Synthesis of benzodithiol-2-yl-substituted nucleoside derivatives as lead compounds having anti-bovine viral diarrhea virus activity

Article abstract of DOI:10.1021/jm049677d

Nucleoside derivatives having a benzodithiol-2-yl (BDT) group were synthesized and examined for their anti-bovine viral diarrhea virus (BVDV) activities. Other substituents structurally similar to the BDT group such as 1,3-benzodioxol-2-yl, benzimidazol-2

Full text of DOI:10.1021/jm049677d

J . Med. Chem. 2004, 47, 5265-5275
5265
Syn th esis of Ben zod ith iol-2-yl-Su bstitu ted Nu cleosid e Der iva tives a s Lea d
Com p ou n d s Ha vin g An ti-Bovin e Vir a l Dia r r h ea Vir u s Activity
Kohji Seio,†,| Takahide Sasaki,‡ Koichirou Yanagida,§ Masanori Baba,§ and Mitsuo Sekine*,‡,|
Frontier Collaborative Research Center and Department of Life Science, Tokyo Institute of Technology,
Nagatsuta, Midori-ku, Yokohama 226-8501, Division of Antiviral Chemotherapy, Center for Chronic Viral Diseases,
Faculty of Medicine, Kagoshima University, Kagoshima 890-8520, J apan, and CREST,
J ST (J apan Science and Technology Agency), Nagatsuta-cho, Midori-ku, Yokohama 226-8501, J apan
Received May 1, 2004
Nucleoside derivatives having a benzodithiol-2-yl (BDT) group were synthesized and examined
for their anti-bovine viral diarrhea virus (BVDV) activities. Other substituents structurally
similar to the BDT group such as 1,3-benzodioxol-2-yl, benzimidazol-2-yl and 1-oxo-benzodithiol-
2-yl groups were not effective as the pharmacophore. The anti-BVDV assay revealed that
2′-O-BDT-guanosine and 2′-O-BDT-inosine had the strongest anti-BVDV activity among the
nucleoside derivatives synthesized in this study. Since BVDV has been recognized as a surrogate
for human hepatitis C virus (HCV), the BDT-modified nucleosides might become a new class
of lead compounds to find nucleoside-type anti-HCV agents such as ribavirin.
Sch em e 1. Structure of the Thymidine Derivatives
Having a BDT Group
In tr od u ction
Hepatitis C virus is a member of the Flaviviridae
family.1 It was estimated that approximately 170 million
people are infected by this virus and the infection per-
sisted in more than 80% of the infected population.2
Moreover, 4-5% of the chronically infected patients will
develop liver cirrhosis and hepatocellular carcinoma
within 20-30 years after infection.3 Currently the most
effective treatment of HCV infection is the combined use
of interferon-R and the antiviral agent ribavirin.4 How-
ever, the treatment is frequently accompanied by severe
adverse effects such as fever, depression and atonia,
whereas the response is at best around 40%. Therefore,
alternative agents for the treatment of HCV have to be
discovered.
assay system. In this paper, we report significant anti-
BVDV activity of new nucleoside derivatives having a
1,3-benzodithiol-2-yl group introduced into their hy-
droxyl groups.
Resu lts a n d Discu ssion
Although there are a number of molecular targets for
the development of anti-HCV drugs,5 the efficiency of
these compounds against the viral replication should be
confirmed in cell culture systems. However, the inability
to propagate HCV in culture cells apparently hampers
the discovery of effective anti-HCV agents. Instead of
such cell culture systems, several research groups have
developed model systems that can surrogate the cell
culture systems of HCV. Among the model systems,
bovine viral diarrhea virus (BVDV) is a popular system
with which antiviral agents can be evaluated for poten-
tial activity against HCV.6
BVDV is a Pestivirus member which also belongs to
the Flaviviridae family. The amino acid sequences coded
on the BVDV genome have high homology to those of
HCV.1 Since the anti-HCV agent rivabirin showed
strong activity against BVDV replication in vitro, it is
expected that novel nucleoside-type anti-HCV agents
can be discovered through the screening by use of this
Syn th esis a n d An ti-BVDV Activities of BDT-
Mod ified Deoxyn u cleosid es. The lead compound,
3′-O-(benzodithiol-2-yl)thymidine (1), was first discov-
ered in random screening of our chemical libraries to
find first-stage drug candidates that showed inhibitory
effects against the BVDV cell culture system described
above. The compound was previously reported by Sekine
et al. as an intermediate of a synthetic unit for oligode-
oxyribonucleotide synthesis.7 Compound 1 showed weak
anti-BVDV activity (EC50 ) 46 µM) and weaker cyto-
toxicity (CC50 ) 72 µM). In the same assay, ribavirin
showed much stronger activity (EC50 ) 1.5 µM) and
much weaker cytotoxicity (CC50 > 100 µM). Interest-
ingly, a regioisomer of 1, 5′-O-(benzodithiol-2-yl)thymi-
dine7 (Scheme 1), showed no anti-BVDV activity. There-
fore, we focused our interest on nucleoside derivatives
modified by benzodithiol-2-yl (BDT) on their 3′-hydroxyl
groups to improve the anti-BVDV activity of 1.
Several 2′-deoxynucleosides having a BDT group (4a -
c) on their 3′-oxygens were synthesized, as shown in
Scheme 2. In addition, the ring-opened analogues of 1,
i.e., compounds 6a -d were synthesized by reduction of
the intermediates 3a -d with tributyltin hydride fol-
lowed by the methylation and the deprotection.8 (Scheme
3)
* To whom correspondence should be addressed. phone: +81-45-
924-5706; fax: +81-45-924-5772; e-mail: msekine@bio.titech.ac.jp
† Frontier Collaborative Research Center, Tokyo Institute of Tech-
nology.
‡ Department of Life Science, Tokyo Institute of Technology.
§ Kagoshima University.
| CREST.
10.1021/jm049677d CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/10/2004

5266 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Seio et al.
Sch em e 2. Synthesis of Deoxynucleoside Derivatives Having a BDT Group
Sch em e 3. Synthesis of Deoxynucleoside Derivatives Having a MPTM Group
Sch em e 5. Oxidation of 1a
Ta ble 1. Anti-BVDV Activity of Nucleoside Derivatives Having
BDT and MPTM Groupsa
rel
compound
EC50 (µM)
EC50
1
CC50 (µM)
CC50/EC50
ribavirin
1
1.5
46.8
63.8
79.4
69.1
>100
72.4
> 67
1.5
1.1
1.3
1.0
> 1.1
-
31.2
42.5
52.9
46.1
59.7
-
4a
4b
4c
6a
6b
6c
6d
16
68.4
>100
69.7
89.6
>100
>100
>100
>100
81.3
>100
>100
>100
86.7
-
-
-
-
57.8
0.93
a
rel
EC50 ) EC50(compound)/EC50(ribavirin)
.
Sch em e 4. Synthesis of 2-Methoxybenzodithiol 7
a
Reagents: (a) mCPBA (1 equiv), NaHCO3 (1 equiv), CH2Cl2,
r.t., 2 h; (b) mCPBA (2 equiv), NaHCO3 (2 equiv), CH2Cl2, r.t., 2
h; (c) mCPBA (4.1 equiv), NaHCO3 (4.1 equiv), CH2Cl2, r.t., 90
min.
Sch em e 6. Synthesis of Benzodioxole (12) and
Benzimidazole (15) Analogue of 1
The anti-BVDV activities of 1, 4a -c and 6a -d thus
obtained are listed in Table 1. As shown in Table 1,
ribavirin showed potent activity and no cytotoxicity up
to 100 µM. The deoxynucleosides modified by the BDT
group (1, 4a -c) showed weaker but significant activity.
In contrast, the deoxynucleosides modified by the MPTM
group (6a -d ) showed much weaker or undetectable
activity against BVDV replication. From these observa-
tions, it was evident that the BDT group was essential
for the anti-BVDV activity of the modified deoxynucleo-
sides. Despite the anti-BVDV activity of the BDT-
modified deoxynucleosides, they are considered to be
inferior to ribavirin in terms of their activity and
cycotoxicity. Therefore, further structural modifications
were examined to improve these drawbacks.
BDT moieties were indispensable for the activity of the
BDT-modified deoxynucleosides.
Im p or ta n ce of th e Nu cleosid e Moiety for An ti-
BVDV Activity. The importance of the nucleoside
moiety was evaluated by testing the anti-BVDV activity
of a simple BDT-containing compound 7 (Scheme 4). The
anti-BVDV assay revealed that compound 7 had neither
anti-BVDV activity nor cytotoxicity. Therefore, it was
unambiguously proved that both the nucleoside and
To clarify more detailed structural requirement for
the BDT group, several thymidine derivatives were
synthesized incorporating other bicyclic substituents
which were structurally similar to that of the BDT
group. Shown in Schemes 5 and 6 are the synthesis of
the sulfoxide and sulfonyl derivatives of 1 prepared by
oxidation by use of m-CPBA, and 3′-O-benzodioxol-2-yl

Nucleoside Derivatives Having Anti-BVDV Activity
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5267
Ta ble 2. Anti-BVDV Activity of Ribonucleoside Derivatives
Having the BDT Group
Sch em e 7. Synthesis of 5′-Deoxy Derivative of 1
rel
compound
EC50 (µM)
EC50
CC50 (µM)
CC50/EC50
ribavirin
19
20
0.67
50.6
56.7
<48.0
28.5
1
75.5
84.6
<71.69
42.5
58.5
>100
73.9
67.3
48.0
70.4
74.3
73.2
59.2a
60.1b
150<
1.46
1.19
<1
21
23a
23b
23c
26
2.47
1.89
5.09
3.21
1.19
(12) and 3′-O-benzimidazol-2-yl (15) derivatives of thy-
midine. However, all the compounds lacking the BDT
moiety showed neither anti-BVDV activity nor cytotox-
icity. It should be noted that replacement of the sulfur
atoms of 1 to oxygen atoms resulted in loss of the anti-
BVDV activity of 12. Therefore, on the basis of the
structure-activity relationship described above, we con-
cluded that the bicyclic structure and the two divalent
sulfur atoms were essential for expression of the anti-
BVDV activity of the BDT-containing deoxynucleosides.
