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 agonist, 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 coli, 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 coil 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.

1 Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), an inhibitor of P2x-purinoceptormediated responses in rabbit vas deferens, was investigated for its ability to antagonize contractions evoked by a,B-methylene ATP (a,P-MeATP), carbachol and electrical field stimulation in the rabbit urinary bladder detrusor muscle.2 PPADS (1-30 gM) caused concentration-dependent inhibition of contractions to the stable P2X-purinoceptor agonist, cx,,-MeATP, decreasing the maximum response to a,4-MeATP (30 JAM) at concentrations of 3-30 JiM. The pD2 value for a,4-MeATP in the absence of PPADS was 6.52 ± 0.10 (8). In the presence of PPADS at concentrations of 1, 3, 10 and 30 gM the negative log concentrations of a,4-MeATP that cause the same contractile response as the pD2 value were significantly different from control, being respectively 6.17 ± 0.09 (8), 5.64 ± 0.12 (7), 5.15 ± 0.23 (7) and 4.78 ± 0.22 (5). 3 PPADS (1-30IM) caused concentration-dependent inhibition of contractions to stimulation of intramural purinergic nerves (1 -32 Hz). There was a greater inhibition at lower frequencies (1-8 Hz)than at higher frequencies (16-32 Hz). PPADS, 30 JAM, did not produce significantly greater antagonism than 1OIJM. 4 PPADS (30 jAM) had no significant influence on the contractile potency of carbachol: the pD2 values of carbachol in the absence and presence of PPADS were not significantly different being 6.42 ± 0.16 (5) and 6.33 ± 0.18 (5), respectively. However, PPADS caused a small, but significant, suppression of the maximal response of carbachol, reducing it by approximately 9%. 6 Thus, PPADS (1-30 JM) antagonized responses mediated via P2X-purinoceptors in the rabbit urinary bladder. It was selective for P2-purinoceptor-mediated contractions rather than those mediated via muscarinic receptors. Binding studies demonstrated that the antagonistic effect of PPADS is via a direct interaction with P2x-purinoceptors.

The structure-activity relationships for a variety of adenine nucleotide analogues at P 2x -and P 2Y -purinoceptors were investigated. Compounds formed by structural modifications of the ATP molecule including substitutions of the purine ring (C2, C8, N1, and N 6 -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 P 2Y -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 P 2X -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 P 2X -or the P 2Y -purinoceptors. Certain analogues displayed selectivity or specificity within the P 2X -or P 2Y -purinoceptor superfamilies, giving hints about possible subclasses. For example, 8-(6-aminohexylamino)ATP and 2′,3′-isopropylidene-AMP were selective for endothelial Pzypurinoceptors over P 2Y -purinoceptors in the guinea pig taenia coli, rabbit aorta, and turkey erythrocytes. These compounds were both inactive at P 2X -purinoceptors. The potent agonist N 6 -methyl ATP and the somewhat less potent agonist 2′-deoxy-ATP were selective for P 2Y -purinoceptors in the guinea pig taenia coli, but were inactive at P 2X -purinoceptors and the vascular P 2Y -purinoceptors. 3′-Benzylamino-3′-deoxyATP was very potent at the P 2X -purinoceptors in the guinea pig vas deferens and bladder, but not in the rabbit saphenous artery and was inactive at P 2Y receptors. These data suggest that specific compounds can be developed that can be utilized to activate putative subtypes of the P 2X -and P 2Y -purinoceptor classes.

1 Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), a P2-purinoceptor antagonist, was investigated for its ability to antagonize: (1) P2x-purinoceptor-mediated contractions of the rabbit central ear artery and saphenous artery evoked by either a,p-methylene ATP (Qx,-MeATP) or electrical field stimulation (EFS); (2) P2Y-purinoceptor-mediated relaxations of the rabbit mesenteric artery; (3) endothelium-dependent and endothelium-independent, P2Y-purinoceptor-mediated relaxations of the rabbit aorta.2 a,4-MeATP (0.1-1001JM) caused concentration-dependent contractions of the rabbit ear and saphenous arteries. The negative log[a,4-MeATP] that produced a contraction equivalent to the EC25 for noradrenaline (ear artery) or histamine (saphenous artery) in the absence of PPADS was 6.60 ± 0.18 (9) and 6.18 ± 0.17 (9) in the ear artery and saphenous artery, respectively. These effects of exogenous a,P-MeATP were concentration-dependently inhibited by PPADS 4 PPADS (30 JM) had no effect on relaxations to 2-methylthio ATP (3 nM-3 JAM) in rabbit mesenteric artery and to ATP (1 JAM-I mM) in rabbit aorta (with endothelium intact or removed). In addition, PPADS (30 JM) had no significant influence on the contractile potency of noradrenaline and histamine in rabbit ear and saphenous artery, respectively. 5 In conclusion, these results support the evidence that PPADS is a selective antagonist of P2X-purinoceptor-mediated responses.

