5′-O-Masked 2′-deoxyadenosine analogues as lead compounds for hepatitis C virus (HCV) therapeutic agents

Article abstract of DOI:10.1016/j.bmc.2007.08.025

On the basis of our previous study on antiviral agents against the severe acute respiratory syndrome (SARS) coronavirus, a series of nucleoside analogues whose 5′-hydroxyl groups are masked by various protective groups such as carboxylate, sulfonate, and

Full text of DOI:10.1016/j.bmc.2007.08.025

Bioorganic & Medicinal Chemistry 15 (2007) 6882–6892
50-O-Masked 20-deoxyadenosine analogues as lead compounds
for hepatitis C virus (HCV) therapeutic agents
Masahiro Ikejiri,a Takayuki Ohshima,a Keizo Kato,a Masaaki Toyama,a
Takayuki Murata,b Kunitada Shimotohnob and Tokumi Maruyamaa,*
aFaculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, 1314-1 Shido, Sanuki, Kagawa 769-2193, Japan
bDepartment of Viral Oncology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
Received 18 July 2007; revised 9 August 2007; accepted 14 August 2007
Available online 22 August 2007
Abstract—On the basis of our previous study on antiviral agents against the severe acute respiratory syndrome (SARS) coronavirus,
a series of nucleoside analogues whose 50-hydroxyl groups are masked by various protective groups such as carboxylate, sulfonate,
and ether were synthesized and evaluated to develop novel anti-hepatitis C virus (HCV) agents. Among these, several 50-O-masked
analogues of 6-chloropurine-20-deoxyriboside (e.g., 50-O-benzoyl, 50-O-p-methoxybenzoyl, and 50-O-benzyl analogues) were found
to exhibit effective anti-HCV activity. In particular, the 50-O-benzoyl analogue exhibited the highest potency with an EC50 of 6.1 lM
in a cell-based HCV replicon assay. Since the 50-O-unmasked analogue (i.e., 6-chloropurine-20-deoxyriboside) was not sufficiently
potent (EC50 = 47.2 lM), masking of the 50-hydroxyl group seems to be an effective method for the development of anti-HCV
agents. Presently, we hypothesize two roles for the 50-O-masked analogues: One is the role as an anti-HCV agent by itself, and
the other is as a prodrug of its 50-O-demasked (deprotected) derivative.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
To date, a number of nucleoside analogues, including
ribavirin, have been synthesized and tested for anti-
HCV activity.6,7 Among these, some 20-modified ribonu-
cleoside analogues such as 20-C-methyl analogues 1,6a
20-fluoro analogues 2,6b and 20-O-methyl analogue 36c
exhibit potent anti-HCV activities in a cell-based HCV
replicon assay (Fig. 1). Particularly, Valopicitabine, a
prodrug of 1c, is currently in phase II clinical trials.8 Re-
cently, the anti-HCV properties of 40-substituted ribo-
nucleosides such as 4 have also been reported by the
Roche group.6d Concerning the antiviral mechanism of
common nucleoside analogues, it is known that most of
these analogues are converted to their corresponding 50-
triphosphates by cellular and/or viral enzymes and then
compete with natural triphosphates as substrates for
The hepatitis C virus (HCV), a member of the family
Flaviviridae, is an enveloped, positive-sense, single-
strand RNA virus.1 The virus is a major causative agent
of non-A, non-B hepatitis and infects an estimated 170
million people worldwide.2 In most cases, acute phase
infection with HCV is asymptomatic; however, the virus
frequently establishes chronic hepatitis in up to 80% of
the infected individuals and persists for decades with a
substantially high risk of developing liver cirrhosis and
hepatocellular carcinoma.3 Currently, there is no vac-
cine available against HCV, and the approved drugs
are combinations of interferon-a (IFN-a) and ribavirin
(1-b-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide).
Although the use of pegylated IFN-a instead of the
unmodified one has resulted in improved therapeutic
effectiveness, the sustained virological response is still
poor (41–55%); moreover, in some cases, significant side
effects can be caused.4 Therefore, more efficacious ther-
apies are urgently required for ensuring public health.5
NH2
N
NH2
N
HO
Base
O
HO
HO
N3
N
O
N
O
O
O
R
HO OH
HO X
HO OH
1a: Base = A
1b: Base = G
1c: Base = C
2a: R = H, X = F
2b: R = Me, X = F
3: R = H, X = OMe
4
Keywords: Hepatitis C virus; HCV; Antiviral agent; Nucleoside.
*
Corresponding author. Tel.: +81 87 894 5111x6404; e-mail:
maruyama@kph.bunri-u.ac.jp
Figure 1. Examples of anti-HCV nucleoside analogues.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.025

M. Ikejiri et al. / Bioorg. Med. Chem. 15 (2007) 6882–6892
6883
pounds 10, 12, and 15–19 were carried out by the treat-
ment of diol 9 or 11 with the corresponding acyl or
sulfonyl chloride (i.e., benzoyl chloride for 10 and 12;
pivaloyl chloride, butyryl chloride, p-methoxybenzoyl
chloride, 2,4,6-trimethylbenzoyl chloride, and benze-
nesulfonyl chloride for 15–19, respectively), as shown
in Scheme 1. In the case of compound 18, conventional
conditions (trimethylbenzoyl chloride, DMAP, and
pyridine) required a long reaction time and led to the
gradual decomposition of 9 and 18, probably because
the 6-chloropurine moiety reacted with the amine bases
by degrees due to its electrophilic nature. This side
reaction was slightly suppressed by the use of a low-
nucleophilic base, N,N-diisopropylethylamine instead
of pyridine and DMAP.
Cl
N
R
EC50 (SARS-CoV)
N
N
N
5: Benzoyl (Bz)
6: H
14.5 µM
RO
> 300 µM
O
OH
Bz =
Figure 2. Anti-SARS-coronavirus (CoV) activities of carbocyclic
oxetanocin analogues.
incorporation into viral nucleic acids during viral replica-
tion.9 Therefore, the 50-hydroxyl group is indispensable
to the anti-HCV activity of these nucleoside analogues.10
Recently, we have reported that a carbocyclic oxetanocin
analogue 5, one of whose hydroxyl groups (that corre-
sponds to the 50-hydroxyl group of the ribonucleoside)
was masked by a benzoyl group, showed antiviral activity
against the severe acute respiratory syndrome (SARS)
coronavirus (Fig. 2).11 Interestingly, the antiviral activity
of 5(EC50 = 14.5 lM)wasmuchmoreefficaciousthanthat
of the unmasked 6 (almost no activity, EC50 > 300 lM),
which seems to indicate an inconsistency with the necessity
of the 50-hydroxyl group to this compound.
Compound 10 was also prepared via an alternative route
illustrated in Scheme 2A. Regioselective protection of
the 50-hydroxyl group of 20-deoxyadenosine (26) yielded
the benzoate 23 in 65% yield,12c which was subsequently
converted to the silyl ether 27 in 93% yield. After trans-
formation to the 6-chloropurine derivative 28 with a
58% yield,13 the TBS group was cleaved to afford 10 in
72% yield. Compound 28 was also used as an intermedi-
ate in the synthesis of 25, as shown in Scheme 2B. Expo-
sure of 28 to sodium methylthiolate resulted in the
formation of the debenzoylated 6-SMe analogue 29 with
a 67% yield, which was reacylated into 30 in 97% yield.
Unfortunately, the use of thiourea in place of sodium
methylthiolate led to the decomposition of the resultant
products. Finally, the removal of the TBS group affor-
ded 25 in 88% yield. The synthesis of 24 is illustrated
in Scheme 2C. The diacetate 31, which was prepared
as described in a previous report,11 was treated with
dimethylamine to furnish 32 in 62% yield.14 Subse-
quently, regioselective benzoylation of the 50-hydroxyl
group in 32 afforded 24 in 77% yield.
This result attracted our interest and led us to expect
that this unique trend could contribute to developing
anti-HCV agents as well. Thus, several nucleoside ana-
logues, including 5 and 6, were designed as candidate
compounds for the agents (Fig. 3). In this paper, we re-
port the syntheses of these nucleoside analogues and
their biological evaluations as anti-HCV agents.
2. Results and discussion
2.1. Chemistry
Meanwhile, the syntheses of 20 and 21 turned out to be
unexpectedly difficult because of the instability of these
Compounds 7–9, 11, 13, 14, 22, and 23 were prepared
based on the previous reports.12 The syntheses of com-
Cl
N
Cl
N
Cl
Cl
N
N
N
N
N
N
N
N
N
N
N
R2O
R1O
R3O
R4O
N
N
O
O
O
N
HO
HO
9: R2 = H
HO OH
OH
7: R1 = H
11: R3 = H
12: R3 = Bz
13: R4 = H
14: R4 = Bz
8: R1 = Bz
10: R2 = Bz
Cl
X
N
N
N
N
N
R5O
BzO
N
N
N
O
O
15: R5 = t BuC(=O)- (Pivaloyl, Pv)
16: R5 = n PrC(=O)- (Butyryl)
17: R5 =
p-Methoxybenzoyl
18: R5 = 2,4,6-Trimethylbenzoyl
19: R5 = PhSO2- (Benzenesulfonyl)
20: R5 = PhCH2- (Benzyl, Bn)
21: R5 = CH2=CHCH2- (Allyl)
HO
HO
22: X = OH
23: X = NH2
24: X = NMe2
25: X = SMe
Figure 3. The candidate compounds for anti-HCV agents.

