Synthesis and biological evaluation of novel 9-substituted-8-hydroxyadenine derivatives as potent interferon inducers

Article abstract of DOI:10.1021/jm051089s

Recently we reported the adenine derivatives 3a-d as novel interferon (IFN) inducers. In the present study, we conducted a detailed structure-activity relationship study of analogues of 3a-d with respect to their IFN-inducing activity, mainly focusing on the N(9)-position of the adenine. From this study, we found that introduction of the 3-pyridylmethyl moiety was effective to increase in vitro activity, and compound 9ae was identified as being the most potent IFN inducer. This compound gave a minimum effective concentration (MEC) of 3 nM, which is comparable with that of R-848, a second generation IFN inducer. Compound 9ae also demonstrated potent IFN-inducing activity at a dose of 0.1 mg/kg by oral administration in mice. Furthermore, compound 9ae induced IFN in monkeys in a dose dependent manner, with a potency superior to that of R-848. In addition, 9ae did not cause emesis in ferrets even at a dose of 30 mg/kg. In this study the maximum plasma concentration of 9ae was 1019 ng/mL (ca. 3.1 μM), which was approximately 1000-fold higher than the MEC value. Therefore, with respect to both the efficacy and the safety margin, compound 9ae (SM-276001) is considered to be a promising compound as an orally active IFN inducer.

Full text of DOI:10.1021/jm051089s

2088
J. Med. Chem. 2006, 49, 2088-2095
Synthesis and Biological Evaluation of Novel 9-Substituted-8-Hydroxyadenine Derivatives as
Potent Interferon Inducers
Yoshiaki Isobe,*,† Ayumu Kurimoto,† Masanori Tobe,† Kazuki Hashimoto,† Tomoaki Nakamura,† Kei Norimura,†
Haruhisa Ogita,† and Haruo Takaku‡
Chemistry Research Laboratories and DiscoVery Research Laboratories II, Dainippon Sumitomo Pharmaceuticals Company Ltd., 1-98,
Kasugade Naka, 3-chome, Konohana-ku, Osaka 554-0022, Japan
ReceiVed October 27, 2005
Recently we reported the adenine derivatives 3a-d as novel interferon (IFN) inducers. In the present study,
we conducted a detailed structure-activity relationship study of analogues of 3a-d with respect to their
IFN-inducing activity, mainly focusing on the N(9)-position of the adenine. From this study, we found that
introduction of the 3-pyridylmethyl moiety was effective to increase in vitro activity, and compound 9ae
was identified as being the most potent IFN inducer. This compound gave a minimum effective concentration
(MEC) of 3 nM, which is comparable with that of R-848, a second generation IFN inducer. Compound 9ae
also demonstrated potent IFN-inducing activity at a dose of 0.1 mg/kg by oral administration in mice.
Furthermore, compound 9ae induced IFN in monkeys in a dose dependent manner, with a potency superior
to that of R-848. In addition, 9ae did not cause emesis in ferrets even at a dose of 30 mg/kg. In this study
the maximum plasma concentration of 9ae was 1019 ng/mL (ca. 3.1 µM), which was approximately 1000-
fold higher than the MEC value. Therefore, with respect to both the efficacy and the safety margin, compound
9ae (SM-276001) is considered to be a promising compound as an orally active IFN inducer.
Chart 1. Structures of 1 (Imiquimod), 2 (R-848), and 3a-d
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 significant clinical problem
affecting an estimated 150 million patients worldwide. Chronic
infection can lead to liver cirrhosis and associated development
of hepatocellular carcinoma within 10-15 years.2 At present
the recommended treatment for patients with HCV is a
combination of pegylated interferon-R (IFN-R) and ribavirin.
The sustained virological response rate of this combination
therapy is 42-48% for patients with genotype 1 after a course
of 48 weeks and 80% for patients with genotype 2 or 3 after a
course of 24 weeks.3,4 IFN-R therapy is very expensive, and
the formation of antibodies against IFN-R is observed in some
patients. Treatment with IFN-R over a 6 month period enhances
the efficacy and reduces the occurrence of hepatocellular
carcinoma,5 though the patients have to suffer regular injections
of IFN-R once a week.
Orally available IFN inducers offer the possible advantages
of convenience, prolonged action, and avoidance of immuno-
genicity. Therefore, IFN inducers are expected to be a new
therapeutic class drug for treating viral infections. Although a
number of IFN inducers including polyribonucleotide com-
plexes,6 fluorenones,7 and pyrimidones8 have been investigated,
no results have so far been reported for IFN production in
humans.9
a topical agent in the treatment of papilloma viral infections
possibly to avoid such side effects.
In our previous papers we reported on a series of 8-hydroxy-
adenine derivatives as novel structural IFN inducers,13-16
especially focusing on the substituent at the C(2)-position of
the adenine. In the course of the structure-activity relationship
(SAR) study we found that compounds 3a-d exhibited moder-
ate IFN-inducing activities which were superior to that of
imiquimod (1) but not to that of R-848 (2) (Chart 1). Our
previous work suggested that the amino group at C(6)-position
and the hydroxy at C(8)-position were essential to demonstrate
IFN-inducing activity.13 To find compounds having potent IFN-
inducing activities both in vitro and in vivo, we now report a
detailed SAR study on the modification of the N(9)-substituent
based on 3a.
Imiquimod (1), developed by 3M Pharmaceuticals, is a low
molecular weight IFN inducer and has been reported to induce
IFN production in humans.10 However, the potential systemic
usefulness of 1 was limited because it was emetic in clinical
study.11 The emetic activity of it was also reported in the
experimental animal model.12 However, imiquimod is used as
Chemistry
The synthetic routes to 9-substituted-8-hydroxyadenine de-
rivatives are shown in Schemes 1-4. The reaction of 4 with an
appropriate alkyl or benzyl chloride in the presence of potassium
carbonate gave corresponding compounds 5 in good yield.
Compounds 6 were obtained by heating 5 with butylamine in
an autoclave at 120 °C (method A). Compounds 6 could be
also prepared using method B. The replacement of the chlorine
atom at the C(2)-position in 4 with butylamine was carried out
* Corresponding author. Tel: +81-6-6466-5185. Fax: +81-6-6466-5483.
E-mail: yoshiaki-isobe@ds-pharm.co.jp.
† Chemistry Research Laboratories.
‡ Discovery Research Laboratories II.
10.1021/jm051089s CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/15/2006

9-Substituted-8-Hydroxyadenine DeriVatiVes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2089
Scheme 1. Synthetic Route to Compounds 9a-i,l-aja
a Reagents and conditions: (a) R-CH2Cl, K2CO3, DMF; (b) BuNH2, H2O; (c) BuNH2, PrOH, 120 °C; (d) Br2, CHCl3; (e) 12 N HCl, 1,4-dioxane, 110
°C.
Scheme 2. Synthetic Route to Compounds 9j,k,ak and 13a
a Reagents and conditions: (f) NaOH/MeOH, reflux; (g) 12 N HCl, room temp; (h) 10% Pd(OH)2/C, HCO2H.
Scheme 3. Synthetic Route to Compounds 20-22a
a Reagents and conditions: (c) X-NH2, PrOH, 120 °C; (d) Br2, CHCl3; (e) 12 N HCl, 1,4-dioxane, 110 °C.
by heating in an autoclave at 180 °C to give 7. This was
Scheme 4. Synthetic Route to Compound 23a
followed by a reaction with an appropriate alkyl or benzyl
chloride in the presence of potassium carbonate to give
corresponding compounds 6. As shown, compound 7 was
considered as a common intermediate, so method B was more
convenient than method A to use to make various derivatives.
The bromination of 6 was easily achieved by the addition of
bromine to give 8. The desired compounds (9a-i,l-aj) were
obtained by heating the corresponding 8 in 12 N HCl. In the
case of compounds 9j,k,ak, heating of the corresponding 8 in
12 N HCl brought about decomposition of the products.
a Reagent and condition: (i) Me2NH, 140 °C.
