5-HT2C Receptors in the Pathophysiology of CNS Disease (The Receptors, Vol. 22)

The Receptors

For other titles published in this series, go to www.springer.com/series/7668

Giuseppe Di Giovanni  •  Ennio Esposito Vincenzo Di Matteo Editors

5-HT2C Receptors in the Pathophysiology of CNS Disease

Editors Editor-in-Chief Giuseppe Di Giovanni University of Malta Department of Physiology and Biochemistry Msida, Malta [email protected]

Assistant Editor Vincenzo Di Matteo Consorzio Mario Negri Sud Centro di Ricerche Farmacologiche e Biomediche Chieti, Italy [email protected]

Assistant Editor Ennio Esposito Consorzio Mario Negri Sud Centro di Ricerche Farmacologiche e Biomediche Chieti, Italy [email protected]

ISBN 978-1-60761-940-6 e-ISBN 978-1-60761-941-3 DOI 10.1007/978-1-60761-941-3 Springer New York Dordrecht Heidelberg London © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

To my wife Samantha, For her abundant support, for her patience and understanding, and for her love G. Di Giovanni

Preface

Accepting the challenge of editing the first book on a subject is a risky business, particularly if you attempt to produce an overview in the research of 5-HT2C receptors, an area now going back over 25 years with so productive results. Nevertheless, I have embarked on this unique editorial enterprise with the aim of giving pleasure to the readers and researchers and not least to myself, confident that it will be instrumental in future research in 5-HT2C pathophysiology. A few years ago Bryan Roth edited, for the same series “The Receptors,” a very fine volume on serotonin receptors. The present volume is a further development of this and is a thorough examination of the 5-HT2C receptor subclass, needed to cover the extraordinary amount of research into nearly every aspect of 5-HT2C receptor function that has recently emerged. This is not surprising, considering that the 5-HT2C receptor is a prominent central serotonin receptor subtype, widely expressed within the central and the peripheral nervous system and is thought to play a major role in the regulation of a plethora of behaviors. Therefore, it has been shown by experimental and clinical observation that it may represent a possible therapeutic target for the development of drugs for a range of CNS disorders such as schizophrenia, depression, drug abuse, eating disorders, Parkinson’s disease, and epilepsy, to cite but a few. The book, a result of the efforts of an international group of authors, has the aim of providing an update of the functional status of the 5-HT2C receptor, covering molecular, cellular, anatomical, biochemical, and behavioral aspects, to highlight its distinctive regulatory properties, the emerging functional significance of constitutive activity and RNA-editing in vivo, and the therapeutic potentiality in different diseases that are singled-out in different chapters. While covering the latest research, for obvious reasons, this volume cannot be exhaustive and it has been impossible to include a number of authors of obvious merit. I hope that more volumes on the subject will be possible in the future. I want to thank all the authors who have responded very willingly and contributed their time and expertise in preparing their individual contribution to a consistently high standard. My thanks go to Vincenzo Di Matteo and Ennio Esposito who have contributed to the realization of this book. I am indebted to Philippe De Deurwaerdère, who has unselfishly dedicated his time and expertise to insightful and helpful reading of this text and Dr Clare Austen for reviewing the English style of these manuscripts. vii

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Finally, I would like to express my sincere appreciation to Kime Neve, series editor, and Matthew Giampoala, Springer publishing editor, for their help in driving the book’s development and eventual publication. I hope that the contents of this volume will further inspire and stimulate discussions and new interdisciplinary research on the 5-HT2C receptor. May 2010

Giuseppe Di Giovanni University of Malta Msida, Malta

Contents

1 The Making of the 5-HT2C Receptor........................................................ Jose M. Palacios, Angel Pazos, and Daniel Hoyer 2 Serotonin 5-HT2C Receptors: Chemical Neuronatomy in the Mammalian Brain........................................................................... Guadalupe Mengod 3 The Medicinal Chemistry of 5-HT2C Receptor Ligands.......................... Marcello Leopoldo, Enza Lacivita, Paola De Giorgio, Francesco Berardi, and Roberto Perrone 4 Insights into 5-HT2C Receptor Function Gained from Transgenic Mouse Models................................................................ Stephen J. Bonasera

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5 Serotonin 5-HT2C Receptor Signal Transduction.................................... Maria N. Garnovskaya and John R. Raymond

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6 Homology Modeling of 5-HT2C Receptors............................................... Nicolas Renault, Amaury Farce, and Philippe Chavatte

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7 5-HT2C Receptor Dimerization.................................................................. 129 Katharine Herrick-Davis and Dinah T. Farrington 8 RNA Editing of 5-HT2C Receptor and Neuropsychiatric Diseases................................................................. 157 Kazuya Iwamoto, Miki Bundo, and Tadafumi Kato 9 Serotonergic Control of Adult Neurogenesis: Focus on 5-HT2C Receptors....................................................................... 169 Annie Daszuta

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10 The Constitutive Activity of 5-HT2C Receptors as an Additional Modality of Interaction of the Serotonergic System...................................................................... 187 Sylvia Navailles and Philippe De Deurwaerdère 11 The 5-HT2C Receptor Subtype Controls Central Dopaminergic Systems: Evidence from Electrophysiological and Neurochemical Studies................................. 215 Giuseppe Di Giovanni, Ennio Esposito, and Vincenzo Di Matteo 12 The Role of 5-HT2C Receptors in the Pathophysiology and Treatment of Depression.......................... 249 Eliyahu Dremencov, Joost H.A. Folgering, Sandra Hogg, Laurence Tecott, and Thomas I.F.H. Cremers 13 5-HT2C Receptors and Suicidal Behavior............................................... 261 Fabio Panariello, Naima Javaid, and Vincenzo De Luca 14 The 5-HT2C Receptor as a Target for Schizophrenia............................ 275 Herbert Y. Meltzer, Liwen Sun, and Hitoshi Hashimoto 15 Serotonin and Reward-Related Behavior: Focus on 5-HT2C Receptors..................................................................... 293 Paul J. Fletcher and Guy A. Higgins 16 Tat-3L4F: A Novel Peptide for Treating Drug Addiction by Disrupting Interaction Between PTEN and 5-HT2C Receptor.................................................................... 325 Amy Hu, Lintao Jia, Jean-Christian Maillet, and Xia Zhang 17 The Role of Serotonin in Eating Behavior: Focus on 5-HT2C Receptors..................................................................... 339 Jason C.G. Halford 18 Physiological and Pathophysiological Aspects of 5-HT2c Receptors in Basal Ganglia..................................................... 351 Philippe De Deurwaerdère, Laurence Mignon, and Marie-Françoise Chesselet 19 Modeling Tardive Dyskinesia: Predictive 5-HT2C Receptor Antagonist Treatment................................................ 383 Richard M. Kostrzewa

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20 The Role of 5-HT2A/2C Receptors in Sleep and Waking......................... 393 Jaime M. Monti and Héctor Jantos 21 Role of Alternative Splicing of the 5-HT2C Receptor in the Prader–Willi Syndrome............................................... 413 Shivendra Kishore and Stefan Stamm 22 The Role of 5-HT2C Receptor in Epilepsy.............................................. 429 Rita Jakus and Gyorgy Bagdy 23 The Role of Serotonin on Attentional Processes and Executive Functioning: Focus on 5-HT2C Receptors..................... 445 Eleftheria Tsaltas and Vasileios Boulougouris 24 5-HT2C Receptors in Learning................................................................ 461 López-Vázquez Miguel Ángel, Gutiérrez-Guzmán Blanca Érika, Cervantes Miguel, and Olvera-Cortés María Esther 25 The Role of 5-HT2C Polymorphisms in Behavioral and Psychological Symptoms of Alzheimer Disease............................. 509 Antonia Pritchard 26 Ocular Hypotension: Involvement of Serotonergic 5-HT2 Receptors...... 523 Najam A. Sharif Index.................................................................................................................. 545

Chapter 1

The Making of the 5-HT2C Receptor Jose M. Palacios, Angel Pazos, and Daniel Hoyer

1.1 Introduction 1.1.1 5-HT2C Receptors in the Context of Neurotransmitter Receptor Multiplicity: 1983–1988 1.1.1.1 The Concept of Receptor This chapter is a personal account of how the 5-HT2C receptor was discovered and characterized. The discovery of 5-HT2C receptor took place more than 25 years ago, in the middle of a revolutionary period of change in the receptor concept: the realization that single messengers (such as neurotransmitters) were acting through a multiplicity of receptors. The concept of receptor was initially postulated more than 100  years ago by Paul Ehrlich as “selected binding sites for chemotherapeutic agents” and evolved though the work of J.N. Langley who formulated the concept of “receptive substances” (Bennett 2000). For many decades, receptors were defined mainly through functional assays based on responses in isolated tissues to chemical series and translated in pharmacological effects in animal models and from there extended to the therapeutic use of such new molecular entities. This concept is the basis for the development of more than 45% of the about 500 known therapeutic targets and for the discovery of numberless drugs. Despite many decades of receptor research, whether the receptors existed as actual molecular entities was still debated in the 1970s. It was the introduction of radioligand and affinity labeling that led to the solubilization and purification of receptor proteins opening the “molecular era of receptor research” and to the cloning of the first receptors, the cholinergic nicotinic and the b2-adrenergic receptors J.M. Palacios (*) BrainCo Biopharma, Parque Tecnológico de Vizcaya, Edificio 801, 48160 Derio (Bilbao), Spain e-mail: [email protected]

G. Di Giovanni et al. (eds.), 5-HT2C Receptors in the Pathophysiology of CNS Disease, The Receptors 22, DOI 10.1007/978-1-60761-941-3_1, © Springer Science + Business Media, LLC 2011

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representing the two big families of receptors, i.e., ligand gated ­channels and GPCRs (Lefkowitz 2004). The development of the identification of receptors in cell-free preparations using radiolabeled ligands, a simple tool for the characterization of the recognition sites with the properties of the receptors, in a simple and fast manner, opened new possibilities to study reception. Radioligand binding was expanded later to intact tissue sections for the localization at the light microscope level. These new methodologies revealed many unexpected features of drug and neurotransmitter receptors.

