Potent P2X₇ Receptor Antagonists: Tyrosyl Derivatives Synthesized Using a Sequential Parallel Synthetic Approach

Novel analogs of 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN-62,1) were synthesized and found to be potent antagonists in a functional assay, inhibition of ATP-induced K⁺ efflux in HEK293 cells expressing recombinant human P2X₇ receptors. Antagonism of murine P2X₇ receptors was also observed. The analogs consisted of L-tyrosine derivatives, of the general structure R₁-Tyr(OR₂)-piperazinyl-R₃, in which three positions were systematically varied in structure through facile acylation reactions. Each of the three positions was optimized in sequence through parallel synthesis alternating with biological evaluation, leading to the identification and optimization of potent P2X₇ antagonists. The optimal groups at R₁ were found to be large hydrophobic groups, linked to the α-amino position through carbamate, amide, or sulfonamide groups. The benzyloxycarbonyl (Cbz) group was preferred over most sulfonamides and other acyl groups examined, except for quinoline sulfonyl. At R₂, an arylsulfonate ester was preferred, and the order of potency was p-tolyl, p-methoxyphenyl, phenyl > α-naphthyl, β-naphthyl. A benzoyl ester was of intermediate potency. Aliphatic esters and carbonate derivatives at the tyrosyl phenol were inactive, while a tyrosyl O-benzyl ether was relatively potent. The most potent P2X₇ receptor antagonists identified in this study contained Cbz at the R₁ position, an aryl sulfonate at the R₂ position, and various acyl groups at the R₃ position. At R₃, t-butyloxycarbonyl- and benzoyl groups were preferred. The opening of the piperazinyl ring to an ethylene diamine moiety abolished antagonism. In concentration-response studies, a di-isoquinolinyl, Boc derivative, 4 (MRS2306), displayed an IC₅₀ value of 40 nM as an antagonist of P2X₇ receptor-mediated ion flux and was more potent than the reference compound 1. N^α-Cbz, Boc-piperazinyl derivatives, 11 (MRS2317), 22 (MRS2326), and 41 (MRS2409) were less potent than 1, with IC₅₀ values of 200-300 nM.