6884
M. Ikejiri et al. / Bioorg. Med. Chem. 15 (2007) 6882–6892
Cl
A
N
N
NH2
N
N
N
a
RO
N
N
O
9
BzO
N
O
c
2´-deoxy-
adenosine
(26)
a
HO
R1O
23: R1 = H
27: R1 = TBS
10: R = Bz
15: R = Pv
16: R = Butyryl
17: R = p-Methoxybenzoyl
18: R = 2,4,6-Trimethylbenzoyl
19: R = Benzenesulfonyl
b
Cl
N
N
BzO
N
N
O
d
10
b
11
12
TBSO
28
Scheme 1. Reagents and conditions: (a) for 10, BzCl, pyr, 0 ꢁC, 85%;
for 15, PvCl, DMAP, pyr, 0 ꢁC to rt, 81%; for 16, butyryl chloride, pyr,
0 ꢁC to rt, 68%; for 17, p-methoxybenzoyl chloride, pyr, 0 ꢁC, 72%; for
18, 2,4,6-trimethylbenzoyl chloride, N,N-diisopropylethylamine,
MeCN, rt, 21%; for 19, benzenesulfonyl chloride, pyr, 0 ꢁC to rt,
40%; (b) BzCl, pyr, CH2Cl2, 0 ꢁC, 59%.
B
C
SMe
N
N
R2O
N
N
e
g
O
28
25
TBSO
29: R2 = H
30: R2 = Bz
f
molecules under both strongly basic and acidic condi-
tions; for example, the exposure of 9 to BnBr–NaH or
BnOC(@NH)CCl3–CF3SO3H afforded a complex mix-
ture. Thus, a stepwise protection–deprotection process
was required, which is illustrated in Scheme 3 (3A: syn-
thesis of 20, 3B: synthesis of 21). The monoacetate 33,
which was prepared from 26 by employing a partly mod-
ified Santaniello’s procedure,15 was converted to the silyl
ether 34 in 94% yield. The use of a mixed solvent (pyri-
dine–DMF) effectively reduced the competitive silyla-
tion of the 6-amino group. Protection of the amino
group with pivaloyl chloride followed by the removal
of the acetyl group in 35 afforded 36 with a 98% yield
in two steps. The addition of DMAP and MeOH in
the N-pivaloylation step was effective in converting the
N,N-dipivaloylated by-product to the desired 35.
Chemoselective benzylation of the 50-hydroxyl group
in 36 was successfully carried out by using 2.5 equiv of
potassium tert-butoxide and 1.1 equiv of benzyl bro-
mide in THF to provide 37 in 97% yield. After the re-
moval of the pivaloyl group (76% yield), the resultant
amino group of 38 was substituted with a chloro group
to provide 39, which was subsequently deprotected to
afford 20 with a 34% yield in two steps. Compound 21
was synthesized employing almost the same method as
that for 20 described above (Scheme 3B, 20% overall
yield from 36).
Cl
N
N
N
N
N
N
N
AcO
HO
h
N
i
N
O
O
24
OAc
OH
32
31
Scheme 2. Reagents and conditions: (a) BOPDC, BzOH, pyr, rt, 65%
(Ref. 12c); (b) TBSCl, imidazole, pyr, rt, 93%; (c) BuONO, Et4NCl,
t
CCl4–CH2Cl2, 0–50 ꢁC, 58%; (d) TBAF, AcOH, THF, rt, 72%;
(e) MeSNa, water–DMF, rt, 67%; (f) BzCl, Et3N, DMAP, CH2Cl2,
0 ꢁC, 97%; (g) TBAF, THF, 0 ꢁC, 88%; (h) Me2NH, water–dioxane, rt,
62%; (i) BzCl, pyr, 0 ꢁC to rt, 77%.
tetrazolium (XTT)-based assay according to the manu-
facturer’s protocol.
As expected, the benzoylated carbocyclic oxetanocin
analogue 5 exhibited a stronger anti-HCV activity than
that shown by the unprotected 6 (EC50 = 6.4 lM and
46.1 lM, respectively). This trend seems to be consistent
with that of the anti-SARS-coronavirus activity, as men-
tioned above (Fig. 2; EC50 = 14.5 lM and >300 lM,
respectively). However, compound 5 displayed a cyto-
toxic effect at
a level close to its EC50 value
2.2. Biological evaluation
(CC50 = 20.0 lM; antiviral index = 3.1); therefore, fur-
ther evaluation of this compound was not undertaken.
The above synthesized nucleoside analogues were as-
sayed for their ability to inhibit HCV RNA replication
in a subgenomic replicon Huh7 cell line (LucNeo#2),16
and the result is shown in Table 1. These cells contain
a HCV subgenomic replicon RNA encoding a luciferase
reporter gene as a marker. The potency of the analogues
against the HCV replicon is expressed as EC50, which
was quantified by a luciferase assay after a two-day
incubation period with the corresponding compound.
In addition, the associated cytotoxicity, which is
expressed as CC50 in Table 1, was evaluated in a
2.2.1. Structure–activity relationship (SAR) of the sugar
moiety. To evaluate the effect of the sugar moiety, sev-
eral 6-chloropurine derivatives that possessed the ribo-
furanosyl structure (7 and 8), or deoxyribofuranosyl
structure (20-deoxy: 9 and 10; 30-deoxy: 11 and 12), or
an acyclic backbone (13 and 14) were tested. Among
them, the 20- deoxyribonucleoside derivative 10 exhib-
ited the highest potency against the HCV replicon with
an EC50 of 6.1 lM, which is ninefold potent over that
of ribavirin, and a CC50 of 111 lM. Additionally, the

M. Ikejiri et al. / Bioorg. Med. Chem. 15 (2007) 6882–6892
6885
Table 1. Inhibitory potency (EC50) and cytotoxicity (CC50) of com-
pounds 5–25 and ribavirin (positive control) in HCV replicon assaya
A
NHPv
NH2
N
N
N
Antiviral indexd
b
c
N
N
N
N
Compound
EC50 (lM)
CC50 (lM)
AcO
AcO
R1O
c
a
O
N
5
6.4
46.1
31.0
41.0
47.2
6.1
20.0
>200
>200
97.3
173
3.1
4.3
O
26
6
7
>6.5
2.4
TBSO
35
8
33: R1 = H
34: R1 = TBS
9
3.7
b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Ribavirin
111
18.2
>3.1
1.9
64.1
38.1
80.8
13.3
28.0
30.3
8.4
>200
70.5
>200
108
X1
NHPv
N
N
N
N
N
N
>2.5
8.1
R2O
BnO
d
N
N
f
O
O
177
6.3
>200
76.4
91.0
146
>6.6
9.1
TBSO
TBSO
36: R2 = H
37: R2 = Bn
38: X1 = NH2
39: X1 = Cl
>50
28.8
17.7
24.5
>100
105
<1.8
5.1
g
e
178
140
10.1
5.7
h
20
—
—
>200
>200
—
>1.9
>9.0
—
B
X2
22.3
>100
54.8
40: X2 = NHPv
41: X2 = NH2
42: X2 = Cl
21
N
N
N
i
j
>200
>3.7
36
O
N
O
k
l
a The same experiment was performed at least three times independently.
b Average of 50% effective concentrations.
TBSO
c Average of 50% cytotoxic concentrations.
d Antiviral index was defined as the 50% toxic dose divided by the 50%
effective dose.
Scheme 3. Reagents and conditions: (a) lipase (CAL), vinyl acetate,
MS4A, THF, 60 ꢁC, 89%; (b) TBSCl, DMAP, pyr–DMF, rt, 94%;
(c) PvCl, Et3N, CH2Cl2, rt, and then DMAP, MeOH, rt; (d) K2CO3,
MeOH, 0 ꢁC, 98% from 34; (e) BnBr, tBuOK, THF, 0 ꢁC, 97%:
t
(f) K2CO3, MeOH, rt, 76%, (g) BuONO, Et4NCl, CCl4–CH2Cl2, 0–
50 ꢁC(h) TBAF, AcOH, THF, rt, 34%from 38; (i) allyl bromide, tBuOK,
THF, 0 ꢁC; (j) K2CO3, MeOH, rt, 73% from 36; (k) tBuONO, Et4NCl,
CCl4–CH2Cl2, 0–50 ꢁC (l) TBAF, AcOH, THF, rt, 28% from 41.
able increase in cytotoxicity (CC50 = 76.4 lM). Notably,
other types of protective groups such as ether groups
(compounds 20 and 21) and a sulfonate group (com-
pound 19) also exhibited good anti-HCV activity
(EC50 = 17.7, 24.5, and 28.8 lM, respectively). Overall,
it appears that protective groups containing a phenyl
ring are preferable for the antiviral activity.
