Effects of introduction of hydrophobic group on ribavirin base on mutation induction and anti-RNA viral activity

Article abstract of DOI:10.1021/jm7009952

One of the possible mechanisms of antiviral action of ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, 1) is the accumulation of mutations in viral genomic RNA. The ambiguous incorporation of 5′-triphosphate of ribavirin (RTP, 8) by a viral RNA-dependent RNA polymerase (RdRp) is a key step of the mutation induction. We synthesized three ribavirin analogues that possess hydrophobic groups, 4-iodo-1-β-D- ribofuranosylpyrazole-3-carboxamide (7a), 4-propynyl-1-β-D- ribofuranosylpyrazole-3-carboxamide (7b), and 4-phenylethynyl-1-β-D- ribofuranosylpyrazole-3-carboxamide (7c), and the corresponding triphosphates (9a, 9b, and 9c, respectively). Steady-state kinetics analysis of the incorporation of these triphosphate analogues by a poliovirus RdRp, 3D ^pol, revealed that while the incorporation efficiency of 9a was comparable to RTP, 9b and 9c showed lower efficiency than RTP. Antipolioviral activity of 7a and 7b was much more moderate than ribavirin, and 7c showed no antipolioviral activity. Effects of substituting groups on the incorporation efficiency by 3D^pol and a strategy for a rational design of more active ribavirin analogues are discussed.

Full text of DOI:10.1021/jm7009952

J. Med. Chem. 2008, 51, 159–166
159
Effects of Introduction of Hydrophobic Group on Ribavirin Base on Mutation Induction and
Anti-RNA Viral Activity
Kei Moriyama,*,†, Tetsuya Suzuki,‡ Kazuo Negishi,‡ Jason D. Graci,§ Corinne N. Thompson,§ Craig E. Cameron,§ and
Masahiko Watanabe†
Shujitsu UniVersity, School of Pharmacy, Nishigawara, Okayama 703-8516, Japan, Department of Genomics and Proteomics, Okayama
UniVersity AdVanced Science Research Center, Tsushima, Okayama 700-8530, Japan, and Department of Biochemistry and Molecular Biology,
The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802
ReceiVed August 9, 2007
One of the possible mechanisms of antiviral action of ribavirin (1-ꢀ-D-ribofuranosyl-1,2,4-triazole-3-
carboxamide, 1) is the accumulation of mutations in viral genomic RNA. The ambiguous incorporation of
5′-triphosphate of ribavirin (RTP, 8) by a viral RNA-dependent RNA polymerase (RdRp) is a key step of
the mutation induction. We synthesized three ribavirin analogues that possess hydrophobic groups, 4-iodo-
1-ꢀ-D-ribofuranosylpyrazole-3-carboxamide (7a), 4-propynyl-1-ꢀ-D-ribofuranosylpyrazole-3-carboxamide
(7b), and 4-phenylethynyl-1-ꢀ-D-ribofuranosylpyrazole-3-carboxamide (7c), and the corresponding triph-
osphates (9a, 9b, and 9c, respectively). Steady-state kinetics analysis of the incorporation of these triphosphate
analogues by a poliovirus RdRp, 3Dpol, revealed that while the incorporation efficiency of 9a was comparable
to RTP, 9b and 9c showed lower efficiency than RTP. Antipolioviral activity of 7a and 7b was much
more moderate than ribavirin, and 7c showed no antipolioviral activity. Effects of substituting groups on
the incorporation efficiency by 3Dpol and a strategy for a rational design of more active ribavirin analogues
are discussed.
Introduction
Ribavirin (1-ꢀ-D-ribofuranosyl-1,2,4-triazole-3-carboxamide,
1) is a guanosine analogue that has broad-spectrum anti-RNA
viral activity. It has been clinically used in combination with
interferon R against hepatitis C virus (HCV) infection. Since
the discovery of ribavirin in 1972,1,2 several mechanisms for
the antiviral activity have been reported. After uptake into a
host cell, ribavirin is metabolized to ribavirin 5′-monophosphate
(RMPa) by a cellular adenosine kinase. It has been shown that
RMP inhibits an inosine monophosphate dehydrogenase
(IMDPH) in a host cell.3,4 Further phosphorylation of RMP
by cellular nucleotide kinases yields ribavirin 5′-diphosphate
and ribavirin 5′-triphosphate (RTP). It has also been reported
that RTP inhibits viral RNA polymerases5 and that viral RNA
transcription is suppressed by producing defective 5′-cap
structures containing RTP.6 These inhibitory actions of
Figure 1. Ambiguous base pairing of ribavirin (1) with cytosine (C)
and uracil (U). R is ribose.
ribavirin metabolites on cellular or viral enzymes have been
considered to be related to the antiviral action of ribavirin.
Recently, another mechanism of ribavirin antiviral action is
reported. Ribavirin is a viral mutagen.7 Exceeding an error
threshold by the accumulation of mutation in viral genomic RNA
leads to virus population extinction. This is so-called the lethal
mutagenesis or error catastrophe.8,9 The base moiety of ribavirin,
1,2,4-triazole-3-carboxamide, can base-pair with both cytosine
(C) and uracil (U) by a rotation of the carboxamide group
ambiguous incorporation of RTP by a viral RNA polymerase
is a key step of the mutation induction by ribavirin. Therefore,
investigation of the structure–activity relation between RTP
derivatives and their incorporation by viral RdRp is important
to design more potent antiviral drugs derived from ribavirin.
It is reported that providing a hydrophobic property on the base
moiety of nucleoside 5′-triphosphate improves its incorporation
efficiency by DNA polymerase.10 Introduction of a propynyl group
to dPaTP, an unnatural deoxyribonucleoside 5′-triphosphate,
elevated the incorporation efficiency opposite the partner unnatural
base, Q on template. Base recognition by DNA polymerase was
decreased when a propynyl group was introduced on the Pa base
because the incorporation efficiency was increased evenly opposite
all types of the template base. Introduction of a hydrophobic
propynyl group on base moiety of the triphosphate results in high
incorporation efficiency and low base recognition. These properties
could be advantageous for the mutation induction. Thus, we
hypothesized that by introducing a hydrophobic substitution such
(Figure 1).7 Poliovirus RNA-dependent RNA polymerase (3Dpol
)
incorporates RTP opposite both C and U in template RNA. This
* To whom correspondence should be addressed. Phone: +81-88-665-
2126. Fax: +81-88-665-5392. E-mail: k_moriyama@research.otsuka.co.jp.
†
Shujitsu University.
