Synthesis and structure-activity relationships of 2-substituted-8-hydroxyadenine derivatives as orally available interferon inducers without emetic side effects

Article abstract of DOI:10.1016/S0968-0896(03)00369-9

Recently we reported the adenine derivatives (2-4) as new interferon (IFN) inducers. In the present study, we conducted a detailed structure and activity relationship study of 4 and its related derivatives on IFN inducing activity. From this study, we found that compound 4 exhibited the most potent IFN inducing activity in vitro with a minimum effective concentration of 0.01 μM, and 4 also showed strong IFN-inducing activity at doses of more than 0.3 mg/kg by oral administration in mice. This potency was 10-fold stronger than that of Imiquimod. Moreover, 4 did not cause emesis in ferrets even at doses as high as 10 mg/kg, whereas, 80% of animals were emetic when orally administered with the same dose of Imiquimod. These results indicate that compound 4 is superior to Imiquimod with respect to efficacy and safety.

Full text of DOI:10.1016/S0968-0896(03)00369-9

Bioorganic & Medicinal Chemistry 11 (2003) 3641–3647
Synthesis and Structure–Activity Relationships of 2-Substituted-8-
hydroxyadenine Derivatives as Orally Available Interferon
Inducers without Emetic Side Effects
Yoshiaki Isobe,a Masanori Tobe,a Haruhisa Ogita,a Ayumu Kurimoto,a Tetsuhiro Ogino,a
Hajime Kawakami,a Haruo Takaku,a Hironao Sajiki,b Kosaku Hirotab
and Hideya Hayashia,*
aResearch Division, Discovery Research Laboratories II, Sumitomo Pharmaceuticals Co. Ltd., Konohana-ku,
Osaka 554-0022, Japan
bDepartment of Medicinal Chemistry, Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502-8585, Japan
Received 10 March 2003; accepted 23 May 2003
Abstract—Recently we reported the adenine derivatives (2–4) as new interferon (IFN) inducers. In the present study, we conducted
a detailed structure and activity relationship study of 4 and its related derivatives on IFN inducing activity. From this study, we
found that compound 4 exhibited the most potent IFN inducing activity in vitro with a minimum effective concentration of
0.01 mM, and 4 also showed strong IFN-inducing activity at doses of more than 0.3 mg/kg by oral administration in mice. This
potency was 10-fold stronger than that of Imiquimod. Moreover, 4 did not cause emesis in ferrets even at doses as high as 10 mg/kg,
whereas, 80% of animals were emetic when orally administered with the same dose of Imiquimod. These results indicate that
compound 4 is superior to Imiquimod with respect to efficacy and safety.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
Orally available IFN inducers offer the possible advan-
tages of convenience, prolonged action and avoidance
of immunogenicity. According to its expected low price,
it could be used for the treatment of other viral infec-
tions. Therefore, IFN inducers are expected to be a new
therapeutic class drug for viral infections. Although a
number of IFN inducers including polyribonucleotide
complexes,4 fluorenones,5 and pyrimidones6 have been
investigated, no results have been reported for IFN
production in humans.7
Hepatitis C virus (HCV) is a small enveloped RNA
virus that belongs to the Flavviridae family. It causes
chronic liver disease, including chronic active hepatitis
in up to 80% of infected patients, as well as cirrhosis
and hepatocellular carcinoma.1 HCV infection is a sig-
nificant clinical problem affecting an estimated 150 mil-
lion patients worldwide. Chronic infection can lead to
liver cirrhosis and associated development of hepato-
cellular carcinoma within 10–15 years.2 The current
therapy using interferon-a (IFN-a) alone or in combi-
nation with Ribavirin is not highly efficacious, especially
against genotype 1b viral infections. IFN-a therapy is
very expensive and the formation of antibodies against
IFN-a is observed in some patients. Treatment with
IFN-a over a 6-month period enhances the efficacy and
reduces the occurrence of hepatocellular carcinoma,3
though, the patients have to suffer regular injections of
IFN-a, once or twice a week.
Imiquimod (1), developed by 3M Pharmaceuticals, is a
low molecular weight IFN inducer (MW 240) and has
been reported to induce IFN production in humans.8
However, the clinical trial for HCV or HBV treatment
was discontinued possibly due to its serious side effects
such as vomiting and liver dysfunctions. Such side
effects have not been previously seen in IFN therapy,
therefore, it may be estimated that such side effects are
dependent on the action of Imiquimod itself.9 In fact,
Imiquimod is used only for the treatment of papilloma
viral infections as external agent to avoid such side
effects.
*Corresponding author. Tel.: +81-6-6466-5189; fax: +81-6-6466-
5483; e-mail: hayashih@sumitomopharm.co.jp
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00369-9

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Y. Isobe et al. / Bioorg. Med. Chem. 11 (2003) 3641–3647
We have recently reported a series of 8-hydroxyadenines
as novel IFN inducers (Fig. 1).10 Compound 2 exhibited
the most potent IFN-inducing activity, but the max-
imum plasma concentration (Cmax) was low (80 ng/mL)
when 2 was orally administered to rats at a dose of
10 mg/kg, and 3 also did not demonstrate a good oral
absorption. On the other hand, 4 had a moderate IFN-
inducing activity with a high Cmax value (490 ng/mL).
To find compounds having potent IFN-inducing activ-
ities with good oral absorption, we conducted a detailed
structure and activity relationship study on the modifi-
cation of 4.
