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A series of new analogues of the arecaidine propargyl ester (CAS 35516-99-5, APE, 1a) with alcohols consisting of 4 or 5 carbon atoms were investigated at muscarinic receptor subtypes. The muscarinic activity of the quaternary and tertiary salts of the APE-related compounds were assayed on the isolated guinea-pig ileum (M 3 receptor subtype) and guinea-pig left atria (M 2 receptor subtype) as well as on rabbit isolated vas deferens (M 1 receptor subtype). The structural variations made in the APE molecule, replacing the triple bond in the ester side chain with structures such as double bond, an allene moiety, a single bond, a cyclopropyl group or two triple bonds should alter the selectivity and potency in favour of the M 2 subtype. Enhanced, though modest, selectivity for M 2 receptors was achieved with the 2-butynyl ester 2a. The other structural variations resulted in a loss of potency, but not necessarily of efficacy.

Extracellular adenine and uracil 5'-nucleotides are important signalling molecules that exert a great variety of effects in numerous tissues and cell types through the activation of P2 receptors. In the past eight years, an extended series of P2 receptors (P2X(17), ionotropic subunits; P2Y(1,2,4,6,11,12), metabotropic receptors) has been cloned from vertebrate tissues. In this rapidly expanding field, one of the main current challenges is to relate the cloned P2 receptor subtypes to the diverse physiological responses mediated by the pharmacological phenotypes of native P2 receptors. Unfortunately, subtype-selective P2 ligands, especially potent and selective antagonists, have been only slowly forthcoming, and this acts as a considerable impediment to progress. However, a number of new P2 receptor antagonists have recently been described which to some degree are more potent and more selective than earlier antagonists like suramin or pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). This work moves us closer to the ideal goal of classifying the recombinant and native P2 receptor subtypes on the basis of antagonist profiles. This review begins with a brief account of the current status of P2 receptors and their ligands. It then focuses on structure-activity relationships of PPADS and suramin analogues and will finish with a brief discussion of some related therapeutic possibilities.

The present study was designed to determine to determine the in vitro affinity profile of (R)-(-)-dimethindene and (S)-(+)-dimethindene at muscarinic receptor subtypes using both functional and binding assays. In addition, the racemate was investigated in functional studies. The functional muscarinic receptors studied were putative M1 receptors in rabbit vas deferens and rat duodenum, M2 receptors in guinea-pig left atria and rabbit vas deferens, as well as M3 receptors in guinea-pig ileum and trachea. Furthermore, the histamine H1 antagonism by (R)-(-)- and (S)-(+)-dimethindene has been examined in guinea-pig ileum. Muscarinic binding selectivity was assessed in homogenates from human neuroblastoma NB-OF 1 cells (M1), rat heart (M2), pancreas (3) and striatum (M4). The results demonstrate that (S)-(+)-dimethindene is a potent M2-selective muscarinic receptor antagonist (pA2 = 7.86/7.74; pKi = 7.78) with lower affinities for the muscarinic M1 (pA2 = 6.83/6.36; pKi = 7.08), the M3 (pA2 = 6.92/6.96; pKi = 6.70) and the M4 receptors (pKi = 7.00), respectively. The (S)-(+)-enantiomer was more potent (up to 41-fold) than the (R)-(-)-enantiomer in all muscarinic assays. In contrast, the stereoselectivity was inverse at histamine H1 receptors, the (R)-(-)-enantiomer being the eutomer (pA2 = 9.42; pA2/(S)-isomer = 7.48). In conclusion, (S)-(+)-dimethindene is a useful tool to investigate muscarinic receptor heterogeneity. In addition, this lipophilic compound might become the starting point for the development of M2-selective muscarinic receptor antagonists useful as diagnostic tools for quantifying muscarinic M2 receptors in the central nervous system with positron emission tomography imaging, and to test the hypothesis that muscarinic M2 receptor antagonists show beneficial effects in the treatment of cognitive disorders.

