Synthesis of 6-substituted 9-benzyl-8-hydroxypurines with potential interferon-inducing activity

Article abstract of DOI:10.1248/cpb.51.608

Various 6-substituted 9-benzyl-8-hydroxypurines were synthesized in order to investigate the structure-activity relationship at the 6-position of 9-benzyl-8-hydroxyadenine (1), which is a lead compound for the screening of interferon (IFN)-inducing activity. 6-Unsubstituted, mercapto-, methylthio- and hydroxy-9-benzyl-8-hydroxypurines (2 - 5) were prepared from 5-amino-1-benzyl-4-cyano-2-hydroxyimidazole (9). Synthesis of a 6-methoxy analog (6) was conducted from 5-amino-4-benzylamino-6-chloropyrimidine (13). 6-Alkylamino and acylaminopurines (7 and 8) were also prepared by alkylation and acylation of 1, respectively. Since these compounds (2 - 8) indicated no activity, it was found that a free amino group of 1 is required for the expression of IFN-inducing activity.

Full text of DOI:10.1248/cpb.51.608

608
Notes
Chem. Pharm. Bull. 51(5) 608—611 (2003)
Vol. 51, No. 5
Synthesis of 6-Substituted 9-Benzyl-8-hydroxypurines with Potential
Interferon-Inducing Activity
Kazunori KAZAOKA, Hironao SAJIKI, and Kosaku HIROTA*
Laboratory of Medicinal Chemistry, Gifu Pharmaceutical University; Mitahora-higashi, Gifu 502–8585, Japan.
Received January 30, 2003; accepted March 4, 2003
Various 6-substituted 9-benzyl-8-hydroxypurines were synthesized in order to investigate the structure–ac-
tivity relationship at the 6-position of 9-benzyl-8-hydroxyadenine (1), which is a lead compound for the screening
of interferon (IFN)-inducing activity. 6-Unsubstituted, mercapto-, methylthio- and hydroxy-9-benzyl-8-hydroxy-
purines (2—5) were prepared from 5-amino-1-benzyl-4-cyano-2-hydroxyimidazole (9). Synthesis of a 6-methoxy
analog (6) was conducted from 5-amino-4-benzylamino-6-chloropyrimidine (13). 6-Alkylamino and acylamino-
purines (7 and 8) were also prepared by alkylation and acylation of 1, respectively. Since these compounds (2—8)
indicated no activity, it was found that a free amino group of 1 is required for the expression of IFN-inducing ac-
tivity.
Key word interferon-inducing activity; 8-hydroxypurine; 9-benzyl-8-hydroxyadenine; 5-amino-4-cyano-2-hydroxyimidazole;
reductive alkylation
8-Hydroxypurine derivatives have been reported to have a hauser effect (NOE) between N1-methyl and C2-hydrogens as
wide range of biological activities, such as corticotropin-re- shown in Chart 1. Therefore, the synthesis of 6 was carried
leasing hormone receptor antagonism,1,2) anti-rhinovirus ac- out using 5-amino-4-benzylamino-6-chloropyrimidine (13)9)
tivity,3) xanthine oxidase inhibiting activity4) and excellent as the starting material (Chart 2). 6-Chloro-8-hydroxypurine
binding affinity to a benzodiazepine receptor.5) Recently, we (14) was easily prepared by cyclization of 13 with triphos-
found a novel type of lead compound, 9-benzyl-8-hydroxy- gene in 88% yield. While conversion of 14 into 6-methoxy-
adenine (1), possessing potent interferon (IFN)-inducing ac- purine (6) in the presence of sodium methoxide was unsuc-
tivity and conducted substituent modifications at the 2-, 8- cessful with recovery of 14, 5-amino-4-benzylamino-6-
and 9-positions of 1 to clarify the structure–activity relation- methoxypyrimidine (15), which was prepared by methanoly-
ships.6) Consequently, we found that the 8-hydroxyl and 9- sis of 13 using sodium methoxide, was successfully cyclized
benzyl groups of 1 are required for the expression of IFN-in-
ducing activity and the introduction of an appropriate alkyl-
chain substituent at the 2-position of 1 remarkably increases
the activity.6) The focus of our attention was turned to the ef-
fect of the 6-amino group of 1 on the IFN-inducing activity.
In this report, the synthesis of various 6-substituted 9-benzyl-
8-hydroxypurines (2—8) shown in Fig. 1 and evaluation of
their IFN-inducing activity are described.
Preparation of 6-substituted 9-benzyl-8-hydroxypurines
was carried out as shown in Charts 1—3. Reaction of imida-
zole 97) with thiobenzamide8) afforded 4-thiocarbamoyl imi-
dazole (10) in 69% yield, followed by cyclization with for-
mamidine hydrochloride in refluxing 2-methoxyethanol to
give 6-mercapto derivative (3) in 87% yield. Subsequently, 3
was selectively S-methylated with methyl iodide in the pres-
ence of potassium carbonate to give 6-methylthio derivative
(4). 9-Benzyl-8-hydroxypurine (2) was obtained by desulfur-
ization of 3 with Raney Ni. 6-Hydroxy derivative (5) was
synthesized by hydrolysis of the cyano group of 9 under
acidic conditions and subsequent cyclization of the resulting
11 with formamidine hydrochloride. Methylation of 5 did not
give the desired 6-methoxypurine (6) but 1-methylhypoxan-
thine (12), whose structure was confirmed by nuclear Over-
Chart 1
Fig. 1. 6-Substituted 9-Benzyl-8-hydroxypurines
* To whom correspondence should be addressed. e-mail: hirota@gifu-pu.ac.jp
© 2003 Pharmaceutical Society of Japan

May 2003
609
compounds required for more than 1 IU/ml induction of IFN.
