Nucleotide prodrugs of 2′-deoxy-2′-spirooxetane ribonucleosides as novel inhibitors of the HCV NS5B polymerase

Article abstract of DOI:10.1021/jm4015422

The limited efficacy, in particular against the genotype 1 virus, as well as the variety of side effects associated with the current therapy for hepatitis C virus (HCV) infection necessitates more efficacious drugs. We found that phosphoramidate prodrugs of 2′-deoxy-2′-spirooxetane ribonucleosides form a novel class of HCV NS5B RNA-dependent RNA polymerase inhibitors, displaying EC₅₀ values ranging from 0.2 to >98 μM, measured in the Huh7-replicon cell line, with no apparent cytotoxicity (CC₅₀ > 98.4 μM). Confirming recent findings, the 2′-spirooxetane moiety was identified as a novel structural motif in the field of anti-HCV nucleosides. A convenient synthesis was developed that enabled the synthesis of a broad set of nucleotide prodrugs with varying substitution patterns. Extensive formation of the triphosphate metabolite was observed in both rat and human hepatocyte cultures. In addition, after oral dosing of several phosphoramidate derivatives of compound 21 to rats, substantial hepatic levels of the active triphosphate metabolite were found.

Full text of DOI:10.1021/jm4015422

Article
pubs.acs.org/jmc
Nucleotide Prodrugs of 2′-Deoxy-2′-spirooxetane Ribonucleosides as
Novel Inhibitors of the HCV NS5B Polymerase
Tim H. M. Jonckers,*,† Koen Vandyck,† Leen Vandekerckhove,†,§ Lili Hu,† Abdellah Tahri,†
Steven Van Hoof,†,∥ Tse-I Lin,†,⊥ Leen Vijgen,† Jan Martin Berke,† Sophie Lachau-Durand,† Bart Stoops,†
Laurent Leclercq,† Gregory Fanning,† Bertil Samuelsson,‡ Magnus Nilsson,‡,# Åsa Rosenquist,‡
Kenny Simmen,† and Pierre Raboisson†
†Janssen Infectious Diseases − Diagnostics BVBA, Turnhoutseweg 30, 2340 Beerse, Belgium
‡Medivir AB, P.O. Box 1086, SE-141 22 Huddinge, Sweden
S
* Supporting Information
ABSTRACT: The limited efficacy, in particular against the genotype 1 virus, as well as
the variety of side effects associated with the current therapy for hepatitis C virus (HCV)
infection necessitates more efficacious drugs. We found that phosphoramidate prodrugs
of 2′-deoxy-2′-spirooxetane ribonucleosides form a novel class of HCV NS5B RNA-
dependent RNA polymerase inhibitors, displaying EC50 values ranging from 0.2 to >98
μM, measured in the Huh7-replicon cell line, with no apparent cytotoxicity (CC50
>
98.4 μM). Confirming recent findings, the 2′-spirooxetane moiety was identified as a
novel structural motif in the field of anti-HCV nucleosides. A convenient synthesis was
developed that enabled the synthesis of a broad set of nucleotide prodrugs with varying
substitution patterns. Extensive formation of the triphosphate metabolite was observed in both rat and human hepatocyte
cultures. In addition, after oral dosing of several phosphoramidate derivatives of compound 21 to rats, substantial hepatic levels of
the active triphosphate metabolite were found.
Scheme 1. Marketed Nucleoside Derivatives Used in the
Fields of Virology and Oncology
INTRODUCTION
■
All living organisms store and convey their genetic information
in the form of nucleic acids that are built from structural
monomers called nucleosides. It remains astounding that nature
is able to generate such a breadth of versatility in the millions of
different species that occupy our planet just by a mere
combination of only eight different nucleosides. A profound
understanding of the fundamental role that nucleosides and
their derivatives have in naturally occurring processes has urged
scientists to focus their efforts into the design and preparation
of novel non-natural nucleoside analogues and the assessment
of their biological activities. The observation that some
synthetic nucleoside derivatives (e.g., Emtricitabine (1) and
Lamivudine (2)) demonstrate activity against multiple
pathogens (HIV and HBV) underlines the potential of this
compound class. Discovered more than 50 years ago,
Idoxuridine1 (3) was the first marketed antiviral nucleoside
used for the treatment of herpes simplex virus, and since then,
nucleoside-based treatment regimens started to emerge as real
game-changers in therapeutic settings, especially in the fields of
oncology and virology. Scheme 1 depicts the structure of six
important nucleosides and their field of application.
patients, for which no less than five different nucleoside
analogues are marketed for clinical use (Lamivudine 2,
On a global scale, approximately 175 million people are
infected with hepatitis C virus (HCV). The infection is believed
to be the major cause of liver diseases including fibrosis,
cirrhosis, and hepatocellular carcinoma.2 At least six major
HCV genotypes and multiple subtypes have been identified.3 In
contrast to the treatment options for hepatitis B-infected
Special Issue: HCV Therapies
Received: October 3, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm4015422 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Scheme 2. Structures of Clinical HCV Candidates 7b, 8b, 9b and Other Base- and Ribose-Modified Anti-HCV Active
Nucleosides
Adefovir, Tenofovir, Telbivudine, and Entecavir), the only
approved antiviral nucleoside analogue for the treatment of
hepatitis C patients is Ribavarin. This broad spectrum antiviral
needs to be combined with pegylated interferon-α and an HCV
NS3/4A protease inhibitor (PI) like Telaprevir (VX-950)4,5 or
Boceprevir (SCH 503034)6 to achieve a sustained virological
response (SVR) of about 75% in genotype 1 patients. Despite
the recent improvements seen in response rates and therapy
adherence as a result of the introduction of more efficacious
combination therapies including PI’s, there remains a need for
new and more effective treatment options for several HCV
patient populations (e.g., partial or null responders). In
addition, the emergence of treatment-resistant mutant viruses
as a result of the use of PI-containing regimens remains a risk.
The HCV genome consists of a 9.6 kb positive-sense, single-
stranded RNA that encodes at least 10 proteins, including both
structural and nonstructural (NS) elements. The NS enzymes
are known to be involved in the replication processes of the
virus.7 Within the HCV genome, the HCV NS5B RNA-
dependent RNA polymerase is considered to be a privileged
target for the development of novel HCV inhibitors. This
enzyme catalyzes the synthesis of positive (genomic)- and
negative (template)-strand HCV RNA.8,9 Similar to other
polymerases, the NS5B enzyme has a typical right-hand three-
dimensional motif with a thumb and fingers domain
surrounding the catalytic site in the base of the palm domain.10
Six different classes of NS5B inhibitors have been identified,
including non-nucleoside inhibitors (NNI) that bind at five
distinct allosteric binding sites (NNI-1, NNI-2, NNI-3, NNI-4,
and NNI-5).11 After metabolism to their 5′-triphosphate
derivatives by cellular kinases, anti-HCV active nucleoside
inhibitors (NI’s) exert their antiviral activity by competing with
the binding of natural nucleotide 5′-triphosphates in the NS5B
active site. Incorporation of an unnatural nucleoside 5′-
triphosphate into the nascent nucleic acid prevents further
growth of the elongating RNA chain, a process called chain
termination. Given the relatively strict conservation of the
nucleoside binding site across HCV genotypes, it is generally
assumed that NI’s show a high genotypic coverage.12 In vitro
experiments have shown that NI’s were found to have a higher
genetic barrier to the development of resistance than NNI’s and
protease inhibitors.13
The structure of the NS5B polymerase has been studied
comprehensively,14 although a detailed understanding of the
structural basis for nucleotide recognition has not yet emerged.
RNA polymerases are believed to interact with the 2′-α-
hydroxy moiety of ribonucleoside-5′-triphosphates (NTP’s),
thereby discriminating among 2′-deoxyribonucleoside-5′-tri-
phosphates (dNTP’s),15,16 but chain-terminating 2′-deoxynu-
cleotides that are incorporated into nascent RNA have been
described.17
Interestingly, the HCV NS5B polymerase appears to allow
diversity in both the base and pentose moieties of the
nucleotide, which has resulted in various inventively designed
derivatives like 7a, 8a, 9a, 10, and 11 (Scheme 2).18,19 Clinical
proof of concept has been demonstrated for the 4′-azido
ribonucleoside prodrug R-1626 (7b) and the 2′-modified
ribonucleoside prodrugs NM-283 (8b) and R-7128 (9b)
(Mericitabine). The prodrug approach was used to improve
the bioavailability of the parent nucleosides that are formed in
vivo via esterase-mediated cleavage of the prodrugs. Unfortu-
nately, the development of these compounds has been halted as
a result of various toxicity concerns.