Results and Discussion
The IFN-inducing activities of the prepared compounds
in vitro are shown in Table 1. Mouse splenocytes were
cultured with compounds 4 or 9, and the concentrations
of IFN induced by compounds 4 or 9 in the supernatant
were measured by bioassay using L929 cells with vesi-
cular stomatitis virus.12,13
Table 1. IFN inducing activity in mouse splenocytes
Chemistry
The synthetic route to C(2)-substituted-9-benzyl-8-
hydroxyadenine derivatives is shown in Scheme 1. 5-
Amino-1-benzylimidazole-4-carboxamide (5),11 pre-
pared from 5-aminoimidazole-4-carboxamide with ben-
zyl chloride in the presence of sodium hydroxide, was
heated with an appropriate ethyl ester in the presence of
sodium ethoxide to obtain hypoxanthine derivatives
(6e–u) in good yields. Compounds (6e–u) were then
chlorinated with phosphorous oxychloride, and ami-
nated with ammonia in an autoclave to give adenine
derivatives (7e–u). The reaction of 7e–u with bromine in
the presence of sodium acetate gave compounds 8e–u.
The desired compounds (9e–u) were obtained by heating
8e–u in 12N HCl.
Compd
R
IFN (U/mL)
Concentration of test compound (mM)
0.01 0.03
0.1
0.3
1
10
9a15
H
Me
Et
Pr
Bu
Pen
Hep
i-Pr
—
1.8
9b10
—
6
4.2 14.6
11.8
9c10
—
1.6
12.4
15.8
9d10
—
1.4 13.6
12.2 8.2
4
9e
9f
9g
9h
9i
—
4.4
9.8
—
11.8
13.7 4.5
2.4 8.8
—
—
11.4
4.6
c-Pr
i-Bu
—
15.8
9j
9k
9l
9m
9n
9o
9p
9q
9r
9s
9t
9u
c-Hex
CH2-c-Pen
CH2-c-Hex
Phenyl
3-Pyridyl
Benzyl
4-F-Benzyl
CF3
CHF2
CF2CF3
(CF2)2CF3
(CH2)2CF3
—
—
9.4
5.2
14.4 15.4
12.4 12
10.2
—
—
12.2 12
—
—
6.4
4.8
—
—
—
—
1.8
11.6
13
1.9
—
—
4.2
—
15
13
14.4
6.7 8.8
5.4 14
1.1 8.3
16.4
3.4 5.8 5.8
—
Imiquimod
IFN concentration was measured by the method as described in the
Experimental. Each value represents the mean of duplicate assay.
—Not detected (detection limit was 0.4 IU/mL).
Figure 1. Structures of 1 (Imiquimod) and 2–4.
Scheme 1. Synthetic route to compounds 9e–u. Reagents and conditions. (a) R-COOEt, Na, EtOH, reflux; (b) POCl3, reflux; (c) NH3 (28% in
water), BuOH, 130 ꢀC in autoclave; (d) Br2, AcONa, AcOH, 110 ꢀC; (e) 12 N HCl, 100 ꢀC.

Y. Isobe et al. / Bioorg. Med. Chem. 11 (2003) 3641–3647
3643
The IFN-inducing activity was enhanced by elongation
of the methylene chain length at the C(2)-position, and
the most suitable methylene length was found to be 4 or
5 (4, 9e). The minimum effective concentration (MEC)
of 4 was 0.01 mM, which was 30-fold stronger than that
of Imiquimod. Branched or cyclic alkyl groups (9g–l)
exhibited moderate activities, but were inferior to 4.
Compared to alkyl substituents, aromatic substituents
(9m,14 9n) showed decreased activities (MEC=10 mM).
However, the activities of the benzyl compounds (9o,
9p) were moderate (MEC=0.1 mM), and were the same
as that of the cyclohexylmethyl compound (9l). These
results show that a directly bound aromatic moiety did
not result in potent activity. As the activities of 9o and
9p were 10-fold weaker than that of 4, we did not
conduct further studies on other arylalkyl substituents,
such as 2-phenylethyl. The MEC of the trifluoromethyl
compound (9q) was 0.1 mM, which was 10-fold potent
than that of the methyl compound (9b: MEC=1 mM),
and considering the results of straight alkyl compounds
(9b–d), we expected that the elongated fluorinated alkyl
groups would improve the activity. But the elevation of
the potency could not be achieved, for example, the pen-
tafluoroethyl compound (9s: 0.3 mM), and the hepta-
fluoropropyl compound (9t: 0.3 mM).
above. In the preliminary experiment, the IFN concen-
tration in mouse plasma reached a maximum 2 h after
oral administration of the test compounds (data not
shown), so, the IFN concentration was measured at this
point. As shown in Table 2, compound 4 showed the
most potent IFN-inducing activity and the order of
potency was found to be closely correlated with that
found in the in vitro studies. The potency of compound
4 was 10-fold higher than that of Imiquimod. The in
vivo activities of compounds 9e, 9q, and 9u seemed to be
weaker compared with their in vitro activities. The dis-
crepancy of these results may be due to differing oral
bioavailabilities.
In general, it is reported that the IFN concentration in
plasma is required to be controlled between the level of
50–100 U/mL in treatment of the HCV infection. In
fact, the clinical trials of Imiquimod for treatment of
HCV or HBV infections, were conducted at a dosage
ranging between 100 and 200 mg per day (2–4 mg/kg),
and the reduction of virus titer and the improvement of
liver function were observed in patients. The result of
this study in mice (67 U/mL @ 3 mg/kg of Imiquimod)
was corresponded well with that of the clinical study.
According to the present comparative study of Imiqui-
mod and 4 in mice, it is estimated that 4 could induce
IFN at a dose of 0.3 mg/kg in humans corresponding to
a dose that is 10-fold lower than that of Imiquimod.
Before the in vivo studies were started, we confirmed
that the subtype of IFN induced by the prepared com-
pounds was identified to be mainly alpha IFN by using
anti-human neutralizing antibodies to various types of
IFN. Briefly, the anti-viral activity of IFN was neu-
tralized by more than 90% by addition of the antibody
against alpha IFN into the supernatant of mouse
splenocytes. Whereas, the addition of the beta IFN
antibody had no effect on the anti-viral activity (data
not shown).
Emesis, one of the serious side effects, was observed in
the clinical study of Imiquimod. Therefore, we chose a
ferret emesis model to evaluate the emetic side effect.
