Synthesis and evaluation of 2-substituted 8-hydroxyadenines as potent interferon inducers with improved oral bioavailabilities

Article abstract of DOI:10.1016/j.bmc.2003.12.008

In order to create novel compounds which possess potent interferon (IFN) inducing activities with excellent oral bioavailabilities, a series of 8-hydroxyadenines, which have various alkoxy or alkylthio moieties at the adenine C(2)-position, were synthesiz

Full text of DOI:10.1016/j.bmc.2003.12.008

Bioorganic & Medicinal Chemistry 12 (2004) 1091–1099
Synthesis and evaluation of 2-substituted 8-hydroxyadenines as
potent interferon inducers with improved oral bioavailabilities
Ayumu Kurimoto,a,* Tetsuhiro Ogino,a Shinji Ichii,a Yoshiaki Isobe,a Masanori Tobe,a
Haruhisa Ogita,a Haruo Takaku,a Hironao Sajiki,b Kosaku Hirotab
and Hajime Kawakamia
aResearch Division, Discovery Research Laboratories II, Sumitomo Pharmaceuticals Co. Ltd., Konohana-ku, Osaka 554-0022, Japan
bLaboratory of Medicinal Chemistry, Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502-8585, Japan
Received 23 October 2003; accepted 5 December 2003
Abstract—In order to create novel compounds which possess potent interferon (IFN) inducing activities with excellent oral bio-
availabilities, a series of 8-hydroxyadenines, which have various alkoxy or alkylthio moieties at the adenine C(2)-position, were
synthesized and evaluated. The introduction of hydrophobic groups was not considered to be effective for potentiating the IFN-
inducing activity, but several compounds having hydrophilic groups were effective. Among the compounds tested, compound 13f
induced IFN from the dosage of 0.03 mg/kg, which was approximately 100-fold more potent than that of Imiquimod, and showed
an excellent oral bioavailability (F=40%) which was 10-fold improved over 5, a lead compound (F=4%).
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
compounds is one possible approach and orally bio-
available IFN inducing agents are envisaged as a new
therapeutic class of drugs for virus infections.
Infection by the hepatitis C virus (HCV), which is a
member of the Flaviviridae family of viruses, has been
recognized as one of the leading causes of liver impair-
ment such as cirrhosis and hepatocellular carcinoma in
humans. It is estimated that more than 170 million
people worldwide are infected, and of those infected,
approximately 20% are likely to develop liver cirrhosis
and 4% hepatocellular carcinoma, in the next decade.1
The currently approved treatments for chronic hepatitis
C are based on interferon-alpha (IFN-a), alone or in
combination with ribavirin. Although sustained
response rates are markedly improved using combi-
nation therapies, at least 50% of patients fail to show a
sustained response.2 Furthermore, IFN therapy is asso-
ciated with significant problems of patient compliance, a
loss of therapeutic efficacy as a result of the formation
of neutralizing antibodies against exogenous IFN.3 The
development of effective therapies to treat HCV infec-
tion is therefore highly important. Induction of endo-
genous IFN synthesis with small molecular-weight
In recent years, several natural and synthetic molecules
have been shown to induce IFN.4 For example, low
molecular-weight compounds including the fluor-
enones,5 pyrimidinones,6 and anthraquinones7 induced
IFN in various animal species. However, none of these
agents is capable of inducing high levels of IFN in
humans.8 Imiquimod (1), discovered and developed by
3M Pharmaceuticals, is a low molecular weight agent
(MW 240) with a potent IFN inducing property in
humans. It was launched in the US in 1997 for the
treatment of external genital and perianal warts/con-
dyloma acuminata.9 However, in the clinical trial of
Imiquimod for HCV, serious side effects such as vomit-
ing and liver dysfunction were observed in some
patients.10 Therefore, its further development was dis-
continued probably owing to these side effects.
Recently, we reported the discovery of a series of 8-
hydroxyadenines as novel IFN inducing agents.11 The
IFN inducing activities were remarkably improved by
the introduction of substituents into the adenine C(2)-
position (Table 1) and these analogues could be classi-
fied to following four types (2–5; Fig. 1).
Keywords: 8-hydroxyadenines; Orally active; Interferon inducer; HCV;
Imiquimod.
* Corresponding author. Tel.: +81-6-6466-5185; fax: +81-6-6466-
5483; e-mail: akurimot@sumitomopharm.co.jp
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.12.008

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A. Kurimoto et al. / Bioorg. Med. Chem. 12 (2004) 1091–1099
Scheme 1. Reagents and conditions: (a) NaSR, DMF, 110 ꢀC or NaOR, ROH, reflux; (b) Br2, CH2Cl2, rt; (c) 6N HCl, reflux; (d) NaOMe, MeOH,
reflux; (e) 12N HCl, rt
Scheme 2. Reagents and conditions (a) BzNCS, THF, rt; (b) 2N NaOH, THF, reflux; (c) RBr or RCl, K2CO3, DMF, rt
We have also conducted and disclosed the results of the
structure and activity relationship studies (SAR) based
on 2 and 3, but great improvement of the activities both
in vitro and in vivo could not be achieved.12,13 Compared
to compounds 2 and 3, compounds 4 and 5 demonstrated
potent in vitro activities. However, these compounds did
not show good oral absorption, their oral bioavailabilities
in rats were 3% and 4%, respectively. In order to create
compounds having potent IFN-inducing activities with
improved oral bioavailabilities, we conducted a detailed
SAR study based on 4 and 5 by the introduction of
various hydrophobic or hydrophilic groups into the 8-
hydroxyadenine C(2)-position.
2. Chemistry
The 2-alkylthio derivatives 11b–i, k, l and the 2-alkoxy
derivatives 13a–j were prepared by the method outlined
in Scheme 1. Treatment of 614 with appropriate sodium
thioalkoxides or alkoxides afforded the corresponding
2-alkylthioadenines 7 or 2-alkoxyadenines 8. Bromin-
ation at the C(8)-position was selectively carried out
with bromine to provide the 8-bromoadenines 9 or 10.
The 2-alkylthio derivatives 11b–i, k, and l were obtained
by the treatment of 9 with 6N HCl at reflux. In contrast,
the same treatment of 2-alkoxy-8-bromoadenines 10
gave only 13a. In order to obtain the 2-alkoxy deriv-
atives 13b–j, we employed another synthetic route, that
is, the reaction of 10 with sodium methoxide which
afforded the corresponding 8-methoxydenines 12 which
were then treated with 12N HCl at room temperature.
This route allowed the synthesis of the desired 2-alkoxy
derivatives 13b–j without any accompanying deal-
kylation product (13a).