The 6-unsubstituted and 6-substituted purines (2—8, 14)
were inactive at the maximum concentration tested (10 mM),
while lead compound 1 showed MEC of 10 mM.6)
In conclusion, we have synthesized various 6-substituted
9-benzyl-8-hydroxypurine derivatives and evaluated their
IFN-inducing activities. We revealed that the 6-amino group
of the lead compound 1 is essential for the expression of the
IFN-inducing activity. These results may provide useful in-
formation for mechanistic pharmaceutical studies of the 8-
hydroxyadenine series.
Experimental
Melting points were determined on a Yanagimoto melting-point apparatus
and are uncorrected. UV absorption spectra were recorded on a Shimadzu
260 spectrophotometer. IR spectra were measured using Perkin Elmer 1640
FT-IR spectrometer. 1H-NMR spectra were recorded on a JEOL GX-270
(270 MHz) or JEOL JNM EX-400 (400 MHz) spectrometer using DMSO-d6
as a solvent. Chemical shifts are given in ppm (d) relative to internal solvent
signals. Coupling constants (J) are given in Hz and splitting patterns are
designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multi-
plet; br, broad; *, deuterium exchangeable. 13C-NMR spectra were recorded
on a JEOL JNM EX-400 (100 MHz) spectrometer using DMSO-d6 as a sol-
vent. Chemical shifts are given in ppm (d) relative to internal solvent sig-
nals. Mass spectra were recorded on a JMS-SX 102A spectrometer. Elemen-
tal analyses were performed on Yanagimoto MT-3, and the results (C, H, N)
were within Ϯ0.3% of the theoretical values. Thin-layer chromatographic
(TLC) analyses were carried out on 0.25 mm Silica Gel 60 F254 plates (Art
5715, Merck). The silica gel used for column chromatography was Silica
Gel 60 (230—400 mesh, Merck).
Chart 2
5-Amino-4-cyano-2-hydroxy-4-thiocarbamoylimidazole (10) A mix-
ture of 5-amino-1-benzyl-4-cyano-2-hydroxyimidazole (9, 200 mg, 0.93
mmol)7) and thiobenzamide (641 mg, 4.67 mmol) was heated under Ar at-
mosphere at 140 °C for 15 min. The residue was triturated with MeOH (3
ml) and the resulting precipitate was filtered to give 10 (159 mg, 69%): mp
1
237—239 °C (washed with AcOEt); H-NMR: 4.82 (2H, s, 1-CH2), 7.24—
7.36 (5H, m, 1-Ph), 7.39 (2H, br s*), 8.14 (2H, s*), 9.76 (1H, s*); 13C-NMR:
42.2, 96.8, 127.0, 127.4, 128.5, 136.4, 146.2, 148.6, 174.2; IR (KBr) n
cmϪ1: 3344, 1715, 1624, 1539, 1296, 736; UV (EtOH) lmax 269.0 nm (e
8400), 352.4 nm (e 21600); MS (EI) m/z 248 (Mϩ), 214, 189, 91; HR-MS
(EI) Calcd for C11H12N4OS (Mϩ): 248.0732. Found: 248.0734; Anal. Calcd
for C11H12N4OS·1/10H2O: C, 52.83; H, 4.92; N, 22.40. Found: C, 53.00; H,
4.91; N, 22.24.
Chart 3
9-Benzyl-8-hydroxy-6-mercaptopurine (3) A mixture of 10 (400 mg,
1.61 mmol) and formamidine hydrochloride (519 mg, 6.45 mmol) in 2-
methoxyethanol (10 ml) was refluxed under Ar atmosphere for 60 h. The sol-
vent was removed under reduced pressure and the residue was triturated with
with triphosgene to give 6.
N6-Alkyl-9-benzyl-8-hydroxyadenine derivatives (7a—e) H2O (10 ml). The resulting precipitate was filtered to give 3 (363 mg, 87%):
mp 277—279 °C (recrystallized from EtOH); 1H-NMR: 4.94 (2H, s, 9-CH2),
were synthesized from 9-benzyl-8-hydroxyadenine (1)7) by
7.24—7.33 (5H, m, 9-Ph), 8.16 (1H, s, 2-H), 11.59 (1H, s*), 13.83 (1H, br
reductive alkylation using an appropriate aldehyde and
s*); 13C-NMR: 42.7, 121.9, 127.3, 127.5, 128.6, 136.4, 141.9, 145.5, 152.4,
sodium cyanoborohydride (Chart 3).10) The alkylation of 1
161.3; IR (KBr) n cmϪ1: 3098, 1730, 1622, 1500, 1379, 1174; UV (EtOH)
proceeded in methanol at 50 °C to give 7b—d in 51—78%
yields. In the case of isobutyraldehyde, the reaction was slow
and the corresponding product 7e was obtained in 35% yield
together with recovery of 1 in 52% yield. Although the treat-
ment of 1 with paraformaldehyde in the presence of NaBH4
gave only N6-methoxymethyladenine derivative (16) in 69%
yield instead of the desired product 7a, 16 could be easily
converted into 7a by simple reduction using NaBH4 as a re-
ductant under acidic conditions.11) Synthesis of N6-acylated
derivatives (8a—c) was achieved by chemoselective acyla-
tion of the 6-amino group of 1 using acid anhydride.
