Synthesis and biological evaluation of novel hexahydro-pyrido[3',2':4,5]pyrrolo[1,2-a]pyrazines as potent and selective 5-HT(2C) receptor agonists.

Further lead optimization efforts on previously described 1,2,3,4,10,10a-hexahydro-1H-pyrazino[1,2-a]indoles led to the new class of 5,5a,6,7,8,9-hexahydro-pyrido[3',2':4,5]pyrrolo[1,2-a]pyrazines culminating in the discovery of (5aR,9R)-2-[(cyclopropylmethoxy)methyl]-5,5a,6,7,8,9-hexahydro-9-methyl-pyrido[3', 2':4,5]pyrrolo[1,2-a]pyrazine 18 as a potent, full 5-HT(2C) receptor agonist with an outstanding selectivity profile and excellent hERG and phospholipidosis properties.

Identification of 4-methyl-1,2,3,4,10,10a-hexahydropyrazino[1,2-a]indoles as 5-HT2C receptor agonists.

Synthesis and evaluation of the activity of new 4-methyl-1,2,3,4,10,10a-hexahydropyrazino[1,2-a]indoles as 5-HT(2C) receptor agonists are described. Appropriately substituted, several analogs displayed selectivity against the other 5-HT(2) receptor subtypes of 1 order of magnitude or more. Selectivity was improved for several compounds versus the lead 1, increasing the therapeutic interest in this series of 5-HT(2C) receptor agonists.

Indoline derivatives as 5-HT(2C) receptor agonists.

A series of 1-(1-indolinyl)-2-propylamines was synthesised and evaluated as 5-HT(2C) receptor agonists for the treatment of obesity. The general methods of synthesis of the precursor indoles are described. The functional efficacy and radioligand binding data for all of the compounds at 5-HT(2) receptor subtypes are reported. A number of compounds were found to reduce food intake in rats after oral administration.

Discovery of nonxanthine adenosine A2A receptor antagonists for the treatment of Parkinson's disease.

During a program to investigate the biochemical basis of side effects associated with the antimalarial drug mefloquine, the authors made the unexpected discovery that the (-)-(R,S)-enantiomer of the drug is a potent adenosine A2A receptor antagonist. Although the compound was ineffective in in vivo animal models of central adenosine receptor function, it provided a unique nonxanthine adenosine A2A receptor antagonist lead structure and encouraged the initiation of a medicinal chemistry program to develop novel adenosine A2A antagonists for the management of Parkinson's disease (PD). The authors have synthesized and screened more than 2,000 chemically diverse and novel adenosine A(2A antagonists. Early examples from two distinct chemical series are the thieno[3,2-dy]pyrimidine VER-6623 and the purine compounds VER-6947 and VER-7835, which have high affinity at adenosine A2A receptors (K(i) values 1.4, 1.1, and 1.7 nmol/L, respectively) and act as competitive antagonists. In particular, VER-6947 and VER-7835 demonstrate potent in vivo activity reversing the locomotor deficit caused by the D2 receptor antagonist haloperidol, with minimum effective doses comparable with that of KW6002 (0.3 to 1 mg/kg). In conclusion, the authors have discovered potent, selective, and in vivo active nonxanthine adenosine A2A antagonists that have considerable promise as a new therapy for PD.

Solubilisation and immunoprecipitation of rat striatal adenosine A(2A) receptors.

In the present study, we have sought to solubilise adenosine A(2A) receptors from rat striatal membranes using a variety of different detergents. Of the detergents tested, 1% CHAPS (3-[(3-deoxycholic acid (cholamidopropyl) dimethylammonio]-1-propanesulfonate) yielded optimal conditions for solubilisation (in the presence of 3 mg/ml protein, 44% of receptor was solubilised, 50% of total protein was solubilised). An antipeptide antibody was raised against a 15 amino-acid sequence within the predicted third intracellular loop region of the human and rat adenosine A(2A) receptor. The antibody was coupled to protein A immobilised on sepharose CL-4B and used to immunoprecipitate adenosine A(2A) receptors from solubilised rat striatal preparations. Radioligand-binding studies were performed using the selective adenosine A(2) antagonist [(3)H]ZM 241385 (4-(2-[7-amino-2-(2-fury1)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol). Using [(3)H]ZM 241385, the pharmacology of immunoprecipitated adenosine A(2A) receptors was composed to striatal membrane bound adenosine A(2A) receptors and detergent solubilised adenosine A(2A) receptors. [(3)H]ZM 241385 labelled a single saturable binding site with high affinity in all three preparations (membrane bound K(d) 2.7 nM+/-1.0; solubilised K(d) 1.9 nM+/-0.3; immunoprecipitated K(d) 2.2 nM+/-0.7). Additionally, all three assays confirmed a rank order of potency for displacers consistent with adenosine A(2A) receptor pharmacology: ZM 241385>KW 6002 ((E)-8-[2-(3,4-dimethoxyphenyl)ethynyl]-1-3-diethyl-3,7-dihydro-7-methyl-1-purine 2,6 dione)>CGS 21680, (2-(4-(2 carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamidoadenosine)>DPCPX (8-cyclopentyl-1,3-dipropylxanthine). We conclude that we have solubilised and immunoprecipitated adenosine A(2A) receptors from rat striatum and that their pharmacology is consistent with native striatal adenosine A(2A) receptors.

