Synthesis and evaluation of S-Acyl-2-thioethyl esters of modified nucleoside 5′-monophosphates as inhibitors of hepatitis C virus RNA replication

Article abstract of DOI:10.1021/jm0495172

Several triphosphates of modified nucleosides (1-6) were identified as inhibitors (IC₅₀ -0.08-3.8 μM) of hepatitis C virus RNA-dependent RNA polymerase (RdRp). Although the initial SAR developed by determining the ability of the triphosphates t

Full text of DOI:10.1021/jm0495172

J. Med. Chem. 2005, 48, 1199-1210
1199
Synthesis and Evaluation of S-Acyl-2-thioethyl Esters of Modified Nucleoside
5′-Monophosphates as Inhibitors of Hepatitis C Virus RNA Replication
Thazha P. Prakash,*,† Marija Prhavc,† Anne B. Eldrup,† P. Dan Cook,† Steven S. Carroll,‡ David B. Olsen,‡
Mark W. Stahlhut,‡ Joanne E. Tomassini,‡ Malcolm MacCoss,# Sheila M. Galloway,# Catherine Hilliard,§ and
Balkrishen Bhat†
Department of Medicinal Chemistry, ISIS Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, California 92008, Department of
Biological Chemistry and Department of Safety Assessment, Merck Research Laboratories, West Point, Pennsylvania 19486,
and Department of Medicinal Chemistry, Merck Research Laboratories, Rahway, New Jersey 07065
Received June 20, 2004
Several triphosphates of modified nucleosides (1-6) were identified as inhibitors (IC50 ) 0.08-
3.8 µM) of hepatitis C virus RNA-dependent RNA polymerase (RdRp). Although the initial
SAR developed by determining the ability of the triphosphates to inhibit the in vitro activity
of the HCV RdRp identified several potent inhibitors, none of the corresponding nucleosides
exhibited significant inhibitory potency in a cell-based replicon assay. To improve upon the
activity, bis(tBu-S-acyl-2-thioethyl) nucleoside 5′-monophosphate esters (7-12) were synthe-
sized, and these derivatives exhibited improved potency compared to the corresponding
nucleosides in the cell-based assay. Analysis of the intracellular metabolism demonstrated that
the S-acyl-2-thioethyl (SATE) prodrug is metabolized to the 5′-triphosphate 40- to 155-fold
more efficiently compared to the corresponding nucleoside. The prodrug approach involving
bis(tBuSATE)cytidine 5′-monophosphate ester significantly reduced the deamination of cytidine
derivatives by cellular deaminases. Additionally, chromosomal aberration studies with the SATE
prodrug in cells showed no statistically relevant increase in aberrations compared to the
concurrent controls.
Introduction
teins have been recently reported.6,7 RdRp (RNA-
dependent RNA polymerase) is essential for viral rep-
lication and proliferation. Hence, designing specific
inhibitors of HCV RdRp has become one of the impor-
tant approaches for developing novel drugs against HCV
infection. Several non-nucleoside inhibitors of RdRp
have been identified as anti- HCV agents.8-11 Recently,
a series of nucleoside analogues have also been devel-
oped as RdRp inhibitors.12-15 Though triphosphates of
several of these nucleoside analogues exhibited excellent
RdRp inhibitory potency, only few nucleoside derivatives
have exhibited biological activity in cell culture as-
says.12,15 This might be due to poor cellular penetration
coupled with insufficient metabolism to 5′-triphosphates
of these nucleoside derivatives. Here, we report the
results of the prodrug approach that resulted in im-
proved activity of HCV inhibitors in a cell-based replicon
assay.
Hepatitis C virus (HCV) is a positive-strand RNA
virus that was unambiguously identified in 1989.1 The
HCV RNA genome is approximately 10 kb in length and
shares similarities with the genomes of flaviviruses and
pestiviruses. HCV is responsible for most cases of non-A
and non-B hepatitis, a chronic disease that often leads
to liver cirrhosis and hepatocellular carcinoma. Ap-
proximately 170 million infected individuals are at risk
of developing significant morbidity and mortality.2 HCV
infection has become the primary reason for liver
transplantations among adults in Western countries.3
Thus, HCV has attracted a tremendous amount of
attention from academic and pharmaceutical industry
research.4 Current therapies for HCV infection based
on administering combinations of interferon-R and
ribavirin are suboptimal, especially in patients infected
with HCV genotype 1, and are poorly tolerated and have
less than 50% overall response rates.5 These drugs do
not act directly on the virus, and their mode of action
is poorly understood. Therefore, there is an urgent
medical need for more efficient and better tolerated
antiviral agents for HCV.
Previously we reported that 2′-O-Me-C was able to
inhibit HCV viral RNA replication in the context of a
cell-based replicon12 assay. Here, we report the synthe-
sis and potency evaluation of S-acyl-2-thioethyl (SATE)
ester prodrugs of 2′-O-Me-C and several closely related
nucleoside monophosphates.
Detailed accounts of the HCV genome and gene
products including processing into multiple viral pro-
Results and Discussion
Several triphosphates of modified nucleosides (Figure
1) were designed and synthesized and were evaluated
for the ability to inhibit HCV RdRp.12 This effort yielded
some very potent inhibitors (Table 1). However, when
the cell-based replicon assay became available,7 none
of these nucleosides exhibited comparable intracellular
potency (Table 1).
* To whom correspondence should be addressed. Phone: 760-603-
2590. Fax: 760-603-4654. E-mail: tprakash@isisph.com.
† ISIS Pharmaceuticals.
‡ Department of Biological Chemistry, Merck Research Laboratories,
PA.
§ Department of Safety Assessment, Merck Research Laboratories,
PA.
# Merck Research Laboratories, NJ.
10.1021/jm0495172 CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/01/2005

1200 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Prakash et al.
Table 1. Inhibitory Potency (Replicon EC50) of Nucleoside
Analogues 1-6 and Their 5′-Bis(tBuSATE)monophosphate
Esters (7-12) in HCV Replicon Assay in HBI10A Cells and
Inhibitory Potency (RdRp IC50) of 5′-Triphosphates of
Nucleoside Analogues 1-6 on HCV NS5B-Mediated RNA
Synthesisa
replicon
RdRp
IC50,
µM
toxicity
MTSb
CC50
EC50
,
compd
chemistry
2′-O-Me-G
2′-O-Me-7-deaza-G
3′-deoxy-G
µM
1
2
3
>50
1.2 ( 0.7
0.35 ( 0.08
0.6 ( 0.3
0.08 ( 0.01
3.8 ( 0.3
1.2
>50
>50
>50
21 ( 3
25
4
5
6
3′-deoxy-7-deaza-G
2′-O-Me-C
3′-deoxy-C
>100
>50
7
8
9
10
11
12
5′-SATE-2′-O-Me-G
>50
>50
>100
>50
>50
>100
>100
5′-SATE-2′-O-Me-7-deaza-G >50
5′-SATE-3′-deoxy-G 23
5′-SATE-3′-deoxy-7-deaza-G 7.2
5′-SATE-2′-O-Me-C
5′-SATE-3′-deoxy-C
3 ( 1
1.4 ( 0.9
a Compounds were incubated in cell culture for 24 h prior to
determination of the relative amount of HCV replicon RNA with
the in situ ribonuclease protection assay. b Compounds cytotoxicity
was determined by MTS assay on parallel samples at the same
time.
Figure 1. HCV RNA inhibitors.
steps, Gosselin and Imbach designed the bis(SATE)-
bearing monophosphate prodrugs for several nucleoside
analogues for HIV and demonstrated impressive anti-
viral activity compared to corresponding nucleoside
analogues.21-24 These protected neutral monophosphate
esters have been shown to enter the cell much more
readily than corresponding monophosphate deriva-
tives.18 After entering the cell, the pronucleotide should
metabolize via a sequence of enzymatic thioester hy-
drolyses, followed by subsequent fragmentation with the
simultaneous release of episulfide and its corresponding
5′-monophosphate.21
All of the triphosphates of the nucleoside analogues
1-6 inhibit the catalytic activity of purified HCV RNA
polymerase with IC50 values ranging from 0.08 to 3.8
µM (Table 1). However, nucleosides 1-6 were very poor
inhibitors of RNA replication in cell-based systems
(Table 1). The lack of reasonable activity of the nucleo-
sides 1-6 in the cell-based assay in comparison to the
potency observed in the biochemical enzyme assay may
be due to previously noted reasons such as poor cellular
penetration or the inability of these nucleosides to be
metabolized to the corresponding triphosphates. We
envisaged that 5′-bis(S-pivaloyl-2-thioethyl) nucleoside
monophosphate esters 7-12 (S-pivaloyl-2-thioethyl ab-
breviated as tBuSATE, Figure 2) should improve cell
penetration and would generate the monophosphate
intracellularly by following the decomposition pathway
previously described for SATE prodrugs.21
Chemistry. The 2′-O-methylguanosine 1 and 2′-O-
methylcytidine 5 were procured from commercial sources.
The 3′-deoxyguanosine (3) and 3′-deoxycytidine (6) were
synthesized using the reported procedures.25
The 2-amino-7-(2-O-methyl-â-D-ribofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidin-4(3H)-one (2) was synthesized
via the route described in Scheme 1. The 5-O-tert-butyl-
dimethylsilyl-2,3-O-isopropylidene-D-ribofuranose26 14
(Scheme 1) was treated with carbon tetrachloride in
hexamethylphosphorus triamide in THF at -76 °C
to generate in situ 1-choloro-5-O-tert-butyldimethyl-
silyl-2,3-O-isopropylidene-D-ribofuranose. The sodium
Figure 2. Bis(S-pivaloyl-2-thioethyl]phosphotriester deriva-
tives of HCV RNA inhibitors 1-6.
All these nucleoside analogues have no intrinsic
antiviral activity. They must be metabolized to their
respective 5′-triphosphate by nucleotidase, kinases, and/
or other activating enzymes present naturally in cells
to act as RdRp inhibitors.17 Of the three anabolic steps
in the biochemical pathway to convert nucleoside ana-
logues to the active triphosphates, the monophos-
phorylation step is considered as generally being the
rate-limiting step, though there are notable exceptions
such as AZT.18 The presence and the activity of intra-
cellular enzymes involved in the monophosphorylation
of nucleoside analogues depend on the host species, the
cell type, and the phase in the cell cycle.19,20 The
dependence on phosphorylation for activation of the
nucleoside analogues may therefore be problematic in
cells where the phosphorylating enzyme activity is
known to be low or even lacking. Another factor that
may be contributing to the lack of activity of nucleoside
analogues in cell-based assays would be their poor
cellular uptake. To circumvent these two metabolic