Mod ifica tion on th e Su ga r Moiety. As mentioned
above, the BDT group must be left intact to maintain
the anti-BVDV activity of modified nucleosides. There-
fore, we next tried to modify the structure of the
deoxyribose moiety in order to improve the antiviral
activity. Initially, we attempted to clarify the impor-
tance of the 5′-hydroxyl group using a 5′-deoxythymi-
dine9 derivative (16) (Scheme 7). The anti-BVDV and
cytotoxicity of 16 are listed in Table 1. Although the 5′-
deoxy derivative 16 still retained weak anti-BVDV
activity, the cytotoxicity appeared at the lower concen-
tration range in comparison with the antiviral activity.
This result suggested that the intact 5′-hydroxyl group
must remain in order to improve the antiviral activity
without intensifying the cytotoxicity.
On the other hand, the anti-BVDV activity of 16
provided some implication on the action mechanism of
the nucleoside derivatives having a BDT group inde-
pendent of its 5′-phosphorylation. It is well-known that
some nucleoside-type antiviral agents act after their
conversion to the corresponding 5′-monophosphate or
5′-triphosphate derivatives in cells. For example,
ribavirin elicits its antiviral activity as an inosine
5′-monophosphate (IMP) dehydrogenase inhibitor after
its conversion to the corresponding 5′-monophosphate,10
and AZT and acyclovir act as chain terminators after
similar transformation to the 5′-triphosphates.11
39.2
14.4
21.5
18.4a
50.3b
15.3a
82.5b
30
a
In this case, the EC50 and CC50 for ribavirin were 1.2 and
b
>100, respectively. In this case, the EC50 and CC50 for ribavirin
were 0.61and 90.2, respectively.
lation of the nucleosides. Moreover, Borowski and co-
workers13 discovered the in vitro inhibitory activity of
the same nucleosides against the RNA helicase and
NTPase activity of nonstructural protein 3 (NS3) of HCV
and related viruses. Considering the importance of the
RNA helicase and NTPase activity of NS3 protein in
BVDV replication14 and the anti-BVDV activity of
5′-deoxynucleoside analogue 16, the BDT-nucleosides
described here might have a possibility that they have
an anti-NS3 protein activity similar to those of haloga-
nated â-D-ribosylbenzimidazoles.
If the 2′-deoxynucleosides derivatives described above
act as anti-BVDV agents according to a mechanism
similar to that of the haloganated â-D-ribosylbenzimi-
dazoles, ribonucleoside derivatives must be more active
than deoxynucleoside derivatives. Therefore, we exam-
ined the synthesis of the ribonucleosides modified by
BDT groups on their 2′ and 3′ hydroxyl groups. Accord-
ingly, 3′-O-BDT-uridine (19) was synthesized as shown
in Scheme 8, and 2′-O-BDT-uridine (20) and 2′, 3′-O-
bis-BDT-uridine (21) were synthesized, as reported
previously.15
As shown in Table 2, uridine derivatives 19 and 20
having a BDT group showed weaker anti-BVDV activity
as compared to the lead compound 1 as judged from
their relative EC50 values to that of ribavirin, whereas
the ratios of CC50/EC50 of 19 and 20 were comparable
to that of 1. On the contrary, the uridine derivative 21
bearing two BDT groups showed much higher toxicity.
From these results, it was postulated that a BDT
could be introduced either on the 2′ or the 3′ position of
ribonucleosides without losing the CC50/EC50 ratio and
that introduction of two BDT groups in a ribonucleoside
could induce severe cytotoxicity.
However, recent studies revealed another type of
reaction mechanism of nucleoside derivatives having
antiviral activities. Drach and co-workers12 revealed
that the anti-HCMV activity of haloganated â-D-ribo-
sylbenzimidazoles was independent of the phosphory-
Sch em e 8. Synthesis of Uridine Derivatives Having BDT Groups

5268 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Seio et al.
Sch em e 9. Synthesis of Cytidine, Adenosine and
Guanosine Derivatives Having a BDT Group
study, has been known to form stable base pairs with
both the cytosine and uracil bases,17,18 and this stable
mismatch base pair may induce mutation in viral
genome. Therefore, a similar effect on viral genome
mutation was expected for the mismatch-forming nu-
cleoside 30 if the mechanism of action of BDT-nucleo-
sides is the same as that of ribavirin.
The anti-BVDV activities of the ribonucleoside deriva-
tives are shown in Table 2. As judged from the relative
EC50 values (EC50rel), the inosine 26 and the guanosine
23c derivatives showed the best and the second best
antiviral activities, respectively. In both cases, the anti-
viral activities were stronger than that of the lead com-
Therefore, we further examined the synthesis of other
ribonucleoside derivatives having a BDT group on their
2′ position. The 2′ position was chosen because it can
be modified more easily via the selective protection of
the 3′- and 5′-hydroxyl groups by the 1,1,3,3-tetraiso-
propyldisiloxan-1,3-di-yl (TIPDS) group (Scheme 9).
The 2′-BDT derivatives of cytidine, adenosine and
guanosine were synthesized starting from the fully
protected ribonucleosides, 22a , 22b and 22c, respec-
tively. The 2′-BDT derivatives of inosine 26 and 4-N-
methoxycarbonyldytidine 30 were synthesized, as shown
in Scheme 10 and Scheme 11.
rel
pound 1 (EC50 ) 31.2). On the other hand when the
CC50/EC50 values were used to evaluate the overall effic-
acy, the guanosine derivative 23c was apparently the
most preferable among the 2′-deoxyribo- and ribonucleo-
side derivatives reported in this study. Since the CC50/
EC50value of 23c was significantly smaller than those of
1 and other nucleoside derivatives, the guanosine moiety
must play some important role to enhance the antiviral
activity without stimulating the cytotoxicity. On the con-
trary, the mismatch-forming nucleoside 30 showed only
weak antiviral activity and rather strong cytotoxicity.
Compound 30 was designed based on our previous
finding16 that 4-N-methoxycarbonylcytosine could form
a stable base pair with both guanine and adenine. The
antiviral agent ribavirin, a positive control used in this
Sch em e 10. Synthesis of Inosine Derivative Having a BDT Group
Sch em e 11. Synthesis of 4-N-Methoxycarbonylcytidine Derivative (30)a
a
Reagents: (i) HMDS (3 equiv), TMSCl (cat.), CH3CN, 60 °C, 1 h; (ii) methyl chloroformate (1.5 equiv), pyridine, r.t., 15 min; (iii)
concentrated NH3-pyridine (1:1, v/v), r.t., 50 min. (iv) 1,3-dichloro-1,1,3,3-tetraisopropyl-1,3-disiloxane (1.2 equiv), pyridine, r.t., 3 h; (v)
1,3-benzodithiolium tetrafluoroborate (1 equiv), pyridine, r.t., 4 h; (vi) tetra-n-butylammonium fluoride (2.5 equiv), THF, r.t., 15 min.

Nucleoside Derivatives Having Anti-BVDV Activity
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5269
mmol). The resulting mixture was stirred at ambient temper-
ature. After 12 h, to this solution was added triethylamine (5.4
mL, 38.5 mmol), and the resulting solution was stirred for an
additional 15 min. The reaction mixture was washed three
times with water (100 mL), dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was chro-
matographed on a column of silica gel (120 g) with hexanes-
ethyl acetate (85:15, v/v) containing 0.5% pyridine to give 3a
(5.0 g, 69%) as a white foam: 1H NMR (CDCl3) δ -0.10 (3H,
s), -0.05 (3H, s), 0.77 (9H, s), 1.80-1.94 (4H, m), 2.38-2.45
(1H, m), 3.52 (1H, dd, J ) 1.9 Hz, 11.2 Hz), 3.75 (1H, dd, J )
1.9 Hz, 11.1 Hz), 4.13-4.19 (2H, m), 6.23 (1H, m), 6.89 (1H,
s), 7.03-7.32 (5H, m), 9.21 (1H, br); 13C NMR (CDCl3) δ - 5.5,
- 5.4, 12.6, 18.2, 25.9, 39.4, 62.8, 75.4, 84.7, 85.5, 88.8, 110.8,
121.8, 121.9, 125.6, 135.0, 135.6, 135.7, 150.2, 163.7. MS m/z
calcd for C23H33N2O5S2Si+: 509.1600, found 509.1594.
Con clu sion
In this paper we reported new nucleoside derivatives
modified by a BDT group as anti-BVDV agents. Since
BVDV is similar to HCV in terms of their genome struc-
ture and amino acid sequences, anti-BVDV agents are
expected to become good lead compounds for anti-HCV
agents. As discussed in the text, some of the 2′-deoxy-
ribo- and ribonucleosides having a BDT group on their
hydroxyl functions showed significant anti-BVDV activ-
ity. The BDT groups could not be replaced by other het-
eroaromatic substituents such as benzodioxol-2-yl, ben-
zimidazole-2-yl and the S-oxide derivatives of BDT.
Therefore, the BDT could be considered as the essential
structural component to elicit the anti-BVDV activities.
Among all the nucleosides reported newly in this paper,
the guanosine derivative 23c showed the most prefer-
able properties in terms of the high anti-BVDV activity
and rather low cytotoxicity. Although the inosine de-
rivative 26 showed much stronger anti-BVDV activity
than 23c, it was not superior to 23c because of its higher
cytotoxicity. The fact that both the guanosine and ino-
sine derivatives showed rather strong anti-BVDV activi-
ties while the adenosine derivative 23b did not exhibit
any activity suggested that the carbonyl group and the
imino group of the positions 6 and 1 of the purine ring
might be important to enhance the anti-BVDV activity.
The detailed mechanism of action of nucleoside deriva-
tives having a BDT group has not been clarified yet.
However, the involvement of the mechanism indepen-
dent of the 5′-phosphorylation was indicated by the anti-
BVDV activity of the 5′-deoxy derivative 16. Such, 5′-
phosphorylation independent mechanisms have been
recently reported for the activity of haloganated benz-
imidazole nucleosides on HCV and related viruses.12,13
Inhibition of the RNA helicase and NTPase activity of
NS3 protein is considered important in such a mecha-
nism.14 Therefore, the new pharmacophore, BDT, found
in this study might provide a new drug design strategy
to develop nucleoside-type anti-BVDV agents having
RNA helicase and/or NTPase activity of NS3 protein.
However, further extensive studies are needed to clarify
if BDT-modified nucleoside derivatives show the anti-
BVDV activity in a similar mode.
3′-O-(1,3-Ben zod ith iol-2-yl)-5′-O-(ter t-bu tyld im eth ylsi-
lyl)-6-N-p iva loyl-d eoxya d en osin e (3b). 5′-O-(tert-Butyldim-
ethylsilyl)-6-N-pivaloyl-deoxyadenosine (4.5 g, 10.0 mmol) was
dissolved in anhydrous CH2Cl2 (100 mL). To this solution were
added 1,3-benzodithiolium tetrafluoroborate (3.6 g, 15.0 mmol)
and anhydrous pyridine (2.4 mL, 30.0 mmol). The resulting
mixture was stirred at ambient tempetarure. After 4.5 h, to
this solution was added triethylamine (8.4 mL, 60 mmol), and
the resulting solution was stirred for an additional 15 min.
