Synthesis of 3-fluoro-6-S-(2-S-pyridyl) nucleosides as potential lead cytostatic agents

Article abstract of DOI:10.1016/j.bioorg.2010.08.001

The 3-deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-β-d-glucopyranosyl nucleoside analogs 7 were prepared via two facile synthetic routes. Their precursors, 3-fluoro-6-thio-glucopyranosyl nucleosides 5a-e, were obtained by the sequence of deacetylation of 3-deoxy-3-fluoro-β-d-glucopyranosyl nucleosides 2a-e, selective tosylation of the primary OH of 3 and finally treatment with potassium thioacetate. The desired thiolpyridine protected analogs 7a-c,f,g were obtained by the sequence of deacetylation of 5a-c followed by thiopyridinylation and/or condensation of the corresponding heterocyclic bases with the newly synthesized peracetylated 6-S-(2-S-pyridyl) sugar precursor 13, which was obtained via a novel synthetic route from glycosyl donor 12. None of the compounds 6 and 7 showed antiviral activity, but the 5-fluorouracil derivative 7c and particularly the uracil derivative 7b were endowed with an interesting and selective cytostatic action against a variety of murine and human tumor cell cultures.

Full text of DOI:10.1016/j.bioorg.2010.08.001

Bioorganic Chemistry 38 (2010) 285–293
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of 3-fluoro-6-S-(2-S-pyridyl) nucleosides as potential lead
cytostatic agents
Evangelia Tsoukala a, Niki Tzioumaki a, Stella Manta a, Alexandra Riga a, Jan Balzarini b, Dimitri Komiotis a,
⇑
a Department of Biochemistry and Biotechnology, Laboratory of Organic Chemistry, University of Thessaly, 41221 Larissa, Greece
b Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, K.U.Leuven, B-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 6 July 2010
Available online 17 August 2010
The 3-deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-b-
via two facile synthetic routes. Their precursors, 3-fluoro-6-thio-glucopyranosyl nucleosides 5a-e, were
obtained by the sequence of deacetylation of 3-deoxy-3-fluoro-b- -glucopyranosyl nucleosides 2a-e,
D
-glucopyranosyl nucleoside analogs 7 were prepared
D
selective tosylation of the primary OH of 3 and finally treatment with potassium thioacetate. The desired
thiolpyridine protected analogs 7a-c,f,g were obtained by the sequence of deacetylation of 5a-c followed
by thiopyridinylation and/or condensation of the corresponding heterocyclic bases with the newly syn-
thesized peracetylated 6-S-(2-S-pyridyl) sugar precursor 13, which was obtained via a novel synthetic
route from glycosyl donor 12. None of the compounds 6 and 7 showed antiviral activity, but the 5-fluo-
rouracil derivative 7c and particularly the uracil derivative 7b were endowed with an interesting and
selective cytostatic action against a variety of murine and human tumor cell cultures.
Ó 2010 Elsevier Inc. All rights reserved.
Keywords:
3-Fluoro-6-S-(2-S-pyridyl) nucleosides
Thiopyridinylation
Cytostatic agents
1. Introduction
candidates for use as both antiviral and antineoplastic agents
[21,24,27], with the latter being assessed as prodrugs able to inter-
Nucleoside analogs markedly contribute to the chemotherapy
of cancer and viral diseases [1–4]. However, novel applications
are still being explored and new nucleoside structures have
reached the clinic as effective agents for the treatment of AIDS
[5], herpes virus infections [6] and viral hepatitis [7].
Fluorine-containing nucleosides and their analogs are attractive
compounds and have drawn special attention [8–11], with the
fluorine incorporated either on the base moiety or on the sugar
part frequently leading to a drastic change in biological activity,
stability and bioavailability [8,12,13]. Our recent data have re-
vealed, that new classes of uncommon fluorinated pyranonucleo-
sides have a promising potential in combating the rotaviral
infections, in the treatment of colon cancer and are efficient antitu-
mor growth inhibitors [10,11,14,15]. Experimental data also sug-
gested, that human Poly(A)-specific ribonuclease is among the
molecular targets of these compounds and could act therapeuti-
cally by lowering the mRNA turnover rate [16].
fere with HIV reverse transcription [21,22,26]. Particularly, nucleo-
sides in which the primary hydroxyl group is replaced by a thiol- or
an alkylthio-group, are promising antiviral agents since they dis-
rupt essential viral macromolecular methylation processes [28,29].
In view of the above data and our recent findings on the biology
of modified nucleosides, as templates for antiviral and anticancer
activity [10,11,14–16,30–32], we turned our attention to the possi-
ble improvement of the biological properties of such molecules.
We thus synthesised a series of glucopyranosyl nucleoside analogs,
where we combined both a pharmacologically compatible fluorine
and a sulfur/thiopyridine moiety in the sugar part. In this paper, we
report on two efficient methods for their preparation and supply
data on their biological activity.
2. Experimental
In the field of nucleosides, sulfur chemistry appears particularly
fruitful in the development of therapeutic agents [17,18]. Consider-
able interest has been drawn in the synthesis of thionucleoside
analogs [18–21] and nucleosidyl disulfides [22–26], attractive
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). 1H, 19F and 13C NMR
spectra were obtained at room temperature with a Brucker 400
⇑
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 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2010.08.001

286
E. Tsoukala et al. / Bioorganic Chemistry 38 (2010) 285–293
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, d = doublet,
dd = doublet doublet, dtr = doublet triplet and m = multiplet. Mass
spectra were obtained with a Micromass Platform LC (ESI-MS).
Infrared spectra were obtained with a Perkin–Elmer Model 1600
FT-IR spectrophotometer. Optical rotations were measured using
an Autopol I polarimeter. All reactions were carried out in dry sol-
vents. Acetonitrile was distilled from calcium hydride and stored
over 3E molecular sieves. Dimethylformamide (DMF) was also
stored over 3E molecular sieves, and pyridine was stored over
potassium hydroxide pellets.
Rf = 0.4 in hexane/AcOEt, 3:7) as white solid. m.p. 175–177 °C;
+ 5.26 (c 0.1, CHCl3); kmax 260 nm (
6760); 1H NMR (CDCl3):
½
a 2D2
ꢀ
e
d 8.20 (br s, NH), 7.76 (d, 1H, J5,6 = 8.2 Hz, H-6), 7.38–7.22 (m, 4H,
tosyl group), 5.79 (d, 1H, H-5), 5.73 (d, 1H, J1 ,2 = 9.6 Hz, H-10),
0
0
5.26–5.15 (m, 2H, H-20 and H-40), 4.70 (dtr, 1H, JF,3 = 51.7 Hz,
0
J2 ,3 = J3 ,4 = 9.0 Hz, H-30), 4.22–4.06 (m, 2H, H-6a0,6b0), 3.85 (m,
1H, Y-50), 2.45 (s, 3H, ArCH3), 2.10 and 2.06 (2s, 6H, 2OAc); Anal.
Calcd for C21H23FN2O10S: C, 49.03; H, 4.51; N, 5.45. Found: C,
49.19; H, 4.65; N, 5.62. ESI-MS (m/z): 515.49 (M+H+).
0
0
0
0
2.2.5. 1-(2,4-Di-O-acetyl-3-deoxy-3-fluoro-6-O-p-toluenesulfonyl-b-
D-glucopyranosyl)5-fluorouracil (4c)
5-Fluorouracil derivative 4c was synthesized from 3c [15] by
the similar procedure as described for 4a. Purified by flash chroma-
tography (hexane/AcOEt, 4:6) derivative 4c was obtained (1.18 g,
60%, Rf = 0.4 in hexane/AcOEt, 4:6) as white solid. m.p. 198–
2.2. Synthesis of 1-[3-deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-b-
glucopyranosyl] nucleosides (7a-c) from 5a-c and/or 6a-c
D-
200 °C; ½a 2D2
ꢀ
+ 5.32 (c 0.1, CHCl3); kmax 260 nm (e
7486); 1H NMR
(CDCl3): d 8.28 (br s, NH), 7.50 (d, 1H, J6,F5 = 5.85 Hz, H-6), 7.37–
7.20 (m, 4H, tosyl group), 5.74 (d, 1H, J1 ,2 = 9.4 Hz, H-10), 5.27–
0
0
5.15 (m, 2H, H-20 and H-40), 4.75 (dtr, 1H, JF,3 = 51.5 Hz, J2 ,3 = 9.2,
0
0
0
2.2.1. 1-(3-Deoxy-3-fluoro-b-
D
-glucopyranosyl)cytosine (3f)
A mixture of methanolic ammonia (140 mL) and N4-benzoyl-
cytosine derivative 2d [11] (1.70 g, 3.36 mmol) stirred for 4 h at
room temperature. The reaction mixture was concentrated and
crude 3f was obtained (0.83 g, 90%, Rf = 0.15 in AcOEt) as colorless
J3 ,4 = 9.0 Hz, H-30), 4.21–4.06 (m, 2H, H-6a0,6b0), 3.90 (m, 1H, Y-
50), 2.45 (s, 3H, ArCH3), 2.11 and 2.07 (2s, 6H, 2OAc); Anal. Calcd
for C21H22F2N2O10S: C, 47.37; H, 4.16; N, 5.26. Found: C, 47.54; H,
4.29; N, 5.42. ESI-MS (m/z): 533.48 (M+H+).