We previously reported the discovery of 2′-deoxy-2′-
spirocyclopropyl cytidine (12) as a novel inhibitor of the
HCV NS5B polymerase.20 This 2′-substitution pattern hints at
a new type of electrostatic interaction as an alternative to the
known approaches that use a 2′-α-hydroxyl or a 2′-α-fluoro
substituent (as in 8 and 9).21,22 Further evaluation of the 2′
region of the ribose moiety suggested that the 2′-deoxy-2′-
spirooxetane motif could be a promising novel isosteric variant
of the known 2′-substitution patterns. The oxetane moiety
would have a profound impact on the physicochemical
characteristics of organic molecules, illustrated by the favorable
modulation of parameters like lipophilicity, solubility, potential
for hERG channel inhibition, and so forth.23 Our rationale for
B
dx.doi.org/10.1021/jm4015422 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 3. Synthesis of 2′-Deoxy-2′-spirooxetane Cytidine 13
a
(a) BzCl, Et3N, DMAP, CH2Cl2, rt, 4 days; 63%; (b) persilylated uracil, SnCl4, rt 1 h, then reflux, 1.5 h; (c) NaOMe, MeOH; rt, 1.5 days, 76% from
15; (d) (i) NaIO4, OsO4, rt, 2 h, (ii) NaBH4, THF/H2O, rt, 77%; (e) MsCl, dry pyridine, rt, 2 h; (f) NaH, dry THF, rt, 79% from 18; (g) Pd(OH)2,
H2 atm, MeOH, rt, overnight, 56%; (h) TBDMSCl, imidazole, DMF, rt, 1.5 days; (i) (i) TPSCl, DMAP, Et3N, CH3CN, rt, 2 h, (ii) NH4OH,
CH3CN, rt, 1.5 h, 87% from 21; (j) TBAF, THF, rt, 1.5 h, 52%
examining 2′-deoxy-2′-spirooxetane nucleosides as novel
potential inhibitors of the HCV NS5B polymerase is not
based on physicochemical reasons, but it originates from steric
and electronic considerations. Initially, the cytosine derivative
(13) (Scheme 3), where the isomer having its oxygen atom
located at the α-face of the ribose, was considered to be a
valuable new target. Literature analysis revealed that several
annelated oxetane nucleosides were already known; however,
the only nucleoside analogue having a 2′-deoxy-2′-spirooxetane
motif reported was a thymine derivative. This compound was
made to study conformationally restricted oligonucleotides that
were found to be inactive against the HIV virus.24 While this
manuscript was in preparation, Du et al. reported on the HCV-
inhibiting properties of nucleosides bearing different 2′-
spirocyclic ether motifs including oxetanes, with the main
focus on the guanosine (G)-based derivatives thereof.25
confirming that compound 13 is less potent then its congeners,
8a, 9a, and 12.
Table 1. Inhibition of HCV Replication (EC50, Luciferase
Assay) and Cytotoxicity (CC50) in Huh-7 Replicon Cells for
Reference Nucleosides 8a, 9a, and 12 and Oxetane
Derivatives 13 and 21
a
a
compound
EC50 (μM)
CC50 (μM)
8a
9a
12
13
21
4.6
>98
>98
>98
>98
>98
1.9
7.3
17.1
>98
a
Values reported are the average of at least three repeats.
It is possible that the nucleoside triphosphate metabolite of
CHEMISTRY
■
13 is a less efficient inhibitor of the HCV NS5B polymerase
enzyme because of an unfavorable bound conformation. The
ring pucker of the ribose moiety is known to be influenced by
the substitution pattern on it, and the spirooxetane motif forces
the NTP to adopt a C2′-endo (Southern) conformation as
opposed to the C3′-endo (Northern) conformation.20 This
would prevent the correct orientation of the interacting
residues in the active site. However, an alternative explanation
appears more likely. In essence, nucleoside analogues like 13
act as prodrugs whose efficacy highly depends on their
conversion, via cellular kinases, to the potentially active
triphosphate (TP). Limited formation of the triphosphate,
resulting from one or more suboptimal phosphorylation steps,
would lead to an apparently weaker or even inactive nucleoside
inhibitor. In particular, the formation of the nucleoside
monophosphate is considered as the rate-limiting step toward
generating the NTP.27 This might also be the case for
compound 13.
To evaluate our hypothesis, spirooxetane derivative 13 was
prepared. The synthetic approach toward this new derivative is
outlined in Scheme 3. Starting from D-ribose, compound 14
was synthesized in five steps according to a literature
procedure.26 Treatment with BzCl resulted in glycosylation
precursor 15, which was reacted with persilylated uracil in the
presence of SnCl4, resulting in uridine derivative 16 stereo-
selectively. After debenzoylation, alkene 17 was converted into
diol 18 by consecutive oxidation and reduction. The primary
alcohol was then treated with methylsulfonylchloride, yielding
mesylated compound 19 that was converted into oxetane 20 by
NaH-mediated cyclization. Reductive debenzylation resulted in
2′-spirooxetane uridine derivative 21. This compound was
reprotected as a TBDMS ether (compound 22) and converted
into 2′-spirooxetane cytidine derivative 13 by consecutive
treatment with 2,4,6-triisopropylbenzenesulfonyl chloride
(TPSCl) and aminolysis and finally deprotection of inter-
mediate compound 23.
The concept of using aryloxy phosphoramidate (PA)
derivatives as monophosphate prodrugs is well-documented.
Introduced in the 1990s by McGuigan et al., the pronucleotide
(Protide) methodology has established itself as one of the
preferred techniques to circumvent hampered nucleotide
Following successful synthesis of compound 13, its inhibitory
activity and potential cytotoxicity in the cell-based HCV
replicon assay were measured (Table 1). The EC50 value was
found to be in accordance with recently published results,25
C
dx.doi.org/10.1021/jm4015422 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
formation.28 Being able to bypass the rate-determining first
phosphorylation step has resulted in the evaluation of uracil-
based NTP derivatives as potential drugs. 4′-Azidouridine does
not show any HCV-inhibiting activity in the HCV replicon
assay (EC50 > 100 μM), whereas phosphoramidate derivatives
thereof are active inhibitors in vitro.29 The same strategy was
applied in the design of Sofosbuvir (25), which is the most
advanced nucleoside candidate in the field of HCV.30 Scheme 4
obvious explanation at present.34 Furthermore, 25, which has
an identical phosphoramidate substituent pattern, was found to
have an EC90 value of 0.254 μM in the same assay, similar to
the reported value.30 Further SAR analysis revealed that, in
most cases, aryloxy variations including p-Cl-Ph and Naphthyl
(Naph) were well-tolerated (see 29b, 30b, 30c, 30d, 30g, and
30i), with the exception being 2-i-Pr-5-Me-Ph derivative 30q,
for which cellular toxicity was observed. Variation of the amino
acid fragment showed that L-alanine derivatives and (S)-ethyl
glycine derivatives (see 30k, 30l, and 30o) displayed good
inhibition, whereas (S)-phenyl glycine derivative 30t and
dimethylglycine derivative 30s were found to be less potent.
In general, the impact of the ester functionality on the antiviral
activity was limited because both straight and branched
aliphatic chains as well as benzyl derivatives were allowed.
This is an important observation because the enzymatic
hydrolysis of the ester moiety is the putative first step in the
phosphoramidate metabolism.28 In contrast to what was
reported for 25 and its diastereomer (PSI-7976),31 no
substantial difference in antiviral activity between the individual
diastereomers of several derivatives was observed (see pairs 29c
and 29d, 30g and 30i, 30k and 30l, and 30f and 30n). Finally,
the activity of compound 30j was measured in a transient
replicon HCV assay wherein the NS5B enzyme bore the S282T
mutation. This has been reported to inflict a 3−6-fold loss of in
vitro sensitivity to other 2′-modified nucleosides.35 Because the
S282 position is in close proximity to the enzyme’s catalytic
site, it is believed that a reduced activity toward this mutant
constitutes an indirect proof of the mode of action (i.e.,
targeting the active site of the HCV NS5B polymerase).
