Serotonin Receptors and their Ligands

PHARMACOCHEMISTRY LIBRARY- VOLUME 27 SEROTONIN RECEPTORS AND THEIR LIGANDS PHARMACOCHEMISTRY LIBRARY ADVISORY BOARD ...

Author: Berend Olivier | I. Van Wijngaarden | W. Soudijn

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PHARMACOCHEMISTRY LIBRARY- VOLUME 27

SEROTONIN RECEPTORS AND THEIR LIGANDS

PHARMACOCHEMISTRY LIBRARY ADVISORY BOARD T. Fujita E. Mutschler

Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan Department of Pharmacology, University of Frankfurt, Frankfurt, F.R.G.

N.J. de Souza Research Centre, Hoechst India Ltd., Bombay, India D.T. Witiak College of Pharmacy, The Ohio State University, Columbus, OH, U.S.A. F.J. Zeelen

Organon Research Centre, Oss, The Netherlands

PHARMACOCHEMISTRY

LIBRARY

E d i t o r : H. T i m m e r m a n

V o l u m e 27

SEROTONIN RECEPTORS AN D THEIR LIGAN DS Edited by" B. Olivieff ,2~

"Solvay Duphar B.V., CNS Research, Weesp, The Netherlands 2~University of Utrecht, Faculty of Pharmacy, Utrecht, The Netherlands

I. van W i j n g a a r d e n

Solvay Duphar B.V., CNS Research, Weesp, The Netherlands

W. Soudijn

Leiden~Amsterdam, Center for Drug Research, Leiden, The Netherlands

ELSEVIER A m s t e r d a m - L a u s a n n e - N e w Y o r k - O x f o r d - S h a n n o n - T o k y o 1997

ELSEVIER SCIENCE B.V. P.O. Box 1527 1000 BM Amsterdam, The Netherlands

ISBN 0-444-82041-8 91997 Elsevier Science B.V. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Science B.V., Copyright & Permissions Department, P.O.Box 521, 1000 AM Amsterdam, The Netherlands. Special regulations for readers in the USA- This publication has been registered with the Copyright Clearance Center Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside of the USA, should be referred to the publisher. No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. This book is printed on acid-free paper. Printed in The Netherlands

PHARMACOCHEMISTRY LIBRARY, edited by H. Timmerman Other titles in this series Volume 19 Small Peptides. Chemistry, Biology and Clinical Studies edited by A.S. Dutta Volume 20 Trends in Drug Research. Proceedings of the 9th Noordwijkerhout-Camerino Symposium, Noordwijkerhout (The Netherlands), 23-27 May, 1993 edited by V. Claassen Volume 21 Medicinal Chemistry of the Renin-Angiotensin System edited by P.B.M.W.M. "13mmermans and R.R. Wexler Volume 22 The Chemistry and Pharmacology of Taxol| and its Derivatives edited by V. Farina Volume 23 QSAR and Drug Design: New Developments and Applications edited by T. Fujita Volume 24 Perspectives in Receptor Research edited by D. Giardin~, A. Piergentili and M. Pigini. Volume 25 Approaches to Design and Synthesis of Antiparasitic Drugs by Nitya Anand Volume 26 Stable Isotopes in Pharmaceutical Research edited by Thomas R. Browne

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vii

Preface The book is the result of the efforts of an international group of authors to produce an overview of the progress made in the medicinal chemistry of compounds (selectively) acting at serotonin receptors or serotonin transporters either as agonists, partial agonists or antagonists. Pharmacological assays in vitro and in vivo are described and structureaffinity, and structure-activity relationships are reported. The tremendous impact of molecular biology on medicinal chemistry research is obvious. By developing elegant techniques of cloning and expression of serotonin receptor subtypes, their mutants and chimeras, an unique opportunity was offered to study the binding mode of serotoninergic ligands to their receptors and transporters. The distribution, structure and homologies ofserotonin receptor subtypes and the structure of the serotonin transporter are also taken into account. The (potential) therapeutic applications of ligands of the different subtypes are described. The technical assistance of Marijke Mulder in the preparation of the manuscript is gratefully acknowledged. Without her help this volume would not have appeared.

The Editors.

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ix

CONTENTS

Preface

vii

SEROTONIN RECEPTOR SUBTYPES (Chapter 1) 5-Hydroxytryptamine receptor subtypes S.J. Peroutka

5-HTIA RECEPTORS (Chapter 2) 5-HT1A Receptor ligands L van Wijngaarden, W. Soudijn and M.Th.M. Tulp

17

Structural characteristics of 5-HT1Areceptors and their ligands W. Kuipers

45

5-HT1AReceptor coupling to G-proteins W. Soudijn

65

Ligand binding assays M.Th.M. Tulp and I. van Wijngaarden

67

5-HT1A Behavioural models J. Mos and B. Olivier

73

Therapeutic applications 5-HT1A receptor ligands I. van Wijngaarden

81

5=HTIBRECEPTORS (Chapter 3) 5-HT m Receptor ligands I. van Wijngaarden and W. Soudijn

87

5-HTIB Receptors W. Kuipers

97

5-HTlv RECEPTORS (Chapter 4)

5-HTID Receptors D.N. Middlemiss, M.S. Beer and V.G. Matassa

101

5-HT1E, 5-HTw RECEPTORS (Chapter 5)

5-HT1E and 5-HTIF Receptors G. McAllister and J.L. Castro

141

5-HT2A, 5-HT2B and 5-HT2c RECEPTORS (Chapter 6)

5-HT2A, 5-HT2B and 5-HT2c Receptor ligands L van Wijngaarden and W. Soudijn

161

The 5-HT2-type receptor family E. Ronken and B. Olivier

199

5-HT 2 Receptor antagonists: (potential) therapeutics W. Soudijn

215

5-HT 3 RECEPTORS (Chapter 7)

5-HT 3 Receptors H. Gozlan

221

5-HT 4 RECEPTORS (Chapter 8)

5-HT4 Receptors A. Dumuis, H. Ansanay, C. Waeber, M. Sebben, L. Fagni and J. Bockaert

261

5-HT 5, 5-HT 6 and 5-HT 7 RECEPTORS (Chapter 9)

The 5-HT 5, 5-HT 6 and 5-HT 7 Receptors R. Grailhe, U. Boschert and R. Hen

311

5-HT TRANSPORTER (Chapter 10)

5-HT Transporter W. Soudijn and L van Wijngaarden

327

Index

363

Chapter 1 SEROTONIN RECEPTOR SUBTYPES 5-Hydroxytryptaminereceptor subtypes

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Serotonin Receptors and their Ligands B. Olivier, I. van Wijngaarden and W. Soudijn (Editors) 1997 Elsevier Science B.V. All rights reserved.

5-Hydroxytryptamine receptor subtypes Stephen J. Peroutka Director of Neuroscience, Palo Alto Institute for Molecular Medicine, 2462 Wyandotte Street, Mountain View, CA 94043, U.S.A., (415) 574-2246, (415) 5716433 (Fax).

INTRODUCTION Alterations in 5-hydroxytryptamine (5-HT) neurotransmission have been implicated in a number of human disorders such as migraine, depression and anxiety as well as in normal human functions such as sleep, sexual activity and appetite. Unfortunately, the scientific association between 5-HT and these disorders has been largely suggestive rather than definitive. Nonetheless, recent advances in the understanding of 5-HT receptor subtypes have strengthened the ability to document specific links between modulation of 5-HT neurotransmission and human disease states. This brief chapter will present an overview of the current status of 5-HT receptor subtypes. Table 1 Overview of 5-HT receptor subtypes G protein-coupled receptors 5-HT 1 "Family":

5-HT1A, 5-HTIB, 5-HTxD, 5-HT m, 5-HT1F,

5-HT7 "Family"" 5-HT~ "Family": 5-HT2 "Family": 5-HT6: 5-HT4:

5-HTd~o~, 5-HTd,o2B, 5-HT..~I 5-HT7, 5-HTa,ol 5-HTsA, 5-HTsB 5-HT2A, 5-HT2B, 5-HT2c 5-HT6 5-HT4s, 5-HT4L

Ligand-gated ion channels 5-HT3 Transporters 5-HT uptake site 5-HT receptors consist of at least 3 distinct types of molecular structures: G protein-couples receptors, ligand-gated ion channels and transporters (Table 1) [62].

Prior to the introduction of molecular biological techniques, the classification of 5-HT receptor was based predominantly on the pharmacological properties of the receptors. For example, "5-HT~" receptors were defined as membrane binding sites which displayed nanomolar affinity for [3H]5-HT [1]. Subsequently, "5-HT~like" receptors were defined by their susceptibility to antagonism by methiothepin and/or methysergide, resistance to antagonism by 5-HT2 antagonists and potent agonism by 5-carboxamidotryptamine (5-CT) [2]. Thus, these classification systems were dependent upon the availability of selective pharmacological agents. THE EVOLUTION SUB2TPES

OF

G.PROTEIN-COUPLED

5-HT

RECEPTOR

Molecular biological data have unequivocally confirmed the existence of multiple 5-HT receptors (Table 2 and 3). Indeed, the multiplicity of 5-HT receptor subtypes, both within and between species, has exceeded most of the predictions that might have been made on the basis of pharmacological data. Within the group of G protein-coupled 5-HT receptors, the evolutionary relationships between the known 5-HT receptor subtypes were determined by a phylogenetic tree analysis (figure 1) [3]. The aligned sequences of all identified G protein-coupled 5-HT receptors were compared and a phylogenetic tree was constructed [4]. The length of each '~ranch" of the phylogenetic tree (figure 1) correlates with the evolutionary distance between receptor subpopulations. Thus, the primordial G protein-coupled 5-HT receptor differentiated into 3 clearly discernible major subtypes as indicated by the three major receptor '"vranches" within the phylogenetic tree: 5-HT 1 receptors (which include 5-HT5 and 5-HT7 receptors), 5-HT2 receptors and 5-HTe receptors. The low level of homology (approximately 25%) between the major branches suggests the various 5-HT receptor subtypes diverged from a common ancestor gene early in evolution. An evolutionary perspective allows these data to be placed in context. Based on the fact that most invertebrate homologs of vertebrate G protein-coupled receptors are approximately 50% identical, then the major subtypes of 5-HT receptors are likely to have evolved prior to the divergence of vertebrates and invertebrates. The differentiation of vertebrates and invertebrates is believed to have occurred approximately 500-600 million years ago. Thus, all groups of mammalian G protein-coupled 5-HT receptor subtypes which display <50% homology are likely to have invertebrate analogs. Indeed, invertebrate homologs of 5-HT~ receptors have been identified in snail [5] and drosophila [6]. Based on this prediction, it is likely that 5-HT 2 and 5-HTe receptor homologs also exist in invertebrates. For the purposes of this review, a receptor "family" is defined as any group of G protein-coupled receptors sharing at least a 50% sequence homology. The 5-HT~ receptor "family a n d the inhibition of adenylylcyclase. The 5-HT 1 receptor "family" or "branch" includes 5-HT1A, 5-HT1B, 5-HTm, 5HTIE and 5-HT1F receptors and certain 5-HT receptors identified in invertebrates

Serotonin 1 B: mouse [--~

1B: rat

I

1B: human

I

1 B: opossum 1D: human 1 D: rat 1D: canine Pseudo: human 1 F: mouse

,~

1F:rat 1 F: human 1E: human 1A: rat 1

,,.

~ ,

1A:rat 2 1A: human

..........

dro 2A: drosophila

.....

dro 2B: drosophila snail 7: mouse 7: rat 7: human dro 1" drosophila 5B: mouse

t

5B: rat 5A: mouse

"l

5A: rat 2A: mouse

~

t ~ 2A:rat 2A: hamster 2A: human 2C: mouse

~

-~

2C:rat 2C:human 2B: mouse

't

~

2B: rat 6: rat

800

7~

6~

5~

400

3~

2~

1~

0

Millions of years ago

Figure 1. Phylogenetic tree of 5-HT receptors (early 1994). The phylogenetic tree was constructed according to the method of Feng and Doolittle [4]. The length of each "branch" of the tree correlates with the evolutionary distance between receptor subpopulations.

Table 2. G protein-coupled 5-HT receptors

second receptor messenger

species cloned

a.a. sequence

=

5-HT~A

5-HT m

5-HTxD

-AC

-AC

-AC

human

422

rat 1 rat 2 mouse

422 422

human

390

rat

386

mouse

386

human

377

canine guinea pig rat mouse

377 374

accession primary nnmber references M83181 X57829 J05276

[26-28] [29]

[30] [31] -

[32]

M89478 M83180 M81590 M75128 X62944 M89954 M85151

[33] [28] [34] [35] [36] [33] [37]

M89955 M81589 X14049

[38] [34] [39]

-

[40]

M89953

[33]

-

[40]

L06179

[7]

5-HT 1

pseudogene

human

5-HTlz

-AC

human

365

Z11166 M92826 M91467

[41] [42] [43]

5-HT1F

-AC

human

366

rat mouse

366 366

L05597 L04962 L05596 Z14224

[44] [45] [44] [46]

-AC

drosophila

834

Z11489

[6]

5-HT~o2n -AC

drosophila

645

Z11490

[6]

5-HT~m

snail

509

L06803

[5]

5-HT~2A

Table 2 (continued). G protein-coupled 5-HT receptors second receptor messenger

5-HTdrol

+AC

5-HTsA

-

species cloned

a.a. sequence

accession primary number references

drosophila

564

M55533

[ 10]

huInan

357 357 357

L10073 Z18278

[59] [12] [11,13]

rat mouse

human

371 370

L10072 X69867

[-] [12] [11]

rat

mouse

5-HTsB

-

5-HT7

+AC

human rat rat mouse guinea pig

445 448 404 448 446

5-HT2A

+PI

human rat hamster mouse

471 471 471 471

X57830 M30705 X53791 -

[27] [47] [48] [49]

5-HT2c

+PI

huInan rat mouse

458 460 459

M86841 M21410 A43951

[27] [50] [51]

rat mouse

479 504

X66842 Z15119

[52] [53]

huinan rat rat rat

440 436 437 438

L41147 L19656 L03202 L41146

[54] [55] [16] [54]

5-HT2B

+PI

5-HTe

+AC

[56] [57] [8] [9] [58]

5-HT4

+AC

human

390

[60]

5-HT4s

+AC

rat

387

U20906

[61]

5-HT4L

+AC

rat

406

U20907

[61]

such as the 5-HTdro2A, 5-I-ITdro2Band 5-HTs~ l receptors [5,6]. The 5-HT~ and 5-HTdro2 receptors share the ability to inhibit adenylylcyclase and are approximately 50% homologous to one another. The next evolutionary differentiation within this "family" of subtypes occurs when 5-HT1^ receptors branch from a receptor group which eventually evolved into 5-HT1B, 5-HT~D, 5-HT~E and 5-HT~F receptors as well as an apparent human 5-HT~ receptor pseudogene [7]. For all identified members of the 5-HT 1 receptor "family", the interspecies variation is minimal (i.e. >90% identity between species homologs) as indicated by the very short branches linking these subtypes in the phylogenetic tree (figure 1).

The 5-HT7 receptor "fsmil3f' and the stimulation of adenylylcyclase. Recently, 5-HT 7 receptors have been identified in rat [8,57] mouse [9], guinea pig [58] and human [56]. Although designated "5-HT7" receptors, these 5-HT receptors are most closely homologous to mammalian 5-HT~ and 5-HT5 receptors. 5-HT 7 receptors are positively coupled to adenylylcyclase [8,9]. The mammalian 5HT 7 receptors may be the vertebrate homologs of the 5-HTdrol receptor [10], as can been seen in figure 1. The observed phylogenetic relationship between these receptors is further supported by the fact that both mammalian 5-HT 7 receptors and 5-HTd~ol receptors are positively linked to adenylylcyclase. The 5-HT s receptor "family". The 5-HTsA and 5-HTsB [11-13,59] receptors appear to have differentiated early in evolution since they are equally homologous to both the vertebrate 5-HT1 and 5-HT5 receptors and the invertebrate 5-HTd~o and 5-HTsn~l receptors. Biochemical studies have shown that 5-HT5 receptors do not alter the levels of cAMP or inositol phosphates. Therefore, the second messenger system of 5-HT5 receptors remains to be identified. The 5-HT~ receptor "family" and the stimulation of phosphatidyl inositol turnover. The 5-HT 2 "family" or "branch" of G protein-coupled 5-HT receptors includes 5HT2A, 5-HT2c and 5-HT2B receptors. These receptors share a significant number of molecular biological, pharmacological and biochemical characteristics [14,15]. However, their characteristics are quite distinct from all other 5-HT receptor subtypes. Based on their homologies to each other, these three 5-HT receptor subtypes are likely to have differentiated from one another approximately 500 million years ago. 5-HTe receptors. In early 1993, the cloning and expression of a third major subtype of a G protein-coupled 5-HT receptor was reported [6]. The receptor appears to have differentiated from the primordial 5-HT receptor just prior to the differentiation of the 5-HT 1 and 5-HT2 receptor "families" (figure 1). This receptor has, thus far, had been identified only in rat but was recently cloned and shown to be present in human brain [54]. 5-HT e receptors are positively linked to adenylylcyclase, thus making them functionally similar to 5-HT7 receptors. However, based on their

molecular structure and pharmacological properties, they appear to represent the first identified member of an extremely ancient 5-HT receptor "family". 5-HT 4 receptors.

The human and rat 5-HT4 receptor was recently cloned and expressed [60,61]. Two splice variants of the 5-HT~ receptor of the rat have been isolated: 5-HT4s encoding 387 amino acids and 5-HT4L encoding 406 amino acids. The long isoform contains a protein kinase C phosphorylation site in the C-terminal which is not present in the short isoform [61]. The 5-HT4 receptors are positively coupled to adenylylcyclase. Functionally the 5-HT4 receptors are similar to the 5-HT8 and 5HT7 receptors. Table 3 Other 5-HT receptors.

second receptor messenger 5-HT 3

5-HT

5-HT 3

Cation channel

Transporter

RECEPTOR

species cloned

a.a. sequence

accession primary number references

human rat mouse 1 mouse 2

487 483

M74425 X72395

human

630

LO5568

LIGAND-GATED

ION

[21] [23] [17] [24] [20J [25]

CHANNELS

AND

5-HT

TRANSPORTERS In contrast to the G protein-coupled 5-HT receptors which modulate cell activities via second messenger systems, 5-HT3 receptors directly activate a 5-HTgated cation channel which rapidly and transiently depolarizes a variety of neurons. In late 1991, the primary amino acid structure of a 5-HT a receptor subunit was reported [17]. Like other ligand gated ion channels, the 5-HTs receptor consists of 4 transmembrane segments and a large extracellular Nterminal region incorporating a cysteine-cysteine loop and potential Nglycosylation sites. The generation of a functional 5-HTa receptor with a single clone indicates that homomeric receptors exist. A third major molecular recognition system for 5-HT are the transporter proteins. Transporters consist of 12 membrane spanning proteins and represent a large gene family encoding Na § and Cl dependent transport proteins. For 5-HT, the first transporter was identified in rat in 1991 [18,19]. The human 5-HT

lO transporter was identified in human placental cDNAs. Transfection of the 5-HT transporter cDNA yielded a high affinity (I~=460 nM) transport activity in HeLa cells which was Na § and Cl" dependent [20].

OTHER 5.HT RECEPTORS At the present time, the differentiation of 5-HT receptors into 3 molecular types of receptors appears to be the most relevant classification system (Table 1). A variety of data indicate that multiple other, as yet unidentified, 5-HT receptor subtypes also exist. Further studies are needed to determine whether the 5-HT4 receptor represents yet another major "family" of 5-HT receptors or, since the site is clearly linked to the stimulation of adenylylcyclase, whether it will eventually be classified as a member of the 5-HTs or 5-HT7 "families". The number of such "orphan" receptors is likely to decrease in the years ahead as a result of the rapidly evolving molecular biological data. THE CLASSIFICATION OF 5-HT RECEPTORS Recent discussions have focused on the need for a "new approach to receptor characterization" for 5-HT receptors [22]. One group has recommended that both operational (i.e. pharmacological) and transductional information are required in order to categorize receptors. However, a more practical approach to receptor classification could be based on molecular structure and the inferred evolutionary relationships between the receptor subtypes. This molecular evolutionary approach to receptor classification allows for the inclusion of additional G protein-coupled 5-HT receptors as their sequences are identified, independent of the availability of selective pharmacological agents of functional assay systems. Moreover, this classification system does not preclude the addition of pharmacological and transductional information for each receptor. Indeed, a molecular evolutionary nomenclature system allows the pharmacological and transductional information to be determined at any time, either prior to or subsequent to the identification of the structure of the receptor. Humprey et al. [22] stated recently that their proposed nomenclature system for 5-HT receptors was "not rigid and will certainly have to be modified as more information is gained and our understanding evolves". By contrast, a nomenclature system based on molecular evolution will not change unless new mutational events occur in the millennia ahead.

Acknowledgements I thank Tiffany A. Howell for excellent editorial assistance. This work was supported in part by NIH Grant NS 25360-07.

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Chapter 2 5-HTIA R E C E P T O R S 5-HT~A Receptor ligands Structural characteristics of 5-HT1A receptors and their ligands 5-HT1A Receptor coupling to G-proteins Ligand binding assays 5-HT~A Behavioural models Therapeutic applications 5-HT~AReceptor ligands

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Serotonin Receptors and their Ligands B. Olivier, I. van Wijngaarden and W. Soudijn (Editors) (~) 1997 Elsevier Science B.V. All rights reserved.

17

5-HT1AReceptor ligands I. van Wijngaarden a, W. Soudijn b and M.Th.M. Tulp a ~Solvay Duphar B.V., P.O.Box 900, 1380 DA Weesp, The Netherlands. bLeiden/Amsterdam, Center for Drug Research, P.O.Box 9502, 2300 RA Leiden, The Netherlands.

INTRODUCTION For the 5-HTIA receptors many potent and selective ligands, belonging to different chemical classes, such as aminotetralins, indolylalkylamines, ergolines, aporphines, arylpiperazines and aryloxyalkylamines, are available (for reviews see [I-5]). The majority of these compounds are agonists or partial agonists. Pure antagonists, devoid of any agonistic activity at presynaptic receptors (somatodendritic autoreceptors) or postsynaptic receptors were identified only very recently (for details of the definition of agonists, partial agonists and antagonists, see [6]).

Aminotetralins The best known member of this class is 8-OH-DPAT (Table 1). For more than a decade 8-OH-DPAT is the most frequently used tool to characterize 5-HT receptors [7]. 8-OH-DPAT displays a high affinity for 5-HTIA receptors and weak (or no) affinity for other 5-HT receptor subtypes or transmitter receptors tested [8,4]. 8-OH-DPAT is a racemate, but the compound and its enantiomers are all potent 5-HT1A ligands [9] (Table 1). In functional tests, however, the enantiomers behave differently: (R)-8-OH-DPAT is a full agonist and (S)-8-OH-DPAT is a partial agonist [1]. Replacement of the N,N-di-n-propyl groups of 8-OH-DPAT or 8-MeO-DPAT by smaller or larger di-n-alkyl substituents results in a significant drop in affinity [10]. The rankorder of potency is di-n-propyl > di-n-ethyl > di-n-butyl > di-nmethyl (Table 1). For the smaller di-n-alkyl substituted analogues (C,-C~), the (R)enantiomers are more potent than the (S)-enantiomers. This stereo selectivity however, is reversed if larger di-n-alkyl substituents are introduced. The (S)enantiomer of the di-n-butyl compound is about two times more potent than the (R)-enantiomer (Table 1). Without loss in affinity one of the n-propyl groups can be omitted. The second n-propyl is essential for high binding. The unsubstituted analogues, such as 8MeO-AT, display only a moderate affinity for the 5-HTIA receptors [11]. Potent compounds are obtained if the aminogroup is monosubstituted with relatively large substituents. Even the presence of an extra N-methyl group is well tolerated [12,13] (Table 1).

18 Fixation of the N,N-di-n-propyl groups of 8-MeO-DPAT into a piperidine ring decreases affinity by a factor of 29 [11]. Table 1 Effect of substitution of the m~no group of aminotetralins on 5-HTI^ affinity

91

\

R

COMPOUND

_+8-OH-DPAT (R)-8-OH-DPAT (S)-8-OH-DPAT (R)-8-OH-DMAT (S)-8-OH-DMAT (R)-8-OH-DEAT (S)-8-OH-DEAT (R)-8-OH-DBAT (S)-8-OH-DBAT • 8-MeO-DPAT • 8-MeO-PAT • 8-MeO-AT

N-R2

R

R1

R2

pI~.

Ref

OH OH OH OH OH OH OH OH OH OMe OMe OMe OMe OMe OMe

n-Pr n-Pr n-Pr Me Me Et Et n-Bu n-Bu n-Pr n-Pr H (CH2)sPh (CH2)sPh (CH~)~PTH**

n-Pr n-Pr n-Pr Me Me Et Et n-Bu n-Bu n-Pr H H H Me H

8.5 8.4 8.2 7.1" 6.2* 8.0* 6.9* 7.0* 7.3* 8.6 8.3 6.8 8.6 8.7 8.0

[9] [9] [9] [10] [10] [10] [10] [ 10] [ 10] [11] [11] [11] [12] [12] [13]

Data are expressed as pI~. values. * pICso; ** PTH = 2-phthalimido The position of the monohydroxy group at C-8 of the aminotetralins is crucial for 5-HT~^ affinity. The other monophenolic regio-isomers are pure dopamine agonists [14]. Binding to 5-HT~^ receptors is only slightly influenced if the 8hydroxy function is replaced by a methoxy group. 8-MeO-DPAT is about as potent as the parent compound, indicating that the proton of the 8-hydroxy group is not essential for drug-receptor interaction [1,15] (Table 2).

19 Other favourable C-8 substituents are acetyl and methoxycarbonyl. The corresponding carbamoyl analogue is somewhat less potent. A carboxyl group however, is detrimental for 5-HTI^ affinity [1,15] (Table 2). High affinity is also obtained by the introduction of a chlorine atom at position C-8. A fluorine atom is less favourable [1]. Large substituents such as phenyl,2-furyl or benzylthio are well tolerated [15,16,1] (Table 2). For most derivatives the (R)-enantiomers are more potent than the (S)-enantiomers. Table 2 Effect of C-8 substitution of DPAT on 5-HT1^ affinity

k_ \ f N

R2 COMPOUND (racemic)

R1

R2

pICso (RS)

pI~. (R)

pKi (S)

Ratio R/S

LY 133610 LY 198354 LY 198342 LY 198743 8-OH-DPAT 8-MeO-DPAT

H F C1 CH s OH OCH3 COCHs COOCH3 COOH CONH 2 CsH~ C4HsO** SCH2CeH5 OH

H H H H H H H H H H H H H F

7.5 7.1 8.0 7.4 8.6 8.3 9.0* 8.6 i.a. 8.1 7.9*

7.7 n.d n.d 7.5 8.9 8.8 8.7 8.4 n.d n.d 8.1 8.0 n.d 8.2

7.3 n.d n.d 7.2 8.7 8.6 9.2 8.8 n.d n.d 7.6 8.7 n.d 7.3

3.3

LY 198968 LY 161610 LY 233178 UH-301

7.9 n.d

1.8 1.4 1.9 0.4 0.4 3.1 0.2 8.5

Data, expressed as pICso are from [1]; data, expressed as pKi values are from [15,16]; IXH-301 data are from [17]. * pI~.; ** 2-furyl.

20 The stereoselectivity is reversed in the C-8-methoxycarbonyl,C-8-acetyl - and 2furyl substituted analogues, i.e. the (S)-enantiomers are more potent than the (R)antipodes [15,16]. Substitution of (R)-8-OH-DPAT with a fluoro atom at position C-5 (UH-301) decreases the 5-HT~A affinity slightly (pI~.=8.2) [17]. In functional studies the (R)enantiomer behaves as a partial agonist [1]. The (S)-enantiomer (S)-UH301 is about nine times less potent than the corresponding (R)-enantiomer (Table 2). But in contrast to (R)-UH301, the (S)-enantiomer is a pure antagonist at both pre- and postsynaptic receptors [17]. Unfortunately (S)-UH301 is not highly selective for 5HT~A receptors. The compound has also affinity for dopamine D2 receptors (p~=6.4). Modification of the non-aromatic ring of the 2-amino-tetralins, such as ring contraction (indamines) [18], or ring expansion (benzocycloheptamines) [19] decreases 5-HT~ affinity. Replacement of the C-4 by oxygen (aminochromans) does not influence affinity and selectivity [20,21]. Substitution at C-1 of the non-aromatic ring of tetralin with methyl introduces high stereoselectivity. Of the C-l-methylated 8-OH-DPAT enantiomers, only cis (2R)-3 displays high affinity for 5-HT~Areceptors (Table 3). The antipode cis (2S)-4 is hardly active. The trans racemate (5,6) shows low affinity [9]. Cis (2R)-3 however is not selective for 5-HT~A receptors. The compound displays also affinity for dop_amine D2 receptors. In functional tests cis (2R)-3 behaves as a mixed partial 5-HT1A receptor agonist/D~ receptor antagonist [22]. Restriction of the conformation of 1-Me-8-OH-DPAT by incorporation of the C-1 methyl and the C-2 nitrogen into a four-membered azetidine or five-membered pyrrolidine ring enhances 5-HT~A affinity [23,24] (Table 3). These more rigid four/six and five/six fused angular tricyclic 2-aminotetralins were N-substituted with either n-propyl or its bioequivalent 2-propenyl. The cis racemate of the azetidines 7 and 8 is about ten times more potent than cis(• (Table 3). A similar high affinity is also obtained in the cis pyrrolidine analogues. In the cis series the (3aR)-enantiomer 9 shows the highest affinity. The trans racemate of 11 and 12 possesses also a good affinity for 5-HT,A receptors, although weaker than the corresponding cis analogues. Replacement of the 8-OH in the azetidines or 9-OH in the pyrrolidine series by 8-MeO and 9-MeO respectively did hardly affect the 5-HT~A affinity. Of the fused tricyclic 2-aminotetralins only the cis analogues substituted with a hydroxyl group on the aromatic ring are selective 5-HT~A ligands. The corresponding trans analogues and all methoxy-substituted compounds are mixed 5-HT~A agonists and dopamine D~ antagonists [23,24]. In the methoxy-substituted cis series high 5-HT,A selectivity could be obtained by placing an extra methyl group on the pyrrolidine ring at the C-2 position. The (• CHs-substimted analogues (pI~.=9.0) are more potent than the corresponding (• CHs compounds (pI~.=7.S) [24]. Incorporation of the C-1 methyl and the C-2 nitrogen into a more flexible sixmembered piperidine ring (Table 3, 13-16) is less favourable for 5-HT1A affinity [9,75]. In these six/six fused angular tricyclic 2-aminotetralins, the trans enantiomers 15 and 16 are more potent than the cis antipodes 13 and 14.

21 This is in contrast to the pyrrolidine series, where the cis analogues are more potent th an the trans analogues. There is a decrease in 5-HT1A affinity of more than three-orders of magnitude by incorporation the C-1 methyl and C-2 nitrogen into a cis six/six fused instead of a cis five/six fused tricyclic 2-aminotetralin. Table 3 5-HTI^ ligands derived from 8-OH-DPAT

9

1

HO

pKi = 8.4

2

pKi = 8.2

C

3

pKi = 8.7

4

pKi = 5.5

_]

c_N/"

--'~

pKi = 9.5

T

T

8

cC.~_/- cA._/9

pKi---10

6

pKi = 6.0

Y

c_N/"

7

5

N

10

r-~ N .../-

pKi = 8.5

cA._/= 11

c,,-~_/-

C

cANS 12

pKi = 8.3

~ _ N/

9

C

c

Z-x _/-N

"OC__

.

13

p[Cso=6.6

14

15

pKi=7.5

16

pKi = 8.4

22 This fall in 5-HTI^ affinity is not seen in the corresponding trans analogues (Table 3). Alkyl substitution at C-3 of the tetralin ring of 8-OH-DPAT is unfavourable for high 5-HT1^ affinity (Table 3, compounds 17-20). Only the trans (2S) enantiomer 1__99displays a moderate affinity, being ten times less potent than (R)-8-OH-DPAT. The other three enantiomers are weak 5-HTI^ ligands [9]. In corporation of the C-2 nitrogen and C-3 methyl into a five-membered pyrrolidine ring does not enhance 5-HT~^ affinity [25]. These five/six fused linear tricyclic 2-aminotetralins are only moderately active (Table 3, compounds 21-24). The cis racemate (21, 22) is equipotent to the trans racemate (23, 24). Adding an extra methyl on the C-2 of the pyrrolidine ring of racemic cis 21, 22 enhances 5-HT~^ affinity when placed in the 2~-position (pI~.=8.0) but decreases affinity when placed in the 2a-position (pI~.=6.4) [25]. Affinity for dopamine D 2receptors was not observed in these five/six linear tricyclic 2-aminotetralines. Incorporation of the C-2 nitrogen and C-3 methyl in a six membered piperidine ring is accompanied by a further decrease in 5-HT~^ affinity [9] (Table 3, compounds 25-28). Only the cis (4aS, 10aR) enantiomer 26 displays a moderate affinity for 5-HT~^ receptors. The other enantiomers 25, 27, 28 are not or weakly active. Table 3 (continued)

"0

~

17

CO

21

~jlc

pKi = 5.7

~

/

pKi =

"Od:> "00 c

18

CO

8.8

22

i.a

26

pKi = 6.8

20

~

23

"

,,Ic

pKi = 7.3

c

lOa . N ' ~

25

19

pKi = 6.4

c

pKi =

C

7.0

24

.o 27

pKi = 5.9

"5 i.a

28

pKi = 5.6

Data, expressed pK 1 values are from [9] (8-OH-DPAT; C-1 and C-3 alkylated 8OH-DPA~, 6-6 fused analogues); [24,25] (6-5 fused analogues); [23] (6-4 fused analogues) and [75] (6-6 fused analogues 13, 14).

23 A different six/six fused angular tricyclic of 2-aminotetralin is obtained by incorporation of the 8-oxygen atom and C-7 into a six membered pyran ring [26]. This modification, however, reduces affinity. The (R)-enantiomer (p~=7.7) is about five times less potent than (R)-8-OH-DPAT and the (S)-enantiomer (pI~.=6.9) is even twenty times less active than (S)-8-OH-DPAT.

Table 4 5-HT,A ligands derived from 2-aminotetralins 94

R2

UH92016A

(R) or (S)

R,

(R) (S) (R) (S) (R) (S) (R) (S) (R) (S) (R) (S) (R) (S) (R) (S)

H H CHO CHO CHO CHO H H H H CN CN H H H H

\

R~

R2

H H H H H H CONH2 CONH2 CN CN H H CN CN CONH 2 CONH 2

]

R3

n-Pr n-Pr n-Pr n-Pr n-Pr n-Pr n-Pr n-Pr n-Pr n-Pr n-Pr n-Pr n-Pr n-Pr n-Pr n-Pr

N--- R3

R4

n-Pr n-Pr n-Pr n-Pr GIB* GIB n-Pr n-Pr n-Pr n-Pr n-Pr n-Pr GIB GIB GIB GIB

5-HTIA

D2

(pI~.)

(pI~.)

8.2 7.9 8.9 8.7 9.2 8.8 8.8 8.7 10 9.4 8.6 8.5 9.7 8.6 9.2 7.0

7.0 6.8 6.4 5.4 7.2 6.1 6.7 <6.0 7.5 6.1 6.4 5.1 7.4 <6.5 7.4 6.0

Data, expressed as pK i values are from [27,28,31] (R1 substituted compounds) and [29] (1~ substituted compounds). *GIB 4,4'-diakylglutarimidebutyl.

24

A further decrease in affinity is seen in compounds bearing an annelated pyrrole ring in which the indole N H moiety is in the same position as the hydroxy group of 8-OH-DPAT [27,28]. However the isosteric 6,7,8,9-tetrahydro-N,N-di-npropyl-3H-benzindole-8-amines display high 5-HTL~ affinity(Table 4). The affinity and selectivityfor 5-HTI^ receptors improves further by substitution of the pyrrole ring at C-1 with a formyl or cyano group [27] (Table 4). Also substitution at the C-2 of the pyrrole with a carboxamlde or cyano function is favourable for 5-HTIA affinity [29]. In the C-1 and C-2 substituted series the (R)-enantiomers display a high affinity for the 5-HTI^ receptors and only a moderate affinity for the dopamine D 2 receptors. The (S)-enantiomers are somewhat less potent but even more selective 5-HT~^ ligands. An unsubstituted indole-NH moiety is crucial for the interaction with the 5-HTI^ receptor. The N- methyl compounds are significantly less potent [30]. Without loss in 5-HT~A affinity one of the propyl groups can be replaced by a variety of large substituents such as glutarimidebutyl [29,31] (Table 4). In functional tests most of the (R)-enantiomers behave as full agonists, whereas the corresponding (S)-enantiomers are partial agonists. Indolylalkylamines The prototype of this class is serotonin (5-HT), a potent but non-selective 5HTIA agonist [8] (Table 5). The hydroxyl group at position C-5 is necessary for high affinity.The unsubstituted analogue tryptamine is thirty times less potent than 5-HT. The 5-hydroxy group can be replaced by 5-methoxy (5-MeOT) or 5carboxamide (5-CT) without loss in 5-HTI^ affinity [11]. N,N-di-alkylation enhances selectivity without affecting the 5-HTI^ affinity.The N,N-di-n-propyl analogue of 5-CT (DP-5-CT) is a potent and selective5-HTIA agonist [32,4] (Table 5). Alkylation of 5-HT at the C-1- and C-2-positions decreases affinityby a factor of 60 and 400 respectively [33,8]. Incorporation of the alkylamine side-chain of 5-HT and analogues into a 4substituted tetrahydropyridine ring lowers affinity [34]. The tetrahydropyridylindole derivative of DP-5-CT (TD 60, Table 5), for instance is about 60 times less potent than the parent compound [1]. Incorporation of the side-chain of DP-5-CT into a 3-substituted tetrahydropyridine (TD59, Table 5) results in a smaller decrease in affinity. Replacement of the 5-carboxyamido function of TD59 by e.g. methoxy, cyano or hydrogen lowers the 5-HTIA receptor affinity 50-100 fold [79]. TD59 is a full agonist and TD60 a partial agonist [1].

Ergolines

Combining the structural elements of the indolealkylamines with the 2aminotetralins into the ergoline skeleton results in potent and selective 5-HTIA ligands [35]. The S A R at C-6 of this partial ergoline structure is similar to the S A R at the C-8 position of the 2-aminotetralins. From the series LY228729 displays the highest potency (Table 5). The other antipode LY228730 is 2 times less potent. In functional tests LY228729 is an agonist at pre- and postsynaptic 5-HTRA receptors [36].

25 In contrast to the tricyclic partial ergolines, the tetracyclic ergolines such as dLSD (pI~.=8.7), lisuride (pI~.=9.1) or pergolide (pI~.=8.7) bind with high affinity to 5-HT1A receptors but they lack selectivity. Table 5 Indole containing 5-HT1A ligands

NH2 H

H2N -

---~N j -

O !1 C/~/

H 5-HT

pK~

H = 8.4

DP-5-CT

pKi = 9.5

k

pKi = 7.8

TD-59

pKi = 8.4

\ O

\ \_]--

\

H

H TD-60

_

S

._/-

H2N -

~Nf H LY228729

H pKi = 9.9

PERGOLIDE

pKi = 8.7

Data, expressed as pI~. values are from [8] (5-HT); [32] (DP-5-CT); [1] (TD59, TD60), [35] (LY228729); [23] (pergolide).

26

Aporphines An other aminotetralin containing compound is (R)-(-)-10-methyl-11hydroxyaporphine (MHA) a potent 5-HT,A agonist [37] (Table 6). In contrast to the majority of aporphines MHA is devoid of dopaminergic activity. The corresponding (S)-enantiomer is 10 times less potent and an antagonist at postsynaptic 5-HT1^ receptors [38]. The 10-methyl-ll-hydroxy substitution pattern is a prerequisite for high affinity. The positional isomers 10-hydroxy-ll-methyl-, 9-hydroxy-10-methyl and 9-methyl-10-hydroxyaporphines are inactive [39]. However, the related (R)-(-)8,11-dimethoxyaporphine is in contrast to MHA a potent 5-HT1^ antagonist [40]. Table 6 5-HT1^ ligands derived from aporphines

-C

-C

H

IH

.

/

co_{L oc C

MHA

C

pKi = 8.5

pKi = 7.4

pKi = 8.1

Data, expressed as pI~. values are from [38,40]. Arylpiperazines The best known member of the class of the 1-(2-pyrimidinyl) piperazines is the anxiolytic buspirone, a relatively potent but non-selective 5-HT,A ligand (Table 7). Since the discovery of buspirone many long-chain arylpiperazines have been published as 5-HTL~ agents (for review see [2]). A number of representative examples are gepirone, ipsapirone and zalospirone (Table 7). Of this series ipsapirone is the most potent and selective 5-HT, Aligand. In functional tests these compounds act as partial agonists. In the buspirone analogues the 1-(2-pyrimidinyl) piperazine moiety is coupled via an alkyl chain to an amide function. The bulkiness of the amide containing part of the molecules hardly influences the 5-HT1^ affinity (i.e. zalospirone vs gepirone). Shortening the side chain of buspirone reduces affinity. The rankorder of potency is C-4 > C-2 > C-3 (pIC5o values 7.5, 6.8 and 6.6 respectively) [41]. Such a decrease in 5-HTL~ atfinity is not seen in the ipsapirone series.The C-3 analogue is as potent as ipsapirone [42]. The unsubstituted 1-(2-pyrimidinyl) piperazine is only weakly active (pI~.=5.9) [43].

27 Replacement of the aryl moiety ofbuspirone by 1-(2-methoxyphenyl)piperazine, yielding BMY8227, enhances affinity (Table 8). Shortening the butyl side chain to propyl (BMY7924) reduces affinity, whereas the corresponding ethyl analogue (BMY7378) is as potent as the parent compound [41]. Furthermore BMY7378 is rather selective for 5-HT1A receptors [44] (Table 8). In functional tests these compounds behave as partial agonists (for review see [3]). Table 7 5-HT1A ligands derived from buspirone 5-HTIA

D2

al

7.8

7.4

6.2

7.2

5.6

5.7

8.3

6.4

6.6

7.6

5.8

5.8

7.8

6.5

n.d

//o

/

N -C4-N

'b

BUSPIRONE

\

/

\

/

\

/

\

/

N

N

O

GEPIRONE

X% /

N -C4-N

N -C4-N

N

N

N

.

IPSAPIRONE

o

o o N ~.~/

-C4-N

TANDOSPIR

o

-C4-N

ZALOSPIRONE

o

\

/

N

N

N

Data, expressed as pKi. values are from [8] (buspirone, ipsapirone); [76] (gepirone, tandospirone) and [77] (zalospirone).

28 High 5-HT1A at~nity is maintained by replacement of the azaspirodecanedione moiety of BMY8227 by a N-phthalimido group. This compound NAN-190 however is again not selective (Table 8). Shortening the alkyl side chain decreases the affinity significantly. In contrast to the azaspirodecanedione containing compounds a N-phthalimido substituent is not tolerated at a distance of two carbon atoms [2] (Table 8 continued). In a series of2-[4-(o-methoxyphenyl)piperazin- 1-yl)alkyl- 1,3-dioxoperhydroimidazo [1,5-a]pyridine derivatives, the rankorder of potency is propyl > butyl >ethyl (pKi values 8.4, 8.1 and 7.3 respectively). All compounds are non-selective with respect to a~, D 2 and 5-HT2A receptors. Interestingly the methyl analogue is a moderately potent (pI~.=7.5) but selective presynaptic agonist [80]. High affinity and selectivity is obtained in a series of non-cyclic amides [45,46] (Table 8). Without loss in affinity the secondary amide function can be placed at a distance of four carbon atoms (RK-153) or of two carbon atoms from the basic nitrogen atom of the arylpiperazine (Table 8). The corresponding benzamide is somewhat less potent and non-selective [47] (Table 8). A more potent and selective 5-HT~^ ligand is WAY 100635 (Table 8). The compound displays a high affinity for the 5-HT~^ receptor (pI~.=9.1), a weak affinity for a~-adrenoceptors (pI~.=6.4) and no affinity for the other receptors tested [48] (Table 8). In functional tests WAY 100635 acts as an antagonist at both presynaptic somatodendritic and postsynaptic 5-HT~A receptors [49]. [3H]-WAY 100635 labels both free and bound 5-HT~A receptors [81]. At present [HC]-WAY 100635 is available to study 5-HT~A receptors in vivo [82]. The presence of an amide function is not a prerequisite for high affinity [50]. The phenylbutyl, phenylpropyl and phenylethyl are all potent 5-HTIA ligands (Table 9). Substitution of the phenetyl analogue at the a-carbon with a N-tert.butylamine group (WAY-100135) reduces affinity (Table 9). However WAY-100135 is the first selective 5-HT1A ligand which acts as a pure antagonist at both presynaptic somatodrendritic and postsynaptic 5-HT~^ receptors [51,52,53]. WAY-100135 is a racemate. The 5-HT~A affinity resides predominantly in the (S)-enantiomer, being 28 times more potent than the (R)-enantiomer. (S)-WAY-100135 (pIC5o=7.8) is as selective as the racemate. A new arylpiperazine derivative was recently published as a pure antagonist at 5-HT1A receptors. This compound, containing a benzotriazole ring at a distance of three carbon atoms of the basic nitrogen atom, however is not highly selective with respect to al-adrenoceptors (ratio 5-HT~=4.6) [54] (Table 9). Replacement of the benzotriazole ring by a tetralin moiety results in potent 5HT~A ligands [55] (Table 10). Removal of the methoxy group of 2-methoxyphenyl piperazine enhances the selectivity significantly. A similar effect is observed by replacement of the 2-methoxyphenyl moiety by 2-pyridyl (Table 10). Introduction of an amino or amido function in the alkyl chain or opening of the piperazine ring is unfavourable for high 5-HT~A receptor affinity [83, 84].

29 Table 8 Long-chain arylpiperazines as 5-HTI^ ligands D2

O:1

8.6"

7.0"

6.7"

9.2

8.0

9.2

5-HT1A O

BMY 8227

/

N-C4- N

\

/

8.7 *

N

0

OC

0 //

BMY 7924

/

N-C3- N

\

I

N

O

7.1 *

--

OC

0 -C2"

BMY 7378

N

\

/

N

0

-OC

0

-C4- N \

NAN-190

/

N

0

OC

-- C 3 " N

\

0

/

N

7.7 [ OC

Data, expressed as p ~ values or pICso* are from [41,44] (BMY compounds); [2] (Nan-190, C-3 and C-2 analogues of NAN-190); [8] (NAN-190; D 2 and a~ affinities); [45] (RK-153); [47] (C-2 analogue and benzamide analogues of RK-153) and [48] (WAY 100635).

30 Table 8 (continued) Long-chain arylpiperazines as 5-HTI^ ligands 5-HTIA

D2

o(,1

O -02- N \

/

c - N --04-- N \

/

6.0

N

0

/ oc

o

RK-153

"~~--.

o

"

C - N -C2- N \

/

N

9.4

8.6

9.5

7.3

8.8

7.9

9.1

i.a

7.2

/ oc

N / oc

---

O C- N-C2- N \

o WAY 100635 ~

/

N

---

/ OC

/

~; - N - C 2 - N \ N

/

N

6.4

/ OC

Data, expressed as pI~. values or pICso* are from [41,44] (BMY compounds); [2] (Nan-190, C-3 and C-2 analogues of NAN-190); [8] (NAN-190; D2 and a~ affinities); [45] (RK-153); [47] (C-2 analogue and benzamide analogues of RK-153) and [48] (WAY 100635).

31

Table 9 Arylalkyl arylpiperazines as 5-HT1A ligands 5 - HT1A

C-C -C -C -N

\

..../\N - - ~

9.0

OC

8.4

NAN - 664 OC

C-C-N

\

/

8.7

N

OC

WAY-

C-C-N

100135

i

C=O i Nit

7.5 *

N

X_._../ OC

.4, N~

\

/

~_)

7.8

N-C-C-C-

o~

Data, expressed as pI~. values or pICso* value are from [50] (Nan-664 analogues); [51] (WAY-100135); [54] (benztriazole).

32 Table 10 Arylalkyl arylpiperazines as 5-HT1A ligands

co-C) (

\ X - Y - C - C - N ,'

/

\

/

N-L

B

X-Y

L

5-HT1^

D2

al

(E)C=CH (E)C=CH CH-CH 2 CH-CH 2 CH-CH 2

A C A B C

9.3 9.5 9.5 9.2 9.4

7.9 6.8 7.7 6.8 7.0

8.2 7.1 8.7 7.5 7.1

Data, expressed as pI~. values, are from [55]. Incorporation of the methoxygroup of 1-(2-methoxyphenyl)-piperazine (pI~.=6.8) into an annelated benzodioxane or benzofurane ring enhances affinity (pI~. values 7.4 and 7.9 respectively) [56] (Table 11). Small N-4-alkyl groups up to n-propyl influences the 5-HT~A affinity only slightly. Elongation of the hydrocarbon chain increases the affinity with a local maximum for the N-n-hexyl analogues (pI~. values 9.3). Introduction of an oxygen atom in the alkylchain decreases affinity 3fold. Branching of the hydrocarbon chain near the basic nitrogen atom is unfavourable. The cyclohexyl analogue for instance is significantly less potent than the corresponding cyclohexylmethyl and cyclohexylethyl derivatives (Table 11). A similar effect is seen in the corresponding phenylalkyl series [57]. However the indan-2-yl piperazine analogue (S15535) is a potent 5-HTI^ partial agonist [58]. The 4-fluorobenzamide ethyl and azaspirodecanedione ethyl analogues are again highly potent [57,59].

33 Table 11 Heterobicyclic phenylpiperazines as 5-HT~A ligands

/ R-N

\

/

N

-/ o

\

\ /

\

\

/

N

o

R

(S15535)

/

R-N

H C3 Ce (c-Cell11) (c-CeHll)-C1 (c-CeHll)-C2 indane-2-yl Cells-C1 C6H5-C2 C6H5-C4 Cells-O-C3 * CeH4F-C=O-NH-C2 ** ASD-C2

5-HTIA

5-HT1^

7.4 7.1 9.3 n.d 8.0 9.6 8.8 7.6 9.3 9.5 9.0 9.5 9.2

7.9 7.5 9.3 7.2 8.1 n.d 7.7 9.4 n.d 9.1 9.8

Data, expressed as pK i values, are from [56,57,59]. S15535 is from [58]. * parafluorobenzamide; ** ASD=azaspirodecanedione. In the benzamide series the annelated ring of the arylpiperazine moiety can be substituted with small alkyl, alkoxy or hydroxyalkyl groups without significant loss in 5-HTI^ affinity. Introduction of a methyl in the C-2 or C-3 position of the benzodioxane ring for instance hardly influences the 5-HT1A affinity (Table 12). However there is a remarkable loss in selectivity notable in the 2-methyl substituted analogue. Selectivity is regained by oxidation of the 2-methyl group to the primary alcohol function. Resolution of this compound shows that the R(+) enantiomer (flesinoxan) is 10 times more potent than the S(-) antipode. Flesinoxan is somewhat less active but more selective than the unsubstituted benzodioxane compound (Table 12). In functional tests flesinoxan is potent 5-HTI^ agonist at pre- and postsynaptic 5-HT1A receptors.

34 Table 12 Heterobicyclic phenylpiperazines as 5-HTI^ ligands

5-HTIA

F

o:1

o

,~~,_~

II H C--N-C2 0

\

-

F

D2

9.5

7.8

7.5

R = 2-C

9.1

8.5

8.2

R= 3-C

8.8

7.3

7.1

R (+)

8.8

6.9

6.4

s (-)

7.8

6.4

6.5

9.2

7.7

7.3

6.1

6.2

6.4

0

/

O

C--N-C2--N

--

\

N

/

o

o

\312/

!N

o F

C-N-C2--N

_

\ \

FLESINOXAN

N

/

o

\

/

O

"//CH2OH N-C4--N

\

O

/

o

F

_

\ --/

SDZ 216-525

~

N

O II H l C-N-C2--N \

NH

\COOC

\

\oJ

Data, expressed pK 1 values are from [47] (flesinoxan analogues) and [61] (SDZ 216-525).

35 The benzodioxane moiety can be replaced by a variety of other heterobicyclic rings without loss in affinity [60]. A recent example is SDZ 216-525. This compound in which the N-4 of the indolepiperazine is substituted with the saccharinebutyl chain of ipsapirone is a potent and rather selective antagonist at postsynaptic 5-HT~A receptors [61] (Table 12). At the somatodendritic 5-HTsA autoreceptors SDZ 216-525 behaves as a partial agonist [62]. Shifting the annelated ring of the arylpiperazine moiety to the meta and para positions of the phenylring is detrimental for 5-HT1A affinity. In the benzodioxane analogues the decrease in affinity is 2500 fold (Table 12).

Aryloxyalkylamines MDL 72832 and binospirone (MDL 73005 EF) are conformationally constrained aryloxyalkylamines (Table 13). The 2-aminomethylbenzodioxane moiety is bioisosteric to 2-methoxyphenylpiperazine. Similar to the phenylpiperazines, high 5-HT1A affinity is obtained by substitution of the nitrogen atom with long chains, such as azaspirodecanedione butyl (MDL 72832) or azaspirodecanedione ethyl (binospirone) (review [63]) (Table 13). For comparison see BMY 8227 and BMY 7375 (Table 8). The high 5-HT1A affinity of MDL 72832 resides mainly in the S()enantiomer (piC5o=9.2). MDL 72832 however, is not selective with respect to a~adrenoceptors (Table 13). This high as-affinity could be destroyed by shortening the side chain of MDL 72832 with 2 carbon atoms. The resulting compound binospirone is a potent and selective 5-HTIA ligand. In functional tests both compounds behave as partial agonists, but binospirone is predominantly antagonistic [63]. An other aminomethylbenzdioxan containing 5-HT1Aligand is the antipsychotic spiroxatrine. This compound displays a high affinity for the 5-HT1A affinity (pI~.=9.1) but is not selective with respect to various other receptors, such as dopamine D 2(pI~.=9.0), morphine ~ (pI~.=8.2) receptors, and a~-adrenoceptors (pI~.= 7.1) [47] Table 13. Spiroxatrine is a racemate. The affinity for 5-HT1A receptors resides, similar to MDL 72832, mainly in the S(-)-enantiomer [64]. In functional tests spiroxatrine acts as a partial agonist [3]. Structurally related to spiroxatrine are the aryloxypropanolamines R28935 and R29814 possessing two centers of asymmetry. Both diastereoismers possess a high affinity for 5-HT1A receptors, the threo-form being twice as potent as the erythroform (Table 13). In comparison to spiroxatrine the affinity for dopamine D 2 receptors is modest, but the affinity for a~-adrenoceptors is high. Inspite of the presence of the aryloxypropanol side chain of ~-adrenoceptor antagonists, the affinity for ~adrenoceptors is not high (pI~.-values 7.3 and 6.7 respectively) [47]. An example of a flexible aryloxyalkylamine is S14063, claimed as a potent 5HT~A antagonist devoid of ~-adrenoceptor blocking activity [65] (Table 13). The activity of $14063 on somatodendritic autoreceptors is not yet published. A rather simple aryloxyalkylamine is MEP125, synthesized in a program for 5H T m antagonists lacking the 5-HT1B and ~-adrenergic affinity of propranolol [66] (Table 13). MEP125 binds with the same 5-HTsA afl~nity but greater selectivity

SPIROXATRINE

BINOSPIRONE ( MDL 73005EF )

MDL 72832

[ ~ O

OH

~ ~-c-\

/'NJ

N ~

NH

[~O)_oc-~-~ [~~i~O)_o c-~-~'-~ 0

9.1 9.4

erythro threo

9.1

6.0

7.1

9.0

6.4*

6.8*

9.1"

8.6*

D2

5HTIA

n.d

8.4

7.1

5.6*

7.8*

P_

x

,r

0~

>"

p.L

Q

I~~ r

o~ Q

=..

t=

~o

t~

~

I,---6

="

37 Table 13 (continued) 5-HT1A ligands derived from aryloxyalkylamines

S 14063

n.d

~ ~.... c _ c _ N - C

I

c

/-~.c-

MEP 125

c-

N

\

7.4

\

OH PROPANOLOL

~

N

S(-)

7.3

Data, expressed as pK 1 values or pICso* values are from [63] (MDL compounds); [47] (spiroxatrine, R28935, R29814, S(-)propranolol); [65] (S14063) and [66] (MEP 125). Besides propranolol, various ~-adrenoceptor antagonists display a moderately to high affinity for 5-HT1A receptors (Table 14). The affinity resides predominantly in the (S)-(-)-enantiomers, being more potent than the (R)-(+)-enantiomers [32]. This enantio-selectivity is similar to that assessed for the ~-adrenoceptor. The majority of ~-adrenoceptor blockers has also affinity for the 5-HT, Breceptors. The ratio 5-HT1A/5-HT1B ranges from 1 ((S)-(-)-propranolol) to 20 ((S)-(-)-tertatolol) [67,68]. The rankorder of potency for 5-HT1Areceptors is cyanopindolol > levopenbutolol > (S)-(-)-tertatolol > (S)-(-)pindolol > (S)-(-)-propranolol > oxprenolol (Table 14). ~-adrenoceptor antagonists substituted at the para-position with respect to the oxypropanolamine chain are inactive at the 5-HT1A (and 5-HT1B) receptors (Table 14). In functional models the (S)-(-)-enantiomers of propanol, pindolol, penbutolol (levopenbutolol) and tertatolol behave as antagonists at both pre- and postsynaptic 5-HT1A receptors [3,69,70].

38 Table 14 5-HT1A ligands derived from ~-adrenoceptor antagonists

I

o

H

o

"T"

I

(-) - PROPRANOLOL

5 - H T I A = 7.3

I I I I I

5 - H T 1 B = 7.3 131,2 = 9.2

(-) - PINDOLOL

5 - H T 1 A = 7.9 5 - H T 1 B = 7.0 ~1,2 = 9.7

I I

OH

LEVO PENBUTOLOL

CN

I

!

CYANOPINDOLOL

5 - H T I A = 8.6 5-HT1B-

8.4

5 - H T I A = 8.7 5 - H T I B = 8.5

1~1,2 = n.t **

~1,2 = 9.7

OH H

OXPRENOLOL

5 - H T I A = 7.0

I

5 - H T I B = 6.2

I a

1~1,2 = 7.9 OH

( - ) - TERTATOLOL

5 - H T I A = 8.2 5 - H T 1 B = 6.9

13~,2 = 9.0

T I

OH H

METOPROLOL

5 - H T 1 A = i.a 5 - H T 1 B = i.a 1~1,2 = 6.5

I a

ATENOLOL

5 - H T I A = i.a 5 - H T I B = i.a 1~1,2 =<6.3

Data, expressed as pK i values are from [67] (5-HT1A, 5-HT1B); [47] (~1,2); except oxprenolol [78] and levopenbutolol (not in our files, nt**). Data of (-)-tertatolol are from [68].

39 Table 15 Miscellaneous structures as 5-HT1A ligands

5-HTIA

o

OC

F

. ~ 1 1

O

o

H C-N -C 2

_

,)

H II C--N-- C

9.2

7.3 *

s

8.1

>_

N

( 1R,2S )

8.1

( 1S,2R )

6.0

Data, expressed pK I or pICso* are from respectively [71]; [72]; [73]; [9].

40 Miscellaneous s t r u c t u r e s Novel 5-HTIA ligands were obtained in a series of trans-fused hexahydroindeno [2,1-c]-pyridines, combining structural elements from both the aminotetralins and arylpiperazines [71]. Similar to these reference classes an appropriate long-chain on the basic nitrogen enhances the affinity. The highest affim'ty was obtained in the 4-fluorobenzamide ethyl analogue (pKi.=9.2) (Table 15). This new class of 5HT1A ligands is not selective with respect to 5-HT 2 receptors. T h e c i s - f u s e d analogues are less potent at 5-HT~^ receptors. Structurally unrelated to known 5-HT~A ligands are the 1,2,3,4-tetrahydro [1] benzothieno [2,3-c] pyridines [72] (Table 15). A lipophilic substituent is necessary for binding to 5-HT~^ receptors. The 5-HT~^ affinity resides mainly in the (R)enantiomers. The compounds are only moderately active. The highest affinity is obtained if the C-3 substituent is N-cyclohexylmethyl-carboxAmide (pIC5o=7.7). The corresponding N-cyclopropyl-carbox~mide is inactive. From the series the substituted benzyl analogue was selected for further testing. This compound is moderately potent (pIC5o=7.3) but a selective 5-HT~^ partial agonist. An other rather unusual structure is the imidazole MDL102181 displaying high affinity for 5-HT~^ receptors [73] (Table 15). Structurally however, there is some resemblance with N,N-di-n-propyl-orthohydroxy-trans-2-phenylcyclopropylamine, one of the most simple structures possessing high 5-HTI^ affinity [74]. The affinity of this compound resides predominantly in the (1R,2S)-enantiomer (pI~.=8.1). The (1S,2R)-antipode is 100 times less potent (pI~.=6.0) [9] (Table 15). The high affinity of the trans phenylcyclopropylamino derivative is destroyed by separation of the cyclopropane ring and the nitrogen atom by one carbon atom [85]. REFERENCES

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42 37 Cannon JG, Mohan P, Bojarski J, Long JP, et al. J Med Chem 1988; 31: 313318. 38 Cannon JG, Moe ST, Long JP. Chirality 1991; 3: 19-23. 39 Cannon JG, Flaherty PT, Ozkutlu U, Long JP. J Med Chem 1995; 38: 18411845. 40 Cannon JG, Jackson H, Long JP, Leonard P, et al. J Med Chem 1989; 32: 1959-1962. 41 Yocca FD, Smith DW, Hyslop DK, Maayani S. Soc Neurosci, abstracts, 1986; 12: 422. 42 LtJscher W, Witte U, Fredow G, Traber J, et al. Naunyn-Schiedebergs Arch Pharmacol 1990; 342: 271-277. 43 Glennon RA, Naiman NA, Lyon RA, Titeler M. J Med Chem 1988; 31: 19681971. 44 Yocca FD, Hyslop DK, Smith DW, Maayani S. Eur J Pharmacol 1987; 137: 293-294. 45 Raghuphathi R, Rydelek-Fitzgerald L, Teitler M, Glennon RA. J Med Chem 1991; 34: 2633-2638. 46 Orjales A, Alonso-Cires L, Labeaga L, Corcdstegui R. J Med Chem 1995; 38: 1273-1277. 47 Tulp MThM, unpublished results. 48 Gozlan H, Thibault S, Laporte AM, Lima L, et al. Eur J Pharmacol 1995; 228: 173-186. 49 Fletcher A, Bill DJ, Cliffe IA, Foster EA, et al. Br J Pharmacol 1994; 112 (Proceed Suppl), 91P. 50 E1-Bermawy M, Raghupathi R, Ingher SP, Teitler M, et al. Med Chem Res 1992; 2: 88-95. 51 Cliffe IA, Brightwell CI, Fletcher A, Forster EA, et al. J Med Chem 1993; 36: 1509-1510. 52 Fletcher A, Bill DJ, Bill SJ, Cliffe IA, et al. Eur J Pharmacol 1993; 237: 283291. 53 Routledge C, Gurling J, Wright IK, Dourish CT. Eur J Pharmacol 1993; 239: 195-202. 54 Mokrosz JL, Paluchowska MH, Chornacka-Wojcik E, Malgorzata F, et al. J Med Chem 1994; 37: 2754-2760. 55 Perrone R, Berardi F, Colabufo NA, Leopoldo N, et al. J Med Chem 1995; 38: 942-949. 56 Van Steen BJ, Van Wijngaarden I, Tulp MThM, Soudijn W. J Med Chem 1993; 36: 2751-2760. 57 Van Steen BJ, Van Wijngaarden I, Tulp MThM, Soudijn W. J Med Chem 1994; 37: 2671-2773. 58 Millan MJ, Canton H, Gobert A, et al. J Pharm Expt Ther 1994; 268: 337-352. 59 Van Steen BJ, Van Wijngaarden I, Tulp MThM, Soudijn W. J Med Chem 1995; 38: 4303-4308. 60 Hartog J, Van Wijngaarden I, Wouters W. EP 0138280. 61 Schoeffter Ph, Fozard JR, Stoll A, Siegl H, et al. Eur J Pharmac Mol Pharmac Section 1993; 244: 251-257.

43 62 Gurling J, Ashworth-Preece MA, Hartley JE, Fletcher A, et al. Brit J Pharmacol 1993; 108: 255P. 63 Hibert M, Moser PC. Drugs Fut 1990; 15: 159-170. 64 Nikam SS, Martin AR, Nelson DL. J Med Chem 1988; 31: 1965-1968. 65 Dabire H, Bajjou R, Chaouch-Teyara K, Fournier B, et al. Eur J Pharmacol 1991; 203: 323-324. 66 Pierson ME, Lyon RA, Titeler M, Kowalski P, et al. J Med Chem 1989; 32: 859-863. 67 Langlois M, Br6mont B, Rouselle D, Gaudy F. Eur J Pharmacol 1993; 244: 7787. 68 Prisco S, Cagnotto A, Talone D, De Blasi A, et al. J Pharm Expt Ther 1993; 265: 739-744. 69 Hjorth S, Sharp T. J Pharm Expt Ther 1993; 265: 707-712. 70 Lejeune F, Rivet J-M, Gobert A, Canton H, et al. Eur J Pharmacol 1993; 240: 307-310. 71 Meyer MD, De Benardis JF, Hancock AA. J Med Chem, 1994; 37: 105-112. 72 Kawakubo H, Takagi S, Yamaura Y, Katoh S, et al. J Med Chem 1993; 36: 3526-3532. 73 Romero AG, McCall RB. Ann Reports Med Chem 1992; 27: 21-29. 74 Arvidsson L-E, Johansson AM, Hacksell U, Nilsson JLG, et al. J Med Chem 1988; 31: 92-99. 75 WikstrSm H, personal communication. 76 Hamik A, Oksenberg D, Fischette C, Peroutka SJ. Biol Psychiatry 1990; 28: 99-109. 77 Abou-Gharbia M, Patel UR, Webb MB, Moyer JA, et al. J Med Chem 1988; 31: 1382-1392. 78 Bilezikian JP, Dornfeld AM, Gammon DE. Biochem Pharmacol 1978; 27: 14551461. 79 Dahlgren T, Dean RL, Gharat LA, Johansson AM, et al. Bioorg Med Chem Lett 1995; 5: 2963-2968. 80 L6pez-Rodriquez, Morcillo MJ, Rosado ML, Benhamu B, et al. Bioorg Med Chem Lett 1996; 6: 689-694. 81 Gozlan H, Thibault S, Laporte AM, Lima L, et al. Eur J Pharmacol 1996; 288: 173-186. 82 Pike VW,McCarron JA, Lammersma AA, Hume SP, et al. Eur J Pharmacol 1995; 283: R1-R3. 83 Perrone R, Berardi F, Leopoldo M, Tortorella V. J Med Chem 1996; 39: 31953202. 84 Berardi F, Colabufo NA, Giu7ice G, Peronne R, et al. J Med Chem 1996; 39: 176-182. 85 Appelberg U, Mohell N, HackseU U. Bioorg Med Chem Lett 1996; 6: 415-420.

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Serotonin Receptors and their Ligands

B. Olivier, I. van Wijngaardenand W. Soudijn (Editors) 1997 Elsevier ScienceB.V, All rights reserved.

45

Structural characteristics of 5-HT~A receptors and their ligands W. Kuipers

Solvay Duphar B.V., Dept. of Medicinal Chemistry, P.O.Box 900, 1380 DA Weesp, The Netherlands.

R E C E P T O R STRUCTURE AND MOLECULAR BIOLOGY

Receptor Structure The 5-HT1A receptors belongs to the class of G-protein coupled receptors (GPCRs). The members of this class all share a number of characteristics which are essential for their structure and second-messenger activation. Although no 3D-structures of GPCRs have been published yet, these receptors are believed to consist of 7 membrane-bound a-helices, connected by three extracellular and three cytoplasmic loops (see Figure 1). The amino-terminus is located on the outside of the cell, and the carboxyl-terminus on the inside. The lipophilic part of the transmembrane regions is in contact with the membrane while the more polar and highly conserved residues, located on the inside, form a hydrophilic receptor core. This topology for GPCRs was initially based on sequence and hydrophobicity analyses, and later experimentally corroborated (reviews e.g. Savarese and Fraser [1], Lee and Karlavage [2], Strader et al. [3] and Schwartz [4]). Figure 2 shows the sequences of the putative transmembrane domains of the rat 5-HT m receptor. All GPCRs use an intermediary G-protein, connected to the receptor at the cytoplasmic side to activate the second messenger system. GPCRs have a number of characteristic amino acid patterns in common. These highly conserved residues are believed to play a special role in receptor structure and cell signalling. The residues involved in ligand binding are less conserved, as different receptor subclasses recognize structurally different ligands. The specific function (for structure or ligand-binding) of a number of more or less conserved residues was revealed by mutation studies. Residues involved in receptor structure and function

N-glycosylation sites at the N-terminal end may be important for proper binding of the receptor to the cell membrane [1]. The N-terminus of the 5-HT1A receptor contains three sites with the consensus sequence for N-glycosylation of Asn residues: AsnoXaa-Ser/Thr (i.e. NNTT: both Asn residues, and NVTS, see Figure 1).

45

Figure 1. Schematic representation of the putative seven transmembrane domains of the rat 5-HT~A receptor as defined by Kuipers et al 1994. Solid circles represent amino acids with a special function for receptor conformation or ligand-binding. Amino acids marked Y are putative N-glycosylation sites. Sites marked * are sites that may be phosphorylated by protein kinase C (PKC).

47 In helix I the combination Gly-Asn is highly conserved, like the Leu-Ala-X-XAsp-Leu motif at the cytoplasmic side of helix II. The highly conserved aspartate residue in this last motif was found to influence agonist binding in a number of investigated GPCRs (e.g. Savarese and Fraser [1] and Schwartz [4]), but hardly affects the affinities of the antagonists tested. In analogy, mutation of Asp82 in the 5-HT~A receptor decreases 5-HT (i.e. 5-hydroxytryptamine, serotonin) agonist affinity [5, 6], while the mutant's affinity for the antagonist pindolol equals that of the wild-type [5]. Variations in [Na § and pH were shown to alter agonist affinity via the highly conserved Asp-residue in helix II [7, 8]. This residue probably allosterically modulates the receptor conformation by which the agonist binding site is altered. Oliveira et al. [9] postulated that the highly conserved Asp in helix II may interact with the Arg of the highly conserved 'DRY' motif at the cytoplasmic side of helix III. According to their hypothesis this interaction would prevent coupling of the G-protein. Interestingly, the Asp of 'DRY' was shown to interact with the G-protein in a number of receptors [1]. Decoupling of the G-protein is known to decrease the affinity for the neurotransmitter. Mutagenesis studies have shown that C- and N-terminal sides of the 3~a cytoplasmic loop are important not only for G-protein coupling, but may also determine G-protein selectivity. The exact mechanism of G-protein binding and specificity has not been elucidated yet, as also other parts of the cytoplasmic loops seem to contribute to the binding process [1, 2]. Two highly conserved cysteines appear to be important for proper protein folding, probably by forming a disulfide bond between the second extracellular loop (between helices IV and V) and the N-terminal end of helix III. Mutation studies concerning these residues in ~-adrenergic, bovine rhodopsin and muscarinic acetylcholine receptors provided strong evidence for this hypothesis [10, 11, 12]. In the 5-HT~A receptor, these residues correspond with Cysl09 at the top of helix III at the extracellular side and Cys187 in the second extracellular loop. Highly conserved proline residues are found in helices V, VI and VII of most GPCRs. Several of these prolines are surrounded by a number of highly conserved other residues, like in the PFF motif in helix VI, or the NP motif in helix VII. Prolines are known to produce kinks in a-helices of membrane proteins [13, 14]. Therefore these highly conserved prolines in GPCRs are believed to be required for a proper receptor structure [15]. A highly conserved NP (Asn-Pro) motif is located at the intracellular side of helix VII. The Asn of this motif probably directly interacts with the highly conserved Asp in helix II, which indicates that helices II and VII are located next to each other [16, 17]. The importance of the Asn396 of the NP motif for agonist binding to the 5-HT~A receptor was illustrated by Chanda et al. [6]. In human 5-HT~A receptors only a highly conservative Asn~Gln mutation was allowed without loss of affinity for the agonist 8-OH-DPAT. However, replacements of this Asn396 with Ala, Phe or Val were detrimental for 5-HT~A receptor affinity for 8-OH-DPAT. Other residues known to be involved in ligand binding are located far from the NP motif.

48 5-HT~^ 5-HT~B 5-HTI~ 5-HT2^ 5-HT2c

r r h r r

38

I L S W W

T L L P P

S V A A A

L A V L L

L L V L S

L L L T I

G A S T V

T L V V V

L I I V I

I T T I I

F L L I I

C A A L M

A T T T T

V T V I I

L L L A G

G S S G G

N N N N N

A A A I I

C F F L L

V V A A V I A T V L T T V I M A V I M A

5-HT~A 5-HTIs 5-HTI~ 5-HT2^ 5-HT2c

r r h r r

74

L L L F F

I I I L L

G A G M M

S S S S S

L L L L L

A A A A A

V V T I I

T T T A A

D D D D D

L L L M M

M L L L L

V V V L L

S S S G G

V I I F F

L L L L L

V V V V V

L M M M M

P P P P P

M I I V V

A S S S S

A T I M M

5-HTI^ 5-HTIB 5-HTI~ 5-HT2^ 5-HT2c

r r h r r

i09 C C C C C

D D D A P

L F I I V

F W W W W

I L L I I

A S S Y S

L S S L L

D D D D D

V I I V V

L T T L L

C C C F F

C C C S S

T T T T T

S A A A A

S S S S S

I I I I I

L M L M M

H H H H H

L L L L L

C C C C C

A I V I V I A I A I

5-HTI^ 5-HTIs 5-HTI~ 5-HT2^ 5-HT2c

r r h r r

153 A A A A A

A A A F I

A I T L M

L M M K K

I I I I I

S V A I A

L L I A I

T V V V V

W W W W W

L V A T A

I F I I I

G S S S S

F I I V I

L S C G G

I I I I V

S S S S S

I L I M V

P P P P P

P P P I I

M F L P P

L F F V V

5-HTI^ 5-HTIB 5-HTI~ 5-HT2^ 5-HT2c

r r h r r

195 Y Y Y F F

T T T V V

I V I L L

Y Y Y I I

S S S G G

T T T S S

F V C F F

G G G V V

A A A A A

F F F F F

Y Y Y F F

I L I I I

P P P P P

L T S L L

L L V T T

L L L I I

M L L M M

L I I V V

V A I I I

L L L T T

Y Y Y Y Y

5-HTI^ 5-HTIB 5-HTI~ 5-H%^ 5-HT2c

r r h r r

347

L L L L L

G G G G G

I I I I I

I I I V V

M L L F F

G G G F F

T A A L V

F F F F F

I I I V L

L V I V I

C C C M M

W W W W W

L L L C C

P P P P P

F F F F F

F F F F F

I I V I I

V I V T T

A S S N N

L L L I I

V V V M L

L W L A S

P P P V V

F I I I L

5-HTIA 5-HTIB 5-HTI~ 5-HT2A 5-HT2c

r r h r r

381

L L L L L

G G F L L

A D D N N

I F F V V

I F F F F

N N T V V

W L G Y S N W L G Y L N W L G Y L N W I G Y L S W I G Y V C

S S S S S

L L L A G

L I I V I

N N N N N

P P P P P

V I I L L

I I I V V

Y Y Y Y Y

A T T T T

Y F M S V F LF L F

N N N N N

L M A L L

Y Y Y T T

II

III

IV

VI

VII

Figure 2. Sequences of the putative transmembrane domains of the rat 5-HT~A receptor [28]. Residues marked bold were found in the active site of serotonin in the model of Trumpp-KaUmeyer et al. [51]. Underlined residues play a role in agonists or antagonist binding in the 5-HTIA receptor model by Kuipers et al. [49, 52], and residues printed in italics were found to be part of the binding site of MHA in the model by Hedberg et al. [55]. Therefore, allosteric modulation of the agonist binding site by mutation of Asn396 seems a more likely explanation for the observed effects than a direct involvement of this residue in agonist binding. This hypothesis is in agreement with the putative contact of the Asn of the NP motif with the Asp in helix II, which was also shown to influence agonist binding allosterically. Phosphorylation disrupts the coupling between the receptor and the Gprotein, which is required for high agonist affinity, thus desensitizing the

49 receptor for agonist activation. Phosphorylation by protein kinase C (PKC) of probably two or three sites has been reported as a possible mechanism for desensitization of the 5-HT~A receptor [18]. Its putative PKC-sites, having consensus sequence Serfrhr-X-Arg~ys, are located in the second and third cytoplasmic loop (see also Figure 1).

Residues involved in ligand binding An Asp116 cognate in helix III is conserved in all cationic neurotransmitter receptors, but absent in other GCPR. It is, therefore, believed to interact with the ligand's basic nitrogen via an ionic H-bond interaction. Many mutagenesis experiments have confirmed the importance of this residue for agonist and antagonist binding (reviews e.g. by Savarese and Fraser [1] and Schwartz [4]). In the 5-HT1A receptor, 5-HT affinity was markedly reduced by an Aspll6Asn mutation [5], but surprisingly the affinity of the antagonist pindolol was not affected. For several receptors hydrogen bonding residues in helix V have been shown to be important for agonist affinity. In the 5-HT1A receptor, Ser199Val mutation decreases 5-HT affinity, but has no effect on the affinity for the antagonist pindolol [5]. Thr200Val replacement renders a receptor devoid of measurable 5HT affinity, although still a signal can be obtained by 5-HT stimulation of the mutant receptor. Therefore, both Ser199 and Thr200 on helix V are believed to be involved in binding serotonin to the 5-HT1A receptor [5]. The OH-group of Thr200 is conserved in many other GPCRs. Trumpp-Kallmeyer et al. [15] postulated that aromatic residues that are highly conserved in GPCR may be involved in signal transduction as well as ligand binding. Site-directed mutagenesis studies in the ~2-adrenoceptor and the 5-HT2A receptor showed the importance of the Phe residues in the 'PFF' motif of helix VI for ligand binding [19, 20]. In the rat 5-HT~A receptor these residues correspond with Phe361 and Phe362, respectively. Asn386 in helix VII was shown to be crucial for receptors which bind aryloxypropanol antagonists like pindolol and propranolol (i.e. ~-adrenergic, 5-HT~A and rodent 5-HT~B receptors). In the rat 5-HT~A receptor Asn386Val mutation decreases the receptors affinity for pindolol, while 5-HT binding is not changed [21]. Several other mutation studies have further substantiated the importance of this residue for aryloxypropanol affinity [22-25]. Chanda et al. [6] investigated the importance of a number of serine residues in helix VII for 5-HT1A agonist binding. The mutation Ser393Ala dramatically decreased receptor affinity for 8-OH-DPAT. Apparently, the highly conserved Ser393 is either directly involved in agonist-binding, or is required for a proper receptor conformation. This residue is one turn above the NP motif. No effect on the receptor affinity for 8-OH-DPAT was found by replacement of Ser391 into Ala. Thus this less conserved residue does not appear to be crucial for 5HT~A receptor affinity of 8-OH-DPAT.

Receptor Cloning

The human 5-HTIA receptor has been cloned and expressed by Kobilka et al.

