Synthesis and anti-hepatitis C virus (HCV) activity of 3′-C- substituted-methyl pyrimidine and purine nucleosides

Article abstract of DOI:10.1016/j.bmc.2010.05.002

On the basis of potent anti-hepatitis C virus (HCV) activity of 2′-C-hydroxymethyladenosine, 3′-C-substituted-methyl-ribofuranosyl pyrimidine and purine nucleosides were designed and synthesized from d-xylose. Among compounds tested, all adenine analogues, 4a, 4d, and 4g showed significant anti-HCV activity in a replicon-based cell assay irrespective of the substituent (Y = OH, N₃, or F) at the 3′-C-substituted methyl position, among which 4g (Y = N₃) was the most potent, but it is also cytotoxic. This study guarantees the 3′-C-substituted-methyl nucleoside serves as a new template for the development of new anti-HCV agents.

Full text of DOI:10.1016/j.bmc.2010.05.002

Bioorganic & Medicinal Chemistry 18 (2010) 4812–4820
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anti-hepatitis C virus (HCV) activity of 30-C-substituted-methyl
pyrimidine and purine nucleosides
Won Jun Choi a,b, Yu Min Kim a, Hea Ok Kim a, Hyuk Woo Lee a,b, Dong-Eun Kim c, Kwang-su Park c,
Youhoon Chong c, Lak Shin Jeong a,b,
*
a Department of Bioinspired Science, Ewha Womans University, 11-1 Seodaemun-gu, Daehyun-dong, Seoul 120-750, Republic of Korea
b Laboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University, 11-1 Seodaemun-gu, Daehyun-dong, Seoul 120-750, Republic of Korea
c Department of Bioscience & Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
On the basis of potent anti-hepatitis C virus (HCV) activity of 20-C-hydroxymethyladenosine, 30-C-substi-
Article history:
Received 19 March 2010
Revised 30 April 2010
Accepted 1 May 2010
Available online 31 May 2010
tuted-methyl-ribofuranosyl pyrimidine and purine nucleosides were designed and synthesized from D-
xylose. Among compounds tested, all adenine analogues, 4a, 4d, and 4g showed significant anti-HCV
activity in a replicon-based cell assay irrespective of the substituent (Y = OH, N3, or F) at the 30-C-substi-
tuted methyl position, among which 4g (Y = N3) was the most potent, but it is also cytotoxic. This study
guarantees the 30-C-substituted-methyl nucleoside serves as a new template for the development of new
anti-HCV agents.
Keywords:
30-C-Hydroxymethyl nucleosides
30-C-Azidomethyl nucleosides
30-C-Fluoromethyl nucleosides
Anti-hepatitis C virus (HCV) activity
Neighboring group effect
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Many compounds have been synthesized to inhibit the HCV spe-
cific RNA polymerase.12 Non-nucleoside derivatives13 were discov-
The disease infected with hepatitis C virus (HCV) is lethal in that
its chronic infection is gradually developed to liver cirrhosis and
hepatocellular carcinoma (HCC).1,2 Since about 170 million people
worldwide suffer from this disease, much efforts have been made
to cure HCV infection, but only ribavirin in combination with inter-
ered as the inhibitors binding at the allosteric site, while nucleoside
analogues14 are inhibitors acting at the catalytic site, inhibiting vir-
al RNA synthesis. 20-C-Methylcytidine (1, EC50 = 0.27 0.04
l
M)5
M15
and 20-C-methyladenosine (2, EC50 = 0.60 0.07
l
M5 or 0.3
l
)
are the representatives binding at the catalytic site of HCV, showing
potent and selective anti-HCV activity (Fig. 2). They are incorpo-
rated into proviral RNA after being converted to their triphosphates,
resulting in the viral RNA chain termination.16 The 20-methyl group
prevents the incorporation of the natural substrate, nucleoside tri-
phosphate (NTP), leading to the viral RNA chain termination.16 On
the basis of potent anti-HCV activity of 1 and 2, we reported the
synthesis and anti-HCV activity of 20-C-hydroxymethyladenosine
(3).17 Compound 3 also showed potent anti-HCV activity in a
feron-
a
or pegylated interferon-
a
is being used in the treatment of
HCV infection.3 Interferon-
a increases the nonadaptive antiviral
response in cells.4
Ribavirin (Fig. 1) alone inhibits HCV subgenomic replicon repli-
cation in HuH6 cells with an EC50 of 87 22
M.5 Ribavirin is con-
l
verted to the mono-, di-, and triphosphate inside of cells.6 This
triphosphate is incorporated into a HCV RNA chain by a viral
RNA polymerase, mutagenizing the genome and decreasing the
yield of infectious virus.7 Ribavirin also shows anti-HCV activity
by inhibiting inosine-50-monophosphate (IMP) dehydrogenase,
resulting in depleting purine nucleotide pools.8
O
The HCV specific NS5b RNA dependent RNA polymerase9,10 rep-
licating a single strand genomic RNA into double strand RNA is
one of the key enzymes in the life cycle11 of HCV. Inhibition of this
enzyme makes the HCV unable to replicate. Thus, it has been a prom-
ising therapeutic target for the development of new anti-HCV
agents.
H2N
N
N
N
HO
O
HO
OH
* Corresponding author. Tel.: +82 2 3277 3466; fax: +82 2 3277 2851.
E-mail address: lakjeong@ewha.ac.kr (L.S. Jeong).
Figure 1. Structural formula of ribavirin.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.002

W. J. Choi et al. / Bioorg. Med. Chem. 18 (2010) 4812–4820
4813
the 6-chloropurine derivative 9 as a single diastereomer, due to the
neighboring group participation by 20-acetoxy group (Scheme 3).
The 6-chloropurine derivative 9 was treated with methanolic
ammonia and aqueous methylamine to give adenine derivative
10 and N6-methyladenine derivative 11, respectively, along with
concomitant removal of the acetyl protecting group. Treatment of
10 and 11 with palladium black in 5% formic acid in methanol
afforded 4d and 4e, respectively. For the synthesis of the cytosine
derivative 4f, the glycosyl donor 8 was condensed with silylated
N4-benzoylcytosine to give the N4-benzoylcytosine derivative 12
as a single stereoisomer. Removal of the benzyl group of 12 with
palladium black following the treatment with methanolic ammo-
nia produced the final cytosine derivative 4f.
B
HO
B
O
HO
Y
O
X
HO
OH
HO
OH
1 (X = CH3, B = cytosine-1-yl)
2 (X = CH3, B = adenine-9-yl)
3 (X = CH2OH, B = adenine-9-yl)
4 (Y = OH, F, N3, or NH2)
B = pyrimidines and purines
Figure 2. The rationale for the design of the target nucleoside 4.
cell-based replicon assay, indicating that 20-C-hydroxymethyl
group might induce the hydrogen bonding interaction with HCV
RNA polymerase and/or the steric repulsion like the 20-methyl
group. Based on these findings, we designed and synthesized bio-
isosteric compound 4 (Y = OH)18 shifting the hydroxymethyl substi-
tuent from 20-position to 30-position and compared the
conformation of 4 (Y = OH) with that of 3. We also synthesized
the analogues 4 (Y = N3, NH2, or F) in bioisosteric relationship with
compound 4 (Y = OH). Herein, we report the synthesis, anti-HCV
activity, and conformational analysis of 30-C-substituted pyrimi-
30-C-Azidomethyl nucleosides were also synthesized from the
same intermediate 5 via the glycosyl donor 15 (Scheme 4). Treat-
ment of 5 with diphenylphosphoryl azide (DPPA), triphenylphos-
phine (PPh3), and diisopropyl azodicarboxylate (DIAD) in THF
produced the 3-C-azidomethyl derivative 14.19 Compound 14
was converted into the diacetate 15 by the same method used in
Scheme 2.
Another glycosyl donor 15 was condensed with silylated 6-
chloropurine and N4-benzoylcytosine using the same conditions
used in Scheme 3 to give the 6-chloropurine derivative 16 and
N4-benzoylcytosine derivative 18, respectively (Scheme 5). Palla-
dium black in 5% formic acid in methanol used to remove the ben-
zyl protecting group in Scheme 3 could not be used for compounds
16 and 18 as the azido group was reduced to the amino group un-
der this condition. Alternatively, boron trichloride and boron tri-
bromide were used, but tertiary benzyl group was inert under
these conditions, only giving mono debenzylated products, 17
and 19 as major products. To solve this problem, another strategy
to oxidize the benzyl group to the benzoyl group was employed, as
shown in Scheme 6.
Oxidation of 16 with catalytic RuCl3 and excess NaIO4 in co-sol-
vents (CH3CN/CCl4/H2O = 2:2:3) afforded 20 as a mixture of the de-
sired dibenzoyl and partially oxidized monobenzoyl derivatives.20
Treatment of the mixture 20 with methanolic ammonia yielded
the final adenine derivative 4g and its 30-benzyl derivative 21. Sim-
ilarly compound 20 was converted to the final N6-methyladenine
derivative 4h and its 30-benzyl derivative 22. However, the same
oxidation on the cytosine derivative 18 resulted in the decomposi-
tion instead of giving the desired dibenzoate 23. Thus, we em-
ployed alternative procedure to synthesize the cytosine
derivative 4j as depicted in Scheme 7.
dine and purine nucleosides 4 from D-xylose.
