Discovery of {4-[4,9-bis(ethyloxy)-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl]-2-fluorophenyl}acetic acid (GSK726701A), a novel EP4 receptor partial agonist for the treatment of pain.

A novel series of EP4 agonists and antagonists have been identified, and then used to validate their potential in the treatment of inflammatory pain. This paper describes these novel ligands and their activity within a number of pre-clinical models of pain, ultimately leading to the identification of the EP4 partial agonist GSK726701A.

A novel series of benzimidazole NR2B-selective NMDA receptor antagonists.

A series of novel benzimidazoles are discussed as NR2B-selective N-methyl-d-aspartate (NMDA) receptor antagonists. High throughput screening (HTS) efforts identified a number of potent and selective NR2B antagonists such as 1. Exploration of the substituents around the core of this template identified a number of compounds with high potency for NR2B (pIC(50) >7) and good selectivity against the NR2A subunit (pIC(50) <4.3) as defined by FLIPR-Ca(2+) and radioligand binding studies. These agents offer potential for the development of therapeutics for a range of nervous system disorders including chronic pain, neurodegeneration, migraine and major depression.

Synthesis and biological activity of a series of tetrasubstituted-imidazoles as P2X(7) antagonists.

A series of analogues of the pyrazole lead 1 were synthesized in which the heterocyclic core was replaced with an imidazole. A number of potent antagonists were identified and structure-activity relationships (SAR) were investigated both with respect to activity at the P2X(7) receptor and in vitro metabolic stability. Compound 10 was identified as a potent P2X(7) antagonist with reduced in vitro metabolism and high solubility.

Structure-activity relationships and in vivo activity of (1H-pyrazol-4-yl)acetamide antagonists of the P2X(7) receptor.

Structure-activity relationships (SAR) of analogues of lead compound 1 were investigated and compound 16 was selected for further study in animal models of pain. Compound 16 was shown to be a potent antihyperalgesic agent in both the rat acute complete Freund's adjuvant (CFA) model of inflammatory pain [Iadarola, M. J.; Douglass, J.; Civelli, O.; Naranjo, J. R. rain Res.1988, 455, 205] and the knee joint model of chronic inflammatory pain [Wilson, A. W.; Medhurst, S. J.; Dixon, C. I.; Bontoft, N. C.; Winyard, L. A.; Brackenborough, K. T.; De Alba, J.; Clarke, C. J.; Gunthorpe, M. J.; Hicks, G. A.; Bountra, C.; McQueen, D. S.; Chessell, I. P. Eur. J. Pain2006, 10, 537].

Synthesis and structure-activity relationships of a series of (1H-pyrazol-4-yl)acetamide antagonists of the P2X7 receptor.

High-throughput screening identified compound 1 as a potent P2X(7) receptor antagonist suitable for lead optimisation. Structure-activity relationships (SAR) of a series of (1H-pyrazol-4-yl)acetamides were investigated and compound 32 was identified as a potent P2X(7) antagonist with enhanced potency and favourable physicochemical and pharmacokinetic properties.

Identification of 2-oxo-N-(phenylmethyl)-4-imidazolidinecarboxamide antagonists of the P2X(7) receptor.

A backup molecule to compound 2 was sought by targeting the most likely metabolically vulnerable site in this molecule. Compound 18 was subsequently identified as a potent P2X(7) antagonist with very low in vivo clearance and high oral bioavailability in all species examined. Some evidence to support the role of P2X(7) in the etiology of pain is also presented.

Discovery and structure-activity relationships of a series of pyroglutamic acid amide antagonists of the P2X7 receptor.

A computational lead-hopping exercise identified compound 4 as a structurally distinct P2X(7) receptor antagonist. Structure-activity relationships (SAR) of a series of pyroglutamic acid amide analogues of 4 were investigated and compound 31 was identified as a potent P2X(7) antagonist with excellent in vivo activity in animal models of pain, and a profile suitable for progression to clinical studies.

Decavanadate, a P2X receptor antagonist, and its use to study ligand interactions with P2X7 receptors.

In this study we have studied decavanadate effects at P2X receptors. Decavanadate competitively blocked 2'- and 3'-O-(4benzoylbenzoyl) ATP (BzATP) stimulated ethidium accumulation in HEK293 cells expressing human recombinant P2X7 receptors (pK(B) 7.5). The effects of decavanadate were rapid (minutes) in both onset and offset and contrasted with the much slower kinetics of pyridoxal 5-phosphate (P5P), Coomassie brilliant blue (CBB) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62). Decavanadate competitively blocked the slowly reversible, or irreversible, blockade of the P2X7 receptor produced by P5P and oxidised ATP suggesting competition for a common binding site. However, the interaction between decavanadate and KN62 was non-competitive. Decavanadate also blocked P2X2 and P2X4 receptors but with slightly lower potency. These data demonstrate that decavanadate is the first reversible and competitive antagonist of the P2X7 receptor and is a useful tool for studying the mechanism of interaction of ligands with the P2X7 receptor.

Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain.

