Design, synthesis, and antiviral properties of 4′-substituted ribonucleosides as inhibitors of hepatitis C virus replication: The discovery of R1479

Article abstract of DOI:10.1016/j.bmcl.2007.02.004

A series of 4′-substituted ribonucleoside derivatives has been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell culture. The most potent and non-cytotoxic derivative was compound 28 (4′-azidocytidine, R1479) with an

Full text of DOI:10.1016/j.bmcl.2007.02.004

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2570–2576
Design, synthesis, and antiviral properties of 40-substituted
ribonucleosides as inhibitors of hepatitis C virus replication:
The discovery of R1479
David B. Smith,a,* Joseph A. Martin,a,b Klaus Klumpp,e Stewart J. Baker,b
Peter A. Blomgren,a Rene Devos,c Caroline Granycome,c,e Julie Hang,e
Christopher J. Hobbs,b Wen-Rong Jiang,c,e Carl Laxton,c Sophie Le Pogam,e
Vincent Leveque,e Han Ma,e Graham Maile,b John H. Merrett,b
Arkadius Pichota,a Keshab Sarma,d Mark Smith,a Steven Swallow,a
Julian Symons,e David Vesey,b Isabel Najerac,e and Nick Cammacke
aMedicinal Chemistry, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
bMedicinal Chemistry, Roche Discovery Welwyn, Broadwater Road, Welwyn Garden City, Herts AL7 4AY, UK
cViral Diseases TA, Roche Discovery Welwyn, Broadwater Road, Welwyn Garden City, Herts AL7 4AY, UK
dProcess Research, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
eViral Diseases TA, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
Received 22 December 2006; revised 31 January 2007; accepted 2 February 2007
Available online 4 February 2007
Abstract—A series of 40-substituted ribonucleoside derivatives has been prepared and evaluated for inhibition of hepatitis C virus
(HCV) RNA replication in cell culture. The most potent and non-cytotoxic derivative was compound 28 (40-azidocytidine, R1479)
with an IC50 of 1.28 lM in the HCV replicon system. The triphosphate of compound 28 was prepared and shown to be an inhibitor
of RNA synthesis mediated by NS5B (IC50 = 320 nM), the RNA polymerase encoded by HCV. Data on related analogues have
been used to generate some preliminary requirements for activity within this series of nucleosides.
Ó 2007 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) is a major causative agent of
chronic liver disease and it is estimated that more than
170 million individuals worldwide are infected with this
pathogen.1 After initial infection, the majority of HCV
infections become chronic which can lead to cirrhosis
of the liver, hepatocellular carcinoma, and eventually
death. Furthermore, HCV infection is a leading cause
of liver disease, necessitating organ transplantation.
Currently, interferon-based therapies are the only
FDA-approved regimens for the treatment of HCV
infection. Response rates are in the 50–90% range
depending on the genotype of the infecting virus. In
addition, response rates are markedly reduced in indi-
viduals co-infected with HIV, with cirrhosis, and in
older patients. Most individuals experience undesirable
side effects with the currently available therapies and
in many cases patients choose to discontinue their treat-
ment. In addition, there are significant numbers of indi-
viduals who do not respond to interferon-based
therapies, therefore, there is an urgent need to discover
and develop new HCV therapeutic agents that are more
effective either alone or in combination with interferon.
Historically, the majority and most successful antiviral
agents in terms of efficacy and side-effect profile have
been targeted against viral-encoded enzymes. The larg-
est group of antiviral agents approved by regulatory
authorities inhibit viral nucleic acid polymerases, and
within that group nucleoside analogues represent the
majority, exemplified by agents approved to treat HIV,
hepatitis B (HBV), and the herpes group of viruses.
All of these are inhibitors of DNA synthesis catalyzed
by the virally encoded DNA polymerases. In fact, the
active entity is the triphosphate of the nucleoside
Keywords: Hepatitis C; HCV; R1479; Antiviral; Nucleoside; Ribonu-
cleoside; Replicon; NS5B.