50 [26] and has later been characterized by Fargin et al. [27]. This receptor contains 422 amino acid residues, and shows 89% amino acid homology with the rat analogue, which also has 422 residues [6, 28, 29]. The sequence of the 5-HT~A receptor by Fujiwara et al. [29] differs in one residue from that of Albert et al. [28], but this may not cause significant pharmacological differences [30]. The mouse 5-HT~A gene has also been localized [31], but no sequence data have been published. MODELS OF 5-HTL~ LIGANDS AND T H E I R INTERACTION WITH THE RECEPTOR The 5-HT~A receptor is the most intensively studied serotonin receptor, as a result of the early discovery of 8-OH-DPAT, the (R)-enantiomer being a highly potent and selective 5-HT~A agonist. Several models that rationalize affinities and functional properties of 5-HT~A ligands have been published. Experimental studies in GPCRs indicate that agonists and antagonists occupy partially overlapping, but different binding sites on the receptor [1, 32]. Superimposing antagonists on agonists should therefore be avoided, although their structures may look very similar. For agonists, the hypothesis that ligands bind at a similar site at the receptor is supported by experimental data. Mutagenesis data indicate that all amine neurotransmitters bind in a region between the helices III, V and VI, close to the extracellular side (for a review see Schwartz [4]). Structure-affinity relationships (SARs) of most 5-HT1A receptor agonists show highly similar trends between structurally different classes. This allows the superimposition of these ligands in model building procedures. For antagonists, however, this hypothesis seems less substantiated. In several cases it was shown that antagonists may respond differently to receptor mutations or changes in receptor conformation, for instance as a result of the decoupling of the Gprotein (e.g. Neve et al. [8]). These findings indicate that antagonists do not address identical binding regions at the receptor [33]. Therefore it is erroneous to superimpose structurally very different antagonists without taking SARs into consideration.

Agonist models

5-HT1Aagonist pharmacophores Hibert et al. [34] derived a crude 5-HTI^ pharmacophore by fitting potent 5HT1A agonists of different structural classes (aminotetralins, ergotamines, tryptamines, arylpiperazines and RU24969). This pharmacophore gives the relative orientation of an aromatic ring and a basic nitrogen atom (see Figure 3a). These two elements are the only features shared by all 5-HT~A agonists. Both ends of a normal through the centre of the aromatic ring and a dummy atom (O') in the direction of the nitrogen lone pair were used as fitting elements.

51

a

N

-~ § y[ ~

-~ O'

Figure 3. Relative position of the essential benzene ring with respect to the basic nitrogen in 5-HTIA ligand models by a) Hibert et al. [34] and b) Mellin et al.

[35].

a) In the pharmacophore by Hibert et al. [34], for potent agonists the distance between the aromatic centre and the basic nitrogen d= 5.2/~, and the height of the basic nitrogen with respect to the aromatic plane h= 0.9 .~. b) The pharmacophore by Mellin et al. [35] describes the spatial orientation of the aromatic ring with respect to a dummy atom O'. This dummy O' represents an oxygen atom (of an Asp at the receptor) which forms an ionic hydrogen bond with the ligand's protonated basic nitrogen. The distance of O' to the aromatic plane is defined as y. X is the distance between O' and the normal through the centre of the aromatic plane. Vector V results from summation of the two vectors connecting the basic nitrogen atoms of the two enantiomers 15 and 16 with O'. The angle a represents the angle between this vector V, and the N-dummy vector of the compound. The angle ~ is the angle between the aromatic planes of the compound and that of the enantiomers 15 and 16. For potents agonists, the pharmacophore is defined by : x= 5.2-5.7 A. y= 2.1-2.6 .~, a = -,- 28 ~ ~= _+4".

52 These dummy atoms represent putative aromatic and hydrogen bonding groups at the receptor. In potent compounds, the nitrogen atom is nearly coplanar with the aromatic ring, and the electron lone pair is almost perpendicular to it. Mellin et al. [35] further refined this pharmacophore model with affinity data of a number of chiral aminotetralin-derived agonists. She compared the relative position of the aromatic ring and the nitrogen lone pair of each compound with respect to the enantiomers of a semi-rigid six/six fused angular 2-aminotetralin (15 and 16, respectively), as depicted in Figure 3b. Each compound was fitted on these enantiomers by using the same dummy atoms as Hibert et al. [34]: the ends of a normal through the aromatic ring centre, and a dummy atom (O') in the direction of the nitrogen lone pair. In the model by Mellin et al. [35], receptor excluded volume was defined by areas which are unfavourable for aliphatic substituents. Chidester et al. [36] presented a model based on a series of new tricyclic aminotetralin-related compounds. In the defined pharmacophore the hydroxy group of 8-OH-DPAT acts as a hydrogen bond acceptor. For most compounds in their study a relative orientation with respect to the aminotetralin moiety of (R) 8-OH-DPAT was presented. This model was further corroborated by Jain et al. [37], who used similar compounds for the construction of a quantitative model with the Compass method. This method searches for the best conformation and alignment of the molecules investigated. The preferred orientations of the compounds as found by Jain et al. [37] were consistent with the Chidester study [36]. Figure 4a shows the interacting groups which were described in various ligand-fitting models [34, 36, 38]. For high 5-HT~^ receptor affinity, besides an aromatic ring and a basic nitrogen atom, additional interacting groups are required. The hydroxy group in both serotonin and 8-OH-DPAT is essential for high 5-HT~A affinity. This group may be replaced with methoxy without loss of affinity, and probably acts as a hydrogen bond acceptor [36]. The pyrrole ring in serotonin seems to be i m p o ~ t for its high affinity for 5-HT 1 receptors [34]. This ring can have an additional aromatic interaction and the pyrrole NH may form a hydrogen bond. In 8-OH-DPAT, which lacks a pyrrole ring, affinity for the 5-HT~A receptor is regained by one of the N-n-propyl chains. According to ligand models this essential group must be located in a hydr0Phobic pocket that is unique for the 5-HT~^ receptor with respect to other serotonin subtypes. The size of the pocket is limited to non-branched chains smaller than n-butyl.

Modeling of 8-OH-DPAT and analogues The two enantiomers of 8-OH-DPAT both have high affinity for the 5-HT~A receptor, but the (R)-enantiomer 1 is a full agonist, while the (S)-enantiomer 2 is a partial agonist (see page *, Table 3). The two different ways of superimposing the two enantiomers as suggested by Hibert et al. [34] and Mellin et al. [35] are shown in Figure 5.

53

o

-"-',,'*"'-X o ,z" ~ - /

"

Figure 4. Schematic representation of a) pharmacophore elements as defined in various 5-HT~A agonist models [34, 36, 38] and b) the binding site of agonists like serotonin and 8-OH-DPAT in the 5-HT~A receptor model by Kuipers et al. [52].

54

|

Figure 5. Fit of (R) and (S) 8-OH-DPAT according to a) Hibert et al. [34] and b) MeUin et al. [35]. In both fits the nitrogen lone pairs coincide. In a) the hydroxy groups are also fitted, but not the aromatic centers. In contrast, in b) the aromatic centers are fitted, while the hydroxy groups are located on opposite sides of the benzene ring. As a result, in a) the N-substituents of both enantiomers are more distant than in b).

Y OH

eq

O(

essential n-propyl

9

- , e q

7

Figure 6. Schematic representation of (R) 8-OH-DPAT 1 in the putative bioactive conformation. The basic nitrogen is equatorially positioned at the tetralin ring, which is in a half-chair conformation. The preferred torsion angle Hc2C2-N-H N amounts approximately 180 ~ Methyl substituents t r a n s C1, C2, and cis and t r a n s C3 are unfavourable for 5-HTI^ receptor affinity, while a similar substitution at the c/s C1 position is indifferent.

55 In the fit presented by Hibert et al. [34] (see Figure 5a), the hydroxy groups and the dummy atoms in the direction of the lone pairs coincide and Nsubstituents of the (R) and (S) enantiomers are further apart. This fit seems to be in good agreement with 5-HT1A structure-affinity relationships. As an example, effects of substitution at the 8-positions of the enantiomers of dipropylaminotetralins show similar trends [39]. This indicates that the 8positions of both (R) and (S) enantiomers may have a fairly identical surrounding at the receptor. In addition, the effect of N-substitution of 8hydroxy aminotetralin derivatives is not the same for (R) and (S) enantiomers [40]. Possibly the N-alkyl substituents of (R) and (S) 8-hydroxy aminotetralin enantiomers do not occupy similar binding sites at the receptor. In this respect the fit of (R) and (S) 8-OH-DPAT (1 and 2) as presented by Mellin et al. [35] seems to be less satisfactory. In this fit (see Figure 5b) the aromatic parts and the dummy atoms in the direction of the nitrogen lone pairs coincide, while the hydroxy groups are located on opposite sides of the aromatic centre. It is also possible that both fits are partly correct (i.e. two binding modes for the (S)enantiomer), which might account for the partial agonist character of the (S)enantiomer 2. Structure-affinity relationships of the aminotetralin class are rather complex (Page *, Table 3). Substituents may directly affect the interaction with the receptor, but may also cause a conformational change of the compound. For example t r a n s (equatorial) C1 methyl substitution (see also Figure 6) as in compound 5 has a negative effect on 5-HT1A receptor affinity. However, compounds with t r a n s C1 substituents incorporated into a fused ring, like compounds 11 and 15, are considerably more potent than compound 5. Therefore, the C1 methyl group in 5 is unlikely to occupy essential receptor volume. Instead steric repulsion between the C1 substituent and C~-atoms of the propyl chain may be responsible for the low 5-HT~A receptor affinity of compound 5. As a result of this hindrance the compound may not be able to adopt the bioactive conformation. In the bioactive conformation as defined in ligand-fitting models, the nitrogen atom is equatorially positioned at the tetralin ring, which is in a half-chair conformation. The propyl C~-atoms are approximately in the plane of the aromatic ring, and the torsion angle Hcg-C2N-HN amounts 180". Indeed aminotetralins with t r a n s equatorial C1 methyl substituents were shown to prefer conformations with a torsion angle Hc2-C2N-H N of-60" [41, 42]. The aminotetralin t r a n s C1 substituents may also force the 8-hydroxy group into a conformation in which it cannot accept a hydrogen bond. This effect may play a role in the affinity decrease of compounds 5 and 15 when compared to compound 1. C1 substituents positioned c/s (axially) with respect to N, like in compound 3, or the small 4,6,6 and 5,6,6 fused ring analogues 7 and 9, are tolerated or even enhance 5-HT1A receptor affinity and maintain agonist activity. In the bioactive conformation of the aminotetralin moiety as defined in ligand models, these substituents do not interfere with either the N-propyl chains or the 8hydroxy group [42, 43]. The corresponding 6,6,6 fused ring analogue 13 is unexpectedly only weakly active. The calculated (MM2) energy minimum for

55 compound 13 deviates from the calculated (bioactive) conformation of compound 3 by a different direction of the nitrogen lone pair, which may account for its rather low 5-HTIA receptor affinity [42]. 5-HT1^ receptor ~ i t y is lowered by the introduction of axial 2- and 3- (cis) methyl (compound 17) substituents at the aminotetralin ring of (R) 8-OHDPAT, 1. These substituents may occupy essential receptor volume and thus prevent a good fit at the receptor. The 2- and 3- axially positioned methyl groups may also cause hindrance with (e.g. C~ or C~ atoms of) the essential propyl chain in the bioactive conformation. Similar explanations may account for the negative effect on 5-HT~^ receptor affinity of the trans (equatorial) C3 methyl substituents in compound 19. As aforementioned, the torsion angle Hc2C2-N-H N in the putative bioactive conformation amounts approximately 180". However, compounds with equatorial C3 methyl substituents were shown to prefer conformations with a torsion angle Hc2-C2-N-HN of 60 ~ [42]. It is also possible that aminotetralin trans C3 substituents cause direct hindrance with the receptor. This last hypothesis would account for the 10w 5-HT~A receptor affinity of compounds with trans C3 substituents incorporated into a ring system. In compound 27 the conformation of the essential propyl chain is constrained into a fused ring. Apparently, this conformation is not the same as the bioactive conformation of the free propyl chain.

Moon-22 X=CH2

OCH3

Moon-24 X=NH NH

HN

U24969

Figure 7. Structures of 5-HT~^ receptor ligands.

57 As a result, the lipophilic interaction may not be optimal and the ring may even cause steric hindrance with the receptor. Similar arguments may explain the low 5-HT1A receptor affinity of compound 23. In the model by Chidester et al. [36] the fit of several aminotetralin-derived compounds was discussed. The aminotetralin ring of most compounds was oriented similar to that of (R) 8-OH-DPAT. In contrast, ACA ('Angular, Cis, Ring Closure Away from O', see Figure 7) was fitted on the model in the same orientation as the (S) enantiomer of 8-OH-DPAT according to Mellin et al. [35]. In this fit, the 8-OH group of ACA is located close to the indole NH of serotonin. This orientation accounts for the preferred stereochemistry (R) of the compound. The preference for hydroxy over methoxy subsituents indicates that the 8-hydroxy~ group of ACA, like the pyrrole NH of serotonin, may act as a hydrogen bond donor. Compound Moon-24 (Figure 7) was also fitted in this orientation. The racemate of Moon-24 was reported to possess full agonist activity [44]. According to Chidester et al. [36], the activity of Moon-24 probably resides in the (R) enantiomer. In agreement with the role as a hydrogen bond acceptor in the model of Figure 4a, the oxygen atom was shown to increase 5HTIA receptor affinity 20-fold. The amide NH of Moon-24 seems to have no special function, as replacement with CH2 in Moon-22 was shown to be indifferent [44].

Fitting 5-HTIA agonists of other structural classes With the use of the defined common interacting groups, structurally different 5-HT1A agonists can be superimposed. For aminotetralins, tryptamines and ergotamines this can be done rather straightforwardly, as is shown in Figure 8a. The aromatic nuclei, the basic nitrogens and the nitrogen lone pairs of the compounds can be fitted. As a result, the hydroxy groups shown to be essential for high 5-HT1A affinity, occupy the same spacial position. Hibert and co-workers [34] argued that for RU24969 two different orientations with respect to ergotamines are possible. The benzene ring of this compound may be fitted on the benzene or the pyrrole ring of the indole moiety. Substitution patterns at the 2- and the 5-positions of the indole ring of tryptamines are similar to observations for RU24969 analogues [45, 46]. These findings justify a straightforward fit of the indole rings of the two classes, as shown in Figure 8b. The way of fitting other classes may be somewhat more ambiguous. For instance, the structure of MHA (page *, Table 6 (5-HT1A ligands)) can be considered an aminotetralin-analogue. However, MHA cannot be superimposed on 8-OH-DPAT in an atom-to-atom fit with full comprehension of its 5-HT1A profile. Westkaemper and Glennon [38] showed that MHA may well be fitted on 8-OH-DPAT as depicted in Figure 8c. In this orientation, the aromatic nuclei, the basic nitrogens and the nitrogen lone pairs of the two compounds coincide, and the hydroxy groups are located close to each other. The (R) enantiomer of MHA is preferred, as the (S) enantiomer of MHA cannot be superimposed on (R)-8-OH-DPAT 1 satisfying all four criteria. Thus the stereoselectivity of MHA regarding 5-HT~Aaffinity is accounted for.

58 a

b

d

J

F i g u r e 8.

Superimpositions of a) 8-OH-DPAT and 5-HT, b) RU24969 and 5-HT, c) MHA and 8-OH-DPAT, and d) arylpiperazines and 5-HT.

Fitting the class of arylpiperazines also appeared to be somewhat puzzling [47]. Like flesinoxan, many potent agonists that have been reported in this class owe their high 5-HT~^ receptor affinity to a large N4-substituent. Because of their flexibility they were left out of most modelling studies [34, 48]. Without these substituents, however, most compounds show only moderate 5-HT~A receptor affinity. In a recent study by Kuipers et al [49] new highly potent N 4unsubtituted arylpiperazines were reported. It was shown that arylpiperazines that display agonist affinity probably bind to the receptor in a relatively coplanar conformation with a plane angle of approximately 30 ~ between the aryl and piperazine rings. The fit as in Figure 8d was shown to agree best with structure-affinity relationships of bicyclic arylpiperazines.

Receptor-ligand interactions Models of GPCR are used for the study of receptor-ligand interactions, as for no member of the GPCR family experimentally determined high-resolution 3D structures are available. Most of these models are based on the high-resolution structure of bacteriorhodopsin which is not coupled to a G-protein, but shows

59 functional resemblance with the GPCR rhodopsin [4, 33, 50]. In these studies an attempt was made to combine receptor-binding data with knowledge from other experimental data concerning receptor structure and function. The strength and weaknesses of GPCR models were discussed and evaluated by Hibert et al. [33]. Several models that rationalize 5-HT~A receptor-ligand interactions have been published. As an example Trumpp-Kallmeyer et al. [51] presented a docking study of several natural ligands in their corresponding receptor models. The 5-HT~A receptor was one of the subtypes investigated. In the 5-HT~A receptor model Aspll6 in helix III interacts with the agonists basic nitrogen. The indole NH of serotonin points towards Gly164 in helix IV, while its 5-OH group forms a hydrogen bonding interaction with Thr200 of helix V. Aromatic interactions occur between serotonin and Phe204 and Phe362 in helices V and VI, respectively. Kuipers et al. [52] rationalized affinities of 5-HT~A agonists of several structural classes (e.g. tryptamines, aminotetralins, aporphines) with a docking study of these compounds in a 5-HT~A receptor model. Figure 4 shows a comparison of interactions as defined in this model (b) which were based on mutation data, with those from ligand-fitting models (a) [34, 36, 38]. The space in the ligand models that may contain large substituents at the basic nitrogen atom, overlaps with the available space in the core of the 5-HT~A receptor model. Sterically hindered positions in the receptor map derived from ligandfitting, coincide with the positions of the backbones of helices IV and V. The hydroxy group interacts with Thr200 in helix V. This residue has a dual character as it contains a hydroxy and a methyl group which enables the formation of hydrogen bonds and hydrophobic contacts, respectively. Thus methoxy groups in the 5-position of tryptamines and the 8-position of aminotetralins may also have favourable hydrophobic contacts with this residue. The indole NH of tryptamines (e.g. serotonin) as well as the hydroxy groups of compounds homochiral with (S) 8-OH-DPAT may form a hydrogen bonding interaction with Ser199 in helix V. In the model by Kuipers et al. [52], the lipophilic pocket which is essential for the high 5-HT~A receptor affinity of 8-OH-DPAT may consist of the residue combination Valll7-Cysl20 (see Figure 2). In 5-HT2 receptors Cysl20 is replaced by a polar Ser. According to the receptor model, the propyl chain of 8-OH-DPAT does not attribute to affinity for 5-HT 2 receptors because a lipophilic pocket is lacking, in 5-HT1B.D receptors Valll7 is replaced by a more bulky Ile, which may cause steric hindrance. This hypothesis is corroborated by SAR data, which indicate that 8-OH-DPAT has low affinity for 5-HT 2 receptors because it lacks a pyrrole ring, while steric hindrance of its propyl substituents causes low affinity for 5-HT m and 5-HT~D receptors [53, 54]. The area in which aromatic groups are favourable in ligands, is surrounded with aromatic residues in the receptor model. For instance, the second benzene ring of MHA may have an additional aromatic interaction with residues Phe361 and Phe362 in helix VI [52]. The binding mode of MHA was also subject of a docking study by Hedberg et al. [55], who analyzed differences in aporphine binding to 5-HT~A and D2

6O receptors. In this model, the basic nitrogen interacts with Asp116 and the essential benzene ring of MHA has an aromatic interaction with Phe362 in helix VI. The selectivity of MHA for 5-HTI^ receptors is explained by the C10methyl substituent of MHA being located in a lipophilic cavity unique for the 5HT1A receptor. The binding site of agonists appears to be rather similar in all three receptor models, as several residues found in the active sites are the same (see Figure 2). For instance, Phell2, Aspll6 (TM III), Gly164 (TM IV), Ser199, Thr200 (TM V) and Trp358, Phe361, Phe362 (TM VI) are involved in agonist binding in two or all three of the models. However some differences exist. In ligandmodelling studies it was shown that MHA can be fitted on 8-OH-DPAT and serotonin with overlap of the hydroxy groups (see Figure 4). In the model by Hedberg et al. [55] the hydroxy group of MHA interacts with Ser199. However, in the models by Trumpp-Kallmeyer et al. [51] and Kuipers et al. [52] the hydroxy group of the agonists interacts with Thr200. This last interaction accounts for the effect of Thr200-~Ala substitution which completely abolishes serotonin affinity [5]. This effect is not explained by the Hedberg model [55]. In the model by Kuipers et al. [52] Ser199 interacts with the indole NH of serotonin. The occurrence of this interaction is supported by SAR data of tryptamines [56, 57] and mutation data [5]. In this aspect the model differs from that of Trumpp-Kallmeyer et al. [51] in which the indole NH is directed towards Gly164. This last model provides no explanation for the effect of Ser199-~Ala substitution which decreases 5-HT1A receptor affinity for serotonin. The model by Hedberg et al. [55] explains the difference in 5-HT1A/D2 receptor binding profile of MHA. The C10-methyl group of MHA may bind in a lipophilic pocket formed by Ala203 and Ile205 of the 5-HT~A receptor. This lipophilic [5~pocket is absent in D2 receptors because Ala203 is replaced by a Ser residue. However, in the model by Kuipers et al. [52] a similar lipophilic pocket that can accommodate the C10-methyl group may be formed by Aia203, Thr200 and Leu366 [49]. This lipophilic pocket is also absent in Dg. receptors as these residues have been replaced by two polar serines and an isoleucine, respectively. Thus both orientations of MHA as in the models by Hedberg et al. [55] and Kuipers et al. [49, 52] account for its selectivity for 5-HT1A with respect to D2 receptors. Probably further mutation experiments will be required to elucidate the exact binding mode of this compound.

Antagonist models The pharmacophore derived by Hibert et al. [58] by superimposition of antagonists of different structural classes contains the same elements as the agonist pharmacophore, namely an aromatic ring and a basic nitrogen atom. As might be expected, the spacial requirements differ from the agonist pharmacophore: d= 5.6/~ and h= 1.6~. Unfortunately, the model was built with only moderately active compounds. For validation of this model more SARs of the concerning classes should become available.

61

oo

"r

Figure 9. Schematic representation of a) a ligand model for aryloxypropanol amines by Langlois et al. [59] and b) interactions of this class of antagonists in a 5HT~A receptor model by Kuipers et al. [52].

62 Figure 9a shows a schematic representation of a model of aryloxypropanolamines, which are potent ~-adrenoceptor antagonists but also display high affinity for 5-HT~A and (rodent) 5-HT~B receptors [59]. This model seems to be in good agreement with the suggested binding site of these compounds in the 5-HT~^ receptor model by Kuipers et al. [52] (Figure 9b). The preference for lipophilic and aromatic ring substituents R may be explained by the surrounding with aromatic (Tyr96, Tyr390, Trp387 and Phell2) and lipophilic aliphatic (Met92 and Leu43) residues in the receptor model. The sterically unfavourable region in the ligand model coincideswith the backbone of helix I in the receptor model. The (double) interaction of the oxypropanol moiety with the essential Asn386 in helix VII makes it very unlikely that these compounds are capable of occupying the area between the helices III, V and VI in which agonists bind. Thus the antagonistic properties of this class may be explained.

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Savarese TM, Fraser CM. Biochem J 1992 (a); 283; 1-19. Lee NH, Kerlavage NR. Tr Biomed Res 1993; 6; 488-497. Strader CD, Fong TM, Tota MR, Underwood D. Ann Rev Biochem 1994; 63: 101-132. Schwartz T. Curr Opinion Biotech 1994; 5: 434-444. Ho BY, Karschin A, Branchek T, Davidson N, et al. FEBS Lett 1992; 312: 259-262. Chanda PK, Minchin MCW, Davis AR, Greenberg L, et al. Mol Pharmacol 1993; 43: 516-520. Horstman DA, Brandon S, Wilson AL, Guyer CA, et al. J Biol Chem 1990; 35: 21590-21595. Neve KA, Cox BA, Henningsen RA, Spanoyannis A, et al. Mol Pharmacol 1991; 39: 733-739. Oliveira L, Paiva ACM, Sander C, Vriend G. TIPS 1994; 15: 170-172. Dixon RAF, Sigal IS, Candelore MR, Register RB, et al. EMBO J 1987; 6: 3269-3275. Karnik SS, Khorona HG. J Biol Chem 1990; 265: 17520-17524. Savarese TM, Wang CD, Fraser CM. J Biol Chem 1992 (b); 267: 1143911448. Henderson R, Baldwin JM, Ceska TA, Zemlin D, et al. J Mol Biol 1990; 213: 899-929. Heijne von G. J Mol Biol 1991; 218: 499-503. Trumpp-Kallmeyer S, Hoflack J, Bruinvels A, Hibert M. J Med Chem 1992; 35: 3448-3462. Zhou W, Flanagan C, Ballesteros JA, Konvicka K, et al. Mol Pharmacol 1994; 45: 165-170. Sealfon SC, Chi L, Ebersole BJ, Rodic V, et al. J Biol Chem 1995; 270: 16683-16688.

63 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Raymond JR. J Biol Chem 1991; 266: 14747-14753. Strader CD, Sigal IS, Dixon RAF. FASEB J 1989 (a); 3: 1825-1832. Choudhary MS, Graigo S, Roth BL. Mol Pharmacol 1993; 43: 755-761. Guan X, Peroutka SJ, Kobilka BK. Mol Pharmacol 1992; 41: 695-698. Suryanarayana S, Daunt DA, Zastrow M von, Kobilka BK. J Biol Chem 1991; 266: 15488-15492. Oksenberg D. Marsters SA, O'Dowd BF, Jin H, et al. Nature 1992; 360: 161-163. Metcalf MA, McGuffin RW, Hamblin MW. Biochem Pharmacol 1992; 44: 1917-1920. Adham N, Tamm JA, Salon JA, Vaysse PJJ, et al. Neuropharmacol 1994; 33: 387-391. Kobilka BK, Frielle T, Collins S, Yang-Feng T, et al. Nature 1987; 329: 7579. Fargin A, Raymond JR, Lohse MJ, Kobilka BK, et al. Nature 1988; 335" 358-360. Albert PR, Zhou Q, Tol HHM van, Bunzow JR, et al. J Biol Chem 1990; 265: 5825-5832. Fujiwara Y, Nelson DL, Kahsihara K, Varga E, et al. Life Sci 1990; 47" 127-132. Peroutka SJ. Synapse 1994; 18: 241-260. Oakey RJ, Caron MG, Lefkowitz RJ, Seldin MF. Genomics 1991; 10: 338344. Strader CD, Candelore MR, Hill WS, Dixon RAF, et al. J Biol Chem 1989 (b); 264: 16470-16477. Hibert, M, Trumpp-Kallmeyer, S, Hoflack, J, Bruinvels A. TIPS 1993; 14: 7-12. Hibert MF, McDermott I, Middlemiss DN, et al. Eur J Med Chem 1989; 24: 31-37. Mellin C, Vallgarda J, Nelson DL, et al. J Med Chem 1991; 34: 497-510. Chidester CG, Lin C-H, Lahti RA, et al. J Med Chem 1993; 36: 1301-1315. Jain AN, Harris NL, Park JY. J Med Chem 1995; 38: 1295-1308. Westkaemper RB, Glennon RA. Pharmacol Biochem Behav 1991; 40: 10191031. Hacksell U, Liu Y, et al. Drug Design Disc 1993; 9: 287-297. BjSrk L, Backlund B, H85k, et al. J Med Chem 1989; 32: 779-783. Mellin C, BjSrk L, Karl~n A, et al. J Med Chem 1988; 31: 1130-1140. WikstrSrn H, Andersson B, et al. J Med Chem 1987; 30: 1567-1573. Arvidsson LE, Johansson AM, et al. J Med Chem 1987; 30: 2105-2109. Moon MW, Morris JK, Heier RF, et al. J Med Chem 1992; 35: 1076-1092. Glennon RA, Naimann NA, Pierson ME, Smith JD, et al. J Med Chem 1989; 32: 1921-1926. Taylor EW, Nikam, SS, Lambert G, Martin AR, et al. Mol Pharmacol 1988; 34: 42-53. Glennon RA. Drug Dev Res 1992; 26: 251-274. Sleight AJ, Peroutka SJ. Naunyn-Schmiedeberg's Arch Pharmacol 1991;

64 343: 109-116. 49 Kuipers W, Van Wijngaarden I, Kruse CG, Ter Horst-Van Amstel M, et al. J Med Chem 1995; 38: 1942-1954. 50 Baldwin JM. EMBO J 1992; 12: 1693-1703. 51 Trumpp-Kallmeyer S, Bruinvels A, Hoflack J, et al. Neurochem Int 1991; 19/4: 397-406. 52 Kuipers W, Van Wijngaarden I, IJzerman AP. Drug Design Disc 1994; 11: 231-249. 53 Glennon RA, Titeler M, Lyon RA, Slusher RM. J Med Chem 1988; 31: 867870. 54 Slaughter JL, Harrington MA, Peroutka SJ. Life Sci 1990; 47: 1331-1337. 55 Hedberg MH, Johansson AM, Nordvall G, Yliniemel~i A, et al. J Med Chem 1995; 38: 647-658. 56 Hoyer D.In: Fozard JR, ed. The peripheral actions of 5-hydroxytryptamine. New York: Oxford University Press, 1989; 72-99. 57 Macor JE, Fox CB, Johnson C, Koe K, et al. J Med Chem 1992; 35: 36253632. 58 Hibert MF, Gittos MW, Middlemiss DN, et al. J Med Chem 1988; 31: 10871093. 59 Langlois M, Br~mont B, Rouselle D, et al. Eur J Pharm - Mol Pharm Sec 1993; 244: 77-87.

Serotonin Receptors and their Ligands B. Olivier, I. van Wijngaarden and W. Soudijn (Editors) 9 1997 Elsevier Science B.V. All rights reserved.

65

5-HT~A Receptor coupling to G-proteins W. Soudijn Leiden/Amsterdam, Center for Drug Research, P.O.Box 9502, 2300 RA Leiden, The Netherlands.

A majority of the different receptors in the cell membrane including the 5,HTIA receptor, is coupled to a G-protein associated with the cytoplasmic surface of the cell membrane. The G-protein transduces the signal from the activated receptor to an effector molecule resulting in the stimulation or inhibition of a second messenger molecule or in the modulation of an ion channel. G-proteins (guanine nucleotide binding proteins) are heterotrimeric proteins consisting of three subunits, a, ~ and T of different amino acid composition and relative molecular mass of about 40.5, 37.4 and 8.4 kDa respectively. The a subunit is the guanine nucleotide binding unit that also has intrinsic GTP-ase activity. Mechanism

of activation of the G-protein

In the inactivated state GDP is tightly bound to the a subunit of the G-protein. Interaction of the receptor with an agonist results in a conformational change of the receptor and a coupling of the G-protein via its a unit to the receptor. Then GDP dissociates from the a unit followed by an immediate association of GTP. The next step is dissociation of the whole complex with the formation of: - the receptor in a low affinity state for agonist binding - the a unit with bound GTP - the ~/T dimeric unit The a unit with bound GTP activates the proper effector e.g. adenylylcyclase and the second messenger c-AMP is produced. The ~/T complex may in some cases at least interact with another effector or with the same effector as the a unit thereby modulating its activity. The GTP-ase activity of the a unit eventually hydrolyses the bound GTP to GDP. The a unit dissociates from the effector and reassociates with the ~/T complex so that the inactivated G-protein is reinstated [11-13]. There is a host of different G-proteins with different functions generating different second messengers and modulating different ion channels. For reviews on these subject see [1-5]. The 5-HTIA receptor is coupled to a Gi-protein which means that activation of this protein by 5-HT1A agonists results in inhibition of the production of the second messenger c-AMP by adenylylcyclase [6].

66 The mechanism of this inhibition is still a matter of debate [7] but it is clear that not only the Gi~ subunits but also the ~/~,dimeric subunits are involved in the regulation of adenylylcyclase activity. As there exist at least six distinct isoforms of adenylylcyclase [8] different patterns of regulation are to be expected. It was shown by Taussig et al. [9] that under the experimental conditions used the different Gia subunits Gi~, Gi~2and Gi~ all inhibit the adenylylcyclase isoforms type 1, 5 and 6 but not type 2. High concentrations of G~ activate type 2 and also type 1 adenylylcyclase. Both types also interact with the ~/~ dimeric subunit of the G protein. Type 1 is inhibited and type 2 is activated in the presence of G~ the a subunit of a G~ protein that transduces a signal for the stimulation of c-AMP production by adenylylcyclase. There is consensus that stimulation of 5-HT1A receptor usually leads to a decrease in the production of c-AMP. However the degree in which this occurs depends on a number of factors as cell and tissue type, concentration of 5-HT~A receptors and G~ proteins, the agonist concentration in the receptor compartment, the abundance of the different isoforms of adenylylcyclase in the cell and the presence and activation state of other types of G proteins. Partial agonists couple to G~.~ subunits with a lower efficacy than full agonists [10]. Antagonists binding to the 5-HT~A receptor prevent the association of the G~ protein with the receptor and thereby block the signal transduction.

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13

Gilman AG. Ann Rev Biochem 1987; 56: 615-649. Spiegel AM. Ann Repts Med Chem 1988; 23: 235-242. Spiegel AM. Med Research Revs 1992; 12" 55-71. Hepler JR, Gilman AG. TIBS 1992; 17: 383-387. Sternweis PC, Smrcka AV. TIBS 1992; 17: 502-506. Zifa E, Fillion G. Pharmacol Revs 1992; 44: 401-458. Simon MI, Strathmann MP, Gautam N. Science 1991; 252: 802-807. Iyengar R, FASEB J 1993; 7: 768-775. Taussig R, Tang WJ, Hepler JR, Gilman AG. J Biol Chem 1994; 269: 60936100. Gettys ThW, Fields TA, Raymond JR. Biochem 1994; 33: 4283-4290. Lambright DG, Sondek J, Bohm A, Skiba NP, et al. Nature 1996; 379: 311319. Sondek J, Bohm A, Lambright DG, Harem HE, et al. Nature 1996; 379: 369374. Clapham DE. Nature 1996; 379: 297-300.

Serotonin Receptors and their Ligands B. Olivier, I. van Wijngaarden and W. Soudijn (Editors) 9 1997 Elsevier Science B.V. All rights reserved.

67

Ligand binding assays M.Th.M.Tulp and I.van Wijngaarden Solvay Duphar B.V., P.O.Box 900, 1380 DA Weesp, The Netherlands.

INTRODUCTION The history of serotonin receptor binding started nearly three decades ago with the pioneering work of Marehbanks [1] who studied interactions of [3H]-serotonin with synaptosomes from rat brain. Farrow and Vanukis [2] were the first who showed that [3H]-d-LSD labels serotonin receptors, using an equilibrium dialysis technique. Application of rapid filtration techniques confirmed that [3H]-d-LSD labels serotonin receptors with high affinity [3]. In 1976 for the first time [3H]-5HT was used to label 5-HT receptors using modern binding techniques [4]. The observation that [3H] d-LSD consistently labels more receptors than [3H]-5-HT does, made the authors to assume that these ligands labelled two different 'states' of the 5-HT receptor. Binding studies with [3H]-serotonin showed clearly biphasie displacement curves with a number of compounds, suggesting a heterogeneous population of receptors for this neurotransmitter [5]. The availability of [3H]spiperone soon confirmed this hypothesis, and for the first time it was proposed on the basis of binding studies that different 5-HT-reeeptors exist: 5-HT 1 and 5HT 2 [6]. In 1981 the name '5-HT1A' appeared in the literature: this name was used to distinguish 5-HT receptors for which both 5-HT and spiperone had a high affinity from '5-HT1B' receptors for which spiperone had a low affinity [7]. The discovery of the tetraline derivative 8-OH-DPAT [8] was an extremely important development in the history of 5-HT1A receptor research. Shortly, the compound became available as [3H]-ligand, and allowed direct binding studies on 5-HT1A receptors [9]. Despite the fact that since then at least a dozen other radioligands have been used to label this receptor (see Table 1), [3H]-OH-DPAT is still the ligand of choice, and used by the majority of investigators. Extensive studies on the pharmacological and biochemical properties of 5-HT1A receptors in various brain regions revealed no real differences between somatodendritic autoreeeptors within the dorsal raphe nucleus and the postsynaptie receptors in the septum and hippoeampus [24]. 5-HT1Areceptors from different species, including man, have been cloned, and found to be so similar that until now it is generally accepted that 5-HT1A receptors, at least 5-HTIA binding sites, are identical [25]. In Table 2 affinity constants have been collected of all compounds which are listed in at least two of the first articles in which the different radioligands for 5HT1A receptors have been introduced. From this table it is evident that the data generated with the different ligands are largely identical.

68 Table 1 Radioligands used to label 5-HT,A receptors

Radioligand [3H]-serotonin [3H]-8-OH-DPAT [3H]-ipsapirone [3H]-WB 4101 [3H]-PAPP [3H]-p-azido-PAPP [3H]-5-MeO-DPAC [3H]-spiroxatrine [12sI]-BH-8-MeO-N-PAT [3H]-rauwolscine [3H].buspirone 9 [3H]-NAN-190 [3H]-5-methylurapidil [3H]-flesinoxan ** [3H]-tandospirone *** [3H]-WAY 100635

K~(nM) -.4.29 -.3.8 2.9 1.0 1.04 2.21 ... 13.1 1.9 0.91 1.7 11.2 0.37

First Reference Nelson et al. 1978 Gozlan et al. 1983 Dompert et al. 1985 Norman et al. 1985 Ransom et al. 1986 a Ransom et al. 1986 b Cossery et al. 1987 Nelson et al. 1987 Gozlan et al. 1988 Convents et al. 1989 Bruning et al. 1989 Rydelek-Fitzgerald et al. 1990 Gross et al. 1990 Schipper et al. 1991 Tanaka et al. 1991 Khawaja et al. 1995

Ref. nr. [ ] [5] [9] [10] [11] [12] [~3] [14] [15] [16] [17] [18] [19] [2O] [21] [22] [23]

* labels dopamine-D 2 rather than 5-HT,A receptors. ** exclusively used for autoradiography *** essentially autoradiography, no quantitative displacement data. In the excellent review by Zifa & Fillion [26] a very large table is given in which a large number of affinity constants of many different compounds are listed. From this table it is even more obvious there are no essential differences between I~.-values for 5-HT1A receptors, irrespective of ligand, species or tissue. As stated above, [3H]-8-OH-DPAT is still the ligand of choice to label 5-HT1A receptors. It is first of all very potent and very selective, it has a low non-specific binding (specific binding is usually reported to be between 80 and 90%), it is commercially available and the combined data published on labelling 5-HT1A receptors with this ligand form a very extensive and reliable reference framework. Why then all those other labels? Of course there is a sound scientific rationale for labelling any 5-HT1^ ligand: it allows direct autoradiographical comparisons with [3H]-8-OH-DPAT and thus produces the most convincing evidence about the mechanism of action which can be obtained. Examples of such studies are e.g. those with [3H]-flesinoxan and [3H]-tandospirone. Both ligands were used primarily for this reason, and were not claimed to be competitors for [3H]-8-OHDPAT in 5-HT~A binding studies. Flesinoxan is equipotent with 8-OH-DPAT, and has a comparable selectivity. For this reason it could be used as 5-HTIA ligand, too.

Table 2 Ki- or ICso-values for 5-HT~A receptors (all in nM) refi ligand:

9 DPAT

10 ipsa

11 4101

12 PAPP

13 azid

14 DPAC

15 spir

16 NPAT

17 rauw

19 NAN

20 5-M-u

COMP:

~

~

!~C~

~

~

~

~

~

_~

~

~

8-OH-DPAT 3.0 5-HT 6.8 RU24969 9.8 bufotenine 21 buspirone 30 metergoline 35 5-MeO-N,N 40 methysergide 71 methiothepine 89 yohimbine 1300 quipazine 3400 haloperidol 3600 ketanserine 4200 dopamine 17000 noradrenaline 170000 d-LSD ipsapirone spiroperidol W B 4101 prazosine TFMPP mianserine gepirone mesulergide quinpirole clonidine propranolol S C H 23390

0.4 4.3

6.7

2.8 18

14 70

6.7 5.2

1.4 4.1

8.9 20.7

23 20 15

1.2 5.0 5.5 3.5 25

16 2.0 3.2

2.0 4.8

7.0 10

100

23 WAY

5.6 15 97 19

66 28

8.3

40 79

95 32

1170 7000 38000 0.31 2.2 23

920 1290 3750 28100 74 3.8 1295

>10000 1000 1589 >10000 >10000 2.5 6.1 8.4 40 38

230

>10000 235 550

2130

2840 7680 62900 8.2

1800 2637

>I0000

2500 57600

40.9 67.9 23.4

7900 140000 9.6 450

8960

11000 230

97 680 2210 2710

56 460 2600 1800 260 280

250

400

82

6.0 13 8

8.3 550

2820 180 3890

250 320

69

70 Tandospirone is 5-HT~ selective, but about ten times less potent than DPAT. 5HT, the natural ligand has the obvious disadvantage that (by definition) it labels all 5-HT receptor subpopulations with approximately equally high affinity: it can not be used to label 5-HTI^ receptors unless all other 5-HT receptors are blocked. For all practical purposes, [aH]-ipsapirone is also a good ligand for 5-HT1A receptors, although it does not have any advantage over 8-OH-DPAT. The same holds true for [3H]-5-MeO-DPAC and [I=I]-BH-8-MeO-N-PAT. The ligands [SH]PAPP and [aH]-p-azido-PAPP are also 5-HT~^ selective, but both feature a high non-specific filterbinding which results in a specific binding of only half that of [3H]-8-OH-DPAT. [3H]-WB 4101 and [aH]-5-methylurapidil can only be used to label 5-HTL~ receptors if al-adrenergic receptors are blocked; in order to use [~I-I]-NAN-190 not only al-adrenergic but also dopamine-D 2 receptors need to be blocked. DopamineD2 receptors also have to be blocked to allow the use of [3H]-spiroxatrine or [3H]buspirone. [SH]-Rauwolscine not only has a modest potency (about 1110 of DPAT), but it is also necessary to block a2-adrenergic receptors. In summary: most of the ligands given in Table 2 have a very clear disadvantage when compared with [3H]-8-OH-DPAT, either in potency, selectivity, or percentage specific binding. REFERENCES

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Marchbanks RM. J Neurochem 1966; 13: 1481-1493. Farrow JT, Van Vanukis H. Nature 1972; 237: 164-166. Bennet JL, Aghajanian GK. Life Sci 1974; 15: 1935-1944. Bennet JP, Snyder SH. Mol Pharmacol 1976; 12: 373-389. Nelson DL, Herbet A, Bourgoin S, Glowinski J, et al. Mol Pharmacol 1978; 14: 983-995. Peroutka SJ, Snyder SH. Mol Pharmacol 1979; 16: 687-689. Pedigo NW, Yamamura HI, Nelson DL. J Neurochem 1981; 36: 220-226. Hjorth S, Carlsson A, Lindberg P, Sanchez D, et al. J Neural Transm 1982; 55: 169-188. Gozlan H, E1 Mestikawy S, Pichat L, Glowinski J, et al. Nature 1983; 305: 140-142. Dompert WU, Glaser T, Traber J. Naunyn-Schmiedeberg's Arch Pharmacol 1985; 328: 467-470. Norman AB, Battaglia G, Morrow AL, Creese I. Eur J Pharmacol 1985; 106: 461-462. Ransom RW, Asarch KB, Shih JC. J Neurochem 1986a; 46: 68-75. Ransom RW, Asarch KB, Shih JC. J Neurochem 1986b; 47: 1066-1072. Cossery JM, Gozlan H, Spampinato U, Perdicakis C, et al. Eur J Pharmacol 1987; 140: 143-155. Nelson DL, Monroe PJ, Lambert G, Yamamura HI. Life Sci 1987; 41: 15671576.

71 16 Gozlan H, Ponchant M, Daval G, Verg6 D, et al. J Pharmacol Exp Ther 1988; 244: 751-759. 17 Convents A, De Keyser J, De Backer JP, Vauquelin G. Eur J Pharmacol 1989; 159: 307-310. 18 Bruning G, Kaulen P, Schneider U, Baumgarten HG. J Neural Transm 1989; 78: 131-144. 19 Rydelek-Fitzgerald L, Teitler M, Fletcher PW, Ismaiel AM, et al. Brain Res 1990; 532: 191-196. 20 Gross G, Schfittler K, Xin X, Hanft G. J Cardiovasc Pharmacol 1990; 15: $8S16. 21 Schipper J, Tulp MThM, Berkelmans B, Mos J, et al. Human Psychopharmacol 1991; 6: $53-61. 22 Tanaka H, Shimizu H, Kumasaka Y, Hirose A, et al. Brain Res 1991; 546: 181-189. 23 Khawaja X, Evans N, Reilly Y, Ennis C, et al. J Neurochem 1995; 64: 27162726. 24 Radja F, Daval G, Hamon M, Verg~ D. J Neurochem 1992; 58: 1338-1346. 25 Humphrey PPA, Hartig P, Hoyer D. TIPS 1993; 14: 233-236. 26 Zifa E, Fillion G. Pharmacol Rev 1992; 44: 401-458.

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73

5-HT~A Behavioural models J. Mos ~ and B.Olivier 1'2~ ~Solvay Duphar B.V., CNS Research, P.O.Box 900, 1380 DA Weesp, The Netherlands. 2~Jniversity of Utrecht, Faculty of Pharmacy, Dept. of Psychopharmacology, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

Behavioural models 5-HTaA receptor agonists have been tested in a wide variety of animal models indicative of CNS functions. These range from models for motion sickness and emesis to models predictive for antipsychotic drugs. In most of these tests 5-HTxA receptor agonists appear to be active, although the degree of specificity varies. 5HTxA receptor agonists influence the total serotonin neurotransmission by acting on the somatodendritic autoreceptor as well as by acting on postsynaptic receptors. From many behavioural effects of 5-HT1A receptor agonists the precise molecular mechanism of action remains elusive and it is quite conceivable that similar behavioural effects can be induced by various manipulations of the serotonin neurotransmission. In order to avoid confusion on the specificity of the behavioural effects we limit our discussion of behavioural models to those effects which are almost certainly derived from direct effects on the 5-HT~A receptor and which represent behavioural changes unique for specific 5-HTIA receptor agonists. Thus although the 5-HTIA receptor plays a more ubiquitous role than delineated below, the chosen behaviours are the most predictive of the 'pure' 5-HTIA related effects.

5-HTL~ receptor agonist and feeding Early experiments with 8-OH-DPAT in rats revealed the stimulatory effects on feeding [1, 2]. Subsequent experiments confirmed the increase in feeding after application of other 5-HT1A agonists. Gilbert and Dourish [3] reported that buspirone, ipsapirone and gepirone, specific but partial 5-HT1A agonists, also increased feeding in rats [4]. Similarly MDL 72832 was found to increase food intake; the stereospecificity of the effects supported the functional role of 5-HTaA receptors in feeding responses [5]. The effects on feeding seem quite robust as various authors have reported reliable effects of 8-OH-DPAT. Only one study found strain differences in response to 8-OH-DPAT [6]. Shepherd and Rodgers [7] demonstrated that 8-OH-DPAT effects on food intake were not limited to rats only. They demonstrated the specificity of 8-OH-DPAT effects on feeding in mice using a behavioural competition paradigm. Despite these consistent effects, some controversy arose regarding the behavioural specificity of feeding induced by 8-OH-DPAT. For example, the drug

74 did not increase the intake of liquid diets and elicited gnawing on wooden blocks in the absence of solid food, suggesting that it's hyperphagic action may be secondary to stimulation of gnawing or non-specific increase in arousal [8, 9]. However, in other laboratories, 8-OH-DPAT did increase liquid feeding [57, 58]. Moreover, young and old rats differ [10], the taste and food texture affect feeding [11] as well as novelty [12]. In summary 5-HT~A agonist increase food intake in free feeding rats, although several (behavioural) factors are of importance. Studies on the site of action strongly suggest an effect mediated via the somatodendritic autoreceptor. Direct injection of 8-OH-DPAT into the dorsal and medial raphe nucleus enhanced feeding [13, 14, 15]. Similarly 5-HT depletion by PCPA antagonized 8-OH-DPAT effects on feeding, pointing to the autoreceptor as the site of action [16]. Pharmacological antagonism studies have resulted in variable effects. Hutson et al. [17] showed that metergoline blocked 8-OH-DPAT-induced feeding, but methysergide, ketanserin, MDL 77222 and ICS 205930 had no effect. Since ()pindolol and spiperone also blocked the effects of 8-OH-DPAT, it was suggested that 5-HT~A receptors, rather than 5-HT 2 or 5-HT3 receptors are involved. With regard to the involvement (direct or indirect) of dopamine, the results appear contradictory. Muscat et al. [18] and Fletcher and Davies [19] suggest significant effects of dopamine antagonists on 8-OH-DPAT induced feeding, but such effects were not observed by Hutson et al. [17]. Most likely these studies have all been hampered by the fact that specific 5-HT1A receptor antagonists were not yet available. Recent studies with the specific 5-HTIA antagonist WAY 100635 [20] have unambiguously demonstrated the crucial role of the 5-HT~A receptor in feeding. L o w e r Lip R e t r a c t i o n (LLR) in rats Stimulation of 5-HT receptors in the brain of rats induced a characteristic behavioural pattern, the so-called 5-HT syndrome [21]. This syndrome may consist of one or more of the following symptoms: lower lip retraction, fiat body posture, hindlimb abduction, spreadpaws, arched back, head weaving, wet dog shakes, penile erections and purposeless chewing. Some distinct components of this syndrome have been associated with the activation of specific subtypes of the 5-HT receptor. Lower lip retraction is related to the selective activation of 5-HT1A receptors as had been described by Berendsen et al. [22] and Molewijk et al. [23]. Treatment with the 5-HT~A receptor agonist 8-OH-DPAT affects the musculature of the lower lip of rats, thus causing the lower incisors to become visible (albeit that close inspection is needed). Berendsen et al. [22] tested a wide variety of serotonergic antagonists, but none was able to effectively block the 8-OH-DPAT induced LLR. Partial 5-HT1A agonists like ipsapirone and buspirone also induce LLR, but several compounds with a high affinity for the 5-HT~Areceptor, like 5-MeODMT did not induce LLR by itself; only when other serotonergic receptor antagonists were co-administered, 5-MeODMT was able to induce lower lip retraction. This suggests that there is a delicate interplay between the various serotonergic receptors, some of which may interfere

75 with the full expression of LLR. By and large their experiments strongly suggested that the 5-HT1A receptor is specifically involved in the mediation of LLR. Later developments have yielded specific 5-HT1A antagonists, notably WAY 100,135; WAY 100,635 and (S)-UH-301. These silent and specific 5-HT~A receptor antagonists have no effects on their own, but totally block the LLR induced by 8OH-DPAT or flesinoxan, two of the most full agonists at the 5-HT~A receptor (own unpublished data). An intriguing question is the site of action of 5-HT~A receptor agonists to induce LLR. 5-HT~A receptor agonists may act postsynaptically at the receptor, or may affect the somatodendritic receptor in the raphd nuclei. Furthermore, it is of interest which brain area is involved, i.e. is the dorsal or the medial raph4 responsible and which of the projection areas is crucial for the induction of the LLR. Local application studies in the dorsal or medial raphd suggested a preferential involvement of the medial raph~ nucleus [59], but higher doses of 8OH-DPAT were effective in inducing LLR after local application into the dorsal raphd. However, our own experiments did not confirm this idea, since both injection of 8-OH-DPAT in the dorsal as well as in the median raphd nucleus induced lower lip retraction (Bouwknecht et al. unpublished data). Local application of 8-OH-DPAT in the raph~ nuclei reduces the firing rate of serotonergic neurons by acting on the somatodendritic autoreceptor. This in turn leads to a reduction of the serotonergic neurotransmission, i.e. less serotonin is released in the synaptic clett. It is not known which of the postsynaptic receptors is critically involved in the mediation of LLR. As far as we know, no local application studies have been performed in the major projection areas of the serotonin system. In summary, most experiments point to the 5-HT~A receptor as playing an important role in the induction of the LLR, although interactions with other serotonergic receptors may interfere. Antagonist studies support the specificity of the role of the 5-HT~A receptor in the LLR. Although the studies are by no means exhaustive, the provisional conclusion is that the site of action of 5-HT~A agonists is presynaptically in the raphd nuclei.

5-HT~ agonist and male sexual behaviour in rats The first report that linked 5-HT,~ agonist to sexual behaviour in male rats was published in 1981. Ahlenius et al. [24] reported that 8-OH-DPAT and 8-OMeDPAT reduced the number of intromissions preceding ejaculation and shortened the ejaculation latency. In addition 8-OH-DPAT reduced the postejaculatory interval. Finally, they described that 8-OH-DPAT and 8-OMe-DPAT partly or completely restored sexual behaviour in castrated male rats. At this time 8-OH-DPAT was viewed as a serotonin agonist, just like DOM, LSD and quipazine, none of which resulted in a facilitation of male sexual male behaviour. Only later, receptor binding experiments showed the unique qualities of 8-OH-DPAT in being a potent and selective 5-HT~A agonist [25]. Subsequent experiments corroborated and extended the findings with 8-OHDPAT. 8-OH-DPAT facilitated copulating behaviour in penile desensitized male rats, affected ultrasonic communication associated with sexual activities [26],

76 restored sexual behaviour in neonatally ATD-treated rats [27] and reversal of sexual exhaustion [28]. Thus it appears that the 5-HT1A receptor 8-OH-DPAT is a powerful mediator of rat male sexual behaviour. Other studies revealed that 5-HTI^ full flesinoxan and partial agonists [29], buspirone [30] and ipsapirone [31] also facilitated male sexual behaviour in rats. By contrast, male mice showed no facilitation of male sexual behaviour, but rather an inhibition [32]. In ferrets, 8-OH-DPAT, similarly inhibited masculine sexual behaviour [33]. In rhesus monkeys, however, facilitatory effects on male sexual behaviour were observed, albeit in more limited dose range for 8-OH-DPAT than for ipsapirone [34]. The effects of several 5-HTI^ agonists, notably the aminotetralins, appeared to be stereo selective, which is well in agreement with the effects of 5-HT~^ receptors

[35]. Initial antagonism studies with metergoline and methiotepine [36] were unsuccessful, i.e. 8-OH-DPAT was not antagonized. However, using (-)alprenolol and (-)pindolol the effects of 8-OH-DPAT could be antagonized (Ahlenius and Larsson, ch. 16). Subsequent studies confirmed that pindolol was an effective antagonist [37; 38]. The most convincing evidence that 5-HT~A receptors are responsible for the observed effects of 8-OH-DPAT comes from studies by Johansson et al. [39] who used the specific and silent 5-HT~A antagonist (S)-UH301. This drug dose dependently antagonized the effects of 8-OH-DPAT, but had no effects of its own. Various investigations have addressed the issue of the site of action of 5-HT~A receptor agonist on sexual behaviour of the rat. The results are quite complex since not only intracerebral, but also intrathecal administration of buspirone [40] as well as 8-OH-DPAT [41] affected genital reflexes and mating. Most injection studies have been performed in projection areas of the serotonergic system and in the raph~ nuclei from which serotonergic fibers emanate. Hillegaart et al. [42] reported that 8-OH-DPAT injected into the nucleus accumbens produced a facilitation of the male rat sexual behaviour, as evidenced by a decrease in number of mounts and intromissions to ejaculation, as well as by a decrease in the postejaculatory interval. Injections into the olfactory tubercle had no effects on sexual behaviour. Fern~mdez-Guasti et al. [43] confirmed the stimulatory effects of 8-OH-DPAT after local application into the nuclear accumbens, but also found similar effects for medial preoptic area injections. They found no effects after dorsal raph~ administration, in line with Hillegaart et al. [42]. 8-OH-DPAT, however, did facilitate male sexual behaviour after local administration into the medial raph~ nucleus [42]. In summary, no single site of action can be pinpointed where 5-HT1A agonists can be said to exclusively facilitate male sexual behaviour. The facilitatory effects of 5-HT~A agonists on male sexual behaviour in rats are pronounced, intriguing and quite specific; a potential clinical application has not yet extensively studied.

Drug discrimination studies Although drug discrimination is different from the other models for 5-HT1A effects described, it is important to realize the significance of this test. Briefly,

77 animals are trained to discriminate a drug from vehicle. Drug and vehicle are given in a balanced fashion and animals gradually learn to respond on one lever when given the drug and another when receiving saline. The interoceptive cue that a drug gives is "translated" into a choice for a lever, which is rewarded when pressed correctly. One of the attractive features of this experimental design is that related drugs can be tested to investigate whether these are recognized as having the same cue. Most experiments have been performed with rats and pigeons, but humans can also learn to discriminate different compounds. Intensive studies have shown that rats learn to discriminate various doses of 8-OH-DPAT from vehicle [44;45;46;47] and that flesinoxan, buspirone, ipsapirone and other 5-HT1A agonists are recognized by 8-OH-DPAT trained animals. It has also successfully been tried to train animals on flesinoxan [48], buspirone [49] and ipsapirone [50]. These animals again showed that 5-HT1A drugs substitute for each other. Not only full agonist can be used to train animals, also partial agonists are useful tools. Moreover, in substitution tests partial agonists also show dose dependent generalization. Although some contradictory findings have emerged with regard to oh adrenoceptor antagonists, Sanger and Schoemaker [51] successfully showed that the cue of 8-OH-DPAT is largely mediated by activity at 5-HT1A receptors. Many other psychoactive drugs have been tested and almost all drugs fail to substitute for the 5-HT1A cue. Test results strongly suggest that drug discrimination studies can be successfully applied to detect potential 5-HTIA agonistic properties of a drug. This does not imply that all compounds with a high affinity for the 5-HT~A receptor fully substitute for e.g. 8-OH-DPAT [52], because secondary interferences may prevent full recognition. Antagonism studies have been performed in rats and pigeons. Barrett and Gleeson [53] reported that NAN-190 effectively antagonized the 5-HTIA cue in pigeons trained to discriminate 8-OH-DPAT from saline. Similar results on NAN190 as well as WAY 100,635 were obtained in pigeons trained on the 5-HT1A agonist flesinoxan (van Hest et al. submitted). Partial agonist often block the cue of a full 5-HT1A agonist, but they do lead to (some) generalization when given alone. In rats, pindolol produced some antagonism of the 8-OH-DPAT cue [54]. NAN190 also blocked the 8-OH-DPAT cue in rats. However, these drugs were not as effective as in pigeons. WAY 100,635 and (S)-UH 301, two new and putative silent antagonists at the 5-HT1A receptor, have now been tested in flesinoxan-trained rats and found full antagonists [55]. A brief note with respect to the site of action of 5-HTIA agonists in drug discrimination studies. Schreiber and de Vrij [56] have performed the most extensive study using 8-OH-DPAT as specific 5-HT~A cue. They found that both pre- and postsynaptic mechanisms were involved in the 5-HT~A cue, i.e. local administration in the raphe nuclei and in the hippocampus resulted in drugappropriate responding. Although it is puzzling to understand these multiple sites of action, the effects of local application could be antagonized by NAN-190 suggesting that the effects were indeed 5-HT~A mediated.

78 In summary, various behavioural models exist that are specific in the response to 5-HT1A agonists. Using these models full and partial agonists have been evaluated and this lead to a further contribution in our knowledge of the role of 5-HT1A agonists in the CNS and to the mechanism of action. REFERENCES

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Dourish CT, Hutson PH, Curzon G. Brain Res Bull 1985a; 15: 377-384. Dourish CT, Hutson PH, Curzon G. Psychopharmacol 1985b; 86: 197-204. Gilbert F, Dourish CT. Psychopharmacol 1987; 93: 349-352. Gilbert F, Dourish CT, Brazell C, McClue S, et al. Psychoneuroendocrinol 1988; 13: 471-478. Neill JC, Cooper SJ. Eur J Pharmacol 1988; 151: 329-332. Aulakh CS, Hill JL, Murphy DL. Pharmacol Biochem Behav 1989; 31: 567571. Shepherd JK, Rodgers RJ. Psychopharmacol 1990; 101: 408-413. Fletcher PJ. Psychopharmacol 1987; 92: 192-195. Montgomery AMJ, Willner P, Muscat R. Psychopharmacol 1988; 94: 110-114. Chaouloff F, Serrurrier B, M~rino D, Laude D, et al. Eur J Pharmacol 1988; 151: 267-273. Fletcher PJ, Zack MH, Coscina DV. Psychopharmacol 1991; 104: 302-306. Fletcher PJ, Davies M. Psychopharmacol 1990c; 102: 301-308. Bendotti C, Samanin R. Eur J Pharmacol 1986; 121: 147-150. Fletcher PJ, Davies M. Psychopharmacol 1990b; 100: 188-194. Hutson PH, Dourish CT, Curzon G. Eur J Pharmacol 1986; 129: 347-352. Dourish CT, Hutson PH, Curzon G. Psychopharmacol 1986; 89: 467-471. Hutson PH, Dourish CT, Curzon G. Eur J Pharmacol 1988; 150: 361-366. Muscat R, Montgomery AMJ, Willner P. Psychopharmacol 1989; 99: 402-408. Fletcher PJ, Davis M. Br J Pharmacol 1990a; 99: 519-525. Harley JE, Forster EA, Fletcher A. Br J Pharmacol 1994; 113: 125P. Jacobs BL. Life Sci 1976; 19: 777-786. Berendsen HHG, Jenck F, Broekkamp CLE. Pharmacol Biochem Behav 1989; 33: 821-827. Molewijk HE, Van der Heyden JAM, Olivier B. Eur J Neurosci 1989; $2: 64.23. Ahlenius S, Larsson K, Svensson L, et al. Pharmacol Biochem Behav 1981; 15: 785-792. Gozlan H, Mestikawy EL, Pichat L, Glowinski J, et al. Nature 1983; 305: 140142. Mos J, Van Logten J, Bloetjes K, Olivier B. Neurosci & Biobehav Rev 1991; 15: 505-510. Brand T, Kroonen J, Mos J, Slob K. Hormones and Behavior 1991; 25: 323341. Rodriquez-Mauzo G, Fernandez-Guasti A. Behav Brain Res 1994; 62: 127-134. Ahlenius S, Larsson K, WijkstrSm A. Eur J Pharmacol 1991; 200: 259-266.

79 30 Ahlenius S, Larsson K. J Psychopharmacol 1988;2: 47-53. 31 Glaser T, Dompert WU, Schuurman T, Spencer DG, et al. Brain 5-HT~A receptors, Ellis Horwood 1987; pp 106-119. 32 Svensson K, Larsson K, Ahlenius S, Arvidsson LE, et al. Brain 5-HT1A receptors, Ellis Horwood 1987; pp 199-210. 33 Paredes RG, Kica E, Baum MJ. Psychopharmacol 1994; 114: 591-596. 34 Pomerantz SM, Hepner BC, Wertz JM. Eur J Pharmacol 1993; 243: 227-234. 35 Ahlenius S, Larsson K, Arvidsson L-E. Pharmacol Biochem Behav 1989; 33: 691-695. 36 Ahlenius S, Larsson K. Eur J Pharmacol 1984; 99: 279-286. 37 Andersson G, Larsson K. Eur J Pharmacol 1994; 255: 131-137. 38 Ahlenius S, Larsson K. J Neur Transm 1989; 77: 163-170. 39 Johansson CE, Meyerson BJ, Hacksell U. Eur J Pharmacol 1991; 202: 81-87. 40 Mathes CW, Smith ER, Popa BR, Davidson JM. Pharmacol Biochem 1990; 36: 63-68. 41 Lee RL, Smith ER, Mas M, Davidson JM. Physiol Behav 1990; 47: 665-559. 42 Hillegaart V, Ahlenius S, Larsson K. Behav Brain Res 1991; 42: 169-180. 43 Fern~ndez-Guasti A, Escalate AL, Ahlenius S, Hillegaart V, et al. Eur J Pharmacol 1992; 210: 121-129. 44 Glennon RA. Pharmacol Biochem Behav 1986; 25: 135-139. 45 Cunningham KA, CaUahan PM, Appel JB. Eur J Pharmacol 1987; 138: 29-36. 46 Tricklebank MD, NeiU J, Kidd EJ, Fozard JR. Eur J Pharmacol 1987; 133: 4756. 47 Ybema CE, Slangen JL, Olivier B, Mos J. Behav Pharmacol 1993; 4: 610-624. 48 Ybema CE, Slangen JL, Olivier B, Mos J. Pharmacol Biochem Behav 1990; 35: 781-784. 49 Rijnders HJ, Slangen JL. Psychopharmacol 1993; 111: 55-61. 50 Spencer DG, Traber J. Psychopharmacology 1987; 91: 25-29. 51 Sanger DJ, Schoemaker H. Psychopharmacology 1992; 108: 85-92. 52 Rabin RA, Winter JC. Eur J Pharmacol 1993; 235: 237-243. 53 Barrett JE, Gleeson S. Eur J Pharmacol 1992; 217: 163-171. 54 Winter JC, Rabin RA. Pharmacol Biochem Behav 1993; 44: 851-855. 55 Gommans J, Hijzen TH, Maes RA, Mos J, et al. 1995; 284: 135-140. 56 Schreiber R, de Vrij J. J Pharmacol Exp Therap 1993; 265: 572-579. 57 Dourish CT, Clark ML, Iversen SD. Psychopharmacol 1988; 95: 185-188. 58 Dourish CT, Cooper SJ, Gilbert F, Coughlan J, et al. Psychopharmacol 198; 194: 58-63. 59 Berendsen HHG, Jenck F, Broekkamp CLE. Pharmacol Biochem Behav 1989; 33: 821-827.

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Therapeutic applications 5=HTIAreceptor ligands I. van Wijngaarden Solvay Duphar B.V., P.O.Box 900, 1380 DA Weesp, The Netherlands.

ANXIETY The first compound showing anxiolytic activity was buspirone. Originally buspirone was under development as a new type of antipsychotic, possessing dopamine D~ antagonistic activity without inducing catalepsy. Clinically however, buspirone was not very effective and further development was stopped. After the discovery of its taming properties in aggressive rhesus monkeys, buspirone was reintroduced as anxiolytic. Subsequently it was demonstrated that the anxiolytic activity of buspirone was due to its interaction with central 5-HT1A receptors (for review see [1]). This finding initiated the search for more potent and selective 5-HT~Areceptor agents. At present a variety of 5-HT1A receptor ligands are in clinical trials for the indication anxiety. Most of these compounds e.g. gepirone, ipsapirone, tandospirone and binospirone, act like buspirone as partial agonists at the 5-HTxA receptor. Flesinoxan is a full agonist. Pure antagonists, such as WAY 100.635 are still in the preclinical phase of development. Buspirone, gepirone and ipsapirone, all azapirones have been proven to be effective in generalized anxiety disorders. The compounds maintain the level of efficacy during the period of treatment. The time lag to onset of action is two weeks. The side effects of these drugs (gastrointestinal complaints dizziness and headache) are totally different from those of the benzodiazepines (sedation, memory-loss and withdrawal syndrome). The short half-life of these compounds, requiring multiple daily dosing, is a serious drawback (for review see [2]). At present a sustained release preparation for buspirone is in Phase III, clinical trial. Tandospirone, an other azapirone is awaiting registration in Japan [3]. Flesinoxan is in Phase III, clinical development for use in anxiolytic disorders. Flesinoxan has in contrast to the azapirones a favourable pharmacokinetic profile.

Depression The early finding that chronic treatment of rats with buspirone induced a down-regulation of central 5-HT 2receptors initiated the extensive testing of 5-HT1A receptor ligands, such as buspirone, gepirone, ipsapirone and 8-OH-DPAT, in animal models for depression. All these compounds displayed anti-depressive

82 activity, indicating a possible role of central 5-HT1A receptors in depression (for review see [1]). Clinically buspirone and gepirone have been proven to be effective in major depressive disorder, especially in the melancholic subtype. This subtype improved significantly from week 1 of treatment with buspirone. The side effects are similar too, but less severe than the 5-HT reuptakeinhibitors (for review see [2]). A sustained release formulation of buspirone is in Phase III clinical trial. The development of gepirone for the indication depression is discontinued. Tandospirone is awaiting registration for the indication depressive neurosis in Japan [3]. Ipsapirone and flesinoxan are in Phase III clinical trial. The mechanism by which the azapirones display both anxiolytic and antidepressive activity is explained by their partial agonistic properties at postsynaptic 5-HTI^ receptors. In anxiety, characterized by an excessive stimulation of serotonin receptors, the azapirones displace 5-HT from its postsynaptic 5-HT1A receptors and act as antagonists. In depression, characterized by a defiency in serotonergic neurotransmission the azapirones do not have to compete with the full agonist 5-HT and act as agonists with moderate intrinsic activity. Flesinoxan is a full agonist at postsynaptic 5-HT1^ receptors. As antidepressant flesinoxan will be more effective than the azapirones which are partial agonists. The anxiolytic activity of flesinoxan will probably not involve post-synaptic 5-HT1A receptors. Presynaptically the azapirones and flesinoxan act as full agonists at somatodendritic autoreceptors. Repeated administration of these compounds induces a down-regulation of the autoreceptors resulting in a normalization of serotonin cell firing. This may be the mechanism by which both the azapirones and flesinoxan are anxiolytic (for reviews see [2, 4]). The results of the flesinoxan study will decide whether a full 5-HT1A agonist is to be preferred to a partial agonist in the treatment of anxiety. 5-HT~A receptor antagonists are probably useful to accelerate the onset of antidepressive action of selective serotonin reuptake inhibitors (SSRI's). SSRI's inhibit the reuptake of 5-HT by blocking the 5-HT transporter. This results in an increase in extra-cellular concentration of 5-HT in the brain. Recent in vivo microdialysis studies in rats have demonstrated that a single administration of SSRI's markedly increase the concentration of 5-HT in the vicinity of the somatodendritic 5-HT1A autoreceptors of the serotonergic neurones of the raphe nuclei. In brain areas rich in nerve-endings such as the frontal cortex the increase was rather slight. Apparently stimulation of the 5-HT~Aautoreceptors results in the inhibition of the fi~ng activity in 5oHT neurons, 5-HT synthesis and 5-HT release from nerve-endings. Chronic administration of SSRI's gradually desentisize the 5-HT~A autoreceptors and gradually increase the extra-cellular concentration of 5-HT in the nerve-endings. Co-administration of 5-HT~A autoreceptor antagonists and SSRI's prevent the inhibition on the 5-HT release leading to a faster increase in the concentration of 5-HT in nerve ending (for review see [5]). Indeed treatment of patients with major depression with SSRI's in combination with pindolol, a non-selective 5-HT1A antagonist, shortened the lag-time to onset of action significantly. The combination was also more efficacious (for review see

[6]).

83 Other i n d i c a t i o n s The azapirones have been tested in panic disorder, obsessive compulsive disorder (OCD), drug-abuse and alcoholism. Gepirone, but not buspirone, was superior to placebo on panic attacks. Buspirone produced a significant improvement in OCD and reduced the craving and cigarette smokers, cocaine and phencyclidine users and alcoholists to some extent (for review see [2]).

REFERENCES

1 2 3 4 5 6

New JS. Med Res Reviews 1990; 10: 283-326. Pecknold JC. CNS Drugs 1994; 2: 234-251. Barradell LB, Fitton A. CNS Drugs 1996; 5: 147-153. De Vrij J. Psychopharmac 1995; 121: 1-26. Gardier AM, Malagi~ I, Trillat AC, Jacquot C, et al. Fundam Clin Pharmac 1996; 10: 16-27. Artigas F, Romero L, De Montigny C, Blier P. Trends in Neurosci 1996; 9: 378-383.

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Chapter 3 5"HTIB RECEPTORS 5-HT1BReceptor ligands 5-HTm Receptors

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87

5-HT1B Receptor ligands I. van Wijngaarden a and W. Soudijn b aSolvay Duphar B.V., P.O.Box 900, 1380 DA Weesp, The Netherlands. bLeiden/Amsterdam, Center for Drug Research, P.O. Box 9502, 2300 RA Leiden, The Netherlands.

INTRODUCTION Many compounds, belonging to different chemical classes, such as indolylalkylamines, 3-tetrahydropyridylindoles, ergolines, arylpiperazines and aryloxypropanolamines, display affinity for the rodent 5-HT m receptors (for reviews see [1-4]). Unfortunately most of these ligands are non-selective. The first selective 5HTIB ligand CP 93,129 was published by [5], nine years after the discovery of the 5-HT m receptor. No new selective agents have been reported since. As 5-HT m receptors could not be identified in human the search for selective 5-HTm ligands faded away. However, recent cloning experiments have demonstrated that the human homologue of the rodent 5-HT m receptor exists (for review see [2]). The human 5HT1B receptor is termed 5-HTID~ as its pharmacological profile is very similar to that of the 5-HTIDa receptor and quite distinct from that of the rodent 5-HTIB receptor. Responsible for this discrepancy in pharmacological profile is the presence of an asparagine at position 355 in the rat 5-HTIB receptor and a threonine at the same position in the human 5-HT1D~ receptor. Exchanging threonine 355 in the human 5-HT1D~ receptor for asparagine, results in a human mutant of the 5-HTID~ receptor with a pharmacological profile typical for the rat 5-HT m receptor [2] (Table 1). It is obvious that these new findings will stimulate the search for compounds selective for the human 5-HTID~ receptor.

In dolylalkylamines The prototype of this class is serotonin (5-HT) a potent but non-selective 5-HT1B agonist [6,2] (Table 2). The hydroxyl group at position C-5 is a prerequisite for high affinity. The unsubstituted analogue tryptamine is hardly active [7]. Replacement of the 5-hydroxy group by methoxy (5-MeOT) reduces affinity by two decades. Interestingly elongation of the alkyl group of 5-MeOT to nonyl enhances affinity 30-fold [8] (Table 2). The 5-nonyloxy-tryptamine (NOT) is only five times less potent than 5-HT, indicating that the nonyl group reaches an accessory binding site resulting in a favourable interaction with the 5-HT receptor. The 5-methyltryptamine analogue displays only a weak affinity. Also sumatriptan bearing a 5methylaminosulphonylmethyl group is moderately active in rat brain.

88 Table 1 Pharmacological profile of cloned rat 5-HT1B and human 5-HTlm receptors and effect of mutation T355N on the profile of human 5-HTlm receptors. Rat 5-HT~B Human 5-HTlm Mutant 5-HTID~ (T355N) 5-HT 5-CT dihydroergotamine methiothepin

7.8 - 8.5 7.7 - 8.5 8.4 7.9

7.5 - 8.4 8.4- 8.6 8.2 7.9

7.9- 8.4 8.5 - 8.8 8.7 7.4

RU 24969 (_+)-cyanopindolol (-)-pindolol (-)-propranolol

8.68.66.87.2-

8.8 9.6 7.2 7.5

7.2 - 7.4 7.0 4.6- 5.0 5.0- 5.1

8.6- 8.7 9.2 7.3- 7.7 7.8- 7.9

DP-5-CT sumatriptan metergoline CGS 12066B

<5 6.3 6.9 6.8- 7.0

6.4 7.0 - 8.0 7.6 8.8

5.0 6.2 - 6.9 6.7 - 6.8 7.7

Data, expressed as pI~. values are taken from references in [2].

A more favourable C-5 substituent is a carboxamide group. This compound (5CT) is nearly as potent as 5-HT [7] (Table 2). Alkylation of the amine function of tryptamine is unfavourable for 5-HT1B affinity. The N,N-di-n-propyl analogue of 5-CT (DP-5-CT) for instance has no affinity for the 5-HTIB receptor (pI~.=4.9) [1]. In functional tests the tryptamine derived 5-HT1B ligands such as 5-CT, sumatriptan and NOT are like 5-HT full agonists of the 5-HT m receptor [2,8]. For the human 5-HTID~ receptor 5-HT and 5-CT display affinities identical to that of the rat 5-HT~B receptor. Sumatriptan and NOT [8] are about 10 fold more potent in human than in rat. 3-Tetrahydropyridylindoles

Incorporation of the alkylamine side-chain of tryptamine into a tetrahydropyridine ring increases 5-HT1B receptor affinity by more than 3 decades (Table 3). In contrast to the tryptamines, the 5-position of the indole nucleus of the 3-tetrahydropyridylindoles can be substituted by a variety of substituents without loss in affinity [9]. The most potent compound of the series is the carboxamide analogue being 25 times more active than 5-CT (pI~. values 9.7 and 8.3 respectively). Unfortunately none of the compounds is selective. However, there is a preference for the 5-HTla receptor over the 5-HT1A receptor. Depending on the nature of the substituent the ratio 5-HT1A/ 5-HT1Bvaries from 2-56 (Table 3). The most selective compound is CP-96,501, the propyloxy-analogue of RU 24969.

89 However, CP-96,501 is less selective with respect to the rat 5-HT m receptor (ratio 5-HT1D over 5-HTm = 28). Table 2 5-HT~B ligands derived from indolylalkylamines NH2 R

N H

Compound

R

5-HT1B*

Tryptamine (T) Serotonin (5-HT) 5-Methoxy-T (5-MeOT) 5-Nonyloxy-T (NOT) 5-Methyl-T (5-MET) Sumatriptan 5-Carboxamide-T (5-CT)

H OH OC OC9 C CSO2NHC CONH2

5.0 8.6 6.4 7.9 5.8 6.8 8.3

Data expressed as pI~. values are from [7]; [6] (5-HT, sumatriptan); [8] (NOT). *rat brain membranes. In functional tests CP-96,501 behaves as an agonist. The observation that compounds with small (H, F, OH) or directional (CN) 5-substituents displayed increasing selectivity led to the design of CP-93,129, the amide tautomer of 3tetrahydropyridine-5-OH-4-azaindole [5] (Table 3). In this rotationally restricted bio-isostere of 5-OH indole, the hydrogen-bond accepting interactions occur exclusively in the plane of the aromatic ring. This type of interaction is highly favourable for the 5-HT m receptor and detrimental for interactions with other 5HT receptor subtypes like 5-HT1A, 5-HT1D, 5-HT2c, 5-HT2A [9]. Functionally, CP93,129 acts as a 5-HT m agonist [10]. For the human 5-HT~m receptor RU 24969 displays a lower affinity than for the rat 5-HT1B receptor (Table 1). The 5-HT1D~ receptor affinity of the more selective 5-HT m compound CP-93,129 has not yet been reported.

90 Table 3 5-HT m ligands derived from 3-tetrahydropyridylindoles

R ~5

4

.__..

NH

\

/

2

H

H

I Compound

RU 24969 CP-96,501 II

CP-93,129

II Ratio A/B

R

5-HT]B*

5-HT~A*

H F C1 C CN CONH 2 NO2 OH OC OC~

8.7 9.0 8.8 8.5 9.4 9.7 8.7 9.2 9.0 9.1

7.1 7.5 7.9 7.9 7.8 9.3 7.7 7.8 8.2 7.3

39 31 8 4 37 2 9 23 7 56

8.1

5.8

185

Data expressed as pI~. values are from [9]. *rat brain membranes.

Ergot alkaloids A number of peptide-type ergot alkaloids such as ergotamine and dihydroergotamine are potent, non-selective 5-HT1B ligands [1] (Table 4). The complex structure seems necessary for high affinity. Simple ergolines (i.e. metergoline, LSD) are about 2 decades less active. In functional tests the ergotamines act as agonists [4]. For the human 5-HT1D~ receptor dihydroergotamine displays a similar high affinity as for the rodent 5HT1B receptor. Metergoline is more potent in human (Table 1).

91 Table 4 5-HT1B ligands derived from ergot alkaloids

5-HTB* C O II H ~ C-.NII"

O

OH :,,,'~._ ~ .,""

8.7

ERGOTAMINE

\N/ H DIHYDROERGOTAMINE

9.1 O

H II

C-N-C--O--C

METERGOLINE

Hie.

""

H

7.4

I

C

O

II LSD

/

6.8

H

D a t a e x p r e s s e d as pI~. values are from [1]. *rat brain.

92 Table 5 5-HT1B ligands derived from arylpiperazines Compound

Structure

5-HTIB*

F3C

TFMPP

N

:[- NP

N

/

6.6

NH

\

O

7.3

NH

x

/

O

7.3

ELTOPRAZINE N

NH

\

/

QUIPAZlNE

6.2 N

\

F3C nor - CGS12066B

CGS12066B

> (\

\,

NH

,/~ N

/

~

// ~N \

N

/

R= H

8.0

R=CH3

7.1

S-C

METHIOTHEPIN

N

\

/

N- C

7.3

Data expressed as pKi. values are from [6] (TFMPP, quipazine); [7] (1-NP, methiothepin); [12] (eltoprazine); [9] (nor-CGS 12066B). *rat brain.

