Progress in Medicinal Chemistry, Volume 38

Progress in Medicinal Chemistry 38

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Progress in Medicinal Chemistry 38

Editors: F.D. KING,

B.SC., D.PHIL., C C H E M . , F.R.S.C

SmithKIine Beecham Pharmaceuticals New Frontiers Science Park (North) Third Avenue Harlow, Essex CM19 5 A W United Kingdom and

Consultant in Medicinal Chemistry P.O. Box 151 Royston SG8 5 Y Q United Kingdom

2001

ELSEVIER AMSTERDAM-LAUSANNE-NEW YORK*OXFORD*SHANNON*SINGAPORE~TOKYO

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 21 1, 1000 AE Amsterdam, The Netherlands

0 2001

Elsevier Science B.V. All rights reserved.

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@ T h e paper used in this publication meets the requirements of ANSI/NISO 239.48-1992 (Permanence of Paper). Printed in The Netherlands

V

Preface The interplay of chemistry, biochemistry, pharmacology and related disciplines is well exemplified in the seven chapters of this volume which describe exciting advances in new drug molecules and in the application of new technologies to drug discovery and development. Chapter 1 provides a comprehensive account of p38 mitogen-activated protein kinase inhibitors, a new class of anti-inflammatory agents with potential application to rheumatoid arthritis, inflammatory bowel disease and psoriasis, as well as other disorders with an inflammatory component. The extent to which modern techniques have addressed the problems of the first generation compounds, that of selectivity, in vivo activity and interaction with cytochrome P450, is also discussed. A timely review of ligands for the adenosine A3 receptor in Chapter 2 focuses on potent and selective agonists and partial agonists derived from adenosine, but demonstrates the wide diversity in the range of structural classes displaying high affinity as A3 antagonists. The binding of antagonists is discussed in terms of the ligand-receptor models that have been postulated.

The classification of purinoreceptors of the P2 family, their biological properties, selective ligands, SAR and clinical applications is extensively reviewed in Chapter 3. Drug targets include both ligand gated ion channels and GPCRs for which there are already very promising compounds in development. There is an intriguing account in Chapter 4 describing the use of radioligands for brain 5-HT1A receptors in clinical research programmes involving PET (positron emission tomography) or SPET (single photon emission tomography) to investigate neuropsychiatric disorders and drug action. One of the more significant advances of recent years in the treatment of asthma has been the introduction into medicine of LTD4 antagonists. A fascinating account of the discovery process leading to montelukast, one of the first generation of these agents, is described in Chapter 5.

vi

PREFACE

In marked contrast to histamine HI and H2-receptor ligands none of the ligands for H3 receptors have so far been registered for therapeutic use despite it being known for nearly two decades that H3 is a presynaptic autoreceptor for histaminergic neurones. Chapter 6 reviews progress that has been achieved in this area and speculates on the more likely therapies that may be anticipated from this research. Isothermal titration calorimetry is a general technique for measuring the generation or absorption of heat when substances bind. The collection and interpretation of this data for a ligand-protein interaction can provide insight into the 3-D structure of the molecular complex so it is of value in drug design. The scope and limitations of the methodology are comprehensively reviewed in Chapter 7. We thank all the authors of this volume for committing so much of their time and effort to evaluating the extensive literature which is required to compile these articles. We also thank the staff of the publishers for their continuing support and encouragement to the series. July 2000

F.D. King A.W. Oxford

vii

Contents Preface List of Contributors

1 p38 MAP Kinase: Molecular Target for the Inhibition of Pro-inflammatory Cytokines Jerry L. Adams, Alison M. Badger, Sanjay Kumar and John C. Lee 2 The Adenosine A3 Receptor and its Ligands Jacqueline E. Van Muijlwijk-Koezen, Henk Timmerman and Adriaan P. Ijzerman

V

ix 1

61

3 The Medicinal Chemistry of the P2 Receptor Family 115 Simon D. Guile, Francis Ince, Anthony H. Ingall, Nicholas D. Kindon, Premji Meghani and Michael P. Mortimore 4 Radioligands for the Study of Brain 5-HTIA Receptors In Vivo Victor W. Pike, Christer Halldin and HBkan V. Wikstrom

189

5 Discovery of Montelukast: a Once-a-Day Oral Antagonist

249

of Leukotriene D4 for the Treatment of Chronic Asthma Robert N. Young

6 The Histamine H3 Receptor and its Ligands Holger Stark, Jean-Michel Arrang, Xavier Ligneau, Monique Garbarg, C. Robin Ganellin, Jean-Charles Schwartz and Walter Schunack

279

7 Isothermal Titration Calorimetry in Drug Discovery Walter H.J. Ward and Geoffrey A. Holdgate

309

Subject Index

317

Author Index (Vols. 1-38)

383

Subject Index (Vols. 1-38)

389

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IX

List of Contributors Jerry L. Adams SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, U.S.A. Jean-Michel Arrang Unite de Neurobiologie et Pharmacologie Moleculaire (U. 109), Centre Paul Broca de I’INSERM, 2 ter rue d’Alesia, 75014 Paris, France Alison M. Badger SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, U.S.A. C. Robin Ganellin Department of Chemistry, Christopher Ingold Laboratories, University College London, 20 Gordon Street, London WClH OAJ, U.K. Monique Garbarg Unite de Neurobiologie et Pharmacologie Moleculaire (U. 109), Centre Paul Broca de I’INSERM, 2 ter rue d’Alksia, 75014 Paris, France Simon D. Guile Department of Medicinal Chemistry, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire, LE11 5RH, U.K. Christer Halldin Karolinska Institutet, Department of Clinical Neuroscience, Psychiatry Section, Karolinska Hospital, S-17176 Stockholm, Sweden Geoffrey A. Holdgate AstraZeneca, R&D Mereside, Alderley Park, Macclesfield, Cheshire, SKlO 4TG, U.K. Adriaan P. Ijzerman Leidenl Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Universiteit Leiden, PO Box 9502, 2300 RA Leiden, The Netherlands

X

LIST OF CONTRIBUTORS

Francis Ince Department of Medicinal Chemistry, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire, LEI 1 5RH, U.K. Anthony H. Ingall Department of Medicinal Chemistry, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire, L E l l 5RH, U.K. Nicholas D. Kindon Department of Medicinal Chemistry, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire, L E l l 5RH, U.K. Sanjay Kumar SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, U.S.A. John C. Lee SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, U.S.A. Xavier Ligneau Laboratoire Bioprojet, 30 rue des Francs-Bourgeois, 75013 Paris, France Premji Meghani Department of Medicinal Chemistry, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire, LEI 1 5RH, U.K. Michael P. Mortimore Department of Medicinal Chemistry, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire, LEI 1 5RH, U.K. Present address: Vertex Pharmaceuticals (Europe) Ltd, 88 Milton Park, Abingdon OX14 4RY, U.K. Jacqueline E. van Muijlwijk-Koezen Leidenl Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