Present address: Otsuka Pharmaceutical Co., Ltd., Hiraishi Ebisuno,
Kawauchi-cho, Tokushima 771-0182, Japan.
‡
Okayama University.
The Pennsylvania State University.
§
aAbbreviations: RTP, ribavirin 5′-triphosphate; RMP, ribavirin 5′-
monophosphate; RdRp, RNA-dependent RNA polymerase.
10.1021/jm7009952 CCC: $40.75
2008 American Chemical Society
Published on Web 12/08/2007

160 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Moriyama et al.
5 and protected ꢀ-D-ribose was successful using the procedure
reported by Manfredini et al.12 with a minor modification.
We found that the coupling product (6a) was obtained in
better yield (92%) with a smaller amount of byproduct when
the reaction was performed at room temperature instead of
under reflux conditions as described in the literature.12
Palladium-catalyzed coupling reactions (6a to 6b and 6c) and
the conversion of nucleosides to its corresponding 5′-
triphosphates were carried out as described previously.10,19
This coupling reaction is very important because various
ribavirin analogues as well as 6b and 6c can be synthesized
from 6a by the palladium-catalyzed reaction. 6a could be a
key intermediate for synthesizing ribavirin analogues. Hy-
drophobicity of RTP and RTP analogues (9a-c) was evalu-
ated from these retention times (tR) from reverse-phase HPLC
analyses under HPLC condition II (see Experimental Section).
The tR values of these triphosphates are listed in Table 1.
The order of hydrophobicity was RTP < 9a ) 9b < 9c.
Incorporation Efficiency of Hydrophobic Ribavirin 5′-
Triphosphate Analogues by Poliovirus RNA Dependent
RNA Polymerase, 3Dpol. Ribavirin can form base pairs with
both C and U by the rotation of carboxamide group (Figure
1),7 and this results in the ambiguous incorporation of
ribavirin 5′-triphosphate (RTP, 8), an intracellular metabolite
of ribavirin, opposite template C and U by 3Dpol. Conse-
quently, ribavirin induces mutation on viral genomic RNA
and causes error catastrophe.7–9 The incorporation assay of
RTP (8) and its analogues (9a-c) opposite C and U by 3Dpol
was performed with a template primer called “sym/sub-C”
and “sym/sub-U”, respectively (Figure 3).7 First, 5′-triphos-
phates (8, 9a-c) were applied to time course analysis of 3Dpol
incorporation with sym/sub-U and sym/sub-C (Figure 4).
Incorporation of iodinated RTP analogue (9a) and propynyl
RTP analogue (9b) was slower than that of RTP opposite
template C (Figure 4A). No elongated product was observed
for phenylethynyl RTP analogue (9c) on sym/sub-C. In
contrast, opposite U, the incorporation time course of 9a was
almost identical with that of RTP (8) (Figure 4B). 9b was
incorporated slightly more slowly than 9a, and 9c was rarely
incorporated opposite U as well as opposite C. To evaluate
these incorporation efficiencies, steady-state kinetics analyses
were carried out. Results are summarized in Table 2, and
the incorporation efficiencies (kcat/KM) of 5′-triphosphates
opposite C and U are shown in the bar graph (Figure 5).
kcat/KM of 9a opposite U was 2.0 × 10-5 (µM-1 s-1), ∼2-
fold larger than that of RTP (8) (1.1 × 10-5 (µM-1 s-1)).
On the other hand, kcat/KM of 9a opposite C (4.4 × 10-6 (µM-1
s-1)) was ∼2-fold smaller than that of RTP (8.4 × 10-6 (µM-1
s-1)). These results indicate that 9a is comparable to RTP in its
potential to be incorporated into viral genomic RNA. The KM values
of 9a on both sym/sub-C and sym/sub-U were smaller than that
of RTP, which indicates that 9a was superior to RTP in the affinities
with sym/sub-3Dpol complexes. 9b showed almost equal incorpora-
tion efficiency to RTP opposite U but ∼3-fold less than RTP
opposite C. 9c provides no detectable elongated product. Although
kinetic parameters of correct NTP incorporations (GTP on sym/
sub-C and ATP on sym/sub-U) were not determined because of
rapid reactions with these template-NTP combinations in our
experimental condition, we can refer to previously reported pre-
steady-state kinetics data on the same sym/sub-C and sym/sub-U,
which revealed that RTP incorporation by 3Dpol was 3 × 105 fold
slower than GTP incorporation opposite C7 and 2 × 104 fold slower
than ATP incorporation opposite U.20
Figure 2. Structures of RTP (8) and hydrophobic RTP analogues
(9a-c).
as propynyl group on a ribavirin base, the mutagenicity would
become higher than ribavirin, which would lead to an increase of
anti-RNA viral activity of ribavirin.
Here, we report chemical syntheses of RTP analogues that
possess hydrophobic groups on the base moiety (Figure 2)
and their incorporation efficiency by 3Dpol, and the effects
of introduction of these substituting groups on the activity
of ribavirin are discussed.
Results
Chemical Synthesis. All synthetic pathways are sum-
marized in Scheme 1. Iodination of pyrazole and its deriva-
tives has been carried out previously by boiling them with
iodine and potassium iodide in alkaline aqueous solution.13,18
To accomplish this in milder conditions, we applied a
trifluoroacetic acid catalyzed iodination with N-iodosuccin-
imide11 for iodination of methyl pyrazole-3-carboxylate.
Methyl 4-iodopyrazole-3-carboxylate (5) was obtained in a
sufficient yield (87%), and an iodine substitution at position
4 of the pyrazole ring was confirmed by the disappearance
1
of the H4 signal on H NMR (data not shown). Coupling of

Effects of Hydrophobic Group
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 161
Scheme 1. Chemical Synthesis of Hydrophobic Ribavirin Analogues (7a-c) and RTP Analogues (9a-c) .a
a Reagents and conditions: (a) KMnO4, H2O, reflux; (b) H2SO4, MeOH, room temp; (c) N-iodosuccinimide, trifluoroacetic acid, CH3CN, room temp; (d)
ꢀ-D-ribofuranose-1-acetate-2,3,5-tribenzoate, 1,1,1,3,3,3-hexamethyldisilazane, (NH4)2SO4, Me3SiCl, CF3SO3H, CH3CN, room temp; (e) Pd(PPh3)4, CuI,
Et3N, tributyl(1-propynyl)tin, room temp; (f) Pd(PPh3)4, CuI, Et3N, ethynylbenzene, room temp; (g) NH3, MeOH, room temp; (h) (1) POCl3, Proton Sponge,
(CH3O)PO, 0°C, (2) tri-n-butylamine, bis(tri-n-butylammonium)pyrophosphate, DMF, 0°C.