Table 1. Activities of IFN induction
Compound
MECa (mM)
MEDb (mg/kg)
2
3
4
5
1
0.03
0.1
0.01
0.001
1
0.3
0.3
0.3
0.03
3
a MEC: Minimum Effective Concentration (mouse spleen cells).
b MED: Minimum Effective Dose (mouse, po).
The 2-thiol 11a and the 2-alkylthio derivatives 11j, m–z
were prepared as outlined in Scheme 2. The reaction of 14
with benzoyl isothiocyanate followed by treatment with
sodium hydroxide afforded 2-mercaptoadenine 11a.15
Subsequent S-alkylation with alkyl halides proceeded
selectively to give the 2-alkylthio derivatives 11j, m–z.
Figure 1. Structures of 1 (Imiquimod) and 2–5.

A. Kurimoto et al. / Bioorg. Med. Chem. 12 (2004) 1091–1099
1093
3. Results and discussion
effects of the phenyl ring substituents were investigated
using 11l. The analogues possessing electron-with-
drawing groups, like chloro (11n, o), were less active
than the parent compound (11l), the analogues posses-
sing electron-donating groups, like methoxy (11p, q)
showed equipotent activities with the parent compound
(11l). Thus, lead compound (4) exhibited the most
potent activity and the improvement of the IFN-indu-
cing activity was not achieved by the introduction of
hydrophobic groups into the C(2)-position. Next, in
order to evaluate the influence of hydrophilic groups at
the C(2)-position on the IFN-inducing activities, we
synthesized compounds containing ether (11r–u), dime-
thylamino (11v, w) and hydroxy (11x–z) groups at the
C(2)-alkyl side chain.
For in vitro studies, mouse spleen cells were cultured
with the prepared compounds, and the amounts of IFN
in supernatant were measured by a bioassay system
using L929 cells infected with vesicular stomatitis
virus.16 The results of the ability of the prepared
compounds to induce IFN are summarized in Tables 2
and 3.
In the case of the 2-alkylthio derivatives (Table 2), IFN-
inducing activity was improved by the elongation of the
alkyl chain, and the most suitable alkyl chain length was
3 or 4, (4, 11d). The minimum effective concentrations
(MECs) of these compounds were 0.01 mM, which was
approximately 100-fold stronger than that of Imiqui-
mod (1). The alkyl groups with the linear configuration
(4, 11d) were more potent than those of the branched
(11f–h) and cycloalkyl ones (11i, j). From the results of
compound 11k–m, directly binding an aromatic group
to the adenine was not preferable (11k), and the optimal
alkyl chain length between the adenine and the phenyl
group was found to be one (11l). While the electronic
Among the ether analogues, 11r showed a potent activ-
ity and the MEC was equivalent with the propyl analo-
gue (11d). Further elongation of the side chain resulted
in decreased activities (11s–u). In the dimethylamino
analogues, 11v showed a weak activity, but the one
methylene elongated compound (11w) exhibited a mod-
erate activity. Considering the results of the straight
alkyl analogues (4, 11b–e) and the ether analogues (11r–
u), further elongation of the methylene chain seemed
not to be effective in obtaining potent activities. From
the results of these types of analogues, it seemed that the
substituent at the C(2)-position required a suitable
chain length. Different from the above mentioned
results, all the compounds having hydroxy groups (11x–
z) showed the same MECs (0.1 mM). The alkyl chain
length had no influence on the activity in this case.
Table 2. IFN inducing activities of 2-alkylthioderivatives in mouse
spleen cells
Next, we investigated the activities of the 2-alkoxy deri-
vatives (Table 3). Among the straight chain alkyl
derivatives (5, 13b–e), the most potent activity was
observed in the butyl derivative (5), this is a similar
Compd
R
IFN (IU/mL)a
Drug concentration (mM)
0.001
0.01
0.1
1
10
11a
11b
11c
11d
4
H
Me
Et
n-Pr
n-Bu
n-Pent
iso-Pr
iso-Bu
s-Bu
<1.8
3.4
<1.8 92.6
<1.8 114.0
46.3 30.5
15.9 17.8
33.7 17.8
29.5 40.9
23.0 22.9
38.3 28.4
Table 3. IFN inducing activity of 2-alkoxy derivatives in mouse
spleen cells
<1.8
<1.8
4.1
13.6
66.0
68.3
19.4
42.4
87.7
73.9
7.8
11.8
<1.8
68.4
2.0
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
11o
11p
11q
11r
11s
11t
11u
11v
11w
11x
11y
11z
1
<1.8
<1.8
<1.8
<1.8
<1.8
<1.8
26.0
9.2
22.0 16.8
53.9 57.7
28.1 20.0
79.5 48.3
59.1 35.4
27.3 43.4
47.2 45.2
Cyclohexyl
Cyclohexylmethyl
Ph
Compd
R
IFN (IU/mL)a
Bn
<1.8
<1.8
(CH2)2Ph
3-Chlorobenzyl
4-Chlorobenzyl
3-Methoxybenzyl
4-Methoxybenzyl
CH2OMe
(CH2)2OMe
(CH2)2OEt
(CH2)3OEt
(CH2)2NMe2
(CH2)3NMe2
(CH2)2OH
(CH2)3OH
(CH2)4OH
Drug concentration (mM)
<1.8
<1.8
12.4
13.9
21.3
28.4
17.8
58.1
15.8
ntb
0.001
0.01
0.1
1
10
<1.8
<1.8
2.2
<1.8
<1.8
<1.8
28.3 17.0
35.8 40.0
13.1 10.5
13.6 11.8
13a
13b
13c
13d
5
13e
13f
13g
13h
13i
13j
1
H
Me
Et
n-Pr
n-Bu
n-Pent
(CH2)2OMe
(CH2)2OEt
(CH2)3OEt
(CH2)2OH
(CH2)3OH
<1.8
<1.8
<1.8
43.0
33.0
36.9
1.9
22.6
110.0
123.0
71.1
56.5
29.8
46.6
25.4
<1.8
<1.8
<1.8
<1.8
23.8
26.3
23.8
23.1
24.0
24.6
25.9
18.0
23.3
26.5
49.4
26.2
21.7
16.8
15.3
16.1
20.4
19.8
24.9
17.2
25.0
41.3
18.3
19.6
<1.8
<1.8
2.4
<1.8
<1.8
16.9
9.5
38.4 19.7
<1.8 14.6
30.2 30.3
14.0 13.3
12.0 12.7
18.5 20.0
26.2 19.6
<1.8
<1.8
<1.8
<1.8
1.8
32.8
30.5
21.5
<1.8
<1.8
a Each value represents the mean of duplicate assay (IU/mL).
b nt: not tested.
a Each value represents the mean of duplicate assay (IU/mL).

