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

77 downloads 1166 Views 17MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

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

This Page Intentionally Left Blank

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.


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


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 "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 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 =_

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

1 2 3 4 5

Nelson DJ. Pharmacol Biochem and Behav 1991; 40: 1041-1051. Glennon RA. Drug Devl Res 1992; 26: 251-274. Cliffe IA, Fletcher A. Drugs of the Future 1993; 18: 631-642. Boess FG, Martin IL. Neuropharmac 1994; 33: 275-317. Hoyer D, Clarke DC, Fozard JR, Hartig PR, et al. Pharmacol Reviews 1994; 46: 157-203. 6 Fletcher A, Cliffe IA, Dourish CT. TIPS 1993; 14: 441-448. 7 Middlemiss DN, Fozard JR. Eur J Pharmacol 1983; 90: 151-153. 8 Van Wijngaarden I, Tulp MThM, Soudijn W. Eur J Pharmacol Molec Pharmac 1990; 188: 301-312. 9 Mellin C, Vallg~rda J, Nelson DL, BjSrk L, et al. J Med Chem 1991; 34: 497510. 10 BjSrk L, Backlund H88k B, Nelson DL, And~n N, et al. J Med Chem 1989; 32: 779-783. 11 Hibert MF, McDermott I, Middlemiss DN, Mir AK, et al. Eur J Med Chem 1989; 24: 31-37.

41 12 Naiman N, Lyon RA, Bullock AE, Tydelek LT, et al. J Med Chem 1989; 32: 253-256. 13 Glennon RA, Naiman NA, Pierson ME, Smith JD, et al. J Med Chem 1989; 32: 1921-1926. 14 Arvidsson L-E, Hacksell U, Nilsson JLG, Hjorth S, et al. J Med Chem 1981; 24: 921-923. 15 Liu Y, Yu H, Svensson BE, Cortizo L, et al. J Med Chem 1993; 36: 4221-4229. 16 Liu Y, Cortizo L, Yu H, Svensson BE, et al. Eur J Med Chem 1995; 30: 277286. 17 Hillver S-E, BjSrk L, Li YL, Svensson B, et al. J Med Chem 1990; 33: 15411544. 18 Hacksell U, Arvidsson LE, Svensson U, Nilsson JL, et al. J Med Chem 1981; 24: 429-434. 19 Liu Y, MeUin C, Bjork L, Svensson B, et al. J Med Chem 1989; 32" 2311-2318. 20 Thorberg S-O, Hall H, Akesson C, Svensson K, et al. Acta Pharm Suec 1987; 24: 169-182. 21 Podona T, Guardiola-Lemaitre B, Caignara D-H, Adam G, et al. J Med Chem 1994; 37: 1779-1793. 22 Liu Y, Yu H, Mokell N, Nordrall G, et al. J Med Chem 1995; 38: 150-160. 23 Chidester CG, Lin C-H, Lathi RA, Haadsma-Svensson SR, et al. J Med Chem 1993; 36: 1301-1315. 24 Lin C-H, Haadsma-Svensson SR, Lahti RA, Mc Call RB, et al. J Med Chem 1993; 36: 1053-1068. 25 Lin C-H, Haadsma-Svensson SR, Philips G, Lahti RA, et al. J Med Chem 1993; 36: 1069-1083. 26 Backlund H55k B, Yu H, Mezei T, BjSrk L, et al. Eur J Med Chem 1991; 26: 215-220. 27 StjernlSf P, GuUme M, Elebring T, Andersson B, et al. J Med Chem 1993; 36: 2059-2065. 28 StjernlSf P, Elebring Th, Nilsson J, Andersson B, et al. J Med Chem 1994; 37: 3263-3273. 29 Romero AG, Leiby JA, McCall RB, Piercey MF, et al. J Med Chem 1993; 36: 2066-2074. 30 StjernlSf P, Ennis MD, Hansson LO, Hoffman RL, et al. J Med Chem 1995; 38: 2202-2216. 31 Ennis MD, StjernlSfP, Hoffman RL, Ghazal NB, et al. J Med Chem 1995; 38: 2217-2230. 32 Hoyer D. In: Fozard JR ed. The peripheral actions of 5-HT. Oxford University Press 1989; pp 72-99. 33 Hoyer D, Schoeffter P. J Recept Res 1991; 11: 197-214. 34 Taylor EW, Nikam SS, Lambert G, Martin AR, et al. Mol Pharmacol 1988; 34: 42-53. 35 Slaughter FL, Harrington MA, Peroutka SJ. Life Sci 1990; 47: 1331-1337. 36 Foreman MM, Fuller RW, Leander JD, Benvenga MJ, et al. J Pharm Expt Ther 1993; 267: 58-71.

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.


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.


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

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

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.


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

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.



81

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.


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



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

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

NMe2 MeaN

o ,-o ~L.~jL,>

NMe2 O

H

o ~ ' ....

H

O

H

l(sumatriptan) . ~ ~ " N~.,.,,(-"-)

R-K~-I

,~ I-

O

2 NH2

~ MeSO2NH

H

4a(L-694,247)

H

N

H

N

/ H

'-(-)

m

,.,,,.,.NMe2

~

8

NH2

~ N 10

'

k"~iNMe ~ N

H

7

H

5

%..>~..,,>

6

NMe2

N

Me

"o

",E'I.

H 3

NH2

II \'~

4

9

N~

,) H NMe2

~ 11

II \?

~'~.,~N" H

I="

O..:

NMe2

s

NMe2

>

.

H 12(CP 110,330)

,. .o~

NH2

~...~N,>

H

~e~ H

~

~ NMeH

15(S-CM-GTNH2)

/~NMe ~N H

H

16(GR 85548)

17(RU 24969)

(~~e

Me

M e - " N \ s ~

o~o ~L~L.'>

~N H"

H 21(BRL 56905)

NH2

19(CP 122,288)

o

N

H

H

18([R]-18:CP 108,509) NH2

H2N~[~

14

13(CP 146,662)

O ~ .. O

M

O

N H

o

OONH~(~. .

O =.,

H

2

/

N

o

NH2

L

H2N

~ H

22("easterly")

H 23 ("northerly")

N(Pr)2

r

OH ~ ~

NH

./N(Pr)2

H

H N

O ~

N----_9 H 24a

24

cF a H

N

O

24b(8-OH-DPAT)

H

0

F~

OMe H

NH2

N.x~ N

H

H

25 ~

N""~

Me O

OMe

26(MDL 100,687) NMe2

N

H

27(BMS 181,101) NH2 O

CI

Nx..~ N H 28

MeNHSO,

Me\

29

2~NMe2

\ O ~ N

3O H I /N~ /~ ~ ~ ~ ~NMe2 Me .S" -h// .-h/ ~-h/ v

"o

H 31

32

33

~r

o ~cF3

Me

H

N

t-

t~ 0 IJo

t~

34 N/

Me

35(CGS 12066B) /

H

36(Oxymetazoline) [~ /Me

CI , 3 ~ H 37

38

39 O~,~.O

~

"

CI O/N

r

J

/

o ~N

NMe2

NMe2

40(SR 9 27592)

41(SR 28734)

42

sMe

~.

"r9/

Z / ~ . ~

I

!

