2,5-Dihydroxymethyl-3,6-dimethylpyrazine (liguzinediol) has been recently discovered as a potential agent for treatment of heart failure with low safety risk. In the present study, four main metabolites of liguzinediol were synthesized and their positive inotropic activities were evaluated. Synthetic compounds were identical with the isolated metabolites of liguzinediol. Pharmacological examinations showed that the four major metabolites were not observed positive inotropic activity, and revealed that the positive inotropic activity of liguzinediol was essentially attributed to the parent agent.