2. Results and discussion
2.1. Chemistry
Our synthetic strategy to the final nucleosides was first to syn-
thesize the glycosyl donor and then to condense with pyrimidine
or purine base. The 30-C-hydroxymethyl-ribofuranosyl pyrimidine
and purine nucleosides 4a–c were synthesized from D-xylose via
the key intermediate 5 according to our previously reported proce-
dure18 (Scheme 1).
For the synthesis of 30-C-fluoromethyl nucleosides, the glycosyl
donor 8 was synthesized from the key intermediate 5, as shown in
Scheme 2. 3-C-Hydroxymethyl derivative 5 was converted to the
mesylate 6 which was heated with tetra-n-butylammonium fluo-
ride (TBAF) in DMF at 100 °C to give the 3-C-fluoromethyl deriva-
tive 7. Acid-catalyzed hydrolysis of 7 followed by acetylation of
resulting diol yielded the diacetate 8 in good yield, which is ready
for the condensation with pyrimidine or purine bases.
For the synthesis of the adenine derivatives 4d and 4e, the gly-
cosyl donor 8 was condensed with silylated 6-chloropurine to give
NH2
N
NHR
N
N
N
BnO
O
ref. 18
ref. 18
N
O
N
D-Xylose
HO
HO
HO
HO
O
HO
O
O
BnO
O
HO
OH
5
HO
OH
4c
4a (R = H)
4b (R = Me)
Scheme 1.
BnO
F
BnO
BnO
BnO
b
O
c
O
O
a
O
OAc
85%
88%
58%
O
O
O
HO
F
MsO
BnO
O
BnO
O
BnO
OAc
BnO
O
5
7
8
6
Scheme 2. Reagents and conditions: (a) MsCl, pyridine, rt, overnight; (b) TBAF, DMF, 100 °C, overnight; (c) (i) 80% AcOH, 90 °C, overnight; (ii) Ac2O, pyridine, rt, overnight.

4814
W. J. Choi et al. / Bioorg. Med. Chem. 18 (2010) 4812–4820
Cl
NHR
N
NHR
N
N
N
N
N
N
N
N
d
b or c
N
N
N
BnO
F
BnO
F
HO
F
O
O
O
61%
BnO OAc
BnO OH
HO OH
4d (R = H) (80%)
4e (R = Me) (79%)
9
10 (R = H) (90%)
11 (R = Me) (88%)
BnO
F
a
O
OAc
BnO OAc
8
NHBz
N
NH2
N
NH2
N
83%
N
O
N
O
N
O
BnO
F
BnO
F
HO
F
b
83%
d
73%
O
O
O
BnO OAc
BnO OH
HO OH
12
13
4f
Scheme 3. Reagents and conditions: (a) persilylated bases, TMSOTf, DCE, rt; (b) NH3/MeOH, 80 °C, 1 d; (c) 40% NH2Me in H2O, 80 °C, 1 d; (d) Pd black, 5% HCO2H in MeOH,
reflux, overnight.
BnO
N3
BnO
HO
BnO
N3
O
O
O
b
a
OAc
66%
72%
O
O
BnO
OAc
BnO
O
BnO
O
15
14
5
Scheme 4. Reagents and conditions: (a) Ph3P, DIAD, DPPA, THF, rt, 10 h; (b) (i) 80% AcOH, 90 °C, overnight; (ii) Ac2O, pyridine, rt, 7 h.
Cl
N
Cl
N
N
N
N
N
N
N
65%
b
HO
N3
BnO
N3
O
O
68%
BnO OAc
BnO OAc
BnO
N3
a
O
17
16
OAc
BnO OAc
NHBz
N
NHBz
15
N
b
N
O
N
O
HO
N3
BnO
N3
50%
O
O
64%
BnO OAc
BnO OAc
18
19
Scheme 5. Reagents and conditions: (a) persilylated bases, TMSOTf, DCE, rt, 2 h; (b) BCl3 or BBr3, CH2Cl2, ꢀ78 °C, 1.5 h to ꢀ40 °C, 3 h.
Oxidation of the dibenzylate 14 under the same oxidation con-
ditions used in Scheme 6 gave an inseparable mixture 24 of the de-
sired dibenzoate and partially oxidized monobenzoyl derivative.
Treatment of 24 with sodium methoxide produced diol 25a
(R = H) and 30-benzyl derivative 25b (R = Bn), which were easily
separated by silica gel chromatography. The diol 25a was treated
with acetic anhydride in pyridine at 80 °C to give the diacetate
26. Hydrolysis of 1,2-acetonide of 26 under the same condition
used in previous Schemes 2 and 4 followed by acetylation of result-
ing diol afforded the undesired pyranosyl acetate as a major prod-
uct and the desired furanosyl acetate 27 as a minor product in 9:1
ratio. However, treatment of 26 with acetic acid, acetic anhydride,
and c-H2SO4 gave the desired glycosyl donor 27 and the undesired
pyranosyl acetate in 4.5:1 ratio. The glycosyl donor 27 was con-
densed with persilylated N4-benzoylcytosine to give the N4-ben-
zoylcytosine derivative 28 as the single b-anomer in 51% yield.
The 30-C-azidomethyl-substituted cytosine derivative 4j was ob-
tained by treating 28 with methanolic ammonia.
2.2. Anti-HCV activity
All synthesized final nucleosides 4a–j were tested for anti-HCV
activity in a replicon-based cell assay.21 Antiviral activity and cyto-
toxicity of 4a–j are shown in Table 1. All adenine analogues, 4a, 4d,

W. J. Choi et al. / Bioorg. Med. Chem. 18 (2010) 4812–4820
4815
Cl
N
Cl
R
N
R
N
N
N
N
N
N
N
N
N
N
N
N
N
BnO
N3
BzO
N3
a
b or c
HO
N3
O
O
+
N
O
HO
N3
O
BnO OAc
RO OAc
BnO OH
HO OH
4g (R = NH2) (23%)
4h (R = NHMe) (25%)
4i (R = OH)
16
20 (R = Bz and Bn)
21 (R = NH2) (34%)
22 (R = NHMe) (20%)
d
48%
NHBz
NHBz
N
N
a
N
O
N
O
BnO
N3
BzO
decomposed
O
O
N3
BnO
OAc
BzO
OAc
18
23
Scheme 6. Reagents and conditions: (a) RuCl3 ꢁ H2O, NaIO4, rt, 2 d; (b) NH3/MeOH, rt, overnight; (c) 40% NH2Me in H2O, 80 °C, 1 d; (d) NaNO2, AcOH,H2O, rt to 40 °C, 2.5 h.
HO
N3
BzO
N3
AcO
98% N3
BnO
N3
c
O
a
b
O
O
O
43%
from 14
O
O
O
O
RO
O
RO
O
AcO
O
BnO
O
14
26
24 (R = Bz and Bn)
25a (R = H)
25b (R = Bn)
53%
d
NH2
N
NHBz
N
AcO
N3
O
OAc
e
f
N
O
N
O
HO
N3
AcO
N3
O
51%
77%
O
AcO OAc
27
HO OH
AcO OAc
4j
28
Scheme 7. Reagents and conditions: (a) RuCl3 ꢁ H2O, NaIO4, rt, 2 d; (b) NaOMe, MeOH, rt, 18 h; (c) Ac2O, pyridine, 80 °C, 1 d; (d) c-H2SO4, AcOH, Ac2O, 30 °C, 5 h; (e)
persilylated N4-Bz-cytosine, TMSOTf, DCE, rt, 4 h; (f) NH3/MeOH, 80 °C, overnight.
and 4g showed significant anti-HCV activity irrespective of the
substituent (Y = OH, N3, or F) at the 30-C-substituted methyl posi-
tion, among which 4g (Y = N3) was the most potent, but they are
also cytotoxic. This result indicates that a hydrogen bonding accep-
tor such as oxygen, nitrogen or fluorine at the 30-C-substituted
methyl position may be essential for anti-HCV activity. In case of
N6-methyladenine derivatives, 30-C-hydroxymethyl derivative 4b
(Y = OH) also showed toxicity-dependent anti-HCV activity, while
30-C-azido- and 30-C-fluoro-methyl derivatives (Y = N3 and F) were
(C30-endo) conformation exhibited anti-HCV activity among 20-
and 30-C-methyladenosine series, but the compounds, 4a, 4b, 4d,
and 4g synthesized in this study adopting Southern (C20-endo) con-
formations also showed significant anti-HCV activity, indicating
that the substituent such as OH, F, or N3 at the 30-C-substituted
methyl position might induce the hydrogen bonding interaction
with HCV RNA polymerase and/or the steric repulsion15 like the
20-C-methyl group.
inactive up to 100
the cytosine derivatives 4f (Y = F) and 4j (Y = N3) did not show any
lM. The hypoxanthine derivative 4i (Y = N3) and
3. Conclusion
In this work, we have accomplished the synthesis of 30-C-substi-
significant anti-HCV activity up to 100 lM.
tuted methyl pyrimidine and purine nucleosides from D-xylose as
potent anti-HCV agents. Although highly active anti-HCV agents
could not be discovered in this study, finding of 30-C-substituted-
methyl nucleosides showing significant anti-HCV activity
guarantees that they can be utilized as new templates for the
development of other anti-HCV agents.