1094
A. Kurimoto et al. / Bioorg. Med. Chem. 12 (2004) 1091–1099
result with that of the 2-alkylthio derivatives. The MEC
of 5 was 0.001 mM, which was approximately 1000-fold
more potent than that of Imiquimod (1). In addition, we
investigated the activities of the compounds having
ether (13f–h) and hydroxy (13i, j) groups, and obtained
similar results with those of the 2-alkylthio derivatives.
absorptions of them caused those results. The straight
alkyloxy derivatives (5, 13b–e) also showed similar
results with those of the alkylthio derivatives. In addi-
tion to 5, the ether type compound (13f) exhibited a
most potent IFN-inducing activity in vivo. Among these
tested compounds, 5, 11d and 13f had induced IFN
from the dosage of 0.03 mg/kg, which was approxi-
mately 100-fold more potent than that of Imiquimod.
The in vivo activity of compound 13f seemed to be
stronger compared with its in vitro potential. The dis-
crepancy of this result may be due to the excellent oral
absorption.
The amounts of induced IFN by all these derivatives
including Imiquimod (1) exhibited bell-shaped dose–
response curves. Cytotoxicity could be considered as
one of the possibilities for the bell-shape phenomenon,
however, cytotoxicity was not observed in our assay
system. It is reported that Imiquimod exhibited a
bell-shape type IFN-induction,17 but the reason for
this bell-shape is still not clear.
Finally, several compounds described above were tested
in a rat pharmacokinetic model and we found that 13f,
in particular, showed a good oral absorption. The oral
bioavailability of 13f was calculated as 40% (Table 6).
Thus, we were able to find a compound that exhibited
potent IFN-inducing activity both in vitro and in vivo
and possessing excellent oral bioavailability.
In addition to the in vitro study, the compounds with
potent activities were further evaluated in an in vivo
study to select compounds for further investigations.
The results of the in vivo studies are shown in Table 4
and Table 5. The test compounds were orally adminis-
tered to male Balb/c mice and then the concentration of
IFN in plasma 2 h after dosing was measured by the
bioassay as mentioned above. In the preliminary
experiment, IFN concentration in mouse plasma
reached a maximum at 2 h after oral administration of
the compounds (data not shown). In the straight alkyl-
thio derivatives (4, 11c–e), IFN-inducing activities in
vivo correlated well with the results of the in vitro study,
among them 11d showed the most potent activity.
Compounds 11r and 11x exhibited potent activities as
expected from their in vitro potentials. However, in
other alkylthio derivatives the expected in vivo activities
were not observed. We considered that poor oral
Akira et al. has recently reported that Imiqimod (1)
induces IFN via Toll-like receptor 7 (TLR7) signaling
pathway.18 Therefore, the action mechanisms of our
compounds toward TLR7 is under investigation.
In conclusion, a series of 8-hydroxyadenines, having
various alkoxy or alkylthio moieties at the adenine C(2)-
position, were synthesized and evaluated for their ability
to induce IFN both in vitro and in vivo. We found that
Table 5. IFN inducing activity 2-alkoxy derivatives in mouse (in
vivo)
Table 4. IFN inducing activity of 2-alkylthioderivatives in mouse (in
vivo)
Compd
R
IFN (IU/mL)a
Dose (mg/kg)
0.3
0.03
0.01
1
3
Compd
R
IFN (IU/mL)a
Dose (mg/kg)
13a
13b
13c
13d
5
13e
13f
13g
1
H
Me
Et
n-Pr
26
142
55
59
293
318
317
356
95
703
569
318
908
28
20
186
610
314
<
836
330
877
894
2872
0.03
99
0.01
0.3
1
3
n-Bu
60
<
75
n-Pent
(CH2)2OMe
(CH2)2OEt
11c
11d
4
Et
n-Pr
n-Bu
n-Pent
iso-Pr
iso-Bu
143
308
<
786
452
918
2387
225
935
107
<
496
285
2171
1085
64
11e
11f
11g
11i
11j
11l
11r
11s
11w
11x
11y
11z
1
a Each value shows the mean of three mice (IU/mL).
51
516
<
<
71
Cyclohexyl
Cyclohexylmethyl
Bn
CH2OMe
(CH2)2OMe
(CH2)3NMe2
(CH2)2OH
(CH2)3OH
(CH2)4OH
<
322
50
542
152
1827
456
<
Table 6. Pharmacokinetic parameters after oral administration of Bf
to rats at 3 mg/kga
226
169
393
389
44
Compd
F
(%)
Cmax
(ng/mL)
Tmax
(hr)
MRT
(min)
CL
(l/hr/kg)
Vd
(l/kg)
13f
40
41.0
0.5
46.4
2.74
2.12
64
a The results are expressed as the mean of three rats.
a Each value shows the mean of three mice (IU/mL).

A. Kurimoto et al. / Bioorg. Med. Chem. 12 (2004) 1091–1099
1095
compound 13f (SM-360320) induced IFN from the
dosage of 0.03 mg/kg, which was approximately 100-
fold more potent than that of Imiquimod, and showed
good oral bioavailability (F=40%). Additional studies
are underway to further evaluate the therapeutic poten-
tial of these derivatives.
brine, dried over Na2SO4 and evaporated. The residue
was purified by silica gel column chromatography
(0.5% MeOH–CHCl3) to give 9b (10 mg, 8%) as a col-
orless solid: 1H NMR (CDCl3) d 7.34 (5H, m), 5.64 (2H,
s), 5.33 (2H, s), 2.57 (3H, s); MS (EI) m/z 350 (MH+).
4.2.3. 9-Benzyl-8-bromo-2-methoxyadenine (10b). To a
solution of 8b (118 mg, 0.462 mmol) in CH2Cl2 (50 mL)
was added bromine (0.5 mL, 9.76 mmol) dropwise and
the mixture was stirred at room temperature for 5 h.
The reaction mixture was poured into 10% aq Na2S2O3.
The organic layer was separated, washed with brine,
dried over Na2SO4 and evaporated. The residue was
purified by silica gel column chromatography (1%
MeOH–CHCl3) to give 10b (90 mg, 58%) as a colorless
solid: 1H NMR (DMSO-d6) d 7.48 (2H, br s), 7.39–7.24
(5H, m), 5.26 (2H, s), 3.82 (3H, s); MS (EI) m/z 334
(MH+).
4. Experimental
4.1. Chemistry
Melting points were measured on a Thomas Hoover
capillary melting point apparatus and are uncorrected.
Nuclear magnetic resonance (NMR) spectra were
recorded at ambient temperature on a JEOL JNM LA-
300 or a Bruker AVANCE 400 FT NMR spectrometer.