Compound 30j showed an average (n = 8) EC50 value of 18.9
μM, which is a 4.8-fold difference compared to the WT activity.
For 25, an EC90 value of 7.8 μM is reported, which is an 18.6-
fold change compared to its WT EC90 value.31 These data
supported our assumption that the triphosphate metabolite
formed upon metabolic conversion of phosphoramidate
prodrug 3j most likely exerts its antiviral activity by binding
in the active site of the HCV NS5B polymerase. This was
confirmed in a biochemical assay using WT HCV RdRp. A Ki
value of 2.4 μM was obtained (average of two independent
experiments) for the NTP derivative of 21, which is about 6-
fold higher than the reported value for the NTP metabolite of
25.22
Scheme 4. Phosphoramidate Compounds Reaching the
Clinical Stage
shows several Protide derivatives that have progressed into
clinical trials. As potential anti-HCV drugs, only 25 and VX-135
(structure not disclosed yet) are still under active clinical
development because others were discontinued as a result of
toxicity concerns or PK/PD variability.31−33
On the basis of these considerations, we pursued
phosphoramidate derivatives of compound 13 and of
corresponding uridine nucleoside 21. The synthesis of 5′-
phosphoramidates, obtained as diastereoisomeric mixtures, was
accomplished as illustrated in Scheme 5.
Scheme 5. Synthesis of Phosphoramidate Derivatives of 13
and 21
IN VITRO AND IN VIVO EVALUATION
■
Although both cytosine and uracil derivatives of the 2′-deoxy-
2′-spirooxetane ribonucleoside scaffold were found to be
inhibitors of the HCV NS5B polymerase, we considered the
uracil derivatives as the most promising. Cytidine deaminase-
mediated metabolism of the cytosine derivatives could
potentially complicate the biological and preclinical evaluation
of these derivatives.36 Therefore, we opted to focus solely on
the uracil analogues. It is known that the conversion of a
phosphoramidate prodrug into the active triphosphate
metabolite involves multiple steps including passive or
transporter-mediated cellular uptake, hydrolytic and/or ester-
ase-mediated cleavage, and double kinase-mediated phosphor-
ylation.28 In principal, one expects a more active derivative to
generate higher intracellular NTP levels, and the quantification
thereof in replicon cells might contribute to a better SAR
understanding. However, it is not guaranteed that the observed
SAR trends in the replicon system will transfer to similar in vivo
Unprotected nucleosides 13 and 21 were reacted with a
selection of phosphorochloridates (28a−m) in the presence of
N-methyl imidazole (NMI) as base and cosolvent, resulting in
compounds 29a−d and 30a−u. Table 2 shows the antiviral
results obtained. Gratifyingly, phosphoramidate prodrug
derivatives of both 13 and 21 showed inhibitory activity in
the HCV replicon assay, with EC50 values ranging from 0.2 to
>98 μM. Uracil derivatives 30e and 30m were found to be
more active than corresponding cytosine analogues 29c and
29d. In contrast, compound 29a showed submicromolar
activity, which is about 30-fold more potent than its cytosine
derivative, 30r. Notably, the reported activity of 29a is
significantly lower,25 a discrepancy for which we have no
D
dx.doi.org/10.1021/jm4015422 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 2. Replicon Activity (EC50) and Cellular Toxicity (CC50) for Phosphoramidate Derivatives 29a−d and 30a−u
a
b
compound
R1
R2
R3
R4
EC50 (μM)
CC50 (μM)
29a
29b
Ph
i-Pr
Bn
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
0.74
2.6
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
29.6
>98
>98
>92.8
>98
p-Cl-Ph
c
29c
Ph
Bn
35.8
41.5
0.23
0.32
1.3
c
29d
Ph
Bn
30a
30b
30c
30d
Ph
i-Bu
Bn
p-Cl-Ph
p-Cl-Ph
Bn
Me
Me
Me
Me
Me
Me
Me
Me
Et
Naph
Bn
2.0
c
30e
Ph
Bn
2.1
c
30f
Ph
n-Bu
Bn
2.2
c
30g
Naph
2.3
30h
Ph
i-Pen
Bn
3.5
c
30i
Naph
3.7
30j
Ph
n-Bu
Bn
3.9
c
30k
Ph
4.6
c
30l
Ph
Bn
Et
4.9
c
30m
Ph
Bn
Me
Me
Et
5.0
c
30n
Ph
n-Bu
Bn
5.3
30o
30p
30q
30r
30s
30t
Ph
5.7
Ph
Bn
Me
Me
Me
Me
Ph
5.7
2-i-Pr-5-Me-Ph
Bn
7.5
Ph
Ph
Ph
Ph
i-Pr
Me
Bn
20.9
37.9
>87.2
>96.4
30u
i-Pr
Et
a
b
c
Values reported are the average of at least three repeats. CC50 in Huh-7cells. Pure diastereoisomer.
a
Table 3. Formation of the Triphosphate Metabolite in Fresh Rat and Human Hepatocytes
30d
30j
30o
30p
time (h)
rat
human
rat
human
rat
human
rat
human
2
8
293
107
1713
3373
152
75
351
215
87
202
112
81
304
2730
1480
2457
a
Values reported are expressed in nanograms per milliliter and are the average of at least three repeats for the human hepatocyte data; two data
points were used for rat hepatocytes.
results given the multistep mechanism of phosphoramidate
prodrug metabolism.
Table 4. PK Parameters of the Triphosphate Metabolite in
Rat Liver after Oral Dosing of Phosphoramidate Prodrugs
30d, 30j, 30o, and 30p
a
We studied NTP formation in freshly prepared hepatocyte
cultures and in rat livers for a structurally diverse set of
analogues with varying R1, R2, and R3 groups that showed
similar in vitro potencies. On the basis of the negligible potency
difference between diastereomeric pairs, these studies were
performed with diastereomeric mixtures of compounds 30d,
30j, 30o, and 30p.37 In the hepatocyte studies, compounds
were incubated at a concentration of 10 μM, and the formation
of TP was determined by LC−MS analysis of the cell lysates
after 2 and 8 hours (Table 3).
c
30d
30j
894
30o
30p
Cmax (ng/g)
754
6
419
6
775
2
b
tmax (h)
6
AUC(0−last) (ng h/mL)
AUC(0−inf) (ng h/mL)
8427
9142
9871
10 480
1108
ND
3263
ND
a
Compounds were dosed as an oral solution in 20% HP-β-
cyclodextrin except for 30d, for which 40% HP-β-cyclodextrin was
b
used. Livers were removed at 1, 2, 4, 6, and 24 h. ND, not defined.
c
Indicative values because of bioanalytical reasons.
Formation of active TP was seen for the four derivatives
tested, with values for compound 30d being the highest. A
substantial difference was observed when comparing the levels
of TP formed in human hepatocytes compared to rat
hepatocytes. Such a difference has been previously reported
for related prodrugs.30 Encouraged by the observed levels of
active triphosphate being generated in hepatocyte cultures, we
performed a PK experiment in rats to study the in vivo
formation of TP metabolite. Sprague−Dawley rats were given a
single oral dose of 50 mg/kg of phosphoramidate prodrug, and
the hepatic levels of triphosphate were determined (Table 4).
As can be seen from Table 4, the highest liver concentrations
of TP were obtained after oral dosing of compounds 30d and
30j, which showed measurable TP concentrations in the liver
up to 24 h postdosing (data not shown). For compounds 30o
and 30p, the overall hepatic levels of TP were substantially
lower. In none of the experiments was unreacted phosphor-
amidate prodrug found in the plasma. Comparison between the
in vitro rat hepatocyte and in vivo rat PK results showed a poor
correlation between the two data sets. This is not surprising
because the extent of TP formation is dependent on both the
E
dx.doi.org/10.1021/jm4015422 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Hz), 4.56 (s, 1H), 4.53−4.40 (m, 3H), 4.05−4.11 (m, 1H), 3.32−3.53
(m, 4H), 3.44 (s, 3H), 2.37 (dd, 1H, J = 14.3, 6.7 Hz), 2.25 (dd, 1H, J
= 14.3, 7.6 Hz).
inherent permeability of each phosphoramidate derivative itself
as well as its rate of metabolism.
