The design, synthesis, and antiviral activity of 4′-azidocytidine analogues against hepatitis C virus replication: The discovery of 4′-azidoarabinocytidine

Article abstract of DOI:10.1021/jm800981y

4′-Azidocytidine 3 (R1479) has been previously discovered as a potent and selective inhibitor of HCV replication targeting the RNA-dependent RNA polymerase of hepatitis C virus, NS5B. Here we describe the synthesis and biological evaluation of several der

Full text of DOI:10.1021/jm800981y

J. Med. Chem. 2009, 52, 219–223
219
The Design, Synthesis, and Antiviral Activity of 4′-Azidocytidine Analogues against Hepatitis C
Virus Replication: The Discovery of 4′-Azidoarabinocytidine
David B. Smith,† Genadiy Kalayanov,‡ Christian Sund,‡ Anna Winqvist,‡ Pedro Pinho,‡ Tatiana Maltseva,‡
Veronique Morisson,‡ Vincent Leveque,† Sonal Rajyaguru,† Sophie Le Pogam,† Isabel Najera,† Kurt Benkestock,‡
Xiao-Xiong Zhou,‡ Hans Maag,† Nick Cammack,† Joseph A. Martin,† Steven Swallow,† Nils Gunnar Johansson,‡
Klaus Klumpp,† and Mark Smith*,†
Roche Palo Alto LLC, 3431 HillView AVenue, Palo Alto, California 94304, MediVir AB, Department of Medicinal Chemistry, Lunastigen 7,
SE-141 44 Huddinge, Sweden
ReceiVed August 1, 2008
4′-Azidocytidine 3 (R1479) has been previously discovered as a potent and selective inhibitor of HCV
replication targeting the RNA-dependent RNA polymerase of hepatitis C virus, NS5B. Here we describe
the synthesis and biological evaluation of several derivatives of 4′-azidocytidine by varying the substituents
at the ribose 2′ and 3′-positions. The most potent compound in this series is 4′-azidoarabinocytidine with an
IC50 of 0.17 µM in the genotype 1b subgenomic replicon system. The structure-activity relationships within
this series of nucleoside analogues are discussed.
Introduction
Hepatitis C virus (HCV) is a causative agent of chronic liver
disease, estimated to affect over 170 million people worldwide.1
HCV establishes a chronic infection in the majority of affected
people, which is associated with reduced liver function, fibrosis,
and hepatocellular carcinoma. Currently, the standard treatment
for HCV infection is pegylated interferon R in combination with
the nonspecific nucleoside analogue ribavirin. This treatment
regimen effects a sustained viral response in approximately
40-60% of the genotype-1 (GT-1) population.2 Current HCV
treatment for GT-1 infected patients requires 48 weeks and is
associated with undesirable side effects. Therefore, a significant
unmet clinical need remains and warrants the development of
new HCV specific treatment options.
Viral-encoded polymerases have proven to be excellent
molecular targets for human chemotherapeutic intervention in
several viral mediated diseases, including human immunode-
ficiency virus, herpes simplex virus, and hepatitis B virus.
Recently, a number of nucleoside analogues have been reported
to inhibit HCV replication.3-6 Among these, 2′-ꢀ-methylcytidine
1,3 2′-R-fluoro-ꢀ-methylcytidine 24 and 4′-azidocytidine 35,6
have been evaluated in clinical studies for the treatment of
chronic HCV infection (Figure 1). Previously, we reported the
design and synthesis of 4′-substituted ribonucleosides as po-
tential antiviral inhibitors including a number of modifications
at the 4′ position.5 The 4′-azido group was found to confer high
antiviral potency and selectivity relative to cellular polymerases.
The triphosphate of 3 is a substrate of HCV polymerase and
inhibits RNA chain extension after incorporation as a non-
obligatory chain terminator of RNA synthesis mediated by the
NS5B polymerase.6 A prodrug of 4′-azidocytidine, 3, is currently
in phase II clinical development for the treatment of HCV
infection.7
Figure 1. Structures of 2′-ꢀ-methylcytidine, 2′-R-fluoro-ꢀ-methylcy-
tidine, and 4′-azidocytidine.
moiety causing misalignment of the 3′-hydroxyl group in the
polymerase active site.6,8 The 4′-azido moiety conferred the
highest anti-HCV activity among a series of 4′-substituted
nucleosides. The lower potency of other compounds in this series
was partly related to poor phosphorylation by cellular kinases
or the reduced efficiency of modified nucleotides as substrates
of HCV NS5B polymerase.5,8-10 The goal toward more potent
nucleoside inhibitors of viral replication was, therefore, to
increase substrate efficiencies of nucleoside analogues for human
kinases and those of the corresponding triphosphates for viral
polymerases.
This paper describes a series of 4′-azidocytidine analogues
with modifications at the 2′- and 3′-carbon and their effects on
HCV replication in vitro.
Chemistry
The synthesis of 4′-azidoxylocytidine was performed as
outlined in Scheme 1. Thus, the reaction of 3, prepared
according to the literature,11 with benzoyl chloride, followed
by partial deprotection, resulted in a mixture of derivatives.