LIST OF CONTRIBUTORS

xi

Victor W. Pike Chemistry and Engineering Group, MRC Cyclotron Unit, Imperial College School of Medicine, Hammersmith Hospital, Ducane Road, London W12 ONN. U.K. Walter Schunack Freie Universitat Berlin, Institut fur Pharmazie, Konigin-Luise-Strasse 2+4, 14195 Berlin, Germany Jean-Charles Schwartz Unite de Neurobiologie et Pharmacologie Moleculaire (U. 109), Centre Paul Broca de I’INSERM, 2 ter rue d’Alksia, 75014 Paris, France Holger Stark Freie Universitat Berlin, Institut fur Pharmazie, Konigin-Luise-Strasse 2+4, 14195 Berlin, Germany Henk Timmerman Leiden/ Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands Walter H.J. Ward AstraZeneca, R&D Mereside, Alderley Park, Macclesfield, Cheshire, SKI0 4TG. U.K. HIkan V. Wikstrom University Centre for Pharmacy, University of Groningen, A. Deusinglaan 1, NL-9713 AV Groningen, The Netherlands Robert N. Young Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe Claire - Dorval, Quebec, Canada H9R 4P8

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Progress in Medicinal Chemistry - Vol. 38, Edited by F.D. King and A.W. Oxford 0 2001 Elsevier Science B.V. All rights reserved

1 p38 MAP Kinase: Molecular Target for the Inhibition of Pro-inflammat ory Cytokines JERRY L. ADAMS, ALISON M. BADGER, SANJAY KUMAR and JOHN C. LEE SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, P A 19406, U.S.A.

INTRODUCTION

2

BACKGROUND Discovery of p38 kinase Relationship of p38 to the MAP kinases

4

BIOCHEMISTRY p38 Kinase family Upstream activators Downstream substrates Kinase substrates of p38 Transcription factor substrates of p38 Enzymology

6 6 8 9

INHIBITORS O F p38 KINASE Medicinal chemistry preceding the discovery of p38 p38 SAR Pyridinyliniidazoles Non-imidazole 5-membered ring .scaffolds Other heterocyclic core ~tructures New structural classes of p38 inhibitors Structural basis of kinase selectivity Selectivity data X-ray crystallography Mutagenesis PHARMACOLOGY O F p38 INHIBITORS In vitro studies Regulation of protein synthesis

4 5

10

12 13 15

15 17 17 21 22 24 25 26 27 29

30 30

30

2

p38 MAP KINASE

Translational regulu tion of cytokines p38 M A P kinase and apoptosis Pulmonary physiology A ngiogenesis Viral infection Bone biology Cartilage biology Animal models A djuvan t-induced arthritis Collagen-induced arthritis Pulmonary inflammation Stroke Psoriasis Ischaemia/reperfusion Immune function Endotoxin shock

31 32 36 36 37 37 38 40 40 41 42 42 43 43 43 44

PRECLINICAL AND CLINICAL DEVELOPMENT Oral activity P450 inhibition Clinical status

45 45 41 41

CONCLUSIONS AND FUTURE DEVELOPMENTS

48

REFERENCES

49

INTRODUCTION The network of immune and inflammatory responses is comprised of a variety of cell types. Co-ordination of this network occurs through both direct cell-cell contact and by way of intercellular signalling molecules. These signalling molecules (which include lymphokines, cytokines, chemokines and growth factors) regulate the growth, differentiation and function of a variety of target cells. Understanding the structure and function of these molecules has provided new and important insights into the fundamental biology of immunity and inflammation and has led to the identification of new strategies for the development of more effective medicines for the treatment of a variety of autoimmune and inflammatory diseases. The pluripotent pro-inflammatory cytokines, interleukin-1 (IL-1) and tumour necrosis factor alpha (TNFa ) appear to play particularly important roles in disease [l-31. Although these proteins and their cellular receptors are structurally unrelated, they elicit a similar profile of pro-inflammatory responses. Because IL-1 and T N F are produced early in response to

J.L. ADAMS ET AL.

3

pro-inflammatory signals and because of their central role in mediating this response, they have often been termed the master cytokines. The expectation that blockade of cytokine action would be of clinical utility has been realized with the introduction of protein antagonists, such as soluble TNF-receptor, chimeric anti-TNFcc -monoclonal antibody and IL- 1 receptor antagonist (IL-IRA) for the treatment of rheumatoid arthritis and Crohn's disease [4-61. While the success of these therapies has validated the importance of IL-1 and T N F in promoting disease, they also highlight the need for improved therapies that do not suffer from the disadvantages of proteinaceous macromolecules which must be administered parenterally and are inherently more expensive to produce than small molecule drugs. To date, no orally active low molecular weight cytokine receptor antagonist has emerged from clinical trials. However, in the last decade several new strategies to interrupt the synthesis and signalling of these cytokines have emerged. One of the first of these targets to be elucidated is the stress-activated protein kinase, p38.