The reaction mixture was washed three times with water (100
mL), dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was chromatographed on a
column of silica gel (N60, 160 g) with hexanes-ethyl acetate
(55:45, v/v) to give 3b (4.3 g, 71%) as a white foam: 1H NMR
(CDCl3) δ -0.06, -0.04 (6H, 2s), 0.79 (9H, s), 1.36 (9H, s),
2.56-2.62 (2H, m), 3.60 (1H, dd, J ) 3.0), 3.76 (1H, dd, J )
3.0, 11.1 Hz), 4.20 (1H, m), 4.43 (1H, m), 6.38 (1H, m), 6.78
(1H, s), 7.07-7.34 (4H, m), 8.09 (1H, s), 8.46 (1H, s), 8.65 (1H,
s); 13C NMR (CDCl3) δ -5.5, -5.4, 18.3, 25.9, 27.5, 39.7, 40.5,
62.7, 75.5, 84.3, 85.8, 89.0, 121.9, 122.0, 122.8, 125.6, 135.4,
135.5, 140.8, 149.2, 151.0, 152.3, 175.3. MS m/z calcd for
C
28H40N5O4S2Si+: 602.2291, found 602.2285.
3′-O-(1,3-Ben zod ith iol-2-yl)-5′-O-(ter t-bu tyld im eth ylsi-
lyl)-2-N-isob u t yr yl-d eoxygu a n osin e (3c). 5′-O-(tert-Bu-
tyldimethylsilyl)-2-N-isobutyryl-deoxyguanosine (3.5 g, 7.8
mmol) was dissolved in anhydrous CH2Cl2 (78 mL). To this
solution were added 1,3-benzodithiolium tetrafluoroborate (2.8
g, 11.6 mmol) and anhydrous pyridine (1.9 mL, 23.3 mmol).
The resulting mixture was stirred at ambient temperature.
After 4 h, to this solution was added triethylamine (6.5 mL,
46.5 mmol), and the resulting solution was stirred for an
additional 15 min. The reaction mixture was washed three
times with water (80 mL), dried over Na2SO4, filtered and
concentrated under reduced pressure. The residue was chro-
matographed on a column of silica gel (C300, 180 g) with
hexanes-ethyl acetate (50:50, v/v) to give 3c (2.9 g, 62%) as a
white foam: 1H NMR (CDCl3) δ -0.08 (6H, s), 0.77 (9H, s),
1.20 (6H, m), 2.33-2.81 (3H, m), 3.56 (1H, m), 3.68 (1H, dd, J
) 2.2, 11.1 Hz), 4.16 (1H, m), 4.33 (1H, m), 6.06 (1H, dd, J )
6.6, 6.6 Hz), 6.77 (1H, s), 7.05-7.33 (4H, m), 7.77 (1H, s), 9.82
(1H, br), 12.19 (1H, br); 13C NMR (CDCl3) δ - 5.6, - 5.4, 18.3,
19.0, 19.1, 25.9, 62.8, 75.5, 83.6, 85.7, 89.0, 120.8, 121.9, 122.0,
125.7, 135.4, 135.5, 136.4, 147.5, 148.0, 155.8, 178.8. MS m/z
calcd for C27H38N5O5S2Si+ 604.2084, found 604.2089.
3′-O-(1,3-Ben zodith iol-2-yl)-5′-O-(isobu tyloxycar bon yl)-
4-N-p iva loyl-d eoxycytid in e (3d ). 5′-O-(Isobutyloxycarbo-
nyl)-4-N-pivaloyl-deoxycytidine (600 mg, 1.5 mmol) was dis-
solved in anhydrous CH2Cl2 (15 mL). To this solution were
added 1,3-benzodithiolium tetrafluoroborate (700 mg, 2.9
mmol) and anhydrous pyridine (0.35 mL, 4.4 mmol). The
resulting mixture was stirred at ambient tempetarure. After
7 h, to this solution was added triethylamine (1.2 mL, 8.8
mmol), and the resulting solution was stirred for an additional
15 min. The reaction mixture was washed three times with
water (10 mL), dried over Na2SO4, filtered and concentrated
under reduced pressure. The residue was chromatographed
on a column of silica gel (C200, 20 g) with hexanes-ethyl
acetate (60:40, v/v) containing 0.5% pyridine (v/v) to give 3c
Exp er im en ta l Section
3′-O-(1,3-Ben zod ith iol-2-yl)th ym id in e (1). Compound 3a
(5.0 g, 9.8 mmol) was dissolved in tetrahydrofuran (100 mL).
To this solution was added tetra-n-butylammonium fluoride
(3.8 g, 14.7 mmol), and the resulting solution was stirred at
ambient temperature for 30 min. The solvent was removed
under reduced pressure, and the residue was dissolved in
CHCl3 (100 mL), washed three times with water (100 mL),
dried over Na2SO4, filtered and concentrated under reduced
pressure. The residue was chromatographed on a column of
silica gel (C200, 150 g) with hexanes-ethyl acetate (35:65, v/v)
containing 0.5% triethylamine (v/v) to give 1 (3.8 g, 98%): 1H
NMR (CDCl3) δ 1.77 (3H, s), 2.22-2.41 (2H, m), 3.63 (1H, dd,
J ) 2.7, 12.0 Hz), 3.80 (1H, dd, J ) 2.7, 12.3 Hz), 4.01 (1H,
m), 4.26-4.31 (1H, m), 6.05 (1H, dd, J ) 6.8, 6.8 Hz), 6.69
(1H, s), 7.05-7.35 (5H, m, 6-H); 13C NMR (CDCl3) δ 12.5, 38.2,
61.5, 74.7, 84.8, 86.1, 89.3, 111.0, 121.9, 122.1, 125.6, 135.3,
135.5, 136.5, 150.3, 163.8. MS m/z calcd for C17H19N2O5S2
395.0735, found 395.0738.
3′-O-(1,3-Ben zod ith iol-2-yl)-5′-O-(ter t-bu tyld im eth ylsi-
lyl)th ym id in e (3a ). 5′-O-(tert-Butyldimethylsilyl)thymidine
(5.0 g, 14 mmol) was dissolved in anhydrous CH2Cl2 (140 mL).
To this solution were added 1,3-benzodithiolium tetrafluorobo-
rate (3.7 g, 15.4 mmol) and anhydrous pyridine (2.5 mL, 30.8
+
:

5270 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Seio et al.
(700 mg, 85%) as a white foam: 1H NMR (CDCl3) δ 0.84 (6H,
d, J ) 6.9 Hz), 1.17 (9H, s), 1.83-2.68 (3H, m), 3.81 (2H, d, J
) 6.8 Hz), 4.05-4.27 (4H, m), 6.06 (1H, dd, J ) 5.9, 5.9 Hz),
6.64 (1H, s), 6.97-7.24 (5H, m, 5-H), 7.77 (1H, s, J ) 7.6 Hz),
8.09 (1H, s); 13C NMR (CDCl3) δ 18.7, 26.8, 26.9, 27.5, 65.5,
73.7, 74.1, 82.2, 86.6, 86.7, 89.0, 95.7, 121.7, 121.8, 125.3, 134.9,
135.1, 143.4, 154.3, 154.4, 161.7, 177.5. MS m/z calcd for
and the resulting solution was stirred at ambient temperature
for 4 h. The reaction mixture was diluted by adding CHCl3
(50 mL), washed five times with water (50 mL), dried over
Na2SO4, filtered and concentrated to dryness under reduced
pressure. The residue was chromatographed on a column of
silica gel (C200, 20 g) with hexanes-ethyl acetate (80:15. v/v)
to give 5a (894 mg, 85%): 1H NMR (CDCl3) δ 0.09 (6H, s),
0.90 (9H, s), 1.89-2.00 (4H, m), 2.33-2.42 (4H, m), 3.75 (1H,
dd, J ) 2.2, 11.2 Hz), 3.84 (1H, dd, J ) 2.2, 11.2 Hz), 4.05
(1H, m), 4.56 (1H, m), 4.93 (1H, d, J ) 11.9 Hz), 5.02 (1H, d,
J ) 11.9 Hz), 6.23-6.28 (1H, m), 7.07-7.50 (5H, m, ArH), 9.46
(1H, br); 13C NMR (CDCl3) δ -5.4, -5.3, 12.6, 15.8, 18.4, 25.9,
37.7, 63.5, 73.6, 77.1, 84.8, 110.8, 125.26, 125.28, 127.9, 131.6,
132.4, 135.1, 140.5, 150.3, 163.8. MS m/z calcd for C24H37N2O5S2-
Si+: 525.1913, found 525.1911.
C
26H34N3O7S2+: 564.1838, found 564.1829.
3′-O-(1,3-Ben zod ith iol-2-yl)d eoxya d en osin e (4a ). Com-
pound 3b (602 mg, 1.0 mmol) was dissolved in 2 M NH3/CH3-
OH (10 mL), and the resulting solution was stirred at ambient
temperature for 21 h. The solvent was removed under reduced
pressure, and the residue was dissolved in CHCl3 (10 mL). The
solution was washed three times with water (10 mL) and the
solvent was removed under reduced pressure. The residue was
dissolved in tetrahydrofuran (10 mL). To this solution was
added tetra-n-butylammonium fluoride (392 mg, 1.5 mmol),
and the resulting solution was stirred at ambient temperature
for 2 h. The solvent was removed under reduced pressure, and
the residue was dissolved in CHCl3 (10 mL) and washed three
times with water (10 mL). The aqueous layer was extracted
with CHCl3 (10 mL), and the all the organic layers were
combined, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The resulting precipitates were collected by
filtration to give 4a (309 mg, 77%): 1H NMR (DMSO-d6) δ
2.77-2.87 (2H, m), 3.46-3.61 (2H, m), 4.08 (1H, m), 4.57 (1H,
m), 5.41 (1H, t, J ) 5.5 Hz), 6.25 (1H, m), 6.99 (1H, s), 7.14-
7.19 (2H, m), 7.35 (2H, s), 7.46-7.51 (2H, m), 8.09 (1H, s), 8.26
(1H, s); 13C NMR (DMSO-d6) δ 36.7, 61.6, 77.8, 84.1, 85.2, 89.1,
119.2, 122.4, 125.5, 135.2, 135.3, 139.5, 148.6, 152.2, 156.0.