l
max 240.0 nm (e 12100), 315.6 nm (e 12400), 336.6 nm (e 11700); MS (EI)
m/z 258 (Mϩ), 91; HR-MS (EI) Calcd for C12H10N4OS (Mϩ): 258.0575.
Found: 258.0570; Anal. Calcd for C12H10N4OS: C, 55.80; H, 3.90; N, 21.69.
Found: C, 55.61; H, 4.00; N, 21.50.
9-Benzyl-8-hydroxy-6-methylthiopurine (4) A mixture of 3 (100 mg,
0.39 mmol) and K2CO3 (64 mg, 0.47 mmol) in dry N,N-dimethylformamide
(DMF) (8 ml) was stirred under Ar atmosphere at room temperature for 1 h.
To the mixture was added methyl iodide (29 ml, 0.47 mmol) and the mixture
was stirred at room temperature for 12 h. After evaporation, the residue was
triturated with H2O (3 ml). The resulting suspension was neutralized with
10% NaHSO4 solution and extracted with AcOEt (10 ml). The organic layer
was washed with brine (10 ml) and dried over MgSO4. The solvent was
evaporated and the residue was chromatographed on silica gel (ether/
hexaneϭ2/1) to give 4 (21 mg, 20%): mp 250—253 °C; 1H-NMR: 2.60 (3H,
s, 6-CH3), 4.97 (2H, s, 9-CH2), 7.24—7.33 (5H, m, 9-Ph), 8.47 (1H, s, 2-H),
11.73 (1H, br s*); 13C-NMR: 11.5, 42.6, 118.3, 127.4, 127.5, 128.6, 136.6,
144.3, 147.0, 150.5, 152.8; IR (KBr) n cmϪ1: 3446, 1709, 1612, 1482; UV
(EtOH) lmax 222.2 nm (e 15700), 295.4 nm (e 16000); MS (EI) m/z 272
The in vitro assay for IFN-inducing activities of 9-benzyl-
8-hydroxypurine derivatives (2—8, 14) synthesized above
was examined by a typical method for determining IFN titer
check.12,13) The activity is indicated by MEC (minimum ef-
fective concentration), which is the concentration of the test (Mϩ), 91; HR-MS (EI) Calcd for C13H12N4OS (Mϩ): 272.0732. Found:

610
Vol. 51, No. 5
272.0725.
ture was diluted with H2O (20 ml) and the solution was neutralized with 1 N
9-Benzyl-8-hydroxypurine (2) To a solution of 3 (300 mg, 1.16 mmol) HCl solution. The resulting solution was extracted with AcOEt (50 ml) and
in MeOH (25 ml) and 28% NH4OH solution (5 ml) was added Raney Ni (1 the organic layer was washed with brine (30 ml) and dried over MgSO4. The
ml of suspension in H2O) and the mixture was refluxed for 4 h. After re- solvent was evaporated off and the residue was chromatographed on silica
moval of the catalyst by filtration using celite cake, the filtrate was concen- gel (CHCl3) to give 15 as a crude product, which was used for the next reac-
1
trated in vacuo. The residue was chromatographed on silica gel tion without further purification: H-NMR: 3.83 (3H, s, 6-CH3), 4.18 (2H,
1
(CHCl3/MeOHϭ66/1) to give 2 (95 mg, 36%): mp 197—198 °C; H-NMR: s*, 5-NH2), 4.59 (2H, d, Jϭ5.9 Hz, 4-CH2), 6.66 (1H, t*, Jϭ5.9 Hz, 4-NH),
5.00 (2H, s, 9-CH2), 7.24—7.32 (5H, m, 9-Ph), 8.26 (1H, s, 2-H), 8.57 (1H, 7.20—7.30 (5H, m, 4-Ph), 7.70 (1H, s, 2-H).
s, 6-H), 11.50 (1H, br s*); 13C-NMR: 42.4, 122.0, 127.47, 127.54, 128.6,
9-Benzyl-8-hydroxy-6-methoxypurine (6) To a solution of crude prod-
132.8, 136.4, 149.5, 150.5, 153.0; IR (KBr) n cmϪ1: 3422, 3125, 1719, uct 15 and triethylamine (92 ml, 0.66 mmol) in dry THF (5 ml) was added
1493, 1379, 702; UV (EtOH) lmax 239.4 nm (e 3600), 279.0 nm (e 10900); triphosgene (98 mg, 0.33 mmol) in dry THF (2 ml) and the mixture was
MS (EI) m/z 226 (Mϩ), 197, 121, 91; HR-MS (EI) Calcd for C12H10N4O stirred under Ar atmosphere at room temperature for 1.5 h. The solvent was
(Mϩ): 226.0855. Found: 226.0844; Anal. Calcd for C12H10N4O: C, 63.71; H, removed under reduced pressure and the solidified product was chro-
4.46; N, 24.77. Found: C, 63.58; H, 4.49; N, 24.65.