1.1.2 The Multiplicity of Receptors for a Single Neurotransmitter One concept that raised tremendous opposition in the beginning was the multiplicity of receptors for a single neurotransmitter. It was a generally accepted concept that the number of receptors for a given neurotransmitter was very limited. The concept moved from the extreme – “one transmitter-one receptor” – to the “liberal” – “one transmitter-two receptors.” Examples include acetylcholinenicotinic and muscarinic, noradrenalin-alpha and b-adrenergic receptors, and histamine-H1 and histamine-H2. Proposing additional or more than two receptors was viewed as a heresy, and if the proposal was coming from results based on radioligand binding, it was viewed as doubly heretical. In other words, radioligand binding was considered to represent a rather obscure side product of biochemical pharmacology, as were the effects of guanine nucleotides on agonist binding (Laduron 1984), which were merely associated with detergent-like effects of the GTP and the likes, rather than on active versus inactive states of the receptor (about 20 years later, the Nobel Prize was attributed for the discovery of G proteins). However, as Galileo said e pur si muove, more and more results were accumulating making it difficult to constrain the size of receptor families.

1.1.3 5-HT-M and 5-HT-D Versus 5-HT1 and 5-HT2 Receptors With respect to serotonin receptors, it should be remembered that in those days (1983/1984 – this is when we started working together on this subject: José María (Chema) Palacios [JMP], Angel Pazos [AP] and Danny Hoyer [DH]), 5-HT receptors were considered to form a fairly simple family, and since the cloning era was to start a few years later, there was little structural evidence for further complexity. In the brain, one could distinguish between 5-HT1 and 5-HT2 sites (Peroutka and Snyder 1979), labeled by [3H]5-HT and [3H]spiperone/[3H]ketanserin, respectively. In the periphery, 5-HT-M and 5-HT-D receptors had been known for some time (the effects of 5-HT on this receptor were first reported in the guinea-pig ileum), but they were not labeled by anything,

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p­ rimarily since this was the field of physiologists working in the intestine, and electro-physiologists in the peripheral nervous system (Bradley et  al. 1986; Gaddum and Picarelli 1957; Hoyer et al. 1994). As it was becoming clear that 5-HT2 sites and 5-HT-D receptors were very closely related (Engel et al. 1984), it was decided to reconsider the whole nomenclature, and there was agreement to name these receptors 5-HT1, 5-HT2 (5-HT-D) and 5-HT3 (5-HT-M): The Bradley scheme was born and the first receptor nomenclature party was in its infancy (Bradley et al. 1986).

1.2 [3H]Mesulergine: An Interesting Molecule 1.2.1 CU32085: A Semisynthetic Ergot Compound It is in this conceptual environment that the discovery of the 5-HT1C (later 5-HT2C) receptors took place at Sandoz (now Novartis) in Basel, Switzerland. Sandoz had a long tradition of working with ergot derivatives with multiple useful pharmacological activities. Semisynthetic ergot compounds were being developed at the end of the seventies in many therapeutic areas. One of those was CU32085, also known as mesulergine. This compound presented interesting dopaminomimetic activities in animal models and was being developed as an antiparkinsonian drug. Binding studies carried out by Annemarie Closse at Daniel Hauser’s group, with the technical help of Armand Wanner, using [3H]mesulergine, surprisingly showed a potent binding of this compound to 5-HT2 receptors in the rat brain (Closse 1983).

1.2.2 Receptor Localization Using [3H]mesulergine In 1983, having just set up the autoradiographic methodology for receptor localization developed in Mike Kuhar’s laboratory at the Johns Hopkins University to label brain tissue sections with radioligands, Jose M. Palacios decided to investigate the localization of [3H]mesulergine. At his lab, Rolf Lenherr incubated rat brain tissue sections with the radiolabeled ligand and generated some autoradiograms. When we developed the films, we were disappointed – at first glance the film appeared “empty.” A closer look revealed some repeated dark lines in the film; after a while, we realized that those heavy labeled thready-looking spots corresponded to the choroid plexus (Fig. 1.1) and were filling the brain ventricles in our tissues sections. This labeling was selectively blocked by coincubation with unlabeled serotonin. We knew from our own experience that radiolabeled serotonin also labeled intensely the choroid plexuses in the rat brain, as well as many other brain areas, but no special attention had been paid to them.

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Fig. 1.1  Images from early autoradiograms of sagittal and horizontal rat brain sections incubated with [3H]mesulergine [(a), (b), and (c)] or 3H-5-HT [(d), (e), and (f)] and different unlabeled compounds. (a) Short exposure revealing intense labeling in choroid plexus. (b) and (c) are longer exposures showing binding of [3H]mesulergine to 5-HT2A and 5-HT2C; (b) and (c) show the blockade of 5-HT2A binding by coincubation with ketanserin. (d) shows [3H]5-HT binding to 5-HT1A, 5-HT1B, and 5-HT2C. (e) Binding to 5-HT1A has been blocked with 8-OH-DPAT. (f) 5-HT1B binding was blocked with compound SDZ 21009 (see text for details)

1.2.3 New 5-HT-Related Site Thus, while one could expect some 5-HT2 labeling as well as dopamine D2 labeling with [3H]mesulergine, our first autoradiograms (probably developed in haste) were pointing to a very different pattern. Simple blockade studies suggested that we were in the presence of a “new 5-HT-related site.” Those preliminary autoradiographic studies were further confirmed by Angel Pazos (AP), a postdoctoral fellow who had just joined the JMP group, together with Roser Cortés, a Ph.D. student at the same laboratory. The striking results were first presented at a meeting of the British Pharmacological Society in 1984 in Birmingham and received with amused commentaries from the establishment. It is noteworthy that these were also the days where comments about “the binding of pepperoni to pizza” were made in Trends in Pharmacological Sciences (Guth 1982). Nevertheless, a full description of the pharmacological profile and characteristics of 5-HT1C receptors was published at the end of the year in the European Journal of Pharmacology, and their detailed anatomical distribution compared with 5-HT1A and 5-HT1B and 5-HT2 receptors was published 1 year later in Brain Research. Those studies have been cited extensively (Pazos et al. 1984a, 1985; Pazos and Palacios 1985).

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1.3 Collecting Pig Choroid Plexus: The Characterization of 5-HT1C Sites by means of Membrane Binding 1.3.1 Danny Hoyer Joined Sandoz Classical membrane binding studies are simple to carry out if one wants to study the kinetic characteristics as well as the pharmacological profile with a large number of drugs to fully characterize a new binding site. Daniel Hoyer (DH), who had just joined Sandoz in 1983 from his postdoc at Perry Molinoff´s lab (Philadelphia), came into the picture and, due to his previous experience in membrane binding methodology, led the radioligand characterization of these sites in several species. Angel did not have a previous background in binding procedures – his PhD work had been mainly on in  vivo biological recordings – but under the supervision of Danny, he soon became familiar with all aspects of binding assays. In order to characterize the pharmacological profile of a new binding site, one needs a tissue rich in these sites that can be easily obtained in sufficient amounts.

1.3.2 Collecting Pig Choroid Plexus Since the putative 5-HT receptor had been clearly identified in the choroid plexus (we later demonstrated its presence in other brain regions but to a much lesser extent), we decided to study its pharmacology by applying radioligand binding to membranes of choroid plexus. As the rat choroid plexus are not easy to collect in large quantities in a reasonable manner, we turned to pig brains – it was cheaper to collect the choroid plexus from brains obtained from the local slaughterhouse. However, dealing with tissue preparation was not simple, since the choroid plexus is primarily a tight mix of vessels and harsh connective tissue, but with the initial help of Monique Rigo, a resourceful technician at JMP laboratory, we managed to obtain good enough membrane fractions, perform regular membrane labeling studies, and get decent quality binding data (Pazos et al. 1984a, b; Hoyer et al. 1985a). From there, we started parallel autoradiographic studies to establish what came to be the 5-HT2C receptor, as will be discussed later in section 1.4 in this chapter. The project in fact followed a research plan carried out in parallel in two different buildings: Autoradiographic experiments were done at building 360 (JMP lab), while membrane assays were carried out at building 386 (DH lab). During the autumn of 1983 and the whole year 1984, it become quite usual to Angel to walk daily from one place to the other, bearing tissue samples, autoradiograms and counter prints in his hands, and frequently, a certain level of anxiety in his mind, since we were using old-generation liquid scintillation counters, with rather modest ­throughput, when we were ­preparing hundreds of samples. This meant that they had to be loaded and unloaded one by one, by hand with two or three loading sessions per day, weekends included; every sample was labeled by hand in case of counter failure and to allow recounting.

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1.3.3 The Profiles of [3H]mesulergine, [3H]5-HT and [3H]LSD in the Choroid Plexus Membranes Thus, we compared the profiles of [3H]mesulergine, [3H]5-HT, and [3H]LSD in the plexus membranes. The very first studies with [3H]mesulergine added an additional surprise to the story. We found that mesulergine had a peculiar and unique binding profile in the choroid plexus but that its pharmacology in the brain across various species was not overlapping. Species differences, a new feature in receptor studies, which was to appear repeatedly afterwards in the serotonin receptor field, made its first appearance adding further complexity to the field until it was recognized that such differences are real and dictated by the gene structure of these receptors (Hartig et al. 1996; Hoyer and Middlemiss 1989). After obtaining the initial data with [3H]mesulergine, the three of us spent many hours (in the labs and also at the canteen) talking about what kind of experiments (at that phase, focusing on binding to membranes and sections) had to be carried out, and which compounds – both labeled and unlabeled – were the best ones to define the new site, especially with respect to the classical 5-HT2 site, which was disturbingly close to 5-HT1C, depending on the tools used.