Therefore, compounds 10-12 were prepared as shown in
Scheme 2 by replacement of the bromine atom with a methoxy

2090 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Isobe et al.
Table 1. IFN-Inducing Activities of the Test Compounds Both in Vitro and in Vivo (IU/mL)
in vitro (nM)
in vivo (mg/kg)
compd
X
R
3
10
30
100
0.1
0.3
3a
3b
3c
3d
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
13
9o
9p
9q
9r
9s
Bu
Ph
Ph
Ph
Ph
<0.5
<0.5
3.4
7.1
80.4
9.1
4.5
4.4
7.4
3.6
5.5
2
4.2
15.2
2
2
10.6
11
113
44
203
<5
465
MeO(CH2)2
639
398
73
PhCH2
4-Py-CH2
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
<0.5
<0.5
44.4
2.2
<0.5
<0.5
8.5
<0.5
<0.5
9.2
12.8
2.9
3.9
2
2.8
8.2
3
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
2-Me-Ph
3-Me-Ph
4-Me-Ph
4-F-Ph
4-CF3-Ph
4-NO2-Ph
4-OMe-Ph
4-OBnn-Ph
4-n-Bu-Ph
4-t-Bu-Ph
4-Ph-Ph
4-OH-Ph
3,4-Cl2-Ph
3,4-F2-Ph
2-Cl-4,5-OCH2O-Ph
CH2Ph
1-naphthyl
2-naphthyl
5-thiophen(2-Cl)
CMe2OH
i-propyl
c-hexyl
2-Py
3-Py
4-Py
nda
nda
219
nda
nda
356
313
276
444
933
nda
<5
<5
nda
20
<0.5
7
3
6.2
<0.5
<0.5
<0.5
<0.5
<0.5
28
139
8
61
73
10.7
4.5
4.7
<0.5
<0.5
4.4
11.6
6.5
<0.5
13
<0.5
3.4
3.1
10
<0.5
<0.5
<0.5
12.4
0.8
6.4
<5
35
136
338
<5
nda
39
14.7
<0.5
7.1
2.1
<0.5
15.4
17.4
7.2
0.6
4.1
3.7
9.1
8.1
4.3
4.2
8.3
4.5
5.2
4
<0.5
<0.5
2.2
<0.5
8.9
34.0
7.1
11.3
<0.5
<0.5
1.7
<5
9t
<5
484
<5
nda
nda
<5
327
332
nda
521
<5
492
1714
9u
9v
9w
9x
9y
9z
9aa
9ab
20
21
22
2
34
<0.5
<0.5
32.5
4.2
<0.5
<0.5
7.4
1.6
<0.5
16.6
11.9
2.9
4.8
<0.5
76
21
2-pyrazine
4-F-Ph
4-F-Ph
4-F-Ph
MeO(CH2)2
PhCH2
4-Py-CH2
54
<0.5
2.8
11.1
11
19
1176
7
a nd: not determined.
group with the use of sodium methoxide. The cleavage of the
methoxy group in 10-12 by stirring in 12 N HCl at room
temperature gave the desired compounds (9j,k,ak). The catalytic
hydrogenation of 9k gave 13. As shown in Scheme 3, the
replacement of the chlorine atom at the C(2)-position in 5g with
appropriate amines led to compounds 14-16 which were
converted to the desired products 20-22 in the same way. The
reaction of 8ah with dimethylamine in an autoclave at 140 °C
afforded 23 (Scheme 4).
On the basis of compound 3a, we examined the electronic
effect of substituents by use of a chlorine atom, as an electron-
withdrawing group, or a methyl group, as an electron-donating
group, at various positions on the benzene ring (9a-f). The
results are summarized in Table 1. It can be seen that para-
substituted compounds (9c,f) were 10-fold more potent than
unsubstituted 3a, whereas meta- or ortho-substituted compounds
(9a,b,d,e) proved less favorable. These results indicated that
the electronic component of the substituent was negligible,
whereas the correct regiochemistry of the substituent was
important. Next, we examined compounds containing various
substituents at the para position (9g-n, 13). The activities of
compounds 9k,l-n (MEC ) 30 or 100 nM) suggested that
bulky substituents and long linear alkyl groups were detrimental
to the activity, whereas compounds containing relatively
compact groups (9g-j, 13) were preferred (MEC ) 10 nM).
Dihalogenated compounds (9o,p) showed equipotent activities
with the monohalogenated compounds (9c,g) (MEC ) 10 nM)
showing that a meta halogen group was tolerated. However, it
can be seen that the amount of IFN induced by compounds 9o,p
was inferior to that of 9c,g. Interestingly, in this group of potent
compounds the 4-fluoro analogue (9g) exhibited the most potent
Results and Discussions
The in vitro IFN-inducing activities of the prepared com-
pounds were conducted by the following method. Mouse
splenocytes were cultured with the compounds, and the con-
centrations of IFN induced by the compounds in the supernatant
were measured by bioassay using L929 cells with vesicular
stomatitis virus.17,18 With regard to the in vivo test, we orally
administered the test compounds to male Balb/c mice and then
the concentrations of IFN in the plasma were measured by the
bioassay mentioned above. In the preliminary experiment, the
IFN concentration 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.

9-Substituted-8-Hydroxyadenine DeriVatiVes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2091
Table 2. IFN-Inducing Activities of the Test Compounds Both in Vitro
Table 3. IFN-Inducing Activities of Compounds 2 and 9ae,ah,ak in
and in Vivo (IU/mL)
Cynomolgus Monkeys (IU/mL)
dose (mg/kg)
compd
0.1
0.3
1
9ae
9ah
9ak
2
121 ( 89
0
3 ( 3
7 ( 4
591 ( 411
19 ( 16
244 ( 244
13 ( 10
34810 ( 15915
3602 ( 1619
17422 ( 14108
44037 ( 34055
in vivo
in vitro (nM)
(mg/kg)
30 100 0.1 0.3
76 327
8.3 3.0 186 1462
2.1 4.6
nda
3.7 1.4 393 1891
Table 4. Emetic Activities of Compounds 2, and 9ae,ah,ak) in Ferrets
compd
R
1
3
10
compd
dose (mg/kg)
vomiting
C
max (ng/mL)
9z
3-Py
<0.5
1.6 32.5 16.6 8.1
9ae
0.3
3
30
0.3
3
30
0.3
3
30
0.3
3
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
2/5
5/5
nda
20.8
1019
nda
20.1
275.2
nda
108.9
1199
nda
9ac 2-Me-3-Py
9ad 5-Me-3-Py
9ae 6-Me-3-Py
<0.5 12.8
<0.5
<0.5
1.9
4.4
2.4
7.8
9ah
9ak
2
9af
2-Cl-3-Py
<0.5
3.7 16.3 5.0
<5 94
9ag 4-Cl-3-Py
9ah 6-Cl-3-Py
9ak 6-OMe-3-Py
<0.5 4.2
nda
<0.5
<0.5
<0.5
5.6
6.8
2.6 1.5 811 1826
3.0 1.0 375 1920
8.2 13.4
23
9ai
9aj
2
6-NMe2-3-Py
2-Cl-6-Me-3-Py
2,6-Cl-3-Py
4.9
<0.5
9.4
8.4
5.1 2.7
3.4
<5 403
<5 619
<0.5 14.6 5.7
2.8 11
42 414
1181
<0.5
7
4
1176 1714
a Nd: not determined.
a nd: not determined.
IFN-inducing activity in vivo which we speculated may be due
to its good oral bioavailability.
