反应详情

温度

30 °C

To a 40 mM Tris-hydrocholoride buffer (pH=8) containing magnesium chloride at a final concentration of 5 mM and 2-mercaptoethanol at a final concentration of 3.75 mM were added 200,000 cpm of [3H]-labeled cyclic AMP, 0.125 to 100 μM of cyclic AMP, 1 μg of cyclic AMP-dependent phosphodiesterase and a test compound at various concentrations (the total volume of the reaction mixture was 400 μl). After the reaction was allowed to proceed at 30° C. for 10 minutes, the reaction mixture was treated at 100° C. for 2.5 minutes so as to deactivate the enzyme. After the temperature of the reaction mixture had dropped to room temperature, 50 μg of snake venom (manufactured by Sigma Corp.) was added thereto, and the mixture was heated at 30° C. for 10 minutes to decompose 5'-AMP, which was formed by the action of cyclic AMP phosphodiesterase, to adenosine. To this was added 0.5 ml of a 1:3 Cresin:H2O aqueous suspension of AG1-X2 resin (manufactured by Bio-Rad Co.) to terminate the decomposition, and the reaction mixture was allowed to stand at 4° C. for 15 minutes. Thereafter, the resulting mixture was centrifuged at 12,000 rpm for 2 minutes. The radioactivity of 55 μl of the supernatant was measured with a liquid scintillation counter, and the inhibition activity of the test compound was calculated. In the case of Compound 1, prepared in Example 1 described hereinbelow, marked inhibition activity was exhibited at a concentration of 100 μM.

1. concentrationa test compound at various concentrations (the total volume of the reaction mixture
2. additionthe reaction mixture was treated at 100° C. for 2.5 minutes so as
3. additionAfter the temperature of the reaction mixture had dropped to room temperature
4. addition50 μg of snake venom (manufactured by Sigma Corp.) was added