Synthesis and biological evaluation of 2,8-disubstituted 9-benzyladenines: Discovery of 8-mercaptoadenines as potent interferon-inducers

Article abstract of DOI:10.1016/S0968-0896(03)00234-7

Recently, we have identified 9-benzyl-8-hydroxyadenines bearing an appropriate substituent (a butoxy, propylthio or butylamino group) at the 2-position as potent interferon (IFN)-inducers. Herein we report the design, synthesis, and IFN-inducing activity of 8-substituted 9-benzyladenines possessing such an appropriate substituent at the 2-position. Introduction of the appropriate substituent into the 2-position of the adenine nucleus gave rise to expression of the activity even in 9-benzyladenines bearing no hydroxyl group at the 8-position. An amino group at the 6-position and a hydroxyl or thiol group carrying an acidic proton at the 8-position are required to express excellent IFN-inducing activity. 9-Benzyl-2-butoxy-8-mercaptoadenine (9) indicated the most potent activity with MEC of 0.001 μM.

Full text of DOI:10.1016/S0968-0896(03)00234-7

Bioorganic & MedicinalChemistry 11 (2003) 2715–2722
Synthesis and Biological Evaluation of 2,8-Disubstituted
9-Benzyladenines: Discovery of 8-Mercaptoadenines as
Potent Interferon-Inducers
Kosaku Hirota,* Kazunori Kazaoka and Hironao Sajiki
Laboratory of Medicinal Chemistry, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan
Received 24 February 2003; accepted 3 April2003
Abstract—Recently, we have identified 9-benzyl-8-hydroxyadenines bearing an appropriate substituent (a butoxy, propylthio or
butylamino group) at the 2-position as potent interferon (IFN)-inducers. Herein we report the design, synthesis, and IFN-inducing
activity of 8-substituted 9-benzyladenines possessing such an appropriate substituent at the 2-position. Introduction of the appro-
priate substituent into the 2-position of the adenine nucleus gave rise to expression of the activity even in 9-benzyladenines bearing
no hydroxylgroup at the 8-position. An amino group at the 6-position and a hydroxylor thiolgroup carrying an acidic proton at
the 8-position are required to express excellent IFN-inducing activity. 9-Benzyl-2-butoxy-8-mercaptoadenine (9) indicated the most
potent activity with MEC of 0.001 mM.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
Various compounds possessing IFN-inducing activity
have been hitherto reported.4 Since tilorone was repor-
ted as an IFN inducer,5 a variety of low-molecular-
weight compounds including BL-20803,6 atabrine,7
CP-28888,8 ABMP,9 DRB,10 10-carboxymethyl-9-acri-
done,11 bropirimine12 and imiquimod13,14 have been
reported as IFN inducers. Among them, imiquimod,
which is clinically used in the USA for treatment of
exophylic warts caused by the human papillomavirus, is
especially a potent IFN inducer. However, its serious side-
effects such as vomiting and hepatopathy found during the
clinical trial stage forced abandonment of its further
development as a chemotherapy drug for hepatitis C.
Subsequently, imiquimod analogues such as R-842 (a
hydroxylated metabolite of imiquimod)15 and resiqui-
mod14,16 were found to be more effective IFN inducers
than imiquimod (Fig. 1). However, no IFN inducer has yet
been clinically employed for the treatment of hepatitis C.
Hepatitis C virus (HCV) is a major cause of acute
hepatitis and chronic liver disease, including cirrhosis
and liver cancer. Globally, an estimated 170 million
persons are chronically infected with HCV and 3–4
million persons are newly infected each year.1 It has
been a serious problem of HCV infection that the pro-
portion of progression of hepatitis C to cirrhosis, liver
failure, and liver cancer over a long period is very high.
The major therapeutic option of HCV carriers is inter-
feron (IFN). Various clinical trials have been conducted
to optimize IFN treatment of HCV-infected patients.2
However, administration of recombinant IFN fre-
quently induces neutralizing antibodies, which diminish
3
the antiviralactivity of IFN. Furthermore, the admin-
istration of IFN by intramuscular or subcutaneous
injection causes severe pain and irritation at the site of
injection. The cost of IFN therapy is also very high
because of the inordinately expensive IFN preparations.
To avoid such drawbacks, the development of an IFN
inducer, which enhances the release of endogenous IFN
by oraladministration, has been ardentyl desired for a
long time.
Recently, we found 9-benzyl-8-hydroxyadenine (1) pos-
sessing an IFN-inducing activity as a lead compound in
our first screening17 and its substituent modifications at
the 2-, 6- and 9-positions were conducted to investigate
structure–activity relationships for IFN-inducing activ-
ity.18,19 Consequently, it was found that the introduc-
tion of an appropriate chain substituent at the
2-position remarkably increases the activity. Among
*Corresponding author. Tel.: +81-58-237-8572; fax: +81-58-237-
5979; e-mail: hirota@gifu-pu.ac.jp
various
2-substituted
9-benzyl-8-hydroxyadenines,
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00234-7

2716
K. Hirota et al. / Bioorg. Med. Chem. 11 (2003) 2715–2722
Figure 1. Some advanced low-molecular-weight interferon inducers.
2-butoxy (2), 2-propylthio (3) and 2-butylamino (4)
analogues indicated potent IFN-inducing activity.17 On
the basis of the results mentioned above, our attention
was directed to the chemicalmodification at the 8-posi-
tion of 9-benzyladenines possessing a substituent having
considerable influence on the activity such as a butoxy,
propylthio or butylamino group at the 2-position. In
this report, we describe the synthesis and evaluation of
8-substituted 9-benzyladenines (A) carrying an appro-
priate substituent at the 2-position as shown in Figure 2.
In addition, in order to examine the importance of the
6-amino group, some 6-substituted 9-benzyl-8-hydroxy-
purines possessing such a chain substituent at the
2-position were synthesized and the IFN-inducing
activity was evaluated.
Scheme 1. Reagents and conditions: (a) 10% Pd/C, (C2H5)3N, H2,
CH3OH, rt (94%); (b) ref 18; (c) Al(CH3)3, Ph3P, PdCl2, THF, reflux
(96%); (d) H2NCSNH2, C2H5OH, reflux (86%); (e) CH3I, K2CO3,
DMF, rt (88%).
