Discovery of 8-hydroxyadenines as a novel type of interferon inducer

Article abstract of DOI:10.1021/jm0203581

9-Benzyl-8-hydroxyadenine (6) was found to possess interferon-inducing activity in vitro as a lead compound. Although replacement of the 9-benzyl group of 6 did not improve the activity, the introduction of a substituent such as alkyl, alkylthio, alkylami

Full text of DOI:10.1021/jm0203581

J . Med. Chem. 2002, 45, 5419-5422
5419
ataburine,6 CP-28888,7 ABMP,8 DRB,9 10-carboxy-
methyl-9-acridone,10 bropirimine,11 and imiquimod.12,13
Imiquimod, which is clinically used in the United States
Discover y of 8-Hyd r oxya d en in es a s a
Novel Typ e of In ter fer on In d u cer
Kosaku Hirota,*,† Kazunori Kazaoka,† Itaru Niimoto,†
Hiroshi Kumihara,† Hironao Sajiki,† Yoshiaki Isobe,‡
Haruo Takaku,‡ Masanori Tobe,‡ Haruhisa Ogita,‡
Tetsuhiro Ogino,§ Shinji Ichii,§
Ayumu Kurimoto,§ and Hajime Kawakami§
Laboratory of Medicinal Chemistry, Gifu Pharmaceutical
University, Mitahora-higashi, Gifu 502-8585, J apan,
Pharmaceuticals & Biotechnology Laboratory, J apan Energy
Corporation, Niizo-minami, Toda 335-0026, J apan, and
Research Division, Sumitomo Pharmaceuticals,
for treatment of exophytic warts caused by the human
papillomavirus, is especially a potent IFN inducer.
However, its serious side effects such as vomiting and
hepatopathy found during the clinical trial stage forced
abandonment of its further development for hepatitis
C as a chemotherapy drug. Subsequently, imiquimod
analogues such as R-84214 (a metabolite of imiquimod)
and resiquimod13,15 were found to be more effective IFN
inducers than imiquimod. However, no IFN inducer has
yet been clinically employed for the treatment of hepa-
titis C.
To search for novel IFN inducers, we have focused
on the screening of pyrimidine and purine derivatives
stocked in our compound library. Herein we describe
the discovery of a novel type of lead compound possess-
ing IFN-inducing activity and its chemical modification
and qualitative structure-activity relationships (SAR).
Ch em istr y. 8-Chloro-9-benzyladenine (2) was pre-
pared by chlorination of 9-benzyladenine (1) by using a
combination of titanium tetrachloride and hydrogen
peroxide. Other various 8-substituted 9-benzyladenine
derivatives (3-10) were synthesized from 1 according
to usual methods16 employed for the preparation of
8-substituted purines. 9-Unsubstituted 8-hydroxy-
adenine (11)17 and a variety of 9-substituted 8-hydroxy-
adenine derivatives (12-22)18 were synthesized accord-
ing to the method previously reported.
The methods for the synthesis of 9-benzyl-8-hydroxy-
adenines possessing various substituents at the 2-posi-
tion are shown in Schemes 1 and 2. 5-Amino-1-benzyl-
4-cyano-2-hydroxyimidazole (23) is a useful intermediate
for the synthesis of 8-hydroxyadenines such as 2-methyl
(24), 2-phenyl (28), 2-amino (37), and 2-hydroxy (43)
derivatives.18 Condensation of 23 with substituted imi-
dates afforded the corresponding 2-alkyl-8-hydroxy-
adenines 25-27. Although analogous condensation with
thiourea failed to obtain the expected 2-mercapto-8-
hydroxyadenine (29), the reaction with benzoyl isothio-