~z ~~

z

CO On

II

~o~o ~J

r-

-r

~Z1~l

ro

~

-1-

-1-

z

I

i

ro

~0

.~

~0

\

7~o IJ.,

0

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

Peroutka SJ, Snyder SH. Mol Pharmacol 1979; 16: 687-699. Pedigo NW, Yamamura HI, Nelson DL. J Neurochem 1981; 36: 220-226. Pazos A, Hoyer D, Palacios JM. Eur J Pharmacol 1984; 106: 539-546. Humphrey PPA, Hartig P, Hoyer D. TIPS 1993; 14: 233-235. Heuring RE, Peroutka SJ. J Neurosci 1987; 7: 894-903. Hoyer D, Waeber C, Pazos A, Probst A, et al. Neurosci Letts 1988; 85: 357362. Herrick-Davis K, Titeler M, Leonhardt S, Struble R, et al. J Neurochem 1988; 51: 1906-1912. Waeber C, Schoeffter P, Palacios JM, Hoyer D. Naunyn-Schmiedeberg's Arch Pharmacol 1988; 337: 595-601. Waeber C, Schoeffter P, Palacios JM, Hoyer D. Naunyn-Schmiedeberg's Arch Pharmacol 1989; 340: 479-485. Beer MS, Stanton JA, Bevan Y, Chauhan NS, Middlemiss DN. Eur J Pharmacol 1992; 213: 193-197. Summer MJ, Humphrey PPA. Br J Pharmacol 1989; 98: 29-31. Peroutka SJ. Brain Res 1991; 553: 206-210. Leonhardt S, Herrick-Davis K, Titeler M. J Neurochem 1989; 53: 465-471. Bradley P e t al. Neuropharmacol 1986; 25: 563-576. Harrington MA, Peroutka SJ. Brain Research 1990; 506: 172-174. Mahle CD, Nowak HP, Mattson RJ, Hurt SD, et al. Eur J Pharmacol 1991; 205: 323-324.

133 17 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

Boulenguez P, Chauveau J, Segu L, Morel A, et al. J Pharmacol Exp Ther 1991; 259: 1360-1365. Boulenguez P, Segu L, Chauveau J, Morel A, et al. J Neurochem 1992; 58: 951-959. Bruinvels AT, Landwehrmeyer B, Waeber C, Palacios JM, et al. Eur J Pharmacol 1991; 202: 89-91. Bruinvels AT, Hugues L, Nozulak J, Palacios JM, et al. NaunynSchmiedeberg's Arch Pharmacol 1992; 346: 243-248. Beer MS, Middlemiss DN. Eur J Pharmacol 1993; 242: 195-198. Weinshank RL, Branchek T, Hartig PR. Int Patent Number WO 91/17174, 14-11-1991. Hoyer D, Lery H, Waeber C, Bruinvels AT, Nozulak J, Palacios JM. NaunynSchmiedeberg's Arch Pharmacol 1992; 346: 249-254. Xiong W-C, Nelson DL. Life Sci 1989; 45: 1433-1442. Herrick-Davis K, Titeler M. J Neurochem 1988; 50: 1624-1631. Bruinvels A, Palacios JM, Hoyer D. Naunyn-Schmiedeberg's Arch Pharmacol 1993; 347: 569-582. Weinshank RL, Zgombick JM, Macchi MJ, Branchek TA. Proc Natl Acad Sci 1992; 89: 3630-3634. Hamblin MW, MetcalfMA. Mol Pharmacol 1991; 40: 143-148. Hamblin MW, Metcalf MA, McGuffin RW, Karpells S. Biochem Biophys Res Comm 1992; 184: 752-759. Zgombick JM, Weinshank RL, Macchi M, Schechter LE, et al. Mol Pharmacol 1991; 40: 1036-1042. Veldman SA, Bienkowski MJ. Mol Pharmacol 1992; 42: 439-444. Demchyshyn L, Sunahara RK, Miller K, Teitler M, et al. Proc Natl Acad Sci 1992; 89: 5522-5526. Beer MS, Stanton JA, Bevan Y, Heald A, et al. Br J Pharmacol 1993; 110: 1196-1200. Hartig PR, Branchek TA, Weinshank RL. TIPS 1992; 13: 152-159. Libert F, Parmentier M, Lefort A, Dinsart C, et al. Science 1989; 244: 569572. Maenhaut C, Van Sande J, Massart C, Dinsart C, et al. Biochem Biophys Res Comm 1991; 180: 1460-1468. Oksenberg D, Marsters SA, O'Dowd BF, Jin H, et al. Nature 1992; 360: 161163. Peroutka SJ, Switzer JA, Hamik A. Synapse 1989; 3: 61-66. Waeber C, Dietl MM, Hoyer D, Probst A, et al. Neurosci Letts 1988; 88: 1116. Waeber C, Dietl MM, Hoyer D, Palacios JM. Naunyn-Schmiedeberg's Arch Pharmacol 1989; 340: 486-494. Waeber C, Palacios JM. Synapse 1992; 12: 261-270. Lowther S, De Paermentier F, Crompton MR, Horton RW. Eur J Pharmacol 1992; 222: 137-142. Palacios JM, Waeber C, Bruinvels AT, Hoyer D. Mol Brain Res 1992; 13: 175179.

134 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74

Miller KJ, Teitler M. Neurosci Letts 1992; 136: 223-226. Del Arco C, Galende I, Pazos A. Naunyn-Schmiedeberg's Arch Pharmacol 1993; 347: 248-256. Bruinvels AT, Landwehrmeyer B, Probst A, Palacios JM, et al. Mol Brain Res 1993; 21: 19-29. Price GW, Roberts C, Boyland PS, Hagan JJ, et al. Serotonin Meeting, Birmingham 1991; p35. Waeber C, Palacios JM. Brain Res 1990; 528: 207-211. Waeber C, Zhang L, Palacios JM. Brain Res 1990; 528: 197-206. Hoyer D, Middlemiss DN. TIPS 1989; 10: 130-132. Bruinvels AT, Palacios JM, Hoyer D. Neurosci 1993; 53: 465-473. Jin H, Oksenberg D, Ashkenazi A, Peroutka SJ, et al. J Biol Chem 1992; 267: 5735-5738. Hamel E, Fan E, Linville D, Ting V, et al. Mol Pharmacol 1993; 44: 242-256. Bruinvels AT, Landwehrmeyer B, Gustafson EL, Durkin MM, et al. Neuropharmacol 1994; 33: 367-386. Bruinvels AT, Maynard KI, Landwehrmeyer B, Probst A, et al. In Press. Feniuk W, Humphrey PPA. Drug Dev Res 1992; 26: 235-240. Branchek T. Curr Biol 1993; 3: 315-317. Ward TJ. Curr Drugs 1993; 3: 417-423. Glennon RA, Westkaemper RB. Drug News & Perspectives 1993; 6: 390-405. Schoeffter P, Hoyer D. Naunyn-Schmiedeberg's Arch Pharmacol 1989; 340: 135-138. Welcome Foundation Ltd. W0 9118897-A. Welcome Foundation Ltd. EP 0313397-A. Street LJ, Baker R, Castro JL, Chambers MS, et al. J Med Chem 1993; 36: 1529-1538. Castro JL, Matassa VG, Broughton HB, Mosley RT, et al. Bioorg Med Chem Letts 1993; 3: 993-997. Street LJ, Matassa VG, Baker R, Reeve AJ, et al. 7th RSC-SCI Med Chem Symp, Cambridge, England 1993. Merck, Sharpe and Dohme, WO 9321182. Merck, Sharpe and Dohme, WO 9320066-A1. Castro JL, Matassa VG, Russell MGN, Baker R, et al. 7th Fechem Conference on Heterocycles in Bio-organic Chemistry, Chicago, 1993. Koe BK, Level LA, Nowakowski JT, Fox CB, et al. ACS Meeting Chicago 1993. Pfizer Inc. W0 9311106-A1. Connor HE, O'Shaughnessy CT, Feniuk W, Perren MJ, et al. Br J Pharmacol 1993; 108: 99P. Perren MJ, Connor HE, Feniuk W, North PC, et al. Br J Pharmacol 1993; 108: 260P. Kempsford RD, Lacey LF, Keene ON, Thomas M. Br J Pharmacol 1993; 108: 124P. Macor JE, Burkhart CA, Heym JH, Ives JL, et al. J Med Chem 1990; 33: 2087-2093.