Chemistry
First, synthesis of several8-substituted 9-benzy-2l-
butoxyadenines, whose 2-butoxy substituent induces the
highest IFN-inducing activity in the 9-benzyl-8-hydroxy-
adenine series,17 was attempted. Most of the 8-sub-
stituted 9-benzyl-2-butoxyadenines were prepared from
the corresponding 8-bromoadenine (5)18 as shown in
Scheme 1. 8-Unsubstituted 9-benzyl-2-butoxyadenine
(6) was prepared in 94% yield by hydrogenolysis of the
8-bromo group using a Pd/C catalyst in the presence of
triethylamine. 8-Methoxy analogue (7) was prepared by
nucleophilic substitution of 5 with sodium methoxide as
previously reported.18 The synthesis of 8-methyl( 8) and
Scheme 2. Reagents and conditions: (a) NCS, THF, rt (87%); (b)
H2NCONH2, neat (78%); (c) C4H9Br, K2CO3, DMF, rt (66%).
8-mercapto (9) derivatives was achieved according to
20,21
the usualmethods.
Cross-coupling of 5 with tri-
methylaluminum using a palladium catalyst20 gave
8-methyladenine (8) in 96% yield, and refluxing of 5 and
thiourea in ethanol21 afforded 8-mercaptoadenine (9) in
86% yield. Subsequently, 9 was selectively S-methylated
with methyliodide in the presence of potassium car-
bonate to give 8-methylthio analogue (10) in 88% yield.
8-Chloro analogue (14) was prepared from 5-amino-1-
benzyl-4-cyanoimidazole (11)22 by the following three
steps: chlorination of 11, cyclization of 12 with urea and
selective O-butylation of 13 (Scheme 2).
Scheme 3. Reagents and conditions: (a) C3H7CHO, NaBH3CN,
CH3OH, rt (16: 84%, 18: 85%).
Some 2-butylaminoadenine derivatives were also pre-
pared, in which the synthetic methods of 8-unsub-
stituted (16) and 8-methoxy (18) analogues are
illustrated in Scheme 3. 2-Amino-9-benzyladenine (15),
which was easily prepared by the cyclization of imida-
zole 1122 with guanidine, was treated with butyr-
aldehyde in the presence of sodium cyanoborohydride
Figure 2. Strategy for structure-optimization of 9-benzyladenines.

K. Hirota et al. / Bioorg. Med. Chem. 11 (2003) 2715–2722
2717
to afford the desired 9-benzyl-2-butylaminoadenine (16)
via regioselective reductive alkylation.18 Similarly, the
synthesis of 9-benzyl-2-butylamino-8-methoxyadenine
(18) was carried out by reductive alkylation of 2-amino-
9-benzyl-8-methoxyadenine (17), which was prepared by
cyclization of 5-amino-1-benzyl-2-bromo-4-cyanoimi-
dazole18 with guanidine and subsequent methanolysis of
the 8-bromo group of the resulting 2-amino-9-benzyl-8-
bromoadenine. The synthetic sequence of 8-mercapto-
adenine (22) is shown in Scheme 4. 2-Mercaptoimida-
the 6-ethylthio group in 2-amino-9-benzyl-6-ethylthio-8-
hydroxypurine (23)18 using Raney Ni, followed by
reductive alkylation using butyraldehyde afforded 25.
N6-ethylanaol gue ( 27) containing a propylthio group at
the 2-position was obtained by the reaction of 9-benzyl-
8-hydroxy-2-propylthioadenine (26)18 with acetaldehyde
in the presence of sodium cyanoborohydride in 69%
yield (Scheme 6).
zole (20), which is
a key intermediate for the
Results and Discussion
preparation of 22, was synthesized by treatment of
aminomalononitrile with benzyl isothiocyanate and
subsequent known ring-transformation23 of the result-
ing thiazole (19) into imidazole (20) in alkali medium.
Cyclization of 20 with guanidine gave 8-mercaptoade-
nine (21), which was selectively alkylated at the 2-amino
group to yield the expected 9-benzyl-2-butylamino-8-
mercaptoadenine (22). On the other hand, cyclization of
thiazole 19 with guanidine did not give thiazolo[5,4-
d]pyrimidine (B), but 8-mercaptoadenine 21 in 76%
yield. This reaction would involve the ring-transforma-
tion caused by the basicity of guanidine.
An in vitro assay for IFN-inducing activities of 2,8-dis-
ubstituted 9-benzyladenines (5–10, 14, 16, 18, 22) and
2,6-disubstituted 9-benzyl-8-hydroxypurines (25, 27)
synthesized above was performed by a typicalmethod
for determining IFN titer check.24,25 The activity is
indicated by MEC (minimum effective concentration),
which is the concentration of test compounds required
for more than 0.9 IU/mL induction of IFN.
The in vitro IFN-inducing activities of 8-substituted
9-benzyl-2-butoxy or 2-butylaminoadenine derivatives
(5–10, 14, 16, 18, 22) are summarized in Table 1. In the
series of 2-butoxyadenines, 8-unsubstituted (6), chloro
(14), bromo (5) and methyl( 8) analogues, which possess
no hydroxylgroup at the 8-position, were 10 times more
Preparation of 9-benzyl-2-butylamino-8-hydroxypurine
(25) was performed as shown in Scheme 5. Removalof
active than the originaellad compound 1. It was
recently reported that 9-benzyl-8-mercaptoadenine, an
8-thio analogue of 1, indicated an equivalent activity of
Scheme 6. Reagents and conditions: (a) CH3CHO, NaBH3CN,
CH3OH, 50 ꢀC (69%).
Table 1. IFN-inducing activities of 2,8-disubstituted 9-benzylade-
nines
Scheme 4. Reagents and conditions: (a) PhCH2NCS, THF, 40 ꢀC,
.
(86%); (b) 5% Na2CO3, reflux (84%); (c) H2NC(NH)NH2 HCl,
NaOC2H5, C2H5OH, reflux (from 19: 76%, from 20: 69%); (d)
C3H7CHO, NaBH3CN, CH3OH, rt (72%).
Compd
R1
R2
MECa (mM)
6
14
5
8
718
9
10
16
18
22
1
2
OC4H9
OC4H9
OC4H9
OC4H9
OC4H9
OC4H9
OC4H9
NHC4H9
NHC4H9
NHC4H9
H
H
Cl1
Br
CH3
OCH3
SH
SCH3
H
OCH3
SH
OH
OH
OH
1
1
1
10
0.001
10
1
>10
0.01
10
OC4H9
NHC4H9
0.001
0.1
1
4
Imiquimod
aMinimum effective concentration (mice spleen cells): concentration of
compounds required for more than 0.9 IU/mL induction of IFN.
Scheme 5. Reagents and conditions: (a) Raney Ni, NH4OH, CH3OH,
reflux (61%); (b) C3H7CHO, NaBH3CN, CH3OH, rt (40%).