1.3.4 Discovery of 5-HT1C In rat cortex [3H]mesulergine-labeled sites with a profile indistinguishable to that of [3H]ketanserin-labeled sites (thus 5-HT2), whereas in porcine and human cortex, [3H]mesulergine binding sites were different from [3H]ketanserin-labeled sites. On the other hand, in the choroid plexus of the three species, [3H]mesulergine binding was different from 5-HT2 but equivalent to [3H]5-HT- and [3H]LSD-labeled sites. These sites were named 5-HT1C since our results clearly demonstrated that they were also different from the 5-HT1A and 5-HT1B subtypes, already well characterized as separate entities at that time in our labs, and since they were labeled by [3H]5-HT, the prototypical 5-HT1 radioligand at that time. Obviously, as more receptor families appeared, it became clear that [3H]5-HT was by no means specific for the 5-HT1 family (Hoyer et al. 1994).

1.4 Visualizing 5-HT Receptors at the Microscopic Level: The Power of the Anatomical Resolution 1.4.1 5-HT1C Autoradiographic Characterization In parallel to the studies carried out in membranes, we performed a detailed autoradiographic characterization. After gaining enough expertise with Chema´s advice,

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Angel was dealing for a long time with the challenges and possibilities of brain anatomy across species. Studies were first designed to identify brain areas specially enriched in the different subtypes of 5-HT1 receptors proposed in order to reinforce the singularity of 5-HT1C sites as a separate entity from the others. At that time, there was already quite some evidence that 5-HT1 binding was divided into 5-HT1A and 5-HT1B (Pedigo et al. 1981) and 8-OH-DPAT had just been reported as a 5-HT1A selective agonist (Gozlan et al. 1983; Hjorth et al. 1982; Middlemiss and Fozard 1983).

1.4.2 The Independence of 5-HT1C Sites with Respect to 5-HT1A and 5-HT1B In addition, [3H]OH-DPAT was shown to label a more restricted population of sites in the brain than [3H]5-HT (Pazos and Palacios 1985; Hoyer et  al. 1986a). By using [3H]5-HT and [3H]mesulergine as radioligands and 8-OH-DPAT, the betablocker (SDZ 21-009, a very useful compound picked by Guenter Engel), and mesulergine itself as the main displacers, we succeeded in identifying anatomical areas particularly enriched in each 5-HT1 subtype: Some examples are the dentate gyrus of the hippocampus for the 5-HT1A, the substantia nigra (reticular) for the 5-HT1B, and of course, the choroid plexus for the “new” 5-HT1C. These studies contributed to establishing the independence of 5-HT1C sites with respect to 5-HT1A and 5-HT1B.

1.4.3 The Pharmacological Profile of the New 5-HT1C Receptor Subsequently, our work addressed the characterization of the pharmacological profile of the new receptor at the microscopic level, by constructing autoradiographic competition curves. These studies revealed a pharmacological profile fully comparable to the one found in parallel in membranes (see section 1.5). They also showed the first picture of the distribution of this subtype throughout the rat brain. In addition to the choroid plexuses that presented the highest autoradiographic density by far, clearly detectable levels of the new 5-HT sites were mainly localized over the olfactory system, hippocampus (CA1 field), thalamic nuclei, substantia nigra, and spinal cord (external); lower levels were detected in neocortex (piriform, cingulate, frontal), putamen, globus pallidus, hypothalamus (ventromedial), and several nuclei at brainstem (i.e., spinal trigeminal nucleus). In general, our autoradiographic studies illustrated very well the power of adding the anatomical dimension to the binding strategies at that time. In fact, without the initial identification of a region enriched in only one class of receptor site, it would be rather unlikely to go for the detailed studies in choroid plexuses membranes that eventually led to the full description of the renamed 5-HT2C receptors.

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1.5 More Binding, More Fun 1.5.1 Using Beta-Blockers We subsequently published evidence (Hoyer et  al. 1985a) that brain 5-HT1/ [3H]5-HT binding could be displaced in a triphasic manner by a beta-blocker (SDZ 21-009), which was characterized by high affinity for 5-HT1B sites, intermediate for 5-HT1A sites, and very low affinity for 5-HT1C sites. Our autoradiographic data had also demonstrated the ability of this compound to selectively bind to 5-HT1B sites in specific brain areas (Hoyer et al. 1985a, b). Some further indole beta-blockers turned out to be very important tools for the delineation of 5-HT1 receptor subtypes. Indeed, we noticed that ICYP, [125I]iodocyanopindolol, a very potent antagonist at b-adrenoceptors, which is still the most popular radioligand for labeling these receptors (Engel et al. 1981; Hoyer et al. 1982), was also binding in the brain to sites sensitive to 5-HT and other serotoninergic ligands. Although the radioligand was initially described (in 1981) as highly specific for beta-receptors, we had to admit that it was perfectly suitable to label 5-HT1B receptors in rodents but not in other species.

1.5.2 Using Other Compounds Thus, using an iterative process, we started to put bits and pieces together by comparing the binding profiles of radioligands known to interact with 5-HT/Dopamine D1-D2/5-HT2 or mixed 5-HT2/D2, beta-receptor ligands, and the one that seems to label pretty much everything, [3H]LSD and its derivative [125I]LSD. In fact, [125I] SCH23982 and [125I]LSD were characterized in the choroid plexus and found to label 5-HT1C sites (Hoyer et al. 1986b). We also used [3H]OH-DPAT, which at the time was (and still is) an exquisite tool to define 5-HT1A sites, whereas [3H]ketanserin turned out to be another valuable tool for labeling 5-HT2 receptors. Both membrane and autoradiographic studies allowed us to define the pharmacological profile of affinities on the new receptor, with the drugs available at that time – in addition to 5-HT and mesulergine, LSD, methysergide, and mianserin were among the compounds showing high or very high affinity for 5-HT1C receptors; ketanserine, pirenperone, and methergine bound to the new site with an intermediate affinity; finally, 8-OH-DPAT (a 5-HT1A drug), the beta-blocker and 5-HT1B compound (−) 21009 and spiperone demonstrated low or very low affinity for the new recognition site (Hoyer et al. 1985a). The iterations were multiple: Radioligand binding was performed in brain membranes, including choroid plexus, receptor autoradiography in brain slices of various species, and whichever other functional models could be used, e.g., contractions of the guinea-pig ileum (Engel et al. 1984; Kalkman et al. 1986), inhibition of 5-HT release in the cerebral cortex (Engel et al. 1986), stimulation or inhibition of cAMP

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production in hippocampus (Markstein et al. 1986; Schoeffter and Hoyer 1989a) in the substantia nigra (Schoeffter and Hoyer 1989b), stimulation of PLC production in choroid plexus (Hoyer et al. 1989) or in smooth muscle cells, and contraction or relaxation of various vessels (Hoyer 1988a).

1.6 Labeling 5-HT Receptors in Human Brain 1.6.1 5-HT1C Receptors in Postmortem Human Brain Tissue After characterizing the properties of the new site in animal (rat, pig) brain, we then went on with the characterization and localization of 5-HT1C receptors in postmortem human brain tissue. Taking advantage of the fact that Dr. Alphonse Probst, a pathologist at the University of Basel, shared a collaboration with Chema´s group aimed to visualize and analyze neurotransmitter receptors in human tissue, we could use similar experimental procedures both in membranes and sections from a series of human brains, although confronting the specific limitations associated to the work with postmortem material. The 360–386 buildings connection worked again fairly well and by the end of 1985 the anatomical distribution and pharmacological profile of 5-HT1 and 5-HT2 receptors in the human brain were obtained, proving to be relatively similar to that reported in animals (choroid plexuses starring again), although species differences were evident – in fact, the marked speciesdependent differences in 5-HT1B pharmacology complicated for quite a while the exact delineation of the distribution of 5-HT1C sites in non-choroid plexus areas (Hoyer et al. 1986c, d; Pazos et al. 1987a, b).

1.7 Looking for a Physiological Role for 5-HT Receptors in the Choroid Plexus The enormous concentration of 5-HT1C receptors in the choroid plexus of all the species investigated was calling for an examination of the role of these receptors in controlling one of the functions of this tissue, namely, the volume and composition of cerebrospinal fluid. Monique Rigo cannulated rat cerebral ventricles, examined using perfusion with artificial CSF, and radiolabeled inulin alterations in the volume of CSF. We found effects of 5-HT and other drugs, but the system was too complex to carry out detailed pharmacological studies, and we did not progress enough as to get publishable data. Along the same lines in 1988, LindvallAxelsson and colleagues (Lindvall-Axelsson et  al. 1988) from the University of Lund in Sweden published a detailed study in the rabbit, a better suited model, described the inhibition of CSF production by 5-HT and its blockade by ketanserin.

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The role of 5-HT1C (now 5-HT2C) receptors in choroid plexus function has been the object of intense research, even in outer space. In the NASA Website, one can find a description of experiments proposed by Dr. J. Gabrion (Université Pierre et Marie Curie, Paris, France) aimed at studying the role of several molecules including 5-HT2C receptor in choroidal regulation in space/light conditions.

1.8 The Cloning of 5-HT2C Receptor: Bye Bye 5-HT1C 1.8.1 The 5-HT2C Receptor Signal Transduction Around that time, it became clear that 5-HT2 receptors were acting via the PLC/ PKC/calcium pathway (whereas 5-HT1 receptors were modulating cAMP production), and since there was no evidence for cAMP modulation in the choroid plexus (Palacios et  al. 1986), we searched and found that modulation: PLC, which was indeed positive for 5-HT1C, as did others (Hoyer et al. 1989; Conn et al. 1986). We also suggested that 5-HT1C receptors were present in the stomach fundus and attempted to correlate both activities (5-HT1C binding and 5-HT-mediated contraction of the fundus). However, this is another story since it was the 5-HT2B receptor that is expressed in the fundus (Foguet et al. 1992a, b).

1.8.2 The Cloning of 5-HT2C Receptor Since our initial studies, the pharmacological similarity between 5-HT1C and 5-HT2 in terms of pharmacological profile was rather striking for us (Pazos et al. 1984a, b; Hoyer et al. 1985a, b). Eventually these similarities suggested these receptors to be closely linked (Hoyer 1988b); this was indeed the case when both receptors were finally cloned: 5-HT1C receptor was cloned first (Lubbert et al. 1987) although the full length sequence came a bit later (Julius et al. 1988), and almost simultaneously the 5-HT2 receptor was also cloned (Pritchett et al. 1988). The 5-HT2-receptor family was then extended when the fundus receptor was also cloned, first named 5-HT2F, but then renamed 5-HT2B. Finally, the serotonin receptor nomenclature committee agreed that subtypes existed for 5-HT1 and 5-HT2 receptors (cloning had of course strongly supported these views)(Humphrey et al. 1993). It was then logically decided to allocate 5-HT1C to the 5-HT2 family and 5-HT1C was renamed 5-HT2C, which was least disruptive.