Anal. Calcd for C17H17N5O3S2‚1/10H2O: C, 50.38; H, 4.28; N,
17.28; S, 15.82. Found: C, 50.33; H, 4.52; N, 17.09; S, 15.55.
3′-O-[[2-(Meth ylth io)ph en yl]th iom eth yl]th ym idin e (6a).
Compound 5a (894 mg, 1.7 mmol) was dissolved in tetrahy-
drofuran (17 mL). To this solution was added tetra-n-buty-
lammonium fluoride (667 mg, 2.55 mmol). The resulting
solution was stirred at ambient temperature for 1 h. The
solvent was removed under reduced pressure, and the residue
was dissolved in CHCl3 (20 mL), washed three times with
water (20 mL), dried over Na2SO4, filtered and concentrated
under reduced pressure. The residue was chromatographed
on a column of silica gel (C200, 30 g) with hexanes-ethyl
acetate (55:45, v/v) to give 6a (550 mg, 79%): 1H NMR (CDCl3)
δ 1.86 (3H, s), 2.26-2.32 (2H, m), 2.43 (3H, s), 3.06 (1H, s),
3.70-3.88 (2H, m), 4.02 (1H, m), 4.61-4.65 (1H, m), 4.93 (1H,
d, J ) 11.7 Hz), 5.06 (1H, d, J ) 11.7 Hz), 7.06-7.49 (5H, m,
6-H), 9.47 (1H, s); 13C NMR (CDCl3) δ 12.6, 15.8, 37.0, 62.3,
74.0, 84.7, 86.4, 110.9, 125.2, 125.3, 128.0, 131.7, 132.2, 136.2,
140.5, 150.3, 163.9. MS m/z calcd for C18H23N2O5S2+: 411.1048,
found 411.1049.
3′-O-(1,3-Ben zod ith iol-2-yl)d eoxygu a n osin e (4b). Com-
pound 3c (700 mg, 1.16 mmol) was dissolved in 2 M NH3/CH3-
OH (12 mL), and the resulting solution was stirred at ambient
temperature for 48 h. The solvent was removed under reduced
pressure, and the residue was dissolved in N,N-dimethylfor-
mamide (10 mL)-tetrahydrofuran (10 mL). To this solution
was added tetra-n-butylammonium fluoride (455 mg, 1.74
mmol), and the resulting solution was stirred at ambient
temperature for 3 h. To this solution as added ethyl acetate
(10 mL), and the resulting solution was washed twice with
water (10 mL). The organic layer was removed and the white
precipitation separated from the aqueous layer was collected
by filtration to give 4b (345 mg, 71%): 1H NMR (DMSO-d6) δ
2.37-2.65 (2H, m), 3.47 (2H, m), 3.99 (1H, m), 4.49 (1H, m),
5.06 (1H, s), 6.00 (1H, m), 6.48 (2H, br), 7.14-7.50 (4H, m),
7.86 (1H, s, 8-H), 10.66 (1H, br, 1-NH); 13C NMR (DMSO-d6)
δ 36.7, 61.3, 77.9, 82.4, 84.7, 89.1, 116.5, 122.5, 125.6, 135.0,
135.2, 150.7, 153.6, 156.6. Anal. Calcd for C17H17N5O4S2: C,
48.67; H, 4.08; N, 16.70; S, 15.29. Found: C, 48.37; H, 3.91;
N, 16.50; S, 15.29.
3′-O-[[2-(Meth ylth io)p h en yl]th iom eth yl]d eoxya d en o-
sin e (6b). Compound 3b (3.0 g, 5.0 mmol) was dissolved in
benzene (50 mL). To this solution were added tri-n-butyltin
hydride (3.4 mL, 12.5 mmol) and AIBN (1.6 g, 10 mmol). The
resulting solution was stirred under reflux for 90 min. The
reaction mixture was cooled to ambient temperature and the
solvent was removed under reduced pressure. To this residue
were added anhydrous N,N-dimethylformamide (67 mL) and
methyl iodide (3.1 mL, 50 mmol), and the resulting solution
was stirred at ambient temperature for 2 h. The reaction
mixture was diluted by adding CHCl3 (50 mL), washed seven
times with water (50 mL), dried over Na2SO4, filtered and
concentrated to dryness under reduced pressure. The residue
was chromatographed on a column of silica gel (C300, 60 g)
with hexanes-ethyl acetate (60:40. v/v) to give crude product
5b. The crude 5b was dissolved in 2 M NH3/CH3OH (23 mL),
and the resulting solution was stirred at ambient temperature
for 4 days. The solvent was removed under reduced pressure,
and the residue was dissolved in tetrahydrofuran (25 mL). To
this solution was added tetra-n-butylammonium fluoride (890
mg, 3.4 mmol), and the resulting solution was stirred at
ambient temperature for 7 h. To this solution was added CHCl3
(30 mL), washed twice with water (30 mL). The organic layer
was dried over Na2SO4, filtered and concentrated under
reduced pressure. The white precipitation separated during
the evaporation was collected by filtration to give 6b (640 mg,
32%): 1H NMR (DMSO-d6) δ 2.44 (3H, s), 2.49 (1H, m), 2.77-
2.87 (1H, m), 3.51-3.70 (2H, m), 4.04 (1H, m), 4.68-4.69 (1H,
m), 5.16 (2H, s), 5.45 (1H, m), 6.25-6.30 (1H, m), 7.13-7.57
(6H, m, ArH), 8.14 (1H, s), 8.33 (1H, s); 13C NMR (DMSO-d6)
δ 14.9, 36.3, 38.6, 61.8, 72.6, 77.7, 84.2, 85.2, 119.2, 124.8,
125.0, 127.6, 130.7, 132.0, 139.4, 139.8, 148.6, 152.2, 156.0.
Anal. Calcd for C18H21N5O3S2: C, 51.53; H, 5.05; N, 16.69; S,
15.29. Found: C, 51.65; H, 5.02; N, 16.56; S, 15.24.
3′-O-(1,3-Ben zodith iol-2-yl)deoxycytidin e (4c). Compound
3d (600 mg, 1.06 mmol) was dissolved in concentrated NH3/
CH3OH (10 mL), and the resulting solution was stirred at
ambient temperature for 11 h. The solvent was removed under
reduced pressure and the residue trituated with CH3OH. The
white precipitation was collected by filtration to give 4c (316
mg, 78%): 1H NMR (DMSO-d6) δ 1.97-2.07 (1H, m), 2.27-2.49
(1H, m), 3.50 (2H, s), 4.35 (1H, s), 5.06 (1H, t, J ) 4.9 Hz),
5.71 (1H, d, J ) 7.6 Hz), 6.04-6.09 (1H, m), 6.93 (1H, s), 7.14-
7.19 (4H, m), 7.46-7.49 (2H, m), 7.72 (1H, d, J ) 7.6 Hz); 13
C
NMR (DMSO-d6) δ 37.5, 61.1, 77.4, 84.3, 84.85, 84.93, 89.0,
94.06, 94.15, 122.4, 125.5, 135.20, 135.24, 140.8, 154.8, 165.3.
MS m/z calcd for C16H18N3O4S2+: 380.0739, found 380.0739.
5′-O-(ter t-Bu tyld im eth ylsilyl)-3′-O-[[2-(m eth ylth io)p h e-
n yl]th iom eth yl]th ym id in e (5a ). Compound 3a (1.0 g, 2.0
mmol) was dissolved in toluene (20 mL). To this solution were
added tri-n-butyltin hydride (1.3 mL, 5.0 mmol) and AIBN (492
mg, 3.0 mmol). The resulting solution was stirred at 100 °C
for 90 min. The reaction mixture was cooled to ambient
temperature, and the solvent was removed under reduced
pressure. To this residue were added anhydrous N,N-dimeth-
ylformamide (20 mL) and methyl iodide (1.3 mL, 20 mmol),
3′-O-[[2-(Meth ylth io)p h en yl]th iom eth yl]d eoxygu a n o-
sin e (6c). Compound 3c (1.5 g, 2.4 mmol) was dissolved in
benzene (24 mL). To this solution were added tri-n-butyltin
hydride (2.3 mL, 8.4 mmol) and AIBN (788 g, 4.8 mmol). The
resulting solution was stirred under reflux for 2.5 h. The
reaction mixture was cooled to ambient temperature, and the
solvent was removed under reduced pressure. To this residue

Nucleoside Derivatives Having Anti-BVDV Activity
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5271
were added anhydrous N,N-dimethylformamide (24 mL) and
methyl iodide (1.5 mL, 24 mmol), and the resulting solution
was stirred at ambient temperature for 75 min. The reaction
mixture was diluted by adding CHCl3 (50 mL), washed five
times with water (50 mL), dried over Na2SO4, filtered and
concentrated to dryness under reduced pressure. The residue
was chromatographed on a column of silica gel (C300, 40 g)
with hexanes-ethyl acetate (45:55, v/v) to give crude product
5c. The crude 5c was dissolved in 2 M NH3/CH3OH (10 mL)
and the resulting solution was stirred at ambient temperature
for 2 days. The solvent was removed under reduced pressure,
and the residue was dissolved in tetrahydrofuran (10 mL). To
this solution was added tetra-n-butylammonium fluoride (290
mg, 1.1 mmol), and the resulting solution was stirred at
ambient temperature for 20 min. To this solution was added
CHCl3 (30 mL), and it was washed three times with water (30
mL). The organic layer was dried over Na2SO4, filtered and
concentrated under reduced pressure. The white precipitation
separated during the evaporation was collected by filtration
to give 6c (230 mg, 22%): 1H NMR (DMSO-d6) δ 2.18-2.66
(5H, m), 3.54 (2H, m), 3.96 (1H, m), 4.60 (1H, m), 5.08-5.14
(3H, m), 6.04 (1H, dd, J ) 5.9, 8.1 Hz), 6.48 (2H, br), 7.13-
7.55 (4H, m), 7.94 (1H, s), 10.68 (1H, s); 13C NMR (DMSO-d6)
δ 14.9, 36.4, 61.6, 72.5, 77.5, 82.7, 84.8, 116.6, 124.8, 125.0,
127.6, 130.6, 132.0, 135.1, 139.7, 150.7, 153.5, 156.6. MS m/z
calcd for C18H22N5O4S2+: 436.1113, found 436.1125.
