Stereospecific synthesis and biological evaluations of β-L-pentofuranonucleoside derivatives of 5-fluorouracil and 5-fluorocytosine

Article abstract of DOI:10.1016/S0223-5234(01)01238-7

In the search for new chemotherapeutic agents, we have focused our work on the synthesis and the study of several unnatural β-L-nucleoside analogues. In this paper, we report on the synthesis of β-L-pentofuranonucleosides (and their 2′-deoxy derivatives) of 5-fluorouracil and their inhibitory effects on the proliferation of several murine and human tumor cells. The corresponding 5-fluorocytosine derivatives were also synthesized and their anti-HIV and anti-HBV activities have been evaluated.

Full text of DOI:10.1016/S0223-5234(01)01238-7

Eur. J. Med. Chem. 36 (2001) 447–460
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01238-7/FLA
Original article
Stereospecific synthesis and biological evaluations of b-
L
-pentofuranonucleoside
derivatives of 5-fluorouracil and 5-fluorocytosine
Jean-Franc¸ois Griffona, Christophe Mathe´a,*, Abdesslem Farajb, Anne-Marie Aubertinc,
Erik De Clercqd, Jan Balzarinid, Jean-Pierre Sommadossie, Gilles Gosselina,b
aLaboratoire de Chimie Organique Biomole´culaire de Synthe`se, UMR 5625 CNRS-UM II, Universite´ Montpellier II,
case courrier 008, Place Euge`ne Bataillon, F-34095 Montpellier, France
bLaboratoire Coope´ratif Novirio, CNRS-UM II, Universite´ Montpellier II, France
cUniversite´ Louis Pasteur, INSERM U 74, F-67000 Strasbourg, France
dRega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
eNovirio-Pharmaceuticals, Inc., Cambridge, MA, USA
Received 22 January 2001; revised 29 March 2001; accepted 4 April 2001
Abstract – In the search for new chemotherapeutic agents, we have focused our work on the synthesis and the study of several
unnatural b-
L
-nucleoside analogues. In this paper, we report on the synthesis of b-L-pentofuranonucleosides (and their 2%-deoxy
derivatives) of 5-fluorouracil and their inhibitory effects on the proliferation of several murine and human tumor cells. The
corresponding 5-fluorocytosine derivatives were also synthesized and their anti-HIV and anti-HBV activities have been evaluated.
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS
b-L-nucleoside analogues / 5-fluorouracil / anti-tumoral activity / 5-fluorocytosine / anti-HIV activity / anti-HBV activity
1. Introduction
(dTTP). The 2%-deoxy-b-
D
-ribofuranonucleoside of 5-
fluorouracil (b- -5FdU) is a very potent cytotoxic
D
5-Fluorouracil (1) (5FU) is widely used in the treat-
ment of various solid tumors. This 5-fluorinated ana-
logue of the natural pyrimidine base uracil exerts its
anti-tumoral activity through various mechanisms [1,
2]. In fact, 5FU can be converted intracellularly
agent in cell culture systems, but it suffers in vivo
from substantial degradation by thymidine phospho-
rylase to the pyrimidine base 5FU and deoxyribose-1-
phosphate before reaching the target tissues [4]. In the
search for new anti-neoplastic agents, many sugar-
modified nucleoside analogues of 5FU have been syn-
thesized earlier and their interactions with TS or
thymidine phosphorylase studied [5–8]. Only very few
into
5-fluoro-2%-deoxyuridine
5%-monophosphate
(FdUMP), which is a mechanism-based inhibitor of
the thymidylate synthase (TS) [3]. TS is an essential
enzyme in pyrimidine de novo biosynthesis; it cataly-
ses the methylation of 2%-deoxyuridine 5%-monophos-
phate (dUMP) to 2%-deoxythymidine 5%-monophos-
phate (dTMP) with concomitant conversion of
N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4-
folate) to 7,8-dihydrofolate (H2-folate). The direct
effect of TS inhibition is a depletion of dTMP, and
subsequently, of 2%-deoxythymidine 5%-triphosphate
studies have dealt with the unnatural b- nucleoside
analogues of 5FU. Holy´ et al. [9, 10] have reported
the anti-bacterial effects and the enzymatic degrada-
L
tion of the b-
nosyl derivatives. Recently, Weis et al. [11] have
evaluated the inhibitory effect of the b- -2%-de-
L
-2%-deoxyribo-, ribo- and arabino-fura-
L
oxyribo- and ribofuranosyl derivatives on the prolif-
eration of several human tumor cell lines. These
compounds were found to be either weakly active or
not active at all.
* Correspondence and reprints
E-mail address: cmathe@univ-montp2.fr (C. Mathe´).

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J.-F. Griffon et al. / European Journal of Medicinal Chemistry 36 (2001) 447–460
(HIV) and anti-hepatitis B virus (HBV) drug and its
5-fluoro derivative [(−)-FTC] was also found to ex-
hibit not only anti-HIV but also anti-HBV activities
[13–15]. Additionally, b-
L
-2%,3%-dideoxycytidine (b-
L
-
ddC) and its 5-fluoro derivative (b-
L
-FddC), widely
studied by our group [16–20] and others [21–24]
show potent anti-HIV and anti-HBV activities in cell
culture systems.
As part of our ongoing research on b-
analogues, the stereospecific syntheses of the unnatu-
ral b- -pento- and 2%-deoxy-b- -pentofuranonu-
cleosides of 5FU and 5-fluorocytosine (figure 2) are
-Nucleoside derivatives of
5FU were evaluated for their inhibitory effect on the
proliferation of several murine and human tumor
L
-nucleoside
Figure 1. Cytosine or 5-fluorocytosine nucleoside analogues
endowed with the unnatural
viral activity.
L
L
L configuration with potent anti-
presented in this work. b-
L
Nucleoside analogues endowed with the unnatural
L
configuration have drawn considerable attention as
cells, while b- -nucleoside derivatives of 5-fluorocy-
L
potential anti-viral drugs. The possibility that
cleoside derivatives may be more efficient than the
corresponding -counterparts, owing to their power-
L
-nu-
tosine were evaluated for their anti-HIV and anti-
HBV activities.
D
ful anti-viral activity and favorable toxicity profile,
has been demonstrated for some of them [12]. Particu-
larly, those bearing a cytosine or 5-fluorocytosine
base moieties were found to possess very potent anti-
2. Chemistry
2.1. i-
15)
L
-Pentofuranonucleosides of 5FU (4, 7, 11 and
viral activity (figure 1). For instance, b- (-)-2%,3%-
L
dideoxy-3%-thiacytidine (3TC, Lamivudine,) was
approved recently by the Food and Drug Administra-
tion (FDA) as an anti-human immunodeficiency virus
From a synthetic viewpoint, the dissymetrically pera-
cylated 1-O-acetyl-2,3,5-tri-O-benzoyl-b- -ribofuranose
L
Figure 2. b-
L
-Pento- and 2%-deoxy-b-L-pentofuranonucleosides of 5FU and 5-fluorocytosine.