1.8.3 In situ Hybridization Histochemistry in Brain The cloning of 5-HT2C receptors allowed for the first visualization of mRNA coding for this receptor by in situ hybridization histochemistry in brain (Mengod et  al.

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1990), showing that 5-HT2C binding and mRNA distributions were largely overlapping (Pompeiano et al. 1994) and selective 5-HT2C agonists and antagonists were synthesized, some of which are in clinical development (see reference Millan et al. 2003), although none of them has been demonstrated to bind to pizza (Guth 1982)! The progress in the field has been compelling, due to major advances in molecular biology, the availability of selective tools, and their judicious, occasionally “out of the box” thinking.

1.8.4 Gene Encoding the 5-HT2C Receptor The gene encoding the 5-HT2C receptor is extraordinarily complex, which explains why it has taken quite some time to obtain the full sequence (see reference Lubbert et al. 1987). There are three splice variants of the 5-HT2C receptor: the full length receptor and two severely truncated forms (Canton et al. 1996), thought to be inactive. In addition, it has become clear that the primary transcript undergoes RNA editing (Burns et al. 1997), something that is unheard of on the GPCR field but was described for ionotropic glutamate receptors of the AMPA type. In rodents there are four editing sites within the coding region, whereas in humans a fifth editing site is present; together they may produce up to 32 different mRNAs and 24 different proteins. The 5-HT2C receptor is characterized by constitutive activity, the level of which decreases as editing increases (Herrick-Davis et al. 1999). Editing also leads to a loss of the active state of the receptor (Niswender et al. 1999) and a delay in agonist-stimulated calcium release in the fully edited isoforms (Price and SandersBush 2000). The fully edited receptor couples to both Gq/11 and G13, whereas editing reduces or eliminates coupling to G13 (Price et al. 2001). Thus, editing may serve to stop constitutive activity by reducing coupling to G proteins.

1.9 A New Player in the Serotonin Field 1.9.1 Summary We had initially noticed that [3H]mesulergine, a ligand with high affinity for 5-HT2 and dopamine D2 receptors (Closse 1983; Enz et  al. 1984; Markstein 1983), did strongly label the choroid plexus. By using binding techniques to both tissue membranes and sections, we had also shown the presence of a [3H]5-HT binding site in the choroid plexus which was neither 5-HT1A, 5-HT1B, nor 5-HT2A. Indeed, the choroid plexus was not labeled by [3H]8-OH-DPAT (5-HT1A) or [125I]ICYP (5-HT1B), nor was it labeled by [3H]ketanserin, [3H]spiperone, or classical 5-HT2 or 5-HT2/D2 ligands. In contrast, [3H]LSD and [125I]LSD did strongly label the choroid plexus. We named this site 5-HT1C, since it was also labeled by [3H]5-HT: by definition [3H]5-HT binding was defining 5-HT1 sites (later however, it was renamed 5-HT2C, for operational, transductional, and structural reasons as mentioned in section 1.8).

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1.9.2 Posttransductional Changes As previously mentioned, to believe that the 5-HT1C receptor was greeted with general acceptance is a vast overstatement. The area was complex, new, and rather hotly debated. It actually took years to be acknowledged by the 5-HT “experts”: A famous 5-HT researcher at the time suggested that we go back to the books, since “there were only two 5-HT receptors (S1 and S2),” as it was well know that “there were only two dopamine receptors (D1 and D2)”; therefore, any additional complexity was considered superfluous. Needless to say, the statements made by eminent experts had also an influence on internal acceptance of the value of that type of research. For them the worst was still to come, and it arrived with the irruption of the molecular biology techniques. In fact, the family of serotonin receptors ended up with 14 children. The cloning of GPCRs, including that of multiple 5-HT receptors, the use of receptor KOs, and the development of selective tools/drugs facilitated the eventual acceptance of these strange binding sites as receptors. However, cloning was only to start only 3–4 years later, a long time in the career of a young scientist. The first member of the 5-HT2 family to be cloned was the 5-HT1C receptor. Due to its transductional and structural characteristics, and according to the agreement of the Serotonin Receptor Nomenclature Committee, the site was then renamed 5-HT2C.

1.10 A Receptor Looking for a Therapeutic Use The clinical relevance of 5-HT2C receptor editing has been linked in association studies to suicidality (Niswender et  al. 2001), schizophrenia (Sodhi et  al. 2001), anxiety (Hackler et al. 2006), depression (Iwamoto et al. 2005), and spatial memory (Du et al. 2007). However, these data need confirmation, as is common in the field. 5-HT2C receptors have been shown to modulate mesolimbic dopaminergic function, where they exert a tonic inhibitory influence over dopamine neurotransmission (Di Giovanni et al. 1999; Bubar and Cunningham 2007) and, therefore, the interest in this receptor as a therapeutic target for treating abuse (Bubar and Cunningham 2006). The 5-HT2C receptor is also believed to mediate, in part, the effects of antidepressants, e.g., mirtazapine or agomelatine (Cremers et al. 2007), possibly by stimulating neurogenesis, as well as that of atypical antipsychotics (Herrick-Davis et al. 2000). 5-HT2C receptors are expressed in the amygdala, and functional magnetic resonance imaging fMRI data have demonstrated that 5-HT2C receptor agonists produce its neuronal activation (Hackler et  al. 2007). Other potential indications relate to obesity and epilepsy (Tecott et al. 1995; Tecott and Abdallah 2003), but it will be a challenge to produce selective agonists for the 5-HT2C receptor (Bonhaus et al. 1997) that do not interact with the other 5-HT2 subtypes, especially 5-HT2B, which has serious liabilities (see reference Fitzgerald et al. 2000). In any case, 5-HT2C receptors have already made a relevant contribution to the modern pharmacology: The development of selective 5-HT2C receptor drugs has been pioneer in the demonstration of the capability of certain ligands to ­differentially

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activate different signal transduction pathways (mainly inositol phosphate accumulation versus arachidonic acid release) (Berg et al. 2003). This evidence has been instrumental for the concept of “ligand-dependent functional selectivity.” In addition to challenging the dogma of classical pharmacology, this concept will have a clear impact on drug discovery (Millan et al. 2003).  cknowledgments  We thank the members of the JMP and DH labs (M. Rigo, R. Lehnherr, A M. Girod, E. Schuepbach, D. Fehlmann), who were instrumental in training and supporting our PhD students and postdocs active in the labs in the 5-HT1C times (C. Waeber, R. Cortés, M. Dietl, A. Karpf, H. Davies, S. Srivatsa, A. Bruinvels, P. Schoeffter, H. Neijt, V. Doyle, J. Creba, I. SahinErdemli), which in turn contributed in discussions, shared lab and counter spaces, and helped in membrane preparations and some other atypical experiments. We also need to thank our colleagues (G. Engel, K.H. Buchheit, R. Markstein, H. Kalkman, H.G.W.M. Boddeke, A. Enz, A. Fargin, A.K. Dixon, A. Closse, E. Mueller-Schweinitzer, U. Ruegg, K.H. Wiederhold, B.P. Richardson, J.R. Fozard, M.P. Seiler, G. Mengod, E. Schlicker, M. Goethert, A. Probst, D. Middlemiss as well as various members of the 5-HT receptor nomenclature committee) for help and discussing our data in a critical and constructive manner. Finally, we thank Daniel Hauser and Manfred Karobath heads of Preclinical Research at Sandoz, which supported us all along those years.