*
Corresponding author. Tel.: +1 650 852 1126; fax: +1 650 855
6908; e-mail: Dave.Smith@Roche.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.004

D. B. Smith et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2570–2576
2571
NH2
NH2
N
NH2
N
NH2
N
NH2
N
N
N
HO
HO
HO
HO
N
HO
N
N
N
N
N
O
O
O
O
O
O
O
O
O
HO
F
HO OMe
HO OH
HO
F
HO OH
3
4
1
2
5
Figure 1. Recently reported nucleoside inhibitors of NS5B.
analogue, which is formed by metabolism within the
infected cell. The polymerase encoded by HCV (NS5B)
is an RNA dependent RNA polymerase, and since there
are no marketed inhibitors of viral RNA polymerases,
this provides both a challenge and an opportunity to dis-
cover potent and selective inhibitors of HCV replication
that could form the basis of new treatment regimens.
further chain elongation, an alternate avenue for chain
termination after incorporation would have to be
operative. In order to qualitatively explore the possible
structure–activity relationships, we targeted a range of
40-substituents which varied in size, shape, and polarity.
Here we describe the SAR of cytidine and uridine
analogues from this series (Fig. 2).
There are recent reports of 20-modified ribonucleoside
analogues that inhibit HCV replication (Fig. 1).2 One
example is 20-deoxy-20-a-fluorocytidine 12a–c, but this
and other examples of 20-deoxy-20-a-fluoronucleosides
have been shown to have low selectivity for HCV, which
can probably be attributed to their ability to inhibit both
viral and cellular polymerases. Because of their poor
selectivity this class of nucleosides is unlikely to form
the basis of a useful drug candidate. The 20-O-meth-
ylcytidine analogue 2 has also been reported as an inhib-
itor of HCV, although the cellular activity reported for
this compound is an order of magnitude lower than that
of compounds currently in clinical development.2d
Another group of nucleosides that possess a 20-b-methyl
substituent has provided several examples of inhibitors
of HCV replication, and the corresponding triphos-
phates have been shown to be inhibitors of HCV poly-
merase. While 32d and its 7-deaza analogue2e have
been reported to have interesting preclinical properties,
of particular interest is 20-b-methylcytidine 42f which is
currently in Phase II clinical trials for the treatment of
HCV infection. Another recent disclosure combines ele-
ments of both of the above structural classes, that is, 20-
deoxy-20-b-methyl-20-a-fluorocytidine 5.2c,g
Synthetic routes used to prepare the target molecules
were chosen according to the nature of the 40-substituent
and were based on procedures described in the litera-
ture. Cytidine derivatives were in general prepared from
the corresponding uridine analogues.
Compound 10 (40-azidouridine) was prepared as out-
lined in Scheme 1, which is a modification of the proce-
dures described by Verheyden and Moffatt.3 Treatment
of 64 with iodine azide, generated in situ from iodine
chloride and sodium azide, gave compound 7 which
was benzoylated with benzoyl chloride in pyridine to
give 8. Oxidative de-iodination of compound 8 with
m-chloroperbenzoic acid gave 9, which after deprotec-
tion with methanolic ammonia gave the desired 40-azi-
douridine 10 in good overall yield.
40-Propynyluridine 15 was readily prepared as illustrat-
ed in Scheme 2 from compound 115 by treatment with
propynyl magnesium bromide followed by ozonolysis
of the isopropenyl group to give as a key intermediate
the hemiacetal 12. Removal of the isopropylidine pro-
tecting group from 12 with acetic acid, followed by per-
acetylation with acetic anhydride in pyridine, gave
compound 13. Condensation of 13 with persilylated ura-
Here, we present our early studies directed toward the
design and discovery of the totally different class of 40-
substituted ribonucleoside analogues that are potent
and selective inhibitors of NS5B and of HCV
replication.
cil using the conditions of Vorbruggen6 gave the protect-
¨
ed uridine 14, which upon treatment with methanolic
ammonia gave the target uridine 15 in good yield. Com-
pound 16 was also prepared from 11 in a similar manner
as described for 15, but using ethyl Grignard in the first
step. For the coupling step, persilylated N-benzoyl
All of the marketed HIV, HBV and herpes antiviral
nucleoside analogues inhibit viral DNA synthesis
mediated through virally encoded polymerases. The
majorities of these are either 30-deoxy- or 20,30-dide-
oxy-nucleoside analogues, and thus act as obligate chain
terminators. Chain termination is effected by these mol-
ecules after the corresponding nucleoside analogue has
been phosphorylated and becomes incorporated into
the growing chain of DNA. Since there is no 30-hydroxy
group, further elongation of the growing nucleic acid
chain is not possible. For a 40-substituted ribonucleo-
side, which possesses the 30-hydroxyl necessary for
O
NH2
N
NH
HO
HO
N
N
O
O
O
O
R
R
HO OH
HO OH
Figure 2. Ribonucleosides targeted in this work.

