Application of the phosphoramidate ProTide approach to 4′- azidouridine confers sub-micromolar potency versus hepatitis C virus on an inactive nucleoside

Article abstract of DOI:10.1021/jm0613370

We report the application of our phosphoramidate ProTide technology to the ribonucleoside analogue 4′-azidouridine to generate novel antiviral agents for the inhibition of hepatitis C virus (HCV). 4′-Azidouridine did not inhibit HCV, although 4′-azidocyti

Full text of DOI:10.1021/jm0613370

1840
J. Med. Chem. 2007, 50, 1840-1849
Application of the Phosphoramidate ProTide Approach to 4′-Azidouridine Confers
Sub-micromolar Potency versus Hepatitis C Virus on an Inactive Nucleoside
Plinio Perrone,† Giovanna M. Luoni,† Mary Rose Kelleher,† Felice Daverio,† Annette Angell,† Sinead Mulready,†
Costantino Congiatu,† Sonal Rajyaguru,‡ Joseph A. Martin,‡ Vincent Leveˆque,‡ Sophie Le Pogam,‡ Isabel Najera,‡
Klaus Klumpp,‡ David B. Smith,‡ and Christopher McGuigan*,†
Welsh School of Pharmacy, Cardiff UniVersity, King Edward VII AVenue, Cardiff CF10 3XF, UK, and Roche Palo Alto, 3431 HillView AVenue,
Palo Alto, California 94304
ReceiVed NoVember 17, 2006
We report the application of our phosphoramidate ProTide technology to the ribonucleoside analogue 4′-
azidouridine to generate novel antiviral agents for the inhibition of hepatitis C virus (HCV). 4′-Azidouridine
did not inhibit HCV, although 4′-azidocytidine was a potent inhibitor of HCV replication under similar
assay conditions. However 4′-azidouridine triphosphate was a potent inhibitor of RNA synthesis by HCV
polymerase, raising the question as to whether our phosphoramidate ProTide approach could effectively
deliver 4′-azidouridine monophosphate to HCV replicon cells and unleash the antiviral potential of the
triphosphate. Twenty-two phosphoramidates were prepared, including variations in the aryl, ester, and amino
acid regions. A number of compounds showed sub-micromolar inhibition of HCV in cell culture without
detectable cytotoxicity. These results confirm that phosphoramidate ProTides can deliver monophosphates
of ribonucleoside analogues and suggest a potential path to the generation of novel antiviral agents against
HCV infection. The generic message is that ProTide synthesis from inactive parent nucleosides may be a
warranted drug discovery strategy.
Introduction
triphosphate was described as a competitive inhibitor of
cytidylate incorporation by HCV polymerase and a potent
inhibitor of native, membrane-associated HCV replicase in
vitro.6
The hepatitis C virus (HCVa) was identified for the first time
in 1989 as a single-stranded positive sense RNA virus of the
Flaviviridae family.1 According to the World Health Organiza-
tion (WHO), more than 170 million people are estimated to be
chronically infected by this virus, which is a major cause of
severe liver disease.2
Interestingly, the corresponding uridine analogue, 4′-azido-
uridine (1), was inactive as an inhibitor of HCV replication in
the cell-based replicon system.7
At present, treatment options comprise immunotherapy using
recombinant interferon (often pegylated) in combination with
ribavirin. The clinical benefit of this treatment is limited, and a
vaccine has not yet been developed. The development of
selective inhibitors of essential viral enzymes such as the serine
protease NS3 or the RNA-dependent RNA polymerase NS5b
are expected to improve the potency and tolerability of future
treatment options for HCV infected patients.3,4
Nucleoside analogues have already been validated as an
important class of polymerase inhibitors of other viral targets,
such as HCMV, HSV, HIV, and HBV.5 All antiviral agents
acting via a nucleoside analogue mode of action need to be
phosphorylated, most of them to their corresponding 5′-
triphosphates, by cellular and/or viral enzymes. The nucleotide
triphosphate analogues will then inhibit the requisite polymerase
and/or compete with natural nucleotide triphosphates as sub-
strates for incorporation into viral nucleic acid during viral
replication.5
It was hypothesized that (1) (1, Figure 1) may be a poor
substrate for phosphorylation by cellular enzymes. The first
phosphorylation step to produce the 5′-monophosphate has often
been found to be the rate-limiting step in the pathway to
intracellular nucleotide triphosphate formation, suggesting that
nucleoside monophosphate analogues could be useful antiviral
agents. However, as unmodified agents, nucleoside monophos-
phates are unstable in biological media and they also show poor
membrane permeation because of the associated negative
charges at physiological pH.8,9
Our aryloxy phosphoramidate ProTide approach allows
bypass of the initial kinase dependence by intracellular delivery
of the monophosphorylated nucleoside analogue as a membrane
permeable “ProTide” form.10,11 This technology greatly increases
the lipophilicity of the nucleoside monophosphate analogue with
a consequent increase of membrane permeation and intracellular
availability. Previously we have demonstrated the success of
our approach with the aryloxy-phosphoramidate derivatives of
ddA,10 d4T,11,12 LCd4A,13 and d4A.10,14 These nucleotide
monophosphate analogues were shown to exhibit greatly
enhanced activity against HIV compared to the parent nucleoside
analogues in vitro. In contrast to the parent nucleosides, full
antiviral activity of the monophosphate analogues was retained
in kinase-deficient cell lines, which was consistent with an
efficient bypass of the first phosphorylation step in HIV infected
cells. Aryloxy-phosphoramidates are considered to be efficient
lipophilic prodrugs of the corresponding 5′-monophosphate
species in which the two masking groups are represented by an
amino acid ester and an aryl moiety. After passive diffusion
Recently, 4′-azidocytidine was discovered as a potent inhibitor
of HCV replication in cell culture. The corresponding 5′-
* Corresponding author: Tel. +44 29 20874537; fax: +44 29 20874537;
mcguigan@cardiff.ac.uk.
† Cardiff University.
‡ Roche Palo Alto.
a Abbreviations: NS3, nonstructural protein 3; NS5B, nonstructural
protein 5B; HSV, herpes simplex virus; HCV, hepatitis C virus; HIV, human
immunodeficiency virus; HBV, hepatitis B virus; d4A/d4T, 2′,3′-dideoxy-
2′,3′-didehydroadenosine/thymidine; ddA, 2′,3′-dideoxyadenosine; LCd4A,
(1R,cis)-4-(6-Amino-9H-purin-9-yl)-2-cyclopentene-1-methanol; BVdU, E-5-
(2-bromovinyl)-2′-deoxyuridine; HCMV, human cytomegalovirus; AZU,
4′-azidouridine; NMI, N-methylimidazole.
10.1021/jm0613370 CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/17/2007

Phosphoramidate ProTide Approach to 4′-Azidouridine
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1841
compounds were always isolated as mixtures of two diastereo-
isomers. The presence of these diastereoisomers in the final
1
preparations was confirmed by 31P (two peaks), H, and 13C
NMR. A total of 22 phenyl phosphoramidates were synthesized
as reported in Table 1.
We have previously reported extensive structure-activity
relationship (SAR) studies of anti-HIV phosphoramidates
exploring the amino acid region, including natural amino acid
variation,21 un-natural R,R-dialkyls,22 stereochemical variation,23
and amino acid extensions24 and replacements.25 In general,
L-alanine and the unnatural amino acid R,R-dimethylglycine
showed the best activity for the d4T parent molecule versus
HIV.11,12
Figure 1. Structure of AZU and its corresponding phenyl-phos-
phoramidate ProTide.
through cell membranes, the suggested activation pathway15
involves initial enzyme-catalyzed cleavage of the carboxylic
ester, followed by the internal nucleophilic attack of the acid
residue on the phosphorus center, displacing the aryloxy group.
The putative transient, cyclic mixed-anhydride is then rapidly
hydrolyzed to the corresponding amino acid phosphomonoester.
Last, a suggested phosphoramidase activity catalyzes the cleav-
age of the P-N bond to free the nucleoside monophosphate
intracellularly. In the current study we tested the possibility to
apply our ProTide approach to the inactive 4′-azidouridine (1)
in order to achieve bypass of the first phosphorylation step and
thereby generate novel antiviral agents (2) with potent activity
against HCV.
Using the previously described method (Scheme 1), we
synthesized the L-alanine (12), R,R-dimethylglycine (18),
cyclopentylglycine (20), L-phenylalanine (22), L-leucine (27),
L-methionine (29), ethyl L-glutamate (31), and L-proline (28)
phenylphosphoramidates of 1, each bearing an ethyl ester.
Further investigations on the amino acid variation were con-
ducted on a series of benzyl esters: L-alanine (17), R,R-
dimethylglycine (19), cyclopentylglycine (21), L-phenylalanine
(23), L-valine (24), and glycine (25). We further compared the
importance of the stereochemistry at the amino acid position
by preparing a D-alanine benzyl ester phosphoramidate (26).