2.3. Conformational analysis
The relative energies of Northern- and Southern-type conform-
ers were calculated for 20-C-hydroxymethyladenosine (3) and 30-C-
hydroxymethyladenosine (4a). As shown in Table 2, 20-C-hydrox-
ymethyladenosine (3) displayed a strong preference for a Northern
(C30-endo) conformation similar to the ribo configuration of aden-
osine, whereas 30-C-hydroxymethyladenosine (4a) showed to pre-
fer a Southern (C20-endo) conformation. These results are in
agreement with those of calculation of conformational preference
for 20-C-methyladenosine (2) and 30-C-methyladenosine.15 It was
reported that only 20-C-methyladenosine showing a Northern
4. Experimental section
4.1. General methods
1H NMR Spectra (CDCl3, CD3OD or DMSO-d6) were recorded on
Varian Unity Invoa 400 MHz. The 1H NMR data are reported as

4816
W. J. Choi et al. / Bioorg. Med. Chem. 18 (2010) 4812–4820
Table 1
mined on Jasco III in methanol. UV spectra were recorded on U-
3000 made by Hitachi in methanol. Infrared spectra were recorded
on FT-IR (FTS-135) made by Bio-Rad. The IR data were reported as s
for sharp. Elementary analyses were measured on EA1110. Melting
points were measured on B-540 made by Buchi. Reactions were
checked with TLC (Merck precoated 60F254 plates). Spots were de-
tected by viewing under a UV light, colorizing with charring after
dipping in anisaldehyde solution with acetic acid, sulfuric acid
and methanol. Column chromatography was performed on Silica
Gel 60 (230–400 mesh, Merck). Reverse column chromatography
Anti-HCV activity of the 30-C-substituted-methyl nucleosides (4a–j)a
R
NH2
N
N
N
N
N
O
N
HO
Y
HO
Y
O
O
HO
OH
HO
OH
4c,f,j
4a,b,d,e,g-i
was performed on YMC-GEL ODS-A (12 nm S-150 lm, AA12SA5).
All the anhydrous solvents were distilled over CaH2, P2O5 or so-
dium/benzophenone prior to the reaction.
b
c
Compounds
EC50
(l
M)
CC50
(l
M)
SId (CC50/EC50
)
4a (Y = OH, R = NH2)
4b (Y = OH, R = NHMe)
4c (Y = OH)
18.6 10.8
37.8 11.2
ND
37.3 11.3
58.8 11.9
ND
2.0
1.6
—
4.2. Chemistry
4d (Y = F, R = NH2)
4e (Y = F, R = NHMe)
4f (Y = F)
15.3 5.0
>100
>100
11.8 3.8
>100
>100
18.9 9.8
>100
>100
12.3 2.8
>100
>100
1.2
—
—
1.0
—
—
4.2.1. 3b-C-Methanesulfonyloxymethyl-3,5-di-O-benzyl-1,2-O-
isopropylidene-a-D-ribofuranose (6)
4g (Y = N3, R = NH2)
4h (Y = N3, R = NHMe)
4i (Y = N3, R = OH)
4j (Y = N3)
To a stirred solution of 5 (505 mg, 1.26 mmol) in pyridine
(10 mL), methanesulfonyl chloride (0.19 mL, 2.52 mmol) was
added and the mixture was stirred at room temperature for 18 h.
The solvent was removed and the residue was purified by silica
gel column chromatography (hexane/ethyl acetate = 3:1) to give
>100
0.3
>100
ND
—
—
2e
a
Each experiment was repeated three times and data are expressed as mean
values standard deviation.
Inhibitor concentration needed to reduce viral replication to 50%. Interferon
-2b was used as a (positive) reference compound (10000 Units/well) and reduced
the signal in the viral RNA (luciferase) assay to background levels without any
cytotoxic activity.
6 (532 mg, 88%) as a white solid: mp 88–91 °C; ½a D20
ꢀ42.63 (c
ꢂ
1.37, MeOH); 1H NMR (CDCl3) d 7.34–7.26 (m, 10H), 5.87 (d, 1H,
J = 4.0 Hz), 4.78 (dd, 2H, J = 10.8, 32.4 Hz), 4.76 (d, 1H, J = 4.0 Hz),
4.56 (d, 2H, J = 4.0 Hz), 4.42 (s, 2H), 4.34 (dd, 1H, J = 2.8, 4.8 Hz),
3.76 (dd, 1H, J = 3.2, 11.2 Hz), 3.66 (dd, 1H, J = 3.2, 11.2 Hz), 2.85
(s, 3H), 1.61 (s, 3H), 1.39 (s, 3H); 13C NMR (CDCl3) d 138.53,
137.93, 128.65, 128.50, 128.12, 128.00, 127.88, 127.79, 113.28,
104.18, 83.78, 79.73, 78.81, 73.97, 69.78, 69.07, 66.94, 37.40,
26.86, 26.79; Anal. Calcd for C24H30O8S: C, 60.23; H, 6.32. Found:
C, 60.45; H, 6.19.
b
a
c
Inhibitor concentration that inhibits cell growth by 50%.
Selectivity index.
Ref. 15.
d
e
peak multiplicities: s for singlet, d for doublet, dd for doublet of
doublets, t for triplet, q for quartet, br s for broad singlet and m
for multiplet. Coupling constants are reported in hertz. 13C NMR
(CDCl3, CD3OD or DMSO-d6) were recorded on Varian Unity Inova
100 MHz. 19F NMR (CDCl3, CD3OD) were recorded on Varian Unity
Inova 376 MHz. The chemical shifts were reported as parts per mil-
lion (d) relative to the solvent peak. Optical rotations were deter-
4.2.2. 3b-C-Fluoromethyl-3,5-di-O-benzyl-1,2-O-isopropylidene-
a
-D-ribofuranose (7)
To a stirred solution of 6 (781 mg, 1.63 mmol) in DMF (50 mL),
tetrabutylammonium fluoride (4.89 mL, 4.89 mmol, 1.0 M solution
in THF) was added and the mixture was stirred at 100 °C for 18 h.
Table 2
Lowest-energy conformations of 20-C-hydroxymethyladenosine (3) and 30-C-hydroxymethyladenosine (4a)
Compound
20-C-Hydroxymethyladenosine (3)
30-C-Hydroxymethyladenosine (4a)
a
[(EN ꢀ ES)/kcal molꢀ1
]
ꢀ2.92
+5.03
Lowest-energy conformation
Northern, C30-endo conformer
Southern, C20-endo conformer
NH2
NH2
N
N
N
N
HO
HO
H
N
N
H
N
N
O
O
OH
HO
OH
HO
OH
OH
a
Calculated energy difference between North and South conformers. Conformational search algorithm implemented in MacroModel (v. 7.5.106, Schrödinger Inc.) was
performed using the MMFFs force field in the presence of water as a solvent. Starting from both north and south conformers provided the same lowest-energy conformers.

W. J. Choi et al. / Bioorg. Med. Chem. 18 (2010) 4812–4820
4817
The reaction mixture was evaporated and the residue was purified
by silica gel column chromatography (hexane/ethyl acetate = 8:1)
(s, 1H), 7.40–7.22 (m, 10H), 6.12 (d, 1H, J = 7.2 Hz), 6.05 (br s,
2H), 5.07 (s, 1H), 4.96 (s, 1H), 4.89 (dd, 2H, J = 11.2, 42.0 Hz), 4.73
(d, 1H, J = 7.2 Hz), 4.65 (t, 1H, J = 2.4 Hz), 4.57–4.55 (m, 2H), 3.84
(s, 2H); 13C NMR (CDCl3) d 155.59, 152.77, 150.09, 139.06,
137.88, 136.97, 128.84, 128.79, 128.68, 128.44, 128.36, 128.15,
128.10, 127.95, 127.87, 127.77, 119.57, 88.53, 85.58, 83.85, 83.47,
to give 7 (380 mg, 58%) as a colorless syrup: ½a D20
ꢂ
+13.92 (c 1.20,
MeOH); 1H NMR (CDCl3) d 7.45–7.31 (m, 10H), 5.91 (d, 1H,
J = 4.0 Hz), 4.81 (dd, 2H, J = 12.0, 22.4 Hz), 4.80 (d, 1H, J = 4.0 Hz),
4.69 (dd, 1H, J = 2.4, 11.2 Hz), 4.59–4.56 (m, 1H), 4.57 (s, 2H),
4.35–4.32 (m, 1H), 3.77 (dd, 1H, J = 2.8, 11.2 Hz), 3.63 (dd, 1H,
J = 4.8, 11.2 Hz), 1.65 (s, 3H), 1.41 (s, 3H); 13C NMR (CDCl3) d
138.65, 137.98, 128.64, 128.47, 128.33, 128.08, 127.80, 127.74,
127.68, 127.63, 112.92, 104.31, 84.93, 83.18, 80.72, 79.95, 73.70,
82.06, 74.23, 69.71, 67.72; 19F NMR (CDCl3)