Chromatographic separation of the mixture gave 8 in a yield of
26%. No attempts were made to optimize the deprotection step.
Treatment of 8 with trifluromethansulfonic anhydride, in the
presence of pyridine, followed by water, provided a mixture of
4′-azido nucleosides with different numbers of benzoyl groups.
The ability of 4′-azido ribonucleosides to prevent chain
elongation may be related to reduced nucleophilicity of the 3′-
hydroxyl group or to a conformational change of the furanose
1
As follows from H NMR analysis of the mixture, one of the
constituents was 1-(4′-azido-2′,3′-O-dibenzoyl-ꢀ-D-xylofurano-
syl)-4-N-benzoylcytosine. Deprotection of the whole mixture
with ammonia in methanol furnished the desired 4′-azidoxylo-
cytidine 9 as a single isomer in 63% yield. It can be concluded
from this data that the inversion of configuration at the
3′-position is due to neighboring group participation of the 5′-
* To whom correspondence should be addressed. Phone: +1 650 855
5076. Fax: +1 650 852 1132. E-mail: Mark.Smith@Roche.com.
†
Roche Palo Alto LLC.
Medivir AB.
‡
10.1021/jm800981y CCC: $40.75
2009 American Chemical Society
Published on Web 12/05/2008

220 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Brief Articles
Scheme 1a
a (a) I2, PPh3, imidazole, THF; (b) NaOMe, MeOH; (c) [Bn(Et)3]N3:I2, NMO; (d) BzCl, DMAP, NMP, THF; (e) m-CPBA, m-CBA, (NH4)HSO4, CH2Cl2;
(f) NH3, MeOH; (g) triazole, POCl3, Et3N, MeCN; (h) NH3, MeOH; (i) BzCl, pyridine; (j) MeONa, THF; (k) Tf2O pyridine, CH2Cl2 then H2O; (l) NH3,
MeOH.
Scheme 2a
Scheme 3a
a (a) (PhO)2CO, NaHCO3, DMF, ∆; (b) NaOH, EtOH; (c) Ac2O, pyridine;
(d) triazole, POCl3, Et3N, MeCN; (e) NH4OH.
O-benzoyl group with the activated 3′-triflate intermediate via
an acyloxonium ion.
a (a) I2, PPh3, imidazole, THF; (b) NaOMe, MeOH; (c) [Bn(Et)3N]Cl,
NaN3:I2, CH3CN, NMO, THF; (d) BzCl, DMAP, NMP, THF; (e)
(Bu4N)HSO4, K2HPO4, m-CPBA, m-CBA, CH2Cl2; (f) NH3, MeOH; (g)
Ac2O, DMAP; (h) 1H-tetrazole, Cl2P(O)OC6H4Cl, pyridine; (i) NH3,
dioxane; (j) NH3, MeOH.
4′-Azidoarabinocytidine 13 was prepared as outlined in
Scheme 2. Herein, 2,2′-anhydro-4′-azido-1-(ꢀ-arabinofurano-
syl)uracil 11 was prepared in 96% yield from 4′-azidouridine
10 according to Reese’s adaptation of Moffat’s method.12
Treatment of 11 with 1 M NaOH in ethanol gave the 4′-
azidoarabinouridine 12 in 93% yield. Standard conversion from
uridine to cytidine via the triazole method, developed by Divakar
and Reese, yielded 4′-azidoarabinocytidine 13.13
Scheme 4a
4′-Azido-3′-deoxycytidine 21 was prepared according to
Scheme 3. Herein, the olefin 16 was synthesized according to
a method originally developed by Verheyden and Moffat.14
Treatment of 16 with iodine and benzyltriethylammonium azide,
prepared from the corresponding chloride and sodium azide,
followed by benzoylation, provided 18 in 40% yield as a 1:1
mixture of isomers. Oxidative nucleophilic substitution of 5′-
iodine with m-chlorobenzoic acid/m-chloroperoxybenzoic acid
followed by deprotection and acetylation gave a 1:1 mixture of
the 2′,5′-diacetates. At this stage, the isomers were separated
by silica gel chromatography to give the diacetate 20 in moderate
yield. Compound 20 was readily converted to 4′-azido-3′-
deoxycytidine 21 by treatment with 1H-tetrazole and 4-chlo-
rophenyldichlorophosphate followed by ammonia.
a (a) TIPDSCl, pyridine; (b) BzCl, pyridine; (c) Dess-Martin, CH2Cl2;
(d) AlMe3, THF; (e) HCl, MeOH; (f) NH3, MeOH.
a 1:1 mixture of 2′-methyl isomers 24 and 25, which were
separated by preparative HPLC.
