A facile, alternative synthesis of 4'-thioarabinonucleosides and their biological activities

Article abstract of DOI:10.1021/jm9701536

4'-Thioarabinonucleosides, which are potential antiviral agents, were synthesized from D-glucose. 1,4-Anhydro-4-thioarabitol (8), which can be derived from diacetone glucose in nine steps, was subjected to Pummerer rearrangement after protection of the hy

Full text of DOI:10.1021/jm9701536

J . Med. Chem. 1997, 40, 2177-2183
2177
A F a cile, Alter n a tive Syn th esis of 4′-Th ioa r a bin on u cleosid es a n d Th eir
Biologica l Activities
Yuichi Yoshimura,*,† Mikari Watanabe,† Hiroshi Satoh,† Noriyuki Ashida,† Katsushi Ijichi,† Shinji Sakata,†
Haruhiko Machida,† and Akira Matsuda‡
Research and Development Division, Yamasa Corporation, 2-10-1 Araoicho, Choshi, Chiba 288, J apan, and Faculty of
Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060, J apan
Received March 10, 1997X
4′-Thioarabinonucleosides, which are potential antiviral agents, were synthesized from
D-glucose. 1,4-Anhydro-4-thioarabitol (8), which can be derived from diacetone glucose in nine
steps, was subjected to Pummerer rearrangement after protection of the hydroxyl groups to
give 1-O-acetyl-4-thioarabinose (11), which was condensed with nucleobases to give 4′-
thioarabinonucleosides. The 5-substituted-4′-thioaraU (6a -e) derivatives showed anti-HSV-1
activity (ED50 ) 0.43-3.50 µg/mL). 4′-ThioaraG (6h ) and 2,6-diaminopurine 4′-thioarabino-
nucleoside (4′-thioaraDAP, 6g) showed antiviral activity against several herpes viruses and
were particularly potent against human cytomegalovirus (0.010 and 0.022 µg/mL, respectively).
Ch a r t 1
In tr od u ction
The development of new effective antiviral agents is
essential for overcoming diseases that are caused by
viruses, such as acquired immunedeficiency syndrome
(AIDS). The first effective antiviral agents were 5-sub-
stituted-2′-deoxyuridines, such as 5-iodo-2′-deoxyuri-
dine1 (5-IdUrd, 1; Chart 1) and 5-(trifluoromethyl)-2′-
deoxyuridine2 (5-CF3dUrd, 2), which are used for the
treatment of herpes simplex virus (HSV) infections.
Since then, many efforts have been made to synthesize
various nucleoside antimetabolites. As a consequence,
some 2′-modified derivatives, e.g., 5-(bromovinyl)-1-(â-
D-arabinofuranosyl)uracil3 (BVaraU, 3) and 5-iodo-1-(2-
deoxy-2-fluoro-â-D-arabinofuranosyl)cytosine4 (FIAC, 4),
have been shown to have potent anti-HSV activities.
The former is an arabinosyl nucleoside, and the latter
is a unique fluorine-containing nucleoside. Further-
more, both derivatives have 2′-“up” stereochemistry
(arabino configuration), which is important for the
recognition of virus-encoded thymidine kinase, but not
host kinases.
In 1991, another novel class of antiviral agents was
independently reported by Walker5 and Secrist.6 The
2′-deoxy-4′-thionucleosides 5, 4′-thio congeners of the
first generation of antivirals described above, were
shown to have potent antiviral activities and cytotox-
icities.5,6 These findings triggered the further investi-
gation of 4′-thionucleosides as potential therapeutic
agents for treating virus infection and cancer. Although
there were reports of the synthesis of some 4′-thio-
nucleosides before 1991,7-11 most of these studies
focused only on their antitumor activities.9,10 Welcome’s
group recently illustrated that 2′-deoxy-4′-thiopurine
nucleosides possess potent anti-human cytomegalovirus
activity12 and 2′-deoxy-4′-thiopyrimidine nucleosides
possess anti-HSV-1 activity.13
agents: some 2′-modified 4′-thionucleosides, such as 4′-
thioarabinonucleosides and 2′-deoxy-2′-fluoro-4′-thio-
arabinonucleosides, which represent the 4′-thio coun-
terparts mentioned above, could be promising. Although
the synthesis of the former group has been reported
previously,8,9,11 their biological effects, including their
antiviral activities, remain unknown, except for those
of 4′-thioaraC.9 Recently, we explored a new strategy
for the synthesis of 2′-substituted-4′-thionucleosides.14
This method should be applicable to the synthesis of 4′-
thioarabinonucleosides. Hence, we report here a facile,
alternative synthesis of 4′-thioarabinonucleosides 6,
beginning from D-glucose. Their antiviral activities are
also discussed.15
Resu lts a n d Discu ssion
Ch em istr y. We previously reported that 1,4-anhy-
dro-4-thioarabitol (8) could be derived from diacetone
glucose (7) in nine steps.14 Compound 8, a versatile
intermediate for the synthesis of the target nucleosides,
was benzylated to give the tribenzyl derivative 9 in good
yield. Previously, we reported the trimethysilyl triflate
(TMSOTf)-catalyzed Pummerer-type glycosylation reac-
These results drew our attention to the design and
synthesis of 4′-thionucleosides as potential antiviral
* Corresponding author: tel, 479-22-0095, ext. 384; fax, 479-22-9821;
e-mail, yuichi96@choshinet.or.jp.
† Yamasa Corp.
‡ Hokkaido University.
X Abstract published in Advance ACS Abstracts, J une 15, 1997.
S0022-2623(97)00153-2 CCC: $14.00 © 1997 American Chemical Society

2178 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Yoshimura et al.
Sch em e 1
Ta ble 1. Chemical Yield and R,â-Selectivity of Glycosylation
Reaction
Sch em e 2
compd
no.
yield from 11 (%)
R/â
R
(isolated yield of â-anomer, %) ratioa
6a
6b
6c
6d
6e
Et
Me
I
35 (11)
50 (14)
45 (22)
37 (11)
52 (13)
2.30
2.16
1.58
2.36
2.22
-CHdCHCl-(E)
-CHdCHBr-(E)
a
R,â-Ratios were determined by HPLC analysis of free nucleo-
sides.
tion using sulfoxides as glycosyl donors for the prepara-
tion of 4′-thioDMDC and 4′-thiogemcitabine.14 How-
ever, application of this approach to sulfoxide 10,
obtained from 9 by m-CPBA oxidation, gave the R-4′-
thionucleoside exclusively (data not shown). Therefore,
we selected 1-acetate 11, in place of 10, for the glyco-
sylation reaction. The tribenzyl derivative 9 was sub-
jected to Pummerer rearrangement (m-CPBA oxidation
followed by treatment with acetic anhydride) to give 11
as an anomeric mixture (1.7:1). Next, Vorbru¨ggen
glycosylation16 between 11 and persilylated 5-ethyl-
uracil was investigated. The reaction of silylated 5-ethyl-
uracil with 11 in the presence of TMSOTf in 1,2-di-
chloroethane proceeded efficiently (Scheme 1). How-
ever, the resulting R,â-anomers of protected nucleosides
12a could not be separated using a silica gel column.
