1064
O. Vitse et al. / Bioorg. Med. Chem. 7 (1999) 1059±1065
chromatography on a silica gel column eluted with
methylene chloride to give 0.7 g (2.4 mmol, 82%) of the
desired compound. H NMR (CDCl3): d 7.92 (s, 1H),
7.51 (s, 1H), 7.16 (br m, 1H), 6.07 (br m, 1H), 3.12 (d,
3H, J=5.0 Hz), 2.95 (d, 3 H, J=5.0 Hz). Anal. calcd for
C9H10N5OBr: C, 38.05; H, 3.55; N, 24.65. Found: C,
37.96; H, 3.60; N, 24.74.
ene chloride gave 0.14 g (0.55 mmol, 25%) of 25 and
0.2 g (0.88 mmol) of unreacted product. 1H NMR
(CDCl3): d 10.28 (s, 1H), 8.65 (s, 1H), 8.08 (s, 1H), 6.27
(br m, 1H), 3.15 (d, 3H, J=5.0 Hz). Anal. calcd for
C8H7N4OBr: C, 37.67; H, 2.77; N, 21.96. Found: C,
37.67; H, 2.84; N, 22.01.
1
6-Bromo-8-(methylamino)-3-nitroimidazo[1,2-a]pyrazine
(26). Compound 2 (2.2 mmol) was dissolved in cold
( 15 ꢀC) 98.2% H2SO4 (3.5 mL) and HNO3 (0.35 mL,
d=1.38) was added dropwise with stirring. The solution
was left to stand for 1 h at 0 ꢀC and 2 h at room tem-
perature. Then, the resulting mixture was poured onto
ice, neutralized with Na2CO3 and extracted with
CH2Cl2. After drying with Na2SO4, the extracts were
evaporated and the solid residue was puri®ed by column
chromatography on silica gel eluted with methylene
8-Amino-6-bromo-3-(hydroxymethyl)imidazo[1,2-a]pyrazine
(21). A mixture of 1 g of 6-bromo-3-(hydroxymethyl)-8-
methoxyimidazo[1,2-a]pyrazine 209 (3.8 mmol) in 100 mL
of a 30% NH4OH solution was heated to 120 ꢀC for 2 h
in a 250 mL autoclave. Extraction with methylene
chloride followed by chromatography on a silica gel
column eluted with 5% methanol-methylene chloride
aorded 0.24 g (1 mmol, 25%) of the desired compound
21 and 0.5 g (1.9 mmol) of unreacted product. 1H NMR
(CDCl3): d 7.67 (s, 1H), 7.47 (s, 1H), 5.64 (br s, 2H),
4.91 (d, 2H, J=2.0 Hz). Anal. calcd for C7H7N4OBr: C,
34.59; H, 2.90; N, 23.05. Found: C, 34.48; H, 3.00; N,
22.97.
1
chloride. Yield 65%. H NMR (CDCl3): d 8.45 (s, 1H),
7.57 (s, 1H), 3.11 (d, 3H, J=5.0 Hz). Anal. calcd for
C7H6N5O2Br: C, 30.90; H, 2.22; N, 25.74. Found: C,
31.01; H, 2.31; N, 25.77.
6-Bromo-8-(ethylamino)-3-(hydroxymethyl)imidazo[1,2-
a]pyrazine (23). This agent was prepared in an analo-
gous manner with a 70% ethylamine aqueous solution.
Elution with 5% methanol±methylene chloride yielded
6-Bromo-8-(ethylamino)-3-nitroimidazo[1,2-a]pyrazine
(27). This derivative was prepared, in an analogous
manner, from 3 (yield 55%). H NMR (CDCl3): d 8.43
(s, 1H), 7.56 (s, 1H), 3.75 (m, 2H, J=7.0 Hz), 1.34 (t,
3H, J=7.0 Hz). Anal. calcd for C8H8N5O2Br: C, 33.59;
H, 2.82; N, 24.48. Found: C, 33.64; H, 2.94; N, 24.53.