Finally, the synthesis of 4′-azido-2′-ꢀ-methylcytidine 24 and
4′-azido-2′-R-methylarabinocytidine 25 is outlined in Scheme
4. Thus, protection of 3 with TIPDSCl and BzCl provided 22
in 25% overall yield. Oxidation with Dess-Martin periodinane
gave 2′-ketone 23 in 83% yield. The ketone 23 was reacted
with trimethylaluminum followed by total deprotection to give
Results and Discussion
Compounds were characterized as inhibitors of HCV replica-
tion in the subgenomic replicon assay system using the 2209-
23 cell line as described previously.5,6,8 It was also determined
if any of the compounds inhibited cell proliferation, an indication

Brief Articles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 221
kinase 1 (UCK-1), respectively.8 The combination of the
structural features of 2′-hydroxy-2′-methyl and 4′-azido moieties
on a single molecule 24 may have therefore resulted in the
exclusion at both the dCK and UCK-1 active sites.
Table 1. IC50 and CC50 Determination in the HCV Replicon System
GT-1b stable
cytotoxicity
proliferation
replicon (2209-23) (2209-23) CC50 (3H-Thy incorporation)
compd
IC50 (µM)
(µM)
IC50 (µM)
3
9
1.28
27.8
<2000
<100
<1000
nd
nd
nd
<100
nd
<100
nd
nd
nd
0.029
Conclusions
13
21
24
25
AraC
0.171
<100
<100
<100
0.020
The data presented here shows that 4′-azido-arabinocytidine 13
is an unexpectedly potent and selective inhibitor of HCV replication
despite the inversion of a 2′-hydroxyl group, whereas 3′ modified
derivatives of 4′-azidocytidine lose substantial potency. Pharma-
cologic studies have recently been published with 13 and further
preclinical investigations are currently underway.8
<100
of potentially reduced selectivity against human polymerases.
For this, the effect of compounds on the incorporation of tritiated
thymidine into cellular DNA was measured using the SPA [3H]-
thymidine incorporation assay system from Amersham Bio-
sciences.5,6,8 Cytotoxicity was determined using the WST cell
viability assay as described previously.5,6,8 Results are sum-
marized in Table 1.
Experimental Section
All starting materials, solvents, and reagents were reagent grade
and used as purchased. Chromatography solvents were HPLC grade
and used without further purification. Flash column chromatography
was performed on Merck KGaA Silica gel 60 F254 (particle size
0.040-0.063 mm) unless otherwise stated. Reactions were moni-
tored by thin layer chromatography (TLC) analysis using Merck
KGaA precoated glass plates (silica gel 60 F254).
None of the compounds tested showed significant cytotoxic or
cytostatic properties with the exception of the control compound
AraC (arabinocytidine or cytarabine), which was a potent inhibitor
of cell proliferation inhibiting tritiated thymidine incorporation with
an IC50 value of 29 nM. AraC is a known cytostatic agent and is
used for the treatment of acute myelogenous lymphoma. AraC also
interfered with HCV RNA replication in 2209-23 cells, but the
maintenance of the replicon RNA in these cells is dependent on
cell proliferation. Therefore, cytostatic agents nonspecifically reduce
HCV RNA levels in this cell line. Consistent with this, the apparent
antiviral IC50 value and the IC50 value of inhibition of cell
proliferation were similar and the antiviral potency AraC can not
be assessed in this system. The introduction of a 4′-azido group
converted AraC to 13, the most potent compound in this series.
13 inhibited HCV replication with an IC50 value of 171 nM in
2209-23 cells but did not inhibit cell proliferation. Therefore, the
4′-azido moiety conferred more than 3000-fold increased selectivity
to 13 as compared to AraC. Furthermore, the high potency of 13
as an inhibitor of HCV replication was unexpected as it lacks a
2′-R-hydroxy group, which was thought to be necessary for
recognition by RNA polymerases.8 13 was shown to be a substrate
of HCV polymerase causing efficient chain termination after
incorporation despite the presence of a 3′-hydroxyl group.8 This
was similar to results obtained with 4′-azidocytidine, suggesting
that the 4′-azido group may generally lead to misalignment of the
3′-position in the HCV polymerase active site.6 It is unknown if
chain termination will occur under all cellular and/or in vitro
conditions or if mutations within HCV polymerase may affect chain
termination efficiency of nucleoside analogues. 3′-Deoxy-CTP has
previously been described as an efficient substrate of HCV
polymerase and obligatory chain terminator. Therefore, we syn-
thesized 21, 4′-azido-3′-deoxycytidine, to evaluate the possibility
to derive an obligatory chain terminator from 4′-azidocytidine 3.
Interestingly, 21 was completely inactive in the HCV replicon
system (Table 1). Similar to what is known for 3′-deoxycytidine,
21 may therefore not be phosphorylated by cellular kinases to the
5′-triphosphate derivative. In contrast to 21, 9 (4′-azidoxylocytidine)
did show measurable antiviral potency in the replicon system (IC50
) 27.8 µM). However, this potency was more than 20-fold lower
than that of 3, consistent with an important role of the 3′-hydroxyl
group for the formation of active nucleoside triphosphate analogues.