Table 1. Retention Times of RTP (8) and Hydrophobic RTP Analogues
(9a-c) on Reverse-Phase HPLC
NTP
retention time (min)a
8
7.71
10.56
10.14
16.68
9a
9b
9c
a Retention time from HPLC condition II (see Experimental Section).
Wavelength for detection varied with 5′-triphosphates: 220, 250, 260, and
271 nm for 8, 9a, 9b, and 9c, respectively.
Figure 3. Sym/sub-C (A) and sym/sub-U (B). Underlined bases were
the templating bases for 3Dpol assays.
Antipolioviral Activities of Hydrophobic Ribavirin Ana-
logues. Antipolioviral activities of ribavirin (1) and hydrophobic
analogues (7a-c) were tested, and results were summarized in
Table 3. Each compound was applied to poliovirus infected
HeLa cells at the final concentrations of 0.5 and 2 mM in culture
medium. Iodine- and propynyl-substituted ribavirin analogues
(7a and 7b, respectively) showed very moderate antipolioviral

162 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Moriyama et al.
Figure 4. Incorporation of 5′-triphosphates by 3Dpol into 32P-labeled sym/sub-C (A) and sym/sub-U (B) templates. Products of 3Dpol assays were
separated by 20% polyacrylamide denaturing gel electrophoresis and visualized by Personal Molecular Imager FX (Bio-Rad).
Table 2. Kinetic Parameters for Incorporation of RTP (8) and
Hydrophobic RTP Analogues (9a-c) by 3Dpol a
kcat/KM (×10-6
)
NTP
KM (µM)
kcat (×10-4) (s-1
)
(µM s-1
)
sym/sub-C
ATP
8
9a
9b
9c
110 ( 30b
69 ( 18
40 ( 5
51 ( 16
ndc
0.82 ( 0.11
5.8 ( 0.9
1.8 ( 0.2
1.3 ( 0.3
ndc
0.74
8.4
4.4
2.6
ndc
sym/sub-U
GTP
8
9a
9b
9c
56 ( 20
34 ( 9
24 ( 2
35 ( 6
ndc
5.0 ( 1.1
3.7 ( 0.3
4.7 ( 0.7
3.1 ( 0.6
ndc
8.9
11
20
9.1
ndc
a Initial rate of reaction was determined as {(band intensity of g11-
mer)/[(band intensity of g11-mer) + (band intensity of 10-mer)]}(0.5/120)
(µM s-1) for each concentration of NTP. kcat and KM values were calculated
from Lineweaver–Burk plot. b Mean ( standard deviation (n ) 3). c No
elongated product was detected.
Figure 5. Incorporation efficiencies (kcat/KM from steady-state kinetics)
of RTP and analogues into sym/sub-C and sym/sub-U.
activity when compared to ribavirin. No antipolioviral effect
was observed on phenylethynyl-substituted ribavirin analogue
(7c).
and the corresponding 5′-triphosphates (9a, 9b, and 9c, respec-
tively). Steady-state kinetics of RTP and 9a-c incorporation
by purified poliovirus RdRp, 3Dpol revealed that the incorpora-
tion efficiencies of 9a opposite C and U were ∼2-fold lower
and ∼2-fold higher, respectively, than RTP (Table 2 and Figure
5). This indicates that 9a is comparable to RTP in terms of in
vitro incorporation into viral genomic RNA.
Judging from the retention time on HPLC, the hydrophobici-
ties of synthesized 5′-triphosphates increased in the order of
RTP < 9a ) 9b < 9c (Table 1). Contrary to our expectation,
the incorporation efficiencies by 3Dpol were decreased in the
Discussion
On the basis of the observation that a substitution by a
hydrophobic group on the base moiety of dNTP results in an
increase of the incorporation efficiency accompanied with a
decrease of base recognition by DNA polymerase,10 we designed
ribavirin 5′-triphosphate analogues that possessed hydrophobic
groups on the base to attempt to increase the rate of mutation
induction. We synthesized iodine-, propynyl-, and phenylethy-
nyl-substituted ribavirin analogues (7a, 7b, and 7c, respectively)

Effects of Hydrophobic Group
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 163
Table 3. Antipolioviral Activity of Ribavirin (1) and Hydrophobic
Ribavirin Analogues (7a-c)
of 7a was much lower than that of ribavirin (Table 3). A reason
for this difference is the mutagenicity of 9a. One factor that
might influence mutagenicity is if the substitution affects
rotation of the carboxamide group due to steric or electronic
hindrance since the mutagenic property of ribavirin is based
on this rotation.7 The incorporation efficiency of 9a is better
than ribavirin opposite U, and 9b is about the same as
ribavirin in that context. However, both are worse than
ribavirin in the context of a templating C. This suggests that
the analogues might favor the “A-like” form and that the
“G-like” form might be less prevalent. It is possible that the
substitution of position 4 of the ribavirin base makes the “A-
form” more stable and the “G-form” less stable. Thus, even
if they are incorporated efficiently, they may not induce
mutation at a high enough rate to show activity in the antiviral
assay. Another reason for the low antipolioviral activity of
7a is that it might be poorly phosphorylated in the infected
cells. Ribavirin is phosphorylated by cytosolic 5′-nucleotidase
II as well as adenosine kinase.25 It is necessary for ribavirin
analogues to be phosphorylated intracellularly by these
enzymes to behave as a viral mutagen. Investigation of the
structure–activity relation between ribavirin analogues and
phosphorylation activity of these enzymes as well as the
property of rotation of the carboxamide group should garner
further attention.
nucleoside
relative PFU (%)a
none
100
40
4
1 (0.5 mMb)
1 (2 mM)
7a (0.5 mM)
7a (2 mM)
7b (0.5 mM)
7b (2 mM)
7c (0.5 mM)
7c (2 mM)
76
68
94
57
122
91
a Virus titer after 6 h of infection in the presence of nucleosides compared
to an untreated control (defined as 100%). The standard deviation of each
titer is typically less than 40% based on our prior experiments. b Final
concentration in culture medium.