J.-F. Griffon et al. / European Journal of Medicinal Chemistry 36 (2001) 447–460
449
(2) [25] or 1,2-di-O-acetyl-3,5-di-O-benzoyl-b-
L
-xylo-
furanose (5) [26] were, respectively, condensed with the
commercially available 5-fluorouracil 1, under Vo¨rbrug-
gen [27] conditions, using (trimethylsilyl)trifluoro-
methane sulfonate (TMSTf) as a catalyst, to exclusively
give (owing to 2-O-acyl participation [28]) the corre-
sponding fully protected b-
3). These compounds were deacylated with saturated
methanolic ammonia to afford the desired 1-(b- -ribo-
furanosyl)-5-fluorouracil (4) and 1-(b- -xylo-furanosyl)-
L
-nucleosides 3 and 6 (figure
L
L
5-fluorouracil (7) after silica gel column chromatography.
Regioselective 2%-O-deacylation (figure 4) of the fully
protected xylo-nucleoside 6 with hydrazine hydrate gave
8, which was then subjected to an oxidation–reduction
process to obtain the arabino-nucleoside derivative as re-
ported earlier by Gosselin et al. [29]. Oxidation of com-
pound 8 was accomplished with dimethylsulfoxide
(DMSO), dicyclohexylcarbodiimide (DCC) and dichloro-
acetic acid. The resulting 2%-keto nucleoside 9a undergoes
an epimerization at C-3%, via a keto–enol equilibrium as
shown in square brackets (figure 4), to give the 2%-keto in-
termediate 9c. Reduction of 9c with sodium borohydride
(NaBH4) proceeded stereoselectively leading to the for-
mation of 10, which was then debenzoylated with sodium
methanolate in a methanol–toluene mixture to form 1-
Figure 3. Synthesis of compounds 4 and 7. Reagents and
conditions: (a) HMDS, (NH4)SO4, reflux; (b) TMSTf,
(CH2Cl)2, r.t.; (c) MeOHꢀNH3, r.t.
(b-
tion on a silica gel column.
Additionally, 1-(b- -lyxo-furanosyl)-5-fluorouracil
(15) was obtained in several steps from the b- -xylo-fura-
nonuc1eoside (7) (figure 5), according to a strategy de-
scribed earlier by Fox et al. [30] in the b- -pyrimidine
L
-arabino-furanosyl)-5-fluorouracil (11) after purifica-
L
L
D
series. Thus, the reaction of 7 with 2,2-dimethoxypropane
and p-toluene sulfonic acid (p-TsOH) gave 3%,5%-O-iso-
propylidene nucleoside (12), which was treated with
methanesulfonyl chloride (MsCl) followed by hydrolysis
with 80% aqueous acetic acid to form the 2%-O-mesyl
derivative (13). Treatment of 13 with water under reflux
allowed the inversion of configuration at C-2% via 2,2%-O-
anhydro intermediate (14) and led to the formation of 1-
(b-
L
-lyxo-furanosyl)-5-fluorouracil (15) after silica gel
column chromatography.
2.2. 2%-Deoxy-i-
L
-pentofuranonucleosides of 5FU (17
and 20)
1-(2-Deoxy-b-L-threo-pentofuranosyl)-5-fluorouracil
(17) was obtained from 8 via a Barton–McCombie
reductive process [31]. Thus, the treatment of 8 (figure 6)
with O-phenyl chloro(thio)formate [PhOC(ꢁS)Cl] and
4-(dimethyamino)pyridine (DMAP) formed the corre-
Figure 4. Synthesis of compound 11. Reagents and conditions:
(a) H2NNH2·H2O, AcOH, pyridine, r.t.; (b) DCC, DMSO,
Cl2CHCO2H, benzene, pyridine, r.t.; (c) NaBH4, EtOH, ben-
zene, r.t.; (d) MeONa, MeOH, benzene, r.t.

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J.-F. Griffon et al. / European Journal of Medicinal Chemistry 36 (2001) 447–460
sponding 2%-O-[phenoxy(thiocarbonyl)] intermediate,
which was subsequently deoxygenated with tris-
(trimethylsilyl)silane [32] in the presence of a,a%-
azoisobutyronitrile (AIBN). Finally, debenzoylation
with saturated methanolic ammonia formed 17 as a
crystalline solid after silica gel column chromatography.
To prepare compound 20, 2%-deoxy-b-L-threo-pentofura-
nonucleoside (17) was selectively converted into the
5%-O-benzoyl derivative (18). Inversion of the hydroxyl
group at C-3% was achieved via the Mitsunobu reaction
Figure 7. Synthesis of compounds 21 and 22. Reagents and
conditions: (a) Ac2O, pyridine, r.t.
[33], using as incoming nucleophile benzoic acid. Thus,
the reaction of 18 with diethyl azodicarboxylate
(DEAD), triphenyl phosphine (PPh3) and benzoic acid
in anhydrous tetrahydrofuran (THF) at 0 °C gave nu-
cleoside 19 which upon deprotection with methanolic
ammonia formed 1-(2-deoxy-b-L-erythro-pentofura-
nosyl)-5-fluorouracil 20 as a crystalline solid after purifi-
cation on silica gel column.
Figure 5. Synthesis of compound 15. Reagents and conditions:
(a) 2,2-dimethoxy-propane, p-TsOH, DMF, r.t.; (b) (1) MsCl,
pyridine, 0 °C, (2) AcOH 80%, reflux; (c) H2O, reflux.
2.3. i-L-Pento- and 2%-deoxy-i-L-pentofurano-
nucleosides of 5-fluorocytosine (23–28)
The synthesis of the 5-fluorocytosine nucleosides was
carried out from the corresponding 5FU peracylated
nucleosides. For the arabino- and xylo-nucleoside
derivatives, 10 and 16, an additional step of acetylation
with acetic anhydride was necessary to obtain the fully
protected nucleosides 21 and 22 (figure 7).
Conversion of peracylated 5FU derivatives 3, 6, 16,
19, 21, and 22 to the corresponding 5-fluorocytosine
nucleosides 23–28 was carried out via a treatment with
Lawesson’s reagent [34, 35], followed by the treatment
of 4-thioamide intermediates with methanolic ammonia
at 100 °C in a stainless-steel bomb (figure 8).
3. Biological results
The b- -nucleosides analogues of 5FU, 4, 7, 11, 15,
L
Figure 6. Synthesis of compounds 17 and 20. Reagents and
conditions: (a) (1) DMAP, PhO(CꢁS)Cl, CH3CN, r.t, (2)
(Me3Si)3SiH, AIBN, dioxane, 100 °C; (b) MeOHꢀNH3, r.t; (c)
BzCl, pyridine, DMF, 0 °C; (d) DEAD, benzoic acid, PPh3,
THF, 0 °C.
17 and 20 (table I) were tested for their in vitro
inhibitory effect on the proliferation of murine
leukemia cells (L1210), murine mammary carcinoma
cells (FM3A) and human T-lymphocyte cells (Molt4/

J.-F. Griffon et al. / European Journal of Medicinal Chemistry 36 (2001) 447–460
451
only b-
(EC50 =4 mM for HBV RI, EC50 =5 mM for HBV
virion). In the same assays, the others b- -nucleoside
analogues were inactive (up to a concentration of 10
-nucleoside derivatives of 5-fluorocy-
tosine did not show any cytotoxicity in Hep-G2 cells
(up to a concentration of 200 mM).
L
-5FdC (26) showed anti-viral activity
C8 and CEM). 5FU 1 and b-
reference compounds. From these data, it appears
-xylo-5FU (7) shows some activity, al-
beit restricted to the murine L1210 and FM3A cells.
The weak activity of compound 7 and the lack of an
D
-5FdU were used as
L
that only b-
L
mM). All b-
L
inhibitory effect for the other b- -nucleoside deriva-
L
tives of 5FU highlights the fact that these compounds
are probably not able: (i) to release 5FU, via a chem-
ical or enzymatic cleavage of the glycosidic bound,
from the parent nucleoside; (ii) to serve as substrates
for intracellular enzymes involved in the anabolic
pathway.
4. Conclusion
The b- -pento- and 2%-deoxypentofuranonu-
L
cleosides of 5FU, 4, 7, 11, 15, 17 and 20, were
stereospecifically and conveniently prepared by fol-
lowing multi-step reaction sequences. When evaluated
for their inhibitory effect on the proliferation of sev-
eral murine and human tumor cells, it appeared that
b- -Nucleoside analogues of 5-fluorocytosine 23–
L
28 were evaluated for their in vitro inhibitory effect
on the replication of HIV-1 in CEM-SS and MT-4
cell systems, but none of them showed any anti-viral
activity or cytotoxicity (up to 100 mM, data not
shown). When evaluated in anti-HBV assays in the
HBV DNA-transfected Hep-G2 cells (2.2.15 cells),
only b-
ity. The b-
thesized from the corresponding 5-fluorouracil
L
-xylo-5FU (7) showed some inhibitory activ-
L
-5-fluorocytosine derivatives (23–28), syn-
Figure 8. General synthetic procedure for compounds 23–28. Reagents and conditions: (a) (1) Lawesson’s reagent, (CH2Cl)2, reflux;
(2) MeOHꢀNH3, 100 °C.
Table I. Inhibitory effect of b-
L-nucleoside analogues of 5-fluorouracil on the proliferation of murine leukemia cells (L1210), murine
mammary carcinoma cells (FM3A) and human T-lymphocyte cells (Molt4/C8 and CEM).
Compound
IC50 a(mM)
L1210
FM3A
Molt4/C8
CEM
b-
D
-5FdU
0.00190.0001
0.28290.143
294995
4395.6
0.00390.0004
0.18390.090
\500
6798.4
\500
\500
1192.7
2393.0
\500
\500
\500
\500
\500
\500
0.01490.001
8990.43
\500
1 (5-fluorouracil)
4 (b-
7 (b-
11 (b-
15 (b-
17 (b-
20 (b-
L
-5FU)
-xylo-5FU)
L
3959148
\250
L
L
L
L
-ara-5FL)
-lyxo-5FU)
-threo-5FdU)
-5FdU)
\500
\500
3199133
135953
\500
465961
129961
\500
\250
a IC50 value represents the drug concentration (mM) required to inhibit the proliferation of malignant cells by 50%.