24 was generated as a hybrid of 1 and 3, both compounds
that have progressed into clinical development. Surprisingly,
24 was found to be inactive in the replicon system, most likely
due to lack of phosphorylation to the 5-triphosphate. As reported
previously, 1 and 3 are phosphorylated by different cellular
enzymes, deoxycytidine kinase (dCK) and uridine-cytidine
1-(4′-Azido-2′,5′-O-dibenzoyl-ꢀ-D-ribofuranosyl)-4-N-benzoyl-
cytosine (8). To a solution of azidocytidine 311 (0.426 g, 1.5 mmol)
in pyridine (5 mL) was added benzoyl chloride (0.68 g, 4.8 mmol)
at 0 °C. The reaction mixture was stirred at ambient temperature
for 2 h, quenched with water (1 mL), and evaporated. The residue
was dissolved in ethyl acetate washed with water, dilute HCl,
saturated aqueous NaHCO3 solution, and brine. The organic layer
was dried (MgSO4) and evaporated to dryness under reduced
pressure. The residue was coevaporated several times with anhy-
drous THF. The intermediate tetrabenzoyl derivative was dissolved
in anhydrous THF (50 mL) and treated with sodium methoxide
(0.162 g, 3 mmol). The reaction mixture was stirred at ambient
temperature for 1 h, concentrated, and diluted with ethyl acetate.
The organic phase was washed with water, brine, and evaporated
to dryness under reduced pressure. Chromatography (0-10%
methanol in CH2Cl2) yielded 8 (0.232 g, 26%). 1H NMR (methanol-
d4): δ 8.12 (d, J ) 7.5 Hz, 1H), 8.09-8.04 (m, 2H), 7.98-7.91
(m, 4H), 7.67-7.60 (m, 2H), 7.56-7.48 (m, 5H), 7.45 (d, J ) 7.5
Hz, 1H), 7.39-7.30 (m, 4H), 6.10 (d, J ) 2.8 Hz, 1H), 4.67-4.62
(m, 2H), 4.58-4.48 (m, 2H).
1-(4′-Azido-ꢀ-D-xylofuranosyl)cytosine (9). To a solution of 8
(0.060 g, 0.1 mmol) in anhydrous CH2Cl2 (1.7 mL) and pyridine
(0.12 mL) was added triflic anhydride (0.038 mL, 0.3 mmol) at
-30 °C. The reaction mixture was stirred at -30 °C for 0.5 h then
warmed up to room temperature and stirred for 1.5 h. Water was
added and the reaction mixture was stirred at room temperature
overnight. Solvents were evaporated under reduced pressure and
the residue was treated with saturated ammonia in methanol (2 mL)
for 14 h. The organic solvent was evaporated to dryness under
reduced pressure. Chromatography (8% methanol in CHCl3) gave
1
9 (0.018 g, 63%) as white solid. H NMR (DMSO-d6): δ 7.73 (d,
J ) 7.5 Hz, 1H, 6-H), 7.31; 7.21 (2s, 2H, 4-NH2), 6.10 (d, J ) 7.3
Hz, 1 H, 1′R-H), 5.77 (d, J ) 7.5 Hz, 1H, 5-H), 5.63 (d, J ) 6.1
Hz, 1H, 3′ꢀ-OH), 5.61 (t, J ) 6.1 Hz, 1H, 5′-OH), 5.51 (d, J ) 6.2
Hz, 1H, 2′R-OH), 4.19-4.15 (m, 2H, 2′ꢀ-H, 3′R-H), 3.52-3.39 (m,
2H, 5′-H, 5”-H). HRMS (ES+) calcd for C9H13N6O5 [M + H]+
285.0947; found 285.0938.
2,2′-Anhydro-1-(4′-azido-ꢀ-D-arabinofuranosyl)uracil (11). A
mixture containing 10 (1.00 g, 3.50 mmol), diphenyl carbonate (0.83
g, 3.85 mmol), sodium hydrogen carbonate (0.030 g, 0.35 mmol),
and N,N-dimethylformamide (1 mL) was heated at 100 °C for 14 h.
The reaction mixture was cooled to room temperature and diluted
with CH3CN (5 mL). The resulting precipitate was collected by
1
filtration to provide 11 (0.80 g, 85%) as off-white solid. H NMR
(DMSO-d6): δ 7.89 (d, J ) 7.5 Hz, 1H), 6.56 (br. d, 1H), 6.40 (d,
J ) 6.1 Hz, 1H), 5.86 (d, J ) 7.5 Hz, 1H), 5.48 (br. t, 1H), 5.30
(dd, J ) 2.4 and 6.0 Hz, 1H), 4.44 (br. s, 1H), 3.41 (ddd, J ) 5.2,
12.1 and 26.6 Hz, 2H).