The P2X(7) purinoceptor is a ligand-gated cation channel, expressed predominantly by cells of immune origin, with a unique phenotype which includes release of biologically active inflammatory cytokine, interleukin (IL)-1beta following activation, and unique ion channel biophysics observed only in this receptor family. Here we demonstrate that in mice lacking this receptor, inflammatory (in an adjuvant-induced model) and neuropathic (in a partial nerve ligation model) hypersensitivity is completely absent to both mechanical and thermal stimuli, whilst normal nociceptive processing is preserved. The knockout animals were unimpaired in their ability to produce mRNA for pro-IL-1beta, and cytometric analysis of paw and systemic cytokines from knockout and wild-type animals following adjuvant insult suggests a selective effect of the gene deletion on release of IL-1beta and IL-10, with systemic reductions in adjuvant-induced increases in IL-6 and MCP-1. In addition, we show that this receptor is upregulated in human dorsal root ganglia and injured nerves obtained from chronic neuropathic pain patients. We hypothesise that the P2X(7) receptor, via regulation of mature IL-1beta production, plays a common upstream transductional role in the development of pain of neuropathic and inflammatory origin. Drugs which block this target may have the potential to deliver broad-spectrum analgesia.

Mechanism of action of species-selective P2X(7) receptor antagonists.

BACKGROUND AND PURPOSE: AZ11645373 and N-{2-methyl-5-[(1R, 5S)-9-oxa-3,7-diazabicyclo[3.3.1]non-3-ylcarbonyl]phenyl}-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide hydrochloride (compound-22) are recently described P2X(7) receptor antagonists. In this study we have further characterized these compounds to determine their mechanism of action and interaction with other species orthologues. EXPERIMENTAL APPROACH: Antagonist effects at recombinant and chimeric P2X(7) receptors were assessed by ethidium accumulation and radioligand-binding studies. KEY RESULTS: AZ11645373 and compound-22 were confirmed as selective non-competitive antagonists of human or rat P2X(7) receptors respectively. Both compounds were weak antagonists of the mouse and guinea-pig P2X(7) receptors and, for each compound, their potency estimates at human and dog P2X(7) receptors were similar. The potency of compound-22 was moderately temperature-dependent while that of AZ11645373 was not. The antagonist effects of both compounds were slowly reversible and were not prevented by decavanadate, suggesting that they were allosteric antagonists. Indeed, the compounds competed for binding sites labelled by an allosteric radio-labelled P2X(7) receptor antagonist. The species selectivity of AZ11645373, but not compound-22, was influenced by the nature of the amino acid at position 95 of the P2X(7) receptor. N(2)-(3,4-difluorophenyl)-N(1)-[2-methyl-5-(1-piperazinylmethyl)phenyl]glycinamide dihydrochloride, a positive allosteric modulator of the rat receptor, reduced the potency of compound-22 at the rat receptor but had little effect on the actions of AZ11645373. CONCLUSIONS: AZ11645373 and compound-22 are allosteric antagonists of human and rat P2X(7) receptors respectively. The differential interaction of the two compounds with the receptor suggests there may be more than one allosteric regulatory site on the P2X(7) receptor at which antagonists can bind and affect receptor function.

Discovery of novel, non-acidic 1,5-biaryl pyrrole EP1 receptor antagonists.

Replacement of the carboxylic acid group in a series of previously described 1,5-biaryl pyrrole EP1 receptor antagonists led to the discovery of various novel non-acidic antagonists. Several analogues displayed high binding affinity and high binding efficiency indices.

1,5-Biaryl pyrrole derivatives as EP1 receptor antagonists: Structure-activity relationships of 4- and 5-substituted benzoic acid derivatives.

This paper details the SAR of 1,5-biaryl pyrrole derivatives with substituents in the 2-, 4-, and 5-positions of the benzoic acid group as EP1 receptor antagonists. Substitution at the 2-position was poorly tolerated, whereas only fluorine was tolerated at the 4-position. In contrast, a range of substituents at the 5-position were discovered which enhanced the in vitro affinity and led to compounds with promising oral exposure. Three derivatives showed efficacy in a preclinical model of inflammatory pain when dosed orally to rats.

1,5-Biaryl pyrrole derivatives as EP1 receptor antagonists. Structure-activity relationships of 6-substituted and 5,6-disubstituted benzoic acid derivatives.

Herein we describe the SAR of 1,5-biaryl pyrrole derivatives, with substituents in the 6-position of the benzoic acid moiety, as EP(1) receptor antagonists. Substitution at this position was well tolerated and led to the identification of several analogues with high affinity for the EP(1) receptor that displayed good efficacy in the established FCA model of inflammatory pain. Furthermore, several analogues were prepared which combined substitution at the 5- and 6-positions as well as derivatives with an aromatic ring fused to the 5- and 6-positions.

The discovery of 6-[2-(5-chloro-2-{[(2,4-difluorophenyl)methyl]oxy}phenyl)-1-cyclopenten-1-yl]-2-pyridinecarboxylic acid, GW848687X, a potent and selective prostaglandin EP1 receptor antagonist for the treatment of inflammatory pain.