93 Arylpiperazines Compounds belonging to the class of arylpiperazines display in general a moderate affinity for the 5-HT m receptor. Only the tricyclic piperazine nor-CGS 12066B shows high affinity (Table 5). CGS 12066B and methiothepin are tertiary amines, which is unfavourable for high 5-HT~Breceptor affinity. Nor-methiothepin is expected to display a higher affinity for the 5-HT m receptor. CGS 12066B has a higher affinity for the human 5-HT~D~ receptor than for the rat 5-HT m receptor. Methiothepin does not discriminate between both receptors (Table 1). None of the arylpiperazines is selective for 5-HT m receptors [6,7,1,11,9]. Aryloxypropanolamines Several ~-adrenoceptor antagonists display high affinity for the rodent 5-HT~B receptor. The affinity resides predominantly in the (S)-(-)-enantiomers being approximately 2 orders of magnitude more potent than the (R)-(+)-enantiomers [7]. This enantioselectivity is similar to that assessed for the ~-adrenoceptor. Table 6 5-HT,B ligands derived from aryloxypropanolamines

,<

0

CN

.T,

0

0

(+) - I -

CYANOPINDOLOL

N

0

5-HT1B =

9.5

5-HTIA =

8.3

( - ) - S D Z 21 - 0 0 9

N

5-HT1B =

9.4

5-HT1A =

8.0

OH

Ny. H

(-) - TERTATOLOL

5-HT1B =

6.9

5-HT1A =

8.2

METOPROLOL

5-HTIB =

i.a

5-HT1A =

i.a

The 5-HT1A and 5-HT m data expressed as p ~ values are from [3] (Icyanopindolol,(-)-SDZ 21-009); [13] ((-)-tertatolol) and [14] (metoprolol).

94 The most potent 5-HT~ ligand is iodo-cyanopindolol (Table 6). As a racemate the compound is 10 and 50 times more active than its congeners (_+)-cyanopindolol and (-)-pindolol [7]. Iodo-cyanopindolol shows a 10 fold preference for the 5-HT1B over the 5-HT~^ receptor (Table 6). Cyanopindolol and (-)-pindolol display similar affinities for both 5-HT~ sub-types [7]. Another pindolol derived 5-HTm ligand is (-)-SDZ 21-009. The affinities for 5-HT~B and 5-HT~^ receptors is very similar to that ofiodo-cyanopindolol (Table 6). Not all ~-adrenoceptor antagonists are potent 5-HTm ligands. (-)-Tertatolol shows a 20 folds preference for the 5-HT~^ receptor and ~-adrenoceptor antagonists substituted at the para-position with respect to the aryloxypropanol amine chain are inactive at the 5-HTm (and 5-HT~^) receptors [13,14] (Table 6). In functional models the aryloxypropanololamine derived 5-HT~B ligands are antagonists [1]. The affinity of aryloxypropanololamines for the human 5-HT~D~ receptor is about 2-orders of magnitude lower than for the rat 5-HTm receptor (Table 1). Table 7 5-HTln ligands derived from miscellaneous structures Compound

Structure

/ Oxymetazoline

i = 1 _ ~%1---/ \~k /

R

5-HTm*

OH

8.0

/..N -O

oxymetazoline

]

C-'C / \ C C

xylometazoline H

7.8

Data expressed as p ~ values are from [12]; oxymetazoline, xylometazoline. *rat brain. Miscellaneous structures

The adrenergic compounds oxymetazoline and xylometazoline are potent nonselective 5-HT m ligands (Table 7). Rouselle et al. [15] reported the extraction and purification of an endogenous factor from rat brain tissues selectively interacting with the 5-HT m receptor. The purified fraction had no significant affinity for the 5-HT1A,E,F, 5-HT2A or 5HT s receptors neither for the a~, ~, D2, H~, cholinergic (muscarinic), opiate and

95 benzodiazepine receptors nor for the 5-HT, NE, DA, gaba, or choline transport systems. The chemical structure of the purified fraction was identified as the tetrapeptide leu-ser-ala-leu, LSAL. Synthetic LSAL inhibits the binding of [all]-5 HT to the 5-HT1B receptor at an IC~o=0.1 nM and shows the same selectivity profile as the purified fraction. The binding of [3H]-5-HT to the 5-HT1D~ receptor of bovine cortex membranes is inhibited at a rather similar IC5o of 0.07 nM. The inhibition of 5-HT binding to the 5-HT m receptor by LSAL is a noncompetitive-one possibly by action via an allosteric binding site. Recently it was demonstrated that LSAL is implicated in the regulation of 5HT~a receptor activity [16]. It is quite possible that other small peptides do have a similar role in the regulatory mechanisms of other G-protein coupled receptors.

REFERENCES

1 2 3 4 5 6 7

9 10 11 12 13 14 15 16

Hoyer D, Schoeffter P. J Recept Res 1991; 11: 197-214. Boess FG, Martin IL. Neuropharmacol 1994; 33: 275-317. Hoyer D, Clarke DE, Fozard JR, Hartig PR, et al. Pharmac Rev 1994; 46: 157203. Martin GR, Humphrey PPA. Neuropharmac 1994; 33: 261-273. Macor JE, Burkhart CA, Heym J, Ives JL, et al. J Med Chem 1990; 33: 20872093. Van Wijngaarden I, Tulp MThM, Soudijn W. Eur J Pharm Mol Pharm Section 1990; 188:301-312. Hoyer D. In: Fozard JR, ed. The Peripheral actions of 5-HT. Oxford University Press 1989; 72-99. Glennon RA, Hong SS, Dukat M, Teitler M, et al. J Med Chem 1994; 18: 28282830. Koe BK, Lebel; LA, Fox CB, Macor JE. J Neurochem 1992; 58: 1268-1276. Pauwels PJ, Palmier CH. JPET 1994; 270: 938-945. Schipper J, Tulp MThM, Sijbesma H. Reviews on Drug metabolism and drug interactions 1990; 8 (1-2): 85-114. Tulp MThM, unpublished results. Prisco S, Cagnotto A, Talone D, De Blasi A, et al. J Pharm Exept Ther 1993; 265: 739-744. Langlois M, Br6mont B, Rouselle D, Gaudy F. Eur J Pharmacol 1993; 244: 7787. RousseUe JE, Massot O, Delepierre M, Zifa E, et al. J Biol Chem 1996; 271: 726-735. Massot O, Rousselle JE, Fillion MP, Grimaldi B, et al. Mol Pharmac 1996; 50: 752-762.

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97

5-HTm Receptors W. Kuipers

Solvay Duphar B.V., Dept. of Medicinal Chemistry, P.O.Box 900, 1380 DA Weesp, The Netherlands.

RECEPTOR STRUCTURE AND MOLECULAR BIOLOGY Receptor Cloning 5-HT m receptors have been found in rat [1, 2], mouse [3] and man [4, 5, 6]. The latter consists of 390 amino acid residues, while both rodent 5-HT1B receptors consist of 386 residues. The rodent 5-HT m receptors display high affinity for (cyano)pindolol, propranolol and 5-carboxamidotryptamine. In contrast, the reported human 5-HT m receptors display very low affinity for pindolol and propranolol (I~. varies from 640-17000 nM). Pharmacologically, the human 5-HTm receptor resembles 5-HT~D receptors and is, therefore, also referred to as the 5-HT~D~receptor. Adham et al. [2] postulated that 5-HT m and 5-HTID receptors are species homologues. Both receptors are coupled to the inhibition of adenylate cyclase and their species distribution seems to be complementary: 5-HTm in rodent and 5-HTID in human and bovine tissues. Furthermore, the rat 5-HT m and the human 5-HT1D~ display a striking sequence homology, especially in the transmembrane domains (overall homology is 93%). The pharmacological difference between these receptors appeared to be caused by just one residue difference in helix VII. Rodent 5-HT1B receptors contain an Asn in this position, which is replaced by a Thr in 5-HTxDa and 5-HT1D~ receptors. A single Thr355Asn mutation was shown to change the binding profile of the human 5-HT1D~ receptor to that of the rat 5-HT m receptor [7, 8, 9]. The T ~ N mutants display higher affinities for cyanopindolol, pindolol and propranolol and lower affinity for sumatriptan than the wild-type receptor. 5-HT m receptors belong to the class of G-protein coupled receptors (GPCRs). The structure and functions that members of this family have in common are more extensively described in the chapter "Structural Characteristics of 5-HTIA Receptors and their Ligands" (chapter 2). The human 5-HTIB receptor was shown to be phosphorylated and palmytoylated [10]. Consensus serine and threonine residues for phosphorylation were found in the first and third intracellular loop. A cysteine residue in the short carboxyl tail may be involved in palmytoylation. REFERENCES 1

Voigt MM, Laurie DJ, Seeburg PH, Bach A. EMBO J 1991; 10: 4017-4023.

98 2

Adham N, Romanienko P, Hartif P, Weinshank RL, et al. Mol Pharmacol 1992; 41: 1-7. 3 Maroteaux L, Saudou F, Amlaiky N, Boschert U, et al. Proc Natl Acad Sci USA 1992; 89: 3020-3024. Jin H, Oksenberg D, Ashkenazi A, Peroutka SJ, et al. J Biol Chem 1992; 267: 5735-5738. 5 Mochizuld D, Yuyama Y, Tsujita R, Komaki H, et al. Biochem Biophys Res Comm 1992; 185: 517-523. 6 Hamblin MW, Metcalf, MA, McGuffin RW, Karpells S. Biochem Biophys Res Comm 1992; 184: 752-759. 7 Oksenberg D. Marsters SA, O'Dowd BF, Jin H, et al. Nature 1992; 360: 161163. 8 MetcalfMA, McGuffin RW, H~mblin MW. Biochem Pharmacol 1992; 44: 19171920. 9 Parker EM, Grisel DA, Iben LG, Shapiro RA. J Neurochem 1993; 60: 380-383. 10 Ng GYK, George SR, Zastawny RL, Caron M, et al. Biochemistry 1993; 32: 11727-11733.

Chapter 4 5-HTID RECEPTORS

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101

5-HT1DReceptors D.N. Middlemiss, M.S. Beer and V.G. Matassa SmithKline Beecham Pharmaceuticals, Coldharbour Road, The Pinnacles, Harlow, Essex, CM19 5AD; Merck Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR Author for Correspondence: Dr. DN Middlemiss, SmithKline Beecham Pharmaceuticals, Coldharbour Road, The Pinnacles, Harlow, Essex, CM19 5AD. Tel:(44) 279 622303, Fax:(44) 279 622230

RADIOLIGAND BINDING STUDIES The heterogeneity of receptors mediating 5-HT responses was apparent long before their definitive identification using cloning techniques, largely due to radioligand binding studies. Hence, as early as 1979 5-HT recognition sites were subdivided into 2 classes. Those that had a high affinity for agonists and were labelled with [3H]5-HT were termed 5-HT 1binding sites and those that displayed a high affinity for antagonists and were labelled with [aH]spiperone were called 5-HT 2 binding sites [1]. In 1981 Pedigo et al. [2] demonstrated that spiperone displaced [aH]5-HT in a complex manner and hence that the 5-HT1 class comprised at least two subgroups. The component having a high affinity for spiperone was called a 5-HTIArecognition site (labelled with [~H]8-OH-DPAT) and the remaining component the 5-HTIB binding site (subsequently labelled with [125I]ICYP (iodocyanopindolol) in the presence of isoprenaline). This was followed in 1984 by the work of Pazos et al. [3] identifying a third 5-HT 1receptor in pig. This subtype is located throughout the brain but is present at high density in the choroid plexus and was designated a 5-HTlc receptor (labelled with [3H]mesulergine). This receptor, although displaying a low nanomolar affinity for 5-HT, has since been reclassified into the 5-HT 2 receptor family based on its amino acid sequence and also its secondary messenger system and given the appellation 5-HT2c receptor [4]. In 1987 Heuring and Peroutka [5] published on a fourth 5-HTl-like binding site with a unique pharmacological profile. The binding site, which they named a 5HT1D recognition site, was found throughout the bovine brain and indeed appeared to be the most abundant 5-HTl-like binding site present. Heuring and Peroutka [5] noted that both 8-OH-DPAT and mesulergine displaced [~H]5-HT from pig caudate membranes in a biphasic manner. The high affinity component for the 8-OH-DPAT and mesulergine displacement curves comprised just 7% and 8% of the total specific binding respectively. Hence, in the

102 presence of 100nM 8-OH-DPAT and 100nM mesulergine all 5-HT1A and 5-HT2c recognition sites were essentially blocked out without significantly affecting the remaining (85-90%) [3H]5-HT-labelled sites. Under these assay conditions, using 10~M 5-HT to define non-specific binding, saturation studies indicated saturable, high affinity (KD 1.8nM), stereo selective (LSD, yohimbine) binding to a homogeneous population of recognition sites. The binding was shown to be guanine nucleotide and calcium sensitive. Although located in all brain regions studied, this site was most dense in the basal ganglia (caudate, globus pallidus). Competition studies using a series of compounds, from varying structural classes, yielded monophasic displacement curves. The compounds displaying the highest affinities at this recognition site were tryptamine derivatives such as 5-CT, 5-HT and 5-methoxytryptamine (and sumatriptan). These were followed closely by ergot derivatives such as metergoline and methysergide. In addition RU 24969 and yohimbine displayed a moderately high affinity. 5-HT1Areceptor (WB4101), 5-HT,B receptor (TFMPP) and 5-HT2c receptor (mianserin) selective agents were relatively weak whereas 5-HT2A receptor (spiperone) and 5-HT 8 receptor (MDL 72222) selective compounds were essentially inactive as were dopamine and epinephrine. These findings were confirmed and extended by a number of groups who identified similar binding sites in other species. Using similar radioligand binding conditions, Hoyer et al. identified a high affinity [3H]5-HT recognition site in human cortex and caudate [6]. Again these binding sites made up a large proportion of the total [3H]5-HT binding site population in both areas (60% in human cortex and >90% in human caudate). Herrick-Davis et al. [7], substituting 100nM 8-OH-DPAT for I~M pindolol, demonstrated the presence of [3H]5-HT binding sites in human prefrontal cortex, human caudate and that the binding in prefrontal cortex was guanyl nucleotide but not adenyl nucleotide sensitive. Waeber et al., carrying out similar radioligand binding studies in pig, calf and human brain [8] and also in guinea pig cortex and pigeon telenchephalon [9], were the first group to indicate that, whereas most compounds, including 5-HT, yield monophasic displacement curves, some, in particular 5-CT, yield curves which were best fit to a two site model. This suggested that under such assay conditions [3H]5-HT bound to two sites or to two affinity states of a single site. These findings were confirmed by Beer et al. [10] in brain homogenates from dog, guinea pig, rabbit, pig, human, hsmster and calf. 5-HT yielded monophasic displacement curves in these studies whereas 5-CT and sumatriptan consistently yielded biphasic curves. Monophasic 5-HT displacement curves and biphasic 5-CT displacement curves were also reported by Sumner and Humphrey [11], using similar assay conditions, in neonatal porcine caudate. Sumatriptan, however, yielded monophasic curves which plateaued at 52% inhibition suggesting that two binding sites were present and that 5-CT binds to both of these sites whereas sumatriptan binds to only one. Studies reported by Peroutka in 1991 [12], carried out in bovine, porcine, guinea pig and human caudate and cortex, also resulted in complex 5-CT displacement curves as well as monophasic sumatriptan competition curves which fail to fully displace the [ZH]5-HT. This sumatriptan insensitive component,

103 although similar to that observed by Sumner and Humphrey, comprised a much smaller proportion of the total specific binding (7-20%). Possible explanations for this discrepancy are differing assay conditions or the age of the animals used. The situation was clarified by a comprehensive study investigating this multiple component binding, in human cortical tissue, by Leonhardt et al. [13]. Initial studies demonstrated that 5-CT and ergotamine displaced [3H]5-HT, in the presence of lpM pindolol and 100nM mesulergine, in a biphasic manner. The high affinity component (55%) yielded a pharmacology consistent with a 5-HT1Dbinding site and the low affinity component (45%) yielded affinities some 500-800-fold lower. Studies were then carried out replacing lpM pindolol with 100nM 5-CT to block out 99% of the 5-HT1D binding sites as well as 5-HT1Abinding sites. Under these assay conditions a novel binding site with a distinct pharmacological profile (5-HT > methysergide > ergotamine > DOB = TFMPP > DPAT > 5-CT >> ketanserin = ICS 205,930) was defined and named a 5-HT1Ereceptor. This binding site was GTPTS and Gpp(NH)p but not ATPTS or App(NH)p sensitive indicating a receptor-GTP-binding protein interaction. The pharmacological characteristics of this site have more recently been confirmed by the cloning, from human tissue sources, of the cDNA encoding this receptor by three independent groups using differing low-stringency screening strategies (see chapter 5 of this monograph). Hence these studies indicate that under the assay conditions first described by Heuring and Peroutka [SH]5-HT labels two recognition sites. The high affinity component reflects binding to the 5-HT1D binding site and the low affinity component to the 5-HT1p. binding site. The 5-HT1D recognition site falls into the 5HTl-like category as described by Bradley et al. [14] in that it shows a high affinity for 5-CT, 5-HT and methiothepin. More specifically the 5-HT~Dbinding site is characterised by having a high affinity (nanomolar) for 5-HT and related tryptamines (5-CT > 5-HT > 5-methoxytryptamine) and some ergolines (metergoline > methysergide) and a moderate affinity for RU24969 >/= yohimbine > mianserin >/= methiothepin. It displays a low affinity for 5-HT1A selective agents (8-OH-DPAT > ipsapirone > buspirone), 5-HTm selective agents (SDZ 21-009), 5-HT2A selective agents (ketanserin, cinanserin), 5-HT2c selective agents (mesulergine). 5-HT S receptor antagonists such as MDL 72222 are essentially inactive at this binding site. In 1990 Harrington and Peroutka carried out detailed saturation studies in bovine caudate, comparing the effects of GTP and its non-hydrolyzable analogue GTPTS, on the binding of [3H]5-HT to the 5-HT1D recognition site [15]. This work demonstrated that saturation studies carried out in the presence of GTP lowered the affinity of [3H]5-HT whereas similar studies in the presence of GTPTS resulted not only in a lowered affinity but also induced persistent changes in the 5-HT1D receptor-G-protein interactions which was reflected in a decrease in the density of the binding site. Their explanation is that GTP shifts the affinity state, at least one of which may not bind agonists. In contrast, GTPTS may interrupt the interconvertability of the binding and non-binding states. Similar effects have been described for the 5-HT~A binding site and this may reflect linkage to the same Gjprotein.

104 Work carried out in guinea pig striatum and frontal cortex, reported by Mahle et al. [16] suggests further that, using the Heuring and Peroutka [5] 5-HTID receptor radioligand binding assay conditions, 5-CT binds to at least three distinct recognition sites. Using [3H]5-HT as radioligand, in agreement with previously published work, 5-HT yielded monophasic displacement curves whereas 5-CT and sumatriptan yielded biphasic displacement curves, reflecting binding to 5-HTiDlike and 5-HT~E recognition sites. Similar studies using [3H]5-CT at sufficiently low concentrations to avoid labelling the 5-HT~e binding site (and also 5-HTIF sites) were also carried out. Under these conditions 5-CT yielded monophasic displacement curves in both the striatum and frontal cortex. 5-HT yielded monophasic curves in the striatum but biphasic curves in the frontal cortex, the high affinity component comprising 87% of the total specific binding. Conversely sumatriptan yielded biphasic displacement curves in both the striatum and the frontal cortex, the high affinity component consisting of 48% of the total specific binding in the former tissue and 77% in the latter. Mahle et al. suggest that, under the assay conditions described, [3H]5-CT binds to two recognition sites, as the ligand concentration is probably too low to occupy a low affinity state, and that under 5-HT~D radioligand binding assay conditions 5-CT binds to at least three distinct sites, two high affinity 5-HT~D-like sites and a low affinity 5-HT1E site. This may explain discrepancies seen with binding data from cloned 5-HT~D receptors and may indicate the existence of more than one 5-HT~D receptor. However, these two sites do not correspond to the cloned 5-HTID~ receptors as these do not discriminate between 5-HT or sumatriptan. Boulenguez et al. [17] described a new serotonin derivative, serotonin-Ocarboxymethyl-glycyl-tyrosinamide (GTI) which is selective for and has high affinities at 5-HT~B and 5-HT~Dbinding sites (IC~os 28 and 67nM respectively). The same group linked a 12~I to the tyrosine residue of GTI [18] and the resulting radioligand [~25I]GTI was used in quantitative autoradiographical studies in rat and guinea pig brain. Binding was saturable and reversible with K D values of 1.3nM and 6.4nM respectively. The heterogeneous distribution of the binding paralleled the anatomical distribution of the 5-HT m receptor in the rat and the 5HT~D receptor in the guinea pig. This radioligand has been more fully characterised by Bruinvels et al. [19, 20] carrying out radioligand binding studies in human substantia nigra and caudate, dog caudate and whole brain, monkey caudate and whole brain, calf caudate and guinea pig cortex. Saturation studies in human substantia nigra suggests this radioligand binds to a single population of high-affinity recognition sites yielding a PKD of 9.48. This radioligand yields steep monophasic displacement curves and the rank order of potencies for twenty five standard compounds reflects binding to a 5-HT1D receptor. Beer and Middlemiss [21] carried out similar [~25I]GTI radioligand binding studies in human cortex and confirmed the findings of Bruinvels et al. Also, however, a comparison of their data with the published affinity values for the same standard compounds at the human cloned 5-HT~D~and 5-HT~D0receptors [22] demonstrated, largely due to the fact that both ketanserin and ritanserin display some selectivity for the 5-HTID~ receptor (70 fold and 20 fold respectively), that

105 [125I]GTI binding, in human cerebral cortex reflects binding to the 5-HT~m rather than the 5-HTxDa receptor. Hoyer et al. [23] used [~25I]GTI to further investigate the claims of Xiong and Nelson that rabbit brain contains a novel 5-HT~-like receptor subtype which they named the 5-HT~a receptor [24]. Hoyer et al. describe saturation studies in rabbit whole brain homogenates in which high affinity, saturable binding is seen yielding a pK D of 8.5. Displacement studies yielded a pharmacological profile largely consistent with a 5-HT1D receptor and, although outlying data was obtained with some compounds e.g. spiperone, this was probably due to species variations as is seen at other receptor subtypes i.e. with mesulergine at 5-HT2A receptors. To further verify this notion Hoyer et al. also carried out autoradiographical studies using [~H]5-HT in the presence of 100nM 8-OH-DPAT and 100nM mesulergine (reflecting binding to 5-HT~D receptors) and [~25I]iodocyanopindolol ([~25I]ICYP) in the presence of 3~M isoprenaline and 100nM 8-OH-DPAT (reflecting binding to 5-HT~B receptors). They were able to demonstrate that 5-HT~B receptors, as defined with [~25I]ICYP, are not present in rabbit brain and that the pattern of [3H]5-HT binding was very similar to that described for the 5-HT1D recognition sites in other species (see below) and conclude that rabbit brain expresses a 5-HT~D receptor similar to other species and not a species specific receptor subtype. Although radioligand binding studies suggested that the 5-HT1D receptor is absent in rat and mouse brain Herrick-Davis and Titeler [25], as long ago as 1988, carrying out studies in rat cortex and striatum, characterised a [3H]5-HT radioligand binding site, identified in the presence of I~M pindolol and 100nM mesulergine, which yielded a 5-HT~D-like receptor pharmacology. This binding site, for which [~H]5-HT yielded a KD of 3.2nM in cortex and 4.2nM in striatum, was widespread throughout the brain. (5-CT yielded an affinity of 33-48nM which was too low for a 5-HT~D binding site and too high for a 5-HT~E binding site but could possibly represent binding to a mixture of these two sites.) The presence, in rat, of a receptor with a 5-HT~D-like pharmacology has been confirmed by Bruinvels et al. [26] using autoradiographical studies employing [~25I]GTIin the presence of the 5-HT m selective compound CP93129 (see below). This binding site probably represents the 5-HT~D~ receptor subtype which has recently been cloned from rat (see below). Two genes have now been identified, in human, dog and rat, that encode two variants of the pharmacological 5-HT~Dreceptor and have been designated 5-HT~D~ and 5-HT~D~receptors (see below). The 5-HT~D~ receptor is the species homologue of the rat 5-HT~B receptor [27,28,29,30,31,32]. Both receptor genes are intronless and have a high degree of amino acid sequence homology (63% for human receptors [27]). These receptors have been stably transfected into various cell lines i.e. murine LM(tk-) fibroblasts [27,30] CHO cells [28,31,32] and HeLa cells [29] and shown to have low nanomolar affinity for [~H]5-HT, ranging from 1.5 to 8nM. The rank order of potency for ligands competing for this [3H]5-HT binding site in both receptors is essentially the same as that observed for the native receptors in brain homogenates; 5-CT > 5-HT > yohimbine > 8-OH-DPAT > spiperone > zacopride. As mentioned above, ketanserin and ritanserin are the only compounds, to date, which have been shown to discriminate between these two receptor

106 subtypes displaying a 70 and 20-fold selectivity for the 5-HT~D~ receptor respectively [22]. Further tritiated radioligands, which are relatively selective for the 5-HT1D receptor, are now becoming commercially available i.e. [3H]sumatriptan and [ZH]L694,247 [33] and these should prove invaluable for future investigations at the native receptor subtype. R E C E P T O R STRUCTURE A excellent review article on the characterisation of cloned 5-HT1D receptors and their relationship with other cloned 5-HTl-like receptors, with particular reference to the 5-HT1B receptor, has been written by Hartig et al [34]. A clone encoding the 5-HT1D receptor was first isolated in 1989 from a canine thyroid cDNA library by Libert et al. [35] using a low stringency screening strategy. This clone, RCD4, one of four new members of the G-protein-coupled receptor family described, presented a strong homology to the 5-HT~A receptor. It was characterised as encoding a 5-HT~D receptor in 1991 [30,36] when the human gene encoding the species homologue (MA6A) was also isolated [28]. The receptors encoded by these genes contain structural features which are characteristic of serotonin receptors. These include conserved aspartate residues in transmembrane regions II and III, an Asp-Arg-Tyr sequence at the end of transmembrane region III and conserved proline residues in transmembrane regions IV-VII. The human clone contains an intronless open reading frame encoding a 377-amino acid polypeptide chain with seven hydrophobic, putative transmembrane domains characteristic of G-protein-linked receptors. The deduced amino acid sequence reveals further features commonly associated with the 5-HT receptor gene family. These include three consensus N-linked glycosylation sites (Ash 5,17,21) on the proposed extracellular N-terminal region and one on the presumed second extracellular loop (191) and a large third cytoplasmic loop (83 amino acids). This loop has two consensus cAMP-dependent protein kinase sites (Thr 240, Ser 292) and a number of other serine and threonine residues that might serve as phosphorylation sites but no strong consensus recognition sites for protein kinase C or for ~-adrenergic receptor kinase. The receptor also possesses a particularly short carboxyl-terminal putative intracellular tail (14-21 residues) a feature often associated with receptors linked to the inhibition of adenylyl cyclase via Gi. This tail lacks the cysteine residue often conserved in members of this receptor family. The MA6A deduced amino acid sequence is 88%, 43%, 33% and 31% identical to that for RDC4, human 5-HT~A receptor, rat 5-HT2c and rat 5-HT2A receptors respectively, paralleling their pharmacological similarities. In 1992 work on the cloning of a human cDNA encoding a second human 5HT1D receptor, possessing a 59% homology with the original human cloned 5-HT1D receptor, was reported [27,29]. This second gene displays all the characteristic features of the originally cloned cDNA i.e. free of introns in the coding region. The two expressed receptor subtypes display similar pharmacological binding profiles (with the exception of ketanserin and ritanserin, see above) and were designated 5-HT1D~and 5-HTID~receptors. The proteins encoded by these genes exhibit a 63%

107 amino acid homology. The gene encoding the human 5-HT1D~ receptor has been localised to chromosome 1 and that for the 5-HT1D~ receptor to chromosome 6 and species variants of both have been isolated from dog, rat and mouse. The rat (and mouse) 5-HTIB receptor is the species homologue of the human 5-HTlm receptor sharing a 92% amino acid homology and similar distribution i.e. striatum, hippocampus and frontal cortex. These two receptors, however, display quite different pharmacologies in particular with regard to ~-adrenoceptor antagonists (pindolol, propranolol) ~which show relatively high affinities at the rat 5-HT~B receptor and (z-adrenoceptor antagonists (yohimbine, rauwolscine) which display relatively high affinities for the human 5-HT1D~receptor. These differences are due to a single amino acid substitution from a Thr (human) to Asn (rat) at position 355 in transmembrane region VII (37). The rat and human 5-HT1D,receptors share a 91% overall homology and yield similar typical 5-HT~D receptor pharmacological binding profiles. The overall expression level of the 5-HT~D~receptor in rat is low relative to the 5-HTID0receptor and this probably hampered its identification using conventional brain homogenate radioligand binding techniques. RECEPTOR DISTRIBUTION STUDIES It was apparent, from the initial work of Heuring and Peroutka [5], that non-5HTI~I~2C receptors comprised, by far, the largest proportion (85%) of 5-HTl-like receptor in pig caudate. Similar findings were reported by Hoyer et al., Waeber et al. and Peroutka et al. [6,8,38] in human brain: caudate (>/=90%) and cortex (>/=60%) substantia nigra (>/=90%), frontal cortex (>90%) and globus pallidus (>90%). Early 5-HTID receptor in vitro autoradiographical mapping studies were carried out by Waeber's group initially in human brain [39] followed by a more comprehensive study comparing binding in pigeon, rat, mouse, guinea pig, cat, dog, monkey and human brain [40] and in rabbit, hamster and opossum brain [41]. All of these studies were carried out essentially using the binding conditions as described by Heuring and Peroutka [5], that is using [aH]5-HT in the presence of 100nM 8-OH-DPAT to block out 5-HTIA sites, 30nM (+/-)SDZ 21-009 to block out 5-HT1B sites and 100nM mesulergine to block out 5-HT2c sites. Under such assay conditions it was found that high densities of non-5-HT1A, non-5-HT2c [aH]5-HT binding sites were found in all the animal species investigated and that these sites exhibit a relatively well preserved regional distribution, being particularly concentrated in the basal ganglia (globus pallidus and striatum) and associated regions such as the substantia nigra and the superior colliculus. These findings indicate that non-5-HTl~2C sites, which have a high affinity for ~ blockers (pharmacological 5-HTIB sites), are confined to the brains of rat, mouse and hamster (myomorph rodents) and the phylogenetically distant species the opossum. Pharmacologically defined 5-HT1D binding sites display a similar distribution in the remaining species including guinea pig (hystricomorph rodent), rabbit (lagomorph rodent), pigeon, monkey, cow, pig dog, cat and human.

108 Although the pigeon brain contains 5-HT1A and 5-HT~c binding sites, by far the majority of [3H]5-HT binding was of the 5-HT1D class and these sites were particularly enriched in homologous areas to the basal ganglia eg telencephalon. Hence the anatomical distribution of 5-HTI~/~D binding site is evolutionarily well conserved. In human tissue these non-5-HT1A, non-5-HT2c binding sites represent a high proportion of the total [3H]5-HT binding in the basal ganglia, substantia nigra, both medial and lateral parts of the globus pallidus and to a lesser extent the nucleus accumbens. A lower proportion was observed in the hippocampus and raphe nucleus. We now know that the binding site, as defined by the conditions described above, does not necessarily reflect binding to a single population of receptors and may well include non 5-HTl~z~2c 5-CToinsensitive (5-HT1E/1F) sites as well as pharmacologically defined 5-HT~D binding sites. Indeed it has been possible from radioligand binding studies, yielding two component displacement curves, to estimate the proportions of these 5-HT m versus 5-CT-insensitive binding sites in various discrete regions of the brain. Hence from the studies of Beer et al [10] the 5-CT-insensitive component of the '5-HT~D binding site' was shown to comprise approximately 50% of the binding in dog, guinea pig, rabbit, pig and human cortex, and approximately 30-40% in pig and calf caudate and from the studies of Lowther et al. [42] approximately 50% in human frontal cortex and caudate and 15% in human globus pallidus etc. In order to carry out "true" 5-HT m receptor localisation studies Palacios et al. [43] compared [3H]5-HT (in the presence of 100nM 8-OH-DPAT and 100nM mesulergine) autoradiographical studies, in human brain, with those using the 5HTI~m selective radioligand [~2SI]GTI. Both assay conditions gave similar anatomical distribution patterns. As seen previously the receptors labelled with [3H]5-HT were enriched in the pars reticulata of the substantia nigra, basal ganglia, both the lateral and medial parts of the globus pallidus, the caudateputamen and the nucleus accumbens. [~25JGTI binding was most dense in the substantia nigra, pars compacta and reticulata and again both lateral and medial parts of the globus pallidus. Less dense binding was observed in the claustrum, hippocampus, amygdala and nucleus accumbens. A different approach was taken by Miller and Teitler [44] and also by Lowther et al. [42]. Both groups compared the binding seen with [3H]5-HT in the presence of blocking concentrations of pindolol and mesulergine (non-5-HTlv2C) with that in the presence of blocking concentrations of 5-CT and mesulergine (non-5HT~c~m) in discrete regions of human brain. Miller and Teitler carried out autoradiographical analyses in cortex, putamen, globus pallidus, cerebellum and thalamus whereas Lowther et al. carried out radioligand binding displacement and saturation studies in the frontal cortex, hippocampus, amygdala, globus pallidus and caudate putamen. Both studies indicate a clear distinction between the globus pallidus and the functionally and anatomically related caudate putamen. 5-HT~D binding sites were most dense in the globus pallidus and least dense in the putamen. These studies also suggest that the previously designated 5-HT1D binding in the frontal cortex and amygdala did in fact comprise a large percentage

109 of 5-CT-insensitive binding sites unlike the pallidal and thalamic regions which contained relatively little 5-CT-insensitive binding sites. Similar autoradiographical studies have been carried out by Del Arco et al. [45] in guinea pig comparing the binding patterns seen with [aH]5-HT in the presence of 8-OH-DPAT and mesulergine (non-HT~2c binding) with its use in the presence of 5-CT (non-5-HT1A/2C/1D binding). 5-HT1D binding sites were very high in the claustrum, globus pallidus, caudate putamen, accumbens, substantia nigra, superficial layer of the superior colliculus and central grey, high in the ventromedial nucleus, lateral hypothalamic area of the hypothalamus and the marnmillary bodies, intermediate in the external layers of the cortex, the paraventricular nucleus and the lateral geniculate nucleus. These findings were confirmed by Waeber and Palacios [41] in rabbit, hamster and opossum brain where most of the non-5-HT~2c [3H]5-HT binding, which displayed a high affinity for 5-CT, was present in the substantia nigra. Also Bruinvels et al. [46], utilising GTI displacement of [3H]5-HT (in presence of 8-OH-DPAT and mesulergine), demonstrated that the majority of recognition sites in guinea pig and human substantia nigra, globus pallidus, claustrum, caudate, superficial grey layer of the superior colliculus, central grey, subiculum, CA4 field of the hippocampus and frontal cortex of the guinea pig were of the 5-HTID subtype. The high densities of 5-HT~D receptors in basal ganglia and substantia nigra suggest an involvement in motor activity. Their concentration in the superficial layer of the superior colliculus may indicate their involvement in the regulation of visual activity. A number of groups have carried out lesioning studies aimed at determining the cellular pre- and postsynaptic localisation of 5-HTiDreceptors. Price et al. [47] lesioned guinea pigs with p-chloroamphetamine and detected a significant decrease in [aH]paroxetine radioligand binding but no corresponding decrease in [3H]5-HT binding. They concluded that only a very small proportion of the 5-HT1D binding sites in the cortex correspond to the presynaptic autoreceptor. Autoradiographical studies have also been used to determine the pre- and postsynaptic localisation of 5-HT1D receptors. The guinea pig superior colliculus is an area known to be rich in 5-HTID binding sites (see above). Primary retinal afferents project into the superficial grey layer of the superior colliculus and the terminals of this area arise from the nucleus raphe dorsalis. Waeber and Palacios [47] first demonstrated that [aH]5-HT binding in this area can be completely blocked by 10nM 5-CT indicating a lack of 5-HTm/1F recognition sites and then went on to show that unilateral enucleation leads to a nearly complete depletion of the [~H]5-HT binding in the contralateral superior colliculus. These results indicate that these 5-HT m receptors may be presynaptically located on the terminals of the non-serotonergic primary retinal afferents (non-serotonergic neuronal pathways) and may be involved in the release ofneurotransmitters other than 5-HT. This same group have also carried out pre- and postsynaptic localisation studies in the guinea pig striatonigral pathway [49]. Although the 5-HT1D receptor population in this study was defined with [~H]5-HT in the presence of 8-OH-DPAT and mesulergine, later studies using [~2~I]GTI and [3H]5-HT in the presence of 5-

110 CT indicated that the 5-HT1E/1F receptor component is minor in the areas investigated in this study, the caudate-putamen. This area is densely innervated by serotonergic fibres arising from the mesencephalic raphe nucleus, the globus pallidus, substantia nigra pars reticulata. Quinolinic acid lesioning of (medial striatum) striatal intrinsic neurons led to a marked decrease in 5-HT1D binding sites in the striatum and the medial aspects of the pars reticulata of the substantia nigra, an area known to be innervated by striatal afferents, on the side ipsilateral to the lesion. In animals given unilateral 6-hydroxydopamine lesions of the nigral dopaminergic cell bodies, no significant decrease in 5-HT1D receptor binding was observed in any of the components of the striatonigral pathway. These results indicate a presynaptic localisation of 5-HT~D receptors on the terminals of the striatal neurons projecting to the pars reticulata of the substantia nigra. Hence, these studies in guinea pig suggest that, in addition to their presynaptic location on serotonergic nerve terminals where they act as 5-HT autoreceptors [50], 5-HT~D receptors have at least two further possible cellular localisations. They are located on the terminals of non-serotonergic neurons where they may act as presynaptic heteroreceptors i.e. in the superficial grey layer of the superior colliculus and in the substantia nigra and they are postsynaptically located on the dendrites or cell bodies of neurons in other brain areas i.e. the caudate-putamen and globus pallidus. As mentioned above a cDNA encoding a receptor displaying a 5-HT1D receptor pharmacology, as well as one encoding a 5-HT1B receptor pharmacology, has been isolated from rat tissue and Bruinvels et al. [51] have been able to demonstrate the presence of this receptor in native tissue. Initially this group used GTI to displace [3H]5-HT from n o n - 5 - H T ~ c binding sites and obtained monophasic displacement curves in the substantia nigra, dorsal subiculum and globus pallidus but biphasic curves in the caudate-putamen and the frontoparietal cortex. It was concluded that biphasic displacement curves occurred in areas where 5-HT~B and 5-HT~D-like binding sites were present and monophasic curves in areas lacking 5HT~D-like receptors. Unpublished data (Bruinvels et al.) suggests that this rat 5HT~D-like binding sites comprises about 30% of the 5-HT1B/~Dreceptor population in the substantia nigra and approximately 25% in the globus pallidus. These 5HT~D-like binding sites probably represent the 5-HT1D~receptor which has recently been cloned from rat tissue and which displays a typical 5-HTID receptor pharmacology. Bruinvels et al. [26] then went on to localise this 5-HT1D receptor in rat autoradiographically, comparing [~SI]GTI binding with that of [~sI]ICYP in the presence of 8-OH-DPAT and isoprenaline to block out 5-HT~A and ~-adrenoceptors respectively. Both assay conditions were designed to look at 5-HTm receptor binding and as expected gave largely similar distribution patterns. Displacement of [l~SI]GTI in globus pallidus and substantia nigra, with 5-HT1B selective compounds, i.e. CP93129 and (-)pindolol, however led to biphasic competition curves with a majority of high affinity sites whereas displacement with 5-HT~D selective compounds i.e. PAPP and sumatriptan yielded biphasic competition curves with a minority of high affinity sites. Subsequent autoradiographical studies, using [~SI]GTI in the presence of 100nM CP93129, demonstrated a low

111 density binding, probably representing the 5-HT~D~ recognition site in globus pallidus, ventral pallidum, caudate-putamen, subthalamic nucleus, entopeduncular nucleus, substantia nigra pars reticulata, nucleii of optic tract and geniculate body and the frontoparietal cortex. In all cases these receptors were present in much lower densities than the 5-HT m recognition site. With the cloning of the 5-HTm~ and 5-HT1D~receptors it is now possible to carry out studies to investigate the localisation of the mRNA encoding these receptors. Northern blot analysis has revealed mRNA transcripts for the 5-HT~D~ receptor in human tissue. These are abundant in striatum, moderately expressed in hippocampus and frontal cortex and barely detectable in pituitary and cerebellum [52, 32]. Similarly 5-HT~m mRNA has been isolated from monkey frontal cortex, striatum medulla and amygdala [32]. This 5-HT1D~ distribution correlates well with the localisation of 5-HT~D-like receptor localisation in mammalian brain with the highest levels in limbic regions and basal ganglia. In situ: hybridisation studies by the same group were in good agreement with the northern blot assays. The 5-HTm~ transcripts were found to be widespread in human and Rhesus monkey brain. Caudate putamen revealed high levels ofhybridisation signal which were homogeneously distributed. In addition a band with high levels of hybridisation, corresponding to lamina V, was observed in neocortical areas. The cerebellar cortex also showed hybridisation, particularly in the Purkinje cell layer. The enrichment of these transcripts in the basal ganglia is in good agreement with the presence of high densities of [~H]5-HT binding sites in these brain regions and in the areas receiving afferents from the basal ganglia i.e. the globus pallidus and the substantia nigra (see above). Hamel et al. [53] carried out northern blot hybridisation studies and demonstrated the presence of 5-HTID~ mRNA in bovine caudate nucleus and cerebral cortex, and in human cerebral cortex. 5-HT1D~ receptor hybridisation was not observed with RNA extracts from pial vessels in either species. Hybridisation with the 5-HT~D~receptor probe revealed the presence ofmRNA transcripts in both human and bovine cerebral tissue and pial vessels. This work indicating the presence of 5-HT~D~, but not 5 - H T ~ , receptor mRNA in bovine and human cerebral arteries located outside the brain parenchyma, but neither type in intraparenchymal microvessels or capillaries suggests that the anti-migraine effects seen with sumatriptan, if vascular related, are probably mediated via 5-HTtD~ receptors. Finally Bruinvels et al. [54] carried out in situ: hybridisation studies using 5HT~B and 5-HT~D~ receptor probes in mouse and rat. Although the 5-HT~D~ signal revealed overall low densities of this mRNA in these species it was most dense in the caudate-putamen and cortical areas. 5-HT~B mRNA was also present, in high densities, in the hippocampus and cerebellum. Comparisons of the localisation of mRNA with the regional distribution of 5-HT1B/1Dbinding sites in rat brain reveal that both receptor subtypes could be putative presynaptic heteroreceptors modulating release of various neurotransmitters. The same group have also demonstrated the presence of 5-HT~ and 5-HTID~ mRNA in rat trigeminal ganglia and 5-HT~D~ and 5-HT~D~mRNA in human trigeminal ganglia and have suggested

112 that both receptor subtypes may be involved in mediating the anti-migraine action of sumatriptan [55].

5"HT1D RECEPTOR LIGANDS The emergence of novel ligands - particularly agonists - for the 5-HTID receptor has been greatly stimulated by the knowledge that the Glaxo 5-HT1D receptor agonist sumatriptan (1) is efficacious in the treatment of migraine [56]. The clinical success of sumatriptan has provided the impetus for the development of 5-HT~D receptor agonists which possess improved in vitro and in vivo properties. The discovery of two subtypes (5-HT~D~and 5-HT1D~) of the human 5-HT~Dreceptor, between which sumatriptan has little selectivity, has intensified interest. The concept of a "selective" 5-HT1D receptor ligand is constantly being revised at present, as the superfamily of serotonin receptors continues its explosive growth [57]. At the time of writing, there are no truly selective 5-HT1D receptor ligands. This review will highlight developments in the structures of ligands for the 5-HT~D receptor beyond sumatriptan [58], and will concentrate on the in vitro binding and functional properties of molecules. An excellent review of earlier literature has appeared [59]. The emphasis will be on receptor agonists, reflecting the current focus of pharmaceutical laboratories, but novel, potent antagonists for the 5-HT1D receptor have been reported, and these structures will be discussed in the final part of the chapter. The binding and functional data quoted in this chapter are from disparate sources and will therefore be only qualitatively comparable. 1. INDOLES

i) C5 substitutents Sumatriptan (1) is a N,N-dimethyltryptamine with a sulphonamidomethyl group at the indole C5 position and is a moderately potent 5-HT1Dreceptor agonist with affinity and functional potency (Ki 29nM, calf caudate (CC); ECso 525nM, inhibition of adenylate cyclase on calf substantia nigra (CSN)) similar to that of 5-HT [60]. It has limited selectivity over 5-HT1Areceptors (Ki 740nM) [60]. Several groups of workers have shown that the sulphonamide can be successfully replaced with heterocycles. Wellcome [61] claim five-membered non-aromatic cyclic ureas and cyclic urethanes (and related thia-analogues), either substituted or unsubstituted. For example, the oxazolidinone (2) is a 5-HT1D agonist in contracting rabbit saphenous vein (RSV) (ECho 100nM). Bulky groups are tolerated on the ring, but generally do not lead to significantly higher potency - thus, the benzyl imidazolidinedione [62] (3)(ECho 32nM, RSV) is only three-fold more potent than (2). Wellcome reportedly have a compound (BW311C90) in late clinical development, the structure of which has not been disclosed, but which may be related to (2). Generally, in the 5-HT1D agonist field, compounds containing the N,N-dimethyltryptamine group are somewhat less potent than the primary tryptamines, but the former, being metabolically more stable, are often the preferred pharmaceutical entities (cf 1).

113 Based on the structures of the 5-HT1D receptor agonists 5-HT, 5-carboxamido tryptamine (5-CT) and sumatriptan, Merck have examined aromatic and nonaromatic five-membered heterocycles as the indole C5 substituent in tryptamines, and reportedly have a compound (MK-462) in clinical trials, the structure of which (45) was recently disclosed [179]. A full account of structure/activity relationships (SAR) in the oxadiazole series has been published [63]. The methyloxadiazole (4, R = Me, n = 0), where the oxadiazole and indole are conjugated, has comparably high affinity (ICso 10nM, pig caudate (PC)) and efficacy (ECho 250nM, RSV) to 5HT (ICso 10nM, PC; ECso 160nM, RSV) at the 5-HT1D receptor. The affinity of this compound is similar to that of sumatriptan (IC~o 20nM, PC), which suggests that a hydrogen bond acceptor group (the oxadiazole or the sulphonamide), and not a donor, is required at the 5-HT1D receptor. However, the precise positioning of the hydrogen bond donor group with respect to the indole would appear not to be critical, since the methylene-linked (4, R = Me, n = 1) (IC~o 32nM) and propylenelinked (4, R = Me, n = 3) (ICso 5nM) analogs have similar affinities to the parent conjugated molecule (4, n = 0). The introduction of a methylene spacer between the indole and oxadiazole rings, which breaks the conjugation of the aromatic rings, reduces logP by about an order-of-magnitude. For example 4 (R = Me, n = 0) has logP -0.4 (pH 7.4) whereas 4 (R = Me, n = 1) is much more hydrophilic (logP -1.7, pH 7.4). Such considerations are important in the design of ligands which do not penetrate the blood-brain barrier. The methyl group in (4) (R = Me, n = 0) may be replaced by bulkier groups - eg, ethyl (IC~o 13nM), cyclopropyl (ICso 7.9nM) or benzyl (IC~o 6.3nM) - without compromising affinity, or by an amino group to give 4 (R = NH2, n = 0) (IC~o 50nM) with somewhat reduced affinity. The methylenelinked aminooxadiazole 4 (R = NH2, n = 1) has good affinity (ICso 25nM). A study of SAR for substitution of the phenyl ring of the benzyloxadiazole 4 (R = Bn, n = 0) led to the discovery that affinity could be increased by about an order-ofmagnitude by the introduction of amide or sulphonamide functionality at the para position. One of the most potent compounds to emerge from this study was the para-sulphonamido benzyloxadiazole L-694,247 (4a) (IC~o 0.3nM, PC; ECso 16nM, RSV). Taken together with the Wellcome compound of the type (3), the high afffinity of(4a) suggests the existence of an extensive binding pocket at the 5-HT1D receptor binding site which is capable of accomodating a large indole C5 substitutent. A detailed description of the profile of L-694,247 has been published [33] and the compound is now commercially available in radiolabelled (tritiated) form. Interestingly, the oxazole [63] (5) has high affinity (ICso 7.9nM, PC) at the 5HT1D receptor, suggesting that the oxadiazole N2 lone pair in (4) may not be engaged in hydrogen bonding during the receptor binding event. An intruiging observation [64] was that the thiadiazole (6) (X = S) had twenty-fold higher affinity (ICso 1.2nM, PC) and fifty-fold higher functional potency (ECs• 10nM, RSV) compared to its oxadiazole analog (6) (X = O) (ICso 25nM, PC; ECso 500nM, RSV) at the 5-HT1D receptor. In probing this unexpected effect, molecular electrostatic potential maps were constructed which suggest that the distribution of electron density is significantly different in the oxygen- and sulphur-containing rings, and

114 a tentative conclusion is that the thiadiazole may have better electrostatic complementarity with the 5-HT1D receptor than its oxadiazole counterpart. Tetrazole analogues of the oxadiazoles have been reported [65]. While the parent (zwitterionic) tetrazole (7) (R = H) has low affinity (IC~o 4,000nM, PC) at the 5-HT1D receptor site, the N1 and N2 alkylated analogs show good affinity and potency. For example, the ethyltetrazole (7) (R = N-1 Et) has good affinity (ICso 25nM, PC) and functional activity (ECho 200nM, RSV) at the 5-HT m receptor, and shows good selectivity over other 5-HT receptors (5-HT2c, 5-HT2, 5-HT~ IC~o > 10,000nM), although it has affinity for the 5-HT1A receptor (ICso 320nM), as has come to be expected of 5-HT m receptor ligands. The isomer 7 (R = N-2 Et) (ICso 160nM, PC) and related triazole 8 (ICso 25 nM, PC) likewise have useful affinity at the 5-HT m receptor [65]. Interestingly, although the parent tetrazole (7) (R = H) referred to above had low affinity for the 5-HT~D receptor, suggesting that a negatively charged group is not well tolerated at that part of the receptor, the compound has reasonably high affinity for the 5-HT1A site (ICso 63nM) and shows good serotonin receptor selectivity, being devoid of affinity (IC~o >10,000nM) at 5HT~c, 5-HT 2 and 5-HT 3 receptors. Merck have reported heteroatom-linked series of heterocyclic compounds - for example the aminothiadiazole [66] (9) and the aminotriazole [67] (10). A series of cyclic sulphamides has been reported [68]. These may be viewed either as conformationally-constrained sulphonamides or as hybrid molecules between sulphonamides and the heterocyclic series (vide supra). As an example, the methylsulphamide (11) (m = n = 1) shows moderate affinity (IC~o 63nM, PC) and functional potency (ECho 630nM, RSV) at the 5-HT~Dreceptor. This level of activity is similar to that of sumatriptan, reinforcing the notion that the sulphonamide proton in sumatriptan may not be necessary for hydrogen bonding at the receptor. (11) (m = n = 1) is devoid of affinity at 5-HT~ and 5-HT~ receptors (IC~o's > 10,000nM), although, not surprisingly, it has limited selectivity (ca. 10fold) over the 5-HT~A receptor (ICso 790nM). The analog in which the sulphamide and indole rings are directly linked (11, m = 1, n = 0) has comparable 5-HT1D receptor affinity (ICso 100nM, PC) to the methylene-linked analog, but surprisingly shows much lower functional activity (ECso 3200nM, RSV). A similar observation was made for the six-membered sulphamide (11, m = 2, n = 1) (IC~o 63nM, PC; ECso 2500nM, RSV). Pfizer have reported thiazolyltryptamines related to the Merck series [69,70]. The aniline CP-110,330 (12) has high affinity for the 5-HT~D receptor (ICso 1.9nM, bovine caudate (BC)). However, 12 has no selectivity over 5-HT1A sites (IC~o 1.5nM). In a further variation of this series, the 5-aminoindole pyrrolidine 13 (CP146,662) was shown to have high affinity for the 5-HT m site (ICso 1.1nM, BC), but, like CP-110,330, CP-146,662 shows little or no selectivity over the 5-HTIA receptor (IC~o 3.1nM). Oxygen- and sulphur-linked analogs are also claimed. Both CP110,330 and CP-146,662 are potent dopamine uptake inhibitors. Pfizer also claim (nitropyridyl)indole derivatives (eg, 14) as 5-HT m receptor agonists [70]. The 5-HT conjugate serotonin-O-carboxymethylglycyltyrosinamide (S-CMGTNH2, (15)) has been reported [17] as a selective new ligand for the 5-HT~D site. Affinity for a selection of 5-HT receptors was assessed using quantitative

115 autoradiography on rat and guinea pig brain sections. In the guinea pig, the affinity of (15) for 5-HT~D sites (IC~o 67nM) is twenty-fold higher than at 5-HT1A sites (ICso 1400nM). The structural and functional complexity of the indole C5 substituent present in (15) is a further illustration of the tolerance of the 5-HT1D receptor to ligand structure in the "western" region of tryptamines. (15) has been radio-iodinated to give serotonin-O-carboxymethyl-glycyl [12~I]tyrosinamide ([~25I]GTI) which has been used for radioligand binding studies at the 5-HT w receptor site [19]. [12~I]GTI labelled a single site population of high-affinity recognition sites in human substantia nigra with Bm,x39.4fmol/g protein and PKD 9.48. The rank order of affinity of the ligands tested was in good agreement with that determined earlier in binding studies performed with [~H]5-HT in caudate membranes of various species. However, the affinity values were typically somewhat higher using [125I]GTI compared with those using [aH]5-HT. It is suggested that this may be due to [~H]5-HT being capable of labelling high and low affinity states of the receptor, the observed affinities with [SH]5-HT therefore being an underestimation of the true value for the high affinity site [19]. ii) C3 a m i n o c h a i n v a r i a t i o n s A variety of cyclic amine analogs of the serotonin aminoethyl side chain have been reported. Glaxo have discussed the properties of the piperidine GR85548 (16) [71,72,73]. GR85548 has high affinity for the 5-HT1D site (I~. 8nM, guinea pig striatum), is ten-fold selective over 5-HT1A sites (I~. 80nM), has weak affinity at the 5-HT3 receptor (I~. 1260nM), and is about four-fold more potent than sumatriptan in contracting dog basilar artery and dog saphenous vein preparations in vitro. The increased bulk of the piperidine ring (compared to the ethylamine) is therefore well tolerated at the 5-HTID receptor, as might have been anticipated from the known good affinity of RU24,969 (17) at the 5-HT1D receptor [74]. In addition, the piperidine ring can be viewed as a conformationally-restricted tryptamine, and, as such, provides useful information about possible preferred orientations for the (charged) amino group - which is crucial for receptor recognition - with respect to the indole ring, at the 5-I-IT1D receptor. In vivo, GR85548 has higher oral bioavailability in rat (71%) and in dog (95%) than sumatriptan, perhaps reflecting improved metabolic stability of the piperidine, and has a tl/2 of 1.7h in both species. The safety, tolerability and pharmacokinetics of subcutaneous GR85548 in man showed that doses up to 5mg were well tolerated, with about half the dose recovered in urine. The enantiomers of 5-methoxy-3-[(N-methylpyrrolidin-2-yl)methyl]indole (18), which contains another conformationally-constrained tryptamine replacement, have been prepared and a preliminary pharmacological profile has been reported by the Pfizer group [75]. The (R)-enantiomer of(18) (CP-108,509) (ICso 24nM, BC) proved to be eighteen-fold higher in affinity than the (S)-enantiomer (IC~o 420nM, BC), an interesting stereogenic differentiation which could help define the preferred location of the amino group for optimal 5-HT1D receptor recognition. (R)18 had comparable affinity to 5-methoxy-N,N-dimethyltryptamine (IC~o49nM, BC), and was a 5-HT~D receptor agonist in inhibiting adenylate cyclase (ECso 43nM, guinea pig substantia nigra), being somewhat less potent than 5-HT (ECho 5.2nM).

116 The (R)-(pyrrolidin-2-ylmethyl) analog of sumatriptan (19; CP-122,288) has been reported, and is claimed to have a similar in vitro profile to sumatriptan [76]. Merck have claimed azetidine analog[Cs oftryptamines (eg, the imidazole 20)as 5-HT1D agonists [77]. The profile of the interesting (racemic) tetrahydrocarbazole BRL 56905 (21), an analogue of the potent 5-HT1D receptor agonist 5-carboxamidotryptamine (5-CT) in which the tryptamine chain is conformationally frozen, has been described by the SmithKline Beecham group [78]. This molecule probes the preferred spatial disposition of the amino group with respect to the indole nucleus for 5-HT1D and 5-HT~A receptors. BRL 56905 has high affinity (KD 10nM, PC) for the 5-HT1D receptor, although somewhat lower than that of 5-CT (KD 1.6nM, PC). The affinity of BRL 56905 at the 5-HT1A (KD 500nM) receptor is markedly lower than that of 5-CT (KD 0.3nM), however. In functional assays on dog saphenous vein (ECso 70nM) and rabbit basilar artery (ECso 280nM), BRL 56905 was a partial agonist with significantly greater potency than sumatriptan (ECso'S 400nM and 2400nM, respectively). It may be concluded from this work that the preferred binding orientation of the amino chain in 5-CT at the 5-HT1D receptor is as shown in 22 ("easterly" orientation) and not as in 23 ("northerly" orientation). The latter seems to be preferred for 5-HT~A receptor binding. Molecular modeling studies of BRL 56905 in conjunction with Pfizer stereogenic pyrrolidines (R)- and (S)-18 and the piperidines (eg, GR85548, 16) would provide further insights into the preferred positioning of the amino group at the 5-HT~D receptor. In contrast to the conformationally-locked tetrahydrocarbazole BRL 56905, Lilly claim heterocyclic tetrahydrobenz[cd]indoles (eg, 24) and the corresponding indolines in which the amino group is held in an alternative arrangement, as 5HT1D (and 5-HT1A) receptor agonists [79, 80]. The heterocycle can be five- or sixmembered. In these structures, the amino group is arguably constrained in a "disfavoured ....northerly" arrangement for 5-HT1D receptor binding, reminiscent of that portrayed in 23. Alternatively, this series could be considered as being based (cf 24a) on the 5-HT~A agonist structure 8-hydroxydipropylaminotetralin (8-OHDPAT, 24b). Consistent with this notion and other literature precedent [81], 24 is selective for the 5-HT~A receptor (ICso 0.6nM) over 5-HT1D (ICso 18nM, BC). KaliChemie have claimed indolo-aminolactams (eg, 25) as 5-HT1D agonists [82]. Merrell Dow have reported analogs of 5-HT wherein the tryptamine nitrogen carries an elongated anilide group [83,84]. MDL-100,687 (26) has moderate affinity for the 5-HT1D receptor on bovine caudate (ICso 34nM), but has higher affinity at the 5-HT~A receptor (ICso 12nM), and retains affinity for the 5-HT2c (ICso 637nM) and 5-HT2 (ICso 450nM) recognition sites. Bristol-Myers Squibb have reported an analogous series of indoles in which there is a simple substituent (eg, F) at C5 of the indole and a pyrimidinylpiperazine at C3 [85]. BMS 181,101 (27), a representative of this class, has high affinity for the 5-HT~D site (ICso 3nM, BC) and moderate affinity at 5HT1A (ICso 59nM) and 5-HT 2 (ICso 120nM) sites. An additional feature of BMS 181,101 is its potent blockade of 5-HT reuptake sites (ICso 0.1nM). This combined 5-HT1D agonist/5-HT reuptake blocker profile may have potential for (fast-acting) antidepressant activity. The sumatriptan analogue 28 has also been reported [86],

117 and has significantly higher affinity (ICso 0.1nM, BC) than 27. Like structures 4a and 15 with their elaborate C5 substitutents, compounds 26-28 demonstrate the remarkable tolerance of the 5-HTm receptor for ligands which are substantially larger than the native neurotransmitter (5-HT), this time at the indole C3 position. Previous SAR had suggested limited bulk tolerance in this part of the tryptamine structure [87]. iii) C4-Substituted i n d o l e s SmithKline Beecham have claimed 4,5-disubstituted tryptamines (eg, the 4chloroserotonin analogue 29) as 5-HTm receptor agonists [88]. These compounds also have 5-HT2 receptor properties.

2. INDOLE REPLACEMENTS The Merck benzofuran analog (30) [89] of 5-CT retains significant affinity (ICso 20nM, PC) and functional activity (ECho ~200nM, RSV) at the 5-HT m receptor, albeit somewhat lower than that of 5-CT itself (ICso 4nM; ECso 20nM). This data shows that the indole NH is not a necessary pre-requisite for 5-HT1D receptor recognition and activation, and that the benzofuran ring is a viable indole isostere. Both 30 and 5-CT have high affinity at the 5-HT~Areceptor (ICso 5nM and 0.3nM, respectively). Merck also claim indazole as an isostere for the indole ring (eg, the oxadiazole 31)[90]. Merrell Dow have claimed tetralin analogs of sumatriptan (eg, 32) as 5-HT~D receptor agonists [91]. This series is structurally related to 8-OH-DPAT, and as such would be expected to possess 5-HT1Areceptor properties. Adir et Compagnie have claimed the naphthalene analog of sumatriptan (33) as a 5-HT~D ligand vasoconstrictor [92]. 33 shows weak functional potency (ECso 1000nM, dog basilar artery). Merrell Dow claim benzodioxan analogs (eg, 34) of their amino anilide 26 as 5-HT~D and 5-HT~Areceptor agonists [83]. 3. OTHER S T R U C T U R E S

The arylpiperazine 5-HT m receptor agonist [93]GS 12066B (35), has been shown to be a potent 5-HT m receptor agonist (ICso 32nM, CC; ECso 78nM, CSN), but is non-selective with respect to the 5-HT1A site (ICso 65nM) [94]. CGS 12066 is a member of a family of arylpiperazines which bind at the 5-HT1D receptor. The a-adrenoceptor agonist oxymetazoline (36) was recently shown to have good affinity (Ko 5nM, CC) and potency (ECho 45nM, CSN) as a 5-HT~D receptor agonist [95]. However, oxymetazoline has equally high affinity and efficacy at 5-HT1A receptors, and is also non-selective with respect to a2-adrenoceptors. BASF have claimed pyrrolo[4,3e]benzazepines [96] (eg, 37; ICso 30nM, bovine frontal lobe (BFL)) and dibenzoheptenes [97] (eg, 38; ICso 3nM, BFL) as ligands for the 5-HT~D receptor with higher affinity than sumatriptan (ICso 50nM, BFL).

118 These structures have a strong structural resemblance to the non-selective 5-HT1D receptor antagonist methiothepin (39) and may therefore be receptor antagonists. Sanofi have reported a novel series of thieno-indanone oximes as 5-HT~D agonists [98,99]. Like CGS 12066B and oxymetazoline, the compounds from this series bear little overt structural resemblance to 5-HT, and as such are intellectually stimulating. Two compounds from this series have been highlighted as having higher potency and better selectivity than sumatriptan for the 5-HT~D receptor. Thus SR 27592 (40) (ICso 16nM, BC; ECso 15nM, dog saphenous vein; sumatriptan ICso 60nM; ECho 570nM) and SR 28734 (41) (IC5o 10nM; EC5o 30nM) are potent 5-HTm receptor agonists. SR 27592 is sixty-fold selective for 5-HT~D sites over 5-HT~A. These compounds are likely to be lipophilic and brain-penetrant. SmithKline Beecham have claimed tetrahydrobenzazepines ([100], 42) as 5HT1D receptor agonists. This series possesses 5-HT~ receptor properties.

5-HTm receptor subtypes The cloning, deduced amino acid sequences, pharmacological properties, and second-messenger coupling of two human receptor genes (designated 5-HT~D~and 5-HT~D0) have been reported [27]. The relative binding affinities of a range of serotonergic ligands reveals a rank order of potency which is consistent with a 5HT m receptor pharmacological profile for both clones: thus 5-CT > 5-HT > yohimbine > 8-OH-DPAT > spiperone > zacopride. Sumatriptan (I~. 3.4nM, 5-HT~D~; I~. 7.7nM, 5-HTID0) has high affinity for both subtypes and showed about a two-fold selectivity for the a-subtype. A number of compounds showed better than seven-fold selectivity for the a-subtype, including methysergide, 5methoxytryptamine, tryptamine and spiperone. 5-HT1D agonists like sumatriptan inhibited forskolin-stimulated increases in c-AMP production in these clonal cells (sumatriptan: ECso 3.2nM, 5-HT1D~; ECho 5.2nM, 5-HT1D~). The binding properties of the 5-HT~D receptor subtypes are very similar, a linear correlation coefficient of 0.96 being obtained in the comparison of log I~. values of 19 compounds [27]. Receptor models for the 5-HT~D~ and 5-HTlm receptors have recently been constructed [59], which complements earlier work on the 5-HTID receptor [101]. In accord with the conclusion from binding studies, these receptor molecular models indicate a very close similarity between the two subtypes, which should make the discovery of selective ligands a challenging task.

5-HTm receptor antagonists Glaxo have reported a series of arylpiperazinyl benzamides as potent 5-HT1D receptor antagonists [102,103,104]. GR 127,935 (43) is claimed to have high affinity for the human cloned 5-HTID~ subtype (~ 0.13nM) and to have ten-fold selectivity over the 5-HT1D~ subtype (I~. 1.3nM) [105]. GR 127,935 has good selectivity for 5-HTID over other serotonin receptors, having only modest affinity for 5-HT1A ( ~ 126nM), 5-HT2c ( ~ 400nM) and 5-HT2 ( ~ 250nM) receptors. This compound is therefore substantially more potent and more selective than existing 5-HT~D receptor antagonists like ketanserin or methiothepin and represents a significant breakthrough. Functionally, GR 127,935 was capable of antagonising

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124 sumatriptan-induced contraction of dog basilar artery in vitro, and was orally active in blocking 5-HTID agonist-induced contralateral rotation in guinea pig. FUNCTIONAL ASSAYS FOR 5-HTID RECEPTORS In vitro b i o c h e m i c a l assays The 5-HT1D receptors possess a high degree of sequence homology to the other 5-HTl-like receptors and in particular display a long 3rd intracellular loop sequence [34], a characteristic which is often associated with a negative coupling to adenylyl cyclase. This prediction has been borne out in practice with both native and cloned receptors being negatively linked to adenylyl cyclase activity. Thus both the calf and guinea-pig substantia nigra contain 5-HT~Dreceptors which can be quantified by the determination of the inhibition of forskolin-stimulated adenylyl cyclase [106,107]. To date, the sub-type of 5-HT~D receptor which subserves this action is unknown although, given the predominance of 5-HT~D~ receptors in the mammalian CNS, this receptor may correspond to the latter subtype. These discoveries on the linkage of 5-HT~D receptors have also led to the identification of a comparable biochemical response in the dog saphenous vein [108], an observation which is compatible with the presence of a functional, contractile 5-HTID receptor in this preparation.

Table 1 Receptor subtype of the human cortex terminal 5-HT autoreceptor Observation

Conclusion

Ref

Agonists 5-CT>RU24969>8-OH-DPAT

5-HT~-like [ 115] not 5-HT1A, 5-HT1E, 5-HT~F 5-HT>sumatriptan>8-OH-DPAT>DOI not 5-HT1A, 5-HT2A, 5-HT2B, 5-HT2c, 5-HT1D? [116] Antagonists Blocked by Metitepine, Metergoline

5-HTl-like

Not blocked by ketanserin (I~M) Metitepine enhances release

5-HT1D~? endogenous tone at receptor

[114, 115,116] [116] [114]

125 The cloned 5-HT~D receptors are also negatively linked to adenylyl cyclase activity. Thus both the human 5-HT~D~ and 5-HTlm as well as the dog RCD4 [109] (human homologue: 5-HT1D.) have been shown to possess this activity although, interestingly, there is one report of a cloned canine 5-HT1D receptor which is positively linked to adenylyl cyclase [36]. The reasons for the apparent discrepancy of the latter coupling are not clear but may be related to an excess of the appropriate Gs proteins over the Gi proteins in the particular cell line used. As is the case for the rodent 5-HT m receptor (species homologue of the human 5-HTID~ receptor), 5-HT~D(~?)receptors are represented as terminal 5-HT autoreceptors in several species. These include the guinea-pig [110,111], pig [112], rabbit [111,113] and human [114,115,116]. In the human cortex, the known pharmacological profile of this inhibitory terminal 5-HT autoreceptor is consistent with a 5-HT~D~ subtype (see Table 1). Although the present data are consistent with the terminal 5-HT autoreceptor in the afore mentioned species being of the 5-HT w subclass [50], the presence of the mRNA for all the 5-HTl-like receptors in the raph6 nucleus [117] leads to the possibility that other members of this receptor family may be represented as terminal autoreceptors. Indeed the presence of two inhibitory terminal 5-HT autoreceptors in the guinea pig CNS has been postulated on the basis of the apparent pA2 of metitepine in antagonising the actions of either 5-HT or sumatriptan in frontal cortex slices [118] and as a result of a detailed study of the dose-response relationship of the inhibitory effect of 5-HT, 5-CT and sumatriptan on 5-HT release in cortex slices [119].

In vitro pharmacological assays A large number of functional assays of a pharmacological origin have been delineated since the discovery of the 5-HT m recognition site in 1987. The best characterised of these are the endothelium-intact pig coronary artery [120] and guinea-pig jugular vein [121], the rabbit [122,123,126] and human [124,125] saphenous vein and the human pial arteriole [127]. In many other less well characterised preparations, sumatriptan and/or metitepine have been used to postulate the presence of 5-HTl-like (5-HT1D?) receptors on isolated pharmacological preparations. These include the dog saphenous vein [128,129], the dog [130], rabbit [131], sheep [132], primate and human [130,133] basilar artery, the isolated perfused rat kidney [134], the guinea pig ileac artery [135], the dog and human coronary artery [136-141] and the human hand vein [142] and dural [143,144] artery. The exact relationship of these (mainly) contractile responses to the 5-HTID receptors is not fully resolved since in many cases the studies were performed with a limited range of agonists and in most cases metitepine was a more potent antagonist of the response than metergoline, an observation at variance with the observed radioligand binding affinity of these drugs for 5-HTID~ or 5-HT1D~ receptors [145]. To date there is only one report of the use of the selective 5-HT1D receptor antagonist, GR 127935, as a tool to define 5-HT~D receptors. Thus the dog isolated basilar artery preparation is antagonised by GR 127935 in the range of 1-10nM [105] and this observation is consistent with this response being mediated by 5-

126 HTID receptors. GR 127935 is somewhat selective for the 5-HTaD~ over the 5-HTxD~ receptor [105] but the non-competitive nature of its antagonism of the dog isolated basilar artery preparation precludes a conclusion as to the exact subtype of 5-HT1D receptor which subserves this effect. Nevertheless GR 127935 is now the best tool to define the presence of functional 5-HT1D receptors both in vitro and in vivo and will undoubtedly supersede the use of metitepine in this context. In vivo functional

assays

The best characterised in vivo functional assay, mediated by 5-HT1D receptors, is the induction of contralateral rotation in the guinea-pig elicited by direct injection of 5-HT1D receptor agonists into the substantia nigra [146]. This model was developed as a result of observations that, in the rat, direct injection of 5-HT receptor agonists into the substantia nigra induces rotation and that given the high density of 5-HT1D receptors in this brain area in the guinea-pig, such an effect may be mediated by 5-HT m receptors in this species [49]. The initial report of this model established an induction of turning behaviour by the direct injection of the 5-HTl-like receptor agonists 5-CT and sumatriptan but only weakly by the 5-HTla receptor agonist, 8-OH-DPAT [146]. Subsequent studies using another 5-HTl-like receptor agonist, GR 56764, showed that the rotation induced by this agonist w a s potently (0.3 mg/kg p.o.) blocked by the selective 5-HTID receptor antagonist, GR127935 [104], thereby validating this model of central 5-HTtD receptor function. The characterisation of the aforementioned model of in vivo 5-HTID receptormediated function has relied on the availability of the 5-HT~D receptor antagonist, GR127935. Previous studies, which have attempted to delineate such in vivo functional correlates, have tended to rely on the non-selective 5-HTl-like receptor antagonist, metitepine and the 5-HT1D receptor agonist, sumatriptan. The former compound, whilst acting as a potent 5-HTt-like receptor antagonist in vivo, also has high affinity for other neurotransmitter receptors such as dopamine, aadrenergic and histamine [147] and, as such, is a poor tool to explore the functional consequences of 5-HT~D receptor activation in vivo. The latter compound displays high affinity for 5-HT~D~ and 5-HT1D~ receptors but has only moderate selectivity with respect to 5-HT~A and 5-HT1F receptors [148] and any studies which utilise sumatriptan in order to characterise the response as being mediated by 5-HTaD receptors must therefore be treated with caution. For this reason many reports have referred to the "5-HT~-like receptor agonist", sumatriptan rather than referring to its 5-HT1D receptor agonist properties. This is no where better exemplified by the studies of Humphrey and his co-workers on the haemodynamic effects of sumatriptan. Thus in experimental animals sumatriptan causes a selective vasoconstrictor effect in the dog common carotid artery which is antagonised by metitepine [149] but subsequent studies cast doubt as to whether this action of sumatriptan w a s mediated by 5-HT~D receptors [150]. In parallel studies in the pig, in which sumatriptan causes a reduction in cranial arteriovenous anastomotic shunting [151], a similar conclusion as to the lack of mediation of the effect by 5-HT~D receptors was arrived at [152]. Extensive studies using the even less selective 5HT1D receptor agonists, ergotamine and dihydroergotamine, have also failed to

127 define the locus of action of these drugs as being mediated by 5-HTxD receptors [153,154]. These vasoconstrictor actions of sumatriptan in animals led to the evaluation and demonstration of the acute anti-migraine activity of sumatriptan in man [155161]. Subsequent attempts to demonstrate a vasoconstrictor action of sumatriptan in human cerebral vessels has led to equivocal results [156] and no attempt to define the receptor(s) involved has been reported. Similarly the receptor subtype mediating the vasopressor response in the human systemic and pulmonary arterial circulation and the coronary artery vasoconstrictor effects of sumatriptan [158] have not been delineated although it is possible that such effects are mediated by activation of 5-HTID receptors. Another area of research into the functional actions of 5-HTID receptors, which has heavily relied on sumatriptan as a tool, is that of blockade of neurogenic plasma extravasation in the dura mater of the rat and guinea-pig [162,163]. Sumatriptan is selectively active in this putative animal model of migraine in that it can block plasma extravasation in the dura mater but not extracranial vessels such as the conjunctiva, eyelid and lip [162]. Interestingly, in a study which compared the actions of the selective 5-HT1B receptor agonist, CP-93,129 with sumatriptan in both the rat and guinea-pig, it was concluded that blockade of plasma extravasation in the rat dura mater may be mediated by 5-HTm receptors whereas the 5-HTID receptor may be more pertinent to the guinea-pig [163]. The latter conclusion remains to be validated using the selective 5-HT1D receptor antagonist, GR127935. In the course of these studies on neurogenic plasma extravasation, Moskowitz has identified two further functional responses to sumatriptan which may be mediated by 5-HTm receptors. Thus sumatriptan can induce an increase in c-foslike immunoreactivity in the rat which may be mediated by 5-HT m receptor activation [164] and, by analogy, 5-HTID~ receptor activation in other species. The second functional response of interest is an observation made in the course of the studies of trigeminal nerve stimulation which is a sumatriptan-induced decrease in CGRP levels in rat plasma [165]. Such observations may be akin to the reduction in CGRP levels induced by sumatriptan in man during a migraine attack [166] and again remains to be shown to be mediated by 5-HTID receptor activation. A range of other functional responses may be mediated by 5-HTID receptor activation although definitive proof using either selective 5-HTID receptor agonists or antagonists is, as yet, lacking. Thus the in vivo correlate of the in vitro activation of the terminal 5-HT autoreceptor may lie in the decrease seen in 5-HT release induced by 5-CT or sumatriptan in the guinea-pig frontal cortex as measured by intracerebral dialysis [167,168]. Finally a number of studies of the behavioural effects of 5-HTx-like receptor agonists have raised the possibility that these actions may be mediated by 5-HT1D receptors. These include the hypothermic effects of 5-HT1D receptor agonists in the guinea pig [169], and several behaviours in the rat including hindlimb scratching induced by 5-methoxytryptamine [170], suppresion of penile erection by 5-HT receptor agonists [171] and 5-CT-induced drinking [172].

128 THERAPEUTIC APPLICATIONS

5-HT1D receptor agonists The most obvious therapeutic application of a 5-HTaD receptor agonist is as a potential acute treatment for migraine headache. This interest was triggered by the introduction of sumatriptan into clinical practice, and has been the subject of intense activity in both the pharmaceutical industry (see section on receptor ligands: this chapter) and also in studies of the mode of action of sumatriptan [for useful reviews of the latter see [173]. The early assumption that sumatriptan was a reasonable selective 5-HT1D receptor agonist has not, however, been born out in practice since this drug displays affinity for both subtypes of 5-HT~D receptor and 5-HT1B, 5-HT1A and 5-HT~v receptors. This lack of specificity of sumatriptan means that any mode of action studies using the drug as a tool are difficult to interprete in the absence of a selective 5-HT~D receptor antagonist. As was discussed earlier such a tool, GR127935 has recently become available and many of the original studies on the in vitro and in vivo pharmacology of sumatriptan will need to be reevaluated using this 5-HT1D receptor antagonist. In addition it is important to attempt to differentiate the receptor subtype(s) which subserve the presumed clinical effects of sumatriptan (cerebral vasoconstriction, inhibition of plasma extravasation) from the unwanted clinical actions (coronary vasoconstriction, tingling, growth hormone secretion [174]). In this regard a combination of in situ hybridisation, quantitative analysis of mRNA by PCR technology, radioligand binding and functional studies will have to be performed on target and non-target tissues in an attempt to define the critical functional receptors subserving the actions of sumatriptan. Such studies have begun using in situ hybrisation techniques to define the mRNA present in the rat trigeminal ganglion [55] and reports of the receptor subtype subserving the vasoconstrictor action of sumatriptan in the human isolated coronary artery. Clearly the definition of these receptors is an important target for preclinical research which may eventually lead to the development of more selective 5-HT1D receptor agonists with a better side-effect profile. 5-HTxD Receptor antagonists Until recently the only 5-HT1D receptor antagonist available was metitepine which, because of its non-selectivity for 5-HT1D receptors, was a poor tool with which to define the therapeutic potential of this class of drug. This situation has been dramatically changed by the disclosure of the relatively selective 5-HT1D receptor antagonist, GR127935 [104], which, at least in the patent applications, has been claimed to have therapeutic potential in depression, anxiety disorders and Parkinson's disease. Clearly this area from the clinical standpoint remains to be justified and the results of the first clinical trials with such compounds are eagerly awaited as is preclinical guidance which may point to a peripheral utility for such a class of antagonist.

129 nerve impulse PRE-SYNAPTIC

NEURONE

Q " _ ~

" 9

re-uptake

storage vesicle

autoreceptor

site

POST-SYNAPTIC

NEURONE

onward transmission

Fig. 1. Synapse of a 5-HT neuron.

There is, however, some preclinical basis to justify the use of 5-HT1D receptor antagonists in the treatment of depressive disorders. It has been known for some time that facilitation of 5-HT neurotransmission can be achieved by blockade of the presynaptic reuptake site by selective serotonin reuptake inhibitors such as paroxetine and fluoxetine and that such an action results in clinically useful antidepressant properties. It is now believed that the concentration of 5-HT at the terminal is controlled not only by the uptake site but also by an inhibitory terminal 5-HT autoreceptor (see Figure 1) which in higher species of animals is likely to be of the 5-HTxD subclass (see earlier in this chapter). This inhibitory 5HT autoreceptor is normally activated by the endogenous release of 5-HT and antagonism of this receptor would lead to disinhibition of the neurone and a facilitation of 5-HT release. Since the net effect of this action would be to provide a rapid increase in 5-HT release, it has been postulated that a 5-HT~D receptor antagonist could have antidepressant properties.

