Synthesis and studies of 3'-C-trifluoromethyl nucleoside analogues bearing adenine or cytosine as the base.

Article abstract of DOI:10.1016/S0968-0896(02)00216-X

3'-Deoxy-3'-C-CF3, 2',3'-dideoxy-3'-C-CF3 and 2',3'-unsaturated-3'-C-CF3 nucleoside derivatives of adenosine and cytidine have been synthesized. All these derivatives were prepared by glycosylation of adenine and uracil with a suitable peracylated 3-trifluoromethyl sugar precursor. The resulting protected nucleosides were subject to appropriate chemical modifications to afford the target nucleoside derivatives. Additionally, the chemical stability in acidic and neutral media of the 2',3'-dideoxy-3'-C-CF3 and 2',3'-unsaturated-3'-C-CF3 nucleoside derivatives of adenosine was compared to that of their parent nucleosides 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxy-2',3'-didehydroadenosine (d(4)A). Our results confirm that addition of a trifluoromethyl group at C-3' on such nucleoside derivatives appears to confer increased chemical stability toward acid-catalyzed cleavage of the glycosidic bond comparatively to their parent counterparts. When evaluated for their antiviral activity in cell culture experiments, two compounds, namely, 2',3'-dideoxy-3'-C-CF3-adenosine and 2',3'-dideoxy-2',3'-didehydro-3'-C-CF3-cytidine exhibited moderate anti-HBV activity with EC50 values of 10 and 5 microM, respectively.

Full text of DOI:10.1016/S0968-0896(02)00216-X

Bioorganic & Medicinal Chemistry 10 (2002) 3153–3161
Synthesis and Studies of 30-C-trifluoromethyl Nucleoside
Analogues Bearing Adenine or Cytosine as the Base§
Frederic Jeannot,a Gilles Gosselin,a,b David Standring,c
Martin Bryant,c,, Jean-Pierre Sommadossi,c
Anna Giulia Loi,d Paolo La Collad and Christophe Mathea,*
a
´
`
Laboratoire de Chimie Organique Biomoleculaire de Synthese, UMR 5625 CNRS-UM II,
´
`
´
Universite Montpellier II, case courrier 008, Place Eugene Bataillon, 34095 Montpellier, cedex 5, France
b
´
´
Laboratoire Cooperatif Novirio-CNRS-Universite Montpellier II, Universite Montpellier II,
`
case courrier 008, Place Eugene Bataillon, 34095 Montpellier, cedex 5, France
cNovirio-Pharmaceuticals, Inc., 125 Cambridge Park Drive, Cambridge, MA 02140, USA
`
Dipartimento di Biologia Sperimentale, Universita degli Studi di Cagliari, 09042Cagliari, Italy
d
Received 28 March2002; accepted 7 June 2002
Abstract—30-Deoxy-30-C-CF3, 20,30-dideoxy-30-C-CF3 and 20,30-unsaturated-30-C-CF3 nucleoside derivatives of adenosine and cyti-
dine have been synthesized. All these derivatives were prepared by glycosylation of adenine and uracil with a suitable peracylated
3-trifluoromethyl sugar precursor. The resulting protected nucleosides were subject to appropriate chemical modifications to afford
the target nucleoside derivatives. Additionally, the chemical stability in acidic and neutral media of the 20,30-dideoxy-30-C-CF3 and
20,30-unsaturated-30-C-CF3 nucleoside derivatives of adenosine was compared to that of their parent nucleosides 20,30-dideoxy-
adenosine (ddA) and 20,30-dideoxy-20,30-didehydroadenosine (d4A). Our results confirm that addition of a trifluoromethyl group at
C-30 on such nucleoside derivatives appears to confer increased chemical stability toward acid-catalyzed cleavage of the glycosidic
bond comparatively to their parent counterparts. When evaluated for their antiviral activity in cell culture experiments, two com-
pounds, namely, 20,30-dideoxy-30-C-CF3-adenosine and 20,30-dideoxy-20,30-didehydro-30-C-CF3-cytidine exhibited moderate anti-
HBV activity withEC values of 10 and 5 mM, respectively.
50
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
virus (HIV) infection. One of them, 3TC was also
To date, six nucleoside analogues, namely, 30-azido-30-
deoxythymidine (AZT), 20,30-dideoxyinosine (ddI), 20,30-
licensed by FDA for use in hepatitis B virus (HBV)
therapy. All these 20,30-dideoxynucleoside analogues
share a common mechanism of action. They are meta-
bolized by cellular kinases to their 50-triphosphate
forms, which then exert their biological effect as a virus-
specific polymerase competitive inhibitors or chain ter-
minators because they lack a hydroxyl group at the C0-3
position.1 However, inherent drug resistance2 and toxi-
city3 of the currently used antiviral drugs have promp-
ted the development of new agents possessing more
potent and broad antiviral activities. In order to dis-
cover new nucleoside derivatives withantiviral activity,
modifications of the base and/or sugar moiety of nat-
ural nucleosides can be attempted. As a part of our
ongoing research on this topic, we have synthesized,
from a common trifluoromethyl sugar precursor, var-
ious 30-C-trifluoromethyl nucleoside analogues bearing
adenine and cytosine as the base. Many advantages can
dideoxycytidine
thymidine (d4T),
(ddC),
20,30-didehydro-30-deoxy-
20,30-dideoxy-30-thia-b-l-cytidine
(3TC) and (1S,4R)-4-[2-amino-6-(cyclopropyl)-9H-
purin-9-yl]-2-cyclopentene-1-methanol (Abacavir) have
been approved by the Food and Drug Administration
(FDA) for the treatment of human immunodeficiency
§This work has been presented in preliminary form at the Fourteenth
International Round Table on Nucleosides, Nucleotides and their
Biological Applications, Sept. 10–14, 2000, San Francisco, CA, USA.
For the proceedings, See: Jeannot, F., Mathe, C., Gosselin, G.
Nucleosides, Nucleotides & Nucleic Acids 2001, 20, 755–758.
Deceased on March4, 2002.
*Corresponding author. Tel.: +33-4-67-14-47-76; fax: +33-4-67-04-
20-29; e-mail: cmathe@univ-montp2.fr
,
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00216-X

3154
F. Jeannot et al. / Bioorg. Med. Chem. 10 (2002) 3153–3161
be expected from the presence of a CF3 group on the
sugar moiety of nucleosides, including higher lipophili-
city and increased chemical and/or enzymatic stability.
Herein, we report on the synthesis of 30-deoxy-30-C-CF3,
20,30-dideoxy-30-C-CF3 and 20,30-unsaturated-30-C-CF3
nucleoside derivatives of adenosine (3, 6 and 8) and cyti-
dine (10, 13 and 15), all of them being hitherto unknown
except for 6,4 134,5 and 15.5 In addition, chemical stability
studies within the adenine series are presented for 6 and 8.
