First example of phosphoramidate approach applied to a 4′-substituted purine nucleoside (4′-Azidoadenosine): Conversion of an inactive nucleoside to a submicromolar compound versus hepatitis C virus

Article abstract of DOI:10.1021/jm070362i

We report on the synthesis of the anti hepatitis C virus (HCV) agent 4′-azidoadenosine (1) and the application of the phosphoramidate ProTide technology to this nucleoside. The synthesis of 1 was achieved through an epoxide intermediate followed by regio- and stereoselective ring opening by azidotrimethylsilane in the presence of a Lewis acid. Compound 1 did not inhibit HCV replication in cell culture at concentrations up to 0.1 mM. However, a submicromolar active agent could be derived from 1 by the application of the ProTide technology. All the phosphoramidates prepared were L-alanine derivatives with variations in the aryl moiety and in the ester part of the amino acid. The benzyl ester and the 1-naphthyl phosphate (18) had the best activity in replicon assay. Phosphoramidates (18-21) achieved a significant improvement in antiviral potency over the parent nucleoside (1) with no increase in cytotoxicity.

Full text of DOI:10.1021/jm070362i

J. Med. Chem. 2007, 50, 5463-5470
5463
First Example of Phosphoramidate Approach Applied to a 4′-Substituted Purine Nucleoside
(4′-Azidoadenosine): Conversion of an Inactive Nucleoside to a Submicromolar Compound
versus Hepatitis C Virus
Plinio Perrone,† Felice Daverio,† Rocco Valente,† Sonal Rajyaguru,‡ Joseph A. Martin,‡ Vincent Le´veˆque,‡ 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 March 28, 2007
We report on the synthesis of the anti hepatitis C virus (HCV) agent 4′-azidoadenosine (1) and the application
of the phosphoramidate ProTide technology to this nucleoside. The synthesis of 1 was achieved through an
epoxide intermediate followed by regio- and stereoselective ring opening by azidotrimethylsilane in the
presence of a Lewis acid. Compound 1 did not inhibit HCV replication in cell culture at concentrations up
to 0.1 mM. However, a submicromolar active agent could be derived from 1 by the application of the
ProTide technology. All the phosphoramidates prepared were L-alanine derivatives with variations in the
aryl moiety and in the ester part of the amino acid. The benzyl ester and the 1-naphthyl phosphate (18) had
the best activity in replicon assay. Phosphoramidates (18-21) achieved a significant improvement in antiviral
potency over the parent nucleoside (1) with no increase in cytotoxicity.
Introduction
low or even lacking. The pharmacologically active triphosphate
species cannot be considered as a possible drug candidate
because of high instability and poor cellular permeation. For
many nucleosides the first phosphorylation constitutes the rate-
limiting step in the synthetic pathway to triphosphate formation,
suggesting that monophosphate analogues might be useful
antiviral agents. However, nucleoside monophosphates are also
generally unstable in blood and show poor membrane perme-
ation.9
The hepatitis C virus (HCV) was identified for the first time
in 1989 as a single-stranded positive sense RNA virus of the
flaviviridae family.1 HCV is the most common blood-borne
infection and a major cause of chronic liver disease and liver
transplantation in industrialized countries. According to the
World Health Organization (WHO), more than 170 million
people are estimated to be chronically infected by this virus,
representing the 3.1% of worldwide population.2
In order to bypass this first rate-limiting step required of
nucleoside analogues, our group has developed in the past a
suitable nucleotide delivery strategy. The aryloxy-phosphora-
midate (2) allows the bypass of the initial nucleoside kinase
dependence, by the intracellular delivery of the monophospho-
rylated nucleoside analogue in the membrane permeable “Pro-
Tide” form.10,11 This technology increases the lipophilicity of
the nucleoside monophosphate analogues and the intracellular
availability by passive diffusion. Previously we have reported
the success of the ProTide approach applied to various nucleo-
side analogues including d4A, ddA, L-Cd4A, and d4T.12-14
In these examples the corresponding aryloxy-phosphorami-
dates have shown an enhancement in antiviral activity against
target viruses such as HIV and HBV compared to the parent
nucleoside analogues in Vitro. In contrast to the nucleoside
analogues, the phosphoramidates retained full antiviral activity
in kinase-deficient cell lines.
Current therapy for HCV treatment is both poorly tolerated
and has limited efficacy, with less than 50% response rates
among patients infected with the most prevalent virus genotype.
Therefore, there is a need for more efficient and better tolerated
anti HCV agent.
The HCV genome is a single strand RNA molecule of
approximately 9600 nucleotides,1 containing coding sequences
for structural and nonstructural HCV proteins, flanked by 5′-
and 3′-nontranslated regions (5′-NTR and 3′-NTR)a at the
extremities of the HCV genome.1,3,4
Modified nucleosides already represent an important class
of polymerase inhibitors of other viruses, such as HSV and HIV.
It is notable that nucleoside derivatives generally need to be
phosphorylated to their corresponding triphosphate by host cell
(or in the case of some antiherpetics viral) kinases to be
converted to their corresponding pharmacologically active
species.
However, in many cases nucleoside analogues are poor
substrates for nucleoside kinases. Moreover, the dependence on
phosphorylation for activation of a particular nucleoside may
be a problem in cells where the nucleoside kinase activity is
The putative monophosphate release pathway involves initial
enzyme-catalyzed cleavage of the carboxy ester, followed by
the internal nucleophilic attack of the acid residue on the
phosphorus center, displacing the aryloxy group.13 The putative
transient, cyclic mixed-anhydride is then hydrolyzed to the
corresponding amino acid phosphomonoester which has been
observed both in Vitro and in ViVo.13
4′-Azidocytidine has been identified as a potent inhibitor of
HCV replication in cell culture.8 The corresponding 4′-azidocy-
tidine triphosphate is a competitive inhibitor of RNA synthesis
by HCV polymerase.8 As part of an effort to characterize
structure-activity relationships in this series of 4′-substituted
* Corresponding author. Tel. +44 29 20874537; fax: +44 29 20874537;
e-mail: mcguigan@cardiff.ac.uk.
† Cardiff University.
‡ Roche Palo Alto.
a Abbreviations: 5′-NTR, 5′-nontranslated region; 3′-NTR, 3′-nontrans-
lated; HIV, human immunodeficiency virus; HSV, herpes simplex virus;
d4A/d4T, 2′,3′-dideoxy-2′,3′-didehydroadenosine/thymidine; ddA, 2′,3′-
dideoxyadenosine; L-Cd4A, (1R,cis)-4-(6-amino-9H-purin-9-yl)-2-cyclo-
pentene-1-methanol.
10.1021/jm070362i CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/03/2007

5464 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Perrone et al.