An R,â-mixture of 12a was deprotected using boron
trichloride (BCl3), and the resulting anomers were
separated by HPLC to give R- and â-5-ethyl-4′-thioara-
binouridine (6a ) in yields of 24% and 11% from 11,
respectively. The other 5-substituted-pyrimidine ara-
binonucleosides (6b-e) were synthesized in a similar
manner, and the results are summarized in Table 1. The
structure of these 4′-thioarabinonucleosides (6b-e) was
confirmed by instrumental analysis. The anomeric
configuration of these nucleosides was easily assessed
by comparing the chemical shifts of H-4′ protons; those
of R-anomers were shifted downfield due to the deshield-
ing effect of the C-2 keto group of the uracil moiety.14,17
The R,â-ratios of the glycosylation reactions were ap-
proximately 1.5-2.4:1, and R-isomers were predominant.
We next tried to synthesize purine analogues using
11. In contrast to pyrimidine derivatives, the reactions
between 11 and purine derivatives were complicated.
As a first target, we tried to synthesize 4′-thioarabino-
syladenine (4′-thioaraA). The glycosylation reaction of
pertrimethylsilylated adenine with 11, under the condi-
tions described above (method A), gave 4′-thionucleo-
sides 13, which were debenzylated to give R- and â-14
in yields of 15% and 7.3%, respectively. The results of
the instrumental analysis of R- and â-14 were consistent
with the desired structures, except for their UV spectra,
which showed red shift absorption at 275 and 273 nm,
respectively. This red shift in their UV spectra relative
to that of adenosine suggests that glycosylation had
occurred at the 7-position of the adenine base. None of

Alternative Synthesis of 4′-Thioarabinonucleosides
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2179
Ta ble 2. Antiviral Activities of Pyrimidine 4′-Thioarabinonuleosides
anticell proliferative activity,
antiviral activities, ED50 (µg/mL)
IC50 (µg/mL)
compd no.
R
HSV-1a,e
HSV-2b,e
VZVc,e
HCMVd,e
CCRF-HSB-2 f
R-6a
â-6a
R-6b
â-6b
R-6c
â-6c
R-6d
â-6d
R-6e
â-6e
Et
Et
Me
Me
I
>100
0.43
>100
6.5
>50
>50
>50
>50
44
>50
>50
>50
>50
>50
>50
>100
>100
42
>100
>100
>100
>100
>100
>100
>100
>50
>50
>100
>100
0.77
4.6
6.6
>50
27.1
>50
>50
>50
0.20
ND
0.0013
2.7
>100
3.50
>100
13.6
>100
71
I
-CHdCHCl-(E)
-CHdCHCl-(E)
-CHdCHBr-(E)
-CHdCHBr-(E)
>100
0.97
>100
0.82
>100
58
araC
BVAU
acyclovir
ND
0.036
0.14
ND
62
0.23
ND
>50
6.9
0.0086
>100
>100
a
b
d
VR-3 strain. HSV-2 MS strain. c VZV Oka strain. HCMV AD 169 strain. e Plaque reduction assay. f MTT assay.
Ch a r t 2
Sch em e 3
the 9-glycosylated products were found in the mixture
after careful TLC analyses. We eventually found that
the reaction of adenine, instead of pertrimethylsilylated
adenine, and 1-O-acetyl-4-thioarabinose (11) in the
presence of TMSOTf as a Lewis acid in acetonitrile18
(method B) followed by deprotection of the product 15f
effectively gave R- and â-9-(4-thioarabinosyl)adenine (6f)
in yields of 11% and 18% from 11, respectively. Their
1
UV as well as H NMR spectra and other instrumental
analyses support the structure (Scheme 2). It is note-
worthy that the formation of â-4′-thioaraA (â-6f), in
sharp contrast to the pyrimidine derivatives, was pre-
dominant even though the total yield of the products
decreased. The same conditions were used for the
reaction with thymine. However, none of the desired
4′-thioaraT was obtained (data not shown).
To prepare the guanine derivative, we first attempted
to use 2-amino-6-chloropurine as a nucleobase for the
glycosylation reaction. This reaction gave an anomeric
mixture of the products, which were deprotected by BCl3
to give 17 in a low yield. The structure of 17 shown in
Chart 2 was determined based on the following data:
(1) In the 1H NMR spectrum, anomeric protons (5.26
and 5.51 ppm) were each coupled with a D2O exchange-
able proton. (2) Both of the anomers, which were
separated by HPLC, returned to an initial mixture when
kept in an aqueous solution. (3) In the FAB mass
spectra, both compounds showed molecular ion peaks
at m/ z 318. These results showed that glycosylation
of 2-amino-6-chloropurine unexpectedly occurred at the
2-amino position.
yield, which was deprotected to give the R- and â-ano-
mers of 6g (5% and 9% yields from 11, respectively).
After separation by HPLC, the isolated â-6g was deami-
nated with adenosine deaminase to give 4′-thioara-
binoguanosine 6h in 97% yield from 6g (Scheme 3).
Biologica l Activities. The antiviral effects of the
synthesized pyrimidine and purine 4′-thioarabinonu-
cleosides are summarized in Tables 2 and 3. None of
the pyrimidine derivatives were cytotoxic against hu-
man T-cell leukemia cells (CCRF-HSB-2 cells) up to 100
µg/mL, except for R-4′-thioaraT (R-6a ), which showed
very weak cytotoxicity toward CCRF-HSB-2 (IC50 ) 42
µg/mL). All of the â-anomers of the 5-substituted 4′-
thioaraU derivatives tested in this study were active
against HSV-1 (IC50’s ranging from 0.43 to 3.50 µg/mL)
but showed weak activity against HSV-2 and no activity
against HCMV. The 5-(halogenovinyl)uracil congeners
6d ,e exhibited very weak activity against HSV-2, as did
BVaraU. These results show that â-stereochemistry
Therefore, instead of using 2-amino-6-chloropurine,
we selected 2,6-diaminopurine as a nucleobase for the
glycosylation reaction. The reaction between 2,6-diami-
nopurine and 11 under the conditions described for the
synthesis of 4′-thioaraA gave an anomeric mixture of
2,6-diamino-9-(4-thioarabinosyl)purine (18g) in a low

2180 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Ta ble 3. Antiviral Activities of Purine 4′-Thioarabinonucleosides
Yoshimura et al.
anticell proliferative activity,
antiviral activities, ED50 (µg/mL)
IC50 (µg/mL)
compd no.