The following abbreviations are used in this article. AA, adjuvant arthritis; ANF, atrial natriuretic factor; AMPPNP, adenylyl imidodiphosphate; AP-1, activating protein-I; ATF, activating transcription factor; ATP, adenosine triphosphate; BCR, B cell antigen receptor; CHOP, C/ EBP homologous protein; COPD, chronic obstructive pulmonary disorder; COX, cyclooxygenase; CNS, central nervous system; CREB, c-AMP response element binding protein; CSAID, cytokine-suppressive anti-inflammatory drug; CSBP, CSAID binding protein; g-CSF, granulocyte colony-stimulating factor; EGFR, epidermal growth factor receptor; ERK, extracellular regulated kinase; EST, expressed sequence tag; ETS, E26-transformation specific; FSBA, 5'-p-fluorosulfonylbenzoadenosine; GM-CSF, granulocyteimacrophage colonystimulating factor; HSP, heat shock proteins; ICAM, intercellular adhesion molecule; IFN-?, interferon-gamma; IL-I, interleukin-I; ILI-RA, IL-1 receptor antagonist; iNOS, inducible nitric oxide synthetase; JNK, c-Jun N-terminal kinase; 5-LO, 5-lipoxygenase; LPS, lipopolysaccharide; MAPK, mitogen activating protein kinase; MAPKAP K, MAPK activated protein kinase; MAPKK or MKK. MAPK kinase; MAPKKK, MAPKK kinase; MCAO, middle cerebral artery occlusion; MEF, myocyte-enhancer factor; MEK, MAPK kinase or ERK kinase; MLK, mixed lineage kindse; MNK, MAP kinase-interacting kinase; MMP, matrix metalloproteinase; MRI, magnetic resonance imaging; MSK, mitogen- and stress-activated protein kinase; NGF, nerve growth factor; NSAID, non-steroidal antiinflammatory drug; PAK, p2 1-activated kinase; PMN, polymorphonuclear neutrophil; PTH, parathyroid hormone; PRAK, p38 regulated/activated protein kinase; RANTES, regulated upon activation, normal T-cell expressed and secreted; RSK, ribosomal S6 kinase; SAPK, stress activated protein kinase; SAR, structure activity relationship; SRE, serum response element; STAT, signal transducers and activators of transcription; TGF, transforming growth factor; TNF, tumour necrosis factor; UTR, untranslated region; YVAD, Ac-Tyr-Val-Ala-Aspchloromethylketone; ZVAD, Z-Val-Ala-Asp(0Me)-fluoromethylketone.

4

p38 MAP KINASE

BACKGROUND DISCOVERY OF p38 KINASE

In the late 1980s Lee and co-workers at SmithKline and French Laboratories reported that SK&F 86002 (I), one member of a larger class of antiinflammatory imidazothiazoles, inhibited LPS (lipopolysaccharidestimulated IL-1 and TNF) production in human monocytes [7, 81. SK&F 86002 and related pyridinylimidazoles were also shown to inhibit both cyclooxygenase and 5-lipoxygenase. However, an examination of a series of analogues revealed that the effects on arachidonic acid metabolism were not correlated with the inhibition of cytokine synthesis [9]. When tested at its ICso for cytokine inhibition, SK&F 86002 had no effect on general DNA, RNA or protein synthesis [lo, 111. Importantly, (1) also inhibited IL-1 production in a number of different target cells using a variety of stimuli. In addition to IL-1, other cytokines such as TNF, but not g-CSF or IL-1 receptor antagonist were inhibited [8]. Western blot analysis established that the intracellular levels of IL-1 and TNF were significantly reduced in LPS treated human monocytes, but these results were not paralleled by similar changes in the respective mRNA [12]. These studies established that the CSAID (Cytokine-Suppressive Anti-Inflammatory Drug) properties of (1) were not the result of a non-selective toxicity and that the effects were mechanistically distinct from the corticosteroids. F

(1) SK&F 86002

(2) SB 202190

(3) SB 20671 8

A more complete understanding of the mechanism of action for these compounds required identification of the molecular target [ 131. Towards this end, radiophotoaffinity ligands were prepared and used as probes to search for the molecular target in THP. 1 cells, a human monocytic cell line whose LPS-stimulated cytokine profile was inhibited by (1) in a manner identical to that of human monocytes. The first attempts to configure a radioligand binding assay with a pyrroloimidazole analogue (16) (Table 1.I ) , which was a relative weak inhibitor of IL-1 synthesis (ICso= 0.5 pM),

J.L. ADAMS ET AL.

5

failed [13]. Subsequently, radiolabelled SB 202190 (2), a more potent triaryl imidazole (ICSo= 0.050 pM), was prepared and its uptake in THP. 1 cells was found to be time and temperature dependent, saturable and competitive with unlabelled (2), but not with an inactive analogue (12) [13]. These results suggested that a compound-specific binding site was present in THP.1 cell lysates. A binding assay was configured to quantitate the binding of [3H]-SB 202190 to the cytosolic fraction of THP.1 cells. The binding was specific, time dependent, reversible and of high affinity (Kd N 50 nM) [13]. When many structural analogues were examined, a high degree of correlation was established between cytokine biosynthesis inhibition and competition in the binding assay, thus confirming that the cytosolic binding activity was related to cytokine inhibition. However, it was difficult to purify the target protein more than about 20-fold as determined by the binding assay. Therefore, ['251]-SB 206718 (3), a radio-iodinated aryl azide, was synthesized as an irreversible photoaffinity label. Photolysis of this radiolabel in the presence of the partially purified THP. 1 cytosol predominantly labelled a single 43 kDa protein [ 131. Purification of the labelled protein and sequencing of proteolysis fragments identified two unique peptides which permitted the cloning and expression of the target protein. The molecular targets were identified as a pair of closely related novel serinel threonine protein kinases of the mitogen activating protein (MAP) kinase family. These kinases were termed CSAID binding proteins I and 2 (CSBPI, CSBP2) [13]. CSBP2 is the human orthologue of the murine p38a kinase [14].

RELATIONSHIP OF p38 TO THE MAP KINASES

There are at least three families of MAP kinases which differ in the sequence and size of the activation loop: the extracellular regulated kinases (ERKs) have a TEY motif, the c-Jun NH2 terminal kinases or stress activated protein kinases (JNKs or SAP kinases) which have a TPY motif and the p38 family which have a TGY motif. The ERK kinases are stimulated by mitogens and several 7-transmembrane receptor agonists resulting in the sequential activation of ras, raf, MAP kinase kinase 1 or 2 (MKKl or 2) and ERK, which in turn phosphorylate transcription factors such as Elk1 and STATl, thereby stimulating their transcriptional activity [I 51. Both the JNKs and p38 MAP kinases are primarily activated by stress stimuli including UV [ 161, heat [ 171, chemical or osmotic shock [ 14, 181, IL- 1 [ 19, 201, T N F [21] and endotoxin [13].

