Perspectives in Receptor Research: Proceedings of the 10th Camerino-Noordwijkerhout Symposium, Camerino, Italy, 10-14 September 1996 (Pharmacochemistry Library)

PHARMACOCHEMISTRY LIBRARY- VOLUME 24 PERSPECTIVES IN RECEPTOR RESEARCH

PHARMACOCHEMISTRY LIBRARY- VOLUME 24 PERSPECTIVES IN RECEPTOR RESEARCH

PHARMACOCHEMISTRY LIBRARY, edited by H. Timmerman Other titles in this series Volume 10 QSAR in Drug Design and Toxicology, Proceedings of the Sixth European Symposium on Quantative Structure-Activity Relationships, Portoro~-Portorose (Yugoslavia), September 22-26, 1986 edited by D. Had~i and B. Jerman-Bla~_i~; Volume 11 Recent Advances in Receptor Chemistry. Proceedings of the Sixth CamerinoNoordwijkerhout Symposium, Camerino (Italy), September 6-10. 1987 edited by C. Melchiorre and Giannella Volume 12 Trends in Medicinal Chemistry '88. Proceedings of the Xth International Symposium on Medicinal Chemistry, Budapest, 15-19 August, 1988 edited by H. van der Groot, G. Domany, L. Pallos and H. Timmerman Volume 13 Trends in Drug Research. Proceedings of the Seventh Noordwijkerhout-Camerino Symposium, Noordwijkerhout (The Netherlands), 5-8 September, 1989 edited by V. Claassen Volume 14 Design of Anti-Aids Drugs edited by E. De Clerq Volume 15 Medicinal Chemistry of Steroids by F.J. Zeelen Volume 16 QSAR: Rational Approaches to the Design of Bioactive Compounds. Proceedings of the Eighth European Symposium on Quantitative Structure-Activity Relationships, Sorrento (Italy), 9-13 September, 1990 edited by C. Silipo and A. Vittoria Volume 17 Antilipidemic Drugs- Medicinal, Chemical and Biochemical Aspects edited by D.T. Witiak, H.A.I. Newman and D.R. Feller Volume 18 Trends in Receptor Research. Proceedings of the Eighth Camerino-Noordwijkerhout Symposium, Camerino (Italy), September 8-12, 1991 edited by P. Angeli, U. Giulini and W. Quaglia 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-Angiotesin System edited by P.B.M.W.M. Timmermans and R.R. Wexler Volume 22 The Chemistry and Pharmacology of Taxol| and its Derivatives edited by V. Farina Volume 23 Qsarand Drug Design: New Developments and Applications edited by T. Fujita

PHARMACOCHEMISTRY

LIBRARY

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

Volume

24

PERSPECTIVES IN RECEPTOR RESEARCH Proceedings of the 10th Camerino-Noordwijkerhout Symposium, Camerino, Italy, 10-14 September 1995 Edited by"

DARIO GIARDIN~,

ALESSANDRO PIERGENTILI MARIA PIGINI Department of Chemical Sciences University of Camerino Via S. Agostino 1 62032 Camerino (MC), Italy

ELSEVIER Amsterdam - Lausanne - New York-

O x f o r d - S h a n n o n - T o k y o 1996

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

ISBN 0-444-82204-6 91996 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 ADVISORY BOARD r. Fujita

Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan

E. Mutschler

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

This Page Intentionally Left Blank

~

Vll

PREFACE This proceedings book contains the invited lectures delivered at the 10th CamerinoNoordwijkerhout Symposium, held in Camerino from the 10th to the 14th September 1995. In 1987, the Italian and Dutch scientific groups decided to join forces in organizing meetings, that were to become known as the Camerino-Noordwijkerhout Symposia, on specific aspects of Medicinal Chemistry. Since the beginning, in 1978, a particular feature of the Camerino Symposia has been that of reporting and debating the most recent knowledge and discoveries of chemists, pharmacologists and biologists involved in studying receptors and their mechanism of action. In this 1995 edition, too, an extremely intense exchange of information and opinions took place during the five days of the Symposium, when 27 invited lectures (16 plenary and 1 lmain ones), 8 oral communications, and 90 posters were presented and discussed in depth. Since that first 1978 meeting, Camerino has offered hospitality to 950 scientists, more and more of them intemational, as the proportion of foreign participants has risen from 12% to 60%. In the 1987 and 1991 editions two Nobel prize winners, Sir John Vane and Sir James Black, respectively, gave the opening lectures of the Symposia, and on three occasions, in 1987, 1991, and 1995, Honofis Causa degrees were conferred on Drs B. Belleau, E.J. Miens, and D.J. Tfiggle, respectively. During these 17 years, the existence of distinct receptofial subpopulations has been confirmed, something hypothesized at the 1978 Camerino Symposium by N.J.M. Birdsall, who during his paper "Muscarinic receptors: biochemical and binding studies" by himself, A.S.V. Burgen and E.C. Hulme, when analyzing data from their experiments, affu-med:

"These results are precisely the behaviour predicted for a heterogeneous population of binding sites" In addition, that first Symposium saw the setting up of a work relation between Nigel Birdsall and Rudolf Hammer that was subsequently to lead to the discovery of pirenzepine as a selective ligand for M1 muscarinic receptor subtype. As organizers of the 10th Symposium, we feel extremely gratified by how the exchange of opinion between the various lines of research in our meetings has opened the road to a deeper knowledge of the receptor as a biological target.

viii In conclusion, we hope that other meetings will continue to be held in Camerino, with the same high quality of scientific content and the same high values of friendly human relationships that have characterized all the past ones. The encouraging results obtained so far will thereby be carried even further forward.

Dario Giardin~t Alessandro Piergentili Maria Pigini

ix CONTENTS Giardin~t D, Piergentili A, Pigini M

Preface Triggle DJ Medicinal chemistry: through a glass darkly Klotz K-N, Jesaitis AJ, Lohse MJ

Mechanisms regulating G protein-coupled receptors

11

Shapiro G

Introduction to muscarinic receptors

27

Wess J, Blin N, Yun J, Sch6neberg T, Liu J

Structure-function analysis of muscarinic acetylcholine receptors

31

Mutschler E, Ensinger HA, Gross J, Leis A, Mendla K, Moser U, Pfaff O, Reichel D, Rtihlmann K, Tacke R, Waelbroeck M, Wehrle J, Lambrecht G

Muscarinic receptor subtypes - Search for selective agonists and antagonists

51

Casagrande C, Bertolini G

Perspectives in the design and application of dopamine receptor agonists

67

Branchek TA

Serotonin receptor complexity: relationships and roles

85

Becker DP, Goldstin B, Gullikson GW, Loeffler R, Moormann A, Moummi C, Nosal R, Spangler D, Villamil CI, Yang D-C, Zabrowski DL, Flynn DL

Design and synthesis of agonists and antagonists of the serotonin 5-HT4 receptor subtype Theroux TL, Esbenshade TA, Minneman KP oti-Adrenergic receptor subtypes and signal transduction

99

121

Leonardi A, Testa R, Motta G, De Benedetti PG, Hieble P, Giardinh D O~l-Adrenoceptors: subtype- and organ-selectivity of different agents

135

Jacobson KA, van Rhee AM, Siddiqi SM, Ji X-d, Jiang Q, Kim J, Kim HO

Molecular recognition in adenosine receptors

153

Cristalli G, Camaioni E, Di Francesco E, Vittori S, Volpini R

Chemical and pharmacological profile of selective adenosine receptor agonists

165

IJzerman AP, van der Wenden EM, Roelen HCPF, Math6t RAA, von Frijtag Drabbe Ktinzel JK

Partial agonists for adenosine receptors

181

Leurs R, Timmerman H

New histamine 1-13receptor ligands as pharmacological tools

193

Hibert M, Hoflack J, Trumpp-Kallmeyer S, Paquet J-L, Leppik R, Mouillac B, Chini B, Barberis C, Jard S

Three-dimensional structure of G protein-coupled receptors: from speculations to facts

205

Triggle DJ

Ion channels as targets for drug design

215

Evans JM

Recent advances in potassium channel activators

227

Lodge D, Bond A

Spinal glutamate receptors

241

Froestl W, Mickel SJ, Mondadori C, Olpe H-R, Pozza MF, Waldmeier PC, Bittiger H

GABAB receptor antagonists: new tools and potential new drugs

253

xi DiMaio J, Winocour P, Leblond L, Saifeddine M, Laniyonu A, Hollenberg MD Thrombin inhibitors and thrombin receptor agonists/antagonists

271

Rees DC Structure-activity relationships of non-peptide kappa-opioid analgesics:

a perspective of the last 10 years

291

Portoghese PS Selective nonpeptide ligands as probes to explore ~ opioid receptor

architecture

303

de Costa B R, He X-s, Dominguez C, Williams W, Rice KC, Bowen WD

The role of novel ligands in the biological characterization of sigma receptors

313

Melchiorre C, Angeli P, Bolognesi ML, Budriesi R, Cacciaguerra S, Chiarini A, Crucianelli M, Giardin~ D, Gulini U, Marucci G, Minarini A, Spampinato S, Tumiatti V

Tetraamines as lead compounds for the design of neurotransmitter receptor ligands: focus on o~-adrenergic and muscarinic receptors recognition

321

Lambrecht G, Ardanuy U, B~iumert HG, Bo X, Hoyle CHV, Nickel P, Pfaff O, Ralevic V, Windscheif U, Ziganshin AU, Ziyal R, Mutschler E, Burnstock G

Design and pharmacological characterization of selective Pepurinoceptor antagonists

337

Feldman J, Dontenwill M, Greney H, Bennai F, Bousquet P

Imidazoline receptors: an update

351

Brasili L, Pigini M, Bousquet P, Carotti A, Dontenwill M, Giannella M, Moriconi R, Piergentili A, Quaglia w, Tayebati SK

Discovery of highly selective imidazoline receptor ligands

361

xii Gaviraghi G, Cassar~ P, Corsi M, Curotto G, Donati D, Feriani A, Finch H, Finizia G, Pentassuglia G, Polinelli S, Ratti E, Reggiani A, Tarzia G, Tedesco G, Tranquillini ME, Trist DG, Ursini A

Synthesis and structure-activity relationship of new 1,5-benzodiazepine CCK-B antagonists

375

Author index

389

Subject index

393

Perspective in Receptor Research D. Giardinh, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved. MEDICINAL

CHEMISTRY:

THROUGH

A GLASS

DARKLY

David J. Triggle, State University of New York, Buffalo, New York, USA INTRODUCTION

0 brave new world, That has such people in't! William Shakespeare,

The Tempest.

The patenting of a mouse predisposed to cancer and genetically engineered by Philip Leder and Timothy Stewart at Harvard University in April 1988 generated important scientific, commercial and ethical questions. Some five years later a selfimposed moratorium has ended and the United States Patent and Trademark Office is again issuing patents to a variety of genetically engineered animals, including those that exhibit prostate enlargement, develop insulin-resistant diabetes and produce human proteins including antibodies ( i ) . There is little question but that these transgenic species will accelerate progress towards treatment of a variety of human disorders. Similarly, the production of transgenic plants, whilst generating less immediate ethical attention, will prove of value to the generation of medicinal products of natural origin such as scopolamine through the introduction of the hyoscyamine hydroxylase gene into Atropa belladonna (2). During the same time period there has been a similar increase in human gene therapy (3). Some thirty-seven gene transfer trials have been approved worldwide and eighteen of these are designed to produce immediate therapeutic benefits. These include several trials inserting the gene for adenosine deaminase to alleviate the absence of a natural immune system and interleukin genes for several cancer types (4,5). With continued progress in overcoming the major problems of gene therapy including low gene transfer efficiency, poor cell targeting and premature gene turnoff it is clear that the horizons for human gene therapy will continue to expand. The potential benefits of alleviating dopamine deficiency in the brains of Parkinson's patients by tissue implantation have been widely discussed from both clinical and ethical perspectives. Conventional dopamine replacement therapy suffers from well known and serious limitations. The most recent data indicate that fetal-tissue transplants into patients with idiopathic or MPTP-induced Parkinson's disease are clinically encouraging and may be highly beneficial for at least some patients (6,7). Do these and similar developments in gene and tissue therapy signal the end of medicinal chemistry and the arrival at a solely biotechnology-oriented therapy mall where patients shop for both designer jeans and genes? Or, and more plausibly perhaps, will the molecular and medical sciences continue to adjust to a clinical interface that is positioned by science, economics and ethics?. It is clear that the issues of ethics raised by our rapid approach to the human genome will be of increasing importance to defining new clinical interfaces (8).

THE

HXSTORY

heavenly messengers bringing evil reports of armies on the move and time r u n n i n g short and famines and earthquakes and train wrecks and the tearing down of the wall bob dylan

The search for power and the explanation of natural events led naturally to the attribution of specific properties to plants and to their extracts. These properties were used in murder, magic and medicine, processes inextricably linked in man's history (9). Predictive, perhaps, of contemporary issues drugs were used also for their recreational properties; r e c e n t studies of Egyptian mummies have revealed the presence of cocaine, nicotine and hashish (I0). Frequently, specific features of the plant were associated with and predictive of these properties. Thus the mandrake [ Mandragora o f f i c i n a r u m ] was associated with the human form and special techniques were believed to be necessary in the collection of the plant to avoid madness or death. Those hominids partaking of Aminata m u s c a r i a would have been exposed to both the toxic and h a l l u c i n o g e n i c effects of the muscarine, muscimol and ibotenic acid c o n t a i n e d in this fungus. Aminata may have been the source of the soma of Vedic India and the inspiration for the soma of Aldous H u x l e y ' s Brave New World which had "all of the advantages of C h r i s t i a n i t y and alcohol; none of their defects". One of the plagues of Egypt may well have been caused by the proliferation of d i n o f l a g e l l a t e s causing a red tide, fish killing and water p o i s o n i n g (II). The history of hashish dates back to at least 2000 BC and is associated with a considerable folk-lore. The word "assassin" derives from the Arabic for "hashish-eater" and the i n t r o d u c t i o n of hashish to Europe by Marco Polo initiated investigations that have culminated in the identification of a specific neural receptor for the active constituent c a n n a b i n o i d s of m a r i j u a n a and in the isolation of an endogenous ligand, anandamide, for this receptor (12). The concept of specific receptors and sites of drug action has many origins. However, the work of Thomas Fraser and A l e x a n d e r Crum Brown of Edinburgh University described in their p u b l i c a t i o n of 1869, "On The Connection Between Chemical C o n s t i t u t i o n and Physiological Action", provides both a d e f i n i t i o n of structure-activity relationships and a p i o n e e r i n g example of chemist-pharmacologist collaboration (13). The definition of receptors as specific sites for drug action owes much to the work of John Newton Langley [ 18521925 ] and Paul Ehrlich [ 1854-1915 ]. Their separate w o r k on the autonomic nervous system and toxins and c h e m o t h e r a p e u t i c agents led to the concept of a receptor that possesses both r e c o g n i t i o n and transduction components and of c h e m o t h e r a p e u t i c m o l e c u l e s possessing discrete molecular features s u b s e r v i n g specific functions:

"It is convenient to have a term for the specially excitable constituent and I have called it the r e c e p t i v e substance. It r e c e i v e s the stimulus [ recognition ] and by transmitting it [ transduction ] causes contraction [ response ]" John Newton Langley, 1906

"---they must p o s s e s s a certain definite grouping which is c h e m i c a l l y a l l i e d to one of the c h e m o r e c e p t o r s of the p a r a s i t e . - - - T h e r e must, therefore, be in addition to the f i x i n g g r o u p a n o t h e r which brings about the d e s t r u c t i o n -is to be c h a r a c t e r i z e d as the toxophoric" Paul Ehrlich, 1913

These predictions have been admirably fulfilled by the isolation and characterization of receptors themselves and by the assignment within receptors and individual molecules of domains responsible for such specific functions; these include ligand binding, voltage sensitivity and effector coupling in ion channels and detection, initiation and delivery in the DNA cleaving enediynes (14,15). Although he died during World War 1 Ehrlich was an optimist and writing some two years before his death: "Now that the l i a b i l i t y to, and danger of, disease are to a large extent circumscribed--the effects of c h e m o t h e r a p e u t i c s are directed as far as possible to fill up the gaps left in this ring".

Ehrlich had not, however, contemplated drug resistance, including the recent appearance of drug-resistant tuberculosis (16,17), nor presumably had he contemplated the dual impact of mass migrations and habitat destruction on the emergence of new and lethal infections including Lassa rift valley, Ross River, Crimean-Congo and HI viruses (18). THE P R E S E N T "The a c h i e v e m e n t s of chemical synthesis are firmly b o u n d to our a t t e m p t to break the shackles of disease and poverty" Roald Hoffmann, 1993.

The emergence of both old and new diseases focuses continuing efforts on drug discovery and the central role of medicinal chemistry. These foci are switching increasingly from the empirical and intuitive discovery processes to structureand mechanism-based discovery routes. These changes are coupled to the emergence of new targets and roles for medicinal chemistry that are increasingly at an interface with molecular biology. New cellular messengers with unconventional properties continue to define new targets. Nitric oxide, or a labile derived species, is now established to mediate physiological and pathological events in cardiovascular, secretory and neuronal cells (19-21). The recent postulate that CO may also be a messenger species (22,23) serving, with nitric oxide, a common guanylyl cyclase effector defines new signalling pathways that link space and time to coordinate the activities and functions of cell populations (24). A major role for such species can be envisaged in the regulation of the brain and its multiple neuronal populations. The modulation of the activities of such novel species presents both major opportunities and major problems of site specificity and delivery. The critical role of calcium as a cellular regulator has been recognized for over a century (25). Cyclic adenosine

diphosphate ribose [ cADP-ribose ] is the latest messenger serving to modulate the release of intracellular Ca ~T and perhaps representing the endogenous ligand for the ryanodinesensitive Ca 2+ release channel (26,27). Not only is this m e s s e n g e r an important link between nutrient levels and insulin release in pancreatic beta-cells, b ~ it may serve quite generally to link nutrients with Ca fluxes. The availability of both cADP ribwse and inositol phosphate pathways to control intracellular Ca ~T relwase provides a parallel to the multiple entry processes for Ca ~T during cell excitation and provides an additional base for the spatial and temporal control of this messenger (28). Additionally, this discovery raises again the issue of endogenous ligands for other types of Ca 2+ channels (29). New targets demand new ligands and more subtle definition of the ligand-target interactions. Target definition by X-ray and NMR as structure-based drug design promises to become a predictive component of the discovery process (30). The design of two-fold symmetric inhibitors of HIV protease followed directly from the discovery that the retroviral enzymes are heterodimers (31-33). Other examples are likely to follow for other soluble proteins. Membrane-bound proteins constitute, however, particular problems for both X-ray and N M R m e t h o d s , but the high resolution electron microscopic techniques applied to two-dimensional membrane sheets including bacteriorhodopsin and porin may well be useful for other systems (34,35). The d e t e r m i n a t i o n of the structure of the human growth hormone complexed with the soluble extracellular domain of its receptor suggests an alternative strategy for membrane proteins (36). Additionally, extension to ligand interactions with other m a c r o m o l e c u l e s including DNA as in the glucocorticoid-DNA complex is occurring (37). Gene isolation has been a major force in the characterization, expression and screening of pharmacological receptors. Two lessons learned are that pharmacological specificity may depend upon a single amino acid residue and that for receptors composed of subunits only a limited number of permutations may exist. The antagonist specificity of the rodent 5-HT.IB receptor is determined by a single residue [ asparaglne-355 ] from that of a human brain receptor [ threonine-355 ] with overall sequence homology, although both receptors bind 5-HT equally well (38). The benzodiazepinesensitive GABA A receptor, composed of multiple alpha, beta, gamma and delta subunits derived from some 15 genes and with several hundred thousand possible permutations, actually exists in very restricted distribution and combinations (39,40). The generation of lead structures and their optimization constitutes a central theme of medicinal chemistry. This theme places continuing demands on synthetic chemistry including the specification of new catalytic processes and consideration of chiral interactions (41,42). Increasingly, these demands will interface with biological processes including catalytic antibodies for disfavored chemical reactions (43) and chiral control through enzymatic and biomimetic processes (41). The mimicking of the biological world by chemistry has become extremely productive. The award of the 1987 Nobel Prize in Chemistry to Cram, Lehn and Pedersen was an explicit acknowledgment of the role of molecular recognition processes and of the analogies between host-guest chemistry and ligandreceptor interactions (45,46). Thus, a simple A4B6

cyclooligomer of trimesic acid and R,R-diaminocyclohexane exhibits very high chirality and side-chain specificity in peptide recognition (47). In turn, chemistry is expanding the synthetic capabilities of biological systems. The "natural" biological code specifies only 20 amino acids. Expansion of this code is possible through the creation of new codonanticodon pairs that specify novel residues (48). The nonstandard pair (iso-C)AG:(iso-dG)CU specifies L-iodotyrosine and indicates the possibilities of a semi-synthetic genetic code. A general method for incorporation of unnatural amino acids into proteins derives from replacement of a specific codon by the "blank" nonsense codon TAG through oligonucleotide-directed mutagenesis and use of a suppressor tRNA directed against this codon that has been aminoacylated by the amino acid residue of interest (49). Although ligand design may ultimately be completely rational and predictable from target structures, iterative processes of design and selection will continue to be of major significance despite its traditional heavy consumption of time and money. It is of increasing importance to devise methods that facilitate lead generation and selection A variety of combinatorial techniques are now available that generate libraries of compounds from amino acids or nucleotides. These range from extensions of peptide resin synthesis to the use of fusion phage which express on their surface a single peptide encoded by a randomly mutated region of the phage genome (5054). Affinity-mediated selection followed by physical or sequence-based localization permits the characterization of a lead structure. A combination of these processes entitled "encoded combinatorial chemistry" has been proposed in a theoretical treatment by Brenner and Lerner to consist of parallel syntheses where each monomeric chemical unit is encoded by a nucleotide sequence (55). Active molecules are affinityselected and identity and amplification achieved through the PCR applied to the retrogenetic tag. In principle, this method is not restricted to amino acid or nucleotide precursors and any set of building blocks could be employable. Such molecular construction kits are now becoming available and some are termed, appropriately enough, "Molecular Meccano" (56-58). A final component to the process of ligand selection may be accomplished by closing the circle of variation/mutation, selection and amplification/replication to provide evolutiondirected ligand selection (59-61). THE FUTURE

give me back my broken night my secret room my secret life its lonely here leonard cohen

Advances in synthetic methodologies notwithstanding, the future of medicinal chemistry will make increasing demands on chiral control, on peptidomimetics and on target- and sitespecific delivery. Exploration of specific cellular receptors as surrogate entry loci for peptides and proteins offers significant opportunity for target-specific delivery particularly if combined with mechanisms to selectively enhance the expression

of delivery receptors. Antibodies to the transferrin receptors can cross the blood-brain barrier by internalization through these receptors localized in brain capillary endothelial cells. Antibodies conjugated to nerve growth factor deliver the latter to neurons and enhance the survival of cholinergic and noncholinergic neurons transplanted into the eye (62). Similar techniques may be used to accomplish receptormediated gene delivery (63,64). Gene transfer technology is exploitable to create both target- and site-specific delivery systems. Retroviral vectors may be particularly and selectively useful because of their ability to transfer genes only into dividing cells. Thus, murine fibroblasts expressing a retroviral vector containing the herpes simplex thymidine kinase gene transduced proliferating rat glioma cells. Addition of the anti-herpes drug ganciclovir regressed the gliomas since these cells only expressed the herpes enzyme (65). The goal of peptide mimicry is the discovery of structural leads, or the development of strategies for molecular design, which result in bioavailable peptide analogs (66). The development of a strategy is awaited and most active compounds derive from lead identification and optimization. However, "retrofitting" of such leads as the benzodiazepine nucleus found in ligands for opiate, CCK, gastrin and glutamate receptors (7671) and the 1,4-dihydropyridine structure as a component of drugs active at several voltage-gated ion channels (72) is likely to be productive. The importance of chirality to the drug-receptor interaction has been recognized since at least the work of Cushney (73). An increasing need to consider chirality as a theme in all aspects of drug discovery from synthesis and manufacture to pharmacodynamics, pharmacokinetics and toxicology derives from the new FDA guidelines (74-77). Indeed, since 1985 almost 50% of the optically active molecules developed as drugs have been developed as active isomers and there are probably some 50-60 currently marketed racemates that are prime candidates for chiral switching (76,78). Fortunately, perhaps, for the pharmaceutical industry m o l ~ u l e s such as palytoxin with 64 dissymmetric carbon atoms and i0 isomers are rare (79). However, even simple chiral molecules may present complex pharmacology because of opposing chiral effects of pharmacokinetic and pharmacodynamic factors. This may be particularly so for drugs active at ion channels where statedependent interactions may modify both quantitatively and qualitatively the expression of drug action (80,81) Thus the stereoselectivity of local anesthetic interactions at Na + ' channels varies according to the frequency of stimulus and the activation mode (82,83). At the synthetic level we may expect increased emphasis on enantioselective catalysis using metal complex catalysts as a key component of the manufacturing process (84). For biocatalysts there will unquestionably continue to be increasing interest in the "custom synthesis" of enzymes engineered for specific functions and conditions. The first example of the "ultimate" enzyme has been reported with the synthesis of the all-D form of HIV-I protease (85-87). This enzyme exhibits a chiral specificity opposite to that of the naturally occurring L form and it may be generally predicted that enantiomeric proteins will exhibit reciprocal chiral specificity in all aspects of their interactions. These reciprocal chiral

s p e c i f i c i t i e s of D - a n d L - p r o t e i n s are of c o n s i d e r a b l e interest; they s u g g e s t t h a t in worlds a l t e r n a t i v e to ours their FDA will be the m l r r o r image of ours. On the other hand, .......... ACKNOWLEDGMENTS.

This w o r k was p r e s e n t e d in part as a l e c t u r e d e l i v e r e d to The XII I n t e r n a t i o n a l C o n g r e s s on M e d i c i n a l C h e m i s t r y in Basle, S e p t e m b e r 1992. It was o r i g i n a l l y p u b l i s h e d in " A n n u a l R e p o r t s In M e d i c i n a l Chemistry", V o l u m e 28, 1993 and is r e p r i n t e d h e r e in its e n t i r e t y and w i t h o u t change w i t h the kind p e r m i s s i o n of A c a d e m i c Press Inc, Orlando, Florida.

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Perspective in Receptor Research D. Giardina, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

11

Mechanisms regulating G protein-coupled receptors K.-N. Klotz 1, A.J. Jesaitis 2 and M.J. Lohse I l Institut fCir Pharmakologie und Toxikologie der Universit/it W~irzburg, Versbacher Str. 9, D-97078 W/irzburg, Germany 2Department of Microbiology, Montana State University, Bozeman, MT 59717, U.S.A. INTRODUCFION Receptor-mediated signal transduction plays a pivotal role in cell communication and is essential for a coordinate functioning of the organs of our body. Several classes of receptors can be distinguished on the basis of functional and structural differences. The still growing family of G protein-coupled receptors has been investigted in great detail and a wealth of structural and functional information is available for many members of this family. It has long been recognized that receptor-mediated signal transduction is a highly regulated process but only recently have we begun to understand some of the regulatory events that modulate signal intensity resulting from a given stimulus. In particular different mechanisms of desensitization for short-term as well as for long-term modulation of receptor function have been investigated in great detail (for review see [11). Several receptors including the ~-adrenergic receptor, rhodopsin, muscarinic receptors and formylpeptide receptors served as prototypes to study various forms of desensitization [2-5]. In this overview we will focus on the growing number of components of the [3-adrenergic receptor system that are implicated in modulation of signal transduction. In addition, we will present a mechanism for desensitization of N-formylmethionyl peptide receptor (FPR) which might be specific for regulation of chemoattractant receptors. This mechanism might serve as a model for the aspect of spatial organization of signal transduction that appears to be required for an effective interplay of different partners in this multi-component system.

COMPONENTS OF THE ~-ADRENERGICRECEPTORSYSTEM The ~-adrenergic receptor system comprises the main membrane-bound components of transmembrane signaling, which are the receptors themselves, the G-proteins G s, and adenylyl cyclases. In addition, the receptor system contains several cytosolic proteins which regulate the signaling process (Figure 1). The most important ones of these are the [~-adrenergic receptor kinases and the

12 ~-arrestins, which inhibit receptor/G-coupling, and phosducin, which has been proposed to function as an inhibitor of G-protein activation. Protein kinase A is the effector kinase of the system which modulates cellular functions by phosphorylating key proteins of multiple cellular processes. In addition, it has feedback functions at the level of receptor/G-coupling and of phosducin/G-coupling. COMPONENTS OF THE B-ADRENERGIC RECEPTOR SYSTEM agonist

0~S ::

Figure 1. Components of the s receptor system. The abbreviations denote: ~AR, ~-adrenergic receptor; ~ARK, ~-adrenergic receptor kinase; AC, adenylyl cyclase; PKA, protein kinase A; PhD, phosdudn. For all of these components, several isoforms have been described. The potential specificities of all of these isoforms are as yet ill-defined and do not appear to play a major in the principles of the regulatory mechanisms discussed here. The function of these cytosolic components of the ~-adrenergic receptor system is the tuning of the signaling process. Signalling via ~ - a d r e n e r g i c receptors is subject to a variety of regulatory mechanisms that can affect both the number and the function of these receptors. Most importantly, repeated or prolonged stimulation of the receptors results in a reduction of responsiveness, which is called agonist-induced receptor desensitization (reviewed in [1, 2]). Receptor desensitization involves several molecular mechanisms, which may be operative to varying extents in different cells and different receptor systems. These desensitization mechanisms can be broadly classified into those that alter receptor function and those that affect receptor expression.

13 Receptor expression is regulated over many hours, and changes are usually not visible before =15 min. Agonist-induced changes in receptor expression involve the degradation of the receptor protein as well as reduced receptor synthesis due to reductions in its mRNA levels. In contrast, receptor function can be altered within seconds. It occurs primarily by uncoupling receptors from their G-protein, G s.

DESENSITIZATION ~-ARRESTINS

BY ~-ADRENERGIC

RECEPTOR KINASES

AND

The most specific and rapid mechanism desensitizing [3-adrenergic receptors is triggered via their phosphorylation by [3-adrenergic receptor kinases ([3ARK). This process is initiated by agonist-activation of the receptors and proceeds in two steps: first, the receptors are phosphorylated by [3ARK in an agonist-dependent manner [6, 7]; subsequently, the [3-arrestins bind to the phosphorylated receptors, resulting in inhibition of receptor/Gs-coupling [8-10]. Depending on the cell type this mechanism occurs within seconds after agonist exposure and is complete in less than a minute [11]. The cDNAs of six ~ARK-like kinases, which are more generally called G protein-coupled receptor kinases, have been cloned (reviewed in [12]). However, only two have been shown to be involved in the regulation of ~-adrenergic receptor function, even though all six appear to be capable of phosphorylating the receptors in vitro. These two are termed [3-adrenergic receptor kinase-1 and 2. Both isoforms of [3ARK are cytosolic proteins which need to be translocated to the cell membrane in order to phosphorylate the receptors. This translocation appears to occur via three "membrane anchors": First, the kinases require the agonist-occupied, active conformation of the receptors to which they bind [13]. Second, the kinases bind to the G protein [3y subunits which may, in addition to the translocation, result in activation of the kinases [14, 15]. This process appears to be supported by specific combinations of [3T subunits much better than by others [16]. And third, recent data indicate that the kinase binds to phosphatidylinositol bisphosphate (PIP2) via a C-terminal pleckstrin homology domain [17]. At least the first two modes of membrane anchoring and activation appear to be involved in the strictly agonist-dependent action of [3ARK on [3-adrenergic receptors. Phosphorylation of receptors by [3ARK increases their affinity for the [3-arrestins. The [3-arrestin isoforms 1 and 2 are members of the larger family of arrestin proteins (reviewed in [18]). In contrast to the other arrestins which are localized essentially in the retina, [3-arrestin-1 and 2 are widely distributed and are p r o b a b l y most i m p o r t a n t for desensitization of hormone and

14 neurotransmitter receptors. Binding of these proteins to phosphorylated receptors blocks the interaction between receptors and G proteins [8, 9, 19]. DESENSITIZATION BY PROTEIN KINASES A AND C In addition to phosphorylation by ~ARK, ~-adrenergic receptors can also be phosphorylated by protein kinases A and C. While the phosphorylation by ~ARK occurs on multiple sites of the extended C-terminus of these receptors, these kinases phosphorylate two consensus sites which are located close to the membrane in the third cytoplasmic loop of the receptors and in the C-terminus. This phosphorylation results in direct impairment of receptor-Gs-coupling [20, 21]. However, it appears that this type of receptor uncoupling occurs more slowly and is of smaller extent than the [3ARK/~-arrestin-mediated p a t h w a y [11, 22]. Only GRK-mediated phosphorylation of receptors causes enhanced affinity for arrestins. In contrast, phosphorylation of ~2-adrenergic receptors by PKA or PKC has no effect on the affinity or activity of arrestins [15]. REGULATION OF G-PROTEIN FUNCTION In addition to the modulation of signaling at the level of the receptors signal transduction appears also to be regulated at the next level, the G proteins. Again, regulation can occur either via alterations in the function of a G protein or by alterations in its number. Alterations in the number of G proteins have been described mostly for the G proteins regulating adenylyl cyclase, G s and G i. In these cases there appears to be pronounced 'cross-regulation'. Activation of the Gs-pathway leads to enhanced expression of Gi, and activation of the Gi-pathway causes reduced expression of G i [23, 24]. Such regulation occurs not only in isolated cells, but has also been observed in intact animals and in humans. A well-documented pathophysiological example of such regulation is the increased expression of Gia_2 in heart failure [25]. More recently it has become clear that also the function of G proteins can be altered. There are a small number of proteins that have been found to associate with G proteins and to affect their function. These proteins include the growth cone associated protein GAP-43, which has been found to enhance GTP binding by G o in a manner similar to receptors [26], and a complex between the small GTP-binding protein ras p21 and its GTPase activating protein (ras-GAP) which impair coupling of muscarinic receptors to potassium channels [27]. A regulatory protein which can inhibit signaling via multiple G proteins including G s is the cytosolic 33kDa protein phosducin [28]. Phosducin had earlier been believed to be expressed only in the retina and in the developmentally

15 related pineal gland [29, 30], but its mRNA has more recently been found in many other tissues [28]. Phosducin interacts preferentially with the ~, subunits of G proteins, but probably also with their 0~-subunits [28, 31, 32]. This interaction with the G proteins results in inhibition of the activation of G s, but also of G o, Gi and transducin. This inhibition of G protein activation causes a disruption of signaling from [~-adrenergic receptors to adenylyl cyclase [28]. Protein kinase A can phosphorylate phosducin [33]. This phosphorylation markedly inhibits the effects of phosducin on G proteins [28, 34]. Since phosducin inhibits the function of G s, and protein kinase A prevents this inhibition, G s, adenylyl cyclase, protein kinase A and phosducin form a positive feed-back loop. The biological implication of such positive feed-back are as yet not understood. Phosducin and [3ARK both bind to the ~, subunits of G proteins. Because of its very high affinity for the [3~,subunits phosducin can compete very effectively with ~ARK and thereby prevent its translocation to the plasma membrane [35]. As a consequence, phosducin can inhibit ~ARK-mediated phosphorylation and desensitization of ~-adrenergic receptors. Again, this inhibition is relieved following phosphorylation of phosducin by protein kinase A. This indicates that the cytosolic components of the ~-adrenergic receptor system interact with each other in multiple ways. Together, they form a complex network which regulates the signaling function of the receptor system. N-FORMYLMETHIONYL PEPTIDE RECEPTORS AND MEMBRANE DOMAINS N-formylmethionyl peptide receptors (FPR) are chemoattractant receptors on phagocytic cells like human neutrophils. Stimulation of FPR with N-formylmethionyl peptides induces a variety of responses of these cells including chemotaxis, superoxide production, release of hydrolytic enzymes and reorganization of cytoskeletal structures [36, 37]. Most of these responses appear to be mediated through activation of phospholipase C, however, participation of other effector enzymes such as adenylyl cyclase, phospholipase D and phospholipase A 2 have also been demonstrated [38]. In analogy to other receptor systems desensitization of FPR was observed. N-formylmethionyl peptides induce a transient burst of superoxide production in human neutrophils with a maximum after 1-2 min. In the past, several observations suggested that desensitization of FPR may involve the cytoskeleton [39]: (1) The microfilament-disrupting agent cytochalasin B substantially increased the rate and duration of the superoxide production induced by formyl peptides [40]. (2) FPR in desensitized neutrophils accumulated in a Triton X-100-insoluble fraction of the cells [40, 41]. (3) Plasma membrane fractionation revealed that after desensitization FPR appear to be redistributed to a membrane fraction enriched

16 in cytoskeletal proteins like actin and fodrin that was depleted from G proteins [42]. Two membrane fractions can be distinguished in neutrophil plasma membrane preparations based on different densities. The fraction with the higher density (PM-H) contains cytoskeletal proteins whereas the fraction with the lower density (PM-L) is enriched in G proteins. These membrane fractions are thought to correspond to plasma membrane domains with the respective specific distribution of proteins associated with the inner face of the membrane. In the two different membrane domains FPR were expected to show different coupling to G proteins. Solubilized FPR prepared from the PM-L fraction of neutrophils sedimented in a detergent-containing sucrose density gradient as 7 S particles while FPR prepared from PM-H sedimented with an apparent sedimentation coefficient of 4 S. The 7 S form was shifted to the 4 S form in a GTP~/S-sensitive manner indicating that the 7 S form represents receptorG protein complexes.

RESPONSIVE

PM-L

DESENSITIZED

G proteins

G proteins FPR

PM-H

Cytoskeletal proteins

] I I I FPR

Cytoskeletal proteins FPR

Figure 2. Domains and protein distribution in responsive and desensitized neutrophil plasma membranes. For details see text. These data led to the proposal of a model for desensitization based upon lateral segregation of signaling proteins in the plane of the plasma membrane. FPR in responsive cells are able to transduce a signal in the PM-L compartment that is enriched in G proteins. Activation of receptors would then allow for interaction with their signal transduction partners. In desensitized neutrophils this interaction is thought to be blocked because FPR are laterally segregated into the PM-H domain where only low levels of G protein are observed (Figure 2).

17 Recently, an agonist-induced redistribution of FPR in h u m a n neutrophils consistent with the lateral segregation model was d e m o n s t r a t e d by direct m e a s u r e m e n t of receptor lateral mobility with fluorescence recovery after photobleaching [43]. FPR-COUPLING TO THE MEMBRANE SKELETON

The lateral segregation mechanism of FPR desensitization in neutrophils (Figure 2) may be an attractive model that integrates the receptor-mediated actin polymerization/depolymerization and feed-back regulation of receptor function. What do we know about FPR binding to cytoskeletal proteins and membrane disribution of FPR as a function of the activation state of neutrophils to support such an idea? The initial observation that FPR accumulate in the Triton X-100-insoluble fraction of neutrophils upon desensitization led to a more detailed study of the cellular structures that might be implicated in the regulation of receptor function. The signal transduction steps that involve the receptor protein as well as the crucial desensitization events appear to take place in the plasma membrane because desensitization does occur at 15~ a temperatur that inhibits internalization of receptors [41]. Therefore, a potential interaction of receptors with cytoskeletal proteins on the level of the plasma m e m b r a n e was investigated. An association of FPR with the submenbraneous cytoskeleton, respectively the m e m b r a n e skeleton, was shown by centrifugation of detergent-solubilized membranes from responsive neutrophils in sucrose density gradients. When octylglucoside was used as a detergent all receptors were soluble. In contrast, use of Triton X-100 as a detergent that does not solubilize m e m b r a n e skeletal structures, about 50% of the receptors sedimented to the pellet along with m e m b r a n e skeletal actin. Solubilization with Triton X-100 in the presence of agents that cause actin filaments to depolymerize, caused FPR to be released from the membrane skeletal pellet along with actin (Figure 3, [44]). This indicates that some linkage exists between FPR and membrane skeletal actin. In order to define the functional significance of this linkage of FPR to the membrane skeleton in responsive neutrophils, this association was investigated in cells that were desensitized to various degrees. In cells partially desensitized by incubation with an agonist at 4~ about 70% of the receptors were found in the membrane skeletal pellet. In fully desensitized neutrophils virtually all (>90%) receptors sedimented to the pellet (Figure 3).

18

100

80

60

9 !,,,I

40

20

0

C

PD

FD

KC1

pCMPS

DNAse I

Figure 3. Membrane skeletal association of FPR. Neutrophils were solubilized with Triton X-100 and the extracts were spun over sucrose density gradients. The distribution of photoaffinity labeled FPR was analyzed on SDS-polyacrylamide gels with a Phosphor Imager. Percent receptors that are coupled to the membrane skeleton are shown for responsive control cells (C), partially (PD) and fully (FD) desensitized neutrophils. In addition, values are shown for cells after solubilization in the presence of agents causing depolymerizaiton of actin filaments, like KC1, p-chloromercuriphenylsulfonic acid (pCMPS) or DNAse I. For experimental details see [44]. Previous characterization of FPR revealed three distinct ligand-receptor complexes corresponding to defined activation states of the receptor [45]. The most striking feature of the desensitized state, named LRX in order to describe the interaction of the ligand-receptor complex with an unidentified component X, is the energy-dependent formation upon agonist stimulation with a timecourse similar to the desensitization of the response [45]. Recently we have shown that the agonist-induced coupling FPR of the membrane skeleton is also energy-dependent [46], confirming that the membrane skeleton may indeed represent the unidentified component X that is needed to accomplish

19 desensitization. Table I shows that depletion of adenine and guanine nucleotides results in a dramatically reduced FPR coupling to the membrane skeleton. Table 1 FPR coupling to the membrane skeleton in energy-depleted cells percent FPR in pellet Control 15 min fluoride treatment 60 min fluoride treatment

74.9 + 4.3 30.2 + 4.6 28.9 + 2.7

Neutrophils were energy-depleted by treatment with 40 mM NaF for 15 min (depletion of ATP and GTP) and 60 rain (depletion of ATP and GTP including soluble diphosphates, [45]). After this pretreatment the cells were desensitized by incubation with an agonist. For details see [46]. Taken together, these data suggest that an increased FPR coupling to the membrane skeleton in response to agonist stimulation is a basic mechanism for h o m o l o g o u s desensitization of this receptor. This novel desensitization processes may be specific for chemoattractant receptors as a subclass of G proteincoupled receptors [47]. However, the possibility that phosphorylation-dependent mechanisms similar to those observed during desensitization of the [3-adrenergic receptor play an additional regulatory role in the FPR p a t h w a y cannot be excluded (see below). WHAT IS THE LINK OF FPR TO THE MEMBRANE SKELETON?

A central role of the membrane skeleton in the desensitization of FPR in h u m a n neutrophils has been well documented in recent years (for review see [5, 48]). H o w e v e r , the nature of the molecular link of the receptor to the m e m b r a n e skeleton remained elusive. The release of FPR from m e m b r a n e skeletal pellets with agents that depolymerize actin [44] suggests that F-actin may participate in the immobilization process that prevents the receptor from interaction with G proteins. Although it is difficult to demonstrate a specific interaction of a receptor with a protein as abundant as actin there are a series of observations that suggest a specific and direct interaction of FPR with actin. First, an overlay assay was used to demonstrate binding of photoaffinity labeled FPR to actin from neutrophil cytosol [49]. Second, actin increased the sedimentation rate of Triton X-100-solubilized FPR in a sucrose density gradient [49]. Third, anti-actin antibodies were able to immunosediment FPR suggesting

20 the existence of FPR-actin complexes [44, 49]. Fourth, exogenously added actin p r o m o t e d agonist binding to FPR in actin-stripped membranes or FPR solubilized from actin-stripped membranes [49]. These observations suggest that the FPR is indeed an actin-binding protein. The function of actin would be to serve as a matrix that allows for immobilization of FPR in a membrane domain that is depleted in G proteins and, thus, would interrupt the signaling cascade in the process of desensitization. Similarily, binding to actin of the EGF receptor as a member of the tyrosine kinase receptors has been well documented [50]. However, a function for actin binding of the EGF receptor has not yet been identified. At present it is not known what triggers the binding of FPR to the membrane skeleton. In theory several mechanisms are conceivable. A covalent modification like phosphorylation could change the affinity of FPR for G proteins a n d / o r actin. In such a model actin might have a preference for phosphorylated receptors in analogy to the increased affinity of arrestins for phosphorylated rhodopsin or [3-adrenergic receptors [1]. Another possibility might be a conformational change caused by agonist binding to the receptor. So far, there is no indication for phosphorylation being involved in FPR desensitization via the "membrane skeleton pathway". Okadaic acid and staurosporine, inhibitors of serine/threonine phosphatases and protein kinases A and C, respectively, have no significant effect on FPR coupling to the membrane skeleton [46]. In addition, desensitization of neutrophils does not change the affinity of FPR for G protein [51]. An appealing possibility would be that local actin polymerization is involved in the immobilization process leading to desensitization. The energy-dependence of the membrane skeletal association [46] may serve as a hint to such a possibility. FPR PHOSPHORYLATION

In h u m a n neutrophils receptor phosphorylation does not appear to be necessary for the homologous desensitization pathway that implicates receptor immobilization at the membrane skeleton [46]. Nevertheless, phosphorylation of FPR has been demonstrated in differentiated HL60 cells [52] and in rat basophilic leukemia cells (RBL-2H3) with stably expressed FPR [53]. Both studies showed that an agonist-induced and staurosporine-insensitive phosphorylation occured, suggesting a role for a kinase in homologous desensitization. More recently, phosphorylation of the C-terminus of FPR with a kinase different from protein kinase A, protein kinase C or tyrosine kinases has been demonstrated [54]. This phosphorylation, most likely by GRK2, affects serine and threonine residues in a portion of the C terminus that is involved in receptor-G protein coupling [55].

21 How is this observation related to the desensitization via receptor coupling to the membrane skeleton? At present there is no evidence for a role of phosphorylation in FPR desensitization via the lateral segregation pathway. Although inhibition of protein kinase A and protein kinase C with staurosporine does not affect FPR coupling to the membrane skeleton [46], phosphorylation by a G protein-coupled receptor kinase (GRK) may still trigger FPR binding to actin. However, the finding that inhibition of serine/threonine phosphatases with ocadaic acid has also no significant effect on FPR immobilization [46] argues against such a possibility. It appears more likely that phosphorylation and membrane skeleton coupling are parallel mechanisms with the former representing a general mechanism for all G protein-coupled receptors and the latter serving as a mechanism that is operative for the functional subclass of chemoattractant receptors. RECEPTOR-CLASS DESENSITIZATION The possible existence of a mechanism of desensitization specific for chemoattractant receptors was proposed based on the observation that FPR and C5a receptors exhibited heterologous desensitization whereas no crossdesensitization was observed with c~1 adrenergic receptors although all three receptor subtypes are coupled to phospholipase C as an effector enzyme [47]. The cross-regulation of G protein-coupled receptors in neutrophils appears to be a highly complex process because the susceptibility to cross-desensitization is dramatically different for different Ca2+-mobilizing receptors such as peptide and lipid chemoattractant receptors or receptors for ligands other than chemoattractants [56]. Phosphorylation by PKC might be one mechanism for differential crossregulation because not all receptors coupled to the PLC pathway possess a consensus sequence for phosphorylation by PKC [56]. The complexity of the network that appears to be involved in the cross-talk between various receptors of the PLC pathway suggests the existence of multiple mechanisms to accomplish this complicated regulatory task. It is conceivable that interaction of GRKs with specific G protein ~, subunits allows for differential desensitization of different receptors [16]. However, in addition to the well characterized GRK mechanism the lateral segregation model provides an appealing mechanism to explain some of the observed phenomena [5].

22 SIGNAL TRANSDUCTION AND MEMBRANE ORGANIZATION The discovery of an increasing number of proteins that are involved in fine tuning of the intensity of receptor-mediated signaling raises the general question of the spatial organization of the relevant components. Simple collision coupling by lateral motion of proteins in the plasma membrane might be appropriate to explain receptor-G protein or G protein-effector coupling. For an efficient interplay of all proteins involved in signal modulation an organized distribution of the regulatory components appears to be required. Although a clear picture of such a membrane compartmentation remains to be developed, there are some data that point to an important contribution of cytoskeletal structures in the organizational control of the signal trasnduction machinery. As has been shown for FPR and other receptors diffusion of a receptor protein may be restricted by interaction with the membrane skeleton [5, 50]. On the other hand, interaction of G proteins with cytoskeletal elements has also been demonstrated providing for a means to also control G protein distribution in the plasma membrane (for review see [57]). An intriguing idea explaining distinct membrane distribution of G proteins has been put forward by Rodbell and coworkers who observed polydisperse forms of G proteins caused by polymerization of G a subunits in analogy to tubulin [58, 59]. Formation of these large molecular complexes would result in restricted mobility of the G proteins and could serve as an explanation for membrane domains in neutrophils that are enriched in G proteins (see above). CONCLUSION Receptor-mediated signal transduction is no longer considered to be just a simple interaction of a limited number of proteins. It has become a highly regulated process in a complicated network of an increasing number of components. The increasing complexity of the regulation of receptors suggests that spatial organization of proteins is necessary in order to achieve a highly reliable and efficient regulatory machinery [57]. The proposed model for desensitization of FPR that requires lateral segregation of m e m b r a n e components may serve as a simple example for spatial control of signal transduction. It appears that also in systems like the [~-adrenergic receptor a subtle control of the interaction of all the known components must be operative resulting in a well organized and specific signaling process in the cell.

23 Acknowledgement Work in the authors' laboratories is supported by grants from the American Cancer Society (IRG-172B, KNK); the National Institute of Health (RO1-AI-22735 and DMB 900058P, AJJ); the Deutsche Forschungsgemeinschaft, the European Commission, the Fonds der Chemischen Industrie (MJL). REFERENCES

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Lohse MJ. Biochim Biophys Acta 1993; 1179: 171-188. Hausdorff WP, Caron MG, Lefkowitz RJ. FASEB J 1990; 4: 2881-2889. Hargrave PA, McDowell JH. FASEB J 1992; 6: 2323-2331. Hosey MM, Benovic JL, DebBurman SK, Richardson RM. Life Sci 1995; 56: 951-955. Klotz K-N, Jesaitis AJ. Bioessays 1994; 16: 193-198. Benovic JL, Strasser RH, Caron MG, Lefkowitz RJ. Proc Natl Acad Sci USA 1986; 83: 2797-2801. Benovic JL, DeBlasi A, Stone WC, Caron MG, Lefkowitz RJ. Science 1989; 246: 235-240. Lohse MJ, Benovic JL, Codina J, Caron MG, Lefkowitz RJ. Science 1990; 248: 1547-1550. Lohse MJ, Andexinger S, Pitcher J, Trukawinski S, Codina J, Faure J-P, Caron MG, Lefkowitz RJ. J Biol Chem 1992; 267: 8558-8564. Attramadal H, Arriza JL, Aoki C, Dawson TM, Codina J, Kwatra MM, Snyder SH, Caron MG, Lefkowitz RJ. J Biol Chem 1992; 267: 17882-17890. Roth N, Campbell PT, Caron MG, Lefkowitz RJ, Lohse MJ. Proc Natl Acad Sci USA 1991; 88: 6201-6204. Premont RT, Inglese J, Lefkowitz RJ. FASEB J 1995; 9: 175-182. Chen C-Y, Dion SB, Kim CM, Benovic JL. J Biol Chem 1993; 268: 7825-7831. Haga K, Haga T. J Biol Chem 1992; 267: 2222-2227. Pitcher JA, Inglese J, Higgins JB, Arriza JL, Casey PJ, Kim C, Benovic JL, Kwatra MM, Caron MG, Lefkowitz RJ. Science 1992; 257: 1264-1267. M~iller S, Hekman M, Lohse MJ. Proc Natl Acad Sci USA 1993; 90: 1043910443. Touhara K, Koch WJ, Hawes BE, Lefkowitz RJ. J Biol Chem 1995; 270: 1700017005. Wilson CJ, Applebury ML. Curr Biol 1993; 3: 683-686. Wilden U, Hall SW, Kuhn H. Proc Natl Acad Sci USA 1986; 83: 1174-1178. Benovic JL, Pike LJ, Cerione RA, Staniszewski C, Yoshimasa T, Codina J, Caron MG, Lefkowitz RJ. J Biol Chem 1985; 260: 7094-7101.

24 21 Bouvier MB, Leeb-Lundberg LMF, Benovic JL, Caron MG, Lefkowitz RJ. J Biol Chem 1987; 262: 3106-3113. 22 Lohse MJ, Benovic JL, Caron MG, Lefkowitz RJ. J Biol Chem 1990; 265: 32023209. 23 Hadcock JR, Ros M, Watkins DC, Malbon CC. J Biol Chem 1990; 265: 1478414790. 24 Eschenhagen T, Mende U, Nose M, Schmitz W, Scholz H, Schulte am Esch J, Warnholtz A, Sch/ifer H. Circ Res 1992; 70: 688-696. 25 Eschenhagen T, Mende U, Diederich M, Nose M, Schmitz W, Scholz H, Schulte am Esch J, Warnholtz A, Sch/ifer H. Mol Pharmacol 1992; 42: 773-783. 26 Strittmatter SM, Valenzuela D, Sudo Y, Linder ME, Fishman MC. J Biol Chem 1991; 26: 22465-22471. 27 Yatani A, Okabe K, Polakis P, Halenbeck R, McCormick F, Brown AM. Cell 1990; 61: 769-776. 28 Bauer PH, Mfiller S, Puzicha M, Pippig S, Helmreich EJM, Lohse MJ. Nature 1992; 358: 73-76. 29 Lee RH, Flower A, McGinnis JF, Lolley RN, Craft CM. J Biol Chem 1990; 265: 15867-15873. 30 Reig JA, Yu L, Klein DC. J Biol Chem 1990; 265: 5816-5824. 31 Lee RH, Ting TD, Liebermann BS, Tobias DE, Lolley RN, Ho Y-K. J Biol Chem 1992; 267: 25104-25112. 32 Xu J, Wu D, Slepak VZ, Simon MI. Proc Natl Acad Sci USA 1995; 92: 20862090. 33 Lee RH, Brown BM, LoUey RN. J Biol Chem 1990; 265: 15860-15866. 34 Yoshida T, Willardson BM, Wilkins JE, Jensen GJ, Thornton BD, Bitensky MW. J Biol Chem 1994; 269: 24050-24057. 35 Hekrnan M, Bauer PH, S6hlemann P, Lohse MJ. FEBS Lett 1994; 343: 120-124. 36 Boxer GJ, Curnutte JT, Boxer LA. Hosp Practice 1985; 40: 69-90. 37 Snyderman R, Uhing RJ. In: Gallin JI, Goldstein IM, Snyderman R, eds. Inflammation. New York: Raven Press, 1988; 309-323. 38 Cockcroft S. Biochim Biophys Acta 1992; 1113: 135-160. 39 Jesaitis AJ, Bokoch GM, Allen RA. In: Condeelis J, Lazarides E, Satir P, eds. Signal Transduction in Cytoplasmic Organization and Cell Motility. New York: Alan R Liss, Inc, 1988; 325-337. 40 Jesaitis AJ, ToUey JO, Allen RA. J Biol Chem 1986; 261: 13662-13669. 41 Jesaitis AJ, Naemura JR, Sklar LA, Cochrane CG, Painter RG. J Cell Biol 1984; 98: 1378-1387. 42 Jesaitis AJ, Bokoch GM, Tolley JO, Allen RA. J Cell Biol 1988; 107: 921-928. 43 Johansson B, Wymann MP, Holmgren-Peterson K, Magnusson K-E. J Cell Biol 1993; 121- 1281-1289.

25 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

Klotz K-N, Krotec KL, Gripentrog J, Jesaitis AJ. J Immunol 1994; 152: 801-810. Sklar LA, Mueller H, Omann G, Oades Z. J Biol Chem 1989; 264: 8483-8486. Klotz K-N, Jesaitis AJ. Cell Signal 1994; 6: 943-947. Didsbury JR, Uhing RJ, Tomhave E, Gerard C, Gerard N, Snyderman R. Proc Natl Acad Sci USA 1991; 88: 11564-11568. Jesaitis AJ. Comments Mol Cell Biophys 1992; 8: 97-114. Jesaitis AJ, Erickson RW, Klotz K-N, Bommakanti RK, Siemsen DW. J Immunol 1993; 151: 5653-5665. den Hartigh JC, van Bergen en Henegouwen PMP, Verkleij AJ, Boonstra J. J Cell Biol 1992; 119: 349-355. Klotz K-N, Jesaitis AJ. Biochem Pharmacol 1994; 48: 1297-1300. Tardif M, Mery L, Brouchon L, Boulay F. J Immunol 1993; 150: 3534-3545. Ali H, Richardson RM, Tomhave ED, Didsbury JR, Snyderman R. J Biol Chem 1993; 268: 24247-24254. Prossnitz ER, Kim CM, Benovic JL, Ye RD. J Biol Chem 1995; 270: 1130-1137. Bommakanti RK, Klotz K-N, Dratz EA, Jesaitis AJ. J Leukocyte Biol 1993; 54: 572-577. Tomhave ED, Richardson RM, Didsbury JR, Menard L, Snyderman R, Ali H. J Immunol 1994; 153: 3267-3275. Neubig RR. FASEB J 1994; 8: 939-946. Nakamura S-I, Rodbell M. Proc Natl Acad Sci USA 1990; 87: 6413-6417. Coulter S, Rodbell M. Proc Nail Acad Sci USA 1992; 89: 5842-5846.

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27

Introduction to Muscarinic Receptors

Gideon Shapiro Sandoz Pharma Ltd., Basel Switzerland CH-4002

The muscarinic system is one of the earliest established components of the nervous system. Its pharmacology was already well defined over one hundred years ago using poisons or medicinal agents of the time, the natural products muscarine [1] pilocarpine, atropine and physostigmine. Interestingly the elucidation of acetylcholine [2] as the endogenous neurotransmitter was achieved many decades later highlighting the importance of natural products in medicine and pharmacology which remains with us.

OH

____/

I...../-~"0)'''' OH3 N+ OH3 / ~CH3 CH3

Muscarine Amanita Muscaria

Atropine Belladona

I

N'~I N

Me

o NH o.

/ Me \

Pilocarpine

Pilocarpus Jaborandi

Physostigmine

I

o II ~o "7"" Me

Acetylcholine

Physostigma venenosum

Over the past several decades with the ripening of the discipline of organic synthesis, man made compounds have been prepared which act on the functional unit of the muscarinic system which we now recognize as a receptor protein. Some of these have or remain to be used as therapeutic agents. More recently, the efforts of synthetic organic chemists have yielded muscarinic receptor antagonist ligands such as pirenzepine, AFDX-112, 4-DAMP, and

28 methoctramine which have catalyzed a revival of muscarinic receptor pharmacology. With these tools it has been possible to classify muscarinic receptors in terms of subtypes M1, M2 and M3 [3] which presumably allow subtle regulation of physiological processes. Again basic pharmacological science and the development of therapeutic agents have gone hand in hand, pirenzepine, an agent which inhibits gastric acid release, reaching the marketplace for ulcer treatment [4]. Many other selective agents have since been synthesized which are under investigation for various indications.

N~

~ OH

~N~)

~

/

Pirenzepine(M1)

N(Et)2

AF-DX116 (M2)

Hexahydrosiladifenidol(M3) I

oH

~ N ~

o O " ~ ~ N ~ */

v

N

-O H

1

4-DAMP(M3)

Methoctramine

(M2)

The latest impetus in the muscarinic field has come from molecular biology which has brought the muscarinic receptor, once a hypothetical construct, to the level of an expressed gene with a corresponding protein primary sequence. The cloning of muscarinic receptors has not only confmned but expanded the subtype division from three pharmacologically characterizable to five (ml-m5) molecular entities [3]. Compound screening has been greatly enhanced by the availability of cell lines expressing homogenous populations of specific receptor subtypes on which binding and functional studies can be performed. A great impact may be forseen on the search for therapeutically useful subtype selective compounds. Recent examples of this are the ml selective agonists Xanomeline and PD-151832 which are potential Alzheimer therapeutics. Finally, the structure of the muscarinic receptor(s) may be determined someday in the future ideally as ligand complexes. Until this is achieved the primary sequence used in tandem with mutagenesis experiments [5] and the only structure determined to date of

29 a membrane bound G-protein coupled receptor, namely bacteriorhodopsin [6], will have to continue to guide current efforts [7] to understanding how muscarinic ligands interact with the

~

N "" S

Xanomeline

\

N

\o

PD151832

/.O

receptor. Previously, structure activity relationships and pharmacophore models have been formulated solely on the basis of ligand structure and the receptor has been considered to be a rigid entity. The limitations of this assumption have been pointed out using a simplified hypothetical model for the binding of muscarinic agonists to a segment of transmembrane helix-3 of the ml receptor [8]. Conformation mobility of the receptor is necessary to account for optimal binding for all of the potent agonists studied. Where is the future of muscarinic receptor ligands/drugs headed? High capacity screening technologies using optimized cloned systems may tuna up interesting selective compounds already sitting on the shelf. Selective antagonists may certainly be improved upon while the difficulty in making selective agonists may be of an intrinsic nature. For the design of new therapeutically useful ligands the new insights into muscarinic receptor structure and function provide a sound basis for progress.

References 1. Schmiedeberg O and Koppe R. Das Muscarin, das Giftige Alkaloid des Fliegenpilzes, Leipzig: Vogel, 1869. 2. Dale HH and Dudley FH. J Physiol 1929; 68: 97-123. 3. Receptor and Ion Nomenclature Supplement Trends Pharmacol Sci 1995; 16: 4. 4. Blum AL and Hammer R, eds. Die Behandlung des Ulcus Pepticum mit Pirenzipin, Munich: Demter, 1979. 5. Liu J, Schoeneberg T, Van Rhee M and Wess J. J Biol Chem 1995; 270:1953219539. 6. Henderson R, Baldwin JM, Ceska TA, Zemlin F, Beckmann E and Downing KH. J Mol Biol 1990, 213: 899-929. 7. Hibert MF, Tnmapp-Kallmeyer S, Bruinvels A and Hoflack J. Trends Pharmacol Sci 1993; 14: 7-12. 8. Shapiro G, Floersheim P, Boelsterli J, Amstutz R, Bolliger G, Gammenthaler H, Gmelin G, Supavilai P and Walkinshaw M. J Med Chem 1992; 35:15-27.

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31

Structure-function analysis of muscarinic acetylcholine receptors J. Wess, N. Blin, J. Yun, T. SchSneberg and J. Liu Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bldg. 8A, Room B 1A-09, Bethesda, MD, U.S.A. INTRODUCTION The muscarinic acetylcholine receptors (mAChRs) are typical members of the superfamily of G protein-coupled receptors (GPCRs; refs. 1,2). Following the cloning of five distinct mAChR genes (ml-m5), the last decade has witnessed a flood of new information about how these receptors function at a molecular level. Molecular genetic and biochemical approaches have been applied to identify receptor regions/single amino acids involved in ligand binding, G protein coupling, regulation of receptor activity, and other fundamental aspects of mAChR function. In this chapter, we will describe recent work dealing with the structure and assembly of mAChRs as well as with the molecular mechanisms governing the selectivity of receptor/G protein interactions. A R R A N G E M E N T OF T H E T R A N S M E M B R A N E HELICES

Like all other G protein-coupled receptors (GPCRs), the five mAChRs are predicted to be composed of seven a-helically arranged transmembrane domains (TM I-VII) connected by three extraceUular (o2-o4) and three intracellular (il-i3) loops [1,2]. Several lines of evidence suggest that the seven TM helices are arranged in a ring-like fashion, thus forming a tightly packed helical bundle [3-6]. Site-directed mutagenesis [7,8] and affinity labelling studies [9,10] have shown that residues present on the inner surfaces of different TM helices (facing the cavity enclosed by TM I-VII) are involved in the binding of muscarinic agonists (including the neurotransmitter, acetylcholine) and antagonists. Given the lack of high-resolution structural information on any GPCR, several laboratories have employed mutagenesis approaches to identify molecular interactions between individual TM helices [11-16]. We previously identified a series of hybrid m2/m5 receptors containing m2 receptor sequence in TM VII and m5 receptor sequence in TM I (such as C4 in Fig. 1B) which were unable to bind significant amounts of muscarinic radioligands [12]. However, replacing TM I in these apparently misfolded mutant receptors with the corresponding m2 receptor segment could restore proper ligand binding activity [12], indicating that TM I and TM VII are located directly adjacent to each other and that molecular interactions between these two TM helices are required for proper receptor folding. Immunocytochemical and ELISA studies showed that the pharmacologically inactive m2/m5 hybrid receptors were properly expressed on the cell surface [16], suggesting that the virtual lack of binding activity seen with these mutant receptors is due to a specific folding defect. It seemed reasonable to assume that the amino acids responsible for this folding defect are located at the TM Ifi'M VII

32

A

m2

m5 143v36G47

m, f424,~ ~-A~ ~L428 m32:..:.~.~~'.V~ W427r ,,,- ~C~9 / VI

isles41

B

Y4?..26S~3~

I

m5

~'''l

Ligand Binding Cavity

m2

m5"i'37->m2A30

[3H]NMS Binding Bmax (fmol/mg) K D (pM)

m2 (wt) 698 _+84 53 __.10

m2T423->m5H4 78

C4 < 25

541 +_79

556 +_54

52 _+4

76 +_20

Figure 1. Pharmacological activity of hybrid m2/m5 mAChRs. (A) Helical wheel model of the possible orientation of TM I and VII in mAChRs, assuming an anticlockwise connectivity of TM I-VII. The view is from the extracellular surface of the membrane. Shown here is a pharmacologically inactive hybrid receptor [12] containing m5 receptor sequence in TM I and m2 receptor sequence in TM VII (only the first 18 amino acids of TM I and VII are shown). Amino acids which differ between the m2 and m5 mAChRs (human) are marked with asterisks. The two Thr residues (m5Thr37 and m2Thr423) which face each other at the TM IfVII interface in the pharmacologically inactive hybrid receptors are highlighted. The model shown here slightly differs from the Baldwin projection [4,5] in that TM VII has been rotated by approximately 30 degrees (counterclockwise) to allow m2Tyr426 (underlined) to project into the ligand binding cavity formed by TM III-VII [8,48]. (B) Structure and [3H]NMS binding properties of hybrid m2/m5 mAChRs transiently expressed in COS cells [16].

33 interface and that the identification of these residues might therefore provide useful information as to how TM I and TM VII are oriented relative to each other. Helical wheel models [4,5] of the possible a r r a n g e m e n t of TM I-VII (assuming an anticlockwise connectivity of TM I-VII, as viewed from the extracellular membrane surface) showed that there are only two residues at the TM IfrM VII interface in which the m2 and m5 mAChRs differ [16]. As shown in Fig. 1A, in all misfolded m2/m5 hybrid receptors, m5Thr37 (TM I) is predicted to be located adjacent to m2Thr423 (TM VII). We therefore speculated that a novel hydrogen bond can form between these two Thr residues (which are both located 1-2 helical turns away from the membrane surface), thus interfering with proper helix/helix packing and receptor folding. To test this hypothesis, two single point mutations, m5Thr37->m2Ala30 or m2Thr423->m5His478, were introduced into all pharmacologically inactive hybrid receptors (shown for C4 in Fig. 1B). If the model shown in Fig. 1A is correct, these mutations should "recreate" the TM IfI?M VII interfaces present in the wild type m2 and m5 mAChRs, respectively [16]. Consistent with our working hypothesis, we found that either of the two point mutations resulted in mutant receptors which, when transiently expressed in COS-7 cells, were able to bind significant amounts ofmuscarinic radioligands (shown for C4 in Fig. 1B) and to couple to G proteins [16]. These findings therefore indicated that the model shown in Fig. 1A that guided this mutagenesis study is probably correct. This model was based on the recently proposed "Baldwin projection" [4,5], assuming an anticlockwise connectivity of the TM helices (as viewed from the extracellular membrane surface). In a model assuming a Clockwise arrangement of the TM helices, m5Thr37 (TM I) would still face TM VII, whereas m2Thr423 (TM VII) would lie adjacent to TM VI [16]. Such an a r r a n g e m e n t makes it very difficult to rationalize how mutational modification of either of these two Thr residues can restore a proper receptor fold. Our data therefore strongly support the concept that the TM helices in GPCRs are arranged in an anticlockwise fashion (as viewed from the cell surface). RECEPTOR ASSEMBLY

Previous studies have shown that GPCRs are composed of two major building blocks, one containing TM I-V and the other, TM VI and VII [17-19]. It was found that these two domains, when coexpressed as two separate polypeptides, can fold independently of each other and then properly assemble to form functional receptor complexes [17-19]. Based on these results, we have recently tested the hypothesis that GPCRs are assembled from many, r a t h e r t h a n only two, autonomous folding units. To address this question, the r a t m3 mAChR was "split" in all three intracellular (il-i3) and all three extracellular loops (o2-o4), thus generating six polypeptide pairs (Nil+Cil, No2+Co2, etc.; Fig. 2; ref. 20). As shown in Fig. 3, coexpression of three of the six polypeptide pairs (Ni2+Ci2, No3+Co3, and Ni3+Ci3, respectively) led to the appearance of a significant number of specific high-affinity [3H]NMS binding sites. These data clearly indicated that covalent connections between TM III and IV, TM IV and V, and TM V and VI, respectively, are not essential

34

n3

;llular .

v

Figure 2. The rat m3 mAChR was "split" at the indicated positions (arrows), thus generating six polypeptide pairs [20]. for proper receptor folding. However, it was also noted that the the Ni2+Ci2 and No3+Co3 polypeptide complexes showed significant decreases in binding affinities for most ligands tested (including acetylcholine; Fig. 3), suggesting that the i2 and o3 loops exert indirect conformational effects on the proper arrangement of the TM helical bundle predicted to contain the primary ligand binding site. To investigate whether the Ni2+Ci2, No3+Co3, and Ni3+Ci3 polypeptide complexes were still capable of activating G proteins, their ability to mediate carbachol-induced phosphoinositide (PI) hydrolysis was examined [20]. As shown in Fig. 4, the No3+Co3 and Ni3+Ci3 complexes were able to stimulate PI hydrolysis to a similar maximum extent as the wild type m3 mAChR. In contrast, the Ni2+Ci2 complex displayed only residual functional activity, indicating that the structural integrity of the i2 loop is essential for efficient G protein coupling [20]. Immunocytochemical and ELISA studies showed that all N- and C-terminal receptor fragments capable of forming functional receptor complexes in the cotransfection experiments (see above) were stably inserted into lipid bilayers, even when expressed alone. Similar to the wild type m3 mAChR, the various receptor polypeptides were found both intraceUularly (ER/Golgi complex) as well as in the plasma membrane [20]. These observations suggest that the presence of the full-length receptor protein is not essential for membrane insertion and proper intracellular tr~fScking. Taken together, these results strongly support the notion that mAChRs and, most likely, other GPCRs [21] are composed of multiple (rather than only two) individually stable, autonomous folding domains. The folding and assembly of GPCRs therefore appears to occur via a two-step mechanism, similar to that previously described for the bacterial membrane protein, bacteriorhodopsin [22]. In step I, individually stable folding units are established across the lipid bilayer; in step II, these domains interact with each other to form functional receptor complexes.

35 [3H]NMS

m3 (wt)

Ni2 ~

Jill

No3

N i 3 ~

!111Oq + fi::!::iii::!ii!illi||ill::i::iii::i::itCi2 i~llh qlll I

:::::::::::::::::::::::::::::::::::::::::::::::::::: Co3

+

,-il--

+

I::i::i::i::~i::i:J Ci3

Bmax (fmol/mg) KD (pM)

4-DAMP K i (nM)

Carbachol K i (~M)

Acetylcholine K i (~M)

960+120

12+-3

0.47 + 0.03

7.28+0.88

1.40+0.17

44+3

28+6

1.43+0.13

35.0 + 3.7

11.0+2.1

52 + 16

43 + 7

9.20 + 0.48

33.9 +__3.6

15.4 + 0.8

22+__2

0.92+0.08

6.94+0.44

1.16 + 0.04

122+7

Figure 3. Ligand binding properties of polypeptide complexes formed by coexpressed m3 mAChR fragments. All studies were carried out in transiently transfected COS-7 cells (data taken from ref. 20).

100 -

I--

12.

._= ~

80~

o ._ r

60-

40"~"

200

%

%

x

%

%

•

%

Figure 4. Functional properties of polypeptide complexes formed by coexpressed m3 mAChR fragments. The ability of the wild type m3 mAChR (Emax = 100%) and three polypeptide complexes (for fragment structures, see Fig. 3) to mediate carbachol-induced PI hydrolysis was studied in transiently transfected COS-7 cells. For these experiments, the wild type m3 mAChR was expressed at Bmax levels similar to those found with the coexpressed polypeptides (data taken from ref. 20).

36

RECEPTOPJG PROTEIN COUPLING Characteristically, each GPCR can couple only to a limited set of the many structurally similar G proteins expressed in a cell [23]. The same is also true for the individual members of the mAChR family: the ml, m3, and m5 mAChRs are known to preferentially couple to G proteins of the Gq/ll family [2,25,27] which mediate the activation of different isoforms of PLC~ (biochemical response: PI hydrolysis) [24,26,27]. The m2 and m4 mAChRs, on the other hand, are selectively linked to G proteins of the Gi]o class [25,28] which, at a biochemical level, mediate the inhibition of adenylyl cyclase [27].

Receptor residues/domalns critical for G protein coupling selectivity Studies with chimeric m2/m3 mAChRs have shown that the N-terminal portion of the i3 loop is intimately involved in G protein recognition [29-31]. When this receptor segment was exchanged between the m2 and m3 mAChRs, the resultant hybrid receptors gained the ability to interact with the same type of G proteins as the wild type receptor from which this short sequence was derived [29-31], although with clearly reduced efficiency [30]. It was also noted that a m u t a n t m2 mAChR which contained m3 mAChR sequence at the beginning of the i3 loop still retained the ability to inhibit adenylyl cyclase [30], suggesting that the N-terminal segment of the i3 loop is an important but clearly not the sole structural element determining the selectivity of mAChR/G protein interactions (see below). Computational methods [32] as well as site-directed mutagenesis studies [3338] suggest that the N-terminal portion of the i3 loop of mAChRs and other GPCRs forms an amphipathic a-helical extension of TM V (shown for the rat m3 mAChR in Fig. 5) and that the noncharged side of this helical segment represents an important G protein binding surface. Detailed mutational analysis of this segment of the m3 mAChR showed that a Tyr residue (which is conserved among the m l , m3, and m5 mAChRs; m3Tyr254 in Fig. 5) is of particular importance for efficient Gq recognition [35,36]. We could show, for example, that substitution of Tyr254 into a mutant m3 mAChR in which the first 16 ~mino acids of the i3 loop were replaced with the corresponding m2 mAChR sequence (CR1; Fig. 6) was able to confer on this mutant receptor (which does not couple to Gq) the ability to efficiently activate the PI pathway (Fig. 6; ref. 36). However, when Tyr254 was substituted into the homologous position of the wild type m2 mAChR, the resulting m2(Ser210->Tyr) did not gain the ability to efficiently couple to Gq (Fig. 6; ref. 36), thus providing additional support for the notion that other intracellular domains, besides the N-terminus of the i3 loop, also contribute to the specificity of mAChR]G protein interactions. To identify such regions, we systematically substituted distinct intracellular domains of the m3 receptor into the m2(Ser210->Tyr) mutant receptor (= m2Y) and into the wild type m2 receptor [39]. As shown in Fig. 7, introduction of the i l loop or the C-terminal tail of the m3 receptor into the wild type m2 or the m2Y mutant receptor did not lead to a significant stimulation of PLC activity (PI hydrolysis). In contrast, substitution of the i2 loop of the m3 mAChR into the wild type m2 receptor (as well as into m2-Y) led to a hybrid receptor which could stimulate PI hydrolysis in a fashion similar to a mutant m2 receptor (m2-Ni3) in which the

37 K262 K255 E2~

259

A265

R252 E263

T261

E256 i/01

L264 1253

, i--~0

Figure 5. Helical wheel projection of the N-terminal segment of the i3 loop of the rat m3 mAChR (Arg252-Ala265), viewed from the N- to the C-terminus. This model predicts that one side of this putative helix consists of exclusively charged residues (plain), whereas the other side is formed by exclusively noncharged residues (bold). Tyr254 (boxed) is conserved among all PI-coupled mAChRs (ml, m3, and m5) and is required for efficient activation of G proteins of the Gq/ll family [35,36]. N-terminal 21 ~mino acids of the i3 loop were replaced with m3 sequence (Emax ca. 40% of wild type m3; Fig. 7). This finding clearly indicates that the i2 loop also makes a critical contribution to proper recognition of Gq/ll proteins. Introduction of the C-terminal 30 amino acids of the i3 loop of the m3 receptor (Lys464-Ser493) into the wild type m2 mAChR (resulting in m2-Ci3) did not improve coupling to Gq/ll proteins. However, introduction of the additional Ser210->Tyr point mutation into this chimeric construct (resulting in m2-Y-Ci3) led to a mutant receptor which, similar to m2-i2 and m2-Ni3, gained coupling to PI turnover (Fig. 7). This result suggested that the N-terminus of the i3 loop (where m3Tyr254 is located) acts in a cooperative fashion with a region at the C-terminus of the same loop to allow proper recognition of Gq/ll proteins. Following these initial results, detailed site-directed mutagenesis studies were carried out to identify individual amino acids within the i2 loop and the C-terminal segment of the i3 loop of the m3 mAChR required for efficient coupling to Gq [39]. We found that substitution of Ser168, Argl71, Arg176, and Arg183 (residues located in the i2 loop of the m3 receptor; Fig. 8A) into the wild type m2 mAChR yielded a mutant receptor that showed functional properties very similar to the m2-i2 mutant receptor (Fig. 8B). These four amitm acids therefore fully account for the contribution of the i2 loop of the m3 mAChR to Gq coupling efficiency. Single amino acid substitutions showed that m3Arg176 which is located in the middle of the i2 loop is of particular importance for Gq coupling (Fig. 8B). However, experiments with double and triple point mutants (data not shown) suggested that all four m3 receptor residues are required for optimum coupling to Gq [39].

38 I

I m2

CR1

~

m3

CR1-Y

m2(Ser210->Tyr)

12o- I 9 m3(wild type) [] CR1-Y

E-Q 80 (D (D

>

(!) (~ f~

co~

o m2(wiid type) 9 m2(Ser210->Tyr) .. CR1

40

-8

-7

-6

-5

-4

-3

-2

Carbachol, log M

Figure 6. Importance of Tyr254 (rat m3 receptor sequence) for mAChR -mediated stimulation of PI hydrolysis [35,36]. All studies were carried out with transiently transfected COS-7 cells. In CR1, the first 16 amino acids of the i3 loop of the r a t m3 mAChR (RIYKETEKRTKELAGL) were replaced with the corresponding human m2 receptor sequence (HISRASKSRIKKDKKE). In CR1-Y and m2(Ser210->Tyr), a Tyr residue (corresponding to Tyr254 in the rat m3 mAChR) replaces Ser210 located at the N-terminus of the i3 loop of the m2 mAChR. The maximum stimulation of PI hydrolysis mediated by the wild type m3 receptor was set at 100%. The C-terminal segment of the i3 loop was analyzed in a similar fashion to identify additional amino acids required for efficient coupling to Gq. Substitution of Ala488, Ala489, Leu492, and Ser493 into m2(Ser210->Tyr) resulted in a m u t a n t receptor which quantitatively mimicked the PI response mediated by the m2-Y-Ci3 hybrid receptor (Figs. 7, 9). More detailed mutagenesis studies (Fig. 9B; ref. 39) showed that all four residues (which are located at the i3 loop/ TM VI junction) are required for optimum Gq activation. Secondary structure prediction programs [32] suggest that the region at the i3 loopfrM VI junction of mAChRs and other GPCRs is a-helically arranged. If this is correct, m3Ala488, m3Ala489, m3Leu492, and m3Ser493 are located on one side of an a-helical receptor domain (Fig. 10A). Based on this notion, there are two patches of primarily hydrophobic amino acids located at the N- and Ctermini of the i3 loop, respectively. Given the predicted spatial proximity of the N- and C-terminal segments of the i3 loop, it is therefore likely that these two

39

il I

i2 II

III

Max.

i4 IV

i n c r e a s e in I P1 (% above basal)

VI VII

v

m

m

+++

m3 m2

.

m2-Y

f

..,

~ ,

--{ -

rn2-il m2-Y-il m2-i2

..~ i

.: ~

:'i' I

~

.

.;

.u

i

m2-Y-i2

~

m2-Ni3

~

m2-Ci3 m2-Y-Ci3

~ ,

m2-i4

~

m2-Y-i4

~

|

,_

I

(+) (+) (+) (+) ++ ++ ++

I ,L

Y

I

(+) ++

(+) (+)

Y (+) + + +++

< 15 % 40-60 % 100%

Figure 7. Carbachol-induced PI hydrolysis mediated by hybrid m2/m3 mAChRs transiently expressed in COS-7 cells. The Tyr residue (Y) present at the beginning of the i3 loop in several of the chimeric constructs corresponds to m3Tyr254 and replaces m2Ser210 (data taken from ref. 39). receptor sites form a contiguous hydrophobic binding surface critical for G protein recognition and activation. Additional mutagenesis studies [39] with mutant m2 mAChRs containing m3 receptor sequences in multiple intracellular domains showed that the residues important for Gq coupling (located in the i2 loop and at the N- and C-termin_i of the i3 loop of the m3 mAChR; see above) act in a cooperative fashion to allow efficient Gq activation. It was found that Ser168, Argl71, Arg176, Arg183 (present in the i2 loop of the m3 mAChR), together with Ala488, Ala489, Leu492, and Ser493 (present at the i3 loopfrM VI junction of the m3 mAChR) and the Nterminal segment of the i3 loop, quantitatively account for the G protein coupling preference of the m3 mAChR (Fig. 11). These residues are conserved among all Gq-coupled mAChRs (ml, m3, and m5; Figs. 8A, 9A) but replaced with different residues in the m2 and m4 receptors. It will be of interest to examine whether amino acids at the corresponding positions in other GPCRs have a similar

40

A

i2

ml m5 mj

DRY~LSY~RTP~ DRY~S~~LT~KRTP~ DRY~~LTY~AKRTT~

m4

120]

I"

1 00 -=

e-

"7~

r

o e-

~ v

t-~

171

176

183

127

132

1~

801 / 9

r

168

124

DRY~LT~RRTT~ ****# *#** *#

B n

164 120

60

**

#

m3 residues substituted into the m2 mAChR

s=serlosR i2 = Asp164-Arg 183

R. = Arg171 = Arg176 R** = Arg 183

40 20

Figure 8. Four 3mino acids in the i2 loop of the m3 mAChR are critical for receptor-mediated PI hydrolysis [39]. (A) Amino acid sequences of the i2 loop of the ml-m5 mAChRs (rat=human). *, Positions at which all five receptors have identical residues. #, Positions at which the ml, m3, and m5 receptors have identical residues (boxed) which differ from those present in the m2 and m4 receptors. Numbers refer to amino acid positions in the human m2 and rat m3 mAChRs, respectively [49]. (B) The ability of the wild type m2 and m3 mAChRs (wild type m3: Emax = 100%) and a series of m u t a n t m2/m3 receptors to mediate carbachol-induced increases in IP1 levels was studied in transiently transfected COS-7 cells [39].

41

A

VI

ml

I-T-1 I-T--I R A G K G Q K P R O K E Q L A K R K T F S a V E m K ~ k ~ L : ~ . T ~ ~z~.I a n iii iii

m3

L~R-

m2

V A R K - IVKMTK- Q PAKKKP- P P S R E K K ~ ~ T ~

m5

m4

B

TM

Ci3

FALKTRSQ I TKRK~'VlSL mK E K ~ i Q ~ I ~ ~ A r LL

I I I

V~K-

I I

AILL

FAS I~e~QVRKKRQM- ~ R E R ~ I R T i Z l ~ A I EL * *# #* * # # * # # * * * *

120 -]

m3 residues substituted into the m2 mAChR

1

100 c- ~

a~ ~ m o -g ~ o~ ~'~

"

Y = Tyr254

8060 40

" .

~

-T~ ~

--]~

Ci3 = A A*= L = S =

Lys464-Ser493 Ala488 Ala489 Leu492 Ser493

20

Figure 9. Four amino acids at the C-terminus of the i3 loop of the m3 mAChR play an important role in receptor-mediated PI hydrolysis [39]. (A) Amino acid sequences of the C-terminal portion of the i3 loop of the ml-m5 mAChRs (except for m3 [rat], human sequences are shown). Boxed residues were targeted by sitedirected mutagenesis [39]. For additional explanations, see legend to Fig. 8. (B) see Fig. 8B (data taken from ref. 39).

42

494 "k41~7 490

B

4~l.k

.384

~

)2 "k

4t

O

0

488

493

O 489

385

4~6~r 492

390

O 386

393 ~'L

389

Figure 10. Helical wheel models of the m3 (A) and m2 mAChR (B) sequences located at the i3 loopfrM VI junction (for linear sequences, see Fig. 9A). The direction of view is from the N- to the C-terminus. Residues highlighted in black are thought to be critically involved in recognition of Gq/ll (m3 mAChR; ref. 39) and Gi/o (m2 mAChR; ref. 46), respectively. *, Positions at which all five mAChRs have identical residues.

I

A F K V N K Q L

II

N N V T K

III

y F

IV

RD

(~K 183 T T

. E:

1~O

IT

171

V

O 172

IR 9

T

254

48~(i~

o A K Te 9

P L T Y 176

i2

VI

K'. EL

FT

E

i3 eOeeooee272 S

K

N

RTTFKT

K

E.

R~ T 9

VII

K I L

AG L Q A S GT - " 2 0 8 a a

LCQ LL 25 aa

i4

OI A

,,~ (COoH)

Cytoplasm

Figure 11. Residues in the m3 mAChR (rat) critically involved in selective recognition of Gq/ll [39]. Only the intracellular receptor regions and a few ~mino acids of TM I-VII are shown. Besides the nine ~mino acids highlighted in black, optimum activation of Gq/ll proteins appears to require one or more additional residues in the N-terminal portion of the i3 loop (stippled sequence; ref. 39).

43 functional role in determining the selectivity of G protein recognition as described here for a Gq-coupled mAChR. I d e n t i f i c a t i o n of a receptor/G p r o t e i n c o n t a c t site To better understand how receptors and G proteins interact with each other at a molecular level, the regions on the G proteins with which the various intracellular receptor domains (see above) can functionally interact need to be identified. Biochemical studies suggest that at least three distinct regions on the GTPase domain of the G protein a-subunits, including the N- and the C-terminus, can directly contact the receptor [40,41]. These regions clearly differ from those predicted to be involved in the activation of effector enzymes such as adenylyl cyclase [42] or phospholipase C [43]. The C-terminus of the G protein a-subunits (Ga) is known to play a key role in receptor/G protein interactions [40,41,44]. Con]din et al. [45] recently showed that m u t a n t versions of Gaq in which the last five amino acids of aq were replaced with the corresponding sequences derived from different members of the Gai/o protein family (ai2, ao, or az) allowed stimulation of PLC by receptors (A1 adenosine and D2 dopamine receptors) that otherwise are exclusively coupled to G proteins of the Gi]o class which cannot activate PLC efficiently. Recently, we obtained essentially similar results with the m2 mAChR (Fig. 12; ref. 46). In COS-7 cells, the m2 mAChR, a prototypical Gi/o-coupled receptor, stimulates PI hydrolysis only poorly, even in the presence of overexpressed wild type Gaq (q(wt); Figs. 12, 13). However, coexpression of the m2 receptor with qi5 or qo5 (mutant Gaq-subunits in which the last five amino acids of q(wt) were replaced with the corresponding ai2 or ao sequences, respectively) led to a pronounced stimulation (4-8-fold) of PLC activity (Figs. 12, 13; ref. 46). The specificity of this interaction could be demonstrated by the relative lack of PLC activity observed after coexpression of the m2 receptor with a mutant aq-subunit (qs5) that contained five amino acids of as sequence at its C-terminus [46]. Taken together, these results suggest that the m2 mAChR as well as other Gi/o-coupled receptors contain a structural motif that can specifically recognize the C-terminus of Gai/o-subunits and that this interaction is critical for the specificity of receptor/ G protein interactions and G protein activation. To identify this receptor site, a series of mutant m2 receptors were created in which distinct intracellular segments were systematically replaced with the corresponding sequences derived from receptors (m3 receptor, Gq-coupled; ~2adrenergic receptor, Gs-coupled) that couple to G proteins different from Gi/o. It seemed reasonable to assume that structural modification of the m2 receptor region(s) that can contact the C-terminus of Gai/o-subunits should prevent m2 receptor-mediated activation of m u t a n t aq-subunits such as qo5 or qi5. We found [46] that the ability of the m2 receptor to productively interact with qo5 or qi5 was not affected by substitutions involving the il and the i2 loops, the Nterminal segment of the i3 domain, or the C-terminal tail (i4). In contrast, replacement of the C-terminal portion of the i3 loop of the m2 receptor (residues 361-390) with the homologous m3 or ~2-adrenergic receptor sequence almost completely abolished qo5/qi5-mediated PLC stimulation [46]. This observation suggested that the C-terminal segment of the i3 loop of the m2 mAChR plays a specific role in the recognition of the C-terminus of Gai/o-subunits. To further test this hypothesis, a series of gain-of-function mutagenesis

44

Receptor

Max. increase in IP1 (above basal)

G protein a-subunit

m2(wt)

+

I

m2(wt)

+

I

II

G a i 2 or G a o sequence

G a q (=

I

q(wt))

< 2-fold

i

qi5 or qo5

4-8-fold

Figure 12. Functional interaction of the wild type m2 mAChR with mutant G protein aq-subunits [46]. COS-7 cells were cotransfected with expression plasraids coding for the wild type m2 mAChR and wild type Gaq (q(wt)) or mutant Gaq-subunits in which the C-terminal five amino acids of Gaq (EYNLV) were replaced with the corresponding sequences from Gai2 (qi5; DCGLF) or Gao (qo5; GCGLY). Cells were then stimulated with the agonist carbachol (1 raM), and increases in intracellular IP1 levels were determined [46].

m2 i i m2 (wt) CR2

'

CR13 CR14 CR15

, , i

CR16

I

Max. increase in IP1 (above basal)

m3 r-~ tl

ill

iv

v

I

'

'

+

i '

qo5 +-I-++

+

+

+

++++

~.

+

++++

l,~, VT~IL

+

+-I--I-+

.,j);

VT CR17

q(wt)

vt vtt

,

~..

+

+

+

+

IL.

Figure 13. Functional interaction of mutant m2/m3 mAChRs with wild type Gaq (q(wt)) or the mutant Gaq-subunit, qo5. COS-7 cells were cotransfected with the indicated mutant receptors and wild type Gaq (q(wt)) or a mutant Gaq-subunit in which the last five amino acids of Gaq were replaced with the corresponding sequence from Gao (qo5; see Fig. 12). Cells were then stimulated with the agonist carbachol (1 raM), and increases in intracellular IP1 levels were determined [46]. The degree of PLC stimulation mediated by the various mutant receptors upon coexpression with q(wt) or qo5 is denoted by + (~2-fold) and ++++ (4-8-fold). In CR15, Va1385, Thr386, Ile389, and Leu390 (m2 receptor sequence) were substituted into CR2 (thus replacing Ala488, Ala489, Leu492, and Ser493, respectively; m3 receptor sequence).

45 studies were designed. Initially, the C-terminal segment of the i3 loop of the m2 mAChR (residues 359-390) was substituted into a mutant m3 receptor, CR2 (Fig. 13), to study whether the resulting hybrid receptor (CR13; Fig. 13) gained the ability to productively interact with qo5. Whereas CR2 was unable to efficiently stimulate PI hydrolysis when coexpressed with either q(wt) or qo5, CR13 gained the ability to productively interact with qo5 (Fig. 13). Further mutational modification of CR13 showed that substitution into CR2 of only four m2 receptor residues (Va1385, Thr386, Ile389, and Leu390), predicted to be located at the i3 loopfrM VI junction, was sufficient to confer on the resulting mutant receptor (CR15) the ability to potently stimulate PLC activity when coexpressed with qo5 (Fig. 13). Coexpression of CR15 with qi5, but not coexpression with q(wt) or qs5, also resulted in a pronounced activation of the PLC pathway [46]. Substitution into CR2 of m2Va1385Thr386 or m2Ile389Leu390 alone resulted in m u t a n t receptors (CR16 and CR17, respectively) which failed to productively interact with qo5 (Fig. 13). Introduction of the %VrIL motif' (m2Va1385, m2Thr386, m2Ile389, and m2Leu390) directly into the wild type m3 receptor (thus replacing m3 receptor residues Ala488, Ala489, Leu492, and Ser493, respectively) yielded a hybrid receptor (m3(AALS->VTIL))which was able to stimulate PI hydrolysis with high efficiency when coexpressed with qo5 or qi5 (5.5_+0.3-fold stimulation of PLC activity; carbachol EC50 = 0.42+0.07 ~M; Fig. 14). In contrast, the interaction of this mutant receptor with q(wt) was clearly less efficient (3.8_+0.3-fold stimulation of PLC activity; carbachol EC50 = 4.7+_1.5 ~M). This finding is consistent with the observation that the "AALS" motif at the C-terminus of the i3 loop of the m3 mAChR is required for efficient coupling to Gq (see above; ref. 39). Moreover, coexpression experiments with wild type Gai2 showed that the m3(AALS->VTIL) m u t a n t receptor also gained the ability to inhibit adenylyl cyclase (29+3% inhibition of stimulated cAMP levels; wild type m2: 41_+4% inhibition) (Fig. 15; ref. 46). Consistent with these gain-of-function experiments, replacement of the '~VTIL motif' in the wild type m2 mAChR with the corresponding m3 receptor residues resulted in a mutant receptor (m2(VTIL->AALS)) that almost completely lost the ability to functionally interact with qo5 (Fig. 16) or to mediate inhibition of adenylyl cyclase (Fig. 15; ref. 46). Taken together, these results strongly support the concept that the '%ZTIL motif' (formed by m2 receptor residues Va1385, Thr386, Ile389, and Leu390) can specifically recognize the C-terminus of Gai/o-subunits and that this interaction is required and sufficient for Gi/o activation. Except for an m2Leu390->Phe substitution, these four amino acids are also present in the Gi/o-coupled m4 mAChR (but not in the Gq-coupled mAChRs; Fig. 9A). In analogy to the proposed arrangement of the corresponding residues in the m3 mAChR (see previous section), Va1385, Thr386, Ile389, and Leu390 (m2 mAChR sequence) are predicted to be located at the i3 loop/TM VI junction on one side of an a-helical receptor segment (Fig. 10B). It is therefore conceivable that these four amino acids form a receptor surface which can recognize the Cterminus of Gai/o-subunits, most likely via hydrophobic interactions. This interaction is likely to induce conformational changes in the C-terminus of the Gai/osubunits which eventually lead to GDP release and G protein activation. This concept is supported by NMR studies on a C-terminal peptide derived from

46 750 " (D (D

m3(AALS->VTIL)

qo5 + qi5

9 + 9

500 12..Q

o

.=_ ~>

+

q(~)

o

250 -

(D L,_

f: v I

-10

I

-8

I

I

-6

I

I

-4

I

I

-2

Carbachol, log (M)

Figure 14. Functional interaction of the m3 (AALS->VTIL) mutant receptor with wild type Gaq (q(wt)) and mutant Gaq-subunits, qo5 or qi5 (see Fig. 12). In the m3 (AALS->VTIL) mutant receptor, Ala488, Ala489, Leu492, and Ser493 (m3 receptor sequence) were replaced with the corresponding residues present in the m2 mAChR (Va1385, Thr386, Ile389, and Leu390, respectively). COS-7 cells coexpressing the m3 (AALS->VTIL) mutant receptor and wild type Gaq (q(wt)) or mutant Gaq-subunits, qi5 or qo5 (see Fig. 12), were incubated with the indicated concentrations of carbachol, and increases in intracellular IP1 levels were determined. Data are taken from a representative experiment [46]. 5O "~

40

~~

3o

8-~ 20 ~
m2

m3

m2(VTIL m3(AALS ->AALS) ->VTIL)

Figure 15. Inhibition of adenylyl cyclase by wild type and mutant m2/m3 mAChRs. COS-7 cells cotransfected with mAChR DNA and plasmids coding for the V2 vasopressin receptor and wild type Gai2 were studied for their ability to mediate carbachol-induced (0.1 raM) inhibition of [Arg]vasopressin-stimulated cAMP levels [46]. The maximum stimulation of cAMP levels induced by [Arg]vasopressin (0.5 nM) amounted to 13+3-fold above basal levels (100 %). In the m2(VTIL->AALS) and m3(AALS->VTIL) mutant receptors, Va1385, Thr386, Ile389, and Leu390 (m2 receptor sequence) and Ala488, Ala489, Leu492, and Ser493 (m3 receptor sequence) were exchanged between the two wild type receptors. Data are taken from a representative experiment [46].

47 IP1 levels 16

[-7

-

Control (no carbachol) + 1 mM Carbachol

|

o12 X

8

E d. d

4-

0

"

m2(wt) + qo5

m2(VTIL->AALS) + qo5

Figure 16. Functional interaction of the wild type m2 mAChR and the m2(VTIL>AALS) m u t a n t receptor with the mutant Gaq-subunit, qo5 (see Fig. 12). In the m2(VTIL->AALS) m u t a n t receptor, Va1385, Thr386, Ile389, and Leu390 (m2 mAChR sequence) were replaced with the corresponding residues present in the m3 mAChR (Ala488, Ala489, Leu492, and Ser493, respectively). COS-7 cells coexpressing the m2(VTIL->AALS) mutant receptor and the m u t a n t Gaq-subunit, qo5 (see Fig. 12), were incubated with 1 mM carbachol, and increases in intracellular IP1 levels were determined. Data are taken from a representative experiment [46]. at (transducin; another member of the Gai/o protein family) demonstrating that the last four amino acids of Gai/o-subunits form a type II' ~-turn (centered around a glycine residue at position -3 which is specific for all members of the Gai/o family) which is broken upon interaction with the ligand-occupied receptor [47]. In conclusion, this is the first report describing a specific, functionally relevant site of interaction between a GPCR and its cognate Ga-subunits. The receptor/G protein contact site described here may serve as a useful anchoring point for the delineation of three-dimensional models of the receptor/G protein interface. It should be borne in mind, however, that optimum_ receptor/G protein coupling is thought to involve several intracellular receptor domains (see above) and at least three sites on the G protein a-subunits (including the C-terminus; refs. 40,41,44). It is therefore likely that additional interactions, besides the one described above, modulate the specificity of receptor/G protein coupling and further increase the efficiency of G protein activation. It will be interesting to examine whether GPCRs that couple to G proteins different from Gi/o operate through a molecular mechanism similar to that described here for the m2 mAChR.

48 REFERENCES

1 Hulme EC, Birdsall NJM, Buckley NJ. Annu Rev Pharmacol Toxicol 1990; 30:633-673. 2 Wess J. Trends Pharmacol Sci 1993; 14:308-313. 3 Schertler GFX, Villa C, Henderson R. Nature 1993; 362:770-772. 4 Baldwin JM. EMBO J 1993; 12:1693-1703. 5 Baldwin JM. Curr Opin Cell Biol 1994; 6:180-190. 6 Schwartz TW. Curr Opin Biotechnol 1994; 5:434-444. 7 Fraser CM, Wang C-D, Robinson DA, Gocayne JD, et al. Mol Pharmacol 1989; 36:840-847. 8 Wess J, Gdula D, Brann, MR. EMBO J 1991; 10:3729-34. 9 Curtis CAM, Wheatley M, Bansal S, Birdsall NJM, et al. J Biol Chem 1989; 264:489-495. 10 Spalding TA, Birdsall NJM, Curtis CAM, Hulme EC. J Biol Chem 1994; 269:4092-4097. 11 Suryanarayana S, von Zastrow M, Kobilka BK. J Biol Chem 1992; 267:21991-1994. 12 Pittel Z, Wess J. Mol Pharmacol 1994; 45:61-64. 13 Zhou W, Flanagan C, Ballesteros JA, Konvicka K, et al. Mol Pharmacol 1994; 45:165-170. 14 Rao VR, Cohen GB, Oprian DD. Nature 1994; 367:639-642. 15 Elling CE, Nielsen SM, Schwartz TW. Nature 1995; 374:74-77. 16 Liu J, Sch~ineberg T, van Rhee M, Wess J. J Biol Chem 1995; 270:1953219539. 17 Kobilka BK, Kobilka TS, Daniel K, Regan JW, et al. Science 1988; 240:1310-1316. 18 Maggio R, Vogel, Z, Wess J. FEBS Lett 1993; 319:195-200. 19 Maggio R, Vogel, Z, Wess J. Proc Natl Acad Sci USA 1993; 90:3103-3107. 20 Sch/ineberg T, Liu J, Wess J. J Biol Chem 1995; 270:18000-18006. 21 Ridge KD, Lee SSJ, Yao LL. Proc Natl Acad Sci USA 1995; 92:3204-3208. 22 Popot J-L, Engelman DM. Biochemistry 1990; 29:4031-4037. 23 Hedin, KE, Duerson, K, Clapham, DE. Cell Sig 1993; 5:505-518. 24 Berstein G, Blank JL, Smrcka AV, Higashijima T, et al. J Biol Chem 1992; 267:8081-8088. 25 Offermanns S, Wieland T, Homann D, Sandmann J, et al. Mol Pharmacol 1994; 45:890-898. 26 Smrcka AV, Hepler JR, Brown KO, Sternweis PC. Science 1991; 251:804807. 27 Peralta EG, Ashkenazi A, Winslow JW, Ramachandran J, et al. Nature 1988; 334:434-437. 28 Parker EM, Kameyama K, Higashijima T, Ross EM. J Biol Chem 1991; 266:519-527. 29 Wess J, Brann MR, Bonner TI. FEBS Lett 1989; 258:133-136. 30 Wess J, Bonner TI, D6oe F, Brann MR. Mol Pharmacol 1990; 38:517-523. 31 Lechleiter J, Hellmiss R, Duerson K, Ennulat D, et al. EMBO J 1990; 9:4381-4390. 32 Strader CD, Sigal IS, Dixon RAF. FASEB J 1989; 3:1825-1832.

49 33 Cheung AH, Huang R-RC, Strader CD. Mol Pharmacol 1992; 41:10611065. 34 Arden JR, Nagata O, Shockley MS, Philip M, et al. Biochem Biophys Res Commun 1992; 188:1111-1115. 35 Blfiml K, Mutschler E, Wess J. J Biol Chem 1994; 269:402-405. 36 Blfiml K, Mutschler E, Wess J. J Biol Chem 1994; 269:11537-11541. 37 Bliiml K, Mutschler E, Wess J. Proc Natl Acad Sci USA 1994; 91:79807984. 38 HSgger P, Shockley MS, Lameh J, Sadee W. J Biol Chem 1995; 270:74057410. 39 Blin N, Yun J, Wess J. J Biol Chem 1995; 270:17741-17748. 40 Harem HE, Deretik D, Arendt A, Hargrave PA, et al. Science 1988; 241:832-835. 41 Co~klin BR, Bourne HR. Cell 1993; 73:631-641. 42 Berlot CH, Bourne HR. Cell 1992; 68:911-922. 43 Arkinstall S, Chabert C, Maundrell K, Peitsch M. FEBS Lett 1995; 364:45-50. 44 Rasenick MM, Watanabe M, Lazarevic MB, Hatta S, et al. J Biol Chem 1994; 269:21519-21525. 45 Conklin BR, Farfel Z, Lustig KD, Julius D, Bourne HR. Nature 1993; 363:274-276. 46 Liu J, Conklin BR, Blin N, Yun J, Wess J. Proc Natl Acad Sci USA (in press). 47 Dratz EA, Furstenau JE, Lambert CG, Thireault DL, et al. Nature 1993; 363:276-281. 48 Matsui H, Lazareno S, BirdsaU NJM. Mol Pharmacol 1995; 47:88-98. 49 Bonner TI, Buckley NJ, Young AC, Brann MR. Science 1987; 237:527-532.