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J.-F. Griffon et al. / European Journal of Medicinal Chemistry 36 (2001) 447–460
nucleosides, were evaluated against HIV-1 in cell cul-
tures, but none of them showed any anti-viral activ-
ity. When evaluated in anti-HBV assays, only
with methanol. The crude material was dissolved in
water and the resulting solution was washed four times
with methylene chloride. The aqueous layer was evapo-
rated under reduced pressure and the residue was
purified using silica gel column chromatography (eluent:
stepwise gradient of methanol (0–10%) in chloroform or
gradient of methanol (0–5%) in ethyl acetate). Finally,
the appropriate fractions were evaporated under re-
duced pressure, diluted with methanol and filtered
through a unit Millex HV-4 (0.45 mm, Millipore).
b- -5FdC (26) showed moderate anti-viral activity
L
without concomitant cytotoxicity.
5. Experimental
5.1. Chemistry
Melting points (m.p. (dec.)) were determined in open
capillary tubes on a Gallenkamp MFB-595-010 M ap-
paratus and are uncorrected. The UV absorption spec-
tra were recorded on an Uvikon 931 (KONTRON)
spectrophotometer in ethanol. 1H NMR spectra were
run at room temperature (r.t.) in DMSO-d6 with a
Bruker AC 250 or 400 spectrometer. Chemical shifts are
given in ppm, DMSO-d5 being set at 2.49 ppm as
reference. Deuterium exchange, decoupling experiments
or 2D-COSY spectra were carried out to confirm the
proton assignments. Signal multiplicities are represented
by s (singlet), d (doublet), dd (doublet of doublets), t
(triplet), q (quadruplet), br (broad), m (multiplet). All
J-values are in Hz. FAB mass spectra were recorded in
the positive- (FAB>0) or negative- (FAB<0) ion mode
on a JEOL DX 300 mass spectrometer; the matrix was
3-nitrobenzyl alcohol (NBA) or a mixture (50:50, v/v) of
glycerol and thioglycerol (GT). Specific rotations were
measured on a Perkin–Elmer 241 spectropolarimeter
5.1.2. General procedure for the preparation of
compounds 23–28
Lawesson’s reagent (0.7 equiv.) was added under ar-
gon to a solution of the peracylated 5-fluorouracil nu-
cleoside in anhydrous 1,2-dichloroethane (ca. 25
mL mmol−1) and the reaction mixture was stirred
overnight under reflux. The solvent was then evaporated
under reduced pressure and the residue was purified
using silica gel column chromatography (eluent: step-
wise gradient of methanol (0–2%) in chloroform) to give
the corresponding 4-thio intermediate. A solution of the
4-thio intermediate in methanolic ammonia (saturated
beforehand at −10 °C and stoppered tightly), (ca. 25
mL mmol−1) was heated at 100 °C in a stainless-steel
bomb for 3 h, and then cooled to r.t. The solution was
evaporated to dryness under reduced pressure and the
residue was co-evaporated several times in methanol.
The crude material was dissolved in water and the
resulting solution was washed with methylene chloride.
The aqueous layer was evaporated under reduced pres-
sure and the residue was purified using silica gel column
chromatography (eluent: stepwise gradient of methanol
(0–15%) in chloroform or ethyl acetate). The appropri-
ate fractions were pooled and evaporated under reduced
pressure, diluted with methanol and filtered through a
unit Millex HV-4 (0.45 mm, Millipore).
(path length,
1 cm) and are given in units of
10−1 deg cm2 g−1. Elemental analyses were carried out
by the ‘Service de Microanalyses du CNRS, Division de
Vernaison’ (France). Analyses indicated by the symbols
of the elements or functions were within 0.4% of the
theoretical values. Thin layer chromatography was per-
formed on precoated aluminum sheets of Silica Gel 60
F254 (Merck, Art. 5554), visualization of products being
accomplished by UV absorbency followed by charring
with 10% ethanolic sulphuric acid and heating. Column
chromatography was carried out on Silica Gel 60
(Merck, Art. 9385) at atmospheric pressure.
5.1.3. 1-(2,3,5-Tri-O-benzoyl-i-L-ribofuranosyl)-5-
fluorouracil (3)
A suspension of 5-fluorouracil (1) (387 mg, 297 mmol)
was treated with hexamethyldisilazane (HMDS, 20 mL)
and a catalytic amount of ammonium sulfate during 18
h under reflux. After cooling to r.t., the mixture was
evaporated under reduced pressure, and the residue
obtained as a colorless oil was diluted with anhydrous
1,2-dichloroethane (20 mL). 1-O-Acetyl-2,3,5-tri-O-ben-
5.1.1. General procedure for the preparation of
compounds 4, 7, 11, 17 and 20
A solution of the protected nucleoside in methanolic
ammonia (saturated earlier at −10 °C and stoppered
tightly), (ca. 25 mL mmol−1) was stirred overnight at r.t.
The solution was evaporated to dryness under reduced
pressure and the residue co-evaporated several times
zoyl-b- -ribofuranose (2) [25] (1.0 g, 1.98 mmol) in
L
anhydrous 1,2-dichloroethane (11 mL) was added to the
resulting solution followed by the addition of TMSTf

