Phosphoramidate dinucleosides as hepatitis C virus polymerase inhibitors

Article abstract of DOI:10.1021/jm800617c

GC dinucleosides exhibiting a phosphoramidate internucleosidic linkage with neutral, amphiphile, positively or negatively charged side chains were synthesized. Their potential inhibitory effect on the hepatitis C virus (HCV) NS5B polymerase was evaluated

Full text of DOI:10.1021/jm800617c

J. Med. Chem. 2008, 51, 5745–5757
5745
Phosphoramidate Dinucleosides as Hepatitis C Virus Polymerase Inhibitors
Ivan Zlatev,†, He´le`ne Dutartre,‡, Ivan Barvik,§ Johan Neyts,| Bruno Canard,‡ Jean-Jacques Vasseur,† Karine Alvarez,*,‡ and
Franc¸ois Morvan*,†
Institut des Biomole´cules Max Mousseron (IBMM), UMR 5247 CNRSsUniVersite´ Montpellier 1sUniVersite´ Montpellier 2,
Place Euge`ne Bataillon, CC1704, 34095 Montpellier Cedex 5, France, Department of Structural Virology, AFMB UMR 6098,
CNRS UniVersite´ de la Me´diterrane´e, Case 925, 163 AVenue de Luminy, 13288 Marseille, France, Institute of Physics, Charles UniVersity, Ke
KarloVu 5, Prague 12116 2, Czech Republic, Rega Institute for Medical Research, Katholieke UniVersiteit LeuVen, Minderbroedersstraat 10,
B-3000 LeuVen, Belgium
ReceiVed May 23, 2008
GC dinucleosides exhibiting a phosphoramidate internucleosidic linkage with neutral, amphiphile, positively
or negatively charged side chains were synthesized. Their potential inhibitory effect on the hepatitis C virus
(HCV) NS5B polymerase was evaluated in vitro and in HCV replicon containing cells. Whereas the
amphiphile and the positively charged analogues were found to be inactive, the neutral (1) and the negatively
charged (4) ones inhibited enzyme activity when tested as a diastereoisomeric mixture. The most potent
inhibitor proved to be the Sp isomer of the 5′-thiophosphorylated dinucleotide bearing the carboxylic side
chain (8) (IC50 of 25 µM in vitro and an EC50 of 9 µM in HCV subgenomic replicon). Molecular modeling
suggests that the phosphoramidate dinucleoside (8) is stabilized in the active site by interactions with
magnesium ions and lysine and arginine residues of the polymerase.
Introduction
polymerase (RdRp) able to replicate RNA without a primer in
a so-called de novo synthesis mechanism.5 In vitro, however,
the C-terminal truncated recombinant polymerase can use a wide
range of hetero- or homopolymeric templates in a primer-
dependent or primer-independent fashion.6 During de novo RNA
synthesis, the RdRp has to synthesize first its RNA primer and
then copy the entire RNA template. RNA synthesis can thus be
separated into two steps7,8 that are structurally and biochemically
distinct: the initiation step leading to the RNA primer synthesis
and the elongation step of the neosynthezised RNA up to the
end of the template.
The hepatitis C virus (HCVa) represents a global health
problem; about 3% of the world’s population is chronically
infected and at risk of developing liver cirrhosis and/or
hepatocellular carcinoma. HCV infection is the leading reason
for liver transplantation in the West.1 Current therapy consists
of a combination of interferon-R with the nucleoside analogue
ribavirin and results in 40-70% of sustained virological
responses depending on the genotype.2 However, this therapy
is associated with significant side effects,3 and there is no
approved therapeutic alternative for nonresponders. There is thus
an urgent need for new therapeutic molecules active against
the HCV infection.
HCV is an enveloped, positive-strand RNA virus; the 9.6 kb
genome encodes a single large polyprotein, which is cleaved
by cellular and viral proteases into four structural (Core, E1,
E2, and p7) and six nonstructural proteins (NS2, NS3, NS4A,
NS4B, NS5A, and NS5B). The nonstructural proteins assemble
into a replication complex that is responsible for the viral RNA
genome synthesis (for a review see ref 4). Because they bear
essential enzymatic activities for viral replication, the NS3
protease-helicase and the NS5B polymerase are targets of choice
for antiviral intervention. Moreover, the NS5B polymerase is
the key component of the replicative complex. It is a endoplas-
mic reticulum membrane-associated RNA-dependent-RNA-
Intensive drug screening campaigns have led to the discovery
of many NS5B inhibitors, some of them being evaluated in
clinical settings (reviewed in refs 9, 10). Two main classes of
NS5B RdRp inhibitors have been reported: non-nucleoside
inhibitors (NNI) and nucleoside inhibitors (NI). NNIs are
allosteric, noncompetitive inhibitors that target the alloenzyme,
free of RNA substrates (reviewed in ref 11). For most of them,
the mode of action requires the binding to the apoenzyme before
engagement of the enzyme into an elongative complex. For NIs,
the corresponding triphosphates (TPs) are competitors of the
natural nucleoside triphosphates and can be used as substrates
by the polymerase. After their incorporation into the nascent
RNA, they act as nonobligate chain terminators.12 Alternatively,
compounds that target the active site before the enzyme has
entered into the elongation steps of RNA synthesis may be
potent inhibitors. For example, it has been reported that
dinucleotides can be used more efficiently as primer molecules
than mononucleotides in in vitro RNA replication assay.6 On
the basis of these results modified dinucleotides, lacking the
3′-hydroxyl necessary for the polymerization reaction, were
synthesized and were shown able to decrease the RNA synthesis
measured in vitro, although with modest IC50 values.13
* To whom correspondence should be addressed. For F.M.: phone, +33
467 144 961; fax, +33 467 042 029; E-mail: morvan@univ-montp2.fr. For
K.A.: Phone: +33 491 825 571; Fax: +33 491 266 720; E-mail:
karine.alvarez@afmb.univ-mrs.fr.
†
Universite´ Montpellier 2.
Universite´ de la Me´diterrane´e.
Charles University.
Katholieke Universiteit Leuven.
‡
§
|
These authors contributed equally to the work.
a Abbreviations: HCV, hepatitis C virus; NS3, non structural protein 3;
NS5B, non structural protein 5B; RdRp, RNA-dependent-RNA-polymerase;
NNI, non nucleoside inhibitor; NI, nucleoside inhibitor; MP, monophos-
phate; TP, triphosphate; GC, guanosin-3′-yl-cytidin-5′-yl dinucleotide; GMP,
guanosine monophosphate; GTP, guanosine triphosphate; NTP, nucleoside
triphosphate.
We took advantage of this novel approach to synthesize new
dinucleotides bearing modifications on the internucleosidic
linkage in order to increase both their binding to the active site
of the polymerase and their affinity to the RNA template and
hence their inhibition properties. Our target compounds were
10.1021/jm800617c CCC: $40.75
2008 American Chemical Society
Published on Web 08/23/2008

5746 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
ZlateV et al.
Scheme 1. Retrosynthetic Pathway for the Synthesis of Phosphoramidate Dinucleosides I
Scheme 2. Synthetic Pathway for the Synthesis of 14
designed based on several previously published structural data
of the NS5B enzyme in its putative RNA synthesis initiation
conformation.14,15 As it was also previously demonstrated that
the 5′-phosphate group of the dinucleotide was required for
inhibition,13 probably because it allowed hydrogen bonding and
electrostatic interaction with residues present in the active site,
we decided to study this modification thoroughly. Moreover,
such 5′-phosphorylated dinucleotide structures would act as
direct substrate to the enzyme, without the need of any
intracellular 5′-phosphorylation, as required for NIs. As a
modification for the internucleosidic linkage, we chose a
phosphoramidate diester linkage. The latter is a chemical
modification used either in a prodrug approach for nucleoside
analogues16-18 or in modified oligonucleotides19,20 and allows
the introduction of a large variety of side chain functionalities.
Because the 3′-end of the viral RNA negative strand template
of HCV is GC3′, the complementary sequence of the dimer is
5′GC. A 2′-O-methyl guanosine was chosen instead of natural
guanosine to gain higher enzymatic and chemical stability of
the final dinucleotide. The phosphoramidate linkage is also more
resistant to nucleases,21 hence the resulting dinucleotides would
have higher stability in biological media. Thus, we first designed
and synthesized 5′-phosphorylated dinucleotides made of a 2′-
O-methyl guanosine and a cytidine, linked together with a
phosphoramidate function bearing different side chains.20 To
establish a structure-activity relationship (SAR), we used side
chains with neutral, cationic, amphiphile, and anionic groups
exhibiting different properties. Then, to study the influence of
the 5′-environment, phosphoramidate dinucleotide analogues
with a 5′-hydroxyl group or bearing a 5′-phosphorothioate
monoester instead of the 5′-phosphate monoester were prepared.
Then, we evaluated their ability to inhibit the NS5B polymerase
activity in vitro. A molecular model of the NS5B polymerase
and the most potent dinucleotide analogue was constructed to
explain the mechanism of inhibition. Finally, the potential
inhibitory effect on HCV replicon replication in cell cultures
was evaluated.
Results and Discussion
Chemistry. The preparation of these 2′-O-Me-GC phospho-
ramidate dinucleosides proceeded by a strategy using convergent
and divergent syntheses. First, a convergent synthesis using 3′-
OH-2′-O-Me guanosine derivatives (9-11) and the 5′-H-
phosphonate monoester of N4,2′,3′-O,O-tribenzoyl cytidine (14)
was applied, affording common intermediates 15-17 with a
H-phosphonate diester linkage between the 2′-O-Me guanosine
and the cytidine moieties. Second, the divergent strategy
concerned the amidative oxidation,22 allowing, from the com-
mon intermediates 15-17, the preparation of the different dimers
I with various phosphoramidate side chains (Scheme 1).
The building block 9 is commercially available. The building
blocks 10 and 11 were synthesized from N2-isobutyryl-2′-O-
methyl guanosine23 by a selective 5′-phosphitylation using solid-
supported reagents as previously reported.24,25
The N4,2′,3′-O,O-tribenzoyl-5′-H-phosphonate monoester of
cytidine (14) was prepared according to a five-step procedure
with a 63% overall yield (Scheme 2). Cytidine was first
selectively benzoylated on the N4 position by means of benzoic
anhydride in DMF.26 After replacement of DMF by pyridine,
the crude was directly dimethoxytritylated on the 5′-hydroxyl
(90%) by addition of dimethoxytrityl chloride (Scheme 2). After
purification, the nucleoside 12 was benzoylated on both 2′ and
3′ hydroxyls by means of benzoyl chloride, and after workup,
the crude was directly treated with benzene sulfonic acid,
affording after purification the N4,2′,3′-O,O-tribenzoyl cytidine
13 (88%). Finally, 13 was treated with diphenylphosphite
followed by aqueous hydrolysis,27 affording the expected 5′-
H-phosphonate monoester of N4,2′,3′-O,O-tribenzoyl cytidine
14 (80%).

Phosphoramidate Dinucleosides as HCV Polymerase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5747
Scheme 3. Coupling of 9-11 and 14 Affording H-Phosphonate Diester Dimers 15-17
Scheme 4. Amidative Oxidations of 15 and Deprotection Affording 1, 2, 3, and 4
The coupling of the two building blocks 14 and 9-11 to form
H-phosphonate diesters 15-17 was carried out using a solid-
supported acyl chloride activator28 (Scheme 3). This coupling
proceeded in two steps, first, the H-phosphonate monoester
function of 14 reacted with the solid-supported acyl chloride
and, second, the formed mixed anhydride was displaced by the
3′-hydroxyl of 9-11 to yield 15-17 in solution. By this way,
the excess of 14 was trapped on the solid support and only
dimers 15-17 and pyridinium salt remained in solution. A
simple filtration, an aqueous extraction, and a precipitation
in diethyl ether afforded the dimers 15-17 with >85% HPLC
purity (Figure S1 in Supporting Information). Because this
coupling reaction is nonstereoselective, the H-phosphonate
diesters were all a ∼1:1 mixture of Rp and Sp diastereoiso-
mers on the chiral phosphorus center and their presence was
confirmed by 31P NMR as two peaks (δP-H between 7.3 and
9.8 ppm).
was added. The amidative oxidation occurred to form the two
diastereoisomers (Sp and Rp) of the phosphoramidate I with
some minor formation of phosphodiester linkage corresponding
to a side-oxidation due to trace of water. First, the oxidation of
15 was performed in the presence of four different amines:
2-methoxyethylamine, 3-(dimethylamino)-1-propylamine, his-
tamine, or 6-aminocaproic acid methyl ester to afford, after
deprotection, the corresponding dimers bearing neutral (1),
cationic (2), amphiphile (3), or anionic (4) side chains,
respectively (Scheme 4). Pure target compounds were obtained
after two chromatographic steps (ion exchange and reverse
phase). Finally, sodium ion-exchange was performed, yielding
the desired compounds as their sodium salt.
To further establish the role of the 5′-terminal function group
in a SAR, dimers 16 and 17 were also oxidized in presence of
2-methoxyethylamine and 6-aminocaproic acid methyl ester,
affording, after deprotection, phosphoramidate dimers with a
neutral or an anionic chain, 5-6 and 7-8, respectively, bearing
The crude H-phosphonate diesters 15-17 were dissolved in
dry pyridine, and a mixture of carbon tetrachloride and amine