Unfortunately, against our expectations the in vitro activities
of compounds 9ae,ah were evaluated as 3 nM, which were
equivalent with that of 9z. These results were not consistent
with those of para-substituted benzyl compounds (9c,f) where
an increase in potency was observed. In addition, the results of
compounds 9ac,af,ag indicated that substitution at the C2- or
C4-positions of the pyridine ring did not lead to an increase in
activity. These results are similar to those of ortho-substituted
benzyl compounds (9a,d). Among the compounds tested here,
compounds 9ae,ah,ak showed potent IFN-inducing activities
at a dose of 0.1 mg/kg by oral administration: The induced
amounts of IFN were 393 IU/mL for 9ae, 811 IU/mL for 9ah,
and 375 IU/mL for 9ak, respectively. Their potencies were
severalfold greater than 3a. The compound R-848 (2),19
developed by 3M Pharmaceuticals, also exhibited potent in vitro
IFN-inducing activity, and it induced 1176 IU/mL of IFN at an
oral dose of 0.1 mg/kg. This in vivo potency was greater than
our compounds, and we speculate that is the case because R-848
might possess superior oral bioavailability than our compounds.
Further biological evaluations of compounds 9ae,ah,ak were
carried out with comparison to compound 2. Test compounds
were orally administered to cynomolgus monkeys, and the
amount of IFN in plasma was measured during 0-24 h after
dosing. The results of maximum concentrations of IFN in plasma
are summarized in Table 3. It can be seen that compound 9ae
induced IFN in a dose dependent manner, and it is more potent
than 9ah and 9ak. The compoound R-848 (2) also exhibited
potent IFN-inducing activity at a dose of 1 mg/kg, but it did
not show the clear dose-dependency. Therefore, the activity of
compound 9ae was considered to be superior to that of R-848
at low doses.
In our previous paper15 we disclosed compounds 3b-d;
therefore, here we substituted the N(9)-benzyl moiety present
in 3b-d with a 4-fluorobenzyl moiety with the expectation of
an improvement in the activity. Unfortunately, as shown in Table
1, compound 20 demonstrated a 3-fold improvement in the in
vitro activity compared with 3b, whereas 21 and 22 did not
improve the activity in comparison with 3c and 3d, respectively.
However, the in vivo activity of 20 was not significantly
improved; therefore, the 4-fluorobenzyl group could not be
considered to be a generally useful N(9)-substituent to increase
the activity.
As shown in Table 1, although compound 9g was 10-fold
more potent than 3a in vitro, this did not translate into an
increase in the in vivo activity as measured by the quantity of
IFN in plasma (139 IU/mL versus 113 IU/mL). To find
compounds having activities more potent than 9g, both in vitro
and in vivo, we continued further examination by the replace-
ment of the phenyl ring in 3a with other moieties. Alkyl
compounds (9r,v-x) gave weak or diminished activities, while
lipophilic aromatic compounds (9s,u) exhibited potencies
comparable with 9g (MEC ) 10 nM). Unfortunately, again these
compounds did not show potent IFN-inducing activity in vivo,
as determined by the amount of IFN induced at a dose of 0.1
mg/kg in comparison to 9g. The in vivo results of para-
substituted compounds 9c,f-j or 9s,u suggested that lipophilic
substituents at N(9)-positions were unfavorable for potent in
vivo activity which we suggest may be due to a problem in
oral bioavailability. Next we investigated the pyridyl derivatives
as examples of hydrophilic aromatic substituents (9y-aa). As
shown in Table 1, the order of the potency was 3-pyridyl (9z,
3 nM) > 4-pyridyl (9aa, 10 nM) > 2-pyridyl (9y, 30 nM). It is
noteworthy that 9z was 30-fold more potent than the benzyl
compound 3a; however, the 2-pyrazine (9ab) was equipotent
with the 2-pyridyl (9y), suggesting that the nitrogen atom at
the ortho position might not be advantageous in increasing the
potency further. Although compound 9z induced IFN when
orally administered (76 IU/mL at 0.1 mg/kg), this result was
unexpectedly inferior to that of 3a or 9g. The SAR of 9z was
further investigated by the introduction of substituents onto the
pyridine ring, and the results are summarized in Table 2.
In the clinical study of imiquimod (1), one serious side effect,
emesis, was observed.11 Compound R-848, a structurally similar
compound to 1, is being developed as topical agent, and to date
no report of any emesis has been published. We chose a ferret
emesis model to evaluate this side effect which was evaluated
by the absence or presence of vomiting behavior within 6 h
after administration of the compounds. As shown in Table 4,
when compounds 9ae,ah,ak were orally administered at doses
of 0.3-30 mg/kg, no vomiting was observed in any of the
ferrets, while the administration of 2 at a dose of 3 mg/kg caused
vomiting in all animals. The plasma concentrations of these

2092 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Isobe et al.
compound as a pale yellow solid (0.62 g, 63%): 1H NMR (DMSO-
d6) δ 7.76 (1H, s), 7.50 (1H, dd, J ) 7.6, 1.4 Hz), 7.32 (2H, m),
7.00 (1H, brs), 6.69 (2H, s), 6.26 (1H, t, J ) 5.4 Hz), 5.28 (2H, s),
3.19 (2H, q, J ) 6.8 Hz), 1.44 (2H, m), 1.28 (2H, m), 0.83 (3H, t,
J ) 7.6 Hz).
Method B. 2-Butylaminoadenine (7). A suspension of 4 (5.0
g, 30 mmol) and butylamine (30 mL) in H2O (20 mL) was heated
at 180 °C for 11 h in an autoclave. The reaction mixture was
concentrated, and water was added to the residue to give the title
compound as a dark green solid (5.6 g, 92%): 1H NMR (DMSO-
d6) δ 12.01 (1H, brs), 7.62 (1H, s), 6.53 (2H, s), 6.07 (1H, t, J )
5.6 Hz), 3.18 (2H, q, J ) 6.8 Hz), 1.48 (2H, m), 1.31 (2H, m),
0.88 (3H, t, J ) 7.2 Hz).
2-Butylamino-9-(4-methylbenzyl)adenine (6f). To a suspension
of 7 (118 mg, 0.57 mmol) and potassium carbonate (59 mg, 0.43
mmol) in DMF (5 mL) was added 4-methylbenzyl chloride (120
mg, 0.86 mmol), and the mixture was stirred at room temperature
for 18 h. The reaction mixture was concentrated, and the residue
was partitioned with water and CHCl3, dried over MgSO4, and
concentrated. The residue was purified by silica gel chromatography
by use of 2% MeOH in CHCl3 as an eluent to give the title
compound as a white solid (125 mg, 71%): 1H NMR (DMSO-d6)
δ 7.75 (1H, s), 7.21 (2H, d, J ) 7.6 Hz), 7.12 (2H, d, J ) 7.6 Hz),
6.61 (2H, s), 6.21 (1H, t, J ) 6.0 Hz), 5.11 (2H, s), 3.22 (2H, q, J
) 6.8 Hz), 2.25 (3H, s), 1.48 (2H, m), 1.31 (2H, m), 0.88 (3H, t,
J ) 7.6 Hz).
8-Bromo-2-butylamino-9-(2-chlorobenzyl)adenine (8a). To a
suspension of 6a (600 mg, 1.8 mmol) in CHCl3 (10 mL) was added
bromine (350 mg, 2.2 mmol) in an ice cooling condition, and the
reaction mixture was stirred at ambient temperature for 3 h. The
resulting precipitate was collected to give the title compound as a
pale yellow solid (0.81 g, 91%): 1H NMR (DMSO-d6) δ 9.42 (1H,
brs), 8.54 (1H, brs), 7.55 (2H, m), 7.36 (2H, m), 6.95 (1H, t, J )
6.8 Hz), 5.33 (2H, s), 3.24 (2H, m), 1.45 (2H, m), 1.26 (2H, m),
0.83 (3H, t, J ) 7.2 Hz).