On the basis of the L-alanine phenyl phosphoramidate backbone,
we also explored the SAR of different esters including methyl
(11), ethyl (12), butyl (13), 2-butyl (14), isopropyl (15), tert-
butyl (16), and benzyl (17). In order to have an indirect proof
of phenyl phosphoramidate metabolism, we synthesized the
N-methylglycine (30) and â-alanine (32) analogues, which were
considered unfavorable substrates according to the postulated
mechanism of activation.15
Results and Discussion
Chemistry. The synthesis of 1 has been previously de-
scribed.16 To prepare monophosphate prodrugs (2) of 1 we
initially followed the previously described phosphorochloridate
chemistry for the synthesis of ProTides developed in our
laboratory, using 1-methylimidazole (NMI) as the coupling
agent.14,17,18 Several attempts were performed using different
conditions (different amino acid esters, different reaction
conditions) without successful isolation of the corresponding
aryloxy-phosphoramidate. These initial unsuccessful attempts
might be explained considering the presence of a bulky group
(azido) at the 4′-position adjacent to the coupling site at the
5′-position; in all previously published ProTide examples the
4′-position was unsubstituted.
The method of Uchiyama was investigated next.19 This
approach is based on the treatment of a nucleoside with 1 equiv
of a strong organometallic base, such as a solution of tert-
butylmagnesium chloride (tBuMgCl), to form the corresponding
metal alkoxide. In the case of (1), this reaction was observed
to be very rapid and gave yields between 3% and 20% of desired
products. In the first instance, we synthesized 4′-azidouridine
phosphoramidates starting from an unprotected nucleoside. The
apparent reactivity at the 2′- and 3′-positions was low, suggesting
high regioselectivity for the reaction at the 5′-position. In this
way it was also possible to synthesize compounds 13, 21, and
26. In order to achieve higher solubility in the reaction solvent
(tetrahydrofuran) and increase reactivity at the 5′ position, the
2′- and 3′-positions of 1 were protected with a cyclopentyl
group.20 The final synthetic pathway (Scheme 1) involves the
coupling of phenyl dichlorophosphate with different amino acid
ester salts (5) to give the corresponding phenyloxy-phospho-
rochloridates (6), which were purified by flash chromatography
and then coupled with the 2′,3′-O,O-cyclopentylidene derivative
7 of (1) in the presence of tBuMgCl (1 M solution in THF).
Recently we noted an increase of in Vitro potency of a
1-naphthyl-phosphoramidate analogue compared to the corre-
sponding phenyl derivative while investigating the anticancer
activity of BVdU phosphoramidates.26 Therefore, similar phos-
phoramidate analogues were also generated for (1). The
synthesis of the 1-naphthyl phosphoramidate (33) was performed
by reacting 1-naphthol with phosphorus oxychloride in an almost
quantitative reaction to give the corresponding phosphoro-
dichloridate (Scheme 3), which was then coupled to an amino
acid ester and the nucleoside analogue according to our standard
procedures. In this case, the separation of the two phosphate
diastereoisomers (34 and 35) was achieved by using a semi-
preparative HPLC purification with elution conditions of 70%
water/30% acetonitrile. The 31P NMR spectrum showed the
presence of only one peak for the first of the two fractions
separated, and the 1H NMR spectrum supported the suggestion
of a single diastereoisomer in this case. The second fraction
contained an excess of the second diastereoisomer together with
a minor proportion (estimated at 7% by 31P NMR integration)
of the first diastereoisomer (see Supporting Information for
data).
Antiviral Activity. The phenyl phosphoramidates described
above (11-32) were characterized in Vitro as inhibitors of HCV
replication in a HCV replicon assay as previously reported.6,7
Data are presented in Table 1 as EC50 values (representing the
concentration of compounds reducing HCV replication by 50%)
and CC50 values (representing the concentration of compounds
reducing cell viability by 50%) as determined using the WST
assay. All compounds showed CC50 values greater than 100 µM.
The parent compound 1 did not inhibit HCV replication
significantly in the replicon system (EC50 > 100 µM). In
contrast, a number of phosphoramidate derivatives showed
The deprotection step was performed with a solution of 80%
formic acid in water for 4 h at room temperature (Scheme 2).
Due to the stereochemistry at the phosphorus center, the final

1842 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Perrone et al.
Scheme 1. General Synthetic Pathway for the Synthesis of AZU Aryloxy-phosphoramidates
Scheme 2. General Synthetic Pathway for the Deprotection of 2′-3′-cyclopentylidene AZU Phenyl-phosphoramidates
potent inhibition of HCV replication. Assuming that 4′-
azidouridine-5′-triphosphate is the active HCV polymerase
inhibitor, these results support the notion that the active
phosphoramidates successfully delivered 4′-azidouridine mono-
phosphate intracellularly, that 4′-azidouridine (1) is inefficiently
phosphorylated to the monophosphate in replicon cells, and that
4′-azidouridine monophosphate can be phosphorylated to the
5′-triphosphate in replicon cells. As shown in Table 2, 4′-
azidouridine triphosphate notably inhibited recombinant HCV
polymerase NS5b in vitro, and did so with similar sub-
micromolar potency, like that of the previously described NS5b
inhibitor R1479-TP (4′-azidocytidine triphosphate).6 Therefore,
the application of our phosphoramidate approach shown to be
a successful tool in overcoming the phosphorylation block of 1
and converting an inactive nucleoside analogue to a potent
inhibitor of HCV replication, thus accessing the full potential
of the 4′-azidouridine triphosphate.
As shown in Table 1, L-alanine derivatives represented a
series of active antiviral phosphoramidates (11-17). Low or
sub-micromolar activity was noted in marked contrast to the
inactiVe nucleoside parent (1). The tert-butyl ester (16) was the
least active of the series, which was in agreement with the SARs
previously obtained in the d4T series24 and may relate to the
relative stability of tertiary esters to enzyme-mediated hydrolysis.
The isopropyl ester (15) showed high potency and represented
one of the most active phosphoramidates prepared. Similarly,
the 2-butyl ester (14) was highly active in our assay in contrast
to previous observations with other nucleoside analogues.
Together with the benzyl analogue (17), these three esters
provided the most potent compounds of HCV replication

Phosphoramidate ProTide Approach to 4′-Azidouridine
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1843
Table 1. Anti HCV Activity and Cytotoxicity Data for (1) and Phenyl
Table 3. Anti HCV Activity and Cytotoxicity Data for (1) and
Phosphoramidate Nucleotide Analogues
1-Naphthyl Nucleotide Analogues
compound
amino acid
ester
EC50 (µM)
CC50 (µM)
phosphorus
configuration
amino
acid
EC50
(µM)
CC50
(µM)
compound
ester
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
Me2Gly
Me2Gly
cPntGly
cPntGly
Phe
Phe
Val
Gly
D-Ala
Leu
Me
Et
3.1
1.3
1.2
0.63
0.77
5.1
0.61
10.3
3.4
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
33
34
35
S/R
R
S
L-Ala
L-Ala
L-Ala
L-Ala
Bn
Bn
Bn
Bn
0.22 >100
0.39 >100
0.43 >100
0.61 >100
Bu
2-Bu
iPr
tBu
Bn
Et
Bn
Et
Bn
Et
Bn
Bn
Bn
Bn
Et
Et
Et
Et
Et
Et
-
17 (Phenyl ProTide)
4′-azidouridine (1)
S/R
>100
>100
activity, displaying an EC50 value of 3.4 µM. Therefore, matrix-
based optimization of amino acid and ester functions may be
preferred over stepwise approaches.
The inactivity of the â-alanine (32) and of the N-methyl
glycine (30) compounds might underline the presence of an
R-amino acid and a free NH as a minimum requirement in the
amino acid structure to enable the metabolic activation of
aryloxy-phosphoramidates. However, the proline compound
(with a blocked NH) did show modest (28) activity, pointing
to a complex amino acid SAR.
In conclusion, ester variation was widely tolerated except for
the tert-butyl which gave a slight reduction in potency in the
L-alanine series (16) and the benzyl in the case of the
L-phenylalanine derivative (23). L-Alanine remained the most
effective amino acid, with glycine and D-alanine showing only
slightly reduced potency. Dimethylglycine, L-leucine, and L-
proline also provided compounds with antiviral potencies in a
low micromolar range. It therefore appears that the amino acid
core could be considerably varied to give antiviral agents with
potencies within a 10-fold range in replicon cells. Importantly,
potency optimization requires consideration of both amino acid
and ester moieties as most clearly shown for the ethyl and benzyl
esters of the L-phenylalanine analogues. Moreover, quite distinct
SARs emerged from this family versus HCV as compared to
our prior studies in other families.
