The Biginelli reactions. A round-bottom flask was charged
with aldehyde (0.50 mmol), urea (1.00 mmol), acetoacetate
(1.00 mmol), n-butyl acetate (0.70 mL) and the crude earthworm
extract (75 mg), to which deionized water (0.30 mL) was
introduced. The resultant mixture was stirred at 45uC and
monitored by TLC. After a specified reaction time, ethanol
(20 mL) was added to the reaction mixture to dissolve the crude
products with stirring. The mixture was then filtrated (with
buchner funnel and qualitative filter paper), and ethanol (20 mL)
was employed to wash the filter cake. The filtrate was concentrated
under reduced pressure. The residue was dissolved in ethyl
acetate, and washed with saturated brine to remove the excess
urea. The organic phase was dried over anhydrous Na2SO4, and
the solvents were then removed under reduced pressure. The solid
residue was recrystallized from ethyl acetate-petroleum ether to
give the first part of the product. The mother liquor was
concentrated under reduced pressure, and the residue was then
purified by silica gel flash column chromatography (petroleum
ether/ethyl acetate) to give the second part of the product. The reaction yield refers to the combination of the two parts of product
above.