Synthesis and anti-viral activity of a series of D- and l-2′-deoxy-2′-fluororibonucleosides in the subgenomic HCV replicon system

Article abstract of DOI:10.1016/j.bmc.2004.12.011

Based on the discovery of (2′R)-D-2′-deoxy-2′- fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of D- and L-2′-deoxy-2′-fluororibonucleosides with modifications at 5- and/or 4-positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The key step in the synthesis, the introduction of 2′-fluoro group, was achieved by either fluorination of 2,2′-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compound, namely (2′R)-D-2′-deoxy-2′,5-difluorocytidine (13), demonstrated potent anti-HCV activity and toxicity to ribosomal RNA. The replacement of the 4-amino group with a thiol group resulted in the loss of activity, while the 4-methylthio substituted analogue (25) exhibited inhibition of ribosomal RNA. As N⁴-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, we combined the two functionalities of the N⁴-hydroxyl and the 2′-fluoro into one molecule, resulting (2′R)-D-2′-deoxy- 2′-fluoro-N⁴-hydroxycytidine (23). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the L-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot always predict anti-HCV activity.

Full text of DOI:10.1016/j.bmc.2004.12.011

Bioorganic & Medicinal Chemistry 13 (2005) 1641–1652
Synthesis and anti-viral activity of a series of D- and
L-20-deoxy-20-fluororibonucleosides in the subgenomic
HCV replicon system
Junxing Shi,a,* Jinfa Du,a Tianwei Ma,b Krzysztof W. Pankiewicz,a Steven E. Patterson,a
Phillip M. Tharnish,a Tamara R. McBrayer,a Lieven J. Stuyver,a Michael J. Otto,a
Chung K. Chu,b Raymond F. Schinazic and Kyoichi A. Watanabea
aPharmasset, Inc., 1860 Montreal Rd., Tucker, GA 30084, USA
bCollege of Pharmacy, The University of Georgia, Athens, GA 30602-2352, USA
cDepartment of Pediatrics, Emory University School of Medicine, Atlanta, GA 30323,
and Veterans Affairs Medical Center, 1670 Clairmont Road, Decatur, GA 30033, USA
Received 15 November 2004; accepted 7 December 2004
Available online 11 January 2005
Abstract—Based on the discovery of (20R)-D-20-deoxy-20-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of
D- and L-20-deoxy-20-fluororibonucleosides with modifications at 5- and/or 4-positions were synthesized and evaluated for their in
vitro activity against HCV and bovine viral diarrhea virus (BVDV). The key step in the synthesis, the introduction of 20-fluoro
group, was achieved by either fluorination of 2,20-anhydronucleosides with hydrogen fluoride–pyridine or potassium fluoride, or
a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compound, namely
(20R)-D-20-deoxy-20,5-difluorocytidine (13), demonstrated potent anti-HCV activity and toxicity to ribosomal RNA. The replace-
ment of the 4-amino group with a thiol group resulted in the loss of activity, while the 4-methylthio substituted analogue (25) exhib-
ited inhibition of ribosomal RNA. As N4-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, we combined
the two functionalities of the N4-hydroxyl and the 20-fluoro into one molecule, resulting (20R)-D-20-deoxy-20-fluoro-N4-hydroxycyti-
dine (23). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the L-forms of the analogues were devoid of
anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot always predict
anti-HCV activity.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
NM283, has shown potent anti-HCV activity,5 and is
in Phase II clinical trials by Idenix Pharmaceuticals.
Earlier, we discovered that a sugar-fluorinated nucleo-
side, (20R)-D-20-deoxy-20-fluorocytidine (1), had potent
anti-HCV activity.6 Based on the activity of this com-
pound, we synthesized a series of D- and L-analogues,
and evaluated them against bovine viral diarrhea virus
(BVDV) and HCV in the replicon system. In addition,
our discovery of a base-modified nucleoside, D-N4-
hydroxycytidine (NHC),7 possessing anti-HCV activity
prompted us to combine these two features in one mole-
cule. Studying the structure–activity relationship of this
class of nucleosides would increase our knowledge of the
structural requirements for anti-viral agents for HCV,
and would aid in the search for better anti-HCV agents.
Herein we report the synthesis and the biological evalu-
ation of D- and L-20-deoxy-20-fluororibonucleosides.
Hepatitis C virus (HCV) is an important pathogen
affecting nearly 170 million people worldwide.1 HCV
infections become chronic in about 50% of cases,2 and
about 20% of these chronic patients develop liver cirrho-
sis that can lead to hepatocellular carcinoma.3 The cur-
rent therapy, based on interferon-alpha (IFN-a), alone
or combination with ribavirin, is only moderately effec-
tive.4 Therefore, there is a need for more effective anti-
HCV agents. Recently, a ribonucleoside analogue,
Keywords: HCV; BVDV; 20-Deoxy-20-fluororibonucleosides; 20-Fluoro-
ribosides; Fluorination.
*
Corresponding author. Tel.: +1 678 395 0037; fax: +1 678 395
0030; e-mail: jshi@pharmasset.com
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.12.011

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J. Shi et al. / Bioorg. Med. Chem. 13 (2005) 1641–1652
2. Results and discussion
a similar result (about 40% yield). From this com-
pound, several other 5-halogenated cytidine analogues
(3–5) were prepared by halogenation (Scheme 1).
However, this fluorination did not work on D-2,20-
anhydro-5-fluorocytidine and D-2,20-anhydro-5-meth-
ylcytidine. The replacement of potassium fluoride/
crown ether with hydrogen fluoride–pyridine was also
unsuccessful.
(20R)-D-20-Deoxy-20-fluorouridine, the first 20-deoxy-20-
fluororibonucleoside, was described by Cordington
et al. four decades ago.8 Since then, several 20-deoxy-
20-fluororibonucleosides have been prepared.9 The syn-
thesis of 20-deoxy-20-fluororibonucleosides can be
achieved primarily by three strategies: (i) fluorination
of an appropriate nucleoside; (ii) condensation of an
appropriate 2-fluorosugar and a nucleobase; and (iii)
transglycosylation of a 20-deoxy-20-fluororibonucleoside
with a nucleobase. Currently, there are two main ap-
proaches for the fluorination of nucleosides: one is
through the substitution of anhydronucleosides with
hydrogen fluoride-based reagents, and the other is by
an SN2 substitution of arabinonucleosides either directly
with diethylaminosulfur trifluoride (DAST) or with tet-
rabutylammonium fluoride (TBAF) via a sulfonate
intermediate. The first approach, the fluorination of
anhydronucleosides, is a simple, short, and direct meth-
od to prepare carbohydrate-modified nucleosides, and
the second approach, fluorination through an arabino-
nucleoside, is a relatively lengthy route. For the fluorina-
tion of anhydronucleosides, earlier efforts were focused
on the use of the dangerous anhydrous hydrogen fluo-
ride. More recently, the use of hydrogen fluoride–pyri-
dine has been widely adopted. In our synthesis, the
20-fluorine atom of L-20-deoxy-20-fluororibonucleosides
was introduced by the fluorination of arabinonucleo-
sides with DAST, while various fluorination approaches
were used for the synthesis of D-analogues, based mainly
on the accessibility.
For the synthesis of (20R)-D-20-deoxy-20,5-difluorouri-
dine (11), a DAST fluorination approach was utilized.
Thus, D-5-fluorouridine (6) was cyclized, protected,
and hydrolyzed to the arabinose 9. The fluorination
and deprotection of 9 yielded (20R)-D-20-deoxy-20,5-
difluorouridine (11) in excellent yield, and the latter
afforded (20R)-D-20-deoxy-20,5-difluorocytidine (13)
after acetylation and amination (Scheme 2).
(20R)-D-20-Deoxy-20-fluorouridine (18) was prepared
from D-2,20-anhydrouridine by fluorination with hydro-
gen fluoride–pyridine, as described by Kawasaki et al.13
In a similar manner, (20R)-D-20-fluorothymidine (19)
was also prepared in 31% yield. Acetylation followed
by amination converted 19 to the 5-methylcytidine ana-
logue 22 (Scheme 3). Following a literature process10
4-thio analogue 24 and 4-methylthio analogue 25 were
synthesized, and (20R)-D-20-deoxy-20-fluoroadenosine
(26) was prepared according to the published proce-
dures.14 Unfortunately, the fluorination of the 5-
fluoro-substituted anhydronucleoside with hydrogen
fluoride–pyridine failed.
As both D-N4-hydroxycytidine (NHC) and (20R)-D-20-
deoxy-20-fluorocytidine (1) have shown potent anti-
HCV activity, it was of interest to combine the two func-
tionalities into one molecule to improve the anti-viral
potency and reduce cytotoxicity. Therefore, (20R)-D-20-
deoxy-20-fluoro-N4-hydroxycytidine (23) was synthe-
sized from (20R)-D-20-deoxy-20-fluorouridine (18), by
acetylation, sulfonation, hydroxyamination, and depro-
tection, as depicted in Scheme 3.