Chemical shifts are expressed in d values (ppm) relative
to tetramethylsilane as an internal standard, and signals
are expressed s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), or br (broad). Mass spectra
(MS) were measured on a JEOL JMS-AX505W or
JEOL JMS-SX102A mass spectrometer. Elemental
analyses were performed by Sumika Chemical Analysis
Service, Osaka, Japan. Commercial reagents and sol-
vents were of reagent grade and used without further
purification. Thin-layer chromatography (TLC) was
performed on Merk Kieselgel 60 F254 precoated plates
and components were visualized using UV light. Flash
chromatography was conducted using Merk Kieselgel
60 F254 or Cica-Reagent Silica Gel 60.
4.2.4. 9-Benzyl-8-hydroxy-2-methylthioadenine (11b). A
solution of 9b (10 mg, 0.026 mmol) in 6N HCl (10 mL)
was heated to reflux for 4 h. After evaporation, water
(10 mL) was added to the residue and the solution
was basified with 28% aq NH3. The resulting
precipitate was collected by filtration to give 11b (8 mg,
96%) as a colorless solid: mp 298–300 ꢀC; 1H NMR
(DMSO-d6) d 9.60 (1H, s), 7.31 (5H, m), 6.53 (2H, s),
4.88 (2H, s), 2.42 (3H, s); MS (FAB) m/z 288 (MH+);
HRMS calcd for C13H14N5OS 288.0919, found 288.0915.
.
Anal. calcd for C13H13N5OS 1/10H2O: C, 54.00; H,
4.60; N, 24.22. Found: C, 53.75; H, 4.61; N, 24.13.
Compounds 11c–i, 11k and 11l were prepared using
similar procedures as for 11b.
4.2. 9-Benzyl-2-methylthioadenine (7b)19
A mixture of 6 (100 mg, 0.385 mmol) and sodium thio-
methoxide (270 mg, 3.852 mmol) in DMF (10 mL) was
heated at 110 ꢀC for 3.5 h. The mixture was evaporated
in vacuo and the residue was partitioned with water (50
mL) and ethyl acetate (150 mL). The organic layer was
separated, dried over Na2SO4 and evaporated. The
residue was purified by silica gel column chromato-
graphy (1% MeOH–CHCl3) to give 7b (64 mg, 61%) as
4.2.5. 9-Benzyl-2-ethylthio-8-hydroxyadenine (11c). A
colorless solid (4% for 3 steps from 6), mp 292–293 ꢀC;
1H NMR (DMSO-d6) d 10.09 (1H, s), 7.31 (5H, m), 6.51
(2H, s), 4.88 (2H, s), 2.97 (2H, q, J=7.3 Hz), 1.25 (3H,
t, J=7.3 Hz); MS (FAB) m/z 302 (MH+); HRMS calcd
for C14H16N5OS 302.1076, found 302.1086. Anal.
.
calcd for C14H15N5OS 1/10H2O: C, 55.46; H, 5.05; N,
23.10. Found: C, 55.39; H, 4.99; N, 23.16.
1
a colorless solid: H NMR (CDCl3) d 7.63 (1H, s), 7.34
(5H, m), 5.45 (2H, s), 5.31 (2H, s), 2.58 (3H, s).
4.2.6. 9-Benzyl-8-hydroxy-2-propylthioadenine (11d). A
colorless solid (12% for 3 steps from 6), mp 288–290 ꢀC;
1H NMR (DMSO-d6) d 10.19 (1H, s), 7.31 (5H, m), 6.55
(2H, s), 4.87 (2H, s), 2.98 (2H, t, J=6.9 Hz), 1.61 (2H,
m), 0.94 (3H, t, J=7.2 Hz); MS (FAB) m/z 316 (MH+);
HRMS calcd for C15H18N5OS 316.1232, found
316.1241. Anal. calcd for C15H17N5OS 1/10H2O: C,
56.80; H, 5.47; N, 22.08. Found: C, 56.75; H, 5.43; N,
22.11.
4.2.1. 9-Benzyl-2-methoxyadenine (8b).14a A solution of
6 (200 mg, 0.770 mmol) and sodium methoxide (208 mg,
3.850 mmol) in MeOH (20 mL) was heated to reflux for
30 h. The mixture was evaporated in vacuo and the
residue was partitioned with water (50 mL) and CHCl3
(150 mL). The organic layer was separated, dried over
Na2SO4 and evaporated. The residue was purified by
silica gel column chromatography (2% MeOH–CHCl3)
.
1
to give 8b (151 mg, 77%) as a colorless solid: H NMR
(DMSO-d6) d 8.05 (1H, s), 7.37–7.25 (7H, m), 5.26 (2H,
s), 3.81 (3H, s).
4.2.7. 9-Benzyl-8-hydroxy-2-pentylthioadenine (11e). A
colorless solid (11% for 3 steps from 6), mp 270–272 ꢀC;
1H NMR (DMSO-d6) d 10.05 (1H, s), 7.30 (5H, m), 6.50
(2H, s), 4.88 (2H, s), 2.99 (2H, t, J=7.3 Hz), 1.59 (2H,
m), 1.30 (4H, m), 0.84 (3H, t, J=7.3 Hz); MS (FAB)
m/z334 (MH+); HRMS calcd for C17H22N5OS
4.2.2. 9-Benzyl-8-bromo-2-methylthioadenine (9b). To a
solution of 7b (100 mg, 0.369 mmol) in CH2Cl2 (100
mL) was added bromine (0.5 mL, 9.76 mmol) dropwise
and the mixture was stirred at room temperature for 3
h. The reaction mixture was poured into 10% aq
Na2S2O3. The organic layer was separated, washed with
334.1545,
found
.
334.1523.
Anal.
calcd
for
C17H21N5OS 1/4H2O: C, 58.68; H, 6.23; N, 20.13.
Found: C, 58.52; H, 6.17; N, 20.31.

1096
A. Kurimoto et al. / Bioorg. Med. Chem. 12 (2004) 1091–1099
4.2.8. 9-Benzyl-8-hydroxy-2-isopropylthioadenine (11f).
A cꢀolorless solid (13% for 3 steps from 6), mp 310–
This crude product was used next step without further
purification. The crude product (30 mg) was recrysta-
lized from ethyl acetate to give 11a (10 mg) as a yellow
solid: mp 276–278 ꢀC; 1H NMR (DMSO-d6) d 12.10
(1H, br s), 10.06 (1H, s), 7.36–7.24 (5H, m), 6.74 (2H, s),
4.85 (2H, s); MS (FAB) m/z 274 (MH+); HRMS calcd
for C12H12N5OS 274.0763, found 274.0777.