B
HSV-1a,e
HSV-2b,e
VZVc,e
HCMVd,e
CCRF-HSB-2 f
R-6f
â-6f
R-6g
â-6g
6h
A
A
DAP
DAP
G
>100
18.4
>100
>100
13.5
>100
>50
1.85
>50
0.11
0.11
>50
>100
14.8
>100
1.36
>50
0.52
0.49
0.40
0.59
0.022
0.010
0.20
0.29
araA
araDAP
araG
17.1
49
55
6.6
>100
78
1.17
9.2
10.1
0.21
6.45
12.9
16.8
0.21
12.9
1.7
2.8
DHPG
0.016
0.039
17.0
a
b
d
VR-3 strain. HSV-2 MS strain. c VZV Oka strain. HCMV AD 169 strain. e Plaque reduction assay. f MTT assay.
around the glycosidic conformation is essential for
phosphorylation by both virus-encoded and cellular
kinases. On the other hand, 5(E)-(bromovinyl)-4′-thio-
araU (â-6e) showed anti-VZV activity 10 times higher
than acyclovir but 150 times lower than BVaraU. 5-
(E)-(Chlorovinyl)-4′-thioaraU (â-6d ), unlike â-6e, did not
show any activity against VZV, whereas it did have
significant activity against HSV-1. This is consistent
with reports regarding 5-(chlorovinyl)-2′-deoxy-4′-thiou-
ridine.13 In 4′-oxy derivatives, however, both 5-(bromo-
vinyl)- and 5-(chlorovinyl)araU were almost equally
active against both VZV and HSV-1.3 These differences,
which could be related to the substrate specificity of the
kinase, require further investigation.
â-Purine 4′-thioarabinonucleosides showed marked
biological activities. All of the R-nucleosides were
inactive, as in the case of the pyrimidine series described
above. 4′-ThioaraA (â-6f), which behaved like araA, had
moderate activity against VZV and HCMV and rela-
tively weak activity against HSV-1, HSV-2, and the
growth of CCRF-HSB-2 cells (Table 3). On the other
hand, both the guanine (â-6h ) and diaminopurine (â-
6g) derivatives had potent anti-HCMV activities (ED50
) 0.010 and 0.022 µg/mL, respectively). It is noteworthy
that â-6h and â-6g were highly active antiviral agents,
although both parental 4′-oxy congeners (araG and
araDAP) were inactive or had only relatively weak
activities. Thus, it appears as though the “4′-thio”
substituents of â-6h and â-6g have greatly improved
antiviral profiles. 4′-ThioaraG (â-6h ) was 20 times and
the diamino derivative (â-6g) 10 times more potent than
ganciclovir, although both compounds were highly cy-
totoxic to CCRF-HSB-2 (IC50 ) 0.29 and 0.20 µg/mL,
respectively). It is likely that 4′-thioaraGTP is an active
metabolite in both cases because the diamino derivative
â-6g was easily converted to 4′-thioaraG (â-6h ) by
adenosine deaminase. Recently, 2′-deoxy-4′-thio-2-
amino-6-subsituted-purine nucleosides were reported to
have potent anti-human hepatitis B virus and HCMV
activities.12 Among these, 2′-deoxy-4′-thioguanosine
was also reported to be highly toxic to leukemic cells.12
Although compounds â-6g and â-6h were cytotoxic, they
had over a 10-fold greater effect on HCMV. Thus, it is
possible that purine 4′-thioarabinonucleosides may lead
to a new anti-HCMV agent. Future studies are needed
to identify less cytotoxic and more selective active
derivatives.
derivatives of 4′-thioarabinonucleosides exhibited potent
anti-HCMV activity and cytotoxicity. Although 4′-
thioaraG was cytotoxic, it had a 30-fold greater effect
on HCMV. Thus, it could lead to a new class of anti-
HCMV agents.
Exp er im en ta l Section
Gen er a l Meth od s. Physical data were measured as fol-
lows: Melting points were determined on a Yanagimoto MP-
500D micromelting point apparatus and are uncorrected. 1H
NMR spectra were recorded on a J EOL J NM-GSX-400 instru-
ment in CDCl3 or DMSO-d6 as the solvent with tetramethyl-
silane as internal standard. UV spectra were recorded with
a Shimadzu UV-160A spectrophotometer. Low- and high-
resolution mass spectra were taken on a J EOL J MS-AX500
spectrometer. THF was freshly distilled under argon from
sodium/benzophenone before use, whereas dichloromethane
was distilled from calcium hydride. All the reactions described
below were performed under argon atmosphere and monitored
by thin layer chromatography (TLC). TLC and preparative
TLC were carried out on Merck precoated plates (Kieselgel
60F254). Silica gel for chromatography was Merck Kieselgel
60. BVaraU, acyclovir, ganciclovir, araA, araDAP, and araG
were synthesized in the laboratory of Yamasa Corp. Adenosine
deaminase (from bovine spleen, EC 3.5.4.4) was purchased
from Sigma Co., Ltd., and used without further purification.
1,4-An h ydr o-2,3,5-tr i-O-ben zyl-4-th io-D-ar abitol (9). So-
dium hydride (60%, 4.16 g, 104 mmol) was added to a solution
of 1,4-anhydro-3-O-benzyl-4-thio-D-arabitol (8)14 (5.0 g, 20.8
mmol) in DMF (100 mL) at 0 °C, and the mixture was stirred
for 1 h. To this mixture were added DMF (50 mL) and benzyl
chloride (16.8 mL, 146 mmol), and the whole was stirred at
room temperature overnight. The reaction mixture was
poured into ice water (150 mL) and extracted with AcOEt (×2).
The organic phase was washed with brine and dried (Na2SO4).
The filtrate was concentrated under reduced pressure, and the
residue was purified on column chromatography over silica
gel (14% AcOEt in hexane) to give 9 (5.54 g, 63%, syrup): 1H
NMR (CDCl3) δ 7.35-7.25 (15H, m), 4.90 (1H, m), 4.72-4.45
(6H, m), 4.11 (1H, m), 3.69 (1H, dd, J ) 7.3, 8.8 Hz), 3.56 (1H,
ddd, J ) 3.4, 6.4, 7.3 Hz), 3.50 (1H, dd, J ) 6.4, 8.8 Hz), 3.08
(dd, 1H, J ) 4.9, 11.2 Hz), 2.90 (1H, dd, J ) 4.4, 11.2 Hz); EI
MS m/ z 420 (M+). Anal. (C26H28O3S) C, H.