d
ꢀ230.04 (t,
J = 47.0 Hz); Anal. Calcd for C25H26FN5O4: C, 62.62; H, 5.47; N,
14.61. Found: C, 62.95; H, 5.51; N, 14.73.
68.90, 67.06, 26.76, 26.73; 19F NMR (CDCl3)
J = 48.5 Hz); Anal. Calcd for C23H27FO5: C, 68.64; H, 6.76. Found:
d
ꢀ229.18 (t,
4.2.6. 9-(3b-C-Fluoromethyl-3,5-di-O-benzyl-b-D-ribofuranosyl)
-N6-methyladenine (11)
C, 68.80; H, 6.87.
A solution of 9 (257 mg, 0.475 mmol) in 40% aqueous MeNH2
(8 mL) was heated at 80 °C for 1 d and the reaction mixture was
evaporated. The resulting residue was purified by silica gel column
chromatography (CH2Cl2/MeOH = 40:1–20:1) to give 11 (205 mg,
4.2.3. 3b-C-Fluoromethyl-3,5-di-O-benzyl-1,2-di-O-acetyl-a,b-D-
ribofuranose (8)
A solution of 7 (619 mg, 1.53 mmol) in 80% aqueous acetic acid
(10 mL) was stirred at 90 °C overnight and the solvents were re-
moved. To a solution of crude residue in pyridine (8 mL), acetic
anhydride (0.73 mL, 7.69 mmol) was added, and the reaction mix-
ture was stirred at room temperature for 18 h. The solvents were
evaporated, and the residue was purified by silica gel column chro-
matography (hexane/ethyl acetate = 5:1) to give 8 (585 mg, 85%) as
a colorless syrup.
88%) as a white foam: UV (MeOH) kmax 264.5 nm; ½a D20
ꢀ64.55 (c
ꢂ
2.35, MeOH); 1H NMR (CDCl3) d 8.31 (s, 1H), 7.98 (s, 1H), 7.41–
7.19 (m, 10H), 6.11 (d, 1H, J = 6.8 Hz), 5.07 (s, 1H), 4.95 (s, 1H),
4.90 (dd, 2H, J = 11.2, 44.0 Hz), 4.73 (d, 1H, J = 7.2 Hz), 4.63 (t, 1H,
J = 2.8 Hz), 4.55 (d, 1H, J = 7.2 Hz), 4.56–4.54 (m, 2H), 4.55 (d, 1H,
J = 7.2 Hz), 3.82 (d, 2H, J = 2.0 Hz), 3.15 (br s, 3H); 13C NMR (CDCl3)
d 155.39, 153.06, 138.27, 137.91, 137.01, 128.81, 128.72, 128.68,
128.38, 128.18, 128.13, 127.91, 127.82, 119.96, 88.82, 85.41,
82.33, 77.84, 74.23, 69.71, 67.78; 19F NMR (CDCl3) d ꢀ230.12 (t,
J = 47.4 Hz); Anal. Calcd for C26H28FN5O4: C, 63.27; H, 5.72; N,
14.19. Found: C, 63.40; H, 5.79; N, 14.27.
Major isomer: 1H NMR (CDCl3) d 7.37–7.27 (m, 10H), 6.24 (d, 1H,
J = 2.4 Hz), 5.48 (d, 1H, J = 2.0 Hz), 4.89 (dd, 2H, J = 8.0, 47.2 Hz),
4.62–4.54 (m, 4H), 4.50–4.47 (m, 1H), 3.74 (s, 2H), 2.08 (s, 3H),
2.04 (s, 3H); Anal. Calcd for C24H27FO7: C, 64.56; H, 6.10. Found:
C, 64.31; H, 6.28.
4.2.7. 9-(3b-C-Fluoromethyl-b-D-ribofuranosyl)adenine (4d)
To a stirred solution of 10 (110 mg, 0.229 mmol) in 5% formic
acid in MeOH (10 mL) was added Pd black (98% Pd, 34 mg, catalytic
amount) and the reaction mixture was refluxed overnight. The
mixture was filtered through a pad of Celite and washed with
MeOH several times. After evaporation of solvents, the residue
was purified by silica gel column chromatography (CH2Cl2/
MeOH = 20:1–10:1) to give 4d (55 mg, 80%) as a white solid: mp
4.2.4. 9-(3b-C-Fluoromethyl-3,5-di-O-benzyl-2-O-acetyl-b-D-
ribofuranosyl)-6-chloropurine (9)
A mixture of 6-chloropurine (423 mg, 2.74 mmol), (NH4)2SO4
(5 mg, catalytic amount), and hexamethyldisilazane (15 mL) was
refluxed overnight under N2 atmosphere, and the mixture was
evaporated. The resulting solid was dissolved in anhydrous 1,2-
dichloroethane (10 mL). To this mixture was added a solution of
8 (612 mg, 1.37 mmol) in 1,2-dichloroethane (8 mL) and the reac-
tion mixture was cooled to 0 °C. Trimethylsilyl trifluoromethane-
sulfonate (0.5 mL, 2.74 mmol) was added dropwise at the same
temperature, and the reaction mixture was stirred at room temper-
ature for 2 h. After quenched with aqueous NaHCO3 solution, the
mixture was extracted with CH2Cl2. The organic layer was dried
over anhydrous MgSO4, filtered, and evaporated. The residue was
purified by silica gel column chromatography (hexane/ethyl ace-
tate = 3:1) to give 9 (450 mg, 61%) as a white foam: UV (MeOH)
210–216 °C (dec); UV (MeOH) kmax 259.0 nm; ½a D20
ꢀ22.88 (c
ꢂ
1.04, MeOH); 1H NMR (CD3OD) d 8.29 (s, 1H), 8.18 (s, 1H), 6.00
(d, 1H, J = 7.6 Hz), 4.93 (dd, 1H, J = 9.6, 46.4 Hz), 4.77 (d, 1H,
J = 8.0 Hz), 4.64 (dd, 1H, J = 9.6, 46.4 Hz), 4.20 (t, 1H, J = 2.4 Hz),
3.90 (dd, 1H, J = 2.8, 13.2 Hz), 3.82 (dd, 1H, J = 2.8, 13.2 Hz); 13C
NMR (CD3OD) d 157.86, 153.54, 150.12, 142.72, 121.39, 91.03,
89.01, 87.15, 85.49, 79.87, 79.70, 75.49, 75.42, 62.65; 19F NMR
(CDCl3) d ꢀ238.02 (t, J = 47.4 Hz); Anal. Calcd for C11H14FN5O4: C,
44.15; H, 4.72; N, 23.40. Found: C, 43.92; H, 4.84; N, 23.56.
kmax 263.5 nm; ½a D20
ꢂ
ꢀ63.40 (c 2.05, MeOH); 1H NMR (CDCl3) d
4.2.8. 9-(3b-C-Fluoromethyl-b-D-ribofuranosyl)-N6-
methyladenine (4e)
8.70 (s, 1H), 8.50 (s, 1H), 7.41–7.31 (m, 10H), 6.54 (d, 1H,
J = 7.2 Hz), 5.92 (d, 1H, J = 7.6 Hz), 4.97 (dd, 2H, J = 10.8, 47.6 Hz),
4.81 (dd, 2H, J = 2.4, 11.2 Hz), 4.64 (d, 2H, J = 2.4 Hz), 4.58 (dd,
2H, J = 2.0, 7.6 Hz), 3.83 (t, 1H, J = 2.4 Hz), 2.02 (s, 3H); 13C NMR
(CDCl3) d 170.24, 152.32, 151.28, 143.89, 138.05, 136.48, 131.99,
129.01, 128.81, 128.69, 128.66, 128.45, 128.26, 128.18, 128.03,
127.93, 127.40, 85.93, 84.74, 83.53, 83.02, 76.66, 74.33, 69.31,
67.52, 20.69; 19F NMR (CDCl3) d ꢀ226.90 (t, J = 47.0 Hz); Anal.
Calcd for C27H26ClFN4O5: C, 59.95; H, 4.84; N, 10.36. Found: C,
60.11; H, 4.95; N, 10.29.
