromatography (hexane/AcOEt, 2:8) and compound 4b (1.17 g,
(m, 2H, H-5 and H-10), 5.30–5.14 (m, 2H, H-20 and H-40), 4.75
(dtr, 1H, JF,3 = 51.6 Hz, J2 ,3 = 9.0 Hz, J3 ,4 = 9.1 Hz, H-30), 3.50 (m,
1H, H-50), 3.40–3.16 (m, 2H, H-6a0,6b0), 2.18 and 2.08 (2s, 6Y,
2JAc); Anal. Calcd for C14H16FIN2O7: C, 35.76; H, 3.43; N, 5.96.
Found: C, 36.05; H, 3.18; N, 6.10. ESI-MS (m/z): 471.18 (M+H+).
0
0
0
0
0
74%, Rf = 0.39 in AcOEt) was obtained as a white foam. ½a D22
ꢁ 3:0
ꢀ
(c 0.50, CHCl3); kmax 280 nm (e
17712); 1H NMR (CDCl3): d 8.83
and 8.26 (2s, 2H, H-2,8), 8.05–7.53 (m, 5Y, Bz), 5.87 (d, 1H,
J1 ,2 = 9.3 Hz, H-10), 5.66 (m, 1H, H-20), 5.19 (m, 1H, H-40), 4.78
0
0
2.4.3. 1-(2,4-Di-O-acetyl-3,6-dideoxy-3-fluoro-b-
glucopyranosyl)uracil (4c)
Uracil derivative 4c was synthesized from 3c by the same meth-
odology as described for the synthesis of 4a. Purified by flash chro-
matography (hexane/AcOEt, 1:1) and compound 4c (0.67 g, 70%,
Rf = 0.22 in hexane/AcOEt, 1:1) was obtained as a white foam.
D-
(dtr, 1H, JF,3 = 51.8 Hz, J2 ,3 = J3 ,4 = 8.9 Hz, H-30), 3.83 (m, 1H, H-
0
0
0
0
0
50), 2.18 and 1.83 (2s, 6Y, 2JAc), 1.31 (d, 3Y, J5 ,6 = 5.5 Hz, H-60);
Anal. Calcd for C22H22FN5O6: C, 56.05; H, 4.70; N, 14.86. Found:
C, 55.83; H, 4.83; N, 14.61. ESI-MS (m/z): 472.42 (M+H+).
0
0
2.3.4. 9-(4-O-Acetyl-3,6-dideoxy-3-fluoro-b-
benzoyl adenine (5b)
D
-glucopyranosyl)-N6-
½
a 2D2
ꢀ
þ 4:0 (c 0.50, CHCl3); kmax 255 nm (
e
7832); 1H NMR (CDCl3):
d 8.45 (br s, 1H, NH), 7.34 (d, 1H, J5,6 = 8.2 Hz, H-6), 5.82 (d, 1H, H-
5), 5.75 (d, 1H, J1 ,2 = 9.6 Hz, H-10), 5.22 (m, 1H, H-20), 5.02 (m, 1H,
0
0
Adenine derivative 5b was synthesized from 4b by the same
methodology as described for the synthesis of 5a. Purified by flash
chromatography (AcOEt) and compound 5b (0.56 g, 53%, Rf = 0.29
H-40), 4.68 (dtr, 1H, JF,3 = 51.8 Hz, J2 ,3 = J3 ,4 = 9.1 Hz, H-30), 3.70
(m, 1H, H-50), 2.16 and 2.07 (2s, 6Y, 2JAc), 1.27 (d, 3Y,
0
0
0
0
0
in AcOEt) was obtained as an oil. ½a D22
ꢀ
þ 16:0 (c 0.50, CHCl3); kmax
J5 ,6 = 6.2 Hz, H-60); Anal. Calcd for C14H17FN2O7: C, 48.84; H,
4.98; N, 8.14. Found: C, 48.52; H, 5.14; N, 8.45. ESI-MS (m/z):
345.28 (M+H+).
0
0
280 nm (e
14468); 1H NMR (CDCl3): d 8.52 and 8.24 (2s, 2H, H-
2,8), 7.99–7.50 (m, 5Y, Bz), 5.73 (d, 1H, J1 ,2 = 8.1 Hz, H-10), 5.17
0
0
(m, 1H, H-40), 4.80–4.66 (m, 2H, H-20 and H-30), 3.79 (m, 1H, H-
50), 2.19 (s, 3Y, JAc), 1.28 (d, 3Y, J5 ,6 = 5.4 Hz, H-60); Anal. Calcd
for C20H20FN5O5: C, 55.94; H, 4.69; N, 16.31. Found: C, 55.67; H,
4.80; N, 16.19. ESI-MS (m/z): 430.42 (M+H+).
0
0
2.4.4. 1-(2-J-Acetyl-4-O-benzoyl-3,6-dideoxy-3-fluoro-b-
glucopyranosyl)uracil (8c)
D-
To a stirred solution of 1-(2-O-acetyl-3,6-dideoxy-3-fluoro-b-D-
glucopyranosyl)uracil (7c) [32] (1.50 g, 4.96 mmol) in pyridine
(24.8 mL) was added benzoyl chloride (BzCl) (2.88 mL, 24.8 mmol).