222 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Brief Articles
1-(4′-Azido-ꢀ-D-arabinofuranosyl)uracil (12). To a flask con-
taining 7 (0.80 g, 3.0 mmol) and EtOH (10 mL) was added 1 M
NaOH solution (3 mL). The resulting solution was stirred at room
temperature for 3 h and then acidified with Amberlyst 15 ion-
exchange resin, filtered, and evaporated to dryness under reduced
mmol) was added and the resulting solution was cooled on an
ice-water bath. A solution of iodine (1.81 g; 7.14 mmol) in
anhydrous THF (39 mL) was added dropwise over 60 min. The
reaction mixture was stirred at 0-5 °C for another 2 h to produce
1-(4′-azido-5′-iodo-3′-deoxy-ꢀ-D-ribofurnaosyl)uracil (17) as a
mixture of diastereoisomers. N-Acetyl-L-cysteine (0.069 g; 0.035
mmol) was added to destroy excess azide and the solution was
stirred until bubbling subsided. 4-Methylmorpholine (1.2 mL, 10.5
mmol) and DMAP (0.05 g, 0.42 mmol) were added, followed by
a dropwise addition of benzoyl chloride (0.55 mL, 4.62 mmol).
The reaction mixture was stirred at 0-5 °C for 1 h and quenched
with water to destroy excess of benzoyl chloride and sodium sulfite
to destroy residual iodine. The mixture was extracted with ethyl
acetate. The organic phase was then washed with 5% citric acid,
brine, and evaporated to dryness under reduced pressure. Chroma-
tography (0-2% methanol in CH2Cl2) gave 18 as a 1:1 inseparable
mixture of diastereoisomers that were taken through to the next
step without further purification (0.811 g, 40%).
1-(4′-Azido-3′-deoxy-ꢀ-D-ribofuranosyl)uracil (19). A solution
of 18 (0.81 g, 1.67 mmol) in CH2Cl2 (75 mL) was combined with
a mixture of tetrabutylammonium hydrogensulfate (0.397 g; 1.17
mmol) and m-chlorobenzoic acid (0.173 g, 1.1 mmol) in 1.75 M
aqueous K2HPO4 (26 mL). The two-phase system was stirred
vigorously at room temperature and two portions of a commercially
available reagent mixture containing 55% m-chloroperbenzoic acid,
10% m-chlorobenzoic acid, and 35% H2O (2 × 1.72 g, 2 × 1.58
equiv) was added with a 1.5 h interval. The mixture was stirred
vigorously at room temperature for a further 18 h. A solution of
Na2S2O3 ·5H2O (17 g) in saturated aqueous NaHCO3 (240 mL) was
added and the mixture was then vigorously stirred at room
temperature for 30 min. The organic layer was separated and the
aqueous layer washed with CH2Cl2. The combined organic layers
were washed with saturated aqueous NaHCO3 solution, dried
(Na2SO4), filtered, and evaporated to dryness under reduced
pressure. The crude product was passed through a silica column
eluting with 2-5% methanol in CH2Cl2 and the combined fractions
evaporated to dryness. The residue was redissolved in saturated
ammonia in methanol (10 mL) and stirred for 24 h in order to ensure
deprotection of the alcohols. The crude reaction mixture was
evaporated to dryness under reduced pressure. Chromatography
(10% methanol in CH2Cl2) gave 19 (0.125 g, 33%) as a 1:1
inseparable mixture of isomers that was taken as such into the next
step. MS (ES+) [M + H]+ m/z 270.
1
pressure to give 12 (0.83 g, 97%) as an off-white foam. H NMR
(methanol-d4): δ 7.74 (d, J ) 8.1 Hz, 1H), 6.36 (d, J ) 5.8 Hz,
1H), 5.67 (d, J ) 8.1 Hz, 1H) 4.39 (t, J ) 5.9 Hz, 1H), 4.18 (d, J
) 6 Hz, 1H), 3.82 (s, 2H).
1-(4′-Azido-ꢀ-D-arabinofuranosyl)cytosine (13). DMAP (cata-
lytic) was added to a stirred solution of 12 (0.80 g, 2.84 mmol) in
acetic anhydride (10 mL) and pyridine (10 mL). The reaction
mixture was left to stir under nitrogen at room temperature for 20 h.
The organic mixture was evaporated to dryness under reduced
pressure and the residue was dissolved in CH3CN (30 mL). To
this was added triazole (3.09 g, 44.87 mmol) and triethylamine (7.81
mL, 56.09 mmol). The contents of the flask were placed under
nitrogen and cooled to ∼5 °C (ice bath). POCl3 (1.04 mL, 11.21
mmol) was added to the flask, and the resulting mixture was left
to stir at room temperature for 24 h. The solvents were evaporated
to dryness under reduced pressure, dissolved in ethyl acetate (∼100
mL), and washed with saturated aqueous NaHCO3 solution, dried
(MgSO4), and evaporated to dryness under reduced pressure to give
the crude triazole intermediate. The residue was dissolved in dioxane
(5 mL) and treated with saturated NH4OH (2 mL). After stirring
for 12 h the reaction mixture was evaporated to dryness under
reduced pressure. Chromatography (10-30% CH3CN in H2O;
reverse phase C-18 column) provided 13 (0.21 g, 26%) as a white
1
solid; mp: 188.0-189.0 °C. H NMR (DMSO-d6): δ 7.47 (d, J )
7.4 Hz, 1H), 7.18-7.05 (br d, 2H), 6.31 (d, J ) 5.7 Hz, 1H), 5.90
(d, J ) 5.7 Hz, 1H), 5.72-5.67 (m, 1H), 5.48 (d, J ) 6 Hz, 1H),
4.19 (dd, J ) 5.4 and 10.8 Hz, 1H), 4.05 (t, J ) 5.6 Hz, 1H), 3.65
(d, J ) 6.1 Hz, 2H). HRMS (ES+) calcd for C9H12N6O5 [M + H]+
285.0941; found 285.0939.