2718
K. Hirota et al. / Bioorg. Med. Chem. 11 (2003) 2715–2722
Table 2. IFN-inducing activities of 2,6-disubstituted 9-benzyl-8-
hydroxypurines
Experimental
Chemistry
Melting points were determined on a Yanagimoto
melting-point apparatus and are uncorrected. UV
absorption spectra were recorded on a Shimadzu 260
spectrophotometer. IR spectra were measured using
Perkin Elmer 1640 FT-IR spectrometer. 1H NMR
Compd
R1
R2
MECa (mM)
spectra were recorded on
a JeolJNM EX-400
(400 MHz) spectrometer using DMSO-d6 as a solvent.
Chemicalshifts are given in ppm ( d), coupling constants
(J) are given in Hz, and splitting patterns are designated
as follows: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad; *, deuterium exchangeable. 13C
NMR spectra were recorded on a JeolJNM EX-400
(100 MHz) spectrometer using DMSO-d6 as a solvent.
Chemicalshifts are given in ppm ( d) relative to internal
solvent signals. Mass spectra were recorded on a JMS-
SX 102A spectrometer. Elemental analyses were per-
formed on Yanagimoto MT-3, and the results (C, H, N)
were within Æ0.3% of the theoreticalvaul es. Thin-al yer
chromatographic (TLC) analyses were carried out on
0.25 mm Silica Gel 60 F254 plates (Art 5715, Merck).
The silica gel used for column chromatography was
Silica Gel 60 (230–400 mesh, Merck).
25
27
3
NHC4H9
SC3H7
SC3H7
H
NHC2H5
NH2
1
10
0.01
0.1
1
4
Imiquimod
NHC4H9
NH2
aMinimum effective concentration (mice spleen cells): concentration of
compounds required for more than 0.9 IU/mL induction of IFN.
1.17 Similarly, 2-butoxy-8-mercapto analogue (9) pos-
sessed activity equalto that of the corresponding
8-hydroxylanaolgue 2 with MEC of 0.001 mM. How-
ever, S-methylation of 9 resulted in a drastic decrease
of the activity (10, MEC=10 mM). An analogous ten-
dency was observed in the 2-butylamino series.
8-Unsubstituted analogue (16) showed the activity
with MEC of 1 mM, which was equipotent with the
2-butoxy analogue 6. 8-Methoxy analogue (18) was
inactive (MEC=>10 mM) unlike the case of 2-butoxy
analogue 7. Interestingly, the activity of 2-butylamino-
8-mercapto analogue (22, MEC=0.01 mM) was 10-fold
more potent than that of the corresponding
8-hydroxylanaolgue 4.
9-Benzyl-2-butoxyadenine (6). After two vacuum/H2
cycles to remove air from the reaction flask, the mixture
of 518 (100 mg, 0.27 mmol), 10% Pd/C (10% the weight
of 5) and Et3N (44 mL, 0.32 mmol) in MeOH (5 mL) was
hydrogenated at ambient pressure (balloon) and tem-
perature for 2 h. The reaction mixture was filtered using
a Celite cake and the filtrate was evaporated. The resi-
due was chromatographed on silica gel (CHCl3/
MeOH=100/1) to give 6 (75 mg, 94%). Mp 182–183 ꢀC
(washed with hexane) (lit.,26 174–175 ꢀC); UV (EtOH)
lmax 268.8 nm (e 13,200); IR (KBr) n cmÀ1: 3307, 3150,
As shown in Table 2, removalof the 6-amino group
from 2-butylamino-8-hydroxyadenine 4 resulted in
some reduction of IFN-inducing activity (25,
MEC=1 mM). On the other hand, the introduction of
an ethylgroup into the 6-amino group of 8-hydroxy-2-
propylthioadenine 3 markedly decreased the activity
(27, MEC=10 mM).
1
1660, 1599, 1338; H NMR d 8.02 (1H, s, 8-H), 7.25–
7.35 (5H, m, 9-Ph), 7.16 (2H, brs*, 6-NH2), 5.25 (2H, s,
9-CH2), 4.20 (2H, t, J=6.6 Hz, 2-CH2), 1.60–1.67 (2H,
m, 2-CH2), 1.34–1.43 (2H, m, 2-CH2), 0.91 (3H, t,
J=7.3 Hz, 2-CH3); 13C NMR d 161.5, 156.7, 151.2,
139.3, 137.2, 128.6, 127.6, 115.0, 65.7, 45.9, 30.6, 18.7,
13.7; MS (EI) m/z 297 (M+), 241, 240, 91; HRMS (EI)
calcd for C16H19N5O (M+): 297.1590. Found: 297.1595;
Anal. calcd for C16H19N5O: C, 64.63; H, 6.44; N, 23.55.
Found: C, 64.55; H, 6.38; N, 23.42.
Conclusion
In summary, we have synthesized 8-substituted 9-ben-
zyladenines and 6-substituted 9-benzyl-8-hydroxy-
purines bearing an appropriate chain substituent such
as a butoxy, propylthio or butylamino group at the
2-position and evaluated their IFN-inducing activities in
order to investigate the structure–activity relationships
of 2-substituted 9-benzyl-8-hydroxyadenines (2–4) and
to explore highly active compounds. Among them,
2-butoxy-8-mercaptoadenine derivative (9), having an
acidic proton at the 8-position like 8-hydroxyadenine 2,
emerged as having the most potent activity. The typical
characteristics of the qualitative structure–activity rela-
tionship at the 8-position of the adenine nucleus are as
follows: OH, SH> >H, halogene, CH3>OCH3, SCH3.
It has been found that both of a 6-amino group and an
8-hydroxylor thiolgroup are requisite to express exce-l
lent IFN-inducing activity and that the presence of the
2-butoxy group brings the most significant increase of
the activity.
9-Benzyl-2-butoxy-8-methyladenine (8). A mixture of 518
(100 mg, 0.27 mmol), 1 M hexane solution of AlMe3
(0.53 mL, 0.53 mmol), PdCl2 (2.4 mg, 0.01 mmol) and
Ph3P (7.0 mg, 0.03 mmol) in dry THF (5 mL) was
refluxed under Ar atmosphere for 10 h. After evapora-
tion, the residue was triturated with CHCl3 (10 mL) and
the resulting suspension was filtered using a Celite cake.