The effect was evaluated by the absence or presence of
vomiting behavior during 6 h after administration of the
compounds. When 4 was orally administered at a dose
of 10 mg/kg, no vomiting was observed in any of three
ferrets, while the administration of Imiquimod at the
same dose caused vomiting in four out of five ferrets.
The plasma concentrations of these compounds at 6 h
postdosing were 2.5 mM for 4 and 4 mM for Imiquimod.
Thus, although Imiquimod induced IFN production, it
was strongly emetic. These results indicate that com-
pound 4 exerts a highly potent IFN-inducing activity
and is superior to Imiquimod in terms of the margin
between the IFN-inducing and emetic doses. As for the
possible reason for this result, we hypothesize that 4 is
not able to transmigrate into the brain effectively due to
the presence of a polar group (aromatic hydroxy), but
the exact details including the action mechanism of
emesis are still unclear. From these results, compound 4
is expected to be a useful IFN inducer for treatment of
viral infections such as HCV and HBV. At present,
further optimization studies of the substituents present
in 4 are still underway.
The results of the in vivo studies are shown in Table 2.
The test compounds were orally administered to male
Balb/c mice and then the concentrations of IFN in the
plasma were measured by the bioassay mentioned
Table 2. IFN concentrations in mouse plasma 2 h after oral
administration
Compd
R
IFN (U/mL)
Dose (mg/kg)
3
0.3
30
9a
9b
9c
9d
4
9e
9g
9k
H
Me
Et
Pr
Bu
Pen
i-Pr
<7
510Æ172
3302Æ310
3208Æ1027
2204Æ661
1146Æ132
6054Æ1792
1015Æ204
1157Æ278
10Æ5
<7
76Æ23
859Æ133
230Æ117
1476Æ146
942Æ265
259Æ64
205Æ61
<7
173Æ41
21Æ6
CH2-c-Pen
Phenyl
Benzyl
4-F-benzyl
CF3
<7
9m
9o
9p
9q
9u
Imiquimod
250Æ113
458Æ160
174Æ59
62Æ2
2545Æ863
Experimental
Chemistry
<7
1747Æ67
426Æ23
CH2CH2CF3
—
67Æ21
1672Æ614
General. All reagents and solvents were obtained from
commercial suppliers and were used without further
purification. Melting points were measured with a Buchi
IFN concentration was measured by the method as described in the
Experimental. Each value shows the meanÆSE of three mice.

3644
Y. Isobe et al. / Bioorg. Med. Chem. 11 (2003) 3641–3647
535 melting point apparatus and were uncorrected.
Proton NMR spectra were recorded on a JEOL
GSX270 FT NMR spectrometer. Chemical shifts were
given in parts per million (ppm) using tetramethylsilane
as an internal standard for spectra obtained in DMSO-
d6. TOF MS (time-of-flight mass spectrometry) was
recorded on a Kompact MALDI 3 V4.0.0 spectrometer.
Elemental analyses were performed at the Toray
Research Center. Wakogel C-200 (Wako; 70–150 mm)
was used for column chromatography. Monitoring of
reactions was carried out using Merck 60 F254 silica gel,
glass-supported TLC plates, and visualization with UV
light (254 and 365 nm).
(2H, s), 3.95 (2H, s); MS (TOF) m/z 317 (M+H)+. 6p:
1H NMR (DMSO-d6) d 12.41 (1H, s), 8.12 (1H, s), 7.28–
7.39 (7H, m), 7.11–7.18 (2H, m), 5.28 (2H, s), 3.94 (2H,
s); MS (TOF) m/z 336 (M+H)+. 6q: 1H NMR (DMSO-
d6) d 7.88 (1H, s), 7.25–7.36 (5H, m), 5.26 (2H, s); MS
1
(TOF) m/z 295 (M+H)+. 6r: H NMR (DMSO-d6) d
13.15 (1H, s), 8.32 (1H, s), 7.28–7.39 (5H, m), 6.84 (1H,
t, J=52.4 Hz), 5.41 (2H, s); MS (TOF) m/z 277
(M+H)+. 6s: 1H NMR (DMSO-d6) d 7.87 (1H, s), 7.32
(5H, m), 5.23 (2H, s); MS (TOF) m/z 345 (M+H)+. 6t:
1H NMR (DMSO-d6) d 13.74 (1H, s), 8.57 (1H, s), 7.28–
7.36 (5H, m), 5.45 (2H, s); MS (TOF) m/z 395
(M+H)+. 6u: 1H NMR (DMSO-d6) d 7.87 (1H, s),
7.23–7.36 (5H, m), 5.26 (2H, s), 2.63–2.82 (4H, s); MS
(TOF) m/z 323 (M+H)+.
9-Benzyl-2-butylhypoxanthine. Ethyl valerate (6.0 g,
46 mmol) and 5 (1.3 g, 5.6 mmol) was added to a solu-
tion of sodium (1.1 g, 47 mmol) in EtOH (30 mL), and
the reaction mixture was refluxed for 10 h. The reaction
mixture was concentrated, water (30 mL) was added to
the residue and neutralized by 12 N HCl. The pre-
cipitate was collected to give the title compound as a
9-Benzyl-2-butyladenine. A suspension of 9-benzyl-2-
butylhypoxanthine (1.4 g, 5 mmol) in POCl3 (15 mL)
was refluxed for 3 h, and the reaction mixture was con-
centrated. Next, it was poured into crashed ice and
extracted with CH2Cl2. The organic layer was washed
with water and brine, dried over Na2SO4, and con-
centrated. The residue was purified by silica gel chro-
matography using CH2Cl2 as an eluent to give 9-benzyl-
2-butyl-6-chloropurine as a pale yellow oil (1.39 g,
93%). A suspension of 9-benzyl-2-butyl-6-chloropurine
(1.38 g, 5 mmol) and 28% ammonium solution (12 mL)
in EtOH (30 mL) was heated at 130 ꢀC for 12 h in an
autoclave. The reaction mixture was concentrated to
give the title compound as a pale yellow solid (1.08 g,
1
white solid (1.4 g, 89%); H NMR (DMSO-d6) d 12.13
(1H, s), 8.11 (1H, s), 7.28–7.37 (5H, m), 5.32 (2H, s),
2.62 (2H, t, J=7.3 Hz), 1.61–1.72 (2H, m), 1.23–1.36
(2H, m), 0.88 (3H, t, J=7.3 Hz); MS (TOF) m/z 283
(M+H)+.
