Y. Miyazaki et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1967–1971
1971
labelled inhibitor, and a variable concentration of test
compound were incubated together to reach thermody-
namic equilibrium under conditions such that in the
absence of test compound, the fluorescent inhibitor is
significantly (>50%) enzyme bound and in the presence of
a sufficient concentration (>10 Ki, where Ki = dissociation
constant for inhibitor binding) of a potent inhibitor the
anisotropy of the unbound fluorescent ligand is measur-
ably different from the bound value. Inhibitor binding to
GSK-3b was assessed by a fluorescence anisotropy com-
petitive binding assay. All components were dissolved in
buffer of composition 50 mM Hepes, pH 7.4, 1 mM
CHAPS, 1 mM DTT, 10 mM MgCl2 with final concen-
trations of 65 nM GSK-3b and 5 nM fluorescently labelled
inhibitor. This reaction mixture is added to wells contain-
ing various concentrations of test compound (typically
24 pM–25 lM final) or DMSO vehicle (<3% final) in black
384-well microtitre plates and equilibrated for 60–180 min
at ambient temperature. Fluorescence anisotropy was
measured in a Molecular Devices Acquest spectrofluorim-
eter (excitation wavelength 485 nm: emission wavelength
535 nm). The error of the assay was estimated to be within
0.2 log units, based on the median standard deviation of
compounds which have been tested more than eight times.
9. IC50 value of 4-amino-5,6-bis(4-methoxylphenyl)-furo[2,3-
d]pyrimidine in GSK-3 enzyme assay was over 15 lM.
Replacement of pyridine of compound 12 with 4-meth-
oxylphenyl reduced the GSK-3 inhibitory activity
(IC50 > 15 lM). These results indicated interaction of
pyridine plays one of the key roles to exhibit activity.
10. EC50 value of compound 12 in the glycogen accumulation
assay in L6 cells was 3.2 lM. The method was described
by: Peat, A. J.; Garrido, D.; Boucheron, J. A.; Schweiker,
S. L.; Dickerson, S. H.; Wilson, J. R.; Wang, T. Y.;
Thomson, S. A. Bioorg. Med. Chem. Lett. 2004, 14, 2127.
11. Davis, S. P.; Reddy, H.; Caivano, M.; Cohen, P. Biochem.
J. 2000, 351, 95.
(3.68 g ) were added. The mixture was stirred at 38 ꢁC
(inner temp) for 22 h. The mixture was concentrated in
vacuo until the total volume reached to ca. 300 ml, diluted
with ethyl acetate (1200 ml), and washed with water
(200 ml) and brine (2· 400 ml). Aqueous layers were re-
extracted with ethyl acetate (800 ml then 400 ml). Com-
bined organic layers were dried over anhydrous sodium
sulfate and concentrated in vacuo to give crude 5: 1H
NMR (DMSO-d6) ppm 10.36 (s, 1H), 7.88 (m, 2H), 7.71
(m, 2H), 4.99 (m, 1H), 4.73 (m, 2H), 2.09 (s, 3H) as a solid
(7.17 g, 95%), which was used for the next step without
purification.
(b) 5-(4-Acetamidophenyl)-4-amino-furo[2,3-d]pyrimidine
(7). 4-(4-Acetamidophenyl)-3-cyano-2-[(ethoxymethylid-
ene)amino]furane (10.26 g, 34.5 mmol), which was
obtained from compound 6 by ethoxymethylidenation
of the amine, was suspended in a mixture of EtOH
(410 ml) and THF (410 ml). Into the mixture cooled in
an ice-water bath and under vigorous stirring, NH3 gas
was bubbled for 40 min. The suspension became a clear
solution at once, and subsequently it began to make a
precipitation. The mixture in the sealed reaction vessel
was stirred at room temperature for 3 h, concentrated in
vacuo, and dried under reduced pressure. The residual
solid was triturated with small amount of methanol,
filtrated, and dried under reduced pressure to give 4-(4-
acetamidophenyl)-2-[(aminomethylidene)amino]-3-cyano-
1
furan (6.16 g). H NMR (DMSO-d6): ppm 10.06 (s, 1H),
8.21 (br, 2H), 7.87 (br, 1H), 7.64 (d, 2H, J = 8 Hz), 7.63
(s, 1H), 7.52 (d, 2H, J = 8 Hz), 2.06 (s, 3H); MS(ESI) m/z
269 (M+1)+. Thus, obtained solid (6.00 g, 22.4 mmol)
was suspended in a mixture of EtOH (180 ml) and THF
(180 ml). To the mixture was added dropwise 27.3 ml
(0.9 N, 24.6 mmol) of sodium ethoxide (freshly prepared
from sodium in ethanol). The suspension gradually
dissolved and gave a clear solution during stirring at
room temperature. After stirring for 4 h, the mixture was
12. Compound 12 showed IC50 value of 1120 nM against
VEGFR2.
concentrated in vacuo to give a solid, which was
triturated with water, collected by filtration, washed with
water, and dried under reduced pressure to give 7 (6.01 g)
as a brown solid. 1H NMR (DMSO-d6): ppm 10.12 (s,
1H), 8.25 (s, 1H), 7.93 (s, 1H), 7.73 (d, 2H, J = 8 Hz),
7.45 (d, 2H, J = 8 Hz), 6.55 (br, 2H), 2.09 (s, 3H);
MS(ESI) m/z 269 (M+1)+.
13. Experimentals for some intermediates are as follows.
(a) 40-Acetamido-2-hydroxyacetophenone (5). To a sus-
pension of 40-acetamido-2-bromoacetophenone (10.0 g,
39 mmol) in EtOH (800 ml), aqueous potassium formate
(35.4 g in 200 ml ) and subsequently sodium bicarbonate