The filtrate was partitioned between CHCl3 (10 mL) and
H2O (10 mL), whose organic layer was washed with
brine (10 mL) and dried over MgSO4. After filtration
and evaporation, the residue was chromatographed on
silica gel (CHCl3/MeOH=200/1), which was triturated
with hexane (2 mL) to give 8 (80 mg, 96%). Mp
135–136 ꢀC (washed with hexane); UV (EtOH) lmax
269.4 nm (e 13,600); IR (KBr) n cmÀ1: 3313, 3148, 2958,

K. Hirota et al. / Bioorg. Med. Chem. 11 (2003) 2715–2722
2719
1
1654, 1600, 1465, 1342; H NMR d 7.18–7.34 (5H, m,
5-Amino-1-benzyl-2-chloro-4-cyanoimidazole (12). To a
solution of 1122 (700 mg, 3.53 mmol) in dry THF
(15 mL) was added N-chlorosuccinimide (519 mg,
3.89 mmol) in dry THF (15 mL), and the reaction mix-
ture was stirred under Ar atmosphere at room tem-
perature for 10 h. After evaporation, the resulting solid
was partitioned between AcOEt (50 mL) and saturated
NaHCO3 (50 mL) solution. The organic layer was
washed with brine (30 mL) and dried over MgSO4. After
filtration, the solvent was evaporated off, and the soli-
dified product was chromatographed on silica gel
(CHCl3/MeOH=50/1) to give 12 (713 mg, 87%): mp
206–208 ꢀC (recrystallized from benzene); UV (EtOH)
lmax 249.0 nm (e 14,000); IR (KBr) n cmÀ1: 3334, 3179,
2222, 1650, 1596, 1502, 1195, 725; 1H NMR d 7.28–7.38
(3H, m, 1-Ph), 7.13 (2H, d, J=7.8 Hz, 1-Ph), 6.69 (2H,
s*, 5-NH2), 5.12 (2H, s, 1-CH2); 13C NMR d 149.2,
135.2, 128.7, 127.7, 126.5, 124.7, 116.2, 88.8, 45.8; MS
(EI) m/z 234 (M++2), 232 (M+), 91; HRMS (EI) calcd
for C11H9ClN4 (M+): 232.0516. Found: 232.0512; anal.
calcd for C11H9ClN4: C, 56.78; H, 3.90; N, 24.08.
Found: C, 56.91; H, 4.00; N, 24.13.
9-Ph), 7.06 (2H, brs*, 6-NH2), 5.24 (2H, s, 9-CH2), 4.19
(2H, t, J=6.6 Hz, 2-CH2), 2.34 (3H, s, 8-CH3),
1.60–1.67 (2H, m, 2-CH2), 1.34–1.43 (2H, m, 2-CH2),
0.90 (3H, t, J=7.3 Hz, 2-CH3); 13C NMR d 161.0,
155.8, 152.4, 146.8, 136.9, 128.7, 127.5, 127.0, 113.7,
65.7, 44.7, 30.7, 18.7, 13.7; MS (EI) m/z 311 (M+), 282,
268, 255, 240, 91; HRMS (EI) calcd for C17H21N5O
(M+): 311.1746. Found: 311.1754; Anal. calcd for
C17H21N5O: C, 65.57; H, 6.80; N, 22.49. Found: C,
65.34; H, 6.71; N, 22.31.
9-Benzyl-2-butoxy-8-thioadenine (9). A mixture of 518
(200 mg, 0.53 mmol) and thiourea (81 mg, 1.06 mmol) in
dry EtOH (8 mL) was refluxed under Ar atmosphere for
24 h. After evaporation, the residue was partitioned
between AcOEt (20 mL) and H2O (20 mL). The organic
layer was washed with brine (20 mL) and dried over
MgSO4. After filtration, the residue was chromato-
graphed on silica gel (CHCl3/MeOH=50/1) to give 9
(151 mg, 86%). Mp 268–269 ꢀC (recrystallized from
EtOH); UV (EtOH) lmax 241.2 nm (e 15,000), 267.8 nm
(e 7600), 314.8 nm (e 28,000); IR (KBr) n cmÀ1: 3396,
1
3171, 1678, 1616, 1486, 1354; H NMR d 12.18 (1H, s*,
9-Benzyl-8-chloro-2-hydroxyadenine (13). A mixture of
12 (650 mg, 2.79 mmol) and urea (1.68 g, 27.94 mmol)
was heated under Ar atmosphere at 160 ꢀC for 24 h. An
additionalamount of urea (1.68 g, 27.94 mmo)l was
added to the reaction mixture and the resulting mixture
was heated at 160 ꢀC for 12 h. The residue was triturated
with H2O (20 mL) and the resulting precipitate was fil-
tered to give 13 (604 mg, 78%): mp >300 ꢀC (washed
8-SH), 7.23–7.38 (5H, m, 9-Ph), 6.85 (2H, brs*, 6-NH2),
5.26 (2H, s, 9-CH2), 4.17 (2H, t, J=6.3 Hz, 2-CH2),
1.58–1.65 (2H, m, 2-CH2), 1.32–1.41 (2H, m, 2-CH2),
0.89 (3H, t, J=7.1 Hz, 2-CH3); 13C NMR d 166.0,
161.3, 150.9, 148.6, 136.3, 128.3, 127.8, 127.4, 102.6,
66.1, 45.0, 30.4, 18.6, 13.6; MS (EI) m/z 329 (M+), 273,
240, 182, 91; HRMS (EI) calcd for C16H19N5OS (M+):
329.1310. Found: 329.1304; Anal. calcd for
C16H19N5OS: C, 58.34; H, 5.81; N, 21.26. Found: C,
58.28; H, 5.74; N, 21.34.
1
with hot MeOH and hot H2O); H NMR d 10.63 (1H,
brs*, 2-OH), 7.63 (2H, brs*, 6-NH2), 7.22–7.36 (5H, m,
9-Ph), 5.13 (2H, s, 9-CH2); MS (EI) m/z 277 (M++2),
275 (M+), 240, 91; HRMS (EI) calcd for C12H10ClN5O
(M+): 275.0574. Found: 275.0567.
9-Benzyl-2-butoxy-8-methylthioadenine (10). A mixture
of
9
(100 mg, 0.30 mmol) and K2CO3 (44 mg,
0.32 mmol) in dry DMF (6 mL) was stirred under Ar
atmosphere at room temperature for 0.5 h. To the mix-
ture was added methyliodide (23 mL, 0.36 mmol) and
the mixture was stirred at room temperature for 3 h.
The solvent was removed under reduced pressure and
the residue was triturated with H2O (5 mL). The
resulting suspension was neutralized with 10%
NaHSO4 solution and extracted with AcOEt (20 mL).