6

p38 MAP KINASE

BIOCHEMISTRY p38 KINASE FAMILY

The p38 family includes four different isoforms (p38a/Ply/6). In addition, p38a and p38p have several splice variants (CSBPI, CSBP2/p38a, Mxi-2, p38p and p38p2). A complete amino acid alignment of all members of the p38 MAPK family is presented in (Figure 1.1). CSBPl, CSBPZ ( ~ 3 8 and ~ ) Mxi-2

As mentioned earlier, the human p38a enzyme was identified as a target of CSAIDs and the two variants were initially termed CSBPl and 2 [13]. CSBP2 is now widely referred to as p38a and differs from CSBPl in an internal sequence of 25 amino acids. The two isoforms are formed by the differential splicing of two exons that are conserved in both human and mouse [22]. CSBPl and p38a are expressed ubiquitously and there are no known differences between the two isoforms with respect to tissue distribution, activation profile, substrate preference and CSAID binding. A shorter C-terminal truncated form of p38a known as Mxi-2 has also been identified in a yeast two-hybrid screen based on its association with the transcription factor Max [23]. p38g and ~ 3 8 8 2

A search of GenBank EST database resulted in the identification of p38p with over 70% identity to p38a [24]. A highly related but distinct isoform ~~

~~

Figure 1.1. Multiple sequence alignment of p38 MAPK and its various homologues. The alignment was generated using MegAlign program from LASERGENE using the clustul algorithm. Residues identical to p 3 8 ~are hidden and shaded. Boxed residues in p388 represent the 8 amino acids insertion in p388 not found in p38p 2 and other homologues. Amino acids in loop 12 (Asp-168 10 Tyr-182) that are responsible for interaction with the upstream kinase and Thr-180 and Tyr-182, iwo residues that are phosphorylaied by the upstream kinase and ere essential for activation are indicated by a straight line and stars. respectively. Residues (Thr-106 to Leu-108) are essential for sensitivity to pyridinylimidazole compounds are shown by solid circles. Residues (Asn-114 to His-126) are involved in binding and phosphorylation of MAPKAP K2 and K3 are indicated by u dotted line. Amino acids (Arg-149, Asp-150, Lys-152, Arg-189, Leu-195, Asn-196, His-199, Tyr-200, Asn-201) which affects substrate phosphorylation are indicated by solid squares. Amino acid numbering are f r o m p38u.

J.L. ADAMS ET A L .

8

p38 MAP KINASE

of p38p was identified as p38p2 or p38-2 which lacks the 8 amino acid insertion found in p38p [25-271. Between the two variants, p38p2 is now believed to be the major form as p38p is catalytically less active and is difficult to isolate by RT-PCR [25]. p38a and p38p2 are expressed in many different tissues. In monocytes and macrophages p38a is the predominant p38 kinase activity [13, 24, 281. SAPK3 (p38y) and SAPK4 (p386)

SAPK3 and 4 were also identified by an EST database search [25, 29-35]. These enzymes are -63% and 61% identical to p38a, respectively. SAPK3 is expressed predominantly in skeletal muscle whereas SAPK4 is expressed more widely in testes, pancreas, prostate, small intestine and in certain endocrine tissues [25, 351. The expression of SAPK4 is also regulated during development [361. N

UPSTREAM ACTIVATORS

All p38 homologues and splice variants contain a 12 amino acid activation loop (L12 linker) between kinase subdomains VII and VIII that constitutes the activation lip and includes a Thr-Gly-Tyr motif (Figure 1.1). Dual phosphorylation of both Thr-180 and Tyr-182 (p38a numbering) in the TGY motif by a dual specificity upstream kinase is essential for the activation of p38 resulting in >1000-fold increase in specific activity of these enzymes [37]. Dual phosphorylation of p38 and other homologues is effected by MKK6 and under certain conditions by a related enzyme, MKK3 [27]. Recent studies suggest that functional complexes which are formed between MKK3, MKK6 and various isoforms of p38 are determined by the presence of a specific docking site in the N-terminus of MKK3/6 and by the selective recognition of the activation loop of p38 isoforms [38, 401. MKK3 and MKK6 belong to the family of enzymes termed MAPKK (mitogen activating protein kinase kinase) which are in turn activated by MAPKKK (mitogen activating kinase kinase kinase), also known as MAP3K. Several MAP3Ks have been identified that are activated by a wide variety of stimuli including environmental stress, inflammatory cytokines and other factors. MEKK4/MTK1 (MAP or ERK kinase kinase/MAP three kinase-1), ASK1 (apoptosis stimulated kinase) and TAKl (TGF-8-

J.L. ADAMS ET A L .

9

activated kinase) are some of the enzymes that have been identified as the upstream activators of MAP3K. MEKK4/MTKl is believed to be activated by several GADD45-like genes that are induced in response to environmental stimuli, such as UV radiation, and which eventually lead to p38 activation [41]. TNF-stimulated activation of p38m is believed to be mediated via recruitment of TRAF2 (TNF receptor associated factor) and the Fas adaptor protein, Daxx, which results in the activation of ASK1 and subsequently p38 and JNK. Similarly, TAKl has also been shown to activate MKK6 in response to TGF-P and perhaps to some other signals as well. TAKl is also believed to associate with TRAF6 in an IL-1-dependent manner suggesting an involvement of TAKl in IL-1 -mediated p38 activation [42]. Mixed lineage kinase-3 or MLK-3 physically associates with MKK3 and MKK6 and is believed to be involved in activation of p38 by Ste-20-like kinases [43]. Recently, MEKK3 was also reported to directly activate MKK6 and MKK7 [44]. A simplified p38 MAPK pathway with upstream activators and downstream substrates is shown in (Figure 1.2.) Small G proteins of the Rho family have also been shown to activate the p38 pathways. Over expression of constitutively active forms of Cdc42 and Racl in mammalian cells results in activation of p38 via the activation of p21 -activated kinase (PAK). Conversely, the expression of dominant negative Cdc42 and Rac inhibits IL-1-mediated p38 activation [45, 461.

DOWNSTREAM SUBSTRATES

The availability of highly selective inhibitors of p38 MAPK and the intense focus applied to this field in the past few years has greatly enhanced our understanding of the p38 MAPK pathways. Several substrates of p38 have been identified including other kinases (MAPKAP K2/3/5, PRAK, MNKl/2, MSKl /RLPK, and RSK-B), transcription factors (ATF2/6, MEF2A/C, CHOP, Elk1 and Sap-lal) and others (cPLA2, p47phox, etc.). The concept of short peptide sequence docking sites, which are found in substrates and which can dramatically enhance the phosphorylation efficiency and selectivity, has been proposed. A conserved 1 1-residue region containing 5-6 concensus amino acids has been identified for MNKl/2, MSK/2, RSK-B, PRAK and MAPKAP K2/3 [47, 481. Similarly, distinct regions in p38 have been identified that are responsible for binding and phosphorylation of its substrates [38, 491 (Figure 1.I).

p38 MAP KINASE

10

EnvironmentalStress I Inflammatory Cytokines

...................... .............~ ' ............................ & . ' . " ' i

" ' ~ l i ! ~ ~ ~ ~ ~ ~ ~ ~ " ~ , , .

I

Ras

?

TRAF2

Cdc42IRac

1 TAKl

4

PAK

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

MAPKK

Substrates

p38p2

,P38a

ATF1/2/6 CHOP1 MEF2ClA

t

i'

!