Compounds 6e–u were also prepared from appropriate
commercially available esters using the same procedure
described above.
1
84%); H NMR (DMSO-d6) d 8.18 (1H, s), 7.26–7.33
1
6e: H NMR (DMSO-d6) d 12.13 (1H, s), 8.11 (1H, s),
(7H, m), 5.34 (2H, s), 2.66 (2H, t, J=7.6 Hz), 1.67–1.73
(2H, m), 1.27–1.35 (2H, m), 0.89 (2H, t, J=7.6 Hz); MS
(TOF) m/z 282 (M+H)+.
7.28–7.35 (5H, m), 5.33 (2H, s), 2.61 (2H, t, J=7.3 Hz),
1.69 (2H, tt, J=7.3, 7.3 Hz), 1.23–1.33 (4H, m), 0.85
(3H, t, J=7.3 Hz); MS (TOF) m/z 297 (M+H)+. 6f: 1H
NMR (DMSO-d6) d 12.14 (1H, s), 8.12 (1H, s), 7.26–
7.37 (5H, m), 5.33 (2H, s), 2.59 (2H, t, J=7.3 Hz), 1.68
(2H, tt, J=7.3, 7.3 Hz), 1.23–1.25 (8H, m), 0.84 (3H, t,
J=7.3 Hz); MS (TOF) m/z 325 (M+H)+. 6g: 1H NMR
(DMSO-d6) d 12.10 (1H, s), 8.12 (1H, s), 7.27–7.36 (5H,
m), 5.32 (2H, s), 2.94 (1H, m), 1.24 (6H, d, J=6.8 Hz);
MS (TOF) m/z 269 (M+H)+. 6h: 1H NMR (DMSO-d6)
d 12.39 (1H, s), 8.07 (1H, s), 7.26–7.38 (5H, m), 5.24
(2H, s), 2.02 (1H, tt, J=5.9, 5.9 Hz), 1.04 (4H, m); MS
Compounds 7e–u were also prepared using the same
procedure described above.
1
7e: H NMR (DMSO-d6) d 8.14 (1H, s), 7.24–7.33 (5H,
m), 7.07 (2H, s), 5.33 (2H, s), 2.63 (2H, t, J=7.6 Hz),
1.68–1.74 (2H, m), 1.26–1.30 (4H, m), 0.85 (3H, t,
1
J=7.6 Hz); MS (TOF) m/z 296 (M+H)+. 7f: H NMR
(DMSO-d6) d 8.17 (1H, s), 7.19–7.33 (7H, m), 5.33 (2H,
s), 2.64 (2H, t, J=7.6 Hz), 1.70–1.73 (2H, m), 1.24–1.27
(8H, m), 0.84 (3H, t, J=7.6 Hz); MS (TOF) m/z 324
(M+H)+. 7g: 1H NMR (DMSO-d6) d 8.14 (1H, s),
7.26–7.38 (5H, m), 7.05 (2H, s), 5.32 (2H, s), 2.92 (1H,
m), 1.24 (6H, d, J=6.8 Hz); MS (TOF) m/z 268
(M+H)+. 7h: 1H NMR (DMSO-d6) d 8.10 (1H, s),
7.25–7.37 (5H, m), 7.03 (2H, s), 5.24 (2H, s), 2.02 (1H,
m), 1.04 (4H, t, J=2.7 Hz); MS (TOF) m/z 266
(M+H)+. 7i: 1H NMR (DMSO-d6) d 8.14 (1H, s),
7.20–7.33 (5H, m), 7.08 (2H, s), 5.33 (2H, s), 2.18 (2H,
m), 0.88 (6H, d, J=6.5 Hz); MS (TOF) m/z 282
(M+H)+. 7j: 1H NMR (DMSO-d6) d 8.14 (1H, s),
7.27–7.35 (5H, m), 7.00 (2H, s), 5.32 (2H, s), 2.50–2.63
(1H, m), 1.51–1.91 (7H, m), 1.15–1.40 (3H, m); MS
1
(TOF) m/z 267 (M+H)+. 6i: H NMR (DMSO-d6) d
12.14 (1H, s), 8.12 (1H, s), 7.32 (5H, m), 5.33 (2H, s),
2.48 (2H, m), 2.05–2.20 (1H, m), 0.89 (6H, d,
1
J=6.5 Hz); MS (TOF) m/z 283 (M+H)+. 6j: H NMR
(DMSO-d6) d 12.06 (1H, s), 8.11 (1H, s), 7.29–7.36 (5H,
m), 5.32 (2H, s), 2.58–2.69 (1H, m), 1.49–1.89 (7H, m),
1.14–1.37 (3H, m); MS (TOF) m/z 309 (M+H)+. 6k:
1H NMR (DMSO-d6) d 12.13 (1H, s), 8.11 (1H, s), 7.28–
7.32 (5H, m), 5.32 (2H, s), 2.60 (2H, d, J=7.8 Hz), 2.26–
2.36 (1H, m), 1.46–1.72 (6H, m), 1.12–1.24 (2H, m); MS
1
(TOF) m/z 309 (M+H)+. 6l: H NMR (DMSO-d6) d
12.12 (1H, s), 8.13 (1H, s), 7.28–7.34 (5H, m), 5.32 (2H,
s), 1.77–1.87 (1H, m), 1.58–1.66 (6H, m), 1.46–1.72 (6H,
m), 0.88–1.26 (6H, m); MS (TOF) m/z 323 (M+H)+.