2-Butylamino-9-(2-chlorobenzyl)-8-hydroxyadenine (9a). A
solution of 8a (350 mg, 0.7 mmol) and 12 N HCl (10 mL) in 1,4-
dioxane (10 mL) was heated at 110 °C for 6 h. The reaction mixture
was concentrated, and the residue was neutralized with 1 N NaOH
aqueous solution. The precipitate was collected and purified by silica
gel chromatography by use of 2% MeOH in CHCl3 as an eluent to
give the title compound as a white solid (165 mg, 67%): recrystal-
lized from MeOH, mp 241-244 °C; 1H NMR (DMSO-d6) δ 10.45
(1H, s), 7.70 (2H, s), 7.47 (1H, d, J ) 6.8 Hz), 7.30 (3H, m), 7.15
(1H, s), 5.00 (2H, s), 3.18 (2H, q, J ) 6.4 Hz), 1.42 (2H, m), 1.25
(2H, m), 0.83 (3H, t, J ) 7.6 Hz). Anal. (C16H19ClN6O) C, H, N.
Compounds 9b-i,l-aj, and 20-22 were also prepared using
the similar procedure described above.
2-Butylamino-9-(3-chlorobenzyl)-8-hydroxyadenine (9b). Yield
94%; recrystallized from MeOH, mp 244-246 °C; 1H NMR
(DMSO-d6) δ 9.67 (1H, s), 7.38 (3H, m), 7.25 (1H, m), 6.24 (1H,
t, J ) 6.8 Hz), 6.04 (2H, s), 4.81 (2H, s), 3.16 (2H, q, J ) 6.8 Hz),
1.45 (2H, m), 1.28 (2H, m), 0.86 (3H, t, J ) 7.6 Hz). Anal. (C16H19-
ClN6O‚0.18H2O) C, H, N.
2-Butylamino-9-(4-chlorobenzyl)-8-hydroxyadenine (9c). Yield
85%; recrystallized from EtOH, mp 278-280 °C; 1H NMR
(DMSO-d6) δ 9.56 (1H, s), 7.40 (2H, d, J ) 9.2 Hz), 7.29 (2H, d,
J ) 9.2 Hz), 6.22 (1H, t, J ) 5.6 Hz), 6.02 (2H, s), 4.79 (2H, s),
3.15 (2H, q, J ) 6.8 Hz), 1.43 (2H, m), 1.27 (2H, m), 0.86 (3H, t,
J ) 7.6 Hz). Anal. (C16H19ClN6O) C, H, N.
2-Butylamino-8-hydroxy-9-(2-methylbenzyl)adenine (9d). Yield
74%; recrystallized from MeOH-H2O, mp 195-196 °C; 1H NMR
(DMSO-d6) δ 10.25 (1H, s), 7.16 (7H, m), 4.83 (2H, s), 3.17 (2H,
q, J ) 6.8 Hz), 2.41 (3H, s), 1.45 (2H, m), 1.27 (2H, m), 0.85 (3H,
t, J ) 7.2 Hz). Anal. (C17H22N6O‚HCl‚0.35H2O) C, H, N.
2-Butylamino-8-hydroxy-9-(3-methylbenzyl)adenine (9e). Yield
67%; recrystallized from MeOH, mp 238-240 °C; 1H NMR
(DMSO-d6) δ 9.62 (1H, s), 7.19 (1H, t, J ) 7.2 Hz), 7.08 (3H, m),
6.20 (1H, t, J ) 5.6 Hz), 6.00 (2H, s), 4.76 (2H, s), 3.16 (2H, q, J
) 6.8 Hz), 2.26 (3H, s), 1.47 (2H, m), 1.27 (2H, m), 0.87 (3H, t,
J ) 7.2 Hz). Anal. (C17H22N6O‚0.1H2O) C, H, N.
compounds achieved a sufficient level to induce IFN. The Cmax
value of 9ae at 30 mg/kg was nearly the same with that of 2 at
3 mg/kg. These results indicated that compound 9ae exerted a
highly potent IFN-inducing activity and was superior to 2 in
terms of the margin between the IFN-inducing and emetic doses.
As for the possible reason for this, we hypothesize that 9ae is
not able to transmigrate into the brain effectively because of
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 9ae (SM-276001) is
expected to be a useful IFN inducer in treatment of viral
infections such as HCV and HBV. It was reported that 1 and 2
induced IFN via the activation of Toll-like receptor-7 (TLR-
7).20 With regard to the mechanism of action of 9ae, we have
identified it as being a TLR-7 agonist and will report further
detailed information in appropriate opportunity in the future.
Conclusion
In this study the search for a potent IFN inducer was
addressed. Replacement of the benzyl moiety of 3a at the N(9)-
position by para-substituted benzyl groups was shown to
increase the in vitro activities by 10-fold (9c,f-l). However,
their in vivo activities were not improved in comparison to 3a,
possibly due to poor oral bioavailability. The examination of
various lipophilic compounds resulted in no improvement in
activities. Among the compounds having a hydrophilic N(9)-
substituent, the 3-pyridylmethyl moiety (9z) was identified to
be a very potent compound with in vitro activity in the
nanomolar range. From the SAR study of 9z, we identified
compounds 9ae,ah,ak as potent IFN inducers (Table 2).
Especially, compound 9ae demonstrated IFN induction in
monkeys in a clear dose-dependent manner by oral administra-
tion (Table 3). Furthermore, it did not cause emesis in a ferret
model, whereas a literature compound R-848 showed emesis
(Table 4). From the viewpoint of the margin between activity
and toxicity (emesis) compound 9ae (SM-276001) is expected
to be a promising compound as a novel IFN inducer.
Experimental Section
All reagents and solvents were obtained from commercial
suppliers and were used without further purification. Melting points
were measured with a Thomas-Hoover capillary melting point
apparatus and were uncorrected. Proton NMR spectra were recorded
on a Brucker Avance 400 FT NMR spectrometer. Chemical shifts
were given in parts per million (ppm) using tetramethylsilane as
an internal standard for spectra obtained in DMSO-d6. Elemental
analyses were performed at Sumika Chemical Analysis Service,
Osaka, Japan. Wakogel C-200 (Wako; 70-150 mm) was used for
column chromatography. The 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).
Method A. 2-Chloro-9-(2-chlorobenzyl)adenine (5a). To a
suspension of 4 (0.68 g, 4 mmol) and potassium carbonate (0.7 g,
5 mmol) in DMF (10 mL) was added 2-chlorobenzyl chloride (1.0
g, 6.2 mmol), and the mixture was heated at 75 °C for 4 h. The
reaction mixture was concentrated, and water was added to the
residue. The resulting precipitate was collected and washed with
diethyl ether to give the title compound as a pale yellow solid (1.03
g, 85%): 1H NMR (DMSO-d6) δ 8.24 (1H, s), 7.86 (2H, s), 7.53
(1H, d, J ) 7.8 Hz), 7.38 (2H, m), 6.95 (1H, dd, J ) 7.6, 1.4 Hz),
5.43 (2H, s).
2-Butylamino-9-(2-chlorobenzyl)adenine (6a). A solution of
5a (0.88 g, 3 mmol) and butylamine (10 mL) in n-PrOH (30 mL)
was heated at 130 °C for 8 h in an autoclave. The reaction mixture
was concentrated, and water was added to the residue. The resulting
precipitate was collected and was purified by silica gel chroma-
tography using 1% MeOH in CHCl3 as an eluent to give the title

9-Substituted-8-Hydroxyadenine DeriVatiVes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2093
2-Butylamino-8-hydroxy-9-(4-methylbenzyl)adenine (9f). Yield
98%; recrystallized from MeOH, mp 282-283 °C; 1H NMR
(DMSO-d6) δ 9.63 (1H, s), 7.18 (2H, d, J ) 8.0 Hz), 7.10 (2H, d,
J ) 8.0 Hz), 6.18 (1H, t, J ) 5.6 Hz), 5.99 (2H, s), 4.74 (2H, s),
3.15 (2H, q, J ) 7.2 Hz), 2.25 (3H, s), 1.44 (2H, m), 1.28 (2H, m),
0.87 (3H, t, J ) 7.6 Hz). Anal. (C17H22N6O‚0.2H2O) C, H, N.