The muscarinic pharmacology of C1-methyl-substituted chiral compounds related to McN-A-343 and of (R)- and (S)-dimethindene has been studied. Among the McN-A-343 analogues, the (S)-enantiomers were more potent and had higher affinity than the (R)-isomers. The quaternary compound (S)-BN 228 was found to be the most potent M1-selective agonist known today (pEC50: M1/rabbit vas deferens = 7.83; M2/guinea-pig atria = 6.35; M3/guinea-pig ileum = 6.29). In both the atria and ileum the tertiary carbamate, (S)-4-F-MePyMcN, was a competitive antagonist (pA2 value = 7.39 and 6.82, respectively). In contrast, in rabbit vas deferens (S)-4-F-MePyMcN was a potent partial agonist (pEC50 = 7.22; apparent efficacy = 0.83). These results indicate that (S)-4-F-MePyMcN might be a useful tool to study M1 receptor-mediated effects involved in central cholinergic function. (S)-Dimethindene was a potent M2-selective antagonist (pA2 = 7.86/atria; pKi = 7.8/rat heart) with lower affinities for the M1 (pA2 = 6.36/rat duodenum; pKi = 7.1/NB-OK 1 cells), M3 (pA2 = 6.92/guinea-pig ileum; pKi = 6.7/rat pancreas) and M4 receptors (pKi = 7.0/rat striatum). It was more potent (up to 41-fold) than the (R)-isomer. In contrast, the stereoselectivity was inverse at ileal H1 receptors (pA2: (R)-isomer = 9.42; (S)-isomer = 7.48). Thus, (S)-dimethindene could be a valuable agent to test the hypothesis that M2 antagonists show beneficial effects in the treatment of cognitive disorders. It might also become the starting point for the development of diagnostic tools for quantifying M2 receptors in the CNS with PET imaging.

Pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonat e) (PPNDS) potently antagonized P2X(1) receptor-mediated responses in rat vas deferens (pK(B)=7.43) and Xenopus laevis oocytes (pIC(50)=7. 84). It showed lower (up to 20,000-fold) inhibitory potency on ecto-nucleotidase in Xenopus oocytes and on P2Y(1) receptors in guinea-pig ileum (pA(2)=6.13). PPNDS did not interact with alpha(1A)-adrenoceptors, adenosine A(1) and A(2B), histamine H(1) and muscarinic M(3) receptors. Thus, PPNDS is a novel, specific P2 receptor antagonist and represents the pyridoxal-5'-phosphate derivative with the highest potency at P2X(1) receptors.

The role of ATP-stimulated P2X1 receptors in human platelets is still unclear. They may act alone or in synergy with other pathways, such as P2Y1 or P2Y12 receptors, to accelerate and enhance calcium mobilisation, shape change and aggregation. To date very few pharmacological means of selectively inhibiting platelet P2X1 receptors have been described, although recent work has shown that suramin is a useful lead compound for the development of high-affinity P2X1 antagonists. We therefore investigated the effects of a series of bivalent and tetravalent suramin analogues on alphabeta meATP (P2X1 receptors)-induced or ADP (P2Y1 receptors)-induced intracellular calcium increases and shape change, as well as on ADP-induced aggregation (P2Y1P2Y12 receptors) in human platelets. Changes in intracellular calcium were measured using standard fluorescence techniques, while shape change and aggregation were determined by turbidimetry. The novel tetravalent compound NF864 (8,8',8'',8'''-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-naphthalene-1,3,5-trisulfonic acid-dodecasodium salt) proved to be the most potent platelet P2X1 antagonist reported to date, blocking alphabeta meATP-induced Ca2+ increases and shape change in a concentration-dependent manner, with a pA2 of 8.17 and 8.49, respectively. The ability to inhibit the platelet P2X1 receptor displayed the following order : NF864NF449or = NF110NF023 = MK-HU1 = suramin. A different antagonistic profile was observed for ADP-induced Ca2+ increases, shape change and aggregation; however, overall four compounds showed sufficient ability to selectively inhibit P2X1 responses, with the order NF110NF449or = NF864or = MK-HU1. Therefore, these compounds should prove useful tools for investigating the functional significance of platelet P2X1 receptors in thrombosis and haemostasis, NF864 being the most promising compound.