135 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108

Macor JE, Blake J, Fox CB, Johnson C, et al. J Med Chem 1992; 35: 45034505. Macor JE, Blank DH, Post RJ, Ryan K. Tetrahedron Letts 1992; 33: 80118014. Merck, Sharp and Dohme. WO 9318029. King FD, Brown AM, Gaster LM, Kaumann AJ, et al. J Med Chem 1993; 36: 1918-1919. Eli Lilly & Co EP 0506363 A. Eli Lilly & Co EP 0506369 A. Glennon RA. J Med Chem 1987; 30: 1-12. Kali-Chemie Pharma. EP-525584-A1. Merrell Dow Pharm Inc. EP 0478954 A. Sprouse J et al. Amer Soc Neurosci 1992; 18: 580.8. Bristol-Myers Squibb Co. EP-464558-A. Bristol-Myers Squibb Co. US-810661. Glennon RA, Ismaiel AM, Chaurasia C, Titeler M. Drug Dev Res 1991; 22: 25-36. Smith Kleine, Beecham. WO 9300333-A1. Russell MGN, Castro JL, Matassa VG, Beer MS, et al. Bioorg Med Chem Letts 1994; 4: 1207-1212. Merck Sharpe and Dohme Ltd. EP-494774-A. Merrell Dow Pharm Inc EP 0451008 A. Adir et Compagnie EP 553016-A1. Neale RF, Fallon SL, Boyer WC, Wasley JWF, et al. J Pharmacol 1987; 136: 1-9. Schoeffter P, Hoyer D. Naunyn-Schmiedeberg's Arch Pharmacol 1989; 339: 675-683. Schoeffter P, Hoyer D. Eur J Pharmacol 1991; 196: 213-216. BASF AG WO 9211014. BASF AG WO 9211013. Frehel D, De Cointet P, Badorc A, Carpy A, et al. GESA XXIII Annecy, France 1993. Elf Sanofi. EP 0534856-A1. Smith Kline, Beecham. WO 9300094-A2. Tnnnpp-Kallmeyer S, Bruinvels A, Hoflack J, Hibert M. Neurochem Int 1991; 19: 397-406. Glaxo Group Research Ltd. EP 0533268 A1. Glaxo Group Research Ltd. EP 0533266 A1. Glaxo Group Research Ltd. EP 0533267 A1. Skingle M, Beattie DT, Scopes DIC, Starkey SJ, et al. Behav Brain Res 1996; 73: 157-161. Schoeffter P, Waeber C, Palacios J, Hoyer D. Naunyn-Schmiedeberg's Arch Pharmacol 1988; 337: 602-608. Waeber C, Schoeffter P, Palacios J, Hoyer D. Naunyn-Schmiedeberg's Arch Pharmacol 1989; 340: 479-485. Sumner M, Humphrey P. Br J Pharmacol 1990; 99: 219-220.

136 109 Zgombick J, Weinshank R, Macchi M, Schechter L, et al. Mol Pharmacol 1991; 40: 1036-1042. 110 Middlemiss D, Bremer M, Smith S. Eur J Pharmacol 1988; 157: 101-107. 111 Limberger N, Deicher R, Starke K. Naunyn-Schmiedeberg's Arch Pharmacol 1991; 343: 353-364. 112 Schlicker E, Fink K, Gothert M, Hoyer D, et al. Naunyn-Schmiedeberg's Arch Pharmacol 1989; 340: 45-51. 113 Feurerstein T, Lupp A, Hertting G. Neuropharmacol 1992; 31: 15-23. 114 Schlicker E, Brandt F, Classen K, Gothert M. Brain Res 1985; 331: 337-341. 115 Galzin A, Poirier M, Lista A, Chodkiewicz J, et al. J Neurochem 1992; 59: 1293-1301. 116 Maura G, Thellung S, Andriolo G, Ruelle A, et al. J Neurochem 1993; 60: 1179-1182. 117 Beer M, Middlemiss D, McAllister G. TIPS 1993; 14: 228-231. 118 Wilkinson L, Middlemiss D. Naunyn-Schmiedeberg's Arch Pharmacol 1992; 345: 696-699. 119 Price G, Watson J, Roberts C, Jones B. Br J Pharmacol 1993; 108: 101P. 120 Schoeffter P, Hoyer D. J Pharmacol Exp Ther 1990; 252: 387-395. 121 Gupta P. Br J Pharmacol 1992; 106: 703-709. 122 Martin G, MacLennan S. Naunyn-Schmiedeberg's Arch Pharmacol 1990; 342: 111-119. 123 Ormandy G, Wilson D, Wren P, Barrett V, et al. Br J Pharmacol (in press). 124 Bax A, Van Heuven-Nolsen D, Bos E, Simoons M, et al. NaunynSchmiedeberg's Arch Pharmacol 1992; 345: 500-508. 125 Molderings G, Werner J, Likungu J, Gothert M. Naunyn-Schmiedeberg's Arch Pharmacol 1990; 342: 371-377. 126 Heuven-Nolsen D, Tysse Klasen T, Luo Q, Saxena P. Eur J Pharmacol 1990; 191: 375-382. 127 Hamel E, Bouchard D. Br J Pharmacol 1991; 102: 227-233. 128 Humphrey P, Feniuk W, Perren M, Connor H, et al. Br J Pharmacol 1988; 94: 1123-1132. 129 Cohen M, Schenck K, Nelson D, Robertson D. Eur J Pharmacol 1992; 211: 43-46. 130 Connor H, Feniuk W, Humphrey P. Br J Pharmacol 1989; 96: 379-387. 131 Parsons A, Whalley E. Eur J Pharmacol 1989; 174: 189-196. 132 Gaw A, Wadsworth R, Humphrey P. Eur J Pharmacol 1990; 179: 35-44. 133 Parsons A, Whalley E, Feniuk W, Connor H, et al. Br J Pharmacol 1989; 96: 434-449. 134 Bond R, Craig D, Charlton K, Ornstein A, et al. J Auton Pharmac 1989; 9: 201-210. 135 Sahin-Erdemil I, Hoyer D, Stoll A, Seiler M, et al. Br J Pharmacol 1991; 102: 386-390. 136 Parsons A, Stutchbury C, Raval P, Kaumann A. Naunyn-Schmiedeberg's Arch Pharmacol 1992; 346: 592-596. 137 Conner H, Feniuk W, Humphrey P. Eur J Pharmacol 1989; 161: 91-94.

