Abstract: The functional group primary or secondary amines of DPP-IV inhibitors provide critical hydrogen bonds to Glu205/Glu206 of DPP-IV and constitute the key factor for an effective inhibition of the enzyms.A docking study showed that the active hydrogen at tertiary amine of DPP-IV inhibitor could interact with the carbonyl at Glu-motif (Glu205-Glu206) of the DPP-IV enzyme.Fourteen potential DPP-IV inhibitors containing piperazine group were synthesized,and determined by MS and ¹H NMR.In vitro inhibitory activity of these compounds against DPP-IV was evaluated.The assay results indicated that the inhibitory activity of the tertiary amines compounds against DPP-IV enzyme was lost.Therefore,it seems that hydrogen atoms in the primary or secondary amines of DPP-IV inhibitors may play a crucial role for the inhibition of DPP-IV enzyme.