1
1
0.5 g (1.9 mmol, 50%) of 23. H NMR (CDCl3): d 7.62
(s, 1H), 7.29 (s, 1H), 5.99 (br s, 1H), 4.82 (s, 2H), 3.58±
3.50 (m, 2H, J1=6.0 Hz, J2=7.0 Hz), 1.25 (t, 3H,
J=7.0 Hz). Anal. calcd for C9H11N4OBr: C, 39.87; H,
4.09; N, 20.67. Found: C, 39.88; H, 3.99; N, 20.84.
Pharmacology: guinea pig isolated trachea preparation
Adult male Dunkin±Hartley guinea pigs (Ia Credo,
France), weighing 400±500 g, were killed by a blow to
the head. Tracheae were excised and cleaned of adher-
ing adipose and connective tissue. The contractility of
tracheal segments (four tracheal rings in all cases) was
measured by adapting the method previously descri-
bed.24 Tissue segments were suspended in Krebs' solu-
tion maintained at 37 ꢀC and gassed with a mixture of
95% O2, 5% CO2. All tissues were connected to an iso-
metric force-displacement transducer under a basal ten-
sion of 0.5 g. The force-displacement transducer
provided input to a Physiograph Narco Bio-system. The
bronchoconstrictor agent (1 mM carbachol) induced a
contraction which reached a plateau within 5 min.
Cumulative log±concentration response curves were
constructed for each test compound, relaxation being
measured as the percentage reduction in carbachol-
induced contraction. Relaxant potency of each com-
pound was expressed as the negative log EC50 (pD2),
where EC50 is the concentration producing 50% inhibi-
tion of the contraction. The EC50 values were calculated
by linear regression analysis applied to the linear por-
tion of each concentration±response curve. The results
presented in Table 2 are meansSEM of six to eight
determinations.
6-Bromo-3-(hydroxymethyl)-8-(methylamino)imidazo[1,2-
a]pyrazine-2-carbonitrile (24). A mixture of 6-bromo-8-
(methylamino)imidazo[1,2-a]pyrazine-2-carbonitrile 16
(1 g, 4 mmol), sodium acetate (1.4 g, 17 mmol), acetic
acid (1 mL, 17 mmol), and 10 mL (129 mmol) of a 37%
solution of formaldehyde in water was heated in an
autoclave at 120 ꢀC for 2 h. On cooling, 30 mL of water
was added to the reaction mixture. After alkalinization
with Na2CO3 and extraction with methylene chloride,
the organic phase was dried and evaporated. The resi-
due was puri®ed by chromatography on a silica gel col-
umn eluted with 2% methanol±methylene chloride to
give 0.4 g (1.4 mmol, 35%) of the desired compound. 1H
NMR (CDCl3): d 8.19 (s, 1H), 6.23 (br m, 1H), 5.00 (d,
2H, J=6.0 Hz), 3.14 (d, 3H, J=5.0 Hz). Anal. calcd for
C9H8N5OBr: C, 38.32; H, 2.86; N, 24.83. Found: C,
38.41; H, 2.84; N, 25.00.
6-Bromo-3-formyl-8-(methylamino)imidazo[1,2-a]pyrazine
(25). Phosphorus oxy-bromide (7.5 g, 26 mmol) was
added dropwise, with stirrring, to cooled (0 ꢀC), dry
dimethylformamide (10 mL). 0.5 g (2.2 mmol) of com-
pound 2 in 15 mL of dry dimethylformamide was then
slowly added to the stirred mixture. After being allowed
to warm to 120 ꢀC, the mixture was stirred for 2 h at
85 ꢀC. After cooling on ice, 20 mL of 35% HCl were
added and the cooled solution was then basi®ed with an
aqueous solution of sodium hydroxide (2 M), extracted
with dichloromethane, dried and evaporated. The
resulting oil was washed with diethylether. Chromato-
graphy on silica gel, eluted with 2% methanol±methyl-
Biochemistry: measurement of inhibitory activity against
cyclic nucleotide phosphodiesterase (PDE) isoenzymes
Isoenzymes of cyclic nucleotide PDE were isolated from
guinea pig cardiac ventricles (type III) and bovine
trachealis (type IV) as previously described.25 The iden-