Thioethyl Esters as Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1201
Scheme 1a
Scheme 2a
a Reagents and conditions: (i) (a) H2O/CH3CN, R-acetoxyiso-
butyryl bromide; (b) MeOH, Dowex OH-; (ii) LiEt3BH in THF, 0
°C; (iii) 2 N NaOH, reflux.
a Reagents and conditions. TBDMS: tert-butyldimethylsilyl; (i)
(a) hexamethylphosphorus triamide, THF, CCl4, 2-amino-6-chloro-
1H-pyrrolo[2,3-d]pyrimidine, CH3CN, NaH, -76 to -20 °C; (b)
Dowex WX 8-400; (ii) MeI, NaH, DMF; (iii) NaOH, H2O, reflux.
salt of 2-amino-4-chloro-1H-pyrrolo[2,3-d]pyrimidine
in acetonitrile was coupled to 1-choloro-5-O-tert-butyl-
dimethylsilyl-2,3-O-isopropylidene-D-ribofuranose to give
2-amino-4-chloro-7-(5-O-tert-butyldimethylsilyl-2,3-
O-isopropylidene-â-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine and R anomer (R/â 1:3). The two isomers
were separated by flash silica gel column chromatog-
raphy. The â isomer was treated with Dowex WX8-400
resin to deprotect the 5′-silyl and 2′,3′-ispropylidine
groups to yield 2-amino-4-chloro-7-(â-D-ribofuranosyl)-
7H-pyrrolo[2,3-d]pyrimidine (15). The treatment of 15
with methyl iodide in the presence of sodium hydride
in DMF yielded 2-amino-4-chloro-7-(2′-O-methyl-â-D-
ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (16) and the
3′-O-methyl derivative. Two regioisomers were sepa-
Figure 3. Bis(S-pivaloyl-2-thioethyl) N,N-diisopropylphos-
phoramidite.
60%). To assign the position of the hydroxyl group, 2D
correlation NMR spectroscopy experiments of 18 in
DMSO-d6 were performed. The presence of strong
symmetrical cross-peaks (data not shown) in the 2D
NMR spectrum, due to the coupling of the hydroxyl
proton and proton at the 2′-position as well as the
absence of cross-peaks from the coupling of hydroxyl
proton and 3′-proton, confirms that the hydroxyl group
was at the 2′-position of 18. Compound 18 was refluxed
with 2 N aqueous sodium hydroxide and neutralized
with 2 N aqueous hydrochloric acid to give 4 in 80%
yield.
The P(III) chemistry involving phosphoramidite in-
termediates is the most efficient method to prepare
phosphate derivatives, and this approach has been used
to synthesize several SATE prodrugs.21 We used similar
strategy to synthesize nucleoside-5′-bis(tBuSATE) mono-
phosphate esters 7-12 (Figure 2). The phosphitylation
reagent bis(tBuSATE)-N,N-diisopropylphosphoramidite
13 (Figure 3) with thioester group was synthesized
according to the literature procedure.21 Coupling of the
appropriately protected nucleosides with 13 in the
presence of 1H-tetrazole followed by in situ oxidation
yielded the nucleoside-5′-bis(tBuSATE) monophosphate
esters.
The 2′-O-methylguanosine 5′-[bis(tBuSATE)]phos-
phate 7 and 2-amino-7-(2-O-methyl-â-D-ribofuranosyl)-
7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one 5′-[bis(tBuSATE)]-
phosphate 8 were synthesized from 2′-O-methylguanosine
(1) and 2-amino-7-(2-O-methyl-â-D-ribofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidin-4(3H)-one (2), respectively
(Scheme 3). Compounds 1 and 2 were silylated with 1,3-
dichloro-1,1,3,3-tetraisopropyldisiloxane in anhydrous
pyridine to yield 19 and 20 (80-85%), respectively.
These were subsequently treated with 4-monomethoxy-
trityl chloride in the presence of DMAP in anhydrous
1
rated by column chromatography. H NMR spectrum
of compound 16 exhibited only one singlet at δ 3.25 for
three protons. These data suggested that there was only
one methyl group in the molecule. To assign the position
of methyl group, two-dimensional (2D) 1H-1H correlated
NMR spectroscopy experiments of 16 in DMSO-d6 were
performed. In the 2D correlated NMR spectrum, strong
symmetrical cross-peaks due to coupling of hydroxyl
protons and 3′- and 5′-protons were observed whereas
no coupling between hydroxyl proton and 2′-proton was
observed (data not shown). These data indicated that
compound 16 was a 2′-O-methyl derivative. Compound
16 on saponification with aqueous sodium hydroxide
followed by neutralization afforded 2′-O-methyl-7-deaza-
guanosine 2.
The synthesis of 2-amino-7-(3-deoxy-â-D-ribofurano-
syl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one (4) was
achieved as described in Scheme 2. Treatment of
compound 15 with R-acetoxyisobutyryl bromide in
acetonitrile and water gave a mixture of 2′- and 3′-
bromoarabino nucleosides.27 This mixture on treatment
with Dowex OH- resin in methanol gave 2-amino-7-(2,3-
anhydro-â-D-ribofuranosyl)-4-methoxy-7H-pyrrolo[2,3-
d]pyrimidine 17 (94% yield).26 The chlorine at the
6-position of the nucleoside base was displaced with a
methoxy group under this condition. Reduction of the
2′,3′-anhydro derivative 17 with triethyllithium boro-
hydride in THF yielded 2-amino-7-(3-deoxy-â-D-ribo-
furanosyl)-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine (18,

1202 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Prakash et al.
Scheme 3a
Scheme 4a
a Reagents and conditions. DMT: 4,4′-dimethoxytrityl. (i) for
23, 25, and 26, tert-butyldimethylsilyl chloride, imidazole, DMF,
room temperature; for 24, acetic anhydride, triethylamine, aceto-
nitrile, DMAP; (ii) (a) p-anisylchlorodiphenylmethane (for 27-28)
or 4,4′-dimethoxytrityl (for 29, 30), DMAP, pyridine; (b) for 27,
29, and 30, triethylamine trihydrofluoride, triethylamine, THF,
room temperature; for 28, 2 N aqueous NaOH, dioxane, room
temperature; (iii) (a) 13, 1H-tetrazole, CH3CN; (b) 3-chloroper-
benzoic acid, CH2Cl2, -40 to -10 °C; (c) acetic acid/H2O/MeOH,
3:1:6, 55 °C.
a Reagents and conditions. MMT: p-anisyldiphenylmethyl; (i)
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, pyridine, room tem-
perature; (ii) (a) p-anisylchlorodiphenylmethane, DMAP, pyridine;
(b) triethylamine trihydrofluoride, triethylamine, THF, room
temperature; (iii) (a) 13, 1H-tetrazole, CH3CN; (b) 3-chloroper-
benzoic acid, CH2Cl2, -40 to -10 °C; (c) acetic acid/H2O/MeOH
(3:1:6), 55 °C.
pyridine to protect the exocyclic amino group at the
2-position of the nucleosides. The protected nucleosides
were desilylated on treatment with triethylamine tri-
hydrofluoride and triethylamine in THF to yield 21 and
22 (72-75% yield). The coupling of 21 and 22 with
phosphoramidite 13 in the presence of 1H-tetrazole
followed by oxidation with 3-chloroperbenzoic acid gave
a mixture of 5′- and 3′-bis(tBuSATE) monophospates of
nucleosides in a 3:2 ratio. The mixture of 5′- and
3′-regioisomers were then heated with a solution of
acetic acid/water/methanol (3:1:6) at 55 °C to remove
the trityl protecting group from the exocyclic amino
group. The 5′-regioisomers (7 and 8, 28-39% yield) were
separated from 3′-regioisomers by high-pressure liquid
chromatography (HPLC). The structure of the two
isomers was assigned using two-dimensional 1H-1H
correlated NMR spectroscopy experiments in DMSO-
d6. The 2D NMR spectra showed coupling of the proton
at the 3′-hydroxyl group and the proton at the 3′-position
(data not shown), and no coupling was observed between
the hydroxyl proton and the 5′-proton. These data
suggested that phosphate ester group was at 5′-position
of the compounds 7 and 8.
The 5′-bis(tBuSATE) monophosphate esters of 3′-
deoxyguanosine (9), 2-amino-7-(3-deoxy-â-D-ribofurano-
syl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one (10), 2′-O-
methylcytidine (11), and 3′-deoxycytidine (12) were
synthesized (Scheme 4) using a similar synthetic strat-
egy described for 7 and 8. The nucleosides 3, 5, and 6
were treated with tert-butyldimethylsilyl chloride in
DMF in the presence of imidazole to yield respective
silylated nucleosides 23, 25, and 26 (52-98% yield). The
nucleoside 4 was treated with acetic anhydride in
acetonitrile in the presence of triethylamine to yield
2′,5′-O-bis(acetyl) derivative 24 in 95% yield. The exo-
cyclic amino group of the nucleosides 23-26 was
protected with trityl groups on treatment with p-
anisylchlorodiphenylmethane (MMT-Cl) or 4,4′-dimeth-
oxytrityl chloride (DMT-Cl) in anhydrous pyridine. The
fully protected nucleosides 23, 25, and 26 were then
treated with triethylamine trihydrofluoride and tri-
ethylamine in THF at room temperature to remove the
tert-butyldimethyl silyl group to yield 27, 29, and 30.
The 2′,5′-O-bis(acetyl)-N2-(p-anisyldiphenylmethyl) pro-
tected nucleoside 24 was treated with 2 N aqueous
sodium hydroxide at room temperature to yield 28.
Compounds 27-30 were phosphitylated with 13 and
1H-tetrazole. The nucleoside phosphites thus formed
were oxidized to phosphate using 3-chloroperbenzoic
acid in dichloromethane followed by deprotection of the
trityl group at the exocyclic amine, yielding the nucleo-
side 5′-bis(tBuSATE)phosphates 9-12. The mixture of
5′ and 3′ isomers were separated by HPLC. The 5′
regioisomers were identified using 2D NMR correlation
spectroscopy (COSY) experiments in DMSO-d6. The 2D
NMR spectra showed coupling of proton at the 2′- or
3′-hydroxyl group and the proton at the 2′- or 3′-position,
whereas no coupling was observed between hydroxyl
proton and 5′-proton. These data indicated that phos-
phate ester group was at the 5′-position of compounds
9-12.
To evaluate the intracellular metabolism of SATE
prodrugs, we have synthesized the tritiated version of
11 using 5-bromo-2′-O-methylcytidine 5′-[bis(tBuSATE)-
monophosphate] 33. Compound 33 was synthesized
according to the route described in Scheme 5. The 3′,5′-
O-bis(tert-butyldimethylsilyl)-2′-O-methylcytidine 25 was
treated with lithium bromide and ceric(IV) ammonium
nitrate in acetonitrile to yield the 5-bromo derivative
31 (83% yield).28 The tert-butyldimethylsilyl group at
the 5′-postion of 31 was selectively removed on heating
with 80% acetic acid in water at 50 °C for 6 h to yield

Thioethyl Esters as Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1203
Scheme 5a
nucleoside triphosphates that proved to be submicro-
molar to low micromolar (IC50 ) 0.08-3.8 µM) inhibitors
of HCV NS5B mediated RNA synthesis.
The lack of activity of the nucleosides 1-6 in the
replicon assay, in comparison to the enzyme assay, may
be due to the inability of these nucleosides to be
metabolized to triphosphates or poor cellular penetra-
tion or a combination of both. To address these issues,
bis(tBuSATE) monophosphate esters 7-12 (Figure 2)
were synthesized and were tested for inhibitory activity
in the cell-based replicon assay.
The tBuSATE derivatives of 2′-O-methylcytidine-5′-
monophosphate (11, Table 1) and 3′-deoxycytidine-5′-
monophosphate (12, Table 1) showed improved activity
in the assay at 24 h with EC50 values of 3 and 1.4 µM,
respectively, which was comparable to the correspond-
ing IC50 values of triphosphates in enzymatic assay. The
replicon potencies of these prodrugs were 7- to 18-fold
greater than the corresponding nucleosides. These
results suggested that the poor activity of nucleosides
(5 and 6 Table 1) in the cell-based assay was due to
inefficient conversion of these nucleosides into active
triphosphates. The improved activity of prodrugs also
suggested that the tBuSATE monophosphate esters
were delivered intracellularly and that the monophos-
phates are released resulting in accumulation of phos-
phorylated forms of the nucleoside derivatives. In
contrast, the tBuSATE derivative of 2′-O-methyl-
guanosine-5′-monophosphate (7) and 2′-O-methyl-7-
deazaguanosine-5′-monophosphate (8) were inactive in
the cell-based assay whereas 3′-deoxyguanosine-5′-
monophosphate (9) and 3′-deoxy-7-deazaguanosine-5′-
monophosphate (10) showed improved activity com-
pared to the corresponding nucleosides (3 and 4). The
poor activities of compounds 7 and 8 may be due to poor
cellular penetration of guanosine nucleosides or inef-
ficient phosphorylation of the 2′-O-methylguanosine 5′-
monophosphates generated after the intracellular me-
tabolism of 7 and 8.
a Reagents and conditions. (i) LiBr, ammonium ceric(IV) nitrate,
CH3CN; (ii) 80% acetic acid in water, 50 °C; (iii) (a) 13, 1H-
tetrazole, CH3CN; (b) 3-chloroperbenzoic acid, CH2Cl2, -40 to -10
°C; (c) triethylamine trihydrofluoride, triethylamine, THF.
5-bromo-3′-O-[(tert-butyldimethyl)silyl]-2′-O-methyl-
cytidine (32, 40%). Compound 32 was coupled with
phosphoramidite 13 in the presence of 1H-tetrazole
followed by oxidation, giving the 5-bromo-3′-O-[(tert-
butyldimethyl)silyl]-2′-O-methylcytidine 5′-[bis(tBu-
SATE)phosphate. This, on treatment with triethylamine
trihydrofluoride to remove tert-butyldimethylsilyl group,
yielded 33 (37%). To develop a catalytic hydrogenation
method for the conversion of 33 into the tritiated version
of 11, compound 33 and 10% palladium on carbon in
methanol were stirred under a normal atmosphere of
hydrogen at room temperature for 24 h. The product
formed was analyzed by spectroscopic methods (1H and
31P NMR and mass spectrometry) and was identical to
compound 11. Compound 33 was subjected to similar
reduction conditions in the presence of tritium to
synthesize the tritiated version of 11.
To investigate the intracellular metabolism of nucleo-
side analogues, we chose compound 11 as our model
compound for further studies. Therefore, a tritiated
derivative of 11 was synthesized to understand the
metabolic fate of its tBuSATE nucleoside prodrug.
All compounds were characterized by 1H and 13C
NMR and mass spectroscopic analysis. The purity of all
the compounds tested in biological assay was deter-
mined by HPLC and was >95%.
Intracellular Metabolism of the 2′-O-methyl-
cytidine 5′-Bis(tBuSATE)-phosphate (11). The in-
tracellular metabolism of the tritiated version of 5′-bis-
(tBuSATE)-2′-O-methylcytidinephosphate 11 was stud-
ied in Huh-7 and HBI10A replicon cell lines. [5-3H]-2′-
O-Methylcytidine and the [5-3H]-tBuSATE prodrug 11
were prepared using 5-bromo-2′-O-methylcytidine and
the 5-bromo derivative of prodrug 33 (Scheme 5) by the
Tritium Custom Preparation group at Amersham Phar-
macia Biotech Ltd. (Cardiff, Wales). The Huh-7 and
HBI10A cells were incubated with compounds for 3 or
23 h at 2 µM prior to extraction and HPLC analysis.
The results are summarized in Table 2. We observed
that the 5′-bis(tBuSATE)-2′-O-methylcytidine mono-
phosphate ester was more efficiently converted to the
corresponding triphosphate compared to 2′-O-methyl-
cytidine nucleoside. No detectable amounts of 2′-O-
methyluridine 5′-triphosphate (2′-O-Me UTP), cytidine
5′-triphosphate (CTP), or uridine 5′-triphosphate (UTP)
were formed when Huh-7 or HBI10A cells were incu-
Antiviral Activity. The triphosphates of modified
nucleosides 1-6 were screened against the purified
HCV RdRp for their ability to inhibit HCV NS5B
mediated RNA synthesis (RdRp, Table 1). The corre-
sponding nucleosides were screened in a cell-based,
subgenomic replicon assay for their ability to inhibit
RNA replication in a stable Huh-7 human hepatoma cell
line designated HBI10A, which supports the replication
of HCV RNA and protein. The effect of the nucleosides
upon RNA replication was directly detected by an in situ
ribonucleoside protection assay.12 Thus, in the cell-based
assay, activity would be subject to cellular uptake and
metabolism to the nucleoside triphosphate. The replicon
data indicated that none of the modified nucleosides (1-
6) demonstrated significant activity in the cell-based
assay, with EC50 values ranging from 20 to >50 µM
(replicon, Table 1), in contrast to their corresponding