1-O-Ace t yl-2,3,5-t r i-O-b e n zyl-D -4-t h ioa r a b in ofu r a -
n ose (11). To a solution of 9 (2.88 g, 6.85 mmol) in CH2Cl2
(40 mL) was added dropwise a solution of m-CPBA (80%, 1.48
g, 6.85 mmol) in CH2Cl2 (40 mL) at -78 °C. The mixture was
stirred at the same temperature for 30 min. The reaction was
quenched by saturated NaHCO3, and the whole was extracted
with CHCl3 (×2). The organic phase was washed with 10%
sodium thiosulfate solution, saturated NaHCO3 (×2), and brine
and dried (Na2SO4). The solvent was removed under reduced
pressure, and the residue was dissolved in acetic anhydride
(34 mL). The mixture was kept at 100 °C for 3 h and then
evaporated to dryness. The residue was purified on column
chromatography over silica gel (9% AcOEt in hexane) to give
11 (1.79 g, 55%, syrup): 1H NMR (CDCl3) δ 7.35-7.24 (15H,
m), 6.07 (0.63H, d, J ) 3.9 Hz), 5.98 (0.37H, d, J ) 2.9 Hz),
In summary, we have developed a new and facile
synthesis of 4′-thioarabinonucleosides. Among these
compounds, (E)-5-(bromovinyl)-4′-thioarabinosyluracil
showed potent anti-HSV-1 and anti-VZV activity in
vitro. In addition, 2,6-diaminopurine and guanine

Alternative Synthesis of 4′-Thioarabinonucleosides
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2181
4.83-4.48 (6H, m), 4.26 (0.37H, dd, J ) 2.9, 4.9 Hz), 4.18
(0.63H, dd, J ) 3.9, 8.8 Hz), 4.12 (0.63H, dd, J ) 6.8, 8.8 Hz),
4.03 (dd, 0.37H, J ) 4.9, 6.4 Hz), 3.76 (0.37H, m), 3.73-3.44
(2H, m), 3.40 (0.63H, m), 2.04 (3H, s); FAB MS m/ z 435 (M+
- COCH3). Anal. (C28H30O4S‚0.75H2O) C, H.
HPLC (YMC-PACK D-ODS-5 20 × 250mm, YMC Co., Ltd.,
J apan; 7% CH3CN in H2O, flow rate 8 mL/min; retention time
R, 42 min, â, 46 min) to give R-6c (92 mg, 23%) and â-6c (87
mg, 22%). â-Anomer (â-6c): mp 189-190 °C (crystallized from
H2O); UV λmax (H2O) 292 nm (ꢀ 8300), 217 (ꢀ 11 400); 1H NMR
(DMSO-d6) δ 11.67 (1H, s, D2O exchangeable), 8.55 (1H, s),
6.34 (1H, d, J ) 5.9 Hz), 5.79 (1H, d, J ) 5.4 Hz, D2O
exchangeable), 4.97 (1H, d, J ) 4.4 Hz, D2O exchangeable),
4.32 (1H, t, J ) 4.9 Hz, D2O exchangeable), 3.99 (1H, dt, J )
5.8, 5.9 Hz), 3.95-3.93 (1H, m), 3.71 (1H, dd, J ) 5.4, 11.2
Hz), 3.66 (1H, dd, J ) 5.4, 11.2 Hz), 3.18-3.16 (1H, m); EI
MS m/ z 386 (M+). Anal. (C9H11IN2O5S) C, H, N. R-Anomer
(R-6c): mp 196-198 °C (crystallized from H2O); UV λmax (H2O)
5-Eth yl-1-(4-th io-D-a r a bin ofu r a n osyl)u r a cil (6a ). A so-
lution of 5-ethyluracil (841 mg, 6.0 mmol) in 1,1,1,3,3,3-
hexamethyldisilazane (HMDS; 8.4 mL) and ammonium sulfate
(8 mg) was kept under reflux overnight and concentrated to
dryness. To this residue were added a solution of 9 (925 mg,
1.93 mmol) in 1,2-dichloroethane (37 mL) and TMSOTf (0.62
mL, 3.2 mmol). After the mixture stirred for 1.5 h at room
temperature, saturated NaHCO3 was added to the reaction
mixture. The whole was extracted with CHCl3, and the
organic phase was washed with water (×2) and brine and dried
(Na2SO4). The filtrate was concentrated under reduced pres-
sure. The residue was purified by column chromatography
over silica gel (2% MeOH in CHCl3) to give 12a (841 mg, 78%).
Compound 12a (199 mg, 0.356 mmol) was dissolved in CH2-
Cl2 (3 mL). To this mixture was added a CH2Cl2 solution of
BCl3 (1 M, 2.14 mL, 2.14 mmol) at -78 °C. After 30 min of
stirring at -78 °C, the mixture was allowed to warm to -20
°C. After 2 h of stirring at -20 °C, the reaction was quenched
by saturated NaHCO3 and Celite filtration and then extracted
with CHCl3. The separated water phase was concentrated
under reduced pressure. The residue was purified on column
chromatography over silica gel (10-20% MeOH in CHCl3), and
the anomers were separated by HPLC (YMC-PACK SIL-06 20
× 250mm, YMC Co., Ltd., J apan; 7% MeOH in CHCl3, flow
rate 7 mL/min; retention time R, 23 min, â, 22 min) to give
R-6a (34 mg, 24%) and â-6a (12 mg, 11%) as amorphous solids.
â-Anomer (â-6a ): UV λmax (H2O) 272 nm (ꢀ 9600); 1H NMR
(DMSO-d6) δ 11.25 (1H, s, D2O exchangeable), 7.91 (1H, s),
6.08 (1H, d, J ) 5.9 Hz), 5.73 (1H, d, J ) 5.4 Hz, D2O
exchangeable), 5.44 (1H, d, J ) 4.9 Hz, D2O exchangeable),
5.24 (1H, t, J ) 4.4 Hz, D2O exchangeable), 3.99 (1H, q, J )
5.4 Hz), 3.94 (1H, q, J ) 5.8 Hz), 3.74 (1H, dd, J ) 5.4, 11.2
Hz), 3.67 (1H, dd, J ) 4.4, 11.2 Hz), 3.15 (1H, dt, J ) 4.4, 5.4
Hz), 2.21 (2H, q, J ) 7.3 Hz), 1.04 (3H, t, J ) 7.3 Hz); EI MS
m/ z 288 (M+). Anal. (C11H16N2O5S‚0.5H2O) C, H, N. R-Ano-
mer (R-6a ): UV λmax (H2O) 271 nm (ꢀ 10 200); 1H NMR (DMSO-
d6) δ 11.25 (1H, s, D2O exchangeable), 7.78 (1H, s), 5.77 (1H,
d, J ) 7.3 Hz), 5.69 (1H, d, J ) 5.4 Hz, D2O exchangeable),
5.52 (1H, d, J ) 4.9 Hz, D2O exchangeable), 4.91 (1H, t, J )
5.1 Hz, D2O exchangeable), 4.01 (1H, q, J ) 7.3 Hz), 3.85 (1H,
dt, J ) 3.9, 10.7 Hz), 3.66-3.61(1H, m), 3.56 (1H, ddd, J )
3.4, 3.9, 7.8 Hz), 3.44 (1H, dt, J ) 7.8, 10.7 Hz), 2.27 (2H, q, J
) 7.5 Hz), 1.05 (3H, t, J ) 7.5 Hz); EI MS m/ z 288 (M+). Anal.