J.-F. Griffon et al. / European Journal of Medicinal Chemistry 36 (2001) 447–460
453
(0.72 mL, 3.96 mmol). The solution was stirred for 6 h
at r.t. under argon atmosphere, then diluted with chlo-
roform (150 mL), washed with the same volume of a
saturated aqueous sodium hydrogen carbonate solution
and finally with water (2×100 mL). The organic phase
was dried over sodium sulfate, and then evaporated
under reduced pressure. The resulting crude material
was purified using silica gel column chromatography
(eluent: stepwise gradient of methanol (0–4%) in meth-
ylene chloride) to give pure 3 (1.08 g, yield: 95%), which
was crystallized from chloroform; m.p.=200–202 °C
compound 3, and outlined for compound 6 (11.0 g,
Yield: 84%) as a white foam; m.p.=96–98 °C; UV:
umax (nm) (m, M−1 cm−1) (ethanol): 228 (25 900), 266
(9000), umin (nm): 250 (7200); H NMR (DMSO-d6): l
11.1 (br s, 1H, NH), 8.05 (1H, H-6, J6-F5=6.8 Hz),
1
7.9–7.4 (m, 10H, 2C6H5CO), 5.99 (d, 1H, H-1%, J1%-2%
=
3.1 Hz), 5.74 (dd, 1H, H-3%, J3%-2%=4.2 Hz, J3%-4%=2.3
Hz), 5.54 (t, 1H, H-2%, J=2.9 Hz), 4.8–4.6 (m, 3H,
H-4%, H-5%, H-5¦); FABMS (FAB>0); m/z (GT): 513
[M+H]+, 383 [S]+, 105 [C6H5CO]+; FABMS (FAB<0);
m/z (GT): 511 [M−H]−, 469 [M−CH3CO]−, 129 [B]−,
121 [C6H5CO2]−; [a]D20= −91 (c 0.88, DMSO); Anal.
C25H21FN2O9 (C, H, N).
(lit. value=204 °C [10]); UV: umax (nm) (m, M−1 cm−1
)
(ethanol): 230 (37 400), 266 (11 500), umin (nm): 253
1
(10 200); H NMR (DMS0-d6): l 13.0 (br s, 1H, NH),
8.19 (d, 1H, H-6, J6-F5=6.8 Hz), 8.0–7.4 (m, 15H, 3
C6H5CO), 6.16 (d, 1H, H-1%, J1%-2%=3,3 Hz), 5.9–5.8 (m,
2H, H-2%, H-3%), 4.8–4.6 (m, 3H, H-4%, H-5%, H-5¦);
FABMS (FAB>0); m/z (GT): 1149 [2M+H]+, 575 [M+
H]+, 445 [S]+, 105 [C6H5CO]+; FABMS (FAB<0); m/z
(GT): 1721 [3M−H]−, 1147 [2M−H]−, 665 [M+G−H]−,
573 [M−H]−, 129 [B]−, 121 [C6H5CO2]−; [a]2D0= +45 (c
0.94, DMSO) (lit. value= +76.8 (c 0.50, DMF) [10]);
Anal. C30H23FN2O9·0.1CH3Cl (C, H, N).
5.1.6. 1-(i-L-xylo-furanosyl)-5-fluorouracil (7)
Compound 6 (500 mg, 0.98 mmol) was deacylated by
treating with methanolic ammonia following the general
procedure to form 7 (240 mg, Yield: 94% ), which was
crystallized from ethanol; m.p.=161–162 °C (lit.
value=203 °C for 1-(b-D-xylo-furanosyl)-5-fluorouracil
[10]); UV: umax (nm) (m, M−1 cm−1) (ethanol): 268
1
(10 200), umin (nm): 234 (2500); H NMR (DMSO-d6): l
11.9 (br, s, 1H, NH), 7.99 (d, 1H, H-6, J6-F5=7.4 Hz),
5.78 (d, 1H, OH-2%, J=4.2 Hz), 5.6 (br s, 1H, H-1%),
5.50 (d, 1H, OH-3%, J=3.5 Hz), 4.78 (t, 1H, OH-5%,
J=5.6 Hz), 4.1–4.0 (m, 1H, H-4%), 3.98 (d, 1H, H-2%),
3.9 (m, 1H, H-3%), 3.8–3.6 (m, 2H, H-5%, H-5¦); FABMS
(FAB>0); m/z (GT): 263 [M+H]+, 133 [S]+, 131 [BH2]+;
FABMS (FAB<0); m/z (GT): 261 [M−H]−, 129 [B]−;
[a]2D0= +21 (c 0.87, DMSO); Anal C9H11FN2O6·
0.25EtOH (C, H, N).
5.1.4. 1-(i- -Ribofuranosyl)-5-fluorouracil (4)
L
Compound 3 (350 mg, 0.61 mmol) was deacylated by
treating with methanolic ammonia following the general
procedure to form 4 (130 mg, Yield: 82%), which was
crystallized from
a diethyl ether–ethanol mixture;
m.p.=180–182 °C (lit. values=184 °C [10], 208–
209 °C [11]); UV: umax (nm) (m, M−1 cm−1) (ethanol):
1
269 (8900), umin (nm): 235 (1900); H NMR (DMSO-d6):
l 11.9 (br s, 1H, NH), 8.27 (d, 1H, H-6, J6-F5=7.3 Hz),
5.72 (dd, 1H, H-1%, J1%-2%=4.5 Hz, J1%-F5=1.7 Hz), 5.4
(br s, 1H, OH-2%), 5.3 (br s, 1H, OH-5%), 5.1 (br s, 1H,
OH-3%), 4.0–3.9 (m, 2H, H-2%, H-3¦), 3.8 (m, 1H, H-4%),
3.7–3.4 (m, 2H, H-5%, H-5¦); FABMS (FAB>0); m/z
(GT): 525 [2M+H]+, 355 [M+G+H]+, 263 [M+H]+, 133
[S]+, 131 [BH2]+; FABMS (FAB<0); m/z (GT): 523
[2M−H]−, 353 [M+G−H]−, 261 [M−H]−, 129 [B]−;
[a]2D0= −20 (c 0.60, DMSO) ([a]2D0= +18 (c 1.00,
5.1.7. 1-(3,5-Di-O-benzoyl-i-
5-fluorouracil (8)
Hydrazine hydrate (80%, 2.80 mL, 57.4 mmol) was
L-xylo-furanosyl)-
added to a solution of 1-(2-O-acetyl-3,5-di-O-benzoyl-b-
L
-xylo-furanosyl)-5-fluorouracil (6) (9.80 g, 19.1 mmol)
in acetic acid (35 mL) and pyridine (150 mL). The
resulting solution was stirred overnight at r.t. Acetone
(50 mL) was added and the mixture was stirred continu-
ously for 2 h. The reaction mixture was concentrated to
a small volume and partitioned between ethyl acetate
(200 mL) and water (200 mL). Ther layers formed were
separated and the organic phase was washed with a
saturated aqueous sodium hydrogen carbonate solution
(2×200 mL) and finally with water (2×200 mL). The
organic phase was dried over sodium sulfate, and then
evaporated under reduced pressure. The residue was
purified using silica gel column chromatography (eluent:
stepwise gradient of methanol (0–5%) in methylene
DMSO) for commercial b-
D-5-fluorouridine [Fluka,
Ref. 47 576]); Anal. C9H11FN2O6·0.25H2O (C, H, N).
5.1.5. 1-(2-O-Acetyl-3,5-di-O-benzoyl-i-
L-xylo-
furanosyl)-5-fluorouracil (6)
Condensation of 5-fluorouracil (1) (5.0 g, 38.4 mmol)
-xylo-furanose
with 1,2-di-O-acetyl-3,5-di-O-benzoyl-b-
L
(5) [26] (11.3 g, 25.6 mmol) was carried out by following
the same procedure as described for the preparation of

454
J.-F. Griffon et al. / European Journal of Medicinal Chemistry 36 (2001) 447–460
(FAB>0); m/z (NBA): 471 [M+H]+, 341 [S]+; [a]D20=
−22 (c 1.01, DMSO); Anal. C23H19FN2O8 (C, H, N).
chloride) to give pure 8 (7.82 g, Yield: 87%), which was
crystallized from methylene chloride; m.p.=93–97 °C;
UV: umax (nm) (m, M−1 cm−1) (ethanol): 227 (22 800), 267
1
5.1.9. 1-(i- -Arabino-furanosyl)-5-fluorouracil (11)
L
(8200), umin: 249 (5900); H NMR (DMSO-d6): l 11.9
Sodium (0.87 g, 37.8 mmol) was added to a solution
of 10 (8.05 g, 17.1 mmol) in a mixture of anhydrous
toluene (80 mL) and methanol (160 mL). The resulting
solution was stirred at r.t. under argon for 2 h, and then
neutralized by the addition of Dowex 50 W X 2 resin
(H+ form). The resin was filtered and washed with warm
methanol, and the combined filtrates were evaporated to
dryness. Column chromatography of the residue on
silica gel (eluent: stepwise gradient of methanol (0–5%)
in ethyl acetate) and filtration through a unit Millex
HV-4 (0.45 mm, Millipore) gave 11 (3.81 g, Yield: 85%),
which was crystallized from a methylene chloride–
methanol mixture; m.p.=183–185 °C (lit. value=l87–
(br s, 1H, NH), 8.06 (d, 1H, H-6, J6-F5=6.9 Hz),
8.0–7.4 (m, 10H, 2C6H5CO), 6.35 (d, 1H, OH-2%, J=3.8
Hz), 5.77 (d, 1H, H-1%, J1%-2%=3.3 Hz), 5.43 (dd, 1H,
H-3%, J3%-2%=3.1 Hz, J3%-4%=1.9 Hz), 4.8–4.6 (m, 3H,
H-4%, H-5%, H-5¦), 4.43 (t, 1H, H-2%, J=2.3 Hz);
FABMS (FAB>0); m/z (GT): 941 [2M+H]+, 471 [M+
H]+, 341 [S]+, 131 [BH2]+, 105 [C6H5CO]+; FABMS
(FAB<0); m/z (GT): 939 [2M−H]−, 469 [MH]−, 129
[B]−, 121 [C6H5CO2]−; [a]2D0= −110 (c 1.55, DMSO);
Anal. C23H19FN2O8·1.1CH2C12 (C, H, N).
5.1.8. 1-(3,5-Di-O-benzoyl-i-L-arabino-furanosyl)-
5-fluorouracil (10)
188 °C for 1-(b-D-arabino-furanosyl)-5-fluorouracil
DCC (5.26 g, 25.5 mmol) was added to a solution of
8 in a mixture of anhydrous benzene (75 mL), DMSO
(50 mL) and pyridine (0.68 mL). The resulting solution
was stirred at r.t. under argon for 4 h and diluted with
ethyl acetate (300 mL). Oxalic acid (2.3 g, 25.5 mmol)
dissolved in methanol (6.8 mL) was added and the
reaction mixture was then stirred at r.t. for 1 h and
filtered to eliminate dicyclohexylurea. The filtrate was
washed with brine (3×300 mL), saturated aqueous
sodium hydrogen carbonate solution (2×300 mL), water
(3×200 mL), dried over sodium sulfate and evaporated
under reduced pressure. The resulting residue was co-
evaporated several times with absolute ethanol and dis-
solved in a mixture of absolute ethanol (45 mL) and
anhydrous benzene (15 mL). The resulting solution was
then cooled to 0 °C after which NaBH4 (0.48 g, 12.8
mmol) was added. The reaction mixture was stirred at
r.t. under argon for 1 h and diluted with ethyl acetate
(300 mL) and then filtered. The filtrate was washed with
brine (3×300 mL), water (2×200 mL), dried over
sodium sulfate and evaporated under reduced pressure.
The resulting residue was purified using silica gel
column chromatography (eluent: stepwise gradient of
ethyl acetate (0–50%) in chloroform) to give pure 10
(2.41 g, Yield: 60%), which was crystallized from
methanol; m.p.=223–224 °C; UV: umax (nm) (m,
M−1 cm−1) (ethanol): 231 (29 100), 268 (11 900), umin
[36]); UV: umax (nm) (m, M−1 cm−1) (ethanol): 270 (9100),
1
umin (nm): 235 (1600); H NMR (DMSO-d6): l 11.8 (br
s, 1H, NH), 7.94 (1H, H-6, J6-F5=7.4 Hz), 5.94 (d, 1H,
H-1%, J1%-2%=3.9 Hz), 5.6 (br s, 1H, OH-2%), 5.5 (br s, 1H,
OH-3%), 5.2 (br s, 1H, OH-5%), 4.0 (br s, 1H, H-2%), 3.9
(br s, 1H, H-3%), 3.7 (m, 1H, H-4%), 3.6 (m, 2H, H-5%,
H-5¦); FABMS (FAB>0); m/z (GT): 787 [3M+H]+, 525
[2M+H]+ 447 [M+2G+H]+, 263 [M+H]+, 133 [S]+, 131
[BH2]+; FABMS (FAB<0); m/z (GT): 1309 [5M−H]−,
1047 [4M−H]−, 785 [3M−H]−, 523 [2M−H]−, 369 [M+
TꢀH]−, 261 [M−H]−, 129 (B)−; [a]2D0= −127 (c 1.02,
DMSO); Anal. C9H11FN206 (C, H, N).
5.1.10. 1-(3,5-O-Isopropylidene-i-L-xylo-furanosyl)-
5-fluorouracil (12)
2,2-Dimethoxypropane (10.8 mL, 87.7 mmol) and
p-TsOH (170 mg, 0.88 mmol) were added to a solution
of 1-(b- -xylo-furanosyl)-5-fluorouracil (7) (2.3 g, 8.77
L
mmol) in anhydrous DMF (60 mL). The resulting solu-
tion was stirred at r.t. under argon for 2 h, and then
evaporated under reduced pressure (0.02 mbar, 45 °C).
The residue was purified using silica gel column chro-
matography (eluent: stepwise gradient of methanol (0–
8%) in chloroform) to give pure 12 (2.04 g, 77%), which
was crystallized from methanol; m.p.=112–114 °C;
UV: umax (nm) (m, M−1 cm−1) (ethanol): 270 (9200), umin
1
(nm): 235 (1700); H NMR (DMSO-d6): l 11.67 (br s,
1
(nm): 251 (8600); H NMR (DMSO-d6): l 11.9 (br s,
1H, NH), 7.96 (d, H-6, J6-F5=7.5 Hz), 5.84 (br d, 1H,
H-1%, J1%-2%=3.1 Hz), 5.40 (d, 1H, OH-2%, JOH-2%=1.3
Hz), 4.0–3.8 (m, 4H, H-3%, H-4%, H-5%, H-5¦), 3.79 (s,
1H, H-2%), 1.20 (s, 3H, CꢀCH3), 1.06 (s, 3H, CꢀCH3);
FABMS (FAB>0); m/z (GT): 907 [3M+H]+, 605 [2M+
1H, NH), 8.1–7.5 (m, 11H, 2C6H5CO, H-6), 6.2 (br s,
1H, OH-2%), 6.13 (d, 1H, H-1%, J1%-2%=3.6 Hz), 5.36 (d,
1H, H-3%, J3%-4%=1.8 Hz), 4.8 (m, 1H, H-5%), 4.6 (m, 1H,
H-5¦), 4.5 (m, 1H, H-4%), 4.4 (m, 1H, H-2%); FABMS