1-(3′,5′-Dideoxy-5′-iodo-ꢀ-D-ribofuranosyl)uracil (15). A mix-
ture of 14 (2.04 g, 8.96 mmol), imidazole (732 mg, 10.7 mmol),
and triphenylphosphine (2.82 g, 10.7 mmol) in anhydrous THF (30
mL) was cooled on an ice-water bath, and a solution of iodine
(2.50 g, 9.85 mmol) in anhydrous THF (10 mL) was added dropwise
over 10 min. The reaction mixture was stirred at 0-5 °C for another
10 min. The ice-water bath was removed and the reaction mixture
was stirred at room temperature for 18 h. The reaction mixture
was then diluted with CH2Cl2 (200 mL) and washed with 0.5 M
Na2S2O3 in saturated aqueous NaHCO3 (150 mL). The aqueous
layer was washed with CH2Cl2 and the combined organic layers
were dried (Na2SO4), filtered, and evaporated to dryness under
reduced pressure. Chromatography (1-5% methanol in CH2Cl2)
1-(4′-Azido-2′,5′-O-diacetyl-3′-deoxy-ꢀ-D-ribofurnosyl)uracil (20).
A 1:1 mixture of 1-(4′-azido-3′-deoxy-ꢀ-d-ribofuranosyl)uracil 19
and its diastereomer (0.12 g, 0.44 mmol) was treated with acetic
anhydride (2 mL) in the presence of catalytic amount of DMAP
for 15 h. The excess of acetic anhydride was destroyed with water
(3 mL) and the solvents were evaporated to dryness. The isomers
were separated by means of preparative HPLC using a HyperCarb
column and 10-100% CH3CN in H2O to afford the first eluting
isomer 20 (0.047 g, 30%) and second eluting isomer, the diaste-
1
gave 15 (2.18 g, 72%). H NMR (CDCl3): δ 7.78 (d, J ) 8.2 Hz,
1H), 5.78-5.75 (m, 2H), 4.52-4.48 (m, 1H), 4.42-4.38 (m, 1H),
3.55-3.40 (m, 2H), 2.26-2.20 (m, 1H), 1.95-1.85 (m, 1H).
1-(5′-Deoxy-erytho-ꢀ-D-pent-4-enofuranosyl)uracil (16). 15
(1.76 g, 5.22 mmol) was dissolved in a solution of sodium
methoxide in methanol (0.16 M, 80 mL). The reaction mixture was
stirred at 60 °C for 20 h to complete the elimination and was then
cooled on an ice-water bath. Pyridinium form DOWEX H+
[DOWEX H+ was treated with pyridine (10 mL/g resin), filtered,
and washed with methanol prior to use] was added portion wise
until pH of the mixture was neutral (total 6 g). The ice-water bath
was removed and the mixture was stirred at room temperature for
5 min. The resin was removed by filtration and washed with
methanol (100 mL). The organic solvent was evaporated to dryness
under reduced pressure. Chromatography (6-7% ethanol in CH2Cl2)
gave 16 (1.07 g, 98%) (not completely pure, contaminated with
4′-methoxyuridine epimers). MS (ES+) [M + H]+ m/z 211.
1
reomer of 20 (0.042 g, 27%). H NMR data for 20 (CDCl3): δ
8.42 (br. s, 1H), 7.20 (d, J ) 8.2 Hz, 1H), 5.78-5.72 (m, 2H),
5.45-5.41 (m, 1H), 4.40 (s, 2H), 2.80-2.88 (m, 1H), 2.18 (s, 3H),
2.17 (s, 3H), 2.16-2.12 (m, 1H).