Originally, (20R)-D-20-deoxy-20-fluorocytidine (1) was
prepared from (20R)-D-20-deoxy-20-fluorouridine via
amination.10 To the best of our knowledge, none of
the (20R)-20-deoxy-20-fluorocytidine analogues were
prepared by direct substitution of the 2,20-anhydrocyti-
dine with hydrogen fluoride-based reagents. However,
there was a report on the preparation of (20R)-D-20-
deoxy-20-fluorocytidine by the heating of the 2,20-
anhydrocytidine hydrofluoride salt.11 The direct con-
version of D-2,20-anhydrocytidine to (20R)-D-20-deoxy-
20-fluorocytidine was achieved by Mengel and Gus-
chlbauer utilizing potassium fluoride and crown
ether.12 This represents a simple and direct method
for the preparation of (20R)-20-deoxy-20-fluorocytidine
nucleosides. We performed this reaction for the prepa-
ration of D-20-deoxy-20-fluorocytidine (1) and achieved
For the synthesis of the L-series of 20-deoxy-20-fluorori-
bonucleosides, the fluorination of arabinonucleosides
with DAST reagent was adopted, and the corresponding
arabinonucleosides were prepared either by HolyÕs
method15 or Vorbruggen sugar-base condensation.16
¨
Thus, L-2,20-anhydrouridine (27) was protected, and
hydrolyzed to give arabino-nucleoside 29. Through a
NHHCl
N
NH2
NH2
N
R
N
ii
i
N
N
O
N
O
HO
HO
O
HO
O
O
O
OH
OH
F
OH
F
2
1
3 R = Cl
4 R = Br
5 R = I
Scheme 1. Reagents and conditions: (i) KF, dibenzo[18]crown-6, DMF, 120 ꢁC, 4 h; (ii) BzCl, m-CPBA, DMF, rt, 30 min; or pyridine, Br2, CCl4, rt,
25 min; or MeOH, ICl, 50 ꢁC, 4 h.

J. Shi et al. / Bioorg. Med. Chem. 13 (2005) 1641–1652
1643
O
O
O
N
F
F
F
NH
N
O
N
O
i
ii
N
N
O
HO
HO
THPO
O
O
O
OH OH
OH
THPO
6
7
8
iii
O
O
O
F
F
F
NH
NH
NH
v
iv
O
O
O
N
O
N
N
HO
THPO
THPO
O
O
OH
OH
11
F
THPO
F
THPO
10
9
vi
O
N
NH2
N
F
F
NH
vii
O
O
N
AcO
HO
O
O
OAc F
OH
13
F
12
Scheme 2. Reagents and conditions: (i) HMPA, Ph2CO, NaHCO3, 135 ꢁC, 40 min; (ii) DMF, 3,4-dihydro-2H-pyran, p-TsOH, 0 ꢁC, 4 h; (iii) NaOH,
MeOH, H2O, rt, 2 h; (iv) DAST, CH2Cl2, pyridine, À70 to reflux, 4 h; (v) p-TsOH, MeOH, rt, 3 h; (vi) pyridine, Ac2O, rt, overnight; (vii) (a) CH3CN,
Et3N, TIPCl, DMAP, rt, 1 d; (b) NH4OH, rt, 1 d.
O
N
O
N
O
N
R
R
R
NH
N
O
NH
i
ii
O
O
HO
HO
HO
O
O
O
OH OH
OH
16 R = H
OH
F
14 R = H
18 R = H
15 R = CH3
17 R = CH3
19 R = CH3
v
iii
NHOH
N
NH2
O
H3C
R
N
NH
iv
N
N
N
O
O
O
HO
HO
AcO
O
O
O
OH
23
F
OH
22
F
OAc F
20 R = H
21 R = CH3
Scheme 3. Reagents and conditions: (i) HMPA, Ph2CO, NaHCO3, 150 ꢁC, 30 min; (ii) HF–pyridine, dioxane, 120–125 ꢁC, 18 h; (iii) Ac2O, pyridine;
(iv) (a) CH3CN, Et3N, TIPCl, DMAP, rt, 1 d; (b) NH4OH, rt, 1 d; (v) (a) CH3CN, Et3N, TIPCl, DMAP, rt, 1 d; (b) NH2OH-HCl, rt, 1 d; (c) NH3/
MeOH, rt, 14 h.
fluorination with DAST, followed by deprotection and
acetylation, protected (20S)-L-20-deoxy-20-fluorouridine
31 was prepared in 41% yield. Deprotection of 31 affor-
ded (20S)-L-20-deoxy-20-fluorouridine (32), and amina-
tion of 31 yielded (20S)-L-20-deoxy-20-fluorocytidine
(40). Halogenation of 32 followed by deprotection pro-
duced 5-halogenated analogues 33–35. 5-Bromo and 5-
iodo analogues 34–35 were converted to their cytidine
analogues 38–39 by acetylation and amination, whereas
the chlorination of (20S)-L-20-deoxy-20-fluorocytidine
(40) yielded the 5-chlorocytidine analogue 41 (Scheme
4).
Because of the difficulty of the direct introduction of
5-methyl and 5-fluoro groups to the pyrimidine nucleo-
sides, these nucleosides had to be prepared using a

1644
J. Shi et al. / Bioorg. Med. Chem. 13 (2005) 1641–1652
O
N
O
O
O
N
N
O
N
O
HN
HN
iii
i
ii
N
N
O
O
OTHP
OTHP
OH
OTHP
O
O
O
O
HO
OTHP
OH
OTHP
F
OTHP
27
28
29
30
iv
NH2
O
N
O
N
O
X
X
X
N
HN
HN
HN
viii
vii
vi
N
F
N
O
O
O
O
OH
OAc
OH
OAc
O
O
O
O
OH
F
OAc
F
OH
F
OAc
38 X = Br
39 X = I
36 X = Br
37 X = I
33 X = Cl
34 X = Br
35 X = I
31
vii, viii
v
NH2
NH2
N
O
Cl
N
HN
ix
N
N
N
O
O
O
OH
OH
OH
O
O
O
F
OH
F
OH
F
OH
41
40
32
Scheme 4. Reagents and conditions: (i) DMF, 3,4-dihydro-2H-pyran, p-TsOH, 0 ꢁC, 4 h; (ii) NaOH, MeOH, H2O, rt, 2 h; (iii) DAST, CH2Cl2,
pyridine, À70 ꢁC to reflux, 4 h; (iv) (a) p-TsOH, MeOH, rt, 3 h; (b) pyridine, Ac2O, rt, overnight; (v) NH3/MeOH, rt, overnight; (vi) NCS, HOAc, 80–
90 ꢁC, 3 h; or HOAc, Ac2O, Br2, rt, overnight; or CH2Cl2, ICl, reflux, 5 h; (vii) Ac2O, pyridine, rt, overnight; (viii) (a) 1,2,4-triazole, pyridine, POCl3,
rt, 17 h; (b) NH4OH, rt, 4 h; (c) NH3/MeOH, rt, 12 h; (ix) DMF, HCl, m-CPBA, rt, 2 h.
Vorbruggen condensation of L-riboside and a silylated
¨
(20S)-L-20-deoxy-20, 5-fluorouridine (56). Through an
acetylation and an amination, the uridine analogues
were converted to the cytidine analogues 59 and 60
(Scheme 5).
nucleobase. The condensation products 43 and 44 were
deprotected, and cyclized to anhydronucleosides 47
and 48. Protection by tetrahedronpyranyl group fol-
lowed by hydrolysis gave the arabinonucleosides 51
and 52, and fluorination with DAST followed by depro-
tection afforded (20S)-L-20-fluorothymidine (55) and
5-Ethynyl, ethyl, and bromovinyl groups were converted
from 5-iodouridine nucleosides via
a
palladium-
O
N
O
O
N
O
N
X
X
X
X
HN
HN
N
O
N
O
OBz
O
AcO
i
ii
iii
iv
N
O
O
OBz
OH
OH
OTHP
O
O
O
O
OAc OBz
OAc OBz
HO
OH
OH
42
OTHP
43 X = Me
44 X = F
45 X = Me
46 X = F
47 X = Me
48 X = F
49 X = Me
50 X = F
v
NH2
O
N
O
N
O
O
X
X
X
X
X
N
O
HN
HN
HN
HN
ix
viii
vii
vi
N
N
N
O
O
O
O
OTHP
OTHP
OH
OAc
OH
O
O
O
O
O
HO
F
OH
F
OAc
F
OH
F
OTHP
OTHP
59 X = Me
60 X = F
57 X = Me
58 X = F
55 X = Me
56 X = F
53 X = Me
54 X = F
51 X = Me
52 X = F
Scheme 5. Reagents and conditions: (i) (a) HMDS, nucleobase, reflux; (b) CH3CN, TMSOTf; (ii) NaOMe, MeOH, rt, overnight; (iii) DMF, Ph2CO,
NaHCO3, 150 ꢁC, 1 h; (iv) DMF, 3,4-dihydro-2H-pyran, p-TsOH, 0 ꢁC, 4 h; (v) NaOH, MeOH, H2O, rt, 2 h; (vi) DAST, CH2Cl2, pyridine, À70 ꢁC
to reflux, 4 h; (vii) p-TsOH, MeOH, rt, 3 h; (viii) pyridine, Ac2O, rt, overnight; (ix) (a) 1,2,4-triazole, pyridine, POCl3, rt, 17 h; (b) NH4OH, rt, 4 h; (c)
NH3/MeOH, rt, 12 h.