Scheme 1. Initial Pathway for the Synthesis of 4′-Azidoadenosine (1)a
a 1. See Table 1; 2. tBuOK, pyridine; 3. ICl, NaN3, DMF; 4. BzCl, pyridine; 5. a. MCPBA, CH2Cl2/H2O, b. MeONa, MeOH.
Another attempt was performed using pyridine as solvent.
1
In this case, after purification by column chromatography, H
NMR and 31P NMR showed clear evidence of the presence of
triphenylphosphine oxide. An extraction in EtOAc/H2O was
explored as a purification method without success.
In order to use a different source of iodine, we tried the
iodination reaction using CI4 and triphenylphosphine in a
solution of adenosine and hexamethylphosphoric acid triamide
(HMPA).19 The difficulty of removing the solvent was a
limitation of this reaction on large scale.
Figure 1. 4′-Azidoadenosine (1) and general structure of its corre-
sponding aryloxy-phosphoramidate.
As it was proving difficult to avoid the presence of tri-
phenylphosphine mixed with the desired product, the same
reaction was performed using triphenylphosphine supported on
resin (polystyrene, PS-triphenylphosphine). In this case the
desired product (5) was obtained pure and in reasonable yield.
Because of the high polarity of 5, this route required large
amounts of EtOAc to achieve extraction.
Table 1. Conditions Attempted for the Synthesis of 5′-Deoxy-5′-
iodoadenosine
source of
solvent
reagent
Ph3P
Ph3P/imidazole
Ph3P
iodine
conditions
dioxane/pyridine
NMP
HMPT
I2
I2
CI4
I2
r.t.
r.t.
r.t.
r.t.
In an attempt to increase the lipophilicity of the desired 5′-
iodo derivative the same reaction was also performed using as
starting material the 2′,3′-isopropylidene-protected adenosine (4)
with PS-triphenylphosphine. In this case, the reaction required
an increase in temperature to 50 °C. After purification by
extraction, the pure desired product (6) was obtained in poor
yield (13%). We noted the presence of a high polar compound:
it was isolated and fully characterized, and by H NMR we
attribute the structure to the cyclic product of intramolecular
nucleophilic substitution in the 5′-position from the nitrogen in
3-position (10, Scheme 2).20-22
pyridine
Ph3P
pyridine
PS-Ph3P
I2
50 °C
nucleosides, 4′-azidoadenosine (1) was synthesized and evalu-
ated as a potential inhibitor of HCV replication.16 The ProTide
approach was applied to 1 to explore monophosphate delivery
and a potential increase of the antiviral activity. In addition it
was envisaged that delivering the adenosine analogue as a
monophosphate derivative could reduce the extent of intracel-
lular metabolism by deamination.
Because of the impossibility to avoid the use of the column
chromatography to purify 5, this first step was repeated on a
large scale using column chromatographic purification. The
elimination step was performed according the reported procedure
(Scheme 1) in the presence of a solution of potassium tert-
butoxide in pyridine.17 The regio- and stereoselective addition
of iodo-azide resulted successful only in the first attempt. The
final three steps involved the full protection of adenosine
followed by iodine displacement and deprotection with metha-
nolic ammonia (Scheme 1).
Results and Discussion
Chemistry. The synthesis of 4′-azidoadenosine (1) was
initially performed using the same conditions reported in the
literature (Scheme 1).17 The selective 5′-iodination of adenosine
was first attempted in the presence of dioxane-pyridine as
solvent, but because of the low solubility of the starting material
(3), the yield was poor. In an attempt to increase the yield,
different conditions were attempted (Table 1). The first alterna-
tive was to use triphenylphosphine, iodine, and an excess of
imidazole in a solution of N-methylpyrrolidinone (NMP).18 This
procedure resulted in only a modest 26% yield with suboptimal
purity of the desired product 5′-deoxy-5′-iodoadenosine (5).18
A different synthetic pathway (Scheme 3) was envisioned in
which the introduction of the azido group at 4′-position was
achieved trough an epoxide intermediate. In the literature this

Submicromolar ActiVe Nucleosides against Hepatitis C Virus
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5465
Scheme 2. Iodination Attempted with PS-Ph3P and Structure of the Side Product Isolated (10)
Scheme 3. Alternative Pathway for the Synthesis of 2′,3′-Cyclopentylidene-4′-azidoadenosinea
a 1. a. I2, pyridine, Ph3P; b. TBDMSCl, imidazole, pyridine; 2. tBuOK, pyridine; 3. pivaloyl chloride, DIEA, DCM; 4. DMDO 1 M in acetone; 5. TMSN3,
SnCl4; 6. TBAF, THF;.7. a. dimethoxycyclopentane; b. NH3/MeOH.
method has never been applied to introduce an azido group to
a purine nucleoside.
The synthesis of the phosphoramidate was performed fol-
lowing the Uchiyama procedure in the presence of tert-butyl
magnesium chloride (1 M solution in THF).24 The deprotection
step was performed in the presence of a solution of 80%
HCOOH in water for 4 h at room temperature (Scheme 4).
Because of the stereochemistry at the phosphorus center, the
final compounds were isolated as mixtures of two diastereo-
isomers. The confirmation of the presence of two diastereo-
The modified synthetic pathway started with the selective 5′-
iodination followed by protection of 2′ and 3′-OH with tert-
butyl dimethylsilyl groups. The elimination of HI was performed
in the same conditions described above. The key step was the
synthesis of the epoxide (14) and the subsequent ring opening
reaction. In order to avoid N1-oxidation with DMDO, it was
necessary to acylate the 6-NH2 group (13). The unsaturated
derivative of adenosine (13) was converted to the corresponding
epoxide (14) in the presence of 1 M solution of dimethoxy-
dioxirane, which has been previously reported as stereoselective
reagent for such epoxidation reactions.23 The ring opening
reaction was then performed in the presence of a Lewis Acid
(tin tetrachloride) and azidotrimethylsilane. The obtained product
(15) was subsequently deprotected in 2′- and 3′-positions in
presence of a solution of tetrabuthylammonium fluoride (TBAF).
For the synthesis of the 5′-phosphoramidate the protection with
a cyclopentylidine ketal group of 2′ and 3′-OH was performed
(16). This protection guaranteed a selective 5′-phosphorylation
and an increased in the solubility of the nucleoside in THF,
which was the solvent used in the synthesis of the phosphora-
midate.
1
isomers was shown by 31P NMR (two peaks) and H NMR
(splitting of many of the nucleoside signals).