In conclusion, we report here on the identification of 2′-
deoxy-2′-spirooxetane ribonucleosides and phosphoramidate
prodrug derivatives thereof as novel HCV NS5B polymerase
inhibitors. The majority of the derivatives showed EC50 values
in the low micromolar range. In the in vitro hepatocyte studies,
the formation of the triphosphate metabolite in both rat and
human hepatocytes was observed. For two compounds,
substantial hepatic levels of triphosphate metabolite were
observed after oral dosing to rats. Results of the further
characterization of this 2′-deoxy-2′-spirooxetane nucleoside
class will be reported in future communications.
Synthesis of (2S,3R,4R,5R)-3-Allyl-4-(benzyloxy)-5-(benzy-
loxymethyl)-2-methoxytetrahydrofuran-3-yl benzoate (15).
To a solution of 14 (5.0 g, 13 mmol) in dry DCM (60 mL) at
room temperature were added Et3N (26.2g, 260 mmol) and catalyst
DMAP (0.5 eq, 6.5 mmol). Then, benzoyl chloride (5.4 g, 38.6 mmol)
was added in one portion. The mixture was stirred for 4 days. The
reaction mixture washed with saturated aqueous NaHCO3 followed by
brine. After drying with MgSO4, filtration, and evaporation of the
volatiles, the solvent was removed, and the residue was isolated by
column chromatography (heptane to 15% ethylacetate in heptane) to
afford 15 (4 g, 8.2 mmol, 63%) as an oil. HPLC condition A, tR = 3.41
1
min, m/z = 457 (M − OMe)+. H NMR (400 MHz, CDCl3) δ 8.07
(d, 2H, J = 8.0 Hz), 7.51−7.60 (m, 1H), 7.38−7.47 (m, 2H), 7.17−
7.37 (m, 10H), 5.63−5.78 (m, 1H), 5.22 (s, 1H), 5.07−5.13 (m, 1H, J
= 10.0 Hz), 4.90−5.01 (m, 2H), 4.42−4.57 (m, 2H), 4.36 (d, 1H, J =
11.9 Hz), 4.14−4.19 (m, 1H), 3.87 (d, 1H, J = 4.5 Hz), 3.51−3.58 (m,
1H), 2.37 (dd, 1H, J = 14.3, 6.7 Hz), 2.25 (dd, 1H, J = 14.3, 7.6 Hz).
Synthesis of 1-((2R,3R,4R,5R)-3-Allyl-4-(benzyloxy)-5-(ben-
zyloxymethyl)-3-hydroxytetra-hydrofuran-2-yl)pyrimidine-
2,4(1H,3H)-dione (17). BSA (29.2 mL, 118 mmol) was added to a
mixture of 15 (14.0 g, 23.1 mmol) and uracil (5.99 g, 53.4 mmol) in
anhydrous acetonitrile (300 mL). The reaction mixture was refluxed
for 1 h, and the clear solution was allowed to cool to room
temperature. Perchlorostannane (11.55 mL, 99 mmol) was added
dropwise at room temperature, and the mixture was further stirred for
1 h. The mixture was then stirred at reflux for 1.5 h and again cooled
to room temperature. EtOAc (250 mL) was added followed by
saturated aqueous NaHCO3 (250 mL), and the mixture was stirred for
15 min. After filtration through Celite, the organic phase was separated
and washed with saturated aqueous NaHCO3 (250 mL). The
combined aqueous phase was extracted with EtOAc (250 mL), and
the combined organic phase was dried (MgSO4), filtered, and
evaporated to dryness under reduced pressure. The resulting yellow
oil containing compound 16 was dissolved in MeOH, and 25%
MeONa (25 mL) was added. Stirring continued overnight. More 25%
NaOMe (15 mL) was added, and stirring was further continued
overnight. Acetic acid (30 mL) was added, and the solvent was
removed. The residue was purified by column chromatography with
heptane/EtOAc 50:50 to 100% EtOAc. Compound 17 (9.38 g, 76%)
was obtained as a colorless oil. HPLC condition A, tR = 2.49 min, m/z
= 465 (M + H)+. 1H NMR (400 MHz, CDCl3) δ 8.39 (1H, NH), 7.75
(d, 1H, J = 8.0 Hz), 7.22−7.43 (m, 10H), 6.05 (s, 1H), 5.71−5.84 (m,
1H), 5.35 (d, 1H, J = 8.0 Hz), 5.00−5.11 (m, 2H), 4.70 (d, 1H, J =
11.5 Hz), 4.53 (d, 1H, J = 11.5 Hz), 4.47 (d, 1H, J = 11.1 Hz), 4.47 (d,
1H, J = 11.1 Hz), 4.11−4.16 (m, 1H), 4.04 (d, 1H, J = 8.0 Hz), 3.81−
3.87 (m, 1H), 3.45−3.52 (m, 1H), 3.17 (br s, OH), 2.15−2.33 (m,
2H).
Synthesis of 1-((2R,3R,4R,5R)-4-(Benzyloxy)-5-(benzyloxy-
methyl)-3-hydroxy-3-(2-hydroxyethyl)tetrahydrofuran-2-yl)-
pyrimidine-2,4(1H,3H)-dione (18). To a stirred solution of 17 (7.8
g, 16.79 mmol) in a mixture of THF (10 mL) and H2O (10 mL) was
added sodium periodate (11.17 g, 52.2 mmol) followed by
osmium(VIII) oxide (2 mL, 2.5 w/v % in t-BuOH, 0.168 mmol)
and stirring was continued for 2 h at room temperature. H2O was
added, and extraction was performed with EtOAc (2×). The organic
phase was washed with saturated aqueous NaHCO3 (2×). The
combined aqueous phase was extracted with EtOAc, and the combined
organic phase was dried (Na2SO4), filtered, and evaporated to dryness
under reduced pressure. The oily residue obtained was dissolved in a
mixture of THF (100 mL) and H2O (20 mL), and sodium
borohydride (1.361 g, 36.0 mmol) was added. The reaction mixture
was stirred overnight at room temperature, H2O was added, and
extraction was performed with EtOAc (2×). The combined organic
phase was washed with saturated aqueous NaHCO3, the combined
aqueous phase was extracted with EtOAc, and the combined organic
phase was dried (Na2SO4), filtered, and evaporated to dryness under
reduced pressure. The oily residue obtained was purified by column
EXPERIMENTAL SECTION
■
NMR spectra were recorded on a Bruker Avance 400 spectrometer,
1
operating at 400 MHz for H NMR and 100 MHz for 13C NMR.
Chemical shifts are given in ppm, and J values, in Hz. Multiplicity is
indicated using the following abbreviations: s, singlet; d, doublet; t,
triplet; m, multiplet, and so forth. If possible, for individual
diastereomers, the stereochemistry of the P center was assigned on
the basis of ref 38. The purity of final compounds was determined via
one of the following methods and was greater than 95%. Method A:
Waters Alliance 2695; column: Waters XTerra 2.5 μm, 4.6 × 50 mm;
column temp.: 55 °C; flow: 2 mL/min; mobile phase A: 10 mM
NH4OOCH + 0.1% HCOOH in H2O; mobile phase B: CH3CN,
gradient: 0 min: 85:15 A/B, to 3.0 min: 5:95 A/B, to 4.20 min: 5:95
A/B. Method B: column: Hypercar 3 μ, 4.6 × 50 mm, mobile phase C:
10 mM NH4OAc in H2O/CH3CN 1:9; mobile phase D: 10 mM
NH4OAc in H2O/CH3CN 9:1; column temp: 50 °C; flow gradient: 2
mL/min; 0 min: 0:100 C/D, to 3.0 min: 100:0 C/D, to 4.20 min:
100:0 C/D. Method C: column: SunFire C18 3.5 μ, 4.6 × 100 mm;
mobile phase A: 10 mM NH4OOCH + 0.1% HCOOH in H2O,
mobile phase B: MeOH operating at a column temperature of 50 °C
using a flow rate of 1.5 mL/min. Gradient conditions: t = 0 min: 65%
A, 35% B; t = 7 min: 5% A, 95% B; t = 9.6 min: 5% A, 95% B; t = 9.8
min: 65% A, 35% B; and t = 12 min:, 65% A, 35% B. Optical rotation
20
was measured using a PerkinElmer 341 polarimeter. [α]D indicates
the optical rotation measured with light at the wavelength of the D-line
of sodium (589 nm) at a temperature of 20 °C. The cell path length is
1 dm. Next to the actual value, the concentration and solvent of the
solution that was used to measure the optical rotation are mentioned.