5748 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
ZlateV et al.
and increasing the concentration of the phosphoramidate di-
nucleosides allowed determining the IC50 values of the different
phosphoramidate dinucleosides (Figure 2B-C). The phospho-
ramidate dinucleosides harboring either positively charged (2)
or amphiphile, with a more hindered imidazolylethyl (3), side
chains have no significant effect on pppGC formation. However,
when the phosphoramidate linkage is either neutral (1) or
negatively charged (4), a significant and reproducible inhibition
was observed, with the best effect measured with the negatively
charged phosphoramidate dinucleoside 4 (IC50 ) 69 µM).
Therefore, only compounds 1 and 4 were selected for further
study.
We then studied the influence of the chemical nature of the
5′-moieties on the inhibitory effect of the inhibitors. In the first
series, the phosphoramidate dinucleosides were synthesized with
a 5′-phosphate monoester. Indeed, it was shown that guanosine
monophosphate (GMP) was able to correctly initiate the RNA
synthesis with a better efficiency than the natural guanosine
triphosphate (GTP),31 suggesting a higher affinity of mono-
phosphate compounds for the active site. For SAR studies, the
5′-nonphosphorylated and the 5′-thiophosphate analogues were
also synthesized and evaluated for their inhibition in vitro. As
expected,13 the removal of the phosphate monoester group at
the 5′-position of 1 or 4 resulted in a loss of activity, with an
increase of IC50 to >200 µM and 160 µM for compounds 5
and 7, respectively (not shown). However, the 5′-thiophosphate
substitution increased the inhibition potency of the phosphora-
midate dinucleosides (Figure 3) regardless of the nature of the
phosphoramidate side chain; for both neutral (6) and negatively
charged (8) phosphoramidate dinucleosides, the IC50 value is
decreased up to 2-fold (Figure 3A: 52 µM for 8 and 47 µM for
6) compared to the 5′-monophosphate phosphoramidate di-
nucleotides (Figure 3A: 69 µM for 4 and 106 µM for 1).
Figure 1. Schematic Structure of Phosphoramidate Dimers 5-8.
a 5′-hydroxyl function for 5 and 7 and a 5′-phosphorothioate
monoester function for 6 and 8 (Figure 1).
For each target compound, a mixture of two diastereoisomers
exhibiting the Sp and the Rp absolute configurations on the
asymmetric phosphorus atom of the phosphoramidate linkage
was obtained and this was confirmed by NMR and sometimes
visible on the HPLC spectra (as two peaks). The mixture was
directly tested for inhibitory evaluation. Henceforth, the more
potent compounds tested as isomeric mixture were further
separated by HPLC on a preparative reverse phase C18 column,
and each diastereoisomer was isolated pure (see Supporting
Information for HPLC and NMR examples). For all isolated
diastereoisomers, a clear correlation was established between
the retention times on RP-HPLC and the 31P NMR chemical
shift of the phosphoramidate bond, as previously noticed29 (see
Table S1 in Supporting Information). This correlation was also
observed in the inhibition assays. In analogy to the established
behavior of dinucleoside phosphoramidate diastereoisomers,29
the isomer with the shorter retention time on RP-HPLC (named
“fast” or F) and with the more upfield 31P chemical shift of the
P-N bond (named “uf”) was assigned as the diastereoisomer
with the Rp configuration, while the other (named “slow” or S
and “df”) was assigned to the one with the Sp configuration.
The validity of this assignment is supported by the results of
the molecular dynamics (MD) experiments (see below).
In Vitro Determination of IC50. In a previous work using a
polyC template, we studied the RNA polymerization catalyzed
by NS5B and identified three kinetically distinct steps.7 First,
the very first phosphodiester bond formation from G1 to G2
occurs rapidly, and G2 products accumulate over time. Second,
products from G2 up to G6 are formed. Subsequently, a third
phase occurs in which G6 products are elongated in a processive
fashion into template-length, runoff products. The formation of
G2, of G3 to G6, and of >G6 products has been referred to as
initiation, transition, and elongation of RNA synthesis, respec-
tively.7 Bearing GC nucleobases complementary to the 3′-end
of a synthetic oligonucleotide template corresponding to the 3′-
end of the negative strand of the HCV genome, the phospho-
ramidate dinucleosides were designed to target the initiation step
of RNA synthesis. This initiation phase is defined as the
formation of the first phosphodiester bond between two nucle-
otides, leading to a ribodinucleotide primer.
For phosphoramidate dinucleosides 1, 4, 6, and 8, the Sp and
Rp diastereoisomeric mixture was separated and distinct isomers
were tested. The IC50 values show that the best inhibition
potency is always observed for the slowly eluting isomers (i.e.,
with longer retention time in RP-HPLC) (Figure 3B, noted S
for “slow” in the figure). The “slow” dimer is always 2- to 3-fold
more potent than the “fast” one (Figure 3B, for example 54
µM for 8F compared to 25 µM for 8S). Moreover, this tendency
is observed irrespective to the nature of the phosphoramidate
linkage. Finally, the separation of the diastereoisomers did not
change the SAR trend obtained with the mixtures: series 1 is
always the less potent, followed by the negatively charged 4,
the 5′-thiophosphorylated 6 and 8 series being the most potent.
MolecularDynamicsStudyofthePolymerase-Phosphora-
midate Dinucleoside Complex. Structural studies of the HCV
polymerase have indicated that the active site is encircled by a
peculiar secondary structure, the flap, on one side, and the fingers
on the other.14,15 Therefore, the orientation of the phosphora-
midate modification may influence the accommodation of the
dinucleotide in this extremely closed and constrained active site.
We measured the production of the pppGC product and the
ability of phosphoramidate dinucleosides to inhibit this reaction
(Figure 2A). As reported by others for such a template,30 the
HCV polymerase is able to initiate RNA synthesis at two sites,
giving rise to the desired product pppGC and a minor pppCC
byproduct, with no effect on the calculations because the CTP
concentration was kept constant throughout all the experiments.
When a phosphoramidate dinucleoside is added in the reaction
(up to 500 µM), the accumulation of pppGC product is
decreased, reflecting the inhibition of RNA synthesis (Figure
2A). The concentration of the pppGC product can be quantified,
Indeed, only the Sp isomer was straightforwardly docked
within the active site of the HCV RdRp (Figure 4). As expected,
important hydrogen bond and/or electrostatic interactions be-
tween the 5′-phosphate/thiophosphate moiety and three arginine
residues (ARG386, ARG394, ARG158) can be observed in our
model. In the Sp absolute configuration, the nonbridging oxygen
atom of the internucleosidic linkage is situated in a pseudo-
equatorial position, directed toward the two magnesium ions
present in the polymerization site, thus allowing complexation
between them. Moreover, the only way to accommodate
smoothly the long phosphoramidate side chain into the HCV

Phosphoramidate Dinucleosides as HCV Polymerase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5749
Figure 2. Phosphoramidate dinucleosides decrease the formation of initiation products (pppGC and pppCC) synthesized by wild-type NS5B. (A)
Time course of the formation of initiation products by the NS5B polymerase using an RNA template corresponding to the 3′ end of the HCV strand
(-) genome in the presence of phosphoramidate dinucleoside 4 at the concentration of 50 µM. Initiation products were separated by electrophoresis
on denaturated sequencing gel. (B) pppGC products obtained for various concentrations of compound 4 were quantified and values reported as a
function of concentration. Curves from 2 independent experiments are shown, and IC50 values calculated from these curves are indicated under the
arrow. (C) pppGC products obtained for various concentrations of compounds 1-4 were quantified as described in (B). IC50 values were calculated
and are represented as a bar for each phosphoramidates dinucleosides. All data represents average values for at least three separated experiments.
Figure 3. 5′-Thiophosphate modification and separation of the diastereoisomers increase the inhibition potency of phosphoramidate dinucleosides.
(A) IC50 values of negatively charged (4 and 8) or neutral (1 and 6) phosphoramidate dinucleosides. (B) In vitro evaluation of separated diastereoisomers
of negatively charged (4 and 8) or neutral (1 and 6) phosphoramidate dinucleosides. “F” stands for “fast”, meaning the fast eluting isomer on
RP-HPLC, and “S” stands for “slow”, meaning the slowly eluting isomer.
RdRp active site is to direct it toward the nucleotide entry
channel, again in the Sp absolute configuration. Thus, the
negatively charged side chain interacts with arginine and lysine
residues (ARG158, ARG48, LYS155), situated on the incoming
pathway for nucleoside triphosphates (NTPs) during polymer-
ization. In a steric clash way, this configuration may block the
access of NTPs to the polymerization site, suggesting an
explanation to the observed inhibitory effect. In the other
orientation, the Rp absolute configuration, the phosphoramidate
linkage would be in conflict with Mg2+ ions in the active site.
Possibly, Rp oriented phosphoramidate linkage could evict Mg2+
ions and create direct hydrogen bonds with ASP residues in
the active site. Such stabilizing interaction would be probably
less efficient than the strong Coulombic attraction originating
from sandwiching of Mg2+ ions between ASP residues and the
oxygen from the phosphoramidate group.
The phosphoramidate dinucleoside mimics the presence of
the initiation GC dinucleotide product in the active site of the
HCV RdRp. Transition from the initiation to the later phase is
initiated by repositioning of the GC product and enables the
accommodation of another NTP into the active site. It seems,
however, to be disabled by the cooperative action of three potent
anchors: the 5′-phosphate/thiophosphate moiety, the carboxylic
side chain and the nonbridging oxygen atom from the phos-
phoramidate linkage both positioned in the Sp absolute con-
figuration. Moreover, the intrusion of some NTPs into the active
site seems to be obstructed by the long carboxylate side chains
of the phosphoramidate, creating a net of hydrogen bonds with
positively charged ARG/LYS residues, which normally paved
the way for incoming NTPs into the active site. In that way,
ARG/LYS residues interacting with the phosphoramidate side
chains are crippled in serving as clips for negatively charged

5750 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
ZlateV et al.
Figure 4. Molecular modeling of 8 in the Sp configuration with its interactions with amino acid residues of the NS5B polymerase in the active site.
Figure 5. (A) Evaluation of compounds 1, 4, 6, and 8 in Huh-5-2 cells, containing HCV subgenomic replicon, cultured with increasing amount
of the corresponding phosphoramidate dinucleoside. (B) Evaluation of the separated diastereoisomers of compounds 4 and 8. HCV replication was
analyzed by quantification of the luciferase activity reporter protein after 72 h, and the signals for each individual concentration of the different
compounds were expressed as a percentage of untreated cells and used to calculate the EC50. All data represents average values for at least three
independent experiments.
triphosphate moieties of incoming NTPs. Interestingly, it was
reported recently that the 5′-phosphoramidate of deoxyadenosine
bearing an L-aspartic amino acid could act as a substrate for
HIV-1 reverse transcriptase.32,33 In this case, aspartic acid
mimics the pyrophosphate of NTP, showing that such a
carboxylic function could interact with amino acids of the
polymerase in the active site.
µM, not shown). In contrast to the biochemical studies,
compounds 1 and 6 (diasteroisomeric mixtures) did not inhibit
replicon replication at the highest concentration (60 µM)
evaluated. The diasteroisomeric mixture of anionic phosphora-
midate dinucleosides 4 and 8 resulted in inhibition of HCV
replicons with EC50 values of ∼40 µM (Figure 5A). The “slow”
diastereoisomers (4S and 8S) were about 2-fold more active
(Figure 5B, 56 and 9 µM, respectively) than the “fast” ones
(4F and 8F, >60 and 21 µM, respectively).
The best inhibition was obtained with both isomers of the
5′-thiophosphate negatively charged phosphoramidate dinucleo-
side (8S and 8F, EC50: 9 and 21 µM, respectively), which
appeared to be more potent than the active isomer (4S) of the
5′-monophosphate analogue (EC50: 56 µM). This difference may
be the result of a better enzymatic stability of the 5′-thiophos-
phate analogue.34 To our knowledge, no similar inhibitory effect
on HCV replication in replicon containing cells was ever
reported in the literature using dinucleotides as inhibitors.
As an addition to the in vitro SAR developed, the model of
the polymerase-phosphoramidate dinucleoside complex refined
by molecular dynamics simulations settles the better inhibitory
activity observed for 5′-phosphorylated/thiophosphorylated, ne-
gatively charged phosphoramidates in the Sp absolute config-
uration (slow eluting diastereoisomers) as the whole of these
modifications revealed to be important in our model by crucial
interactions with amino acid residues within the binding site.
Activity of Phosphoramidate Dinucleosides on HCV sub-
genome Replicon replication. The more potent dinucleotides
active against the NS5B polymerase were evaluated in Huh-
5-2 cells, a hepatocyte cell line stably carrying the HCV replicon
I389luc-ubi-neo/NS3-3′/5.1. This replicon contains a firefly lucif-
erase-ubiquitin-neomycin phosphotransferase fusion protein used
as a reporter gene, and its expression is strictly dependent on
the level of replicon replication. The expression of the non-
structural NS3-5B HCV polyprotein is driven by the EMCV-
IRES. The activities of compounds 1, 4, and their 5′-thio-
phosphate analogues 6, 8 were evaluated in this model (Figure
5). All compounds proved relatively noncytotoxic (CC50 >60
Conclusion
In our search for new potent HCV polymerase inhibitors, we
have synthesized different phosphoramidate dinucleosides and
evaluated their inhibition activity in in vitro analysis on purified
recombinant NS5B polymerase. Our SAR study revealed that
the highest inhibition potency was obtained when the phospho-
ramidate dinucleosides bear both a negatively charged side chain
and a 5′-thiophosphate modification. Moreover, Sp diastereoi-