1204 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Prakash et al.
Table 2. Intracellular Metabolism 5-[3H] Derivative of 2′-O-Methylcytidine 5 and 5′-Bis(tBuSATE)-2′-O-methylcytidine
Monophosphate Ester 11 in Cell Culturea
cell
culture
incubation
time, h
triphosphate,
major metabolic
byproducts observed
compd
pmol/million cellsb
5c
2′-O-Me-C
Huh-7
3
23
3
23
3
23
3
23
0.17
0.61
0.05
0.18
7.02
1.88
15.9
5.8
CTP and UTP
CTP, UTP, 2′-O-MeUTP
HBI10A
Huh-7
CTP, UTP, 2′-O-Me UTP
CTP, UTP, 2′-O-Me UTP
none
none
none
none
11
5′-SATE-2′-O-Me-C
HBI10A
a Compounds were incubated in cell culture for times indicated, prior to extraction and analysis by ion pair reverse-phase HPLC. Data
are averages of at least three separate experiments. b Passage number for Huh-7 is 27; for HBI10A, it is 15. c See ref 12.
Table 3. Evaluation of Genotoxicity of 5′-Bis(tBuSATE)-2′-O-methylcytidine Monophosphate Ester 11. Assay for Chromosomal
Aberrations in Vitro in Chinese Hamster Ovary Cellsa
treatment/sampling time, h
treatment type dose, µM
20/20
20/30
20/42
growth
% aberrant cells
growth
% aberrant cells
growth
% aberrant cells
DMSO,
1% v/v
10000
550
100
65
59
51
27
21
6
1.5
9.5b
0.0
2.0
Ns
Ns
Ns
100
101
82
57
46
6
1.5
8.0b
3.5
1.5
4.5
Ns
100
105
95
88
90
47
0
2.0
3.0
2′3′-dideoxycytidine
11
600
1.5
3.0
3.5
Ns
650
700
800
0
Ns
a Ns ) not scored because of toxicity. Growth is measured as population doublings from the beginning of the experiment as a percentage
of controls, with 200 cells scored per point. b P < 0.01 compared with concurrent controls.
bated with the SATE prodrug of 2′-O-methylcytidine
(Table 2). In contrast, when cells were incubated with
2′-O-methylcytidine, very little triphosphate was formed
(Table 2) and considerable amounts of 2′-O-Me UTP,
CTP, and UTP were detected.12
(CHO) cells. The tBuSATE prodrug 11 was tested with
and without a microsomal enzyme activation system (S-
9) prepared from the livers of xenobiotic-treated rats.
A solution of 11 in dimethyl sulfoxide was used for the
study. Known chromosomal breaking agents, cyclophos-
phamide (CP) with S-9 activation and mitomycin C
(MMC) without S-9 activation, were tested as positive
controls. 2′,3′-Dideoxycytidine was also used as a posi-
tive control. The doses selected were based on limiting
solubility in culture medium and on suppression of cell
growth. The protocol used was previously shown to be
suitable for detection of aberration induction by nucleo-
side analogues and included long treatment (20 h) with
and without recovery times of 10 and 22 h. The 3 h
treatments were also done with sampling at 20 h.
The 3 h treatments with 11 with and without S-9 did
not induce aberrations at 700 and 800 µM, respectively
(data not shown). These were the highest does that could
be scored and had little growth suppression, whereas 1
mM doses suppressed growth completely. Positive con-
trols cyclophosphamide at 5 µM and mitomycin C and
1.5 µM induced 25% and 48% of cells with aberrations,
respectively. The results from the long (20 h) treatment
are shown in Table 3. The top doses selected for scoring
reduced cell growth by about 50%. There were no
statistically significant increases in aberrations over
concurrent controls. Dideoxycytidine induced an in-
crease in aberrations after 20 h of treatment, at 20 and
30 h (Table 3).
Cytidine deaminase (CDA) is a widely distributed
enzyme that catalyzes the hydrolytic deamination of
cytosine nucleosides to the corresponding uracil deriva-
tives.29,30 The observed formation of 2′-O-methyl UTP
during incubations with 2′-O-methylcytidine is possibly
due to CDA activity. It has been reported that 5′-
monophosphates of cytidine nucleoside derivatives are
poor substrates for the deaminases.31,32 The tBuSATE
monophosphate ester generates monophosphate intra-
cellularly, which probably helps to protect the cytidine
derivative from deamination by CDA. The fact that no
2′-O-methyl UTP was formed when radiolabeled 11 was
incubated with cells suggests that the tBuSATE mono-
phosphate ester is metabolized into 2′-O-methylcytidine
5′-monophosphate and at the same time the 5′-mono-
phosphate nucleoside is stable to CDA activity. The 2′-
O-methylcytidine was metabolized extensively to UTP
and CTP because of base swapping in the cells.12
However, the tBuSATE derivative of 2′-O-methylcyti-
dine (11) was not metabolized to CTP or UTP (Table
3). This suggests that 11 and the subsequently gener-
ated 5′-monophosphate are poor substrates for the
nucleoside pyrophosphorylase responsible for these con-
versions. These observations suggest that the tBuSATE
derivative enhanced the half-life of 2′-O-methylcytidine
and may in part account for the improved potency of
prodrug 11 (EC50 ) 3 µM, Table 1) in the cellular assay
compared to 2′-O-methylcytidine (5, EC50 ) 21 µM).
Evaluation of Genotoxicity of tBuSATE Pro-
drug: In Vitro Chromosomal Aberrations in Chi-
nese Ovary Cells. To assess the genotoxicity of the
tBuSATE prodrug, we evaluated its potential to cause
chromosomal aberrations in Chinese hamster ovary
Conclusions
We have identified several nucleoside analogues as
inhibitors of RNA-dependent RNA polymerase (NS5B)
of hepatitis C virus. Bis(tBuSATE) nucleoside 5′-mono-
phosphate ester derivatives 7-12 improved the activity
of these nucleosides in a cell-based assay. Analysis of
the intracellular metabolism of a radiolabeled tBuSATE
prodrugs demonstrated that the prodrugs were metabo-