50-O-benzoyl analogue 10 was more potent than the
corresponding unprotected analogue 9 (EC50: 6.1 lM
(50-OBz, 10) vs 47.2 lM (50-OH, 9)). This trend was also
observed in the acyclic analogues 13 and 14 (EC50:
13.3 lM (ꢀOBz, 14) vs 80.8 lM (ꢀOH, 13)). In
contrast, in case of the ribofuranosyl structure, the
50-hydroxyl analogue 7 was slightly more potent as
2.2.3. SAR at position 6 of the purine base. The effect of
the substituents at the purine 6-position was investi-
gated by the evaluation of compounds 22–25. Among
these analogues, the 6-dimethylamino derivative 24
exhibited a good potency with an EC50 of 22.3 lM
and low cytotoxicity (CC50 > 200 lM), while the
compared with the 50-O-benzoyl analogue 8 (EC50
=
31.0 and 41.0 lM, respectively). These results indicate
the importance of the masked 50-hydroxyl group in
the 20-deoxyribofuranosyl structure. It is also interesting
to note that the 20-deoxyribonucleoside derivative 10
was more potent than the corresponding ribonucleoside
derivatives such as 7 and 8 in spite of the fact that HCV
is an RNA virus and most of the anti-HCV agents bear
ribofuranosyl structures (or their bioisosteres).6,7
6-amino derivative 23 showed
a weak potency
(EC50 = 105 lM). Unfortunately, the others (i.e., 6-hy-
droxyl (hypoxanthine) analogue 22 and 6-methylthio
analogue 25) did not show any significant potency
(EC50 > 100 lM). Accordingly, the chloro group at the
purine 6-position was considered to be important for
the anti-HCV activity.17
2.2.2. SAR of the 50-O-moiety. As a next step, we evalu-
ated the inhibitory activities of compounds 15–21,
whose 50-hydroxyl groups were masked with various
protective groups (i.e., pivaloyl, butyryl, trimeth-
ylbenzoyl, benzenesulfonyl, benzyl, and allyl groups)
to examine the effect of the substituent group at the
50-position. Among these compounds, the EC50 value
of the close structural analogue 17 (p-methoxybenzoyl
analogue) was comparable to that of 10 (EC50 = 8.4
and 6.1 lM, respectively); however, 17 led to an undesir-
The luciferase assay described above revealed that com-
pound 10 exhibited the maximum potency among the
analogues. Next, in order to confirm the anti-HCV
activity of 10, the replicon RNA levels were quantified
by performing real-time RT-PCR analysis.16 Fig. 4
shows the result obtained with 10 and ribavirin (positive
control). Compound 10 reduced the replicon RNA
amount up to approximately 45% at 12.5 lM and 6%
at 25 lM, which is almost consistent with the result of
the luciferase assay (EC50 = 6.1 lM).

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M. Ikejiri et al. / Bioorg. Med. Chem. 15 (2007) 6882–6892
4. Experimental
4.1. Chemistry
4.1.1. General methods. The chemicals used were of com-
mercial origin (Wako chemicals, TCI, Kanto Kagaku,
nacalai tesque, and Aldrich) and were employed without
further purification. The progress of all reactions was
monitored by TLC (silica gel 60F254, Merck). The col-
umn chromatography was performed with a Combi-
Flash Companion system (Teledyne Isco, Inc.). The
melting points were measured on a Yanaco melting
point apparatus and are uncorrected. The 1H NMR
and 13C NMR spectra were recorded on a Bruker
Figure 4. The quantity of HCV subgenomic replicon RNA. The RNA
amounts were determined by real-time RT-PCR analysis after a two-
day incubation period with compound 10 or ribavirin.
1
AV400M spectrometer. The solvent H and 13C signals
Among these molecules, two compounds can be
hypothesized to be species with real antiviral activity.
One is the 50-O-masked 20-deoxyadenosine analogue it-
self such as 10, and the other is the deprotected 9 (or
its activated form, 50-triphosphate) since carboxylic
ester bonds are often hydrolyzed in cultured cells, as
reported earlier.18 However, the chemically stable 50-
O-masked analogues 20 and 21 (Bn and allyl ether,
respectively) showed anti-HCV potency to some extent.
Moreover, the deoxyadenosine derivative 23, which
would be transformed to the inactive 20-deoxyadeno-
sine after the hydrolysis, also showed potency. Thus,
taking these results into consideration, it would be rea-
sonable to consider that some 50-O-masked analogues
themselves possess the anti-HCV potency. On the other
hand, it would also be reasonable to consider that
compound 9 (or its 50-triphosphate) is one of the real
active species since 9 also exhibited anti-HCV activity,
though only moderately; in other words, compound 10
operates as a prodrug of 9.19 Therefore, at this stage,
we believe that both compounds (e.g., 9 and 10) would
function as the species with antiviral activity in the
cells.
were used as internal standards (DMSO-d6: 2.50 and
39.5 ppm; acetone-d6: 2.05 and 29.8 ppm; CD3OD:
3.31 and 49.0 ppm; CDCl3: 7.26 and 77.0 ppm, respec-
tively). The mass spectra were recorded on a Bruker
Apex-Qe FT-ICR MS spectrometer.
4.1.2. Synthesis of 9-(5-O-benzoyl-b-D-2-deoxyribofur-
anosyl)-6-chloropurine (10). To a stirred solution of 9
(50 mg, 0.18 mmol) in pyridine (0.5 mL) was added ben-
zoyl chloride (32 lL, 0.28 mmol) at ice-water tempera-
ture, and the mixture was stirred at the same
temperature for 1.5 h. Subsequent to the addition of sat-
urated NaHCO3 solution, the mixture was extracted
with ethyl acetate. The combined organic layer was
washed with water and brine, dried over Na2SO4, and
concentrated under reduced pressure. The resultant res-
idue was purified by silica gel column chromatography
(ethyl acetate/hexane, 2:1 to 3:1) to give 10 (59 mg,
1
85%) as a white solid. Mp 111.0–111.5 ꢁC. H NMR
(DMSO-d6) d: 2.47 (1H, m), 2.99 (1H, dt, J = 13.6,
6.4 Hz), 4.19 (1H, q-like, J = 4.4 Hz), 4.44 (1H, dd,
J = 12.0, 6.0 Hz), 4.56 (1H, dd, J = 12.0, 4.0 Hz), 4.68
(1H, quintet-like, J = 4.8 Hz), 5.61 (1H, br d,
J = 2.8 Hz), 6.50 (1H, t, J = 6.4 Hz), 7.48 (2H, br t,
J = 7.6 Hz), 7.64 (1H, br t, J = 7.6 Hz), 7.85 (2H, br d,
J = 7.6 Hz), 8.74 (1H, s), 8.86 (1H, s); 13C NMR
(DMSO-d6) d: 38.4, 64.1, 70.2, 84.2, 84.3, 128.7, 129.1,
129.3, 131.5, 133.4, 146.2, 149.3, 151.2, 151.6, 165.4;
HRMS (ESI) calcd for C17H15ClN4NaO4 (M+Na+)
397.0680, found 397.0664.
3. Conclusion
A series of nucleoside analogues 5–25 were synthesized
and their abilities to inhibit HCV RNA replication were
evaluated. Among these, several 50-O-masked analogues
of 6-chloropurine-20-deoxyriboside, such as 10, 17, and
20, exhibited effective anti-HCV activity. In particular,
50-O-benzoyl analogue 10 exhibited the highest activity.
Presently, we hypothesize two roles for these 50-O-
masked analogues: One is the role as an anti-HCV agent
by itself, and the other is as a prodrug of its 50-O-de-
masked (deprotected) derivative.