Characterisation of agonist binding on human 5-HT2C receptor isoforms.

The 5-HT2C receptor is expressed in different isoforms as a result of mRNA editing. Both INI (unedited) and VSV (a fully edited version) isoforms are abundant in rat brain. The VSV isoform lacks the high affinity recognition site for 5-HT, which may be caused by low efficiency coupling to G-proteins. In this study we have investigated the pharmacology of the agonist binding site of these two isoforms of the 5-HT2C receptor. The VSV isoform was expressed in Chinese hamster ovary cells (CHO) and the INI isoform in both Chinese hamster ovary cells and human embryonic kidney cells (HEK-293). Saturation analysis using [3H]5-HT revealed high and low affinity recognition sites on the INI isoform in both cell types whilst the VSV isoform did not have the high affinity binding site for [3H]5-HT. Displacement studies were undertaken using [3H]5-HT to label the receptors. In these studies the affinity of agonists (5-HT, Ro600175 ((S)-2-(6-Chloro-5-fluoroindol-1-yl)-1-methylethylamine), MK212 (6-Chloro-2-(piperazinyl) pyrazine), mCPP (1-(m-chlorophenyl)-piperazine), TfMPP (N-(m-trifluoromethylphenyl)piperazine), DOI (1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane), DOB (1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane) and 8OH-DPAT (8-hydroxy-2-(di-N-propylamino)tetralin) was higher at the INI isoform, whilst antagonist affinity (ketanserin and mesulergine) did not change between the two receptor isoforms. There were no differences between the INI isoform expressed in the CHO and HEK-293. This suggests that the INI isoform of the 5-HT2C receptor is pharmacologically similar to the VSV form of the 5-HT2C receptor but that it couples more efficiently to G-proteins.

Monospecific antibodies as probes for the stoichiometry of recombinant GABA(A) receptors.

GABA(A) receptors composed of alpha1beta3 gamma2 and alpha1beta3 subunits were expressed in insect Sf9 cells and solubilized in 1% Triton X100. In sucrose density gradients, [3H]-Ro15-1788 binding activity, in the case of alpha1beta3 gamma2, and [3H]-muscimol binding activity, in the case of alpha1beta3 containing receptors sedimented as a single sharp peak suggesting the formation of receptors containing a defined number of subunits. When alpha1beta3gamma2 -containing receptors were incubated with an alpha-subunit specific antibody (bd24), a single class of antibody receptor complex was formed irrespective of the receptor-antibody ratio. This is consistent with two alpha subunits cross-linked within the receptor by the antibody. Similar results were obtained using a beta-subunit specific antibody (bd17). Several classes of antibody-receptor complex were formed when receptors were pre-incubated with a gamma specific antibody (anti gamma(2) 1-15 Cys). This profile is consistent with the presence of a single gamma subunit in each complex. Experiments with alpha1beta3 subunit containing receptors and antibody bd24 produced a profile similar to that seen with alpha1beta3 gamma2 receptors, consistent with two alpha subunits per receptor complex. In this case, the anti-beta subunit antibody, bd17, produced a unique and complex profile consistent with three beta subunits per receptor. This method permits the rapid determination of subunit stoichiometries of homogeneous receptor populations

Molecular size of recombinant alpha1beta1 and alpha1beta1gamma2 GABAA receptors expressed in Sf9 cells.