0
0
oil. Product 3f was used without further purification. ½a D22
ꢀ
+ 5.00 (c
0.1, MeOH); kmax 268 nm (
e
7162); ESI-MS (m/z): 276.26 (M+H+).
2.2.6. 1-(2,4-Di-O-acetyl-3-deoxy-3-fluoro-6-O-p-toluenesulfonyl-b-
D
-glucopyranosyl)-N4-benzoyl cytosine (4d)
2.2.2. 9-(3-Deoxy-3-fluoro-b-
D
-glucopyranosyl)adenine (3g)
N4-benzoyl cytosine derivative 4d was synthesized from 3d [11]
Adenine derivative 3g was synthesized from N6-benzoyl ade-
nine derivative 2e [11] by the similar procedure as described for
3f. Compound 3g was obtained (0.85 g, 88%, Rf = 0.12 in AcOEt)
as colorless oil and it was used without further purification.
by the similar procedure as described for 4a. Purified by flash chro-
matography (hexane/AcOEt, 4:6) derivative 4d was obtained
(1.41 g, 62%, Rf = 0.4 in hexane/AcOEt, 4:6) as white foam.
½
a 2D2
ꢀ
+ 12.00 (c 0.5, CHCl3); kmax 263 nm (
e
21,906); 1H NMR
½
a 2D2
ꢀ
+ 4.00 (c 0.1, MeOH); kmax 259 nm (
e 4930); ESI-MS (m/z):
(CDCl3): d 8.66 (br s, NH), 7.88–7.31 (m, 11H, Bz, H-6, H-5 and tosyl
300.27 (M+H+).
group), 6.01 (d, 1H, J1 ,2 = 9.4 Hz, H-10), 5.30–5.14 (m, 2H, H-20 and
0
0
H-40), 4.74 (dtr, 1H, JF,3 = 51.6 Hz, J2 ,3 = 9.1, J3 ,4 = 9.0 Hz, H-30),
4.24–4.08 (m, 2H, H-6a0,6b0), 3.86 (m, 1H, Y-50), 2.45 (s, 3H, ArCH3),
2.12 and 2.04 (2s, 6H, 2OAc); Anal. Calcd for C28H28FN3O10S: C,
54.45; H, 4.57; N, 6.80. Found: C, 54.84; H, 4.29; N, 6.62. ESI-MS
(m/z): 618.62 (M+H+).
0
0
0
0
0
2.2.3. 1-(2,4-Di-O-acetyl-3-deoxy-3-fluoro-6-O-p-toluenesulfonyl-b-
D-glucopyranosyl)thymine (4a)
A solution of compound 3a [15] (1.10 g, 3.70 mmol) in 4.50 mL
of dry pyridine was cooled to 0 °C, and p-toluenesulfonyl chloride
(1.10 g, 5.58 mmol) in 4.50 mL of dry pyridine was added dropwise
with stirring. The reaction mixture was stored at room tempera-
ture overnight, and then was concentrated. The residual gum was
dissolved in pyridine (49 mL), acetic anhydride was added
(25 mL) and the resulted mixture stirred for 3 h at room tempera-
ture. MeOH (0.40 mL) was added to quench the reaction and the
mixture was concentrated under high vacuum to remove the sol-
vents. The mixture was extracted with two 200 mL portions of
ethyl acetate and neutralized with aqueous NaHCO3. The organic
layer was washed with NaHSO4 (20 mL), dried over anhydrous so-
dium sulfate and evaporated to dryness. Purification by flash chro-
matography (hexane/AcOEt, 4:6), gave 4a (1.20 g, 62%, Rf = 0.35 in
2.2.7. 9-(2,4-Di-O-acetyl-3-deoxy-3-fluoro-6-O-p-toluenesulfonyl-b-
D
-glucopyranosyl)-N6-benzoyl adenine (4e)
N6-benzoyl adenine derivative 4e was synthesized from 3e [11]
by the similar procedure as described for 4a. Purified by flash chro-
matography (hexane/AcOEt, 3:7) compound 4e was obtained
(1.52 g, 64%, Rf = 0.3 in hexane/AcOEt, 3:7) as white foam.
½
a 2D2
ꢀ
ꢁ 2.00 (c 0.2, CHCl3); kmax 278 nm (
e
17,393); 1H NMR (CDCl3):
d 9.05 (br s, NH), 8.84 and 8.15 (2s, 2H, H-2,8), 8.14–7.51 (m, 9H, Bz
and tosyl group), 5.84 (d, 1H, J1 ,2 = 9.5 Hz, H-10), 5.64 (m, 1H, H-20),
0
0
5.35 (m, 1H, H-40), 4.81 (dtr, 1H, JF,3 = 51.5 Hz, J2 ,3 = J3 ,4 = 9.1 Hz,
H-30), 4.23–4.11 (m, 2H, H-6a0,6b0), 4.01 (m, 1H, Y-50), 2.42 (s, 3H,
ArCH3), 2.16 and 1.84 (2s, 6H, 2OAc); Anal. Calcd for C29H28FN5O9S:
C, 54.29; H, 4.40; N, 10.92. Found: C, 54.34; H, 4.59; N, 10.78. ESI-
MS (m/z): 642.64 (M+H+).
0
0
0
0
0
hexane/AcOEt, 4:6) as white solid. m.p. 145–147 °C; ½a D22
ꢀ
+ 10.52 (c
0.1, CHCl3); kmax 260 nm (e
8836); 1H NMR (CDCl3): d 8.60 (br s,
NH), 7.37–7.20 (m, 4H, tosyl group), 7.19 (s, 1H, H-6), 5.75 (d,
1H, J1 ,2 = 9.5 Hz, H-10), 5.29–5.20 (m, 2H, H-20 and H-40), 4.70
0
0
(dtr, 1H, JF,3 = 51.7 Hz, J2 ,3 = 9.1, J3 ,4 = 9.0 Hz, H-30), 4.21–4.07
(m, 2H, H-6a0,6b0), 3.84 (m, 1H, Y-50), 2.45 (s, 3H, ArCH3), 2.10
and 2.05 (2s, 6H, 2OAc), 1.96 (s, 3H, 5-CH3); Anal. Calcd for
2.2.8. 1-(2,4-Di-O-acetyl-6-S-acetyl-3-deoxy-3-fluoro-6-thio-b-
copyranosyl)thymine (5a)
D
-glu-
0
0
0
0
0
The tosylate 4a (1.20 g, 2.30 mmol) was heated with potassium
thioacetate (0.36 g, 3.10 mmol) in DMF (8.70 mL) at 100 °C for 2 h.
The reaction mixture was neutralized with aqueous NaHCO3. After
that, the mixture was concentrated under high vacuum pump to
eliminate the DMF. The residue was partitioned between water
and EtOAc, the organic extract was dried over anhydrous sodium
sulfate, filtered, and evaporated to dryness. The residue was
purified by flash chromatography (hexane/AcOEt, 4:6) to give com-
pound 5a (0.75 g, 78%, Rf = 0.4 in hexane/AcOEt, 4:6) as white solid.
C
22H25FN2O10S: C, 50.00; H, 4.77; N, 5.30. Found: C, 50.26; H,
4.92; N, 5.47. ESI-MS (m/z): 529.51 (M+H+).
2.2.4. 1-(2,4-Di-O-acetyl-3-deoxy-3-fluoro-6-O-p-toluenesulfonyl-b-
D-glucopyranosyl)uracil (4b)
Uracil derivative 4b was synthesized from 3b [15] by the similar
procedure as described for 4a. Purified by flash chromatography
(hexane/AcOEt, 3:7) compound 4b was obtained (1.10 g, 58%,

E. Tsoukala et al. / Bioorganic Chemistry 38 (2010) 285–293
287
m.p. 204–206 °C; ½a D22
ꢀ
+ 5.21 (c 0.1, CHCl3), kmax 260 nm (
e
9219);
2.23 and 1.84 (2s, 6H, 2OAc); Anal. Calcd for C24H24FN5O7S: C,
52.84; H, 4.43; N, 12.84. Found: C, 52.68; H, 4.38; N, 13.09. ESI-
MS (m/z): 546.56 (M+H+).