4.1.3. 6-Chloro-9-(5-O-pivaloyl-b-D-2-deoxyribofurano-
syl)purine (15). To a stirred solution of 9 (63 mg,
0.23 mmol) and DMAP (3 mg, 10 mol%) in pyridine
(0.5 mL) was added pivaloyl chloride (43 lL,
0.35 mmol) at ice-water temperature, and the mixture
was stirred at the same temperature for 30 min and then
at room temperature for 15 min. The work-up process
was performed in the same manner as described for
10. The crude material was purified by silica gel column
chromatography (ethyl acetate/hexane, 1:1 to 2:1) to
give 15 (67 mg, 81%) as a white solid. Mp 111.5–
112.0 ꢁC. 1H NMR (DMSO-d6) d: 1.06 (9H, s), 2.45
(1H, m), 2.91 (1H, dt, J = 13.6, 6.0 Hz), 4.04 (1H, m),
4.16 (1H, dd, J = 12.0, 6.0 Hz), 4.26 (1H, dd, J = 12.0,
4.4 Hz), 4.52 (1H, m), 5.54 (1H, br d, J = 2.8 Hz), 6.47
(1H, t, J = 6.4 Hz), 8.80 (1H, s), 8.84 (1H, s); 13C
NMR (DMSO-d6) d: 26.7, 38.1, 38.4, 63.6, 70.1, 84.1,
There are two notable structural features of these potent
compounds: one is the masked 50-hydroxyl group, and
the other is the 20-deoxyribofuranosyl structure. In rela-
tion to the substituent group at position 6 of the purine
base, the chloro group seems to be preferable. These un-
ique features are rarely seen in common anti-HCV
agents; therefore, although some issues such as improve-
ment of the antiviral potency remain to be resolved, we
hope that the present study will contribute to developing
a new class of HCV therapeutic agents.

M. Ikejiri et al. / Bioorg. Med. Chem. 15 (2007) 6882–6892
6887
84.2, 131.5, 146.0, 149.3, 151.2, 151.6, 177.2; HRMS
(ESI) calcd for C15H19ClN4NaO4 (M+Na+) 377.0993,
found 377.0978.
65.1, 72.4, 85.9, 86.0, 129.0, 131.8, 133.0, 135.6, 140.0,
146.1, 150.9, 152.3, 152.4, 169.9; HRMS (ESI) calcd
for C20H21ClN4NaO4 (M+Na+) 439.1149, found
439.1127.
4.1.4. 9-(5-O-Butyryl-b-D-2-deoxyribofuranosyl)-6- chloro-
purine (16). To a stirred solution of 9 (100 mg,
0.37 mmol) in pyridine (1.0 mL) was added butyryl chlo-
ride (61 lL, 0.59 mmol) at ice-water temperature, and
the mixture was stirred at the same temperature for
4.5 h. The work-up process was performed in the same
manner as described for 10. The crude material was
purified by silica gel column chromatography (ethyl
acetate/hexane, 1:1 to 3:2) to give 16 (97 mg, 68%) as a
colorless oil. 1H NMR (acetone-d6) d: 0.88 (3H, t,
J = 7.6 Hz), 1.56 (2H, sextet, J = 7.6 Hz), 2.26 (2H, t,
J = 7.6 Hz), 2.61 (1H, m), 3.03 (1H, dt, J = 13.6,
6.4 Hz), 4.21 (1H, q-like, J = 4.4 Hz), 4.31 (1H, dd,
J = 12.4, 5.6 Hz), 4.34 (1H, dd, J = 12.4, 4.8 Hz), 4.70–
4.75 (2H, m), 6.59 (1H, t, J = 6.4 Hz), 8.66 (1H, s),
8.73 (1H, s); 13C NMR (acetone-d6) d: 13.7, 18.9, 36.2,
40.3, 64.3, 72.0, 85.8, 85.9, 133.0, 145.9, 150.9, 152.4,
173.4; HRMS (ESI) calcd for C14H17ClN4NaO4
(M+Na+) 363.0836, found 363.0821.
4.1.7. 9-(5-O-Benzenesulfonyl-b-D-2-deoxyribofuranosyl)-
6-chloropurine (19). To a stirred solution of 9 (100 mg,
0.37 mmol) in pyridine (1.0 mL) was added benze-
nesulfonyl chloride (94 lL · 4 times at intervals of
30 min, 0.74 · 4 mmol) at room temperature, and the
mixture was stirred at the same temperature for 3 h in
total. The work-up process was performed in the same
manner as described for 10. The crude material was
purified by silica gel column chromatography (metha-
nol/chloroform, 1:20) to give 19 (60 mg, 40%) as a white
semi-solid. 1H NMR (acetone-d6) d: 2.57 (1H, ddd,
J = 14.0, 6.8, 4.4 Hz), 3.00 (1H, dt, J = 14.0, 6.4 Hz),
4.21 (1H, m), 4.33 (1H, dd, J = 10.8, 6.0 Hz), 4.40
(1H, dd, J = 10.8, 4.0 Hz), 4.73 (1H, m), 4.81 (1H, br
d, J = 4.0 Hz), 6.56 (1H, t, J = 6.8 Hz), 7.56 (2H, t,
J = 8.0 Hz), 7.68 (1H, t, J = 8.0 Hz), 7.85 (2H, d,
J = 8.0 Hz), 8.57 (1H, s), 8.66 (1H, s); 13C NMR (ace-
tone-d6) d: 39.8, 70.8, 72.0, 85.5, 86.0, 128.5, 130.2,
133.1, 134.8, 136.7, 146.1, 150.9, 152.3, 152.3; HRMS
(ESI) calcd for C16H15ClN4NaO5S (M+Na+) 433.0349,
found 433.0330.
4.1.5. 6-Chloro-9-(5-O-p-methoxybenzoyl-b-D-2-deoxyri-
bofuranosyl)purine (17). To a stirred solution of 9
(100 mg, 0.37 mmol) in pyridine (1.0 mL) was added
p-methoxybenzoyl chloride (61 lL, 0.44 mmol) at ice-
water temperature, and the mixture was stirred at the
same temperature for 1 h. The work-up process was per-
formed in the same manner as described for 10. The
crude material was purified by silica gel column chroma-
tography (ethyl acetate/hexane, 3:1 to 1:0) to give 17
4.1.8. 9-(5-O-Benzoyl-b-D-3-deoxyribofuranosyl)-6-chlo-
ropurine (12). To a stirred solution of 11 (600 mg,
2.22 mmol) in CH2Cl2 (15 mL) were added pyridine
(3.5 mL) and benzoyl chloride (268 lL, 2.31 mmol) at
ice-water temperature, and the mixture was stirred at
the same temperature for 2 h. Subsequent to the addi-
tion of saturated NaHCO3 solution, the mixture was ex-
tracted with ethyl acetate. The combined organic layer
was washed with water and brine, dried over Na2SO4,
and concentrated under reduced pressure. The resultant
residue was purified by silica gel column chromatogra-
phy (ethyl acetate/hexane, 1:1 to 2:1) to give 12
1
(108 mg, 72%) as a white solid. Mp 148.5–149.0 ꢁC. H
NMR (DMSO-d6) d: 2.47 (1H, m), 2.98 (1H, dt,
J = 13.2, 6.4 Hz), 3.83 (3H, s), 4.17 (1H, dt, J = 6.0,
4.0 Hz), 4.38 (1H, dd, J = 12.0, 6.0 Hz), 4.52 (1H, dd,
J = 12.0, 4.4 Hz), 4.67 (1H, quintet, J = 5.2 Hz), 5.59
(1H, br d, J = 4.4 Hz), 6.49 (1H, t, J = 6.4 Hz), 6.98
(2H, br d, J = 8.8 Hz), 7.79 (2H, br d, J = 8.8 Hz),
8.76 (1H, s), 8.84 (1H, s); 13C NMR (DMSO-d6) d:
38.4, 55.5, 63.7, 70.2, 84.3, 84.4, 113.9, 121.5, 131.2,
131.5, 146.1, 149.3, 151.2, 151.6, 163.2, 165.1; HRMS
(ESI) calcd for C18H17ClN4NaO5 (M+Na+) 427.0785,
found 427.0767.
1
(488 mg, 59%) as a white solid. Mp 160.5–161.0 ꢁC. H
NMR (DMSO-d6) d: 2.12 (1H, ddd, J = 13.2, 6.0,
2.0 Hz), 2.47 (1H, m), 4.50 (1H, dd, J = 12.0, 5.6 Hz),
4.57 (1H, dd, J = 12.0, 2.8 Hz), 4.73 (1H, m), 4.87
(1H, m), 5.85 (1H, br s), 6.06 (1H, d, J = 2.0 Hz),
7.47–7.51 (2H, m), 7.66 (1H, m), 7.85–7.87 (2H, m),
8.75 (1H, s), 8.80 (1H, s); 13C NMR (DMSO-d6) d:
34.6, 65.4, 74.1, 78.3, 91.7, 128.7, 129.1, 129.3, 131.4,
133.4, 145.6, 149.2, 151.1, 151.6, 165.5; HRMS (ESI)
calcd for C17H16ClN4O4 (M+H+) 375.0860, found
375.0867.
4.1.6.
