The P2X(7) receptor is involved in several processes relevant to inflammation (cytokine release, NO generation, killing of intracellular pathogens, cytotoxicity); thus, it may be an appealing target for pharmacological intervention. The characterization of native and recombinant P2X(7) receptor continues to be hindered by the lack of specific and subtype-selective antagonists. However, a tyrosine derivative named KN-62 exhibits selective P2X(7) receptor-blocking properties. The present study was designed to evaluate the functional antagonistic properties of a novel series of KN-62-related compounds characterized by the presence of different phenyl-substituted piperazine moieties. Antagonistic activity of KN-62 derivatives was tested on HEK293 cells transduced with the human P2X(7) receptor and monocyte-derived human macrophages, a cell type well-known for the high level of expression of this receptor. The biological responses investigated were ATP-dependent Ca(2+) influx across the plasma membrane, ethidium bromide uptake, and secretion of the cytokine interleukin-1beta. KN-62 was characterized by the presence of a phenylpiperazine moiety, and the presence of a one-carbon linker between the piperazine nitrogen and the phenyl ring (compound 61) increases the activity, while a two-carbon linker (compound 62) decreases biological activity 10-fold. Also, the nature and the position of substituents on the phenyl ring tethered to the piperazine seemed to exert a fundamental influence on the biological activity. In the series of synthesized compounds, the presence of a fluorine in the para position gives the most potent compound (63), while the same atom in the ortho position reduces potency by 3-fold. When the p-fluorine was replaced in the same position with other halogens, such as chlorine (compound 64) or iodine (compound 65), the activity decreased dramatically. We then tested the activity of the four most potent KN-62 derivatives on ATP-stimulated secretion of IL-1beta from monocyte-derived human macrophages, a key cell type in inflammation and innate immunity. Interestingly, compound 68 and 71 caused a complete inhibition of IL-1beta release, while with KN-62, 63, and 85, there was a small residual cytokine secretion even at concentrations exceeding 100 nM. None of the compounds tested on IL-1beta release had any effect on isolated CaMII kinase activity up to 20 microM (not shown).