2-Butylamino-9-(4-fluorobenzyl)-8-hydroxyadenine (9g). Yield
77%; recrystallized from MeOH, mp 266-267 °C; 1H NMR
(DMSO-d6) δ 9.67 (1H, s), 7.33 (2H, dd, J ) 8.4, 5.6 Hz), 7.14
(2H, d, J ) 8.4, 5.6 Hz), 6.21 (1H, t, J ) 5.6 Hz), 6.01 (2H, s),
4.78 (2H, s), 3.16 (2H, q, J ) 6.0 Hz), 1.44 (2H, m), 1.29 (2H, m),
0.86 (3H, t, J ) 7.7 Hz). Anal. (C16H19FN6O‚0.1H2O) C, H, N.
2-Butylamino-8-hydroxy-9-(4-trifluoromethylbenzyl)ade-
nine (9h). Yield 85%; recrystallized from MeOH, mp 264-266
°C; 1H NMR (DMSO-d6) δ 9.73 (1H, s), 7.69 (2H, d, J ) 8.0 Hz),
7.46 (2H, d, J ) 8.0 Hz), 6.21 (1H, t, J ) 5.6 Hz), 6.04 (2H, s),
4.89 (2H, s), 3.14 (2H, q, J ) 6.8 Hz), 1.41 (2H, m), 1.26 (2H, m),
0.83 (3H, t, J ) 7.6 Hz). Anal. (C17H19F3N6O‚0.2H2O) C, H, N.
2-Butylamino-8-hydroxy-9-(4-nitrobenzyl)adenine (9i). Yield
45%; recrystallized from MeOH, mp 216-218 °C; 1H NMR
(DMSO-d6) δ 9.73 (1H, s), 8.19 (2H, d, J ) 8.8 Hz), 7.50 (2H, d,
J ) 8.8 Hz), 6.23 (1H, t, J ) 5.6 Hz), 6.05 (2H, s), 4.94 (2H, s),
3.12 (2H, q, J ) 6.8 Hz), 1.40 (2H, m), 1.24 (2H, m), 0.83 (3H, t,
J ) 7.4 Hz). Anal. (C16H19N7O3‚1.75H2O) C, H, N.
2-Butylamino-9-(4-butylbenzyl)-8-hydroxyadenine (9l). Yield
91%; mp 248-249 °C; 1H NMR (DMSO-d6) δ 9.61 (1H, s), 7.19
(2H, d, J ) 8.2 Hz), 7.11 (2H, d, J ) 8.2 Hz), 6.20 (1H, t, J ) 5.6
Hz), 5.99 (2H, s), 4.76 (2H, s), 3.14 (2H, m), 2.51 (2H, m), 1.49
(4H, m), 1.28 (4H, m), 0.87 (6H, m). Anal. (C20H28N6O‚0.12H2O)
C, H, N.
2-Butylamino-9-(4-tert-butylbenzyl)-8-hydroxyadenine (9m).
Yield 96%; mp 256-257 °C; 1H NMR (DMSO-d6) δ 9.66 (1H, s),
7.32 (2H, d, J ) 8.4 Hz), 7.22 (2H, d, J ) 8.4 Hz), 6.23 (1H, t, J
) 5.6 Hz), 6.06 (2H, s), 3.17 (2H, m), 1.45 (2H, m), 1.28 (2H, m),
1.24 (9H, s), 0.87 (3H, t, J ) 7.2 Hz). Anal. (C20H28N6O) C, H, N.
9-(4-Biphenylmethyl)-2-butylamino-8-hydroxyadenine (9n).
Yield 98%; recrystallized from EtOH, mp 285-287 °C; 1H NMR
(DMSO-d6) δ 9.67 (1H, s), 7.62 (4H, m), 7.46 (2H, m), 7.35 (3H,
m), 6.24 (1H, t, J ) 5.6 Hz), 6.03 (2H, s), 4.85 (2H, s), 3.17 (2H,
q, J ) 6.8 Hz), 1.46 (2H, m), 1.28 (2H, m), 0.87 (3H, t, J ) 7.6
Hz). Anal. (C22H24N6O‚0.27H2O) C, H, N.
(2H, m), 1.41 (2H, m), 1.25 (2H, m), 0.83 (3H, t, J ) 7.2 Hz).
Anal. (C20H22N6O) C, H, N.
2-Butylamino-8-hydroxy-9-(2-naphthylmethyl)adenine (9t).
1
Yield 93%; mp 225-227 °C; H NMR (DMSO-d6) δ 10.08 (1H,
s), 7.88 (3H, m), 7.78 (1H, brs), 7.49 (3H, m), 6.85 (2H, s), 5.00
(2H, s), 3.21 (2H, m), 1.46 (2H, m), 1.26 (2H, m), 0.82 (3H, t, J
) 7.6 Hz). Anal. (C20H22N6O‚1.65H2O) C, H, N.
2-Butylamino-9-(5-chlorothiophene-2-ylmethyl)-8-hydroxyad-
enine (9u). Yield 61%; recrystallized from MeOH-CHCl3, mp
1
268-269 °C; H NMR (DMSO-d6) δ 9.65 (1H, s), 6.96 (2H, dd,
J ) 20.8, 3.6 Hz), 6.28 (1H, t, J ) 5.6 Hz), 6.03 (2H, s), 4.87 (2H,
s), 3.19 (2H, q, J ) 6.8 Hz), 1.48 (2H, m), 1.30 (2H, m), 0.88 (3H,
t, J ) 7.2 Hz). Anal. (C14H17ClN6OS) C, H, N.
2-Butylamino-8-hydroxy-9-(2-hydroxy-2,2-dimethylethyl)ad-
1
enine (9v). Yield 92%; mp 214-216 °C; H NMR (DMSO-d6) δ
9.65 (1H, s), 6.26 (1H, t, J ) 5.6 Hz), 6.12 (2H, s), 5.20 (1H, s),
3.63 (2H, s), 3.15 (2H, m), 1.44 (2H, m), 1.29 (2H, m), 1.09 (6H,
s), 0.87 (3H, t, J ) 7.2 Hz). Anal. (C13H22N6O2‚0.2H2O) C, H, N.
2-Butylamino-9-(2,2-dimethylethyl)-8-hydroxyadenine (9w).
Yield 62%; mp 213-215 °C; 1
H NMR (DMSO-d6) δ 9.57 (1H, s),
6.16 (1H, t, J ) 5.6 Hz), 5.95 (2H, s), 3.42 (2H, d, J ) 7.2 Hz),
3.17 (2H, q, J ) 6.8 Hz), 2.14 (1H, m), 1.46 (2H, m), 1.29 (2H,
m), 0.85 (9H, m). Anal. (C13H22N6O‚0.2H2O) C, H, N.
2-Butylamino-9-cyclohexylmethyl-8-hydroxyadenine (9x). Yield
70%; mp 230-231 °C; 1H NMR (DMSO-d6) δ 9.56 (1H, s), 6.17
(1H, t, J ) 5.6 Hz), 5.95 (2H, s), 3.45 (2H, d, J ) 7.2 Hz), 3.17
(2H, q, J ) 6.8 Hz), 1.82 (1H, m), 1.66 (2H, m), 1.55 (2H, m),
1.46 (2H, m), 1.29 (2H, m), 1.13 (3H, m), 0.95 (3H, m), 0.87 (3H,
t, J ) 7.2 Hz). Anal. (C16H26N6O‚0.1H2O) C, H, N.
2-Butylamino-8-hydroxy-9-(2-pyridylmethyl)adenine (9y). Yield
67%; recrystallized from MeOH, mp 172-174 °C; 1H NMR
(DMSO-d6) δ 9.67 (1H, s), 8.46 (1H, m), 7.73 (1H, dt, J ) 7.6,
1.8 Hz), 7.25 (1H, 7.12 (1H, d, J ) 8.0 Hz), 6.18 (1H, t, J ) 5.6
Hz), 5.98 (2H, s), 4.91 (2H, s), 3.09 (2H, q, J ) 6.8 Hz), 1.38 (2H,
m), 1.20 (2H, m), 0.82 (3H, t, J ) 7.2 Hz). Anal. (C15H19N7O‚
H2O) C, H, N.