The organic layer was washed with brine (20 mL) and
dried over MgSO4. After filtration, the solvent was
evaporated and the residue was chromatographed on
silica gel (CHCl3/MeOH=200/1) to give 10 (87 mg,
83%): mp 158–159 ꢀC (recrystallized from EtOH); UV
9-Benzyl-2-butoxy-8-chloroadenine (14). A mixture of 13
(400 mg, 1.45 mmol) and K2CO3 (602 mg, 4.35 mmol) in
dry DMF (8 mL) was stirred under Ar atmosphere at
room temperature for 1 h. To the mixture was added
butylbromide (0.47 mL, 4.35 mmo)l and the mixture
was stirred at room temperature for 2 days. The solvent
was removed under reduced pressure and the residue
was triturated with H2O (15 mL). The resulting suspen-
sion was neutralized with 10% NaHSO4 solution and
extracted with AcOEt (30 mL). The organic layer was
washed with brine (30 mL) and dried over MgSO4. After
filtration, the solvent was evaporated and the residue
was chromatographed on silica gel (CHCl3). The
obtained oily product was solidified with ether (3 mL)
and filtered to give 14 (319 mg, 66%): mp 144–145 ꢀC
(washed with hexane and MeOH); UV (EtOH) lmax
270.6 nm (e 14,600); IR (KBr) n cmÀ1: 3488, 3114, 2958,
1639, 1594, 1348, 731; 1H NMR d 7.37 (2H, brs*,
6-NH2), 7.24–7.36 (5H, m, 9-Ph), 5.25 (2H, s, 9-CH2),
4.21 (2H, t, J=6.6 Hz, 2-CH2), 1.61–1.68 (2H, m,
2-CH2), 1.34–1.43 (2H, m, 2-CH2), 0.90 (3H, t,
J=7.3 Hz, 2-CH3); 13C NMR d 161.6, 155.8, 152.0,
135.9, 134.1, 128.7, 127.8, 127.2, 113.7, 66.0, 45.6, 30.6,
18.7, 13.7; MS (EI) m/z 333 (M++2), 331 (M+), 275,
91; HRMS (EI) calcd for C16H18ClN5O (M+):
331.1200. Found: 331.1208; Anal. calcd for
(EtOH) lmax 282.4 nm (e 16,700); IR (KBr) n cmÀ1
:
3490, 3101, 1641, 1595, 1354, 1265, 1194, 732; 1H
NMR d 7.22–7.34 (5H, m, 9-Ph), 7.10 (2H, s*,
6-NH2), 5.16 (2H, s, 9-CH2), 4.19 (2H, t, J=6.6 Hz,
2-CH2), 2.61 (3H, s, 8-CH3), 1.60–1.67 (2H, m,
2-CH2), 1.34–1.43 (2H, m, 2-CH2), 0.90 (3H, t,
J=7.3 Hz, 2-CH3); 13C NMR d 160.9, 154.9, 153.3,
146.4, 136.3, 128.6, 127.6, 127.3, 115.1, 65.8, 45.2,
30.6, 18.7, 14.3, 13.7; MS (EI) m/z 343 (M+), 287,
196, 91; HRMS (EI) calcd for C17H21N5OS (M+):
343.1467. Found: 343.1462; Anal. calcd for
C17H21N5OS: C, 59.45; H, 6.16; N, 20.39. Found: C,
59.42; H, 6.22; N, 20.31.

2720
K. Hirota et al. / Bioorg. Med. Chem. 11 (2003) 2715–2722
C16H18ClN5O: C, 57.92; H, 5.47; N, 21.11. Found: C,
57.90; H, 5.50; N, 21.10.
washed with brine (20 mL) and dried over MgSO4. After
filtration, the solvent was removed under reduced pres-
sure, and the product was chromatographed on silica
2-Amino-9-benzyladenine (15). To a solution of Na
(0.65 g, 28.25 mmol) in dry EtOH (30 mL) was added
guanidine hydrochloride (3.37 g, 35.31 mmol) and the
mixture was stirred at room temperature for 5 min. The
resulting precipitate was filtered off and the filtrate was
refluxed with 1122 (0.70 g, 3.35 mmol) under Ar atmo-
sphere for 10 h. The solvent was removed under reduced
pressure and the residue was chromatographed on silica
gel(CHCl 3/MeOH=50/1) to give 15 (840 mg, 99%),
which was used for the next reaction without further
purification: mp. 192–193 ꢀC (lit.,27 180–181 ꢀC); 1H
NMR d 7.77 (1H, s, 8-H), 7.20–7.34 (5H, m, 9-Ph), 6.69
and 5.80 (each 2H, each s*, 2-NH2 and 6-NH2), 5.18
(2H, s, 9-CH2); 13C NMR d 160.5, 156.2, 151.8, 137.7,
137.4, 128.6, 127.4, 127.1, 113.1, 45.4.
gel(CHCl /MeOH=100/1) to give 17 (271 mg, 70%):
3
mp 239–241 ꢀC (washed with ether); UV (EtOH) lmax
251.6 nm (e 10,600), 283.0 nm (e 9600); IR (KBr) n
cmÀ1: 3486, 3322, 3182, 1604, 1401, 1337, 1242; 1H
NMR d 7.23–7.33 (3H, m, 9-Ph), 7.16 (2H, d,
J=7.3 Hz, 9-Ph), 6.34 and 5.64 (each 2H, each s*, 2-
NH2 and 6-NH2), 4.97 (2H, s, 9-CH2), 3.97 (3H, s, 8-
CH3); 13C NMR d 159.2, 154.0, 152.4, 151.5, 137.1,
128.5, 127.3, 126.8, 107.7, 56.5, 43.3; MS (EI) m/z 270
(M+), 255, 179, 91; HRMS (EI) calcd for C13H14N6O
(M+): 270.1229. Found: 270.1224; anal. calcd for
C13H14N6O: C, 57.77; H, 5.22; N, 31.09. Found: C,
57.89; H, 5.23; N, 30.93.
9-Benzyl-2-butylamino-8-methoxyadenine (18). Com-
pound 18 was prepared by the reaction of 17 with
butyraldehyde in the presence of NaBH3CN according
to the synthetic procedure for the preparation of 16 (for
2 days): Yield 85%; mp 134–135 ꢀC (recrystallized from
toluene); UV (EtOH) lmax 253.6 nm (e 11,700),
288.0 nm (e 7800); IR (KBr) n cmÀ1: 3332, 3197, 2932,
9-Benzyl-2-butylaminoadenine (16). To a suspension of
15 (100 mg, 0.42 mmol) and NaBH3CN (157 mg,
2.50 mmol) in MeOH (5 mL) was added butyraldehyde
(0.30 mL, 3.33 mmol) and the mixture was stirred under
Ar atmosphere at room temperature for 2 days. After
evaporation, the residue was chromatographed on silica
1
1608, 1528, 1399; H NMR d 7.22–7.33 (5H, m, 9-Ph),
gel(CHCl /MeOH=100/1) to give 16 (104 mg, 84%):
3
6.27 (2H, s*, 6-NH2), 6.04 (1H, t*, J=6.3 Hz, 2-NH),
4.96 (2H, s, 9-CH2), 3.98 (3H, s, 8-CH3), 3.19 (2H, q,
J=6.3 Hz, 2-CH2), 1.42–1.49 (2H, m, 2-CH2), 1.25–1.34
(2H, m, 2-CH2), 0.86 (3H, t, J=7.3 Hz, 2-CH3); 13C
NMR d 158.5, 153.8, 152.2, 151.4, 137.1, 128.4, 127.3,
127.1, 107.5, 56.4, 43.4, 40.7, 31.5, 19.7, 13.8; MS (EI)
m/z 326 (M+), 297, 283, 270, 91; HRMS (EI) calcd for
C17H22N6O (M+): 326.1855. Found: 326.1862.
