Currently, there are different approaches for introducing fluorescent labels into living organisms. One approach is to carry out click reactions between an azido-modified biomolecule and a fluorescent and strained cycloalkyne. The reaction proceeds independently of the presence of other functional groups, with a high yield, rather quickly and under mild conditions. The problem lies in the small number of available cycloalkynes for click reactions. Therefore, this work is devoted to the development of new cycloalkynes that are sufficiently stable, but at the same time active for conducting click reactions in living organisms and subsequent visualization of cells. During the project, a general synthetic route for cycloalkynes was developed. This synthetic approach allows for variations in the natures of both the heterocycle and heteroatom to reach the optimal stability/reactivity balance for these new strained systems. Three new cycloalkynes active in click reactions were synthesized, the applicability of heterocyclononynes for in vitro bioconjugation was exemplified by labeling and visualization of HeLa cancer cells isocoumarin-fused azacyclononyne IC9N-BDP-FL.