matographed on silica gel to give 6 (36 mg, 11% in 2 steps): mp 266—267
5-Amino-1-benzyl-4-carbamoyl-2-hydroxyimidazole (11) To concen- °C; 1H-NMR: 3.99 (3H, s, 6-CH3), 4.98 (2H, s, 9-CH2), 7.23—7.33 (5H, m,
trated H2SO4 (3 ml) in an ice bath was added 9 (300 mg, 1.40 mmol) and the 9-Ph), 8.30 (1H, s, 2-H), 11.58 (1H, s*); 13C-NMR: 42.7, 53.7, 106.3, 127.4,
mixture was stirred at room temperature for 2 h. The mixture was diluted 127.5, 128.5, 136.7, 149.8, 149.9, 151.3, 152.8; IR (KBr) n cmϪ1: 3027,
with ice water (15 ml) and neutralized with 28% NH4OH solution. The mix- 1706, 1647, 1376, 1096; UV (EtOH) lmax 266.2 nm (e 13600); MS (EI) m/z
ture was kept standing overnight in a refrigerator (at 5 °C). The resulting 256 (Mϩ), 227, 151, 91; HR-MS (EI) Calcd for C13H12N4O2 (Mϩ):
precipitate was filtered to give 11 (294 mg, 91%): mp 206—208 °C (washed 256.0960. Found: 256.0966; Anal. Calcd for C13H12N4O2: C, 60.93; H, 4.72;
1
with AcOEt); H-NMR: 4.76 (2H, s, 1-CH2), 6.10 (2H, s*), 6.45 (2H, s*), N, 21.86. Found: C, 60.70; H, 4.78; N, 21.62.
7.23—7.34 (5H, m, 1-Ph), 9.42 (1H, s*); 13C-NMR: 41.8, 90.5, 127.0,
Typical Procedure for the Synthesis of 7b—e To a suspension of 1
127.2, 128.4, 137.2, 140.2, 149.6, 162.3; IR (KBr) n cmϪ1: 3372, 3204, (0.42 mmol) and NaBH3CN (2.45 mmol) in dry MeOH (20 ml) was added an
1685, 1551, 1403, 739, 697; UV (EtOH) lmax 289.2 nm (e 14300); MS (EI) aldehyde [3.32 mmol except for the case of the preparation of 7b (acetalde-
m/z 232 (Mϩ), 189, 91; HR-MS (EI) Calcd for C11H12N4O2 (Mϩ): 232.0960. hyde was used 14.09 mmol)] and the mixture was stirred under Ar atmos-
Found: 232.0966; Anal. Calcd for C11H12N4O2·1/7H2O: C, 56.27; H, 5.27; phere at 50 °C for 8 days. After evaporation, the residue was chro-
N, 23.86. Found: C, 56.37; H, 5.24; N, 23.67.
9-Benzyl-6,8-dihydroxypurine (5) A mixture of 11 (300 mg, 1.29
matographed on silica gel.
9-Benzyl-N6-ethyl-8-hydroxyadenine (7b): Yield 78%; mp 253—255 °C
1
mmol) and formamidine hydrochloride (416 mg, 5.17 mmol) in 2- (recrystallized from 1,4-dioxane); H-NMR: 1.16 (3H, t, Jϭ7.1 Hz, 6-CH3),
methoxyethanol (8 ml) was refluxed under Ar atmosphere for 60 h. The sol- 3.40—3.46 (2H, m, 6-CH2), 4.91 (2H, s, 9-CH2), 6.46 (1H, t*, Jϭ5.4 Hz, 6-
vent was removed under reduced pressure and the residue was triturated with NH), 7.22—7.32 (5H, m, 9-Ph), 8.08 (1H, s, 2-H), 10.18 (1H, s*); 13C
H2O (10 ml). The resulting precipitate was filtered to give 5 (124 mg, 40%): NMR: 15.0, 34.9, 42.4, 103.4, 127.4, 128.5, 137.1, 145.9, 146.5, 151.1,
mp Ͼ300 °C (recrystallized from EtOH); 1H-NMR: 4.91 (2H, s, 9-CH2), 151.9; IR (KBr) n cmϪ1: 3242, 1710, 1638, 1348, 890, 780, 697; UV
7.23—7.33 (5H, m, 9-Ph), 7.95 (1H, s, 2-H), 11.36 (1H, s*), 12.53 (1H, br (EtOH) lmax 273.6 nm (e 16700); MS (EI) m/z 269 (Mϩ), 254, 178, 91; HR-
s*); 13C-NMR: 42.6, 107.8, 127.3, 127.4, 128.5, 137.0, 144.8, 145.1, 150.7, MS (EI) Calcd for C14H15N5O (Mϩ): 269.1277. Found: 269.1270; Anal.