cyanate and successive treatment with sodium hydrox-
ide resulted in the successful formation of it. Subsequent
alkylation of 29 with alkyl halides selectively on the
sulfur atom proceeded to give 2-alkylthio analogues 30-
35. Oxidation of the 2-butylthio derivative (33) with
m-chloroperbenzoic acid afforded the corresponding
S-oxide (36). 2-Alkylamino analogues 39-42 were pre-
pared by the reductive alkylation of 2-aminoadenine (37)
with aliphatic aldehydes and sodium cyanoborohydride.
The alkylation was proceeded regioselectively at the
2-amino group. On the other hand, alkylation of 2-hy-
Kasugade-naka, Konohana-ku, Osaka 554-0022, J apan
Received August 19, 2002
Abstr a ct: 9-Benzyl-8-hydroxyadenine (6) was found to pos-
sess interferon-inducing activity in vitro as a lead compound.
Although replacement of the 9-benzyl group of 6 did not
improve the activity, the introduction of a substituent such
as alkyl, alkylthio, alkylamino, and alkoxy groups into the
2-position of the adenine ring resulted in
a remarkable
increase in the activity. The 2-alkylthio (30-32), 2-butylamino
(41), and 2-butoxy (47) analogues indicated the highest activi-
ties by oral administration to mice.
In tr od u ction . Hepatitis C is induced by infection
with the hepatitis C virus (HCV) through contact with
blood products, which might occur by accidentally being
stuck with a dirty (used) needle, using IV drugs and
sharing needles, or getting a blood transfusion. The
major problems of HCV infection are the progression of
hepatitis C to cirrhosis, liver failure, and liver cancer
after a long period of time. All current treatment
protocols for hepatitis C are based on the use of various
preparations of interferon alpha (IFN-R) alone or in
combination with ribavirin.1 Recombinant (synthetic)
IFNs, which currently dominate the IFN market, are
genetically engineered and consequently are sometimes
recognized as “foreign” by the body’s immune system.
Treatment with recombinant IFN may cause unfavor-
able immune responses and the formation of neutral-
izing antibodies that reduce the effectiveness of a
particular therapy.2 Furthermore, as IFN preparations
are administered by intramuscular or subcutaneous
injection, the treatment with IFN causes pain and
irritation at the site of injection. The cost of treatment
is very high because of the inordinately expensive IFN
preparations. Therefore, enhancing the release of en-
dogenous IFN by the oral administration of small-
molecular-weight compounds is one approach to anti-
HCV therapeutics. Development of small-molecule IFN
inducers has been ardently desired for a long time to
avoid such drawbacks of IFN injections.
Various compounds possessing IFN-inducing activity
have been hitherto reported.3 Among them, small-
molecule IFN inducers include tilorone,4 BL-20803,5
* To whom correspondence should be addressed. Phone: +81-58-
237-8572. Fax: +81-58-237-5979. E-mail: hirota@gifu-pu.ac.jp.
† Gifu Pharmaceutical University.
‡ J apan Energy Corporation.
§ Sumitomo Pharmaceuticals.
10.1021/jm0203581 CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/09/2002