order of RTP ) 9a > 9b > 9c (Table 2 and Figure 5). This
contrasts with a finding on DNA polymerase (Klenow fragment
of DNA polymerase I) incorporation of dNTP analogues.10 This
difference of the behavior of hydrophobic 5′-triphosphate
between DNA and RNA polymerase might be explained from
the structure of polymerases. The crystal structure of the Klenow
fragment21 revealed that the base of incoming dNTP interacts
with aromatic amino acids, His881 or Tyr766. This means that
the interaction between the base of dNTP and polymerase active
site is dependent on π-π interaction. Since a propynyl
substitution on the base expands the distributing area of the π
electron, π-π interaction of dNTP and polymerase would be
increased and thus the incorporation efficiency of a propynyl
substituted dNTP becomes higher. In contrast, the base of
incoming NTP interacts with Arg174 of the polymerase active
site of poliovirus 3Dpol by cation-π interaction.22,23 One of
the possible mechanisms for the decrease in the incorporation
efficiency by propynyl and phenylethynyl substitution is that a
substitution with these electron-withdrawing groups (the elec-
tronegativity of these groups is ∼3.0)24 would reduce the π
electron density of the base and that cation-π interaction
between 9b or 9c and 3Dpol would be weakened. As a result,
the affinity of these 5′-triphosphates and 3Dpol is decreased. 1-ꢀ-
D-Ribofuranosyl-3-nitropyrrole 5′-triphosphate (3-NPNTP) also
showed much lower incorporation efficiency than RTP.17 The
same mechanism could be underlying this observation because
a nitro group is a strong electron-withdrawing group. As for
iodine substitution, since the electronegativity of iodine is 2.5,
which is the same as that of carbon, the substituted iodine
behaves as an electron-donating group because of the electron-
donating resonance effect (+R effect) of lone pair electrons on
iodine, enriches π electron density of pyrazole ring, and
strengthens cation-π interaction. It is possible that the decrease
of the KM value of 9a compared with RTP arises from this effect.
Beyond the electronic contributions, the bulkiness of the
substituting group could greatly affect the incorporation ef-
ficiency of RTP analogues. The phenylethynyl group might be
too bulky to fit the active site structure of 3Dpol. Taking all
these effects into consideration, the introduction of a substituting
group on the ribavirin base such as a small and electron-donating
group might be more effective to increase incorporation
efficiency by 3Dpol. We are now trying to seek novel RTP
analogues that possess more potent mutagenicity on the basis
of findings in this work.
A rational design of RTP analogues that aims to increase the
incorporation efficiency and the mutation induction ability from
the viewpoint of NTP and polymerase interaction in the active
site may be a useful strategy for discovering ribavirin analogues
that are more active against RNA viruses. In parallel, rotation
of the carboxamide group and intracellular phosphorylation of
the analogues must be studied precisely to understand the
antiviral and mutagenic properties of ribavirin analogues in the
infected cells.
Experimental Section
General Procedures. 1H, 13C, and 31P NMR. Spectra were
recorded on ECA500 spectrometer with Delta software (JEOL).
Except where noted, the temperature for NMR measurement was
25 °C.
High-Resolution Mass Spectra (HR-MS). HR-MS were
obtained on MicroTOF LC (Bruker Daltonics). Spectra were
calibrated with an internal standard “Tune Mix solution” (Bruker
Daltonics).
Ultraviolet–Visible Spectra. UV–vis spectra for synthesized 5′-
triphosphates over the range 220-400 nm were recorded on GE
Healthcare Ultrospec 1100 pro.
Silica Gel Column Chromatography. Chromatography was
performed on Wakogel C-200 (Wako). Solvents were described in
each procedure.
HPLC. HPLC data were collected from a JASCO HPLC system
(HPLC pump, PU-2089Plus; UV detector, UV-2075Plus) with HSS-
2000 software. HPLC condition I is as follows: column, Symme-
tryPrep C18 7 µm (column size L 19 mm × 150 mm, Waters);
solvent A, H2O; solvent B, 50% (v/v) CH3CN in H2O; 0–10 min,
0–100% B; 10–15 min, 100% B; 15–16 min, 100–0% B; 16–21
min, 0% B; flow rate, 10 mL/min; detection, UV 254 nm. HPLC
condition II is as follows: column, Cosmosil 5C18-MS-II (column
size L 4.6 mm × 250 mm, Nacalai tesque); solvent A, 100 mM
tetraethylammonium acetate (TEAA) in H2O; solvent B, 100 mM
TEAA and 50% (v/v) CH3CN in H2O; 0–15 min, 0–60% B; 15–16
min, 60–100% B; 16–21 min, 100% B; 21–22 min, 100–0% B;
22–27 min, 0% B; flow rate, 1 mL/min. HPLC condition III is as
follows: column, Cosmosil 5C18-MS-II (column size L 4.6 mm ×
250 mm, Nacalai tesque); solvent A, 100 mM TEAA in H2O;
solvent B, 100 mM TEAA and 50% (v/v) CH3CN in H2O; 0–15
Antipolioviral activity of ribavirin and analogues (1, 7a-c)
was not wholly consistent with the incorporation efficiency of
the corresponding 5′-triphosphates (8, 9a-c) judged from
steady-state kinetics of incorporation by 3Dpol in vitro (Table 2
and Figure 5). While 9a showed in vitro incorporation efficiency
comparable to RTP as described above, antipolioviral activity

164 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Moriyama et al.
min, 0–100% B; 15–20 min, 100% B; 20–21 min, 100–0% B;
21–26 min, 0% B; flow rate, 1 mL/min.
53.50, 52.23. ESI-MS (positive) for C31H25N2O9INa ([M + Na]+):
calcd, 719.05; found, 718.97.
