Functionalized Congeners of Tyrosine-Based P2X₇ Receptor Antagonists:  Probing Multiple Sites for Linking and Dimerization

Abstract

Chemically funtionalized analogues of antagonists of the P2X 7 receptor, an ATP-gated cation channel, were synthesized as tools for biophysical studies of the receptor. These functionalized congeners were intended for use in chemical conjugation with retention of biological potency. The antagonists were L-tyrosine derivatives, related to [N-benzyloxycarbonyl-O-(4-arylsulfonyl)-Ltyrosyl]benzoylpiperazine (such as MRS2409, 2). The analogues were demonstrated to be antagonists in an assay of human P2X 7 receptor function, consisting of inhibition of ATP-induced K + efflux in HEK293 cells expressing the recombinant receptor. The analogues were of the general structure R 1 -Tyr(OR 2 )-piperazinyl-R 3 , in which three positions (R 1 -R 3 ) were systematically varied in structure through introduction of chemically reactive groups. Each of the three positions was designed to incorporate a 3-or 4-nitrophenyl group. The nitro groups were reduced using NaBH 4 -copper(II) acetylacetonate to amines, which were either converted to the isothiocyanate groups, as potential affinity labels for the receptor, or acylated, as models for conjugation. An alternate route to N α -3-aminobenzyloxycarbonyl functionalization was devised.The various positions of functionalization were compared for effects on biological potency, and the R 2 and R 3 positions were found to be most amenable to derivatization with retention of high potency. Four dimeric permutations of the antagonists were synthesized by coupling each of the isothiocyanate derivatives to either the precursor amine or to other amine congeners. Only dimers linked at the R 2 -position were potent antagonists. In concentration-response studies, two derivatives, a 3-nitrobenzyloxycarbonyl derivative 18 and a 4-nitrotoluenesulfonate 26b, displayed IC 50 values of roughly 100 nM as antagonists of P2X 7 receptor-mediated K + flux.

Molecular Probes for P2X7 Receptor Studies

“…tyrosine), 2) R 2 (the tyrosine side chain), and 3) R 3 (the substituent attached to the N-piperazine) (Fig. 15) [137][138][139].…”

“…15 ) on the P2X 7 R antagonistic properties was compared on the basis of inhibition of K + release [137,138]. At the R 1 position, large hydrophobic moieties linked to the amino position through sulfonamide (34a-b) or carbamate (34c) groups were preferable for P2X 7 R inhibition.…”

Agonists and Antagonists for P2 Receptors

“…The selective P2X 2,3 /P2X 3 receptor antagonist A-317491 has antinociceptive properties and has been developed as a radioligand, but suffers from low bioavailability . Derivatives of the isoquino-line KN-62, such as MRS2427 (Chen et al 2002) and a high affinity radioligand (Romagnoli et al 2004), and novel 4,5-diarylimidazolines and other antagonists identified through screening of chemical libraries are selective for the P2X 7 receptor (Merriman et al 2003, Baxter et al 2003.…”

Molecular Recognition at Purine and Pyrimidine Nucleotide (P2) Receptors

“…KN-62 antagonizes the human P2X 7 receptor more potently than mouse or rat homologues. The tyrosine derivative MRS 2409 30a (IC 50 of 200 nM at hP2X 7 ) and its derivatives [25] act as potent antagonists of both mouse and human P2X 7 receptors. A functionalized congener approach to the design of P2X 7 receptor antagonists [25] has resulted in the amine-functionalized antagonist MRS 2483 30b.…”

“…The tyrosine derivative MRS 2409 30a (IC 50 of 200 nM at hP2X 7 ) and its derivatives [25] act as potent antagonists of both mouse and human P2X 7 receptors. A functionalized congener approach to the design of P2X 7 receptor antagonists [25] has resulted in the amine-functionalized antagonist MRS 2483 30b. Baraldi and coworkers have explored the SAR in this series of P2X 7 receptor antagonists, identifying 31 as a particularly potent congener with an IC 50 value of 1.3 nM at hP2X 7 receptors [26].…”