Compound 11 (160 mg, 0.324 mmol) was converted to N6-
methyladenine derivative 4e (80 mg, 79%) as
a white solid
according to the same procedure used in the preparation of 4d:
mp 135–140 °C; UV (MeOH) kmax 265.0 nm; ½a D20
ꢀ50.60 (c 0.67 ,
ꢂ
MeOH); 1H NMR (CD3OD) d 8.18 (s, 2H), 5.97 (d, 1H, J = 7.6 Hz),
4.94 (dd, 1H, J = 10.0, 46.8 Hz), 4.86 (d, 1H, J = 5.6 Hz), 4.75 (d,
1H, J = 7.6 Hz), 4.65 (dd, 1H, J = 10.0, 46.8 Hz), 4.20 (t, 1H,
J = 2.0 Hz), 3.93–3.81 (m, 2H), 3.07 (br s, 3H); 13C NMR (CD3OD) d
156.93, 153.45, 148.80, 142.13, 121.86, 91.01, 88.94, 87.17, 85.49,
75.38, 62.63, 27.80; 19F NMR (CDCl3) d ꢀ238.12 (t, J = 47.5 Hz);
Anal. Calcd for C12H16FN5O4: C, 46.01; H, 5.15; N, 22.35. Found:
C, 46.13; H, 5.21; N, 22.18.
4.2.5. 9-(3b-C-Fluoromethyl-3,5-di-O-benzyl-b-D-ribofuranosyl)
adenine (10)
A solution of 9 (147 mg, 0.272 mmol) in saturated methanolic
ammonia (5 mL) was stirred in a glass bomb at 80 °C for 1 d and
the volatiles were evaporated. The residue was purified by silica
gel column chromatography (CH2Cl2/MeOH = 40:1–20:1) to give
10 (117 mg, 90%) as a white foam: UV (MeOH) kmax 258.5 nm;
4.2.9. 1-(3b-C-Fluoromethyl-3,5-di-O-benzyl-2-O-acetyl-b-D-
ribofuranosyl)-N4-benzoylcytosine (12)
The glycosyl donor 8 (140 mg, 0.314 mmol) was condensed
with silylated N4-benzoylcytosine (134 mg, 0.690 mmol) according
to the same procedure used in the preparation of 9, to give 12
½
a 2D0
ꢂ
ꢀ75.87 (c 1.06, MeOH); 1H NMR (CDCl3) d 8.23 (s, 1H), 8.07

4818
W. J. Choi et al. / Bioorg. Med. Chem. 18 (2010) 4812–4820
(158 mg, 83%) as white foam: UV (MeOH) kmax 308.0 nm; ½a D20
ꢂ
138.06, 128.59, 128.46, 128.02, 127.87, 127.79, 113.12, 104.07,
84.11, 80.24, 79.39, 73.80, 68.43, 67.72, 52.10, 26.89, 26.75; IR
(KBr) 2101.27 cmꢀ1 (s); Anal. Calcd for C23H27N3O5: C, 64.93; H,
6.40; N, 9.88. Found: C, 64.74; H, 6.56; N, 10.05.
+42.08 (c 1.15, MeOH); 1H NMR (CDCl3) d 8.56 (br s, 1H), 8.25 (d,
1H, J = 7.2 Hz), 7.89 (d, 2H, J = 6.8 Hz), 7.62–7.28 (m, 14H), 6.60
(d, 1H, J = 7.2 Hz), 5.40 (d, 1H, J = 7.2 Hz), 4.93 (dd, 1H, J = 11.2,
47.6 Hz), 4.85 (dd, 1H, J = 11.2, 47.6 Hz), 4.88–4.82 (m 2H), 4.69
(d, 1H, J = 11.6 Hz), 4.59 (d, 1H, J = 11.2 Hz), 4.55 (t, 1H,
J = 2.4 Hz), 3.92–3.82 (m, 2H), 2.09 (s, 3H); 13C NMR (CDCl3) d
170.51, 162.26, 144.92, 138.09, 136.60, 133.28, 129.14, 129.05,
128.79, 128.75, 128.60, 128.55, 128.45, 128.37, 128.26, 127.88,
127.77, 127.36, 127.32, 86.99, 84.24, 83.73, 83.48, 82.51, 74.26,
4.2.13. 3b-C-Azidomethyl-3,5-di-O-benzyl-1,2-di-O-acetyl-a,b-
D-ribofuranose (15)
A solution of 14 (2.0 g, 4.70 mmol) in 80% aqueous acetic acid
(10 mL) was stirred at 90 °C overnight and the solvents were re-
moved. To a solution of crude residue in pyridine (15 mL), acetic
anhydride (2.22 mL, 13.5 mmol) was added, and the reaction mix-
ture was stirred at room temperature for 18 h. The solvents were
evaporated, and the residue was purified by silica gel column chro-
matography (hexane/ethyl acetate = 4:1) to give 15 (1.49 g, 66%) as
a colorless syrup.
74.08, 69.28, 67.50, 20.83; 19F NMR (CDCl3)
J = 47.0 Hz); Anal. Calcd for C33H32FN3O7: C, 65.88; H, 5.36; N,
d
ꢀ228.19 (t,
6.98. Found: C, 66.11; H, 5.42; N, 7.06.
4.2.10. 1-(3b-C-Fluoromethyl-3,5-di-O-benzyl-b-D-
ribofuranosyl)cytosine (13)
Compound 12 (218 mg, 0.362 mmol) was converted to pro-
tected cytosine derivative 13 (137 mg, 83%) as a white solid
according to the same procedure used in the preparation of 10:
Major isomer: 1H NMR (CDCl3) d 7.40–7.25 (m, 10H), 6.27 (d, 1H,
J = 2.8 Hz), 5.49 (d, 1H, J = 2.8 Hz), 4.68 (s, 2H), 4.56 (dd, 2H, J = 3.2,
7.6 Hz), 4.54 (t, 1H, J = 2.4 Hz), 3.75 (d, 2H, J = 7.2, 13.2 Hz), 2.06 (s,
3H), 2.05 (s, 3H).
mp 181–184 °C; UV (MeOH) kmax 271.0 nm; ½a D20
ꢂ
+55.43 (c 1.51,
MeOH); 1H NMR (CD3OD) d 7.91 (d, 1H, J = 7.6 Hz), 7.44–7.24 (m,
10H), 6.26 (d, 1H, J = 7.6 Hz), 5.66 (d, 1H, J = 7.6 Hz), 5.04 (s, 1H),
4.89 (dd, 2H, J = 11.6, 44.4 Hz), 4.64 (d, 1H, J = 10.4 Hz), 4.54 (d,
1H, J = 10.4 Hz), 4.50 (t, 1H, J = 2.8 Hz), 4.18 (d, 1H, J = 7.6 Hz),
3.88–3.81 (m, 2H); 13C NMR (CD3OD) d 167.55, 159.11, 142.91,
142.24, 140.27, 138.98, 129.88, 129.67, 129.44, 129.39, 129.17,
128.89, 128.84, 128.67, 128.54, 96.73, 89.39, 86.05, 84.34, 83.18,
75.01, 70.95, 68.45; 19F NMR (CD3OD) d ꢀ232.96 (t, J = 47.4 Hz);
Anal. Calcd for C24H26FN3O5: C, 63.29; H, 5.75; N, 9.23. Found: C,
63.46; H, 5.89; N, 9.13.
4.2.14. 9-(3b-C-Azidomethyl-3,5-di-O-benzyl-2-O-acetyl-b-D-
ribofuranosyl)-6-chloropurine (16)