+

: :

/

ASK1

i

./ GADD45slGADD153

> i

f {'

1

MEKK4IMTKl ......... ......................

MKKG,

p38ylSAPK3 p38USAPK4

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

Transcriptional regulation

i

i

i

MAPKAP K?/315 MSKl MNKl I i PRAK RSK-B

&

,. ?

i ' i

1

<MKK3

?

!

i

TAB

MAPK

TRAF6

Daxx

Upstream activators

MAPKKK

...................p.. ...........

,

P47 phox cPLA, Stathrnin MBP

........................ , .................+ j

b

Cytokine production Other effects

Figure 1.2. A simpli3edpathways representing thep38 MAPKfamily. A s a result of environment stress and inflammatory cytokines. upstream activators are stimulated which then activate the M A P K cascade involving MAPKKK, MAPKK, and MAPK. The dotted and solid lines indicate indirect or direct interactions, respectively. For simplicity, both MKK3 and MKK6 as well as all p38 family members are shown to phosphorylate all substrates. In reality, M K K 3 and MKK6 dijferentially phosphorylates various p38 M A PK family members which in turn phosphorylate dflerent substrates with varying efjciency. The components of p38 MAPK pathways responsible for cytokine production are italicized and indicated in bold.

Kinase substrates of p38

MAPKAP K2 was the first kinase to be identified as a specific substrate of p38 [17, 501. It is a Ser/Thr kinase in the RSK family which is activated by p38 in response to environmental stress and by ERK in response to growth factors and phorbol esters. Treatment of cells with the p38 inhibitor, SB 203580 (33 in Table 1.I ) , completely abolishes stress-induced activation of MAPKAP K2 [51, 521. Mice engineered to lack MAPKAP K2 do not produce T N F in response to LPS. Production of several other cytokines such as IL-1, IL-6, IFN-y and IL-10 is also partially inhibited [53]. Similarly,

J.L. ADAMS ET AL.

11

MAPKAP K2 of embryonic stem cells from p38a null mice was not activated in response to stress and these cells did not produce IL-6 in response to IL-1 [54]. These data indicate that MAPKAP K2 is essential not only for T N F and IL-I production but also for signalling induced by these cytokines. Two related protein kinases MAPKAP K3/3PK and MAPKAP K5 have also been identified as potential p38 substrates [52, 55, 561. Both MAPKAP K2 and K3 are phosphorylated by p38a and ~ 3 8 8 2but not by p38y /SAPK3 or p386 /SAPK4 [25-27, 331. MAPKAP K2 contains a nuclear localization signal and in resting cells MAPKAP K2 and p38 appear to exist as a complex in the nucleus. Activation of the p38 pathway and subsequent phosphorylation of MAPKAP K2 results in the export of p38-MAPKAP K2 complex to the cytoplasm [57]. Thus MAPKAP K2 is not only a substrate of p38 but it also regulates the cellular localization of p38 and therefore the availability of cytosolic and nuclear substrates of both enzymes. The small heat shock proteins HSP 25 and HSP27, which are involved in cytoskeletal reorganization, appear to be regulated by MAPKAP K2/3 which phosphorylate these proteins on both Ser and Thr residues [58,59]. Recently, activated MAPKAP K2 has also been shown to phosphorylate serum response factor (SRF) on Ser- 103 with efficiency similar to that of HSP25/27 [60]. MAP kinase interacting kinases (MNKI and 2) are Ser/Thr protein kinases related to the MAPKAP K2 family. Both MNKl and 2 bind strongly to and are activated by ERK, whereas only MNKl is activated by p 3 8 ~MNKl is activated by agents that stimulate both the ERK and the p38 pathways, and can be differentially inhibited by inhibitors of the ERK and p38 pathways. Thus, MNKI could be an integration point for ERK and p38 pathways. Activated MNKl has been shown to phosphorylate initiation factor eIF-4E [61]. p38 regulated/activated kinase (PRAK) is another Ser/Thr kinase in the MAPKAP K2 family which has -20-30'% identity to other MAPKAP Ks [62]. Similar to the MAPKAP Ks, PRAK is activated by environmental stress and inflammatory cytokines via p38a and p38B 2 and activated PRAK phosphorylates small heat shock protein HSP27 [62]. Mitogen- and stress-activated kinase I (MSK1) is a Ser/Thr kinase which differs from other MAPKAP Ks in that it contains two kinase domains in a single polypeptide [63]. Both ERK and p38 activate MSKl in response to mitogenic factors or environmental stress, respectively. Activated MSK 1 phosphorylates CREB and ATFl. The activation of MSK1/2 by LPS in RAW264 cells is completely prevented by a combination of a MEK inhibi-

12

p38 MAP KINASE

tor (PD 098059) and a p38 inhibitor (33) (SB 203580), but is only partially inhibited by either inhibitor alone [64]. The LPS-stimulated activation of ATFl and CREB, and the transcriptional induction of COX-2 and IL-lP were also inhibited in the presence of both drugs. Thus, LPS-induced activation of MSK1/2 and CREB/ATFl and the subsequent induction of COX-2IIL-18 requires both ERK and p38 MAPKs [64]. Ribosomal S6 kinase-B (RSK-B) was identified due to its ability to interact with p38 in a yeast two hybrid screen. Similar to MSK1, RSK-B also contains two kinase domains within one polypeptide and activated RSK-B can phosphorylate CREB and c-Fos peptides [65]. In cellular assays, RSK-B drives CAMP and AP- 1-dependent reporter gene expression. Transcription factor substrates of p38

Activation transcription factor 2 (ATF2) was one of the first in vitro substrates to be identified for both p38 and JNK [16,18]. Heterodimers of ATF2 and c-Jun constitute the AP-1 factor and bind to the AP-1 site present in AP-1 responsive genes such as c-jun [66].ATF2 is phosphorylated by p38a and ~ 3 8 8 2on Thr-69 and Thr-71 resulting in an increased transcriptional activity which can be blocked by the p38 inhibitor SB 203580 [18]. ATF2 is also phosphorylated by p38y /SAPK3 and p386/ SAPK4 [25, 331. Recently, ATF6 was also shown to be efficiently phosphorylated by p38. ATF6 binds SRF at the serum response element (SRE) present in the atrial natriuretic factor (ANF) gene promoter. Thus p38 may regulate ANF gene expression in cardiomyocyte via phosphorylation of ATF6 [67]. Myocyte-enhancer factor 2C (MEF2C) was identified as a p38 interacting protein in a yeast two hybrid screen using p38u as a bait. In LPS-stimulated monocytes MEF2C is activated by p38a phosphorylation which results in an increased expression of c-jun. It is possible that p38a stimulated c-jun gene expression is part of the host defence and response to inflammation to maintain a balance of c-Jun consumed during infection [68]. Phosphorylation and activation of MEF2C may also be involved in increased skeletal muscle differentiation [69]. Recently, MEF2A was also found to be a substrate of p38a and ~ 3 8 8 2[70]. C/EBP homologous protein (CHOP), also known as GADD153, is a member of the C/EBP family of transcription factors which mediate the effects of environmental stress on cell growth and differentiation. CHOP has been shown to be phosphorylated on Ser-78 and Ser-81 in cells subjected

J.L. ADAMS ET AL.