The residue was chromatographed on a column of silica gel
(C200, 200 g) with hexane containing 1% triethylamine (v/v)
to give 7 (6.3 g, 85%): 1H NMR (CDCl3) δ 3.20 (1H, s), 6.76
(1H, s), 7.07-7.36 (4H, m); 13C NMR (CDCl3) δ 51.2, 90.9,
+
121.68, 121.71, 125.2, 136.0. MS m/z calcd for C8H9OS2
:
185.0095, found 185.0026.
3′-O-(1-Oxo-1,3-ben zod ith iol-2-yl)th ym id in e (8). Com-
pound 1 (394 mg, 1.0 mmol) was dissolved in anhydrous CH2-
Cl2 (10 mL). To this solution were added m-chlorobenzoic
peroxide (240 mg, 1.0 mmol) and NaHCO3 (84 mg, 1.0 mmol).
The resulting suspension was stirred vigorously at ambient
temperature for 2 h. The reaction mixture was washed three
times with saturated aqueous NaHCO3, and the organic layer
was dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was chromatographed on silica
gel (C200, 12 g) with CHCl3-CH3OH (99:1, v/v) containing
0.5% triethylamine (v/v) to give 8 (191 mg, 47%): 1H NMR
(DMSO-d6) δ 1.76 (3H, s), 2.29 (2H, m), 3.58 (2H, m), 3.92-
3.96 (1H, m), 4.68 (1H, m), 5.17 (1H, m), 6.03 (1H, dd, J )
3.0, 3.0 Hz), 6.53 (1H, s), 7.34-8.08 (5H, m, ArH), 11.30 (1H,
s); 13C NMR (CDCl3-DMSO-d6, 9:1, v/v) δ 12.1, 36.9, 37.2, 61.0,
77.2, 102.3, 102.4, 110.1, 110.2, 124.0, 124.1, 125.95, 125.98,
128.3, 128.4, 133.2, 135.1, 135.2, 139.8, 142.1, 149.99, 150.01,
+
163.6. MS m/z calcd for
C
17H19N2O6S2 411.0685, found
411.0684. IR (KBr) 1055 cm-1 (SO).
3′-O-(1,3-Dioxo-1,3-ben zodith iol-2-yl)th ym idin e (9). Com-
pound 1 (394 mg, 1.0 mmol) was dissolved in anhydrous CH2-
Cl2 (10 mL). To this solution were added m-chlorobenzoic
peroxide (480 mg, 2.0 mmol) and NaHCO3 (168 mg, 2.0 mmol).
The resulting suspension was stirred vigorously at ambient
temperature for 2 h. The reaction mixture was washed three
times with saturated aqueous NaHCO3, and the separated
white precipitate was collected by filtration to give 9 (100 mg,
23%) as a diastreomeric mixture: 1H NMR (DMSO-d6) δ 1.77
(3H, s), 2.27-2.44 (2H, m), 3.64 (2H, m), 4.14 (1H, m), 4.80
(1H, m), 5.21 (1H, s), 6.16 (1H, dd, J ) 8.6, 8.6 Hz), 6.54 (1H,
s), 7.71 (1H, s), 7.85-8.23 (4H, m), 11.33 (1H, s); 13C NMR
(DMSO-d6) δ 12.4, 37.0, 61.3, 83.5, 83.6, 84.4, 85.2, 105.1,
109.5, 109.6, 111.2, 128.4, 129.2, 133.4, 135.8, 135.9, 141.6,
142.97, 143.00, 143.3, 150.27, 150.29, 163.49, 163.51; IR (KBr)
3′-O-[[2-(Meth ylth io)p h en yl]th iom eth yl]-4-N-p iva loyl-
d eoxycytid in e (6d ). Compound 3d (380 mg, 0.7 mmol) was
dissolved in toluene (6.7 mL). To this solution were added tri-
n-butyltin hydride (0.45 mL, 1.7 mmol) and AIBN (330 g, 2.0
mmol). The resulting solution was stirred at 100 °C for 2 h.
The reaction mixture was cooled to ambient temperature, and
the solvent was removed under reduced pressure. The residue
was chromatographed on silica gel (N60, 15 g) with hexanes-
ethyl acetate (70:30, v/v) to remove remaining starting mate-
rial. The fractions containing stanylated intermediated were
collected and concentrated under reduced pressure. To this
residue were added anhydrous N,N-dimethylformamide (6.7
mL) and methyl iodide (417 µL, 6.7 mmol), and the resulting
solution was stirred at ambient temperature for 24 h. The
reaction mixture was diluted by adding CHCl3 (10 mL), washed
five times with water (10 mL), dried over Na2SO4, filtered and
concentrated to dryness under reduced pressure. The residue
was chromatographed on a column of silica gel (C200, 15 g)
with hexanes-ethyl acetate (60:40. v/v) to give crude product
5d . The crude 5d was dissolved in 2 M NH3/CH3OH (2 mL),
and the resulting solution was stirred at ambient temperature
for 22 h. The solvent was removed under reduced pressure,
and the residue was dissolved in CHCl3 (10 mL) and washed
three times with water (10 mL). The organic layer was dried
over Na2SO4, filtered and concentrated under reduced pres-
sure. The residue was chromatographed on a column of silica
gel (C200, 40 g) column with CHCl3-CH3OH (96:4. v/v) to give
6d (75 mg, 30%): 1H NMR (CDCl3) δ 2.42-2.46 (3H, m), 2.95
(1H, br), 3.71-3.93 (2H, m), 4.07 (1H, m), 4.63-4.68 (1H, m),
4.93 (1H, d, J ) 11.6 Hz), 5.12 (1H, d, J ) 11.6 Hz), 5.35 (2H,
2H, br), 5.68 (1H, d, J ) 7.6 Hz), 5.99 (1H, dd, J ) 6.5, 6.5
Hz), 7.10-7.53 (4H, m), 7.67 (1H, d, J ) 7.6 Hz); 13C NMR
(DMSO-d6) δ 14.9, 37.1, 61.4, 72.5, 77.2, 84.4, 85.0, 94.0, 124.8,
125.0, 127.5, 130.6, 132.0, 139.6, 140.7, 154.8, 165.3. Anal.
Calcd for C17H21N3O4S2‚3/10H2O: C, 50.93; H, 5.43; N, 10.48;
S, 16.00. Found: C, 50.99; H, 5.27; N, 10.23; S, 15.96.
1051 cm-1 (SO). MS m/z calcd for C17H19N2O7S2 427.0634,
+
found 427.0638.
3′-O-(1,1,3,3-Tetr a oxo-1,3-ben zod ith iol-2-yl)th ym id in e
(10). Compound 1 (394 mg, 1.0 mmol) was dissolved in
anhydrous CH2Cl2 (10 mL). To this solution were added
m-chlorobenzoic peroxide (983 mg, 4.1 mmol) and NaHCO3
(344 mg, 4.1 mmol). The resulting suspension was stirred
vigorously at ambient temperature for 90 m. Ethyl acetate (10
mL) was added, and the reaction mixture was washed three
times with water (20 mL). The organic layer was dried over
Na2SO4, filtered and concentrated under reduced pressure. The
residue was trituated with CH3OH and C2H5OH, and the
separated white precipitate was collected by filtration to give
10 (418 mg, 91%): 1H NMR (DMSO-d6) δ 1.77 (3H, s), 2.27-
2.48 (2H, m), 3.63 (2H, m), 4.06 (1H, m), 4.23 (1H, m), 6.13
(1H, dd, J ) 4.6, 4.6 Hz), 6.11-6.16 (1H, m), 6.95 (1H, s), 7.71
(1H, s), 8.07-8.29 (4H, m), 11.31 (1H, s); 13C NMR (DMSO-
d6) δ 12.4, 37.0, 61.1, 83.5, 84.3, 85.7, 93.9, 109.6, 123.5, 135.7,
136.0, 136.1, 136.4, 150.3, 163.5; IR (KBr) 1178, 1353 cm-1
+
(SO2). MS m/z calcd for C17H19N2O9S2 459.0532, found
459.0537.
3′-O-(1,3-Ben zod ioxol-2-yl)-5′-O-(ter t-bu tyld im eth ylsi-
lyl)th ym id in e (11). 5′-O-(tert-Butyldimethylsilyl)thymidine
(1.0 g, 2.8 mmol) was dissolved in anhydrous 1,4-dioxane (28
mL). To this solution were added molecular sieves 4A (3.0 g),
2-methoxy-1,3-benzodioxol (4.3 g, 28 mmol) and p-toluene-
sulfonic acid hydrate (1.1 mL, 5.6 mmol). The resulting
mixture was stirred under reflux for 29 h. The reaction mixture
was cooled to ambient temperature and triethylamine (39 mL,
14 mmol) was added. After 15 min, molecular sieves was
removed by filtration and the filtrate was concentrated under
reduced pressure. The residue was dissolved in CHCl3 (30 mL),
washed three times with water (30 mL), dried over Na2SO4,
filtered and concentrated under reduced pressure. The residue
2-Meth oxyben zod ith iol (7). 1,3-Benzodithiolium tetraflu-
oroborate (9.6 g, 40 mmol) was dissolved in anhydrous pyridine
(80 mL). To this solution was added anhydrous methanol (8.1
mL, 200 mmol), and the resulting solution was stirred at
ambient temperature for 1 h. Triethylamine (22 mL, 240
mmol) was added, and the mixture was stirred for 15 min.
The solvents were removed under reduced pressure, and the
residue was dissolved in ethyl acetate (100 mL). The solution
was washed three times with water (100 mL). The organic
layer was collected and concentrated under reduced pressure.

5272 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Seio et al.
was chromatographed on a column of silica gel (C200, 40 g)
with hexanes-ethyl acetate (70:30, v/v) containing 0.5%
pyridine to give 3a (198 mg, 15%) as a white foam: 1H NMR
(CDCl3) δ -0.99, -0.05 (6H, 2s), 0.73 (9H, s), 1.85-2.01 (4H,
m), 2.44-2.52 (1H, m), 3.56 (1H, dd, J ) 1.9, 11.6 Hz), 3.80
(1H, dd, J ) 1.9, 11.6 Hz), 4.19 (1H, m), 4.44 (1H, m), 6.31-
6.36 (1H, m), 6.82-6.96 (5H, m), 7.38 (1H, s), 9.50 (1H, br);
13C NMR (CDCl3) δ -5.7, -5.5, 12.5, 18.2, 25.8, 39.2, 62.9, 73.2,
84.5, 85.5, 108.2, 108.3, 110.9, 117.9, 121.9, 122.0, 134.9, 145.3,
145.5, 150.3, 163.8. MS m/z calcd for C23H33N2O7Si+: 477.2057,
found 477.2064.