J. Shi et al. / Bioorg. Med. Chem. 13 (2005) 1641–1652
1645
TMS
O
N
O
N
analogue (23) showed neither activity against HCV
nor inhibitory activity to ribosomal RNA.
I
HN
HN
i
In the L-series, none of the analogues, including (20S)-L-
20-deoxy-20-fluorocytidine (40), had any anti-HCV activ-
ity or anti-BVDV activity.
O
O
OAc
OAc
O
O
OAc
F
OAc
F
35
61
20-Deoxy-20-fluororibonucleoside analogues are known
to be active against some RNA viruses. Several ana-
logues of this class showed high activity against influ-
enza A and B, and parainfluenza 1.20 Also, (20R)-D-20-
deoxy-20-fluorocytidine has demonstrated inhibitory
activity against herpes simplex virus type 1 and 2, pseu-
dorabies and equine abortion virus.21 It is surprising that
our findings indicate that 20-deoxy-20-fluororibonucleo-
sides possess anti-HCV activity, as some earlier works
on HCV polymerase concluded that in order to be rec-
ognized by HCV RNA dependent RNA polymerase, a
ribonucleoside was needed.22 The discovery that 20-
deoxy-20-fluorocytidine possesses potent anti-HCV
activity suggests that the 20-fluoro instead of 20-hydroxyl
group is recognized by the HCV RNA polymerase. In
terms of Van der Vaar radii, the fluorine atom is closer
to a hydrogen atom than a hydroxyl group. However,
the fluorine atom may mimic the hydroxyl group, in
terms of electronegativity and hydrogen bonding. The
conformational study of 20-fluorinated nucleosides also
suggests that 20-deoxy-20-fluororibonucleosides are more
like ribonucleosides than 20-deoxyribonucleosides, since
it has been confirmed that both 20-deoxy-20-fluororibo-
nucleosides and ribonucleosides adopt a 30-endo confor-
mation.23,24 However, the fact that the anti-HCV
activity of 20-deoxy-20-fluorocytidine can be abolished
by the addition of 20-deoxycytidine, not by cytidine,6
demonstrates that 20-deoxy-20-fluororibonucleosides
are recognized as 20-deoxynucleosides in at least one
step in the catabolic pathway in HCV replicon cells.
ii
O
N
O
N
C2H5
HN
HN
iii
O
O
OH
OH
O
O
F
OH
F
OH
63
62
Scheme 6. Reagents and conditions: (i) Et3N, CuI, (Ph3P)2PdCl2,
TMSCCH, 50–60 ꢁC, 7 h; (ii) NaOMe, MeOH, rt, 15 h; (iii) EtOH,
10% Pd–C, H2, 1 atm, 1 h.
mediated reaction.17 From acetylated 5-iodo analogue
35, after ethynylation followed by deprotection, 5-ethyn-
yl analogue 62 was produced. Hydrogenation of 62
yielded 5-ethyl analogue 63 (Scheme 6). (20S)-L-5-
Bromovinyl-20-deoxy-20-fluorouridine (64) was prepared
from 5-iodouridine analogue 35, by
procedure.18
a published
The above synthesized 20-deoxy-20-fluororibonucleo-
sides were evaluated in BVDV and HCV subgenomic
replicon RNA-containing Huh7 cells, as described previ-
ously,7,19 and the results of the selected nucleosides are
shown in Table 1. For the BVDV assay, all the tested
compounds showed no inhibitory activity, including
the lead compound (20R)-D-20-deoxy-20-fluorocytidine
(1). For in vitro anti-HCV activity, in the D-series,
among the 5-substituted cytidine analogues, only
(20R)-D-20-deoxy-20,5-difluorocytidine (13) exhibited po-
tent anti-HCV activity and inhibition of ribosomal
RNA. The 5-chloro, 5-bromo, 5-iodo, and 5-methyl
substituted 20-deoxy-20-fluorocytidine analogues showed
no anti-HCV activity. Similarly, the uridine analogues,
It seems that the anti-HCV activity resides with the D-
nucleosides. To the best of our knowledge, until now,
no L-enantiomer has been reported to possess any spe-
cific anti-HCV activity in vitro. As more L-nucleosides
are evaluated against HCV in vitro, this hypothesis will
be further tested.
(20R)-D-20-deoxy-20-fluorouridine,
(20R)-D-20-deoxy-
20,5-difluorouridine, and (20R)-D-20-fluorothymidine
were not active against HCV. The replacement of
the amino with a thiol group at the 4-position also
resulted in an inactive compound 24. However, the 4-
methylthio analogue 25 demonstrated inhibition of ribo-
somal RNA. Unfortunately, the N4-hydroxylamino
The fact that all the tested compounds showed no anti-
BVDV activity may be due to the inadequate phosphor-
ylation of these nucleosides in MBDK cells, as it has
been shown that gemcitabine, a potent anti-cancer agent
with nanomolar inhibition in most human cells, did not
Table 1. In vitro anti-BVDV and anti-HCV activity of selected 20-deoxy-20-fluororibonucleosides
Compound
Configuration
Base
4-Substitution
5-Substitution
EC90 (lM) BVDV
EC90 (lM) HCV
CC50 (lM) rRNA
1
13
D
D
D
D
C
C
C
U
>100
ND
ND
>100
5.4
5.6
9.4
>100
<1
F
23
25
NHOH
SCH3
>100
>100
5.0
>100
8.7
NHC
Ribavirin
>100
14.4
1.5
P100
All the other nucleosides had values of EC90 and CC50 over 100 lM; ND: not determined; EC90: effective concentration required for reducing the
HCV RNA or BVDV levels by 90% in 96 h or 72 h.
CC50: cytotoxic concentration required for reducing the rRNA levels by 50% in 96 h.

1646
J. Shi et al. / Bioorg. Med. Chem. 13 (2005) 1641–1652
exhibit an inhibitory affect in this cell line.7 This may
suggest that the MDBK-cpBVDV assay is not always
a reliable model for predicting anti-HCV activity in a
replicon system.
H-6), 7.93, 7.28 (2s, 2H, NH2), 5.84 (d, J = 16.4 Hz,
1H, H-10), 5.56 (d, J = 6.4 Hz, 1H, OH-30), 5.37 (t,
J = 4.8 Hz, 1H, OH-50), 4.90 (dd, J = 4.0 and 52.8 Hz,
1H, H-20), 4.21–4.09 (m, 1H, H-30), 3.88 (d,
J = 8.4 Hz, 1H, H-40), 3.80, 3.60 (2m, 2H, H-50). MS
(FAB+) m/e 286 [(M+Li)+]; HRMS (FAB+) calcd for
(C9H11ClFN3O4+Li): 286.0582, found: 286.0577. Anal.
Calcd for C9H11ClFN3O4+0.5H2O: C 37.51, H 4.02, N
14.58. Found: C 37.78, H 3.99, N 13.94.
3. Conclusions
A series of D- and L-20-deoxy-20-fluororibonucleosides
were synthesized and evaluated for in vitro anti-HCV
and anti-BVDV activity, as well as their inhibition of
ribosomal RNA. The study revealed that (20R)-D-20-
deoxy-20,5-difluorocytidine (13) showed potent anti-
HCV and inhibitory activity to ribosomal RNA. All of
the other 5-modified D-nucleosides were not active
against HCV, and all the L-series compounds were de-
void of anti-HCV activity. The 4-methylthio analogue
25 showed inhibitory activity to ribosomal RNA. None
of the tested compounds demonstrated anti-BVDV
activity. Furthermore, (20R)-D-20-deoxy-20-fluoro-N4-
hydroxylcytidine (23) was neither active nor toxic to
liver cells.
4.1.2. (20R)-b-D-5-Bromo-20-deoxy-20-fluorocytidine (4).