IR (Nujol): 1697 (SAc) cmꢁ1
;
1H NMR (CDCl3): d 8.10 (br s, NH),
7.12 (s, 1H, H-6), 5.75 (d, 1H, J1 ,2 = 9.5 Hz, H-10), 5.29–5.18 (m,
0
0
2H, H-20 and H-40), 4.71 (dtr, 1H, JF,3 = 51.8 Hz, J2 ,3 = 9.1
0
0
0
J3 ,4 = 9.0 Hz, H-30), 3.78 (m, 1H, Y-50), 3.30–3.11 (m, 2H, H-6a0,6b0),
2.37 (s, 3H, SAc), 2.21 and 2.08 (2s, 6H, 2OAc), 1.98 (s, 3H, 5-CH3);
Anal. Calcd for C17H21FN2O8S: C, 47.22; H, 4.89; N, 6.48. Found: C,
47.39; H, 4.78; N, 6.54. ESI-MS (m/z): 433.44 (M+H+).
2.2.13. 1-[3-Deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-b-D-glucopyran-
osyl]thymine (7a) from 5a
0
0
To a solution of thioacetate 5a (1.10 g, 2.50 mmol) in methano-
lic ammonia (104 mL), 2,2-dipyridyl-disulfide (DTDP) (2.60 g,
12 mmol) dissolved in MeOH/H2O (1:1, 22 mL) was added drop-
wise and the mixture was stirred at room temperature. After stir-
ring for 20 h, the reaction mixture was concentrated. The crude
was finally purified by flash chromatography (AcOEt) to give com-
pound 7a (0.71 g, 68%, Rf = 0.30 in AcOEt) as white foam.
2.2.9. 1-(2,4-Di-O-acetyl-6-S-acetyl-3-deoxy-3-fluoro-6-thio-b-
copyranosyl)uracil (5b)
D-glu-
Uracil derivative 5b was synthesized from 4b by the similar
procedure as described for 5a. Purified by flash chromatography
(hexane/AcOEt, 3:7) and obtained (0.64 g, 76%, Rf = 0.45 in hex-
½
a 2D2 9093); 1H NMR
+ 16.00 (c 0.5, MeOH); kmax 266 nm (e
(CD3OD): d 8.09 (br s, NH), 8.41 (d, H-6 of pyridine), 7.83–7.58
ꢀ
ane/AcOEt, 3:7) as white solid. m.p. 158–160 °C; ½a D22
ꢀ
+ 5.18 (c
0.1, CHCl3); kmax 260 nm (
e
6283); IR (Nujol): 1697 (SAc) cmꢁ1
;
(m, H-4 and H-5 of pyridine), 7.29–7.12 (m, H-3 of pyridine and
1H NMR (CDCl3): d 8.30 (br s, NH), 7.30 (d, 1H, J5,6 = 8.3 Hz, H-6),
H-6), 5.40 (d, 1H, J1 ,2 = 9.4 Hz, H-10), 4.29 (dtr, 1H, JF,3 = 52.3 Hz,
0
0
0
5.82 (d, 1H, H-5), 5.75 (d, 1H, J1 ,2 = 9.5 Hz, H-10), 5.26–5.15 (m,
J2 ,3 = 8.5 Hz, J3 ,4 = 8.4 Hz, H-30), 3.92–3.79 (m, 1H, Y-20), 3.56–
3.45 (m, 2H, H-6a0 and H-40), 3.44–3.35 (m, 1H, 6b0), 2.98–2.89
(m, 1H, H-50); 19F NMR: d ꢁ65.00; 13C NMR (CD3OD): d 163.73,
160.52, 150.11, 148.90, 136.95, 132.92, 121.11, 119.18, 110.23,
92.18, 85.49, 72.36, 70.12, 67.95, 33.63, 12.41; Anal. Calcd for
0
0
0
0
0
0
2H, H-20 and H-40), 4.69 (dtr, 1H, JF,3 = 51.8 Hz, J2 ,3 = J3 ,4 = 9.1 Hz,
H-30), 3.80 (m, 1H, Y-50), 3.27–3.11 (m, 2H, H-6a0,6b0), 2.35 (s, 3H,
SAc), 2.19 and 2.07 (2s, 6H, 2OAc); Anal. Calcd for C16H19FN2O8S:
C, 45.93; H, 4.58; N, 6.70. Found: C, 45.89; H, 4.64; N, 6.82. ESI-
MS (m/z): 419.41 (M+H+).
0
0
0
0
0
C
16H18FN3O5S2: C, 46.26; H, 4.37; N, 10.11. Found: C, 46.41; H,
4.51; N, 10.32. ESI-MS (m/z): 416.47 (M+H+).
2.2.10. 1-(2,4-Di-O-acetyl-6-S-acetyl-3-deoxy-3-fluoro-6-thio-b-
D-glu-
copyranosyl)5-fluorouracil (5c)
2.2.14. 1-[3-Deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-b-D-glucopyran-
5-Fluorouracil derivative 5c was synthesized from 4c by the
similar procedure as described for 5a. Purified by flash chromatog-
raphy (hexane/AcOEt, 4:6) and obtained (0.65 g, 75%, Rf = 0.47 in
osyl]uracil (7b) from 5b
Uracil derivative 7b was synthesized from thioacetate 5b by the
similar procedure as described for 7a. Purified by flash chromatog-
raphy (AcOEt) and obtained (0.70 g, 70%, Rf = 0.27 in AcOEt) as
hexane/AcOEt, 4:6) as white solid. m.p. 200–202 °C; ½a D22
ꢀ
+ 5.26 (c
0.1, CHCl3); kmax 260 nm (
e
8232); IR (Nujol): 1697 (SAc) cmꢁ1
;
white foam. ½a 2D2
ꢀ
+ 6.00 (c 0.1, MeOH); kmax 262 nm (e
6478); 1H
NMR (CD3OD): d 8.53 (br s, NH), 8.32 (dd, H-6 of pyridine), 7.76–
7.73 (m, H-4 and H-5 of pyridine), 7.66 (m, H-3 of pyridine), 7.57
(d, 1H, J5,6 = 8.1 Hz, H-6), 5.68 (d, 1H, H-5), 5.46 (d, 1H,
1H NMR (CDCl3): d 8.28 (br s, NH), 7.36 (d, 1H, J6,F5 = 5.3 Hz, H-6),
5.71 (d, 1H, J1 ,2 = 9.3 Hz, H-10), 5.23–5.11 (m, 2H, H-20 and H-40),
0
0
4.70 (dtr, 1H, JF,3 = 51.5 Hz, J2 ,3 = 9.2, J3 ,4 = 9.0 Hz, H-30), 3.80 (m,
1H, Y-50), 3.29–3.10 (m, 2H, H-6a0,6b0), 2.36 (s, 3H, SAc), 2.19 and
2.08 (2s, 6H, 2OAc); Anal. Calcd for C16H18F2N2O8S: C, 44.04; H,
4.16; N, 6.42. Found: C, 44.28; H, 4.29; N, 6.34. ESI-MS (m/z):
437.41 (M+H+).
0
0
0
0
0
J1 ,2 = 9.4 Hz, H-10), 4.29 (dtr, 1H, JF,3 = 52.3 Hz, J2 ,3 = 8.7,
0
0
0
0
0
J3 ,4 = 8.6 Hz, H-30), 3.83–3.77 (m, 1H, Y-20), 3.64–3.60 (m, 1H, H-
6a0), 3.57–3.52 (m, 1H, H-40), 3.42–3.39 (m, 1H, 6b0), 3.01–2.96
(m, 1H, H-50); 19F NMR: d ꢁ63.20; 13C NMR (CD3OD): d 163.34,
160.43, 150.19, 147.96, 142.56, 132.74, 120.95, 119.26, 101.76,
91.98, 84.77, 71.83, 70.31, 67.62, 33.64; Anal. Calcd for
0
0
2.2.11. 1-(2,4-Di-O-acetyl-6-S-acetyl-3-deoxy-3-fluoro-6-thio-b-
D-glu-
copyranosyl)-N4-benzoyl cytosine (5d)
C
15H16FN3O5S2: C, 44.88; H, 4.02; N, 10.47. Found: C, 44.71; H,
N4-benzoyl cytosine derivative 5d was synthesized from 4d by
the similar procedure as described for 5a. Purified by flash chroma-
tography (hexane/AcOEt, 2:8) to give compound 5d (0.82 g, 68%,
Rf = 0.32 in hexane/AcOEt, 2:8) as solid. m.p. 277–279 °C;
4.22; N, 10.52. ESI-MS (m/z): 402.46 (M+H+).
2.2.15. 1-[3-Deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-b-
osyl]5-fluorouracil (7c) from 5c
D-glucopyran-
½
a 2D2
ꢀ
+ 16.00 (c 0.1, CHCl3); kmax 263 nm (
e
20,626); IR (Nujol):
5-Fluorouracil derivative 7c was synthesized from correspond-
ing thioacetate 5c by the similar procedure as described for 7a.