mp 147–148 ꢀC (washed with hexane); UV (EtOH) lmax
259.0 nm (e 9900), 289.4 nm (e 9500); IR (KBr) n cmÀ1
:
3322, 2956, 1599, 1539, 1482, 721; 1H NMR d 7.77 (1H,
s, 8-H), 7.23–7.33 (5H, m, 9-Ph), 6.60 (2H, brs*,
6-NH2), 6.19 (1H, t*, J=6.7 Hz, 2-NH), 5.16 (2H, s,
9-CH2), 3.22 (2H, q, J=6.7 Hz, 2-CH2), 1.43–1.51 (2H,
m, 2-CH2), 1.25–1.34 (2H, m, 2-CH2), 0.87 (3H, t,
J=7.3 Hz, 2-CH3); 13C NMR d 159.6, 155.9, 151.7,
137.6, 137.1, 128.5, 127.6, 127.5, 113.0, 45.6, 40.7, 31.5,
19.7, 13.9; MS (EI) m/z 296 (M+), 267, 253, 240, 91;
HRMS (EI) calcd for C16H20N6 (M+): 296.1749.
5-Amino-2-benzylamino-4-cyanothiazole (19). To a sus-
pension of aminomalononitrile p-toluenesulfonate
(2.00 g, 7.90 mmol) in dry THF (20 mL) was added
N,N-diisopropylethylamine (1.10 mL, 6.32 mmol) and
the mixture was stirred under Ar atmosphere at room
temperature for 5 min. To the solution was added ben-
zylisothiocyanate (2.09 mL, 15.79 mmo)l in dry THF
(20 mL) and the mixture was stirred at 40 ꢀC for 72 h.
The solvent was removed under reduced pressure, and
the residue was partitioned between AcOEt (100 mL)
and H2O (100 mL). The organic layer was washed with
brine (50 mL) and dried over MgSO4. The solvent was
evaporated and the residue was chromatographed on
silica gel (CHCl3) to give 19 (1.57 g, 86%): mp 143–
145 ꢀC (washed with hexane); UV (EtOH) lmax
.
Found: 296.1765; Anal. calcd for C16H20N6 1/8H2O: C,
64.35; H, 6.84; N, 28.14. Found: C, 64.40; H, 6.77; N,
28.08.
2-Amino-9-benzyl-8-methoxyadenine (17). The reaction
of 5-amino-1-benzyl-2-bromo-4-cyanoimidazole18 with
guanidine under the same conditions as those for the
preparation of 15 gave 2-amino-9-benzyl-8-bromoade-
nine (for 9 h): Yield 90%; mp 274–275 ꢀC (washed with
ether); UV (EtOH) lmax 260.2 nm (e 9800), 286.0 nm (e
12700); IR (KBr) n cmÀ1: 3466, 3310, 3191, 1595, 1473;
1H NMR d 7.25–7.35 (3H, m, 9-Ph), 7.16 (2H, d,
J=6.8 Hz, 9-Ph), 6.88 (2H, brs*, 2-NH2 or 6-NH2), 5.96
(2H, s*, 2-NH2 or 6-NH2), 5.16 (2H, s, 9-CH2); 13C
NMR d 160.5, 155.1, 153.1, 136.4, 128.7, 127.5, 126.7,
121.2, 113.4, 45.8; MS (EI) m/z 320 (M++2), 318
(M+), 239, 91; HRMS (EI) calcd for C12H11BrN6
(M+): 318.0229. Found: 318.0233; Anal. calcd for
C12H11BrN6: C, 45.16; H, 3.47; N, 26.33. Found: C,
45.09; H, 3.43; N, 26.18. A mixture of 2-amino-9-ben-
zyl-8-bromoadenine (459 mg, 1.44 mmol) in MeOH
(10 mL) and 1 N NaOH solution (10 mL) was refluxed
for 2 days. After evaporation, the residue was triturated
with H2O (20 mL) and the mixture was neutralized with
10% NaHSO4 solution. The resulting mixture was
extracted with AcOEt (30 mL) and the organic layer was
252.2 nm (e 7600), 305.0 nm (e 7300); IR (KBr) n cmÀ1
:
3410, 3300, 3198, 2212, 1612, 1547, 1213, 699; 1H NMR
d 7.54 (1H, t*, J=5.9 Hz, 2-NH), 7.22–7.34 (5H, m,
2-Ph), 6.45 (2H, s*, 5-NH2), 4.31 (2H, d, J=5.9 Hz,
2-CH2); 13C NMR d 153.6, 153.4, 139.2, 128.3, 127.4,
126.9, 116.9, 95.8, 46.7; MS (EI) m/z 230 (M+), 91;
HRMS (EI) calcd for C11H10N4S (M+): 230.0626.
Found: 230.0633; Anal. calcd for C11H10N4S: C, 57.37;
H, 4.38; N, 24.33. Found: C, 57.21; H, 4.23; N, 24.10.
5-Amino-1-benzyl-4-cyano-2-mercaptoimidazole (20).
The suspension of 19 (500 mg, 2.17 mmol) in 5%
Na2CO3 solution (15 mL) was refluxed for 2 h. The mix-
ture was neutralized with 1 N HCl solution and the pre-

K. Hirota et al. / Bioorg. Med. Chem. 11 (2003) 2715–2722
2721
cipitated solid was collected, which was chromatographed
on silica gel (CHCl3/MeOH=50:1) to give 20 (420 mg,
84%): mp 280–283 ꢀC (recrystallized from MeOH); UV
Celite cake, the filtrate was concentrated in vacuo. The
residue was chromatographed on silica gel (CHCl3/
MeOH=40:1) to give 24 (56 mg, 61%): mp 268–269 ꢀC;
(EtOH) lmax 289.4 nm (e 17,600); IR (KBr) n cmÀ1
:
UV (EtOH) lmax 314.0 nm (e 8300); IR (KBr) n cmÀ1
:
3329, 3183, 2216, 1656, 1468, 1334, 1171, 696; 1H NMR
d 12.39 (1H, s*, 2-SH), 7.25–7.33 (5H, m, 1-Ph), 6.77
(2H, s*, 5-NH2), 5.21 (2H, s, 1-CH2); 13C NMR d 160.0,
147.1, 135.6, 128.3, 127.3, 127.0, 113.1, 75.1, 45.1; MS
(EI) m/z 230 (M+), 91; HRMS (EI) calcd for
C11H10N4S (M+): 230.0626. Found: 230.0619; Anal.
calcd for C11H10N4S: C, 57.37; H, 4.38; N, 24.33.