The glycosyl donor 15 (430 mg, 0.916 mmol) was condensed
with silylated 6-chloropurine (284 mg, 1.84 mmol) according to
the same procedure used in the preparation of 9, to give 16
(338 mg, 65%) as a white foam: UV (MeOH) kmax 263.5 nm; ½a D20
ꢂ
ꢀ76.92 (c 0.91, MeOH); 1H NMR (CDCl3) d 8.70 (s, 1H), 8.53 (s,
1H), 7.44–7.30 (m, 10H), 6.50 (d, 1H, J = 7.2 Hz), 5.99 (d, 1H,
J = 7.2 Hz), 4.90 (d, 1H, J = 10.8 Hz), 4.80 (d, 1H, J = 10.8 Hz), 4.66
(d, 1H, J = 12.0 Hz) 4.57 (t, 1H, J = 2.4 Hz), 4.55 (d, 1H, J = 12.0 Hz),
3.87 (dd, 1H, J = 2.4, 12.0 Hz), 3.80 (s, 2H), 3.76 (dd, 1H, J = 2.4,
11.2 Hz), 2.06 (s, 3H); 13C NMR (CDCl3) d 169.90, 152.05, 151.94,
150.98, 143.69, 137.56, 136.15, 131.82, 128.82, 128.55, 128.35,
128.20, 127.93, 127.30, 86.01, 84.04, 83.85, 73.99, 68.79, 67.03,
51.76, 20.61; Anal. Calcd for C27H26ClN7O5: C, 57.50; H, 4.65; N,
17.38. Found: C, 57.60; H, 4.81; N, 17.53.
4.2.11. 1-(3b-C-Fluoromethyl-b-D-ribofuranosyl)cytosine (4f)
To a stirred solution of 13 (79 mg, 0.173 mmol) in 5% formic
acid in MeOH (8 mL) was added Pd black (98% Pd, 5 mg, catalytic
amount) and the reaction mixture was refluxed overnight. The
mixture was filtered through a pad of Celite and washed with
MeOH several times. After evaporation of solvents, the resulting
residue was purified by C18 reverse phase column chromatography
(0–3% acetone in water) to give 4f (35 mg, 73%) as a white solid:
4.2.15. 9-(3b-C-Azidomethyl-3,5-di-O-benzoyl-2-O-acetyl-b-D-
ribofuranosyl)-6-chloropurine (20, R = Bz) and 9-(3b-C-azido-
methyl-5-O-benzoyl-3-O-benzyl-2-O-acetyl-b-D-ribofuranosyl)-
6-chloropurine (20, R = Bn)
mp 230–235 °C (dec); UV (MeOH) kmax 271.0 nm; ½a D20
ꢂ
+88.91 (c
0.51, MeOH); 1H NMR (CD3OD) d 7.99 (d, 1H, J = 7.2 Hz), 6.00 (d,
1H, J = 7.6 Hz), 5.91 (d, 1H, J = 7.2 Hz), 4.71 (dd, 1H, J = 10.0,
46.8 Hz), 4.54 (dd, 1H, J = 10.0, 48.0 Hz), 4.24 (d, 1H, J = 7.2 Hz),
4.08 (t, 1H, J = 2.8 Hz), 3.83–3.74 (m, 2H); 13C NMR (CD3OD) d
167.47, 158.75, 144.22, 96.67, 91.20, 87.93, 86.80, 85.11, 79.62,
79.44, 75.94, 75.88, 62.06; 19F NMR (CD3OD) d ꢀ236.47 (t,
J = 47.4 Hz); Anal. Calcd for C10H14FN3O5: C, 43.64; H, 5.13; N,
15.27. Found: C, 43.69; H, 5.22; N, 15.31.
To a stirred heterogeneous solution of 16 (434 mg, 0.770 mmol)
in co-solvents (17.5 mL, CH3CN/CCl4/H2O = 2:2:3) were added
RuCl3 ꢁ H2O (16.6 mg, catalytic amount) and NaIO4 (1.3 g,
6.16 mmol) at 0 °C and the mixture was stirred at room tempera-
ture for 2 d. The mixture was diluted with CH2Cl2 and extracted
with CH2Cl2 (three times). The combined organic layers were dried
over anhydrous MgSO4, filtered, and evaporated. The residue was
prepared for the next step without further purification.
4.2.12. 3b-C-Azidomethyl-3,5-di-O-benzyl-1,2-O-
isopropylidene-a-D-ribofuranose (14)
Compound 20 (R = Bz): 1H NMR (CDCl3) d 8.59 (s, 1H), 8.23 (s,
1H), 8.14–8.05 (m, 2H), 7.69–7.38 (m, 8H), 6.46 (d, 1H,
J = 7.2 Hz), 6.32 (d, 1H, J = 7.2 Hz), 5.08 (t, 1H, J = 4.0 Hz), 5.00
(dd, 1H, J = 4.0, 12.4 Hz), 4.63 (dd, 2H, J = 4.8, 12.8 Hz) 4.53 (d,
1H, J = 12.8 Hz), 4.11 (d, 1H, J = 7.2 Hz), 2.05 (s, 3H).
To a stirred solution of PPh3 (1.57 g, 5.99 mmol) in THF (50 mL),
were added diisopropyl azodicarboxylate (1.21 g, 5.99 mmol) and
diphenylphosphoryl azide (1.65 g, 5.99 mmol) at 0 °C. The mixture
was stirred for 10 min at the same temperature, and white precip-
itation was formed. The solution of 5 (2.01 g, 4.99 mmol) in THF
(50 mL) was added to the mixture at the same temperature and
the reaction mixture was stirred at room temperature for 18 h.
The solvents were removed and the residue was purified by silica
gel column chromatography (hexane/ethyl acetate = 8:1–5:1) to
4.2.16. 9-(3b-C-Azidomethyl-b-D-ribofuranosyl)adenine (4g)
and 9-(3b-C-Azidomethyl-3-O-benzyl-b-D-ribofuranosyl)
adenine (21)
A solution of 20 (crude 116 mg, 0.211 mmol) in saturated meth-
anolic ammonia (8 mL) was stirred in a glass bomb at 80 °C over-
night and the volatiles were evaporated. The residue was purified
by silica gel column chromatography (CH2Cl2/MeOH = 20:1–10:1)
to give 4g (28 mg, 23% for two steps) as a white solid and 21
(58 mg, 34% for two steps) as a slightly yellowish solid.
Compound 4g: mp 213–217 °C (dec); UV (MeOH) kmax
give 14 (1.52 g, 72%) as a pale yellowish syrup: ½a D20
ꢂ
+64.10 (c
1.46, MeOH); 1H NMR (CDCl3) d 7.41–7.21 (m, 10H), 5.80 (d, 1H,
J = 4.0 Hz), 4.78 (dd, 2H, J = 2.4, 13.2 Hz), 4.61 (d, 1H, J = 4.0 Hz),
4.57 (dd, 2H, J = 6.8, 11.2 Hz), 4.53 (t, 1H, J = 2.4 Hz), 4.40 (dd, 1H,
J = 3.2, 6.8 Hz), 3.76 (dd, 1H, J = 3.2, 11.2 Hz), 3.63 (dd, 1H, J = 6.8,
11.2 Hz), 1.61 (s, 3H), 1.37 (s, 3H); 13C NMR (CDCl3) d 138.49,
257.5 nm; ½a 2D0
ꢂ
ꢀ25.66 (c 0.643, MeOH); 1H NMR (CD3OD) d 8.27

W. J. Choi et al. / Bioorg. Med. Chem. 18 (2010) 4812–4820
4819
(s, 1H), 8.16 (s, 1H), 5.95 (d, 1H, J = 7.6 Hz), 4.65 (d, 1H, J = 8.0 Hz),
4.17 (t, 1H, J = 2.0 Hz), 3.87 (dd, 1H, J = 2.4, 13.2 Hz), 3.81 (dd, 1H,
J = 2.0, 13.2 Hz), 3.69 (s, 2H); 13C NMR (CD3OD) d 157.87, 153.48,
150.04, 142.77, 129.50, 91.02, 89.02, 80.96, 76.62, 62.64, 55.53;
IR (KBr) 2101.33 cmꢀ1 (s); Anal. Calcd for C11H14N8O4: C, 40.99;
H, 4.38; N, 34.77. Found: C, 41.13; H, 4.51; N, 34.67.
phy (hexane/ethyl acetate = 8:1–5:1) to give a mixture of 24
(R = Bz and Bn).
Compound 24 (R = Bz): 1H NMR (CDCl3) d 8.12–8.09 (m, 2H),
8.05–8.01 (m, 2H), 7.61–7.52 (m, 2H), 7.45–7.28 (m, 4H), 5.89 (d,
1H, J = 3.6 Hz), 4.94 (d, 1H, J = 4.0 Hz), 4.63 (dd, 2H, J = 4.0,
8.0 Hz), 4.29 (d, 1H, J = 13.2 Hz), 3.62 (d, 1H, J = 13.2 Hz), 1.50 (s,
3H), 1.34 (s, 3H); Anal. Calcd for C23H23N3O7: C, 60.92; H, 5.11;
N, 9.27. Found: C, 61.12; H, 5.03; N, 9.41.
Compound 21: mp 86–89 °C; 1H NMR (CD3OD) d 8.28 (s, 1H),
8.16 (s, 1H), 7.48 (d, 2H, J = 7.6 Hz), 7.36 (t, 2H, J = 6.8 Hz), 7.29
(d, 1H, J = 7.6 Hz), 6.05 (d, 1H, J = 8.0 Hz), 5.02 (d, 1H, J = 11.2 Hz),
4.87 (d, 1H, J = 8.0 Hz), 4.84 (d, 1H, J = 11.2 Hz), 4.40 (t, 1H,
J = 2.4 Hz), 4.10 (d, 2H, J = 2.4 Hz), 3.94 (dd, 1H, J = 2.4, 13.2 Hz),
3.85 (dd, 1H, J = 2.0, 13.2 Hz).
4.2.20. 3b-C-Azidomethyl-1,2-O-isopropylidene-a-D-
ribofuranose (25a)
To a stirred solution of 24 (crude 540 mg, 1.27 mmol) in MeOH
(10 mL) was added NaOMe (171 mg, 3.10 mmol) and the mixture
was stirred at room temperature for 18 h. The mixture was ex-
tracted with CH2Cl2 (two times) and ethyl acetate (two times).