Considering the established SAR previously reported, we
decided to focus on L-alanine phosphoramidates with ester and
aryl moiety variation. In previous work L-alanine had shown
the best activity with 1-naphthyl as aryl moiety and the benzyl
group as ester. We choose to have a variation of three ester
(tert-butyl, ethyl, and benzyl) and 2 aryl moieties (1-naphthyl
and phenyl).14
Antiviral Activity. The phosphoramidates (18-23) were
characterized in Vitro as inhibitors of HCV replication in the
HCV replicon assay using similar conditions as described.8 Data
are presented in Table 2 as EC50 values representing the
concentration of compound reducing HCV replication by 50%
and CC50 values representing the concentration of compound
reducing cell viability by 50% as determined using the WST
assay. All of the compounds showed CC50 values >100 µM.
The parent compound (1) did not inhibit HCV replication (EC50
The pivaloyl group was removed in presence of a saturated
solution of ammonia in methanol to give the desired product
(17) in high yield (87%).

5466 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Scheme 4. Synthesis of 4’-Azidoadenosine Phosphoramidatesa
Perrone et al.
a Compound 18 has been synthesized using 1, and it did not need deprotection. Compound 19, 20, 21, 22, 23 have been synthesized using
2′,3′-cyclopentylidene-4′-azidoadenosine (17).
Table 2. Anti-HCV Activity and Cytotoxicity Data for AZA and Aryl
Among the three esters tested, the tert-butyl esters (22, 23)
Phosphoramidate Nucleoside Analogues
were found to be inactive as inhibitors of HCV replication,
EC50
CC50
whereas benzyl and ethyl esters were potent inhibitors with
either 1-naphthyl or phenyl as aryl moiety (18, 19). The lack
of antiviral activity of tert-butyl ester phosphoramidates may
be related to the relative stability of tertiary esters to enzyme-
mediated hydrolysis. Comparing compound 18 and 19, we
noticed an increase of antiviral activity of 20 fold for the
1-naphthyl relative to phenyl as aryl moiety. This difference
might be related to the higher lipophilicity and/or the decrease
of pKa of 1-naphthyl relative to phenyl. In the case of ethyl
ester, instead the difference observed between 1-naphpthyl (20)
and phenyl phosphoramidate (21) was not significant. There
was no significant difference in antiviral potency between the
ethyl and benzyl esters, indicating that both of these esters are
good substrates for the esterase involved in phosphoramidate
conversion.
compd
ester
aryl moiety
(µM)
(µM)
18
19
20
21
22
23
benzyl
benzyl
ethyl
ethyl
tert-butyl
tert-butyl
R-naphthyl
phenyl
R-naphthyl
phenyl
R-naphthyl
phenyl
0.22
>100
>100
>100
>100
>100
>100
>100
4.00
0.59
1.50
>100
>100
>100
4-azidoadenosine (1)
> 100 µM). In contrast, a number of phosphoramidate deriva-
tives (18-21) showed potent inhibition of HCV replication.
Assuming that 4′-azidoadenosine-5′-triphosphate is the active
HCV polymerase inhibitor, these results support the notions that
(a) the active phosphoramidates successfully delivered 4′-
azidoadenosine monophosphate to the replicon cells, that (b)
4′-azidoadenosine (1) is a poor substrate for kinases in replicon
cells and that (c) 4′-azidoadenosine monophosphate is efficiently
phosphorylated to the 5′-triphosphate in replicon cells. There-
fore, the phosphoramidate approach could successfully overcome
the first phosphorylation of 1 and converted an inactive
nucleoside analogue into a potent inhibitor of HCV replication.
In conclusion, the most potent compounds in this series of
L-alanine ProTides were the 1-naphthyl derivatives with either
benzyl (18) or ethyl ester moieties (20). These compounds
inhibited HCV replication in the HCV replication system with
EC50 values of 0.22 and 0.59 µM, respectively. In contrast the
parent nucleoside 4′-azidoadenosine (1) was inactive as an
antiviral agent in this system (EC50 > 100 µM). Therefore, the
ethyl and benzyl phosphoramidates provided a more than 3 log
improvement in antiviral potency of the ProTide as compared
to the parent nucleoside in the cell-based system.
Considering previously reported SAR of phosphoramidates,
we chose to focus our attention on L-alanine which previously
provided the most potent pharmacological activity in a range
of antiviral and antiproliferative assays.

Submicromolar ActiVe Nucleosides against Hepatitis C Virus
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5467
removed under reduced pressure, and the black solid was purified
by column chromatography using as eluent a mixture of CHCl3/
MeOH 9:1, and then 8:2 and 7:3. The product was obtained as
brown solid (18.54 g, 0.074 mol, 80%). δH (d6-(CH3)2SO): 8.37
(1H, s, H2), 8.16 (1H, s, H8), 7.32 (2H, s, NH26), 6.16 (1H, d,
H1′, J ) 5.3 Hz), 5.73 (1H, s, OH2′), 5.58 (1H, s, OH3′), 4.83
(1H, t, H2′), 4.73 (1H, t, H3′), 4.31 (1H, s, H5′), 4.21 (1H, s, H5′).
Synthesis of 4′-Azido-5′-deoxy-5′-iodoadenosine (8). Sodium
azide (11.73 g, 0.180 mol) was added to a solution of ICl (14.65 g,
0.090 mol) in DMF (50 mL) and stirred at 30 °C for 20 min. Then
a solution of 1-(5-deoxy-â-D-erythro-pent-4-enofuranosyl)-adenine
(7, 9 g, 0.036 mol) in DMF (200 mL) was added dropwise in 30
min. After 1 h a saturated solution of NasS2O3 was added, and the
solvent was removed under reduced pressure. The solid was
dissolved in MeOH, and the precipitate was removed by filtration.
The solvent was removed under reduced pressure, and the yellow
solid was purified by column chromatography using as eluent
CHCl3/MeOH 9:1. The desired product was obtained as a yellow
solid (19 g, 0.045 mol, >100%). δH (d4-CH3OH): 8.86 (1H, s,
H2), 8.78 (1H, s, H8), 6.26 (1H, d, H1′, J ) 5.1 Hz), 5.28 (1H, m,
H2′), 4.73 (1H, t, H3′), 3.71 (1H, s, H5′), 3.68 (1H, s, H5′). For
C-13 NMR data see SI.
Synthesis of N,N-Dibenzoyl-2′,3′-di-O-benzoyl-4′-azido-5′-
deoxy-5′-iodoadenosine (9). To a solution of 4′-azido-5′-deoxy-
5′-iodoadenosine (8, 18 g, 0.040 mol) in pyridine, benzoyl chloride
(33 mL, 0.320 mol) and dimethylaminopyiridine (4.9 g, 0.040 mol)
were added. After 15 h the solvent was removed under reduce
pressure, and the dark solid obtained was purified by column
chromatography using as eluent EtOAc/hexane 3:7. The desire
product was obtained as a yellow solid (8.5 g, 0.010 mol, 35%).