>100
<100
1.37
<100
<100
1.6
1.2
2.3
6.0
14
Pro
Met
N-MeGly
EtGlu
â-Ala
-
>100
>100
>100
>100
4′-azidouridine (1)
Scheme 3. Synthetic Pathway for the Synthesis of
1-naphthylphosphorodichloridate
Table 2. Inhibition of HCV Polymerase (NS5B) Activity in Vitro
IC50 [µM]
We also explored the possibility to replace the phenyl
substituent on the phosphate with a more hydrophobic moiety,
1-naphthyl. Previously, we noted an increase of in Vitro potency
of 1-naphthyl-phosphoramidates compared to the corresponding
phenyl phosphoramidates when investigating BVdU phospho-
ramidates in an anticancer assay.26 We synthesized 33, the
1-naphthyl analogue of 17 (L-alanine benzyl ester). As shown
in Table 3, compound 33 inhibited HCV replication with an
EC50 of 0.22 µM, leading to a further increase in antiviral
activity (>450-fold) in comparison to 4′-azidouridine (Table
3). One of the two phosphorus diastereoisomers could be
purified using a C-18 reverse phase semipreparative HPLC. One
of the two main fractions obtained showed only one 31P NMR
peak. The second fraction was less pure, although the second
diastereoisomer appeared as the major component of the
mixture. We have previously reported a method for the
prediction of the phosphorus configuration of such diastereo-
isomers based on a different 1H NMR profile of the methylene
protons of the benzyl ester.26 Applying this concept to com-
pounds 34 and 35, we noticed that in one case (more polar, 35)
a clear AB-system was observed while, for the other diastereo-
isomer (less polar, 34), the two protons displayed an apparent
doublet. Conformational studies were performed using the Sybyl
7.0 software package. The lowest energy conformation found
for each diastereoisomer is shown in Figure 2. These differences
in proton profiles can be explained by the ability of one, but
not the other, diastereoisomer to form π-π interactions between
the naphthyl and the phenyl group of the benzyl ester resulting
in a constrained conformation. This interaction can only occur
with the S phosphorus configuration (35) with the two methylene
enzyme
R1479-TP (4′-azido CTP)
4′-azido UTP
NS5B570n-BK
NS5B570-Con1
0.29 ( 0.13
0.32 ( 0.11
0.23 ( 0.01
0.22 ( 0.02
inhibitors in the L-alanine series, all having µM inhibition of
HCV. The antiviral activity of these three phosphoramidates
was exceptional if compared to the parent compound 1 (EC50
> 100 µM), providing strong support for the notion of ProTide-
mediated kinase bypass.
In the benzyl ester family, L-alanine (17) provided the most
active compound with D-alanine (26) and glycine (25) being
only slightly less potent. These results were striking when
compared to the 60-70 fold reduction in anti-HIV potency for
d4T ProTides with an L-alanine to glycine replacement and a
20-40 fold reduction for the corresponding abacavir Pro-
Tides.21,27 This reinforces our earlier conclusion that a separate
ProTide motif optimization process is needed for each nucleo-
side analogue versus a given target. It may be that cell line
dependent enzyme expression may determine different phos-
phoramidate SARs.
The presence of a methyl (D- and L-alanine, 26 and 17) or
R,R-dimethyl (19) enhanced the activity if compared to larger
and hydrophobic amino acid side chain residues such as L-valine
(24), L-phenylalanine (23), and cyclopentylglycine (21), which
were weakly active in the replicon assay.
An unexpected correlation was found between amino acid
and ester function. While the L-phenylalanine derivative was
substantially inactive as a benzyl ester (23), the corresponding
ethyl ester (22) showed a significantly increased antiviral

1844 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Perrone et al.
(q), broad signal (br), doublet of doublet (dd), doublet of triplet
(dt), or multiplet (m). Chemical shifts for 31P spectra are quoted in
parts per million relative to an external phosphoric acid standard.
Many proton and carbon NMR signals were split due to the presence
of (phosphate) diastereoisomers in the samples. The mode of
ionization for mass spectroscopy was fast atom bombardment (FAB)
using MNOBA as matrix. Column chromatography refers to flash
column chromatography carried out using Merck silica gel 60 (40-
60 µM) as stationary phase.
For convenience, standard procedures have been given, as similar
procedures were employed for reactions concerning the synthesis
of precursors and derivatives of ProTides. Variations from these
procedures and individual purification methods are given in the
main text. Preparative and spectroscopic data on individual precur-
sor, blocked nucleosides are given as Supporting Information only
(see below), excluding only the first example.
Figure 2. Lowest energy conformations of compounds 34 and 35.
protons becoming nonmagnetically equivalent (AB system). For
the diastereoisomer with R phosphorus configuration (34), this
interaction does not occur and the higher degree of flexibility
around the methylene renders its protons more magnetically
similar (apparent doublet). The biological activities of the
separated diastereoisomers (34 and 35) were comparable to each
other and to the mixture (33) (Table 3).
Interestingly, application of similar ProTide methods to the
active 4′-azidocytidine gave little or no boost in anti-HCV
activity (data not shown), implying a rather efficient phospho-
rylation of this nucleoside analogue, with which ProTide
methods presumably cannot compete.
Standard Procedure 1: Preparation of 2′,3′-O,O-Cyclopen-
t
tylidene-4′-azidouridine Phosphoramidates. BuMgCl (2.0 mol
equiv) and 2′,3′-O,O-cyclopentylidene-4′-azidouridine (1.0 mol
equiv) were dissolved in dry THF (31 mol equiv) and stirred for
15 min. Then a 1 M solution of the appropriate phosphorochloridate
(2.0 mol equiv) in dry THF was added dropwise and then stirred
overnight. A saturated solution of NH4Cl was added, and the solvent
was removed under reduced pressure to give a yellow solid, which
was consequently purified by chromatography.
Standard Procedure 2: Deprotection of 2′,3′-Protected 4′-
Azidouridine Phosphoramidates. The appropriate 2′,3′-O,O-
cyclopentylidene-4′-azidouridine phosphoramidate was added to a
solution 80% of formic acid in water. The reaction was stirred at
room temperature for 4 h. The solvent was removed under reduced
pressure, and the obtained oil was purified by chromatography.
Standard Procedure 3: Preparation of 4′-Azidouridine Phos-
phoramidates via Free Nucleoside. tBuMgCl (2.0 mol equiv) and
4′-azidouridine (1.0 mol equiv) were dissolved in dry THF (31 mol
equiv) and stirred for 15 min. Then a 1 M solution of the appropriate
phosphorochloridate (2.0 mol equiv) in dry THF was added
dropwise and then stirred overnight. A saturated solution of NH4-
Cl was added, and the solvent was removed under reduced pressure
to give a yellow solid, which was purified by chromatography.
HPLC Method Used for the Separation of Compound 34 and
35. Varian ProStar instrument using a Polaris C18-A 10u column;
elution was performed using a mobile phase consisting of water/
acetonitrile 70% H2O/30% CH3CN, 17 min elution time with a flow
of 20 mL/min. Optimal loading on column: 8 mg of phosphora-
midate per run.
Conclusion
A series of phosphoramidate ProTides of 4′-azidouridine were
prepared and evaluated as inhibitors of HCV replication in Vitro.
The phosphoramidate approach provided novel compounds with
highly increased potency in the replicon assay when compared
to the inactive parent compound, corresponding to boosts in
anti-HCV potency of >450-fold. All phosphoramidates tested
were nontoxic in the replicon assay (CC50 > 100 µM). The most
active compound prepared in the series was the 1-naphthyl
L-alanine benzyl ester phosphoramidate with an EC50 of 0.22
µM in the replicon assay. The diastereoisomers of this com-
pound were separated by HPLC and their absolute phosphorus
configurations predicted by modeling and NMR. However, they
did not show any differences in biological activity. This report
demonstrates the ability of the ProTide approach to successfully
bypass the rate limiting initial phosphorylation of a ribonucleo-
side analogue and thus confer significant antiviral activity on
an inactive parent nucleoside.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(methyloxy-L-alaninyl)] Phosphate (Methyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-alaninate). Prepared according
to the standard procedure 1, from 2′,3′-O,O-cyclopentylidene-4′-
Experimental Section
Biology. HCV replicon assay was performed in the stable
replicon cell line 2209-23 derived from Huh-7 cells stably trans-
fected with a bicistronic HCV replicon (genotype 1b) expressing
the renilla luciferase reporter gene, as described.7 The RNA
synthesis activity of recombinant HCV polymerase proteins was
measured as incorporation of radiolabeled UMP into acid-insoluble
RNA products using HCV genome derived cIRES RNA as a
template in a primer-independent RNA synthesis assay.7 Recom-
binant proteins used were truncated at amino acid position 570 and
derived from genotype 1b strain BK (NS5B570n-BK) or Con1
(NS5B570-Con1).
Chemistry. General Procedures. All experiments involving
water-sensitive compounds were conducted under scrupulously dry
conditions. Anhydrous tetrahydrofuran and dichloromethane were
purchased from Aldrich. Proton, carbon, and phosphorus Nuclear
Magnetic Resonance (1H, 13C, 31P NMR) spectra were recorded
on a Bruker Avance spectrometer operating at 500, 125, and 202
MHz, respectively. All 13C and 31P spectra were recorded proton-
decoupled. All NMR spectra were recorded in CD3OD at room
temperature (20 °C ( 3 °C). Chemical shifts for 1H and 13C spectra
are quoted in parts per million downfield from tetramethylsilane.
Coupling constants are referred to as J values. Signal splitting
patterns are described as singlet (s), doublet (d), triplet (t), quartet
t
azidouridine (150 mg, 0.427 mmol), BuMgCl (0.85 mL, 1 M
solution in THF, 0.854 mmol), and phenyl(methyloxy-L-alaninyl)
phosphorochloridate (0.85 mL of solution 1 M in THF, 0.854
mmol). The crude product was purified by column chromatography,
using as eluent CHCl3/MeOH (95/5). The pure product was a white
solid (156 mg, 0.263 mmol, 61%). δP (d4-CH3OH): 3.14, 3.04; δH
(d4-CH3OH): 7.66 (1H, t, H6-uridine), 7.35 (2H, t, 2 CH-phenyl),
7.28-7.19 (3H, m, 3 CH-phenyl), 5.97 (1H, dd, H1′-uridine), 5.70
(1H, dd, H5-uridine), 5.12-5.04 (2H, m, H2′-uridine, H3′-uridine),
4.31-4.27 (2H, m, H5′-uridine), 4.01 (1H, m, CHR), 3.70 (3H, d,
CH3-methyl), 2.21-2.11 (2H, m, CH2-cyclopentyl), 1.79-1.73 (6H,
m, 3 CH2-cyclopentyl), 1.37 (3H, t, CH3-alanine, J ) 9.5 Hz).