137 138 Borton M, Neligan M, Wood F, Dervan P, et al. Br J Clin Pharmac 1990; 30: 107S-108S. 139 Chester A, Martin G, Bodelsson M, Arneklo-Nobin B, et al. Cardiovase Res 1990; 24: 932-937. 140 Terron J-A. Eur J Pharmacol 1996; 300: 109-112. 141 Bax W, Renzenbrink G, Van Heuven-Nolsen D, Thijssen E, et al. Eur J Pharmacol 1993; 239: 203-210. 142 Bodelsson M, Tornebrandt K, Bertilsson I-L, Arneklo-Nobin B. Eur J Pharmacol 1992; 219: 455-460. 143 Humphrey P, Feniuk W, Montevalian M, Parsons A, et al. Serotonin: molecular biology. Receptors and functional effects 1991; 421-429. 144 Jansen I, Edvinsson L, Mortensen A, Olesen J. Cephalalgia 1992; 12: 202205. 145 Bruinvels A, Lery J, Nozulak J, Palacios J, et al. Naunyn-Schmiedeberg's Arch Pharmacol 1992; 346: 243-248. 146 Higgins G, Jordon C, Skingle M. Br J Pharmacol 1991; 102" 305-310. 147 Peroutka S, Havlik S, Oksenberg D. Headache 1993; 33: 347-350. 148 Adham N, Kao H-T, Schechter LE, Bard J, et al. Proc Natl Acad Sci 1993; 90: 408-412. 149 Feniuk W, Humphrey P, Perren M. Br J Pharmacol 1989; 96: 83-90. 150 Perren M, Feniuk W, Humphrey P. Br J Pharmacol 1991; 102: 191-197. 151 Den Boer M, Villal6n C, Heiligers J, Humphrey P, et al. Br J Pharmacol 1991; 102: 323-330. 152 Den Boer M, Villal6n C, Saxena P. Naunyn-Schmiedeberg's Arch Pharmacol 1992; 345: 509-515. 153 Mfiller-Schweinitzer E. Cardiovasc Drugs Ther 1990; 4: 1455-1460. 154 Saxena P, Ferrari M. Cephalalgia 1992; 12: 187-196. 155 Doenicke A, Brand J, Perrin V. Lancet 1988; 1309-1311. 156 Friberg L, Olesen J, Iversen H, Sperking B. Lancet 1991; 338: 13-17. 157 Ferrari M, Haan J, Blokland A, Minnee P, et al. Migraine and other headaches: The vascular mechanisms 1991: 245-248. 158 MacIntyre P, Bhargava B, Hogg K, Gemmill J, et al. Br J Clin Pharmac 1992; 34: 541-546. 159 Scott A, Grimes S, NG K, Critchley M, et al. Br J Clin Pharmacol 1992; 33: 401-404. 160 Caekebeke H, Ferrari M, Zwetsloot C, Jansen J, et al. Neurology 1992; 42: 1522-1526. 161 Moskowitz M. Cephalalgia 1992; 12: 5-7. 162 Gabriella-Buzzi M, Moskowitz M. Br J Pharmacol 1990; 99: 202-206. 163 Matsubara T, Moskowitz M, Byun B. Br J Pharmacol 1991; 104: 3-4. 164 Nozaki K, Moskowitz M, Boccalini P. Br J Pharmacol 1992; 106: 409-415. 165 Gabriella-Buzzi M, Carter W, Shimizu T, Heath H, et al. Neuropharmacol 1991; 30: 1193-1200. 166 Goadsby PJ, Edvinsson L, Ekman D. Ann Neurology 1990; 28: 183-187. 167 Sleight A, Cervenka A, Peroutka S. Neuropharmacol 1990; 29: 511-513. 168 Lawrence A, Marsden C. J Neurochem 1992; 58: 142-146.

138 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186

Skingle M, Higgins GA, Feniuk W. J Psychopharmacol 1994; 8: 14-21. Berendsen H, Broekkamp C. Eur J Pharmacol 1991; 199: 107-109. Finberg J, Vardi Y. Eur J Pharmacol 1990; 183: 693-694. Simansky K. Pharmacol Biochem Behav 1991; 38: 459-462. Humphrey P, Feniuk W. TIPS 1991; 12: 444-446. Brown E, Endersby C, Smith R, Talbot J. Eur Neurol 1991; 31: 339-344. Clitherow JW, Scopes DIC, Skingle M, Jordan CC, et al. J Med Chem 1994; 37: 2253-2257. Walsh DM, Beattie DT, Connor HE. Eur J Pharmacol 1995; 287: 79-84. Pauwels PJ, Colpaert FC. Eur J Pharmacol 1996; 300: 141-145. Pauwels PJ, Palmier Ch, Wurch Th, Colpaert FC. Naunyn-Schmiedeberg's Arch Pharmacol 1996; 353: 144-156. Street LJ, Baker R, Davey WB, Guiblin AR, et al. J Med Chem 1995; 38: 1799-1810. Sternfeld F, Baker R, Broughton HB, Guiblin AR, et al. Bioorg Med Chem Letts 1996; 6: 1825-1830. Macor JE, Blank DH, Fox CB, Lebel LA, et al. J Med Chem 1994; 37: 25092512. Macor JE, Blank DH, Desai K, Fox CB et al. Bioorg Med Chem Letts 1995; 5: 2391-2396. Glennon RA, Hong S-S, Bondare M, Law H, et al. J Med Chem 1996; 39: 314322. Glennon RA, Hong S-S, Dukat M, Teitler M, et al. J Med Chem 1994; 37: 2828-2830. Watson JM, Burton MJ, Price GW, et al. Eur J Pharmacol 1996; 314: 365372. Zgombick JM, Schechter LE, Adham SA, et al. Naunyn-Schmiedeberg's Arch Pharmacol 1996; 354: 226-236.

Chapter 5

5-HT1E , 5HTlr RECEPTORS



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.


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


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 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.


Chapter 7

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



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

S 9 EROTONIN

"