1
(TOF) m/z 308 (M+H)+. 7k: H NMR (DMSO-d6) d
1
6n: H NMR (DMSO-d6) d 12.70 (1H, s), 9.27 (1H, s),
8.72–8.74 (1H, m), 8.44–8.49 (1H, m), 8.26 (1H, s),
8.14 (1H, s), 7.24–7.33 (5H, m), 7.07 (2H, s), 5.32 (2H,
s), 2.63 (2H, d, J=7.8 Hz), 2.30–2.50 (1H, m), 1.44–1.69
(6H, m), 1.15–1.27 (2H, m); MS (TOF) m/z 308
(M+H)+. 7l: 1H NMR (DMSO-d6) d 8.15 (1H, s),
7.25–7.35 (5H, m), 7.11 (2H, s), 5.32 (2H, s), 2.54 (1H,
7.55–7.60 (1H, m), 7.30–7.39 (5H, m), 5.44 (2H, s); MS
1
(TOF) m/z 304 (M+H)+. 6o: H NMR (DMSO-d6) d
12.41 (1H, s), 8.14 (1H, s), 7.23–7.34 (10H, m), 5.30

Y. Isobe et al. / Bioorg. Med. Chem. 11 (2003) 3641–3647
3645
m), 1.79–1.93 (6H, m), 1.14–1.25 (4H, m), 0.86–1.01
1
(M+H)+. 8n: 1H NMR (DMSO-d6) d 9.53 (1H, d,
J=1.9 Hz), 9.12–9.16 (1H, m), 8.91–8.93 (1H, m), 8.01–
8.06 (1H, m), 7.83 (2H, brs), 7.28–7.40 (5H, m), 5.49
(2H, m); MS (TOF) m/z 322 (M+H)+. 7n: H NMR
(DMSO-d6) d 9.50 (1H, d, J=2.3 Hz), 8.60–8.64 (1H,
m), 8.32 (1H, s), 7.26–7.53 (8H, m), 5.45 (2H, s); MS
1
(2H, s); MS (TOF) m/z 382 (M+H)+. 8o: H NMR
1
(TOF) m/z 303 (M+H)+. 7o: H NMR (DMSO-d6) d
(DMSO-d6) d 7.18–7.39 (12H, m), 5.32 (2H, s), 3.97
1
8.18 (1H, s), 7.18–7.34 (10H, m), 5.33 (2H, s), 3.96 (2H,
s); MS (TOF) m/z 316 (M+H)+. 7p: 1H NMR (DMSO-
d6) d 8.18 (1H, s), 7.28–7.33 (7H, m), 7.19 (2H, s), 7.04–
7.12 (2H, m), 5.32 (2H, s), 3.95 (2H, s); MS (TOF) m/z
(2H, s); MS (TOF) m/z 395 (M+H)+. 8p: H NMR
(DMSO-d6) d 7.21–7.41 (9H, m), 7.05–7.12 (2H, m),
5.31 (2H, s), 3.96 (2H, s); MS (TOF) m/z 414 (M+H)+.
1
8q: H NMR (DMSO-d6) d 8.12 (2H, s), 7.31–7.40 (3H,
1
335 (M+H)+. 7q: H NMR (DMSO-d6) d 8.45 (1H, s),
s), 7.21–7.24 (2H, m), 5.40 (2H, s); MS (TOF) m/z 373
(M+H)+. 8r: 1H NMR (DMSO-d6) d 7.89 (2H, s),
7.30–7.38 (3H, m), 7.19–7.22 (2H, m), 6.69 (1H, t,
J=54.8 Hz), 5.39 (2H, s); MS (TOF) m/z 355 (M+H)+.
7.91 (2H, s), 7.26–7.39 (5H, m), 5.42 (2H, s); MS (TOF)
m/z 294 (M+H)+. 7r: 1H NMR (DMSO-d6) d 8.37 (1H,
s), 7.26–7.39 (5H, m), 7.11 (2H, s), 6.67 (1H, t,
J=52.4 Hz), 5.41 (2H, s); MS (TOF) m/z 276 (M+H)+.
1
8s: H NMR (DMSO-d6) d 8.11 (2H, s), 7.25–7.36 (3H,
1
1
7s: H NMR (DMSO-d6) d 8.47 (1H, s), 7.29–7.36 (5H,
m), 5.38 (2H, s); MS (TOF) m/z 423 (M+H)+. 8t: H
m), 7.03 (2H, s), 5.40 (2H, s); MS (TOF) m/z 344
(M+H)+. 7t: 1H NMR (DMSO-d6) d 8.46 (1H, s),
7.28–7.36 (5H, m), 7.05 (2H, s), 5.45 (2H, s); MS (TOF)
m/z 394 (M+H)+. 7u: 1H NMR (DMSO-d6) d 8.21
(1H, s), 7.25–7.37 (7H, m), 5.35 (2H, s); MS (TOF) m/z
322 (M+H)+.
NMR (DMSO-d6) d 8.12 (2H, s), 7.24–7.36 (5H, m),
5.38 (2H, s); MS (TOF) m/z 473 (M+H)+. 8u: 1H
NMR (DMSO-d6) d 7.46 (2H, s), 7.22–7.38 (5H, m),
5.34 (2H, s); MS (TOF) m/z 401 (M+H)+.