Purified by flash chromatography (AcOEt) and obtained (0.72 g,
1697 (SAc) cmꢁ1 1H NMR (CDCl3): d 7.88 (d, 1H, J5,6 = 7.2 Hz, H-
;
6), 7.84–7.48 (m, 6H, Bz and H-5), 6.03 (d, 1H, J1 ,2 = 9.4 Hz, H-10),
0
0
5.27–5.17 (m, 2H, H-20 and H-40), 4.75 (dtr, 1H, JF,3 = 51.8 Hz,
69%, Rf = 0.25 in AcOEt) as white foam. ½a D22
ꢀ
+ 2.00 (c 0.5, CHCl3);
0
J2 ,3 = 9.1 Hz, J3 ,4 = 9.0 Hz, H-30), 3.83–3.77 (m, 1H, H-50), 3.29–
3.11 (m, 2H, H-6a0,6b0), 2.35 (s, 3H, SAc), 2.20 and 2.05 (2s, 6H,
2OAc); Anal. Calcd for C23H24FN3O8S: C, 52.97; H, 4.64; N, 8.06.
Found: C, 52.70; H, 4.80; N, 8.22. ESI-MS (m/z): 522.54 (M+H+).
kmax 263 nm (e
5885); 1H NMR (CD3OD): d 8.34–8.32 (m, H-6 of
0
0
0
0
pyridine), 8.28 (br s, NH), 7.83–7.78 (m, H-4 and H-5 of pyridine),
7.17–7.13 (m, H-3 of pyridine), 5.44 (d, 1H, J1 ,2 = 9.0 Hz, H-10), 4.28
0
0
(dtr, 1H, JF,3 = 52.3 Hz, J2 ,3 = 8.7, J3 ,4 = 8.6 Hz, H-30), 3.82–3.76 (m,
1H, Y-20), 3.64–3.60 (m, 1H, H-6a0), 3.56–3.51 (m, 1H, H-40), 3.43–
3.39 (m, 1H, H-6b0), 3.01–2.96 (m, 1H, H-50); 19F NMR: d ꢁ64.30,
ꢁ63.20; 13C NMR (CD3OD): d 162.34, 157.89, 150.41, 148.76,
138.11, 132.91, 127.45, , 120.32, 119.07, 94.41, 85.29, 72.37,
70.11, 68.65, 33.47; Anal. Calcd for C15H15F2N3O5S2: C, 42.95; H,
3.60; N, 10.02. Found: C, 42.81; H, 3.48; N, 10.28. ESI-MS (m/z):
420.44.
0
0
0
0
0
2.2.12. 9-(2,4-Di-O-acetyl-6-S-acetyl-3-deoxy-3-fluoro-6-thio-b-
D-glu-
copyranosyl)-N6-benzoyl adenine (5e)
N6-benzoyl adenine derivative 5e was synthesized from 4e by
the similar procedure as described for 5a. Finally it was purified
by flash chromatography (hexane/AcOEt, 2:8) to give compound
5e (0.61 g, 60%, Rf = 0.24 in hexane/AcOEt, 2:8) as solid. m.p.
118–120 °C; ½a 2D2
ꢀ
+ 4.00 (c 0.1, CHCl3); kmax 279 nm (
e 12,567); IR
(Nujol): 1697 (SAc) cmꢁ1
;
1H NMR (CDCl3): d 9.07 (br s, NH),
2.2.16. Bis-[1-(3-deoxy-3-fluoro-6-thio-b-
D-glucopyranosyl)thymine]-
8.77 and 8.19 (2s, 2H, H-2,8), 7.98–7.42 (m, 5H, Bz), 5.88 (d, 1H,
6,6-disulfide (6a)
J1 ,2 = 9.5 Hz, H-10), 5.70–5.61 (m, 1H, H-20), 5.40–5.31 (m, 1H, H-
A mixture of methanolic ammonia (72.30 mL) and thionucleo-
side 5a (0.75 g, 1.73 mmol) stirred for 4 h at room temperature.
The reaction mixture was concentrated and disulfide 6a was
0
0
40), 4.81 (dtr, 1H, JF,3 = 51.7 Hz, J2 ,3 = 9.3 Hz, J3 ,4 = 8.9 Hz), 3.95–
0
0
0
0
0
3.89 (m, 1H, H-50), 3.32–3.15 (m, 2H, H-6a0,6b0), 2.35 (s, 3H, SAc),

288
E. Tsoukala et al. / Bioorganic Chemistry 38 (2010) 285–293
obtained (0.95 g, 90%, Rf = 0.20 in AcOEt) as white foam. ½a D22
ꢀ
+ 2.00
2.3. Synthesis of 1-[3-deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-b-
glucopyranosyl] nucleosides (7a-c,f,g) from glycosyl donor 13
D-
(c 0.5, MeOH); kmax 262 nm (e
19,126); 1H NMR (CD3OD): d 7.88 (br
s, NH), 7.49 (s, 1H, H-6), 5.55 (d, 1H, J1 ,2 = 9.4 Hz, H-10), 4.37 (dtr,
0
0
1H, JF,3 = 52.3 Hz, J2 ,3 = 8.5, J3 ,4 = 8.3 Hz, H-30), 4.14–4.04 (m, 1H,
Y-20), 3.72–3.55 (m, 1H, H-40), 3.35–3.23 (m, 2H, H-6a0,6b0),
2.92–2.80 (m, 1H, H-50), 1.89 (s, 3H, 5-CH3); Anal. Calcd for
0
0
0
0
0
2.3.1. 3-Deoxy-3-fluoro-1,2-O-isopropylidene-6-O-p-toluenesulfonyl-
a-D-glucofuranose (9)
C22H28F2N4O10S2: C, 43.27; H, 4.62; N, 9.18. Found: C, 43.18; H,
To a solution of diol 8 [33] (4.00 g, 18 mmol) in dry pyridine
4.84; N, 9.07. ESI-MS (m/z): 611.64.
(46.20 mL) was added p-toluenesulfonyl chloride (4.60 g,
24 mmol) and kept for 2 h at room temperature. After neutraliza-
tion (NaHCO3) and extraction with ethyl acetate (4 ꢂ 500 mL),
the combined extracts were dried over anhydrous sodium sulfate
and evaporated to dryness. Purification of the residue by flash
chromatography (hexane/AcOEt, 1:1) yielded the title compound
(5.08 g, 74%, Rf = 0.45 in hexane/AcOEt, 1:1) as colorless oil.
2.2.17. Bis-[1-(3-deoxy-3-fluoro-6-thio-b-
6,6-disulfide (6b)
Uracil derivative 6b was synthesized from thionucleoside 5b
following similar procedure as described for 6a. Disulfide 6b was
obtained (0.89 g, 88%, Rf = 0.16 in AcOEt) as white foam.
D-glucopyranosyl)uracil]-
½
a 2D2
ꢀ
ꢁ 8.00 (c 0.5, CHCl3); 1H NMR (CDCl3): d 7.38 and 7.82 (dd,
½
a 2D2
ꢀ
+ 3.00 (c 0.5, MeOH); kmax 258 nm (
e
14,339); 1H NMR
4H, tosyl group), 5.92 (d, 1H, J1 ,2 = 3.2 Hz, H-10), 5.09 (dd, 1H,
0
0
J3 ,F = 49.3 Hz, H-30), 4.70 (dd, 1H, J2 ,F = 10.3 Hz, H-20), 4.34 (d, 1H,
H-40), 4.13–4.06 (m, 2H, H-5a0,5b0), 2.47 (s, 3H, ArCH3), 1.48 (s,
3H, CH3), 1.33 (s, 3H, CH3); Anal. Calcd for C16H21FO7S: C, 51.06;
H, 5.62. Found: C, 51.14; H, 5.46. ESI-MS (m/z): 377.42 (M+H+).
(CD3OD): d 7.66 (d, 1H, J5,6 = 8.2 Hz, H-6), 7.24 (br s, NH), 5.64 (d,
0
0
1H, H-5), 5.56 (d, 1H, J1 ,2 = 9.5 Hz, H-10), 4.38 (dtr, 1H,
0
0
JF,3 = 52.0 Hz, J2 ,3 = 8.6 Hz, J3 ,4 = 8.1 Hz, H-30), 3.95–3.81 (m, 1H,
Y-20), 3.74–3.53 (m, 1H, H-40), 3.34–3.22 (m, 2H, H-6a0,6b0), 2.92–
2.81 (m, 1H, H-50); Anal. Calcd for C20H24F2N4O10S2: C, 41.23; H,
4.15; N, 9.62. Found: C, 41.38; H, 4.34; N, 9.78. ESI-MS (m/z):
583.57.