2572
D. B. Smith et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2570–2576
O
O
O
NH
NH
NH
O
ICl, NaN3
BzCl
I
I
N
N
N
O
O
O
O
O
N3
N3
BzO OBz
HO OH
HO OH
6
7
8
m-CPBA
O
O
NH
NH
NH3/MeOH
HO
N3
BzO
N3
N
N
O
O
O
O
HO OH
HO OBz
10
9
Scheme 1.
TBDMSO
TBDMSO
O
TBDMSO
AcO
1) HOAc
2) Ac2O
O
OH
O
OAc
1) propynyl-MgBr
O
O
O
O
2) O3
OAc
13
11
12
TMS-uracil
TMSOTf
O
O
NH2
N
NH
NH
O
TBDMSO
NH3/MeOH
HO
N
HO
N
N
O
O
O
O
O
AcO OAc
14
HO OH
15
HO OH
16
Scheme 2.
O
1) DMT-Cl
2) TBDMS-Cl
3) HOAc
O
O
1) Ph3P=CHCl
2) nBuLi
NH
N
NH
O
NH
O
HO
HO
TBDMSO
4) Swern
TBDMSO
N
O
O
N
O
O
O
TBDMSO OTBDMS
TBDMSO OTBDMS
TBDMSO OTBDMS
17
19
22
1) NH2OH.HCl/pyr.
2) Ac2O/NaOAc
3) nBu4NF
1) Ph3P=CH2
2) nBu4NF
nBu4NF
nBu4NF
O
O
O
O
NH
N
NH
NH
NH
HO
HO
N
HO
HO
HO
N
N
N
O
O
O
O
O
O
O
O
HO OH
HO OH
HO OH
HO OH
18
20
21
23
Scheme 3.

D. B. Smith et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2570–2576
2573
O
O
O
1) allyl-TMS
1) TBDMS-Cl
NH
NH
O
SnCl4
NH
HO
N
O
N
N
O
O
O
O
O
O O
2)
HO OH
TBDMSO OTBDMS
TBDMSO OTBDMS
6
24
25
O
nBu4NF
NH
HO
N
O
O
HO OH
26
Scheme 4.
O
NH2
N
O
1) 1,2,4-triazole
Et3N, POCl3
Ac2O/pyr.
NH
NH
HO
HO
N
AcO
N
N
O
O
O
O
O
O
2) NH4OH
3) NH3 / MeOH
R
R
R
HO OH
HO OH
28 (R = -N3)
AcO OAc
10 (R = -N3)
27
15 (R = -CCMe)
18 (R = -CH2OH)
29 (R = -CCMe)
30 (R = -CH2OH)
20 (R = -CN)
21 (R = -CH=CH2)
31 (R = -CN)
32 (R = -CH=CH2)
23 (R = -CCH)
26 (R = -CH2CH=CH2)
33 (R = -CCH)
34 (R = -CH2CH=CH2)
Scheme 5.
cytidine was used, and the corresponding uridine deriv-
ative was not prepared.
40-Allyluridine was prepared according to Scheme 4.
Protection of the previously described olefin 6 with
TBDMS chloride followed by reaction with dimethyldi-
oxirane gave the b-epoxide 24. Treatment of the epoxide
with allyl trimethylsilane in the presence of anhydrous
stannic chloride gave the protected uridine 25.10 Depro-
tection of 25 with TBAF afforded the target uridine,
compound 26.
A range of other 40-substituted uridines were prepared
as shown in Scheme 3 from the key starting material
17.7 Deprotection of compound 17 with TBAF (tetrabu-
tylammonium fluoride) gave 40-hydroxymethyl uridine
188 in excellent yield. Selective protection of the 40-a-hy-
droxymethyl substituent of compound 17 was achieved
by treatment with dimethoxytrityl chloride in pyridine,
and was followed by protection of the 40-b-hydroxy-
methyl group with TBDMS chloride. Removal of the
dimethoxytrityl group with acetic acid, followed by
Swern oxidation,9 gave the aldehyde derivative 19. Con-
densation of the aldehyde function in compound 19 with
hydroxylamine gave the corresponding oxime which was
dehydrated with acetic anhydride in the presence of
sodium acetate to give the nitrile. Global deprotection
with TBAF gave 40-a-cyanouridine 20. Alternatively,
treatment of 19 with methylene triphenylphosphorane
under Wittig conditions followed by deprotection with
TBAF gave 40-a-vinyl-uridine 21. Next, intermediate
19 was condensed with a chloromethylene triphenyl-
phosphorane Wittig reagent, which was followed by
dehydrohalogenation with butyl lithium to provide com-
pound 22. Deprotection with TBAF was conducted to
afford the target molecule, 40-a-ethynyluridine 23.