Thioethyl Esters as Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1205
residue obtained was purified by flash silica gel column
chromatography and eluted with 5% MeOH in CH2Cl2. A
mixture of 5′- and 3′-[bis(S-pivaloyl-2-thioethyl)]phosphate
derivative (6:4 ratio) was eluted together (0.18 g, 28% yield).
13P NMR (80 MHz, CDCl3) δ -0.70, -1.37; MS (API-ES) m/z
936.32 [M - H]-. The mixture (0.18 g, 0.19 mmol) was
dissolved in acetic acid/MeOH/H2O, 3:6:1 (10 mL), and was
heated at 55 °C for 24 h. Solvent was removed, and the residue
was purified by HPLC on a reverse-phase column (Hamilton
PRP-1, 250 mm × 22 mm, A ) acetonitrile, B ) H2O, 20-
100% B in 65 min, flow rate of 10 mL min-1). Fractions
containing 3′- and 5′-isomers were pooled together and evapo-
rated. The 3′- and 5′-isomers were characterized by 1H-1H
COSY experiments in DMSO-d6. The 5′-isomer 7 was isolated
in 16% yield (0.11 g). HPLC retention time was 18.64 min
(Phenomenex aqua, C-18, 150 mm × 4.6 mm, 5 µm, A ) 100
mM triethylammonium acetate, pH 7, B ) 90% acetonitrile
in A, 2-100% B in 25 min, flow rate of 1 mL min-1, λ ) 260
lized to the 5′-triphosphate 40- to 155-fold more ef-
ficiently compared to the corresponding nucleosides. The
prodrug approach involving bis(tBuSATE)cytidine 5′-
monophosphate ester helped to protect the cytidine
derivative from deamination. The chromosome aberra-
tion studies of cells treated with bis(tBuSATE)cytidine
5′-monophosphate ester showed no statistically signifi-
cant increase in aberrations over the concurrent con-
trols.
Experimental Section
General Procedures. All the reagents and anhydrous
solvents were purchased from Aldrich and were used without
further purification. Compounds 1 and 5 were procured from
R. I. Chemical Inc., CA. Thin-layer chromatography was
performed on precoated plates (silica gel 60 F254, EM Science,
NJ) and visualized with UV light and spraying with a solution
of p-anisaldehyde (6 mL), H2SO4 (8.3 mL), and CH3COOH (2.5
mL) in C2H5OH (227 mL) followed by charring. 1H NMR
spectra were referenced using internal standard (CH3)4Si, and
31P NMR spectra were referenced using external standard 85%
H3PO4. Mass spectra were recorded by Mass Consortium, San
Diego, CA, and the College of Chemistry, University of
California, Berkeley. Combustion analyses were performed by
Quantitative Technologies Inc., NJ.
1
nm). H NMR (DMSO-d6) δ 1.17 (s, 18H), 3.07 (m, 4H), 3.33
(s, 3H), 3.95-4.06 (m, 6 H), 4.14-4.21 (m, 2H), 4.3 (m, 1H),
5.42 (d, J ) 5.4 Hz, 1H), 5.81 (d, J ) 5.8 Hz, 1H), 6.49 (br s,
2H), 7.86 (s, 1H), 10.66 (br s, 1H); 13C NMR (50 MHz, CDCl3)
δ 27.2, 27.3, 28.5, 46.4, 46.5, 58.8, 66.4, 68.02, 68.8, 69.7, 82.3,
83.1, 84.6, 87.6, 116.5, 135.8, 151.4, 154.1, 160.0, 205.7; 13P
NMR (80 MHz, DMSO-d6) δ -0.71; MS (API-ES) m/z 664.2
[M - H]-; HRMS (FAB) calcd for C25H41N5O10PS2 666.7232,
+
found 666.7283.
2-Amino-7-(2-O-methyl-â-D-ribofuranosyl)-7H-pyrrolo-
[2,3-d]pyrimidin-4(3H)-one (2). A solution of the compound
16 (2.0 g, 6.65 mmol) in NaOH/H2O (4.09 g/51.1 mL) was
heated at reflux for 7 h. The pH of the solution was adjusted
to 7 with 1 N HCl, and the solvent was removed under reduced
pressure. The residue obtained was purified by flash silica gel
column chromatography and eluted with 20% MeOH in ethyl
acetate to afford 2 (1.79 g, 86%) as a white solid. 1H NMR (200
MHz, DMSO-d6) δ 3.25 (s, 3H), 3.52 (m, 2H) 3.81 (m, 1H), 4.00
(m, 1H), 4.19 (m, 1H), 5.10 (br s, 2H), 5.95 (d, J ) 2.5 Hz, 1H),
6.27 (d, J ) 4 Hz, 1H), 6.33 (br s, 2H), 6.95 (d, J ) 4 Hz, 1H),
10.55 (br s, 1H); 13C NMR (100 MHz, D2O/CD3OD, 9:1) δ 57.8,
60.5, 69.2, 71.7, 82.3, 85.2, 100.9, 102.9, 119.3, 151.5, 153.9,
2-Amino-7-(2-O-methyl-â-D-ribofuranosyl)-7H-pyrrolo-
[2,3-d]pyrimidin-4(3H)-one 5′-[Bis-(S-pivaloyl-2-thioeth-
yl)phosphate] (8). Compound 8 (0.16 g, 29% yield) was syn-
thesized from 7-deaza-N2-(4-monomethoxytrityl)-2′-O-methyl-
guanosine 22 (0.47 g, 0.82 mmol) according to the procedure
used for the synthesis of 7 from 21. 1H-Tetrazole (0.044 g, 0.63
mmol), bis(S-pivaloyl-2-thioethyl) N,N-diisopropylphosphor-
amidite 13 (0.29 g, 0.63 mmol), acetonitrile (11 mL), 3-chloro-
perbenzoic acid (0.21 g, 1.26 mmol, 57-80%) in CH2Cl2 (5.2
mL), and a solution of acetic acid/MeOH/H2O, 3:6:1 (20 mL),
were also used for the synthesis. HPLC retention time was
11.23 min (Phenomenex aqua, C-18, 150 mm × 4.6 mm, 5 µm,
A ) 100 mM triethylammonium acetate, pH 7, B ) 90%
acetonitrile in A, 2-90% B in 9 min, 90% B till 15 min, flow
rate of 1 mL min-1, λ ) 260 nm). 1H NMR (200 MHz, DMSO-
d6) δ 1.14 (s, 18H), 3.06 (m, 4H), 3.31 (s, 3H), 3.96-4.26 (m,
9H), 5.35 (d, J ) 2.6 Hz, 1H), 5.99 (d, J ) 6.6 Hz, 1H), 6.27
(m, 3H), 6.86 (d, J ) 3.6 Hz, 1H), 10.39 (s, 1H); 13C NMR (50
MHz, CDCl3) δ 27.2, 28.4, 28.5, 46.4, 46.5, 58.5, 66.3, 66.4,
67.2, 69.7, 81.9, 82.9, 86.3, 101.3, 103.2, 118.2, 151.5, 152.3,
161.12, 205.6, 205.7; 13P NMR (80 MHz, DMSO-d6) δ -0.72;
MS (API-ES) m/z 663.20 [M - H]-; HRMS (MALDI) calcd for
C26H42N4O10PS2 665.2074, found 665.2071. Anal. (C26H41O10PS2)
H, N. C: calcd, 46.98; found, 46.17.
+
161.7; HRMS (FAB) calcd for C12H17N4O5 297.1193, found
297.1199.
2-Amino-7-(3-deoxy-â-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidin-4(3H)-one (4). Compound 18 (0.4 g, 1.4 mmol)
was refluxed in 2 N aqueous NaOH (40 mL) for 3 h. The
solution was cooled in an ice bath, neutralized with 2 N
aqueous HCl, and evaporated to dryness. The residue obtained
was suspended in MeOH and mixed with silica, and the solvent
was evaporated to give a dry powder. The dry powder was
loaded onto a flash silica gel column and eluted with 10-20%
MeOH in CH2Cl2 to yield 4 (0.3 g, 80%) as white solid. 1H NMR
(400 MHz, DMSO-d6) δ 1.85 (m, 1H), 2.12 (m, 1H), 3.46 (m,
1H), 3.54 (m, 1H), 4.18 (m, 1H), 4.28 (m, 1H), 4.85 (t, J ) 5.6
Hz, 1H), 5.42 (d, J ) 4.4 Hz, 1H), 5.82 (d, J ) 2.4 Hz, 1H),
6.19 (s, 2H), 6.23 (d, J ) 3.6 Hz, 1H), 6.87 (d, J ) 3.6 Hz, 1H),
10.31 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 36.0, 63.8, 75.4,
80.1, 90.3, 110.7, 102.8, 117.7, 151.2, 153.6, 159.3; HRMS
3′-Deoxyguanosine 5′-[Bis(S-pivaloyl-2-thioethyl)phos-
phate] (9). Compound 9 (0.067 g, 30% yield) was synthesized
from compound 27 (0.20 g, 0.35 mmol) following a similar
procedure used for the synthesis of 7 from compound 21. 1H-
Tetrazole (0.02 g, 0.27 mmol), bis(S-pivaloyl-2-thioethyl) N,N-
diisopropylphosphoramidite 13 (0.12 g, 0.27 mmol), 3-chloro-
perbenzoic acid (0.12 g, 0.7 mmol, 57-80%), CH2Cl2 (2.2 mL),
and a solution of acetic acid/water/methanol (5 mL, 3:1:6) were
also used for the synthesis. HPLC retention time was 10.93
min (Phenomenex aqua, C-18, 150 mm × 4.6 mm, 5 µm, A )
100 mM triethylammonium acetate, pH 7, B ) 90% aceto-
nitrile in A, 2-90% B in 9 min, 90% B from 9 to 11 min, flow
+
(FAB) calcd for C11H15N4O4 267.1043, found 267.1239.
2′-O-Methylguanosine 5′-[Bis(S-pivaloyl-2-thioethyl)-
phosphate] (7). N2-(4-Monomethoxytrityl)-2′-O-methylgua-
nosine 21 (0.6 g, 1.05 mmol) was mixed with 1H-tetrazole (0.05
g, 0.7 mmol) and dried over P2O5 in vacuo overnight. The
mixture was suspended in anhydrous acetonitrile (13.8 mL),
bis(S-pivaloyl-2-thioethyl) N,N-diisopropylphosphoramidite 13
(0.32 g, 0.7 mmol) was added, and the reaction mixture was
stirred at ambient temperature for 8 h under an inert
atmosphere. Solvent was removed under reduced pressure.
The residue was cooled to -40 °C, and a solution of 3-chloro-
perbenzoic acid (0.2 g, 1.4 mmol) in CH2Cl2 (10 mL) was added.
The solution was allowed to warm to room temperature over
1 h. Aqueous sodium hydrogen sulfite (10 wt %, 2 mL) was
added to reduce the excess of 3-chloroperbenzoic acid. The
organic phase was separated, diluted with CH2Cl2 (20 mL),
washed with saturated aqueous NaHCO3 (10 mL) and water
(10 mL), dried over Na2SO4, and evaporated to dryness. The
1
rate of 1 mL min-1, λ ) 260 nm). H NMR (DMSO-d6) δ 1.15
(s, 18H), 1.92-2.01 (m, 1H), 2.17-2.28 (m, 1H), 3.04 (t, J )
6.2 Hz, 4H), 3.91-4.23 (m, 6 H), 4.37-4.55 (m, 2H), 5.67 (m,
2H), 6.45 (br s, 2H), 7.75 (s, 1H), 10.61 (s, 1H); 13P NMR (80
MHz, DMSO-d6) δ -0.75; MS (API-ES) m/z 634.2 [M - H]-.
+
HRMS (FAB) calcd for C24H39N5O9PS2 636.1923, found
636.1913. Anal. (C24H38N5O9PS2) H, N. C: calcd, 45.35; found,
44.37.
2-Amino-7-(3-deoxy-â-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidin-4(3H)-one 5′-[Bis(S-pivaloyl-2-thioethyl)-
phosphate] (10). Compound 10 (0.08 g, 23% yield) was