To a solution of 1 (72 mg, 0.29 mmol) in anhydrous pyri-
dine (10 mL) at room temperature was added a solution
of bromine (75 mg. 0.47 mmol) in CCl4 (360 lL). The
resulting orange colored solution was stirred at room
temperature for 25 min, and then the solvent was evap-
orated to dryness. The residue was purified by flash
chromatography on silica gel, eluting with CH2Cl2/
MeOH (4:1), to give 80 mg (84%) of product as a pale
yellow powder. Mp 165–167 ꢁC (dec); 1H NMR
(DMSO-d6) d 8.42 (s, 1H, H-6), 7.93, 7.06 (2s, 2H,
NH2), 5.84 (d, J = 17.2 Hz, 1H, H-10), 5.56 (d,
J = 6.4 Hz, 1H, OH-30), 5.37 (t, J = 4.4 Hz, 1H, OH-
50), 4.90 (dd, J = 4.0 and 53.2 Hz, 1H, H-20), 4.21–4.09
(m, 1H, H-30), 3.88 (d, J = 8.4 Hz, 1H, H-40), 3.80,
3.60 (2m, 2H, H-50). MS (FAB+) m/e 330 [(M+Li)+];
HRMS (FAB+) calcd for (C9H11BrFN3O4+Li):
330.0077, found: 330.0071.
4. Experimental
4.1. Materials
All reagents were used as received unless stated other-
wise. Anhydrous solvents were purchased from Aldrich
Chemical Company, Inc. (Milwaukee). Melting points
(mp) were determined on an electrothermal digit melting
point apparatus and are uncorrected. 1H and 13C
NMR spectra were taken on a Varian Unity Plus 400
spectrometer at room temperature and reported in parts
per million downfield from internal tetramethylsilane.
Mass spectra were recorded at the Emory University
Mass Spectrometry Center (Atlanta, GA). Polarimetries
were measured on a Jasco DIP-370 digital polarimeter.
Elemental analyses were performed by Atlantic Micro-
lab Inc. (Norcross, GA). Analytic TLC was performed
on Whatman LK6F silica gel plates. HPLC identifica-
tion and purification were conducted with a Varian
Prostar system. (20R)-D-20-Deoxy-20-fluorocytidine (1),12
D-2,20-anhydrouridine (16),25 (20R)-D-20-deoxy-20-fluoro-
uridine (18),13 (20R)-D-20-deoxy-20-fluoro-4-thiouridine
(24),10 (20R)-D-20-deoxy-20-fluoro-4-methylthiouridine
(25),10 (20R)-D-20-deoxy-20-fluoroadenosine (26),14 and
(20S)-L-5-bromovinyl-20-deoxy-20-fluorouridine (64)18
were prepared according to the published procedures.
4.1.3. (20R)-b-D-20-Deoxy-20-fluoro-5-iodocytidine (5). To
a solution of 1 (123 mg, 0.5 mmol) in anhydrous MeOH
(4 mL) was added ICl (163 mg, 1 mmol), and the solu-
tion was stirred at 50 ꢁC under an argon atmosphere
for 4 h. The solvent was evaporated to dryness and the
residue was purified by flash chromatography on silica
gel, eluting with CH2Cl2/MeOH (4:1), to give, after crys-
tallization from EtOH/CHCl3/hexane, 73 mg (39%) of
product as a pale yellow crystalline solid. Mp 180–
182 ꢁC; 1H NMR (DMSO-d6) d 8.45 (s, 1H, H-6),
7.90, 6.68 (2s, 2H, NH2), 5.84 (d, J = 17.2 Hz, 1H, H-
10), 5.55 (d, J = 6.4 Hz, 1H, OH-30), 5.36 (t,
J = 4.8 Hz, 1H, OH-50), 4.88 (dd, J = 4.4 and 53.2 Hz,
1H, H-20), 4.21–4.09 (m, 1H, H-30), 3.87 (d,
J = 8.4 Hz, 1H, H-40), 3.80, 3.58 (2m, 2H, H-50). MS
(FAB+) m/e 378 [(M+Li)+]. Anal. Calcd for C9H11FI-
N3O4: C 29.13, H 2.99, N 11.32. Found: C 29.07, H
3.02, N 11.15.
4.1.4. b-D-2,20-Anhydro-5-fluorouridine (7). To a solution
of 6 (1.60 g, 6.10 mmol) and diphenyl carbonate (1.74 g)
in anhydrous HMPA (6 mL) was added NaHCO3
(40 mg, mmol), and the mixture was stirred at 135 ꢁC
for 40 min. The resulting solution was cooled to room
temperature, and poured into cold water (80 mL). The
mixture was washed with CHCl3 (3 · 40 mL), and
concentrated in vacuo. The residue was crystallized from
MeOH to give 575 mg (39%) of product as a white
solid. Mp 222–224 ꢁC; 1H NMR (DMSO-d6) d 8.28
(d, J = 3.6 Hz, 1H, H-6), 6.31 (d, J = 5.6 Hz, 1H, H-
10), 5.91 (d, J = 3.2 Hz, 1H, H-20), 5.25 (d, J =
6.4 Hz, OH-30), 5.00 (t, J = 4.8 Hz, 1H, OH-50), 4.40
(s, 1H, H-30), 4.12 (m, 1H, H-40), 3.31, 3.25 (2m, 2H,
H-50).
4.1.1. (20R)-b-D-5-Chloro-20-deoxy-20-fluorocytidine (3).
To a solution of 1 (123 mg, 0.5 mmol) in DMF (1.5 mL)
at room temperature was added BzCl (63 lL), followed
by a solution of m-CPBA (125 mg) in DMF (0.5 mL).
The solution was stirred at room temperature for
30 min, and then poured into cold water (10 mL). The
resulting precipitate was filtered off and washed with
water. The combined filtrates were evaporated in vacuo
to dryness, and the residue was purified by flash chroma-
tography on silica gel, eluting with CH2Cl2/MeOH (4:1),
to give 33 mg (24%) of product as a white solid. Mp
1
176–178 ꢁC (dec); H NMR (DMSO-d6) d 8.35 (s, 1H,

J. Shi et al. / Bioorg. Med. Chem. 13 (2005) 1641–1652
1647
4.1.5. b-D-2,20-Anhydro-5-fluoro-30,50-di-O-tetrahydro-
pyranyluridine (8). To solution of (560 mg,
(1 mL, mmol), and the solution was stirred at room tem-
perature overnight. After the removal of the solvent by
evaporation, the residue was partitioned between water
and CHCl3. The organic layer was separated, dried
(Na2SO4), evaporated, and co-evaporated with toluene
to give 497 mg (93%) of product as a brown solid. This
crude product was used for the next reaction without
further purification.
a
7
2.31 mmol) and p-TsOH (431 mg) in anhydrous DMF
(10 mL) was added 3,4-dihydro-2H-pyran (5.6 mL,
mmol), and the solution was stirred at 0 ꢁC for 4 h.
The solution was neutralized by the addition of Et3N,
and then the volatile was evaporated to dryness. The resi-
due was dissolved in EtOAc (50 mL), washed with sat-
urated aqueous NaHCO3, dried over Na2SO4, and
evaporated. The residue was triturated with hexane to
give 640 mg (76%) of product as a white solid. This
crude product was used for the next reaction without
further purification.
4.1.10. (20R)-b-D-20-Deoxy-20,5-difluorocytidine (13). To
a solution of 12 (497 mg, 1.43 mmol) in anhydrous
CH3CN (20 mL) and Et3N (0.80 mL, 5.71 mmol) at
0 ꢁC was added TIPSCl (892 mg, 2.85 mmol), followed
by DMAP (174 mg, 1.43 mmol). The solution was stir-
red at room temperature for 24 h, and then concentrated
NH4OH (50 mL) was added. After stirring at room
temp for 24 h, the solvent was evaporated, and the res-
idue was purified by flash chromatography on silica
gel, eluting with CH2Cl2/MeOH (4:1), to give 101 mg
(27%) of product as a pale yellow solid. Mp 164–
166 ꢁC; 1H NMR (DMSO-d6) d 8.24 (d, J = 3.6 Hz,
1H, H-6), 7.84, 7.58 (2s, 2H, NH2), 5.83 (d,
J = 17.2 Hz, 1H, H-10), 5.57 (d, J = 6.4 Hz, 1H, OH-
30), 5.35 (t, J = 4.8 Hz, 1H, OH-50), 4.87 (dd, J = 4.4
and 53.2 Hz, 1H, H-20), 4.20–4.08 (m, 1H, H-30), 3.87
(d, J = 8.0 Hz, 1H, H-40), 3.80, 3.60 (2m, 2H, H-50);
MS (FAB+) m/e 270 [(M+Li)+]; HRMS (FAB+) calcd
for (C9H11F2N3O4+Li): 270.0878, found: 270.0869.
Anal. Calcd for C9H11F2N3O4+H2O: C 38.44, H 4.66,
N 14.94. Found: C 38.82, H 4.61, N 14.78.
4.1.6. b-D-30,50-Di-O-tetrahydropyranyl-5-fluoro-arabino-
uridine (9). A suspension of 8 (640 mg, 1.77 mmol) in
MeOH (9 mL) and NaOH aqueous solution (1 N,
3 mL) was stirred at room temperature for 2 h. The solu-
tion was neutralized by 1 N HOAc. The mixture was
evaporated and co-evaporated with EtOH. The residue
was purified by flash chromatography on silica gel, elut-
ing with CH2Cl2/MeOH (95:5), to give 680 mg (quant.)
of product as a pale yellow solid. This crude product
was used for the next reaction without further
purification.