152.2; IR (KBr) n cmϪ1: 3446, 3037, 1721, 1551, 1452, 1370; UV (EtOH) Calcd for C14H15N5O: C, 62.44; H, 5.61; N, 26.01. Found: C, 62.37; H, 5.70;
l
max 257.8 nm (e 10400); MS (EI) m/z 242 (Mϩ), 91; HR-MS (EI) Calcd for N, 25.98.
C12H10N4O2 (Mϩ): 242.0804. Found: 242.0812; Anal. Calcd for C12H10N4O2·
9-Benzyl-8-hydroxy-N6-propyladenine (7c): Yield 51%; mp 226—228 °C
(recrystallized from toluene); 1H-NMR: 0.91 (3H, t, Jϭ7.3 Hz, 6-CH3),
1.51—1.60 (2H, m, 6-CH2), 3.37 (2H, q, Jϭ6.7 Hz, 6-CH2), 4.91 (2H, s, 9-
1/3H2O: C, 58.06; H, 4.33; N, 22.57. Found: C, 58.17; H, 4.27; N, 22.62.
Methylation of 9-Benzyl-6,8-dihydropurine 5 (Preparation of 12)
A
mixture of 5 (274 mg, 1.13 mmol) and K2CO3 (188 mg, 1.36 mmol) in dry CH2), 6.48 (1H, t*, Jϭ6.7 Hz, 6-NH), 7.24—7.32 (5H, m, 9-Ph), 8.08 (1H,
DMF (20 ml) was stirred under Ar atmosphere at room temperature for 1 h. s, 2-H), 10.20 (1H, s*); 13C-NMR: 11.3, 22.5, 41.7, 42.4, 103.3, 127.3,
To the mixture was added methyl iodide (85 ml, 1.36 mmol) and the mixture 127.4, 128.4, 137.1, 146.0, 146.5, 151.0, 151.9; IR (KBr) n cmϪ1: 3358,
was stirred at room temperature for 24 h. After evaporation, the residue was 1703, 1641, 1458, 1371, 699, 632; UV (EtOH) lmax 274.4 nm (e 16700);
triturated with H2O (5 ml). The resulting suspension was neutralized with MS (EI) m/z 283 (Mϩ), 268, 254, 241, 192, 91; HR-MS (EI) Calcd for
10% NaHSO4 solution and extracted with AcOEt (20 ml). The organic layer C15H17N5O (Mϩ): 283.1433. Found: 283.1427; Anal. Calcd for C15H17N5O:
was washed with brine (10 ml) and dried over MgSO4. The solvent was C, 63.59; H, 6.05; N, 24.72. Found: C, 63.32; H, 5.90; N, 24.69.
evaporated and the residue was chromatographed on silica gel (CHCl3/
9-Benzyl-N6-butyl-8-hydroxyadenine (7d): Yield 58%; mp 201—203 °C
MeOHϭ100/1) to give 12 (119 mg, 41%): mp 269—270 °C; 1H-NMR: 3.47 (recrystallized from toluene); 1H-NMR: 0.90 (3H, t, Jϭ7.3 Hz, 6-CH3),
(3H, s, 1-CH3), 4.91 (2H, s, 9-CH2), 7.23—7.33 (5H, m, 9-Ph), 8.27 (1H, s, 1.31—1.40 (2H, m, 6-CH2), 1.49—1.56 (2H, m, 6-CH2), 3.41 (2H, q, Jϭ6.5
2-H), 11.39 (1H, s*); 13C-NMR: 33.8, 42.5, 107.1, 127.3, 127.4, 128.5, Hz, 6-CH2), 4.91 (2H, s, 9-CH2), 6.44 (1H, t*, Jϭ6.5 Hz, 6-NH), 7.24—7.32
137.0, 144.3, 147.7, 150.7, 152.3; IR (KBr) n cmϪ1: 3448, 3140, 1702, (5H, m, 9-Ph), 8.08 (1H, s, 2-H), 10.18 (1H, s*); 13C-NMR: 13.6, 19.5, 31.3,
1539, 1457, 1346, 726; UV (EtOH) lmax 259.0 nm (e 8700), 286.6 nm (e 42.4, 103.3, 127.3, 127.4, 128.4, 137.1, 146.0, 146.5, 151.0, 151.8; IR (KBr)
4200); MS (EI) m/z 256 (Mϩ), 91; HR-MS (EI) Calcd for C13H12N4O2 (Mϩ): n cmϪ1: 3353, 2957, 1702, 1642, 1456, 698, 632; UV (EtOH) lmax 274.2 nm
256.0960. Found: 256.0958; Anal. Calcd for C13H12N4O2: C, 60.93; H, 4.72; (e 16800); MS (EI) m/z 297 (Mϩ), 281, 268, 254, 241, 91; HR-MS (EI)
N, 21.86. Found: C, 60.87; H, 4.82; N, 21.63.