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

,,CH3

0

CH,

LIGANDS

C l ~.,~,,~~ / ~

c,

H2 N'~ ~'~'// ~ ~.,. H

CI

CH3 BEMESETRON

CHs ONDANSETRON

ZACOPRIDE

CH3

/CH,

o

S/ "h

H2 N NH

/~CO

TROPISETRON

/

I II ~1,,~.,,~ NCH:I

BMY 33464

GR 65630 CHs

o c, 0

o!~_c~

~~~

~/~-'N H

N CHs

ZATOSETRON

DOLASETRON

GR 67330

c~h

m.o~~~ I~ L-683877

CI

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

P P P P

D D D D

I L L T

217

!:i i:! i:i !:i !:! i:! !:! !:! !.T.':"i-'."!7 :::::::::::::::::::::::::::22Q ......

183 I s S

185 T G G

j[12_20

i@I H|

K K V E

225

s K F

FT

T G A Y

Y D Y T T

OOH

286 D D

S G -iE R V F K S GE K I S LG S G - E'K M T L S

D A

A

P A IR V

G L

G

I L L -IL , G Y, ,S , V F L I V S I TVLiLS L T V F M L L V A E I S V LIL S LIT V F L L V I V

I T T V !L i T M IT T Q

S

S G

D E

S R A

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.

Acknowledgements: I thank Dr Igal Nevo for his thoughtful comments and for assistance in correcting the manuscript. REFERENCES 1 2 3 4 5 6 7 8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Hoyer D, Martin GR. Behav Brain Res 1996; 73: 263-268. Gaddum JH, Picarelli ZP. Br J Pharmacol 1957; 12: 323-328. Kilpatrick GJ, Bunce KT, Tyers MB. Med Res Rev 1990; 10: 441-475. Costall B, Naylor RJ, Tyers MB. Pharmacol Ther 1990; 47: 181-202. Barnes JM, Barnes NM, Cooper SJ. Neurosc Behav Rev 1992; 16: 107-113. Peters JA, Malone HM. Lambert JJ. Trends Pharmacol Sci 1992; 13: 391-397, Zifa E, Filion G. Pharmacol Rev 1992; 44: 401-458. Hamon M. In: Hamon M (ed) Central and pheripheral 5-HT3 receptors. London: Acad Press 1992. Greenshaw AJ. Trends Pharmacol Sci 1993; 14: 265-270. Apud JA. Neuropsychopharmacol 1993; 8: 117-130. Emerit MB, Riad M, Fattaccini CM, Hamon M. Neurochem 1993; 60: 20592067. Richardson BP, Engel G, Donatsch P, Stadler PA. Nature 1985; 316: 126-131. Van Wijngaarden I, Tulp MTM, Soudijn WT. Eur J Pharmacol Mol Pharmacol 1990; 188: 301-312. Fozard JR. In: Saxena PR. Wallis DI, Wouters W, Bevan P (eds) Cardiovascular pharmacology of 5-hydroxytryptamine. Dordrecht: Kluwer Academic Publishers 1990; 101-115. Craig DA, Eglen RM, Walsch KLM, Perkins LA, et al. Naunyn Schmiedeberg's Arch Pharmacol 1990; 342: 9-16. Ireland SJ, Tyers MB. Br J Pharmacol 1987; 90: 229-238. Kilpatrick GJ, Butler A, Burridge J, Oxford AW. Eur J Pharmacol 1990; 182: 193-197. Boess FG, Sepulveda MI, Lummis SCR, Martin IL. Neurochem Int 1992; 31: 561-564. Bachy A, H~aulme M, Giudice A, et al. Eur J Pharmacol 1993; 237: 299-309. Glennon RA, Young R, Dukat M. Pharmacol Biochem Behav 1992; 41: 361364. Schmidt CJ, Black CK. Eur J Pharmacol 1989; 167: 309-310. Kean PE, Bachy A, Giudice A, Michaud JC, et al. Third IUPHAR Satellite meeting on Serotonin, Chicago, USA 1993; p 82. Richardson BP, Buchheit KM. In: Osborne NN, Hamon M (eds) Neuronal Serotonin, Chichester: John Wiley & Sons 1988; 465-506.

250 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 49 50 51

Ruat M, Traiffort E, Arrang J-M, et al. Biochem Biophys Res Commun 1993; 193: 268-276. Shen Y, Monsma FJ Jr, Metcalf MA, Jose PA, et al. Biol Chem 1993; 268: 18200-18204. Bockaert J, Fozard JR, D~_lmuis A, Clarke DE. Trends Pharmacol Sci 1992; 13: 141-145. Rosen T, Seeger TF, McLean S, et al. J Med Chem 1993; 33: 3020-3023. Rasmussen K, Stockton ME, Czachura JR. Eur J Pharmacol 1991; 205: 113116. Gozlan H, Langlois M. In: H~mon M (ed) Central and pheripheral 5-HT3 receptors. London: Aacdemic Press 1992; 59-88. Fozard JR, Mobarok Ali ATM, Newgrosh G. Eur J Pharmacol 1979; 59: 195210. Fozard JR, Mobarok Ali ATM. Eur J Pharmacol 1978; 49: 109-112. Rosen T, Nagel AA, Rizzi JP, et al. J Med Chem 1990; 33: 2715-2720. Swain CJ, Baker R, Kneen C, et al. J Med Chem 1991; 35: 1019-1031. Swain CJ, Baker R, Kneen C, et al. J Med Chem 1991; 34: 140-151. Collin S, E1 Tayar N, Van De Waterbeemd H, et al. Eur J Med Chem 1989; 24: 163-169. King FD, Dabbs S, Bermudez J, Sanger GJ. J Med Chem 1990; 33: 29422944. Bradley G, Ward TJ, White JC, Coleman J, et al. J Med Chem 1992; 35: 1515-1520. Hibert MF, Hoffmann R, Miller RC, Cart AA. J Med Chem 1990; 33: 15941600. Rizzi JP, Nagel AA, Rosen R, McLean S, et al. J Med Chem 1990; 33: 27212725. Evans SM, Galdes A, Gall M. Pharmnacol Biochem Behav 1991; 40: 10331040. Kilpatrick GJ, Jones BJ, Tyers MB. Nature 1987; 330: 746-748. Kilpatrick GJ, Butler A, Hagen RM, Jones BJ, et al. Naunyn Schmiederberg's Arch Pharmacol 1990; 342: 22-30. Wong EHF, Bonhaus DW, Wu I, Stefanich E, et al. J Neurochem 1993; 60: 921-930. Gehlert DR, Schober DA, Gackenheimer SL, Mais DE, et al. Neurochem Int 1993; 23: 373-383. Nelson DR, Thomas DR. Biochem Pharmacol 1989; 38: 1693-1695. Barnes JM, Barnes NM, Champaneria S, Costall B, et al. Neuropharmacol 1990; 29: 1037-1045. Wong DT, Robertson DW, Reid LR. Eur J Pharmacol 1989; 166: 107-110. Barnes NM, Costall B, Naylor RJ. J Pharm Pharmacol 1988; 40: 548-551. Wafting KJ, Aspley S, Swain CJ, Saunders J. Eur J Pharmacol 1988; 149: 397-398. Hoyer D, Neijt HC. Eur J Pharmacol 1987; 143: 291-292. Milburn CM, Peroutka SJ. J Neurochem 1989; 52: 1787-1792.

251 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82

