Histone deacetylase 1 and 2 (HDAC1/2) inhibitors are potentially useful as tools for probing the biological functions of the isoforms and as therapeutic agents for cancer and neurodegenerative disorders. To discover potent and selective inhibitors, we screened a focused library synthesized by using click chemistry and obtained KPZ560 as an HDAC1/2-selective inhibitor. Kinetic binding analysis revealed that KPZ560 inhibits HDAC2 through a two-step slow-binding mechanism. In cellular assays, KPZ560 induced a dose- and time-dependent increase of histone acetylation and showed potent breast cancer cell growth-inhibitory activity. In addition, gene expression analyses suggested that the two-step slow-binding inhibition by KPZ560 regulated the expression of genes associated with cell proliferation and DNA damage. KPZ560 also induced neurite outgrowth of Neuro-2a cells and an increase in the spine density of granule neuron dendrites of mice. The unique two-step slow-binding character of o-aminoanilides such as KPZ560 makes them interesting candidates as therapeutic agents.