4.1.7. (20R)-b-D-20-Deoxy-20,5-difluoro-30,50-di-O-tetra-
hydropyranyl-uridine (10). To a stirred mixture of 10
(680 mg, 1.77 mmol) in anhydrous CH2Cl2 (10 mL)
and anhydrous pyridine (1.7 mL) at À70 ꢁC was added
DAST (887 mg, 5.5 mmol) and the mixture was allowed
to warm up to room temperature. The mixture was
heated at reflux for 4 h. The reaction was quenched by
the addition of saturated aqueous NaHCO3 (2 mL)
and ice water (10 mL). The mixture was extracted with
CH2Cl2 (3 · 20 mL). The combined extracts were
washed with saturated aqueous NaHCO3, dried
(Na2SO4), and concentrated to give 735 mg (96%) of
product as brown oil. This crude product was used for
the next reaction without further purification.
4.1.11. b-D-2,20-Anhydro-5-methyluridine (17). To a mix-
ture of 15 (5.16 g, 20 mmol) and diphenyl carbonate
(5.72 g) in anhydrous HMPA (35 mL) was added NaH-
CO3 (130 mg, mmol), and the mixture was stirred at
150 ꢁC for 30 min. The resulting solution was cooled
to room temperature, and poured into cold water
(120 mL). The mixture was washed with CHCl3
(3 · 50 mL), and concentrated in vacuo. The residue
was crystallized from MeOH to give 3.07 g (64%) of
1
product as a white solid. H NMR (DMSO-d6) d 7.75
4.1.8. (20R)-b-D-20-Deoxy-20,5-difluorouridine (11).26 To
a solution of 10 (735 mg, 1.70 mmol) in anhydrous
MeOH (15 mL) was added p-TsOH (500 mg, mmol),
and the solution was stirred at room temperature for
3 h. After the removal of the solvent by evaporation,
the residue was co-evaporated with pyridine three times
to give 427 mg (95%) of product as a brown solid. From
this crude product, a sample of pure compound was ob-
tained by recrystallization from MeOH/CH2Cl2/hexane,
while the crude product was used for the next reaction
without further purification. The NMR data was in
agreement with that which was previously reported.26
(s, 1H, H-6), 6.29 (d, J = 6.0 Hz, 1H, H-10), 5.88 (s,
1H, H-20), 5.17 (d, J = 6.0 Hz, OH-30), 4.97 (t,
J = 5.2 Hz, 1H, OH-50), 4.37 (s, 1H, H-30), 4.06 (m,
1H, H-40), 3.24, 3.17 (2m, 2H, H-50), 1.79 (s, 3H, CH3).
4.1.12. (20R)-b-D-20-Fluorothymidine (19).14 A solution
of 17 (3.0 g, 12.5 mmol) in anhydrous 1,4-dioxane
(150 mL) and HF–pyridine (50 mL) was sealed in a tight
stainless bomb and heated at 120–125 ꢁC for 18 h. After
cooling to room temperature, the solution was neutral-
ized with solid CaCO3, and stirred at room temperature
overnight. The solid was filtered and washed with
MeOH (50 mL). The combined filtrates were evaporated
and the residue was purified by flash chromatography
on silica gel, eluting with CH2Cl2/MeOH (4:1), to give
1.02 g (31%) of product as a white solid. Mp 185–
187 ꢁC; 1H NMR (DMSO-d6) d 11.41 (s, 1H, NH),
7.80 (s, 1H, H-6), 5.91 (d, J = 6.0 Hz, 1H, H-10), 5.62
(d, J = 6.0 Hz, OH-30), 5.26 (t, J = 5.2 Hz, 1H, OH-50),
5.02 (d, J = 52.8 Hz, 1H, H-20), 4.21–4.11 (m, 1H, H-
30), 3.85 (d, J = 6.8 Hz, 1H, H-40), 3.76, 3.59 (2m, 2H,
H-50), 1.75 (s, 3H, CH3).
1
mp 135–137 ꢁC; H NMR (DMSO-d6) d 11.90 (s, 1H,
NH), 8.34 (d, J = 7.2 Hz, 1H, H-6), 5.86 (d,
J = 16.4 Hz, 1H, H-10), 5.63 (d, J = 8.0 Hz, 1H, OH-
30), 5.41 (t, 1H, OH-50), 5.02 (dd, J = 53.0 Hz, 1H, H-
20), 4.17 (m, 1H, H-30), 3.90 (m, 1H, H-40), 3.72 (m,
2H, H-50).
4.1.9. (20R)-b-D-20-Deoxy-30,50-di-O-acetyl-20,5-difluoro-
uridine (12). To a solution of 11 (404 mg, 1.53 mmol) in
anhydrous pyridine (5 mL) at 0 ꢁC was added Ac2O

1648
J. Shi et al. / Bioorg. Med. Chem. 13 (2005) 1641–1652
4.1.13. (20R)-b-D-20-Deoxy-30,50-di-O-acetyl-20-fluorouri-
dine (20). In an analogous manner to the preparation of
12, the title compound 20 was prepared in the yield of
88%. This crude product was used for the next reaction
without further purification.
and filtered. The filter cake was washed with hexanes
and dried to give (20S)-b-L-2,20-anhydro-30,50-di-O-tet-
rahydropyranyluridine (28) as a white solid (27.5 g,
93.0%), which was pure enough for the next reaction.
A suspension of 28 (23.0 g, 58.5 mmol) in MeOH
(300 mL) and 1 N NaOH (100 mL) was stirred at room
temperature for 2 h, then neutralized with dilute acetic
acid. The mixture was evaporated to dryness and the
residue was loaded to a silica gel pad and eluted with
EtOAc to give (20S)-b-L-30,50-di-O-tetrahydropyranyl-
arabinouridine (29) as a white solid (22.6 g, 94.0%).
4.1.14.
(20R)-b-D-30,50-Di-O-acetyl-20-fluorothymidine
(21). In an analogous manner to the preparation of 12,
the title compound 21 was prepared in a quantitative
yield. This crude product was used for the next reaction
without further purification.
4.1.15.
(20R)-b-D-20-Deoxy-20-fluoro-5-methylcytidine
(22). In an analogous manner to the preparation of 13,
the title compound 22 was prepared in the yield of
57%. Mp 185–187 ꢁC (dec); 1H NMR (DMSO-d6) d
7.75 (s, 1H, H-6), 7.39, 6.87 (2s, 2H, NH2), 5.88 (dd,
J = 1.6 and 18.4 Hz, 1H, H-10), 5.53 (d, J = 6.8 Hz,
1H, OH-30), 5.20 (t, J = 5.2 Hz, 1H, OH-50), 4.93, 4.79
(2m, 1H, H-20), 4.20–4.05 (m, 1H, H-30), 3.84 (m, 1H,
H-40), 3.77, 3.60 (2m, 2H, H-50), 1.81 (s, 3H, CH3).
MS (FAB+) m/e 266 [(M+Li)+]; HRMS (FAB+) calcd
for (C10H14FN3O4+Li): 266.1128, found: 266.1117.
Anal. Calcd for C10H14FN3O4+0.5H2O: C 44.86, H
5.46, N 15.69. Found: C 45.87, H 5.39, N 15.81.