An efficient method for the conversion of uridines to the
corresponding cytidines has been described by Divakar
and Reese,11 and was used in this work as shown in
Scheme 5. Accordingly, each of the uridines 10, 15, 18,
20, 21, 23, and 26 was peracetylated with acetic anhy-
dride in pyridine to give the corresponding protected
derivatives represented by the generic structure 27. Acti-
vation of the 4-keto group using phosphorus oxychlo-
ride and triazole was followed by treatment with
aqueous ammonia to afford the corresponding triacety-
lated cytidine derivatives, which after treatment with
methanolic ammonia gave the desired cytidines 28–34,
respectively, in good overall yield.
Finally, the synthesis of 40-ethoxycytidine is presented in
Scheme 6. For this compound, we did not discretely pre-
pare the corresponding uridine analogue, but utilized a
minor variation of the procedure reported by Verheyden

2574
D. B. Smith et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2570–2576
O
O
NH2
N
1) I2, EtOH
PbCO3
NH
NH
O
1) Ac2O
N
N
N
BzO
HO
O
O
O
O
O
2) POCl3
triazole
3) NH3
2) BzCl
3) mCpBA
EtO
HO
EtO
HO
HO
OH
OBz
OH
6
36
35
Scheme 6.
and Moffatt.3b Starting from 6, addition of iodine in
ethanol in the presence of lead carbonate installed the
requisite 40-ethoxy group. Following protection of the
20- and 30-hydroxyl groups with benzoyl chloride, dis-
placement of the iodide as previously described for the
conversion of 8–9 in Scheme 1 furnished 35. Acetylation
of the newly liberated 30-hydroxy was followed by con-
version of uridine to cytidine in the usual manner, with
global deprotection using ammonia providing the target
compound 36.
Table 1. Initial evaluation of activity and cytotoxicity in the Huh-7
replicon assay
Compound
(base C/U)
40-a-Substituent
(R)
Replicon
inhibition
at 20 lM
Cytotoxicity
at 20 lM
10 (U)
28 (C)
16 (C)
36 (C)
18 (U)
30 (C)
20 (U)
31 (C)
23 (U)
33 (C)
15 (U)
29 (C)
26 (U)
34 (C)
21 (U)
32 (C)
N3
N3
13%
97%
20%a
11%
1%
17%
13%
20%a
0%
Et
EtO
CH2OH
CH2OH
C„N
0%
2%
13%
0%
HCV replicon assays.12 The evaluation of compounds,
inhibitory activity at 20 lM and/or the determination
of the inhibitory concentrations at which a 50% reduc-
tion in replicon replication was observed (IC50) were ob-
tained using replicon cell line 2209-23. This cell line was
established from the Huh-7 cells expressing a HCV
genotype 1b subgenomic replicon including Renilla
luciferase as a reporter gene. Quantification of Renilla
luciferase activity was performed using the Renilla lucif-
erase assay kit (Promega) according to manufacturer’s
instructions. The WST-1 or MTT assays (Roche Diag-
nostics) were used to measure cell viability.
0%
C„N
99%
0%
100%
0%
0%
19%a
C„CH
C„CH
C„CMe
C„CMe
CH2CH@CH2
CH2CH@CH2
CH@CH2
CH@CH2
3%
76%a
11%
N.D.b
0%
0%
N.D.b
0%
0%
50%a
53%a
0%
C, cytidine; U, uridine.
a Activity at 100 lM.
b IC50 and CC50 are reported in Table 2.
Replicon proliferation assay.12 The effect of compounds
on the incorporation of tritiated thymidine into cellular
DNA was measured using the SPA [3H]-thymidine
Table 2. IC50, CC50, and CT50 determination
Compound
Replicon Cytotoxicity Cytostaticity
IC50 (lM) CC50 (lM) CT50 (lM)
incorporation
Biosciences.
assay
system
from
Amersham
28 40-Azido-cytidine 1.28
>2000
39
>100
>100
N.D.
0.8
26 40-Allyl-uridine
1 20-Deoxy-20-a-F-
cytidine
22
0.58
HCV polymerase assay.12 The enzymatic activity of
NS5B570-BK proteins was measured as incorporation
of radiolabeled nucleoside monophosphate into acid-in-
soluble RNA products using HCV cIRES RNA as a
template.
