Synthesis and anti-HCV activity of 3′,4′-oxetane nucleosides

Article abstract of DOI:10.1016/j.bmcl.2010.06.025

Hepatitis C virus afflicts approximately 180 million people worldwide and currently there are no direct acting antiviral agents available to treat this disease. Our first generation nucleoside HCV inhibitor, RG7128 has already established proof-of-concept

Full text of DOI:10.1016/j.bmcl.2010.06.025

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4539–4543
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-HCV activity of 30,40-oxetane nucleosides
Wonsuk Chang a, Jinfa Du a, Suguna Rachakonda a, Bruce S. Ross a, Serge Convers-Reignier b, Wei T. Yau b,
Jean-Francois Pons b, Eisuke Murakami a, Haiying Bao a, Holly Micolochick Steuer a, Phillip A. Furman a,
Michael J. Otto a, Michael J. Sofia a,
*
a Pharmasset, Inc., 303A College Road East, Princeton, NJ 08540, USA
b Evotec (UK) Ltd, 114 Milton Park, Abingdon, Oxfordshire OX14-4SA, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 February 2010
Revised 2 June 2010
Accepted 4 June 2010
Available online 8 June 2010
Hepatitis C virus afflicts approximately 180 million people worldwide and currently there are no direct
acting antiviral agents available to treat this disease. Our first generation nucleoside HCV inhibitor,
RG7128 has already established proof-of-concept in the clinic and is currently in phase IIb clinical trials.
As part of our continuing efforts to discover novel anti-HCV agents, 30,40-oxetane cytidine and adenosine
nucleosides were prepared as inhibitors of HCV RNA replication. These nucleosides were shown not to be
inhibitors of HCV as determined in a whole cell subgenomic replicon assay. However, 20-mono/diflouro
analogs, 4, 5, and 6 were readily phosphorylated to their monophosphate metabolites by deoxycytidine
kinase and their triphosphate derivatives were shown to be inhibitors of HCV NS5B polymerase in vitro.
Lack of anti-HCV activity in the replicon assay may be due to the inability of the monophosphates to be
converted to their corresponding diphosphates.
Keywords:
Antivirals
Hepatitis C
HCV
RG7128
Ó 2010 Elsevier Ltd. All rights reserved.
NS5B polymerase
Bicyclic nucleosides
Hepatitis C virus (HCV) is a leading cause of chronic liver dis-
ease.1 Nearly 1.6% of the U.S. population and an estimated 180 mil-
lion people worldwide are infected with HCV.2 Approximately 80%
of infected individuals become chronically infected; this chronic
infection can lead to liver cirrhosis and hepatocellular carcinoma
in a significant number of patients.3 The current standard of care
(SOC) for those infected with HCV includes administration of pegy-
lated interferon alpha and ribavirin. However, only 50% of patients
infected with genotype 1 virus respond to treatment with SOC
therapy. In addition, the SOC regimens are associated with various
intolerable adverse side effects.4 To date no vaccine has been
developed to treat HCV due to the multiple genotypes of HCV
and to the relatively high mutation rate associated with this virus.5
Consequently, there is an urgent need for the identification of new
small molecule direct acting antiviral agents to effectively treat
chronic HCV infection.
(2) is an alternative substrate of the HCV RdRp and acts as a non-
obligate chain terminator.7,8
Other than the 20- -fluoro-20-C-methyl class of nucleosides rep-
a
resented by PSI-6130 (1), several other nucleoside classes have
been reported to exhibit in vitro anti-HCV activity. These other
nucleosides include 20-deoxy-20- -fluorocytidine,9 20-
a a-O-methyl
substituted nucleosides, 20-C-methyl ribonucleosides,10,11 and 40-
substituted nucleoside derivatives where 20-substitution can be
either a
-OH, b-OH, b-F or diF.12,13 What is also known about these
nucleosides is that the 40-substituted nucleosides possess a differ-
ent conformational preference (20-endo) relative to the other clas-
ses which prefer a 30-endo conformation ( Fig. 2), yet they still
demonstrate good in vitro potency as inhibitors of HCV.7,12 There-
fore, we were interested in taking key features of each of these
NH2
N
NH2
N
RG7128 is a nucleoside prodrug of b-D a
-20-deoxy-20- -fluoro-20-
C-methylcytidine 1 (PSI-6130, Fig. 1) and is a specific inhibitor of
the HCV NS5B RNA dependent RNA polymerase (RdRp). Recently,
we reported exceptional clinical efficacy of RG7128 as an anti-
HCV direct acting antiviral agent.6 Since 1 is a nucleoside, in order
to inhibit the HCV RdRp, it must be phosphorylated by cellular ki-
nases to its 50-triphosphate form. The 50-triphosphate of PSI-6130
O
O
P
O
N
O
N
O
HO
HO P O
O P O
O
O
OH OH OH
HO
F
HO
F
2
1, PSI-6130
Ki = 0.059 µM for HCV NS5B
Figure 1.
EC90 = 4.6 µM
* Corresponding author. Tel.: +1 609 613 4123; fax: +1 609 613 4150.
E-mail address: michael.sofia@pharmasset.com (M.J. Sofia).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.025