δH (d4-CH3OH): 8.76 (1H, s, H2), 8.67 (1H, s, H8), 8.06-7.86
(10H, m, benzoyl), 7.63-7.40 (10H, m, benzoyl), 6.88 (1H, d, H3′,
J ) 3.0 Hz), 6.60-6.59 (2H, m, H2′, H1′-adenosine), 3.93 (1H,
m, H5′).
Synthesis of 4′-Azidoadenosine (1). To a solution of N,N-
dibenzoyl-2′,3′-di-O-dibenzoyl-4′-azido-5′-deoxy-5′-iodoadenos-
ine (9, 2.20 g, 2.64 mmol) in CH2Cl2 (20 mL, saturated with 1%
of water) was added 85% MCPBA (m-chloroperbenzoic acid) (3.63
g, 15.84 mmol), and the reaction was stirred at 40 °C for 1 h. EtOAc
was added and then washed with a saturated solution of NasS2O3.
The organic layer was dried using MgSO4, and then the solvent
was removed under reduced pressure. The yellow solid was
dissolved in a solution 1 N MeONa in MeOH (6 mL) for 1 h. The
product (200 mg, 0.649 mmol, 24%) was obtained after purification
by column chromatography using as eluent CHCl3/ MeOH 9:1 with
1% NH4OHconcd δH (d4-CH3OH): 8.31 (1H, s, H2), 8.19 (1H,s, H8),
6.25 (1H, d, H1′, J ) 6.4 Hz), 5.00 (1H, t, H2′), 4.53 (1H, d, H3′),
3.77 (1H, d, H5′, J ) 12.2 Hz), 3.60 (1H, d, H5′, J ) 12.2 Hz).
MS (ES) m/e: 331.1 (MNa+, 100%); Accurate mass: C10H12N8O4-
Na required 331.0879, found 331.0886.
Experimental Procedures
Biology. The HCV replicon assay was performed in the stable
replicon cell line 2209-23 derived from Huh-7 cells stably
transfected with a bicistronic HCV replicon (genotype 1b) express-
ing the renilla luciferase reporter gene, as described.8
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 CDCl3 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
(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 because of 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.
Standard Procedure 1: Preparation of 2′,3′-Di-O-cyclopen-
tylidene-4′-azidoadenosine Phosphoramidates. tBuMgCl (2.0 mol
equiv) and 2′,3′-di-O-cyclopentylidene-4′-azidoadenosine (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 subsequently purified by column chromatography.
Standard Procedure 2: Preparation of 4′-Azidoadenosine
Phosphoramidates. The appropriate 2′,3′-di-O-cyclopetylidene-
4′azidoadenosine phosphoramidate was added to a solution 80%
formic acid in water. The reaction was stirred at room temperature
for 4 h. The solvent was removed under reduced pressure, and the
obtained yellow oil was subsequently purified by column chroma-
tography.
Standard Procedure 3: Preparation of 4′-Azidoadenosine
Phosphoramidates. tBuMgCl (2.0 mol equiv) and 4′-azidoadenos-
ine (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 column chromatog-
raphy.
Synthesis of 2′,3′-Di-O-isopropylidene-5′,N3-anhydro-5′-
deoxyadenosine (10). δH (DMSO): 9.41 (1H, s, H2), 9.31 (1H, s,
H8), 8.54 (1H, NH6), 5.87 (1H, s, H1′), 5.07-5.01 (3H, m, H2′,
H5′, H5′), 4.59-4.51 (1H, m, H3′, H4′), 1.45 (3H, s, 1 CH3-
isopropylidene), 1.21 (3H, s, 1 CH3-isopropylidene).
Synthesis of 2′,3′-Di-O-tert-butyldimethylsilyl-5′-deoxy-5′-io-
doadenosine (11). Iodine (14.22 g, 0.056 mol) and triphenylphos-
phine (14.22 g, 0.056 mol) were added to a solution of adenosine
(3, 10 g, 0.037 mol) in pyridine (45 mL). After 2 h tert-
butyldimethylsilyl chloride (24.54 g, 0.149 mol) and imidazole
(20.36 g, 0.299 mol) were added at 0 °C. After 30 min, the reaction
was slowly warmed at room temperature and stirred for 15 h. The
solvent was removed under reduced pressure, and the crude was
purified by column chromatography using as eluent a mixture of
EtOAc/hexane in gradient: 1:9, 8:2, 7:3, 65:35, 50:50, and 6:4.
The pure product (10 g, 0.0165 mol, 44%) was a white solid. δH
(CDCl3): 8.38 (1H, s, H2), 7.92 (1H, s, H8), 5.87 (1H, d, H1′, J )
5.2 Hz), 5.25 (2H, NH26), 5.23 (1H, t, H2′), 4.42 (1H, m, H3′),
4.16 (1H, m, H4′), 3.73 (1H, m, H5′), 3.41 (1H, m, CH5′), 1.00
(9H, s, 3 CH3-tert-butyl), 0.81 (9H, s, 3 CH3-tert-butyl), 0.21 (3H,
Synthesis of 5′-Deoxy-5′-iodoadenosine (5). Iodine (35.43 g,
0.140 mol) and triphenylphosphine (36.80 g, 0.140 mol) were added
to a solution of adenosine (3, 25 g, 0.093 mol) in pyridine (200
mL). After 2 h a saturated solution of NasS2O3 was added; the
solvent was removed under reduced pressure, and the yellow solid
was purified by column chromatography using as eluent CHCl3/
MeOH 9:1. The obtained product (60 g, 0.159 mol, >100%) was
pure enough for the following reaction. δH (d6-(CH3)2SO): 8.82
(2H, s, NH26), 8.59 (1H, s, H2), 8.36 (1H, s, H8), 5.95 (1H, d,
H1′, J ) 5.6 Hz), 4.75 (1H, t, H2′), 4.16 (1H, t, H3′), 4.01 (1H, m,
H4′), 3.60 (1H, m, H5′), 3.46 (1H, m, H5′).
Synthesis of 1-(5-Deoxy-â-D-erythro-pent-4-enofuranosyl)-
adenine (7). tBuOK (47.0 g, 0.418 mol) was added to a solution
of 5′-deoxy-5′-iodoadenosine (5, 35 g, 0.093 mol) in pyridine (200
mL), and the reaction was stirred for 1 h at 80 °C. The solvent was

5468 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Perrone et al.
s, CH3-silyl), 0.16 (3H, s, CH3-silyl), -0.10 (3H, s, CH3-silyl),
-0.27 (3H, s, CH3-silyl).
