Synthesis and Structure-Activity Relationships of Pyridoxal-6-arylazo-5'-phosphate and Phosphonate Derivatives as P2 Receptor Antagonists.

Novel analogs of the P2 receptor antagonist pyridoxal-5'-phosphate-6-phenylazo-2',4'-disulfonate (PPADS) were synthesized. Modifications were made through functional group substitution on the sulfophenyl ring and at the phosphate moiety through the inclusion of phosphonates, demonstrating that a phosphate linkage is not required for P2 receptor antagonism. Substituted 6-phenylazo and 6-naphthylazo derivatives were also evaluated. Among the 6-phenylazo derivatives, 5'-methyl, ethyl, propyl, vinyl, and allyl phosphonates were included. The compounds were tested as antagonists at turkey erythrocyte and guinea-pig taenia coli P2Y(1) receptors, in guinea-pig vas deferens and bladder P2X(1) receptors, and in ion flux experiments by using recombinant rat P2X(2) receptors expressed in Xenopus oocytes. Competitive binding assay at human P2X(1) receptors in differentiated HL-60 cell membranes was carried out by using [(35)S]ATP-?-S. A 2'-chloro-5'-sulfo analog of PPADS (C(14)H(12)O(9)N(3)ClPSNa), a vinyl phosphonate derivative (C(15)H(12)O(11)N(3)PS(2)Na(3)), and a naphthylazo derivative (C(18)H(14)O(12)N(3)PS(2)Na(2)), were particularly potent in binding to human P2X(1) receptors. The potencies of phosphate derivatives at P2Y(1) receptors were generally similar to PPADS itself, except for the p-carboxyphenylazo phosphate derivative C(15)H(13)O(8)N(3)PNa and its m-chloro analog C(15)H(12)O(8)N(3)ClPNa, which were selective for P2X vs. P2Y(1) receptors. C(15)H(12)O(8)N(3)ClPNa was very potent at rat P2X(2) receptors with an IC(50) value of 0.82 ?M. Among the phosphonate derivatives, [4-formyl-3-hydroxy-2-methyl-6-(2-chloro-5-sulfonylphenylazo)-pyrid-5-yl]methylphosphonic acid (C(14)H(12)-O(8)N(3)ClPSNa) showed high potency at P2Y(1) receptors with an IC(50) of 7.23 ?M. The corresponding 2,5-disulfonylphenyl derivative was nearly inactive at turkey erythrocyte P2Y(1) receptors, whereas at recombinant P2X(2) receptors had an IC(50) value of 1.1 ?M. An ethyl phosphonate derivative (C(15)H(15)O(11)N(3)PS(2)Na(3)), whereas inactive at turkey erythrocyte P2Y(1) receptors, was particularly potent at recombinant P2X(2) receptors

Structure activity relationships for derivatives of adenosine-5?-triphosphate as agonists at P2 purinoceptors: Heterogeneity within P2x and P2y subtypes

The structure-activity relationships for a variety of adenine nucleotide analogues at P2x- and P2Y-purinoceptors were investigated. Compounds formed by structural modifications of the ATP molecule including substitutions of the purine ring (C2, C8, N1, and N6-substituents, and a uridine base instead of adenine), the ribose moiety (2′ and 3′-positions), and the triphosphate group (lower phosphates, bridging oxygen substitution, and cyclization) were prepared. Pharmacological activity at P2Y-purinoceptors was assayed in the guinea pig taenia coli, endothelial cells of the rabbit aorta, smooth muscle of the rabbit mesenteric artery, and turkey erythrocyte membranes. Activity at P2X-purinoceptors was assayed in the rabbit saphenous artery and the guinea-pig vas deferens and urinary bladder. Some of the analogues displayed selectivity, or even specificity, for either the P2X- or the P2Y-purinoceptors. Certain analogues displayed selectivity or specificity within the P2X- or P2Y-purinoceptor superfamilies, giving hints about possible subclasses. For example, 8-(6-aminohexylamino)ATP and 2′,3′-isopropylidene-AMP were selective for endothelial Pzypurinoceptors over P2Y-purinoceptors in the guinea pig taenia coli, rabbit aorta, and turkey erythrocytes. These compounds were both inactive at P2X-purinoceptors. The potent agonist N6-methyl ATP and the somewhat less potent agonist 2′-deoxy-ATP were selective for P2Y-purinoceptors in the guinea pig taenia coli, but were inactive at P2X-purinoceptors and the vascular P2Y-purinoceptors. 3′-Benzylamino-3′-deoxyATP was very potent at the P2X-purinoceptors in the guinea pig vas deferens and bladder, but not in the rabbit saphenous artery and was inactive at P2Y receptors. These data suggest that specific compounds can be developed that can be utilized to activate putative subtypes of the P2X- and P2Y-purinoceptor classes