The reaction mixture was stirred at room temperature for 2 h, di-
luted with AcOEt and washed several times with saturated aqueous
NaHCO3 solution. The organic layer was dried over anhydrous so-
dium sulfate, filtered and evaporated to dryness. The residue was
purified by flash chromatography (hexane/AcOEt, 1:1) and com-
pound 8c (1.59 g, 79%, Rf = 0.33 in hexane/AcOEt, 1:1) was obtained
2.3.5. 9-(4,6-Dideoxy-3-fluoro-b-
ulose)-N6-benzoyl adenine (6b)
Adenine derivative 6b was synthesized from 5b by the same
methodology as described for the synthesis of 6a. Purified by flash
chromatography (AcOEt) and compound 6b (0.29 g, 60%, Rf = 0.26
in AcOEt) was obtained as a yellow powder: mp. 211–213 °C;
D-glycero-hex-3-enopyranosyl-2-
½
a 2D2
ꢀ
ꢁ 12:0 (c 0.50, CHCl3); kmax 280 nm (
e
10491); 1H NMR
(CDCl3): d 8.81 and 8.12 (2s, 2H, H-2,8), 8.08–7.55 (m, 5Y, Bz),
asa whitefoam. ½a D22
ꢀ
ꢁ 6:0 (c 0.50, CHCl3);kmax 255 nm (
e
19627);1H
6.69 (d, 1H, JF,4 = 10.7 Hz, H-40), 6.53 (s, 1H, H-10), 5.08 (m, 1H,
NMR (CDCl3): d 8.13–7.94 (m, 5H, Bz), 7.53 (d, 1H, J5,6 = 8.3 Hz, H-6),
0
H-50), 1.55 (d, 3Y, J5 ,6 = 5.3 Hz, H-60); 13C NMR (CDCl3): d 190.31,
164.28, 156.35, 157.29, 151.60, 150.01, 147.32, 136.11, 133.27,
129.28, 127.69, 124.33, 116.37, 85.91, 67.39, 22.15; 19F NMR: d
ꢁ63.2; Anal. Calcd for C18H14FN5O3: C, 58.85; H, 3.84. N, 19.07.
Found: C, 59.07; H, 3.73; N, 19.38. ESI-MS (m/z): 368.34 (M+H+).
5.98 (d, 1H, H-5), 5.82 (d, 1H, J1 ,2 = 9.3 Hz, H-10), 5.36–5.26 (m, 2H,
0
0
0
0
H-20 and H-40), 4.90 (dtr, 1H, JF,3 = 51.8 Hz, J2 ,3 = J3 ,4 = 9.0 Hz, H-
0
0
0
0
0
30), 3.88 (m, 1H, Y-50), 2.07 (s, 3Y, JAc), 1.37 (d, 3Y, J5 ,6 = 6.2 Hz,
H-60); Anal. Calcd for C19H19FN2O7: C, 56.16; H, 4.71; N, 6.89. Found:
C, 56.28; H, 4.59; N, 7.02. ESI-MS (m/z): 407.35 (M+H+).
0
0

S. Manta et al. / Bioorganic Chemistry 38 (2010) 48–55
51
2.4.5. 1-(4-O-Benzoyl-3,6-dideoxy-3-fluoro-b-
(9c)
D-glucopyranosyl)uracil
2.5.3. 1-(2,4-Di-O-acetyl-3,6-dideoxy-3-fluoro-b-
glucopyranosyl)thymine (4d)
D-
Compound 8c (1.59 g, 3.92 mmol) was dissolved in EtOH-pyri-
dine (78.4 + 35.5 mL), 2 M NaOH (2.75 mL) was added and the mix-
ture stirred at room temperature for 30 min. Amberlite IR-120 (H+)
wasaddedto neutralize thebase. Thesuspensionwasfiltered, there-
sin was washed with EtOH and pyridine (30 + 30 mL) and the filtrate
was evaporated. The solid residue was purified by flash chromatog-
raphy (hexane/AcOEt, 3:7) and compound 9c (0.93 g, 65%, Rf = 0.34
Thymine derivative 4d was synthesized from 3d by the same
methodology as described for the synthesis of 4a. Purified by flash
chromatography (hexane/AcOEt, 6:4) and compound 4d (0.71 g,
67%, Rf = 0.5 in hexane/AcOEt, 4:6) was obtained as a white foam.
½
a 2D2
ꢀ
þ 2:0 (c 0.50, CHCl3); kmax 261 nm (
e
10114); 1H NMR (CDCl3):
0
0
d 8.43 (br s, 1H, NH), 7.16 (s, 1H, H-6), 5.75 (d, 1H, J1 ,2 = 9.5 Hz, H-
10), 5.25 (m, 1H, H-20), 5.04 (m, 1H, H-40), 4.68 (dtr, 1H, JF,3 = 51.9 Hz,
0
in hexane/AcOEt, 3:7) was obtained as a yellowish foam. ½a D22
ꢀ
þ 4:0
J2 ,3 = 9.0 Hz, J3 ,4 = 9.1 Hz, H-30), 3.70(m, 1H, H-50), 2.16and2.06(2s,
0
0
0
0
(c 0.50, CHCl3); kmax 261 nm (e
3936); 1H NMR (CDCl3): d 8.07–
6Y, 2JAc), 1.96 (s, 3H, 5-CH3), 1.28 (d, 3Y, J5 ,6 = 6.2 Hz, H-60); Anal.
Calcd for C15H19FN2O7: C, 50.28; H, 5.34; N, 7.82. Found: C, 50.47;
H, 5.12; N, 7.98. ESI-MS (m/z): 359.31 (M+H+).
