Silent 5-HT1A receptor antagonists: utility as research tools and therapeutic agents.

The 5-hydroxytryptamine 5-HT1A receptor has been the focus of considerable research effort for over a decade. However, the definitive classification of this receptor and the full characterization of its pharmacology have awaited the development of highly selective 5-HT1A receptor antagonists. The only compounds available until recently have been either nonselective or partial 5-HT1A receptor agonists (or a combination of both). Confusion has arisen owing to the use of different pharmacological models in examining the functional activity of 5-HT1A receptor ligands. Several partial agonists display only antagonist activity in models of postsynaptic 5-HT1A receptor function, whereas their agonist properties are revealed in models of presynaptic, somatodendritic 5-HT1A autoreceptor function. In view of these considerations, the term 'silent antagonist' has been introduced to distinguish true 5-HT1A receptor antagonists from partial agonists. Allan Fletcher and colleagues review the pharmacological properties of the first selective silent 5-HT1A receptor antagonists that have been recently discovered and discuss the potential therapeutic utility of these novel compounds.

Oral hypoglycemic agents. Pyrimido[1,2-a]indoles and related compounds.

A series of pyrimido[1,2-a]indoles were synthesized and studied for their hypoglycemic activity following oral administration at a standard dose of 100 mg/kg to fed rats. The effect of 10-alkoxyalkyl, 10-alkyl, 10-aryl, and 3,3-dialkyl substitution on the activity of 10-hydroxypyrimido[1,2-a]indoles was investigated. Relative potencies of a number of the most active compounds were defined by three-point dose-response studies. The most potent compounds were those with either 3,3-dimethyl substituents, compounds 21, 22, and 38, or 3,3-spirocyclohexane substituents, compounds 39 and 49. 10-Aminopyrimido[1,2-a]indoles were in general less active than the 10-hydroxy analogues, and potency was further decreased by derivatizing the 10-amino group. The most potent 10-amino derivatives were 57 and 58.

Electrophysiological, biochemical, neurohormonal and behavioural studies with WAY-100635, a potent, selective and silent 5-HT1A receptor antagonist.

Although considerable progress has been made in characterising the 5-HT1A receptor using agonists, partial agonists or non-selective antagonists, further studies of 5-HT1A receptor function have been hindered by the lack of highly selective antagonists. The term 'silent' antagonist has been used for such compounds in order to distinguish them unequivocally from several 5-HT1A receptor partial agonists which were initially designated 'antagonists'. In this report we provide a comprehensive review of the biochemical, pharmacological and behavioural properties of the first potent, selective and silent 5-HT1A receptor antagonist, WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-(2- pyridinyl)cyclohexanecarboxamide trihydrochloride). WAY-100635 had an IC50 (displacement of specific [3H]8-OH-DPAT binding to 5-HT1A receptors in the rat hippocampus) of 1.35 nM and was > 100-fold selective for the 5-HT1A site relative to a range of other CNS receptors. [3H]WAY-100635 was also characterised as the first 5-HT1A antagonist radioligand, displaying the same regional distribution of binding sites as [3H]8-OH-DPAT in rat brain. As would be expected for the binding of an antagonist to a G-protein-coupled receptor, the Bmax of [3H]WAY-100635 specific binding was consistently 50-60% greater than that of the agonist radioligand, [3H]8-OH-DPAT. Mn2+, but not guanine nucleotides, inhibited [3H]WAY-100635-specific binding. [3H]WAY-100635 was also shown to bind selectively to brain 5-HT1A receptors in vivo, following intravenous administration to mice. In vitro electrophysiological studies demonstrated that WAY-100635 had no 5-HT1A receptor agonist actions, but dose-dependently blocked the effects of agonists at both the postsynaptic 5-HT1A receptor in the CA1 region of the hippocampus, and the somatodendritic 5-HT1A receptor located on dorsal raphe 5-HT neurones. In vivo, WAY-100635 also dose-dependently blocked the ability of 8-OH-DPAT to inhibit the firing of dorsal raphe 5-HT neurones, and to induce the '5-HT syndrome', hypothermia, hyperphagia and to elevate plasma ACTH levels. In the mouse light/dark box anxiety model, WAY-100635 induced anxiolytic-like effects. WAY-100635 had no intrinsic effect on cognition in the delayed-matching-to-position model of short-term memory in the rat, but reversed the disruptive effects of 8-OH-DPAT on motor motivational performance. These data clearly demonstrate that WAY-100635 is the first potent, selective and silent 5-HT1A receptor antagonist. Furthermore, [3H]WAY-100635 is the first antagonist radioligand to become available for 5-HT1A receptor binding studies both in vitro and in vivo. The positive effects of WAY-100635 in an anxiety model also indicate that a postsynaptic 5-HT1A receptor antagonist action may contribute to the anxiolytic properties of 5-HT1A receptor partial agonists.