(C11H16N2O5S‚0.5H2O) C, H, N.
1
291 nm (ꢀ 8300), 217 (ꢀ 11 900); H NMR (DMSO-d6) δ 11.69
(1H, s, D2O exchangeable), 8.42 (1H, s), 5.74 (1H, d, J ) 6.8
Hz), 5.73 (1H, d, J ) 6.8 Hz, D2O exchangeable), 5.50 (1H, d,
J ) 4.9 Hz, D2O exchangeable), 4.93 (1H, t, J ) 4.9 Hz, D2O
exchangeable), 4.05 (1H, dt, J ) 6.4, 6.8 Hz), 3.84 (1H, ddd, J
) 4.2, 4.9, 11.2 Hz), 3.72 (1H, dt, J ) 6.4, 7.3 Hz), 3.47 (1H,
ddd, J ) 4.2, 7.3, 7.8 Hz), 3.43 (1H, ddd, J ) 4.9, 7.8, 11.2
Hz); EI MS m/ z 386 (M+). Anal. (C9H11IN2O5S‚H2O) C, H, N.
(E)-5-(2-Ch lor ovin yl)-1-(4-t h io-D-a r a b in ofu r a n osyl)-
u r a cil (6d ). From 11 (462 mg, 0.967 mmol), R- and â-6d were
obtained as described in the synthesis of 6a . Anomers were
separated by HPLC (YMC-PACK ODS-AQ 20 × 250 mm, YMC
Co., Ltd., J apan; 15% CH3CN in H2O, flow rate 8 mL/min;
retention time R, 61 min, â, 56 min) to give R-6d (80 mg, 26%)
and â-6d (34 mg, 11%). â-Anomer (â-6d ): mp 241-243 °C
(crystallized from H2O); UV λmax (H2O) 297 nm (ꢀ 7300), 248
1
(ꢀ 9700); H NMR (DMSO-d6) δ 11.56 (1H, s, D2O exchange-
able), 8.34 (1H, s), 7.18 (1H, d, J ) 13.4 Hz), 6.62 (1H, d, J )
13.4 Hz), 6.07 (1H, d, J ) 5.9 Hz), 5.74 (1H, d, J ) 5.9 Hz,
D2O exchangeable), 5.44 (1H, d, J ) 4.9 Hz, D2O exchange-
able), 5.33 (1H, t, J ) 5.1 Hz, D2O exchangeable), 4.01 (1H,
dt, J ) 5.9, 6.4 Hz), 3.95 (1H, dt, J ) 5.9, 6.4 Hz), 3.77-3.73
(2H, m), 3.17-3.14 (1H, m); EI MS m/ z 320 (M+). Anal.
(C11H13ClN2O5S‚0.5H2O) C, H, N. R-Anomer (R-6d ): mp 234-
238 °C (crystallized from H2O); UV λmax (H2O) 295 nm (ꢀ
1
12 200), 249 nm (ꢀ 15 600); H NMR (DMSO-d6) δ 11.60 (1H,
s, D2O exchangeable), 8.25 (1H, s), 7.26 (1H, d, J ) 13.4 Hz),
6.73 (1H, d, J ) 13.4 Hz), 5.76 (1H, d, J ) 7.3 Hz), 5.74 (1H,
d, J ) 5.9 Hz, D2O exchangeable), 5.59 (1H, d, J ) 4.4 Hz,
D2O exchangeable), 4.94 (1H, t, J ) 5.4 Hz, D2O exchangeable),
4.04 (1H, dt, J ) 7.3, 7.8 Hz), 3.87 (1H, dt, J ) 3.9, 11.2 Hz),
3.68 (1H, dt, J ) 7.8, 8.3 Hz), 3.61 (dt, 1H, J ) 3.9, 8.3 Hz),
3.42-3.39 (1H, m); EI MS m/ z 320 (M+). Anal. (C11H13
ClN2O5S) C, H, N.
-
(E)-5-(2-Br om ovin yl)-1-(4-th io-D-a r a bin ofu r a n osyl)u r a -
cil (6e). From 11 (619 mg, 1.29 mmol), R- and â-6e were
obtained as described in the synthesis of 6a . The residue was
purified on column chromatography over silica gel (15% MeOH
in CHCl3), and anomers were separated by HPLC (Wakosil-II
5C18 HG 20 × 250 mm, Wako Pure Chemicals Industries, Ltd.,
J apan; 20% CH3CN in H2O, flow rate 8 mL/min; retention time
R, 34 min, â, 32 min) to give R-6e (187 mg, 39%) and â-6e (63
mg, 13%). â-Anomer (â-6e): mp 216 °C dec (crystallized from
50% aqueous EtOH); UV λmax (H2O) 297 nm (ꢀ 10 400), 251 (ꢀ
14 100); 1H NMR (DMSO-d6) δ 11.58 (1H, s, D2O exchange-
able), 8.36 (1H, s), 7.25 (1H, d, J ) 13.5 Hz), 6.89 (1H, d, J )
13.5 Hz), 6.06 (1H, d, J ) 5.9 Hz), 5.75 (1H, d, J ) 5.9 Hz,
D2O exchangeable), 5.45 (1H, d, J ) 4.9 Hz, D2O exchange-
able), 5.34 (1H, t, J ) 5.1 Hz, D2O exchangeable), 4.02 (1H,
dt, J ) 5.9, 6.8 Hz), 3.94 (1H, dt, J ) 3.4, 6.8 Hz), 3.77 (1H,
dt, J ) 4.9, 11.2 Hz), 3.73 (1H, dt, J ) 5.4, 11.2 Hz), 3.15 (1H,
5-Meth yl-1-(4-th io-D-ar abin ofu r an osyl)u r acil (6b). From
11 (925 mg, 1.93 mmol), R- and â-6b were obtained as
described in the synthesis of 6a . Anomers were separated by
HPLC (YMC-PACK SIL-06 20 × 250mm, YMC Co., Ltd.,
J apan; 15% MeOH in CHCl3, flow rate 8 mL/min; retention
time R, 20 min, â, 18 min) to give R-6b (137 mg, 36%) and
â-6b (53 mg, 14%). â-Anomer (â-6b): mp 108-111 °C (crys-
tallized from H2O); UV λmax (H2O) 272 nm (ꢀ 10 500); 1H NMR
(DMSO-d6) δ 11.27 (1H, s, D2O exchangeable), 7.95 (1H, s),
6.09 (1H, d, J ) 5.4 Hz), 5.73 (1H, d, J ) 5.4 Hz, D2O
exchangeable), 5.44 (1H, d, J ) 4.4 Hz, D2O exchangeable),
4.02 (1H, dt, J ) 5.4, 6.4 Hz), 3.95 (1H, dt, J ) 5.9, 6.4 Hz),
3.78 (1H, dd, J ) 4.9, 11.2 Hz), 3.70 (1H, dd, J ) 5.9, 11.2
Hz), 3.17-3.14 (1H, m), 1.78 (3H, s); EI MS m/ z 274 (M+).