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Perspective in Receptor Research D. Giardin~, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

51

Muscarinic Receptor Subtypes - Search for Selective Agonists and Antagonists

E. Mutschler a, H. A. Ensingerb, J. Gross a, A. Leis c, K. Mendla b, U. Moser a, O. Pfai~, D. Reichel d, K. ROhlmannc, R. Tacke d, M. Waelbroecke, J. Wehrle a and G. Lambrecht a aDepartment of Pharmacology, Biocentre Niederursel, University of Frankfiart, Marie-CurieStr. 9, D-60439 Frankfurt/Main, Germany bBoehringer Ingelheim KG, Binger Str., D-55216 Ingelheim, Germany Clnstitute of Inorganic Chemistry, Technical University of Dresden, Mommsen-Str. 13, DO1069 Dresden, Germany dInstitute of Inorganic Chemistry, University of Wiarzburg, Am Hubland, D-97074 W0rzburg, Germany eDepartment of Biochemistry and Nutrition, Medical School, Free University of Brussels, B1070 Brussels, Belgium ABSTRACT Since the late eighties five muscarinic receptor subtypes (ml - m5) have been cloned and four of them (M 1 - M4) have also been pharmacologically characterized. However, there is still a lack of potent muscarinic agonists and antagonists, which are highly selective for one muscarinic receptor subtype over all other subtypes. For the treatment of Alzheimer's disease, Ml-selective agonists capable of penetrating into the CNS are needed. It is hypothezised that such substances would not only improve memory and cognitive ability, but also delay the progression of the disease. In our laboratory, the functionally M 1-selective quaternary ammonium compound McN-A-343 has been used as a starting point for the design of such CNS active muscarinic ligands. Structure-activity relationship studies led to the tertiary amine 4-(4-fluorophenylcarbamoyloxy)-2-butynylpyrrolidine (4-FPyMcN), which was found to stimulate M 1 receptors with some functional selectivity. In order to increase the potency and selectivity of 4-F-PyMcN several new derivatives were synthezised and pharmacologically characterized in different functional assays as well as in binding and biochemical (PI turnover) studies. The most promising results were obtained with (S)4-(4-fluorophenylcarbamoyloxy)-l-methyl-2-butynylpyrrolidine (4-F-MePyMcN). Due to its potent partial agonistic activity at M 1 receptors and its M2-antagonistic properties leading to an increase of acetylcholine release by blockade of M2 autoreceptors, this compound may be considered as an important tool for future drug research of cognitive disorders. M 2 receptor antagonists may also be used for the treatment of Alzheimer's disease, furthermore in the therapy of supraventricular bradycardia and for quantifying M2 receptors in the CNS with PET imaging. In the search for antagonists which clearly differentiate M 2 from other muscarinic receptors, we investigated the two enantiomers of the widely used Hi-antihistaminic drug dimethindene. (S)-Dimethindene proved to be a potent M2-selective antagonist with lower affinities for the M1, M 3 and M4 receptors. In addition, the (S)-enantiomer

52 was more potent than the (R)-enantiomer in all muscarinic assays. Interestingly, the stereoselectivity was inverse at histamine H1 receptors, the (R)-enantiomer being the eutomer. M 3 receptor antagonists may be useful in the treatment of spastic disorders of the gastrointestinal, urogenital and respiratory tract as well as for the relief of glandular hypersecretion. In previous studies, hexahydro-difenidol (HHD) and its sila-analogue, hexahydro-sila-difenidol (I-~SiD), as well as the antiparkinsonian drug trihexyphenidyl (THP) were found to be valuable tools for the discrimination of M 3 and M 2 receptors. In order to further assess the structural requirements (including stereochemical aspects) of the above-mentioned compounds for potency and selectivity, a series of ttHD and THP analogues as well as of the corresponding silicon and germanium derivatives (sila- and germa-substitution) were studied. The (R)-enantiomers displayed higher affinities and selectivities than the corresponding (S)-isomers. The enantioselectivity of some of these analogues is best explained by the concept of the fourbinding-subsite model suggesting that the differences in affinity of the (R)- and (S)-enantiomers at muscarinic receptors are due to opposite binding of the phenyl and the cyclohexyl ring to the preferring subsites. Surprisingly, there was no significant difference between the Si and Ge analogues indicating a strongly pronounced Si/Ge bioisosterism in this series of compounds. The related carbon derivatives, however, showed higher receptor affinities as well as greater stereoselectivities at all muscarinic receptors studied compared with the silicon and germanium analogues. INTRODUCTION Four muscarinic receptor subtypes (M 1 - M4) have been defined by pharmacological studies. On the other hand, receptor cloning studies revealed the existence of five different but highly homologous muscarinic receptor gene products (ml - m5). The pharmacological classification of muscarinic receptor subtypes is mainly based on the relative sensitivities of the receptor subtypes to key muscarinic antagonists such as pirenzepine (M1 > M4 > M3 > M2), 11-[[2-[(diethylamino)methyl]- 1-piperidinyl]acetyl]-5,11-dihydro6H-pyrido-[2,3-b][1,4]-benzodiazepin-6-one (AF-DX 116) and related compounds, methoctramine and himbacine (M2 > M 4 > M 1 > M3) as well as hexahydro-sila-difenidol and its pfluoro derivative (M 3 > M 1 > M 4 > M2) [for recent reviews see 1-4]. In addition, muscarinic receptors may be differentiated on the basis of their stereoselectivity to chiral antagonists [57]. However, it should be noted that in contrast to antagonists of other neurotransmitters, e.g. of histamine or serotonin, most of these antimuscarinic agents are not highly selective for one receptor subtype over all other subtypes. Thus, the search for subtype-selective muscarinic and antimuscarinic ligands is still of great importance. Ml-selective agonists capable of penetrating into the CNS may be important for the treatment of Alzheimer's disease [8-10]. It is hypothezised that such substances would not only improve memory and cognitive ability, but also delay the progression of the disease by enhancing the formation of the neuroprotective secreted amyloid precursor protein (APPs) and by inhibiting the production of the neurotoxic amyloid B-protein [11-13]. M 2 receptor antagonists may be applied also for the treatment of Alzheimer's disease [9, 14-16], furthermore in the therapy of supraventricular bradycardia [17-19] and for quanti~ing M2 receptors in the CNS with PET imaging [20].

53

M 3 receptor antagonists may be useful in the treatment of spastic disorders of the gastrointestinal, urogenital and respiratory tract as well as for the relief of glandular hypersecretion [21-241. In the following we will concentrate on Ml-selective agonists as well as on M 2- or M3-selective antagonists, respectively. METHODS

Agonism and antagonism of the compounds for muscarinic receptors have been measured in functional and binding experiments using the preparations listed below:

Functional experiments (pD 2 and pA2 values) M1 :Rabbit vas deferens (RVD) [25] Rat duodenum (RD) [26] M2: Guinea-pig left atria (GPA) Rabbit vas deferens [27] M3: Guinea-pig ileal longitudinal muscle (GPI) Guinea-pig trachea (GPT) [28]

Binding experiments (pK i values) Binding affinities were determined in NB-OK 1 cells (M 1 receptors) and rat heart (M2 receptors), pancreas (M 3 receptors) and striatum (M4 receptors) homogenates using [~H]-Nmethylscopolamine as radioligand [29]. Binding affinities of the compound (S)-F-MePyMcN were measured in membrane preparations from CHO-cells transfected with human ml to m4 receptors using [3H]-N-methylscopolamine as radioligand, too. The assay was carried out according to [7].

Biochemical experiments PI turnover was measured in CHO-cells transfected with human ml receptors [30]. RESULTS AND DISCUSSION

M 1 agonists with additional M2-antagonistic properties~ M1 receptors are particularly enriched in two regions of the central nervous system, the cortex and the hippocampus, known to play an important role in learning and memory processes. Activation of these postsynaptically located muscarinic receptors causes cellular excitation. In contrast, M 2 receptors appear to be most often expressed as presynaptic autoreceptors mediating the inhibition of acetylcholine release [31 ]. Based on this knowledge, an ideal compound for the treatment of Alzheimer's disease would act as a selective postsynaptic M1 agonist and as a selective presynaptic M 2 antagonist. The functional Ml-selective agonist McN-A-343 (Figure 1) has been used in our laboratory as a starting point for the design of CNS-active muscarinic ligands, the primary objective being

54 to replace the quaternary ammonium group of McN-A-343 by a tertiary amino moiety. The chemical structures of the new compounds are given in Figure 1.

X , ~ H

0 II

R1 I

N-C-O-C-C-C-CH2-Am I

H

R1 = H, CH 3, C2Hs

X 3-CI, -F

Am (R2= H, CH3) *NR2(CH3)2 /\

4-Br, -CI, -F

+NR2CH3C2Hs

3,4-dichloro

*NR2(C2Hs)2

Figure 1. Chemical structure of tertiary and quaternary analogues of McN-A-343 [X = 3-C1, Am = +N(CH3)3, R1 = H], 4-F-MePyMcN +" X = 4-F, Am = N-methylpyrroli-dinium, R 1 = CH3; 4-F-MePyMcN: X = 4-F, Am = pyrrolidino, R 1 = CH3; 4-CI-McN-A-343" X = 4-C1, Am = +N(CH3)3, R 1 = H; 4-F-PyMcN: X = 4-F, Am = pyrrolidino, R 1 = H; 4-F-PyMcN+: X = 4-F, Am = N-methylpyrrolidinium, R1 = H. The functional investigations of these compounds revealed that all agonist responses were muscafinic in nature in that their activities were blocked by: pirenzepine (0.1 ~tM; pA 2 = 8.14 8.51) in rabbit vas deferens (M1), AQ-RA 741 (100 - 300 riM; pA 2 = 8.31 - 8.49) in guineapig atria (M2) and p-F-HHSiD (0.5 ~tM, pA 2 = 7.51 - 7.91) in guinea-pig ileum (M3). Tetrodotoxin (0.1 ~tM) and hexamethonium (100 ~tM) did not block agonist activities in guinea-pig atria and ileum. The potency (pEC50), apparent efficacy (a.e.) and affinity (PA2) for McN-A343 and some of its analogues are shown in Table 1. From these data it becomes evident that the effects of McN-A-343 and of most of its analogues differ clearly in the three functional assays. McN-A-343 and its 4-chloro derivative had comparable potency in rabbit vas deferens, whereas in both atria and ileum they behaved as partial or full agonists, being more potent and more efficacious in the M 3 assay. The incorporation of chirality into this series of acetylenic McN-A-343 derivatives had a beneficial effect upon the potency (affinity) and muscafinic receptor subtype-selectivity, the (S)-enantiomers consistently showing higher activity than the corresponding (g)-isomers.

55 Table 1

In vitro functional activity of McN-A-343 analogues at M 1 receptors in rabbit vas deferens (RVD), M 2 receptors in guinea-pig atria (GPA) and M 3 receptors in guinea-pig ileum (GPI). Abbreviations for the muscarinic agems are explained in the legend of Figure 1 RVD/M 1 pEC50 a.e. a

pA 2

GPA/M 2

GPI/M 3

pECs0 a.e.b-- pA 2 '

pEC50 a.e.--c

pA2

5.51g 5.71 . -

5.76_h 6.42_.h_h 5.29 h 6.82h

i

McN-A-343 d 4-CI-McN-A-343 d (R)-a-F-MePyMcN + (S)-4-F-MePyMcN + (R)-4-F-MePyMcN (S)-4-F-MePyMcN

6.77 e 7.13 6.37 7.25 7.22

1.00 1.00 0.27 0.25 0.83

-

5.65 h 7.26_.h_h 5.41 h 7.11h

4.87-I5.26 -

0.49 0.77 _ _ -

5.90 h 6.33 h 5.60_h 7.39h--

0.83 1.00 _ _ -

a,b_,_c Apparent efficacy: the maximum response to McN-A-343 a and to arecaidine propargyl esterb-,-c = 1.00. d Data taken from Lambrecht et al. [32]. e pK A (= 5.17) was estimated using irreversible antagonism [33]. _f,g pK A values determined by the technique of Waud [34] and using arecaidine propargyl ester as full agonist, were: M 2 = 4.79, M 3 = 5.17. h Slopes of Schild plots were not significantly different from unity (P>0.05). Among the amino terminally modified analogues of McN-A-343, the tertiary amine, (S)-4F-MePyMcN, was a potent partial agonist at M 1 receptors in rabbit vas deferens. In contrast, at M 2 receptors in atria and at ileal M 3 receptors (S)-4-F-MePyMcN lacked efficacy and was shown to act as a competitive antagonist with pA 2 values of 7.4 and 6.8, respectively. Unexpectedly, the corresponding quaternary analogue, (S)-4-F-MePyMcN +, was found to be a relatively potent M 1-selective antagonist. The (R)-enantiomers of 4-F-MePyMcN and 4-FMePyMcN + were weak non-selective muscarinic antagonists (pA 2 values = 5.3 - 5.9). The stereoselectivity ratios (= antilogs of the difference of respective pA 2 values) for 4-FMePyMcN at M 1, M 2 and M 3 receptors were very similar [(S)/(R) = 50, 63 and 32, respectively]. In contrast, these ratios of the quaternary analogue, 4-F-MePyMcN +, differed in the three functional pharmacological assays. They were very low at M 2 and M 3 [(S)/(R) = 2.5 and 4, respectively], but high at M 1 receptors [(S)/(R) = 40]. This implies that the stereochemical requirements of the muscarinic receptor subtypes are different for the enantiomers of 4-F-MePyMcN +, being most stringent at M 1 receptors. The most promising compound (S)-4-F-MePyMcN was further assessed for its affinity to muscarinic receptor subtypes, m l - m4, in binding experiments as well as for its agonistic properties at ml receptors expressed in CHO-K1 cells by measuring the stimulation of the PI turnover. The binding affinities (pK i values: ml = 7.16, m2 = 7.21, m3 = 6.71, and m4 = 7.04) were very similar to the affinities obtained in functional studies (Table 1). The muscarinic receptor-mediated PI turnover in CHO-hml cells of this compound was found to be 40 % of the maximum effect evoked by carbachol, the EC50 value being in the lower nanomolar range. Thus, the high potency and functional M 1 selectivity of (S)-4-F-MePyMcN make this compound suitable for the study of muscarinic receptor mechanisms. Due to its M l-agonistic and M2-antagonistic properties (S)-4-F-MePyMcN may be considered as an important tool for future drug research in the field of cognitive disorders. Its partial agonistic nature may be ad-

56 vantageous in therapeutic applications, as chronic administration of the drug would be less likely to result in receptor desensitization.

M 2 receptor antagonists. Muscarinic M 2 receptors have been reported to be depleted in post mortem brains from patients with Alzheimer's disease [9]. This has raised the interest of developing M2-selective muscarinic receptor antagonists, capable of penetrating into the central nervous system, that could be useful both as diagnostic tools for quantif~ng the loss of muscarinic M 2 receptors with positron emission tomography imaging [20], and as therapeutic agents (see introduction). Most of the substances described in the literature as M2-selective muscarinic antagonists, however, cannot distinguish between M 2 and M 4 receptors. In the search for antagonists which clearly differentiate M 2 from M1, M 3 and M 4 receptors we investigated the two enantiomers of the widely used Hi-antihistaminic drug dimethindene (Figure 2) [35, 36]. It has been found that the enantiomers of dimethindene behaved as simple competitive muscarinic antagonists. Functional (pA 2 values) and binding (pK i values) affinities for the two enantiomers are shown in Table 2 and 3. In general, (S)-dimethindene was more potent than the (R)-enantiomer in all muscarinic assays. However, the stereoselectivity ratios were found to be different at the four muscarinic receptor subtypes, being greatest at M 2 receptors (32- to 41-fold). In contrast, the stereoselectivity was inverse at histamine H I receptors, (R)-dimethindene being the eutomer (Table 2). The pA 2 values for (S)-dimethindene at M 1 receptors in rat duodenum and rabbit vas deferens were not significantly different (P>0.05) either from each other or from pA 2 values determined at M 3 receptors mediating contractions of the guinea-pig ileum and trachea or from the pK i value determined at M 4 receptors in rat striatum. However, the pA 2 (PKi) values for (S)-dimethindene at muscarinic M 2 receptors were significantly (P<0.05) greater (up to 32fold) than those obtained at M 1, M 3 and M4 receptors. The functional affinity estimates (Table 2) of(R)- and (S)-dimethindene at M1, M 2 and M 3 receptors were highly correlated (r = 0.98) with their binding affinities (Table 3) in NB-OK 1 cells and rat heart and pancreas. This correlation supports the suggestion that the receptor subtypes studied in the functional preparations are equivalent to the muscarinic binding sites in the tissues investigated. As far as muscarinic receptor specificity is concerned, no specific binding (up to a concentration of 10 laM) was found for (S)-dimethindene by Nicholson et al. [37] at ~2 and 13 adrenoceptors, 5I-IT1 receptors as well as at benzodiazepine receptors. With respect to ~ 1 adrenoceptors, 5HT 2 receptors and dopamine D 2 receptors, interactions of (S)-dimethindene occur only at concentrations in the micromolar range (PKi: c~1 = 6.3; 5-HT 2 = 5.7; D 2 = 6.5).

CH2--- CH2--N

/ \

CH 3 CH3

H3

Figure 2. Chemical structure of (R)/(S)-dimethindene. The asterisk denotes the centre of chirality.

57 Table 2

Affinity estimates (pA2 values) for (R)- and (S)-dimethindene at various functional muscarinic receptor subtypes PA2 value~ Subtype M1 M1 M2 M2 M3 M3 HI

Preparation

(R)-Dimethindene

Rabbit vas deferens-b-b Rat duodenun~c Guinea-pig atriad Rabbit vas deferens-e Guinea-pig ileumd Guinea-pig trachead Guinea-pig ileum-f

5.81 5.49 6.25 6.22 5.61 5.59 9.42

(S)-Dimethindene

ii

6.83 6.36 7.86 7.74 6.92 6.96 7.48 ,i

a Slopes of Schild plots were not significantly different from unity (])>0.05). Thus, pA2 values were calculated from regression lines whose slopes were constrained to 1.00. b.c,d,e,_f, Agonists used: 4-CI-McN-A-343 b (Figure 1), 4-F-PyMcN+-c (Figure 1), arecaidine propargyl esterd-, carbachol -e, histaminef. In conclusion, the experiments demonstrate that (S)-dimethindene is a novel M2-selective muscarinic antagonist. Since studies in man showed that (S)-dimethindene penetrates readily into the brain [35-37], this compound could be a valuable tool to test the hypothesis that lipophilic M2-selective antagonists show beneficial effects in the treatment of Alzheimer's disease. (S)-Dimethindene might also become the starting point for the development of appropriate PET ligands useful as antemortem diagnostic tools for quantifying the loss of M 2 receptors in the brain of Alzheimer's patients. One interesting aspect of using chiral antagonists, such as dimethindene, in PET studies is the availability of the less potent optical isomer, which can be used to measure nonspecific binding under the imaging condition. Table 3

Apparent affinities (pK i values) for (R)- and (S)-dimethindene at muscarinic binding sites in various tissues and cell lines pKi values Subtype

Tissue/Cell Line

(R)-Dimethindene

(S)-Dimethindene

M1 M2 M3 M4

NB-OK 1 cells Rat heart Rat pancreas Rat striatum

5.6 6.3 5.6 6.0

7.1 7.8 6.7 7.0

Muscarinic binding sites were labelled using [3H]N-methylscopolamine. Hill coefficients of the competition curves were not significantly different from unity (P>0.05).

58

Antagonists with high affinity for M 3 receptors. The muscarinic antagonist (R)-hexahydro-difenidol [(R)-I: HHD] and especially its racemic sila-analogue hexahydro-sila-difenidol (2: HHSiD) are widely used as selective antagonists for the classification of muscarinic receptor subtypes. Both compounds exhibit up to 50-fold higher affinity to M1 and M 3 (as well as to M4) receptors than to M 2 receptors (Figure 3) [38, 39]. In addition, the configurationally stable enantiomers of HHD display high stereoselectivity at M 1 and M 3 receptors (up to 500fold), but ~'ather low stereoselectivity at M 2 receptors (16-fold)[38]. Another compound structurally related to this series is the aminoethyl analogue of (K)HHD, i.e. the (K)-enantiomer of the antiparkinsonian drug trihexyphenidyl (7, THP). (R)-THP proved to be an Ml-selective muscarinic antagonist with intermediate affinity at M 3 and at M 4 and low affinity at M 2 receptors. However, its stereoselectivity profile 0US) of MI=M3=M4 > M 2 is veq' similar to that of HHD (Figure 4) [39]. In contrast to the enantiomers of the carbinols, e.g. HHD or THP, the enantiomers of silanols, e.~. sila-procyclidin, racemize quickly in aqueous solution [40] and are therefore unsuitable for the pharmacological investigation of enantioselectivity. Thus, we wanted to further assess the structural requirements of this kind of compounds for affinity and selectivity on muscarinic receptor subtypes. Accordingly, we replaced the OH group of (R)- and ( S ) - ~ as well as of (R)- and (S)-1vd:P at the central atom by a CH2OH (3, 8), a COOH (5) or CONH 2 (6, 11) group and investigated the influence of this structural change on antimuscarinic potency and stereoselectivity. Furthermore, the exchange of the central carbon atom in the hydroxymethyl analogue o f ~ by a silicon atom (--> 4) as well as of THP by a silicon (-> 9) or a germanium atom (--> 10) led to enantiomers, which were expected to be configurationally stable and therefore valuable for the stereochemical characterization of the interaction of antagonists at muscarinic receptors (Affinity data of compounds 4a, 9 and 9a have already been published [41, 42]). From previous investigations it was postulated, that muscarinic receptors possess four different subsites for antagonist binding, shown e.g. for the carbinols procyclidine and hexahydro-difenidol [43, 44]. Therefore, another aim of this study was to compare the antimuscarinic properties of the enantiomers of the hydroxymethyl-derivatives described above (3, 4, 8, 9) with their related diphenyl- (3a, 4a, $a, 9a) as well as their dicyclohexyl analogues (3b, 4b, $b, 9b) in order to interpret their stereoselective interaction with the different muscarinic receptor subtypes. The following results were obtained: In all cases where the absolute configuration was determined the (R)-enantiomers (eutomers) exhibited higher affinities and stereoselectivities at muscarinic receptor subtypes than the corresponding (S)-enantiomers (distomers) (Figure 3, 4). For the compounds 8, 10 and 11 the configuration is not yet known. However, with respect of the high structural homology and the unique enantioselectivity data [(R) over (S) at muscarinic receptors] found for all compounds in this study, we hypothesize that the absolute configurations of the eutomers of 8, 10 and 11 is (R), too. In the following, eutomers and distomers of these compounds will be given the prefix I and U, respectively. Substitution of the OH group by a CH2OH, a COOH or a CONI-I2 group in the eutomers of HHD and THP resulted in a decrease of affinity, receptor selectivity and stereoselectivity (except stereoselectivity of compound 5) (Figure 5). Nevertheless, all eutomers of compounds 3 - 6 and $ - 12 displayed a selectivity profile of M 1 = M 3 > M 2. In addition, most of these enantiomers exhibited also a stereoselectivity profile of M 1 = M 3 > M 2. Compound 5 with a

59 COOH group - instead of a OH group - at the central carbon atom displayed a reduced affinity at all three receptors, but the enantioselectivity ratios were slightly increased, compared to the parent compound HHD. These results are not in agreement with Pfeiffer's role. Additionally, the eutomer of this carboxylic acid (1-5) displayed higher affinities than its amide 6, and the stereoseleetivity ratios were higher for 5 than for 6. Shortening of the aminopropyl to an aminoethyl chain in the eutomers of the HHD-derivatives (3---)11, 4---)9, 6 ~ 1 1 ) did not change significantly the affinities. In contrast, the stereoselectivity ratios for the enantiomers of these aminopropyVaminoethyl pairs were changed. It was found, that the stereoseleetivity ratios for 3 (aminopropyl series) were higher than those for the related compound in the aminoethyl series (8). Vice versa, the amide of the HHD type (6) exhibited lower eudismic ratios than the amide of the THP type (11).

* /

El

CH2.--CH2.--CH2--N

4

\

5

6

pA2 9.0-

8.0

8.7

r---i (R)-isomer

8.0

7.9

-

E! C Si C Si C C

Compd HHD'I HHSiD: 2

R

8.2

L ~ racemate

II

8.2

R OH OH CH20H CH20H COOH CONH 2

(s)-isomer 7.9

7.7 .,.....,-.

7~ 7,.0

x x

-

7.3

3.7

7.0

7.0

K~

6,,0

5.0

K~

-

K

•

~

x

51.11

-

,cx

~

9 x

4.0

C~

-

M1 M2 M3 1 !

121131

I M1 M2 M3 M1 M2 M3 M1 M2 M3 ! 4 II S !1 6 1

Figure 3. Structures and affinity profiles (pA2 values) of (R)- and (S)-enantiomers of compounds 1 - 6 at muscarinic M 1 (rabbit vas deferens), M 2 (guinea-pig atria) and M 3 receptors (guinea-pig ileum); *center of chirality.

60 Compd R2

./

PA2 10.0 -

,,

R1 9.6

El

THP: 7 8

/

\

CH2_CH2.__ N \ "

-

8~

-

/

6.0

-

5.0

-

Com -I

10 11 Fenpipramide: 12

R2 OH CH20H CH20H CH20H CONH 2 CONH2

~

distomer -b

8.2

IK~

73 9

7.1

7.3 --

7.1

7.2

ii

M1 M2 M3 M1 M2 M3 M1 M2 M3 M1 M2 M3 M1 M2 M3

7

1 l 8

9.1

8.: ~>

7.4 -

9

racemate

I eutomer -a

8.2

7.0

\

R1 c-Hex c-Hex c-Hex c-Hex c-Hex Ph

9.4

I 9.0

,

El C C Si Ge C C

I !

'

I I

10

I

I

11

!

K~x

Ig>

KY

Ix> lg>

g:~ K)<

IK) la;)

gx K)<

Ix>

g~

Ig>

K)<

D(>

KX

!~

Kx

IX'>

~-)<

iIx~

<x

M1 M2 M3 I

12

I

Figure 4. Structures and affinity profiles (pA2 values) of enantiomers of compounds 7 - 11 and of the achiral compound 12 at muscarinic M1 (rabbit vas deferens), M 2 (guinea-pig atria) and M 3 receptors (guinea-pig ileum); *center of chirality; a and b (R)- and (S)-isomers of compounds 7, 9 and 10, respectively; Ph = Phenyl; c-Hex = Cylohexyl; THP = trihexyphenidyl. Replacement of the central carbon atom by a silicon atom in the (R)-enantiomers of the hydroxymethyl analogue of HHD (3-->4) and THP (8->9) lowered atfmity and stereoselectivity, whereas the receptor selectivity for compounds 3 and 4 was unchanged. In contrast, the corresponding (R)- and (S)-enantiomers of the Si/Ge analogues exhibited very similar affinities to the different muscarinic receptors indicating a strongly pronounced Si/Ge bioisosterism. Replacement of the cyclohexyl by a phenyl ring (-~ diphenyl analogues: 3a, 4a, 8a, 9a) or of the phenyl by a cyclohexyl ring (--> dicyclohexyl analogues: 3b, 4b, 8b, 9b) of the hydroxymethyl C/Si analogues of HIK) (3, 4) and THP (8, 9) diminished affinity to all muscarinic receptors studied compared to the corresponding eutomers (Figures 5 and 6).

61

R1 x

R2"

C

/ \

CH=--CH2--CH2"-'N /

pA2 8.5-

R1

Compd

CH2-OH

\

8.2

'~)

/

V-q

R2

Ph c - H e x (R)-3 Ph Ph 3a 3b c - H e x c-Hex Ph 1s1-3 c - H e x 8.2

8.07.5-

7.1

7.06.56.0-

ApA2

5.5 1.1 1.4 2.1 2.5

AI" [(R)-31 - [3al A 2 : [ ( R ) - 3 1 - [3b] E.I.: [[R)-3] - [(S)-3] A I + A 2 = expected E.I.:

RI

X

CH2-OH

/

,, Si \

R2

CH2--CH2--CH2--N

pA 2 7.5

I~

/

Compd

RI

R2

(R)-4 4a 4b

Ph

c-Hex

Ph Ph c-Hex c-Hex

I---! (s)-4 c-Hex

\

7.2

0.9 1.3 2-2 2-2

0.4 0.9 1.1 1.3

Ph

7.3

7.0 6.5

6.2

6.05.5-

ApA 2

5.0 -

A 1" [(R)-4] - [4a] A2: [(R)-4] - [4b] EJ.: [(R)-4] - [(S)-41 A I + A 2 = expected E.I.:

0.9 0.8 1.0

0.2 0.6 0.8

0.5 0.8 1.0

1.7

0.8

1.3

Figure 5. pA 2 and ApA2 values of (R)- and (S)-isomers of compounds 3 and 4 as well as of related achiral compounds 3a, 3b, 4a and 4b at muscarinic M 1 (rabbit vas deferens), M 2 (guinea-pig atria) and M 3 receptors (guinea-pig ileum); Ph = Phenyl; c-Hex = Cylohexyl; E.I. = Eudismic index.

62 R1

\

CH2-OH

/

II

/ CX

R2

fl)-8

8a 8b I----1 (11)-8

CH 2-- CH2 --N/~

pA 2 8 . 5 -

Compd

8.2

R1

R2

Ph

c-Hex

Ph

Ph

c-Hex c-Hex c-Hex Ph 8.2

8.0--

7.5

7.2 7.2

7.06.56.0-

APA2 A 1" [(!)-8] -

5.5 [8a]

R1 \

R2 pA2/pKi

1.5 1.6

/

/

0.0 0.7 0.7 0.7

0.6 1.0

A2: [(I)-8] - [8b] E.I.: [(I)-8] - [(11)-8] AI+A2 = expected E.I.:

SiX

2.0

CH2-OH I~

CH2--CH2 - N

7.4

7.5

0.9 1.1 1.6

7.3

7.0

Compd

R1

R2

(R)-9

Ph

c-Hex

9a

Ph

Ph

9b c-Hex c-Hex r---I (s}-9 c-Hex Ph 7.2

6.8 ~ 7

6.5

'

6.0 5~

APA2/PKi

5.0

A 1" [(R)-9] -

[9a]

-

A2: [[R)-9] - [9b] E.I.: [(R)-9] - [ ( S ) - 9 ] • 1 + A 2 = expected E.I.:

0.4 0.6 0.9

0.1 0.6 0.5

0.5 0.5 1.1

0.4 0.4 0.8

1.0

0.7

1.0

0.8

Figure 6. pA 2 (PKi) and ApA2 (ApKi) values of the enantiomers of compounds 8 and 9 as well as of related achiral compounds 8a, 8b, 9a and 9b at muscarinic M 1 (rabbit vas deferens), M 2 (guinea-pig atria) and M 3 receptors (guinea-pig ileum); Ph = Phenyl; c-Hex = Cylohexyl; E.I. = Eudismic index.

63 However, these achiral analogues displayed still higher affinities to the muscarinic receptors than the corresponding distomers. In contrast to these results, the diphenyl derivative with a CONH 2 moiety (fenpipramide, 12) showed up to 220-fold higher atY~ties than the related eutomer I - l l at the muscarinic receptors. According to the concept of the four-binding-site model [43, 44], it is suggested that muscarinic antagonists of the given structure (except 11) might be recognized by muscarinic receptors with subsites for the ammonium group, the hydroxyl group and the phenyl and cyclohexyl moiety (Figure 7). As can be seen in Figure 5 and 6, the experimentally obtained eudismic indices for 3, 4, 8 and 9 [pA2(eutomer ) - pA2(distomer)] were in most cases very similar to the expected eudismic indices {[PA2(eutomer) - PA2(diphenyl analogue)] + [PA2(eutomer) - PA2(dicyclohexyl analogue)]} at all muscarinic receptors. Only the expected and the observed eudismic index for compound 4 at M 1 receptors showed a remarkable difference. These results suggest that in most cases the stereoselective interaction of the two enantiomers of these antagonists with muscarinic receptors is based on opposite binding of the phenyl and cyclohexyl ring to site 1 and site 2 as well as on equal binding of the hydroxyl and the ammonium group to site 3 and site 4, respectively (Figure 7). Thus, the stereoselectivity ratios of these compounds are best explained by weaker binding of the phenyl and the cyclohexyl ring of the distomers.

Site I

Site 3

Site I

Site 3

8+...X

.~1~~ CH=-OH

H

CH2-CH=-CH::~Nx - - +,_._., ~ X"

Site 2

_ ~'I"'"ElXcH=-CH=-cH=H~,N+~

LR

f

~lUet4

"~

Figure 7. Interaction of the protonated (R)- and (S)-isomers of compounds 3 (El = C) and 4 (El = Si) with four subsites of muscarinic receptors. Site 1: phenyl-preferring hydrophobic subsite; Site 2: cyclohexyl-preferring hydrophobic subsite; Site 3: polar subsite for the hydroxyl group; Site 4: ionic subsite for the protonated amino group; *center of chiraliV. In contrast to these results, the enantiomers of the amide 11 exhibited an aJLtagonistic binding behaviour which could not be interpreted by the concept of the four-binding-site model. It is therefore suggested that the amides 11 and 12 may interact with other ~;ubsites of muscarinic receptors than the related chiral compounds 3, 4, 8 and 9. In conclusion, the substitution of the hydroxyl moiety in HHD and HHSiD as well as in THP by a CH2OH or a CONH2 group decreased affinity, subtype-selectivity and stereoselectivity. The enantiomers of the carboxylic acid derivative 5 showed the highest sterecselectivity of the compounds tested in this study. However, all these derivatives are able to differentiate M1 and M 3 receptors from M 2 receptors. In addition, most of the stereoselectivirf ratios of

64 these compounds follow the same profile of MI=M3>M2, and may therefore used as an additional parameter to characterize muscafinic receptor subtypes. Their enantioselectivities are best explained by the four-binding-subsite model suggesting that the differences in affinity of (R)- and (S)-enantiomers at muscafinic receptors are due to opposite binding of the phenyl and the cyclohexyl ring to the preferring subsites. ACKNOWLEDGEMENTS The authors thank the Fonds der Chemischen Industrie (Germany), the Deutsche Forschungsgemeinschafl, the Boehringer Ingelheim KG, Ingelheim, and the Fonds for Medical Scientific Research (Belgium) for financial support and gratefully acknowledge the donation of drugs [(R)- and (S)-dimethindene: Zyma GmbH, Munich; (R)- and (S)-trihexyphenidyl: Dr. A. Aasen, Oslo]. REFERENCES

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Hulme EC, Birdsall NJM, Bucldey NJ. Annu Rev Pharmacol Toxicol 1990; 30: 633-673. Levine RR, Birdsall NJM. Life Sci 1993; 52: 405-597. Levine RR, BirdsaU NJM. Life Sci 1993; 56: 801-1053. CaulfieldMP. Pharmac Ther 1993; 58: 319-379. Feifel R, Wagner-R6der M, Strohmann C, Tacke R, et al. G Br J Pharmacol 1990; 99: 455-460. Waelbroeck M, Camus J, Tastenoy M, Mutschler E, et al. Eur J Pharmacol - Mol Pharmacol Sect 1992; 227: 33-42. D6rje F, Wess J, Lambrecht G, Tacke R, et al. J Pharmacol Exp Ther 1991; 256: 727733. Baker R, MacLeod AM. In: Kozikowski AP, eds. Drug Design for Neuroscience, New York: Raven, 1993; 61-85. Quirion R, Aubert I, Lapchak PA, Schaum RP, et al. Trends Pharmacol Sci 1989; 10 (Suppl.): 80-84. Bartus RT, Dean RL, Beer B, Lippa AS. Science 1982; 217: 408-417. Nitsch KM, Wurtmann RJ, G-rowdon JH. Arzneim -Forsch/Drug Res 1995; 3a (Suppl): 435-438. Gandy S, Greengard P. Trends Pharrnacol Sci 1992; 13" 108-112. CordeUB. Annu Rev Pharmacol Toxicol 1994; 34: 69-89. Aubert I, Araujo DM, Cecyre D, Robitaille Y, et al. J Neurochem 1992; 58: 529-541. Gitler MS, Reba RC, Cohen VI, Rzeszotarski WJ, et al. Brain Res 1992; 582: 253-260. Doods H, Entzeroth M, Ziegler I-I, Schiavi G, et al. Eur J Pharrnacol 1993; 242: 23-30. Schulte B, Volz-Zang C, Mutschler E, Home C, et al. Clin Pharrnacol Ther 1991; 50: 372-378. Goyal RK. New Engl J Med 1989; 321: 1022-1029. Mutschler E, Feifel R, Moser U, Tacke R, et al. Eur J Pharmacol 1990; 183" 117-119. Dewey SL, Volkow ND, Logan J, MacGregor KR, et al. J Neurosci Res 1990; 27: 569575. Barnes PJ. Life Sci 1993; 52 521-527.

65 22 Barnes PJ, Belvisi MG, Mak JCW, Haddad E-B, O'Connor B. Life Sci 1995; 56: 853859. 23 Bungardt E, Mutschler E. Spasmolytics. In: Ullmann's Encyclopedia of Industrial Chemistry, 1993; Vol. A 24. VCH Publishers, Inc., Weinheim, New York, pp 515-528. 24 Minette PA, Barnes PJ. Am Rev Respir Dis 1990; 141: S 162-S 165. 25 Eltze M, Gmelin G, Wess J, Strohmann C, et al. Eur J Pharmacol 1988; 158: 233-242. 26 PfaffO, Gross J, Waelbroeck M, Mutschler E, Lambrecht G. Life Sci 1995; 56: 1038. 27 Eltze M. Eur J Pharmacol 1988; 151: 205-221. 28 Emmerson J, Mackay D. J Pharm Pharmacol 1979; 31: 798. 29 Waelbroeck M, Tastenoy M, Camus J, Christophe J. Mol Pharmacol 1990; 38: 267-273. 30 Ensinger HA, Doods HN, Immel-Sehr H ~ Kuhn FJ, et al. Life Sci 1993; 52: 473-480. 31 Doods HN. Drugs ofthe Future 1995; 20: 157-164. 32 Lambrecht G, Moser U, ~ U, PfaffO, et al. Life Sci 1993; 52: 481-488. 33 Micheletti R, Schiavone A. J Pharmacol Exp Ther 1990; 253: 310-314. 34 Waud DR. J Pharmacol Exp Ther 1969; 170:117-122. 35 Cicurel L, P6chadre J-C, Grandjean E, Duch~ne-Marullaz P. Agents Actions 1992; SPC: C440-C443. 36 Casy AF, Drake AF, Ganellin CR, Mercer AD, Upton C. Chirality 1992; 4: 356-366. 37 Nicholson AN, Pascoe PA, Turner C, Ganellin CR, et al. Br J Pharmacol 1991; 104: 270276. 38 Lambrecht G, Feifel R, Moser U, Wagner-ROder M, et al. Trends Pharmacol Sci 1989; 10 (Suppl.):60-64. 39 Waelbroeck M, Camus J, Tastenoy M, Mutschler E, et al. Eur J Pharmacol Mol Pharmacol Sect 1992; 227: 33-42. 40 Tacke R, Linoh H, Ernst L, Moser U, et al. Chem Ber 1987; 120: 1229-1237. 41 Tacke R, Reichel D, Kropfgans M, Jones PG, et al. J Organomet Chem 1995; 14: 251262. 42 Tacke R, Kropfgans M, Tafel A, Wiesenberger F, et al. Z Naturforsch 1994; 49B: 898910. 43 Waelbroeck M, Camus J, Tastenoy M, Lambrecht G, et al. Eur J Pharmacol Mol Pharmacol Sect 1990; 189:135-142. 44 Waelbroeck M, Camus J, Tastenoy M, Mutschler E, et al. Chirality 1991; 3:118-123.

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Perspective in Receptor Research D. Giardinh, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

67

Perspectives in the design and application of dopamine receptor agonists Cesare Casagrande, Giorgio Bertolini Department of Organic and Industrial Chemistry, University of Milan Italfarmaco Research Centre, Milan Introduction

The era of dopamine receptor pharmacology began around 1960, heralded by the discovery of the neurotransmitter role of dopamine, previously regarded only as a metabolic precursor of noradrenaline. In the early years fundamental discoveries were made, such as the dopamine defect as a cause of Parkinson disease and the substitutive therapeutic value of levodopa, the involvement of dopamine receptors in the mode of action of existing neuroleptics, e.g., chlorpromazine, leading to the hypothesis that schizofrenia may originate from a disfunction of neuronal dopaminergic pathways, and the unique vasodilator and natriuretic properties of dopamine with respect to the other catecholamines. These discoveries were made possible by the use of the few available drugs 9in addition to dopamine itself, applicable "in vivo" only by intravenous infusion and not entering the CNS upon peripheral administration, and to levodopa, actively transported into the CNS and converted into dopamine, apomorphine (!) and some ergot derivatives, such as bromocriptine 2(2) and codergocrine were soon recognized to exert some of the actions of dopamine and became useful tools for pharmacological investigations. These drugs were also the leads of medicinal chemistry, either indicating the way to new prodrugs of dopamine as carriers to the cardiovascular-renal system or to the CNS, or being suggestive of new active molecules by the recognition of a dopamine-like moiety buried in the rigid structures of aporphines and ergolines. Indeed, all dopaminergic pharmacology has been imprinted by these early findings: as a matter of fact, Parkinson disease, psychotic disorders, and cardiovascular disease, such as congestive heart failure and hypertension, remain the main therapeutic targets, and medicinal chemists have elaborated most of their efforts to develop new agonists, and some of the antagonists, on structural relationships with dopamine. Only one drug of this "first generation", piribedil (3) [1], developed for its vasodilator properties and somewhat later shown to act by stimulation of dopamine receptors, indicated the possibility to work out new leads for agonists not related to dopamine. Investigation of dopamine receptors in the central nervous system led to the identification of two subtypes, D 1 and D 2 showing distinctive ligand binding profiles [2]; at the same time, two similar subtypes DA 1 and DA 2 were characterized in peripheral tissues, mostly by "in vivo" pharmacological responses, and were kept on a separate denomination, by suggestion of L.I. Goldberg [3], until an unequivocal demonstration of correspondence could be achieved.*

* Investigation of D 1 vs DA1, and D 2 vs DA 2 receptors later reconciled their belonging to the same subtype, and the denomination D l_like and D2.1ike became comprehensive of these receptors and of the new subtypes (])3, D4, D5). For the sake of simplicity, in the following we shall use the D1-D 2 denomination for discussion of literature prior to the discovery of the latter subtypes, and the D 1 to D 5 denomination in the section "Investigation of new subtypes" and in the following ones when referring to recent literature making relevant distinction of the subtypes.

68 O HN H I

1

CH3 O CH(CI'I~)2 F-----I

H

"

%o

"NH'I ~I u

"CH3

!

I0, X : OH

3

'~ #c,,

H

Ho.

CH2CH(CH3)2" ~ /

;, U

A

~

I~

"~

4, R=H 5, R =nPr

NR2 ~

N

HO- y OH

, ~ Z

~,x=oso~cF, I- I1

8, X=H 9, X= OH

HO

2 Br

r

N ~

12, X = SO2CH3

R

2

H

v 6, R=H 7, R=nPr

~ d,~H Nit

NO X

13, X = Y = Z = R = H 14, X= CIY = OHZ= R = H 15, X= CIY= HZ= R = CH3

These findings opened the way in the late 1970 to the design of a second generation of agonists and to the definition of structure-activity relationships including selectivity for the receptor subtypes. The progress registered in the following decade has been extensively reviewed [ 1, 3-6]; we shall point out here the main pharmacological and clinical achievements, as well as the essential results of the search of pharmacophores and of tentative receptor models, in order to provide the basis for discussing the ongoing efforts towards a "third generation" of dopamine receptor agonists.

The "second generation" of dopamine receptor agonists Simple modifications of the dopamine structure showed that N-substituents, apart from N-methyl, abolished D 1 activity. D 2 activity also decreased by mono-substitution but was preserved or increased in disubstituted compounds, when one of the substituents was a small alkyl, optimally n-propyl; activity markedly decreased with n-butyl, or with branched groups. A modest D 1 activity (twenty times less than dopamine) reappeared in N,N-di-n-propyl and Nn-propyl-N-n-butyldopamine. At the same time, it was shown that D 2 activity was preserved in compounds having only the m-hydroxy group in the aromatic ring, while D 1 activity required the unchanged catechol system. Several groups become engaged in the synthesis of rigid dopamine analogues, focussing on the peculiar pharmacological properties of 6,7-ADTN and 5,6-ADTN (racemic _4 and 6_., respectively), whereby it was confirmed that one hydroxy group was required for D 2 activity, respectively in 7 or 5 position, in both cases coincident with the m-position of dopamine structure, while two hydroxy groups were required for D 1 activity. In the two isomeric series, the dopamine side chain was fixed in two different rotameric conformations, respectively denominated 13 and oL While the D 2 activity increased in both isomeric series from the primary amines to the N,N-di-n-propyl derivatives (racemic _5 and 7_), and D 1 activity was significantly expressed only in the N-unsubstituted 6,7-ADTN, it was surprisingly observed that in the 5,6-ADTN series D 1 activity was elicited only by the N,N-din-propyl derivative 7(].) and not by the primary amine. These observations were in good part made rational by an investigation of McDermed et al. [7], revealing that in the 6,7- and 5,6ADTN series, as well as in their monophenolic analogues, the pharmacological activity was respectively expressed by the (R)- and (S)-enantiomers (respectively 4_, _5 and _6, 7_); they

69

C HO

\ H~

~-----~~ "~ /

\

i~/"=-FN OH3

Fig. 1. Receptor models by McDermed [7] ( A red, 4-5_.; black, 6-7; B " red, (+)isoapomorphine; black, 1_) and Kaiser [4,5] (C, 13 with phenyl group in pseudoequatorial or pseudoaxial (dotted lines) conformation). keenly proposed the first model of the dopamine receptor (Fig. 1A), accomodating in an almost superimposable way the active enantiomers, thus accounting for the activity of both ctand 13-rotameric forms of dopamine. Furthemore, the model provided an explanation of the activity of apomorphine and the inactivity of (RS)-isoapomorphine (9,10-dihydroxyaporphine) (Fig. 1B). The latter was related to a steric impairement in the bottom of the receptor cleft, rather than to its 13-rotameric conformation, as previously suggested. The model could not account for the peculiarity of the activity of N,N-di-n-propyl-5,6-ADTN with respect to the D 1 receptor. Investigation of the role of the catechol system, using in part previous experience on agonists of 13-adrenergic receptors, pointed out, among various cyclic and non cyclic analogues carrying NH acidic groups in lieu of the m-hydroxy group of dopamine, the indolone SK&F 89124 ~ as the most potent and selective D 2 agonist [8]. The analogue devoid of the paralike hydroxy group (ropirinole, 8_), although markedly less potent, showed a good bioavailability and pharmacological activity in vivo. The latter may be due to its metabolical conversion into 9. Ropirinole has been initially investigated as an antihypertensive agent, and later on in Parkinson disease. Clinical studies are currently in progress; ropirinole has recently been shown to be also active on D 3 receptors. Swedish academic and industrial investigators [9] decided to exploit the new information on the role of presynaptically located neurotransmitter autoreceptors, including those of dopamine, in controlling, by a feed-back mechanism, the release of the neurotransmitter in the synapse. They suggested that a selective agonist of the presynaptic dopamine autoreceptor could provide a pharmacological alternative to the blockade of postsynaptic receptors in the treatment of psychotic conditions. Their prototype D 2 presynaptic agonist, preclamol 0 ~ , was preferred to the corresponding racemate, which had generated some confounding results, and it was promoted to clinical investigations, which have not yet reached a full conclusion, but have stimulated new research in this direction. The discovery of the selective vasodilation produced by dopamine in the renal circulation and of its natriuretic effects, both mediated by D 1 receptors, respectively in renal arteries and in renal tubules, prompted research of new D 1 selective and peripherally acting agonists, potentially useful in the treatment of congestive heart failure and of hypertension. In the most severe forms of the former clinical condition, dopamine is used by intravenous infusion, taking

70 advantage of dopamine receptor mediated effects at low doses, as well as of cardiac stimulation via 13-adrenergic receptor stimulation at high doses. The interest in the exploitation of the renal effect of dopamine in hypertension is linked to the complex control exerted by the kidney on the circulation [ 10, 11 ]. Derivatives of 7,8-dihydroxy-3-benzazepine, which enclose a constrained, but not rigid, dopamine-like structure, show only modest dopamine-like activity. Addition of a phenyl ring in the 1-position afforded SK&F 38393 (racemic 1_3), showing a remarkably selective activity at the D 1 receptor. A large number of" analogues was investigated [4, 12], pointing out that appropriate structural modifications can increase the intrinsic activity, producing full agonists, while (+)-13 shows a partial agonist profile in some assays, or can decrease selectivity and specificity by inducing either D 2 agonistic activity or affinity for adrenergic or serotoninergic receptors. Modification of the benzazepines has also produced a class of D 1 selective antagonists, the prototype of which is SCH23390. Among the derivatives, fenoldopam, the racemic form of the 6-chloro-4'-hydroxy analogue ( ! ~ , showing predominant distribution in peripheral tissues, was selected for in-depth investigation and became a unique tool for studying the cardiovascular implication of stimulation of D 1 receptor in the kidney and the circulation. Clinical investigations were promoted in congestive heart failure and hypertension [ 13 ]. Development was restricted to short-term intravenous administration in the treatment of hypertensive crises. The oral use in chronic treatment was discouraged in part by the low bioavailability and short duration of action, and in part by the observation that the efficacy rapidly decreased due to reflex sympathetic activation, consequent to the potent vasodilator effects of the drug, and also due to increase of plasma renin activity. The latter has been linked to an increase in renin secretion dependent upon the stimulation of specific D 1 receptors located in the juxtaglomerular cells in the kidney. The interest in the use of dopamine agonist in cardiovascular therapy was also the basis of investigation of dopexamine ~ at Fisons laboratories [14]. Starting from the biscatecholamine structure of hexoprenaline, a tocolytic 132-agonist, a pair of dopamine agonistic structures were joined by variable length links between the amino groups. It was shown, as could be expected, that only one moiety carrying the essential features for dopamine receptor stimulation was sufficient for the activity, but it was also shown that a six-atom link was optimal, and that the basicity of the second amino group and the second aromatic ring were essential for D 1 receptor stimulation. The selected compound, dopexamine, expressed a marked activity at D 1 receptors, unusual in N-mono-substituted dopamines, suggesting that useful interactions involving counterparts of the second amino group and aromatic ring in a region of the receptor far away from the points of interaction of dopamine may be involved. A lesser (1/10) activity at D2 receptors and a 132-adrenergic agonistic action were shown to be present. The latter was considered a useful complement of the pharmacological profile, providing additional vasodilation and possibly a positive inotropic effect. Dopexamine has been clinically compared with i.v. dopamine, showing a dose-dependent vasodilator effect, at difference from that of dopamine which is limited, at very high doses, by the counteraction of the ot-adrenergic vasoconstrictor activity. The intravenous infusion of dopexamine is in clinical use in some countries in conditions of low cardiac output [ 13 ]. As in the case of fenoldopam, the effects of a prolonged administration, such as an infusion prolonged up to 24-48 hours, tended to wear off, because of reflex sympathetic activation, which was amplified in this case by a specific ability of the drug to block the uptake of norepinephrine, thus sustaining the high circulating level produced by the synaptic spillover. Elaborations of the ergoline structural lead were prevailingly addressed to the development of new antiparkinsonian agents, since bromocriptine, acting as a D 2 agonist, had

71

xN~HN

H N!

~

R 16, % - nPr, Rsb - CH2SCH 3

17, R6 = allyl, R8b = N-- CONHEt |

(CH2)3N(CH3)2

18

~

X~ ~ ~ ~ , v ~ , s 19

N

20, X - H

21, X OH 22, X = COOH

shown its clinical utility, either in delaying the initiation of levodopa therapy, or in complementing the efficacy of moderate doses of levodopa. Levodopa, although markedly effective, had shown undesirable side effect in a number of patients, and frequently a decrease of efficacy after some years of therapy, along with the induction of "on-off' fluctuations of effects and of motory disturbances. Bromocriptine interacts with adrenergic and serotoninergic receptors. The pharmacological profile of pergolide ~ [15] showed increased, although not complete, specificity for dopamine receptors, and also involved both D 1 and D 2 agonistic action. Cabergoline (17.) [16] showed increased specificity for the dopamine receptors, marked D 2 agonistic activity and long duration of action. Owing to its scarce entrance in the CNS, its clinical application has been preferentially addressed to the reduction of hyperprolactinemia, an effect elicited via the D 2 receptors located in the anterior pituitary, which is not protected by the blood-brain barrier. High prolactin secretion has clinical consequences, such as galactorrhea, amenorrhea and increased risk of breast cancer relapse. In the ergoline structure, two blocked heteroaryl-ethylamine moieties can be assumed to have relevance in dopamine receptor interaction : a 4-indoleethylamine, in which the NHindole group may surrogate the m-hydroxy group of dopamine in donating ahydrogen bond, or a 3-pyrroleethylamine also containing the NH-group suitable for a similar interaction. This ambiguity has stimulated different approaches of molecular dissection, such as the one represented by N,N-dipropylindoleethylamine ( ~ ) [1], which was shown to be significantly active (1/10 of pergolide) at D 2 receptors, and the other one by quinpirole (racemic 1_.9), a pyrazoloquinoline endowed with marked D 2 activity, in common with the pyrrolo-quinoline analogue directly derived from ergoline structure modification. Quinpirole, originally investigated as an antihypertensive agent, showed some paradoxical hypertensive effects in normal volunteers; its pharmacological profile is being reconsidered in view of its D 3 agonistic activity [9]. Active D 2 agonists, having an indolebutylamine structure not strictly related with the ergolines, were discovered when compound EMI) 23348 2(!0.) was submitted, in a frame of pharmacological screening, to the Ungerstedt test of controlateral rotation in rats with unilateral nigrostriatal lesions. Roxindole ~ and carmoxirole ~ were generated from this new lead and respectively investigated as potential antiparkinson and antihypertensive agents, in the latter case because of the exclusively peripheral distribution of carmoxirole [ 17]. Intensive discussion of structure-activity relationships and features of pharmacophores and receptor models accompanied the development of the "second generation" of dopamine agonists. There was agreement in pointing out, as the key features of the pharmacophores, the distance between the m-hydroxy group and the amine nitrogen, corresponding to a fully extended conformation of the side chain of dopamine, and the directionality of the lone pair orbital of the nitrogen. Among various hypotheses, the model of Kaiser [4,5] shown in Fig. 1C implemented the basic concepts of McDermed [7] and included all possible, and yet not fully defined, interactions of the D 1 and D 2 agonists. There were considered : 1) an anionic site

72 (COO-), binding the amino nitrogen; 2) the M-site, binding the m-hydroxy group; 3) the Psite, binding the p-hydroxy group; 4) an N-site, located at about 2.0 A distance from the Psite, interacting with the NH group of the pyrrole ring in the ergoline structure; 5) a I'I 1 site providing a rI-rI interaction with the aromatic ring of the catecholamine/ergoline structure; 6) a I-I2 site, at the bottom of the receptor pocket, allowing a 1-I-rI interaction of the pseudoaxial conformation of (R)-I3 (dotted-line phenyl), or the alternative site 1-I2.. The latter appeared as most likely in view of the pseudoequatorial conformation of (R)-fenoldopam ~ derived from X-ray diffraction [12]; 7) a I"I3 site, corresponding to the second aromatic ring of apomorphine; 8) a C3-site, as a lipophilic cavity suitable to fit a N-substituent not larger than n-propyl; 9) a large lipophilic site (LL) suitable to enclose a larger N-substituem.

Dopamine prodrugs Levodopa holds the main role in the therapy of Parkinson disease, complemented by bromocriptine and the new ergolines. The use as a prodrug of this endogenous precursor of dopamine showed, in addition to side effects, drawbacks dependent upon irregular absorption and peripheral decarboxylation. The latter problem was effectively overcome by the coadministration of inhibitors of levodopa decarboxylase having distribution limited to peripheral tissues, such as benserazide and carbidopa, allowing an increased and more regular access of the prodrug to the CNS. So far attempts to design prodrugs of dopamine or levodopa, which could effectively improve the delivery of the active agent in the central nervous system, were not successful. Analyses by various kinetic models of the factor governing the selective delivery of a drug in a target compartment by a suitably designed prodrug [18], applicable to the CNS and the kidney, brought into light the main determinants of site-specificity: a small volume of distribution of the prodrug in the central and other non-target compartments, an active uptake of the prodrug into the target compartment, a fast conversion of the prodrug into the drug in the target compartment, and the retention there of the drug. Retention or binding of the prodrug in the target compartment may also contribute, as far as the conversion to the drug is not hindered. The fast diffusion of the prodrug to the target compartment gives only modest contribution, since it produces similarly fast distribution towards non-target compartment and tends to decrease selectivity. The key advantage of levodopa as a prodrug is represented by its transport via the physiological mechanism of aromatic aminoacids uptake into the brain, particularly when peripheral metabolization/activation is drastically reduced. Up to now no practical progress with new prodrugs has been achieved; use of soluble prodrugs of levodopa has been suggested as a means of continuous administration, approaching a zero-order rate of absorption, in order to limit the incidence of kinetical factors on-off phenomena in severe patients. A device for retention of a dopamine prodrug into the central nervous system has been designed by the synthesis on N-methyl-dihydronicotinoyl amides of dopamine 3,4-0-diesters, such as 23, which are converted into a quaternary pyridinium derivative by oxidation, and thereby retained inside the blood-brain barrier [19]. Only modest dopamine-like activity was observed, and this may due to a slow rate of enzymatic hydrolysis of the amide bond joining the dopamine and the pyridinium moieties. A new intriguing approach to a prodrug retained by quatemarization in the CNS has been reported recently [20]. Exploiting a mechanism previously used in the vitamin prodrug thiamine disulphide, lipophilic compound 2_4,4has been synthetized by coupling a 3,4-0-diacyl ester of levodopa with the open-ring precursor of a thiazolium compound. In this case, the quaternary derivative is generated by a reductive cleavage of the disulphide, involving

73

~

H~J~

U9

--N--

AH~

23

N CH3

I I

0

I.,

~O~

L~"

O" ~(.~ I~

CH3 OH3.N~ ~ . / O , .,~ I

~

/I

NH~ ,~'1"1 ~" L~x I ~

Oy~y~'~,~. NHCH3HO~ ~ ~ ,,,~/

24

~

,

~~L. ~

"~

O

-o

O O / 1 ~Y

0

@H3

,

,~

"1.

o-

T

27

HO~

p -0 I' o

NHCH3 26

~ -NH ,

~

H

--

T/

I~ ..~

HO~ ' ~

IT v

" ~og.

"C,OOH

2s

H NHCOCH3

O rO y'vNHC' 'SOH O O "~ /

O"~'O

28

O

glutathione; thereafter, the release of levodopa appears to be fast, and produces by i.v. administration in rats brain level AUC's 30 times larger than an equimolecular dose of levodopa. At difference from the pyridinium amides, an easily hydrolyzed ester bond joins levodopa with the quaternary moiety. It has however to be investigated whether this prodrug can overcome first-pass modification by the glutathione pool in the liver, and which consequences can derive from the burden of the thiazolium compound in the brain. Specific delivery of dopamine to the kidney represents an interesting pharmacological target in hypertension and in congestive heart failure. Investigations in this direction have been inspired by the presence of dopamine receptors in tubular cells, in which both active transport of organic anions and high local concentrations of enzymes amenable to prodrug activation are available. Gludopa (2~ has been assumed to be taken up, hydrolyzed to levodopa by Tglutamyltranspeptidase, and then decarboxylated "in situ" to dopamine. A group of phosphoric esters of dopamine, including as the most active compound fosopamine (N-methyldopamine 40-phosphate) (~6.), were investigated assuming uptake and dephosphorylation by alkaline phosphatase in the brush border membrane of the tubular cell [ 18]. In laboratory investigation 26 showed a potent, dose-related renal vasodilator effect in dogs on oral administration even in low doses of 0.6 mg/kg, but no dopamine-like effect was observed in preliminary studies in humans. It has to be concluded that the prodrug, which appeared to reach the kidney for local activation in significant amounts in laboratory animals, was most probably completely dephosphorylated in the gut wall in humans. Gludopa, although not absorbed orally, was investigated in a number of studies in healthy volunteers and in selected hypertensive patients by i.v. infusion, indicating that it can correct the fault of sodium excretion, and improve renal function without modifying renin plasma levels [21 ]. Its effects appeared to be dependent both on D 1 and D 2 receptor stimulation. The dose required was high (10-50 times the dose of i.v. dopamine). It has recently been shown in animal experiments [22] that the effects of 25 are not selective for the renal district; this may be due to extensive metabolization in other tissues, particularly in the intestinal wall. Thus, both attempts failed because of scarce organ selectivity in the metabolic activation step, and the quest of a renal selective dopaminergic drug, or prodrug, remains unsolved. At difference from these results, the design of prodrugs suitable to produce by oral administration all the cardiovascular and renal effects of dopamine met success with ibopamine 2(~7.) and with docarpamine 2(2~. Ibopamine, a diester of N-methyldopamine, appeared to be orally absorbed and rapidly activated in peripheral tissues, but did not induce central dopaminergic effect in laboratory animals, at difference from levodopa. It was compared with

74 dopamine i.v. infusion in short term studies, showing similar hemodynamic and renal effects, and it was thereafter investigated in the chronic treatment of congestive heart failure [ 13]. In long term studies, its efficacy appeared to be dependent not only upon D 1 receptor related effects, but also on effects mediated by the stimulation of peripheral D 2 receptors, in particular decreased noradrenaline release via presynaptic receptors, reduction of overstimulated aldosterone release via D 2 receptors located in the adrenal zona glomerulosa, and lack of increase of renin plasma level, since D 2 presynaptic action counteracts the release induced via D 1 receptors. This pattern of neurohumoral effects has clinical relevance in congestive heart failure, particularly for sustained therapeutical efficacy. Docarpamine was also shown to be an orally effective peripheral dopamine prodrug, at doses higher than ibopamine [13, 23], and it is used in Japan for weaning patients from i.v. dopamine infusion in severe heart failure.

New dopamine agonists in cardiovascular therapy The therapeutic properties of ibopamine appear to be dependent upon its combined effects on D 1 and D 2 receptors, avoiding unwanted neurohumoral activation and development of tolerance, at difference from selective D 1 agonists. This suggests that an advancement in cardiovascular and renal applications of peripherally acting dopamine agonists may be represented by balanced D1-D 2 activity in a drug devoid of the ot-adrenergic and 13-adrenergic effects that dopamine and N-methyldopamine can elicit in high doses. In our approach to such a goal, we were attracted by the potent dopaminergic activity of the isomeric ADTN and by the possibility of modulating it, as well as the adrenergic activity present in these compounds [4], by structural modifications of the nitrogen substituents. In particular, we considered the use of ortho-substituted-aryloxyethyl groups [24, 25], which could revert the a-adrenergic action into an antagonistic effect this result was indeed achieved by the synthesis of N-2,6dichlorophenoxyethyl-N-n-propyl-5,6-ADTN ~ , which showed antihypertensive properties on oral administration in spontaneous hypertensive rats. Investigation of the enantiomeric pairs of 33 and of the monosubstituted analogue 32 showed, as it was expected, that only the (S)-enantiomers were endowed with D 2 agonistic activity, but also pointed out a new, interesting feature whereas the N-propyl derivatives 33 was endowed with agonistic activity, 32 showed D 1 antagonist properties [26]. It appears that in the 5,6-ADTN series the receptor interaction provided by the N-propyl cleft is not only compatible with, but required for D 1 agonistic activity, confirming that (S)-5,6-ADTN and (R)-6,7-ADTN, although similar as shown in the model of McDermed, still behave very differently in the interaction of the Npropyl substituents with the D 1 receptor. We and others [27] thereafter applied similar structural modification in a series of dopexamine analogues, were significant D 1 activity had been previously reported [14], and achieved the synthesis of Z 1046 ~ . Compound 34 appeared to be endowed with D 1 activity comparable to dopexamine and with a marked D 2 activity, as well as with c~1-adrenergic antagonistic activity and with a moderate ot2-agonistic action, while it was devoid of any affinity for the 13-adrenergic receptors [28]. Vasodilatory and antihypertensive effects were observed on oral administration in rats, dogs, and monkeys [29], and were shown to depend on both D 1 and D 2 receptor stimulation, since they were fully antagonized only by the combination of SCH 23390 and domperidone. Thus ~ showing the desired pharmacological profile, may represent an interesting candidate for clinical investigation in congestive heart failure and in hypertension. Other groups have been continuing the pursuit of D 1 selective agonists as cardiovascular drugs, aiming at new compounds having full agonistic properties and increased potency with

75 OH

OH

HO

"~

HO

R

29, R = (CH2)6NH(CH2)2Ph, R 1 = H

31, R

~"/~~..'/N

CI ' ~

,

34

(CH2)6NH(CH2)2Ph' R1 =

OH

H~O

HO-

v

NH2

H

36, R = phenyi 37, R = 1-adamantyl

HO

HO-

v

v

HO HO

38, X = H 39, X = OH

-OH

35

NH

HO ~

~/CI-I ~ , '~,s-- N H ' ~

/(CH2)e

x

HO~,IH

|

k,,,,,,,,/

32, R = H 33, R = nPr

.~cH~

30, R = H, R 1 = ~

I. IJ

0~

H,

HO - v

v 40

H H

HO C ~ C l ~ . HO- v

'NH

41

respect to fenoldopam. A research programme at Abbott laboratories exploited new methods for 3D-database search [30]. At first a common pharmacophore for a number of D 1 and D 2 agonists was defined, relating the positions of the amino nitrogen and the catechol oxygen atoms, substantially in accordance with previous findings, then an aromatic ring was added to define a region accessible to D 1 agonists, but not to D 2 agonists, as suggested by the results of fenoldopam and related benzazepines. Among various attempts, a very active and selective benzopyran derivative, A-68930 ~ , was obtained by a relatively simple synthesis [3i]. Unexpectedly, the substitution of the phenyl ring by a branched alkyl substituent was shown to maintain activity, and the 1-adamantyl derivative, A-77636 ~ , showed potent activity and selectivity [3 2]. Other attempts to achieve D 1 selectivity by adding an aromatic ring to a flexible or rigid dopamine structure led to the synthesis of30 [33], 40 (dihydrexidine) [34], and 41 [35], while previous observations concerning the 3',4'-dihydroxy derivative of nomifensine were brought to a rational definition by the synthesis of 38 [6] and 39 (YM 43 5) [36]. Further modifications of the dopexamine structure led to FPL-63012 ~ [37], a selective D 1 agonist, and RS45496 35 [38], a mixed D1-D 2 agonist. The results of molecular modelling studies of these selective agonists are altogether consistent with the revised model of Kaiser (Fig. 1C), as far as lipophilic interactions, rather than H-H-stacking, are considered to be involved in the D 1-selecting site. The catechol moiety is preserved in all selective and non selective D 1 agonists considered so far, with the exception of pergolide ~ and some related ergolines. Compound 43, a monophenolic benzo(g)quinoline analogue of 7, was also shown to act as a D 1 and D 2 agonist, with 50% intrinsic efficacy ad D 1 receptors, by the work of group of investigators at Sandoz. This group set to prepare hybrids of dopamine and ergoline structures [6, 39], in the search of a common pharmacophore. The actions of 43 was totally shifted to D 2 selectivity by the insertion of the side chain of etisulergine 4(~), resulting in quinagolide ~ clinically effective as a prolactin release inhibitor. On the other hand, the addition of a fused aromatic ring to the ergoline structure resulted in compound CY 208-243 ~ , which was shown to be the first selective non-catechol D 1 agonist. Among these new D 1 agonists, clinical development in cardiovascular medicine is reported only for 39, while 37 and 45 are investigated for their potential antiparkinsonian properties.

76 X

.,, NHSO2NE

HN

H

H I CH3

H

42

H2N

I

//NH~ 48

H

H I CH3 45, X = H 46, x = OH

_

43, X= H 44, X = NHSO2NEt2 N

47

H

~X

r ~

H2N

CH3 49

50

H%~"

Investigation of new receptor subtypes and of their pharmacological relevance Starting from 1990, cloning of dopamine receptors of human and animal tissues, their expression in cultured cell lines, the analysis of their structure, as well as the availability of techniques of receptor imaging, boosted a new wave of investigation of dopamine pharmacology. The attention is now focussed on two subtypes of the D l_like family, namely D 1 and D 5, and three subtypes of the D2_like family, namely D2, D3, and D4, while other subtypes are on the horizon [40, 41]. The search for selective ligands, both agonists and antagonists, is inspired by different anatomical locations of the subtypes and by different pharmacological responses. The understanding of the latters is made complex by the uncommon wealth of transduction mechanisms that the dopamine receptors appear to be endowed with [42]. Hopefully, a "third generation" of precisely targeted and therapeutically useful dopaminergic agents shall arise from current research efforts. A first glimpse to the interaction of the main classes of dopaminergic agonists and antagonists with the new subtypes is provided by the extensive tabulation of affinity data reported by Seeman and van Tol [43], some of which are shown in Table 1. No selectivity was detected between D 1 and D 5 in these studies, but an over-simplified description of the Dl_like family appears undue. For instance, the selective benzazepine SK&F 83959 ~ showed high affinity, but variable action (from weakly agonistic to antagonistic) on adenylate cyclase stimulation in D 1 receptor preparations from different brain areas, and still potently induced Dl_like behavioural effects in laboratory animals [44, 45]. Interesting differences emerged in the ratio D2/D 3 and D3/D 4, both for agonists and antagonists. As for the latter, current interest for antipsychotic agents is directed to selective D 3 or D 4 agents, for different reasons. D 4 receptor density has been found to be markedly increased in postmortem examination of the brain of schizophrenic patients, and clozapine, an atypical neuroleptic having reduced risk of side effects, shows a good degree of selectivity for D 4 receptor, suggesting that at therapeutic levels in tissues (10-20 nM) it may act almost exclusively on this subtype [43]. D 3 receptors, on the other hand, are specifically located in the mesolimbic area of the brain, presiding to motivation, emotion, and cognition, whereas D 2 and D 4 are more evenly distributed and are present in striatal areas, presiding to locomotion [46]. Thus selective D 3 antagonists should be less liable to produce locomotor side effects, as well as disturbing side effects in female patients, such as amenorrhea and galactorrhea. The latters depend upon the inhibition of specific D 2 receptors in the anterior pituitary. As for agonists, compounds selective for the the various subtypes are investigated as potential antiparkinsonian drugs. For instance, D 1 agonists could be preferable, since they should not induce nausea and emesis. Still, in order to really ascertain the therapeutic value of

77

T a b l e 1. D i s s o c i a t i o n c o n s t a n t (K i, nM) f o r a g o n i s t s (A) a n d a n t a g o n i s t s (B). All v a l u e s refer to t h e h i g h - a f f i n i t y state, e x c e p t f o r D 3. M o d i f i e d with p e r m i s s i o n f r o m ref. 43

Receptor subtype A

B

Dopamine Apomorphine Bromocriptine Pergolide SKF 38393 7-OH-DPAT Chlorpromazine Haloperidol Clozapine (S)-Sulpiride SCH 23390

Dl-like

D2-1ike

D1

D5

D2

D3

D4

0.9 -- 0.7 - 440 0.8 1 ~ 5000

< 0.9

~ 7 ~ 0.7 ~ 8 - 0.8 - 150 10

~ 4 ~ 32 ~ 5 ~ 1.5 ~ 5000 ~ 1

~ 30 ~ 4 - 290 - 1000 650

3 1.2 ~ 230 -- 15 - 1100

4 - 7 ~ 170 ~ 13 ~ 800

35 2.3 21 1000 ~ 3000

~ 90 ~ 80 - 170 ~ 45 000 ~ 0.2

~ 450 -- 0.5 ~ 130 ~ 100 - 330 77 0 0 0 0.3

selective agonists, either alone or in combination with levodopa, a large effort in clinical trials appears mandative. On the side of psychopharmacology, the hypothesis of employing a selective agonist of the central presynaptic D 2 autoreceptors, such as preclamol (1..O), as an antipsychotic agent, is currently under new focus. Although it is now recognized that presynaptic and postsynaptic D 2 receptors are not structurally different, the presynaptic "autoreceptors" appear to be more sensitive to low concentration of agonists, and particularly of partial agonists. It has been proposed that the responsiveness to partial agonists depends on a considerably high reserve of the autoreceptors. Considerable interest is raised by the observation that presynaptic receptors in the mesolimbic area of the brain pertain to the D 3 subtype [47], and that preclamol ~ is equally active on this subtype. Analogues, such as the triflate 11 and the sulphoxide 12 [48] were found to act as antagonists, and to be somewhat selective for the D 3 with respect to the D 2 subtype, indicating that modifications of the phenolic group of 1..9.0 suppress its partial agonist character. On the contrary, it was unexpectedly shown that marked agonistic activity was preserved in the triflate ester of the full agonist (S)-5-hydroxy-N,N-di-n-propyl-2-aminotetralin [49]. Other structural modifications of the aminotetralins produced antagonists endowed with D 3 vs D 2 selectivity, such as (+)-UH 232 (53) [50] and (+)-S 14297 ~ [51], the latter with 301 ratio. On the other hand, the 13rotameric (R)-(+)-7-OH-DPAT ~ [46] and its rigid analogue PD 198907 (5~ [52] did show a similarly high selectivity, acting as agonists.

HO. N HOrN i/~

51

~,

52

~H~

OCH 3

~

53

54

Other previously "known D 2 agonists, such 8_, 16, ~ 21, and 48 [53] were also found to bind with high affinity to D 3 receptors. The last compound, pramipexole, had been structurally derived from talipexole ( 4 ~ [1], a D 2 agonist discovered within a series of ot2-adrenergic agonists. The stereochemistry of 4...S8and 19 suggests that they may fit the receptor in a similar way of (S)-5,6-ADTN (Fig. 1A) but the different activity of (+)- and (-)-49 [54], having

78 unsolved absolute configuration and respectively behaving as an autoreceptor agonist and a postsynaptic antagonist, raises new questions. A broad programme of investigation of potential D3/D 2 autoreceptor agonists at Parke Davis laboratories followed leads unrelated to dopamine, including piribedil (3), 20, ~ and features of antagonists of the butyrophenone and benzamide classes. PD 143188 (~3 emerged from these studies as a candidate antipsychotic agent [55]. The lack of hydrogen bond donor group in 50, as well as in the triflate of (S)-5-hydroxy-N,N-di-n-propyl-2-aminotetralin cast doubt on the requirement of such bonds for agonistic activity.

Progress in dopamine receptor models A new avenue of investigation of the interactions of agonistic and antagonistic ligands with receptor models directly derived from the structure of the protein is opened by the recent advancements. However, although the amino acidic sequences have been defined, tridimensional structures are not available for any of the members of the G-protein coupled family, to which dopamine receptors pertain. As a consequence, direct models built using theoretical methods of protein modelling and/or criteria of analogy are not exempt from criticism and controversy. Thus, progress in indirect modelling, based on steric and electronic properties of the ligands, plays an important role not only in the design of new active compounds, but also in the improvement and the validation of the direct models. In the search of indirect (ligand-based) models no substantial progress has been recorded for D 2 receptors with respect to the proposals of McDermed [7] and Kaiser [4, 5]. There is agreement among several investigators [56] on the main features, such as a plane defined by the oxygen atoms and the aromatic ring of dopamine, the protonated amine nitrogen approximately on the same plane, with its salt bridge to the binding site directed backwards and its two substituents extending far from the aromatic ring and slightly in front of the plane, respectively towards the "n-propyl cleft" and towards a region which can accomodate large groups. The distance from the m-oxygen and the nitrogen atom ranges between 6.2 and 7.3 A. In these models, it has been generally accepted that at least one phenolic or equivalent function, such as the indole NH group of the ergolines, acts as a hydrogen bond donor with respect to a site of the receptor. The recent discovery of potent dopamine agonists devoid of such functions suggests that recognition modes other than hydrogen bonding may be involved in the corresponding region of the receptor. Interesting considerations have been derived from molecular electrostatic potential (MEP) studies of the ligand. Subsequently to studies focusing on sets of structurally homogeneous ligands, a recent comparison of agonists pertaining to different classes [57] pointed out, in addition to the global minimum corresponding to the amino group, a second minimum located in the region of the aromatic/heteroaromatic moiety. The good matching of both minima in these agonists supports a role in the interaction with the receptor. In the case of the aromatic/heteroaromatic moieties, their I-I or lone-pair electron dense regions could interact either with a proton-donor group or with a H-stacking heterocyclic ring in the receptor. A suggestion that the phenyl ring of PD 143188, or the 2-pyridyl and p-hydroxyphenyl ring of two related, and somewhat more active analogues [58], interact in this way with Ser-194 and Ser-197 appears consistent with the hypothesis. The proposal of Kaiser [4, 5] for 1-arylbenzazepines, revised by using the most likely equatorial conformation (Fig. 1C) established the basis for a number of investigations of D 1 iigand-based, indirect models, analysing the position of the aryl substituent, considered as the selectivity-inducing feature, excluded by a steric barrier from interaction with the D 2

79 receptor. A recent comparison of the D 1 agonists of different structural classes [59] used quantum-mechanical methods and superimposed the lone pair of the amine nitrogen and the catechol ring. A site for the aryl ring could not be precisely defined, leading to the assumption that it is probably involved in a directionally non critical hydrophobic interaction in a lipophilic pocket, rather than a specific rI-rI stacking. The assumption is supported by the finding of selective agonists carrying an adamantyl, such as 37, or cyclohexyl moiety [31]. The close matching of the dipole vectors of structurally diverse agonists indicates a significant role of the electronic distribution in the recognition process [59]. An analysis of the MEP o f D 1 and D 2 selective and non-selective agonists [60] showed similarity of main features, regarded as primary requirements for binding, in agreement with Kocjan et al. [57]; it was pointed out that potentials in the proximity of the catechol ring are consistently lower in D2-selective than in D 1-selective compounds, suggesting that the difference may have some relevance in modulating selectivity. Accomodation of ergoline derivatives in D 1 and D 2 indirect models raised questions about the positioning of their heteroaromatic system, related both to the lack of phenolic groups and the ambiguity of the pyrrolethylamine/phenethylamine moiety participating in the pharmacophore. Seiler et al. [6, 39] proposed a superimposition of the ergolines, different from that provided by the model of Kaiser (Fig. 1C). In their model the NH group lays between the m- and p-phenolic groups not only in D 2 selective and D1-D 2 non selective ergolines, but also in the D 1-selective benzergoline 45. The hydroxy derivative 4_..66,devoid of selectivity, being 10 times more effective as a D 2 than a D l-agonist, was suggested to assume a position similar to that predicted by the Kaiser model. It is interesting to recall that 13hydroxy-lergotrile, a metabolite having a phenolic group in a position equivalent to 4...66was shown to be 10 times more active than its parent compound in the inhibition of prolactin release, a D 2 mediated effect [61 ]. On the contrary, the results of Alkorta and Villar [59] for the benzergoline 45 were in accordance with the model of Kaiser. Our analysis of six D 1 selective agonists, 14, 30, 3..66,38,

Fig. 2. D 1 receptor pharmacophore (amine nitrogen, blue; m-oxygen, red; centroid of the selectivity inducing aromatic ring, grey) with superimposed agonists. A 93..88,blue; 40, grey; 41, green; B " ~ blue; 36, grey; 30, green. A and B 945, red. See text for explanations.

80 40, and 41 by a systematic constrained search with Tripos' program RECEPTOR led to similar conclusions. The compounds were superimposed (Fig. 2) on a three-centres pharmacophore including the amine nitrogen, the oxygen atom corresponding to the m-position of dopamine and the centroid of the selectivity inducing aromatic ring. They fitted together in an almost identical way, both when equal geometrical weight was given to the three centres (as shown in the Fig. 2 A and B) and when the weight of the oxygen atom was decreased to 1/100. When the indole NH of 45 was assumed to be equivalent to the m-oxygen atom, and given equal weight, it was superimposed in accordance to Seiler et al. [6, 39], but the aromatic ring protruded out of the common aryl region (Fig. 2 B). On the other hand, when NH was released by assigning predominant weight to the other two centres, 45 was superimposed (Fig. 2 A) in accordance to Kaiser and to Alkorta and Villar. Direct (receptor-based) models of dopamine receptors, as well as of other G-protein coupled receptors, have been built on the analogy with bacteriorhodopsin, an evolutionarily related protein, whose tridimensional structure is known, although with moderate refinement. Notwithstanding the scarce sequence homology, the similarity of the topology of the seven transmembrane helices (TM1-7) was the starting point for the development of a number of Gprotein coupled receptors [62]. Models of dopamine receptors were built using primary sequence analysis, site-directed mutagenesis, and ligand structure-activity relationships [6265], frequently taking advantage of the similarity with the 132-adrenergic receptor. A main difference in the modelling approach was the use of the structural data of bacteriorhodopsin, either employed only as a 3D template for building the transmembrane helices from helical sequences otherwise assigned [62-64, 66], or also taken as the basis for the identification of the helical sequences [65]. Site-directed mutagenesis indicated that the aspartate residue in TM3 (Asp-114 in D 2 *) is essential for binding of both agonists and antagonists [67]. All models provide for a ionic interaction between Asp-114 and the charged amino group of the ligand. Most models also involve hydrogen bonding of the catechol system of dopamine and of related functional group of other agonists to Ser-193 (or Ser-194) and Ser-197 in TM5 [62-65], as in the examples of D 2 receptors shown in Fig. 3, B and C. In the model ofMalmberg et al. [64], shown in Fig. 3 B with 7_ as the ligand, the ionic interaction at Asp-114 is reinforced by surronding aromatic residues, as previously suggested [62], and Ser-193 is involved in the hydrogen bonding interaction with the 5-hydroxy group. Aromatic residues enclosing the binding pocket are Phe390, Phe-389, and Trp-386 at the bottom. The n-propyl cleft is defined by the residues Leu113 and Met-117 in TM3, Ph-389 in TM6, and Tyr-408 and Thr-412 in TM7. The aromatic moieties of larger aralkyl substituents, e.g. in compound 33, pointing in the extracellular direction can favourably interact with the aromatic moieties of Phe-110 in TM3 and Tyr-408 in TM7. The comparison of this model with the analogous model built on the D 3 sequence failed to point out important differences; it was suggested that the distance between Asp-110 and Ser192, being 1 A larger than the corresponding distance of Asp- 114 and Ser- 193 in D 2, may be more suitable to accomodate (R)-7-OH-DPAT, a D 3 70-fold selective agonist 5(5~, which shows a 0-7 to N distance of 7.4 A, vs 6.6 A of the somewhat less selective (S)-5-OH-DPAT. In the model of Teeter et al. [65] (Fig.3 C), similar features were pointed out; the hydrogen bonds were suggested to involve Ser-194 and Ser-197 in TM5, and the aromatic residues

* In the following and in Fig. 3 residues are numbered according to the human D 2 long isoform sequence (443 residues), unless otherwise annotated.

81

A

B

EXTRACELLULARSPACE

~.NH2

1M4 lU51

TM4

TM2

~'~

TM7 I Vd91

TU6

Asp114 Ile391 Pro388 Leu387 /'~ 11e384 Cys282 _..~

11tl !

Va187 ,Lcu4-1ql /./~o' 'Phi/3191 os

c~4151 kspso ~181

I

Asn124 Asr~221 : Set75

'"-~-

~

m5

Ash,52 I

3

~

~s

"x~ 'lrl~

o rpN6

/ /

,._

AspS0

.......

I

Fig. 3. Schematic representation of direct receptor models by 9A) Hutchins [66] Malmberg et al [64] C) Teeter et al [65], respectively holding compounds 36, 7, and 1.

B)

Trp-160 in TM4, Phe-389, Phe-390, and Trp-386 to build the flanks and bottom of the binding pocket. An ancillary site, flanked by Phe-110 in TM3 and Tyr-408 in TM7, suitable to accomodate large N-substituents, such as phenethyl, projecting upward and parallel to the helical axes, was similarly suggested. On the contrary, substituents longer than n-propyl projecting perpendicular to the axes appeared not to be accepted due to the hindrance by Thr412 in TM7. The aspartate residue in TM2 (Asp-80) is considered to be crucial for receptor activation and triggering of signal transduction [68], but its function at the molecular level is not clear. It was considered by Teeter et al. [65] to be involved in receptor regulation by Na +, along with Ser-121 and Asn-124 in TM3, and Ser-419 and Asn-422 in TM7, forming a square piramidal coordination site, directed from the middle to the intracellular face of the membrane. Hutchins [66] took a completely different approach, suggesting that Asp-80 actually interacts with the catechol system of dopamine (Fig. 3 A). In this way, the interaction of the catechol system with the serine residues on TM5 was disregarded, pointing out the scarce consistency of mutagenesis studies of these residues, but, on the other hand, a mechanism for receptor activation was hypothesized. Hydrogen bonding of the m-hydroxy group, or a functionally equivalent group, to Asp-80 would release some existing interactions of the latter with a set of residues, such as Asn-52 in TM1, Ser-75 in TM2, Asn-124 in TM3, and Asn-422 in TM7, located near to the intraceliular side of the membrane. This could start a conformational change relaying a signal transduction message to the G-protein. In this model the n-propyl site has been identified in a region projecting between TM3 and TM7 towards Val-87 and Val-91 on TM2. D 1 selectivity was analyzed, pointing out an additional pocket between TM6 and

82 TM7 accomodating the phenyl substituent. As indicated for 36 in Fig. 3 A, in the D 1 receptor, differing from D 2 for Cys-283 instead of Ile-384, and Phe-319 instead of Leu-414, the pocket has a suitably open shape, fixed by hydrogen bond linking of Cys-283 to the asparagine residue corresponding to Asn-418 in D 2. It has to be noticed that these models are limited to the transmembrane domains, necessarily neglecting the intracellular loops, which can anyhow exert an important role in binding and in signal transduction. For instance, chimeric modifications of the intracellular loop between TM5 and TM6 have shown its relevance in modulating agonist affinity and selectivity for D 2 and D 3 receptors (Robinson, 1994). Some remarks, and new questions

In the last few years significant progress has been recorded in the investigation of structural requirements for agonist selectivity on the new receptor subtypes, although there are yet no hints for selectivity at D 5 vs D1, and D 4 vs D 2 or D 3 receptors. As for D4, (+)-apomorphine has unexpectedly been shown to act as a selective antagonist [70], pointing out the interest of a new search for agonists in the previously fertile field of the aporphines. Selective D 3 agonists have been identified and are on the way to clinical investigation, particularly because of their action on mesolimbic autoreceptors. In the analysis of the interaction of dopamine agonists with the receptors old dogmas have been gradually dismantled, including the one of hydrogen bond donation, at least in D 2 and D 3 receptors. A recent report [71] also suggested, according to results of affinity modulation by GTP, that 13-(3-pyridyl)-N,N-di-n-propylphenethylamine, which is devoid of hydrogen donor groups and has nanomolar affinity for D 1 receptors, may behave as an agonist. Functional response data are desirable in order to confirm this interesting observation. The finding that recognition can involve electron rich, or polarizable, regions of agonist molecules widens the space of controversy and of further investigation on direct and indirect receptor models, including the possibility that either different sites or different conformations of receptors may fit different classes of agonists. In the case of the D 1 receptor, the most selective feature for recognition appears to be the occupation of a lipophilic site, forbidden for D 2 activity, near to the "mouth" of the receptor pocket, but other features also appear to favour D 1 activity : 1) the catechol system, although no longer considered as essential; 2) a far-away site in the receptor, accomodating an aryl group and a second amine nitrogen at two-three atom distance, in compounds such as dopexamine 2(~), 31, 34, and 35; and 3) a specific role of the n-propyl group in N,Ndisustituted derivatives of 5,6-ADTN such as 7, 33, and 43, which is supported by the observation that ~ the N-despropyl analogue of 33, behaves as an antagonist. These features frequently appear to cooperate in increasing the activity, suggesting that multiple interactions may help in a mutual conformational accomodation of some ligands and the D 1 receptor. The existence of different modes of interaction for different classes of agonists appears as a likely explanation in the case of the above 5,6-ADTN derivatives with respect to the primary amine 6,7-ADTN (4). The therapeutic targets of the development of a new generation of dopaminergic agonists remain mainly those that were identified for the first generation, where there is still a medical need of improved drugs. We can anyhow expect that the availability of potent and selective agonists for pharmacological and clinical investigations will lead to the discovery of new applications. Some suggestions, by way of example, may be derived by recent reports: a D 3 receptor agonist showed gastroprotective and antisecretory effects in rats [72]; dopamine

83 agonists may find application in conditions of alcohol [73] and opiate [74] dependence; D 4 receptors are expressed in relatively high density in cardiac tissues [75, 76], and their function awaits to be elucidated.

Acknowledgment : We are indebted to Dr. Andrea Zaliani for molecular modelling studies and for useful discussion.

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 30 31

Cannon JP. In: Jucker E, ed. Progress in Drug Research. Basel: Birkh~iuser Verlag 1985; 29: 303-412 Seeman P. Biochem Pharmacol 1982; 31:2563-2568 Goldberg LI, Kohli JD. In: Casagrande C, ed. Experimental Cardiology: Linking Laboratory and Clinical Research. Cormano - Milan: CNM 1993; 81-100 Kaiser C, Jain T. Med Res Rev 1985; 5:145-229 Katermopoulos HE, Schuster DI. Drugs of Future 1987; 12:223-253 Seiler MP, B61sterli JJ, Floersheim P, et al. In: Testa B, Kyburr E, Fuhrer W, Giger R, eds. Perspectives in Medicinal Chemistry. Basel: Verlag Helvetica Chimica Acta 1993; 221-237 McDermed JD, Freeman HS. In: Kohsaka M, Shohmoki T, Tsukada Y, Woodruff GN, eds. Advances in Dopamme Research. Oxford: Pergamon Press 1982; 179-187 De Marmis R, Hieble JP. Drags of Future 1989; 14:781-797 Wikstrom H. In: Ellis GP, Luskombe DK, eds. Progress in Medicinal Chemistry. Amsterdam: Elsevier 1992; 185-216 Soares-da-Silva P, ed. Cardiovascular and renal actions of dopamme. Oxford: Pergamon Press 1993 Casagrande C. Herz 1991; 16:102-115 Weinstock J, Hieble JP, Wilson III J. Drugs of Future 1985; 10:646-697 Casagrande C. In: Barnett DB, Pouleur H, Francis GS, eds. Congestive heart failure: pathophysiology and treatment. New York: M Dekker Inc 1993; 251-286 Brown RA, Brown RC, Hall JC, et al. In: Lambert RW, ed. Third SCI-RSC Medicinal Chemistry, Symposium. London: Royal Society of Chemistry 1985; 169-192 Fuller RW, Clemens JA. Life Sci 1991; 49:925-930 Pontiroli AE, Cammelli L, Baroldi P, Pozza G. J Clin Endocrmol Metab 1987; 65:1057-1059 Seyfried CA, Boettcher H. Drugs of Future 1990; 15:819-832 Casagrande C. In: ref. 10; 203-216 Simpkins JW, Bodor N, Enz A. J Pharm Sci 1985; 74:1033-1036 Ishikura T, Senou T, Ishidara H, et al. Int J Pharm 1995; 116:51-63 Lee MR. Triangle 1987; 26:11-21 Drieman JC, van Kaan FJPM, Thijsen HHW, et al. Brit J Pharmacol 1994; 111:1117-1122 Nishiyama D, Yoshikawa M, Yamaguchi I. Cardiovasc Drag Rev 1992; 10:101-116 Augstein J, Austin WC, Boscott RJ, et al. J Med Chem 1965; 8:365-371 Casagrande C, Galli A, Femni R, Miragoli G. Farmaco Ed Sci 1972; 27:445-470 Casagmnde C, Bertolini G, Montanafi S, Santangelo F. Colloque de Chimie Organique Fine, ERAI, Lyon 1991; 11 Santangelo F, Bertolmi G, Casagrande C, et al. U.S. Pat. 5.407956 Pocchiari F, AUievi L, Zanzottera D, et al. Can J Physiol Pharmacol 1994; 72 (suppl 1):132 Abs. P.1.9.61 Marchini F, Pradella L, Miragoli G, Semeraro C. Can J Physiol Pharmacol 1994; 72 (suppl 1): 131 Abs. P1.9.60 Martin Y. J Med Chem 1992; 35:2145-2154 DeNinno MP, Schoenleber R, Pemer RJ, et al. J Med Chem 1991; 34:2561-2569

84 32 33 34 35 36 37 38 39 40 41 42 43 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 75 76

Kebabian JW, Briton DR, DeNinno MP, et al. Eur J Pharmacol 1992; 229:203-209 Springthorpe B, Dixon J., Inee F, et al. 12th Int Syrup Med Chem. Basel 1992; P-144-A Brewster WK, Nichols DE, Riggs RM, et al. J Med Chem 1990; 33:1756-1764 Mori N, Ishihara H, Matsuoka T, et al. Eur J Pharmacol 1990; 183:1049 Anan H, Tanaka A, Tsuzuki R, et al. Chem Pharm Bull 1991; 39:2910-2914 Smith GW, Farmer JB, Ince F, et al. Br J Pharmacol 1990; 100:295-300 McClelland DL, Lakatos I, Rosenkranz RP. Faseb J 1990; 4: A608, Abs. 1981 Seiler MP, Floersheim P, Markstein R, Widmer A. J Med Chem 1993; 36:977-984 Civelli O, Bunrow JR, Grandy DK. Ann Rev Pharmacol Toxieol 1993; 32:281-307 Jackson DM. Pharrnaeol Ther 1994; 64:261-369 Memo M. Mol Neurobiol 1990; 4:181-195 Seeman P, van Tol HHM., Trends Pharmacol Sci 1994; 15:264-270 Arnt J, Hyttel J, Sgmchez C. Eur J Pharmacol 1992; 213:259-267 Gnanalingham KK, Hunter AJ, Jenner P, Marsden CD. Biochem Pharmacol 1995; 49: 11851193 Levesque D, Diaz J, Pilon C, et al. Proc Natl Acad Sci USA 1992; 89:8155-8159 Murray AM, Ryoo HL, Gurevich E, Joyce JN. Proc Natl Acad Sci USA 1994; 91:11271-1"1275 Sonesson C, Lin CH, Hansson L, et al. J Med Chem 1994; 37:2735-2753 Sonesson C, Boije M, Svensson K, et al. J Med Chem 1993; 36:3409-3416 Haadsma-Svensson SR, Smith MW, Lin CH, et al. Bioorg Med Chem Letters 1994; 4:689-694 Rivet JM, Audinot V, Gobert A, et al. Eur J Pharmaeol 1994;265:175-177 Dijkstm D, Damsma G, Wikstr6m H, et al. 12th Int Symp Med Chem. Paris, 1994; P302 Lecrubier Y. Neuropsychopharmacol 1994; 10: (suppl 3) Abs. S- 16-91 Meltzer LT, Caprathe BW, Christoffersen CL, et al. J Pharmacol Exp Ther 1994; 266:11771189 Wright JL, Caprathe BW, Downing DM, et al. J Med Chem 1994; 37:3523-3533 For a review, see Johansson AM, Grol CJ, Karlen A, Hacksell U. Drug Design and Discovery 1994; 11:159-174 Kocjan D. J Med Chem 1994; 37:2851-2855 Downing DM, Wright JL, Mirzadegan T, et al. 206th ACS National Meeting, Chicago, August 1993, Abstract MEDI 172 Alkorta I, Villar HO. J Computer-Aided Molecular Design 1993; 7:659-670 Alkorta I, Villar HO. J Med Chem 1994; 37:210-213 Parli CJ, Schmidt B, Shaar CJ. Biochem Pharmacol 1978; 26:1405-1408 Trumpp-Kallmeyer S, Hoflack J, Bruinvels A, Hibert M. J Med Chem 1992; 35:3448-3462 Livingstone CD, Strange PG, Naylor LH. Biochem J 1992; 287:277-282 Malmberg A, Nordvall G, Johansson AM, et al. Mol Pharmacol 1994; 46:299-312 Teeter MM, Froimowitz M, Stec B, DuRand CJ. J Med Chem 1994; 37:2874-2888 Hutchins C. Endocr J 1994; 2:7-23 Mansour A, Meng F, Meador-Woodruff JH, et al. Eur J Pharmacol 1992; 227:205-214 Neve KA, Cox BA, Hermmgsen RA, Spanoyannis A, Neve RL. Mol Pharmacol 1991; 39: 733739 Robinson SW, Jarrie KR, Caron MG. Molecular Pharmacol 1994; 46:352-356 Seeman P, Van Tol HHM. Eur J Pharmacol 1993; 233:173-174 Claudi F, Cingolani GM, Giorgioni G et al. Eur J Med Chem 1995; 30:415-421 Glavin GB. Life Sci 1995; 56:287-293 Lawford BR, Young RMcD, Rowell JA, et al. Nature Med 1995; 1:337-341 Defonseca FR, Rubio P, Martmcalderon JL, et al. Eur J Pharmacol 1995; 274:1-3 O'Malley KL, Harmon S, Tang L, Todd RD. New Biol 1992; 4:137-146 Amenta F, Ricci A. 5th Int Conf Peripheral Dopamine, Kyoto, 1994; pg 22, Abs L-4

Perspective in Receptor Research

D. Giardin~, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All fights reserved.

85

Serotonin Receptor Complexity: Relationships and Roles T.A. Branchek Department of Pharmacology, Synaptic Pharmaceutical Corporation, 215 College Road, Paramus NJ 07652, United States of America

INTRODUCTION The serotonergic innervation of the brain arises primarily from a small and discreet set of raphe nuclei which send diffuse afferents to innervate virtually all levels of the CNS. This distribution suggests that 5-HT can influence neural activity in a wide variety of brain regions and may exert modulatory or integrative functions. In addition to its actions in the CNS, 5-HT also acts on cells and tissues throughout the rest of the body including smooth muscle, endothelia, and neurons. This heterogeneity of action appears to be served through a complementary complexity of receptor subtypes via which 5-HT exerts these actions. The rules for relating receptor subtypes to roles in serotonergic fimction remain elusive. Although the ubiquity of serotonin responses has been apparent for several decades, the precise definition of the molecular substrates underlying this apparent complexity is a recent achievement. At present genes for at least fourteen distinct serotonin receptor subtypes have been cloned and their expressed proteins evaluated by a variety of techniques. Progress has been made in the understanding of the structural relationships of these subtypes, residues in the amino acid sequences that are important for ligand recognition, their localization in the body, their signal transduction mechanisms, and some of their possible roles in physiology and pathophysiology. However, simple generalizations about this receptor system cannot be made. For example, there is no definitive evidence that points to any receptor subtype as having an exclusively CNS or peripheral location or action. For a given preparation whether it be a single enteric neuron, raphe neuron, or smooth muscle cell, there is not a single receptor subtype but often sets of 4-5 different ones. How signals mediated through these diverse subtypes are integrated with each other as well as with those from receptors which converge onto the same signaling pathways remains an active area of investigation. The more global understanding of the exact function of each of these subtypes awaits the discovery of selective chemical and molecular tools with which to refine our hypotheses.

STRUCTURAL RELATIONSHIPS INTRACELLULAR SIGNALING

OF

5-HT

RECEPTORS

AND

THEIR

Receptor subtypes for serotonin have been divided into seven distinct classes based on operational, structural and transductional properties [1]. There are five 5-HT~ receptor subtypes, three 5-HT 2 subtypes; one 5-HT 3 receptor, one 5-HT 4 receptor, two 5-HT 5 subtypes, one 5-HT6 receptor and one 5-HT7 receptor. Splice variants or additional

86 subtypes are likely for the 5-HT2c [2] and 5-HT2A[3] and have already been demonstrated for the 5-HT3 [4] and 5-HT 4 [5] receptors. With the exception of the 5-HT3 receptor which forms a ligand-gated ion channel [6], all other cloned 5-HT receptors are members of the G-protein coupled receptor superfamily. The ligand binding and activation or "operational" properties of these receptors has been extensively reviewed [1,7]. In general, the task to create sets of selective agonists and antagonists for each subtype remains a significant challenge for the future. The salient features of the categorization of 5-HT receptors based on commonality in structure and signalling are addressed below.

5-HT 1 Receptors

The five 5-HT~ receptors are termed 5-HTIA, 5-HTI o~, 5"HT~o~, 5-HT~E and 5-HT1F [1,7]. They each contain similar numbers of amino acids as follows: 421,377, 390, 365, 366, respectively for the human homologues. Each receptor is encoded by an intronless gene. At the structural level these proteins all share a "homology cluster" wherein there is mutual conservation of greater than half of their amino acid residues in the transmembrane spanning region which contains the ligand binding pocket for many biogenic amine receptors [8,9]. In contrast, the extracellular amino termini and loop regions of these seven helix transmembrane spanning proteins as well as their intracellular loops and carboxy termini lack observable homology that would facilitate categorization or prediction of their common intracellular coupling. Each of these 5-HT~ receptors couples to G-proteins which leads to the inhibition of adenylate cyclase activity and thus cAMP production [1]. The 5-HT~A [10], 5-HT~D~ [11], 5-HTIDp [11], and 5-HT~F [12] subtypes, also modulate intracellular Ca** through PTX-sensitive G-proteins. In cases where it has been evaluated, the 5-HTzE receptor has not been shown to do so [13]. In addition to these pathways, each of the 5-HT I receptors, with the exception of 5-HT1E [13] has been shown to increase the turnover of inositol phosphates [11,12,14,15]. Physiologically, the initial mediator of the action of these receptors may be through Gprotein-gated channels such as KGA [16]. Thus far, coupling of cloned 5-HT~ receptors to G-protein-gated K§ or Ca** channels has only been demonstrated for the 5-HTIA receptor [17]. Coupling of the 5-HT~A [14], 5-HT1D~ [18,19], and 5-HT~E [13] receptors to the stimulation of adenylate cyclase has also been demonstrated. However, this signaling pathway appears to be detected when relatively high levels of expression are attained relative to levels which can elicit a cyclase inhibition in the same cell host, or in the absence of a cyclase inhibitory response in a particular host. Therefore, it is tempting to speculate that these ancillary coupling pathways may result from a simple law of mass action in which all of the relevant Gi alpha subunits for a given receptor subtypes are "unavailable" and thus there is overflow to alternative pathways. Such a speculation is consistent with observation of preferential receptor-G-protein interaction when assayed using immunoprecipitation [20]. Future directions for the study of 5-HTI receptor signal transduction include quantitative analysis of subtype-G-protein interactions in reconstituted systems which do not rely on the variability of G-protein expression in host cells selected

87 for transfection based on incidental properties. In addition, coupling of the remaining cloned 5-HT I receptor subtypes to channels requires investigation.