Low-resolution mass spectra (LRMS) were performed on a single
quadrupole (Waters ZQ) or a time-of-flight (Waters LCT) mass
spectrometer using electrospray ionization (ESI) in positive or
negative mode. For a limited set of pairs (29c and 29d, 30e and
30m, 30g and 30i, 30k and 30l, and 30f and 30n), diastereomers were
separated via chromatography on silica gel using the indicated solvent
or via supercritical fluid chromatography (SFC) on a Multigram II
Supercritical Fluid Chromatography system from Berger instruments
(Newark, DE, USA) using a Chiralpak Diacel OJ 20 × 250 mm
column. The mobile phase used was CO2, EtOH with 0.2% iPrNH2.
Synthesis of (2S,3R,4R,5R)-3-Allyl-4-(benzyloxy)-5-(benzy-
loxymethyl)-2-methoxytetrahydrofuran-3-ol (14). To a solution
of (2S,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxydihy-
drofuran-3(2H)-one (obtained as in ref 26) in dry THF (400 mL)
at −78 °C under argon atmosphere, was added allylmagnesium
bromide (400 mL, 400 mmol; 1.0 M in Et2O). After the reaction
mixture was stirred for 4 h at −78 °C, the reaction mixture was
allowed to stir at room temperature for 2 h. The reaction was carefully
quenched with saturated aqueous ammonium chloride. The mixture
was extracted with dichloromethane, and the organic layer was washed
with brine. The solvent was removed, and the residue was purified by
silica gel chromatography (600 g silica) by gradient elution with 15−
20% ethyl acetate in hexane to give 14 as a colorless oil (32.9 g, 70%).
HPLC condition A, tR = 2.97 min, m/z = 402 (M + NH4)+. 1H NMR
(400 MHz, CDCl3) δ 7.38−7.20 (m, 10H), 5.84−5.97 (m, 1H), 5.12
(m, 1H, J = 10.2 Hz), 5.01 (m, 1H, J = 17.2 Hz), 4.74 (d, 1H, J = 12.3
F
dx.doi.org/10.1021/jm4015422 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
chromatography [0−10% (v/v) MeOH in CH2Cl2 then 10%
isocratic], affording reaction product 18 as white foam (4.8 g, 57%).
HPLC condition A, tR = 2.12 min, m/z = 469 (M + H)+.
min, m/z = 270 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 7.80 (d,
1H, J = 7.4 Hz), 7.13−7.28 (m, NH2), 6.12 (s, 1H), 5.69 (d, 1H, J =
7.4 Hz), 5.35 (br s, OH), 5.12 (br s, OH), 4.31−4.44 (m, 2H), 3.72−
3.82 (m, 2H), 3.50−3.62 (m, 2H), 2.40−2.49 (m, 1H), 2.29−2.39 (m,
1H).
Synthesis of 1-((4R,5R,7R,8R)-8-(Benzyloxy)-7-(benzyloxy-
methyl)-1,6-dioxaspiro[3.4]octan-5-yl)pyrimidine-2,4(1H,3H)-
dione (20). Methanesulfonyl chloride (0.800 mL, 10.34 mmol) was
added to 18 (4.32 g, 9.22 mmol) in dry pyridine (100 mL). After 1 h
15 min, 0.1 equiv more of methanesulfonyl chloride was added, and
the mixture was further stirred at room temperature for 45 min. Then,
a small amount of MeOH was added, and the mixture was evaporated
to dryness. The residue was dissolved in CH2Cl2/EtOAc and washed
with saturated NaHCO3 (2×). The combined aqueous phase was
extracted with EtOAc. The combined organic phase was dried with
Na2SO4 and concentrated in vacuo. The obtained residue containing
compound 19 was dissolved in dry THF, and 95% NaH (932 mg, 36.9
mmol) was added at once at room temperature. After stirring for 2 h at
room temperature, saturated NH4Cl (30 mL) was slowly added
followed by addition of CH2Cl2 (250 mL). The separated organic
phase was washed with saturated aqueous NaHCO3 (2 × 100 mL),
and the combined aqueous phase was extracted with CH2Cl2 (250
mL). The combined organic phase was dried (Na2SO4), filtered, and
evaporated to dryness under reduced pressure. The residue obtained
was purified by column chromatography (heptane to EtOAc and then
EtOAc), affording 20 (3.27 g, 79%) as a foam. HPLC condition A, tR =
2.33 min, m/z = 451 (M + H)+.
General Procedure for Synthesis of Phosphochloridates
28a−m. Phosphorchloridates 28a−m were synthesized using an
analogous procedure as that described for methyl 2-(chloro(phenoxy)-
phosphorylamino)-2-methylpropanoate 28k. A solution of phenyl
phosphorodichloridate (1.0 eq., 13.0 mmol, 1.9 mL) and methyl α-
aminoisobutyrate hydrochloride (1.0 equiv, 13.0 mmol, 2.0 g) in
CH2Cl2 (80 mL) was cooled to −80 °C. Dry DIPEA (2.0 equiv, 26.0
mmol, 4.3 mL) was added dropwise. After 2 h, the reaction was
warmed to room temperature, and the solvent was removed under
reduced pressure. Dry diethylether was added, and the precipitate was
filtered off and washed twice with dry diethylether under an argon
atmosphere. The filtrate was evaporated to dryness to give the
phosphochloridate 28k, which was stored as a 0.90 M solution in
tetrahydrofuran (THF) at −18 °C and used as such.
General Procedure for the Synthesis of Phosphoramidates
30a−u. Phosphoramidate derivatives 30a−u were synthesized using a
similar procedure as that described for the preparation of methyl 2-
((((4R,5R,7R,8R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-8-hy-
droxy-1,6-dioxaspiro[3.4]octan-7-yl)methoxy)(phenoxy)-
phosphorylamino)-2-methylpropanoate 30s.
Synthesis of 1-((4R,5R,7R,8R)-8-Hydroxy-7-(hydroxymethyl)-
1,6-dioxaspiro[3.4]octan-5-yl)pyrimidine-2,4(1H,3H)-dione
(21). To a stirred solution of 20 (5.0 g, 11.1 mmol) in THF (1.0 L)
was added 20% Pd(OH)2 (2.5 g). The mixture was degassed several
times with argon and placed under a hydrogen atmosphere. The
mixture was hydrogenated at 15 psi for 18 h at 25 °C. The catalyst was
removed by filtration on decalite, and the solvent was removed from
the filtrate by evaporation. The resulting residue was purified by silica
gel chromatography, eluting with 10% methanol in EtOAc to give 21
as white powder (2.3 g, 8.51 mmol, 77%). HPLC condition B, tR =
To a solution of 21 (1.0 equiv, 0.28 mmol, 75 mg) in dry THF (3
mL) was added NMI (12.0 equiv, 3.33 mmol, 0.27 mL) at room
temperature. A solution of the phosphorchloridate (1.4 equiv, 0.39
mmol, 0.9 M, 0.43 mL) was added dropwise, and the mixture was
stirred at room temperature for 1 h. The reaction mixture was washed
three times with 0.5 M HCl. The organic phase was dried with MgSO4
and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (0−10% methanol in CH2Cl2), resulting
in compound 30s (24 mg, yield = 15%, purity = 95%) as a mixture of
diastereomers. HPLC condition A, tR = 1.49 min, m/z = 526 (M +
1
1.98 min, m/z = 271 (M + H)+. H NMR (400 MHz, DMSO-d6) δ
1
H)+. H NMR (400 MHz, DMSO-d6) δ 1.33 (s, 3H), 1.37 (s, 3H),
2.35−2.44 (m, 1H), 2.52−2.58 (m, 1H), 3.52−3.63 (m, 2H), 3.71−
3.80 (m, 1H), 3.85 (t, J = 8.58 Hz, 1H), 4.30−4.48 (m, 2H), 5.12 (t, J
= 4.95 Hz, 1H), 5.42 (d, J = 8.14 Hz, 1H), 5.62 (d, J = 7.92 Hz, 1H),
6.03 (s, 1H), 7.82 (d, J = 8.14 Hz, 1H), 11.44 (s, 1H).