Anal. (C10H14N2O5S‚0.75H2O) C, H, N. R-Anomer (R-6b): an
1
dt, J ) 3.4, 4.9 Hz); EI MS m/ z 364, 366 (M+). Anal. (C11H13
-
amorphous solid; UV λmax (H2O) 271 nm (ꢀ 10 500); H NMR
(DMSO-d6) δ 11.29 (1H, s, D2O exchangeable), 7.85 (1H, s),
5.76 (1H, d, J ) 7.8 Hz), 5.69 (1H, d, J ) 5.9 Hz, D2O
exchangeable), 5.54 (1H, d, J ) 4.9 Hz, D2O exchangeable),
5.25 (1H, t, J ) 5.1 Hz, D2O exchangeable), 4.91 (1H, t, J )
5.1 Hz, D2O exchangeable), 4.01 (1H, dt, J ) 7.8, 8.3 Hz), 3.87-
3.83 (1H, m), 3.65 (1H, dt, J ) 3.4, 8.3 Hz), 3.56 (1H, dt, J )
3.4, 8.3 Hz), 3.39-3.36 (1H, m), 1.84 (3H, s); EI MS m/ z 274
(M+). Anal. (C10H14N2O5S‚0.5H2O) C, H, N.
BrN2O5S) C, H, N. R-Anomer (R-6a ): mp 201-203 °C (crystal-
lized from 50% aqueous EtOH); UV λmax (H2O) 296 nm (ꢀ
12 300), 251 (ꢀ 15 300); 1H NMR (DMSO-d6) δ 11.61 (1H, s,
D2O exchangeable), 8.28 (1H, s), 7.33 (1H, d, J ) 13.7 Hz),
6.99 (1H, d, J ) 13.7 Hz), 5.77 (1H, d, J ) 7.3 Hz), 5.74 (1H,
d, J ) 3.4 Hz, D2O exchangeable), 5.60 (1H, d, J ) 4.4 Hz,
D2O exchangeable), 4.95 (1H, t, J ) 5.1 Hz, D2O exchangeable),
4.01 (1H, dt, J ) 7.8, 7.3 Hz), 3.86 (1H, dd, J ) 3.9, 11.2 Hz),
3.68 (1H, dt, J ) 7.8, 8.3 Hz), 3.61 (1H, dt, J ) 3.9, 8.3 Hz),
3.43 (1H, dd, J ) 8.3, 11.2 Hz); EI MS m/ z 364, 366 (M+).
Anal. (C11H13BrN2O5S‚0.5H2O) C, H, N.
5-Iod o-1-(4-th io-D-a r a bin ofu r a n osyl)u r a cil (6c). From
11 (503 mg, 1.05 mmol), R- and â-4c were obtained as
described in the synthesis of 6a . Anomers were separated by

2182 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Yoshimura et al.
7-(4-Th io-D-a r a bin ofu r a n osyl)a d en in e (14). From 11
(462 mg, 0.967 mmol), R- and â-14 were obtained as described
in the synthesis of 6a . The residue was purified on column
chromatography over silica gel (15% MeOH in CHCl3), and
anomers were separated by HPLC (Wakosil-II 5C18 HG 20 ×
250 mm, Wako Pure Chemicals Industries, Ltd., J apan; 20%
CH3CN in H2O, flow rate 8 mL/min; retention time R, 34 min,
â, 32 min) to give R-14 (42 mg, 15%) and â-14 (15 mg, 7.3%).
â-Anomer (â-14): mp 163-167 °C (crystallized from H2O); UV
exchangeable), 5.51 (1H, brt, J ) 3.9 Hz), 5.47 (1H, d, J ) 4.9
Hz), 5.27 (1H, d, J ) 4.9 Hz, D2O exchangeable), 4.87 (1H, t,
J ) 4.9 Hz, D2O exchangeable), 4.03 (1H, dt, J ) 5.4, 6.4 Hz),
3.97 (1H, dt, J ) 3.9, 6.4 Hz), 3.79 (1H, dt, J ) 4.9, 10.7 Hz),
3.46 (1H, ddd, J ) 4.9, 7.8, 10.7 Hz), 3.13 (1H, ddd, J ) 4.9,
5.4, 7.8 Hz); FAB MS m/ z 318 (M+). Anal. (C10H12N5O3-
SCl‚0.2EtOH‚0.8H2O) C, H, N. R-Anomer (R-17): 1H NMR
(DMSO-d6) δ 13.07 (1H, br), 8.17 (1H, s), 7.78 (1H, br, D2O
exchangeable), 5.39-5.37 (2H, m, D2O exchangeable), 5.26 (1H,
brdt, J ) 6.8 Hz), 4.75 (1H, t, J ) 5.1 Hz, D2O exchangeable),
3.99 (1H, dt, J ) 6.8, 7.3 Hz), 3.81 (1H, ddd, J ) 4.2, 5.1, 10.5
Hz), 3.64 (1H, dt, J ) 7.3, 7.8 Hz), 3.37 (1H, ddd, J ) 5.1, 8.1,
10.5 Hz), 3.30 (1H, ddd, J ) 4.2, 7.8, 8.1 Hz); FAB MS m/ z
318 (M+). Anal. (C10H12N5O3SCl‚0.8H2O) C, H, N.