Methyl 4-Propynyl-1-(2′,3′,5′-tribenzoyl-ꢀ-D-ribofuranosyl)pyra-
zole-3-carboxylate (6b). To a solution of 223 mg (0.321 mmol)
of 6a, 9.8 mg (0.051 mmol) of CuI, and 18.5 mg (0.016 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 1.6 mL of dry CH3CN
were added 67 µL (0.481 mmol) of triethylamine and 146 µL (0.481
mmol) tributyl(1-propynyl)tin under argon atmosphere, and the
solution was stirred at room temperature for 8 h. The reaction
mixture was then partitioned with EtOAc/H2O, and the organic layer
was washed three times with saturated NaCl aqueous solution, dried
over Na2SO4, filtrated, and evaporated. An amount of 146 mg (0.24
mmol, 75%) of 6b was obtained by silica gel column chromatog-
raphy (solvent, n-hexane/EtOAc ) 4: 1 (v/v)). 1H NMR (500 MHz,
CDCl3) δ (ppm) 8.06 (d, 2H, J ) 7.5 Hz), 7.97 (d, 2H, J ) 7.5
Hz), 7.91 (d, 2H, J ) 6.9 Hz), 7.80 (s, 1H), 7.58–7.53 (m, 3H),
7.44 (t, 2H, J ) 7.7 Hz), 7.40 (t, 2H, J ) 8.0 Hz), 7.35 (t, 2H, J
) 8.0 Hz), 6.22 (d, 1H, J ) 2.9 Hz), 6.11 (dd, 1H, J ) 5.2 Hz, 3.4
Hz), 6.02 (t, 1H, J ) 5.4 Hz), 4.83–4.77 (m, 2H), 4.64 (dd, 1H, J
) 12.0, 4.0 Hz), 3.91 (s, 3H), 2.04 (s, 3H). 13C NMR (126 MHz,
CDCl3) δ (ppm) 166.27, 165.19, 165.00, 161.64, 144.42, 133.85,
133.74, 133.36, 132.92, 129.96, 129.88, 129.44, 128.76, 128.66,
128.63, 128.57, 108.21, 92.45, 90.18, 80.78, 75.29, 71.31, 68.91,
63.68, 52.17, 4.67. HR-MS (ESI-positive) for C34H28N2O9Na ([M
+ Na]+): calcd, 631.1687; found, 631.1705.
Methyl 4-Phenylethynyl-1-(2′,3′,5′-tribenzoyl-ꢀ-D-ribofura-
nosyl)pyrazole-3-carboxylate (6c). 6c was synthesized in a similar
procedure to 6b by the palladium-catalyzed coupling reaction. To
a solution of 491 mg (0.705 mmol) of 6a, 21.5 mg (0.113 mmol)
of CuI, and 40.7 mg (0.035 mmol) of tetrakis(triphenylphosphine)-
palladium(0) in 3.5 mL of dry CH3CN were added 147 µL (1.06
mmol) of triethylamine and 116 µL (1.06 mmol) ethynylbenzene
under argon atmosphere, and the solution was stirred at room
temperature for 9 h. Work-up was carried out as described above.
An amount of 368 mg (0.548 mmol, 78%) of 6c was obtained by
silica gel column chromatography (solvent, n-hexane/EtOAc ) 4:1
(v/v)). 1H NMR (500 MHz, CDCl3) δ (ppm) 8.08 (d, 2H, J ) 7.5
Hz), 7.98 (d, 2H, J ) 7.5 Hz), 7.94 (s, 1H), 7.92 (d, 2H, J ) 7.5
Hz), 7.59–7.33 (m, 14H), 6.27 (d, 1H, J ) 3.4 Hz), 6.15 (dd, 1H,
J ) 5.0, 3.6 Hz), 6.05 (t, 1H, J ) 5.7 Hz), 4.86–4.81 (m, 2H),
4.66 (dd, 1H, J ) 11.7, 3.7 Hz), 3.94 (s, 3H). 13C NMR (126 MHz,
CDCl3) δ (ppm) 166.28, 165.19, 165.02, 161.44, 144.69, 133.88,
133.76, 133.45, 132.84, 131.69, 129.98, 129.88, 129.86, 129.41,
128.71, 128.65, 128.59, 128.48, 128.37, 123.18, 107.53, 93.40,
92.57, 80.89, 78.95, 75.35, 71.27, 63.58, 52.24. HR-MS (ESI-
positive) for C39H30N2O9Na ([M + Na]+): calcd, 693.1844; found,
693.1866.
General Procedure for Ammonia Treatment of Protected
Nucleoside (6a-c). Ammonia treatment of protected nucleoside
6a-c converts the 3-carboxylate group to the 3-carboxiamide group
as well as benzoyl-protected 2′-, 3′-, and 5′-hydroxyl groups to free
hydroxyl groups. The protected nucleosides were dissolved in 10
mL of saturated (∼7 N) ammonia in methanol (Sigma-Aldrich) and
stirred at room temperature overnight. Ammonia and solvent were
removed under reduced pressure, and deprotected nucleosides 7a-c
were purified by a silica gel column chromatography (solvent,
0-10% methanol in CH2Cl2) followed by preparative HPLC
purification.
4-Iodo-1-ꢀ-D-ribofuranosylpyrazole-3-carboxamide (7a). An
amount of 319 mg (0.458 mmol) of 6a was treated with ammonia
as described above. Following silica gel column chromatography,
7a was purified by two-step HPLC purifications: first, HPLC
condition I (see General Procedures); second, 5% (v/v) CH3CN
in H2O isocratic conditions (flow rate 10 mL/min). Yield, 82.8
mg (0.224 mmol, 49%). 1H NMR (500 MHz, DMSO-d6) δ (ppm)
8.22 (s, 1H), 7.46 (bs, 1H), 7.27 (bs, 1H), 5.61 (d, 1H, J ) 4.6
Hz), 5.43 (d, 1H, J ) 5.7 Hz), 5.08 (d, 1H, J ) 5.7 Hz), 4.89
(t, 1H, J ) 5.7 Hz), 4.28 (q, 1H, J ) 5.0 Hz), 4.07 (q, 1H, J )
5.0 Hz), 3.87 (q, 1H, J ) 4.4 Hz), 3.61–3.56 (m, 1H), 3.49–3.45
(m, 1H). 13C NMR (126 MHz, DMSO-d6) δ (ppm) 163.12,
145.45, 137.13, 94.48, 85.97, 75.15, 70.64, 61.83, 59.22. HR-
All synthetic reactions were monitored by thin layer chroma-
tography (TLC) with silica gel sheet 60 F254 (Merck).