2.42−2.43 (m, 2H), 3.56 (s, 3H), 3.70−3.79 (m, 1H), 3.80−3.88 (m,
0.4H), 3.88−3.96 (m, 0.6H), 4.09−4.20 (m, 1H), 4.26−4.48 (m, 3H),
5.50−5.56 (m, 1H), 5.61−5.69 (m, 1H), 5.88−5.97 (m, 1H), 5.97−
6.04 (m, 1H), 7.12−7.24 (m, 3H), 7.31−7.41 (m, 2H), 7.44 (d, J =
8.22 Hz, 0.4H), 7.52 (d, J = 8.02 Hz, 0.6H), 11.49 (br s, 1H).
In a similar way, compounds 30d, 30j, 30o, and 30p were prepared.
Their analytical data are reported below. For the other derivatives, all
characterization data can be found in the Supporting Information.
(2S)-Benzyl-2-(((((4R,5R,7R,8R)-5-(2,4-dioxo-3,4-dihydropyri-
midin-1(2H)-yl)-8-hydroxy-1,6-dioxa-spiro[3.4]octan-7-yl)-
methoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate
Synthesis of 4-Amino-1-((4R,5R,7R,8R)-8-(tert-butyldime-
thylsilyloxy)-7-((tert-butyldimethylsilyloxy)methyl)-1,6-
dioxaspiro[3.4]octan-5-yl)pyrimidin-2(1H)-one (23). To 21 (750
mg, 2.78 mmol) in dry DMF (20 mL) were added TBDMSCl (900
mg, 6.0 mmol) and imidazole (500 mg, 7.3 mmol). The mixture was
stirred at room temperature overnight. More TBDMSCl (900 mg) and
imidazole (600 mg) were added, and stirring was again continued
overnight. The mixture was quenched with a small amount of MeOH
and stirred for 5 min. Water was added, and the mixture was extracted
with EtOAc (2×). The combined organic layer was washed with 1 N
HCl and dried on Na2SO4. After filtration, the solvent was removed,
resulting in a colorless oil. This was dissolved in CH2Cl2 and filtered
on silica (rinced with 80:20 CH2Cl2/EtOAc). After evaporation, the
resulting oil (1.6 g) was dissolved in dry acetonitrile (40 mL), and
2,4,6-triisopropylbenzenesulfonyl chloride (1.7 g, 5.6 mmol) and
DMAP (686 mg, 5.6 mmol) were added immediately followed by
Et3N (0.78 mL, 5.6 mmol). The mixture was stirred at room
temperature. After 2 h, NH4OH (20 mL) was added. The mixture was
stirred at room temperature for 1.5 h. The solvent was removed, and
the residue was purified by column chromatography with EtOAc to
EtOAc/MeOH 90:10, resulting in 23 as a glassy solid (1.123 g, 87%).
HPLC condition A, tR = 3.18 min, m/z = 498 (M + H)+.
Synthesis 4-Amino-1-((4R,5R,7R,8R)-8-Hydroxy-7-(hydroxy-
methyl)-1,6-dioxaspiro[3.4]octan-5-yl)pyrimidin-2(1H)-one
(13). To 23 (1213 mg, 2.437 mmol) in THF (20 mL) was added
TBAF (5.1 mL, 1 M in THF, 2.1 equiv), and the mixture was stirred at
room temperature. After 1.5 h, the solvent was removed. The mixture
was purified by reversed-phase chromatography (A: CH3CN, B: H2O;
0 min, 0% A; 7 min, 25% A; 10 min, 95% A; column: Atlantis PrepT3
OBD 5 μm, 30 × 50 mm). Compound 13 was obtained as a white
solid (350 mg, 52%) after lyophilization. HPLC condition B, tR = 1.81
1
(30d). HPLC condition A, tR = 3.06 min, m/z = 638 (M + H)+. H
NMR (400 MHz, CDCl3) δ 1.23−1.41 (m, 2H), 1.66 (br s, 0H), 1.93
(br s, 1H), 2.37 (m, J = 12.56, 12.56, 8.63, 6.24 Hz, 1H), 2.54−2.75
(m, 1H), 3.44−3.67 (m, 1H), 3.78 (dd, J = 9.27, 1.85 Hz, 1H), 3.86−
4.03 (m, 1H), 4.05−4.17 (m, 1H), 4.18−4.28 (m, 1H), 4.31−4.41 (m,
1H), 4.41−4.66 (m, 3H), 4.96−5.16 (m, 2H), 5.32 (d, J = 8.20 Hz,
0H), 5.44 (d, J = 8.20 Hz, 0H), 6.11 (s, 1H), 7.08 (d, J = 8.19 Hz, 0H),
7.18−7.41 (m, 6H), 7.44−7.57 (m, 3H), 7.64 (d, J = 8.00 Hz, 1H),
7.78−7.89 (m, 1H), 8.01−8.18 (m, 1H), 9.37 (br s, 1H).
(2S)-Butyl-2-(((((4R,5R,7R,8R)-5-(2,4-dioxo-3,4-dihydropyri-
midin-1(2H)-yl)-8-hydroxy-1,6-dioxa-spiro[3.4]octan-7-yl)-
methoxy)(phenoxy)phosphoryl)amino)propanoate (30j).
1
HPLC condition A, tR = 1.88 min, m/z = 554 (M + H)+. H NMR
(400 MHz, DMSO-d6) δ 0.77−0.93 (m, 3H), 1.14−1.25 (m, 3H),
1.24−1.37 (m, 2H), 1.41−1.62 (m, 2H), 2.35−2.47 (m, 2H), 3.63−
3.92 (m, 3H), 3.92−4.06 (m, 2H), 4.05−4.21 (m, 1H), 4.23−4.46 (m,
3H), 5.48−5.59 (m, 1H), 5.59−5.72 (m, 1H), 5.89−6.15 (m, 2H),
7.09−7.25 (m, 3H), 7.31−7.41 (m, 2H), 7.43−7.52 (m, 1H), 11.51
(br s, 1H).
(2S)-Benzyl-2-(((((4R,5R,7R,8R)-5-(2,4-dioxo-3,4-dihydropyri-
midin-1(2H)-yl)-8-hydroxy-1,6-dioxaspiro[3.4]octan-7-yl)-
methoxy)(phenoxy)phosphoryl)amino)butanoate (30o). HPLC
condition A, tR = 2.00 min, m/z = 602 (M + H)+. 1H NMR (400 MHz,
DMSO-d6) δ 0.71−0.83 (m, 3H), 1.45−1.73 (m, 2H), 2.33−2.48 (m,
G
dx.doi.org/10.1021/jm4015422 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2H), 3.59−3.80 (m, 2H), 3.79−3.96 (m, 1H), 4.04−4.19 (m, 1H),
4.24−4.47 (m, 3H), 4.98−5.14 (m, 2H), 5.47−5.57 (m, 1H), 5.58−
5.73 (m, 1H), 5.96−6.03 (m, 1H), 5.96−6.03 (m, 1H), 7.09−7.22 (m,
3H), 7.27−7.39 (m, 7H), 7.44 (d, J = 8.02 Hz, 0.5H), 7.48 (d, J = 8.22
Hz, 0.5H), 11.50 (br s, 1H).
(2) Ghobrial, R. M.; Steadman, R.; Gornbein, J.; Lassman, C.; Holt,
C. D.; Chen, P.; Farmer, D. G.; Yersiz, H.; Danino, N.; Collisson, E.;
Baquarizo, A.; Han, S. S.; Saab, S.; Goldstein, L. I.; Donovan, J. A.;
Esrason, K.; Busuttil, R. W. A 10-year experience of liver trans-
plantation for hepatitis C: Analysis of factors determining outcome in
over 500 patients. Ann. Surg. 2001, 234, 384−393.