0
0
7.63 (m, 5H, Bz), 7.37 (d, 1H, J5,6 = 8.2 Hz, H-6), 5.78 (m, 2H,
J1 ,2 = 9.7 Hz, H-5 and H-10), 5.22 (m, 1H, H-40), 4.81 (dtr, 1H,
0
0
JF,3 = 52.1 Hz, J2 ,3 = J3 ,4 = 9.0 Hz, H-30), 4.01–3.85 (m, 2H, H-20 and
0
0
0
0
0
Y-50), 1.29 (d, 3Y, J5 ,6 = 6.1 Hz, H-60); Anal. Calcd for C17H17FN2O6:
C, 56.04; H, 4.70; N, 7.69. Found: C, 56.15; H, 4.55; N, 7.88. ESI-MS
(m/z): 365.34 (M+H+).
2.5.4. 1-(2-O-Acetyl-4-O-benzoyl-3,6-dideoxy-3-fluoro-b-
D-
0
0
glucopyranosyl)thymine (8d)
Thymine derivative 8d was synthesized from 1-(2-O-acetyl-3,6-
dideoxy-3-fluoro-b- -glucopyranosyl)thymine (7d) [32] by the
D
same methodology as described for the synthesis of 8c. Purified
by flash chromatography (hexane/AcOEt, 6:4) and compound 8d
(1.45 g, 73%, Rf = 0.65 in hexane/AcOEt, 4:6) was obtained as a
2.4.6. 1-(4,6-Dideoxy-3-fluoro-b-
ulose)uracil (6c)
D-glycero-hex-3-enopyranosyl-2-
Uracil derivative 6c was synthesized from 9c by the same meth-
odology as described for the synthesis of 6a. Purified by flash chro-
matography (hexane/AcOEt, 3:7) and compound 6c (0.37 g, 60%,
Rf = 0.35 in CH2Cl2/MeOH, 9.5:0.5) was obtained as a white powder:
white solid: mp. 288–290 °C; ½a D22
ꢁ 14:0 (c 0.50, CHCl3); kmax
ꢀ
260 nm (e
18654); 1H NMR (CDCl3): d 8.25 (br s, 1H, NH), 8.09–
0
0
7.49 (m, 5H, Bz), 7.22 (s, 1H, H-6), 5.84 (d, 1H, J1 ,2 = 9.5 Hz, H-
10), 5.38–5.29 (m, 2H, H-20 and H-40), 4.88 (dtr, 1H, JF,3 = 52.0 Hz,
0
mp. 174–176 °C; ½a D22
ꢀ
ꢁ 6:0 (c 0.50, CHCl3); kmax 260 nm (
e
1224); 1H
J2 ,3 = J3 ,4 = 9.0 Hz, H-30), 3.87 (m, 1H, H-50), 2.09 (s, 3Y, JAc),
0
0
0
0
NMR(CDCl3):d 8.31(br s, 1H, NH), 7.82(d, 1H, J5,6 = 8.1 Hz, H-6), 7.07
2.00 (s, 3H, 5-CH3), 1.35 (d, 3Y, J5 ,6 = 6.2 Hz, H-60); Anal. Calcd
for C20H21FN2O7: C, 57.14; H, 5.04; N, 6.66. Found: C, 56.89; H,
4.93; N, 6.88. ESI-MS (m/z): 421.37 (M+H+).
0
0
(d, 1H, JF,1 = 8.1 Hz, H-10), 6.58 (dd, 1H, JF,4 = 11.4 Hz, J4 ,5 = 1.4 Hz,
0
0
0
0
H-40), 5.79 (d, 1H, H-5), 4.94 (m, 1H, H-50), 1.51 (d, 3Y, J5 ,6 = 6.7 Hz,
H-60); 13C NMR (CDCl3): d 187.05, 164.31, 157.26, 148.37, 140.02,
117.35, 103.09, 86.28, 68.71, 20.19; 19F NMR: d ꢁ64.3; Anal. Calcd
for C10H9FN2O4: C, 50.01; H, 3.78; N, 11.66. Found: C, 50.12; H,
3.57; N, 11.85. ESI-MS (m/z): 241.17 (M+H+).
0
0
2.5.5. 1-(4-O-Benzoyl-3,6-dideoxy-3-fluoro-b-
D-
glucopyranosyl)thymine (9d)
Thymine derivative 9d was synthesized from 8d by the same
methodology as described for the synthesis of 9c. Purified by flash
chromatography (hexane/AcOEt, 4:6) and compound 9d (0.89 g,
68%, Rf = 0.23 in hexane/AcOEt, 4:6) was obtained as a clear viscous
2.5. Synthesis of 1-(4,6-dideoxy-3-fluoro-b-
enopyranosyl-2-ulose)thymine (6d)
D-glycero-hex-3-
oil. ½a 2D2
ꢀ
þ 3:0 (c 0.50, CHCl3); kmax 263 nm (
e
7658);1HNMR (CDCl3):
d 9.95 (br s, 1H, NH), 8.07–7.41 (m, 5H, Bz), 7.22 (s, 1H, H-6), 5.81 (d,
1H, J1 ,2 = 9.2 Hz, H-10), 5.26 (m, 1H, H-40), 4.88 (dtr, 1H,
0
0
2.5.1. 1-(3-Deoxy-3-fluoro-6-iodo-b-
Thymine derivative 2d was synthesized from 1-(3-deoxy-3-flu-
oro-b- -glucopyranosyl)thymine (1d) [32] by the same methodol-
D-glucopyranosyl)thymine (2d)
JF,3 = 52.0 Hz, J2 ,3 = 8.7 Hz, J3 ,4 = 9.0 Hz, H-30), 4.06–3.89 (m, 2H,
0
0
0
0
0
H-20 and H-50), 1.93 (s, 3H, 5-CH3), 1.31 (d, 3Y, J5 ,6 = 6.0 Hz, H-60);
Anal. Calcd for C18H19FN2O6: C, 57.14; H, 5.06; N, 7.40. Found: C,
57.44; H, 4.93; N, 7.58. ESI-MS (m/z): 379.36 (M+H+).