J.-F. Griffon et al. / European Journal of Medicinal Chemistry 36 (2001) 447–460
455
H]+, 303 [M+H], 245 [M−[(CH3)2CO]+H], 173 [S]+, 131
[BH2]+, 59 [[(CH3)2CO]+H]+; FABMS (FAB<0); m/z
(GT): 1309 [5M−H]−, 1207 [4M−H]−, 905 [3M−H]−,
603 [2M−H]−, 301 [M−H]−, 129 [B]−.
4.36 (q, 1H, H-2%, J=11.0 Hz, J=5.8 Hz), 4.0 (m, 1H,
H-3%), 3.8 (m, 1H, H-4%), 3.7–3.5 (m, 2H, H-5%, H-5¦);
FABMS (FAB>0); m/z (GT): 263 [M+H]+, 131 [BH2]+;
FABMS (FAB<0); m/z (GT): 261 [M−H]−, 129 [B]−;
[a]2D0= −120 (c 1.00, DMSO); Anal. C9H11FN2O6 (C, H,
N).
5.1.11. 1-(2-O-Mesyl-i-L-xylo-furanosyl)-5-fluorouracil
(13)
MsCl (1.86 mL, 24.0 mmol) was added dropwise to a
stirred suspension of 12 (2.07 g, 6.85 mmol) in dry
pyridine (100 mL) at 0 °C. The reaction mixture was
stirred overnight at 0 °C, then poured into icewater (300
mL) and extracted with chloroform (3×150 mL). Com-
bined extracts were washed with 5% aqueous sodium
hydrogen carbonate solution (2×300 mL), water (2×300
mL), dried over sodium sulfate and evaporated under
reduced pressure. The crude material was treated with
80% acetic acid (55 mL) for 40 mn under reflux, cooled
to r.t. and evaporated under reduced pressure. The
residue was purified using silica gel column chromatog-
raphy (eluent: stepwise gradient of methanol (7.5–15%)
in chloroform) and filtered through a unit Millex HV-4
(0.45 mm, Millipore) to give pure 13 (1.75 g, Yield: 75%),
which was crystallized from ethanol; m.p.=188–
189 °C; UV: umax (nm) (m, M−1 cm−1) (ethanol): 267
5.1.13. 1-(2-Deoxy-3,5-di-O-benzoyl-i-
furanosyl)-5-fluorouracil (16)
O-phenyl chlorothioformate (6.80 mL, 49.1 mmol)
and DMAP (12.0 g, 98.2 mmol) were added to a solu-
L-threo-pento-
tion of 1 - (3,5 - di - O - benzoyl - b - L - xylo - furanosyl) - 5-
fluorouracil (8) (15.4 g, 32.7 mmol) in anhydrous
acetonitrile (650 mL). The resulting solution was stirred
at r.t. under argon for 1 h, and then evaporated under
reduced pressure. The residue was dissolved in methyl-
ene chloride (350 mL) and the solution was successively
washed with water (2×250 mL), ice-cold 0.5 N hydro-
chloric acid (250 mL), water (2×250 mL), dried over
sodium sulfate and evaporated under reduced pressure.
The crude material was co-evaporated several times with
anhydrous dioxane and dissolved in the same solvent
(265 mL). To the resulting solution were added under
argon tris(trimethylsilyl)silane (12,1 mL, 39.3 mmol) and
AIBN (1.74 g, 10.8 mmol). The reaction mixture was
then heated and stirred at 100 °C for 2.5 h under argon.
The mixture was then cooled to r.t. and evaporated
under reduced pressure. The residue was purified using
silica gel column chromatography (eluent: stepwise gra-
dient of methanol (0–2%) in chloroform) to give pure 16
(13.0 g, Yield: 87%), which was crystallized from a
diethyl ether–methanol mixture; m.p.=182–184 °C;
UV: umax (nm) (m, M−1 cm−1) (ethanol): 229 (25 800), 269
1
(10 100), umin (nm): 233 (2400); H NMR (DMSO-d6): l
12.0 (br s, 1H, NH), 7.97 (d, 1H, H-6, J6-F5=7.2 Hz),
6.04 (d, 1H, OH-3%, J=4.1 Hz), 5.9 (br s, 1H, H-1%), 5.0
(m, 2H, H-2%, OH-5%), 4.3 (m, 1H, H-3%), 4.1 (m, 1H,
H-4%), 3.7 (m, 2H, H-5%, H-5¦), 3.33 (s, 3H, CH3SO2);
FABMS (FAB>0); m/z (GT): 773 [2M+G+H]+, 681
[2M+H]+, 525 [M+2G+H]+, 433 [M+G+H]+, 341 [M+
H]+, 131 [BH2]+; FABMS (FAB<0); m/z (GT): 679
[2M−H]−, 339 [M−H]−, 129 [B]−, 95 [CH3SO3]−; [a]5D0=
−15 (c 1.01, DMSO).
1
(9300), umin (nm): 251 (6500); H NMR (DMSO-d6): l
11.8 (br s, 1H, NH), 8.05 (d, 1H, H-6, J6-F5=7.0 Hz),
5.1.12. 1-(i-
L
-lyxo-Furanosy1)-5-fluorouracil (15)
8.0–7.4 (m, 10H, 2C6H5CO), 6.15 (d, 1H, H-1%, J1%-2%=
A suspension of 13 (1.70 g, 5.00 mmol) in water (100
mL) was boiled overnight under reflux. The mixture was
then cooled to r.t. and evaporated under reduced pres-
sure. The residue was purified using silica gel column
chromatography (eluent: stepwise gradient of methanol
(7.5–15%) in ethyl acetate) and filtered through a unit
Millex HV-4 (0.45 mm, Millipore) to give pure 15 (1.12
g, Yield: 81%), which was crystallized from ethanol;
m.p.=208–209 °C; UV: umax (nm) (m, M−1 cm−1) (etha-
nol): 269 (9300), umin (nm): 234 (1800); 1H NMR
(DMSO-d6): l 11.8 (br s, 1H, NH), 8.15 (1H, H-6,
7.4 Hz), 5.68 (t, 1H, H-3%, J=4.2 Hz), 4.8–4.6 (m, 2H,
H-5%, H¦-5), 4.6 (m, 1H, H-4%), 3.0–2.8 (m, 1H, H-2%),
2.40 (d, 1H, H-2¦, J2¦-2%=14.8 Hz); FABMS (FAB>0);
m/z (GT): 455 [M+H]+, 325 [S]+, 131 [BH2]+, 105
[C6H5CO]+; FABMS (FAB<0); m/z (GT): 452 [M−H]−,
129 [B]−; [a]2D0= −125 (c 1.05, DMSO); Anal.
C23H19FN2O7 (C, H, N).
5.1.14. 1-(2-Deoxy-i-L-threo-pentofuranosyl)-5-
fluorouracil (17)
Compound 16 (3.25 g, 7.15 mmol) was debenzoylated
by treating with methanolic ammonia following the
general procedure to give 17 (1.60 g, Yield: 91%), which
was crystallized from methanol; m.p.=198–200 °C;
J6-F5=7.7 Hz), 6.01 (dd, 1H, H-1%, J1%-2%=6.9 Hz, J1%-
F5=1.8 Hz), 5.50 (d, 1H, OH-2%, J=4.2 Hz), 5.40 (d,
1H, OH-3%, J=3.7 Hz), 4.77 (t, 1H, OH-5%, J=5.1 Hz),