Autoradiographic localization of 5-HT1A receptors in the post-mortem human brain using [3H]WAY-100635 and [11C]way-100635.

The distribution of 5-HT1A receptors was examined in the post-mortem human brain using whole hemisphere autoradiography and the selective 5-HT1A receptor antagonist [3H]WAY-100635 ([O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2- pyridinyl)cyclohexanecarboxamide trihydrochloride). The autoradiograms showed very dense binding to hippocampus, raphe nuclei and neocortex. The labeling in neocortex was slightly lower than in the hippocampus and was mainly at superficial layers, although a faintly labeled band could be seen in deeper neocortical layers. Other regions, such as the amygdala, septum and claustrum, showed low densities caudatus and putamen, in cerebellum or in structures of the brain stem except in the raphe nuclei. The labeling of human 5-HT1A receptors with [3H]WAY-100635 was antagonised by the addition of 5-HT1A receptor ligands, 5-HT, buspirone, pindolol or 8-OH-DPAT (10 microM), leaving a very low background of non-specific binding. Saturation analysis of semiquantitative data from several human regions indicated that [3H]WAY-100635 has a Kd of approximately 2.5 nM. The selective labeling of 5-HT1A receptors with [3H]WAY-100635 clearly show that this compound is useful for further studies of the human 5-HT1a receptor subtype in vitro [11C]WAY-100635 is used for the characterization of 5-HT1A receptors with positron emission tomography (PET). WAY-100635 was also radiolabeled with the short-lived positron-emitting radionuclide carbon-11 (t1/2 = 20 min) and used for in vitro autoradiography on human whole hemisphere cryosections. [11C]WAY-100635 gave images qualitatively similar to those of [3H]WAY-100635, although with a lower resolution. Thus, the hippocampal formation was densely labeled, with lower density in the neocortex. Buspirone, pindolol or 8-OH-DPAT (10 microM), blocked all binding of [11C]WAY-100635. The in vitro autoradiography of the distribution of 5-HT1A receptors obtained with radiolabeled WAY-100635 provide detailed qualitative and quantitative information on the distribution of 5-HT1A-receptors in the human brain. Moreover, the studies give reference information for the interpretation of previous initial results at much lower resolution in humans with PET and [11C]Way-100635. These data provide a strong basis for expecting [11C]WAY-100635 to behave as a highly selective radioligand in vivo.

Evaluation of [O-methyl-3H]WAY-100635 as an in vivo radioligand for 5-HT1A receptors in rat brain.