Phosphoramidate Dinucleosides as HCV Polymerase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5751
nonradioactive cell proliferation assay (MTS, Promega). In this
assay, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-
2-(4-sulfophenyl)-2H-tetrazolium (MTS) was bioreduced by cells
into a formazan that is soluble in tissue. The number of cells
correlates directly with the production of the formazan. The MTS
stained cultures were quantified in a plate reader.
somers were always found to be more active than Rp diaste-
reoisomers, irrespective to the nature of the phosphoramidate
linkage. This was tentatively explained by molecular modeling
of the more potent phosphoramidate dinucleoside, 8S, in the
HCV polymerase active site. Finally, this compound proved also
more potent to inhibit the HCV replication as measured in Huh-
5-2 replicon containing cells. Thus, phosphoramidate dinucleo-
sides bearing a 5-carboxypentyl side chain on the internucleo-
sidic linkage show promising inhibiting activities, on both NS5B
polymerase and HCV subgenomic replication, which makes
them new potential candidates for anti-HCV therapy.
General Procedures. TLC was performed on Merck silica coated
plates 60F254 (art. 5554). Compounds were revealed on UV light
(254 nm) and after spraying with 10% sulfuric acid ethanol solution
and heating. Phosphorus containing compounds were revealed after
spraying with the Hanes molybdate reagent35 and heating. Column
chromatography was performed on silica gel Merck 60 (art. 9385).
Flash chromatography was performed using a Biotage SP1 ap-
paratus and Biotage silica gel columns. Ion exchange purifications
were carried using Pharmacia Sephadex DEAE A-25 resin (art.
170), ISCO TRIS Pump and UA-6 detector (254 nm). Triethylam-
monium bicarbonate (TEAB) 1 M, pH 7.5, was prepared from
triethylamine, milli Q water (18.2 MΩ·cm) and CO2 gas. Ion
exchange reactions were performed using DOWEX 50WX8-200
H+ resin (Aldrich), different ion forms were obtained after washing
with corresponding 2 M aqueous solutions and then water until
neutrality. Polystryrene acyl chloride resin was prepared according
to previously published procedure28 and vaccuum-dried over P2O5.
Analytical HPLC were performed using a Nucleosil C18 column,
(150 mm × 4.6 mm, 5 µm) equipped with a prefilter and a
precolumn (Nucleosil, C18, 5 µm) on a Waters apparatus using a
Waters-616 pump and a Waters 966 photodiode array detector.
Compounds were eluted using a linear gradient 0-80% of aceto-
nitrile in a 50 mM triethylammonium acetate buffer (pH 7) with a
1 mL/min flow rate. Preparative HPLC purifications were performed
with a Waters DeltaPak C18 preparative column (7.8 mm × 300
mm, 15 µm) using the analytical HPLC conditions and a 2 mL/
min flow rate. All moist-sensitive reactions were carried under
anhydrous conditions using dry glassware, anhydrous solvents and
Argon atmosphere. Acetonitrile was distilled from CaH2 and stored
over activated 3 Å molecular sieves. Pyridine and triethylamine
were distilled from CaH2. Dichloromethane and carbon tetrachloride
were distilled from P2O5. All commercially available reagents were
used as received. Three Å molecular sieves were dried in a 300 °C
oven during 3 h prior to use. FAB-MS and FAB-HRMS spectra
were recorded on a JEOL SX102 mass spectrometer using a mixture
of glycerol/thioglycerol [50/50, v/v, (G-T)] as a matrix. MALDI/
Tof-MS spectra were recorded on a Perspective Biosystems
Voyager DE spectrometer using a mixture of 2,4,6-trihydroxyac-
etophenone/ammonium citrate [10/1, m/m, saturated solution in
water/acetonitrile, 1/1, v/v, (THAP/cit)] as a matrix. ESI-HRMS
spectra were recorded on a Q-Tof Micromass spectrometer. 1H and
13C NMR spectra were recorded at room temperature on a Bru¨ker
spectrometer at 200, 400, or 300 MHz (1H) and 100 or 75 MHz
(13C). Chemical shifts are given in ppm referenced to the solvent
residual peak (CDCl3 -7.27 and 77.0 ppm; DMSO-d6 -2.49 and
39.5 ppm; D2O -4.79 ppm + drop of CH3OH at 49.5 ppm as
internal reference36). Coupling constants are given in hertz. Signal
splitting patterns are described as singlet (s), doublet (d), triplet
(t), quartet (q), broad signal (br), doublet of doublet (dd), doublet
of triplet (dt), doublet of quartet (dq), pseudo (p), and multiplet
(m). Signal assignments were based on 2D homo-1H/1H, NOE or
heteronuclear-1H/13C correlations and D2O exchange. 31P NMR
spectra were recorded at 101 and 121 MHz on proton-decoupled
mode and on proton-coupled mode for H-phosphonates. Chemical
shifts are given in ppm referenced to external H3PO4-80%; coupling
constants are given in hertz. UV spectra were recorded on a Varian
Cary 300 Bio UV/vis spectrometer.
Experimental Section
HCV 1b Polymerase Plasmid Constructs, Enzyme Prepara-
tion, and Reagents. NS5B-∆55 gene was tagged with six C-terminal
histidines and expressed from the pDest 14 vector (Invitrogen) in
Escherichia coli BL21(DE3) cells (Novagen). NS5B protein was
purified as described previously.7 RNA oligonucleotides were
obtained from MWG-Biotech. R-32P-labeled cytosine 5′-triphos-
phate (3000 Ci/mmole) was purchased from Amersham.
Determination of Inhibitory Concentration 50% (IC50). An RNA
oligonucleotide corresponding to the 3′ end of the negative strand
of the HCV genome (RNA H (-) 5′-UCGGGGGCUGGC-3′) was
used to analyze the synthesis of the first phosphodiester bond and
its inhibition induced by the modified phosphoramidate dinucle-
otides. The modified dinucleotides concentration, leading to 50%
inhibition of NS5B-mediated RNA synthesis, was determined in
RdRp buffer (50 mM HEPES pH 8.0, 10 mM KCl, 2 mM MnCl2,
1 mM MgCl2, 10 mM DTT, 0.5% Igepal CA630) containing 10
µM of RNA template, 100 µM R-32P-CTP (1 µCi), 50 µM GTP,
and various concentration of dinucleotides (0, 5, 10, 20, 50, 100,
200, 500 µM). Reactions were initiated by the addition of 1 µM
NS5B, incubated at 30 °C for 15 min, and quenched with EDTA/
formamide. Products were separated using sequencing gel electro-
phoresis and quantified using photostimulated plates and a Fuji
Imager (Fuji). IC50 was determined using the equation:
% of active enzyme ) 100 ⁄ (1 + (I)2 ⁄ IC50)
(1)
where I is the concentration of the inhibitor. IC50 was determined
from curve-fitting using Kaleidagraph (Synergy Software).
Evaluation of Antiviral Activity and Cytostatic Activities of
Selected Compounds in HCV Genotype 1b Subgenomic Repli-
con Carrying Huh-5-2 Cells. Huh-5-2 cells were cultured in RPMI
medium (GIBCO) supplemented with 10% fetal calf serum, 2 mM
L-glutamine (Life Technologies), 1× nonessential amino acids (Life
Technologies), 100 IU/mL penicillin and 100 µg/mL streptomycin,
and 250 µg/mL G418 (Geneticin, Life Technologies). Cells were
seeded at a density of 7000 cells per well in a 96-well View Plate
(Packard) in medium containing the same components as described
above, except for G418. Cells were allowed to adhere and proliferate
for 24 h. At that time, culture medium was removed and five serial
dilutions (5-fold dilutions starting at 50 µg/mL or around 60 µM)
of the test compounds were added in culture medium lacking G418.
Interferon R-2a (500 IU) was included as a positive control in each
experiment for internal validation. Plates were further incubated at
37 °C and 5% CO2 for 72 h. Replication of the HCV replicon in
Huh-5-2 cells resulted in luciferase activity in the cells. Luciferase
activity was measured by adding 50 µL of 1× Glo-lysis buffer
(Promega) for 15 min followed by 50 µL of the Steady-Glo
luciferase assay reagent (Promega). Luciferase activity was mea-
sured with a luminometer and the signal in each individual well is
expressed as a percentage of the untreated cultures. The 50%
effective concentrations (EC50) were calculated from these data sets.
Parallel cultures of Huh-5-2 cells, seeded at a density of 7000 cells/
well of classical 96-well cell culture plates (Becton-Dickinson) were
treated in a similar fashion except that no Glo-lysis buffer or Steady-
Glo luciferase reagent was added. The effect of the compounds on
the proliferation of the cells was measured 3 days after addition of
the various compounds by means of The CellTiter 96 AQueous
For convenience, general procedures have been given for the
coupling, oxidation, and deprotection reactions. Variations from
these procedures and individual purification methods are given in
the main text for each compound. Preparative and spectroscopic
data for citydine monomer compounds 12-14 is given as Sup-
porting Information only.
General Procedure for H-Phosphonate Coupling. In a dry flask
and under argon, PS acyl chloride resin (6 mol equiv) was

