2970 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Kurimoto et al.
added, and the organic layer was separated, dried over Na2SO4,
and evaporated. The residue was purified by silica gel column
chromatography using MeOH-CHCl3 as eluent to give com-
pound 7k (597 mg, 55%) as a colorless solid. 1H NMR (CDCl3)
δ 7.95 (1H, dd, J = 6.7 Hz, 2.4 Hz), 7.61 (1H, s), 7.48 (1H, m),
7.12 (1H, dd, J = 10.3 Hz, 8.6 Hz), 5.55 (2H, brs), 5.27 (2H, s),
4.33 (2H, t, J = 6.6 Hz), 3.93 (3H, s), 1.78 (2H, tt, J = 7.6 Hz, 6.6
Hz), 1.51 (2H, tq, J = 7.6 Hz, 7.4 Hz), 0.97 (3H, t, J = 7.4 Hz).
{5-[(6-Amino-2-butoxy-9H-purin-9-yl)methyl]-2-fluorophenyl}-
methanol (11k). To a suspension of lithium aluminum hydride
(121 mg, 3.20 mmol) in THF (20 mL) was added dropwise a
solution of compound 7k (597 mg, 1.60 mmol) in THF (40 mL)
at 0 °C and stirred for 1.5 h. The reaction mixture was quenched
with H2O (0.72 mL) and 1N NaOH (0.54 mL) and filtered
through celite. The filtrate was concentrated in vacuo and the
residue triturated with CHCl3. The precipitate was collected by
filtration to give compound 11k (536 mg, 97%) as a colorless
solid. 1H NMR (DMSO-d6) δ 8.03 (1H, s), 7.47 (1H, dd, J = 7.1
Hz, 2.2 Hz), 7.28 (1H, m), 7.11 (1H, dd, J = 10.2 Hz, 8.6 Hz),
5.23 (2H, s), 4.49 (2H, s), 4.21 (2H, t, J = 6.6 Hz), 1.65 (2H,
tt, J = 7.5 Hz, 6.6 Hz), 1.38 (2H, tq, J = 7.5 Hz, 7.4 Hz), 0.91
(3H, t, J = 7.4 Hz).
compound 9k (321 mg, 93%) as a colorless solid; mp 272-
274 °C. 1H NMR (DMSO-d6) δ 9.96 (1H, brs), 7.29-7.23 (2H,
m), 7.14 (1H, dd, J = 9.7, 8.4 Hz), 6.46 (2H, brs), 4.82 (2H, s),
4.14 (2H, t, J = 6.6 Hz), 3.70 (2H, s), 3.60 (3H, s), 1.62 (2H, m),
1.37 (2H, m), 0.90 (3H, t, J = 7.4 Hz). Anal. (C19H22FN5O4)
C, H, N.
Compound 9j was prepared from compound 6 using a similar
procedure as for compound 9k.
Methyl{3-[(6-amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-
9-yl)methyl]-4-methoxyphenyl}acetate (9j). Yield 31%; mp
282-287 °C. 1H NMR (DMSO-d6) δ 10.00 (1H, brs), 7.13
(1H, d, J = 8.4 Hz), 6.97 (1H, d, J = 8.4 Hz), 6.67 (1H, s),
6.47 (2H, brs), 4.80 (2H, s), 4.08 (2H, t, J = 6.6 Hz), 3.83 (3H, s),
3.53 (3H, s), 3.50 (2H, s), 1.59 (2H, m), 1.33 (2H, m), 0.87 (3H, t,
J = 7.4 Hz). Anal. (C20H25N5O5) C, H, N.
Compound 9h was prepared from compound 7e using a
similar procedure as for compound 9k.
Methyl 3-{3-[(6-Amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-
9-yl)methyl]phenyl}propanoate (9h). Yield 44%; mp 269-
1
271 °C. H NMR (DMSO-d6) δ 9.99 (1H, brs), 7.15 (4H, m),
6.46 (2H, brs), 4.81 (2H, s), 4.14 (2H, t, J = 6.6 Hz), 3.54
(3H, s), 2.80 (2H, t, J = 7.6 Hz), 2.58 (2H, t, J = 7.6 Hz), 1.62
(2H, m), 1.36 (2H, m), 0.90 (3H, t, J = 7.3 Hz). Anal.