N-(2-(4-(2-Methoxyphenyl)-1-piperazinyl)ethyl)-N-(2- pyridyl)cyclohexanecarboxamide trihydrochloride (WAY-100635) is a new, potent and selective 5-HT1A receptor antagonist. We have evaluated radiolabelled WAY-100635 as a prospective radioligand for positron emission tomography (PET) by studying biodistribution in rat ex vivo. After intravenous injection, [O-methyl-3H]WAY-100635 cleared rapidly from plasma but was retained in brain. Specific binding was quantified from kinetic studies, using a reference-tissue compartment model, fitting for binding potential (k3/k4). The regional variation in binding potential correlated with the known distribution of 5-HT1A receptors. Saturation studies gave Bmax values in vivo that were consistent with those reported in vitro. At 60 min after injection, the ratio of radioactivity in 5-HT1A receptor-rich regions (e.g. septum, entorhinal cortex and hippocampus) to that in cerebellum reached approximately 16. Pre-dosing the rats with WAY-100635 (2 mg/kg) reduced this ratio to one, whereas similar pre-dosing with citalopram (5-HT uptake site inhibitor), prazosin (alpha 1A-adrenoceptor antagonist) or idazoxan (alpha 2-adrenoceptor antagonist) caused little or no reduction. Substantial (77%) blockade of [3H]WAY-100635 binding was achieved with the 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), and the partial agonists, ipsapirone and buspirone. Thus, the properties of WAY-100635 are such that, when labelled with carbon-11, it could provide a radioligand suitable for clinical and pharmacological investigations of central 5-HT1A receptors in man using PET.

A pharmacological profile of the selective silent 5-HT1A receptor antagonist, WAY-100635.

WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2- pyridinyl)cyclohexanecarboxamide trihydrochloride) is an achiral phenylpiperazine derivative that binds with high affinity and selectivity to the 5-HT1A receptor. WAY-100635 displaced specific binding of the 5-HT1A radioligand, [3H]8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), to rat hippocampal membranes with a pIC50 of 8.87. This represented a greater than 100-fold selectivity relative to binding at other 5-HT receptor subtypes and major neurotransmitter receptor, reuptake and ion channel sites. In functional assays, WAY-100635 was a potent 5-HT1A receptor antagonist, with no evidence of any 5-HT1A receptor agonist or partial agonist activity. In the isolated guinea-pig ileum WAY-100635 was a potent and, at high concentrations, an insurmountable antagonist of the 5-HT1A receptor agonist action of 5-carboxamidotryptamine, with an apparent pA2 value (at 0.3 nM) of 9.71. WAY-100635 blocked the inhibitory action of 8-OH-DPAT on dorsal raphe neuronal firing in the anaesthetised rat at doses which had no inhibitory action per se. In behavioural models, WAY-100635 itself induced no overt behavioural changes but potently antagonised the behavioural syndrome induced by 8-OH-DPAT in the rat and guinea-pig (minimum effective dose = 0.003 mg/kg s.c. and ID50 = 0.01 mg/kg s.c., respectively). WAY-100635 also blocked the hypothermia induced by 8-OH-DPAT in the mouse and rat with ID50 values of 0.01 mg/kg s.c. These data indicate that WAY-100635 will be used as a standard antagonist in further studies of 5-HT1A receptor function.

WAY100135: a novel, selective antagonist at presynaptic and postsynaptic 5-HT1A receptors.

The novel phenylpiperazine derivative, (+/-)-WAY100135 (N-tert-butyl-3-(4-(2-methoxyphenyl)piperazin-1-yl)-2-phenylpro pionamide dihydrochloride), is a selective antagonist at both somatodendritic and postsynaptic 5-HT1A receptors. The IC50 of (+/-)-WAY100135 at the rat hippocampal 5-HT1A receptor was 34 nM, whereas its IC50 at a range of other receptor sites was > 2 microM. Up to a dose of 2.5 mg/kg i.v. (+/-)-WAY100135 induced a maximum 30% inhibition of raphe neuronal firing and (at 0.5 mg/kg i.v.) antagonised the inhibition of firing induced by 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin) in anaesthetised rats. (+/-)-WAY100135 antagonised the action of 5-carboxamidoiodotryptamine in the guinea-pig ileum, with a pA2 of 7.2. (+/-)-WAY100135 had no agonist-like behavioural effects but antagonised the behavioural syndrome and hypothermia induced by 8-OH-DPAT in the rat and mouse, respectively. The interaction of (+/-)-WAY100135 with the 5-HT1A receptor was stereoselective; the (+)-enantiomer being markedly more active in binding, functional and behavioural studies. These data indicate that (+/-)-WAY100135 is the first highly selective antagonist at both somatodendritic and postsynaptic 5-HT1A receptors.