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(methyloxy-L-alani-
nyl)] Phosphate (Methyl N-[{1-(2R,3S,4R,5R)-5-Azido-tetrahy-
dro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-2,4-
(1H,3H)-dione} (Phenoxy)-phosphoryl]-L-alaninate) (11). Prepared
according to the standard procedure 2, from 2′,3′-O,O-cyclopen-
tylidene-4′-azidouridine 5′-O-[phenyl(methyloxy-L-alaninyl)] phos-
phate (135 mg, 0.222 mmol), and a solution 80% of HCOOH in
water (10 mL). The crude was purified by column chromatography,

Phosphoramidate ProTide Approach to 4′-Azidouridine
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1845
using as eluent CHCl3/MeOH (8/2). The obtained pure product was
a white solid (65 mg, 0.116 mmol, 54%). δP (d4-CH3OH): 3.50,
3.31; δH (d4-CH3OH): 7.65 (1H, dd, H6-uridine), 7.38 (2H, m, 2
CH-phenyl), 7.28-7.20 (3H, m, 3 CH-phenyl), 6.15 (1H, m, H1′-
uridine), 5.72 (1H, m, H5-uridine), 4.42-4.36 (2H, m, H2′-uridine,
H3′-uridine), 4.26-4.18 (2H, m, H5′-uridine), 4.00 (1H, q, CHR),
3.70 (3H, d, CH3-methyl), 1.35 (3H, dd, CH3-alanine). MS (E/I):
549.1124 (MNa+); C19H23N6O10NaP requires 549.1111. Anal.
(C19H23N6O10P) C, H, N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(ethyloxy-L-alaninyl)] Phosphate (Ethyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-cy-
clopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-di-
one} (Phenoxy)-phosphoryl]-L-alaninate). See Supporting Infor-
mation for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(ethyloxy-L-alani-
nyl)]Phosphate (Ethyl N-[{1-(2R,3S,4R,5R)-5-Azido-tetrahydro-
3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-2,4-
(1H,3H)-dione} (Phenoxy)-phosphoryl]-L-alaninate) (12). Pre-
pared according to the standard procedure 2, from 2′,3′-O,O-
cyclopentylidene-4′-azidouridine 5′-O-[phenyl(ethyloxy-L-alaninyl)]-
phosphate (135 mg, 0.222 mmol), and a solution 80% of HCOOH
in water (10 mL). The crude product was purified by column
chromatography, using as eluent CHCl3/MeOH (8/2). The obtained
pure product was a white solid (65 mg, 0.116 mmol, 54%). δP (d4-
CH3OH): 3.56, 3.35; δH (d4-CH3OH): 7.65 (1H, m, H6-uridine),
7.37 (2H, m, 2 CH-phenyl), 7.29-7.21 (3H, m, 3 CH-phenyl), 6.17
(1H, m, H1′-uridine), 5.70 (1H, m, H5-uridine), 4.41-4.35 (2H,
m, H2′-uridine, H3′-uridine), 4.26-4.15 (4H, m, H5′-uridine, CH2-
ethyl), 3.97 (1H, m, CHR), 1.35 (3H, m, CH3-ethyl), 1.26 (3H, m,
CH3-alanine). MS (E/I): 563.1267 (MNa+), C20H25N6O10NaP
requires 563.1257. Anal. (C20H25N6O10P) C, H, N.
uridine, H3′-uridine), 4.22-4.15 (2H, m, H5′-uridine), 3.97 (1H,
q, CHR), 1.61-1.56 (2H, m, CH2-2-butyl), 1.36 (3H, d, CH3-
alanine, J ) 5.4 Hz), 1.22 (3H, t, CH3-2-butyl, J ) 2.1 Hz), 0.92
(3H, CH3-2-butyl, J ) 7.3 Hz). MS (E/I) 591.1580 (MNa+),
C22H29N6O10NaP requires 591.1580. Anal. (C22H29N6O10P) C, H,
N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-Azidouridine 5′-
O-[Phenyl(isopropyloxy-L-alaninyl)] Phosphate (2-Propyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-alaninate). See Supporting In-
formation for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(isopropyloxy-L-
alaninyl)] Phosphate (2-Propyl N-[{1-(2R,3S,4R,5R)-5-Azido-
tetrahydro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrim-
idine-2,4(1H,3H)-dione} (Phenoxy)-phosphoryl]-L-alaninate) (15).
Prepared according to the standard procedure 2, from 2′,3′-O,O-
cyclopentylidene-4′-azidouridine 5′-O-[phenyl(isopropyloxy-L-alani-
nyl)] phosphate (171 mg, 0.275 mmol), and a solution 80% of
HCOOH in water (10 mL). The crude product was purified by
column chromatography, using as eluent CHCl3/MeOH (8/2). The
obtained pure product was a white solid (144 mg, 0.260 mmol,
94%). δP (d4-CH3OH): 3.59, 3.38; δH (d4-CH3OH): 7.64 (1H, dd,
H6-uridine), 7.37 (2H, d, 2 CH-phenyl), 7.28-7.22 (3H, m, 3 CH-
phenyl), 6.15 (1H, dd, H1′-uridine), 5.70 (1H, dd, H5-uridine), 5.00
(1H, q, CH-isopropyl), 4.40-4.35 (2H, m, H2′-uridine, H3′-uridine),
4.24-4.18 (2H, m, H5′-uridine), 3.94 (1H, q, CHR), 1.34 (3H, dd,
CH3-alanine), 1.24 (6H, m, 2 CH3-isopropyl). MS (E/I) 577.1427
(MNa+), C21H27N6O10NaP requires 577.1424. Anal. (C21H27N6O10P)
C, H, N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(tert-butyloxy-L-alaninyl)] Phosphate (tert-Butyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-alaninate). See Supporting In-
formation for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(butoxy-L-alaninyl)]
Phosphate (Butyl N-[{1-(2R,3S,4R,5R)-5-Azido-tetrahydro-3,4-
dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-alaninate) (13). Prepared ac-
cording to the standard procedure 3, from 4′-azidouridine (300 mg,
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(tert-butyloxy-L-
alaninyl)] Phosphate (tert-Butyl N-[{1-(2R,3S,4R,5R)-5-Azido-
tetrahydro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrim-
idine-2,4(1H,3H)-dione} (Phenoxy)-phosphoryl]-L-alaninate) (16).
Prepared according to the standard procedure 2, from 2′,3′-O,O-
cyclopentylidene-4′-azidouridine 5′-O-[phenyl(tert-butyloxy-L-alani-
nyl)] phosphate (180 mg, 0.283 mmol), and a solution 80% of
HCOOH in water (10 mL). The crude product was purified by
column chromatography, using as eluent CHCl3//MeOH (8/2). The
obtained pure product was a white solid (90 mg, 0.158 mmol, 56%).
δP (d4-CH3OH): 3.63, 3.59; δH (d4-CH3OH): 7.65 (1H, m, H6-
uridine), 7.37 (2H, m, 2 CH-phenyl), 7.28-7.20 (3H, m, 3 CH-
phenyl), 6.14 (1H, m, H1′-uridine), 5.71 (1H, m, H5-uridine), 4.41-
4.34 (2H, m, H2′-uridine, H3′-uridine), 4.24-4.19 (2H, m, H5′-
uridine), 3.87-3.84(1H, m, CHR), 1.46 (9H, s, 3 CH3-tert-butyl),
1.32 (3H, d, CH3-alanine, J ) 7.4 Hz). MS (E/I) 591.1580 (MNa+),
C22H29N6O10NaP requires 591.1586. Anal. (C20H25N6O10P) C, H,
N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(benzyloxy-L-alaninyl)] Phosphate (Benzyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-alaninate). See Supporting In-
formation for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(benzyloxy-L-alani-
nyl)] Phosphate (Benzyl N-[{1-(2R,3S,4R,5R)-5-Azido-tetrahy-
dro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-2,4-
(1H,3H)-dione} (Phenoxy)-phosphoryl]-L-alaninate) (17). Prepared
according to the standard procedure 2, from 2′,3′-O,O-cyclopen-
tylidene-4′-azidouridine 5′-O-[phenyl(benzyloxy-L-alaninyl)] phos-
phate (140 mg, 0.209 mmol), and a solution 80% of HCOOH in
water (10 mL). The crude was purified by column chromatography,
using as eluent CHCl3/MeOH (8/2). The obtained pure product was
a white solid (70 mg, 0.116 mmol, 55%). δP (d4-CH3OH): 3.53,
3.28; δH (d4-CH3OH): 7.61 (1H, m, H6-uridine), 7.36 (6H, m, 3
t
0.986 mmol), BuMgCl (2.46 mL 1 M solution of THF, 2.465
mmol) and phenyl(butoxy-L-alaninyl) phosphorochloridate (2.46 mL
of solution 1 M in THF, 2.465 mmol). The crude product was
purified by column chromatography, using as eluent CHCl3/MeOH
(95/5), a preparative TLC using as eluent CHCl3/MeOH (85/15).