4540
W. Chang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4539–4543
stricted nucleoside derivatives. Introducing
a conformational
Base
HO
HO
Base
restriction by the use of fused ring system is a well-established ap-
proach to enhance a specific biological activity of a nucleoside.14
This modification has been extensively pursued not only for anti-
sense oligonucleotides,15,16 but also for direct-acting therapeutic
agents such as antituberculosis,17 antitumor,18 and antivirals.19 In
pursuing this concept of conformationally restricted nucleosides
we chose to investigate the 30-O,40-C methylene (30,40-oxetane)
nucleoside template. The ribofuranose ring of a 30,40-oxetane
nucleoside is known to prefer the C20-endo (Southern-type) con-
formation similar to the 20-deoxy-40-azido substituted anti-HCV
nucleosides.12,16 However, the biological activity of this class of
molecules against HCV remains unexplored. Therefore, we pro-
ceeded to investigate the anti-HCV activity of the 30,40-oxetane
nucleosides introducing substitution at the 20-position known to
be compatible with anti-HCV activity (Fig. 3).
O
O
HO
R
R
OH
C2'-endo
C3'-endo
Figure 2. C20- and C30-endo conformations of nucleosides.
HO
HO
Cytosine
R2
Base
R2
O
O
R1
R1
O
HO
3, Base = cytosine, R1 = F; R2 = Me
9, R1 = F; R2 = H
10, R1 = H; R2 = F
11, R1 = F; R2 = F
4, Base = cytosine, R1 = F; R2 = H
R1 = H; R2 = F
R1 = F; R2 = F
5, Base = cytosine,
6, Base = cytosine,
Oxetane nucleoside 3 was prepared from 20-deoxy-20-fluoro-20-
C-methyluridine 12 (Scheme 1). The 50- and 30-hydroxyl groups
were sequentially protected with a dimethoxytrityl group and a
tert-butyldimethylsilyl group. After deprotection under acidic con-
ditions, the primary alcohol at C50 was oxidized via the Pfitzner–
Moffatt reaction20 to give aldehyde 16. Subsequent aldol reaction
with formaldehyde followed by reduction of the aldehyde provided
7, Base = cytosine, R1 = OMe; R2 = H
8, Base = adenine, R1 = F; R2 = H
Figure 3.
nucleosides and developing a novel series of compounds as poten-
tial inhibitors of HCV replication.
In exploring the potential of novel nucleosides as anti-HCV
agents, we were interested in investigating conformationally re-
diol 17. Then, the less hindered C50-primary alcohol on the
a-face
of diol 17 was selectively protected with a dimethoxytrityl group.
The other primary alcohol was then protected with TBDPS. Subse-
O
O
N
O
N
NH
NH
a
NH
b
O
N
O
O
O
O
O
O
HO
DMTrO
TBSO
DMTrO
12
14
HO
HO
O
13
F
F
F
O
N
O
N
O
NH
NH
NH
c
d
e
HO
HO
O
N
O
O
O
O
HO
TBSO
TBSO
TBSO
17
15
16
F
F
F
O
O
O
NH
NH
NH
h
g
f
HO
DMTrO
TBDPSO
DMTrO
TBDPSO
HO
N
O
N
O
N
O
O
O
O
TBSO
TBSO
19
Bz
N
TBSO
18
20
F
F
F
Bz
N
HN
N
O
HN
N
NH
i
j
k
TBDPSO
TfO
TBDPSO
TfO
O
N
O
O
O
O
O
HO
21
22
23
TBSO
F
TBSO
O
F
F
l
3
Scheme 1. Reagents and conditions: (a) DMTrCl, pyridine, 0 °C to rt, 8 h, 93%; (b) TBSCl, imidazole, rt, 16 h, 92%; (c) 3% TFA in DCM, rt, 3 h, 66%; (d) EDC, DMSO, pyridine, TFA,
rt, 30 min, 50%; (e) CH2O, NaOH, aq dioxane, rt, 30 min; NaBH4, 0 °C to rt, 1 h, 45%; (f) DMTrCl, pyridine, 0–10 °C, 8 h, 67%; (g) TBDPSCl, imidazole, rt, 18 h, 46%; (h) CAN,
}
}
MeCN, rt, 16 h, 83%; (i) Tf2O, 2,6-lutidine, DCM, 16 h, 91%; (j) TIPBSCI, Hunig’s base, DMAP, CH3CN, rt, 1 h; NH4OH, rt, 0.5 h; BzCl, Hunig’s base, DMAP, rt, 1 h, 65%; (k) TBAF,
THF, reflux, 3 h, 58%; (l) 7 N NH3 in MeOH, rt, 6 h, 62%.