The combined organic layers were dried over anhydrous MgSO4,
filtered, and evaporated. The residue was purified by silica gel col-
umn chromatography (hexane/ethyl acetate = 5:1–1:1) to give 25a
4.2.17. 9-(3b-C-Azidomethyl-b-D-ribofuranosyl)-N6-methylad-
enine (4h) and 9-(3b-C-Azidomethyl-3-O-benzyl-b-D-
ribofuranosyl)-N6-methyladenine (22)
A solution of 20 (crude 143 mg, 0.260 mmol) in 40% aqueous
MeNH2 (8 mL) was heated at 80 °C overnight and the reaction mix-
ture was evaporated. The resulting residue was purified by silica
gel column chromatography (CH2Cl2/MeOH = 20:1) to give 4h
(20 mg, 25% for two steps) as a white solid and 22 (41 mg, 20%
for two steps) as a slightly yellowish solid.
(205 mg, 43% for two steps) as a white solid: mp 131–133 °C; ½a D20
ꢂ
+1.78 (c 0.95, MeOH); 1H NMR (CDCl3) d 5.74 (d, 1H, J = 3.6 Hz),
4.44 (d, 1H, J = 4.0 Hz), 3.96 (dd, 1H, J = 2.8, 7.2 Hz), 3.79 (dd, 1H,
J = 3.2, 12.4 Hz), 3.61 (dd, 1H, J = 3.2, 12.4 Hz), 3.44 (d, 1H,
J = 13.2 Hz), 3.12 (d, 1H, J = 13.2 Hz), 1.55 (s, 3H), 1.35 (s, 3H); 13C
NMR (CDCl3) d 113.78, 105.04, 83.22, 82.16, 60.69, 53.63, 26.90,
26.78; IR (KBr) 2096.08 cmꢀ1 (s); Anal. Calcd for C9H15N3O5: C,
44.08; H, 6.17; N, 17.13. Found: C, 43.94; H, 6.31; N, 17.09.
Compound 4h: mp 222–226 °C (dec); UV (MeOH) kmax
263.0 nm; ½a 2D0
ꢂ
ꢀ51.93 (c 0.31, MeOH); 1H NMR (CD3OD) d 8.20
(s, 2H), 5.94 (d, 1H, J = 7.6 Hz), 4.64 (d, 1H, J = 8.0 Hz), 4.57 (br s,
1H), 4.17 (t, 1H, J = 2.0 Hz), 3.87 (dd, 1H, J = 2.0, 13.2 Hz), 3.81
(dd, 1H, J = 2.0, 13.2 Hz), 3.69 (s, 2H), 3.10 (br s, 3H); 13C NMR
(CD3OD) d 157.08, 153.45, 148.82, 142.22, 121.98, 103.87, 91.05,
88.98, 80.94, 76.58, 62.66, 55.53; IR (KBr) 2104.58 cmꢀ1 (s); Anal.
Calcd for C12H16N8O4: C, 42.86; H, 4.80; N, 33.32. Found: C,
42.70; H, 4.67; N, 33.44.
4.2.21. 3b-C-Azidomethyl-3,5-di-O-acetyl-1,2-O-isopropylidene-
a-D-ribofuranose (26)
To a stirred solution of 25a (238 mg, 0.971 mmol) in pyridine
was added acetic anhydride (1.0 mL, excess amount) and the mix-
ture was stirred at 80 °C for 1 d. The solvents were evaporated, and
the residue was purified by silica gel column chromatography
(hexane/ethyl acetate = 4:1–3:1) to give 26 (314 mg, 98%) as a col-
Compound 22: mp 94–96 °C; 1H NMR (CD3OD) d 8.23 (s, 1H),
8.16 (s, 1H), 7.37–7.31 (m, 5H), 6.10 (d, 1H, J = 7.6 Hz), 4.57 (s,
2H), 4.54 (d, 1H, J = 7.6 Hz), 4.25 (t, 1H, J = 2.4 Hz), 3.77–3.65 (m,
2H), 3.50–3.47 (m, 2H), 3.12 (br s, 3H).
orless syrup: ½a D20
ꢂ
ꢀ93.46 (c 1.47, MeOH); 1H NMR (CDCl3) d 5.78
(d, 1H, J = 3.6 Hz), 4.73 (d, 1H, J = 4.0 Hz), 4.51 (dd, 1H, J = 2.0,
11.2 Hz), 4.22 (dd, 1H, J = 2.0, 11.2 Hz), 4.19 (t, 1H, J = 2.8 Hz),
4.09 (d, 1H, J = 13.2 Hz), 3.31 (d, 1H, J = 13.2 Hz), 2.14 (s, 3H),
2.11 (s, 3H), 1.54 (s, 3H), 1.33 (s, 3H); 13C NMR (CDCl3) d 170.73,
169.86, 133.57, 130.15, 128.50, 112.88, 103.74, 83.74, 80.47,
62.18, 48.78, 26.56, 26.43; Anal. Calcd for C13H19N3O7: C, 47.41;
H, 5.82; N, 12.76. Found: C, 47.63; H, 5.92; N, 12.65.
4.2.18. 9-(3b-C-Azidomethyl-b-D-ribofuranosyl)hypoxanthine
(4i)
Compound 4g (34 mg, 0.108 mmol) was dissolved in H2O (3 mL)
and NaNO2 (254 mg, 3.69 mmol) and AcOH (0.3 mL) were added to
this solution. The mixture was stirred at room temperature for 1 h,
and at 40 °C for 1.5 h. The reaction mixture was evaporated and the
resulting residue was purified by C18 reverse phase column chro-
matography (0–7% acetone in water) to give 4i (17 mg, 48%) as a
4.2.22. 3b-C-Azidomethyl-1,2,3,5-tetra-O-acetyl-a,b-D-
ribofuranose (27)
pale yellowish foam: UV (MeOH) kmax 243.0 nm; ½a D20
ꢀ70.88 (c
ꢂ
0.45, MeOH); 1H NMR (DMSO-d6) d 12.41 (br s, 1H), 8.34 (s, 1H),
8.08 (s, 1H), 5.90 (d, 1H, J = 7.6 Hz), 5.72 (d, 1H, J = 6.4 Hz), 5.47
(s, 1H), 5.37 (dd, 1H, J = 4.8, 5.6 Hz), 4.34 (dd, 1H, J = 6.8, 7.6 Hz),
4.01 (t, 1H, J = 2.8 Hz), 3.62 (d, 1H, J = 13.2 Hz), 3.43 (d, 1H,
Compound 26 (511 mg, 1.55 mmol) was dissolved in a solution
of acetic acid (8 mL) and acetic anhydride (2 mL, excess amount).
After the mixture was cooled to 0 °C, c-H2SO4 (0.04 mL) was added
to this mixture. The reaction mixture was stirred at 30 °C for 5 h
and poured into saturated NaHCO3 solution. The mixture was ex-
tracted with CH2Cl2. The organic layer was dried over anhydrous
MgSO4, filtered, and evaporated. The residue was purified by silica
gel column chromatography (hexane/ethyl acetate = 3:1–1:1) to
J = 13.2 Hz); 13C NMR (DMSO-d6)
d 156.48, 148.45, 146.04,
138.90, 124.36, 86.74, 86.43, 79.03, 75.90, 60.51, 53.45; IR (KBr)
2103.01 cmꢀ1 (s); Anal. Calcd for C11H13N7O5: C, 40.87; H, 4.05;
N, 30.33. Found: C, 40.54; H, 4.16; N, 30.17.
give a,b-mixture of 27 (308 mg, 53%) as a colorless syrup and the
corresponding pyranosyl derivative (65 mg, 12%).
Compound 27 (b-isomer, major): 1H NMR (CDCl3) d 6.09 (d, 1H,
J = 2.0 Hz), 5.43 (d, 1H, J = 2.0 Hz), 4.59 (dd, 1H, J = 2.8, 12.0 Hz),
4.48 (dd, 1H, J = 2.8, 6.4 Hz), 4.20 (d, 1H, J = 13.2 Hz), 4.14 (dd,
1H, J = 6.4, 12.0 Hz), 3.80 (d, 1H, J = 13.2 Hz), 2.14 (s, 3H), 2.11 (s,
3H), 2,10 (s, 3H), 2.09 (s, 3H); Anal. Calcd for C14H19N3O9: C,
45.04; H, 5.13; N, 11.26. Found: C, 44.98; H, 5.27; N, 11.33.