Total deprotection of 2 withmethanolic ammonia pro-
vided crystalline nucleoside 3. In order to prepare the
target compounds 6 and 8, regioselective 20-O-deacyl-
ation of 2 with hydrazine hydrate8 was accomplished to
give the key derivative 4. The latter was then treated
with O-phenyl chloro(thio)formate (C6H5OC(S)Cl) and
4-dimethylaminopyridine (DMAP) in acetonitrile. The
corresponding 20-O-[phenoxy-(thiocarbonyl)] intermediate
was subsequently deoxygenated withtris(trimeythl-
silyl)silane9 in dry toluene in the presence of a,a0-azoi-
sobutyronitrile (AIBN) to yield the protected 20,30-
dideoxy-30-C-CF3-nucleoside derivative 5. Removal of
the benzoyl group with methanolic ammonia afforded the
desired dideoxynucleoside 6 as a crystalline solid in 83%
after purification on silica gel column. On the other hand,
introduction of a double bond between the 20 and 30 posi-
tions was achieved from 4 via a base-promoted b-elimina-
tion. The first step involved the preparation (by treatment
of 4 withmesyl chloride) of the corresponding 2 0-O-mesyl
ester, which upon reaction with tetrabutylammonium
fluoride9 (TBAF) in tetrahydrofuran (THF) gave com-
pound 7. Finally, treatment withmethanolic ammonia
provided the desired 20,30-unsaturated-30-C-CF3-nucleo-
side derivative 8 in 70% yield.
Results and Discussion
The synthesis began with the preparation of an appro-
priate trifluoromethyl sugar precursor, namely, 1,2-di-
O-acetyl-5-O-benzoyl-3-deoxy-3-C-trifluoromethyl-b-d-
ribofuranose5 (1) which was obtained from commer-
cially available diacetone-d-glucose following a mod-
ified procedure initially developed by Lavaire et al.6 The
syntheses of the 30-deoxy-30-C-CF3-nucleoside deriva-
tives of adenosine (3, 6 and 8) are depicted in Scheme 1.
A glycosylation reaction withadenine and 1 using stan-
nic [tin(IV)] chloride as a catalyst7 afforded 9-(2-O-ace-
tyl-5-O-benzoyl-3-deoxy-3-C-trifluoromethyl-b-d -
ribofuranosyl)adenine (2) in 71% yield after purification
by silica gel column chromatography. The structure of 2
was fully established from 1H, 13C and UV spectra.
The syntheses of the cytidine nucleoside derivatives 10,
13 and 15 were achieved via the preparation of the
30-deoxy-30-C-CF3-nucleosides of uridine10 (9, 12 and
14), followed by conversion into their corresponding
cytidine derivatives as depicted in Scheme 2. Briefly,
glycosylation reaction of uracil withsugar 1 was carried
out under Vorbruggen conditions using (trimethylsilyl)
trifluoromethane sulfonate (TMSOTf) as a catalyst11 in
anhydrous 1,2-dichloroethane and afforded 1-(2-O-ace-
tyl-5-O-benzoyl-3-deoxy-3-C-trifluoromethyl-b-d-ribo-
furanosyl)uracil (9) in 63% after silica gel column
chromatography. Compound 9 was regioselectively
deacylated to provide the key intermediate 11. In order
to obtain the 20,30-dideoxy-30-C-CF3 derivative 12,
compound 11 was then treated with [C6H5OC(S)Cl] in
the presence of DMAP (4 equiv). After conventional
work-up, the crude material was not purified but
directly subjected to a radical reductive process to yield,
after column chromatography, an inseparable mixture
of compounds 12 and 14 (ratio 12/14=7/3, as deter-
mined by 1H NMR). Formation of the 20,30-unsaturated
nucleoside as a side-product is probably due to a
b-elimination reaction of the labile transperiplanar
H-30b from the 20-O-[phenoxy(thiocarbonyl)] inter-
mediate promoted by DMAP. To overcome this side
reaction, DMAP was replaced by pyridine, and thus,
the 20,30-dideoxy-30-C-CF3 derivative 12 was obtained as
the sole product in 80% overall yield from 11. Addi-
tionally, the 20,30-unsaturated-30-C-CF3-nucleoside 14
was prepared by a sequence similar to that developed
for the synthesis of the didehydro derivative 7 of ade-
nosine. Finally, compounds 9, 12 and 14 were converted
into their corresponding cytidine derivatives 10, 13 and
15 by using the nitrophenylation-ammonolysis proce-
dure12 in 72, 78 and 82% overall yield, respectively.
Scheme 1. (a) Adenine, SnCl4, CH3CN, rt; (b) NH3/MeOH, rt; (c)
H2NNH2, AcOH, pyridine, rt; (d) (i) DMAP, C6H5O(S)Cl, CH3CN,
rt; (ii) (Me3Si)3SiH, AIBN, toluene, reflux; (e) (i) MsCl, pyridine, rt;
(ii) TBAF, THF, 50 ꢀC.
Modifications of the sugar moiety of natural nucleosides
have led to the discovery of the 20,30-dideoxynucleoside

F. Jeannot et al. / Bioorg. Med. Chem. 10 (2002) 3153–3161
3155
analogues family, and some of them have demonstrated
potent anti-HIV and anti-HBV activities. However,
20,30-dideoxynucleosides and their 20,30-unsaturated
counterparts are chemically less stable in acidic media
than their ribosyl forms due to the lack of the electron-
withdrawing inductive effect of the hydroxyl groups. In
particular, the 20,30-dideoxypurine nucleosides are by far
more sensitive to chemical hydrolysis13 than the dideoxy
pyrimidine nucleosides.14 Owing to the high electron
withdrawing power of the CF3 group15 and its potential
stabilizing effect on the lability of a glycosyl-purine
bound, we have examined the chemical stability of the
20,30-dideoxy-30-C-CF3 and 20,30-unsaturated-30-C-CF3
nucleoside derivatives 6 and 8 of adenosine compara-
tively to 20,30-dideoxyadenosine (ddA) and 20,30-
dideoxy-20,30-didehydroadenosine (d4A). The stabilizing
effect of the trifluoromethyl group has been stated in
previous publications5,6 but, until now, it has never been
demonstrated on nucleoside structures. The cleavage of
the glycosidic bond was studied at pH 2 and 7.2 buffer
solutions and 37 ꢀC. Time-dependent degradation of the
nucleosides to the free adenine base was analyzed by
high performance liquid chromatography (HPLC) of
the samples taken at different time intervals. As expec-
ted, ddA and d4A showed chemical instability. This
phenomenon was illustrated by the relative high rates of
decomposition of ddA and d4A at pH 2 witha half-life
(t1/2) of 20 min (Fig. 1, panel A) and less than 1 min
(Fig. 2, panel A), respectively. Additionally, d4A was
also been found to present chemical instability at pH 7.2
(t1/2=2.2 days, Fig. 3, panel A), in accordance to pre-
viously reported data.16 Introduction of the electron
withdrawing CF3 group at the 30-position increased in a
striking way the stability in acidic and neutral media of
the nucleoside analogues, as demonstrated for com-
pounds 6 and 8. In our study, the 20,30-dideoxy-30-C-CF3
nucleoside derivative 6 proved to be over 250-fold more
stable (t1/2=3.7 days, Fig. 1, panel B) at pH 2 than its
corresponding 20,30-dideoxynucleoside (ddA) counterpart.
In a similar manner, the 20,30-unsaturated-30-C-CF3
Scheme 2. (a) Silylated uracil, TMSOTf, 1,2-dichloroethane, rt; (b) (i)
1-methylpyrrolidine, CH3CN, (CF3CO)2O, 0 ꢀC; (ii) 4-nitrophenol,
0 ꢀC; (iii) concd aq NH3/dioxane, 55 ꢀC; (iv) NH3/MeOH, rt; (c)
H2NNH2, AcOH, pyridine, rt; (d) (i) pyridine, C6H5O(S)Cl, CH2Cl2,
rt; (ii) (Me3Si)3SiH, AIBN, toluene, reflux; (e) (i) MsCl, pyridine, rt;
(ii) TBAF, THF, 50 ꢀC.