0
0
0
0
0
2.3.2. 3-Deoxy-3-fluoro-1,2-O-isopropylidene-5-O-tetrahydropyranyl-
6-O-p-toluenesulfonyl-a- -glucofuranose (10)
D
To a 0 °C solution of 9 (5.00 g, 13.30 mmol) and dried p-toluene-
sulfonic acid (0.25 g, 1.33 mmol) in anhydrous CH2Cl2 (26.60 mL)
was added 3,4-dihydro-2H-pyran (1.80 mL, 19.90 mmol). After
being kept for 2 h in room temperature, the reaction mixture was
neutralized with aqueous NaHCO3 and extracted with CH2Cl2
(2 ꢂ 1 L). The organic layers were combined, dried over Na2SO4
and concentrated in vacuo. The residue was chromatographed
through a column of silica gel (hexane/AcOEt, 3:7). Fractions con-
taining the product were combined and concentrated to afford
10 (4.78 g, 78%, Rf = 0.48 in hexane/AcOEt, 3:7) as off-white oil.
2.2.18. Bis-[1-(3-deoxy-3-fluoro-6-thio-b-
uracil]-6,6-disulfide (6c)
5-Fluorouracil derivative 6c was synthesized from thionucleo-
side 5c by the similar procedure as described for 6a. Compound
6c was obtained (0.95 g, 89%, Rf = 0.13 in AcOEt) as white foam.
D-glucopyranosyl)5-fluoro-
½
a 2D2
ꢀ
+ 2.00 (c 0.5, MeOH); kmax 263 nm (
e
15,056); 1H NMR
(CD3OD): d 7.89 (d, 1H, J6,F5 = 6.4 Hz, H-6), 7.35 (br s, NH), 5.56
(d, 1H, J1 ,2 = 9.3 Hz, H-10), 4.38 (dtr, 1H, JF,3 = 52.5 Hz, J2 ,3 = 8.7,
0
0
0
0
0
J3 ,4 = 8.6 Hz, H-30), 3.90–3.84 (m, 1H, H-20), 3.70–3.66 (m, 1H, H-
6a0), 3.61–3.56 (m, 1H, H-40), 3.29–3.26 (m, 1H, H-6b0), 2.88–2.83
(m, 1H, Y-50); Anal. Calcd for C20H22F4N4O10S2: C, 38.84; H, 3.59;
N, 9.06. Found: C, 38.72; H, 3.47; N, 9.12. ESI-MS (m/z): 619.56.
0
0
½
a 2D2
ꢀ
ꢁ 6.00 (c 0.5, CHCl3); 1H NMR (CDCl3): d 7.84–7.80 (four sep-
arate s, 2H, HC8 + HC2), 7.35–7.28 (m, 5H, Ph), 5.98 (d, 1/2H,
J1 ,2 = 2.8 Hz, H-10), 5.89 (d, 1/2H, J1 ,2 = 2.9 Hz, H-10), 2.47 (s, 3H,
0
0
0
0
ArCH3), 1.50 (s, 3H, CH3), 1.33 (s, 3H, CH3); Anal. Calcd for
C
21H29FO8S: C, 54.77; H, 6.35. Found: C, 54.64; H, 6.42. ESI-MS
(m/z): 461.53 (M+H+).
2.2.19. 1-[3-Deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-b-D-glucopyran-
osyl]thymine (7a) from 6a
2.3.3. 3-Deoxy-3-fluoro-1,2-O-isopropylidene-5-O-tetrahydropyranyl-
To a solution of 6a (1.83 g, 3.00 mmol) in MeOH (26 mL), DTDP
(3.16 g, 14.46 mmol) dissolved in MeOH/H2O (1:1, 26 mL) was
added dropwise. The reaction was carried out at room temperature
and within 12 h. After disappearance of the starting material (TLC),
the solvents were removed under diminished pressure and the yel-
low residue was purified by flash column chromatography (AcOEt)
to give compound 7a (0.88 g, 71%, Rf = 0.30 in AcOEt) as white
foam.
6-S-acetyl-6-thio-a- -glucofuranose (11)
D
The tosylate 10 (4.50 g, 9.80 mmol) was heated with potassium
thioacetate (1.50 g, 13.50 mmol) in DMF (39.20 mL) at 100 °C for
1 h. The reaction mixture was neutralized with aqueous NaHCO3.
After that, the mixture was concentrated under high vacuum pump
to eliminate the DMF. The residue was partitioned between water
and EtOAc, the organic extract was dried over anhydrous sodium
sulfate, filtered, and evaporated to dryness. The residue was purified
by flash chromatography (hexane/AcOEt, 4:6) to give compound 11
(2.71 g, 76%, Rf = 0.42 in hexane/AcOEt, 4:6) as yellow syrup.
2.2.20. 1-[3-Deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-b-
osyl]uracil (7b) from 6b
Uracil derivative 7b was synthesized from 6b by the similar
procedure as described for 7a. Purified by flash chromatography
(AcOEt) and obtained (0.83 g, 69%, Rf = 0.27 in AcOEt) as white
foam.
D-glucopyran-
½
a 2D2
ꢀ
ꢁ 8.00 (c 0.1, CHCl3); IR (Nujol): 1697 (SAc) cmꢁ1
;
1H NMR
(CDCl3): d 7.84–7.80 (4 separate s, 2H, HC8 + HC2), 7.35–7.28 (m,
5H, Ph), 5.96 (d, 1H, J1 ,2 = 3.7 Hz, H-10), 2.37 (s, 3H, SAc), 1.52 (s,
3H, CH3), 1.34 (s, 3H, CH3); Anal. Calcd for C16H25FO6S: C, 52.73;
H, 6.91. Found: C, 52.84; H, 6.72. ESI-MS (m/z): 365.47 (M+H+).
0
0
2.3.4. 1,2,4-Tri-O-acetyl-3-deoxy-3-fluoro-6-S-acetyl-6-thio-D-gluco-
2.2.21. 1-[3-Deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-b-D-glucopyran-
pyranose (12)
osyl]5-fluorouracil (7c) from 6c
A solution of thioacetate 11 (2.50 g, 6.90 mmol) in 90% TFA
(172.50 mL) was stirred for 20 min at room temperature. The reac-
tion mixture was concentrated under diminished pressure and
crude was obtained as a colorless oil. Acetylation of the residue
with Ac2O-pyridine (1:2, 36.50 mL) and chromatography of the
5-Fluorouracil derivative 7c was synthesized from 6c by the
similar procedure as described for 7a. Purified by flash chromatog-
raphy (AcOEt) and obtained (0.86 g, 68%, Rf = 0.25 in AcOEt) as
white foam.

E. Tsoukala et al. / Bioorganic Chemistry 38 (2010) 285–293
289
product (hexane/AcOEt, 4:6) gave compound 12 (1.77 g, 70%,
added and the reaction mixture was refluxed for 5 h, cooled, neu-
tralized with aqueous sodium bicarbonate, and extracted with
CH2Cl2 (500 mL). The organic layer was washed with water
(3 ꢂ 10 mL) and dried over anhydrous sodium sulfate, evaporated
to dryness. The crude underwent deacetylation with methanolic
ammonia (550 mL) within 4 h, concentrated and finally purified
by flash chromatography (AcOEt) to give compound 7c (3.89 g,
70%, Rf = 0.25 in AcOEt) as white foam.
Rf = 0.45 in hexane/AcOEt, 4:6) as colorless syrup. ½a D22
ꢀ
+ 12.00
(c 0.1, CHCl3); IR (Nujol): 1697 (SAc) cmꢁ1 1H NMR (CDCl3): d
;
6.30 (br s, 1H, H-10), 5.62 (d, 1H, H-40), 5.33–5.04 (m, 1H, H-20),
4.80 (dtr, 1/2H, J3 ,F = 53.3 Hz, J3 ,4 = 9.2 Hz, H-30), 4.58 (dtr, 1/2H,
0
0
0
J3 ,F = 51.9 Hz, J3 ,4 = 9.0 Hz, H-30), 4.03–3.98 (m, 1/2H, H-50), 3.70–
3.64 (m, 1/2H, H-50), 3.28–3.21 and 3.15–3.08 (2 m, 2H, H-6a0,6b0),
2.36 (s, 3H, SAc), 2.17, 2.14, 2.12, 2.10 (4s, 12H, 4OAc); Anal. Calcd
for C14H19FO8S: C, 45.90; H, 5.23. Found: C, 46.04; H, 5.16. ESI-MS
(m/z): 367.38 (M+H+).