5-HT 2 Receptors The 5-HT 2 receptors include 5-HT2A, 5-HTzB and 5-HT2c [ 1]. The proteins are similar in length, containing 471, 479, and 458 amino acids, respectively, for the human homologues. These subtypes possess a high degree of conservation in their amino acid sequences, contain over 70% identical amino acids in their transmembrane regions, and show highly conserved intron-exon boundaries [21]. All 5-HT 2 receptors couple to Gproteins which elicit phospholipase C activation and Ca++ mobilization. However, it appears that there may be some differences in the efficiency of coupling of these subtypes. Whereas both the 5-HTzA and 5-HTzc receptors show large stimulation of PI hydrolysis in many types of host cell, the 5-HTzB receptor gives a weaker response under similar conditions [21]. This recently cloned 5-HTzB receptor can also be observed in native systems such as the rat stomach fundus where it was originally described [22], and more recently in a teratocarcinoma cell line [23]. In this cell line, the 5-h 22B subtype couples to PI hydrolysis via a Gq protein. Coupling to both adenylate cyclase activation and inhibition has been reported for the 5-HT2c subtype [24] at high expression levels only. Interestingly, coupling to PI hydrolysis in the rat stomach fundus has never been demonstrated, although in this tissue it is clear that the receptor couples to Ca *+ influx through voltage sensitive Ca ++ channels, intracellular Ca+* release, and activation of PLC [25].

5-HT3 Receptors The 5-HT 3 receptor is a ligand-gated ion channel. Although there are differences in the literature in the apparent operational properties of this receptor in native preparations, they appear to be related to species differences. There has been no report of novel subtypes of 5-HT 3 receptor despite searching by many excellent investigators. The 5-HT 3 receptor was originally cloned from the mouse and the sequence predicts a protein of 487 amino acids [6]. This molecular mass is consistent with that reported for the solubilized receptorchannel complex [26]. An interesting structural property of the 5-HT 3 receptor is that it appears to be a homo-oligomeric receptor which distinguishes it from other ligand-gated ion channels such as those for acetylcholine or glutamate which are heteropentameric. The 5-HT3 receptor has also been cloned from the human and contains 478 amino acids [27]; however, no other characterization is available.

88 5-HT 4 Receptors

Cloning of the 5-HT4 receptor has finally been reported [5,28]. So far no subtypes of this receptor have been identified. However, a splice variant has been found in the rat which differs only in the carboxy terminus. The variants contain 387 and 406 amino acids for the rat receptors. No obvious operational differences have been detected in binding studies. Functionally, the cloned 5-HT4 receptor couples to the stimulation of adenylate cyclase activity. The two isoforms differ in their rates of desensitization, consistent with differences in phosphorylation sites in the carboxy termini [29]. No coupling to cyclase inhibition or to PI turnover has been detected.

5-HT 5 Receptors

The 5-HT5 receptors include 5-HT5A and 5-HT5B in rodents [1, 30, 31]. The rat receptors are proteins containing 357 and 371 amino acids. They share >75% amino acid identity in the transmembrane regions. In the human, the 5-HT5A has been cloned [32] and it has 84% overall amino acid identify to its murine homologue. Only an untranscribed fragment (one of the two exons) of the human 5-HT5B receptor has been detected [32] . If the absence of a full length cDNA for the human 5-HT5B subtype can be proven, it would be the first G-protein coupled receptor found in lower species but absent in man. Quite recently, a different group has reported the second exon of the human sequence and has found that it is interrupted by several stop codons [33]. Both Southern blot and PCR experiments indicate that this is the only homologue of the rodent 5-HTsB gene and thus, there will be no functional 5-HT5B receptor in the human. Since the function of the 5-HT 5 receptor in rodents is unknown, it is unwise to speculate on the significance of its possible loss in evolution. Although the signal transduction pathway of the vast majority of Gprotein coupled receptors is demonstrated readily after transfection into several host cells, coupling of the 5-HT 5 subtypes has remained somewhat mysterious until recently. Evidence now indicates that these subtypes may couple to the inhibition of adenylate cyclase activity [34]. Details of the response are not yet available. However, if the cyclase coupling is a robust and primary response, it is likely that the receptor will also couple to mobilization of intracellular Ca++.

5-HT 6 Receptors

The rat 5-HT 6 receptor is a protein of 436 amino acids [35, 36]. The human homologue contains 440 amino acids [36a]. This sequence contains at least one intron, located in the third intracellular loop. However, no isoforms have been cloned. Like the 5-HT4 receptor,

89 5-HT 6 receptor also couples to adenylate cyclase stimulation. Despite this similarity in coupling, however, there is no significant homology in the amino acid sequence of these two subtypes [5]. A detailed functional analysis has not been reported for the cloned 5HT 6 receptor. However, a native receptor which is detected in N 18TG2 cells, displays a rank order of agonist potency in cAMP assays which is similar to the affinity values for these compounds determined by radioligand binding in the same cell line [37]. It is likely that this site is similar or identical to the 5-HT 6 receptor. A detailed analysis of the N18TG2 cells using molecular techniques has not been reported.

5-HT 7 Receptors

The 5-HT 7 receptor is a third adenylate cyclase stimulatory subtype [1]. The human sequence encodes a protein of 445 amino acids [38]. The sequence appears to contain an intron in the carboxy terminus. Like the 5-HT 6 receptor, the 5-HT 7 subtypes lacks any homology to the other cloned 5-HT stimulatory receptors [5]. No inhibition of adenylate cyclase activity or coupling to PLC has been demonstrated for this subtype.

FUNCTIONS OF 5-HT RECEPTORS: STUDIES, TRANSGENIC ANIMALS

SELECTIVE LIGANDS, ANTISENSE

Although the majority of 5-HT receptor subtypes have been cloned, the task ahead remains to discover their distinct and/or interacting roles in physiology and pathophysiology. In order to do so, three main methods are under investigation. The classical approach is the development of subtype selective ligands with which to probe receptor function. The fastest approach is to employ antisense methodology to "knockdown" expression a discreet subtype of receptor. The third approach is to manipulate gene expression by homologous recombination and thus to "knock-out" or overexpress receptors.

USE OF SELECTIVE LIGANDS TO REVEAL RECEPTOR FUNCTION: 5-HT l

The clearest demonstration of 5-HT, receptor function which can be linked to therapeutic potential has come from the development of drugs which target the 5-HT~A and 5-HT~D receptors. The 5-HT~A partial agonists such as buspirone are clinically useful in the treatment of anxiety [39]. In addition, it has been suggested that antagonists of the 5-HTIA receptor, such as pindolol, may lead to a more rapid onset of antidepressant activity when combined with a selective serotonin reuptake inhibitor [40]. Although this particular

90 compound has pharmacological actions at receptors other than the 5-HT~A site, preliminary clinical observations indicate that the approach may hold promise [41]. Agonists of the 5-HT~D receptor subtypes have been developed for the acute treatment of migraine. Experimental data indicate that drugs such as sumatriptan may act on extracranial blood vessels to reduce vasodilation and/or on the terminals of the trigeminal-vascular afferent s to inhibit the release of peptides which mediate the sterile inflammatory response [42]. Clinically, sumatriptan is useful in the relief of acute migraine. Possible functions of the 5-HT~ and 5-HTlr receptor await the development of selective compounds or other approaches.

USE OF SELECTIVE LIGANDS TO REVEAL RECEPTOR FUNCTION:

5-HT 2

Much evidence has accumulated to link activation of the 5-HT2A receptor to hallucinations [43]. Agonists of this receptor subtype correlate best with the potency of several classes of hallucinogenic compounds including tryptamines and phenylalkyamines to induce hallucinations in people. Since this is not a clinically useful profile, focus has been placed on the development of antagonists for the 5-HTEA receptor. Many such compounds have been developed but their benefit has been elusive. Ketanserin, once thought to be a new antihypertensive agent via a 5-HT2A receptor mechanism, is now thought to exert that action via an alpha-1 receptor subtype. Several compounds with affinity for 5-HT2 receptors are now under investigation for a collection of CNS disorders including anxiety, depression, sleep disorders and drug abuse [44]. The 5-HTEAantagonists risperidone, sertindole, and amperozide all have been tested clinically as atypical antipsychotics [44]. These compounds combine 5-HT2A antagonist with D2 antagonist action and are reported to have a benefit for the negative symptoms of schizophrenia as well as a lower rate of extrapyramidal side effects. Drugs such as methysergide, used in the prophylactic treatment of migraine, are now speculated to act on 5-HT2s receptors [45]. However, there are many compounds which have mixed 5-HT2B and 5-HT2c activity and thus it is still difficult to assign a single subtype to the observed activities of these compounds. Potential therapies mediated by one or both subtypes include anxiety, depression, sleep disorders, and feeding disorders.

USE OF SELECTIVE LIGANDS TO REVEAL RECEPTOR FUNCTION: 5-HT 3

The 5-HT 3 antagonists, such as ondansetron and granisetron, have found clinical utility in the treatment of emesis, particularly for radiation or chemotherapy-induced cases [46]. Application to post-surgical and other forms of emesis are less well documented. 5-HT 3 receptors have also been speculated to be involved in cognition, anxiety, migraine, and pain. However, clinical proof of their role in these states is still lacking.

91 USE OF SELECTIVE LIGANDS TO REVEAL RECEPTOR FUNCTION: 5-HT4 Possible functions of the 5-HT4 receptor have been recemly reviewed [47,48,49]. In the CNS, 5=HT4 activation increases cAMP in the hippocampus [50]. This observation has led to behavioral studies using cognition models. 5-HT4 receptor activation induces relaxation of the esophagus [51,52] and increases cAMP accumulation in this tissue [53]. These data indicate a possible role for the 5-HT4 receptor in GERD. A selective 5-HT4 partial agonist, cisapdde, has been used clinically for gastroprokinesis for many years prior to the elucidation of its molecular site of action. Functional 5 - H T 4 receptors have also been demonstrated in the guinea pig ileum [54] and in the cardiac atrium, in which increases in cAMP accumulation can also be detected [55]. Such functions indicate possible roles of the 5-HT4 receptor in irritable bowel syndrome and in arrhythmia.

USE OF SELECTIVE LIGANDS TO REVEAL RECEPTOR FUNCTION: 5-HT 5, AND $-HT6 At present there are no selective ligands to evaluate 5-HT5 function. Possible i.n vivo roles of the 5-HT6 receptor may be related to neuropsychiatrie functions, based primarily on the high affinity of atypical antipsyehotic drugs and for several tricyclic antidepressants for this receptor [56]. Thus, it may modulate affective state. The localization of mRNA for the 5=HT6 receptor in the limbic system is consistent with this speculation.

USE OF SELECTIVE LIGANDS TO REVEAL RECEPTOR FUNCTION: 5-HT 7 There are no selective ligands available to study the 5-HT7 receptor. However, based on the activity profiles of a set of known compounds (which have additional receptor activity), the 5-HT7 receptor may play a role in neuropsychiatric processes. Like the 5-HT6 receptor, the 5=HT7 subtype has high affinity for several antipsychotic compounds and antidepressants [56]. In addition to these actions, many smooth muscle responses to 5-HT which result in relaxation [57,58,59] may be mediated by 5=HT7 receptors. In several tissues, positive linkage to cyclase stimulation has been detected. A recent study of relaxant responses of the ileum has [60] revealed pharmacological properties which are nearly identical to those of the cloned guinea pig 5=HT7 receptor [61]. Thus the 5-HT7 receptor may play a role in smooth muscle relaxation in irritable bowel syndrome or angina.

92 The possible roles of the 5-HT7 receptor in circadian phase shifts [62] has been hampered by the lack of selective compounds. However, based on the observation that 5CT, DPAT and 5-HT, compounds with affinity for both 5-HT~A and 5-HT7 receptor subtypes, can phase advance neuronal firing activity in hypothalamic slices in culture [63], and that a 5-HT~A antagonist was unable to block this response [20], it remains a plausible hypothesis. Whole cell voltage clamp recordings from rat suprachiasmatic nuclei neurons [64] also support a role for the 5-HT 7 receptor in circadian function. Proof of the role of 5-HT 7 receptors in such processes awaits the development of subtype selective tools.

USE OF ANTISENSE AND TRANSGENIC RECEPTOR FUNCTION: 5-HT 1 RECEPTORS

APPROACHES

TO

REVEAL

Antisense oligonucleotides can be administered in vivo in an attempt to uncover the function of receptors when their sequences first become available. As such, it is an attractive approach which has been applied to the study of G-protein coupled receptors over the last several years [65]. Employing such a methodology, there is no time lag for selective ligands or for the production of transgenic animals, both of which can be quite lengthy processes. The application of this technology to the study of 5-HT ! receptors has only come quite recently. In a preliminary report, antisense oligos directed against the 5HT~A receptor have been injected into rats in order to evaluate the role of this subtype in anxiety [66]. Behavioral data are not yet available. For the 5-HT~w~Da receptor, a transgenic knockout mouse has been produced and partially characterized at the behavioral level [67]. These animals display an increase in aggressive behavior in an intruder paradigm. Many other studies employing these animals are underway.

USE OF ANTISENSE AND TRANSGENIC RECEPTOR FUNCTION: 5-HT 2 RECEPTORS

APPROACHES

TO

REVEAL

Antisense oligonucleotides have been used to disrupt the function of 5-HT2Areceptors [68]. In a learned helplessness model for depression, treatment of animals with such tools led to a significant difference in the treatment vs the control group, indicating a possible role for 5-HT2A antagonists in depression. Transgenic animals have also been constructed which knock out the 5-HT2A receptor [69]. Unfortunately, this knock out leads to disruptions in cranial facial structure and results in lethality. Behavioral analysis of the heterozygotes was not complete due to these malformation. The future use of inducible knockouts may facilitate the study of the function of this receptor subtype using the heterologous recombination technology. More success has been obtained in animals with targeted disruptions of the 5-HT2c receptor [70]. These animals display profound disruptions of brain function. They are prone to sudden death as a result of seizure activity

93 and also display a significantly greater body weight than their wild type litter mates. Such an observation is consistent with previous pharmacological data linking 5-HT2c antagonists to reduction in food intake [70].

USE OF ANTISENSE AND TRANSGENIC APPROACHES RECEPTOR FUNCTION: 5-HT 5 AND 5-HT 6 RECEPTORS

TO

REVEAL

Antisense oligonucleotides targeted to the 5-HT 6 receptor subtype have been administered to rats and evaluated behaviorally [71 ]. In these studies, the animal exhibited a behavioral phenotype consisting of an increase in the number of yawns and stretches. This behavior was blocked by atropine, suggesting a role of the 5-HT6 receptor in the control of cholinergic neurotransmission. For the 5-HT 5 receptor, knockout mice are in progress but no behavioral data have been reported [33].

SUMMARY Many advances have been made in understanding the receptor substrates for the multiple actions of 5-HT. However, many fruitful areas of exploration remain ahead. The generation of receptor subtype selective approaches including ligands, radioligands, antibodies, antisense oligonucleotides, and transgenic animals has only begun. Even "wellcharacterized" receptors may hold new surprises. Further investigation of the molecular basis of ligand recognition are largely unexplored. Only a rudimentary understanding of signal transduction of these subtypes is extant. The next decade will likely see enormous progress in the understanding of this complex signaling system.

ACKNOWLEDGMENTS Many thanks to my colleagues at Synaptic Pharmaceutical Corporation and on the Serotonin Nomenclature Committee for helpful discussions and sharing preliminary information. Thanks to Ms. Lisa Gonzalez and Elinor Bernstein for preparation of the manuscript.

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Perspective in Receptor Research D. Giardin/L, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

99

Design and Synthesis of Agonists and Antagonists of the Serotonin 5-HT4 Receptor Subtype Daniel P. Beckerw Bella Goidstin (t), Gary W. Gullikson(t), Richard Loeffler (t), Alan Moormannw Chaffiq Moummi(t ), Roger Nosalw Dale Spanglerw Clara I. Villamilw Dai-C. Yang (t), Daniel L Zabrowskiw and Daniel L. Flynnw Departments of Medicinal Chemistryw and Gastrointestinal Diseases Research(t ), Searle Research and Development, Skokie, IL USA 60077

Introduction Serotonin is unsurpassed among monoamine neurotransmitters in the number of receptor subtypes reported to-date. Fourteen subtypes of seven major serotonin receptor classes have been identified in the central nervous system, the peripheral nervous system, myeloid/immune cell types, and smooth musculature of mammals [1]. While discreet, endogenous release of serotonin is able to manifest a variety of responses and behaviours, exogenous administration of serotonin (and nonselective mimetics) suffers from indiscriminate actions at all populations of accessible receptors. Hence, the discovery of subtype-specific agonists and/or antagonists to the various serotonin receptors have been sought as specific therapeutic agents. For some time, it has been known that serotonin exerts profound effects in the enteric nervous system (ENS). Gaddum and Picarelli originally characterized the serotonin-induced contractions of guinea pig ileal smooth muscle as being direct (D-receptor) and indirect (M-receptor) [2]. While the former receptor was later clearly defined as being the 5HT2 site, the ENS-mediated indirect contractions of the GP ileum were less clearly defined. In 1989, Dumuis et al reported that a series of prokinetic benzamides were found to a c t as agonists at the newly defined CNS 5-HT4 receptor subtype [3], which had been identified in mouse CNS colliculi neurons [4-5]. Subsequent studies by independent investigators confirmed the 5-HT4 agonist properties of benzamides, and demonstrated that the peristaltic reflex induced in the GP ileum by these benzamides was attributed to 5-HT4 agonism within the myenteric plexus [6-7]. While many benzamides have been disclosed over the last two decades, these compounds were designed either empirically or by rational

lO0

design to block other monoamine receptors, including the serotonin 5-HT3 receptor and the dopamine D-2 receptor. Therefore, while many of these compounds, including metoclopramide and cisapride, do interact with 5HT4 receptors, they also interact at other serotonergic, adrenergic, and dopaminergic sites. Our research goal was to design potent gastrointestinal prokinetic agents based upon a 5-HT4 receptor agonist mechanism. A parallel goal was to demonstrate that a class of 5-HT4 agonists could be selective as GI prokinetic agents, lacking untoward effects in other CNS [8] and cardiac tissues [9] where 5-HT4 receptors have been functionally described. Finally, an optimal goal was to identify a class of chemistry which would afford therapeutic dual 5-HT4 agonism/5-HT3 antagonism at such a dose which would be free of actions at other monoamine receptors, most notably antagonism at dopamine D-2 receptors. Such a dual 5-HT4 agonist/5-HT3 antagonist was considered ideal for the treatment of a variety of GI dysmotilities, including irritable bowel syndrome, gastroparesis, nonulcer dyspepsia, gastroesophageal reflux disease, emesis, and other abnormal states characterized by altered ENS sensory reflex mechanisms.

Molecular

Approach

Several indole analogs of serotonin were evaluated for 5-HT4 agonism in the recently described rat esophageal tunica muscularis mucosae (RatTMM) preparation [10]. Serotonin acts at 5-HT4 receptors in this preparation to relax the esophageal smooth muscle" pre-contracted with carbachol. We, and others [10-12] demonstrated that 5-HT [(1), EC50 = 10 nM] and 5-methoxytryptamine [(2), EC50 = 148 nM] are potent full agonists at the 5-HT4 receptor. However, tryptamine [(3), EC5o - 8000 nM] and 2-methyl-5HT [(4), EC50 - 20,000 nM] are much less active. See table 1. The activity of the phenolic-containing (1) and the methoxyl-containing (2) --- but dramatic decrease in activity of the 5-unsubstituted analog (3) --- suggested that a hydrogen-bond acceptor interaction is required at the R s-position of 5-HT4 agonists. The very weak agonist properties of 2-methyl-5HT (4) suggested an unfavorable A1,3 strain interaction between the 2-methyl group and the ethylamine side chain in the conformation(s) required for 5-HT4 agonism. Such A1,3 strain would predominate in conformers wherein Tau 1 = 180 ~ Figure 1 illustrates this set of conformers, wherein the side chain amino group may be positioned at rotamer sites A, B, and C [13].

101

Table 1 Agonist activities

of various

indoles NH2

R5

Proposed

Figure 1 5-HT4 Bioactive

D = 8.0 A for B, C rotamers; 7.6 A for A rotamer. .A.

R2

R2

R5

D N->L.P., A

A

L.P.

H Compd

Conformers

I

5-HT4 EC50, l.tM

1

H

OH

10

2

H

OMe

148

3

H

4

Me

H

-8,000

OH

~ 20,000

HIO ~ ~ ~ _ _ NR H Illustration of Tau 1 = 180 ~ conformers of 5-HT (R = H) and 2-Me 5HT (R = Me). The asterisks indicate the bonds defining the Tau 1 torsional angle. The amino group is oriented at rotameric sites A, B, or C.

Figure 2 illustrates a Ramachadran plot of energetically accessible conformations (< 2 Kcal/mole above global minimum) available to both The two extended/gauche serotonin (R = H) and 2-Me5HT (R = OH3)[14].

102

conformations and one fully extended conformation illustrated in Figure 1 are available to serotonin but not available to 2-Me5HT (compare local minima for Tau 1 = 180 o). When mapped as distances between the amino group and the proximal phenolic hydroxyl lone electron pair, D[N ~L.P.], the three exclusive conformations of 5-HT have distances out to 8 A for the two mirror image extended/gauche conformers (rotamers B and C), and out to 7.6 A for the fully-extended conformer (rotamer A). These distances are noted in figure 1.

Original

Aza-Adamantane

Series

We then modeled several of the previously reported benzamides shown to exhibit 5-HT4 agonism. Most benzamides, typified by the modest agonists metaclopramide and zacopride, exhibited D[N ~L.P.] of --- 7.1 A-- somewhat shorter than the distance of 7.6-8.0 A modeled with serotonin in the conformations proposed in figure 1. An initial series of azaadamantylcontaining benzamides of varying D[N-~L.P.] distances were produced which both confirmed our conformational analysis and provided a foundation upon which to design further analogs. Figure 3 illustrates the proposed functional group distance relationships for these benzamides, wherein the distal carbonyl lone electron pair serves as a surrogate for the serotonin

103

5-OH lone pair. Among the four syn- and anti-substituted adamantyl derivatives, the syn-aminomethyl compound (SC-54750) was predicted to be the most active analog due to the optimal DIN ~L.P.] of 8.1 ,&,. Evaluation of these four analogs confirmed our prediction. The synaminomethyl analog SC-54750 exhibits an EC50 = 74 nM in the ratTMM assay. See table 2. A second notable relationship emerged. While the syn-aminomethylene (n = 1) analog is the most potent analog, the next most potent member is the epimeric anti-normethylene (n = 0) analog SC51718. This suggested that a similar spatial orientation of the basic bridgehead 3 ~ amine is achievable for this set of syn/anti-epimeric analogs.

Table 2 A comparison of various aza-adamantyl ratTMM 5-HT4 agonist assay

containing

n ~

0

benzamides

in the

Anti ,n

n2rq Compound SC-51718 SC-51717 SC-54750 SC-54815

IMe Type Anti Syn Syn Anti

n 0 0 1 1

Distance, A 7.0 7.3 8.1 8.8

5-HT4 Agonism* 262 (107) 538 (36) 74 (12.8) >1000

~'ECso (SEM), nM. Rat TMM functional assay. Scaffold

Optimization

With the aza-adamantyl prototype in hand, we undertook a systematic effort to probe for subtle variations in 1) scaffold bulk, and 2) spatial orientation of the bridgehead nitrogen with respect to the benzamide attachment site. Additionally, the syn/anti-epimeric analog relationship at the attachment site was further developed. Figure 4 illustrates the target aza-noradamantane scaffolds 1-5. Each of these scaffolds is the

104

result of an enantiotopic removal of a bridging methano group from the parent aza-adamantane scaffold (I). The target number represents that compound produced by removal of the corresponding-numbered methano group from the orignal aza-adamantane scaffold.

R

benzamide acyl moiety

//

fl\

1 Aza-adamantane

1

n

-

pT

RHN

3_ _2 RHN

n

n

N

RHN

" /n /

RHN

Figure

Target Aza-noradamantane

4

\

/

4

scaffolds

selected

for

evaluation

Removal of the enantiotopic methano bridges 1 and 2 produced the corresponding aza-noradamantanes as shown in table 3. Compound SC52491 of Type 1 is very potent in the ratTMM assay, EC50 = 51 nM [15]. However, the enantiomer SC-52490 of Type 2 is 70-fold less active. This indicated that the combination of removal of the C-1 methano bridge and retention of the C-2 methano bridge leads to an aza-noradamantane with a

105 5-fold improvement in potency relative to the parent aza-adamantane SC51718 (cf. table 2).

Table 3 Aza-noradamantanes

of T y p e s

1 and

2:

SC-52491

Scaffold Q~

o .,,~,. A

Typel NH-R

H2N

R

I

~

....

Me

Compound

R,,

Type2

Type

Distance, ,i.

5-HT4 Agonism*

SC-52491

1

7.0

SC-52490

2

7.0

3,870 (1061)

7.0

262 (107)

SC-51718 (table 2)

51 (6.6)

* EC50 (SEM), nM. Rat TMM functional assay.

Removal of the C-3 methano bridge of the parent aza-adamantane afforded an opportunity not only to probe the importance of this methylene, but also to probe a different spatial orientation of the 3 ~ bridgehead amine lone pair (with respect to the benzamide attachment site). See figure 4, target (3). Moreover, this new aza-noradamantane (3) retains the desirable plane of symmetry of the parent aza-adamantane (I), obviating the requirement for asymmetric synthesis or resolution of racemic intermediates. Evaluation of both nor-methylene (n = O) and methylene (n = 1 ) s y n - / a n t i - pairs of this symmetric azanoradamantane again revealed that the epimeric analogs syn-(3C)/anti-(3A) exhibit the more potent 5-HT4 agonism. See table 4. However, these two compounds exhibit potencies similar to each other even though the 7.1 A distance of (3A) is considerably shorter, than the more ideal 7.9 A distance of (3C). Moreover, the potency of (3C), EC50 = 421 nM, is reduced compared to the analogous syn-aminomethyl aza-adamantane SC-54750, EC50 = 74 nM (cf.

106

table 2). The distorted spatial orientation of the 3 ~ amine lone pair compared to the aza-adamantyl system may explain this discrepency. In the event, however, this meso-azanoradamantyl system (3) offered no advantage over the original aza-adamantane series. Table

4

Meso-Azanoradamantyl

scaffold

of Type

o

3 Anti

n H

Syn

I Me

Type

n

3A

Anti

0

7.1

382 (24)

3B

Syn

0

7.3

712 (84)

3C

Syn

1

7.9

421 (87)

3D

Anti

1

8.6

660 (126)

Compound

Distance, ~.

5-HT4 Agonism*

* EC5o (SEM), nM. Rat TMM functional assay. As illustrated in figure 4, removal of the enantiotopic C-4 and C-5 methano bridges from the azadamantane series (I) would afford the 03, 7bridged pyrrolizidines of Types (4) and (5). Again, these derivatives afforded an opportunity not only to probe the role of these scaffold methylenes, but also to probe another unique spatial orientation of the 3 ~ bridgehead amine lone pair. Unfortunately, resolution efforts were unsuccessful in providing enantiopure derivatives (4) and (5) separately, requiring that comparisons be made using racemates. 5-HT4 agonist activites of this series is shown in table 5. The epimeric analogs exo (4/5-C)/endo (4/5-A) exhibit the more potent 5-HT4 agonism as anticipated. Additionally, the racemic exo-aminomethyl analog (4/5-C) having the closest distance D[N ~L.P.] (7.8 A) to that modeled for serotonin (8.0 A) exhibits the greatest 5-HT4 agonist potency, EC50 = 176 nM. Assuming that one of the (4/5-C) antipodes is responsible for activity,

107

this C3,7-bridged pyrrolizidine offers potency similar to the parent azaadamantane SC-54750, EC50 = 74 nM (cf. table 2). Table

5

C3,7-bridged

pyrrolizidines

0

/

CI H2N

'l

of

Types

(4)

and

Type 4

n

(5) Scaffolds

Type 5

exo

N H' ' ~ scaffold

exo

endo Me

Type

n

endo/exo

Distance, ,~

5-HT4 Agonism*

4/5 A

0

endo

7.0

542 (151)

4/5 B

0

exo

7.2

1518 (167)

4/5 C

1

exo

7.8

176 (58)

4/5 D

1

endo

8.4

415 (70)

* EC50 (SEM), nM. Rat TMM functional assay. Transition to Pyrrolizidine Scaffold There were two considerations which led us to probe this pyrrolizidinecontaining series more fully. First, we sought a pyrrolizidine system wherein enantiomerically pure isomers could be evaluated. Second, given the observation that the aza-noradamantane series of Type 1 (lacking the C-1 bridging methylene; see table 3), afforded the more potent compound SC-52491 (EC50 = 51 nM), we were interested if a similar improvement in potency would be obtained by removal of the analogous "C-1 like" bridging methano group from series (4/5).

Results from the enantiomeric pyrrolizidine types (6) and (7) are shown in table 6. With one exception ( e n d o 6/7-D), each of the exo- and e n d o- analogs was prepared by asymmetric synthesis as previously reported [16]. The type 6 antipodal series afforded the more potent 5-

108

HT4 agonists. Again, the epimeric analog pair (exo-6C), n = 1, and (endo6A), n = 0, are more potent than other members. Compound (exo-6C), SC53116, exhibits potency (EC50 = 17 nM) approaching that observed for serotonin (EC5o = 10 nM) in this rat TMM model. The key distance D[N--->L.P.]= 8.0 .A, of SC-53116 matches that proposed for serotonin (see figure 1, rotamers B and C). Table Potent

6 5-HT4 agonsim

by pyrrolizidine series:

SC-53116 Scaffold

Type 6 O

n NH

H2N

Me

COMPOUND TYPE SC-55967

SC-53116

Endo/Exo

Type 7

en0o -- - 7 exo

n

DISTANCE, A 0

7.0

endo

5-HT4 AGONISM*

6A

Endo

167 (27)

6B

Exo

0

7.1

6C

Exo

1

8.0

17 (4)

6/7-D

Endo

1

8.4

280 (38)

2369 (560)

7A

Endo

0

7.0

>3,000

7B

Exo

0

7.1

>3,000

7C

Exo

1

8.0

323 (46)

* EC5o (SEM), nM. Rat TMM functional assay. Selective ( $ 0 - 5 3 1 1 6 ) and Dual 5-HT3 blocking (SC-52491) Serotonin 5-HT4 Agonists Taken together, the rigorous analysis of the parent aza-adamantyl scaffold (I) led to the identification of azanoradamantane Type 1, SC52491 (EC5o = 51 nM) and pyrrolizidine of Type 6C, SC-53116 (EC5o = 17

109

nM) as potent 5-HT4 agonists. Receptor binding profiles for these two agents are shown in Table 7. Not surprisingly, both SC-52491 and SC53116 exhibit potent Ki constants, 29 nM and 5.2 nM, respectively) in a radioligand 5-HT4 receptor binding assay [17]. Neither compound exhibits affinity for 5-HTl-like, 5-HT2, dopaminergic, or adrenergic receptors up to 10 ~M [18]. SC-52491 exhibits potent affinity for the serotonin 5-HT3 receptor (Ki = 1.2 nM) [19]. However, SC-53116 is much less potent for 5HT3 receptors (Ki = 152 nM), affording a selectivity ratio of 30 in favor of 5-HT4 affinity. The affinity of both compounds for 5-HT3 receptors functionally results in pure antagonism by analysis in the von BezoldJarisch reflex model [20]. Thus, SC-52491 is regarded as a dual 5-HT4 agonist/5-HT3 antagonist, while SC-53116 is a more selective 5-HT4 agonist.

Table

7

Receptor

Profiles

C' o H2N

of

SC-52491

o

and

SC-53116

c,._

Me

SC-52491

Me

,yJ. o

COMPOUND

5HT-4*

5HT-4

5HT-3

SC-52491

51 (6.6)

29 (3)

1.2 (0.3)

SC-53116

17 (4)

5HT-1, 5HT-2, D-l, D-2, r >10K

5.2 (0.5) 152.0(1.0)

* EC50 (SEM), nM in rat TMM assay. appropriate binding assays.

SC-53116

>IOK

All other values are Ki (SEM), nM in

Development of 5-HT4 Antagonists The high 5-HT4 potency of the pyrrolizidine system prompted us to consider other applications of this scaffold. The N-methyl quaternary salt of SC-53116 was prepared and evaluated in both 5-HT4 ligand binding and functional assays. As shown in table 8, the quaternary salt SC-53785 is eight-fold less potent than the parent SC-53116 in the ratTMM functional assay (compare EC50s of 129 and 17 nM). However, radioligand binding studies revealed a divergent trend for 5-HT4 receptor affinity.

110

The quaternary salt is very potent, Ki = 350 pM, being more than an order of magnitude higher affinity than the parent SC-53116. This result suggested that the pyrrolizidine ring system could possibly be manipulated to afford 5-HT4 antagonists. The pyrrolizidine ester SC-55822 confirmed this hypothesis. While this compound did exhibit very high potency in the functional ratTMM assay, ECs0 = 1.5 nM, its maximal efficacy was only 57% that of serotonin. Binding studies revealed very potent affinity, Ki = 183 pM, for this compound.

Table 8 5-HT4 functional activity and affinity of pyrrolizidine analogs of SC-53116 ..... 5.HT4 ..... ECs0 (SEM), nM RatTMM Ki (SEM), n M

COMPOUND SC-53116

O

H 17.0 (3)

5.2 (0.5)

129 (10)

0.35 (0.05)

1.5 (0.2) 57 % E of 5-HT

0.183 (0.033)

Me SC.53785

o

CI~~o, H2N

NH Me

SC.55822 _

Me

le

H

Me

Encouraged by these results, we sought other aromatic acid ring systems for coupling to our pyrrolizidine scaffold. A series of indole-

lll

type pyrrolizidine esters was prepared and evaluated both for 5-HT4 agonist and/or antagonist activity using a modification of the ratTMM assay [12]. Among all compounds investigated, the N-methyl indazole (8) (pA2 = 8.56), 5-fluoroindole (9) (pA2 = 8.50), and the N-methylindole (10) (pA2 = 9.20) are particularly noteworthy. These compounds are illustrated in table 9. None of these antagonists exhibit agonist properties up to a ceiling test concentration of 10 ~M in this assay. Compound (10), SC56184, is comparable in potency to the recently described Glaxo antagonist GR-113808 (pA2 = 9.39) [21].

Table 9 Indole-like

pyrrolizidine

esters

as

Compound

(8)

O

5-HT4

antagonists 5-HT4 Antagonism

pA2, Rat TMM

H 8.56

Me

(9)

O

H 8.50

(10) O SC-56184 ~.~

H 9.20

Me

One short-coming of most previously reported 5-HT4 antagonists is that the aminergic-containing side chain is connected to the aromatic nucleus by an ester bond (e.g. GR-113808, SC-56184 and others). While this linkage is of little consequence for in vitro use, we considered that

112

5-HT4 antagonists wherein this ester linkage is replaced with the more metabolically stable amide bond might find broader utility for those studies requiring the in vivo administration of a 5-HT4 antagonist. After screening several chemical series, we were gratified to find that a series of imidazopyridine amides afforded moderately potent antagonists. It has been previously appreciated that 5-HT4 agonism requires a planar orientation of the amide bond and the aromatic ring system. As illustrated in figure 5, this feature contributes to the potent agonist properties of the ortho-alkoxy benzamides i, wherein intramolecular hydrogen-bonding maintains such a planar conformation. Esters ii have much less tendency to exist in this planar conformation due to the replacement of the H-bond donor amide with an ester oxygen. Although the imidazopyridine series iii has retained the amide NH group, it has less tendency toward intramolecular H-bonding due to the relatively weak Hbond acceptor properties of the imidazopyridine 2-N lone pair and also due to its distorted orientation by virtue of the strain invoked by the fused 5membered imidazole ring. o

o s, ec a,n

I

Alkyl

o side chain

s,Oec a,n

I

Alkyl

~N~

Figure 5 i, Benzamides

ii, Benzoates

iii, Imidazopyridines

intramolecular H-bond. 5-HT4 agonism

No intramolecular H-bond. 5-HT4 antagonism

No intramolecular H-bond. 5-HT4 antagonism

Table 10 illustrates the structures of two of these antagonists. Notably, the amide (11), SC-53606, is ten-fold more potent in the ratTMM assay than the corresponding ester (12); compare pA2 values of 8.13 and 7.0, respectively. We have recently reported that SC-53606 exhibits similar pA2 values in this preparation regardless of the 5-HT4 agonist used in the Schild analysis [12]. Thus, values of 8.13, 7.67, 7.63, and 7.68 have been observed for SC-53606 vs. 5-HT, 5-methoxytryptamine, renzapride, and SC-53116, respectively.

113

Table 10 5-HT4 antagonist

properties

(11) SC-53606 O

H NH

(12)

O

of selected

SC-53606

imidazopyridines

5-HT4 AGONISM ECso, RAT TMM

5-HT4 ANTAGONISM pA2 (SEM)

>10,000 nM

8.13 (0.06)

>10,000 nM

7.0 (0.05)

H

Profiles of 5-HT4 a n t a g o n i s t s Table 11 provides receptor profiling data on the partial agonist SC-55822, and the full 5-HT4 antagonists SC-56184 (indole ester) and SC-53606 (imidazopyridine amide). For comparison, data for the reference antagonist GR-113808 are also shown. The partial agonist SC-55822 exhibits potent affinity (Ki = 183 pM) for the 5-HT4 receptor in the ligand-binding assay, with a 280-fold selectivity vs. the serotonin 5-HT3 receptor (Ki = 51 nM). The full antagonist SC-53606 exhibits good potency in the 5-HT4 binding assay (Ki = 1.4 nM), with a 185-fold selectivity vs. the 5-HT3 receptor (Ki = 259 nM). The indole ester SC-56184 is a more potent 5-HT4 antagonist (Ki = 233 pM), but less selective (21-fold) vs. the 5-HT3 receptor. By comparison, GR-113808 exhibits a Ki = 57 pM with a 24,000-fold selectivity vs. the 5-HT3 receptor when evaluated under identical conditions. None of the compounds exhibit appreciable affinity for 5-HTl-like, 5-HT2, dopamineric, or adrenergic receptors. In vivo properties of the amide-containing antagonist SC-53606 will be reported separately.

114

Table

11

Receptor profiles of selected 5-HT4 antagonists O

H

Me O

Cl

SC-55822 H

H

NHH ~ , , . ~ ~ N

SC-53606

SC-56184 Me

COMPOUND

5HT-4

5HT-3

5HT-2

CZ-2

SC-55822

0.183 (0.033)

51.6 (5)

1,700 (100)

7,400 (800)

>10K

SC-53606

1.40 (0.5) pA2 8.13 (0.06)

259.0 (28)

> 10 K

>10K

>10K

SC-56184

0.233 (0.033) pA2 9.2

5.0 (1.0)

> 10 K

>10K

>IOK

1,400 (200)

> 10 K

>10K

>10K

GR-113808 0.057 (0.003) pA2 9.39 (0.10)

5HT-1, ~-1, [3-1/2, D-l/D-2

Ki (SEM) nM, except otherwise noted for pA2 values. Design

of

a

Scalable

Synthesis

of

SC-52491

Given the desired 5-HT4/5-HT3 receptor profile and pharmacological efficacy of SC-52491, a scalable synthesis was developed. The azanoradamantane ring system found in this agent possesses four contiguous chiral centers within its tricyclic framework. An asymmetric route was deemed to be superior to resolution of racemates, as previous efforts along the latter pathway resulted in difficult separations of fairly endstage intermediates [22]. A route based on an initial asymmetric Diels-Alder reaction is summarized in Scheme 1. Cycloaddition of a chiral fumarate ester (A)

115

with cyclopentadiene would afford the adduct (B). Regioselective Hofmann rearrangement of a derived exo amide would give the exo-protected amine (C), wherein all relative and absolute stereochemistry required for the elaboration of the substituted azanoradamantane is intact. Ozonolysis of olefin (C), with reductive work-up and subsequent primary amide formation, would afford the highly-substituted diol (D). Reduction of the amide, followed by a double-cyclization protocol, would afford the desired aza-noradmantane (F). Scheme 1 Asymmetric Synthetic

Design

to

Prepare

Azanoradamantane

F NHTos

~

'

A

B

OH

OH

,,,,,,,,,,~

H,N....rF,,,," ---~ (~

NHTos

D

CO2H

CO2R*

C

OH

,E-),,,,,,,,,,I,

....

OH

,

..

BOCHN

,

NHTos

E

F

Following the key finding of Helmchen [23], the bis-(S)-ethyl lactate ester of fumaric acid was prepared from fumaryl chloride, as illustrated in Scheme 2. Cycloaddition of this dienophile with cyciopentadiene to give (3) required some experimental modification in order to eliminate halogenated solvents. A survey of solvents and reaction conditions revealed that triethylamine as solvent allows both a reasonable reaction rate and high diastereomeric excess (d.e. = 93%) when the reaction is conveniently conducted at room temperature.

116

Scheme 2 Asymmetric

Diels-Alder

reaction

CH3

CIOC j

90%

R,O2C.j

1

TEA 2

3

CO2R*

d.e. = 9 3 %

The exo ester function was regioselectively manipulated as illustrated in Scheme 3. Basic hydrolysis of (3), followed by iodo-lactonization of the endo-acid onto the olefin, afforded the highly crystalline (4). Recrystallization of (4) afforded material of > 99% ee in 75% over-all yield from (2). The exo-primary amide (5) was prepared to set the stage for the subsequent rearrangement. Koser's method [24], utilizing hydroxytosyloxy-iodobenzene (HTIB), was employed for the modified Hofmann rearrangement. Tosylation of the exo-amine gave the tosylamide (6) in 72% overall yield. Reductive elimination of the iodolactone afforded norbornene (7)in high yield. Scheme 3 Synthesis of key norbornene (7) CO2R.

1) LiOH/H20 =

I

CO2H

1) S O C I

96%

CO2R* 75% from D.A.

5

4

I.

~

1) HTIB 76%

2) TsCI/py 95%

ONH 2

=

2) NH3

2) KI/12/H20 3

2

O ~)~ ~ " 0 6

NHTos

Zn/HOAc

~

95%

O

NHTos

C02H 7

ll7

Finally, the substituted norbornene (7) was transformed into the desired aza-noradamantane (11) as shown in Scheme 4. Ozonolysis of (7), with sodium borohydride work-up, gave the bicyclic lactone (8) quantitatively. Ammoniolysis of (8) afforded the primary amide (9), which was then reduced with borane/THF to afford (10) after BOC protection. Double cyclization was performed based on a modification of our earlier reported procedure [22]. Bis-tosylation, followed by TFA mediated removal of the BOC group, afforded the cyclization precursor in situ. Double-cyclization was effected by employing di-isopropylethylamine in acetonitrile at RT. Chromatography afforded azanoradamantane (11) in 78% yield. This material was conveniently stored for future use. After calcium metal reductive removal of the tosylamide group, the free amine (11, Tos = H) was coupled to the commercially available 5-chloro-2-methoxy-4-aminobenzoic acid to give SC-52491 as previously reported [22b]. This scalable synthesis of the azanoradamantane (11) is asymmetric, takes place with only one chromatography, and does not utilize halogenated solvents. Scheme 4 Completion of synthesis.

~

NHTos

OH

/,,,....... r~

O 3 0

CO2H

Aza-noradamantane (11)

//"" ~

NaBH4 100%

O

........../

NHTos

OH

/,,,,,,,,,,~ OH

NH3/MeOH 100~

H2N_...rr,,,,'" - - ~ / / ~ O NHTos 9

OH /

OH

1) TosCl/py

..........

2) BOC20 70%

I BOCHN

NHTos 10

H__~ ,

T o . . .

3) Hunig's/MeCN 4) chrom 78%

11

118

Conclusion A series of aza-adamantyl containg benzamides were initially made based upon a computationaily-derived model of 5-HT4 agonism. Further refinement of this series led to the very potent Type 1 aza-noradamantane SC-52491 (5-HT4 agonist EC50 = 51 nM; Ki = 29 nM). Other azanoradamantane types were not as active as those derived from Type 1. Further scaffold reduction led to a series pyrrolizidine 5-HT4 agonists, highlighted by SC-53116 (5-HT4 agonist EC50 = 17 nM, Ki = 5.2 nM). Pyrrolizidine containing 5-HT4 antagonists were also identified. SC56184 (Ki = 233 pM; pA2 = 9.2) and SC-53606 (Ki = 1.4 nM; pA2 = 8.14) were shown to exhibit no agonist activity up to 10pM concentration. Both compounds also are 5-HT3 antagonists. Otherwise the compounds are quite selective vs. other serotonergic, adrenergic, and dopaminergic receptors. Finally, a scalable synthesis of SC-52491 was developed to facilitate its preclinical development. This synthesis is highlighted by an asymmetric Diels-Alder reaction and a novel double-cyclization step to produce the aza-noradamantane ring system. No halogenated solvents are employed and only one chromatography is incurred late in the synthesis. The overall yield for this process currently stands at 25%.

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14.

Conformational analysis was performed for serotonin and 2-methyl serontonin varying tau 1 and tau 2 at 15 degree increments. For each conformation all other coordinates were optimized using MacroModel/BatchMin Version 3.1 with a modified MM2 force field (C. Still, Columbia University).

15.

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Grossman CJ, Kilpatrick GJ, Bunce KT. 624.

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Br J Pharmacol 1993; 109: 618-

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19.

[3H] zacopride was used as the radioligand. Pinkus LM, Sarbin NS, Barefoot DS, Gordon JC. Eur J Pharmacol 1989; 168: 355-362.

20.

Saxena PR, Lawang A. Arch Int Pharmacodyn 1985; 277: 235-252.

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Flynn DL, Becker DP, Spangler DP, Nosal R, et al. Presentation at 206th National American Chemical Society Meeting, Division of Medicinal Chemistry Symposium: Antagonists to the Serotonin 5-HT4 Receptor Which are Based on a Pyrrolizidine Scaffold. Chicago, Illinois Aug 22-27,1993

22.

a) Flynn DL, Zabrowski DL, Nosal R. Tet Lett 1992; 33: 7281-7282. b) Flynn DL, Becker DP, Nosal R, Zabrowski DL. Tet kett 1992; 33: 7283-7286.

23.

Helmchen G, Goeke A, Lauer G, Urmann M, et al. Angew Chem Int Ed Engl 1990; 29: 1024-1025.

24.

Lazbin IM, Koser GF. J Org Chem 1986; 51: 2669-2671.

Perspective in Receptor Research

D. Giardin~, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

121

t~l-Adrenergic R e c e p t o r Subtypes and Signal T r a n s d u c t i o n T.L. Theroux, T.A. Esbenshade, and K.P.Minneman Department of Pharmacology, Emory University, Atlanta, GA 30322, USA

ABSTRACT Two pharmacologically distinct ~l-adrenergic receptors (ttXA and alB) have been described in animal and human tissues using selective antagonists and site-directed alkylating agents, whereas three different al-adrenergic receptor subtypes have been cloned (ttlb. Gilt, and ttl~/d), all of which are members of the G protein linked receptor superfamily. The relationship between native and cloned subtypes was a source of much confusion. However, recently a consensus was reached that the ale clone encodes the pharmacological a ~g subtype, and that both the axb and ttl~/d clones encode receptors with ~tlB-like pharmacology. It is now agreed that the cloned and native receptors should be called ~a/alg, aXb/~B, and ttld/~m, and the tz~cdesignation has been dropped. Initial studies in isolated smooth muscle tissue suggested that the pharmacologically defined {Xlg subtype preferentially activates voltage-gated Ca 2+ influx while the ttlB subtype stimulates formation of inositol (1,4,5) trisphosphate (IP3*) and mobilizes intracellular Ca 2+ stores. Although these two signalling pathways are generally associated with different receptor subtypes, further work with both native and recombinant subtypes has provided exceptions to this rule. For example, tt~g-adrenergic receptors are linked to inositol phosphate (InsP) formation and mobilization of intracellular Ca 2+ in human SK-N-MC neuroblastoma cells, and tz~b- and a ld-adrenergic receptors are linked to voltage-gated Ca 2+ influx in rat medullary thyroid carcinoma (rMTC 6-23) cells. The exact mechanisms by which tZladrenergic receptor subtypes initiate these signals in native cells and tissues are still unclear. When expressed in mammalian cells, all three cloned subtypes can stimulate InsP formation and release intracellular Ca 2+, however the efficiency with which each subtype couples to this signalling system appears to vary (ttla>ttlb>ald). No model system is currently suitable for similar studies examining tt~-adrenergic receptor modulation of voltagegated Ca 2+ influx in response to transfection of the cloned subtypes. These subtypes also activate other second messenger systems following transfection, including phospholipase A2 (PLA2) and adenylate cyclase. Use of inducible vectors to titrate the density of each cloned subtype in transfected cell lines allows direct determination of the efficacy of each subtype in activating second messenger responses.

*Abbreviations used are as follows: AR, adrenergic receptor; [Ca2+]i, intracellularcalcium; CEC, chloroethylclonidine: DAG, diacylglycerol;EPI, epinephrine; CNS, central nervous system; HEK 293, human embryonic kidney 293 cells; IPTG, isoproply-13-D-thiogalactoside;InsP, inositol phosphate; IP3, inositol (1,4,5) trisphosphate; NE, norepinephrine; PI, phosphatidylinositol; PLA2,phospholipaseA2; PLC, phospholipaseC; PLD, phospholipase D; PKC, protein kinase C; rMTC 6-23, rat medullarythyroid carcinoma cells; VDCC, voltage-dependent Ca2§channel.

122 INTRODUCTION Norepinephrine (NE) and epinephrine (EPI) act as neurotransmitters and hormones in both the peripheral and central nervous systems (CNS). NE is released from neurons throughout the CNS and periphery to participate in a variety of physiological functions, while both NE and EPI are released from the adrenal medulla in response to stress. NE and EPI modulate fluid homeostasis, cardiac function, energy metabolism, and may play a role in depression. At the cellular level, these actions are mediated by multiple adrenergic receptor (AR) subtypes and second messenger systems. Adrenergic receptors are part of a large family of G protein coupled receptors. There are three well defined classes of ARs: 13-ARs, al-ARs, and ~2-ARs. The 13- and iz2-ARs are positively and negatively coupled to adenylyl cyclase through Gs and Gi, respectively. The al-ARs trigger an increase in intracellular Ca2+ concentration ([Ca2+].), through two pathways. a l-ARs couple to the Gq class of G proteins to activate PLC, generating I P 3 and causing a release of intracellular Ca 2+ stores, a l-ARs can also couple to voltage-dependent Ca 2+ channels (VDCC) in the plasma membrane allowing influx of extracellular Ca 2+. Members of the a x-AR family have been characterized based on antagonist selectivity, tissue distribution, coupling mechanisms, and molecular cloning. Selective antagonists distinguish between ~IA and a lB-AR subtypes [1] in various tissues and cell lines (uppercase letters designate pharmacologically defined subtypes while lowercase letters indicate cloned subtypes), a IA-ARs are expressed in a wide variety of tissues and generally couple to VDCCs [2,3], although this subtype can also couple to InsP formation and the release of intracellular Ca 2+ stores in certain tissues [4] and cell lines [5]. ~IB-ARs generally couple to InsP production and release of intraceUular Ca 2+ stores, although this subtype also couples to VDCCs in certain in vitro systems [6]. Molecular cloning has revealed three distinct subtypes: ala, ~lb, and a~d [7,8,9,10]. In expression systems, the three subtypes demonstrate distinct pharmacological profiles. The a la shows a lA-like antagonist pharmacology, while both the a lb and kid clones demonstrate pharmacological characteristics more similar to the pharmacologically def'lned tt IB-AR. New compounds have recently been developed which distinguish between the a lb and a ld subtypes [11]. All three clones couple to phospholipase C (PLC) and InsP generation in a variety of expression systems. The ability of each clone to couple to VDCCs has not yet been examined, since there is as yet no good expression system for examining this coupling. Other a~-AR subtypes have also been proposed. In particular, Muramatsu et al. [12] described an afAR subtype exhibiting a low affinity for prazosin. This subtype has recently been proposed to mediate the contraction of prostatic smooth muscle [18] and therefore could play a key role in the development of more effective drug therapies to treat benign prostatic hypertrophy. Unfortunately, it has been difficult to identify this subtype with radioligand binding approaches, and its relationship to the cloned subtypes is currently uidmown. More selective compounds, further functional studies, and the cloning of this receptor subtype will help to clarify this issue. The existence of multiple a l-AR subtypes coupled to multiple second messenger systems and co-expressed in a variety of tissues complicates studies aimed at defining the role of each receptor subtype. Expression of the cloned subtypes and the development of subtype selective compounds will help to evaluate the function of this class of receptors.

123 P H A R M A C O L O G I C A L CHARACTERIZATION OF CZlA-AND cxm-AR SUBTYPES Morrow and Creese [ 1] demonstrated multiple ,t~-AR subtypes by differences in their affinities for phentolamine or WB 4101 (both rigA-selective compounds) and prazosin (nonselective) in various rat tissues. The high and low affinity binding sites for these selective compounds are designated a lh- and *tlB-ARs, respectively. Other antagonists, such as (+)niguldipine and 5-methylurapidil, also have a relatively high affinity for the ,tlA-AR subtype as compared to the tt 1B-AR subtype [ 13, 14]. Chloroethylclonidine (CEC) is currently the only known antagonist exhibiting ,t 1Bselectivity. CEC binds to both receptor subtypes, but alkylates only the *tlB and not the a~A subtype [15]. Using these pharmacological tools, tissues were characterized as expressing heterogeneous or homogeneous populations of these two a l-AR subtypes and distinct biochemical and physiological responses were attributed to each subtype. Functional studies revealed tZlA-ARs are commonly expressed in the vasculature mediating the contractile responses to NE and EPI. Other tissues expressing primarily ,t 1A-ARs, as assessed by binding assays, include regions of the brain, vas deferens, the submaxillary gland, and prostate, a mARs were found to be the major subtype expressed in spleen and liver (rat), also assessed through binding studies. Functional studies revealed that the ,tin subtype consistently couples to InsP generation and release of intracellular Ca 2§ stores. Recently, more selective drugs have been identified or synthesized, allowing a more precise pharmacological analysis and reevaluation of the functional differences between these subtypes (Table 1).

Table 1.

Highly selective antagonists for a t-AR subtypes. Antagonist (+)niguldipine 5-methylurapidil BMY 7378 RS 17053 SNAP 5089 chloroethylclonidine

Selectivity a 1g a 1A axd tXla

axa tt lb and a 10

Reference [16] [ 17] [11] [18] [19] [20]

CLONING OF al~-, a lb- AND IXld-Ag SUBTYPES The first al-AR subtype to be cloned was from a hamster DDT1 MF-2 cell line [7]. When expressed in heterologous systems, this clone demonstrates classical ~m-AR pharmacology, with a low affinity for tt 1A-selective antagonists and a high sensitivity to CEC inactivation. Northern blot analysis also demonstrated that this clone shows an tim-like tissue distribution, mRNA expression and the pharmacological profile of this clone led to its identification as the a lb-AR. The a la-AR was first cloned from bovine brain [8], but was originally thought to

124 encode a novel subtype called a lc. When expressed in COS-7 cells the agonist and antagonist pharmacology of this subtype was characteristic of the defined GtIA-AR, with the exception of CEC. The expressed receptor showed a marked sensitivity to CEC inactivation, unlike the native UIA subtype. In addition, Northern blot analysis and in situ hybridization of various bovine, rat, rabbit, and human tissues revealed expression only in human dentate gyms and rabbit liver. In contrast, the pharmacologically defined ~xA-AR is known to be widely expressed in many tissues based on both antagonist binding and functional assays. Such observations led to the classification of this clone as a novel subtype (Ulc)- However, when the human [21] and rat [22] homologs were cloned, rnRNA levels were detected in the tissues previously characterized as expressing a~A-ARs (vas deferens and submaxillary gland, among others), contradicting the previous findings for the bovine clone. This a~-AR sequence was renamed a~a. The a ld-AR was cloned from rat [9, 10] and, although there was some initial confusion concerning the identity of this receptor based on initial mRNA distribution studies [9], this clone was found to have an ~B-like pharmacology. Like the a lb clone, expression of this clone resulted in a receptor with a low affinity for many a~A-selective antagonists (phentolamine, WB4101, (+)niguldipine, and 5-methylurapidil) and a high sensitivity to alkylation by CEC. Recently, BMY 7378 has been found to be selective for the a xd-AR subtype, giving us a tool to better characterize this receptor. Cloning of these subtypes and their human homologs allows for a more definitive pharmacological characterization, and facilitates investigation of both their physiological functions and biochemical coupling mechanisms.

IS THERE A SUBTYPE WITH A LOW AFFINITY FOR PRAZOSIN? An al-AR subtype with a relatively low affinity for prazosin was proposed by Flavahan and Vanhoutte [23] in 1984. The pharmacology of this subtype was further described in 1990 by Muramatsu et al. [12] based on contractile studies of various blood vessels from four different species: dog, rabbit, guinea-pig, and rat. The a I-AR subtypes expressed in vascular smooth muscle from these sources were classified into three groups based on the difference between the pA2 values for prazosin and for either WB4101 or HV723. One of these groups, classified as the a lL-AR subtype, shows a low affinity for all three compounds. Evidence that this subtype is also expressed in human prostate was found by Ford et al. [18]. A new ala selective antagonist, RS 17053, demonstrates an approximately 100-fold selectivity in binding at the a la-AR subtype over the a lb and ttxa subtypes (based on studies with both cloned and native subtypes). This compound potently inhibits NE-stimulated contraction of isolated perfused kidney (expressing the tt 1A-AR subtype) and weakly inhibits the NE stimulated contraction of aortic rings (expressing the a lB or alD-AR subtype), verifying its predictable activity in functional studies. In contrast, RS 17053 only weakly inhibits the contraction of prostatic smooth muscle. This is surprising, since previous correlation studies had concluded that the a lA-AR is the predominant subtype expressed in prostate and is thought to mediate contraction of prostatic smooth muscle [24]. To resolve these apparently contradictory studies, RS 17053 has been proposed to distinguish a subset of the prostatic a ~A-AR subtype population, namely the a 1L-

125 AR subtype. This compound may serve as a useful pharmacological tool in establishing the existence of an additional subtype and defining its pharmacological profile.

PARALLEL COMPARISON OF ANTAGONIST PHARMACOLOGY OF THE CLONED r SUBTYPES Currently, the most valuable approach for directly comparing the al-AR subtypes is to express the three cloned receptors individually in heterologous expression systems. Many of the compounds previously described as being =lA-selective have nearly identical low aff'mities for both the alb and =ld subtypes. We investigated the binding selectivity of a variety of drugs by expressing the bovine r hamster r ~b, and rat a ld in human embryonic kidney 293 (HEK 293) cells [20]. We used the radioligand [3H]tamsulosin to label the cloned subtypes and examined a variety of compounds for their ability to compete with this ligand. Tamsulosin, in its unlabeled form, is being developed for the treatment of benign prostatic hypertrophy and demonstrates some selectivity for the alk subtype. Like its unlabeled counterpart, [3H]tamsulosin labels all three cloned receptor subtypes, although with slightly different affinity constants. Association and dissociation curves of [3H]tamsulosin binding to membranes from HEK 293 cells expressing the human a xa-AR subtype is shown in Fig. 1. This compound has an association rate constant (k0 of 1.3 x 109 1/mole-min and a dissociation rate constant (k.~) of 0.0185 min-~; with a calculated KD of 14 pM (k.x/kl) (compared to a similar value of 46 pM from saturation binding curves) at the cloned r la-AR subtype. [3H]Tamsulosin demonstrates lower affinities at the =lb and =~d binding sites, with

100. E

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20.: 100

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Figure I. Association and dissociation curves for [3H]tamsulosin in HEK 293 cells expressing the cloned human ala receptor. Binding assays were performed at 30~ [3H]Tamsulosin binding at 40 min is used for the maximal binding in association studies. In dissociation studies, the membranes were incubated for 40 rain in the presence of [3H]tamsulosinto allow equilibrium to be reached prior to measuring the dissociation of the ligand. Points represent the mean +_S.E.M. of two experiments performed in duplicate.

126 KDS of 534 pM and 209 pM, respectively. In screening a number of =IA selective antagonists, such as 5-methylurapidil and ( + )niguldipine, for inhibition of [3H]tamsulosin binding, we found that the r selective compounds had high affinities at the cloned r and much lower affinities at both the r and r subtypes. CEC, a compound previously established as an =m-selective alkylating agent, was closely examined to determine its selectivity for inactivating the cloned r Inhibition of [3H]tamsulosin binding by pretreating membranes with CEC showed that all three cloned subtypes were partially sensitive to alkylation, although to varying degrees (Fig.2). At low concentrations of CEC (I~M), 40% of the eta sites were blocked, and 70% of each the r and aid sites were blocked. Therefore the traditional pharmacological tools previously used to distinguish the r and =IB subtypes are insufficient to distinguish the cloned r from the cloned r BMY 7378, initially described as a serotonin receptor agonist with selectivity for the 5-HT~A subtype [25 26] has recently ' ' been suggested to be selective for the r subtype as well [11]. We tested this compound for its selectivity at the expressed human e x-AR clones. In [3H]tamsulosin binding inhibition curves, BMY 7378 potently inhibited binding at the =ld subtype with a ten-fold selectivity over the =~b-AR subtype and one hundred-fold selectivity over the r subtype (see Fig. 3). Other newly developed compounds, such as SNAP 5089 [19], are also being screened for their selectivity in binding to each subtype. Characterization of these compounds and the development of other selective compounds will be valuable in future studies of the function of each receptor subtype in intracellular signalling and physiological function.

10080~

,-

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o

~

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Figure 2. CEC inhibition of [ 3 H ] t a m s u l o s i n bindingin HEK 293 cells expressing a~-AR subtypes. Membranes from transfected cells were incubated with 1~tM CEC for 10 min at 37~ Membranes were washed and binding of a 90% saturating concentration of [3H]tamsulosin determined. Values are expressed as a % of specific binding in membranes pretreated under identical conditions in the absence of CEC, and are the mean + S.E.M. of 5 experiments performed in duplicate.

T 11o.1 1 0

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F i g u r e 3. Inhibition of [3H]tamsulosin binding by BMY 7378 at cloned human c~-AR subtypes. Values are the mean + S.E.M. of 3 experiments each performed in duplicate. The concentration of [3H]tamsulosin was adjusted for each subclone to give 90% saturating conditions: for cx~a:0.52nM, for a~b:5.2 nM, and for CX~d:1.9 nM.

127

=I-AR SIGNALLING VIA M I ~ T I P L E SECOND MESSENGER PATHWAYS The r are known to couple to the Gq class of G proteins to activate phospholipase C (PLC). PLC activation catalyzes the cleavage of phosphatidylinositol (4,5) bisphosphate to form inositol (1,4,5) trisphosphate (IP3) and diacylglycerol (DAG). IP3 mediates the release of intracellular Ca 2§ stores and DAG potently activates protein kinase C (PKC) which leads to activation of a number of downstream effectors. r can also activate other members of the G protein superfamily. EPI stimulates GTP[a32p] labeling of Gh, a high molecular weight G protein [27]. Gh couples el-ARs purified from rat liver to PLC in a reconstituted system [28]. In addition to PLC coupling, r also couple to second messenger systems apparently through both PLC dependent and independent pathways. Multiple G proteins may contribute to the diversity seen in r second messenger signalling (Fig. 4). Evidence of =~-AR coupling to multiple signalling pathways have come from both in vivo and in situ studies. r couple to Ca2§ influx through VDCCs in the vasculature [3] and in rat medullary thyroid carcinoma cells (rMTC 6-23) [6]. r can couple to phospholipase A2 (PLA2) through two parallel, PLC-independent and -dependent pathways. In the MDCK [29] and FRTL-5 cell lines [30], and in transfected COS-1 cells [31], r mediated PLA2 stimulation can occur independent of PLC activation. For example, in transfected COS-1 cells, PLA2 activation is dependent upon Ca2§ influx and is blocked by the VDCC antagonist, nifedipine. In contrast, studies in rat pineal gland [32] and MDCK cells [33] demonstrate that PKC is able to ~,-AR mimic the activation of PLA2 seen with r tAR I stimulation. This suggests that PLC is able to I '1 G~ G, mediate the coupling to PLA2 through PKC activation and/or release of intracellular Ca 2§ I I stores. PLC VDCC Similar to PLA2 coupling, I i = phospholipase D (PLD) coupling is stimulated DAG IP~ through two parallel but independent pathways by r [34]. Evidence for this PKC [C +1, includes f'mdings from experiments in rat ....i>-::; cerebral cortical slice preparations. The effects of NE and phorbol 12-myristate 13PLA, PLD acetate, a potent activator of PKC, are additive in stimulating PLD, and inhibitors of Figure 4. Schematic diagram of ctl-AR PKC do not inhibit PLD activation by NE. intracellular signalling. Solid lines represent the Similar results were found in MDCK cells established signalling pathways while dotted lines show pathways supported by some experimental [35] supporting the idea that r couple to evidence. Abbreviations used are as follows: multiple second messenger systems through PLC - phospholipase C; VDCC - voltage divergent but interacting pathways. dependent Ca 2§ channel; DAG - diacylglycerol; In addition to these second messenger IP3 - inositol (1,4,5) trisphosphate; PKC - protein systems, a l-ARs activate other downstream kinase C; PLA2 - phospholipase A2, PLD phospholipase D. effectors through mechanisms that are not well understood, a l-ARs potentiate isoproterenol

128 (a 13-AR agonist) stimulated adenylate cyclase [2], increase N-acetyltransferase [36] activity, induce c-fos expression [37, 38], and trigger release of adenine nucleotides [39]. Defining the role of each aI-AR subtype in catecholamine signalling is complicated not only by the multiplicity of second messenger pathways coupled to r but also by the limited availability of subtype selective compounds. Molecular biological techniques and the development of subtype selective compounds are now allowing us to clarify subtype pharmacology, tissue expression, and the individual roles of each subtype in cellular signalling.

NATIVELY EXPRESSED D I F F E R E N T CA 2+ P O O L S

al-AR

SUBTYPES

COUPLE

SELECTIVELY

TO

al-ARs are known to couple to increases in intracellular Ca 2+ through both Ca 2+ influx and the release of intracellular Ca 2+ stores. Each subtype was evaluated for its ability to interact with each of these pools with the use of antagonists selective for the NIF 15o pharmacologically def'med a lA and a lB-AR subtypes. In various rat blood vessels, there is a direct correlation between expression of the e~A-AR subtype and the importance of extracellular Ca 2§ in muscle contraction [40]. In rabbit aorta, similar results were NE found for the alA-AR-extracellular Ca 2§ 0 dependence [41]. In the same study, a o 2'0 4'o o'o 8'0 16o ~o ~.o parallel correlation was observed for e~Btime (sec) ARs coupling to PLC and mobilization of intracellular Ca 2§ stores. These observations led to the hypotheses that r Figure 5. NE stimulation of Ca2§through VDCC in ARs couple to Ca 2§ influx while r rMTC 6-23 cells. Fura-2 loaded cells were exposed couple to phosphatidyl inositol (4,5) to 30~tM NE followed by 1~tM nifedipine (NIF). bisphosphate (PI) hydrolysis and release of [Ca2§ determinations were made by measuring the intracellular Ca 2§ stores to trigger blood emission wavelength at 510nm when the cells were exposed to excitation wavelengths of 340 and vessel contraction. 380nm. Data are from a single experiment, Exceptions to this generalization representative of three experiments. Fura-2 loaded have since been observed in both SK-N-MC cells were prepared as follows: Cell suspensions and rMTC 6-23 cell lines. SK-N-MC ceils, were incubated in DMEM with 5 ~tM fura-2/AM for a human neuronal cell line, express a lA30 min at 37~ Cells were washed twice and resuspended in BSS (130mM NaC1, 5mM KC1, lmM ARs which couple to PI hydrolysis and the MgCI2, 1.5mM CaCI2, 20mM HEPES, 10mM mobilization of intracellular Ca z§ stores glucose, 0.1mM glucose, 0.1% BSA). Fura-2 loaded [5]. rMTC 6-23 cells demonstrate both PI cells were then transferred to a cuvette for hydrolysis and Ca 2§ influx through VDCCs spectrofluorometric analysis. (see Figs. 5 and 6). However, Northern blot analysis, binding inhibition curves,

129

10.0

, . .

7.5

o ~9

5.0

2.5

0.0 basal NE

Figure 6. Analysisof [3H]inositolphosphate formation in rMTC 6-23 cells in response to 30~tM NE. Cells were prelabeled with myo-[3H]inositoland production of [3H]InsPswas determined as follows: cells were washed with Krebs buffer containing 10raM LiCl and incubated with or without 30~tMNE for lh. The medium was removed and the reaction stopped with methanol. [3H]InsPswere isolatedby extraction and anion exchange chromatography.

and sensitivity to CEC show that rMTC 623 cells do not express ~A-ARs [6]. In addition, these two pathways appear to be independently activated by NE. With an 80% inhibition of PI hydrolysis by U-73122, a potent PLC inhibitor, there is no decrease in NE stimulated Ca 2+ influx through VDCC. In addition, potassium chloride, which depolarizes rMTC 6-23 cells and triggers Ca 2§ influx through VDCC, does not stimulate PI hydrolysis. These exceptions have required a re-evaluation of the previous theories on how a~-ARs couple to signal transduction machinery. Recent studies suggest that the subtypes, although able to couple to multiple signalling pathways, do so with different efficiencies. To conduct such investigations, a thorough understanding of agonist pharmacology is necessary.

COMPARISON OF AGONIST PHARMACOLOGY AT CLONED

II1-A.Rs

Agonist profiles are another useful means to classify receptors and provide additional evidence for differential coupling efficiencies of the a l-AR subtypes to various second messenger systems. We evaluated a number of agonists for affinities and intrinsic efficacies at each of the cloned receptors expressed in HEK 293 clonal cell lines [42]. In radioligand binding assays, the endogenous catecholamines, NE and EPI, showed high affinities for the ald-AR subtype, intermediate affinities for the a~b-AR subtype, and low aff'mities for the ~laAR subtype with nearly a 100-fold difference between the a~a and a ld subtypes. Surprisingly, this binding selectivity was not reflected in functional assays (InsP accumulation). NE and EPI were equipotent in stimulating PI hydrolysis through each of the three cloned a l-AR subtypes. Many widely used synthetic agonists appear to be highly selective for the tt~a-AR subtype. Methoxamine and phenylephrine are partial agonists at the ~b- and ~t~d-ARsubtypes, but act as full agonists at the a la-AR subtype. Cirazoline and SKF 89784 bind to all three subtypes, but only activate InsP generation in ~tla-AR expressing cells, causing low levels of activation only at extremely high agonist concentrations in a~b- and ~ld-AR expressing cells. One possible explanation of these findings focuses on the ability of an agonist to activate a second messenger response through the different receptor subtypes. Although agonists may be able to fully activate each receptor subtype, each activated subtype may not be able to equally activate G proteins resulting in differences in the magnitude of the second messenger response. Conversely, the agonists may indeed show selectivity in their ability to

130 activate the different receptor subtypes. Other explanations for the discrepancies between agonist affinity and efficacy in eliciting a response were investigated. To test the possibility that differential receptor reserves influenced the results, we used the non-selective alkylating agent phenoxybenzamine to decrease receptor density and knock out any receptor reserve. Treatment with phenoxybenzamine caused little or no rightward shift in the dose-response curves to NE indicating very little or no receptor reserves in any of the transfected cell lines. In a system where there is no receptor reserve, measuring the maximum response to agonist is a fairly direct measurement of the relative efficacy of the agonist at a particular receptor subtype. We also investigated possible effects of the conditions under which radioligand binding measurements were performed to see if the affinity of agonists for the receptors was changed in any way. Receptors can exhibit multiple affinity states when bound by an agonist. These multiple affinity states reflect the dynamic properties of agonists. When an agonist binds to a receptor, the conformation of the receptor is changed and the affinity for the agonist can be affected. In addition, the local ionic and osmotic environment can play an important role in determining agonist-receptor interactions. However, in this system the presence of Mg 2§ (to modulate the ionic environment of the receptor) or GTP (to shift receptor equilibrium to a low affinity conformation), NE and EPI still demonstrate selectivity for the a~d-AR subtype. Neither receptor reserve nor multiple affinity states of the receptors can account for these findings. Our evidence suggests that all three cloned a~-AR subtypes do, in fact, couple to Gq and the PLC pathway, although possibly with different efficiencies.

D ~ ' F E R E N T EFFICIENCIES OF COUPLING OF u l-ARs TO PLC

PLC

More direct evidence for differential ! I I I I coupling of ~I-AR subtypes to PLC is seen in a study by Schwinn et al. [43] where the ttla-AR Gq G,1 G,4 Gls subtype appears to couple to PLC with a greater efficiency than the alb-AR, similar to our findings in HEK 293 cells. When a la- and a lb-ARs were (~la (~la ~la l~lb independently, stably expressed in HeLa cells at 700 (~,lb (~lb (~,lb fmol/mg and 1500 fmol/mg, respectively, NE (~,ld (~,ld stimulated PI hydrolysis in both cell lines. Surprisingly, the a~a transfected cell line produced an InsP response roughly twice the magnitude of the Figure 7. G protein subtypes mediating a lb cell line even though it expressed less than half InsP response to NE in Cos-7 cells the level of receptors. Further investigation into transfected with different subtypes as determined by Wu and colleagues [44]. this question will require use of inducible expression vectors to directly examine how the modulation of receptor density affects the intracellular signalling. Wu et al. also demonstrated selectivity in the coupling of ~I-AR subtypes to different G protein subtypes [44]. a l-ARs couple to PLC through the Gq class of G proteins which include Gq, Ga~, G14, and G16 (as well as the recently described G15 not used in this study).

I

I

i

I

131 Wu and colleagues cotransfected each of the three a x-AR subtypes with each of the four cloned Ga subunits into Cos-7 cells. They found that the a subunits of both Gq and Gll couple all three a l-AR subtypes to PI hydrolysis upon NE stimulation. In contrast, the a subunits of G~4 and G16 exhibit some selectivity in coupling to each of the subtypes. NE stimulates PI hydrolysis in a lb-AR transfected cells when either G14 or G16 is cotransfected into the system. In ald-AR transfected cells, neither G~4 nor G16 is able to mediate NE stimulated PI hydrolysis. NE stimulates PI hydrolysis through a la-AR when cotransfected with G~4 but not G~6 (fig. 7). These findings suggest an additional mechanism by which signal transduction pathways can be selectively activated by the a~-AR subtypes.

COMPARING RECEPTOR DENSITY TO SECOND MESSENGER RESPONSE Esbenshade et al. investigated the InsP response to NE in hamster DDT1 MF-2 smooth muscle cells [45]. These cells natively express the a~B-AR subtype and are the original source for the cloning of this subtype [7]. When transfected with an IPTG inducible vector containing the hamster a Ib-AR clone, a range of expression levels was achieved, from 860 fmol/mg protein in the absence of IPTG to 2300 fmol/mg protein in the presence of IPTG [45]. NE was able to stimulate PI hydrolysis at all levels of receptor expression with an equal potency, indicating there was no receptor reserve even at high levels of receptor expression. Instead, there was an increase in the maximal response to NE that paralleled the increase in receptor density. Modulation of a IB-AR density by the cell would provide a mechanism to directly modulate the magnitude of the ImP response. With other a l-AR subtypes there may not be a direct relationship between receptor density and the InsP response. A receptor that is very efficiently coupled to a second messenger response would rapidly develop a receptor reserve as the receptor density is increased. In this case there would be a heightened sensitivity to NE and EPI. Comparisons similar tO this a lb-AR-InsP study will help to evaluate the ability of each subtype to stimulate second messenger responses.

CONCLUSIONS Our understanding of a 1-AR subtypes has dramatically changed in recent years with the cloning of three receptor subtypes. Molecular based pharmacological studies have required a re-evaluation of the traditional pharmacology based on the natively expressed subtypes. Inconsistencies between the pharmacological profiles of the subtypes based on these two approaches raises the possibility that there may be other subtypes that have yet to be cloned. The very existence of multiple subtypes, well conserved among many species also raises the question of why so many subtypes should be expressed if they have very similar functions. Current research now focuses on distinguishing the subtypes and determining what unique role they play in receptor signalling at the cellular level and in mediating a physiological responses. Experimental evidence suggests that the answer may lie in subtype-G protein coupling. Although the receptor subtypes apparently couple to the same multiple signalling pathways,

132 subtle differences in coupling efficiencies may cause a range of physiological responses to NE and EPI depending upon which subtype, and which G protein subunits, are expressed. The use of molecular biological techniques and the development of more selective compounds is now allowing us to critically analyze these questions.

ACKNOWLEDGEMENTS Supported by Grant NS 32706 from the National Institutes of Health.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. .

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

Morrow AL, Creese I. Mol Pharm 1986; 29: 321-330. Minneman KP. Pharm Rev 1988; 40:87-119. Nelson MT, Standen NB, Brayden JE, Worley III JF. Nature 1988; 336: 382-385. Chiu AT, Bozarth JM, Timmermans PBMWM. J Pharm Exp Ther 1987; 240: 123-127. Esbenshade TA, Han C, Murphy TJ, Minneman KP. Mol Pharm 1993; 44: 76-86. Esbenshade TA, Theroux TL, Minneman KP. Mol Pharm 1994; 45: 591-598. Cotecchia S, Schwinn DA, Randall RR, Letkowitz ILl, Caron MG, Kobilka BK. Proc Natl Acad Sci USA 1988; 85: 7159-7163. Schwinn DA, Lomasney JW, Lorenz W, Szldut PJ, Fremeau Jr. RT, Yang-Feng TL, Caron MG, Lefkowitz RJ, Cotecchia S. J Biol Chem 1990; 265:8183-8189. Lomasney JW, Cotecchia S, Lorenz W, Leung WY, Schwinn DA, Yang-Feng TL, Brownstein M, Lefkowitz RJ, Caron MG. J Biol Chem 1991; 266" 6365-6369. Perez DM, Piascik MT, Graham RM. Mol Pharm 1991; 40: 876-883. Goetz AS, King HK, Ward SDC, True TA, Rimele TJ, Saussy DL. Eur J Pharm 1995; 272: R5-R6. Muramatsu I, OhmuraT, Kigoshi S, Hashimoto S, Oshita M. Br J Pharm 1990; 99:197201. BoerR, GrasseggerA, SchudtCH, GlossmanH. EurJPharm 1989; 172: 131-145. Minneman KP, Atkinson B. Mol Pharm 1991; 40: 523-530. Minneman KP, Han C, Abel PW. Mol Pharm 1988; 33: 509-514. Schwinn DA, Lomasney JW. Eur J Pharm 1992; 227: 433-436. Gross G, Hanft G, Rugevics C. Eur J Pharm 1988; 151: 333-335. Ford APDW, Arredondo NF, Blue DR, Bonhaus DW, Kava MS, Williams TJ, Vimont RL, Zhu QM, Pfister JR, Clarke DE. BrJ Pharm 1995; ll4(supp): 24P. Wetzel JM, Miao SW, Forray C, Borden LA, Branchek TA, Gluchowski C. J Med Chem 1995; 38: 1579-1581. Hart C, Hollinger S, Theroux TL, Esbenshade TA, Minneman KP. Pharm Comm 1995; 5:117-126. Hirasawa A, Horie K, Tanaka T, Takagaki K, Murai M, Yano J, Tsujimoto G. Biochem Biomed Res Comm 1993; 195: 902-909. Laz TM, Forray C, Smith KE, Bard JA, Vaysse JJ, Branchek TA, Weinshank RL. Mol Pharm 1994; 46: 414-422.

133 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

Flavahan NA, Vanhoutte PM. Trends Pharm Sci 1986; 7: 347-349. Forray C, Bard JA, Wetzel JM, Chiu G, Shapiro E, Tang R, Lepor H, Hartig PR, Weinshank RL, Branchek TA, Gluchowski C. Mol Pharm 1994; 45: 703-708. Stubbs CM, Trezise D, Connor HE, Feniuk W. J Auton Pharm 1991; 11: 237-245. Higgins GA, Elliot PJ. Eur J Pharm 1991; 193: 351-356. Baek KJ, Das T, Gray C, Antar S, Murugesan G, Im MJ. J Biol Chem 1993; 268: 2739027397. Das T, Baek KJ, Gray G, Im MJ. J Biol Chem 1993; 268: 27398-27405. Slivka SR, Insel PA. J Biol Chem 1987; 262: 4200-4207. Burch RM, Luini A, Axelrod J. Proc Natl Acad Sci USA 1986; 83: 7201-7205. Perez DM, DeYoung MB, Graham RM. Mol Pharm 1993; 44: 784-795. Ho AK, Klein DC. J Biol Chem 1987; 262:11764-11770. Weiss BA, Insel PA. J Biol Chem 1991; 266: 2126-2133. Llahi S, Fain JN. J Biol Chem 1992; 267: 3679-3685. Slivka SR, Meier KE, Insel PA. J Biol Chem 1988; 263: 12242-12246. Klein DC, Sugden D, Weller JL. Proc Natl Acad Sci USA 1983; 80: 599-603. Thonberg HS, Zhang SJ, Tvrdik P, Jacobsson A, Nedergaard J. J Biol Chem 1994; 269: 33179-33186. Okazaki MZ, HuZW, FujinagaM, HoffmanBB. J Clinlnv 1994; 94: 210-218. Shinozuka KM, Hashimoto M, Masumura S, Bjur RA, Westfall DP, Hattori K. Br J Pharm 1994; 113: 1203-1208. Hart C, Li J, Minneman K_P. Eur J Pharm 1990; 190: 97-104. Suzuki E, Tsujimoto G, Tamura K, Hashimoto K. Mol Pharm 1990; 38: 725-736. Minneman KP, Theroux TL, Hollinger S, Han C, Esbenshade TA. Mol Pharm 1994; 46: 929-936. Schwinn DA, Page SO, Middleton JP, Lorenz W, Liggett SB, Yamamoto K, Lapetina EG, Caron MG, Lefkowitz ILl, Cottecchia S. Mol Pharm 1991; 40:619-626. Wu D, Katz A, Lee CH, Simon MI. J Biol Chem 1992; 267: 25798-25802. Esbenshade TA, Wang X, Williams NG, Minneman KP. Eur J Pharrn 1995; 289: 305-310.

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Perspective in Receptor Research D. Giardin/~, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

135

tXl-ADRENOCEPTORS: SUBTYPE- AND ORGAN-SELECTIVITY OF DIFFERENT AGENTS A. Leonardi a, 1LTesta a, G. Motta a, P.G. De Benedettib, P. Hieble c, D. Cfiardin~d a

b

R & D Division R,ecordati S.p.A., Via M.Civitali 1, 20148 Milano, Italy Dip. Chimica, Un. Modena, Via Campi, 183 - 41100 Modena, Italy Div. PharmacoL Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania, 19406, U.S.A. Dip. Scienze Chimiche, Un. Ca,merino, Via S. Agostino, 1 - 62032 Camerino, Italy.

INTRODUCTION The subclassitication of the a-adrenergic receptor (a-AR) into the al" and a2-subtypes [ 1, 2] made possible the development of effective antihypertensive drugs that either selectively block the ctl-AR or activate the ct2-subtype. About ten years after this subclassification, the first suggestions for fimher subdivision were made based on functional and radioligand binding data [3-5]. Currently, the presence of at least three distinct al-ARs has been established through molecular biology, radioligand binding and fimctional studies [6]. The functional evidence of further distinct subtype(s) showing low affinity for prazosin is still awaiting confirmation from molecular biology [3, 7, 8]. The presence of these different subtypes of the ctl-AR in blood vessels and other smooth muscles offers the oppommity for selective drug action. Selective antagonists are perhaps the most useful tools for receptor characterization and classification. The aim of this short review is to update the knowledge on tx~-AR subtypes selectivity of known and novel a~-AR antagonists, based on radioreceptor binding study results. By this technique, the affinity of most of the antagonists for several other G-protein-coupled receptors was also investigated. Functional correlates to the observed selectivity patterns were investigated by studying the effects of selected antagonists on blood vessels and lower urinary tract organs on in vitro and in vivo models. Antagonists endowed with high selectivity for the lower urinary tract are presented.

r

SUBTYPES

The initial subdix~'on of the a~-AR into subtypes was based primarily on the selectivity of WB-4101 for the c~subtype [4] and the selective alkylation of the era-subtype shown by chloroethylclonidine (CEC) [5]. Other antagonists selective for the ct~-AR have been identified, such as 5-methylurapidil [9] and (S)-(+)-niguldipine [10].

136 Molecular biology techniques allowed to identify cDNAs encoding three afARs, namely the rat ala/d, the hamster alb and the bovine a~c [ 11-15]. Originally, the pharmacological characteristics of the cloned subtypes appeared to be inconsistent with the pharmacological properties of the native subtypes, the existence of four subtypes being also suggested [15]. It is now clear that the three recombinant a~-ARs which have been cloned to date correlate closely with the three a f A R subtypes, that have been identified in native tissues and which mediate their functional responses [16-18]. These receptors are now designated as a m (ala), alB (alb) and aiD (a~d), with lower case subscripts being used to designate the recombinant receptors, and upper case subscripts to denote the native receptors [6]. The recombinant receptor previously designated as the a~c-AR has now been shown to correspond to the native atA-AR [16, 19-21], and as such, the terms a~c- or alc-ARs are no longer used [22]. Furthermore, the atD-AR has now been characterized in detail, and antagonists capable of discriminating between this receptor and the a~A-AR are now available, confirming that these two receptors represent distinct a f A R subtypes [23, 24]. As such, the previously used designation of am/o-AR is now inappropriate. In addition to these am, alB, and atD-AR subtypes, which share a high affinity for prazosin, the existence of additional al-ARs has been proposed. These are called a~L-ARs and are characterized by a low functional affinity for prazosin, intermediate affinity for WB 4101 and 5-methylurapidil and resistance to CEC alkylation [3, 7, 8]. No related recombinant ax-AR has yet been identified and the radioreceptor binding characterization of these sites is still difficult, perhaps due to the lack of a suitable radioligand. Nevertheless, there is clear evidence from functional studies for the existence of these ax-ARs in blood vessels and other smooth muscles [8], and these prazosin-low affinity al-ARs may represent an additional therapeutic target for selective drug action. CLASSIFICATION OF al-ADRENOCEPTORS ANTAGONISTS The considered a f A R antagonists were classified according to their affinity for the different receptors and cloned subtypes, determined by radioreceptor binding studies. Tritiated prazosin was utilized to assess the affinity for the native a f A R (rat cerebral cortex [4]), the UtA (rat hippocan~us pretreated with CEC [25]) and the a~B (rat liver [25]) adrenergic subtypes, as well as for the recombinant bovine Ula (formerly a~c [12]), hamster alb [11], and rat a~d (formerly a~a/d [13]) transiently expressed in COS-7 cells. Transfected COS-7 cells were provided by Dr. S. Cotecchia (University of Lausanne, Switzerland). The same ligand was utilized to investigate the affinity of a few compounds on the human recombinant afsubtypes expressed in CHO cells [20]. Where not specifically stated, the affinity for the cloned subtypes in Tables 1 and 2 was determined in the animal clones. The rat cerebral cortex data are reported here in order to maintain a link with the data in the older literature and the native subtypes data for comparison with the results obtained on the cloned subtypes. The other G-protein coupled receptors investigated by us were the ae-AR ([3H]rauwolscine, rat cerebral cortex [26]), the 5-HT~-serotonergic ([3H]-8-OHDPAT, rat hippocan~us [27]),

137 the 5-HTzA-serotonergic ([3H]ketanserine, rat cerebral cortex [28]), the D2-dopaminergic ([3H]spiperone, rat striatum [29]). For the 1,4-dihydropyridines, also the binding to the 1,4-DHP binding site was investigated ([3H]isradipine, rat brain [30]). For most of the data, at least 2 determinations (in triplicate) were performed. Regression analysis of the affinity obtained for the native atA vs the cloned C~a and for the native a~B VS the cloned a~b-subtypes gave the following equations (n=45): pKi~a = 0.91 pKitA + 0.34 (R e = 0.89) pKi~b = 0.90 pKi~B + 0.94 (R2 = 0.93) Only for 9 compounds a difference in pKi higher than 0.5 was experienced, this difference being in the range 0.5-0.7, thus confirming the good agreement between the two data-sets. Representative examples from the known classes of cxl-AR antagonists, as reported in several reviews [31-36], have been evaluated by radioreceptor binding screening. In addition, compounds from more recently introduced classes were also included. The results are shown in Table 1. The structure of considered compounds, grouped by class, is shown in Figure 1.

Poorly subtype-selective al-antagonists (Table 1) Yohimbanes : corynanthine The most constrained and less potent among the known a~-AR antagonists confirmed its low potency and showed little subtype selectivity. Although corynanthine is reported to be a selective al-adrenoceptor antagonist, affinities for a~- and oq-ARs were nearly equal.

Ergot alkaloids: dihydroergocryptine Another very rigid molecule which unexpectedly showed the same high affinity for the a~b, and the other receptors tested.

ala ,

Ouinazolines This is one of the most investigated classes in this field, due to the high selectivity for the al-AR vs the ~ - and the other receptors, with prazosin being the prototype drug. Subtype selectivity is very low for the "classical" quinazolines and in some cases also al/a 2 selectivity proved low. The main interaction with the receptor has been proposed [37] and later confirmed [38] to occur through a charge-reinforced H-bond between an anionic site in the receptor (aspartate ion on the 3rd transmembrane a-helix) and the protonated quinazoline N 1. The presence of a substituted amino group at position 2 contn'butes to protonability and stabilization of the charge on the N 1 whereas N 3 is not mandatory for activity [39], as supported in Table 1 by the data for abanoquil, a very potent but non subtype-selective c~l-blocker, in comparison to its quinazoline analogue I [37]. The modelling and QSAR for a series of quinazoline derivatives has recently been published [40]. Structural modification of the amino group at position 2 afforded novel derivatives endowed with some selectivity for the a~B-AR subtype, discussed below.

N-A rylpiperazines This is another class of compounds extensively investigated mainly for their activity at a1-[34 ]

138

and 5-HT~-receptors [41]. The older representatives, urapidil and A R C 239, listed in Table 1, s h o w no relevant subtype selectivity and no separation o f the affinity for the t w o receptors. F u r t h e r w o r k on this class led to m o r e selective c o m p o u n d s , discussed below.

Table 1. Binding affinity o f

RECEPTOR

ct~-AR antagonists (Ki, nM).

~1

0[~IA 0{~IB 0['la 0~lb

0{~ld 0['2

5-HTtA 5-HT2A D2

Ca++

Poorly selective Corynanthine Dihydroergocriptine Prazosin Terazosin

367 3.7 0.7 3.4 0.1 0.2

424 5.8 0.9 5.8 0.1 0.2

671 9.9 0.4 3.5 0.7 0.1

128 4.8 0.7 26 0.2 0.1

1.0a

2.0a

0.4 a

855

272

429

290

1302

Phentolamine SNAP 5036

31

16

89

3.2 4.4 c

Tamsulosin WB 4101 Benoxathian (+)-SPAL (-)-SPAL Indoramm RS 17053

3 8.7 5.3 121 10 29

0.2 4 1.3 26 2.0 57 44 619 1.2 60 28 43 0.7 e,f

Compd 2

10 28 4.0 10 2.8 13 4.3 2.1 28

Compd 1 Abanoquil ARC 239 Urapidil

1070 10 0.5 26 0.2 0.1

287 33 1.4 35 0.2 0.1

2167 2.4 2357 > 2325 2489

868 13 > > 7800 717

1818 3.5 > > 2474 2207

1658

437 1.7 147 174 11 43 10a,b 2343

213

8445

3181

89 72c

67 155 c

40 126c,d

1263

432

>

0.2 0.6 0.7 4.1 0.4 5.0 0.3 f

6.7 57 55 159 21 24 14f

1.8 6.3 21 140 6.8 274 17f

237 15 109 1504 2035 1634

4.4 7.3 5.3 890 510 74

> 716 182 890 577 243

220 144 281 822 97 315

0.6 2 3.5 0.3 1.3 0.4 0.4 0.2 0.4 1.8c

16 775 12 52 5.1 118 44 18 25 85c

4.1 175 39 24 43 14 5.3 7.6 82 191 c

23 432 12 92 193 151 19 110 1611 372 c,g

125 1.2 93 11 162 22 7.4 54 >

158 > 364 1257 1882 915 197 952 712

69 1051 369 100 153 101 37 100 295

0.4 h

230h

427 h

562 h'g

0.5 1.3h

76 269 h

468 9415 1047 h 1318h'g

0.6 h

324h

CXla-AR selective

5-Me-urapidil

Compd 3 Compd 4 Compd 5 Compd 6 Compd 7 Compd 8 (S)(+)-Niguldipine

1.9 4.7 1.4 1.3 1.7 0.9 0.5 0.3 6.4

15 220 3.2 11 13 62 20 10 380

Compd 9 SNAP 5089

28

2.6

229

Compd 10 Compd 11

0.8 h 2754 h

31 h >

2385

899

(+)-Cyclazosm AH l l l l 0 A Spiperone Risperidone

652 794 h

204 h'g

>h

4467 h 1072d,h

>h

631 h

CZlb-AR selective Compd 12 Compd 13

0.3 9.1 c

1.4 173 5.8

2.2 503 25

1.1 23 0.2

7.5 424 33 161 2511 i

0.5 101 0.7 40 76 i

10 622 27 382 2574 i

1561 4804 613

4498 > >

1364 > > >

8005 3557 3533 >

7.0 9.3

38 4.7

1.6 2.2

13 2.8

7.1 7.7

22 25

136 14

25 371

2.3 0.5

0.6 6.6

139

Table 1-Follows RECEPTOR

r

~1

I~IA 0~IB (~la O~lb O~ld 0['2 5"HTxA 5-HT2A D2 Ca++

selective

BMY 7378 Compd 14 Compd 15 SKF 104856 SKF 106686 (-)-Discretamine

282 422 314 0.7 0.8

273 1.2

495 426 43 1038 1.2 0.2 36a 29a 58a 15a 6171 3631

5.7 474 0.1 1.6a 1.2a 251

657 > 3.4a'b 15a'b

0.4 287 >

493 >

53 > 29

Notes to Table 1. > means > 10000 nM. a) human clones; b) Ot2b-Subtype, see ref. [24] for method; c) human clones, see ref. [43]; d) c~b-subtype; e) rat submaxillary gland; f) see ref [46]; g) a2csubtype; h) human clones, see ref. [52]; i) see ref. [57]; 1) functional data, see ref. [64].

a~a-Adrenoceptor selective antagonists (Table 1) lmidazolines : phentolamine Based on our results, this well known compound can be considered endowed with a slight selectivity for the C~a over the C~b- and eqd-AR, in agreement with the affinity for the cloned oq-subtypes recently reported for it [42]. Phenylalkylamines Although BE 2254 is devoid of subtype and ~ / % selectivity, slight modification of its structure afforded SNAP 5036 [43], showing moderate selectivity for the a~a-AR. A relevant decrease in affinity for the %-AR must also be noticed. Of the other phenylalkylamines, tamsulosin was confirmed to be very potent at the C~a-AR and more selective over the C~b- than the Oqd-Subtype. A significant affinity for the serotonin 5-HT~A receptor is also present. Benzodioxanes In our hands, WB 4101 confirmed its high potency at the Oqa-AR with a moderate selectivity respect to the C~ad-and a pharmacolo~cally relevant difference with regard to the Oqb-Subtype. Significant affinity for the ~ - A R and the 5-HT~A receptor was also found. Its thio analog, benoxathian, gave approximately the same profile, but with a lower affinity for the ~-AR. Introduction of a 4-tolyl group at position 3 of the benzodioxane nucleus of WB 4101 afforded a very interesting compound whose trans isomer showed a unique selectivity for c~over ~ - A R [44]. This compound was resolved [45] and the two enantiomers, (+)- and (-)-SPAL proved stereoselective at the c~-subtypes and D2-dopaminergic receptors, maintaining a remarkable C~a-subtype selectivity and practically losing the affinity for the ~ - A R and the serotonergic 5-HT~A-receptor present in WB 4101.