9-Benzyl-2-butyl-8-hydroxyadenine (4). A suspension of
9-benzyl-8-bromo-2-butyladenine (1.85 g, 12.0 mmol) in
12 N HCl (27.8 mL) was heated at 100 ꢀC for 8 h. The
reaction mixture was concentrated, water (5 mL) was
added to the residue and neutralized by 10% NaOH
aqueous solution. The precipitate was collected and
purified by silica gel chromatography using 3% MeOH
in CH2Cl2 as an eluent to give the title compound as a
9-Benzyl-8-bromo-2-butyladenine (8d). A suspension of
9-benzyl-2-butyladenine (330 mg, 1.2 mmol), bromine
(2.8 g, 18 mmol), and NaOAc (1.6 g, 20 mmol) in AcOH
(20 mL) was heated at 110 ꢀC for 2 h. The reaction mix-
ture was concentrated, water was added to the residue
and extracted with CH2Cl2. The organic layer was
washed with water, dried over Na2SO4, and con-
centrated. The residue was purified by silica gel chro-
matography using 1% MeOH in CH2Cl2 as an eluent to
give the title compound as a pale yellow solid (215 mg,
ꢀ
1
white solid (246 mg, 62%), mp 280–283 C; H NMR
(DMSO-d6) d 10.24 (1H, s), 7.23–7.30 (5H, m), 6.42
(2H, s), 4.89 (2H, s), 2.56 (2H, t, J=6.6 Hz), 1.61–1.66
(2H, m), 1.24–1.32 (2H, m), 0.87 (3H, t, J=6.6 Hz); MS
(TOF) m/z 298 (M+H)+. Anal. calcd for
1
51%); H NMR (DMSO-d6) d 7.22–7.37 (7H, m), 5.32
.
(2H, s), 2.65 (2H, t, J=7.6 Hz), 1.64–1.75 (2H, m), 1.24–
1.38 (2H, m), 0.88 (2H, t, J=7.6 Hz); MS (TOF) m/z
361 (M+H)+.
C16H19N5O 1.4H2O; C, 59.57; H, 6.37; N, 21.71.
Found: C, 59.36; H, 6.23; N, 21.48.
Compounds 9e–u were also prepared by using the same
procedure as for 4.
Compounds 8e–u were also prepared using the same
procedure described above.
9-Benzyl-8-hydroxy-2-pentyladenine (9e). White solid
8e: 1H NMR (DMSO-d6) d 7.22–7.37 (7H, m), 5.33 (2H,
s), 2.61–2.75 (2H, m), 1.66–1.76 (2H, m), 1.28 (4H, m),
0.85 (3H, t, J=7.3 Hz); MS (TOF) m/z 375 (M+H)+.
8f: 1H NMR (DMSO-d6) d 7.21–7.36 (7H, m), 5.32 (2H,
s), 2.63 (2H, t, J=7.6 Hz), 1.65–1.75 (2H, m), 1.23–1.26
(8H, m), 0.84 (3H, t, J=7.6 Hz); MS (TOF) m/z 403
(yield 65%), mp 251–253 ꢀC; H NMR (DMSO-d6) d
1
10.09 (1H, s), 7.24–7.29 (5H, m), 6.34 (2H, s), 4.88 (2H,
s), 2.55 (2H, t, J=7.2 Hz), 1.65 (2H, m), 1.26 (4H, m),
0.84 (3H, t, J=7.2 Hz); MS (TOF) m/z 312 (M+H)+.
Anal. calcd for C17H21N5O: C, 65.57; H, 6.80; N, 22.49.
Found: C, 65.54; H, 6.77; N, 22.59.
1
(M+H)+. 8g: H NMR (DMSO-d6) d 7.25–7.38 (7H,
m), 5.32 (2H, s), 2.89 (1H, m), 1.23 (6H, d, J=6.8 Hz);
MS (TOF) m/z 347 (M+H)+. 8h: 1H NMR (DMSO-d6)
d 7.24–7.40 (7H, m), 5.32 (2H, s), 2.11 (1H, m), 1.07
(4H, t, J=2.7 Hz); MS (TOF) m/z 345 (M+H)+. 8i: 1H
NMR (DMSO-d6) d 7.22–7.36 (7H, m), 5.32 (2H, s),
2.54 (2H, m), 2.13–2.23 (1H, m), 0.88 (6H, d,
9-Benzyl-2-heptyl-8-hydroxꢀyadenine (9f). White solid
1
(yield 72%), mp 218–219 C; H NMR (DMSO-d6) d
10.08 (1H, s), 7.24–7.29 (5H, m), 6.33 (2H, s), 4.89 (2H,
s), 2.56 (2H, t, J=7.5 Hz), 1.62–1.67 (2H, m), 1.22–
1.24 (8H, m), 0.84 (3H, t, J=7.5 Hz); MS (TOF) m/z
340 (M+H)+. Anal. calcd for C19H25N5O: C, 67.23;
H, 7.42; N, 20.63. Found: C, 67.11; H, 7.42; N,
20.51.
1
J=6.5 Hz); MS (TOF) m/z 361 (M+H)+. 8j: H NMR
(DMSO-d6) d 7.24–7.39 (7H, m), 5.32 (2H, s), 2.50–2.59
(1H, m), 1.50–1.91 (7H, m), 1.18–1.40 (3H, m); MS
1
(TOF) m/z 367 (M+H)+. 8k: H NMR (DMSO-d6) d
9-Benzyl-8-hydroxy-2-isopropyladenine (9g). White solid
(yield 59%), mp 269–271 ꢀC; H NMR (DMSO-d6) d
1
7.23–7.37 (5H, m), 5.32 (2H, s), 2.64 (2H, d, J=7.3 Hz),
2.30–2.50 (1H, m), 1.44–1.71 (6H, m), 1.15–1.27 (2H,
m); MS (TOF) m/z 367 (M+H)+. 8l: 1H NMR
(DMSO-d6) d 7.24–7.37 (7H, m), 5.32 (2H, s), 2.54 (1H,
m), 1.80–1.93 (1H, m), 1.59–1.62 (6H, m), 1.13–1.25
(4H, m), 0.89–1.02 (2H, m); MS (TOF) m/z 401
10.13 (1H, s), 7.41 (5H, m), 6.39 (2H, s), 4.89 (2H,
s), 2.15 (2H, m), 1.51 (6H, d, J=6.8 Hz); MS (TOF)
m/z 284 (M+H)+. Anal. calcd for C15H17N5O: C,
63.59; H, 6.05; N, 24.72. Found: C, 63.49; H, 6.01;
N, 24.51.