13

to stress stimuli and the same residues are also phosphorylated by p38a in vitro [71]. SB 203580 blocked the stress inducible phosphorylation of CHOP in cells. Ternary complex factor (TCF) together with SRF, bind to the SRE in the promoter of serum responsive genes such as c-fos. Elkl and Sap-la are ETS domain transcription factors and are components of TCF. Elkl is poorly phosphorylated on Ser-383 and Ser-389 by p38, but Sap-la is efficiently phosphorylated on the homologous Ser-381 and Ser-387 residues [72]. ERK is also known to phosphorylate both Elkl and Sap-la suggesting a convergence of mitogenic and stress signals in the regulation of serum responsive genes [73].

ENZYMOLOGY

Three groups have reported on the mechanism of inhibition of recombinant activated p38a by the pyridinylimidazoles [74-761. Young and co-workers have described a p38 kinase assay which uses the EGF receptor peptide fragment T699 as the peptide substrate and constitutively active p38a expressed in Saccharomyces cerevisiae [74, 771. Using this assay, the inhibition of p38a kinase activity by a series of pyridinylimidazoles was correlated with their ability to compete in the THP.l binding assay [78]. As pyridinylimidazole binding had previously been correlated to the inhibition of pro-inflammatory cytokine production from LPS-activated monocytes [ 131, these data causally linked p38u kinase and cytokine inhibition. The inhibition of p38a by SB 203580 (33) was shown to be competitive with respect to ATP with a Ki of 21 nM and a K, for ATP of 200 pM. The kinetic mechanism of fully activated p38a has been described by LoGrasso et al. at Merck employing GST-ATF2 as the protein substrate [75] and by Chen et al. at Vertex using the T699 substrate [76]. Interestingly, the two groups obtained different results. The Merck group determined that substrate binding was ordered with the phosphate acceptor binding before ATP. The Vertex group also determined an ordered mechanism, but in their case ATP bound prior to peptide. Assuming both results are valid, then these data imply that the kinetic mechanism is substrate-dependent. Relative to a peptide, the physiologically more relevant protein substrate, GST-ATF2, would be expected to make extensive contacts with p38 and hence bind more tightly (GST-ATF2 K m = 6 pM).

14

p38 MAP KINASE

In fact, in cells p38 kinase is known to exist in complexes with several of its substrates [40]. The Merck finding that a protein substrate binds prior to ATP is consistent with the existence of preformed substrate-kinase complexes. In contrast to this, the non-physiologic T699 peptide substrate used by Chen has a very poor affinity for p38 (Km = 812pM). Under these circumstances ATP (Km of 100 pM) can effectively compete for binding to p38 and thereby reverse the order of binding preferred for protein substrates. The overall rate of the reaction is similar for both substrates as the Kcat of 19 s-l for the T699 peptide is much higher than that of 0.08 s-l observed with GST-ATF2. Both groups found SB 203580 to be a competitive inhibitor with respect to ATP with a Ki of 34 nM (Merck) and 100 nM (Vertex). Additional experiments described by Young and co-workers on the unactivated kinase offer useful insights into the mechanistic details of inhibitor binding [74]. Using microcalorimetry analysis, the binding of SB 203580 to unphosphorylated p38a kinase expressed in E. coli was determined. SB 203580 bound to p38a with approximately 1:l stoichiometry, a KD = 15 nM and a AH = -12 kcal at 30°C. In agreement with the observed ATP competitive kinetics, no binding of SB 203580 was observed when unactivated p38a was reacted with FSBA, a covalent ATP-site inhibitor of kinases. Although E. coli expressed p38a has no detectable protein kinase activity, the protein does possess an intrinsic ATPase activity in the absence of substrate (k,,, 0.006 s-' and ATP KM of 9.6 mM). The ATPase activity was completely inhibited by one mole equivalent of SB 203580, Ki 5 100 nM. Using fully activated p38a a much more robust ATPase activity was observed by Chen et al. (kcat 0.4 s-' and ATP KM of 170pM) [76]. In summary, these studies indicate that activation of p38a greatly enhances affinity of the enzyme for ATP. Activation of p38a also enhances the efficiency of the enzymatic reaction as measured both by the hydrolysis of ATP (- 70 fold increase in Kcat) and the phosphorylation of peptide and protein substrates. Unlike ATP, the affinity of the pyridinylimidazole SB 203580 is unaffected by the activation state of the enzyme (Kd -37 nM for both unactivated and activated enzyme) [79]. These data indicate significant differences in the manner in which these two ligands interact with p38a . However, the finding of ATP competitive inhibition for SB 203580 suggests that the binding sites of ATP and SB 203580 overlap, a result which has been confirmed by crystallographic studies. Thus, although ATP competitive the pyridinylimidazoles are clearly not ATP mimetics like AMPPNP

J.L. ADAMS ET A L .

15

(adenylyl imidodiphosphate), whose affinity for the kinase would be expected to mimic that of ATP. Considering the large number of kinases and other ATP utilizing enzymes and hence potential for non-selective interactions for an ATP mimetic, the suggested lack of ATP mimicry for the pyridinylimidazoles is a highly desirable feature. In general, the pyridinylimidazoles have ICso values for inhibition of LPS-stimulated IL-1 synthesis in human monocytes that are in close agreement with the ICs0 values for p38 inhibition [78]. This is not the expected result for an ATP competitive kinase inhibitor, whose cellular activity should be significantly attenuated in the face of the high intracellular concentration of ATP (estimated to be 2-5 mM) [80]. In an attempt to provide an explanation for this unexpected equivalency of purified p38a and cell-based assay ICso values, it has been suggested that the inhibition of p38 activation, and not the ATP competitive inhibition of activated p38, is the operative mechanism of action for SB 203580 in cells [79]. In support of this proposal Frantz ef al. presented data demonstrating that SB 203580 inhibited both LPS- and TNF-induced p38 activation in THP.l cells. However, several earlier studies found that SB 203580 did not inhibit activation of p38 [74, 81, 821. Most recently, Kumar et al. have confirmed in a variety of cell lines and using several activation stimuli that SB 203580 does not inhibit p38a activation [83].