0
0
D
ogy as described for the synthesis of 2a. Purified by flash
chromatography (CH2Cl2/MeOH, 9:1) and compound 2d (1.43 g,
69%, Rf = 0.4 in CH2Cl2/MeOH, 9:1) was obtained as a white foam.
½
a 2D2
ꢀ
þ 2:0 (c 0.50, MeOH); kmax 262 nm (
e
5394); 1H NMR (CD3OD):
2.5.6. 1-(4,6-Dideoxy-3-fluoro-b-D-glycero-hex-3-enopyranosyl-2-
ulose)thymine (6d)
0
0
d 8.06 (br s, 1H, NH), 7.50 (s, 1H, H-6), 5.63 (d, 1H, J1 ,2 = 9.4 Hz, H-
10), 4.43 (dtr, 1H, JF,3 = 52.1 Hz, J2 ,3 = 8.8 Hz, J3 ,4 = 9.1 Hz, H-30),
3.91 (m, 1H, H-20), 3.65–3.41 (m, 3H, H-40 and H-6a0,6b0), 3.22
(m, 1H, H-50), 1.93 (s, 3H, 5-CH3); Anal. Calcd for C11H14FIN2O5:
C, 33.02; H, 3.53; N, 7.00. Found: C, 33.26; H, 3.36; N, 7.18. ESI-
MS (m/z): 401.15 (M+H+).
Thymine derivative 6d was synthesized from 9d by the same
methodology as described for the synthesis of 6a. Purified by flash
chromatography (CH2Cl2/MeOH, 9.5:0.5) and compound 6d (0.35 g,
58%, Rf = 0.36 in CH2Cl2/MeOH, 9.5:0.5) was obtained as a white foam.
0
0
0
0
0
½
a 2D2
ꢀ
ꢁ 40:0 (c 0.50, CHCl3); kmax 260 nm (
e
2542); 1H NMR (CDCl3): d
8.84 (s, 1H, H-6), 6.88 (s, 1H, H-10), 6.58 (d, 1H, JF,4 = 11.1 Hz, H-40),
0
4.94 (m, 1H, H-50), 1.93 (s, 3H, 5-CH3), 1.51 (d, 3Y, J5 ,6 = 6.8 Hz, H-
60); 13C NMR (CDCl3): d 189.36, 162.98, 156.01, 149.00, 136.12,
116.31, 111.90, 84.92, 69.26, 20.21, 11.98; 19F NMR: d ꢁ65.5; Anal.
Calcd for C11H11FN2O4: C, 51.97; H, 4.36; N, 11.02. Found: C, 51.83;
H, 4.58; N, 10.77. ESI-MS (m/z): 255.22 (M+H+).
0
0
2.5.2. 1-(2,4-Di-O-acetyl-3-deoxy-3-fluoro-6-iodo-b-
glucopyranosyl)thymine (3d)
D-
Thymine derivative 3d was synthesized from 2d by the same
methodology as described for the synthesis of 3a. Purified by flash
chromatography (hexane/AcOEt, 6:4) and compound 3d (1.43 g,
83%, Rf = 0.5 in hexane/AcOEt, 4:6) was obtained as a white pow-
der: mp. 160–162 °C; ½a D22
ꢀ
þ 2:0 (c 0.44, CHCl3); kmax 261 nm (
e
2.6. Synthesis of 1-(4,6-dideoxy-3-fluoro-b-
D-glycero-hex-3-
enopyranosyl-2-ulose)-N4-benzoyl cytosine (6e)
7617); 1H NMR (CDCl3): d 8.37 (br s, 1H, NH), 7.17 (s, 1H, H-6),
5.85 (d, 1H, J1 ,2 = 9.5 Hz, H-10), 5.32–5.14 (m, 2H, H-20 and H-40),
0
0
4.74 (dtr, 1H, JF,3 = 51.7 Hz, J2 ,3 = J3 ,4 = 9.1 Hz, H-30), 3.51 (m, 1H,
H-50), 3.39–3.16 (m, 2H, H-6a0,6b0), 2.18 and 2.07 (2s, 6Y, 2JAc),
1.97 (s, 3H, 5-CH3); Anal. Calcd for C15H18FIN2O7: C, 37.21; H,
3.75; N, 5.79. Found: C, 37.51; H, 3.63; N, 5.98. ESI-MS (m/z):
485.20 (M+H+).
0
0
0
0
0
2.6.1. 1-(3-Deoxy-3-fluoro-6-iodo-b-
cytosine (2e)
D
-glucopyranosyl)-N4-benzoyl
Cytosine derivative 2e was synthesized from 1-(3-deoxy-3-flu-
oro-b-
D
-glucopyranosyl)-N4-benzoyl cytosine (1e) [30] by the