0
0
0
2.3.9. 1-[3-Deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-b-
osyl]cytosine (7f)
D-glucopyran-
2.3.5. 1,2,4-Tri-O-acetyl-3-deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-
glucopyranose (13)
D
-
A mixture of N4-benzoyl cytosine (3.99 g, 18.55 mmol), HMDS
(4.80 mL, 23 mmol) and saccharine (0.16 g, 0.85 mmol) in anhy-
drous CH3CN (69 mL) was refluxed for 30 min. Triacetylated thio-
pyridyl sugar 13 (5.74 g, 13.25 mmol) and trimethylsilyl
trifluoromethane-sulfonate (3.10 mL, 17.22 mmol) were then
added and the reaction mixture was refluxed for 5 h, cooled, neu-
tralized with aqueous sodium bicarbonate, and extracted with
CH2Cl2 (500 mL). The organic layer was washed with water
(3 ꢂ 10 mL) and dried over anhydrous sodium sulfate, evaporated
to dryness. The crude underwent deacetylation with methanolic
ammonia (550 mL) within 4 h, concentrated and finally purified
by flash chromatography (AcOEt) to give compound 7f (3.60 g,
To a solution of thiosugar 12 (2.00 g, 5.46 mmol) in methanolic
ammonia (228 mL), DTDP (9.55 g, 43.70 mmol) dissolved in
MeOH:H2O (1:1, 47 mL) was added dropwise and the mixture
was stirred at room temperature. After stirring for 20 h, the reac-
tion mixture was concentrated. Acetylation of the residue with
Ac2O-pyridine (1:2, 109 mL) and chromatography of the product
(hexane/AcOEt, 4:6) gave compound 13 (1.67 g, 70%, Rf = 0.45 in
hexane/AcOEt, 4:6) as colorless syrup. ½a D22
ꢀ
+ 4.00 (c 0.1, CHCl3);
kmax 283 nm (
e
5510); 1H NMR (CDCl3): d 5.62 (d, 1/2H, H-10),
5.26–5.03 (m, 2H, H-20 and H-40), 4.79 (dtr, 1/2H, J3 ,F = 51.9 Hz,
0
J3 ,4 = 9.2 Hz, H-30), 4.53 (dtr, 1/2H, J3 ,F = 51.7 Hz, J3 ,4 = 9.0 Hz, H-
30), 4.19–4.14 (m, 1/2H, H-50), 3.75–3.70 (m, 1/2H, H-50), 3.42–
3.35 (m, 2H, H-6a0,6b0), 2.14, 2.12, 2.10, 2.08, 2.07, 2.05 (6s, 18H,
6OAc); Anal. Calcd for C17H20FNO7S2: C, 47.10; H, 4.65; N, 3.23.
Found: C, 47.24; H, 4.46; N, 3.35. ESI-MS (m/z): 434.49.
68%, Rf = 0.32 in AcOEt) as white foam. ½a D22
ꢀ
+ 2.00 (c 0.5, MeOH);
0
0
0
0
0
kmax 269 nm (e
7400); 1H NMR (CD3OD): d 8.23 (m, H-6 of pyri-
dine), 7.78–7.69 (m, H-4 and H-5 of pyridine), 7.51 (d, 1H, H-5),
7.46 (d, 1H, J5,6 = 7.3 Hz, H-6), 7.08–7.03 (m, H-3 of pyridine),
5.52 (d, 1H, J1 ,2 = 9.4 Hz, H-10), 4.22 (dtr, 1H, JF,3 = 52.5 Hz,
0
0
0
J2 ,3 = 9.0 Hz, J3 ,4 = 8.9 Hz, H-30), 3.73–3.66 (m, 1H, Y-20), 3.53–
3.42 (m, 2H, H-40 and H-6a0), 3.34–3.30 (m, 1H, H-6b0), 2.92–2.87
(m, 1H, H-50); 19F NMR: d ꢁ64.33; 13C NMR (CD3OD): d 165.54,
160.32, 154.28, 148.46, 137.56, 132.91, 122.03, 120.12, 99.36,
92.38, 85.62, 72.39, 70.64, 70.22, 33.63; Anal. Calcd for
0
0
0
0
2.3.6. 1-[3-Deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-b-
osyl]thymine (7a)
D-glucopyran-
A mixture of thymine (2.33 g, 18.55 mmol), HMDS (4.80 mL,
23 mmol) and saccharine (0.16 g, 0.85 mmol) in anhydrous CH3CN
(69 mL) was refluxed for 30 min. Triacetylated thiopyridyl sugar 13
(5.74 g, 13.25 mmol) and tin chloride (1.94 mL, 17.22 mmol) were
then added and the reaction mixture was refluxed for 5 h, cooled,
neutralized with aqueous sodium bicarbonate, and extracted with
CH2Cl2 (500 mL). The organic layer was washed with water
(3 ꢂ 10 mL) and dried over anhydrous sodium sulfate, evaporated
to dryness. The crude underwent deacetylation with methanolic
ammonia (550 mL) within 4 h, concentrated and finally purified
by flash chromatography (AcOEt) to give compound 7a (3.53 g,
64%, Rf = 0.30 in AcOEt) as white foam.
C15H17FN4O4S2: C, 44.99; H, 4.28; N, 13.99. Found: C, 44.82; H,
4.42; N, 13.52. ESI-MS (m/z): 401.48.
2.3.10. 9-[3-Deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-b-D-glucopyran-
osyl]adenine (7g)
A mixture of N6-benzoyl adenine (2.93 g, 12.25 mmol), HMDS
(3.10 mL, 15.19 mmol) and saccharine (0.10 g, 0.56 mmol) in anhy-
drous CH3CN (46 mL) was refluxed for 30 min. Triacetylated thio-
pyridyl sugar 13 (3.80 g, 8.75 mmol) and tin chloride (1.38 mL,
12.25 mmol) were then added and the reaction mixture was re-
fluxed for 5 h, cooled, neutralized with aqueous sodium bicarbon-
ate, and extracted with CH2Cl2 (500 mL). The organic layer was
washed with water (3 ꢂ 10 mL) and dried over anhydrous sodium
sulfate, evaporated to dryness. The crude underwent deacetylation
with methanolic ammonia (512 mL) within 4 h, concentrated and
finally purified by flash chromatography (AcOEt) to give compound
2.3.7. 1-[3-Deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-b-
osyl]uracil (7b)
D-glucopyran-
A mixture of uracil (2.08 g, 18.55 mmol), HMDS (4.80 mL,
23 mmol) and saccharine (0.16 g, 0.85 mmol) in anhydrous CH3CN
(69 mL) was refluxed for 30 min. Triacetylated thiopyridyl sugar 13
(5.74 g, 13.25 mmol) and trimethylsilyl trifluoromethane-sulfonate
(3.10 mL, 17.22 mmol) were then added and the reaction mixture
was refluxed for 5 h, cooled, neutralized with aqueous sodium
bicarbonate, and extracted with CH2Cl2 (500 mL). The organic layer
was washed with water (3 ꢂ 10 mL) and dried over anhydrous so-
dium sulfate, evaporated to dryness. The crude underwent deacet-
ylation with methanolic ammonia (550 mL) within 4 h,
concentrated and finally purified by flash chromatography (AcOEt)
to give compound 7b (3.29 g, 62%, Rf = 0.27 in AcOEt) as white foam.
7g (3.32 g, 64%, Rf = 0.32 in AcOEt) as white foam. ½a D22
ꢀ
+ 4.00 (c 0.5,
MeOH); kmax 263 nm (e
9860); 1H NMR (CD3OD): d 8.58 and 8.35
(2s, 2H, H-2,8), 8.33–8.25 (m, H-6 of pyridine), 7.63–7.18 (m, H-
0
0
4, H-5, H-3 of pyridine), 6.96 (br s, NH2), 5.88 (d, 1H, J1 ,2 = 9.1 Hz,
H-10), 4.01 (dtr, 1H, JF,3 = 52.3 Hz, J2 ,3 = 8.7 Hz, J3 ,4 = 8.6 Hz, H-30),
3.79–3.67 (m, 1H, Y-20), 3.59–3.53 (m, 1H, H-6a0), 3.51–3.47 (m, 1H,
H-40), 3.42–3.37 (m, 1H, H-6b0), 2.96–2.81 (m, 1H, H-50); 19F NMR: d
ꢁ63.20; 13C NMR (CD3OD): d 160.54, 155.75, 152.31, 149.86,
147.75, 145.76, 136.71, 120.37, 120.23, 116.98, 91.76, 89.64,
72.19, 69.70, 66.64, 33.87; Anal. Calcd for C16H17FN6O3S2: C,
45.27; H, 4.04; N, 19.80. Found: C, 45.31; H, 4.12; N, 19.62. ESI-
MS (m/z): 425.51.