λ
max (H2O) 273 nm (ꢀ 9300), 249 (sh, ꢀ 6300); 1H NMR (DMSO-
d6) δ 8.89 (1H, s), 8.15 (1H, s), 6.80 (2H, br, D2O exchangeable),
6.08 (1H, d, J ) 5.9 Hz), 5.78 (1H, d, J ) 5.9 Hz, D2O
exchangeable), 5.46 (1H, d, J ) 5.9 Hz, D2O exchangeable),
5.33 (1H, t, J ) 4.6 Hz, D2O exchangeable), 4.14-4.10 (1H,
m), 3.87-3.83 (1H, m), 3.81-3.79 (2H, m), 3.16 (1H, ddd, J )
4.4, 7.8, 8.3 Hz); FAB MS m/ z 284 (M + H+). Anal.
(C10H13N5O3S‚0.75H2O) C, H, N. R-Anomer (R-14): mp 247-
249 °C (crystallized from H2O); UV λmax (H2O) 275 nm (ꢀ 9400),
251 (sh, ꢀ 6200); 1H NMR (DMSO-d6) δ 8.62 (1H, s), 8.22 (1H,
s), 6.96 (2H, br, D2O exchangeable), 6.01 (1H, br, D2O
exchangeable), 5.93 (1H, d, J ) 7.3 Hz), 5.63 (1H, d, J ) 4.4
Hz, D2O exchangeable), 5.03 (1H, t, J ) 5.1 Hz, D2O exchange-
able), 4.16 (1H, dt, J ) 7.3, 8.3 Hz), 3.91 (1H, ddd, J ) 3.4,
5.1, 10.7 Hz), 3.80 (1H, t, J ) 8.3 Hz), 3.63 (1H, ddd, J ) 3.4,
7.8, 8.3 Hz), 3.53 (1H, ddd, J ) 5.1, 7.8, 10.7 Hz); FAB MS
m/ z 284 (M + H+). Anal. (C10H13N5O3S‚0.55H2O) C, H, N.
9-(4-Th io-D-a r a bin ofu r a n osyl)a d en in e (6f). A mixture
of 11 (489 mg, 1.02 mmol), adenine (261 mg, 1.93 mmol),
TMSOTf (0.75 mL, 3.88 mmol), and 4A molecular sieves (897
mg) was stirred at room temperature for 1 h. The reaction
was quenched by saturated NaHCO3. The whole was ex-
tracted with CH2Cl2, and the organic phase was washed with
saturated NaHCO3 and dried (Na2SO4). After the solvent was
removed under reduced pressure, the residue was purified on
column chromatography over silica gel (5% MeOH in CHCl3),
giving 378 mg of 15f (0.697 mmol) which was dissolved in CH2-
Cl2 (5 mL).
To this mixture was added a CH2Cl2 solution of BCl3 (1 M,
4.2 mL, 4.2 mmol) at -78 °C. After 1 h of stirring at -78 °C,
the mixture was allowed to warm to -20 °C. After 2 h of
stirring at -20 °C, the reaction was quenched by saturated
NaHCO3, and the mixture was extracted with CHCl3. The
separated water phase was concentrated under reduced pres-
sure. The residue was purified on column chromatography
over silica gel (17% MeOH in CHCl3), and the anomers were
separated by HPLC (Wakosil-II 5C18 HG 20 × 250 mm, Wako
Pure Chemicals Industries, Ltd., J apan; 10% CH3CN in H2O,
flow rate 8 mL/min; retention time R, 13 min, â, 15 min) to
give R-6f (32 mg, 11%) and â-6f (52 mg, 18%). â-Anomer (â-
6f): mp 138-140 °C (crystallized from H2O); UV λmax (H2O)
261 nm (ꢀ 13 300); 1H NMR (DMSO-d6) δ 8.36 (1H, s), 8.13
(1H, s), 7.22 (2H, br, D2O exchangeable), 6.05 (1H, d, J ) 5.4
Hz), 5.72 (1H, br, D2O exchangeable), 5.51 (1H, d, J ) 2.9 Hz,
D2O exchangeable), 5.19 (1H, br, D2O exchangeable), 4.18-
4.11 (2H, m), 3.87 (1H, dd, J ) 3.9, 11.2 Hz), 3.78 (1H, dd, J
) 6.6, 11.2 Hz), 3.25 (1H, ddd, J ) 3.9, 5.9, 6.6 Hz); EI MS
m/ z 283 (M+). Anal. (C10H13N5O3S‚2H2O) C, H, N. R-Anomer
(R-6f): mp 250 °C (crystallized from H2O); UV λmax (crystallized
from H2O) 261 nm (ꢀ 13 600);1H NMR (DMSO-d6) δ 8.41 (1H,
s), 8.15 (1H, s), 7.24 (2H, br, D2O exchangeable), 5.79 (1H, d,
J ) 4.9 Hz, D2O exchangeable), 5.73 (1H, d, J ) 7.3 Hz), 5.61
(1H, d, J ) 4.4 Hz, D2O exchangeable), 4.93 (1H, t, J ) 4.6
Hz, D2O exchangeable), 4.56 (1H, dt, J ) 4.4, 7.3 Hz), 3.89
(1H, dt, J ) 3.9, 10.7 Hz), 3.75 (1H, dt, J ) 4.4, 7.8 Hz), 3.66
(1H, ddd, J ) 3.9, 7.8, 8.1 Hz), 3.50 (1H, dt, J ) 8.1, 10.7 Hz);
EI MS m/ z 283 (M+). Anal. (C10H13N5O3S) C, H, N.
2,6-Dia m in o-9-(4-th io-D-a r a bin ofu r a n osyl)p u r in e (6g).
From 11 (489 mg, 1.06 mmol), R- and â-6g were obtained as
described in the synthesis of 6f. Anomers were separated by
HPLC (Wakosil-II 5C18 HG 20 × 250 mm, Wako Pure
Chemicals Industries, Ltd., J apan; 10% CH3CN in H2O, flow
rate 8 mL/min; retention time R, 13 min, â, 14 min) to give
R-6g (17 mg, 5%) and â-6g (28 mg, 9%). â-Anomer (â-6g): mp
292-295 °C (crystallized from H2O); UV λmax (H2O) 282 nm (ꢀ
10 900), 258 (ꢀ 9300); 1H NMR (DMSO-d6) δ 7.93 (1H, s), 6.67
(2H, br, D2O exchangeable), 5.93 (1H, d, J ) 5.4 Hz), 5.77 (2H,
br, D2O exchangeable), 5.74 (1H, d, J ) 4.9 Hz, D2O exchange-
able), 5.51 (1H, d, J ) 4.9 Hz, D2O exchangeable), 5.19 (1H,
br, D2
O exchangeable), 4.12 (1H, dt, J ) 5.9, 6.8 Hz), 4.04 (1H,
dt, J ) 5.4, 6.8 Hz), 3.83 (1H, dd, J ) 4.9, 10.7 Hz), 3.68 (1H,
dd, J ) 6.8, 10.7 Hz), 3.22 (1H, ddd, J ) 4.9, 5.9, 6.8 Hz); EI
MS m/ z 298 (M
+). Anal. (C10H14N6O3S‚0.75H2O) C, H, N.