2-Butylamino-8-hydroxy-9-(3-pyridylmethyl)adenine (9z). Yield
60%; recrystallized from MeOH-H2O, mp 216-218 °C; 1H NMR
(DMSO-d6) δ 9.57 (1H, s), 8.55 (1H, d, J ) 1.6 Hz), 8.47 (1H, dd,
J ) 4.8, 1.6 Hz), 7.68 (1H, d, J ) 7.6 Hz), 7.34 (1H, m), 6.23
(1H, t, J ) 5.6 Hz), 6.02 (2H, s), 4.83 (2H, s), 3.15 (2H, q, J ) 7.0
Hz), 1.44 (2H, m), 1.28 (2H, m), 0.85 (3H, t, J ) 7.6 Hz). Anal.
(C15H19N7O) C, H, N.
2-Butylamino-8-hydroxy-9-(4-pyridylmethyl)adenine (9aa).
Yield 75%; recrystallized from MeOH-H2O, mp 205-207 °C; 1H
NMR (DMSO-d6) δ 9.72 (1H, s), 8.49 (1H, dd, J ) 4.4, 2.0 Hz),
7.20 (2H, d, J ) 7.0 Hz), 6.22 (1H, t, J ) 5.6 Hz), 6.05 (2H, s),
4.83 (2H, s), 3.12 (2H, q, J ) 7.0 Hz), 1.39 (2H, m), 1.25 (2H, m),
0.83 (3H, t, J ) 7.6 Hz). Anal. (C15H19N7O‚0.5H2O) C, H, N.
2-Butylamino-8-hydroxy-9-(2-pyrazylmethyl)adenine (9ab).
Yield 44%; recrystallized from MeOH-H2O, mp 188-190 °C; 1H
NMR (DMSO-d6) δ 9.67 (1H, s), 8.61 (31H, s), 8.54 (2H, dd, J )
8.8, 2.4 Hz), 6.17 (1H, t, J ) 5.6 Hz), 6.02 (2H, s), 4.98 (2H, s),
3.08 (2H, q, J ) 6.8 Hz), 1.35 (2H, m), 1.22 (2H, m), 0.81 (3H, t,
J ) 7.2 Hz). Anal. (C14H18N8O‚0.65H2O) C, H, N.
2-Butylamino-9-(3,4-dichlorobenzyl)-8-hydroxyadenine (9o).
Yield 96%; mp 269-270 °C; 1H NMR (DMSO-d6) δ 9.73 (1H, s),
7.57 (2H, m), 7.24 (2H, dd, J ) 8.8, 1.8 Hz), 6.22 (1H, t, J ) 5.6
Hz), 6.05 (2H, s), 4.80 (2H, s), 3.14 (2H, q, J ) 6.8 Hz), 1.43 (2H,
m), 1.26 (2H, m), 0.85 (3H, t, J ) 7.6 Hz). Anal. (C16H18Cl2N6O‚
0.15H2O) C, H, N.
2-Butylamino-9-(3,4-difluorobenzyl)-8-hydroxyadenine (9p).
Yield 100%; recrystallized from MeOH-(CH3)2CO, mp 217-219
1
°C; H NMR (DMSO-d6) δ 10.54 (1H, s), 7.69 (2H, s), 7.40 (2H,
m), 7.16 (1H, t, J ) 4.0 Hz), 4.85 (2H, s), 3.16 (2H, m), 1.47 (2H,
m), 1.29 (2H, m), 0.84 (3H, t, J ) 7.6 Hz). Anal. (C16H18F2N6O‚
HCl‚0.3H2O) C, H, N.
2-Butylamino-9-(2-chloro-4,5-methylenedioxylbenzyl)-8-hy-
droxyadenine (9q). Yield 65%; recrystallized from EtOH, mp
2-Butylamino-8-hydroxy-9-(2-methylpyridine-3-ylmethyl)ad-
enine (9ac). Yield 66%; recrystallized from MeOH, mp 215-217
1
214-215 °C; H NMR (DMSO-d6) δ 9.70 (1H, s), 7.10 (1H, s),
1
6.59 (1H, brs), 6.24 (1H, t, J ) 5.6 Hz), 6.05 (2H, s), 6.03 (2H, s),
4.79 (2H, s), 3.13 (2H, q, J ) 7.0 Hz), 1.42 (2H, m), 1.27 (2H, m),
0.85 (3H, t, J ) 7.4 Hz). Anal. (C17H19ClN6O3) C, H, N.
2-Butylamino-8-hydroxy-9-(2-phenylethyl)adenine (9r). Yield
93%; recrystallized from MeOH-H2O, mp 204-205 °C; 1H NMR
(DMSO-d6) δ 9.52 (1H, s), 7.11-7.59 (5H, m), 6.22 (1H, t, J )
5.6 Hz), 5.98 (2H, s), 3.86 (2H, m), 3.17 (2H, m), 2.98 (2H,m),
1.47 (2H, m), 1.31 (2H, m), 0.88 (3H, t, J ) 7.6 Hz). Anal.
(C17H22N6O‚1.35H2O) C, H, N.
°C; H NMR (DMSO-d6) δ 9.70 (1H, m), 8.32 (1H, d, J ) 3.1
Hz), 7.37 (1H, d, J ) 7.7 Hz), 7.14 (1H, dd, J ) 7.7, 3.1 Hz), 6.20
(1H, t, J ) 6.4 Hz), 6.00 (2H, s), 4.82 (2H, s), 3.12 (2H, m), 2.60
(3H, s), 1.39 (2H, m), 1.25 (2H, m), 0.84 (3H, t, J ) 7.1 Hz).
Anal. (C16H21N7O‚0.25H2O) C, H, N.
2-Butylamino-8-hydroxy-9-(5-methylpyridine-3-ylmethyl)ad-
enine (9ad). Yield 79%; recrystallized from MeOH, mp 221-222
1
°C; H NMR (DMSO-d6) δ 9.65 (1H, m), 8.35 (1H, s), 8.31 (1H,
s), 7.50 (1H, s), 6.23 (1H, m), 6.02 (2H, s), 4.80 (2H, s), 3.16 (2H,
m), 2.25 (3H, s), 1.44 (2H, m), 1.28 (2H, m), 0.86 (3H, t, J ) 7.2
Hz). Anal. (C16H21N7O‚1.2H2O) C, H, N.
2-Butylamino-8-hydroxy-9-(1-naphthylmethyl)adenine (9s).
Yield 100%; recrystallized from MeOH, mp 249-251 °C; 1H NMR
(DMSO-d6) δ 9.72 (1H, s), 8.39 (1H, d, J ) 7.8 Hz), 7.95 (1H,
m), 7.86 (1H, m), 7.56 (2H, m), 7.43 (1H, t, J ) 7.6 Hz), 7.35
(1H, s), 6.19 (1H, t, J ) 5.6 Hz), 6.03 (2H, s), 5.28 (2H, s), 3.12
2-Butylamino-8-hydroxy-9-(6-methylpyridine-3-ylmethyl)ad-
enine (9ae). Yield 98%; recrystallized from MeOH, mp 248.5-
252 °C; 1H NMR (DMSO-d6) δ 9.78 (1H, m), 8.42 (1H, d. J ) 2.2

2094 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Isobe et al.
Hz), 7.57 (1H, dd, J ) 8.0, 2.2 Hz), 7.19 (1H, d, J ) 8.0 Hz), 6.22
(1H, t, J ) 7.1 Hz), 6.09 (2H, s), 4.78 (2H, s), 3.16 (2H, m), 2.41
(3H, s), 1.44 (2H, m), 1.28 (2H, m), 0.87 (3H, t, J ) 7.2 Hz).