0
0
0
0
0
2.3.8. 1-[3-Deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-b-
osyl]5-fluorouracil (7c)
D-glucopyran-
A
mixture of 5-fluorouracil (2.41 g, 18.55 mmol), HMDS
(4.80 mL, 23 mmol) and saccharine (0.16 g, 0.85 mmol) in anhy-
drous CH3CN (69 mL) was refluxed for 30 min. Triacetylated thio-
pyridyl sugar 13 (5.74 g, 13.25 mmol) and trimethylsilyl
trifluoromethane-sulfonate (3.10 mL, 17.22 mmol) were then
2.4. Biological assays
The antiviral assays were based on the inhibition of virus-in-
duced cytopathicity in confluent cell cultures, and the cytostatic

290
E. Tsoukala et al. / Bioorganic Chemistry 38 (2010) 285–293
assays on inhibition of tumor cell proliferation in exponentially
growing tumor cell cultures according to previously described
methodology [15,32].
13b, 13c and 5-FU, respectively, 20.1, 20.1 and 2.4 min. UV-based
detection was performed at 267 nm.
2.4.1. Antiviral activity assays
3. Results and discussion
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 confluent 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
3.1. Synthesis
Two main strategies were used for the synthesis of 3-fluoro-6-
S-(2-S-pyridyl) nucleosides 7a-c,f,g.
Our first approach (Scheme 1) was focused on the preparation of
3-fluoro-6-thio-glucopyranosyl nucleosides of thymine 5a, uracil
5b, 5-fluorouracil 5c, N4-benzoyl cytosine 5d and N6-benzoyl ade-
nine 5e as useful precursors toward the synthesis of 6-S-nucleosidyl
S-pyridine disulfides. 1,2:5,6-di-O-isopropylidene-3-deoxy-3-flu-
oro-a-D-glucofuranose (1) [34]was readilytransformed into the cor-
responding 1,2,4,6-tetra-O-acetyl-3-deoxy-3-fluoro-glucopyranose
the presence of varying concentrations (200, 40, 8, . . .
lM) of the test
[35], which upon condensation with purine and pyrimidine bases
compounds. Viral cytopathicity was recorded as soon as it reached
completion in the control virus-infected cell cultures that were not
treated with the test compounds. 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 (ꢃ3 ꢂ 105 cells/cm3) cells were infected with
afforded 3-deoxy-3-fluoro-b-D-glucopyranosyl nucleosides 2a-e
[14]. Deprotection of nucleosides 2a-e in methanolic ammonia gave
the fully unprotected derivatives 3a-c,f,g, while the base protected
benzoylated derivatives 3d,e were obtained when 2d,e were treated
with NaOH-ethanol-pyridine [10,11,14]. Treatment of 3a-e with p-
toluenesulphonyl chloride in dry pyridine [35] and direct acetyla-
tion of the free hydroxyls at C-20 and C-40 with acetic anhydride/pyr-
idine afforded the desired acetylated 6-O-tosylated derivatives 4a-e
in very good yields. Subsequently, the tosylated derivatives 4a-e,
after treatment with potassium thioacetate in hot N,N-dimethyl-
formamide (DMF) [36–38], were converted into the corresponding
100 CCID50 of HIV(IIIB) or HIV-2(ROD)/mL and seeded in 200 lL
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.
thioacetates,
thio-b- -glucopyranosyl)thymine (5a), 1-(2,4-di-O-acetyl-6-S-acet-
yl-3-deoxy-3-fluoro-6-thio-b- -glucopyranosyl)uracil (5b), 1-(2,4-
di-O-acetyl-6-S-acetyl-3-deoxy-3-fluoro-6-thio-b- -glucopyrano-
syl)5-fluorouracil (5c), 1-(2,4-di-O-acetyl-6-S-acetyl-3-deoxy-3-
fluoro-6-thio-b-
-glucopyranosyl)-N4-benzoyl cytosine (5d) and
9-(2,4-di-O-acetyl-6-S-acetyl-3-deoxy-3-fluoro-6-thio-b- -gluco-
1-(2,4-di-O-acetyl-6-S-acetyl-3-deoxy-3-fluoro-6-
2.4.2. Cytostatic/toxic activity assays
D
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,
D
D
FM3A), 75,000 (CEM) or 20,000 (HeLa) cells per 200
l
L-well in
D
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%.
D
pyranosyl)-N6-benzoyl adenine (5e), respectively. Deprotection of
the b-protected thionucleosides 5a-e by methanolic ammonia
and in situ thiopyridinylation in H2O, MeOH and 2,2-dipyridyl
disulfide (DTDP) at room temperature [22], led to the desired deriv-
atives 7a-c. These reactions were completed within 20 h and the
isolated yields of the 6-S-nucleosidyl S-pyridine disulfides were
generally higher than 65%. Attempts to fully unprotect thionucleo-
sides 5a-c resulted to the formation of their corresponding sym-
metrical disulfides 6a-c, the thiopyridinylation of which also led
to the desired 6-S-nucleosidyl S-dipyridine disulfides 7a-c. It is
noteworthy that only decomposition products were obtained after
deprotection/thiopyridinylation of N4-benzoyl cytosine and N6-
benzoyl adenine derivatives, 5d and 5e, respectively.
2.4.3. Thymidine and uridine phosphorylase assays
The conversion of dThd to thymine, Urd to uracil and com-
pounds 13b and 13c to the free base by human recombinant thy-
midine phosphorylase and uridine phosphorylase type 1 (kindly
provided by Dr. T.P. Roosild, Las Vegas, Nevada, USA) was mea-
sured by high-pressure liquid chromatography (HPLC) analysis.
To determine the conversion activity, a high amount of recombi-
nant enzyme was incubated with 100 lM of dThd or 13b and
13c in TP-buffer (10 mM Tris.HCl, pH 7.6, 1 mM EDTA, 2 mM
KH2PO4/K2HPO4 and 150 mM NaCl) or UP-buffer (same as TP-buf-
fer, but 300 mM NaCl instead of 150 mM). At 20, 40 and 60 min,
In order to circumvent this difficulty we chose to investigate a
route in which the thionucleosides could be generated from a suit-
able thiopyridine sugar precursor. Our alternative synthetic path-
way, in which compound 12 was envisioned as an appropriate
glycosyl donour for the preparation of the suitable sugar, is de-
scribed in Scheme 2. Initially, selective removal of the 5,6-O-iso-
100 lL aliquots of the reaction mixtures were withdrawn and
heated at 95 °C for 3 min to inactivate the enzyme. dThd and Urd
were separated from thymine and uracil and 13b and 13c were
separated from 5-FU and uracil on a reverse-phase RP-8 column
(Merck, Darmstadt, Germany) and quantified by high-pressure li-
quid chromatography (HPLC) analysis (Alliance 2690, Waters, Mil-
ford, MA). The separation was performed by a linear gradient from
98% buffer B (50 mM NaH2PO4 and 5 mM heptane sulfonic acid, pH
3.2), to 20% buffer B + 80% acetonitrile (8 min 98% buffer B + 2%
acetonitrile; 5 min linear gradient of 98% buffer B + 2% acetonitrile
to 20% buffer B + 80% acetonitrile; 10 min 20% buffer B + 80% aceto-
nitrile, followed by equilibration at 98% buffer B + 2% acetonitrile).
Retention times for thymine and dThd were, respectively, 5.1 and
10.8 min, for uracil and Urd, respectively, 2.1 and 2.4 min and for
propylidene group of starting material
1 [34] led to the
formation of diol 8 [33]. Selective sulfonylation of 8 with p-tolu-
enesulphonyl chloride in pyridine led to compound 9, which was
treated with dihydropyran (DHP) and p-toluenesulphonic acid
monohydrate in dry CH2Cl2 in order to give compound 10 in very
satisfactory overall yield. Displacement of the tosyl group with
the thioacetyl moiety proceeded without difficulties to afford com-
pound 11. Precursor 12 was readily available from glucofuranose
11 through hydrolysis with 90% aqueous trifluoroacetic acid at
room temperature and direct acetylation. Deprotection of the gly-
cosyl donor 12 using methanolic ammonia, direct thiopyridinyla-

E. Tsoukala et al. / Bioorganic Chemistry 38 (2010) 285–293
291
H3C
H3C
O
O
AcO
HO
B
B
O
O
O
ii
i
F
F
F
O
CH3
CH3
O c
A
OH
O
OAc
OH
3 a - g
1
2 a - e
iii
S
AcS
TsO
B
B
B
O
O
O
vi
iv
F
F
F
OH
OAc
OAc
OH
OAc
OAc
2
6 a - c
vii
5 a - e
4 a - e
v
S
N
S
B
O
a: Thymine
b: Uracil
c: 5-Fluoro-Uracil
d: Bz-Cytosine
e: Bz-Adenine
f: Cytosine
g: Adenine
F
OH
OH
7 a - c
Scheme 1. (i) (a) MeOH/H2O/amberlite IR 120(H+); (b) Ac2O/pyridine; (c) silylated base, CH3CN, trimethylsilyl trifluomethane-sulfonate or tin chloride; (ii) pyridine/MeOH/
amberlite IR 120(H+)/NaOH or methanolic ammonia; (iii) (a) pyridine, p-toluenesulfonyl chloride; (b) Ac2O/Pyridine; (iv) KSAc/DMF/100 °C; (v) methanolic ammonia/H2O/
MeOH/2,2-dipyridine-disulfide; (vi) methanolic ammonia; (vii) H2O/MeOH/2,2-dipyridine-disulfide.
tion [22] (8 mol excess of DTDP/MeOH/H2O) followed by acetyla-
tion led to the desired thiopyridine sugar 13. In order to prepare
not only the target 6-S-nucleosidyl S-dipyridine disulfides 7f,g,
but the previously synthesized 6-S-(2-S-pyridyl) nucleosides 7a-c
as well, glycosylation reactions with the corresponding purine
and pyrimidine bases and the 6-thiopyridine sugar 13 followed
by direct deacetylation were employed.
acetoxyl group and the presence of 2-S-pyridyl moiety, whose pro-
ton peaks appeared between 8.4 and 7.1 ppm.