(2S)-Benzyl-2-(((((4R,5R,7R,8R)-5-(2,4-dioxo-3,4-dihydropyri-
midin-1(2H)-yl)-8-hydroxy-1,6-dioxaspiro[3.4]octan-7-yl)-
methoxy)(phenoxy)phosphoryl)amino)propanoate (30p).
(3) Simmonds, P. Genetic diversity and evolution of hepatitis C virus
− 15 years on. J. Gen. Virol. 2004, 85, 3173−3188.
1
HPLC condition A, tR = 1.92 min, m/z = 588 (M + H)+. H NMR
(4) McHutchison, J. G.; Manns, M. P.; Muir, A. J.; Terrault, N. A.;
Jacobson, I. M.; Afdhal, N. H.; Heathcote, E. J.; Zeuzem, S.; Reesink,
H. W.; Garg, J.; Bsharat, M.; George, S.; Kauffman, R. S.; Adda, N.; Di
Bisceglie, A. M. Telaprevir for previously treated chronic HCV
infection. N. Engl. J. Med. 2010, 362, 1292−1303.
(5) Revill, P.; Serradell, N.; Bolos, J.; Rosa, E. Telaprevir. Drugs
Future 2007, 32, 788.
(6) Jensen, D. M. A new era of hepatitis C therapy begins. N. Engl. J.
(400 MHz, DMSO-d6) δ 1.19−1.29 (m, 3H), 2.38−2.46 (m, 2H),
3.53−3.97 (m, 3H), 4.06−4.20 (m, 1H), 4.26−4.46 (m, 3H), 5.05−
5.14 (m, 2H), 5.49−5.59 (m, 1H), 5.61−5.73 (m, 1H), 5.88−6.05 (m,
1H), 6.07−6.18 (m, 1H), 7.09−7.23 (m, 3H), 7.30−7.40 (m, 7H),
7.43−7.51 (m, 1H), 11.51 (br s, 1H).
ASSOCIATED CONTENT
* Supporting Information
■
S
Med. 2011, 364, 1272−1274.
Experimental details, analytical data of phosphoramidates, and
general procedure for the synthesis of phosphoramidates 29a−
d. This material is available free of charge via the Internet at
http://pubs.acs.org.
(7) Lindenbach, B. D.; Rice, C. M. Unravelling hepatitis C virus
replication from genome to function. Nature 2005, 436, 933−938.
(8) Behrens, S.-E.; Tomei, L.; De Francesco, R. Identification and
properties of the RNA-dependent RNA polymerase of hepatitis C
virus. EMBO J. 1996, 15, 12−22.
(9) Lohmann, V.; Korner, F.; Herian, U.; Bartenschlager, R.
Biochemical properties of hepatitis C virus NS5B RNA-dependent
RNA polymerase and identification of amino acid sequence motifs
essential for enzymatic activity. J. Virol. 1997, 71, 8416−8428.
(10) Lesburg, C. A.; Cable, M. B.; Ferrari, E.; Hong, Z.; Mannarino,
A. F.; Weber, P. C. Crystal structure of the RNA-dependent RNA
polymerase from hepatitis C virus reveals a fully encircled active site.
Nat. Struct. Biol. 1999, 6, 937−943.
(11) Legrand-Abravanel, F.; Nicot, F.; Izopet, J. New NS5B
polymerase inhibitors for hepatitis C. Expert Opin. Invest. Drugs
2010, 19, 963−975.
(12) Sarrazin, C.; Zeuzem, S. Resistance to direct antiviral agents in
patients with hepatitis C virus infection. Gastroenterology 2010, 138,
447−462.
(13) McCown, M. F.; Rajyaguru, S.; Le Pogam, S.; Ali, S.; Jiang, W.-
R.; Kang, H.; Symons, J.; Cammack, N.; Najera, I. The hepatitis C
virus replicon presents a higher barrier to resistance to nucleoside
analogs than to non-nucleoside polymerase or protease inhibitors.
Antimicrob. Agents Chemother. 2008, 52, 1604−1612.
(14) Kim, J.; Chong, Y. Understanding the RNA-specificity of HCV
RdRp: Implications for anti-HCV drug discovery. Bull. Korean Chem.
Soc. 2006, 27, 59−64.
(15) Tan, S.-L. Hepatitis C Viruses: Genomes and Molecular
Biology; Horizon Bioscience: Wymondham, UK, 2006.
AUTHOR INFORMATION
Corresponding Author
*E-mail: tjoncker@its.jnj.com. Tel: +32 014601168.
■
Present Addresses
§Vlaamse Milieumaatschappij Afdeling Rapportering Water,
Dienst Laboratorium, Raymonde de Larochelaan 1, B-9051
Sint-Denijs-Westrem, Belgium.
∥Universiteit Hasselt, TechTransfer Office, Martelarenlaan 42,
3500 Hasselt, Belgium.
⊥Roche R&D China Ltd. 720 Cai Lun Rd, Building 5, Pudong,
201203 Shanghai, P. R. China.
#AstraZeneca R&D Molndal, Pepparedsleden 1, SE-431 83
̈
Molndal, Sweden.
̈
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Rudy Strijbos, Lieven Dillen, Liesbeth Vereycken,
■
Koen Wuyts, and Karin Gohlin for help with the in vitro and in
̈
vivo experiments.
ABBREVIATIONS USED
■
(16) Bressanelli, S.; Tomei, L.; Rey, F. A.; De Francesco, R. Structural
analysis of the hepatitis C virus RNA polymerase in complex with
ribonucleotides. J. Virol. 2002, 76, 3482−3492.
HCV, hepatitis C virus; NS5B, nonstructural protein 5B; RNA,
ribonucleic acid; Huh-7, hepato cellular carcinoma cell line; TP,
triphosphate; HIV, human immunodeficiency virus; HBV,
hepatitis B virus; NS3/4A, nonstructural proteins 3 and 4A;
BSA, bis(trimethylsilyl)acetamide; PI, protease inhibitor; NS,
nonstructural; NNI, non-nucleoside inhibitor; hERG, human
(17) Klumpp, K.; Kalayanov, G.; Ma, H.; Le Pogam, S.; Leveque, V.;
Jiang, W.-R.; Inocencio, N.; De Witte, A.; Rajyaguru, S.; Tai, E.;
Chanda, S.; Irwin, M. R.; Sund, C.; Winqist, A.; Maltseva, T.; Eriksson,
S.; Usova, E.; Smith, M.; Alker, A.; Najera, I.; Cammack, N.; Martin, J.
A.; Johansson, N. G.; Smith, D. B. 2′-Deoxy-4′-azido nucleoside
analogs are highly potent inhibitors of hepatitis C virus replication
despite the lack of 2′-alpha-hydroxylgroups. J. Biol. Chem. 2008, 283,
2167−2175.
̀
Ether-a-go-go-Related Gene; BzCl, benzylchloride; NaH,
sodium hydride; TBDMS, tert-butyl dimethylsilyl; DMAP,
dimethylaminopyridine; rt, room temperature; TBAF, tetrabu-
tylammonium fluoride; PA, phosphoramidate; dNTP, 2′-
deoxyribonucleoside 5′ triphosphate; NTP, ribonucleoside 5′
triphosphate; TPSCl, 2,4,6-triisopropylbenzenesulfonyl chlor-
ide; PK, pharmacokinetic; PD, pharmacodynamics; NMI, N-
methyl imidazole; Naph, naphthyl; WT, wild type; LC−MS,
liquid chromatography−mass spectrometry
(18) Gunic, E.; Girardet, J.-L.; Ramasamy, K.; Stoisavljevic-Petkov,
V.; Chow, S.; Yeh, L.-T.; Hamatake, R. K.; Raney, A.; Hong, Z. Cyclic
monophosphate prodrugs of base-modified 2′-C-methyl ribonucleo-
sides as potent inhibitors of hepatitis C virus RNA replication. Bioorg.
Med. Chem. Lett. 2007, 2452−2455.
(19) 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. A 7-deaza-adenosine analog is a potent and
selective inhibitor of hepatitis C virus replication with excellent
REFERENCES
■
(1) Prusoff, W. H. Synthesis and biological activities of iododeox-
yuridine, an analog of thymidine. Biochim. Biophys. Acta 1959, 32,
295−296.