Calcd for C16H19N5O (Mϩ): 297.1590. Found: 297.1597; Anal. Calcd for
9-Benzyl-6-chloro-8-hydroxypurine (14) To a solution of 5-amino-4- C16H19N5O: C, 64.63; H, 6.44; N, 23.55. Found: C, 64.79; H, 6.42; N, 23.51.
benzylamino-6-chloropyrimidine (13, 416 mg, 1.77 mmol)9 and triethy-
9-Benzyl-8-hydroxy-N6-isobutyladenine (7e): Yield 35%; mp 206—208
1
lamine (0.49 ml, 3.55 mmol) in dry tetrahydrofuran (THF) (10 ml) was °C (recrystallized from toluene); H-NMR: 0.91 (6H, d, Jϭ6.4 Hz, 6-CH3),
added triphosgene (526 mg, 1.77 mmol) in dry THF (5 ml) dropwise and the 1.77—1.87 (1H, m, 6-CH), 3.25 (2H, t, Jϭ6.1 Hz, 6-CH2), 4.91 (2H, s, 9-
mixture was stirred under Ar atmosphere at room temperature for 1 h. After CH2), 6.47 (1H, t*, Jϭ6.1 Hz, 6-NH), 7.24—7.30 (5H, m, 9-Ph), 8.07 (1H,
evaporation, the resulting residue was chromatographed on silica gel s, 2-H), 10.22 (1H, s*); 13C-NMR: 20.0, 28.0, 42.4, 47.3, 103.3, 127.3,
(CHCl3/MeOHϭ200/1) to give 1414) (408 mg, 88%): mp 256—257 °C 127.4, 128.4, 137.1, 146.1, 146.5, 150.9, 151.8; IR (KBr) n cmϪ1: 3247,
1
(washed with ether); H-NMR: 5.00 (2H, s, 9-CH2), 7.24—7.32 (5H, m, 9- 2960, 1708, 1632, 1354, 891, 782, 734; UV (EtOH) lmax 273.6 nm (e
Ph), 8.43 (1H, s, 2-H), 12.19 (1H, s*); 13C-NMR: 43.0, 119.7, 127.5, 127.6, 17100); MS (EI) m/z 297 (Mϩ), 282, 254, 241, 91; HR-MS (EI) Calcd for
128.6, 134.6, 136.1, 150.2, 150.6, 152.8; IR (KBr) n cmϪ1: 3424, 3007, C16H19N5O (Mϩ): 297.1590. Found: 297.1582; Anal. Calcd for C16H19N5O:
1715, 1493, 1375, 1158, 735; UV (EtOH) lmax 246.4 nm (e 3700), 281.2 nm C, 64.63; H, 6.44; N, 23.55. Found: C, 64.89; H, 6.37; N, 23.35.
(e 12400); MS (EI) m/z 262 (Mϩϩ2), 260 (Mϩ), 231, 155, 91; HR-MS (EI)
9-Benzyl-8-hydroxy-N6-methoxymethyladenine (16) A mixture of 1
Calcd for C12H9ClN4O (Mϩ): 260.0465. Found: 260.0458; Anal. Calcd for (300 mg, 1.24 mmol), paraformaldehyde [1.27 g, 42.30 mmol (calculated as
C12H9ClN4O: C, 55.29; H, 3.48; N, 21.49. Found: C, 55.23; H, 3.38; N, formaldehyde)] and NaBH4 (282 mg, 7.46 mmol) in dry MeOH (30 ml) was
21.63.
stirred under Ar atmosphere at 50 °C for 8 d. The solvent was removed under
5-Amino-4-benzylamino-6-methoxypyrimidine (15) To a solution of reduced pressure, and the residue was chromatographed on silica gel
Na (300 mg, 13.05 mmol) in dry MeOH (20 ml) was added 13 (300 mg, 1.28 (CHCl3/MeOHϭ100/1) to give 16 (246 mg, 69%): mp 225—227 °C (recrys-
mmol) and the mixture was refluxed under Ar atmosphere for 3 d. The mix- tallized from MeOH); 1H-NMR: 3.22 (3H, s, 6-CH3), 4.87 (2H, d, Jϭ7.3 Hz,

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6-CH2), 4.94 (2H, s, 9-CH2), 7.23—7.33 (5H, m, 9-Ph), 7.38 (1H, t*, Jϭ7.3
14900); MS (EI) m/z 345 (Mϩ), 317, 105, 91, 77; HR-MS (EI) Calcd for
C19H15N5O2 (Mϩ): 345.1226. Found: 345.1219; Anal. Calcd for C19H15N5O2·
1/3H2O: C, 64.95; H, 4.49; N, 19.93. Found: C, 64.99; H, 4.33; N, 20.02.
IFN Induction in Mouse Splenocyte Cultures Male C3H/HeJ mice
(Clea Japan Inc.) aged 8 weeks were sacrificed, spleens were removed from
Hz, 6-NH), 8.14 (1H, s, 2-H), 10.25 (1H, s*); 13C-NMR: 42.4, 54.6, 71.9,
103.9, 127.4, 128.5, 137.0, 145.1, 147.7, 150.7, 152.0; IR (KBr) n cmϪ1
:
3240, 3043, 1717, 1635, 1368, 1070; UV (EtOH) lmax 273.8 nm (e 15500);
MS (EI) m/z 285 (Mϩ), 253, 224, 150, 91; HR-MS (EI) Calcd for
C14H15N5O2 (Mϩ): 285.1226. Found: 285.1222; Anal. Calcd for C14H15N5O2: 6 mice. Spleens were meshed in phosphate buffered saline (PBS) and filtered
C, 58.94; H, 5.30; N, 24.55. Found: C, 58.67; H, 5.30; N, 24.39.