INHIBITORS OF p38 KINASE MEDICINAL CHEMISTRY PRECEDING THE DISCOVERY OF p38

The first and most widely described class of p38 inhibitors is the pyridinylimidazoles of which SK&F 86002 (1) is the prototypical CSAID. The rationale that led to the synthesis of (1) was the search for a new class of anti-inflammatory compounds. Towards this end, the pharmacophore of an immunomodulator levamisole (4) was fused with a cyclooxygenase inhibitor flumizole (5). The resulting hybrid, SK&F 81 114 (6), proved to have both these properties and additional analogues were prepared to further investigate this activity [84]. In an effort to improve aqueous solubility, a series of regioisomeric 4-pyridyl-containing dihydroimidazothiazoles were prepared. The set of regioisomers to which SK&F 86002 (1) belongs was effective at reducing inflammation in the rat

16

p38 MAP KINASE

adjuvant-induced arthritis model, whereas the regioisomers, which included SK&F 86055 (7), were inactive [85].

(7) SK&F 86055

(8) S K&F 105809

The potent anti-inflammatory activity of SK&F 86002 was initially attributed to its ability to inhibit both the cyclooxygenase (COX-I) and 5-lipoxygenase (5-LO) pathways [86]. However, further studies revealed an additional anti-inflammatory mechanism for SK&F 86002, namely the inhibition of LPS-induced IL-1 production in human monocytes [7, 81. During preclinical development several shortcomings of (1) were identified in rat safety assessment studies. These included induction of hepatocellular hypertrophy, gastric ulceration, acute CNS effects, ovarian dysfunction and testicular atrophy [87]. Based on these results and on additional rat toxicology studies with related compounds, it was concluded that certain toxicities were related to cyclooxygenase inhibition (ovarian and gastric) [88J or were compound specific (convulsions and testicular atrophy). Further studies with (1) demonstrated the compound was a potent P450 inducer, thus offering a plausible explanation for the observed hepatocellular hypertrophy [89]. SK&F 86002 also proved to be a potent inhibitor of both rat and human P450 enzymes. Because compounds that inhibit P450 enzymes are also often P450 inducers, it was hypothesized that the observed hepatotoxicity could be eliminated by reducing the P450 inhibitory activity of these compounds.

J.L. ADAMS ET AL.

17

Following our efforts to develop these compounds as dual 5-LOICOX-1 inhibitors, we chose to focus on cytokine inhibition as a potential pharmacologically important mechanism. The rationale for this change in focus was two fold. Most importantly, there was a growing body of evidence in the literature that cytokines, such as IL-1 and TNF, had profoundly potent pro-inflammatory activity. Secondly, mechanistic data for dual 5-LO/COX-l inhibitors pointed to a common redox mechanism for inhibition of these enzymes which was related to the redox potential of the inhibitors. Indeed, mechanistic studies with (1) and (8) were supportive of the involvement of a redox component [90, 911. Furthermore, there were several reports associating potential toxicity (methemoglobinemia) with redox 5-LO inhibitors [92]. p38 SAR

Prior to the discovery of p38, the SAR of the pyridinylimidazoles for cytokine inhibition was first established in LPS-stimulated human monocytes. Subsequently, data was reported using the THP. 1 cytosolic radioligand binding (CSBP binding) and p38a kinase inhibition assays. Because similar ICso values for the pyridinylimidazoles were observed in all three assays, data from all three assays is used in the description of the SAR [78]. Pyridiny lim idazoles The minimal structural requirement for p38 inhibition is a 4-(pyridin-4-yl)-5-phenylimidazole(9) (CSBP binding ICso = 0.21 pM) [78]. That both the vicinal aryl and 4-pyridyl groups are required for activity is illustrated by the following data from a series of triaryl imidazoles. Substitution of the vicinal aryl of (10) (CSBP binding ICSo= 0.15 pM) [78] with either cyclohexyl (11) (p38 ICs0 >10pM) [93] or ethyl (12) (CSBP binding ICso >30pM) abolishes p38 inhibition, as does replacement of the 4-pyridyl with phenyl (13) (CSBP binding lCs0 >lOpM). In addition to tolerating substitution of the imidazole at C-2 (lo), substitution is allowed on the nitrogen adjacent to the pyridyl(l4) (CSBP binding ICso = 0.050 pM). However, substitution of the imidazole nitrogen adjacent to the aryl abolishes activity (1 5) (CSBP binding ICsO > 10 pM). This regioselectivity requirement was first noted in the rat adjuvant arthritis assay in which ( I ) , but not the regioisomer (7) was active [ 8 5 ] . The anti-inflammatory data was later correlated to the relative abilities of (1) and (7) to inhibit LPS-stimulated IL-1

18

p38 MAP KINASE

synthesis in human monocytes, IC50 of 0.50 pM and >5 pM, and the affinity for CSBP in the binding assay, ICs0 of 0.26 pM and 10 pM, for (1) and (7) respectively [ 1 11.

x3c I

Y

(l0)R1=4-FPh,R 2 = H (1 1) R' = cyclohexyl. R2 = H (1 2 ) Rl = Et. R2 = OMe

(13)

/

(14)X =N, V = CH (15)XsCH. Y = N

The dependency of p38 affinity on the substitution of the vicinal aryl group is illustrated in Table f . f for three different classes of pyridinylimidazole inhibitors [78]. The most potent inhibitors have fluorine (17,25 and 33) or chlorine (18, 19,26 and 34) at the meta or para positions. Potency is maintained for 3,4-dichlorophenyl (27) (CSBP binding ICso = 0.32 pM), but lost with the more bulky 3,5-bis(trifluoromethyl) phenyl (31) (CSBP binding ICs0= 114pM). As the size of the aryl substituents at the 4 position increases, potency decreases F, CI > Br > OMe, SMe, CF3 > SEt. The polar 4-sulfinyl methyl group is highly unfavourable (8) (CSBP binding ICso > 10 pM). Likewise, compounds with polar groups at the 3 position, sulfoxide (32) and sulfonamide (37), are weakly active. Substitution of phenyl with other aromatics is allowed, but these are less favourable (38, 39 and 40). As noted above, the 4-pyridyl group is essential for p38 inhibition. Unfortunately, potent inhibition of cytochrome P450 enzymes is also associated with CSAIDs containing the 4-pyridyl group [94]. The nitrogen lone pair of pyridine is known to ligate the ferric, heme iron of cytochrome P450 and compounds containing sterically accessible pyridyl nitrogens are often potent inhibitors of P450 enzymes. Thus the 4-pyridyl group, in addition to being required for binding to p38, is also an important factor contributing to potent P450 inhibition. Hence, the requirements for this portion of the pharmacophore were investigated in search of 4-pyridyl replacements which retained p38, but not P450 inhibition. A facile synthesis of the required analogues was performed using an adoption of the van Leusen imidazole synthesis to prepare a series of 1 -(N-morpholinylpropy1)- 4-(4-fluorophenyl) imidazoles (Table 1.2) [94]. Replacement of the 4-pyridyl(25) with either 2or 3-pyridyl (41 and 42) dramatically reduced affinity for p38 (Table f . 2 ) . Other modifications of the 4-pyridyl group (43-47) were unsuccessful, as