Identification of potent, selective P2Y-purinoceptor agonists: structure-activity relationships for 2-thioether derivatives of adenosine 5'-triphosphate

Study of P2-purinoceptor subtypes has been difficult due to the lack of potent and selective ligands. With the goal of developing high affinity P2-purinoceptor-selective agonists, we have synthesized a series of analogues of adenine nucleotides modified on the purine ring as chain-extended 2-thioethers or as N6-methyl-substituted compounds. Chemical functionality incorporated in the thioether moiety included cyanoalkyl, nitroaromatic, amino, thiol, cycloalkyl, n-alkyl, and olefinic groups. Apparent affinity of the compounds for P2Y-purinoceptors was established by measurement of P2Y-purinoceptor-promoted phospholipase C activity in turkey erythrocyte membranes and relaxation of carbachol-contracted smooth muscle in three different preparations (guinea pig taenia coil, rabbit aorta, and rabbit mesenteric artery). Activity at P2X-purinoceptors was established by measurement of contraction of rabbit saphenous artery and of the guinea pig vas deferens and urinary bladder. All 11 of the 2-thioethers of ATP stimulated the production of inositol phosphates with K0.5 values of 1.5–770 nM, with an (aminophenyl)ethyl derivative being most potent. Two adenosine diphosphate analogues were equipotent to the corresponding ATP analogues. Adenosine monophosphate analogues were full agonists, although generally 4 orders of magnitude less potent. ATP 2-thioethers displayed pD2 values in the range of 6–8 in smooth muscle assay systems for activity at P2Y-receptors. There was a significant correlation for the 2-thioether compounds between the pK0.5 values for inositol phosphate production and the pD2 values for relaxation mediated via the P2Y-purinoceptors in the guinea pig taenia coli, but not for the vascular P2Y-receptors or for the P2X-receptors. At P2X-receptors, no activity was observed in the rabbit saphenous artery, but variable degrees of activity were observed in the guinea pig vas deferens and bladder depending on distal substituents of the thioether moiety. N6-Methyl-ATP was inactive at P2X-receptors, and approximately equipotent to ATP at taenia coli P2Y-receptors. This suggested that hybrid N6-methyl and 2-thioether ATP derivatives might be potent and selective for certain P2Y-receptors, as was shown for one such derivative, N6-methyl-2-(5-hexenylthio)-ATP

Synthesis and structure-activity relationships of pyridoxal-6-arylazo-5?-phosphate and phosphonate derivatives as P2 receptor antagonists

Novel analogs of the P2 receptor antagonist pyridoxal-5′-phosphate-6-phenylazo-2′,4′-disulfonate (PPADS) were synthesized. Modifications were made through functional group substitution on the sulfophenyl ring and at the phosphate moiety through the inclusion of phosphonates, demonstrating that a phosphate linkage is not required for P2 receptor antagonism. Substituted 6-phenylazo and 6-naphthylazo derivatives were also evaluated. Among the 6-phenylazo derivatives, 5′-methyl, ethyl, propyl, vinyl, and allyl phosphonates were included. The compounds were tested as antagonists at turkey erythrocyte and guinea-pig taenia coli P2Y1 receptors, in guinea-pig vas deferens and bladder P2X1 receptors, and in ion flux experiments by using recombinant rat P2X2 receptors expressed in Xenopus oocytes. Competitive binding assay at human P2X1 receptors in differentiated HL-60 cell membranes was carried out by using [35S]ATP-γ-S. A 2′-chloro-5′-sulfo analog of PPADS (C14H12O9N3ClPSNa), a vinyl phosphonate derivative (C15H12O11N3PS2Na3), and a naphthylazo derivative (C18H14O12N3PS2Na2), were particularly potent in binding to human P2X1 receptors. The potencies of phosphate derivatives at P2Y1 receptors were generally similar to PPADS itself, except for the p-carboxyphenylazo phosphate derivative C15H13O8N3PNa and its m-chloro analog C15H12O8N3ClPNa, which were selective for P2X vs. P2Y1 receptors. C15H12O8N3ClPNa was very potent at rat P2X2 receptors with an IC50 value of 0.82 μM. Among the phosphonate derivatives, [4-formyl-3-hydroxy-2-methyl-6-(2-chloro-5-sulfonylphenylazo)-pyrid-5- yl] methylphosphonic acid (C14H12-O8N3ClPSNa) showed high potency at P2Y1 receptors with an IC50 of 7.23 μM. The corresponding 2,5-disulfonylphenyl derivative was nearly inactive at turkey erythrocyte P2Y1 receptors, whereas at recombinant P2X2 receptors had an IC50 value of 1.1 μM. An ethyl phosphonate derivative (C15H15O11N3PS2Na3), whereas inactive at turkey erythrocyte P2Y1 receptors, was particularly potent at recombinant P2X2 receptors