To a stirred mixture of 29 (20.6 g. 50.0 mmol) in CH2Cl2
(300 mL) and pyridine (50 mL) at À60 ꢁC was added
DAST (25.0 g, 155 mmol) under N2. It was slowly
warmed up to room temperature and then refluxed for
4 h. The reaction was quenched by saturated NaHCO3
and ice-water, then extracted with CH2Cl2
(100 mL · 3), washed with saturated NaHCO3, and
dried (MgSO4). Removal of solvent gave a dark-brown
syrup (18.9 g), which was redissolved in MeOH
(300 mL). To this solution was added p-toluenesulfonic
acid (6 g) and the mixture was stirred at room tempera-
ture for 3 h. It was neutralized with pyridine (50 mL),
co-evaporated with pyridine (50 mL · 2), and then redis-
solved in pyridine (100 mL). To this solution was added
Ac2O (20 mL) and the mixture was stirred at room tem-
perature for 20 h. The removal of the solvent and recrys-
tallization from EtOH gave 31 as a white solid (6.8 g,
4.1.16. (20R)-b-D-20-Deoxy-20-fluoro-N4-hydroxycytidine
(23). To a solution of 20 (430 mg, 1.3 mmol) in anhy-
drous CH3CN (25 mL) and Et3N (0.72 mL, 5.2 mmol)
at 0 ꢁC was added TIPSCl (813 mg, 2.6 mmol), followed
by DMAP (159 mg, 1.3 mmol). The solution was stirred
at room temperature for 24 h, and then NH2OH–HCl
(185 mg, 2.6 mmol) was added. After stirring at room
temperature for 24 h, the solvent was evaporated, and
the residue was passed through a silica gel column, elut-
ing with CH2Cl2/MeOH (95:5). The eluent was concen-
trated to give white foam, to which NH3/MeOH (2.0 M
solution, 25 mL) was added. The mixture was stirred in
a stoppered flask at room temperature for 14 h. After
the removal of the solvent by evaporation, the residue
was purified by flash chromatography on silica gel, elut-
ing with CH2Cl2/MeOH (5:1), to give 133 mg (39%) of
product as a white solid. Mp 201–203 ꢁC (dec); 1H
NMR (DMSO-d6) d 10.02 (s, 1H, NHOH), 9.65 (s,
1H, NHOH), 7.06 (d, J = 8.0 Hz, 1H, H-6), 5.86 (d,
J = 3.6 Hz, 1H, H-10), 5.59 (d, J = 8.0 Hz, 1H, H-5),
5.57 (d, J = 6.4 Hz, 1H, OH-30), 5.11 (t, J = 5.2 Hz,
1H, OH-50), 5.04, 4.91 (2m, 1H, H-20), 4.17–4.07 (m,
1H, H-30), 3.81 (m, 1H, H-40), 3.67, 3.55 (2m, 2H, H-
50). MS (FAB+) m/e 268 [(M+Li)+]. Anal. Calcd for
C9H12FN3O5: C 41.38, H 4.63, N 16.09. Found: C
41.62, H 4.66, N 16.05.
1
41% from 29): H NMR (CDCl3) d 8.62 (s, 1H, NH,
D2O exchangeable), 7.33 (d, IH, H-6, J = 8.1 Hz). 5.72
(dd, 1H, H-10, J1 ,2 = 2.0, J1 ,F = 25.0 Hz 5.70 (d, 1H,
0
0
0
H-5, J = 8.1 Hz), 5.30 (ddd, 1H, H-20, J2 ,F = 52.2 Hz
0
5.08 (ddd, 1H, H-30, J3 F = 17.9 Hz), 4.31 (m, 3H, H-
0
40, H-50a,b), 2.07, 2.03 (2s, 6H, Ac).
4.1.18. (20S)-b-L-20-Deoxy-20-fluorouridine (32). Com-
pound 31 was treated with satd NH3/MeOH at room
temperature for 20 h to give, after crystallization from
25
water, 32 as a white solid (83%): mp 150–152 ꢁC; ½aꢀ
D
1
À50.26 (c 0.19, MeOH); H NMR (DMSO-d6) d 11.39
(s, 1H NH, D2O exchangeable), 7.91 (d, 1H H-6,
0
J = 8.1 Hz), 5.87 (br d, 1H, H-10, J1 F = 17.5 Hz), 5.62
(d, 1H 30-OH, D2O exchangeable), 5.61 (d. 1H H-5,
J = 8.1 Hz), 5.20 (br s, 1H 50-OH, D2O exchangeable),
5.00 (dt, 1H H-20, J2 F = 53.1 Hz), 4.13 (dm, 1H H-30,
0
J3 F = 20.8 Hz), 3.86 (m, 1H H-40), 3.66 (m, 2H, H-
0
50a,b). Anal. Calcd for C9H11FN2O5+0.2H2O: C 43.24,
H 4.48, N 11.21. Found: C 43.18, H 4.60, N 11.06.
4.1.19. (20S)-b-L-5-Chloro-20-deoxy-20-fluorouridine (33).
A mixture of 31 (1.0 g, 3.0 mmol) and N-chlorosuccin-
imide (0.8 g, 6.0 mmol) in AcOH (30 mL) was stirred at
80–90 ꢁC for 3 h. It was then poured into ice-water, ex-
tracted with CHCl3 (50 mL · 3), washed with saturated
NaHCO3 and dried MgSO4. After the removal of the
solvent, the resulting white foam was treated with
0.2 N NaOCH3/CH3OH at room temperature over-
night. After co-evaporation with EtOH, product 33
4.1.17. (20S)-b-L-30,50-Di-O-acetyl-20-deoxy-20-fluorouri-
dine (31). To a suspension of b-L-2,20-anhydrouridine
(27) (17.0 g, 75.0 mmol) in DMF (300 mL) and 3,4-
dihydropyran (180 mL) at 0 ꢁC was added p-toluenesulf-
onic acid (14.0 g) and the mixture was stirred at 0 ꢁC for
4 h when a clear solution was obtained. It was neutral-
ized with Et3N (30 mL) and then evaporated to dryness.
The residue was redissolved in EtOAc, washed with sat-
urated NaHCO3, and dried (MgSO4). Removal of sol-
vent gave a residue, which was triturated with hexanes
was obtained as a white powder (75%): mp 159–
25
161 ꢁC; ½aꢀ : À17.60 (c 0.29, MeOH); 1H NMR
D
(DMSO-d6) d 8.47 (s, 1H, H-6), 5.86 (br d, 1H, H-10,

J. Shi et al. / Bioorg. Med. Chem. 13 (2005) 1641–1652
1649
25
tone gave a yellow solid: mp 182–184 ꢁC; ½aꢀ À6.66
J1 F = 15.6 Hz) 5.64 (d, 1H, 30-OH, D2O exchangeable),
0
D
1
5.44 (t, 1H, 50-OH, D2O exchangeable), 5.04 (dd, 1H, H-
(c 0.79, MeOH); H NMR (DMSO-d6) d 8.46 (s, 1H,
H-6), 7.91, 6.70 (2s, 2H, NH2, D2O exchangeable),
20, J2 F = 52.9 Hz) 4.19 (dm, 1H, H-30, J3 F = 23.8 Hz),
3.91 (m, 1H, H-40), 3.73 (m, 2H, H-50a,b). Anal. Calcd
for C9H10ClFN2O5+0.8H2O: C 36.61, H 3.93, N 9.49.
Found: C 16.96, H 4.02, N 9.17.
0
0
5.84 (d, 1H, H-10, J1 ,F = 17.0 Hz), 5.56 (d, 1H, 30-OH,
0
D2O exchangeable), 5.36 (t, 1H, 50-OH, D2O exchange-
able), 4.90 (dd, 1H, H-20, J2 ,F = 53.2 Hz), 4.15 (dm, 1H,
0
H-30, J3 ,F = 24.2 Hz), 3.88 (m, 1H, H-40), 3.70 (m, 2H,
0
4.1.20. (20S)-b-L-5-Bromo-20-deoxy-20-fluorouridine (34).
To a stirred mixture of 31 (1.0 g, 3.0 mmol) in Ac2O (20
mL) and AcOH (10 mL) was added Br2 (1 mL) in AcOH
(3.0 mL), and the mixture was sealed and stirred at
room temperature overnight. To this was added EtOH
slowly, and the mixture was evaporated to dryness and
collaborated with EtOH until no more fume formed.
The residue was then purified by silica gel column chro-
H-50a,b). Anal. Calcd for C9H11FIN3O4: C 29.13, H
2.99, N 11.32. Found: C 29.02, H 3.00, N 11.20.
4.1.24. (20S)-b-L-20-Deoxy-20-fluorocytidine (40). To a
stirred mixture of 31 (1.32 g, 4.0 mmol) and 1,2,4-triaz-
ole (2.20 g, 32.0 mmol) in pyridine (20 mL) was added
POCl3 (0.75 mL, 8.0 mmol), and the mixture was stirred
at room temperature for 17 h. The solvent was evapo-
rated and the residue was treated with NH4OH–dioxane
at room temperature for 4 h followed by satd NH3/
CH3OH at room temperature for 12 h. The removal of
the solvent gave a residue, which was recrystallized from
matography (9:1 CHCI3–CH3OH) to give a white
25
D
solid (0.89 g, 92%): mp 164–166 ꢁC; ½aꢀ À8.66 (c 0.47,
MeOH); 1H NMR (DMSO-d6) d 11.89 (s, 1H, NH,
D2O exchangeable), 8.50 (s, 1H, H-6), 5.86 (br d, 1H,
H-10, J1 ,F = 16.4 Hz), 5.63 (br s, 1H, 30-OH, D2O
cold water to give 40 as a white solid (750 mg, 77%): mp
0
0
25
164–167 ꢁC; ½aꢀ À68.85 (c 0.35, MeOH); 1H NMR
exchangeable), 5.44 (br s, 1H, 50-OH, D2O exchange-
D
able), 4.99 (dd, 1H, H-20, J2 ,F = 53.1 Hz), 4.18 (dm,
(DMSO-d6) d 7.90 (d, 1H, H-6, J = 7.3 Hz), 7.24 (br d,
0
1H, H-30, J3 ,F = 23.7 Hz), 3.90 (m, 1H, H-40), 3.73 (m,
2H, NH2, D2O exchangeable), 5.99 (d, 1H, H-10,
0
2H, H-50a,b). Anal. Calcd for C9H10BrFN2O5+0.6H2O:
C 32.15, H 3.33, N 8.34. Found: C 32.03, H 3.27, N 8.34.