2-Butoxy-9-[3-(chloromethyl)-4-fluorobenzyl]-9H-purin-6-
amine (12k). To a suspension of compound 11k (535 mg, 1.55
mmol) in CHCl3 (16 mL) was added SOCl2 (0.57 mL, 7.80
mmol) and refluxed for 1 h. The reaction mixture was neutra-
lized with saturated aq NaHCO3 and separated. The organic
layer was dried over Na2SO4 and evaporated to give compound
12k (535 mg, 95%) as a pale-yellow solid. 1H NMR (CDCl3) δ
7.61 (1H, s), 7.40 (1H, dd, J = 6.9 Hz, 2.2 Hz), 7.25 (1H, m), 7.05
(1H, dd, J = 9.0 Hz, 8.8 Hz), 5.84 (2H, brs), 5.25 (2H, s), 4.59
(2H, s), 4.33 (2H, t, J = 6.6 Hz), 1.78 (2H, tt, J = 7.6 Hz, 6.6 Hz),
1.50 (2H, tq, J = 7.6 Hz, 7.4 Hz), 0.97 (3H, t, J = 7.4 Hz).
{5-[(6-Amino-2-butoxy-9H-purin-9-yl)methyl]-2-fluorophenyl}-
acetonitrile (13k). To a solution of compound 12k (535 mg, 1.47
mmol) in DMF (15 mL) was added NaCN (220 mg, 4.50 mmol)
and stirred at room temperature for 15 h. The reaction mixture
was neutralized with 1N HCl and concentrated in vacuo. Water
and CHCl3 were added, and the organic layer was separated,
dried over Na2SO4, evaporated, and the residue triturated with
CHCl3-hexane. The precipitate was collected by filtration to
give compound 13k (457 mg, 88%) as a colorless solid. 1H NMR
(CDCl3) δ 7.61 (1H, s), 7.44 (1H, dd, J = 7.0 Hz, 2.1 Hz), 7.29
(1H, m), 7.08 (1H, dd, J = 9.0 Hz, 8.8 Hz), 5.54 (2H, brs), 5.26
(2H, s), 4.34 (2H, t, J = 6.6 Hz), 3.75 (2H, s), 1.79 (2H, tt, J = 7.6
Hz, 6.6 Hz), 1.51 (2H, tq, J = 7.6 Hz, 7.4 Hz), 0.97 (3H, t, J =
7.4 Hz).
(C20H25N5O4 0.25H2O) C, H, N.
3
{3-[(6-Amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-9-yl)methyl]-
phenyl}acetic Acid (16). To a solution of compound 9e (100 mg,
0.259 mmol) in MeOH (50 mL) was added 1N NaOH (20 mL)
and stirred at room temperature for 2 h. The reaction mixture
was neutralized with 1N HCl and concentrated in vacuo. Water
was added to the residue and the precipitate collected by
filtration to give compound 16 (92 mg, 96%) as a colorless
solid; mp 259-262 °C. 1H NMR (DMSO-d6) δ 12.31 (1H, brs),
10.03 (1H, brs), 7.22 (4H, m), 6.47 (2H, brs), 4.83 (2H, s),
4.14 (2H, t, J = 6.8 Hz), 3.50 (2H, s), 1.60 (2H, m), 1.38 (2H,
m), 0.90 (3H, t, J = 7.0 Hz). Anal. (C18H21N5O4 0.25H2O)
3
C, H, N.
Ethyl {3-[(6-Amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-
9-yl)methyl]phenyl}acetate (9l). To a suspension of compound
16 (58 mg, 0.159 mmol) in EtOH (20 mL) was added H2SO4
(1 mL) under ice-cooling and refluxed for 3 h. After cooling to
room temperature, the mixture was neutralized with aq NH3
and extracted with CHCl3. The organic layer was dried over
Na2SO4 and evaporated. The residue was purified by silica gel
column chromatography using MeOH-CHCl3 as eluent to give
compound 9l (53 mg, 85%) as a colorless solid; mp 269-270 °C.