Synthesis of 6N-Pivaloyl-4′-azidoadenosine (16). To a solution
of 6N-pivaloyl-2′,3′-di-O-tert-butyldimethylsilyl-4′-azidoadenosine
(15, 2.8 g, 4.52 mmol) in THF (30 mL) was added a 1 M solution
of TBAF (tetrabutylammonium fluoride, 9.0 mL, 9.0 mmol), and
the reaction was stirred at room temperature for 1 h. The solvent
was removed under reduced pressure to give a yellow oil that was
purified by column chromatography using as eluent a mixture of
CHCl3/MeOH 85:15. The pure product was obtained as white solid
(1.15 g, 2.93 mmol, 76%). δH (d6-(CH3)2SO): 10.23 (1H, s, NH6),
8.77 (1H, s, H2), 8.74 (1H, s, H8), 6.33 (1H, d, H1′, J ) 6.1 Hz),
5.97 (1H, d, OH2′, J ) 4.7 Hz), 5.82 (1H, d, OH3′, J ) 5.96 Hz),
5.65 (1H, m, OH5′), 4.94 (1H, m, H2′), 4.48 (1H, m, H3′), 3.67
(1H, m, H5′), 3.51 (1H, m, H5′), 1.33 (9H, s, 3 CH3-pivaloyl).
Synthesis of 2′,3′-Di-O-cyclopentylidene-4′-azidoadenosine
(17). 6N-Pivaloyl-2′,3′-di-O-cyclopentylidene-4′-azidoadenosine (1.12
g, 2.44 mmol) was dissolved in a solution of MeOH saturated with
NH3. The solution was sealed at room temperature overnight. The
solvent was removed under reduced pressure, and the crude was
purified by column chromatography using as eluent a mixture of
CHCl3/MeOH in gradient: 100:0, 98:2, and 90:10. The pure product
was obtained as white solid (0.80 g, 2.13 mmol, 87%). δH
(CDCl3): 8.44 (1H, s, H2), 8.00 (1H, s, H8), 6.23 (1H, d, H1′, J )
5.1 Hz), 6.20 (2H, s, NH26), 5.46 (1H, m, H2′), 5.21 (1H, d, H3′,
J ) 5.6 Hz), 3.99 (1H, d, H5′, J ) 12.3 Hz), 3.75 (1H, d, H5′, J
) 12.3 Hz), 2.39-2.22 (2H, m, CH2-cyclopentyl), 2.00-1.85 (6H,
m, CH2-cyclopentyl).
Synthesis of 2′,3′-Di-O-tert-butyldimethylsilyl-1-(5-deoxy-â-
D-erythro-pent-4-enofuranosyl)-adenine (12). tBuOK (0.627 g,
4.95 mmol) was added to a solution of 2′,3′-di-O-tert-butyldim-
ethylsilyl-5′-deoxy-5′-iodoadenosine (11, 1.0 g, 1.65 mmol) in
pyridine (15 mL), and the reaction was stirred for 3 h at room
temperature. The solvent was removed under reduced pressure, and
the solid was purified by column chromatography using as eluent
a mixture of EtOAc/hexane in gradient: 7:3, 65:35, 50:50, and then
8:2 and 7:3. The pure product was obtained as white solid (0.700
g, 1.46 mmol, 89%). δH (CDCl3): 8.22 (1H, s, H2), 7.72 (1H, s,
H8), 5.96 (1H, d, H1′, J ) 6.2 Hz), 5.52 (2H, s, NH26), 4.92 (1H,
m, H2′), 4.44 (1H, d, H3′, J ) 4.2 Hz), 4.37 (1H, d, H5′, J ) 2.3
Hz), 4.13 (1H, d, H5′, J ) 2.3 Hz), 0.80 (9H, s, 3 CH3-tert-butyl),
0.61 (9H, s, 3 CH3-tert-butyl), 0.00 (3H, s, CH3-silyl), -0.22 (3H,
s, CH3-silyl), -0.45 (3H, s, CH3-silyl), -0.27 (3H, s, CH3-silyl).
Synthesis of 6N-Pivaloyl-2′,3′-di-O-tert-butyldimethylsilyl-1-
(5-deoxy-â-D-erythro-pent-4-enofuranosyl)-adenine (13). To a
solution of 2′,3′-di-O-tert-butyldimethylsilyl-1-(5-deoxy-â-D-erythro-
pent-4-enofuranosyl)-adenine (12, 3.6 g, 7.53 mmol) in anhydrous
DCM (145 mL) were added tBuOCl (tert-butanoyl chloride, 1.86
mL, 15.10 mmol) and diisopropylethylamine (2.63 mL, 15.10
mmol), and the reaction was stirred for 3 h at room temperature.
The reaction was washed three times with a saturated solution of
NaHCO3. The organic layer was dried with MgSO4, and the solvent
was removed under reduced pressure. The crude was purified by
column chromatography using as eluent a mixture of EtOAc/hexane
in gradient: 1:10, 1:5, 1:1. The pure compound was obtained as
white solid (4.0 g, 7.13 mmol 95%). δH (CDCl3): 8.62 (1H, s, H2),
8.42 (1H, s, NH6), 7.90 (1H, s, H8), 6.02 (1H, d, H1′, J ) 6.2
Hz), 4.91 (1H, m,), 4.42 (1H, d, H3′, J ) 4.2 Hz), 4.38 (1H, d,
H5′, J ) 2.3 Hz), 4.15 (1H, d, H5′, J ) 2.3 Hz), 1.26 (9H, s, 3
CH3-pivaloyl), 0.80 (9H, s, 3 CH3-tert-butyl), 0.60 (9H, s, 3 CH3-
tert-butyl), 0.00(3H, s, CH3-silyl), -0.22 (3H, s, CH3-silyl), -0.47
(3H, s, CH3-silyl).
Synthesis of 6N-Pivaloyl-2′,3′-di-O-tert-butyldimethylsilyl-
4′,5′-epoxy-adenosine (14). 6N-Pivaloyl-2′,3′-di-O-tert-butyldim-
ethylsilyl-1-(5-deoxy-â-D-erythro-pent-4-enofuranosyl)-adenine (13,
2.0 g, 3.56 mmol) was added to a 0.1 solution of DMDO
(dimethoxydioxirane) in acetone (110 mL). The reaction was stirred
for 15 min at room temperature, and then the solvent was removed
under reduced pressure to give a the pure white product (2.05 g,
3.56 mmol, 100%). δH (CDCl3): 8.70 (1H, s, H2), 8.07 (1H, s,
NH6), 8.07 (1H, s, H8), 6.11 (1H, d, H1′, J ) 5.2 Hz), 4.94 (1H,
m, H2′), 4.12 (1H, d, H3′, J ) 3.8 Hz), 3.10 (1H, d, H5′, J ) 3.5
Hz), 2.94 (1H, d, H5′, J ) 3.5 Hz), 1.31 (9H, s, 3 CH3-pivaloyl),
0.85 (9H, s, 3 CH3-tert-butyl), 0.71 (9H, s, 3 CH3-tert-butyl), 0.03
(3H, s, CH3-silyl), 0.00 (3H, s, CH3-silyl), -0.14 (3H, s, CH3-
silyl), -0.33 (3H, s, CH3-silyl).