J1 F = 17.8 Hz), 5.88 (d, 1H, H-5, J = 7.3 Hz), 5.73 (d,
0
1H, 30-OH, D2O exchangeable), 5.16 (t, 1H 50-OH,
D2O exchangeable), 4.89 (dd, 1H H-20, J2 F = 53.3 Hz),
0
4.1.21. (20S)-b-L-20-Deoxy-20-fluoro-5-iodouridine (35). A
mixture of 31 (3.3 g, 10 mmol) and ICl (2.4 g, 15 mmol)
in CH2C12 (100 mL) was stirred at reflux for 5 h. It was
then diluted with CH2C12 (150 mL), washed successively
with NaHSO3 (100 mL · 3), saturated NaHCO3
(50 mL · 2) and dried (MgSO4). After removal of the sol-
vent, the residue was treated with NaOCH3/CH3OH.
Trituration in ether followed by recrystallization from
4.11 (dm, IH, H-30, J3 F = 22.4 Hz), 3.86 (m, 1H, H-
0
40), 3.68 (m, 2H, H-50a,b). Anal. Calcd for
C9H12FN3O4+2H2O: C 38.41, H 5.69, N 14.94. Found:
C 38.49, H 5.72, N 14.87.
4.1.25.
(20S)-b-L-5-Chloro-20-deoxy-20-fluorocytidine
(41). To a solution of 40 (200 mg, 0.8 mmol) in DMF
(5 mL) was added a solution of HCl/DMF (2 M,
0.6 mL) followed by a solution of m-CPBA (350 mg,
1.6 mmol) in DMF (1 mL). The mixture was stirred at
room temperature for 2 h, then evaporated to dryness.
The residue was suspended in H2O and MeOH, filtered,
and washed with H2O. The combined filtrate was trea-
ted with concentrated NH4OH and then evaporated to
dryness. The residue was purified by silica gel column
chromatography (10:1 CHCI3/CH2OH) and collabo-
water gave 35 as a white solid (78%): mp 217–218 ꢁC;
25
½aꢀ +9.41 (c 0.36, MeOH); 1H NMR (DMSO-d6) d
D
11.75 (s, 1H, NH, D2O exchangeable), 8.54 (s, 1H, H-
6), 5.86 (br d, 1H, H-10, J1 ,F = 16.8 Hz), 5.62 (d, 1H,
0
30-OH, D2O exchangeable), 5.41 (t, 1H, 50-OH, D2O
exchangeable), 5.04 (dd, 1H, H-20, J2 ,F = 53.1 Hz), 4.18
0
(dm, 1H, H-30, J3 ,F = 23.4 Hz), 3.90 (m, 1H, H-40),
0
3.72 (m, 2H, H-50a,b). Anal. Calcd for C9H10FIN2O5:
C 29.05, H 2.71, N 7.53. Found: C 29.11, H 2.85, N 7.33.
rated with Et2O to give 41 as a white solid (65 mg,
25
D
28.5%): mp >105 ꢁC (dec); ½aꢀ À47.74 (c 0.19, MeOH);
4.1.22.
(20S)-b-L-5-Bromo-20-deoxy-20-fluorocytidine
1H NMR (DMSO-d6) d 8.54 (s, 1H, H-6), 8.54, 8.26 (2s,
(38). Compound 34 was treated with Ac2O in pyridine
to give 36, which was further treated in a similar manner
as described for the synthesis of 40. Trituration of
2H, NH2, D2O exchangeable), 5.82 (d, 1H, H-10,
J1 ,F = 16.4 Hz), 4.94 (dd, 1H, H-20. J2 ,F = 53.1 Hz),
0
0
4.14 (dm, 1H, H-30, J3 ,F = 25.42 Hz), 3.91 (m, 1H, H-
0
the crude product with acetone gave 38 as a white
40), 3.72 (dm, 2H, H-50a,b). Anal. Calcd for
C9H11ClFN3O4+HCl+0.5H2O: C 33.21, H 4.00, N
12.92. Found: C 32.98, H 3.75, N 12.86.
25
D
solid (38%): mp >145 ꢁC (dec); ½aꢀ À30.06 (c 0.54,
MeOH); 1H NMR (DMSO-d6) d 8.43 (s, 1H, H-6),
7.94, 7.08 (2s, 2H, NH2, D2O exchangeable), 5.85 (d,
1H, H-10, J1 ,F = 17.1 Hz), 5.56 (d, 1H, 30-OH, D2O
4.1.26. b-L-1,2-Di-O-acetyl-3,5-di-O-benzoylribofuranose
0
exchangeable), 5.38 (br s, 1H, 50-OH, D2O exchange-
(42).
l,2-Di-O-isopropylidene-3,5-di-O-benzoyl-a-L-
able), 4.91 (dd, 1H, H-20, J2 ,F = 53.1 Hz), 4.16 (dm,
ribofuranose27 (50 g, 125 mmol) was stirred in AcOH
(500 mL), Ac2O (1.50 mL), and concentrated H2SO4
(10 mL) at room temperature for 16 h. It was then
poured into ice-water, extracted with CHCl3, washed
with satd NaHCO3, dried (MgSO4). The evaporation
of the solvent gave a syrup, from which, a white crystal
was obtained (35 g, 63%), as well as a syrup of 20 g
0
1H, H-30, J3 ,F = 24.3 Hz), 3.86 (m, 1H, H-40), 3.72 (m,
0
2H, H-50a,b). Anal. Calcd for C9H11BrFN3O4+
0.5C2H5OH: C 34.57, H 4.03, N 12.09. Found: C
34.36, H 3.78, N 11.78.
4.1.23. (20S)-b-L-20-Deoxy-20-fluoro-5-iodocytidine (39).
Compound 39 was prepared from 35 in 40% yield as de-
scribed for 40. Trituration of the crude product in ace-
1
(36%): H NMR (CDCl3) d 8.10, 7.41 (m, 10H, Bz),
6.29 (br s, 1H, H-l), 5.80 (t, 1H, H-2), 5.58 (d, 1H,

1650
J. Shi et al. / Bioorg. Med. Chem. 13 (2005) 1641–1652
25
1
H-3), 4.77, 4.50 (m, 3H, H-4, H-5a,b), 2.07, 2.00 (2s, 6H,
Ac).
½aꢀ À60.35 (c 0.14, MeOH); H NMR (DMSO-d6) d
D
11.91 (s, 1H, NH, D2O exchangeable), 8.34 (d, 1H, H-
6, J = 7.1 Hz), 5.86 (d, 1H, H-10, J1 ,F = 16.4 Hz), 5.63
0
4.1.27. b-L-2,20-Anhydro-5-methyluridine (47). A suspen-
sion of thymine (2.52 g, 20.0 mmol) in 1,1,1,3,3,3-hexa-
methyldisilazane (30 mL) was stirred at reflux under
argon for 3 h to give a clear solution, which was then
evaporated to dryness in vacuo to give an oil. To this
was added 42 (4.42 g, 10.0 mmol) under argon followed
by CH3CN (40 mL), and the mixture was stirred at 0 ꢁC,
while TMSOTf (3.88 mL, 20.0 mmol) was added
through a syringe, and the mixture was stirred at room
temperature overnight. It was then poured into ice-
water, extracted with CHCl3 and washed with saturated
NaHCO3 and dried (MgSO4). The removal of the sol-
vent followed by recrystallization in MeOH gave 43 as
a white solid (3.7 g, 73%).
(d, 1H, 30-OH, D2O exchangeable), 5.41 (t, 1H, 50-OH,
D2O exchangeable), 5.02 (dd, 1H, H-20, J2 ,F = 53.0 Hz),
0
4.16 (dm, 1H, H-30, J3 ,F = 22.5 Hz), 3.90 (m, 1H, H-40),
0
3.72 (m, 2H, H-50a,b). Anal. Calcd for C9H10F2N2O5+-
H2O: C 38.27, H 4.25, N 9.92. Found: C 38.42, H 4.33,
N 9.90.
4.1.31.
(20S)-b-L-20-Deoxy-20-fluoro-5-methylcytidine
(59). The title compound was prepared from 57 by using
a similar procedure as described for the synthesis of 38;
59, 32% yield after crystallization from acetone: mp
25
1
>143 ꢁC (dec); ½aꢀ À49.68 (c 0.25, MeOH); H NMR
D
(DMSO-d6) d 7.76 (s, 1H, H-6), 7.42, 6.91 (2s, 2H,
NH2, D2O exchangeable), 5.89 (dd, 1H, H-l0,
J1 ,F = 18.1 Hz), 5.54 (d, 1H, 30-OH, D2O exchange-
0
Compound 43 (3.5 g, 6.9 mmol) was treated with
NaOMe (1.5 g) in MeOH (50 mL) at room temperature
overnight. It was then neutralized with Dowex 50w · 8
(H+) resin and then filtered and washed with MeOH.