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Hoyer D, Srivatsa S, Pazos A, et al (1986) [125I]LSD labels 5-HT1C recognition sites in pig choroid plexus membranes. Comparison with [3H]mesulergine and [3H]5-HT binding. Neurosci Lett 69:269–274. Hoyer D, Pazos A, Probst A, et al (1986) Serotonin receptors in the human brain. I. Characterization and autoradiographic localization of 5-HT1A recognition sites. Apparent absence of 5-HT1B recognition sites. Brain Res 376:85–96. Hoyer D, Pazos A, Probst A, et  al (1986) Serotonin receptors in the human brain. II. Characterization and autoradiographic localization of 5-HT1C and 5-HT2 recognition sites. Brain Res 376:97–107. Hoyer D, Waeber C, Schoeffter P, et al (1989) 5-HT1C receptor-mediated stimulation of inositol phosphate production in pig choroid plexus. A pharmacological characterization. Naunyn Schmiedebergs Arch Pharmacol 339:252–258. Hoyer D, Clarke DE, Fozard JR, et al (1994) International Union of Pharmacology classification of receptors for 5- hydroxytryptamine (Serotonin). Pharmacol Rev 46:157–203. Humphrey PPA, Hartig P, Hoyer D (1993) A proposed new nomenclature for 5-HT receptors. Trends Pharmacol Sci 14:233–236. Iwamoto K, Nakatani N, Bundo M, et al (2005) Altered RNA editing of serotonin 2C receptor in a rat model of depression. Neurosci Res 53:69–76. Julius D, MacDermott AB, Axel R, et al (1988) Molecular characterization of a functional cDNA encoding the serotonin 1c receptor. Science 241:558–564. Kalkman HO, Engel G, Hoyer D (1986) Inhibition of 5-carboxamidotryptamine-induced relaxation of guinea-pig ileum correlates with [125I]LSD binding. Eur J Pharmacol 129:139–145. Laduron PM (1984) Criteria for receptor sites in binding studies. Biochem Pharmacol 33:833–839. Lefkowitz RJ (2004) Historical review: a brief history and personal retrospective of seven-­ transmembrane receptors. Trends Pharmacol Sci 25:413–422. Lindvall-Axelsson M, Mathew C, Nilsson C, et al (1988) Effect of 5-hydroxytryptamine on the rate of cerebrospinal fluid production in rabbit. Exp Neurol 99:362–368. Lubbert H, Hoffman BJ, Snutch TP, et al (1987) cDNA cloning of a serotonin 5-HT1C receptor by electrophysiological assays of mRNA-injected Xenopus oocytes. Proc Natl Acad Sci USA 84:4332–4336. Markstein R (1983) Mesulergine and its 1,20-N,N-bidemethylated metabolite interact directly with D1- and D2-receptors. Eur J Pharmacol 95:101–107. Markstein R, Hoyer D, Engel G (1986) 5-HT1A-receptors mediate stimulation of adenylate cyclase in rat hippocampus. Naunyn Schmiedebergs Arch Pharmacol 333:335–341. Mengod G, Nguyen H, Le H, et al (1990) The distribution and cellular localization of the serotonin 1C receptor mRNA in the rodent brain examined by in situ hybridization histochemistry. Comparison with receptor binding distribution. Neuroscience 35:577–591. Middlemiss DN, Fozard JR (1983) 8-Hydroxy-2-(di-n-propylamino)-tetralin discriminates between subtypes of the 5-HT1 recognition site. Eur J Pharmacol 90:151–153. Millan MJ, Gobert A, Lejeune F, et al (2003) The novel melatonin agonist agomelatine (S20098) is an antagonist at 5-hydroxytryptamine2C receptors, blockade of which enhances the activity of frontocortical dopaminergic and adrenergic pathways. J Pharmacol Exp Ther 306:954–964. Niswender CM, Copeland SC, Herrick-Davis K, et al (1999) RNA editing of the human serotonin 5-hydroxytryptamine 2C receptor silences constitutive activity. J Biol Chem 274:9472–9478. Niswender CM, Herrick-Davis K, Dilley GE, et al (2001) RNA editing of the human serotonin 5-HT2C receptor. Alterations in suicide and implications for serotonergic pharmacotherapy. Neuropsychopharmacology 24:478–491. Palacios JM, Markstein R, Pazos A (1986) Serotonin-1C sites in the choroid plexus are not linked in a stimulatory or inhibitory way to adenylate cyclase. Brain Res 380:151–154. Pazos A, Palacios JM (1985) Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin-1 receptors. Brain Res 346:205–230. Pazos A, Hoyer D, Palacios JM (1984) The binding of serotonergic ligands to the porcine choroid plexus: characterization of a new type of serotonin recognition site. Eur J Pharmacol 106:539–546.

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Pazos A, Hoyer D, Palacios JM (1984a) Mesulergine, a selective serotonin-2 ligand in the rat cortex, does not label these receptors in porcine and human cortex: evidence for species differences in brain serotonin-2 receptors. Eur J Pharmacol 106:531–538. Pazos A, Cortés R, Palacios JM (1985) Quantitative autoradiographic mapping of serotonin receptors in the rat brain. II. Serotonin-2 receptors. Brain Res 346:231–249. Pazos A, Probst A, Palacios JM (1987) Serotonin receptors in the human brain. III. Autoradiographic mapping of serotonin-1 receptors. Neuroscience 21:97–122. Pazos A, Probst A, Palacios JM (1987) Serotonin receptors in the human brain. IV. Autoradiographic mapping of serotonin-2 receptors. Neuroscience 21:123–139. Pedigo NW, Yamamura HI, Nelson DL (1981) Discrimination of multiple [3H]5-hydroxytriptamine-binding sites by the neuroleptic spiperone in rat brain. J Neurochem 36:220–226. Peroutka SJ, Snyder SH (1979) Multiple serotonin receptors: differential binding of [3H]5-hydroxytryptamine, [3H]lysergic acid die ethylamide and [3H]spiroperidol. Mol Pharmacol 16:687–699. Pompeiano M, Palacios JM, Mengod G (1994) Distribution of the serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5-HT2C receptors. Brain Res Mol Brain Res 23:163–178. Price RD, Sanders-Bush E (2000) RNA editing of the human serotonin 5-HT(2C) receptor delays agonist-stimulated calcium release. Mol Pharmacol 58:859–862. Price RD, Weiner DM, Chang MS, et al (2001) RNA editing of the human serotonin 5-HT2C receptor alters receptor-mediated activation of G13 protein. J Biol Chem 276:44663–44668. Pritchett DB, Bach AW, Wozny M, et al (1988) Structure and functional expression of cloned rat serotonin 5HT-2 receptor. EMBO J 7:4135–4140. Schoeffter P, Hoyer D (1989) 5-Hydroxytryptamine 5-HT1B and 5-HT1D receptors mediating inhibition of adenylate cyclase activity. Pharmacological comparison with special reference to the effects of yohimbine, rauwolscine and some beta-adrenoceptor antagonists. Naunyn Schmiedebergs Arch Pharmacol 340:285–292. Schoeffter P, Hoyer D (1989) 5-Hydroxytryptamine 5-HT1B and 5-HT1D receptors mediating inhibition of adenylate cyclase activity. Pharmacological comparison with special reference to the effects of yohimbine, rauwolscine and some beta-adrenoceptor antagonists. Naunyn Schmiedebergs Arch Pharmacol 340:285–292. Sodhi MS, Burnet PW, Makoff AJ, et al (2001) RNA editing of the 5-HT(2C) receptor is reduced in schizophrenia. Mol Psychiatry 6:373–379. Tecott LH, Abdallah L (2003) Mouse genetic approaches to feeding regulation: serotonin 5-HT2C receptor mutant mice. CNS Spectr 8:584–588. Tecott LH, Sun LM, Akana SF, et al (1995) Eating disorder and epilepsy in mice lacking 5-HT2c serotonin receptors [see comments]. Nature 374:542–546.

Chapter 2

Serotonin 5-HT2C Receptors: Chemical Neuronatomy in the Mammalian Brain Guadalupe Mengod

2.1 Introduction Serotonin 5-HT2C receptors belong to the 5-HT2 family, which includes 5-HT2A and 5-HT2B receptors. All three share similarities in their molecular structure, pharmacology, and signal transduction pathways (Barnes and Sharp 1999; Hoyer et al. 1994). Initially named 5-HT1C, based on the conventions for naming serotonin receptors at the time of its discovery, it was later renamed 5-HT2C receptors after the cloning of its gene and that of 5-HT2A (Pazos et al. 1984a; Prichett et al. 1988; Julius et al. 1988, 1990; Hoyer et  al. 1985; Lubbert et  al. 1987; Pazos and Palacios 1985) (see also Palacios et  al., Chap. 1, this volume). Chemical neuroanatomical techniques were pivotal in the discovery of 5-HT2C receptors, which were first identified by autoradiography after labeling of rat brain sections with the 5-HT2A and dopamine D2 receptor ligand mesulergine. This was followed by a thorough pharmacological characterization performed in pig brain choroid plexus, and the binding site was differentiated from the 5-HT2A receptor. The combined use of membrane receptor binding/pharmacology and brain slice autoradiography allowed its differentiation from 5-HT1A, 5-HT1B, and 5-HT2A receptors; it was then named 5-HT1C. The pharmacology and distribution of the new site differed from the existing knowledge about other receptors (Pazos et al. 1984a, b; Hoyer et al. 1985). The 5-HT1C receptor was cloned soon thereafter (Julius et al. 1988). The knowledge of the messenger ribonucleic acid (mRNA) sequence and the derived protein sequence of these receptors allowed the development of new important tools for the study of their chemical neuroanatomy. The autoradiographic localization of 5-HT2C receptors had suffered from the lack of selective ligands. While mesulergine remains the ligand of choice, 5HT2C sites can also be labeled by other ligands such as 5-HT self, LSD (lysergic acid diethylamide), and DOI (1-(2,5-dimethoxy-4-iodophenyl-2-aminopropane), but

G. Mengod (*) Departamento Neuroquímica y Neurofarmacología, IIBB/CSIC-IDIBAPS-CIBERNED, Barcelona, Spain e-mail: [email protected] G. Di Giovanni et al. (eds.), 5-HT2C Receptors in the Pathophysiology of CNS Disease, The Receptors 22, DOI 10.1007/978-1-60761-941-3_2, © Springer Science+Business Media, LLC 2011

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always in combination with unlabeled ligands to block additional sites labeled by these ligands. The ability to combine in situ hybridization with radioligand binding autoradiography has allowed the establishment of the anatomical distribution of 5-HT2C receptors in the brain of many animal species by different anatomical and pharmacological manipulations (Pazos and Palacios 1985; Eberle-Wang et al. 1997; Hoffman and Mezey 1989; Mengod et  al. 1990a, b; Molineaux et  al. 1989; Pompeiano et  al. 1994). Immunohistochemistry with antibodies against parts of 5-HT2C receptors has also been used to visualize the receptor protein (Clemett et al. 2000; Abramowski et al. 1995). In this chapter I will review some of the main findings concerning the anatomical and cellular distribution of 5-HT2C receptors in the brain of rodents, primates, and humans with special emphasis on recent studies on the characterization of the phenotype of the brain cells expressing these receptors and the significance of these findings for the understanding the role of the 5-HT2C receptors in brain function.