130 ADDENDUM re GR127,935 (43)

The SAR of a series of substituted di-Me-aminopropylbenzanilides and piperazinyl benzanilides [175] resulted in the discovery of the potent, selective and orally active 5-HT,D antagonist GR127,935 (43). Although the compound shows no agonistic activity in a wide variety of in vitro and in vivo experiments [105], it behaves as antagonist in Hela cells stably transfected with human 5-HT~, and 5-HT,D ~ receptors inhibiting the forskolin stimulated c-AMP production with pIC~o values of 7.9 and 8.0 respectively [176]. Its congener GR55562 (44) behaves in this study as a modestly potent antagonist devoid of any agonistic activity. A study on human 5-HT,D~ and 5-HT,D ~ receptors stably transfected in C6-glial cells of the rat showed that GR127,935 is an agonist at the 5-HT,D~ receptors with a piC5o=6.98 but behaves as a potent antagonist at the 5-HT,D~ receptor versus the 5-HT~D agonist naratriptan, also reducing the maximal response of the latter [177]. The same group showed that GR127,935 at a concentration of 10SM is devoid of agonistic activity at the 5-HT,Da receptor. The receptor density in both cell lines was the same, 350 fmol per mg protein. The agonistic activity at the 5-HT~D~ receptor was tested at two different receptor densities (1050 and 350 fmol per mg protein) and proved to be virtually identical with pICso'S of 6.88 and 6.85 respectively [178]. The results suggest that the shift from antagonistic to agonistic activity is not due to differences in receptor density (see also [185, 186]). The group of Middlemiss has shown that GR 127,935 is a partial agonist when interacting with human 5-HT,~ or 5-HT1D~ receptors stably expressed in CHO cells [185]. re MK 462 (45)

Street et al. [179] showed that MK 462 (45) has a fairly high affinity for the 5HT,D receptor in pig caudate membranes (pIC~o=7.3, PC) and is a full agonist with moderate potency (pEC5o=6.6) at the 5-HT m receptor in the rabbit saphenous vein (RSV). The compound is selective in its affinity for the 5-HT1D receptor compared with 5-HT,A,2A,2C and 5-HT 3 receptors. Direct linkage of the triazole ring of MK 462 to the C-5 of the indole moiety as in compound (46) results in an increase in both the affinity and the potency (piC5o=7.7, pEC5o=7.2). Exchanging the triazolering in (46) for a 2-Me-imidazole ring as in compound (47) increases the affinity somewhat further (pICso=8.1) but not the potency (pEC5o=6.8). Both compounds are full agonists in the RSV test and do not differ in selectivity for the 5-HT,D receptor from MK 462 except compound (46). The affinities of (46) for the 5-HT m and the 5-HT,A receptor are identical while its selectivity in regard to the 5-HT2A,~c and 5-HT 3 receptors is maintained. Sternfeld et al. [180] published the synthesis and pharmacological profile of L741,604 (48) a new potent and orally active analogue of compound (46). It has a

131 high affinity for the 5-HT~D receptor (pIC~o=8.7), a 20 fold selectivity in regard to the 5-HT~A receptor and a more than 1000 fold selectivity compared to the 5HT2A,2c and 5-HT a receptors. In vitro L741,604 has a two fold higher potency (pEC5o=7.5 RSV) than its analogue (46). Restricting the conformation of the ethylamino side chain in L741,604 by replacement by a N-Me piperidinyl group results in a four fold decrease in affinity for the 5-HTlo receptor while the potency is retained (pEC5o=7.4 RSV). re 5 - ( n i t r o p y r i d y l ) a m i n o i n d o l e d e r i v a t i v e s

The affinity and potency at the 5-HT~D and 5-HT1A receptors of 5-(nitropyridyl) amino indole derivatives was published by Macor et al. [181]. The binding experiments were performed on bovine caudate membranes using [3H]-5-HT as radioligand (5-HT1D) or on rat cortex membranes using [3H]-5-OH-DPAT (5-HT1A). The 5-HT agonist activity was measured by testing the inhibition of forskolin stimutated c-AMP production in guinea-pig substantia nigra (5-HTID) or guinea-pig hippocampus preparations (5-HT1A). Compound (14)=CPl13,113 has a high affinity for the 5-HT1D receptor (pICso=8.0) and a 7 fold lower affinity for the 5-HT1A receptor. The 5-HT~D agonist potency of CPl13,113 (pEC5o=8.7) is 85 times higher than its 5-HTIA agonist potency. Stereo selective ring closure between the di-Me amino group in the side chain of (14) and the a-C atom results in the pyrrolidine derivative (49, CP135,807) and its nor analogue (50, CP123,803). Both compounds (49) and (50) have a high affinity for the 5-HT~D receptor (pICso'S of 8.5 and 8.2 respectively) and an 11 and 19 fold lesser affinity for the 5HT1A receptor. The potencies of both compounds at the 5-HT1D receptor are the same pEC~o=8.9 but the selectivity ratio 5-HTIJ5-HT1D=36 for compound (49) and 170 for compound (50). Non stereo selective ring closure between the di-Me-amino group in (14) and the [~-C atom of the side chain results in the racemate CP124,439 (structure not shown). This compound has a pIC5o=7.8 for the 5-HT~D receptor and a 21 fold lower affinity for the 5-HT1A receptor. The potencies at the 5-HT1D receptor of CP124,439 and the parent compound (14) are the same, pEC~o=8.7. However, its selectivity compared to the potency at the 5-HT1A receptor is much higher 440 fold versus 7 fold for the parent compound (14). Reduction of the nitrogroup in (14) followed by ring closure results in the pyridoimidazole derivative (52) [182]. The affinity of (52) for the 5-HT1D receptor is the same as that of the parent compound (14) whereas its selectivity ratio 5HTIA/5-HTID is slightly higher 19 versus 7 for (14). Both the 5-HTID agonist potency of (52) and the 5-HT1A/5-HT~D selectivity ratio in this test are lower than those of parent compound (14): pECso'S 7.85 versus 8.7 and ratios 32 versus 85 [182]. CP161,242 (51) is a potent centrally active receptor agonist [182]. The compound has a high affinity for the 5-HTID receptor (pIC~o=8.9) and a moderate selectivity ratio 5-HT~J5-HT~D of 15. However, the potency in the in vitro forskolin

132 test (pEC5o=10.4) is very much higher than can be expected from the binding data and the selectivity ratio is 620. The authors [182] propose that a tentative explanation of this phenomenon could be that the affinity binding site of the compound may differ from the activity binding site.

Alkyloxytryptamines A SAR study on 5-alkyloxy tryptamines was published by Glennon et al. [183]. The length of the unbranched alkylchain ranged from 1 to 11 C atoms. The affinities for the 5-HT~D~ receptor of the methoxy- to the nonyloxyderivative are all comparable with I~ values smaller than 5nM. The affinities of the decyl- and undecyloxy analogues are 10 fold lower. The selectivity ratio 5-HTIA/5-HT~D~=315 of the nonyloxy derivative is the highest of all compounds tested. The methoxy derivative is not selective at all. The other compounds have selectivity ratio's ranging from 16 to 44. 5-(nonyloxy) tryptamine has a high affinity for the 5-HT1D~ receptor (pICso=9) [183] and acts as a full agonist in the inhibition of c-AMP production stimulated by forskolin in CHOKM 6 cells transfected with the human 5-HT1D~ receptor gene (pEC5o=7.2) [184].

REFERENCES

10 11 12 13 14 15 16

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Chapter 5

5-HT1E , 5HTlr RECEPTORS

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Serotonin Receptors and their Ligands B. Olivier, I. van Wijngaarden and W. Soudijn (Editors) 9 1997 Elsevier Science B.V. All rights reserved.

141

5-HTm and 5-HT1FReceptors G. McAllister and J.L. Castro Merck, Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, United Kingdom.

INTRODUCTION The recent advances in gene cloning techniques has led to an explosion of information about the design of the nervous system, and has altered the approach of scientists to the process of drug discovery. The ability to isolate individual genes encoding particular receptors has revealed a level of complexity in the brain not always appreciated by the traditional techniques of pharmacology. It seems that most neurotransmitters have not just one target receptor but many, and as this book demonstrates, serotonin, in particular, has a plethora of receptors to interact with. The challenge for scientists today are to understand why so many receptors have evolved, understand their function in vivo, and use this information to develop novel drugs to interact with these receptors in a more directed, selective way than has been possible until now. As discussed in previous chapters five 5HTl-like receptors, termed 5-HTIA, 5-HT1B, 5-HT1D- 5-HTIF have been described. The 5-HTlc subtype is generally agreed to belong in the 5-HT2 family and the 5HTIB receptor in rodents is the species homologue of the 5-HTID~ receptor in man [for review see 32]. All of these 5-HTl-like receptors have relatively high affinity for serotonin and when activated can inhibit forskolin stimulated adenylyl cyclase activity in transfected mammalian cells. The question of why nature has evolved several receptors that have similar affinity for the endogenous ligand, serotonin, and apparently couple to the same effector system is particularly intriguing and awaits the development of subtype selective ligands to find an answer. This chapter will deal with two of these 5-HTl-like receptors: 5-HTIE and 5HT1F. Radioligand binding studies initially identified a putative receptor, termed 5-HT1E, in human cortex and putamen which was able to bind [3H]5-HT even in the presence of concentrations of5-carboxamidotryptamine (5-CT) and mesulergine that would block 5-HTIA.D sites [1]. Recently, two receptor subtypes, 5-HT m [2,3,4] and 5-HTIF[5,6,7] that have pharmacological profiles similar to this 5-HT m binding site have been cloned by several groups. RECEPTOR STRUCTURE

The discovery of the existence of a G protein coupled receptor superfamily [8] has greatly facilitated the isolation and characterisation of serotonin receptor clones. With the exception of 5-HT3 receptors (see chapter 7), all of the cloned

142 serotonin receptors are members of this family and analysis of their primary structures reveals the characteristic seven putative transmembrane domains and large regions of sequence homology indicating their common evolutionary origins. The 5-HT1A receptor was the first of the 5-HTl-like receptors to be cloned and this was achieved because of its homology with the [~2-adrenergic receptor (see chapter 2). Since then several groups, have used variations of homology cloning to obtain the other 5-HTl-like receptor clones. All of these receptors are encoded by intronless genes, a feature which distinguishes them from the 5-HT 2 receptors, and a feature which has allowed the direct amplification of novel receptors from genomic DNA using polymerase chain reaction (PCR) techniques. This has accelerated the characterisation of this subfamily of receptors because clones can be isolated without identifying a tissue source that contains the receptor. The first group to isolate a 5-HT1E receptor clone did so by homology screening of a human genomic DNA library using oligonucleotide probes derived from the cloned 5-HT~A and 5-HTlc receptors [3]. The authors termed this human gene $31 and demonstrated that when expressed in mammalian cells it mediated the inhibition of adenylyl cyclase activity. However, no radioligand binding data accompanied this finding so $31 was not identified as a 5-HTIE receptor initially. However, soon afterwards it emerged that several groups had independently isolated this gene and confirmed that its pharmacology was very similar to the 5HT~E binding site found in human brain [2,4,9]. More recently another receptor was cloned, again by several groups, which also had a 5-HT1E-like pharmacology, but was clearly encoded by a separate gene. The mouse version of this gene was termed 5-HT1E~ [5], whereas the human gene was termed 5-HTIF [6] or MR77, a 5-HT~E-like receptor [7]. However, based on sequence comparisons some differences in the pharmacology of this receptor it has been proposed that it is sufficiently different to warrant its own subtype therefore it will be referred to as the 5-HTIF receptor in the rest of this chapter. As can be seen from the dendrogram in figure 1 the 5-HT~E and 5-HTIF receptors are more closely related to each other than to other 5-HT receptors. The 5-HTIE receptor, within the highly conserved transmembrane (TM) domains, exhibits approximately 52%, 40%, 64% and 70% amino acid identity to the 5-HT1A, 5-HT m, 5-HT1D subtypes, and 5-HTIF receptors, respectively. The 5-HT~F receptor exhibits approximately 53%, 40%, 63% and 70% amino acid identity to the 5-HT1A, 5-HTlc, 5-HT1D subtypes, and 5-HT1E receptors, respectively. Both the 5-HT~p. and the 5-HTLF receptors are of similar length (365 and 366 amino acids respectively) and share other common features with the serotonin receptor family, such as conserved aspartate residues in transmembrane (TM) regions 2 and 3 and a single conserved serine residue in TM5, potential glycosylation sites in the NH2-terminal domain, and consensus phosphorylation sites particularly in the third intracellular loop regions. Comparing the sequences of the serotonin family as a whole it can be seen that those most related at the amino acid sequence level (eg. 5-HT~D~and 5-HT1D~ or 5-HT 2 and 5-HTlc) also share similar properties (eg. same affinity for 5-HT, same effector systems, similar pharmacological profile). These closely related members (or subtypes) display amino identity values of 75% or more in their TM regions, whereas less related members (eg. 5-HT1A and 5-HT2) display

143 values closer to 45% amino acid identity. The 70% identity between the 5-HTI~. and 5-HTIv receptors makes it difficult to decide on structural criteria alone whether they belong to the same subtype or represent two new subtypes. Indeed, following this analysis, the 5-HT m and 5-HT~ receptors could both be considered distantly related members of the 5-HT~D subtype.

5-HT2

[2A]

5-HT1c [2C] 5-HT1D 5-HT1B 5-HT1F 5-HT1E 5-HT1A Figure 1. Dendrogram. The human 5-HT1A [10], 5-HTI~. [2], 5-HTIF [6], 5HTID~[ll], rat 5-HTIB [12], 5-HT~c [13] and 5-HT~ [14] receptors were compared based on their sequence similarity. The relative lengths of the bars are inversely proportional to their sequence homology.

This raises the interesting question of why so many 5-HTi-like receptors have evolved and been maintained in the genomes of several different species. There are two schools of thought on this subject. The first suggests thatbecause they exist, they must be doing something important or there would be many more pseudogenes of the family. This implies distinct functions for each subtype despite the fact that they are often expressed in the same regions and even cell types (see later). Possible differences in function may arise from different midpoints of activation by serotonin under physiological conditions, different efficiencies of coupling to adenylyl cyclase inhibition, coupling to other effector pathways (eg. directly to ion channels) or discrete spatial or temporal expression. A second way of looking at this question turns the argument on its head. Perhaps there are so many different receptors because it is relatively easy to duplicate intronless genes

144 and any one of the receptors can replace the others functionally. It has been postulated that many of the genes encoding the G protein linked receptor family evolved from a single precursor gene (possibly an opsin gene) that lost its introns approximately 1 billion years ago [15]. Since then, gene duplication events have resulted in many related genes. These intronless genes are so small that they are more likely to be functional, when duplicated, than large intron-containing genes and have, therefore, diverged into a large related family of functional receptors. These events would, therefore, allow an increase in the diversity of receptors available and correspondingly in the level of complexity possible, particularly in the brain, conferring an evolutionary advantage. The extended subfamily of 5-HT~like receptors m a y represent duplicated genes that have not yet evolved to have differentfunctions, or that they represent duplicated genes with discrete functions. The development of subtype specific ligands that can dissect the function(s) of these receptors in vivo will enable us to decide which of these two arguments is correct. R E C E P T O R LOCALIZATION

As mentioned earlier,5-HT~z receptors were firstdescribed as a binding site in h u m a n cortical tissue and putamen homogenates that displayed high affinity for [3H]5-HT even in the presence of concentrations of 5-CT and mesulergine that would block binding to the other 5-HTl-like receptors [1]. Taking advantage of this, Beer and colleagues carried out autoradiography studies in rat and guinea pig brain (see figure 2) indicating that 5-CT insensitive 5-HT~-like receptors (5HTlz / 5-HTI~), as defined with [3H]5-HT in the presence of 100nM 5-CT and 300nM mesulergine, are most densely concentrated in the caudate putamen and olfactory tubercle [16, 33]. Interestingly,in the guinea pig but not the rat, these receptors are also abundant in the claustrum, a littlestudied brain area thought to be involved in visual attention [17]. This study cannot distinguish between 5HT~z and 5-HT~F receptors as both have low affinity for 5-CT and mesulergine. [aH]Sumatriptan however, having a high affinityfor the 5-HT~F receptor and low affinity for 5-HTIz receptors (Table 2) can be used to distinguish between both receptors [34-37]. A comparison with the 5-HTIE site in h u m a n cortex which also shows low affinityfor sumatriptan suggests that this siteis predominantly 5-HTIE rather than 5-HT~F [2].Another quantitative autoradiography study [18],looking at 5-CT sensitive and insensitive sitesin h u m a n brain regions indicated that while 5-HTID and 5-HT~z sites were relativelyequal and abundant in frontal cortex and globus pallidus (140 - 220 fmol/mg) there was ten times more 5-HTIE sites (224 fmol/mg) than 5-HT~D sitesin the putamen (28 frnol/mg).As pointed out above this binding site is likely to represent 5-HT~E binding rather than 5-HTIF binding but does not rule out the possibility of yet more as yet uncharacterised receptors contributing to the total binding sites.In situ hybridization studies of the 5-HT~z receptor in h u m a n brain revealed expression in cortical areas, caudate, putarnen and amygdala areas [19]. All these areas have been shown to contain 5-CTinsensitive 5-HT~-like binding sites.

145 More information is available for the 5-HTI~ receptor as its mRNA expression has been examined using both PCR and in situ hybridization techniques. Amlaiky and colleagues reported that the mouse 5-HT1F receptor mRNA (5-HT1E~) was not detectable on Northern blots of poly(A)*RNA suggesting a relatively low level of expression in mouse brain [5]. However, using more sensitive PCR techniques, a signal was observed in spinal cord and brain, predominantly in forebrain. Further analysis of the mouse brain, using in situ hybridization techniques, showed that a signal was only found in the pyramidal neurons of the CA1-3 layers of the hippocampus. In contrast, Adham and colleagues carried out in situ hybridization studies in the guinea pig [6], and found 5-HTI~ mRNA in lamina V of frontal cortex, again in large pyramidal cells as well as moderate labelling in the hippocampus. Moderate labelling was also detected over layer VI nonpyramidal neurons. In layer V and VI, the strongest signal was found in dorsal sensorimotor neocortex and in cingulate and retrosplenal cortices. Pyramidal cells in the piriform cortex and large neurons in the raphe nuclei were also heavily labelled and in contrast to the mouse study some labelling was seen in the granule cells of the dentate gyrus. The differences in distribution observed by these two groups may represent species differences or differences in sensitivity in their respective in situ hybridization studies. The regional distribution of guinea pig 5-HT~F receptor mRNA is very similar to that of 5-HTI~ receptors labelled with [3H]sumatriptan [34, 35]. The detection of 5-HTa~ transcripts in the dorsal raphe nucleus indicates a possible role as an autoreceptor regulating neurotransmitter release. However, 5-HTm~ and 5-HTm~ transcripts are also expressed in this nucleus. Whether one or all of these receptors can be autoreceptors will be answered only when selective ligands for these receptors become available. In the same study, the authors also examined the distribution of 5-HT~F mRNA in various human tissues by PCR techniques. Intriguingly, as well as in brain, they also found transcripts in the uterus and the mesentery. The possible role of this receptor in uterine or vascular function is very interesting, particularly as the 5-HT~ receptor has such high affinity for the antimigraine drug, sumatriptan. The mechanisms involved in a migraine attack are still unknown but the intense unilateral and throbbing headache characteristic of migraine is likely to be vascular origin as the brain itself is largely insensitive to pain. Two models have been proposed suggesting that either migraine is caused by vasodilation of intra and/or extracranial arteries leading to activation of sensory nerves and pain, or that the initiating factor is a neuronal disorder leading to neurogenic inflammation of the same blood vessels (reviewed in 20). In both cases the 5-HT~D receptor subtypes have been implicated as the target of efficacious antimigraine compounds such as sumatriptan based on a correlation of their affinities at the 5HTID receptors and their clinically effective doses. However, the discovery of the 5-HT~F receptor and its expression in at least some vascular tissues raise the possibility that 5-HT1~ receptors may also play a role in this disorder and could therefore be a potential target for novel, more selective antimigraine drugs [38].

146

Figure 2. Autoradiography of the distribution of 5-CT-insensitive sites in a coronal section of guinea pig brain. Sites were labelled with [3H]5-HT in the presence of 100nM 5-CT and 300nM mesulergine to block out other 5-HT,-like receptors. Highest density of labelling was observed in the claustrum (C1), olfactory tubercle (Tu) and caudate putamen (Cpu). Data provided by MS Beer. R E C E P T O R BINDING ASSAYS Receptor binding assays of the 5-HT,E and 5-HT,F receptors using tissue preparations are made difficult because no selective compound is available for use as a radioligand. Analysis of the cloned receptors expressed in mammalian cells is much simpler because the cell lines chosen for expression have no endogenous 5-HT,-like receptors present. Therefore it is possible to use a non-discriminating radioligand to characterise the pharmacological profile of the receptor. These studies are very useful, of course, because they may allow the experimenter to identify binding conditions specific for a particular subtype that would be useful for tissue studies. In the case of the 5-HT,E / 5-HT,F (or 5-CT insensitive) binding site, the binding assay predated the characterisation of the cloned receptors by

147 some three years. As mentioned previously, Leonhardt and colleagues first suggested in 1989 the existence of 5-HT1E receptors when they found evidence of heterogeneity in the pharmacology of the 5-HT m binding site in human brain [1]. They carried out radioligand binding studies using [~H]5-HT at a concentration (2nM) that would allow binding to all of the 5-HTl-like receptors known at the time (i.e. 5-HT1A - 5-HT1D). To examine the 5-HT,D binding only, they included in their assay lmM pindolol (to block 5-HT,A and 5-HT m receptors) and 100nM mesulergine (to block 5-HTIc and 5-HT 2 receptors). However, two binding sites were observed in the presence of these blockers. One of these sites demonstrated high affinity for 5-CT and ergotamine, consistent with the known pharmacology of the 5-HT1D site and the second site demonstrated low affinity for these two compounds. The high affinity (or 5-CT sensitive) site represented some 55% of the total specific [3H]5-HT binding in these human cortical tissue homogenates and the low affinity (5-CT insensitive) site comprised the other 45% of binding sites. Further analysis of the low affinity site, termed 5-HT m was carried out by replacing pindolol with 100nM 5-CT in the binding assay. This concentration of 5CT would prevent binding to 5-HT m sites as well as to 5-HT1A and 5-HT m sites. These studies demonstrated that the 5-HTm binding site displayed a Ka of 5.3nM for [3H]5-HT, was GTP- but not ATP-sensitive and had a unique pharmacological profile, the most distinguishing feature being a relatively low affinity for 5-CT and ergotamine. Table 1 A comparison of the I~ values (nM) of serotoninergic ligands at the cloned human 5-HTm and 5-HTI~ receptors and the 5-HT,~ binding site in human cortex.

5-HT1E

5-HT 5-CT Sumatriptan Methysergide Methiothepin Ergotamine Metergoline

6 3300 2090 220 120 540 776

5-HT1F*

Human Cortex

10 717 23 34 650 171 341

6 2000 1300 170 1500 800 426

Values taken from McAllister et al. [2] and Adham et al. *[6]. A comparison of published I~. values for the cloned 5-HTIE [2,4,9] and 5-HT1F [5,6,7] receptors and the values originally found for the human cortex 5-HT1E binding site [1] shows that either or both cloned receptors could in principle

148 correspond to the native receptor. However, McAllister and colleagues extended the pharmacological analysis of the human cortex site in direct comparison with the cloned human 5-HT~E receptor [2]. In particular, this study demonstrated that in cortex the 5-HTIE site had a relatively low affinity for the antimigraine drugs sumatriptan ( ~ 1300nM) and ergotamine (I~. 800nM), a profile much closer to the cloned 5-HT~ receptor than to the cloned 5-HTI~ receptor as shown in Table 1. As previously noted, sumatriptan has approximately 100-fold higher affinity for the 5-HTI~ receptor than the 5-HT1~ receptor suggesting that inclusion of 200nM sumatriptan in future autoradiography studies would eliminate the potential problem of also labelling the 5-HT1F receptor. Further evidence that the "5-HTI~" site labelled in tissue is predominantly 5-HT~F. rather than 5-HT~F comes from similar displacement studies carried out by Beer and colleagues on a variety of species [21]. They demonstrated that in contrast to 5-HT which was mono-phasic, 5-CT and sumatriptan displayed very similar biphasic distribution curves when they were used to displace ['~H]5-HT binding (in the presence of cyanopindolol and mesulergine to block 5-HTIA, 5-HT~B and 5-HT~c receptors) in the cortex and caudate of dog, guinea pig, human, hamster, rabbit, pig and calf. The high affinity component of these biphasic curves corresponds to 5-CT and sumatriptan binding to 5-HT1D receptors and the low affinity component is likely to correspond to 5HT m receptors as 5-CT and sumatriptan show similar displacements. The proportion of sites with low affinity for 5-CT and sumatriptan would be different if significant numbers of 5-HT~p receptors were present. A contribution of the more recently discovered 5-HT receptors (5-HTn.~, 5-HT~b, 5-HT6 and 5-HT 7) to 5-HTm binding site can be ruled out based on their pharmacological profiles (see later Chapters). However, other, as yet undiscovered, subtypes obviously cannot be discounted. It is not immediately obvious how a similar strategy could be used to specifically label the 5-HTI~ sites in tissue preparations, so direct visualization of native 5-HT~.. receptor binding sites will require the development of more specific ligands. LIGANDS Although no selective ligands for 5-HTI,~ or 5-HT1F receptors have been reported so far, several interesting trends in structure-affinity relationships can be extracted from the published binding of several tryptamine derivatives and related analogues. For the purpose of the present discussion, comparisons will be made, where appropriate, with the 5-HT~D,~and 5-HT~t~ receptors because they present the highest homology with the 5-HT1~:.11.~receptors within the TM domains and, as mentioned earlier, the 5-HT1F receptor has been suggested as a potential target for the antimigraine drug sumatriptan. Selectivity with respect to other 5-HT receptors will not be discussed. Inspection of the data in Table 2 reveals that 5-HT remainsthe highest affinity ligand for both 5-HT1~~ and 5-HTI~.~ receptors reported to date, and that simple modifications of this structure can result in dramatic changes in affinity. Particularly notable is the 100-fold reduction in affinity on methylation of the 5-

149 Table 2 A p p a r e n t dissociation c o n s t a n t s (Ki values; nM) of various drugs for cloned h u m a n 5-HT1E, 5-HT1F, 5-HTIDa and 5-HT1D~ receptors. . Compound a

5-HTIE b

5-HT1F c

5-HT1Da d

5-HT1D~ d

5.0 (11")

10

3.9

4.3

Tryptamine

316

2409

86

521

5-MeOT

630

1166

4.8

34

5-BnOT

794

9.6

19

5-MeO-DMT

100

37

4.4

21

a-Me-5-HT

121"

184

211

133

2-Me-5-HT

817"

413

915

860

5-CT

3980

717

0.70

1.6

> 10,000 *

1613

13

42

1995

23

3.4

7.7

5-HT

DP- 5- CT Sumatriptan RU-24,969

63

..........

TFMPP

1995

1002

64

114

1-NP

207*

54

7.4

12

NAN-190

.....

203

194

652

> 10,000

73

5198

Ketanserin

> 10,000

Mianserin

100

Metitepin

126

Cyproheptadine

790

8-OH-DPAT

.......... 652

11

25

..........

3160

1772

120

260

89*

31

0.86

2.9

200 (228*)

34

3.6

25

Ergotamine

125

171

..........

Dihydroergotamine

316

..........

> 10,000

..........

Methylergonovine Methysergide

Bromocriptine Yohimbine

398

92

22

27

a For the s t r u c t u r e s of the compounds discussed in this article see Figure 3. b Values t a k e n from B e e r et al. [17]. c Values t a k e n from A d h a m et al. [6]. d Values t a k e n from W e i n s h a n k et al. [22]. * Ki values t a k e n from Zgombick et al.

[4].

150

/ MeO~~

NH2

NMe2

NH2

N

H 5-MeOT

N

H 5-BnOT

5-MeO-DMT

HO

H2N

.o.~~~Me

Me H

H 5-CT: R= H DP-5-CT: R= npr

N

2-Me-5-HT

jJ

Sumatriptan

a.Me-5-NT

H ~XN

NMe2 MeHN._A ~

NH2

NH2

NR2 O

.L

MeO ~~N

H

H RU-24,969

H

Pindolol

H HO

H

TFMPP

8-OH-DPAT

1-NP

H _Nr--~N__~

F

~

N~N O

Ketanserin

k__/\ \OMe NAN-190

Figure 3" Structures of compounds discussed in this chapter

O

O

151

N

SMe

~NMe Metitepin

Mianserin

Me Cyproheptadine ,,,~

HO.

0

H ~ N ~

,170 HC~ O."~N ~ N " Me H

N'MeH

H

Ergotamine

..,,,~ ipr

~

,.Me

H

R Methylergonovine: R= H Methysergide: R=Me

He O~I~'N~ ip~ H

Ph

O

N"•Me

.,,~ Ph

~N

..Me H

Br H

Dihydroergotamine

Bromocriptine

H

MeOOC OH Yohimbine

Figure 3 (continued)" Structures of compounds discussed in this chapter

H

152 hydroxy group of 5-HT (to give 5-methoxytryptamine, 5-MeOT) for both 5-HT,z and 5-HT,v , a transformation which has little consequence for 5-HT,D receptors. The fact that tryptamine (T) binds with the same affinity as 5-MeOT suggests that the 5-hydroxy functionality in 5-HT is acting as a hydrogen bond donor (and not an acceptor) group at 5-HT,~.,I.~ receptors (with Ser186 of 5-HT,E or Ser185 of 5HT1F in TM V?) [23-25] but as a hydrogen bond acceptor group at 5-HTID receptors [26] (compare 5-HT, T and 5-MeOT). In marked contrast to 5-HT,D receptors, 5carboxamidotryptamine (5-CT) also has low affinity for 5*HT,E,,F receptors, a result which would appear to indicate that the excellent hydrogen bond acceptor capability of its carboxamido group is being utilized when binding to 5-HT1D receptors but is not relevant for binding at 5-HT1E,,F. Interestingly, sumatriptan, which has comparably low affinity to 5-CT for the 5-HT,~ receptor, binds with high affinity to 5-HT~. Thus, at least in the latter case, effective complementarily (hydrogen bond interactions?) can be achieved with functionalities which are further away from C~ of the tryptamine. It is also noteworthy that large arylalkyl groups are tolerated at C~ of the tryptamine (compare 5-BnOT and 5-MeOT) although, by virtue of the similar affinities, the benzyl group of 5-BnOT does not contribute to binding. By direct analogy to 5-HT,D receptors, 2-methylation of the indole nucleus, as in 2-Me-5-HT, greatly reduces the affinity for both 5-HT,E and 5-HT,F receptors (70 to 80-fold) whereas ~-methylation of the ethylamino side chain (compare a-Me5-HT and 5-HT) is somewhat less detrimental (10 to 20-fold). Assuming that the ergot derivatives bind at the same site in the receptor as 5-HT, comparison of methylergonovine and methysergide, would appear to suggest that N 1methylation of tryptamines might be slightly unfavourable (2-fold) for 5-HT,E receptors but of little consequence for 5-HT,F. Similar trends were reported for 5-HT1D receptors [27] and should be easy to confirm with commercially available 1methyltryptamine. There is the indication, however, that not all modifications result in reduced affinities. In particular, N,N-dimethylation of 5-MeOT to give 5-MeOT-DMT improves the affinity to 5-HT,E by 6-fold to 5-HT,~ by 30-fold. The slightly detrimental effect of larger, N,N-di-alkyl groups for 5-HT~E,~ (compare 5-CT and DP-5-CT) could either reflect a limited space being available for binding at this part of the receptor (steric) or be a direct consequence of the increased conformational freedom of these groups (entropic). Moreover, replacement of the ethylamino side chain by a 1,2,5,6-tetrahydropyridine moiety affords a 10-fold improvement in 5-HT~E binding affinity (compare RU-24,969 and 5-MeOT). The binding of RU-24,969 to 5-HT,~ receptors has not been reported and is awaited with great interest. The fact that ergotamine and dihydroergotamine bind to 5-HT,E and 5-HT,F receptors, although with less affinity than to 5-HT1D, shows that there are regions of bulk tolerance at both receptors. The poor affinity of bromocryptine for 5-HT~z receptors may reflect the detrimental effect of 2-substitution on the indole nucleus as noted above. The presence of an indole moiety does not appear to be a requirement in order to produce moderate to high affinity 5-HTjE.,F receptor ligands. Thus, although a

153 simple arylpiperazine such as TFMPP has micromolar affinities for both receptors, the combination of a naphthyl nucleus and a piperazine ring as in 1napthylpiperazine (1-NP) results in a good mimic of the tryptamine core (compare 1-NP and T). In the case of the 5-HTI~. receptor, this replacement even leads to some 40-fold increase in affinity. The more elaborate 2-methoxyphenylpiperazine analogue NAN-190 also has respectable (200nM) affinity for the 5-HT1F receptor. Other unselective, non-indolic 5-HT receptor ligands which also bind with moderate affinity to 5-HT~E include the structurally related tricyclic/tetracyclic compounds metitepin, mianserin and cyproheptadine. Finally, ~-adrenergic agents such as pindolol bind with very low affinity to 5HTI~,~F receptors. This is perhaps not surprising in view of the fact that, in contrast to 5-HT~A and 5-HT~ receptors which bind [~-adrenergic antagonists with high affinity [28], the 5-HTiE and the 5-HT~.~ receptors lack a key residue in the seventh transmembrane domain (Asn385 in 5-HT1A and Asn351 in 5-HTm) which has been suggested to participate in hydrogen bond interactions with the aryl oxygen of [3-blockers. In the 5-HTtE receptor this Asn residue is replaced by Thr330 and by Ala333 in the 5-HT~ receptor. Indeed, it has recently been shown [29] that replacement of these two residues by Ash affords 5-HT~ and 5-HT~ mutants which bind pindolol and other ~-blockers with significantly improved affinities (>100-fold), although the binding of the endogenous neurotransmitter 5-HT is not affected. In conclusion, although no selective ligands are yet available for either 5-HT m or 5-HT~ receptors, the steadily increasing understanding of their molecular architectures through the combined utilization of pharmacophore mapping, receptor modelling and site directed mutagenesis studies will no doubt lead to the discovery of useful pharmacological tools in the near future. FUNCTIONAL ASSAYS Based on the high degree of sequence homology among the 5-HTl-like receptors and the characteristic long third intracellular loop and short carboxyl-terminal domain of both the 5-HT~ and 5-HT~ receptors it would be predicted that both subtypes are negatively coupled to adenylyl cyclase activity. This prediction was supported by the original characterisation of the cloned 5-HT1E and 5-HTI~ receptors expressed heterologously in various mammalian cell lines. Activation of 5-HT~E receptors in Ltk', Y-1 or HEK cells resulted in a relatively weak (20-35%) inhibition of forskolin-stimulated cAMP levels [2,4,9]. This weak inhibition may be due to a lack, or low levels, of the appropriate G-protein or other component of the signal transduction system being present in these cell lines. Indeed, increased levels of inhibition were observed by reducing the levels of free Mg ~ [9] or by expressing the 5-HTj~ receptor in BS-C-1 cells [30]. Intriguingly, in BS-C-1 cells expressing high levels of the receptor (5 pmol/mg of protein) activation of the receptor led to both the inhibition and potentiation of forskolin-stimulated cAMP accumulation. Pretreatment of cells with pertussis toxin or cholera toxin eliminated agonist induced inhibition and potentiation of cAMP levels respectively.

154 The potentiation of forskolin-stimulated cAMP accumulation appears to be a direct effect as no changes in PI metabolism or Ca2+mobilization were observed. Agonists displayed higher affinity for the inhibitory response suggesting an interesting potential mechanism of regulation of these receptors in which higher 5-HT concentrations would counter the initial inhibition of cAMP levels by stimulating cAMP production. The physiological significance of this finding is unclear. It seems to be a receptor density-dependent feature as cell lines expressing somewhat less receptors (2 pmol/mg of protein) only couple to the inhibition of cAMP levels. However, as the authors point out, there is likely to be both a high concentration of endogenous ligand and a high density of receptors present at the synapse. This is not the first description of 5-HTl-like receptors apparently coupling to more than one second messenger system. For example, cloned 5-HTxD receptors were recently reported to couple to both inhibitory adenylyl cyclase activity and the elevation of intracellular Ca2§ levels via pertussis toxin-sensitive G-proteins [31]. Further enlightenment awaits the development of sub-type specific ligands. The overlapping pharmacology of the 5-HT1D and 5-HTxE / 5-HT~F receptors makes it impossible, at the moment, to unambiguously identify the in vivo function of 5HT~z and 5-HT1F receptors. This problem is compounded by the fact that most ligands developed for these receptors have been agonists and are subject to the problems of receptor reserve in interpreting data. Ideally, subtype specific antagonists will be developed to give a clearer understanding of the functional roles of these receptors. Alternatively, the effects of antagonists can be mirrored by the development of transgenic mice devoid of particular receptors or by the application of antisense oligonucleotides to investigate the function(s) of these receptors in vivo. THERAPEUTIC APPLICATIONS Agonists The successful clinical use of s u m a t r i p ~ as an acute treatment for migraine headache has intensified interest in its mode of action. It was originally thought to be a relatively selective 5.HT~D receptor agonist (see previous chapter). However, that hypothesis turned out to be an oversimplification as sumatriptan also demonstrated affinity for the 5-HTu~, 5-HT m and 5-HT~v receptors in addition to the two 5-HT~D subtypes. It has poor affinity for 5-HTaE receptors so its action is unlikely to be mediated by that receptor subtype. The recent development of the 5-HT1D receptor antagonist, GR127935, may help clarify the role of these various receptor subtypes although the selectivity of this antagonist over the 5-HT~z and 5-HT~F receptors has not yet been reported. However, it may yet be that 5-HT~E receptor agonists are also useful in the treatment of migraine. The full elucidation of which 5-HT receptor subtypes are present on the target tissues of an antimigraine drug and their role(s) in mediating the proposed desirable effects of such a drug (cerebral vasoconstriction, inhibition of plasma extravasation) remains to be discovered. The role of 5-HT1v receptors in particular will be interesting as it has been shown to have a high affinity for sumatriptan (I~. 23nM) and a vascular

155 distribution [6]. It may also be that some of the less desirable effects of sumatriptan (coronary vasoconstriction etc.) could be reduced by avoiding activation of certain subtypes, therefore the distribution of 5-HT receptors in nontarget tissues such as coronary artery will also be of great interest.

Antagonists There are no clear therapeutic indications for 5-HT1E or 5-HTI~ antagonists so far. However, as this chapter has emphasized, the lack of selective antagonists makes it difficult to assign particular functions to a given receptor subtype. In general then, it appears that anything a 5-HT1D antagonist might be proposed for may also be a potential target for a selective 5-HTm or 5-HTIF compound. It is thought that treatment with selective serotonin re-uptake inhibitors (SSRIs), such as paroxetine or fluoxetine, leads to the facilitation of 5-HT neurotransmission. This is the proposed mechanism of action for the antidepressant properties of this class of drug. An alternative method of facilitating 5-HT neurotransmission is to block the inhibitory terminal 5-HT autoreceptor, normally activated by the release of 5-HT. It is proposed that blockade of this autoreceptor would stop the inhibition of 5-HT release, thus increasing synaptic 5-HT concentration and facilitating neurotransmission. The question is, which of these receptor subtypes can act as an autoreceptor? There is some evidence suggesting that a 5-HT~D receptor subtype is the autoreceptor (see previous chapter) and the expression of mRNA encoding both the 5-HT1D~ and 5-HTIDI3 receptors in the guinea pig dorsal raphe nucleus adds support to this idea. However, 5-HTI,.~ mRNA has also been found in this nucleus and the presence of 5-HT~E has not been excluded [14]. Therefore, it is possible that both 5-HT~,~ and 5-HT1~ receptors may act as autoreceptors and are still potential therapeutic targets for a novel antidepressant. However, no mutations in the human 5-HT~, receptor gene of patients suffering from schizophrenia and bipolar affective disorder were detectable, indicating that 5HTlr receptors are not commonly involved in the etiology of these diseases [39]. Interestingly, it appears that fluoxetine is now being successfully used to treat some patients suffering from anxiety. The mechanism of action of this effect is unclear. It could be that an autoreceptor antagonist could mimic this effect or it may be that fluoxetine treatment is causing down regulation of a postsynaptic receptor. Whichever is the case, the possible role(s) of 5-HTm and 5-HT~F receptors in anxiety should also be investigated.

REFERENCES 1 2 3 4

Leonardt S, Herrick-Davis K, Titeler M. J Neurochem 1989; 53: 465-471. McAllister G, Charlesworth A, Snodin C, Beer MS, et al. Proc Natl Acad Sci 1992; 89: 5517-5521. Levy FO, Gudermann T, Birnbaumer M, Kaumann AJ, et al. FEBS Lett 1992; 296:201-206. Zgombic JM, Schechter LE, Macchi M, Hartig PR, et al. Mol Pharm 1992; 42: 180-185.

156 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Amlaiky N, Ramboz S, Boschert U, Plassat JL, et al. J Biol Chem 1992; 267: 19761-19764. Adham N, Kao HT, Schechter LE, Bard J, et al. Proe Natl Acad Sci 1993; 90: 408-412. Lovenberg TW, Erlander MG, Baron BM, Racke M, et al. Proe Natl Acad Sci 1993; 90: 2184-2188. O'Dowd BF, Leikowitz RJ, Caron MG. Ann Rev Neurosci 1989; 12: 67-83. Gudermann T, Levy FO, Birnbaumer M, Birnbaumer L, et al. Mol Pharm 1993; 43: 412-418. Fargin A, Raymond JR, Lohse MJ, Kobilka BK, et al. Nature 1988; 335: 358360. Hamblin M, Metc~f M. Mol Pharm 1991; 40: 143-148. Voigt MM, Laurie DJ, Seeburg PH, Bach A. EMBO J 1991; 10: 4017-4023. Julius D, MacDermott AB, Axel R, Jessell TM. Science 1988; 241: 558-564. Pritchett DB, Bach AWJ, Wozny M, Taleb O, et al. EMBO J 1988; 7: 41354140. Doolittle R. In: Of Urfs and Orfs. University Sci Books 1986; 37-47. Beer MS, Stanton JA, Hawkins LM, Middlemiss DN. Eur J Pharm 1993; 236: 167-169. Beer MS, Middlemiss DN, McAllister G. Trend Pharmac Sci 1993; 14: 228-231. Miller KJ, Teitler M. Neurosci Lett 1992; 136: 223-226. Bruinvels AT, Landwehrmeyer B, Gustafson EL, Durkin MM, et al. Neuropharmacol 1994; 33: 367-386. Humphrey PPA, Feniuk W. Trends Pharmac Sci 1991; 12: 444-446. Beer MS, Stanton JA, Bevan Y, Chauhan NS, et al. Eur J Pharmacol 1992; 213: 193-197. Weinshank RL, Zgombic JM, Macchi MJ, Branchek TA, et al. Proc Natl Acad Sci 1992; 89: 3630-3634. Hibert MF, Tr~_~mpp-Kallmeyer S, Bruinvels AT, Hoflack J. Mol Pharm 1991; 40: 8-15. Tnmlpp-Kallmeyer S, Bruinvels AT, Hoflack J, Hibert MF. Neurochem Int 1991; 397-406. Lee NH, Kerlavage A. Drugs News and Perspectives 1993; 6: 488-497. Street I.J, Baker R, Castro JL, Chamberts MS, et al. J Med Chem 1993; 36: 1529-1538. Glennon RA, Ismaiel AM, Chaurasia C, Titeler M. Drug Dev Res 1991; 22: 2536. Glennon RA, Westkaemper RB. Drugs News and Perspectives 1993; 6: 390405. Adham N, Tamm JA, Salon JA, Vaysse PJJ, et al. Neuropharmaeol 1994; 33: 387-392. Adham N, Vaysse PJJ, Weinshank RL, Branchek TA. Neuropharmacol 1994; 33: 403-410. Zgombick JM, Borden LA, Cochran TL, Kucharewicz SA, et al. Mol Pharm 1993; 44: 575-582. Hoyer D, Martin GR. Behav Brain Res 1996; 73: 263-268.

157 33 Stanton JA, Middlemis DN, Beer MS. Neuropharmacol 1996; 35: 223-229. 34 Rhodes VLH, Reilly YC, Bruinvels AT. Brit J Pharmacol 1995; 114: Proc. Suppl 364P. 35 Waeber C, Moskowitz MA, Naunyn Schmiedeberg's Arch Pharmacol 1995; 352: 263-275. 36 Pascual J, Del Arco C, Romon T, Del Omo E, et al. Eur J Pharmacol 1996; 295: 271-274. 37 Pascual J, Del Arco C, Romon T, Del Omo E, et al. Cephalalgia 1996; 16: 317322. 38 Bouchelet I, Cohen Z, Case B, Seguela P, et al. Mol Pharm 1996; 50: 219-223. 39 Shimron-Abarbanell D, Harms H, Erdman J, Albus M, et al. Am J Med Genet Neuropsych Genet 1996; 67: 225-228.

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Chapter 6

5-HT2A , 5-HT2B and 5-HT2c RECEPTORS 5-HT2A , 5-HTEB and 5-HT2c Receptor ligands The 5-HT2-type receptor family 5-HT2 -type Receptor antagonists: (potential) therapeutics

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Serotonin Receptors and their Ligands B. Olivier, I. van Wijngaarden and W. Soudijn (Editors) O 1997 Elsevier Science B.V. All rights reserved.

161

5-HT2A, 5-HT2Band 5-HT2c Receptor ligands I. van Wijngaarden 1) and W.Soudijn 2) 1)Solvay Duphar B.V., P.O.Box 900, 1380 DA Weesp, The Netherlands. 2~Leiden/Amsterdam, Center for Drug Research, P.O.Box 9502, 2300 RA Leiden, The Netherlands.

INTRODUCTION For the 5-HT2 receptors many potent ligands, belonging to different chemical classes, such as phenylalkylamines, indoles, ergots, 4-aryl(alkyl) piperidines, 4aryl(alkyl)piperazines and diarylurea, are available. However, the majority of these compounds is not selective and displays besides affinity for 5-HT 2 receptors also affinity for other 5-HT subtypes and/or other neurotransmitter receptors (for reviews see [1-6]. Moreover, the 5-HT2A, 5-HT2B and 5-HTec receptors are closely related making it rather difficult to design ligands selective for one of the subtypes. Many of the well-known 5-HT 2ligands, originally thought to be selective for 5-HT2A receptors, display high affinity for all three subtypes. Representative examples are the (partial) agonists DOI and mCPP and the antagonists methysergide, ritanserin and mianserin (Table 1). But not all 5-HT2 ligands show high affinity for the 5-HT2B and 5-HT2c receptors. The 5-HT 2 antagonist spiperone for example retains selectivity for the 5-HT2A receptor (Table 1). However, spiperone is not selective with respect to dopamine-D 2 receptors (I~.=0.71 nM) and al-adrenoceptors (I~=100 nM) [2]. At present only a small number of selective ligands for the 5-HT2A or 5-HT2B receptor has been published. For the 5-HT2c receptor these ligands are still lacking. Point mutations in cloned and expressed 5-HT2A receptors provides new information on the molecular 5-HT2A ligand-receptor interactions.

5-HTzA, 5-HTzB and 5-HTzc RECEPTOR LIGANDS 4-Arylalkylpiperidines The best known member of this class is ketanserin (Table 2). For more than a decade ketanserin is the most widely used tool to characterize 5-HT2 receptors [7]. Ketanserin displays a high affinity for the 5-HT2A receptor, a moderate affinity for the 5-HT2c receptor and a weak affinity for the 5-HT2B receptor (Tables 1,2). Ketanserin is not selective for the 5-HT2A receptor and binds with high affinity to al-adrenoceptors and moderate affinity to histamine receptors [2]. In functional tests ketanserin acts as an antagonist.

162 Table 1 Affinity of 5-HT2 ligands for cloned 5-HT2^, 5-HT2n and 5-HT2c receptors 5-HT2A DOI

oc

1.00

J~

5-HT~B 5-HT~c 27.5

6.46

NH2

OC

mCPP

41.0 ~ _

f-"X

N

26.8

24.0

NH

/ CI Methysergide

c- " ~ ~ ~

N "~ H

I C

3.98

7.94

1.58

0.01

5.01

0.25

C-N- -C-OH c-c

F

Ritanserin o

F

Mianserin

N

N-C

3.98

20.0

5.01

163 Table 1 (continue) Affinity of 5-HT2 ligands for cloned 5-HT2A, 5-HT2B and 5-HT2c receptors 5-HTg^ F

Ketanserin

5-HT~B

5-HT2c

3.16

630

200

1.00

1585

>1000

o

Spiperone o F

o

C--C3--N

Data, expressed as Ki in nM are from [51] (DOI, mCPP: 5-HT2A, 5-HT2c receptors rat); [18] (DOI, mCPP: 5-HT2B receptor rat); [52] (methysergide, ritanserin, mianserin, ketanserin, spiperone: 5-HT2A, 5-HT2B and 5-HT2c receptors human). The quinazolindione ethyl part of ketanserin can be replaced by a variety of other heteroaryl containing side chains all with little effect on 5-HT2A affinity (Table 2). Replacement of the quinazoline nucleus by a pyridopyrimidine results in pirenperone, a potent non-selective 5-HT~A antagonist. The benzisothiazole-3carboxamide ethyl derivate is somewhat less active [8]. Combining benzoylpiperidines with the tetrahydrocarbazolone methyl moiety of the 5-HT3 antagonist ondansetron is highly favourable for 5-HT2A receptor affinity [9]. Interestingly affinity for 5-HT8 receptors is absent. In functional tests the compound acts as an antagonist. The (-)-enantiomer is 148 times more potent than the (+)-antipode (Table 2). An unusual structure is the naphthosultam derivative possessing high affinity for 5-HT2A receptors [10]. This napthosultam is more selective than ketanserin with respect to al-adrenoceptors (Table 2).

154 Table 2 5-HT2A ligands derived from 4-benzoylpiperidines

5-HT2A 5-HT2B 5-HT2c a, Ketanserin

N ~

~

I

j

~

H1

D2

Ref. [2~18]

FI 2.00

3548

100

7.94

100

398

1.58

2138

12.6 8.30

2.19

16.0 [3~51]

n.d

n.d

15.7" n.d

9.36+ 7.19+

n.d n.d

n.d n.d

8.08+ 6.74+ n.d 7.28+ 6.82+ n.d

[9] [9]

0.1"

n.d

n.d

7.9*

[10]

O Pirenperone

o N

o

17.0"

286* [8]

H

o (-) (+) I CH3 o II s--o

i

Data are expressed as I~. in nM; ICso in nM* or pKB+.

n.d

41"

165 Further structure-activity relationships studies of ketanserin show that the 4(4-fluorobenzoyl) piperidine moiety hardly binds to the 5-HT2A receptor [11] (Table 3). N-substitution with small alkyl groups improves the affinity slightly. However the n-pentyl analogue displays a good affinity for the 5-HT2A receptor (Table 3). High affinity is obtained in the phenylethyl, phenylbutyl and butyrophenone analogues, being as potent as ketanserin [12]. Ring opening of the quinazolinone nucleus into the corresponding benzamide is less favourable than ring opening into the corresponding phenylurea (Table 3). These results show that the quinazolinone ring is not essential for binding to 5-HT2A receptors. Table 3 5-HT2A ligands derived from 4-benzoylpiperidines

L

Ketanserin

H/F

Q*-C2 H C, C2 C5 Phe-C2 Phe-C4

Phe-C(=O)-C3 Phe-C(=O)NH-C2 PheNH-C(=O)NH-C2

F H F F H F F F H F H F F F

5-HT2A 5-HT2c

3.5 6.5 430 125 600 260 30 8.5 9.6 5.3 10 6.5 16 4.3

50 760 1100 1510 i.a 3160 n.d 145 800 620 2400 350 1610 200

5-H~' /5-HT2c

Ref

14 115 2 12

[ 11] [11] [11] [11] [12] [11] [12] [12] [12] [12] [12] [ 11] [11] [11]

12 17 83 120 240 50 100 50

Data are expressed as I~. in nM. *=Q=quinazoline-2,4-dione; i.a=inactive; n.d=not determined.

166 All compounds display a weak to very weak affinity for 5-HT2c receptors. Striking is the decrease in 5-HT2c receptor affinity in the desfluoro-analogues (Table 3). From the series is the N-(4-phenylbutyl)-4-(benzoyl) piperidine analogue the most selective 5-HT2A ligand (selectivity ratio 5-HT2A/5-HT2c = 240), being 17 times more selective than ketanserin (selectivity ratio 5-HT2A/5-HT2c = 14) (Table 3). Replacement of the benzylic carbonyl oxygen of ketanserin by hydrogen or phenyl reduces affinity 5 fold [11,12]. For the phenylethyl- and phenylbutyl analogues is the decrease in affinity 2 and 12 times respectively [12]. Reduction of the benzylic carbonyl group of desfluoro-ketanserin decreases the affinity for 5-HT2A receptors 100 times [11] (Table 4). The N-ethyl substituted analogue even lost all affinity. However contrary to the expectations high affinity is maintained in the phenylethyl derivatives [12] (Table 4). Table 4 5-HT2A ligands derived from 4-carbinolpiperidines OH

R

R

5-HT2A 5-HT2c 5 - ~ /5-HT2c

MDL MDL MDL MDL

Q*-C2 C2 28,161 Phe-C2 11,939 Phe-C2 26,508 Phe-C2 100,907 R(+) 4FPhe-C2 Phe-C4 Phe-C4

H 655 4-F i.a 4-F 3.0 H 2.5 2,3-di-OC 2.3 2,3-di-OC 0.36 4-F 126 H 265

i.a n.d 1520 830 170 105 6600 i.a.

Data are expressed as ~ in nM. *Q=quinazoline-2,4-dione; i.a=inactive; n.d=not determined.

460 330 74 292 52

Ref [11] [11] [12] [12] [13] [15] [12] [12]

167 These compounds (MDL 28,161 and MDL 11,939) are even 3-4 times more potent and 4-27 more selective than the corresponding ketones (cf Table 3). The 2,3dimethoxy analogue MDL 26,508 is as potent as but less selective than MDL 28,161 and MDL 11,939 [13]. MDL 11,939 is the first truly selective 5-HTzAligand. Besides the high affinity for 5-HT2A receptors MDL 11,939 displays low or negligible affinity for the other 5-HT receptors as well as other neurotransmitter receptors tested [14, 13]. In functional tests MDL 11939 behaves as an antagonist [14]. MDL 11,939 is a racemate. Resolution of the 4-fluorophenylethyl analogue of MDL 26,508 into its enantiomers shows that the 5-HT~ affinity resides predominantly in the (R)-enantiomer MDL 100,907. MDL 100,907 is even more potent and more selective than MDL 11,939 [15, 73]. Lengthening the phenylethyl side chain of MDL 28,161 and MDL 11,939 to phenylbutyl results in a significant drop in affinity [12] (Table 4). This decrease in affinity is absent in the corresponding ketones (Table 3). The non-parallel structure affinity relationships between the N-substituted benzoylpiperidines and corresponding phenylcarbinolpiperidines indicate that both series bind differently to the 5-HT~ receptor. Bioisosteric to the 4-benzoylpiperidines are the 3-(4-piperidinyl)-l,2benzoxazoles. The best known member of this class is risperidone, a potent but non-selective 5-HT2A antagonist [ 1 6 ] (Table 5). Replacement of the tetrahydropyridopyrimidinone ethyl side chain of risperidone by (aryloxy)propyl results in iloperidone [17]. This compound is less potent at 5-HT2A receptors and more active at a~-adrenoceptors than risperidone (Table 5). The benzisoxazole-3-earboxamide ethyl derivative (compound 1) displays the same affinity for 5-HT~ receptors as the analogous 4-benzoylpiperidine [8] (Tables 5, 2). In functional tests the compounds act as antagonists.

Indoles The prototype of this class is the neurotransmitter serotonin (5-HT). 5-HT displays a high affinity for the high affinity state (KH) of all 5-HT 2 receptor subtypes (Table 6). The affinity for the low affinity state of the receptors is significantly lower [3]. 5-HT is not selective with respect to other 5-HT receptors [2, 41. The affinity of 5-HT for the 5-HT2B receptor is higher at 0~ than at 37~ indicating that the binding is enthalpy driven [18]. The hydroxyl group of 5-HT is not essential for high affinity and can be replaced by methoxy, halogen or lower alkyl with little effect on the affinity for 5HT2A, 5-HT2B and 5-HT2c receptors. Replacement of the hydroxylgroup by hydrogen (T) or carboxamide (5-CT) lowers affinity for all 5-HT 2 receptors (Table 6). Methylation of 5-HT at the C-1 position is unfavourable for the 5-HT2A and 5-HT2B receptors, but not for the 5HT2c subtype. A methyl group at the C-2 position of 5-HT is not tolerated by any of the 5-HT 2 receptors [3]. Alkylation of the side-chain of 5-HT at the a-position (a-Me-5-HT) has little effect on the affinity of all sub-types (Table 6).

168 Table 5 5-HT2^ ligands derived from 3-[piperidinyl]-l,2-benzisoxazoles

_0 o

Risperidone

Iloperidone

H3C e,~~ 0

5-HTu 5-HT2c

al

oh

D2

0.16

2.4

7.5

3.1

0.4*

n.d

54

n.d

n.d

197

n.d

48

3.1

n.d

17.6

n.d

H1 2.1

~

,,

0

OH3

Compound 1

104

o

o~N

OH3

Data expressed as I~. in nM or ICso* in nM are from [16] (risperidone); [17] (iloperidone) and [8] (compound 1).

Introduction of one or two alkyl groups on the basic nitrogen atom of 5-HT or 5-MeO-T does not influence the 5-HTu affinity but lowers the affinity for 5-HT2B receptors [19, 20, 18]. The 5-HT2^ receptor affinity is even enhanced in the N-(4bromobenzyl) derivative [20] (Table 6). The N-benzyl analogues display a weak affinity for the 5-HT2c receptor (Table 6). Unfortunately [3H]-mesulergine was used as radioligand. As no data of agonism or antagonism are reported yet, no conclusions on selectivity can be drawn. Replacement of the hydroxyl group of a-Me-5-HT by a 2-thienylmethoxy group results in BW 723C86 claimed as a selective 5-HT2B agonist [69] (Table 6).

169 Table 6 5-HT 2 ligands derived from tryptamine

/ R4

R I ~ ~ R 2 N~R3 R1 T 5-HT 5-F-T 5-C1-T 5-Br-T 5-Me-T 5-MeO-T 5-CT

H OH F C1 Br Me MeO NH2CO r OH BW 723 C86 C4H3SCH20 Br OH MeO MeO MeO RU 24969 MeO

R2

Ra,R4

5-HT2A

H H,H 37.2 H H,H 4.4O H H,H 6.03 H H,H n.d H H,H n.d H H,H 5.89 H H,H 4.78 H H,H 87.1 Me H,H 8.71 Me H,H <5.4*** H H,n-Pr n.d H di-n-Pr 1.00 H di-n-Pr 7.08 H H,C-Ph 5.3 H H,C-Ph-4-Br 0.1 H THP**** 151

5-HT2B

5-HT2c

113" (3.75)** 16.6 10.2 (0.19) 3.10 5.7 (n.d) 3.72 5.1 (n.d) n.d 39.3 (0.42) n.d 32.8 (0.95) 5.62 9.2 (0.26) 7.08 150 (1.48) 208 10.5 (0.45) 5.12 7.9*** >126 131 (17.2) n.d 61.0 (7.26) n.d 169 (14.1) n.d n.d 370 n.d 100 17.1 47.9

Affinities for the agonist-labeled 5-HT 2 receptors, expressed as I~. in nM or pECso*** values are from [51] (5-HT2A, 5-HT2c); [18] (5-HT2B); [69] (BW723C86); [19] (5-HT2A, 5-OH and 5-MeO-N1,Nl-dipropyltryptamine); [20] 5-HT2A (ago-label), 5-HT2c (anta-label); arylalkyltryptamines. *I~. at 37~ , 9* * ~ at 0~ * * * * THP=tetrahydropyridine.

Ring closure of the methoxy group of 5-MeO-T into a pyrano [3,2-e] indole has little effect on the affinity for 5-HT2A and 5-HT2c receptors. Ring closure to a pyrano [2,3-f] indole is unfavourable for 5-HT2A and 5-HT2c receptor affinity [21].

170 Incorporation of the alkylamine side-chain of 5-MeO-T into a 4-substituted tetrahydropyridine ring results in RU 24969, a non-selective 5-HT ligand [2, 18, 4] (Table 6).

Table 7 5-HT2A ligands derived from 1-phenyl-3-(4-piperidinyl)-lH-indoles o

~ / N ~ R

N \

9k

N--R4 /

R3 R2 Rl

LU 23-086

H F C1 CH 3 CFs i-Pr CH3 CHs CHs CH 3 CH~ H

R2

4-F 4-F 4-F 4-F 4-F 4-F H 2-F 3-F 4-F 4-C1 4-F

R3

R4

H H H H H H H H H H H CH 3

H H H H H H i-Pr i-Pr i-Pr i-Pr i-Pr H

5-HT2A

0.72 0.73 1.4 0.82 1.7 18 2.0 2.5 11 1.6 6.0 0.59

D2

18 36 56 270 260 2600 500 730 3200 190 620 380

(Z1

3.0 2.2 9.6 24 33 250 120 370 670 85 450 76

Data, expressed as ICso in nM are from [22]. An interesting series of 5-HT2 antagonists are the 1-[2-[4-(1H-indol-3-yl)-lpiperidinyl]ethyl]-2-imidazolidinones [22] (Table 7). The prototype is Lu 23-086 a potent, but non-selective 5-HT2A ligand. The 6-position of the indole nucleus can be substituted with a fluorine or chlorine atom with little effect on affinity and selectivity. Introduction of a methyl group at the 6-position is highly favourable for potency and selectivity (Table 7). The 6-trifluoromethyl analogue is somewhat less selective. A bulky group such as isopropyl is not well tolerated. Substitution of the imidazolidinone ring with isopropyl lowers the 5-HT2A receptor affinity 2-fold, but increases the selectivity with respect to al-

171 adrenoceptors by a factor 3 (Table 7). The 1-phenyl ring, which is essential for high 5-HT2A receptor affinity, may be unsubstituted or substituted at the 2 and 4 position with fluorine. The 4-chloro and 3-fluoro analogues are less active. Introduction of a methyl group at the 2-position of the indole nucleus favours potency and selectivity. This compound is the most selective one of the series (ratio D2/5-HT2A = 644; (z,/5-HT2A = 129) (Table 7). Table 8

5-HT2Aligands derived from 1-phenyl-3-(4-piperidinyl)-lH-indoles O

2, CI

R,

LU 26-042

R2

x_--./ M_.__../ H CH3 CH~

CHa H H

R3 H i-Pr CH3 CH3 i-Pr

5"HT2A 1.4 1.5 1.3 1.6 2.2

D2 56 130 27 82 74

al 9.6 70 29 16 26

Data, expressed as IC~o in nM are from [22]. Ring opening of the imidazolidinone into the corresponding urea has no influence on the 5-HT2A receptor affinity but lowers selectivity (Table 8). Ring opening of the piperidine ring lowers the affinity for 5-HT2c receptors. The 2-aminoethyl derivative 1-[3-[[2-[6-chloro-l-(4-fluorophenyl)-lH-indol-3-yl] ethyl] methylamino]propyl]-2-imidazolidinone for instance is about 80 times more potent at 5-HT2A than 5-HT2c receptors [74]. Lu 26-042 which displays a high affinity for 5-HT2A and 5-HT2c receptors [75] was selected for further pharmacological evaluation. Interchanging the nitrogen atom, and the C-3 atom of the indole nucleus results in the isosteric 3-phenyl-l-(4-piperidinyl)-lH-indoles [23] (Table 9). These

172 compounds are slightly less potent but more selective with respect to dopamine-D 2 and a,-adrenoceptors t h a n the corresponding 1-phenyl-3-(4-piperidinyl)lH-indoles (cf. Table 8). The SAR in both series is very similar. Substitution of the indole nucleus at the 5- a n d / o r 2-position with methyl results in potent and selective 5-HT2A ligands. The most selective compound is the 2,5-dimethyl substituted analogue (selectivity ratio's D2/5-HT2^=2029 and a,/5-HT2^=676) (Table 9).

Table 9 5-HT2A ligands derived from 3-phenyl-3-(4-piperidinyl)-lH-indoles 0

N

N ~

N

2,

I,

N---R 5 s

I

R3-" R4 R

R1

H F C1 CH 3 CF 3 CH3 CH 3 CH 3 CH 3

R2

R3

R4

R5

5-HT2~

D2

H H H H H H CH 3 H H

\ \ \ \ \ ~ \ H C

] 7 / 1 / / J CH3 H

H H H H H i-Pr H CH3 CH 3

3.1 3.2 5.7 1.6 9.3 2.7 3.4 2.3 2.2

120 250 410 920 2500 1100 6900 260 1200

(zl

6.3 19 67 71 190 280 2300 89 54

Data, expressed as ICso in nM are from [23].

The piperidine group can be replaced by 1,2,3,6-tetrahydro-4-pyridinyl without loss in affinity and selectivity. Also the replacement of the imidazolidinonylethyl side chain by a methyl group has little effect on the affinity and selectivity for 5-

173

HT2A receptors [23]. In vivo the compounds act as potent 5-HT2A antagonists. Data on the affinity for 5-HT2c receptors have not been published yet. Novel 5-HT2A ligands were obtained by incorporation of the tryptamine structure into a bridged 7-carboline. The most potent compounds of the series are substituted on the basic nitrogen atom with a butyrophenone chain [24]. The 7S,10R enantiomer displays the highest affinity for the 5-HT2A receptor. The 7R,10S enantiomer is slightly less active but more selective with respect to dopamine-D 2 receptors [25] (Table 10).

Table 10 5-HT2A ligands derived from bridged 7-carbolines

,o

II N -'- (CH2)n -- C

F

I R

7S,10R 7R,10S 7S,10R 7R,10S rac* rac

R

n

H H H H CH3 Ph

3 3 4 4 3 3

5-HT2A

0.80 2.56 1.87 109 2.83 330

D2

D2 / 5-HT2A

99.3 1440 2.59 755 287 840

124 562 1.38 6.93 101 2.55

Data expressed as I~. values in nM are from [24,25]. *desfluoroindole. Lengthening the side-chain with one methyl group has little effect on the 5HT2A affinity of the 7S,10R enantiomer. However, the affinity for dopamine-D2 receptors is significantly increased, making this enantiomer less selective than the corresponding butyrophenone analogue (Table 10). The 7R,10S enantiomer of the valerophenone analogue displays a weak affinity for 5-HT2A and dopamine-D2 receptors. The indole nitrogen can be substituted with a methyl group with only a 3-fold decrease in 5-HT2A receptor affinity. However substitution with a phenyl ring is not tolerated [24] (Table 10).

174

Ergots A rich source for potent 5-HT2 ligands are the ergolines. The best known member of this class is the hallucinogenic agent (§ LSD displays a high affinity for the 5-HT2^ and 5-HT~c receptors but lacks selectivity with respect to other 5-HT (5-HTIA.D, 5-HTIF, 5-HT5, 5-HT 6, 5-HTT) and nourotransmitter (a~, oh, D2) receptors [4, 5, 1] (Table 11). More selective for 5-HT2 receptors is mesulergine, which does not interact with 5-HT~^.F, 5-HT5 and 5-HT 6 receptors [4, 2]. Table 11 5-HT 2 ligands derived from ergolines .

.

R 1

-

_

I R2

R1

R2

d-LSD*

(R)-C(=O)N(C2Hs)2 (S)-NHS(=O)zCHOHCH3

H CH3

LY 53857

(R)-C(=O)OCH(CHz)CHOHCHs CH(CH~)2 7.20

(30.6)

LY 86057

(R)-C(=O)OCH(CH3)CHOHCH3 H

(3.30)

12.2

n.d

Amesergide

(R)-C(=O)NHc.C6H12

(42.1)

10.7

n.d

Metergoline (S)-CH2NHC(=O)OCH~C6H6

5-HT~A 11.2 8.20 (48.5)

55.9

CH(CH3)2 6.90 CH3

0.76 (1.20)

5-HT2B5-HT2c n.d 35.9

3.8O 2.51

6.87 8.32

6.62 0.12

Data expressed as ICso in nM are from [33] (5-HT2A rat, 5-HT2A human (between brackets)). Data expressed as I~. in nM from [51] (d-LSD: 5-HT2A, 5-HT2c rat); [18] (5-HT2B rat); [66] (mesulergine 5-HTzc rat); [28] (LY 53857, metergoline 5-HT~c pig). *9,10-didehydroergoline; n.d.=not determined.

175 Ergolines lacking prominent affinity for dopamine D 2 and a,-adrenoceptors are the (8-~l)-ergoline-8-carboxylic acid esters. The prototype is LY 53857, which displays a high affinity for the 5-HT2A, 5-HT2B and 5-HT2c receptors and no or weak affinity for other 5-HT and neurotransmitter receptors tested [26, 27, 3, 18, 28] (Table 11). Structurally LY 53857 possesses five centers of asymmetry. Three of these centers reside in the ergoline structure and are fixed in the RRR stereochemical configuration. The other two asymmetric centers are located at the butanediolester side chain and are not resolved in LY 53857. However the stereochemical configuration of the side chain is not critical for 5-HT2A activity and selectivity. All four diastereomers of LY 53857 are potent 5-HT2A antagonists [29]. The ester group of LY 53857 can be replaced by a variety of ester moieties [30, 31]. Maximum affinity for 5-HT2A receptors is obtained by the cyclohexyl esters bearing an oxygen atom in or near the 4-position of the cyclohexyl ring. Replacement of the cyclohexyl ester group by a cyclohexylamide results in amesergide, being about 10 fold more potent than the corresponding ester [32]. Amesergide displays high affinity for both 5-HT2A and 5-HT2B receptors (Table 11). Table 12 5-HT 2 ligands derived from ergopeptines

R1 II H ~ O OH , , , ~ C-N ll"'P ~..";" O

""A

~

.'j

H

H R20

H

R,

R2

X

5-HT2A 5-HT2B

5-HT2c

Ergotamine

Me

CH2Ph

H

20.9

60.3

Dihydroergotamine*

Me

CH2Ph

H

21.9

Bromocriptine

i-Pr

i-Bu

Br

72.4

n.d 145 n.d

14.8 53.7

Data expressed as I~. in nM are from [51] (5-HT2A, 5-HT2c rat) and [67] (5-HT2B rat). * 9,10-dihydro (10a-H).

176 A methyl substituent at the N-6 position of the ergoline is optimal for high affinity. The affinity decreases with larger N-6 alkyl substituents. Omitting of the N-6 methyl group destroys the affinity for 5-HT2A receptors [31]. The N-1 of the indole nucleus can be substituted with small alkyl groups [30, 31, 32]. In the rat the N-1 alkyl substituted ergolines mesulergine, LY 53857 and amesergide are more potent than in the human. Conversely the N-1 unsubstituted analogues such as Ly 86057 display a higher affinity for the human 5-HT2Areceptors compared to the rat 5-HT2A receptor [33]. No species difference is observed for metergoline, a potent non-selective 5-HT2 antagonist [33, 4, 5, 1] (Table 11). These results indicate that metergoline binds in a different way to the 5-HT2A receptor (vide infra). Beside ergolines also ergopeptines, bearing a bulky peptide-like tricyclic ring system at the C-8 position of the ergoline skeleton, bind to 5-HT 2 receptors. Representative examples are ergotamine, dihydroergotamine and bromocriptine (Table 12). The affinity for the rat 5-HT2^ receptors is very similar to that of the N-1 unsubstituted ergoline LY 86057 (cf. Table 11). Data of the human 5-HT2A receptors have not been published yet. For the 5-HT2s and 5-HT2c receptors are the ergopeptines less potent. The ergopeptines are not selective for 5-HT2 receptors. They display a moderate to high affinity for the majority of 5-HT receptor subtypes such as 5-HT1A.F, 5-HT,7 [4, 34, 5] as well as a high affinity for al, oh-adrenoceptors and dopamine-D2 receptors [1]. In functional tests LSD acts as a partial agonist of 5-HT2A and 5-HT2c receptors [35] and mesulergine, metergoline, Ly 53857 and analogues as antagonists [5, 30, 32]. Phenylalkylamines The best known members of this class are the hallucinogenic agents (_+)DOB and (_+)DOI. They display a high affinity for the high aiFmity state of the 5-HT2A and 5-HT2c receptors. DOI also binds to 5-HT2B receptors (Table 13). Structureactivity relationships studies show that a lipophilic substituent at the 4-position of the phenyl ring is essential for high affinity [36, 37]. The rank order of potency for the 5-HT2A receptor is n.Hex >I>Br>Bu>Et=Me>OC>H (Table 13). A similar rank order is observed for the 5-HT2c receptor. No data for the 5-HT2B receptor have been reported yet. a-Desmethyl DOB is as potent as DOB, indicating that the a-methyl group does not contribute to the 5-HT2A or 5-HT2c binding [20] (Table 13). Introduction of one methyl group on the basic amine of DOB lowers the affinity 2 fold. The N-monopropyl substituted analogue is hardly active at all. Interestingly high affinity for 5-HT2A receptors is regained in the N-benzyl substituted compounds. In functional tests act (_+)DOB and (+_)DOI as partial agonists of the 5-HT2A and 5-HT2c receptors [65, 38]. The N-benzyl substituted derivatives are not yet tested functionally.

177 Table 13 5-HT2 ligands derived from phenylalkylamines

oc R~

R1 H OC (_+) DOB Br (+_) DOI I (+_) DOM Me (_+) DOET Et Bu n.Hex Br Br Br Br Br Br

R2

/ /

/

OC

R2

R3

5-HT2A

Me H Me H Me H Me H Me H Me H Me H Me H H H Me Me Me n-Pr Me CH2-Ph H CH2-Ph H CH2-Ph4-Br

5200 1250 41 19 100 100 58 2.5 34 80 i.a 10 16 1.6

N \

H

5-HT2~

5-HT2c

n.d 1217 n.d 2666 (1.00) n.d 60 (3.00) (0.71) (27.5)(1.44)* 45 (3.00) n.d 370 (41.0) n.d 101 n.d 26 n.d n.d (0.1) n.d 36 n.d 100 n.d 2460 n.d 440 (0.3) n.d 90 (0.4) n.d 48

Affinities expressed as ~ in nM for the antagonistic or agonistic (between brackets) site of 5-HT 2 receptors are from [36,37,20] (5-HT2A, 5-HT2c); [ 18] (5-HT2B) and [66] (5-HT2cDOB, DOI, DOM). n.d=not determined.

1-Arylpiperazines In the series of N-4 unsubstituted arylpiperazines 7-methoxy-1naphthylpiperazine is the most potent 5-HT2A ligand (I~.=1.8 nM). The 2-methoxy analogue is more than 2 decades less active. This result indicates that the N-1 phenyl ring of 1-naphthylpiperazine mimics the pyrrole ring of 5-HT. The benzfused phenyl ring of 1-naphthylpiperazine corresponds to the phenyl ring of 5-HT [39]. 1-Naphthylpiperazine (1-NP) is not selective and binds with high affinity to 5HT2A (I~.=18 nM), 5-HT2B (Ki=5.25 nM), 5-HT2c (6.31 nM), 5-HTIA (~=11 nM), 5HT1D=14 nM) and 5-HT 7 (I~.=20 nM) receptor. The affinity for the other 5-HT

178 receptor subtypes is 20-50 fold less than the affinity for 5-HT 2 receptors [28, 5, 39, 3, 4]. Removal of the benz-fused phenyl ring of 1-NP resulting in 1-phenylpiperazine is detrimental for 5-HT 2 receptor affinity. However substitution of the phenyl ring at the 3-position with trifluoromethyl (TFMPP) or chlorine (mCPP) enhances the affinity significantly. These compounds are only about 10 times less potent than 1-NP [3]. Like 1-NP, TFMPP and mCPP are non-selective [4, 5]. In functional tests these arylpiperazines are partial agonists of 5-HT~ and 5-HT2c receptors [40]. For the 5-HT2B receptor 1-NP acts as an antagonist and TFMPP and mCPP as partial agonists [19, 6]. Table 14 5-HT2A ligands derived from 1-arylpiperazines O

\\/!

O

- - -

n

RP 62203*

3 3 3 3 3 3 3 3 3 2 4

(CH2) n - - N

\

R

4-F 3-F 2-F H 4-C1 4-OH 4-OCH~ 4-CHs 4-CF 8 4-F 4-F

N

/

5-HT2^

0.5 18 3.0 2.6 5.0 0.3 25 4.0 275 1.0 3.7

al

103 688 52 141 i.a 110 i.a 700 i.a 64 11

D2

i.a i.a i.a i.a i.a 508 i.a i.a i.a n.d 300

Data, expressed as IC~o in nM are from [10]. i.a=inactive; n.d=not determined. *note: The receptorbinding data of RP 62203 published by [41] and [42] result in al/5-HT~ ratio's of 35.8 and 66.7 respectively.

179 Structurally unrelated to the well-known 5-HT 2 antagonists are the naphthosultam derivates [10]. This new class of5-HT2 antagonists was discovered by chance. The prototype is RP 62203 displaying high affinity for the 5-HT2A receptors (Table 14). Structure-activity relationships studies show that a fluoro atom at the 4-position of the phenyl is optimal for 5-HT2A receptor affinity and selectivity with respect to r and dopamine D~ receptors. The 2fluoro and 3-fluoro analogues are less potent and less selective (Table 14). At the 4-position of the phenyl a hydroxy substituent is also favourable for 5-HT2A affinity. Other substituents reduce the affinity significantly. The rankorder of potency is OH~F>CH3~CI>OCHs>CF3 (Table 14). Extension or reduction of the alkaline side-chain of RP 62203 with one carbon atom decreases 5-HT2A affinity and selectivity (Table 14). The phenylpiperazine moiety can be replaced by 4-phenyl-l,2,3,6tetrahydropyridine or by 4-phenylpiperidine without loss in affinity and selectivity. The 4-(4-fluorobenzoyl) piperidine analogue is potent but less selective (cf. Table 2). Table 15 5-HT2A ligands derived from trans-l-piperazines-3-phenylindans O

1~'"'11 N~==/N/~ R /--k

R1 C1 Br CF3 CH3 SCH3 Cl C1 C1 C1 C1

N

N

k__J

R2 4-F 4-F 4-F 4-F 4-F H 2-F 3-F 4-C1 4-CH 3

Data, expressed as ICso in nM are from [43].

5-HT2A (_+) (+_) (_+) (_+.) (_) (_) (_+) (_+) (_+) (_+)

3.9 6.4 3.7 3.0 4.0 9.9 3.5 26 15 11

D2

{~1

280 260 240 270 510 370 450 120 720 250 750 510 920 670 490 970 280 180 450 60

180 Table 16 5-HT2Aligands derived from trans-l-piperazino-3-phenylindans . . . .

IIN

\

N

~

L

I

5-HT2A

=N-i-Pr o ,,~

--N X \

X =NH =N-Me =N-Et =N-Ph =CH2 =0

/

D2

(Z,

(_+) (+) (-) (_+) (_+) (_+) (_+) (_+) (_+)

3.9 75 1.1 2.9 2.8 2.6 44 10 23

280 1300 200 360 190 260 300 500 550

260 340 210 200 600 240 310 370 140

(+) (-) (_+)

21 0.75 1.5

490 33 67

350 67 52

(_+)

5.6

110

66

(+_)

3.8

93

78

65

24

S

--N

NH

O --N

n

=2

n

=3

NH

O

H

o

cN~~N~~S~ H3 Data, expressed as IC~0 in nM are from [43].