456
J.-F. Griffon et al. / European Journal of Medicinal Chemistry 36 (2001) 447–460
UV: umax (nm) (m, M−1 cm−1) (ethanol): 267 (8500), umin
(nm): 233 (2100); H NMR (DMSO-d6): l 11.8 (br s,
and solvents were removed under reduced pressure. The
residue was purified using silica gel column chromatog-
raphy (eluent: stepwise gradient of methanol (0–2.5%)
in chloroform) to give 19 (483 mg, Yield: 60%), which
was precipitated in chloroform; m.p.=243–244 °C (lit.
1
1H, NH), 8.16 (d, 1H, H-6, J6-F5=7.4 Hz), 6.05 (dd,
1H, H-1%, J1%-2%=6.5 Hz, J1%-F5=1.8 Hz), 5.38 (d, 1H,
OH-3%, J=3.3 Hz), 4.72 (t, 1H OH-5%, J=5.4 Hz), 4.22
(q, 1H, H-3%, J=3.1 Hz, J=7.5 Hz), 3.8–3.6 (m, 3H,
H-4%, H-5%, H¦-5), 2.6 (m, 1H, H-2%), 1.83 (d, 1H, H-2¦,
J2¦-2%=14.7 Hz); FABMS (FAB>0); m/z (GT): 339 [M+
G+H]+, 247 [M+H]+, 131 [BH2]+, 115 [S]+; FABMS
(FAB<0); m/z (GT): 245 [M−H]−, 129 [B]−; [a]D20= +16
(c 0.86, DMSO); Anal. C9H11FN2O5 (C, H, N).
value >250 °C for the D-enantiomer [9]); UV: umax (nm)
(m, M−1 cm−1) (ethanol): 231 (31 200), 268 (11 500), umin
1
(nm): 251 (8500); H NMR (DMSO-d6): l 11.9 (br s,
1H, NH), 8.1–7.4 (m, 11H, 2C6H5CO, H-6), 6.21 (t, 1H,
H-1%, J=6.6 Hz), 5.6 (m, 1H, H-3%), 4.6–4.5 (m, 2H,
H-5%, H¦-5), 4.5 (m, 1H, H-4%), 2.7–2.5 (m, 2H, H-2%,
H-2¦); FABMS (FAB<0); m/z (GT): 453 [M−H]−, 129
[B]−, 121 [C6H5CO2]−.
5.1.15. 1-(2-Deoxy-5-O-benzoyl-i-L-threo-pento-
furanosyl)-5-fluorouracil (18)
A solution of benzoyl chloride(1.39 mL, 12.0 mmol)
in anhydrous pyridine (22 mL) was added dropwise to a
suspension of 17 (2.68 g, 10.9 mmol) in anhydrous
pyridine (88 mL) and DMF (22 mL) at 0 °C. The
reaction mixture was stirred for 3 h at 0 °C, then
poured onto icewater (200 mL) and extracted with chlo-
roform (3×100 mL). Combined extracts were washed
with a saturated aqueous sodium hydrogen carbonate
solution (150 mL), water (2×150 mL), dried over
sodium sulfate and evaporated under reduced pressure.
The residue was purified using silica gel column chro-
matography (eluent: stepwise gradient of methanol (0–
5%) in methylene chloride) to give pure 18 (3.0 g, Yield:
81%), which was crystallized from methylene chloride;
m.p.=187–189 °C; UV: umax (nm) (m, M−1 cm−1) (etha-
5.1.17. 1-(2-Deoxy-i-
5-fluorouracil (20)
L-erythro-pentofuranosyl)-
Compound 19 (190 mg, 0.42 mmol) was debenzoy-
lated by treating with methanolic ammonia following
the general procedure, giving 20 (95 mg, Yield: 92%),
which was crystallized from ethanol; m.p.=148–
150 °C (lit. value 147–149 °C [10]); UV: umax (nm) (m,
M−1 cm−1) (ethanol): 269 (11 100), umin (nm): 234 (1800);
1H NMR (DMSO-d6): l 11.8 (br s, 1H, NH), 8.20 (1H,
H-6, J6-F5=7.2 Hz), 6.11 (t, 1H, H-1%, J=6.6 Hz),
5.3–5.1 (m, 2H, OH-5% and OH-3%), 4.2 (br d, 1H, H-3%,
J=3.0 Hz), 3.8 (m, 1H, H-4%), 3.6–3.5 (m, 2H, H-5%,
H-5¦), 2.0 (m, 2H, H-2%, H-2¦); FABMS (FAB>0); m/z
(GT): 247 [M+H]+, 131 [BH2]+, 117 [S]+; FABMS
(FAB<0); m/z (GT): 245 [M−H]−, 129 [B]−; [a]D20= −44
(c=0.77, DMSO) (+37 (c 1.00, DMSO)) for the com-
1
nol): 228 (16 100), 269 (8500), umin (nm): 217 (6000); H
NMR (DMSO-d6): l 11.8 (br s, 1H, NH), 8.21 (d, 1H,
H-6, J6-F5=7.3 Hz), 8.0–7.5 (m, 5H, C6H5CO), 6.13
(dd, 1H, H-1%, J1%-2%=6.6 Hz and J6–5F=1.7 Hz), 5.72
(d, 1H, OH-3%, J=3.5 Hz), 4.6–4.5 (m, 2H, H-5% et
H¦-5), 4.37 (dd, 1H, H-3%, J=7.8 Hz), 4.2–4.1 (m, 1H,
mercial 2%-deoxy-b-
D-5-fluorouridine [Pharma-Waldhof
Gmbh, Ref. 6717 60]; Anal. C9H11FN205 (C, H, N).
5.1.18. 1-(2-O-Acetyl-3,5-di-O-benzoyl-i-
furanosyl)-5-fluorouracil (21)
L-arabino-
H-4%), 2.7–2.6 (m, 1H, H-2%), 1.97 (d, 1H, H-2¦, J2¦-2%
=
Acetic anhydride (0.17 mL, 1.81 mmol) was added to
solution of 1-(3,5-di-O-benzoyl-b- -arabino-fura-
14.8 Hz); FABMS (FAB>0); m/z (GT): 701 [2M+H]+,
351 [M+H]+, 221 [S]+, 131 [BH2]+, 105 [C6H5CO]+;
FABMS (FAB<0); m/z (GT): 699 [2M−H]−, 349 [M−
H]−, 121 [C6H5CO2]−; [a]2D0= −46 (c 1.10, DMSO); Anal.
C16H15FN2O6·0.2CH2C12 (C, H, N).
a
L
nosyl)-5-fluorouracil (10) (709 mg, 1.51 mmol) in dry
pyridine (11 mL) under argon at 0 °C. The resulting
mixture was stirred at r.t. for 22 h. Ethanol was then
added and the solvents were removed under reduced
pressure. The residue was purified using silica gel
column chromatography (eluent: stepwise gradient of
methanol (0–4%) in chloroform) to give pure 21 (518
mg, Yield: 67%) as a white foam; m.p.=94–95 °C; UV:
umax (nm) (m, M−1 cm−1) (ethanol): 231 (27 400), 267
5.1.16. 1-(2-Deoxy-3,5-di-O-benzoyl-i-L-erythro-
pentofuranosyl)-5-fluorouracil (19)
DEAD (0.83 mL, 5.30 mmol) and benzoic acid (647
mg, 5.30 mmol) in anhydrous THF (7.5 mL) were added
dropwise to a solution of 18 (619 mg, 1.77 mmol) and
PPh3 (1,39 g, 5.30 mmol) in anhydrous THF (28.5 mL)
at 0 °C. The reaction mixture was stirred for 2 h at r.t.
The reaction was quenched by the addition of ethanol
1
(10 300), umin (nm): 251 (8400); H NMR (DMSO-d6): l
12.0 (br s, 1H, NH), 8.1–7.5 (m, 11H, 2C6H5CO, H-6),
6.35 (d, 1H, H-1%, J1%-2%=4.9 Hz), 5.7 (m, 1H, H-3%), 5.6
(m, 1H, H-2%), 4.7 (m, 2H, H-5%, H-5¦), 4.5 (br s, 1H,