First delineation of 5-HT1A receptors in human brain with PET and [11C]WAY-100635.

The selective 5-HT1A receptor radioligand, [11C]WAY-100635 ([11C]N-2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-2- pyridyl)cyclohexanecarboxamide), has been injected intravenously into healthy male volunteers and studied by PET (positron emission tomography). The results provide the first delineation of 5-HT1A receptors in living human brain and demonstrate the potential to use [11C]WAY-100635 for the study of central 5-HT1A receptors in patients with psychiatric and neurological disorders and for the investigation of the pharmacology of drugs acting on the central nervous system.

Exquisite delineation of 5-HT1A receptors in human brain with PET and [carbonyl-11 C]WAY-100635.

The 5-HT1A receptor antagonist, WAY-100635 [N-(2-(4-(2-methoxyphenyl)- 1-piperazinyl)ethyl)-N-(2-pyridyl) cyclohexanecarboxamide], was labelled in its carbonyl group with carbon-11 (t1/2 = 20.4 min), injected intravenously into healthy male volunteers and studied with positron emission tomography (PET). The acquired data provide exquisite delineation of 5-HT1A receptors in brain, with the ratio of radioactivity uptake in receptor-rich regions, such as medial temporal cortex, to that in receptor-devoid cerebellum reaching 25 by 60 min after radioligand injection. Application of biomathematical modelling to the data revealed high values (7.8) for binding potential, a measure of Bmax/Kp, in receptor-rich regions. Only very polar radioactive metabolites were present in plasma, a finding consistent with the low level of nonspecific binding seen in cerebellum. [carbonyl-11C]WAY-100635 is concluded to be far superior to the previously reported [0-methyl-11C]WAY-100635 as a radioligand for PET studies of 5-HT1A receptors in human brain.

A retrospect on the discovery of WAY-100635 and the prospect for improved 5-HT(1A) receptor PET radioligands.

Abstract. An outline is given of the developments that led to the identification of [O-methyl-(11)C]WAY-100635 (4) as the first useful PET ligand for imaging serotonin(1A) (5-HT(1A)) receptors in the living human brain. Recent attempts to develop 5-HT(1A) receptor radioligands superior to 4 are reviewed, and [carbonyl-(11)C]WAY-100635 (6) has been shown to be the best currently available radioligand for human studies. Of other (11)C-radiolabelled compounds, [O-methyl-(11)C](R,S)-CPC-222 (9), DWAY (8), and [(11)C]NAD-299 (14) all demonstrate specific binding to 5-HT(1A) receptors in animals and warrant further expedited studies in humans. The trans-fluorocyclohexane, 12, and fluorobenzene, [(18)F]p-MPPF 13, are highlighted as examples of promising (18)F-labelled ligands.

Characterization of the radioactive metabolites of the 5-HT1A receptor radioligand, [O-methyl-11C]WAY-100635, in monkey and human plasma by HPLC: comparison of the behaviour of an identified radioactive metabolite with parent radioligand in monkey using PET.