9-Benzyl-8-hydroxy-N6-methyladenine (7a) To a solution of 16 (100 tonic treatment with 0.2% NaCl solution, and washed twice with PBS.
mg, 0.35 mmol) and NaBH4 (80 mg, 2.10 mmol) in dry THF (15 ml) was
Splenocytes were resuspended at a concentration of 2ϫ106 cells/ml in MEM
through nylon mesh. The cell suspension was freed of erythrocytes by hypo-
added trifluoroacetic acid (0.3 ml, 3.89 mmol) and the mixture was stirred supplemented with 5% fetal calf serum, 100 U/ml of penicillin, and 100
under Ar atmosphere at room temperature for 3 h. After evaporation, the mg/ml of streptomycin. The test compounds were dissolved in DMSO and
residue was triturated with H2O (5 ml) and the resulting suspension was neu-
tralized with 1 N NaOH solution. The solution was extracted with AcOEt (20
diluted to 500-fold with supplemented MEM.
Above splenocytes suspension (0.5 ml) and various concentration of the
ml) and the organic layer was washed with brine (10 ml) and dried over test compounds solution (0.5 ml) were mixed in 24-well plates, and cultured
MgSO4. The solvent was evaporated off and the residue was chro- in a humidified 5% CO2/95% air atmosphere at 37 °C for 18 h. Supernatants
matographed on silica gel (CHCl3/MeOHϭ50/1) to give 7a (24 mg, 27%): were then collected, filter sterilized, and stored at Ϫ80 °C until they were an-
mp 242—243 °C (washed with MeOH); 1H-NMR: 2.93 (3H, d, Jϭ4.4 Hz, 6- alyzed for IFN.
CH3), 4.92 (2H, s, 9-CH2), 6.46 (1H, br q*, Jϭ4.4 Hz, 6-NH), 7.24—7.30
IFN Analysis Mouse IFN titer in supernatants of splenocytes was quan-
titated by measuring its antiviral activity in a bioassay using mouse L929
cell monolayers challenged with vesicular stomatitis virus.12) Results are ex-
(5H, m, 9-Ph), 8.10 (1H, s, 2-H), 10.21 (1H, s*); 13C-NMR: 27.1, 42.4,
103.6, 127.3, 128.4, 137.1, 146.4, 146.6, 151.0, 152.0; IR (KBr) n cmϪ1
:
3447, 1696, 1653, 1455, 1375; UV (EtOH) lmax 273.2 nm (e 15600); MS pressed as IFN IU/ml in terms of the international mouse IFN standard13)
(EI) m/z 255 (Mϩ), 226, 164, 150, 91; HR-MS (EI) Calcd for C13H13N5O obtained from the National Institute of Health, Bethesda, MD, U.S.A.
(Mϩ): 255.1120. Found: 255.1123; Anal. Calcd for C13H13N5O·1/5 MeOH:
C, 60.59; H, 5.32; N, 26.76. Found: C, 60.73; H, 5.29; N, 26.55.
Acknowledgements The authors thank Dr. H. Hayashi and his co-work-
ers, Japan Energy Corporation for measurements of IFN-inducing activities.
N6-Acetyl-9-benzyl-8-hydroxyadenine (8a) To a solution of acetic an-
hydride (5 ml) and concentrated H2SO4 (10 ml) was added 1 (200 mg, 0.83
mmol) and the mixture was stirred at room temperature for 30 min. The re- References and Notes
action mixture was dropped into ice water (10 ml) and the solution was neu-
tralized with 28% NH4OH solution. The precipitated product was filtered
off, which was chromatographed on silica gel (CHCl3/MeOHϭ100/1) to
1) Beck J. P., Arvanitis A. G., Curry M. A., Rescinito J. T., Fitzgerald L.
W., Gilligan P. J., Zaczek R., Trainor G. L., Bioorg. Med. Chem. Lett.,
9, 967—972 (1999).
2) Cocuzza A. J., Chidester D. R., Culp S., Fitzgerald L., Gilligan P.,
Bioorg. Med. Chem. Lett., 9, 1063—1066 (1999).
3) Kelley J. L., Linn J. A., Selway J. W. T., J. Med. Chem., 34, 157—160
(1991).
4) Biagi G., Costantini A., Costantino L., Giorgi I., Livi O., Pecorari P.,
Rinaldi M., Scartoni V., J. Med. Chem., 39, 2529—2535 (1996).
5) Kelley J. L., McLean E. W., Linn J. A., Krochmal M. P., Ferris R. M.,
Howard J. L., J. Med. Chem., 33, 196—202 (1990).