The present study examines the physical properties of recombinant human GABAA receptors. The baculovirus/Sf9 cell system was used to express combinations of human GABAA receptor subunits: alpha 1 alone, alpha 1 with beta 1, and alpha 1 with beta 1 and gamma 2. Receptors were solubilized using 1% Triton X-100. In sucrose density gradients containing 150mM NaCl, alpha 1 beta 1 receptor-detergent complexes sedimented more slowly than alpha 1 beta 1 gamma 2 constructs (sedimentation coefficient = 7.00 +/- 0.32 and 8.63 +/- 0.48 S, respectively). Stokes' radii for the two receptor-detergent complexes were determined by gel filtration in Sephacryl S-300. These experiments were performed in the presence of 1 M sodium chloride to prevent aggregation. The Stokes' radii for alpha 1 beta 1 and alpha 1 beta 1 gamma 2 receptor-detergent complexes were 9.06 +/- 0.23 and 7.91 +/- 0.19 nm, respectively. Sedimentation experiments in 1 M NaCl revealed similar sedimentation coefficients for alpha 1 beta 1 and alpha 1 beta 1 gamma 2 receptor-detergent complexes (8.79 +/- 0.59 and 8.46 +/- 0.72 S, respectively). The molecular weight of the alpha 1 beta 1 receptor excluding detergent was estimated to be 281 +/- 19 kDa, that of the alpha 1 beta 1 gamma 2 receptor, 247 +/- 21 kDa. This difference is not statistically significant. Given subunit molecular weights which are close to 50 kDa, this suggested a pentameric structure for the majority of alpha 1 beta 1 gamma 2 receptors, and that alpha 1 beta 1 receptors are not"assembly intermediates" with fewer subunits.

The pharmacology of adenylyl cyclase modulation by GABAB receptors in rat brain slices.

GABAB receptor activation inhibits forskolin-stimulated adenylyl cyclase activity but augments noradrenaline-stimulated adenylyl cyclase activity. The present study investigated the pharmacology of these two GABAB receptor mediated responses. In a cross-chopped rat cortical slice preparation, it was confirmed that (-)baclofen inhibited forskolin-stimulated adenylyl cyclase activity and augmented noradrenaline-stimulated adenylyl cyclase activity. The potency of five further agonists was investigated (SKF97541, CGP47656, CGP44533, 3-APA and CGP44532). Of these agonists two compounds were significantly more potent as inhibitors of forskolin-stimulated adenylyl cyclase than as augmenters of noradrenaline-stimulated adenylyl cyclase activity, these were (-)baclofen (pEC50 = 6.07 +/- 0.29 and 5.04 +/- 0.17, respectively (p < 0.05)), and CGP47656 (pEC50 = 6.44 +/- 0.05 and 4.48 +/- 0.26, respectively (p < 0.05)). It is possible to explain this difference in potency by proposing that these compounds have low intrinsic efficacy, and the augmentation of noradrenaline-stimulated adenylyl cyclase has a low receptor reserve. In addition six antagonists (CGP49311A, CGP46381, CGP45024, CGP45397, CGP36742) were also tested for their ability to antagonize 10 microM (-)baclofen in these two assays. These antagonists ranged in potency as inhibitors of forskolin-stimulated adenylyl cyclase activity from CGP49311A (pEC50 = 5.45 +/- 0.30) to CGP36742 (pEC50 = 3.87 +/- 0.16). Each antagonist had similar potency in the two assays, suggesting that these two responses are mediated by pharmacologically similar receptors.

GABA receptors in rats with spontaneous generalized nonconvulsive epilepsy.

We have used the technique of autoradiography to study the binding of [3H]-GABA to GABAA and GABAB receptors in brains taken from rats that are genetically predisposed to petit mal type seizures. A range of concentrations of [3H]-GABA were employed to test the hypothesis that this predisposition was due to regional changes in either the number of GABAA or GABAB receptors, or affinity of GABA for these receptors. We found no statistical difference in the levels of radioligand binding to GABAA and GABAB receptors in animals susceptible to seizures compared to control animals in any of the brain regions studied over the concentration range 25 nM to 400 nM. This indicated that there was no change in either the Kd (affinity) or Bmax (receptor number) in these animals and that the pharmacological basis for the efficacy of GABAB antagonists in this seizure condition probably lies elsewhere.

The human sigma site, which resembles that in NCB20 cells, may correspond to a low-affinity site in guinea pig brain.