6-Chloro-9-(5-O-(2,4,6-trimethylbenzoyl)-b-D-2-
deoxyribofuranosyl)purine (18). To a stirred solution of
9 (100 mg, 0.37 mmol) and N,N-diisopropylethylamine
(478 lL, 2.96 mmol) in acetonitrile (4.0 mL) was added
2,4,6-trimethylbenzoyl chloride (303 lL, 1.85 mmol) at
ice-water temperature, and the mixture was stirred at
room temperature for 3 h. The work-up process was per-
formed in the same manner as described for 10. The
crude material was purified by preparative thin layer
chomatography (Merck, 113895) (ethyl acetate/hexane,
4.1.9. 50-O-Benzoyl-30-O-(tert-butyldimethylsilyl)-20-deoxy-
adenosine (27). To a stirred solution of 23 (915 mg,
2.45 mmol) in pyridine (12 mL) were added imidazole
(1.00 g, 14.7 mmol) and tert-butyldimethylsilyl chloride
(1.11 g, 7.36 mmol) at room temperature, and the mix-
ture was stirred overnight at the same temperature. Sub-
sequent to the addition of water, the mixture was stirred
for 15 min. After concentration under reduced pressure,
the residue was dissolved in ethyl acetate. The ethyl ace-
tate solution was washed with water, 1 M aqueous HCl,
saturated NaHCO3 solution and brine, dried over
Na2SO4, and concentrated under reduced pressure.
1
2:1) to give 18 (33 mg, 21%) as a white semi-solid. H
NMR (acetone-d6) d: 2.14 (6H, s), 2.25 (3H, s), 2.62
(1H, ddd, J = 13.6, 6.8, 4.0 Hz), 3.11 (1H, dt, J = 13.6,
6.0 Hz), 4.34 (1H, m), 4.56 (1H, dd, J = 12.0, 4.4 Hz),
4.59 (1H, dd, J = 12.0, 6.0 Hz), 4.79–4.86 (2H, m),
6.61 (1H, t, J = 6.4 Hz), 6.84 (2H, br s), 8.60 (1H, s),
8.66 (1H, s); 13C NMR (acetone-d6) d: 19.7, 21.1, 40.0,

6888
M. Ikejiri et al. / Bioorg. Med. Chem. 15 (2007) 6882–6892
The resultant residue was purified by silica gel column
chromatography (ethyl acetate/hexane, 4:1 to 1:0) to
give 27 (1.07 g, 93%) as a white solid. Mp 129.5–
130.0 ꢁC. H NMR (CDCl3) d: 0.14 (6H, s), 0.93 (9H,
1:3 to 1:2) to give 29 (122 mg, 67%) as a white solid.
Mp 96.0–96.5 ꢁC. 1H NMR (DMSO-d6) d: 0.12 (6H,
s), 0.90 (9H, s), 2.33 (1H, ddd, J = 13.2, 6.4, 3.2 Hz),
2.67 (3H, s), 2.87 (1H, ddd, J = 13.2, 7.6, 6.0 Hz), 3.51
(1H, m), 3.61 (1H, dt, J = 11.6, 5.2 Hz), 3.88 (1H, m),
4.62 (1H, m), 5.04 (1H, t, J = 5.6 Hz), 6.42 (1H, t,
J = 6.8 Hz), 8.67 (1H, s), 8.74 (1H, s); 13C NMR (ace-
tone-d6) d: ꢀ4.7, ꢀ4.6, 11.6, 18.5, 26.1, 41.5, 63.0,
74.0, 86.5, 90.0, 133.2, 143.7, 148.6, 152.0, 162.3; HRMS
(ESI) calcd for C17H28N4NaO3SSi (M+Na+) 419.1549,
found 419.1558.
1
s), 2.49 (1H, ddd, J = 13.2, 6.4, 4.8 Hz), 2.97 (1H, dt,
J = 13.2, 6.0 Hz), 4.28 (1H, q-like, J = 4.0 Hz), 4.50
(1H, dd, J = 12.4, 4.8 Hz), 4.62 (1H, dd, J = 12.4,
4.4 Hz), 4.80 (1H, m), 5.75 (2H, br s), 6.39 (1H, t,
J = 6.4 Hz), 7.42 (2H, t, J = 8.0 Hz), 7.56 (1H, t,
J = 8.0 Hz), 7.94 (1H, s), 7.97 (2H, d, J = 8.0 Hz), 8.32
(1H, s); 13C NMR (CD3OD) d: ꢀ4.7, ꢀ4.5, 18.8, 26.3,
40.5, 64.5, 73.4, 86.0, 86.2, 120.7, 129.6, 130.5, 130.9,
134.4, 141.5, 150.2, 153.8, 157.3, 167.6; HRMS (ESI)
calcd for C23H31N5NaO4Si (M+Na+) 492.2043, found
429.2021.
4.1.13. 50-O-Benzoyl-30-O-(tert-butyldimethylsilyl)-20-
deoxy-6-S-methyl-6-thioinosine (30). To a stirred solu-
tion of 29 (122 mg, 0.31 mmol) in CH2Cl2 (4 mL) were
added triethylamine (255 lL, 1.86 mmol), DMAP
(4 mg, 10 mol%), and benzoyl chloride (71 lL, 0.62
mmol) at ice-water temperature, and the mixture was
stirred at the same temperature for 2 h. Subsequent to
the addition of saturated NaHCO3 solution, the mixture
was extracted with ethyl acetate. The combined organic
layer was washed with water and brine, dried over
Na2SO4, and concentrated under reduced pressure.
The resultant residue was purified by silica gel column
chromatography (ethyl acetate/hexane, 1:4) to give 30
4.1.10. 9-[5-O-Benzoyl-3-O-(tert-butyldimethylsilyl)-b-D-
2-deoxyribofuranosyl]-6-chloropurine (28). To a stirred
solution of 27 (245 mg, 0.52 mmol) in CCl4 (10 mL)
were added a solution of tetraethylammonium chloride
(346 mg, 2.08 mmol) in CH2Cl2 (2.5 mL) and then
tert-butyl nitrite (313 lL, 2.60 mmol) at ice-water tem-
perature. The mixture was stirred for 1 h at the same
temperature, warmed to room temperature for 0.5 h,
and stirred at 50 ꢁC for 2 h. The solvent was removed
under reduced pressure, and the resultant residue was
purified by silica gel column chromatography (ethyl ace-
tate/hexane, 1:3 to 1:2) to give 28 (147 mg, 58%) as a
1
(150 mg, 97%) as a colorless oil. H NMR (DMSO-d6)
d: 0.12 (3H, s), 0.12 (3H, s), 0.90 (9H, s), 2.40–2.55
(1H, overlapping with DMSO-d6), 2.66 (3H, s), 3.10
(1H, dt, J = 13.2, 6.4 Hz), 4.17 (1H, q-like, J = 4.4 Hz),
4.41 (1H, dd, J = 12.0, 5.2 Hz), 4.55 (1H, dd, J = 12.0,
4.8 Hz), 4.91 (1H, m), 6.46 (1H, t, J = 6.4 Hz), 7.46–
7.50 (2H, m), 7.65 (1H, m), 7.84–7.87 (2H, m), 8.64
(1H, s), 8.68 (1H, s); 13C NMR (acetone-d6) d: ꢀ4.7,
ꢀ4.6, 11.5, 18.5, 26.1, 40.1, 64.5, 73.2, 85.5, 85.7,
129.3, 130.2, 130.8, 133.0, 134.0, 143.7, 148.8, 152.3,
161.9, 166.5; HRMS (ESI) calcd for C24H32N4NaO4SSi
(M+Na+) 533.1811, found 533.1818.
1
pale yellow oil. H NMR (DMSO-d6) d: 0.12 (3H, s),
0.13 (3H, s), 0.90 (9H, s), 2.47 (1H, m), 3.09 (1H, dt,
J = 13.2, 6.0 Hz), 4.19 (1H, q-like, J = 4.8 Hz), 4.42
(1H, dd, J = 12.0, 5.2 Hz), 4.57 (1H, dd, J = 12.0,
4.8 Hz), 4.92 (1H, q, J = 5.6 Hz), 6.50 (1H, t,
J = 6.4 Hz), 7.46–7.50 (2H, m), 7.64 (1H, m), 7.82–7.85
(2H, m), 8.74 (1H, s), 8.87 (1H, s); 13C NMR (ace-
tone-d6) d: ꢀ4.7, ꢀ4.6, 18.5, 26.1, 40.1, 64.4, 73.1,
85.9, 85.9, 129.4, 130.1, 130.7, 133.2, 134.0, 146.5,
151.0, 152.3, 152.3, 166.4; HRMS (ESI) calcd for
C23H29ClN4NaO4Si
511.1517.
(M+Na+)
511.1544,
found
4.1.14. 50-O-Benzoyl-20-deoxy-6-S-methyl-6- thioinosine
(25). To a stirred solution of 30 (150 mg, 0.30 mmol) in
THF (3 mL) was added 1 M THF solution of TBAF
(360 lL, 0.36 mmol) at ice-water temperature, and the
mixture was stirred at the same temperature for 1.5 h.