1206 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Prakash et al.
synthesized from 2-(4-monomethoxytrityl)amino-7-(3-deoxy-â-
D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one 28 (0.30
g, 0.52 mmol) according to the procedure used for the synthesis
of 7 from 21. 1H-Tetrazole (0.028 g, 0.40 mmol), bis(S-pivaloyl-
2-thioethyl) N,N-diisopropylphosphoramidite 13 (0.18 g, 0.40
mmol), 3-chloroperbenzoic acid (0.14 g, 0.8 mmol, 57-80%),
and a solution of acetic acid/water/methanol (10 mL, 3:1:6)
were also used for the synthesis. HPLC retention time was
34.78 min (Hamilton RP-1, 250 mm × 21.5 mm, 10 µm, A )
water, B ) acetonitrile, 20-100% B in 65 min, flow rate of 10
mL min-1, λ ) 260 nm). 1H NMR (200 MHz, DMSO-d6) δ 1.16
(s, 18H), 1.91-2.01 (m, 1H), 2.17-2.25 (m, 1H), 3.05 (t, J )
6.2 Hz, 4H), 3.92-4.2 (m, 6 H), 4.35 (br s, 2H), 5.56 (d, J )
4.2 Hz, 1H), 5.76 (d, J ) 2.4 Hz, 1H), 6.24 (m, 3H), 6.77 (d, J
) 3.6 Hz, 1H), 10.36 (s, 1H); 13C NMR (50 MHz, CDCl3) δ 27.2,
28.5, 34.9, 46.5, 46.5, 66.3, 68.9, 75.6, 90.4, 100.0, 102.4, 117.4,
150.8, 152.3, 161.12, 205.6, 205.7; 13P NMR (DMSO-d6) δ
-0.89; HRMS (MALDI) calcd for C25H40N4O9PS2 635.1969,
found 635.1964. Anal. (C24H38N5O9PS2) C, H, N.
2′-O-Methylcytidine 5′-[Bis(S-pivaloyl-2-thioethyl)-
phosphate] (11). Compound 11 (0.12 g, 22% yield) was
synthesized from N4-(4,4′-dimethoxytrityl)-2′-O-methylcytidine
29 (0.48 g, 0.86 mmol) using a similar procedure used for the
synthesis of compound 7 from compound 21. 1H-Tetrazole
(0.06 g, 0.86 mmol), anhydrous acetonitrile (6 mL), bis(S-
pivaloyl-2-thioethyl) N,N-diisopropylphosphoramidite 13 (0.39
g, 0.86 mmol), 3-chloroperbenzoic acid (0.3 g, 1.72 mmol, 57-
80%) in CH2Cl2 (5.5 mL), and a solution of acetic acid/water/
methanol (10 mL, 3:1:6) were also used for the synthesis.
HPLC retention time was 11.12 min (Phenomenex aqua, C-18,
150 mm × 4.6 mm, 5 µm, A ) 100 mM triethylammonium
acetate, pH 7, B ) 90% acetonitrile in A, 2-90% B in 9 min,
90% B from 9 to 12 min, flow rate of 1 mL min-1, λ ) 260
nm). 1H NMR (DMSO-d6) δ 1.18 (s, 18H), 3.12 (t, J ) 6.4 Hz,
4H), 3.39 (s, 3H), 3.69 (t, J ) 4.2 Hz, 1H), 3.93-4.3 (m, 8H),
5.29 (d, J ) 6.2 Hz, 1H), 5.72 (d, J ) 7.4 Hz, 1H), 5.86 (d, J )
4 Hz, 1H), 7.21 (br s, 2H), 7.58 (d, J ) 7.4 Hz, 1H); 13C NMR
(50 MHz, CD3CN) δ 27.4, 29.2, 47.1, 58.9, 66.4, 67.1,67.4, 69.4,
82.3, 84.0, 89.5, 95.0, 141.9, 156.4, 167.1, 206.4; 13P NMR (80
MHz, CD3CN) δ -0.64; MS (AP-ES) m/z 625.69 [M + H]+;
HRMS (MALDI) calcd for C24H40N3O10PS2Na+ 648.1785, found
648.1804. Anal. (C24H40N3O10PS2) H, N. C: calcd, 46.07; found,
45.54.
tion of 5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-D-ribo-
furanose 14 (13.3 g, 44 mmol), anhydrous THF (135 mL), and
CC14 (5.6 mL, 58 mmol) under N2 at -76 °C. After 30 min,
the temperature was raised to -20 °C. In a separate flask, a
suspension of 2-amino-4-chloro-1H-pyrrolo[2,3-d]pyrimidine
(15 g, 89 mmol) in CH3CN (900 mL) was treated with 60%
NaH (3.60 g., 90 mmol) at 15 °C. The reaction mixture was
stirred for 30 min, whereupon the previous reaction mixture
was cannulated with vigorous stirring. The reaction mixture
was stirred for 16 h and then concentrated under reduced
pressure. To the residue water (200 mL) was added and
extracted with ethyl acetate (3 × 200 mL). The organic phase
was dried over anhydrous Na2SO4 and evaporated. The result-
ing oil was purified by flash silica gel column chromatography
and eluted with 50% ethyl acetate in hexane to afford an oil.
The oil was dissolved in MeOH/H2O (200 mL/100 mL), Dowex
WX8-400 (4.8 g) was added, and the mixture was stirred for
16 h at room temperature. The resin was filtered off and the
filtrate was evaporated to yield 15 (10.7 g, 81% yield) as a
white solid. 1H NMR (400 MHz, DMSO-d6) δ 3.47-3.60 (m,
2H), 3.83 (q, J ) 3.6 and 4 Hz, 1H), 4.04 (m, 1H), 4.29 (q, J )
6.4 and 5.2 Hz, 1H), 4.95 (t, J ) 5.6 Hz, 1H), 5.06 (d, J ) 4.8
Hz, 1H), 5.26 (d, J ) 6.4 Hz, 1H), 5.97 (d, J ) 6.4 Hz, 1H),
6.35 (d, J ) 4 Hz, 1H), 6.67 (s, 2H), 7.37 (d, J ) 4 Hz, 1H); MS
(ES) m/z 301.70 [M + H]+.
2-Amino-4-chloro-7-(2-O-methyl-â-D-ribofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidine (16). A solution of the compound
15 (3.0 g, 9.9 mmol) in anhydrous DMF (300 mL) at 15 °C
was treated with 60% NaH (0.42 g, 10.5 mmol). After 30 min,
iodomethane (1.4 g, 9.9 mmol) was added dropwise to the
reaction mixture and stirred at room temperature for 16 h.
The solvent was removed under reduced pressure. The residue
was purified by flash silica gel column chromatography and
eluted with 5-10% MeOH in dichloromethane to afford 16
1
(2.43 g, 80% yield). H NMR (400 MHz, DMSO-d6) δ 3.25 (s,
3H), 3.54 (m, 2H), 3.87 (m, 1H), 4.07 (m, 1H), 4.22 (m, 1H),
5.01 (m, 1H), 5.16 (d, J ) 6.4 Hz, 1H), 6.07 (d, J ) 6.4 Hz,
1H), 6.37 (d, J ) 4 Hz, 1H), 6.70 (s, 2H), 7.40 (d, J ) 4 Hz,
1H); MS (ES) m/z 316.00 [M + H]+.
2-Amino-7-(2,3-anhydro-â-D-ribofuranosyl)-4-methoxy-
7H-pyrrolo[2,3-d]pyrimidine (17). To a mixture of com-
pound 15 (1.8 g, 6.0 mmol) in acetonitrile (80 mL) a solution
of H2O/CH3CN (1:9, 1.08 mL) and R-acetoxyisobutyryl bromide
(3.5 mL, 24 mmol) was added. After 2 h of stirring at room
temperature, saturated aqueous NaHCO3 (170 mL) was added
and the mixture was extracted with ethyl acetate (2 × 200
mL). The combined organic phase was washed with brine (100
mL), dried over anhydrous Na2SO4, and evaporated. The pale-
yellow residue obtained was suspended in anhydrous MeOH
(80 mL) and stirred overnight with 25 mL of DOWEX OH-
resin (previously washed with anhydrous MeOH). The resin
was filtered and washed thoroughly with MeOH, and the
combined filtrate was evaporated to yield 17 (1.82 g, 94%) as
a pale-yellow foam. 1H NMR (200 MHz, DMSO-d6) δ 3.51 (m,
2H), 3.94 (s, 3H), 4.10 (t, J ) 5.76 Hz, 1H), 4.19 (d, J ) 2.6
Hz, 1H), 4.25 (d, J ) 2.62 Hz, 1H), 5. 02 (t, J ) 5.4 Hz, 1H),
6.20 (s, 1H), 6.31 (m, 3H), 7.13 (d, J ) 3.76 Hz, 1H); MS (ES)
m/z 279.1 [M + H]+.
3′-Deoxycytidine 5′-[Bis(S-pivaloyl-2-thioethyl)phos-
phate] (12). Compound 12 (0.07 g, 22% yield) was synthesized
from N4-(4,4′-dimethoxytrityl)-3′-deoxycytidine 30 (0.3 g, 0.57
mmol) following a similar synthetic procedure used for the
synthesis of compound 7 from compound 21. 1H-Tetrazole
(0.04 g, 0.57 mmol), acetonitrile (4 mL), bis(S-pivaloyl-2-
thioethyl) N,N-diisopropylphosphoramidite (0.52 g, 1.14 mmol),
3-chloroperbenzoic acid (0.2 g, 1.14 mmol, 57-80%) in CH2Cl2
(3.6 mL), and acetic acid/water/methanol (10 mL, 3:1:6) were
also used for the synthesis. HPLC retention time was 12.79
min (Phenomenex aqua, C-18, 250 mm × 4.6 mm, 5 µm, A )
100 mM triethylammonium acetate, pH 7, B ) 90% aceto-
nitrile in A, 2-80% B in 15 min, flow rate of 1 mL min-1, λ )
1
260 nm). H NMR (200 MHz, DMSO-d6) δ 1.17 (s, 18H), 1.84
(m, 2H), 3.11 (t, J ) 6.4 Hz, 4H), 3.93-4.31 (m, 8H), 4.39 (m,
1H), 5.55 (d, J ) 4.2 Hz, 1H), 5.67 (dd, J ) 7.4 and 1.8 Hz,
2H), 7.1 (br s, 2H), 7.56 (d, J ) 7.4 Hz,1H); 13C NMR (50 MHz,
CD3CN) δ 27.5, 29.3, 29.4, 34.0, 47.2, 67.1, 67.2, 69.0, 69.1,
76.7, 79.8, 94.9, 141.7, 157.3, 167.3, 206.5; 13P NMR (80 MHz,
CD3CN) δ -0.71; MS (AP-ES) m/z 596.1 [M + H]+; HRMS
(MALDI) calcd for C23H38N3O9PS2Na+ 618.1679, found 618.1600.
2-Amino-7-(3-deoxy-â-D-ribofuranosyl)-4-methoxy-7H-
pyrrolo[2,3-d]pyrimidine (18). A solution of LiEt3BH/THF
(1 M, 75 mL, 75 mmol) was added dropwise at 0 °C (ice bath)
to a deoxygenated (argon, 15 min) solution of compound 17
(1.92 g, 6.90 mmol) under argon atmosphere. Stirring was
continued for 4 h at 0 °C. The reaction mixture was acidified
with 5% aqueous acetic acid (110 mL), then purged with argon
for 1 h and evaporated. The residue obtained was purified by
flash silica gel column chromatography and eluted with 10-
15% MeOH in CH2Cl2 to yield 18 (1.01 g, 60%) as a colorless
foam. 1H NMR (400 MHz, DMSO-d6) δ 1.87 (m, 1H), 2.15 (m,
1H), 3.51 (m, 2H), 3.90 (s, 3H), 4.21 (m, 1H), 4.33 (m, 1H),
4.88 (t, J ) 5.64 Hz, 1H), 5.44 (d, J ) 4.44 Hz, 1H), 5.92 (d, J
) 2.80 Hz, 1H), 6.15 (s, 2H), 6.23 (d, J ) 3.60 Hz, 1H), 7.05
Bis(S-pivaloyl-2-thioethyl) N,N-Diisopropylphosphor-
amidite (13). Bis(S-pivaloyl-2-thioethyl) N,N-diisopropylphos-
phramidite was synthesized according to the literature pro-
cedure.21 1H NMR (200 MHz, CDCl3) δ 1.15-1.28 (m, 30H),
3.10 (t, J ) 6.5 Hz, 4H), 3.45-3.83 (m, 6H); 13C NMR (50 MHz,
CDCl3) δ 24.5, 27.3, 30.0, 42.8, 43.1, 46.3, 62.3, 206.1; 13P NMR
(80 MHz, CDCl3) δ 147.12.
2-Amino-4-chloro-7-(â-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (15). Hexamethylphosphorous triamide (10.65
mL, 55 mmol) was added portionwise over 30 min to a solu-
+
(d, J ) 4.0 Hz, 1H); HRMS (FAB) calcd for C12H17N4O4
281.1244, found 281.1252.

