Abstract: Heterocyclic enediynes are promising analogues of natural enediyne antibiotics. Here, we report on the synthesis of a benzothiophene-fused azaenediyne with an iso-position of NTs function within the enediyne cycle. The iso-N-enediyne was obtained using the iodoalkyne approach, i.e. iodocyclization of the functionalized iodoalkyne followed by two subsequent one-pot Sonogashira reactions with diiodobenzothiophene, and then the Nicholas cyclization. Despite the successful synthesis of starting acyclic enediyne, the synthetic accessibility of cyclic iso-N-enediyne was poorer than that of its known isomer due to lower regioselectivity during the cobalt complexation stage. The obtained iso-N-enediyne exhibits lower reactivity in Bergman cyclization and it is less cytotoxic against the HeLa cell line. Therefore, the previously reported N-enediyne has advantages over the newly synthesized iso-N-enediyne for the further development of anticancer agents.