5752 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
ZlateV et al.
suspended in 10 mL/mmol of anhydrous pyridine/dichloromethane
1:1 (v/v) solution. In another dry flask, a mixture of the appropriate
guanosine 3′-hydroxy monomer 9-11 (1 mol equiv) and the
cytidine 5′-H-phosphonate monomer 14 (1.2 mol equiv) were
dissolved in 10 mL/mmol of anhydrous pyridine/dichloromethane
solution after drying by azeotropic distillation with anhydrous
pyridine. The nucleoside containing solution was then introduced
to the resin containing flask, and the mixture was shaken at room
temperature for 2 h. The reaction mixture was then diluted with
dichloromethane; the resin was filtered off and well rinsed. Filtrates
were evaporated to half-volume and then washed twice with water.
Organic layers were dried over Na2SO4, filtered, and evaporated
to dryness. The residue was coevaporated three times with
acetonitrile, providing the crude dimer as a white foam (HPLC
purity >85%) in a 1:1 diastereomeric mixture. Compounds were
used in oxidation steps without further purification.
Oxidation Method A. In a dry flask, appropriate H-Phosphonate
diester (1 mol equiv) was dried by azeotropic distillation with
anhydrous pyridine and dried under vacuum with P2O5 for 30 min.
It was then dissolved in anhydrous pyridine (5 mL/mmol). To this
solution were simultaneously added: anhydrous CCl4 (25 mol
equiv, 2.5 mL/mmol), corresponding amine (10 mol equiv), or
10 M solution of histamine (50 mol equiv) in anhydrous pyridine.
Reaction color turned to bright yellow and the mixture was
stirred at room temperature for 1 h. The solvents were removed
under reduced pressure and the residue was coevaporated twice
with acetonitrile. Compounds were directly involved in depro-
tection steps.
for each compound. After purification, the TEAAc buffer was
removed after several lyophilizations from water. Collected pure
isomers were analyzed by analytical HPLC and retention times for
each isomer are given after a coinjection of an equimolar mixture.
According to retention times on RP-HPLC, the names “fast eluting”
and “slow eluting” were attributed to each isomer.
Synthesis of O-(N4,2′,3′-O,O-Tribenzoylcytidin-5′-yl)-O-{N2-
isobutyryl-2′-O-methyl-5′-O-[bis-(2-cyanoethyl)phosphoryl]guanosin-
3′-yl} Hydrogenophosphonate Diester (15). Prepared according to
general procedure of coupling using monomers 14 (0.35 g, 0.48
mmol, 1.2 equiv) and 10 (0.25 g, 0.4 mmol, 1 equiv), dry PS-acyl
chloride resin (1.2 g, 2.5 mmol, 6 equiv), and 10 mL of anhydrous
dichloromethane/pyridine 1:1 (v/v). The residue was finally pre-
cipitated in ether to give crude 15 as white foam (HPLC purity
>85%) (0.30 g, 60%). tR-HPLC: 16.6 min, 10-80% of acetonitrile
in 20 min. 1H NMR (CDCl3, 200 MHz, peaks of both diastereomers
are described) δ 12.24 (2H, s, br, NH-G1), 10.52 (2H, s, br, NHibu),
10.38 (1H, s, NHBz), 9.14, 9.04 (2H, 2s, H-G8), 8.68 (1H, d,
H-C6), 7.96-7.87 (12H, m, Hortho), 7.65-7.33 (19H, m, Hpara,
1
Hmeta, H-C5), 7.27 (1H, d, H-P, JH-P ) 744.6 Hz), 7.19 (1H,
1
d, H-P, JH-P ) 737.3 Hz), 6.37, 6.26 (2H, 2d, H-C1′, J ) 4.4
Hz), 5.99-5.83 (6H, m, H-G1′, H-C2′, H-C3′), 5.04, 4.99 (2H,
m, H-G3′), 4.65-4.19 (20H, m, H-C4′, H-G4′, H-C5′,5′′,
H-G5′,5′′, HRCNE, HR′CNE), 3.40, 3.37 (6H, 2s, G2′-O-CH3), 2.89
(4H, t, HꢀCNE, J ) 5.7 Hz), 2.71 (4H, t, Hꢀ′CNE, J ) 5.9 Hz),
1.26-1.21 (12H, 2d, CH3ibu, J ) 6.9 Hz). 31P NMR (CDCl3, 101
MHz) δ 9.4 (1/4, dq, 1JH-P ) 743.8 Hz, 3JH-P ) 7.5 Hz), 8.4 (1/4,
dq, 1JH-P ) 746.5 Hz, 3JH-P ) 8.2 Hz), -2.8 (1/4, m, 3JH-P ) 7.0
Hz), -3.1 (1/4, m). MALDI+ (THAP/cit) m/z 1155 (M + H)+.
Synthesis of O-(N4,2′,3′-O,O-Tribenzoylcytidin-5′-yl)-O-[N2-
isobutyryl-2′-O-methyl-5′-O-(4,4′-dimethoxytrityl)guanosin-3′-yl] Hy-
drogenophosphonate Diester (16). Prepared according to general
procedure of coupling from monomer 14 (0.35 g, 0.48 mmol, 1.2
equiv), N2-isobutyryl-2′-O-methyl-5′-O-DMTr guanosine 9 (0.27
g, 0.4 mmol, 1 equiv), dry PS-acyl chloride resin (1.2 g, 2.5 mmol,
6 equiv), and 10 mL of anhydrous dichloromethane/pyridine 1:1
(v/v). Crude 16 was a white foam (HPLC purity >90%) (0.39 g,
77%). tR-HPLC: 14.1 min, 14.3 min, 10-80% of acetonitrile in 10
min. 1H NMR (CDCl3, 200 MHz, peaks of both diastereomers are
described) δ 12.07, 12.02 (2H, 2s, br, NH-G1), 9.45 (1H, s, br,
NHBz), 8.99 (2H, 2s, H-G8), 8.64 (1H, d, H-C6), 7.95-6.76 (60H,
Preparation of 6-Aminocaproic Acid Methyl Ester Hydrochlo-
ride.37 First, 400 mg of 6-aminocaproic acid (3 mmol, 1 mol equiv)
were suspended in 40 mL of 2,2-dimethoxypropane (10-15 mL/
mmol). To the mixture was added 3 mL of 37% HCl and the
solution was stirred at room temperature for 18 h. After removal
of the solvents under reduced pressure, the residue was precipitated
in acetonitrile and recrystallized from acetonitrile/ether. After
filtration, the white crystals were dried under vacuum with P2O5 at
1
refluxing ethanol for 2 days (490 mg, 90%). H NMR (D2O, 300
MHz) δ 3.60 (3H, s, CO2CH3), 2.90 (2H, t, CH2-6, J ) 7.4 Hz),
2.33 (2H, t, CH2-2, J ) 7.3 Hz), 1.64 (4H, m, CH2-3, CH2-5),
1.32 (2H, q, CH2-4, J ) 7.3 Hz). 13C NMR (D2O, 75 MHz) δ
177.3 (CO2CH3), 52.1 (CO2CH3), 39.3 (CH2-6), 33.3 (CH2-2),
26.3 (CH2-5), 25.0 (CH2-3), 23.6 (CH2-4).
Oxidation Method B. In a dry flask, appropriate H-phosphonate
diester (1 mol equiv) was dried by azeotropic distillation with
anhydrous pyridine and dried under vacuum with P2O5 for 30 min.
It was then dissolved in anhydrous pyridine (2.5 mL/mmol). To
this solution were simultaneously added: anhydrous CCl4 (25 mol
equiv, 2.5 mL/mmol), 4 M solution of 6-aminocaproic acid methyl
ester hydrochloride (10 mol equiv) in anhydrous pyridine, and
nhydrous triethylamine (10 mol equiv). Reaction color turned to
bright yellow, and the mixture was stirred at room temperature for
1 h. The solvents were removed under reduced pressure, and the
residue was coevaporated twice with acetonitrile. Compounds were
directly involved in deprotection steps.
Deprotection Method A. Appropriate compound was dissolved
in THF/H2O 1:1 (v/v) (14 mL/mmol), and 28% aqueous ammonia
solution was added (70 mL/mmol). The flask was hermetically
closed, and the mixture was stirred 7 h at 50 °C. The solvents were
removed under reduced pressure to give the corresponding com-
pound as a brown oil, which was further purified by chromatography.
Deprotection Method B. Appropriate compound was dissolved
in methanol (12 mL/mmol), and 80% acetic acid solution (62 mL/
mmol) was added; then the mixture was stirred 2 h at room
temperature. It was finally diluted with 10 mL of water and washed
with dichloromethane (5 times) and ether (3 times). Aqueous layer
was evaporated to dryness, and then the residue was purified by
chromatography.
1
m, Harom), 7.34 (1H, d, H-P, JH-P ) 740.0 Hz), 7.02 (1H, d,
1
H-P, JH-P ) 729.8 Hz), 6.42, 6.28 (2H, 2d, H-C1′, J ) 4.2
Hz), 5.91-5.83 (6H, m, H-G1′, H-C2′, H-C3′), 4.96, 4.82 (2H,
m, H-G3′), 4.86-4.38 (12H, m, H-C4′, H-G4′, H-C5′,5′′,
H-G5′,5′′), 3.78-3.76 (12H, 4s, O-CH3DMTr), 3.52, 3.50 (6H, 2s,
G2′-O-CH3), 1.26 (12H, s, CH3ibu). 31P NMR (CDCl3, 101 MHz)
1
3
δ 9.4 (1/2, dq, JH-P ) 740.4 Hz, JH-P ) 7.6 Hz), 7.3 (1/2, dq,
1JH-P ) 739.1 Hz, 3JH-P ) 8.0 Hz). MALDI+ (THAP/cit) m/z 1272
(M + H)+, 969 (M + H-DMTr)+.
Synthesis of O-(N4,2′,3′-O,O-Tribenzoylcytidin-5′-yl)-O-{N2-
isobutyryl-2′-O-methyl-5′-O-[bis-(2-cyano-1,1-dimethylethyl)thiono-
phosphoryl]guanosin-3′-yl} Hydrogenophosphonate Diester (17).
Prepared using general procedure for coupling from monomers 14
(0.25 g, 0.33 mmol, 1.2 equiv), 11 (0.17 g, 0.28 mmol, 1 equiv),
dry PS-acyl chloride resin (0.7 g, 1.7 mmol, 6 equiv), and 7 mL of
anhydrous dichloromethane/pyridine 1:1 (v/v). Crude 17 was a
white foam (HPLC purity >93%) (0.23 g, 70%). tR-HPLC: 15.9
min, 10-80% of acetonitrile in 15 min. 1H NMR (CDCl3, 200
MHz, peaks of both diastereomers are described) δ 12.18-12.16
(2H, 2s, br, NH-G1), 9.59, 9.84 (2H, 2s, br, NHibu), 8.67, 8.65
(2H, 2s, H-G8), 8.15, 8.08 (2H, 2d, H-C6, J ) 7.6 Hz), 7.97-7.87
(12H, m, Hortho), 7.66-7.34 (19H, m, Hpara, Hmeta, H-C5), 7.35
(1H, d, H-P, 1JH-P ) 744.2 Hz), 7.22 (1H, d, H-P, 1JH-P ) 736.6
Hz), 6.47, 6.31 (2H, 2d, H-C1′, J ) 4.3 Hz), 5.97-5.83 (6H, m,
H-G1′, H-C2′, H-C3′), 5.61, 5.48 (2H, m, H-G3′), 4.87-4.34
(12H, m, H-C4′, H-G4′, H-C5′,5′′, H-G5′,5′′), 3.46, 3.43 (6H,
2s, G2′-O-CH3), 3.11-2.71 (8H, m, CH2CN), 2.68 (2H, m, CHibu),
1.73-1.66 (24H, m, C(CH3)2), 1.26-1.21 (12H, 2d, CH3ibu, J )
6.8 Hz). 31P NMR (CDCl3, 101 MHz) δ 48.9, 48.7 (1/2, 2t, 3JH-P
HPLC Method for Purification and/or Separation of Diastere-
oisomers. Waters DeltaPak C18 preparative column was used on a
Waters apparatus with a 2 mL/min flow rate (see above). Gradient
conditions, run times, and maximal loading of the column are given
1
3
) 7.3 Hz), 9.8 (1/4, dq, JH-P ) 744.7 Hz, JH-P ) 7.6 Hz), 8.0