J.-F. Griffon et al. / European Journal of Medicinal Chemistry 36 (2001) 447–460
457
H-4%), 1.90 (s, 3H, CH3CO); FABMS (FAB>0); m/z
(GT): 513 [M+H]+, 383 [S]+, 105 [C6H5CO]+, 43
[CH3CO]+; FABMS (FAB<0); m/z (GT): 1023 [2M−
H]−, 511 [M−H]−, 129 [B]−, 121 [C6H5CO2]−, 59
[CH3CO2]−; [a]2D0= −49 (c 1.01, DMSO).
5.1.21. 1-(i-
Thiation of 1-(2-O-acetyl-3,5-di-O-benzoyl-b-
L
-xylo-Furanosyl)-5-fluorocytosine (24)
-xylo-
L
furanosyl)-5-fluorouracil (6) (500 mg, 0.976 mmol), with
Lawesson’s reagent (276 mg, 0.68 mmol), was carried
out following the general procedure giving the corre-
sponding 4-thio intermediate (469 mg, Yield: 91%) as a
yellow foam. The 4-thio intermediate (410 mg, 0.78
mmol) was treated with methanolic ammonia following
the general procedure to give 24 (180 mg, Yield: 89%),
which was crystallized from methanol; m.p.=221–
225 °C; UV: umax (nm) (m, M−1 cm−1) (ethanol): 238
5.1.19. 1-(2,3,5-Tri-O-acetyl-i-L-lyxo-furanosyl)-
5-fluorouracil (22)
Acetylation of 1-(b-L-lyxo-furanosyl)-5-fluorouracil
(16) (0.75 g, 2.86 mmol) with acetic anhydride (2.03 mL,
21.5 mmol), was carried out following the same proce-
dure as described for the preparation of 21 giving com-
pound 22 (1.00 g, Yield: 90%) as a white foam;
m.p.=84–86 °C; UV: umax (nm) (m, M−1 cm−1) (etha-
nol): 266 (9400), umin (nm): 233 (2400); 1H NMR
(DMSO-d6): l 11.9 (br s, 1H, NH), 7.82 (1H, H-6,
1
(8700), 280 (8100), umin (nm): 226 (8000), 261 (6100); H
NMR (DMSO-d6): l 7.84 (d, 1H, H-6, J6-F5=7.4 Hz),
7.7–7.4 (br d, 2H, NH2), 5.70 (d, 1H, OH-2%, J=4.2
Hz), 5.6 (br s, 1H, H-1%), 5.37 (d, 1H, OH-3%, J=3.5
Hz), 4.75 (t, 1H, OH-5%, J=5.6 Hz), 4.1–4.0 (m, 1H,
H-4%), 3.9–3.8 (m, 2H, H-2%, H-3¦), 3.7–3.6 (m, 2H,
H-5%, H-5¦); FABMS (FAB>0); m/z (GT): 1045 [4M+
H]+, 784 [3M+H]+, 523 [2M+H]+, 262 [M+H]+, 130
[BH2]+; FABMS (FAB<0); m/z (GT): 521 [2M−H]−,
260 [M−H]−, 128 [B]−; [a]2D0= +10 (c 0.90, DMSO);
Anal. C9H12FN3O5 (C, H, N).
J6-F5=7.1 Hz), 6.19 (dd, 1H, H-1%, J1%-2%=6.5 Hz, J1%-
F5=1.7 Hz), 5.69 (dd, 1H, H-2%, J2%-3%=5.1 Hz), 5.46
(dd, 1H, H-3%, J3%-4%=3.3 Hz), 4.4–4.3 (m, 3H, H-4%,
H-5%, H-5¦), 2.06 (s, 3H, CH3CO), 2.04 (s, 3H, CH3CO),
1.90 (s, 3H, CH3CO); FABMS (FAB>0); m/z (GT): 777
[2M+H]+, 388 [M+H]+, 329 [M−[CH3CO2]]+, 259 [S]+,
131 [BH2]+, 43 [CH3CO]+; FABMS (FAB<0); m/z (GT):
775 [2M−H]−, 387 [M−H]−, 345 [M−[CH3CO]]−, 59
[CH3C02]−; [a]2D0= −66 (c 1.05, DMSO).
5.1.22. 1-(2-Deoxy-i-
fluorocytosine (25)
Thiation of 1-(2-deoxy-3,5-di-O-benzoyl-b-L-threo-
L-threo-pentofuranosyl)-5-
pentofuranosyl)-5-fluorouracil (16) (5.0 g, 11.0 mmol),
with Lawesson’s reagent (3.1 g, 7.70 mmol), was carried
out following the general procedure, giving the corre-
sponding 4-thio intermediate (4.6 g, Yield: 80% as a
yellow foam. The 4-thio intermediate (1.0 g, 2.13 mmol)
was treated with methanolic ammonia following the
general procedure to give 25 (0.44 g, Yield: 84%), which
was crystallized from an ethyl acetate–methanol mix-
5.1.20. 1-(i-
Thiation of 1-(2,3,5-tri-O-benzoyl-b-
L
-Ribofuranosyl)-5-fluorocytosine (23)
-ribofuranosyl)-
L
5-fluorouracil (3) (720 mg, 1.25 mmol), with Lawesson’s
reagent (355 mg, 0.88 mmol), was carried out following
the general procedure giving the corresponding 4-thio
intermediate (530 mg, Yield: 72%) as a yellow foam. The
4-thio intermediate (500 mg, 0.85 mmol) was treated
with methanolic ammonia following the general proce-
dure to give 23 (188 mg, Yield: 85% ) as a white solid;
m.p.=70–71 °C; UV: umax (nm) (m, M−1 cm−1) (etha-
nol): 241 (7600), 284 (6900), umin (nm): 226 (6600), 264
(5200); 1H NMR (DMSO-d6): l 8.20 (d, 1H, H-6,
ture; m.p.=199–201 °C; UV: umax (nm) (m, M−1 cm−1
)
(ethanol): 226 (7700), 281 (8500), umin (nm): 262 (6300);
1H NMR (DMSO-d6): l 7.99 (d, 1H, H-6, J6-F5=7.4
Hz), 7.7–7.4 (br d, 2H, NH2), 5.98 (d, 1H, H-1%, J1%-2%
=
8.1 Hz), 5.25 (d, 1H, OH-3%, J=3.4 Hz), 4.71 (t, 1H,
OH-5%, J=5.6 Hz), 4.2 (m, 1H, H-3%), 3.8–3.6 (m, 3H,
H-4%, H-5%, H-5¦), 2.5 (m, 1H, H-2%), 1.8 (m, 1H, H-2¦);
FABMS (FAB>0); m/z (GT): 491 [2M+H]+, 246 [M+
H]+, 130 [BH2]+; FABMS (FAB<0); m/z (GT): 489
[2M−H]−, 244 [M−H]−, 128 [B]−; [a]2D0= −21 (c 0.92,
DMSO); Anal. C9H12FN3O4 (C, H, N).
J6-F5=7.4 Hz), 7.7–7.5 (br d, 2H, NH2), 5.69 (dd, 1H,
H-1%, J1%-2%=3.6 Hz, J1%-F5=1.8 Hz), 5.35 (d, 1H, OH-2%,
J=5.1 Hz), 5.20 (t, 1H, OH-5%, J=4.9 Hz), 4.99 (d, 1H,
OH-3%, J=5.5 Hz), 4.0–3.9 (m, 2H, H-2%, H-3¦), 3.8 (m,
1H, H-4%), 3.7–3.5 (m, 2H, H-5%, H-5¦); FABMS (FAB>
0); m/z (GT): 523 [2M+H]+, 262 [M+H]+, 130 [BH2]+;
FABMS (FAB<0); m/z (GT): 260 [M−H]−, 128 [B]−;
[a]2D0= −28 (c 0.96, DMSO); Anal. C9H12FN3O5·2H2O
(C, H, N).
5.1.23. 1-(2-Deoxy-i-
fluorocytosin (26)
Thiation of 1-(2-deoxy-3,5-di-O-benzoyl-b-
pentofuranosyl)-5-fluorouracil (19) (670 mg, 1.47
L-erythro-pentofuranosyl)-5-
L-erythro-