Ribavirin (1-ꢀ-D-ribofuranosyl-1,2,4-triazole-3-carboxamide,
1). Ribavirin (1) was synthesized and crystallized according to the
literature.2 1H NMR (500 MHz, DMSO-d6) δ (ppm) 8.83 (s, 1H),
7.79 (bs, 1H), 7.58 (bs, 1H), 5.78 (d, 1H, J ) 3.4 Hz), 5.53 (d, 1H,
J ) 4.6 Hz), 5.15 (d, 1H, J ) 4.6 Hz), 4.87 (t, 1H, J ) 5.5 Hz),
4.32 (q, 1H, J ) 4.4 Hz), 4.10 (q, 1H, J ) 5.0 Hz), 3.91 (q, 1H,
J ) 4.4 Hz), 3.62–3.57 (m, 1H), 3.49–3.44 (m, 1H).
Methyl Pyrazole-3-carboxylate (4). Methyl pyrazole-3-car-
boxylate (4) was synthesized by oxidation of 3-methylpyrazole (2)
followed by ester formation. Briefly, to a solution of 4.11 g (50.0
mmol) of 3-methylpyrazole (Tokyo Kasei) in 150 mL of H2O was
added slowly 17.38 g (110 mmol) of KMnO4 in 100 mL of H2O,
and the solution was heated to reflux conditions for 4 h. After the
reaction mixture was cooled to room temperature, insoluble material
was removed by filtration. The filtrate was evaporated to ∼30 mL,
and then pyrazole-3-carboxylic acid (3) (2.68 g, 23.9 mmol, 48%)
was crystallized by adding concentrated HCl to give pH ∼2. To a
solution of 2.04 g (18.2 mmol) of 3 in 36 mL of dry methanol was
added 2.9 mL (54.4 mmol) of H2SO4, and the solution was stirred
at room temperature overnight under an argon atmosphere. The
solution was evaporated, and the residue was dissolved in 50 mL
of H2O. NaHCO3 was added to neutralize. The solution was
extracted with EtOAc, dried over Na2SO4, filtrated, and evaporated.
An amount of 2.00 g (15.9 mmol, 87%) of 4 was obtained by silica
gel chromatography (solvent, 5-10% methanol in CH2Cl2) as a
white solid. 1H NMR (500 MHz, DMSO-d6, 55 °C) δ (ppm) 13.40
(bs, 1H), 7.75 (bs, 1H), 6.72 (s, 1H), 3.78 (s, 3H).
Methyl 4-Iodopyrazole-3-carboxylate (5). An efficient iodina-
tion of aromatic compounds in mild conditions is reported by
Castanet et al.11 According this procedure, 4 was converted to the
4-iodinated form 5. Amounts of 1.74 g (13.8 mmol) of 4 and 3.41 g
(15.2 mmol) of N-iodosuccinimide were dissolved in 55 mL of dry
CH3CN under argon atmosphere. To the stirring solution, 320 µL
(4.14 mmol) of trifluoroacetic acid was added, and the solution
was stirred for 3 h. After evaporation, the residue was partitioned
with EtOAc/5% NaHCO3. The organic layer was dried over
Na2SO4, filtrated, and evaporated. 5 (3.03 g, 12.0 mmol, 87%)
was crystallized from n-hexane/EtOAc. 1H NMR (500 MHz,
DMSO-d6, 55 °C) δ (ppm) 13.74 (bs, 1H), 7.98 (bs, 1H), 3.79
(s, 3H).
Methyl 4-Iodo-1-(2′,3′,5′-tribenzoyl-ꢀ-D-ribofuranosyl)pyra-
zole-3-carboxylate (6a). A coupling reaction of 5 and sugar moiety
was carried out according to the literature12 with minor modifica-
tions. To a solution of 2.91 g (11.5 mmol) of 5 in 115 mL of dry
CH3CN was added 8.74 g (17.3 mmol) of ꢀ-D-ribofuranose 1-acetate
2,3,5-tribenzoate (Tokyo Kasei), 2.68 mL (12.7 mmol) of 1,1,1,3,3,3-
hexamethyldisilazane, and 152 mg (1.15 mmol) of ammonium
sulfate under argon atmosphere, and the solution was stirred at room
temperature. An amount of 1.76 mL (13.9 mmol) of chlorotrim-
ethylsilane followed by 2.45 mL (27.7 mmol) of trifluoromethane-
sulfonic acid was added, and the solution was stirred at room
temperature for 3 h. Mixed with 115 mL of CH2Cl2, the solution
was washed with saturated NaHCO3 aqueous solution. The organic
layer was subsequently washed with saturated NaCl aqueous
solution, dried over Na2SO4, filtrated, and evaporated. An amount
of 7.38 g (10.6 mmol, 92%) of 6a was obtained by silica gel column
chromatography (solvent, n-hexane/EtOAc ) 4: 1 (v/v)). 1H NMR
(500 MHz, CDCl3) δ (ppm) 8.05 (d, 2H, J ) 6.9 Hz), 7.97 (d, 2H,
J ) 6.9 Hz), 7.92 (d, 2H, J ) 6.9 Hz), 7.81 (s, 1H), 7.59–7.53 (m,
3H), 7.45 (t, 2H, J ) 7.7 Hz), 7.40 (t, 2H, J ) 8.0 Hz), 7.36 (t,
2H, J ) 7.7 Hz), 6.26 (d, 1H, J ) 2.9 Hz), 6.11 (dd, 1H, J ) 5.2
Hz, 3.4 Hz), 6.02 (t, 1H, J ) 5.4 Hz), 4.83–4.81 (m, 2H), 4.64
(dd, 1H, J ) 13.5 Hz, 4.9 Hz), 3.90 (s, 3H). 13C NMR (126 MHz,
CDCl3) δ (ppm) 166.23, 165.20, 165.04, 161.53, 144.38, 136.04,
133.90, 133.78, 133.51, 129.97, 129.87, 129.84, 129.37, 128.79,
128.72, 128.65, 128.60, 92.60, 80.96, 75.30, 71.33, 63.54, 61.47,

Effects of Hydrophobic Group
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 165
MS (ESI-positive) for C9H12N3O5INa ([M + Na]+): calcd,
391.9714; found, 391.9702.
(ppm) -9.38 (d, 1H, J ) 18.6 Hz), -10.21 (d, 1H, J ) 20.1
Hz), -21.73 (t, 1H, J ) 20.9 Hz). HR-MS (ESI-negative) for
C12H17N3O14P3 ([M - H]-): calcd, 519.9918; found, 519.9920.