1 The effect of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on the relaxant response to adenine nucleotides was examined in the carbachol-contracted guinea-pig taenia coli and rat duodenum, two tissues possessing P2y-purinoceptors. In addition, in the taenia coli PPADS was investigated for its effect on relaxations evoked by adenosine, noradrenaline and electrical field stimulation. In order to assess the selectivity of PPADS between P2-purinoceptor blockade and ectonucleotidase activity, its influence on ATP degradation was studied in guinea-pig taenia coli. 2 The resulting rank order of potency for the adenine nucleotides in guinea-pig taenia coli was: 2-methylthio ATP> >ATP>a,,B-methylene ATP with the respective pD2-values 7.96 + 0.08 (n = 23), 6.27 ± 0.12 (n = 21) and 5.88 ± 0.04 (n = 24).3 In guinea-pig taenia coli, PPADS (10-100 yM) caused a consistent dextral shift of the concentrationresponse curve (CRC) of 2-methylthio ATP and ATP resulting in a biphasic Schild plot. A substantial shift was only observed at 100 JM PPADS, the respective pA2-values at this particular concentration were 5.26 ± 0.16 (n = 5) and 5.15 ± 0.13 (n = 6). Lower concentrations of PPADS (3-30 pM) antagonized the relaxant effects to a,fi-methylene ATP in a surmountable manner. An extensive shift of the CRC was produced only by 30 gM PPADS (pA2 = 5.97 ± 0.08, n = 6), and the Schild plot was again biphasic. 4 The relaxant responses to electrical field stimulation (80 V, 0.3 ms, 5 s, 0.5-16 Hz) in guinea-pig taenia coli were concentration-dependently inhibited by PPADS (10-100 uM).5 In guinea-pig taenia coli, the potency of ATP in inducing relaxation appeared to be independent of its rate of degradation by ecto-nucleotidases, since the Km-value (366 pM) obtained in the enzyme assay was much higher than the functional EC50-value (0.45 pM) of ATP. PPADS (3-100 gM) was only weakly active in inhibiting ecto-nucleotidase activity leaving a residual activity of 81.8 ± 5.1% at 100 pM.Enzyme inhibition by PPADS was concentration-independent and non-competitive. 6 In rat duodenum, the rank order of potency was: 2-methylthio ATP > ATP> >ca,,B-methylene ATP, the respective pD2-values being 6.98 ± 0.04 (n = 76), 6.26 ± 0.02 (n = 6) and 4.83 ± 0.02 (n = 6). Among these agonists, 2-methylthio ATP displayed the lowest apparent efficacy.7 The CRC of 2-methylthio ATP in rat duodenum was shifted to the right by PPADS (10-100 gM) in a concentration-dependent manner, and Schild analysis gave a pA2-value of 5.09 ± 0.06 (slope = 1.02, n=14). 8 PPADS was without any effect on the carbachol-induced contraction in guinea-pig taenia coli or rat duodenum and on the relaxation to noradrenaline or adenosine in guinea-pig taenia coli. 9 In conclusion, the antagonistic properties of PPADS at the taenia coli and rat duodenum P2y-purinoceptors were different from those recently described at the P2k-subtype: inhibition of P2y-purinoceptor-mediated responses was observed at higher concentrations (3-100 gM vs. 1-10 (30) gM). Furthermore, we conclude th...

Extracellular ATP can p r o d u c e various effects acting via P,-purinoceptors. ATP i s rapidly b r o k e n down b y ecto-ATPase a n d other ecto-enzymes that limit its effect. Further, adenosine, a metabolite of ATP breakdown, can p r o d u c e its own effect acting via P,-purinoceptors, sometimes masking the effects of ATP. An inhibitor o f ATP degradation would be a useful pharmacological tool to discriminate between effects of ATP a n d its metabolites, as well as to potentiate its actions. Diverse c o m p o u n d s that have b e e n claimed to b e inhibitors o f ATP-metabolising ectoenzymes are evaluated, but specific a n d selective Ca2+/Mg2+-dependent ecto-ATPase inhibitors still appear to b e lacking. o 1994 WiIey-Liss, Inc.

Ecto-ATPase activity of Xenopus oocytes was studied by measuring the production of inorganic phosphate (Pi) from the breakdown of extracellular ATP. Enzyme activity involved Ca2+/Mg(2+)-dependent and Ca2+/Mg(2+)-independent dephosphorylation of ATP. Ca2+/Mg(2+)-dependent ecto-ATPase was active over a limited range of 0.01-1.0 mM ATP, while Ca2+/Mg(2+)-independent ATPase activity was active over a range of 0.1-30 mM ATP. Total enzyme activity was insensitive to changes in buffer pH (pH 7.0-9.0), but increased in a relatively linear manner with: (1) time of reaction (0-90 min), (2) number of cells (1-20 oocytes), and (3) temperature (10-37 degrees C). Ecto-ATPase activity was unaffected by ouabain (100 microM), sodium azide (100 microM), and oligomycin (5 micrograms/ml) (as inhibitors of endo-ATPases) and beta-glycerophosphate (10 mM) and p-nitrophenyl phosphate (10 mM) (as inhibitors of non-specific alkaline phosphatase). Total ecto-ATPase activity was reduced significantly in defolliculated oocytes, suggesting that the enzyme was located mainly on the enveloping follicle cell layer. The range order of preferential substrates was: ATP>GTP, ITP, UTP, CTP, TTP, 2-methylthioATP>ADP, 2-methylthioADP, AMP>>alpha, beta-methylene ATP, beta, gamma-methylene ATP, in the presence of divalent ions (where G is guanosine, I is inosine, U is uridine, C is cytidine and T is ribosylthymine). The P2-purinoceptor antagonist suramin [8-(3-benzamido-4-methylbenzamido)napthalene-1,3,5-trisul phonic acid), 100 microM] significantly inhibited total ecto-ATPase activity; this inhibition was competitive for the Ca2+/Mg(2+)-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

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 (C14H12O8N3ClPSNa) 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. Drug Dev. Res. 45:52–66, 1998. Published 1998 Wiley‐Liss, Inc.