2.2 5-HT2C Receptors: Neuroanatomical Localization by Radioligand Binding Autoradiography, In situ Hybridization, and Immunohistochemistry in the Rodent Brain 5-HT2C receptor localization is restricted to the central nervous system (CNS), unlike that of 5-HT2A and 5-HT2B receptors. Radioligands that label 5-HT2C receptors are: [3H]mesulergine (in the presence of a selective 5-HT2A antagonist), [3H]5-HT (with adequate protection with a cocktail of 5-HT1 ligands), [125I]SCH23982 (also dopamine D1), and [125I]LSD (in the presence of adequate 5-HT2A selective drugs). The remarkable concentration of 5-HT2C receptors in the mammalian choroid plexus somehow obscures its presence throughout the CNS. Autoradiographic studies have identified this receptor in anterior olfactory nucleus, olfactory tubercle, lateral amygdaloid nucleus, cortex, nucleus accumbens, hippocampus, amygdala, caudate, and substantia nigra in addition to the choroid plexus in rat brain (Pazos and Palacios 1985). 5-HT2C receptor mRNA is very abundant in the pyramidal cell layer of the ventral and posterior part of CA (cornu ammonis) 1 and CA2 fields of the hippocampus and of the anterior part of the CA3, while it is very low in the pyramidal cell layer of the dorsal and anterior region of CA1 and CA2, posterior part of CA3, and the granule cell layer of the dentate gyrus. 5-HT2C binding sites show a regional distribution in agreement with that of the mRNA, being concentrated on the pyramidal cell layer of CA1 and CA2 at ventral levels and the granule cell layer of the dentate gyrus. However, receptors are also seen in the stratum lacunosum molecular of the CA1 and CA3 fields at anterior and dorsal level (Palacios et al. 1991). These localizations are illustrated in Fig. 2.1. The distribution of [3H]mesulergine binding sites in rat brain (Pazos and Palacios 1985) are very similar to those found in mouse brain (Mengod et  al. 1990a). In mouse brain, [3H]mesulergine binding sites (in the presence of spiperone to block

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Fig. 2.1  5-HT2C receptors in the mouse brain. Regional distribution of [3H]mesulergine binding sites and 5-HT2C receptor mRNA in adjacent sections of the mouse brain. (a) and (c) Receptor binding sites labeled by 5 nmol/L [3H]mesulergine. (b) and (d) Hybridization signal obtained with a 33P-labeled oligonucleotide probe complementary to the mRNA encoding 5-HT2C receptors. Pictures are digital photographs from film autoradiograms. Amy indicates amygdala, CA1 CA1 field of the hippocampus, CgCx cingulate cortex, ChP choroid plexus, CPu caudate-putamen, Hp hippocampus, Hy hypothalamus, PAG periaqueductal gray, Pn pontine nuclei, Pyr pyriform cortex, Rt reticular nucleus of the hypothalamus, SN substantia nigra. Scale bar: 1 mm

binding of the radioligand to 5-HT2A receptors) (Mengod et al. 1990a) are present at high densities in choroid plexus, where it is predominant, although the presence of low/very low specific [3H]mesulergine signals can be detected in nucleus accumbens, patches of the caudate putamen, olfactory tubercle, claustrum, septum, cingular cortex, amygdala, dentate gyrus, periaqueductal gray, entorhinal cortex, and several brainstem motor nuclei. This binding is not detected in the 5-HT2C receptor knockout (KO) mouse brain (López-Giménez et al. 2002), indicating that 5-HT2C receptors are indeed present in the brain although at much lower densities than 5-HT2A receptors (with the remarkable exception of the choroid plexus). The distribution of mRNA is very similar to that of protein or binding sites, except for high levels in the habenular nucleus; where binding site levels are very low (Mengod et al. 1990a; López-Giménez et al. 2001a). There are multiple splice and editing variants of 5-HT2C receptors (Fitzgerald et al. 1999; Niswender et al. 1998), which are beyond the scope of this chapter; they are not discriminated, so far as is known, by the antagonist radioligands used in autoradiographic studies. In monkey brain, 5-HT2C mRNA is present in choroid plexus, in layer V of most cortical regions, and in nucleus accumbens, ventral anterior caudate and putamen, septal

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nuclei, diagonal band, ventral striatum, and extended amygdala (López-Giménez et al. 2001a). Several thalamic, midbrain, and brainstem nuclei also ­contain 5-HT2C mRNA. [3H]Mesulergine binding and mRNA show a good correlation across the brain supporting a predominant somatodendritic localization of 5-HT2C receptors. However, in a few instances, a lack of correlation between both patterns of signal suggests a possible location of 5-HT2C receptors on axon terminals. Examples of poor correlation are the septal nuclei and horizontal limb of the diagonal band (presence of mRNA with apparent absence of binding sites) and interpeduncular nucleus (presence of binding sites with apparent absence of mRNA).

2.3 Species Differences in 5-HT2C Receptors Distribution: Rodent Versus Human and Nonhuman Primates The pharmacological profile for 5-HT2C receptors is very similar for human, pig and rat (Hoyer et al. 1985, 1986; Pazos et al. 1984b, 1987) both radioligand binding and autoradiographic procedures in frontal cortex, hippocampus, and choroid plexus of these species using [3H]mesulergine. The distribution of 5-HT2C receptor binding sites in the human brain is somewhat different from that found in the rat brain (Pazos and Palacios 1985; Hoyer et  al. 1986). In the rat hippocampus, 5-HT2C receptor binding sites are located in the stratum lacunosum molecular, whereas in the human hippocampus the pyramidal layer is enriched in these receptors. There are also differences in [3H]mesulergine binding sites in human brain when compared with monkey (Macaca fascicularis) brain. High densities of binding sites are observed in the human globus pallidus and substantia nigra (Pazos et al. 1987), whereas they are absent in monkey globus pallidus and low in substantia nigra (López-Giménez et al. 2001a). In monkey neocortex, low levels of [3H]mesulergine binding sites are detected on layer V, whereas in human cortical areas binding sites are located predominately in layer III. Although the distribution of 5-HT2C receptor mRNA in monkey brain (LópezGiménez et  al. 2001a) is very similar to that in rat (Eberle-Wang et  al. 1997; Pompeiano et  al. 1994; Wright et  al. 1995), mouse (Mengod et  al. 1990a), and human brain (Pasqualetti et al. 1999), there are some differences. In the neocortex of mouse and rat the 5-HT2C receptor mRNA are found at detectable levels only in prefrontal, cingulate, and retrosplenial cortices, whereas in monkey, mRNA is present in all neocortical areas except in the calcarine sulcus within the occipital cortex. The CA3 subfield of the hippocampus is another region of divergence: rat, mouse, and human brain contain this mRNA, and no signal is found in the monkey CA3. The rat entopeduncular nucleus contains 5-HT2C receptor mRNA, whereas its equivalent in primate, the internal segment of the globus pallidus, is devoid of it. In the striatum, the hybridization signal is uniformly intense in human, rat, and mouse brain, whereas in monkey brain this signal is not uniform and is restricted to ventral aspects of the anterior striatum. The substantia nigra is another brain region that presents differences in the distribution of 5-HT2C receptor mRNA among the ­species.

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In human brain the presence of this mRNA is detected predominantly in the pars compacta of this nucleus, whereas monkey brain cells showing the hybridization signal were confined in the lateral part of substantia nigra. As in the rodent brain, there is in general a good correlation between mRNA and binding sites in human and monkey brains with the exceptions identified above. Some of these exceptions are now discussed in relationship to the cellular localization of these receptors.

2.4 Phenotype of Cells Expressing 5-HT2C Receptors The widespread distribution of 5-HT2C receptors in the brain of the mammalian species studied until now suggests, as is the case for other neurotransmitter receptors, that 5-HT2C receptors are expressed by neurons with different neurotransmitter phenotypes. In this section evidence is presented that suggests the presence of these receptors in neuropeptidergic, cholinergic, serotonergic, and GABAergic neurons, as well as recent studies showing the interaction of 5-HT2C receptors with the cannabinoid and the dopaminergic systems.

2.4.1 5-HT2C Receptors and Neuropeptidergic Neurons In nucleus accumbens and striatum, 5-HT2C receptor mRNA was found localized with each of the neuropeptides (enkephalin, substance P, and dynorphin) as shown by Ward and Dorsa (Ward and Dorsa 1996). The level of colocalization was similar among the three neuropeptides but varied by region: high levels of colocalization were observed (from 64% to 89%) ventrally, medially and scattered in patches with high expression of the receptor in the striatum, whereas lower levels of colocalization (43–54%) were observed in matrix-like areas of lower receptor expression. According to the authors this colocalization could provide an anatomical basis for earlier observations that alterations in serotonergic input can lead to changes in the levels of striatal neuropeptides (Kondo et al. 1993).

2.4.2 5-HT2C Receptors and Serotonergic or GABAergic Neurons The cellular localization of 5-HT2C receptor mRNA in relation to serotonergic and GABAergic neurons has been studied in the anterior raphe nuclei of the rat (Serrats et al. 2005). In the dorsal and median raphe nuclei, 5-HT2C receptor mRNA is not detected in serotonergic cells identified as those expressing serotonin (5-HT) transporter mRNA. In contrast, 5-HT2C receptor mRNA is found in the majority of GABAergic cells of the anterior raphe nuclei, mainly located in the lateral and intermediolateral parts of the dorsal raphe and lateral part of the median raphe, supporting

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previous hypotheses that proposed a negative-feedback loop involving reciprocal connections between GABAergic interneurons bearing 5-HT2A/2C receptors and 5-HT neurons in the dorsal raphe and surrounding areas. According to this model, the excitation of GABAergic interneurons through these 5-HT2C (and also 5-HT2A) receptors would result in the suppression of 5-HT cell firing. The finding of 5-HT2C-immunoreactive cells in the raphe nuclei has led to the proposal that some 5-HT neurons might express these receptors (Clemett et  al. 2000). In contrast, electrophysiological data suggest that 5-HT2C receptors are located on local GABAergic neurons, inside or close to the dorsal raphe (Liu et al. 2000), being part of a local negative-feedback circuit that would involve reciprocal connections between GABAergic and 5-HT neurons. This model has been proposed to explain the increases in the frequency of inhibitory postsynaptic currents (IPSCs) induced by 5-HT and the 5-HT2A/2C agonist DOI [1-(2,5)-dimethoxy-4-iodophenyl-2-aminopropane] when applied to rat brain slices containing the dorsal raphe. The localization of the 5-HT2C receptor in GABAergic cells has been also described in other brain areas. Immunohistochemical analyses on the 5-HT2C receptor reveals that this receptor is mainly expressed in deep layers of the rat medial prefrontal cortex (Liu et al. 2007) and cortex (Abramowski et al. 1995) in agreement with the presence of the mRNA coding for this receptor in layers IV and V of PFC of mice (Mengod et al. 1990a), rat (Pompeiano et al. 1994), monkey (LópezGiménez et  al. 2001a), and human (Pasqualetti et  al. 1999). Around 50% of the neurons expressing 5-HT2C receptor immunoreactivity in the prelimbic region of the medial prefrontal cortex also expressed GAD67 immunoreactivity (Liu et  al. 2007), a marker of GABAergic interneurons. We have detected abundant expression of 5-HT2C receptor mRNA in layer V cells of the mouse cingulate cortex that were not GABAergic. A detail of these results is shown in Fig. 2.2.