140 Figure 1 - Structure o f the investigated CZl-amagonists, grouped by class (the letter close to the structure shows the c~I-AR subtype selectivity, if any) Yohimbanes:

H

~

o~,~o~

Ergot alkaloids: H,c CH, OH

~N

O ":'H

I~-C~~ \"o

,--/ "H

H..r

Dibenzoquinolizines:

OH

HO

CH~

OCH,

Dihydroergocrypfine

Corynanthine Quinazolines:

R= --N

R=

~N

~

N X__J

O

Terazosin

~>.~

H3CO1 v ~

,.~o,es:

Abanoquil

,~o~~~o~ H,NO~S

H

H,C

SNAP5036(a) Benzodioxanes:

"N"

(+)-Cyclazosin (b)

[~~ OCH3

H

OC o Compd 12~o)

H3CO~

Compd 13 (b)

~~~.~~

Prazosin

Compd I

OCHs

Phenylalk~lamines:

o

(-)-Discretamine (d)

~- ~ ~ - ~ o ~

"~OCH3

R=/N ~

o~

N k..._./

R=

_~

OCH,

OvClA

Tamsulosin(a)

lmidazolines: N/"7

~

-NxH

, c ~Phentolamine ~ - ~ (a)~

OCH3

~H Indoramin (a)

X= O R= H WB 4101 (a) X= S R= H Benoxathian (a) X= O R= 4-tolyl (trans) SPAL (a)

;~ ~ o _ ~

H,C C%

"H

RS 17053 (a)

o~.~~ c,,

H.CO

Compd 2 (a)

141 Figure 1- Follows

H3C

N-Arylpiperazines:

x

ARC 239

R o

~

o H~C CH3 ci~ n I J O~~N~

Urapidil

OCH3 Compd 3 Comlxl 4 (a) OCH3

o

N~

N

5-Me-Urapidil (~c .~N~~[~ C~ o BMY 7378 (a)

OCH3

o

~N---

Ph

Compd 6 (a)

t-Bu

o ~

Various structures:

F

~

2-OCH3

Compd $ (a) Cy-Hex

2-OCH3;5-C1

1,4-D~ydropyridines:

H

~N ~.O

~'~N'~ H

%c- -~--c% n

CI

(S)(+)-Niguldipine (a) Compd 9 (a) SNAP 5089 (a)

NO 2

H

OH NH

Spiperone %c ...2-/~

Risperidone

R 3-NO2 3-NO 2 4-NO 2

",,..,,,~Ph Ph X O NH NH

o@o

O

H

C

2-OCH3

Compd 7 (a) Cy-Hex

OCH3 I

O

o-N I 'k_/~

2-OCH3;5-C1

OCH3

~N--

Compd 15

Ph Ph

Compd 5 (a)

o o Compd 14

R1 H 2-OCH3

R

%c

O_

o

Eompd 10 (a) Compd 11 (a)

Ph

R2 R6 R4 c2r% Crt2OCH2Crt2~ 2 t,h CH3 CH 3 COOCH3

s~N-c%

Thienobenzazepines: Cl

R

R

SKF 104856 (d) CH2=CH2 SKF 106686 (d) C2H5

142 Indoles The first example in this class, indoramin~ exhibits a substantial selectivity for the {~la-Al{ over the a~d-subtype, lndoramin has also significant affinity for the serotonin 5-HT~A and histamine H~ receptors. Another indole bearing a basic alkyl chain at position 3 is KS 17053 [46] which proved more than 40-fold more potent at a~a- than at a~b- and a~d-AR. A different substitution pattern is present in compound 2 [47], showing a moderate a~a- over a~b-AR selectivity but an interesting lower affinity for the serotonergic 5-HTtA-receptor, in spite of the presence of the N-(2-methoxyphenyl)piperazinyl group in the ether chain. N-A rylp ipe razi nes 5-Methylurapidil is the best known of this class and was confirmed to be higly selective both for the aL~- and a~a-AR. Very high affinity for the 5-HTtA-serotonergic receptor was also confirmed. The effect of different substituents on the phenyl ring of the arylpiperazine moiety was investigated in a small series of 8-flavonecarbamylalkyl derivatives, compounds 3-5 [48, 20] where the nature and length of the spacer had previously been optimized. With regard to subtype selectivity, the 2-methoxy group gave the best results. The introduced substituents gave lower affinity for the ~ - A R with regard to the unsubstituted derivative 3. Interestingly, the introduction of a chlorine atom para to the methoxy group gave a satisthctory selectivity for the ala-AR with regard to the 5-HTm-serotonergic (compound 5, more than 100-fold difference). This trend is confirmed also by the results obtained with compounds 7 and 8, differing from the above series for the presence a cyclohexyl instead of a phenyl group at position 2 of the chromone ring. The best Ctla/alb selectivity in this series was obtained with the introduction of a tert-butyl group at position 2 of the chromone ring (compound 6). Based on our results and literature findings [49] it can be stated that careful modulation, series by series, of the substitution pattern of the aryl and alkyl substituents on the two piperazine nitrogen atoms can result in selective ax-antagonists. 1, 4-Dihydropyridines (S)-(+)-Niguldipine was the first 1,4-dihydropyridine to combine tx~-AR and Ca~-chatmel blockade. Studies on its action in native tissues showed selectivity for the auc vs the CqB-A1L Due to its very lipophilic nature both its absolute potency and subtype selectivity may vary significantly between laboratories [50, 17, 20, 22, 51]. Formal transformation of the aminoalkyl ester of niguldipine into the corresponding N-aminoalkyl carboxamide afforded compound 9 [52], endowed with striking selectivity for the human axa-AR with regard to the a~b- and a~d-subtype , but still active on the Ca~-channel. ala-Selectivity was maintained and affinity for the Ca~-channel was greatly reduced in compound SNAP 5089 [52, 53], which is the 4-nitrophenyl isomer of 9. Extensive structural modification was performed on the 4-(4-nitrophenyl)- 1,4-dihydropyridine structure [52] which led to compounds which can be considered specific for the human axa-AR, based on the reported results (see for example compounds 10-11 in Table 1).

143

a~b-Adrenoceptor selective antagonists (Table 1) In contrast with the ala- and axd-ARs , highly selective axb-AR antagonists have not yet been identified. A very promising agent, (+)-cyclazosin, is introduced here, whereas the selectivity of compounds initially reported to be a~B-selective was not confirmed in our study.

Novel quinazolines Screening a series of novel 4-amino-6,7-dimethoxy-2-substituted quinazolines [54], compounds 12 and 13 were identified, showing a moderate albeit inconsistent selectivity. The acylpiperazine derivative 12 showed a moderate selectivity for the recombinant Crib-subtype , whereas it proved non-selective when native ARs where used. On the contrary, the tetrahydropapaverine derivative 13 exhibited moderate selectivity on native but not on recombinant subtypes, in analogy with spiperone (see below). (+)-Cyclazosin was obtained by one of the authors (D.G.) by resolution of the racemic compound [55], which showed moderate selectivity for both rat liver CqB- and ham~er c~xb-AR [56]. This (+)-enantiomer can be considered the first al~-AR selective antagonist, being on the order of 100-fold selective vs the native c ~ and about 40-fold vs the recombinant a~a and Cqd-AK Research to assess its absolute stereochemistry is in progress. Various structures The novel amidine AH l l l l 0 A has been recently reported to show more than 30-fold selectivity for the hamster Ct~b-AR, compared to the ~a- or Ctld-subtypes [57]. In our hands, this compound showed a much lower selectivity on the same clones. Analogous results were obtained for spiperone, which proved about 20-fold selective on the native subtypes and non-selective on the clones, in agreement with the results from other laboratories [58, 22]. Also another dopamine receptor antagonist, risperidone, was reported to show some selectivity for the eqB-AR [59], which was not confirmed in our studies. These problems of reproducibility might be ascribed to the marginal selectivity of the currently investigated compounds. a~d-Adrenoceptor selective antagonists (Table 1) N-A rylpiperazines BMY 7378 is by far the most selective C~ld-AR antagonist reported [23]. Its high selectivity was confirmed also in our studies, together with its relevant affinity for the serotonergic 5-HT~-receptor. On replacing the spirocyclic imidoyl group of BMY 7378 with a phthalimidoyl group (compound 14 [60]) both selectivity and affinity went lost, suggesting a specific interaction of the spirocyclic group with the AK Compound 15 [49] shows a very high affinity for the C~ld-AR but also a very low subtype selectivity. Structural modification of 15 afforded compounds endowed with very high selectivity for both ~ld- and ~a" versus ~lb-AR [61]. Hetero-fused 3-benzazepines This is a novel class of compounds showing moderate selectivity for the human recombinant C~ld-Clone. The thienobenzazepines SKF 104856 and 106686 show a high potency and a

144 moderate selectivity, more marked vs the ala" than the O~lb-and ~b-AR [62]. Di be nzoqumolizines : (-) -discre tamme This alkaloid was extracted from the plant Fissisti~a glaucescens [63] and its select'wiry for the a~D-AR was assessed through functional studies where antagonism to NA-induced contraction of rat aorta (a~D), spleen (alB) and vas deferens (au0 was determined [64]. The results available so far strongly suggest further evaluation of this (class of) compound(s).

LIGAND-RECEPTOR INTERACTION MODEI.J.JNG AND QSARs The heuristic-direct QSAR approach introduced by one of the authors [65] was applied to the study of the interaction of selected ligands with the three ~I-AR subtypes. This approach, applicable when the atomic resolved structure of the target is unknown but predictable by means of the available experimental information, is able to handle the heterogeneous experimental information on the ligands and their targets, and to synthesize and translate them into QSAK models. The first step of this procedure consisted of the computer-aided three dimensional (3-D) model building of the seven transmembrane (7-TM) helix bundles of the three aI-AR subtypes. The seven helices, selected from the human primary sequences [66, 51, 67], were initially packed according to the seven-helix wheel projection models proposed by J. Baldwin [68]. The packing preferences of the 7-TMs were then investigated by comparative Molecular Dynamics (MD) simulations (QUANTA-CHARMm) [69]; the structures averaged over the last 100 picosecond time period of the MD simulation and minimized are reported in Figure 2. The 7-TM models of the three al-ARs show different dynamic behaviours and different topographies of the binding sites, which are however mainly constituted by conserved residues. The structure/dynamic analysis of the three models shows that the r site is more flexible and structurally different with respect to that of the other two subtypes. The second step in our approach involved docking simulations with selected ligands maximizing the complementarity between ligand and receptor. The following ligands in their protonated forms were considered: (-)-noradrenaline (Kiocaa--4530.2; Kia~b = 9264.7; Kia~d = 770.4), WAY 100635 (Kia~a = 143.4; Kialb = 183.7; KiOt,d = 63.1), prazosin, spiperone, urapidil, compound 12, BMY 7378, phentolamine, tam_~osin, WB 4101, compound 4, (S)-(+)-niguldipine and 5-methylurapidil. These ligands were docked into the three a~-AR subtype binding sites. For each complex several rninimirations were performed in order to optimize several fundamental interactions i.e. the charge reinforced H-bonds between the protonated nitrogen atom of the ligands and the carboxylate of the Asp307 present in the TM-3 (the numbering is arbitrary: the first digit indicates the helix and the next two digits indicate the position of the residue in the helix). In the final step, a detailed extensive correlation analysis between the computed binding energies (BE) and the experimental binding aff-mities determined on the cloned bovine a~a-, hamster a~b" and rat ~qd-AR subtypes were carried out in order to evaluate the consistency of the QSAR models proposed [70].The binding energies (BE) were computed according to the following formula: BE = IE (receptor-ligand) + E (receptor) + E (ligand), where IE (receptor-ligand) is the total interaction energy of the ligand-receptor complex, and E (receptor) and E (ligand) are the distortion energies of the receptor and of the ligand,

145

Figure 2. Minimi7ed average structures of the three All, subtypes. The helix bundles are seen from the intracellular side in a direction parallel to the main helix axes.

/

.t

X,

Figure 3. Details of the interaction between the antagonist 5-methylurapidil and the conserved amino acids of the three AK subtypes, postulated to make the major contribution to antagonist-receptor interaction. The green color labels for favourable interactions while the red color labels for unfavourable interactions.

146 respectively, calculated as differences between the energies of the bound and of the flee optimized molecular forms. Very satisfactory correlations were obtained: pKodla = 5.109 (+ 0.346) - 0.094 (+ 0.010) BEaaa, n=13, R=0.94 pKicqb = 3.692 (+ 0.381) - 0.136 (+ 0.014) BEa~b, n=13, R=0.95 pKkxld = 4.645 (+ 0.390) - 0.084 (+ 0.010) BECZxd, n=13, R=0.91 Very similar correlations have also been obtained by plotting the binding affinities of nine ligands ((-)-noradrenaline, prazosin, spiperone, urapidil, phentolamine, tamsaflosin, WB-4101, compound 4 and 5-methylurapidil) measured on human cloned al subtypes versus the computed BE (pKia~a vs BEaxa: n=9, R=0.96; pKi(Xlb vs BEalb: n=9, R=0.95; pKiald vs BEard: n=9, R=0.78). By analyzing the ligand-receptor complexes we found that compounds endowed with high affinity and no selectivity, like prazosin, interact mainly with the same conserved residues of the three subtype binding sites without inducing relevant distortions. Potent and selective ala-AR antagonists like 5-methylurapidil interacts without inducing significant distortions only with the (Xta-AR binding site, as indicated by the green color which labels the binding site residues of the (Zxa-AR subtype in Figure 3. In contrast, 5-methylurapidil induces significant unfavourable distortions into the a~b" and the a~d-AR binding sites, as indicated by the red color. On these bases, the molecular determinants for affinity and selectivity can be postulated. VITRO AND I N VIVO SELECTIVITY

~-Antagonistic activity on different organs was investigated for selected compounds, with the final aim of verifying how the subtype selectivity observed in the radioreceptor binding studies translates into functional results. Rat aorta, rabbit aorta and urethra, and human prostate tissues were utilized. Prototypic tissues for the functional investigation of the different al-AR subtypes are under continuous investigation, and the correspondence of rat aorta with the Cqd-, rat spleen with the alb" and rat kidney with the (Z~a-subtype seems currently the most accepted [19,71,64,72]. For other tissues, in particular those of the lower urinary tract, the situation is, in our opinion, less clear. From one side there is convincing evidence of the functional relevance for human prostate of the atA-AR [17,18,73], in addition to analysis ofmRNA expression [74,75] and to radioreceptor binding studies [20,76]. On the other hand, resistance to CEC and low affinity for prazosin suggest that the functional affinity to this tissue could be due to the (ZlL-AR [8,77,46]. Listed in Table 2 are the functional and receptor binding results of some selected antagonists, in an attempt to clarify this issue. A good correlation is present between affinity for the aid-subtype and potency in inh~iting the contraction of rat aorta (R2 = 0.696), confirming our previous findings [72]. With regard to human prostate, the very poor functional results obtained by other authors with KS 17053 and abanoquil, despite their high potency in inh~iting NA-induced contractions in rat isolated kidney and vas deferens, respectively, and their high affinity for the cloned and native ala-ARs [46,78], destroy the relatively good correlation present with the other antagonists (Figure 4A), and suggest the relevance of other receptors, possibly the alL-AK

147 In our screening procedure in a program aiming to identify selective al-AR antagonists for use in BPH, we used the rabbit urethra preparation for screening in vitro functional activity of the compounds. Table 2

~ty

of S d ~ e d I~I-ARanta~oni~Lsfor the recombinant animal ~x-AR subtypes (pKi= -loga0Ki, nM) and functional affinity for the %-ARs of vascular and lower urinary tract tissues (pKb = -lOgl0 Kb, nM, for inhibition of NA-mduced contraction). R.A.= rat aorta; RB.A.= rabbit aorta; RB.U. = rabbit urethra; H.P.=human prostate. The in vivo effects after i.v. administration in the dog model are also listed. Data represent the doses ( ~ t ~ ) active in inhibiting by 50% the urethral contractions reduced by NA (UP), the doses active in lowering diastolic blood pressure (DBP) by 25%, and the ratio between them. The ratios between the in vitro potency (Kb) on rabbit aorta and urethra are also reported (1/2).

COMPD

O~la

Otlb

r

S.A.

pig

pKi

pig

pKb

RB. A. pKb 1

RB. U.

pKb 2

H.P. UP

DBP DBP/

1/2

UP

pKb ED~ ED~

Prazosm

9.14

9.34

8.86

9.90

9.00

8.11

8.25

3.6

6.6

1.83

0.13

Terazosm

7.58

7.52

7.46

8.58

7.92

6.95

7.38

21

61

2.9

0.11

Tamsulosm

9.82

8.17

8.75

9.35

9.54

9.31

9.20

1.1

11

10

0.59

5-Methylur.

8.69

6.10

6.80

7.65

7.52

8.04

7.93

1.4

10.8

7.7

3.31

SNAP 5089 9.33

7.12

6.33

6.65

<5.00

5.53

310

Compd 12

8.12

9.35

7.99

8.74

7.49

6.20

65

183

2.8

0.05

Compd 16a

8.85

8.20

7.76

6.94

7.49

8.26

8.47

5.7

141

24.7

5.89

Compd 7

9.39

7.36

8.27

8.74

7.82

8.57

8.17

1.8

97

53.9

5.62

Compd 4

9.54

7.29

7.61

8.62

7.70

8.64

8.57

2.4

243

101.2

8.71

Abanoquil

10.1

9.89

10.2

7.30 b

RS 17053c

9.52

7.84

7.76

6.89

>3000 >10

>3.40

a) N-[3-[4-(2-hydroxyphenyl)- 1-piperazinyl]propyl]-3-methyl-4-oxo-2-phenyl-4H1-benzopyran-8-carboxamide; b) extrapolated from ref. [78]; c) from ref. [46]. This tissue has initially been characterized as am-subtype dependent [25]. However the results obtained by us with compound 12 and SNAP 5089, both showing much lower functional affinity than expected based on their binding affinity for the ala-AR (see Figure 4B), and the low pKb of prazosin, suggest that also in this case the presence of ctxL-AR must be accounted for. In our view, this tissue could be considered as (XlL-AR dependent and SNAP 5089 and compound 12 may be very poorly active on this subtype. We investigated organ selectivity of our compounds by utili~ng, in addition to rabbit urethra, rabbit aorta which is reported to be dependent on alL" and (XlB-AR population [8]. The classification of this tissue seems difficult with the data presented here. In any case, we can observe that among the tested compounds three groups can be considered, based on the results on rabbit urethra and aorta. The quinazoline derivatives prazosin, terazosin and compound 12 show higher affinity for the vascular tissue than for the urethra; tamsulosin and

148 10,5 -

10.5 -

~'

9.5

A

-

D,.

s 9162149 ~l

o.

== 8 . 5

4-- 8 . 5 W

Rra'zosin

r

o. 7.5 -

(D t._

abanoquil

r a

s

E

RS

17053

r

-

L_

9 9

O L_

,.r=~

/is/

B

9.5 -

9

=

9

5.5

!

I

I

I

6-5

7.5

8.5

9.5

,

00

9P r a z o s i n

/ 9

-9 6 . 5 -

5.5

9

is/S 9

7.5

==

6.5 -

9

9

.."

."

/

compd

/

5.5

10.5

,

5.5

12 9 , SNAP

6.5

7.5

cloned o l = - A R (pKi)

I

5089

==

'~1

8.5

I

9.5

10.5

cloned o T , - A R (pKi]

Figure 4. Correlation between the binding affinity (pKi) for the cloned ala-AR and the functional affinity (pKb) for the cxl-adrenoceptors in human prostate (A) and rabbit urethra (B). Dashed line represents the line of identity.

100

-

o

80-

/

/

Z

0

.,.._

z

50

-

40

-

/

-

60r

40-

/

0

/

30

-

I

0.1

'

'

' t'"'l

'

'

' t'"'l

1

,

n

u inJ=n !

10 DOSE

[pglkg

100

=

,

n lUnnl !

,

m Z

O I--

20

o D C3

LU I:Z:

"

s

/

20-

-

/

o.. 1:3

-

I--

IZ]

-

-

10

-

0

1000

i.v.)

Figure 5. Effects of cumulative i_v. injection of increasing doses of compound 4 (squares) and prazosin (circles) on NA-induced increase of urethral pressure (filled symbols), and decrease in DBP (open symbols). Each point represents the mean + s.e. of the responses obtained in 9 and 6 dogs, respectively.

149 5-methylurapidil show roughly the same potency on the two tissues, and the tlavone derivatives 4, 7 and 16 exhibit higher potency on urethra than on aorta, suggesting functional selectivity for the lower urinary tract. The in vivo effects of selected a~-AR antagonists were studied, after intravenous admmi~ration, in a dog model [79] where the active doses inh~iting by 50% the urethral contractions induced by k a. NA injection, and the doses active in lowering diastolic blood pressure by 25% were simultaneously evaluated in the same dog. The results obtained are listed in Table 2 together with m vitro and in vivo selectivity indices. A typical dose-response curves for prazosin and compound 4 are shown in Figure 5. In this model, prazosin, terazosin and compound 12 proved devoid of selectivity, whereas tam~axlosin and 5-methylurapidil exhl"oited limited selectivity. SNAP 5089 showed some selectivit3, but its potency against the urethral contractile response was unexpectedly very low. On the contrary, the novel N-arylpiperazine derivatives (in particular compounds 4 and 7) were endowed with very high potency and selectivity for the lower urinary tract. Interestingly, a good correlation between the in vitro potency on rabbit urethra and the in vivo potency in inhibiting the urethral contractions induced by i.a. injection of the agonist in the dog was found (R2 = 0.925). In summary, compounds endowed with high potency in inh~iting contractions of isolated rabbit urethra (a ~tL-AR dependent tissue) showed high potency in inh~iting also the NA induced contraction of the dog urethra in vivo, and in vitro selectivity could be correlated to m vivo selectivity. CONCLUSIONS The growing interest in the field of r subtypes and their antagonists has yielded a number of novel compounds, whose pharmacological evaluation allowed an update of the classification of the tz~-antagonists based on radioreceptor binding studies. A novel class of 4-(4-nitrophenyl)-l,4-dihydropyridines, originated from niguldipine's structure modification, represents the most selective ala-AR antagonists available, highly selective for this subtype versus both other a~-ARs and the other neurotransmitters receptors studied. The N-arylpiperazine derivatives represent the more flexa'ble class which, upon "calibrated" modifications, afforded very selective ~la" and cirri-antagonists. Modification in the benzodioxane group also proved whorthwile, with the identification of t~la-Subtype selective derivatives with a cleaner profile with regard to other receptors. The quinazoline derivatives were confirmed to be ctl-AR selective and unexpectedly afforded a compound si,~nificantly selective for the tZ~b-subtype. The combined use of comparative structure/dynamics simulations and QSAR analysis applied to a set of complexes between structurally heterogenous ligands and the tz~-AR subtypes allowed to obtain energy-minimized models of the ligand-AR interaction. The single residues involved in this interaction have been identified and their effects quantitated, allowing for a more rational design of novel ligands. Correlation of the radioreceptor binding affinity for the tX~d-AR and rat aorta was straightforward, whereas selectivity for other tissues was not so clear. In particular, activity on the tissues of the lower urinary tract of different species, formerly thought to be tzL~-dependent was not confirmed, mainly by using selective Ct~a-AR antagonists. The functional relevance of

150 the atL-AR subtype both in vitro and in vivo could not be ruled out, suggesting the involvement o f this subtype in the pathopysiological response o f the prostate in BPI-I. Although a~L-AR selective antagonists are not yet known, their involvement in the effects on the lower urinary tract in vivo can be anticipated. The screening procedure adopted in this project, based on radioreceptor binding and in vitro and in vivo functional assays, allowed us to select compound 4, which is n o w undergoing Phase II clinical trials for the therapy o f B P H with the codes g e e 15/2739 or SB 216469. Receptor subtyping and development o f selective agents are the means to novel and improved drugs.

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152

68 69 70 71 72

73 74 75 76 77 78 79

Baldwin JM. E M B O J 1993; 12: 1693-1703. Fanelli F, Menzian/MC, Cocchi M, De Benedett/PG. J Mol Stmct (Theochem) 1995; 333: 49-69. Fanelli F, Menzian/MC, Cocchi M, Leonardi A, De Benedetti PG. J Mol Struct (Theochem) 1994; 314: 265-276. Graham RM, Perez DM, Piascik MT, Riek RP, Hwa J. Pharmacol C o m m 1995; 6: 15-22. Testa R, Destefan/C, Guarner/L, Poggesi E, Simonazzi I, Taddei C, Leonarcli A. Life Sci 1995; PL 159-PL 163. Forray C, Chiu G, Wet,zel ~ Bard JA, Weinshank RL, Branchek TA, Hartig P, Gluchowski C. J Urol 1994; 151: 267A. Price D, Schwinn DA, Lomasney JW, Allen LF, Caron MG, Lefkowitz RJ. J Urol 1993; 150: 546-551. Faure C, Pimoule C, Vallancien G, Langer SZ, Graham D. Life Sci 1994; 54: 1595-1605. Goetz AS, Lutz M W , R/mele TJ, Saussy D L Jr. J Pharmacol Exp Ther 1994; 271: 1228-1233. Hieble JP, Caine M, Zalaznik E. Eur J Pharmacol 1985, 107: 111-117. Marshall I, Burr RP, Anderson PO, Chapple CR, Greengrass PM, Johnson GI, Wyllie MG. Br J Pharmacol 1992; 107: 327P. Imagawa J, Akima M, Saka/K. J Phannacol Meth 1989; 22:103- II I.

Perspective in Receptor Research D. Giardinh, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All fights reserved.

153

M o l e c u l a r R e c o g n i t i o n in A d e n o s i n e R e c e p t o r s K.A. Jacobson,* A.M. van Rhee, S.M. Siddiqi, X.-d. Ji, Q. Jiang, J. Kim, and H.O. Kim Molecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, MD 20892 USA. INTRODUCTION Extracellular adenosine acts as a modulator within many physiological systems [1], in general, to compensate for excessive activity of a given organ and to protect it against the detrimental effects of stress. This is a negative feedback loop, by which locally produced adenosine (originating from degradation of either intracellular or extracellular ATP) both reduces the energy demand and increases the oxygen supply. Adenosine is particularly critical m maintaining the homeostasis of essential organs such as the brain, heart, and kidneys, but is also very important for the immune system. In the brain, exogenously administered adenosine agonists have proven exceptionally efficient in neuroprotection [2] (in part, by counteracting the damage caused by excessive glutamate release), and adenosine has been shown to be revolved in pain, cognition, movement, and sleep [3]. Thus, there is a tremendous impetus for the development of therapeutic agents based on selective interactions with adenosine receptor subtypes. Four subtypes of adenosine receptors have been cloned: A 1, A2A, A2B, and A3 [4]. Adenosine agonists, which are almost exclusively derivatives of adenosine, have been sought as potential anti-arrhythmic, anti-lipolytic' (thus anti-diabetic), and cerebroprotective agents (A1), and hypotensive and anti-psychotic agents (A2A). Adenosine antagonists, of which xanthines and a number of fused heterocyclic compounds are representative [1], have been under development as antiasthmatic, anti-depressant, anti-arrhythmic, renal protective, anti-Parldnson's, and cognition enhancing (h~gs. The adenosine receptors are members of the G protein-coupled receptor family, having seven transmembrane helical regions [5]. Site-directed mutagenesis of the A 1 and A2A receptors [6,7] has yielded much insight into structure function relationships. The emerging receptor models [7-9] are approaching a degree of resolution that promises to assist in the design of improved ligands. The discovery of a novel and distinct adenosine receptor subtype, the A 3 receptor [10], has opened new therapeutic vistas m the purmoceptor field. This receptor subtype has a unique SAR (structure activity relationship) profile, tissue distribution, and effector coupling. Reports on A 3 selective agonists have recently appeared [11-13], but the physiological role of A 3 receptors remains to be clarified.

154 In spite of the massive effort to develop selective ligands, a number of agents that initially looked promising did not survive clinical trials [1]. Nevertheless, the interest in adenosine-based therapy has not waned. On the contrary, as our knowledge of the biological effects of adenosine advances, promising, newly envisioned therapeutic applications have become evident. Recently, the use of adenosine agonists in treating stroke has come into focus [2], since, m acute clinical use the interference by some of the previously documented side effects may be tolerable. RECENTLY-DEVELOPED SELECTIVE LIGANDS Highly selective ligands for adenosine A 1 and A2A receptors [1] have been designed both by classical medicinal chemical approaches and by a functionalized congener approach [14]. By the latter approach, a chemically functionalized chain is incorporated at a specific site on a pharmacophore leading to increased flexibility of substitution and enhancement of potency/selectivity via distal interactions at the receptor. In general, for adenosine agonists (Table 1), modification of the N6-position with hydrophobic moieties (such as CHA, N6-cyclohexyladenosine, 9, and CPA, N 6cyclopentyladenosine, 1 0 ) h a s provided selectivity for A1 receptors (and thus cerebroprotective properties). Substitution at the C2-position (such as occurs in CGS 21680, 2-[4- [(2-cm'boxyethyl)phenyl] ethylamino]-5'-N-ethylcarboxamidoadenosine, 11, and its ethylenediamine conjugate, APEC) has resulted in A2A selectivity. A thio- substitution of the 4'-oxygen of 2-chloroadenosine was found to enhance affinity selectively at A2A receptors [23]. Selective antagonists for A 1 receptors (Figure 2) include many 8-aryl and 8-cycloalkyl xanthine derivatives, such as the amine congener XAC, 12, and CPX, 13, which is -500-fold selective for A1 receptors. A thio group may be substituted at the 2-position, but not 6-position, carbonyl group of the xanthine with retention of high affinity. Selectivity for A2A receptors in xanthines has been more difficult to achieve. However, 8-styrylxanthines, such as CSC (8-(3chlorostyryl)caffeine), 14, are A2A selective. The non-xanthine ZM241385, 15, is the most selective A2.,~ antagonist (6800-fold) yet reported [15]. The most recent challenge in the medicinal chemistry of adenosine receptors has been to produce A3 selective agents. A 3 selective antagonists have not yet been reported. One principle of achieving A3 selectivity among adenosine agonists is the combination of optimal substitutions at the N 6- and 5'-positions of adenosine [9,11]. Specifically, among alkyl, cycloalkyl, and aralkyl N 6substituents, a benzyl group is favored, due to its diminished potency at A1 and A2A receptors (Table 1). The A3-selectivity enhancing effects of N6-benzyl

155 Table 1. Affinities (~tM) of adenosine derivatives at rat brain A 1, A2A, and A 3

NLN

receptors, arranged in order of decreasing affinity at rat A3 receptors.a NHR 3

R2

,>

N

!

HO OH

Compound 1. b 2. c 3. d 4. 5. 6. e 7. f 8. g 9. h 10. i 11. J

R1

R2

R3

CH3NHCO C1 3-I-Bz CH3NHCO H 3-I-Bz CH3NHCO H 3-I-4-NH2-Bz CH3NHCO CH3S 3-I-Bz CH3NHCO CH3NH 3-I-Bz C2HsNHCO H Bz CH3NHCO H H C2HsNHCO H H HOCH 2 H cyclohexyl HOCH 2 H cyclopentyl C2HsNHCO NH(CH2)2-pH Ph(CH2)2-

Ki(A1)

Ki(A2A)

0.82 0.054 0.018 2.14 4.89 0.087 0.0836 0.0063 0.0013 0.00059 2.6

0.47 0.056 0.197 3.21 4.12 0.095 0.0668 0.0103 0.514 0.462 0.015

Ki(A3) 0.00033 0.0011 0.0013 0.0023 0.00312 0.0068 k 0.072 k 0.113 k 0.167 k 0.24 k 0.584 k

a. Ki+ S.E.M. determined in radioligand binding assays expressed in ~M, using the following radioligands: A1, [3H]N6-R-phenylisopropyladenosine in rat cortical membranes; A2A, [3H]CGS 21680, 11, binding in rat striatal membranes; A 3, [i25I]AB-MECA, 3, binding, unless noted, in membranes of CHO cells stably transfected with the rat A3-cDNA; b. CI-IB-MECA; c. IBMECA; d. I-AB-MECA; e. Bz-NECA; f. MECA; g. NECA; h. CHA; i. CPA; j. CGS 21680; k. A 3 affinity determined versus [125I]APNEA (N 6aminophenylethyladenosine) binding.

156 modification are additive with the A3-affinity enhancing effects of the 5'uronamido function, as in NECA (adenosine-5'-N-ethyluronamide), 8. The first such hybrid molecule to show A3 selectivity [9] was NG-benzyl-NECA, 6. In a comparison of various 5'-uronamido groups in mono-substituted adenosine derivatives, the 5'-N-methyluronamide, 7 [11], had particularly favorable A3 receptor vs. A1/A2A affinity. Empirical and QSAR studies [11,20] of substituent effects on the N6-benzyl group have shown that substitution at the 3-position with sterically bulky groups, such as the iodo group, is optimal, leading to the development of the highly potent A3 agonist N6-(3-iodobenzyl)-adenosine-5'-Nmethyluronamide (IB-MECA), 2, which is 50-fold selective for A3 vs. either A1 or A2 receptors in vitro [11] and appears to be highly A 3 selective in vivo [21]. [125I]I-AB-MECA, 3, was introduced as a high affinity radioligand for A 3 receptors [21]. 2-Substitution in combination with modifications at N 6 and 5'-positions was found to further enhance A 3 selectivity [12]. Adenosine derivatives bearing an N6-(3-iodobenzyl) group, reported to enhance the affinity of adenosine-5'uronamide analogues as agonists at A3 adenosine receptors, were synthesized starting from 1-O-methyl ~-D-ribofuranoside in 10 steps [12]. 2-Chloro-N6-(3 iodobenzyl)-adenosine-5'-N-methyluronamide, 1, which displayed a Ki value of 0.33 nM, was selective for A3 vs. A1 and A2A receptors by 3600- and 2000-fold, respectively. Compound 1 was 66,000-fold selective for A3 receptors vs. the Na +independent adenosine transporter, as indicated in its weak displacement of [3H]S-(4-nitrobenzyl)thioinosine binding at this transporter in rat brain membranes. In a functional assay of rat A3 receptors expressed in CHO cells, it inhibited adenylyl cyclase with an IC50 of 67 nM. 2-Methylthio-N6-(3 iodobenzyl)adenosine-5'-N-methyluronamide, 4, and 2-methylamino-N6-(3 iodobenzyl)-adenosine-5'-N-methyluronamide, 5, were less potent, but nearly as selective for A3 receptors.

DO XANTHINES BIND TO A3 RECEPTORS? The A3 adenosine receptor cloned from rat [10] was shown to be unique among the subtypes in that agonist action is not antagonized by xanthines, such as theophylline. The affinity of certain xanthines, such as BWA522 (Figure 1), 16, is greater in the human and sheep homologues of the A 3 receptor [16,17] than in the rat. Typical Ki values at rat A 3 receptors of roughly 10 .4 M have been obtained for many xanthines that have nearly nanomolar potency at the A1 or A2A subtypes. X A C , 12, for example at 1 pM, has been used in

157

Pr\

N

O .LL

H I~I I

O LI

H ~

O,-~N /

--~N

Pr\

/

oc

I

I

Pr

~

-

Pr

12, XAC

13, CPX

O OH3 CH3.N.,~ _~1~1

C!

.o

~

CH3

NH.,J,,.-N,,,J~N/~--'<"O'~

14, CSC

O

Pr\

.LL

15, ZM241385

H

~

0

.~" ~,~=-~o~coo~ c~ .~ _~

O " ~ N ~ -~N

"~"

I @ (0H2)2

16, BWA522

S"/~N ~ ~N

,

NH2

CH3 17

I

Figure 1. Xanthine (12-14,16,17) and non-xanthine (15) adenosine antagonists.

Bu

~,u 0

o~~.~> I

Bu

o~~~> I

H 18

19

]'1'

HO OH

20

~-~ HO OH

Ki (~M) at Rat A 3 Receptors:

143

6.03

0.229

A1 Receptors:

0.50

4.19

37.3

Figure 2. Ribose moiety at the 7-position anchors xanthine in the A 3 binding site.

158 pharmacological experiments in vitro and in rodents [2] for distinguishing A3 receptors from A1 and A2A receptors. In an effort to synthesize A3 antagonists, we have attempted to maximize the affinity of xanthine derivatives at the binding site [18]. One such xanthine is compound 17, which had a K i value at rat A3 receptors of 9.4 ~M. The presence of an anionic group on the xanthine tended to diminish the affinity at A1 and A2A receptors. Thus, compound 17 is 7-fold selective for rat A3 vs. A2A receptors. Molecular modeling of adenosine receptors (supported by mutagenesis) suggests that two histidine residues in the sixth and seventh transmembrane domains (TM6 and TM7) are important for ligand recognition [5-8]. We have proposed [9] that the ribose moiety of adenosine, that is relatively more important for high affinity binding to A3 receptors than at other subtypes, is coordinated to a conserved histidine residue in TM7 (His278 of hA2A, see below). Consequently, we tested the hypothesis that a means of anchoring xanthines in the A 3 binding site is by adding a sugar moiety at the 7-position to form xanthine-7-ribosides (Figure 2). Several members of this class of compounds, the 1,3-dialkylxanthine-7-ribosides, have been synthesized and were previously found by IJzerman and colleagues to bind weakly to A1 receptors [19]. At rat brain A3 receptors, 1,3-dibutylxanthine-7-riboside (DBXR), 19, was found to bind with a Ki value of 6.03 ~M [9],. whereas the parent xanthine, 1,3dibutylxanthine, 18, displayed a Ki value of 143 p~M. Thus, the presence of the ribose moiety enhances affinity of xanthines at rat A3 receptors, while at A 1 receptors the xanthine-7-riboside derivatives are, as a rule, less potent than the parent xanthines. Functionally, 1,3-d~butylxanthine-7-riboside, 19, as a structural hybrid of classical A1/A2A agonist and antagonist molecules, appeared to act as a partial agonist at rat A 3 receptors [9], providing hope that this was a means of designing antagonists. However, upon structural modification that increased the potency and selectivity of the xanthine ribosides at A 3 receptors, full agonism was achieved. Specifically, the structural parallel between adenosine derivatives and the xanthine-7-ribosides was maintained with respect to A 3 receptor affinity. This parallel lead to the design of 1,3-dibutylxanthine-7riboside-5'-N-methylcarboxamide (DBXRM, Figure 2), 20, having a Ki value of 229 nM at A3 receptors with 1G0-fold and >400-fold selectivity for A3 vs. A1 and A2A receptors, respectively. The selectivity of this compound is a result of incorporation of the 5'-methyluronamide group, found to enhance A 3 selectivity in IB-MECA, 2, and optimization of the alkyl chain length at positions 1 and 3. Unlike 1,3-dibutylxanthine-7-riboside, DBXRM acted as a full agonist in the rat A3 receptor-mediated inhibition of adenylyl cyclase. Thus, there was a tendency

159 towards the increase of efficacy as the affinity increased within the same series of compounds.

SITE-DIRECTED MUTAGENESIS AND MOLECULAR MODELING OF ADENOSINE R E C E P T O R S In addition to chemical probing of the binding sites of adenosine receptors, we have also used molecular biological approaches to determine structure function relationships for human A2A adenosine receptors [7,22]. Amino acid residues were mutated and the constructs expressed in COS-7 cells. Numerous residues in transmembrane domains 3, 5, 6 and 7, individually replaced with alanine and other amino acids, were identified as essential for ligand recognition. An immunological method [8] was used to determine whether the pharmacologically inactive mutant receptors were properly oriented at the cell surface and not simply retained in an intracellular compartment. Thus, an epitope tag was attached near the N-terminus of the receptor to allow detection with an antibody. This extra l 1-amino acid sequence did not interfere with ligand binding or adenylyl cyclase activation by the human A2A receptor. The pharmacological properties of mutant receptors were determined both m radioligand binding experiments and through stimulation of adenylyl cyclase. Specific binding of [3H]CGS 21680, 11, and [3H]XAC, 12, an A2A agonist and antagonist, respectively, was measured. Although A 1 selective in the rat, XAC has greatly enhanced affinity at human A2A receptors, and it is useful as a nonselective radiotracer in this species. High affinity binding of either agonist or antagonist was absent m the following Ala mutants: F182A, H250A, N253A, I274A, H278A and $281A, although they were well expressed in the plasma membrane. The hych'oxy group of $277 is required for high affinity binding of agonists, but not antagonists, since the $277A mutant bound [3H]XAC but not [3H]CGS 21680 similar to the ~dld type receptor. /h~ N181S mutant lost affinity for adenosine agonists substituted at the N 6 or C-2, but not at the C-5' position. The mutant receptors I274A, $277A, H278A showed full stimulation of adenylyl cyclase at high concentrations of CGS 21680. The functional agonist potencies at mutant receptors that lacked radioligand binding were >30-fold less than those at the wild type receptor. A molecular model based on the structure of rhodopsm was developed concurrently to mutagenesis studies in order to visualize the environment of the ligand binding site. We have found that the low resolution structure of rhodopsin serves as a more versatile template for G protein-coupled receptors (GPCRs) than the coordinates of bacteriorhodopsin [7]. The A2A receptor model was composed in steps including: construction and energ~ minimization of each helix individually, composition of the helical bundle based on consideration of

160

161 Figure 3 [upper left]. Vicinity of the bound ligand NECA in the rhodopsin-based molecular model of the human A2A receptor. Docking of the ligand was based on mutagenesis results, e.g. single amino acid substitution of the receptor, followed by energy minimization [7]. A putative pocket consisting of four aromatic residues shown m space f'filing form surrounds the adenine moiety of adenosine. Figure 4 [lower left]. Vicinity of the ribose moiety of NECA to hydrophilic residues of TM7 m the rhodopsin-based molecular model of the human A2A receptor [7]. Mutation to Ala of each of the residues shown in space filling form prevents the high affinity binding of [3H]CGS 21680.

Figure 5. Putative overlay of the agonist NECA, 8, and the antagonist XAC, 12, h~ the human A2A receptor model according to the 'N6/C8' hypothesis. Transmembrane helical regions are represented as ribbon structures. NECA was docked and minimized as above [7]. The amino group of XAC rests near the exofacial side of the receptor.

162 homology among GPCRs and rotation of amphipathic helices such that predominantly hydrophobic faces are pointed towards the lipid bilayer. Only the final step in construction of the model, i.e. docking of a ligand, NECA, took into account the results of mutagenesis. Thus, the receptor portion of the model, based only on computational methods and previous pharmacological structural insights into adenosine receptors [8,9] and other GPCRs, was highly predictive of which residues were potentially facing m the direction of the binding site. Most of the positions at which alanine mutation was not tolerated, were calculated to have site chains pointing into the binding cleft. This helped to define the placement of the ligand in the binding site. The current model takes into consideration only the contribution of the transmembrane helical domains, however a mutagenesis study by Olah et al. [24] has revealed that a portion of the second extracellular loop is also involved m antagonist recognition by A 1 receptors. Some of the residues targeted in this study may be involved in the direct interaction with ligands in the human A2A adenosine receptor. Four aromatic side chains in TM6 and TM7 appear to form a cluster in the receptor model that constitutes a putative binding pocket for the adenine moiety (Figure 3). The residues are H250, Y257, and F182 (all found to be essential by mutagenesis) and Y183 (not mutated in our study) [7]. H250 in TM6 appears to be a required component of a hydrophobic pocket, and H-bonding to this residue is not essential since the H250F mutant receptor binds radioligand like wild type. On the other hand replacement of H278 in TM7 with other aromatic residues was not tolerated in ligand binding, since hydrophilic groups at this position are required. According to the molecular model N253 is in proximity to form an Hbond with the exocyclic amino group of adenosine, h~ fact, the N253A mutant receptor did not bind either radioligand with high affinity. The xibose moiety appears to span a region of hydrophilic residues located in TM3 and TM7. Figure 4 shows the proposed interaction of the ribose moiety of NECA with hydrophilic residues of TM7. h~ our A2A adenosine receptor model the 5'-NH in NECA is hych'ogen bonded to $277 and H278. We have found that the T88A (TM3) mutant receptor binds antagonists like wild type receptors, but fails to bind agonists with high affinity [22]. In the biogenic amine receptors, an aspartate residue conserved in TM3 is essential as a counterion to the charged ammonium group of the ligand. In the adenosine receptors a Val residue occurs at the position of this Asp, and the essential T88 occurs roughly one helical turn closer to the cytoplasmic side of the helix. In the model, the ri.bose region is in proximity to T88. Certain mutant A2-x receptors have revealed differences in affinity shifts between agonists and antagonists, for example: $281N (agonists become more potent and antagonists less potent) and mutations of T88 (agonists become much less potent). Therefore, a partially overlapping set of amino acid residues

163 in the receptor are proposed to be involved in agonist vs. antagonist binding. We attempted to contrast the agonist and antagonist binding domains in the A2A receptor model. Based on SAR insights, the nitrogens of xanthines and adenosine derivatives, although both purines, certainly do not occupy similar coordinates in the receptor-bound state. At least three models for mapping of agonists onto antagonists have been proposed [25]. Extending this analysis to the entire receptor model clearly favors one of the three hypotheses, i.e. the 'N6/C8 ' model. In this hypothesis, the C-8 substituent of xanthines overlays the N 6 substituent of adenosine analogues. Figure 5 shows the relative orientation when the antagonist XAC is docked into our NECA-occupied receptor model according to this hypothesis, whereas in applying the 'flipped' hypothesis the C8substituent side chain of XAC protrudes into the helical segment (not shown), which is energetically untenable.

REFERENCES 1 Jacobson KA, van Galen PJM, Williams M. J Med Chem 1992; 35: 407-422. 2 von Lubitz DKJE, Lin RC-S, Popik P, Carter MF, Jacobson KA. Eur J Pharmacol 1994; 263:59-67. 3 von Lubitz DKJE, Jacobson KA In: Bellardinelli, L., Pelleg, A., Eds. Adenosine and Adenine Nucleotides: From Molecular Biology to Integrative Physiology; Kluwer: Norwell, 1995; 489-498. 4 Jacobson MA. In: Bellardinelli, L., Pelleg, A., Eds. Adenosine and Adenine Nucleotides: From Molecular Biology to Integrative Physiology; Kluwer: Norwell, 1995; 5-13. 5 van Galen PJM, Stiles GL, Michaels G, Jacobson KA. Med Res Rev 1992" 12:423-471. 6 Olah ME, Ren HZ, Ostrowski J, Jacobson KA, Stiles GL. J Biol Chem 1992; 267"10764-10770. 7 Kim J, Wess J, van Rhee AM, Sch6neberg T, Jacobson K. J Biol Chem 1995 270:13987-13997. 8 IJzerman AP, van Galen PJM, Jacobson KA. Drug Des Discov 1992; 9:49-67. 9 van Galen PJM, van Bergen AH, Gallo-Rodriquez C, Melman N, Olah ME, IJzerman AP, Stiles GL, Jacobson KA. Mol Pharmacol 1994; 45: 1101-1111. 10 Zhou QY, Li CY, Olah ME, Johnson RA, Stiles GL, Civelli O. Proc Natl Acad Sci USA 1992" 89:7432-7436. 11 Gallo-Rodriguez C, Ji XD, Melman N, Siegman BD, Sanders LH, Orlina J, Fischer B, Pu QL, Olah ME, van Galen PJM, Stiles GL, Jacobson KA. J Med Chem 1994" 37:636-646. 12 Kim HO, Ji XD, Siddiqi SM, Olah ME, Stiles GL, Jacobson KA. J Med Chem 1994 373614-3621.

164

13 Kim HO, Ji XD, Melman N, Olah ME, Stiles GL, Jacobson KA. J. Med. Chem. 1994; 37:4020-4030. 14 Jacobson KA, Ukena D, Padgett W, Kirk KL, Daly JW. Biochem Pharmacol 1987; 36:1697-707. 15 Poucher SM, Keddie JR, Singh P, Stoggall SM, Caulkett PWR, Jones G, Collis MG. Br J Pharmacol 1995; 115:1096-1102. 16 Linden J, Taylor HE, Robeva AS, Tucker AL, Stehle JH, Rivkees SA, Fink JS, Reppert SM. Mol Pharmacol 1993; 44:524-532. 17 Salvatore CA, Jacobson MA, Taylor HE, Linden J, Johnson RG. Proc Natl Acad Sci U S A 1993; 90:10365-10369. 18 Kim HO, Ji XD, Melman N, Olah ME, Stiles GL, Jacobson KA. J Med Chem 1994; 37:3373-3382. 19 IJzerman AP, van der Wenden EM, von Frijtag Drabbe Kiinzel J, MathSt RAA, Danhof M, Borea PA, Varani K. Naunyn Schmiedebergs Arch Pharmacol 1994; 350:638-645. 20 Siddiqi SM, Pearlstein RA, Sanders LH, Jacobson KA. Bioorg Med Chem 1995, 3:1331-1343. 21 Jacobson KA, Nikodijevic O, Shi D, Gallo-Rodriguez C, Olah ME, Stiles GL, Daly JW. FEBS Lett 1993; 336:57-60. 22 Jiang Q, van Rhee AM, Kim J, Yehle S, Wess J, Jacobson IC J Biol Chem 1995, submitted. 23 Siddiqi SM, Jacobson KA, Esker JL, Melman N, Tiwari KN, Secrist JA, Schneller SW, Cristalli G, Johnson CA, IJzerman AP. J Med Chem, 1995; 38:1174-1188. 24 Olah ME, Jacobson KA, Stiles GL, J Biol Chem 1994; 269:24692-24698. 25 van der Wenden EM, Price SL, Apaya RP, IJzerman AP, Soudijn W. J Comp Aided Mol Design 1995; 9:44-54.

Perspective in Receptor Research D. Giardin~,, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

C H E M I C A L AND P H A R M A C O L O G I C A L ADENOSINE RECEPTOR AGONISTS

165 P R O F I L E OF S E L E C T I V E

G. Cristalli, a E. Camaioni,b E. Di Francesco, b S. Vittorib and R. Volpinib aDipartimento di Scienze Farmaceutiche, Universi~ di Modena, 41100 Modena bDiparfimento di Scienze Chimiche, Universit~ di Camerino, 62032 Camerino, Italy INTRODUCTION There is evidence that the purine nucleoside adenosine specifically modulates neurotransmission through the interaction with four cell surface receptors recently classified as A1, A2a, A2b, and A3 [1 ]. These receptor subtypes have been cloned and characterized as belonging to the supeffamily of receptors with seven transmembrane helices that couple to G proteins [2]. Over the last decade a large number of adenosine analogs with high affinity and selectivity for A1 and A2a and, more recently, A3 receptors have been synthesized [3]. However, the existence of at least four AR subtypes necessitates the generation of more discriminating compounds which may ultimately have therapeutic use. More in detail, selective A I receptor agonists might be of therapeutical interest as antiarrhythmic and cardioprotective agents since: a) the cardiodepressant effects of adenosine appear to be important in the pathogenesis of arrhythmias in ischemic heart desease [4,5]; b) adenosine exerts Al-mediated cardioprotective action in preconditioning, a phenomenon by which one or more brief periods of ischemia result in improved functional recovery after substained ischemia [6,7]. Selective A2a receptor agonists have a potential for the treatment of cardiovascular disorders such as hypertension, ischemic heart desease, and atherosclerosis since it is known that stimulation of adenosine A2a receptors leads to vasodilation, inhibition of platelet aggregation, and neutrophil adhesion to vascular endothelium, and reduction in generation of oxygen free radicals by activated neutrophils [8,9]. Selective A3 receptor antagonists have potential use in the treatment of asthmatic and other allergic conditions, and may afford cardioprotection from prolonged ischemia [10]. In this paper our contribution both to the improvement of structure-activity relationships and to the development of A1 and A2a potent and selective agonists will be reported.

166

~~

N

.~ HO OH

HO OH 1-DeazaADO

ADO

C, N; X0-HO OH

1-DeazaCCPA

N

HO-~ HO OH CCPA

H O = ~ i~ HO OH CPA

Figure 1 CCPA We have developed interest in adenosine receptors by investigating the role of the purine nitrogens on adenosine activity. Several deaza analogues of adenosine were therefore synthesized and tested for affinity at A1 and A2a receptors in radioligand binding studies [11 ], and for their activity as inhibitors of human platelet aggregation [12]. We found that only 1-deazaadenosine (1-deazaADO) displayed appreciable aff'mity for adenosine receptors with a 25 fold A 1-selectivity, and presented moderate activity as inhibitor of human platelet aggregation induced by ADP (IC50 = 65 ktM). In a search for more potent and selective A1 adenosine receptor agonists, and taking in mind that at A1 receptor the most active analogues were N 6 substituted adenosines, a series of N6-cycloalkyl-1-deazaadenosine derivatives were synthesized and evaluated in radioligand binding studies and adenylate cyclase assays [13]. N6Cyclopentyl-2-chloro-1-deazaadenosine (1-deazaCCPA) showed the highest affinity at A1 receptor with more than two fold higher A1 selectivity than N 6cyclopentyladenosine (CPA).

167 Table 1 Effects of 1-deazaADO, 1-deazaCCPA, CCPA, and CPA on radioligand binding, and adenylate cyclase assays. Adenylate cyclase c Binding assay b Compd. a

A1 (Ki, nM)d

A2a (Ki, nM)d

A1 (IC50, nM)d

A2a (ECs0, nM)d

1-DeazaADO

115 (91-144)

2 900 (2400-3500)

6 7 00 (4100-11000)

18000 (11000-28000)

1-DeazaCCPA

1.6 13200 360 28000 (1.1-2.5) (10900-15900) (220-660) (20000-39000) CCPA 0.4 3900 33 3500 (0.2-0.7) (2500-4700) (22-51) (3200-3800) CPA 0.8 2000 58 2200 (0.6-1.0) (140o-2900) (24-140) (1500-320o) aThe structure of compounds is reported in Figure 1. bKi values were calculated from competition for [3H]PIA binding to rat brain membranes (A1 receptor), and for [3H]NECA binding to rat striatal membranes in the presence of 50 nM CPA (A2a receptor), cECs0 and IC50 values were calculated from the inhibition of rat fat cell adenylate cyclase (A1 receptor), and the stimulation of human platelet adenylate cyclase (A2a receptor), dData are means from at least three separate experiments; 95% confidence limits are in parenthesis [13,14]. The finding that the contemporary presence of a chlorine in 2-position and a cyclopentyl substituent on the N-6 amino group increased both A1 affinity and selectivity prompted us to synthesize the 2-chloro derivative of CPA, namely CCPA [141. In Table 1, the radioligand binding and the adenylate cyclase data referred to 1deazaADO, 1-deazaCCPA, CCPA, and CPA are reported. CCPA proved to be both more active and A 1-selective than the reference compound, showing A1 affinity in the subnanomolar range and a selectivity three fold that of CPA. CCPA is now available in the market also in the tritiated form, obtained by catalytic reduction in a tritium atmosphere of the corresponding cyclopentenyl derivative [15]. A detailed pharmacological profile of CCPA in binding, functional, and in vivo models has been recently reported by Monopoli et al. [16], and some data are shown in Table 2.

168 (,t:x~,)~,coox

Nx,

Nx,

1CH212 -HN-"~N/ " N" CH3-1CH213-'C--EtHNC~ EtHN~,~CO

EtHN~ HO

.,.

OH

HO

NECA

OX

CGS 21680

NH2

cx3-(cx2N~c EIXN~..~ HO OH

THENECA

NH2

EIHN~ HO OH

(R)-PHPNECA

HO

OH

HENECA

NH2

l~'~J EtHN~ HO OH

(S)-PHPNECA

Figure 2 I-IENECA As far as A2a receptor, over the last few years there has been a considerable effort directed toward both discovery and characterization of potent and selective agonists and antagonists of this subtype [17]. A major advance in the search of A2a adenosine agonists has been made with the introduction of C-2 substituents in the adenosine structure [18]. Selectivity and potency of the C-2 substituted adenosine analogs are greatly improved by replacing the 5'-hydroxyl group with other substituents, in particular with the N-ethylcarboxamide group. For example, the prototypical A2a agonist 5'-N-ethylcarboxamidoadenosine (NECA) showed little or no A2a selectivity [19] whilst [2-[4-(carboxyethyl)phenethyl]amino]adenosine-5'-N-ethylcarboxamide (CGS21680) is a moderately selective A2a agonist which is widely used as a reference compound in biochemical and pharmacological studies [20]. On this basis, we have reported the synthesis of the 2-hexynyl derivative of NECA, identified as HENECA, which showed high affinity at A2a adenosine receptors, and good A2a vs A1 selectivity. HENECA, in addition to having good A2a selectivity similar to that of CGS21680, shows higher inhibitory activity on platelet aggregation [21 ].

Table 2 Activity of some adenosine agonists on in vitro assays. Binding assaya Adenylate cyclaseb Compd. (fi,Me (EC50, n W e Rat brain Rat striatum Human PC12 cell A1 A2a platelet 130 2.2 14 4. I HENECA ( 1 16-145)

CGS21680

569 ( 5 1 1-634)

NECA

10.4 (9.4-1 1.5)

CCPA

I .3 (1.1-1.4)

(1.9-2.6)

11.0 (9.4- 12.9)

7.8 (6.6-9.1)

650 (555-762)

(13-15)

83 (7 8- 8 8)

Functional activityc (EC50,

Rat atria A1

>lOpM

(3.7-4.6)

72

(244-1460)

> 10pM

(66-78)

99

54.8

(82-1 16)

(30- 150)

1200

1200

(1000-1400) ( 1 170-1230)

115 (53-251)

( 1 10-112)

110

Rat aorta A2a 596

8.2

3 94

Antiaggregatory activityd (Ic50, nMIe Rabbit Human plateletf plateletb 70 50 (30- 150)

2160 (1620-3620)

210

(4 1-60)

820 (640-1 100)

3 60

(209-742)

(160-280)

(350-380)

>l o w

>lOpM

>lOpM

(4.4-15.3)

aReceptor binding affinity at A1 and A2a receptors was determined using (3HICHA and [3H]CGS21680 as radioligands, respectively [ 16,261. bECso values were calculated from the stimulation of PC12 adenylate cyclase and human platelet adenylate cyclase (A2a receptors) [ 151. CNegative chronotropic activity was studied on rat atria induced by cumulative addition of an agonist, and was recorded by measuring the isometric contractions: vasodilating effects were calculated on rat aorta precontracted by PGF2a (3 pM) [26]. dPlatelet aggregation was induced by ADP. eData are means from at least three separate experiments; 95% confidence limits are in parenthesis. ‘Data are from Sandoli et al. [24].

170 A detailed characterization of HENECA in comparison with CGS21680, NECA, and CCPA is reported in Table 2. In this Table, in addition to the binding data, the activity on the stimulation of adenylate cyclase in platelet and PC12 cells is shown. The results of the cyclase assays are in good agreement with the higher affinity of HENECA for the A2a receptor in comparison with the other adenosine agonists [22]. In the same Table the negative chronotropic activity in rat atria and the vasodilating activity in rat aorta of the four compounds are reported [23]: the profile of selectivity accounts for the higher chronotropic effect of CCPA in rat atria. The adenosine analogues were also evaluated as inhibitors of rabbit [24] and human [22] platelet aggregation induced by ADP. The ICs0 of NECA is about 200 nM in the rabbit, and about 350 nM in the human platelets. CCPA is a very poor inhibitor, according to its A1 selectivity; CGS21680 is less potent than NECA, while HENECA resulted from 3 to 7 times more potent than the model compound. Moreover, Dionisotti and coworkers evaluated in vivo rabbit platelet accumulation in response to ADP, given either before or after drug infusion. NECA, and, to higher extent, HENECA clearly decreased platelet accumulation in the rabbit. This antiaggregatory activity is of short duration, and does not appear to be due to hemodynamic changes, but rather is a result of selective drug action on platelet adenosine receptors [25]. In the same experiment the selective A1 adenosine agonist CCPA resulted inactive. The predominant effects on the cardiovascular system elicited by the same compounds in spontaneously hypertensive rats have been investigated by Monopoli et al. [ 16,26]. All the compounds induced a dose-dependent decrease in sistolic blood pressure. As expected, the effect of HENECA is due to vasodilation, and is accompanied by slight tachycardia. According to its A1 profile, CCPA markedly reduced heart rate and did not show any relevant vasodilating properties. The non selective agonist NECA induced bradicardia only at high doses. All tested drugs appear to penetrate into the brain. This is demonstrated by the general protection of pentylenetetrazole-induced lethal seizures in rats. HENECA showed protective effects at doses 10-fold higher than those active on the cardiovascular system, but the sleep-waking cycle was not influenced by the compound over the dose range examined. Conversely NECA and, to a lower extent CCPA, altered the sleep pattern mostly decreasing the REM sleep stage at high doses [16,26].

171 ALKYNYL AND ALKENYL DERIVATIVES OF NECA The therapeutic potential of HENECA for the treatment of cardiovascular diseases, and the need for more selective A2a agonists prompted us to synthesize a number of new 2-alkynyl derivatives of NECA bearing hydroxyl, amino, chloro, cyano, and heterocyclic groups or substituted aromatic or heteroaromatic tings in the side chain [27,28]. For the synthesis of the 2-alkynyl derivatives, three different approaches have been used, owing to different reactivity of the side chains or separative problems during purification. Briefly, from the common intermediate, the 5'-carboxyl-2',3'isopropyliden derivative, the two carboxamido derivatives I and 5 were obtained as starting material for the synthetic routes a-d depicted in Scheme 1. In any case the introduction of the alkynyl chain was carried out by a modification of the classical cross-coupling reaction catalysed by palladium. Moreover, some alkenyl and alkyl derivatives of NECA were also synthesized as classical modification of the triple bond [29]. In the case of the alkenyl derivatives, it is possible to design two geometric isomers (cis or Z, and trans or E). They were obtained by two different approaches illustrated in Scheme 1. The synthesis of Z-alkenyl derivatives were carried out by partial reduction of triple bond with the Lindlar catalyst (route f). The fully saturated derivatives were obtained starting from the same alkynes by using palladium on charcoal as a catalyst (route g). The synthesis of the E-alkenyl derivatives was accomplished by reaction of the corresponding alkynes with catecholborane followed by cross-coupling reaction of this product with the protected 2-iodoNECA. Deprotection in acidic medium gave the desired isomers (route a and b). The interaction of the new 2-alkynyl and 2-alkenyl derivatives of NECA with the adenosine receptors was evaluated using both radioligand binding technique and functional assays. The same compounds were also tested as inhibitors of platelet aggregation induced by ADP. The results of the binding experiments and antiaggregatory tests in the alkynyl series are reported in Table 3. From the binding data it is possible to draw some structure-activity relationships: -The presence of an o~-hydroxyl group in the alkynyl chain of NECA derivatives accounts for the A2a agonist potency, leading to compounds endowed with subnanomolar affinity in binding studies. However, these analogues also possess good A1 receptor affinity resulting in low A2a selectivity.

172 Scheme 1

NH2

N~ ' ~

N.H2

N

EtNH~~I~

N.H2

N~

(a)

N

R EtNHCO

N~

(b)

I

N

H--C=C.~NI~N~J HO

l

OH

3

(c)

NH2

Nx2 N~

N

N"~'"

EtNH~II~

NH2

N~ ' ~

N

.L. II ij R-C--C N,'~ z

HO

N

OH 59

EtNX~ HO OH 7-58

T NHz

(e)

NX2 ,.L-N i ~ Y

HO

OH 5

(a)

x

NH2 "

HO

OH

.'-(c

6

HO

OH 60

(a) Alkenyl catacholeborane derivatives, CH3CN, tetrakis, and K3CO3 at 90~ (b) CF3COOH or 50% HCOOH. (c) Alkynes, CH3CN or DMF, (Ph3P)2PdC12, CuI, and Et3N. (d) 1) Trimethylsylylacetilene, CH3CN or DMF, (Ph3P)2PdC12, CuI, and Et3N; 2) KOH. (e) Aromatic halide (Br or I), DMF, (Ph3P)2PdC12, CuI, and Et3N. (f) H2 18 psi, Lindlar catalyst. (g) H2 40 psi, 10% Pd/C.

173 -Aromatic or heteroaromatic tings conjugated to the triple bond conferred in general weaker activity and lower selectivity than aliphatic chains -Introduction of methylene groups between the triple bond and the phenyl ring markedly increased the A2a binding affinity resulting in compound 47 endowed with a 180-fold A2a selectivity, when three methylenes are present in the side chain (Table 4). -Reduction of triple to double bond led to compounds whose activity is strictly related to cis-trans isomerism, the trans isomers being more active and selective than the cis ones. Fully saturation of the side chain markedly reduced adenosine receptor affinity. The results of radioligand binding experiments performed on cis and trans isomers of 2-hexenylNECA, and on 2-hexylNECA are reported in Table 5. In comparison with HENECA, trans 2-hexenylNECA (THENECA, 3) showed similar aff'mity at AEa receptor, and a 3-fold higher selectivity for this subtype. Hence THENECA proved to be about 160-fold A2a selective with a Ki of 1.6 nM at A2a receptors. The antiaggregatory effect of the new alkynyl derivatives of NECA on rabbit platelet aggregation induced by ADP is reported in Table 3 as potency ratio calculated versus NECA. Some compounds resulted more potent than HENECA itself as inhibitors of platelet aggregation. All of them bear a hydroxyl group or a heteroatom in the side chain, and it is evident that introduction of a polar group in the side chain increased both affinity for A1 receptors and activity as platelet aggregation inhibitors. It is interesting to notice that PHPNECA (16) is about 15-times more active than NECA. More in detail, the most active compounds in the series present an a-hydroxyl group (Table 3, compds 11-13, 15, and 16), or a 5-hydroxyl group (10) which, however, may be so flexible to mimic the a-position. The importance of the hydroxyl group is stressed by comparing the three benzyl derivatives shown in Table 6. Introduction of an hydroxyl group on the methyl ct to the triple bond greatly improved the antiaggregatory potency in comparison with the first compound in the Table (45), resulting in the ct-hydroxybenzyl derivative PHPNECA (16), the most potent inhibitor in the series with IC50 of 13 nM. However, the contemporary amethylation markedly reduced the antiplatelet activity without affecting the A2a binding affinity, as shown by compounds 17 and 14 in Table 6. From these data it is clear that the hydroxyl group did not influence the A2a binding affinity while it is crucial for the antiaggregatory activity.

174 Table 3 In vitro pharmacological activity of 2-alkynyl derivatives of NECA. Compd.

Binding assay a (Ki, nM) b

R

Antiaggregatory activity (potency

Rat brain

Rat striatum

ratio vs NECA) c

A1

AZa

Rabbit platelets

6

H

35.4 (21.9-57.2)

57.8 (48.9-68.3)

0.30

7

CH2OH

14.1 (7.8-256)

9.1 (6.0-13.7)

2.30

8

(CH2)2OH

47.3 (42.8-52.4)

10.8 (9.8-12.0)

1.10

9

(CH2)aOH

99.9 (89.6-111)

11.3 (10.1-12.5)

2.20

10

(CH2)4OH

42.1 (39.9-44.5)

6.8 (6.0-7.7)

4.80

11

CH(OH~H3

11.1 (10.1-12.2)

7.6 (6.6-8.7)

4.70

12

CH(OH)CH2CH3

20.4 (18.5-22.6)

12.4 (10.8-14.2)

14.10

13

CH2CH(OH)CH3

69.6 (64.7-74.9)

56.4 (52.3-60.8)

3.20

14

C(OH,CHa)-[But

3.0 (2.5-3.6)

0.5 (0.4-0.6)

0.54

15

1-Hydroxycyclopentyl

4.0 (3.5-4.5)

0.6 (0.5-0.7)

5.30

16

CH(OH)Ph

2.5 (2.2-2.9)

0.9 (0.7-1.3)

15.70

17

C(OH, CH3)Ph

32.7 (29.7-35.9)

1.7 (1.6-1.8)

0.26

18

(CH2)2OPyr d

267 (191-375)

5.0 (4.8-5.2)

0.30

19

CH(OCH2CH3) 2

656 (515-835)

20

CH2NH2

48.6 (31.8-74.4)

38.3 (25.4-57.7)

0.40

21

CH2N(CH3)2

27.9 (25.0-31.1)

2.3 (2.2-2.4)

2.30

22

1-Aminocyclohexyl

39.4 (25.2-61.7)

0.60

23

(CH2)3C1

1.0 (o.8-1.2)

2.30

24

(CH2)aCN

184 (167-2o4)

4.7 (4.1-5.5)

2.10

25

1-Methylvinyl

167 (111-252)

27.3 (22.1-33.7)

0.66

26

1-Cyclohexen

3295 (3068-3538)

61.3 (55.5-67.6)

0.03

27

Cyclohexyl

236 (203-274)

9.2 (7.4-11.4)

0.66

28

Ph

698 (611-798)

29

p-PhCH3

500 (421-594)

36.3 (30.7-42.8)

0.013

30

p-PhCH2CN

1603 (1522-1690)

54.8 (49.0-61.4)

0.032

31

p-PhOCH3

414 (404-425)

52.4 (34.2-80.4)

0.004

398 (363-436) 37.9 (34.1-42.o)

441 (336-578)

120 (112-128)

0.02

0.010

175 Table 3 (Continued) 32

p-PhOH

497 (412-599)

20.6 (18.6-22.7)

0.006

33

p-PhNH2

599 (522-687)

113 (lO-123)

0.007

34

p-PhCF3

2432 (2308-2563)

315 (173-575)

0.01

35

p-PhF

3312 (3078-3563)

36

p-PhCONHe

7498 (6752-8325)

37

p-PhCOCH3

38

o-PhCHO

39

81.4 (63.5-104)

0.009

1500 (1411-1596)

0.003

187 (165-213)

0.019

6864 (6489-7261)

1158 (883-1518)

0.006

m-PhCHO

1435 (1127-1826)

176 (139-221)

0.004

40

p-PhCHO

1000 (911-1097)

6.3 (5.8-6.9)

0.110

41

p-PhNOe

900 (815-993)

21.5 (21.2-29.7)

0.014

42

p-Ph(CH)2COOtBu

>10~M

743 (698-792)

0.009

43

p-Ph(CH2)2COOH

>1000

>1000

0.008

44

1-naphtyl

488 (448-530)

0.003

45

CH2Ph

46

(CHE)2Ph

47

>10 gM

4463 (4077-4885)

1.6 (1.5-1.7)

0.01

448 (373-537)

7.0 (4.4-11.1)

0.19

(CHz)aPh

209 (194-226)

1.2 (1.1-1.4)

0.35

48

2-pyridyl

114 (100-131)

89.8 (79.7-101)

0.01

49

3-pyridyl

139 (116-167)

50

4-pyridyl

428 (372-492)

51

2-furyl

310 (267-359)

52

2-thienyl

597 (536-665)

53

2-thiazolyl

54

CH2-N-imidazolyl

55

CH2-N-piperidyl

56 57

27.4 (25.8-29.1)

234 (175-311) 87.0 (67-113) 130 (118-144)

0.06 0.11 0.01

19.5 (17.5-21.7)

0.023

41.3 (37.2-45.8)

0.44

16.5 (9.1-29.8)

3.5

27.5 (22.2-34.1)

4.3 (3.2-5.8)

4.7

CH2-N-piperazyl-4-CH3

35.9 (32.3-40.0)

19.1 (15.4-23.7)

nd

CH2-N-morpholyl

90.5 (77.7-105)

27.4 (15.5-48.2)

2.6

85.4 (80.3-90.9) 178 (166-191)

58 CH2-N-thiomorpholyl 52.8 (44.1-63.3) 5.9 (4.4-7.9) 2.9 aReceptor binding affinity at A1 and A2a receptors was determined using [3H]CHA and [3H]CGS21680 as radioligands, respectively, bData are means from at least three separate experiments; 95% confidence limits in parenthesis, cplatelet aggregation was induced by ADP. The potency ratio was calculated using the concentration of the test compound close to the IC50 value. In our experimental conditions the IC50 value for NECA was 0.2 gM; data are means from three separate experiments, dpyr = 2tetrahydro(2H)pyrane [27,28].

176 Table 4 Structure-activity relationships in some alkynyl derivatives Binding assay (Ki, nM) A1 A2a

Compd.

Selectivity A1/Aza

NECA-~C-(CH2)3-CH 3

130

28

NECA-C~C~

700

45

NECA-~C-CH 2~

27

1.6

17

46

NECA-C~C-(CH2)2 ~

450

7.0

64

47

NECA-C-':-=C(CH2)3~

210

1.2

175

2.2

60

120

Table 5 Structure-activity relationships in some alkynyl and alkenyl derivatives. Binding assay (Ki, nM) A1 A2a

Compd.

59

NECA- C~C-- (CH2)3-CH3

130

NECA..____~(CH2)3-CH3

950

H...L--I.=,,H

NECA..

~H

ir'~C~ (CH213-CH3

60

NECA-(CH2)5-CH3

250

3580

2.2

72

1.6

140

Selectivity A1/A2a 60

13

160

26

177 Table 6 Structure-activity relationships in some benzyl derivatives Compd.

~CH

45

16

A2a Binding assayb (Ki, nM)

Antiaggreg. activity c (IC50, nM)

2-

1.6

1100

H-

0.9

13

1.7

810

0.5

390

Ra

17 3 ox

14

CH3-CH-CH2-~CH3

aThe whole structure of compounds is reported in Figure 2 and in Scheme 1. bReceptor binding affinity A2a receptors was determined using [3H]CGS21680 as radioligand [27]. cplatelet aggregation was induced by ADP; data are means from at least three separate experiments. The IC5o values are calculated from the potency ratio listed in Table 3, the IC50 value for NECA is 0.2 ~tM. In conclusion, these findings give further support to the hypothesis that the A2 receptor on the platelets is not a typical A2a site. From our data it is possible to draw some structure-activity relationships for the platelet receptor: -The presence of the 5'-ethylcarboxamido group improved the activity in comparison with the corresponding adenosines. -The importance of the alkynyl chain in C-2 is strictly related to the substituents on the carbon in ot to the triple bond. -The introduction of polar groups in this position markedly increased the potency. -The contemporary presence of a lipophilic side chain improved the activity. -The fully substitution of the carbon in ~ to the triple bond led to much weaker inhibitors.

178 PHPNECA As already shown, the c~-hydroxybenzyl derivative PHPNECA is the most active

agonist which inhibits platelet aggregation. Since this compound bears a chiral carbon in the side chain, the enantiomeric resolution was undertaken to assess the enantioselectivity of A2a adenosine receptors. Separation of the two diastereoisomers was analytically achieved by chiral HPLC. To obtain the two isomers on a preparative scale, the suitable R and S alkynes were alternatively coupled to iodoNECA (5) in the usual cross coupling conditions to give the desired diastereoisomers S and R PHPNECA, respectively. The enantiomeric purity was assessed by analytical chiral HPLC, and by 1H NMR techniques. The binding affinity and the functional activity of the enantiomers in comparison with those of the racemic mixture are shown in Table 7. The S-diastereoisomer proved to be more potent than the R-diastereoisomer, and than the mixture as well, in all model systems. However, the most important achievement is that the platelet receptor clearly discriminates between the two compounds since R-PHPNECA is equiactive with NECA, whereas the S diastereoisomer shows an antiaggregatory potency about 30-fold that of NECA, i.e. about 7 nM, resulting in the most potent inhibitor so far known in the adenosine agonist derivatives [30]. Table 7 Preliminary in vitro activity of diastereomeric PHPNECA compounds. Binding assays b Functional activityc Antiaggr. activity (1 Compd. a

(Ki, nM) Rat brain Rat striatum

ECs0 (nM) Rat atria Rat aorta

A1

A~

A1

R-PHPNECA

6

2.6

85

132

200

S-PHPNECA

4

0.6

52

30

7

2.5

0.9

110

123

13

(R,S)-PHPNECA

A2a

(IC50, nM) Rabbit platelets A2

aThe whole structure of compounds is reported in Figure 2. bReceptor binding affinity at A1 and A2a receptors was determined using [3H]CHA and [3H]CGS21680 as radioligands, respectively.CNegative chronotropic activity induced by cumulative addition of an agonist was studied on rat atria, and was recorded by measuring the isometric contractions; vasodilating effects were calculated on rat aorta precontracted by PGF2a (3 btM) [27] dPlatelet aggregation was induced by ADP. dData are means from at least three separate experiments.

179 Acknowledgement This work was supported by grants from MURST, CNR, and Schering-Plough S.p.A. We also thank M. Brandi, F. Lupidi, and G. Rafaiani for technical assistance. The autors are involved in the concerted action ADEURO (EU, Biomed I). REFERENCES 1 Fredholm B, Abbracchio MP, Burnstock G, Daly JW, Harden TK, Jacobson KA, LeffP, Williams M. Pharmacol Rev 1994; 46 (2): 143-213. 2 Jacobson MA. In: Belardinelli L, Pelleg A, eds. Adenosine and Adenine Nucleotides: from Molecular Biology to Integrative Physiology. Boston: Kluwer Academic Publishers, 1995; 5-13. 4 Lerman BB, Belardinelli L. Circulation 1991; 83: 1499-1509. 5 Pelleg A. Coronary Artery Dis 1993; 4: 109-115. 6 Liu GS, Thornton J, van Winkle DM, Stanley AW, Olson RA, Downey JM. Circulation 1991; 84: 350-356. 7 Shen W-K, Kurachi Y. Mayo Clin Proc 1995; 70: 274-291. 8 Hori M, Kitakaze M. Hypertension 1991; 18: 565-574. 9 Cronstein BN. J Applied Physiol 1994; 76: 5-13. 10 Linden J. Trends Pharmac Sci 1994; 15: 298-306. 11 Cristalli G, Grifantini M, Vittori S, Balduini W, Cattabeni F. Nucleosides & Nucleotides 1985; 4: 625-639. 12 Antonini I, Cristalli G, Franchetti P, Grifantini M, Martelli S, Petrelli F. J Pharm Sci 1984; 73: 366-369. 13 Cristalli G, Grifantini M, Vittori S, Klotz K-N, Lohse MJ. J Med Chem 1988; 31: 1179-1183. 14 Lohse MJ, Klotz K-N, Schwabe U, Cristalli G, Vittori S, Grifantini M. NaunynSchmiedeberg's Arch Pharmacol 1988; 337: 687-689. 15 Lohse MJ, Klotz K-N, Schwabe U, Cristalli G, Vittori S, Grifantini M. NaunynSchmiedeberg's Arch Pharmacol 1989; 340: 679-683. 16 Monopoli A, Conti A, Dionisotti S, Casati C, Camaioni E, Cristalli G, Ongini E. Arzneim-Forsch/Drug Res 1994; 44 (12): 1350-1312. 17 van Galen PJM, Stiles GL, Michaels G, Jacobson KA. Med Res Rev 1992; 12: 423-471. 18 Matsuda A, Ueda T. Nucleosides & Nucleotides 1987; 6: 15-94. 19 Bruns RF, Lu GH, Pugsley TA. Mol Pharmacol 1986; 29: 331-346.

180 20 21 22 23 24 25 26 27 28 29 30

Hutchison AJ, Webb RL, Oei HH, Ghai GR, Zimmerman MB, Williams M. J Pharmacol Exp Ther 1989; 251: 47-55. Cristalli G, Eleuteri A, Vittori S, Volpini R, Lohse MJ, Klotz K-N. J Med Chem 1992; 35: 2363-2368. Cristalli G, Vittori S, Thompson RD, Padgett WL, Shi D, Daly JW, Olsson RA. Naunyn-Schmiedeberg's Arch Pharmacol 1994; 349: 644-650. Conti A, Monopoli A, Gamba M, Borea PA, Ongini E. Naunyn-Schmiedeberg's Arch Pharmacol 1993; 348: 108-111. Dionisotti S, Zocchi C, Varani K, Borea PA, Ongini E. Naunyn-Schmiedeberg's Arch Pharmacol 1992; 346: 673-676. Sandoli D, Chiu PJ S, Chintala M, Dionisotti S, Ongini E. Eur J Pharmacol 1994; 259: 43-49. Monopoli A, Conti A, Zocchi C, Casati C, Volpini R, Cristalli G, Ongini E. Arzneim-Forsch/Drug Res 1994; 44 (12): 1296-1304. Cristalli G, Volpini R, Vittori S, Camaioni E, Monopoli A, Conti A, Dionisotti S, Zocchi C, Ongini E. J Med Chem 1994; 37: 1720-1726. Cristalli G, Camaioni E, Vittori S, Volpini R, Borea PA, Conti A, Dionisotti S, Ongini E, Monopoli A. J Med Chem 1995; 38: 1462-1472. CristaUi G, Camaioni E, Vittori S, Volpini, Dionisotti S, Ongini E, Monopoli A. J Med Chem (submitted). Cristalli G, Camaioni E, Vittori S, Volpini R, Dionisotti S, Ongini E, Monopoli A. Biorg Med Chem (submitted).

Perspective in Receptor Research

D. Giardinh, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

181

Partial agonists for adenosine receptors A.P. IJzerman, E.M van der Wenden, H.C.P.F. Roelen, R.A.A. Math& and J.K. von Frijtag Drabbe Ktinzel Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, PO Box 9502, 2300RA Leiden, The Netherlands.

INTRODUCTION Adenosine is generally considered as a 'local hormone' with profound physiological activity. It is thought to mediate a large variety of effects, as diverse as vasodilation in the cardiovascular system, inhibition of lipolysis in fat cells and depression of neuronal activity in the CNS. Most of its effects are mediated by membrane-bound receptors, called Px-purinoceptors, of which currently three subclasses have been defined: A l, A: and A 3. All three classes have been cloned, and are coupled to the enzyme adenylate cyclase, A~ and A 3 adenosine receptors in an inhibitory, and A2 receptors, of which two further subtypes A2a and A2b exist, in a stimulatory fashion [1]. A great number of both A 1 and A2a selective ligands are available, both agonists and antagonists [2]. This is not yet the case for the A2b receptor. Very recently selective agonists for the A 3 receptor have been reported [3]. The ubiquity of adenosine receptors in mammalian tissues suggests a vast array of therapeutic options. For example, it has recently been found that stimulation of the Azb receptors on human blood cells leads to complete inhibition of TNF-~ release, a crucial feature in interfering with septic shock [4]. A drawback, however, to a possible therapeutic use of adenosine receptor agonists is the common hypotensive action of this class of compounds. 1000

14.0 120

T E E v G_ <

1O0

~ J

100

80 10 60

< & 9

40

20 4'

1'

0 -15

HO

Fig. 1.

OH

E u~ f-. v

,

,

i 0

,

,

.

.

.

.

.

.

15

30

time

(min)

.

.

0.1

. 45

60

Chemical structure, effect on mean arterial blood pressure ( M A P ; - ) and (CPA) concomitant blood concentrations (.) of N-cyclopentyladenosine 6 after a 5 min infusion (black bar) of 200 ~tg/kg to a freely moving rat.

182 In Fig. 1 the tremendous decrease in mean arterial blood pressure induced by the potent and A~-selective full agonist N6-cyclopentyladenosine (CPA) in an unrestrained, conscious normotensive rat is shown in relation to the blood concentrations of the compound [5]. The hypotensive effects of adenosine receptor agonists probably are of limited (commercial) use, in view of the overwhelming number of therapeutic strategies already in clinical practice. On the contrary, the potentially beneficial metabolic, antiarrhythmic and CNS effects also elicited by these compounds are confounded by the cardiovascular actions. Thus, the clinical development of an adenosine receptor agonist as a novel antipsychotic was discontinued due to its cardiovascular toxicity [6]. Partial agonists may potentially avoid these problems, since virtually all agonists synthesized so far are full agonists [7-9]. In general, the variation between tissues in the density and coupling efficiency o~' a given receptor may result in a differential expression of agonist activity amongst those tissues [10]. Conceptually, partial agonists for the adenosine receptor with less outspoken cardiovascular activity may be very useful in e.g., the treatment of arrhythmias. Also, receptor downregulation and desensitization, a common problem and drawback associated with the use of agonists for G protein-coupled receptors in general, and also demonstrated for both A~ [11] and A2a adenosine receptors [12], may be circumvented to some extent. As an example, partial agonists for 13-adrenergic receptors have met considerable success. Such compounds have been developed for the 131adrenergic receptor, displaying a positive inotropic action without the unwanted but usually concurrent positive chronotropy. The reference full agonist isoproterenol displays both actions [ 13].

HOW TO 'SCREEN' FOR PARTIAL AGONISTS

Radioligand binding studies are a rapid means to determine the affinities of reference and novel ligands for a receptor. It is less realised, however, that this technique can equally be applied as a protocol for the discrimination between agonists and antagonists. Therefore some attention in this chapter will be paid to this methodology and its biochemical background. Lefkowitz and coworkers, in their pioneering work on the 13-adrenergic receptor [14,15], demonstrated significant differences between agonists, partial agonists and antagonists in their behaviour in radioligand binding studies. The displacement of a radiolabelled antagonist by an agonist yielded shallow curves with slope factors or pseudo Hill coefficients (nil) less than unity. Curves with 'normal' steepness (nil = 1) were observed for antagonists. Computer-assisted analysis of the shallow curves showed that agonists recognise two sites on or states of the receptor, one with higher (KH), the other with lower affinity (KL). Subsequently it was shown that both the ratio KL/KH and the percentage of high affinity binding sites (%RH) correlated well with the intrinsic activity (as determined on the enzyme adenylate cyclase in the same membrane preparation) of a series of 13-adrenergic receptor agonists [14]. The addition of GTP to the assay yielded monophasic curves with affinities similar to K L. This latter finding suggested the involvement of G proteins which led to the development of the 'ternary complex' model [ 15]. It also led to the determination of the GTP shift as yet another parameter to identify potential partial agonists. This GTP shift is defined as the ratio of the agonist's apparent affinities determined in the presence and absence of GTP, and also correlates well with intrinsic activity [16].

183 Another way of discriminating between agonists and antagonists in radioligand binding studies is based on experiments at different temperatures. This thermodynamic analysis makes use of two formulas relating the change in Gibbs free energy AG ~ = AH ~ -

TAS

and A G o = -RT

llIg a

=

RT

lnK i

in which AG ~ represents the change in free energy, AH~ the change in enthalpy, AS~ the change in entropy, R the gas constant, K~ the equilibrium association constant, and ~ the equilibrium dissociation constant. Combination of these two equations yields: InK~ = ( A H

~

)(I/T )

-

AS ~

In such a thermodynamic analysis, ligand binding experiments are carried out at different temperatures. The values of AH~ AS~ and AG~ can be obtained from a Van 't Hoff plot of lnK i against 1/T. Thus, the temperature dependency of binding can be separated in enthalpy stabilisation, representing the formation of new bonds, and entropy stabilisation, associated with displacement of ordered water and hydrophobic interactions. Again, this approach was first tried on the 13-adrenergic receptor [17,18], allowing such a thermodynamic evaluation of binding data. For the 13-adrenergic receptor binding of agonists appeared mainly enthalpy driven, whereas antagonist binding was entropy driven. From later studies reviewed by Hitzemann [19] it appeared that these findings cannot be generalized to all G protein-coupled receptors. As an example entropy stabilisation of agonists was found for ~t-opiate receptors. In any case, partial agonists are expected to display an intermediate thermodynamic behaviour between agonists and antagonists for a certain receptor subfamily. Third, modulation of the affinity and activity of ligands by monovalent cations was demonstrated for several receptors linked to adenylate cyclase in an inhibitory or stimulatory way [20,21]. In particular, sodium ions inhibit agonist binding strongly, but may increase antagonist binding to some extent. These effects of Na § are presumably not mediated via G protein coupling, because guanine nucleotides and Na § can decrease agonist affinity synergistically. An aspartate residue in transmembrane helix II conserved in virtually all G protein-coupled receptors appears to be involved. The so-called Na § shift (the ratio of the agonist's affinities in the presence and absence of e.g., 100 mM Na § was shown to be correlated with the intrinsic activity of ligands [22], and can therefore be useful too in the in v i t r o determination of intrinsic activity.

APPLICATION RECEPTORS

OF

THESE

'SCREENING'

METHODS

TO

ADENOSINE

The sensitivity of adenosine receptors towards GTP was noticed soon after the introduction of the first radioligands [23-26]. The use of tritiated 1,3-dipropyl-8cyclopentylxanthine ([3H]DPCPX), a radiolabelled adenosine receptor antagonist with high AI receptor selectivity, high specific activity and low non-specific binding, facilitated the quantification of the GTP sensitivity. Despite different experimental conditions (species, tissues and guanine nucleotide concentrations) a remarkable unanimity emerged [27-29].

184 The displacement of the radioligand by full agonists, such as CPA and the R- and Sstereoisomers of N6-phenylisopropyladenosine (PIA), yielded shallow curves with ratio's of KL/KH between 25 and 50 and a percentage of high affinity states (RH) over 50. In the presence of GTP (100 ~tM - 1 mM) the displacement curves were shifted to the right with steeper appearance and most receptors apparently in the low affinity state. These data correspond to GTP shift values roughly between 5 and 10 for full agonists. Displacement of the radioligand by adenosine receptor antagonists was not affected by GTP, yielding (by definition) GTP shift values of 1. Consequently, shifts with values significantly lower than found for full agonists may be indicative for partial agonism. We have used this criterion in our studies, also to select compounds for in vivo evaluation. Murphy and Snyder were the first to study the temperature dependence of ligand binding to adenosine A I receptors [30]. They established that agonist binding is largely entropy driven, whereas enthalpy is the driving force for antagonists. This is typical for adenosine receptors, resembling the la-opiate receptors mentioned above. These results were corroborated by Lohse et al [26] who additionally found that the binding of agonists to the low affinity state of the A~ receptor resembled antagonist binding, i.e. driven by enthalpy. The thermodynamic differences between agonists and antagonists are so outspoken that they can be used to identify partial agonists. Elaborating on these principles Borea et al [31-33] tested series of agonists and antagonists, both on adenosine A x and A2a receptors. Unequivocally agonist binding was again governed by changes in entropy, whereas antagonist binding was enthalpy driven. In this way theophylline-7-riboside [34] and N6-methyladenosine [35] were identified as partial agonists for the adenosine A~ receptor. The discovery of theophylline-7-riboside will be outlined below. The effects of sodium ions on adenosine receptor binding have also been studied. Goodman et al noticed that at 150 mM NaC1 radiolabelled agonist binding to guinea pig membranes was decreased by 40% from control values, whereas virtually no change was observed for radiolabelled antagonist binding [36]. Green obtained comparable findings on rat hippocampal membranes, noticing an almost twofold increase in KD value at 200 mM NaC1 for the radiolabelled agonists (with little change in Bronx), again with negligible effect on radiolabelled antagonist binding [37]. On bovine brain membranes Na § effects appeared somewhat more outspoken, although they vanished when the membrane preparation was solubilised [38]. The synergistic effects of guanine nucleotides and sodium ions were also convincingly demonstrated in the latter study. It was concluded that guanine nucleotides facilitate a change in the equilibrium between the two distinct agonist states whereas the sodium ions decrease affinity of both states. When present simultaneously, they induce a new (very) low affinity state. This latter phenomenon was exploited to a full extent by Linden and coworkers who were able to obtain a 1000-fold difference in agonist affinities, whereas antagonist affinities changed only slightly [39].

THE DESIGN OF PARTIAL AGONISTS FOR ADENOSINE RECEPTORS

In this paragraph our endeavours to obtain partial agonists for adenosine receptors will be described. We followed two lines of reasoning that are based on the assumption that the ribose moiety is essential for agonist activity. This assumption has been validated on many occasions. As an example N6-cyclopentyl-9-methyladenine, i.e. the ribose in CPA substituted for a methyl group, is an adenosine receptor antagonist with modest affinity [40]. One approach was to start from an antagonist and 'add' a ribose moiety, eventually yielding xanthine-7-ribosides. The other was to use CPA and other N6-substituted

185 adenosines as the starting point and 'remove' hydroxyl functionality in the ribose group in a consecutive way. The rationale here was that in receptor modelling studies the 2'-, 3'and 5'-hydroxy groups appear to govern the interaction between the adenosine receptor (helix VII in particular) and the ribose moiety [41,42]. Also, 2',3'-dideoxy-N 6cyclohexyladenosine proved to be an antagonist on A 1 adenosine receptors [43]. xanthine-7-ribosides

With the aid of molecular modelling methods we have developed and compared models for the antagonist binding site of the receptor, based on steric, electrostatic and hydrophobic properties of various adenosine receptor antagonists [44,45]. In one of the favoured models theophylline binds to the adenosine receptor in a flipped orientation, i.e. the ring atoms N1, N3, N7 and N9 in adenosine coincide with C2, C6, N9 and N7, respectively, in theophylline (Fig. 2). This implicates that the domain where the ribose moiety of adenosine binds must be adjacent to N7 in xanthines.

N~~ N NH 2

I

0 4'

R I

R

RINT" H

T

I HO

OH

R = CH 3, C 3 H 7, C 4 H 9

[ I

HO

OH

Figure 2. Chemical structures of adenosine (left), xanthines (middle) and xanthine-7ribosides (right) used in this study.

Subsequently, we synthesized the 7-ribosides of theophylline, 1,3-dipropylxanthine and 1,3-dibutylxanthine [46]. The I~ values at 25 ~ of the three xanthines, the corresponding xanthine-7-ribosides and the reference full agonist R-PIA were obtained in radioligand binding studies with the tritiated agonist N6-cyclohexyladenosine (CHA) on rat brain membranes. The results are summarized in Table 1. Next, we performed radioligand binding studies at various temperatures to obtain thermodynamic parameters (AG~ AH~ and AS~ that characterize the binding process. Van 't Hoff plots for the xanthines and their 7-ribosides are shown in Fig. 3, from which we derived the thermodynamic parameters, also gathered in Table 1. From this table it is obvious that theophylline-7-riboside has values for both AH~ and AS~ that are intermediate between the full agonist R-PIA and the xanthine antagonists. The other two xanthine-7ribosides resembled antagonists more. This thermodynamic analysis with the radioliabelled agonist appeared more sensitive than the determination of GTP shifts with the radiolabelled antagonist [3H]DPCPX due to the wider range of values. The GTP shifts for

186 the xanthines as well as the xanthine-7-ribosides were not significantly different from unity, except for theophylline-7-riboside on calf, not rat, brain tissue (1.6 +_.0.1).

Table 1. Thermodynamic equilibrium parameters for displacement of [3H]CHA by xanthines, xanthine-7-ribosides and R-PIA. Results are from three independent experiments performed in duplicate. AG~ AH~ and AS~ values were calculated from ~ values (S.E.M. < 15%, see also In K A values in Fig. 3) determined at different temperatures. 7-r: 7riboside Compound

I~, 25 ~ ([3H]CHA)

theophylline-7-r dipropylxanthine-7-r dibutylxanthine-7-r theophylline dipropylxanthine dibutylxanthine R-PIA

25 I

AG~ (KJ/mol)

45 _ 5 ~/I -24.7 23 _ 3 pM -26.4 5.9 _ 0.8 laM -29.7 11 __. 2 HM -28.2 0.51 +_ 0.06 laM -35.7 0.43 _+ 0.05 plVl -36.2 0.60 _+ 0.03 nM -52.0

20

10

I

I

T(~

0

9

1615<:

3r

.5

14~31211-

-----"0----

1043-

93.3

i

!

3.t.

3.5 lIT

( 103/K )

i

3.6

AH~ (KJ/mol)

AS~ (J/mol/degree)

9.0 - 14.4 - 13.0 -30.3 -35.4 -32.9 30.1

113.1 40.4 56.3 -7.3 1.0 10.8 276.0

Figure 3. Van 't Hoff plots showing the effect of temperature on the equilibrium association constants, KA, of both xanthines ( l : theophylline; O: 1,3-dipropylxanthine; x: 1,3dibutylxanthine) and xanthine-7ribosides (I-q: theophylline-7riboside; o: 1,3-dipropylxanthine-7-riboside; zx: 1,3dibutylxanthine-7-riboside). All plots were essentially linear (r > 0.95). Reproduced with permission of principal author and publisher of ref. [34].

Based on these findings, theophylline-7-riboside was selected for in vivo studies. In Fig. 4 the effects of an intravenous infusion of theophylline-7-riboside (80 mg/kg rat) in 5 minutes are represented. In pilot studies it was found that this dosage yielded maximal effects. Infusion of the vehicle alone, despite the high concentration of DMSO present, did not change the baseline heart rate and blood pressure significantly. The decrease in MAP (Fig. 4a) observed with theophylline-7-riboside, although substantial from 100 mm Hg to

187 70 mm Hg, is less than observed with reference full agonists (from approx. 110 to 40 mm Hg for CPA on A~ or from 110 to 70 mm Hg for 2-p-(2-carboxyethyl)phenethylamino-5'N-ethylcarboxamidoadenosine (CGS 21680) on m2a receptors, respectively; data not shown).

..-...

140

500

T

E 120

r~-

co

-

~"

"I

T,

100

~

40o

O. 80

g E

3oo

6O

40 -5

'

'

' 30

0

t~me

'

' 45

'

'

200 60

I - 15

(rain)

.

.

.

. 0

~ 1.5 time

,

,

,. 30

,

,

, 45

,

, 60

(rain)

Figure 4. Effect of a 5 min infusion of theophylline-7-riboside (80 mg/kg) in unrestrained, conscious and normotensive rats on a) mean arterial pressure and b) heart rate. Results are the means (+_ SE) of data obtained in 3 rats. Rats were observed during 5.5 h after administration, with no changes in parameters after 1 h. Reproduced with permission of principal author and publisher of ref. [34].

The rise in heart rate (Fig. 4b) observed within the first minutes of infusion is probably best described as a reflex tachycardia. The same phenomenon has also been observed with the Aza-selective full agonist CGS 21680. The latter compound, however, reached peak values (data not shown) as high as 550 beats per minute (bpm), contrasting to the mean highest value for theophylline-7-riboside of 425 bpm. CPA in the same experimental setup caused a decrease in heart rate to as low as 150 bpm. Thus, theophylline-7-riboside behaved as a partial agonist on parameters that are strictly A1 receptor mediated (heart rate) or both Al and A 2 (mean arterial pressure). In conclusion, theophylline-7-riboside, developed from the antagonist theophylline, may be a useful tool as a partial agonist for adenosine receptors. Its low affinity is a potential drawback, which warrants the development of other compounds for which theophylline-7-riboside may be a lead.

deoxyribose analogues of N-substituted 6 adenosines In this paragraph the contribution to affinity and intrinsic activity of the hydroxyl functions in the ribose moiety are explored. Taylor et al [47] reported that deoxy analogues of R-PIA were less potent than the parent compound in both in vitro and in vivo studies, without discriminating between affinity and intrinsic activity. We first synthesized the N6-cyclopentyl -, cyclohexyl- and R- and S-phenylisopropyl analogues of 2'- and 3'deoxyadenosine (Fig. 5).

188 N.H2

Figure 5. Synthesis of N 6substituted deoxyadenosine analogues.

.NH2 O

O

pyridine

(DMAP)

X2 Xl

Y2 Y1 CH212 hv

R'-NH

H

I

4

RNH2 ethanol

X2 X1 a: b:

~NO

X1 = H and X2 = OH X1 = OH and X2 = H

~ ~ AcO-~ Y2 Y1 a: Y1 = H and Y2 = OAc b: Y1 = OAc and Y2 = H

The route described in the literature for the synthesis of deoxy analogues of R-PIA is not very convenient: it involves a multistep deoxygenation of R-PIA, which leads to a mixture of the 2'- and 3'-deoxy analogues [47]. We used a practical short route to convert 2'- and 3'-deoxyadenosine into their N6-substituted analogues. The route of synthesis, summarised in Fig. 5, included a three-step reaction: protection of the hydroxyl groups, reductive deamination and combined amination and deprotection. The non-optimised overall yield of the products was approximately 30%. The second step was used to replace the N6-amino group by a better leaving group by treating it with isopentylnitrite to give a diazo group, and substituting it by an iodo substituent with diiodomethane. We tried this reaction first with acetonitrile as solvent and a small excess of the reactants, as described by Nair et al for 2-amino substituted purines [48] but this gave very low yields. We found that the yield is much higher if the reaction is carried out with the two reactants, isopentylnitrite and diiodomethane, in excess as solvent, similar to the reaction described by Nair et al with n-pentylnitrite [49]. The use of tungsten lamps with this reaction led to an approximately doubled yield of our product after two hours. However, we are not certain whether the improved yield is due to the light itself by increasing the rate of the proposed radical reaction, or by an additional heating of the mixture by the 150 W tungsten lamps close to the flask. The last step in the synthesis of the N6-substituted deoxy analogues, the combined deprotection and amination, had to be carried out at ambient temperature for approximately two weeks. Refluxing was not possible for the deoxypurineribosides, due to decomposition of the products at higher temperatures. For adenosine itself, however, this step in the reaction could easily be carried out by refluxing the mixture for several hours. These compounds and the 5'-deoxy analogues of CPA and R-PIA (obtained as gifts) together with three reference compounds were tested in vitro for their affinity for the adenosine A~ and A2a receptors, and their GTP shift on the A~ receptors (Table 2).

189 Table 2 Adenosine A~ (~-values in presence and absence of GTP and GTP shifts) and affinities of the N6-substituted deoxy analogues.

A2a receptor

Ligand

GTP shift

I~ A2ab

6.0_+1.5

0.58_+0.12

0.062_+0.004 14.7_+2.8

5.5+_2.5 1.2-+0.4

0.14+_0.01 8.60+_0.56

7.60 (7.18-8.01) 22.8 (22.5-23.1) 14.4 (14.0-14.7)

4.1_+1.2 2.6_+0.8 1.7+_0.6

-

-

CPA

I~ A1 - GTP ~

(rtM)

I~ A x + GTP ~

(0M)

0.00590

0.0352

(0.00578-0.00602)

(0.0297-0.0407)

(~tM)

R-PIA 9-methyladenine

0.011+_0.005 12.5+_2.9

2'-deoxy-CPA 2'-deoxy-CHA 2'-deoxy-R-PIA 2' -deoxy-S-PIA

1.87_+0.52 8.94+_2.77 8.45+_2.26

3'-deoxy-CPA 3'-deoxy-CHA 3'-deoxy-R-PIA 3'-deoxy-S-PIA

0.11_+0.03 0.31_+0.11 0.35-+0.06 8.26_+2.25

0.47_+0.04 1.26_+0.16 1.01_+0.12 17.5_+2.8

4.3_+1.2 4.1__.1.5 2.9_+0.6 2.1_+0.7

18.6_+6.8 22.1-+6.4 7.36-+0.53 57.2_+9.7

5'-deoxy-CPA 5'-deoxy-R-PIA

0.07__.0.02 0.30-+0.08

0.45___0.02 1.07_-+0.09

6.4_+1.9 3.6_+1.0

10.1 _+0.4 3.67-+0.08

(91%)*

(88%)* (90%)* (86%)* (91%)*

[3H]DPCP X binding on rat brain membranes b [3H]CGS21680 binding on rat striatal membranes - not determined. * percentage radiolabelled ligand bound to the receptor in the presence of 10.5 M ligand (n=l). a

Apparently, removal of the 2'-hydroxy group led to the more substantial drop in A1 receptor affinity (300-800 fold), whereas the 3'- and 5'-deoxy derivatives retained significant affinity for this receptor subtype (10-30 fold decrease). A similar pattern was observed for the Aza receptor. The GTP shifts of the deoxy analogues range between those found for reference full agonists (5.5 - 6.0) and antagonists (9-methyladenine: 1.2), with values between 6.4 and 1.7. The hydroxy groups affected affinity and GTP shifts in a similar way. Affinity and GTP shifts tend to be low for the 2'-deoxy derivatives, intermediate for the 3'-deoxy analogues, whereas the 5'-deoxy analogues reflect the behaviour of full agonists. CPA and its deoxy analogues were further used in in vivo experiments [50]. The purpose was again to establish the relationship between blood concentration and the effect on heart rate in conscious, normotensive rats for the four compounds. The pharmacokinetics and pharmacodynamics were determined after a single intravenous infusion of each substance by monitoring the reductions in both heart rate (Fig. 6) and blood pressure (data not shown) and taking serial arterial blood samples for analysis of drug concentration.

190

m

AA

400 v

300

~

200

100 0.3

,

1

~

10

'

100

~

1000

10000

blood concentration(ng/ml)

Figure 6. Blood concentration - heart rate relationship of four individual rats which had received an intravenous infusion of 20 mg/kg 2'deoxyCPA (-), 1.2 mg/kg 3'-deoxyCPA (11), 0.80 mg/kg of 5'-deoxyCPA (v) and 0.20 mg/kg of CPA (o) over 15 min. Solid lines represent the sigmoidal Emax model fitted to the data [51]. The no-drug response (E0) values were fixed.

In Table 3 the averaged pharmacodynamic parameters for the heart rate of 6 - 7 rats are summarized. For the bradycardic effects the ECs0.u values (based on free drug concentrations, i.e. not bound to plasma proteins) were equivalent to 1.6 nM, 19 nM, 56 nM and 830 nM for CPA, 5'-deoxyCPA, 3'~deoxyCPA and 2'-deoxyCPA, respectively. The Emax values of 2'- and 3'-deoxyCPA were lower than those of the other compounds. ECs0 and ECs0.u were significantly different between the four compounds. The in vivo potencies (ECs0.u) of the compounds correlated well with their apparent adenosine A~ receptor affinities as determined in the absence of GTP (Table 2). With respect to intrinsic activity a fair correlation between relative GTP shifts and Emax values was observed. The 2'- and 3'-deoxy analogues of CPA proved partial agonists in vitro and in vivo.

Table 3 Pharmacodynamic parameter estimates yielding E 0 (no-drug response), Emax (maximum effect), ECs0 (blood concentration inducing half-maximal effect), ECso,u (ECs0, corrected for plasma protein binding) and N (Hill factor). Analysis of the reduction in heart rate was according to the sigmoidal Emax model [51] after intravenous infusion of CPA, 5'deoxyCPA, 3'-deoxyCPA and 2'-deoxyCPA to conscious normotensive rats. Values are means +__SE. compound

n

CPA 5'-deoxyCPA 3'-deoxyCPA 2'-deoxyCPA

6 7 7 7

Eo (bpm) 355 368 349 338

_ 13 _+ 14 +_ 17 _+. 18

Emax (bpm) -208 -215 - 167 -65

_+ 19 +_ 31 -!-_16 +__12

ECs0 (ng/ml) 1.8 +_.0.5 19 -4-_ 6 48 +_ 8 650-4-160

ECs0,u (ng/ml) 0.54 5.9 18 260

-4-0.15 _ 1.7 __. 4 _+. 70

N

1.4 1.4 1.4 2.3

-4-_0.4 _+ 0.3 _+ 0.3 +_ 0.6

191 CONCLUSIONS According to two lines of reasoning we have designed and synthesized partial agonists for the adenosine receptor. With theophylline-7-riboside we were able to convert a reference antagonist (theophylline) into a partial agonist. By making deoxy analogs of a reference full agonist (N6-cyclopentyladenosine) we also succeeded in obtaining partial agonists. For the compounds studied a good correlation was observed between in vitro and in vivo parameters. It should be kept in mind, however, that intrinsic activities as determined in the present study are both drug and tissue dependent. Nevertheless it appears that for the adenosine A 1 receptor the GTP shift and/or the thermodynamic parameters AH~ and AS~ are useful indicators of cardiovascular partial agonism in vivo.