The solvent was removed under reduced pressure, and
the resultant residue was purified by silica gel column
chromatography (ethyl acetate/hexane, 1:1 to 2:1) to
give 25 (102 mg, 88%) as a white solid. Mp 148.0–
4.1.11. An alternative synthesis of compound 10. To a
stirred solution of 28 (330 mg, 0.67 mmol) in THF
(5 mL) was added 1 M THF solution of TBAF–AcOH
(1:1, 810 lL, 0.81 mmol) at ice-water temperature, and
the mixture was stirred at the same temperature for
5 h and then at room temperature for 1 h. The solvent
was removed under reduced pressure, and the resultant
residue was purified by silica gel column chromatogra-
phy (ethyl acetate/hexane, 2:1 to 3:1) to give 10
(181 mg, 72%) as a white solid.
1
148.5 ꢁC. H NMR (DMSO-d6) d: 2.46 (1H, m), 2.66
(3H, s), 2.99 (1H, dt, J = 13.6, 6.4 Hz), 4.17 (1H, m),
4.43 (1H, dd, J = 12.0, 6.0 Hz), 4.55 (1H, dd, J = 12.0,
4.4 Hz), 4.68 (1H, m), 5.58 (1H, br d, J = 4.4 Hz), 6.47
(1H, t, J = 6.4 Hz), 7.49 (2H, t, J = 7.6 Hz), 7.65 (1H,
t, J = 7.6 Hz), 7.87 (2H, d, J = 7.6 Hz), 8.62 (1H, s),
8.69 (1H, s); 13C NMR (DMSO-d6) d: 11.5, 40.0, 65.1,
72.2, 85.5, 85.7, 129.4, 130.2, 130.9, 132.9, 134.0,
143.3, 148.9, 152.4, 161.9, 166.5; HRMS (ESI) calcd
for C18H18N4NaO4S (M+Na+) 409.0946, found
409.0929.
4.1.12. 30-O-(tert-Butyldimethylsilyl)-20-deoxy-6-S-methyl-
6-thioinosine (29). To a stirred solution of 28 (225 mg,
0.46 mmol) in DMF (3.5 mL) was added a solution of
methyl mercaptan sodium salt (15% in water, 860 lL,
1.84 mmol) at ice-water temperature, and the mixture
was stirred at room temperature for 3 h. After dilution
of the mixture with ethyl acetate, the organic layer was
washed with water, saturated NaHCO3 solution and
brine, dried over Na2SO4, and concentrated under re-
duced pressure. The resultant residue was purified by sil-
ica gel column chromatography (ethyl acetate/hexane,
4.1.15. N6,N6-Dimethyl-20-deoxyadenosine (32). To a
stirred solution of 31 (200 mg, 0.56 mmol) in dioxane
(1 mL) was added 50% dimethylamine–water solution
(8 mL) at room temperature, and the mixture was stirred

M. Ikejiri et al. / Bioorg. Med. Chem. 15 (2007) 6882–6892
6889
overnight at the same temperature. The solvent was re-
moved under reduced pressure, and the resultant residue
was washed with ether several times, and purified by sil-
ica gel column chromatography (methanol/chloroform,
J = 6.8 Hz), 7.29 (2H, br s), 8.14 (1H, s), 8.32 (1H, s);
13C NMR (DMSO-d6) d: ꢀ5.1, ꢀ4.9, 17.6, 20.5, 25.6,
38.4, 63.4, 72.4, 83.3, 84.1, 119.2, 139.7, 149.0, 152.5,
156.1, 170.0; HRMS (ESI) calcd for C18H30N5O4Si
(M+H+) 408.2067, found 408.2082.
1
1:10) to give 32 (98 mg, 62%) as a white solid. The H
and 13C NMR spectra and the mass spectrum were iden-
tical to the reported values.14
4.1.19. 30-O-(tert-Butyldimethylsilyl)-20-deoxy-N-(2,2-di-
methyl-1-oxopropyl)adenosine (36). To a stirred solution
of 34 (300 mg, 0.74 mmol) and triethylamine (408 lL,
2.94 mmol) in CH2Cl2 (6 mL) was added pivaloyl chlo-
ride (207 lL, 1.70 mmol) at ice-water temperature, and
the mixture was stirred at room temperature for 1 h.
Subsequent to the addition of DMAP (9 mg, 10 mol%)
and methanol (2 mL), the mixture was stirred overnight
at room temperature. After dilution of the mixture with
ethyl acetate, the organic layer was washed with water,
5% KHSO4 solution, saturated NaHCO3 solution and
brine, dried over Na2SO4, and concentrated under re-
duced pressure to leave the crude compound 35
(385 mg), which was employed in the next reaction with-
out purification.
4.1.16. 50-O-Benzoyl-N6,N6-dimethyl-20-deoxyadenosine
(24). To a stirred solution of 32 (290 mg, 1.04 mmol)
in pyridine (5 mL) was added benzoyl chloride
(181 lL, 1.56 mmol) at ice-water temperature, and the
mixture was stirred at room temperature for 4 h. Subse-
quent to the addition of saturated NaHCO3 solution,
the mixture was extracted with ethyl acetate. The com-
bined organic layer was washed with water and brine,
dried over Na2SO4, and concentrated under reduced
pressure. The resultant residue was purified by silica
gel column chromatography (ethyl acetate/hexane, 3:2
to 4:1) to give 24 (306 mg, 77%) as a white semi-solid.
1H NMR (acetone-d6) d: 2.53 (1H, ddd, J = 13.6, 6.8,
4.0 Hz), 3.05 (1H, dt, J = 13.6, 6.8 Hz), 3.49 (6H, br s),
4.30 (1H, q-like, J = 4.4 Hz), 4.54 (1H, dd, J = 12.0,
5.6 Hz), 4.62 (1H, dd, J = 12.0, 4.4 Hz), 4.70 (1H, br
d, J = 4.0 Hz), 4.85 (1H, m), 6.50 (1H, t, J = 6.8 Hz),
7.49 (2H, t, J = 8.0 Hz), 7.64 (1H, t, J = 8.0 Hz), 7.99
(2H, d, J = 8.0 Hz), 8.14 (1H, s), 8.20 (1H, s); 13C
NMR (acetone-d6) d: 38.4, 40.1, 65.3, 72.3, 72.4, 85.1,
85.5, 121.3, 129.4, 130.2, 130.9, 134.0, 138.4, 151.2,
152.9, 155.7, 166.6; HRMS (ESI) calcd for
C19H21N5NaO4 (M+Na+) 406.1491, found 406.1475.
To a stirred solution of 35 (385 mg) in methanol (5 mL)
was added potassium carbonate (203 mg, 1.48 mmol) at
ice-water temperature, and the mixture was stirred at the
same temperature for 0.5 h. Subsequent to the addition
of acetic acid (150 lL) and water (3 mL), the organic
solvent was removed under reduced pressure. The resul-
tant mixture was extracted with ethyl acetate, and the
organic layer was washed with saturated NaHCO3 solu-
tion and brine, dried over Na2SO4, and concentrated un-
der reduced pressure. The resultant residue was purified
by silica gel column chromatography (ethyl acetate/hex-
ane, 3:1 to 4:1) to give 36 (324 mg, 98% from 34) as a
white solid. Compound 35: 1H NMR (DMSO-d6) d:
0.13 (6H, s), 0.91 (9H, s), 1.28 (9H, s), 1.98 (3H, s),
2.40 (1H, ddd, J = 13.2, 6.4, 4.0 Hz), 3.01 (1H, dt,
J = 13.2, 6.4 Hz), 4.03 (1H, m), 4.14 (1H, dd, J = 12.0,
6.0 Hz), 4.25 (1H, dd, J = 12.0, 4.8 Hz), 4.73 (1H, m),
6.46 (1H, t, J = 6.8 Hz), 8.61 (1H, s), 8.69 (1H, s),
10.14 (1H, br s). Compound 36: Mp 86–87 ꢁC. 1H
NMR (DMSO-d6) d: 0.12 (6H, s), 0.91 (9H, s), 1.28
(9H, s), 2.34 (1H, ddd, J = 13.6, 6.4, 3.6 Hz), 2.89 (1H,
ddd, J = 13.6, 7.2, 6.0 Hz), 3.52 (1H, dt, J = 12.0,
5.2 Hz), 3.61 (1H, dt, J = 12.0, 5.2 Hz), 3.89 (1H, m),
4.63 (1H, m), 5.05 (1H, br t, J = 5.2 Hz), 6.45 (1H, dd,
J = 7.2, 6.4 Hz), 8.64 (1H, s), 8.69 (1H, s), 10.14 (1H,
br s); 13C NMR (acetone-d6) d: ꢀ4.7, ꢀ4.6, 18.5, 26.1,
27.5, 40.7, 41.4, 63.0, 74.0, 86.5, 90.0, 126.1, 143.7,
151.3, 152.2, 176.0; HRMS (ESI) calcd for
C21H35N5NaO4Si (M+Na+) 472.2356, found 472.2363.