1
313 C; H NMR (DMSO-d6) d 10.09 (1H, s), 7.32 (5H,
m), 6.50 (2H, s), 4.87 (2H, s), 3.78 (1H, m), 1.30 (6H, d,
J=6.9 Hz); MS (FAB) m/z 316 (MH+); HRMS calcd
for C15H18N5OS 316.1232, found 316.1240. Anal.
.
calcd for C15H17N5OS 2/5H2O: C, 55.85; H, 5.56; N,
21.71. Found: C, 55.76; H, 5.31; N, 21.91.
4.2.15. 9-Benzyl-2-cyclohexylmethylthio-8-hydroxyade-
nine (11j). To a suspension of crude 11a (200 mg) and
K2CO3 (102 mg, 0.738 mmol) in DMF (60 mL) was
added cyclohexylmethyl bromide (130 mg, 0.734 mmol)
and the mixture was stirred at room temperature for 9
h. The mixture was evaporated in vacuo and the residue
was purified by silica gel column chromatography (3%
MeOH–CHCl3) to give 11j (93mg, 24% for 2 steps from
14) as a colorless solid: mp 285-288 ꢀC; 1H NMR
(DMSO-d6) d 10.09 (1H, s), 7.30 (5H, m), 6.50 (2H, s),
4.87 (2H, s), 2.93 (2H, d, J=6.6 Hz), 1.78–0.88 (11H,
m); MS (FAB) m/z 370 (MH+); HRMS calcd for
C19H24N5OS 370.1702, found 370.1702. Anal. calcd for
4.2.9. 9-Benzyl-8-hydroxy-2-isobutylthioadenine (11g). A
colorless solid (20% for 3 steps from 6), mp 276–281 ꢀC;
1H NMR (DMSO-d6) d 10.10 (1H, s), 7.35–7.26 (5H,
m), 6.51 (2H, s), 4.87 (2H, s), 2.93 (2H, d, J=6.6 Hz),
1.83 (1H, m), 0.93 (6H, d, J=6.6 Hz); MS (FAB) m/z
330 (MH+); HRMS calcd for C16H20N5OS 330.1389,
found 330.1382.
4.2.10. 9-Benzyl-2-(s-butylthio)-8-hydroxyadenine (11h).
A colorless solid (2% for 3 steps from 6), mp 299–
301 ꢀC; 1H NMR (DMSO-d6) d 10.09 (1H, s), 7.35–7.24
(5H, m), 6.50 (2H, s), 4.87 (2H, s), 3.65 (1H, m), 1.61
(2H, m), 1.28 (3H, d, J=7.0 Hz), 0.93 (3H, t, J=7.3
Hz); MS (FAB) m/z 330 (MH+); HRMS calcd for
C16H20N5OS 330.1389, found 330.1366. Anal. calcd
.
C19H23N5OS 1/5H2O: C, 61.17; H, 6.32; N, 18.77.
Found: C, 61.12; H, 6.24; N, 19.05.
.
for C16H19N5OS 1/10H2O: C, 58.02; H, 5.84; N, 21.14.
Found: C, 57.97; H, 5.83; N, 21.32.
Compounds 11m–z were prepared using similar proce-
dures as for 11j.
4.2.11. 9 - Benzyl - 2 - cyclohexylthio - 8 - hydroxyadenine
(11i). A colorless solid (24% for 3 steps from 6), mp
4.2.16. 9-Benzyl-8-hydroxy-2-(2-phenylethylthio)adenine
(11m). A colorless solid (44% for 2 steps from 14), mp
304–307 ꢀC; H NMR (DMSO-d6) d 10.09 (1H, s), 7.31
271–272 ꢀC; H NMR (DMSO-d6) d 10.13 (1H, s), 7.28
1
1
(5H, m), 6.49 (2H, s), 4.87 (2H, s), 3.62 (1H, m), 2.00
(2H, m), 1.68 (2H, m), 1.62–1.56 (1H, m), 1.35 (5H, m);
MS (FAB) m/z 356 (MH+); HRMS calcd for
C18H22N5OS 356.1545, found 356.1560. Anal. calcd
for C18H21N5OS 2/5H2O: C, 59.61; H, 6.06; N, 19.31.
Found: C, 59.38; H, 5.90; N, 19.58.
(10H, m), 6.56 (2H, s), 4.92 (2H, s), 3.22 (2H, t, J=6.6
Hz), 2.89 (2H, t, J=6.6 Hz); MS (FAB) m/z 378
(MH+); HRMS calcd for C20H20N5OS 378.1389, found
378.1394. Anal. calcd for C20H19N5OS 1/10H2O: C, 63.34;
H, 5.10; N, 18.47. Found: C, 63.27; H, 5.10; N, 18.58.
.
.
4.2.17. 9-Benzyl-2-(3-chlorobenzylthio)-8-hydroxyadenine
(11n). Aꢀcolorless solid (33% for 2 steps from 14), mp
4.2.12. 9-Benzyl-8-hydroxy-2-phenylthioadenine (11k). A
colorless solid (8% for 3 steps from 6), mp 281–283 ꢀC;
1H NMR (DMSO-d6) d 9.50 (1H, s), 7.55 (2H, m), 7.46
(3H, m), 7.28 (3H, m), 7.13 (2H, m), 6.55 (2H, s), 4.67
(2H, s); MS (FAB) m/z 350 (MH+); HRMS calcd for
C18H16N5OS 350.1076, found 350.1069.
1
276–277 C; H NMR (DMSO-d6) d 10.14 (1H, s), 7.45
(1H, s), 7.27 (8H, m), 6.61 (2H, s), 4.90 (2H, s), 4.30
(2H, s); MS (FAB) m/z 398 (MH+); HRMS calcd for
C19H17ClN5OS 398.0842, found 398.0837. Anal. calcd
for C19H16ClN5OS: C, 57.35; H, 4.05; N, 17.60. Found:
C, 57.12; H, 4.05; N, 17.68.
4.2.13. 9-Benzyl-2-benzylthio-8-hydroxyadenine (11l). A
colorless solid (3% for 3 steps from 6), mp 284–287 ꢀC;
1H NMR (DMSO-d6) d 10.12 (1H, s), 7.34–7.19 (10H,
m), 6.58 (2H, s), 4.91 (2H, s), 4.29 (2H, s); MS (FAB)
m/z 364 (MH+); HRMS calcd for C19H18N5OS
364.1232, found 364.1225.
4.2.18. 9-Benzyl-2-(4-chlorobenzylthio)-8-hydroxyade-
nine (11o). A colorless solid (27% for 2 steps from 14),
mp 305-306 ꢀC; H NMR (DMSO-d6) d 10.13 (1H, s),
1
7.29 (9H. m), 6.59 (2H, s), 4.91 (2H, s), 4.26 (2H, s); MS
(FAB) m/z 398 (MH+); HRMS calcd for
C19H17ClN5OS 398.0842, found 398.0824. Anal. calcd
.