Figure 1. Relative concentration versus time curves for ddA (^, panel A) and 20,30-dideoxy-30-C-CF3 adenosine (6, ~, panel B) in Glycine/HCl
buffer (pH 2) at 37 ꢀC.

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F. Jeannot et al. / Bioorg. Med. Chem. 10 (2002) 3153–3161
Figure 2. Relative concentration versus time curves for d4A (&, panel A) and 20,30-dideoxy-20,30-didehydro-30-C-CF3 adenosine (8,*, panel B) in
Glycine/HCl buffer (pH 2) at 37 ꢀC.
tested (generally 100 mM, data not shown). The anti-
HBV activity of the synthesized nucleosides was eval-
uated in 2.2.15 cells (HBV DNA-transfected Hep-G2
0
cells) and in suchexperiments 2 ,30-dideoxy-30-thia-b-l-
cytidine (3TC) was included as a reference control. In
our cell culture experiments, 20,30-dideoxy-30-C-CF3-
adenosine 6 and 20,30-dideoxy-20,30-didehydro-30-C-CF3-
cytidine 15 exhibited moderate anti-HBV activity,
although their EC50 (10 mM for 6 and 5 mM for 15)
were higher than 3TC (EC50=0.05 mM). It is note-
worthy that 6 and 15 (as 3TC) did not show cyto-
toxicity (CC50) in Hep-G2 cells up to a concentration
of 200 mM. In the same assays, the others nucleoside
analogues were inactive (data not shown) and not
cytotoxic.
Conclusion
The syntheses of a series of 30-deoxy-30-C-CF3 nucleo-
sides bearing adenine and cytosine as the base were
undertaken to discover new nucleoside derivatives as
potential anti-HIV and anti-HBV drugs. As demon-
strated, we found that introduction of a trifluoromethyl
group at C-30 on acid sensitive purine dideoxy- and
didehydronucleosides was able to confer better stability
toward chemical hydrolysis compared to their non-tri-
fluoromethylated counterparts. When evaluated against
HIV-1 in cell cultures, none of the 30-deoxy-30-C-CF3
nucleosides showed any antiretroviral activity. How-
ever, when evaluated in anti-HBV assays, two com-
pounds 20,30-dideoxy-30-C-CF3 adenosine 6 and 20,30-
dideoxy-20,30-didehydro-30-C-CF3 cytidine 15, showed
moderate antiviral activity without concomitant cyto-
toxicity. Evaluation of the compounds against a broad
range of other viruses, as well as, the synthesis of novel
trifluoromethylated nucleoside derivatives bearing other
purine and pyrimidine bases are currently in progress in
our laboratory.
Figure 3. Relative concentration versus time curves for d4A (&) and
20,30-dideoxy-20,30-didehydro-30-C-CF3 adenosine (8, *) in phosphate
buffer (pH 7.2) at 37 ꢀC.
nucleoside 8 appeared to be over 500-fold more stable
(t1/2=8.9 h, Fig. 2, panel B) in acidic medium than its
20,30-didehydro nucleoside parent (d4A). At pH 7.2 and
in contrast to d4A, compound 8 was completely stable
withno decrease in concentration of 8 or formation of
adenine over a 7-day period (Fig. 3, panel B). Also, no
decomposition of ddA or nucleoside 6 were detected at
pH 7.2 over a 7-day period (data not shown).
The 30-C-CF3 nucleoside derivatives of adenine (3, 6 and
8) and cytosine (10, 13 and 15) were tested for their inhi-
bitory effects on the replication of HIV-1 in MT-4 cells.
None of the compounds showed significant anti-HIV
activity nor cytotoxicity at the highest concentration

F. Jeannot et al. / Bioorg. Med. Chem. 10 (2002) 3153–3161
3157
Experimental
sico chemical properties were similar to those previously
described:5 mp 127 ꢀC (lit.5 126.4–126.6 ꢀC); [a]2D0=ꢁ20
(c 0.95 in DMSO). Anal. calcd for C17H17O7F3: C,
52.31; H, 4.39; F, 14.60. Found: C, 52.15; H, 4.52; F,
14.52.
General methods
Evaporation of solvents was carried out on a rotary
evaporator under reduced pressure. Melting points were
determined in open capillary tubes on a Gallenkamp
MFB-595-010 M apparatus and are uncorrected. UV
spectra were recorded on an Uvikon 931 (Kontron)
9-(2-O-Acetyl-5-O-benzoyl-3-deoxy-3-C-trifluoromethyl-
ꢀ-D-ribofuranosyl)adenine (2). Stannic chloride (1.32
mL, 11.3 mmol) was added cautiously to a stirred sus-
pension of adenine (0.83 g, 6.15 mmol) and 1,2-di-O-
acetyl-5-O-benzoyl-3-deoxy-3-C-trifluoromethyl-d-ribo-
furanose 1 (2 g, 5.1 mmol) in dry acetonitrile (46 mL) at
room temperature. After 14 h, pyridine (9 mL) was
added to the resultant solution. The white precipitate
was filtered and washed with chloroform (2 ꢂ 50 mL).
The combined filtrates were washed with a solution of
saturated sodium hydrogen carbonate (2 ꢂ 50 mL),
water (2 ꢂ 50 mL), dried over sodium sulfate and eva-
porated. Silica gel column chromatography of the resi-
due using a stepwise gradient of methanol (0–5%) in
dichloromethane afforded the title compound 2 as a
white foam (1.7 g, 71%): [a]2D0=ꢁ34 (c 1.06 in DMSO);
UV lmax (EtOH)/nm 260 nm (e 14,500), 232 nm (e
15,600), lmin (EtOH)/nm 245 (e 11,100); 1H NMR
(DMSO-d6) d 2.10 (3H, s, CH3CO), 4.41–4.46 (2H, m,
1
spectrophotometer. H NMR spectra were recorded at
400 MHz, 13C NMR spectra at 100 MHz and 19F NMR
at 235 MHz in (CD3)2SO at ambient temperature witha
Bruker DRX 400. Chemical shifts (d) are quoted in
parts per million (ppm) referenced to the residual sol-
vent peak, [CD3)CD2H)SO] being set at d-H 2.49 and
d-C 39.5 relative to tetramethylsilane (TMS).19F chemi-
cal shits are reported using trichlorofluoromethane as
external reference. Deuterium exchange and COSY
experiments were performed in order to confirm proton
assignments. Coupling constants, J, are reported in
Hertz. 2D H–13C heteronuclear COSY were recorded
1
for the attribution of 13C signals. FAB mass spectra
were recorded in the positive-ion or negative-ion mode
on a JEOL SX 102. The matrix was a mixture (50:50, v/
v) of glycerol and thioglycerol (G-T). Specific rotations
were measured on a Perkin-Elmer Model 241 spectro-
polarimeter (pathlength1 cm), and are given in units of
H-50 and H-30), 4.68 (1H, dd, H-500, J5 ,4 =2.6,
00
0
ꢀ
0
0
10ꢁ1 cm2 gꢁ1. Elemental analyses were carried out by
J5 ,5 =12.6), 4.80 (1H, m, H-4 ), 6.24 (1H, d, H-1 ,
00
0
0
0
0
0
0
the Service de Microanalyses du CNRS, Division de
Vernaison (France). Thin-layer chromatography was
performed on precoated aluminium sheets of Silica Gel
60 F254 (Merck, Art. 5554), visualization of products
being accomplished by UV absorbency followed by
charring with 5% ethanolic sulfuric acid and heating.