(_+)

19

181 Modification of the naphthosultam group such as expansion or opening of the sultam ring lowers 5-HT2A receptor affinity and selectivity. New receptor binding studies confirm that RP62203 is a potent 5-HT2A ligand [41,42]. However the selectivity with respect to al-adrenoceptors is in these studies only 35.8 and 66.7 instead of 206 as reported in [10]. RP62203 is selective with respect to other receptors tested (including the 5-HT2c receptor). In functional tests RP62203 acts as a potent 5-HT2A antagonist [41]. The trans-l-piperazine-3-phenylindan derived 5-HT 2 antagonists are related to the 1-phenyl-3-(4-pi peridinyl )-and 3-phenyl- 1-(4-pi peri dinyl )- 1H-indoles (Table 15 cf Tables 7,9). Substitution of the indan ring at the 5-position with halogen, alkyl or thioalkyl results in potent 5-HT2A ligands [43]. The compounds display a weak affinity for the dopamine-D2 and al-adrenoceptors. The most selective compound is the 5-CFa substituted analogue (ratio D2/5-HT2A=138, ratio al/5-HT2A=100). The 3-phenyl group, which is essential for high 5-HT2A receptor affinity, may be substituted with halogen or alkyl. The rankorder of potency is 2F=4F>4CHa~4CI>3F (Table 15). The 2-fluoro analogue is about 3-fold more selective than the 4-fluoro analogue. In vivo the 2-fluoro derivative is rather weak. The 4-fluoro derivative displays the best overall profile. Resolution of this compound shows that the 5-HTzA receptor affinity resides mainly in the (-)-enantiomer. The (-)-enantiomer is also highly selective with respect to dopamine-D 2 and a~-adrenoceptors (ratio D2/5-HT2A=182; a~/5-HT2A=191) (Table 16). Data on the affinity for 5-HT2c receptors have not been published yet. Replacement of the isopropyl group of the imidazolidinone ring by hydrogen, methyl or ethyl has little effect on potency and selectivity [43] (Table 16). A phenyl group is not well tolerated. Exchanging of the imidazolidinone ring for a pyrrolidinone or oxazolidinone ring reduces activity and selectivity. The imidazolidine-2-thione and tetrahydropyrimidine-2-thione analogues are more potent but less selective 5-HT2A antagonists than the corresponding oxygen containing congeners. Replacement of the imidazolidinone ring by a benzimidazolone or quinazolinedione results in potent, non-selective 5-HTzA ligands. Striking is the rather low 5-HT~A receptor affinity of the compound bearing the ritanserin side chain (Table 16).

Diarylurea From the class of the diarylurea originates the first selective 5-HT2B antagonist [44]. This compound SB 204741 displays a good affinity for the 5-HT2B receptors, a low affinity for the 5-HT2c receptors and no affinity for the 5-HTzA receptors (Table 17). SB 204741 is also selective with respect to other receptors tested. The lead compound for SB 204741 was the [3-(trifluoromethyl)phenyl] urea analogue, having a 100-fold greater potency for the 5-HT receptors in rat stomach fundus than in rat jugular preparations [45] (Table 17, compound 1). Replacement of the trifluoromethylphenyl moiety by a 3-pyridyl group and removal of the alkyl substituents at the 2- and 3-positions of the indole nucleus increases the affinity of the rat stomach fundus receptors 2-fold (Table 17, SB 200646).

182 Table 17 5-HTJ5-HT2c ligands derived from diarylurea

Compounds

5-HT2A 5-HT2B* 5-HT2B 5-HT2c

1 OH3 -- CH2

H

H

CH3

5.18"

7.16"

n.d

n.d

CF3

SB 200646A

H

H

i.a

7.41"

631

110

H

H

i.a

7.95*

79

1513

6310

7.82*

n.d

417

/ CH3

SB 204741

/ CH3 2

H

H

H

H

CH3

/ CH3

Data, expressed as I~. in nM or pA2* are from [45] (compound 1); [44] (pA2 values, 5-HTzA/5-HT2c affinities) and [52] (5-HT2D affinities). n.d=not determined; i.a=inactive.

183 The compound displays also affinity for the cloned 5-HT2c receptors but is selective with respect to the 5-HT2A and other receptors tested [46]. Further optimalisation of SB 200646A resulted in SB 204741, obtained by replacement of the 3-pyridyl moiety by an isothiazole ring.This modification is favourable for 5-HT28 affinity and decreases the affinity at 5-HT~c receptors (Table 17). The indole moiety can be replaced by a benzothiophene ring without any affect on the activity at 5-HT2B receptor. The affinity for 5-HT~c receptors however regains, making the compound less selective than SB 204741. There is a drop in affinity and selectivity in the isoxazole analogue (Table 17, compound 3). Without loss in 5-HT2B activity the indole (C5) urea nitrogen atom can be incorporated into angularly or linearly fused tricyclic ring systems [47] (Table 18). The highest 5-HT2B affinity is seen in the five/six/five fused analogues. These tetrahydropyrroloindoles are 2 times (compound 1) and 10 times (SB 206553) more potent than the parent indolylurea SB 200646A. There is a parallel increase in affinity for the 5-HT2c receptor (Table 18). Table 18 5-HT2B/5-HT2c ligands derived from diarylurea 0

(CH2)n

"N ~

~

/

-

(CH2)m

CHa

Compounds

n

m

5-HT2A

SB 200646 A

0

0

i.a

1

2

0

2

3

0

0

2

0

3

SB 206553 3

2884 i.a 1622 i.a

5-HT2B* 5-HT2c 7.40*

138

7.75*

66

7.22*

i.a

8.48*

10

7.27*

4073

Data expressed as Ki. in nM or pA2* are from [47]. i.a=inactive.

184

The five/six/six fused analogues compounds 2 and 3, are for the 5-HT2B receptors as potent as the non-cyclic indole urea. However, in contrast to the five/six/five fused analogues are the tetrahydropyrroloquinolines inactive at the 5HT2c receptors (Table 18). This fall in 5-HT2c receptor affinity is probably due to the loss in coplanarity of the fused five/six/six ring system [47]. The affinity for the 5-HT2A receptor of the cyclized urea derivatives is weak or absent (Table 18). Miscellaneous structures The atypical antipsychotic clozapine displays nanomolar affinity for the 5-HT2A and 5-HT2c receptors, but is not selective with respect to other neurotransmitter receptors (Table 19). Org-5222, a congener of mianserin belongs to the most potent antagonists of 5HT2A and 5-HT2c receptors known. The compound however is not selective [48] (Table 19). A novel 5-HT 2 antagonist is the indolonaphthyridine SDZ SER 082 discovered by general screening [49]. The compound has a good affinity for 5-HT2B and 5-HT2c receptors and weak or no affinity for 5-HT2A, 5-HTI.a and neurotransrnitter receptors tested (Table 19). An other polycyclic structure displaying high affinity for 5-HT 2 receptors is the well-known antihistaminic agent cyproheptadine (Table 19). A rather selective 5-HT2A antagonist is cinanserine. It displays a high affinity for 5-HT2A receptors a weak affinity for the 5-HT2B and 5HT2c receptor subtypes and no affinity for the neurotransmitter tested (Table 19). Structurally related to cinanserine is (R)-M-1 the active metabolite of sarpogrelate ((R, S)- 1-[2-[ 2-( 3- me th oxyphenyl )-ethyl] p he no xy]- 3- (dime thyl amino )- 2- pro pyl hydrogen succinate hydrochloride) [50] (Table 19). (R)-M-I is a potent 5-HT2A antagonist possessing only weak activity for the (z~-adrenoceptors and histamineH 1receptors. (R)-M-I lacks prominent affinity for the ~-adrenoceptors (pK~=5.84). The other enantiomer (S)-M-I is nearly as active (pKn=8.84) as the (R)-enantiomer but slightly less selective.

Table 19 5-HT2A ligands derived from miscellaneous structures

5-HT2A 5-HT2B 5-HT2c

D2

a,

H,

M,

69.0

11.1

6.50

3.19

cI

Clozapine ~

~~

H.

24.0 N Il _

\ /

/"k

N

N --- C H 3

n.d

8.13

185 Table 19 (continued) 5-HT2, ligands derived from miscellaneous structures

H1

5-HT2A 5-HT2B 5-HT2c D2

1.3

0RG-5222

n.d

0.05

5.7

0.42

M1

7.7

i.a

n.d

n.d

1.51

3.5

i.a

i.a

Cl

H - -

SDZ SER-082

CH3

631

7.34*

15.8

i.a 2512

H

--- C H 3

Cyproheptadine

5.60

22.4

22.9

41.0

58

--- C H 3

Cinanserin

5.00

HN~'~ /CH3 S-C-C-C-N 0 ~CH3

:)

1659

200

i.a

i.a

186 Table 19 (continued) 5-HT2^ ligands derived from miscellaneous structures 5-HT2^ 5-HT2B 5-HT2c D2

(R)-M-1

9.04*

OHI

/CH3

H

~CH3

-C-C-C-N

n.d

n.d

n.d

a:

H1

6.58* 6.49*

M1

5.14"

OCH3

Data, expressed as Ki in nM or PKB* are from [51] (5-HT2A, 5-HT2C, D2, {zl, H~, M1); [3] (5-HT2B); [48] (ORG 5222); [49] (SDZ SER-082); [50] (R)-M-1). MOLECULAR INTERACTIONS OF LIGANDS AND 5-HT2 RECEPTORS

Site-directed mutations of the 5-HT~ receptor Wang [53] studied the role of three aspartate residues (Asp-120, Asp-155 and Asp-172) of the 5-HT2A receptor of the rat in the binding of ligands and the activation of the G-protein. Three asparagine mutants (Asn- 120, Asn- 155 and Asn172) were constructed and stably expressed in NIH 3T3 fibroblasts as was the cloned wild type receptor. ~25I-LSD was used in radio ligand binding studies (Kd, Bm~) and in competition experiments (I~.) by agonists and antagonists. The functional properties of the mutant receptors were tested by determination of the effect of, 5-HT stimulation on the production of 3H-inositolphosphates (IP) from the hydrolysis of SH-phosphatidylinositol as the result of G-protein activation. I: The Asn-120 mutant receptor (transmembrane helix 2). The affinity of the agonist 5-HT and the partial agonist DOI is substantially decreased compared to the wild type receptor. The GTP sensitivity of both agonists is lost, which suggests a G-protein uncoupled state of the receptor. Interaction of 5-HT with the mutant receptor did not result in signal transduction and IP production. The affinity of the antagonists ketanserin and mianserin decreased in about the same degree as the agonists, whereas for spiperone only a modest decrease was observed.

187

II: The Asn-155 mutant receptor (helix 3). The decrease in binding affinity for both agonists and antagonists was the largest by far. The binding of both agonists was GTP sensitive and the production of IP on stimulation of the receptor by 5-HT was restored although the ECso was increased owing to the low affinity of 5-HT compared to the wild type receptor. III: The Asn-172 mutant receptor (interface helix 3 and second intracellular loop). Compared to the wild type receptor the binding affinity of both agonists was moderately (5x) decreased. The binding was GTP sensitive and the IP production was restored. The ECso for 5-HT stimulated IP production was twice as high in comparison with the wild type receptor. There was a fivefold decrease in the binding affinity ofmianserin and spiperone whereas the affinity of ketanserin was the same as that for the wild type receptor. Conclusions: The data suggest that Asp-120 is essential for coupling with and activation of the G-protein. Asp-155 is necessary for a high affinity of the binding of basic ligands but not essential for binding per se. The binding sites of the different antagonists are probably different or partially overlapping. Sealfon [54] described the effect on functioning and ligand binding by mutation of the Asp-120 [helix 2] and Asn-376 [helix 7] residues of the cloned human 5-HT2A receptor transiently expressed in COS-1 cells. Stimulation of the Asn-120, Ala-120 and Ala-376 mutant receptors by 5-HT did not result in G-protein activation and IP production. Stimulation of the Asp-376 mutant however restores the IP production to about 95% albeit that there is a 4-fold increase in ECso compared to the wild type receptor. Double mutation that is interchanging the positions of Asp-120 and Asn-376 (Asp-120 -~ Asn-120, Asn-376 ~ Asp-376) partly restores the IP production to 50% whereas the EDso value increases 9-fold. Molecular dynamics simulation indicated that stimulation of the wild type receptor by 5-HT resulted in a change in the direction of helix 5 and 6. The directional change was less and in an opposite direction when the non active Asn-120 mutant interacted with 5-HT. Antagonists did not cause a directional change at all. The binding affinity of 5-HT, DOI and LSD and of the antagonists mianserin, haloperidol and 5-hydroxygramine for aH-ketanserin labeled sites in the Asn-120 mutant and in the double mutant Asn-120, Asp-376 is about the same as the affinity for the wild type receptor. The data generated by the foregoing experiments lead to the conclusion that both Asp-120 and Asn-376 are part of a hydrogen bonding region in the same micro environment. Choudhary [55] investigated the role of the conserved phenylalanine residues in helix 6 (Phe 339, Phe 340) in the binding of agonists, partial agonists, antagonists and especially the ergolines and ergopeptines [56]. Several different

188 mutants were constructed and transiently transfected in COS-7 cells but the mutants of direct interest were Leu-339 and Leu-340. The change of Phe-340 to Leu-340 results in a severe loss in affinity for the mutant receptor of 5-HT, DOI and the eight ergolines tested with no effect on the affinity of ketanserin, ritanserin and the four ergopeptines tested except for ergocornine where the loss is moderate (about 5 fold). The losses in affinity of 5-HT and the ergolines are largely recuperated by the use of the Leu-339 mutant receptor whereas the affinity of ketanserin has decreased about 10 fold. The affinities for the Leu-339 mutant receptor of the two ergopeptines tested ergotamine and ergocryptine did not differ from those for the wild type receptor. These results show that the Phe-340 residue is essential for optimal binding of ergolines probably because the phenyl moiety offers the opportunity of interaction with the indole moiety of the ergolines be it by stacking or by edge to face interaction of the aromatic rings. The same kind of interaction may also hold for the binding of 5-HT. The ergopeptines probably owing to the size and conformation of the tricyclic substituent in the 8-position may bind to a (partly) different site on the receptor. The Phe-339 residue is important for the binding of ketanserin. The conclusions based on the effects on binding by the mutant receptors are only valid if the conformations of the mutants and the wild type receptor are similar. Some ergolines show a significantly higher affinity for the rat- than for the human 5-HTgA receptor whereas the reverse is true for other ergolines, see [33, 57] and references therein. Point mutation studies in rat- and human 5-HT2Areceptors as reported by Kao [58] and Johnson [59] offered an elegant explanation for this phenomenon. Kao [58] demonstrated that by exchanging the serine residue Ser-242 (helix 5) of the human 5-HT2A receptor for an alanine Ala-242 as present in the rat receptor the affinity of mesulergine for this mutant is greatly increased and has become equal to the affinity for the rat cortex membrane receptor. The cloned human receptor was stably expressed in murine fibroblast cells (LMCTK)cells whereas the Ala-242 mutant receptor was expressed in COS-7 cells. In addition Johnson [59] showed that by exchanging the alanine residue Ala242 of the rat 5-HT~ receptor for a serine Ser 242 residue as present in the human receptor the affinity of mesulergine for this mutant has greatly decreased and has now become similar to the affinity for the human receptor. Johnson [59] also constructed two other functionally active mutants (threonine Thr-242 and valine Val-242) of the rat 5-HT2A receptor. Cloned receptors and their mutants were expressed in an AV-12 cell line. A series of 11 ergolines was tested for affinity for native and cloned rat- and cloned human receptors. Nine of the ergolines were tested for affinity for the mutant receptors. The series consisted of compounds with an unsubstituted indole nitrogen (N1-H) and of their alkyl substituted congeners (N~-Me, N~-iPr). 3H-DOI and 3H-ketanserin were used to label the binding sites.

189 The unsubstituted ergolines (N-H) had a higher affinity for the human wild type- and the Ser-242 mutant receptors than for the rat wild type receptor. A higher affinity for the Thr-242 mutant receptor than for the Val-242 mutant receptor was found for the two N-H ergolines from the series that were tested with these mutants. The affinities of the N-substituted ergolines are the inverse of the affinities of the unsubstituted ergolines, a higher affinity for the (cloned) rat receptor and a lower affinity for the cloned human- and Ser-242 mutant receptor. A lower affinity for the Thr-242 mutant receptor and a higher affinity for the Val-242 mutant receptor was found for the three (one N-Me and two N-iPr) ergolines that were tested with these mutants. The results suggest that the higher affinity ofunsubstituted (N-H) ergolines for the human 5-HT2A receptor is due to stabilization of the binding by hydrogen bonding to the hydroxyl group of Ser-242 whereas in the rat receptor where the serine is replaced by alanine this stabilization is not possible. The lesser affinity of the N-alkyl substituted ergolines for the human receptor than for the rat receptor is due to an unfavourable interaction of the alkylsubstituents with the more hydrophilic serine moiety.

5-HT2 receptor chimeras Roth [60] and Choudhary [55] reported the binding affinity of a variety of structurally different antagonists for 5-6 chimeras of the 5-HT2A- and the 5-HT2c receptor and concluded that ligands do not bind to a common site on the 5-HT2 receptor. This makes the molecular description of the binding sites ofligands of different chemical structures fairly complicated. Oksenberg [61] constructed a chimera of the human 5-HT1B receptor and the third intracellular loop, i3 of the human 5-HT2A receptor. The rat 5-HT2A receptor was stably transfected in NIH3T3 cells whereas the cloned 5-HTm receptor and the chimera were transiently transfected in the human embryonic kidney 293 cell line. The affinities of the agonists 5-HT, RU 24969 and sumatriptan and of the 5~antagonists methiothepin, ketanserin and spiperone for both the 5-HT m receptor and its chimera are comparable but differ significantly from the affinities for the 5-HT2A receptor with the exception of RU 24969 where the affinity for the three receptor types is similar. The affinity of sumatriptan for the 5-HT2A receptor is two orders of magnitude lower than for the other two receptors. On the other hand the affinity of the antagonists ketanserin and spiperone for the 5-HT2A receptor is three orders of magnitude higher than for the 5-HT m receptor and its chimera. The results show that the internal loop i3 of the 5-HT2A receptor in the 5-HT m chimera has no effect on the binding characteristics of the 5-HTm receptor. Stimulation of the native 5-HT m receptor by HT agonists results in a diminished production of the second messenger c-AMP caused by inhibition of adenylylcyclase. The increase in the production of c-AMP by stimulation of adenylylcyclase by forskolin was counteracted by 5-HT, sumatriptan and RU

190 24969 in the 5-HT~B receptor transfected cells but not in the chimera- and 5-HT~A receptor transfected cells. Stimulation of the native 5-HT~ receptor results in the activation of phospholipase C. This enzyme catalyzes the hydrolysis ofphosphatidylinositol 4,5diphosphate producing diacylglycerol and inositol 1,4,5 triphosphate IP 3. IP3 mobilizes Ca 2§ from its intracellular stores. In the 5-HT2A receptor transfected cells there was an increase in [Ca2§ after stimulation with 5-HT. In the cells expressing the 5-HTIB chimera receptor the [Ca2§ i was also increased after stimulation with 5-HT, sumatriptan or RU 24969. In cells expressing the 5-HT~B receptor there was no mobilization of Ca 2§ ions after stimulation with the 5-HT agonists. It is evident that the i3 loop is an important factor in determining specific effector coupling as demonstrated for the 5-HT~B- and 5-HT2A receptors with their respective effectors adenylylcyclase and phospholipase C.

Molecular modeling Recently molecular dynamics simulation studies on the binding of 5-HT, ritanserin and ketanserin to the 5-HT2^ receptor have been extensively described [62, 63] and the results were compatible with those of the mutagenesis studies. The molecular modeling of the 5-HT 2 receptor subtypes and the interaction of 5-HT, DOB and LSD with the 5-HT2s receptor was reported by Westkaemper and Glennon [64]. In their model are the amino acid residues contributing to a direct interaction between 5-HT and its receptor: Asp-155 (helix 3) forming an ionic bond with the basic NH2 group of 5-HT, Ser-239 (helix 5) forming a hydrogen bond with the 5-hydroxy group, Phe-243 (helix 5) forming a ring stacking interaction with the indole ring, and lastly Phe-340 (helix 6) which is perpendicular to the indole ring and situated between the ring and the aliphatic side chain. Ser-207 (helix 4) is considered to be too distant from the indole NH for hydrogen bond formation. The interaction of DOB (1-(2,5 dimethoxy-4-bromophenyl) isopropylamine) and 5-HT with the 5-HTzs receptor is very similar: Ionic bonding with Asp-155, hydrogen bonding of the 2-OMe group with Ser-239 and stacking of the phenyl group and Phe-243. In this case Ser-207 (helix 4) may donate a hydrogen bond to the 5-OMe group. The 4-Br substituent can be accommodated in a "lipophilic pocket" formed by Ala-242 (helix 5) Phe-243 (helix 5) and Val-204 (helix 4). When the phenyl group of DOB is rotated 180 ~ the interactions of the OMe groups with the serine moieties are reversed i.e hydrogen bonding of 5-OMe with Ser-239 and of 2-OMe with Set-207. According to this report LSD binds in a similar way as 5-HT but is shifted towards the extracellular side and the Ca-C~ bonds of Asp-155 and of Phe-340 have to be rotated in order to accommodate a larger bulk.

191 ADDENDEM re MOLECULAR MODELING

Molecular dynamics simulation was used in the molecular modeling study by Kristiansen and Dahl on the interaction of ritanserin and ketanserin with the 5HT2c receptor and differences in the interaction with the model of the 5-HT~A receptor were discussed [68]. Almaula et al. [72] investigated the binding site pocket of the cloned human 5HT2A receptor expressed in COS-1 cells using mutation studies and computational dynamic simulations. It was shown that the full agonist 5-HT binds with its cationic primary amine group to Asp-155 (helix 3) and by a hydrogen-bond type interaction with Ser-159 (helix 3) whereby serine acts as a H-acceptor. In the competition binding experiments 3H-ketanserin was used as radioligand and it was shown that ketanserin has comparable affinities for the wild-type - and the mutant receptors. The affinity of 5-HT for the Ser-159-Cys mutant was only 5-fold lower than for the wild-type receptor whereas the affinity for the Ser-159-Ala mutant was 18-fold lower. The affinity of N,N-dimethyl-5HT=bufotenine - a partial agonist- for the Ser159-Ala m u t a n t was only 4 fold less than for the wild type receptor whereas its affinity for the Ser-159-Cys mutant was about the same as for the wild-type receptor. The partial agonist LSD has the same affinity for all three receptor types. Molecular dynamic simulation showed that although the three agonists interact with the aspartate residue in helix 3 only 5-HT can interact with both aspartate and cysteine residues. In the functional test (stimulation of phosphatidyl-inositol hydrolysis) bufotenine acts as a partial agonist at the wild-type receptor and as a full agonist at the Ser-159-Ala mutant receptor.

re 5-HT 2 RECEPTOR

LIGANDS

Aryloxyalkylimidazolines

A novel chemical class - the aryloxyalkylimidazolines - with affinities in the nanomolar range for the rat 5-HT2A and 5-HT2c receptors was recently described by Siegel et al. [70]. The structures and affinities of these compounds are shown in Table 20. The rat 5-HT2A and 5-HT2c receptors were cloned and stably expressed in NIH 3T3 mouse fibroblasts. Binding and competition experiments were performed on membranes prepared from cell cultures using [125I]LSD as radioligand. Inhibition of 5-HT stimulated phosphoinositide hydrolysis was used to determine the antagonistic potencies of the compounds. The agonistic properties of the compounds and their relative efficacies (relative to serotonin that is) were assessed

192 by their potency in stimulating the phospholipase C mediated phosphoinositide hydrolysis.

Table 20 5-HT2 ligands derived from aryloxyimidazolines

B

A

c

compound

A/B

R

n

5-HTzA

5-HT2c

MDL MDL MDL MDL MDL

A A A A B

BnO BnO BnO CH3S

1 2 3 3

4 70 211 223 144

5 44 102 45 29

101,600 103,097 102.588 100.971 101.156

I~. in nM, radioligand [12SI]LSD, [70], BnO=benzyloxy

MDL 101.600 is a potent antagonist with equal affinity for both receptor subtypes but a slightly higher potency in the functional test with the 5-HT2A receptor than with the 5-HT2c receptor (ICso=25 -+ 13 nM vs ICso=70 -+ 16 nM). Lengthening of the sidechain of MDL 101.600 by one C-atom as in MDL 103.097 (n=2) results in a decrease in affinity and antagonistic potency for both receptor subtypes (ICso 5-HT2A=ca 1000 nM, 5-HT2c= ca 1650 nM). Further lengthening of the sidechain by one C-atom yields MDL 102.588 (n=3) a compound with a rather different pharmacological profile. The compound has a moderate affinity for both receptor subtypes and shows agonistic characteristics at the 5-HT2c receptor and antagonistic characteristics at the 5-HT2A receptor both of a modest potency (EC~o=6468 _+ 779 nM, relative efficacy=0.7 ICso=ca 4000 nM). Replacement of the ortho-benzyloxygroup of MDL 102.588 by a SCH 3 group results in the partial agonist MDL 100.971 with a 5 fold higher affinity for the 5HT2c receptor than for the 5-HT2A receptor (I~.=45 nM vs ca 223 nM). The ECso for

193 the 5-HT2c receptor is about 447 nM and the relative efficacy is 0.85 while for the 5-HT2A receptor the ECso=Ca 2200 nM and the relative efficacy=0.7. MDL 101.156 a structural analogue of MDL 103.097 with a 1-naphthyloxy group instead of an ortho-substituted phenoxygroup is a moderately potent full agonist with a 5-fold selectivity in affinity as well as in potency for the 5-HT2c receptor (ECso 5-HT2c=237 _+ 36 nM, 5-HT2A=1157 _+ 134 nM, relative efficacies

1.oo). Tetrahydrobetacarbolines A series of potent and selective antagonists of the 5-HT2B receptor was recently reported by Audia et al. [71]. A selection of the more potent tetrahydrobetacarbolines derivatives is presented in Table 21. The apparent dissociation constants KB of the antagonists determined by inhibition of the 5-HT induced contractile responses of smooth muscle strips of the rat stomach fundus (5-HT2B) or jugular vein (5-HT2A) were expressed as their negative logarithm pKB. For the binding experiments cloned rat 5-HT2A or mouse 5-HT2c receptors were stably expressed in Syrian hamster fibroblast cells (AV12). The radio ligands used were 3H-ketanserin (5-HT2A) and SH-mesulergine (5-HT2c). p ~ = t h e negative logarithm of the inhibition constant I~.. The unsubstituted tetrahydrobetacarboline has a very low affinity for the 5HT2B receptor (pI~. <5,5) [71]. Substitution at the 1-position by a benzylgroup raises the affinity more than 100 fold (pI~.=7.61, Table 21). Substitution of the benzylgroup at the 3- or 4-position by a methoxygroup caused a further 3-fold increase in affinity. Substitution by methoxygroups at both the 3- and 4-position results in a ca 13 fold increase in affinity compared to the monosubstituted compounds. The introduction of a Cl-atom at the 2-position of the 3.4-diMeO-benzyl moiety does not result in a significant further increase in affinity whereas the 3.4.5 triMeO analogue suffers a 12 fold decrease compared to its 3.4-diMeO congener [71]. The introduction of methylgroups in the indole moiety of 3.4-diMeObenzyltetrahydrobetacarbolines induces a significant increase in affinity. For example the increase is 5-fold for the 6-Me-3'.4'-diMeO derivative and even 9-fold for the 7,8-diMe-3'.4'-diMeO congener (Table 21). The 5.7, 6.7, and 6.8-diMe derivatives have comparable affinities with pKB's of 9.34, 9.71 and 9.61 respectively [71]. Introduction of a 6-methylgroup in the 2'-C1-3'.4'-diMeO derivative causes a moderate 3-fold increase in affinity (Table 21). The compounds shown in table 21 are highly potent 5-HT2B receptor antagonists and from the data available it is clear that a number of compounds are highly selective compared to their affinities for the 5-HT2A and 5-HT~c receptors in the binding- as well as in the functional tests.

194 Table 21 5-HT2B ligands derived from tetrahydrobetacarboline

4

NH

31

5 6 7 8

compound

pKB

pI~.

ratio

5-HT2B 5-HT2A 5-HT2A 5 - H T 2 c

H 4'MeO 3'MeO 3'.4'MEO a 6Me3'.4'MeO 7.8Me3'.4'MeO b 6Me2'C1 3'.4'MEO c 2'C1 3'.4'MEO

7.61 8.09 8.04 9.17 9.86 10.12 9.80 9.27

7.43 7.21 8.14

7.18 7.65 8.06 7.71

6.90 7.92 8.11 7.61

2B/2A

2B/2C

98 162 115 123

186 87 102 155

Codenames" a=Ly 23728; b=Ly 287375; c=Ly 266097. REFERENCES

1 2 3 4 5 6 7 8

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196 35 Burris KD, Breeding M, Sanders-Bush C. J PET 1991; 258: 891-896. 36 Seggel MR, Yousif MY, Lyon RA, Titeler M, et al. J Med Chem 1990; 33: 10321036. 37 Glennon RA, Raghupathi R, Bartyzel P, Teitler M, et al. J Med Chem 1992; 35: 734-740. 38 Sanders-Bush E, Breeding M. Psychopharmac 1991; 105: 340-346. 39 Glennon RA, Dukat M. Pharmacol Biochem Behav 1991; 40: 1009-1017. 40 Conn PJ, Sanders-Bush E. J PET 1987; 242: 552-557. 41 Doble A, Girdlestone D, Piot O, Betschart J, et al. Brit J Pharmac 1992; 105: 27-36. 42 Malgouris Ch, Flamand F, Doble A. Eur J Pharmac 1993; 233: 29-35. 43 Boges~ KP, Arnt J, Hyttel P, Pedersen H. J Med Chem 1993; 36: 2761-2770. 44 Forbes IT, Jones GE, Murphy OE, Holland V, et al. J Med Chem 1995; 38: 855-857. 45 Fludzinski P, Wittenauer LA, Schenk KW, Cohen ML. J Med Chem 1986; 29: 2415-2418. 46 Forbes IT, Kennett GA, Gadre A, Ham P, et al. J Med Chem 1993; 36: 11041107. 47 Forbes IT, Ham P, Booth DH, Martin RT, et al. J Med Chem 1995; 38: 25242530. 48 De Boer Th, Tonnaer JADM, De Vos CJ, Van Deli~ AML. Arzneim Forsch Drug Res 1990; 40: 550-554. 49 Nozulak J, Kalkman HO, Floersheim Ph, Hoyer D, et al. J Med Chem 1995; 38: 28-33. 50 Pertz H, Elz S. J Pharm Pharmacol 1995; 47: 310-316. 51 Tulp MTHM, unpublished results. 52 Bonhaus DW, Bach Ch, De Souza A, Salazar FHR, et al. Brit J Pharm 1995; 115: 622-628. 53 Wang Ch-D, Gallaher TK, Shih JC. Mol Pharmacol 1993; 43: 931-940. 54 Sealfon SC, Chi L, Ebersole BJ, et al. J Biol Chem 1995; 270: 16683-16688. 55 Choudhary MS, Craigo S, Roth BL. Mol Pharmacol 1993; 43: 755-761. 56 Choudhary MS, Sachs N, Uluer A, Glennon RA, et al. Mol Pharmacol 1995; 47: 450-457. 57 Johnson MP, Audia JE, Nissen JS, Nelson DL. Eur J Pharmacol 1993; 239: 111-118. 58 Kao HT, Adham N, Olsen MA, et al. FEBS Letters 1992; 307: 324-328. 59 Johnson MP, Longcharich RJ, Baez M, Nelson DL. Mol Pharmacol 1994; 45: 277-286. 60 Roth BL, Craigo S, Choudhary S. Med Chem Res 1992; 2: 329-341. 61 Oksenberg D, Havlik S, Peroutka SJ. Ashkenazi A. J Neurochem 1995; 64: 1440-1447. 62 Edvardsen ~, Sylte I, Dahl SG. Mol Brain Research 1992; 14: 166-178. 63 Kristiansen K, Edvardsen ~, Dahl SG. Med Chem Res 1993; 3: 370-385. 64 Westkaemper RB, Glennon RA. Med Chem Res 1993; 3: 317-334. 65 Sanders-Bush E, Burris KD, Knoth K. J PET 1988; 246: 924-928. 66 Leonardt S, Gorospe E, Teitler M. Molec Pharmac 1992; 42: 328-335.

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Serotonin Receptors and their Ligands B. Olivier, I. van Wijngaarden and W. Soudijn (Editors) 9 1997 Elsevier Science B.V. All rights reserved.

199

The 5-HT2-type receptor family E. Ronken 1 and B. Olivier 1'2 1)Solvay Duphar B.V., Dept. of CNS Pharmacology, P.O.Box 900, 1380 DA Weesp,

The Netherlands. 2~trecht University, Dept. of Psychopharmacology, Faculty of Pharmacy, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

INTRODUCTION The 5-HT 2 receptor family currently consists of three different receptor subtypes, i.e. the 5-HT2A receptor (formerly known as the 5-HT2 receptor) has been discovered on basis of pharmacological criteria. Secondly, the 5-HT2c receptor (formerly known as the 5-HTlc receptor) was first recognized on basis of deviant pharmacological behavior in neuroanatomical studies. Finally, the 5-HT2B receptor (which has also been denoted as 5-HT2F receptor), has been found with molecular genetic techniques, finally describing a receptor subtype that is known for 4 decades now. To date, all three receptor types have been cloned for various species and are members of the seven-transmembrane domain receptor super-family [1, 2, 3, 4, 5, 6, 7]. These receptors share a large degree of homology in molecular structure and in pharmacological properties (see for recent review [8]). Furthermore, at signal transduction level, all three receptor types are coupled to phospholipase C in a stimulatory manner, i.e. receptor activation results in increased hydrolysis of membrane phospholipids into inositol-trisphosphate (IPs) and diacylglycerol [9, 10]. Together, these unifying criteria, i.e. molecular genetic information, signal transduction biochemistry and pharmacology, were proposed by the nomenclature committee of 'the serotonin club', and used to define the 5HT2 receptor family [11, 12]. In this chapter we will attempt to provide an overview of anatomical, physiological and pharmacological data that currently exist on 5-HT2-type receptors. Furthermore, with the recent cloning of the 5HT2B receptor and the imminent development of more selective agonists and antagonists as well as the availability of clinical data pertaining to 5-HT2-type antagonists, we may reconsider some of the implications of 5-HT 2 receptors for application in psychiatric disorders.

Anatomical distribution of 5-HT2-type receptors a) 5-HT2A receptors: Using various tritiated radioligands, such as ketanserin, mesulergine, LSD and spiperone, extensive mapping of 5-HT2A receptors has been done in a variety of species, including rat [13] and man [14]. With [3H]ketanserin 5HT2A receptors were found to occur widely throughout the brain with particularly high densities in layer IV of frontal and cingulate cortex, in the mamillary nuclei,

200 claustrum and enthorhinal cortex [13]. In general, receptor expression seems to be more abundant in telencephalic regions, whereas the more caudally located mesencephalic regions and brain stem and spinal cord have quite low densities of 5-HT~A receptors [13, 15]. In the human brain, binding of [3H]ketanserine was found in layers II, III and V of the frontal cortex, as well as in hippocampus (particularly the pyramidal layers of CA1 and CA3, as well as the dentate gyrus) [16]. In addition, high levels of [~H]ketanserine binding were found in caudateputamen and in the dorsal raphe, but neither were found to be subject to displacement with serotonergic drugs. This may be explained at least in part by the binding of ketanserin to monoamine transporters that are abundantly present in dopaminergic terminals in striatum [17]. Initial studies for anatomical distribution of 5-HT2A mRNA in brain and peripheral organs indicated the abundant expression of 5-HT2A receptor mRNA in rat cortex and olfactory bulb [3, 18], whereas no detectable levels of mRNA were found in various other brain region extracts, nor in peripheral organs such as heart, lung and spleen [3]. The particularly high expression levels in cortex is in close agreement with receptor autoradiographic studies using tritiated ketanserine [13]. Finally receptor binding studies indicated high affinity binding of [3H]ketanserine in membrane preparations of a number of peripheral tissues, including, platelets [19, 20], uterus [21], and vesselwalls [99, 100].

5-HT2s receptors: This is the most recently cloned and identified member of the 5HT2-type receptors, which shares a number of pharmacological properties with 5-HT2A and 5-HT2c receptors [22, 4, 7, 6]. In man, 5-HT2B receptor mRNA is expressed in a variety of peripheral tissues, i.e. heart, ovaries, lung, skeletal muscle, kidneys, trachea, testes, and is abundantly expressed in the small intestine and uterus [7]. In the central nervous system, 5-HT2n receptors are also widely expressed, including in thalamus, hypothalamus, amygdala, hippocampus, cortex and cerebellum [7], albeit in very low levels. In the central nervous system, 5-HT2B receptor expression has been demonstrated in the developing mouse brain as early as embryonic day 10.5, and gradually declines [4]. In human brain samples, signal amplification by polymerase chain reaction (PCR) is necessary to detect 5-HT2B receptor expression [7]. With regard to the developmental expression pattern of the mouse 5-HT2B receptor, it can be suggested that 5HT2B receptors may play a role in neural development [4]. Stable transfection of 5-HT2c receptors into fibroblasts resulted in receptor-induced cell transformation, i.e. cell growth occurred in foci [2]. Although it was recognized that the receptor was brought into an unnatural environment, still its potential capacity to induce cell transformation, might substantiate its putative role in neural development, i.e. cell growth and differentiation. In conjunction with the receptor cloning, selective ligands are only now becoming available and may allow the elucidation of 5-HT~B receptor localization and function using receptor autoradiography [23]. However, at present no such information exists.

201

5-HT2creceptors: Similar to 5HT2Areceptors, the anatomical distribution of 5-HT2c receptors have been described using in vitro receptor autoradiography. First studies were performed using tritiated ligand such as 5HT, mesulergine and LSD [14, 24, 25]. Using excess of various selective non-radioactive competitive ligands, the occurrence of 5-HT2c receptors could be defined. 5-HT2c density is particularly high in epithelial cells of the choroid plexus in all ventricular systems [25]. In addition, low abundance of 5-HT2c occurs in layer III of the cortex, olfactory tubercles, globus pallidus and substantia nigra [25]. Anatomical localization of 5-HT2c mRNA, using specific oligonucleotide probes with in situ hybridisation, revealed the presence of receptor-expressing cells distributed widely through the central nervous system, including thalamus (lateral habenulae), hypothalamus, hippocampus, basal ganglia and spinal cord, with the highest expression in the choroid plexus epithelial cells. In these studies, northern blots revealed no detectable amounts of 5-HT2c receptor mRNA was found in peripheral organs such as liver, kidneys, heart, lung and intestine [1, 18, 26, 27, 34]. Furthermore, 5-HT2c receptor mRNA has been found in the lateral amygdala, enthorhinal cortex, nucleus accumbens, striatum, septum, subiculum, and substantia nigra [18]. The substantia nigra contains the dopaminergic cell bodies of the A9 dopamine system, whereas the other areas are all innervated by dopaminergic projections, both arising from the substantia nigra or the ventral tegmental area [28]. In addition, abundant 5HT2c receptor mRNA has been found in the suprachiasmatic nucleus (SCN), whereas only few scattered cells expressed 5-HT2A receptor mRNA [29]. This suggests a role of 5-HT2c receptors in maintenance of biological rhythm. Notably, it was found recently that disruption of 5-HT2-type receptor neurotransmission using the non-selective antagonist ritanserine resulted in an increase in slow wave sleep [30]. Finally, in the human brain low levels of 5-HTzc receptor binding was observed in layer III of the frontal cortex and the pyramidal layers of hippocampal CA1 and CA3. Again, the highest receptor density for 5-HT2c receptors was found in the choroid plexus [16]. Ontogenetic studies revealed that 5-HT2c receptors emerge relatively early in embryonic development, i.e earlier than for instance 5-HT1A and 5-HT1B receptors [31]. The possible involvement of 5-HTzc receptors, like that of 5-HT2B receptors, in developmental signals is supported by the finding that 5HT~c binding is high in the visual cortex (area 17, layer IV) of the cat during development of ocular dominance [32]. Transient expression occurred in columnar structures between postnatal days (PD) 45 and PD90 but disappeared thereafter. Interestingly, 5-HT2 expression remained elevated until PD120 but seems to be confined to the upper part of layer IV of areas 17 and 18. Increased receptor densities appeared to be organized in the same columnar structures as are the 5-HT2c receptors [32]. Association of 5-HT2c receptors with growth and regulation of cell division has been suggested [33, 2].

5-HT2c and the choroid plexus The highly enriched expression of 5-HT2c receptors on epithelial cells of the choroid plexus suggest a role in conveying serotonergic innervation to epithelial cell function.

202 The choroid plexus constitutes a cell lining between blood and cerebrospinal fluid (CSF) and selectively controls transport processes from the blood into the CSF [36]. Choroid plexus endothelial cells exclusively express 5-HT2c receptors and are densely innervated with 5-HT-containing fibres, originating from the raphe nuclei [37]. CSF is produced by ultrafiltration through the endothelial lining and the endothelial cells secrete a number of proteins into the ultraliltrate, thereby forming the CSF. These proteins include transferrin, transthyretin and ceruloplasmine [35]. Synthesis and secretion of transferrin, and possibly various other secreted proteins as well, appeared to be subject to regulation by 5-HT2c receptors, i.e. the 5-HT2c receptor agonists 5-HT, MK212 and LSD, the latter in a stereoselective manner, stimulated secretion oftransferrin by cultured choroid plexus endothelial cells without altering intracellular steady-state levels of transferrin [35]. In these preparations, transferrin production was attenuated by incubation with 8Br-cAMP, suggesting that expression oftransferrin in choroid plexus endothelial cells reflect a balance between the stimulatory 5-HT moiety and an inhibitory part that is conveyed through cAMP. Thus 5-HT-stimulated PI-turnover was found to be antagonized by mianserine and ritanserine, whereas spiperone was inactive in this respect. However, these antagonists all failed to block the stimulatory 5-HT effects on transferrin expression [35]. Although it has been discussed that 5-HT2c receptors might not be the sole receptor mediating the 5HT response, it has not been considered that these studies consisted of relatively long treatments (4 days) in contrast to studies addressing PI-turnover (60 rain). It is at present unknown how long receptors should be stimulated in order to increase transferrin expression. Furthermore, even at high antagonist concentrations, there is a (small) fraction of receptors unoccupied that may mediate stimulatory effects on transferrin expression. Finally, intermittent receptor stimulation, rather than continuous receptor activation, may yield prolonged effects on gene transcription. Molecular b i o l o g y o f 5-HT 2 r e c e p t o r s With the cloning of the 5-HT 2 receptor cDNAs it has become apparent that all three receptor types share a great deal of homology in the overall receptor coding sequence and in particular in the conserved membrane-spanning regions. All three 5-HT2 belong to the so-called seven transmembrane receptor superfamily [1, 2]. These receptors are highly homologous in that they share 51% of the overall amino acid sequence, whereas more than 80% sequence homology is found in the transmembrane-conserved-regions [8, 38, 7, 5, 39].

a) 5-HT2Areceptors: Rat 5-HT~Areceptor cDNA was identified using probes, derived from the previously cloned 5-HT2c receptor [3]. The coding sequence encodes a 471 amino acid membrane protein with a hydropathy plot suggesting a seventransmembrane receptor structure. Subsequently, the 5-HT2A receptor was cloned from man [5], mouse [40] and hamster [41]. The human 5-HT2A receptor is 90% homologous to the rat 5HT2A receptor [5] and as high as 98% in the membranespanning domains. The human receptor was brought to expression in Swiss 3T3 cells for further pharmacological characterization [38]. It was found that the

203 receptor-coding sequence was interrupted by two introns. Intron I consists of about 2.9kb, which is inserted directly adjacent to coding sequence of transmembrane domain (TM) II. A potential splice site has been identified at the codon for Gly138 (position 412 of mRNA). Intron II was found at position 613, in the centre of the region coding for TM IV and is at least 3.7kb long [38]. These data are completely in line with the genomic organization of the mouse 5-HT2A receptor [40]. Furthermore, it was found that whereas the third cytoplasmic loop usually displays large variability when different receptors are compared, as little as 2 amino acid residues are different between rat and man, indicating a strong conservation of the receptor [5]. Expression levels of 5-HT2~ receptor mRNA is subject to control of 5-HT~A receptor stimulation and PI hydrolysis in granule cells of the cerebellum [42]. In P l l cells, derived from the rat pituitary tumor cell line 7315a, 5-HT~^ receptor expression was studied [43]. Receptor stimulation using 10 ~Vl 5HT induced a transient upregulation of 5-HT~A receptor mRNA, due to both increase in gene expression as well as a doubling in the half-life of 5-HT2A receptor mRNA. These effects were clearly receptor-controlled as the effects could be antagonized by ketanserine. Interestingly, the effects could be mimicked by activation of protein kinase C (PKC) using phorbol esters, indicating that PI hydrolysis and intracellular signalling is critical to 5-HT2A receptor regulation.

b) 5-HT28 receptors: Recently, [4] identified a mouse cDNA, encoding for a receptor which was 60% homologous to the mouse 5-HT2A receptor and carried 59% homology to the mouse 5-HT2c receptor. This receptor was denoted to be the 5HT2B receptor and found to be homologous to the cloned 5-HT~F (F for fundus) receptor [6,7]. The 5-HT2~ receptor has pharmacological properties similar to those of 5-HT2A and 5-HT2c receptors. Thus, whereas 5-HT2-type agonists tend to display a higher affinity towards 5-HT2c receptors than to 5-HT2A and 5-HT~B receptors. Conversely, 5-HT2B affinity for various antagonists tend to converge to 5-HT2c receptor values more when compared with their affinity towards 5-HT2A receptors [22, 44, 4]. At present, 5-HT2B receptors have been cloned for mouse [4], rat [45, 7, 6], and man [7]. The human receptor had an overall homology of 82% with rat 5-HT2B coding sequence, which amounted to 92% for the transmembrane regions. Similar to 5-HT2A and 5-HT2c genes, the 5-HT2B receptor gene contains 2 introns.

c) 5-HT2c receptors: The coding sequence for the rat 5-HT2c receptor was first identified in a cDNA library of choroid plexus, the brain region where 5-HT2c receptors are abundantly expressed. Using an expression vector system in conjunction with Xenopus oocytes, a cDNA was found with a 1380bp open reading frame [1]. The encoded receptor displayed pharmacological characteristics similar to those observed with 5-HT2c receptors in rat choroid plexus. The cDNA encoded for a 460 amino acid polypeptide, and deduced amino acid sequence was found to have strong homology to other known seven transmembrane spanning receptors. Stable transfection in NIH-3T3 fibroblasts yielded specific binding with x25ILSD, weak affinity for spiperone and high affinity for the mixed 5-HT2J5-HT2c

204 antagonist mianserin [1]. Furthermore, use of 5HT2c agonists, such as DOI or 5HT, produces a strong, concentration-dependent stimulation of PI hydrolysis, with pharmacological properties identical to what has previously been observed with rat choroid plexus. The 5-HT2c receptor has been cloned for rat [1], mouse [46], and man [5]. There is close homology between rat and human 5-HT2c receptor: for the transmembrane spanning domains 98% homology exists, whereas the N-terminal tail of the 5-HT2c receptor is 78% homologous and the third cytosolic loop bears 'only' 71% homology between rat and human receptor [ 1]. The human 5-HT2c receptor-coding sequence has been localized on the X-chromosome [47]. Comparison between human 5-HT~A and 5-HT2c receptors indicated 50% overall homology, suggesting an evolutionary close relationship [5] as was already suggested from the identical signal-transduction pathways and the close similarity in pharmacological profile [10, 1]. As with the 5-HT2A receptor, preliminary data have been obtained that the human 5HT2c gene has at least 2 introns. P h a r m a c o l o g y o f 5-HT 2 r e c e p t o r s

Clinically, 5-HT 2 receptors have been implicated in a variety of regulatory functions. Peripherally, 5-HT 2 receptors contribute to sympathetic innervation of the cardiovascular system, for instance by mediating 5-HT-induced contractions of smooth muscles of the vascular walls [48, 49, 101]. In addition, 5-HT2 receptors contribute to regulation of plasma glucose levels [50, 102] as well as to regulation of the hypothalamus-pituitary-adrenal axis [51, 52]. In the central nervous system 5-HT 2 receptors are implicated in food intake, depression, anxiety and psychosis [53, 54, 55, 56, 57]. Furthermore, 5-HT 2 receptors are thought to convey the hallucinogenic properties of LSD [58, 59]. Several of these roles merit the search and development of 'selective' agonists and antagonists.

a) Receptor binding: First steps were set with the identification of 5-HT 2 antagonism in the neuroleptic spiperone and the subsequent development of ketanserin as a 5-HT2A antagonist lacking dopamine D 2 antagonism [60, 61]. Both ketanserin and spiperone were shown later to lack affinity towards 5-HT2c receptors [62, 63]. The first 5-HT~c antagonist to be identified was mesulergine [62], although mesulergine also displays strong antagonism at 5-HT2A receptors. In summary, it seems that 5-HT2c antagonists, like mesulergine, ritanserin and mianserin, are also very potent 5-HT~A antagonists, whereas conversely, 5-HT2A antagonists, like spiperone, ketanserin and their analogues, have no affinity towards 5-HT2c receptors at all. However, with the recent description of a series of pyridylurea derivatives [23], a class of compounds seems to arise where at least 5-HT2c receptor antagonism exceeds that for 5-HT2A receptors. The search for selective 5-HT2-type receptor agonists, which might discriminate between 5-HT2A and 5-HT2c receptors, has not been easy as well. First, 5-HT2 agonists may have hallucinogenic properties, since several 5-HT 2receptor agonists like LSD and the various phenylalkylamines, like DOI, DOB and DOM, have high affinity for 5-HT2A as well as for 5-HT2c receptors [59, 64]. Hallucinogenic properties seem to be selectivity as both LSD and the phenylalkylamines display

205 similar affinity towards both receptor subtypes. Nor is it related to intrinsic activity as LSD is a partial agonist to both 5-HT2A and 5-HT2c receptors, whereas for instance DO I is a full agonist on both 5-HT2A and 5-HT2c receptors. Until recently, hallucinogenic properties of LSD and phenylalkylamines were considered to correlate with binding affinities towards 5-HT2A receptors and much less to those for 5-HT2c receptors [59, 65]. Recent experiments however, using choroid plexus and cortex preparations, it was shown that both LSD and hallucinogenic phenylalkylamines, such as DOI, DOB, DOM, and DMA were potent 5-HT2A as well as 5-HT2c receptor agonists in a stereoselective manner [58, 64]. Moreover, stimulation of PI-turnover in choroid plexus preparations correlated well with observed binding affinities using [~H]mesulergine as radioligand [64]. 5-HT 2 receptors belong to the superfamily of G-protein-coupled receptors, indicating that agonist-induced conformational changes in the receptor protein is conveyed to its associated G-protein which exchanges its GDP-molecule for a molecule of GTP. The G-protein dissociates not only from its receptor but also splits into an a-subunit (the GTPase-moiety) and a l~7-complex, and these proteins activate a variety ofintracellular effector systems such as phospholipase C [66, 67, 68]. Association of the receptor protein with a GDP-loaded G-protein stabilizes the receptor binding site in a high-affinity conformation as measured with agonist radioligand binding or competition experiments [69]. G-protein activation can be mimicked by incubating membrane preparations with GTP or nonhydrolyzable GTP-analogs like GTPTS or Gpp(NH)p, thereby inducing the uncoupling of the receptor from its G-protein. Subsequent receptor binding reveals a low-affinity conformational state of the receptor when radioligand binding or competition os performed with agonists (see for instance [70]). In conclusion, the coupling state of a receptor has important consequences for the perceived affinity of a variety of agonists. However, binding affinity is not always reflected in functional properties as far as agonists are considered. Comparison of [3H]ketanserin binding and ['25I]DOI binding in rat frontal cortex membranes yields that on average only 5% of 5-HT2A receptors are in a high-affinity state [71]. Furthermore, agonist competition of either [3H]ketanserin or [3H]DOB from its binding sites yields considerable differences [72, 70, 71]. Thus, agonists were 10-100-fold more potent in competing with [3H]DOB for 5-HT2A binding sites as compared to their potency in competing with the antagonist [3H]ketanserin. In these experiments, perceived antagonist affinity did not depend on agonist or antagonist nature of the radioligand. Interestingly, 5-HT2c receptor binding has been done almost exclusively with [3H]5HT or ['25I]DOI binding to choroid plexus membranes, whereas 5-HT2A affinity is usually determined with [~H]spiperone or [~H]ketanserin binding in frontal cortex membranes. Comparison of affinities that are assessed with competition with agonist radioligand on 5-HT2c receptors and antagonist radioligand binding for 5-HT2A receptors has yielded a dramatic underestimation of agonist affinity for 5-HT2A receptors. By consequence, agonists have been reported to be 'selective' for 5-HT2c receptors without appreciable affinity towards 5-HT2A receptors [69]. Using cell lines that stably express 5-HT2A and 5-HT2c

206 receptors in radioreceptor binding as well as for functional studies, agonist selectivity has become less apparent. The 5-HT~D receptor binding is performed using [~H]-5-HT in cloned rat 5-HT~B receptors [103]. b) Functional tests: Definition of drug potencies in pharmacology is always related to species and tissue preparation, used to study the effects. Species differences might underlie observed differences in drug potencies due to significant deviation in receptor structure [73]. Furthermore, differences in drug potencies might be due to the presence of spare receptors in a given preparation, a phenomenon which strongly affects observed pD 2. Finally, the presence of pharmacologically related receptors in a preparation may confound the conclusions regarding properties of new compounds. In case of 5-HT2 receptors, there seems to be little species variation with regard to the primary receptor structure. This is also reflected in only minor variations in apparent binding affinity towards 5-HT2A or 5-HT2c receptors. The presence of spare receptors may, however, strongly affect observed pD 2 values for agonists. For choroid plexus this was resolved using the irreversible antagonist phenoxybenzamine. It was found that rat choroid plexus cells expressed a small receptor reserve, constituting about 50% extra, thereby allowing a slightly more potent effect of 5-HT on PI-turnover [64]. Furthermore, it was demonstrated that partial agonists, such as LSD and TFMPP, were strongly affected in observed intrinsic activity. Partial agonists such as LSD may be found as full agonists, partial agonists or antagonists, depending on the amount of spare receptors for 5HT 2 receptors. The occurrence of receptor reserve may yield different conclusions with respect to description of new pharmacological compounds (see [74] for example). Finally, the presence of pharmacologically related receptors makes it difficult to discriminate between specific receptor effects. Inasmuch as cardiovascular research is concerned, the contribution of 5-HTz~ receptors to blood pressure through contraction of smooth muscle cells, seems firmly established. 5-HT-stimulated rise in blood pressure or in vitro arterial contractions, is subject to ketanserin and spiperone [75, 48, 49]. However, it was also shown that a close correlation exists with al-antagonist properties, also exerted by ketanserin. Furthermore, the mixed 5HT2~2c antagonist ritanserine was much less effective in mimicking the vascular relaxing properties of ketanserin [75]. In tracheal preparations, 5-HT-stimulated PI-turnover was partially blocked by ketanserin [76]. Therefore these studies do not rule out the possibility of other receptor types, since for instance the a~-adrenoceptor is coupled to PI-turnover as well. Finally, in aorta preparations, 5-HT-stimulated PI-turnover was subject to antagonism by ketanserin [77], however, the mixed 5-HT2~ c antagonist mianserin displayed a biphasic profile, indicating that 5-HT2~3or 5-HT2c receptors might also mediate some of the 5-HT-induced effects.

Signal transduction Both 5-HT2A and 5-HT~c receptors preferentially couple with phospholipase C

207 (PLC) through Gq proteins [58, 66, 78]. Agonist stimulation of these receptors increase activity of PLC, resulting in increased hydrolysis of the membranephospholipid phosphatidylinositol-bisphosphate (PIP 2) into inositol(1,3,4)triphosphate (IP 3) and diacylglycerol (DAG) [67]. Intracellular IP3 can subsequently promote the transient release of calcium that is stored in intracellular organelles such as the endoplasmic reticulum, golgi and mitochondria. The calcium spikes can be considered as third messengers and may elicit its own effects through its association with calmodulin. One of these actions is the stimulation of protein kinase C (PKC), together with DAG. Activated PKC will phosphorylate a number ofintracellular protein substrates, thereby producing an altered cellular state which can remain for a prolonged period of time and may be reversed by various phosphatases. Recently, intracellular cyclic GMP levels in choroid plexus were found to respond to 5-HT2c receptor stimulation as well [79]. The formation of cGMP by guanylate cyclase was completely dependent on calcium, suggesting a sequential appearance of IP 3 and cGMP. In this respect, Gq has been implicated in the activation of PLC. At present it is not clear whether cGMP formation is a direct consequence of IPa-stimulated rise ofintracellular Ca 2§ can activate NO synthase, producing NO of arginine, which in turn is an activator of cytosolar guanylate cyclase [80]. In this respect cGMP might function in modulating calcium fluxes in epithelial cells. 5-HT2-type receptors couple positively to phospholipase C (PLC) as the principal means of conveying extracellular stimuli into intracellular responses [9]. Moreover, this type of signal transduction has become one of the hallmarks in use for classification of the 5-HT2 receptors [11, 12]. Hydrolysis of PIP 2 into IP3 and diacylglycerol (DAG) results in a cascade of intracellular responses, including mobilization ofintracellular calcium, sequestered in golgi and endoplasmic reticulum [67,81]. Release of calcium transients is due to interaction with formed IP 3 and an IP~-specific receptor (which is Ca §247 sensitive). In addition, Ca§247 and DAG act in conjunction to activate protein kinase C (PKC) which will phosphorylate a number of proteins. To complicate intracellular signalling, DAG not only activates PKC, but in addition, is substrate to the formation of arachidonic acid by action of the membrane-bound enzyme diacylglycerol-lipase [82, 83]. Arachidonic acid freely diffuses out of cells and may elicit a variety of responses. In addition, arachidonic acid can be converted by cyclooxygenase and lipogenase enzymes into a variety of eicosanoid products, including prostaglandins and leuktrienes which induce vasoconstriction and are implicated in a number of physiological processes such as thermoregulation, vasoconstriction, and inflammation. The complex interactions of 5-HT2-type receptor-mediated effects in conjunction with arachidonic acid species have not yet been studied into any detail [79]. 5-HT~-type r e c e p t o r s a n d b e h a v i o u r

Analysis of 5-HT2-type receptor-mediated behaviour completely depends on the use of selective agonists and antagonists, i.e~ the affinity of used compounds for

208 5HT 2 receptors should be at least one order of magnitude greater than those for other, possibly interfering receptors. For 5-HT2A receptors, agonist-induced behavioural changes should be sensitive to ketanserin antagonism. Thus, it was shown in rats that ketanserin-sensitive head-twitches and wet dog shakes can be induced by the non-selective full 5-HT 2 agonist DOI, as well as by the non-selective partial 5-HT2A agonists mCPP, MK212, and TFMPP [84, 85, 86]. Thus, head-twitches and wet dog shakes are likely to be 5-HT2A receptor-mediated. mCPP-induced head twitches were significantly reduced when the central 5-HT system was lesioned using the neurotoxins 5,7-DHT or pCPA [87]. As lesions with 5,7DHT does not change the number of ketanserin binding sites in rat brain [17] one can conclude that the 5HT2A receptors are postsynaptically located and that receptor expression is unlikely to be controlled by tonic 5-HT-innervation. Changes in responsivity to mCPP, however, may be due to either slight variations in the amount of spare receptors, bearing in mind that mCPP is a partial agonist, or that coactivation of other receptors, such as 5-HTIA receptors, to which mCPP also acts as an agonist. As lesioning of the 5-HT system was done 10 days prior to behavioural testing, it is likely that adaptive changes in 5-HT signalling has occurred, including supersensitivity of 5-HT~c receptors [88]. 5-HT2c-receptor-mediated effects has been proposed for feeding behaviour [89, 90], locomotion [44] and penile erection [84]. Pharmacological assessment of involvement of 5-HT2c receptors is largely based on 5-HT2-type receptor agonistinduced behavioural changes that appear to be insensitive to the 5-HT2A-selective antagonist ketanserin, but can be dose-dependently antagonized by non-selective 5-HT2-receptor antagonists such as mianserin and ritanserine [44, 84]. With the recent advent of 5-HT2n receptors and putatively selective antagonists, some of the behavioural test models may have to be reevaluated as claims have been made that several paradigms may be mediated through 5-HT2B receptors. In this respect mCPP-induced decrease in social interactions as well as feeding behaviour was found to be sensitive to the 5-HT2~/2c-selective antagonist SB200646A [44, 53]. As there are at present no selective 5-HT2c antagonists available, the matter of multiple receptors contributing to a certain effect cannot be resolved easily through pharmacological methods. However, we recently found that 5-HT2A and 5-HT2c receptors are differentially vulnerable to the irreversible antagonist EEDQ [91, unpublished results]. Using EEDQ, 5-HTA receptors are easily alkylated in a concentration- and time-dependent manner, completely abolishing 5-HT2A binding properties and functional responses [72]. Interestingly, 5-HT2c receptors remain unaffected under similar conditions. Thus, EEDQ might be instrumental to assessing 5-HT2c contribution to an effect after challenge of 5HT 2 receptors with a non-selective agonist like DOI or mCPP. In this respect, some caution must be taken as several other receptors are vulnerable to EEDQ as well, including various adrenergic receptors, muscarinic and dopaminergic receptors. Several in vivo models have been used to describe functional 5-HT2A and 5-

209 HT2c responses. Although only weakly selective, m-CPP has been used extensively in analyzing 5-HT~c contribution in animal models for anxiety, eating disorder and obsessive-compulsive behaviour, m-CPP induces hypophagia in food-deprived rats or in schedule-induced feeding paradigms, which can be attributed, at least in part, to the activation by mCPP of 5-HT2c or 5-HT2~ receptors [92, 90, 86, 44]. Interestingly, the recently developed 5-HT.~c knock-out mouse strain suggests multiple receptor functions. Mice, lacking 5-HT~c receptors become obese, i.e. have increased (up to 50%) fat stores without concomitant increase in body size [89]. Furthermore, the animals show a decrease in threshold doses of metrazol-induced convulsions. Thus, 5-HT2c receptors are clearly involved in appetite regulation as well as in controlling activity of neuronal networks, i.e. regulation of GABA and glutamate neurotransmission [89]. In this respect, the decrease in seizurethreshold due to absence of 5-HT2c receptors may be related to the proconvulsive effects of chronic treatment with antipsychotics [93]. Thus, adaptive changes in 5HT2c receptors, due to chronic treatment with antipsychotics [94, 95] yields a decrease in the number of 5-HT2c binding sites without an appreciable effect on maximal receptor-mediated PI hydrolysis [95]. Thus, it would seem that the amount of receptor reserve is decreased, thereby diminishing the 5HT2c-receptormediated control of amino acid neurotransmission. T h e r a p e u t i c options for 5-HT2-1ike receptors in psychiatric disorders 5-HT2-type receptors have been a focus for investigations into its putative clinical importance for treatment of psychiatric disorders. 5-HT2-type receptors have been implicated in several disorders like anxiety, depression and psychosis. As we are presently in the dark with respect to the mechanisms underlying the development of these disorders, future pharmacotherapy largely aims at alleviating symptoms. To that end various compounds, which have antagonistic properties on 5-HT2-type receptors, are currently tested in clinical studies. For instance, it has been suggested that LSD-induced hallucinations might resemble those experienced during psychotic episodes. Although todate it is recognized that LSD-induced hallucinations are clearly distinct, one can still consider that somehow receptors that display high affinity for LSD interact with conveying and interpreting sensory information. Although LSD is not highly selective towards 5-HT2-type receptors, the phenylalkylamines like DOI, DOB and DOM seem to be more selective, and these too are hallucinogenic. Thus, it can be argued that 5-HT2-type receptors distort sensory information which are experienced as hallucinations. Therefore, 5-HT.~ receptor antagonists may compensate for these distortions. Interestingly, clozapine is a successful drug in treatment of both positive and negative symptoms of schizophrenia. One current theory is that these properties come from the combination of dopamine D2 and 5HT 2 antagonism. Although clozapine displays moderate affinity towards a plethora of other receptors, the antipsychotic properties of clozapine seem to be retained in much more cleaner compounds such as risperidone [96], YM-8018 [97] and olanzepine, which are close relatives of clozapine. It is claimed that combined dopamine D2 and 5-HT2-type receptor antagonism is effective in treatment of both positive and negative

210 symptoms of schizophrenia and reduces the incidence of extrapyramidal symptoms. At present, however, it remains to be established whether the positive contribution of 5-HT2-type receptors arises from 5-HT2A, 5-HT2B or 5-HT2c receptors, or a combination. 5-HT2-antagonism is likely to be a clean contribution to antipsychotics, and indeed other CNS drugs, as 5-HT 2 receptors seem to mainly convey adverse effects when stimulated by agonists, i.e. increased blood pressure, hypoglycaemia, hallucination, etc. These are unlikely to be found in drugs having 5HT 2 antagonism. For antidepressant and anxiolytic properties of 5-HT 2 antagonists a number of data are currently available, although in vivo behavioural pharmacology is still carried out with non-selective drugs, such as mCPP. Specific functions however, can be elucidated by use of specific antagonists such as ketanserin (5-HT2A) and the recently developed SB200646 and SB204741 [23, 44]. Thus, in animal models, the anxiogenic properties of mCPP [86, 85] can be antagonized by SB200646, but remains insensitive to ketanserin [44]. Thus it has been argued that 5-HT2A receptors do not seem to be involved in the anxiogenetic mechanism of mCPP. Furthermore, 5-HT2a and 5-HT2c receptors seem to be involved in eating disorders [44]. Interestingly, the recent development of 5-HT2c-deficient mice [89] are obese. These data strongly support the notion that 5-HT2n and 5-HT2c receptors are involved in anxiety, OCD and depression. In that respect, a possible role for 5-HT A receptors is less well supported.

Summary 5-HT2-type receptors occur throughout the central nervous system. These receptors exert more subtle effect on the brain as measured in a variety of behavioural paradigms. Furthermore, behavioral effects are not easily attributed to one receptor subtype alone, although this is for a large part due to lack of truly selective agonists and antagonists. It can be expected that a better understanding of the neurobiology of 5-HT2-type receptors through development of selective agonists and antagonists may help in disclosing the design of truly selective 5HT2-type receptor agonists and antagonists for CNS purposes. Furthermore, the development of knock-out mice for the 5-HT2c receptor is likely to be followed closely by other knock-out mouse strains and strains that over-express certain receptors. These animals will stand model for psychiatric disorders, as the behavioral deficits will become more apparent. Needless to say that compensatory mechanisms are likely to occur as the brain is highly plastic and there is redundant control in most, if not all systems. With respect to 5-HT2a and 5-HT2c receptors, this is likely, as evidence for their involvement in cell growth and differentiation has already accumulated. Thus, anatomical and functional deficits are likely to arise at the embryonic stage already. This might be one of the underlying factors in the increased vulnerability to seizures in 5HT2c knockout mice [89]. In this respect, the development of inducible knockouts may provide additional model systems for acutely developing disorders, which have no 'organic' origin. The onset of receptor deficit or over-expression can be wholly controlled and studied [98]. This type of progress will prove to be important tools for both neurobiological

211 research as well as for the development pharmacotherapy. In this respect a less laborious approach, i.e. antisense infusion into the brain which produces (partial) knockouts, has already produced data on the importance of a variety of receptors.

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Sanders-Bush E, Burris KD, Knoth K. J Pharmacol Exp Ther 1988; 246: 924928. Titeler M, Lyon RA, Glennon RA. Psychopharmacol 1988; 94: 213-216. Leysen JE, Niemegeers CJE, Tollenaere JP, Laduron PM. Nature 1978; 272: 163-166. Leysen JE, Awouters F, Kennis L, Laduron PM, et al. Life Sci 1981; 28: 10151022. Pazos A, Hoyer D, Palacios JM. Eur J Pharmacol 1985c; 106: 539-546. Herndon JL, Ismaiel A, Ingher SP, Teitler M, et al. J Med Chem 1992; 35: 4903-4910. Sanders-Bush E, Breeding M. Psychopharmacol 1991; 105: 340-346. Glennon RA, Dukat M, El-Bermawy M, Law H, et al. J Med Chem 1994; 37: 1929-1935. Milligan G. The Humana Press Inc 1992; 20: 1-56. Berridge MJ, Irvine RF. Nature 1989; 341: 197-204. Lo WWY, Hughes J. FEBS lett 1987; 224: 1-3. Leonhardt S, Gorospe E, Hoffman BJ, Teitler M. Mol Pharmacol 1992; 42: 328-335. Shannon M, Battaglia G, Glennon RA, Titeler M. Eur J Pharmacol 1984; 102: 23-29. Lyon RA, Davis KH, Titeler M. Mol Pharamcol 1987; 31: 194-199. Battaglia G, Shannon M, Titeler M. J Neurochem 1984; 43: 1213-1219. Kenakin TP. Pharmacol Rev 1984; 36: 165-222. Mylecharane EJ. Kluwer Academic Publishers, Dordrecht, 1990; pp 81-100. Blauw GJ, Van Brummelen P, Van Zwieten PA. Kluwers Academic Publishers, Dordrecht, 1990; pp. 391-399. Yang CM, Yo Y-L, Hsieh JT, Ong R. Br J Pharmacol 1994; 111: 777-786. Roth BL, Nakaki T, Chuang D-M, Costa E. J Pharamcol Exp Ther 1986; 238: 480-485. Fain JN, Wallace MA, Wojcikiewicz JH. FASEB J 1988; 2: 2569-2574. Kaufman MJ, Hartig PR, Hoffman BJ. J Neurochem 1995; 64: 199-205. Knowles RG, Palacios M, Palmer RMJ, Moncada S. Proc Natl Acad Sci 1989; 86:5159-5162. Krause K-H. FEBS lett 1991; 285: 225-229. Bazan NG. Ann NY Acad Sci 19.. vo! 559; pp. 1-16. Mukherjee AB, Miele L, Pattabiraman N. Biochem Pharmacol 1994; 48: 1-10. Berendsen HHG, Jenck F, Broekkamp CLE. Psychopharmacol 1990; 101: 5761. Curzon G, Kennett GA. Trends Pharmacol Sci 1990; 11: 181-182. Kennett GA, Whitton P, Shah K, Curzon G. Eur J Pharmacol 1989; 164: 445454. Berendsen HHG, Broekkamp CLE. Behav Neural Biol 1991; 55: 214-226. Conn PJ, Janowsky A, Sanders-Bush E. Brain Res 1987; 400: 396-398. Tecott LH, Sun LM, Akana SF, Strack AM, et al. Nature 1995; 374: 542-546. Kennett GA, Curzon G. Psychopharmacol 1988; 96: 93-100. Ronken E, Menkveld A, Van Dijk R, Kodden L. 1996 (unpublished studies).

214 92 93 94 95 96 97 98 99 100 101 102

103

Aulakh CS, Zohar J, Wozniak KM, Hill JL, et al. Psychopharmacol 1989; 98: 448-452. McInerney SC, Hofman DC, Donovan H, Cornfield LJ, et al. Neuroscience meeting, 1994; poster 726.6. Canton H, Verri~le LMillen MJ. Neurosci Lettl 1994; 181: 65-68. Knoppam~iki M, P~ilvimtiki E-P, Sjv/~lahti E, Hietala J. Eur Pharmacol 1994; 255: 91-97. Rabasseda X, Mealy N, Peuskens J. Drugs of Today 1993; 29: 535-553. Ishibashi T, Ohno Y, Ishida K, Ikeda K, et al. Abstract 726.13, 24th Society for Neuroscience 1994, Miami. Lucas JJ, Hen R. Trends in Pharmacol Sci 1995; 16: 246-252. Ullmer C, Schmuck K, Kalkman HO, Lfibbert H. FEBS Lettt 1995; 370: 215221. Schmuck K, Ullmer C, Kalkman HO, Probst A, Lfibbert H. Eur J Neurosci 1996; 8: 959-967. Watts SW, Bael M, Webb CR. J Pharmacol Exp Ther 1996; 277: 1103-1113. Sugimoto Y, Yamada J, Yoshihawa T, Hurisaka K. Eur J Pharmacol 1996; 307: 75-80. Wainscott DB, Cohen ML, Schenck KW, Audia JE, et al. Mol Pharm 1993; 43: 419-426.

Serotonin Receptors and their Ligands B. Olivier, I. van Wijngaarden and W. Soudijn (Editors) 9 1997 Elsevier Science B.V. All rights reserved.

215

5-HT 2 R e c e p t o r antagonists: (potential) t h e r a p e u t i c s W.Soudijn Leiden/Amsterdam, Center for Drug Research, P.O. Box 9502, 2300 RA Leiden, The Netherlands.

THERAPY Treatment of schizophrenia with typical neuroleptics like for instance chlorpromazine, haloperidol or fluphenazine has a beneficial effect on the positive symptoms of schizophrenia (delusions, hallucinations and positive thought disturbances) but little effect on the negative symptoms (social withdrawal, blunted affect and apathy). Extrapyramidal side effects (motor disturbances EPS) are commonly encountered. It is generally accepted that blockade of dopamine receptors is probably responsible for the effect on the positive symptoms as well as for causing EPS. The discovery that antipsychotics with a relatively high affinity for 5-HT 2 receptors had a positive effect on the negative symptoms and caused little or no EPS led to the development of a large number of compounds combining antiserotonergic and antidopaminergic activity in different ratios. Based on the examination of binding data of 37 antipsychotics Meltzer et al. [1] proposed that the ratio of 5-HT~A/D2 receptor binding affinity determines whether a compound can be classified as an atypical or a typical antipsychotic. Atypical antipsychotics have a higher affinity for 5-HT2Areceptors than for D 2 receptors and the reverse is true for typical antipsychotics [1]. Some caution however is recommended. Clozapine for instance has such a low affinity for D2-receptors that though the ratio seems sufficiently large an effect on the positive symptoms at the clinical doses used is not to be expected. However, clozapine has a 10-fold higher affinity for the D4-receptor that may explain its efficacy on the positive symptoms of schizophrenia [2-4]. Ritanserin has a 10-fold higher affinity for both D 2- and 5-HT2A receptors than clozapine [5]. Its clinical usefulness as an antipsychotic is not clearly established. However as comedication with haloperidol - a typical neuroleptic - ritanserin has a significant effect on the negative symptoms. Risperidon with an affinity for the D 2 receptors comparable to that of haloperidol and an affinity for the 5-HT2A receptor 1.5 fold that of ritanserin [5] is clinically effective on both positive and negative symptoms in the dose range 4-8 mg dd without causing EPS. Higher doses than 10 mg however may cause EPS. The question arises whether 5-HT2A antagonism per se may be sufficient to treat positive as well as negative symptoms in schizophrenia without causing EPS.

216 MDL 100,907 seems a likely candidate for answering this question as the compound is a potent and selective 5-HT2A antagonist with a I~. for the 5-HT~A receptor of 0.36 nM, for the 5-HT2c receptor of 105 nM, for the al receptor of 545 nM and with I~.'s >1000 nM for oh, D2 5-HT~A, 5-HT1D, 5-HT3, H1, muscarine, gaba, glycine and benzodiazepine receptors [6]. However for a conclusive answer to the question the affinity for the D4 receptor which to our knowledge has not yet been reported should be negligible. The pharmacology of MDL 100,907 was summarized in a mini review [7]. The question whether there is a causal relationship between 5-HT~ antagonism and a beneficial effect on EPS is still not unequivocally resolved [12, 13]. New potential antipsychotics with a low potential for EPS and a beneficial effect on negative symptoms are presently under clinical investigation e.g. sertindole, olanzapine, seroquel and ziprasidone [14, 15]. The ratios of the airmities for the 5-HT 2 and D2 receptor of these compounds are quite different. The affinities for the 5-HT and dopamine receptor subtypes also differ as well as their affinity for e.g. the a~-adrenergic receptor. A potentially useful drug in the treatment of generalized anxiety disorder was recently reported [8,11]. SB 206553 a 5-HT2c~B receptor antagonist selective over 5-HT2A and other 5-HT receptors tested is a ring closed congener of SB 200646 A the first selective 5-HT2caB antagonist. SB 206553 however has a considerably higher activity in receptor binding - as well as in functional tests both in vitro and in vivo. The affinity for other receptors (D2, D3, a~, H~, A1) is negligible. For the role of 5-HT in depression and anxiety see [9] and references herein. Ketanserin a 5-HT2A antagonist with al-adrenolytic activity is clinically used as an antihypertensive agent. The precise molecular mechanism of action is still a matter of debate. The potential usefulness of ketanserin in the treatment of portal hypertension, airway obstruction, acute respiratory failure, carcinoid syndrome and neurogenic bladder is still under investigation. An in depth review of the clinical pharmacological aspects of ketanserin also in relation to the proposed mechanism of action has been published recently. [10]. REFERENCES

1 2 3 4 5 6

Meltzer HY, Matsubara S, Lee J-C. J Pharmacol Exp Therap 1989; 251: 238246. Van Tol HHM, Bunzow JR, Guan HC, et al. Nature 1991; 350: 614-619. Van Tol HHM, Wu CM, Guan HC, et al. Nature 1991; 358: 149-152. Breier A. Schizophrenia Research 1995; 14: 187-202. Leysen JE, Janssen PMF, Schotte A, Luyten WHML, et al. Psychopharmac 1993; 112: $40-$54. Palfreyman MG, Schmidt CJ, Sorensen SM, Dudley MW, et al. Psychopharmac 1993; 112: $60-$67.

217 7 8 9 10 11 12 13 14 15

Schmidt ChJ, Sorensen SM, Kehne JH, et al. Life Sci 1995; 56: 2209-2222. Forbes IT, Ham P, Booth DH, Martin RT, et al. J Med Chem 1995; 38: 25242530. Baldwin D, Rudge S. Int Clin Psychopharmacology 1995; 9 suppl 4: 41-45. Frishman WH, Huberfeld S, Okin S, et al. J Clin Pharmacol 1995; 35: 541572. Kennett GA, Wood MD, Bright F, Cilia J, et al. Brit J Pharm 1996; 117: 427434. Kaput S. Psychopharmacology 1996; 124: 35-39. Kapur S, Remington G. Am J Psychiatry 1996; 153: 466-476. Gerlach J, Peacock L. Int Clin Psychopharmacology 1995; 10 Suppl 3: 39-48. Tamminga CA, Lahti AC. Int Clin Psychopharmacology 1996; 11 Suppl 2: 7376.

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Chapter 7

5-HT 3 R E C E P T O R S

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Serotonin Receptors and their Ligands B. Olivier, I. van Wijngaarden and W. Soudijn (Editors) (~) 1997 Elsevier Science B.V. All rights reserved.

221

5-HT3 Receptors H.Gozlan INSERM U-29, H6pital de Port-Royal, 123, Boulevard de Port-Royal, 75014 Paris, France.