52
S. Manta et al. / Bioorganic Chemistry 38 (2010) 48–55
H-20 and H-50), 1.33 (d, 3Y, J5 ,6 = 6.2 Hz, H-60); Anal. Calcd for
C24H22FN3O6: C, 61.67; H, 4.74; N, 8.99. Found: C, 61.95; H, 4.63; N,
9.17. ESI-MS (m/z): 468.46 (M+H+).
0
0
same methodology as described for the synthesis of 2a. Purified by
flash chromatography (CH2Cl2/MeOH, 9:1) and compound 2e
(1.14 g, 59%, Rf = 0.27 in CH2Cl2/MeOH, 9:1) was obtained as a yel-
lowish foam. ½a 2D2
ꢀ
þ 1:0 (c 0.50, MeOH); kmax 261 nm (
e
6517); 1H
NMR (CD3OD): d 8.68 (br s, 1H, NH), 7.79–7.38 (m, 7H, Bz, Y-5
2.6.6. 1-(4,6-Dideoxy-3-fluoro-b-
D-glycero-hex-3-enopyranosyl-2-
and H-6), 5.89 (d, 1H, J1 ,2 = 9.2 Hz, H-10), 4.67–4.43 (m, 2H, H-20
and H-30), 3.67 (m, 1H, H-40), 3.59–3.37 (m, 2H, H-6a0,6b0), 3.22
(m, 1H, H-50); Anal. Calcd for C17H17FIN3O5: C, 41.73; H, 3.50; N,
8.59. Found: C, 42.09; H, 3.64; N, 8.42. ESI-MS (m/z): 490.22
(M+H+).
ulose)-N4-benzoyl cytosine (6e)
0
0
Cytosine derivative 6e was synthesized from 9e by the same
methodology as described for the synthesis of 6a. Purified by flash
chromatography (hexane/AcOEt, 2:8) and compound 6e (0.40 g,
58%, Rf = 0.62 in CH2Cl2/MeOH, 9.5:0.5) was obtained as a white
powder: mp. 226–228 °C; ½a D22
ꢁ 16:0 (c 0.50, CHCl3); kmax 261 nm
ꢀ
(e
18216); 1H NMR (CDCl3): d 8.66 (br s, 1H, NH), 8.12–7.44 (m, 7H,
2.6.2. 1-(2,4-Di-O-acetyl-3-deoxy-3-fluoro-6-iodo-b-
glucopyranosyl)-N4-benzoyl cytosine (3e)
D-
Bz, Y-5 and H-6), 6.58 (dd, 2H, JF,4 = 11.4 Hz, J4 ,5 = 1.7 Hz, H-40 and
0
0
0
H-10), 4.98 (m, 1H, H-50), 1.53 (d, 3Y, J5 ,6 = 6.8 Hz, H-60); 13C NMR
(CDCl3): d 191.36, 166.25, 165.22, 157.01, 155.19, 142.93, 134.13,
131.87, 129.43, 127.89, 117.07, 101.32, 83.96, 66.04, 22.66; 19F
NMR: d ꢁ65.0; Anal. Calcd for C17H14FN3O4: C, 59.47; H, 4.11; N,
12.24. Found: C, 59.65; H, 3.99; N, 11.96. ESI-MS (m/z): 344.30
(M+H+).
Cytosine derivative 3e was synthesized from 2e by the same
methodology as described for the synthesis of 3a. Purified by flash
chromatography (hexane/AcOEt, 4:6) and compound 3e (1.1 g,
0
0
82%, Rf = 0.62 in AcOEt) was obtained as a yellow oil. ½a D22
þ 16:0
ꢀ
(c 0.50, CHCl3); kmax 262 nm (e
28431); 1H NMR (CDCl3): d 8.76
(br s, 1H, NH), 7.91–7.43 (m, 7H, Bz, Y-5 and H-6), 6.14 (d, 1H,
J1 ,2 = 9.4 Hz, H-10), 5.31–5.19 (m, 2H, H-20 and H-40), 4.80 (dtr,
0
0
1H, JF,3 = 51.6 Hz, J2 ,3 = J3 ,4 = 9.1 Hz, H-30), 3.53 (m, 1H, H-50),
3.42–3.19 (m, 2H, H-6a0,6b0), 2.19 and 2.06 (2s, 6Y, 2JAc); Anal.
Calcd for C21H21FIN3O7: C, 43.99; H, 3.69; N, 7.33. Found: C,
44.24; H, 3.55; N, 7.21. ESI-MS (m/z): 574.32 (M+H+).
0
0
0
0
0
2.7. 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, vesicular sto-
matitis virus, cytomegalovirus (HCMV) and varicella-zoster virus
(VZV)], Vero (parainfluenza-3, reovirus-1, sindbis virus and cox-
sackie 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 con-
fluent for MDCK cells) 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 h virus adsorption period, residual
virus was removed, and the cell cultures were incubated in the pres-
2.6.3. 1-(2,4-Di-O-acetyl-3,6-dideoxy-3-fluoro-b-
N4-benzoyl cytosine (4e)
D-glucopyranosyl)-
Cytosine derivative 4e was synthesized from 3e by the same
methodology as described for the synthesis of 4a. Purified by flash
chromatography (hexane/AcOEt, 2:8) and compound 4e (0.59 g,
69%, Rf = 0.39 in AcOEt) was obtained as
þ 22:0 (c 0.50, CHCl3); kmax 262 nm (
(CDCl3): d 8.65 (br s, 1H, NH), 7.90–7.50 (m, 7H, Bz, Y-5 and H-
a
white foam.