W. Chang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4539–4543
4541
quent removal of the DMTr protection and derivatization as the tri-
flate gave intermediate 21. Conversion to the cytidine analog 22
was accomplished by mild ammonolysis of the 2,4,6-tri-
isopropylbenzenesulfonyl ester. Deprotection of 30-silyl group trig-
gered the key ring closure to afford oxetane 23. Finally, removal of
the 6-N-benzoyl group gave the desired 30,40-oxetane cytidine
modified nucleoside 3. Cytidine nucleosides 4 to 7 were prepared
via similar synthetic sequences starting from corresponding uri-
dine nucleosides.
same substitution pattern at C20 as the potent HCV inhibitor, 1
did not show significant anti-HCV activity. As previously reported,
20-
a-fluorocytidine 9 showed effective inhibition of HCV; however,
its corresponding oxetane derivative 4 did not significantly inhibit
HCV.9 The 40-azido-20-deoxy-20-b-fluorocytidine was previously
shown to be a potent inhibitor of HCV but, again, the 20-deoxy-
20-b-fluoro-30,40-oxetane nucleoside 5 was inactive as an HCV
inhibitor.13 An attempt to gain activity by combining both an
a
- and b-fluoro group at 20 gave oxetane 6. However no HCV inhib-
itory activity was observed for this compound when assayed at
50 -methoxy derivative 7 also did not show activity
M. The 20-
at 50 M. To assess if a base modification could induce anti-HCV
The 20-deoxy-20-C-methyl adenosine analog was shown to be a
potent inhibitor in a HCV replicon assay.10 Therefore, we wished to
prepare a purine analog of a 30,40-oxetane nucleoside in addition to
the pyrimidines. The synthesis began with N-6 protection of 20-
deoxy-20-b-fluoro-adenosine (Scheme 2). After a series of protec-
tion and deprotection steps to give the nucleoside 28 containing
a protected 30-hydroxyl group, the free 50-hydroxyl group was oxi-
dized to give aldehyde 29, which upon aldol condensation and
reduction provided diol 30. Unlike the case of the uridine analog
l
a
l
activity when combined with an oxetane template, the adenosine
derivate 8 was evaluated, and again no significant activity was ob-
served. It is also noteworthy that none of these oxetane nucleo-
sides was shown to be cytotoxic when evaluated at
a
-40-
Table 1
HCV clone A replicon data
17, selective derivatization of the hydroxyl group on the
hydroxymethylene moiety was not observed in diol 30.21 Instead,
cyclicsulfate 31 was prepared as an intermediate for oxetane ring
closure. After removal of the silyl protecting group on the 30-oxy-
gen, intermediate 32 was treated under basic conditions to effect
oxetane ring formation. Hydrolysis of the sulfonic acid salt and re-
moval of N-benzoyl protection group completed the sequence and
afforded the desired 30,40-oxetane adenine nucleoside 8 in 67%
from cyclic sulfate 31.
Compound
EC90
(l
M)
CC50 (lM)
1
3
4
5
6
7
8
9
10
11
4.6
>50
>50
>50
>50
>50
>50
>50
>50
>50
<0.1
>50
>50
>50
>50
>50
>50
5.21
>50
<1
Anti-HCV activity of the 20-substituted-30,40-oxetane nucleo-
sides was determined using the subgenomic replicon assay (Table
1).22 The 20-b-methyl-20-
a
-fluoride derivative 3 which shares the
NHBz
NHBz
NH2
N
N
N
N
N
N
a
N
N
N
N
b
N
O
O
O
N
HO
DMTrO
HO
24
26
25
F
F
F
HO
HO
NHBz
HO
NHBz
N
N
N
N
N
N
N
c
d
e
O
O
N
DMTrO
TBSO
HO
27
28
F
F
TBSO
NHBz
NHBz
NHBz
N
N
N
N
N
N
N
N
N
N
N
N
O
f
g
HO
O
S
O
O
O
O
O
O
TBSO
HO
TBSO
NHBz
29
30
31
F
F
F
TBSO
NHBz
N
N
N
N
N
N
O
O
h
j
i
S
N
O
O
O
N
8
KO3SO
O
33
32
O
F
HO
F
Scheme 2. Reagents and conditions: (a) TMSCl, pyridine, rt, 1 h, then, BzCl, 0 °C to rt, 3 h, 85%; (b) DMTrCl, pyridine, 0 °C to rt, 3 h, 94%; (c) TBSCl, imidazole, DMF, rt, 16 h; (d)
TsOH, DCM/MeOH, 0 °C, 3 h, 70% in two steps; (e) Dess–Martin periodinane, DCM, 0 °C to rt, 4 h, 71%; (f) CH2O, NaOH, aq dioxane, rt, 10 h; NaBH4, EtOH, 0 °C to rt, 1 h, 47%; (g)
SOCl2, pyridine, DCM, ꢀ10 °C to 0 °C; RuCl3–xH2O, NaIO4, DCM/MeCN/H2O, rt, 1 h, 81%; (h) TBAF, THF, rt, 1 h, 76%; (i) KOtBu, THF, 0–10 °C, 30 min; (j) H2SO4, rt, 48 h; NH3,
MeOH, rt, 16 h, 67% in three steps.