Thioethyl Esters as Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1207
2′-O-Methyl-3′,5′-O-[1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl]guanosine (19). The 2′-O-methylguanosine 1 (10 g,
36.46 mmol) was dried over P2O5 under reduced pressure
overnight. It was then dissolved in anhydrous pyridine (160
mL), 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (17.2 mL,
54.69 mmol) was added, and the reaction mixture was stirred
at room temperature for 8 h under an inert atmosphere. The
solvent was removed under reduced pressure, and the residue
was partitioned between diethyl ether (200 mL) and water (200
mL). The mixture was then stirred for 5 min, and the
precipitate formed was filtered. The sticky solid obtained was
triturated with dichloromethane (100 mL), and the white solid
obtained was filtered and washed with diethyl ether and dried
under reduced pressure to yield 19 (16.23 g, 87% yield) as
white solid. 19. Rf ) 0.38 (10% MeOH in CH2Cl2); 1H NMR
(200 MHz, DMSO-d6) δ 1.05 (m, 28H), 3.35 (s, 3H), 3.90-4.15
(m, 4H), 4.47 (m, 1H), 5.77 (s, 1H), 6.51 (br s, 2H), 7.74 (s,
1H), 10.68 (s, 1H); 13C NMR (50 MHz, DMSO-d6) δ 12.0, 12.2,
12.3, 12.6, 16.8, 17.1, 17.2, 58.7, 60.1, 69.8, 80.8, 82.7, 86.1,
116.7, 133.8, 150.3, 153.8, 156.6; MS (AP-ES) m/z 538.7 [M -
H]-. Anal. (C23H41N5O6Si2) C, H, N.
2-Amino-7-[2-O-(methyl)-3,5-O-(1,1,3,3-tetraisopropyl-
disiloxane-1,3-diyl)-â-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidin-4(3H)-one (20). Compound 20 (2.7 g, 85%) was
synthesized from 2-amino-7-[2-O-(methyl)-â-D-ribofuranosyl)-
7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one 2 (1.7 g, 6.13 mmol),
anhydrous pyridine (27 mL), and 1,3-dichloro-1,1,3,3-tetra-
isopropyldisiloxane (3.85 mL, 12.26 mmol) following the
procedure used for the synthesis of 19 except that the residue
obtained after workup was purified by flash silica gel column
chromatography and eluted with 5-10% MeOH in CH2Cl2. Rf
) 0.3 (5% MeOH in CH2Cl2). 1H NMR (200 MHz, DMSO-d6) δ
1.00 (m, 28H), 3.30 (s, 3H), 3.81-4.10 (m, 4H), 4.27 (m, 1H),
5.88 (d, J ) 1.4 Hz, 1H), 6.25 (d, J ) 3.7 Hz, 1H), 6.28 (br s,
2H), 6.75 (d, J ) 3.7, 1H), 10.40 (s,1H); 13C NMR (50 MHz,
CDCl3) δ 12.5, 13.0, 13.2, 16.9, 17.1, 17.3, 59.2, 60.1, 69.9, 80.8,
84.4, 87.4, 101.3, 102.3, 117.9, 150.5, 152.6, 161.1; MS (AP-
ES) m/z 537.3 [M - H]-. Anal. (C24H42N4O6Si2) C, H. N: calcd,
10.40; found, 9.80.
8.33 mmol), and triethylamine (0.60 mL, 4.20 mmol) in THF
(17 mL) were also used. The residue obtained after workup
was purified by flash silica gel column chromatography and
eluted with 5% MeOH in CH2Cl2. Rf ) 0.2 (5% MeOH in
CH2Cl2). 1H NMR (DMSO-d6) δ 2.85 (s, 3H), 3.32-3.64 (m, 4H),
3.71 (s, 3H), 3.97 (q, J ) 6.20 and 5.40 Hz, 1H), 4.84 (m, 2H),
5.28 (d, J ) 3.4 Hz, 1H), 6.19 (d, J ) 3.6 Hz, 1H), 6.85 (m,
2H), 6.88 (d, J ) 3.6 Hz, 1H), 7.14-7.30 (m, 12H), 7.43 (s,
1H), 10.38 (s, 1H); 13C NMR (50 MHz, CDCl3) δ 55.1, 57.8,
62.0, 69.6, 70.2, 82.5, 83.7, 87.2, 101.6,102.9, 113.2, 118.8,
127.0, 127.9, 128.5, 128.6, 129.9, 136.4, 144.4, 144.6, 149.5,
150.1, 158.3, 159.8; MS (AP-ES) m/z 567.2 [M - H]-.
2′,5′-O-Bis(tert-butyldimethylsilyl)-3′-deoxygua-
nosine (23). The 3′-deoxyguanosine 3 (1.45 g, 5.43 mmol) was
mixed with imidazole (0.92 g, 13.6 mmol) and dried over P2O5
under reduced pressure overnight. The reaction mixture was
dissolved in anhydrous DMF (50 mL) and tert-butyldimethyl-
silyl chloride (2.05 g, 27.15 mmol) was added. The resulting
mixture was stirred at room temperature for 12 h under argon
atmosphere. The reaction mixture was diluted with water (100
mL) and extracted with ethyl acetate (2 × 60 mL). The organic
phase was dried over anhydrous Na2SO4, and solvent was
removed under reduced pressure. The residue obtained was
purified by flash silica gel column chromatography and eluted
with 5% MeOH in CH2Cl2 to yield 23 (1.42 g, 53% yield).
2-Amino-7-[2,5-O-bis(acetyl)-3-deoxy-â-D-ribofuranosyl)-
7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one (24). Compound 4
(0.5 g, 1.9 mmol) was mixed with DMAP (0.09 g, 0.7 mmol)
and dried under reduced pressure overnight. The sample was
suspended in acetonitrile (10 mL) and triethylamine (0.63 mL,
3.8 mmol), and acetic anhydride (0.36 mL, 3.8 mmol) was
added. The reaction mixture was stirred at room temperature
for 5 h under argon atmosphere. The solvent was removed
under reduced pressure, and the oily residue obtained was
partitioned between water (15 mL) and CH2Cl2 (15 mL). The
organic phase was separated and extracted with brine (20 mL),
dried over anhydrous Na2SO4, and evaporated to yield 24 (0.63
1
g, 95% yield). H NMR (400 MHz, DMSO-d6) δ 1.99 (s, 3H),
2.05 (s, 3H), 2.08-2.14 (m, 2H), 4.04 (dd, J ) 6.4 and 5.6 Hz,
1 H), 4.23 (dd, J ) 2.8 and 9.2 Hz, 1H), 4.35 (m, 1H), 5.34 (d,
J ) 6.8 Hz, 1H), 5.97 (d, J ) 2 Hz, 1H), 6.27 (m, 3H), 6.84 (d,
J ) 3.6 Hz, 1H), 10.39 (s, 1H); MS (FAB) m/z 349.23 [M -
H]-.
N2-(4-Monomethoxytrityl)-2′-O-methylguanosine (21).
Compound 19 (5.32 g, 10.2 mmol) was mixed with DMAP (1.25
g, 10.2 mmol) and dried under reduced pressure over P2O5.
The mixture was dissolved in anhydrous pyridine (102 mL),
p-anisylchlorodiphenylmethane (9.44 g, 30.6 mmol) was added,
and the reaction mixture was stirred at room temperature
under argon atmosphere for 16 h. The solvent was removed
under reduced pressure, and the residue obtained was co-
evaporated with CH2Cl2 (3 × 50 mL). To the residue, a mixture
of triethylamine trihydrofluoride (8.45 mL, 51 mmol) and
triethylamine (3.56 mL, 25.5 mmol) in THF (102 mL) was
added. The reaction mixture was stirred at room temperature
for 24 h, diluted with ethyl acetate (150 mL), and washed with
water (100 mL), aqueous NaHCO3 (5%, 100 mL), and brine
(100 mL). The organic phase was dried over anhydrous Na2SO4
and concentrated to dryness, and the residue obtained was
dissolved in 5% MeOH in dichloromethane (20 mL) and added
dropwise into vigorously stirring hexane (800 mL). White
precipitate formed was filtered and dried to yield 21 (7.81 g,
3′,5′-O-Bis(tert-butyldimethylsiliyl)-2′-O-methylcyti-
dine (25). The 2′-O-methylcytidine 5 (3 g, 11.7 mmol) was
mixed with imidazole (7.94 g, 116.6 mmol) and dried under
reduced pressure overnight. The mixture was dissolved in
anhydrous DMF (8 mL), tert-butyldimethylsilyl chloride (8.82
g, 58.5 mmol) was added, and the reaction mixture was stirred
at room temperature for 18 h under argon atmosphere. The
reaction mixture was diluted with water (100 mL) and washed
with ethyl acetate (2 × 60 mL). The organic phase separated,
dried over anhydrous Na2SO4, and evaporated. The residue
obtained was purified by flash silica gel column chromatog-
raphy and eluted with 60% ethyl acetate in hexane to yield
25 (5.52 g, 97.5%). Rf ) 0.33 (10% MeOH in CH2Cl2). 1H NMR
(200 MHz, DMSO-d6) δ 0.05 (s, 6H), 0.08 (s, 6H), 0.87 (m, 18H),
3.30 (s, 3H), 3.64 (m, 1H), 3.71 (br s, 1H), 3.84-3.95 (m, 2H),
4.17 (t, J ) 6.6 and 5 Hz, 1H), 5.68 (d, J ) 7.4 Hz, 1H), 5.82
(d, J ) 2.4 Hz, 1H), 7.17 (br s, 2H), 7.82 (d, J ) 7.4 Hz 1H);
13C NMR (50 MHz,CDCl3) δ -5.6, -5.4, -5.1, -4.6, 18.0, 18.3,
25.6, 25.9, 58.2, 60.3, 67.9, 82.0, 84.1, 88.1, 93.9, 141.2, 155.7,
166.0; MS (ES) m/z 484.3 [M - H]-. Anal. (C22H43N3O5Si2) C,
H, N.
2′,5′-O-Bis(tert-butyldimethylsilyl)-3′-deoxycytidine (26).
Compound 26 (1.27 g, 79% yield) was synthesized from
3′-deoxycytidine 6 (0.8 g, 3.54 mmol), imidazole (2.41 g, 35.4
mmol), DMF (2.5 mL), and tert-butyldimethylsilyl chloride
(2.68 g, 17.78 mmol) according to the procedure used for the
synthesis of compound 25. Rf ) 0.24 (5% MeOH in CH2Cl2).
1H NMR (200 MHz, DMSO-d6) δ 0.05 (m, 6H), 0.09 (s, 6H),
0.80 (s, 9H), 0.84 (s, 9H), 1.6-1.70 (m, 1H), 1.79-1.93 (m, 1H),
3.74 (dd, J ) 2.2 and 9.6 Hz, 1H), 4.00 (dd, J ) 2.4 and 9.4
Hz, 1H), 4.22 (m, 1H), 4.33 (m, 1H), 5.61 (m, 2H), 7.07 (br s,
1
72%). Rf ) 0.45 (10% MeOH in CH2Cl2). H NMR (DMSO-d6)
δ 2.87 (s, 3H), 3.35-3.53 (m, 2H), 3.61-3.68 (m, 2H), 3.71 (s,
3H), 3.91 (q, J ) 5.2 and 6.52 Hz, 1H), 4.91 (m, 2H), 5.17 (d,
J ) 3.3 Hz, 1H), 6.86 (m, 2H), 7.14-7.34 (m, 12H), 7.64 (s,
1H), 7.88 (s, 1H), 10.66 (s, 1H); 13C NMR (50 MHz, DMSO-d6)
δ 55.0, 57.2, 60.4, 68.3, 69.7, 82.6, 83.9, 85.3, 113.0, 117.3,
126.5, 127.4, 127.6, 128.4, 129.8, 135.8, 136.6, 144.3, 144.7,
149.0, 150.9, 156.4, 157.7; MS (AP-ES) m/z 568.1 [M - H]-.
Anal. (C31H31N5O6) C, H. N: calcd, 12.30; found, 11.80.
2-[(4-Monomethoxytrityl)amino]-7-[2-O-(methyl)-â-D-
ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one (22).
Compound 22 (0.72 g, 75% yield) was synthesized from
compound 20 (0.9 g, 1.67 mmol) according to the procedure
used for the synthesis of 21. DMAP (0.20 g, 1.67 mmol),
anhydrous pyridine (16 mL), p-anisylchlorodiphenylmethane
(1.03 g, 3.34 mmol), triethylamine trihydrofluoride (1.37 mL,

