Functionalized congeners of tyrosine-based P2X(7) 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)-L-tyrosyl]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(alpha)-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.

Figure 1

Effects of tyrosyl derivatives on P2X₇ receptor-activation in hP2X₇–HEK cells. Cells were preincubated with or without the indicated concentrations of selected antagonists (26b MRS2447; 32 MRS2452; 18 MRS2464; and 30 MRS2483) prior to stimulation with 3 mM ATP. Data points represent the mean (± SD) K⁺ contents from triplicate wells in a single experiment. IC₅₀ values mentioned in the text are rough estimates from visual inspection of the concentration–response relationships. Hill coefficients were not determined, since previous studies have shown that the lead compound (KN-62) represses P2X₇ receptor function via complex mechanisms that are not readily amenable to standard ligand-binding analyses.

Scheme 1

Synthesis of P2X₇ Receptor Antagonists Consisting of L-Tyrosine Derivatives Differing in the Cinnamoyl or Urethane Substituent Present at the Nα Position

Scheme 2

Synthesis of P2X₇ Receptor Antagonists Consisting of L-Tyrosine Derivatives Containing a Nitro Substitution at Either the Nα-Cinnamoyl Substituent, the Aryl Sulfonate Group Present on the Tyrosyl Side Chain, or the Cα-Benzoylpiperazine Substituent

Scheme 3

Synthesis of P2X₇ Receptor Antagonists Consisting of L-Tyrosine Derivatives Containing a Nitro Substitution at the Nα-Benzyloxycarbonyl Substituent or of the Cα-Benzoylpiperazine Substituent

Scheme 4

Synthesis of P2X₇ Receptor Antagonists Consisting of L-Tyrosine Derivatives Containing a Nitro Substitution at the Aryl Sulfonate Group Present on the Tyrosyl Side Chain

Scheme 5

Synthesis of P2X₇ Receptor Antagonists Consisting of L-Tyrosine Derivatives Containing 3-Nitro Substitution at the Nα-Benzyloxycarbonyl Substituent

Scheme 6

Functional Group Replacement in P2X₇ Receptor Antagonists

Scheme 7

Dimerization of P2X₇ Receptor Antagonists through Formation of Thiourea Linkages