C(O)C(N)(CO)CO.Cl.[Mg+2].[Cl-].[Cl-].C(S)[C@@H](O)[C@H](O)CS.[CH:21]1[N:25]([C@@H:26]2[O:30][C@@H:29]3[CH2:31][O:32]P(O)([O:35][C@H:28]3[C@H:27]2[OH:37])=O)[C:24]2[NH:38][C:39]([NH2:43])=[N:40][C:41](=[O:42])[C:23]=2[N:22]=1.C(N(CC(O)=O)CC(O)=O)COCCOCCN(CC(O)=O)CC(O)=O>CS(C)=O.O>[C@@H:26]1([N:25]2[C:24]3[NH:38][C:39]([NH2:43])=[N:40][C:41](=[O:42])[C:23]=3[N:22]=[CH:21]2)[O:30][C@H:29]([CH2:31][OH:32])[C@@H:28]([OH:35])[C@H:27]1[OH:37]To 100 μL of a buffer D (40 mmol/L Tris-HCl, pH: 7.4, 10 mmol/L MgCl2, 1 mM DTT, 2 μM cGMP) solution containing [3H]-cGMP (0.5 pa/mL), 10 μL of a compound solution for evaluation (a solution in which a compound was dissolved in DMSO and diluted so that the DMSO concentration became 5%) and 90 μL of a solution prepared by diluting the PDE9 enzyme solution prepared in the above with a buffer E (40 mmol/L Tris-HCl, pH: 7.4, 10 mmol/L MgCl2, 1 mM DTT, 1 mmol/L EGTA) were added under ice cooling. The resultant mixed solution was incubated at 30° C. for 10 min, and thereafter heated for 2 min in boiled water to stop the enzyme reaction of the PDE9. Then, the resultant was returned to room temperature; 50 μL of 5′-Nucleotidase (Biomol GmbH, 10 units/mL) was added thereto; and the resultant was incubated at 30° C. for 10 min to thereby convert [3H]-5′-GMP formed in the previous reaction to [3H]-guanosine. 500 μL of an anion exchange resin (Bio-Rad AG1-X2 resin, mesh size: 200-400, H2O:resin=2:1) was added to the resultant reaction liquid, and allowed to stand for 10 min, and thereafter centrifuged (2,000 rpm, 10 min); and a supernatant in which the [3H]-guanosine was present was transferred to a LumaPlate (PerkinElmer, Inc.), and the radioactivity was measured by a TopCount NXT microplate scintillation and luminescence counter (PerkinElmer, Inc.).
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