The obtained pure product was a white solid (16.8 mg, 0.030 mmol,
3%). δP (d4-CH3OH): 4.91, 4.35; δH (d4-CH3OH): 7.65 (1H, m,
H6-uridine, J ) 8.1 Hz), 7.46-7.22 (5H, m, CH-phenyl), 6.16 (1H,
m, H1′-uridine, J ) 3.6 Hz), 5.72 (1H, m, H5-uridine, J ) 8.1
Hz), 4.39-4.27 (2H, m, H2′-uridine, H3′-uridine), 4.24-4.09 (5H,
m, CHR, H5′-uridine, CH2-butyl), 1.67-1.59 (3H, m, CH3-alanine),
1.48-1.30 (4H, m, 2 CH2-butyl), 0.95 (3H, m, CH3-butyl). δC dept
(d4-CH3OH): 143.60, 142.98 (1C, C6-uridine), 131.57, 131.32 (1C,
CH-phenyl), 121.72, 121.65 (2C, CH-phenyl), 104.04 (1C, C5-
uridine), 92.68-92.38 (1C, C1′-uridine), 74.25, 74.08 (1C, C3′-
uridine), 73.95, 73.78 (1C, C2′-uridine), 71.77 (1C, CH2-butyl),
69.30 (1C, C5′-uridine), 68.86 (1C, CH2-butyl), 52.12, 51.90 (1C,
CH-R), 32.15 (1C, CH2-butyl), 20.92, 20.83 (1C, CH3-lateral chain),
20.71 (1C, CH2-butyl), 14.41 (1C, CH3-butyl). Anal. (C22H29N6O10P)
C, H, N.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(2-butoxy-L-alani-
nyl)] Phosphate (2-Butyl N-[{1-(2R,3S,4R,5R)-5-Azido-tetrahy-
dro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-2,4-
(1H,3H)-dione} (Phenoxy)-phosphoryl]-L-alaninate) (14). Prepared
according to the standard procedure 2, from 2′,3′-O,O-cyclopen-
tylidene-4′-azidouridine 5′-O-[phenyl(2-butyloxy-L-alaninyl)] phos-
phate (135 mg, 0.208 mmol), and 10 mL of a solution 80% of
HCOOH in water. The crude product was purified by column
chromatography, using as eluent CHCl3/MeOH (8/2). The obtained
pure product was a white solid (110 mg, 0.189 mmol, 94%). δP
(d4-CH3OH): 3.59, δP (d4-CH3OH): 3.60, 3.40; δH (d4-CH3OH):
7.65 (1H, dd, H6-uridine), 7.38 (2H, br, 2 CH-phenyl), 7.28-7.22
(3H, m, 3 CH-phenyl), 6.15 (1H, dd, H1′-uridine), 5.70 (1H, m,
H5-uridine), 4.80 (1H, m, CH-2-butyl), 4.38-4.34 (2H, m, H2′-

1846 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Perrone et al.
CH-phenyl, 3 CH-benzyl), 7.31-7.19 (4H, m, 2 CH-phenyl, 2 CH-
benzyl), 6.13 (1H, m, H1′-uridine), 5.68 (1H, m, H5-uridine), 5.15
(2H, s, CH2-benzyl), 4.36 (2H, m, H2′-uridine, H3′-uridine), 4.21-
4.14 (2H, m, H5′-uridine), 4.05 (1H, m, CHR), 1.37 (3H, m, CH3-
alanine). MS (E/I) 625.1424 (MNa+), C25H27N6O10NaP requires
625.1426. Anal. (C25H27N6O10P) C, H, N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-Azidouridine 5′-
O-[Phenyl(ethyloxy-dimethylglycinyl)] Phosphate (Ethyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-dimethylglycinate). See Support-
ing Information for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(ethyloxy-dimeth-
ylglycinyl)] Phosphate (Ethyl N-[{1-(2R,3S,4R,5R)-5-Azido-
tetrahydro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrim-
idine-2,4(1H,3H)-dione} (Phenoxy)-phosphoryl]-dimethylglycinate)
(18). Prepared according to the standard procedure 2, from 2′,3′-
O,O-cyclopentylidene-4′-azidouridine 5′-O-[phenyl(ethyloxy-dim-
ethylglycinyl)] phosphate (179 mg, 0.288 mmol), and a solution
80% of HCOOH in water (10 mL). The crude product was purified
by column chromatography, using as eluent CHCl3/MeOH (8/2).
The obtained pure product was a white solid (144 mg, 0.260 mmol,
90%). δP (d4-CH3OH): 1.90, 1.87; δH (d4-CH3OH): 7.64 (1H, dd,
H6-uridine), 7.39-7.35 (2H, m, 2 CH-phenyl), 7.26 (2H, d, 2 CH-
phenyl), 7.20 (1H, d, CH-phenyl), 6.14 (1H, d, H1′-uridine, J )
2.6 Hz), 5.67 (1H, dd, H5-uridine), 4.40-4.37 (2H, m, H2′-uridine,
H3′-uridine), 4.33 (2H, br, H5′-uridine), 4.23-4.15 (1H, m, CH2-
ethyl), 1.49 (6H, d, 2 CH3-lateral chain), 1.27 (3H, t, CH3-ethyl).
MS (E/I) 577.1431 (MNa+), C21H27N6O10NaP requires 577.1424.
Anal. (C21H27N6O10P) C, H, N.
OH): 7.69 (1H, dd, H6-uridine), 7.40-7.36 (2H, m, 2 CH-phenyl),
7.27-7.20 (3H, m, 3 CH-phenyl), 6.16 (1H, d, H1′-uridine, J )
5.3 Hz), 5.67 (1H, m, H5-uridine), 4.40-433 (2H, m, H2′-uridine,
H3′-uridine), 4.26-4.16 (4H, m, 2 H5′-uridine, CH2-ethyl), 2.15-
1.96 (4H, m, 2 CH2-cyclopentyl), 1.75-1.62 (4H, m, 2 CH2-
cyclopentyl), 1.25 (3H, q, CH3-ethyl). MS (E/I) 603.1585 (MNa+),
C23H29N6O10NaP requires 603.1580. Anal. (C23H29N6O10P) C, H,
N.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(benzyloxy-L-cyclo-
pentylglycinyl)] Phosphate (Benzyl N-[{1-(2R,3S,4R,5R)-5-Azido-
tetrahydro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrim-
idine-2,4(1H,3H)-dione} (Phenoxy)-phosphoryl]-cyclopentylgly-
cinate) (21). Prepared according to the standard procedure 3, from
t
4′-azidouridine (300 mg, 1.052 mmol), BuMgCl (2.10 mL of
solution 1 M in THF, 2.10 mmol), and phenyl(benzyloxy-L-
cyclopentylglycinyl) phosphorochloridate (6.7) (2.10 mL of solution
1 M in THF, 2.10 mmol). The crude was purified by column
chromatography, using as eluent CHCl3/MeOH (95/5) and then a
preparative TLC using as eluent CHCl3/MeOH (9/1). The obtained
pure product was a white solid (130 mg, 0.202 mmol, 20%). δP
(d4-CH3OH): 3.77, 3.74; δH (d4-CH3OH): 7.58 (1H, m, H6-uridine,
J ) 8.13 Hz), 7.28 (7H, m, 5 CH-phenyl, 2 CH-benzyl), 7.15 (3H,
m, CH-benzyl), 6.09 (1H, m, H1′-uridine), 5.55 (1H, m, H5-uridine,
J ) 8.13 Hz), 5.08 (2H, s, CH2-phenyl), 4.29 (1H, m, H2′-uridine),
4.24 (1H, m, H3′-uridine), 4.09 (2H, m, H5′-uridine), 2.04 (2H,
m, CH2-cyclopentyl), 1.98 (2H, m, CH2-cyclopentyl), 1.64 (2H,
m, CH2-cyclopentyl), 1.55 (2H, m, CH2-cyclopentyl); δC (d4-CH3-
OH): 176.83 (1C, CdO ester), 166.20 (1C, C4-uridine), 152.62,
152.48, 152.39 (1C, C2-uridine), 143.00, 142.88 (1C, C6-uridine),
137.75, 137.73 (1C, C-phenyl), 131.22 (2C, CH-phenyl), 129.98
(1C, C-benzyl), 129.73 (2C, CH-phenyl), 129.69 (1C, CH-phenyl),
126.74 (2C, CH-benzyl), 122.00 (1C, CH-benzyl), 121.98, 121.93
(2C, CH-benzyl), 103.99, 103.96 (1C, C5-uridine), 99.23, 99.20,
99.06 (1C, C4′-uridine), 92.32, 92.13 (1C, C1′-uridine), 74.86 (1C,
C3′-uridine), 69.32 (1C, C2′-uridine), 69.25, 68.79, (1C, C5′-
urdine), 68.75, 68.62 (2C, CH2-benzyl), 40.35, 40.24 (1C, CH2-
cyclopentyl), 40.07, 39.55 (1C, CH2-cyclopentyl), 25.10 (1C, CH2-
cyclopentyl), 24.99 (1C, CH2-cyclopentyl). Anal. (C28H31N6O10P)
C, H, N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(benzyloxy-dimethylglycinyl)] Phosphate (Benzyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-dimethyglycinate). See Supporting
Information for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(benzyloxy-dimeth-
ylglycinyl)] Phosphate (Benzyl N-[{1-(2R,3S,4R,5R)-5-Azido-
tetrahydro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrim-
idine-2,4(1H,3H)-dione} (Phenoxy)-phosphoryl]-dimethylglycinate)
(19). Prepared according to the standard procedure 2, from 2′,3′-
O,O-cyclopentylidene-4′-azidouridine 5′-O-[phenyl(benzyloxy-di-
methylglycinyl)] phosphate (148 mg, 0.217 mmol) and a solution
80% of HCOOH in water (10 mL). The crude product was purified
by column chromatography, using as eluent CHCl3/MeOH (8/2).