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J.-F. Griffon et al. / European Journal of Medicinal Chemistry 36 (2001) 447–460
mmol), with Lawesson’s reagent (417 mg 1.03 mmol),
was carried out following the general procedure, and
forming the corresponding 4-thio intermediate (636 mg,
Yield: 92%) as a yellow foam. The 4-thio intermediate
(275 mg, 0.58 mmol) was treated with methanolic am-
monia following the general procedure to give 26 (125
mg, Yield: 87%), which was crystallized from ethanol;
m.p.=200–201 °C; UV: umax (nm) (m, M−1 cm−1) (etha-
nol): 242 (9500), 284 (9000), umin (nm): 226 (7200), 264
(6600); 1H NMR (DMSO-d6): l 8.09 (d, 1H, H-6,
with methanolic ammonia following the general proce-
dure to give 28 (202 mg, Yield: 63% ), which was
crystallized from an ethyl acetate–methanol mixture;
m.p.=78–80 °C; UV: umax (nm) (m, M−1 cm−1) (etha-
nol): 241 (7700), 283 (7700), umin (nm): 226 (6600), 262
1
(5600); H NMR (DMSO-d6): l 7.99 (1H, H-6, J6-F5
=
7.6 Hz), 7.7–7.4 (br d, 2H, NH2), 6.03 (dd, 1H, H-1%,
J1%-2%=6.5 Hz, J1%-F5=2.0 Hz), 5.38 (d, 1H, OH-2%, J=
5.7 Hz), 5.33 (d, 1H, OH-3%, J=3.6 Hz), 4.81 (t, 1H,
OH-5%, J=5.4 Hz), 4.27 (br d, 1H, H-2%, J=5.4 Hz), 4.1
(m, 1H, H-3%), 4.0 (m, 1H, H-4%), 3.7–3.5 (m, 2H, H-5%,
H5¦); FABMS (FAB>0); m/z (GT): 523 [2M+H]+, 262
[M+H]+, 130 [BH2]+; FABMS (FAB<0); m/z (GT): 521
[2M−H]−, 260 [M−H]−, 128 [B]−; [a]2D0= −33 (c 1.00,
DMSO).
J6-F5=7.2 Hz), 7.8–7.5 (br s, 2H, NH2), 6.10 (t, 1H,
H-1%, J=6.1 Hz), 5.21 (d, 1H, OH-3%, J=4.0 Hz), 5.09
(t, 1H, OH-5%, J=4.9 Hz), 4.2 (br s, 1H, H-3%), 3.9 (br
s, 1H, H-4%), 3.6–3.5 (m, 2H, H-5%, H-5¦) 2.2 (m, 1H,
H-2%), 2.0 (m, 1H, H-2¦); FABMS (FAB>0); m/z (GT):
736 [3M+H]+, 491 [2M+H]+, 338 [M+G+H]+, 246 [M+
H]+, 130 [BH2]+, 117 [S]+; FABMS (FAB<0); m/z (GT):
244 [M−H]−, 128 [B]−; [a]2D0= −71 (c 0.82, DMSO);
Anal. C9H12FN3O4 (C, H, N).
5.2. Biology
5.2.1. Inhibition of cell proliferation
The test compounds were evaluated for their cyto-
static activity against murine leukemia L1210, murine
mammary carcinoma FM3A and human T-lymphocyte
Molt4/C8 and CEM cells in 96-microtiter plates as
described earlier [37–39]. Briefly, 100 ml of an appropri-
ate dilution of the test compound and 100 ml of 5×104–
7.5×104 tumor cells were added to each 200 ml well.
After a 48 h (L1210 and FM3A) or 72 h (Molt4/C8,
CEM) incubation period at 37 °C in a humidified CO2-
controlled atmosphere, cells were counted using an au-
tomated Coulter counter. The IC50 represents the
compound concentration required to inhibit cell prolif-
eration by 50%.
5.1.24. 1-(i-
Thiation of 1-(2-O-acetyl-3,5-di-O-benzoyl-b-
L
-Arabino-furanosyl)-5-fluorocytosine (27)
-ara-
L
bino-furanosyl)-5-fluorouracil (21) (750 mg, 1.46 mmol),
with Lawesson’s reagent (414 mg, 1.02 mmol), was
carried out following the general procedure forming the
corresponding 4-thio intermediate (610 mg, Yield: 80%)
as a yellow foam. The 4-thio intermediate (308 mg, 0.58
mmol) was treated with methanolic ammonia following
the general procedure to give 27 (126 mg, Yield: 83%),
which was crystallized from methanol; m.p.=237–
239 °C (lit. value 232–233 °C for the
D-enantiomer
[36]); UV: umax (nm) (m, M−1 cm−1) (ethanol): 240 (7800),
1
285 (7600), umin (nm): 226 (6800), 264 (5100); H NMR
(DMSO-d6): l 7.76 (1H, H-6, J6-F5=7.2 Hz), 7.7–7.4
(br d, 2H, NH2), 6.0 (m, 1H, H-1%), 5.4 (m, 2H, OH-2%,
OH-3%), 5.1 (br s, 1H, OH-5%), 4.0 (br S, 1H, H-2%), 3.9
(m, 1H, H-3%), 3.7 (m, 1H, H-4%), 3.6 (m, 2H, H-5%,
H-5¦); FABMS (FAB>0); m/z (GT): 523 [2M+H]+,
262 [M+H]+, 130 [BH2]+; FABMS (FAB<0); m/z (GT):
260 [M−H]−; [a]D20= −15 (c 0.89, DMSO); Anal.
C9H12FN3O5 (C, H, N).
5.2.2. Anti-viral assays
The anti-HIV and anti-HBV assays in cell cultures
were carried out following the procedures established
earlier [40].
5.2.2.1. Anti-HIV evaluation
Briefly, in MT-4 cells, the determination of the anti-
viral activity of compounds was based on a reduction of
HIV-1IIIB-induced cytopathogenicity, the metabolic ac-
tivity of the cells being measured by the property of
mitochondrial deshydrogenase to reduce MTT into for-
mazan. For CEM-SS cells, the production of virus
HIV-1LAI was measured by quantifying the reverse tran-
scriptase activity associated with the virus particle re-
leased into the culture supernatant. In parallel
experiments, cytotoxicity of the test compounds was
5.1.25. 1-(i-
Thiation of 1-(2,3,5-tri-O-acetyl-b-
L
-lyxo-Furanosyl)-5-fluorocytosine (28)
-lyxo-furanosyl)-
L
5-fluorouracil (22) (0.96 g, 2.47 mmol), with Lawesson’s
reagent (0.70 g, 1.73 mmol), was carried out following
the general procedure giving the corresponding 4-thio
intermediate (0.97 g, Yield: 97%) as a yellow foam. The
4-thio intermediate (500 mg, 1.24 mmol) was treated

J.-F. Griffon et al. / European Journal of Medicinal Chemistry 36 (2001) 447–460
459
Schinazi R.F., Painter G.R., Antimicrob. Agents Chemother.
36 (1992) 2686–2692.
measured after incubating the uninfected cells for 5 days
in their presence using the colorimetric MTT test.
[14] Wilson J.E., Martin J.L., Borroto-Escoda K., Hopkins S.,
Painter G., Liotta D.C., Furman P.A., Antimicrob. Agents
Chemother. 37 (1993) 1720–1722.
5.2.2.2. Anti-HBV evaluation
Briefly, the 2.2.15. cells (HBV DNA-transfected hu-
man hepatoblastoma-derived Hep-G2 cells) were cul-
tured and the inhibition of HBV extracellular DNA
(HBV virion) or HBV intracellular DNA(HBV replica-
tive intermediate, HBV RI) was determined. Cytotoxic-
ity assays were conducted in Hep-G2 cells. Each
compound was tested in four concentrations in triplicate
cultures and the median inhibitory concentration (IC50)
was determined.
[15] Mansour T.S., Jin H., Wang W., Hooker E.U., Ashman C.,
Cammack N., Salomon H., Belmonte A.R., Wainberg M.A.,
J. Med. Chem. 38 (1995) 1–4.
[16] Faraj A., Agrofolio L.A., Wakefield J.K., McPherson S.,
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(1994) 2300–2305.
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These investigations were supported by the Associa-
tion pour la Recherche contre le Cancer (ARC, France)
and the Agence Nationale de Recherche sur le SIDA
(ANRS, France). One of us (J.-F.G.) is particularly
grateful to ARC for the doctoral fellowship.
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