Koscielniak T, Ponchant M, Laporte A-M, et al. CR Acad Sci Paris 1990; 311: 231-237. Kidd EJ, Levy J-C, Nielsen M, Hamon M, et al. Eur J Pharmacol Mol Pharmacol 1993; 247; 45-56. Schmidt AW, Hurt ST, Peroutka SJ. Eur J Pharmacol 1989; 171: 141-143. Meyerson LR, Park H. Drug Dev Res 1991; 23: 27-33. Steward LJ, West KE, Baker J, Lummis SCR, Barnes NM. In: 2nd Int. Symp on Serotonin: Huston 1992: 41P. Sharif NA, Wong EHF, Loury DN, et al. Br J Pharmacol 1991; 102: 919-925. Barnes JM, Barnes NM, Costall B, Ironside JW, et al. J Neurochem 1989; 53: 1787-1793. Kilpatrick GJ, Barnes NM, Chen CHK, Costall B, et al. Neurochem Int 1991; 19: 389-396. Kilpatrick GJ, Jones BJ, Tyers MB. Eur J Pharmacol 1989; 159: 157-164. Buffer A, Elswood CJ, Burridge J, et al. Br J Pharmacol 1990 101: 591-598. Richardson BP, Engel G. Trends Neurosci 1986; 9: 424-428. Newberry NR, Cheshire SH, Gilbert MJ. Br J Pharmacol 1991; 102: 615-620. Bolafios FJ, Schechter LE, Miquel MC, et al. Biochem Pharmacol 1990; 40: 1541-1550. Waeber C, Dixon K, Hoyer D, Palacios JM. Eur J Pharmacol 1988; 151: 351352. Perry DC. Eur J Pharmacol 1990; 187: 75-85. Barnes JM, Barnes NM, Costall B, et al. Br J Pharmacol 1992; 105: 500-504. Barnes JM, Barnes NM. Biochem Pharmacol 1993; 45: 2155-2158. Gozlan H, Kidd E, G~rard C, Emerit MB, et al. In: 2nd Int Symp Serotonin, Houston 1992; 32P. Neijt HC, Te Duits IJ, Vijverberg HPM. Neuropharmacol 1988; 27: 301-307. Sancilio LF, Pinkus LM, Jackson CB, Munson HR Jr. Eur J Pharmacol 1990; 181: 303-306. Barnes JM, Barnes NM, Costall B, et al. Biochem Behav 1990; 37: 717-727. Bradley PB, Engel G, Peniuk W, et al. Neuropharmacol 1986; 6: 563-575. Fozard JR. In: Peripheral actions of 5-hydroxytryptamine. Oxford: Oxford Univ Press 1989. Pinkus LM, Sarbin NS, Gordon JC, Munson HR Jr. Eur J Pharmacol 1990; 179: 231-235. Hoyer D, Waeber C, Korf A, Neijt H, et al. Naunyn Schmiedeberg's Arch Pharmacol 1989; 340: 396-402. Kilpatrick GJ, Jones BJ, Tyers MB. Neurosci Lettr 1988; 94: 156-159. Hamon M, GaUissot MC, Menard F, Gozlan H, et al. Eur J Pharmacol 1989; 164: 315-322. Reynolds DJM, Leslie RA. Grahame-Smith DG, Harvey JM. Eur J Pharmacol 1989; 174: 127-130. Waeber C, Hoyer D, Palacios JM. Neurosci 1989; 31: 393-400. Pratt GD, Bowery NG. Neuropharmacol 1989; 28: 1367-1376. Waeber C, Pinkus LM, Palacios JM. Eur J Pharmacol 1990; 181: 283-287.

252 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112

Barnes JM, Barnes NM, CostaU B, Naylor IL, et al. Naunyn Schmiedeberg's Arch Pharmacol 1990; 342: 17-21. Leslie RA, Reynolds DJM, Andrews PLR, Grahame-Smith DG, et al. Neurosci 1990; 38: 667-673. Reynolds DJM, Leslie RA, Grahame-Smith DG, Harvey JM. Neurochem Int 1991; 18: 69-73. Gehlert DR, Gackenheimer SL, Wong DT, Robertson DW. Brain Res 1991; 553: 149-154. Laporte AM, Koscielniak T, Ponchant M, Vergd D, et al. Synapse 1992; 10: 271-281. Palacios JM, Waeber C, Mengod G, Hoyer D. Neurochem Int 1991; 18: 17-25. Merahi N, Orer HS, Laporte A-M, Gozlan H, et al. Neurosci 1992; 1: 91-100. Miller AD, Nonaka S. J Pharmaeol Exp Ther 1992; 260: 509-517. Hoyer D, Gozlan H, Bolafios F, Schechter LE, et al. Eur J Pharmacol 1989; 171: 137-139. Reiser G, Hamprecht B. Brain Res 1989; 479: 40-48. Derkach V, Surprenant A, North RA. Nature 1989; 339: 706-709. Yakel JL, Jackson MB. Neuron 1988; 1: 615-621. Yang J. J Gen Physiol 1990; 96: 1177-1198. Yang J, Mathei A, Hille B. J Physiol (Lond) 1992; 448: 237-256. Yakel JL, Shao XM, Jackson MBT. Brain Res 1990; 533: 46-52. Peters JA, Hales TG, Lambert JJ. Eur J Pharmacol 1988; 151: 491-495. Maricq AV, Peterson AS, Brake AJ, Myers RM, et al. Science 1991; 254: 432437. Shao XM, Yakel JL, Jackson MB. J Neurophysiol 1991; 65: 630-638. Hope AG, Downie DL, Sutherland L, Lambert JJ, et al. Eur J Pharmacol Mol Pharmacol 1993; 245: 187-192. Yakel JL, Lagrutta A, Adelman JP, North RA. Proc Natl Acad Sci USA 1993; 90: 5030-3033. Miquel M-C, Emerit MB, Gozlan H, Hamon M. Biochem Pharmacol 1991; 42: 1453-1461. Gordon JC, Sarbin NS, Barefoot DS, Pinkus LM. Eur J Pharmacol Mol Pharmacol 1990; 188: 313-319. McKernan RM, Biggs CS, Gillard N, Quirk K, et al. Biochem J 1990; 269: 623-628. McKernan RM, Gillard NP, Quirk K, et al. J Biol Chem 1990; 265: 1357213577. Miller K, Weisberg E, Fletcher PW, Teitler M. Synapse 1992; 11: 58-66. Boess FG, Lummis SCR, Martin IL. J Neurochem 1992; 59: 1692-1701. Lummis SCR, Martin IL. Mol Pharmacol 1992; 41: 18-23. Gozlan H, Schechter LE, Bolafios F, et al. Eur J Pharmacol 1989; 172: 497500. Miquel M-C, Emerit MB, Bolafios FJ, Schechter LE, et al. J Neurochem 1990; 55: 1526-1536. Lummis SCR, Nielsen M, Kilpatrick GJ, Martin IL. Eur J Pharmacol Mol Pharmacol 1990; 189: 229-232.

253 113 Changeux J-P, Devillers-Thi~ry A, Galzi J-L, Bertrand D. Trends Pharmacol Sci 1992; 13: 299-301. 114 Vandenberg RJ, Handford CA, Schofield PR. Neuron 1992; 9: 491-496. 115 Hibert MF, Trumpp-Kalmeyer S, Bruinvels A, Hoflack J. Mol Pharmacol 1991; 40: 8-15. 116 Revah F, Bertrand D, Galzi J-L, et al. Nature 1991; 353: 846-849. 117 Kilpatrick GJ, Tyers MB. Biochem Soc Trans 1992; 20: 118-121. 118 Hartig PR, Branchek TA, Weinshank RL. Trends Pharmacol Sci 1992; 13: 152-159. 119 Metcalf MA, McGuffin RW, Hamblin MW. Biochem Pharmacol 1992; 44: 1917-1920. 120 Oksenberg D, Marsters SA, O'Dowd BF, et al. Nature 1992; 360: 161-163. 121 Galzi J-L, Devillers-Thi~ry A, Hussy N, Bertrand S, et al. Nature 1992; 359: 500-505. 122 Bertrand D, Galzi JL, Devillers-Thi~ry A, Bertrand S, et al. Proc Natl Acad Sci, USA 1993; 90: 6971-6975. 123 Blier P, Bouchard C. Br J Pharmacol 1993; 108: 13-22. 124 Glaum SR, Anderson EG. Eur J Pharmacol 1988; 156: 287-290. 