Column chromatography was carried out on Silica
Gel 60 (Merck, Art. 9385). Analytical HPLC studies
were carried out on a Waters Assoc. unit (600E
multisolvent delivery system, 600E system gradient
controller, 717 autosampler injector, 996 photodiode
array detector and a Millenium data workstation)
using a reverse-phase analytical column (Nucleosil,
C18, 150 ꢂ 4.6 mm, 5 mm) equipped witha prefilter, a
precolumn (Nucleosil, C18, 5 mm) and a photodiode
array detector (detection at 260 nm). The compound to
be analyzed was eluted using a linear gradient of 0–80%
acetonitrile in 20 mM triethylammonium acetate buffer
(TEAC, pH 7) programmed over a 40-min period with
a flow rate of 1 mL/min. All moisture-sensitive reac-
tions were carried out under rigorous anhydrous con-
ditions under an argon atmosphere using oven-dried
glassware. Solvents were dried and distilled prior to use
and solids were dried over P2O5 under reduced pres-
sure.
J1 ,2 =3.0), 6.28 (1H, dd, H-2 , J2 ,3 =6.7), 7.39 (2H, br
s, NH2), 7.45–7.86 (5H, m, C6H5CO), 8.08 (1H, s, H-2),
8.29 (1H, s, H-8); 13C NMR (DMSO-d6) d 21.3
(CH3CO), 45.2 (C-30, q, JC,F=27.4), 64.1 (C-50), 73.9
2
(C-20), 76.8 (C-40), 88.4 (C-10), 119.9 (C-5), 126.0 (q,
1
CF3, JC,F=277.0), 129.6–134.4 (C-Arom), 141.0 (C-8),
149.6 (C-4), 153.8 (C-2), 157.1 (C-6), 166.1 (CO), 170.1
(CO); 19F NMR (DMSO-d6) dꢁ62.0 (d, CF3,
JF,H=9.4) ; m/z (FAB>0) 466 (M+H)+, 331 (S)+, 136
(BH2)+, 105 (C6H5CO)+; m/z (FAB<0) 464 (MꢁH)ꢁ,
134 (B)ꢁ, 121 (C6H5CO2)ꢁ. Anal. calcd for
C20H18F3N5O5: C, 51.62; H, 3.90; N, 15.05; F, 12.25.
Found: C, 51.42; H, 3.82; N, 14.85; F, 12.01.
9-(3-Deoxy-3-C-trifluoromethyl-ꢀ-D-ribofuranosyl)ade-
nine (3). A solution of 2 (0.350 g, 0.75 mmol) in metha-
nolic ammonia (previously saturated at ꢁ10 ꢀC and
tightly stoppered) (20 mL) was stirred for 14 h at room
temperature, then evaporated to dryness. The residue
was subjected to silica gel column chromatography with
a stepwise gradient of methanol (0–10%) in dichloro-
methane to afford the title compound 3 (0.235 g, 97%)
which was crystallized from methanol: mp 256 ꢀC;
[a]2D0=ꢁ42 (c 1.01 in DMSO); UV lmax (EtOH)/nm 260
(e 12,600); 1H NMR (DMSO-d6) d 3.41–3.54 (2H, m, H-
50 and H-30), 3.74–3.78 (1H, m, H-500), 4.40 (1H, m, H-
1,2-Di-O-acetyl-5-O-benzoyl-3-deoxy-3-C-trifluorome-
thyl-D-ribofuranose (1). Compound (1) was prepared
from commercially available diacetone-d-glucose fol-
lowing a modified procedure initially developed by
Lavaire et al.6 for the synthesis of 1,2,5-tri-O-acetyl-3-
deoxy-3-C-trifluoromethyl-d-ribofuranose. During the
course of our studies, the synthesis of 1 has been descri-
bed witha different strategy starting from d-xylose.5 An
analytical sample of 1 was obtained after crystallization
from petroleum ether providing the b-anomer. Its phy-
40), 4.97 (1H, m, H-20), 5.49 (1H, t, OH-50, J=4.7), 5.91
0
(1H, d, H-10, J1 ,2 =4.3), 6.27 (1H, d, OH-2 , J=5.7)
7.35 (2H, br s, NH2), 8.14 (1H, s, H-2), 8.38 (1H, s,
0
0
H-8); 13C NMR (DMSO-d6)
d
45.9 (q, C-30,
2JC,F=25.2), 62.6 (C-50), 73.5 (C-20), 79.3 (C-40), 90.4
(C-10), 120.0 (C-5), 126.8 (q, CF3, JC,F=278.7),
1
140.4 (C-8), 149.7 (C-4), 153.4 (C-2), 157.0 (C-6); 19F
NMR (DMSO-d6) dꢁ61.8 (d, CF3, JF,H=10.1); m/z
(FAB>0) 320 (M+H)+, 136 (BH2)+ . Anal. calcd for
ꢃ
C11H12F3N5O3 0.5 CH3OH: C, 41.20; H, 4.21; N,

3158
F. Jeannot et al. / Bioorg. Med. Chem. 10 (2002) 3153–3161
20.89; F, 17.00. Found: C, 41.05, H, 3.80; N, 21.24; F,
16.98.
6.36 (1H, dd, H-10), 7.30 (2H, br s, NH2), 7.46–7.88 (5H,
m, C6H5CO), 8.11 (1H, s, H-2), 8.32 (1H, s, H-8); 13C
NMR (DMSO-d6)
2JC,F=27.6), 65.4 (C-50), 77.5 (C-40), 84.6 (C-10), 120.1
d
31.4 (C-20), 43.9 (q, C-30,
9-(5-O-Benzoyl-3-deoxy-3-C-trifluoromethyl-ꢀ-D-ribofur-
anosyl)adenine (4). Hydrazine hydrate (0.51 mL, 10.5
mmol) was added to a stirred solution of 2 (1.63 g, 3.5
mmol) in an acetic acid–pyridine (v/v, 1:4, 33.3 mL)
mixture. After 60 h, acetone (10 mL) was added and the
solution was stirred at room temperature for 30 min. A
solution of saturated sodium hydrogen carbonate (50
mL) was then added, and the aqueous phase was
extracted with dichloromethane (2 ꢂ 50 mL). The
organic phase was washed with water (2 ꢂ 50 mL),
dried over sodium sulfate and evaporated to dryness.
Silica gel column chromatography of the residue using a
stepwise gradient of methanol (0–6%) in dichloro-
methane afforded the title compound 4 as a white foam
(1.13 g, 76%) which was crystallized from acetonitrile:
mp 190 ꢀC; [a]2D0=ꢁ39 (c 1.27 in DMSO); UV lmax
(EtOH)/nm 260 nm (e 14,000), 232 nm (e 14700), lmin
(EtOH)/nm 245 (e 10,500); 1H NMR (DMSO-d6) d 3.98
1
(C-5), 127.7 (q, CF3, JC,F=277.4), 129.0–134.3 (C-
Arom), 140.7 (C-8), 149.7 (C-4), 153.5 (C-2), 157.0 (C-
6), 166.2 (CO); 19F NMR (DMSO-d6) dꢁ67.9 (d, CF3,
JF,H=9.4); m/z (FAB>0) 408 (M+H)+, 136 (BH2)+,
105 (C6H5CO)+; m/z (FAB<0) 406 (M- H)ꢁ, 134 (B)ꢁ.
ꢃ
Anal. calcd for C18H16F3N5O3 0.9 CH3OH: C, 52.04;
H, 4.53; N, 16.06, F, 13.07. Found: C, 52.21; H, 4.17; N,
16.39; F, 12.71.