4 20-b-Me-cytidine
1.23
>33
>100
toxic effect of the same order of its replicon potency,
indicating the replicon activity observed is likely due
to reduction in cell viability. Compound 1 showed cyto-
static activity at levels similar to the IC50 value. Cyto-
static activity is consistent with inhibition of DNA
synthesis and of cell proliferation that has been shown
to result from non-specific inhibition of HCV replicon
RNA replication.13
Table 1 shows the results obtained with the compounds
prepared and evaluated for inhibition of HCV replica-
tion. Only compounds 28 and 31 were active in the rep-
licon system, with >50% inhibition at 20 lM.
Importantly, compound 28 was not cytotoxic up to
>2 mM, but compound 31 displayed a level of cytotox-
icity similar to its inhibitory activity in the replicon. We
concluded that the activity of compound 31 is due to
inhibition of cell viability; therefore, further evaluation
of this compound was not undertaken.
Table 3. Inhibition of the RNA polymerase activity of NS5B
Compound TP
NS5B inhibition (IC50 lM)
In Table 2, we present comparative data for compounds
28, 26, 1, and 4. Compounds 28 and 4 displayed good
activity in the replicon assay with no measurable cyto-
toxic or cytostatic effect. Compound 26 showed a cyto-
10 TP
28 TP
33 TP
0.30
0.29
2.7

D. B. Smith et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2570–2576
2575
In order to elucidate the mechanism of action of com-
Acknowledgment
pound 28, the corresponding triphosphate derivative
was prepared. The triphosphate of 28 inhibited RNA
synthesis by HCV polymerase in a template dependent
manner, consistent with competitive inhibition of
CMP incorporation by HCV polymerase (Table 3).12
These data are consistent with compound 28 being effi-
ciently metabolized to the corresponding triphosphate
in cell culture and inhibition of HCV replication being
mediated through competitive inhibition by the triphos-
phate. Once incorporated into the nascent RNA chain, a
terminal 28 acts as a chain terminator with efficiency
similar to that of 30-deoxy-CTP.12 Our favored mecha-
nism to explain how a nucleoside bearing the 30-hydrox-
yl can act as a chain-terminator is both conformational
and steric in nature. Based on related work in 20-deoxy
systems, the 40-a-azido substituent introduced into the
carbohydrate moiety of a nucleoside will prefer to exist
in a pseudo-axial orientation, which in turn will induce
the nucleoside to exist predominantly in the northern
conformation.14 In this conformation, the 30-hydroxyl
group resides in a pseudo-equatorial orientation and is
closely flanked by the adjacent 40-azido group, which
would serve to encumber the ability of the 30-hydroxyl
to act as a nucleophile.
We thank Hans Maag for many helpful discussions
regarding this work.
References and notes
1. Wasley, A.; Alter, M. J. Semin. Liver Dis. 2000, 20, 1.
2. (a) Stuyver, L. J.; McBrayer, T. R.; Whitaker, T.;
Tharnish, P. M.; Ramesh, M.; Lostia, S.; Cartee, L.; Shi,
J.; Hobbs, A.; Schinazi, R. F.; Watanabe, K. A.; Otto, M.
J. Antimicrob. Agents Chemother. 2004, 48, 651; (b) Shi, J.;
Du, J.; Ma, T.; Pankiewicz, K. W.; Patterson, S. E.;
Tharnish, P. M.; McBrayer, T. R.; Stuyver, L. J.; Otto, M.
J.; Chu, C. K.; Schinazi, R. F.; Watanabe, K. A. Bioorg.