REFERENCES

9 10

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

Jacobson MA. In: Belardinelli L, Pelleg A, eds. Adenosine and adenine nucleotides: from molecular biology to integrative physiology. Boston: Kluwer, 1995; 5-13 Jacobson KA, Van Galen PJM, Williams M. J Med Chem 1992; 35: 407-422. Kim HO, Ji X, Siddiqi SM, Olah ME, Stiles GL, Jacobson KA. J Med Chem 1994; 37: 3614-3621. a) Jacobson MA, Chin J, Salvatore CA, Johnson RG, Kostura MJ. Drug Dev. Res. 1994; 31: 282. b) Thiel M, Chouker A, Dieterich HJ, Brosig A, Bardenheuer HJ. Drug Dev. Res. 1994; 31: 328. Math6t RAA, Van Schaick EA, Soudijn W, Breimer DD, IJzerman AP, Danhof M. J. Pharmacol. Exp. Ther. 1994; 268: 616-624. Metz AL, Dominick MA, Suchanek G, Gough AW. Toxicol Pathol 1991; 19: 98107. Ukena D, Olsson RA, Daly JW. Can J Physiol Pharmacol 1987; 65:365-376 Pirovano IM, IJzerman AP, Van Galen PJM, Soudijn W. Eur J Pharmacol - Mol Pharmacol Sect 1989; 172:185-193 Daly JW, Padgett WL. Biochem Pharmacol 1992; 43:1089-1093 Kenakin TP. In: Pharmacologic analysis of drug-receptor interaction, 2nd ed. New York: Raven, 1987; 455-459. Parsons WJ, Stiles GL. J Biol Chem 1987; 262: 841-847. Webb RL, Sills MA, Chovan JP, Peppard JV, Francis JE. J Pharm Exp Ther 1993; 267: 287-295. Barlow JJ, Main BG, Snow HM. J Med Chem 1981; 24: 315-322. Kent RS, De Lean A, Lefkowitz RJ. Mol Pharmacol 1980; 17: 14-23. DeLean A, Stadel JM, Lefkowitz RJ. J Biol Chem 1980; 255:7108-7117. Ehlert FJ. Mol Pharmacol 1985; 28: 410-421. Weiland GA, Minneman KP, Molinoff PB. Nature 1979; 281: 114-117. Weiland GA, Minneman KP, Molinoff PB. Mol Pharmacol 1980; 18: 341-347. Hitzemann R. TIPS 1988; 9:408-411. Pert CB, Snyder SH. Mol Pharmacol 1974; 10: 868-879. Horstman DA, Brandon S, Wilson AL, Guyer CA, Cragoe EJ Jr, Limbird LE. J Biol Chem 1990; 265: 21590-21595. Tsai BS, Lefkowitz RJ. Mol Pharmacol 1978; 14: 540-548. Bruns RF, Daly JW, Snyder SH. Proc Natl Acad Sci USA 1980; 77: 5547-5551.

192 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

Goodman RR, Cooper MJ, Gavish M, Snyder SH. Mol Pharmacol 1982; 21: 329335. Yeung SMH, Green RD. J Biol Chem 1983; 258: 2334-2339. Lohse MJ, Lenschow V, Schwabe U. Mol Pharmacol 1984; 26: 1-9. Bruns RF, Fergus JH, Badger EW, Bristol JA, Santay LA, Hartman JD, Hays SJ, Huang CC. Naunyn-Schmiedeberg's Arch Pharmacol 1987; 335: 59-63. Lohse MJ, Klotz KN, Lindenborn-Fotinos J, Reddington M, Schwabe U, Olsson RA. Naunyn-Schmiedeberg's Arch Pharmacol 1987; 336: 204-210. Math6t RAA, Van den Aarsen BCFM, Von Frijtag Drabbe Ktinzel JK, Danhof M, IJzerman AP. Naunyn-Schmiedeberg's Arch Pharmacol 1994; 350: 109-112. Murphy KMM, Snyder SH. Mol Pharmacol 1982; 22: 250-257. Borea PA, Varani K, Malaguti V, Gilli G. J Pharm Pharmacol 1991; 43: 866-868. Borea PA, Varani K, Guerra L, Gilli P, Gilli G. Mol Neuropharmacol 1992; 2: 273-281. Borea PA, Dalpiaz A, Varani K, Guerra L, Gilli G. Biochem Pharmacol 1995; 49: 461-469. IJzerman AP, Van der Wenden EM, Von Frijtag Drabbe Ktinzel JK, Math6t RAA, Danhof M, Borea PA, Varani K. Naunyn-Schmiedeberg's Arch Pharmacol 1994; 350: 638-645. Borea PA, Varani K, Dalpiaz A, Capuzzo A, Fabbri E, IJzerman AP. Eur J Pharmacol - Mol Pharmacol Sect 1994; 267: 55-61. Goodman RR, Cooper MJ, Gavish M, Snyder SH. Mol Pharmacol 1982; 21: 329335. Green RD. J Neurosci 1984; 4: 2472-2476. Stiles GL. J Neurochem 1988; 51: 1592-1598. Linden J, Patel A, Earl CQ, Craig RH, Daluge SM. J Med Chem 1988; 31: 745751. Ukena D, Padgett WL, Hong O, Daly JW, Daly DT, Olsson RA. FEBS Lett 1987; 215:203-208 IJzerman AP, Van Galen PJM, Jacobson KA. Drug Design and Discovery 1992; 9: 49-67 IJzerman AP, Van der Wenden EM, Van Galen PJM, Jacobson KA. Eur J Pharmacol - Mol Pharm Sect 1994; 268:95-104 Lohse MJ, Klotz K-N, Diekmann E, Friedrich K, Schwabe U. Eur J Pharmacol 1988; 156:157-160 Van Galen PJM, Van Vlijmen HWT, IJzerman AP, Soudijn W. J Med Chem 1990; 33:1708-1713 Van der Wenden EM, IJzerman AP, Soudijn W. J Med Chem 1992; 35:629-635 Van Galen PJM, IJzerman AP, Soudijn W. Nucleosides & Nucleotides 1990; 9: 275-291 Taylor MD, Moos WH, Hamilton HW, Szotek DS, Patt WC, Badger EW, Bristol JA, Bruns RF, Heffner TG, Mertz TE. J Med Chem 1986; 29:346-353 Nair V, Turner GA, Buenger GS, Chamberlain SD. J Org Chem 1988; 53: 30513057 Nair V, Richardson SG. J Org Chem 1980; 45:3969-3974 Math6t RAA, Van der Wenden EM, Soudijn W, IJzerman AP, Danhof M. Br J Pharmacol, in press Danhof M, Mandema JW, Hoogerkamp A, Math6t RAA. J Drug Metab Pharmacokin 1993; 18:41-47

Perspective in Receptor Research D. Giardin~, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

193

N e w h i s t a m i n e H 3 r e c e p t o r l i g a n d s as p h a r m a c o l o g i c a l tools R. Leurs and H. Timmerman Leiden/Amsterdam Center for Drug Research Department of Pharmacochemistry, Vrije Universiteit De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

INTRODUCTION In various brain areas neuronal histamine release and synthesis is regulated presynaptically by histamine H3 receptors [1]. Recent studies reveal that H3 receptors not only act as autoreceptors to regulate the release and synthesis of histamine [2, 3], but also modulate the release of other neurotransmitters, like acetylcholine [4], serotonin [5], dopamine [6] and noradrenaline [7]. In view of these widespread modulatory activities of the histamine H3 receptors, important roles for this receptor subtype in the various mammalian brain functions have been indicated [8, 9]. Consequently, selective ligands for the H 3 receptor have been suggested to be benificial in the treatment of e.g. epilepsia, Alzheimer disease, sleeping and attention-deficit disorders [10, 11, 12, 13, 14, 15, 16]. Concomitant with the increased knowledge on the role of H 3 receptors in (patho)physiological processes medicinal chemists have been developing highly selective ligands for the H 3 receptor [17, 18, 24]. In 1987 Arrang et al. introduced (R)a-methylhistamine and thioperamide as respectively a highly selective H 3 receptor agonist and antagonist [20]. Recently a large number of new histamine H 3 receptor ligands have been developed by several laboratories, resulting in the availability of a variety of H 3 receptor agonists and antagonists. These agents proved to be very useful for a further pharmacological definition of the H 3 receptor and for the development of new H 3 receptor radioligands as research tools [21].

H 3 r e c e p t o r agonists Potent agonists for the H 3 receptor are obtained by simple modifications of the histamine molecule. The imidazole ring is very important for H 3 agonistic activity [ 17]. Methylation of one of the nitrogens or replacements with other aromatic ring systems are not tolerated [22]. In contrast, subtle changes of the ethylamine side chain have proven to be very useful [ 17].

194

NH2 NH,,,,~ N

~ NH,,~N

histamine ~~.~ NH,,,~ N

S NH imetit

NH2 H CH3

(R)-a-methylhistamine

/

NH

\

H immepip

Figure 1. Structures of histamine and several potent H 3 receptor agonists

Branching of the ethylamine side chain can be very favourable for H 3 receptor agonistic activity [20, 23, 19]. A methyl group at the a-position drastically increased the potency at the H 3 receptor [20, 23]. This increased activity _almost completely resided in the R-isomer (Fig. 1) [20, 23]. In combination with its less active S-isomer (R)-a-methylhistamine is very useful for the pharmacological characterization of H 3 receptor-mediated effects. It was observed by various laboratories that for potent H 3 receptor agonism the protonated amine function of hist_~mine may be replaced by other protonated groups [25, 26, 27, 28]. This observation resulted in the development of the potent and highly selective H 3 receptor agonist imetit (Fig. 1) [25, 26, 27]. With the introduction of an isothiourea group a full and highly potent H 3 receptor agonist was obtained [25, 26, 27]. As for (R)-a-methylhistamine, imetit is active in low doses in vivo [25] and has become an important pharmacological tool for the H 3 receptor. Comparing the structures of histamine and imetit it is apparent that the distance between the imidazole ring and the protonated group on the sidechain are different. Whereas in histamine the distance between the ring system and the protonated amine function is 2-methylene groups (ca 4.5 A) the distance between the imidazole moiety and the charged isothiouronium group of imetit is much larger (ca 8,~). To obtain more information on the optimal location of the postively charged group relative to the imidazole group we recently prepared a

195 series of histamine analogues in which we incorporated the amino group in various ring structures. For one of these compounds (4(5)-lH-imidazolylmethylpiperidine, immepip, VUF 4708) the alkyl side chain was extended to four methylene groups, by the incorporation of the amino function in a piperidine ring (Fig. 1), resulting in a distance of ca 7.5A between the amino group and the imidazole ring [28]. Immepip is an highly effective H 3 receptor agonist in the guinea-pig intestine [28]; a pD 2 value of 8.0 was established for immepip, w h e r e a s for (R)-amethylhistamine a pD 2 value of 7.8 was observed.

F r o m i m e t i t to p o t e n t H 3 antagonists Previously H 3 receptors in the brain have been succesfully labelled with tritiated H 3 agonists. With the initial description of (R)-a-methylhistamine as a selective agonist for the H 3 receptor, Arrang et al. (1987) introduced the tritiated analogue as a radioligand for the H 3 receptor [20]. Binding of [3H]R-(a)methylhistamine to membranes of rat cerebral cortex is reversible and saturable [20, 29]. The binding site was pharmacologically characterized as an H 3 receptor. As observed in the histamine release experiments, the [ 3 H ] R - ( a ) methylhistamine binding was stereoselectively inhibited by the enantiomers of amethylhistamine [29]. Inhibition of the binding by burimamide appeared however to be complex; shallow competition curves with a Hill coefficient of 0.48 were observed [29]. Moreover, whereas the affinity of the antagonists corresponded rather well with their potency in the histamine release assay, agonists showed a 5- to 10-fold higher activity in the [3H]R-(a)-methylhistamine binding assay. This last finding may readily be explained by the selective labelling of the high affinty state of the G-protein-coupled receptor. Also [3H]Na-methylhistamine has been evaluated as a radiolabelled probe for the H 3 receptor. Since this compound has been labelled to a high specific activity, this radioligand provides a higher sensitivity than [3H](R)-a-methylhistamine. Competition binding studies revealed that [3H]Na-methylhistamine labels H 3receptors in guinea-pig brain [30]. In the rat cerebral cortex studies with [3H]Namethylhistamine [31, 32] resulted in biphasic displacement curves for the H 3 antagonists thioperamide and burimamide. This observation led West et al. (1990) to suggest the existence of H3A- and H3B-receptor subtypes, which, according to their hypothesis, are responsible for respectively the observed H 3 receptor mediated inhibition of histamine release and the H 3 receptor mediated inhibition of histamine synthesis. This subclassification was based on the biphasic displacement with thioperamide (I~ ratio 13) and burimamide (I~ ratio 36) [32] and the observation of a small difference of the thioperamide potency (8fold) against (R)-a-methylhistamine effects in either the histamine release and synthesis assay [2, 20]. For e.g. burimamide (5-fold) and impromidine (4-fold)

196 small differences in the potencies in these two assays are noticed as well [20]. Yet, for these ligands the affinities for the putative H3A- and H3B-receptor subtypes (burimamide I~ ratio 36; impromidine t~ ratio 1) do not follow the differences in potencies in the two functional assays [31, 32]. Despite the complex binding characteristics, the use of the radiolabelled agonists has provided i m p o ~ t information on the presence of H3-receptors in the brain [30, 33] and peripheral tissues, like guinea-pig lung, intestine and pancreas [30]. To resolve the complexity related to the use of agonists as radioligands a search for a radiolabelled antagonist was initiated. Already in the first paper on the H 3 receptor in 1983 relatively potent H 3 receptor antagonists were identified [22]. Both the H 2 agonist impromidine and the H 2 receptor antagonist burimamide displayed H 3 receptor antagonism [22] and these ligands were in the early years of the H 3 receptor pharmacology usefull tools. In 1987 Arrang et al. introduced the prototype H 3 receptor antagonist thioperamide. Despite an affinity in the nanomolar range so far no succesfull reports on the use of thioperamide (analogues) as H 3 receptor radioligand appeared. In search for new and potent H 3 receptor antagonists we noticed that methylation of the isothiourea moiety or lengthening the alkyl sidechain of the H 3 agonist imetit gave rise to moderate H 3 receptor antagonism [27]. After a detailed exploration of these findings, clobenpropit was introduced as one of the most potent H 3 receptor antagonist known at this time (pA2 = 9.9) [27]. Within the same series of compounds an iodonated analogue, iodophenpropit, was also identified as a potent H 3 receptor antagonist (pA 2 = 9.1) and subsequently radiolabelled [34]. The resulting [125I]iodophenpropit was the first radiolabelled H 3 receptor antagonists that was suitable for the detection of the H 3 receptor protein in rat cerebral cortex [35, 36].

197

NH

NH,,,,,~N

NH imetit (pD2 = 8.7)

HNx,,~N

Cl

clobenpropit (pA2 = 9.9)

HN,,,,~N [12Sl]iodophenpropit (pKi = 9.2) Figure 2. Structures of imetit and the related H 3 receptor antagonists clobenpropit and iodophenpopit. Between the parentheses the H 3 receptor activity at the guinea-pig intestine or the affinity for the H 3 receptor in rat brain is indicated.

It shows saturable, readily reversible, high affinity binding to rat cortex membranes. Inhibition of the [z25I]iodophenpropit binding by various H 3 agonists results in GTPyS-sensitive, shallow, biphasic competition curves, indicating the coupling to G-proteins (Fig. 3) [35, 36]. GTP~-insensitive, biphasic displacement by some H 3 receptor antagonists was also observed (burimamide and dimaprit), but, in contrast to the studies with [3H]Na-methylhistamine, for thioperamide only monophasic displacement curves were obtained [35, 36]. These results further question the H3A-H3B subtype extension on the basis of [3H]N amethylhistamine binding, but also reinforce the observation of complex receptorligand interactions. Definite proof for the hypothesis that these complex binding data reflect receptor heterogeneity ultimately should however come from the demonstration of the presence of distinct H 3 receptor gene products or, more classically, from the comparison of drug potencies in different functional studies.

198

120t

!

,

I

,

C~ e-

m

~D E: .O

80 ~

o

6O

m

o l tl--

,

I

,

I

i

I

I

? 1 ~M GTP T S

t

m

.

B

control

o m

L~

O. o~

I

4O

Kill= 4 nM .

K iL = 0.2 pM

20'

0

-11

!

-10

'

I

-9

A

'

I

'

I

'

I

-8 -7 -6 Iog[immepip]

'

"1"

-5

i

-4

Figure 3. Displacement of the specific [125I]iodophenpropit binding to rat cerebral cortex membranes by immepip in the absence or presence of 1 pM G T P ~ . Data were taken from Jansen et al. [36].

Recently also another radiolabelled H 3 antagonist, in which an ether function has been incorporated as polar group in the side chain, has been described [37]. The [125I]iodoproxyfan labels with high affinity the H 3 receptor in rat brain; the binding can be displaced by various H 3 agonists for approximately 60% [37]. Yet, most H 3 antagonists fully displaced the [125I]iodoproxyfan binding [37]. Apparently [125I]iodoproxyfan labels with high affinity a secondary site in rat striatum; most H 3 antagonists also show a high affinity for this site [37], complicating the analysis of antagonist displacement curves.

F r o m i m m e p i p to p o t e n t H 3 a n t a g o n i s t s ; the i d e n t i f i c a t i o n of impent~mine As mentioned before, the distance between the imidazole ring and the amino function in histamine and the H a agordst immepip is quite different (Fig. 1). Following this observation we recently synthetized a new series of histamine homologues which only differed in the length of the alkylamineo sidechain [38].

199 Decreasing or increasing the alkyl sidechain of histamine with one methyl group completely eliminates the H 3 agonistic activity at the guinea-pig jejunum. In contrast, lengthening the sidechain of histamine results in H 3 antagonistic activity. The optimal sidechain length for H 3 antagonism of the histamine homologues at the guinea-pig jejunum appeared to be five methylene groups [38] (Fig. 4); this analogue VUF 4702, impentamine, has a pA 2 of 8.4 at the H 3 receptor of the guinea-pig jejunum. Further increasing the sidechain length resulted in a diminished H 3 antagonistic activity [38]. Comparing the agonist immepip with a cyclic sidechain of four methylene groups and the corresponding histamine homologue with the linear sidechain (Fig. 4) the difference in pharmacological activity at the guinea-pig jejunum is striking. Apparently there are very strict structural demands for H 3 agonism in this preparation. Therefore, immepip and/or (cyclic) histamine analogues can be of great use for a computational chemistry approach of H 3 receptor agonists.

120

NH ,,,,,,~N

/--\ NH,~~

/ NH.~~

immepip

~ N H 2 VUF 4701 (PA2 = 7.7)

\~ N H 2 VUF 4702 (pA2 = 8.4)

C O "~_.

=

I

~

1

=

I

~

I

,

1

'

I

100 -

"F, 8 0 .

9 I

60

"~ 4O E o

20

o

-9

-8

!

I

-6 -7 log [RAMH] (tool/i)

"

'

-5

I

-4

Figure 4. Structures of newly developed histamine analogues. The H 3 receptor activity at the guinea-pig jejunum is indicated between parentheses. The doseresponse curves for (R)-a-methylhistamine (RAMH), as measured as the inhibition of electrically-induced neurogenic contractions of the guinea-pig jejunum, in the absence or presence of three different concentrations of impentamine (VUF 4702) are shown.

I m p e n t a m i n e acts as H 3 r e c e p t o r a g o n i s t in the b r a i n Following our observation of the potent S 3 receptor antagonism of the

200 histamine homologues in the guinea-pig intestine, we extensively investigated these new H 3 ligands in other H 3 receptormodels [39]. This is specifically of importance, since these compounds do not contain an (iso)thiourea moiety and therefore might be of a potential therapeutic value (reduced risk of toxicity). Initially we determined the affinity of impentamine for the H 3 receptor in the rat brain, using [125I]iodophenpropit.

0s - e~

120-

o

100-

o

8O

m

60

o

~

,

c

mm

c

o--

.Q 0

o Q.

cn

40200

r

-12

I

'

I

'

I

-10 -8 -6 log [impentamine]

'

I

-4 (mol/l)

'

I

-2

Figure 5. Displacement of the binding of [125I]iodophenpropit to rat cerebral cortical membranes by impentamine in the absence (filled circles) and presence of 10 ~ GTP~S. Data were taken from Leurs et al. [39].

A biphasic displacement of the [125I]iodophenpropit binding to rat cerebral cortex was observed (Fig. 5), but in contrast to the biphasic displacement of the H 3 antagonists burimamide and dimaprit, the displacement curve of impen~mine was shifted rightwards after the inclusion of GTP~S (Fig. 5). As mentioned before the GTP~S-induced shift of the displacement curve was previously also found for several H 3 receptor agonists (Fig. 4). Our results for the [125I]iodophenpropit binding suggested that in rat brain tissue impentamine was acting as a H 3 agonist. We therefore investigated impentamine in a functional assay in mouse brain [39]. In this model system [3H]noradrenaline release after electrical stimulation

201 can be inhibited for approximately 70% by several H 3 agonists (Fig. 6). As expected for a H 3 receptor mediated effect the [3H]n0radrenaline release was stereoselectively inhibited by the two isomers of a-methylhistamine. However, impentamine was also able to inhibit the [3H]noradrenaline release from mouse brain slices with a high potency (pD 2 = 8.2) (Fig. 6). The partial inhibition of the n e u r o t r a n s m i t t e r release by impentamine was rightward shifted by the H 3 antagonist clobenpropit, yielding an apparent pA 2 value of 9.4. This value closely corresponds to the expected values for clobenpropit at the H 3 receptor [31].

110-

0 Q

IO0-

o

90

>

~ "0 Q

80 c 0

-~o

70

Q

>'6

60

-~

5O

"=

40

0

o o Q

I,U

30

,

-10

'

-g

-8

-7

-6

I

-5

log [agonist] (mol/I)

'

1

-4

J

t

-3

Figure 6. Inhibition of the electrically evoked [3H] noradrenaline release from mouse brain slices by (R)- (filled squares) and (S)-a-methylhistamine (open squares) and impentamine in the absence (open circles) or presence (closed circles) of 10 nM clobenpropit. Data were taken from Leurs et al. [39].

subtypes? Within a series of closely related histo_m~ne analogues we have thus identified several H 3 receptor ligands with different pharmacological characteristics. Small modifications of the alkylamine side chain have a great impact on the H 3 agonistic activity at the guinea-pig jejunum. I m p e n t a m i n e , with a pentyleneamine sidechain, is a highly potent H 3 antagonist at the guinea-pig jejunum. In contrast, the same compound appears to be a H 3 agonist in the rat H 3 receptor

202 and mouse brain. These data provide prelimnary evidence for the existence of p u t a t i v e H 3 receptor subtypes (Table 1). However, before a definite subclassification of the H 3 receptor can be accepted a careful investigation of the pharmacological profile of several impentamine analogues is needed. Moreover, the identification of distinct H 3 receptor genes would provide ultimate proof. Yet, sofar no molecular biological information of the H 3 receptor is available.

Table 1 S u m m a r y of the H 3 receptor activities of the H 3 agonist (R)-a-methylhistamine, the H 3 antagonist thioperamide and the new agonist/antagonist impentamine [39]. compound

guinea-pig [3H]-NA release intestine mouse brain

(R)-a-methylhistamine pD 2 = 7.8

pD 2 = 7.9

impentamine

pA2 = 8.4

pD 2 = 8.2 (a = 0.6)

thioperamide

pA2 = 8.9

pA2 = S.4

[125I]IPPbinding rat cortex PitH = 8.46 PilL = 5.92 pI~.H = 8.50 PilL = 6.60 =S.37

Acknowledgement The work of Dr Leurs has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Schwartz JC, Arrang JM, Garbarg M, Pollard H, Ruat M. Physiol Rev 1991; 71: 1-51. Arrang JM, Garbarg M, Schwartz JC. Neurosci 1987; 23: 149-157. Van der Werf JF, Bast A, Bijloo GJ, Van der Vliet A, Timmerman H. Eur J Pharmacol 1987; 138: 199-206. Clapham J, Kflpatrick GJ. Br J Pharmacol 1992; 107: 919-923. Fink K, Schlicker E, Neise A, GSthert M. Arch Pharmacol 1990; 342:513519. Schlicker E, Fink K, Detzner M, Gothert M. J Neural Transm 1993; 93: 1-10. Schlicker E, Fink K, Hinterthaner M, GSthert M. Arch Pharmacol 1989; 340: 633-638.

203 8. Timmerman H. J Med Chem 1990; 33: 4-11. 9. Van der Werf JF, Timmerman H. Trends Pharmacol Sci 1989; 10: 159-162. 10. Barnes JC, Clapham J, Dennes RP, Kilpatrick GL, Marshall FH, O'Shaughnessy CT, Cavicchini E. Soc Neurosci Abstr 1993; 19: P1813. 11. Lin JC, Sakai K, Louvet M. C R Acad Sci 1986; 303: 469-474. 12. Lin JS, Sakai K, Jouvet M. Eur J Neurosci 1994; 6: 618-625. 13. Lin JS, Sakai K, Vannimercier G, Arrang JM, Garbarg M, Schwartz JC, Jouvet M. Brain Res 1990; 523: 325-330. 14. Smith CPS, Hunter AJ, Bennett GW. Psychopharmacology 1994; 114: 651656. 15. Yokoyama H, Onodera K, Iinuma K, Watanabe T. Eur J Pharmacol 1993; 234: 129-133. 16. Yokoyama H, Onodera K, Maeyama K, Sakurai E, Iinuma K, Leurs R, Timmerman H, Watanabe T. Eur J Pharmacol 1994; 260: 23-28. 17. Leurs R, Timmerman H. Prog Drug Res 1992; 39: 127-165. 18. Leurs R, Van der Goot H, Timmerman H. In: Testa B, ed. Advances in Drug Research, Vol. 20. London: Academic Press, 1991; 217-304. 19. Lipp R, Arrang JM, Garbarg M, Luger P, Schwartz JC, Schunack W. J Med Chem 1992; 35: 4434-4441. 20. Arrang JM, Garbarg M, Lancelot JC, Lecomte JM, Pollard H, Robba M, Schunack W, Schwartz JC. Nature 1987; 327: 117-123. 21. Leurs R, Smit MJ, Timmerman H. Pharmacol Ther 1995; 66: 413-463. 22. Arrang JM, Garbarg M, Schwartz JC. Nature 1983; 302: 832-837. 23. Arrang JM, Schwartz JC, Schunack W. Eur J Pharmacol 1985; 117: 109114. 24. Lipp R, Stark H, Schunack W. In: Schwartz JC, Haas HL, eds. The Histamine Receptor. New York: Wiley-Liss, Inc., 1992; 57-72. 25. Garbarg M, Arrang JM, Rouleau A, Ligneau X, Tuong MDT, Schwartz JC, Ganellin CR. J Pharmacol Exp Ther 1992; 263: 304-310. 26. Howson W, Parsons ME, Raval P, Swayne GTG. Bioorg Med Chem Lett 1992; 2: 77-78. 27. Van der Goot H, Schepers MJP, Sterk GJ, Timmerman H. Eur J Med Chem 1992; 27:511-517. 28. Vollinga RJ, De Koning JP, Jansen FP, Leurs R, Menge WMPB, T~mmerman H. J Med Chem 1994; 37: 332-333. 29. Arrang JM, Roy J, Morgat J, Schunack W, Schwartz JC. Eur J Pharmacol 1990; 188: 219-227. 30. Korte A, Myers J, Shih N, Egan R, Clark M. Biochem Biophys Res Commun 1990; 168: 979-986.

204 31. Kathmann M, Schlicker E, Detzner M, Timmerman H. Arch Pharmacol 1993; 348: 498-503. 32. West RE, Zweig A, Shih N, Siegel MI, Egan RW. Mol Pharmacol 1990; 38: 610-613. 33. Kilpatrick GJ, Michel AD. In: Timmerman H, Van der Goot H, eds. New Perspectives in Histamine Research. Basel: Birkh~iuser Verlag, 1991; 69-75. 34. Menge WMPB, Van der Goot H, Timmerman H, Eersels JLH, Herscheid JDM. J Labelled Comp Radiopharm 1992; 31: 781-786. 35. Jansen FP, Rademaker B, Bast A, Timmerman H. Eur J Pharmacol 1992; 217: 203-205. 36. Jansen FP, Wu TS, Voss HP, Steinbusch HWM, VoUinga RC, Rademaker B, Bast A, Timmerman H. Br J Pharmacol 1994; 113: 335-362. 37. Ligneau X, Garbarg M, Vizuette ML, Diaz J, Purand K, Stark H, Schunack W, Schwartz JC. J Pharmacol Exp Ther 1994; 271: 452-459. 38. Vollinga RC, Menge WMPB, Leurs R, Timmerman H. J Med Chem 1995; 38: 266-271. 39. Leurs R, Kathmann M, Vollinga RC, Menge WMPB, Schlicker E, Timmerman H. J Pharmacol Exp Ther 1996; in press.

Perspective in Receptor Research D. Giardin~t, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

Three-dimensional structure f r o m s p e c u l a t i o n s to facts.*

205

of G p r o t e i n - c o u p l e d r e c e p t o r s :

M. Hibert a, J. Hoflack a, S. Trumpp-Kallmeyer a, J.-L. Paquet a, R. Leppik a, B. Mouillac b, B. Chini b, C. Barberis b, S. Jard b aMarion Merrell Dow Research Institute, Strasbourg, France.

16 rue d'Ankara, 67000

bCCIPE, rue de la Cardonille, 34000 Montpellier Cedex.

INTRODUCTION G protein-coupled receptors (GPCR) represent very important targets for drug design in most therapeutic areas. More than 200 different GPCRs have been cloned and expressed over the last 8 years, allowing a better characterization of their structure and function. We review here briefly the biochemical features of GPCRs and the three-dimensional model of their complex with different ligands. The experimental validation of these models and their putative usefulness for drug design are discussed.

GPCRs : TARGETS FOR DRUG DESIGN G protein-coupled receptors belong to the same functional and structural protein family. These receptors are found in cell membrane (Figure 1). They are activated upon binding of an extracellular endogeneous ligand. This most probably induces a receptor conformational change leading to the activation of an intracellular protein, the G protein. The G protein is a heterotrimer which dissociates into alpha and beta-gamma subunits upon activation. The alpha subunit can subsequently interact with ion channels or enzymes to generate intracellular second messengers. Overall, GPCRs act as selective signal transmitters and amplifiers. GPCR endogenous ligands present a striking structural diversity (Figure 1). The challenge for the medicinal chemist is to understand how molecules as different as small cationic neorotransmitters, small cyclic or non-cyclic peptides, proteins and glycoproteins can interact with structurally related receptors.The access to a three-dimensional structure of GPCRs would represent a major step towards the understanding of receptor structure-function and ligand structure-activity relationships. Since access to an experimental 3D structure of a GPCR still remains a challenge, homology protein modelling is currently the only alternative strategy to gain some structural insight into receptor activation and function.

206 ~F

HORMONE

~kA

Io. IArach.

Second

G protein

PAF GABA

glutamate cyclic AMP pheromone M prostaglandin thromboxane

IP G

PEPTIDE

SMALL MOLECULE

adrenaline acetylcholine dopamine histamine serotonin adenosine cannabinol

Efl'ector

ACTH

angiotensin II bradyklnin CCK endothelin N formyl peplide born besin substance P substance K neuromedin neurotensin

Messengers

PROTEIN NPY

opiold oxytocin parathyroid hormone secrelln somatostatln vasopressin

VIP

TRH thrombln

C5A chemotaclic ca,atonln glucagon

SENSORY

olfactory opsins

IL--8

melanotrop|n

TSH FSH LH

Figure 1 9G protein-coupled receptors and some of their ligands.

T H R E E - D I M E N S I O N A L M O D E L S OF G P C R s Structural information derived from sequence analysis, chemical labelling, fluorescence, circular dichroism, accessibility to enzymes and antibodies, ligand structure-activity relationship, chimeric receptors, site-directed mutagenesis, etc.. has been taken into consideration to propose generic 2D and 3D models of GPCRs. In a first step, such studies had led to the approximate localisation of the ligand binding site (transmembrane region) and of the G protein coupling domain [1,2]. The next step consisted in proposing a more detailed 3D model. This has been achieved as follows [3,4]. The reference protein chosen is bacteriorhodopsin. This protein shows no sequence or functional homology with GPCRs. However, it presents an activation s y s t e m very similar to mammalian opsins (which are GPCR members) and contains seven transmembrane, a-helical domains as postulated for GPCRs [5]. A low resolution (10 A) crystallographic structure of bacteriorhodopsin [5] has been used as a scaffold to position the seven ahelical domains of GPCR relative to each other. For a given receptor, each of the seven hydrophobic stretches of aminoacids was built as an a-helix with the SYBYL programme and energy minimized with AMBER [6] and TRIPOS [7] force fields. The seven helices have then been assembled using bacteriorhodopsin as a template. The relative orientation of helices fulfills three independent criteria : the helix hydrophobic surface points to the outside, towards the hydrophobic membrane environment ; the most conserved residues are found in the core

n

Figure 2 : Three-dimensional model of acetylcholine in the muscarinic m l receptor.

Figure 3 : Three-dimensional model of vasopressin in the VIA vasopressin receptor.

208 of the protein ; the ligand binding residues converge to the inside of the protein. Loops were built with the loop search programme in SYBYL. The whole structure has then been extensively energy minimized without or with ligands. More recently, the model has been refined (eg Figures 2 and 3) in order to obtain a perfect match between the theoretical projection map of the model and the experimental projection map of bovine opsin, the first crystallographic data related to a GPCR [8]. The optimised model differs from the original one by the relative tilt of transmembrane a-helices. However the two models are qualitatively similar. These models led to the proposal of a number of novel hypotheses : 9The activation site of GPCR appears to be buried in the structure at about 15 .~ from the extracellular surface of the receptor. 9 The putative binding sites of several important neurotransmitters have been described [3,4] (Figures 4-6). For instance, residues likely to interact directly with dopamine, adrenaline, serotonin, histamine, acetylcholine, adenosine, substance P, TSH, etc.., have been identified [3,4,9-11]. 9All endogenous ligands (small or huge, cationic or not, peptidic or not) can interact with a homologous region of the receptor transmembrane domain. 9 A number of hypotheses regarding the receptor activation mechanisms and its dynamics have been proposed [3,4]. The proposed model was in good qualitative agreement with the existing experimental results. However, the next crucial step was to further support this model with new experiments and to examine their predictive value.

FROM SPECUI~TION

TO FACTS: E X P E R I M E N T A L V A L I D A T I O N

Four years after the first disclosure of GPCR 3D models, it is interesting and necessary to examine their relevance and level of predictability in the light of more recent experimental results. Briefly : 9 The predicted backbone architecture is in very good agreement with the first crystallography data on a GPCR (bovine opsin) published recently [8]. GPCR have indeed seven alpha-helical transmembrane domains, bundled very similarly to the bacteriorhodopsin transmembrane domain. 9 The depth of the binding site of small cationic ligands is at least 11 .~ as predicted and as demonstrated by fluorescence quenching experiments [12]. 9 The binding site of a number of neurotransmitters has been probed by site directed mutagenesis. More than 100 mutants have been studied [13]. The observed shifts in ligand affinity are in most cases in good qualitative agreement with the models. 9 Structure-activity relationships are in good agreement with the 3D models and have been better understood [14-18]. Although one should be cautious in interpreting binding affinity shifts in terms of direct ligand-protein interaction changes, site directed mutagenesis remains the easiest way to probe a model. We have u n d e r t a k e n to probe the proposed models of GPCR in exchanging systematically residues predicted to be responsible for ligand binding [1922]. We decided to probe two classes of GPCRs: receptors of cationic

209

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/

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3

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TT,~,,~ 6

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N---El/_ H'--.;er4o6I

5

4

3

Pro615 TrP613 6

Figure 4 : S c h e m a t i c r e p r e s e n t a t i o n of predicted n e u r o t r a n s m i t t e r binding sites : A : d o p a m i n e ; B : a d r e n a l i n e ; C : acetylcholine ; D : serotonin. Helix axes are r e p r e s e n t e d as v e r t i c a l lines and n u m b e r e d 1 to 7. R e s i d u e s s u r r o u n d i n g t h e n e u r o t r a n s m i t t e r are s c h e m a t i c a l l y d i s p l a y e d a n d n u m b e r e d in a coded m a n n e r : the first digit indicates the t r a n s m e m b r a n e domain an the next two digits correspond to the rank of the residue in this domain (reproduced w i t h the a u t h o r i s a t i o n of the authors and publishers, from Medecine/Science 1993, 9, 31-40.).

210 neurotransmitters and receptors of neuropeptides. Hence, the binding of acetylcholine to the human m l muscarinic receptor has been studied. A schematic model of the acetylcholine binding site had been proposed (Figure 2 and 4). Most amino acids in the putative direct neighbourhood of the ligand have been mutated. The binding affinity of a number of agonists, partial agonists and antagonists for these mutants has been measured. The binding shifts observed have already been reported [19] and suggest comments important for structure-activity relationship understanding and drug design. In brief: 9 Agonist, partial agonist and antagonist binding is generally affected by mutations around this region of the receptor while it is not by similar mutations in other regions. This indicates that the considered cholinergic ligands bind indeed to the postulated binding domain. 9 Highly homologous receptor subtypes (ml, m2, m3; more than 95 % similarity) interact in a different way with the same ligand. These results suggest that in spite of their sequence similarity and binding site identity, these receptor subtypes differ in some way : in their extracellular loops, in their folding, in the packing of helices or, most probably, in their dynamics. This result also highlights the limits of homology protein modelling if one wants to account for subtle receptorligand interactions. 9 The same receptor residue contributes to a different extent to the binding affinity of ligands binding to the same receptor region. 9 Compounds with highly similar structures bind differently to the same receptor. This observation raises the issue of the relevance of any structureactivity relationship study based on ligand comparison. 9 Taken together, these experimental results demonstrate that the ligand binding sites and the residues likely to be involved in the recognition process had been correctly identified from the model. However, as anticipated [23], the model could not account for the subtle differences in interaction reported above. Similarly, a three-dimensional model of the vasopressin/oxytocin receptor has been defined and the binding mode of the neuropeptides has been analysed (Figure 3 and 5). Residues likely to contribute to the affinity, efficacy and selectivity have been proposed from the model. These qualitative predictions have subsequently been confirmed by site directed mutagenesis [20-22]. A number of general comments can be made : 9A large cyclic neuropeptide can indeed penetrate the core transmembrane domain of its receptor. 9The bottom of the binding pocket is in a position equivalent to the binding site of small cationic neurotransmitters (dopamine, adrenaline, serotonin, acetylcholine,etc.) and to the ionone ring of retinal in opsins. 9 Peptidic and non-peptidic antagonists binding is not affected by the mutations which led to a decrease in agonist affinity. 9The selectivity of vasopressin for its receptor is due to a single interaction between the side-chain of Arg8 on the ligand and T y r l l 5 on the receptor. 9 Primary sequence analysis of neuropeptide receptors and SAR study of their ligands suggest t h a t the residue in the first extracellular loop corresponding to Tyr115 of the vasopressin receptor might contribute to binding affinity and selectivity. This would seem to be the case notably for angiotensin, cholecystokinin, neuropeptide Y and neurokinin receptors [22].

211 YII5 L

60

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Figure 5 : S c h e m a t i c r e p r e s e n t a t i o n of vasopressin binding to the V1A receptor. Dotted lines indicate the postulated interactions between the ligand and the receptor residues located in the t r a n s m e m b r a n e domain of the receptor. The residue n u m b e r i n g is coded as in Figure 4, with the exception of Tyr115 which corresponds to the official VIA receptor label. The site directed m u t a t i o n and the decrease (x fold) in affinity of vasopressin for this m u t a n t are indicated near each residue.

Figure 6 9Similarity in the binding mode of GPCR ligands, as predicted from the model [9]. Reproduced from reference [9] with permission of the authors and publishers.

212 QUALITATIVE VERSUS QUANTITATIVE VALUE OF THE MODEL The experimental results reported and discussed above and many others not reported here indicate that GPCR 3D models are QUALITATIVELY excellent. We moved from the "Lock and Key" model to an atomic representation which led to novel working hypotheses. Many of these hypotheses have a posteriori received support from experiments. However, as feared [23], these models have in general little, if any, QUANTITATIVE predictive value. It seems indeed unlikely t h a t the site directed mutagenesis results reported above could have been predicted from the model. In order to avoid to expect too much from these models, it is important to understand how complex GPCRs are, compared to soluble enzymes for instance. This complexity is illustrated in Figure 7. We are in fact dealing with a multimolecular system and many inter-dependent equilibria : ligand<->receptor ; water<->receptor ; ligand<->water ;resting r e c e p t o r < - > a c t i v a t e d receptor ; receptor<->G protein ; receptor<> i n t r a c e l l u l a r allosteric cation ; r e c e p t o r < - > p h o s p h o r y l a s e ; G protein<->GDP ; alpha subunit<->beta-gamma subunit ; etc. Additional parameters such as the nature of the phospholipidic constituents of the membrane, receptor concentration, G protein nature and concentration, acidity of the extracellular membrane surface can modulate the measured ligand binding affinity. Finally, the intrinsic dynamics of the receptor is most probably determinant for ligand binding and function. The hurdle thus appears very high for those aiming at predicting quantitatively subtle molecular interactions in these systems.

9 9

Ligand

H20 Sialic acid

/\

,

,,

Ro (~

(,_jb Kinase

,o p Y

--'~ ' %

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e

cation

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Figure 7 : Schematic representation of the molecular equilibria involved in G protein-coupled receptor activation ( reproduced with permission from publishers and authors of Hibert M, et al. Sanz F, ed. Trends in QSAR and Molecular Modelling '94, Barcelona : Prous Sciences, 1995).

213 CONCLUSION For one century, medicinal chemists tried to design ligands for receptors without knowing how these receptors look like. With the recent progress in molecular biology, molecular pharmacology, biophysics, SAR, a n d theoretical chemistry, it became possible to propose a threedimensional representation of GPCRs and to predict qualitatively the binding mode of very structurally different ligands. Originally based on very w e a k hypotheses, these models could progressively be refined and have received support from experimental data. In fact, they proved to be a very v a l u a b l e tool to g e n e r a t e novel h y p o t h e s e s on receptor functional architecture and led to conceive and perform new interesting experiments. The next important milestone will probably be the access to a high resolution experimental structure of GPCRs. However, these static pictures might not even be sufficient to provide scientists with definitive answers on receptor-ligand interaction since recent studies have already demonstrated the importance of the kinetic parameters for the neurotransmission process and the extraordinary complexity of this system.

REFERENCES

1 Lefkowitz EJ, Caron MG. J Biol Chem 1988; 263: 4993-4996. 2 Dixon RAF, Strader CD. Annu Rep Med Chem 1988; 23: 221-223. 3 Hibert MF, Trumpp-Kallmeyer S, Bruinvels A, Hoflack J. Mol Pharmacol 1991; 40: 8-15. 4 Trumpp-Kallmeyer S, Hoflack J, Bruinvels A, Hibert M. J Med Chem 1992; 35: 3448-3462. 5 Henderson R, Baldwin JM, Ceska TA, Zemlin F, Beckmann E, Downing KH. J Mol Biol 1990 ; 213: 899-929. 6. Weiner SJ, Kollman PA. J Comput Chem 1986; 7: 230-252. 7 Sybyl Molecular Modelling Software, Tripos Associates inc., St Louis MO, USA 8 Schertler GFX, Villa C, Henderson R. Nature 1993; 362: 770-772. 9 Hoflack J, Trumpp-Kallmeyer S, Hibert M. In: Kubinyi H, eds. 3D QSAR and Drug Design. Theory, Methods and Applications. Leiden: ESCOM, 1993; 355-372 10 Hoflack J, Hibert MF, Trumpp-Kallmeyer S. Drug Des Discov 1993; 10: 157-171. 11 Trumpp-Kallmeyer S, Hoflack J, Hibert M. In: Buck SH, eds. The Tachykinin Receptors. New York: Humana Press, 1993; 237-255. 12 Tota M, Candelore M, Dixon RAF, Strader C. Trends Pharmacol. Sci. 1991; 12: 4-6. 13 Savarese TM, Fraser CM. Biochem J 1992; 283: 1-19. 14 Lewell XQ.Drug Des Discov 1992; 9: 29-48.

214 15 IJzerman AP, Van Galen PJ, Jacobson KA. Drug Des Discov 1992" 9: 49-67. 16 Nordvall G, Hacksell UJ. J Med Chem 1993; 36: 967-976. 17 Fanelli F, Menziani M, Carotti A, De Benedetti. Bioorg Med Chem 1994; 2: 195211. 18 Yamamoto Y, Kamiya K, Terao S. J Med Chem 1993; 36: 820-825. 19 Hibert M, Hoflack J, Trumpp-Kallmeyer S, Paquet J'L, Leppik R, Barberis C, Mouillac B, Chini B, Jard S. Eur J Med Chem 1995; 30:189-199. 20 Chini B, Mouillac B, Ala Y, Balestre MN, Trumpp-Kallmeyer S, Hoflack J, Elands J, Hibert M, Manning M, Jard S, Barberis C. EMBO J 1995; 14: 2176-2182. 21 Mouillac B, Chini B, Balestre MN, Elands J, Trumpp-Kallmeyer S, Hoflack J, Hibert M, Jard S, Barberis C. J Biol Chem 1995; 270: 25771-25777.. 22 Trumpp-Kallmeyer S, Chini B, Mouillac B, Barberis C, Hoflack J, Hibert M. Pharm Acta Helvetiae 1995; 70: 255-262. 23 Hibert MF, Trumpp-Kallmeyer S, Hoflack J, Bruinvels A. Trends Pharmacol Sci 1993; 14: 7-12. *This article is adapted and incremented from reference [19].

Perspective in Receptor Research D. Giardinb., A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved. ION

CHANNELS

David J. School 14260

AS

TARGETS

FOR

DRUG

215 DESIGN

Triggle

of Pharmacy,

State University

of New York,

Buffalo,

NY

Abstract

Ion channels are a heterogeneous group of p h a r m a c o l o g i c receptors that respond to a variety of i n f o r m a t i o n a l inputs by controlling in rapid and specific fashion the m o v e m e n t s of ions across cell membranes. Under p a t h o l o g i c conditions the disorderly movements of ions may c o n t r i b u t e to cell pathology and death. Drugs that regulate ion channel function in both voltage- and l i g a n d - g a t e d c a t e g o r i e s represent both molecular tools w i t h w h i c h to dissect channel function and therapeutic agents for, inter alia, c a r d i o v a s c u l a r and neuronal disorders. These aspects are well i l l u s t r a t e d by the Ca 2+ channel antagonist class of drugs. GENERAL

PROPERTIES

OF

ION CHANNELS

Introduction Ion channels are one group of p h a r m a c o l o g i c receptors. As such ion channels: I. Exist as homologous protein families 2. Possess specific drug binding sites which define s t r u c t u r e - a c t i v i t y relationships for both activators and antagonists 3. Have binding sites for both endogenous and exogenous ligands 4. May be regulated by homologous and h e t e r o l o g o u s influences including disease states. Ion channels are one important cell signalling process by w h i c h e x c i t a b l e cells respond to a variety of informational signals i n c l u d i n g neurotransmitters, hormones and sensory inputs [ 1 ] .

The cell maintains an asymmetric distribution of ions t h r o u g h the operation of a variety of pumps and p e r m e a b i l i t y barriers. The net result is the m a i n t e n a n c e of a m e m b r a n e p o t e n t i a l that is controlled by the selective and c o o r d i n a t e d o p e n i n g and closing of discrete ion channels [ Figure 1 ]. Quite generally, the selective opening of an ion channel will drive the m e m b r a n e potential towards the e q u i l i b r i u m p o t e n t i a l for that ion. Thus, the opening of Na § and Ca 2§ channels or the c l o s i n g of K § channels will depolarize the cell and m e d i a t e e x c i t a t i o n and, the opening of K § and Cl-channels will move the resting membrane potential in a h y p e r p o l a r i z i n g d i r e c t i o n to m e d i a t e inhibitory responses. These c o n s i d e r a t i o n s underlie the actions of drugs that may act s e l e c t i v e l y at one or other channel class. It should be e m p h a s i z e d that under physiological conditions ion channels c o o p e r a t e to regulate membrane potential and i n t r a c e l l u l a r ionic c o n c e n t r a t i o n s . Thus, the cardiac action potential is made up of a dozen or more ionic conductances functioning in h i g h l y c o o r d i n a t e d fashion.

216

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K.=--90mV

Ec~§=+120mY

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[K+in,] = - 1 5 5 m M

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0

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Figure I. Ionic concentrations and equilibrium potentials Na +, K + and Ca 2+ and the effects of drugs [ a n t a g o n i s t / a g o n i s t s ] that regulate the ion channels.

IT T Lipid bilayer

TTT

for

Remote Sensor

G Pr

Intermediate

Figure 2. A schematic representation of ion channel structure depicting the principal components that determine channel function.

217 D y s r e g u l a t i o n of one or more of these conductances may p e r t u r b c r i t i c a l l y this coordination leading to a variety of arrhythmic situations. The

S t r u c t u r e a n d C l a s s i f i c a t i o n of Ion C h a n n e l s The schematic r e p r e s e n t a t i o n of Figure 2 depicts ion channels as m e m b r a n e p r o t e i n s that possess regulatory sensors, both e x t r a c e l l u l a r and intracellular, that respond to i n f o r m a t i o n a l inputs, gates that open and close and a selectivity filter a s s o c i a t e d w i t h the channel pore and which controls ion permeation. The sensors may be components of the channel protein[s] itself or may be remote and linked to channel function by c y t o s o l i c or membrane signalling devices. The u n d e r l y i n g m o l e c u l a r bases of channel structures are i n c r e a s i n g l y well defined through biochemical and m o l e c u l a r biology studies [ 2,3 ]. Ion channels may be c l a s s i f i e d by several criteria including the nature of the permeant ion, the e l e c t r o p h y s i o l o g i c a l characteristics including c o n d u c t a n c e and opening and closing rates, the principal informational input as a ligand- or v o l t a g e - g a t e d channel and the p h a r m a c o l o g i c a l s e n s i t i v i t y of the channel [ Figure 3 ]. The latter c l a s s i f i c a t i o n is of particular importance to issues of drug development. Ion Channels

As

Targets

For D r u g s

As m a j o r

classes

of

p h a r m a c o l o g i c a l receptors ion channels are automatic loci for drug action. However, ion channels are p a r t i c u l a r l y a t t r a c t i v e targets for several additional reasons [ 4 ] : i. They are loci for integrated cellular c o m m u n i c a t i o n 2. They are extremely rapid and efficient signalling devices 3. They exist in several major classes and subclasses 4. They are multiple pharmacologic receptors, each channel typically possessing several specific drug b i n d i n g sites 5. The p r o p e r t i e s of drug binding are often m o d u l a t e d by state-dependent interactions whereby the a f f i n i t y or access of a drug for its binding site is d e t e r m i n e d by the state - open, closed or i n a c t i v a t e d - of the channel. Collectively, these properties define o p p o r t u n i t i e s for m u l t i p l e control mechanisms of a single physiological or p a t h o l o g i c a l process. Thus, pathologies that require n e u r o p r o t e c t i v e agents may find pharmacological s a t i s f a c t i o n in at least four discrete groups of agents - e x c i t a t o r y amino acid antagonists, Ca 2§ channel antagonists, Na + channel a n t a g o n i s t s and activators of ATP-dependent K § channels [ Figure 4 ]. Additionally, each channel typically p o s s e s s e s several d i f f e r e n t drug binding sites or receptors that amplify c o n s i d e r a b l y the potential for pharmacologic control [ Figure 5 ]. These o p p o r t u n i t i e s have been explored and i n c r e a s i n g l y realized w i t h several ion channel types including voltagegated Ca 2+ channels [ 5 ].

218

O"

-"

H O ' / ~ " ~ ~ ' / ~ ~ NH > I~H2 OH OH

H2Nco/O~3 H

H21~"~Y ~HN" ~--NH

/10 12~ OH 11 OH

a

+

NEt 4

b

~NO2 MeOOC~ COOMe d

F i g u r e 3. D r u g s t h a t s e l e c t i v e l y i n t e r a c t w i t h ion c h a n n e l s a,b - tetrodotoxin a n d s a x i t o x i n for Na § c h a n n e l s ; c tetraethylammonium for K § c h a n n e l s ; d - n i f e d i p i n e for Ca 2+ channels.

EAA Antagonists I NMDAJ AMPA I

Ca 2+ Antagonists

Phenytoin BW 1003C87

Cromakalin Pinacidil

Na + Antagonists

K+ATPActivators

CNS 1102 NBQX Remacemide GYKI52466 HA966

F i g u r e 4. The c l a s s e s of ion c h a n n e l in n e u r o p r o t e c t i o n strategies.

Nimodipine Conotoxins

drugs

that

are

effective

219

CGP 40116 LY 233053

<

i

Dizocilpine CNS 1102 Remacemide

HA 966 LY-687,414 L-701,252 ~

Glycine

J

"~

<

f

Ifen prodil Nitroglycerin - . ~

Other

Figure 5. The classes of drugs active at excitatory amino acid receptor-channels.

Pr i

MeO MeO OMe

~

S N

I

OCOCH3

Me2NCH2CH2 Diltiazem

, -q-

Me

~(CH2 )3N(CH2)2 CN

Verapamil

OMe OMe

MeOOL~O~OMe Nifedipine

Figure 6. Drugs that interact at the L-type voltage-gated Ca 2+ channel and a schematic representation of the three principal drug binding sites and their allosteric association.

220 VOLTAGE-GATED

C a 2+ C H A N N E L S

Cellular Calcium Regulation Calcium plays critical roles in cellular communication and regulation. The normally very low resting free ionized concentration of Ca 2§ is maintained by a variety of ion channels, pumps, and intracellular storage processes. The elevation of intracellular Ca 2§ levels during cell stimulation serves to couple information with cellular response -stimulus-response coupling. The control of Ca 2+ homeostasis represents, therefore, a potentially powerful control of cellular excitability and response [ 5 ]. C a 2+ C h a n n e l

Antagonists The clinically available Ca 2+ channel antagonists, including the first generation verapamil, nifedipine and diltiazem, control Ca 2§ movements dominantly in the cardiovascular system by interacting at one particular class of voltage-gated Ca 2§ channel - the L-type channel [ 5,6 ]. Their interactions at this channel define a typical multiple pharmacologic receptor with three distinct drug binding sites linked allosterically one to the other and to the permeation and gating machinery of the channel [ Figure 6 ]. The 1,4-dihydropyridine binding site accommodates both nifedipine and a number of second-generation 1,4-dihydropyridines including amlodipine, felodipine, nimodipine etc, that represent structural variations on a theme [ Figure 7 ] with putatively enhanced or more desirable properties relative to nifedipine [ 7 ]. Additionally, and consistent with the definition of ion channels as pharmacologic receptors, this site also accommodates 1,4dihydropyridine Ca z§ channel activators [ 5,8,9 ].

The available Ca 2§ channel antagonists represent chemically, pharmacologically and therapeutically heterogeneous agents that define a common end-point inhibition of Ca 2+ current through the L-class of channel. An important expression of the selectivity of action of these antagonists is seen in their relative cardiac and vascular effects [ Figure 8 ]. Verapamil, and to a lesser extent, diltiazem, posses both vasodilating and cardiac depressant properties over similar therapeutic concentration ranges. In contrast, nifedipine and all other 1,4-dihydropyridines are dominantly vasodilating drugs which exhibit normally modest cardiac depressant properties. These differences in v a s c u l a r : c a r d i a c selectivity define their respective therapeutic profiles whereby verapamil [ and diltiazem ] possess vasodilating [ hypertension and angina ] and selective antiarrhythmic properties and nifedipine possesses dominantly vasodilating properties [ 6,10 ]. The vascular:cardiac selectivity of action of the Ca 2+ channel antagonists has several origins: i. Pharmacokinetic properties 2. Source of Ca 2+ mobilized 3. Class and subclass of Ca 2+ channel 4. State-dependent interactions 5. Pathology of tissue and channel regulation. These origins have been discussed previously [ 7,11 ]. They assume further importance with the second-generation 1,4-

221

%.0

MeOOC~ . ~ , Me"

~

COOMe

MeOOC- ~ , ~

"Me

Me "

Nifedipine ~

~

COOEt "Me

Me "

Nitrendipine

~

Me2CHOOC

COOCH2CH2OMe

Me/ ~ " M e Nimodipine

Cl COOEt

MeOOC

Me" ~ " M e Felodipine

C a 2.

"Me

j-NO 2

Mei ~9 " Me Nicardipine

Figure 7. Structural i, 4 -dihydropyridines.

~

COOCH2CHMe2

Nisoldipine

NO2

OOC~./~/COOCH2CH2N < Me CH2Ph

MeOOC

MeOOC" ~ ' ~

Cl COOEt

Me H CH2OCH2CH2NH2 Amlodipine

formulae of first- and second-generation

Antagonist Classification Vasodilation

Verapamil Diltiazem

Contractility

++ ++

+++ ++

+++

+ 1

++++

0

A-V Conduction +++ ++

1,4-Dihydropyridines Nifedipine Nicardipine Isradipine Amlodipine Felodipine

Figure 8. Classification of Ca ~§ channel antagonists of their cardiovascular profile.

in terms

222 dihydropyridines which may exhibit enhanced vascular selectivity relative to nifedipine [ 12,13; Figure 8 ]. Such selectivity may be important to defining differences in therapeutic and side-effect profile between first- and secondgeneration 1,4-dihydropyridines [ 14,15 ] .

State-Dependent Interactions One important determinant of v a s c u l a r : c a r d i a c selectivity is voltage-dependence of drug interaction, whereby the affinity of a drug is modulated by membrane potential and stimulus frequency [ 16,17; Figure 9 ]. The Ca 2§ antagonists have been well defined to exhibit such interactions in a number of electrophysiological investigations [ 18,19 ]. The frequency- and voltagedependent interactions exhibited by verapamil and nifedipine respectively define their differential anti-arrhythmic and vasodilating properties respectively. These state-dependent interactions are also analyzed in radioligand binding techniques in intact cells under membrane potential control [ 20,21 ]. These techniques have demonstrated for the 1,4-dihydropyridine pharmacophore that voltage-dependence of interaction is highly dependent upon structure. In the two series of 1,4-dihydropyridines depicted in Figure i0 voltage-dependent binding in the antagonist series exists and correlates with antagonist activity measured in a smooth muscle preparation; in contrast, a series of 1,4dihydropyridine activators exhibits little or no voltagedependent binding regardless of potency. These data demonstrate that the substituents on a 1,4-dihydropyridine nucleus not only control qualitatively and quantitatively pharmacologic activity, but also differentially control the modulation of drug-receptor interactions by membrane potential. The clinical relevance of these observations is also apparent in the observations that the second-generation 1,4dihydropyridines exhibit voltage-dependent binding the extent of which varies with structure and that this correlates well with an experimental measure of vascular:cardiac selectivity [ 22; Figure Ii ]. These data indicate that one important, although not exclusive, determinant of tissue selectivity is defined by state-dependent interactions, that these statedependent interactions are sensitive to small changes in chemical structure and that therapeutic differences in vascular and regional vascular selectivity between first- and second-generation 1,4-dihydropyridines have a definable molecular basis. These differences may translate to greater efficacy and safety for these second-generation 1,4dihydropyridines [ 14,15 ].

223

(~

. X

R --~ O

Repetitive

-." (!6')

Persistent kl/k_l

.~,,"

al

k2/k-2~..~,~'" q~,~," 3/k-3

X K

Polar, hydrophilic ~ pathway

R--~

O

*,-.,.

Nonpolar,hydrophobic pathway

Figure 9. The "modulated receptor" concept of drug action at ion channels. Drugs may bind selectively to or access p r e f e r e n t i a l l y b i n d i n g sites in the resting, open or i n a c t i v a t e d states of the channel. Equilibrium between these channel states is determined by several factors including m e m b r a n e potential.

100 0o

(_) k_ O

I

c.w

o Lo

10

,,,,..

LO s

6

9

-Log EC50 or IC50 in Toil Artery Figure 10. The relationship between voltage-dependent binding of activator [ o ] and antagonist [ @ ] 1,4-dihydropyridines in cardiomyocytes and pharmacologic activity in vascular smooth muscle. Data from Zheng et al., [ 21 ].

224

o ~ 750

:4~

r

o

Nitrendipine

t"

m m 500

. ~

~_

~'r"

N 9

c"

~

- Felodipine

CI ~X: 250 ~

..

/

9Nicardipine

/ ~ m io~J~l~iepine J

I

I

250 500 750 Vascular: Cardiac Selectivity [IC50 Papillary Muscle/IC50 Tail Artery]

I

1000

Figure II. The relationship between voltage-dependent binding for clinically available 1,4-dihydropyridines and vascular.cardiac selectivity. Data from Sun and Triggle [22]. REFERENCES .

9

O

4.

.

9

O

8. 9

I0. ii.

12. 13.

Hille, B. Ionic Channels In Excitable Membranes. 2nd Edition, Sinauer Associates, Sunderland, MA, 1992. Rampe D, Triggle DJ [ Editors ],Ion Channels As Targets For Drug Action. Drug Dev. Res. 33: 189-372, 1994. Catterall WA, Ann. Rev. Biochem. 64: 493-532, 1995. Triggle DJ, Drugs Acting On Ion Channels In Membranes. In, Comprehensive Medicinal Chemistry [ Editors, Hansch, C, Emmett JC, Kennewell PD, Ramsden CA, Sammes PG, Taylor JB. 3: 647-699, 1990. Janis RA, Silver P, Triggle DJ. Adv. Drug Res., 16: 309591, 1987. Fleckenstein A. Calcium Antagonism in Heart and Smooth Muscle. J. Wiley & Sons, New York, NY. 1983. Triggle DJ, Clev. Clin. J. Med., 59; 617-627, 1992. Triggle, DJ, Langs DA, Janis RA Med. Res. Revs. 9:123180, 1989. Golclmann BS, Stoltefuss J, Ang. Chemie [ Int. Ed. Eng. ] 30: 1559-1578, 1991. Triggle DJ, Calcium Antagonists, In, Cardiovascular Pharmacology [ Ed. Antonaccio M ], 3rd. edition pp. 107160. Raven Press, New York, NY 1990. Triggle, DJ, Biochemical and Pharmacologic Differences Among Calcium Channel Antagonists: Clinical Implications 9 In Calcium Antagonists in Clinical Medicine [ Epstein M, Ed. ], pp. 1-27. Hanley and Belfus, Inc. Philadelphia, PA, 1992. Freedman DD, Waters DD, Drugs 34: 578-598, 1987. Opie LH, Cardiovas. Drugs Therap. 6: 19-24, 1992.

225 14. 15. 16. 17. 18. 19.

20. 21. 22.

Psaty BM, Heckbert SR, Koepsell TD et al., JAMA 274: 620625, 1995. Yusuf S, C i r c u l a t i o n 92: 1079-1082, 1995. Hille B, J. Gen. Physiol., 69: 497-515, 1977. H o n d e g h e m LM, Katzung, BG, Annu. Rev. Pharmacol. Toxicol., 24: 387-423, 1985. Lee K, Tsien RW, Nature 302; 790-794, 1983. T r i g g l e DJ, S t r u c t u r e - F u n c t i o n Correlations of 1,4D i h y d r o p y r i d i n e C a l c i u m channel Antagonists and Activators, In M o l e c u l a r and Cellular M e c h a n i s m s of A n t i a r r h y t h m i c Agents [ H o n d e g h e m LM, Ed ] pp. 269-291, Futura Press, Mt. Kiscoe, NY 1989. Wei XY, Rutledge A, Triggle DJ. Mol. Pharmacol. 35: 541552, 1989. Zheng W, Stoltefuss, Goldmann S, Triggle, DJ, Mol. Pharmacol. 41; 535-541, 1992. Sun J, Triggle DJ, J Pharmacol. Exp. Therap. 274; 419426, 1995.

This Page Intentionally Left Blank

Perspective in Receptor Research D. Giardin~, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

227

R e c e n t A d v a n c e s in P o t a s s i u m C h a n n e l A c t i v a t o r s John M. Evans SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, United Kingdom

INTRODUCTION Potassium channels have been the subject of extensive research for some time, but the use of the techniques of electrophysiology and molecular biology coupled w i t h t h e emergence of compounds that modulate these channels has hastened the process [1,2]. In particular, the last decade since the discovery [3] of cromakalim 1 has seen a flurry of interest in the development of compounds that open the ATP-sensitive K + channel (KATP) and their pharmacological effects in a range of tissue types. Subsequently, compounds have been discovered that activate K + channels other than KATP. This review of potassium channel activators (KCAs), or openers, encompasses the range of different compound types but, after a brief description of the earlier compounds, concentrates on the more recent advances in compound structure modification and resultant pharmacological effect and especially on the search for compounds selective for a particular type of K + channel.

~'~o 1 cromakalim 2 (3S,4R)levcromakalim

L E V C R O M A K A L I M - CLINICAL P R O F I L E The 3S,4R active enantiomer of cromakalim, levcromakalim 2 [4], is in the late stages of development as an antihypertensive agent. In clinical trials in moderately hypertensive patients, supine diastolic blood pressure, typically at the 95-114 mmHg level, was lowered by 5-10 mmHg following administration

228 once daily of 0.5 to 1.5 mg of 2. Measurements were made 24 hours after administration at intervals during 4-8 weeks. These decreases were not accompanied by significant changes in pulse rate. The lipid profiles of fasting blood samples from a sub-population of these patients with baseline total cholesterol > 6.5 mmol/L were compared with those from a placebo group, where all the lipid parameters measured (see Table 1) increased over the 8 week period of administration. Encouragingly, in patients treated with levcromakalim 2, significant reductions in total cholesterol, LDL and triglycerides were observed and the HDL:total cholesterol ratio, which is a good indicator of atherogenic potential, was improved. These data are indicative of a potential for improvement of two risk factors in hypertensive patients with hypercholesterolemia, not generally observed with other antihypertensive agents. Table 1 Effects of levcromakalim 2 or placebo (once daily oral doses for up to 8 weeks) on plasma lipid profile (% change) in patients with baseline total cholesterol > 6.5mmoU1

Total Cholesterol HDL LDL Triglycerides HDL/cholesterol * p<0.05

Placebo (n=31) +1.2 +7.1 +4.1 +5.5 +6.1

0.5rag (n=26) -3.0 +17.6 -0.1 -19.6"** +21.4 **p<0.01

1.0rag (n=22) -5.7* +28.8 -11.7"* -8.8 +38.1 ***p<0.0001

1.5rag (n=21) -6.3*** +27.0 -7.5* -24.4*** +37.2*

S T R U C T U R A L L Y DISTINCT FAMILIES OF KCAs

Shortly after the discovery of cromakalim 1 and its novel mode of pharmacological action [5], other KCAs of differing structural types were identified. Indeed, several of these compounds were already known, but their mode of pharmacological action remained undetermined. One exception was nicorandil 3, as it had already been reported to possess the dual action of K + channel activation [6] and guanylate cyclase activation [7]. One of the key elements in the pharmacological study of these compounds was the observation that their KATP channel activating effects could be inhibited by the insulinreleasing sulphonylurea, glibenclamide 4 [ 1 ] . Thus, to the established compounds such as minoxidil sulphate 5 and diazoxide 6 were added RP 49356 7, the racemic form of the active 1R,2R enantiomer, aprikalim 8, and pinacidil 9.

229 There t h e n followed a period of high activity when m a n y pharmaceutical companies initiated studies to develop KCAs, principally of the benzopyran based series. NH 2 N~,~

CONH(CH2)2ONO2

/N

--OSO: NH2

3 nicorandii

5 minoxidil sulphate OMe

CONH OH , O NHOON

/

CI'

4 glibenclamide o,

'~

CN ~

/~

6 diazoxide

H

N

7 (+) RP 49356

9 pinacidii

8 (1 R,2R) aprikalim Such developments have been reviewed [8-10] and examples 10 - 16 of this second wave of KCA molecules are depicted in Figure 1. The m a i n indications claimed to be treatable by these compounds are those t h a t can be alleviated by smooth muscle relaxation, such as hypertension, bronchial a s t h m a and u r i n a r y incontinence, although there is no evidence to suggest t h a t these earlier compounds are tissue selective. THE SEARCH FOR TISSUE SELECTIVITY Some of the most potent compounds have been discovered in the aprikalim 8 series of KCAs and the 4-thioamide substituted benzopyran series. Thus in the former series, the 2S-benzoate ester RP 66471 17 had an IC90=0.3 nM in inhibiting 20 mM KC1 induced contractions in rat isolated aorta [11]. In the latter series, KC-399 18 (pEC50=9.85) was found to be approximately a thousandfold more potent t h a n cromakalim 1 (pEC50=6.77) in relaxing rat isolated aorta precontracted with 30 mM KC1 [12].

230

•N,•N•

O

NC

~,,,

Me

~N~N~O

T

OH

11 EMD57283

10 emakalim

C N CN N

{ ~

T

12 FR 119748

O

o...JL

13 Y 27152

N II

+

N~-~O

0 o

/N- - ~ , , ,

OH

Me

NC

~O-

Me

14 celikalim

15 NIP 121

16 Ro31-6930

Figure 1. KCAs developed from the prototype cromakalim 1 However, despite such impressive levels of potency, these compounds are not reported to possess the highly desirable property of tissue selectivity. The search for KCAs that are selective for indications other than that of hypertension has been pursued in several different ways, for example by identifying compounds that open different or additional K + channels, or possess additional mechanisms of pharmacological action. In addition, some success has been achieved by the identification of novel structures that open K + channels and by structural variation in the existing series of KCA molecules. The remainder of this article is therefore devoted to the application of these strategies and the interesting results that have accrued from them.

231 ;SNHCH2CH2CN

~

PhCO2 17 RP 66471

Cl--~F CH2F

18 KC-399

Cardioprotective KCAs The standard KCAs, such as cromakalim 1 and pinacidil 9, have been shown in animal models to have direct cardioprotective properties that are independent of their vasodilator effects [13]. In the search for KCAs that offer cardioprotective effects without lowering blood pressure, a series of phenylcyanoguanidine benzopyrans was identified that possessed reduced vascular relaxant properties [14]. SAR studies revealed that inhibition of methoxamine-induced contraction in rat isolated aorta was not paralleled by anti-ischaemic potency (expressed as an EC25 - the concentration required to increase time to contracture by 25%) in rat isolated hearts. Thus, BMS-180448 19 had a vasorelaxant potency (IC50=1.8 ~tM) about fiftyfold less than cromakalim 1 (IC50=0.032 ~M) and an antiischaemic potency (EC25=2.5 ~tM) approximately four times greater than that of 1 (EC25=8.9 ~tM). Glibenclamide (0.1 ~tM), while having no effect of its own, completely abolished the cardioprotective effect of the des-chloro analogue of compound 19, thus suggesting the involvement of KATP. Further SAR studies supported the distinction of antihypertensive and cardioprotective properties and has resulted in the identification of the racemic C(6)-trifluoro analogue 20 with approximately twofold enhanced selectivity for the myocardium compared with compound 19 [15]. These observations were accounted for by the proposal that there are differing K + channel subtypes in vascular smooth muscle and in cardiac muscle. H~C(=NCN)NHPh-p-CI \,,OH

19 BMS-180448 (3S,4R) R=CN

20 (.t) R=CF3

232 A i r w a y s - s e l e c t i v e KCAs When it was appreciated that KCAs such as cromakalim 1 could produce relaxation of different tissues, airways selectivity became a highly desirable goal for this class of compound. Some success has been achieved in tissue selectivity for guinea-pig isolated trachealis over rat isolated aorta in a series of N-cyanoamidines exemplified by KC-!28 21, the pECs0s being 7.29 and <4.5, respectively. It is interesting to note that replacement of the methyl groups of the N-dimethyl side chain by hydrogen or ethyl diminishes selectivity, while, most interestingly, the nitro substituent can be replaced only by trifluoromethyl and trifluoromethoxy groups and not by the phenylsulphonyl and cyano groups found in other KCAs when selectivity and potency are attenuated [16].

~0 C2F'5"~~ x Me~(Me ,

21KC-128

~, O H

22BRL55834

In contrast, BRL 55834 22 does not appear to be airways-selective in vitro as it showed a similar magnitude of inhibition of 30 mM KCl-induced tone in guineapig isolated portal vein and of spontaneous tone and histamine-induced tone in guinea-pig isolated tracheal spirals. But, the key finding for this compound is its in vivo selectivity for the airways over the vasculature compared with levcromakalim 2 when administered by a variety of routes to the guinea-pig. Thus, BRL 55834 22 (100 ~g/kg) had a similar magnitude of effect to 2 (500 ~g/kg) in lowering histamine-induced airways resistance on intraduodenal administration to the anaesthetised animal, while blood pressure falls were almost half those observed for 2 [17]. The reason for this approximately tenfold selectivity is not fully understood. Mechanistic studies using patch clamp techniques have suggested that 22 is able to activate a large, 243 pS conductance, calcium-dependent K + channel (BKCa) in addition to the 30 pS KATP channel activated by 2 in bovine trachea [18]. This finding may account for the profile difference between BRL 55834 and levcromakalim. Opening of the charybdotoxin-sensitive BKCa channel in guinea-pig tracheal spirals is part of the pharmacological profile demonstrated by SCA40 23 that also has phosphodiesterase inhibitory properties [19]. The high density of BKCa channels in airways smooth muscle suggests that this compound may be particularly suitable as a bronchodilating agent, but as these channels are present in a range of tissues, selectivity for the channel population in airways, or

233 in vivo selectivity needs to be demonstrated before the compound can said to have true potential in airways related disease.

Screening of the fermentation products of an unidentified coelomycete MF5717, has produced a novel dihydroxyisoprimane, maxikdiol 24 [20], that inhibits the binding of 125I-charybdotoxin to the BKca channel in bovine isolated aortic sarcolemmal membrane vesicles. The structural similarity to forskolin 25, a blocker of a delayed rectifier K + channel in mouse neurones in culture [21], is quite striking. Maxikdiol 24 [20] had no effect on 125I-charybdotoxin binding to voltage-dependent K + channels in rat brain synaptic plasma membranes, indicating specificity for the former tissue. The report [21] that this compound may have a potential use in airways dysfunction also requires further substantiating data.

Me

Br

i-

~.

-5

o

'

CN MeMe 23 SCA40

24 maxikdiol

MeMe

OH

25 forskolin

B l a d d e r - s e l e c t i v e KCAs

4-Phenyl-dihydropyridines are well known as vasodilator antihypertensive agents acting via antagonism of L-type calcium channels. Since one of the enantiomers of niguldipine 26, a calcium channel antagonist, activates the BKCa channel in vascular smooth muscle [22], it was only a matter of time before certain dihydropyridines were discovered that act principally via K + channels [23]. Thus the relaxant effect (IC50=0.16 ~M - fourfold more potent than cromakalim 1) of compound 27 in guinea-pig isolated bladder strips, precontracted with 15 mM KC1, was reversed by glibenclamide 4, indicative of KATP channel involvement. In comparison, compound 27 relaxed precontracted (15 mM KC1) guinea-pig isolated portal vein with an IC50=0.009 ~M which is twice as potent as that of cromakalim 1. Such a finding is possibly indicative of a high degree of homology of the calcium channels modulated by the dihydropyridines and BKCa and KATP channels. Further screening for KCAs that might be useful for the alleviation of urinary incontinence has identified a class of antihypertensive anilide carbinol antiandrogen compounds [24]. Of particular interest are the members of the class

234 that are reported to be bladder selective, for example the preclinical compound ZD-6169 28 (IC5o=1.6 ~M for relaxation of 15 mM KC1 induced contraction of guinea-pig isolated bladder strip). This compound attenuated micturition frequency (ED50=0.16 mg/kg po) in rats with minimal effects on blood pressure and heart rate (ED20=30 mg/kg). In dogs, ZD-6169 28, at 3 mg/kg po, resulted in a reduction in the number of bladder contraction events without affecting blood pressure and heart rate. In contrast, similar bladder effects could only be achieved by cromakalim at dose levels that caused falls in blood pressure [25].

O S

26 niguldipine O

U

....

28 ZD-6169

0

ir

O

27'

NCk

A

O~

/,~0

29

A recent patent [26] describes compounds of the pinacidil 9 series where the cyanoguanidine segment has been replaced by the diamino-cyclobutenedione group. Thus compound 29, is the most potent (IC50=0.52 I~M) of the compounds tested for their ability to inhibit 15 mM KCl-induced contractions in rat isolated bladder strips. Reports of the cardiovascular profile of this compound are awaited with interest to determine whether it has sufficient selectivity for potential in the treatment of urinary incontinence. In the course of a programme designed to produce bladder selective KCAs related to celikalhn 14, the sulphonamide 30 was found to relax 15 mM KC1induced contractions in rat isolated detrusor strips (IC50=4.6 ~M) and rat isolated aorta (IC50=8.0 ~M). It was observed that replacement of the trifluoromethyl group with larger groups such as phenyl, or N-substitution, caused an unexpected pharmacological effect as the compounds elicited a contractile response [27]. Both enantiomers of compound 30 were virtually

235 equipotent as relaxants and the effect was insensitive to glibenclamide, thus excluding the possibility that this was mediated by an effect on KATP channels. It is interesting to note that recently, the closely related sulphonamide 31 has been described as a potent (IC50=0.6 ~M) stereospecific inhibitor of chloride ion conductance in rabbit isolated distal colon, by apparently inhibiting the cAMPregulated K + ion conductance [28].

CF30

HNSO2C 3F ",,~,,,O:e

~

)'~Me

MeNSO2Et NC~ , , , OH 31

30

Central N e r v o u s S y s t e m Examples of a series of benzimidazole derivatives were described [29] as able to open the high conductance (154 pS) BKCa channel in cerebellar granule cell membrane. Hence, it was thought that, despite opening similar channels in bovine and guinea-pig cultivated tracheal cells, these compounds had a potential for selectively alleviating CNS disorders. Subsequently NS-004 32 [30], an example of the series, was found also to be cardioprotective and vasorelaxant. The former action was not blocked by iberotoxin (50 nM) nor by glibenclamide (1 ~M), thus excluding BKCa and KATP channel activation as its pharmacological mechanism. Additional studies showed that the mechanism of action involved Ltype calcium current blockade. In contrast, other workers [31] consider that the activation of BKCa channel in rat isolated coronary smooth muscle is masked by the modulation of other types of K + channel. These additional findings suggest that compound 32 lacks potential in CNS disorders, although its mechanism of action remains of interest.

32 NS-O04

~

o/

OH

OEt

H

H

Me

33 Bay x9227

Whereas both enantiomers of pinacidil possess cardiovascular properties, bioisosteric replacement of the cyanoguanidine group by a nitroethenyl group and of the pyridine moiety by an ethoxyphenyl group has produced a disparity in the properties of the enantiomers [32]. Electrophysiological examination of the

236 (-)-enantiomer, Bay x9227, of compound 33 demonstrated a dose-dependent hyperpolarising activity of high (EC50=3 pM) potency in rat cultivated hippocampal neurones. It also showed a thousandfold selectivity over activity in rat vascular smooth muscle cells. In marked contrast, the (+)-enantiomer showed the typical profile associated with a KATP channel activator, hyperpolarising cell membranes in a glibenclamide-sensitive manner. The consequences of this, as yet undefined, mechanism of pharmacological action of compound (-)-33 are inhibition of elevated intracellular Ca ++ concentration and protection from glycine-induced neurotoxicity, both highly desirable properties for potential neuroprotective agents. In considering how the cell membrane hyperpolarising action of cromakalim 1 could be of potential application in CNS disorders, it was reasoned t h a t the outcome could be a decrease in neuronal intracellular Ca ++ ion concentration and a possible attenuation of the release of brain excitatory aminoacids. A consequence of this might well be an application of KCAs in diseases such as epilepsy. Indeed, while our studies were underway, advances by other workers lent support to this concept. Thus, cromakalim 1 and levcromakalim 2 were shown to inhibit both MCD-induced seizures in rats and spontaneous seizures in genetically epileptic rats on intracerebroventricular administration. The effect is stereospecific, the (+)-3R,4S enantiomer of 2 being ineffective at doses at which 2 was effective [33]. In contrast, it was found that 1 when administered systemically (orally or intraperitoneally) did not inhibit seizure activity in a mouse electroshock model, even at a dose of 10 mg/kg, which is much greater than t h a t required to elicit antihypertensive activity [3]. Although the models used in both investigations are different, a possible explanation for the lack of activity of 1 is its poor ability to penetrate the CNS. In a search for compounds that are potentially more brain penetrant than compound 1, a series of more lipophilic analogues of 1 was tested orally in the mouse maximal electroshock threshold (MEST)model [34]. Thus, typically, replacement of the nitrile group of cromakalim by the more lipophilic t-butyl group or elimination of the 3-hydroxyl group to produce the chromene, failed to generate anticonvulsant activity, while replacement of the 4pyrrolidinone group by a fluoro-benzoylamino substituent surprisingly introduced the requisite activity.

237 16:)-.--14) ----

Compound 35 Mean Diastolic Blood Pressure (mmHg)

12).--I(D -.-8:) ,

Compound 37

9

Time (minutes) Post-Dose Figure 2. Effect of enantiomers 35 and 37 in SHRs (10 mg/kg po). [Diastolic bp was monitored using a tail cuff at 5-10 min intervals following po administration of test compounds in suspension in 1% w/v methylcellulose to groups of 3 SHRs. Standard errors were determined and fell within the limits of the data points.]

Table 2 Effect of compounds 34-39 in the mouse MEST model (10 mg/kg po) H_~ . , , .

Cpd. No.

Fluorine position

34 35 36 37 38 39

p m p m p p

Stereochemistry

F

% I n c r e a s e in S e i z u r e

Threshold 3R, 4S trans 41" 3R,4S trans 50* 3S, 4R trans 17 3S,4R trans 12" 3S, 4S cis 28* 3R, 4R cis 0 *: p<0.05 compared to vehicle controls

238 Very surprisingly, however, when the individual enantiomers 34-37 of m e t a and p a r a t r a n s - f l u o r o b e n z a m i d e s (see Table 2) were examined, it was found t h a t the anticonvulsant activity resided predominantly in the 3R,4S enantiomers 34 and 35. These compounds have the opposite stereochemistry to that of levcromakalim 2 and compound 35 did not significantly lower blood pressure at 10 mg/kg orally in the spontaneously hypertensive rat (SHR; see figure 2). Thus it seems very unlikely t h a t the anticonvulsant activity of 34 and 35 is a result of modulation of the type of KATP channel with which levcromakalim 2 interacts. In contrast, the 3S,4R enantiomer 37, which has the same stereochemistry as levcromakalim 2, lowered diastolic blood pressure to about half of the predose value. Attention was then focused on cis fluorobenzamides. The chemical means of generating cis benzamides from their trans isomers was already available [35]. Our most recent work demonstrated that this synthetic route [35] provided highly enantiomerically pure cis isomers from their highly enantiomerically pure t r a n s isomers with inversion of the hydroxyl group at the 3-position of the benzopyran ring [36]. On examination of these compounds, it was surprising to observe t h a t the 3S,4S benzamide 38 was almost as potent as its 3R,4S isomer 34 in increasing the threshold to shock in the mouse MEST model (see table 2) [36]. Interestingly, there is a similar stereochemical differentiation in the cis series to that seen in the t r a n s series because the 3R,4R enantiomer 39, unlike compound 38, did not demonstrate any anticonvulsant activity at the dose of 10 mg/kg employed. Moreover, compound 38 had little effect on blood pressure (-5 + 3%) after an i.v. infusion of 10 mg/kg over 0.5 hr to anaesthetised Hooded Lister rats, whereas its enantiomer 39 caused a fall of 36 + 7% at the same dose. Comparison of all four isomers leads to the conclusion that anticonvulsant activity is associated with the presence of an S configured fluorobenzoylamino substituent at position C(4), while the hydroxl group at C(3) can be of either configuration. In contrast, the presence of an R configured fluorobenzoylamino substituent at position C(4) is associated with antihypertensive activity and the opening of KATP channels. Further reports of the interesting 4S substituted compounds, particularly those of a mechanistic nature, are keenly awaited. CONCLUSION Although certain of the earlier compounds, such as levcromakalim 2, described in this brief review of advances in potassium channel activators, do possess an attractive profile as antihypertensive agents, several strategies have been devised in attempts to enhance tissue selectivity of the later generations of compounds. The strategies involving structural variation of existing classes of KCAs and the search for novel structures that open either KATP and/or BKCa

239 channels have in certain instances achieved the desired profile. In particular, BMS-180448 19 may have potential in cardiac ischaemia, BRL 55834 22 in airways disease and ZD6169 28 in urinary incontinence. In terms of a possible change in pharmacological action from the usual KATP activation observed for benzopyrans, one of the most interesting findings is that of the anticonvulsant fluoro-benzoylaminobenzopyrans, where recognition of the influence of stereochemistry has proved to be the critical feature in achieving selectivity. With the advances that are continually being made in pharmacological screening techniques, molecular biology and mechanistic studies, it is very likely that the goal of highly selective KCAs for the range of channels and currents will eventually be attained and may provide potentially useful drugs. REFERENCES

9

10

11 12 13 14 15 16 17

Longman SD, Hamilton TC. Med Res Rev 1992; 12:73-148 and references therein. Weston AH, Hamilton TC, eds. Potassium Channel Modulators, pharmacological, molecular and clinical aspects. Oxford: Blackwell, 1992. Evans JM, Stemp G. Chem in Britain 1991; 27: 439-442. Hamilton TC, Beerahee A, Moen JS, Price RK, et al. Cardiovasc Drug Rev 1993; 11: 199-222. Hamilton TC, Weir SW, Weston AH. Br J Pharmacol 1986; 88: 103-111. Furukawa K, Itoh T, Kajiwara M, Kutamura K, et al. J Pharmacol Exptl Ther 1981; 218: 248-259. Holzmann S. J Cardiovasc Pharmacol 1983; 5: 364-370. Hirst P, ed. Potassium Channel Modulators (from November 1991). London: Current Drugs. Atwal KS. Drug Dev Res 1994; 33: 250-262. Evans JM, Taylor SG. In: Ellis GP, Luscombe DK, eds. Progress in Med Chem. Amsterdam: Elsevier, 1994; 411-446. Hart TW, Guillochon D, Perrier G, Sharp BW, et al. Tet Letts 1992; 33: 7211-7214. Sato H, Koga H, Ishizawa T, Makino T, et al. Bioorg Med Chem Lett 1995; 5: 233-236. Auchampach JA, Maruyama M, Cavero I, Gross GJ. Circulation 1992; 86: 311-319. Atwal KS, Grover GJ, Ahmed SZ, Ferrara FN, et al. J Med Chem 1993; 36: 3971-3974. Atwal KS, Grover GJ, Ferrara FN, Ahmed SZ, et al. J Med Chem 1995; 38: 1966-1973. Ishizawa T, Koga H, Sato H, Makino T, et al. Bioorg Med Chem Lett 1995; 4: 1995-1998. Bowring NE, Arch JRS, Buckle DR, Taylor JF. Br J Pharmacol 1993; 109: 1133-1139.

240 18 19

Ward JPT, Taylor SG, Collier ML. Br J Pharmacol 1992; 107: 49P. Laurent F, Michel A, Bonnet PA, Chapat JP, et al. Br J Pharmacol 1993; 108: 622-626. 20 Singh SB, Goetz MA, Zink DL, Dombrowski AW, et al. J Chem Soc Perkin Trans 1 1994; 3349-3352. 21 Grega DS, Werz MA, Macdonald RL. Science 1987; 235: 345-348. 22 K15ckner U, Isenberg G. Br J Pharmacol 1989; 97: 957-967. 23 Frank CA, Forst JM, Grant T, Harris RJ, et al. Bioorg Med Chem Lett 1993; 3: 2725-2726. 24 Grant T, Frank CA, Kau ST, Li JH, et al. Bioorg Med Chem Lett 1993; 3: 2723-2724. 25 Ohnmacht CJ, Russell K, Brown FJ, Frank C, et al. Abs Pap 207th ACS meeting, San Diego, 1994; Ptl: MEDI 218. 26 Butera JA, Antane SA. US Pat Appln, 1995; 5,401,753. 27 Soll RM, Dollings PJ, McCaully RJ, Argentieri TM, et al. Bioorg Med Chem Lett 1994; 4: 769-773. 28 Lohrmann E, Burhoff I, Nitschke RB, Lang HJ, et al. Pflugers Arch - Eur J Physiol 1995; 429: 517-530. 29 Olesen S-P, Watjen F. Eur Pat Appln, 1992; 0 477 819. 30 Sargent CA, Grover GJ, Antonaccio MJ, McCullough JR. J Pharmacol Exp Ther 1993; 266: 1422-1429. 31 Xu X, Tsai TD, Wang J, Lee EW, Lee KS. J Pharmacol Exp Ther 1994; 271: 362-369. 32 Hunnicutt EJ, Davis JN, Chisholm JC. Eur J Pharmacol 1994; 261: R1-R3. 33 Gandolfo G, Romettino S, Gottesman C, van Luijtelaar G, et al. Eur J Pharmacol 1989; 167:181-183 and references therein. 34 Blackburn TP, Buckingham RE, Chan WN, Evans JM, et al. Bioorg Med Chem Lett 1995; 5: 1163-1166. 35 Burrell GE, Evans JM, Jones GE, Stemp G. Tet Letts 1990; 31: 3649-3652. 36 Brown TH, Campbell CA, Chan WN, Evans JM, et al. unpublished work.

Perspective in Receptor Research D. Giardin~, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

241

Spinal glutamate receptors David Lodge and Ann Bond Lilly Research Centre, Erl Wood Manor, Windlesham, Surrey GU20 6PH, United Kingdom

INTRODUCTION Glutamate, and possibly other acidic amino acids, are now accepted as the major mediators of excitatory neurotransmission in the brain and spinal cord. The initial discovery of glutamate's stimulatory effects on neurones of the central nervous system (CNS) stems from work in the 1950s 1,2]. Despite its ubiquitous function in general cellular metabolism, the transmitter role of glutamate was slowly accepted during the 1960s and 70s. This was aided by separate but linked observations in pioneering laboratories. 1. Neurones in different parts of the brain and spinal cord showed different sensitivities to derivatives of glutamate [3,4]. These included synthetic derivatives such as N-methyl-D-aspartate (NMDA) and natural products such as quisqualate and kainate [5]. 2. Secondly, other synthetic derivatives were found to have antagonistic actions against some of the above glutamate analogues and against synaptic excitations in the CNS [6-9]. These included 1-hydroxy-3-aminopyrrolidone-2 (HA-966), D-a-amino-adipate, ~/-glutamyl-amino-methyl-sulphonate (GAMS) and glutamate diethyl ester (GDEE). The former two selectively reduced responses to NMDA, a pharmacological feature which was shared with several divalent cations, such as magnesium [10]. GAMS and GDEE were shown to have selective effects versus quisqualate and kainate induced responses respectively. Additionally, short latency monosynaptic responses were reduced by the quisqualate antagonists whereas longer latency polysynaptic responses were reduced by NMDA antagonists both in vitro and in vivo [11-13]. 3. Neurochemical studies using radiolabelled glutamate and some of the above analogues showed specific binding sites and uptake mechanisms [3, 14, 15]. The presence of these presumed postsynaptic receptors and transporter systems provided key support for the electrophysiological evidence for the transmitter role of L-glutamate. The selectivity of many of these early agonists and antagonists was poor but nevertheless a concept of three glutamate receptor subtypes developed [7, 8, 11, 16, 17], and despite close examination over the years, this subdivision still holds generally true today (see below). NMDA receptors. The NMDA receptor has received considerable attention, largely because of the early development of selective tools for its study. NMDA itself is a specific ligand for this receptor with little action at other types of glutamate receptor. In particular, the discovery of a highly selective and competitive NMDA antagonist

242

in D-2-amino-5-phosphonopentanoate (AP5) [17, 18] allowed many aspects of the physiological and pathological role of this receptor type to be elucidated. The discovery that arylcyclohexylamines such as ketamine and phencyclidine and benzomorphans such as cyclazocine and N-allyl-normetazocine (SKF10,047) were non-competitive channel-blocking NMDA antagonists, was particularly helpful for whole animal electrophysiological and behavioural studies, since these compounds, unlike the competitive antagonists, crossed the blood-brain barrier quickly [19,20]. Three other key discoveries provided insights into the physiology of the NMDA receptor. Firstly, the block of the NMDA receptor by magnesium [10] was shown to provide the key voltage-dependent property of this receptor [21]. At resting membrane potentials around -70 to -60 mV, normal extracellular levels of magnesium provide a brake on the permeability of the receptor, but as neurones depolarise due to synaptic inputs (or to metabolic challenges) the magnesium braking action is relieved and the channels conduct current freely. Secondly, the NMDA receptor was shown to be highly permeable to calcium [22]. The resulting increase in intracellular calcium is important not only for physiological, but also for pathological, facilitation of calcium dependent enzymic functions. Thirdly, allosteric modulation of NMDA receptors by glycine acting at a separate recognition domain on the NMDA receptor complex [23]. It is now known that the NMDA receptor requires co-activation of both the glutamate and glycine recognition sites and indeed a co-transmitter role for glycine has been proposed. In the presence of low amounts of glycine the NMDA receptor appears to undergo desensitisation which can be reversed by increasing the external concentration of glycine [24, 25]. It is still a matter of some debate as to whether extracellular levels of glycine are sufficient to fully saturate this site. Nevertheless because of the requirement for co-activation of the glycine site, considerable pharmacological effort has been made to produce partial agonists and full anatgonists at this receptor [25]. Alongside these largely electrophysiological studies, others showed that NMDA receptors mediated profound neurotoxicity [26-30] and have a central role in epileptiform discharges [31-32] in vitro and in vivo. As a result, the therapeutic potential of NMDA receptor antagonists has emerged and several agents are under development by pharmaceutical companies [33]. The first NMDA receptor subunit (now known as NMDA-R1) was cloned and expressed as a functional NMDA receptor channel complex by Moriyoshi, Nakanishi and colleagues [34]. By homology cloning, NMDA-R2 subunits, A,B,C and D, were also identified. NMDA-R2 subunits, unlike NMDA-R1, do not, however, form functional homomeric channels. It has subsequently been shown that native NMDA receptors consist of heteromeric complexes of NMDAR1 associated with NMDA-R2A-D, presumably in a pentameric structure. All such known NMDA receptor configurations have pharmacology in general agreement with that described above, although variations in kinetics and pharmacology have been reported. Constructs with NMDA-R2C are relatively insensitivite to glycine and to Mg 2+, which may correlate with the pharmacology of NMDA receptors of the cerebellum where 2C subunits are highly expressed. As NMDA receptors are not the major subject of this chapter, readers are referred to recent reviews of their molecular biology and pharmacology [13, 3537].

243 AMPA and kainate receptors. Until recently there has been a lack of pharmacological tools for other glutamate receptors. The original subdivision of non-NMDA receptors depended on the agonist selectivity of quisqualate, which has subsequently been shown also activate phospholipase-coupled metabotropic receptors [38]. The synthesis of a new and more selective ligand was therefore a crucial step. The discovery of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) as a GDEE sensitive agonist [39], led to the re-naming of the non-NMDA receptors as AMPA and kainate receptors [16, 40]. Furthermore radioligand binding experiments have allowed separate high affinity [3H]AMPA and [3H] kainate binding sites to be distinguished [14, 40, 41]. Some cross displacement, particularly of [3H]AMPA by unlabelled kainate, however, confirmed electrophysiological observations of some non-selectivity of these two agonists, which still remains a problem. The characteristic pharmacology of AMPA was subsequently corellated, following the use of patch clamp techniques, with its kinetic profile; thus, AMPA evoked rapidly desensitising responses in central neurones whereas those to kainate were relatively non-desensitising [42, 43]. Block of AMPA currents, however, by prior application of kainate suggested a common site but with different mechanisms, AMPA acting as a partial agonist and kainate as a full agonist [42, 44-46]. On this basis, the separation between kainate and AMPA receptors was unlikely to be achieved without other selective agents or molecular biological identification of distinct receptor subunits. Both these advances have been made in the last five years.

Competitive AMPA receptor antagonists.

The low margin of selectivity between AMPA and kainate responses with the early antagonists such as GDEE and GAMS especially in in vitro studies required the development of more potent and selective competitive antagonists. The first such compounds showing selective AMPA antagonist activity were the 6-cyano-7-nitro- and 6,7-dinitro-quinoxalinediones, CNQX and DNQX, [47] but their selectivity for AMPA over kainate was not more than five-fold. A further synthetic development led to 3-dihydroxy-6-nitro-7-sulphamoyl-benz(F)quinoxaline (NBQX) which has a 30-fold greater selectivity at displacing the binding of [3H]AMPA, rather than that of [3H]kainate [48]. NBQX also has similar 30 fold higher potency as an antagonist of AMPA-evoked depolarisations on cortical slices in vitro [48, 49]. In a later development of competitive AMPA receptor antagonists, LY293558, a decahydroisoquinoline with a tetrazole group substituted at the 6 position, was shown to selectively displace [3H]AMPA binding on rat brain membranes and to antagonise AMPA-induced depolarisations on cortical slices and, following systemic administration, to block central effects of AMPA receptor agonists [50]. This compound is also interesting from a medicinal chemistry stand-point. In LY293558 the decahydroisoquinoline is separated from the tetrazole by 2 carbons [50]; a single carbon in this chain yields a selective NMDA antagonist, LY233536 [51] whereas a direct carbon linker gives a non-selective NMDA and AMPA antagonist, LY246492 [52].

244 Similar data to that with LY293558 has been reported for two analogues of AMPA called AMOA and AMNH which respectively are substituted with a 3carboxymethoxy and a 2-methylisoxazole on the isoxazole ring of AMPA [53]. AMOA selectively displaces [3H]AMPA binding whereas AMOA displaced [3H]AMPA and low affinity [3H]kainate binding more or less equally; neither compound displaced high affinity [3H]kainate binding. On cortical slices AMOA and AMNH were weakly selective for AMPA- and kainate- induced depolarisations respectively [53]. AMPA r e c e p t o r channel blockers A number of invertebrate polyamine toxins have recently been shown to be open channel blockers of glutamate receptors initially at the neuromuscular junction of locusts [54] and recently on central mammalian neurones [54-57]. These toxins include argiotoxin 636, Joro spider toxin and philanthotoxin. Although these polyamine toxins block NMDA receptors at least in some preparations, in others they also block responses mediated by AMPA receptors more selectively [58-60].

Allosteric modulators of AMPA receptors. In addition to the above competitive antagonists, a 2,3-benzodiazepine, GYKI 52466 [61-63] has highlighted a new mechanism of modulating non-NMDA receptors in a non-competitive manner. GYKI 52466, selectively reduces responses to AMPA and not to NMDA, and is about one fifth as potent against kainate responses, on rat cortical slices [63]. This is, therefore, another useful tool for separating between AMPA and kainate receptors. LY300164 (GYKI 53655), the 3-methyl-carbamoyl derivative of GYKI 52466, is approximately 10 times more potent than GYKI 52466 on AMPA receptors but less potent on kainate receptors [64, 65]. As desensitisation is a prominent feature of AMPA receptors, a plant lectin, concanavilin A, previously shown to block desensitisation of insect glutamate receptors [54], was tested and found to enhance AMPA and kainate responses on hippocampal [43, 66], retinal [67] and dorsal root ganglion (DRG) [68] neurones. More recently, a series of benzothiazides including diazoxide and cyclothiazide, were shown to reduce glutamate receptor desensitisation and hence increase AMPA responses on hippocampal [69, 70] and cortical [71] neurones. As will be discussed below, it has become clear that concanavilin A and cyclothiazide act selectively on rapidly inactivating kainate and AMPA responses respectively. Molecular biology of AMPA and kainate receptors. An AMPA receptor, GluR1 (or GluRA) was the first glutamate receptor to be cloned and expressed [72]. Our concepts of AMPA and kainate receptors have been revolutionised over the past six years through the application of molecular biology techniques to this area. In the ensuing five years, the total number of non-NMDA receptor subunits has increased to nine. They can be divided into two major types: a) the AMPA receptors, GluR1-4, which bind AMPA with high affinity and have a rapidly desensitising response to this agonist and a smaller but non-desensitising response to kainate. These channels with the exception of GluR2 are permeable to calcium [73]. In the proposed TM1 region of GluR2 an

245 arginine occurs at the site at which glutamine is positioned in GluR1,2 and 4. This so called Q/R site has been shown by site directed mutagenesis studies to be a major determinant of calcium permeability [35-37]. b) the kainate receptors, GluR5-7 and KA1-2 which bind kainate with medium and high affinity respectively [35-37] Only GluR5-6 form homomeric channels whereas the others require heteromeric expression with GluR5 or 6 to become functional. GluR5 and 6 also have the Q/R site in the TM2 region and in this case editing appears to be variable throughout the CNS [35-37]. Only a small part of the molecular biology will be covered here; more comprehensive reviews are recommended [35-37, 74, 75]. Although the molecular pharmacology of glutamate receptors is still in its infancy, some interesting data are emerging:1. The calcium permeability of non-NMDA receptors is controlled largely by the so called Q/R site in the putative second transmembrane segment (TM2). This site is occupied by glutamine (Q) in GluRsl, 3 and 4 but by an arginine (R) in GIuR2. This arginine accounts for the low permeability to calcium of both homomeric or hetromeric channels with GluR2. The equivalent site in the NMDA receptors is occupied by an asparagine which may account for the calcium permeability and magnesium block of the NMDA channel. The genomic DNA codes for a glutamine at this Q/R site even in GluR2; so editing of RNA (CAG to CIG by adenosine deaminase) results in an arginine in the GluR2 protein. 2. Desensitisation is controlled in part by alternative splicing of the so-called flip and flop region in the loop between TM3 and TM4 [76]. The presence of glycosylation sites [77-79] and at least part of the agonist binding domain [80] in the same loop suggests its extracellular location. 3. The topology of the glutamate receptor as a result of such studies with m u t a n t receptors is now thought to be quite different from the four transmembrane crossing model suggested for nicotinic receptors, etc. [74,77-79]. TM2 is thought to loop within the membrane rather than crossing it, which in turn leads to an extracellular TM3-4 loop and an intracellular carboxy terminus. The Q/R site is thought to be near the apex of the intramembrane TM2 loop. This model, which has similarities with voltage-dependent ion channels, highlights the limitations of interpretations based solely on hydrophobicity plots. 4. NBQX and CNQX are relatively non-selective antagonists across all the recombinant non-NMDA receptors. Thus NBQX blocks GluR1-4 receptors in the 100nM region [81, 82] and GluR5 and 6 in the 2mM region [83, 84]. 5. By contrast LY293558, tested on human recombinant receptors, displaces GluR1-4 binding with 2-40uM potency, and 900nM potency on GluR5 but is almost inactive (mM) on GluR6 (R.J. Kamboj - unpublished observations). This selectivity is also seen in patch clamp studies [84]. Thus LY293558 could be used to differentiate between GluR5 and GluR6 based kainate receptors, although the situation in GluR6 heteromerics is unkown. 6. GYKI 52466, and other more potent 2,3-benzodiazepines, are active on GluR1-4 with only small effects on the kainate preferring subtypes. Thus, LY300164 (GYKI 53655) is effective at lmM as an antagonist of recombinant GluR1-4 and native AMPA receptors whereas 100uM is required to block native and recombinant kainate receptors (64,65, D. B l e a k m a n unpublished observations).