2.4.3 5-HT2C Receptors and Cholinergic Neurons In our studies on the distribution of 5-HT2C receptor mRNA in the Macaca brain (López-Giménez et al. 2001a) we remarked that several regions where cholinergic cell groups are located also contained mRNA for 5-HT2C receptor. These regions of codistribution include several forebrain areas [medial septal nucleus (cholinergic group Ch1), vertical nucleus of diagonal band (Ch2), horizontal nucleus of diagonal band (Ch3), and nucleus basalis of Meynert (Ch4)], several mesencephalic nuclei [pedunculopontine nucleus (Ch5), laterodorsal tegmental nucleus (Ch6), parabigeminal nucleus (Ch8), oculomotor and trochlear nuclei], and motor nuclei of the brainstem cranial nerve. This correspondence is also observed between the distribution of 5-HT2A receptor mRNA (López-Giménez et al. 2001b) and several mesencephalic and brainstem cholinergic cell groups, particularly in the latter region where the different cranial nerve motor nuclei are highly enriched in both 5-HT2A receptor mRNA and ChAT mRNA.

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Fig. 2.2  Cellular visualization of 5-HT2C receptors mRNA in the cingulate cortex. (a) Macroscopic visualization of 5-HT2C receptor mRNA in the mouse coronal section. The inset in (a), corresponding to the cingulate cortex, is shown at higher magnification in (b). (b) Cellular localization of 5-HT2C receptors mRNA (labeled with 33P, black silver grains) in the cells of cingulate cortical layers. GABAergic cells (GAD mRNA expressing cells seen as a brown precipitate) do not display 5-HT2C receptor mRNA hybridization signal. Scale bars: (a) 1 mm; (b) 20 mm

In fact, the interaction of cholinergic and serotonergic systems has been extensively studied, especially those aspects relating to the modulation of central cholinergic function by serotonin and its possible cognitive implications (See the review in Cassel and Jeltsch (1995)). Regarding 5-HT2C receptors and cholinergic function, several microdialysis studies carried out in rat brain showed the effect of 1-(3-chlorophenyl)piperazine (mCPP) (an unselective 5-HT2C receptor agonist) on the release of acetylcholine in rat cortex (Zhelyazkova-Savova et  al. 1997) and hippocampus (Zhelyazkova-Savova et al. 1999). This effect consisted of an increase of acetylcholine release, which was shown to be mediated by 5-HT2C receptors, especially in the case of the cortex, providing corroborating evidence that the effect was produced particularly via the nucleus basalis magnocellularis (Zhelyazkova-Savova et al. 1997).

2.4.4 5-HT2C Receptors and the Cannabinoid Receptors System The interaction of 5-HT2C receptors with the cannabinoid system through the CB1 receptor has been recently studied (Aso et  al. 2009). CB1 KO mice exhibited a reduction in the expression of the 5-HT2C receptor in dorsal raphe, nucleus accumbens, and paraventricular nucleus, among other brain areas. In contrast, 5-HT2C receptor expression was higher in the CA3 field of the ventral hippocampus of CB1 KO mice, suggesting different roles of this receptor in these brain areas. The decreased expression of the 5-HT2C receptor in the dorsal raphe of CB1 mutant mice

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could lead to a reduction in the inhibitory effect exerted by the 5-HT2C receptor on 5-HT neurons through the GABAergic mechanism (Boothman et al. 2006), which supports the increased 5-HT extracellular levels in the brain areas receiving projections from the dorsal raphe observed in CB1 KO mice. Likewise, the decreased levels of 5-HT2C mRNA in CB1 KO mice, observed within the nucleus accumbens and the paraventricular nucleus of the hypothalamus, could indicate a diminished capacity of this receptor to inhibit dopamine activity (Dremencov et al. 2006) and to stimulate corticotrophin-releasing factor release (Heisler et  al. 2007), respectively.

2.4.5 5-HT2C Receptors and the Dopaminergic Receptors System Dopaminergic nuclei, such as retrorubral area, substantia nigra pars compacta, ventral tegmental area and periaqueductal gray, and dorsal striatum and nucleus accumbens, express 5-HT2C receptor mRNA (Eberle-Wang et al. 1997; Mengod et  al. 1990a; Pompeiano et  al. 1994; Ward and Dorsa 1996). Pharmacological activation of 5-HT2C receptors inhibits firing rates of ventral tegmental area neurons and dopamine release within the nucleus accumbens (Prisco et al. 1994; Di Giovanni et al. 1999; Di Matteo et al. 1998). The implication of 5-HT 2C receptors in the regulation of nigrostriatal dopaminergic function has been a subject of debate mainly due to controversial results obtained with different 5-HT2C receptor acting molecules (Di Matteo et  al. 2001; Porras et al. 2002; De Deurwaerdère et al. 2004; Navailles et al. 2004). Very recently (Abdallah et al. 2009) by using the 5-HT2C receptor null mutant mice, previously generated by Tecott and collaborators (Tecott et al. 1995), it has been studied in a more direct manner the influence of this receptor subtype on functions mediated by the nigrostriatal dopaminergic pathway. Based on results generated by the combination of electrophysiological, pharmacological, neurochemical, and behavioral methods, Abdallah and coworkers have recently described that 5-HT2C receptor null mutant mice displayed (1) an increment in the activity of the dopaminergic neurons of substantia nigra pars compacta, (2) an increment in the extracellular dopamine in the dorsal striatum and nucleus accumbens, (3) increased syntactic grooming chain failures and altered grooming behaviors, and (4) increased sensitivity to the stereotypic behavioral effects following selective dopamine transporter blockade. All these responses occur without phenotypic differences in the elevation of druginduced striatal extracellular dopamine concentration, suggesting that the loss of 5-HT2C receptor function may be accompanied by enhanced behavioral responses to released dopamine. The phenotypic differences these authors observe in stereotypic behavior following selective stimulation of dopamine D1 receptors with an agonist support this hypothesis. All these findings suggest that 5-HT2C receptors play a significant role in the control of nigrostriatal physiology and behavior.

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2.5 Conclusions The studies reviewed here show that 5-HT2C receptors are extensive but distributed heterogeneously in the mammalian brain. Although this distribution shows a remarkable similarity among the species, there are nevertheless significant differences that have been identified. Many different neuronal populations including neuropeptidergic, cholinergic, serotonergic and GABAergic as well as the cannabinoid and dopaminergic systems have been shown to express 5-HT2C receptor mRNA and/or protein. In addition, the distribution suggests the involvement of other transmitter systems such as the glutamatergic and dopaminergic, although colocalization data are still missing. All these chemical neuroanatomical studies clearly point to the role of these receptors in the functions of many brain pathways. The modulation through selective agonists or antagonists of 5-HT2C receptors, an area of intensive research, will reveal the importance of these receptors as therapeutic tools.

References Abdallah L, Bonasera SJ, Hopf FW, et al (2009) Impact of serotonin 2C receptor null mutation on physiology and behavior associated with nigrostriatal dopamine pathway function. J Neurosci 29:8156–8165. Abramowski D, Rigo M, Duc D, Hoyer D, Staufenbiel M (1995) Localization of the 5-hydroxytryptamine2C receptor protein in human and rat brain using specific antisera. Neuropharmacology 34:1635–1645. Aso E, Renoir T, Mengod G, et al (2009) Lack of CB(1) receptor activity impairs serotonergic negative feedback. J Neurochem 109:935–944. Barnes NM and Sharp T (1999) A review of central 5-HT receptors and their function. Neuropharmacology 38:1083–1152. Boothman L, Raley J, Denk F, Hirani E, Sharp T (2006) In vivo evidence that 5-HT(2C) receptors inhibit 5-HT neuronal activity via a GABAergic mechanism. Br J Pharmacol 149:861–869. Cassel JC, Jeltsch H (1995) Serotonergic modulation of cholinergic function in the central nervous system: cognitive implications. Neuroscience 69:1–41. Clemett DA, Punhani T, Duxon MS, Blackburn TP, Fone KCF (2000) Immunohistochemical localisation of the 5-HT2C receptor protein in the rat CNS. Neuropharmacology 39:123–132. De Deurwaerdère P, Navailles S, Berg KA, Clarke WP, Spampinato U (2004) Constitutive activity of serotonin2C receptor inhibits in  vivo dopamine release in the rat striatum and nucleus accumbens. J Neurosci 24:3235–3241. Di Giovanni G, De Deurwaerdère P, Di Matteo V, Di Mascio M, Esposito E, Spampinato U (1999) Selective blockade of serotonin2C/2B receptors enhances mesolimbic and mesostriatal dopaminergic function: a combined in  vivo electrphysiological and microdialysis study. Neuroscience 91:587–597. Di Matteo V, Di Giovanni G, Di Mascio M, Esposito E (1998) Selective blockade of serotonin2C/2B receptors enhances dopamine release in the rat nucleus accumbens. Neuropharmacology 38:1195–1205. Di Matteo V, De Blasi A, Di Giulio C, Esposito E (2001) Role of 5-HT2C receptors in the control of central dopamine function. Trends Pharmacol Sci 22:229–232. Dremencov E, Weizmann Y, Kinor N, Gispan-Herman I, Yadid G (2006) Modulation of dopamine transmission by 5HT2C 5HT3 receptors: a role in the antidepressant response. Curr Drug Targets 7:165–175.

