Martyn C. Coldwell

1,3-Biarylureas as Selective Non-peptide Antagonists of the Orexin-1 Receptor

This communication reports SARs for the first orexin-1 receptor antagonist series of 1-aryl-3-quinolin-4-yl and 1-aryl-3-naphthyridin-4-yl ureas. One of these compounds, 31 (SB-334867), has excellent selectivity for the orexin-1 receptor, blood-brain barrier permeability and shows in vivo activity following ip dosing.

Pharmacological characterisation of human 5-HT2 receptor subtypes.

Prompted by conflicting literature, this study compared the pharmacology of human 5-hydroxytryptamine2 (5-HT2) receptors expressed in SH-SY5Y cells using a fluorometric imaging plate reader (FLIPR) based Ca2+ assay. 5-Hydroxytryptamine (5-HT) increased intracellular calcium concentration ([Ca2+]i) at 5-HT2A, 5-HT2B and 5-HT2C receptors (pEC(50)=7.73+/-0.03, 8.86+/-0.04 and 7.99+/-0.04, respectively) and these responses were inhibited by mesulergine (pKB=7.42+/-0.06, 8.77+/-0.10 and 9.52+/-0.11). A range of selective agonists and antagonists displayed the expected pharmacology at each receptor subtype. Sodium butyrate pretreatment increased receptor expression in SH-SY5Y/5-HT2B (15-fold) and SH-SY5Y/5-HT2C cells (7-fold) and increased agonist potencies and relative efficacies. In contrast, sodium butyrate pretreatment of SH-SY5Y/5-HT(2A) cells did not affect receptor expression. The present study provides a direct comparison of agonist and antagonist pharmacology at 5-HT(2) receptor subtypes in a homogenous system and confirms that agonist potency and efficacy varies with the level of receptor expression.

Comparison of the functional potencies of ropinirole and other dopamine receptor agonists at human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells

The aim of the present study was to characterize functional responses to ropinirole, its major metabolites in man (SKF‐104557 (4‐[2‐(propylamino)ethyl]‐2‐(3H) indolone), SKF‐97930 (4‐carboxy‐2‐(3H) indolone)) and other dopamine receptor agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) receptors separately expressed in Chinese hamster ovary cells using microphysiometry. All the receptor agonists tested (ropinirole, SKF‐104557, SKF‐97930, bromocriptine, lisuride, pergolide, pramipexole, talipexole, dopamine) increased extracellular acidification rate in Chinese hamster ovary clones expressing the human D2, D3 or D4 receptor. The pEC50s of ropinirole at hD2, hD3 and hD4 receptors were 7.4, 8.4 and 6.8, respectively. Ropinirole is therefore at least 10 fold selective for the human dopamine D3 receptor over the other D2 receptor family members. At the hD2 and hD3 dopamine receptors all the compounds tested were full agonists as compared to quinpirole. Talipexole and the ropinirole metabolite, SKF‐104557, were partial agonists at the hD4 receptor. Bromocriptine and lisuride had a slow onset of agonist action which precluded determination of EC50s. The rank order of agonist potencies was dissimilar to the rank order of radioligand binding affinities at each of the dopamine receptor subtypes. Functional selectivities of the dopamine receptor agonists, as measured in the microphysiometer, were less than radioligand binding selectivities. The results show that ropinirole is a full agonist at human D2, D3 and D4 dopamine receptors. SKF‐104557 the major human metabolite of ropinirole, had similar radioligand binding affinities to, but lower functional potencies than, the parent compound.

Pharmacological characterization of extracellular acidification rate responses in human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells

This study characterized pharmacologically the functional responses to agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) zreceptors separately expressed in cloned cells using the cytosensor microphysiometer. Dopaminergic receptor agonists caused increases in extracellular acidification rate in adherent Chinese hamster ovary (CHO) clones expressing hD2, hD3 or hD4 receptors. Acidification rate responses to agonists in other cell lines expressing these receptors were smaller than those in adherent CHO cells. The time courses and maximum increases in acidification rate of the agonist responses in adherent CHO cells were different between the three dopamine receptor clones. Responses were blocked by pretreatment of cells with pertussis toxin or amiloride analogues. Most agonists had full intrinsic activity at each of the dopamine receptor subtypes, as compared to quinpirole, however both enantiomers of UH‐232 and (−)3‐PPP were partial agonists in this assay system. The functional potency of full agonists at each of the three receptors expressed in CHO cells was either higher than, or similar to, the apparent inhibition constants (Ki) determined in [125I]‐iodosulpride competition binding studies. Functional selectivities of the agonists were less than radioligand binding selectivities. The rank orders of agonist potencies and selectivities were similar, but not identical, to the rank orders of radioligand binding affinities and selectivities. The dopamine receptor antagonists, iodosulpride and clozapine, had no effect on basal acidification rates but inhibited acidification responses in CHO cells to quinpirole in an apparently competitive manner. Antagonist potencies closely matched their radioligand binding affinities in these cells.

