Three series of novel heterocyclic azoles derivatives containing pyrazine (5a-5k, 8a-8k and 11a-11k) have been designed, synthesized, structurally determined, and their biological activities were evaluated as potential telomerase inhibitors. Among the oxadiazole derivatives, compound 5c showed the most potent biological activity against SW1116 cancer cell line (IC(50)=2.46 μM against SW1116 and IC(50)=3.55 μM for telomerase). Compound 8h performed the best in the thiadiazole derivatives (IC(50)=0.78 μM against HEPG2 and IC(50)=1.24 μM for telomerase), which was comparable to the positive control. While compound 11f showed the most potent biological activity (IC(50)=4.12 μM against SW1116 and IC(50)=15.03 μM for telomerase) among the triazole derivatives. Docking simulation by positioning compounds 5c, 8h and 11f into the telomerase structure active site was performed to explore the possible binding model. The results of apoptosis demonstrated that compound 8h possessed good antitumor activity against HEPG2 cancer cell line. Therefore, compound 8h with potent inhibitory activity in tumor growth inhibition may be a potential antitumor agent against HEPG2 cancer cell. Therefore, the introduction of oxadiazole, thiadiazole and triazole structures reinforced the combination of our compounds and the receptor, resulting in progress of bioactivity.