3′-O-(1,3-Ben zod ioxol-2-yl)th ym id in e (12). Compound 11
(190 mg, 0.4 mmol) was dissolved in tetrahydrofuran (4 mL).
To this solution was added tetra-n-butylammonium fluoride
(157 mg, 0.6 mmol), and the resulting solution was stirred at
ambient temperature for 30 min. The solvent was removed
under reduced pressure, and the residue was dissolved in
CHCl3 (10 mL) and washed three times with water (10 mL).
The aqueous layer was extracted twice with CHCl3 (10 mL),
and all the organic layer was combined, dried over Na2SO4,
filtered and concentrated under reduced pressure. The residue
was chromatographed on a column of silica gel (C200, 12 g)
with hexanes-ethyl acetate (50:50, v/v) containing 0.5%
triethylamine to give 12 in quantitative yield: 1H NMR
(DMSO-d6) δ 1.75 (3H, s), 2.17-2.34 (2H, m), 3.49-3.62 (2H,
m), 3.96-3.98 (2H, m), 4.58-4.62 (1H, m), 5.15 (1H, t, J ) 4.9
Hz), 6.08-6.13 (1H, m), 6.89-7.05 (4H, m), 7.21 (1H, s), 7.65
(1H, s), 11.3 (1H, s); 13C NMR (CDCl3) δ 12.4, 38.1, 61.7, 73.4,
85.2, 86.4, 108.4, 108.5, 110.9, 118.1, 121.9, 136.8, 145.2, 150.4,
164.0. MS m/z calcd for C17H19N2O7+: 363.1192, found 363.1192.
3′-O-(1-Ben zylb en zim id a zol-2-yl)-5′-O-(ter t-b u t yld im -
eth ylsilyl)th ym id in e (13). 5′-O-(tert-Butyldimethylsilyl)thy-
midine (2.0 g, 5.6 mmol) was dissolved in anhydrous N,N-
dimethylformamide (50 mL). To this solution was added
sodium hydride (470 mg, 12 mmol), and the resulting mixture
was stirred at ambient temperature for 1 h. 1-Benzylbenzimi-
dazol-2-yl (2.7 g, 11 mmol) was added, and the resulting
mixture was stirred for 28 h. The reaction was quenched by
adding CH3OH (10 mL), and the resulting solution was stirred
for 15 min. CH2Cl2 (50 mL) was added, and the solution was
washed four times with water (30 mL), dried over Na2SO4,
filtered and concentrated under reduced pressure. The residue
was chromatographed on a column of silica gel (C200, 60 g)
with hexanes-ethyl acetate (75:25, v/v) to give 13 (1.3 g, 41%)
as a white foam: 1H NMR (CDCl3) δ 0.17, 0.18 (6H, 2s), 0.95
(9H, s), 1.94 (3H, s), 2.24-2.70 (2H, m), 3.96 (1H, dd, J ) 1.9,
11.5 Hz), 4.11 (1H, dd, J ) 1.9, 11.3 Hz), 4.31 (1H, m), 5.17
(1H, s), 6.41 (1H, dd, J ) 5.4, 9.2 Hz), 7.12-7.53 (9H, m), 7.59
(1H, s), 9.38 (1H, br); 13C NMR (CDCl3) δ -5.3, -5.2, 12.6,
18.4, 26.0, 38.5, 45.9, 63.7, 80.9, 84.7, 85.3, 108.7, 111.1, 117.9,
121.2, 126.9, 127.8, 128.8, 133.6, 134.9, 135.7, 139.8, 150.4,
155.7, 163.7. MS m/z calcd for C30H39N2O5Si+: 563.2690, found
563.2685.
3′-O-(Ben zim id a zol-2-yl)-5′-O-(ter t-bu tyld im eth ylsilyl)-
th ym id in e (14). Compound 13 (710 mg, 1.3 mmol) was
dissolved in acetic acid (23 mL). To this solution were added
palladium hydroxide (1.2 g), and the resulting mixture was
stirred under hydrogen atmosphere at ambient temperature
for 5 d. The catalyst was removed by filtration using Celite,
and the filtrate was concentrated under reduced pressure. The
residue was dissolved in CHCl3 (30 mL), washed three times
with water (30 mL), dried over Na2SO4, filtered and concen-
trated under reduced pressure. The residue was chromato-
graphed on a column of silica gel (C300, 20 g) with hexanes-
ethyl acetate (70:30, v/v) to give 14 (375 g, 63%) as a white
foam: 1H NMR (CDCl3) δ 0.10 (6H, s), 0.89 (9H, s), 1.92 (3H,
s), 2.13-2.59 (2H, m), 3.89-4.05 (2H, m), 4.29 (1H, m), 5.56
(1H, m), 6.35 (1H, dd, J ) 4.9, 9.5 Hz), 7.09-7.46 (4H, m),
7.56 (1H, s), 10.03, 10.10 (2H, 2s); 13C NMR (CDCl3) δ -5.42,
-5.37, 12.5, 18.3, 25.9, 38.3, 63.7, 80.5, 84.6, 85.0, 110.0, 111.3,
117.2, 121.3, 132.3, 135.0, 140.6, 150.9, 156.6, 164.2. MS m/z
calcd for C23H33N4O5Si+: 473.2220, found 473.2227.
To this solution was added tetra-n-butylammonium fluoride
(311 mg, 1.2 mmol), and the resulting solution was stirred at
ambient temperature for 90 min. The separated precipitate
was dissolved by adding CHCl3 and CH3OH. To this solution
was added silica gel (C300, 2.5 g), and the solvent was removed
under reduced pressure. The silica gel containing the materials
was placed on
a column of silica gel (C300, 10 g) and
chromatographed with CHCl3-CH3OH (97.5:2.5, v/v) to give
15 (218 mg, 77%): 1H NMR (DMSO-d6) δ 1.79 (3H, s), 2.36-
2.50 (2H, m), 3.71 (1H, dd, J ) 3.2, 11.6 Hz), 3.78 (1H, dd, J
) 3.2, 12.0 Hz), 4.20 (1H, m), 5.31 (1H, br), 6.24-6.30 (1H,
m), 7.01-7.30 (4H, m), 7.79 (1H, s), 11.38 (1H, s), 11.96 (1H,
s); 13C NMR (DMSO-d6) δ 12.4, 36.9, 61.5, 80.1, 83.7, 84.6,
109.7, 120.7, 135.7, 150.3, 157.0, 163.5. MS m/z calcd for
C
17H19N4O5+: 359.1355, found 359.1348.
3′-O-(1,3-Ben zod ith iol-2-yl)-5′-d eoxyth ym id in e (16). 5′-
Deoxythymidine (452 g, 2 mmol) was dissolved in anhydrous
pyridine (10 mL). To this solution were added 1,3-benzodithio-
lium tetrafluoroborate (960 mg, 4.0 mmol), and the resulting
mixture was stirred at ambient temperature for 24 h. Triethyl-
amine (1.7 mL, 12 mmol) was added, and the resulting mixture
was stirred for 15 min. The solvents were removed under re-
duced pressure, and the residue was dissolved in CHCl3 (10
mL), washed three times with water (10 mL), dried over
Na2SO4, filtered and concentrated under reduced pressure. The
residue was chromatographed on a column of silica gel (C200,
20 g) with hexanes-ethyl acetate (65:35, v/v) containing 0.5%
pyridine (v/v) to give 16 (520 mg, 69%) as a white foam. 1H
NMR (CDCl3) δ 1.25 (3H, d, J ) 6.5 Hz), 1.83 (3H, s), 2.00-
2.07 (1H, m), 2.42-2.51 (1H, m), 3.74-3.80 (1H, m), 3.99-
4.03 (1H, m), 6.05-6.10 (1H, m), 6.74 (1H, s), 6.87-7.31 (5H,
m), 9.45 (1H, br); 13C NMR (CDCl3) δ 12.7, 18.5, 38.5, 78.3,
80.0, 84.4, 89.1, 111.0, 121.8, 122.0, 125.6, 134.6, 135.4, 135.7,
150.1, 163.6. MS m/z calcd for C17H19N2O4S2+ : 379.0786, found
379.0784.
3′-O-(1,3-Ben zod ith iol-2-yl)-N-3-ben zoyl-2′,5′-O-bis(ter t-
bu tyld im eth ylsilyl)u r id in e (17). N-3-Benzoyl-2′, 5′-O-bis-
(tert-butyldimethylsilyl)uridine (1.4 g, 2.4 mmol) was dissolved
in anhydrous CH2Cl2 (24 mL). To this solution were added 1,3-
benzodithiolium tetrafluoroborate (1.3 g, 5.3 mmol) and pyri-
dine (864 mL, 11 mmol), and the resulting mixture was stirred
at ambient temperature for 3 days. Triethylamine (1.9 mL,
13 mmol) was added, and the resulting mixture was stirred
for 15 min, washed three times with water (20 mL), dried over
Na2SO4, filtered and concentrated under reduced pressure. The
residue was chromatographed on a column of silica gel (C200,
50 g) with hexanes-ethyl acetate (97:3, v/v) containing 0.5%
triethylamine (v/v) to give 17 (1.5 g, 85%) as a white foam:
1H NMR (CDCl3) δ 0.06-0.13 (12H, m), 0.87, 0.92 (18H, 2s),
3.59-3.63 (1H, m), 3.88-3.95 (2H, m), 4.16-4.26 (2H, m), 5.63
(1H, d, J ) 8.4 Hz), 5.79 (1H, d, J ) 2.7 Hz), 6.80 (1H, s),
7.08-7.92 (9H, m), 7.98 (1H, d, J ) 8.4 Hz); 13C NMR (CDCl3)
δ -5.5, -5.3, -4.7, -4.4, 18.1, 18.5, 25.7, 26.0, 61.4, 72.2, 75.5,
82.6, 89.1, 89.5, 101.6, 121.89, 121.92, 125.7, 126.0, 128.9,
130.4, 131.4, 134.9, 135.3, 135.7, 139.4, 148.8, 162.0, 168.4.
MS m/z calcd for C28H45N2O6S2Si2+: 625.2258, found 625.2255.