Synthesis of 6N-Pivaloyl-2′,3′-di-O-tert-butyldimethylsilyl-4′-
azidoadenosine (15). To a solution of 6N-pivaloyl-2′,3′-di-O-tert-
butyldimethylsilyl-4′,5′epoxy-adenosine (14, 3.09 g, 5.34 mmol)
in anhydrous DCM (200 mL) were added TBSN3 (tert-butylazi-
dosilane, 2.10 mL, 16.02 mmol) and SnCl4 (tin chloride, 3.4 mL,
16.02 mmol) at -78 °C. The reaction was stirred for 30 min, and
then it was slowly warmed at room temperature and stirred for other
30 min. A saturated solution of NaHCO3 was added until the
reaction reached pH 7. The white emulsion formed was filtered on
a celite pad, and the solution was washed three times with a
saturated solution of NaHCO3. The organic layer was dried with
MgSO4. The solvent was removed under reduced pressure, and the
crude was purified by column chromatography using as eluent a
mixture of EtOAc/hexane in gradient: 50:50 and 70:30. The pure
product was obtained as white solid (1.6 g, 52%). δH (CDCl3): 8.58
(1H, s, H2), 8.50 (1H, s, NH6), 7.91 (1H, s, H8), 6.26 (1H, m,
OH5′), 5.84 (1H, d, H1′, J ) 7.9 Hz), 5.08 (1H, m, H2′), 4.22
(1H, d, H3′, J ) 4.4 Hz), 3.64 (1H, m, H5′), 3.31 (1H, m, H5′),
1.24 (9H, s, 3 CH3-pivaloyl), 0.83 (9H, s, 3 CH3-tert-butyl), 0.59
(9H, s, 3 CH3-tert-butyl), 0.03(3H, s, CH3-silyl), 0.00 (3H, s, CH3-
silyl), -0.32 (3H, s, CH3-silyl), -0.88 (3H, s, CH3-silyl).
Synthesis of 4′-Azidoadenosine 5′-O-[r-Naphthyl(benzyloxy-
l-alaninyl) Phosphate (18). Prepared according to Standard
t
Procedure 3, from 4′-azidoadenosine (165.6 mg, 0.537 mmol), -
BuMgCl (1.34 mL 1 M solution of THF, 1.343 mmol), and
R-naphthyl(benzyloxy-l-alaninyl) phosphorochloridate (1.34 mL of
solution 1 M in THF, 1.343 mmol). The crude was purified by
column chromatography, using as eluent CHCl3/MeOH (85:15), and
preparative HPLC. The obtained pure product was a white solid
(20.2 mg, 0.0306 mmol, 6%). δP (d4-CH3OH): 3.71, 3.67; δH (d4-
CH3OH): 8.26 (1H, d, H2), 8.17 (1H, s, H8), 8.17 (1H, s, CH-
naphthyl), 7.88 (1H, d, CH-naphthyl, J ) 7.9 Hz), 7.69 (1H, m,
CH-naphthyl), 7.53-7.43 (4H, m, 3 CH-naphthyl, 1 CH-phenyl),
7.38-7.25 (5H, CH-naphthyl, 4 CH-phenyl), 6.28 (1H, d, H1′, J
) 5.1 Hz), 5.05 (2H, m, CH2-benzyl), 4.95 (1H, m, H2′), 4.70 (1H,
d, H3′, J ) 5.4 Hz), 4.40 (2H, m, H5), 4.05 (1H, m, CHR), 1.28
(3H, m, CH3-alanine). MS (E/I) 698.1852 (MNa), C30H30N9O8NaP
requires 698.1853. Anal. (C30H30N9O8NaP) C, H, N.
Synthesis of 2′,3′-Di-O-cyclopentylidene-4′-azidoadenosine 5′-
O-[Phenyl(benzyloxy-l-alaninyl)] Phosphate (19a). Prepared ac-
cording to Standard Procedure 1, from 2′,3′-di-O-cyclopentylidene-
t
4′-azidoadenosine (17, 150 mg, 0.40 mmol), BuMgCl (1.00 mL,
1 M solution in THF, 1.00 mmol), and phenyl(benzyloxy-L-alaninyl)
phosphorochloridate (1.00 mL of solution 1 M in THF, 1.00 mmol).
The crude was purified by column chromatography, using as eluent
CHCl3/MeOH (95:5). The obtained pure product was a white solid
(200 mg, 0.290 mmol, 72%). δP (CDCl3): 2.28; δH (CDCl3): 8.22
(1H, s, H2), 7.90 (1H, s, H8), 7.27-7.19 (6H, m, 1 CH-phenyl, 5
CH-benzyl), 7.13-7.10 (3H, m, 2 CH-phenyl), 7.97-6.98 (2H, m,
2 CH-phenyl), 6.33 (1H, s, H1′), 6.20 (1H, s, NH26), 5.12-4.98
(4H, CH2-benzyl, H2′, H3′), 4.42 (2H, m, NH-alanine), 4.23 (1H,
m, H5′), 4.13 (1H, m, H5′), 3.98 (1H, m, CHR), 2.16-2.03 (2H,
m, CH2-cyclopentyl), 1.68-1.62 (6H, m, 3 CH2-cyclopentyl), 1.25
(3H, d, CH3-alanine, J ) 6.9 Hz).
Synthesis of 4′-Azidoadenosine 5′-O-[Phenyl(benzyloxy-l-
alaninyl)] Phosphate (19). Prepared according to Standard Pro-
cedure 2, from 2′,3′-di-O-cyclopentylidene-4′-azidoadenosine 5′-
O-[phenyl(benzyloxy-l-alaninyl)] phosphate (19a, 200 mg, 0.290
mmol) and a solution 80% of HCOOH in water (10 mL). The crude
was purified by column chromatography, using as eluent
CHCl3/MeOH (95:5). The obtained pure product was a white solid
(100 mg, 0.159 mmol, 55%). δP (d4-CH3OH): 3.38. 3.21; δH
(d4-CH3OH): 8.30 (1H, s, H2), 8.21 (1H, s, H8), 7.44-7.33 (1H,
m, CH-phenyl), 7.32-7.28 (7H, m, 2 CH-phenyl, 5 CH-benzyl),
7.25-7.15 (2H, m, 2 CH-phenyl), 6.30 (1H, d, H1′, J ) 5.1 Hz),
5.12 (2H, m, CH2-benzyl), 4.96 (1H, m, H2′), 4.68 (1H, d, H3′, J

Submicromolar ActiVe Nucleosides against Hepatitis C Virus
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5469
) 5.4 Hz), 4.32 (1H, m, H5′), 4.23 (1H, m, H5′), 3.92 (1H, m,
CHR), 1.28 (3H, m, CH3-alanine). MS (E/I) 648.1696 (MNa+),
C26H28N9O8NaP requires 648.1696. Anal. (C26H28N9O8P) C, H, N.