Phosphoramidate Dinucleosides as HCV Polymerase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5753
1
3
(1/4, dq, JH-P ) 737.2 Hz, JH-P ) 8.1 Hz). MALDI+ (THAP/
washed twice with ether. Aqueous layer was evaporated to dryness
and purified on a DEAE-A25 Sephadex column (linear gradient:
TEAB 10-3 to 0.3 M). High purity final compound was obtained
after purification on preparative RP-HPLC (gradient: acetonitrile
7-16% in 20 min; maximal loading of the column: 6.4 mg of crude
product per run). Residue was then dissolved in 3 mL of 1 M NaCl
solution and eluted on a small RP-18 column (gradient: H2O 100%
to 25% of acetonitrile) After lyophilization from water, the suitable
salt form of 2 was a white spongy solid (18 mg, 30%) epimeric
mixture (ratio ∼1:1). tR-HPLC: 7.6 min, 0-40% acetonitrile in 15
min. UV: (H2O) λmax ) 255 nm (ε ) 14800). 1H NMR (D2O, 400
MHz, peaks of both diastereomers are described) δ 8.20 (2H, s,
br, H-G8), 7.58 (2H, d, H-C6, J ) 7.3 Hz), 5.93 (1H, pd, H-G1′,
J ) 5.0 Hz), 5.83-5.66 (5H, m, H-C1′,H-G1′, H-C5), 5.12, 5.01
(2H, m, H-G3′), 4.56 (2H, m, H-G2′), 4.45-4.06 (12H, m, H-G4′,
H-C2′, H-C3′, H-C4′, H-C5′,5′′), 3.89 (4H, m, H-G5′,5′′), 3.48,
3.47 (6H, 2s, G2′-O-CH3), 3.12-2.81 (8H, m, CH2-PNH, CH2-
N(CH3)2), 2.76 (12H, s, N(CH3)2), 1.89 (4H, m, -CH2-). 13C NMR
(D2O, 75 MHz) δ 166.9 (C-C4), 156.5 (C-C2), 148.0 (C-G4),
141.9 (C-C6), 118.1 (C-G5), 96.5 (C-C5), 91.7, 90.6 (C-C1′,
C-G1′), 81.9 (C-4′), 81.9, 81.7 (C-2′, C-4′), 75.1 (C-C3′), 74.6
(C-G3′), 69.8, 69.4 (C-C5′, C-G5′), 59.2, 59.1 (G2′-OCH3), 55.8
(CH2), 43.4 (N(CH3)2), 38.8 (CH2), 27.4 (CH2). 31P NMR (D2O,
101 MHz) δ 10.98, 10.87 (1/2, P-N), 3.7 (1/2, P-O). FAB+ (GT)
m/z 811 (M-Cl)+; 789 (M-NaCl + H)+; 767 (M-NaCl-Na + 2H)+;
FAB- (GT) m/z 787 (M-NaCl-H)-; 765 (M-2Na-Cl)-. HRMS:
FAB- (GT) m/z calcd for (C25H39N10O14P2)- 765.2122; obsd,
765.2129.
cit) m/z 1227 (M + H)+.
Synthesis of O-(Cytidin-5′-yl)-N-(2-methoxyethyl)-O-(2′-O-meth-
yl-5′-O-phosphorylguanosin-3′-yl) Phosphoramidate, Sodium Salt
(1). Prepared according to oxidation method A from H-phosphonate
diester 15 (0.07 mmol, 1 equiv), 0.5 mL of anhydrous CCl4, 0.5
mL of anhydrous pyridine, and 2-methoxyethylamine (60 µL, 0.7
mmol, 10 equiv) and according to deprotection method A using 1
mL of THF/H2O 1:1 (v/v) and 5 mL of 28% aqueous ammonia.
Residue was dissolved in water and washed twice with ether.
Aqueous layer was evaporated to dryness and purified on a DEAE-
A25 Sephadex column (linear gradient: TEAB 10-3 to 0.3 M). High
purity final compound was obtained after purification on preparative
RP-HPLC (gradient: acetonitrile 5-40% in 20 min; maximal
loading of the column: 10 mg of crude product per run). Target
compound was obtained as a sodium salt after elution on a
DOWEX-Na+ column with water. After lyophilization from water,
1 was a white spongy solid (28 mg, 47%) epimeric mixture (ratio
∼1:1). tR-HPLC: 8.2 min; 8.5 min, 0-40% acetonitrile in 15 min.
UV (H2O) λmax ) 255 nm (ε ) 15100). 1H NMR (D2O, 300 MHz,
peaks of both diastereomers are described) δ 8.19 (2H, s, H-G8),
7.71, 7.64 (2H, 2d, H-C6, J ) 7.5 Hz), 5.94 (1H, d, H-G1′, J )
6.4 Hz), 5.90 (4H, pd, H-C1′, H-G1′, H-C5), 5.73 (1H, d, H-C5,
J ) 7.5 Hz), 5.16 (2H, m, H-G3′), 4.66 (2H, m, H-G2′), 4.52 (2H,
m, H-G4′), 4.42-4.18 (10H, m, H-C2′, H-C3′, H-C4′, H-C5′,5′′),
4.02 (4H, m, H-G5′,5′′), 3.52 (3H, s, G2′-O-CH3), 3.49 (4H, pt,
CH2OMe, J ) 5.1 Hz), 3.47 (3H, s, G2′-O-CH3), 3.36, 3.33 (6H,
2s, CH2-OCH3), 3.18, 3.16 (4H, m, PNH-CH2, J ) 5.1 Hz). 13C
NMR (D2O, 75 MHz) δ 168.2 (C-C4), 161.1 (C-G6), 157.7
(C-C2), 152.5 (C-G4), 141.6, 141.4 (C-C6), 138.0 (C-G8), 118.1
(C-G5), 96.7, 96.3 (C-C5), 91.1, 90.8 (C-C1′), 85.2, 84.7 (C-
G1′), 83.9 (C-G4′), 82.2 (C-C4′), 81.9, 81.8 (C-G2′), 75.1 (C-C3′),
74.8 (C-G3′), 72.8 (CH2OMe), 66.9, 66.3 (C-C5′), 63.7 (C-G5′),
58.9, 58.6 (G2′-OCH3, CH2-OCH3), 40.8 (PNH-CH2). 31P NMR
(D2O, 121 MHz) δ 11.4 (1/4, P-N), 11.0 (1/4, P-N), 3.3 (1/4,
P-O), 3.2 (1/4, P-O). FAB+ (GT) m/z 784 (M + H)+; 762 (M-
Na + H)+; FAB- (GT) m/z 760 (M-Na)-; 738 (M-2Na + H)-.
HRMS: FAB- (GT) m/z calcd for (C23H34N9O15P2)-, 738.1802;
obsd, 738.1778.
Synthesis of O-(Cytidin-5′-yl)-N-[2-(imidazol-4-yl)ethyl]-O-(2′-
O-methyl-5′-O-phosphorylguanosin-3′-yl) Phosphoramidate, Tri-
ethylammonium Salt (3). Prepared according to oxidation method
A from H-phosphonate diester 15 (0.07 mmol, 1 equiv), 0.15 mL
of anhydrous CCl4, 0.15 mL of anhydrous pyridine, and dry
histamine base (0.33 g, 3 mmol, 50 equiv) in solution in 0.3 mL of
anhydrous pyridine and according to deprotection method A using
1 mL of THF/H2O 1:1 (v/v) and 5 mL of 28% aqueous ammonia.
Solvents were evaporated to dryness; the residue was dissolved in
water and washed twice with ether. Aqueous layer was evaporated
to dryness and purified on a DEAE-A25 Sephadex column (linear
gradient: TEAB 10-3 to 0.3 M). High purity final compound (at
this stage, the compound was contaminated with 45% of C4-
transaminated by histamine, cytosine analogue) was obtained after
purification on preparative RP-HPLC (gradient: acetonitrile 6-11%
in 30 min; maximal loading of the column: 1 mg of crude product
per run). Compound 3 was obtained as a white spongy solid bis-
triethylammonium salt (15 mg, 22%) epimeric mixture (ratio ∼1:
1). Final compound for enzyme assays was obtained as sodium
salt after elution on a DOWEX-Na+ column with water and
lyophilization. tR-HPLC: 7.8 min, 0-40% acetonitrile in 15 min.
UV: (H2O) λmax ) 255 nm (ε ) 12300). 1H NMR (D2O, 300 MHz,
peaks of both diastereomers are described) δ 8.45, 8.43 (2H, 2s,
H-Im2), 8.04, 8.01 (2H, 2s, H-G8), 7.52, 7.49 (2H, 2d, H-C6, J
) 7.5 Hz), 7.18 (2H, s, H-Im5), 5.82 (1H, d, H-G1′, J ) 5.6 Hz),
5.75 (3H, m, H-C1′,H-G1′), 5.68, 5.57 (2H, 2d, H-C5, J ) 7.4
Hz), 4.93, 4.84 (2H, 2m, H-G3′), 4.49 (2H, m, H-G2′, J ) 5.2
Hz), 4.38, 4.31 (2H, 2m, H-G4′), 4.21-3.98 (10H, m, H-C2′,
H-C3′, H-C4′, H-C5′,5′′), 3.90 (4H, m, H-G5′,5′′), 3.41, 3.35
(6H, 2s, G2′-O-CH3), 3.18 (4H, m, PNHCH2), 3.09 (4H, q, CH2CH3,
J ) 7.1 Hz), 2.82 (4H, t, Im-CH2, J ) 6.4 Hz), 1.16 (12H, t,
CH2CH3, 3J ) 7.3 Hz). 13C NMR (D2O, 100 MHz) δ 165.8
(C-C4), 158.9 (C-G6), 157.1 (C-C2), 153.9 (C-G4), 140.9, 140.8
(C-C6), 139.0 (C-G8), 133.2 (C-Im4), 133.1 (C-Im2), 116.5, 116.2
(C-Im5), 95.7, 95.5 (C-C5), 90.7 (C-C1′), 84.8, 84.1 (C-G1′),
81.5, 81.4 (C-2′, C-4′), 74.0 (C-C3′), 73.9 (C-G3′), 68.8, 68.6
(C-C5′, C-G5′), 58.2, 58.2 (G2′-O-CH3), 46.6 (CH2CH3), 39.8,
39.7 (PNHCH2), 26.4 (CH2-Im), 8.2 (CH2CH3). 31P NMR (D2O,
101 MHz) δ 10.4, 10.1 (1/2, P-N), 2.2, 2.1 (1/2, P-O). FAB+
(GT) m/z 776 (M-2TEAH + 3H)+; FAB- (GT) m/z 774 (M-
The two diastereoisomers of the obtained mixture were separated
by preparative RP-HPLC using the general conditions described
above. Gradient: acetonitrile 7-10% in 30 min; maximal loading
of the column: 1 mg of mixture per run.
Fast Eluting Isomer of 1. tR - HPLC: 8.2 min - 0 to 40%
acetonitrile in 15 min. H NMR (D2O, 300 MHz) δ 8.19 (1H, s,
1
H-G8), 7.67 (1H, d, H-C6, J ) 7.5 Hz), 5.91 (1H, d, H-G1′, J )
6.9 Hz), 5.89 (1H, d, H-C1′, J ) 2.7 Hz), 5.77 (1H, d, H-C5, J
) 7.8 Hz), 5.20-5.15 (1H, m, H-G3′), 4.66 (1H, pt, H-G2′, J )
5.4 Hz), 4.53 (1H, m, H-G4′), 4.42-4.19 (5H, m, H-C2′, H-C3′,
H-C4′, H-C5′,5′′), 4.04 (2H, m, H-G5′,5′′), 3.55-3.51 (5H, m,
G2′-O-CH3, CH2OMe), 3.37 (3H, s, CH2-OCH3), 3.17 (2H, dt,
PNH-CH2, JH-H ) 5.1 Hz, JH-P ) 12.0 Hz). 31P NMR (D2O,
3
3
121 MHz) δ 10.5 (1/2, P-N), 2.0 (1/2, P-O).
Slow Eluting Isomer of 1. tR-HPLC: 8.4 min, 0-40% acetonitrile
1
in 15 min. H NMR (D2O, 300 MHz) δ 8.22 (1H, s, H-G8), 7.75
(1H, d, H-C6, J ) 7.5 Hz), 5.98-5.90 (3H, m, H-G1′, H-C1′,
H-C5), 5.21-5.16 (1H, m, H-G3′), 4.68 (1H, pt, H-G2′), 4.55 (1H,
m, H-G4′), 4.47-4.27 (5H, m, H-C2′, H-C3′, H-C4′, H-C5′,5′′),
4.03 (2H, m, H-G5′,5′′), 3.52 (2H, t, CH2OMe, J ) 5.4 Hz), 3.48
(3H, s, G2′-O-CH3,), 3.35 (3H, s, CH2-OCH3), 3.17 (2H, dt, PNH-
CH2, JH-H ) 5.4 Hz, JH-P ) 11.7 Hz). 31P NMR (D2O, 121
3
3
MHz) δ 10.8 (1/2, P-N), 3.1 (1/2, P-O).
Synthesis of O-(Cytidin-5′-yl)-O-(2′-O-methyl-5′-O-phospho-
rylguanosin-3′-yl)-N-[3-(N,N-dimethylamino)propyl] Phosphora-
midate, Hydrochloride, Sodium Salt (2). Prepared according to
oxidation method A from H-phosphonate diester 15 (0.07 mmol, 1
equiv), 0.5 mL of anhydrous CCl4, 0.5 mL of anhydrous pyridine,
and 3-(N,N-dimethylamino)propylamine (100 µL, 0.7 mmol, 10
equiv) and according to deprotection method A using 1 mL of THF/
H2O 1:1 (v/v) and 5 mL of 28% aqueous ammonia. Solvents were
evaporated to dryness; the residue was dissolved in water and
2TEAH
+
H)-. HRMS: FAB+ (GT) m/z calcd for
(C25H36N11O14P2)+, 776.1918; obsd, 776.1915.