R-Anomer (R-6g): mp 241-247 °C (crystallized from H2O); UV
1
λmax
(H2O) 281 nm (ꢀ 10 900), 259 (ꢀ 9600); H NMR (DMSO-
d6
br, D2O exchangeable), 5.75 (1H, br, D2O exchangeable), 5.58
O exchangeable), 5.56 (1H, d, J ) 7.3 Hz), 4.90 (1H,
) δ 8.00 (1H, s), 6.67 (2H, br, D2O exchangeable), 5.78 (2H,
(1H, br, D2
br, D2O exchangeable), 4.45 (1H, t, J ) 7.3 Hz), 3.86 (1H, dt,
J ) 1.0, 11.0 Hz), 3.70 (1H, t, J ) 7.8 Hz), 3.63 (1H, m), 3.45
(1H, dt, J ) 8.1, 11.0 Hz); EI MS m/ z 298 (M+). Anal.
(C10H14N6O3S‚H2O) C, H, N.
9-(4-Th io-â-D-a r a bin ofu r a n osyl)gu a n in e (â-6h ). A solu-
tion of 6g (20 mg, 0.067 mmol) and adenosine deaminase (43
µL, 10 units) in Tris-HCl buffer (5 mL, pH 7.0) was kept at 55
°C for 7.5 h. After dilution, the solution was applied to a
column of adsorption resin (10 mL, Sepabeads SP 206, Mit-
subishi Chemical Corp., J apan). After it was washed with 200
mL of water, the eluate of 10% aqueous EtOH was collected
and concentrated under reduced pressure to leave crystalline
â-6h (19.5 mg, 97%): mp 260-264 °C (crystallized from H2O);
UV λmax (H2O) 273 nm (ꢀ 10 500), 256 (ꢀ 14 000);1H NMR
(DMSO-d6) δ 10.56 (1H, br), 7.92 (1H, s), 6.44 (2H, br, D2O
exchangeable), 5.86 (1H, d, J ) 5.4 Hz), 5.71 (1H, d, J ) 5.4
Hz, D2O exchangeable), 5.49 (1H, d, J ) 4.9 Hz, D2O exchange-
able), 5.14 (1H, t, J ) 5.4 Hz, D2O exchangeable), 4.07 (1H,
dd, J ) 5.4, 11.0 Hz), 4.03 (1H, dd, J ) 6.6, 11.0 Hz), 3.83
(1H, dt, J ) 5.4, 5.9 Hz), 3.67 (1H, dt, J ) 5.4, 5.9 Hz), 3.21
(1H, dt, J ) 5.4, 6.6 Hz); FAB MS m/ z 300 (M + H+). Anal.
(C10H13N5O4S‚H2O) C, H, N.
An tivir a l Assa ys. HEL cells and the following virus
strains were used: HSV-1 VR-3 strain, HSV-2 MS strain, VZV
Oka strain, and HCMV AD 169 strain. The origins of viruses
and cells have been described previously.3,19
Antiviral activi-
ties against these herpes viruses were determined by the
plaque reduction assay as described earlier.3,20
Cell Gr ow th In h ibition Test. A cell suspension of human
T-cell acute lymphoblastoid leukemia cells, CCRF-HSB-2,
containing 1.1 × 105 cells/mL was prepared in RPMI 1640
medium supplemented with 10% fetal bovine serum; 90 µL of
the cell suspension was seeded in a 96-multiwell plate, and
10 µL of medium or phosphate-buffered saline (PBS) contain-
ing test compound in serial 0.5 log10 dilution was added. Cells
were incubated in a 5% CO2 incubator at 37 °C. After 3 days,
10 µL of MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphen-
yltetrazolium bromide, Sigma Chemical Co., St. Louis, MO),
prepared in a concentration of 5 mg/mL in PBS, was added to
each well. After another 4 h incubation at 37 °C, 100 µL of
extraction buffer (0.02 N HCl solution of 50% DMF containing
6-Ch lor o-2-[(4-t h io-D-a r a b in ofu r a n os-1-yl)a m in o]p u -
r in e (17). From 11 (461 mg, 0.967 mmol), R- and â-17 were
obtained as described in the synthesis of 6f. The residue was
purified on column chromatography over silica gel (15% MeOH
in CHCl3), and anomers were separated by HPLC (Wakosil-II
5C18 HG 20 × 250 mm, Wako Pure Chemicals Industries, Ltd.,
J apan; 20% CH3CN in H2O, flow rate 8 mL/min; retention time
R, 34 min, â, 32 min) to give R-17 (18.7 mg, 6.2%, amorphous
solid) and â-17 (35.9 mg, 12%, amorphous solid). â-Anomer
(â-17): 1H NMR (DMSO-d6) δ 8.12 (1H, s), 6.96 (1H, br, D2O

Alternative Synthesis of 4′-Thioarabinonucleosides
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2183
(7) Reist, E. J .; Gueffroy, D. E.; Goodman, L. Synthesis of 4-thio-D-
and -L-ribofuranose and the corresponding adenine nucleosides.
J . Am. Chem. Soc. 1964, 86, 5658-5663.
20% SDS) was added to each well to solubilize MTT-formazan.
Absorbance at 570 nm (test wavelength) and 690 nm (reference
wavelength) were measured using a microplate reader (MPR-
A4i, Tosoh Co.). The percentage of cell growth inhibition was
calculated by the following formula:
(8) Reist, E. J .; Fisher, L. V.; Goodman, L. Thio sugars. Synthesis
of the adenine nucleosides of 4-thio-D-xylose and 4-thio-D-
arabinose. J . Org Chem. 1968, 33, 189-192.
(9) Ototani, N.; Whistler, R. L. Preparation and antitumor activity
of 4′-thio analogs of 2,2′-anhydro-1-â-D-arabinofuranosylcytosine.
J . Med. Chem. 1974, 17, 535-537.
inhibition (%) ) [1 - (Tx - C0/Cx - C0)] × 100
(10) Bobek, M.; Bloch, A.; Parthasarathy, R.; Whistler, R. L. Syn-
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where Tx is absorbance at the end of incubation with test drug,
Cx is absorbance at the end of incubation without drug, and
C0 is absorbance at beginning of incubation. IC50 of the test
compound was determined graphically from a dose-inhibition
curve.
Ack n ow led gm en t. The authors would like to thank
Dr. A. Kuninaka, Yamasa Corp., for his critical reading
of the manuscript and acknowledge Mr. M. Morozumi
for his helpful discussions. The authors also thank Mr.
T. Miyashita for his collaboration in synthesizing araG.
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