The Nicholas-type macrocyclization through NH-tosyl functional group has been found to be an efficient technique for the synthesis of a 10-membered azaenediyne system annulated with a benzothiophene. To compare the activity of azaenediyne synthesized with similar oxa- and carbocyclic enediynes the Bergman cyclization activation energies and the ability of enediynes to cleave DNA (pBR322 plasmid) were investigated. The order of reactivity predicted by DFT calculations (N-enediyne < C-enediyne < O-enediyne) was confirmed by DSC analysis data. Surprisingly azaenediyne was found to be more active in the DNA cleavage assay than the C-analogue.