Synthesis of 2′,3′-Di-O-cyclopentylidene-4′-azidoadenosine 5′-
O-[r-naphthyl(ethyloxy-l-alaninyl)] Phosphate (20a). Prepared
according to Standard Procedure 1, from 2′,3′-di-O-cyclopentyl-
idene-4′-azidoadenosine (17, 150 mg, 0.40 mmol), tBuMgCl (1.00
mL, 1 M solution in THF, 1.00 mmol), and R-naphthyl(ethyloxy-
l-alaninyl) phosphorochloridate (1.00 mL of solution 1 M in THF,
1.00 mmol). The crude was purified by column chromatography,
using as eluent CHCl3/MeOH (95:5). The obtained pure product
was a white solid (250 mg, 0.369 mmol, 92%). δP (CDCl3): 2.74;
δH (CDCl3): 8.19 (1H, s, H2), 7.91 (1H, m, CH-naphthyl), 7.83
(1H, s, H8), 7.80 (1H, d, CH-naphthyl, J ) 4.85 Hz), 7.52 (1H, d,
CH-naphthyl, J ) 8.2 Hz), 7.44-7.29 (3H, m, CH-naphthyl), 7.24
(1H, m, CH-naphthyl), 7.42-7.21 (1H, m, CH-naphthyl), 6.17 (1H,
d, H1′, J ) 2.3 Hz), 6.03 (1H, s, NH26), 5.06 (1H, m, H2′), 4.96
(1H, d, H3′, j) 6.5 Hz), 4.35, 4.25 (2H, m, NH-alanine, H5′), 4.22
(1H, m, H5′), 4.00-3.91 (2H, m, CHR, CH2-ethyl), 2.18-2.03 (2H,
m, CH2-cyclopentyl), 1.67-1.60 (6H, m, 3 CH2-cyclopentyl), 1.23
(3H, d, CH3-alanine), 1.08 (3H, CH3-ethyl).
Synthesis of 4′-Azidoadenosine 5′-O-[r-naphthyl(ethyloxy-l-
alaninyl)] Phosphate (20). Prepared according to Standard Pro-
cedure 2, from 2′,3′-di-O-cyclopentylidene-4′-azidoadenosine 5′-
O-[R-naphthyl(ethyloxy-l-alaninyl)] phosphate (250 mg, 0.369
mmol), and a solution 80% of HCOOH in water (10 mL). The
crude was purified by column chromatography, using as eluent
CHCl3/MeOH (95:5). The obtained pure product was a white solid
(100 mg, 0.169 mmol, 51%). δP (d4-CH3OH): 3.72, 3.68; δH (d4-
CH3OH): 8.27 (1H, s, H2), 8.13 (1H, s, H8), 8.07 (1H, m, CH-
naphthyl), 7.83 (1H, m, CH-naphthyl), 7.66 (1H, m, CH-naphthyl),
7.52-7.43 (3H, m, 3 CH-naphthyl), 7.38-7.32 (1H, m, CH-
naphthyl), 6.30 (1H, d, H1′-adenosine, J ) 5.1 Hz), 4.97 (1H, m,
H2′), 4.75 (1H, d, H3′, J ) 5.5 Hz), 4.43 (1H, m, H5′), 4.36 (1H,
m, H5′), 4.06-3.89 (2H, m, CH2-ethyl), 3.73 (1H, m, CHR), 1.27
(3H, m, CH3-alanine), 1.14 (3H, CH3-ethyl). MS (E/I) 636.1682
(MNa+), C25H28N9O8NaP requires 636.1696. Anal. (C25H28N9O8P)
C, H, N.
tylidene-4′-azidoadenosine (17, 150 mg, 0.40 mmol), tBuMgCl (1.00
mL, 1 M solution in THF, 1.00 mmol), and R-naphthyl(tert-
butyloxy-l-alaninyl) phosphorochloridate (1.00 mL of solution 1
M in THF, 1.00 mmol). The crude was purified by column
chromatography, using as eluent CHCl3/MeOH (95:5). The obtained
pure product was a white solid (220 mg, 0.308 mmol, 77%). δP
(CDCl3): 2.96; δH (CDCl3): 8.17 (1H, s, H2-adenosine), 8.05 (1H,
m, CH-naphthyl), 7.82 (1H, s, H8), 7.72-7.65 (2H, m, CH-
naphthyl), 7.54-7.44 (2H, 2 CH-naphthyl), 7.42-7.21 (2H, m, 2
CH-naphthyl), 6.47 (1H, s, NH26), 6.14 (1H, d, H1′), 4.97 (2H, m,
H2′, H3′), 4.61 (1H, m, NH-alanine), 4.30 (1H, m, H5′), 4.19 (1H,
m, H5′), 3.94 (1H, m, CHR), 2.13-2.01 (2H, m, CH2-cyclopentyl),
1.65-1.56 (6H, m, 3 CH2-cyclopentyl), 1.36 (9H, 3 CH3-tert-butyl),
1.23 (3H, m, CH3-alanine).
Synthesis of 4′-Azidoadenosine 5′-O-[r-naphthyl(tert-buty-
loxy-l-alaninyl)] Phosphate (22). Prepared according to Standard
Procedure 2, from 2′,3′-di-O-cyclopentylidene-4′-azidoadenosine 5′-
O-[R-naphthyl(tert-butyloxy-l-alaninyl)] phosphate (220 mg, 0.308
mmol), and a solution 80% of HCOOH in water (10 mL). The
crude was purified by column chromatography, using as eluent
CHCl3/MeOH (95:5). The obtained pure product was a white solid
(100 mg, 0.169 mmol, 51%). δP (d4-CH3OH): 3.85, 3.73; δH (d4-
CH3OH): 8.25 (1H, s, H2), 8.21 (1H, s, H8), 8.10 (1H, m, CH-
naphthyl), 7.84 (1H, m, CH-naphthyl), 7.64 (1H, m, CH-naphthyl),
7.51-7.48 (2H, m, 2 CH-naphthyl), 7.45-7.32 (2H, m, 2 CH-
naphthyl), 6.29 (1H, d, H1′, J ) 5.1 Hz), 4.96 (1H, m, H2′), 4.72
(1H, d, H3′, J ) 5.54 Hz), 4.42 (1H, m, H5′), 4.36 (1H, m, H5′),
3.85 (1H, m, CHR), 1.35 (9H, s, 3 CH3-tert-butyl), 1.25 (3H, d,
CH3-alanine, J ) 6.3 Hz). MS (E/I) 664.2015 (MNa+), C27H32N9O8-
NaP requires 664.2009. Anal. (C27H32N9O8P) C, H, N.