The discovery of a series of selective EP1 receptor antagonists based on a 1,2-diarylcyclopentene template is described. After defining the structural requirements for EP1 potency and selectivity, heterocyclic rings were incorporated to reduce logD and improve in vitro pharmacokinetic properties. The 2,6-substituted pyridines and pyridazines gave an appropriate balance of potency, in vivo pharmacokinetic properties and a low potential for inhibiting a range of CYP450 enzymes. From this series, GW848687X was shown to have an excellent profile in models of inflammatory pain and was selected as a development candidate.

Identification of novel glycine sulfonamide antagonists for the EP1 receptor.

A high-throughput screen targeting the EP(1) receptor identified non-acidic glycine sulfonamide derivative 2a with a pK(i) of 6.2. Analogue synthesis allowed a thorough investigation of the structure-activity relationship (SAR) and led to a 100-fold increase in recombinant potency.

Structure-activity relationships of 1,5-biaryl pyrroles as EP1 receptor antagonists.

The preliminary SAR of a series of novel 1,5-biaryl pyrrole EP1 receptor antagonists derived from compound 1 is described. Replacement of the benzyl group of 1 with isosteric groups was investigated. The most effective replacement was found to be the isobutyl group. The cyclopentylmethyl and cyclohexylmethyl groups were also effective benzyl replacements. The cyclohexylmethyl derivative 19 demonstrated the lowest metabolic clearance within this series. In addition, several high affinity substituted benzyl analogues were also identified. Compound 39 was found to have good bioavailability in rats and demonstrated efficacy in the established FCA preclinical model of inflammatory pain with a calculated ED50 of 9.2mg/kg.

Discovery of novel biaryl heterocyclic EP1 receptor antagonists.

We describe the generation of novel EP(1) receptor antagonists by investigation of thiophene isosteres. In addition, we disclose preliminary in vitro and in vivo DMPK for selected compounds.

Characterization and channel coupling of the P2Y(12) nucleotide receptor of brain capillary endothelial cells.

Rat brain capillary endothelial (B10) cells express an unidentified nucleotide receptor linked to adenylyl cyclase inhibition. We show that this receptor in B10 cells is identical in sequence to the P2Y(12) ADP receptor ("P2Y(T)") of platelets. When expressed heterologously, 2-methylthio-ADP (2-MeSADP; EC(50), 2 nm), ADP, and adenosine 5'-O-(2-thio)diphosphate were agonists of cAMP decrease, and 2-propylthio-D-beta,gamma-difluoromethylene-ATP was a competitive antagonist (K(B), 28 nm), as in platelets. However, 2-methylthio-ATP (2-MeSATP) (EC(50), 0.4 nm), ATP (1.9 microm), and 2-chloro-ATP (190 nm), antagonists in the platelet, were also agonists. 2-MeSADP activated (EC(50), 0.1 nm) GIRK1/GIRK2 inward rectifier K(+) channels when co-expressed with P2Y(12) receptors in sympathetic neurons. Surprisingly, P2Y(1) receptors expressed likewise gave that response; however, a full inactivation followed, absent with P2Y(12) receptors. A new P2Y(12)-mediated transduction was found, the closing of native N-type Ca(2+) channels; again both 2-MeSATP and 2-MeSADP are agonists (EC(50), 0.04 and 0.1 nm, respectively). That action, like their cAMP response, was pertussis toxin-sensitive. The Ca(2+) channel inhibition and K(+) channel activation are mediated by beta gamma subunit release from a heterotrimeric G-protein. G alpha subunit types in B10 cells were also identified. The presence in the brain capillary endothelial cell of the P2Y(12) receptor is a significant extension of its functional range.

AICA riboside both activates AMP-activated protein kinase and competes with adenosine for the nucleoside transporter in the CA1 region of the rat hippocampus.

5-Aminoimidazole-4-carboxamide riboside (AICA riboside; Acadesine) activates AMP-activated protein kinase (AMPK) in intact cells, and is reported to exert protective effects in the mammalian CNS. In rat cerebrocortical brain slices, AMPK was activated by metabolic stress (ischaemia > hypoxia > aglycaemia) and AICA riboside (0.1-10 mm). Activation of AMPK by AICA riboside was greatly attenuated by inhibitors of equilibrative nucleoside transport. AICA riboside also depressed excitatory synaptic transmission in area CA1 of the rat hippocampus, which was prevented by an adenosine A1 receptor antagonist and reversed by application of adenosine deaminase. However, AICA riboside was neither a substrate for adenosine deaminase nor an agonist at adenosine receptors. We conclude that metabolic stress and AICA riboside both stimulate AMPK activity in mammalian brain, but that AICA riboside has an additional effect, i.e. competition with adenosine for uptake by the nucleoside transporter. This results in an increase in extracellular adenosine and subsequent activation of adenosine receptors. Neuroprotection by AICA riboside could be mediated by this mechanism as well as, or instead of, by AMPK activation. Caution should therefore be exercised in ascribing an effect of AICA riboside to AMPK activation, especially in systems where inhibition of adenosine re-uptake has physiological consequences.