The combined filtrate was evaporated to dryness and
the residue was redissolved in water (50 mL), washed
with Et2O (50 mL · 2) and then evaporated to dryness.
The residue was collaborated with EtOH to give 45 as
a white foam (1.7 g, 96%).
able), 5.22 (t, 1H, 50-OH, D2O exchangeable), 4.87
(dd, 1H, H-20, J2 ,F = 53.5 Hz), 4.14 (dm, 1H, H-30,
0
J3 ,F = 22.4 Hz), 3.85 (m, 1H, H-40), 3.68 (m, 2H, H-
0
50a,b), 1.77 (s, 3H, CH3). Anal. Calcd for
C10H14FN3O4+1.5H2O: C 41.92, H 5.94, N 14.68.
Found: C 41.50, H 5.37, N 15.10.
4.1.32. (20S)-b-L-20-Deoxy-20,5-difluorocytidine (60).
The title compound was prepared from 58 by using a
similar procedure as described for the synthesis of 38;
To a stirred solution of 45 (1.6 g, 6.0 mmol) in DMF
(10 mL) was added diphenylcarbonate (2.0 g, 11 mmol)
and NaHCO3 powder (100 mg). The mixture was stirred
at 150 ꢁC for 1 h and then cooled down to room temper-
ature and poured into Et2O (50 mL). After 30 min, the
precipitate formed was filtered and washed with Et2O
(10 mL · 2). The filter cake was recrystallized twice from
EtOH to give 47 as a white solid (1.35 g, 91.0%): mp
222–225 ꢁC.
60, 69% after silica gel column chromatography (10:1/
25
D
5:1 CHCl3/CH3OH): hygroscopic solid; ½aꢀ À90.68 (c
1
0.16, MeOH); H NMR (DMSO-d6) d 8.25 (d, 1H, H-
6, J = 7.4 Hz), 7.85, 7.59 (2s, 2H, NH2, D2O exchange-
able), 5.84 (d, 1H, H-10, J1 ,F = 17.0 Hz), 5.58 (d, 1H,
0
30-OH, D2O exchangeable), 5.36 (t, 1H, 50-OH,
D2O exchangeable), 4.89 (dd, 1H, H-20, J2 ,F = 53.2 Hz),
0
4.14 (dm, 1H, H-30, J3 ,F = 23.6 Hz), 3.88 (m, 1H,
0
H-40), 3.72 (m, 2H, H-50a,b). Anal. Calcd for
C9H11F2N3O4+0.8H2O: C 38.90, H 4.54, N 15.13.
Found: C 38.99, H 4.50, N 15.02.
4.1.28. b-L-2,20-Anhydro-5-fluorouridine (48). The title
compound was prepared from 5-fluorouracil and 42 in
a similar way as described for the synthesis of 47; 81%
yield for 44, 94% yield for 46, and 62% yield for 48:
mp 193–196 ꢁC.
4.1.33.
(20S)-b-L-20-Deoxy-20-fluoro-5-ethynyluridine
(62). Argon was bubbled through Et3N (100 mL) for
0.5 h. To this was added compound 35 (1.22 g,
2.00 mmol), followed by CuI (50 mg), (Ph3P)2PdC12
(50 mg), and trimethylsilyl acetylene (0.71 mL,
5.0 mmol). The mixture was stirred at 50–60 ꢁC for 7 h
and then evaporated to dryness. The residue was redis-
solved in CHCl3 (100 mL), washed with 5% EDTA solu-
tion (50 mL · 3) and satd NaHCO3, dried (MgSO4).
Evaporation of solvent and purification on silica gel col-
umn (100:1 CHCI3/CH3OH) followed by crystallization
from CH3OH gave a white solid (530 mg). To the solid
(480 mg) was added 0.2 N NaOCH3/CH3OH and the
solution was stirred at room temperature for 15 h. It
was then neutralized with Dowex 50w · 8 (H+) resin
and filtered, washed with MeOH. The combined filtrate
was evaporated to dryness, triturated with Et2O to
give 62 as a pale yellow solid (205 mg, 42%): mp
4.1.29. (20S)-b-L-20-Fluorothymidine (55). The title com-
pound was prepared from 47 by using a similar proce-
dure as described for the synthesis of 32; 55, white
25
solid from water (83%): mp 185–188 ꢁC; ½aꢀ À31.88
D
1
(c 0.26, MeOH); H NMR (DMSO-d6) d 11.41 (s, 1H,
NH, D2O exchangeable), 7.80 (s, 1H, H-6), 5.92 (dd,
1H, H-10, J1 ,F = 17.6 Hz), 5.62 (d, 1H, 30-OH, D2O
0
exchangeable), 5.26 (t, 1H, 50-OH, D2O exchangeable),
5.02 (ddd, 1H, H-20, J2 ,F = 53.2 Hz), 4.18 (dm, 1H, H-
0
30, J3 ,F = 20.1 Hz), 3.87 (m, 1H, H-40), 3.68 (m, 2H,
0
H-50a,b), 1.77 (s, 3H, CH3). Anal. Calcd for
C10H13FN2O5: C 46.16, H 5.04, N 10.77. Found: C
45.99, H 5.09, N 10.78.
25
D
1
4.1.30. (20S)-b-L-20-Deoxy-20,5-difluorouridine (56). The
title compound was prepared from 48 by using a similar
procedure as described for the synthesis of 32; 56, white
powder (81%) after silica gel column chromato-
graphy (15:1/8:1 CHCI3/CH3OH): mp 134–136 ꢁC;
>189 ꢁC (dec); ½aꢀ À8.15 (c 0.32, MeOH); H NMR
(DMSO-d6) d 11.71 (s, 1H, NH, D2O exchangeable),
8.46 (s, 1H, H-6), 5.86 (br d, 1H, H-l0, J1 ,F = 16.7 Hz),
0
5.62 (br d, 1H, 30-OH, D2O exchangeable), 5.39 (br s,
1H, 50-OH, D2O exchangeable), 5.04 (dd, 1H, H-20,

J. Shi et al. / Bioorg. Med. Chem. 13 (2005) 1641–1652
1651
J2 ,F = 53.0 Hz), 4.18 (dm, 1H, H-30, J3 ,F = 23.5 Hz),
control (TaqMan Ribosomal RNA control Reagents,
Applied Biosystems, CA) were amplified in a single-step
multiplex RT-PCR protocol, as described.19 Recombi-
nant interferon alfa-2a (INF-a-2a; Roferon-A, Hoffman
La Roche Inc, NJ) served as a positive control.
0
0
4.10 (s, 1H, CCH), 3.86 (m, 1H, H-40), 3.50 (m, 2H,
H-50a,b). Anal. Calcd for C11H11FN2O5: C 48.89, H
4.10, N 10.37. Found: C 48.63, H 4.15, N 10.37.
4.1.34. (20S)-b-L-20-Deoxy-20-fluoro-5-ethyluridine (63).
Compound 62 (108 mg, 0.40 mmol) was stirred in EtOH
(10 mL) with 10% Pd–C (50 mg) under H2 at 1 atm for
1 h, then filtered and washed with EtOH. The combined
filtrate was evaporated to dryness and co-evaporated
Acknowledgements
HCV replicon RNA-containing Huh 7 cells were pro-
vided by Apath, LLC, St. Louis, MO. C.K.C. and
R.F.S. are supported in part by NIH grant RO1-AI-
32351. C.K.C. is supported in part by NIH grant
1-RO1-AI-25899. R.F.S. is supported by NIH grant
2P30-AI-50409, and the Department of Veterans
Affairs. R.F.S. and C.K.C. are the founders and consul-
tants of Pharmasset Ltd, and did not receive any fund-
ing from the company for these studies.
with ether to give 63 as a white solid (110 mg, 100%):
25
mp 158–160 ꢁC; ½aꢀ À21.79 (c 0.46, MeOH); 1H
D
NMR (DMSO-d6) d 11.35 (s, 1H, NH, D2O exchange-
able), 7.78 (s, 1H, H-6), 5.90 (br d, 1H, H-10, J1 ,F
=
0
17.6 Hz), 5.71 (br s, 1H, 30-OH, D2O exchangeable),
5.34 (br s, 1H, 50-OH, D2O exchangeable), 5.01 (dd,
1H, H-20, J2 ,F = 53.2 Hz), 4.18 (dm, 1H, H-30,
0
J3 ,F = 20.9 Hz), 3.85 (m, 1H, H-40), 3.76 (m, 2H, H-
0
50a,b), 2.18 (q, 2H, CH2CH3), 1.06 (t, 3H, CH2CH3).
Anal. Calcd for C11H15FN2O5: C 48.18, H 5.51, N
10.21. Found: C 48.24, H 5.47, N 10.14.
References and notes
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