H
dx.doi.org/10.1021/jm4015422 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
pharmacokinetic properties. Antimicrob. Agents Chemother. 2004, 48,
3944−3953.
S.; Kim, C. U. Discovery of the first C-nucleoside HCV polymerase
inhibitor (GS-6620) with demonstrated antiviral response in HCV
infected patients. J. Med. Chem. [Online early access] DOI: 10.1021/
jm400201a. Published Online: April 2, 2013.
(34) A minor difference in experimental setup (96-well format with a
4 day incubation vs 384-well format with a 3 day incubation) was
noted when comparing the work of Du et al.25 and our protocol.
However, this cannot explain these differences.
(35) Ali, S.; Leveque, V.; Le Pogam, S.; Ma, H.; Philipp, F.;
Inocencio, N.; Smith, M.; Alker, A.; Kang, H.; Najera, I.; Klumpp, K.;
Symons, J.; Cammack, N.; Jiang, W.-R. Selected replicon variants with
low-level in vitro resistance to the hepatitis C virus NS5B polymerase
inhibitor PSI-6130 lack cross-resistance with R1479. Antimicrob Agents
Chemother. 2008, 52, 4356−4369.
(36) Chung, S. J.; Fromme, J. C.; Verdine, G. L. Structure of human
cytidine deaminase bound to a potent inhibitor. J. Med. Chem. 2005,
48, 658−660.
(20) Jonckers, T. H. M.; Lin, T.; Buyck, C.; Lachau-Durand, S.;
Vandyck, K.; Van Hoof, S.; Vandekerckhove, L. A. M.; Hu, L.; Berke, J.
M.; Vijgen, L.; Dillen, L. L. A.; Cummings, M. D.; de Kock, H.;
Nilsson, M.; Sund, C.; Rydegard, C.; Samuelsson, B.; Rosenquist, A.;
Fanning, G.; Van Emelen, K.; Simmen, K.; Raboisson, P. 2′-Deoxy-2′-
spirocyclopropylcytidine revisited: A new and selective inhibitor of the
hepatitis C virus NS5B polymerase. J. Med. Chem. 2010, 53, 8150−
8160.
(21) Pierra, C.; Benzaria, S.; Amador, A.; Moussa, A.; Mathieu, S.;
Storer, R.; Gosselin, G. NM 283, an efficient prodrug of the potent
anti-HCV agent 2′-C-methylcytidine. Nucleosides, Nucleotides Nucleic
Acids 2005, 24, 767−770.
(22) Murakami, E.; Niu, C.; Bao, H.; Micolochick Steuer, H. M.;
Whitaker, T.; Nachman, T.; Sofia, M. A.; Wang, P.; Otto, M. J.;
Furman, P. A. The mechanism of action of β-D-2′-deoxy-2′-fluoro-2′-
C-methylcytidine involves a second metabolic pathway leading to β-D-
2′-deoxy-2′-fluoro-2′-C-methyluridine 5′-triphosphate, a potent inhib-
itor of the hepatitis C virus RNA-dependent RNA polymerase.
Antimicrob. Agents Chemother. 2008, 52, 458−464.
(23) Burkhard, J. A.; Wuitschik, G.; Rogers-Evans, M.; Muller, K.;
Carreira, E. M. Oxetanes as versatile elements in drug discovery and
synthesis. Angew. Chem., Int. Ed. 2010, 49, 9052−9067.
(24) Babu, B. R.; Keinicke, L.; Petersen, M.; Nielsen, C.; Wengel, J.
2′-Spiro ribo- and arabinonu-cleosides: Synthesis, molecular modelling
and incorporation into oligodeoxynucleotides. Org. Biomol. Chem.
2003, 1, 3514−3526.
(37) Determination of NTP levels formed in hepatocytes after
incubation of compounds 30a and 30b was not performed because of
their limited availability at the time of the hepatocyte experiments.
(38) Perrone, P.; Luoni, G. M.; Kelleher, M. R.; Daverio, F.; Angell,
̂
A.; Mulready, S.; Congiatu, C.; Rajyaguru, S.; Martin, J. A.; Leveque,
V.; Le Pogam, S.; Najera, I.; Klumpp, K.; Smith, D. B.; McGuigan, C.
Application of the phosphoramidate ProTide approach to 4′-
azidouridine confers sub-micromolar potency versus hepatitis C virus
on an inactive nucleoside. J. Med. Chem. 2007, 50, 1840−1849.
(25) Du, J.; Chun, B.-K.; Mosley, R.; Bansal, S.; Bao, H.; Lam, A.;
Espiritu, C.; Murakami, E.; Niu, C.; Micolochick, S.; Furman, P.; Sofia,
M. Use of 2′-spirocyclic ethers in HCV nucleoside design. J. Med.
Chem. [Online early access]. DOI: 10.1021/jm401224y. Published
Online: Sept 30, 2013.
(26) Li, N.-S.; Lu, J.; Piccirilli, J. A. Efficient synthesis of methyl 3,5-
di-O-benzyl-α-D-ribofuranoside and application to the synthesis of 2′-
C-β-alkoxymethyluridines. Org. Lett. 2007, 9, 3009−3012.
(27) Van Rompay, A. R.; Johansson, M.; Karlsson, A. Phosphor-
ylation of nucleosides and nucleoside analogs by mammalian
nucleoside monophosphate kinases. Pharmacol. Ther. 2000, 87,
189−198.
(28) Mehellou, Y.; Balzarini, J.; McGuigan, C. Aryloxy phosphor-
amidate triesters: A technology for delivering monophosphorylated
nucleosides and sugars into cells. ChemMedChem 2009, 4, 1779−1791.
(29) Klumpp, K.; Martin, J. A.; McGuigan, C.; Smith, D. B. PCT Int.
Appl. WO 2007020193 A2 20070222, 2007.
(30) Sofia, M. J.; Bao, D.; Chang, W.; Du, J.; Nagarathnam, D.;
Rachakonda, S.; Ganapati Reddy, P.; Ross, B. S.; Wang, P.; Zhang, H.;
Bansal, S.; Espiritu, C.; Keilman, M.; Lam, A. M.; Micolochick Steurer,
H. M.; Niu, C.; Otto, M. J.; Furman, P. A. Discovery of a β-D-2′-deoxy-
2′α-fluoro-2′-β-C-methyluridine nucleotide prodrug (PSI-7977) for
the treatment of hepatitis C virus. J. Med. Chem. 2010, 53, 7202−7218.
(31) Lackey, D. B.; Groziak, M. P.; Sergeeva, M.; Beryt, M.; Boyer,
C.; Stroud, R. M.; Sayre, P.; Park, J. W.; Johnston, P.; Slamon, D.;
Shepard, H. M.; Pegram, M. Enzyme-catalyzed therapeutic agent
(ECTA) design: Activation of the antitumor ECTA compound
NB1011 by thymidylate synthase. Biochem. Pharmacol. 2001, 61, 179−
189.
(32) Vernachio, J. H.; Bleiman, B.; Dawn Bryant, K.; Chamberlain, S.;
Hunley, D.; Hutchins, J.; Ames, B.; Gorovits, E.; Ganguly, B.; Hall, A.;
Kolykhalov, A.; Liu, Y.; Muhammad, J.; Raja, N.; Robin Walters, C.;
Wang, J.; Williams, K.; Patti, J. M.; Henson, G.; Madela, K.; Aljarah,
M.; Gilles, A.; McGuigan, C. INX-08189, a phosphoramidate prodrug
of 6-O-methyl-2-C-methyl guanosine, is a potent inhibitor of hepatitis
C virus replication with excellent pharmacokinetic and pharmacody-
namic properties. Antimicrob. Agents Chemother. 2011, 55, 1843−1851.
(33) Cho, A.; Zhang, L.; Xu, J.; Lee, R.; Butler, T.; Metobo, S.;
Aktoudianakis, V.; Lew, W.; Ye, H.; Clarke, M.; Doerffler, E.; Byun, D.;
Wang, T.; Babusis, D.; Carey, A. C.; German, P.; Sauer, D.; Zhong, W.;
Rossi, S.; Fenaux, M.; McHutchison, J. G.; Perry, J.; Feng, J.; Ray, A.
I
dx.doi.org/10.1021/jm4015422 | J. Med. Chem. XXXX, XXX, XXX−XXX