246 7. Interestingly the two blockers of desensitisation, cyclothiazide and concanavilin A, show complimetary selectivities. Cyclothiazide blocks the desensitisation on recombinant GluR1-4 but, if anything, reduces responses on on kainate receptors, GluR5 and 6 [85]. Concanavilin A on the other hand markedly potentiates responses on these kainate receptors but is only weakly effective on the recombinant AMPA receptors [85]. Such results parallel those from the GluR1-4-dominated hippocampal neurones and the GluR5-dominated dorsal root ganglion cells [64, 68, 86, 87] and show the usefulness of these compounds in differentiating between AMPA and kainate receptors. 8. Argiotoxin 636, Joro spider toxin and philanthotoxin blocks AMPA receptors GluR1, 3 and 4 but is much less active on the homomeric or heteromeric channels containing GluR2 and GluR6 [88-90]. GluR6 may also be edited to an arginine at the Q ~ site. Hence this differential effect of toxins has been related to the Q/R site editing of GluR2 and unedited, glutaminecontaining, versions of GluR2 and GluR6 are sensitive to Joro spider toxin. Similarly conversion to an arginine at the Q/R site of GluR4 renders it insensitive to argiotoxin. So the two properties of calcium permeability and toxin efficacy appear to be controlled at least partially by the molecular entities in the channel.

Metabotropic glutamate receptors.

In addition to ion channel coupled receptors, glutamate also activates second messenger systems via G-protein coupled receptors. The molecular biology and pharmacology of metabotropic glutamate receptors (mGluRs) will not be covered in this article and the following reviews are suggested [37, 91-93]. Suffice it to say that much of the pharmacology of mGluRs has been elucidated on the hemisected rat spinal cord p r e p a r a t i o n with pre- and post- synaptic receptor subtypes being pharmacologically identified [92, 94]. SPINAL CORD PHARMACOLOGY

Methods.

Two basic techniques have contributed much to the advances in the pharmacology of glutamate receptors. The first was the in vivo spinal cord preparation using the technique of microelectrophoresis to administer compounds into the region of single neurones of cats and rats whilst making extracellular recordings of their action potential discharges [2, 19]. In this way neurones can be excited by a number of excitatory amino acids and test compounds can then be administered locally or systemically to study any specific effects on particular receptors. Compounds may also be administered while recording synaptic responses so that receptors mediating neurotransmission can be examined. The second technique is the hemisected spinal cord in vitro, initially of amphibia but now more commonly of neonatal rats [6]. Grease-seals or suction electrodes are used to record DC potentials or synaptic responses from ventral or dorsal roots. Since compounds are usually added to the bathing solution, more quantitative information from this in vitro preparation can be obtained than from microelectrophoresis experiments in vivo.

247 Much of the basic pharmacology of glutamate receptors was elucidated from these two spinal cord preparations in vivo or in vitro. Thus, the differential sensitivity of neurones to kainate, quisqualate and NMDA [3], the differential antagonistic effects of DAA, HA-966, GAMS, GDEE, D-AP5, Mg 2+ and ketamine [6-9, 19] and the effects of these antagonists on synaptic transmision [11-13, 95] were initially described in these spinal preparations.

Results. Competitive non-NMDA antagonists. NBQX reduces responses of rat spinal neurones to electrophoretically administered AMPA and kainate. There is no differential effect on these two agonists but responses to NMDA are unaffected [49]. Part of the depolarising response to kainate of the hemisected rat spinal cord in vitro and cortical slices is, however, resistant to NBQX and is interestingly sensitive to the barbiturate, methohexitone [48, 49]. Similarly electrophoretically administered LY293558 [50] and AMOA [53] reduce AMPA and kainate responses in parallel on spinal neurones in vivo whereas, on cortical slices, these two antagonists preferentially reduce responses to AMPA. LY293558 is also active following systemic administration; thus 2, 5 and 10 mg/kg i.v. produced 18+2%, 49+4% and 86+7% inhibitions respectively of responses of spinal neurones to AMPA. The 10mg/kg close reduced NMDA responses by only 8+4%. C h a n n e l blockers. Philanthotoxin, from the Egyptian digger wasp, selectively and potently blocks responses of spinal neurones in vivo to AMPA and kainate but not to NMDA [60]. This observation was extended to argiotoxin 636, Joro spider toxin and Nephila spider toxin [60]. With the exception of argiotoxin, all the toxins showed good selectivity for non-NMDA receptors relative to those for NMDA, but again did not differentiate between AMPA and kainate. The onset of block and recovery was considerably slower than that of electrophoretically administered competitive antagonists and the recovery was clearly dependent on frequency of agonist application suggesting that, as in other preparations, these act as open channel blockers [54, 57]. Allosteric modulators. The 2,3-benzodiazepine, GYKI 52466, was first shown to block spinal reflexes [61] and subsequently to block responses to AMPA on spinal neurones in vivo [63]. Following electrophoretic ejection of GYKI 52466, responses to kainate were reduced in parallel with those to AMPA whereas those to NMDA were unaffected. The block is clearly non-competitive on cortical slices and presumably will also be on spinal cord tissue when tested in vitro. LY300164 (GYKI 53655) was a more potent AMPA antagonist t h a n GYKI 52466 on spinal neurones but had the same selectivity [96]. For example, following intravenous (5mg/kg) or oral (10mg/kg) administration, LY300164 reduces AMPA responses by 80-100% with only minor effects on responses to NMDA. Cyclothiazide administered elctrophoretically increased response to AMPA

248 and kainate to a similar extent with no effect on those to NMDA [97]. Given intravenously lmg/kg, cyclothiazide enhanced responses of 6 spinal neurones to AMPA by 119 + 28 % and those to NMDA by an insignificant 16 + 6%. Again with cyclothiazide, AMPA responses were enhanced to a greater extent than those of kainate on cortical slices [71]. Concanavilin A has not been tested in vivo but on dorsal root ganglion neurones and dorsal root fibres in vitro responses to kainate are enhanced by this plant lectin but not by cyclothiazide [64, 68, 86, 87]. Concanavilin A also enhances responses of low doses of kainate on cortical wedges to a greater extent t h a n those of AMPA (D. Lodge - unpublished observations). Interestingly when tested in the presence of either NBQX or LY300164, cyclothiazide reversed the effects of LY300164 but not those of NBQX [97]. Thus cylothiazide changed the reduction of responses to AMPA by NBQX from 69+5% to 50+6% whereas those by LY300164 were changed from 68+5% to 21+7%. An i n t e r a c t i o n , therefore, seems likely b e t w e e n the cyclothiazide and 2,3benzodiazepine sites on the AMPA receptor complex. DISCUSSION.

On spinal as on other neurones there is a clear separation between nonNMDA and NMDA receptors. Responses to AMPA and kainate are, however, not easily separated pharmacologically in vivo by competitive (NBQX, LY293558 & AMOA) and non-competitive (GYKI 52466, LY300164 & polyamine toxins) antagonists nor by cyclothiazide, a blocker of desensitisation. Hence it appears t h a t AMPA and kainate excite spinal neurones via the same or similar receptors. The effects of 2,3-benzodiazepines and cyclothiazide make it likely that this is an AMPA receptor of the GluR1-4 type, since cyclothiazide and 2,3-benzodiazepines have little effect on the kainate (GluR5-6) subtype of receptors [64, 65, 68, 8587]. F u r t h e r m o r e the activity of Joro spider and other polyamine toxins suggest t h a t the AMPA receptors contain only a low proportion of GluR2 subunits, since receptors including this subunit are insensitive to these toxins [88-90]. Because the effectiveness of Joro spider toxin is dependent on the presence of glutamine r a t h e r t h a n arginine at the Q ~ site on TM2, another possibility is that GluR2 s u b u n i t s are not fully edited in the r a t spinal cord. Both of these two possibilities suggests t h a t AMPA receptors on these spinal neurones are permeable to calcium and hence could mediate calcium induced cell death when glutamate levels are increased as in spinal ischaemia or trauma. The reversal of 2,3-benzodiazepine, b u t not NBQX, a n t a g o n i s m by cyclothiazide is interesting. Initially this might suggest an interaction between the binding sites of these two compounds but it is strange that the response of AMPA during the NBQX administration is not enhanced. In experiments in cortical slices, a similar phenomenon is seen with cyclothiazide shifting the doseresponse curve of 2,3-benzodiazepines, but not of NBQX, to the right but in a non-parallel fashion. In patch clamp experiments on hippocampal neurones the interaction between cyclothiazide and 2,3-benzodiazepines has been suggested to be competitive [98] but on AMPA-evoked noradrenaline release cyclothiazide did

249 not change the IC50 either of these non-competitive or of competitive AMPA antagonists [99]. With such divergent data more experimentation is required. There are two obvious outstanding questions which require attention. Firstly, what is the role of kainate receptor subunits in the spinal cord? These have been demonstrated in localisation experiments. There is high level of GluR5 expression in the dorsal root ganglion [85] and of this and other kainate subunits in the cord proper [100]. On dorsal root ganglia and C fibres, kainate, rather than AMPA, produces depolarisations with desensitising response which was reversed by concanavilin A [64, 68, 86, 87]. This may then represent a pure population of kainate receptors? In the present in vivo experiments, however, no responses to kainate insensitive to the AMPA receptor selective compounds described above were observed, suggesting that there are few true kainate receptors on or near the neuronal soma from which recordings were made. It seems likely that kainate receptors therefore have a dendritic, or more likely a presynaptic, location. In this position they presumably act as autoreceptors. There is supportive evidence of this in the literature both from electrophysiological studies on dorsal root fibres and from release experiments [see 101 & 102]. The NBQX resistant component in grease seal preparations of the neocortex [101] and spinal cord [102] are compatible with this, since depolarisation of terminals would contribute to the signal seen in such experiments. S e c o n d l y , it should be remembered that the original separation of AMPA and kainate receptors came from spinal cord experiments using GDEE and GAMS. Why did these two rather weak antagonists separate between AMPA and kainate responses when the newer, more potent and selective AMPA antagonists do not? One possibility would be that GDEE and GAMS change the receptor configuration into kainate- and AMPA- preferring states respectively which might be equivalent to a desensitised and non-desensitised state. The similarity of the effects of cyclothiazide on response to AMPA and kainate make this seem unlikely. Unfortunately there has been no extensive study of GDEE and GAMS on recombinant receptors which might shed some light on the conundrum.

Acknowledgements:

We would like to thank those Drs Paul Ornstein, Tage Honore and Istvan Tarnawa for supplies of LY293558, NBQX, and GYKI 52466 respectively, and our earlier colleagues Martyn Jones, Sophie Zeman and Andrew Palmer for allowing us to represent their published data.

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Honore T, Davies SN, Drejer J, Fletcher EJ, et al. Science 1988; 241:701703. Sheardown MJ, Nielsen EO, Hansen AJ, Jacobsen P, Honore T. Science 1990; 247:571-574. Lodge D, Jones MG, Palmer AJ. Can J Physiol Pharmacol 1991; 69:1123. Ornstein PL, Arnold MB, Augenstein NK, Lodge D, et al. J Med Chem 1993; 36:2046-2048. Ornstein PL, Schoepp DD, Arnold MB, Augenstein NK, et al. J Med Chem 1992; 35:3547-3560. Ornstein PL, Arnold MB, Augenstein NK, Leander JD, et al. J Med Chem in press. Krogsgaard-Larsen P, Ferkany JW, Nielsen EO, Madsen U, et al. J Med Chem 1991; 34:123-130 Jackson H, Usherwood PNR. Trends Neurosci 1988; 11:276-283. Eldefrawi A, Eldefrawi T, Konno ME, Mansour NA, et al. Proc Natl Acad Sci 1988; 85:4010-4013. Parks TN, Mueller AL, Artman LD, Albensi BC, et al. J Biol Chem 1991; 266:21523-215529. Priestley T, Woodruff GN, Kemp JA. Br J Pharmacol 1989; 97:1315-1323. Ashe JH, Cox CL, Adams ME. Brain Res 1989; 480:234-241. Saito M, Kawai N, Miwa A, Pan-Hou H, Yoshioka M. Brain Res 1985; 481:16-24. Jones MG, Lodge D. Eur J Pharmacol 1991; 204:203-209. Tarnawa I, Farkas S, Bersenyi P, Pataki A, Andrasi, F. Eur J Pharmacol 1990; 167:193-199. Ouardouz M, Durand J. Neurosci Lett 1991; 125:5-8. Lodge D, Jones MG, Palmer AJ, Zeman S. In: Drug Research related to Neuroactive Amino Acids, Schousboe, A., Diemer, N.H. and Kofod, H. (eds,) Munksgaard, Copenhagen. 1992; 153-160. Wilding TJ, Huettner JE. Mol Pharmacol 1995; 47:582-587. Paternain AV, Morales M, Lerma J. Neuron 1995; 14:185-189. Zorumski CF, Thio LL, Clark DB Neuron 1990; 5:61-66. O'Dell TJ, Christensen BN. J Neurophysiol 1989; 61:1097-1109. Huettner JE. Neuron 1990; 5:255-266. Yamada KA, Rothman SM. J Physiol 1992; 458:409-423. Patneau DK, Vyklicky L Jr, Mayer ML. J Neurosci 1993; 13:3496-3509. Palmer AJ, Lodge D. Eur J Pharmacol-Mol Pharmacol 1993; 244:193-194. Hollmann M, O'Shea-Greenfield A, Rogers S, Heinemann S. Nature 1989; 343:643-648. Hollman M, Hartley M, Heinemann S. Science 1991; 252:851-853. Dani JA, Mayer ML. Curr Opin Neurobiol 1995; 5:310-317. Bettler B, Mulle C. Neuropharmacology 1994; 24:123-139. Sommer B, Keinanen K, Verdoorn TA, Wisden W, et al. Science 1990; 249:1580-1585. Wo ZG, Oswald RE. Trends Neurosci 1995; 18: 161-168. Hollmann M, Maron C, Heinemann S. Neuron 1994; 13:1331-1343. Bennett JA, Dingledine R. Neuron 1995; 14:373-384. Stern-Bach Y, Bettler B, Hartley M, Sheppard PO et al. Neuron 1994; 13:1345-1357.

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Lambolez B, Audinat E, Bochet P, Crepel F, Rossier J. Neuron 1992; 9:247258. 82 Stein E, Cox JA, Seeburg PH, Verdoorn TA. Mol Pharmacol 1992; 42:864871. 83 Sommer B, Burnashev N, Verdoorn TA, Keinanen K, S et al. EMBO J 1992; 11:1651-1656. 84 Bleakman D, Pearson K, H a m a n S, Kamboj R, Schoepp D. Br J. Pharmacol 1995; 115:112P. 85 Partin KM, Patneau DK, Winters CA, Mayer ML, Buonanno A. Neuron 1993; 11:1069-1082. 86 Wong LA, Mayer ML. Mol Pharmacol 1993; 44:504-510. 87 Pook P, Brugger F, Hawkins NS., Clark KC, et al. Br. J. Pharmacol. 1993; 108;179-184. 88 Herlitze S, Raditsch M, Ruppersberg JP, Jahn W, et al. Neuron 1993; 10:1131-1140. 89 Blaschke M, Keller BU, Rivosecchi R, Hollmann M, et al. Proc Natl Acad Sci USA 1993; 90:6528-6532. 90 Brackley PTH, Bell DR, Choi S-K, Usherwood PNR. J Pharmacol Exp Ther 1993; 266:1573-1580. 91 Schoepp DD, Conn PJ. Trends Pharmacol Sci 1993; 14:13-20. 92 Watkins JC, Collingridge GL Trends Pharmacol Sci 1994; 15:333-342. 93 Pin J-P, Duvoisin R. Neuropharmacology 1995; 34: 1-26. 94 Roberts PJ Neuropharmacology 1995; 34:813-819 95 Headley PM, Grillner, S Trends Pharmacol Sci 1989; 11:205-211. 96 Lodge D, Bond A, Palmer AJ. Br J Pharmacol 1994; 112:13P. 97 Lodge D, Bond A, Birds D, Palmer AJ. Neurosci Soc Abs 1993; 19:124.4. 98 Zorumski CF, Yamada K, Price MT, Olney JW. Mol Pharmacol 1991; 40: 45-51. 99 Desai MA, Burnett JP, Ornstein PL, Schoepp DD. J Pharmacol Exp Ther 1995; 272:38-43. 100 Tolle TR, Berthle A, Zieglgansberger W, Seeburg PH, Wisden W. J Neurosci 1993; 13:5009-5028. 101 Palmer AJ, Zeman S, Lodge D. Eur J Pharmacol 1992; 221:205-209. 102 Zeman S, Lodge D. Br J Pharmacol 1992; 106:367-372.

Perspective in Receptor Research D. Giardin~t, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

253

GABAB Receptor Antagonists: New Tools and Potential New Drugs W. Froestl, S.J. Mickel, C. Mondadori, H.-R. Olpe, M.F. Pozza, P.C. Waldmeier, and H. Bittiger Research and Development Department, Pharmaceuticals Division, CIBA-GEIGY AG, K-136.5.25, CH-4002 Basel, Switzerland

INTRODUCTION

The most abundant inhibitory neurotransmitter in the mammalian central nervous system is y-aminobutyric acid (GABA), which interacts with two types of receptors designated GABAA and GABAB by Hill and Bowery in 1981 [1]. GABAA receptors are linked to chloride channels and transmit fast synaptic inhibition. The actions of GABAB receptors are mediated indirectly through heterotrimeric G-proteins of the subtypes Go and Gil [2]. Agonists for GABAB receptors are GABA and the (R)-(-)enantiomer [3] of the antispastic agent baciofen (Fig. 1) synthesized in 1962. GABAB receptor subtypes are located on postsynaptic as well as on presynaptic sites. Postsynaptic GABAB receptors are coupled via pertussis toxin (PTX) sensitive Gproteins to potassium channels [4,5] of the GIRK class (G-protein activated inwardly rectifying K + channels) [6-8]. One of the physiological roles of the postsynaptic GABAB receptors is to mediate the late inhibitory postsynaptic potential (IPSPB) [4,9,10]. An important function of presynaptic GABAB receptors is modulation of the release of many neurotransmitters, as GABA, glutamate, aspartate, noradrenaline, dopamine, serotonin and of various neuropeptides, such as substance P, cholecystokinin and somatostatin. Activation of GABAB autoreceptors causes an inhibition of GABA release, an effect dependent on PTX sensitive Go proteins linked to potassium and N-type and P-type calcium channels [11,12]. GABAB receptors on excitatory nerve endings are also associated with PTX sensitive G proteins. Some effects on glutamate release, however, are mediated by PTX insensitive G proteins, whose links to ion channels are still unknown [12].

GABAB RECEPTOR ANTAGONISTSWITH MICROMOLARAFFINITY Selective GABAB receptor antagonists have been elusive for many years, in 1987 Kerr et al. introduced phaclofen (Fig. 1), the phosphonic acid analogue of baclofen as the first peripheral and central baclofen antagonist [13,14]. However, neither phaclofen (IC50 = 130 I~M), nor the more potent sulfonic acid derivatives of baclofen [15-17], saclofen (Fig. 1; IC50 = 26 I~M) or 2-hydroxysaclofen (Fig. 1; IC50 = 12 I~M; IC50's: inhibition of binding of [3H]CGP27492, Fig. 2) were capable of penetrating the blood-brain barrier and were inactive in vivo as central nervous system agents. A new class of GABAB receptor modulators was discovered simultaneously by replacing the carboxylic acid groups of GABA, GABOB (i.e. y-amino-13-hydroxy-

254

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R = CO2H R = PO3H2 R = SO3H R = SO3H

X= H GABAB receptor agonist X = H GABAB receptor antagonist X = H GABAB receptor antagonist X = OH GABAB receptor antagonist

Figure 1. Structures of GABAB receptor modulators butyric acid) or baclofen, respectively, by potentially isosteric phosphinic acid residues [18,19], an idea, which was first explored for naturally occurring o~-amino acids [20]. The small phosphinic acid derivatives with residues R equal to hydrogen (Fig. 2; CGP27492, IC5o = 2.4 nM) or R equal to methyl (Fig. 2; CGP35024, IC50 = 6.6 nM, identical to SK&F97541 [21]) displayed properties of very potent GABAB receptor agonists and even surpassed the affinities of GABA (IC5o = 25 nM) and of baclofen (IC50 = 35 nM; IC5o's: inhibition of binding of [3H]baclofen) [22]. [3H]CGP27492 [23,24] offers several advantages over [3H]baclofen or [3H]GABA as a radioligand for GABAB receptor binding assays: 15 times higher potency, high specific binding and the possibility to carry out filtration assays. All 3-aminopropyl-phosphinic acids with larger residues bound to phosphorus, i.e. R > ethyl (Fig. 2, CGP36216, IC50 = 2 pM) including the diethoxymethyl-derivative CGP35348 [25,26] (Fig. 2; IC5o = 27 pM) antagonized the actions of baclofen in various biochemical and electrophysio!ogical paradigms [27]. Interestingly, CGP47656 (Fig. 2; R = CHF2) with a substituent, the size of which is intermediate between that of a methyl and an ethyl group, showed properties of a GABAB receptor partial agonist as was demonstrated in GABA release experiments [22]. When GABA B receptor agonists interact with GABA B autoreceptors they cause an inhibition of the electrically induced release of [3H]GABA from rat cortical slices. When the slices were stimulated at a frequency of 0.125 Hz CGP47656 inhibited [3H]GABA release (IC50 = 10.6 pM), whereas upon stimulation at 2 Hz it increased the release of [3H]GABA (EC15 o = 62 pM, i.e. the concentration causing a 50% increase) [22].

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R = CHF2 R2 = H CGP47656 GABA B receptor partial agonist CGP 36216 GABA B receptor antagonist R = Et R2 = H R = CH(OEt)2 R2 = H CGP 35348 GABA B receptor antagonist R = n-Bu R2 = H CGP 36742 GABA B receptor antagonist R = CH2C6H11 R2 = H CGP 46381 GABA B receptor antagonist R = CH206H11 R2 = OH CGP 51176 GABAB receptor antagonist

Figure 2. Structures of GABAB receptor modulators Penetration of the blood-brain barrier was demonstrated for CGP35348 after i.v. or i.p. administration and for the n-butyl-phosphinic acid derivative CGP36742 [28] (Fig. 2; IC50 = 38 I~M) and the cyclohexylmethyl-phosphinic acid derivative

255

CGP46381 (Fig. 2; IC50 = 4 I~M; IC50's: inhibition of binding of [3H]CGP27492) after oral administration in an electrophysiological paradigm in vivo by antagonizing the response to baclofen applied iontophoretically to rat cerebral cortex [28,29]. Microdialysis analysis in vivo confirmed that CGP36742, after i.v. administration, rapidly entered the frontal cortex and the CSF of rats with peak levels at 1 hr [30].

THERAPEUTIC POTENTIAL OF GABAB RECEPTOR ANTAGONISTS Despite their weak affinities to GABAB receptors some of the orally active GABAB receptor antagonists (Fig. 2) showed remarkable - and very different - in vivo activities in a multitude of pharmacological tests. CGP36742, but not CGP46381 or CGP51176 showed learning and memory improving effects in a variety of paradigms indicative for amelioration of cognitive functions. CGP36742 at doses of 0.3, 3 and 30 mg/kg orally produced an improved retention performance of mice in a one-trial stepdown passive avoidance paradigm [31,32]. CGP35348 also showed memory improving effects in the same paradigm provided the compound was administered i.p. Chronic treatment of 27 months old rats with 0.3, 3 or 30 mg/kg of CGP 36742 for 30 days resulted in an improved learning performance in a multiple trial one-way active avoidance test. After chronic treatment with 0.5 mg/kg of CGP36742, orally, Rhesus monkeys performed better in a "conditional spatial color" task, where learning of the placement of a food reward hidden under an upturned beaker in relationship to the color of the beaker was required [31-34]. Memory facilitation was observed in a radial maze task in mice after chronic treatment at doses of 10 and 100 mg/kg of CGP36742, i.p. administration, whereas (R)-(-)-baclofen at doses of 2 mg/kg i.p. induced a significant impairment of performance thus implying that the learning and memory effects are mediated by GABAB receptor mechanisms [35]. All GABAB receptor antagonists active in vivo (CGP's 35348, 36742, 46381, 51176 and others) suppressed spontaneous 3 Hz spike and wave discharges in various animal models of absence epilepsy [36-41] probably due to blockade of postsynaptic GABAB receptors, which are present in high density in the medial lateral part of the thalamus. The oscillating spike and wave discharges may be caused by GABAB receptor-mediated IPSP's (vide infra) preparing thalamocortical cells for burst firing by activated low-threshold calcium potentials [42]. CGP36742 showed effects in the forced-swimming test [43] indicating potential antidepressant activity, which may be due to some extent to significant upregulation of GABAB receptors after chronic treatment of rats with this drug [44]. CGP51176 ([3-amino-2(R)-hydroxypropyl]-cyclohexylmethyl-phosphinic acid) showed pronounced antidepressant effects [45] in the chronic stress model of Willner [46]. The broad spectrum of pharmacological activity of these chemically quite similar GABAB receptor antagonists may originate from their different affinities to presynaptic GABAB receptor subtypes influencing the release of excitatory and inhibitory neurotransmitters in different ratios [47,48]. There are also significant differences in their interactions with postsynaptic GABAB receptors as was shown in electrophysiological investigations [28].

256

GABAB RECEPTOR ANTAGONISTS AS TOOLS TO ELUCIDATE MECHANISMS OF INDUCTION OF LONG-TERM POTENTIATION GABAB receptors influence a multitude of modulatory synaptic processes in the central nervous system [49-51] as well as in the periphery [50,52]. GABAB receptor antagonists have been used extensively to elucidate the underlying mechanisms, which is reflected in more than hundred papers describing various biological effects of CGP35348, alone. In order to illustrate the usefulness of the new GABAB receptor antagonists as pharmacological tools, the investigations on the roles of pre- and postsynaptic GABAB receptors for the induction of long-term potentiation are described in more detail. Long-term potentiation is the use-dependent strengthening of glutamate-mediated excitatory synaptic transmission [53]. it can be induced by delivering high frequency tetanic stimulation to selected pathways, e.g. the Schaffer collateral-commissural fibers in the hippocampus. The frequency most commonly employed is 100 Hz, a frequency reached by discharging (firing) pyramidal neurons [54], which causes an enhanced release of glutamate. Interaction of glutamate with AMPA receptors results in an enhanced influx of Na+ ions through the AMPA receptor-linked ion channel leading to a depolarization of the postsynaptic membrane. The depolarization relieves the block of Mg 2+ ions from the NMDA receptor-linked ion channel allowing an influx of Ca2+ ions. Therefore the NMDA receptor functions as a molecular coincidence detector that allows calcium ion influx only in conjunction with

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257

depolarization in the target dendrite, as Donald Hebb of McGill University postulated in 1949 [55]. The receptor systems involved in the induction processes of LTP are shown in Figure 3 [53]. Via interaction with calmodulin the enhanced concentration of Ca2+ ions effects an activation of the Ca2+/calmodulin-dependent protein kinase I1. Long-term memory storage processes lasting for days and weeks, require gene transcription involving immediate early genes such as c-fos and zif/268 and transcription factors as the cAMP response element binding protein [53,56]. GABAB Receptor Antagonists Facilitate Induction of Long-term Potentiation Experiments in hippocampi of rats, in vitro and in vivo, showed that the blood-brain barrier penetrating GABAB receptor antagonist CGP35348 was capable of facilitating the induction of homosynaptic LTP [57,58], which was induced by tetanic stimulation. The results of the LTP experiments in vitro are presented in Figure 4 [58]. LTP was induced by two consecutive trains of rectangular pulses (train interval 5 s) lasting in total for 200 ms, delivered via the stimulation electrode placed on the Schaffer collaterai-commissural fibers. Individual pulses lasted for 0.2 ms. LTP was induced using 30 Hz (left panel) or 100 Hz (right panel) tetanic stimulation. Twenty minutes following LTP induction the mean population spike increase amounted to 120% using 30 Hz and to 145% using 100 Hz tetanic stimulations. In both cases, slices exposed to 1 mM CGP35348 showed a significantly stronger LTP, an increase of the population spike of up to 150% with 30 Hz and of up to 185% with 100 Hz tetanic stimulations. The differences were statistically significant after 5, 10, 15 and 20 minutes following LTP induction.

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Figure 4 The facilitatory action of CGP35348 on LTP induction by 30 Hz (left panel) or 100 Hz (right panel) in rat hippocampal slices in vitro. Data normalized for mean amplitudes prior to tetanic stimulation (=100%). Solid line: control; dashed line: in presence of 1 mM CGP35348; 30 Hz: n=14; 100 Hz: n=15; *p<0.005, **p<0.001, x~_ SEM. (Reproduced with permission of principal investigator and editor of [58])

258

The facilitatory effect of CGP35348 on the induction of LTP in vivo is shown in Figure 5 [58]. 100 mg/kg of CGP35348 (or 0.9% NaCI) were administered intravenously to chloral hydrate anaesthetized rats (n=12). Fifteen minutes later LTP was induced using a series of 100 Hz tetanic stimuli of increasing duration given at intervalls of 30 minutes as indicated in Figure 5 [58]. In the animals treated with CGP35348 LTP was facilitated significantly. The increase in population spike amplitude amounted to 350% after 30 and 60 minutes and reached 450% after further tetanic stimulations at 90, 120 and 150 minutes.

600 -

% increase in population spike amplitude ............................

500 - ........................................

300 200

100

0

',

I

t

!

I

t

1

30

60

90

120

150

180

210rain

t

t

t

1 xl00ms

t

2xl00ms

t

1 x20Oms

2x200ms 1 xl000ms

t

2x1000ms

stimulation frequency 100Hz

Figure 5. The facilitation of LTP by CGP35348 in rat hippocampus in vivo. *p<0.05, * p<0.01, 9 ***p<0.005, ****p<0.001. (Reproduced with permission of principal investigator and editor of [58]). One of the physiological roles of postsynaptic GABAB receptors is the mediation of the late inhibitory postsynaptic potential (IPSP, or current, IPSC) [4,9,10,25,26]. Brief electrical stimulation of the Schaffer collateral-commissural fibers and intracellular recording from a CA1 pyramidal neuron in rat hippocampal slices elicited the following potentials: First the excitatory postsynaptic potentials (EPSP's), which are mediated by AMPA and NMDA receptors, as they can be blocked by the AMPA receptor antagonist CNQX and the NMDA receptor blocker D-AP5, followed by a large hyperpolarization consisting of two components: the early inhibitory postsynaptic potential, with an onset of 3 ms or less, with a peak at 20 ms after the stimulus, lasting for about 200 to 250 ms [10], which is mediated by GABAA receptors. It can be blocked selectively by the GABAA antagonist bicuculline and the chloride channel blocker picrotoxin. Blockade of the early IPSP (or IPSPA) results in epileptic-like discharges of the slice preparation. The second component is the late

259

IPSP, with an onset of about 30 ms, with a peak at 150 to 200 ms lasting for about 700 ms to 1 s [10]. The late IPSP can be blocked selectively by 100 I~M CGP35348, leaving the IPSPA unaffected (Fig. 6) [26]. Blockade of the late IPSP (or IPSPB) did not result in epileptic-like discharges of the slice preparation.

J

J control

J

CGP35 348 lOOpM (l 0 rain)

'

Catp=l~:

5 mV/~ mJ

. , , . , f ~

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Figure6. The blocking action of 1001~M CGP35348 on the late inhibitory postsynaptic potential (IPSP) recorded intracellularly from a rat CA1 pyramidal neuron. (Reproduced with permission of authors and editor of [26]). CGP35348 selectively blocks the IPSPB by blocking its underlying potassium conductance, thus removing a large hyperpolarization, which is responsible for maintaining the block of Mg 2+ ions at the NMDA receptor-linked ion channels [59]. The whole sequence of biological events is shown in Figure 7 [53].

GABAB Receptor Antagonists Impede Induction of Long-term Potentiation

GABAB receptor antagonists facilitate the induction of L TP only, when LTP is induced by tetanic high frequency stimulation employing frequencies of 30 Hz or 100 Hz. In contrast, GABAB receptor antagonists inhibit the induction of L'/P, when induced by primed-burst stimulation [58-64]. In this paradigm LTP is induced by two consecutive primed-bursts delivered at an interval of 5 s. Each primed-burst consists of a priming pulse followed 200 ms later by four primed pulses given at 100 Hz. Individual pulses last for 0.2 ms. This protocol integrates frequencies of both 5 Hz and 100 Hz. As shown in Figure 8 [58] the result is a stable potentiation of the

260

population spike (solid line) similar in magnitude to the potentiation observed with the high frequency stimulation paradigm (see Fig. 4 [58]). in the presence of I mM of

EPSP

nal

p 2 L-Glu

~

~

Dend~e

"

!

L

)G Inhibitonjintemeuron

,

i

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Figure 7. Suppression of the GABAB mediated late IPSP by GABAB receptor antagonists facilitates the induction of LTP [58]. (Drawing reproduced with permission of both authors and editor of [53]).

200-' * 190- ~ , ~ * * 180g

c

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Figure 8. CGP35348 applied at a concentration of 1 mM impaired the development of LTP induced by two primed-bursts. (Reproduced with permission of principal investigator and editor of [58]).

261

CGP35348 (dashed line) there was only an immediate transient potentiation lasting for 5 to 10 minutes but no LTP was induced [58]. At longer intervals the population spike was even weakly depressed. Why are the effects of GABAB receptor antagonists on the induction of LTP by the primed-burst paradigm different from their effects on the induction of LTP by high frequency stimulation? Within a frequency range of 0.5 to 20 Hz repetitive stimuli of identical strength cause a significant depression of the amplitudes of both the early and late IPSP's, a phenomenon called paired-pulse depression or fatigue (or fading) of synaptic inhibition. Figure 9 shows that the decline in IPSP amplitude is most pronounced for the second response, clearly visible in the superimposed drawing of the first and second IPSP (lower right side) [64]. Subsequent stimuli caused smaller reductions and reach a steady state after 3 stimuli. B.

tOO m s

Superimposed

C.

100 mJ

50 ms

Figure 9. Fading of the mossy fiber-evoked inhibitory postsynaptic potentials recorded with the cell hyperpolarized to the reversal potential of IPSPB after stimulation at 5 Hz. (Reproduced with permission of the principal investigator and editor of [64]). The most pronounced effect of paired-pulse depression occurs at an interstimulus interval of 200 ms [64]. The impact of paired-pulse depression is significantly larger for the GABAB receptor mediated inhibitory postsynaptic currents. The IPSCB evoked by the second stimulus was depressed by 62+8%, whereas the IPSCA evoked by the second stimulus was depressed by 37+4% as shown in Fig. 10 [64]. Paired-pulse depression is due to a feedback mechanism involving GABAB autoreceptors [10,59-66]. With the priming stimulus GABA is released into the synaptic cleft and builds up the priming early and late IPSP/C's by interaction with postsynaptic GABAA and GABAB receptors. The GABA concentration in the narrow synaptic cleft (<15 nm) is sufficiently high to feed back to GABAB autoreceptors and thus depresses its own release, which results in depressed amplitudes of the second

262

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.

120

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so

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400

600

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Figure 10. Time course of paired-pulse depression of the amplitudes of both inhibitory postsynaptic currents IPSCA and IPSCB. (Reproduced with permission of principal investigator and editor of [64]).

CONTROL

a

,A

A

1O0 ~

-A

CGP 35348

A

1 mM CGP 35348

A

A

0.5 nA 100 ms

X A

A

i

A

i

Figure 11. CGP35348 blocks the GABAB receptor mediated IPSC and reverses paired-pulse (A) and primed-burst (B)induced depressions of monosynapticallyactivated GABAA receptor-mediated IPSC's. (Reproduced with permission of principal investigator and editor of [60]).

263

and subsequent IPSP/CA'S and IPSP/CB'S. CGP35348 interacts with postsynaptic GABAB receptors and blocks the IPSP/CB's of the priming and the subsequent primed pulses to an extent of 93%. It had no effect of the IPSP/CA of the priming pulse. Figure 11a (upper panel) shows that CGP35348 reversed the paired-pulse depression of the amplitude of the IPSCA of the second pulse by 36% at a concentration of 100 I~M and by 92% at a concentration of 1 mM by blocking GABAB autoreceptors thus restoring the release of GABA to the initial value of the priming stimulus [60]. The induction of LTP according to the primed-burst stimulation protocol is a result of fatigue of synaptic inhibition [60]. Due to paired-pulse depression of both early and late IPSP's the summation of hyperpolarizations of the primed pulses is smaller. The reduction of the sum of hyperpolarizations is equivalent to a depolarization, large enough to relieve the block of Mg 2+ ions from the NMDA receptor-linked channels. The effects of 100 I~M and 1 mM concentrations of the GABAB receptor antagonist CGP35348 on the sum of four primed pulses are shown in Figure 11b (lower panel). At 100 I~M the peak amplitudes of the primedburst IPSC's amount to 121+7%, at 1 mM to 164+10% of control, respectively. The reversal of the paired-pulse depression of the GABAA receptor mediated early IPSC's, (Fig. 11a, upper panel), therefore, outweighs the blockade of the GABAB receptor mediated late IPSC's. By blockade of the GABAB autoreceptors at 1 mM CGP35348 provides a sufficient increase of hyperpolarization to reinforce the block of Mg2+ ions at the NMDA receptor-linked channels and to impede the induction of LTP. The whole course of biological events in the case of induction of LTP via the primed-burst stimulation is summarized in Figure 3 (vide supra, [53]).

GABAB Receptor Antagonists and Long-term Depression

One paper describes the effects of GABAB receptor antagonists on homosynaptic long-term depression (LTD) [67], a persistent activity-dependent decrease of synaptic transmission, while no information is available yet on the effects of GABAB receptor antagonists on heterosynaptic, associative or cerebellar LTD [68]. Homosynaptic LTD was elicited by low frequency (1 Hz) stimulation to the Schaffer coilateral-commissural fibers in the CA1 region of rat hippocampus for 15 minutes. In naive slices of young animals (16 to 22 clays old) a robust LTD was induced, reducing the slope of the field EPSP to 73+_1%. LTD was attenuated by 1 mM of CGP35348 to the extent of 88+_2%. CGP35348 acted primarily by blocking the paired-pulse depression of the GABAA receptor mediated early IPSP's. LTD was completely (95+_5%) blocked by 100 I~M of the NMDA blocker D,L-AP5 confirming that this form of I_TD is dependent upon NMDA receptor activation. However, the situation was different after low frequency stimulation of slices, which had received prior inductions of LTP by high frequency stimulation. The resulting depression is called depotentiation. When LTP was induced by three high frequency trains of 100 pulses of 100 Hz spaced 30 s apart followed by low frequency stimulation 30 minutes later the induced depotentiation amounted to 39_-+6% of the originally potentiated level. CGP35348 (1 raM) had no effect on depotentiation (45+_5%), whereas D,L-AP5 (100 taM) attenuated depotentiation (74_+6%).

264

GABAB RECEPTORS AND ALZHEIMER'S DISEASE GABAB receptors are heavily affected in brains of patients suffering from dementia of the Alzheimer type (DAT). Post mortem studies showed that the density of GABAB receptors was reduced by 50% to 70% in several areas of the hippocampus (Fig. 12, [69]) and the frontal cortex (Fig. 13, [70]). The reduction of the density of GABAA receptor.s was significantly smaller. This may be a consequence of the loss of presynaptic nerve terminals carrying GABAB receptors. In contrast, GABAA receptors are only located on postsynaptic sites.

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Figure 12. Histograms of Bmax values for GABAA and GABAB binding to control (open bars) and DAT (hatched bars) hippocampus. (Reproduced with permission of principal investigator and editor of [69]).

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Figure 13. Histograms of Bmax values for GABAA and GABAB binding to control (open bars) and DAT (stippled bars) frontal gyrus. (Reproduced with permission of principal investigator and editor of [70]).

265

GABAB RECEPTOR ANTAGONISTS WITH NANOMOLAR AFFINITY Despite extensive work on structure activity relationships (SAR) by varying the substituents on the phosphorus and the carbon atoms of 3-aminopropyl- alkyl-, cycloalkyl- or arylalkyl-phosphinic acids (Fig. 2) the affinities of the new GABAB receptor antagonists never surpassed the micromolar range. A significant - and quite unexpected -improvement of the potency of this class of compounds was discovered by substituting the nitrogen of various 3-aminopropyl-phosphinic acids with selected benzyl substituents [71-75]. This finding was very unusual, as it was known from many baclofen analogues, i.e. GABA B receptor agonists, that any substitution on the nitrogen invariably led to a significant loss of affinity. An example: baclofen displays a high affinity to GABAB receptors (IC50 = 35 nM; inhibition of binding of [3H]baclofen), whereas the IC50 of N-monobenzylbaclofen (CGPl1970) is only 16 I~M. However, substitution on the nitrogen of the weak GABA B receptor antagonist CGP35348 (Fig. 2, IC50 = 27 I~M) with a 4-chlorobenzyl residue led to CGP51783 (Fig. 14) displaying an enhanced affinity to GABAB receptors (IC50 = 975 nM). Extensive SAR work, first by varying the substituents on the benzene ring using the operational scheme of Topliss [76] and second by attaching selected substituents to the 3aminopropyl- side chain and to the (z-position of the benzyl-substituent finally led to CGP52432 (IC50 = 55 nM), CGP55845 (IC50 = 6 nM) and CGP54626, the latter also available as [3H]ligand (IC5o = 6 nM, KD = 1.5 nM, [77]). These compounds (Fig. 14) have been used as pharmacological tools by many neuroscientists of very diverse backgrounds in several recent investigations [66,78-85].

~

1..~

CGP51783 RI = H CGP52432 RI = H R1=Me CGP54626 R1 = Me

R20 -

H~i'R

R1

CGP58848

OH

R2 =H R2 =H R2=OH R2 = OH

X=H X=CI X=CI X = CI

R=CH(OEt)2 R=CH(OEt)2 R=CH2C6H5 R = CH2C6Hll

Figure 14. Structures of potent GABAB receptor antagonists None of the potent GABAB receptor antagonists of Figure 14 was active in vivo. Only significantly more hydrophilic derivatives were capable of penetrating the gutblood and blood-brain barriers providing potent GABAB receptor antagonists active after systemic, including oral, administration in several in vivo paradigms, as e.g. CGP57250 (IC50 = 6 nM) or CGP56999 (IC50 = 2 nM; both Fig. 15). COOH

R1

OH

R = CH(OEt)2 R = CH(OEt)2 R = CH2CsH11 R = CH2C6H11

R1 = (R)-Me R1 = (S)-Me R1 = (R)-Me R1 = (S)-Me

Figure 15. Structures of potent GABAB receptor antagonists

CGP57250 CGP56571 CGP56999 CGP56433

266

Interestingly, only in the benzoic acid series were compounds with (R)stereochemistry of the methyl goup in the benzylic position considerably more potent than the corresponding diastereoisomers bearing an (S)-methyl substituent, as CGP56571 (Fig. 15; IC50 = 260 nM) or CGP56433 (Fig. 15; IC50 = 80 nM). In the dichiorobenzyl- as well as in other series, the diastereoisomers with (S)-methyl substituents, e.g. CGP55845 or CGP54626 (both Fig. 14) displayed higher affinities to GABAB receptors than the corresponding (R)-diastereoisomers.

SUBTYPES OF PRESYNAPTIC GABAB RECEPTORS Neurotransmitter release experiments showed that the new GABAB receptor antagonists interacted with several presynaptic GABAB receptor subtypes influencing the release of GABA, glutamate, somatostatin or cholecystokinin. Selected GABAB receptor antagonists were able to block the inhibition of release of various neurotransmitters from potassium ion depolarized rat cerebral cortex synaptosomes elicited by 10 I~M (R)-(-)-baclofen. Based on these experiments a new classification of multiple presynaptic GABAB receptor subtypes (Fig. 16) has been proposed by Raiteri et al. [47,48,78] (Structures: phaclofen: Fig. 1, CGP's 27492, 35348 and 47656: Fig. 2 and CGP52432: Fig. 14). However, experiments in electrically stimulated rat cortical slices did not confirm the results in potassium stimulated rat cortical synaptosomes [86].

GABAB1

GABAB2

CGP27492-sensitive, baclofen-sensitive: cerebral cortex

CGP27492-sensitive, baclofen-insensitive: spinal cord

pKb Drugs

GABABIr Autoreceptor GABA

Phaclofen 5.1 CGP35348 inactive CGP52432 7.9 CGP47656 6.1 a n.d. not determined [48]

GABABII~ Heterorec. Glutamate inactive 6.2 5.7 n.d. a

GABABI~ Heterorec. SRIF

4.9 6.1 6.2 full agonist

GABABI~ Heterorec. CCK 4.8 6.1 7.6 inactive

Figure 16. Raiteri's classification of multiple presynaptic GABAB receptor subtypes [48]

267

CONCLUSION

The discovery of potent, readily water soluble GABA B receptor antagonists capable of penetrating the blood-brain barrier after systemic including oral administration has stimulated many scientists of very diverse backgrounds to use these new pharmacological tools to explore different aspects of GABAB receptor research. Out of many, one example was selected to show in detail how instrumental these compounds proved to be in order to elucidate the mechanisms of induction of long-term potentiation after tetanic or primed-burst stimulation. Extensive in vivo investigations suggest that GABA B receptor antagonists may have therapeutic potential for the treatment of cognition deficits, absence epilepsy, anxiety and depression. REFERENCES

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73 74 75 76 77 78 79 80 81 82 83 84 85 86

PC, Bittiger H. In: Testa B, Kyburz E, Fuhrer W, Giger R, eds. Perspectives in Medicinal Chemistry. Basel: Verlag Helvetica Chimica Acta, 1993; 259-272 Froestl W, Mickel SJ, Bittiger H. Curr Opin Ther Pat 1993; 3: 561-567. Bittiger H, Froestl W, Mickel SJ, Olpe H-R. Trends Pharmacol Sci 1993; 14: 391-393. Froestl W, Mickel SJ. In: Enna S, Bowery NG, eds. The GABA Receptors. 2nd ed. Totowa, N. J.: Humana Press, (in press). Topliss JG. J Med Chem 1972; 15:1006-1011. Bittiger H, Reymann N, Froestl W, Mickel SJ. Pharmacol Commun 1992; 2: 23. Lanza M, Fassio A, Gemignani A, Bonanno G, Raiteri M. Eur J Pharmacol 1993; 237: 191-195. Brugger F, Wicki U, Olpe H-R, Froestl W, Mickel S. Eur J Pharmacol 1993; 235: 153-155. Lacey G, Curtis DR. Exp Brain Res 1994; 101: 59-72. Turgeon SM, Albin RL. Neurosci 1994; 62: 59-72. Davies CH, Pozza MF, Collingridge GL. Neuropharmacol 1993; 32: 1071-1073. Blake JF, Cao CQ, Headley PM, Collingriclge GL, Brugger F, Evans RH. Neuropharmacol 1993; 32: 1437-1440. Jarolimek W, Demmelhuber J, Bijak M, Misgeld U. Neurosci Lett 1993; 154: 3134.. Lambert NA, Wilson WA. Neuron 1993; 11: 1057-1067. Waidmeier PC, Wicki P, Feldtrauer J-J, Mickel SJ, Bittiger H, Baumann PA. Br J Pharmacol 1994; 113: 1515-1521.

Perspective in Receptor Research

D. Giardinh, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

271

Thrombin inhibitors and thrombin receptor agonists/antagonists

J. DiMaio a, P. Winocour a, L. Leblond a, M. Saifeddine b, A. Laniyonub and M.D. Holienberg b aBioChem Therapeutic Inc., 275 Armand-Frappier Boulevard, Laval, Qu6bec H7V 4A7, Canada bDepartment of Pharmacology and Therapeutics, The University of Calgary, Faculty of Medicine, Calgary, Alberta T2N 4N1, Canada SUMMARY The information presented in this article illustrates the feasibility of designing low molecular weight protein mimetics by exploiting the three dimensional array of functional binding elements of the parent protein. In the case of thrombin, the unique mechanism of enzyme inhibition exerted by hirudin is preserved in low molecular weight peptides that mimic the concerted binding to two remote recognition sites on the enzyme surface. Optimization yields compounds having equilibrium dissociation constants in the low picomolar range. The high affinity translates into high efficacy in rodent models of thrombosis. The inhibitors can be tailored to be cleared or metabolized rapidly such that biological activity is terminated. The design of selective thrombin receptor agonists and antagonists, based on the thrombin-revealed self-activation sequence has proved problematic because of unexpected tissue differences in the relative potencies of TRAP agonists and differences in the intrinsic activities of putative antagonists in different assay systems. Remarkably a putative antagonist that could block thrombin's action in a platelet aggregation assay turned out to be an agonist in a smooth muscle contractile assay; and a peptide that displayed partial agonist/antagonist activity in a vascular contractile assay proved to be a full agonist in a platelet aggregation assay. Clearly the complexities of the thrombin receptor system will have to be better understood to assist the design of effective and selective receptor antagonists for therapeutic use. INTRODUCTION Aberrant thrombus formation and deposition on blood vessel walls underlies the pathogenesis of acute cardiovascular disease states which remain the principal cause of morbidity and mortality in the industrialized world [1,2,3]. Plasma proteins, proteases and specific cellular receptors that participate in hemostasis have emerged as important risk considerations in thrombosis and thromboembolic disorders. The clinical manifestations of the above disease states include acute coronary artery and cerebrovascular syndromes, peripheral arterial occlusion, deep vein thrombosis and pulmonary/renal embolism [3]. The most dilabilitating acute events precipitated by these disorders are myocardial infarction and stroke. In addition, the interplay between hemostatic factors and hypertension (4) or atherosclerosis (5) dramatically enhances the manifestation of these pathologic states. Hemostasis is controlled by a delicate balance of finely tuned reactions in the blood coagulation cascade [6-8] and fibrinolytic system requiting zymogen to enzyme activation steps for propagation. This prerequisite, together with the protective (nonthrombogenic)

272 surface provided by the endothelial cell lining [9] implies that hemostasis is biased toward negative regulation. However, aberrations stemming from vascular injury provide a stimulus for explosive activation of surface coagulation with positive feedback amplification through proteolytic activation of required cofactors. Concomitant with this primary procoagulant event is the adhesion and aggregation of platelets [10,11], recruitment of inflammatory cells [12] and proliferation of smooth muscle cells underlying the basement membrane [ 13]. Thus, depending on the severity of injury, acute or recurring thrombosis is a multifactorial process implicating enzymatic, cellular and humoral mediators that respond to the initial injury; but whose chronic presence exacerbate the pathologic states. Relevance of thrombin as a therapeutic target

The enzyme thrombin plays a pivotal role in the formation of thrombus [14] and propagation of disease [15]. Thrombin is a dimeric serine proteinase derived from prothrombin through a single proteolytic step mediated by factor Xa [16,17]. The primary role of thrombin is widely recognized as a procoagulant enzyme through the proteolytic conversion of its substrate fibrinogen to insoluble fibrin (Figure 1) [18]. Despite its FP B

14 U ,:E-G-V-N.D-N.E-E-G.F-F.S.A.Arl~- Gly.H-R.P

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95-98

95-95

:

.

Figure 1. Schematic diagram of the fibrinogen dimer illustrating the cleavage site within the tx and 13chains as well as the RGD motifs that crosslink platelets through the GP IIb, IIIa receptor. "trypsin-like" primary selectivity, thrombin specifically cleaves only two Arg-Gly bonds located on the N-terminal domain of fibrinogen At~ and B~ chain, resulting in the release of two mole equivalents of fibrinopeptide-A and fibrinopeptide-B, respectively [19]. The enzyme also mediates feedback amplification of its action through proteolytic activation of the required cofactors Va, VIIIa as well as the fibrin transglutaminase factor XIIIa that serves to crosslink and strengthen fibrin polymer [20,21]. Fibrin (nogen) contains several RGD sequences within its primary structure that serve as a recognition motif for the integrin GP IIb-IIIa receptor and crosslinks platelets [22-23]. Thus platelets and fibrin (ogen) form an integrated mesh or hemostatic plug. The activated cofactor Va also binds to platelets and serves as a high affinity co-receptor for factor Xa [24]. This prothrombinase complex involving also C a +2 and phospholipid enhances the proteolytic rate exerted by Xa on prothrombin by over five orders of magnitude. Thus, highly

273 concerted proteolytic reactions localized on platelet or endothelial cell surface provide an efficient mechanism for thrombin generation [25]. The role of thrombin in the development of venous thrombosis, wherein thrombi consist mainly of fibrin and erythrocytes can be explained by its primary action on fibrin (ogen) as well as the high volume and low shear forces in small blood vessels. On the other hand, the etiology of arterial thrombosis is more complex and the role of thrombin has remained equivocal, especially in view of alternate mediators of platelet activation including prostaglandin endoperoxides, thromboxane A2, catecholamines, PAF and collagen [26]. This controversy has been resolved in part through the cloning of a specific thrombin receptor which has been shown to be present on numerous cell types including platelets [27], endothelial cells [28] fibroblasts [29,30] vascular smooth muscle cells [31] and macrophages [32]. Accordingly, the cellular actions of thrombin as a mitogenic and chemotactic factor, secretagogue and powerful aggregating agent can be explained as being manifest by classical cell surface receptors. In addition, the demonstration of thrombin and thrombin receptors in brain is highly provocative vis-/l-vis a functional role in neurodegenerative diseases [33-37]. There is also an intriguing association between cancer metastasis and abnormal coagulopathy [38,39] and the importance of thrombin in malignancy has been shown consistently. In one study it was shown that thrombin-treated human melanoma cells enhance pulmonary metastasis in vivo [40] by promoting tumor cell adhesion to endothelial cells and extracellular matrix [41 ]. The thrombin receptor, although belonging to the superfamily of G-protein linked receptors, defines a new paradigm in signal transduction as outlined below. Receptor activation requires proteolysis of its extracellular N-terminal domain [42,43] between residues Arg 41 and Ser42. This proteolytic step unmasks a new amino terminal sequence that is tethered onto the receptor but folds into a recognition site in a classical ligandreceptor association process [27]. Clearly then, thrombin pathophysiology can be rationalized by its coagulant and cellular fimctions which are inextricably linked to its proteolytic activity. Accordingly, direct acting thrombin inhibitors or thrombin receptor antagonists may have therapeutic benefit in a plethora of pathologic states. THROMBIN INHIBITORS Endogenous factors If the action of thrombin is central to the processes of hemostasis and thrombosis then thrombin activity and production must be tightly controlled. Indeed one can distinguish two principal deactivating mechanisms: the principal neutralizing determinant in human plasma is antithrombin III (ATIII) [44]. ATIII belongs to the serpin superfamily of proteinase inhibitors [45] but does not exhibit signficant selectivity towards its target proteinases [46]. Its complexation with heparin-like molecules drastically accelerates the inactivation of thrombin, Factor Xa as well as other enzymes higher in the cascade. Indeed heparin remains the mainstay drug for the treatment of thrombosis and its sequelae including embolic complications, coronary disease and stroke as well as for surgical procedures requiring extracorporeal conduits [47]. Despite its widespread use, heparin is not without drawbacks and its application in certain arterial disease states having a large measure of platelet component is regarded as producing subclinical efficacy.

274 The other mechanism relies on thrombin binding to thrombomodulin, resulting in an unusual substrate specificity switch from its circulating substrate fibrinogen to proteinC, which in turn deactivates cofactors Va and VIII (48). The net result is the conversion of thrombin from coagulant to anticoagulant enzyme. Protein antithrombotics Hematophagous species secrete a plethora of proteinaceous molecules that interfere with discrete biochemical events and adversely affect the host's hemostatic process [49]. These include hirudin [50] and antistasin [51] which inhibit thrombin and factor Xa, respectively. Proteins exhibiting fibrinolytic and antiplatelet activity have also been isolated [52-54]. The most potent natural inhibitor of thrombin known is hirudin, a family of isoproteins isolated from the glandular secretions of the leech Hirudo Medicinalis [50, 55, 56]. Native Hirudin is a 65-amino acid residue protein that is O-sulfated on Tyr 63 of each variant (Figure 2) and forms a high affinity non-covalent complex having an equilibrium dissociation constant in the order of 20 fM toward human a-thrombin [57]. The tight binding properties and absolute specificity of hirudin toward its target enzyme stem from the cumulative interactions with multiple and remote regions on the thrombin surface [5860]. However, the specific interactions responsible for the tight binding properties of hirudin have remained equivocal until the recent reports describing the crystal structure of the bimolecular complex formed by two recombinant hirudin variants with human (zthrombin [61,62]. Detailed analysis of the crystal structure which is shown schematically in Figure 2 confmns previous hypotheses regarding the mechanism of inhibition but also raises new questions. For example, the densely negatively charged COOH-terminal domain of hirudin has been reported to be indispensable for tight binding interactions with the putative complementary "fibrinogen anion binding exosite" (FRE) of thrombin; yet the crystal structure revealed that the bimolecular contacts are primarily hydrophobic with a large electronegative cluster directed toward bulk solvent. In keeping with site directed mutagenesis studies of Lys 47 [63], the crystal structure demonstrates that high affinity binding is neither governed nor does it necessitate insertion of a basic amino acid side

Disulfide Core

Thr 45

l \

Y YY Apolar

groove

. . specificity

. pOCKet

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nio

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"

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"

Figure 2. Schematic illustration of the association of himdin with a-thrombin in the region of the active site and "anion" exosite. The diagram also shows that slicing the protein at residue 45 exposes a cryptic recognition region complementary to the catalytic site.

275 chain in the $1 specificity pocket of the enzyme. Instead, the three N-terminal residues participate in hydrophobic interactions with an apolar binding site adjacent to the specificity pocket. As shown schematically in Figure 2, hirudin may be regarded as a "bifunctional" inhibitor wherein two remote ends of the protein bind distinctly to complementary widely separated regions of the enzyme surface.

Low molecular weight "bifunctionar' thrombin inhibitors. The design of small molecular weight enzyme inhibitors that retain the high affinity and specificity of their protein counterpart toward the macromolecular target constitutes a formidable challenge in protein chemistry. It is also regarded as an essential task in the discovery path of drug development. Conceivably, such a protein mimetic would be "multifunctional" insofar as it may integrate two or more recognitive elements that are spatially disposed so as to preserve and simulate the simultaneous interactions exerted by the parent protein inhibitor [64]. During the course of our investigations on hirudin peptides, it was observed that the fragment corresponding to hirudin 45-65 (Figure 3) 45 50 55 NH2Thr-Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-AspPhe-Glu-Glu-Ile-Pro-Glu-Glu-Yyr-Leu-Gln 60 65

Figure 3. Primary amino acid sequence of the fragment corresponding to native desulfato hirudin 45-65. weakly inhibited the thrombin-mediated hydrolysis of a synthetic substrate (Tos-Gly-ProArg-X; where X is p-nitrophenyl or 7-amino-4-methyl coumarin). This anomalous result necessitates that some component of the hirudin fragment competes for and blocks access to substrate in thrombin's catalytic site. Initially, the result was also at variance with previous studies which concluded that the truncated fragment hirudin 45-65 inhibited fibrin clot formation but is not sufficient for inhibition of the amidolytic activity of thrombin toward synthetic substrates [65,66]. The divergent results could be explained by the unexpected kinetic behaviour of the truncated inhibitor as shown in Figure 4 where the resulting Dixon plot exhibits saturating reciprocal velocity as a function of inhibitor concentration up to a limiting value above which no further inhibition could be observed. Accordingly, if substrate concentrations greater than five times its Km are used then no inhibition is observed. The combined Dixon and Lineweaver-Burk (Figure 4A) plots are characteristic of partial competitive inhibition represented by schematic equation (Figure 4B) with kinetic parameters K~ = 0.11 + 0.003 gM and tx = 4 toward human a-thrombin [67]. Ultimately, this finding could be explained by the crystal structure of hirudin bound to thrombin. In the complex, the region comprising hirudin residues Thr45-Pro46-Lys47Pro 48 does not participate in any apparent contact with the enzyme; however the deletion of the preceding 44 residues must expose a new recognitive element that is spatially predisposed to bind in the active site as depicted in Figure 2 and functionally substitute for the entire globular N-terminal hirudin domain. Therefore the initial interaction with

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(B) Schematic equation that may explain the kinetic behaviour.

exosite E, unique to thrombin, acts as a nucleating point that orients the second inhibiting component in the vicinity of the catalytic site (CS).

Optimization and biological activity

The working model described above was used as a point of departure to design low molecular weight inhibitors with the intention of recapturing the almost 105 loss in affinity, obtaining compounds suitable for X-ray crystallography and evaluating biological activity in clinically relevant animal models. Tripeptides of the type X-Pro-Y-Z that are derived from the NH2 terminal sequence of the fibrinogen a-chain have been widely investigated as active-site directed thrombin inhibitors [68]. For maximum affinity X requires a hydrophobic residue in the R configuration and Z is a non hydrolyzable function. An arginyl residue is optimal for Y

277 since thrombin prefers a guanidyl function in its $1 specificity pocket. In order to test whether the newly exposed N-terminal component of the native hirudin 45-65 fragment interacts with the active site, the preferred features described above were incorporated. Table 1 illustrates that this compound Na-acetyl [D-Phe 45, Arga7]hirudin 45-65 is a potent thrombin inhibitor having a Ki= 2.8 + 0.9 nM. In addition, the inhibition is purely competitive and specific for a-thrombin, therefore validating the above model of simultaneous dual binding mechanism [67,69]. Paradoxically, inhibitor 3 in Table 1 is slowly cleaved b e t w e e n mrg47 and Pro 48 by plasma proteases in vitro and by thrombin itself, despite the presence of an imino acid (Proline) in the Pl' position. Recognizing the proteolytically sensitive scissile bond, we introduced homologous ketomethylene arginyl pseudopeptides as surrogates of the scissile PI--PI' residues [70,71]. The bifunctional inhibitor 4 is representative of this class wherein a further affinity enhancement was observed which may be ascribed to a likely tetrahedral character of the ketone group. However, in the crystal structure of the complex between inhibitor 4 and human (zthrombin [72] the carbonyl oxygen of the PI--PI' residue was located in the oxyanion hole formed by the NH groups of Gly 193 and Ser 195 in thrombin, but the carbonyl carbon was 2.8A away from the 0v of Ser 195 and did not form a hemiketal.

Table 1

Inhibitory dissociation constants for human ct-thrombin mediated hydrolysis of the synthetic substrate Tos-Gly-Pro-Arg-AMC by r-hirudin and modified fragments proteases

1. 2. 3. 4. 5.

Inhibitor

Ki nM

r-hirudin HV2 hirudin 55-65 hirudin 45-65 N%acetyl [(D) Phe 45, Arg 47] hirudin 45-65 Na-acetyl [(D) Phe 45, Arg 47~g (COCH2) CH2CO] hirudin 45-65 N%acetyl [(D) Cha 4s, Arg 47~g (COCH2)CHACO] hirudin 45-65

0.0038 + 0.0005 not inhibited 110 • 2.8 • 0.9 0.37 • 0.03 0.013 • 0.002

SAR studies in this series affords bifunctional inhibitors having low picomolar inhibitory dissociation constants. This is easily achieved by saturating the D-Phe 45 aromatic ring (5, Ki = 13 pM). Removal of the Na-acetyl group or increasing the hydrophobic character of Leu64 (Leu64Cha) within the FRE binding component enhances affinity further. In each case the affinity enhancement can be explained by the bimolecular crystal structure [72]. Remarkably, the six intervening residues in 4 or 5 that connect the active site-directed component and the FRE segment can be substituted with a linear chain of one or more trans 8-amino pentenoyl units. The FRE-directed segment can be tnmcated to six residues, provided that the native Leu 64 residue is transposed to position 61. This exercise yields the series of compounds 6-8 illustrated in Figure 5. When n = 4, one spacer

278 unit is not sufficient to fix the two recognitive components in place; however with n > 2 the inhibitory dissociation constant decreases one hundred fold. NH2 HN=C

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Biological activity In view of the overwhelming evidence implicating thrombin as the primary mediator of arterial thrombosis (vide supra), numerous thrombin inhibitors have been evaluated in various experimental models of thrombosis [73,74] while hirudin and HirulogTM have progressed to advanced stages of clinical development [75]. Using an arteriovenous shunt model of thrombosis in the rat, compounds 4 and 7 were evaluated for their ability to maintain patency of the extracorporeal circuit which serves as a thrombogenic surface [76]. Table 2 shows that on a gravimetric basis r-hirudin and inhibitor 4, were equipotent [r-hirudin ED15 = 1.4 mg/kg; 4 EDI5= 1.2 mg&g i.v. bolus]. The thrombin inhibitor 7 was less effective in this assay having an EDI5 = 6.3 mg/kg. The in vivo efficacy of the bifunctional thrombin inhibitors 4 and 7 was also assessed in a chemically-induced thrombosis model where deep arterial injury to the rat carotid artery is mediated by FeC13 [79]. Stenosis of the blood vessel resulting from FeCI3mediated injury results in decreased blood flow and an abrupt temperature drop across the stenosed area due to arterial occlusion. Both bifunctional thrombin inhibitors 4 and 7 doubled the mean occlusion time (MOT) at an i.v. bolus dose of 1 mg/kg. At a dose of 2 mg/kg i.v. of compound 7, the vessel remained patent for over 60 minutes compared to saline treated control rats (MOT = 19 + 1 minute), r-hirudin was slightly more potent, doubling the mean occlusion time at an i.v. dose of 0.5 mg/kg. Inhibitors 4 and 7 and the reference compounds were also administered by intravenous infusion calibrated to provide a two to three fold increase in activated partial thromboplastin time (aPTT). Using this drug regimen both compounds 4 and 7 maintained vessel patency over thirty minutes while a significantly larger degree of anticoagulation

279 was required for heparin to achieve an equivalent antithrombotic effect. This result validates the superior efficacy observed previously in various experimental models using direct-acting thrombin inhibitors. Previous characterization of the arterial injuD" area by scanning electron microscopy showed extensive deposition of platelets, leukocytes and fibrin [79]. In our own studies, the carotid artery segments were examined by electron microscopy which showed partial or total denuded endothelium. However, in contrast to saline treated control animals, drug treated arterial segments showed significantly reduced or absence of platelets and fibrin. The inhibitors were also evaluated in a rat venous thrombosis model caused by balloon injury followed by stasis in the vena cava. The doses required to effectively abolish thrombus formation paralleled the Ki values reflecting the direct activity of thrombin on fibrin formation in this experimental model. In addition to its intrinsic reduced affinity for thrombin, the higher doses required for inhibitor 7 may be correlated to its pharmacokinetics and metabolic susceptibility. The t 89 of r-hirudin and inhibitor 7 were approximately the same in the rat (25-30 minutes) while for the low molecular weight inhibitor 7 it is less than 3 minutes. It is well known that the kidney serves as the primary site of elimination of many polypeptide based drugs [77]. Indeed hirudin is excreted intact in the urine of several species [78]. We found that differences in the rate of elimination between inhibitors 4 and 7 may be explained in part by differences in the metabolic susceptibility toward rat kidney membrane proteases. The principal result from these studies is that the mmcated inhibitor 7 is much more proteolytically labile compared to 4 in vitro. In 7, the proteolytic products arise rapidly (< 5 min) from the cleavage of the Phe56-Glu57 bond followed by cleavage of AspSS-Phe 56, both of which are sufficient to inactivate the peptide. In inhibitor 4, three principal proteolytic sites were identified, two of which are within the spacer residues: 1) Leu 64Gln 65 ; 2) Asn 52 -Asp 53 and 3) Gln 49 -Ser 50 . However, intact compound could still be detected after 30 minutes.

Table 2

Antithrombotic effect of r-hirudin, heparin and experimental compounds in various models of thrombosis in the rat

Compound

Arteriovenous shunt ED15 mg/kg

Injury and stasis in vena cava minimum dose to achieve patency mg/kg

FeC13-mediated injury to carotid artery bolus dose (mg/kg) + infusion (gg/kg/min)

Mean time* to occlusion (minutes)

heparin 15 U/kg 25 U/kg + 0.5 U/kg/min 29 r-hirudin 1.4 0.15 + 15 31 + 4 4 1.2 0.25 0.15 + 20 35 + 8 7 6.3 1.0 1 + 125 46 + 9 *Mean occlusion time (MOT) in saline treated animals is 19 + 1 min. All drugs were administered by the intravenous route.

280 Despite the ubiquitous nature of serine proteases, it is possible to design specific thrombin inhibitors by taking advantage of the accessory regulatory binding sites on the enzyme surface. The inibitors are efficacious in several experimental models of thrombosis, and above all, illustrate the feasibility of low molecular weight protein mimetics.

THROMBIN RECEPTOR AGONISTS/ANTAGONISTS Thrombin receptor mechanisms As outlined above, it has been known for some time that in addition to its ability to clot blood, thrombin can affect a variety of cellular functions ranging from the aggregation of platelets to the stimulation of cell proliferation and the modulation of smooth muscle contractility [80-87]. It was recognized early on that these actions of thrombin were due to the interaction of the enzyme with the cell surface and that most (but not all) of thrombin's cellular effects required its proteolytic activity [88]. Nonetheless, the complex mode of binding of thrombin to the cell surface and to extracellular matrix constituents [88,89] made it difficult to identify the thrombin receptor using the ligand binding approach that had succeeded in characterizing receptors related to hormone action [90]. Relatively recently, as alluded to above, this difficulty was circumvented using an expression cloning approach to isolate the thrombin receptor eDNA from human and hamster cell sources [27,29]. It is now widely accepted that thrombin's ability to regulate the fimction of many target tissues is due to the proteolytic activation of its specific G-protein-coupled receptor. The novel mechanism described above, whereby thrombin activates its receptor comprises the proteolytic exposure of a "tethered" receptor-activating sequence, beginning with serine-42 in the human receptor [27,91,92]. Remarkably, synthetic peptides derived from this cryptic N-terminal receptor sequence, containing up to 14 amino acids (i.e. S42FLLRNPNDKYEPF, or P14, from the human sequence) have been observed on their own to activate the thrombin receptor, so as to mimic many of the diverse cellular actions of thrombin, ranging from platelet aggregation [27,93,94] to the contraction of vascular and gastric smooth muscle [95-99]. Activation of the thrombin receptor can cause both Gq-protein-triggered phosphoinositide hydrolysis (with a consequent elevation of cytoplasmic calcium) and a Gi-protein-mediated inhibition of adenylate cyclase [100]. These G-protein-coupled events could explain many of the rapid actions of thrombin via its receptor. The mechanism(s) whereby thrombin receptor activation regulates cell growth remains an open question (e.g. see [101]). Evidently in certain instances, activation of the thrombin receptor using the receptor-activating peptide derivatives (so-called TRPs or TRAPs) does not result in a response equivalent in all respects to the response caused by thrombin itself. It would appear that peptide sequences in thrombin apart from its catalytic domain can also affect the function of cells such as macrophages, via a mechanism that does not involve the cloned thrombin receptor [83,102]. Structure-activity studies with thrombin receptor-activating peptides (TRAPs). Although the biological activity of the receptor-derived peptide, P14, was originally evaluated using a human platelet aggregation assay [27], in our own work, we quickly established that this peptide, along with other so-called TRAPs, was active in vascular and

281 gastric smooth muscle bioassay systems (Figure 6) [95,96]. In brief, the TRAPs can cause a contractile response in either gastric longitudinal smooth muscle or in an endotheliurn-free rat

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vascular preparation [96-99], and the TRAPs can cause a relaxation response in precontracted vascular tissue that has an intact endothelium [95,103]. We have made extensive use of the several smooth muscle preparations (both gastric and vascular) to evaluate the actions of a number of TRAPs for comparison with structure-activity data that have been obtained using a human platelet assay [93,94,104-106]. From such studies it has become evident that a pentapeptide comprising either SFLLR (P5, from the human receptor sequence) or SFFLR (from the rat or hamster receptor sequence) retains full intrinsic activity for activating the thrombin receptor [93,107]. Further, it is clear that in both the platelet and smooth muscle assay systems, the phenylalanine at the second position of the pentapeptide plays a key role in receptor activation [93,94,103-107]. A much more extensive structure-activity study based on this pentapeptide motif, using a platelet activation assay, has fully substantiated the requirement for an aromatic residue at position 2 of the pentapeptide and has further delineated the side chain requirements for the activation of platelets by the pentapeptide, P5 [108]. In our own work, we detemained that amidation of the carboxyl at the C-terminal end of the TRAPs could increase the activity appreciably [103]. For that reason, we have routinely used the TRAP, SFLLR-NH2 (or P5-NH2) as a probe for thrombin receptor function in a variety of assay systems and we have used a spectrum of TRAPs having either amidated or free carboxy-terminus residues (e.g. SFLLR/SFLLR-NH2 or P5/P5-NH2 and SFLLRNP/SFLLRNP-NH2 or P7/P7-NH2) to screen receptor structure-activity profiles in a number of tissue response systems [99,103,109].

282 Using the four TRAP agonists mentioned above (P5, P5-NH2, P7 and P7--NH2), we have now obtained structure-activity profiles for a variety of response systems, including rat and guinea pig gastric longitudinal smooth muscle, rat and guinea pig aorta preparations, human umbilical and placental vascular preparations and human platelets [95,96,99,103,107,109]. One main finding of our surveys is that in a number of the different assay systems, even those derived from the same species, the relative potencies of P7, P7NH2, P5 and P5-NH2 have turned out to be markedly different. For instance, in the rat aorta relaxation assay (reflecting the function of the endothelial thrombin receptor), the potency of the four peptides was: P7-NH2 > P5-NH2 > P5 > P7. In contrast in the rat gastric longitudinal muscle contraction assay (reflecting the gastric smooth muscle receptor), the potency series for the same four peptides was: P7-NH2 > P5-NHa > P7 > P5 [103]. In a similar fashion, the potency series for a human placental artery contractile preparation was: P7-NH2 > P7 > P5-NH2 > P5, whereas in the human umbilical vein contraction assay, the potency order was P5-NH2 >> P7-NH2 = P7 >> P5; and these two vascular potency series differed in turn from the one observed for a human platelet aggregation assay: P5-NH2 > P7-NH2 > P7 >> P5 [ 109]. Taken together, these distinct orders of agonist potencies for the same set of agonists in the different assay systems, as summarized in Table 3, point to distinct

Table 3 Relative potencies of TRAPs in different assay systemsa

Tissue

Response

Potency series (P7-NH2, P7, P5-NH2, P5)

Rat aorta Rat gastric LM Human placental artery Human umbilical vein Human platelets

relaxation contraction contraction contraction aggregation

P7-NH2 > P5-NH2 > P5 _>P7 P7-NH2 > P5-NH2 > P7 > P5 P7-NH2 > P7 > P5-NH2 > P5 P5-NH2 >> P7-NH2 = P7 >> P5 P5-NH2 > P7-NH2 > P7 > P5

a

Data from Hollenberg et al. [ 103,109]

functional receptor subtypes, according to classical criteria [ 110]. The question to be asked, however is: do the functionally distinct receptor subtypes characterized by different structure-activity profiles correspond to different thrombin receptor sequences in the different tissues? To deal with this question, we used a reverse-transcriptase/polymerase chain reaction (RT-PCR) approach to analyze thrombin receptor mRNA present in human placental artery and umbilical vein tissues, which exhibited very distinct structure-activity profiles (Table 3). Somewhat to our surprise, the two tissues yielded exactly the same cDNA sequence for the thrombin receptor. Our finding of the same thrombin receptor mRNA in tissues that possess pharmacologically distinct receptors according to their structure-activity profiles may be rationalized by a number of possibilities. First, it may be that distinct posttranslational processing of the product of the same receptor mRNA in different tissues may yield functionally distinct receptor subtypes. Such a situation appears to be the case for the

283 murine bradykinin receptor expressed by transfection in COS cells [111]. A second possibility is that the expression in different tissues of markedly different types and amounts of the G-protein, to which the thrombin receptor couples could, in theory, alter relative agonist potency, as has been discussed by Kenakin and Morgan [ 112]. A third alternative to consider is that the TRAPs, such as P5-NH2, in addition to activating the thrombin receptor, might also be able to activate the recently cloned protease-activated receptor, PAR-2 [113,114], which could coexist along with the thrombin receptor in a number of tissues. Further work willbe required to evaluate these several possibilities. From a practical point of view, nonetheless, the distinct structure-activity profiles we have observed in different assay systems suggest that it might be possible to design selective agonists or antagonists for specific tissue targets. In this regard it can be pointed out that TRAPs, such as P5-NH2, which are relatively potent in activating the thrombin receptor in rat gastric and vascular preparations [98,99,103] are not able to activate rat platelets, which are otherwise stimulated by thrombin via an as-yet uncharacterized thrombin receptor [ 115]. Similarly, the TRAP, P5, has been reported to regulate canine coronary artery contractility, but is unable to aggregate canine platelets that are otherwise responsive to thrombin [ 116]. Such absolute selectivity for the TRAPs between vascular and platelet assay systems has not been observed for human tissue samples.

Thrombin receptor antagonists Given the pathophysiologic importance of thrombin and its receptor, as outlined above, it is no surprise that considerable effort has been aimed at developing thrombin receptor antagonists. To date however, only a few peptide antagonists have been described, primarily resulting from a human platelet assay screen of peptides based on the TRAP, P 14, sequence. One of these peptides, YFLLRNP, can antagonize the aggregation of human platelets stimulated by low concentrations of ct-thrombin or the TRAP, P7; however, this peptide was a partial agonist in the platelet assay [117]. An alternative novel series of antagonists based on the TRAP motif, beginning with mercaptopropionic acid (Mpr) instead of serine and having the two leucine side chains replaced with a cyclohexylalanyl residue (Cha), (Mpr F Cha Cha R...) have been observed to inhibit both thrombin and P5-induced platelet aggregation, with ICs0s ranging from about 5 to 80 ~tM [118,119]. One of these Mpr analogues (Mpr F Cha Cha RKPNKD-NH2 or Mpr-P 10-NH2), exhibiting an IC50 of 5 ~tM in a thrombin-induced human platelet aggregation assay [ 118] has also been reported to inhibit the relaxant and contractile effects of thrombin and P7 in porcine coronary artery ring preparations [120]. Nonetheless, the data reported by Tesfamariam [120] would indicate that in the porcine coronary preparations Mpr-P10-NH2 was a desensitizing partial agonist. A peptide based on the P5 sequence, SFLVR-NH2 (or VR-NH2) has also been reported to act as a TRAP antagonist in a coronary artery preparation, shifting the concentration-effect curve for P5 to the right [121]. Another intriguing independent method for the discovery of thrombin receptor antagonists has used a phage display approach, coupled with a platelet binding recovery procedure to identify the sequence, MSRPACPNDKYE, as an inhibitor of TRAP- and thrombin-induced platelet aggregation [ 122]. In our own work, we wished to evaluate the actions of the putative thrombin receptor antagonist, Mpr-P 10-NH2 in the rat gastric longitudinal muscle contraction assay; and we wished to assess the actions of VR-NH2 both in the gastric contractile assay and in the platelet aggregation assay. As indicated by the data in Figure 7, Mpr-P10-NH2 which was an

284

RAT STOMACH L M 140

...I 0 ~-120

& 9P5-NHz Mpr-PIO-NH

l

z

ElO0 o 143 80

z O 60 D I-O .~ 40 I--z 20 O O o

9

!

0.1

,

i

1

,

10

i

|

100

CONCENTRATION

Figure 7. Contractile activity of MprP10-NH2 compared with P5-NH2 in the rat gastric LM assay. The contractile activities of Mpr-P10-NH2 (&) and PSNH2 (A) in the rat gastric longitudinal muscle strip were expressed as a percentage (% 50 mM KC1) of the contractile response to 50 mM KC1.

IOO0

(pM)

antagonist in the platelet aggregation assay, was a relatively potent contractile agonist in the rat gastric LM assay, with an activity comparable to that of P5-NH2. In the rat aorta relaxation assay, Mpr-P 10-NH2 was a weak agonist, without antagonist activity (not shown). In contrast, VR-NH2, which was a desensitizing partial agonist in the canine coronary assay, was a full agonist in the platelet assay and was a non-desensitizing full agonist in the rat gastric LM assay (Figure 8). Thus, the main findings of our work with the peptides reported to be thrombin receptor antagonists were that the observation of antagonist activity depends heavily on the type of assay system used for the evaluation. Clearly, in the search for thrombin receptor antagonists, it will be necessary to use a matrix of bioassay systems to evaluate in depth the complete pharmacologic properties of any lead compound.

120 ~. ~---100 ~

120

P5-NH 2 (&.A} VR-NH z (O,e)

~00

80

8O

x 3;

~ 8o

60

~ ,o

40 ~

:~ 20

20

O

Z O u,l

0

0

0.1

1

10

CONCENTRATION

100 (pM}

1000

O

Figure 8. Agonist activity of SFLVRNH2 in the rat gastric LM and human platelet assays. The contractile (relative to 50 mM KC1) and platelet aggregation (% max) activities of PS-NH2 (A, &) and VR-NH2 (0, O) were measured using rat gastric longitudinal muscle (open symbols) and washed human platelets (solid symbols) essentially as described elsewhere [103,28].

285 ACKNOWLEDGEMENTS

Work reported in this article was supported in part by a Medical Research Council of Canada University-Industry grant awarded to M.D.H. in conjunction with BioChem Therapeutic Inc. The authors are grateful to the staff of the Pharmaceutical sector of the Biotechnology Research Institute of Montreal (NRC) without whom part of the work described in this article would not have been possible. We are also indebted to Drs. Ren6 Martel and Arshad Siddiqui as well as to Annie St-Pierre, C~line Locas, Micheline Tarazi and Nicole Landry for expert assistance. We are also grateful to Mrs. Sylvie Ouellet for secretarial help. ABBREVIATIONS USED

TRAP, thrombin receptor-activating peptide; P14, SFLLRNPNDKYEPF; P7, SFLLRNP; P7-NH2, SFLLRNP-NH2; P5, SFLLR; P5-NH2, SFLLR-NH2; P5VR-NH2, SFLVR-NH2; Cha, cyclohexylalanyl; Mpr, mercaptopropionyl; LM, gastric longitudinal muscle. REFERENCES

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Perspective in Receptor Research D. Giardinb., A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All fights reserved.

291

Structure-Activity-Relationships of Non-Peptide Kappa-Opioid Analgesics: A Perspective of the Last 10 Years D.C. Rees Organon Laboratories, Newhouse, Scotland, ML1 5SH, UK

INTRODUCTION Opium, obtained from the ripe seed capsule of the poppy plant, Papaver somniferum, has been used to alleviate pain and induce sleep in humans for over two thousand years. During the last two decades research into the pharmacology of opioid receptors has led to the identification of at least three opioid receptor subtypes, designated mu, kappa and delta. Recently the human forms of these three receptor subtypes have been cloned. They are characteristic of the seven transmembrane domain structure of the G-protein coupled receptor superfamily [la]. Activation of mu receptors is associated with classical morphine-like properties such as centrally mediated analgesia, constipation, respiratory depression and addiction. Activation of kappa receptors appears to be associated with centrally mediated analgesia, sedation and diuresis. This has provided the impetus for the discovery of selective kappa opioid agonists for evaluation as clinically useful analgesic drugs without the limiting side-effects that characterise morphine and all morphine-related pain relieving drugs in use today. Some kappa selective compounds have recently been progressed into clinical trials, although to date results describing their analgesic properties in humans have not been published. Potential side-effects include diuresis, sedation and possibly dysphoria. The discovery of highly selective non-peptide kappa agonists has assisted the pharmacological study of this system and it has become apparent that these compounds also behave as neuroprotective agents in animal models of stroke/cerebral ischaemia. This is of particular current interest because of the lack of any effective medicine for these patients. Further developments in the kappa field have led to patents and publications disclosing selective antagonists and also the identification of agonists with limited access to the central nervous system. The potential therapeutic utility of these agents is currently being investigated. AIM

The aim of this project was to synthesise non-peptide selective kappa opioid agonists for evaluation as new analgesic drugs.

292

KAPPA OPIOID AGONISTS:

EXAMPLES OF PEPTIDOMIMETICS

During the last decade several medicinal chemistry projects have aimed at discovering low molecular-weight, non-peptide ligands which modulate the actions of membrane bound receptors of endogenous peptide neurotransmitters [l b]. The opioids (e.g. morphine) are sometimes regarded as prototype "peptidomimetics" since they act as receptor agonists of the endogenous opioid peptides (e.g. endorphins, enkephalins). In a similar way the small molecule kappa agonists described in this talk can be regarded as peptidomimetics of the dynorphin peptides. Three distinct medicinal chemistry strategies have emerged for the discovery of these non-peptides, firstly broad screening followed by lead optimisation, secondly design incorporating elements of a peptide structure, and thirdly modification of an existing non-peptide lead [1]. The opioids discussed here exemplify the third of these strategies. EARLY SYNTHETIC ACHIEVEMENTS

Ten years ago, when this project began in the Parke-Davis Laboratories there were a number of chemical structures reported in the literature as possessing high affinity for the kappa opioid receptor. Some of these are shown below (fig. 1). They include the endogenous peptide, dynorphin, the benzomorphan derivative EKC, the benzodiazepine tifluadom, and the 1,2 aminoamide U-50488. At this point ! would like to emphasise .the importance of the prototype Upjohn compound, U-50488 [2]. It represents a milestone in kappa opioid research because of its high selectivity for this receptor and its in vivo activity as a non mu-opioid analgesic agent. This compound was selected by the Parke-Davis group (and subsequently by many others) as an attractive chemical lead for synthetic modification. STRUCTURE-ACTIVITY-RELATIONSHIPS OF THE AROMATIC RING IN THE U-50488 SERIES

The dichlorophenyl aromatic ring of U-50488 was extensively modified to probe the SAR of this fragment. Replacement with a bicyclic 10-pi aromatic group rapidly led to the identification of the benzo[b]thiophene series. Five regioisomers of benzo[b]thiophene acetic acid were prepared. Substitution at the 4-position was shown to be optimal in terms of in vitro kappa receptor binding affinity and mu-kappa binding selectivity [3] (see fig. 2). A 4-benzo[b]furanyl ring retains greater than 100-fold mu-kappa selectivity and this group also found favour in later structures. The 4-benzo[b]thiophene derivative PD117302, is typical of these compounds in that it causes naloxone reversible inhibition of electrically evoked contractions of isolated guinea-pig ileum (IC50=l.lnM) and behaves as a naloxone reversible analgesic in a variety of rodent models, for example, mouse tail clip MPE50=2.2 mg/kg sc [4]. Other pharmacological effects are characteristic of a kappa agonist, i.e. naloxone reversible diuresis and locomotor impairment, but no sign of mu-receptor mediated

293 Dynorphin (1 - 17) = Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg--Pro--Lys--Leu-Lys-Trp-Asp-Asn-GI n CH 3 CH2NHCO

o

7

HO

Ethyl ketocyclazocine

Tifiuadom

Cl

u-50488 Figure 1. "Kappa-Selective" Ligands (1985).

~

NCO - Ar

Opiotd __l~c~__~lorBinding

~nnny~ (nu)

4

Kappa

(+.)

Mu Mu/kappa Rat Paw i~ouun~ ratio Assay (iv) MPE,aiomg4cg ,

Substitution Poslton X 3 4 5 6 7

Me

OPIOID BINDING Ki (nM) Kappa Mu Mu/Kappa 69 3.9

$

94

$ S

ClCz # ~

3.4

864

258

HT

~C/-~~

12.7

4340

341

NT

127

>104

0.37

244

859

0.014

1.2

273

227

0.43

,

S S

146 17

1,400 410 475 425 650

21 110 5 3 39

Figure 2. Structure-affinity relationships of aromatic ring.

,

~C/~~

NT

Figure 3. Conforrnationally restricted aromatic side chain.

294 respiratory depression, inhibition of gastrointestinal motility or naloxone induced withdrawal jumping in mice. Having concluded that steric properties of the aromatic ring are an important factor in determining receptor selectivity the study was extended by incorporating a methyl group on the o~-carbon between the aromatic ring and the amide carbonyl. This significantly reduced kappa receptor affinity and after consulting molecular models it was suggested that this inactivity may be caused by the methyl group disfavouring the biologically active conformation. One such conformation is when the methyl group interacts unfavourably with the C-3 proton on the aromatic ring. If this is the conformation required for kappa receptor binding then it should be possible to increase affinity by locking the aromatic group in this orientation. To test this hypothesis a rigid tricyclic acenaphthene moiety was prepared. The results, given in the table below (fig. 3), show that one of the acenaphthene diastereoisomers has 10fold greater affinity than the corresponding conformationally flexible napthalene analogue and behaves as a potent analgesic in the rat paw pressure test [5].

CI-977 (ENADOLINE)

Credit has already been given to the Upjohn compound, U50488 as being the prototype 1,2-amino amide selective kappa agonist: A second development in the SAR of these compounds discovered by Upjohn involved incorporating a spiroether group at the C-4 position of the cyclohexane ring to increase the kappa analgesic activity [6] (fig. 4). The compound with which Upjohn performed clinical trials, U62066 (spiradoline), contained this structural modification. However, U 62066 is a racemic mixture of two enantiomers and has only a modest mu/kappa selectivity ratio (approx. 10-fold) [7]. The compound which Parke-Davis selected for clinical evaluation was CI-977 (enadoline), a single enantiomer of the 4-benzofuranyl derivative of spiradoline and a very selective, potent kappa agonist [8], see (fig. 5,6). The diuretic effects, pharmacokinetic effects and safety of CI-977 in 16 healthy human volunteers have been reported over a dose range of 5-25 l~g intramuscular injection [9]. CI-977 Crnaxand AUC(0-oo) values increased in proportion to dose and significant dose-related decreases in negative free water clearance and urine osmolality were observed, in accordance with the diuretic effects previously seen in rodents. Adverse effects include dizziness and fatigue. In another rising, singledose tolerance study in humans doses up to 40 ~g were administered intramuscularly. The highest well tolerated dose was 15 ~g. No euphoria or dysphoria occurred [10].

295

Upjohn's Spiroether Derivatives .=

(_+)

CH31 _ ..,N- ~1" CH=

CI

~

CI

U-62066 (Spiradoline) 913 x more potent analgesic than U-50488 9(-) enantiomer more potent and more selective 9

chloro derivative is highly kappa selective (U-69593) Vonvoigtlanderand Lewis,1988

Figure 4. Upjohn's spiroether derivatives.

Receptor binding affinity (K, nM) Compound kappa

mu

delta

CI-97"/ Spiradoline U-69593 EKC

100 44 2460 1.02

1040 4530 9900 7.0

0.11 0.35 0.67 0.21

Smooth muscle (ICs0,nM)

mu/kappa 905 125 3670 4.9

GP!

LVD

0.087 0.88 3.4 0.26

3.3 29 89 22

Figure 5. Opioid activity in vitro for CI-977 and reference compounds.

296

MPEs0 (mg/kg) (tail clip, s.c.)

Mouse

(paw pressure,i.v.) (tooth pulp, i.m.)

Rat

Dog

(tail immersion i.m)

C I-977

0.03

Oo02

0.00026

0.001

Spiradoline

0.81

0.38

0.0022

NT

Morphine

2.4

1.3

NT

1.0

Compound

Monkey

Figure 6. Antinociceptive activity of CI-977 and reference compounds.

Me I

MeO2CN,,~

~

( . . ~ . ~ NCOCH," - ' ~ o

,/~CI NCOCH~ - - ~ - - C l

.H I. CI-977

2. GR 103545

O

v

"N~

~

"CI

Me HO

3.

4.

5. EKC

Figure 7. Kappa Opioid Agonists used in molecular modelling study.

297

DESIGN OF NOVEL STRUCTURES (UNRELATED TO U-50488) During the late 1980's a number of laboratories reported preliminary data on a variety of kappa agonists based on the 1,2-amino amide, U-50488. Some of these are shown in fig. 7. We decided to use a molecular modelling approach to probe the chemical similarities of these non-peptide agonists together with the benzomorphan, EKC, in order to construct a template which could be used to design novel structures for evaluation as kappa ligands [11]. The compounds that were overlayed with each other according to a 3-point pharmacophore model (basic nitrogen atom and two aromatic ring centroids) are shown in fig. 8. This generated a proposed binding model for these kappa agonists. Novel structures could then be examined via computer modelling to see which fitted the model. In this design three factors were considered. Firstly, the designed molecules should have appropriately orientated aromatic and amino groups. Secondly they should as far as possible occupy space within that occupied by the known kappa ligands. Thirdly, they should be as simple as possible in terms of chemical synthesis. Three series, a pyrrolizidine, a macrocyclic iactam and an aryl amine, were then selected for synthesis (fig. 9). The pyrrolizidine (PD 146795) (kappa K~=940 nM, mu/kappa ratio=3.5) and the arylamine (kappa K~=160 nM, mu/kappa ratio >60) represent novel chemical leads for kappa opioids and they illustrate the success of this molecular modelling strategy for designing new receptor ligands. Despite the macrocyclic lactam (PD 146884) fitting the model quite well it is essentially devoid of affinity indicating that this criterion alone is not sufficient for activity. During this study we synthesised and tested fewer than ten compounds based on our receptor binding model yet one of these (PD 148282) has 160 nM affinity for the kappa opioids receptor and is selective with respect to the mu receptor. Unlike almost all other kappa selective ligands reported at the time it is not a 1,2-aminoamide. Since this study was performed a number of other kappa ligands have been reported. A selection of some of these are shown in figs. 10 and 11. ACKNOWLEDGEMENTS i would like to acknowledge the numerous scientists at the Parke-Davis Research Unit who contributed this project over many years. In particular I note the senior staff who initiated the project, Dr. David Horweil (Medicinal Chemistry), Dr. Ray Hill and Professor John Hughes (Pharmacology). Dr. John Hunter (Pharmacology) and Dr. Geoff Woodruff (Pharmacology) also made leading contributions.

298 !

Figure 8. Relaxed stereoview of the spatial overlay of compounds 1-5. N and N 1 are the basic nitrogens of EKC and the aryl acetamides respectively. The three points which define our binding model are labelled p.

kappa

mu

mu/kappa ratio ,

940

3350

,,

3.5

6 Oploid receptor btndlng affinity

(KI, .M)

0

CONMe

~

~

>10 000

>10 000

160

>10 000

>60

Figure 9. Novel structures designed to fit binding model.

299

OyMe

C02Me I

OH O~~~--

Cl

SO=Me peripherally selective agonist LVD ICso= 0.9nM (Glaxo, 1992)

O

CI

GR 89696 LVD IC~ = 0.041nM (Glaxo, 1993)

CH3 ~' CHPh2

~ N (

0 ' ~ 0 H .HCI

kappa binding K~= 47nM (SB-Italy, 1994)

EMD 61753 kappa binding = 2nM (E. Merck, 1994)

Figure 10. Recentlyreportedselective kappa agonists based on U-50488.

300

HO

N~COOCH3 CCB (Ronsisvaile, 1993)

HN

O NMe CO2Me

(Borsodi, 1994)

Figure 11. Recently reported selective kappa ligands not based on U-50488.

301

REFERENCES

l(a) Knapp RJ, Malatynska E, Collins N, Fang L, Wang JY, Hruby V J, Roeske WR and Yamamura HI, Faseb J, 1995; 9: 516-525. l(b) Rees D: In: Bristol J, ed. Ann Rep Med Chem 1993; 28:59-68 and references cited therein. 2(a) Szmuszkovicz J and Von Voigtlander PF, J Med Chem 1982; 25: 1126-1127; (b) Lahti RA, Von Voigtlander PF and Barsuhn, C Life Sci 1982; 31:, 22572260; (c) Katz JL, Woods JH, Winger GD, Jacobson AE, Life Sci 1982; 31: 2375-2378. 3. Clark CR, Halfpenny PR, Hill RG, Horwell DC, Hughes J, Jarvis TC, Rees DC and Schofield D, J Med Chem 1988; 31: 831-836. 4. Clark CR, Birchmore B, Sharif NA, Hunter JC, Hill RG and Hughes J, Br J Pharmacol 1988; 93: 618-626. Leighton GE, Johnson MA, Meecham KG, Hill RG and Hughes J, Br J Pharmacol 1987; 92: 915-922. 5. Halfpenny PR, Horwell DC, Hughes J, Humblet C, Hunter JC, Neuhaus D and Rees, DC J Med Chem 1991 ; 34: 190-194. 6. Lahti RA, Mickelson MM, McCall JM and Von Voigtlander PF, Eur J Pharmacol 1985; 109: 281-284. 7. Meecham KG, Boyle SJ, Hunter J and Hughes J, Eur J Pharmacol, 1989, 173, 151-158. Von Voigtlander PF and Lewis RA, J Pharmacol Exp Ther 1988; 246: 259-262. 8. Halfpenny PR, Horwell DC, Hughes J, Hunter JC and Rees DC, J Med Chem 1990; 33: 286-291. Hunter JC, Leighton GE, Meecham KG, Boyle SJ, Horwell DC, Rees DC and Hughes J, Br J Pharmacol 1990; 101: 183-189. Davis RE, Callahan MJ, Dickerson M, Downs D, J Pharmacol Exp Ther 1992; 261: 10441049. 9. Reece PA, Sedman A J, Rose S, Scott Wright D, Dawkins R, Rajagopalan R, J Clin Pharmacol 1994; 34:1126-1132. 10. Dawkins R, Lebsack ME, Scott Wright D, Posvar EL, J Clin Pharmacol, 1991; 31 : 841-871 (Abstr. 97). 11. Higginbottom M, Nolan W, O'Toole J, Ratcliffe GS, Rees DC, BioMed Chem Lett 1993; 3: 841-846.

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Perspective in Receptor Research D. Giardinh, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

303