5754 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
ZlateV et al.
Synthesis of O-(Cytidin-5′-yl)-O-(2′-O-methyl-5′-O-phospho-
rylguanosin-3′-yl)-N-(5-carboxypentyl) Phosphoramidate, Sodium
Salt (4). Prepared according to oxidation method B from H-
phosphonate diester 15 (0.18 mmol, 1 equiv), 0.5 mL of anhydrous
CCl4, 0.4 mL of anhydrous pyridine, dry 6-aminocaproic acid
methyl ester hydrochloride (350 mg, 1.8 mmol, 10 equiv) in 0.5
mL of anhydrous pyridine, and anhydrous triethylamine (0.5 mL,
10 equiv). Reaction mixture was treated with 0.3 M NaOH for 2 h
at room temperature and then quenched with DOWEX-pyridinium
resin. After filtration of the resin, filtrates were evaporated to dryness
and treated with saturated ammonia (23 mL) according to depro-
tection method A. Crude mixture was then purified on a DEAE-
A25 Sephadex column (linear gradient: TEAB 10-3 to 0.3 M). The
two diastereoisomers of the obtained crude mixture (135 mg) were
separated by preparative RP-HPLC using the general conditions
described above. Gradient: acetonitrile 6-8% in 30 min; maximal
loading of the column: 7 mg of crude mixture per run. Each pure
isomer was then obtained as sodium salt (white spongy solid) after
elution on a DOWEX-Na+ column with water and lyophilization.
Fast Eluting Isomer of 4. Yield 45 mg, 58%. tR-HPLC: 8.3 min,
0-40% acetonitrile in 15 min. UV: (H2O) λmax ) 255 nm (ε )
14800). 1H NMR (D2O, 300 MHz) δ 8.15 (1H, s, H-G8), 7.69 (1H,
d, H-C6, J ) 7.5 Hz), 5.95 (1H, d, H-G1′, J ) 6.3 Hz), 5.89 (1H,
d, H-C1′, J ) 2.4 Hz), 5.83 (1H, d, H-C5, J ) 7.5 Hz), 5.17-5.12
(1H, m, H-G3′), 4.58 (1H, pt, H-G2′, J ) 5.0 Hz, J ) 5.5 Hz),
4.54 (1H, m, H-G4′), 4.44-4.25 (5H, m, H-C2′, H-C3′, H-C4′,
H-C5′,5′′), 4.09 (2H, m, H-G5′,5′′), 3.52 (3H, s, G2′-OCH3),
conditions described above. Gradient: acetonitrile 10-16% in 30
min; maximal loading of the column: 6.5 mg of crude product per
run. After lyophilization from water, pure 5 was a white spongy
solid (32 mg, 60%) epimeric mixture (ratio 1:1). tR-HPLC: 9.5 min,
0-40% acetonitrile in 15 min. UV: (H2O) λmax ) 255 nm (ε )
16600). 1H NMR (D2O, 300 MHz, peaks of both diastereomers
are described) δ 7.92, 7.90 (2H, 2s, H-G8), 7.59, 7.52 (2H, 2d,
H-C6, J ) 7.5 Hz), 5.83-5.75 (5H, m, H-C1′,H-G1′, H-C5),
5.63 (1H, d, H-C5, J ) 7.4 Hz), 4.98 (2H, m, H-G3′), 4.50 (2H,
m, H-G2′), 4.34 - 4.06 (12H, m, H-G4′, H-C2′, H-C3′, H-C4′,
H-C5′,5′′), 3.79-3.69 (4H, m, H-G5′,5′′), 3.43-3.36 (10H, m,
G2′-O-CH3, CH2OMe), 3.27, 3.25 (6H, 2s, CH2-OCH3), 3.05 (4H,
m, PNH-CH2). 13C NMR (D2O, 75 MHz) δ 165.9 (C-C4), 158.9
(C-G6), 157.4, 157.2 (C-C2), 153.9 (C-G2), 151.4 (C-G4), 141.3,
140.8 (C-C6), 137.6, 137.2 (C-G8), 116.4 (C-G5), 96.0, 95.5
(C-C5), 90.7, 90.6 (C-C1′), 85.3, 84.8 (C-G1′), 84.3 (C-G4′), 83.9,
83.8 (C-C4′), 81.5, 81.4 (C-G2′), 80.9 (C-C2′), 74.0, 73.7
(C-C3′), 73.4 (C-G3′), 72.1 (CH2OMe), 69.0, 68.9 (C-C5′), 60.7,
60.5 (C-G5′), 58.3-57.9 (G2′-O-CH3, CH2-O-CH3), 40.1 (PNH-
CH2). 31P NMR (D2O, 121 MHz) δ 11.0 (1/2), 10.6 (1/2). FAB+
(GT) m/z 782 (M + Na)+; 760 (M + H)+; FAB- (GT) m/z 658
(M-H)-. HRMS: FAB+ (GT) m/z calcd for (C23H35N9O12P)+,
660.2143; obsd, 660.2120.
Synthesis of O-(Cytidin-5′-yl)-O-(2′-O-methyl-guanosin-3′-yl)-
N-(5-carboxypentyl) Phosphoramidate, Sodium Salt (7). Prepared
according to oxidation method B from H-phosphonate diester 16
(0.08 mmol, 1 equiv), 0.2 mL of anhydrous CCl4, 0.2 mL of
anhydrous pyridine, dry 6-aminocaproic acid methyl ester hydro-
chloride (150 mg, 0.8 mmol, 10 equiv) in 0.3 mL of anhydrous
pyridine, and anhydrous triethylamine (0.1 mL, 10 equiv). Reaction
mixture was treated with 0.3 M NaOH for 2 h at room temperature
and then quenched with DOWEX-pyridinium resin. After filtration
of the resin, filtrates were evaporated to dryness and treated with
saturated ammonia (5 mL) according to deprotection method A,
then according to deprotection method B using 5 mL of 80% acetic
acid solution. The residue was purified by preparative RP-HPLC
using the general conditions described above. Gradient: acetonitrile
10-16% in 30 min; maximal loading of the column: 3 mg of crude
product per run. After lyophilization from water, pure 7 was a white
spongy solid (15 mg, 25%) epimeric mixture (ratio 1.5:1). tR-HPLC:
8.8 min, 8.9 min, 0-40% acetonitrile in 15 min. UV: (H2O) λmax
3
3.02-2.93 (2H, dt, PNH-CH2, JH-H ) 7.5 Hz, 3JH-P ) 11.4 Hz),
2.19 (2H, t, CH2-COO, J ) 7.9 Hz), 1.60-1.27 (6H, m, 3×
-CH2-). 13C NMR (D2O, 100 MHz) δ 183.8 (COO), 165.9
(C-C4), 158.9 (C-G6), 157.3 (C-C2), 153.9 (C-G2), 151.8 (C-
G4), 140.8 (C-C6), 137.3 (C-G8), 116.1 (C-G5) 95.8 (C-C5),
90.4 (C-C1′), 84.2 (C-G1′), 83.3 (pt, C-G4′), 81.7 (d, C-C4′, 3JC-P
3
) 8.5 Hz), 81.2 (d, C-G2′, JC-P ) 3.7 Hz), 74.2 (C-C2′), 74.1
(C-G3′), 68.9 (C-C3′), 65.8 (d, C-C5′, 2JC-P ) 4.8 Hz), 63.1 (d,
C-G5′, 2JC-P ) 3.6 Hz), 58.3 (G2′-O-CH3), 40.8 (CH2-PNH), 37.5
(CH2-COO), 30.8 (d, CH2-CH2PNH, 3JC-P ) 5.0 Hz), 25.9, 25.5
(2× -CH2-). 31P NMR (D2O, 101 MHz) δ 10.8 (1/2, P-N), 0.9
(1/2. P-O). FAB+ (GT) m/z 796 (M-3Na + 4H)+; FAB- (GT)
m/z 794 (M-3Na
+
2H)-. HRMS: ESI- m/z calcd for
(C26H38N9O16P2)-:, 794.1912; obsd, 794.2012.
1
Slow Eluting Isomer of 4. Yield 26 mg, 34%. tR-HPLC: 8.5 min,
0-40% acetonitrile in 15 min. UV: (H2O) λmax ) 255 nm (ε )
14500). 1H NMR (D2O, 300 MHz) δ 8.21 (1H, s, H-G8), 7.75 (1H,
d, H-C6, J ) 7.5 Hz), 5.98 - 5.93 (3H, m, H-G1′, H-C1′, H-C5),
5.19-5.15 (1H, m, H-G3′), 4.61 (1H, pt, H-G2′), 4.55 (1H, m,
H-G4′), 4.46-4.25 (5H, m, H-C2′, H-C3′, H-C4′, H-C5′,5′′),
4.04 (2H, m, H-G5′,5′′), 3.48 (3H, s, G2′-OCH3), 3.01-2.93 (2H,
) 254 nm (ε ) 22000). H NMR (D2O, 300 MHz, peaks of both
diastereomers are described) δ 7.93, 7.92 (2H, 2s, H-G8), 7.61,
7.57 (2H, 2d, H-C6, J ) 7.5 Hz), 5.85-5.77 (5H, m, H-C1′,
H-G1′, H-C5), 5.70 (1H, d, H-C5, J ) 7.2 Hz), 4.98 (2H, m,
H-G3′), 4.50 (2H, m, H-G2′), 4.34-4.03 (12H, m, H-G4′, H-C2′,
H-C3′, H-C4′, H-C5′,5′′), 3.76-3.69 (4H, m, H-G5′,5′′), 3.42,
3.37 (6H, 2s, G2′-O-CH3), 2.87 (4H, m, PNH-CH2), 2.07, 2.05 (4H,
3
dt, PNH-CH2, JH-H ) 7.2 Hz, 3JH-P ) 11.4 Hz), 2.15 (2H, t, CH2-
2t, CH2-COO, J ) 7.2 Hz), 1.45-1.20 (12H, m, 3× -CH2-). 31
P
COO, J ) 7.5 Hz), 1.58-1.25 (6H, m, 3× -CH2-). 13C NMR
(D2O, 100 MHz) δ 183.8 (COO), 166.0 (C-C4), 159.0 (C-G6),
157.5 (C-C2), 153.9 (C-G2), 151.8 (C-G4), 140.9 (C-C6), 137.6
(C-G8), 116.1 (C-G5), 96.2 (C-C5), 89.9 (C-C1′), 84.4 (C-G1′),
83.7 (pt, C-G4′), 81.8 (d, C-C4′, 3JC-P ) 8.1 Hz), 81.2 (d, C-G2′,
3JC-P ) 4.6 Hz), 74.5 (C-G3′), 74.1 (C-C2′), 69.0 (C-C3′), 65.4
(d, C-C5′, 2JC-P ) 5.5 Hz), 63.0 (d, C-G5′, 2JC-P ) 1.5 Hz), 58.3
(G2′-O-CH3), 40.9 (CH2-PNH), 37.5 (CH2-COO), 30.9 (d,
NMR (D2O, 121 MHz) δ 11.4 (1/2), 10.9 (1/2). FAB+ (GT) m/z
738 (M + H)+; 716 (M-Na + 2H)+; FAB- (GT) m/z 736 (M-
H)-; 714 (M-Na)-. HRMS: FAB+ (GT) m/z calcd for
(C26H39N9O13P)+, 716.2405; obsd, 716.2419.
Synthesis of O-(Cytidin-5′-yl)-N-(2-methoxyethyl)-O-(2′-O-meth-
yl-5′-O-thiophosphoryl Guanosin-3′-yl) Phosphoramidate, Sodium
Salt (6). Prepared according to oxidation method A from H-
phosphonate diester 17 (0.06 mmol, 1 equiv), 0.15 mL of anhydrous
CCl4, 0.4 mL of anhydrous pyridine. and 2-methoxyethylamine (55
µL, 0.6 mmol, 10 equiv) and according to deprotection method A
using 2 mL of THF/H2O 1:1 (v/v) and 5 mL of 28% aqueous
ammonia. At this stage, 50% of the second dimethylcyanoethyl
protection was still present, so solvents were removed under reduced
pressure and 6 mL of ammonia solution were added and the mixture
was stirred at 50 °C for 8 more hours. Solvents were evaporated to
dryness; the residue was dissolved in water and washed with ether.
High purity final compound was obtained after purification on
preparative RP-HPLC using the general conditions described above.
Gradient: acetonitrile 6-11% in 30 min; maximal loading of the
column: 4.7 mg of crude product per run. Final compound was
obtained as a sodium salt after elution on a DOWEX-Na+ column
with water. After lyophilization from water, pure 6 was a white
3
CH2-CH2PNH, JC-P ) 5.0 Hz), 25.9, 25.5 (2× -CH2-). 31P
NMR (D2O, 101 MHz) δ 11.3 (1/2, P-N), 2.6 (1/2. P-O). FAB+
(GT) m/z 796 (M-3Na + 4H)+; FAB- (GT) m/z 794 (M-3Na +
2H)-. HRMS: ESI- m/z calcd for (C26H38N9O16P2)-, 794.1912;
obsd, 794.2003.
Synthesis of O-(Cytidin-5′-yl)-N-(2-methoxyethyl)-O-(2′-O-meth-
yl-guanosin-3′-yl) Phosphoramidate (5). Prepared according to
oxidation method A from H-phosphonate diester 16 (0.08 mmol, 1
equiv), 0.2 mL of anhydrous CCl4, 0.5 mL of anhydrous pyridine,
and 2-methoxyethylamine (70 µL, 0.8 mmol, 10 equiv) and
according to deprotection method A using 1.5 mL of THF/MeOH,
1:1 (v/v) and 5 mL of 28% aqueous ammonia, then according to
deprotection method B using 5 mL of 80% acetic acid solution.
The residue was purified by preparative RP-HPLC using the general