125 Paudice P, Raiteri M. Br J Pharmacol 1991; 103: 1790-1794. 126 Tecott LH, Maricq AV, Julius D. Proc Natl Acad Sci, USA 1993; 90: 14301434. 127 Feuerstein TJ, Hertting G. Naunyn Schmiedeberg's Arch Pharmacol 1986; 22: 191-197. 128 Blandina P, Goldfarb J, Walcott J, Green JP. J Pharmacol Exp Ther 1991; 256:341-347. 129 Martin P, Gozlan H, Puech AJ. Eur J Pharmacol 1992; 212: 73-78. 130 Barnes JM, Barnes NM, Costall B, Naylor RJ, et al. Nature 1989; 338: 762763. 131 Bianchi C, Siniscalchi A, Beani L. Neuropharmacology 1986; 25: 1043-1049. 132 Bianchi C, Siniscalchi A, Beani L. Br J Pharmacol 1990; 101: 448-452. 133 Maura G, Andrioli GC, Cavazzani P, Raiteri M. J Neurochem 1992; 58: 23342337. 134 Johnson RM, Inouye GT, Eglen RM, Wong EHF. Naunyn Schmiedeberg's Arch Pharmacol 1993; 347: 241-247. 135 Consolo S, Bertorelli R, Russi G, Zambelli M, et al. J Neurochem 1994; 62: 2254-2261 136 Martin KF, Hannon S, Philips I, Heal DJ. Br J Pharmacol 1992; 106: 139142. 137 Barnes NM, Cheng CHK, Costall B, Ge J, et al. Br J Pharmacol 1992; 107: 233-239. 138 Williams GM, Smith DL, Smith DJ. Neuropharmacol 1992; 31: 725-733. 139 Kidd EJ, Laporte A-M, Langlois X, et al. Brain Res 1993; 612: 289-298. 140 Koulu M, SjSholm B, Lappalainen J, Virtanen R. Eur J Pharmacol 1989; 169: 321-324. 141 Koulu M, Lappalainen J, Hietala J, SjSholm B. Psychopharmacol 1990; 101: 168-171.

254 142 Imperato A, Puglisi-Allegra S, Zocchi A, Scrocco MG, et al. Eur J Pharmacol 1990; 175: 211-214. 143 Hagan RM, Buffer A, Hill JM, Jordan CG, et al. Eur J Pharmacol 1987; 138: 303-305. 144 Carboni E, Acquas E, Leone P, Perezzani L, et al. Eur J Pharmacol 19878; 151: 159. 145 Carboni E, Acquas E, Frau R, DiChiara G. Eur J Pharmacol 1989; 164:515519. 146 Carboni E, Acquas E, Leone P, DiChiara G. Psychopharmacol 1989; 97: 175178. 147 Jiang LH, Ashby CR, Kasser RJ, Wang RY. Brain Res 1990; 513: 156-160. 148 Parsons LH, Justice JB Jr. Brain Res 1993; 606: 195-199. 149 Chen J, Van Praag HM, Gardner EL. Brain Res 1991; 543: 354-357. 150 Jacocks HM, Cox BM. J Pharmacol Exp Ther 1992; 262: 356-364. 151 Harro J, Vasar E, Bradwejn T. Trends Pharmacol Sci 1993; 14: 244-249. 152 Woodruff GN, Hughes J. Ann Rev Pharmacol Toxicol 1991; 31: 469-501. 153 Raiteri M, Paudice P, Vallebuona F. Naunyn Schmiedeberg's Arch Pharmacol 1993; 347: 111-114. 154 Blandina P, Goldfarb J, Carddock-Royal B, Green JP. J Pharmacol Exp Ther 1989; 251: 803-809. 155 Blandina P, Goldfarb J, Green JP. Eur J Pharmacol 1988; 155: 349-350. 156 Yi SJ, Gifford AN, Johnson KM. Eur J Pharmacol 1991; 199: 185-189. 157 Costall B, Domeney AM, Naylor RJ, Tyers MB. Br J Pharmacol 1987; 92: 881-890. 158 Van der Hoek GA, Cooper SJ. Br J Pharmacol 1990; 100: 414P. 159 Sorensen SM, Humphreys TM, Palfreyman MG. Eur J Pharmacol 1989; 163: 115-118. 160 Palfreyman MG, Schmidt CJ, Sorensen SM, et al. Psychopharmaeol 1993; 112: $60-$67. 161 Ashby CR Jr, Jiang LH, Wang RYC. Eur J Pharmacol 1990; 175: 347-350. 162 Wang RY, Ashby CR, Minabe Y. In: Serotonin 1991; Birmingham 1991; P74. 163 Prisco S, Pessia M, Ceci A, Borsini F, et al. Eur J Pharmacol 1992; 214: 1319. 164 Abbott A. Trends Pharmacol Sci 1990; 11: 49-51. 165 Chen J, Paredes W, Van Praag HM, Lowinson JH, et al. Synapse 1992; 10: 264-266. 166 Hagan RM, Kilpatrick GJ, Tyers MB. Psychopharmacol 1993; 112: $68-$75. 167 Bill DJ, Fletcher A, Glenn BD, Knight M. Eur J Pharmacol 1992; 218: 327334. 168 Costall B, Domeney AM, Kelly ME, et al. Eur J Pharmacol 1993; 234: 91-99. 169 Domeney AM, Costall B, Gerrard PA, Jones DNC, et al. Pharmacol Biochem Behav 1991; 38: 169-175. 170 Carey GJ, Costall B, Domeney AM, et al. Pharmacol Biochem Behav 1992; 42: 75-83. 171 Alhaider AA, Lek SZ, Wilcox GL. J Neurosci 1991; 11: 1881-1888. 172 Aapro MS. Drugs 1991; 42: 551-568.

255 173 Hesketh PJ, Gandara DR. J Natl Cancer Inst 1991; 83: 613-620. 174 CostaU B, Doraeney AM, Gerrard PA, Kelly ME, et al. J Pharm Pharmacol 1988; 40: 302-305. 175 Barnes NM, Cheng CHK, Costall B, Ge J, et al. Eur J Pharmaeol 1992; 218: 91-100. 176 Young R, Johnson DN. Eur J Pharmaeol 1991; 201: 151-155. 177 File SE, Andrews N. Eur J Pharmacol 1993; 237: 127-130. 178 Blackburn TP, Baxter GS, Kennett GA, et al. Psychopharmacol 1993; 110: 257-264. 179 Costall B, Kelly ME, Naylor RJ, Tomkins DM. Br J Pharmacol 1989; 96: 325332. 180 Sugita A, Shen KZ, North RA. Neuron 1992; 8: 199-203. 181 Lecrubier Y, Puech AJ, Bailey PE, Lataste X. Psychopharmaeol 1993; 112: 129-133. 182 Lader MH. In: Satel Syrup of 5th World Congr of Biol Psych, Florence 1991; 17-19. 183 McBain SL, Dineen M, Weller M, et al. Schizophr Res 1992; 6: 112. 184 Newcomer JW, Faustman WO, Zipursky RB, Czernansky JG. Arch Gen Psych 1992; 49: 751-752. 185 Sellers EM, Higgins GA, Sobell MB. Trends Pharmae Sci 1992; 13: 69-75. 186 Barnes JM, Costall B, Coughlan J, et al. Pharmacol Biochem Behav 1990; 35: 955-962. 187 Chigh Y, Saha N, Sankaranarayanan A, Sharma PL. Eur J Pharmacol 1991; 203: 121-123. 188 Jiik~il~iP, Sirv5 J, Riekkinen PJ. Gen Pharmacol 1993; 24: 675-679. 189 Crook TH, Lakin M. In: Satel Symp of 5th World Congre of Biol Psych, Florence 1991; 21-23. 190 Hall ST, Ceuppens PR. Psychopharmacol 1991; 104: 315-322. 191 Fozard JR. In: Bevan JA (ed) Proc 5th Int Syrup on Vasc Neuroeff Mech, Amsterdam: Elsevier 1985; p 321-328. 192 Saria A, Javorsky F, Humpel C, Gamse R. Ann NY Aead Sci 1991; 632: 464465. 193 Tramontana M, Guiliani S, Del Bianco E, et al. Br J Pharmacol 1993; 108: 431-435. 194 Loisy C, Beorchia S, Centonze V, Fozard JR, et al. Cephalagia 1985; 5: 79-82. 195 Couturier EGM, Hering R, Foster CA, Steiner TJ. Headache 1991; 31: 296297. 196 Rowat BMT, Merrill CF, Davies A, South V. Cephalagia 1991; 11: 207-213. 197 Angel I, Schoemaker H, Prouteau M, Garreau M, et al. Eur J Pharmaeol 1993; 232: 139-145. 198 Gorcs TJ, Liposit Z, Palay SFL, Chan-Palay V. Proc Natl Acad Sci 1985; 82: 7449-7452. 199 Bhandari P, Bingham S, Andrews PLR. Neuropharmacol 1992; 31: 735-742. 200 Smith WL, Callaham EM, Alphin RS. J Pharm Pharmacol 1988; 40: 142-143. 201 Higgins GA, Kilpatrick GJ, Bunce KT, Jones BJ, et al. Br J Pharmacol 1989; 97: 247-255.