9-(2,3-Dideoxy-3-C-trifluoromethyl-ꢀ-D-erythro-pento-
furanosyl)adenine (6). A solution of 5 (0.250 g, 0.61
mmol) in methanolic ammonia (previously saturated at
ꢁ10 ꢀC and tightly stoppered) (15 mL) was stirred for
14 hat room temperature, then evaporated to dryness.
The residue was subjected to silica gel column chroma-
tography, with a stepwise gradient of methanol (0–10%)
in dichloromethane to afford the title compound 6
(0.155 g, 83%) which was crystallized from methanol/
water: mp 218 ꢀC; [a]2D0=ꢁ29 (c 1.04 in DMSO); UV
(1H, m, H-30), 4.42 (1H, 00 dd, H-50, J5 ,4 =4.7,
0
0
0
00
00
0
J5 ,5 =12.4), 4.63 (1H, dd, H-5 , J5 ,4 =2.8), 4.73 (1H,
m, H-40), 5.21 (1H, m, H-20), 5.98 (1H, d, H-10,
lmax (EtOH)/nm 260 (e 15,400) ; H NMR (DMSO-d6)
1
0
J1 ,2 =4.3), 6.40 (1H, d, 2 -OH, J=5.8), 7.33 (2H, br s,
NH2), 7.46–7.89 (5H, m, C6H5CO), 8.09 (1H, s, H-2),
8.31 (1H, s, H-8); 13C NMR (DMSO-d6) d 46.8 (q, C-30,
2JC,F=25.9), 64.8 (C-50), 73.2 (C-20), 76.5 (C-40), 91.0
d 2.66 (1H, m, H-20), 2.95 (1H, m, H-200), 3.45–3.74 (3H,
m, H-30, H-50 and H-500), 4.22 (1H, m, H-40), 5.35 (1H, t,
OH-50, J=5.3), 6.30 (1H, t, H-10, J=6.5), 7.35 (1H, br s,
NH2), 8.14 (1H, s, H-2), 8.36 (1H, s, H-8) ; 13C NMR
0
0
0
(C-1 ), 120.0 (C-5), 126.4 (q, CF3, JC,F=278.3), 129.6–
134.4 (C-Arom), 140.9 (C-8), 149.8 (C-4), 153.6 (C-2),
157.0 (C-6), 166.2 (CO); 19F NMR (DMSO-d6) dꢁ61.2
(d, CF3, JF,H=9.7) ; m/z (FAB>0) 424 (M+H)+, 136
(BH2)+, 105 (C6H5CO)+; m/z (FAB<0) 422 (M- H)ꢁ,
134 (B)ꢁ, 121 (C6H5CO2)ꢁ. Anal. calcd for
C18H16F3N5O4: C, 51.07; H, 3.81; N, 16.54; F, 13.46.
Found: C, 51.20; H, 3.71; N, 16.28; F, 13.30.
(DMSO-d6) d 31.9 (C-20), 43.6 (q, C-30, JC,F=27.3),
1
2
63.1 (C-50), 80.9 (C-40), 84.7 (C-10), 120.1 (C-5), 128.1 (q,
1
CF3, JC,F=277.8), 140.4 (C-8), 149.7 (C-4), 153.4 (C-
2), 157.0 (C-6); 19F NMR (DMSO-d6) dꢁ68.3 (d, CF3,
JF,H=9.9); m/z (FAB>0) 607 (2M+H)+, 304
(M+H)+, 136 (BH2)+; m/z (FAB<0) 605 (2M- H)ꢁ,
302 (MꢁH)ꢁ, 134 (B)ꢁ; HPLC tR 21.9 min. Anal. calcd
ꢃ
for C11H12F3N5O2 0.4H2O: C, 42.56; H, 4.16; N, 22.56;
F, 18.36. Found: C, 42.76; H, 4.09; N, 22.69; F, 18.36.
9-(5-O-Benzoyl-2,3-dideoxy-3-C-trifluoromethyl-ꢀ-D-ery-
thro-pentofuranosyl)adenine (5). To a stirred solution of
4 (0.450 g, 1.06 mmol) in dry acetonitrile (15 mL) were
added successively DMAP (1.16 g, 9.57 mmol) and
phenoxy(thiocarbonyl) chloride (0.30 mL, 2.12 mmol).
After 14 h, the solvent was removed under reduced
pressure. The residue was dissolved in dichloromethane
(50 mL) and the organic layer was washed with hydro-
chloric acid 0.2 N (3 ꢂ 50 mL), dried over sodium sul-
fate, and evaporated to dryness. The resulting crude
material was co-evaporated withdry toluene, then dis-
solved in the same solvent (21 mL) and a,a0-azoisobu-
9-(5-O-Benzoyl-2,3-dideoxy-3-C-trifluoromethyl-ꢀ-D-gly-
cero-pent-2-eno-furanosyl)adenine (7). Methanesulfonyl
chloride (0.173 mL, 2.23 mmol) was added to a solution
of nucleoside 4 (0.315 g, 0.75 mmol) in dry pyridine (7.5
mL). The resultant solution was stirred at room tem-
perature for 7 h, and a solution of saturated sodium
hydrogen carbonate (15 mL) was then added. The mix-
ture was extracted with dichloromethane (2 ꢂ 15 mL).
The organic phase was dried over sodium sulfate, eva-
porated under reduced pressure and co-evaporated with
toluene. The crude material was then dissolved in
anhydrous THF (15 mL), and a solution of TBAF in
THF (1 M) (1.5 mL, 1.5 mmol) was added. The mixture
was stirred for 21 hat 50 ꢀC, then evaporated to dry-
ness. Chloroform (30 mL) and water (30 mL) were
added. The organic phase was separated, dried and
evaporated to dryness under reduced pressure. The
residue was subjected to silica gel column chromato-
graphy, with a stepwise gradient of methanol (0–3%) in
dichloromethane to afford the title compound 7 (0.242
g, 80%) as a white foam: [a]2D0=ꢁ45 (c 1.00 in DMSO);
UV lmax (EtOH)/nm 260 (e 15,000), 231 (e 16,100), lmin
(EtOH)/nm 245 (e 11,400) ; 1H NMR (DMSO-d6) d 4.48
tyronitrile
(0.052
g,
0.31
mmol)
and
tris(trimethylsilyl)silane (0.4 mL, 1.27 mmol) were
added. The resultant solution was heated under reflux
for 4 h. After cooling to room temperature, the solvent
was removed under reduced pressure. Chromatography
of the residue on a silica gel column using as eluent a
stepwise gradient of methanol (0–5%) in dichloro-
methane afforded the title compound 5 as a white foam
(0.3 g, 70%): [a]D20=ꢁ31 (c 0.95 in DMSO); UV lmax
(EtOH)/nm 260 nm (e 15,100), 232 nm (e 16,000), lmin
(EtOH)/nm 245 (e 11,400); 1H NMR (DMSO-d6) d 2.75
(1H, ddd, H-20a, J2 ,3 =7.3, J2 ,1 =7.4, J2 a,2 b=14.0),
0
0
0
0
0
0
3.13 (ddd, H-20b, J2 ,1 =4.8, J2 ,3 =9.2), 3.99 (1H, m, H-
(1H, dd, H-50, J5 ,4 =4.2, J5 ,5 =12.4), 4.63 (1H, dd,
0
0
0
0
0
0
0
00
30), 4.40 (1H, m, H-50), 4.54 (2H, m, H-40 and H-500),
H-500, J5 ,4 =2.8), 5.49 (1H, m, H-4 ), 7.12 (1H, br s,
0
00
0

F. Jeannot et al. / Bioorg. Med. Chem. 10 (2002) 3153–3161
3159
H-10), 7.23 (1H, br s, H-20), 7.31 (2H, NH2), 7.48–7.86
(5H, m, C6H5CO), 8.09 (1H, s, H-8), 8.14 (1H, s, H-2);
13C NMR (DMSO-d6) d 64.9 (C-50), 82.4 (C-40), 87.9
viously reported. Anal. calcd for C19H17F3N2O7: C,
51.59; H, 3.87; N, 6.33; F, 12.88. Found: C, 51.67; H,
3.94; N, 6.39; F, 12.92.