The obtained pure product was a white solid (110 mg, 0.250 mmol,
82%). δP (d4-CH3OH): 1.86, 1.83; δH (d4-CH3OH): 7.60 (1H, dd,
H6-uridine), 7.36-7.32 (7H, m, 2 CH-phenyl, 5 CH-benzyl), 7.24-
7.17 (3H, m, 3 CH-phenyl), 6.12 (1H, dd, H1′-uridine), 5.65 (1H,
dd, H5-uridine), 5.15 (2H, dd, CH2-benzyl), 4.38-4.29 (2H, m,
H2′-uridine, H3′-uridine), 4.19-4.16 (2H, dd, H5′-uridine), 1.50
(6H, s, 2 CH3-lateral chain). MS (E/I) 639.1574 (MNa+),
C26H29N6O10NaP requires 639.1580. Anal. (C26H29N6O10P) C, H,
N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(ethyloxy-L-cyclopentylglycinyl)] Phosphate (Ethyl
N-[{1-[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-
2,2-cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-cyclopentylglycinate). See Sup-
porting Information for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(ethyloxy-L-cyclo-
pentylglycinyl)] Phosphate (Ethyl N-[{1-(2R,3S,4R,5R)-5-Azido-
tetrahydro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrim-
idine-2,4(1H,3H)-dione} (Phenoxy)-phosphoryl]-cyclopentylgly-
cinate) (20). Prepared according to the standard procedure 2, from
2′,3′-O,O-cyclopentylidene-4′-azidouridine 5′-O-[phenyl(benzyloxy-
L-cyclopentylglycinyl)] phosphate (188 mg, 0.290 mmol), and a
solution 80% of HCOOH in water (10 mL). The crude product
was purified by column chromatography, using as eluent CHCl3/
MeOH (8:2). The obtained pure product was a white solid (100
mg, 0.172 mmol, 59%). δP (d4-CH3OH): 2.57, 2.55; δH (d4-CH3-
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(ethyloxy-L-phenylalaninyl)] Phosphate (Ethyl N-[{1-
[(3aR,4R,6R,6aSS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-phenylalaninate). See Support-
ing Information for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(ethyloxy-L-pheny-
lalaninyl)] Phosphate (Ethyl N-[{1-(2R,3S,4R,5R)-5-Azido-tet-
rahydro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-
2,4(1H,3H)-dione} (Phenoxy)-phosphoryl]-L-phenylalaninate)
(22). Prepared according to the standard procedure 2, from 2′,3′-
O,O-cyclopentylidene-4′-azidouridine 5′-O-[phenyl(ethyloxy-L-
phenylalaninyl) phosphate (190 mg, 0.278 mmol) and a solution
80% of HCOOH in water (10 mL). The crude product was purified
by column chromatography, using as eluent CHCl3/MeOH (8/2).
The obtained pure product was a white solid (152 mg, 0.246 mmol,
88%). δP (d4-CH3OH): 3.28, 3.06; δH (d4-CH3OH): 7.55 (1H, dd,
H6-uridine), 7.34-7.07 (10H, m, 5 CH-phenyl, 5 CH-lateral chain),
6.14 (1H, dd, H1′-uridine), 5.70 (1H, dd, H5-uridine), 4.27-4.23
(2H, m, H2′-uridine, H3′-uridine), 4.15-4.00 (3H, m, H5′-uridine,
CHR), 3.81-3.78 (2H, CH2-ethyl), 3.10 (1H, q, CH2-lateral chain),
2.89 (1H, q, CH2-lateral chain), 1.20 (3H, m, CH3-ethyl). MS (E/I)
639.1594 (MNa+), C26H29N6O10NaP requires 639.1580. Anal.
(C26H29N6O10P) C, H, N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(benzyloxy-L-phenylalaninyl) Phosphate (Benzyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-phenylalaninate). See Support-
ing Information for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(benzyloxy-L-
phenylalaninyl)] Phosphate (Benzyl N-[{1-(2R,3S,4R,5R)-5-

Phosphoramidate ProTide Approach to 4′-Azidouridine
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1847
Azido-tetrahydro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)-
pyrimidine-2,4(1H,3H)-dione} (Phenoxy)-phosphoryl]-L-phenyl-
alaninate) (23). Prepared according to the standard procedure 2,
from 2′,3′-O,O-cyclopentylidene-4′-azidouridine 5′-O-[phenyl(ben-
zyloxy-L-phenylalaninyl) phosphate (163 mg, 0.219 mmol) and a
solution 80% of HCOOH in water (10 mL). The crude product
was purified by column chromatography, using as eluent CHCl3/
MeOH (8/2). The obtained pure product was a white solid (130
mg, 0.192 mmol, 87%). δP (d4-CH3OH): 3.21, 3.02; δH (d4-CH3-
OH): 7.51 (1H, dd, H6-uridine), 7.32-7.23 (6H, m, 2 CH-phenyl,
2 CH-lateral chain, 2 CH-benzyl), 7.18-7.03 (9H, m, 3 CH-phenyl,
3 CH-lateral chain, 3 CH-benzyl), 6.14 (1H, dd, H1′-uridine), 5.64
(1H, dd, H5-uridine), 5.16-5.09 (4H, m, H2′-uridine, H3′-uridine,
CH2benzyl), 4.24-4.12 (3H, m, H5′-uridine, CHR), 3.09 (1H, m,
CH2-lateral chain), 2.91-2.87 (2H, m, CH2-lateral chain). MS (E/
I) 701.1732 (MNa+), C31H31N6O10NaP requires 701.1737. Anal.
(C31H31N6O10P) C, H, N.
mmol), and phenyl(benzyloxy-D-alaninyl) phosphorochloridate (2.10
mL of solution 1 M in THF, 2.10 mmol). The crude product was
purified by column chromatography, using as eluent CHCl3/MeOH
(95/5) and then a preparative TLC using as eluent CHCl3/MeOH
(9/1). The obtained pure product was a white solid (100 mg, 0.1723
mmol, l6%). δP (d4-CH3OH): 4.89, 4.29; δH (d4-CH3OH): 7.61
(1H, m, H6-uridine), 7.36 (7H, m, 2 CH-phenyl, 5 CH-benzyl),
7.25 (3H, m, CH-phenyl), 6.15 (1H, m, H1′-uridine), 5.68 (1H, m,
H5-uridine), 5.17 (2H, s, CH2-benzyl), 4.38 (1H, m, H3′-uridine),
4.32 (1H, m, H2′-uridine), 4.23 (2H, m, H5′-uridine), 4.05 (1H,
m, CHR), 1.36 (3H, m, CH3-alanine); δC (d4-CH3OH): 175.34,
175.29, 175.07, 175.01 (1C, CdO ester), 166.22 (1C, C4-uridine),
152.65, 152.56, 152.40, 152.36, 152.31, 152.27 (1C, C2-uridine),
142.94, 142.86 (1C, C6-uridine), 137.60, 137.54 (1C, C-phenyl),
131.31 (2C, CH-phenyl), 130.00 (2C, CH-phenyl), 129.79, 129.76,
129.72 (2C, CH-phenyl), 126.79 (1C, CH- phenyl), 121.83, 121.77,
121.71, 121.64 (2C, CH-phenyl), 104.03,103.99 (1C, C5-uridine),
99.11, 98.98 (1C, C4′-uridine), 92.69, 92.43 (1C, C1′-uridine),
74.22, 74.16 (1C, C3′-uridine), 74.13, 73.93 (1C, C2′-uridine),
69.28, 69.21 (1C, CH2-benzyl), 68.71, 68.65, 68.54, 68.48 (1C, C5′-
uridine), 52.17, 51.92 (1C, CHR), 20.80, 20.70, 20.59 (1C, CH3-
lateral chain). MS (E/I) 625.1424 (MNa+), C25H27N6O10NaP
requires 625.1424. Anal. (C25H27N6O10P) C, H, N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(benzyloxy-L-valinyl)] Phosphate (Benzyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-valinate). See Supporting In-
formation for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(benzyloxy-L-vali-
nyl)] Phosphate (Benzyl N-[{1-(2R,3S,4R,5R)-5-Azido-tetrahy-
dro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-2,4-
(1H,3H)-dione} (Phenoxy)-phosphoryl]-L-valinate) (24). Prepared
according to Standard Procedure 2, from 2′,3′-O,O-cyclopentyl-
idene-4′-azidouridine 5′-O-[phenyl(benzyloxy-L-valinyl)] phosphate
(173 mg, 0.248 mmol) and a solution 80% of HCOOH in water
(10 mL). The crude product was purified by column chromatog-
raphy, using as eluent CHCl3/MeOH (8/2). The obtained pure
product was a white solid (70 mg, 0.116 mmol, 55%). δP (d4-CH3-
OH): 4.45, 4.14; δH (d4-CH3OH): 7.62 (1H, m, H6-uridine, J )
6.8 Hz), 7.39-7.32 (7H, m, 4 CH-phenyl, 3 CH-benzyl), 7.24-
7.18 (3H, m, CH-phenyl, 2 CH-phenyl), 6.14 (1H, m, H1′-uridine),
5.68 (1H, m, H5-uridine, J ) 6.8 Hz), 5.19-5.10 (2H, m, CH2-
benzyl), 4.37-4.30 (2H, m, H2′-uridine, H3′-uridine), 4.22-4.14
(2H, m, H5′-uridine), 3.76 (2H, m, CHR), 2.07 (1H, m, CH-valine),
0.90 (3H, t, CH3-valine, J ) 8.6 Hz), 0.84 (3H, t, 3 CH3-valine, J
) 7.8 Hz). MS (E/I) 653.1737 (MNa+), C27H31N6O10NaP requires
653.1754. Anal. (C27H31N6O10NaP) C, H, N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(ethyloxy-L-leucinyl)] Phosphate (Ethyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-leucinate). See Supporting In-
formation for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(ethyloxy-L-leuci-
nyl)] Phosphate (Ethyl N-[{1-(2R,3S,4R,5R)-5-Azido-tetrahydro-
3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-2,4-
(1H,3H)-dione} (Phenoxy)-phosphoryl]-L-leucinate) (27). Pre-
pared according to the standard procedure 2, from 2′,3′-O,O-
cyclopentylidene-4′-azidouridine 5′-O-[phenyl(ethyloxy-L-leucinyl)]
phosphate (135 mg, 0.208 mmol) and a solution 80% of HCOOH
in water (10 mL). The crude product was purified by column
chromatography, using as eluent CHCl3/MeOH (8/2). The obtained
pure product was a white solid (111 mg, 0.190 mmol, 91%). δP
(d4-CH3OH): 3.83, 3.47; δH (d4-CH3OH): 7.64 (1H, dd, H6-
uridine), 7.36 (2H, t, 2 CH-phenyl), 7.25-7.20 (3H, m, 3 CH-
phenyl), 6.16 (1H, d, H1′-uridine), 5.72 (1H, dd, H5-uridine), 4.40-
4.35 (2H, m, H2′-uridine, H3′-uridine), 4.22 (2H, br, H5′-uridine),
4.18-4.11 (1H, m, CH2-ethyl), 3.90 (1H, br, CHR), 1.54 (3H, m
CH-lateral chain, CH2-lateral chain), 1.27-1.19 (3H, m, CH3-ethyl),
0.86 (3H, t, CH3-lateral chain), 0.80 (3H, t, CH3-lateral chain). MS
(E/I) 605.1733 (MNa+), C23H31N6O10NaP requires 605.1737. Anal.