3646
Y. Isobe et al. / Bioorg. Med. Chem. 11 (2003) 3641–3647
9-Benzyl-2-cyclopropyl-8-hydroxyadenine (9h). White
solid (yield 69%), mp 263–266 ꢀC; H NMR (DMSO-
9-Benzyl-2-(4-fluorobenzyl)-8-hydroxyadenine
(9p).
White solid (yield 47%), mp 209–211 ꢀC; 1H NMR
(DMSO-d6) d 10.28 (1H, s), 7.25–7.39 (7H, m), 7.04–
7.11 (2H, m), 6.45 (2H, s), 4.89 (2H, s), 3.88 (2H, s); MS
(TOF) m/z (M+H)+. Anal. calcd for C19H16FN5O: C,
65.32; H, 4.62; N, 20.05. Found: C, 65.31; H, 4.59; N,
20.00.
1
d6) d 10.05 (1H, s), 7.25–7.35 (5H, m), 6.30 (2H, s), 4.87
(2H, s), 1.89 (1H, m), 0.82 (4H, m); MS (TOF) m/z 296
+
.
(M+H) . Anal. calcd for C15H15N5O 0.1H2O: C, 63.68;
H, 5.38; N, 24.75. Found: C, 63.59; H, 5.36; N, 25.06.
9-Benzyl-8-hydroxy-2-isobutyladenine (9i). White solid
(yield 76%), mp 262–265 ꢀC; H NMR (DMSO-d6) d
9-Benzyl-8-hydroxy-2-trifluoromethyladenine (9q). White
1
ꢀ
1
7.24–7.31 (5H, m), 6.35 (2H, s), 4.90 (2H, s), 2.45 (2H,
d, J=6.8 Hz), 2.11 (1H, m), 0.86 (6H, d, J=6.8 Hz); MS
(TOF) m/z 298 (M+H)+. Anal. calcd for C16H19N5O:
C, 64.63; H, 6.44; N, 23.55. Found: C, 64.56; H, 6.50; N,
23.53.
solid (yield 78%), mp 263–265 C; H NMR (DMSO-
d6) d 10.69 (1H, s), 7.23–7.35 (5H, m), 7.01 (2H, s), 4.95
(2H, s); MS (TOF) m/z 310 (M+H)+. Anal. calcd for
C13H15F3N5O; C, 50.49; H, 3.26; N, 22.65. Found: C,
50.47; H, 3.37; N, 22.43.
9-Benzyl-2-cyclohexyl-8-hydroxyadenine (9j). White
solid (yield 68%), mp 249–252 ꢀC; H NMR (DMSO-
9-Benzyl-2-difluoromethyl-8-hydroxyadenine (9r). White
solid (yield 40%), mp 253–256 ꢀC; H NMR (DMSO-
1
1
d6) d 10.14 (1H, s), 7.23–7.33 (5H, m), 6.35 (2H, s), 4.89
(2H, s), 1.16–1.85 (11H, m); MS (TOF) m/z 324
d6) d 10.55 (1H, s), 7.24–7.36 (5H, m), 6.84 (2H, s), 6.61
(1H, t, J=54.5 Hz), 4.95 (2H, s); MS (TOF) m/z 292
(M+H)+. Anal. calcd for C13H11F2N5O; C, 53.61; H,
3.81; N, 24.05. Found: C, 53.69; H, 3.91; N, 24.63.
+
.
(M+H) . Anal. calcd for C18H21N5O 0.5H2O: C,
65.04; H, 6.52; N, 21.07. Found: C, 65.09; H, 6.55; N,
21.02.
9-Benzyl-8-hydroxy-2-pentafluoroethyladenine
(9s).
9-Benzyl-2-cyclopentylmethyl-8-hydroxyadenine
(9k).
White solid (yield 72%), mp 235–238 ꢀC; 1H NMR
(DMSO-d6) d 10.62 (1H, s), 7.24–7.32 (5H, m), 7.00
(2H, s), 4.94 (2H, s); MS (TOF) m/z 360 (M+H)+.
Anal. calcd for C14H15F5N5O: C, 46.81; H, 2.81; N,
19.49. Found: C, 46.52; H, 2.91; N, 19.37.
White solid (yield 60%), mp 230–233 ꢀC; 1H NMR
(DMSO-d6) d 10.13 (1H, s), 7.23–7.32 (5H, m), 6.35
(2H, s), 4.90 (2H, s), 2.56 (2H, d, J=7.3 Hz), 2.24–2.35
(1H, m), 1.43–1.68 (6H, m), 1.14–1.24 (2H, m); MS
(TOF) m/z 310 (M+H)+. Anal. calcd for
.
C18H21N5O 0.2H2O: C, 66.11; H, 6.53; N, 21.42.
Found: C, 66.02; H, 6.55; N, 21.43.
9-Benzyl-2-heptafluoropropyl-8-hydroxyadenine
(9t).
White solid (yield 96%), mp 234–236 ꢀC; 1H NMR
(DMSO-d6) d 10.67 (1H, s), 7.24–7.35 (5H, m), 7.03
(2H, s), 4.94 (2H, s); MS (TOF) m/z 410 (M+H)+.
9-Benzyl-2-cyclohexylmethyl-8-hydroxyadenine
(9l).
White solid (yield 73%), mp 239–242 ꢀC; 1H NMR
(DMSO-d6) d 10.12 (1H, s), 7.23–7.31 (5H, m), 6.35
(2H, s), 4.90 (2H, s), 2.45 (2H, d, J=7.3 Hz), 1.80–1.83
(1H, m), 1.56–1.61 (5H, m), 1.12–1.23 (3H, m), 0.86–
0.98 (2H, m); MS (TOF) m/z 324 (M+H)+. Anal. calcd
for C19H23N5O: C, 67.63; H, 6.87; N, 20.76. Found: C,
67.34; H, 6.90; N, 20.41.
Anal. calcd for C15H10F7N5O 0.3H2O: C, 43.45; H,
2.58; N, 16.89. Found: C, 43.58; H, 2.66; N, 16.57.