1-(4′-Azido-3′-deoxy-ꢀ-D-ribofuranosyl)cytosine (21). 4-Chlo-
rophenylphosphorodichloridate (0.047 g, 0.19 mmol) was added
to a cooled (0 °C) solution of 20 (0.046 g, 0.13 mmol) and 1H-
tetrazole (0.027 g, 0.38 mmol) in dry pyridine (1 mL). After stirring
for a further 5 min, the reaction mixture was warmed to room
temperature and stirred for 5 h. The reaction mixture was then
evaporated to dryness under reduced pressure, dissolved into
CH2Cl2, and washed with saturated aqueous NaHCO3 solution. The
aqueous phase was extracted with CH2Cl2 and the combined organic
phase was dried (Na2SO4), filtered, and evaporated to dryness under
reduced pressure. The residue was dissolved in 0.5 M ammonia in
dioxane (5 mL) and stirred for 5 h. The solution was evaporated to
dryness under reduced pressure and then treated with saturated
ammonia in methanol (5 mL) for 15 h at room temperature. The
mixture was evaporated to dryness under reduced pressure and the
1-(4′-Azido-2′-O-benzoyl-5′-iodo-3′-deoxy-ꢀ-D-ribofuranosyl)-
uracil (18). Benzyltriethylammonium chloride (1.91 g, 8.4 mmol)
and sodium azide (0.54 g, 8.4 mmol) were suspended in anhydrous
CH3CN (32 mL) and sonicated for a few minutes. The resulting
fine suspension was stirred at room temperature for 3 h and was
then filtered into a anhydrous THF solution (30 mL) of compound
15 (0.882 g, 4.2 mmol). N-Methylmorpholine (0.14 mL, 0.106

Brief Articles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 223
residue purified by means of preparative HPLC (HyperCarb column
Supporting Information Available: Biology methods, analytical
methods, and HPLC spectral data for compounds 9, 13, 21, 24,
and 25; NMR data for compounds 3 and 10. This material is
available free of charge via the Internet at http://pubs.acs.org.
and 10-100% CH3CN in H2O) to give 21 (0.024 g, 68%). 1H NMR
(DMSO-D ): δ 7.68 (d, J ) 7.5 Hz, 1H), 7.26 (s, 2H), 6.03 (d, J )
6
3.8 Hz, 1H), 5.73 (d, J ) 7.5 Hz, 1H), 5.65 (br. s, 1H), 5.58 (br.
s, 1H), 4.31 (br. s, 1H), 3.55 (q, J ) 11.7 Hz, 2H), 2.52 (dd, J )
7.5 and 14 Hz, 1H), 1.84 (dd, J ) 4.0 and 14 Hz, 1H). HRMS
(ES+) calcd for C9H13N6O4 [M + H]+ 269.0998; found 269.1010.
References
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1-[4′-Azido-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-
ꢀ-D-ribofurnosyl]-4-N-benzoylcytosine (22). A mixture containing
1-(4′-azido-ꢀ-D-ribofuranosyl)-4-N-benzoylcytosine (1.8 g, 4.64
mmol), prepared from 1-(4′-azido-ꢀ-D-ribofurnosyl)cytosine 3,11
and TIPDSCl (2.25 mL, 7.04 mmol) in pyridine (45 mL) was stirred
at room temperature for 48 h. The reaction was quenched with
methanol and the solvents were evaporated to dryness under reduced
pressure. The residue was partitioned between CH2Cl2 and saturated
aqueous NaHCO3 solution. The combined organic layer was dried
(Na2SO4) and evaporated to dryness under reduced pressure.
Chromatography (0.5-2% ethanol in CH2Cl2) gave 22 (0.724 g,
25%). 1H NMR (CDCl3): δ 8.64 (s, 1H), 7.90-7.83 (m, 3H),
7.65-7.51 (m, 4H), 5.75 (s, 1H), 5.03 (d, J ) 6.2 Hz, 1H), 4.49
(dd, J ) 1.8 and 6.1 Hz, 1H), 4.22 (d, J ) 12.2 Hz, 1H), 4.03 (d,
J ) 12.2 Hz, 1H), 3.24 (d, J ) 2.7 Hz, 1H), 1.13-1.06 (m, 28H).
MS (ES+) [M + H]+ m/z 527.
1-[4′-Azido-2′-keto-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)-ꢀ-D-ribofurnosyl]-4-N-benzoylcytosine (23). Dess-
Martin periodinane (0.672 g, 1.59 mmol) was dissolved in CH2Cl2
(40 mL) and added dropwise to a cooled solution of 22 (0.5 g,
0.79 mmol) in CH2Cl2 (48 mL). The resulting mixture was stirred
for 18 h at room temperature. A solution, containing 0.1 M Na2SO3,
in saturated aqueous NaHCO3 (150 mL) was added and the mixture
was vigorously stirred for 10 min. The product was extracted with
CH2Cl2, dried (Na2SO4), and evaporated to dryness under reduced
pressure. Chromatography (0.5-1% ethanol in CH2Cl2) gave 23
(0.414 g, 83%) as a white solid. 1H NMR (CDCl3): δ 8.72 (s, 1H),
7.87-7.85 (m, 2H), 7.65-7.50 (m, 5H), 5.60 (s, 1H), 5.16 (s, 1H),
4.31 (d, J ) 12.1 Hz, 1H), 4.21 (d, J ) 12.1 Hz, 1H), 1.16-1.06
(m, 28H). MS (ES+) [M + H]+ m/z 629.