The stability of the target compounds was assigned after
3 months storage, either in organic solution or in solid phase, by
performing NMR spectroscopy. Comparison of the primarily NMR
spectra of the target molecules with those of the same storable
compounds could clearly reveal identical proton NMR spectra.
All new compounds were well-characterized by NMR and UV
spectroscopy, mass spectrometry and elemental analysis. The 1H
NMR data obtained for the newly synthesized 5a-e revealed that
3.2. Antiviral and cytostatic activity
0
0
these compounds had the b configuration (J1 ,2 ꢄ 8.0 Yz). It must
also be mentioned that the desired compounds 5a-e, showed
strong infra-red absorptions at 1750 cmꢁ1 (OAc) and 1697 cmꢁ1
(SAc), while in their 1H NMR spectra prominent 3-proton methyl
signals appeared, which were ascribed to the acetylthio moiety
and 6-proton methyl peaks, which correspond to the acetoxyl
group. Moreover, data in the 1H NMR spectra, obtained for the
newly synthesized 6-S-(2-S-pyridyl) nucleosides 7a-c,f,g, revealed
the absence of 6-proton methyl peaks, which correspond to the
Compounds 6a-c and 7b,c,f,g were evaluated for their antiviral
activity against a wide variety of DNA and RNA viruses, including
herpes simplex virus type 1 (HSV-1) (strain KOS), HSV-2 (strain
G), vaccinia virus, vesicular stomatitis virus (VSV), varicella-zoster
virus (VZV) strains OKA and 07/1, and human cytomegalovirus
(HCMV) strains AD-169 and Davis in HEL cell cultures, VSV, Cox-
sackie B4 and respiratory syncytial virus (RSV) in HeLa cell cul-
tures, parainfluenza-3 virus, reovirus, Sindbis virus, Coxsackie
virus B4 and Punta Toro virus in Vero cell cultures, influenza A

292
E. Tsoukala et al. / Bioorganic Chemistry 38 (2010) 285–293
H3C
H3C
O
O
R1
R2
AcS
O
O
O
i
v
F
OAc
F
F
O
CH3
CH3
O
CH3
CH3
A
O c
O
O
OAc
1
12
8 R1=OH, R2=OH
10 R11=OTs, R22=OTHP
11 R1=SAc, R2=OTHP
ii
iii
iv
9 R =OTs, R =
OH
vi
S
S
N
N
S
S
B
O
O
F
F
vii
OAc
a: Thymine
b: Uracil
c: 5-Fluoro-Uracil
d: Bz-Cytosine
e: Bz-Adenine
f: Cytosine
g: Adenine
OH
OAc
OH
OAc
7 a - c,f,g
13
Scheme 2. (i) 70% AcOH; (ii) pyridine, p-toluenesulfonyl chloride; (iii) CH2Cl2, p-TsOH, DHP; (iv) KSAc/DMF/100 °C; (v) (a) 90% TFA (b) Ac2O/pyridine; (vi) (a) methanolic
ammonia/H2O/MeOH/2,2-dipyridine-disulfide (b) Ac2O/pyridine; (vii) (a) silylated base, CH3CN, trimethylsilyl trifluomethane-sulfonate or tin chloride, (b) methanolic
ammonia.
Table 1
(H1N1, H3N2) and B virus in MDCK cell cultures and feline corona
virus (FIPV) and feline herpesvirus in CRFK cell cultures. None of
the compounds tested showed an appreciable inhibitory activity
Inhibitory effects of the test compounds on the proliferation of murine leukemia cells
(L1210), murine mammary carcinoma cells (FM3A), human T-lymphocyte cells
(Molt4/C8, CEM) and human cervix carcinoma (HeLa) cells.
at subtoxic concentrations (i.e. 4–20
lM for 7b, 20–100 lM for
a
Compounds
IC50
(lM)
7c and >100 M for the other compounds). The new molecules
l
were also examined for their cytostatic activity against murine leu-
kemia L1210, murine mammary carcinoma FM3A, human lympho-
cyte Molt4/C8 and CEM and human cervix carcinoma HeLa cells.
Compounds 3a-g (Table 1) and the acetyl-substituted 5a-e analogs
(data not shown) did not show appreciable antiproliferative activ-
ity. Likewise, the 6,6-disulfide glucopyranosyl pyrimidine dinucle-
oside derivatives 6a-c did not show appreciable cytostatic action
L1210
FM3A
Molt4/C8
CEM
HeLa
3a
3b
3c
3d
3e
3f
3g
6a
6b
6c
7a
7b
7c
7f
>200
>200
>200
>200
P200
159 58
166 47
>500
302 36
231 34
120 56
9.5 0.0
54 19
>500
>200
>200
>200
>200
>200
>200
>200
>500
>200
>200
>200
>200
>200
>200
>200
>200
120 15
155 64
124 47
>500
>500
>500
106 21
9.2 1.1
43
>500
228 36
81
7
163 21
183 24
>500
320 40
280
against the evaluated tumor cell lines (IC50: 71 to >500 lM) (Table
123 39
71 18
>200
1). However, their corresponding 2-S-pyridyl 6-thioglucopyranosyl
mononucleosides 7 were endowed with a more pronounced cyto-
static activity in cell culture. In particular, the 5-fluorouracil (7c)
and especially the uracil (7b) derivative showed considerable cyto-
static potency against the murine L1210, and human Molt4/C8,
CEM and HeLa cells. Also, 7c and 7b were cytostatic against prolif-
0
84
4
17
35
4
2
8.8 0.7
3
35
3
>500
P500
369
192
7
9
7g
238 41
a
erating human embryonic lung HEL fibroblasts (IC50: 36
9.2 M, respectively), Crandell feline kidney cells (CRFK) (IC50
6.2 and 39 M) and canine Madin-Darby kidney cells (MDCK)
(IC50: 50 M and 11 M, respectively). Conversely, the adenine
(7g) and cytosine (7f) derivatives showed poor, if any cytostatic
activity (192 ꢁ >500 M) (Table 1). It is somewhat intriguing that
lM and
50% Inhibitory concentration or compound concentration required to inhibit
tumor cell proliferation in cell culture by 50%.
l
:
l
l
l
the fluorouracil derivatives is currently unknown. When com-
pounds 7b and 7c were exposed to purified recombinant thymi-
dine phosphorylase (TPase) or uridine phosphorylase type I
(UPase), they were not hydrolyzed to the free pyrimidine base un-
der conditions where thymidine (exposed to TPase) and uridine
(exposed to UPase) were readily converted to thymine and uracil,
respectively (data not shown). These findings indicate that these
molecules exert their potency via their intact nucleoside moiety
(or anabolite derived thereof), rather than a catabolic metabolite
l
among the 2-S-pyridyl 6-thioglucopyranosyl mononucleotides,
only the derivatives containing an uracil/5-fluorouracil base were
markedly more cytostatic than those, which bear a thymine, cyto-
sine or adenine base. The fluorouracil derivatives proved equally
cytostatic against murine and human cells, as well as against leu-
kemia, mammary carcinoma, cervix carcinoma and lymphoma tu-
mor cells. The molecular basis of the antiproliferative activity of

E. Tsoukala et al. / Bioorganic Chemistry 38 (2010) 285–293
293
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cytostatic potency and can be viewed as novel lead compounds
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This work was supported in part by the Postgraduate Pro-
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Environment”, ‘‘Application of Molecular Biology–Molecular
Genetics–Molecular Markers”, Department of Biochemistry and
Biotechnology, University of Thessaly. The authors are also grateful
to K.U. Leuven (GOA 10/014) for financial support.
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