½
a 2D2
ꢀ
e
20073); 1H NMR
6), 6.04 (d, 1H, J1 ,2 = 9.4 Hz, H-10), 5.24 (m, 1H, H-20), 5.07 (m,
0
0
1H, H-40), 4.73 (dtr, 1H, JF,3 = 51.8 Hz, J2 ,3 = 9.0 Hz, J3 ,4 = 9.1 Hz,
H-30), 3.74 (m, 1H, H-50), 2.17 and 2.05 (2s, 6Y, 2JAc), 1.29 (d,
0
0
0
0
0
ence of varying concentrations (200, 40, 8, . . . , lM) of the test com-
pounds. 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. The minimal cytotoxic concentra-
tion (MCC) of the compounds was defined as the compound concen-
tration that caused a microscopically visible alteration of cell
morphology. The methodology of the anti-HIV assays was as fol-
lows: human CEM (ꢂ3 ꢃ 105 cells/cm3) cells were infected with
3Y, J5 ,6 = 6.2 Hz, H-60); Anal. Calcd for C21H22FN3O7: C, 56.37; H,
4.96; N, 9.39. Found: C, 56.65; H, 4.85; N, 9.26. ESI-MS (m/z):
448.43 (M+H+).
0
0
2.6.4. 1-(2-O-Acetyl-4-O-benzoyl-3,6-dideoxy-3-fluoro-b-
D-
glucopyranosyl)-N4-benzoyl cytosine (8e)
Cytosine derivative 8e was synthesized from 1-(2-O-acetyl-3,6-
dideoxy-3-fluoro-b-
-glucopyranosyl)-N4-benzoyl cytosine (7e)
100 CCID50 of HIV(IIIB) or HIV-2(ROD)/mL and seeded in 200-lL
D
wells of a microtiter 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.
[32] by the same methodology as described for the synthesis of
8c. Purified by flash chromatography (hexane/AcOEt, 3:7) and
compound 8e (1.51 g, 80%, Rf = 0.26 in hexane/AcOEt, 3:7) was ob-
tained as a white solid: mp. 308–310 °C; ½a D22
þ 7:0 (c 0.50, CHCl3);
ꢀ
2.8. Cytostatic/toxic activity assays
kmax 262 nm (e
18578); 1H NMR (CDCl3): d 8.65 (br s, 1H, NH),
0
0
8.09–7.47 (m, 12H, 2Bz, Y-5 and H-6), 6.11 (d, 1H, J1 ,2 = 9.5 Hz,
Murine leukemia L1210, murine mammary carcinoma FM3A,
and human lymphocyte CEM and human cervix carcinoma HeLa
cells were seeded in 96-well microtiter plates at 50,000 (L1210,
H-10), 5.40–5.27 (m, 2H, H-20 and H-40), 4.91 (dtr, 1H, JF,3 = 52.0 Hz,
0
J2 ,3 = 9.3 Hz, J3 ,4 = 9.0 Hz, H-30), 3.90 (m, 1H, H-50), 2.07 (s, 3Y,
0
0
0
0
JAc), 1.35 (d, 3Y, J5 ,6 = 6.2 Hz, H-60); Anal. Calcd for C26H24FN3O7:
C, 61.29; H, 4.75; N, 8.25. Found: C, 61.47; H, 4.64; N, 8.12. ESI-MS
(m/z): 510.49 (M+H+).
0
0
FM3A), 75,000 (CEM) or 20,000 (HeLa) cells per 200 lL-well in
the presence of different concentrations of the test compounds.
After 2 (L1210, FM3A), 3 (CEM) or 4 (HeLa) days, the viable cell
number was counted using a Coulter counter apparatus. The 50%
cytostatic concentration (CC50) was defined as the compound con-
centration required to inhibit tumor cell proliferation by 50%.
2.6.5. 1-(4-O-Benzoyl-3,6-dideoxy-3-fluoro-b-
benzoyl cytosine (9e)
D
-glucopyranosyl)-N4-
Cytosine derivative 9e was synthesized from 8e by the same
methodology as described for the synthesis of 9c. Purified by flash
chromatography (hexane/AcOEt, 2:8) and compound 9e (0.95 g,
69%, Rf = 0.32 in hexane/AcOEt, 2:8) was obtained as a yellowish
3. Results and discussion
foam. ½a 2D2
ꢀ
þ 2:0 (c 0.48, CHCl3); kmax 260 nm (
e
11403); 1H NMR
3.1. Synthesis
(CDCl3): d 8.70 (br s, 1H, NH), 8.08–7.40 (m, 12H, 2Bz, Y-5 and H-
6), 5.98 (d, 1H, J1 ,2 = 9.2 Hz, H-10), 5.28 (m, 1H, H-40), 4.83 (dtr, 1H,
Retrosynthetic analysis of the target unsaturated dideoxy fluoro-
ketopyranosyl nucleosides 6a–e suggested that the b-nucleosides
0
0
JF,3 = 51.9 Hz, J2 ,3 = 8.6 Hz, J3 ,4 = 9.0 Hz, H-30), 4.03–3.87 (m, 2H,
0
0
0
0
0

S. Manta et al. / Bioorganic Chemistry 38 (2010) 48–55
53
group of fluoro nucleosides 1a–e [30–32] using triphenylphosphine,
iodine and imidazole [40–43], led to the desired iodo derivatives 2a–
e. Acetylation of the free hydroxyl groups in the 20- and 40-position of
the sugar moiety of the aforementioned nucleosides with acetic
anhydride/pyridine afforded the desired acetylated derivatives 3a–
e in very good yields (3a, 89%, 3b, 82%, 3c, 86%, 3d, 83% and 3e,
82%). Catalytic hydrogenation of 3a–e in the presence of palla-
dium-on-carbon furnished the deoxy nucleosides 4a–e. However,
when regioselectively 20-O-deacylation of the fully protected deoxy
nucleosides 4a–e with hydroxylamine hydrochloride and sodium
acetate [44] was employed, only the partially acetylated analogues
of 5-fluorouracil (5a) and N6-benzoyl adenine (5b) were formed. It
is noteworthy that treatment of the deoxy nucleosides of uracil
(5c), thymine (5d) and N4-benzoyl cytosine (5e) either with hydrox-
ylamine hydrochloride [44] or hydrazine hydrate [45], resulted in
intractable products. In the final step, oxidation of the fluoro acety-
lated dideoxy precursors 5a,b performed by the acetic anhydride/di-
methyl sulfoxide system [46] gave, after a b-elimination reaction,
1a–e [30–32] were the most promising starting materials (Fig. 1).