4542
W. Chang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4539–4543
concentrations up to 50 l
M. Even in the case of the 20-difluoro
Supplementary data
derivative 6 which is an analog of the highly cytotoxic nucleoside
Gemcitabine (11) no cytotoxicity was observed.23
Spectral data for all new 3’,4’-oxetane nucleosides are provided.
Details on NS5B RNA polymerase assays are given. Supplementary
data associated with this article can be found, in the online version,
at doi:10.1016/j.bmcl.2010.06.025.
Since nucleosides must be anabolized to their triphosphate
derivative in order to inhibit the HCV RdRp, it is possible that the
lack of activity exhibited by these oxetane nucleosides may be as
a result of their inability to be phosphorylated by cellular kinases
in the phosphorylation cascade. To investigate this possibility, we
prepared the triphosphates of three 30,40-oxetane nucleosides
(4-TP, 5-TP, and 6-TP).24 These triphosphates were shown to inhi-
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bit the HCV NS5B polymerase in vitro with IC50s from 30 to 80 lM
(Table 2). Since these triphosphates inhibited the HCV RdRp, lack of
anti-HCV activity may be due to a problem with the phosphoryla-
tion of the nucleosides to their corresponding triphosphates.
Therefore, the phosphorylation of the 30,40-oxetane nucleosides to
their monophosphates by deoxycytidine kinase (dCK) was evalu-
ated.25 As shown in Table 3, several 30,40-oxetane nucleosides (4,
5, and 6) were determined to be substrates for dCK and showed
similar or better substrate efficiency compared to the known
HCV inhibitor 1.
There are several known human nucleoside monophosphate
kinases including UMP-CMP kinase, thymidylate kinase, adenylate
kinase, and guaniylate kinase. Typically, UMP-CMP kinase is
responsible for phosphorylation of deoxycytidine monophosphate
and its analogs.26 Therefore, the second phosphorylation step
was studied by treating the 30,40-oxetane nucleosides (4, 5, and
6) with both dCK and UMP-CMP kinase and the phosphorylated
products were detected by thin layer chromatography. Significant
amounts of the monophosphate products were formed due to
phosphorylation of the nucleosides by dCK but very little or no
30,40-oxetane nucleoside diphosphate products were detected, indi-
cating that 30,40-oxetane nucleoside monophosphates are not sub-
strates for UMP-CMP kinase (data not shown). Therefore, we can
conclude that the lack of anti-HCV activity of the 30,40-oxetane
nucleosides studied can most likely be attributed to the fact that
their monophosphates are not substrates for the monophosphate
kinases which are required for the biosynthesis of the active tri-
phosphate in the cell.
In summary, we prepared several novel 30,40-oxetane nucleo-
sides and studied their anti-HCV activities. Although these oxetane
containing nucleosides did not show significant anti-HCV activity
in the whole cell replicon assay, representative triphosphate deriv-
atives (4-TP, 5-TP, and 6-TP) were shown to be inhibitors of the
HCV NS5B RdRp with the triphosphate of 4 being the most potent
(IC50 = 31 lM), Even though representative oxetane derivatives are
substrates for the first kinase in the phosphorylation cascade
(dCK), their lack of whole cell replicon activity can be attributed
to the inability of the monophosphate kinases to phosphorylate
the monophosphates of 4, 5, and 6.
14. For a review, see: Meldgaard, M.; Wengel, J. J. Chem. Soc., Perkin Trans. 1 2000,
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Table 2
Inhibition of HCV polymerase (NS5B) activity in vitro
Compound
IC50 (lM)
4-TP
5-TP
6-TP
2, PSI-6130-TP
30.96 4.75
78.91 5.68
32.76 5.36
5.37 0.50
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Table 3
Steady-state parameters for dCK reactions
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Compound
kcat (sꢀ1
)
Km
(
lM)
kcat/Km (l )
Mꢀ1 sꢀ1
4
5
6
0.0214
0.1221
0.0599
0.016
60.95
81.1
27.6
81.2
3.5 ꢁ 10ꢀ4
1.5 ꢁ 10ꢀ3
2.2 ꢁ 10ꢀ3
1.9 ꢁ 10ꢀ4
1, PSI-6130

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4543
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