4.2.14. 9-Benzyl-8-hydroxy-2-mercaptoadenine (11a). To
a solution of 14 (31.3 g, 146 mmol) in THF (1500 mL)
was added benzoyl isothioyanate (41 mL, 305 mmol)
dropwise and the mixture was stirred at room temper-
ature for 12 h. After evaporation, the residue was tritu-
rated with diethyl ether (150 mL) and the resulting
precipitate was collected by filtration. To a solution of
the obtained solid in THF (1500 mL) was added 2N
NaOH (150 mL) and the mixture was heated to reflux
for 50 h. The reaction mixture was acidified to pH 3
with 10% aq NaHSO4 and the resulting precipitate was
collected by filtration to give a crude product (27.8 g).
for C19H16ClN5OS 1/5H2O: C, 56.84; H, 4.12; N, 17.44.
Found: C, 56.78; H, 4.08; N, 17.61.
4.2.19. 9-Benzyl-8-hydroxy-2-(3-methoxybenzylthio)ade-
nine (11p). A colorless solid (34% for 2 steps from 14),
mp 247–248 ꢀC; H NMR (DMSO-d6) d 10.13 (1H, s),
1
7.28 (5H, m), 7.15 (1H, m), 6.96 (2H, m), 6.79 (1H, m),
6.59 (2H, s), 4.89 (2H, s), 4.27 (2H, s), 3.68 (3H, s); MS
(FAB) m/z 394 (MH+); HRMS calcd for C20H20N5O2S
394.1338,
found
.
394.1334.
Anal.
calcd
for
C20H19N5O2S 1/10H2O: C, 60.77; H, 4.90; N, 17.72.
Found: C, 60.70; H, 4.86; N, 17.93.

A. Kurimoto et al. / Bioorg. Med. Chem. 12 (2004) 1091–1099
1097
.
4.2.20. 9-Benzyl-8-hydroxy-2-(4-methoxybenzylthio)ade-
nine (11q). A ꢀcolorless solid (29% for 2 steps from 14),
for C17H22N6OS 1/3H2O: C, 56.03; H, 6.27; N, 23.06.
Found: C, 55.95; H, 6.12; N, 23.07.
1
mp 2760–278 C; H NMR (DMSO-d6) d 10.13 (1H, s),
7.28 (7H, m), 6.73 (2H, d, J=8.9 Hz), 6.57 (2H, s), 4.92
(2H, s), 4.22 (2H, s), 3.69 (3H, s); MS (FAB) m/z 394
(MH+); HRMS calcd for C20H20N5O2S 394.1338,
found 394.1337. Anal. calcd for C20H19N5O2S 1/10H2O:
C, 60.77; H, 4.90; N, 17.72. Found: C, 60.63; H, 4.84; N,
17.86.
4.2.27. 9-Benzyl-8-hydroxy-2-(2-hydroxyethylthio)ade-
nine (11x). A colorless solid (32% for 2 steps from 14),
ꢀ
1
mp 264–265 C; H NMR (DMSO-d6) d 10.09 (1H, s),
7.32 (5H, m), 6.52 (2H, s), 4.87 (3H, s), 3.59 (2H, q,
J=5.9 Hz), 3.12 (2H, t, J=6.6 Hz); MS (FAB) m/z 318
(MH+); HRMS calcd for C14H16N5O2S 318.1025,
found 318.1026. Anal. calcd for C14H15N5O2S: C, 52.98;
H, 4.76; N, 22.07. Found: C, 52.70; H, 4.60; N, 22.24.
.
4.2.21. 9-Benzyl-8-hydroxy-2-(methoxymethylthio)ade-
nine (11r). Aꢀcolorless solid (48% for 2 steps from 14),
1
mp 272–273 C; H NMR (DMSO-d6) d 10.15 (1H, s),
4.2.28. 9-Benzyl-8-hydroxy-2-(3-hydroxypropylthio)ade-
nine (11y). A colorless solid (43% for 2 steps from 14),
7.31 (5H, m), 6.59 (2H, s), 5.29 (2H, s), 4.89 (2H, s), 3.21
(3H, s); MS (FAB) m/z 318 (MH+); HRMS calcd for
C14H16N5O2S 318.1025, found 318.1034. Anal. calcd
for C14H15N5O2S: C, 52.98; H, 4.76; N, 22.07. Found:
C, 52.80; H, 4.68; N, 22.04.
mp 235-238 ꢀC; H NMR (DMSO-d6) d 10.09 (1H, s),
1
7.31 (5H, m), 6.50 (2H, s), 4.90 (2H, s), 4.50 (1H, t,
J=5.6 Hz), 3.49 (2H, m), 3.07 (2H, t, J=6.6 Hz), 1.75
(2H, m); MS (FAB) m/z 332 (MH+); HRMS calcd for
C15H18N5O2S 332.1181, found 332.1178.
4.2.22. 9-Benzyl-8-hydroxy-2-(2-methoxyethylthio)ade-
nine (11s). A colorless solid (8% for 2 steps from 14),
4.2.29. 9-Benzyl-8-hydroxy-2-(4-hydroxybutylthio)ade-
nine (11z). A colorless solid (4% for 2 steps from 14),
mp 273–275 ꢀC; H NMR (DMSO-d6) d 10.11 (1H, s),
1
ꢀ
1
7.30 (5H, m), 6.55 (2H, s), 4.87 (2H, s), 3.50 (2H, t,
J=6.6 Hz), 3.22 (3H, s), 3.21 (2H, t, J=6.6 Hz); MS
(FAB) m/z 332 (MH+); HRMS calcd for C15H18N5O2S
mp 251–253 C; H NMR (DMSO-d6) d 10.09 (1H, s),
7.31 (5H, m), 6.50 (2H, s), 4.87 (2H, s), 4.40 (1H, t,
J=5.3 Hz), 3.39 (2H, q, J=5.3 Hz), 3.02 (2H, t, J=6.9
Hz), 1.67–1.48 (4H, m); MS (FAB) m/z 346 (MH+);
HRMS calcd for C16H20N5O2S 346.1338, found
332.1181,
found
332.1194.
Anal.
calcd
for
C15H17N5O2S: C, 54.36; H, 5.17; N, 21.13. Found: C,
54.19; H, 5.09; N, 21.24.
.
346.1336. Anal. calcd for C16H19N5O2S 1/3H2O: C,
54.69; H, 5.64; N, 19.93. Found: C, 54.97; H, 5.60; N,
19.63.