INTRODUCTION As is the case for the other monoamine neurotransmitters, serotonin (5-HT) exerts its effects through multiple receptors. The noradrenaline and dopamine receptors belong exclusively to the G protein-coupled receptor family, whereas those of acetylcholine and 5-HT also include ligand-gated ion channel types. At least nine G protein-coupled 5-HT receptors have been pharmacologically characterized, but the genes coding for many more 5-HT receptors of the same family have been cloned and sequenced [1]. In contrast, 5-HT directly stimulates only one ionic channel, and only one ligand-gated receptor has been cloned so far. Indeed, the 5-HT 3 receptor is a cationic channel which mediates some of the excitatory properties of 5-HT and which displays some analogies with the nicotinic receptor. Interestingly, this receptor resembles the M receptor, described long ago by Gaddum and Picarelli [2] in the guinea-pig ileum, and found later in several other peripheral neuronal preparations. The characterization of this receptor was long hampered by the lack of specific agonists and antagonists, until the pioneering work of Fozard and of Richardson led to the discovery of the first potent and specific 5-HTa receptor antagonists, which have been of considerable help in furthering the knowledge in this field. Finally, the identification of central 5-HT a binding sites and the potential therapeutic properties of 5-HT3 receptor antagonists have further stimulated the interest for this unique 5-HT receptor. As a result, considerable data has been accumulated over the past 8 years, and pharmacological, behavioural, and electrophysiological characteristics of 5-HT3 receptors and their ligands have already been the subject of several documented reviews [3-10, 209-213]. 5-HTs R E C E P T O R LIGANDS 5-HT 3 r e c e p t o r agonists The excitatory response of 5-HT through M receptors, initially described in the guinea-pig ileum, has also been found in other species (rabbit, rat) and in other tissue preparations (heart, vagus nerve, superior cervical ganglion). It is also recognized that the well-known von Bezold-Jarisch reflex can be evoked by 5-HTa agonists. More recently the uptake of[14C]-guanidinium in clonal cell lines has also been shown to be a useful functional model [11]. Different classes of drugs-

222 tryptamines, aryl-biguanides and some heterocyclic amines-have been shown to behave as agonists in these different models (figure 1). These include several Nand C-methylated serotonin derivatives and 2-methyl-5-HT. The latter was initially described as more selective for this receptor than 5-HT itself [12], but was later found to also recognize several other 5-HT binding sites [13]. Interestingly, 5-methoxytryptamine, the O-methylated derivative of 5-HT, was completely devoid of activity at 5-HT s receptors [14]. This drug is therefore a discriminative drug, since it does display moderate to high affinities for all the 5-HT receptors identified to date [14,15]. Another useful agonist, 1-phenylbiguanide (PBG), structurally unrelated to tryptamines, was discovered using the rat vagus nerve preparation [16]. Its affinity for 5-HT 3 receptors was dram_atically improved by the introduction of a chlorine atom in the meta position of the aromatic ring, leading to 3-chloro-PBG [17]. Both of these drugs are quite specific for 5-HT 3 sites. However, some limitations for the use of these agonists have been reported, as 2-methyl-5-HT, PBG and 3-chloro-PBG act as partial agonists in different preparations. For instance, 3-chloro-PBG is an agonist in NIE-115 cells, but a partial agonist in NG108 15 cells, two closely related neuroblastoma cell lines [18]. Furthermore, these agonists do not cross the blood brain barrier [12, 19], limiting their usefulness to in vitro experiments (see however, [20]). Additional drawbacks were reported for PBG and 3-chloro-PBG in that these drugs are inactive at 5-HT a receptors from different guinea-pig tissues, and PBG was reported to induce, at high concentrations, a carrier-mediated release of dopamine [21]. Quipazine, a non specific ligand which was initially described as a 5-HTs receptor antagonist also behaves as an agonist in some assays [11]. More recently, SR 57227A was proposed as a high affinity agonist for 5-HT 3 receptors [19, 22]. This compound stimulated the uptake of [14C]guanidinium in NG108-15 cells, elicited the von Bezold-Jarisch reflex in the rat and contracted the guinea-pig ileum, these responses being blocked by selective 5-HT8 receptor antagonists. The most interesting feature of this compound resides in its ability to cross the blood brain barrier and to bind to central 5-HT8 receptors [19, 22]. SR 57227A would therefore be a useful tool for both in vitro and in vivo studies. Novel 5-HT 8 receptor ligands were derived from piperazino-pyrrolothieno pyrazines [214]. One of the most interesting compounds is the piperazinopyrrolo[1,2-a]thieno[3,2-e]pyrazine substituted with benzyl on the N-4 of the piperazine ring. This compound displays high affinity and selectivity for 5HT a receptors. It increases the uptake of [14C]guanidinium into NG 108-15 cells in the nanomolar dose range. This agonistic effect was blocked by the selective 5HTa antagonist ondansetron. When evaluated on the Von Bezold-Jarisch reflex in rats the compound acts as a partial agonist. High affinity and selective for 5-HT3 receptors is also observed in (R)-N(quinuclidin-3-yl)-2-(1-methyl-lH-indol-3-yl)-2-oxo-acetamide. This compound behaves as a potent partial agonist when tested on the Von Bezold-Jarisch reflex in rats [215]. The (S)-enantiomer has a 100-fold lower affinity for the 5-HT8 receptors and is 14 times less potent in vivo. Both enantiomers have interesting behavioural effects in animal models (to be published).

223

5-HT s RECEPTOR LIGANDS

N Hz

/

H

H

I

H O.,

I ,

R

R= H R=CH 3

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2-methyl-5-HT

R= H R=C!

9P B G "3-Chloro-PBG

N H2

QUIPAZiNE

SR 57227A

N

N~N~~

N~Ph

o H'N \ Me

Figure 1: Chemical structure of some 5-HT s receptor agonists. 5-HT3 r e c e p t o r a n t a g o n i s t s In contrast to 5-HT 3 agonists, a great number of specific and selective high affinity antagonists have been described, particularly in the period 1988-1993. Following the introduction ofbemesetron (MDL 72222) and tropisetron (ICS 205930) (see [23]), several other compounds from different chemical classes have been synthesized and shown to possess potent 5-HT 3 receptor antagonist properties. Thus, ondansetron (GR 38032F), granisetron (BRL 43694), and zacopride have also come to be reference 5-HT 3 receptor antagonists (figure 2).

224 In addition, a great number of other 5-HTs receptor antagonists have also been described e.g: azasetron; BMY 33462; BRL 46470A; batanopride; DAT-582; dolasetron; GR 65630; GR 67330; GR 68755; itasetron; L-683,877; litoxetine; LY 278 584; MDL 74156; Q-ICS-205 930; RG 12915; RS-42358-197; WAY 100289; YM060; zatosetron (see figure 2 and list of abbreviations). Most of these 5-HT3 receptor antagonists display a high a/Fruity for 5-HT3 receptors but not for G protein-coupled 5-HT receptors, nor for D2 or benzodiazepine receptors. However, their interactions with the recently cloned 5-HT receptors, and particularly those linked to cAMP accumulation [24, 25, 216], have not yet been completely examined. This is an important point, since some benzamides (zacopride) and indole derivatives (tropisetron) bind to 5-HT4 sites positively coupled to adenylylcyclase (although at concentrations higher than those required for the recognition of 5-HT 3 receptors) [26]. In contrast to the first agonists, 5-HT3 receptor antagonists are likely to diffuse through the blood brain barrier. This property has been clearly established for e.g 4-(2-methoxyphenyl)-2-[4(5)-methyl5(4)imidazolyl]thiazole [27], and zatosetron [28]. In contrast, the quarternized derivative of tropisetron, Q-ICS 205 930, is thought not to cross the blood brain barrier due to its high polarity, and was therefore used to discriminate between central and peripheral mechanisms mediated by 5-HT 3 agonists. S t r u c t u r e o f 5-HT 3 a n t a g o n i s t s

The chemical structure of 5-HT3 receptor antagonists and the interactions of these drugs with the receptor have been analyzed [29, 211]. The structures of these compounds are generally different from those which interact with G protein-coupled 5-HT receptors. Most of them derived from the initial observation that cocaine, an aromatic ester of tropanol, and metoclopramide, an aromatic amide, block neuronal responses in the rabbit heart [29, 30]. Thus, bemesetron and tropisetron, the first specific 5-HT 3 receptor antagonists to be synthesized, are aromatic esters derived from cocaine. Later, aromatic amides devoid of dopaminergic properties, such as granisetron and zacopride, were also found to be potent 5 - H T 3 receptor antagonists. The structure of this initial set of 5 - H T 3 receptor antagonists consists of an aromatic group linked through a n s p 2 carbon (ester or amide) to a rigid ring containing a basic tertiary amine group (figure 2). Further compounds displaying these basic features were also developed as 5-HT3 receptor antagonists. These include several aromatic ester derivatives such as dolasetron (figure 2), MDL 74,156, and compounds in which the ester group has been replaced by an isosteric thiazole [27, 32] or oxazole [33] group, such as L-683,877 (figure 2). A great number of 5-HT 3 receptor antagonists derived from metoclopramide have also been synthesized. Thus, compounds such as zacopride, BMY 33462, zatosetron, azasetron, RS-42358-197 (figure 2), RG 12915 and, batanopride, bear a secondary amide moiety, but have not retained the dopaminergic properties of the parent compound. These drugs also contain an oxygen group in position 2 of the aromatie ring, sometimes incorporated into a rigid ring (zatosetron, azasetron, RG 12915). As shown for 2-methoxylated benzamides [34], a hydrogen bond between this oxygen atom and the hydrogen of the secondary amide group is likely

225 to exist, blocking the amido nitrogen in a pseudo aromatic cycle. This has been recognized as an important point for a better interaction with the receptor [35], and is illustrated by the high affinity of RS 43258-197 for 5-HTs sites (see figure 2 and table 1). This compound is indeed a cyclic tertiary benzamide in which the hydrogen bond has been replaced by a covalent bond. Furthermore, the potent 5-HT 3 antagonist WAY-100289 (figure 2), in which two hydrogen bonds have been identified, can be considered as a tricyclic amido-related derivative [36]. As mentioned above, all these ester or amido derivatives contain a highly hindered tertiary amine (tropane, granatane or quinuclidine), highlighting the importance of the basicity of this group. In addition, it has been shown that the orientation of the lone pair of electrons on this nitrogen is an important factor for an efficient interaction with the 5-HT 3 receptor. This was recognized very early on, as the endo configuration of the tropane ring in cocaine gives rise to less potent 5-HTs receptor antagonists than an exo configuration [23]. This was later confirmed by several other groups on different compounds [29]. The accessibility, of the nitrogen lone pair of electrons is also an important point to consider. Indeed, 2-6 methylated tropane derivatives are considerably less potent than their corresponding unsubstituted homologues. In these compounds, methyl residues borne by the carbons linked to the amino group considerably increase the steric hindrance around the lone pair of electrons of the nitrogen atom [29]. However, with the discovery of a new structural class of 5-HT 8 receptor antagonists, it appears that the structural requirements for the basic nitrogen atom are completely different. Indeed, ondansetron and FK 1052 (figure 2) and several other related compounds (GR 65630, GR 67330, GR 68755) are aromatic ketones which differ from the two other classes of 5-HT 3 receptor antagonists mainly by the absence of a basic nitrogen. In contrast, these compounds contain an imidazole group, and the aromatic nitrogen atom of this group is obviously less basic than the nitrogen atom of cyclic tertiary amines. Interestingly, the thiazole derivative related to tropisetron and sharing this imidazole moiety instead of the tropane group, retains 5-HT3 receptor ligand characteristics, suggesting that the tertiary amino group and the imidazole amino group interact with the same determinant in the receptor. The situation is, however, much more complicated, as illustrated for instance by the partial agonist properties reported for both tertiary amine and imidazole derivatives (see [29]). The exact nature of the interaction(s) of the nitrogen atom with the receptor remains to be clarified, but the modeling of 5-HT3 receptor antagonists provided useful complementary data. Thus, several groups using different approaches have analyzed the three-dimensional structure of several 5-HTs receptor antagonists, assuming that they bind to a single and identical site [34, 38, 39, 40]. Distances between the key interacting parts of the molecule have been determined (figure 3). The electronic and lipophilic characteristics of the pharmacophore were published recently [217]. Furthermore, at the level of the receptor, it has been suggested that, in addition to an aromatic binding area, the interaction with the nitrogen requires a hydrogen bond acceptor group [39], presumably a carboxylate group as suggested for the G protein-coupled receptors.

226

5-HT s RECEPTOR

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c,

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~/~-'N H

N CHs

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CH3

O AZAS ETRON

Figure 2: C h e m i c a l structure of some 5-HT 3 antagonists.

CH3

FK 105 2

227 5-HT a RECEPTOR

LIGANDS o

CH3

o~ o ~ ~ /

o

~'~N

fM~

N

RS-42358-197

CH~ LY 278584

,CH3

O,..H-.~N, ~ II

~

c O

ITASETRON

O~.....~H~ ~ N

I ~C~.o

H CH3

CH3

GRANISETRON

//~

CH3

BRk 4 6470A

WAY 100289

CHa

O.

I

~

N

H2N

'~N \

CH3

DAT-582

O

/ H 3

4

--CH~

5

Figure 2: C h e m i c a l structure of some 5-HT 3 a n t a g o n i s t s (continued)

N/H

228 A further interaction site involves the atom linked to the s p 2 carbon (an oxygen for aromatic esters, amides and ketones). A hydrogen bond donor, presumably a hydroxylated amino acid, was proposed [39], but a histidine residue could play the same role. Distances between these two determinants have been evaluated and the three-dimensional model mainly based on the initial work of Hibert [38] summarized the presently available data on 5-HT3 receptor-ligand interactions (figure 3).

,

Figure 3:

o c( I

Schematic representation of the 5-HT3 receptor antagonist pharmacophore, (a)= 7.7/~ [39], (b)= 5.1/~ [38], (c)=6.7-7.1/k [38]; and (d)=1.7 A [38].

Also the more recently designed 5-HT 3 receptor antagonists fit the 5-HT 3 receptor antagonist pharmacophore. Some examples (figure 2) are the naphthalimide derivative (compound 1) [218] the 3-methyl-l-indolizine derivative (compound 2) [219], the pyrrolo[2,1-c][1,4]benzoxazine-6-carboxamide derivative (compound 3) [220], the benzopyrano[3,4-c]quinoline derivative (compound 4) [221] and the benzimidazole-4-carboxylic acid derivative (compound 5) [222]. These compounds are potent 5-HT 3 receptor antagonists. Compounds 3 and 4 have similar to RG 12915 a large polycyclic aromatic moiety, which is welltolerated by the 5-HT3 receptor. LIGAND BINDING ASSAYS The identification of central 5-HT 3 sites in the rat brain [41] has stimulated the development of various radioligands for studying 5-HT3 sites in tissue preparations. However, as the density of these sites is generally low in forebrain regions, the discovery of high concentrations of 5-HT3 sites on permanent neuronal

229 cell lines provided a more convenient source of these receptors, allowing a reliable evaluation of the pharmacological data of 5-HT~ receptors. Thus, in addition to central and peripheral tissues from various species, a great number of assays have been conducted on membranes from NCB20, NG108 15 and NIE-115 clonal cell lines. To date, several radiolabelled antagonists have been developed for this purpose (table 1), and those displaying a high affinity for 5-HT8 sites were also found to be useful for autoradiographic studies. Commercially available are [SH]GR 65630, [3H]quipazine, [SH]granisetron, [3H]-(S)-zacopride and [SH]-LY 278584. Table 1 Characteristics of labelled radioligands in the rat cortex: RADIOLIGAND

KD

Reference

(riM) -

[3H]YM060 [~H]GR 67330 [~H]RS 42358-197 [125I](S)-zacopride [~H]granisetron [3H](S)-zacopride [3H]GR 65630 [3H]LY278584 [3H](R, S)- zacopri de [3H]Q-ICS-205 930 [~H]tropisetron [3H]quipazine [125I](RS)-zacopride [~H](R)-zacopride

,

.

.

.

.

0.008 0.04 0.12 0.19 0.30 0.31 0.35 0.70 0.76 0.95 1.2" 1.2 4.3 10.5

.

.

.

.

.

.

.

.

.

Akuzawa, et al. 1995, [223] Kilpatrick, et al. 1990, [42] Wong, et al. 1993, [43] Gehlert, et al. 1993, [44] Nelson and Thomas, 1989, [45] Barnes, et al. 1990, [46] Kilpatrick, et al. 1987, [41] Wong, et al. 1989, [47] Barnes, et al. 1988, [48] Wafting, et al. 1988, [49] Hoyer and Neijt, 1987, [50] Milburn and Peroutka, 1989, [51] Koscielniak, et al. 1990, [52] Kidd, et al. 1993, [53]

KD values refer to the original data reported for the first time. *Value determined in NG108 15 membranes. Most of these radioligands reversibly bind in a stereospecif[c manner to a single and saturable population of sites. However, some limitations have been reported for [3H]quipazine, which also labels a 5-HT uptake site [55], as well as for [~H](R)zacopride, which has been reported to bind to an additional site, unrelated to any 5-HT receptors [53]. In addition, [SH]GR 65630 and [3H]GR 67330 were found to label other unidentified sites [41, 42]. Little data is available for [~H]bemesetron, but some results have shown an unusual binding in the guinea-pig hippocampus, limiting the utility of this ligand for the specific labelling of 5-HTs sites [54]. Finally, the labelling of 5-HT~ binding sites has also been attempted with an

230 agonist, [3H]-3-chloro-PBG, but high non-specific binding was observed in the rat brain, and 2 sites were labelled in NIE-115 cells [56]. The binding of various radioligands to 5-HT 3 sites was illustrated by a rather high affinity for several reference 5-HT3 antagonists (zacopride, ondansetron, tropisetron, granisetron) and by a micromolar affinity for the agonists (table 2). This binding was poorly inhibited by selective drugs acting on the other 5-HT binding sites, with the exception of zacopide and tropisetron, as reported above. In addition, a low micromolar affinity of 5-methoxytryptamine for 5-HT3 sites was found [57], confirming previous observations [14]. GTP and other guanine nucleotides neither affect the binding of tritiated radioligands to 5-HT3 sites, nor do they modify the inhibition of this binding by various agonists, indicating that 5-HT 3 sites are not coupled to G proteins. Thus, the selectivity of most of these radioligands is high enough to consider that they exclusively label 5-HT3 sites in different species, including man [58]. Table 2 Pharmacological characteristics of 5-HT3 sites in different tissue preparations: RAT Cortex

NG 108 15 Cells

RABBIT GUINEA-PIG Myenteric Plexus Myenteric Plexus

DRUGS

Ki (nM)

Ki (nM)

Ki (riM)

RS-42358-197 (S)-zacopride GR65630 Tropisetron 3-chloro-PBG Q-ICS 205-930 Ondansetron (R)-zacopride Bemesetron PBG 2-methyl-5-HT (+)tubocurarine Metoclopramide 5-HT

0.15 0.22 0.48 1.0 1.3 1.8 3.3 4.8 30 51 135 234 282 692

0.07 0.13 0.78 0.63 34 1.0 4.8 2.7 10 933 617 66 174 174

0.12 0.12 0.19 0.27 126 0.20 0.79 2.7 6.8 1202 251 851 75.9 191

Ki (nM)

4.0 4.4 81 14.5 2754 112 126 89 380 3388 646 6457 2089 12023

5-HT 3 binding sites in table 2 were labelled with the highly specific ligand [3H]RS 42358-197 in homogenates from different tissues (adapted from [43]). Three main conclusions can be drawn from radioligand studies: 1) Whatever the radioligand used to label brain 5-HT3 sites, essentially the same pharmacological profile was obtained. 2) Central and peripheral 5-HT3 sites

231 display similar pharmacological characteristics [43, 59, 60], but nearly all the drugs were obviously much less potent at 5-HTa sites in guinea-pig tissues than in other species. This has been seen in several peripheral preparations using other techniques [61, 62, 63], but was much clearer in binding studies [43]. Indeed, for the first time, 5-HT 3 sites of the gtfinea-pig mesenteric membranes were labelled with a radioligand, [~H]RS 42358-197, and were compared in the same experiment with those found in other species and clonal cell lines (table 2) (43).The potencies of several agonists and antagonists for 5-HT3 sites were nearly identical in the rat cortex and in several neuronal clonal cell lines, N1E-115, NCB20 and NG108 15 [43, 57, 64, 224]. However, no 5-HT8 binding sites were found in C6 glioma cells and in primary cultures of glial cells of various brain regions of newborn rats [53] (Kidd et al., unpublished observations), confirming the exclusive neuronal localization of 5-HTs sites. Some pharmacological discrepancies do however exist, and have been emphasized by the differences in the affinities of(+)tubocurarine and 3-chloro-PBG for 5-HT8 receptors in different species. Thus, (+)tubocurarine displays a higher affinity for the mouse clonal cell line than for the rat cortex or for other species, and the affinity of 3-chloro-PBG is much lower (by a factor of 2000) in peripheral tissues than in the rat cortex. The same observation applie s for PBG, but to a lesser extent [43]. These observations have also been reported using electrophysiological techniques [6, 63]. Surprisingly, with the exception of autoradiographic studies [65, 66], no binding experiments have been reported in homogenates from the mouse brain. In addition to these interspecies differences, the potency of 3-chloro-PBG evaluated in whole cell patch-clamp studies was about 200 times lower than its affinity for 5-HT 3 receptors in N1E-115 cells [18]. According to the authors, it is likely that the value obtained in binding studies represents the affinity for a desensitized state of the receptor, since a long period of incubation is required for reaching the equilibrium, whereas that obtained in whole cell patch-clamp experiments is more likely related to a non desensitized state of the receptor. Binding studies have also provided some interesting features about the interaction of agonists with 5-HT3 binding sites. Thus, the inhibition of the binding of several tritiated antagonists by 5-HTS agonists, 5-HT, 2-methyl-5-HT, 3-chloro-PBG, and PBG, but also by quipazine [41, 67, 68, 69] and SR 57227A [19], were characterized by a Hill coefficient generally higher than one. This phenomenon, which has not been observed with 5-HTa receptor antagonists, suggested that a cooperative process was required for the activation of 5-HT8 receptors. The same conclusion was drawn from initial electrophysiological studies [70] and confirmed in subsequent works, but also from in vitro studies performed on isolated tissues [23]. Interestingly, when cells or membranes from different origins were labelled with [aH](R,S)-zacopride or [aH](S)-zacopride [64, 68, 69], but not with [3H](R)-zacopride [69], the Hill coefficients calculated for the interaction of several agonists were consistantly close to one. This suggests that the interaction of (S)-zacopride with 5-HT 3 binding sites is not similar to that of the other 5-HT 3 receptor antagonists. This might be relevant as concerns the partial agonist characteristics reported in a limited number of models [71], and the lack

232 of anxiolytic, antidepressant and promnesic properties, generally observed with (R)-zacopride and the other 5-HT3 receptor antagonists [4, 5, 72]. Bonhaus studied the aUosteric interactions of agonists and competitive antagonists at both native and cloned 5-HT3 receptors [225]. He showed that the dissociation of [3H]mCPG, [3H]RS-42358 and [~H]RS-25259, but not [~H]granisetron, from both native and cloned 5-HT3 receptors, was significantly slower in the presence of 5-HT or 2Me-5-HT than in the presence of antagonists. These data suggest a positive cooperation not only between agonists at the 5-HT3 receptor but also between agonists and some antagonists. A model of the 5-HT3 receptor is proposed in which agonists and some antagonists bind to at least two allosterically interacting, pharmacologically equivalent sites. The clinical significance of these allosteric interactions is unknown. However, it is possible that a 5-HTs antagonist with a preference for the agonist-bound conformation of the receptor behaves in a different way than a 5-HT3 antagonist which does not discriminate between the agonist-bound and agonist-unbound conformations of the receptors [225]. R E C E P T O R LOCALIZATION 5-HT 3 receptors have been identified mainly using the pharmacological criteria defined by Bradley et al. [73]: 1) They should be resistant to blockade by antagonists at 5-HTl-like, 5-HT2 (and 5-HT4) receptors; 2) they should be responsive to PBG and 2-methyl-5-HT (but unresponsive to 5-methoxytryptamine); 3) they should be blocked by low concentrations of bemesetron, tropisetron (and the other specific 5-HT3 antagonists, mainly zacopride and granisetron). Thus, 5-HT3 receptors were found exclusively on neuronal membranes in the periphery and CNS. The distribution and the role of peripheral 5-HT3 receptors have been the subject of several reports [3, 23, 74]. Briefly, excitatory 5-HT3 receptors have been found in ganglionic sympathetic and parasympathetic neurons, where they are involved in the release of noradrenaline and acetylcholine respectively. The presence of 5-HT 3 receptors in the enteric system has been established using both electrophysiological and isolated organ studies. These receptors are involved in the regulation of intestinal secretion and contractility, and probably in the control of gastrointestinal motility. The contraction of the guinea-pig ileum induced by the stimulation of 5-HT 3 receptors has been sugested to involve the release of Substance P [23]. In sensory neurons, 5-HT induces a rapid depolarization of 5-HT3 receptors located on C-type neurons in the nodose ganglion and in the vagus nerve. 5-HT 3 receptors located on sensory neurons have also been involved in some reflex responses such as the von Bezold-Jarisch reflex. In addition, a pain reflex resulting from the application of 5-HT to the human skin suggested the presence of 5-HT 3 receptors on sensory neurons, since this effect was reversed by specific 5-HT 3 receptor antagonists [23]. With the development of radioligands, the localisation of 5-HTa receptors in the gastrointestinal tract [59, 75], the vagus

233 nerve [60, 75, 76] and the superior cervical ganglion [76] of several species has been confirmed. In the central nervous system, the use oftritiated or iodinated radioligands has provided a precise picture of the distribution of 5-HT s sites in several species, including the human brain. However, due to the low density of sites and to the presence of these sites in discrete areas of the brain, the only reliable approach is provided by quantitative autoradiography. This approach has been investigated by using different radioligands and various brain tissues [44, 46, 52, 65, 66 and 77-87]. Thus, concordant results indicated that the highest density of 5-HTs sites was found in the medulla oblongata within the nucleus of the solitary tract (NTS) and particularly in the subnucleus gelatinosus, the dorsal motor nucleus of the vagus nerve, the nucleus of spinal tract of the trigeminal nerve, and to a lesser extent, the area postrema. In the human hindbrain, the concentration of 5-HTs sites is in the range of 400-700 fmol/mg protein. In the rat brain, a combined autoradiographic and histological analysis clearly demonstrated that the area postrema contains less 5-HT 3 sites than the NTS [81, 87]. The same distribution is now accepted for all the other species including man and ferret [88], but not in the guinea-pig hindbrain, where no labelling has been reported [66]. [SH]RS 42358-197, one of the few radioligands to label 5-HT3 sites in guinea-pig homogenates [43], would probably be of great interest for the visualisation of these sites in the brain of this species. The synaptic localization of these binding sites has been investigated using several types of lesions. Thus, unilateral ablation of the rat nodose ganglion, which projects to the NTS, induces a dramatic reduction in the density of 5-HT3 sites in the NTS, which was more marked in the ipsilateral nucleus (70-80%) than in the contralateral NTS (25%) [87, 89]. The localization of the 5-HT3 sites on vagal afferents projecting to the NTS was further confirmed by the complete reduction of 5-HTa sites in the whole dorsovagal complex of the cat after bilateral abdominal vagotomy [84, 90]. In the other parts of the brain, the density of 5-HTs sites is quite low (nearly 10-100 times lower than in the dorsal vagal complex), but a detailed distribution has been achieved using selective antagonists with high specific activity [44, 46, 52, 77, 82, 87]. The greatest density of 5-HT~ sites was found in the limbic areas, especially in several nuclei of the amygdala, the hippocampus, the septum, and in the entorhinal and piriform cortices. In addition, some subcortical areas (nucleus accumbens, hypothalamus) were also labelled, although the observed densities were much lower than in the limbic system. No specific binding was detected in the dorsal raphe nucleus, in extrapyramidal regions (substantia nigra, striatum, globus paUidus), in the ventral tegmental area (VTA) or in the thalamus. Some discrepancies have been noted when autoradiographic analysis was performed with [~H]quipazine [65], but the specificity of quipazine for 5-HT 3 sites has been questioned [66, 54, 91]. Finally, 5-HT3 binding sites have been identified in the superficial layers of the rat dorsal horn [78, 86, 87]. The highest proportion of these sites are located on capsaicin-sensitive primary afferent fibers. Indeed, a massive but incomplete reduction ( - 7 0 % ) in 5-HT3 sites was observed after unilateral dorsal ipsilateral rhizotomy [87], or after neonatal treatment with

234 capsaicin [78, 87], suggesting that at least some of these 5-HT S sites are located on interneurones within the dorsal horn. This distribution of 5-HT 3 sites within the central nervous system is in good agreement with the reported effects of 5-HT 3 receptor antagonists. For instance, they have been shown to potently inhibit radiation and cytotoxic chemotherapyinduced emesis. Accordingly, 5-HT3 binding sites were found in high concentration in the dorsal vagal complex of the human brain, considered as a key zone for the initiation of emesis. The presence of 5-HT 3 sites in the limbic system and particularly in the amygdala and the hippocampus supported the anxiolytic and promnesic properties claimed for 5-HT3 receptor antagonists. In relation with the antipsychotic effects of 5-HT 3 receptor antagonists, 5-HT s sites have been visualized in the nucleus accumbens but not in the substantia nigra and the striatum, although a very low [3H]GR 65630 binding has been reported in striatal homogenates [60]. Finally, in agreement with the antinociceptive properties displayed by 5-HT 3 receptor antagonists, 5-HT 3 sites were identified on capsaicinsensitive afferents in the dorsal horn and in the spinal tract of the trigeminal nerve in the medulla oblongata.

F U N C T I O N A L ASSAYS Most of the bioassays used for evaluating the potency of 5-HTs ligands have been performed on peripheral organs and have been previously reviewed [3, 23, 74]. In v i v o a s s a y s Von B e z o l d - J a r i s c h reflex: This reflex results in a vagally-mediated bradycardia and consequent hypotension of short duration. It occurs when C-type afferent nerve endings in the right ventricle are depolarized by various drugs. For instance, capsaicin induces bradycardia which is blocked by atropine. In contrast, the von Bezold-Jarisch reflex induced by the i.v. injection of 5-HT as a bolus into the jugular vein of anaesthetized rats was only blocked by 5-HT 3 receptor antagonists. This model has frequently been used for studying the structure-activity relationship of newly developed drugs. C a n t h a r i d i n - i n d u c e d b l i s t e r in h u m a n : The application for a few hours of Cantharidin, the active irritant in cantharides (Spanish fly), to human skin resulted in the development of a blister. The application of 5-HT to the base of this blister caused pain, which was reversed by the 5-HT 3 receptor antagonists. This model allowed the evaluation of 5-HT 3 drugs on human sensory nerves. In v i t r o a s s a y s 5-HT3 receptors are located on several peripheral organs, and bioassays using the rabbit vagus nerve, the rabbit heart and the guinea-pig ileum have been developed.

235 R a b b i t v a g u s nerve: Extracellular recording allowed the evaluation of the amplitude of C-fiber action potentials. 5-HT~ agonists reduced this amplitude, and their potency could be evaluated using dose-response curves. Antagonists blocked this inhibition in a competitive manner and shii%ed the dose response curve to the right. I s o l a t e d r a b b i t heart: 5-HT depolarized post ganglionic neurons, which induced the release of noradrenaline and acetylcholine. In the isolated rabbit heart perfused by the Langerdorff technique in the presence of muscarinic antagonists, 5-HT dose-dependently induced positive chronotropic and ionotropic effects which are selectively and competitively blocked by 5-HT3 receptor antagonists. I s o l a t e d g u i n e a - p i g ileum: Serotonin dose-dependently stimulated contractile responses of guinea-pig ileum. Part of this effect is mediated through 5-HT3 receptors. The addition of atropine and a 5-HTs receptor antagonist allowed the study of the specific contribution of 5-HT3 receptors. 5-HT 3 receptor antagonists competively shifted the serotonin response curve to the right.

U p t a k e of [x4C]guanidinium in NG108 15 cells: The early observation that 5-HT and Substance P synergistically activate a cation accumulation in various cell lines [92] was extended by Emerit et al. [11]. Thus, in the presence of Substance P, 5-HT activated a cation permeability in NG108 15 cells which can be assessed by measuring the capacity of the cells to accumulate [~4C]guanidinium. 5-HT 3 agonists stimulate this ion accumulation, whereas the response was selectively blocked by 5-HT3 receptor antagonists. Interestingly, in this model, quipazine behaves as an agonist. Although the mechanism of this process is not yet fully elucidated, a clear correlation between the affinity of several drugs on 5-HT~ sites and their potency in stimulating or inhibiting 5-HT-induced guaninidium uptake has been reported, providing a useful and simple biochemical assay for evaluating the functional response of 5-HTa ligands in clonal cell lines. E L E C T R O P H Y S I O L O G Y OF 5-HT s R E C E P T O R S

The electrophysiological characteristics of 5-HT3 receptors have recently been reviewed [6, 209, 212]. Initial studies showed that the 5-HTa receptors display some similarities with the nicotinic receptor, but the most direct evidence that this receptor corresponds to a ligand-gated ion channel was reported by Derkash et al. [93]. The activation of 5-HT 3 receptors induces a rapid depolarization of the membrane and the response desensitizes, although not completely, in the presence of the agonist. The rate of desenisitization seems to depend on the tissue; for instance, in the rabbit or the isolated rat vagus nerve [12, 16], no desensitization has been observed. The depolarization response was completely blocked by specific 5-HT 3 receptor antagonists. The mechanism of the 5-HT-induced desensitization is not known, but increasing cAMP concentrations enhances the rate of the desensitization [94], suggesting that a phosphorylation process might be involved.

236 The opening of the channel probably involves two agonist molecules, since a cooperative process (Hill coefficient significantly higher than one) has been reported [70], in agreement with similar observations assessed by other approaches. Several lines of evidence completely excluded the participation of a G protein in the ionic response. Indeed, the response was rapid (10-100 ms) and could be recorded during several hours in excised outside-out patches [93]. Furthermore, neither activators of G proteins [94], nor inhibitors (pertussis toxin) nor the recording in nucleotide-free medium [93] significantly modify the ionic response. This response has been found to reverse in polarity at a potential close to 0 mV, consistent with the opening of a cation selective ion channel with equal permeability for sodium and potassium. Indeed, several studies have established that the 5-HT~ channel discriminated poorly between monovalent ions (Cs § Li § Rb *) and was permeant to organic ions (NH4 § methylammonium, guanidinium) but also to divalent cations such as Ca 2§ [95-97] (but see [98]). These characteristics were observed in central and peripheral neurons and in different cell lines, as well as in oocytes, where the cloned 5-HT3 receptor [99] was expressed (but see [6, 212]). In contrast, several electrophysiological differences have been registered and have frequently been presented as indications of the existence of multiple 5-HT 3 receptors. Thus, single channel conductances associated with the activation of 5-HT 3 receptors were reported to be dependent on the preparation, with values ranging from 0.59 pS up to 16.6 pS. Larger values have been found for mammalian neurons, whereas generally lower conductances were measured in neuroblastoma cells derived from mouse neuronal tissue [6, 212]. Furthermore, these values also seem to be dependent on the state of differentiation of the clonal cells [6, 100,212]. The presence of external calcium ions can differently modify the amplitude and the duration of the ionic response [97]. This effect was a voltage-independent process, suggesting that the site of action of calcium does not interact with the membrane ion channel [95, 98]. The desensitization rate was increased in the presence of external calcium in N1E-115 cells [98], but the reverse effect was found in NG108 15 cells [97]. The use of (+)tubocurarine to antagonize the ionic responses has also revealed some interspecies differences. Thus, in the mouse nodose ganglion cells in primary culture, 50% of the response induced by 5-HT was blocked by 1 nM (+)tubocurarine, whereas 10 nM and 10000 nM were needed in the same preparation from the rabbit and the guinea-pig, respectively [6]. Finally, some differences concerning the voltage-dependence of the effect of calcium have also been noted between the cloned 5-HT 3 receptor expressed in oocytes and the native 5-HT3 receptor NCB20 [6, 101, 102]. S T R U C T U R E OF 5.HT s R E C E P T O R S Most of the studies were conducted on mouse neuroblastoma clonal cell lines, since they contain a high density of 5-HT 3 receptors with a pharmacological profile close to that observed in mammalian central and peripheral nervous sytems. Even if the eDNA coding for a 5-HT s subunit has been cloned from NCB20 [99] and

237 NIE-115 cells [101], little structural data is available for 5-HT3 receptors. Initial information was drawn from radioligand binding studies. Thus, the use of relatively specific amino acid-modifying agents has shown that a tryptophan residue might be present, in or near the ligand binding site [103]. In contrast, the modification of disulfide bridges or the alkylation of cysteine residues were without influence on the radioligand binding site characteristics. The 5-HT3 receptor was also found to be glycosylated, and this property was used for its purification [103]. Upon solubilization, a high molecular weight (350-600 kDa) has been reported by several groups [103-108], consistent with the idea of the association of several subunits. Further steps leading to a purified receptor revealed by SDS-PAGE analysis, either a single protein band at 54.7 kDa (from N1E-115 cells, [109]), two distinct bands at 38 and 54 kDa (from NC20 cells, [106]) or 4 broad bands at 36, 40, 50 and 76 kDa (from NG108 15 cells, [108]). Interestingly, the radiation-inactivation technique applied to rat cortical tissue [110], NG108 15 cells [64, 111] or N1E-115 cells [112] indicates that the apparent molecular weight of the ligand binding subunit was in the range of 35-49 kDa. Since the molecular weight deduced from the amino acid sequence of the cloned 5-HT3 subunit in its unglycosylated form is 55.9 [99] or 53.1 kDa [101], several questions arose from the above results. Is the 5-HT~ receptor expressed in the brain and in the different cell lines composed of several subunits, which may be different according to the preparation? Based on similar pharmacological and electrophysiological characteristies of the 5-HT3 receptor expressed in these tissues, this seems to be unlikely. However, several differences have been pointed out which might be explained by some point mutations in the sequence of the second transmembrane domain (see discussion on the heterogeneity of 5-HTa receptors below). Is the highest molecular weight band related to the unglycosylated form of the receptor, or does it correspond to an aggregation of lower molecular weight proteins? Does the low moleculair weight component represent a degradation product of the ligand binding subunit, or could it be related to a protein similar to the 43 kDa protein of the nicotinic acetylcholine receptor? This second possibility seems unlikely, as radiation inactivation data suggest that antagonists actually can bind to this protein. Further studies are necessary to obtain a better idea of the structure of this receptor; undoubtedly, the cloning of other subunits will give the answer. To date only one subunit, 5-HTaRA, has been cloned. The first subunit was isolated from NCB20 murine cells [99]. An apparent splice variant, 5-HT~R-As ('s' for short) showing 98% sequence identy, was cloned from N1E-115 murine cells [101]. The main difference between the two cloned sequences was the deletion of 6 amino acid residues in the second putative cytoplasmic loop. Interestingly, both forms of the 5-HT3 receptor have been detected in the same cell (N1E-115 and NG 108 cells, see [101]), again suggesting that they are probably derived from the same gene by an alternative splicing. However the 5-HT~R-As predominates over the 5-HTaR-A in cell lines as well as neuronal tissues [226]. The two 5-HT~ clones display the general characteristics of native 5-HTa receptors, including the cooperativity and the desensitization processes [226]. However, bemesetron and GR 65630 displayed a 10 fold lower affinity for the clone isolated from NCB20 clonal cell lines than for the native

238 receptor in these cells [57]. This suggests that additional subunits are required for a complete expression of 5-HT3 receptor properties. In contrast, differences in the current-voltage curves originally reported [99] could not be reproduced with the same clone [102]. Further pharmacological characterization of the murine 5-HT3R-A and 5-HT3RAs revealed that the efficacy of the agonist 2-Me-5-HT was significantly lower for the 5-HT3R-As variant (9%) than for the 5-HT3R-A subunit (63%) [227]. The other 5-HT 3 receptor agonists and antagonists tested did not discriminate between both subunits. The reason for the low efficacy of 2-Me-5-HT for 5-HT3R-As is not clear. The amino acid sequence of the cloned 5-HT3 receptor displays similarities with ligand-gated ion channel receptors and especially with the nicotinic receptor. Its sequence contains 4 hydrophobic domains with a large N-terminal domain. By analogy with the nicotinic receptor, the ligand binding domain is probably located at the N-terminal level, where a disulfide bridge and several conserved amino acid 'canonical' [113] residues were found. Some of these amino acid residues have been shown to be involved in the ligand binding domain of the nicotinic [113] and glycine receptors [114]. Some of these residues were conserved in the 5-HT3R-A sequence, for instance W TM and W '~ (figure 3). At least one of these two tryptophan residues may correspond to the residue(s) which were found to be sensitive to NBS treatment [103]. The disulfide bridge (C162-C 176) was conserved in the 5-HT~ sequence (figure 4), although its structural role remains to be established for the 5-HT3 receptor, as its reduction has not induced modifications in the affinity of 5-HT 3 ligands [103]. In contrast, the lack of the two consecutive cysteines, which were also shown to be important for the nicotinic receptor, might explain the reported absence of effect ofthiol-modifying reagents [64, 111]. Instead of these two cysteines, an aspartic acid linked to two bulky isoleucine residues were found: I ~29 - D 22~ - 1221. Interestingly, the mapping of the 5-HT 3 receptor binding site has revealed that the interaction of 5-HT3 ligands involves at least 2 domains: one containing a highly hindered carboxylate group and one hydrogen-bond donating group (see above); the amino acid residues D 22~ y225 or H ~s5 (figure 4) may play these roles but this remains to be proven. Finally, in the 5-HT~ sequence (figure 4) several aromatic amino acid residues were found around this aspartie acid, a situation already reported for the G protein-coupled 5-HT receptors [115]. In the nicotinic receptor, the channel was supposed to be formed by the association of the second transmembrane domain of the 5 subunits [113]. Rings of charged (glutamic acid), hydroxylated (serine and threonine) and hydrophobic (leucine) residues from the different subunits have been implicated in several channel properties, including the desensitization process [116]. Although only one 5-HT 3 receptor subunit is available, it is of interest to note that these important residues are conserved in the MII domain of the cloned 5-HT3 protein (figure 4), suggesting that the same type of association also occurs for the 5-HT3 receptor. Indeed, the electron microscopic analysis of the purified 5-HT3 receptor reveals rosette-shaped particles of 8-9 nm diameter, with a 2 nm pore in the center of the molecule, similar to the well known structure of the nicotinic receptor [108, 228].

239

NH2

121 S I D V S I K I

W W W W

V L K T

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Figure 4: Schematic representation of the 5-HT3 receptor subunit. The sphere represents the ligand binding area, in contact with three loops of the receptor subunit. The upper line of the boxed sequences, corresponds to the sequence of the 5-HT 3 receptor subunit [99] and was numbered starting from the first methionine of the signal sequence. The 3 other lines correspond to the nicotinic, glycine and GabaA a-subunit sequences, respectively. In the MII domain, the sequence of the nicotinic a7-subunit was inserted between the 5-HT3 and the three other sequences.

240 At present the 5-HT3R-A subunit have been identified in mouse [99], rat [229] and human [230, 232]. There is a high degree of amino acid sequence homology of the 5-HT3R-A subunits cloned from mouse, rat and human. But the NH2-terminal domain contains a few amino acids which are unique to a particular species homologue. As the ligand binding site(s) are located on the N-terminal it is possible that these species - specific amino acids are responsible for the observed interspecies differences in affinities and activities of 5-HT 3 receptor ligands (see below).

HETEROGENEITY OF 5-HTs RECEPTORS The heterogeneity of G protein-coupled 5-HT receptors is well documented and has been confirmed by the cloning of the genes coding for a greater number of 5HT receptors than those identified on a pharmacological basis [1]. Thus, five 5-HT 1 subtypes negatively coupled to adenylylcyclase activity, three 5-HT 2 subtypes associated with the activation of phospholipase C, and three subtypes (5-HT 4, 5HT6, 5-HTv) positively coupled to adenylylcyclase activity have been cloned so far. In contrast to this high number of G protein-coupled 5-HT receptors, only one 5-HT s receptor subunit has been cloned. However, several pharmacological and electrophysiological studies have suggested a possible heterogeneity of this receptor. Thus,data based on the potency of 5-HTs ligands in different peripheral tissues initially lead Richardson and Engel to propose 3 distinct 5-HT s subtypes [62]. Electrophysiological and ligand binding studies also revealed striking changes in the characteristics of 5-HT3 receptors from different species or clonal cell lines. This is emphasized by the potency of (+)tubocurarine, which displays 2 to 4 orders of magnitude lower affinity for guinea-pig and human receptors than for the other [43, 232]. From several approaches it is quite clear that the 5-HT s sites located on guinea-pig tissues display less sensitivity for 5-HT 3 ligands than those from other species. Interspecies differences are now well established [117,233]. This situation resembles that of the 5-HT1D subtype located in all mammalian brains except in those of rodents, where a similar but distinct 5-HT m subtype has been found. The two receptors display the same functional properties and possess approximately 92% sequence identity in the transmembrane domains involved in the recognition of the ligands, but do present some significant pharmacological differences, at least for some drugs [118]. This has been explained by a single amino acid difference in the 7th transmembrane domain which, upon the correct mutation [119, 120] in one subtype (5-HTIB for instance), allowed a recovery of the complete properties of the other subtype (5-HT1D). Such point mutations are likely to occur in the MII domain of the 5-HT 3 receptor and would account for the electrophysiological differences mentioned above. Indeed, discrete modifications of the sequence of this part of the nicotinic alpha7 subunit have been shown to be associated with changes in the affinity or the intrinsic activity of the ligand, in ion selectivity and in single channel conductances. In addition, changes in the desensitization rate and in its voltage dependence have been observed after mutation of one or several ~mino acids located in the MII domain of the nicotinic alpha7 subunit [116, 121, 122].

241 The recent discovery that the mutation of L2s6 (figure 4) affects the desensitization rate of the 5-HT3 receptor expressed in oocytes [102] is in favor of such a hypothesis, since similar results have been reported for the mutation of the corresponding L24v residue in the nicotinic receptor alpha7 subunit [116]. Interestingly, some point mutations in this MII domain have also been shown to be associated with modifications in calcium permeability [122], which may explain the reported differences in its permeability for the 5-HT3 receptor [95, 97, 98, 102]. However, the situation is not exactly the same for 5-HT1B~ receptors and 5-HT3 receptors, since all the drugs, not just a limited number of them, display a much lower affinity for the guinea-pig 5-HT3 receptor. Since 5-HTa ligands belong to several distinct chemical classes, it appears unlikely that their interactions with the 5-HT 3 receptor always involved the same amino acids. This suggests, in contrast a more important number of variations in the amino acid sequence, presumably at the N-terminal part of the receptor, although changes in the MII domain may also contribute to the affinity of the ligand. Thus, some modification in the sequence of 5-HT3 receptors is probably required for explaining the interspecies differences. The relative importance of these variations would favor either a species difference or a real heterogeneity of 5-HT3 receptors. Intraspecies variations have also been described, although contradictory reports exist. 1) Thus, low concentrations of PBG were reported to enhance the release of [aH]5-HT from guinea-pig frontal cortex slices, although this agonist has regularly been reported to possess a low affinity and a low efficacy for several guinea-pig central and peripheral 5-HT3 receptors. However, this preliminary result has not been confirmed [123]. 2) Unusually high affinities of 5-HT for 5-HTs sites have been reported in the rat. Thus, in rat spinal cord synaptosomal membranes, Glaum and Anderson [124] characterized a 5-HTs binding site which displays a high affinity for 5-HT (Kd= 11.5 nM). Also, even lower concentrations of 5-HT (ICso= 0.4 nM) were found to increase the evoked release of cholecystokinin like immuno-reactivity from rat cerebral cortex synaptosomes [125]. In contrast, in the rat central and peripheral tissues, several studies have established that the affinity or the potency of 5-HT is at least two orders of magnitude lower [43, 64, 78]. Whether these variations can be related to experimental conditions, as suggested by some experimental modifications which considerably increase the affinity of several agonists for 5-HT3 binding sites [ 19], remains to be determined. 3) Messenger RNA messages for 5-HT 3 receptors have been found in several central and peripheral regions of the mouse nervous system [99, 126], except in the intestine [99], where 5-HT3 receptors are present, suggesting that 5-HTs receptors in this tissue might be of a different type. These points are obviously insufficient to conclude a heterogeneity of 5-HT3 receptors, but this cannot be completely excluded. Finally, only the cloning of another 5-HT3gene would provide a clear demonstration of the heterogeneity of 5-HT 3 receptors, but this remains to be performed. Perren [234], however, found no clear evidence of an intra-species difference in mouse tissue.

242 5-HT 3

R E C E P T O R S AND N E U R O T R A N S M I T I ~ R RELEASE

The release of stored neurotransmitters is a consequence of activation of neuronal 5-HT 3 receptors increasing intracellular calcium concentrations. Thus, 5-HT, has long been known to control the release of acetylcholine (ACh) and noradrenaline (NA) in peripheral tissues. In the central nervous system, the release of NA, ACh, 5-HT, dopamine (DA) and cholecystokinin (CCK) have been shown to be modulated by several 5-HT3 drugs. These biochemical effects, in addition to the presence of 5-HT3 sites in expected areas, support behavioural experiments which have established the putative therapeutic characteristics of 5-HT 3 receptor antagonists. Thus, it has been shown that the stimulation of central 5-HT 3 receptors decreases the release of NA and ACh, although this inhibition of the release of neurotransmitter seems to be in contradiction with the depolarization associated with the stimulation of 5-HT 3 receptors. Less data is available for NA than for ACh, but it seems that the blockade of the 5-HT-induced inhibition of [3H]NA release in rabbit hippocampal slices by high concentrations of 5-HT3 receptor antagonists can be largely non specific [127]. In contrast, Blandina reported a possible control of the release of NA from rat hypothalamic slices by 5-HT 3 receptors [128]. Although an indirect effect cannot be excluded, this apparent decrease of NA concentrations provided support for the antidepressant properties exhibited by some 5-HT3 receptor antagonists in the learned helplessness paradigm [129]. The effects of 5-HT 3 receptors on the release of ACh are better documented, but still remain the subject of debate. The initial paper [130] showing that 5-HT3 agonists inhibit the potassium-evoked release of [3H]ACh from rat cortical slices was surprising, as the opposite effect occurs at the periphery and since previous papers have not reported this phenomenon in guinea-pig cortical slices [131]. However, the same group [132] later reported that 5-HT3 agonists do increase the release of ACh in the cortex of freely moving guinea-pig. Moreover, Maura and coworkers have shown that 5-HT and PBG can inhibit the potassium-evoked release of [3H]ACh in synaptosomes prepared from human cortex [133]. This effect was blocked by tropisetron and ondansetron. Finally, these results are in contradiction with experiments performed in the same conditions as those described initially, showing that 5-HT3 agonists did not inhibit the potassiumevoked released of [3H]ACh from Hooded Lister rat entorhinal slices and that ondansetron did not increase this release. The same results were also found with Sprague-Dawley and aged Wistar rats [134]. In contrast activation of 5-HT3 receptors facilitates the release of ACh in vivo from rat hippocampus [135]. Thus, the initial idea that the effect of 5-HT3 receptor antagonists in cognitive performance of adult and aged rats might be mediated by an increase of ACh release need further confirmation. In vitro experiments have shown that 5-HT3 agonists could stimulate the electrically evoked release of [3H]5-HT from rat hypothalamic slices [122] or from different guinea-pig hypothalamic, frontal cortex or hippocampal slices [123]. This response, blocked by selective 5-HT3 receptor antagonists, was shown to

243 desensitize, indicating that endogenous 5-HT can indeed activate 5-HT 3 receptors. These results provided support for the anxiolytic effects of 5-HT3 receptor antagonists which, with respect to the monoaminergic theory of anxiety, antagonize the effects of 5-HT at 5-HT3 receptors and reduce the release of 5-HT diminishing the overall concentration of 5-HT. However, these effects reported on guinea-pig slices have not been observed in the frontal cortex or hippocampus of freely moving rats using in vivo microdialysis [136] (see also [137]). This technique monitors 5-HT release over long time periods, and the desensitization process may have hidden the phenomenon. However, the potassium-evoked release of [3H]5-HT from rat spinal cord synaptosomes with shorter timing analysis, was also unaffected by 5-HT3 receptor antagonists [138]. Nevertheless, 5-HT3 receptors in the rat cortex are unlikely to be located on 5-HT terminals since the total number of cortical and hippoeampal 5-HT3 binding sites was not affected by 5,7-DHT treatments, whereas those located in the amygdala were slightly reduced (18-25%) by the selective degeneration of 5-HT terminals [139]. Considerable data has been accumulated over the past eight years showing that 5-HT3 receptors can modulate the release of dopamine in terminal response of the mesolimbic areas. The mesolimbic dopaminergic system has been involved in the mediation of locomotor activity and has been shown to modulate reward mechanisms. Acute but also chronic in vivo administration of ondansetron failed to modify dopaminergic neuronal activity [140, 141], but an attenuation of dopamine release was evident when dopaminergic pathways were activated first. Thus, 5-HT 3 receptor antagonists partially reversed the activation of the dopaminergic mesolimbic pathway induced by stress procedures [142], by DiMe-C7 [143], a neurokinin agonist, or by several drugs of abuse known to increase the firing rate of dopaminergic cells, including morphine, nicotine, and ethanol [4, 144-146]. The intracerebroventricular administration of 2-methyl-5-HT [147] increased the release of DA, and these effects were reversed by 5-HT 3 receptor antagonists. This suggested that the ventral tegmental area, containing dopaminergic cell bodies, might be the site of action of 5-HT 3 receptor antagonists. However, the perfusion of the nucleus accumbens with 5-HT [148] or PBG [149] also stimulated the release of dopamine. The effect of PBG was reversed by 5-HT3 receptor antagonists [149]. Since serotonin neurons from the dorsal raphe project to the ventral tegmental area as well as to the nucleus accumbens, and since a low density of 5-HT 3 sites (0.9-5.0 fmol/mg prot.) [41, 44, 46] has been found in the nucleus accumbens but not in the ventral tegmental area, 5-HT3 receptors located in the nucleus accumbens, probably on dopaminergic terminals [149], might be involved in the control of the release of dopamine. This assertion, however, remains to be proven. Furthermore, a direct or indirect effect of 5-HT 3 agonists on the dopaminergic transporter might also explain some of the effects of 5-HT3 agonists on dopamine release. This has been shown for PBG in rat striatal slices [21] and more recent experiments clearly indicated that such a mechanism is likely involved in the 5-HT-induced release of [3H]DA from rat striatum and nucleus accumbens slices [150].

244 Finally, the effect of 5-HT3 agonists on the release of dopamine in the nucleus accumbens might also involve CCK, since this peptide is able to release dopamine in this area (see [151, 152]) and since 5-HT 3 agonists enhanced CCK release. Indeed, Raiteri and co-workers have demonstrated that the stimulation by 5-HT and PBG of 5-HT 3 receptors located on CCK cortical and nucleus accumbens terminals of the rat enhances the release of CCK-like immunoreactivity, and this effect was blocked by 5-HT 3 receptor antagonists [153]. In addition, 5-HT3 receptor antagonists were shown to reverse release of CCK-like immunoreactivity evoked by endogenous 5-HT in the frontal cortex of freely moving rats [125]. Only one group has also reported that the release of endogenous dopamine from rat striatal slices can be stimulated by 5-HT 3 agonists, and that this effect was blocked by 5-HT3 receptor antagonists [154, 155]. This was not expected in the striatllm, as no 5-HT 3 receptors have been identified in this region. However, using synaptosomes from rat striat~lm, another group reported that the release of [3H]dopamine was indeed stimulated by 5-HT [156]. However, this effect was not antagonized by ondansetron and bemesetron, and a transport of 5-HT into dopaminergic terminals has been suggested [156]. An effect of 5-HT3 receptor antagonists on the nigrostriatal pathway was also unexpected, since ondansetron was unable to antagonize the stereotypies induced by a systemic injection of amphetamine [157, 158]. However, the effect of 5-HT 3 receptor antagonists on the activation of the nigrostriatal pathway induced by striatal injection of amphetamine is not known. Other studies have compared the responses of A9 (substantia nigra) or A10 (ventral tegmental area) dopaminergic neurons following chronic treatments with 5-HT 3 receptor antagonists. Acute dolasetron (MDL 73147EF) does not affect the finng rate of A9 and A10 neurons [159]. Chronic administration (5mg~g/day i.p.; 3 weeks) resulted in a reduction of the firing rate of both mesolimbic and nigro-striatal neurons [159], indicating that this drug exerts a neuroleptic-like effect, but a greater effect on A10 neurons was reported later [160]. Chronic treatment with granisetron (5mg/kg/day i.p.; 3 weeks), initially reported to be without effect on A9 and A10 neurons [161], was later found to preferentially decrease the finng rate of A10 neurons at lower doses [162]. Acute and chronic administration of low doses of zatosetron (0.1 and 0.3 mg/kg/day i.p. 3 weeks), decreased the number of spontaneous active A10 neurons without affecting A9 cells [28], but this effect seems to be distinct from those obtained with atypical neuroleptics. In contrast, chronic treatment with a very low dose of itasetron (15 ~g/kg/twice daily s.c.; 3 weeks) [163] seems to reproduce the effects of clozapine (20 mg/kg/day s.c.; 3 weeks) with a selective effect on A10 cells, but not those of a chronic treatment with haloperidol, which affected the firing of dopaminergic cells both in the VTA and in the substantia nigra. These results support, it least for itasetron, the suggestion that 5-HT 3 receptor antagonists may represent a new class of antipsychotic drugs [164], but need further confirmation. Finally, in mesocortical areas, it also seems that 5-HT3 receptors might control the release of dopamine, since the administration of PBG directly into the medial prefrontal cortex increases dopamine release; but again, an action through the dopamine transporter has not been excluded [165].

245 THERAPEUTIC APPLICATIONS OF 5-HT a ANTAGONISTS Even before the identification of central 5-HT 3 receptors, 5-HTa receptor antagonists have been suggested to possess striking properties. To date, based on animal studies, several reports have shown that these drugs display anxiolytic, antipsychotic [see 3, 4, 5, 166, 167], promnesic [72, 169, 170], antidepressant [136], antinociceptive [23, 171] and antiemetic [172, 173] properties, generally at low doses and without side-effects [4]. Confirmation of these results in human are necessary before drawing any definitive conclusion since, except for the antiemetic effects, data from clinical trials are few and generally limited to ondansetron. Critical reviews on the subject have recently been published [9, 235-238]. The pioneering work of Costall and coworkers [41] has shown that 5-HTa receptor antagonists display an anxiolytic profile comparable to that of benzodiazepines in several animal models, except in conflict tests, although they have no affinity for benzodiazepine receptors. A bell-shaped dose-response curve has frequently been described for the first 5-HT~ receptor antagonists evaluated in these models, but the loss of anxiolytic effect at high doses was not associated with sedation and other side-effects occurring with benzodiazepine treatments [4]. This biphasic curve, also reported for several types of behaviour, has not been observed for the new 5-HTa receptor antagonists BRL 47470A [178] and RS 42358-197 [168], and again, high doses do not induce sedation [168, 178]. Interestingly, (S)-zacopride, which displays a 10-fold higher affinity for 5-HT 3 sites than (R)-zacopride [64], was devoid of anxiolytic properties, whereas (R)-zacopride was as potent as the other 5-HT~ receptor antagonists [174, 175, 176]. However, an other study does report the anxiolytic effect of (S)-zacopride [177]. Limited studies have supported a central site of action for these anxiolytic properties of 5-HTa receptor antagonists. Thus, the intra-amygdala administration of low doses of 5-HT S receptor antagonists which are ineffective when injected peripherally, produces an anxiolytic effect [179]. This seems to be in agreement with the presence of functional 5-HT3 receptors in this area [180]. However, the same result has been obtained by the administration of 5-HTs receptor antagonists in the dorsal raphe [179], which is devoid of 5-HT3 sites. The density of 5-HT~ receptors in the brain is quite low, even in the amygdala, but considerably higher in the dorsal vagal complex, a region which is apparently devoid of a blood brain barrier. Therefore, as suggested for CCK receptor antagonists [151], some of the anxiolytic properties of 5-HT~ receptor antagonists might result from a peripheral action on 5-HT3 receptors located on vagal afferents. The clinical evaluation of the anxiolytic effect of 5-HT 3 receptor antagonists is less documented. A double-blind, placebo-controlled trial, reported an effect of tropisetron in generalized anxiety disorders [181], and a preliminary report indicated that ondansetron reduces anxiety [182] without any rebound anxiogenic effect upon cessation of the treatment. Further results are awaited [for reviews see 235, 236]. The neurochemical basis of schizophrenia is not known, but the antipsychotic effect of several drugs are thought to be mediated by blocking the hyperactivity of mesolimbic dopaminergic neurons. Therefore, several animal models based on the enhancement of mesolimbic activity were developed for the evaluation of

246 potential antipsychotic properties of new drugs. In each of these models, 5-HTa receptor antagonists were found, at low doses, to completely reverse this locomotor hyperactivity [166]. These antagonists are, however, devoid of affinity for dopaminergic receptors. Interestingly, the rebound hyperactivity generally reported upon cessation of the treatment with neuroleptics was not observed with 5-HT a antagonists [4, 5]. In addition, they, were ineffective in modifying locomotor activity without prior activation of the mesolimbic pathway, suggesting a lack of tonic control of 5-HTa receptors. In contrast to the other types of behaviour involving 5HT a receptor antagonists, (S)-zacopride, and not (R)-zacopride, antagonizes the hyperactivity induced by the injection of amphetamine or the infusion of dopamine into the nucleus accumbens [72]. Finally, very limited behavioural studies have indicated that 5-HT a receptor antagonists do not modulate dopaminergic responses in the nigro-striatal pathway [157, 158, 163], but this has been enough to suggest that 5-HTa receptor antagonists might represent a new class of antipsychotic drugs devoid of extrapyramidal side-effects [164]. Ondansetron and zacopride have been evaluated in acute schizophrenia. A double-blind, placebo-controlled trial with ondansetron has indicated a reduction in both positive and negative symptoms of schizophrenia [183], but one single-blind trial reported that zacopride was less effective [184] [for review see 236]. Drugs of abuse such as nicotine, morphine, and alcohol have rewarding properties associated with an increase in dopaminergic mesolimbic activity. 5-HTa receptor antagonists have been shown to reverse both effects in animals [3, 4, 5, 9, 166], suggesting that they may be useful when withdrawing patients from these drugs. Limited clinical data are available yet, but long term administration (4 weeks) ofondansetron reduces alcohol consumption by alcohol abusers by only 18% [185]. Further clinical investigations are therefore needed to confirm promising experimental results [for reviews see 236, 238]. Experiments conducted on rodents and primates have shown that 5-HTa receptors facilitate basal cognitive performance of 5-HTa receptor antagonists (3, 4, 5, 72, 169, 170, 186-188]. In addition, impaired cognitive performance following scopolamine treatment or the effect of aging were reversed by 5-HTa receptor antagonists [72, 169]. After a 12-week treatment, ondansetron, evaluated in a double-blind clinical trial, was reported to improve some cognitive performance in age-associated memory impairment [ 189]. However, no improvement in cognitive performance was reported in adults (24-40 years old) [190] [for review see 236]. Exogenous 5-HT induces pain in man through activation of 5-HT3 receptors located on subcutaneous terminals of primary afferent sensory fibers [12, 23], but endogenous 5-HT has been suggested to play a key role in producing pain migraine [191]. The presence of 5-HT 3 sites in the superficial layers of the dorsal horn as well as in the substantia gelatinosa of the spinal trigeminal nucleus has suggested a role for these receptors in pain control. The studies of 5-HTa receptors in the release of Substance P and CGRP [192, 193] are in line with this hypothesis. Indeed, in a double-blind, placebo-controlled study, the acute administration of bemesetron to persons suffering from migraine headaches produces a reduction in pain [194]. Furthermore, more recent clinical trials with

247 granisetron have confirmed the substantial reduction of headaches and associated symptoms [195, 196]. Antidepressant properties of ondansetron, tropisetron and zacopride have been suggested on the basis of their response in the learned helplessness paradigm [129]. Recent experiments extended this observation to the (R) isomer of zacopride but not to the (S)-isomer (Martin and Gozlan, unpublished results), in agreement with their relative potencies in most behavioural tests. Of interest are the findings of Poncelet who showed that SR 57227 A, a potent and selective 5-HTa receptor agonist, produces antidepressant-like effects in various animal models for depression [239]. These effects were antagonized by 5-HTa receptor antagonists. Further experimental approaches are needed to confirm the antidepressant effects of 5-HT a receptor ligands. Their therapeutic use is probably less obvious than that of 5-HT uptake inhibitors. In this context, litoxetine, a selective 5-HT inhibitor, also displays some characteristics of a 5-HT 3 receptor antagonist, and inhibits cisplatin-induced emesis in the ferret [197]. This property would probably contribute to a reduction of the gastrointestinal side-effects generally associated with the antidepressive treatment with 5-HT uptake inhibitors, if these effects could be shown to be related to a stimulation of 5-HTa receptors. Indeed, 5-HT~ receptor antagonists are very potent and selective drugs against radiation and cytotoxic chemotherapy-induced emesis, but not against vomiting induced by other drugs, such as dopaminergic agonists and morphine. This property is firmly established in cancer patients and some drugs have already been marketed or will soon be introduced [237]. The central or peripheral site of action of 5-HT 3 receptor, antagonists has been the subject of debate. The central mechanism was initially suggested by the observation that a high density of 5-HT~ sites is seen in the area postrema. The area postrema is a circumventricular organ containing 5-HT [198] and has been implicated in emesis induced by apomorphine and loperamide [199]. The stimulation of the area postrema would release 5-HT, which in turn would trigger the emetic response. Indeed, local application of 5-HTa receptor antagonists in the 4th ventricle reduces cytotoxic-induced emesis in cats [200] and in ferrets [201]. However, local irradiation of the area postrema of cancer patients did not induce emesis [202]. Furthermore, 5-HT administered in the 4th ventricle of the cat does not readily evoke emesis and 2-methyl-5-HT injected either i.v. [203] or directly into the area postrema [201] was not effective in inducing emesis in the ferret. In contrast, the oral administration of 2-methyl-5-HT and PBG evoked emesis in the ferret, and this effect was blocked by tropisetron [71], and the intragastric administration of zacopride to rhesus monkeys significantly inhibited radiationinduced vomiting [204]. It is now accepted that only low concentrations of 5-HTa receptors are present in the area postrema of several species, including man [88]. The highest density was found in the NTS and these sites are exclusively located on vagal afferents [81, 84, 89]. Finally, vagotomy experiments have demonstrated that an intact vagal afferent innervation is required for the development of cytotoxic-induced emesis [205, 206]. Therefore, it can be hypothesized that acute radiation or cytotoxic drug stimulate the release of 5-HT (or another emetogenic substance) from the 5-HT-rich enterochromaffin cells, which in turn could activate

248 5-HT 3 receptors located on both afferent terminals of the vagus nerve, in the dorsal vagal complex, but also in the abdomen. The mechanism of delayed emesis has not been well analyzed and requires further study [for review see 237]. CONCLUSION The unique ligand-gated ion channel receptor of serotonin is now well characterized. Numerous high affinity 5-HT 3 receptor antagonists have been developed, mainly by the pharmaceutical companies. Several of them are useful radioligands which have confirmed the peripheral neuronal localization of 5-HT 3 receptors and demonstrated the presence of low concentrations of 5-HT 3 sites in the central nervous system. Progress has also been registered with the introduction of a new and potent 5-HT3 receptor agonist, SR 52227A. This drug does not present the limitations associated with the use of the other 5-HT 3 receptor agonists and would be a useful tool for further studying 5-HT 3 receptor-mediated responses. The 5-HT3-mediated depolarization occurs within milliseconds and suggests that, in addition to its neuromodulatory properties, 5-HT can also act as a true neurotransmitter in the brain. The increase in intracellular Ca 2§ concentration following 5-HT 3 receptor stimulation triggers several important events, such as the modulation of the release of neurotransmitters. However, it has not been well established whether this increase is a direct consequence of the activation of 5-HT3 receptors or an indirect effect, related to the entry of C a 2§ through voltage-dependent calcium channels. Therefore, some of the cellular responses reported to be associated with 5-HT 3 receptor stimulation, such as the increase of cGMP [207] or phosphoinositide hydrolysis [208], might be considered a consequence of the depolarization of the membrane rather than a direct stimulation of 5-HT 3 receptors. The large number of biochemical and behavioural effects reported to be controlled by 5-HT 3 receptors suggested a multiplicity of 5-HT3 receptors. Species variations are well recognized, but the existence of distinct 5-HT 3 receptors has yet to be proven, although certain data are better explained by a heterogeneity of 5-HT 3 receptors. The molecular biology of 5-HT 3 receptors is just at its beginning and only one subunit has been cloned. However, given the high number of subunits which have been cloned for the other ligand-gated ion channel receptors, it is quite possible that further subunits will also be cloned very soon for the 5-HT 3 receptor. Finally, the involvement of 5-HT 3 receptors in a great number of behavioural effects is very impressive. 5-HT3 receptor antagonists display antiemetic, antinociceptive, antipsychotic, antidepressive, anxiolytic and promnesic properties. The fact that these effects are generally observed at very low doses and without side-effects is also impressive. However, the direct involvement of a central 5-HT3 receptor in most cases must be better assessed. Additionally, studies by independent laboratories and further clinical trials are still required to confirm most of the extraordinary potential therapeutic applications of 5-HT 3 antagonists. This has already been achieved for the antiemetic properties, and the potency of 5-HT 3

249 antagonists against chemotherapy-induced emesis is well established in patients suffering from cancer. Thus, a considerable amount of work has been done since the initial discovery of this receptor, and at least an equivalent amount remains to be performed in order to solve the unanswered questions.

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258 ABBREVIATIONS: ACh: acetylcholine; 2-methyl.5.HT: 2-methyl-5-hydroxytryptamine; 3-chloro-

PBG:l-(3-chloro-phenyl)-biguanide; 5.H~. 5-hydroxytryptamine; Azasetron: N-( 1-az abicyclo [2.2.2. ]oct- 3-yl )-6- chl oro- 4- methyl- 3- oxo- 3,4- dihydr o-2H- 1,4 -benzoxazine-8-carboxamide; Batanopride: 4-amino-5-chloro-N-[2-(diethylamino) ethyl]-2-(1-methyl-2-oxopropylbenzamide) Bemesetron: 1-H-3-a-5-aH-tropan3-yl-3,5-dichlorobenzoate; BMY 33462: (4-amino-N-(1-azabicyclo-[2.2.2]oct3-yl-2-(butan-2-one-3-yl)oxy-5-chlorobenzamide; BRL 4647OA: endo-N-(8-methyl8- azabi cycl o[ 3.2.1. ]oct- 3-yl )2,3-dihydro 3,3- dimethyl-indol e- 1-carboxami de; DAT-582: N-[1-methyl-4-(3-methyl benzyl) hexahydro-lH-1,4-diazepine-6(R) -yl)]-lH-indazole-3-carbox~mide; Dolasetron: (1H-indole-3carboxylic acid, trans-octahydro-3-oxo-2,6-methano-2H-quinolizin-8-yl ester; FK 1052: (+)-8,9"dihydro-10-dihydro- 10-methyl-7-[-(5-methyl-4-imidazolyl)methyl)] pyrido-[ 1,2a] indol-6(7H)-one; GR 65630: (3-(5-methyl- 1H-imidazol-4-yl)- 1(1-methyl- 1H-indol3-yl)-propanone; GR 67330: ( 1,2,3,9-tetrahydro-9-methyl-3[(5-methyl-1H-imidazol4-yl)methyl]-4H-carbazol-4-one; GR 68755: (1,2,3,9-tetrahydro-3-[(5-methyl-lHimidazol-4-yl)methyl]-4H-carbazol-4-one; Granisetron: (end0-N-(methyl-9-azabi cyclo-[3.3.1]-non-3-yl)-1-methyl-indazole- 3-carboxamide; Iodo-zacopride: 4-aminoN(1-azabicyclo-[2.2.2]-oct-3-yl)-5-iodo-2methoxy-berLzamide; Itasetron: (3-atropanyl) 1H-benzimi dazolone-3-carboxami de; L-683,877: (-)(2'-(1-methyl-1H-indol3yl))spiro(1-azabicyclo[2.2.2]octane-3,5'(4H)-oxazole); Litoxetine: Naphthyl-2-4piperidine benzoate; LY 278584: 1-methyl-N-(8-methyl-8-azabicyclo-[3.3.1]-oct-3yl)-lH-indazole-3-carboxamide; MDL 74156: (1H-indole-3-carboxylic acid, transocta-hydro-3hydroxy-2,6-methano-2H-quinolizm-8-yl ester; NA: 1-(3,4-dihydroxy phenyl)-2aminoethanol; NBS: N-bromosuccinimide; Ondansetron: (1,2,3,9-tetrahy dro-9methyl-3[(2-methyl- 1H-imidazol-1-yl(methyl]-4-one; PBG: 1-phenyl-biguanide; Q-ICS 205-930: N-methyl amonium(3-a-tropanyl)lH-indole-3-carboxylic acid ester; Quipazine: 2-(1-piperazinyl)quinoline; RG 12915: N-l-azabicyclo[2.2.2]oct-3-yl-2chloro-5a,6,7,8,9,9a-hexahydro-,[5aS-[4(R*),5aa,9aa]]-4-dibenzofuran carboxamide; RS-42358.197: N-(1-azabicyclo[2.2.2]-oct-3-yl)-2,4,6,tetrahydro-lH-benzo[de] isoquinolin-l-one SR 57227A: 5-amino-(6-chloro-2-pyridyl) 1-piperazine; Tropisetron: (3-a-tropanyl)lH-indole-3-carboxylic acid ester; Tubocurarine: 7',12'-dihy droxy-6,6'-dimethoxy-2,2',2'trimethyl tubocuraranium chloride; WAY 100289: (endo -N[(8-methyl-8-aza bicyclo[3.2.1.]octan-3-yl)amino carbonyl]-2-cyclopropyl-methoxy benzamide; YM060: (R)-5-[(1-methyl-3-indolyl)carbonyl]-4,(,6,7-tetrahydro-lH-benz imidazol; Zacopride: 4-amino-N(1-azabicyclo[2.2.2]-oct-3-yl)-5-chloro-2-methoxybenzamide; Zatosetron: endo 5-chloro-2,3-dihydro-2,2-dimethyl -N-(8-methyl-8-aza bicyclo [3.2.1.]oct-3-yl)-7-benzofura nocarboxamide.

Chapter 8

5-HT 4 RECEPTORS

This Page Intentionally Left Blank

Serotonin Receptors and their Ligands

B. Olivier, I. van Wijngaardenand W. Soudijn (Editors) t~) 1997Elsevier ScienceB.V. All rights reserved.

261

5-HT4 Receptors A.Dumuis a), H.Ansanay a), C.Waeber b), M.Sebben a), L.Fagni ~) and J.Bockaert ~)

a)CNRS UPR 9023, CCIPE, Rue de la Cardonille, 34094 Montpellier Cedex 5, France. b~Present address: Stroke Research Laboratory, Massachusetts General Hospital, Wellman 432,32 Fruit Street, Boston, MA 02114, USA.

INTRODUCTION

A retrospective In 1987, Shenker et al. [ 1] showed, in guinea-pig hippocampal membranes, that the stimulatory effect of 5-HT on adenylylcyclase (AC) involves two receptors. One displaying a high affinity for 5-HT(RH), characterized as a 5-HT,A-like receptor positively coupled to AC. The other having a low affinity for 5-HT(RL), and was not identified. 5-CT was better than 5-HT to distinguish between these two receptors, because of a clear biphasic dose-activation curve obtained with this agonist (Figure 1A). Indeed, 5-CT had a high (13 nM) and a low (3000 nM) affinity for 5-HT(RH) and 5-HT(RL) receptors, respectively [1, 2]. One year later Dumuis and colleagues [2] found in mouse colliculi neurons, a 5-HT receptor (5-HT-R) stimulating AC having a completely different pharmacology from the well known 5-HT,, 5-HT 2 and 5-HTa-Rs [3]. We proposed to call it the 5-HT4-R [2]. We immediately recognized that this receptor positively coupled to AC, shared with the 5-HT(RL) receptor defined by Shenker et al. [1], similar potencies for a series of agonists: 5-HT=5-MeOT> bufotenine> 5-CT> tryptamine (figure 1B, Table 1A). Our conviction that the 5-HT(R class was different from the 5-HT1,2.a classes came from the observation that highly potent and specific 5-HT1,2.3 antagonists (spiperone, a 5-HT1A + 5-HT 2 antagonist; methiothepin, a 5-HT~ + 5-HT 2 antagonist; mesulergine, a 5-HT2c antagonist; ketanserine, a 5-HT2A antagonist; (-)pindolol, a 5-HTiA + 5-HT,B antagonist; MDL72222, a 5-HTa antagonist) were unable to inhibit the 5-HT4-R in colliculi neurons (Figure 1C) [4]. However, we were fortunate to find a weak (mM potency) but competitive inhibitor of 5-HT4-Rs during our very early series of experiments: i.e. tropisetron [4]. The similarities between the 5-HT(RL) receptor in guinea-pig hippocampus and the 5-HT4-R in mouse colliculi neurons was further confirmed when we found that tropisetron (p~=6.1-6.5) also blocked the 5-HT(RL) receptor at mM concentrations in guinea-pig hippocampal membranes [2, 5]. It is worth noting that tropisetron is more potent in antagonizing 5-HT 3 (pI~.=9) than 5-HT 4Rs. Shortly thereafter, we were able to demonstrate that a series of gastrointestinal prokinetic benzamide derivatives, including metoclopramide, renzapride, cisapride and zacopride, acted as agonists at the 5-HT4-R [6, 7]. This

262 observation built an immediate link between the 5-HT,-R described in colliculi neurons and an unclassified 5-HT binding site in the enteric nervous system, which was postulated to mediate the gastrokinetic actions of these compounds [810]. It was shown that in guinea-pig ileum, this non-5-HT,.2.3-R was located in neurons, had high affinity for 5-MeOT, low affinity for 5-CT, was stimulated by benzamides and displayed low affinity for tropisetron (mM).

A

B

120100t Guinea pig hippocampus

100120_ Colliculi neurons

80

802

6o

60-

40

40

20

20

o,..q

~

-

e~

"d ~ r,9

0

8 -

7 6 5 4 Log [Agonistl (M)

9

3

0

8

7 6 5 4 - Log [agonistl (M)

3

(D

C

--

~ 9~

120 ColUculi neurons 100 J/

~

80

N

60

9

9

A B

x

F o 5-HT [ 9 5CT 9 Spiperone Ketanserine 9 Methiothepin ,~ Tropisetron o

o

40

C

20 0

I

I

..

I

,

.

I

.

I

7 6 5 4 - Log [ Antagonist]

3

Figure 1. Effect of 5-HT agonists and 5-HT,,2,3 antagonists on c-AMP formation in guineapig hippocampal membranes and mouse colliculi neurons. (A) Concentration-effect curves of 5-HT and 5-CT on stimulation of AC activity in guinea-pig hippocampal membranes (data from [2]). (B) Stimulation of c-AMP formation by 5-HT and 5-CT, in colliculi neurons (data from [2]). (C) Effect of 5-HT,.2.3-related antagonists on 5-HT (1 mM) stimulation of c-AMP formation in colliculi neurons (data from [4]).

263 It be became likely that this 5-HT-R in guinea-pig ileum was similar, if not identical, to the 5-HT4-R present in guinea-pig hippocampus and mouse colliculi neurons [5, 10-13]. This was the beginning of scientific efforts to find additional arguments to bring about the pharmacological legitimity of the 5-HT4-R that the Nomenclature Committee of the Serotonin Club would recognize this receptor as a nove! 5-HT-R subtype. This was achieved in 1993 at the meeting organized by the Serotonin Club in Houston [ 14]. In 1994 the human and in 1995 the rat 5-HT4R was cloned and expressed [114, 115]. PHARMACOLOGICAL CHARACTERIZATION

Functional pharmacological analysis Until 1993, studies on the 5-HT4-R pharmacology have been hampered by the absence of radioligands. Therefore, most of our knowledge is still based on functional pharmacological analyses.

Models used to study 5-HT4-R pharmacology Three main preparations have been used to characterize the pharmacology of 5-HT4-Rs: mouse colliculi neurons, guinea-pig ileum and tunica muscularis mucosae of rat oesophagus. (1) In mouse colliculi neurons, the activity of 5-HT4-Rs are quantified by measuring c-AMP production [2]; (2) In guinea-pig ileum, 5-HT4-Rs increase the supramaximal twitch response [810, 15]. This latter preparation is complicated by its relative instability, and by the fast that 5-HT stimulates two neuronal receptors leading to a biphasic contraction of the ileum [15]. Low doses of 5-HT (<300 nM) stimulate 5-HT4Rs, whereas higher doses also activate 5-HT~-Rs [15]. However, because of the rapid desensitization of both 5-HT 3 and 5-HT4-Rs [16], Fozard [17] and Craig et al. [18] introduced a useful refinement in the guinea-pig ileum preparation. Pre-treatment with 5-MeOT or 2-Me-5-HT specific 5-HT4 and 5-HTs-R agonists, respectively [2, 10], can be used to desensitize one or the other of these receptors leaving the preparation with only one functional receptor. The quiescent non-stimulated ileum has also been used and appears to be more robust [19]; (3) The third preparation was introduced more recently and constitutes an excellent model for pharmacological studies. This is the tunica muscularis mucosae of the rat oesophagus [20, 21]. Although small differences are observed between the pharmacology of 5-HT4-Rs expressed in these different preparations (see paragraph: Comparative pharmacological characteristics of 5-HT4-Rs), a good correlation was found between the potency of agonists determined in the mouse colliculi neurons, and their potency determined in the tunica muscularis mucosae of the rat oesophagus and quiescent or stimulated guinea-pig ileum [12, 19, 22].