N-(2-(4-(2-Methoxy-phenyl)-1-piperazin-1-yl)ethyl)-N-(2-pyridyl) cyclohexanecarboxamide (WAY-100635), labelled in the O-methyl group with carbon-11 (t1/2 = 20.4 min), is a promising radioligand for application with positron emission tomography (PET) to the study of 5-HT1A receptors in living human brain. An understanding of the metabolism of this new radioligand is crucial to the development of a biomathematical model for the interpretation of the kinetics of radioactivity uptake in brain in terms of receptor-binding parameters. After intravenous injection of [O-methyl-11C]WAY-100635 into humans, radioactivity was found to clear rapidly from blood and plasma. By using established methods for the analysis of radioactivity in plasma, it was found that intravenously injected [O-methyl-11C]WAY-100635 is rapidly metabolised to more polar radioactive compounds in a cynomolgus monkey and in humans. Thus, at 60 min postinjection, parent radioligand represented 40% and 5% of the radioactivity in monkey and human plasma, respectively. In monkey and human, one of the radioactive metabolites was identified as the descyclohexanecarbonyl analogue of the parent radioligand, namely [O-methyl-11C]WAY-100634. This compound is known to have high affinity for 5-HT1A receptors and alpha 1-adrenoceptors. In a PET experiment it was demonstrated that, after IV injection of [O-methyl-11C]WAY-100634 into a cynomolgus monkey, radioactivity was avidly taken up by brain. Uptake of radioactivity was higher in 5-HT1A receptor-rich frontal cortex than in cerebellum, which is devoid of 5-HT1A receptors. Polar radioactive metabolites appeared in plasma. The results suggest that the use of WAY-100635 labelled with carbon-11 in its cyclohexanecarbonyl moiety may provide enhanced signal contrast in PET studies and a possibility to develop a simple biomathematical model for regional brain radioactivity uptake.

Medicinal chemistry--16th international symposium. 18-22 September 2000, Bologna, Italy.

This meeting was part of a biennial series focusing upon topics at the forefront of medicinal chemistry. There was no single therapeutic focus for the meeting, instead, the lectures, arranged with main lectures taking place in three parallel sessions, were spread over a number of themes relevant to drug discovery. These are highlighted in order of presentation.

RSC-SCI--tenth medicinal chemistry symposium. 5-8 September 1999, Churchill College, Cambridge, UK.

This meeting was part of a biennial series focusing upon topics at the forefront of medicinal chemistry. There was no single therapeutic focus for the meeting, but the 24 lectures were spread between various themes: enzyme inhibitors; drug-receptor/ion channel interactions; enabling technologies; and, new topics in drug research. Several of the lectures featured structure-activity relationships of new drugs, outlining the rationale for the selection of drug candidates. It was clear that many pharmaceutical companies now routinely employ in vivo studies of distribution and pharmacokinetics early in the drug discovery process, thereby adding to the traditional criteria of affinity, selectivity and in vivo effect when potential drugs are selected for development. Likelihood of success during later clinical investigation phases is thereby enhanced.

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.

Synthesis, physicochemical properties, anticonvulsant activities, and GABA-ergic and voltage-sensitive calcium channel receptor affinities of alpha-substituted N-benzylamides of gamma-hydroxybutyric acid. Part 4: Search for new anticonvulsant compounds.

In a search for new anticonvulsant compounds, two series of N-benzylamides of alpha-(benzylamino)-gamma-hydroxybutyric acid (series A) and alpha-(2-phenylethylamino)-gamma-hydroxybutyric acid (series B), were investigated in maximal electroshock (MES), subcutaneous metrazole, and rotorod toxicity assays. The most potent anticonvulsant compounds were alpha-(benzylamino)-gamma-hydroxybutyric acid N-benzylamide (3) and N-(2-chlorobenzylamide (4) with median effective (ED50) doses 63.0 mg/kg and 54.0 mg/kg, respectively. alpha-(4-Phenylpiperazinyl)-gamma-hydroxybutyric acid N-(4-methylbenzyl)amide (17) and alpha-(benzylpiperazinyl-gamma-hydroxy-butyric acid N-(4-methylbenzyl)amide (18) were also tested for their ability to potentiate [3H]-muscimol binding and to inhibit [35S]-TBPS binding (as indices of GABA-A receptor potentiation). Amide 17 exhibited activity at the GABA-A complex which may be the mechanism by which the anticonvulsant effect of this compound is mediated. The N-benzylamides of alpha-(benzylamino)-gamma-hydroxybutyric acid (3-9) were also evaluated for their ability to displace [3H]-nitrendipine from voltage-sensitive calcium channel (VSCC) receptors isolated from rat cortex.

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.

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.