Selective N o n p e p t i d e L i g a n d s as Probes to Explore 8 Opioid R e c e p t o r Architecture P.S. Portoghese

Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, USA INTRODUCTION The relatively recent molecular cloning and sequencing of opioid receptors [1-5] has confirmed the mlmerous pharmacologic and structureactivity relationship studies that have suggested the existence of multiple opioid receptors. Presently, there appear to be at least three types of receptors, designated as 8, ~ and ~, in the opioid receptor family which have high homology and are members of the rhodopsin-like class within the superfamily of G protein-coupled receptors. Numerous ligands have been developed as potential opioid receptor probes in an effort to distinguish between different types of opioid receptors. These include peptide [6,7] and nonpeptide [8] agonists and antagonists. Here we discuss the design and structure-activity relationship studies of ~selective nonpeptide ligands and the role of conformation of the "address" moiety in conferring selectivity to the 81 and 82 subtypes of opioid receptors. THE MESSAGE ADDRESS CONCEFI" IN ~

DESIGN OF NALTRINI~!.E

The prototypical nonpeptide 8 opioid antagonist, naltrindole [9] 1 (NTI), is employed widely as a pharmacologic tool [10]. The rationale for the design of NTI was based on the "message-address" concept which was developed by Schwyzer [11] to analyze the structure-activity relationship of ACTH-related peptides. For agonists, the "message" component of the ligand was envisaged to be responsible for triggering signal transduction upon interaction with the receptor. The "address" was considered to target the ligand to a specific receptor.

OH

H

I

H

1 0~TD

This concept was tested initially in the design of selective opioid ligands by the attachment of putative 8 and ~ address fragments of leucine-enkephalin

304 and dynorphin to oxymorphone, which contains the opioid message component [12]. It was found that the enkepbalin-derived sequence conferred 8 selectivity while the sequence obtained from truncated dynorphin afforded a ~selective ligand. NTI contains an indolic benzene moiety that was envisaged to mimic a key element of the 8 address, the Phe 4 phenyl group of enkephalin. In this context the indolic pyrrole moiety was viewed as part of a molecular scaffold to hold the address. ROLE OF ~ MOLECIKAR SCAFFOLD IN DETERMINING DELTA B E C E F T O R SUBTYPE ~.]e~-L'IVI'I-L While the aforementioned studies are consistent with the concept that selective ligands interact with message and address subsites on the 8 opioid receptor, it was unclear whether the recognition sites for agonists and antagonists are the same, are overlapping, or are different. If agonists and antagonists interact with identical recognition sites, then it was expected that substitution of a methyl for the N-cyclopropylmethyl group in NTI should afford a 8 agonist by analogy with naltrexone 2 and oxymorphone 3. N.-R

N..CI'~

OH

H

2, R = CH2CH(CH2)2 3, R=CH3

4 (OMi)

H

The finding that the oxymorphone analogue, oxymorphindole [13] 4 (OMI), is not a 8 agonist and retains potent 8 opioid antagonist activity in vivo [14], raised the possibility that the molectdar scaffold that holds the address i n 4 might play an i m p o ~ t role in promoting 8 antagonism by fixing the address in a specific conformation. This would imply that Phe 4 in the enkephalins plays a dual role, in that it may serve both as a functional but nonessential part of the message and as an essential part of the 8 address. Molecular dynamics simulation of leucine-enkephalin has shown that the conformational space occupied by the phenyl group of Phe 4 is restricted to the region of the indolic benzene moiety [15] (Fig. 1). However, it appeared unlikely that the Phe 4 phenyl group can assume a conformation identical with that of the indolic benzene moiety because superposition of both rings was not observed during the simulations. In this regard, many of the Phe 4 phenyl conformations were nearly perpendicular to the plane of the indole system.

305

Figure 1.

Ten low energy conformations of leucine enkephalin superposed upon OMI. The indolic benzene moiety is indicated by bold lines.

These results contributed to the design of spiroindan-substituted opiates with a a address moiety that is restricted to a conformation that is perpendicular to that of NTI or OMI. This restriction is due to the spiro substitution which prevents torsion of the indan benzene moiety.

N-R OH

H 5. R = Ci-12CH(CH2)2 (SINTX) 6, R = C1-13 (SIOIVl)

7-Spiroindanylnaltrexone 5 (SINTX) was found to be a potent 8 antagonist with approYimately one-sixth the atrmity of NTI for 8 receptors, although its ~selectivity was considerably lower [16]. Significantly, replacement of the cyclopropylmethyl of SINTX with a methyl group afforded a ligand 6 (SIOM) with potent 5 agonist activity [17]. The fact that this is in contrast to OMI 4, which has its address moiety restricted to a conformation that is coplanar to ring C of the opiate, is consistent with the idea that agonists and antagonists bind either to different sites or to overlapping sites on the 5 receptor. Superposition of SIOM upon OMI illustrates the different orientations of the address moiety (Fig. 2).

306

.~OM

Figure 2.

A stereoview of OMI superposed upon SIOM.

INTERACTION OF LIGANDS W i t h CI~NED WH.n-TYPE AND MUTANT DI~.TA OPIOID RECEPTORS Studies of SIOM and NTI binding to mutant and wild type cloned 8 opioid receptors were consistent with different or overlapping recognition sites for agonists and antagonists [18]. The point mutation involved the replacement of Asp95 with Asn in tr~n~membrane s p a n n i n g region 2 (TM2), which is conserved in the rhodopsin class of G protein-coupled receptors. The fact that the Asn95 mutant exhibited a great decrease in at~inity for 8 agonists, while antagonists were unaffected, suggests that their interaction with 8 receptors is fundamentally different (Table 1). Table 1 Inhibition of [3H]NTI binding to wild-tTpe and Asp95Asn mutant 8 receptors ICso (nM) a Ligand 8 A~onists Deltorphin DPDPE Met-enkephalin SIOM (6) 8 Antagonists NTI (1) NTB (9) BNTX (8) ,,,,

aData from [18].

Wild Type 48 245 81

Asp95Asn 552 >I000

>1000

33 40

>1000 544

2.1 0.04 37

1.7 0.09 30

307 Also consistent with the possibility of different binding modes for agonists and antagonists is the finding that the Asp128Asn mutant in TM3 greatly reduced agonist binding to the S receptor, but did not substantially alter antagonist binding [19]. Asp128 is conserved among the subgroup of G protein coupled receptors that recognize aminergic ligands. It has been suggested that this conserved Asp serves as the anionic binding site for cationic protonated amine or quaternary ammonium groups in different pharmacologic classes of agonist ligands [19]. Since the Asn128 residue is incapable of ion-pairing with a cationic group, the mutant should have considerably lower affinity than the wild type 8 receptor for 8 agonists. While this was found to be the case for agonists, the antagonists exhibited minimal differences in binding between mutant and wild-type receptors. This once again suggests that Asp128 is not involved in the recognition of antagonists and that the recognition sites for agonists and antagonists differ. The fact that the Asn128 mutant bound 8 antagonists equally as well as the wild-type receptor might mean that a receptor-based anionic residue is not important for the binding of antagonists. However, another interpretation is that a counterion is necessary, but an anionic residue other than Asp128 is involved in the binding of antagonists. In an effort to distinguish between these possibilities, amide 7 corresponding to NTI was synthesized and found to possess little or no airmity for 8 receptors [20]. This suggested the latter interpretation of the data the more likely possibility. 0

H

I

H

DI~.TA OPIOID RECk--fOR SUBTYPES

Evidence for the existence of subtypes of the 8 opioid receptor (81 and 82) has been acquired through in vivo pharmacologic studies using selective opioid agonists and antagonists [21,22]. [I)-Pen2,D-Pen5]enkeph~lin [23] (DPDPE)is selective for 81, while [D-Ser2,LeuS]-enkephalin Thr 6 [24] (DSLET) and [DAla2]deltorphi~n II [25] are selective for 82. The antagonist ligands employed to distinguish between these 8-receptor subtypes were [D-Ala2,LeuS]enkephalin Cys 6 [26] for 81 and the NTI analogues, naltriben [15] 9 ( N ) and naltrindole5'-isothiocyanate [27] for 82.

308

H

8 (BN'~

9 (N'm)

[~ to

It appears that the conformation of the address may contribute to subtype selectivity, but it is dearly not the only factor. With regard to the role of conformation, the 81-selective antagonist, 7-benzylidenenaltrexone [28] 8 (BNTX), possesses a phenyl group that is orthogonally oriented with respect to ring C of the morphinan structure. The nonpeptide 8 agonist, SIOM 6, has been reported [17] to be al selective and also has a similarly oriented address. This is in contrast to. the coplanar arrangement of the address in the 82selective antagonist NTB 9 (Fig. 3).

Yi~u'e 3.

Superposition of SIOM, BNTX, and NTB.

If conformation were the only factor contributing to a subtype selectivity, NTI would be expected to be a 82-selective antagonist, as its address moiety is superimposable with that of NTB. However, we have found that NTI does not dearly distinguish between a subtypes. Thus, an additional factor may play a role in 8 subtype selectivity. In view of the fact that the 82 antagonists, NTB and N-benzyl-NTI [29] 10, are more lipophilic than NTI, their 8 subtype selectivity might be related in part to their greater lipid solubility. The pharmacologic evaluation of amino acid conjugates (11, 12) of NTI has provided some insight into this possibility, as these compounds are considerably more hydrophilic than NTI [30]. The finding that 11 and 12 are highly potent al-selective antagonists in vivo suggests that subtype selectivity of 8 antagonists may in part be a function of the greater hydrophilic character contributed by their amino acid residues. Thus, the hydrophilic-lipophilic

309 balance of the antagonist ligand might differentially modulate access to different compartments in the CNS that contain 81 and 82 receptors. For example, accessibility to tissue compartments that contain 81 receptors might be favored by more hydrophilic molecules. Conversely, hydrophobic antagonists might gain access to 52 receptors more readily, while access to 81 receptors is impeded.

~176

H

~C;OOH

i

HO

o

H ""R

11, R = H 12, R = CH2COOH

SUMMARY AND CONCLUSIONS Structure-activity relationship studies have suggested that agonist and antagonist ligands bind to separate or overlapping recognition sites on 8 opioid receptors. These sites eThibit different conformational requirements for the address component (phenyl or benzene moiety). In this regard, nonpeptide agonists (e.g., SIOM) require the plane of the aromatic group to be approximately orthogonal to the plane of ring C in the opiate. On the other hand, there are no specific conformational requirements for antagonists, as the aromatic group that comprises the address may be coplanar (e.g., NTI or NTB) to ring C or oriented orthogonally (e.g., SINTX, BNR~). However, an aromatic group that is coplanar with ring C possesses greater antagonist potency than an orthogonally oriented group. Is there any physiologic significance for separate agonist and antagonist binding sites? It seems within the realm of possibility that binding of endogenous opioids (e.g., enkephalin) to discrete recognition sites on the a receptor may play a modulatory role in signal transduction. A model for the existence of separate recognition sites for agonists and antagonists was proposed in 1983 [31] for the g opioid receptor and is presented here in a modified version for the 8 receptor in Figure 4. We suggest that the 8 receptor possesses two discrete recognition sites that are topographically similar, but not identical. The sites are proposed to possess different afAnities for enkephalin. For agonists, binding to the higher atrmity site (site 1) is associated with signal transduction, while occupation of the lower amnity binding site (site 2) decreases the binding to site 1. Site 2 therefore would function as a regulatory site to reduce signal transduction at high concentrations of endogenous peptide.

310 ml

~rrN~

~F

Figure 4.

A conceptual model for distinct agonist (site 1) and antagonist (site 2)recognition sites on opioid receptors. Binding of endogenous agonist to site 1 stabilizes the active state (a). The inactive state is stabilized by binding of agonist to site 2 (b). The efficacy of an agonist is reflected by the steady-state ratio of agonist-bound active and inactive states. A ~pure ~ antagonist binds selectively to site 2 to shift equilibrium almost entirely to the inactive state. The wavy arrow within the receptor signifies receptor activation of G protein, while the straight arrow symbolizes the stabilization of G protein in the inactive state.

The presence of two sites for agonists present some intriguing possibilities for the SAR of opioid ligands. For example, the efficacy of an agonist ligand would be determined by its relative ~fl~nity for sites 1 and 2. High efficacy would be due to much higher affinity for the agonist site (site 1) relative to the regulatory site (site 2). Conversely, low efficacy agonists or partial agonists would be ligands whose atrmity to site 2 relative to site I is greater than that of high efficacy agonists. An antagonist is envisaged to be a ligand whose atrmity for site 2 is much greater than for site 1. Antagonist binding to site 2 would also promote stabilization of the inactive state of the opioid receptor (32-35). Finally, pharmacologic studies point to the existence of 8 receptor subtypes. The antinociceptive effect of SIOM appears to be mediated through 81 opioid receptors. If DPDPE and SIOM bind to the same agonist recognition site on the 81 receptor, this would suggest a similar conformational relationship between the message and address components for these molecules. The conformation of the address in antagonist ligands plays a less i m p o ~ t role in 8 subtype selectivity, inasmuch as attachment of polar residues to NTI

311 changes the subtype selectivity to 51 receptors even though it retains its coplanar aromatic group which appears to favor 62. Thus, 6 subtype selectivity for antagonists may be determined by a combination of factors. While the coplanar orientation of the address and ring C (e.g., NTB) appears to confer ~2 selectivity, increasing the polarity of the molecule favors 81 selectivity. A possible explanation for this change in subtype selectivity may be the localization of 81 and 52 receptors in different tissues or cellular compartments having different accessibility to the antagonist ligands. REFERENCES

10 11 12 13 14 15 16

17 18 19 20 21

Evans CJ, Keith DE, Morrison H, Magendzo K, Edwards RH. Science 1992; 258; 1952-1955. Kieffer BL, Befort K, Gaveriaux-Ruff C, Hirth CG. Proc Natl Acad Sci USA 1992; 89; 12048-12052. Yasuda K, Raynor K, Kong H, Breder CD, Taketa J, Reisine T, Bell G I. Proc Natl Acad Sci USA 1993; 90; 6736-6740. Chen Y, Mestek A, Liu J, Hurley JA, Yu L. Mol Pharmacol 1993; 44; 8-12. Wang JB, IrnAi y, Eppler CM, Gregor P, Spivale CE, Uhl GR. Proc Natl Acad Sci USA 1993; 90; 10230-10234. Schiller PW. Prog Med Chem 1991; 28; 301-340. Hruby VJ and Gehrig C~, Med Chem Res Rev 1989; 9; 343-401. Portoghese PS. In: Herz A, ed. Opioids I. Berlin; Springer Verlag, 1993; 279-293. Portoghese PS, Sul~n~ M, Nagase H, Takemori AE. J Med Chem 1988; 31;281-282. Takemori AE, Portoghese PS. Annu Rev Pharmaeol Toxicol 1992; 32; 239-269. Schwyzer R. Ann NY Acad Sci. 1977; 297; 3-26. Lipkowski AW, Tam SW, Portoghese PS. J Med Chem 1986; 29; 12221225. Portoghese PS, Sultana M, Takemori AE. J Med Chem 1990; 33; 17141720. Takemori AE, Sultana M, Nagase H, Portoghese PS. Life Sci 1992; 50; 1491-1495. Portoghese PS, Nagase H, MaloneyHuss KE, Lin CE, Takemori AE. J Med Chem 1991; 34; 1715-1720. Portoghese PS, Sultana M, Moe ST, Takemori AE. J Med Chem 1994; 37; 579-585. Portoghese PS, Moe ST, Takemori AE. J Med Chem 1993; 36; 2572-2574. Kong H, Raynor K, Yasuda K, Moe ST, Portoghese PS, Bell GI, Reisine T. J Biol Chem 1993; 268; 23055-23058. Reisine T. Neuropharmacol 1995; 34; 463-472. Larson DL, Portoghese PS. Unpublished data. Sofuoglu M, Portoghese PS, Takemori AE. J Pharmacol Exp Ther 1991; 257; 676-680.

312 22 23 24 25 26

27 28 29

30 31 32 33 34 35

Jiang Q, Takemori AE, Sultana M, Portoghese PS, Bowen WD, Mosberg HJ, Porreca F. J Pharmacol Exp Ther 1991; 257; 1069-1075. Mosberg HI, Hurst R, Hruby VI, Gee K, Yamamura HI, Galligan JJ, Burks TF. Proc Nail Acad Sci U S A 1983; 80; 5871-5874. Fournie-Zaluski M-C, Gacel G, Maigret B, Premilat S, Roques BP. Mol Pharmacol 1981; 20; 484-491. Erspamer V, Melchiorri P, Falconieri-Erspamer G, Negri L, Corsi R, Severini C, Barra D, Simmaco M, Kreil G. Proc Nail Acad Sci U S A 1989; 86; 5188-5192. Bowen WD, HeUeweU SB, Keleman M, Huey R, Stewart D. J Biol C h e m 1~7; 262; 13434-13439. Portoghese PS, Sultana M, Takemori AE. J Med Chem 1990; 33; 15471548. Portoghese PS, Sultana M, Nagase H, Takemori AE. Eur J Pharmacol 1992;218; 195-196. Korlipara VL, Takemori AE, Portoghese PS. J Med Chem 1994;37; 18821885. Portoghese PS, Farouz-Grant F, Takemori AE. J Med Chem 1995; 38; 402-407. Portoghese PS, Takemori AE. J Med Chem 1983; 26; 1341-1343. Costa T, Herz A~ Proc Nail Acad Sci 1989; 86; 7321-7325. Costa T, Ogino Y, Munson PJ, Onaran HO, Rodbard D. Mol Pharmacol 1992; 41; 549-560. Sr W, Freissmuth M. Trends Pharmacol Sci 1992; 13; 376-379. IV[ilIigan G, Bond RA, Lee M. Trends Pharmacol Sci 1995; 16; 10-13.

Perspective in Receptor Research D. Giardinh, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

313

THE ROLE OF NOVEL LIGANDS IN THE BIOLOGICAL CHARACTERIZATION OF SIGMA RECEPTORS Brian R. de Costa, a Xiao-shu He,b Celia Dominguez, c Wanda Williams, d Kenner C. Riced and Wayne D. Bowend aMedical Sciences Building, University of Toronto, Toronto, Ontario M5S lAB, Canada. bNeurogen Corporation, 35 Northeast Industrial Road, Branford, CT 06405, U.S.A. CDuPont Merck Pharmaceutical Co., Experimental Station, Cardiovascular Diseases Research, PO Box 80402, Wilmington DE 19880-0353., U. S. A. dLaboratory of Medicinal Chemistry, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 8, Room B1-26, 9000 Rockville Pike, Bethesda, MD 20892., U. S. A. Investigation of the physiological and pharmacological role of sigma receptors is a relatively new area which has been facilitated by the development of high affinity and selective sigma receptor ligands including putative agonists and antagonists [1]. Many different classes of drugs, ranging from steroids such as progesterone to neuroleptics such as haloperidol, exhibit high affinity for sigma receptors. This binding activity of the neuroleptic drugs suggested that sigma receptors may be responsible, at least in part, for the motor sideeffects of these drugs. The involvement of sigma receptors in several biologic and psychiatric effects ranging from psychotic behavior to second messengers have been reviewed in detail elsewhere [1,2]. More recent studies have indicated uses for sigma receptor ligands in the development of neuroprotective and atypical antipsychotic agents [1 ]. Certain sigma ligands appear to inhibit the behavioral effects of cocaine, which suggests other promising uses for these compounds [ 1]. In general terms, sigma receptors can be summarized as follows: proposed in 1976 by Martin et al non-opioid non-dopaminergic high density found in brain areas that control motor behavior found also in periphery (e.g. liver) exist as subtypes found across many species (including reptiles) MW in the 20-30kDa range no known behavioral effects cross react with many different classes of CNS active drugs confused with many different receptor types

314 The diverse biological activities which add to the "sigma enigma" include: neuroprotective activity motor disturbances certain sigma ligands antagonize motor side effects of haloperidol in rats inhibition smooth muscle contraction may be involved in the cytotoxicity of some drugs may be involved in psychotic behavior modulate Pi turnover may represent modulatory sites for other receptors diversity across many different classes of CNS active drugs The existence of at least two sigma receptor subtypes (designated sigma-1 and sigma2) has been confirmed [1 ]. The vast majority of sigma ligands exhibit high selectivity for the sigma-1 subtype. The disubstituted guanidine DTG (1, Figure 1) displays roughly equal affinity for both sigma subtypes. The 5 , 6 , 7 , 8 , 9 , 1 0 - h e x a h y d r o - 7 , 1 0 iminocyclohept[b]indoles (2, Figure 1)[3] and certain benzomorphan derivatives [4] were shown to be selective and potent sigma-2 ligands. Compounds which bind selectively to sigma1 receptors include (+)-benzomorphans such as (+)-pentazocine, (+)-3-phenyl-1propylpiperidine ([3H](+)-3-PPP), haloperidol, (+)-morphinanssuch as dextrallorphan, cis cyclohexane-l,2-diamines and ethylenediamines [1] (see Figures 3-6). As part of our program to investigate the role of sigma-1 and -2 receptor subtypes, we focused our attention on the high affinity ethylenediamine sigma ligand 3 (Figure 1) and used it as a template [5]. Our interest in the development of sigma-2 selective ligands was spurred by evidence that this subtype may be responsible for the motor effects of sigma ligands (Bowen WD et al., personal communication). This is reinforced by the observation that DTG, which binds with lower affinity to sigma-1 receptors than (+)-pentazocine, is nonetheless more effective at causing motor disturbances in rats.

NyN NH~ 1

HN

N/R ~ ~ N 2 Figure 1

3

CI Cl

315

~N@NI

N~ N

n~Ar

I

~

N ~ Ar

I

8

NH2

?

4:n=0 3:n=l 5:n=2 6:n=3 7:n=4

N~

~NN/NAr 14

I

I

N ~

N ~ Ar

9 I

I

N~

I

N~/~ NAAr I

N ~ N ~ N @ A r Ar = ~ ~ [ ~ CI C1

12

~N

~

10

N

I

~

N

I

~

Ar

& N ~ N ~ N I

13 H2N~NH ~ 15

Ar

H

H2N~N~N

H~

16

Ar

H

~vjN/~/N~

~

Ar

H N~

Ar

I 11

17 Figure 2

Binding studies of polyamines 4-1 7 (Figure 2) indicated that several of these compounds were selective for the sigma-2 receptor subtype. For example, 1 3 is 10-fold selective for sigma-2 over sigma-1 whereas 3 is 4-fold selective for sigma-1 over sigma-2 receptor subtypes [5]. These and other compounds are proving invaluable in the biological characterization of sigma receptor subtypes. The vast array of structure activity studies that have been carried out from 1976-present (see below) have provided several potential therapeutic agents with a range of biological activities that can be summarized as follows: Neuroprotective agents Sigma receptor antagonists for reducing the motor side-effects of neuroleptics Antispasmodics, muscle relaxants Cytotoxic agents (high dose) Diagnostic tools Atypical antipsychotic agents (non-dopaminergic) Modulators for the effects of other drugs: e.g. cholinergic agents, opioids... Sigma ligand classes (1 8- 4 7) discovered and developed from 1976-94 are illustrated in the Figures 3-6 below. The vast array of chemical diversity accommodated by sigma receptors suggests that these receptors may be primordial. This is reinforced by high densities of sigma receptors found in more primitive species such as reptiles (Matsumoto RR, personal communication).

316

Figure 3: Sigma ligands (1976-1990). Z N__N

N "

f/"N

N

18

F

1

N,.

X 20

R

"Y

N.

X

19

~

R

21

:.

/R

N

22

R R

N 1~2R 3 23

24

26

27

CI"

v

25

28

Key to Figure 3:1 8: (+)-SKF10,047 [(+)-cis-benzomorphan opiates]; 1: N,N'-di(otolyl)guanidine; 1 9: 1-alkyl-4-arylpiperazines; 2 0: 3-phenylpiperidines; 2 1: octahydrobenzo[f]quinolines; 2 2: (+)-morphinans; 2 3: phenothiazines; 2 4: 4phenylpiperidines; 2 5: haloperidol (and other butyrophenones); 2 6: progesterone (and related steroids); 2 7: phencyclidine (arylcyclohexylamines); 2 8: tetrahydroisoquinolines and related benzylamines

317

Figure 4: Sigma ligands (1976-1990).

cI

MeHNm~~ 29

-

R

Cl

30

31

H

,~ N ~ N ~ H Cl

NI'I2

r~

~N 32

OH

N ,~,

35

34

33

~ .,~

N. R2 36

Key to Figure 4:2 9: cis-N-methyl-2-(1-pyrrolidinyl)arylacetamides; 3 0: sertraline and other 1-phenyl-4-aminotetralins; 3 1" clorgyline (MAOI's); 3 2. ifenprodil; 3 3: ciscyclohexanediamines; 3 4" polyamines; 3 5: tricyclic antidepressants; 3 6" 2-phenethylamines

318

Figure 5: Sigma ligands (1990-94)

~..~~1~ N ,

c'

Cl

3

37

~N/R 38

~ /H R N H 39

40

o

~ , ~ o

HO

41

L~.

42

c, CI

Key to Figure 5:3 : N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1 -pyrrolidinyl)ethylamine and other substituted ethylenediamines; 3 7: N-substituted-5-phenylpentylamines; 3 8: spiropiperidines; 3 9: hexahydrobenz[f]isoquinolines; 4 O: octahydrobenz[f]isoquinolines; 4 1: (aminoalkoxy)chromones; 4 2: 1-[2-(3,4-dichlorophenyl)ethyl]-4-(1 -propyl)piperazine

319

Figure 6: Sigma ligands (1990-94)

N/• 43

R2

Cl

NR1

44

2

i N

Cl

I

N I

45

46

~.-" L ~ LI/.L

-.~-cI

Cl cl

Cl

47

Key to Figure 6:4 3: 1 -benzyl-4-[ 1 -aryl-(1 -ketoethyl)]piperidine; 4 4: 1 -[2-(3,4dichlorophenyl)ethyl]-3-(1-pyrrolidinyl)piperidine; 2: hexahydro-7,10iminocyclohept[b]indoles; 4 5: N,N'-dimethyi-3-[2-(3,4dichlorophenyl)ethylamino]piperidine; 4 6: N-(3,4-dichlorophenylethyl)-N,N'-dimethyl-2(aminomethyl)piperidine; 4 7 1-[2-(3,4-dichlorophenyl)ethyl-2-[(1pyrrolidinyl)methyl]pyrrolidine Sigma receptor research has been greatly facilitated by the development of chemical tools [1,2] ranging from antagonists which allow the probing of sigma receptor function to PET scanning iigands which have allowed visualization of sigma receptors in mammalian brain [6]. The radioligand [3H] azidoDTG has allowed identification of two receptor subtypes in brain [7]. Modeling Several models have been proposed to account for the chemical diversity of sigma iigands: eModels permit a large degree of steric tolerance. oBasic requirements for binding are aromatic ring separated from N by 2 carbon atoms with a bulky lipophilic N substituent. eModeling: (1) Manallack 1988: hypothetical receptor points; (2) Largent 1987: 3- and 4phenyipiperidines with bulky N-substituent; (3) Ablordeppey 1992: 2-phenylpropanolamine oRevision of the above models may be necessary to account for compounds such as the ~carbolines oModels may be inaccurate due the existence of subtypes The weak binding of polyamines (Table 1) to sigma receptors suggests that polyamines may be the biological modulators of sigma receptors [8]:

320 Table 1" Inhibition of [3H](+)-3-PPP binding by polyamines. IC50 (mM+SEM) IC50 (mM+SEM) Compound IC50 (mM+SEM) Rat Forebrain Adrenal Medulla Guinea Pi9 brain 104.8+20.1 82.1+14.4 Spermine 9.2+2.0 345.3+55.0 280.8+88.2 Spermidine 69.9+22.9 1018.2+147.9 716.0+221.8 Cadaverine 398.2+ 103.3 932.2+173.0 1740.8+420.3 Putrescine 410.8+ 180.1 Data extracted from Paul et aL 19 9 0

Conclusion Our own work in the sigma receptor field can be summarized as follows: ediscovered new sigma ligands starting with the kappa opioid agonist U50,488 edeveloped compounds with high potency and selectivity by conformational restriction of N-J2( 3,4-dichlorophenyl)ethyl]-N-methyl-2-( 1 -pyrrolidinyl) ethyla mine edeveloped subtype selective agents edeveloped sigma receptor antagonists oconfirmed the involvement of sigma receptors in the motor side-effects of neuroleptics ediscovered neuroprotective, antidystonic, and even cytotoxic effects among different sigma ligands from diverse chemical classes odesigned research tools to probe the sigma receptor The field of sigma receptor research continues to be an exciting area with a strong potential for the discovery of novel therapeutic agents. With continued and intensive chemical approaches to further characterize this receptor, the "sigma enigma" may soon be solved! References 1 For an update on the structure-activity-relationships of sigma receptor ligands see: de Costa BR, He XS in: Itzhak J, Chapman C, eds. The Sigma Receptors. London: Academic Press, 1994; 45-111. 2 Extensive reviews of sigma receptor pharmacology, biochemistry and physiology are presented in: Walker JM,.Bowen WD, Walker FO, Matsumoto RR, de Costa B Rice KC. PharmacoL Rev. 1990; 542: 355-402. 3 Mewshaw RE, Sherill RG, Mathew RM, Kaiser C, Bailey MA, Karbon EW. J. Med. Chem. 1993; 36: 343-352. 4 Bertha CM, Mattson MV, Flippen-Anderson JL, Rothman RB, Xu H, Cha XY, Becketts K.; Rice KC. J. Med. Chem. 1994; 37: 3163-3170. 5 de Costa BR, He XS, Dominguez C, Cutts J, Williams W, Bowen WD. J. Med. Chem. 1994; 37: 314-321. 6 Lee KS, Weinberger, D et al., unpublished. 7 Kavanaugh MP, Tester BC, Scherz MW, Keana JFW, Weber E. Proc. Natl. Acad. Sci. USA. 1988; 85: 2844-4288. 8 Paul IA, Kuypers G, Youdim M, Skolnick P. Eur. J. Pharmacol. 1990; 184: 203-204.

Perspective in Receptor Research D. Giardin~, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

321

T e t r a a m i n e s as lead c o m p o u n d s for the design of n e u r o t r a n s m i t t e r r e c e p t o r ligands: focus on a-adrenergic and m u s c a r i n i c receptors recognition C. Melchiorre a, P. Angelib, M.L. Bolognesia, R. Budriesi a, S. Cacciaguerra c, A. Chiarini a, M. Crucianelli b, D. Giardin~ b, U. Gulinib, G. Maruccib, A. Minarini a, S. Spampinato c and V. Tumiatti a a Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy b Department of Chemical Sciences, University of Camerino, Via S. Agostino 1, 62032 Camerino (MC), Italy c Department of Pharmacology, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy

A b s t r a c t . The concept that polyamines may represent a passe-partout (master key) for G-protein-coupled receptors recognition is illustrated. Using a linear t e t r a a m i n e as the focus, appropriate structural modifications have allowed to obtain novel tetraamines displaying selectivity for different receptor systems. It is shown that tetraamines have receptor subtype selectivity since they are site-directed, owing to different chain lengths separating the protonable nitrogens and to the presence of those peculiar structural elements which make them able of discriminating at the binding stage.

INTRODUCTION The advent of molecular biological techniques has made possible to identify and chemically characterize many receptor systems. It has been demonstrated that a high percentage of homology exists not only within receptor subtypes but also among different receptor families. For example, a comparison of the t r a n s m e m b r a n e domains of G - p r o t e i n - c o u p l e d receptors reveals t h a t muscarinic receptor subtypes have more than 70% identity, whereas aadrenoreceptor subtypes show a lower identity (about 40%). However, aadrenoreceptor subtypes share about 45% identity with serotoninergic and dopaminergic receptors [1]. It is evident t h a t the high homology among receptors accounts for the difficulty to achieve receptor selectivity which remains one of the most fascinating challenge to medicinal chemists. In fact, little is known about the structural elements that determine selectivity to one receptor subtype, rather than to another because only subtle and unpredictable differences in the binding pockets would account for selective binding for certain ligands.

322 TETRAAMINES AND R E C E P T O R R E C O G N I T I O N Although receptor homology makes inherently difficult to achieve receptor selectivity it may be used indeed to design molecular entities which are able, in principle, to recognize different receptor systems. This working hypothesis derives from the consideration that n e u r o t r a n s m i t t e r receptors are folded polypeptide chains which contain always the same amino acids, albeit in a different proportion and sequence. The sequence of the amino acids constitutes the primary structure which derives by peptide bonds linking the carboxylate groups to the amino groups. The resulting repeat units in the chain, i.e. the peptide bond and the a carbon, form the backbone. Since the backbone of a protein has only a structural role and cannot be considered a target for selectivity, it is evident that only lateral chains play a major role in drugreceptor binding. Among these functionalities aspartate and/or glutamate residues may acquire p a r a m o u n t importance for the binding with cationic ligands by way of a cation-anion interaction. Considering that proteins may bear several carboxylate residues, somewhere in their structure, in principle, it is possible to design a lead compound having a polyamine backbone which is able to recognize multiple anionic sites of a given receptor. Thus, such a ligand may interact with all receptor proteins, provided that the distance separating the amine functions of the ligand fits the distance between the carboxylate residues of the receptor. In other words a polyamine could be considered a "passe-partout" in the drug-receptor recognition process. The question is: What structural parameters of the polyamine backbone do receptor systems recognize in order to respond so differently to such subtle structural differences? Clearly, it is necessary to define the structural elements which cause selectivity for a given receptor rather than to another. It was reported t h a t polyamines, such as spermine, are highly protonated at physiological pH. It turned out that spermine is 97% tetracation, the remainder being the trication [2]. Thus, the ability of a polyamine to interact with biological counterions, that is a set of carboxylate anions fixed to the backbone of a receptor, could well be related to its cationic properties. Consequently, the distance between the cationic nitrogens of a polyamine becomes critical in the recognition step. It is known that increasing the number of interactions t h a t take place between a receptor and a ligand there is more chance to distinguish receptor systems. Thus, an appropriate modification of the chain length separating the nitrogens of a polyamine might give rise to an increase of selectivity, whereas the insertion of N - s u b s t i t u e n t s might improve the affinity by increasing the overall number of contacts between a dl~ug and a receptor. Many naturally occurring or synthetic polyamines with a linear tetraamine backbone are known. These ligands recognize different receptors which may be either activated or inhibited. For example, synthetic tetraamine disulfides proved to be a-adrenoreceptor or neuropeptide Y receptor antagonists [3-5], whereas synthetic polymethylene tetraamines resulted potent and selective muscarinic receptor subtype antagonists [4, 6, 7]. Furthermore, it has been demonstrated that the naturally occurring tetraamine spermine and analogues modulate NMDA receptor function [8]. The polyamine NMDA receptor site may have either agonistic or antagonistic modulatory activity depending on the

323 concentration and the chemical structure of the polyamine. In addition, N alkylated spermine analogues displayed antineoplastic activity against a number of tumor lines [9] and, in general, it can be stated t h a t natural polyamines are in many ways involved in the complex phenomena related to host defense mechanisms [10]. T E T R A A M I N E S VS a - A D R E N O R E C E P T O R S a l - A d r e n o r e c e p t o r s are not homogeneous as clearly evidenced by pharmacological tools and molecular biological techniques [11]. Two native aladrenoreceptor subtypes, that is alA and ~lS, have been so far characterized by functional and binding assays. The ~IA subtype has high affinity for antagonists such as WB 4101, 5-methylurapidil and (+)-niguldipine, and is insensitive to inactivation by chloroethylclonidine (CEC). The alB subtype displays low affinity for the above antagonists, but is preferentially inactivated by the alkylating agent CEC [11]. However, very recently it has been demonstrated that the aladrenoreceptor mediating contraction upon activation in isolated rat aorta may belong to the alp subtype [12, 13]. Cloning studies have confirmed the existence of at least three distinct al-adrenoreceptors, namely the alb, alc and ald subtypes [14-16]. The cloned alb-adrenoreceptor corresponds to the native alB type whereas the alc-adrenoreceptor is similar to the pharmacologically defined alAadrenoreceptor and for this reason it has been named Ula [17, 18]. Thus, aladrenoreceptors have been subdivided into alA (CY.la), (ZlS (alb) and O~ld subtypes [17]. Although the cloned ~qd-adrenoreceptor is not well characterized yet may find a correspondence with the pharmacologically defined aid subtype in rat aorta [12, 13]. Design of novel tetraamine disulfides based on benextramine and prazosin

Tetraamine disulfides, whose main structural feature is a cystamine moiety carrying amino alkyl substituents on the nitrogen atoms, represent a class of non-competitive antagonists of both al- and ~2-adrenoreceptors [3, 4]. It was shown that al-adrenoreceptor inhibition by tetraamine disulfides is the result of covalent bond formation between a receptor target thiol and the disulfide bridge of the antagonist through a disulfide-thiol interchange reaction [3, 4, 19, 20]. It was also found that optimum activity is associated with well defined chain lengths separating the inner from the outer nitrogens and the inner nitrogens from the sulphur atoms, and depends on the type of substituents on the terminal nitrogens [3, 4]. Benextramine resulted the prototype of this class of irreversible antagonists which bears no structural kinship with the other known a-adrenoreceptor antagonists. It has been fairly well investigated in both functional studies and binding assays [4]. Prazosin is a selective competitive al-adrenoreceptor antagonist widely used not only as a pharmacological tool for a l - a d r e n o r e c e p t o r subtypes c h a r a c t e r i z a t i o n but also as an effective agent in the m a n a g e m e n t of h y p e r t e n s i o n . Its a n t i h y p e r t e n s i v e activity depends on a p e r i p h e r a l vasodilatation mediated by a postjunctional ~l-adrenoreceptor blockade. Moreover, given its high al-selectivity, prazosin lacks side effects such as

324 t a c h y c a r d i a and hypereninemia, which are connected with a presynaptic a2adrenolytic action [21-24]. It has been advanced that benextramine and prazosin may interact with the very same site where norepinephrine binds. On the basis of substituents effects and t a k i n g advantage of the finding t h a t norepinephrine at a relatively low concentration (30 btM) afforded complete protection of aortic al-adrenoreceptors s

iI

ss ~

~%%

%

NH 2 ~

,

"r'f"<'CFNN I

,

A L ) A L )A, A

\ MeO"

/.. /

v

"N"

.

%

, " -",

"~

I

i

H

H2N

9. .

.,'N"

S I

Prazosin

9

l

1 !

,

H

N

S--S

[

,'" ,~

"',

NH2

,.

."

~!

e

Benextramine I

MeO

N

T e t r a a m in e b ac kb one

"

MeO

J

II

I

NH 2 N N(CH2)nNH(CH2)2S I

R n = 4 - 12, R = H, Me (Table 1)

Figure 1. Design strategy for the synthesis of hybrid tetraamines by inserting the structural features of prazosin on the terminal nitrogens of the tetraamine backbone of benextramine.

325 against benextramine, it was advanced that the terminal nitrogen atoms of b e n e x t r a m i n e may i n t e r a c t with the anionic site recognized by the neurotransmitter, while the 2-methoxybenzyl substituent would bind to an accessory area [3]. In agreement with this view, it was found that a catecholbearing tetraamine disulfide, that is a molecular combination of norepinephrine and benextramine, was equiactive with benextramine at rat vas deferens aladrenoreceptors [25]. It has been advanced t h a t prazosin binds also with the norepinephrine binding site [26]. This al-adrenoreceptor model focuses on the importance of c h a r g e - r e i n f o r c e d hydrogen bonding between p r o t o n a t e d agonists and antagonists and a common negatively charged carboxylate counterion. Both n e u r o t r a n s m i t t e r and the 2,4-diamino-6,7-dimethoxy-quinazoline moiety of prazosin interact with the receptor ground state in a similar fashion. However, the antagonist is not able to promote the same conformational change induced by the agonist which leads to receptor activation [26]. If the above hypotheses were correct, it derives that the terminal nitrogens of benextramine, prazosin and norepinephrine interact with the same anionic receptor site. Thus, we thought it of interest to obtain hybrid structures by combining the structural features of both benextramine and prazosin. These derivatives might acquire relevance in a-adrenergic pharmacology for receptor subtypes characterization. Benextramine proved to be relatively selective for the axA-adrenoreceptor subtype [27] which may suggest a pivotal role for the tetraamine backbone at the recognition stage. Since the four amine functions of t e t r a a m i n e disulfides have been already shown to be essential for aladrenoreceptor blocking activity, the relatively low affinity of benextramine for a l-adrenoreceptors might be due to a poor fit of the benzyl moiety of benextramine with the accessory binding site [3, 4]. Considering that the quinazoline moiety of prazosin is essential for high affinity toward aladrenoreceptors [26], the replacement of the benzyl moiety of benextramine with a quinazolinyl nucleus might improve affinity while retaining, hopefully, selectivity for aa-adrenoreceptor subtypes. This design strategy finds support, albeit tenuously, in the observation that the piperazine moiety of prazosin may not be essential for activity at al-adrenoreceptors and can be replaced by an a,o~-alkanediamine chain [28]. Furthermore, it was observed that activity and selectivity depend on the length of alkane chain. The most potent compound of this series was the one bearing a 1,6-hexanediamine moiety which allowed us to advance the hypothesis that the ~l-adrenoreceptor incorporates a lipophilic area, located between the binding sites for the quinazoline and the furan rings of prazosin, which is able to accommodate optimally a 1,6-disubstituted hexane moiety [28]. The fact that benextramine has also a 1,6-hexanediamine moiety separating the inner from the outer nitrogens may suggest that this alkane chain interacts with the same lipophilic pocket where prazosin analogues interact. On the basis of these considerations we studied a series of tetraamine disulfides whose design strategy is schematically shown in Figure 1.

Biological profile at ~-adrenoreceptors

The functional activity of hybrid tetraamine disulfides at a-adrenoreceptors was investigated on isolated rat vas deferens using benextramine and prazosin

326 Table 1 Antagonist affinities of tetraamine disulfides at CZl- and ~.2-adrenoreceptors in the isolated r a t vas deferens, and for inhibiting [3H]prazosin binding at aladrenoreceptor subtypes of rat submaxillary gland (aaA) and liver (alB) NH2 MeO MeO

N(CH2)nNH(CH2)2SI 2 R

no. a Benextramine Prazo sin Spiperone 1 2 3 4 5 6 7 8 9 10

n

4 5 6 6 8 8 10 10 12 12

R

H H H Me H Me H Me H Me

pA2 (pICso*) b

pKi (plCso*) c

al

a2

alA

alB

5.12" 8.54

6.09* 5.43

7.01" 9.07 7.45

6.23* 9.14 8.80

6.76 7.32 7.38 7.81 7.32 7.65 6.64 7.91 6.65 7.45

4.17" 4.80* 5.75* 5.34* N.A. a N.A. a N.A. d N.A. e N.A. a N.A. e

7.63 7.30

8.08 8.15 e

7.10 7.91 7.08 7.91 7.20

8.14 8.19 e 7.73 7.75 7.51 e

aAll t e t r a a m i n e disulfides were tetrahydrochloride salts, bpA2 values were determined by Schild plots [29] obtained from the dose ratios at the ECso values of the agonist (-)-norepinephrine calculated at three antagonist concentrations. Each concentration was tested from four to six times and Schild plots were constrained to a slope of-1. The irreversible u-blockade for benextramine and 14 is expressed as ICso values, which were estimated from graphical plots of p e r c e n t i n h i b i t i o n vs log m o l a r concentration and are defined as the c o n c e n t r a t i o n s t h a t produce 50% inhibition of the m a x i m a l response to n o r e p i n e p h r i n e (al) or clonidine (~.2). r are the m e a n of at least three s e p a r a t e experiments performed in triplicate. All Hill n u m b e r s (nil) were not significantly different from unity (p > 0.05), unless otherwise specified, pKi values were derived using the Cheng-Prusoff equation [30]. pKi values are not given for b e n e x t r a m i n e because the ICso values contain a kinetic t e r m for irreversible binding to the [3H]prazosin binding sites. Nonspecific binding was assessed in the presence of 10 pM phentolamine, aN.A. = not active up to a concentration of 50 ~M. eHill numer was significantly different from unity (see Table 2).

327 as the reference compounds, al-Adrenoreceptor blocking activity was assessed by antagonism of (-)-norepinephrine-induced contractions of the epidydimal portion of the vas deferens, whereas a2-blocking activity was determined by antagonism of the clonidine-induced depression of the twitch responses of the field-stimulated prostatic portion of vas deferens as reported previously [28, 31]. We chose to examine the receptor subtype selectivity of selected tetraamines 3, 5-9 by employing receptor binding assays. [3H]Prazosin was used to label alAand alB-adrenoreceptors binding sites of rat submaxillary gland and liver homogenates, respectively. Prazosin, benextramine and the alB-selective antagonist spiperone [11] were used as the standard compounds. The results obtained at rat vas deferens e.-adrenoreceptors are shown in Table 1. Surprisingly enough, all hybrid tetraamines 1-10, unlike benextramine, did not inhibit irreversibly ul-adrenoreceptors, r a t h e r they antagonized competitively n o r e p i n e p h r i n e - i n d u c e d responses like prazosin. However, t e t r a a m i n e s 1-4 inhibited irreversibly, although with a significantly lower potency than benextramine, clonidine-induced responses at a2-adrenoreceptors. This remarkable difference in the observed antagonism at al-adrenoreceptors may have two main sources: a) hybrid tetraamines 1-10 and benextramine may react with two distinct sets of sites or b) they may bind with the same set but t e t r a a m i n e s 1-10 are not able to unmask the buried receptor thiol thus preventing the disulfide-thiol interchange reaction which would lead to irreversible inactivation of the receptor. Unfortunately, it is not possible to distinguish yet between these two possibilities. An analysis of the results reveals t h a t t e t r a a m i n e s 1-10 displayed a significant potency, albeit lower t h a n t h a t of prazosin, toward a ladrenoreceptors whereas, again in contrast with benextramine, they showed only a modest, if any, affinity for a.2-adrenoreceptors. In fact, hybrid tetraamines displayed a significant selectivity (50-1000-fold) for a a - v e r s u s a2adrenoreceptors whereas benextramine showed an a2-selectivity (about 10-fold). Clearly, the replacement of the 2-methoxybenzyl group of benextramine with a quinazoline nucleus affording tetraamines 1-10 alters significantly the biological profile in comparison to benextramine. Concerning the chain length effects, it turned out that optimum potency at e.l-adrenoreceptors was associated with a six-carbon chain as in 3 although the homologues 2 and 5 retained the same level of activity. However, at a2-adrenoreceptors the chain length effects were more pronounced since either a shorter or a longer carbon chain t h a n six methylenes caused a significant drop in activity. Interestingly, N-methylation of the outer nitrogens of 3, 5, 7 and 9 affording the corresponding analogues 4, 6, 8 and 10 caused an increase in affinity for ~.1-but not ~2-adrenoreceptors. This finding parallels the results observed for prazosin analogues bearing a tertiary amine function at position 2 of the quinazoline moiety [28]. Tetraamines 4, 6, 8 and 10 resulted almost equiactive to each other suggesting that the chain length separating the inner from the outer nitrogens may not play an important role in affinity. Tetraamine 8 turned out to be even more selective, although 4 times less potent, than prazosin toward al- versus ~2-adrenoreceptors. Affinity constants, expressed as pKi values, for hybrid t e t r a a m i n e s and reference compounds at ~aa- and ~.lB-adrenoreceptor subtypes are shown in Table 1. These data indicate that, with the exception of 8, hybrid tetraamines

328 are alB-selective, whereas prazosin although being the most potent is not able to distinguish the two subtypes. The greatest selectivity was seen for tetraamine 5 that dispalyed an l l - f o l d selectivity for alB" versus alA-adrenoreceptors. This finding does not parallel the alA-selectivity shown by benextramine [27, this study] indicating that 2-methoxybenzyl and quinazolinyl groups play a different role on selectivity. Interestingly, spiperone, which is considered a selective alBadrenoreceptor antagonist [11], showed only a 4-fold higher affinity at alBadrenoreceptors and a slightly, higher (2-fold) alS/alA selectivity t h a n 5. However, spiperone may not represent a useful tool for the characterization of alB-adrenoreceptors because of the lack of receptor specificity owing to its high affinity for dopamine D2 and 5-HT1A receptors as well. The slopes of the competition curves obtained with hybrid tetraamines, prazosin and spiperone were not significantly different from unity, indicating t h a t the sites labeled with [3H]prazosin in rat submaxillary gland (alAadrenoreceptors) and liver (alB-adrenoreceptors) homogenates are essentially homogeneous. However, in the case of the binding competition curves with t e t r a a m i n e s 3, 6 and 9 in liver membranes, the slopes were significantly different from unity and the data were best fitted to a two-site model, revealing a high affinity site and a low affinity site (Table 2). Clearly, these results do not agree with the view that rat liver represents a pure als-adrenoreceptor tissue [32]. Using the rat alb-adrenoreceptor probe two mRNA species of similar sizes in liver were detected [16, 18, 33]. It has been advanced [18] that the detection of two different alB-adrenoreceptor subtypes in rat liver raises the possibility that such alb-adrenoreceptor isoforms could exhibit different pharmacological profiles. This hypothesis might explain the affinity profile displayed by tetraamines 3, 6 and 9 which are able to distinguish markedly between high and low affinity sites (Table 2). Table 2 Two-site analysis of the interaction of tetraamines 3, 6 and 9 with aladrenoreceptor binding sites in rat liver membranes a High

Low

no.

nH b

pKi

%

pKi

%

high I low c

3

1.19 1.35 0.58

8.12 8.31 7.81

72 47 62

7.30 7.19 6.65

28 53 38

7 13 14

6

9

aData from Table 1 and fitted to a two-site model of binding, bHill numbers (nil) were significantly different from unity (p < 0.05). cThe high/low selectivity ratio is the antilog of the difference between the pKi values at high and low affinity binding sites, respectively.

329 TETRAAMINES VS MUSCARINIC R E C E P T O R S The identification of multiple muscarinic receptor subtypes has stimulated the search for ligands with selectivity for a given receptor subtype [34-36]. Five different subtypes (ml-ms) have been identified so far by molecular cloning. Muscarinic receptors that have been characterized pharmacologically and classified as M1-M4, appear to correspond to cloned ma-n~ receptors. At present, little information is available about the nature and the cellular location of the m5 subtype. Achievement of selectivity is of paramount importance not only for receptor subtype characterization but also for the development of therapeutically useful muscarinic agonists and antagonists [35, 37, 38]. For instance, muscarinic M1 receptor agonists and muscarinic M2 receptor antagonists may be useful to enhance cognitive function [38], whereas muscarinic M3 receptor antagonists have potential for the treatment of airway obstruction. Furthermore, muscarinic M2 receptor antagonists may have application in the treatment of bradycardic disorders. Presently, several antagonists which bind selectively to the pharmacologically characterized muscarinic receptors are available. For example, pirenzepine [39] has high affinity for muscarinic M1 receptors which are mainly located in the central nervous system and peripheral ganglia; polymethylene tetraamines, exemplified by methoctramine [4, 6, 7], display high affinity for muscarinic M2 receptors of cardiac cells, whereas 4-DAMP [40], hexahydrosiladifenidol and its p-fluoro derivative [41-43] show high affinity for muscarinic M3 receptors located in smooth muscle and exocrine glands. Finally, MT3, a toxin isolated from the venom of green mamba, is highly selective for muscarinic M4 receptors [44]. D e s i g n of M1 o a n d Mz-selective t e t r a a m i n e s b a s e d on m e t h o c t r a m i n e Polymethylene tetraamines are a class of competitive antagonists of muscarinic receptors. Their development started from the observation that benextramine also displayed muscarinic antagonistic activity with a significant selectivity for cardiac muscarinic receptors. Using benextramine as the focus, polymethylene tetraamines lacking a disulfide moiety were designed to achieve specific recognition of muscarinic receptors. Detailed structure-activity relationship studies revealed that both affinity and selectivity for muscarinic receptor subtypes are heavily dependent on either the chain length separating the four amine functions or the type of substituent on the nitrogen atoms of polymethylene tetraamines. The development of polymethylene tetraamines, the prototype of which is methoctramine, as muscarinic M2 receptor antagonists has been the subject of review articles [4, 6, 7]. The finding that methoctramine, like all other M2 selective antagonists available, failed to discriminate between muscarinic M2 and M4 receptors, thus preventing a clear pharmacological characterization of muscarinic receptor subtypes, stimulated a research in order to improve affinity and selectivity for muscarinic M2 receptors by modifying methoctramine structure. The starting point of this investigation was the observation t h a t the two amine functions of AQ-RA 741 (11-[[4-[4(diethylamino)-butyl]- 1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][ 1,4] benzodiazepin-6-one), an analogue of pirenzepine displaying selectivity for muscarinic M2 receptors [45], are located to such a distance that they can be

330 superimposed to one inner and one outer nitrogen of methoctramine when comparison is made between the extended conformations of the two prototypes. The observation t h a t diamines, obtained by truncating in two halves methoctramine structure and which are closely related to AQ-RA 741, resulted in analogues almost devoid of affinity and selectivity toward M2 muscarinic receptors was another consideration of paramount importance in the design strategy of our compounds [46, 47]. Clearly, an ll-acetyl-5,11-dihydro-6Hpyrido[2,3-b][1,4]-benzodiazepin-6-one moiety recognizes better t h a n a 2methoxybenzyl group the binding pocket of muscarinic M2 receptors. Thus, we thought that the insertion of a tricyclic system on the terminal nitrogens of a tetraamine would improve affinity for muscarinic M2 receptors. To prevent Nalkylation of the two inner nitrogens, we choose as a common backbone a tetraamine bearing on these nitrogens a methyl group. Furthermore, this choice was dictated by a previous finding that methylation of inner nitrogens of methoctramine affording N,N'-dimethyl methoctramine does not affect both affinity and selectivity for muscarinic M2 receptors [48]. Thus, a series of tetraamines were designed by replacing the terminal 2-methoxybenzyl groups of m e t h o c t r a m i n e with an ll-acetyl-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one moiety [49]. Tripitramine resulted the most potent and the most selective muscarinic M2 receptor antagonist so far available, able to discriminate also between muscarinic M2 and M4 receptor subtypes. The finding that the affinity profile of methoctramine-related tetraamines depends on the type of substituents on the terminal nitrogens of a tetraamine backbone, was the basis for a further modification of methoctramine structure, in order to improve the affinity and selectivity for different muscarinic receptor subtypes. Thus, we designed new tetraamines by substituting the 2methoxybenzyl groups of methoctramine with the structural features of 4DAMP, a relatively selective muscarinic M1/M3 receptor antagonist, and of its analogues spiro-DAMP and hydroxy-DAMP [50]. This study afforded spirotramine that displayed an inverse selectivity profile in comparison to both methoctramine and tripitramine owing to a higher affinity for muscarinic M1 receptors and a significantly lower affinity for all other muscarinic receptor subtypes. The structures of these novel tetraamines are shown in Figure 2 together with those of the parent compounds. Biological profile of novel tetraamines at muscarinic receptor subtypes Functional activity at muscarinic receptor subtypes was determined by the use of the muscarinic M2 receptor-mediated negative inotropism in driven guinea pig left atria (1 Hz) and muscarinic M3 receptor-mediated contraction of guinea pig ileum longitudinal muscle. The muscarinic receptor subtype selectivity was assessed by employing receptor binding assays [49, 51]. [3H]N-methylscopolamine was used to label native muscarinic M2, M3 and M4 receptors binding sites of rat heart, submaxillary gland, and NG 108-15 cell homogenates, respectively, and to label cloned human muscarinic ml-m5 receptors expressed in chinese hamster ovary cells. [3H]Pirenzepine was the tracer to label muscarinic M1 receptors binding sites of rat cerebral cortex.

331

O -COCH 2-N~

~~-'--(CH2)4NEt2

H'N ~ N AQ-RA 741 I~ CH2NH(CH2)6NH(CH2)sNH(CH2)6NHCH2 OMe

MeO Methoctramine

O

Me Me / -C OCH2NH(CH2)6N(CH2)sN(CH2)6N~ CH2CO--N

Tripitramine 0

d\/

Me (CH2)6N(CH2)4

Spirotramine Figure 2. Chemical structures of AQ-RA 741 and of tetraamines methoctramine, tripitramine and spirotramine.

332 Table 3 Antagonist affinities of t r i p i t r a m i n e and spirotramine in comparison to reference compounds a) at muscarinic receptor subtypes in guinea pig left atria (Ma) and ileum longitudinal muscle (M3), and b ) f o r inhibiting [3H]Nmethylscopolamine binding at native muscarinic receptors of rat heart (M2), rat submaxillary gland (M3), NG 108-15 cell (M4) and at cloned human muscarinic m 1-ms receptors, and for inhibiting [3H]pirenzepine binding at native muscarinic M1 receptors of rat cerebral cortex

pA2a

pKi b

M2

M3

M1/ ml

M~ m2

M3/ m3

MJ m4

Methoctramine c

7.82

6.32

Tripitramine c

9.75

6.55

Spirotramine a AQ-RA 741 c 4-DAMPa Pirenzepine c

5.36 8.03 8.53

<5 7.0 9.19

7.43/ 7.30 7.63/ 8.80 7.88 6.98 9.23 8.19

7.84/ 7.84 9.54/ 9.57 6.20 8.49 8.40 6.10

5.96/ 6.56 6.19/ 7.42 5.81 6.60 8.94 6.76

7.58/ 7.42 7.93/ 8.19 6.27 7.82 9.29 7.46

no.

m5

6.50 7.47

apA2 values were determined by Schild plots [29]. bpKi values were derived using the Cheng-Prusoff equation [30]. cData from refs 51 and 52. dData from refs 53 and 54. An analysis of the results shown in Table 3 reveals that 11-acetyl-5,11dihydro-6H-pyrido[2,3-b][1,4]-benzodiazepin-6-one and 2-methoxybenzyl groups play a key role in both affinity and selectivity for muscarinic receptor subtypes. It is evident that an appropriate substitution of the terminal nitrogens of methoctramine affords potent antimuscarinics that display different selectivity profiles. Among the t e t r a a m i n e s so far investigated, t r i p i t r a m i n e and spirotramine possess outstanding properties in comparison to the standards methoctramine, AQ-RA 741, pirenzepine and 4-DAMP in binding assays as graphically shown in Figure 3. Whereas all M2 selective antagonists available to date bind to muscarinic M2 and M4 muscarinic receptors with similar affinities, tripitramine discriminates significantly between these two subtypes with a selectivity ratio value of 41 which is much higher than those of methoctramine and AQ-RA 741 (2 and 5, respectively). Furthermore, tripitramine has an oustanding affinity for muscarinic M2 receptors as revealed by its pA2 value of 9.75 and pKi values of 9.54 (native) and 9.57 (cloned), that were from one to two orders of magnitude higher than those of methoctramine and AQ-RA 741. In addition, t r i p i t r a m i n e distinguishes significantly also between the other

333 muscarinic receptor subtypes (M1 to M3) in binding assays at native receptors as revealed by the selectivity ratio values (M2/M3, 2200; M2/M1, 81). However, tripitramine fails to discriminate between muscarinic M1 and M4 receptors.

10

m M1 D M2 M3 D M4

. . . . , . - , t

pKi

_

_.

t::::Z - _ - - -

-

,-f..-

:: .

.

: :

.. .: .. .: .. : . ; : :9i. :. 1. .:.1. -: .7 : ....

?...

f.,

9

?~:

Methoctramine

AQ-RA 741

Tripitramine

,..:~gg,

I

Spirotramine

Pirenzepine

Figure 3. Affinity constants (pKi) in rat cerebral cortex (M1), heart (M2) and submaxillary gland (M3), and NG 108-15 cells (M4) muscarinic receptors of tripitramine, spirotramine in comparison with methoctramine, AQ-RA 741 and pirenzepine. Furthermore, it turned out that tripitramine has not only an outstanding affinity and selectivity for muscarinic M2 receptors but also a superior specificity compared with methoctramine. In addition to muscarinic receptors, tripitramine inhibited only frog rectus abdominis muscular (pICso value of 6.14) and rat duodenum neuronal (pICso value of 4.87) nicotinic receptors among receptor systems investigated, namely ~.1-, ~.2-, and ~l-adrenoceptors, Ha- and H2histamine receptors, and muscular and neuronal nicotinic receptors [55]. In contrast to tripitramine, spirotramine displayed, among muscarinic receptor subtypes investigated, the highest affinity towards the M1 subtype with a pKi value of 7.88 which was comparable to that observed for pirenzepine (pKi, 8.19). Furthermore, spirotramine possesses a unique binding profile since it is able to discriminate, unlike tl~ipitramine and pirenzepine, between M1 and Ma muscarinic receptors as well as among all other muscarinic receptor subtypes investigated as revealed by the selectivity ratio values (M1/1V[2, 48; M1/M3, 117;

334 M a/M4, 41. Thus, spirotramine appears to be superior to pirenzepine in discriminating M1 and M4, and M1 and M3 muscarinic receptor subtypes. The fact that the antagonists currently used to classify muscarinic receptors lack a clear subtype selectivity may explain the difficulties to characterize with classic pharmacological studies the five cloned muscarinic receptors. The results presented in this paper clearly show that the use of tripitramine combined with that of spirotramine, which is able to discriminate between M1 and M4, might eventually allow the pharmacological identification of muscarinic receptor subtypes. The results discussed above clearly indicate that an appropriate substituent on the terminal nitrogens of a tetraamine backbone leads to compounds, such as tripitramine and spirotramine, which are able to differentiate among muscarinic receptor subtypes while displaying an inverse affinity profile. To our knowledge, tripitramine represents, until now, the most potent and the most selective M2 muscarinic receptor antagonist in both functional (M2 and M3) and binding assays (M1 to M4), whereas spirotramine is a novel antagonist able to discriminate in binding assays between muscarinic M1 receptors and all other pharmacologically characterized muscarinic receptor subtypes. Thus, an appropriate substitution of the terminal nitrogens of the tetraamine backbone of m e t h o c t r a m i n e may constitute the basis for the design of potent antimuscarinics which display different selectivity profiles.

CONCLUSION The concept that tetraamines may represent a passe-partout in receptor recognition has been applied to the development of selective tetraamines which displayed high potency and selectivity for different protein G-coupledreceptors. The selectivity for a given receptor system was achieved by appropriately modifying the chain length between the nitrogens, whereas the affinity was modulated by inserting particular N-substituents which were able to discriminate among receptors. ACKNOWLEDGMENT Supported by Grants from MURST and CNR (Rome). REFERENCES

1 2 3 4

Trumpp-Kallmeyer S, Hoflack J, Bruinvels A, Hibert M. J Med Chem 1992; 35: 3448-3462. Aikens D, Bunce S, Onasch F, Parker R, III, et al. Biophys Chem 1993; 17: 67-74. Melchiorre C. Trends Pharmacol Sci 1981; 2: 209-211. Melchiorre C, Recanatini M, Bolognesi ML, Filippi P, Minarini A. Current Topics in Med Chem 1993, 1: 43-65.

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Doughty MB, Chu SS, Miller DW, Li K, Tessel RE. Eur J Pharmacol 1990; 185: 113-114. Melchiorre C. Trends Pharmacol Sci 1988; 9: 216-220. Melchiorre C. Med Res Rev 1990; 10: 327-349. Bergeron RJ, Weimar WR, Wu Q, Austin JK, Jr, McManis JS. J Med Chem 1995; 38: 425-428. Bergeron RJ, McManis JS, Lin CZ, Feng Y, et al. J Med Chem 1994; 37: 3464-3476. Seiler N, Atanassov C1. In: Jucker E, ed. Progress in Drug Research, vol 43. Basel: Birkhguser Verlag, 1994; 87-141. Bylund DB, Eikenberg DC, Hieble JP, Langer SZ, et al. Pharmacol Rev 1994; 46: 121-136. Ko F-N, Guh J-H, Yu S-M, Hou Y-S, et al. Br J Pharmacol 1994; 112: 11741180. Goetz AS, King HK, Ward SDC, True TA, et al. Eur J Pharmacol 1995; 272: R5-R6. Cotecchia S, Schwinn DA, Randall RR, Lefkowitz RJ, et al. Proc Natl Acad Sci USA 1988; 85: 7159-7163. Schwinn DA, Lomasney JW, Lorenz W, Szklut PJ, et al. J Biol Chem 1990; 265: 8183-8189. Lomasney JW, Cotecchia S, Lorenz W, Leung WY, et al. J Biol Chem 1991; 266: 6365-6369. Ford APDW, Williams TJ, Blue DD, Clarke DE. Trends Pharmacol Sci 1994; 15: 167-170. Faure C, Pimoule C, Arbilla S, Langer SZ, Graham D. Eur J Pharmacol Mol Pharmacol Sect 1994; 268: 141-149. Lippert B, Belleau B. In: Usdin E, Snyder SH, eds. Frontiers in Catecholamine Research. New York: Pergamon, 1973; 369-371. Melchiorre C, Yong MS, Benfey BG, Belleau B. J Med Chem 1978; 21: 1126-1132. Cavero I, Roach AG. Life Sci 19S0; 27: 1525-1540. Langer SZ, Cavero I, Massingham R. Hypertension (Dallas) 1980; 2: 372382. Colucci WS. Ann Intern Med 1982; 97: 67-77. Davey MJ. J Cardiovasc Pharmacol 1980; 2: 287-301. Alvarez M, Granados R, Mauleon D, Rosell G, et al. J Med Chem 1987; 30: 1186-1193. Campbell SF, Davey MJ, Hardstone JD, Lewis BN, Palmer MJ. J Med Chem 1987; 30: 49-57. Sall~s J, Badia A. Eur J Pharmacol Mol Pharmacol Sect 1994; 266: 301308. Giardin~ D, Gulini U, Massi M, Piloni MG, et al. J Med Chem 1993; 36: 690-698. Arunlakshana D, Schild H 9 Br J Pharmacol 1959; 14: 48-58. Cheng YC, PrusoffWH. Biochem Pharmacol 1973; 22: 3099-3180. Quaglia W, Brasi]i L, Cristalli G, Giardin~ D, et al. J Med Chem 1988: 31: 1861-1866. Han C, Abe] PW, Minneman KP. Mo] Pharmacol 1987; 32: 505-510.

336 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

McGehee RE, Jr, Rossby SP, Cornett LE. Mol Cell Endocrinol 1990; 74: 19. Bonner TI. Trends Neurosci 1989; 12: 148-151. Hulme EC, Birdsall NJM, Buckley NJ. Annu Rev Pharmacol Toxicol 1990; 30: 633-673. Caulfield MP. Pharmacol Ther 1993; 58: 319-379. McKinney M, Coyle JT. Mayo Clin Proc 1991; 66: 1225-1237. Gualtieri F, Dei S, Menetti D, Romanelli MN, et al. I1 Farmaco 1995; 50: 489-503. Hammer R, Berrie CP, Birdsall NJM, Burgen ASV, Hulme EC. Nature 1980; 283: 90-92. Barlow RB, Berry KJ, Glenton PAM, Nikolaou NM, Soh KSA. Br J Pharmacol 1976; 58: 613-620. Lambrecht G, Feifel R, Forth B, Strohmann C, et al. Eur J Pharmacol 1988; 152: 193-194. Waelbroeck M, Tastenoy M, Camus J, Cristophe J, et al. Br J Pharmacol 1989; 98: 187-205. Lambrecht G, Feifel R, Wagner-RSder M, Strohmann C, et al. Eur J Pharmacol 1989; 168: 71-80. Jolkkonen M, van Giersbergen PLM, Hellman U, Wernstedt C, Karlsson E. FEBS Lett 1994; 352: 91-94. Eberlein WG, Engel W, Mihm G, Rudolf K, et al. Trends Pharmacol Sci 1989; Suppl IV: 50-54. Melchiorre C, Minarini A, Angeli P, Giardin~ D, et al. Trends Pharmacol Sci 1989; Suppl IV: 55-59. Quaglia W, Giardin~ D, Marucci G, Melchiorre C, et al. I1 Farmaco 1991; 46:417-434. Melchiorre C, Quaglia W, Picchio MT, Giardin~ D, et al. J Med Chem 1989; 32: 79-84. Melchiorre C, Bolognesi ML, Chiarini A, Minarini A, Spampinato S. J Med Chem 1993; 36: 3734-3737. Tumiatti V, Recanatini M, Minarini A, Melchiorre C, et al. I1 Farmaco 1992; 47: 1133-1147. Maggio R, Barbier P, Bolognesi ML, Minarini A, et al. Eur J Pharmacol Mol Pharmacol Sect 1994; 268: 459-462. Minarini A, Bolognesi ML, Budriesi R, Canossa M, et al. J Med Chem 1994; 37: 3363-3372. Melchiorre C, Minarini A, Budriesi R, Chiarini A, et al. Life Sci 1995; 56: 837-844. Melchiorre C, Minarini A, Spampinato S, Tumiatti V. Bioorg Med Chem Lett 1995; 5: 785-790. Chiarini A, Budriesi R, Bolognesi ML, Minarini A, Melchiorre C. Br J Pharmacol 1995; 114: 1507-1517.

Perspective in Receptor Research D. Giardinh, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

Design and Antagonists

Pharmacological

Characterization

337

of

Selective

P2-purinoceptor

G. Lambrecht a, U. Ardanuyb, H. G. Baumert c, X. Bo d, C. H. V. Hoyled, P. Nickel b, O. PfafD, V. Ralevic d, U. Windscheifa, A. U. Ziganshin d, R. Ziyala, E. Mutschler a and G. Burnstock d aDepartment of Pharmacology, Biocentre Niederursel, University of Frankfurt, Marie-CurieStr. 9, D-60439 Franlffurt, Germany bDepartment of Pharmaceutical Chemistry, University of Bonn, An der Immenburg 4, D53121 Bonn, Germany CDepartment of Biochemistry, Biocentre Niederursel, University of Frankfurt, Marie-CurieStr. 9, D-60439 Frankfurt, Germany dDepartment of Anatomy and Developmental Biology and Centre for Neuroscience, University College London, Gower Street, London WC1E 6BT, United Kingdom

Abstract At least five distinct P2-purinoceptor subtypes have been characterized to date, based on the rank order of potency of several ATP analogues: P2X, P2Y, P2U, P2T and P2ZHowever, the characterization of P2-purinoceptor subtypes is hampered by an unavailability of potent, highly selective, competitive antagonists. In the search for selective P2purinoceptor antagonists, the structure-activity relationships for a series of analogues of pyridoxal-5-phosphate and suramin at P2-purinoceptor subtypes were investigated in our laboratories. Two of these compounds were the subject of a more detailed pharmacological characterization: pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid @PADS) and the symmetrical 3'-urea of 8-(benzamido)naphthalene-l,3,5-trisulfonic acid (NF023). The results demonstrate that the two parent compounds, pyridoxal-5-phosphate and suramin, do not differentiate between P2X- and P2y-purinoceptors. In contrast, PPADS and NF023 were found to be selective antagonists of P2x-purinoceptor-mediated responses in several smooth muscle preparations. In addition, PPADS and NF023 were shown to displace competitively the binding of [3H]ot,13-methylene ATP to rabbit and rat bladder membranes, respectively, which indicates that these two compounds act directly on P2x-receptors. PPADS and NF023 were ineffective at P2u-purinoceptors in rat mesenteric arterial bed. P2Tpurinoceptor-mediated platelet aggregation was only affected by PPADS in concentrations higher than 100 ~tM. Suramin and NF023 were inhibitors of ecto-ATPase activity in the same concentration range needed for P2-purinoceptor antagonism. In contrast, PPADS was only very weakly active in inhibiting ecto-ATPase activity. At 100 ~tM, PPADS and NF023 did not interact with Otl-adrenoceptors, adenosine A 1- and A2-, histamine H l- and muscarinic M 1-, M2" and M3-receptors. In conclusion, PPADS and NF023 are specific P2-purinoceptor antagonists showing a high selectivity for the P2x-subtype. These two compounds may prove to be useful starting points in the synthesis of novel, highly potent and selective antagonists at P2-purinoceptor subtypes.

338 INTRODUCTION Intracellular nucleotides play an important role in enzyme and ion channel regulation as well as in energy metabolism and nucleic acid synthesis. There is now widespread appreciation that ATP (and other nucleotides) may also be released into the extracellular fluid by exocytosis from nerve terminals or secretory cells. Thus, extracellular ATP can act as a neurotransmitter or modulator in a variety of peripheral tissues and cells, in autonomic ganglia and in the central nervous system [1-3]. The responses to extracellular ATP are mediated via membrane-bound receptors, termed P2-purinoceptors. Evidence has accumulated indicating heterogeneity of P2-purinoceptors, and it has become apparent that ATP acts on at least five P2-purinoceptor subtypes, i.e. P2X, P2Y, P2U, P2T and P2Z [37]. This classification has relied almost exclusively on the rank order of potency of ATP and a number of analogues, in particular a,13-methylene ATP, 2-methylthio-ATP and ADPI3S. The possible pitfalls of agonist-based receptor classification are well known and are very relevant to the P2-purinoceptor area. It is now clear that the potency of ATP and of some structural analogues may be greatly decreased by sequential dephosphorylation by ectonucleotidase enzymes [8,9]. This may lead to an incorrect determination of agonist potency order, resulting in a possible erroneous characterization of P2-purinoceptor subtypes. The final removal of all these doubts concerning the existence of P2-purinoceptor subtypes demanded the cloning of their genes. This has now been accomplished for the P2Y[10,11] and P2u-purinoceptors [12,13], which belong to the G protein-coupled receptor superfamily [ 14-16]. In addition, genes for the P2x-purinoceptor were identified and found to encode transmitter-gated ion channels [14-19]. This cloning data and results obtained with new nucleotide agonists that can discriminate pharmacologically between P2purinoceptor subtypes [7] suggest P2X" and P2y-purinoceptor heterogeneity. The pharmacological characterization of cloned and native P2-purinoceptor subtypes and an understanding of the role of ATP in neurotransmission continues to be hindered by the lack of potent and selective, competitive antagonists. Although a number of compounds have been tested for this purpose, for example, reactive blue 2, suramin, arylazido aminopropionyl ATP, 2,2'-pyridylisatogen tosylate, 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid, evans blue and trypan blue, none is ideal [3,5,6,20-24]. All compounds are limited in their usefulness by their irreversibility of the antagonism or by their lack of potency, selectivity and P2-purinoceptor specificity. An exception is 2-propylthio-D-13,~/difluoromethylene ATP (FPL 66096) and its dichloro analogue FPL 67085. These two compounds are competitive and highly potent (pKB = 8.7 and 8.9, respectively) P2T" purinoceptor antagonists in human platelets. In addition, FPL 66096 and FPL 67085 show great selectivity for P2T-purinoceptors; for example, FPL 67085 is greater than 30 000-fold selective for P2T- over P2X-, P2Y- or P2u-purinoceptors [25]. In animal and in Phase I clinical studies FPL 67085 (given i.v.) inhibited ADP-induced platelet aggregation e x vivo. Thus, it is possible that FPL 67085 may prove to be an effective antithrombotic agent in humans. In the face of this situation, the development of ideal P2-purinoceptor antagonists will be an ambitious undertaking in receptor pharmacology and medicinal chemistry. With such agents it will be possible to define the important in vivo roles of extracellular ATP in health, and to understand and apply nucleotide agonist or antagonist therapy in disease. To this end, we have synthesized and pharmacologically characterized a series of suramin and pyridoxal-5-phosphate analogues (Figure 1). Two of these compounds, the symmetrical 3'urea of 8-(benzamido)naphthalene-l,3,5-trisulfonic acid (NF023) and pyridoxalphosphate-6-

339 azophenyl-2',4'-disulfonic acid (PPADS), were the subject of a more detailed pharmacological investigation at P2X-, P2Y', P2U" and P2T-purinoceptors. In addition, the ability of PPADS to inhibit ecto-ATPase activity was investigated. Experimental methods will not be described since these are detailed in the literature: P2Xpurinoceptors [26-30]; P2y-purinoceptors [29-31]; P2u-purinoceptors [30]; P2T" purinoceptors [32]; ecto-ATPase assay [31 ]. m

Na03S O~ /H C H O ~

H3C-

0II

CH2-O-P-O'OI

-N" " N = N _ ~ R

O%c/NH

]~S03Na SO3Na

o,

H

H

~N~c/N~ II 0

N2

-2

Figure 1. Chemical structure of PPADS (let~: R 1 = 2'-SO3H, R 2 = 4'-SO3H) and suramin (fight" R 3 = CH 3, X = 1) analogues. NF023 R 3 = H, X = 0; NF037: R 3 = H, X = 1; NF058: R 3 = CH3, X = 0. SURAMIN ANALOGUES Introduced in 1922, suramin has been used in the treatment of trypanosomiasis and onchocerciasis. More recently, this compound has gained clinical interest for the treatment of acquired immunodeficiency syndrome, hepatitis B virus infection and metastatic carcinomas [21 ]. Suramin has been shown to inhibit the receptor binding of a number of growth factors and cytokines, and it inhibits several nuclear and cytoplasmic enzymes as well as ecto-nucleotidases [33-41 ]. These latter enzymes are of special interest to the field ofP 2purinoceptor research (see INTRODUCTION). Dunn and Blakeley [42] were the first to show that suramin antagonizes P2Xpurinoceptor-mediated effects. Suramin has since been found to be an antagonist at P2X" and P2y-receptors on vascular [43,44] and visceral [45,46] preparations as well as on neurons [47]. Furthermore, an antagonistic effect of suramin on ADP-induced platelet aggregation has been reported, indicating that this compound is also an antagonist on P2T" purinoceptors [48]. The results of these studies show that suramin does not distinguish between these three P2-purinoceptor subtypes, having a pA2 value of around 5 in each case. Although the antagonism by suramin does not always appear to be competitive [21,45-47], suramin was shown to displace competitively the binding of [3H]oql3-methylene ATP and

340 ADPI335S to rat bladder and turkey erythrocyte membranes, respectively, which indicates that this compound acts directly on P2X" and P2y-purinoceptors [27, 35]. In order to gain some insights into the structural features that contribute to the antagonistic properties of suramin at P2-purinoceptors, we have synthesized and pharmacologically characterized a series of suramin analogues (Figure 1). We hoped to alter the activity profile of suramin in favour of one of the P2-purinoceptor subtypes. The field-stimulated (0.05 Hz) rabbit isolated vas deferens, where endogenous ATP induces twitch contractions, was used to determine structure-activity relationshipsfor the P2x-purinoceptor subtype [26]. These results were compared with binding affinities obtained at P2y-receptors on turkey erythrocyte membranes [35]. The data for the inhibitory effects by suramin and three of its congeners are given in Figure 2. All compounds tested caused a concentration-dependent inhibition of the neurogenic response in rabbit vas deferens. Suramin (plC50 = 4.7) and the two compounds NF037 (plC50 = 5.1) and NF058 (plC50 = 4.7) were equipotent, whereas the small urea compound NF023 (plC50 = 6.0) was the most potent suramin analogue, being about 8- to 20-fold more potent than the other three agents. Substitution of the methyl group in compound NF058 (R 3 in Figure 1) with more bulky substituents had no great influence on the inhibitory potency at P2X" purinoceptors in rabbit vas deferens: plCsoCt-butyl analogue = 4.4, plCso/phenyl analogue = 4.9. The binding data obtained on turkey erythrocyte membranes [35] demonstrate that methyl substituents (R 3 in Figure 1) are not a prerequisite for affinity to P2y-purinoceptors, as the pK i values of 5.1 and 3.8 for suramin and NF058 are very similar to that obtained for NF037 (pK i = 5.2) and NF023 (pK i = 4.1), respectively. In contrast, the number of amino-

PKi/plCso -6

PKi/plCso 65.1

5.2 5

o,0

4.7

\\

\

\

\

_

~~

4.1 - 4

3.8

3

--

-

Suramin

==

NF037 ,

,,,

NF058

3

NF023

Figure 2. Inhibitory potency of suramin and of three of its analogues (for chemical structure, see Figure 1) at P2X- and P2y-purinoceptors. e - - - e Inhibition of neurogenic contractions O.05 Hz; plCs0 values) in rabbit vas deferens (P2x-receptors). 1 - - I Inhibition of ADPI3 S binding (pK i values) to turkey erythrocyte membranes (P2y-receptors) [35].

341 benzoyl groups seems to be important for obtaining higher affinity towards P2Y" purinoceptors, since shortening the chain of the large urea compounds, suramin and NF037, by two aminobenzoyl groups (---~NF058/NF023) decreases affinity for P2y-receptors substantially (about 10-fold). Similar to suramin, compound NF037 does not distinguish between P2x-receptors in rabbit vas deferens and P2y-receptors in turkey erythrocytes, whereas NF058 (8-fold) and NF023 (79-fold) were found to be selective for the P2X" subtype. NF023

Exogenous a,13-methylene ATP produced concentration-dependent isometric contractions of the rabbit isolated vas deferens (EC50 = 1.4 ~tM) by acting at P2Xpurinoceptors [26]. Increasing concentrations of suramin and NF023 progressively shifted the agonist concentration-response curve to the fight. Schild plots for suramin and NF023 were linear and slopes (suramin = 1.14; NF023 = 1.06) were not significantly different from unity, indicating competitive antagonism. The apparent affinities (pA2 values) for the two antagonists are shown in Table 1. This data shows that NF023 is 4-fold more potent than suramin in inhibiting P2x-purinoceptor-mediated effects in rabbit vas deferens.The P2X" antagonism by suramin (pA 2 = 5.04) and NF023 (pA 2 = 5.54) was confirmed in rat isolated mesenteric arterial bed. In addition, suramin and NF023 were shown to displace competitively the binding of [3H]oc,13-methylene ATP to rat bladder membranes with pK i values of 6.51 and 7.10, respectively, which indicates that these two compounds act directly on P2x-purinoceptors.

Table 1

Antagonistic properties of suramin and NF023 at P2X- and P2y-purinoceptors obtained in functional (pA 2 values) and radioligand binding (pK i values) experiments pA 2 at P2X

pA 2 at P2Y

pK i at P2X

Rabbit vas deferens

Rat mesentery

Guinea-pig taenia coli

Rat bladder

Suramin

5.10

5.04

5.01

6.51

NF023

5.68

5.54

4.18

7.10

The reversibility of NF023 "s inhibitory effect on successive responses of rabbit vasa deferentia to ct,13-methylene ATP at an approximate EC50 concentration of 3 laM was also examined. Following three reproducible control contractions to the agonist at 45-min intervals, the addition of 30 ~tM NF023 induced a reduction in the magnitude of responses to a,13-methylene ATP of 56%. This effect was reversed on repeated washout during 45 min when responses to ~,13-methylene ATP recovered to 100% of their respective pre-NF023 controls. It is worth mentioning that suramin and NF023 caused a blockade of vascular P2X" purinoceptors after intravenous administration in the pithed rat [44,49]. In these in vivo

342 experiments, NF023 was again 4-fold more potent than suramin in antagonizing the vasopressor effects of ot,13-methylene ATP. The antagonistic properties of suramin and NF023 were evaluated further in the guineapig taenia coli, a tissue endowed with P2y-purinoceptors [31,45; but see below]. The two compounds were examined for interactions with the exogenous P2y-selective agonist 2methylthio ATP. When added during the plateau of the contraction elicited by carbachol (50 nM) 2-methylthio ATP produced concentration-dependent relaxations with an EC50 value of 85 nM. These effects were antagonized by suramin (30 JaM) and NF023 (300 laM) in a parallel and surmountable manner, the pA2 values being 5.01 and 4.18, respectively. These atfinity estimates were very similar to that obtained at P2y-purinoceptors in turkey erythrocyte membranes (Figure 2). These results confirm the P2x-purinoceptor selectivity of NF023. The rat mesenteric arterial bed is, in addition to P2X" and P2y-receptors, endowed with P2u-purinoceptors mediating vasoconstriction and vasodilation, respectively [30]. In methoxamine-raised tone preparations, the pyrimidine nucleotide UTP produced decreases in perfusion pressure (ED50 = 2.4 nmol). These responses remained unaffected by NF023 (30 laM), providing evidence for the ineffectiveness of NF023 at vasodilatation-mediating P2u-purinoceptors. Beukers et al. [50] reported on the ability of suramin and a series of its analogues to inhibit ecto-ATPase of human blood cells. The most potent ecto-ATPase inhibitor in this series of compounds was suramin, leaving a residual enzyme activity of 46.3% at 100 laM (Ki = 44 laM). About 51% of ecto-ATPase activity was let~ in the presence of 100 laM NF023. Regarding suramin and NF023, these results indicate that caution should be adopted when using these two compounds in receptor classification studies. The propensity for their receptor antagonistic (Table 1) and ecto-ATPase inhibitory properties to "self-cancel" when metabolically unstable agonists are used can be a basis for misinterpretation. To test the P2-purinoceptor specificity of NF023, we investigated its effects on responses to various agonists in rat vas deferens (Otl-adrenoceptors), rat duodenum (adenosine A2- and muscarinic M 1-receptors), guinea-pig atria (adenosine A 1- and muscarinic M2-receptors ) and guinea-pig ileum (histamine H l- and muscarinic M 3receptors). NF023 (100 laM) had no significant effect on either the potency or maximum responses to the respective agonists used in the different receptor preparations. In addition, NF023 (100 ~tM) did not alter contractions elicited by high K + (40 mM) in rabbit vas deferens. These results indicate that the antagonistic effects of NF023 against ATP, ot,13methylene ATP and 2-methylthio ATP at P2-purinoceptors are specific. Taken together, the results confirm that the parent compound, suramin, does not differentiate between P2X- and P2y-receptor-mediated events. In contrast, the suramin analogue, NF023, is a specific P2-purinoceptor antagonist showing a high selectivity for the P2x-subtype. PYRIDOXAL-5-PHOSPHATE ANALOGUES An early report suggested that suramin can block the nucleotide-dependent calcium pump of rabbit skeletal sarcoplasmic reticulum by inhibition of the calcium uptake and the ATPase activity [51]. These results have been confirmed by Emmik et al. [52]. B~iumert and Heider [53] found that pyridoxal-5-phosphate and a series of its analogues (for chemical structure,

343 see Table 2) also inhibit the calcium pump of rabbit skeletal sarcoplasmic membranes by acting as antagonists at the adenine nucleotide binding site. Given that suramin is also a ligand at P2-purinoceptors, we hypothesized that pyridoxal-5-phosphate and its analogues might act as P2-purinoceptor antagonists. Thus, the stucture-activity relationships for a variety of pyridoxal-5-phosphate analogues at P2x-purinoceptors were investigated. Compounds formed by structural modifications of the pyridoxal-5-phosphate molecule including substitution of the pyridine ring at C6 and replacement of the aldehyde moiety by CH2OH or CH2NH 2 group were prepared. Pharmacological activity at P2x-Purinoceptors was assayed in the rabbit isolated vas deferens (Table 2). Table 2 Inhibitory potency (mean pIC50 values) of pyridoxal-5-phosphate and some of its analogues at P2x-purinoceptors in rabbit isolated vas deferens a

O~C-"H

0 II

H3C" "N" "NRN._~R 2 R1

2'-SO3H

2'-SO3H

H

H

H

2'-NO 2

pyridoxal-

R2

4'-SO3H

H

4'-SO3H

H

4'-NO 2

4'-NO 2

5-phosphate

3.97

4.46

plC50

5.83

>

5.58

>

5.50

=

5.50

>

5.14

>

aVasa deferentia were stimulated with trains of pulses (4 Hz for 30 s; 0.5 ms duration; 40 V) at intervals of 15 min. The purinergic component of the biphasic response (mediated by P2X- and C~l-receptors) was pharmacologically isolated by the continuous presence of the 1-adrenoceptor antagonist prazosin (1 IaM).

Pyridoxal-5-phosphate caused a concentration-related inhibition of the purinergic component, mediated by P2x-purinoceptors, of the neurogenic contraction in rabbit vas deferens with a plC50 value of 4.46 (Table 2). Substitution of the parent compound at C6 with an azophenyl moiety increased the potency by one order of magnitude (plC50 = 5.50), and introduction of a sulfonic acid residue at the 2'- or 4'-position of the phenyl ring produced P2-purinoceptor antagonists equally potent as the unsubstituted phenyl derivative. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), however, was the most potent compound at the vas deferens P2x-receptors (plC50 = 5.83). Introduction of electron withdrawing nitro groups in the phenyl ring decreased antagonist activity, the 2',4'dinitro analogue being two orders of magnitude less potent than PPADS. Removal of the phosphate group from position 5 of the pyridoxal ring abolished the P2Xpurinoceptor antagonist properties of pyridoxal-5-phosphate [54]. Replacement of the aldehyde moiety in pyridoxal-5-phosphate or PPADS with a CH2OH or CH2NH 2 group, to

344 form pyridoxol-5-phosphate, pyridoxamine-5-phosphate and dihydro-PPADS, respectively, also resulted in inactive compounds. These results show that the phosphate and aldehyde moieties of pyridoxal-5-phosphate and its analogues are required for antagonist activity at P2x-purinoceptors in rabbit vas deferens. Among this series of compounds, PPADS was found to be the most interesting P2antagonist, and it was therefore the subject of a more detailed pharmacological investigation. PPADS

The time course of action of PPADS at 5 pM, a concentration which inhibited the effects of endogenous ATP by about 70%, was examined in the field-stimulated rabbit vas deferens. In these experiments, PPADS acted as a slowly-equilibrating and a slowly-reversible P2Xpurinoceptor antagonist. It was found to reach apparent equilibrium in about 120 min, and the tissue took about 90 min to regain its control twitch height by continual washout of PPADS. Importantly, these findings indicate that PPADS is not an irreversible antagonist at P2x-purinoceptors in rabbit vas deferens. Table 3 Antagonistic properties of pyridoxal-5-phosphate (PLP) and PPADS at P2x-and P2Ypurinoceptors obtained in functional (pA2 values) and radioligand binding (pK i values) experiments pA2 at P2X

pA2 at P2Y

pK i at P2X

Rabbit vas deferens

Rat mesentery

Rat duodenum

Rat bladder

PLP

5.20

5.37 a

5.39 b

4.91 c

PPADS

6.34 d

6.38 d

5.09

7.66/4.32 e

aRat vagus nerve, o~,13-methyleneATP as agonist [54]. bGuinea-pig aorta, 2-methylthio ATP as agonist [55]. CRat vas deferens [54]. dApparent PKB, derived from a double-reciprocal regression, epPADS interacted with two affinity states of the P2x-purinoceptor for [3H]ot,13 -methylene ATP. Pyridoxal-5-phosphate antagonized P2x-purinoceptor-mediated contractions to exogenous cz,13-methylene ATP in the rabbit vas deferens in a surmountable and concentration-related manner. The resulting Schild plot was linear yielding a pA 2 value of 5.20 and a slope of 0.95, which was not significantly different from unity, indicating competitive antagonism. A similar pA2 value (5.37) was obtained by Trezise et al. [54] at P2x-receptors in the rat vagus nerve (Table 3). Pyridoxal-5-phosphate competed for high affinity binding of [3H]cx,13-methylene ATP to membranes of the rat vas deferens with a pK i estimate of 4.91 [54], which indicates that this compound acts directly on P2x-receptors. In the guinea-pig aorta, a tissue endowed with P2y-purinoceptors, pyridoxal-5-phosphate inhibited competitively 2-methylthio ATP-induced relaxations, and Schild analysis gave a pA 2 value of 5.39 [55]. These data indicate that pyridoxal-5-phosphate exhibits antagonist

345 Increase

in tension

3-

(g) U

Figure 3. Effect of PPADS on the mechanical response of the rabbit isolated vas deferens to exogenous cz,13-methylene ATP (o~,13-

mATl').

control

I

a

v

'

I

'

v

I

v

'

I

,I - l o g [a,B-mATP] (M) 6

5

Two concentration response curves were constructed non-cumulatively on each preparation, the first in the absence (It) and the second in the presence of PPADS (O 1 ~tM, "k 3 laM, and @ 10 ~tM). In order to prevent desensitization, very high concentrations of ot,13-methylene ATP were avoided in the first concentration-response curve.

actions at P2-purinoceptors, but it does not differentiate between P2X- and P2y-receptormediated events. Figure 3 shows the mean data for the antagonistic effects of PPADS on monophasic contractions to exogenous cx,13-methylene ATP in rabbit isolated vas deferens. PPADS antagonized concentration-dependently the responses to cz,13-methylene ATP, which are mediated by P2x-purinoceptors [26]. The antagonism by PPADS was characterized by a shift to the fight of the agonist concentration-response curve. In addition, there was a progressive reduction in the agonist maximal response. In the presence of 10 laM PPADS, the contractile response to et,13-methylene ATP was abolished. This profile of insurmountable antagonism by PPADS could not be attributed to irreversible binding since the effect of PPADS could be reversed on washing, albeit slowly (see above). It is tempting to interpret these findings as evidence that PPADS acts as a slowly-reversible purinoceptor antagonist at P2x-receptors in rabbit vas deferens leading to a pseudo-equilibrium situation between agonist and antagonist. Therefore, the antagonism by PPADS was analyzed by a double reciprocal plot according to the method of Kenakin [56], yielding a pKB estimate of 6.34. PPADS is, thus, about 10-fold more potent than the parent compound pyridoxal-5phosphate at P2x-purinoceptors in rabbit vas deferens (Table 3). Radioligand binding studies carried out on rabbit bladder membranes with [3H]ct,13-methylene ATP showed that PPADS concentration-dependently inhibited the binding of the tracer to P2x-purinoceptors. Curvelinear fitting of the data revealed that PPADS interacted with two affinity states of the P2x-receptor for [3H]cz,13-methylene ATP (Table 3). The P2x-antagonism by PPADS has been confirmed in a wide range of tissues and cells including guinea-pig vas deferens [57], rabbit urinary bladder detrusor muscle [28], rabbit blood vessels [29], rat mesenteric arterial bed [30] (Table 3), rat isolated perfused kidney [58], rat sympathetic ganglia [59] and xenopus oocytes injected with RNA transcribed from a P2X cDNA clone from rat vas deferens [ 17]. In order to assess the selectivity of PPADS for different P2-purinoceptor subtypes, its effects on the relaxant responses to adenine nucleotides were examined in the rat duodenum [31 ], rat mesenteric arterial bed [30], rabbit isolated blood vessels [29] and guinea-pig taenia coli [31 ], tissues that are endowed with the archetypal P2y-purmoceptor.

346 Relaxant responses to 2-methylthio ATP in the carbachol-contracted rat duodenum were antagonized by PPADS (10 - 100 laM) in a concentration-related manner. The resulting Schild plot was linear and its slope (1.02) was not significantly different from unity, indicating competitive antagonism. The affinity estimate for PPADS in rat duodenum (pA 2 = 5.09; Table 3) was very similar to that obtained for pyridoxal-5-phosphate in guinea-pig aorta (5.39), but clearly lower compared to the apparent pK B values obtained for the P2Xsubtype in rabbit vas deferens (6.34) and rat mesenteric artery (6.38; Table 3). In rat mesenteric arterial bed, the tone of which was raised by methoxamine, vasodilator responses to 2-methylthio ATP were slightly inhibited by 10 laM PPADS (pA 2 = 5.46), a concentration which virtually abolished P2x-purinoceptor-mediated vasoconstriction. It is noteworthy that PPADS (30 laM) was found to be ineffective at vascular P2y-purinoceptors in rabbit mesenteric artery and aorta. These results clearly demonstrate that PPADS is less effective at duodenal and vascular P2y-purinoceptors than at P2x-receptors (Table 3). PPADS was investigated further for its effect on the relaxant responses to exogenous adenine nucleotides and to electrical field stimulation in the guinea-pig taenia coli. 10 - 100 laM PPADS caused a consistent dextral shift of the concentration-response curve of ATP (0.03 - 300 laM; pECs0 = 6.35) and 2-methylthio ATP (1 nM - 3 laM; pECs0 = 7.96) resulting in a biphasic Schild plot (shown for 2-methylthio ATP in Figure 4). A substantial shift was only observed at 100 laM PPADS, the respective pA 2 values at this particular concentration were 5.15 and 5.26. Lower concentrations of PPADS (3 - 30 laM) antagonized the relaxant effects to ct,13-methylene ATP (0.3 - 10 laM; pEC50 = 5.88) in a surmountable manner. An extensive shift of the concentration-response curve of ot,13methylene ATP was produced only by 30 laM PPADS (pA 2 = 5.97), and the Schild plot was again biphasic (Figure 4). These results show that PPADS is considerably less potent against ATP and 2-methylthio ATP than against ot,13-methylene ATP. This difference in potency, together with the biphasic nature of each Schild plot, lend support that there are at least two relaxation-mediating P2-purinoceptors in guinea-pig taenia coli, the classical P2Ypurinoceptor which is largely PPADS-resistant and a distinct, PPADS-sensitive P2purinoceptor subtype. A similar conclusion has been drawn from the observation that the compound 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) was more potent against or, 13-methylene ATP in the taenia coli than against 2-methylthio ATP and ADPI3S [60]. It is also

log (DR- 1)

2-

_

~"

Figure 4. Schild plots for the antagonism by PPADS in the carbachol-contracted guinea-pig taenia coli using ct,13-methylene SATP ATP (ot,13-mATP) and 2-methylthio ATP (2-MeSATP) as agonists. Lines, which connect the mean log ( D R - 1) values at the respective PPADS concentration, are drawn to highlight the biphasic nature of the Schild plots. The slopes of the overall regression lines (not shown) were: 1.46 for ot,13-mATP and 0.58 for 2-MeSATP. These values were significantly different 5 4 from unity. - l o g [ P P A D S ] (M} I

'

I

347 noteworthy, that PPADS exhibited selectivity for the phospholipase C-coupled P2Y" purinoceptor of the turkey erythrocyte, whereas no activity against the adenylyl cyclasecoupled P2y-purinoceptor in C6 glioma cells was observed [61 ]. Relaxant responses to electrical field stimulation (0.5 - 16 Hz) in guinea-pig taenia coli were inhibited by PPADS at the same concentration-range as were the responses to exogenous ATP and 2-methylthio ATP (Figure 4) [31 ]. In the presence of 100 laM PPADS, these neurogenic relaxations were (compared to controls): 10% (0.5 Hz), 6% (2 Hz), 5% (8 Hz) and 4% (16 Hz). This supports the purinergic nerve hypothesis, which postulates ATP or a related compound as NANC-transmitter [2]. The effect of PPADS on P2u-purinoceptors was investigated in the rat mesenteric arterial bed at basal tone and at tone raised by methoxamine. These experiments demonstrated the ineffectiveness of PPADS (10 laM) at both vasoconstriction- and vasodilatation-mediating P2u-receptors [30]. Similar results were obtained in the rat pulmonary vascular bed [62]. In addition, PPADS (10 - 100 laM) was not an antagonist at P2-purinoceptors that mediate UTP-evoked depolarization of the rat superior cervical ganglion, but it produced a concentration-dependent depression of depolarizations evoked by (z,13-methylene ATP [59]. All these observations substantiate the proposal (see above) that PPADS is a selective antagonist at purine- (P2x-receptors) rather than at pyrimidinenucleotide receptors. PPADS was investigated further for its ability to inhibit ADP-induced platelet aggregation [32]. As shown in Figure 5, PPADS (100 and 500 ~tM) was able to inhibit P2T" purinoceptor-mediated responses in human washed platelets. However, comparing the effective threshold concentration of PPADS in platelets (100 laM; Figure 5) with that found for the P2x-subtype (1.0 laM; Figure 3), it becomes obvious that PPADS is highly selective for P2X- over P2T-purinoceptors. Enzyme activity (~;) 100- i

Maximal amplitude (AE) 1.51.27 1.22

1.08

1.0

~x~

-

,-//

Y/ f f . ,

0o5

_~x~__

800.84

/

a

I

control

60-

m~

~x

i

;//A

:'

Figure 5. The effect of PPADS on the maximal amplitude (expressed as AE) of ADPinduced (4 laM) aggregation of washed human platelets [32 ].

82 zTo K)o K><> K x

I

I

I

;/'A

,I

#l

I

v

;=

, K> <X> , K> __

< x.>

<x> <><> <x>

Id

10 100 500 [ PPADS {luM) l

I I

" "A " I/1

40-

//,

79

"/A

i<x>

"7/

84 -'-,

control

10

30

100

I PPADS (pM) I Figure 6. Residual ecto-ATPase activity in guinea-pig taenia coli using ATP (0.1 mM) as substrate in the absence and presence of PPADS [31].

348 Compounds such as suramin and NF023 are known to combine P2-purinoceptor blockade and inhibition of ecto-ATPase (see above). This combination of properties could, in theory, lead to a "self-cancellation" of the potentiating (enzyme inhibitory) and receptor antagonistic properties when metabolically unstable agonists are used. Thus, we examined the ability of PPADS to affect ecto-ATPase activity in the taenia coli. As shown in Figure 6, there was indeed some enzyme inhibition at PPADS concentrations of 10 jaM or higher. However, this effect was not concentration-related and did not exceed 21%. This weak inhibition by PPADS of enzyme activity suggests that effects on ecto-ATPase will not be a complicating factor ifPPADS is used as a P2-purinoceptor antagonist. To test the specificity of PPADS, we compared its blocking activity on P2-purinoceptormediated responses with its effects on responses mediated by: Ctl-adrenoceptors in rat vas deferens, histamine HI-receptors and muscarinic M3-receptors in guinea-pig ileum, adenosine Al-receptors and muscarinic M2-receptors in guinea-pig atria and adenosine A 2receptors and muscarinic Ml-receptors in rat duodenum. PPADS (100 ~tM) had no significant effect on either the potency or maximum responses to the respective agonists used in the various receptor preparations. These results demonstrate that the antagonistic effects of PPADS against purine-nucleotides at P2-purinoceptors are specific. It is noteworthy, that an isomer of PPADS, pyridoxalphosphate-6-azophenyl-2',5'disulfonic acid (iso-PPADS), acts as an antagonist of P2x-purinoceptor-mediated responses with similar potency as PPADS [59, 63, 64]. However, very little is known about the P2purinoceptor selectivity of this compound. CONCLUSION The results demonstrate that the two parent compounds, pyridoxal-5-phosphate and suramin, do not differentiate between P2X" and P2y-receptor-mediated events. In contrast, PPADS and NF023 are specific P2-purinoceptor antagonists showing a relatively high selectivity for the P2x-receptors. Present efforts are being directed towards subtypes of P2X- and P2y-purinoceptors, in order not only to investigate the agents we presently have, but also to prepare derivatives of PPADS and NF023 with greater potency and subtype selectivity and with eventual applications to new therapeutic targets. ACKNOWLEDGEMENTS The authors thank the Fonds der Chemischen Industrie (Germany), the Deutsche Forschungsgemeinschaft and the WeUcome Trust for financial support. The skillful technical assistance of Mrs. Caren Hildebrandt is gratefully acknowledged. REFERENCES 1

2 3