4.1.17. 50-O-Acetyl-20-deoxyadenosine (33). 20-Deoxya-
denosine (26) (540 mg, 2.01 mmol), vinyl acetate
(417 lL, 4.5 mmol), molecular sieves 4 A (500 mg), and
lipase acrylic resin from Candida antarctica (300 mg)
purchased from SIGMA were suspended in THF
(20 mL), and the mixture was stirred at 60 ꢁC for 1.5 h.
The enzyme was filtered off and washed with MeOH,
and the solvents were removed under reduced pressure.
The resultant residue was purified by silica gel column
chromatography (methanol/chloroform, 1:20 to 1:10)
to give 33 (523 mg, 89%) as a white solid. The 1H
NMR spectrum was identical to the reported values.15
4.1.18. 50-O-Acetyl-30-O-(tert-butyldimethylsilyl)-20-deoxy-
adenosine (34). To a stirred solution of 33 (670 mg,
2.29 mmol) in pyridine-DMF (1:1, 12 mL) were added
tert-butyldimethylsilyl chloride (860 mg, 5.71 mmol)
and DMAP (140 mg, 1.15 mmol) at room temperature,
and the mixture was stirred overnight at the same tem-
perature. Subsequent to the addition of water, the mix-
ture was stirred for 20 min and then extracted with ethyl
acetate. The combined organic layer was washed with
water and brine, dried over Na2SO4, and concentrated
under reduced pressure. The resultant residue was puri-
fied by silica gel column chromatography (ethyl acetate)
to give 34 (940 mg, 94%) as a white solid. Mp 145–
146 ꢁC. 1H NMR (DMSO-d6) d: 0.12 (6H, s), 0.90
(9H, s), 1.98 (3H, s), 2.32 (1H, ddd, J = 13.6, 6.8,
4.0 Hz), 2.97 (1H, dt, J = 13.6, 6.4 Hz), 3.99 (1H, q-like,
J = 3.6 Hz), 4.12 (1H, dd, J = 12.0, 6.0 Hz), 4.24 (1H,
dd, J = 12.0, 5.2 Hz), 4.69 (1H, m), 6.34 (1H, t,
4.1.20. 50-O-Benzyl-30-O-(tert-butyldimethylsilyl)-20-deoxy-
N-(2,2-dimethyl-1- oxopropyl)adenosine (37). To a stirred
solution of 36 (340 mg, 0.76 mmol) in THF (7.5 mL)
was added potassium tert-butoxide (210 mg, 1.89 mmol)
at ice-water temperature, and the mixture was stirred at
the same temperature for 1 min. Subsequent to the addi-
tion of benzyl bromide (99 lL, 0.83 mmol) at ice-water
temperature, the mixture was stirred at the same temper-
ature for further 1 h. After dilution of the mixture with
water, the aqueous layer was extracted with ethyl
acetate. The organic layer was washed with water and

6890
M. Ikejiri et al. / Bioorg. Med. Chem. 15 (2007) 6882–6892
brine, dried over Na2SO4, and concentrated under re-
duced pressure. The resultant residue was purified by sil-
ica gel column chromatography (ethyl acetate/hexane,
1:1 to 2:1) to give 37 (397 mg, 97%) as a colorless oil.
1H NMR (DMSO-d6) d: 0.10 (3H, s), 0.10 (3H, s),
0.89 (9H, s), 1.28 (9H, s), 2.37 (1H, ddd, J = 13.2, 6.4,
4.0 Hz), 2.94 (1H, dt, J = 13.2, 6.4 Hz), 3.57 (1H, dd,
J = 10.4, 4.8 Hz), 3.68 (1H, dd, J = 10.8, 4.8 Hz), 4.01
(1H, q-like, J = 4.4 Hz), 4.51 (2H, s), 4.68 (1H, m),
6.46 (1H, t, J = 6.4 Hz), 7.27–7.34 (5H, m), 8.58 (1H,
s), 8.67 (1H, s), 10.14 (1H, br s); 13C NMR (acetone-
d6) d: ꢀ4.7, ꢀ4.6, 18.5, 26.1, 27.5, 40.7, 41.0, 70.6,
73.8, 73.8, 85.1, 87.4, 125.6, 128.3, 128.4, 129.1, 139.2,
143.0, 151.0, 152.5, 175.9; HRMS (ESI) calcd for
C28H42N5O4Si (M+H+) 540.3006, found 540.3017.
(181 mg, 34% from 38) as a colorless oil. Compound 39:
1H NMR (acetone-d6) d: 0.14 (6H, s), 0.93 (9H, s), 2.54
(1H, m), 2.97 (1H, m), 3.71 (1H, dd, J = 10.8, 4.0 Hz),
3.80 (1H, dd, J = 10.8, 4.0 Hz), 4.14 (1H, m), 4.59 (2H,
s), 4.84 (1H, m), 6.58 (1H, t, J = 6.4 Hz), 7.27–7.36
1
(5H, m), 8.66 (1H, s), 8.70 (1H, s). Compound 20: H
NMR (DMSO-d6) d: 2.41 (1H, ddd, J = 13.2, 6.4,
4.0 Hz), 2.83 (1H, dt, J = 13.2, 6.4 Hz), 3.59 (1H, dd,
J = 10.8, 5.2 Hz), 3.69 (1H, dd, J = 10.8, 4.4 Hz), 4.04
(1H, m), 4.47–4.52 (3H, m), 5.45 (1H, br s, J = 4.4 Hz),
6.48 (1H, t, J = 6.4 Hz), 7.23–7.33 (5H, m), 8.78 (1H,
s), 8.80 (1H, s); 13C NMR (acetone-d6) d: 41.2, 71.0,
72.6, 73.8, 85.7, 87.6, 128.4, 128.5, 129.1, 132.8, 139.2,
145.7, 150.7, 152.4, 152.5; HRMS (ESI) calcd for
C17H18ClN4O3 (M+H+) 361.1067, found 361.1077.
4.1.21. 50-O-Benzyl-30-O-(tert-butyldimethylsilyl)-20-deoxy-
adenosine (38). To a stirred solution of 37 (390 mg,
0.72 mmol) in methanol (7 mL) was added potassium
carbonate (299 mg, 2.17 mmol) at ice-water tempera-
ture, and the mixture was stirred at room temperature
for 7 h. After dilution with CH2Cl2, the mixture was fil-
trated. Subsequent to the condensation of the filtrate in
vacuum, the resultant residue was diluted with ethyl ace-
tate. The ethyl acetate layer was washed with water and
brine, dried over Na2SO4, and concentrated under re-
duced pressure. The resultant residue was purified by sil-
ica gel column chromatography (ethyl acetate/hexane,
3:2 to 3:1) to give 38 (250 mg, 76%) as a white solid.
4.1.23. 50-O-Allyl-30-O-(tert-butyldimethylsilyl)-20-deoxy-
adenosine (41). To a stirred solution of 36 (300 mg,
0.67 mmol) in THF (20 mL) was added potassium tert-
butoxide (188 mg, 1.68 mmol) at ice-water temperature,
and the mixture was stirred at the same temperature for
1 min. Subsequent to the addition of allyl bromide
(203 lL, 2.35 mmol) at ice-water temperature, the mix-
ture was stirred at the same temperature for further
2.5 h. After dilution of the mixture with saturated NaH-
CO3 solution, the aqueous layer was extracted with ethyl
acetate. The organic layer was washed with water and
brine, dried over Na2SO4, and concentrated under re-
duced pressure. The resultant residue was purified by sil-
ica gel column chromatography (ethyl acetate/hexane,
1:2 to 1:1) to give 40 (300 mg) with inseparable by-prod-
ucts, which was employed in the next reaction without
further purification.
1
Mp 134–135 ꢁC. H NMR (DMSO-d6) d: 0.09 (3H, s),
0.10 (3H, s), 0.88 (9H, s), 2.30 (1H, ddd, J = 13.2, 6.8,
3.6 Hz), 2.88 (1H, dt, J = 13.2, 6.8 Hz), 3.57 (1H, dd,
J = 10.8, 5.2 Hz), 3.67 (1H, dd, J = 10.8, 5.2 Hz), 3.98
(1H, q-like, J = 4.4 Hz), 4.51 (2H, s), 4.64 (1H, m),
6.33 (1H, t, J = 6.8 Hz), 7.26–7.35 (7H, m), 8.12 (1H,
s), 8.26 (1H, s); 13C NMR (CD3OD) d: ꢀ4.7, ꢀ4.6,
18.8, 26.3, 42.0, 70.7, 74.1, 74.5, 85.7, 88.1, 120.3,
128.9, 129.0, 129.5, 139.2, 140.8, 150.2, 153.8, 157.3;
HRMS (ESI) calcd for C23H34N5O3Si (M+H+)
456.2431, found 456.2444.