Functional effects of the muscarinic receptor agonist, xanomeline, at 5-HT1 and 5-HT2 receptors

1 Xanomeline [3(3‐hexyloxy‐1,2,5‐thiadiazol‐4‐yl)‐1,2,5,6‐tetrahydro‐1‐methylpyridine] has been reported to act as a functionally selective muscarinic partial agonist with potential use in the treatment of Alzheimers disease. This study examined the functional activity of xanomeline at 5‐HT1 and 5‐HT2 receptors in native tissue and/or human cloned receptors. 2 Xanomeline had affinity for muscarinic receptors in rat cortical membranes where the ratio of the displacement affinity of [3H]‐Quinuclidinyl benzilate vs that of [3H]‐Oxotremorine‐M was 16, indicative of partial agonist activity. Radioligand binding studies on human cloned receptors confirmed that xanomeline had substantial affinity for M1, M2, M3, M4, M5 receptors and also for 5‐HT1 and 5‐HT2 receptor subtypes. 3 Carbachol and xanomeline stimulated basal [35S]‐GTPγS binding in rat cortical membranes with micromolar affinity. The response to carbachol was attenuated by himbacine and pirenzepine with pA2 of 8.2, 6.9 respectively consistent with the response being mediated, predominantly, via M2 and M4 receptors. Xanomeline‐induced stimulation of [35S]‐GTPγS binding was inhibited by himbacine with an apparent pKb of 6.3, was not attenuated by pirenzepine up to 3 μm and was inhibited by the selective 5‐HT1A antagonist WAY100635 with an apparent pKb of 9.4. These data suggest the agonist effect of xanomeline in this tissue is, in part, via 5‐HT1A receptors. Similar studies on human cloned receptors confirmed that xanomeline is an agonist at human cloned 5‐HT1A and 5‐HT1B receptors. 4 In studies using the fluorescent cytoplasmic Ca2+ indicator FLUO‐3AM, xanomeline induced an increase in cytoplasmic Ca2+ concentration in SH‐SY5Y cells expressing recombinant human 5‐HT2C receptors. Atropine antagonized this response, consistent with mediation via endogenously‐expressed muscarinic receptors. In the presence of atropine, xanomeline antagonized 5‐HT‐induced cytoplasmic changes in Ca2+ concentration in cells expressing h5‐HT2A, h5‐HT2B and h5‐HT2C receptors with potencies similar to its affinity at these receptors. 5 These studies indicate that xanomeline is a potent agonist at 5‐HT1A and 5‐HT1B receptors and an antagonist at 5‐HT2 receptor subtypes.

N-(substituted-phenyl) piperazines: antagonists with high binding and functional selectivity for dopamine D4 receptors

Abstract A series of N-(substituted-phenyl) piperazine derivatives was prepared as selective antagonists of the dopamine D 4 receptor. Many analogues possessed a binding selectivity of over 100 fold for D 4 over D 2 receptors. In functional studies in the microphysiometer, compound 24 showed a selectivity over dopamine D 2 receptors of greater than 1000 fold.

A novel series of 2-aminotetralins with high affinity and selectivity for the dopamine D3 receptor

Abstract A novel series of N-[4-(4-Phenylbenzoylamino)butyl]-1,2,3,4-tetrahydro-2-naphthylamines with high affinity and selectivity for the dopamine D3 receptor has been prepared. The 5-cyclopropylmethyloxy 3j, methanesulfonyloxy 3k and trifluoromethanesulfonyloxy 3l derivatives represent some of the highest affinity (pKis 8.6–8.9) and most selective (200–320-fold) dopamine D3 receptor antagonists reported to date.

Novel 1,2,3,4-tetrahydroisoquinolines with high affinity and selectivity for the dopamine D3 receptor.

Using clearance and brain penetration studies as a screen, tetrahydroisoquinoline 3 was identified as a lead having low clearance in rats (CLb 20 ml/min/kg). Introduction of a 7-CF3SO2O- substituent into the tetrahydroisoquinoline, followed by replacement of the biphenylamido group of 3 by a 3-indolylpropenamido group gave 31, having high D3 receptor affinity (pKi 8.4) and 150 fold selectivity over the D2 receptor.

The synthesis, and dopamine D2 and serotonin 5-HT3 receptor affinity of 3-aza analogues (pyridyl) of 4-amino-5-chloro-2-methoxybenzamides

Abstract The synthesis of potential metabolically blocked 3-aza analogues (pyridyl) of 4-amino-5-chloro-2-methoxybenzamides is described. Retention of dopamine D2 and serotonin 5-HT3 receptor affinity is observed.

Site-specific splice variation of the human P2X4 receptor

P2X4 receptors are expressed in specific brain areas. We now describe site-specific splice variations of the human P2X4 receptor subunit, occurring at residue [YVIG / WVFV(W)] near the end of the first predicted transmembrane domain. p2X4(b) is formed by the insertion of an additional 16 amino acids. p2X4(C) is formed by deleting a cassette of 130 amino acids, including six of the 10 conserved extracellular cysteine residues. Transfection of P2X4(a), but not p2x4(c), formed functional channels in Xenopus oocytes and human 1321N1 cells. After transfection of p2X4(b) small, inconsistent ATP-evoked responses were detected only in the human cells, but when co-expressed, p2x4(b) may alter the function of P2X4(a) in oocytes. The distribution of splice variant RNA within human brain suggests regionally-dependent expression. These data indicate that the functions of the human P2X4 receptor may be altered by alternative splicing.