264

5-HT4-R agonists Derivatives of three main classes of chemical compounds have been found to be agonists at 5-HT4-Rs; indoles, benzamides, benzimidazolones. Other classes are represented by a quinoline, a naphthalimide, a benzoate and a ketone. Indole derivatives: The order of potency of indoles at 5-HT4-Rs is very similar in colliculi neurons, guinea-pig ileum and rat oesophagus: 5-HT=5-MeOT > bufotenine > 5-CT. Tryptamine, 2-Me-5-HT and 5-MeODMT being very weak or inactive agonists (Table 1A) [12, 22, 23]. The high affinity of 5-HT4-Rs for 5-MeOT has been reported by most investigators, whatever the preparation used, including mouse colliculi neurons, guinea-pig ileum, rat oesophagus, human and pig myocardia and guinea-pig colon [2, 10, 19, 21, 23, 24, 150]. However, in rat oesophagus and the guinea-pig ileum, Humphrey's group found 5-MeOT to be much less potent than 5-HT [20, 25]. The reason for this discrepancy is not clear. It is worth noting that analogs of 5-HT substituted at position 5 of the indole ring, especially 5-MeOT and CT, are very weak 5-HT~ agonists. When the indole group is substituted at both positions 5 and 3, with a methoxy and tetrahydropyridine, respectively, this leads to compounds which are active (RU 28253) or inactive (RU 24969), respectively. When compared with 5-HT1,2,3-Rs, 5-HT4-Rs exhibit a distinct tryptamine profile (see [23]) which further supports the evidence of a fourth different 5-HT-R subtype. Benzamide derivatives: All benzamides bearing the 2-methoxy-4-amino-5-chloro substitution (Table 1B) have been found to be 5-HT 4 ligands, either acting as agonists or antagonists (Tables 1B, 2B). In contrast, the benzamides displaying different substitution groups on the benzamide ring, such as sulpiride (2-methoxy-5-aminosulfonyl) benzamide, or tiapride (2-methoxy-5-methylsulfonyl) benzamide or the cisapride analog: R 60 918 (2-methoxy-4-acetamido) benzamide lack affinity for 5-HT4-Rs [12, 22]. All the benzamide agonists at 5-HT4-Rs are gastroprokinetic drugs. The better known compounds are: metoclopramide, renzapride, zacopride, cisapride and its analogs (R 76 186 and R 66 621) [7, 22, 26]. Since 1989, distinct progress has been made with 5-HT4 agonists. The newer compounds SB-205149 (quaternized renzapride), SC53116: the active isomer of the racemic mixture SC49518, appear to be the most potent selective 5-HT4-R agonists [27-29]. The efficacy of all these benzamides depends on the preparation studied. They are full agonists in colliculi neurons [7] and in the electrically stimulated guinea-pig ileum [10], whereas they are partial agonists in non-stimulated guinea-pig ileum [19], guinea-pig hippocampal membranes [5], guinea-pig ascending colon [25], human and pig heart [30-32]. They may be antagonists at the 5-HT4-Rs controlling slow depolarization and after-hyperpolarization in CA1 neurons [33, 34]. Metoclopramide, one of the first benzamides used as a gastro-prokinetic drug, is a partial agonist in most preparations [12]. All these benzamides are not strictly selective. Most have 5-HT~ antagonistic properties (zacopride, renzapride, metoclopramide) [8, 35]. In addition, metoclopramide has a dopamine D2 antagonistic effect (170 nM) [36],

265 whereas cisapride has a high potency for 5-HT~-Rs (2.5 riM) [37]. Only new benzamides, SB 205149, SC53116 the active isomer and its corresponding racemic mixture: SC49518, have been shown to be potent and selective 5-HT4 agonists (ECso=10 nM; 18 nM and 66 nM, respectively) with weak 5-HT3 antagonistic potency (pI~.=<7) (Table 1B). Be nzimidazolo nes :

In our search for new compounds acting at 5-HT4-Rs, we thought that drugs which have gastrointestinal prokinetic activity, and for which a clear mechanism of action is not yet known, could be good candidates. Among them, the azabicycloalkyl benzamidazolone derivatives [13, 38] were of interest because their effect on electrically stimulated guinea-pig ileum was blocked by mM concentrations of tropisetron (a 5-HT4 characteristic) but not by ondansetron, a selective 5-HT~ antagonist [13, 39]. Indeed, we found in colliculi neurons that BIMU 1 and BIMU 8, which are alkyl substituted benzimidazolones at position 3 (Table 1C) were potent and efficacious agonists at 5-HT4-Rs in colliculi neurons. DAU 6215 which has a proton at position 3 was a weak partial agonist [40]. Introducing a methoxy residue at position 6 on DAU 6215 leads to DAU 6285 which becomes a 5-HT 4 antagonist (Table 2D) [41]. Similar effects of this class of agonists were observed on rat oesophagus [42]. As many benzamides, the benzimidazolones are also both potent 5-HT 4 agonists and 5-HT~ antagonists [38]. Quinolines:

4-amino-5-chloro-2-methoxy benzamide drugs were modified in order to obtain more potent and selective 5-HT4 agonists. Among the compounds synthesized by Blum et al. [43], SDZ 216-908, a quinoline derivative (Table 1D), was identified as a potent gastrointestinal prokinetic benzamide and 5-HT 4 agonist. However, this compound was not a s selective for 5-HTt, it retained 5-HT 3 antagonism. Naphthalimides:

Naphthalimides such as RS 56532 are conformationally restricted analogs of zacopride. The (S)-enantiomer (S)-RS 56532 is a potent 5-HT4-R agonist (Table 1E) with a high intrinsic activity in the rat oesophagus [116]. However, in guinea-pig colon (S)-RS 56532 acts as a potent antagonist (pI~.=9.4) [150]. The (R)-enantiomer acts as a weaker agonist (pECso <6.0) in the rat oesophagus. The stereospecificity at 5-HT4-Rs of RS 56532 is similar to zacopride i.e.(S) >(R). At 5-HT3-Rs however, is the enantiomeric selectivity of RS 56532 converse to that of zacopride i.e. (R) >(S). Both enantiomers of RS 56532 act as antagonists at the 5-HTs-R. Benzoates:

Removing the steric constrains of the quinuclidine ring as present in zacopride is highly favorable for acting at 5-HT4-Rs and unfavorable for acting at 5-HTa-Rs. This was illustrated with ML 10302 (Table 1F), a totally flexible molecule.

266 Table 1 Structural formulae of some 5-HT-R agonists and their potencies at the 5-HT 4R in mouse colliculi neurons, rat oesophagus and guinea-pig ileum. The potencies of compounds were determined by functional analysis data from [12, 21, 23, 26-29, 38, 43-47, 116-119].

A INDOLES [

~

co,,icu,i !1 Rat [[Guinea pig neurons Ileesophagusll ileum

,og~c~oll ,o~

II ,og

Tryptamine derivatives

HO.(~~

NH2

.J

H 5-Hydroxytryptamine 5-HT :

CHaO~

i,, II

NH2

' II ,,o

~N H 5-MeOT

7.93

7.77

7.29

:

5-Meth o ~ r t r ~ t ~ m i n e

NH2 HOrN

CH3 \ H

r

II

6.87

6.44

o~-Me-5-HT :

a-Methyl-g-hydroxytryptamine N(CH3)2 .J

~~ \ H

Bufotenine : 5-HydroxyoN,N~timethyltryptamine

if,so [i ,,2 il

6.66

267 pig II neurons ~176 IIOeSophagusl II ~a' I Guinea ileum

I: ~og EC~oil- ~og ECho J -log EC5o NH2 J

o

5.5

H2N~~N \ H

iil 6.16 il

5.48 ,,.

5-CT : 5-Carboxamidotryptamine J

N(CH3)2

CH30~N \ H

[4.8 !1

L!

<4

5-MeODMT : 5-Methoxy-N,N-dimethyltryptamine

J

NH2

<4

<4

H T: Tryptamine NH2

HO"~N

II ,nac,,ve!1

CHa

inactive

\ H 2-Me-5-HT 9

2-Methyl-5-hydroxytryptamine NH2

._/ CH30~N 6-MeOT :

~ H

6-Methoxytryptamine

I_ .<4

I! inactive . . _

inactive

268

@

N o n - t r y p t a m i n e derivatives N~H

CH30 ~~N

\ H

R U 28253 : 5-Methoxy-3-(1,2,5,6-tetrahydropyridine-3-yl)indol !H N

CH30 \ H R U 24969 : 5-Methoxy-3-(1,2,5,6-tetrahydropyridine-4-yl)indol

B

BENZAMIDES 2-Methoxy-4-amino-5 chloro b e n z a m i d e derivatives OMe 0

CI SC 53116 :

(1-S, 8 - S ) - 4 - a m i n o - 5 - c h l o r o - N - [ e x o - ( h e x a h y d r o 1H-pyrrolizin-l-yl)-methyl]-2-methoxybenzamide,HCl 1-S, 8-S isomer of the racemic mixture" SC 49518

Guinea pig I Colliculi I! Rat ileum neurons II oesophagus ~ 1 - l o g EC50 -log EC5o

269

l

'

u,,ll

....

Rat

llGuinea pigI

neurons ]l~Sophagusll

ileum

I

-,og EC~oll- ,og ECho II-,og EC~ol

OMe 0 H2N"

H

1

1_'-"

T

Cl

R 76186 ( C i s a p r i d e analogue) : Cis-4-amino-5-chloro-N-[ 1-[4-[4-(dimethylamino)1-piperidinyl]-4.oxobutyl]-3-methoxy-4piperidinyl]-2-methoxy-benzamide, HC1

OMe O

~']N

r__nBu

1l 8"O2!!

H2N- T CI 205 149 : [Exo-N-l-butyl-l-azabicyclo [3.3.1]-nonan-3-yl]-4amino-5-chloro-2-methoxy-benzamide, HC1

SB

,OMe 0

H

/F

ilI ,.26 11 7.7 CI Cisapride: Cis-4-amino-5-chloro-N-[ 1-[3-(4-fluoro-phenoxy) propyl]-3-methoxy-4-piperidinyl]-2-methoxyb e n z a m i d e , HC1

OMe

o

!, cI SC 49518 : [N-[ exo-(hexahydro- 1-H-pyrrolizine- 1-yl)-methyl ]2-methoxy-4-amino-5-chloro-benzamide, HC1] A racemic mixture consisting of l-S, 8-S and l-R, 8-R enantiomers

il, 17.2111l

270

I CoIliculiII~176 II neurons

Rat

IIGuineapig ileum

log EC~oll ~ogECho II ~og ECho

OMe 0

,.9, I[ ,.1 !1 ,.98

H

H2N CI

Renzapride : [ (_+)-endo ] - 4 - a m i n o - 5 - c h l o r o - 2 - m e t h o x y - N - [ 1 a z a b i c y c l o - ( 3 . 3 . 1 ) - n o n - 4 - y l ] - b e n z a m i d e , HCI OMe 0

~""" N" i ' ' " f i f ~

6.87 cI Clebopride : 4-amino-5-chloro-2-methoxy-N- [1-phenylmethyl-4p i p e r i d i n y l ] - b e n z a m i d e , HCI OMe 0

H2N"~

fN,,,,,, H

CI

,1, I! ,, I! ,,,

S-Zacopride : S-4-amino-N-(1-azabicyclo [2.2.2]-oct-3-yl)5-chlor~ 2 - m e t h o x y - b e n z a m i d e , HCI

l~'L.

OMe 0

I H

H2N

H

CI

R-Zacopride : R-4-amino-N-(1-azabicyclo [2.2.2 ]-oct-3-yl)-5-chloro2 - m e t h o x y - b e n z a m i d e , HCI

5.5

6.07

5.8

271

I

OMe O H2N,~ CI

Colliculi !1 Rat llGuineapig neurons Ilc~sophagusll ileum -log EC501[,- log EC50 I1" log EC50

~ N'/~'~

6.7

H ~ ~

BRL 20627 : [2-% 6~, 9a~]-(_+)-4-amino-5-chloro-2-methoxy-N(octa-hydro-6-methyl.2H-quinolizin)-benzamide, HC1

OMe O

~~

H2N"

r"'" ~ N ~ -

,3, ii ,1 il ,,

CI

Metoclopramide : 4-amino-5-chloro-2-methoxy-N-(2-diethylamino e t h y l ) - b e n z a m i d e , HC1

Q

Other b e n z a m i d e d e r i v a t i v e s

OMeO

~r'LN~-y~h

H2N/~L'~

H

L--N.J

CI

~ F

6.27

'

AS 4370 :

4-amino-5-chloro-2-ethoxy-N- { [4-(4-fluorobenzyl)2 - m o r p h o l i n y l ] - m e t h y l | - b e n z a m i d e , citric acid

OMe 0 N ' ~

'na~ II cI Sulpiride : 5-(aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl) m e t h y l ] - 2 - m e t h o x y - b e n z a m i d e , HC1

6.47

272 T

C BENZIMIDAZOLONES

0 H ~

ico,,,cu,,,,

IIGu,nea0,0

neurons II oesophagus ileum -log EC5oll -log EC5o -log EC5o

N-CH3

~ -'-N,,H

,1, It ,9

8.16

)-_ BIMU 8 : [ E n d o - N - 8 - m e t h y l - 8 - a z a b i c y c l o [3.2.1]-oct-3-yl]-2,3dihydro-3-isopropyl-2-oxo-lH-benzimidazol-1c a r b o x a m i d e , HC1

.~_/~N-CH3

""~"

o

~N,~ H

,-~ tt

)

8

8.4

BI31U 1 9 [Endo-N-8-methyl-8-azabicyclo-(3.2.1)-oct-3-yl]-2,3d i h y d r o - 3 - e t h y l - 2 - o x o - l H - b e n z i m i d a z o l - 1c a r b o x a m i d e , HC1

.N-CH3 5.6

H DAU 6215 : [ E n d o - N - 8 - m e t h y l - 8 - a z a b i c y c l o [3.2.1]-oct-3-yl]-2,3d i h y d r o - 2 - o x o - 1 H - b e n z i m i d a z o l - 1 - c a r b o x a m i d e , HCI

II inactive

273 - -

, m ,

.

....

,,,

D QUINOLINE 1

I co,,ou, II ! neurons

Rat

IIGuineapig

Iloesophagusll

I-tog ECs01[-,og EC50 I["~

ileum

EC50

H

I!

H2N" T cI SDZ 216 908 : (_+)-endo-5-amino-6-chloro-quinoline-N-(1~ a l ~ c y e l o [3.3.1]-nonan.4-yl)-8-earbox~ide

II

7.5

_E NAPHTHAT:IMiDE' I o

~~

[ i[

H

(S)-RS 56532: (S)-6-amino-5-chloro-2-(1-azabicyclo [2.2.2]octan-3-yl) 2,3-dih ydro- 1H-be nz[ de ] isoquinoline-l,3-dione

F BENZOATE o

H2N-- v

"OMe

ML 10302: 2-(1-piperidinyl)ethyl-4-amino-5-chloro-2methoxybenzoate

E

7.9

i[ ....

274

"L

G

I neurons [I~176

ileum

1-log ECsol[ -log eCso -log ECso

o

IJ 8.4 If

RS 67333: 1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1n-butyl-4-piperidinyl)-l-propane

' '

_

',

I,

._

v

. . . .

.,

:

'

-.

' '

, :

ML 10302 is a potent, but partial agonist in rat oesophagus and guinea-pig ileum [117]. However in colliculi neurons ML 10302 behaves as an antagonist (unpublished results). ML 10302 lacks significant affinity for 5-HT3-Rs. The ester function as present in ML 10302 is a serious drawback for in vivo experiments. K e to n e s :

To improve the in vivo stability, the ester group of RS 23597 (a 5-HT4-R antagonist, Table 2C) was replaced by a ketone moiety. The corresponding ketone however, is a reasonable potent 5-HT4-R partial agonist [118]. Modification of the side-chain resulted in RS67333 (Table 1G), a potent partial agonist. The affinity for 5-HT3-Rs (p~=6.4) is more than two orders of magnitude less than the affinity for 5-HT4-Rs (pI~.=8.7). However RS67333 is not selective with respect to c~ and (~2receptors ( p ~ values 8.9 and 8.0 respectively) [119]. The close analog RS67506 ( 1-(4- amino- 5- chl oro- 2- me thoxyphe nyl)- 3- ( 1- {2- [(me thyl s ul p ho nyl)amino] ethyl)- 4piperidinyl)-l-propanone is as potent as RS67333, but more selective with respect to (~ (p~=7.9) and c2 (p~=7.3) receptors. The involvement of (~ sites may complicate in vivo experiments with these compounds.

5-HT4-R antagonists Rapid progress has been made since the early days when tropisetron was the only available 5-HT4-R antagonist [4] with low affinity for this receptor (p~=6-6.5) (Table 2A) and high affinity for the 5-HT3-R (pI~=9-10 ). The second generation of antagonists included two competitive antagonists: DAU 6285 [41, 48, 49] and SZ 205 557 [49-51] (Table 2D, 2C). The pI~. values of these two drugs for 5-HT4-Rs are between 6.5 to 7.5 [47].

275 The ratio selectivity of SDZ 205 557 for 5-HT 4 vs 5-HT~-Rs depends on the preparation used [51] but this ratio is approximately 5 to 30. SDZ 205 557 has been reported to non competitively interact with some agonists of the benzamide class [50], although experiments in the oesophagus have failed to corroborate such observations [51]. DAU 6285 has a 5-HT 4 vs 5-HT3 ratio selectivity of 1.5 to 10 [41, 47]. It is not more selective than SDZ 205 557 but has the advantage of being more stable in vivo (SZD 205 557 is a hydrolysable ester) [47, 49-51]. The third generation of 5-HT 4 antagonists has been described in 1992: SKB Comp. 2 [52] SC53606 [53] LY 297524 and LY 297582 [54], RS-23597-190 [47] (Table 2). The 5-HT4 versus 5-HT 3 selective ratio [47] has improved since the second generation of antagonists. RS 23597, although possessing low affinity for 5-HT3-Rs, also labels ~1 binding sites, making the compound an unsuitable ligand for 5-HT4-R binding studies [126]. However, the real break-through came with the synthesis of GR 113808 [55]. In guinea-pig ileum, rat oesophagus and colliculi neurons this compound behaves as a competitive 5-HT 4 antagonist (pI~.=9.2, 9.5 and 9.5, respectively) (Table 2A). A similar high activity is observed in guinea-pig colon (p~=9.1) [150]. GR 113808 has a very low affinity for 5-HT~-receptors (pKi=6) [55]. These pharmacological properties lead Grossman et al. [55] to prepare a tritium labelled ligand [3H]-GR 113808 (83.4 Ci/mmol.) that appeared to bind specifically to 5-HT4 sites [55, 56]. It is also a hydrolysable ester and is rapidly degradated in vivo [127]. An other potent and selective 5-HT 4 antagonist is SB 204070, a benzoate derivative (Table 2C). In the guinea-pig ileum SB 204070 at low concentrations (10-100pM) is a surmountable antagonist (pI~.=10.8). At higher doses (>300 pM) non-surmountable antagonism is observed [120]. In the rat oesophagus and guinea-pig colon SB 204070 acts as an unsurmountable antagonist (pI~. values 10.5 and 11.1 respectively) [150]. In binding studies SB 204070 has a high affinity for 5-HT4-Rs (pI~.=10.6) and weak or no affinity for other receptors tested [128]. Due to the ester function SB 204070 has a limited duration of action and poor bioavailability [124]. The first orally active 5-HT 4 antagonists were reported in 1995. One example is SB 207266, an oxazinoindole-carboxamide (Table 2A). In guinea-pig ileum SB 207266 behaves as a competitive antagonist (pI~=10.6) [121]. The compound has a high affinity for 5-HT~-Rs and weak or no affinity for other receptors tested including 5-HT3-Rs (pI~.<6). In the conscious dog Heidenhahn gastric pouch model, SB 207266 is active after i.v. injection (ID5o=1.3 ~g/kg) and oral administration (ID5o=9.6 l~g/kg) [122]. An other orally active 5-HT 4 antagonist, RS39604 (Table 2F) possesses a ketone moiety. In rat oesophagus, guinea-pig ileum and guinea-pig colon RS39604 behaves as a competitive 5-HT 4 antagonist (pI~. values 9.3, 9.1 and 9.0 respectively) [123, 150]. RS39604 displays a high affinity for 5-HT4-Rs (pI~.=9.1), moderate affinity for ~1 (p~=6.8) and ~2 (pI~.=7.8) and weak or no affinity for other receptors tested including 5-HTa-Rs. RS39604 antagonizes the 5-HT induced tachycardia in anaesthetized pigs (ID~o=4.7 ~g/kg, i.v; ID~o=254.5 ~g/kg, i.d).

276 Table 2 Structural formulae of antagonists and their potencies at the 5-HT4-R in mouse colliculi neurons, rat oesophagus and guinea-pig ileum. The potencies of compounds were determined by functional analyses. Results from [2, 4, 12, 41, 47, 48, 50, 53-55, 57, 120-124].

A

INDOLES

O

Rat ]]Guineapig Colliculi neurons oesophagusl] ileum pKi PKi PKi

~~--~ N''nBu H 10.6

SB 207266: N-(1-Butylpiperidin-4-ylmethyl)-3,4dihydro-2H-[1,3]oxazino[3,2-a]indole -10-carboxamide hydrochloride

O~N~NHSO2CH3 CH3 G R 113 808 :

(1-[2-methylsulphonylamino~thyl]~ipiperidinyl]methyl 1-methyl-lH-indo le-3-c arb oxyla te)

9.5

9.5

9.2

277

Guinea pig ileum pKi

Coiliculi neurons pKi 0

I

\ H

7.2

SB 203 186 :

2-(piperidinyl)-ethyl indole-3-carboxylate, HCI

o~

.N-CH 3

6.5 \

H

Tropisetron: (3~-tropanyl) 1H-indole-3-carboxylic acid ester

B

BENZAMIDE /F

inactive

CI

F

R 50 595 :

trans-4-amino-N[ 1-[4,4-bis(4-fluorophenyl)butyl]3-hydroxy-4-piperidinyl]-5-chloro-2-methoxybenzamide, HC1

6.3

278 .

.

.

.

.

.

I

,

I

,

Ill

C,, BENZOATE I cI

A

co,,,co,, I! ,,..

neurons

.K,

.

,

=.,

ilaes~ Ii p~

o Uo

HN~o" ~ N

10.5

o.3

,._

,

.,

If Guineapig ileum

II

pK'

.....!l ,08

SB 204070: (1-n-butylo4-piperidinyl)methyl-8-amino.7 chloro- 1,4-benzodioxane-5-carboxylate

OMe O H2N" T CI

~"

iI

7.8

RS 23 597 -190 :

[3-(piperidin-l-yl)-propyl-4-amino-5-chloro-2methoxy-benzoate, HCI]

OMe O ~

O

~

N

~

cI

!!i,, I[

Ly 297 524 : 2-methoxy-4-amino-5-chloro-benzoic acid-2(diethyl amino)-propyl ester, HCI

~

OMe 0

-

o ~ N ~

H2N" y CI S D Z 205 557 : 2-methoxy-4-amino-5-chloro-benzoic acid-2(diethylamino)-ethyl ester, HCI

7.1

7.5

279

BENZIMIDAZOLONE

D

OH ~

co,,,cu,,ll Rat Ilou,nea0,0

neurons, oesophagus pKi pKi

N-CH3

yN, H MeO,...~N

L~N>=~

,.8 Ii ' II ,.8

H

DAU

6285 :

Endo~6--methoxy-8-methyl-8-azabicyclo [3.2.1]-oct3-yl)-2,3-dihydro-2-oxo-lH-benzimidazole-1carboxylate, HC1

E CI

ileum pKi

IMIDAZOLE o

IL' II SC 5 3 6 0 6 : (1-S,8-S)-N-[(hexahydro-lH-pyrrolizin-l-yl)methyl]-6-chloro-imidazo [1,2-~]-pyridine-8carboxamide, HCI

280

Rat

F

neurons oesophagusI pKi , pKi ,]

KETONES O

H2

OMe

I ii

Guinea pig ileum pKi

11.2

o..)

RS 100235: 1-(8-amino-7-chloro- 1,4-benzodioxan-5-yl)-3-[ [3-(3,4-di me t h o x y p h e n y l ) p r o p- 1 -yl ] piperidin-4-yl]propan-l-one

o

H2CNO

L~/OMe

SO2CH3

OMe

RS 39604: 1-[4-amino-5-chloro-2-(3,5-dimethoxyb e n z y l o x y ) p h e n y l - 3 - ( 1- { 2 - [ ( m e t h y l s u l p h o n y l ) amino]ethyl}-4-piperidinyl)-l-propane

i

9.3

9.1

281 At maximal doses of 30 lag/kg, i.v and 6 mg/kg i.d RS39604 is effective for more than 6 h. In conscious mice RS39604 inhibits the 5-hydroxytryptophan induced diarrhoea (ID5o=81.3 ~g/kg, i.p and ID~o=l.1 mg/kg, p.o). One of the most potent orally active 5-HT 4 antagonists is RS100235 (Table 2F), a ketone related to SB204070 (Table 2C). In rat oesophagus RS100235 acts as an unsurmountable antagonist (pI~.= 11.2) [ 124]. RS100235 displays a high affinity for 5-HT4-Rs (pKi=10.0) and is selective with respect to other receptors tested, including the 5-HTa-R. In anaesthetized pigs RS100235 antagonizes the 5-HT induced tachycardia (ID~o values: 0.55 ~g~g i.v and 1.52 ~g/kg i.d). At a dose of 3 lag/kg, i.v RS100235 is effective for more than 6 hours. The antagonistic activity of RS100235 is after i.v administration 8.5 times and after i.d administration 161 times greater than that of the ketone RS39604. Two non-competitive antagonists of 5-HT4-Rs in guinea-pig ileum have also been described: R 50595 [57, 58], a benzamide derivative (Table 2B) and FK 1052 a dihydropyridoindole derivative [59]. The mode of action of these drugs is not known. Do they pseudo-irreversibly bind to the agonist binding site, or to an allosteric site, or do they block the contraction by acting downstream to the 5-HT 4R? As mentioned above, three main families of 5-HT 4 compounds have been described based on the nature of their aromatic nucleus (indole, benzamide or/and benzoate and benzimidazolone). In addition to these observations, we can note that the structural characteristics of both aromatic nucleus and side chain are required for the selectivity of the 5-HT 4 ligands. In most cases, the chemical structure of the side chain can be summed up in three residues: a piperidine, an azabicyclo or a pyrrolizine.The compounds in which the aromatic nucleus is substituted by a piperidine such as SB 207266, GR 113808 or RS 100235, are highly selective 5-HT 4 antagonists with very low 5-HT~ antagonistic properties. 5-HT4 agonists with a piperidine side-chain such as cisapride, R 76186 (cisapride analog) or clebopride, are potent prokinetic compounds also devoid of 5HTa antagonist activities. When the side chain is an azabicycle whatever the aromatic nucleus, such as ICS 205930 (indole), renzapride, S-and R-zacopride (benzamides), (S)-RS 56532 (naphthalimide) and BIMU 1, BIMU 8 or DAU 6585 (benzimidazolones), compounds having 5-HT 4 agonist and also antagonistic properties, high 5-HT3 antagonistic properties are obtained. It had been recently shown that the n-butyl quaternization of renzapride to give SB 205149 both reduced 5-HTa antagonistic activity and increased 5-HT4-R agonist potency [27]. The pyrrolizine ring found only in new compounds related to substituted benzamides: SC53116 and its racemic mixture SC49518, lead to potent and highly effective 5-HT 4 agonists devoid of potent 5-HT~ antagonistic properties [28, 29].

282

Pharmacology of the [3H]-GR 113808 binding sites in guinea-pig, human striatum and mouse colliculi neurons. [3H]-GR 113808, the potent and specific 5-HT4-R antagonist, binds to a single class of binding sites in specific membrane fractions of guinea-pig and human striatum, as well as colliculi neurons [55, 56, 125]. Table 3 Affinities of various compounds that compete for 0.1 nM [3H]-GR 113808 in homogenates of colliculi neurons [125], human striatum [56] and guinea-pig striatum [55,56].

Compound

No

(PKD)

(PKD

(pKD)

GRl13808 5-HT Renzapride BIMU 8 5-MeOT Tropisetron Cisapride DAU 6285 SDZ 205557 BIMU 1

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

9.5 7.1 7.0 7.6 6.6 6.8 7.3 7.6 8.2 7.3

9.8 6.5 6.6 7.1 6.3 6.7 6.8 7.2 8.2 N.D.

9.5 7.3 7.0 7.9 6.5 7.2 7.5 N.D. 8.2 7.8

Table 3 and Figure 2 compare the affinities of various 5-HT4 agonists and antagonists that compete for the [3H]-GR 113808 binding in these preparations; it is clear that the pharmacology of the [3H]-GR 113808 binding towards those of 5-HT 4 agonists and antagonists are very similar. Figure 3 shows a good correlation between affinities (Table 3) of several 5-HT4-R ligands that compete with [3H]-GR 113808 in three different tissues. Although there is a minor shift in agonist affinities for [3H]-GR 113808 binding sites in the presence of GTP, there is little doubt that these binding sites are similar to those of the 5-HT4-Rs positively coupled to AC in mouse colliculi neurons [2], rat hippocampal membranes [5], heart [24] or oesophagus [45, 46]. This is only one of many examples where a weak effect of GTP, on agonist binding to a G protein coupled receptor, has been reported.

Comparative pharmacological characteristics of 5-HTcRs. Potential heterogeneity We have already discussed the fact that benzamides can be full agonists, partial agonists or even antagonists, depending on the preparation studied. Figure 4

283

B

A 100 t

100

-

80"

8O

:E

60

60

"~

4O

,~

20

-

n

Human

brain

40 20

0 8 -

7 6 5 4 Log [Agonist] (M)

3

0 - t ~ "~- 7 -10 8 6 4 - Log [Antagonist] (M)

o

5-HT

o

GR

n

BIMU8

"

SDZ 205 557

Renzapride

=

Tropisetron

5 O_.____~T Me ___

o

DA.._.~U628__5_5_

9

C 100~~

100 - r

60

60

"~

40

40

'~

20

20

-

113808

7 6 5 4 log [Agonist] (M)

3

Colliculi [

80

;E

8

2

D Colliculi I neurons

80

0

-

0-

I0 8 6 4 - Log [Antagonist] (M)

2

Figure 2 Comparison between the pharmacological characteristics of [3H]-GR 113808 binding on 5-HT4-Rs in human and colliculi neurons. Pannels A and B represent competition experiments carried out in human caudate membranes with various 5-HT, agonists or antagonists (data from [56]). Pannels C and D represent competition experiments carried out in mouse colliculi neurons with the same 5-HT4 agonists or antagonists as in pannels A and B [125].

284

A

10,,

Compound ~',~

9

9~ ' ~

8

~

7

~'~

/

~

r = 0.968

"1

I

6

Cisapride D A U 6285 S D Z 205 557 BIMU 1

"

7

8

"

6

............. ............. ............. ............. ............. ............. ............. ............. ............. .............

G R 113808 5-HT Renzapride BIMU 8 5 MeOT Tropisetron

9

5

N

10

(pKD) 5-HT4-binding affinity

O

(1) (2) (3)

(4) (5) (6) (7) (8) (9) (10)

in colliculi n e u r o n s

C

B

10

10

ol-,4

9-= "~

M.4

~

9-

0 D 01:)

~

10

8

/I.

3

5 ~

6

i

/

r = 0.953 I

7

'

'

8 (pKD)

I

9

'

9 5

3 9

10

6

r = 0.922 I

7

'

/

'

8

I

9

'

10

(pKD)

5-HT4-binding affinity

5-HT4-binding affinity

in colliculi n e u r o n s

in h u m a i n b r a i n

Figure 3 C o r r e l a t i o n s b e t w e e n t h e affinities of a series of a g o n i s t s a n d a n t a g o n i s t s for [3H]-GR 113808 b i n d i n g sites in m o u s e colliculi n e u r o n s , h u m a n a n d g u i n e a - p i g striatal membranes.

285 illustrates the full agonist behaviour ofrenzapride on 5-HT4-Rs in colliculi neurons and its partial agonist behaviour on 5-HT4-Rs in human heart [30].

B

A Colliculi neurons 100

1000

80

750

HUman heart I ~ 5-HT ' ] Renzapride ]

b

60 5oo

~9 40

250

20 0

.../'//_~J w.~p,.~.~

0

I

8

o Renzapride '

'1

9

I

"

I

"

1

7 6 5 4 -Log [ Agonist I (M)

'

0

0

8

7 6 5 4 -Log [Agonistl (M)

'

Figure 4 Comparative effect of 5-HT and renzapride on 5-HT4-Rs in colliculi neurons and in human heart. (A) Stimulation of c-AMP formation in mouse colliculi neurons by 5-HT and renzapride. The results are expressed as the percentage of the maximal stimulatory effect of 5-HT. (B) Effect of 5-HT and renzapride on increased Ca 2§ current (I Ca) in human cardiomyocytes (data from [30]. The naphthalimide (S)-RS56532, an efficacious partial agonist in rat oesophagus, is a potent antagonist in guinea-pig colon [150]. The benzoate ML 10302, a partial agonist in rat oesophagus and guinea-pig ileum behaves as an antagonist in colliculi neurons (vide sopra). The benzoate RS23597 is an antagonist in rat oesophagus and a partial agonist in guinea-pig colon [150]. At present there are no strong indications to postulate 5-HT4-R heterogenicity or species variants. The observed differences in intrinsic activity can also be explained by differences in efficiencies of coupling of 5-HT4-R to the Gs protein. A similar observation has been made for 5-HT1A-R with methysergide, metergoline or 8-OH-DPAT [60, 61]. Differences in potencies of agonists in various tissues may be the result of differences in receptor reserve. Other discrepancies in the pharmacological characterization of 5-HT4-Rs have been reported. For example cisapride is one of the most potent 5-HT4 agonists (Table 1B) and has been reported to be a relatively weak agonist on electricallyinduced guinea-pig ileum contractions by Craig and Clarke [10] but not by other investigators in the same preparation [12, 47]. 5-MeOT is as potent as 5-HT in most preparations but has been reported to be a weak agonist on rat oesophagus and guinea-pig ileum [20, 25]. Although there is no clear explanation for these

286 paradoxal results, it is unlikely that they reflect heterogeneity of 5-HT4-Rs. Only knowing the primary sequence of 5-HT4-Rs in different tissues and species will provide the final answer. 5-HT4-Rs IN CENTRAL AND P E R I P H E R A L NERVOUS SYSTEM Distribution In the central nervous system

Using functional assays, 5-HT4-Rs have been found in colliculi, hippocampal and cortical neurons [1, 2, 5, 34, 62] where they increase c-AMP concentration and inhibit voltage-dependent K § channels [63]. However, the precise regional distribution of 5-HT4-Rs has been obtained using autoradiographical and binding studies with [3H]-GR 113808 and [125I]-SB 207710 in brain tissue from various species including human [129]. Figure 5 illustrates the distribution of 5-HT4-Rs in coronal sections of rat, guinea-pig and human brain and Table 4 provides the overall distribution in the brain of these three different species [55, 56]. The olfactory tubercles and the limbic structures (including nucleus accumbens, and ventral pallidum) appear to be the most intensely labelled structures in the brain. In the cerebral cortex, 5-HT4-Rs appear to be more represented in the retrosplenial cortical area and cingulate gyrus than in the frontal and motor cortex (not shown). In human brain, labelling of 5-HT4-Rs has been observed in the frontal cortex [56]. In addition to the limbic structures, the basal ganglia.appear to be densely labelled. This concerns the dorsolateral and the posterior parts of the striatum and globus pallidus. In rat, only the pars lateralis of substantia nigra expresses a high 5-HT4-R density, whereas in guinea-pig and human, the entire structure contains a high density of these receptors (Figure 5) [55, 56]. In rat, but not guinea-pig brain, the interpeduncular nucleus appears to be one of the brain areas expressing the highest density of 5-HT4-Rs. It is important to mention the rather particular labelling of the intralaminar thalamic nuclei [130],At the hippocampal level, the distribution is dearly laminar, following the CA3, CA2, CA1 pyramidal cell layers and dentate gyrus. As expected from our previous work, superior colliculi, and to a lesser extent, inferior colliculi express, 5-HT4-Rs. We performed a less extended analysis in human that however confirmed the high density of 5-HT4-Rs in the nigro-striatal pathway and hippocampus (Figure 5) [56]. In the peripheral nervous system

In 1985, it was shown that 5-HT4-Rs induced contraction of the guinea-pig ileum was blocked by atropine as well as by tetrodotoxin [15]. Later, Kilbinger [64] showed that 5-HT4-Rs stimulate acetylcholine release.

287

Table 4 Densities of specific binding sites for [3H]-GR 113808 in the adult rat, guineapig and h u m a n brain. Receptor densities are given in fmol/mg protein +_ SEM (assuming a uniform concentration of I mg protein/10 mg tissue). Regions

Rat

Guinea-pig

Olfactory system Olfactory tubercle (Tu) Islands of Calleja (ICj)

301 +_ 28 412 +_ 49

351 +_ 42 501 +_ 45

Basal ganglia Caudate-putamen (CPu) Accumbens nucleus (Acb) Ventral pallidum (VP)

110 +_ 15 177 +- 17 135 +_ 38

240 _+ 34 (cd) 255 _+ 21

107 +_ 8

Hippocampus Dentate gyrus (DG) CA1 field (pyramidal layer) (CA1) CA2 field (pyramidal layer) (CA2) CA3 field (pyramidal layer) (CA3)

114 101 128 121

129 145 298 134

55 _+ 16 103 +- 9

Thalamus Medial habenula (MHb)

153 +_ 21

Cortical areas Frontoparietal cortex (FrPa) Entorhinal cortex (Ent) Retrosplenial cortex (RSpl)

Midbrain Substantia nigra (reticular part) (SNr) Substantia nigra (lateral part (SNI) Interpeduncular nucleus (IP) Central grey (CG) Superficial grey layer of the superior colliculus (SuG) Inferior colliculus (IC)

_+ 20 +- 22 +_ 17 +- 18

_+ 19 _+ 23 +_ 38 +_ 28

Human

47 +- 11

89 _+ 14

42 +-14 83 +-19 59 +- 13

19 +_ 9(FrCX) 70 _+ 15 29+- 9 55+_ 9

40 +_ 17

246 _+ 21

143 _+ 19 271 _+ 38 81 +_11

74 +_ 11 25 +- 11 76 +_ 15

118 +- 14 21+-9

100 _+ 11 4+_ 3

176 +_ 18

288

Figure 5 Autoradiographic localization of [aH]-GR 113808 binding sites at different levels of human, guinea-pig and rat brain. Pictures from autoradiograms generated by incubating human (A-D) guinea-pig (E-G) or rat (H-J) brain sections in the presence of the 5-HT4-R antagonist [3H]GR 113808. The distribution of 5-HT 4 binding sites is comparable in all species: high densities of sites being found in the caudate (Cd), caudate-putamen (CPu), the hippocampal formation (CA1, CA2, CA3, subfields of Ammon's horn, dentate gyrus (DG), and substantia nigra (SNr or SNI) while much lower densities are observed in cortical areas (frontal cortex (FrCx), frontoparietal cortex (FrPr). Some species differences are nevertheless noteworthy. The labelling in the substantia nigra is concentrated over the reticular part in human and guinea-pig, in the lateral part in rat. The interpeduncular nucleus (IP) contains high densities of sites in rats, but not guinea-pig (this region has not been studied in human). Abbreviations are: olfactory tubercle (Tu), islands of Calleja (ICj), entorhinal cortex (Ent), medial habenula (MHb), superficial grey layer of the superior colliculus (SuG), periaqueductal central grey (CG).

289 These results indicated the involvement of the presynaptic cholinergic and/or substance P neurons, where release of the corresponding neurotransmitter stimulated in turn postsynaptic and/or substance P receptors localized on smooth muscles [13, 15, 39, 65, 66]. Activation of these receptors results in muscle contraction. Morphine is also able to inhibit 5-HT4 mediated contraction suggesting that opiate receptors inhibit the 5-HT4-stimulated acetylcholine release [15]. Much less is known about the localization of 5-HT4-Rs in myenteric nervous of human and other species. In vivo, 5-HT4-R agonists are clearly prokinetic in both human and dog [44, 67]. In rat, 5-HT4-Rs have also been described on vagus nerves in which they trigger depolarization [68]. Transduction mechanisms It is now established that in colliculi, hippocampal and cortical neurons, 5-HT 4Rs are positively coupled to AC [2, 5, 62]. In colliculi neurons, 5-HT4-R-mediated c-AMP accumulation leads to blockade of voltage-sensitive K § channels via activation of a c-AMP-dependent protein kinase (PKA) [63]. Figure 6 indicates that a very brief stimulation (1 rain in this figure but indeed a few seconds in most experiments) is sufficient to induce a slowly developing (15-20 rain) inhibition of K § currents. Once established, this inhibition is very long lasting at least 2 hours [131]. This long-term inhibition ofK § channels results in a long-term inhibition of the after hyperpolarization that follows action potential and therefore long-term increase in neuronal excitability [131, 63]. These effects on neuronal excitability are very similar to the long-lasting effects induced by 5-HT in Aplysia neurons which are considered to be a model of synaptic plasticity [69]. In this model, synaptic transmission between a sensory neuron and a motor neuron controlling the gill withdrawal reflex can be potentiated for hours if a facilitatory neuron makes synapse on the sensory neuron is stimulated (Figure 7). This facilitatory neuron releases 5-HT which in turn stimulates c-AMP production in the postsynaptic sensory neuron, activation of PKA and blockade of K + channels [70]. This cascade of events results, as in our model, in an increase in sensory neuron excitability. In Aplysia neurons, blockade of K § channels results in opening of voltage-activated Ca 2+ channels followed by increase in transmitter release [71, 72]. The similarities between the long-term inhibition ofK § channels by 5-HT4-Rs in vertebrate neurons and 5-HT4-Rs in Aplysia is striking (Figure 7). This suggests that 5-HT4-Rs may be involved in some form of long term synaptic plasticity in the mammalian brain. In the CA1 hippocampal pyramidal neurons, a 5-HT-R which displays most of the pharmacological characteristics of 5-HT4-Rs (although benzamides are weak agonists), mediate a slow depolarization and a reduction in the afterhypolarization [33, 34]. The mechanisms by which 5-HT4-Rs mediate these events are not known but it is noteworthy that previous electrophysiological studies have s h o w n t h a t [~-adrenergic and histamine H2 receptors act through AC to produce identical electrophysiological effects to those elicited by 5-HT4-Rs in these cells [73]. 5-HT2-Rs, like 5-HT4-Rs , have been found to induce slow depolarization and reduce after-hyperpolarization in CA1 neurons [74] probably by blocking K § channels. Since 5-HT2-Rs are coupled to phospholipase C, it is likely that in these

290 cells two different receptors elicit a common response t h r o u g h different molecular m e c h a n i s m s [75].

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In Aplysia neurons, the synaptic transmission between a sensory n e u r o n and a motor n e u r o n involved in the gill withdrawal reflex is p o t e n t i a t e d by a facilitatory neuron, releasing 5-HT from this neuron enhances c-AMP production in the sensory neuron, and is followed by a cascade of events: activation of PKA blockade of K § channels - opening of Ca 2§ channels - increase in t r a n s m i t t e r release. In v e r t e b r a t e neurons, the same cascade of events occurs: 5-HT4-R stimulates c-AMP production leading to activation of PKA and blockade of the K § channels for 2 hours. This long-term, blockade of K § channels results in neuronal excitability and, ultimately, n e u r o t r a n s m i t t e r release [129].

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292

Molecular cloning Recently two splice variants of the rat 5-HT4-R were isolated: a long form (5HTtL) with 406 amino acids and a short form (5-HT4s) with 387 amino acids [115]. The amino acid sequence of these receptor isoforms is identical between positions 1 and 359 and differ only in the length and sequence of their carboxy termini. Both isoforms consist of seven hydrophobic putative membrane-spanning regions, a potential N-linked glycosylation site at their amino-terminal end, three potential protein kinase C phosphorylation sites and a potential palmitoylation site located in the cytoplasmic domain of the cell. The 5-HT4L-R carries an additional potential protein kinase C phosphorylation site at position 400. When expressed transiently in COS-7 cells both isoforms bind [~H]-GR 113808 equally well (5-HT4, :pKD=9.24; 5-HT4s:pKD=9.36). The receptor binding profile is very similar for both isoforms and correlated well with that of the 5-HT4-R as defined by functional and radioligand binding assays. The 5-HT4L-R and 5-HT4s-R are positively coupled to adenylylcyclase. However 5-HT is more potent and less efficacious in 5-HT4s-Rs (EC5o=26 nM; Em~=2107 %) than in 5-HT4L-Rs (EC5o=51 nM; Emax=2598 %). The potency of 5-MeOT and a-Me5-HT is also greater for the 5-HT4s. The benzamides behave differently: cisapride is more potent for the 5-HT4s-R and BRL 24924 and zacopride more potent for the 5-HT4L-R. These partial agonists are full agonists in the adenylylcyclase assay. The antagonist ICS 205930 is equipotent as antagonist for both isoforms. The rank order of potencies obtained from the adenylylcyclase test is different from the rank order of potencies obtained from binding studies. Additionally, both isoforms respond differently to GTP)~S. GTP~S has no effect on the 5-HT4s-R, but reduces the affinity of 5-HT for the 5-HTtL-R 2-fold. In rat brain the 5-HTtL-R is detectable throughout the brain, except in the cerebellum. The 5-HT4s-R is restricted to the striatum. Both isoforms are also present in peripheral tissues such as ileum, colon and bladder. The heart atrium expresses only 5-HTs-Rs. Using 5-HT4L-Rs, expressed transiently in COS-7 cells, the agonist high-affinity state K H of the 5-HT(R could be demonstrated [134]. 5-HT displays a 23-fold higher affinity for the K H, labelled with [3H]-5-HT, than for the antagonistic site, labelled with [~H]-GR 113808. Agonists such as 5-MeOT, cisapride or BRL 24924 bind 3.5-9 times better to the K~-sites. Also ICS-205930, pharmacologically defined as antagonist, displays a preference for the agonist-labelled site (3.4-fold). This indicates that in this test ICS 205930 acts as a partial agonist with low intrinsic activity. The human 5-HT4L-R consists of 390 amino acids [114]. The amino acid sequence is 91,8 % identical to the rat sequence. The carboxy terminal tail of the human 5-HT4L-R is 16 amino acids shorter than that of the rat 5-HT4L-R. The h u m a n clone displays a 5-fold higher affinity for [3H]-GR 113808, a-Me-5-HT and zacopride than the rat clone.

293

5-HT4-Rs IN ALIMENTARY TRACT The guinea-pig ileum The physiology of 5-HT-Rs in the intestine is complicated by a great number of distinct receptor subtypes that are distributed on both nerves and muscular elements [13]. If one considers only the myenteric nerve cholinergic components, at least two 5-HT-Rs are involved: a receptor activated by nanomolar concentrations of 5-HT with pharmacological characteristics of 5-HT4-Rs and a receptor activated by higher 5-HT concentrations corresponding to 5-HTaRs (these latter receptors were described by Gaddum and Picarelli as the M receptors [76]) [11, 13, 15]. The 5-HT4-R-mediated [10, 11, 58] gastrointestinal prokinetic effects were first reported with metoclopramide and cisapride [64, 77, 78] and later with benzimidazolone derivatives [64]. The role for 5-HT4-Rs in promoting peristaltic effects in isolated guinea-pig ileum is now dearly established to be mediated by acetylcholine release [79, 80]. However, a substance P-mediated, atropine-resistant peristalsis cannot be excluded [47]. Comparative distribution in different species The physiology of 5-HT effects on the alimentary tract is further complicated by the species heterogeneity of the response (Table 5). Guinea-pig As well as inducing ileum contractions, 5-HT4-Rs induce ascending colon contraction [25, 26]. In this organ, the response to 5-HT is antagonized by tetrodotoxin and atropine, indicating a cholinergic-mediated effect. 5-HT4-Rs are also localized on enterochromaffin cells of guinea-pig. In these cells, endogenous 5-HT stimulated 5-HT~ autoreceptors and inhibits its own release [81]. In guineapig stomach 5-HT4-Rs facilitate electrically-evoked cholinergically-mediated contractions [135]. Rat In the rat, the most studied 5-HT 4 effect is the relaxation of tunica muscularis mucosa of oesophagus [19, 21]. In this tissue, 5-HT4-Rs are more likely to be localized in smooth muscle, as suggested by their TTX-resistant relaxing effects [21]. Such a localization is supported by the observation that c-AMP accumulates in oesophageal smooth muscle cells after 5-HT 4 activation [45, 46]. Such an acculumation is expected to trigger smooth muscle relaxation. Relaxation of rat colon as well as secretion at the colon mucosa levels are also observed [47]. Relaxations of the rat isolated ileum are also mediated via 5-HT4-Rs [136]. Dog In dog stomach, benzamides and benzimidazolones are gastroprokinetic agents, that also promote gastric emptying [67, 82]. This effect is certainly mediated by acetylcholine release [82]. However, the effect of benzamides and in particular cisapride do not involve 5-HT1,2,3,4-Rs [82]. Surprisingly, benzamides and 5-HT do not seem to mediate their effect via the same receptor. In freely moving dog,

294 benzimidazolones and cisapride stimulate denerved gastric pouch contractility [38, 83]. Obviously, the receptor(s) mediating the diverse gastroprokinetic effects of 5HT remain to be established [133]. Table 5 5-HT4-Rs in the vertebrate alimentary tract Guinea-pig

Ileum Colon Enterochromaffin cells Stomach

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Rat

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Human

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References: [12, 47, 86] Human In vivo, 5-HT4 agonists are clearly prokinetic [44, 133] and display a wide range of therapeutic applications in gastrointestinal motility disorders. Whether or not their therapeutic actions are entirely mediated by 5-HT4-Rs is not clear. In isolated human stomach, renzapride has been shown to potentiate electricallyevoked contractions [84]. This effect was antagonized by mM concentrations of tropisetron, suggesting that 5-HT4-Rs are involved [85]. 5-HT4-Rs are also present in human small intestinal mucosa [86] where the non neuronal electrogenic secretory effect of 5-HT appears to be mediated by 5-HT4-Rs. Putative transduction mechanisms The mechanisms by which 5-HT4-Rs stimulate acetylcholine release in myenteric nerves is not known. However, (see paragraph on Transduction mechanisms), one can tentatively propose that 5-HT4-R stimulation induces a rise

295 in c-AMP concentration, which in turn closes K § channels and activates voltagedependent Ca 2§ channels, leading to an increase in acetylcholine release. In conclusion, 5-HT4-Rs seem to be expressed in the enteric neuro-muscular system in a species-dependent manner, indicating a modulatory, rather than an obligatory role in gastrointestinal functions.

5-HT4-Rs IN H E A R T 5-HT has profound effects on cardiac rate and force in a variety of species, including h u m a n [138, 139]. These effects include short-lasting bradycardia, mediated via a Bezold-Jarisch-like reflex, initiated by stimulation of 5-HT3-Rs present in vagal afferents. This cardiac stimulation is mediated by distinct 5-HTRs depending on the species [87, 88]. In pig and human heart, this receptor appears to be of the 5-HT 4 subtype.

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Human heart Figure 8 Effect of 5-HT on current voltage relationship. Increased Ca 2. current (ICa) in the presence of 1 I~M 5-HT. Data from [30].

Tissular and cellular distribution By 1988, it was known that the 5-HT-induced tachycardia in pig was neither mimicked nor antagonized by a variety of 5-HT1,2,a-R ligands, and was therefore necessarily mediated by a new subtype of 5-HT-R [89]. A similar observation was made by K a u m a n n et al. in h u m a n atrium, in which a non-5-HTi,2,3-R increased contractile force via an increase in c-AMP and PKA activity [24]. Since this receptor was blocked by mM concentrations of tropisetron, this suggested t h a t 5HT4-R subtype was involved. Later, it was shown that both right and left h u m a n atria express 5-HT4-Rs where benzamides are partial agonists [32, 90]. The

296 positive inotropic effects of 5-HT4-Rs in human heart were restricted to atria and not present in ventricles [91, 92]. Similarly, in pig, 5-HT4-Rs appear to be localized in atrium but not ventricles [93-95]. In guinea-pig, intravenous injection of 5-HT or benzamides induce tachycardia [96, 97]. Furthermore, it appears that the receptor in cardiac tissues in species dependent (only expressed in piglet and human heart [30, 52, 87]). Transduction mechanisms In heart, as in central neurons and rat oesophagus, 5-HT4-Rs stimulate c-AMP production [24, 32, 90]. This increase in c-AMP production is followed by an increase in PKA activity [24]. Ouadid and collaborators showed in human myocytes, that a pharmacologically identified 5-HT4-R stimulates a PKAdependent increase in L-type calcium currents (Figure 8) [30]. The pharmacology of this 5-HT-R resembles that of the 5-HT4 described in colliculi neurons [2]. One observes that all benzamide derivatives are only partial agonists at this receptor (Figure 4). The increase in Ca 2§ current could be the underlying mechanism of the 5-HT~-mediated positive chronotropic and inotropic cardiac effect. 5-HT4-Rs IN ADRENAL GLANDS 5-HT has been found to stimulate corticosteroid secretion in various models. In addition, 5-HT is present in adrenochromaffin cells, but also in mast cells localized at the adrenocortical level [98, 99]. Therefore, 5-HT released within the adrenal gland may have a paracrine effect on corticoid secretion. In. frog and human, the receptor involved in 5-HT induced cortisol secretion displays the pharmacological profile of a typical 5-HT4-R [98, 99]. In human volunteers the aldosteronestimulating effects were observed after administration of 5-HT4-agonists [137]. 5-HT4-Rs IN URINARY BLADDER 5-HT4-Rs have been demonstrated in the urinary bladder of human [47, 140, 141], guinea-pig [142] and monkey [100]. They are located prejunctionally in the detrusor muscle. When activated they induce contraction (human, guinea-pig) or relaxation (monkey). Activation of 5HT4-Rs in the human urinary bladder may result in an increased micturition frequency. This phenomenon is observed occasionally during cisapride therapy [143].

DESENSITIZATION OF 5.HT4-Rs H o m o l o g o u s d e s e n s i t i z a t i o n in colliculi n e u r o n s 5-HT4-Rs desensitize very rapidly in various preparations [7, 17, 18, 40, 99]. In colliculi neurons we have analyzed the molecular mechanisms of this desensitization [16]. More than 50% desensitization occurred following 5 min

297 exposure to 5-HT as shown in Figure 9B. Continuing the exposure to 5-HT results in a slower desensitization phase leading to a complete loss of 5-HT4-R-mediated response after 3 h. This desensitization was not mediated nor produced by c-AMP or other agonists that increase c-AMP in these cells. This suggested that the desensitization was strictly homologous. In the ~-adrenergic system, homologous desensitization is dependent on phosphorylation of the agonist occupied receptor by a specific kinase: the ~-adrenergic receptor kinase (~ARK) [101]. This kinase is blocked by heparin and Zn2§ Interestingly, heparin and Zn2§ are potent inhibitors of 5-HT4-R desensitization in colliculi neurons [16]. Figure 9C illustrates the existence of a good correlation between the affinities (pEC~o) and the percentage desensitization mediated by the corresponding 5-HT 4 agonists. No correlation was obtained between the efficacities (Emax) of 5-HT 4 agonists and their abilities to desensitize the 5-HT4-Rs [16]. This indicates that homologous desensitization is a function of mean time of occupancy of the receptor by the agonist. This is consistent with the fact that receptor kinases (such as ~ARK) phosphorylate only occupied receptors. Once completely desensitized, at least 24 hours are required to recover a normal 5-HT4 response. A similar desensitization is observed when blockade of K § channels are used to monitor 5-HT4-mediated receptors [131]. [3H]-GR 113808 binding studies show that the rapid desensitization of the 5HT 4 receptor-stimulated adenylylcyclase activity in colliculi neurons is not accompanied by any decrease in the number of binding sites [125]. Longer exposure with 5-HT decreases the [~H]-GR 113808 binding gradually to about 20 % of the control value. These results show that the rapid phase of desensitization is due to a disturbed coupling of the receptor to the Gs-protein. The second phase is the result of loss of binding sites. The residual [~H]-GR 113808 binding is conserved even after 24 h exposure to 5-HT.

Desensitization in the alimentary tract We have already discussed the rapid desensitization of 5-HTa.4-Rs in guinea-pig ileum and how this can be used to discriminate 5-HT3 and 5-HT4-R-mediated contractions of this organ [17, 18]. Indeed, 5-MeOT is able to completely desensitize 5-HT~ responses without affecting 5-HT 3 responses (Figure 9A), whereas 2-Me-5-HT desensitizes 5-HT3 responses without affecting the 5-HT4 responses. The rapid homologous desensitization of 5-HT4-Rs is not restricted to neurons. Also 5-HT4-Rs of rat oesophagus and adrenocortical gland rapidly desensitize [132, 99]. The consequence of low potency of the agonists (like metoclopramide), is that the desensitizion of the receptor has a much slower time course than in the presence of potent agonists (like cisapride). Turconi et al. reported that low desensitizing properties of the partial agonist BIMU 1 may explain its good clinical efficacy in the alimentary tract compared to BIMU 8 which induces rapid desensitization [38]. This may have important therapeutic consequences.

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299 P R E S E N T AND F U T U R E T H E R A P E U T I C DRUGS ACTING AT 5-HT4-Rs In the c e n t r a l n e r v o u s s y s t e m It is fair to say that there is not yet clinical evidence that could help us to propose any precise therapeutic action via 5-HT4-Rs in neurology or psychiatry.

However, various results suggest that specific receptors such as 5-HT4-Rs may lead to the definition of specific therapeutic drugs: (1) 5-HT4-Rs have a unique pharmacological profile which has already provided a way of designing specific drugs which do not act on other 5-HT-Rs; (2) 5-HT4-Rs have a heterogenous brain distribution in rat but also in human brain, which could mean that specific neurological and psychiatrical disorders could benefit from the development of new 5-HT4-R ligands; (3) the cellular mechanisms of action of 5-HT4-Rs both in central and peripheral neurons, indicate a role in neuronal excitability and neurotransmitter release; (4) finally, since 5-HT is involved in illnesses such as affective and neurodegenerative disorders, psychoses, sleep disorders and migraine, it is reasonable to believe that drugs interacting with any one of the 5-HT-Rs is of potential interest to treat these pathological manifestations. Neurological disorders At present a role of 5-HT4-Rs in cognition appears possible [129, 137]. We have already discussed the common factors between the long-term effects of 5-HT4-Rs in mammalian central neurons (via PKA-mediated-inhibition of voltage sensitive K § channels) and Aplysia neurons [69, 72]. The common localization of 5-HT4-Rs in rat, guinea-pig and human hippocampus [55, 56], a structure involved in learning and memory and the inhibitory effects of 5-HT4-Rs on afterhyperpolarization in rat Cal hippocampal neurons is interesting [34]. Indeed, such an inhibition may contribute to induce long-term potentiation (LTP) in hippocampal neurons, a phenomenon that is considered to be a model of long-term plasticity underlying learning and memory [102]. Although the cellular mechanisms involved in LTP are complex, a role for c-AMP and PKA are well documented [103-105]. By increasing c-AMP in these CA1 hippocampal neurons, 5-HT4-Rs may promote this phenomenon. In the unique study carried out in rats in vivo, intra-cerebroventricular injections of substituted benzamides (zacopride and renzapride), increase energy of the low frequency hippocampal theta rhythm as well as other frequency bands [106]. These effects were blocked by tropisetron but not by a selective 5-HT3-R antagonist. Interestingly, they were also blocked by scopolamine, suggesting a cholinergic step in these effects. By microdialysis experiments in rats, it was possible to measure directly the increase in acetylcholine release in the frontal cortex after intraventricular inj ecti ons of the 5-HT4-R agonists B IMU- 1 and B IMU8 [145]. The effect was reversed by the selective antagonists GR 113808 and GR 125487. These data show that 5-HT4-Rs can modulate the cholinergic system. Note that excitation induced by cholinergic agents is assumed to play a role in learning

300 and memory. In a more recent report by Boddeke and Kalkman indicates that the effect of zacopride was not stereo-selective [107], in contrast to what was found for 5-HT4-Rs in colliculi neurons and guinea-pig ileum [12, 35]. The mechanism by which (R)-zacopride is precognitive remains to be resolved. However BIMU-1 and BIMU-8 improve deficits of hypoxia-induced amnesia in mice and RS 66331 and RS 67333 reverse cognitive deficits on rat spatial memory induced by disruptions of the cholinergic transmission. The effect of RS 66331 and RS 67333 were reversed by the selective 5-HT4-R antagonist RS 67532 [137]. The role of 5-HT4-Rs in cognitive processes is further substantiated by the significant loss of 5-HT4-Rs in the hippocampus and frontal cortex of patients with Alzheimer's disease [146]. Note that Alzheimer's disease is associated with changes in hippocampal and cortical function. A number of 5-HT(R agonists is in an early stage of development for the indication cognitive disorders e.g RS 67333 and RS 67506 [137]. Clinical studies are needed to establish if the propensity of 5-HT4-Rs to desensitize and the potential to cause tachycardia and urinary retention are major drawbacks for longterm treatment with potent 5-HT4-R agonists. 5-HT4-Rs are well expressed in the basal ganglia of rat, guinea-pig and human, including striatum, globus pallidus and accumbens nucleus. They are also highly represented in substantia-nigra [55, 56] (Table 4). The role of basal ganglia in the control of voluntary movements can be understood by considering the consequences ofdysfunctionings in this structure. When neostriatum GABA and/or cholinergic interneurons are damaged (as in Huntington's chorea), the movement is exaggerated. Of interest are the findings of Reynolds [146] who demonstrated a profound loss of 5-HT,-Rs in the putamen of patients with Huntington's disease. These data suggest the presence of 5-HT4-Rs on intrinsic striatal GABAergic and/or cholinergic neurons. Damage to the nigro-striatal dopaminergic pathway, as in Parkinson disease, provokes movement that can only be planned with great difficulty. However, no change in 5-HT4-R density is observed in the putamen and substantia nigra of patients with Parkinson's disease [ 146]. These findings indicate that 5-HT4-Rs are not located on the dopaminergic cell bodies or terminals in the substantia nigra or putamen. Also 6-OH-DA lesions in rat striatum had no effect on the density of 5-HT4-Rs, supporting the absence of 5-HT4-Rs on dopaminergic neurons [147]. In contrast 5-HT4-Rs facilitate dopamine release from rat striatum in vitro [148] and in vivo [148, 149]. Further research is needed to elucidate the role of 5-HT4-Rs in dopamine release. But selective 5-HT4-R antagonists may offer a new therapeutic approach for the treatment of disorders of dopamine transmission [129].

Psychiatric disorders The possible roles of 5-HT4-Rs in emotional and rewarding processes are only suggested by their presence in limbic structures such as septum, nucleus accumbens, ventral pallidum, and frontal cortex in human (Table 4, Figure 5) [55, 56]. One has to take in account that steroid release elevates mood and consequently putative behavioural modifications induced by 5-HT4-R agonists could be complicated by these effects [98, 99, 137].

301 Control of emesis Studies have pointed out for a possible role for 5-HT4-Rs in the control of emesis. One study showed that a component of the 5-HT-evoked depolarization of isolated rat vagus nerve was mediated by a putative 5-HT4-R [68]. The other ones provided evidence for a 5-HT 4 component of the vagally-mediated emesis evoked by zacopride and copper sulfate in the ferret [108] and by copper sulfate in dogs [144]. In addition, it has been found that 5-HTt-Rs localized on enterochromaffin cells inhibit 5-HT release [81]. This inhibition could reduce 5-HTs-R-mediated excitation of vagal fibers and may be one of the factors to inhibit the vomiting reflex elicited by this cascade of events [109]. 5-HT4-R antagonists could offer a novel therapeutic approach in the treatment of emesis. In the a l i m e n t a r y tract Benzamides are already used as therapeutic prokinetic agents. They are of benefit in many gastrointestinal motility disorders, such as gastroesophageal reflux, non-ulcer dyspepsia gastroparesis and constipation [110, 111]. Benzamidazolones could also be potential useful drugs to treat such disorders [38]. Although 5-HT 4 rather than 5-HT3-Rs seem to be involved in the therapeutical actions of benzamides, further work is needed to exclude any other target [82, 133]. Since 5-HT4-Rs have a modulatory role in gastrointestinal contractions, one possibility would be to use 5-HTt-R antagonists or partial agonist or even potent but rapidly desensitizing agonists, to treat irritable bowel disease, in which both hyper- and hypo-motility states exist. Indeed administration of 5-HTP, a precursor of 5-HT, to human volunteers produces symptoms resembling those seen in irritable bowel syndrome (IBS). However, in mice 5-HT t antagonists did not relieve the visceral pain induced by colorectal distension, indicating that 5-HT4antagonists may not ameliorate the pain component of IBS. Definite clinical studies in this regard have not been reported yet. But several 5-HT 4 antagonists are in development for the indication IBS e.g GR 125487 and RS 100235 (preclinical stage and SB 207266 (Phase I/II clinical trials). The outcome of these studies may provide evidence for the therapeutic potential of 5-HT 4 antagonists in the treatment of IBS [137]. 5-HT4-R antagonists may also be useful to treat diarrhea, if one considers the prosecretory action of 5-HTt-R agonists at the colonic level [47, 151]. In the h e a r t As already discussed, 5-HT4-Rs are strictly localized in atria, and the increase in force of ventricular contraction may be secondary to rate [94]. It has been noticed that intravenous infusion of cisapride in healthy volunteers evokes tachycardia [112]. Exaggerated responses to 5-HTt agonists have been found in strips isolated from patients chronically treated with ~-adrenoceptor antagonists [113]. These results certainly contra-indicate 5-HT4 agonist inotropic agents in patients with heart failure. However, 5-HT4-R-induced tachycardia encourages the search for 5-HT4-R antagonists as antiarrhythmic therapeutic drugs [139].

302 In the lower urinary tract As activation of 5-HT4-Rs results in contraction of the detrusor muscle, 5-HT4 agonists may be of therapeutic value in the treatment of detrusor hypomotility or overflow incontinence. 5-HT 4 antagonists may be of value in the treatment of urge incontinence.

Table 6 Present (P) and hypothetical (H) therapeutic action of 5-HT 4 ligands. Central nervous system Learning and memory Emotional and rewarding Emesis

(H) (H) (H)

agonist agonist, antagonist agonist

.Alimentary tract Gastroesophageal reflux Non ulcer dyspepsia Gastroparesis Constipation Irritable bowel disease Diarrhea

(P) (P) (P) (P) (H) (H)

agonist agonist agonist agonist antagonist antagonist

Heart Antiarrhythmic drug

(H)

antagonist

.Urinary tract Detrusor hypomotility Urge incontinence

(H) (H)

agonist antagonist

CONCLUSIONS Since the discovery of 5-HT4-Rs in 1988 many advances have been made in the understanding of its role in physiological and pathological processes. Molecular biological information on the 5-HT4-R became available recently. Selective (radio) ligands have been developed. 5-HT4-R research has identified the presence of 5-HT4-Rs in various tissues, including the brain, gastrointestinal tract, heart, adrenal gland and urinary bladder. In the central nervous system 5-HT4-Rs are likely to be involved in cognition and possibly in dopamine transmission. In the periphery 5-HT4-Rs modulate

303 gastrointestinal motility and secretion, increase heart rate, enhance steroid secretion from the adrenal gland and augment bladder emptying (for review see [152]). At present several of the newly designed potent and selective 5-HT4-R ligands are in development: 5-HT4-R agonists for the treatment of gastric hypomotility, amelioration of gastric-oesophagel reflux, augmenting of stomach emptying and cognitive disorders; 5-HT4-R antagonists for the treatment of irritable bowel syndrome. In the near future novel therapeutic areas for 5-HT4-R agonists (e.g. detrusor hypomotility) and antagonists (e.g. disorders of dopamine transmission, diarrhea, arrhythmia, urge-incontinence) will be explored.

Acknowledgements This work was supported by grants from CNRS (Centre National de la Recherche Scientifique), INSERM (Institute National de la Sant~ et de la Recherche M~dicale), Bayer France/Troponwerke (FRG) and Boehringer Ingelheim. We would also like to thank Mireille Passama and Angela TurnerMadeuf for their assistance in the preparation of this manuscript.

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Chapter

9

5-HT 5 , 5-HT 6 , 5-HT 7 RECEPTORS

This Page Intentionally Left Blank

Serotonin Receptors and their Ligands

B. Olivier, I. van Wijngaardenand W. Soudijn(Editors) 9 1997ElsevierScienceB.V. All rights reserved.

The 5-HT5, 5-HTe and

5-HT 7

311

receptors

R. Grailhe ~), U. Boschert b) and R. Hen ~)

~)Columbia University, Center for Neurobiology and Behavior, 722 West 168th Street, New York, NY 10032, USA. b)Glaxo Institute for Molecular Biology, 14 chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland.

Recently, the molecular cloning of 14 different mammalian receptor subtypes revealed an unexpected heterogeneity within 5-HT receptors. The latest classification of 5-HT receptors takes in account not only their pharmacological profile and their coupling with second messengers but also their amino acid sequence (see figure 1, 2 and ref [1]). The 5-HT~ family consists of the 5-HT~A, 5-HTm, 5-HT1D, 5-HT,E and 5-HT~F receptors which can all inhibit adenylylcyclase. The 5-HT2 family includes receptors that stimulate phospholipase C: the 5-HT2A receptor previously called 5HT 2, the 5-HT2c receptor previously known as 5-HTlc and the 5-HT2B receptor previous name 5-HT2F (found in stomach fundus) [1]. The 5-HT 3 receptors are ligand-gated ion channels. The adenylylcyclase stimulatory receptors are an heterogenous group including the 5-HT 4 receptor, the Drosophila 5-HTd~o~ receptor [2] and two mammalian receptors, the 5-HT 6 and 5-HT7 receptor. Despite their common coupling with second messengers, the 5-HT6 and 5-HT7 receptors display little homology with one another as well as with other 5-HT receptors (figure 1). In addition the pharmacological profiles of the 5-HT 6 and 5-HT 7 receptors differ from one another and from the profiles of all other 5-HT receptors including the 5-HT4 receptor. They have therefore not been included in any of the existing families. The 5-HTsA and 5-HTs~ receptors might also constitute a new family of 5-HT receptors. These receptors display little amino acid homology with the 5-HT, 5HT 2, 5-HT~ and 5-HT v receptors. Furthermore, unlike all other G protein-coupled 5-HT receptors, the 5-HT5 receptors do not modulate the activity of adenylylcyclase or phospholipase C, when they are expressed in fibroblasts. Their effector systems are at present unknown. Molecular biological techniques played a crucial role in the identification and characterization of the 5-HTs, 5-HT~ and 5-HT7 receptors for the following reasons. Cloning studies identified these receptors which had not been predicted by pharmacological techniques. The expression of the cloned receptors in "simple" environments such as cell lines allowed the characterization of their pharmacological and functional properties. -

-

312 5-HTIA h u m a n (39) - S L L 5-HTIB m o u s e (48) - V A L 5- HTI D a h u m a n (41 ) - A V V 5-HTIE h u m a n (25) - C M T 5-HTIF m o u s e (26) - S L T 5-HTdro2A (229) - S V L 5-HTdro2B (80 ) - A V V 5- H T L y m (97 ) - T S V 5-HTSA mouse (43)-LTL 5-HT5B m o u s e (55) - V T L 5-HT7 m o u s e (87) -GSI 5-HTdro I (165) - S I V 5-HT6 rat (30) - A A (77) -ALLT~ 5-HT2A rat (56) -ALLI! 5-HT2B rat (57) -ALSI% 5-HT2C rat 5-HTIA human 5-HTIB mouse 5-HTID a human 5-HTIE human 5-HTIF mouse 5-HTdro2A 5-HTdro2B 5 -H TLym 5-HT5A mouse 5-HT5B mouse 5-HT7 mouse 5-HTdrol 5-HT6 rat 5-HT2A rat 5-HT2B rat 5-HT2C rat 5- H T I A human 5-HTIB mouse 5-HTID a human 5-HTIE human 5-HTI F mouse 5-HTdro2A 5-HTdro2B 5-H TLym 5-HT5A mouse 5-HT5B mouse 5-HT7 mouse 5-HTdrol 5-HT6 rat 5-HT2A rat 5-HT2B rat 5-HT2C rat 5-HTIA human 5-HTIB mouse 5-HTID a human 5-HTIE human 5-HTIF mouse 5 - H T d r o 2A 5- H T d r o 2 B 5- H T L y m 5-HT5A mouse 5-HT5B mouse 5-HT7 mouse 5-HTdro I 5-HT6 rat 5-HT2A rat 5-HT2B rat 5-HT2C rat

DPDA EMLD EMSD PPSQ RDDE EQQK EEQH KTGT EE-QR----V GOPI AP~ KEGS HNIT NNTT

%4

CR RA~'~FRIRKT| GR E#~wk. ~ ILKQ GR RA~ ILNP YR KSLYQKR YK FLYHKR WK KRIHRR WK KRIQRR IR SRIRKD WK KFRMGSR WK KFRFGRR YQ ~KSAAKH YQ ~R I V L E E C~ ~KQAVQV FLTIKSLQKEATLC FLTIHAL~KKAYLV FLTIYVL~RQTLML M P T L L G A I IN FHMAIFDFFN IHPALFDFFT VSSEVADFLT ISEEMSNFLA ISDSVASLFL IH T A V A S L F L IPPFAR SFVL VPAIWKSIFL LPPIWKSIFL I PLWVERTCL VPASLSSLFL IS P G L F D V L T VIGALLNVFV T L K T L L Q I FV LMEKLLNVFV

- (I01) (57 ) (55) - (60 ) - (63 ) (316) - (235)(128) (35)- (35 ) (39)(83 ) (30 ) - (41) - (57)- (50)-

-

-

-

AKRKM-ALAR EKKKL- MAAR ERKR I-SAAR ERQQI-SSTR RRQK I-SGTR RKETL-EAKR RRQLL-EAKR TREKL-ELKR TEGDTWREQK TSGDSWREQK RKNIS-SFKR KKLRF-QLAK RR L A T K H SR K~ GRKTMKSISM GKKPAQTISN LRGTMQAINN (7) (6) (6 ) (6) (4) (14 ) (14 ) (9 ) (4) (4) (46 ) (42 ) ( 102 ) (76) (85) (75)

Figure 1. Sequence comparison. Amino acid similarity between 5-HT receptors. Numbers in parentheses are the numbers of amino acids that are not represented. Putative transmembrane domains are indicated (I to VII). Black boxes show positions where more than 12 out of the 16 sequences are identical.

313

- The availability of the nucleotide sequences corresponding to these receptors enabled a precise determination of their pattern of mRNA expression by in situ hybridization. The 5-HT5 family: 5-HTsA and 5-HTsB r e c e p t o r s The 5-HT 5 family contains two receptors, the 5-HTsA and the 5-HTsB which are very similar to one another (77%) but do not resemble the 5-HT~, 5-HT 2, 5-HT a and 5-HT 4 receptors in terms of amino acid sequence (figure 2), pharmacological profile or transduction system. They define therefore a new family of serotonin receptors.

;-HT 1 amily k~Ar

fAC

AC ~-HT 2 :amily ~'PLC

Figure 2. Dendrogram The sequence of 5-HT receptors were compared and clustered with the program CLUSTAL [3]. The comparison was performed with the amino acid sequences presented in figure 1. The lengths of the horizontal lines are inversely proportional to the percentages of homology between receptors or groups of receptors.

314 T h e 5.HTsA r e c e p t o r

Molecular structure. The 5-HTsA receptor gene has been isolated by reverse transcriptase PCR experiments performed on mouse and rat brain RNA using degenerate oligonucleotides derived from t r a n s m e m b r a n e domains III and VI of G proteincoupled 5-HT receptors [4, 5]. Hydropathy analysis of the corresponding protein revealed seven hydrophobic domains. Amino acid sequence comparisons indicated t h a t this receptor was a distant relative of all previously identified 5-HT receptors. The percentages of homology with known receptors are low, the best score being 37% with the Drosophila serotonin receptor 5-HTdroZ~ [6]. The mouse and the rat 5-HTsA receptors contains several potential phosphorylation sites for protein kinase C, cAMP dependent protein kinase and two potential sites of N-linked glycosylation. Analysis of genomic DNA fragments containing the mouse 5-HTsA gene indicated the presence of one intron about 8 kbp. long, located in the middle of the third cytoplasmic loop [7].

Table 1.5-HT~A , 5-HTsB , 5-HT 6 and 5-HT7 receptors. Receptor

Species

amino acids

Locus

Introns in coding sequence

mRNA size (Kb)

mouse

357

5B

yes (one)

5.8 - 5.0 -4.5

rat

357

7q36 (human)

mouse

370

1 E4-1 EG

rat

370

2q11-q13 (human)

5-HTsA

5-HTsB

3.8 - 4.5 yes (one) 1.5 - 1.8 - 3.0

437 5-HT~

rat 436

5-HT 7

mouse rat human

448 448 445

yes (one)

4.2

yes (two) yes (two)

3.1 - 3.9

19 C3-D 10q23.3-q24.3

The gene encoding the 5-HTsA receptor is located on h u m a n chromosome 7 at position 7q36 which is a locus containing also the mutation Holoprosencephaly type III t h a t results in abnormal brain development [7-8] and on mouse chromosome 5 at position 5B [7] (Table 1).

315 Functional expression. When expressed in Cos-7 cells (Table 2) and NIH-3T3, the mouse 5-HTsA receptor displayed a high affinity for [125]LSD (Kd=340 pM) and the following profile" LSD> ergotamine> 5-CT> methysergide> 5-HT=RU24969> bufotenine= yohimbine [4]. This profile did not correspond to the profile of any of the previously characterized serotonin receptors. Binding experiments using [SH]5-CT revealed that the mouse 5-HTsA receptor displayed both a high affinity (Kd= 0.84 nM) and a low affinity (Kd= 13 nM) for this radiolabelled compound [9] and had a pharmacological profile that might correspond to 5-CT-sensitive sites reported by Mahle et al. [10]. When expressed in CosM6 cells, the rat 5-HT~A receptor displayed a similar pharmacological profile [5]. In Cos-7 cells and NIH-3T3 expressing the mouse or the rat receptor [4-5] no effect on adenylylcyclase or phospholipase C activity could be detected. The 5-HTsA receptor might therefore interact with a different signalling system, such as ion channels. Regional distribution. The 5-HTsA receptor is expressed only in the central nervous system (Table 3, figure 3). In the mouse, Northern analysis revealed three transcripts in brain and cerebellum (5.8, 5.0 and 4.5 kb) [4] while in rat two transcripts (3.8 and 4.5 kb) were detected [5]. Quantitative PCR demonstrated the presence of specific fragments only in brain and spinal cord [4]. In situ hybridization experiment performed on mouse brain sections revealed the presence of 5-HTsA transcripts in the cerebral cortex, hippocampus (pyramidal cells of CA1-CA3 layers and granule cells of the dentate gyrus), granule cells of the cerebellum, medial habenula and tufted cells of the olfactory bulb [4]. The rat 5-HTsA mRNA was found in piriformcortex, hippocampus, amygdala, septum and several thalamic nuclei [5]. No signal was detected in kidney, liver, spleen, lung or heart.

The 5-HTsB receptor Molecular structure. The 5-HTs~ receptor gene has been isolated by reverse transcriptase PCR experiments performed on mouse and rat brain RNA using degenerate oligonucleotides derived from transmembrane domains III and VI of G proteincoupled 5-HT receptors. The 5-HTs~ receptor consists of a polypeptidic chain of 370 amino acids both in mouse and rat [7-5-11]. The mouse and rat 5-HT~B receptor contains several potential phosphorylation sites for protein kinase C, cAMP dependent protein kinase and one potential site of N-linked glycosylation. The 5HTsn receptor is highly homologous to the 5-HTsA receptor (77%) [4], whereas the percentages of homology with other known receptors are low (Figure 2). The genomic fragment containing the 5-HTsB gene has been isolated. Partial sequence analysis revealed that the 5-HTsR gene contains one intron located in the middle of the third cytoplasmic loop (Table 1), at exactly the same position as in the 5-HTsA gene [7].