1208 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Prakash et al.
2H), 7.91 (d, J ) 7.4 Hz, 1H); 13C NMR (50 MHz, CDCl3) δ
-5.6, -5.4, -5.3, -4.5, 17.3, 18.4, 25.7, 25.9, 32.6, 62.8, 77.4,
81.5, 93.1, 141.6, 155.9, 165.9; MS (AP-ES) m/z 454.21 [M -
H]-. Anal. (C21H41N3O4Si2) C, H, N.
N4-(4,4′-Dimethoxytrityl)-3′-deoxycytidine (30). Com-
pound 30 (0.36 g, 43% yield) was prepared from 26 (0.67, 1.47
mmol), DMAP (0.18 g, 1.47 mmol), anhydrous pyridine (4 mL),
4,4′-dimethoxytrityl chloride (1.00 g, 2.95 mmol), THF (15 mL),
triethylamine trihydrofluoride (1.21 mL, 7.35 mmol), and
triethylamine (0.51 mL, 3.68 mmol) according to procedure
used for the synthesis of compound 21. Rf ) 0.41 (5% MeOH
in CH2Cl2). 1H NMR (DMSO-d6) δ 1.66 (m, 1H), 1.85 (m, 1H),
3.47 (m, 1H), 3.63 (m, 1H), 3.71 (s, 6H), 4.00 (br s, 1H), 4.19
(m, 1H), 4.96 (t, J ) 5.2 Hz, 1H), 5.39 (br s, 1H), 5.53 (s, 1H),
6.17 (br s, 1H), 6.83 (d, J ) 8.8 Hz, 4H), 7.04-7.22 (m, 9H),
7.77 (d, J ) 7.6 Hz, 1H), 8.27 (br s, 1H); 13C NMR (50
MHz,CD3CN) δ 33.7, 55.9, 63.1, 70.8, 77.0, 82.2, 94.9, 113.8,
114.2, 127.4, 127.7, 128.6, 129.6, 129.0, 130.0, 131.0, 137.58,
138.1, 140.5, 146.0, 156.3, 159.5; HRMS (FAB) calcd for
C30H32N3O6+ 530.2286, found 530.2291. Anal. (C30H31N3O6) H.
C: calcd, 68.04; found, 67.29. N: calcd, 7.93; found, 7.29.
5-Bromo-3′,5′-O-bis[(tert-butyldimethyl)silyl]-2′-O-
methylcytidine (31). Compound 25 (2.76 g, 5.68 mmol) was
dissolved in acetonitrile (19.4 mL), LiBr (0.62 g, 7.18 mmol)
was added, and the reaction mixture was stirred to get a clear
solution. To this, ammonium ceric(IV) nitrate (6.24 g, 11.37
mmol) was added, and the reaction mixture was stirred at
room temperature for 3 h. Solvent was removed under reduced
pressure. The residue obtained was taken in ethyl acetate (100
mL) and washed with water (80 mL). The organic phase was
separated, dried over anhydrous Na2SO4, and evaporated. The
residue obtained was purified by silica gel column chroma-
tography and eluted with 5% MeOH in CH2Cl2 to yield 31 (2.66
g, 83%). Rf ) 0.57 (10% MeOH in CH2Cl2). 1H NMR (200 MHz,
DMSO-d6) δ 0.00 (s, 6H), 0.05 (s, 6H), 0.79 (s, 9H), 0.84 (s,
9H), 3.29 (s, 3H), 3.61-3.87 (m, 4H), 4.12 (t, J ) 5.2 Hz, 1H),
5.74 (d, J ) 3.6 Hz, 1H), 6.99 (br s, 1H), 7.84 (br s, 2H); 13C
NMR (50 MHz,CDCl3) δ -5.3,-4.9, -4.5, 18.1, 18.8, 25.7, 26.3,
58.2, 60.8, 68.2, 83.4, 84.0, 87.6, 88.4, 141.0, 154.4, 162.6; MS
(AP-ES) m/z 565.1 and 566.1 [M + H]+; HRMS (FAB) calcd
for C22H43BrN3O5Si2+ 564.1922, found 564.1925. Anal. (C22H42-
BrN3O5Si2) H, N. C: calcd, 46.60; found, 45.64. Br: calcd,
14.15; found, 15.83.
N2-(p-Anisyldiphenylmethyl)-3′-deoxyguanosine (27).
Compound 23 (0.36 g, 0.73 mmol) was dissolved in anhydrous
pyridine (5.0 mL), and p-anisylchlorodiphenylmethane (0.25
g, 0.81 mmol) was added. The reaction mixture was stirred at
room temperature overnight, evaporated in vacuo, and co-
evaporated once with acetonitrile. The resulting residue was
purified by flash silica gel column chromatography and eluted
with methanol/dichloromethane (1:19). Fractions containing
the product were pooled together and evaporated in vacuo to
yield the desired intermediate (0.56 g). To this compound in
THF (20 mL) was added tetrabutylammonium fluoride on
silica (6.62 g, 10 equiv), and the resulting mixture stirred at
room temperature for 2 h. The supernatant was decanted and
evaporated in vacuo. The crude product obtained was purified
by flash silica gel chromatography and eluted with MeOH/
CH2Cl2 (1:9) to yield 27 (23.4%, 0.65 g). 1H NMR (DMSO-d6) δ
1.17-1.68 (m, 2H), 3.20-3.50 (m, 2H), 3.71 (s, 3H), 3.92 (br s,
1H), 4.01 (m, 1H), 4.85 (t, J ) 5 Hz, 1H), 5.09 (d, J ) 3.6 Hz,
1H), 5.12 (s, 1H), 6.86 (d, J ) 8.6 Hz, 2H), 7.10-7.4 (m, 11H),
7.62 (s, 1H), 7.83 (s, 1H), 10.59 (br s, 1H); 13C NMR (CD3OD)
δ 34.0, 54.7, 63.2, 70.8, 74.6, 81.2, 91.8, 92.1, 112.9, 117.3,
126.8, 127.7, 128.8, 130.3, 136.8, 137.4, 145.0, 145.1, 149.7,
151.2, 158.15, 158.8; MS (API-ES) m/z 540.0 [M + H]+; HRMS
(FAB) calcd for C30H30N5O5+ 540.2276, found 540.2247. Anal.
(C30H29N5O5‚H2O) H, N. C: calcd, 64.64; found, 65.57.
2-[(p-Anisyldiphenylmethyl)amino]-7-[3-deoxy-â-D-ribo-
furanosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one (28).
Compound 24 (0.63 g, 1.8 mmol) was dissolved in anhydrous
pyridine (15.0 mL), and p-anisylchlorodiphenylmethane (1.2
g, 3.6 mmol) was added. The mixture was stirred at room
temperature overnight, evaporated in vacuo, and coevaporated
once with acetonitrile. The residue was purified by flash silica
gel column chromatography using MeOH/CH2Cl2 (1:19) as the
eluent. Fractions containing the product were pooled together
and evaporated in vacuo to give the desired intermediate (0.94
g, 84%). This intermediate was dissolved in 2 N aqueous NaOH
(50 mL) and dioxane (10 mL) and stirred at room temperature
for 18 h. The reaction mixture was neutralized with 0.5 N
aqueous HCl at 0 °C. The solid separated was filtered and
dried under reduced pressure to yield 28 (0.73 g, 74% yield)
as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 1.63 (m, 1H),
1.81 (m, 1H), 3.36-3.42 (m, 1H), 3.42-3.50 (m, 1H), 3.70 (s,
3H), 3.87 (br s, 1H), 4.01 (m, 1H), 4.79 (t, J ) 5.2 Hz, 1H),
4.91 (d, J ) 4.0 Hz, 1H), 5.17 (d, J ) 1.6 Hz, 1H), 6.14 (d, J )
3.6 Hz, 1H), 6.84 (m, 3H), 7.15-7.39 (m, 12H), 10.30 (s, 1H);
13C NMR (100 MHz, DMSO-d6) δ 35.5, 55.6, 63.4, 70.3, 75.5,
80.3, 90.9, 101.4, 102.2, 113.5, 118.5, 127.0, 127.7, 128.1, 128.2,
129.0, 129.1, 130.7, 137.6, 145.8, 146.0, 149.2, 150.2, 158.3,
159.0; HRMS (FAB) calcd for C31H30N4O5Na+ 561.2108, found
561.2116.
5-Bromo-3′-O-[(tert-butyldimethyl)silyl]-2′-O-methyl-
cytidine (32). Compound 31 (2.66 g, 4.7 mmol) was dissolved
in 80% acetic acid in water (44 mL) and heated at 50 °C for 6
h. The solvent was removed under reduced pressure, and the
residue obtained was purified by flash silica gel column
chromatography and eluted with 5% MeOH in CH2Cl2 to yield
1
32 (0.85 g, 40%). Rf ) 0.31 (5% MeOH in CH2Cl2). H NMR
(200 MHz, DMSO-d6) δ 0.07 (s, 6H), 0.86 (s, 9H), 3.39 (s, 3H),
3.44-3.6 (m, 2H), 3.69-3.9 (m, 2H), 4.24 (m, 1H), 5.29 (t, J )
4.4 Hz, 1H), 5.76 (d, J ) 3.2 Hz, 1H), 7.06 (br s,1H), 7.88 (br
s, 1H), 8.39 (s, 1H); 13C NMR (50 MHz, CD3OD) δ -4.9, -4.6,
19.0, 26.2, 58.9, 62.3, 70.2, 73.5, 85.5, 88.6, 90.2, 143.7, 157.3,
164.4; MS (AP-ES) m/z 450.1 and 453.1 [M + H]+.
5-Bromo-2′-O-methylcytidine 5′-[Bis(S-pivaloyl-2-thio-
ethyl)phosphate] (33). 5-Bromo-3′-O-(tert-butyldimethyl)-
silyl-2′-O-methylcytidine 32 (0.09 g, 0.21 mmol) was mixed
with 1H-tetrazole (0.03 g, 0.42 mmol) and dried over P2O5 in
vacuo overnight. To this mixture anhydrous acetonitrile (2 mL)
and bis(S-pivaloyl-2-thioethyl) N,N-diisopropylphosphoramid-
ite 13 (0.2 g, 0.42 mmol) were added at 0 °C. The reaction
mixture was allowed to come to room temperature and stirred
for 4 h under inert atmosphere. Solvent was removed in vacuo,
the residue obtained was cooled to -40 °C, and a solution of
3-chloroperbenzoic acid (0.07 g, 0.42 mmol, 57-80%) in CH2Cl2
(2 mL) was added. The solution was allowed to warm to -10
°C over 2 h. Sodium hydrogen sulfite (10% aqueous solution,
2 mL) was added to reduce the excess of 3-chloroperbenzoic
acid. The organic phase was separated, diluted with CH2Cl2
(30 mL), washed with saturated aqueous NaHCO3 (20 mL) and
water (20 mL), dried over Na2SO4, and evaporated to dryness.
The residue was dissolved in THF (2.1 mL), and triethylamine
trihydrofluoride (0.17 g, 1.1 mmol) was added. The reaction
mixture was stirred at room temperature for 18 h. The solvent
was removed under reduced pressure to yield an oil. The oil
was dissolved in ethyl acetate (30 mL) and was washed with
N4-(4,4′-Dimethoxytrityl)-2′-O-methylcytidine (29). Com-
pound 29 (1.8 g, 87%) was synthesized from compound 25 (1.75
g, 3.6 mmol), DMAP (0.06 g, 0.36 mmol), anhydrous pyridine
(10. 4 mL), 4,4′-dimethoxytrityl chloride (2.44 g, 7.2 mmol),
triethylamine trihydrofluoride (2.90 g, 18 mmol), and triethyl-
amine (0.91 g, 9 mmol) in THF (36 mL) according to the
procedure used for the synthesis of compound 21. The residue
obtained after workup was purified by flash silica gel column
chromatography and eluted with ethyl acetate/hexane, 60:40,
to yield 29. Rf ) 0.41 (5% MeOH in CH2Cl2). 1H NMR (DMSO-
d6) δ 3.29 (s, 3H), 3.55-3.60, (m, 2H), 3.69 (s, 6H), 3.70-3.78
(m, 2H), 3.98 (m, 1H), 5.00 (d, J ) 6.2 Hz, 2H), 5.69 (d, J )
4.6 Hz, 1H), 6.20 (d, J ) 5.2 Hz, 1H), 6.81 (d, J ) 8.8 Hz, 4 H),
7.10-7.60 (m, 9H), 7.75 (d, J ) 7.2 Hz), 8.32 (br s, 1H); 13C
NMR (CDCl3) δ 55.1, 58.5, 59.3, 67.3, 70.0, 83.2, 84.1, 89.5,
94.8, 112.9, 113.4, 127.3, 127.6, 128.3, 128.4, 128.7, 135.7,
135.9, 142.0, 144.1, 155.2, 158.5, 165.4; HRMS (FAB) calcd
for C31H34N3O7+ 560.2325, found 560.2319. Anal. (C31H33N3O7)
C, H, N.