(C-10), 119.5 (C-5), 121.9 (q, CF3, JC,F=270.0), 129.7–
1
129.9 (C-Arom), 133.7 (q, C-30, JC,F=34.6), 134.2 (q,
1-(3-Deoxy-3-C-trifluoromethyl-ꢀ-D-ribofuranosyl)cyto-
sine (10). A solution of compound 9 (0.5 g, 1.13 mmol)
and 1-methylpyrrolidine (1.12 mL, 10.85 mmol) in
anhydrous acetonitrile (5.6 mL) was cooled down to
0 ꢀC. Trifluoroacetic anhydride (0.4 mL, 2.82 mmol)
was then added. After 45 min at 0 ꢀC, 4-nitrophenol
(0.47 g, 3.39 mmol) was added to the solution. After
stirring for 12 hat 0 ꢀC, the solution was then poured
into a solution of saturated sodium hydrogen carbonate
(30 mL), and the resultant mixture was extracted with
dichloromethane (3 ꢂ 20 mL). The combined organic
layers were dried over sodium sulfate and evaporated
under reduced pressure. The residue was dissolved in
dioxane (5.6 mL) and concentrated aqueous ammonia
(d 0.89, 1.13 mL) was added. The reaction mixture was
stirred at 55 ꢀC for 3 h. The resulting yellow solution
was concentrated under reduced pressure and directly
treated withmethanolic ammonia (previously saturated
at ꢁ10 ꢀC and tightly stoppered) (40 mL) for 14 h at
room temperature. After evaporation to dryness under
reduced pressure, the residue was subjected to silica gel
column chromatography, with a stepwise gradient of
methanol (2–10%) in dichloromethane to afford the title
compound 10 (0.240 g, 72%) which was crystallized
from acetonitrile: mp 177 ꢀC ; [a]D20=+ 8.6 (c 1.05 in
DMSO); UV lmax (EtOH)/nm 272 (e 10,100) ; 1H NMR
(DMSO-d6) d 3.07 (1H, m, H-30), 3.50 (1H, m, H-50),
3.78 (1H, m, H-500), 4.33 (1H, m, H-40), 4.41 (1H, m,
H-20), 5.28 (1H, t, OH-50, J=5.0), 5.68 (2H, m, H-10 and
H-5), 6.14 (1H, d, OH-20, J=5.6), 7.15 (2H, br d, NH2),
2
C-20, JC,F=4.8), 134.5 (C-Arom), 139.6 (C-8), 150.0
3
(C-4), 153.9 (C-2), 156.9 (C-6), 166.2 (CO) ; 19F NMR
(DMSO-d6) dꢁ61.4 (s, CF3); m/z (FAB>0) 406
(M+H)+, 136 (BH2)+, 105 (C6H5CO)+; m/z (FAB<0)
404 (MꢁH)ꢁ, 134 (B)ꢁ, 121 (C6H5CO2)ꢁ. Anal. calcd
ꢃ
for C18H14F3N5O3 0.7 CH3OH: C, 52.51; H, 3.96; N,
16.37; F, 13.32. Found: C, 52.25, H, 3.64; N, 16.33; F,
13.23.
9-(2,3-Dideoxy-3-C-trifluoromethyl-ꢀ-D-glycero-pent-2-
enofuranosyl)adenine (8). A solution of 7 (0.210 g, 0.51
mmol) in methanolic ammonia (previously saturated at
ꢁ10 ꢀC and tightly stoppered) (13 mL) was stirred for
14 hat room temperature, then evaporated to dryness.
The residue was subjected to silica gel column chroma-
tography, with a stepwise gradient of methanol (0–10%)
in dichloromethane to afford the title compound 8
(0.110 g, 70%) which was crystallized from methanol/
water: mp 229 ꢀC; [a]D20=+ 63.3 (c 1.01 in DMSO); UV
l
max (EtOH)/nm 260 (e 14,200); 1H NMR (DMSO-d6) d
3.61–3.69 (2H, m, H-50 and H-500), 5.12 (1H, m, H-40),
5.36 (1H, t, OH-50, J=5.0), 6.99 (1H, d, H-20,
0
0
0
J2 ,1 =1.7), 7.06 (1H, br s, H-1 ), 7.31 (1H, br s, NH2),
8.14 (1H, s, H-2), 8.20 (1H, s, H-8); 13C NMR (DMSO-
d6) d 62.1 (C-50), 86.1 (C-40), 87.5 (C-10), 117.9 (C-5),
121.9 (q, CF3, 1JC,F=277.0), 133.0 (q, C-20, 3JC,F=4.7),
135.1 (q, C-30, JC,F=34.6), 140.2 (C-8), 149.8 (C-4),
2
153.6 (C-2), 156.8 (C-6); 19F NMR (DMSO-d6) dꢁ61.4
(s, CF3); m/z (FAB>0) 603 (2M+H)+, 302 (M+H)+,
136 (BH2)+; m/z (FAB<0) 601 (2MꢁH)ꢁ, 300
(MꢁH)ꢁ, 134 (B)ꢁ; HPLC tR 21.2 min. Anal. calcd for
7.88 (1H, d, H-6, J6,5=7.4); 13C NMR (DMSO-d6) d
0
43.2 (C-3 , q, JC,F=25.5), 59.5 (C-50), 72.4 (C-20), 77.6
2
C11H10F3N5O2 0.8H2O: C, 41.86; H, 3.70; N, 22.19; F,
(C-40), 90.2 (C-10), 92.6 (C-5), 123.2 (q, CF3,
1JC,F=277.9), 140.1 (C-6), 154.1 (C-2), 164.6 (C-4); 19F
NMR (DMSO-d6) dꢁ61.1 (d, CF3, JF,H=9.7) ; m/z
(FAB>0) 296 (M+H)+, 112 (BH2)+; m/z (FAB<0)
ꢃ
18.06. Found: C, 42.25; H, 3.44; N, 21.73; F, 17.95.
1-(2-O-Acetyl-5-O-benzoyl-3-deoxy-3-C-trifluoromethyl-
ꢀ-D-ribofuranosyl)uracil (9). A mixture of uracil (1.14
g, 10.2 mmol), hexamethyldisilazane (51 mL) and a
catalytic amount of ammonium sulfate was refluxed for
14 h. The resultant clear solution was concentrated to
dryness under reduced pressure. TMSOTf (1.98 mL,
10.2 mmol) was added to a solution of sugar 1 (1 g, 2.56
mmol) and silylated base in dry 1,2-dichloroethane (25
mL). The reaction mixture was stirred for 8 h at room
temperature, poured into a solution of saturated sodium
hydrogen carbonate and extracted with chloroform (3 ꢂ
30 mL). The organic phase was dried over sodium sul-
fate and evaporated to dryness. Chromatography of the
residue on a silica gel column using as eluent a stepwise
gradient of ethyl acetate (0–60%) in petroleum ether
afforded the title compound 9 as a white foam (0.710 g,
63%) which was crystallized from ethanol: mp 126–
127 ꢀC (lit.5 116–16.9 ꢀC); [a]2D0=ꢁ8.7 (c 1.15 in
DMSO); UV lmax (EtOH)/nm 260 (e 10,800), 231 (e
16600), lmin (EtOH)/nm 247 (e 10,000) ; m/z (FAB>0)
885 (2M+H)+, 443 (M+H)+, 331 (S)+, 113 (BH2)+,
105 (C6H5CO)+; m/z (FAB<0) 883 (2MꢁH)ꢁ, 441
294 (MꢁH)ꢁ. Anal. calcd for C10H12F3N3O4 0.3
ꢃ
CH3CN: C, 41.16; H, 4.27; N, 14.94. Found: C, 40.90;
H, 4.37; N, 15.10.