Anal. (C16H21N7O) C, H, N.
(2H, m), 1.30 (2H, m), 0.88 (3H, t, J ) 7.2 Hz). Anal. (C17H22N6O2‚
0.2H2O) C, H, N.
Compounds 9k,ak were also prepared using the similar procedure
used to prepare 9j.
2-Butylamino-9-(2-chloropyridine-3-ylmethyl)-8-hydroxyad-
enine (9af). Yield 78%; recrystallized from MeOH, mp 235-237
9-(4-Benzyloxybenzyl)-2-butylamino-8-hydroxyadenine (9k).
Yield 92%; mp 260-262 °C; 1H NMR (DMSO-d6) δ 9.60 (1H, s),
7.40 (7H, m), 6.94 (2H, dd, J ) 6.8, 1.6 Hz), 6.20 (1H, t, J ) 5.6
Hz), 5.98 (2H, s), 5.06 (2H, s), 4.73 (2H, s), 3.17 (2H, q, J ) 6.8
Hz), 1.46 (2H, m), 1.28 (2H, m), 0.88 (3H, t, J ) 7.2 Hz). Anal.
(C23H26N6O2) C, H, N.
1
°C; H NMR (DMSO-d6) δ 8.77 (1H, s), 8.33 (1H, dd, J ) 4.4,
2.0 Hz), 7.43 (1H, m), 7.39 (1H, m), 6.27 (1H, t, J ) 5.6 Hz), 6.08
(2H, s), 3.08 (2H, q, J ) 6.8 Hz), 1.38 (2H, m), 1.22 (2H, m), 0.81
(3H, t, J ) 7.2 Hz). Anal. (C15H18ClN7O‚0.9H2O) C, H, N.
2-Butylamino-9-(4-chloropyridine-3-ylmethyl)-8-hydroxyad-
enine (9ag). Yield 47%; recrystallized from MeOH, mp 208-210
°C; 1H NMR (DMSO-d6) δ 9.73 (1H, s), 8.45 (1H, d, J ) 5.6 Hz),
8.34 (1H, brs), 7.56 (1H, d, J ) 5.6 Hz), 6.22 (1H, t, J ) 5.2 Hz),
6.06 (2H, s), 4.94 (2H, s), 3.09 (2H, q, J ) 6.8 Hz), 1.36 (2H, m),
1.23 (2H, m), 0.83 (3H, t, J ) 7.2 Hz). Anal. (C15H18ClN7O‚
0.2H2O) C, H, N.
2-Butylamino-8-hydroxy-9-(6-methoxypyridine-3-ylmethyl)-
1
adenine (9ak). Yield 49%; mp 220-222 °C; H NMR (DMSO-
d6) δ 9.64 (1H, s), 8.15 (1H, d, J ) 2.2 Hz), 7.66 (1H, dd, J ) 8.6,
2.2 Hz), 6.77 (1H, d, J ) 8.6 Hz), 6.22 (1H, t, J ) 5.7 Hz), 6.00
(2H, s), 4.74 (2H, s), 3.80 (3H, s), 3.17 (2H, q, J ) 6.8 Hz), 1.44
(2H, m), 1.29 (2H, m), 0.88 (3H, t, J ) 7.2 Hz). Anal. (C16H21N7O2‚
1.5H2O) C, H, N.
2-Butylamino-9-(6-chloropyridine-3-ylmethyl)-8-hydroxyad-
enine (9ah). Yield 86%; recrystallized from MeOH, mp 256-258
°C; 1H NMR (DMSO-d6) δ 9.71 (1H, s), 8.38 (1H, d, J ) 2.4 Hz),
7.73 (1H, dd, J ) 8.0, 2.4 Hz), 7.48 (1H, d, J ) 8.0 Hz), 6.23 (1H,
t, J ) 5.6 Hz), 6.04 (2H, s), 4.83 (2H, s), 3.14 (2H, q, J ) 6.8 Hz),
1.43 (2H, m), 1.27 (2H, m), 0.86 (3H, t, J ) 7.2 Hz). Anal. (C15H18-
ClN7O) C, H, N.
2-Butylamino-8-hydroxy-9-(4-hydroxybenzyl)adenine (13). To
a suspension of 9k (209 mg, 0.5 mmol) in HCO2H (3 mL) was
added 10% Pd(OH)2/C (30 mg), and the reaction mixture was
refluxed for 5 h. The catalyst was removed by filtration, and the
resulting solution was concentrated. The residue was neutralized
with a 28% ammonium solution, and the resulting solid was
collected and washed with water to give the title compound as a
1
2-Butylamino-9-(2-chloro-6-methylpyridine-3-ylmethyl)-8-hy-
droxyadenine (9ai). Yield 76%; recrystallized from MeOH, mp
white solid (92 mg, 56%): mp 242-245 °C; H NMR (DMSO-
d6) δ 9.57 (1H, s), 9.35 (1H, s), 7.14 (2H, d, J ) 8.2 Hz), 6.67
(2H, d, J ) 8.2 Hz), 6.19 (1H, t, J ) 5.6 Hz), 5.97 (2H, s), 4.67
(2H, s), 3.17 (2H, m), 1.46 (2H, m), 1.29 (2H, m), 0.89 (3H, t, J
) 7.2 Hz). Anal. (C16H20N6O2‚0.5H2O) C, H, N.
1
215-216 °C; H NMR (DMSO-d6) δ 9.74 (1H, s), 7.33 (1H, d, J
) 5.8 Hz), 7.22 (1H, d, J ) 5.8 Hz), 6.24 (1H, m), 6.06 (2H, s),
4.83 (2H, s), 3.09 (2H, m), 2.42 (3H, s), 1.37 (2H, m), 1.25 (2H,
m), 0.82 (3H, t, J ) 5.5 Hz). Anal. (C16H20ClN7O‚0.3H2O) C, H,
N.
2-Butylamino-9-(6-dimethylaminopyridine-3-ylmethyl)-8-hy-
droxyadenine (23). A suspension of 8ah (250 mg, 3 mmol) and
dimethylamine (40% in H2O, 10 mL) was heated at 140 °C for 6.5
h in an autoclave. The reaction mixture was concentrated, and water
was added to the residue. The resulting precipitate was collected
and purified by silica gel chromatography using 7% MeOH in
CHCl3 as an eluent to give the title compound as a white solid
2-Butylamino-9-(2,6-dichloropyridine-3-yl-methyl)-8-hydroxy-
adenine (9aj). Yield 89%; recrystallized from MeOH, mp 232-
1
234 °C; H NMR (DMSO-d6) δ 9.77 (1H, s), 7.60 (1H, m), 7.53
(1H, d, J ) 8.0 Hz), 6.24 (1H, t, J ) 5.2 Hz), 6.07 (2H, s), 4.88
(2H, s), 3.09 (2H, q, J ) 6.8 Hz), 1.36 (2H, m), 1.23 (2H, m), 0.83
(3H, t, J ) 7.2 Hz). Anal. (C15H17Cl2N7O‚0.85H2O) C, H, N.
9-(4-Fluorobenzyl)-8-hydroxy-2-(2-methoxyethylamino)ade-
nine (20). Yield 68%; recrystallized from EtOH, mp 260-262 °C;
1H NMR (DMSO-d6) δ 9.68 (1H, s), 7.34 (1H, dd, J ) 8.6, 5.6
Hz), 7.14 (2H, dd, J ) 8.6, 5.0 Hz), 6.17 (1H, t, J ) 5.6 Hz), 6.07
(2H, s), 4.79 (2H, s), 3.38 (4H, m), 3.24 (3H, s). Anal. (C15H17-
FN6O2) C, H, N.