(C23H31N6O10P) C, H, N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(benzyloxy-glycinyl)] Phosphate (Benzyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-glycinate). See Supporting Infor-
mation for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(benzyloxy-glyci-
nyl)] Phosphate (Benzyl N-[{1-(2R,3S,4R,5R)-5-Azido-tetrahy-
dro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-2,4-
(1H,3H)-dione} (Phenoxy)-phosphoryl]-glycinate) (25). Prepared
according to the standard procedure 2, from 2′,3′-O,O-cyclopen-
tylidene-4′-azidouridine 5′-O-[phenyl(benzyloxy-glycinyl) phos-
phate (173 mg, 0.264 mmol) and a solution 80% of HCOOH in
water (10 mL). The crude product was purified by column
chromatography, using as eluent CHCl3/MeOH (8/2). The obtained
pure product was a white solid (70 mg, 0.116 mmol, 43%). δP (d4-
CH3OH): 3.53, 3.28; δH (d4-CH3OH): 7.63 (1H, m, H6-uridine),
7.36 (6H, m, 3 CH-phenyl, 3 CH-benzyl), 7.34-7.19 (4H, m, 2
CH-phenyl, 2 CH-benzyl), 6.15 (1H, m, H1′-uridine), 5.69 (1H,
m, H5-uridine), 5.18 (2H, s, CH2-benzyl), 4.39-418 (4H, m, H2′-
uridine, H3′-uridine, H5′-uridine), 3.83 (2H, d, CH2-glycine). MS
(E/I) 611.1267 (MNa+), C24H25N6O10NaP requires 611.1271. Anal.
(C24H25N6O10P) C, H, N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(ethyloxy-L-prolinyl)] Phosphate (Ethyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-prolinate). See Supporting In-
formation for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(ethyloxy-L-prolinyl)
Phosphate (Ethyl N-[{1-(2R,3S,4R,5R)-5-Azido-tetrahydro-3,4-
dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-prolinate) (28). Prepared ac-
cording to the standard procedure 2, from 2′,3′-O,O-cyclopentylidene-
4′-azidouridine 5′-O-[phenyl(ethyloxy-L-prolinyl)] phosphate (121
mg, 0.190 mmol) and a solution 80% of HCOOH in water (10
mL). The crude product was purified by column chromatography,
using as eluent CHCl3/MeOH (8/2). The obtained pure product was
a white solid (101 mg, 0.178 mmol, 94%). δP (d4-CH3OH): 1.60,
1.25; δH (d4-CH3OH): 7.65 (1H, dd, H6-uridine), 7.39 (2H, t, 2
CH-phenyl), 7.30-7.23 (3H, m, 3 CH-phenyl), 6.12 (1H, dd, H1′-
uridine), 5.71 (1H, dd, H5-uridine), 4.39-4.29 (2H, m, H2′-uridine,
H3′-uridine), 4.25-4.13 (24H, m, H5′-uridine, CH2-ethyl), 3.42 (1H,
m, CHR), 2.23-2.17 (2H, m, CH2-proline), 2.02-1.84 (4H, m 2
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(benzyloxy-D-alani-
nyl)] Phosphate (Benzyl N-[{1-(2R,3S,4R,5R)-5-Azido-tetrahy-
dro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-2,4-
(1H,3H)-dione} (Phenoxy)-phosphoryl]-D-alaninate) (26). Prepared
according to the standard procedure 3, from 4′-azidouridine (200
mg, 0.701 mmol), tBuMgCl (1.4 mL of solution 1 M in THF, 1.402

1848 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Perrone et al.
CH2-proline), 1.28 (3H, m, CH3-ethyl). MS (E/I) 589.1416 (MNa+),
C22H27N6O10NaP requires 589.1424. Anal. (C22H27N6O10P) C, H,
N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(ethyloxy-L-methioninyl)] Phosphate (Ethyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-methioninate). See Supporting
Information for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(ethyloxy-L-me-
thioninyl)] Phosphate (Ethyl N-[{1-(2R,3S,4R,5R)-5-Azido-tet-
rahydro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-
2,4(1H,3H)-dione} (Phenoxy)-phosphoryl]-L-methioninate) (29).
Prepared according to the standard procedure 2, from 2′,3′-O,O-
cyclopentylidene-4′-azidouridine 5′-O-[phenyl(ethyloxy-L-methioni-
nyl) phosphate (187 mg, 0.280 mmol) and a solution 80% of
HCOOH in water (10 mL). The crude product was purified by
column chromatography, using as eluent CHCl3/MeOH (8/2). The
obtained pure product was a white solid (133 mg, 0.116 mmol,
79%). δP (d4-CH3OH): 3.81, 3.48; δH (d4-CH3OH): 7.65 (1H, t,
H6-uridine), 7.38 (2H, d, 2 CH-phenyl), 7.30-7.21 (3H, m, 3 CH-
phenyl), 6.15 (1H, dd, H1′-uridine), 5.72 (1H, dd, H5-uridine),
4.41-4.35 (2H, m, H2′-uridine, H3′-uridine), 4.27-4.16 (4H, m,
H5′-uridine, CH2-ethyl), 4.08 (1H, t, CHR), 2.53 (1H, m, CH2-
lateral chain), 2.42 (1H, CH2-lateral chain), 2.03 (3H, d, CH3-lateral
chain, J ) 15.3 Hz), 1.88-1.84 (2H, m, CH2-lateral chain), 1.31
(3H, m, CH3-ethyl). MS (E/I) 563.1267 (MNa+) C20H25N6O10NaP
requires 563.1257. Anal. (C20H25N6O10P) C, H, N.
uridine, CH2-ethyl, CH2-ethyl lateral chain), 4.01-3.94 (1H, m,
CHR), 2.42-2.16 (2H, m, CH2-lateral chain), 2.10-1.82 (2H, m,
CH2-lateral chain), 1.28-1.22 (6H, m, CH3-ethyl lateral chain, CH3-
ethyl). Anal. (C24H31N6O10P) C, H, N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(ethyloxy-L-â-alaninyl)] Phosphate (Ethyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-â-alaninate). See Supporting
Information for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(ethyloxy-L-â-alani-
nyl)] Phosphate (Ethyl N-[{1-(2R,3S,4R,5R)-5-Azido-tetrahydro-
3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-2,4-
(1H,3H)-dione} (Phenoxy)-phosphoryl]-L-â-alaninate) (32). Pre-
pared according to the standard procedure 2, from 2′,3′-O,O-
cyclopentylidene-4′-azidouridine 5′-O-[phenyl(ethyloxy-L-â-alan-
inyl)] phosphate (165 mg, 0.272 mmol) and a solution 80% of
HCOOH in water (10 mL). The crude product was purified by
column chromatography, using as eluent CHCl3/MeOH (8/2). The
obtained pure product was a white solid (127 mg, 0.235 mmol,
86%). δP (d4-CH3OH): 3.33, 3.27; δH (d4-CH3OH): 7.62 (1H, dd,
H6-uridine), 7.38 (2H, t, 2 CH-phenyl), 7.26-7.20 (3H, m, 3 CH-
phenyl), 6.12 (1H, d, H1′-uridine, J ) 3.2 Hz), 5.72 (1H, dd, H5-
uridine), 4.39-4.34 (2H, m, H2′-uridine, H3′-uridine), 4.23-4.11
(4H, m, H5′-uridine, CH2-ethyl), 3.27 (1H, m, CH2R), 2.54 (1H,
br, CH2â), 1.25 (3H, m, CH3-ethyl). MS (E/I) 563.1279 (MNa+),
C20H25N6O10NaP requires 563.1267. Anal. (C20H25N6O10P) C, H,
N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(ethyloxy-L-N-methyl-glycinyl)] Phosphate (Ethyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-N-methyl-glycinate). See Sup-
porting Information for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(ethyloxy-L-N-meth-
yl-glycinyl)] Phosphate (Ethyl N-[{1-(2R,3S,4R,5R)-5-Azido-
tetrahydro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrim-
idine-2,4(1H,3H)-dione} (Phenoxy)-phosphoryl]-L-N-methyl-
glycinate) (30). Prepared according to the standard procedure 2,
from 2′,3′-O,O-cyclopentylidene-4′-azidouridine 5′-O-[phenyl(ethyl-
oxy-L-N-methyl-glycinyl) phosphate (172 mg, 0.284 mmol) and a
solution 80% of HCOOH in water (10 mL). The crude was purified
by column chromatography, using as eluent CHCl3/MeOH (8/2).