1
give 8a (197 mg, 84%): mp 291—292 °C; H-NMR: 2.12 (3H, s, 6-CH3),
5.00 (2H, s, 9-CH2), 7.25—7.32 (5H, m, 9-Ph), 8.38 (1H, s, 2-H), 10.18
(1H, s*), 10.84 (1H, s*); 13C-NMR: 23.1, 42.6, 110.5, 127.47, 127.52,
128.6, 136.6, 137.5, 150.0, 150.8, 151.7, 169.4; IR (KBr) n cmϪ1: 3325,
3022, 1693, 1346; UV (EtOH) lmax 289.0 nm (e 12900); MS (EI) m/z 283
(Mϩ), 241, 91; HR-MS (EI) Calcd for C14H13N5O2 (Mϩ): 283.1069. Found:
283.1082; Anal. Calcd for C14H13N5O2: C, 59.36; H, 4.63; N, 24.72. Found:
C, 59.16; H, 4.60; N, 24.52.
9-Benzyl-8-hydroxy-N6-trifluoroacetyladenine (8b) A mixture of 1
(100 mg, 0.41 mmol) and trifluoroacetic anhydride (3 ml) was refluxed under
Ar atmosphere for 19 h. The mixture was dropped into ice water (10 ml) and
the solution was neutralized with 28% NH4OH solution and then extracted
with AcOEt (10 ml). The organic layer was washed with brine (10 ml) and
dried over MgSO4. After evaporation, the solid product was chro-
matographed on silica gel, (CHCl3/MeOHϭ100/1) to give 8b (49 mg, 35%):
mp 243—245 °C; 1H-NMR: 5.02 (2H, s, 9-CH2), 7.25—7.34 (5H, m, 9-Ph),
6) 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., 45, 5419—5422 (2002).
7) Hirota K., Kazaoka K., Niimoto I., Sajiki H., Heterocycles, 55, 2279—
2282 (2001).
8) A method for the preparation of thioamides from nitriles using thioac-
etamide was previously reported. See: Taylor E. C., Zoltewicz J. A., J.
Am. Chem. Soc., 82, 2656—2657 (1960).
9) 5-Amino-4-benzylamino-6-chloropyrimidine (13) was prepared from
5-amino-4,6-dichloropyrimidine in 92% yield according to the modi-
fied literature procedure (refluxed with 1.2 equiv of benzylamine and
1.25 equiv of triethylamine in n-butanol for 1 h). See: Wilde R. G.,
Bakthavatchalam R., Beck J. P., Arvanitis A. G., WO 9901454 (1999).
10) Borch R. F., Bernstein M. D., Durst H. D., J. Am. Chem. Soc., 93,
2897—2904 (1971).
8.51 (1H, s, 2-H), 11.22 (1H, br s*), 12.33 (1H, br s*); IR (KBr) n cmϪ1
:
3362, 1709, 1590, 1496, 1222, 1166; UV (EtOH) lmax 293.2 nm (e 10100);
MS (EI) m/z 337 (Mϩ), 308, 268, 232, 149, 91; HR-MS (EI) Calcd for
C14H10F3N5O2 (Mϩ): 337.0787. Found: 337.0793; Anal. Calcd for
C14H10F3N5O2: C, 49.86; H, 2.99; N, 20.77. Found: C, 49.81; H, 3.00; N,
20.73.
N6-Benzoyl-9-benzyl-8-hydroxyadenine (8c) A mixture of 1 (300 mg,
1.24 mmol) and benzoic anhydride (844 mg, 3.73 mmol) was heated under 11) Barluenga J., Bayón A. M., Campos P., Asensio G., Gonzalez-Nuñez
Ar atmosphere at 180 °C for 30 min. The resulting solid was triturated with
ether (10 ml) and filtered to give 8c (316 mg, 74%): mp 234—235 °C (re-
crystallized from EtOH); 1H-NMR: 5.03 (2H, s, 9-CH2), 7.25—7.34 (5H, m,
9-Ph), 7.54 (2H, t, Jϭ7.3 Hz, 6-Ph), 7.63 (1H, t, Jϭ7.3 Hz, 6-Ph), 8.04 (2H,
d, Jϭ7.3 Hz, 6-Ph), 8.47 (1H, s, 2-H), 10.65 (1H, s*), 11.19 (1H, s*); 13C-
NMR: 42.6, 112.4, 127.5, 127.6, 128.3, 128.4, 128.6, 132.3, 132.9, 136.6,
137.9, 149.8, 150.9, 152.1, 165.4; IR (KBr) e cmϪ1: 3381, 1722, 1676,
E., Molina Y., J. Chem. Soc., Perkin Trans. 1, 1988, 1631—1636
(1988).
12) Watanabe Y., Kawade Y., “Lymphokines and Interferons: a Practical
Approach,” eds. by Clemens M. J., Morris A. G., Gearing A. J. H.,
IRL Press, Oxford, 1987, pp. 1—14.
13) Pestka S., “Method in Enzymology,” ed. by Pestka S., Academic Press,
New York, 1986, pp. 14—23.
1591, 1495, 1331, 700; UV (EtOH) lmax 232.4 nm (e 22500), 298.8 nm (e 14) Altman J., Ben-Ishai D., J. Heterocycl. Chem., 5, 679—682 (1968).