1-(5-O-Benzoyl-3-deoxy-3-C-trifluoromethyl-ꢀ-D-ribofur-
anosyl)uracil (11). Hydrazine hydrate (0.16 mL, 3.29
mmol) was added to a stirred solution of 9 (0.48 g, 1.09
mmol) in a acetic acid–pyridine (v/v 1:4, 10.4 mL). After
3 days, acetone (5 mL) was added and the solution was
stirred at room temperature for 30 min. A solution of
saturated sodium hydrogen carbonate (30 mL) was then
added, and the aqueous phase was extracted with di-
chloromethane (2 ꢂ 30 mL). The organic phase was
washed with water (2 ꢂ 30 mL), dried over sodium sul-
fate and evaporated to dryness. Column chroma-
tography of the residue on silica gel using a stepwise
gradient of methanol (0–2%) in dichloromethane affor-
ded the title compound 11 as a white foam (0.42 g, 96%)
which was crystallized from diethyl ether: mp 182 ꢀC
(lit.5 177–178.5 ꢀC); [a]2D0=ꢁ17.9 (c 1.17 in DMSO); UV
lmax (EtOH)/nm 260 (e 10,700), 231 (e 15,900), lmin
(EtOH)/nm 247 (e 8700) ; m/z (FAB>0) 401 (M+H)+,
289 (S)+, 105 (C6H5CO)+; m/z (FAB<0) 399 (MꢁH)ꢁ,
1
(MꢁH)ꢁ, 121 (C6H5CO2)ꢁ, 111 (B)ꢁ. H, 13C and 19F
NMR spectra (DMSO-d6) were similar to those pre-

3160
F. Jeannot et al. / Bioorg. Med. Chem. 10 (2002) 3153–3161
121 (C6H5CO2)ꢁ, 111 (B)ꢁ. 1H, 13C and 19F NMR
spectra (DMSO-d6) were similar to those previously
reported. Anal. calcd for C17H15F3N2O6: C, 51.01; H,
3.78; N, 7.00; F, 14.24. Found: C, 50.86; H, 3.79, N,
7.12; F, 14.40.
lmax (EtOH)/nm 272 (e 8500); m/z (FAB>0) 280
1
(M+H)+, 112 (BH2)+. H, 13C and 19F NMR spectra
(DMSO-d6) were similar to those previously reported.
HRMS (FAB>0, m/z ) 280.0919 (calcd 280.0909).
1-(5-O-Benzoyl-2,3-dideoxy-3-C-trifluoromethyl-ꢀ-D-gly-
cero-pent-2-eno-furanosyl)uracil (14). Methanesulfonyl
chloride (0.3 mL, 3.75 mmol) was added to a solution of
nucleoside 11 (0.5 g, 1.25 mmol) in a mixture of dry
pyridine (7.5 mL) and dichloromethane (9.4 mL). The
resultant solution was stirred at room temperature for
48 h, and a solution of saturated sodium hydrogen car-
bonate (15 mL) was then added. The mixture was
extracted with dichloromethane (2 ꢂ 15 mL). The
organic phase was dried over sodium sulfate, evapo-
rated under reduced pressure and co-evaporated with
toluene. The crude material was then dissolved in
anhydrous THF (25 mL), and a solution of TBAF in
THF (1.25 mL, 1.25 mmol) was added. The mixture was
stirred for 4 hat 50 ꢀC, then evaporated to dryness.
Ethyl acetate (30 mL) and water (30 mL) were added.
The organic phase was separated, dried over sodium
sulfate and evaporated to dryness under reduced pres-
sure. The residue was subjected to silica gel column
1-(5-O-Benzoyl-2,3-dideoxy-3-C-trifluoromethyl-ꢀ-D-ery-
thro-pentofuranosyl)uracil (12). To a stirred solution of
11 (0.5 g, 1.25 mmol) in dry dichloromethane (7.5 mL)
were added pyridine (0.38 mL, 4.75 mmol) and phe-
noxy(thiocarbonyl) chloride (0.19 mL, 1.37 mmol) suc-
cessively. After 4 h, the solvent was removed under
reduced pressure. The residue was dissolved in di-
chloromethane (50 mL) and the organic layer was
washed with water (2 ꢂ 50 mL), dried over sodium sul-
fate and evaporated to dryness. The resulting crude
material was co-evaporated withdry toluene, then dis-
solved in the same solvent (25 mL) and a,a0-azoiso-
butyronitrile
(0.102
g,
0.62
mmol)
and
tris(trimethylsilyl)silane (0.77 mL, 2.5 mmol) were
added. The resultant solution was stirred under reflux
for 17 h. After cooling to room temperature, the solvent
was removed under reduced pressure. The residue was
purified on a silica gel column using methanol/di-
chloromethane (1:99) as eluent to afford the title com-
pound 12 (0.384 g, 80%) as a white foam which was
crystallized from ethanol/water: mp 140 ꢀC; (lit.5 80.0–
82.0 ꢀC); [a]2D0=ꢁ15.3 (c 0.98 in DMSO); UV lmax
(EtOH)/nm 260 (e 10,000), 231 (e 15,000), lmin (EtOH)/
nm 247 (e 8400); m/z (FAB>0) 385 (M+H)+, 273
(S)+, 113 (BH2)+, 105 (C6H5CO)+; m/z (FAB<0) 383
chromatography
using
methanol/dichloromethane
(1:99) as eluent to afford the title compound 14 (0.383 g,
80%) as a white foam which was crystallized from di-
ethyl ether: mp 153 ꢀC; [a]2D0=ꢁ100 (c 1.05 in DMSO);
UV lmax (EtOH)/nm 260 (e 10,100), 230 (e 16,400), lmin
1
(EtOH)/nm 247 (e 8800); H NMR (DMSO-d6) d 4.42
(1H, dd, H-50, J5 ,4 =3.5, J5 ,5 =12.8), 4.70 (1H, dd,
0
0
0
00
(MꢁH)ꢁ, 121 (C6H5CO2)ꢁ, 111 (B)ꢁ. H, 13C and 19F
H-500, J5 ,4 =2.7), 5.08 (1H, d, H-5, J5,6=8.0), 5.39 (1H,
1
00
0
0
0
NMR spectra (DMSO-d6) were similar to those pre-
viously reported. Anal. calcd for C17H15F3N2O5
br s, H-40), 6.92 (1H, d, H-10, J1 ,2 =1.6), 6.99 (1H, br s,
H-20), 7.39 (1H, d, H-6), 7.54–7.91 (5H, m, C6H5CO),
11.45 (1H, s, NH); 13C NMR (DMSO-d6) d 64.5 (C-50),
81.8 (C-40), 89.4 (C-10), 102.8 (C-5), 121.7 (q, CF3,
1JC,F=270.2), 129.8–130.0 (C-arom), 133.7 (q, C-30,
2JC,F=34.8), 134.4 (q, C-20, 3JC,F=4.7), 134.7
(C-Arom), 141.4 (C-6), 151.1 (C-2), 163.6 (C-4), 166.1
(CO); 19F NMR (DMSO-d6) dꢁ61.5 (s, CF3); m/z
(FAB>0) 765 (2M+H)+, 475 (M+G+H)+, 383
(M+H)+, 113 (BH2)+, 105 (C6H5CO)+; m/z (FAB<0)
473(M+GꢁH)ꢁ, 381 (MꢁH)ꢁ, 121 (C6H5CO2)ꢁ, 111
(B)ꢁ. Anal. calcd for C17H13F3N2O5: C, 53.41; H, 3.43;
N, 7.33; F, 14.91. Found: C, 53.58; H, 3.56; N, 7.43; F,
14.47.