Synthesis of 2′,3′-Di-O-cyclopentylidene-4′-azidoadenosine 5′-
O-[phenyl(tert-butyloxy-l-alaninyl)] Phosphate (23a). Prepared
according to Standard Procedure 1, from 2′,3′-di-O-cyclopentyl-
idene-4′-azidoadenosine (17, 150 mg, 0.40 mmol), tBuMgCl (1.00
mL, 1 M solution in THF, 1.00 mmol), and phenyl(tert-butyloxy-
L-alaninyl) phosphorochloridate (1.00 mL of solution 1 M in THF,
1.00 mmol). The crude was purified by column chromatography,
using as eluent CHCl3/MeOH (95:5). The obtained pure product
was a white solid (210 mg, 0.356 mmol, 71%). δP (CDCl3): 2.43;
δH (CDCl3): 8.28 (1H, s, H2), 7.97 (1H, s, H8), 7.27-7.00 (5H,
m, CH-phenyl), 6.55 (1H, s, NH26), 5.77 (1H, m, H1′), 5.17 (1H,
m, H2′), 5.16 (1H, d, H3′, J ) 2.1 Hz), 3.98 (1H, m, NH-alanine),
4.29 (1H, m, H5′), 4.20 (1H, m, H5′), 3.88 (1H, m, CHR), 2.21-
2.09 (2H, m, CH2-cyclopentyl), 1.76-1.63 (6H, m, 3 CH2-
cyclopentyl), 1.37 (9H, 3 CH3-tert-butyl), 1.26 (3H, d, CH3-alanine,
J ) 7.0 Hz).
Synthesis of 4′-Azidoadenosine 5′-O-[Phenyl(tert-butyloxy-l-
alaninyl)] Phosphate (23). Prepared according to Standard Pro-
cedure 2, from 2′,3′-di-O-cyclopentylidene-4′-azidoadenosine 5′-
O-[phenyl(tert-butyloxy-l-alaninyl)] phosphate (210 mg, 0.356
mmol), and a solution 80% of HCOOH in water (10 mL). The
crude was purified by column chromatography, using as eluent
CHCl3/MeOH (95:5). The obtained pure product was a white solid
(100 mg, 0.169 mmol, 47%). δP (d4-CH3OH): 3.42; δH (d4-CH3-
OH): 8.29 (1H, s, H2), 8.21 (1H, s, H8), 7.32-7.29 (2H, m, 2
CH-phenyl), 7.19-7.14 (3H, m, 3 CH-phenyl), 6.33 (1H, d, H1′,
J ) 5.1 Hz), 4.97 (1H, m, H2′), 4.72 (1H, d, H3′, J ) 5.45 Hz),
4.36 (1H, m, H5′), 4.27 (1H, m, H5′), 3.77 (1H, m, CHR), 1.42
(9H, s, 3 CH3-tert-butyl), 1.25 (3H, d, CH3-alanine). MS (E/I)
614.1839 (MNa+), C23H30N9O8NaP requires 614.1853. Anal.
(C29H30N9O8P) C, H, N.
Synthesis of 2′,3′-Di-O-cyclopentylidene-4′-azidoadenosine 5′-
O-[phenyl(ethyloxy-l-alaninyl)] Phosphate (21a). Prepared ac-
cording to Standard Procedure 1, from 2′,3′-di-O-cyclopentylidene-
t
4′-azidoadenosine (17, 150 mg, 0.40 mmol), BuMgCl (1.00 mL,
1 M solution in THF, 1.00 mmol), and phenyl(ethyloxy-L-alaninyl)
phosphorochloridate (1.00 mL of solution 1 M in THF, 1.00 mmol).
The crude was purified by column chromatography, using as eluent
CHCl3/MeOH (95:5). The obtained pure product was a white solid
(210 mg, 0.335 mmol, 84%). δP (CDCl3): 2.34; δH (CDCl3): 8.31
(1H, s, H2), 7.97 (1H, s, H8), 7.23-7.22 (2H, m, 2 CH-phenyl),
7.12-7.09 (3H, m, 3 CH-phenyl), 6.31 (1H, d, H1′, J ) 2.2 Hz),
6.23 (1H, s, NH26), 5.24 (1H, m, H2′), 5.14 (1H, d, H3′, J ) 6.3
Hz), 4.35-4.30 (2H, m, NH-alanine, H5′), 4.28-4.22 (1H, m, H5′),
4.15-4.11 (2H, m, CH2-ethyl), 3.99 (1H, m, CHR), 2.26-2.14 (2H,
m, CH2-cyclopentyl), 1.76-1.69 (6H, m, 3 CH2-cyclopentyl), 1.33
(3H, CH3-ethyl), 1.23 (3H, d, CH3-alanine).
Synthesis of 4′-Azidoadenosine 5′-O-[Phenyl(ethyloxy-l-alani-
nyl)] Phosphate (21). Prepared according to Standard Procedure
2, from 2′,3′-di-O-cyclopentylidene-4′-azidoadenosine 5′-O-[phenyl-
(ethyloxy-l-alaninyl)] phosphate (210 mg, 0.335 mmol) and a
solution 80% of HCOOH in water (10 mL). The crude was purified
by column chromatography, using as eluent CHCl3/MeOH (95:5).
The obtained pure product was a white solid (100 mg, 0.159 mmol,
48%). δP (d4-CH3OH): 3.44. 3.28; δH (d4-CH3OH): 8.27 (1H, s,
H2), 8.18 (1H, s, H8), 7.36-7.28 (2H, m, 2 CH-phenyl), 7.16-
7.11 (3H, m, 3 CH-phenyl), 6.38 (1H, d, H1′, J ) 5.0 Hz), 4.96
(1H, m, H2′), 4.25 (1H, m, H5′), 4.15 (2H, m, CH2-ethyl), 3.76
(1H, m, CHR), 1.27 (3H, d, CH3-ethyl, J ) 7.2 Hz), 1.22 (3H, m,
CH3-alanine). MS (E/I) 586.1544 (MNa+), C21H26N9O8NaP requires
586.1540. Anal. (C21H26N9O8P) C, H, N.
Acknowledgment. We thank Helen Murphy for excellent
secretarial assistance and the European Union (Descartes prize)
for support.
Supporting Information Available: Microanalytical data of
target compounds and 13C NMR data of intermediates and products.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Synthesis of 2′,3′-Di-O-cyclopentylidene-4′-azidoadenosine 5′-
O-[r-Naphthyl(tert-butyloxy-l-alaninyl)] Phosphate (22a). Pre-
pared according to Standard Procedure 1, from 2′,3′-O-cyclopen-

5470 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Perrone et al.
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