Therefore, our synthetic route was based on the assumption that
the target molecules 6a–e could be reached from the corresponding
6-bromo-4-benzoates II by hydrogenation and subsequent oxida-
tion (Fig. 1). The latter analogues could be obtained from 4,6-O-ben-
zylidene nucleosides I, since the benzylidene acetals could undergo
smooth ring opening by the action of N-bromosuccinimide [35–38].
Although, direct benzylidenation of nucleosides 1a–e [30–32] with
a,a-dimethoxytoluene, under Evans [39] conditions afforded the de-
sired analogues I, the Hanessian–Hullar reaction on 4,6-O-benzyli-
dene acetals failed and only decomposition products were
obtained. It seems that the presence of the electron-withdrawing
fluorine atom a to the benzylidene acetal ring causes destabilization
of the benzylidene radical formed and therefore the ring opening
cannot take place.
Since the N-bromosuccinimide-mediated fragmentation of ben-
zylidene derivatives I failed, an alternative and more versatile plan
was then decided to acquire the target unsaturated carbonyl nucleo-
sides 6a–e (Fig. 2). Thus, monoiodination of the primary hydroxyl
the desired unsaturated 4,6-dideoxy-3-fluoro-b-D-glycero-hex-3-
Br
O
CH3
HO
Ph
B
B
B
B
O
O
O
O
ii
i
F
F
F
H
OBz
O
OH
O
OH
F
OH
OH
1a,b,c,d,e
B= a: 5-Fluorouracil, b: 6 -Benzoyladenine, c: Uracil, d: Thymine, e: 4 -Benzoylcytosine
II
I
6a,b,c,d,e
N
N
Fig. 1. Rationale for the design of the target molecules 6a–e. Reagents and conditions: (i) N,N-dimethylformamide,
a
,
a
-dimethoxytoluene, 68 °C, 1 h. (ii) BaCO3, N-
bromosuccinimide, CCl4, reflux, 3 h.
CH3
CH3
HO
I
B
B
B
B
O
O
O
O
i
iii
iv
F
F
F
F
OAc
OAc
OH
OH
OR
OAc
4a,b,c,d,e
OH
1a,b,c,d,e
OR
5a,b
2a,b,c,d,e R=H
3a,b,c,d,e R=Ac
ii
ref.32
v
CH3
F
CH3
CH3
CH3
F
B
B
B
B
O
O
O
O
vi
v
vii
F
OH
OBz
OBz
F
O
OAc
7c,d,e
OAc
8c,d,e
OH
9c,d,e
6a,b,c,d,e
Fig. 2. Reagents and conditions: (i) iodine, triphenylphosphine, imidazole, tetrahydrofuran, 80 °C, 1 h; 68% of 2a; 66% of 2b; 60% of 2c; 69% of 2d; 59% of 2e. (ii) Pyridine,
acetic anhydride, 2 h; 89% of 3a; 82% of 3b; 86% of 3c; 83% of 3d; 82% of 3e. (iii) H2, 10% Pd/C, triethylamine, AcOEt, EtOH, 20 °C, 24 h; 65% of 4a; 74% of 4b; 70% of 4c; 67% of
4d; 69% of 4e. (iv) Hydroxylamine hydrochloride, sodium acetate, pyridine, 12 h; 50% of 5a; 53% of 5b. (v) Acetic anhydride/dimethyl sulfoxide, 100 °C, 10 min; 55% of 6a; 60%
of 6b; 60% of 6c; 58% of 6d; 58% of 6e. (vi) BzCl, pyridine, 2 h; 79% of 8c; 73% of 8d; 80% of 8e. (vii) EtOH, pyridine, NaOH, 30 min, Amberlite IR-120 (H+) resin; 65% of 9c; 68% of
9d; 69% of 9e.

54
S. Manta et al. / Bioorganic Chemistry 38 (2010) 48–55
enopyranosyl-2-ulose derivatives of 5-fluorouracil (6a) and N6-ben-
zoyl adenine (6b), in 55% and 60% yield, respectively.
In order to overcome the difficulty encountered in the previous
route and to successfully synthesize the desired unsaturated 1-
Table 2
Cytotoxic activity of 6a–e against a panel of tumor cell lines.
Compounds
MCCa
Vero
MDCK
HeLa
HEL
100
100
>100
100
20
(4,6-dideoxy-3-fluoro-b-D-glycero-hex-3-enopyranosyl-2-ulose)
6a
6b
6c
6d
6e
100
100
>100
100
20
ꢄ0.8
20
100
20
100
100
>100
100
nucleosides of uracil (6c), thymine (6d) and N4-benzoyl cytosine
(6e), a different synthetic strategy was then investigated. After sev-
eral subsequent protection and deprotection steps of compounds
1c–e [31,32] and finally catalytic hydrogenation, the dideoxy flu-
oro acetylated nucleosides 7c–e were formed [32] (Fig. 2). Treat-
ment of the latter analogues with BzCl in pyridine afforded the
corresponding 4-benzoates 8c–e. Deacetylation was performed
using NaOH/EtOH/pyridine [47] to give the partially protected
derivatives 9c–e. Finally, oxidation of the fluoro benzoylated dide-
oxy precursors 9c–e using the acetic anhydride/dimethyl sulfoxide
system [46] afforded, after a b-elimination reaction, the desired
4
ꢄ20
a
Minimum cytotoxic concentration that causes a microscopically detectable
alteration of cell morphology.