ꢃ
0.9H2O: C, 50.98; H, 4.23; N, 6.99. Found: C, 51.08; H,
3.82; N, 7.23.
1-(2,3-Dideoxy-3-C-trifluoromethyl-ꢀ-D-erythro-pento-
furanosyl)cytosine (13). A solution of compound 12
(0.300 g, 0.781 mmol) and 1-methylpyrrolidine (0.780
mL, 7.5 mmol) in anhydrous acetonitrile (3.9 mL) was
cooled to 0 ꢀC. Trifluoroacetic anhydride (0.275 mL,
1.95 mmol) was then added. After 45 min at 0 ꢀC,
4-nitrophenol (0.326 g, 2.34 mmol) was added. The
solution was stirred for 18 hat 0 ꢀC, then poured into a
solution of saturated sodium hydrogen carbonate (30
mL). The resultant mixture was extracted with di-
chloromethane (3 ꢂ 20 mL). The combined organic
layers were dried over sodium sulfate and evaporated
under reduced pressure. The residue was dissolved in
dioxane (3.9 mL) and concentrated aqueous ammonia
(d 0.89, 0.781 mL) was added. The reaction mixture was
stirred at 55 ꢀC for 12 h. The resulting yellow solution
was concentrated under reduced pressure and directly
treated withmethanolic ammonia (previously saturated
at ꢁ10 ꢀC and tightly stoppered) (40 mL) for 12 h at
room temperature. After evaporation to dryness under
reduced pressure, the residue was subjected to silica gel
column chromatography, with a stepwise gradient of
methanol (4–10%) in dichloromethane to afford the title
compound 13 (0.170 g, 78%). An analytical sample of
13 was obtained as its chlorohydrate salt: mp 215 ꢀC
(lit.5 214.8–216 ꢀC); [a]2D0=+ 53.3 (c 0.15 DMSO); UV
1-(2,3-Dideoxy-3-C-trifluoromethyl-ꢀ-D-glycero-pent-2-
enofuranosyl)cytosine (15). A solution of compound 14
(0.333 g, 0.871 mmol) and 1-methylpyrrolidine (0.870
mL, 8.36 mmol) in anhydrous acetonitrile (4.4 mL) was
cooled down to 0 ꢀC. Trifluoroacetic anhydride (0.307
mL, 2.17 mmol) was then added. After 45 min at 0 ꢀC,
4-nitrophenol (0.363 g, 2.61 mmol) was added. The
solution was stirred for 5 hat 0 ꢀC, then poured into a
solution of saturated sodium hydrogen carbonate (30
mL), and the resultant mixture was extracted with di-
chloromethane (3 ꢂ 20 mL). The combined organic
layers were dried over sodium sulfate and evaporated
under reduced pressure. The residue was dissolved in
dioxane (4.4 mL) and concentrated aqueous ammonia
(d 0.89, 0.871 mL) was added. The reaction mixture was
stirred at 55 ꢀC for 12 h. The resulting yellow solution

F. Jeannot et al. / Bioorg. Med. Chem. 10 (2002) 3153–3161
3161
was concentrated under reduced pressure and directly
treated withmethanolic ammonia (previously saturated
at ꢁ10 ꢀC and tightly stoppered) (40 mL) for 12 h at
room temperature. After evaporation to dryness under
reduced pressure, the residue was subjected to silica gel
column chromatography, with a stepwise gradient of
methanol (4–10%) in dichloromethane to afford the title
compound 13 (0.200 g, 82%) which was crystallized
from methanol/water: mp 221 ꢀC (lit.5 221.3–221.7 ꢀC);
[a]2D0=+ 23.4 (c 1.07 in DMSO); UV lmax (EtOH)/nm
272 (e 9000) ; m/z (FAB>0) 370 (M+G+H)+, 278
using the colorimetric MTT test. 2.2.15 Cells (HBV
DNA-transfected human hepatoblastoma-derived Hep-
G2 cells) were used for anti-HBV assays. Briefly, cells
were cultured and inhibition of HBV extracellular DNA
(HBV virion) or HBV intracellular DNA (HBV repli-
cative intermediate, HBV RI) was determined. Cyto-
toxicity assays were conducted in Hep-G2 cells. Each
compound was tested in four concentration in triplicate
cultures and the median inhibitory concentration (IC50)
was determined.
1
(M+H)+; m/z (FAB ) 276 (MꢁH)ꢁ. H, 13C and 19F
NMR spectra (DMSO-d6) were similar to those pre-
viously reported. HRMS (FAB>0, m/z) 278.0750 (calcd
278.0753).
Acknowledgements
Frederic Jeannot is particularly grateful to the Ministere
de l’Education Nationale, de la Recherche et de la
Technologie, France, for a doctoral fellowship.
Stability studies
For determination of the chemical stability of nucleo-
sides, pH 7.2 phosphate buffer (0.02 M) and pH 2 Gly-
cine/HCl buffer were used. For eachcompound, a stock
solution (1ꢂ10ꢁ3 M in Milli-Q water) was prepared.
The study at pH 7.2 buffer was performed in the fol-
lowing manner: 0.1 mL of stock solution was added to a
vial and diluted withpH 7.2 buffer to give a final con-
centration of 5ꢂ10ꢁ5 M; a 0.1 mL aliquot was immedi-
ately removed for analysis and the remainder was
incubated at 37 ꢀC. Subsequent 0.1 mL samples were
taken at predetermined times, then frozen in liquid
nitrogen. The concentration of adenine and remaining
nucleoside in eachsample was determined by HPLC
analysis of an 80-mL aliquot. The study at pH 2 buffer
was accomplished in the following way: 0.25 mL of
stock solution was added to a vial, diluted withpH 2
buffer to give a solution withꢀa concentration of
2.5ꢂ10ꢁ4 M and incubated at 37 C. Subsequent 20 mL
samples were taken at different times, diluted with80 mL
of TEAC to give a final concentration of 5ꢂ10ꢁ5 M,
then frozen in liquid nitrogen. Determination of the
concentration of base and remaining nucleoside in each
sample was determined by HPLC analysis of a 80 mL
aliquot.
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Antiviral assays
The anti-HIV and anti-HBV assays in cell cultures were
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the antiviral activity of compounds was based on a
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metabolic activity of the cells being measured by the
property of mitochondrial dehydrogenase to reduce 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bro-
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