1-(4′-Azido-2′-ꢀ-methyl-ꢀ-D-ribofurnosyl)cytosine (24) and
1-(4′-azido-2′-r-methyl-ꢀ-D-arabinofurnosyl)cytosine (25). To a
stirred solution of 2′-ketone 23 (0.05 g, 1 mmol) in THF (5 mL)
was added 2 M solution of trimethylaluminum in toluene (0.55 mL,
1.1 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for
1 h, then quenched with water (0.5 mL) and filtered through a pad
of celite to remove aluminum hydroxide. Solvents were evaporated
to dryness under reduced pressure. The residue was dissolved in
methanol (5 mL) acidified with 1 drop of 4 M HCl in dioxane.
After 1 h, the reaction mixture was saturated with ammonia in
methanol (1 mL) and stirred overnight at room temperature. The
resulting 1:1 mixture of 2′-Me isomers 24 and 25 was separated
by preparative HPLC on a Hypercarb column and 10-100%
gradient CH3CN in H2O.
24 (7.42 mg, 31%). 1H NMR (DMSO-d6): δ 7.79 (d, J ) 7.6 Hz,
1H, 6-H), 7.22; 7.15 (2s, 2 H, 4-NH2), 6.18 (s, 1H, 1′R-H), 5.78 (s,
1H, 3′R-OH), 5.71 (d, J ) 7.5 Hz, 1H, 5-H), 5.29 (s, 1H, 5′-OH), 5.22
(s, 1H, 2′R-OH), 3.89 (s, 1H, 3′ꢀ-H), 3.62-3.53 (m, 2H, 5′-H2), 0.95
(s, 3H, 2′ꢀ-CH3). 13C NMR (DMSO-d6): δ 166.36 (C-4), 157.14 (C-
2), 141.26 (C-6), 98.19 (C-4′), 95.04 (C-5), 94.16 (C-2′), 77.78 (C-
1′), 73.38 (C-3′), 61.89 (C-5′), 19.00 (CH3-2′). HRMS (ES+) calcd
for C10H15N6O5 [M + H]+ 299.1117; found 299.1103.
(8) Klumpp, K.; Kalayanov, G.; Ma, H.; Le Pogam, S.; Leveque, V.; Jiang,
W.-R.; Inocencio, N.; De, Witte.; Rajyaguru, S.; Tai, E.; Chanda, S.; Irwin,
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N. G.; Smith, D. B. 2′-Deoxy-4′-azido nucleoside analogs are highly potent
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Leveˆque, V.; Le Pogam, S.; Najera, I.; Klumpp, K.; Smith, D. B.;
McGuigan, C. Application of the phosphoramidate ProTide ap-
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hepatitis C virus on an inactive nucleoside. J. Med. Chem. 2007,
50, 1840–1849.
(10) Perrone, P.; Daverio, F.; Valente, R.; Rajyaguru, S.; Martin, J. A.;
Le´veˆque, V.; Le Pogam, S.; Najera, I.; Klumpp, K.; Smith, D. B.;
McGuigan, C. First example of phosphoramidate approach applied to
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an inactive nucleoside to a submicromolar compound versus hepatitis
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(11) Connolly, T. K.; Durkin, K.; Sarma, K.; Zhu, J. Processes for Preparing
4′-Azido Nucleoside Derivatives. Patent WO 2005/000864, 2005.
(12) Miah, M.; Reese, C. B.; Song, Q.; Sturdy, Z.; Neidle, S.; Simpson,
I. J.; Read, M.; Rayner, E. 2′,3′-Anhydrouridine. A useful synthetic
intermediate. J. Chem. Soc., Perkin Trans. 1 1998, 3277–3283.
(13) Divakar, K. J.; Reese, C. B. 4-(1,2,4-Triazol-1-yl)-and 4-(3-nitro-1,2,4-
triazo-1-yl)-1-(ꢀ-D-2,3,5-tri-O-acetylarabinofuranosyl)pyrimidin-2(1H)-
ones. Valuable intermediates in the synthesis of derivatives of 1-(ꢀ-
D-arabinofuranosyl)cytosine (AraC). J. Chem. Soc., Perkin Trans. 1
1982, 1171–1176.
25 (8.07 mg, 34%). 1H NMR (DMSO-d6): δ 7.49 (d, J ) 7.6 Hz,
1H, 6-H), 7.15; 7.09 (2s, 2H, 4-NH2), 6.11 (s, 1H, 1′R-H), 5.99 (d, J
) 5.7 Hz, 1H, 3′R-OH), 5.57 (d, J ) 7.5 Hz, 1H, 5-H), 5.49 (s, 1H,
5′-OH), 5.29 (s, 1H, 2′ꢀ-OH), 3.94 (d, J ) 4.6 Hz, 1H, 3′ꢀ-H), 3.66 (s,
2H, 5′-H2), 1.20 (s, 3H, 2′R-CH3). 13C NMR (DMSO-d6): δ ) 166.23
(C-4), 155.95 (C-2), 143.93 (C-6), 98.64 (C-4′), 93.54 (C-5), 89.06
(C-1′), 79.98 (C-2′), 78.52 (C-3′), 64.36 (C-5′), 21.54 (CH3-2′). HRMS
(ES+) calcd for C10H15N6O5 [M + H]+ 299.1117; found 299.1104.
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JM800981Y