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Niswender CM, Sanders-Bush E, Emeson RB (1998) Identification and characterization of RNA editing events within the 5-HT2C receptor. Ann NY Acad Sci 861:38–48. Palacios JM, Waeber C, Mengod G, Pompeiano M (1991) Molecular neuroanatomy of 5-HT receptors. In: Fozard JR, Saxena PR, eds. Serotonin: Molecular Biology, Receptors and Functional Effects, Basel: Birkhäuser Verlag, 5–20. Pasqualetti M, Ori M, Castagna M, Marazziti D, Cassano GB, Nardi I (1999) Distribution and cellular localization of the serotonin type 2C receptor messenger RNA in human brain. Neuroscience 92:601–611. Pazos A, Palacios JM (1985) Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin-1 receptors. Brain Res 346:205–230. Pazos A, Hoyer D, and Palacios JM (1984) The binding of serotonergic ligands to the porcine choroid plexus: characterization of a new type of serotonin recognition site. Eur J Pharmacol 106:539–546. Pazos A, Hoyer D, Palacios JM (1984) Mesulergine, a selective serotonin-2 ligand in the rat cortex, does not label these receptors in porcine and human cortex: evidence for species differences in brain serotonin-2 receptors. Eur J Pharmacol 106:531–538. Pazos A, Probst A, Palacios JM (1987) Serotonin receptors in the human brain. III. Autoradiographic mapping of serotonin-1 receptors. Neuroscience 21:97–122. Pompeiano M, Palacios JM, Mengod G (1994) Distribution of the serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5-HT2C receptors. Brain Res Mol Brain Res 23:163–178. Porras G, Di Matteo V, Fracasso C, et al (2002) 5-HT2A and 5-HT2C/2B receptor subtypes modulate dopamine release induced in vivo by amphetamine and morphine in both rat nucleus accumbens and striatum. Neuropsychopharmacology 26:311–324. Prichett DB, Bach AWJ, Wozny M, et al (1988) Structure and functional expression of cloned rat serotonin 5-HT2 receptor. EMBO J. 7:4135–4140. Prisco S, Pagannone S, Esposito E (1994) Serotonin-dopamine interaction in the rat ventral tegmental area: an electrophysiological study in vivo. J Pharmacol Exp Ther 271:83–90. Serrats J, Mengod G, Cortes R (2005) Expression of serotonin 5-HT2C receptors in GABAergic cells of the anterior raphe nuclei. J Chem Neuroanat 29:83–91. Tecott LH, Sun LM, Akana SF, et al (1995) Eating disorder and epilepsy in mice lacking 5-HT2C serotonin receptors. Nature 374:542–546. Ward RP, Dorsa DM (1996) Colocalization of serotonin receptor subtypes 5-HT2A, 5-HT2C, and 5-HT6 with neuropeptides in rat striatum. J Comp Neurol 370:405–414. Wright DE, Seroogy KB, Lundgren KH, Davis BM, Jennes L (1995) Comparative localization of serotonin1A, 1C, and 2 receptor subtype mRNAs in rat brain. J Comp Neurol 351:357–373. Zhelyazkova-Savova M, Giovannini MG, Pepeu G (1997) Increase of cortical acetylcholine release after systemic administration of chlorophenylpiperazine in the rat: an in vivo microdialysis study. Neurosci Lett 236:151–154. Zhelyazkova-Savova M, Giovannini MG, Pepeu G (1999) Systemic chlorophenylpiperazine increases acetylcholine release from rat hippocampus-implication of 5-HT2C receptors. Pharmacol Res 40:165–170.

Chapter 3

The Medicinal Chemistry of 5-HT2C Receptor Ligands Marcello Leopoldo, Enza Lacivita, Paola De Giorgio, Francesco Berardi, and Roberto Perrone

3.1 Introduction 5-HT2 receptors are major targets for a wide array of psychoactive drugs, ranging from nonclassical antipsychotic drugs, anxiolytics and antidepressants, which have a 5-HT2 antagonistic action, to hallucinogens, which are agonists of the 5-HT2 receptors. The 5-HT2 receptors form a closely related subgroup of G-proteincoupled receptors and show the typical heptahelical structure of an integral membrane protein monomer. They are currently classified as 5-HT2A, 5-HT2B, and 5-HT2C subtypes, based on their close structural homology, pharmacology and signal transduction pathways. The amino acid sequence of the 5-HT2 receptors shares a high degree (>70%) of identity within the transmembrane segments and consequently, it is not surprising that many compounds bind with high affinity to all these three receptor subtypes. The high degree of sequence homology has considerably hampered the identification of selective 5-HT2C antagonists and especially, agonists (Di Giovanni et al. 2006).

3.2 5-HT2C Receptor Antagonists 3.2.1 Indoline-Containing Structures The search for selective 5-HT2C receptor antagonists has been extensively pursued by researchers at GlaxoSmithKline. The evolution of those studies is graphically summarized by the Structures I to VI depicted in Fig. 3.1. Early studies were aimed to obtain 5-HT2C agents with selectivity over 5-HT2A receptors, because both 5-HT2 M. Leopoldo (*) Dipartimento Farmaco-Chimico, Università degli Studi di Bari, via Orabona, 4, 70125, Bari, Italy e-mail: [email protected] G. Di Giovanni et al. (eds.), 5-HT2C Receptors in the Pathophysiology of CNS Disease, The Receptors 22, DOI 10.1007/978-1-60761-941-3_3, © Springer Science + Business Media, LLC 2011

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Fig.  3.1  The evolution of the first template of 5-HT2C receptor antagonists developed by GlaxoSmithKline

3  The Medicinal Chemistry of 5-HT2C Receptor Ligands

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receptor subtypes are localized within the central nervous system, in contrast to the 5-HT2B subtype, which is localized peripherally. Later, the selectivity over 5-HT2B receptor was considered important because this receptor is notably expressed in heart valves and pulmonary arteries and could be implicated in cardiopulmonary toxicity. Particular attention has been devoted to the pharmacokinetic properties of the new chemical entities, and therefore, structural features that were not strictly necessary for the interaction with the receptor have been incorporated. The starting point was the 5-HT2C receptor antagonist 1 (SB-206553) (pKi = 7.9) with 160-fold selectivity over 5-HT2A receptor. This compound suffered from metabolic demethylation to a nonselective compound and therefore, to circumvent its metabolic liability, the replacement of the pyrroloindole ring was investigated. Modeling studies on a range of isomeric N-substituted pyrrole analogues of 1, in which the pyrrole ring was fused across each of the 4-5, 5-6, and 6-7 bonds of the indoline, provided a definition of an “allowed” volume for 5-HT2C receptor affinity, which included a volume that was “disallowed” at the 5-HT2A receptor. These findings were rationalized by considering key differences in the sequences of the 5-HT2C and 5-HT2A receptors in a region adjacent to the indole N-methyl group in the proposed binding mode of 1. These sequence differences would be expected to give rise in the 5-HT2A receptor to a smaller binding pocket which can less easily accommodate the indole N-methyl group of 1, thus leading to the observed selectivity. On such basis, a series of mono- and disubstituted indolines were studied (Fig.  3.1, Structure I). 5,6-Disubstitution pattern gave the best results in term of affinity at 5-HT2C receptor (pKi values >8) and selectivity over 5-HT2A receptor (>30-fold). In particular, an electron-withdrawing group at the 6 position was preferred and a correlation between 5-HT2C affinity and increasing lipophilicity was apparent at the 5 position. The properties of the substituent in 5 position have been evaluated in depth (compounds 2–17, Table 3.1). In particular, the size and shape of the 5-substituent was varied in order to probe the crucial 5-HT2C-allowed/5-HT2A-disallowed region in combination with the small, lipophilic, electron-withdrawing Cl or CF3 substituent in 6 position. The most interesting results were obtained in the case of the 5-alkylthio- and 5-alkyloxy-disubstituted compounds. Several of these compounds combined high 5-HT2C affinity (pKi > 8) with >100-fold selectivities over 5-HT2A. Moreover, it was evidenced, among the 6-CF3-substituted analogues, that the size and shape of the 5 substituent was crucial to the selectivity of the compounds. The best substituents were the thioethyl (11), thiopropyl (12), and i-propoxy (17). A further increase of the size of the substituent led to a drop in affinity and selectivity (compounds 13 and 14). Molecular modeling on the 5-CH3S-6-CF3 derivative 10 (SB-221284) revealed that these substituents optimally interact/occupy the crucial 5-HT2C-allowed/5-HT2A-disallowed volume. The introduction of a 6-substituent also has the added beneficial effect of restricting the rotation of the 5-substituent to favor those conformations in which the alkyl group occupies the crucial region. Compound 10 was manually docked into a model of the 5-HT2C receptor. The proposed binding mode was very similar to that proposed for 1 with the urea carbonyl oxygen double-hydrogen bonding to the hydroxyl side chains of Ser-312 and Ser-315. The 3-pyridyl ring occupies a lipophilic pocket defined by the side

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M. Leopoldo et al.

Table 3.1  Binding affinities of 5-HT2C receptor antagonistsa

Compound

R5

R6

5-HT2C

   pKi 5-HT2A 5-HT2B

2 CH3 Cl 8.2 6.8 – 3 CH2CH3 Cl 8.3 6.4 – 4 CH2CH2CH3 Cl 7.7 5.9 – 5 CH(CH3)2 Cl 7.7 5.7 – 6 C(CH3)3 Cl 7.7 6.2 6.8 7 OCH2CH3 Cl 7.6 5.4 – 8 OCH(CH3)2 Cl 7.8 400-fold selectivity over 5-HT2A and 5-HT2B receptors. However, the compounds belonging to this class presented high in  vivo clearance. The structure–activity relationships of these compounds indicated that the dihalosubstitution on the phenyl ring was preferred over the monosubstitution; the replacement of the piperidine ring by a morpholine or by a dimethylamino was well tolerated; the shifting of the ethoxyamino chain caused a marked loss in affinity; and the introduction of a double bond in the five-membered ring caused a loss in affinity (Goodacre et al. 2005). A series of diaryl substituted pyrrolidinones and pyrrolones, which fitted the ­pharmacophore model described above, has been studied. The most relevant ­compound was 34 (Fig. 3.3), which showed the best compromise between ­pharmacological and pharmacokinetic properties (pKis: 5-HT2C = 8.5; 5-HT2A = 6.1; 5-HT2B