Phosphoramidate Dinucleosides as HCV Polymerase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5755
spongy solid (28 mg, 58%) epimeric mixture (ratio 1:1). tR-HPLC:
8.7 min, 8.5 min, 0-40% acetonitrile in 15 min. UV: (H2O) λmax
H-G2′), 4.31-4.17 (12H, m, H-G4′, H-C2′, H-C3′, H-C4′,
H-C5′,5′′), 4.01 (4H, m, H-G5′,5′′), 3.40, 3.39 (6H, 2s, G2′-O-
CH3), 2.92-2.83 (4H, m, PNH-CH2), 2.12, 2.09 (4H, 2t, CH2-COO,
J ) 7.2 Hz), 1.51-1.41 (8H, m, -CH2-), 1.28-1.16 (4H, m,
-CH2-). 13C NMR (D2O, 75 MHz) δ 182.7 (COO), 165.4 (C-C4),
158.9 (C-G6), 156.7 (C-C2), 153.9 (C-G2), 151.8 (C-G4), 141.1
(C-C6), 137.6 (C-G8), 116.4 (C-G5), 95.8 (C-C5), 90.3 (C-C1′),
84.4, 84.3 (C-G1′), 83.3 (C-C4′, C-G4′), 81.7 (C-G2′), 81.2
(C-C2′), 75.1 (C-C3′, C-G3′), 74.1 (CH2COO), 68.9 (C-C5′),
65.7, 63.5 (C-G5′), 58.3 (G2′-O-CH3), 40.8 (PNH-CH2), 36.6
(-CH2-), 30.7, 25.7, 25.1 (3× -CH2-). 31P NMR (D2O, 121
MHz) δ 45.9 (1/2), 11.2, 10.7 (1/2). FAB+ (GT) m/z 900 (M +
Na)+; 878 (M + H)+; 856 (M-Na + 2H)+; 834 (M-2Na + 3H)+;
FAB- (GT) m/z 754 (M-Na)-; 832 (M-2Na + H)-; 810 (M-3Na
+ 2H)-. HRMS: FAB- (GT) m/z calcd for (C26H38N9O15P2S)-,
810.1683; obsd, 810.1676.
1
) 255 nm (ε ) 17600). H NMR (D2O, 300 MHz, peaks of both
diastereomers are described) δ 7.92, 7.90 (2H, 2s, H-G8), 7.59,
7.52 (2H, 2d, H-C6, J ) 7.5 Hz), 5.83-5.75 (5H, m, H-C1′,
H-G1′, H-C5), 5.63 (1H, d, H-C5, J ) 7.4 Hz), 4.98 (2H, m,
H-G3′), 4.50 (2H, m, H-G2′), 4.34-4.06 (12H, m, H-G4′, H-C2′,
H-C3′, H-C4′, H-C5′,5′′), 3.79-3.69 (4H, m, H-G5′,5′′),
3.43-3.36 (10H, m, G2′-O-CH3, CH2OMe), 3.27, 3.25 (6H, 2s,
CH2-OCH3), 3.12-3.04 (4H, m, PNH-CH2). 13C NMR (D2O, 75
MHz) δ 165.7 (C-C4), 158.9 (C-G6), 157.2 (C-C2), 153.9 (C-
G2), 140.9 (C-C6), 138.1 (C-G8), 96.0, 95.8 (C-C5), 90.3, 90.1
(C-C1′), 84.7 (C-G1′), 84.1 (C-G4′), 83.4 (C-C4′), 81.6 (C-G2′),
74.1 (C-C3′), 72.2 (CH2OMe), 68.9, 68.8 (C-C5′, C-G5′),
58.3-57.9 (G2′-O-CH3, CH2-O-CH3), 40.1 (PNH-CH2). 31P NMR
(D2O, 121 MHz) δ 44.6 (1/2), 10.7, 10.4 (1/2). FAB+ (GT) m/z
822 (M + Na)+; 800 (M + H)+; 778 (M-Na + 2H)+; FAB- (GT)
m/z 798 (M-H)-; 776 (M-Na)-; 754 (M-2Na + H)-. HRMS: FAB-
(GT) m/z calcd for (C23H34N9O14P2S)-, 754.1421; obsd, 754.1377.
The two diastereoisomers of the obtained mixture were separated
by preparative RP-HPLC using the general conditions described
above. Gradient: acetonitrile 8-11% in 30 min; maximal loading
of the column: 1 mg of mixture per run. Up to 15% of an unknown
byproduct was observed after the separation of the diastereoisomers
on the preparative RP-HPLC. Unfortunately, it could not be
removed from the target compound.
The two diastereoisomers of the obtained mixture were separated
by preparative RP-HPLC using the general conditions described
above. Gradient: acetonitrile 6-11% in 30 min; maximal loading
of the column: 1 mg of mixture per run. Up to 5% of an unknown
decomposition byproduct was observed after the separation of the
diastereoisomers on preparative RP-HPLC. Unfortunately, this
byproduct could not be removed from the target compound.
Fast Eluting Isomer of 8. tR-HPLC: 7.6 min, 0-40% acetonitrile
1
in 15 min. H NMR (D2O, 300 MHz) δ 8.30 (1H, s, H-G8), 7.72
(1H, d, H-C6, J ) 7.8 Hz), 5.94 (1H, d, H-G1′, J ) 7.2 Hz), 5.91
(1H, d, H-C1′, J ) 2.7 Hz), 5.87 (1H, d, H-C5, J ) 7.8 Hz),
5.27-5.23 (1H, m, H-G3′), 4.72 (1H, m, H-G2′), 4.56 (1H, m,
H-G4′), 4.42-4.20 (5H, m, H-C2′, H-C3′, H-C4′, H-C5′,5′′),
4.09 (2H, dd, H-G5′,5′′), 3.49 (3H, s, G2′-O-CH3), 3.00-2.92 (2H,
Fast Eluting Isomer of 6. tR-HPLC: 8.1 min, 0-40% acetonitrile
1
in 15 min. H NMR (D2O, 300 MHz) δ 8.30 (1H, s, H-G8), 7.71
(1H, d, H-C6, J ) 7.5 Hz), 5.92-5.90 (2H, m, H-G1′, H-C1′),
5.82 (1H, d, H-C5, J ) 7.8 Hz), 5.30-5.26 (1H, m, H-G3′), 4.74
(1H, m, H-G2′), 4.56 (1H, m, H-G4′), 4.41-4.16 (5H, m, H-C2′,
H-C3′, H-C4′, H-C5′,5′′), 4.08-4.05 (2H, dd, H-G5′,5′′),
3.55-3.50 (5H, m, G2′-O-CH3, CH2OMe), 3.37 (3H, s,
CH2-OCH3), 3.21-3.14 (2H, dt, PNH-CH2, 3JH-H ) 5.4 Hz, 3JH-P
) 12.0 Hz). 31P NMR (D2O, 121 MHz) δ 43.7 (1/2, P-S), 10.4
(1/2, P-N).
3
dt, PNH-CH2, JH-H ) 6.9 Hz, 3JH-P ) 11.7 Hz), 2.18 (2H, t, CH2-
COO, J ) 7.5 Hz), 1.57-1.26 (6H, m, 3× -CH2-). 31P NMR
(D2O, 121 MHz) δ 44.6 (1/2, P-S), 10.7 (1/2, P-N).
Slow Eluting Isomer of 8. tR-HPLC: 8.1 min, 0-40% acetonitrile
1
in 15 min. H NMR (D2O, 300 MHz) δ 8.30 (1H, s, H-G8), 7.76
(1H, d, H-C6, J ) 7.5 Hz), 5.98-5.92 (3H, m, H-G1′, H-C1′,
H-C5), 5.25 (1H, m, H-G3′), 4.72 (1H, m, H-G2′), 4.57 (1H, m,
H-G4′), 4.46-4.25 (5H, m, H-C2′, H-C3′, H-C4′, H-C5′,5′′),
4.08 (2H, dd, H-G5′,5′′), 3.48 (3H, s, G2′-O-CH3), 3.01-2.93 (2H,
Slow Eluting Isomer of 6. tR-HPLC: 8.3 min, 0-40% acetonitrile
1
in 15 min. H NMR (D2O, 300 MHz) δ 8.30 (1H, s, H-G8), 7.76
(1H, d, H-C6, J ) 7.8 Hz), 5.98-5.91 (3H, m, H-G1′, H-C1′,
H-C5), 5.27 (1H, m, H-G3′), 4.73 (1H, pt, H-G2′), 4.58 (1H, m,
H-G4′), 4.44-4.24 (5H, m, H-C2′, H-C3′, H-C4′, H-C5′,5′′),
4.11-4.08 (2H, m, H-G5′,5′′), 3.53 (2H, t, CH2OMe, J ) 5.4 Hz),
3.48 (3H, s, G2′-O-CH3), 3.35 (3H, s, CH2-OCH3), 3.20-3.14
3
dt, PNH-CH2, JH-H ) 6.9 Hz, 3JH-P ) 12.0 Hz), 2.15 (2H, t, CH2-
COO, J ) 7.2 Hz), 1.57-1.27 (6H, m, 3× -CH2-). 31P NMR
(D2O, 121 MHz) δ 43.9 (1/2, P-S), 11.2 (1/2, P-N).
Molecular Dynamics. The model of the HCV RdR polymerase-
phosphoramidate dinucleoside complex is based on the analogy with
the crystal structure of the bacteriophage initiation complex.38
Bacteriophage and HCV RdRp14,39 were superimposed using the
structural alignment method (embedded in the VMD and Chimera
software packages40,41 and nucleic acids from the bacteriophage
initiation complex were incorporated into the active site of HCV
RdRp as proposed in reference.42
3
3
(2H, dt, PNH-CH2, JH-H ) 5.4 Hz, JH-P ) 12.0 Hz). 31P NMR
(D2O, 121 MHz) δ 43.7 (1/2, P-S), 10.7 (1/2, P-N).
Synthesis of O-(Cytidin-5′-yl)-O-(2′-O-methyl-5′-O-thiophospho-
rylguanosin-3′-yl)-N-(5-carboxypentyl) Phosphoramidate, Sodium
Salt (8). Prepared according to oxidation method B from H-
phosphonate diester 17 (0.04 mmol, 1 equiv), 0.1 mL of anhydrous
CCl4, 0.1 mL of anhydrous pyridine, dry 6-aminocaproic acid
methyl ester hydrochloride (100 mg, 0.4 mmol, 10 equiv) in 0.2
mL of anhydrous pyridine, and anhydrous triethylamine (70 µL,
10 equiv). Reaction mixture was first treated with 70 µL of DBU
in 0.25 mL of N,O-bis-(trimethylsilyl)-acetamide and 1.2 mL of
dry pyridine for 2 h, then with 0.3 M NaOH (5 mL) for 2 h at
room temperature, and then quenched with DOWEX-pyridinium
resin. After filtration of the resin, filtrates were evaporated to dryness
and treated with saturated ammonia (5 mL) according to depro-
tection method A. Crude mixture was then purified on a DEAE-
A25 Sephadex column (linear gradient: TEAB 10-3 to 0.3 M). High
purity final compound was obtained after purification on preparative
RP-HPLC (gradient: acetonitrile 6-12% in 30 min; maximal
loading of the column: 5 mg of crude product per run). Final
compound was obtained as a sodium salt after elution on a
DOWEX-Na+ column with water. After lyophilization from water,
8 was a white spongy solid (13 mg, 37%) epimeric mixture (ratio
∼1.8:1). tR-HPLC: 7.9 min, 8.1 min, 0-40% acetonitrile in 15 min.
UV: (H2O) λmax ) 255 nm (ε ) 15500). 1H NMR (D2O, 300 MHz,
peaks of both diastereomers are described) δ 8.19, 8.18 (2H, 2s,
H-G8), 7.67, 7.63 (2H, 2d, H-C6, J ) 7.5 Hz), 5.88-5.77 (6H,
m, H-C1′, H-G1′, H-C5), 5.14 (2H, m, H-G3′), 4.47 (2H, m,
Several necessary modifications (anchoring of the tethers to the
internucleotide linkage as well as addition of the 2′-O-CH3 group
to rG) were made. This required necessary completion and
modification of all_nuc94.in (molecular topology) and parm94.dat
(force field) files. Several bond (NS_P), angle and dihedral angle
(CT-NS-P-OS, X-CT-NS-P) terms were taken from ref 43 in which
the AMBER parameters for a similar internucleotide linkage
modification were developed. Remaining charges, as well as bond,
angle, and dihedral terms for the side chains, were taken from the
AMBER database44 and did not require modification (similarity of
tethers with the GLU side chain was exploited). Such approximation
was judged sufficient for the geometrical factors analyzed here.
Simulated system was surrounded by ∼16769 TIP3P water
molecules,45 which extended to a distance of approximately 10
Å (in each direction) from the HCV RdRp atoms. This gives a
periodic box size of ∼98 Å, ∼77 Å, ∼97 Å. New *.inpcrd (initial
coordinates) and *.prmtop (molecular topology, force field etc.)
files for the whole simulated system including modified residues
were created by use of the TLEAP module (AMBER software
package44).