316 The mouse 5-HTsB gene is localized on chromosome 1 (position 1E4-1EG) whereas its human homologue is on chromosome 2 (position 2qll-q13) [7] (Table 1). Functional expression. The 5-HTsB receptor expressed in Cos-7 cells displayed a high affinity for [125I]LSD (Kd=470 pM) [7] and two affinities for [3H]5-CT, a high ~ t y (Kd=0.6 nM) and a ,low affinity (Kd=14 nM). The high affinity sites for [3H]5CT might correspond to receptors coupled to G proteins as suggested by the fact that a fraction of these sites are displaced by GTP analogs [11,9]. Displacement of bound [12~I]LSD by various serotonergic drugs gave the following rank order of potencies: LSD> ergotamine> methiothepin> 5-CT> methysergide> 5-HT=RU24969> bufotenine (Table 2). Similar results were obtained in CosM6 cells transfected with the rat homologue [5]. Table 2. Pharmacological profile of the 5-HTsA, 5-HTsB, 5-HTe and 5-HT 7 receptors Receptor

5-HTsA

5-HTsB

5-HT8

5-HT7

Radioligand Cell types species

125I LSD Cos7 Mouse

12~I LSD Cos7 Mouse

125I LSD Cos7 Rat

[~H]5-HT Cos7 Mouse

2-Bromo-LSD LSD Ergotamine 5-CT Ritanserine Methylsergide Methiothepin 5-HT RU 24969 Risperidone Bufotenine 8-OH-DPAT TFMPP Clozapine Spiperone Ketanserin Sumatriptan (-) Propranolol Mianserin

8.7 8.5 8.4 7.8

7.8

8 8 7.3 9 7.6 7.9 8.2 8.3 6.9 8.1 7 6.6 6.3 7.4 7.2 6.4 4.7

7.2 7.0 6.6 6.5 6.5 6.0 5.9 5.6 5.3 5.3 4.8 4.8 4.7 5.7

8.5 7.4 6.9 7.8 6.6 6.4

6.1 7.4 6.4 8.7 6.8 6.4

5.8 6.4 5.4 5.5 5.8 5.1 5.2

6.3 7.9 5.8

7.4

7.0

This pKi value are taken from Plassat et al. [4], Matthes et al. [7], Roth et al. [15], Monsma et al. [12], and our unpublished data.

317 Like in the case of the 5-HT~A receptor, the 5-HTsB receptor did not influence the activity of adenylylcyclase or phospholipase C in NIH-3T3, CHO and 293 cells expressing this receptor [5,7,11]. Regional distribution. Expression of the 5-HTsn receptor is restricted to limited regions in the brain (Table 3, figure 3). In the rat, Northern analysis revealed three transcript of 1.5, 1.8 and 3kb [5]. In situ hybridization experiments performed on mouse brain sections revealed the presence of 5-HTsB mRNA only in the CA1 fields of the hippocampus, the medial and lateral habenula and the dorsal raphe nucleus [5,7]. Low levels of expression were also found in the entorhinal and piriform cortex, subiculum and olfactory bulb in the rat [7,11]. During late embryonic stages (El7 and El9) no transcript could be detected except possibly in the nucleus raphe pallidus [11]. At birth a faint signal was detected in the entorhinal cortex [11].

Figure 3 In situ hybridization. 5A, 5B and 7, Dark-field microscopy of the emulsion autoradiogram of horizontal section through an adult mouse brain, hybridized with either the 5-ht~A (5A), the 5-ht~B (5B) [7], and the 5-ht7 probe (7) [our unpublished data]. Cx, cerebral cortex; H, hippocampus; Cb, cerebellum; CA1, CA2-3, hippocampal area; MH, median habenula; LH, lateral habenula; LS, lateral septum, Ent, enthorhinal cortex.

The 5-HT6 receptor Molecular structure. The 5-HT 6receptor has been first isolated by PRC amplification from rat striatal mRNA. This receptor consists of a polypeptidic chain of 437 [12] or 436 amino

318 acids [13]. The two published amino acid sequences differ in their C terminal tail. However, the nucleotide sequences are identical except for one nucleotide which is absent from one of the sequences resulting therefore in a frameshift. The 5-HT 6 receptor contains seven hydrophobic regions and is distant from all other 5-HT receptors as seen in the dendrogram (Figure 2). The third cytoplasmic loop of the 5-HT6 receptor is short (50 amino acids) while the C-terminal tail is long (120 amino acids) (Figure 1). These characteristics are also observed in receptors stimulating adenylylcyclase or phospholipase C activity such as the 5-HT~ro~or the 5-HT 2 receptors. The 5-HT 6 receptor also contains one potential site for N-linked glycosylation and several potential sites for phosphorylation by cAMP dependent protein kinase and protein kinase C. Both groups reported the presence of at least one intron located in the middle of the third cytoplasmic loop, where an intron is also present in the 5-HT5 family [12,13], (Table 1). These molecular characteristics suggest that the 5-HT6 receptor belongs to a new subclass of serotonin receptor. Functional expression. The 5-HT6 receptor was expressed in Cos-7 cells. The pharmacological profile of this receptor (methiothepin> clozapine> 2-Bromo-LSD> ritanserin> 5-HT> 5-CT) corresponded to a serotonin receptor positively coupled to adenylylcyclase that was characterized in the NCB-20 neuroblastoma cell line [14]. Ergoline derivatives such as LSD and lisuride displayed high affinity for the 5HT6 receptor. Interestingly, atypical and typical antipsychotic drugs such as clozapine and loxapine as well as tricyclic antidepressant drugs (amoxapine and clomipramine) exhibited relatively high affinities for the 5-HT 6 receptor. This receptor might therefore be a target for these psychotropic drugs [12,13,15]. Activation of the 5-HT~ receptor in HEK-293 cells or Cos-7 cells resulted in a stimulation of adenylylcyclase [12,13]. In this functional assay, 5methoxytryptamine and 5-carboxamidotryptamine were agonists. Lisuride and dihydroergocriptine were partial agonists while amoxapine, methiothepin and clozapine were antagonists. Regional distribution. Northern analysis of poly(A +) RNA from various tissues revealed that a 4.2 kb transcript is found predominantly in brain. 5-HT6 mRNA is strongly expressed in olfactory tubercles, striatum and nucleus accumbens. Signals of moderate intensity were also found in the hippocampus (CA1, CA2, CA3 fields and dentate gyrus), olfactory bulb and cerebral cortex. In peripheral tissue a low expression was detected in rat stomach and guinea pig adrenals [12,13]. Recently the cloning, characterization and chromosomal localization of the human 5-HT~ receptor was reported [30]. There is a close similarity in amino acid sequence of the rat- and human 5-HT6 receptor. The inhibition constants of the drugs (n=40) competing for [SH]LSD binding sites of the human 5-HT6 receptors in transiently transfected Cos7 cells are also similar to the inhibition constants of the drugs in Table 2 tested on the 5-HT6 receptors of the rat.

319 The gene for the 5-HT 6 receptors was localized on the human chromosome region lp 35-36 and overlaps that of the gene for the 5-HT1D~ receptor. In human brain mRNA expressing the 5-HT~ receptor is mainly found in the caudate nucleus while lower concentrations are detected in the hippocampus and amygdala.

The 5-HT7 receptor Molecular structure. This 5-HT receptor (Table 1) positively coupled to adenylylcyclase has been cloned in human [16], rat [17,18,19,20], mouse [21] and guinea pig [22]. It consists of a polypeptidic chain of 448 amino acids in mouse and rat and 445 amino acids in human. The mouse, rat and human 5-HT7 receptors contain potential phosphorylation sites for protein kinase A and potential N-linked glycosylation sites. The 5-HT 7 receptor is most homologous to the 5-HTdrol receptor (Figure 1) that also activates adenylylcyclase (37%) but is a distant relative of all the other 5-HT receptors (Figure 2). Like the 5-HTdrol and the 5-HT6 receptors, the 5-HT7 receptor possesses a long C-terminal (Figure 1) tail [18,19,20,21]. The 5-HT 7 gene contains at least two introns in its coding sequence [19,20], one in the middle of the third cytoplasmic loop and another one close to the end of the coding region. Lovenberg et al. [17] isolated a 435 aa cDNA that lacks the last exon containing the carboxy terminal tail of the 5-HT 7 receptor. This shorter cDNA might result from an alternative splicing event. In the case of the PACAP receptor such splice variants have been shown to couple to distinct second messenger machineries [23]. The mouse 5-HT7 gene is localized on chromosome 19 at position C3-D white its human homologue is on chromosome 10 at position q23.3-q24.3 (Table 1). Functional expression. When expressed in mammalian cells, the 5-HT7 receptor displayed a high affinity for [3H]5-HT (Kd=3.6 nM) with the following unique pharmacological profile: 5-CT> methiothepin> 5-HT> clozapine> 8-OH-DPAT [17,18,19,20,21], Table 2). This pharmacological profile might correspond to some of the 5-CT-sensitive sites reported in mammalian brain [10] and to "5-HTl-like" receptors positively coupled to adenylylcyclase and found in the cardiovascular and gastrointestinal systems [24,25]. Furthermore, due to the affinity of the 5-HT7 receptor for 8-OHDPAT, this receptor might correspond to 5-HT1A-like receptors positively coupled to adenylylcyclase [26,27,28]. Such receptors have been suggested to play a role in circadian rhythms [17,27]. The high affinity of the 5-HT7 receptor for atypical neuroleptics such as risperidone and clozapine suggests that this receptor might also play a role in certain neuropsychiatric disorders [15]. When the 5-HT7 receptor is transiently expressed in Cos-7 cells or stably expressed in CHO, HEK-293 and Hela cells, its activation leads to an increase in adenylylcyclase activity [17,19,20,21]. This effect could be blocked by non specific 5-HT receptor antagonists such as methiothepin, methysergide and ergotamine but

320 Table 3. Regional distribution of 5-HTsA, 5-HTsB, 5-HT6 and 5-HT v receptor RNAs in the brain.

Areas

Species

Mouse

Mouse

Rat

Mouse

Receptor

5-HT5A

5-HT5B

5-HT6

5-HT7

++ +§ ++ +§

+++ §247 +§ §

Cerebral cortex Cingulate cortex Frontal cortex Parietal cortex Entorhinal cortex Basal ganglia Striatum Accumbens nucleus Globus pallidus Septum Lateral septal nucleus Septohippocampal nucleus Hippocampus CA1 pyramidal cell layer CA2 pyramidal cell layer CA3 pyramidal cell layer Dentate gyrus Tenia tecta Fimbria fornix Hypothalamus Medial mammillary nucleus Lateral mammillary nucleus Thalamus Paraventricular thalamic nucleus Anteroventral th. n. Anteromedial th. n. Mediodorsal th. n. Parafascicular th. n. Ventral th. n. Habenula Medial habenula Lateral habenula Visual system Geniculate nucleus Superior colliculus Olfactory system Olfactory bulb Olfactory Tubercules Amygdala Amygdalophippocampal area MeduIl~ oblongata and pons Dorsal raphe nucleus Cerebellum Granular layer

++ ++ ++ ++

+++ §247

++ +

++ +++

++ ++ ++ ++

+++

++ ++ ++ ++

+++ ++ +++ +++ ++ ++

++ ++ ++ ++ ++ +

++

++§ ++ ++ +

+

++ +++ ++ +

+

++

This data are taken from Plassat et al. [4], Matthes et al. [7], Ruat et al. [19], and our unpublished data.

32! also by the neuroleptics (+)butaclamol and clozapine. LSD was a partial agonist [19]. Regional distribution. The 5-HT 7 receptor is expressed in the central nervous system. Northern analysis of a variety of rat [17,18,19] and guinea pig tissues [22] revealed two mRNAs of approximatively 3.1 and 3.9 kb [17,18,19,22]. In situ hybridization experiments in the mouse and rat detected the 5-HT v mRNA in hippocampus (pyramidal cells of CA2-CA3 layers, tenia tecta and fimbria fornix), hypothalamus, thalamus, amygdaloid complex, cortex, superior colliculus and dorsal and paramedian raphe nuclei [17,18,21,19 and our unpublished data]. In peripheral tissue a low expression was detected in the stomach and ileum of the rat [19,20J and a faint signal was detected in the spleen [20]. Why are t h e r e so m a n y 5.HT r e c e p t o r s ?

To try to answer such a question, it is worth considering what parameters distinguish the various receptor subtypes. The receptor families differ in their effector systems: While the 5-HT 3 receptors are ion channels, the 5-HT! receptors inhibit adenylylcyclase, the 5-HT 4, 5-HT 6 and 5-HT7 receptors stimulate adenylylcyclase, the 5-HT2 receptors stimulate phospholipase C and the 5-HT~ receptors are probably coupled to a different effector system. Why then are there so many 5-HT 1 receptors (5-HTIA, 5-HTI~, 5-HT1D~, 5-HTIE and 5-HTIF)? First these receptors might not always share the same effector systems. The 5HT1Areceptor for example, can couple with adenylylcyclase, phospholipase C or ion channels, depending on the cell type in which it is expressed. The other 5-HT 1 receptors can also inhibit adenylylcyclase in fibroblasts but their neuronal effectors are not known and might be different from those of the 5-HT1A receptor. Second, the 5-HT~ receptors differ markedly in their pattern of expression. While the 5HT1A receptors are expressed in the raphe nuclei and in the hippocampus, the 5HT1B receptors are found predominantly in the basal ganglia. In addition, even when two receptors are expressed by the same neurons, they are not necessarily found in the same subcellular compartment. The 5-HTL~receptors for example, are localized in the somatodendritic compartment of raphe neurons, while the 5-HT m receptors are localized on the axon terminals of these neurons. The same reasoning might also apply to the receptors that stimulate adenylylcyclase such as the 5-HT~, 5-HT~ and 5-HT~ receptors. However in these cases we do not yet know all their possible effector systems not their subcellular localization. The only characteristic which presently differentiates these receptors is their markedly distinct expression pattern. The existence of a large number of receptors with distinct signalling properties and expression patterns, might enable a single substance like 5-HT to generate simultaneously a large panel of effects in many brain structures. Most complex behaviors require the synchronized modulation of several physiological functions.

322 In a flight situation for example, locomotor activity and fear will increase while sexual activity and digestive functions might be slowed down. The fact that several 5-HT receptors have similar pharmacological properties renders the study of their function by classical techniques exceedingly difficult. However, the availability of the genes encoding these receptors makes it possible to create rodent mutants lacking these receptors or to block their expression with specific oligonucleotides [31,32]. Such techniques will hopefully allow us to understand why we have so many 5-HT receptors and what their functions are [32,34]. REFERENCES

1 Hoyer D, Clarke DE, Fozard JR, Hartig PR, et al. Pharmacol Revs 1994; 46: 158-203. Hoyer D, Martin GR. Behav Brain Res 1996; 73: 263-268. 2 Witz P, Amlaiky N, Plassat JL, Maroteaux L, et al. Proc Natl Acad Sci USA 1990; 87: 8940-8944. 3 Higgins DG, Sharp PM. Gene 1988; 73: 237-244. 4 Plassat JL, Boschert U, Amlaiky N, Hen R. EMBO J 1992; 11: 4779-4786. 5 Erlander MG, Lovenberg TW, Baron BM, de Lecea L, et al. Proc Natl Acad Sci USA 1993; 90: 3452-3456. 6 Saudou F, Boschert U, Amlaiky N, Plassat JL, Hen R. EMBO J 1992; 11: 7-17. 7 Matthes H, Boschert U, Amlaiky N, Grailhe R, et al. Mol Pharmacol 1993; 43: 313-319. 8 Hatziioannou AG, Krauss CM, Lewis MB, Halazonetis TD. J Med Genet 1991; 40: 201-205. 9 Amlaiky N, Ghavami A, Matthes H, Boschert U, et al. Soc Neurosci Abstract 1993; 19: 633. 10 Mahle CD, Nowak HP, Mattson RJ, Hurt SD, et al. Eur J Pharmacol 1991; 205: 323-324. 11 Wisden W, Parker EM, Mahle CD, Grisel DA, et al. FEBS 1993; 333: 25-31. 12 Monsma FJ, Shen Y, Ward RP, Hamblin W, et al. Mol Pharmacol 1993; 43: 320-327. 13 Ruat M, Traiffort E, Arrang JM, Tardivel-Lacombe J, et al. Biochem Biophys Res Commun 1993; 193: 268-276. 14 Conner DA, Mansour TE. Mol Pharmacol 1990; 37: 742. 15 Roth BL, Craigo M, Choudhary S, Uluer A, et al. J Pharmacol Exp Therapeutics 1994; 268: 1403-1410. 16 Bard JA, Zgombick J, Adham N, Vaysse PN, et al. J Biol Chem 1993; 268: 23422-23426. 17 Lovenberg TW, Baron BM, de Lecea L, Miller JD, et al. Neuron 1993; 11: 449458. 18 MeyerhofW, Obermuller F, Feh S, Richter D. DNA Cell Biol 1993; 12: 401-409. 19 Ruat M, Traiffort E, Leurs R, Tardivel-Lacombe J, et al. Proc Natl Acad Sci USA 1993; 90: 8547-8551.

323 20 Shen Y, Monsma FJ, Metcalf MA, Jose PA, et al. J Biol Chem 1993; 268: 18200-18204. 21 Plassat JL, Amlaiky N, Hen R. Mol Pharmacol 1993; 44: 229-236. 22 Jakeman LB, Bonaus DW, Ramsey IS, Wong EHF, et al. Soc Neurosci Abstract 1993; 19: 1164. 23 Spengler D, Waeber C, Pantaloni C, Holsboer F, et al. Nature 1993; 365: 170175. 24 Saxena PR, Mylecharane EJ, Heiligers J. Naunyn Schmiedeberg's Arch Pharmacol 1985; 330: 121-129. 25 Connor HE, Feniuk W, Humphrey PPA, Perren MJ. Br J Pharmacol 1986; 87: 417-426. 26 Shenker A, Maayani S, Weinstein H, Green JP. Eur J Pharmacol 1985; 109: 427-429. 27 Markstein R, Hoyer D, Engel G. Naunyn Schmiedeberg's Arch Pharmacol 1986; 333: 335-345. 28 Fayolle C, Fillion MP, Barone P, Oudar P, et al. Fund Clin Pharznacol 1988; 2: 195-214. 29 Prosser RA, Dean RR, Edgar DM, Heller HC, et al. J Biol Rhythms 1993; 8: 116. 30 Kohen R, Metcalf MA, Khan N, Druck T, et al. J Neurochem 1996; 66: 47-56. 31 Bourson A, Borrini E, Austin RH, Monsma FJ, et al. J Pharmacol Exp Ther 1995; 274: 173-180. 32 Sleight AJ, Monsma FJ, Borroni E, Austin RH, et al. Behav Brain Res 1996; 73: 245-248. 33 Lucas JJ, Hen R. Tr Pharmacol Sci 1995; 16: 246-252. 34 Saudou F, Hen R. Neurochem Int 1994; 25: 503-532.

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Chapter 10

5-HT TRANSPORTER

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Serotonin Receptors and their Ligands B. Olivier, I. van Wijngaarden and W. Soudijn (Editors) 9 1997 Elsevier Science B.V. All rights reserved.

327

5-HT Transporter W.Soudijn a) and I. van Wijngaarden b)

a)Leiden/Amsterdam, Center for Drug Research, P.O.Box 9502, 2300 RA Leiden, The Netherlands. b)Solvay Duphar B.V., P.O.Box 900, 1380 DA Weesp, The Netherlands.

INTRODUCTION The hypothesis that serotonin dysfunction may play an important role in depression and the fact that tricyclic antidepressants are monoamine uptake inhibitors but also have a considerable affinity as antagonists for cholinergic, histaminergic and adrenergic receptors and thus may cause unwanted severe sideeffects initiated the search for and development of selective serotonin uptake inhibitors. Impulse transport between neurons is effected by release of neurotransmitters from the presynaptic neuron into the synaptic cleft where they stimulate their receptors on the membrane of the postsynaptic neuron. Stimulation of the postsynaptic neuron is ended by re-uptake of the neurotransmitter into the presynaptic neuron where it is partly enzymatically inactivated and partly stored in presynaptic vesicles. For a minor part the neurotransmitter is taken up in glial cells where it is enzymatically destroyed. This mechanism acts in the inactivation of neurotransmitters as serotonin = 5hydroxytryptamine (5-HT), noradrenaline = norepinephrine (NE) and dopamine (DA). The re-uptake is effected by selective transporters for each neurotransmitter. 5-HT transporters are located in vesicular and synaptic membranes of presynaptic serotonergic neurons, in the membranes of glial cells and blood platelets. Inhibitors of 5-HT uptake transporters enhance the concentration of 5HT in the synaptic cleft thereby intensifying the action of 5-HT on its postsynaptic receptors, a useful property in case of serotonergic dysfunction.

S T R U C T U R E OF THE 5-HT T R A N S P O R T E R

The aminoacid sequence and putative structure of a rat 5-HT transporter was determined by c-DNA cloning strategies and expression of the c-DNA encoding the 5-HT transporter in CV-1 cells [1] and HeLa cells [2]. The transport of 5-HT by the expressed 5-HT transporter was found to be saturable with apparent Michaelis constants I ~ of 529 _+107 nM for the CV-1 cells and 320 nM for the Hela cells.

328 The transport was blocked by selective 5-HT uptake inhibitors at low nanomolar concentrations whereas less selective or NE selective uptake inhibitors only blocked 5-HT at much higher concentrations. Dopamine and norepinephrine had virtually no effect on 5-HT uptake [1]. The cloned 5-HT transporter from rat basophilic leukemia cells by Hoffman et al. [1] consists of 653 amino acids and has a relative molecular mass of about 73 kD whereas the 5-HT transporter from rat midbrain and brainstem cloned by Blakely et al. [2] consists of 607 amino acids and has a relative molecular mass of 68 kD. The amino acid sequences of both cloned transporters diverge mainly in their carboxylic and amino termini. Whether these differences are cloning artifacts or actual differences remains to be established. Lesch et al. [3] reported the cloning and sequence analysis of a c-DNA encoding a 5-HT transporter from human dorsal raphe nuclei consisting of 630 amino acids and having a relative molecular mass of about 70 kD. These results and also the amino acid sequences are in agreement with those reported by Mayser et al. [4] on a rat 5-HT transporter. Both polypeptides are highly homologous differing only in 51 amino acids = 8% mainly located in the N-terminal part of the transporter. Hydropathicity analysis [5] that is the search for regions in the amino acid chain of the transporter of extended hydrophobicity suitable for the formation of membrane spanning domains indicated 12 putative transmembrane domains. The N-terminus of the cloned 5-HT transporters lacks a signal sequence of amino acids [6] necessary for penetration of the membrane and so it is assumed that it is retained in the cytoplasm. Two potential N-glycosylation sites are located on the large external loop between the third and fourth putative transmembrane domains (fig. 1). Amino acid sequence motifs for phosphorylation by proteinkinase [7] are found on both N- and C termini of the cloned 5-HT transporter. On the N-terminus potential sites for phosphorylation by c-AMP dependent proteinkinase and proteinkinase C are present, whereas on the C-terminus only proteinkinase C recognition sites are found. Phosphorylation and dephosphorylation of the 5-HT transporter could either be an essential link in the 5-HT translocation process per se or have a modulatory effect on the activity of the transporter. There is some evidence of the latter as activation ofproteinkinase C in platelets [8] or in endothelial cells [9] results in an inhibitory effect on the 5-HT transporter. It was demonstrated that the de novo synthesis of the 5-HT transporter - at least in the JAR human placental choriocarcinoma cells - is most probably under control of a c-AMP dependent proteinkinase as enhancement of the c-AMP levels in these cells by e.g. choleratoxin resulted in a considerable increase in 5-HT transporter m-RNA and a concomitant increase in transporter density in the plasma membranes [10,11].

329

extracellular

m

m

( )

HOOC

)

NH2

intracellular

Fig. 1

5-HT transport A frequently used model for studying the role of ions in the 5-HT transport process employs plasma membrane vesicles prepared from porcine blood platelets [12-17]. The results showed that for binding of the 5-HT cations to the transporter binding of sodium ions is required. Binding of chlorine ions is needed for translocation of 5-HT from the external to the internal side of the plasma membrane vesicles. After binding of Na § 5-HT § and Cl'a conformational change of the transporter takes place whereby 5-HT*, Na § and Cl are translocated from the exterior to the interior of the vesicle. Optimal conditions for transport are a gradient out > in for both sodium and chlorine ions and a stoichiometry of transport of 5-HT*:Na§ =1:1:1. The return to the initial state of the transporter is K § dependent. After dissociation of 5-HT*, Na § and CI from the transporter K* is bound and translocated from the interior of the vesicle to the exterior where it dissociates from the carrier complex.

330 Whether the potassium ion is bound to the same site as the sodium ion is as yet unknown. In the presence of K* the transport cycle in this platelet model is electroneutral which means t h a t for every translocated sodium ion a potassium ion has to be counter transported.

A similar mechanism of binding and cotranslocation of 5-HT +, N a § and C1 ~was demonstrated by using plasma membrane vesicles prepared from a synaptosomal fraction of mouse cerebral cortex [18,19]. The stoichiometry of transport in this case however was 5-HT§247 As the results suggested an electroneutral mechanism like that in the porcine platelet vesicles a counter location of two K § ions is required. Studies on the sodium stimulated 5-HT uptake in cerebral synaptosomes as for example by Wood using synaptosomes of rat cerebral cortex [20] or by M a n n and Hrdina using synaptosomes of rat diencephalon [21] suggested a 5-HT+:Na+= 1:1 ratio similar to that found using porcine platelet membranes. The cause of discrepancies in the ratio could be differences in tissues, species and methods of preparation. Inhibition of 5-HT uptake is effected by the competitive binding of a 5-HT uptake inhibitor to the 5-HT transporter thereby preventing the binding of 5-HT to its recognition site on the transporter. The binding of potent 5-HT selective inhibitors is monophasic that is they bind with high affinityat a single site on the transporter. The question is whether the different 5-HT uptake inhibitors of different chemical structures all will bind to the same site on the transporter wholly or partially overlapping the 5-HT recognition site or to totally different sites that all prevent competitively the binding of 5-HT. Using porcine and h u m a n platelet membrane vesicles Humphreys et al. concluded on the basis of the results on the displacement of imipramine by norzimeldine, alaproclate or fluvoxamine and on the effect of these inhibitors on 5-HT transport that the 5-HT uptake inhibitors and serotonin all bind to the same or overlapping sites on the 5-HT carrier but that occupation of sites different from the 5-HT recognition site could not be ruled out conclusively [22]. The results on the displacement of 3H-paroxetine from its binding site on the 5-HT transporter in membranes of the rat cerebral cortex by 5-HT or a variety of 5-HT uptake inhibitors and the effectof these compounds in moderate micromolar concentrations on the dissociation kinetics of aH-paroxetine led G r a h a m et al. to the conclusion that 5-HT and the uptake inhibitors bind at a c o m m o n domain at the 5-HT recognition site [23]. However when high micromolar concentrations (200 micro M) of 5-HT or of 5H T uptake inhibitors were used in determining the dissociation half life of 3Himipramine, SH-paroxetine and 3H-citalopram dissociating from h u m a n platelet membrane preparations it appears that not only the dissociation half life was prolonged but also that the prolongation was differentfor each of the three labeled uptake inhibitors [24]. This led to the conclusion that the three labeled ligands each bind to a different domain on the 5-HT transporter.

331 Furthermore the authors showed that the dissociation kinetics of 3H-citalopram in membrane preparations of human platelets and human brain (putamen) both appeared to be affected differently by different 5-HT uptake inhibitors at 200 micromolar concentration. For instance 5-HT, clovoxamine and fluvoxamine had no effect on the dissociation half life while indalpine shortened and other 5-HT uptake inhibitors prolonged the dissociation half life of 3H-citalopram in both transporter preparations [24]. These phenomena were explained by postulating an affinity modulating site at the 5-HT transporter. For the actual preparation of the membranes and the dissociation rate determination see reference [25]. It is to be expected that in the near future point mutations in the cloned and expressed 5-HT transporter together with computer assisted molecular modelling techniques will probably lead to a greater understanding of the exact mode of binding of 5-HT uptake inhibitors of different chemical structures. SELECTIVE 5-HT UPTAKE INHIBITORS: STRUCTURE AND ACTIVITY Tricyclic first generation antidepressants as for example imipramine and dothiepin (fig. 2) not only inhibit 5-HT and NE reuptake but also act as antagonists on histamine-I, cholinergic muscarine, adrenergic alpha and 5-HT2 receptors [26-28].

R Me Me I

N

R = H -- CI =CN Fig. 2

R

Me I

IMIPRAMINE CLOMIPRAMINE CIANOPRAMINE

R=H =CN

DOTHIEPIN OYANODOTHIEPIN

332 Substituted imipramine and dothiepin congeners as clomipramine, cianopramine and cyanodothiepin are compared to their parent compounds potent and selective inhibitors of 5-HT uptake in rat cortical synaptosomes with I~.-s in nM of 5.4, 0.71 and 4.8 and selectivity indices of 5.2, 21 and 124 respectively [26,29]. The selectivity for the 5-HT transporter is maintained in the in vivo experiments although the selectivity index declines as a result of the formation by desmethylation of metabolites with an uptake inhibitory profile different from that of the parent compound [29-31]. Desmethylclopramine compared to clopramine is a very potent NE uptake inhibitor and a much less potent inhibitor of 5-HT uptake [30,32]. The same holds for desmethylcianopramine (Ro 12-5419) versus cianopramine [33]. The desmethylanalog of cyanodothiepin is still a selective 5-HT uptake inhibitor however of a very low potency compared to the parent compound [34]. Like imipramine and dothiepin their substituted analogs clomipr~mine, cianopramine and cyanodothiepin also have a pronounced antagonistic effect on several neurotransmitter receptors which may contribute to the overall pharmacological effect or side-effect of the drugs. Clomipromine has a substantial affinity for histamine-1, cholinergic muscarine, adrenergic alpha-1 and dopamine-2 receptors [27], dopamine-3 and 5-HT-2 receptors [~hlp 1993 personal communication]. The affinity of cianopramine for muscarinic, alpha-l, dopamine-2 and 5-HT-2 receptors is similar to that of clomipramine [29] and there seems no reason to suppose that the affinity for the histamine-1 receptor will be very different. Compared to clomipram~ne and cianopr~mine the affinity of cyanodothiepin for alpha-l, dopamine-1, dopamine-2 and 5-HT-2 receptors is considerably lower [29]. The affinity for the muscarinic receptor is not insignificant but still about four times lower than that of cianopramine. The a/Vmity for the histamine-1 receptor was not reported in this paper. Side effects attributed to the antagonistic interaction of tricyclic antidepressants with muscarinic, histaminic and adrenergic receptors and the notion that reuptake inhibition is the mainstay of their antidepressant effect induced a search for reuptake inhibitors without receptor blocking properties. This resulted in a host of nonselective and selective reuptake inhibitors of different nontricyclic chemical classes [35]. Several of the 5-HT selective reuptake inhibitors are in clinical use today. Fluvoxamine and clovoxamine are nontricyclic monoamine uptake inhibitors structurally related to the tricyclic antidepressant noxiptiline (fig. 3). Both fluvoxamine and clovoxamine are trans isomers. Clovoxamine is a potent but nonselective inhibitor of the 5-HT and NE transporter in a synaptosomal preparation of the rat brain frontal cortex (I~.=5.9 and 7.0 nM respectively) and a weak (Ki=720 nM) inhibitor of the DA transporter in synaptosomes of the corpus striatum [26]. Clovoxamine has a very low ~ t y for histamine-l, muscarine, alpha 1,2 and beta adrenergic, and serotonergic receptors in vitro [27,26].

333 Me

.

/

Of

1.~

N

/

N

Me~

~Me

NOXIPTILINE

f O

N

N

H

H

R= CF3 FLUVOXAMINE = Cl CLOVOXAMINE

Fig. 3 Multi center pharmacotherapeutic trials showed that clovoxamine was a welltolerated and effective antidepressant however fluvoxamine was preferred for further development. The potency of fluvoxamine as a 5-HT uptake inhibitor in vitro is similar to that of clovoxamine (Ki=7 nM vs 5.9 nM for clovoxamine). In contrast to clovoxamine the affinity of fluvoxamine for the NE transporter is low, I~.=500 nM versus 7.0 nM for clovoxamine. The affinity of fluvoxamine for the DA transporter is also lower than that of clovoxamine, I~.=5000 nM versus 720 nM for clovoxamine [26]. The selectivity of fluvoxamine for the 5-HT transporter is maintained in vivo. Uptake of 5-HT by rat brain synaptosomes after in vivo pretreatment with fluvoxamine was inhibited whereas uptake of NE and DA was not [37-39]. Depletion in rat brain of 5-HT induced by H75/12 and H77/77 was antagonized by pretreatment of the rats with fluvoxamine whereas the depletion of NE was unimpaired [37,39]. Fluvoxamine virtually lacks affinity for most receptor types involved in neurotransmission [39 and Tulp personal communication]. Recently an updated review of the pharmacology and the therapeutic use of fluvoxamine in depressive illness has been published [40].

334 Zimeldine an asymmetrical diarylallyl amino compound with the cis (Z) configuration is a fairly selective 5-HT-uptake inhibitor [41,42]. The chemical structure is shown in table 1. A selection from the data of H6gberg et al. [42] on the structure activity relationship of zimeldine, its metabolite norzimeldine and structural analogs of monoamine uptake inhibition in mouse midbrain slices in vitro and ex vivo is presented in table 1. Table 1 Structure activity relationship ofmonoamine uptake inhibition in mouse midbrain slices in vitro and ex vivo by zimeldine and analogs [42]

X N

I

R-N-CH3 pM..vitro X

H 4-Br 4-Br 4-Br 4-Br 4-MeO 3-Br 3-Br 2-Br 2-Br 2.4-diC1 2.4-diC1

a b c d

R

Me Me b Hc Me H Me Me H Me H Me H

Z/E a

Z Z Z E E Z Z Z Z Z Z Z

NE

27 24 1.5 6.1 0.8 >28 4.2 0.9 2.2 0.3 11 2.3

5-HT

3.7 1.7 0.10 6.1 2.5 0.8 0.9 1.4 4.2 0.6 1.5 0.5

Z=cis E=trans zimeldine nor-zimeldine selectivity index IC5o NE / ICso 5-HT

EDso l~mol/kg io Si d

7.3 14 15 1 0.3 >35 4.6 0.6 0.5 0.5 7.3 4.6

5-HT

NE

>107 >98 >102 25 25 >112 66 58 37 20 >101 73

(37%)

(40%)

>107 49 19 >98 102 >112 >98 >102 >98 102 43 18

(23%)

(43%)

(43%)

335 Zimeldine and norzimeldine are both selective 5-HT uptake inhibitors in vitro as well as in vivo. Norzimeldine however is more potent than zimeldine both in NE- and in 5-HT-uptake inhibition. Ross and Renyi [41] showed that zimeldine and norzimeldine are also 5-HT selective when comparing uptake inhibition of DA and 5-HT in homogenates of the corpus striatum of the rat and again norzimeldine is much more potent than zimeldine in 5-HT uptake inhibition. The trans (E) analog of zimeldine is non-selective in vitro but appears to be an NE uptake selective inhibitor in the ex vivo experiment. The trans analog of norzimeldine is a selective inhibitor of NE uptake in vitro as well as in vivo and it is quite possible that the NE selectivity of transzimeldine in vivo is largely caused by its metabolite trans norzimeldine. All regio isomers (3-Br and 2-Br) of zimeldine and norzimeldine are selective NE uptake inhibitors in vivo. The 3-Br analog of zimeldine is the only regio isomer with selectivity for 5-HT uptake inhibition in the in vitro experiments. The in vivo NE selectivity of this compound is probably caused by its metabolite the 3-Br analog of norzimeldine. The potencies for 5-HT uptake inhibition of the regio isomers decrease (with the exception of the 3-Br analog of zimeldine) and increase for NE uptake inhibition. The effect is most pronounced for the 2-Br analog. This could be due to a slight rotation of the phenyl ring out of the plane ofpi-electron conjugation caused by the steric hindrance of the bulky ortho bromine atom. In contrast the 2-4-diC1 analog of zimeldine and norzimeldine are selective 5HT uptake inhibitors in vitro as well as in vivo with potencies similar to those of the "parent" compounds. In this case the ortho-chlorine substituent does not shii~ the selectivity in the direction of NE uptake inhibition. Either the smaller ortho chlorine atom reduces the rotation of the phenyl ring or the para chlorine substituent by interaction with its binding site on the 5-HT transporter prevents or inhibits rotation. Replacement of the 4-Br group in zimeldine by a 4-MeO group results in a highly selective potent 5-HT uptake inhibitor in vitro. In vivo however there is a loss of selectivity and a severe loss in potency. The same holds true for the 4-iPrO and the 4-MeS analogs (data not shown). This seems to indicate that steric properties of the 4-substituent are much more important for 5-HT selectivity and potency than electronic properties are. The unsubstituted compound is only twice less 5-HT selective and twice less potent than zimeldine in vitro but again the 5-HT selectivity is lost and there is a severe loss in potency in the in vivo situation. Biotransformation of the (substituted) phenyl moiety resulting in the formation of non 5-HT selective products of low potency could explain these phenomena. Metabolic pathways for the formation of such potential products are; para hydroxylation of the unsubstituted phenylring, demethylation of the 4-MeO group, oxidation of a methylgroup of the 4-iPrO substituent to a primary alcohol group, demethylation or oxidation of the 4-MeS substituent.

336 Table 2 Inhibition of 3H-paroxetine binding to membrane fragments of cerebral cortex of the rat by zimeldine and analogs [43]

fl

x

N

R-N-CH3 X

4-Br 4-Br 3-Br 2-Br 2.4-diC1 4-MeO

R

Me Me Me Me Me Me

conf.

I~.nM

Z E Z Z Z Z

39 330 120 n.d. 60 190

X

4-Br 4-Br 3-Br 2-Br 2.4-diC1 4-MeO

R

H H H H H H

conf.

~nM 3.3 66 64 22 3.7 n.d.

Z=cis E=trans n.d.=no data Ki=inhibition constant From the data in table I it can be concluded that with the exception of the 3-Br compounds the secondary amines (norzimeldine analogs) are more potent inhibitors of 5-HT uptake in mouse brain slices than their tertiary counterparts (zimeldine analogs) and thus will have a higher affinity for the 5-HT transporter. This is confirmed by the experiments of Marcusson et al. [43] who studied the structure-activity relationship of the inhibition of SH-paroxetine binding to fragments of rat cerebral cortex by a.o. 28 zimeldine analogs. (Paroxetine is a highly selective and potent blocker of the 5-HT transporter). The results presented in table 2 show that the inhibition constants I~. of the secondary amines are always lower than those of the tertiary amines. The primary amino analog of zimeldine has a ~ value of 360 nM [43] which is about 10 times higher than that of the tertiary amine zimeldine and about 100 times higher than that of the secondary amine norzimeldine. It is obvious that at least in the zimeldine series monosubstitution of the side-chain N results in high affinity for the 5-HT transporter and HSgberg et al. [44] showed that the optimal substituent is a methylgroup. The ICso for 5-HT uptake inhibition in mouse brainslices is for

337 the N-Et analog of norzimeldine 20 times and for the N-Pr analog 240 times higher than for norzimeldine (N-Me). In summary these data suggest that basicity of the side chain and steric properties of the substituted amino group are important factors determining the affinity for the 5-HT transporter in the zimeldine series in vitro. Zimeldine proved to be a clinically effective anti-depressant but was with drawn because of its supposed implication in the occurrence of the Guillain-Barr6 syndrome in some patients. Venlafaxine is a virtually non-selective inhibitor of the uptake of 5-HT and NE by rat brain synaptosomes. It is structurally related to gamfexine a compound with antidepressant and psychostimulant properties (fig. 4). The potency of venlafaxine to inhibit in vitro monoamine uptake is the same range as that of imipramine and desipramine under the experimental conditions described by Muth et al. [45] and Yardley et al. [46].

L~ ..Me N "Me

~Me

N,,Me VENLAFAXINE

GAMFEXlNE

Fig. 4 Venlafaxine a racemate has no appreciable affinity for muscarinic, alphaadrenergic, beta-adrenergic, histamine-I, 5-HT-1A, 5-HT-2, dopamine-2, benzodiazepine, or opiate receptors [45] and thus is not expected to induce sideeffects caused by interaction of venlafaxine itself with these receptors. However, the possibility remained that metabolites of venlafaxine could have affinity for the above mentioned neuroreceptors and thereby cause unwanted side-effects. Three metabolites ofvenlafaxine were identified in man; one major metabolite4-OH instead of 4-MeO - and two minor metabolites - NHMe instead of NMe9 and 4-OH, NHMe. All three metabolites virtually lacked affinity for dopamine-2, cholinergic, alpha-l-adrenergic, histamine-1 and opiate receptors [47]. The metabolites were also tested for monoamine uptake properties in vitro [47] (Table

338 3). The major metabolite having a similar potency for 5-HT uptake inhibition as venlafaxine tends to be the more selective 5-HT uptake inhibitor, because the selectivity index (ICso NE/ICso 5-HT) increases from 3 for venlafaxine to 6 for the major metabolite. Both minor metabolites are considerably less potent than the major metabolite in the uptake inhibition of 5-HT, NE and DA. It is plausible that the major metabolite in patients will be a substantial factor in the mechanism of action of venlafaxine as an antidepressant drug. Venlafaxine as already stated is aracemate. Both enantiomers were obtained by resolution of the racemate using chiral d i - p - t o l u y l ~ c acid as a resolving agent. The absolute configuration S of the (+)enantiomer was established by X-ray crystallography of the (+)HBr salt [46]. The results of the N E and 5-HT uptake inhibition [45,46] by the racemate and both enantiomers are shown in table 3.

Table 3 Monoamine uptake inhibition in rat brain synaptosomes by venlafaxine, enantiomers and metabolites in man [45-47]

venlafaxine (+_) S (+) isomer R (-) isomer major metabolite (4-OH) minor metabolite

(NHCH~)

minor metabolite (4 OH, NHCH 3)

NE

5-HT

DA

Si*

ref.

0.64 3.14 0.76

0.21 0.10 0.19

2.8 -

3 31 4

[46,47] [46] [46]

1.16

0.18

13.4

6

[47]

4.7

1.6

21.1

3

[47]

i

[47]

>10

2.8

>30

*Selectivity index ICso NE / ICso 5-HT i=indeterminate The R(-)isomer does not differ significantly from the racemate either in potency in the inhibition of NE and 5-HT uptake or in selectivity index. With a selectivity index of only 3-4 both compounds can be considered as weakly selective at best. The S(+)isomer however is a 5-HT selective uptake inhibitor with a selectivity index of 31 and a higher potency than both the racemate and the R(-)isomer in the 5-HT uptake inhibition test. Yardley et al. published the synthesis and monoamine

339 uptake inhibition of a series of venlafaxine analogues [46]. A selection of these compounds and their activity is shown in table 4. Exchanging the 4-MeO group of venlafaxine for a CF z group results in an increase in selectivity index with a concomitant twofold decrease in affinity for the 5-HT transporter. The potencies and selectivity indices ofvenlafaxine and its regio isomer (3-MeO) are identical. The regio isomer (3-CF3) of the 4-CF3 compound however shows an inversion of selectivity from the 5-HT transporter to the NE transporter. The same phenomenon holds true for other electron withdrawing groups like C1 and Br (data not shown). Ring contraction of the cyclohexyl moiety to a cyclo pentyl group results for venlafaxine and its CFs analogue in a considerable increase in selectivity index with a concomitant moderate reduction in ICso for 5HT uptake inhibition.

Table 4 Structure-activity relationship of monoamine uptake inhibition in rat brain synaptosomes by venlafaxine analogs [46]

R

__••

On

t0

%Me

R

4-OMe 4-CF 3 4-OMe 4-CF 3 3-OMe 3-CF 3 3-C1, 4-C1 3-OMe, 4-OMe

n

NE

1 1 0 0 1 1 1 1

0.64 2.8 5.8 10.4 0.62 0.36 0.07 1.38

5-HT

0.21 0.4 0.4 0.49 0.19 1.44 0.08 0.13

Si

3 7 14.5 20 3 0.25 1 10.6

Except by ring contraction the rather moderate 5-HT selectivity index of 3 of venlafaxine can also be increased by the introduction of a second MeO group in the 3-position.

340 This compound (3-MeO, 4-MeO) with a selectivity index of 10.6 also shows a slight increase in potency as a 5-HT uptake inhibitor (ICso 0.13 ~/I vs 0.21 ~ for venlafaxine). However, selectivity is completely lost if the 3-MeO, 4-MeO phenyl group is replaced by a 3-C1, 4-C1 phenyl group. Table 5 Monoamine uptake inhibition, rat brain synaptosomes. Inhibition of binding ofSH paroxetine (5-HT selective) and SH-tomoxetine (NE selective) to synaptosomes of rat cortical membranes. [53]

fluoxetine norfluoxetine

RS R S RS R S

5-HT

NE

7.69 7.46 7.66 7.35 6.51 7.86

5.91 6.25 5.69 5.62 5.43 5.37

Si a 60 16 93 54 12 309

3H-paroxetine 3H-tomoxetine

8.51 8.46 8.53 8.48 7.58 8.88

6.88 7.08 6.34 5.84 5.91 5.82

a Si=selectivity index = antilog (pI~. 5-HT - p ~ NE) Fluoxetine [48] was the first selective 5-HT uptake inhibitor in clinical use as an antidepressant. The compound has little affinity for alpha- and beta adrenergic receptors, dopamine, muscarinic, histamine-H1 opiate, gaba, and benzodiazepine receptors [49]. The affinity for serotonin subtype receptors, 5-HT1A.D, 5-HT2 and 5HT s is also very low [50]. Chemically fluoxetine, a racemate, belongs to the class of substituted 3-phenoxy 3-phenyl propanamines (fig. 5). The optical isomers were synthesized and the absolute configurations were determined by Robertson et al [51]. The pharmacological properties of the racemate, and its optical isomers were reported by Wong et al. [50,52,53]. A summary of these data on monoamine uptake inhibition and on inhibition of binding by rat brain synaptosomes of the selective 5-HT uptake inhibitor paroxetine and the selective NE uptake inhibitor tomoxetine taken from [53] is shown in table 5. Although the eudismic ratio (potency less active isomer/potency active isomer) of the R and S isomers of fluoxetine is fairly close to one i.e. 1.58, the selectivity index (antilog (pI~. 5-HT - Pl~. NE) of the S isomer is much larger than that of the R isomer. In other words the R isomer of fluoxetine is a less selective 5-HT uptake inhibitor than the S isomer but their potencies are similar. This is also confirmed by the data on the inhibition of 3H-paroxetine and SHtomoxetine binding.

Lt.

c)

(a I.I.

0

7" /

0

,-

< aD co n

Z ,..... tiii X 0

._a It_

!

Z-

o !

Z--

-I-

"1-

0

/

/

0

0

w

i-0_ UJ

kl.l

_z "'

X 0 iii u_

-

-r

--1"-

/

0

(,-,)-

LU ZLU

V--___

~rA

oO0

w~ XW O• :~0

~z

II

II

~ 0 0 r~

II

!

o

~

342 The eudismic ratio of the R- and S isomer for the inhibition of SH-paroxetine is one, so their potencies are the same. However, the selectivity index for the inhibition of SH-paroxetine and 3H-tomoxetine binding is 24 for the R isomer and 155 for the S isomer of fluoxetine indicating that the R isomer is less selective than the S isomer in regard to the affinity of the compounds for the 5-HT transporter versus the NE transporter. The racemate and the S isomer are equipotent 5-HT uptake inhibitors and inhibitors of 3H-paroxetine binding. However as expected the racemate fluoxetine is somewhat less selective than its S isomers in both selectivity experiments. Norfiuoxetine (N-demethyl fluoxetine) the major metabolite of fluoxetine in animals and man is about as potent and selective as its parent compound (table 5). The R isomer of norfluoxetine is about as selective as R fluoxetine but 9 times less potent. The S isomer of norfluoxetine is about as potent as S fluoxetine but three times more selective (table 5). The affinity of norfluoxetine and its enantiomers for 5-HT receptor subtypes and for other receptors of neurotransmitters is very low and similar to that of fluoxetine and its enantiomers [49,50,53]. The selectivity of mono amine uptake inhibition of the phenoxy-propanamines depends upon the position of the substituent on the phenoxy moiety. Monosubstitution in the para position as in fluoxetine results in selective 5-HT uptake inhibition but monosubstitution in the ortho position results in selective NE uptake inhibition as in nisoxetine (o-MeO), tomoxetine (o-Me) or the o-C1 and o-Br analog (fig. 5) [54-56]. Ortho-para disubstitution may lead to highly potent and selective 5-HT uptake inhibitors such as R-4-iodotomoxetine [57,58]. Meta-para disubstitution also can result in potent and selective 5-HT uptake inhibitors as e.g. the R and S 3-Me-4iodo-phenoxy congeners [57]. MDL 28618 A (fig. 5) the cis (+) isomer of MDL 27777 A is a rigid fluoxetine analog with selective 5-HT inhibitory properties. The cis (+) isomer is 10 times more potent than the cis (-) isomer in vitro as well as in vivo [59]. Data are not given. The absolute configuration of the cis (+) isomer was recently established as 1S, 2S [60]. Basically 5-HT selective uptake inhibitors like MDL28618, femoxetine (fig. 5, table 6) and its close structural analog paroxetine (table 6) can all be considered rigidified phenoxypropamine derivatives that may interact with the same recognition site at the 5-HT transporter. Fluoxetine and its rigidified analogs all seem to fit a common template (fig. 5). Computer assisted molecular modelling could establish the relative binding orientations of the different drugs and thus offer some insight in the topography of the binding site on the 5-HT transporter.

343 Table 6 Structure activity relationship of paroxetine and its analogs. ICso=inhibition of 5HT uptake by synaptosomal membranes of the rat forebrain. I~.=inhibition constant of 3H-paroxetine binding to the rat brain membranes [63].

R3 CH20 -R3

R1

H

R2

trans

H

-

H

Me

+ +

F

H

-

+ F

O

-R 2

I[:~1

ME

-

+

ICso

2.2 14 20 20 1.9

250 22 55

b

OCH3

~

ICso

I~.

0.10 2.7 3 7

50 16 285 80 a 20 20 150 130

3.6 2.4 350 20 2.2 14 60 75

0.11

40 1.5 20

I Cso and I~. in nM a = femoxetine b = paroxetine Paroxetine, a potent and selective 5-HT uptake inhibitor was introduced into the market in 1990. The compound is 45 times [61] to 320 times [62] less potent in blocking NE uptake than in inhibiting 5-HT uptake depending on the brain preparation used. The effect on DA uptake is virtually nil. The drug has no or hardly any affinity for alpha- and beta-adrenergic receptors, histamine receptors, serotonin receptors (5-HT1A.D, 5-HT2, 5-HTa) and dopamine receptors and a low affinity for muscarinic receptors [50]. Paroxetine is a member of a series of3-substituted 4-phenylpiperidines, compounds with two asymmetrical C-atoms and consequently existing as two diastereoisomeric - and four optical isomeric forms. Paroxetine is a pure trans (-) isomer with 3S, 4R absolute configuration and a diequatorial conformation of the substituents [63]. The structure-activity (affinity) relationship ofparoxetine, its stereoisomers and its analogs was described by Plenge et al. [63] and later by Mathis et al. [64].

344 A selection of the results is shown in tables 6 and 7. From this selection the cisisomers are omitted as they are significantly less active than the trans-isomers. From table 6 it can be concluded that in the paroxetine seriesthe trans(-)isomers are more potent than the trans (+) enantiomers especially in the case ofparoxetine itself. Paroxetine and its unsubstituted 4-phenyl trans(-)analog have similar potencies and are the most potent compounds of both the paroxetine - and the femoxetine series. Could it be that the unsubstituted 4-phenyl analog was not developed further because of rapid metabolic para-hydroxylation of the 4-phenyl group and concomitant short duration of action of the drug? Although femoxetine was not the most potent compound in its series, it was chosen for further development. It is much less potent than paroxetine and also less selective [61]. In contrast to paroxetine femoxetine is a 3R, 4S trans(+)isomer. However in both compounds the large substituent groups are in the same diequatorial position. In both paroxetine and femoxetine series N-methylation results in a decrease in potency of the secondary parent ~mines with the exception of the trans (+) NMe paroxetine analog. In table 7 the effects of Me substitution in the trans(_+)paroxetine skeleton on the ~ t y for the paroxetine recognition site in cerebral cortex membranes of the rat are shown [64]. The unsubstituted phenylgroup in the trans(_+)series has s similar affinity for the recognition site of the 5-HT uptake complex as the 4fluoroderivative. Methyl substitution of the phenylgroup in para- or metaposition only causes a slight decrease (2x) in affinity compared to the 4-fluoro compound. Ortho substitution however results in a five times larger decrease in affinity presumably owing to a less favorable rotation of the phenyl group caused by steric hindrance by the ortho-methyl group. Methylsubstitution of the methylenedioxybenzene moiety of the trans(_+) paroxetine isomer has a much larger influence on the affinity than methyl substitution of the phenyl group of the trans(_+)paroxetine skeleton. Although the affinity of the compound with a methylgroup in the R~ position is only two times lower than that of the trans(_+)paroxetine isomer the ~ t i e s of the 1~ and R3 substituted analogs are 80 and 40 times lower (table 7) suggesting that ring substitution of the methylene dioxybenzene moiety is not advantageous for obtaining high affinity. Habert et al. [65] and Plenge et al. [63] found a good correlation between the potency in inhibiting 3H-paroxetine binding in rat brain cortical membranes or rat brain membranes and the potency in inhibiting 5-HT uptake in rat brain synaptosomes of a series of 5-HT uptake inhibitors. Recently Cheetham et al. [66] extended the series and investigated a wide range of monoamine uptake inhibitors. Again a very good and highly significant correlation (r=0.946, p<0.001) was found between the potencies in inhibition of3H paroxetine binding and inhibition of 5-HT uptake in synaptosomal preparations of rat frontal cortex. Taking also into account the saturation data of 3H-paroxetine binding it appears that paroxetine binds to a single site on the 5-HT uptake transporter. This is also confirmed by the data reported by Marcusson et al. supporting a single site model of the 5-HT uptake site / antidepressant binding site [67].

345 Table 7 Structure affinity relationship ofparoxetine and its analogs for the recognition site on the 5-HT uptake complex of rat brain cerebral cortex membranes in vitro [64]. I~.=inhibition constant in nM.

~

"

7

R3 R2

\_ / m

trans

N

o

m

-

Me

• • • • • • • •

o

p

R~

R2

R3

Ki. nM

F

0.81

F

4.3 2.1 3.0 11 4.6 4.0 4.7 160 80

F H Me Me Me F F F

Me Me Me

a

a = paroxetine Litoxetine (SL 81.0385, fig. 6) an achiral compound selectively inhibits 5-HT uptake in rat hypothalamic synaptosomes with an IC~o=18 nM and a selectivity index NE/5-HT=89 [68]. The selectivity is maintained in the in vivo experiments in the rat. The affinity of litoxetine for most receptors involved in neurotransmission is negligible. The affinity oflitoxetine for the 5-HT 2 receptor in membranes of the rat cerebral cortex determined by inhibition of 3H-spiperone binding is fairly low (IC5o=6800 nM). The affinity for the 5-HTs receptor determined by inhibition ofSH -

346 quipazine binding in a similar preparation is significant (ICso=220nM~ [69]). Administration of litoxetine in doses of 1 and 10 m g ~ g i.v. to ferrets reduced vomiting caused by cisplatin infusion in a dose dependent way via the same mechanism of action as other antiemetic 5-HT3 receptor blockers for example ondansetron and granisetron [69]. Clinical development however was recently halted.

)

\N ~

Me

O

~Me

DAPOXETINE

LITOXETINE

Fig. 6 Dapoxetine (Ly 243917) a (1-naphthoxy)-1-phenyl propanamine compound is a selective 5-HT uptake inhibitor structurally related to fluoxetine (fig. 6). A major difference in structure is the position of the phenylgroup which is shii~ed from the 3-position in fluoxetine to the 1-position in dapoxetine. Sertraline, cis(+) 1-methyl~mino-4-(3-4-dichlorophenyl)tetralin, absolute configuration 1S, 4S, is a potent and selective 5-HT uptake inhibitor [70] in clinical use as an antidepressant since 1990. The selectivity index in regard to NE uptake inhibition (NE/5-HT) or DA uptake inhibition in vitro is in both cases 20 [61,70]. Sertraline has no significant affinity for alpha- and beta-adrenoceptors nor for muscarinic, histamine-l, 5-HTI^.D, 5-HT2.4 or dopamine D2 receptors [71, Tulp personal communication]. A selection of data on monoamine uptake inhibition in rat brain synaptomes is shown in table 8. Because the comparable trans isomers are either NE selective or not selective at all [72] data are not shown.

347 The results in table 8 indicate that parasubstitution of the phenylgroup of the racemic parent compound by a halogen atom increases both potency in and selectivity for 5-HT uptake inhibition. Potency and 5-HT selectivity are further increased by dichlorosubstitution in the meta- and para-position of the parent compound. However, ortho-para dichloro substitution decreases potency in 5-HT uptake inhibition and 5-HT selectivity tends to change in the direction of selectivity for NE uptake inhibition. Table 8 Monoamine uptake inhibition in rat brain synaptosomes of corpus striatum (5-HT, DA) and hypothalamus (NE) by 1-methylamino-4-phenyl tetralins [72].

H

I Me-N

~

m

o

m

p

5-HT

NE

DA

H

H

H F C1 Br C1 C1

3.50 1.70 0.26 0.19 0.07 0.50

1.86 2.30 1.41 1.40 0.72 0.31

5.10 4.70 1.38 1.60 0.52 1.70

C1 C1

0.06 0.46

1.20 0.30

1.30 0.32

C1 C1 enantiomers (+) sertraline (-)

O

C1 C1

348 Resolution of the racemic cis-3-4-dichloro compound into the cis (+) and cis (-) enantiomers and testing these compounds proved that potency and selectivity of the racemate is mainly caused by the cis (+) enantiomer sertraline table 8. The potency of sertraline in 5-HT uptake inhibition is the same as that of the racemate but the selectivity index NE/5-HT has increased from 10 to 20. The selectivity index DA/5-HT increased from 7.4 to 20. The eutomer cis (+) is 7.7 times more potent a 5-HT uptake inhibitor than the distomer cis (-). The distomer cis (-) is about equipotent in all three monoamine inhibition tests and thus can be considered as non-selective. Selectivity of sertraline for 5-HT uptake inhibition is also maintained in vivo [71,73]. A series of substituted 3-phenyl-l-indanamines structurally related to the substituted 1-methylamine-4-phenyltetralin series also showed similarities in monoamine uptake inhibition. The compounds of the trans-indanamine series are mainly selective NE uptake inhibitors while those of the cis-series are 5-HT selective [74], the same as in the tetralin series. The changes in potency in the inhibition of the 5-HT transporter caused by different substituents on the phenylgroup often correspond qualitatively in both series. The series differ in potency, the indanamines in general being significantly more potent than the tetralins. Compare for instance cis (_+)-3-(3,4-dichlorophenyl)1-indanamine with the corresponding cis (+_)tetralin derivative (table 9). Note that the selectivity index NE/5-HT is virtually the same for both compounds. In experiments in mice selectivity for the 5-HT- or the NE-transporter of the indanamine series as a whole is oi~en lost [74]. Citalopram a disubstituted racemic 1-(3-dimethylaminopropyl)-l-phenylisobenzofuran derivative (fig. 7) is a highly selective and potent inhibitor of the 5HT transporter in vitro as well as in vivo. Citalopram is used in clinical practice as an antidepressant. In vitro citalopram inhibits 3H-5-HT uptake in rat brain synaptosomes at an ICso = 1.8 nM and a very high selectivity index (NE/5-HT) versus 3H-NE uptake of 4888 (Table 10), see the review of Milne and Goa [75] and references cited therein. Demethylcitalopram the main metabolite of citalopram is a four times less potent 5-HT uptake inhibitor in vitro than its parent compound [table 10]. Its potency is similar to that of fluoxetine and femoxetine but its selectivity index is significantly higher 105 versus 55 and 49 respectively [75]. Both citalopram and its main metabolite retain their selectivity in in vivo experiments antagonizing the depletion of serotonin induced by H 75/12 (a-Et-3OH-4Me-phenethylamine) but not the depletion of norepinephrine induced by H 77/77 (a-Me-3OH-4Mephenethylamine) [31,76,77].

349 Table 9 Rat brain synaptosomal monoamine uptake inhibition.

H I N-Me

H J N--Me

A

B CI

CI

CI

CI

I__~ConM cpd A B

5-HT 70 0.44

NE 720 5.2

DA 520 20

ref [72] [74]

The other metabolites didemethyl citalopram and citalopram-N-oxide are still 5-HT selective but much less potent compared to the main metabolite and the parent compound citalopram (table 10). Being minor metabolites these compounds will probably not contribute to the clinical effect of citalopram in contrast to the main metabolite demethylcitalopram. Citalopram and its metabolites have a negligible inhibitory effect on the uptake of DA in rat brain synaptosomes compared to the effect on 5-HT uptake. Citalopram has little affinity for neurotransmitter receptors like muscarinic, adrenergic, dopaminergic, serotonergic (5-HT1A.m 5-HT 2) histaminergic (H 1) benzodiazepine and opioid receptors [75 and references therein].

350

Me '~Me N

NC, ~ ~ 0 CITALOPRAM Fig. 7

Table 10 Inhibition of 3H-monoamine uptake in rat brain synaptosomes by citalopram and metabolites [75 and refs therein].

5-HT

citalopram demethylcitalopram didemethylcitalopram citalopram-N-oxide

1.8 7.4 24 56

NE

8800 780 1500 3200

DA

41000 26000 12000 > 10000

From the structure-activity relationship data presented by Bigler et al. [78] it can be concluded that substitution in both aromatic moieties of the unsubstituted citalopram is essential for high activity in vitro (inhibition of 5-HT uptake in rabbit blood platelets) as well as in vivo (potentiation of the 5-hydroxytryptophan syndrome in mice). The p-F-atom in citalopram can be exchanged for a Cl-atom or a CN-group and the CN group on the 5-position for an F, C1, Dr atom or CF~ group with only a small loss in activity in vitro and/or in vivo.

351 The effect of bulk of the substituents on the activity of the compounds is probably marginal. The authors [78] suggest that the electronic field effect seems to be a highly important factor in determining the compounds activity in vitro and in vivo.

S-Me

I

I

) McN5652-Z (+) trans Ar vs H (6a, 10b~) 6S, 10 bR Fig. 8

McN 5652-Z (fig. 8) was chosen from a large series of hexahydropyrroloisoquinolines [79] for further development. Table 11 shows that the cis (+_) conformer is virtually inactive in vitro as well as in vivo when compared to the trans (_+) compound. The activity of the racemic trans (+_) compound in vitro as well as in vivo is caused by McN 5652-Z its trans (+) enantiomer as the trans (-) enantiomer is comparatively inactive. The eudismic ratio for 5-HT uptake inhibition (I~. trans (-) / I~. trans (+)) is 150 and for NE uptake inhibition 155. The selectivity index of the trans (+) isomer for 5-HT uptake inhibition versus NE uptake inhibition (Ki NE / ~ 5-HT) is a moderate 4.6. Not only in vitro is McN 5652-Z a highly potent inhibitor of 5-HT-uptake, it also in vivo potentiates at very low doses the 5-hydroxytryptophan induced serotonergic head twitch response in mice [80]. After further studies on McN 5652Z as a potential clinically useful antidepressant the development was halted.

352 Table 11 Monoamine uptake inhibition I~. and headtwitch potentiation in mice EDso of subthreshold doses of L-5-hydroxytryptophan [80].

S - Me

I~. nM conf. a

5-HT

NE

cis (_+) trans (_+) trans (+) trans (-)

16.6 0.68 0.39 58.4

127 2.9 1.8 280

ED.~o mg/kg DA

1740 36.8 23.5 1450

twitch

-10 0.081 0.043 --5

a Configuration Ar versus H However Suehiro and coworkers [81] showed that UC-McN 5652 is probably a very useful tool for in vivo mapping of 5-HT-uptake sites in the central nervous system by position emission tomography (PET) and studying changes in these site brought about by pharmacological intervention or otherwise. In mice brain UC-McN 5652 was selectively bound in areas with a high density of 5-HT uptake sites while its trans (-) enantiomer was distributed aselectively and in a much lower concentration. The UC-cis conformer showed a strikingly low capacity for brain penetration and as expected was also distributed aselectively. Indalpine a 3-substituted indole derivative (fig. 9) selectively inhibits 5-HT uptake in synaptosomal preparations of whole brain of immature female rats with

353 ICso values of 40 nM for 5-HT versus 2000 and 4000 nM for NE and DA uptake inhibition respectively [82]. In slices of several different brain areas of these rats the highest values for 5HT uptake inhibition were found in the medulla + pons and midbrain preparations. The selectivity of 5-HT uptake inhibition was maintained in all the areas examined [83].

H

H INDALPINE LM 5008 Fig. 9 Indalpine antagonized 5-HT depletion in male rat brain induced by H75/12 or by p-chloroamphetamine (PCA) at EDso doses of 0.4 and 3 mg&g i.p. [84]. Scatton et al. showed that under these conditions the 5-HT selectivity is also maintained. PCA induced depletion of 5-HT in male rat brain was antagonized at an EDso dose of 5.8 mg/kg i.p. whereas NE depletion induced by alpha-Me-m-tyrosine was antagonized at EDso >50 mg/kg i.p. [68]. Specifically bound 3H-indalpine to slide mounted parasagittal sections of male rat brain was only displaced at low nanomolar concentrations by 5-HT selective uptake blockers and by 5-HT but not by NE and DA [85]. The uptake by immature female rat brain synaptosomes of choline and of amino acids acting as stimulatory or inhibitory neurotransmitters was not inhibited by indalpine at pharmacologically relevant concentrations [83]. The affinity for a wide variety of neuronal receptor types is either absent or very low. [Tulp personal communication]. Nitroquipazine (fig. 10) is a potent and highly selective 5-HT uptake blocker in occipitoparietal cortex slices of the rat brain with an ICso = 81 nM and a selectivity index (NE/5-HT) of 1100 in contrast to its unsubstituted parent compound quipazine which is 65 times less active and nonselective versus NE [86]. These results confirm the data reported by Vaatstra et al. [87] on the inhibition of monoamine uptake by rat brain synaptosomes. Also in the in vivo experiments is nitroquipazine a potent and selective 5-HT uptake blocker [87]. It is striking

354 that of all the receptors for neurotransmission tested, the 5-HTs receptor is the only one that has a very high affinity for nitroquipazine. Nitroquipazine has a pKi = 8.46 for the inhibition of SH-GR 65630 binding to the 5-HT S receptor [Tulp personal communication]. The potential contribution of the interaction with 5-HT S receptors in vivo to the pharmacological action profile of nitroquipazine was not extensively investigated.

k__/ 6- NITROQUIPAZINE Fig. 10 The potency of 6-substituted quipazines in selective 5-HT uptake inhibition decreases gradually in the order from high to low: nitro _>MeO>CI>Br=CFs but all compounds show a high selectivity for the 5-HT transporter. The disubstituted 5.6dichloroquipazine is also a potent and selective 5-HT uptake blocker with a potency similar to that of the 6-MeO analog [unpublished results]. Substitution of the 4-position in 6-nitroquipazine may also lead to potent and selective 5-HT uptake inhibitors as was reported for 4-bromo-6-nitroquipazine [88]. This compound is only twofold less active than 6-nitroquipazine in vitro as well as in vivo. For use in autoradiographic and in vivo imaging studies of the 5-HT transporter z~I-5-iodo-6-nitroquipazine was developed as a potent and selective 5HT uptake inhibitor [89-91]. ~=sI-5-iodo-6-nitroquipazine was administered to the primate Macaca mulatto in testing the suitability of the compound for the in vivo imaging of 5-HT transporter sites in the brain by single photon emission computer tomography SPECT [92]. Although further development of nitroquipazine as a therapeutic agent was discontinued it still has its merits as an effective pharmacological tool.

CONCLUSION The search for selective inhibitors of the neuronal 5-HT transporter has resulted in the development of several compounds of different chemical classes

355 that are effective in the treatment of typical and atypical depression, panic disorders and obsessive-compulsive disorder. The efficacy of these compounds - fluoxetine, norfluoxetine, fluvoxamine, paroxetine, sertraline and citalopram - in the treatment of depression, is comparable to that of the classical tricyclic antidepressants. However owing to the virtual lack of affinity for the neurotransmitter receptors there is a significant improvement in side-effect profile a major advantage being that the risk of adverse effects on cardiac function is absent or only slight at most. The most common side-effects are transient nausea and diarrhoea and insomnia. Highly selective 5-HT uptake inhibitors considered not suitable for clinical use are often excellent tools for the in vivo imaging of central 5-HT transporters in health and disease by PET or SPECT scan methods.

Addendum re: 5-HT-transporter In order to obtain more detailed information on the nature of the interaction sites on the 5-HT transporter that binds substrates and antagonists various chimeras were constructed. An attempt to obtain functional chimeras between the NE and 5-HT carriers was only partly successful [93]. Three functional cross species chimeras between the rat and the human 5-HT transporter transiently expressed in HeLa cells were described recently [94]. This is of interest because some ligands differ in affinity for the 5-HT transporter of both species and the cross species chimeras are used to explain these differences especially when they are considerable. For example the affinities of imipramine and other tricyclic 5-HT uptake blockers are higher for the human -than for the rat 5-HT transporter while the reverse is true for the substrate d-amphetamine. Serotonin and non-tricyclic antagonists tested exhibit no species preference. In the 5-HT uptake inhibition test in transiently transfected HeLa cells the chimera R1272 H273-630 (i.e. R(at) aminoacid sequence H(uman) a.a. sequence) displays the same characteristics as the cloned human 5-HT transporter with regard to the inhibition constants (I~.) of the tricyclic antidepressants and d-amphetamine. The pharmacological properties of chimera H1-362 R363-630 and its ligands are identical to those of the cloned rat 5-HT transporter in this test. The inhibition constants of imipramine and d-amphetamine obtained with chimera H1-362 R363-531 H532-630 in the 5-HT uptake test indicate that this chimera and the human 5-HT transporter are similar in this respect. H532-630 comprises the transmembrane domains TMD 11 and 12 and the intra cellular carboxylterminus. It was reported previously that the carboxylterminus is not involved in the discriminatory properties of the ligands [95]. Using an additional chimera and site-directed mutagenesis Barker and Blakely [116] showed that a single amino acid at position 586 is responsible for the species difference in affinity of the tricyclics for the 5-HT transporter. Conversion of rat V586 to human F586 selectively increased the tricyclic potency to that of the h u m a n 5-HT transporter.

356 re: regulat.ion of the 5-HT transporter Recently it was reported that the increase in 5-HT transport by rat basophilic leukemia cells (RBL 2H3) after stimulation of the adenosine A3 receptors is due to an increase in maximum velocity of uptake (Vmax) [96]. It was demonstrated that the generation of both nitric oxide NO and c-GMP as a sequence of A3 receptor activation is essential to effect the increase in Vm~. The c-GMP produced could activate a c-GMP dependent kinase which then might phosphorylate the transporter either directly or indirectly. Interestingly direct stimulation of protein kinase C by phorbol-12-myristate-13-acetate leads to a decrease in 5-HT uptake by a decrease in Vmax. These data show that in RBL cells 5-HT transport could be regulated differentially by different receptor mediated second messenger production. According to amino acid sequence analysis the 5-HT transporters in the RBL cells and in the brain cells are identical. The presence of nitric oxide synthetase and 5-HT in neurons of the medial dorsal raphe has been demonstrated [97]. So it is tempting to assume that in the brain and in the RBL cells similar mechanisms of regulation of the 5-HT transporter may operate. re: 5-HT transporter: binding sites Using selective 5-HT- and DA uptake blockers and applying binding surface analysis Silverthorn et al. [98] demonstrated that the cocaine analog RTI-55 binds with equal affinities at two sites on the 5-HT transporter of membranes of whole rat brain minus caudate. Other 5-HT- and monoamine uptake blockers do not discriminate between these two binding sites. The selective 5-HT uptake blocker used was paroxetine. The selective DA uptake blockers were RTI-120 3~-(4'-methylphenyl)tropan-2~-carboxylic acid phenyl ester and Lllll 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl) homopiperazine RTI-55 is 3~-(4'-iodophenyl)tropan-2~-carboxylic acid methyl ester. Similar results were obtained by the same group using rat caudate membranes [99]. Recently Schloss and Betz showed that imipramine and citalopram bind to the rat 5-HT transporter expressed in human HEK-293 cells at two distinct interacting binding sites S, and $2 [115]. Imipramine binds with high affinity (I~ = 11 nM) at S, and with very low affinity at S 2. Citalopram only binds at $2 with Kd 1-2 nM. The binding of imipramine is strictly Na § dependent as substitution of Na § by Li + in the incubation medium virtually abolishes its binding. The binding of citalopram on the other hand is Na § independent. The analysis of Scatchard plots of 3H-ligand binding in the absence or presence of different concentrations of the inhibitor indicated that at low concentrations (10 nM --= K~) imipramine inhibits the binding of 3H-citalopram non-competitively (decreases of Bm~ at constant K~) whereas at high concentrations (100 nM) the inhibition is partly competitive, no further decrease of B .... but increase in Kd. Citalopram inhibits 3Himipramine binding only in a non-competitive way. 5-HT inhibits the binding of both radio ligands purely competitively i.e. increase in Kd at constant Bm.x

357 Although the data indicate the presence of two distinct but interacting binding sites on the recombinant rat 5-HT transporter, the authors do not exclude the possibility that the data can also be explained if the transporter can exist in two conformational states. One where the ligands can only bind to the $2 site and the other favored by the presence of Na § ions where imipramine can also bind to the $1 site. re: Ven.l.afaxine On the basis of the finding that venlafaxine in contrast to all other antidepressants caused down regulation of ~-adrenergic receptors in acute as well as in chronic experiments when tested in the rat pineal model [100, 101] a rapid onset of antidepressant action in the clinic seemed plausible. For a review of its therapeutic potential see also reference [102]. re: Fluoxetine and desipramine DM! Recently Tanda et al. [103] showed that the increase of DA in the prefrontal cortex of free moving rats by the 5-HT uptake inhibitor fluoxetine is caused by stimulation of the 5-HT3 receptor by the increase in extra cellular 5-HT. The effect is blocked by the selective 5-HT3 antagonist tropisetron=ICS 205.930=(3(z.tropanyl)-1H-indole-3-carboxylic acid ester. On the other hand the DA increase evoked by the NE selective uptake blocker DMI is not inhibited by tropisetron [103]. It has been suggested that the increase of extra cellular DA caused by selective or non selective NE uptake inhibitors like DMI or imipramine is due to the inhibition of heterologous uptake of DA by NE neurons via the NE transporter [104,105]. It is possible that increasing extra cellular DA in the prefrontal cortex could be an important factor in the mechanism of action of certain classes of antidepressant drugs. re: Duloxetine Dul oxetine =Ly 248686=S(+ )-N-methyl- 3-( 1-naphthal enyloxy)-2-thiophenepropan amine, inhibits the 5-HT and NE uptake in synaptosomal preparations of the rat hypothalamus with IC~o values of 2.6 and 7.0 nM respectively. In synaptosomal preparations of rat cerebral cortex the inhibition constants for 5-HT and NE uptake were 4.6 and 15.6 nM respectively. The inhibition constants for the binding of ~H-paroxetine (5-HT uptake inhibitor) and 3Htomoxetine (NE uptake inhibitor) were 0.53 and 2.1 nM respectively [106]. These data show a slight selectivity of the compound (about 3x) for 5-HT uptake inhibition vs NE uptake inhibition. The affinity for Ach, H-l, al-NE, 5-HTIA.D, 5HT2A, D~ and opiate receptors is in the micromolar range [106]. Kihara and Ikeda recently reported that the extracellular levels of 5-HT, NE and also of DA in the rat frontal cortex determined by in vivo microdialysis were considerably raised dose dependently by administration of duloxetine per os [107]. Behavioural and electroencephalic properties of duloxetine studied in mice and rats indicated that the compound is a potential useful new antidepressant [108].

358 In 1994 and 1995 several short reviews have been published on the role of serotonergic mechanisms in a variety of psychiatric disorders and on the therapeutic use in these disorders of agents interacting with 5-HT transporters or 5-HT receptor subtypes [109-114].

Acknowledgement The expert secreterial help of Mrs.M.Mulder in the layout and preparation of the manuscript in a printable form is gratefully acknowledged.

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363

Index 5-HT receptors (chapter 1) classification 3,10 evolution of subtypes 4,5,8 molecular cloning 6,7,9 transduction mechanisms 6-9 5-HT~ receptors (chapter 2) agonist binding models 50-60 amino acid sequence 45-49 antagonist binding models 60-62 behavioural models 73-78 ligand binding residues 49, 58-60 molecular cloning 49-50 radio ligand binding 67-70 receptor ligand interactions 67-70 sequence homologies 48 structure 45-49 structure affinity relationships of agonists 17-37, 39-40 antagonists 21, 29, 37-39 therapeutic applications of ligands 81-83 transduction mechanism 65-66 5-HT~s receptors (chapter 3) molecular cloning 97 structure 97 structure affinity relationships of agonists 87-93, 94-95 antagonists 93-94 5-HTID receptors (chapter 4) functional assays 124-127 localization 107-112

364 molecular cloning 106-107, 112 radio ligand binding 101-106 structure activity relationships of agonists 112-123, 130-132 antagonists 118, 123, 130 subtypes 110-112, 118 therapeutic applications of ligands 128-129 transduction mechanism 124-125 5-HTxE and 5-HTxF receptors (chapter 5) functional assays 153-154 localization 144-146 molecular cloning 142 radio ligand binding 146-148 sequence homologies 142-143 structure 141-144 structure affinity relationships of ligands 148-153 therapeutic application ofligands 154-155 transduction mechanism 142-143, 153 5 - H T ~ 5-HT~ and 5-HT~c receptors (chapter 6) behavioural models 207-208 chimeras of 5-HT2A receptor 189-190 functional assays 206 localization 199-202 molecular cloning 202-204 molecular modeling 5-HT2A receptor 190-191 mutants 5-HT2Areceptor 186-189 radio ligand binding 204-206 selective 5-HT2B agonists 168-169 selective 5-HT2Aantagonists 166-167 selective 5-HT2B antagonists 181-184, 193-194 sequence homologies 202-204 structure 202-203 structure affinity relationships of non-selective: 5-HT 2 agonists 161-162, 167-170, 176-178

365 5-HT2 antagonists 161-167, 170-186, 191-194 therapeutic applications of ligands 209-210, 215-216 transduction mechanism 206-207 5-HT s receptors (chapter 7) electrophysiology 235-236 functional assays 234-235 heterogeneity 240-241 localization 232-234 molecular cloning 237-238 neurotransmitter release 242-244 radio ligand binding 228-232 structure 236-240 structure activity relationships of agonists 221-224 antagonists 224-228 therapeutic applications of ligands 245-248 transduction mechanism 242-244 5-HT4 receptors (chapter 8) desensitization 296-298 functional assays 263 heterogeneity 282-286 localization 286-289, 293-296 molecular cloning 292 radio ligand binding 282 structure activity relationships of agonists 264-274 antagonists 274-281 therapeutic applications of ligands 299-302 transduction mechanism 289-291, 294-296 5-HTa~ receptors (chapter 9) amino acid sequence 312 localization 315,-317, 320 molecular cloning 314

366 radio ligand binding 315-316 sequence homologies 312-314 structure 314 transduction mechanism 315 5-HTss recep to rs (chapter 9) amino acid sequence 312 localization 317, 320 molecular cloning 315 radio ligand binding 316 sequence homologies 312-313, 315 structure 314-315 transduction mechanism 317 5-HT 6 receptors (chapter 9) amino acid sequence 312 localization 318-320 molecular cloning 317-318 radio ligand binding 316, 318 sequence homologies 312-313 structure 314, 317-318 structure activity relationships of agonists 318 antagonists 318 transduction mechanism 318 5-HT~ receptors (chapter 9) amino acid sequence 312 localization 317, 320-321 molecular cloning 319 radio ligand binding 316, 318 sequence homologies 312-313 structure 314, 319 structure activity relationships of agonists 319 antagonists 319-320

367 transduction mechanism 319 5-HT t r a n s p o r t e r (chapter 10) binding sites 356-357 chimeras 355 localization 327 molecular cloning 327-328 radio ligand binding 330-331 regulation 356 structure 327-329 structure activity relationships of ligands 331-354, 357-358 therapeutic applications of ligands 354-355 translocation mechanism 329-330 transport 329-331,355-356

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