5420 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Letters
Sch em e 1a
Ta ble 1. IFN-inducing Activities of 8-Substituted
9-Benzyladenines
compd
R
MECa (µM)
MEDb (mg/kg)
1
2
3
4
5
6
7
8
9
H
Cl
Br
I
CH3
OH
OCH3
SH
SCH3
NHNH2
>10
>10
>10
>10
>10
10
>10
10
>10
>10
1
10
>30
10
imiquimod
3
a
Minimum effective concentration (mice spleen cells): a con-
centration of compounds required for more than 1 IU/mL induction
of IFN. Minimum effective dose (mice po): a dose of compounds
a
b
Reagents and conditions: (a) RCH(NH)OC2H5‚HCl, Na,
C2H5OH, reflux, 2 d (32-40%); (b) (i) BzNCS, THF, rt, 1 d; (ii) 2
N NaOH-THF (1:10), reflux, 2 d; (c) RX, K2CO3, DMF, rt, 6 h
(34-43% from 23); (d) mCPBA, THF, rt, 2 h (58%).
required for more than 100 IU/mL induction of IFN.
Ta ble 2. IFN-inducing Activities of 9-Substituted
8-Hydroxyadenines
Sch em e 2a
compd
R
MECa (µM)
6
11
12
13
14
15
16
17
18
19
20
21
CH2Ph
H
C4H9
cyclopentyl
Ph
10 (10)b
>10
>10
>10
>10
>10
10
CH2CH2Ph
R-naphthylmethyl
CH2C6H4 (4-F)
CH2C6H4 (4-Cl)
CH2C6H4 (4-CH3)
CH2C6H4 (2-OCH3)
CH2C6H4 (3-OCH3)
CH2C6H4 (4-OCH3)
10 (30)b
10 (30)b
10
a
Reagents and conditions: (a) aldehyde, NaBH3CN, CH3OH,
rt, 2 d (63-87%); (b) RX, LiH, DMF, rt, 1 d (6-13%).
10
10
10
droxyadenine (43) with alkyl halides in the presence of
lithium hydride gave a mixture of 2-O-mono- and 2-O,7-
N-dialkylated products, from which the desired 2-alkoxy
analogues 44-48 were purified in low yields.
22
imiquimod
1 (3)b
a
Minimum effective concentration (mice spleen cells): a con-
centration of compounds required for more than 1 IU/mL induction
of IFN. Value in parentheses is MED (mg/kg) (mice po): a dose
of compounds required for more than 100 IU mL induction of IFN.
Resu lts a n d Discu ssion . First, a lead compound for
the potential IFN-inducing activity was found among
several 8-substituted 9-benzyladenine derivatives (1-
10), whose IFN-inducing activities were evaluated by
using mice spleen cells19 and are shown in Table 1.
9-Benzyl-8-hydroxyadenine (6) and 9-benzyl-8-mercap-
toadenine (8) showed the activities with a minimum
effective concentration (MEC) of 10 µM, although their
activities were weaker than that of the reference drug,
imiquimod. 8-Hydroxyadenine 6 was selected as a lead
compound because the 8-mercapto analogue (8) did not
indicate in vivo activity,19 having a minimum effective
dose (MED) >30 mg/kg, which was measured in mice.
Interestingly, other analogues, 1-5, 7, 9, and 10,
possessing no acidic proton on the 8-substituents, were
inactive.
b
cyclopentyl (13), phenyl (14), and phenethyl (15) ana-
logues were inactive. Introduction of some substituents
onto the benzene ring of the 9-benzyl group had no
significant effect on the activity (compounds 17-22).
To explore the effects of the substituents at the
2-position on the IFN-inducing activity, we tested a
series of 2-substituted derivatives 24-48 and show the
results in Table 3. A dramatic improvement of the
activity was realized by introduction of chain substit-
uents into the 2-position. In the alkyl-substituted series,
we found that the introduction of a longer alkyl chain
led to stronger activity. For example, the 2-butyl
analogue (27) had more than 100-fold greater activity
(MEC ) 0.03 µM) than lead compound 6. On the other
hand, the 2-phenyl analogue (28) was ineffective in
improving the activity. In the 2-alkylthio series, a
similar tendency was indicated. 2-Propylthio (32) and
Variation of substituents at the 9-position of lead
compound 6 was investigated (Table 2). Although the
R-naphthylmethyl derivative (16) indicated an activity
equivalent to that of 6, unsubstituted (11), butyl (12),