The obtained pure product was a white solid (135 mg, 0.250 mmol,
88%). δP (d4-CH3OH): 5.12, 4.93; δH (d4-CH3OH): 7.66 (1H, dd,
H6-uridine), 7.42-7.38 (2H, m, 2 CH-phenyl), 7.27-7.22 (3H, m,
3 CH-phenyl), 6.15 (1H, d, H1′-uridine, J ) 2.1 Hz), 5.70 (1H,
dd, H5-uridine), 4.42-4.29 (3H, m, H2′-uridine, H3′-uridine, H5′-
uridine), 4.26-4.17 (3H, m, H5′-uridine, CH2-ethyl), 4.00 (1H, m,
CH2R), 3.80 (1H, m, CH2R), 2.83 (3H, d, CH3-N), 1.28 (3H, t,
CH3-ethyl, J ) 5.0 Hz). Anal. (C20H25N6O10P) C, H, N.
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[Phenyl(ethyloxy-L-ethyl-glutamyl)] Phosphate (Ethyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (Phenoxy)-phosphoryl]-L-ethylglutamate). See Supporting
Information for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[Phenyl(ethyloxy-L-ethyl-
glutamyl)] Phosphate (Ethyl N-[{1-(2R,3S,4R,5R)-5-Azido-tet-
rahydro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-
2,4(1H,3H)-dione} (Phenoxy)-phosphoryl]-L-ethylglutamate) (31).
Prepared according to the standard procedure 2, from 2′,3′-O,O-
cyclopentylidene-4′-azidouridine 5′-O-[phenyl(ethyloxy-L-ethyl-
glutamyl)] phosphate (197 mg, 0.284 mmol) and a solution 80%
of HCOOH in water (10 mL). The crude product was purified by
column chromatography, using as eluent CHCl3/MeOH (8/2). The
obtained pure product was a white solid (168 mg, 0.268 mmol,
94%). δP (d4-CH3OH): 3.66, 3.39; δH (d4-CH3OH): 7.60 (1H, dd,
H6-uridine), 7.35 (2H, m, 2 CH-phenyl), 7.26-7.19 (3H, m, 3 CH-
phenyl), 6.12 (1H, d, H1′-uridine), 5.72 (1H, dd, H5-uridine), 4.40-
4.32 (2H, m, H2′-uridine, H3′-uridine), 4.29-4.08 (6H, m, H5′-
Synthesis of 2′,3′-O,O-Cyclopentylidene-4′-azidouridine 5′-
O-[1-Naphthyl(benzyloxy-L-alaninyl)] Phosphate (Benzyl N-[{1-
[(3aR,4R,6R,6aS)-4-Azido-tetrahydro-4-(hydroxymethyl)-2,2-
cyclopentylfuro[3,4-d]1,3dioxol-6-yl]pyrimidine-2,4(1H,3H)-
dione} (1-naphthoxy)-phosphoryl]-L-alaninate). See Supporting
Information for preparative and spectroscopic data.
Synthesis of 4′-Azidouridine 5′-O-[1-Naphthyl(benzyloxy-L-
alaninyl)] Phosphate (Benzyl N-[{1-(2R,3S,4R,5R)-5-Azido-tet-
rahydro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-
2,4(1H,3H)-dione} (1-Naphthoxy)-phosphoryl]-cyclopentylgly-
cinate) (33). Prepared according to the standard procedure 2, from
2′,3′-O,O-cyclopentylidene-4′-azidouridine 5′-O-[1-naphthyl(ben-
zyloxy-L-alaninyl)] phosphate (212 mg, 0.324 mmol) and a solution
80% of HCOOH in water (10 mL). The crude product was purified
by column chromatography, using as eluent CHCl3/MeOH (8/2).
The obtained pure product was a white solid (161 mg, 0.246 mmol,
76%). δP (d4-CH3OH): 3.94, 3.76; δH (d4-CH3OH): 8.18 (1H, m,
CH-naphthyl), 7.90 (1H, m, CH-naphthyl), 7.72 (1H, m, H6-
uridine), 7.57-7.30 (11H, m, 6 CH-naphthyl, 5 CH-phenyl), 6.11
(1H, m, H1′-uridine), 5.50 (1H, m, H5-uridine), 5.11 (2H, m, CH2-
benzyl), 4.37 (1H, m, H3′-uridine), 4.31-4.19 (3H, m, H2′-uridine,
H5′-uridine), 4.11 (1H, m, CHR), 1.35 (3H, d, CH3-alanine, J )
7.2 Hz). MS (E/I) 675.1571 (MNa+), C29H29N6O10P requires
675.1575.
The two diastereoisomers obtained were separated by using a
semipreparative HPLC with elution conditions of 70% H2O/30%
CH3CN, 17 min elution time. Optimal loading on column: 8 mg
of phosphoramidate per run.
Less polar diastereoisomer (34): δP (d4-CH3OH): 3.96; δH (d4-
CH3OH): 8.16 (1H, t, CH-naphthyl, J ) 4.2 Hz), 7.91 (1H, t, CH-
naphthyl, J ) 5.0 Hz), 7.73 (1H, d, H6-uridine, J ) 8.1 Hz), 7.58-
7.42 (6H, m, 3 CH-naphthyl, 3 CH-phenyl), 7.32-7.28 (5H, 2 CH-
naphthyl, 3 CH-phenyl), 6.12 (1H, d, H1′-uridine, J ) 5.6 Hz),
5.50 (1H, d, H5-uridine, J ) 8.0 Hz), 5.10 (2H, d, CH2-benzyl),
4.36 (1H, d, H3′-uridine, J ) 5.8 Hz), 4.27-4.21 (3H, m, H2′-
uridine, H5′-uridine), 4.11 (1H, m, CHR), 1.35 (3H, d, CH3-alanine,
J ) 7.1 Hz).
More polar diastereoisomer (35): δP (d4-CH3OH): 3.77
(major); δH (d4-CH3OH): 8.17-8.15 (1H, m, CH-naphthyl), 7.91-
7.89 (1H, m, CH-naphthyl), 7.72 (1H, t, H6-uridine), 7.57-7.41
(6H, m, 3 CH-naphthyl, 3 CH-phenyl), 7.33-7.29 (5H, 2 CH-
naphthyl, 3 CH-phenyl), 6.12 (1H, dd, H1′-uridine), 5.50 (1H, dd,
H5-uridine), 5.13-5.05 (2H, AB system, CH2-benzyl), 4.37 (1H,

Phosphoramidate ProTide Approach to 4′-Azidouridine
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1849
(14) McGuigan, C.; Wedgwood, O. M.; De Clercq, E.; Balzarini, J.
Phosphoramidate derivatives of 2′,3′-didehydro-2′,3′-dideoxyadeno-
sine (d4A) have markedly improved anti-HIV potency and selectivity.
Bioorg. Med. Chem. Lett. 1996, 6, 2359-2362.
(15) Valette, G.; Pompon, A.; Girardet, J. L.; Cappellacci, L.; Franchetti,
P.; Grifantini, M.; LaColla, P.; Loi, A. G.; Perigaud, C.; Gosselin,
G.; Imbach, J. L. Decomposition pathways and in vitro HIV inhibitory
effects of isoddA pronucleotides-toward a rational approach for
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dd, H3′-uridine), 4.29-4.21 (3H, m, H2′-uridine, H5′-uridine),
4.12-4.09 (1H, m, CHR), 1.35 (3H, m, CH3-alanine).
Acknowledgment. We thank Helen Murphy for secretarial
assistance.
Supporting Information Available: Analytical data on target
compounds, preparative and spectroscopic data on blocked nucleo-
side intermediates, and figures of HPLCs of separated diastereo-
isomers. This material is available free of charge via the Internet
at http://pubs.acs.org.
(16) Maag, H.; Rydzewski, R. M.; McRoberts, M. J.; Crawford-Ruth, D.;
Verheyden, J. P. H.; Prisbe, E. J. Synthesis and anti-HIV activity of
4′-azido- and 4′-methoxynucleosides. J. Med. Chem. 1992, 35, 1440-
1451.
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