3′-O-(1,3-Ben zod ith iol-2-yl)-2′,5′-O-d i(ter t-bu tyld im eth -
ylsilyl)u r id in e (18). Compound 17 (850 mg, 1.2 mmol) was
dissolved in saturated NH3-MeOH (12 mL). The solution was
stirred at ambient temperature for 3.5 h. The solvent was
removed under reduced pressure, and the residue was dis-
solved in CHCl3 (15 mL), washed three times with water (20
mL), dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was chromatographed on a
column of silica gel (C200, 15 g) with hexanes-ethyl acetate
(85:15, v/v) containing 0.5% triethylamine (v/v) to give 18 (709
g, 97%) as a white foam: 1H NMR (CDCl3) δ 0.03-0.14 (12H,
m), 0.882-0.889 (18H, m), 3.56-3.60 (1H, m), 3.85-3.92 (2H,
m), 4.11-4.13 (1H, m), 4.22-4.24 (1H, m), 5.54 (1H, d, J )
8.1 Hz), 5.76 (1H, d, J ) 3.2 Hz), 6.78 (1H, s), 7.05-7.31 (4H,
m), 7.85 (1H, d, J ) 7.8 Hz), 9.13 (1H, br); 13C NMR (CDCl3)
δ - 5.5, - 5.4, - 4.8, - 4.5, 18.1, 18.5, 25.7, 26.0, 61.3, 72.2,
75.3, 82.4, 88.9, 89.5, 101.7, 121.86, 121.92, 125.7, 135.4, 135.7,
3′-O-(Ben zim id a zol-2-yl)th ym id in e (15). Compound 14
(375 mg, 0.8 mmol) was dissolved in tetrahydrofuran (8 mL).

Nucleoside Derivatives Having Anti-BVDV Activity
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5273
+
139.6, 149.9, 163.2. MS m/z calcd for
625.2258, found 625.2255.
C
28H45N2O6S2Si2
:
the separated precipitate was collected by filtration to give 23c
(87 mg, 71%): 1H NMR (DMSO-d6) δ 3.45-3.55 (2H, m), 3.85-
3.86 (1H, m), 4.28-4.29 (1H, m), 4.48-4.53 (1H, m), 4.99-
5.03 (1H, m), 5.34 (1H, d, J ) 4.9 Hz), 5.77 (1H, d, J ) 6.8
Hz), 6.35 (2H, s), 6.94 (1H, s), 7.05-7.37 (4H, m), 7.79 (1H, s),
10.62 (1H, s); 13C NMR (DMSO-d6) δ 61.3, 69.5, 78.2, 84.2, 85.7,
89.4, 116.5, 121.8, 122.1, 125.3, 134.7, 135.2, 153.4, 156.5. MS
m/z calcd for C17H18N5O5S2+: 436.0749, found 436.0748.
3′-O-(1,3-Ben zod ith iol-2-yl)u r id in e (19). Compound 18
(648 mg, 1.0 mmol) was dissolved in tetrahydrofuran (10 mL).
To this solution was added tetra-n-butylammonium fluoride
(678 mg, 2.6 mmol), and the resulting solution was stirred at
ambient temperature for 2.5 h. The solvent was removed under
reduced pressure and the residue was dissolved in CHCl3 (10
mL), and water (10 mL) was added. The separated precipitate
was collected by filtration to give 19 (360 mg, 87%): 1H NMR
(DMSO-d6) δ 3.40-3.57 (2H, m), 3.94 (1H, m), 4.13-4.21 (2H,
m), 5.15 (1H, br), 5.60-5.63 (2H, m), 5.71 (1H, d, J ) 5.7 Hz),
6.95 (1H, s), 7.12-7.46 (4H, m), 7.78 (1H, d, J ) 7.8 Hz), 11.32
(1H, br); 13C NMR (DMSO-d6) δ 60.5, 67.0, 72.5, 75.7, 82.6,
87.4, 89.9, 101.8, 122.2, 122.4, 125.4, 125.5, 135.0, 135.6, 140.2,
150.6, 162.9. MS m/z calcd for C16H17N2O6S2+: 397.0528, found
395.0528.
2′-O-(1,3-Ben zod ith iol-2-yl)cytid in e (23a ). 2′-O-(1,3-Ben-
zodithiol-2-yl)-4-N-pivaloyl-3′-5′-(1,1,3,3-tetraisopropyldisiloxan-
1,3-diyl) cytidine (2.1 g, 2.9 mmol) was dissolved in saturated
NH3-MeOH (30 mL). The solution was stirred at ambient
temperateure for 19 h. The solvent was removed under reduced
pressure, and to the residue was added CHCl3 (30 mL), washed
five times with water (30 mL), dried over Na2SO4, filtered and
concentrated under reduced pressure. The residue was dis-
solved in tetrahydrofuran (30 mL), and to this solution was
added tetra-n-butylammonium fluoride (1.9 g, 7.3 mmol), and
the resulting solution was stirred at ambient temperature for
30 min. The solvent was removed under reduced pressure and
the residue was dissolved in CHCl3 (10 mL), and extracted
six times with water (10 mL). The aqueous layer was collected
and the separated precipitate was collected by filtration to give
23a (850 mg, 77%): 1H NMR ((DMSO-d6) δ 3.45-3.64 (2H,
m), 3.77 (1H, m), 4.02-4.13 (2H, m), 5.21 (1H, d, J ) 5.7 Hz),
5.69 (1H, d, J ) 7.3 Hz), 5.86 (1H, d, J ) 3.5 Hz), 7.80 (1H, d,
J ) 7.3 Hz), 7.07-7.44 (7H, m), 7.80 (1H, d, J ) 7.3 Hz); 13C
NMR (DMSO-d6) δ 59.9, 67.9, 79.5, 83.9, 87.0, 89.5, 93.9, 122.2,
122.3, 125.25, 125.35, 135.2, 135.4, 140.8, 155.0, 165.5. MS
2′-O-(1,3-Ben zodith iol-2-yl)-1-N-pivaloyloxym eth yl-3′,5′-
O-(1,1,3,3-tetr a isop r op yld isiloxa n -1,3-d iyl)in osin e (25).
Compound 24 (1.5 g, 2.4 mmol) was dissolved in anhydrous
CH2Cl2 (24 mL). To this solution were added 1,3-benzodithio-
lium tetrafluoroborate (691 g, 2.9 mmol) and pyridine (465 mL,
5.8 mmol), and the resulting mixture was stirred at ambient
temperature for 6.5 h. Triethylamine (2.0 mL, 14.4 mmol) was
added, and the resulting mixture was stirred for 15 min,
washed three times with water (20 mL), dried over Na2SO4,
filtered and concentrated under reduced pressure. The residue
was chromatographed on a column of silica gel (C200, 40 g)
with hexanes-ethyl acetate (75:25, v/v) containing 0.5%
triethylamine (v/v) to give 25 (1.5 g, 80%) as a white foam:
1H NMR (CDCl3) δ 0.93-1.24 (37H, m), 3.90-4.07 (3H, m),
4.56 (1H, m), 4.77 (1H, dd, J ) 5.9, 9.5 Hz), 5.86-5.97 (3H,
m), 6.77-7.20 (5H, m), 7.82, 7.92 (2H, 2s); 13C NMR (CDCl3)
δ 12.6, 12.7, 13.0, 13.4, 16.9, 17.1, 17.19, 17.21, 17.27, 17.31,
17.4, 26.9, 38.9, 59.4, 67.5, 68.6, 76.7, 81.2, 89.4, 89.8, 121.5,
121.6, 124.8, 124.9, 125.0, 134.7, 135.5, 139.7, 146.2, 147.4,
155.5, 178.2. MS m/z calcd for C35H53N4O8S2Si2+: 777.2843,
found 777.2840.
2′-O-(1,3-Ben zod ith iol-2-yl)in osin e (26). Compound 25
(1.5 g, 1.9 mmol) was dissolved in saturated NH3-MeOH (20
mL). The solution was stirred at ambient temperateure for 13
h. The solvent was removed under reduced pressure, and the
residue was dissolved in tetrahydrofuran (20 mL). To this solu-
tion was added tetra-n-butylammonium fluoride (1.26 g, 4.8
mmol), and the resulting solution was stirred at ambient tem-
perature for 45 min. The solvent was removed under reduced
pressure, and the residue was dissolved in CHCl3 (20 mL) and
washed three times with water (20 mL). The aqueous layer
was extracted seven times with CHCl3-pyridine (7:3, v/v), and
all the organic extracts were combined. The solvent was remov-
ed under reduced pressure. The residue was trituated with
CH3OH and diethyl ether, and the separated precipitate was
collected by filtration to give 26 (470 mg, 58%): 1H NMR
(DMSO-d6) δ 3.33-3.57 (2H, m), 3.90 (1H, m), 4.30 (1H, m),
4.56-4.60 (1H, m), 5.02 (1H, br), 5.39 (1H, br), 5.94 (1H, d, J
) 5.9 Hz), 7.00-7.33 (5H, m), 7.89, 8.18 (2H, 2s), 12.35 (1H,
br); 13C NMR (DMSO-d6) δ 61.2, 69.6, 78.1, 85.8, 86.1, 89.4,
+
m/z calcd for C16H18N3O5S2 396.0688, found 396.0687.
2′-O-(1,3-Ben zod it h iol-2-yl)a d en osin e (23b ). 2′-O-(1,3-
Benzodithiol-2-yl)-6-N-pivaloyl-3′-5′-(1,1,3,3-tetraisopropyldisi-
loxan-1,3-diyl)adenosine (1.9 g, 2.6 mmol) was dissolved in
saturated NH3-MeOH (25 mL). The solution was stirred at
ambient temperateure for 2 days. The solvent was removed
under reduced pressure, and the residue was dissolved in
tetrahydrofuran (26 mL). To this solution was added tetra-n-
butylammonium fluoride (1.7 g, 6.4 mmol), and the resulting
solution was stirred at ambient temperature for 30 min. The
solvent was removed under reduced pressure, the residue was
dissolved in CHCl3 (10 mL), and water (10 mL) was added.
The separated precipitate was collected by filtration to give
23b (980 mg, 92%): 1H NMR ((DMSO-d6) δ 3.41-3.60 (2H,
m), 3.94 (1H, m), 4.33 (1H, m), 4.66 (1H, m), 5.38 (1H, d, J )
4.6 Hz), 5.42-5.46 (1H, m), 5.96 (1H, d, J ) 6.2 Hz), 7.00-
7.38 (6H, m, ArH), 7.96 (1H, s), 8.21 (1H, s); 13C NMR (DMSO-
d6) δ 61.6, 67.0, 77.4, 86.3, 86.5, 89.3, 119.3, 121.5, 122.0,
125.199, 125.240, 134.6, 135.1, 139.9, 148.6, 152.1, 156.0. MS
m/z calcd for C17H18N5O4S2+: 420.0800, found 420.0796.