1H NMR (DMSO-d6) δ 9.98 (1H, brs), 7.27 (1H, t, J = 8.0 Hz),
7.16 (3H, m), 6.46 (2H, brs), 4.83 (2H, s), 4.13 (2H, t, J = 6.6
Hz), 4.03 (2H, q, J = 7.1 Hz), 3.58 (2H, s), 1.62 (2H, m), 1.36
(2H, m), 1.14 (3H, t, J = 7.1 Hz), 0.90 (3H, t, J = 7.3 Hz). Anal.
{5-[(6-Amino-8-bromo-2-butoxy-9H-purin-9-yl)methyl]-2-fluoro-
phenyl}acetonitrile (14k). To a suspension of compound 13k
(455 mg, 1.28 mmol) and NaOAc (190 mg, 2.30 mmol) in CHCl3
(37 mL) was added bromine (310 mg, 1.93 mmol) and stirred at
room temperature for 3 h. To the reaction mixture was added
10% aq Na2S2O3 and saturated aq NaHCO3. The organic layer
was separated, dried over Na2SO4, and evaporated. The residue
was triturated with MeOH and the precipitate collected by
filtration to give compound 14k (426 mg, 77%) as a colorless
(C20H25N5O4 0.1H2O) C, H, N.
3
Compound 17 was prepared from compound 5 using a similar
procedure as for compound 8e.
Methyl {3-[(6-Amino-2-methoxyethoxy-8-oxo-7,8-dihydro-
9H-purin-9-yl)methyl]phenyl}acetate (17). Yield 13%; mp
1
238-241 °C. H NMR (DMSO-d6) δ 9.96 (1H, s), 7.27 (1H,
dd, J = 7.6 Hz, 7.6 Hz), 7.20 (1H, s), 7.17 (1H, d, J = 7.6 Hz),
7.15 (1H, d, J = 7.6 Hz), 6.47 (2H, brs), 4.83 (2H, s), 4.25 (2H, t,
J = 4.8 Hz), 3.65 (2H, s), 3.58 (3H, s), 3.58 (2H, t, J = 4.8 Hz),
1
solid. H NMR (CDCl3) δ 7.53 (1H, dd, J = 7.0 Hz, 2.1 Hz),
7.34 (1H, m), 7.06 (1H, dd, J = 9.0 Hz, 8.8 Hz), 6.01 (2H, brs),
5.28 (2H, s), 4.34 (2H, t, J = 6.6 Hz), 3.75 (2H, s), 1.77 (2H, tt,
J = 7.6 Hz, 6.6 Hz), 1.51 (2H, tq, J = 7.6 Hz, 7.4 Hz), 0.97
(3H, t, J = 7.4 Hz).
Methyl{5-[(6-amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-
9-yl)methyl]-2-fluorophenyl}acetate (9k). To a suspension of
compound 14k (372 mg, 0.86 mmol) in MeOH (18 mL) was
added 5N NaOH (9 mL) and refluxed for 5 h. The reaction
mixture was neutralized with 12N HCl and the precipitate
collected by filtration. To the solid was added MeOH (15 mL)
and H2SO4 (1 mL) and then refluxed for 3 h. The reaction
mixture was neutralized with saturated aq NaHCO3 and
water added. The precipitate was collected by filtration to give
3.26 (3H, s). Anal. (C18H21N5O5 0.25H2O) C, H, N.
3
2. Biology. 2.1. TLR7 Reporter Gene Assay. HEK293-
hTLR7 cells, stably transfected with human TLR7 (pUNO
expression vector) and pNiFty2-SEAP reporter plasmid, were
kindly gifted from AstraZeneca. The cells were seeded in 96-well
plates at 2 ꢀ 104 cells/well in DMEM supplemented with 1%
nonessential amino acid, 10 μg/mL blasticidin S (Invitrogen), 10
μg/mL zeocin (Invivogen), and 10% heat-inactivated FCS and
then stimulated with various concentrations of test compounds
and incubated for 20 h at 37 °C in 5% CO2. Then p-nitrophenyl
phosphate was added as a substrate into the plates and incu-
bated at room temperature for 20 min. After the reaction was