1,3-di(2-[5-3H]tolyl)Guanidine ([3H]DTG) was found to bind to a single saturable population of binding sites in human cerebral cortex and NCB20 cells, a second low-affinity site was apparent in guinea pig brain. Displacement studies were performed to determine the pharmacology of the [3H]DTG binding site in these 3 membrane preparations. In human cortical tissue and NCB20 cell membranes the (+)-stereoisomers of benzomorphans displaced binding with Hill coefficients close to one, displayed similar affinity and did not give the biphasic displacement curve characteristic of guinea pig membranes. The pIC50 of the low-affinity component of the sigma binding site in guinea pig brain correlates best with the affinity of drugs for the binding site in human cortex.

Spironolactone causes a rapid down regulation of sigma recognition sites in guinea pig brain and liver.

The effect of dosing guinea pigs with spironolactone (100 mg/kg twice daily for 3 days) upon the sigma recognition site labelled with [3H]-DTG was investigated. Animals were dosed with 100 mg/kg spironolactone twice a day for 3 days. Spironolactone treatment caused a decrease in sigma radioligand binding in membranes prepared from liver and brain but not in adrenals or testes. Saturation analysis of [3H]-DTG radioligand binding in the brain indicated that the decrease in specifically bound [3H]-DTG binding was due to a reduction in receptor number with no change in affinity. This method for the selective depletion of brain and liver sigma recognition sites will provide a useful tool for exploring the functional significance of this site.

The pharmacological properties of the imidazobenzodiazepine, FG 8205, a novel partial agonist at the benzodiazepine receptor.

1. The pharmacological properties of the benzodiazepine receptor ligand, FG 8205 (7-chloro-5,6-dihydro-5-methyl-6-oxo-3-(5-isopropyl-1,2,4-oxadiazol++ +-3-yl)-4H- imidazol[1,5a][1,4]benzodiazepine) have been examined. 2. FG 8205 potently displaced [3H]-flumazenil binding in rat cortical membranes with a Ki of 3.3 nM, but was inactive at 13 neurotransmitter recognition sites. 3. Consistent with a partial agonist profile, the affinity of FG 8205 for the benzodiazepine recognition site was increased in the presence of gamma-aminobutyric acid (GABA, 300 microM) by a degree (-log [IC50 in the presence of GABA/IC50 alone] = 0.34) significantly less than found for diazepam (0.46). FG 8205 also potentiated the inhibitory potency of the GABAA-receptor agonist, isoguvacine, on the hippocampal CA1 population spike and, again, the maximum shift (-log dose-ratio = 0.2) was significantly less than that seen with diazepam (0.4). 4. In anticonvulsant studies, the ED50 doses of FG 8205 and diazepam needed to antagonize seizures induced by pentylenetetrazol (PTZ) or by sound in audiogenic seizure prone mice were similar with values of 0.2-0.3 mg kg-1, i.p. However, even high doses of FG 8205 (50 mg kg-1) did not protect against seizures induced by electroshock. 5. FG 8205 released responding suppressed by footshock in a rat operant conditioned emotional response task over the dose range 0.5-50 mg kg-1 (i.p.). Similar doses of FG 8205 had a marked taming effect in cynomolgus monkeys. However, measures of sedation and ataxia (as measured by rotarod in the mouse, climbing behaviour in the rat, and by scoring arousal and co-ordination in primates) were slight and only transiently affected by FG 8205, and FG 8205 significantly antagonized the rotarod performance deficit induced by diazepam in the mouse. 6. While the potentiation by FG 8205 of the response to isoguvacine in the rat hippocampal slice and the anxiolytic-like effects of the compound in both rats and primates were reversed by the benzodiazepine receptor antagonist, flumazenil, high doses of the antagonist were able only marginally to block the protective effects of FG 8205 against seizures induced by PTZ in the mouse. 7. Thus, FG 8205 does not show the marked motor impairment characteristic of full agonists at the benzodiazepine receptor, consistent with its partial agonist profile in in vitro assay systems. Nevertheless, the compound has sufficient intrinsic activity to maintain high efficacy in anticonvulsant and anxiolytic tests.

Structure and activity of hydrogenated derivatives of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801).

Several hydrogenated derivatives of the potent NMDA antagonist 1 have been prepared and evaluated as competitive inhibitors of [3H]-1 binding. These compounds were also tested for their ability to act as noncompetitive antagonists of NMDA in vitro. These studies indicate that two aromatic rings are not strictly required for high-affinity binding or NMDA antagonism.