1
(100 mg, 39%): recrystallized from MeOH, mp 277-278 °C; H
NMR (DMSO-d6) δ 9.61 (1H, s), 8.10 (1H, d, J ) 2.2 Hz), 7.49
(1H, dd, J ) 8.6, 2.2 Hz), 6.56 (1H, d, J ) 8.6 Hz), 6.18 (1H, t,
J ) 5.7 Hz), 5.97 (2H, s), 4.65 (2H, s), 3.18 (2H, q, J ) 6.8 Hz),
2.97 (6H, s), 1.48 (2H, m), 1.31 (2H, m), 0.89 (3H, t, J ) 7.3 Hz).
Anal. (C17H24N8O‚0.1H2O) C, H, N.
IFN Induction by Mouse Splenocyte Cultures. Male C3H/
HeJ mice (Clea Japan Inc.) aged 8 weeks were sacrificed, and the
spleens were removed from six mice. The 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 × 106 cells/
mL in MEM supplemented with 5% fetal calf serum, 100 IU/mL
of penicillin, and 100 µg/mL of streptomycin. The test compounds
were dissolved in dimethyl sulfoxide and diluted to 500-fold with
supplemented MEM. The 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 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.
IFN Induction in Mice. The test compounds suspended in 0.5%
sodium carboxymethyl cellulose were administered orally to male
BALB/c mice (Charles River Japan Inc.) aged 8-10 weeks. Blood
was collected by cardiac puncture into a heparinized tube, under
ether anesthesia, 2 h after test compounds administration. Plasma
samples were obtained by centrifugation and stored at -80 °C until
they were analyzed for IFN.
2-Benzylamino-9-(4-fluorobenzyl)-8-hydroxyadenine (21). Yield
95%; recrystallized from EtOH, mp 271-273 °C; 1H NMR
(DMSO-d6) δ 9.65 (1H, s), 7.28 (7H, m), 7.07 (2H, m), 6.86 (1H,
t, J ) 5.6 Hz), 6.06 (2H, s), 4.76 (2H, s), 4.40 (2H, d, J ) 6.4 Hz).
Anal. (C19H17FN6O) C, H, N.
9-(4-Fluorobenzyl)-8-hydroxy-2-(4-pyridylmethylamino)ad-
enine (22). Yield 90%; recrystallized from EtOH, mp 227-230
1
°C; H NMR (DMSO-d6) δ 9.67 (1H, s), 8.44 (2H, dd, J ) 4.4,
1.6 Hz), 7.28 (4H, m), 7.01 (3H, m), 6.08 (2H, s), 4.73 (2H, s),
4.40 (2H, d, J ) 6.2 Hz). Anal. (C18H16FN7O) C, H, N.
2-Butylamino-8-methoxy-9-(4-methoxybenzyl)adenine (10). A
suspension of 8i in 5 N NaOH solution (3 mL) and MeOH (3 mL)
was refluxed for 4 h. The reaction mixture was concentrated. Then
the residue was neutralized with 1 N HCl solution, and the resulting
precipitate was collected to give the title compound as a white solid
(298 mg, 84%): 1H NMR (DMSO-d6) δ 7.20 (2H, d, J ) 8.6 Hz),
6.88 (2H, m), 6.27 (2H, s), 6.07 (1H, t, J ) 5.6 Hz), 4.89 (2H, s),
3.99 (3H, s), 3.71 (3H, s), 3.19 (2H, m), 1.48 (2H, m), 1.31 (2H,
m), 0.89 (3H, t, J ) 7.2 Hz).
2-Butylamino-8-hydroxy-9-(4-methoxybenzyl)adenine (9j). A
solution of 10 (178 mg, 0.5 mmol) in 12 N HCl (5 mL) was stirred
at room temperature for 3 h. The reaction mixture was concentrated
and neutralized with 1 N NaOH solution. The resulting precipitate
was collected and washed with water to give the title compound
as a white solid (160 mg, 92%): recrystallized from MeOH, mp
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 Institutes of Health, Bethesda, MD.
1
266-268 °C; H NMR (DMSO-d6) δ 9.59 (1H, s), 7.25 (2H, d, J
) 8.8 Hz), 6.86 (2H, d, J ) 8.8 Hz), 6.20 (1H, t, J ) 5.6 Hz), 5.98
(2H, s), 4.73 (2H, s), 3.71 (3H, s), 3.17 (2H, q, J ) 6.8 Hz), 1.45

9-Substituted-8-Hydroxyadenine DeriVatiVes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2095
(6) Hovanessian, A. G.; Youn, Y. K.; Buffet-Janvresse, C.; Riviere, Y.;
Michelson, M.; Lacour, F. Enhancement of natural killer cell activity
and 2-5A synthetase in operable breast cancer patients treated with
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Science 1970, 169, 1214-1216.
IFN Induction in Monkey. The test compounds suspended in
0.5% sodium carboxymethyl cellulose were administered orally to
male cynomolgus monkeys aged 3-5 years. Blood samples (0.5
mL) were taken from a jugular vein, and plasma samples were
prepared by centrifugation and stored at -80 °C until they were
analyzed for IFN.
Ferret Emesis Analysis. The test compounds suspended in 0.5%
sodium carboxymethyl cellulose were administered orally to male
ferrets (Charles River Japan Inc.) aged 4 months. Then animals
were transferred to individual 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 evaluate emesis.
Blood samples (0.5 mL) were taken from a jugular vein, and the
serum was obtained by centrifugation at 1630g for 10 min at 4 °C
and kept at -20 °C until drug analysis. The concentrations of test
compounds in the plasma were assayed by an HPLC method.
(8) Nichol, F. R.; Weed, S. D.; Underwood, G. E. Stimulation of murine
interferon by a substituted pyrimidine. Antimicrob. Agents Chemother.
1976, 9, 433-437.
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as a therapeutic agent. J. Interferon Res. 1992, 12, 109-118.
(10) Gerster, J. F.; Lindstrom, K. J.; Miller, R. L.; Tomai, M. A.;
Birmachu, W.; Bomersine, S. N.; Gibson, S. J.; Imbertson, L. M.;
Jacobson, J. R.; Knafla, R. T.; Maye, P. V.; Nikolaides, N.; Oneyemi,
F. Y.; Parkhurst, G. J.; Pecore, S. E.; Reiter, M. J.; Scribner, L. S.;
Testerman, T. L.; Thompson, N. J.; Wagner, T. L.; Weeks, C. E.;
Andre, J. D.; Bastard, D. L. Y.; Lupu, M. Synthesis and structure-
activity-relationships of 1H-imidazo[4,5-c]quinolines that induce
interferon production. J. Med. Chem. 2005, 48, 3481-3491.
(11) Witt, P. L.; Ritch, P. S.; Reding, D.; McAuliffe, T. L.; Westrick, L.;
Grossberg, S. E.; Borden, E. C. Phase I trial of an oral immuno-
modulator and interferon inducer in cancer patients. Cancer Res.
1993, 53, 5176-5180.
(12) Strominger, N. L.; Brady, R.; Gullikson, G.; Carpenter, D. O.
Imiquimod-elicited emesis is mediated by the area postrema, but not
by direct neuronal activation. Brain Res. Bull. 2001, 55, 445-451.
(13) 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. Discovery of 8-hydroxyadenines as a novel type
of interferon inducer. J. Med. Chem. 2002, 45, 5419-5422.
(14) Isobe, Y.; Tobe, M.; Ogita, H.; Kurimoto, A.; Ogino, T.; Kawakami,
H.; Takaku, H.; Sajiki, H.; Hirota, K.; Hayashi, H. Synthesis and
structure-activity relationships of 2-substituted-8-hydroxyadenine
derivatives as orally available interferon inducers without emetic side
effects. Bioorg. Med. Chem. 2003, 11, 3641-3647.
Acknowledgment. We thank Dr. Ewan Hume at Dainippon
Sumitomo Pharma and Drs. Thomas McInally, Roger Bonnert,
and Mark Biffen at AstraZeneca Company for their helpful
suggestions.
Supporting Information Available: 1H NMR data of inter-
mediate compounds and elemental analysis data of final compounds.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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