Found: C, 57.39; H, 4.58; N, 24.11.
3462, 1720, 1641, 1453; H NMR d 10.80 (1H, s*, 8-
OH), 7.74 (1H, s, 6-H), 7.24–7.34 (5H, m, 9-Ph), 6.20
(2H, s*, 2-NH2), 4.87 (2H, s, 9-CH2); 13C NMR d 159.2,
153.1, 151.2, 136.8, 134.5, 128.5, 127.4, 127.1, 113.5,
42.0; MS (EI) m/z 241 (M+), 150, 91; HRMS (EI) calcd
for C12H11N5O (M+): 241.0964. Found: 241.0957.
1
9-Benzyl-2-butylamino-8-hydroxypurine (25). Com-
pound 25 was prepared by the reaction of 24 with
butyraldehyde in the presence of NaBH3CN according
to the synthetic procedure for the preparation of 16 (for 2
days): Yield 40%; mp 224–225 ꢀC (washed with ether);
UV (EtOH) lmax 227.4 nm (e 19,600), 324.2 nm (e 7900);
IR (KBr) n cmÀ1: 3354, 2957, 1708, 1639, 1540, 1461,
1132, 698; 1H NMR d 10.75 (1H, s*, 8-OH), 7.77 (1H, s,
6-H), 7.23–7.33 (5H, m, 9-Ph), 6.72 (1H, t*, J=6.3 Hz,
2-NH), 4.86 (2H, s, 9-CH2), 3.18 (2H, t, J=6.3 Hz,
2-CH2), 1.42–1.49 (2H, m, 2-CH2), 1.24–1.33 (2H, m,
2-CH2), 0.86 (3H, t, J=7.3 Hz, 2-CH3); 13C NMR d
158.3, 153.1, 151.1, 136.9, 134.4, 128.5, 127.4, 113.2,
42.0, 40.8, 31.2, 19.7, 13.8; MS (EI) m/z 297 (M+), 268,
254, 241, 91; HRMS (EI) calcd for C16H19N5O (M+):
297.1590. Found: 297.1587; Anal. calcd for C16H19N5O:
C, 64.63; H, 6.44; N, 23.55. Found: C, 64.36; H, 6.44; N,
23.35.
2-Amino-9-benzyl-8-mercaptoadenine (21). Method A.
Compound 21 was prepared by the reaction of 20 with
guanidine according to the synthetic procedure for the
preparation of 15 in 69% yield (for 2 days).
Method B. To a solution of Na (100 mg, 4.34 mmol)
in dry EtOH (3 mL) was added guanidine hydrochlo-
ride (415 mg, 4.34 mmol) and the mixture was stirred
under Ar atmosphere at room temperature for 5 min.
To the resulting mixture was added 19 (100 mg,
0.43 mmol) in dry EtOH (2 mL) and the mixture was
refluxed for 17 h. After evaporation, the residue was
triturated with H2O (10 mL). The mixture was neu-
tralized with AcOH and extracted with AcOEt
(20 mL). The organic layer was washed with brine
(10 mL) and dried over MgSO4. The solvent was eva-
porated to give 21 (90 mg, 76%): mp 290–292 ꢀC; UV
(EtOH) lmax 268.4 nm (e 9100), 315.8 nm (e 22,600); IR
(KBr) n cmÀ1: 3415, 1621, 1476, 1437; 1H NMR d 11.93
(1H, s*, 8-SH), 7.23–7.32 (5H, m, 9-Ph), 6.41 and 6.01
(each 2H, each s*, 2-NH2 and 6-NH2), 5.21 (2H, s, 9-
CH2); 13C NMR d 164.4, 160.4, 151.2, 148.3, 136.5,
128.2, 127.3, 127.1, 100.1, 44.7; MS (EI) m/z 272 (M+),
239, 181, 154, 91; HRMS (EI) calcd for C12H12N6S
(M+): 272.0844. Found: 272.0851.
9-Benzyl-N6-ethyl-8-hydroxy-2-propylthioadenine (27).
To a suspension of 2618 (100 mg, 0.32 mmol) and
NaBH3CN (120 mg, 1.90 mmol) in dry MeOH (20 mL)
was added acetaldehyde (0.46 mL, 8.24 mmol) and the
mixture was stirred under Ar atmosphere at 50 ꢀC for 8
days. After evaporation, the residue was chromato-
graphed on silica gel (CHCl3/MeOH=100:1) to give 27
(75 mg, 69%): mp 214–215 ꢀC (washed with ether); UV
(EtOH) lmax 229.2 nm (e 20,700), 287.2 nm (e 16,600);
IR (KBr) n cmÀ1: 3348, 2965, 1701, 1636, 1452, 1345,
9-Benzyl-2-butylamino-8-mercaptoadenine (22). Com-
pound 22 was prepared by the reaction of 21 with butyr-
aldehyde in the presence of NaBH3CN according to the
synthetic procedure for the preparation of 16 (for 2 days):
Yield 72%; mp 272–274 ꢀC (recrystallized from EtOH);
UV (EtOH) lmax 271.2 nm (e 12,100), 321.2 nm (e
23,100); IR (KBr) n cmÀ1: 3370, 3129, 1625, 1537, 1460,
1
701; H NMR d 10.01 (1H, s*, 8-OH), 7.23–7.33 (5H,
m, 9-Ph), 6.55 (1H, brt*, J=5.4 Hz, 6-NH), 4.86 (2H, s,
9-CH2), 3.37–3.43 (2H, m, 6-CH2), 2.97 (2H, t,
J=7.3 Hz, 2-CH2), 1.58–1.67 (2H, m, 2-CH2), 1.16 (3H,
t, J=7.1 Hz, 6-CH3), 0.93 (3H, t, J=7.1 Hz, 2-CH3);
13C NMR d 161.5, 151.7, 147.3, 146.0, 137.1, 128.5,
127.43, 127.37, 100.3, 42.4, 34.9, 32.2, 22.8, 14.9, 13.3;
MS (EI) m/z 343 (M+), 328, 310, 91; HRMS (EI) calcd
for C17H21N5OS (M+): 343.1467. Found: 343.1463;
Anal. calcd for C17H21N5OS: C, 59.45; H, 6.16; N,
20.39. Found: C, 59.18; H, 6.15; N, 20.14.
1
1369, 696; H NMR d 11.89 (1H, s*, 8-SH), 7.23–7.37
(5H, m, 9-Ph), 6.48 (1H, brt*, J=6.5 Hz, 2-NH), 6.39
(2H, brs*, 6-NH2), 5.20 (2H, s, 9-CH2), 3.17 (2H, q,
J=6.5 Hz, 2-CH2), 1.40–1.47 (2H, m, 2-CH2), 1.23–1.32
(2H, m, 2-CH2), 0.85 (3H, t, J=7.1 Hz, 2-CH3); 13C
NMR d 164.1, 159.5, 151.1, 148.2, 136.7, 128.2, 127.8,
127.3, 99.9, 44.8, 40.6, 31.3, 19.7, 13.8; MS (EI) m/z 328
(M+), 285, 91; HRMS (EI) calcd for C16H20N6S (M+):
328.1470. Found: 328.1462; anal. calcd for C16H20N6S:
C, 58.51; H, 6.14; N, 25.59. Found: C, 58.61; H, 6.15; N,
25.64.