.
9-Benzyl-8-hydroxy-2-(3,3,3-trifluoropropyl)adenine (9u).
White solid (yield 70%), mp 242–245 ꢀC; 1H NMR
(DMSO-d6) d 10.23 (1H, s), 7.23–7.31 (5H, m), 6.47
(2H, s), 4.91 (2H, s), 2.80–2.86 (2H, m), 2.62–2.77 (2H,
m); MS (TOF) m/z 338 (M+H)+. Anal. calcd for
.
9-Benzyl-8-hydroxy-2-phenyladenine (9m). White solid
(yield 60%), mp 231–233 ꢀC; H NMR (DMSO-d6) d
C15H14F3N5O 0.8H2O: C, 51.22; H, 4.24; N, 19.91.
Found: C, 51.12; H, 4.09; N, 19.95.
1
10.31 (1H, s), 8.37 (2H, dd, J=6.0, 1.9 Hz), 7.23–7.44
(8H, m), 6.52 (2H, s), 5.01 (2H, s); MS (TOF) m/z 318
Biology
+
.
(M+H) . Anal. calcd for C18H15N5O 0.3H2O: C,
66.98; H, 4.87; N, 21.70. Found: C, 67.07; H, 4.74; N,
21.50.
IFN induction in mouse splenocyte cultures. Male C3H/
HeJ mice (Clea Japan Inc.) aged 8 weeks were sacrificed,
and spleens were removed from six 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% 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/mL of penicillin, and 100 mg/mL of streptomy-
cin. The test compounds were dissolved in dimethyl-
sulfoxide and diluted to 500-fold with supplemented
MEM.
9-Benzyl-8-hydroxy-2-(3-pyridyl)adenine (9n). Pale Yel-
low solid (yield 36%), mp 260–263 ꢀC; 1H NMR
(DMSO-d6) d 10.55 (1H, s), 9.40 (1H, s), 8.60–8.65 (2H,
m), 7.55–7.59 (1H, m), 7.23–7.41 (6H, m), 6.73 (2H, s),
5.02 (2H, s); MS (TOF) m/z 319 (M+H)+. Anal. calcd
.
for C17H14N6O 1.8H2O: C, 58.21; H, 4.54; N, 23.96.
Found: C, 58.32; H, 4.78; N, 23.66.
2,9-Dibenzyl-8-hydroxyadenine (9o). White solid (yield
ꢀ
1
74%), mp 220–222 C; H NMR (DMSO-d6) d 10.11
(1H, s), 7.16–7.28 (10H, m), 6.40 (2H, s), 4.89 (2H, s),
3.88 (2H, s); MS (TOF) m/z 332 (M+H)+. Anal. calcd
Above splenocytes suspension (0.5 mL) and various
concentrations of the test compounds solution (0.5 mL)
were mixed in 24-well plates, and cultured in a humidified
.
for C19H17N5O 0.2H2O: C, 68.13; H, 5.24; N, 20.91.
Found: C, 68.12; H, 5.06; N, 20.93.

Y. Isobe et al. / Bioorg. Med. Chem. 11 (2003) 3641–3647
3647
5% CO2/95% air atmosphere at 37 ꢀC for 18 h. Super-
natants were then collected, filter sterilized, and stored
References and Notes
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2. Saito, I.; Miyamura, T.; Ohbayashi, A.; Harada, H.;
Katayama, T.; Kikuchi, S.; Watanabe, Y.; Koi, S.; Onji, M.;
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Agents Chemother. 1976, 9, 433.
at À80 ꢀC until they were analyzed for IFN.
IFN induction in mice. The test compounds suspended
in 0.5% sodium carboxymethyl cellulose were adminis-
tered 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 administration. Plasma samples
were obtained by centrifugation, and stored at À80 ꢀC
until they were analyzed for IFN.
7. Dianzani, F. J. Interferon Res. 1992, 12, 109.
8. (a) Weeks, C. E.; Gibson, S. J. J. Interferon Res. 1994, 14,
81. (b) Savage, P.; Horton, V.; Moore, J.; Owens, M.; Witt, P.;
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V.; Moore, J.; Owens, M.; Witt, P.; Gore, M. E. Brit. J. Can-
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IFN analysis. Mouse IFN titer in supernatants of
splenocytes and plasma sample was quantitated by
measuring its antiviral activity in a bioassay using
mouse L929 cell monolayers challenged with vesicular
stomatitis virus. Results are expressed as IFN IU/mL in
terms of the international mouse IFN standard
obtained from the National Institute of Health,
Bethesda, MD, USA.
10. 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.
11. Kelly, J. L.; Linn, J. A.; Selway, J. W. T. J. Med. Chem.
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12. Watanabe, Y.; Kawade, Y. In Lymphokines and Inter-
ferons: A Practical Approach; Clemens, M. J., Morris, A. G.,
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13. Pestka, S. In: Methods in Enzymology; Pestka, S., Ed.;
Academic: New York, 1986; Vol. 119, p 16.
Ferret emesis analysis. The test compounds suspended
in 0.5% sodium carboxymethyl cellulose were adminis-
tered orally to male ferret (Charles River Japan Inc.)
aged 4 months. Then animals were transferred to indi-
vidual cages and observed continuously over 6 h. Their
behavior was recorded by video cameras and tapes were
subsequently read at the end of the experiment to eval-
uate emesis. Blood samples (0.5 mL) were taken from a
jugular vein, and the serum was obtained by cen-
trifugation at 1630 g for 10 min at 4 ꢀC, and kept at
À20 ꢀC until drug analysis. The concentrations of 1 and
2c in the serum were assayed by an HPLC method.
14. Hirota, K.; Kazaoka, K.; Niimoto, I.; Sajiki, H. Hetero-
cycles 2001, 55, 2279.
15. Altman, J.; Ben-Ishai, D. J. Heterocycl. Chem. 1968, 5, 679.