Antagonistic effects of the novel suramin analogue 4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alphabetameATP; mediated by P2X1 receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by alphabetameATP (mediated by P2X3 receptors) or adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y1 receptors), ATP-induced increases of [Ca2+]i in human embryonic kidney (HEK) 293 cells (mediated by P2Y2 receptors), inward currents evoked by ATP in follicle cell-free Xenopus laevis oocytes expressing rP2X1 or rP2X3 receptors and degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. In addition, NF449 was examined for its P2 receptor specificity in rat vas deferens (alpha1A-adrenoceptors) and guinea-pig ileum (histamine H1 and muscarinic M3 receptors). At native (pIC50=7.15) and recombinant (pIC50=9.54) P2X1 receptors, NF449 was a highly potent antagonist. The P2X3 receptors present in guinea-pig ileum (pIC50=5.04) or expressed in oocytes (pIC50 approximately 5.6) were much less sensitive for NF449. It also was a very weak antagonist at P2Y1 receptors in guinea-pig ileum (pIC50=4.85) and P2Y2 receptors in HEK 293 cells (pIC50=3.86), and showed very low inhibitory potency on ecto-nucleotidases (pIC503.5). NF449 (100 microM) did not interact with alpha1A-adrenoceptors or histamine H1 and muscarinic M3 receptors. Thus, the antagonism by NF449 is highly specific for P2 receptors. In conclusion, the subnanomolar potency at rP2X1 receptors and the rank order of potency, P2X1P2X3P2Y1P2Y2ecto-nucleotidases, make NF449 unique among the P2 receptor antagonists reported to date. NF449 may fill the long-standing need for a P2X1-selective radioligand.

We have characterized PPADS (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid) as a novel antagonist which selectively blocks P2 purinoceptor-mediated responses in rabbit vas deferens at pre- and postjunctional sites. PPADS did not interact with alpha 1-adrenoceptors, muscarinic M2 and M3 receptors, histamine H1 and adenosine A1 receptors. Thus, PPADS is a novel and useful pharmacological tool to study co-transmission in tissues where ATP and co-existing neurotransmitters act in concert.

Effects of 6-[(4,6,8-trisulfo-1-naphthyl)iminocarbonyl-1, 3-(4-methylphenylene)iminocarbonyl-1, 3-phenylene-azo]-pyridoxal-5'-phosphate (SB9), a heterodimeric bivalent ligand consisting of pyridoxal-5'-phosphate and the suramin monomer, were studied on contractions of the rat vas deferens elicited by alpha beta-methylene ATP (alpha beta meATP; mediated by P2X(1)-like receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by adenosine 5'-O-(2-thiodiphosphate) (ADP beta S mediated by P2Y(1)-like receptors), and the degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. SB9 (0.1-10 microM) antagonized contractile responses produced by alpha beta meATP or ADP beta S in a concentration-dependent manner. Schild analysis yielded linear regression lines of unit slope, indicating competitive antagonism. From the rightward shifts of the agonist concentration-response curves pA(2) values of 6.05+/-0.13 (vas deferens) and 6.98+/-0.07 (ileum) were derived. In both preparations, SB9 behaved as a slow onset, slow offset antagonist. Incubation of three oocytes in the presence of ATP produced an increase in inorganic phosphate (P(i)) over a 30-min period, which amounted to 35.1+/-1.9 microM P(i) from 100 microM ATP. SB9 (10-1000 microM) reduced this degradation (pIC(50)=4.33+/-0.10). The results illustrate that SB9 is a high-affinity P2Y(1) receptor antagonist with a remarkable selectivity for P2Y(1) vs. P2X(1) receptors (about 10-fold) and ecto-nucleotidases (447-fold). These properties make it unique among the pyridoxal-5'-phosphate and suramin derivatives reported to date.

The muscarinic pharmacology of two novel agonists related to McN-A-343, 4-F-PyMcN and 4-F-PyMcN+, has been studied by the use of pharmacological and radioligand binding techniques. Both compounds were potent agonists at M1 receptors in rabbit vas deferens (pEC50 = 6.24 and 6.96) and rat duodenum (pEC50 = 5.47 and 6.38), but very weak partial agonists or competitive antagonists at guinea-pig cardiac M2 and ileal M3 receptors. There was no receptor reserve for 4-F-PyMcN in rabbit vas deferens, for which the potency (pEC50 = 6.24) and apparent affinity (pKA = 5.99 and 6.21) were similar. 4-F-PyMcN+ showed only limited binding selectivity between four muscarinic receptor binding assays with apparent affinity constants (pKi) of 5.8, 5.2, 5.6 and 5.7 for M1, M2, M3 and M4 muscarinic receptor subtypes. The two novel functionally M1-selective agonists may provide useful tools with which to study muscarinic receptor mechanisms. The non-quaternary compound, 4-F-PyMcN, might become a starting point for the development of drugs that selectively affect M1 receptors involved in central cholinergic function.