2′-O-(1,3-Ben zod ith iol-2-yl)gu a n osin e (23c). 2′-O-(1,3-
Benzodithiol-2-yl)-2-N-benzoyll-3′-5′-(1,1,3,3-tetraisopropyld-
isiloxan-1,3-diyl)guanosine (220 mg, 0.28 mmol) was dissolved
in saturated NH3-MeOH (3 mL). The solution was stirred at
ambient temperateure for 24 h. The solvent was removed
under reduced pressure, and the residue was dissolved in
tetrahydrofuran (2 mL) and N,N-dimethylformamide (1 mL).
To this solution was added tetra-n-butylammonium fluoride
(183 mg, 0.7 mmol), and the resulting solution was stirred at
ambient temperature for 30 min. The solvent was removed
under reduced pressure, and the residue was dissolved in
CHCl3 (10 mL) and washed three times with water (10 mL).
The aqueous layer was extracted six times with CHCl3-
isopropyl alcohol (7:3, v/v), and all the organic extracts were
combined. The solvent was removed under reduced pressure.
The residue was trituated with CH3OH and diethyl ether, and
121.6, 122.0, 124.5, 125.2, 134.7, 135.2, 138.9, 145.6, 147.8,
+
156.4. MS m/z calcd for
C
17H17N4O5S2 421.0640, found
421.0645.
4-N-Meth oxyca r bon ylcytid in e (27). Cytidine (2.4 g, 10.0
mmol) was rendered anhydrous by repeated coevaporation
with anhydrous pyridine and finally dissolved in anhydrous
acetonitrile (100 mL). Hexamethyldisilazane (6.35 mL, 30.0
mmol) and catalytic amount of chlorotrimethylsilane was
added, and the resulting solution was stirred at 60 °C for 1 h.
The reaction mixture was cooled to ambient temperature, and
the solvents were removed under reduced pressure. The
residual hexamethyldisilazane was removed by repeated co-
evaporation with anhydrous pyridine and finally dissolved in
anhydrous CH2Cl2 (100 mL). To this solution was added
anhydrous pyridine (1.45 mL, 18 mmol), and methyl chloro-
formate (1.2 mL, 15 mmol) was added dropwise at 0 °C. The
reaction mixture was warmed to room temperature and stirred
for 15 min. The reaction was quenched by adding small
amount of water, and the reaction mixture was diluted with
CHCl3 (50 mL) and washed twice with saturated aqueous
NaHCO3 (50 mL). The aqueous layer was extracted with CHCl3
(50 mL), and all the organic extracts were combined, dried over
Na2SO4, filtered and concentrated under reduced pressure. To
this residue was added concentrated NH3-pyridine (1:1, v/v,
100 mL), and the solution was stirred at ambient temperature
for 50 min. The solvents were removed under reduced pres-

5274 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Seio et al.
sure, and the residue was dissolved in water (50 mL). The
aqueous solution was washed twice with chloroform (50 mL),
and the organic layer was extracted with water (30 mL). All
the water extracts were combined and concentrated under
reduced pressure. The separated precipitate was collected with
filtration to give 27 (2.75 g, 80%): 1H NMR (DMSO-d6) δ 3.55-
3.76 (3H, m), 3.89-3.95 (3H, m), 5.03 (1H, d, J ) 5.1 Hz), 5.14
(1H, t, J ) 4.9 Hz), 5.47 (1H, d, J ) 4.6 Hz), 5.76 (1H, d, J )
2.4 Hz), 7.01 (1H, d, J ) 7.6 Hz), 8.39 (1H, d, J ) 7.6 Hz),
10.74 (1H, s); 13C NMR (DMSO-d6) δ 52.5, 60.0, 68.7, 74.4, 84.2,
and 100 µg/mL streptomycin (culture medium). MDBK cells
(5 × 105 cells/ml) were seeded in a six-well plastic plate and
incubated overnight at 37 °C. Then, culture medium was
removed, and the cells were infected with approximately 100
focus forming unit of BVDV (nose strain) and incubated in the
presence of various concentrations of the test compounds. After
a 24-h incubation, the virus inoculum and test compound were
removed, and the monolayers were washed with phosphate-
buffered saline, overlaid with culture medium containing
bactoager, and further incubated. After 24 h, the number of
viral foci was counted microscopically.
Cytotoxicity Assa y. MDBK cells (2 × 104/well) were seeded
in a 96-well plate in the presence of various concentrations of
the test compound and incubated at 37 °C. After a 3-day
incubation, the number of viable cells was measured in a
colorimetric assay using a water-soluble tetrazolium dye.
90.0, 94.0, 144.9, 153.6, 154.3, 162.6. MS m/z calcd for
+
C
11H16N3O7 302.0988, found 302.0987.
4-N-Meth oxyca r bon yl-3′,5′-O-(1,1,3,3-tetr a isop r op yld -
isiloxa n -1,3-d iyl)cytid in e (28). Compound 27 (1.4 g, 4.7
mmol) was rendered anhydrous by repeated coevaporation with
anhydrous pyridine and finally dissolved in anhydrous pyridine
(45 mL). 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxan (1.8 mL,
5.6 mmol) was added, and the resulting solution was stirred
at ambient temperature for 3 h. Water was added, the reaction
mixture was stirred for 15 min, and the solvents were removed
under reduced pressure. The residue was dissolved in CHCl3
(50 mL), washed three times with saturated aqueous NaHCO3
(50 mL), dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was chromatographed on a col-
umn of silica gel (C200, 50 g) with hexanes-ethyl acetate
(45:55, v/v) to give 28 (2.0 g, 81%) as a white foam: 1H NMR
(CDCl3) δ 0.95-1.37 (28H, m), 3.11 (1H, br), 3.78 (3H, s), 3.98
(1H, dd, J ) 2.2, 13.2 Hz), 4.16-4.30 (4H, m), 5.79 (1H, s),
7.19 (1H, d, J ) 7.3 Hz), 7.83 (1H, br), 8.15 (1H, d, J ) 7.3
Hz); 13C NMR (CDCl3) δ 12.43, 12.92, 12.94, 13.37, 16.82,
16.90, 16.97, 17.28, 17.30, 17.39, 17.47, 53.04, 60.00, 68.45,
74.96, 77.20, 81.85, 91.59, 94.42, 144.05, 152.79, 154.59,
Ack n ow led gm en t. This work was supported by
Grant-in-Aid for Scientific Research on Priority Areas
(C) “Medical Genome Science" from the Ministry of
Education, Culture, Sports, Science and Technology of
J apan and by CREST of J ST (J apan Science and
Technology Agency). We would like to express our
sincere thanks to one of the referees for his or her
helpful suggestion that the BDT-modified nucleoside
might work as inhibitors of the NTP/helicase activities
of the BVDV virus and also for his or her kind informa-
tion of the related papers cited as refs 12-14.
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C NMR
spectra of all of the newly synthesized compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
+
162.48. MS m/z calcd for C23H42N3O8Si2 544.2510, found
544.2501.
2′-O-(1,3-Ben zod ith iol-2-yl)-4-N-m eth oxyca r bon yl-3′,5′-
O-(1,1,3,3-tetr a isop r op yld isiloxa n -1,3-d iyl)cytid in e (29).
Compound 28 (1.5 g, 2.7 mmol) was dissolved in anhydrous
CH2Cl2 (27 mL). To this solution were added 1,3-benzodithio-
lium tetrafluoroborate (994 mg, 2.76 mmol) and pyridine (668
µL, 8.3 mmol), and the resulting mixture was stirred at
ambient temperature for 4 h. Triethylamine (2.5 mL, 16.6
mmol) was added, and the resulting mixture was stirred for
15 min, washed three times with water (30 mL), dried over
Na2SO4, filtered and concentrated under reduced pressure. The
residue was chromatographed on a column of silica gel (C200,
50 g) with hexanes-ethyl acetate (70:30, v/v) containing 0.5%
triethylamine (v/v) to give 29 (1.7 g, 89%) as a white foam:
1H NMR (CDCl3) δ 0.87-1.03 (28 H, m), 3.78 (3H, s), 3.86-
3.91 (1H, m), 4.11-4.28 (4H, m), 5.80 (1H, s), 7.00-7.35 (5H,
m), 8.17 (1H, d, J ) 7.6 Hz), 8.72 (1H, br); 13C NMR (CDCl3)
δ 12.4, 12.8, 12.9, 13.4, 16.7, 16.8, 17.0, 17.1, 17.3, 17.39, 17.44,
53.1, 59.2, 67.0, 77.2, 79.5, 81.9, 89.4, 90.7, 94.7, 122.3, 122.4,
125.0, 125.1, 134.9, 135.8, 143.5, 152.9, 154.5, 162.8. MS m/z
Refer en ces
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+
calcd for C30H46N3O8S2Si2 696.2265, found 696.2264.
2′-O-(1,3-Ben zod ith iol-2-yl)-4-N-m eth oxyca r bon ylcyti-
d in e (30). Compound 29(1.6 g, 2.3 mmol) was dissolved in
tetrahydrofuran (20 mL). To this solution was added tetra-n-
butylammonium fluoride (1.5 g, 5.7 mmol), and the resulting
solution was stirred at ambient temperature for 15 min. The
solvent was removed under reduced pressure, the residue was
dissolved in ethyl acetate (10 mL), and water (10 mL) was
added. The separated precipitate was collected by filtration
to give 38 (730 mg, 71%): 1H NMR (DMSO-d6) δ 3.50-3.55
(1H, m), 3.69 (4H, m), 3.82-3.85 (1H, m), 4.04-4.11 (1H, m),
4.18-4.21 (1H, m), 5.12 (1H, m), 5.26 (1H, s, J ) 5.9 Hz), 5.93
(2H, d, J ) 2.7 Hz), 7.02 (1H, d, J ) 7.6 Hz), 7.07-7.46 (4H,
m), 8.32 (1H, d, J ) 7.6 Hz), 10.81 (1H, br); 13C NMR (DMSO-
d6) δ 52.6, 59.4, 67.5, 79.8, 84.1, 87.7, 89.7, 94.3, 122.3, 125.4,
135.1, 135.5, 144.5, 153.7, 154.2, 162.8. Ms m/z calcd for
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+
C
18H20N3O7S2 454.0743, found 454.0750.
An ti-BVDV Assa y. Madin-Darby bovine kidney (MDBK)
cells were grown in Dulbecco’s modified Eagle’s medium
(Gibco/BRL, Gaithersburg, MD) supplemented with 10% heat-
inactivated horse serum (Gibco/BRL), 100 U/mL penicillin G,

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