[3H]MK-801 labels a site on the N-methyl-D-aspartate receptor channel complex in rat brain membranes.

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist [3H]MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. [3H]MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-[3H]SKF 10,047. [3H]MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.

The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist.

The compound MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate)] is a potent anticonvulsant that is active after oral administration and whose mechanism of action is unknown. We have detected high-affinity (Kd = 37.2 +/- 2.7 nM) binding sites for [3H]MK-801 in rat brain membranes. These sites are heat-labile, stereoselective, and regionally specific, with the hippocampus showing the highest density of sites, followed by cerebral cortex, corpus striatum, and medulla-pons. There was no detectable binding in the cerebellum. MK-801 binding sites exhibited a novel pharmacological profile, since none of the major neurotransmitter candidates were active at these sites. The only compounds that were able to compete for [3H]MK-801 binding sites were substances known to block the responses of excitatory amino acids mediated by the N-methyl-D-aspartate (N-Me-D-Asp) receptor subtype. These comprised the dissociative anesthetics phencyclidine and ketamine and the sigma-type opioid N-allylnormetazocine (SKF 10,047). Neurophysiological studies in vitro, using a rat cortical-slice preparation, demonstrated a potent, selective, and noncompetitive antagonistic action of MK-801 on depolarizing responses to N-Me-D-Asp but not to kainate or quisqualate. The potencies of phencyclidine, ketamine, SKF 10,047, and the enantiomers of MK-801 as N-Me-D-Asp antagonists correlated closely (r = 0.99) with their potencies as inhibitors of [3H]MK-801 binding. This suggests that the MK-801 binding sites are associated with N-Me-D-Asp receptors and provides an explanation for the mechanism of action of MK-801 as an anticonvulsant.

Effect of angiotensin converting-enzyme inhibition on potassium-mediated aldosterone secretion in essential hypertension.

1. Eight patients with essential hypertension were challenged with an infusion of 32 mmol of potassium chloride in saline before and after control of their blood pressure by the angiotensin converting-enzyme (ACE) inhibitor enalapril. 2. The potassium infusion was associated with similar increases in plasma aldosterone before and during enalapril treatment, although absolute aldosterone levels were lower after enalapril treatment despite higher plasma potassium levels. 3. The handling of the potassium load was altered by ACE inhibition. The area under the curve of a plot of the increase in plasma potassium above baseline against time was greater during enalapril treatment than during treatment with placebo. 4. These observations contrast with data obtained in the dog and demonstrate that in patients with essential hypertension stimulation of aldosterone secretion by potassium is not abolished by chronic suppression of plasma angiotensin II; and although plasma aldosterone remains at a lower level, the homoeostasis of plasma potassium is only mildly impaired.

Bopindolol: a new long acting beta-receptor antagonist; its effects on haemodynamics, and on the renin response to tilting.

Oral administration of single doses of bopindolol (1-4mg) caused significant reductions in the rises of systolic blood pressure and heart rate produced by exercise; only the reduction in the rise of heart rate was significantly dose-related. Resting heart rate was reduced by bopindolol. There were small effects on resting blood pressure. Bopindolol caused a significant attenuation of the rise in plasma renin activity produced by passive head-up tilting to 75-85 degrees. Bopindolol produced a dose-related attenuation of the increase in pulse rate evoked by passive tilting. All effects 1-4 were maintained for at least 24 h. There was no measurable effect on plasma potassium concentration, peak flow rate or forced expiratory volume (FEV1).

The early detection of colorectal cancer.

A review of the American Cancer Society's statistics for colorectal cancer indicates that there has been little improvements in the survival rate for this disease in the past 25 years. Although there have been advances in surgical techniques, radiation therapy, and chemotherapy, the key to improved survival rates is earlier diagnosis. A significant percentage of patients continues to present with regional or distal metastasis at the time of their initial diagnosis. Both proctosigmoidoscopy and guaiac impregnated filter slide paper methods have been productive in diagnosing this disease at an earlier stage. The "Hemoccult" test, however, is inexpensive, can be used on a routine basis, is easier for patients to perform themselves, and is aesthetically pleasing. It represents a significant cost savings compared to proctosigmoidoscopy and should be advocated for routine use in all patients over 40 years of age. Although there continues to be a role for local and regional mass screening programs, significant improvements in colorectal survival rates cannot be expected until routine application of this type of screening is practiced.