Biology
IFN induction in mouse splenocyte cultures. Male C3H/
HeJ mice (Clea Japan Inc.) aged 8 weeks were sacrificed,
spleens were removed from 6 mice. Spleens were meshed
in phosphate buffered saline (PBS) and filtered through
nylon mesh. The cell suspension was freed of ery-
throcytes by hypotonic treatment with 0.2% NaCl
solution, and washed twice with PBS. Splenocytes were
resuspended at a concentration of 2 Â 106 cells/mL in
MEM supplemented with 5% fetal calf serum, 100 U/
2-Amino-9-benzyl-8-hydroxypurine (24). To a suspension
of 2318 (116 mg, 0.38 mmol) in MeOH (10 mL) and 28%
NH4OH solution (2 mL) was added Raney Ni (1 mL of
suspension in H2O) and the mixture was refluxed for
9 h. After removalof the cataylst by fitlration using

2722
K. Hirota et al. / Bioorg. Med. Chem. 11 (2003) 2715–2722
mL of penicillin, and 100 mg/mL of streptomycin. The
test compounds were dissolved in DMSO and diluted to
500-fold with supplemented MEM.
9. Nichol, F. R.; Weed, S. D.; Underwood, G. E. Antimicrob.
Agents Chemother. 1976, 9, 433.
10. Tamm, I.; Sehgal, P. B. J. Exp. Med. 1977, 145, 344.
11. Taylor, J. L.; Schoenherr, C. K.; Grossberg, S. E. Anti-
microb. Agents Chemother. 1980, 18, 20.
12. Wierenga, W.; Skulnick, H. I.; Stringfellow, D. A.; Weed,
S. D.; Renis, H. E.; Eidson, E. E. J. Med. Chem. 1980, 23, 237.
13. Weeks, C. E.; Reiter, M. J. J. Invest. Dermatol. 1989, 93,
584.
14. Dockrell, D. H.; Kinghorn, G. R. J. Antimicrob. Che-
mother. 2001, 48, 751.
15. Weeks, C. E.; Gibson, S. J. J. Interferon Res. 1994, 14,
81.
16. Tomai, M. A.; Gibson, S. J.; Imbertson, L. M.; Miller,
R. L.; Myhre, P. E.; Reiter, M. J.; Wagner, T. L.; Tamulinas,
C. B.; Beaurline, J. M.; Gerster, J. F.; Horton, V. L. Antiviral
Res. 1995, 28, 253.
17. Hirota, K.; Kazaoka, K.; Niimoto, I.; Kumihara, H.;
Sajiki, H.; Isobe, Y.; Takaku, H.; Tobe, M.; Ogita, H.; Ogino,
T.; Ichii, S.; Kurimoto, A.; Kawakami, H. J. Med. Chem.
2002, 45, 5419.
Above splenocytes suspension (0.5 mL) and various
concentration of the test compounds solution (0.5 mL)
were mixed in 24-well plates, and cultured in a humidi-
fied 5% CO2/95% air atmosphere at 37 ꢀC for 18 h.
Supernatants were then collected, filter sterilized, and
stored at À80 ꢀC untilthey were anayl zed for IFN.
IFN analysis. Mouse IFN titer in supernatants of sple-
nocytes was quantitated by measuring its antiviral
activity in a bioassay using mouse L929 cell monolayers
challenged with vesicular stomatitis virus.24 Results are
expressed as IFN IU/mL in terms of the international
mouse IFN standard25 obtained from the National
Institute of Health, Bethesda, MD, USA.
18. Hirota, K.; Kazaoka, K.; Niimoto, I.; Sajiki, H. Org.
Biomol. Chem. 2003, 1, 1354.
Acknowledgements
19. Kazaoka, K.; Sajiki, H.; Hirota, K. Chem. Pharm. Bull.
2003, 51, 608.
20. Hirota, K.; Kitade, Y.; Kanbe, Y.; Maki, Y. J. Org.
Chem. 1992, 57, 5268.
21. Lin, T.-S.; Cheng, J.-C.; Ishiguro, K.; Sartorelli, A. C.
J. Med. Chem. 1985, 28, 1194.
The authors thank Dr. H. Hayashi and his co-workers,
Japan Energy Corporation for measurements of IFN-
inducing activities.
22. (a) Booth, B. L.; Carpenter, R. A.; Morlock, G.; Mah-
mood, Z.; Pritchard, R. B. Synthesis 2001, 2393. (b) Sen, A. K.;
Ray, S. Indian J. Chem. 1976, 14B, 346.
References and Notes
1. World Health Organization. Weekly Epidemiological
Record. 1999, N-49.
2. Damen, M.; Bresters, D. In Hepatitis C Virus: Current
Studies in Hematology and Blood Transfusion No 62; Reesink,
H. W., Ed. Karger Publishers: Basel, 1998.
3. Ronnblom, L. E.; Tiensuu Janson, E.; Perers, A.; Oberg,
K. E.; Alm, G. V. Clin. Exp. Immunol. 1992, 89, 330.
4. Wierenga, W. Annu. Rep. Med. Chem. 1982, 17, 151.
5. Krueger, R. F.; Mayer, G. D. Science 1970, 169, 1213.
6. Siminoff, P.; Bernard, A. M.; Hursky, V. S.; Price, K. E.
Antimicrob. Agents Chemother. 1973, 3, 742.
23. Ring-transformation of 5-amino-2-benzylamino-4-carba-
moylthiazole into the corresponding 2-mercaptoimidazole was
previously reported. See: Sen, A. K.; Ray, S. Indian J. Chem.
1976, 14B, 351.
24. Watanabe, Y.; Kawade, Y. In Lymphokines and Inter-
ferons: A Practical Approach; Clemens, M. J.; Morris, A.
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