mine, N6-benzoyladenine and N4-benzoylcytosine were present
as the heterocyclic base (IC50: 1.4–16 l
M for the 40-keto deriva-
tives [32]). In contrast, the cytostatic activity of the 5-fluorouracil
20-keto and 40-keto derivatives were quite similar (IC50: 0.49–
unsaturated 4,6-dideoxy-3-fluoro-b-D-glycero-hex-3-enopyrano-
3.7 l
M for the 40-keto derivatives [32]). These findings point to
syl-2-ulose derivatives of uracil (6c), thymine (6d) and N4-benzoyl
cytosine (6e) in 60%, 58% and 58% yield, respectively.
an important role of the heterocycle (5-fluorouracil) in the even-
tual cytostatic activity of this compound.
All new compounds were well-characterized by NMR and UV
The explanation for the ‘‘detoxifying” role of the 20-keto, com-
pared to the 40-keto derivative is currently unclear, but may open
interesting perspectives for this type of derivative if antiviral mol-
ecules may emerge from this class of compounds. Therefore, novel
compounds bearing different heterocyclic base parts should be
synthesized in an attempt to reach this goal (antiviral potency
combined with poor cytostatic activity).
spectroscopy, mass spectrometry and elemental analysis. 1H NMR
0
0
0
0
0
0
data obtained for the nucleosides 2a–e (J1 ,2 , J2 ,3 , J3 ,4 P 8.7 Hz),
0
0
0
0
0
0
0
0
0
0
0
0
3a–e (J1 ,2 , J2 ,3 , J3 ,4 P 8.9 Hz), 4a–e (J1 ,2 , J2 ,3 , J3 ,4 P 8.9 Hz), 5a,b
0
0
0
0
0
0
0
0
0
0
0
0
(J1 ,2 , J2 ,3 , J3 ,4 P 8.1 Hz), 8c–e (J1 ,2 , J2 ,3 , J3 ,4 P 9.0 Hz) and 9c–e
0
0
0
0
0
0
(J1 ,2 , J2 ,3 , J3 ,4 P 8.6 Hz) revealed that, as expected, these com-
pounds have the b configuration and that it existed in the 4C1 confor-
mation. The unsaturated dideoxy fluoro ketonucleosides 6a–e have
half-chair conformations [48] and each of the compounds have the
bulky substituent at the C-10 in the equatorial position [49]. Finally,
in compounds 6a–e the presence of the C(O)–CF@CH-system was
ascertained by the disappearance of the signal of H-20 in the 1H
NMR spectra and the deshieldingof other protons, especially the ole-
finic proton H-40.
4. Conclusion
In summary, the synthesis of the unsaturated 4,6-dideoxy-3-
fluoro-b-D-glycero-hex-3-enopyranosyl-2-ulose nucleosides of 5-
fluorouracil, N6-benzoyl adenine, uracil, thymine, and N4-benzoyl
cytosine, respectively was undertaken and the target nucleosides
were evaluated for their antiviral and cytostatic/cytotoxic activity.
The newly synthesized 20-ketopyranosyl derivatives were not
potent antivirals at subtoxic concentrations, and found to be less
inhibitory than the previously synthesized 40-keto congeners
against a panel of tumor cell lines [32]. Therefore, it becomes clear
that the transposition of the keto group from C-40 to C-20 lowers
the cytotoxic activity of this type of molecules.
3.2. Biological activity
Compounds 6a–e have been evaluated against a broad panel of
DNA and RNA viruses in cell culture (mentioned at Experimental
part). Unfortunately, none of the compounds inhibited virus infec-
tion and replication at subtoxic concentrations. When the com-
pounds 6a–e were evaluated for their anti-proliferative activity
against two murine and two human tumor cell lines, moderate to
poor inhibitory activity was noted for 6b–e (IC50: 7.8 to 219 lM,
Acknowledgments
depending on the nature of the compound and the tumor cell line
tested) (Table 1). However, the 5-fluorouracil derivative 6a showed
pronounced cytostatic activity against the murine (L1210, FM3A)
This work was supported in part by the Postgraduate Pro-
grammes ‘‘Biotechnology-Quality assessment in Nutrition and the
Environment”, ‘‘Application of Molecular Biology-Molecular Genet-
ics-Molecular Markers”, Department of Biochemistry and Biotech-
nology, University of Thessaly. Financial support was also provided
by the ‘‘Geconcerteerde Onderzoeksacties” (GOA No. 05/19) of the
K.U. Leuven. The authors are grateful to Mrs. Lizette van Berckelaer,
Leentje Persoons, Frieda De Meyer, Vickie Broeckx, Anita Camps,
Steven Carmans, Lies Van den Heurck and Leen Ingels for dedicated
technical help.
and human (HeLa) tumor cell lines (IC50: 0.21–1.2
pound was moderately inhibitory against CEM cell proliferation
(IC50: 12 M) (Table 1). The new compounds were in general less
cytotoxic to confluent adherent tumor cell cultures (Table 2).
It is striking to see that the 20-keto derivatives presented in this
study are markedly less cytostatic against the tumor cell lines than
their corresponding 40-keto derivatives [32] when the uracil, thy-
lM). The com-
l
Table 1
Cytostatic activity of 6a–e against a panel of tumor cell lines.
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