Zimmermann H. Trends Neurosci 1994; 17: 420-426. Hoyle CHV. In: Burnstock G, Hoyle CHV, eds. Autonomic Neuroeffector Mechanisms. Chur: Harwood Academic Publishers, 1992; 367 - 407. Chen ZP, Levy A, Lightman SL. J Neuroendocrinol 1995; 7: 83-96.

349 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 30 31 32 33 34 35 36 37

Burnstock G, Kennedy C. Gen Pharmacol 1985; 16: 433-440. Fredholm BB, Abbracchio MP, Bumstock G, Daly JW, et al. Pharmacol Rev 1994; 46: 143-156. Dalziel H ~ Westfall DP. Pharmacol Rev 1994; 46: 449-466. Abbracchio MP, Burnstock G. Pharmac Ther 1994; 64: 445-475. Ziganshin AU, Hoyle CHV, Bumstock G. Drug Dev Res 1994; 32: 134-146. Kennedy C, LeffP. Trends Pharmacol Sci 1995; 16: 168-174. Webb TE, Simon J, Krishek BJ, Bateson AN, et al. FEBS Lett 1993; 324: 219-225. Filtz TM, Li Q, Boyer JL, Nicholas RA, et al. Mol Pharmacol 1994; 46: 8-14. Lustig KD, Shiau AK, Brake AJ, Julius D. Proc Natl Acad Sci 1993; 90:5113-5117. Parr CE, Sullivan DM, Paradiso AM, Lazarowski ER. Proc Natl Acad Sci 1994; 91: 3275-3279. Dubyak GR, EI-Moatassim C. Am J Physiol 1993; 265: C577-C606. Dubyak GR. Am J Respir Cell Mol Biol 1991; 4: 295-300. Harden TK, Boyer JL, Nicholas RA. Annu Rev Pharmacol Toxicol 1995; 35: 541-579. Valera S, Hussy N, Evans RJ, Adami N, et al. Nature 1994; 371: 516-519. Brake AJ, Wagenbach MJ, Julius D. Nature 1994; 317: 519-523. Surprenant A, Buell G, North RA. Trends Neurosci 1995; 18: 224-229. Fedan JS, Lamport SJ. Ann NY Acad Sci 1990; 603:182-197. Voogd TE, Vansterkenburg ELM, Wilting J, Janssen LHM. Pharmacol Rev 1993; 45: 177-203. Btiltmann R, Starke K. Br J Pharmacol 1994; 112: 690-694. B01tmann R, Trendelenburg M, Starke K. Br J Pharmacol 1994; 113: 349-354. B01tmann R, Starke K. Naunyn-Schmiedeberg's Arch Pharmacol 1993; 348: 684-687. Humphries RG, Robertson MJ, LeffP. Trends Pharmacol Sci 1995; 16: 179-181. Lambrecht G, Friebe T, Grimm U, WindscheifU, et al. Eur J Pharmacol 1992; 217: 217-219. Bo X, Fischer B, Maillard M, Jacobson KA, et al. Br J Pharmacol 1994; 112:11511159. Ziganshin AU, Hoyle CHY, Bo X, Lambrecht G, et al. Br J Pharmacol 1993; 110:14911495. Ziganshin AU, Hoyle CHV, Lambrecht G, Mutscher E, et al. Br J Pharmacol 1994; 111: 923-929. WindscheifU, Ralevic V, B/iumert HG, Mutscher E, et al. Br J Pharmacol 1994; 113: 1015-1021. WindscheifU, PfaffO, Ziganshin AU, Hoyle CHV, et al. Br J Pharmacol 1995; 115: 1507-1517. WindscheifU, Radziwon P, Breddin H ~ B~umert HG, et al. Arzneim Forsch/Drug Res (in press). Hourani SMO, Hall DA, Nieman CJ. Br J Pharmacol 1992; 105: 453-457. Crack BE, Beukers MW, McKechnie KCW, et al. Br J Pharmacol 1994; 113: 14321438. van Rhee AM, van der Heijden MPA, Beukers MW, IJzerman AP, et al. Eur J Pharmacol Mol Pharmacol Sect 1994; 268: 1-7. Strassmann G, D'Alessandro F, Fong M, Nordman RP, et al. Int J Immunopharmac 1994; 16: 931-93 9. Jentsch KD, Hunsman G, Hartmann H, Nickel P. J Gen Virol 1987; 68: 2183-2192.

350 38 Oesterle R, Jurkiewicz E, Li~ke W, Nickel P. Antiviral Res 1993; 22:107-119. 39 Rubio GJ, Pinedo HM, Virizuela J, van Ark-Otte J, et al. Eur J Cancer 1995; 31A: 244251. 40 Hourani SMO, Chown JA. Gen Pharmacol 1989; 20:413-416. 41 Ziganshin AU, Ziganshina LE, King BF, Burnstock G. Pfliagers Arch - Eur J Physiol 1995; 429: 412-418. 42 Dunn PM, Blakeley AG. Br J Pharmacol 1988; 93: 243-245. 43 LeffP, Wood BE, O'Connor SE. Br J Pharmacol 1990; 101: 645-649. 44 Schlicker E, Urbanek E, GOthert M. J Auton Pharmacol 1989; 9: 357-366. 45 Hoyle CHV, Knight GE, Bumstock G. Br J Pharmacol 1990; 99: 617-621. 46 Mallard N, Marshall R, Sithers A, Spriggs B. Eur J Pharmacol 1992; 220: 1-10. 47 Trezise DJ, Kennedy I, Humphrey PPA. Br J Pharmacol 1993; 110 1055-1060. 48 Hall DA, Hourani SMO. Biochem Pharmacol 1994; 47: 1013-1018. 49 Urbanek E, Nickel P, Schlicker E. Eur J Pharmacol 1990; 175: 207-210. 50 Beukers MW, Kerkhof CJM, van Rhee MA, Ardanuy U, et al. Naunyn-Schmiedeberg's Arch Pharmacol 1995; 351: 523-528. 51 Layton D, Azzi A. Biochem Biophys Res Commun 1974; 59: 322-325. 52 Emmick JT, Kwon S, Bidasee KR, Besch KT, et al. J Pharmacol Exp Ther 1994; 269: 717-724. 53 Baumert HG, Heider P; personal communication. 54 Trezise DJ, Bell NJ, Khakh BS, Michel AD, et al. Eur J Pharmacol 1994; 259: 295-300. 55 Trezise DJ, Bell NJ, Humphrey PPA. Br J Pharmacol 1994; 112: 502P. 56 Kenakin T. Pharmacological Analysis of Drug-Receptor Interactions. New York: Raven Press, 1993. 57 McLaren GJ, Lambrecht G. Mutschler E, Baumert HG, et al. Br J Pharmacol 1994; 111: 913-917. 58 Eltze M. Naunyn-Schmiedeberg's Arch Pharmacol 1995; 351: suppl R93. 59 Connolly GP. Br J Pharmacol 1995; 114:727-731. 60 Dudeck O, B01tmann R, Starke K. Naunyn-Schmiedeberg's Arch Pharmacol 1995; 351: 107-110. 61 Boyer JL, Zohn IF., Jacobson KA, Harden TK. Br J Pharmacol 1994; 113: 614-620. 62 Rubino A, Burnstock G. Br J Pharmacol 1994; 112: 501P. 63 Trezise DJ, Kennedy I, Humphrey PPA. Br J Pharmacol 1994; 112: 282-288. 64 Khakh BS, Michel A, Humphrey PPA. Eur J Pharmacol 1994; 263: 301-309.

Perspective in Receptor Research D. Giardin~, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

IMIDAZO~

351

RECEPTORS : AN UPDATE

J. Feldman, M.Dontenwill, H. Greney, F.Bennai and P. Bousquet Laboratoire de Pharmacologie Cardiovasculaire et R6nale, Universit6 Louis Pasteur, CNRS URA 589, Facult6 de M6decine,ll rue Humann, 67000 STRASBOURG, FRANCE.

INTRODUCTION Twenty years ago, many efforts were made to synthesise structural analogues of clonidine but none of them led to the development of really more active and/or better tolerated central acting antihypertensive drugs. In early 1980s, the concept of imidazoline specific receptors was proposed as a conclusion of a study concerning the mechanism of the central hypotensive action of clonidine-like drugs (fig.l). Since the concept of the imidazoline receptors was proposed, i.e. since 1984, there were new developments in this pharmacological class [1,2]. As a matter of fact, structure-activity relationship studies first, and then functional studies with clonidine-like substances led to the discrimination of the mechanisms underlying the hypotensive effect from that one involved in the sedative action of these compounds [3-5]. Thus, initially, an extensive structure-activity relationship study was performed; the data of this study suggested the existence of a new class of receptors specific for imidazoline-like compounds different from the classical a-adrenergic receptors [1]. Using the stereotaxic method, the direct injection of various catecholamine and imidazoline drugs into the rostroventrolateral part of the brainstem revealed that only imidazolines could reduce blood pressure in a dose-dependent manner, like clonidine did. Those receptors appeared to be involved in the vasomotor tone regulation and in the mechanism of the hypotensive effect of clonidine-like drugs [1,6-9]. This concept is progressively gaining ground; numerous experimental studies now confirm the existence of binding sites specific for the imidazolines in a variety of tissues including the brain, and in various species. As expected, these specific binding sites do not recognise the catecholamines [8,10-19]. At present the imidazoline binding sites are already sub-classified in two groups [20]. The I 1-type is similarly sensitive to clonidine and idazoxan, a selective antagonist of the cardiovascular effects of the clonidine-like drugs whereas the I2-type, which appears to be mainly located on the mitochondria and is perhaps associated to the monoamine oxidases, is sensitive to idazoxan and poorly sensitive to clonidine [20-23].

352 1. SPECIFIC IMIDAZOLINE BINDING SITF~ Thus, since the concept of imidazoline receptors was proposed in the early 1980S, the existence of binding sites specific for imidazolines and related compounds was amply confirmed. In 1987, the first description of imidazoline binding sites was reported by Ernsberger et al.[8].This group used [3H]paminoclonidine in membranes prepared from the bovine RVLM region, the rostro-ventrolateral medulla, the main site of the hypotensive action of clonidine like compounds, and showed that about 30% of the bound [3H]paminoclonidine were insensitive to an excess of noradrenaline but completely displaced by various compounds having an imidazoline s t r u c t u r e . [3 H] idazoxan, another compound with an imidazoline structure, was shown unable to be completely displaced by catecholamines from renal cortical membranes while several imidazolines were able to do so [10]. Since these basic works, many binding assays confirmed the existence of catecholamine-insensitive imidazoline specific binding sites in various tissues including the h u m a n brain. As an example, Bricca et al. showed that about 80% of the [3 H] clonidine bound to h u m a n brainstem m e m b r a n e s were resistant to the endogenous catecholamines [15]. Some authors faced with experimental data from functional studies that did not fulfilled the criteria of definition of I 1 - o r I2-receptors; this was particularly the case in investigations concerning the possible role of pancreatic imidazoline receptors but it was also evoked with regard to sympathetic presynaptic imidazoline receptors. Such sites were provisionally named non-I1, non-I2 receptors [24,25]. It is quite clear that binding sites of the I2-type are localised on the external membrane of the mitochondria [22]. This mitochondrial localisation prompted some authors to suggest a relation between these imidazoline binding sites and the monoamine oxidases (MAO) [26,27]. It was recently suggested that a mitochondrial specific imidazoline binding site could be associated with the catalytic site of the enzyme; this site would therefore be a regulatory site of the enzyme. In fact, yeast transfected with a monoamine oxidase gene coexpressed imidazoline binding sites together with the MAO itself. The sub- cellular localisation of sites of the Il-type is not yet demonstrated, at least in neurones [28]. Several laboratories tempted the purification of imidazoline specific binding proteins. Thus, the mitochondrial imidazoline binding site was purified at apparent homogeneity; a 60 kD protein with a pharmacological profile similar to that of the I2-imidazoline binding sites was identified [22]. The sequence of this protein proved some homologies with that of the monoamine oxidases. Another group purified a 70 kD protein from adrenal chromaffin cells; that protein also exhibited a pharmacological profile similar to that of the I2-type imidazoline binding sites [29]. More recently, a 43 kD protein was isolated and largely purified from human brain extracts. This protein showed interesting binding characteristics similar to that of II-binding sites. It bound [3H] clonidine and [3H]idazoxan [30,31]. A similar protein (45 kD) was also isolated from the rat brain [32].

353 At last, although there is a general agreement concerning the non-coupling of imidazoline receptors to G proteins [19,33], the transduction mechanism associated with these receptors is still obscure. Many efforts are dedicated to identify the coupling mechanism associated with the imidazoline receptors; there are some indications suggesting that ATP dependent K + channels might be associated with imidazoline receptors. In fact, some authors demonstrated that various imidazoline compounds were able to enhance the glucose evoked i n s u l i n secretion in p a n c r e a t i c [3 cells, this effect, insensitive to catecholamines, was in fact mediated by ATP dependent K + channels [34,35]. Imidazoline binding sites were characterised in various species, rabbit, dog, guinea pig, bovine, rat and man. They were also found in several tissues, brain, liver, placenta, adipose tissue, kidney and even u r e t h r a [8,1012,15,18,36,37]. Several cell lines also appeared to express such receptors, RINm5F cells of pancreatic origin were an example [38]. One should note that, with the exception of the adrenal chromaffin cells, a2adrenoceptors and imidazoline receptors usually appeared co-localised [39]. This is particularly true in the central nervous system. It is also noteworthy that none of the available ligands of the imidazoline receptors are really selective enough for those receptors to avoid significant binding to a2-adrenoceptors.

2. LIGANDS Binding sites specific for imidazolines were defined as sites labelled with t r i t i a t e d p-aminoclonidine, clonidine, r i l m e n i d i n e , i d a z o x a n or piodoclonidine but resistant to an excess of catecholamines. These ligands are still largely employed in binding assays with a special mention for tritiated clonidine and idazoxan; nevertheless, two additional ligands were recently proposed, i.e. tritiated cirazoline presently used by an unique group [40] and tritiated 2-(2-benzofuranyl)-2-imidazoline, (2BFI compound) [41] (fig.l). A photoaffinity ligand was also prepared by Lanier's group,(1993) t h a t is the AZIPI compound (2-[3-azidophenoxy]-methylimidazoline) [42] (fig.l). The latter was particularly used as an interesting tool to characterise imidazoline receptive proteins in various tissues and animal species. These tools however are not very selective for the imidazoline receptors as compared to a2adrenoceptors. Because of that relative lack of selectivity and also because a2adrenoceptors and imidazoline receptors usually appeared co-localised, it is still necessary to mask a-adrenoceptors in binding assays devoted to the characterisation of imidazoline receptors [18,19]. Since imidazoline receptors are sub-classified, it is generally assumed that tritiated clonidine labels I 1-subtype of imidazoline binding sites while tritiated idazoxan is a useful tool to label I2-binding sites; in this case, it is however necessary to check whether these sites are insensitive to unlabelled clonidine [20]. In order to investigate these latter sites a tritiated form of a more selective

354

compound, BFI, was recently proposed but there are so far few experimental data proving its high selectivity for imidazoline I2-receptors [41]. Although cirazoline did not allow to distinguish between the different subtypes of imidazoline receptors, it remains the substance exhibiting the highest affinity for these receptors as compared with a2-adrenoceptors [18,19,21,22,36,43-45] .

AGMATINE

H2N~NH

ALPHA-METHYLNORADRENALINE

~NH \ NH2

HO--(I

CI

~)-- CH-- CH- NH2

HOf

N

NH

NH2 ~ N, ' J ~ NH2

H N

AZIPI

AMILORIDE

2BFI

Co

CIRAZOLINE

.(

O

N

- CH2

CLONIDINE

CI

H N

PARA-AMINOCLONIDINE

CI

H

N

PARA -IODOCLONIDINE CI

CI

CI EFAROXAN

CH3 H

u o cH~,"3 , I

CI

IDAZOXAN H

N

%J

MEDETOMIDINE

N~

o CH3

Figure 1.

H N

CH3

355 Numerous attempts to provide us with selective ligands for imidazoline specific receptors were made. With this aim in view, derivatives of idazoxan as well as of cirazoline were developed and tested for their affinities towards imidazoline binding sites and a-adrenoceptors. Although the affinity of idazoxan itself appeared similar on the two binding sites, its 2-methoxy derivative (RX821002) (fig.l) exhibited a 1000 fold higher affinity towards a2adrenoceptors than for [3H]idazoxan imidazoline specific binding sites [46,47]. Cirazoline was described as the most selective ligand for imidazoline binding sites, exhibiting a 10 to 50 fold selectivity [18,36] among the available compounds. Removal of the cyclopropyl group and amination of the benzyl ring led to the synthesis of AMPI which appeared as an interesting compound that discriminates between various types of imidazoline binding sites [45]. AMIPI, the radioiodinated analogue of AMPI ((2-[3-aminophenoxy]methyl imidazoline), was shown to be unable to bind a2-adrenoceptors [44]. Recently, a new series of imidazoline ligands appeared 1000 to 10000 more selective for [3H]idazoxan imidazoline specific binding sites than for a2-adrenoceptors: the RX 801077 or 2-BFI( 2-2-benzofuranyl)-imidazoline [41] and other new compounds ( Brasili et al, this issue). Ligands more selective for I1 receptor subtype over a2-adrenoceptors and I2 subtype remain to be developed. The question of the nature of the endogenous ligand(s) of the imidazoline receptors is not definitely resolved. Brain extracts prepared in several laboratories appeared to contain substances candidate(s) to be endogenous ligand(s) of these receptors [7,9,48,49]. Some of these extracts were shown hypotensive when applied centrally whereas others increased blood pressure when injected directly in the NRL/RVLM region [7,50]. This point will remain necessarily controversial until complete purification and identification will be achieved. Recently, agmatine, a decarboxylated metabolite of arginine, was proposed as an endogenous ligand of imidazoline receptors (fig.l). This suggestion was based on the fact that agmatine was capable of displacing in some extent the specific binding of various ligands from imidazoline receptors [51]. Nevertheless, affinity of agmatine for imidazoline receptors appeared rather low and it did not really fit the criteria of definition of a specific ligand of imidazoline receptors because it also bound to a2-adrenoceptors. Until the identification of the endogenous ligand(s) of imidazoline receptors is achieved, it is impossible to state about the agonist or antagonist properties of the synthetic substances that act on these receptors.

3. FUNCTIONS AND PERSPECTIVE S Blood pressure Recently an imidazoline-like oxazoline was presented as a central acting antihypertensive drug selective for the imidazoline receptors [52] (fig.2). At the present time, this substance, rilmenidine, is the prototype of such drugs. Moxonidine was subsequently also proposed in this line [53] (fig.2).

356 Concerning rilmenidine, it was established that its hypotensive effect mostly originated within the brain [54,55] and that the selectivity of this compound for imidazoline receptors versus a2-adrenoceptors might explain its lack of significant sedative action in animal models as well as in patients. Compared to the reference substance, clonidine, rilmenidine had an improved selectivity for the imidazoline receptors because of its reduced affinity for a 2 adrenoceptors [17]. Moreover, it was reported that the hypotensive effect of rilmenidine was potently antagonised with idazoxan, an antagonist with an imidazoline structure, whereas it was weakly or not at all antagonised with yohimbine, a reference antagonist of the a2-adrenoceptors [54]. Concerning moxonidine, there is still a controversy about its selectivity for imidazoline receptors; for some authors, it even appeared more selective for imidazoline receptors (versus a2-adrenoceptors) than rilmenidine [56]. New central acting antihypertensive drugs with higher selectivity of imidazoline receptors will probably be developed in the near future.

ooh:_

MOXONIDINE H N OH3 ~

NA

RILMENIDINE

N

CH-

C,

~

~

Or-

ANTAZOLINE H

CH2

N

Figure 2. I~Klney Drugs selective for imidazoline receptors might act within the kidney. A direct effect of rilmenidine was observed in the rabbit as well as in the human kidney; it inhibited the Na+/H + exchanger. This effect was opposite to that

357 induced by the activation of renal a2-adrenoceptors [22]. Rilmenidine was shown to increase renal blood flow, potassium excretion, natriuresis associated to an inhibition of sodium reabsorption and diuresis while the sympathetic renal nerve activity was markedly decreased [57]. Insulin secretion Some imidazoline substances were shown to enhance the glucose induced insulin release from pancreatic ~ cells [35,58]. This effect was not mediated by a-adrenoceptors but appeared to involve some specific imidazoline sites [86]. Moreover, these ceils expressed recognition sites specific for imidazolines but different from the I1-or I2-receptors. In fact, imidazoline compounds such as efaroxan and antazoline were shown to potently stimulate insulin secretion ( fig 2). The latter effect was also attributed to a specific action on pancreatic imidazoline receptors [59]. Central non~ardiovascular effects The imidazoline binding site density was shown to be increased in the brain from patients with Alzheimer's disease or endogenous depression [60,61]. Clonidine and related substances were known to be deleterious in some mood disorders such as depression for instance. It was therefore proposed that drugs with antagonist activity towards the effects of clonidine might be of therapeutic relevance in some of these neuro-psychiatric syndromes. In this respect, idazoxan, which is a clonidine antagonist with an imidazoline structure, was extensively studied. Antidepressant effects of idazoxan were studied in clinical trials. The optimal efficacy was observed after 4 weeks of treatment in depressed subjects [62,63]. Idazoxan was thereafter recognised as an effective antidepressant drug, specially in bipolar depression [64]. However, t r e a t m e n t of schizophrenic patients with idazoxan alleviated negative symptoms [65].

CONCLUSION The existence is now fully demonstrated of binding sites specifically recognising the imidazoline structure or similar chemical structures, both in the brain and in certain peripheral tissues, including the kidney, some of which participate in the control of blood pressure. These binding sites are different from a2-adrenoceptors, both functionally and biochemically. The fact that at least medullary binding sites are associated with a precise function, i.e., the modulation of the sympathetic activity of central origin and therefore the regulation of the vasomotor tone, confirms that they are true receptors. Numerous attempts are currently made to develop selective ligands for each sub-type of imidazoline specific binding sites.

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Perspective in Receptor Research D. Giardin~, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

361

Discovery of Highly Selective Imidazoline Receptor Ligands L. Brasilia, M. Piginib, P. BousquetC, A. Carottid, M. DontenwillC, M. Giannella b, R. Moriconi c, A. Piergentili b, W. Quaglia b, S. K. Tayebati b aDipartimento di Scienze Farmaceutiche, Universit& di Modena, Via Campi 183, 41100 Modena, Italy. bDipartimento di Scienze Chimiche, Universit& di Camerino, Via S. Agostino 1, 62032 Camerino (MC), Italy. CLaboratoire de Pharmacologie Cardiovasculaire et R~nale, Universite Louis Pasteur, CNRS URA 589, Facult~ de Medecine, 11 rue Humann, 67000 Strasbourg, France. dDipartimento Farmaco-Chimico, Universit& di Bari, Via E. Orabona 4, 70125 Bari, Italy

INTRODUCTION

Phenylethylamines and imidazolines are traditionally thought of as representing the two major classes of a-adrenergic agonists, yet there is evidence to suggest that these two classes of compounds do not interact with ~adrenergic receptors in an identical manner. Ruffolo [1] has reported a lack of cross-desensitization on rat vas deferens and some time later he showed that the optically-active phenylethylamines conform to the Easson-Stedmaan hypothesis and appear to bind to the a-adrenergic receptors by way of a three-point attachment, whereas imidazolines do so by means of a two point attachment [2]. Bousquet and co-workers [3], however, were the first to present functional evidence in vivo for a possible different mechanism of action of the two groups of o,-adrenergic agonists; in their studies on the central cardiovascular effect of o,adrenergic drugs they demonstrated that a clonidine-like substance may stimulate an imidazoline-preferring site in the nucleus reticularis lateralis region. This site differs from the classical c,2-adrenergic receptors in that catecholamines with high a2-adrenergic receptor affinity do not stimulate it. Since Bousquet's discovery, the Imidazoline (I) receptor concept has been developed [4]. It is now well established that I receptors differ from o,2-adrenergic receptors structurally, functionally and with respect to their distribution among and within organs. Recent studies have shown that i receptors are widely distributed in both the CNS and periphery and may subserve several physiological actions [5-12]. Furthermore, ligand binding data show that, like other receptors, they exist as multiple subtypes [13-15]. Different rank orders of the affinity of ligands indicate the possible existence of at least two classes of I receptors, I1 and 12. I1 preferentially binds [3H]-clonidine [16-18], whereas the 12 type preferentially binds [3H]-idazoxan [16-23]o The identity of these two binding sites is still unresolved owing to differences in autoradiography Iocalisation and to non-competitive interaction between clonidine and idazoxan in the human brain [24-25]. Other I receptors, which seem to differ from both I1 and 12, have also been identified [26-28]. However, a definitive subclassification, with a statement of their physiological function(s), has yet to be decided upon, mainly because the

362

ligands used for their characterization all suffer from a lack of selectivity with respect to o,-adrenergic receptors. Over the years, medicinal chemists have provided a number of selective tools for discriminating between a~- and a2-adrenergic receptors and their respective subtypes, but to date no ligand has been discovered which binds I receptors in preference to the a-adrenergic receptors. Chart 1 lists some examples of the ligands most commonly used, as such or radiolabeled, for I receptor characterization, but all of them are known to interact also with the a-adrenergic system. There was, therefore, an urgent need to develop selective ligands which would enable us to define the structure and function of ! receptors more accurately. Despite the increasing number of papers published in this field, none of them has adopted a medicinal chemistry approach. Our group has, therefore, undertaken a research project using as its starting point cirazoline 1, a very potent ~l-adrenergic receptor agonist [29], which exhibits high affinity for l receptors in a variety of tissues [13, 19, 20, 30-32] and only moderate affinity for o,2-adrenergic receptors [29]. CI N~

R

N~

]

R= H clonidine R = NH2 p-aminoclonidine

I

CI

H N~

OCH3 N---

H

N I

H rilmenidine

moxonidine

N

idazoxan

N I H

I

cirazoline 1 H

Chart 1. Some of the most used ligands for imidazoline characterization.

receptors

363

AGONIST

S E P A R A T I O N OF (Zl RECEPTOR AFFINITY

ACTIVITY

FROM

IMIDAZOLINE

First, we synthesized a series of cirazoline derivatives and evaluated their pharmacological effects on rabbit aorta and their I receptor affinity on rabbit kidney membranes. The results reported in Table 1 show that their ability to interact with a l-adrenergic as opposed to I receptors follows a characteristic pattern. Table 1 Functional (o~1) and radioligand binding (I) studies of cirazoline derivatives

ox4 R

N

N I

H (Xl a

R

R'

pD2

pKA

pKi Ib

I/a1 c

/"1 1 ",4 H 7.47 6.50 8.41 81 2 H H 6.26 5.23 9.05 6,607 3 H CH3 5.57 4 CH3 H 6.70 6.08 9.05 933 5 CH3 CH3 6.65 5.64 6.70 11 6 C2H5 H 7.19 6.70 8.57 74 7 n-C3H7 H 7.14 6.50 8.21 51 8 i-C3H7 H 7.18 6.92 8.66 55 9 i-CsH7 CH3 7.00 6.27 5.30 0.1 10 s-C4H9 H 6.21 5.94 7.30 23 11 t-C4H9 H 6.45 6.11 6.70 4 12 CH2-CH=CH2 H 7.19 6.97 8.85 76 13 O-iCsH7 H 7.46 6.85 7.07 2 14 O-iC3H7 CH3 5.52 15 C6H5 H 6.55 16 CH2C6H5 H 6.70 a Data obtained on rabbit aorta, taken from reference 33. b Receptor binding affinity was determined on rabbit kidney membranes using [3H]-idazoxan. Clt is the antilog of the difference pKi -pKA. Cirazoline exhibits high affinity for I receptors, with a pKi of 8.41 and a selectivity ratio of 81. Removal (2) or replacement of the cyclopropyl ring with a methyl (4), ethyl (6), n -propyl (7) or allyl (12) group conserves the high degree of affinity. If, on the other hand, cyclopropyl is replaced with bulkier alkyl (8,10,11) or alkoxy (13) groups a large decrease in affinity, spanning two orders of magnitude, is

364 observed. Moreover, the introduction of a methyl group on the oxymethylene bridge has little or no effect on either m agonist potency or affinity, while it impairs I receptor recognition. The results obtained with compound 2 are of interest, in that it has the highest affinity for the I receptors (pKi = 9.05) and the lowest affinity for the m-adrenergic receptors (pKA = 5.23). This compound is therefore the most selective for I receptors with a selectivity value of 6,607. Nevertheless, it might be of limited value in view of the fact that it retains m agonist activity. It has been reported for another series of imidazoline derivatives that substitution of the imidazoline ring decrease m-adrenergic activity [34]. This was confirmed in our studies: as it can be seen from Table 2, methylation of either the carbon or nitrogen atom of the imidazoline ring of the unsubstituted compound 2 causes a loss not only of m agonist activity but also of I receptor affinity. Table 2 Functional (o{1) and radioligand binding (I, a2) studies of compounds 2, 17-21

!

R' al a

X R R' pD2 (pKb) i.a. pKi Ib 1 r 7.33 1.00 8.41 2 0 H H 5.26 0.95 9.05 17 0 CH3 H (4.86) 0 5.96 18 0 H CH3 (4.34) 0 IN 1 (3 S H H 4.69 0.60 7.30 20 NH H H 4.89 1.00 7.48 2 1 CH2 H H (4.51) 0 8.60 a Data obtained on rat vas deferens. b As in footnote to table 1. c Data obtained on rat cerebral cortex using [3H]-clonidine. d It is the antilog of the difference pKi I - pKi a2.

pKi a2 c 7.77 7.28 4.72 IN 6.70 7.14 5.70

I/0~2d 4.4 59 17 4 2.2 794

We then turned our attention to the bridge linking the aromatic and the imidazoline rings. Table 2 reports the results of this study. Isosteric substitution of the oxygen atom with a methylene group (21) determines a loss of cu agonistic activity (i.a.=O), while I receptor affinity is maintained. The affinity for a2adrenergic receptors is also decreased and, as a consequence, there is a tenfold increase in selectivity with respect to the (~2-adrenergic receptors. This compound is the first really selective i receptor ligand to be discovered.

365 CONFORMATIONAL

RESTRICTIONS

Having been successful in separating the two activities and in discovering the first selective ligand for I receptors, we then set out to further improve potency and selectivity. The opportunity for receptor selectivity is suggested by the notion that different conformers of the same compound may interact with different receptors. Compound 21 has a substantial degree of flexibility resulting from rotation about the bridge bonds, as shown in the Figure 1. Thus, we designed novel and selective compounds whith restricted conformational flexibility.

21

0

N I

a


b H

b

i H

23

I

H

U, a+b

~ X ~ y

I

H 24

25 26

O-CH2 X - Y= CH2-CH2 X - Y= CH=CH

X - Y=

Figure 1. Different ways to accomplish conformational restriction of compound 21. The results are reported in Table 3. The first attempt was not successful. In fact, bridging the carbon atom in ortho position of the phenyl ring with the distal carbon atom on the bridge (way a in Figure 1)to obtain the benzopyran derivative 22, resulted in a sharp decrease in selectivity due to a decrease in I receptor affinity and an increase in ~2-adrenergic receptor affinity. Another way of

366

restricting conformational freedom is the insertion of a double bond in the bridge (way b in Figure 1). This restriction gave very good results, in fact, compoud 23, in its trans form with a pKi of 8.74, maintains high affinity for I receptors, while at ot2-sites a seven-fold reduction is observed. This results in a i/o~2 selectivity of 7,762 which is unprecedented. The I/0,1 selectivity of 2,344 is also very remarkable. Thus, this compound, w h i c h we have named t r a c i z o l i n e , is the most selective ligand for I receptors reported to date. Since the concept of conformational restriction worked so well, we decided to pursue this idea further, restricting the conformation of tracizoline by carrying out Table 3 Radioligand binding of compounds 21-26, i d a z o x a n and clonidine

21 22 23 24 25 26

tracizoline

X CH2 CH2 CH CH CH CH

= = = =

Y CH2 CH CH C C C -

Z CH2-O CH2-O CH2-CH2 CH=CH

pKi Ib 8.60 7.80 8.74 8.43 7.95 9.07 8.31 6.10

benazoline idazoxan clonidine b, c, d As in footnote to table 2. e Data obtained on rat cortex using [3H]-prazosin.

pKia2 c I/a2 d pKio~l e I/a1 d 5.70 794 6.87 8.5 4.85 7,762 5.37 2,344 6.15 191 5.56 245 4.80 18,621 5.64 2,691 7.71 4 8.31 0.006 -

the bridging described above for the saturated compound 21 (way a+b in Figure 1) to obtain the benzopyrene and the dihydronaphthalene derivatives 24 and 25. In both cases, we observed a moderate reduction in affinity for ! receptors and an increase in affinity for (~2-adrenergic receptors, and therefore a significant decrease in selectivity. Interestingly, the dehydrogenation of 25 gave the best results. In fact, compound 26, which we have named b e n a z o l i n e , showed the highest affinity (pKi = 9.07) and the highest selectivity with respect to both a2(l/a2 = 18,621) and al-adrenergic receptors (l/m = 2,691).

367

N

OCL<

I

I

H (CZ)

cirazoline

I

H tracizoline (TCZ)

benazoline

H (BNZ)

10

;~ t'l

7

I-I in

CZ

TCZ

i

BNZ

Figure 2. Comparison of affinity of c i r a z o l i n e , t r a c i z o l i n e benazoline at o~1-, e~2-, and I receptors. TOLAZOLINE,

and

NAPHAZOLINE, AND MOLECULAR YARDSTICKS

Compound 2 may be regarded as a close derivative of tolazoline (27), another imidazoline-based adrenergic drug, in which the distance separating the phenyl and the imidazoline rings has been varied by the insertion of an oxygen atom. This variation increases affinity for I receptors by more than two order of magnitude, and also increases selectivity about sixty times (Table 4). Accordingly, we set out to discover whether the same structural variation would work as well with naphazoline (28), another well-known imidazoline-based adrenergic drug, and with its 13-isomer isonaphazoline (30). In doing so, we would be able to produce the two naphtoxy derivatives 29 and 31, which might

368

be regarded as molecular yardsticks of the phenoxy derivative 2, and which enable the spatial dimensions of the lipophilic region of the binding cleft to be determined [35]. The results reported in Table 4 show that the insertion of an oxygen atom in the bridges of naphazoline and isonaphazoline has a positive effect on I receptor binding, while it has a negative effect on ~2-adrenergic receptor binding. As a consequence, the selectivity for the former receptor is increased about a hundred times. Comparison between the naphtoxy and phenoxy derivatives shows that a second phenyl group is admitted in the binding process with ! receptors, regardless of its position with respect to the ethereal bridge. The binding to the a2-adrenergic receptors admits the second phenyl ring, but in this case its location with respect to the bridge plays an important role, for the 13-naphtoxy derivative 31 is ten times less active than the a - n a p h t o x y derivative 29. In order to gain further insight into the topography of the binding site, compounds 33 and 34 were prepared as molecular yardsticks of benazoline 26 and its s-isomer 32, respectively. In both cases, fusion with a third phenyl ring causes a drop in affinity and selectivity (Chart 2). Table 4 Radioligand binding of t o l a z o l i n e (27), n a p h a z o l i n e (28), i s o n a p h a z o l i n e (30) and their derivatives 2, 29, 31

N N I

H

a C4H4 C4H4

b 27 2 28 29 3 0 04H4 31 04H4 b, c, d As in footnote to MOLECULAR

X tolazoline O naphazoline O isonaphazoline 0 table 2.

pki Ib 6.70 9.05 7.00 8.72 6.52 8.40

pki~2 c 6.70 7.28 8.56 7.17 7.03 5.96

I/~2 d 1 59 0.03 35 0.31 275

MODELLING

Comparative Molecular Field Analysis (CoMFA) [36] has been successfully applied to a wide range of biological problems [37], affording significant insight at the 3-D level into the main physicochemical interactions underlying molecular binding, recognition and discrimination. The CoMFA approach, as implemented in SYBYL 6.1 (Tripos Ass., St. Louis, MO, USA) was thus chosen as the most promising tool with which to carry out a 3-D QSAR study on the set of I receptor ligands reported in Tables 1-4 and Chart 2.

369

Im

33 pKil = 7.15 pKi a2 = 5.55

32 pKil = 7.65 pKi o~2 = 5.38

~

pKii

35 = 5.54

l

pKil

lm 34 pKil = 6.00 pKi ~2 = 5.76

Im

m 36 = 7.96

pKii

37 = 5.44

m Im

pKii

38 = 7.62

pKil

,39 = 7.57

N~ NI I

pKil

O 40 = 5.68

1

H Chart 2. Other imidazoline-based structures used in molecular modelling study. Molecular superimposition is the most critical step in any CoMFA study and it was therefore thoroughly studied by testing several different alignments. Benazoline was chosen as the template, since it is the most active ligand and has a relatively limited conformational mobility. Its lowest minimum energy conformer, in which the inter-ring torsion angle between naphtyl and imidazoline moieties is -32.3, was selected for the different molecular overlays. Each molecule, in its protonated form, was subjected to a conformational analysis through a systematic search, and all the conformers with an energy up to 8

370

Kcal/mol above the global minimum were superimposed on the template. In the case of the partially saturated bicyclic derivatives idazoxan, 22 and 39, only equatorial conformations, which afforded a much better fit with the template, were screened. In the congeneric series of cirazolines we selected the torsion angle of the o-substituted phenoxy ring (C-O-01,-02,) corresponding to an average value for the minimum energy conformers of about-149 ~ and kept it constant in the subsequent alignments. The alignment yielding the best statistical parameters was retained (see Figure 3) and used to develop significant CoMFA models.

Figure 3. Alignment of I receptor ligands 1-16, 19-40, r

idazoxan, resulting from a multifit procedure and used in CoMFA.

and

The statistics and the isocontour maps of our preliminary CoMFA model are shown in Figure 4. It can be seen that both steric and electrostatic fields contribute to the model and, within the limits of the physicochemical domain explored, the former is prevalent. The steric contour map (right-hand side of Figure 4) revealed a large unfavourable region (shown in red) where the methyl groups on the oxymethylene bridge, as well as some aromatic rings, may be positioned; a similar but smaller region may be seen in the proximity of one chlorine atom of

371

clonidine. Two green zones, indicative of favourable steric interactions, may be also detected: one close to the small ortho substituents of cirazolines and the other close to one of the aromatic rings of the template. The electrostatic map (left-hand side of Figure 4) revealed two zones (cyancoloured), where the increase in negative charge enhances the activity and which are easily occupable by the two aromatic rings of the template, and a smaller yellow region where the increase in negative charge diminishes the activity. The latter region may be occupied by the carbonyl group of the 4chromenone derivative (40) and by the = electron cloud of one phenyl group of the benzydryl congener (35).

Figure 4. Pictorial representation of 3D-QSAR models. The color code is as follows: sterically favourable and unfavourable interactions, green and red regions, respectively; favourable and unfavourable influence of high electron density, cyan and yellow zones respectively. To aid interpretation the template 26, idazoxan compounds 35 and 40 have been added to the electrostatic map, whereas r compounds 5, 8 and 34 are shown in the steric map. n, number of data points; q and r, cross-validated and non-cross-validated correlation coefficient, respectively; s, standard deviation; onc, optimal number of components.

372

CONCLUSIONS Over the last decade or so, since the hypothesis of Bousquet that the hypothensive action of clonidine could be related to its imidazoline structure rather than to its interaction with ~2-adrenergic receptors, the concept of I receptors has gained consensus. Investigations with radiolabelled ligands have shown the existence of imidazoline binding sites in several tissues and species, including humans. Furthermore, functional studies, though still few in number, have demonstrated that these binding sites may represent functional receptors, and the discovery of agmantine [38] as its natural neurotrasmitter seems to support this view. In view of the fact that all the attempts to characterize this new receptor have been carried out with non-selective or scarcely selective ligands, and that despite the large number of reports in this field none has adopted a medicinal chemistry approach, we set out to undertake a research project aimed at developing selective ligands. In this symposium we have reported on several interesting compounds, two of which, tracizoline (23) and benazoline (26), are outstanding for their affinities and unprecedented selectivities. These two compounds will contribute to a better understanding of I receptors and, it is hoped, give a definitive answer to the question of whether or not they can represent a novel therapeutic target. Congruent 3-D QSAR analyses have afforded important insight into the key structural features governing the binding of a large series of I receptor ligands. Further studies are under way to map the I receptors in greater detail and most importantly, to detect the molecular determinants responsible for a high I/a2 receptor selectivity

REFERENCES

10 11 12 13

Ruffolo RR, Turowski BS, Patil PN. J Pharm Pharmacol 1977; 29: 378-380. Ruffolo RR, Rice PJ, Patil PN, Hamada A, Miller DD. Eur J Pharmacol 1983; 86: 471-475. Bousquet P, Feldman J, Schwartz J. J Pharmacol Exp Ther 1984; 230: 232236. Michel MC, Ernsberger P. Trends Pharmacoi Sci 1992;13: 369-370. Morgan MG, Chan SLF, Brown CA. II International Symposium on Imidazoline Receptors. New York, July 19-20, 1994; Abstract $26. Carpene C, Lafontan M. II International Symposium on Imidazoline Receptors. New York, July 19-20, 1994; Abstract $29. Penner SB, Smyth DD. II International Symposium on Imidazoline Receptors. New York, July 19-20, 1994; Abstract $27. Jackson HC, Griffin IJ, Nutt DJ. Br J Pharmacol 1991; 104: 258-262. Bartunen F, Meana JJ, Martin I, Garcia-Sevilla JA. Fundam Clin Pharmacol 1992; 6 (suppl 1): p 27. Sastre M, Garcia-Sevilla JA. Fundam Ciin Pharmacol 1992; 6 (suppl. 1): p 28. Tibirica E, Feldman J, Mermet C, Gonon F, Bousquet P. J Pharmacol Exp Ther 1991 ; 256:606-613. Olmos G, Miralles A, Bartunen F, Garcia-Sevilla JA. J Pharmacol Exp Ther 1992; 260: 1000-1007. Kilpatrick AT, Brown CC, Mac Kinnon AC. Biochem Soc Trans 1992; 20:

373

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

113-118. Michel MC, Insel PA. Trends Pharmacol Sci 1989; 10: 342-344. Reis DJ, Regunathan S, Wang H, Feinstein DL, Meeley MP. Fundam Clin Pharmacol 1992; 6 (Suppl 1): 23-29. Ernesberg P, Meeley MP, Mann JJ, Reis DJ. Eur J Pharmacol 1987; 134: 113 Ernsberger P, Westbrooks KL, Christen MO, Schafer SG. J Cardiovasc Pharmacol 1992; 20( suppl. 4): 1-10. Molderings GJ, Moura D, Fink K, Bonisch H, Gothert M. NaunynSchmiedeberg's Arch Pharmacol 1993; 348: 70-76. Lauchad-Pettiti V, Podevin RA, Chretien Y, Parini A. Eur J Pharmacol Mol Pharmacol Sect 1991;206: 23-31. Miralles A, Olmos G, Sastre M, Bartunen F, Martin I, Garcia-Sevilla JA. J Pharmacol Exp Ther 1993; 264:1187-1197. Michel MC, Brodde OE, Schnepel B, Behrendt J, Tschada R, Motulsky HJ, Insel PA. Mol Pharmacol 1989; 35: 324-330. Langin D, Paris H, Lafontan M. Mol Pharmacol 1990; 37: 876-885. Molderings GJ, Kundt L, Gothert M. Naunyn-Schmiedeberg's Arch Pharmacol 1994; 350: 252-257. Bricca G, Greney H, Dontenwill M, Zhang J, Belcourt A, Bousquet P. Neurochem Int 1993; 22: 153-163. De Vos H, Bricca G, De Keyser J, De Baker JP, Bousquet P, Vauquelin G. Neuroscience 1994; 59: 589-598. Molderings GJ, Gothert M. Naunyn-Schmiedeberg's Arch Pharmacol 1995; 351: 507-516. Molderings GJ, Donecker K, Gothert M. Naunyn-Schmiedeberg's Arch Pharmacol 1995; 351 : 561-564. Chan SLF, Brown CA, Scarpello KE, Morgan NG. Br J Pharmacol 1994; 112: 1065-1070. Ruffolo RR, Waddell JE. J Pharmacol Exp Ther 1982; 222: 29-36. Wikberg IES, Uhl~n S, Chhajlani V. Eur J Pharmacol 1991; 193: 335-340. Renouard A, Widdowson PS, Cordi A. Br J Pharmacol 1993; 109: 625-631. Yablonsky F, Dausse JP. Biochem Pharmacol 1991;41:701-707. Brasili L, Pigini M, Marucci G, Quaglia W, Malmusi L, Lanier SM, Lanier B. Bioorg Med Chem 1995; 3: 1503-1509. Miller DD, Hamada A, Craig EC, Christoph GG, Gallucci JC, Rice PJ, Banning JW, Patil PN. J Med Chem 1983; 26: 957-963. Martin MJ, Trudell ML, Diaz Arauzo H, Allen MS, LaLoggia AJ, Deng L, Schultz CA, Tan YC, Bi Y, Narayanan K, Dorn LJ, Koehler KF, Skolnick P, Cook JM. J Med Chem 1992; 35: 4105-4117. Cramer III RD, Patterson DE, Bunce JD. J Am Chem Soc 1988; 110: 59595967. Thibaut U. In: Kubinyi H, ed. 3D-QSAR in Drug Design: Theory, Methods and Applications. Leiden: Escom, 1993; 661-696. Li G, Regunathan S, Barrow CJ, Eshraghi J, Cooper R, Reis DJ. Science 1994; 263: 966-969.

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Perspective in Receptor Research

D. Giardinh, A. Piergentili and M. Pigini (Editors) 9 1996 Elsevier Science B.V. All rights reserved.

375

Synthesis and Structure-Activity Relationship of New 1,5 Benzodiazepine CCK-B Antagonists G. Gaviraghj a, P. Cassar/l a, M. Corsi a, G. Curotto a, D. Donati a, A. Feriani a, H. Finchb, G. Finizia a, G. Pentassuglia a, S. Polinelli a, E. Ratti a, A. Reggiani a, G. Tarziaa, c, G. Tedesco a, M.E. Tranquillini a, D.G. Trist a and A. Ursini a. aGlaxo Research Laboratories, Via A. Fleming 4, 37135 Verona, Italy bGlaxo Wellcome Medicines Research Centre, Gunnels Wood Road Stevenage, Hertfordshire, SG1 2NY, U.K. Clstituto di Chimica Farmaceutica, Universit/L di Urbino, Piazza Rinascimento 1, 61029 Urbino (PS), Italy

Abstract Cholecystokinin (CCK) is one of a number of peptides that act both as gut hormones and neurotransmitters in the central nervous system (CNS). Through the discovery of selective agonists and antagonists it has been possible to show that CCK acts through at least two receptor subtypes, CCK-A and CCK-B. The former are found mainly in the periphery, whilst the latter are located extensively in the CNS. Evidence from animal studies have suggested the potential utility of CCK-B antagonists in the treatment of CNS disorders such as anxiety and panic, with a seemingly specific advantage of a better safety profile over marketed anxiolytics, at least on the basis of the preclinical data. The purpose of this study was to identify new CCK-B antagonists endowed with a better pharmacological profile, both in terms of potency and selectivity, than existing antagonists (e.g. L-365,260). As a result of our studies, a novel series of 1,5-benzodiazepines bearing either ureidic or carbamic side-chains at C-3 were discovered. Amongst the compounds synthesised, GV150013 was found to be a highly potent (pKB = 9.2) and selective CCK-B receptor antagonist. It showed potent anxiolytic activity in a number of animal models and it has been progressed into development. ..

INTRODUCTION Cholecystokinin (CCK) is a 33 amino acid gastrointestinal peptide hormone of that was originally isolated from porcine gut and was given its name "the gallbladder mover" due to its ability to stimulate bile flow [1 ]. CCK is also found in high concentrations in the brain, but mainly as the C-terminal octapeptide [2,3]. Usually native CCK-8 is sulphated on tyrosine 7 (CCK-8S, figure 1). CCK is the second most abundant peptide in the brain, and exhibits many of the characteristics of a neurotransmitter. It is synthesised in neurons; it is stored in synaptic vesicles; it is metabolised in the brain; it is released upon depolarisation and it has specific binding sites associated with nerve terminals [4].

376 Thus, CCK can be considered to belong to the class of peptides which act both as gut hormones and central neurotransmitters. o II

0 H,N*

o

N

~

o

~

v ~

~c.S

OmS--O I

J s~CH3

0

0 N

~

~ N

~_~~

0 ~

~ N

v N~

o

Figure 1. The structure of CCK-8S (Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2). CCK interacts with at least two types of receptor [5]. These sub-types have been designated CCK-A and CCK-B. The former is located predominantly in the periphery (gut etc.) and the latter is found in high abundance in the brain. The gastrin receptor of the stomach shares similarities with the CCK-B receptor, but can be pharmacologically distinguished from it [6]. Table 1 summarises the present classification of CCK receptors based on operational, structural, genetic and transductional information. It can be seen that in operational terms the two receptors can be distinguished using fragments of CCK as agonists and specific selective, potent competitive antagonists. CCK-8S has high affinity for CCK-A receptors , but the unsulphated form and CCK-4 (the C-terminal tetrapeptide) both have low affinity [7]. A-71623 was found to be a potent CCK-A receptor agonist following a series of modifications of tetrapeptides based on CCK-4 [8]. This was a major departure from previous structure-activity studies which had shown that peptide fragments smaller than CCK-7, whilst maintaining CCKB receptor activity, lost CCK-A receptor potency. The introduction of a (Nt-Cbz)lys residue in place of Met was found to be the important requisite for CCK-A selectivity. The classification of CCK receptors into two sub-types is supported by the use of nonpeptidic, selective competitive antagonists. L-364,718 (devazepide) was the first competitive antagonist found with high affinity for the CCK-A receptor (pK i = 9.3) [9]. This compound was developed from asperlicin (pK i = 6.2) which had been found during the screening of products of microbiological fermentation [10]. Similarly, a number of selective CCK-B receptor antagonists have been discovered such as the peptoid PD-134308 (pK i = 8.5) which was derived from a chemical manipulation of CCK-4 [11 ]. Structurally, CCK-B receptors and gastrin receptors show high sequence homology (-90%) and the human CCK-B receptor was 90% and 91% identical to those of the rat and guinea pig. CCK-A receptors cloned from the rat show only 48% homology with rat brain CCK-B receptors. The human CCK-A receptor cloned from gallbladder has >90% homology to the rat receptor [12]. In the mouse the CCK-A receptor and the CCK-B receptor are also found on different chromosomes, CCK-A on chromosome 5 and CCK-B on chromosome 7.

377 The regional distribution of the two receptors in the brain, together with studies with the selective agonists and antagonists in different animal models have suggested possible therapeutic targets for drugs acting through CCK receptors. These include cognitive processes, emotional states such as anxiety, motivation such as drug seeking behaviour and nociception [13,14]. Tablel Class!fication of CCK receptors. Feature CCK-A

CCK-B / Gastrin

Structure (human)

428 amino acids

447 amino acids

Chromosomal location

mouse Chr 5

mouse Chr 7

Tissue location

Gallbladder, Pancreas, Ileum

CNS, Stomach, Ileum

Selective Agonists

CCK-8S, A-71623

Gastrin, CCK-4

Selective Antagonists

L-364,718, PD-140548

L-365,260, LY262691

Transduction

IP3/Ca 2+

IP3/Ca2+ (Gastrin)

Constructed from references [7,8,9,12] As part of our research aimed at finding new anxiolytic compounds acting as potent and selective CCK-B antagonists devoid of the side effects which are typical of marketed drugs, we investigated a novel class of 1,5-benzodiazepine-2,4-diones. This was considered an attractive template in view of combining the high flexibility toward the substitution pattern and the potentially straightforward synthesis and high yielding processes to be applied for their preparation. CHEMISTRY Initially, compounds bearing an aryl substituent at N-5 and either 3-ureido or 3-carbamic side chains at C-3 (fig 2, X=NH and X=O respectively) were evaluated and, in particular, the nature of the substituent at N- 1 was explored [ 15, 16]. The synthesis of both classes of compounds was accomplished according to the same procedure and here we report detailed results only for the ureido derivatives (fig 2, X=NH). The retrosynthetic analysis outlined in scheme 1 suggested that the reaction of the amine (2) with a readily available arylisocyanate (3) would be the key step in the preparation of the target molecules (1).

378

RI

o

i,.o

O X = NH, O

Figure 2. 1-Substituted-5-Aryl-l,5-Benzodiazepine-2,4-diones: General Structure.

R"

A

~ O O N -.-~/ ~L~

~ ~ . ~ . R' ~

R

O

O~ ,~R' N

R"~

R~

O

2

3

Scheme 1.1-Substituted-l,5-Benzodiazepines: Key Intermediates. It is worth noting that the compounds mentioned above have a stereogenic centre at C-3 and their resolution may be crucial in view of the potentially different pharmacological profile of the pure enantiomers. Since the reaction between the amine and isocyanate is unlikely to cause racemization at C-3, it would be possible to obtain the enantiomerically pure ureas after separation of the corresponding amines, thus highlighting the strategic importance of such a key intermediate. Two main synthetic approaches were used for the preparation of amines (2). A first approach (scheme 2) considered 2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-lH-1,5-benzodiazepine (5), obtained from phenylenedyamine (4) [ 17], as a common intermediate Alkylation at N-1 of the benzodiazepine nucleus using either an alkyl bromide or an alkyl mesylate gave compound (6) in moderate to high yield, depending on the steric hindrance of the alkyl group. An azido group was subsequently introduced at C-3 using 2,4,6triisopropylbenzensulfonyl azide in the presence of a base. The azido group in (7) was then hydrogenated to the corresponding amine of general formula (8) using palladium on calcium carbonate as catalyst.

379

H

R I

0

r.t.,20h

4

N

5 R

I

tBuOK, Ar-SO2N3, THF -78 ~

0

N

r.t., 24h

R

o

I

H2, Pd/CaCO3 - - N * - N - AcOEt-EtOH ~ latm., r.t., 3-4h

7

, N

o NH2

8

Scheme 2. Synthesis of key intermediates: route A The alkylation of the intermediate (5) represents the limiting step of the route described in the scheme 2. Moreover, the synthesis of benzodiazepines with substituents on either the N-5 phenyl or the fused aromatic ring generally required a non commercially available substituted diphenylamine as starting material. Therefore, an alternative route, as shown in scheme 3, was set up. The starting phenylenediamine (12) was prepared by reaction of a 2-fluoronitro benzene (9) with an aniline (10) in the presence of potassium fluoride and subsequent reduction of the nitro derivative (11). The resulting amine (12) was generally alkylated with an alkyl bromide in the presence of sodium iodide to obtain the intermediate (13). However, when bulky groups had to be introduced, the intermediate ( 1 3 ) was conveniently obtained by reductive amination of either aldehydes or ketones. Condensation of (13) with (phenylhydrazono)malonyl dichloride followed by reduction with zinc and acetic acid led to the 3-amino substituted benzodiazepines (15). The final ureido derivatives (1) were obtained from the intermediate amines (e.g. (15)) according to the well known methods outlined in scheme 4. The reaction of (15) with an arylisocyanate was obviously the most convenient method, however other routes involving either the synthesis of 3-isocyanobenodiazepines (16), by reaction with phosgene, or the synthesis of carbamate (17), by reaction with phenyl chloroformate [ 18], proved to be useful when the required substituted arylisocyanates were not commercially available.

380

~

NO2

R,,V" v

NH2

"F

,,

9

KF

~ N O 2

=

180~ , 18h R,,~ v

10

11

I

alk'ylation . ~ N H

NH

/COCI Ph-NH'N=C~cocI THF

NH

12 R I

R

Zn/AcOH r.t., 3-6h Y=60%

N-NH-Ph N

R"-~

50~ 1-3h

13

o

-=R " ~

v

EtOH - H;O r.t., 2-4h

R

R R,?.~~NH2

Na2S204, K2CO3 ,._

"NH

or H2, Pd/C, HCI AcOEt - H20 latm., r.t., 3-4h

~

I

-~R,,,.~

o

N R"~

NI'I2

15

14

Scheme 3. Synthesis of key intermediates; route B R

R"~

I

o

NCO N

COCI2, CH2CIz/

H2, CH2CI2 R 16

I

N

R"

NH2

Ar-NCO,CH3CN

I•N

15phOCOCl,~ R"

R"

o

--NH-CO-NH-Ar O

H2, DMF NHCOOPh

17 Scheme 4. Synthesis of final compounds

O

N

381 Although modulation of the activity was achieved by introducing appropriate substituents on the aromatic rings, the most remarkable increase in activity and B receptor selectivity was found with the resolution of the stereogenic centre at C-3 of the benzodiazepine ring. Initially, the enantiomers of (1) were separated using chiral phase I-IPLC, but the small scale of the process proved to be a great limitation. Therefore a more general and effective chemical method was sought and attention was focused on the separation of the enantiomers of the corresponding amine, particularly taking into account that their transformation to the target ureas generally involves non-racemizing conditions. Several methods for the resolution of racemic amines are reported in the literature, and among them, we at first evaluated the formation and preferential crystallisation of diastereomeric salts with chiral acids (e.g. camphorsulphonic acid) [19]. This method proved to be efficient in many eases, and allowed the recovery of the wanted enantiomerically pure amine aider the opportune neutralisation [ 15]. The use of covalent diastereomeric derivatives (e.g. amides) is generally precluded by the strong conditions required for their cleavage to obtain the resolved enantiomers, but recently the resolution of 3-amino-l,4 benzodiazepines has been achieved by means of the preparation and separation of diastereomeric phenylalanyl amides [20]. This procedure, which is based upon the reaction of the racemic amine with N-Boc protected phenylalanine, followed by separation of the two diastereomeric amides and subsequent Edman degradation, was also applied to our series [21] and the desired enantiomers were obtained with excellent enantiomeric excess. However the total yield of the process was quite low due to a difficult chromatographic separation of the two diastereomers and the low conversion during the Edman degradation step. We developed an alternative method based on the resolution of phenylglycine derivatives [22]. Accordingly, the racemic amine was reacted with a chiral auxiliary, namely the tosyl derivative of ( S)-(+)-methyl mandelate, followed by chromatographic separation of the two diastereomers formed and the subsequent hydrogenation of the separated compounds to give the free amines with good enantiomeric excess (e.e .94% ).

BIOLOGY All the compounds synthesised were tested in vitro according to known methods to determine their affinity for both CCK-A [23] and CCK-B [24] receptor subtypes, and hence their B/A selectivity. The most interesting compounds were also evaluated for anxiolytic activity in vivo[25]. Finally, some compounds were evaluated in comparison with Diazepam for potential side effects such as tolerance and withdrawal. The affinity and selectivity of 1,5-benzodiazepines with different substitution pattern was studied in either guinea-pig cortical membranes (CCK-B receptors) or in rat pancreatic membranes (CCK-A receptors) using radiolabelled binding studies. A selection of results is reported in table 2. It can be seen from this first series (all are racemic mixtures), that the opportune combination of substituents can afford compounds endowed with both high affinity and high selectivity.

382 Table 2 K i values for 1-alkyl-5-aryl-3-ureido-l,5-benzodiazepine-2,4-diones in CCK-B and CCK-A binding assays R

I

,.~ R~

o

N. - ~ NH-CO-NH'-~~ , N"~\O ~ R

entry

R

R'

R"

R'"

1

CH2CH2CH(CH3)2

H

H

H

2

CH2CH2CH(CH3)2

H

H

3

CH2CH2CH(CH3)2 3-NMe2 2-F

4 5

CH2CH2-cyclopentyl

CH2CH2C(CH3)3

pK i CCK-

pK i CCK-B

B/A

6.49

8.81

209

7-C1

6.02

8.31

195

H

6.90

9.60

501

H

2-F

H

7.00

9.14

138

3-CN

H

H

7.01

9.19

151

In order to optimise the in vitro profile, we focused our attention on the nature of the substituent at N-1 and a quantitative structure-activity study was performed on a series of N-1 alkyl derivatives. After selection of variables, the affinity for the CCK-B receptor was related to the calculated values of both lipophilicity [26] and molar refractivity [27] of the substituent and the following equation was derived using PLS analysis implemented in program GOLPE [28] (all parameters are referred to the substituents at N-1): PKiB = 0.49"Pi - 0.13"pi2 - 0.44*MR - 0.20*MR2 + 0.39'Sb - 0.67"D1 + 7.15 where PKiB is the affinity for the CCK-B receptor subtype; pi(R) is the calculated lipophilicity; MR is the calculted molar refractivity; Sb is Austel's parameter [29] and D1 is a dummy variable that accounts for the presence (D =1), or the absence (D = 0), of branching on the first carbon atom of the substituent. As can be seen from the equation, the lipophilicity and the steric hindrance of the substituent are critical parameters in the optimisation of the affinity for CCK-B receptor subtypes with respect to the substitution at N-1. With the aim of improving both the affinity and the selectivity, the effect of variation of the N-1 substituent in the was studied in a series of racemic compounds beating no substituent at phenyl tings. The results reported in table 3 show that the introduction of bulky cyclobridged groups such as adamantyl is well accepted in terms of receptor affinities, while important changes in the selectivity were obtained by changing the position of the cyclobridged group to

383 be linked. Thus, for example, the 1-adamantyl derivative (entry 4) was more B selective than the corresponding 2-adamantyl derivative (entry 3). Although a structure-activity relationship can not be established yet for the whole substitution pattern, some preliminary comments can be made other than those reported for substitution at N-l: 1) a m e t a substituent on the ureidic phenyl, (e.g. dimethyl amino or thiomethyl ) may enhance B receptor affinity (however, this is dependent on the substituent at N-l); 2) introduction of an ortho-F substituent on the N-5 phenyl proved to be beneficial in combination with a m e t a dimethylamino substituent on the ureidic side chain and a branched alkyl at N-l; 3) substitution with halogens at the fused aromatic ring was not as valid as in other series (e.g. carbamates) [30]. Table 3 pK i values for 1-alkyl-5-phenyl-3-ureido-l,5-benzodiazepine-2,4-diones in CCK-B and CCKA binding assays R

I

[~

O

N

NH-CO-NH

entry

R

pK i CCK-A

pK i CCK-B

B/A

1

-CH(CH3)CH2-Cyclopentyl

7.11

8.45

22

2

-CH2-2-Norbomyl

7.13

9.06

85

3

-CH2-2-Adamantyl

7.03

8.71

48

4

-CH2-1-Adamantyl

6.15

8.64

309

Finally, the separation of the isomers of the most interesting compounds revealed that the (+) isomers are more B selective than the corresponding (-) isomers. This effect was observed for the N-5 aryl derivatives and it is particularly relevant in case of the 1-adamantyl methyl series. As a result of this evaluation, GV 150013 (fig 4) has emerged as a potent and selective CCKB antagonist and has been progressed into development. Its profile is given below.

384 H

Figure 4. The structure of GV150013. In Vitro Profile of GV150013 GV150013 has been tested in a number of radiobinding assays using membranes from guinea-pig cortex, rat cortex, rat pancreas and from Hela cells transfected with human cortical CCK-B receptor cDNA. The results are summarised in table 4.

Table 4 pK i Values for GV150013 and reference antagonists obtained in CCK-B and CCK-A binding assays (mean + s.e., n = 3-6 experiments). Compound

hCCK-B

Guinea-Pig -B

Rat-B

Rat-A

GV150013

9.43 + 0.12

9.15 + 0.04

8.55 + 0.05

5.83 + 0.05

L-365,260

8.80 + 0.06

8.53 + 0.11

7.91 + 0.05

6.48 + 0.09

PD134308

9.45 + 0.06

8.73 + 0.06

8.03 + 0.04

6.16 + 0.06

L-364,718

7.53 + 0.10

7.06 + 0.02

6.57 + 0.10

9.83 + 0.05

GV150013 showed high affinity in all three of the CCK-B binding assays. Except in the human recombinant assay, where it was equal to PD 134308, it had higher affinity than both of the other two CCK-B antagonists tested. It also had the highest selectivity. As expected, the selective CCK-A receptor antagonist had low affinity in the CCK-B assays, but high affinity in the rat CCK-A assay. Interestingly, all three of the CCK-B antagonists exhibited their highest affinity against the human receptor and their lowest in the rat assay. The data obtained from radioligand binding has been confirmed in functional assays for CCK-B and CCK-A receptors. In the guinea-pig myenteric plexus where both receptor subtypes are found [31], a pKB of 8.9 + 0.3 was determined for GV150013 against selective CCK-B agonists and of 5.9 _+ 0.2 against a selective CCK-A agonist [32]. In the guinea-pig gallbladder, a CCK-A selective tissue, GV150013 had a pKB of 5.8 _+ 0.1. In the isolated rat gastric mucosa, an assay for gastfin receptors, GV150013 was less potent as an antagonist

385 than in the neuronal assays. A pK B of 7.4 + 0.2 was determined against pentagastrin-induced acid secretion. Thus, GV150013 seems to discriminate between the classical CCK-B receptor and the stomach gastrin receptor. In Vivo Profile of GV150013 GV150013 has shown activity in a number of animal models of anxiety. In the mouse black/white box test GV150013 increased the time that naive mice remain in a brightly illuminated section of an activity box. The effect was dose related and significant increases were measured at 0.1, 0.3 and 1.01ag.kg'l.Similar anxiolytic effects were seen in the rat social interaction test and the marmoset human threat test [25]. The ED50 values are given in table 5. In all of the anxiolytic models used GV150013 displayed similar efficacy to PD134308 and to a standard benzodiazepine (diazepam in the mouse studies and chlordiazepoxide in the rat and marmoset). GVIS0013 did not show tolerance or rebound anxiogenesis upon withdrawal after chronic treatment (7 days at 0.31.tg.kg-1, p.o.) in the mouse black / white box. Diazepam (2.5mgkg "1, p.o.) carded through in parallel exhibited a marked reduction of effect and a significant rebound upon cessation of dosing. Table 5 GV 150013 in animal models of anxiety. Model ED50 (gg.kg- 1)

Route

Mouse black / white box

0.05

p.o.

Mouse black / white box

0.03

i.v.

Rat social interaction #

1.6

p.o.

Marmoset human threat

0.02

s.c.

Rat Vogel

No effect up to 30gg.kg-1

# against FG 7142 10mg.kg -1, i.p. In general pharmacological studies GV150013 showed no effect up to 3mg.kg -1, p.o. in the rota-rod test (motor function), in passive avoidance (learning and memory) and on pentobarbitone sleeping time (interaction with metabolic enzymes). No gross behaviour effects were seen with the compound up to a dose of 10mg.kg "1, p.o..

CONCLUSIONS After an initial evaluation of 1,5-benzodiazepine-l,4-diones as potential CCK-B antagonists, we restricted our interest to compounds bearing an aryl substituent at N-5 in conjunction with

386 an alkyl chain at N-1 and an arylureidic chain at C-3. The molecular structure of compounds belonging to this class can be efficiently manipulated to obtain potent and selective CCK-B antagonists. Among them, GV150013 exhibits: 1) high affinity and high selectivity for the CCK-B receptor; 2) anxiolytic activity in a number of animal models with an activity in the range 0.3 to 30 ~tg/Kg, depending on the species. Moreover, GV150013 is devoid of any significant side effect, and is a compound that might be useful in the treatment of anxiety and panic disorders.

R E F E R E N C E S AND NOTES

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Mutt V, Jorpes JE. Biochem J 1971; 125: 57-58. Vanderhaeghen JJ, Signeau JC, Gepts W. Nature (London) 1975; 257: 604-605. Dockray GJ. Nature (London) 1976; 264:568-570 Dockray GJ. Br. Medical Bulletin 1982; 38:253-258 Gaviraghi G, Feriani A, Marien M, Trist DG. Trends in Receptor Research 1992; 18: 345365 Corsi M, Dal Fomo G, Pietra C, Trist DG. Pharmacological Research (Supplement 1) 1992; 26: 81. Steigerwalt RW, Williams JA. Regul Pept 1984; 8" 51-59. ShiosakiK, Lin CW, Kopecka H, Craig R, Wagenaar FL, Bianchi B, Miller T, Witte D, Nadzan AM. J Med Chem 1990; 33: 2950-2952. Evans BE, Book MG, Rittle KE, Di Pardo RM, Whitter WL, Veber DF, Anderson PS, Freidinger RM. Proc Natl Acad Sci. USA 1986; 83:4918-4922. Chang RSL, Lotti VJ, Monagan RL, Bimbaum J, Stapley EO, Goetz MA, AlbersSchonberg G, Patchett AA, Liesch JM, Hensens OD, Springer JP. Science 1985; 230: 177-179. Horwell DC, Hughes J, Hunter JC, Pritchard MC, Richardson RS, Roberts E, Woodruff GN. JMed Chem 1991; 32:404-414 Wank SA, PisegnaJR, De Weerth A. Annals New York Acad Sci 1994; 713: 49-66. Wang RY, White FJ, Voigt MM. TiPS 1984; 5: 436-438. Fails PL, Komisaruk BR, Mayer DJ, Watkins RL. Science 1983;219: 310-312. Finch H, Trist DG, Tarzia G, Feriani A. WO93/14074 (Glaxo S.p.A., 07.22.93). Trist DG, Pentassuglia G, Tranquillini ME, Ursini A. WO93/14095 (Glaxo S.p.A., 07.22.93). Rossi S, Pirola O, Maggi R. La Chimica e rlndustria 1969; 51:479-483 Lyon PA, Reese CB. J Chem Soc Perkin Trans I 1978:131-137. Reider, P.J., DiPardo R M, Evans B E, Rittle K E, Veber D F, Freidinger R M, Hirshfield Y, Springer JPJ Org Chem 1987: 52; 955-957. a) Bock MG, DiPardo RM, Evans BE, Rittle KE, Veber DF, Freidinger RM, Hirshfield J, Springer JP. J Org Chem, 1987; 52: 3232-3239; b) Bock MG, Dipardo RM, Evans BE, Rittle KE, Whitter WL, Veber DF, Anderson PS, Freidinger RM. J Med Chem 1989; 32: 13-16; c) Rittle KE, Evans BE, Bock MG, DiPardo RM, Whitter WL, Homnick CF, Veber DF, Freidinger RM. Tetrahedron Lett. 1987; 28: 521-522.

387 21 Finizia G, Curotto G, Donati D, Pentassuglia G, Polinelli S, Ratti E, Tarzia G, Tranquillini ME, Ursini A, van Amsterdam FThM. Proceedings of the II Congresso Congiunto Italiano-Spagnolo di Chimica Farmaceutica Ferrara 1995 22 Curotto G, Donati D, Finizia G, Ursini A. Tetrahedron: Asymmetry 1995; 6: 849-852. 23 Innis RB, Snyder SH. Proc Natl Acad Sci USA 1980; 77: 6917-6921. 24 Van Dijk A, Richards JG, Trzeciak A, Gillessen D, Moehler H. J Neuroscience 1984; 4: 1021-1033. 25 Reggiani A, Gerrard PA, Maraia G, Melotto S, Ratti E, Gaviraghi G, Trist DG. Br J Pharmacol 1995; 114: 92P 26 CLOGP v.3.54, Daylight Chemical Information System CA 27 CMR v.3.54, Daylight Chemical Information System, Claremont CA 28 Clementi S, Baroni M, Costantino G, Cruciani G, Riganelli D, Valigi R. Quant Struct -Act Relat 1993; 12; 9-20. 29 Austel V, Kutter E, Kalbfleisch W. Arzneim Forsch/Drug Res 1979; 29: 585-587. 30 Curotto G, Donati D, Pentassuglia G, Ursini A. Bioorg Med Chem Lett (submitted). 31 Dal Fomo G, Pietra C, Urciuoli M, van Amsterdam FThM, Toson G, Gaviraghi G, Trist DG. J Pharmacol ExpTher 1992; 261: 1056-1063. 32 Corsi M, Dal Forno G, van Amsterdam FThM, Feriani A, Ursini A, Ratti E, Gaviraghi G, Trist DG. Br J Pharmacol (Proceedings suppl.) 1995; 114: 91P.

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389

AUTHOR

INDEX

Angeli P 321

Dominguez C 313

Ardanuy U 337

Donati D 375

Barberis C 205

DontenwiU M 351,361

B~iumert HG 337

Ensinger HA 51

Becker DP 99

Esbenshade TA 121

Bennai F 351

Evans JM 227

Bertolini G 67

Feldman J 351

Bittiger H 253

Feriani A 375

BlinN

Finch H 375

31

Bo X 337

Finizia G 375

Bolognesi ML 321

Flynn DL 99

Bond A 241

Froestl W 253

Bousquet P 351,361

Gaviraghi G 375

Bowen WD 313

Giannella M 361

Branchek TA 85

Giardin~ D 135,321

Brasili L 361

Goldstin B 99

Budriesi R 321

Greney H 351

Burnstock G 337

Gross J 51

Cacciaguerra S 321

Gulini U 321

Camaioni E 165

Gullikson GW 99

Carotti A 361

He X-s 313

Casagrande C 67

Hibert M 205

Cassarh P 375

Hieble P 135

Chiarini A 321

Hoflack J 205

Chini B 205

Hollenberg MD 271

Corsi M 375

Hoyle CHV 337

Cristalli G 165

IJzerman AP 181

Crucianelli M 321

Jacobson KA 153

Curotto G 375

Jard S 205

De Benedetti PG 135

Jesaitis AJ 11

de Costa BR 313

Ji X-d

Di Francesco E 165

Jiang Q 153

DiMaio J 271

Kim HO 153

153

390 Kim J

153

Pigini M 361

Klotz K-N 11

Polinelli S 375

Lambrecht G 51,337

Portoghese PS 303

Laniyonu A 271

Pozza MF 253

Leblond L 271

Quaglia W 361

LeisA 51

Ralevic V 337

Leonardi A 135

Ratti E 375

Leppik R 205

Rees DC 291

Leurs R 193

Reggiani A 375

Liu J 31

Reichel D 51

Lodge D 241

Rice KC 313

Loeffler R 99

Roelen HCPF 181

Lohse MJ 11

Rtihlmann K 51

Marucci G 321

Saifeddine M 271

Math6t RAA 181

SchiSneberg T 31

Melchiorre C 321

Shapiro G 27

Mendla K 51

Siddiqi SM 153

Mickel SJ 253

Spampinato S 321

Minarini A 321

Spangler D 99

Minneman KP 121

Tacke R 51

Mondadori C 253

Tarzia G 375

Moormann A 99

Tayebati SK 361

Moriconi R 361

Tedesco G 375

Moser U 51

Testa R 135

Motta G 135

Theroux TL 121

MouiUac B 205

Timmerman H 193

Moummi C 99

Tranquillini ME 375

Mutschler E 51,337

Triggle DJ 1,215

Nickel P 337

Trist DG 375

Nosal R 99

Trumpp-Kallmeyer S 205

01pe H-R 253

Tumiatti V 321

Paquet J-L 205

Ursini A 375

Pentassuglia G 375

van der Wenden EM 181

Pfaff O 51,337

van Rhee AM 153

Piergentili A 361

ViUamil CI 99

391 Vittori S 165 Volpini R 165 von Frijtag Drabbe Kiinzel JK Waelbroeck M 51 Waldmeier PC 253 Wehrle J 51 Wess J 31 Williams W 313 Windscheif U 337 Winocour P 271 Yang D-C 99 Yun J 31 Zabrowski DL 99 Ziganshin AU 337 Ziyal R 337

181

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393

SUBJECT INDEX A-68930, 75, 79, 81, 82

-, molecular recognition, 153-163

A-71623, 376, 377

-, partial agonists, 181-191

A-77636, 75, 79

-, -, theophylline-7-ribosides, 184-187

Abanoquil, 138, 147, 148

-,-, deoxy adenosine analogues, 187190

Absence epilepsy, 255,267 Acetylcholine, 27 -, binding site model, 207-210 -, release, 53

-, radioligand binding studies, 183-189 -, site-directed mutagenesis, 153, 158163

Actin, 16

-, subtypes, 153, 165

Adenine, 162

-, -, selective ligands, 154-156

Adenosine, 153

Adenosine receptor ligands, studies, 183-190

-, agonists, 153, 154, 168, 170

-, in vitro

-, -, as potential therapeutics, 153, 154

-,-, GTP shift, 189, 190

-, antagonists, 153, 157

-, -, Na + shift, 183

-, -, xanthine-derived, 156-158

-,-, radioligand binding studies, 185,

Adenosine receptors, 153, 162, 165, 166,

186, 189

168, 170, 171, 178, 181-185, 187

-, -, termodynamic analysis, 185, 186

-, A1 and A2a, 153, 165-178, 181

-, -, Van't Hoff plot, 185, 186

-, -, selective ligands, 181

-, in v i v o

-, A2a molecular model, 159-163

-, -, heart rate, 187, 190

studies, 186, 187, 189, 190

-, -, N6/C8 hypothesis, 161, 163

-, -, mean arterial pressure, 181, 187

-, A2b, 153, 165, 181

-,-, pharmacokinetics and

-, A3, 153-158, 165, 181 -, -, binding sites, 157, 158 -,-, selective agonists, 153, 158, 181 -, agonists, 153, 154, 166, 168, 170, 181, 182, 184

pharmacodynamics, 189, 190 Adenylyl cyclase, 156 -, inhibition, 36, 45 ADP, 347 cADP-ribose, 4

-, antagonists, 184, 185

ADP~3S, 338, 346

-,-, binding site models, 185

ADp~35S, binding to P2v-purinoceptors,

-, classes, 181

340

-, classification, 165

Adrenergic receptors classes, 122

-, cloned, 153, 165

ot-Adrenergic receptors see

-, molecular modelling, 158-163, 185

cx-Adrenoceptors

394 ~-Adrenoceptors, 321, 361,362 -, antagonists, 322 cq-Adrenoceptors, 56, 121, 135-150, 323, 362-364, 366, 367

-, involvement in prostate BPH, 150 C~e-Adrenoceptors, 353-357, 361,362, 364, 366-368, 372 13-Adrenergic receptors, 11-14 -, desensitizing mechanism, 13, 14

-, agonist, 362 -, antagonists, 135-137

-, phosphorylation, 13, 14, 20 ~-Adrenergic receptor kinase, 11-14

-, -, activity on rat aorta, rabbit aorta and

5, 6-ADTN and derivatives, 68, 69, 74,

-, agonists pharmacology, 129, 130

urethra, human prostate, 146-149 -,-, binding affinity, 138, 139 -, -, binding data and functional results correlation, 146-149 -, -, in vitro and in vivo

selectivity,

146-149 -, -, lower urinary tract selectivity, 135, 149 -, -, organ selectivity, 146-149

77, 82 6, 7-ADTN and derivatives, 68, 69, 74, 82 AF-DX 116, 27, 52 Agmantine, 354, 355, 372 AH l l l l 0 A , 138, 143 AUosteric modulators, 247 N-Allyl-normetazocine (SKF10,047), 242 Alzheimer disease, 51-53, 56, 57, 193, 264, 357

-, -, structures, 140-141

Alzheimer therapeutics, 28

-, ligand interaction modelling and

Amino acid conjugates, 308

QSARs, 144-146 -, poorly selective antagonists, 137, 138 -, signalling, 127, 128 -, subtypes, 121, 122, 135, 136, 323 -,-, cloning, 123, 124 -, -, coupling to different Ca 2§ pools, 128, 129 -,-, signal transduction, 121-132 -, -, selective antagonists, 123, 135, 139, 142-144, 149, 150 -, -, tissues expression, 122, 123 ~IA- and ~lB-Adrenoceptors, pharmacological characterization, 123 ~lL-Adrenoceptor subtype, 124, 136, 146 -, dependent tissue, 149

2- [3-Aminophenoxy]methylimidazoline (AMPI), 355 3-Aminopropyl-phosphinic acids, 254, 265 a-Amino-3-hydroxy-5-methyl-4isoxazole propionic acid (AMPA), 243, 244, 247-249 D-2-Amino-5-phosphonopentanoate (AP5), 242 D-a-Amino-adipate, ~,-glutamyl-aminomethyl-sulphonate (GAMS), 241,243, 247, 249 "~,-Aminobutyric acid (GABA), 253,254, 261,263, 266 Amlodipine, 219-221,224 AMPA analogues, 244 AMPA receptors, 243, 244, 246, 249, 258

395 -, agonists, 243 -, aUosteric modulators, 244 -, antagonists, 243,249, 258 -, molecular biology, 244

Aza-noradamantane, 104-107, 114, 115, 117, 118 2- [3-Azidophenoxy]-methylimidazoline (AZIPI), 353

-, prefemng state, 249 Amphipatic o~-helix, 36

Baclofen, 253

Amyloid precursor protein (APPs), 52

Bacteriorhodopsin, 34, 206

Amyloid l-protein, 52

Bay x9227, 235, 236

Antazoline, 356

Benazoline, 366, 367, 372

Antiarrhythmic, 153

Benextramine, 323

Antiasthmatic, 153

-, binding affinity, 326

Antibodies, 1

-, binding site, 325

Anticonvulsant, 236, 238, 239

-, irreversible antagonism, 323

Antidepressant, 153, 255

-, structure, 324

Antidiabetic, 153 Antihypertensive drugs, 355, 355 Antiparkinson, 76, 153

Benign prostatic hypertrophy (BPH), 122, 147, 150 8-(Benzamido)naphtalene-1, 3, 5,-

Antipsychotic agents, 153

trisulfonic acid, symmetrical 3'-urea

Antisense oligonucleotides, 92, 93

(NF023), 338, 340-342

Antisense studies, 89, 92, 93

-, antagonistic properties, 341,342

Anxiety, 267, 375, 377, 386 -, models, 384, 385

-, selective P2x antagonist, 342 1,5-Benzodiazepines, 375,377, 378, 381-

Anxiolytic activity, 375, 381,385

383, 385

Apomorphine, 67, 69, 81, 82

-, affinities at CCK-A and CCK-B

Aporphines, 67, 82

receptors, 382, 383

Apparent efficacy, 55

-, CCK-B antagonists, 377

Aprikalim, 228, 229

-,-, general structure, 378

AQ-RA741,329, 330

-,-, parameters for affinity and

-, structure, 331

selectivity, 381-383

Arecaidine propargyl ester, 55, 57 ~-Arrestins, 13, 14

-,-, quantitative structure-activity study,

ATP, 153, 338, 344, 347

-,-, SAR, 375-386

Attention-deficit disorders, 193 Aza-adamantane, 102, 103, 105, 106 -, scaffold bulk optimization, 103-107

382 -,-, synthesis, 378-381 2,3-Benzodiazepine, 244, 247, 248

396 2-( 2-B enzofuranyl )imidazoline (2-B FI), 353-355

Cardiovascular disease, 271 Carmoxirole, 71

(+)-Benzomorphans, 314

CCB, 300

Benzomorphan derivatives, 314

CCPA, 166, 167, 169, 170

7-Benzylidenenaltrexone (BNTX), 308, 309

-, in v i t r o -, in v i v o

studies, 166, 169, 170 studies, 167, 170

Bicuculline, 258

Celikalim, 230

Biotechnology-oriented therapy, 1

Cellular calcium regulation, 220

Blood-brain barrier, 254, 265, 266

Cerebroprotective agents, 153

Blood coagulation cascade, 271

CGS 21680, 154, 155

BMS- 180448, 231

Channel binding sites, 217

BMY 7378, 123, 124, 126, 139, 143

Channel blockers, 247

Bradycardia, supraventricular, 51, 52

Chiral antagonists, 52

BRL 55834, 232

Chiral auxiliary, 381

Bromocriptine, 67, 70, 71

Chloroethylclonidine (CEC), 123, 124,

Burimamide, 195, 196 BWA522, 156, 157

126, 135, 136, 146, 323 2-Chloro-N6-(3-iodobenzyl)-adenosine -5'-N-methyluronamide, 156

Ca2§

CHO-cells, 53

-, cellular roles, 220

CHO-hml ceils, 55

-, fluxes, 4 -, influx, 121, 127, 128

CHO-K1 cells, 55

-, intracellular levels, 220

CCK-4, 376, 377

-, intracellular release, 121,128

CCK-8S, 375-377

-, pools, 128, 129

CCK receptors, 376

Ca 2§ channels, 4, 215, 220 -, L-type voltage-gated, 219, 220 Ca 2§ channel antagonists, 217, 220-222, 233 -, classification, 221 -, therapeutic profile, 220 -, vascular: cardiac selectivity, 220, 222, 224

Cholecystokinin (CCK), 375, 376

-, agonists and antagonists, 376, 377 -, as therapeutic targets, 377 -, CCK-A and CCK-B subtypes, 375377 -, -, structural homology, 376 -, classification, 376, 377 CCK-A receptors, 375-377 -, agonist, 376

Cabergoline, 71

-, antagonist, 376, 384

Carbachol, 57

-, binding assays, 382-384

397 -, structure, 377 -, tissues location, 376, 377 CCK-B receptors, 375-377, 385 -, antagonists, 375-377, 383-385 -, -, as potential anxiolytics, 375 -, binding assays, 382-384 -, structure, 377

Cyclazocine, 242 (+)-Cyclazosin, 138, 143 N6-Cyclohexyladenosine, 154, 155 N6-Cyclopentyladenosine (CPA), 181, 182, 184, 187-190 -, deoxy derivatives, 187-190 Cyclothiazide, 244, 246-248

-, tissues location, 376, 377 CI-977, 294

4-DAMP, 27, 329

-, antinociceptive activity, 296

3D-database search, new methods, 75

-, opioid activity, 295

1-DeazaADO, 166

-, safety, 294

1-DeazaCCPA, 166

Cirazoline, 354, 355, 362-364, 367

Depotentiation, 263

Cirazoline derivatives, 363

Diazepam, 381,385

-, conformationally restricted, 365

1, 3-Dialkylxanthine-7-ribosides, 158

-, -, binding affinities, 366 -, S AR at ~-adrenoceptors and I

Diazoxide, 228, 229

receptor, 363,364 4-C1-McN-A-343, 55, 57 Clobenpropit, 196, 197

1, 3-Dibutylxanthine-7-riboside (DBXR), 157, 158 1, 3-Dibutylxanthine-7-riboside-5'-Nmethylcarboxamide, 157, 158

Clonidine, 351-354, 356, 357

Diels Alder, asymmetric, 114, 118

CO, as messenger species, 3

Dihydrexidine, 75, 79

Cocaine, 2

1,4-Dihydropyridines, 219-224, 233

Cognition deficit, 267

-, binding site, 220

Cognition enhancing drugs, 153

-, Ca 2§ channel antagonists, 220

Cognitive ability, 51, 52

-, first and second generation, 220-222

Cognitive disorders, 51

-, structures, 221

Comparative Molecular Field Analysis

-, voltage-dependent binding, 223,224

(CoMFA), 368-370 Concanavilin A, 244, 246, 248 Congestive heart failure, 67, 69, 73, 74

Dihydropyridines, potassium channel activators (KCAs), 233,234 3-Dihydroxy-6-nitro-7-sulphamoyl-

CPX, 154, 157

benz(F)-quinoxaline (NBQX), 243,

Cromakalim, 227-233, 236

245, 247, 248

CSC, 154, 157 CY 208-243, 75, 79, 80

4,4 -Dnsothiocyanatostllbene-2,2 disulfonic acid (DIDS), 338,346

398 Diltiazem, 220 Dimethindene, 51

EMD 57283, 230 Enantioselectivity, 52, 58, 64

(R)- and (S)-Dimethindene, 51, 52

Encoded combinatorial chemistry, 5

-, affinities at muscarinic receptors, 56, 57 1,3-Dipropyl-8-cyclopentyLxanthine

Endorphins, 292 Enkephalins, 292 -, role in signal transduction, 309

(DPCPX), 183

Enthalpy stabilisation, 183

Docarpamine, 73, 74

Entropy stabilisation, 183

Dog urethra, 149

Epilepsia, 193

Dopamine, 67-81

Epinephrine, 122, 123, 127, 129-132

-, intravenous infusion, 67, 69, 70, 73,

Ergolines, 67, 70, 71, 75, 79

74 -, prodrugs, 67, 72-74

Eserine, 27

Dopamine receptors, 67 -, agonists, 67-83 -, antagonists, 67, 69, 70, 74, 76, 77 -, chimeric modifications, 82 -, D1 and DE, 67-82 -, D3, 67, 69, 76-78, 82

Ethylenediamines, 314 Etisulergine, 75 Eudismic index, 61 Eudismic ratios, 59 Felodipine, 219-221,224 Fenoldopam, 70, 72, 75, 79

-, D4 and Ds, 67, 76, 77, 82, 83 -, models, 69, 71, 75, 78-82 -, pharmacophores, 71, 78-82 -, site-directed mutagenesis, 80, 81 Dopexamine, 70, 74, 75, 82 Drug resistance, 3 Dual 5-HT4 agonism/5-HT3 antagonism, 100, 109

Fenpipramide, 60, 63

Dynorphin peptides, 292

4-F-PyMcN§ 54, 57

Fibrin, 272, 273, 279 Fluorobenzamides, anticonvulsant, 236-238 p-Fluoro-hexahydro-sila-difenidol (p-F-HHSiD), 52, 54, 329 4-(4-Fluorophenylcarbamoyloxy)-2butynylpyrrolidine (4-F-PyMcN), 51, 54 (S)-4-(4-Fluorophenylcarbamoyloxy)-

Ecto-ATPase, 339, 342, 347, 348 Ectonucleotidases, 338, 339

1-methyl-2-butynylpyrrolidine, (S)-4-F-MePyMcN, 51, 55

EKC, 292, 295

4-F-MePyMcN, 54, 55

ELISA, 31, 34

4-F-MePyMcN+, 54, 55

Emakalim, 230 EMD 23348, 71

(S)-4-F-MePyMcN+, 55

399 N-Formylmethionyl peptide receptor (FPR), 11, 15-22 Forskolin, 233

-, intracellular trafficking, 34 -, ligand binding sites localisation, 206, 210

Fosopamine, 73

-, mechanisms regulating, 11-22

FPL-63012, 75

-, membrane insertion, 34

FR 119748, 230

-, three-dimensional models, 206-208

G proteins, 122, 182, 205

-, three-dimensional structure, 205

-,-, qualitative/quantitative value, 212 -, activation, 43, 45

-, transmembrane domains, 31

-, binding surface, 36

G protein-coupled receptor kinases, 21

-, coupling domain, 206

G protein-coupled receptor ligands,

-, coupling selectivity, 36 -, Gi/o class, 36, 43 -, Gq activation, 39

binding mode, 211, 213 GABAA receptors, 253, 258, 261-264 -, antagonist, 258

-, Gq class, 122, 127

GABAB autoreceptors, 253,254, 261,263

-, Gq/11 family, 36, 37, 42

GABAB receptors, 253-255, 261-267

-, subtypes, 130, 131

-, agonists, 254, 265

-, subunits, 205

-, -, structures, 254

-, tx-subunits, 43

-, antagonists, 253,255-257, 259-261,

G protein-coupled receptors (GPCRs), 11,

263, 265-267

31-47, 80, 121, 122, 135, 136, 153,

-,-, structures, 254, 265

159, 182, 183, 205-213, 273, 283,

-, -, therapeutic potential, 255, 267

291,306, 321

-, modulators, 253, 254

-, activation equilibria, 212

-, partial agonist, 254

-, activation site, 208

-, postsynaptic, 253, 255, 256, 258, 263

-, arrangement of transmembrane

-, presynaptic, 253, 255, 256, 266

helices, 31 -, assembly, 33, 34 -, autonomous folding domains, 33, 34

-,-, subtypes, 255, 266 Gain-of-function mutagenesis studies, 43, 45 Gastric smooth muscle, 281

-, Baldwin projection, 33

Gastrin, 377

-, folding, 31, 34

Gastrin receptor, 376, 384

-, folding defect, 31

Gastrointestinal spastic disorders, 52, 53

-, helical wheel model, 33

Gene therapy, 1

-, helix/helix packing, 33

Genetically engineered animals, 1

400 Gi/o-coupled receptors, 43

-, analogues, 58-62

Glandular hypersecretion, 52, 53

-,-, carbon/silicon exchange, 58

Glibenclamide, 228, 229, 231

Hexahydro-sila-difenidol

Gludopa, 73

(HHSiD), 52, 58, 329

Glutamate, 241,246

-, analogues, 58, 59, 61

Glutamate diethyl ester (GDEE), 241,243,

-,-, (R/S)-enantiomers, 58, 59, 61

247, 249

Hexamethonium, 54

Glutamate receptor subtypes, 241

Himbacine, 52

GR-113808, 111, 113, 114

Hirudin, 274, 275, 278, 279

GTP shift, 182, 189, 190

-,/~-thrombin complex, 274

GV150013, 375, 383-386

-, bifunctional inhibitor, 275

-, binding and functional assays, 383, 384 -, potent and selective CCK-B antagonist, 383 Guinea pig

r-Hirudin, 278 -, antithrombotic effects, 279 Histamine, 57, 193, 194 Histamine H1 receptors, 52 Histamine H3 receptors, 193

-, brain, 195

-, agonists, 193, 195

-, cortical membranes, 381

-, -, structures, 194

-, gastric longitudinal muscle, 281

-, antagonists, 193, 195, 196, 198, 199

-, ileum, 53-57, 59-62, 292, 332

-,-, histamine homologues, 199

-, intestine, 195

-, -, structures, 197

-, jejunum, 199, 201

-, ligands, 193

-, left atrium, 53-55, 59-62, 332

-, pharmacological tool, 194

-, taenia coli, 341,346, 347

-, presence in tissues, 196

-, trachea, 53, 56, 57

-, radioligands development, 193

GYKI 52466, 244, 245, 247, 248

-, selective agonists, 194, 195 -, subtypes, 195, 197, 202

Hamster hovary cells, 330 Hashish, 2

-,-, H3A and H3B, 195, 197 5-HT receptors

Hemostasis, 271,273

-, functions, 89

HENECA, 168-171,173

-, -, study by antisense and transgenic

-, in vitro and in vivo studies, 168-170

approaches, 92, 93

Heparin, 273, 279

-,-, study by selective ligands, 89-92

Hexahydro-difenidol (HHD), 52, 58

-, structural relationships and

-, (R/S)-enantiomers, 58

intracellular signaling, 85-89

401 -, subtypes, 85, 86

-, Ii-type, 351-353

-,-, 5-HT1, 86 -,-, 5-HT 2

and 5-HT3, 87

- , - , 5 - H T 4 and

-, density, 357

5-HTs, 88

-, I2-type, 351-353 -, -, localization, 351,352

-,-, 5-HT6 and 5-HTT, 88, 89

-, ligands, 353-355

-,-, selective ligands, 89-92

-, -, structures, 354, 356

5-HT4 receptor -, agonism, 99, 100, 102, 103, 105108, 111, 112, 118

Imidazoline receptors, 351-357, 361 -, coupling mechanism, 353 -, distribution, 361

-, -, requirement, 112

-, endogenous ligand for, 355, 372

-, agonists and antagonists

-, functions, 355-357, 361

design and synthesis, 99-118 -, antagonists, 109-114, 118 -, cloning, 88

-, I1 and 12, 352, 355, 361 -, in hypotensive effect mechanism, 351, 355

-, coupling to adenylate cyclase, 88

-, pancreatic, 357

-, partial agonist, 113

-, photoaffinity ligand, 353

-, selective agonist, 108, 109 5-HT4 agonism/5-HT3 antagonism, dual action, 100, 109 Human gene therapy, 1 Human prostate, 146-148 Hypertension, 67, 69, 70, 73, 74

-, subtypes, 361 Imidazoline/c~z-receptors, -, co-localization, 353 -, ligands selectivity, 354, 356 Imidazoline receptor ligands, 361-372 -, conformational analysis, 369, 370

Hypotensive agents, 153

-, conformationally restricted, 365, 366

Iberotoxin, 235

-, 3-D QSAR study, 368-372 -, I/~1 selectivity, 363, 366

IB-MECA, 155, 156, 158

-, I/c~e selectivity, 364, 366, 368, 372

Ibopamine, 73, 74

-, radioligand binding studies, 363, 364,

Ibotenic acid, 2 Idazoxan, 351-357 -, antidepressant effects, 357 Imetit, 194, 195, 197 -, as H3 pharmacological tool, 194

366-368 -, selective, 364, 366-368, 372 -, structures, 362 Immepip, 195, 198 Immunocytochemical studies, 31, 34

Imidazoline binding proteins, 352

Impentamine (VUF4702), 198-202

Imidazoline binding sites, 351-353,355,

Impromidine, 195, 196

357, 372

Inducible vectors, 121, 131

402 Inhibitory postsynaptic potential, 253, 258, 259, 261 Inositol (1,4,5) trisphosphate (IP3) formation, 121, 122, 127

-, airways-selective, 232, 233 -, ATP-dependent, 217 -, bladder-selective, 233-235 -, cardioprotective, 231

Insulin secretion, 357

-, in CNS disorders, 235,236

Intracellular ionic concentrations, 215

-, structure series, 229, 230

Intrinsic activity, 182, 187, 190, 191

Kainate, 241,245,247-249

Iodophenpropit, 196

Kainate receptors, 243, 244, 245, 249

[125I]Iodophenpropit, 196, 197, 200 -, as first H3 radioligand antagonist, 196 -, binding affinity, 197 -, biphasic displacement, 197, 198

-, molecular biology, 244 -, preferring states, 249 Kappa opioid agonists, 291,292, 297 -, as analgesic drugs, 291

[125I]Iodoproxyfan, 198

-, binding model, 297

Ion channels, 215-224

-, conformational requirement, 294

-, as membrane proteins, 217

-, non-peptides, 291,292

-, as target for drugs, 215, 217

-, pharmacophore model, 297, 298

-, binding sites, 215, 217

-, selective, 294, 299

-, classification, 217

-, structure, 296

-, function, 215, 216

Kappa opioid analgesics, SAR, 291-300

-, general properties, 215-217

Kappa receptor, 291,292, 297

-, ligand and voltage-gated, 215, 217

Kappa selective ligands, 293,297, 300

-, modulated receptor concept, 223

KC-128, 232

-, opening and closing, 215, 217

KC-399, 229, 231

-, regulatory sensors, 217

Kidney, 356

-, selective drugs, 218 -, states, 223

L-364,718 (Devazepide), 376, 377, 384

-, structure, 216, 217

L-365,260, 377, 384 Lead generation and selection, 5

K § channel -, ATP-sensitive (KATP), 227

Lead structures, 4 Learning and memory processes, 53

-, charybdotoxin-sensitive, 232

Learning improving effects, 255

-, large conductance, Ca§

Levcromakalim

(BKCa), 232 -, openers, 227 K § channel activators (KCAs), 227-239

-, blood pressure lowering, 232 -, clinical profile, 227, 228 -, inhibition of seizures, 236

403 Levodopa, 67, 71-73

(+)-Morphinans, 314

Long-term depression, 263 Long-term potentiation, 256, 257, 259

Morphine, 292, 296

LY262691, 377

Mouse maximal electroshock model

LY293558, 243,245,247, 248 LY300164, 244, 247, 248

Motor side-effects, 313 (MEST), 236 Moxonidine, 355, 356 Mpr-P 10-NH2, 283, 284

Maxikdiol, 233

MT3, 329

McN-A-343, 51, 53

Multiple second messanger pathways,

-, analogues, 55

127-129

Membrane potential, 215

Mu receptor, 291,297

Membrane skeleton, 17

Mu-kappa selectivity, 292, 294

Memory, 255, 257

Muscarine, 2, 27

Message-address concept, 303

Muscarinic binding sites, 57

Metabotropic glutamate receptors, 246

Muscarinic ligands, CNS active, 51

Methoctramine, 28, 52, 329, 330, 332, 333

Muscarinic receptors, 27, 31-47, 210, 321,

-, structure, 331

329

N-Methyl-D-aspartate (NMDA), 241,247,

-, agonists activity, 53-55

248 o~,13-Methylene ATP, 338, 341,342,

-, antagonist ligands, 27

344446 [3H]o~,13-Methylene ATP, 339, 341, 344, 345

-, -, affinity profiles, 56-63 -,-, structures, 56, 59-62 -, assembly, 33 -, chimeric m2/m3 receptors, 36, 45

[3H]Na-Methylhistamine, 195, 197 (R)-~-Methylhistamine, 193, 195, 202

-, cloning, 28

[3H] (R)-o~-Methylhistamine, 195

-, gene products, 52 -, hybrid m2/m5 receptors, 31

[3H]-N-Methylscopolamine, 53

-, four-binding-site model, 52, 63, 64

2-Methylthio ATP, 338, 342, 344, 346, 347

-, M1,M2, M3, 28, 51-64

5-Methylurapidil, 123, 124, 135~ 138, 145,

-, -, selective, 51, 55

147, 323 Minoxidil sulphate, 228,229 Molecular electrostatic potential, 78, 79 Molecular modelling studies, 75, 79, 80, 185, 206-208, 296, 297

-, M1 agonists, 53-55, 329 -, M2 antagonists, 51-53, 56, 329 -, -, selective, 57, 329-334 -, M2 autoreceptors, 51, 53 -, M3 antagonists, 53, 58, 329 -, M4, 51-53, 329, 330

404 -, ml, acethylcoline binding, 210

-, ribose moiety interaction, 162

-, ml, selective agonist, 28

Neuropeptides binding mode, 210, 211

-, m l-m3, ligand interaction

Neuropepfides receptor, 210

differentiation, 210

Neuroprotecfive activity, 314

-, ml-m5 molecular entities, 28

Neuroprotective agents, 291

-, rn2, 31, 36, 43

Neurotransmitter receptors, 322

-, m3, 33, 36, 43

Neurotransmitters binding sites, 208-211

-,m5, 31-33

NG 108-15 cell, 332

-, receptor fragments, 34

Nicorandil, 228, 229

-, specificity, 56

Nicotine, 2

-, split receptors, 33, 34

Nifedipine, 218-221,224

-, structure-function analysis, 31-47

Niguldipine, 233, 234

-, subtypes, 28, 51, 52, 329

(+)-Niguldipine, 123, 124, 126, 135, 138,

-,-, pharmacological identification, 334 -, -, selective agonists and antagonists, 51-64, 329

141, 142, 323 Nimodipine, 218-221,224 NIP 121, 230

Muscimol, 2

Nitric oxide, 3

Mutagenesis, 28, 158, 159 Mutant ~5opioid receptor, 306

NMDA receptors, 241,242, 258

Myocardial infarction, 271

-, antagonists, 241-243 -,-, therapeutic potential, 242 -, blocker, 258

Na § channel antagonists, 217

-, subunits cloning, 242

Na § shift, 183

-, voltage-dependent property, 242

Naltriben (NTB), 306-309, 311

[3H]NMS binding site, 33

Naltrindole (NTI), 303-310

Non-NMDA receptors, 243, 244, 247, 248

-, amino acid coniugate, 308, 309 Naphazoline, 367, 368 -, and isonaphazoline derivatives, 367, 368 -, -, SAR at cz2- and I-receptors, 368 NB-OK 1 cells, 53, 57 NECA, 156, 161-163, 168 -, alkenyl derivatives, 171-173, 176

-, antagonists, 247 -, calcium permeability, 245 -, subunits, 244 Non-peptide kappa opioid -, agonists, 291,296, 297 -, analgesics, 291-300 Norepinephrine, 122-124, 127, 129-132 NS-004, 235

-, alkynyl derivatives, 171-178 -, N6-benzyl, 156

(S)-5-OH-DPAT, 77, 80

405 (R)-7-OH-DPAT, 77, 80 Opioids, 292 Opioid message, 304 Opioid peptides, endogenous, 292 -, binding to ~5-receptor sites, 309 Opioid receptors, 291,303 -, peptide and non-peptide agonists and antagonists, 303 -, subtypes, 291,303 ~i Opioid receptors, 303-311 -, agonists and antagonists recognition sites, 304-307, 309 -, agonists binding data, 306

-, selective antagonists, 337, 341-343, 347 Pev-purinoceptors, 337, 338 -, SAR at, 340, 341 P5, 281-283 P5-NH2, 281-284 P7, 281-283 P7-NH2, 281,282 P14, 280, 283 Paired-pulse depression, 261-263 Palytoxin, 6 Panic, 375, 386 Parkinson disease, 67, 69, 72, 75

-, antagonists binding data, 306

Passe-partout concept, 321

-, ligands interaction, 306, 307

PD117302, 292

-, model, 309, 310

PD- 140548, 377

-, mutant and wild-type, 306, 307

PD143188, 78

-, selective non-peptide ligands, 303-311

PD148282, 297

-, selectivity for, 304-306

PD-151832, 28, 29

-, subtypes, 307-309 - , - , ~1 and ~52selective agonists, 307

PD198907, 77

-, -, 51 and 52 selective antagonists, 307,

Peptide antagonists, 283

308 Opioid selective ligands design, 303 Opium, 291 Oxymorphindole (OMI), 304-306

(+)-Pentazocine, 314 Peptidomimetics, 292 Pergolide, 71, 75 Pfeiffer's rule, 59 Phaclofen, 253 Pharmacophore identification, 75, 78-80

Pl-purinoceptors, 181

Phentolamine, 124, 138, 139

P2-purinoceptors, 338

Phosducin, 15

-, selective antagonists, 337-348 -, subtypes, 337, 338 P2u-purinoceptors, 337, 338, 342, 347 P2x-purinoceptors, 337, 338 -, SAR at, 340, 343

Phosphatidylinositol (4,5) bisphosphate (PI) hydrolysis, 127, 128 Phosphoinositide (PI) hydrolysis, 34, 36, 43, 45 Phosphoinositide (PI) turnover, 51, 53, 55 Phospholipase A2 (PLA2), 121, 127

406 Phospholipase C (PLC), 122, 127-130 -, ~l-adrenoceptors coupling, 130, 131

Preclamol, 69, 77

PHPNECA, 173

Procyclidine, 58

Primed-burst stimulation, 259, 263, 267

-, platelet aggregation inhibitor, 178

Prodrugs, 67, 72-74

-, R- and S-diastereoisomer activities,

Prolactin, 71, 79 2-Propylthio-D-I],7-difluromethylene

178 Pilocarpine, 27

ATP (FPL 66096), 338

Pinacidil, 228, 229

Protein antithrombotics, 274

Pirenzepine, 27, 28, 52, 54, 329, 334

Psychotic disorders, 67, 69

Piribedil, 67, 68, 78

Pyridoxal-5-phosphate, 342-344

Platelets -, aggregation, 165, 166, 168-171, 173, 175, 177, 178, 271,272, 284 -, -, ADP-induced, 347 -, -, antagonist, 284 -,-, inhibitor, 178, 283 -, -, P2T-purinoceptors, 347

-, analogues, 338, 342-348 Pyridoxalphosphate-6-azophenyl-2',4'disulfonic acid (PPADS), 339, 344348 Pyridoxalphosphate-6-azophenyl-2',5'disulfonic acid (iso-PPADS), 348 Pyrrolizidines, 106-108, 110, 111, 118

PLC activity, 43, 45 PLCI3, 36

Quinagolide, 75

Polyamines, 315, 321,322

Quinpirole, 71

-, as pass-partout, 321,322, 334 -, as sigma receptor modulators, 319 Polymethylene tetraamines, 322, 329 Positron Emission Tomography (PET) -, imaging, 51, 56

Rabbit -, aorta, 146, 147, 149, 363 -, blood vessels, 345 -, kidney membranes, 363, 364, 366, 368

-, ligands and studies, 57 Postsynaptic GABAB receptors, 253, 255, 256, 258,263

-, urethra, 146-149 -, vas deferens, 53-55, 59-62, 341,344,

Pramipexole, 77 Prazosin, 123, 136-138, 323-325 -, binding and functional affinities, 138, 147, 326 -, binding site, 325

345 Racemic amines resolution, 381 Radioligand binding studies, 182, 183 Rat -, aorta, 146, 147, 229, 281

-, competitive antagonism, 323

-, bladder, 341,344

-, structure, 140, 324

-, brain, 198, 200

407 -, cerebral cortex, 195-198 200, 364, 366, 368

RS-17053, 123 RS-45496, 75

-, duodenum, 53, 56, 57, 344 -, gastric smooth muscle, 281

(+)-S 14297, 77

-, heart, 53, 56, 57, 332

Saclofen, 253

-, liver, 123, 136, 326, 327

SC-52491, 104, 105, 107-109, 114, 117

-, mesentery, 341,344

-, scalable synthesis, 114-117

-, pancreas, 56, 57

SC-53116, 108-110, 112, 118

-, pancreatic membranes, 381

SC-53606, 112-114, 118

-, spleen, 123

SC-55822, 110, 113, 114

-, striatum, 56, 57

SC-56184, 111, 113, 114, 118

-, submaxillary gland, 326, 327, 332

SCA40, 232, 233

-, vascular preparation, 281

Scaffold, 103, 104, 110

-, vas deferens, 326, 327, 364

Serotonin, 85, 89

Rec 15/2739, 150 Receptors

Serotonin receptors, -, relationships and roles, 85-93

-, homology, 321

Si/Ge bioisosterism, 52, 60

-, phosphorylation, 20

Sigma enigma, 314, 320

-, recognition, 321,322

Sigma receptors, 313, 315, 319, 320

Receptor-class desensitization, 21 Receptor/G protein,

-, and neuroleptic motor side-effects, 313, 320

-, contact site, 43

-, antagonists, 313, 319, 320

-, coupling, 34, 36

-, biological activities, 314

-, interface, 47

-, characteristics, 313

Recognition process, 210

-, in psychiatric effects, 313

Renal protective, 153

-, models, 319

Respiratory tract disorders, 52, 53

-, subtypes, 314, 319

Rhodopsin, 11, 159 Rilmenidine, 353,355-357 -, biological effects, 355-357 Ro 31-6930, 230

Sigma receptor ligands, 313 -, as neuroprotective agents, 313 -, biological activities, 315 -, classes, 316-319

Ropirinole, 69

-, from U50,488, 320

Roxindole, 71

-, sigma- 1 and sigma-2 selective, 314

RP 49356, 228, 229

Signal transduction, 11,309

RP 66471, 229, 231

Sila-procyclidin, 58

408 SIOM, 305, 306, 308, 309

Terazosin, 138, 147

Site-directed mutagenesis, 31, 37, 80, 153,

Ternary complex model, 182

208, 210, 245

Tetanic stimulations, 256-258

SK&F 38393, 70

Tetraamines, 321,322, 329, 330, 334

SK&F 83959, 76

Tetraamine disulfides, 322, 323

SK&F 89124, 69

-, hybrid, 324, 325

Sleeping disorders, 193

Tetrodotoxin, 54

Smooth muscle preparations, 281

Theophylline, 185, 186

SNAP 5089, 123, 126, 138, 142, 147-149

Theophylline-7-riboside, 184-187

(+)-and (-)-SPAL, 138, 139

Thioperamide, 193, 195, 196, 202

Spastic disorders, 52, 53

Thrombin, 271-274, 278-280, 283

Spinal cord, 241,246-249

-, aggregating agent, 273

Spinal glutamate receptors, 241-249

-, anticoagulant enzyme, 274

Spiperone, 138, 143

-, as therapeutic target, 272, 273

Spiroether derivatives, 294, 295

-, deactivating mechanisms, 273

Spirotramine, 330

-, role, 272, 273

-, binding profile, 332-334 -, structure, 331 State-dependent interactions, 222 Stereoselective interaction, 58, 63 Stereoselectivity, 52, 58, 60, 63 -, ratios, 56, 59, 63

Thrombin inhibitors, 271-284 -, bifunctional, 275-278 -,-, antithrombotic effect, 279 -, -, lability, 279 Thrombin receptor, 271,273 -, activation, 280, 281,283

Stroke, 154, 271,273

-, agonists, 271,280-283

N6-Substituted adenosines

-, antagonists, 271,273,280, 283,284

deoxy derivatives, 187-190

-, function, 281

-, in v i t r o

studies, 189

-, mechanisms, 280

-, in v i v o

studies, 190

-, subtypes, 282

-, synthesis, 188 Suramin, 338-342

Thrombin receptor-activating peptides (TRAPs), SAR, 280-283

-, analogues, 338-342

-, potency series, 282

-,-, structure, 339

-, vascular/platelet selectivity, 283

-, antagonistic properties, 341,342

Thromboembolic disorders, 271

-, as ecto-ATPase inhibitor, 342

Thrombosis, 271,273, 278 Thrombus formation, 271,279

Talipexole, 77

Tissue therapy, 1

409 TNF-o~ release, 181

Voltage-gated Ca 2§ channels, 220

Tolazoline, 367, 368

VR-NH2, 283, 284

Tracizoline, 366, 367, 372

VUF 4701, 199

Transducin, 47 Transgenic animals, 89, 92, 93

WB4101, 123, 124, 135, 136, 138, 323

Transgenic species, 1 Transmembrane domains, 159, 206, 208

XAC, 154, 157, 161, 163

Transmembrane helical regions, 161

Xanomeline, 28, 29

Trihexylphenidyl (THP), 52, 58, 60 -, (R/S)-enantiomers, 52, 58

Xanthines, 154, 156-158, 185, 186 Xanthine-7-ribosides, 158, 185, 186

-, analogues, 52, 58 -,-, Si and Ge derivatives, 52, 58 ,

,

,

(R/S)-enantiomers, 58, 60, 62

Y 27152, 230 YFLLRNP, 283

Tripitramine, 330 -, antagonist affinities, 332

Z 1046, 74, 82

-, selectivity profile, 332-334

ZD-6169, 234

-, structure, 331

ZM241385, 154, 157

U-50488, 292, 293, 295 -, based, kappa agonists, 299 -, not based, kappa ligands, 300 -, selective kappa agonist, 294 -, series, SAR, 292, 293 U-62066 (spiradoline), 294-296 (+)-UH-232, 77 Ulcer treatment, 28 Urogenital spastic disorders, 52, 53 Van't Hoff plots, 185, 186 Vascular: cardiac selectivity, 220, 222, 224 Vascular preparation, 281 Vasopressin, 210, 211 -,/oxytocin receptor, three-dimensional model, 207, 210, 211 Verapamil, 219, 220

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