5756 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
ZlateV et al.
(14) 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, 34823492.
(15) Bressanelli, S.; Tomei, L.; Roussel, A.; Incitti, I.; Vitale, R. L.;
Mathieu, M.; De Francesco, R.; Rey, F. A. Crystal structure of the
RNA-dependent RNA polymerase of hepatitis C virus. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 13034–13039.
(16) McGuigan, C.; Cahard, D.; Salgado, A.; DeClercq, E.; Balzarini, J.
Phosphoramidates as potent prodrugs of anti-HIV nucleotides: Studies
in the amino region. AntiViral Chem. Chemother. 1996, 7, 31–36.
(17) McGuigan, C.; Bidois, L.; Hiouni, A.; Ballatore, C.; De Clercq, E.;
Balzarini, J. Phosphoramidate derivatives of stavudine as inhibitors
of HIV: unnatural amino acids may substitute for alanine. AntiViral
Chem. Chemother. 2000, 11, 111–116.
Fully solvated trajectories (lasting for 4 ns) were computed with
the aid of the NAMD software package.46 Conventional compu-
tational procedures were used: periodic boundary conditions, cut
off distances of 10 Å for the nonbonded interactions, and the
particle-mesh-Ewald method for the summation of the Coulombic
interactions,47 MD time step ) 0.001 ps. Initially, for 5 ps, the
system was heated up to 310 K using a Langevin temperature
equilibration scheme while restraining the position of the solute.
The MD was then continued for 4 ns at constant T and constant P
with all restraints removed.
Figures were produced with the aid of the VMD software
package.41
(18) Iyer, V. V.; Griesgraber, G. W.; Radmer, M. R.; McIntee, E. J.;
Wagner, C. R. Synthesis, in vitro anti-breast cancer activity, and
intracellular decomposition of amino acid methyl ester and alkyl amide
phosphoramidate monoesters of 3′-azido-3 ′-deoxythymidine (AZT).
J. Med. Chem. 2000, 43, 2266–2274.
(19) Dagle, J. M.; Littig, J. L.; Sutherland, L. B.; Weeks, D. L. Targeted
elimination of zygotic messages in Xenopus laeVis embryos by
modified oligonucleotides possessing terminal cationic linkages.
Nucleic Acids Res. 2000, 28, 2153–2157.
(20) Laurent, A.; Naval, M.; Debart, F.; Vasseur, J. J.; Rayner, B. Chiral
and steric effects in the efficient binding of alpha-anomeric deoxyo-
ligonucleosides N-alkylphosphoramidates to ssDNA and RNA. Nucleic
Acids Res. 1999, 27, 4151–4159.
(21) Prakash, T. P.; Manoharan, M.; Fraser, A. S.; Kawasaki, A. M.; Lesnik,
E. A.; Owens, S. R. Zwitterionic oligonucleotides with 2′-O-[3-(N,N-
dimethylamino)propyl]-RNA modification: synthesis and properties.
Tetrahedron Lett. 2000, 41, 4855–4859.
(22) Atherton, F. R.; Openshaw, H. T.; Todd, A. R. Studies on Phospho-
rylation 0.2. The Reaction of Dialkyl Phosphites with Polyhalogen
Compounds in Presence of Bases: A New Method for the Phospho-
rylation of Amines. J. Chem. Soc. 1945, 660–663.
(23) Zlatev, I.; Vasseur, J.-J.; Morvan, F. Convenient synthesis of N2-
isobutyryl-2′-O-methyl guanosine by efficient alkylation of O6-
trimethylsilylethyl-3′,5′-di-tert-butylsilanediyl guanosine. Tetrahedron
2007, 63, 11174–11178.
(24) Dueymes, C.; Schonberger, A.; Adamo, I.; Navarro, A.-E.; Meyer,
A.; Lange, M.; Imbach, J.-L.; Link, F.; Morvan, F.; Vasseur, J.-J. High-
yield solution-phase synthesis of di- and trinucleotide blocks assisted
by polymer-supported reagents. Org. Lett. 2005, 7, 3485–3488.
(25) Zlatev, I.; Kato, Y.; Meyer, A.; Vasseur, J.-J.; Morvan, F. Use of a
solid-supported coupling reagent for a selective phosphitylation of the
primary alcohol of N2-isobutyryl-2′-deoxy or 2′-O-methyl guanosine.
Tetrahedron Lett. 2006, 47, 8379–8382.
(26) Bhat, V.; Ugarkar, B. G.; Sayeed, V. A.; Grimm, K.; Kosora, N.;
Domenico, P. A.; Stocker, E. A Simple and Convenient Method for
the Selective N-Acylations of Cytosine Nucleosides. Nucleosides
Nucleotides 1989, 8, 179–183.
(27) Jankowska, J.; Sobkowski, M.; Stawinski, J.; Kraszewski, A. Studies
on Aryl H-Phosphonates 0.1. An Efficient Method for the Preparation
of Deoxyribonucleoside and Ribonucleoside 3′-H-Phosphonate Mo-
noesters by Transesterification of Diphenyl H-Phosphonate. Tetrahe-
dron Lett. 1994, 35, 3355–3358.
(28) Adamo, I.; Dueymes, U.; Schonberger, A.; Navarro, A. E.; Meyer,
A.; Lange, M.; Imbach, J. L.; Link, F.; Morvan, F.; Vasseur, J. J.
Solution-phase synthesis of phosphorothioate oligonucleotides using
a solid-supported acyl chloride with H-phosphonate chemistry. Eur.
J. Org. Chem. 2006, 436–448.
(29) Tomoskozi, I.; Gacs-Baitz, E.; Otvos, L. Stereospecific conversion of
H-phosphonates into phosphoramidates. The use of vicinal carbon-
phosphorus couplings for configurational determination of phosphorus.
Tetrahedron 1995, 51, 6797–6804.
(30) Shim, J. H.; Larson, G.; Wu, J. Z.; Hong, Z. Selection of 3′-template
bases and initiating nucleotides by hepatitis C virus NS5B RNA-
dependent RNA polymerase. J. Virol. 2002, 76, 7030–7039.
(31) Ranjith-Kumar, C. T.; Gutshall, L.; Kim, M. J.; Sarisky, R. T.; Kao,
C. C. Requirements for de novo initiation of RNA synthesis by
recombinant flaviviral RNA-dependent RNA polymerases. J. Virol.
2002, 76, 12526–12536.
(32) Adelfinskaya, O.; Herdewijn, P. Amino acid phosphoramidate nucle-
otides as alternative substrates for HIV-1 reverse transcriptase. Angew.
Chem., Int. Ed. 2007, 46, 4356–4358.
(33) Adelfinskaya, O.; Terrazas, M.; Froeyen, M.; Marliere, P.; Nauwelaerts,
K.; Herdewijn, P. Polymerase-catalyzed synthesis of DNA from
phosphoramidate conjugates of deoxynucleotides and amino acids.
Nucleic Acids Res. 2007, 35, 5060–5072.
(34) Eckstein, F.; Gish, G. Phosphorothioates in molecular biology. Trends
Biochem. Sci. 1989, 14, 97–100.
(35) Hanes, C. S.; Isherwood, F. A. Separation of the Phosphoric Esters
on the Filter Paper Chromatogram. Nature 1949, 164, 1107–1112.
Acknowledgment. This work was supported by grant from
the French “Agence Nationale de Recherche Contre le Sida”
(ANRS). I.Z. thanks the “Ministe`re National de la Recherche
et de la Technologie” for the award of a research studentship.
H.D. and F.M. are from INSERM. Support from the Ministry
of Education, Youth and Sports of the Czech Republic (project
no. MSM 0021620835) is gratefully acknowledged.
Supporting Information Available: Preparative and spectro-
scopic data on compounds 12-14; correlation table between HPLC
retention time, δ 31P NMR values and suggested absolute config-
uration for separated isomers of compounds 1, 4, 6, and 8. Purity
1
data from HPLC analysis and H NMR and 31P NMR spectra of
all new target compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) World Health Organization. WHO, Hepatitis C. Fact Sheet No. 164;
World Health Organization: Geneva, 2000; www.who.int/mediacentre/
factsheets/fs164/en/.
(2) Manns, M. P.; McHutchison, J. G.; Gordon, S. C.; Rustgi, V. K.;
Shiffman, M.; Reindollar, R.; Goodman, Z. D.; Koury, K.; Ling, M.;
Albrecht, J. K. Peginterferon alpha-2b plus ribavirin compared with
interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis
C: a randomised trial. Lancet 2001, 358, 958–965.
(3) Manns, M. P.; W, H.; Cornberg, M. Treating viral hepatitis C: efficacy,
side effects, and complications. Gut 2006, 55, 1350–1359.
(4) Moradpour, D.; Penin, F.; Rice, C. M. Replication of hepatitis C virus.
Nat. ReV. Microbiol. 2007, 5, 453–463.
(5) Ranjith-Kumar, C. T.; Kim, Y. C.; Gutshall, L.; Silverman, C.;
Khandekar, S.; Sarisky, R. T.; Kao, C. C. Mechanism of de novo
initiation by the hepatitis C virus RNA-dependent RNA polymerase:
role of divalent metals. J. Virol. 2002, 76, 12513–12525.
(6) Zhong, W.; Ferrari, E.; Lesburg, C. A.; Maag, D.; Ghosh, S. K.;
Cameron, C. E.; Lau, J. Y.; Hong, Z. Template/primer requirements
and single nucleotide incorporation by hepatitis C virus nonstructural
protein 5B polymerase. J. Virol. 2000, 74, 9134–9143.
(7) Dutartre, H.; Boretto, J.; Guillemot, J. C.; Canard, B. A relaxed
discrimination of 2′-O-methyl-GTP relative to GTP between de novo
and elongative RNA synthesis by the hepatitis C RNA-dependent RNA
polymerase NS5B. J. Biol. Chem. 2005, 280, 6359–6368.
(8) Dutartre, H.; Bussetta, C.; Boretto, J.; Canard, B. General catalytic
deficiency of hepatitis C virus RNA polymerase with an S282T
mutation and mutually exclusive resistance towards 2′-modified
nucleotide analogues. Antimicrob. Agents Chemother. 2006, 50, 4161–
4169.
(9) De Francesco, R.; Carfi, A. Advances in the development of new
therapeutic agents targeting the NS34A serine protease or the NS5B
RNA-dependent RNA polymerase of the hepatitis C virus. AdV. Drug
DeliVery ReV. 2007, 59, 1242–1262.
(10) De Clercq, E. The design of drugs for HIV and HCV. Nat. ReV. Drug
DiscoVery 2007, 6, 1001–1018.
(11) De Francesco, R.;.; Migliaccio, G. Challenges and successes in
developing new therapies for hepatitis C. Nature 2005, 436, 953–
960.
(12) Ma, H.; Leveque, V.; De Witte, A.; Li, W.; Hendricks, T.; Clausen,
S. M.; Cammack, N.; Klumpp, K. Inhibition of native hepatitis C virus
replicase by nucleotide and non-nucleoside inhibitors. Virology 2005,
332, 8–15.
(13) Zhong, W.; An, H.; Barawkar, D.; Hong, Z. Dinucleotide analogues
as novel inhibitors of RNA-dependent RNA polymerase of hepatitis
C virus. Antimicrob. Agents Chemother. 2003, 47, 2674–2681.

Phosphoramidate Dinucleosides as HCV Polymerase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5757
(36) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts of
common laboratory solvents as trace impurities. J. Org. Chem. 1997,
62, 75127515.
O. A. Characterization of resistance to nonobligate chain-terminating
ribonucleoside analogs that inhibit hepatitis C virus replication in vitro.
J. Biol. Chem. 2003, 278, 49164–49170.
(43) Cieplak, P.; Cheatham, T. E.; Kollman, P. A. Molecular dynamics
simulations find that 3′-phosphoramidate modified DNA duplexes
undergo a B to A transition and normal DNA duplexes an A to B
transition. J. Am. Chem. Soc. 1997, 119, 6722–6730.
(44) Pearlman, D. A.; Case, D. A.; Caldwell, J. W.; Ross, W. S.; Cheatham,
T. E.; Debolt, S.; Ferguson, D.; Seibel, G.; Kollman, P. Amber, a
Package of Computer Programs for Applying Molecular Mechanics,
Normal-Mode Analysis, Molecular-Dynamics and Free-Energy Cal-
culations to Simulate the Structural and Energetic Properties of
Molecules. Comput. Phys. Commun. 1995, 91, 1–41.
(45) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.;
Klein, M. L. Comparison of Simple Potential Functions for Simulating
Liquid Water. J. Chem. Phys. 1983, 79, 926–935.
(46) Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.;
Villa, E.; Chipot, C.; Skeel, R. D.; Kale, L.; Schulten, K. Scalable
molecular dynamics with NAMD. J. Comput. Chem. 2005, 26, 1781–
1802.
(37) Rachele, J. R. Methyl Esterification of Amino Acids with 2,2-
Dimethoxypropane and Aqueous Hydrogen Chloride. J. Org. Chem.
1963, 28, 2898–2898.
(38) Butcher, S. J.; Grimes, J. M.; Makeyev, E. V.; Bamford, D. H.; Stuart,
D. I. A mechanism for initiating RNA-dependent RNA polymerization.
Nature 2001, 410, 235–240.
(39) O’Farrell, D.; Trowbridge, R.; Rowlands, D.; Jager, J. Substrate
complexes of hepatitis C virus RNA polymerase (HC-J4): structural
evidence for nucleotide import and de novo initiation. J. Mol. Biol.
2003, 326, 1025–1035.
(40) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.;
Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF chimera: a
visualization system for exploratory research and analysis. J. Comput.
Chem. 2004, 25, 1605–1612.
(41) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual molecular
dynamics. J. Mol. Graphics 1996, 14, 33.
(47) Cheatham, T. E.; Miller, J. L.; Fox, T.; Darden, T. A.; Kollman, P. A.
Molecular Dynamics Simulations on Solvated Biomolecular Systems:
The Particle Mesh Ewald Method Leads to Stable Trajectories of DNA,
RNA and Proteins. J. Am. Chem. Soc. 1995, 117, 4193–4194.
(42) Migliaccio, G.; Tomassini, J. E.; Carroll, S. S.; Tomei, L.; Altamura,
S.; Bhat, B.; Bartholomew, L.; Bosserman, M. R.; Ceccacci, A.;
Colwell, L. F.; Cortese, R.; De Francesco, R.; Eldrup, A. B.; Getty,
K. L.; Hou, X. S.; LaFemina, R. L.; Ludmerer, S. W.; MacCoss, M.;
McMasters, D. R.; Stahlhut, M. W.; Olsen, D. B.; Hazuda, D. J.; Flores,
JM800617C