4.2.23. 9-Benzyl-2-(2-ethoxyethylthio)-8-hydroxyadenine
(11t). A colorless solid (8% for 2 steps from 14), mp
260–261 ꢀC; H NMR (DMSO-d6) d 10.11 (1H, s), 7.30
4.2.30. 9-Benzyl-8-hydroxy-2,8-dimethoxyadenine (12b).
A solution of 10b (125 mg, 0.374 mmol) and sodium
methoxide (404 mg, 7.479 mmol) in MeOH (10 mL) was
heated to reflux for 10 h. The mixture was evaporated in
vacuo and the residue was partitioned with water (50
mL) and CHCl3 (150 mL). The organic layer was sepa-
rated, dried over Na2SO4 and evaporated. The residue
was purified by silica gel column chromatography (1%
MeOH–CHCl3) to give 12b (83 mg, 78%) as a colorless
1
(5H, m), 6.54 (2H, s), 4.88 (2H, s), 3.54 (2H, t, J=6.9
Hz), 3.43 (2H, q, J=7.0 Hz), 3.18 (2H, t, J=6.6 Hz),
1.08 (3H, t, J=6.9 Hz); MS (FAB) m/z 346 (MH+);
HRMS calcd for C16H20N5O2S 346.1338, found
346.1323.
4.2.24. 9-Benzyl-2-(3-ethoxypropylthio)-8-hydroxyade-
nine (11u). A colorless solid (21% for 2 steps from 14),
mp 246–249 ꢀC; H NMR (DMSO-d6) d 10.09 (1H, s),
solid: H NMR (DMSO-d6) d 7.73–7.23 (5H, m), 6.90
(2H, s), 5.05 (2H, s), 4.04 (3H, s), 3.78 (3H, s); MS (EI)
1
1
7.31 (5H, m), 6.51 (2H, s), 4.87 (2H, s), 3.44-3.34 (4H,
m), 3.03 (2H, t, J=8.9 Hz).1.83 (m, 2H), 1.08 (3H, t,
J=6.9 Hz).; MS (FAB) m/z 360 (MH+); HRMS calcd
for C17H22N5O2S 360.1494, found 360.1485. Anal.
calcd for C17H21N5O2S: C, 56.80; H, 5.89; N, 19.48.
Found: C, 56.83; H, 5.78; N, 19.48.
m/z 286 (MH+).
4.2.31. 9-Benzyl-2,8-dihydroxyadenine (13a). A solution
of 10b (75 mg, 0.224 mmol) in 6N HCl (15 mL) was
heated to reflux for 5 h. After evaporation, water (10
mL) was added to the residue and the solution was
basified with 28% aq NH3. The resulting precipitate
was collected by filtration and purified by silica gel
column chromatography (0.2% NH3-5% MeOH–
CHCl3) to give 13a (12 mg, 21%) as a colorless solid:
mp 254–256 ꢀC; 1H NMR (DMSO-d6) d 9.64 (2H, br s),
7.34–7.22 (5H, m), 6.51 (2H, s), 4.78 (2H, s); MS (FAB)
m/z 258 (MH+); HRMS calcd for C12H12N5O2
4.2.25. 9-Benzyl-2-(2-dimethyaminoethylthio)-8-hydroxy-
adenine (11v). A colorless solid (3% for 2 steps from
14), mp 245–247 ꢀC; H NMR (DMSO-d6) d 10.11 (1H,
1
s), 7.29 (5H, m), 6.53 (2H, s), 4.88 (2H, s), 3.11 (2H, t,
J=7.6 Hz), 2.49 (2H, t, J=7.6 Hz), 2.14 (6H, s); MS
(FAB) m/z 345 (MH+); HRMS calcd for C16H21N6OS
345.1498, found 345.1494.
258.0991,
found
258.1008.
Anal.
calcd
for
.
4.2.26. 9-Benzyl-2-(3-dimethyaminopropylthio)-8-hydroxy-
adenine (11w). A colorless solid (5% for 2 steps from
C12H11N5O2 H2O: C, 52.36; H, 4.76; N, 25.44. Found:
C, 52.11; H, 4.58; N, 25.22.
ꢀ
1
14), mp 252–255 C; H NMR (DMSO-d6) d 10.10 (1H,
s), 7.30 (5H, m), 6.50 (2H, s), 4.87 (2H, s), 3.00 (2H, t,
J=7.6 Hz), 2.26 (2H, t, J=7.3 Hz), 2.08 (6H, s), 1.72
(2H, m); MS (FAB) m/z 359 (MH+); HRMS calcd for
C17H23N6OS 359.1654, found 359.1657. Anal. calcd
4.2.32. 9-Benzyl-8-hydroxy-2-methoxyadenine (13b). A
solution of 12b (53 mg, 0.186 mmol) in 12N HCl (10
mL) was stirred at room temperature for 5 h. After
evaporation, water (10 mL) was added to the residue

1098
A. Kurimoto et al. / Bioorg. Med. Chem. 12 (2004) 1091–1099
and the solution was basified with 28% aq NH3. The
resulting precipitate was collected by filtration to give
13b (38 mg, 74%) as a colorless solid: mp 308–309 ꢀC;
1H NMR (DMSO-d6) d 9.95 (1H, s), 7.35–7.22 (5H, m),
6.50 (2H, s), 4.86 (2H, s), 3.76 (3H, s); MS (FAB) m/z
272 (MH+); HRMS calcd for C13H14N5O2 272.1148,
found 272.1165.
J=6.6 Hz), 3.44 (2H, t, J=6.6 Hz), 3.38 (2H, q, J=7.0
Hz), 1.85 (2H, m) 1.08 (3H, t, J=7.0 Hz); MS (FAB)
m/z 344 (MH+); HRMS calcd for C17H22N5O3 344.1723,
found 344.1720. Anal. calcd for C17H21N5O3: C, 59.46; H,
6.16; N, 20.40. Found: C, 59.29; H, 6.13; N, 20.49.
4.2.39. 9-Benzyl-8-hydroxy-2-(2-hydroxyethoxy)adenine
(13i). A colorless solid (12% for 4 steps from 6), mp 300–
302 ꢀC; 1H NMR (DMSO-d6) d 9.89 (1H, s), 7.30 (5H, m),
6.46 (2H, s), 4.85 (2H, s), 4.79 (1H, t, J=5.6 Hz), 4.15 (2H,
t, J=4.9 Hz), 3.65 (2H, m); MS (FAB) m/z 302 (MH+);
HRMS calcd for C14H16N5O3 302.1253, found 302.1242.
Compounds 13c–j were prepared using similar proce-
dures as for 13b.
