To a solution of 3-carboxy-6-(2-chlorophenyl)-7-(4-chlorophenyl)pyrazolo-[1,5-a]pyrimidine (compound obtained in Reference Example 1-(4); 58 mg) and 1-cyanocyclohexylamine hydrochloride (25 mg) in chloroform (1.0 mL containing amylene) were added a 0.5 M solution of 1-hydroxybenzotriazole monohydrate in chloroform (0.45 mL containing amylene), a 0.5 M solution of 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride in N,N-dimethylformamide (0.45 mL) and triethylamine (63 μL), and the mixture was stirred at room temperature overnight. To the reaction mixture were added an aqueous saturated sodium hydrogencarbonate solution (2 mL), water (2 mL) and chloroform (4 mL), and the mixture was vigorously stirred for 15 minutes. The organic layer was separated, and the aqueous layer was extracted with chloroform (3 mL). The combined organic layer was washed successively with an aqueous saturated sodium hydrogencarbonate solution (3 mL) and brine (3 mL) and concentrated in vacuo. The resultant crude product was purified by liquid chromatograph-mass spectrometer (LCMS, column; XTerra MS C18, solvent; 10 mM ammonium carbonate/methanol 40/60 to 10/90) and lyophilized to give 6-(2-chlorophenyl)-7-(4-chlorophenyl)-3-[N-(1-cyanocyclohexyl)carbamoyl]pyrazolo[1,5-a]pyrimidine (15.6 mg; yield: 21%) as a powder.