To a stirred solution of 40 (300 mg) in methanol (7 mL)
was added potassium carbonate (253 mg, 1.84 mmol) at
ice-water temperature, and the mixture was stirred at
room temperature for 7 h. After dilution with CH2Cl2,
the mixture was filtrated. Subsequent to the condensa-
tion of the filtrate in vacuum, the resultant residue was
diluted with ethyl acetate. The ethyl acetate layer was
washed with water and brine, dried over Na2SO4, and
concentrated under reduced pressure. The resultant res-
idue was purified by silica gel column chromatography
(ethyl acetate/hexane, 1:1 to 4:1) to give 41 (198 mg,
73% from 36) as a white solid. Compound 40: 1H
NMR (acetone-d6) d: 0.16 (6H, s), 0.95 (9H, s), 1.38
(9H, s), 2.49 (1H, m), 2.94 (1H, m), 3.64–3.75 (2H, m),
4.05–4.10 (3H, m), 4.80 (1H, m), 5.15 (1H, d,
J = 10.4 Hz), 5.28 (1H, d, J = 17.2 Hz), 5.93 (1H, m),
6.55 (1H, t, J = 6.4 Hz), 8.45 (1H, s), 8.60 (1H, s), 8.98
(1H, br s). Compound 41: Mp 100.0–100.5 ꢁC. 1H
NMR (DMSO-d6) d: 0.11 (6H, s), 0.90 (9H, s), 2.30
(1H, ddd, J = 13.2, 6.4, 3.6 Hz), 2.87 (1H, dt, J = 13.2,
7.2 Hz), 3.52 (1H, dd, J = 10.8, 5.2 Hz), 3.61 (1H, dd,
J = 10.8, 5.2 Hz), 3.94–3.98 (3H, m), 4.63 (1H, m),
5.14 (1H, dq, J = 10.4, 2.0 Hz), 5.23 (1H, dq, J = 17.2,
2.0 Hz), 5.87 (1H, ddt, J = 17.2, 10.4, 5.6 Hz), 6.33
(1H, t, J = 6.8 Hz), 7.26 (2H, br s), 8.14 (1H, s), 8.29
(1H, s); 13C NMR (CD3OD) d: ꢀ4.7, ꢀ4.6, 18.8, 26.3,
42.0, 70.8, 73.3, 74.0, 85.7, 88.0, 117.5, 120.3, 135.7,
140.9, 150.2, 153.8, 157.3; HRMS (ESI) calcd for
C19H32N5O3Si (M+H+) 406.2274, found 406.2289.
4.1.22. 9-(5-O-Benzyl-b-D-2-deoxyribofuranosyl)-6-chlo-
ropurine (20). To a stirred solution of 38 (250 mg,
0.55 mmol) in CCl4 (10 mL) were added a solution of
tetraethylammonium chloride (364 mg, 2.20 mmol) in
CH2Cl2 (2.5 mL) and then tert-butyl nitrite (326 lL,
2.75 mmol) at ice-water temperature. The mixture was
stirred for 1 h at the same temperature, warmed to room
temperature for 0.5 h, and stirred at 50 ꢁC for 2 h. The
solvent was removed under reduced pressure, and the
resultant residue was purified by silica gel column chro-
matography (ethyl acetate/hexane, 1:3 to 1:2) to give 39
(108 mg) with inseparable by-products, which was em-
ployed in the next reaction without further purification.
To a stirred solution of 39 (108 mg) in THF (2 mL) was
added 1 M THF solution of TBAF–AcOH (1:1, 300 lL,
0.30 mmol) at ice-water temperature, and the mixture
was stirred at the same temperature for 1 h and then at
room temperature for 5 h. The solvent was removed un-
der reduced pressure, and the resultant residue was puri-
fied by preparative thin layer chromatography (Merck,
113895) (methanol/chloroform, 1:10) to give 20

M. Ikejiri et al. / Bioorg. Med. Chem. 15 (2007) 6882–6892
6891
4.1.24. 9-(5-O-Allyl-b-D-2-deoxyribofuranosyl)-6-chloro-
purine (21). To a stirred solution of 41 (235 mg,
0.58 mmol) in CCl4 (11 mL) were added a solution of
tetraethylammonium chloride (384 mg, 2.32 mmol) in
CH2Cl2 (2.6 mL) and then tert-butyl nitrite (345 lL,
2.90 mmol) at ice-water temperature. The mixture was
stirred for 1 h at the same temperature, warmed to room
temperature for 0.5 h, and stirred at 50 ꢁC for 2 h. The
solvent was removed under reduced pressure, and the
resultant residue was purified by silica gel column chro-
matography (ethyl acetate/hexane, 1:6) to give 42
(95 mg) with inseparable by-products, which was em-
ployed in the next reaction without further purification.
and 41) is available online at www.sciencedirect.com.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2007.08.025.
References and notes
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To a stirred solution of 42 (95 mg) in THF (1.5 mL) was
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0.33 mmol) at ice-water temperature, and the mixture
was stirred at the same temperature for 1 h and then
at room temperature for 6 h. The solvent was removed
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purified by silica gel column chromatography (metha-
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1
41) as a colorless oil. Compound 42: H NMR (acetone-
d6) d: 0.16 (6H, s), 0.94 (9H, s), 2.54 (1H, m), 2.95 (1H,
dt, J = 13.2, 6.0 Hz), 3.67 (1H, dd, J = 10.8, 4.0 Hz),
3.74 (1H, dd, J = 10.8, 4.0 Hz), 4.05 (2H, d,
J = 5.6 Hz), 4.12 (1H, m), 4.82 (1H, m), 5.15 (1H, d,
J = 10.4 Hz), 5.27 (1H, d, J = 17.2 Hz), 5.91 (1H, m),
6.59 (1H, t, J = 6.4 Hz), 8.69 (1H, s), 8.72 (1H, s). Com-
pound 21: 1H NMR (DMSO-d6) d: 2.40 (1H, ddd,
J = 13.6, 6.8, 4.4 Hz), 2.81 (1H, dt, J = 13.6, 6.4 Hz),
3.54 (1H, dd, J = 10.4, 5.2 Hz), 3.63 (1H, dd, J = 10.4,
4.0 Hz), 3.95 (2H, dt, J = 5.2, 1.2 Hz), 4.00 (1H, q-like,
J = 4.8 Hz), 4.47 (1H, m), 5.12 (1H, dq, J = 10.4, 2.0
Hz), 5.20 (1H, dq, J = 17.2, 2.0 Hz), 5.46 (1H, br d,
J = 4.4 Hz), 5.84 (1H, ddt, J = 17.2, 10.4, 5.6 Hz), 6.48
(1H, t, J = 6.4 Hz), 8.81 (1H, s), 8.82 (1H, s); 13C
NMR (DMSO-d6) d: 39.0, 69.8, 70.5, 71.3, 84.1, 85.9,
116.5, 131.3, 134.9, 145.7, 149.2, 151.3, 151.6; HRMS
(ESI) calcd for C13H16ClN4O3 (M+H+) 311.0911, found
311.0918.
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4.2. Biological evaluation
Cell culture, luciferase assay, and real-time RT-PCR
analysis were performed as described previously.16 The
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assay according to the manufacturer’s protocol (Cell
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Acknowledgment
This research was partially supported by a Grant-in-Aid
for Young Scientists (B), No. 18790093, from the Japan
Society for the Promotion of Science (JSPS).
Supplementary data
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Clercq, E., Ed.; Jai Press Inc.: Stamford Connecticut,
1999; Vol. 3, pp 69–91, and also see Ref. 5a and references
therein.
10. A prodrug approach involving 50-monophosphate deriv-
atives is also effective in increasing the antiviral potency.
For example: Prakash, T. P.; Prhave, M.; Eldrup, A. B.;
Dan Cook, P.; Carroll, S. S.; Olsen, D. B.; Stahlhut, M.
W.; Tomassini, J. E.; MacCoss, M.; Galloway, S. M.;
58, 2985.
14. Another synthesis of 32: Ueda, T.; Nomoto, Y.; Matsuda,
A. Chem. Pharm. Bull. 1985, 33, 3263.
15. Ciuffreda, P.; Casati, S.; Santaniello, E. Tetrahedron 2000,
56, 3239.
16. Watashi, K.; Hijikata, M.; Hosaka, M.; Yamaji, M.;
Shimotohno, K. Hepatology 2003, 38, 1282; Murata, T.;
Hijikata, M.; Shimotohno, K. Virology 2005, 340, 105;
Goto, K.; Watashi, K.; Murata, T.; Hishiki, T.; Hijikata,
M.; Shimotohno, K. Biochem. Biophys. Res. Commun.
2006, 343, 879.
17. A similar SAR trend was observed in our previous study
(Ref. 11), and some comments are mentioned therein.
18. Parang, K.; Knaus, E. E.; Wiebe, L. I. Nucleosides
Nucleotides 1998, 17, 987, and references therein.
19. Several 50-O-acyl nucleoside analogues have been reported
as prodrugs of the corresponding deacylated analogues.
See Ref. 18.