UV–vis spectrum (in 10 mM sodium phosphate buffer, pH 7.0),
λmax ) 262 nm (ε262 ) 3.4 × 103).
4-Propynyl-1-ꢀ-D-ribofuranosylpyrazole-3-carboxamide (7b).
An amount of 128 mg (0.211 mmol) of 6b was treated with
ammonia as described above. Following silica gel column chro-
matography, 7b was purified by two-step HPLC purifications, first,
HPLC condition I (see General Procedures); second, 10% (v/v)
CH3CN in H2O isocratic conditions (flow rate 10 mL/min). Yield,
4-Phenylethynyl-1-ꢀ-D-ribofuranosylpyrazole-3-carboxam-
ide 5′-Triphosphate (9c). HPLC purification: HPLC condition III
1
(see General Procedures); detection, UV 271 nm. Yield, 7%. H
1
35.7 mg (0.127 mmol, 60%). H NMR (500 MHz, DMSO-d6) δ
NMR (500 MHz, D2O) δ (ppm) 8.18 (s, 1H), 7.52–7.50 (m, 2H),
7.36–7.35 (m, 3H), 5.80 (d, 1H, J ) 5.2 Hz), 4.63–4.59 (m, 1H),
4.48 (t, 1H, J ) 4.6 Hz), 4.28–4.27 (m, 1H), 4.14–4.13 (m, 2H),
3.10 (q, 18H, J ) 7.3 Hz), 1.18 (t, 27H, J ) 7.5 Hz). 31P NMR
(202 MHz, D2O) δ (ppm) -8.72 (bs, 1H), -10.09 (d, 1H, J )
18.6 Hz), -21.25 (bs, 1H). HR-MS (ESI-negative) for
C17H19N3O14P3 ([M - H]-): calcd, 582.0074; found, 582.0070.
UV–vis spectrum (in 10 mM sodium phosphate buffer, pH 7.0),
λmax ) 272 nm (ε272 ) 1.9 × 104).
(ppm) 8.22 (s, 1H), 7.31 (bs, 1H), 7.28 (bs, 1H), 5.59 (d, 1H, J )
4.0 Hz), 5.44 (d, 1H, J ) 5.7 Hz), 5.07 (d, 1H, J ) 5.2 Hz), 4.89
(t, 1H, J ) 5.4 Hz), 4.25 (q, 1H, J ) 5.0 Hz), 4.07 (q, 1H, J ) 5.2
Hz), 3.87 (q, 1H, J ) 4.4 Hz), 3.61–3.57 (m, 1H), 3.49–3.45 (m,
1H), 1.96 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ (ppm) 162.72,
146.67, 134.57, 104.04, 94.38, 88.81, 85.86, 75.21, 71.12, 70.56,
61.78, 4.71. HR-MS (ESI-positive) for C12H16N3O5 ([M + H]+):
calcd, 282.1084; found, 282.1090. HR-MS (ESI-positive) for
C12H15N3O5Na ([M + Na]+): calcd, 304.0904; found, 304.0898.
4-Phenylethynyl-1-ꢀ-D-ribofuranosylpyrazole-3-carboxam-
ide (7c). An amount of 359 mg (0.535 mmol) of 6c was treated
with ammonia as described above. Following silica gel column
chromatography, 7c was purified by two-step HPLC purifications:
first, HPLC condition I (see General Procedures); second, 28% (v/
v) CH3CN in H2O isocratic condition (flow rate 10 mL/min). Yield,
Expression and Purification of 3Dpol. Expression and purifica-
tion of 3Dpol were performed as described previously.14 A single
band of 3Dpol on SDS-PAGE was obtained. The concentration of
purified 3Dpol was determined with BCA protein assay kit
(PIERCE). Purified 3Dpol was stored at -70 °C in 50 mM
HEPES-KOH, pH 7.5, 5 mM MgCl2, 20% glycerol, 0.1% NP-40,
and 1 mM DTT.
1
137 mg (0.398 mmol, 74%). H NMR (500 MHz, DMSO-d6) δ
Preparation of Oligonucleotides for 3Dpol Assay. Sym/sub-U
(ppm) 8.40 (s, 1H), 7.45–7.34 (m, 7H), 5.64 (d, 1H, J ) 4.0 Hz),
5.48 (d, 1H, J ) 5.7 Hz), 5.10 (d, 1H, J ) 5.2 Hz), 4.92 (t, 1H, J
) 5.7 Hz), 4.30 (q, 1H, J ) 5.0 Hz), 4.10 (q, 1H, J ) 5.0 Hz),
3.90 (q, 1H, J ) 4.4 Hz), 3.64–3.59 (m, 1H), 3.52–3.48 (m, 1H).
13C NMR (126 MHz, DMSO-d6) δ (ppm) 162.64, 147.07, 134.80,
131.62, 129.23, 128.98, 123.42, 103.25, 94.53, 91.66, 85.99, 81.59,
75.28, 70.57, 61.79. HR-MS (ESI-positive) for C17H18N3O5 ([M +
H]+): calcd, 344.1241; found, 344.1245. HR-MS (ESI-positive) for
C17H17N3O5Na ([M + Na]+): calcd, 366.1060; found, 366.1065.
General Procedure for the Synthesis of Nucleoside 5′-
Triphosphates (8, 9a-c). The 0.1 mmol scale synthesis of 5′-
triphosphates of ribavirin (1) and its analogues (7a-c) and DEAE
Sephadex A-25 column purification were carried out as described
by Kimoto et al.13 HPLC purification following the DEAE column
provides pure triphosphates. The yield and the molar absorption
coefficient (ε) of these triphosphates at pH 7.0 were determined
by quantitative analysis of the phosphorus in these compounds.13
Ribavirin 5′-Triphoshate (8). HPLC purification: HPLC condi-
tion II (see General Procedures), detection; UV 220 nm. Yield, 28%.