Thioethyl Esters as Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1209
water (20 mL), 5% aqueous NaHCO3, and brine (20 mL). The
organic phase was dried over anhydrous Na2SO4 and evapo-
rated. The residue was dissolved in 20% MeOH in water and
purified by HPLC on C-18 column (Luna C18, 250 mm × 2.12
mm, A ) water, B ) acetonitrile, 20-100% B in 65 mL, flow
rate of 10 mL min-1, λ ) 260 nm) to yield 33 (0.054 g, 37%
yield). HPLC retention time was 11.55 min (Phenomenex aqua,
C-18, 150 mm × 4.6 mm, 5 µm, A ) 100 mM triethylammo-
nium acetate, pH 7, B ) 90% acetonitrile in A, 2-100% B in
9 min,100% B till 25 min, flow rate of 1 mL min-1, λ ) 260
nm). 1H NMR (200 MHz, DMSO-d6) δ 1.17 (s, 18H), 3.11 (t, J
) 6.2 Hz, 4H,), 3.39 (s, 3H), 3.75 (t, J ) 4.8 Hz, 1H), 3.93-4.3
(m, 8H), 5.23 (d, J ) 6.4 Hz, 1H), 5.8 (d, J ) 3.8 Hz, 1H), 7.07
(br s, 1H), 7.89 (s, 1H) 7.94 (br s, 1H); 13C NMR (50 MHz,
CD3CN) δ 27.5, 29.3, 29.4, 47.2, 59.0, 66.8, 66.9, 67.2, 69.0,
82.5, 82.7, 84.2, 87.7, 89.6, 142.5, 155.1, 163.4, 206.5; 13P NMR
(80 MHz, CD3CN) δ -0.34; MS (AP-ES) m/z 702.00 and 704.00
[M - H]-; HRMS (MALDI) calcd for C24H39BrN3O10PS2Na+
726.0890 and 728.0890, found 726.0893 and 728.0860.
Inhibition of HCV RNA Replication in Cells. The assay
previously described in the literature was used to determine
the inhibition of HCV RNA replication in a subgenomic
bicistronic replicon in HBI10A cells by in situ RPA.12
Inhibition of NS5B Activity in Vitro. Inhibition of the
enzymatic activity of HCV NS5B∆21 by nucleoside analogue
triphosphates was determined as previously described.12
Cytotoxicity Assay.33 Cell cultures were prepared by
plating at 2.0 × 104 cells/well of a 96-well plate in 100 µL of
media. The next day 100 µL of cell culture media containing
a 2 × level of compound was added per well and the plates
were incubated at 37 °C and 5% CO2 for a specified period of
time. After the incubation period, 20 µL of CellTiter 96
Aqueous One Solution Cell Proliferation Assay reagent (MTS)
(Promega) was added to each well, the plates were incubated
at 37 °C and 5% CO2 for 1 h, and the absorbance at 490 nm
was read using a plate reader.
Intracellular Metabolism Studies. The intracellular
metabolism of 5-[3H] derivatives of 2′-O-methylcytidine 5 and
5′-bis(SATE)-2′-O-methylcytidine monophosphates ester 11 in
Huh-7 and HBI10A cell lines was studied using the procedure
reported in the literature.12
Assay for Chromosomal Aberrations in Chinese Ham-
ster Ovary Cells. Compound 11 was evaluated for its
potential to cause chromosome aberrations in Chinese hamster
ovary (CHO, clone WBL) cells. Compound 11 was tested with
and without a microsomal enzyme activation system (S-9)
prepared from the livers of xenobiotic-treated rats. A solution
of 11 in DMSO was used in this study and diluted 100-fold
into culture medium. Exponentially growing monolayers were
treated for 3 h with test compound with S-9 (in serum-free
medium) or without S-9 (in medium with 10% fetal bovine
serum), washed and allowed to recover until harvest at 20 h
after beginning of the treatment, when the cells were fixed
for analysis of chromosome aberrations (about 1.5 normal cell
cycle lengths). Three additional sets of cultures (without S-9)
were treated for 20 h and harvested at 20, 30, and 42 h after
dosing. Colcemid was added 1-2 h before harvest to arrest
cells in metaphase. Cytotoxicity was assessed as reductions
in relative cell growth (population doubling) or monolayer
confluence, abnormal cell morphology, and inhibition of cell
division. Known chromosome-breaking agents, Cyclophospha-
mide (CP) with S-9 activation and mitomycin C (MMC) without
S-9 activation, were tested as positive controls with compound
11. 2′,3′-Dideoxycytidine (ddC) was also used as a positive
control. The doses for the chromosome aberration assay of
compound 11 were based on a preliminary solubility study in
culture medium, and top doses for scoring aberrations were
selected to suppress growth by about 50%.
References
(1) Nguyen, T. T.; Gates, A. T.; Gutshall, L. L.; Johnston, V. K.;
Gu, B.; Duffy, K. J.; Sarisky, R. T. Resistance profile of a
hepatitis C virus RNA-dependent RNA polymerase benzothia-
diazine inhibitor. Antimicrob. Agents Chemother. 2003, 47,
3525-3530.
(2) Lamarre, D.; Anderson P. C.; Balley, M.; Beaulleu, P.; Bolger,
G.; Bonneau, P.; Bo¨s, M.; Cameron, D. R.; Cartier, M.; Cordin-
gley, M. G.; Faucher, A.-M.; Goudreau, N.; Kawai, S. H.; KukoiJ,
G.; Lagace, L.; LaPlante, S. R.; NarJes, H.; Poupart, M.-A.;
Rancourt, J.; SentJens, R. E.; George, R. St.; Simoneau, B.;
Stelnmann, G.; Thibeault, D.; Tsantrizos, Y. S.; Weldon, S. M.;
Yong, C.-L.; Llinas-Brunet, M. An NS3 protease inhibitor with
antiviral effects in humans infected with hepatitis C virus.
Nature 2003, 02099, 1-4.
(3) Hoofnagle, J. H. Hepatitis C: the clinical spectrum of disease.
Hepatology 1997, 26, S15-S20.
(4) Tan, S. L.; Arnim, P.; Shi, Y.; Sonenberg, N. Hepatitis C
theraputics: current status and emerging strategies. Nat. Rev.
Drug Discovery 2002, 1, 867-881.
(5) Hu¨gle, T.; Cerny, A. Current therapy and new molecular
approaches to antiviral treatment and prevention of hepatitis
C. Rev. Med. Virol. 2003, 13, 361-371.
(6) Rosenberg, S. Recent advances in the molecular biology of
Hepatitis C virus. J. Mol. Biol. 2001, 131, 451-464.
(7) Lohmann, V.; Korner, F.; Koch, J.-O.; Herian, U.; Theilmann,
L.; Bartenschlager, R. Replication of subgenomic hepatitis C
virus RNAs in a hepatoma cell line. Science 1999, 285, 110-
113.
(8) De Francesco, R. Presented at the 7th International Meeting
on Hepatitis C Virus and Related Viruses (Molecular Virology
and Pathogenisis), Gold Coast, Queensland, Australia, 2000.
(9) Moradpour, D.; Bieck, E.; Hugle, T.; Wels, W.; Wu, J. Z.; Hong,
Z.; Blum, H. E.; Bartenschlager, R. Functional properties of a
monoclonal antibody inhibiting the hepatitis C virus RNA-
dependent RNA polymerase. J. Biol. Chem. 2002, 277, 593-
601.
(10) Tomei, L.; Altamura, S.; Bartholomew, L.; Biroccio, A.; Ceccacci,
A.; Pacini, L.; NarJes, F.; Gennari, N.; Bisbocci, M.; Incitti, I.;
Orsatti, L.; Harper, S.; Stansfield, I.; Rowley, M.; De Francesco,
R.; Migliaccio, G. Mechanism of action and antiviral activity of
benzimidazole-based allosteric inhibitors of the hepatitis C virus
RNA-dependent RNA polymerase. J. Virol. 2003, 77, 13225-
13231.
(11) Summa, V.; Petrocchi, A.; Pace, P.; Matassa, V. G.; De Francesco,
R.; Altamura, S.; Tomei, L.; Koch, U.; Neuner, P. Discovery of
R,γ-diketo acids as potent selective and reversible inhibitors of
hepatitis C virus NS5b RNA-dependent RNA polymerase. J.
Med. Chem. 2004, 47, 14-17.
(12) Carroll, S. S.; Tomassini, J.; Bosserman, B.; Getty, K.; Stahlhut,
M.; Eldrup, A. B.; Bhat, B.; Hall, D.; Simcoe, A.; LaFemina, R.;
Rutkowski, C. R.; Wolanski, B.; Yang, Z.; Migliaccio, G.; De
Francesco, R.; Kuo, L. C.; MacCoss, M.; Olsen, D. B. Inhibition
of hepatitis C virus RNA replication by 2′-modified nucleoside
analogs. J. Biol. Chem. 2003, 278, 11979-11984.
(13) Migliaccio, G.; Tomassini, J. E.; Carroll, S. S.; Tomei, L.;
Altamura, S.; Bhat, B.; Bartholomew, L.; Bosserman, M. R.;
Ceccacci, A.; Colwell, L. F.; Cortese, R.; De Francesco, R.; Eldrup,
A. B.; Getty, K. L.; Hou, X. S.; LaFemina, R. L.; Ludmerer, S.
W.; MacCoss, M.; McMasters, D. R.; Stahlhut, M. W.; Olsen, D.
B.; Hazuda, D. J.; Flores, O. A. Characterization of resistance
to non-obligate chain-terminating ribonucleoside analogs that
inhibit hepatitis C virus replication in vitro. J. Biol. Chem. 2003,
278, 49164-49170.
(14) Bera, S.; Malik, L.; Bhat, B.; Carroll, S. S.; MacCoss, M.; Olsen,
D. B.; Eldrup, A. B. Synthesis and evaluation of optically pure
dioxolanes as inhibitiors of hepatitis C virus RNA dependent
RNA polymerase. Bioorg. Med. Chem. Lett. 2003, 14, 4455-4458.
(15) Eldrup, A. B.; Allerson, C.; Bennett, F.; Bera, S.; Bhat, B.; Bhat,
N.; Brooks, J.; Carroll, S. S.; Cook, P. D.; MacCoss, M.; Mac-
Masters, D.; Olsen, D. B.; Prakash, T. P.; Marija, P.; Song, Q.;
Tommassini, J.; Xia, J. Structure activity relationship of purine
2′-C-methyl ribonucleosides for inhibition of hepatitis C virus
RNA dependent RNA polymerase. J. Med. Chem. 2004, 47,
2283-2295.
(16) Chander, G.; Sulkowski, M. S.; Jenckes, M. W.; Torbenson, M.
S.; Herlong, H. F.; Bass, E. B.; Gebo, K. A. Treatment of chronic
hepatitis C: a systematic review. Hepatology 2002, 36, S135-
S144.
(17) Peter, K.; Gambertoglio, J. G. Intracellular phosphorylation of
zidovudine (ZDV) and other nucleoside reverse transcriptase
inhibitors (RTI) used for human immunodeficiency virus (HIV)
infection. Pharm. Res. 1998, 15, 819-825.
Supporting Information Available: 1H NMR, 31P NMR,
and HPLC data of compounds 7-12. This material is available
free of charge via the Internet at http://pubs.acs.org.

1210 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Prakash et al.
(18) Martin, T. L.; Cretton-Scott, E.; Placidi, L.; FaraJ, A.; Loi, A.
G.; Schinazi, R. F.; McClure, H. M.; Gosselin, G.; Imbach, J. L.;
Sommadossi, J. P. In vitro and in vivo metabolism and phar-
macokinetics of bis[tert-butyl]-S-acyl-2-thioethyl]-â-L-2′,3′-dideoxy-
5-fluorocytidine monophosphates. Nucleosides, Nucleotides
Nucleic Acids 2000, 19, 481-499.
(19) Gao, W.-Y.; Shirasaka, T.; Johns, D. G.; Broder, S.; Mitsuya, H.
Differential phosphorylation of azidothymidine, dideoxycytidine,
and dideoxyinosine in resting and activated peripheral blood
mononuclear cells. J. Clin. Invest. 1993, 91, 2326-2333.
(20) Gao, W.-Y.; Agbaria, R.; Driscoll, J. S.; Mitsuya, H. Divergent
anti-human immunodeficiency virus activity and anabolic phos-
phorylation of 2′,3′-dideoxynucleoside analogs in resting and
activated human cells. J. Biol. Chem. 1994, 269, 12633-12638.
(21) Lefebvre, I.; Pe´rigaud, C.; Pompon, A.; Aubertin, A.-M.; Girardet,
J.-L.; Kirn, A.; Gosselin, G.; Imbach, J.-L. Mononucleoside
phosphotriester derivatives with S-acyl-2-thioethyl bioreversible
phosphate-protecting groups: intracellular delivery of 3′-azido-
2′,3′-dideoxythymidine 5′-monophosphate. J. Med. Chem. 1995,
38, 3941-3950.
(22) Girardet, J.-L.; Perigaud, C.; Aubertin, A.-M.; Gosselin, G.; Kirn,
A.; Imbach, J.-L. Increase of the anti-HIV activity of D4T in
human T-cell culture by the use of the sate pronucleotide
approach. Bioorg. Med. Chem. Lett. 1995, 5, 2981-2984.
(23) Periguard, C.; Aubertin, A.-M.; Benzaria, S.; Pelicano, H.;
Girardet, J.-L.; Kirn, A.; Gosselin, G.; Imbach, J.-L. Equal
inhibition of the replication of human immunodeficiency virus
in human T-cell culture by ddA bis(SATE)phosphotriester and
3′-azido-2′,3′-dideoxythymidine. Biochem. Pharmacol. 1994, 48,
11-14.
(24) Pompon, A.; Lefebvre, I.; Imbach, J. -L.; Kahn, S.; Farquhar, D.
Decomposition pathways of the mono- and bis(pivaloyloxy-
methyl) esters of azidothymidine 5′-monophosphate in cell
extract and in tissue culture medium: an application of the “on-
line ISRP-cleaning” HPLC technique. Antiviral Chem. Chemo-
ther. 1994, 5, 91-98.
(25) Kumar, A.; Khan, S. I.; Manglani, A.; Khan, Z. K.; Katti, S. B.
Synthesis and antifungal activity of 3′-deoxyribonucleosides.
Nucleosides Nucleotides 1994, 13, 1049-1058.
(26) Kaskar, B.; Heise, G. L.; Michalak, R. S.; Vishnuvajjala, B. R. A
convenient large scale synthesis of protected D-ribonolactone
from D-ribose. Synthesis 1990, 11, 1031-1032.
(27) Gong-Xin, H.; Bischofberger, N. A Convenient Preparation of
Protected 3′-Deoxyguanosine from Guanosine. Tetrahedron Lett.
1995, 36, 6991-6994.
(28) Nair, V.; Panicker, S. B.; Augustine, A.; George, T. G.; Thomas,
S.; Vairamani, M. An efficient bromination of alkenes using
cerium(IV) ammonium nitrate (CAN) and potassium bromide.
Tetrahedron 2001, 57, 7417-7422.
(29) Ipata, P. L.; Falcone, G.; Serra, M. C. Regulatory properties of
cytidine deaminase and urideine phosphorylase of Bacillus
cereus. FEBS Lett. 1970, 10, 67-70.
(30) Ipata, P. L.; Cercignani, G.; Balestreri, E. Partial purification
and properties of cytidine deaminase from baker’s yeast. Bio-
chemistry 1970, 9, 3390-3395.
(31) Wang, T. P.; Sable, H. Z.; Lampen, J. O. Enzymatic deamination
of cytosine nucleosides. J. Biol. Chem. 1950, 184, 17-28.
(32) Cacciamani, T.; Vita, A.; Cristalli, G.; Vincenzetti, S.; Natalini,
P.; Ruggieri, S.; Amici, A.; Magni, G. Purification of human
cytidine deaminase: molecular and enzymatic characterization
and inhibition by synthetic pyrimidine analogs. Arch. Biochem.
Biophys. 1991, 290, 285-292.
(33) Cory, A. H.; Owen, T. C.; Barltrop, J. A.; Cory, J. G. Use of an
aqueous soluble tetrazolium/formazan assay for cell growth
assays in culture. Cancer Commun. 1991, 3, 207-212.
JM0495172