4.2.19. 3b-C-Azidomethyl-3,5-di-O-benzoyl-1,2-O-isopropyl-
idene-a-D-ribofuranose (24, R = Bz) and 3b-C-Azidomethyl-5-O-
benzoyl-3-O-benzyl-1,2-O-isopropylidene-a-D-ribofuranose
(24, R = Bn)
To a stirred heterogeneous solution of 14 (120 mg, 0.282 mmol)
in co-solvents (10.5 mL, CH3CN/CCl4/H2O = 2:2:3) were added
RuCl3 ꢁ H2O (10 mg, catalytic amount) and NaIO4 (603 mg,
2.82 mmol) at 0 °C. The mixture was stirred at room temperature
for 2 d. The mixture was extracted with CH2Cl2. The organic layer
was dried over anhydrous MgSO4, filtered, and evaporated. The
resulting residue was purified by silica gel column chromatogra-
4.2.23. 1-(3b-C-Azidomethyl-2,3,5-tri-O-acetyl-b-D-
ribofuranosyl)-N4-benzoylcytosine (28)
The glycosyl donor 27 (308 mg, 0.825 mmol) was condensed
with silylated N4-benzoylcytosine (355 mg, 1.65 mmol) according

4820
W. J. Choi et al. / Bioorg. Med. Chem. 18 (2010) 4812–4820
to the same procedure used in the preparation of 9, to give 28
tions of G418. Serial dilutions of the test compounds in complete
DMEM without G418 were added 24 h after seeding. Cells were al-
lowed to proliferate for three days at 37 °C, after which the cell
number was determined by WST-1 assay.
(192 mg, 51%) as white foam: UV (MeOH) kmax 308.0 nm; ½a D20
ꢂ
ꢀ10.49 (c 1.01, MeOH); 1H NMR (CDCl3) d 8.85 (br s, 1H), 7.87
(dd, 2H, J = 7.2, 7.6 Hz), 7.57 (tt, 1H, J = 6.8, 8.0 Hz), 7.47 (dd, 2H,
J = 7.6, 8.0 Hz), 6.21 (d, 1H, J = 7.2 Hz), 5.50 (d, 1H, J = 6.8 Hz),
4.63 (dd, 1H, J = 3.2, 5.2 Hz), 4.49 (dd, 1H, J = 3.2, 12.4 Hz), 4.39
(dd, 1H, J = 5.2, 12.4 Hz), 4.06–3.99 (m, 2H), 2.17 (s, 3H), 2.12 (s,
3H), 2.08 (s, 3H); 13C NMR (CDCl3) d 171.25, 170.34, 169.99,
166.94, 165.19, 162.69, 154.98, 144.34, 133.39, 133.09, 129.14,
127.80, 97.70, 87.55, 83.36, 81.84, 75.97, 62.43, 60.49, 50.95,
21.64, 21.16, 21.02; Anal. Calcd for C23H24N6O9: C, 52.27; H, 4.58;
N, 15.90. Found: C, 52.55; H, 4.71; N, 16.03.
Acknowledgements
This research was supported by the grant from Seoul R&BD Pro-
gram, Korea (10541) and the WCU project (R31-2008-000-10010-
0) and the NCRC (No. R15-2006-020) of National Research Founda-
tion (NRF), Korea.
References and notes
4.2.24. 1-(3b-C-Azidomethyl-b-D-ribofuranosyl)cytosine (4j)
A solution of 28 (75 mg, 0.142 mmol) in saturated methanolic
ammonia (6 mL) was stirred in a glass bomb at 80 °C overnight
and the volatiles were evaporated. The resulting residue was puri-
fied by C18 reverse phase column chromatography (0–3% acetone
in water) to give 4j (33 mg, 77%) as a white solid: mp 199–
1. Hoofnagle, J. H. Hepatology 1997, 26, 15S.
2. Szabo, E.; Lotz, G.; Paska, C.; Kiss, A.; Schaff, Z. Pathol. Oncol. Res. 2003, 9, 215.
3. Lake-Bakaar, G. Curr. Drug Targets Infect. Disord. 2003, 3, 247.
4. Vile‘ek, J.; Sen, G. C. In Fields Virology; Fields, B. N., Knipe, D. M., Howley, P. M.,
Eds.; Lippincott-Raven Publishers: Philadelphia, 1996; Vol. 1, p 375.
5. Coelmont, L.; Paeshuyse, J.; Windisch, M. P.; De Clercq, E.; Bartenschlager, R.;
Neyts, J. Antimicrob. Agents Chemother. 2006, 50, 3444.
6. Miller, J. P.; Kigwana, L. J.; Streeter, D. G.; Robins, R. K.; Simon, L. N.; Roboz, J.
Ann. N.Y. Acad. Sci. 1977, 284, 211.
7. Crotty, S.; Maag, D.; Arnold, J. J.; Zhong, W.; Lau, J. Y.; Hong, Z.; Andino, R.;
Cameron, C. E. Nat. Med. 2000, 6, 1375.
206 °C (dec); UV (MeOH) kmax 271.0 nm; ½a D20
ꢀ15.15 (c 0.63,
ꢂ
MeOH); 1H NMR (CD3OD) d 8.03 (d, 1H, J = 7.6 Hz), 5.98 (d, 1H,
J = 7.6 Hz), 5.91 (d, 1H, J = 7.2 Hz), 4.20 (d, 1H, J = 7.2 Hz), 4.07 (t,
1H, J = 2.4 Hz), 3.76 (dd, 2H, J = 1.6, 2.4 Hz), 3.61 (d, 1H,
J = 12.8 Hz), 3.53 (d, 1H, J = 12.8 Hz); 13C NMR (CD3OD) d 167.73,
159.16, 144.24, 96.64, 91.30, 83.96, 80.94, 77.23, 62.05, 55.55; IR
(KBr) 2113.37 cmꢀ1 (s); Anal. Calcd for C10H14N6O5: C, 40.27; H,
4.73; N, 28.18. Found: C, 40.11; H, 4.91; N, 28.28.
8. Robins, R. K.; Revankar, G. R.; McKernan, P. A.; Murray, B. K.; Kirsi, J. J.; North, J.
A. Adv. Enzyme Regul. 1985, 24, 29.
9. Behrens, S.-E.; Tomei, L.; De Francesco, R. EMBO J. 1996, 15, 12.
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E.; Andoh, T.; Yoshida, I.; Okayama, H. J. Virol. 1991, 65, 1105.
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14. Gordon, C. P.; Keller, P. A. J. Med. Chem. 2005, 48, 1.
4.3. Antiviral assay
15. Eldrup, A. B.; Allerson, C. R.; Bennett, C. F.; Bera, S.; Bhat, B.; Bhat, N.;
Bosserman, M. R.; Brooks, J.; Burlein, C.; Carroll, S. S.; Cook, P. D.; Getty, K. L.;
MacCoss, M.; McMasters, D. R.; Olsen, D. B.; Prakash, T. P.; Prhavc, M.; Song, Q.;
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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. J. Biol. Chem. 2003, 278, 49164.
4.3.1. Anti-HCV assay
The human hepatoma cell line Huh-7, carrying the subgenomic
HCV genotype 1 replicon with the luc-ubi-neo (reporter/selective)
fusion gene,22 was kindly provided by Dr. Ralf Bartenschlager (Uni-
versity of Heidelberg, Heidelberg, Germany). The cells were grown
as described.22,23 The conditions of the luminescence-based assay
used to test the antiviral activity of the compounds were previ-
ously described.23 Briefly, Huh-5–2 cells were seeded at a density
of 5 ꢁ 103 per well in a tissue culture-treated white 96-well view
17. Yoo, B. N.; Kim, H. O.; Moon, H. R.; Seol, S. K.; Jang, S. K.; Lee, K. M.; Jeong, L. S.
Bioorg. Med. Chem. Lett. 2006, 16, 4190.
18. Pei, X.; Choi, W. J.; Kim, Y. M.; Zhao, L. X.; Jeong, L. S. Arch. Pharm. Res. 2008, 31,
843.
19. Zhang, G.; Shi, L.; Liu, Q.; Wang, J.; Li, L.; Liu, X. Tetrahedron 2007, 63, 9705.
20. Schuda, P. F.; Cichowicz, M. B.; Heimann, M. R. Tetrahedron Lett. 1983, 24, 3829.
21. Lohmann, V.; Korner, F.; Koch, J.; Herian, U.; Theilmann, L.; Bartenschlager, R.
Science 1999, 285, 110.
22. Vroljk, J. M.; Kaul, A.; Hansen, B. E.; Lohmann, V.; Haagmans, B. L.; Schalm, S.
W.; Bartenschlager, R. J. Virol. Methods 2003, 110, 201.
plate in complete DMEM supplemented with 500 lg/mL G418.
After incubation for 24 h at 37 °C (5% CO2), medium was refreshed
(with G418) and DMSO stock of test compounds were added. After
four days of incubation at 37 °C, cell culture medium was removed
and luciferase activity was determined using the Steady-Glo lucif-
erase assay system (Promega, Leiden, The Netherlands).
23. Gozdek, A.; Zhukov, I.; Polkowska, A.; Poznanski, J.; Stankiewicz-Drogon, A.;
Pawlowicz, J. M.; Zagorski-Ostoja, W.; Borowski, P.; Boguszewska-Chachulska,
A. Antimicrob. Agents Chemother. 2008, 52, 393.
4.3.2. Cytostatic effect
Huh-5–2 cells were seeded at a density of 5 ꢁ 103 per well of a
96-well plate in complete DMEM with the appropriate concentra-