256 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230

Alexander E, Siddon RL, Loeffier JS. Surg Neurol 1989; 32: 40-44. Monkovic I, Gylys JA. Prog Med Chem 1990; 27: 297-323. Dubois A, Fiala N, Boward CA, Bogo V. Radiat Res 1988; 115: 595-604. Andrews PLR, Hawthorn J. Neuropharmacol 1987; 26: 1367-1370. Hawthorn J, Ostler KJ, Andrews PLRQ. J Exper Physiol 1988; 73: 7-21. Reiser G. Eur J Biochem 1990; 189: 547-552. Edwards E, Ashby CR, Wang RY. Brain Res 1993; 617: 113-119. Hoyer D, Clarke DE, Fozard JR, Hartig PR, et al. Pharmacol Rev 1994; 46: 157-203. Boess FG, Martin IL. Neuropharmacol 1994; 33: 275-317. King FD. 5-Hydroxytryptamine-3-receptor antagonists; King FD, Jones BJ, Sanger GJ, eds. CRC: Boca Raton FLA 1994; 1-44. Jackson MB, Yakel JL. Ann Rev Physiol 1995; 57: 447-468. Villalon CM, Terron JA, Raminez-San-Juan E, Saxena PR. Arch Med Res 1995; 26: 331-344. Rault S, Lancelot JC, Prunier H, Robba M, et al. J Med Chem 1996; 39: 2068-2080. Clark RD, Muchowski JM, Weinhardt KK, Dillon MP, et al. Bioorg Med Chem Lettr 1995; 16: 1853-1856. Gerald C, Adham N, Kao HT, Olsen MA, et al. EMBO J 1995; 14: 2806-2815. Laguerre M, Dubost JP, Kummer E, Carpy A. Drug Design Disc 1994; 11: 205-222. Langlois M, Soulier JL, Rampillon V, Gallais C, et al. Eur J Med Chem 1994; 29: 925-940. Ohta M, Suzuki T, Ohmori J, Koide T, et al. Chem Pharm Bull 1996; 44: 1000-1008. Kato M, Nishino S, Ito K, Yamakuni H, et al. Chem Pharm Bull 1995; 43: 1358-1363. Anzini M, Cappelli A, Vomero S, Giorgi G, et al. J Med Chem 1995; 38: 26922704. L6pez-Rodrigues ML, Morcillo MJ, Benhamli B, Riaguas MD. Bioorg Med Chem Lett 1996; 6: 1195-1198. Akuzawa S, Miyata K, Fukutomi H. Eur J Pharmac 1995; 281: 37-42. Akuzawa S, Miyake A, Miyata K, Fukutomi H. Eur J Pharmac 1996; 296: 227-230. Bonhaus DW, Stefanich E, Loury DN, Hsu SA, et al. J Neurochem 1995; 65: 104-110. Werner P, Kawashima E, Reid J, Hussy N, et al. Mol Brain Res 1994; 26: 233-241. Downie DL, Hope AG, Lambert JJ, Peters JA, et al. Neuropharmac 1994; 33: 472-483. Boess FG, Beroukim R, Martin IL. J Neurochem 1995; 64: 1401-1405. Isenberg KE, Ukhun IA, Holstad SG, Jafri S, et al. Neuro-Report 1993; 5: 121-124. Belelli D, Balharek JM, Hope AG, Peters JA et al. Mol Pharmac 1995; 48: 1054-1062.

257 231 Miyake A, Mochizuki S, Takemoto Y, Akuzawa S. Mol Pharm 1995; 48: 407416. 232 Hope AG, Peters JA, Brown AM, Lambert JJ, et al. Brit J Pharmac 1996; 118: 1237-1245. 233 Peters JA, Lambert JJ, Malone HM. 5-Hydroxytryptamine-3 receptor antagonists, ed. King FD, Jones BJ, Sanger GJ. CRC: Boca Raton FLA 1994; 115-153. 234 Perren MJ, Roger H, Mason GS, Bull DR, et al. Naunyn Schiedeberg's Arch Pharmac 1995; 351: 221-228. 235 Bentley KR, Barnes NM. CNS Drugs 1995; 3: 363-392. 236 Roca J, Artaiz I, Del Rio J. Exp Opin Invest Drugs 1995; 4: 333-342. 237 Sorbe B. Exp Opin Invest Drugs 1996; 5: 389-407. 238 Wilde MI, Markham A. Drugs 1996; 52: 773-794. 239 Poncelet M, P~rio A, Simiand J, Gout G, et al. J Neural Transm 1995; 102: 83-90.

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


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 ( 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~.=(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!

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.


Chapter 10

5-HT TRANSPORTER



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

Serotonin Receptors and their Ligands

Serotonin Receptors and Their Ligands (Pharmacochemistry Library - Volume 27)

BLyS Ligands and Receptors

Negative Co-Receptors and Ligands

The Chemokine Factsbook: Ligands and Receptors

Serotonin Receptors in Neurobiology (Frontiers in Neuroscience)

Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors

The Serotonin Receptors. from Molecular Pharmacology to Human Theraputics

Death Receptors and Cognate Ligands in Cancer (Results and Problems in Cell Differentiation)

Privileged Chiral Ligands and Catalysts

The Chemokine Receptors (The Receptors)

Growth factors and their receptors in cancer metastasis

Proteases and Their Receptors in Inflammation (Progress in Inflammation Research)

The Cannabinoid Receptors (The Receptors)

The Dopamine Receptors (The Receptors)

Nucleotides and their Receptors in the Nervous System

The Cannabinoid Receptors (The Receptors)

The Chemokine Receptors (The Receptors)

The Melanocortin Receptors (The Receptors)

Ferrocenes Ligands Materials and Biomolecules

Prostacyclin and Its Receptors

Concert For Siren and Serotonin 3

PAF Receptors

Konzert für Sirenen und Serotonin

Reactions of Coordinated Ligands

Somatostatin Receptors

fMLP Receptors

Chiral Auxiliaries and Ligands in Asymmetric Synthesis

TNF Receptors

Activin Receptors

Serotonin Receptors and their Ligands