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5421
Ta ble 3. IFN-Inducing Activities of 2-Substituted
9-Benzyl-8-hydroxyadenines
compd
R
MECa (µM)
MEDb (mg/kg)
6
24
H
CH3
10
1
30
3
25
C2H5
1
3
26
C3H7
1
3
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
C4H9
Ph
SH
SCH3
0.03
10
10
0.3
>30
ntc
0.1
0.1
0.1
0.3
1
0.1
0.1
0.01
0.01
10
0.1
0.01
10
F igu r e 1. Dose-dependent effects of compounds 33 and 47
on IFN-inducing activity in mice by oral administration.
SC2H5
SC3H7
SC4H9
SC5H11
SCH2Ph
S(O)C4H9
NH2
NHCH3
NHC2H5
NHC3H7
NHC4H9
NHC5H11
OH
>1
Optimal IFN-inducing activity is seen when the chain
length of the 2-substituent is 4-6 atoms.
1
ntc
ntc
ntc
1
The in vivo activity19 of the IFN induction was
examined by oral administration to mice to select
candidates possessing an excellent bioavailability. As
shown in Table 3, several compounds such as 2-propyl-
thio (32), 2-butylamino (41), and 2-butoxy (47) ana-
logues, which indicated excellent activities (MED ) 0.1
mg/kg), showed greater than 100-fold improvement of
the in vivo activity over lead compound 6.
1
1
0.1
0.1
0.1
0.1
0.1
0.1
0.01
0.001
0.01
1
0.1
0.3
1
>1
0.3
0.3
0.1
0.3
3
43
44
45
46
47
48
OCH3
OC2H5
OC3H7
OC4H9
OC5H11
The production of IFN in mice by 8-hydroxyadenine
derivatives was dose-dependent as shown in Figure 1.
The 2-butoxy analogue (47) induced IFN at a dose as
low as 0.1 mg/kg. The IFN production by 33 and 47
plateaued between 3 and 10 mg/kg. In general, IFN
concentration in plasma is required to be 50-100 IU/
mL for the treatment of patients infected by HCV.
Compounds 33 and 47 induced IFN more than 250 IU/
mL at doses of 0.3 and 0.1 mg/kg in mice, respectively.
These results indicated that both compounds exhibit
enough activities to treat patients at lower concentra-
tions than that of imiquimod (67(21 IU/mL at a dose
of 3 mg/kg).
imiquimod
a
Minimum effective concentration (mice spleen cells): a con-
centration of compounds required for more than 1 IU/mL induction
b
of IFN. Minimum effective dose (mice po): a dose of compounds
required for more than 100 IU/mL induction of IFN. c Not tested.
2-butylthio (33) derivatives indicated high activities,
with an MEC of 0.01 µM. However, the 2-pentylthio
analogue (34) exhibited not so impressive activity (MEC
) 10 µM). Additionally, the sulfoxide analogue (36) also
showed an excellent activity (MEC ) 0.01 µM). Similar
tendency was also found in the case of the 2-alkylamino
and 2-alkoxy series. Among them, the 2-butoxy analogue
(47) indicated the most potent activity, with an MEC
of 0.001 µM under the in vitro assay conditions used,
and it had a potency more than 10000-fold greater than
that of lead compound 6. Compound 47 also showed a
high activity with an MEC of 0.01 µM in the in vitro
assay using human PBMC (MEC is a concentration of
47 required to induce more than 1 pg/mL IFN). Re-
placement of the 6-amino group of 6 with other substit-
uents such as hydrogen, hydroxy, and mercapto groups
resulted in the disappearance of the activity, although
no data are shown here.
The remarkable features of the qualitative SAR may
be summarized as follows: (1) 9-Benzyl-8-hydroxy-
adenine is required as the simplest structure for the
expression of IFN-inducing activity. (2) At the 9-posi-
tion, a benzyl-type substituent is essential for the
activity. (3) The introduction of several substituents (4-
F, 4-Cl, 4-Me, 2-OMe, 3-OMe, or 4-OMe) on the benzene
ring of the 9-benzyl group did not improve the activity.
(4) The introduction of some alkyl-chain substituents
(i.e., alkyl, alkylthio, alkylamino, alkoxy) at the 2-posi-
tion of the adenine ring strongly enhances the activity.
The cytotoxic activity of compound 47 was also
determined by using the MTS assay. Little cytotoxicity
was identified by 47 at the highest concentration tested
(10 µM) in this assay (detailed data not shown).
Akira et al. has recently reported that imiquimod
induces IFN via stimulation of the toll-like receptor 7.20
Therefore, the action mode of 8-hydroxyadenines toward
the receptor is under investigation.
Con clu sion . In summary, we discovered a novel type
of IFN inducer, 9-benzyl-8-hydroxyadenine (6) as a lead
compound. The substituent modification at the 2-posi-
tion of the adenine ring brought remarkable improve-
ment of IFN induction. In the in vitro assay, the
2-butoxy analogue (47) emerged as having the most
potent activity. The in vivo activity was examined by
oral administration into mice, and 2-alkylthio (30-32),
2-butylamino (41), and 2-butoxy (47) analogues have
been selected for further pharmacological investigation.
Compounds based on this novel template are the
subjects of continuous investigations toward the devel-
opment of therapeutically useful IFN inducers that
avoid side effects such as vomiting.

5422 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
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