Med. Chem. 2005, 13, 1641; (c) Clark, J. L.; Hollecker, L.;
Mason, J. C.; Stuyver, L. J.; Tharnish, P. M.; Lostia, S.;
McBrayer, T. R.; Schinazi, R. F.; Watanabe, K. A.; Otto,
M. J.; Furman, P. A.; Stec, W. J.; Patterson, S. E.;
Pankiewicz, K. W. J. Med. Chem. 2005, 48, 5504; (d)
Carroll, S. S.; Tomassini, J. E.; Bosserman, M.; Getty, K.;
Stahlhut, M. W.; Eldrup, A. B.; Bhat, B.; Hall, D.;
Simcoe, A. L.; LaFemina, R.; Rutkowski, C. A.; Wolan-
ski, B.; Yang, Z.; Migliaccio, G.; De Francesco, R.; Kuo,
L. C.; MacCoss, M.; Olsen, D. B. J. Biol. Chem. 2003, 278,
11979; (e) Olsen, D. B.; Eldrup, A. B.; Bartholomew, L.;
Bhat, B.; Bosserman, M. R.; Ceccacci, A.; Colwell, L. F.;
Fay, J. F.; Flores, O. A.; Getty, K. L.; Grobler, J. A.;
LaFemina, R. L.; Markel, E. J.; Migliaccio, G.; Prhavc,
M.; Stahlhut, M. W.; Tomassini, J. E.; MacCoss, M.;
Hazuda, D. J.; Carroll, S. S. Antimicrob. Agents Chemo-
ther. 2004, 48, 3944; (f) Pierra, C.; Amador, A.; Benzaria,
S.; Cretton-Scott, E.; D’ Amours, M.; Mao, J.; Mathieu,
S.; Moussa, A.; Bridges, E. G.; Standring, D. N.;
Sommadossi, J-P.; Storer, R.; Gosselin, G. J. Med. Chem.
2006, 49, 6614; (g) Clark, J. L.; Mason, J. C.; Hollecker,
L.; Stuyver, L. J.; Tharnish, P. M.; McBrayer, T. R.; Otto,
M. J.; Furman, P. A.; Schinazi, R. F.; Watanabe, K. A.
Bioorg. Med. Chem. Lett. 2006, 16, 1712.
The inactivity of the other derivatives in the replicon
system might be explained through either lack of phos-
phorylation or through non-acceptance or poor fit of
the 40-substituted triphosphate in the active site of the
HCV polymerase. We selected compounds 10 (40-azido-
uridine) and 33 (40-ethynylcytidine) to differentiate
between these possibilities.
Compounds 10 and 33 did not inhibit HCV replica-
tion in the replicon system, but their triphosphates
were inhibitors of NS5B mediated RNA synthesis (Ta-
ble 3). From these data, we conclude that 10 and 33
were inefficiently phosphorylated in Huh-7 cells but
that their triphosphates could productively bind in
the active-site of NS5B.
3. (a) Moffatt, J. G. Nucleoside Analogues. In Walker, R. T.,
De Clerq, E., Eckstein, F., Eds.; Plenum Publishing
Corporation: New York, 1979; p 71; (b) Verheyden, J.
P. H.; Moffatt, J. G. J. Am. Chem. Soc. 1975, 97, 4386.
4. Verheyden, J. P. H.; Moffatt, J. G. J. Am. Chem. Soc.
1966, 88, 5684.
The comparative results with 10 and 28 also suggest
that uridine analogues may be less efficiently phos-
phorylated than cytidine analogues in Huh-7 cells.
The synthesis and evaluation of additional triphos-
phate derivatives of nucleosides described in Table 1
would have helped to further delineate the SAR for
binding of 40-a-substituted nucleoside triphosphates
to HCV polymerase. Unfortunately, the limited supply
of key compounds precluded such studies from being
undertaken.
5. De Voss, J. J.; Hangeland, J. J.; Townsend, C. A. J. Org.
Chem. 1994, 59, 2715.
6. Vorbruggen, H.; Bennua, B. Chem. Ber. 1981, 114, 1279.
¨
7. Nomura, M.; Shuto, S.; Tanaka, M.; Sasaki, T.; Mori, S.;
Shigeta, S.; Matsuda, A. J. Med. Chem. 1999, 42, 2901.
8. Jones, G. H.; Taniguchi, M.; Tegg, D.; Moffatt, J. G.
J. Org. Chem. 1979, 44, 1309.
9. Mancuso, A. J.; Swern, D. Synthesis 1981, 165.
10. Haraguchi, K.; Takeda, S.; Tanaka, H. Org. Lett. 2003, 5,
1399.
11. Divakar, K. J.; Reese, C. B. J. Chem. Soc., Perkin Trans. 1
1982, 1171.
In conclusion, data reported here show that com-
pound 28, designated as R1479, is a potent and highly
specific inhibitor of HCV replication in cell culture,
and that its triphosphate is a potent and highly selec-
tive inhibitor of NS5B mediated RNA synthesis, the
HCV encoded RNA polymerase. These interesting
preclinical results have led to the selection of R1479
as a clinical candidate. Further details of the advance-
ment of R1479 into clinical studies have been reported
elsewhere.15
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