1H NMR (500 MHz, D2O) δ (ppm) 8.72 (s, 1H), 5.93 (d, 1H, J )
4.6 Hz), 4.64–4.61 (m, 1H), 4.49 (t, 1H, J ) 4.9 Hz), 4.30–4.29
(m, 1H), 4.15–4.12 (m, 2H), 3.10 (q, 18H, J ) 7.3 Hz), 1.18 (t,
27H, J ) 7.2 Hz). 31P NMR (202 MHz, D2O) δ (ppm) -9.44 (d,
1H, J ) 18.6 Hz), -10.26 (d, 1H, J ) 18.6 Hz), -21.78 (t, 1H, J
) 19.4 Hz). HR-MS (ESI-negative) for C8H14N4O14P3 ([M - H]-):
calcd, 482.9714; found, 482.9724. UV–vis spectrum (in 10 mM
sodium phosphate buffer, pH 7.0): no λmax was observed over the
range 220-400 nm (ε220 ) 7.1 × 103).
and sym/sub-C (Figure 3)7 were used as template primer for
pol
3D
assay. These 10-mer RNA oligonucleotides purchased
from Greiner Bio-One were further purified on denaturing PAGE
(200 mm × 400 mm × 2 mm, 20% polyacrylamide containing
7 M urea). The concentrations of purified oligonucleotides were
determined from UV absorbance at 260 nm (TE buffer).
5′-32P-Labeling of Oligonucleotides. Temperature of all enzy-
matic reactions was controlled in Thermal Cycler Dice Gradient
(TaKaRa). An amount of 0.1 nmol of sym/sub oligonucleotide was
mixed with 5 µCi [γ-32P]ATP (GE Healthcare) and 10 U of T4
polynucleotide kinase (TaKaRa) in provided T4 PNK buffer (total
volume is 10 µL) and incubated at 37 °C for 1 h. After addition of
30 µL of H2O, labeled oligonucleotide was separated from unreacted
[γ-32P]ATP by a MicroSpin G-25 column (GE Healthcare). Labeled
oligonucleotide was then diluted to one-tenth by mixing 0.9 nmol
of unlabeled oligonucleotide. H2O was added to the total volume
of 100 µL (final concentration of oligonucleotide was 10 µM).
3Dpol Assays. 3Dpol assays were carried out according to
literatures.15,16 Ultrapure ATP and GTP were purchased from GE
Healthcare and used without further purification. Prior to preparing
the reaction mixture of 3Dpol assays, sym/sub oligonucleotide was
annealed by heating to 90 °C for 1 min and cooled slowly (5 °C/
min) to 10 °C. 3Dpol was diluted to 12.5 µM immediately prior to
use in 50 mM HEPES-KOH, pH 7.5, 10 mM 2-mercaptoethanol,
5 mM MgCl2, 60 µM ZnCl2, and 20% glycerol. For time course
experiments, reaction mixture of 50 mM HEPES-KOH, pH 7.5,
10 mM 2-mercaptoethanol, 5 mM MgCl2, 60 µM ZnCl2, 20 U of
RNase inhibitor (Promega), 500 µM NTP, 5 pmol of 32P-labeled
sym/sub, and 1.25 µM 3Dpol in a total volume of 10 µL was
incubated at 30 °C. The reaction was quenched at each time point
by adding 1 µL of 0.5 M EDTA. For steady-state kinetics, reaction
mixture of 50 mM HEPES-KOH, pH 7.5, 10 mM 2-mercaptoet-
hanol, 5 mM MgCl2, 60 µM ZnCl2, 10 U of RNase inhibitor,
10-250 µM NTP, 2.5 pmol of 32P-labeled sym/sub, and 1.25 µM
3Dpol in a total volume of 5 µL was incubated at 30 °C for 2 min.
Reaction was quenched by adding 0.5 µL of 0.5 M EDTA.
Denaturing PAGE Analysis of 3Dpol Products. To each
reaction mixture was added an equal volume of loading buffer (90%
formamide, 0.025% bromophenol blue, and 0.025% xylene cyanol).
After the mixture was heated at 70 °C for 5 min, one-fourth of the
total amount was loaded onto 20% denaturing polyacrylamide gel
(200 mm × 400 mm × 0.5 mm, 7 M urea). Electrophoresis was
carried out in 1× TBE at 40 W. Bands were visualized and
quantitated by Personal Molecular Imager FX with Quantity One
software (Bio-Rad).
4-Iodo-1-ꢀ-D-ribofuranosylpyrazole-3-carboxamide 5′-Tri-
phosphate (9a). HPLC purification: HPLC condition II (see General
1
Procedures); detection, UV 250 nm. Yield, 27%. H NMR (500
MHz, D2O) δ (ppm) 8.08 (s, 1H), 5.76 (d, 1H, J ) 5.2 Hz),
4.64–4.59 (m, 1H), 4.45 (t, 1H, J ) 4.6 Hz), 4.26–4.25 (m, 1H),
4.11–4.10 (m, 2H), 3.10 (q, 18H, J ) 7.3 Hz), 1.18 (t, 27H, J )
7.5 Hz). 31P NMR (202 MHz, D2O) δ (ppm) -9.36 (d, 1H, J )
18.6 Hz), -10.24 (d, 1H, J ) 17.1 Hz), -21.70 (t, 1H, J ) 19.4
Hz). HR-MS (ESI-negative) for C9H14N3O14IP3 ([M - H]-): calcd,
607.8728; found, 607.8724. UV–vis spectrum (in 10 mM sodium
phosphate buffer, pH 7.0), λmax ) 250 nm (ε250 ) 3.0 × 103).
4-Propynyl-1-ꢀ-D-ribofuranosylpyrazole-3-carboxamide 5′-
Triphosphate (9b). HPLC purification: HPLC condition II (see
General Procedures); detection, UV 260 nm. Yield, 20%. 1H
NMR (500 MHz, D2O) δ (ppm) 8.04 (s, 1H), 5.74 (d, 1H, J )
5.7 Hz), 4.61–4.58 (m, 1H), 4.44 (t, 1H, J ) 4.6 Hz), 4.26–4.25
(m, 1H), 4.11–4.10 (m, 2H), 3.10 (q, 18H, J ) 7.3 Hz), 1.94 (s,
3H), 1.18 (t, 27H, J ) 7.2 Hz). 31P NMR (202 MHz, D2O) δ

166 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Moriyama et al.
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a 1 h of incubation at 37 °C, the medium was removed and cells
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(37 °C) medium containing the specified amount of nucleoside was
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incubated for 2 days at 37 °C, at which time the agar was removed
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Acknowledgment. We gratefully acknowledge the NIH
(Grant AI054776 to C.E.C.) for financial support.
Supporting Information Available: 1H NMR spectra of 1, 7a,
7b, and 7c. This material is available free of charge via the Internet
at http://pubs.acs.org.
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