4.2.33. 9-Benzyl-2-ethoxy-8-hydroxyadenine (13c). A
colorless solid (8% for 4 steps from 6), mp 306–308 ꢀC;
1H NMR (DMSO-d6) d 9.96 (1H, s), 7.35–7.23 (5H, m),
6.45 (2H, s), 4.85 (2H, s), 4.19 (2H, q, J=7.1 Hz), 1.25
(3H, t, J=7.1 Hz); MS (FAB) m/z 286 (MH+); HRMS
calcd for C14H16N5O2 286.1304, found 286.1294.
.
Anal. calcd for C14H15N5O3 1/4H2O: C, 54.99; H, 5.11; N,
22.90. Found: C, 54.89; H, 4.92; N, 22.69.
4.2.40. 9-Benzyl-8-hydroxy-2-(3-hydroxypropoxy)adenine
(13j). A colorless solid (10% for 4 steps from 6), mp
294–295 ꢀC; H NMR (DMSO-d6) d 9.95 (1H, s), 7.30
1
4.2.34. 9-Benzyl-8-hydroxy-2-propoxyadenine (13d). A
colorless solid (18% for 4 steps from 6), mp 319–321 ꢀC;
1H NMR (DMSO-d6) d 9.96 (1H, s), 7.35–7.22 (5H, m),
6.45 (2H, s), 4.86 (2H, s), 4.10 (2H, t, J=6.8 Hz), 1.65
(2H, m), 0.93 (3H, t, J=7.3 Hz); MS (FAB) m/z 300
(MH+); HRMS calcd for C15H18N5O2 300.1461, found
300.1474. Anal. calcd for C15H17N5O2 1/10H2O: C,
59.45; H, 5.79; N, 23.12. Found: C, 59.41; H, 5.63; N,
23.16.
(5H, m), 6.45 (2H, s), 4.85 (2H, s), 4.50 (1H, t, J=5.0
Hz), 4.20 (2H, t, J=6.0 Hz), 3.51 (2H, q, J=5.0 Hz),
1.79 (2H, m); MS (FAB) m/z 316 (MH+); HRMS calcd
for C15H18N5O3 316.1410, found 316.1407. Anal. calcd
for C15H17N5O3 1/5H2O: C, 56.49; H, 5.50; N, 21.96.
Found: C, 56.59; H, 5.42; N, 21.94.
.
.
5. Biology
4.2.35. 9-Benzyl-8-hydroxy-2-pentoxyadenine (13e). A
colorless solid (36% for 4 steps from 6), mp 307–308 ꢀC;
1H NMR (DMSO-d6) d 9.97 (1H, s), 7.35–7.24 (5H, m),
6.44 (2H, s), 4.85 (2H, s), 4.13 (2H, t, J=6.6 Hz), 1.62
(2H, m), 1.32 (4H, m), 0.88 (3H, t, J=6.4 Hz); MS (FAB)
m/z 328 (MH+); HRMS calcd for C17H22N5O2 328.1773,
found 328.1769. Anal. calcd for C17H21N5O2 1/10H2O:
C, 62.03; H, 6.49; N, 21.27. Found: C, 62.04; H, 6.44; N,
21.22.
5.1. 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 erythrocytes by hypotonic treatment with
0.2% aq NaCl, and washed twice with PBS. Splenocytes
were resuspended at a concentration of 2Â106 cells/mL
in MEM supplemented with 5% fetal calf serum, 100 U/
mL of penicillin, and 100 mg/mL of streptomycin. The
test compounds were dissolved in dimethylsulfoxide and
diluted to 500-fold with supplemented MEM. Above
splenocytes suspension (0.5 mL) and various con-
centrations 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 -80 ꢀC until they were analyzed for IFN.
.
4.2.36. 9-Benzyl-8-hydroxy-2-(2-methoxyethoxy)adenine
(13f). A colorless solid (16% for 4 steps from 6), mp
290–292 ꢀC; H NMR (DMSO-d6) d 9.97 (1H, s), 7.35-
1
7.23 (5H, m), 6.48 (2H, s), 4.86 (2H, s), 4.26 (2H, t,
J=4.6 Hz), 3.58 (2H, t, J=4.6 Hz), 3.27 (3H, s); MS
(FAB) m/z 316 (MH+); HRMS calcd for C15H18N5O3
.
316.1410, found 316.1410. Anal. calcd for C15H17N5O3 1/
10H2O: C, 56.81; H, 5.47; N, 22.08. Found: C, 56.75; H,
5.34; N, 22.10.
4.2.37. 9-Benzyl-2-(2-ethoxyethoxy)-8-hydroxyadenine
(13g). A colorless solid (26% for 4 steps from 6), mp
5.2. IFN induction in mouse plasma
278–279 ꢀC; H NMR (DMSO-d6) d 9.98 (1H, s), 7.35–
The test compounds suspended in 0.5% sodium car-
boxymethyl cellulose were administered orally to male
BALB/c mice (Charles River Japan Inc.) aged 8–10
weeks. Blood was collected by cardiac puncture into
heparinized tube, under ether anaesthesia, 2 h after test
compounds administeration. Plasma samples were
obtained by centrifugation, and stored at À80 ꢀC until
they were analyzed for IFN.
1
7.23(5H, m), 6.48 (2H, s), 4.86 (2H, s), 4.25 (2H, t,
J=4.6 Hz), 3.62 (2H, t, J=4.6 Hz), 3.45 (2H, q, J=7.0
Hz), 1.11 (3H, t, J=7.0 Hz); MS (FAB) m/z 330
(MH+); HRMS calcd for C16H20N5O3 330.1566, found
.
330.1549. Anal. calcd for C16H19N5O3 1/10H2O: C,
58.03; H, 5.84; N, 21.15. Found: C, 58.27; H, 5.71; N,
21.29.
4.2.38. 9-Benzyl-2-(3-ethoxypropoxy)-8-hydroxyadenine
(13h). Aꢀcolorless solid (33% for 4 steps from 6), mp
7.23 (5H, m), 6.45 (2H, s), 4.84 (2H, s), 4.17 (2H, t,
5.3. IFN analysis
1
297–300 C; H NMR (DMSO-d6) d 9.97 (1H, s), 7.31–
Mouse IFN titer in supernatants of splenocytes and
plasma sample was quantitated by measuring its antiviral

A. Kurimoto et al. / Bioorg. Med. Chem. 12 (2004) 1091–1099
1099
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Male Sprague–Dawley rats (Charles River Japan Inc.)
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The test compound 13f was administered at a dose of 3
mg/kg intravenously (via bolus injection into the tail
vein of rats) or 3 mg/kg orally (via gavage to the sto-
mach). For intravenous administration the test com-
pound was formulated as a solution in DMSO-PEG400-
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0.5% sodium carboxymethyl cellulose. At 0.5, 1, 2, 4, 8
and 24 h after administration, rats were anaesthetized
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40 ꢀC, and then reconstituted in 50% methanol. The
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