J.L. ADAMS ET AL.

19

Table 1.1. THE EFFECT O F ARYL SUBSTITUTION ON CSBP BINDING AND INHIBITION OF LPS-STIMULATED IL- 1 SYNTHESIS IN HUMAN PERIPHERAL BLOOD MONOCYTES [78] IL-I CSBP monocytes binding Compound R Icm W M ICm PM -

(25) SB 210313 (26) (27) (28) (29)

(32)

Ph

0.50

2.1

4-FPh 4-CIPh 3-CIPh 2-CIPh 4-BrPh 4-MeOPh 4-MeSPh 4-EtSPh

0.17

0.70 4.2 2.1 >20

0.066 0.050 0.040 0.25 3.2 3.6 3.4 5.6

4-MeS(O)Ph

110

4-FPh 3-CIPh 3,4-diClPh 3-CF3Ph 4-CF3Ph 4-MeSPh 3,5-di-CF3Ph 3-MeS(O)Ph

~

0.050 -

0.60 -

1.0 >5

>10

0.12 0.21 0.32 0.46 3.0 0.29 114 5.0 ~~

(33) SB 203580 (34)

/

N\

-

0

(37) (38) (39) (40)

4-FPh 3-CIPh 3-MeOPh 2-MeOPh 3-MeS02NHPh 2-Thiophenyl 1-Naphthyl 2-Naphthyl

0.08 0.08 0.59 1.14 >5 -

0.65 0.77

0.042 0.030 0.38 0.88 3.9 0.46 2.3 5.6

reduced P450 inhibition was only seen for compounds with reduced CSBP binding. Substitution of the pyridine with a pyrimidine ring, which retains the hydrogen bonding ability of the 4-pyridyl nitrogen, and is known to be a weak P450 inhibitor relative to pyridine, yielded compounds with the desired profile. Compounds containing a 2-H-, 2-methoxy- and

p38 MAP KINASE

20

Table 1.2. SYNTHESIS AND SAR OF PYRIDINE REPLACEMENTS A N D THE CORRELATION BETWEEN P38 KINASE INHIBITION AND CSBP BINDING [94,276]

CSBP binding

R (25) (41) (42) (43)

ICsv CLM

a a

p38 ICso CLM

R

CSBP binding

p38

1cso CLM

ICsv CLM

1.3

0.12 65

0.30

65

5.5

3.1

2.0 0.48

(45) y&

,Ql# 1.2

(47)

1.9

9.0

1.7

(56)

H1N

n*i

0.22

(57)

& &

3.6 12 7.4

> 17

217

The kinase data for (44 and 45) and CSBP binding data for (47, 51-54, 56, 57) are previously unpublished results.

2-aminopyrimidine (48, 50, 53) demonstrated improved p38 inhibition and reduced P450 inhibition [94]. As discussed later, the oral activity of these compounds was also improved relative to (25). A wide variety of substituents is allowed at C-2 of the imidazole and on the imidazole nitrogen adjacent to the pyridine on the core 4-aryl5-(pyridin-4-yl)imidazole scaffold of (9) (CSBP binding ICSO= 0.21 pM). In many cases these additional groups have little impact (58) (p38 ICSO= 0.23 pM)[95] or slightly enhance potency (60) (p38 ICSO= 0.07 pM) [93]. In contrast to cyclohexyl (58, 60), the basic 4-piperidinyl group pro-

J.L. ADAMS ET AL.

21

vides a more significant enhancement of activity when attached to the imidazole at either N-1 (59) (p38 ICSo=0.06pM) [96] or C-2 (61) (p38 IC50=0.009pM) [93]. The most potent p38 inhibitors reported to date are derived from further optimization of these piperidine containing inhibitors. Thus, SmithKline Beecham has reported SB 238039 (62) (p38 Ki=0.4nM) [97] and Merck (63) (p38 ICS0=0.11 nM) [93]. Other companies which have filed patents for pyridinylimidazole inhibitors of p38 are Aventis [98], Ortho McNeil [99] and Novartis [loo].

(58) X = CH, (59)X=NH.VK-19911, S8 235699. HEP689

(60) X = CH, (61)X=NH

(62) S8 238039

Non-imidazole 5-membered ring scaffolds Although a primary role of the central imidazole is to serve as a scaffold to orient the vicinal aryl and pyridyl rings, this heterocycle does interact with the p38 binding site and can profoundly affect potency. Recognizing the non-essential nature of the central imidazole, de Laszlo et al. at Merck investigated three sets of regioisomeric analogues related to the triaryl imidazole SB 203580 (33) [loll. Little difference in potency was observed for the two regioisomeric furans (65 and 67) (p38 IC50of 0.63 and 0.53 pM, respectively). However, a clear regioisomer preference was seen with the pyrroles, favouring (64) over (66) (p38 ICSo of 0.20 and 1.4pM, respectively) and this preference was even more pronounced with the pyrazolones (68 and 69) (p38 lCso of >0.50 and 0.035pM, respectively). These data guided Merck to synthesize the pyrrole (70) (p38 ICso of 0.005pM) and pyrazolone (71) (p38 ICsO of 0.085pM) analogues of SB 203580 (33). Encouraged by the 40-fold improvement seen with the pyrrole (70) an additional 40 analogues were prepared [loll. The SAR for these pyrroles parallels that seen for the pyridinylimidazoles suggesting a similar mode of binding.

p38 MAP KINASE

22

As outlined in four patent applications, G.D. Searle and Co. has extensively investigated pyrazole replacements of the imidazole [102-1 051. The most comprehensive application, 800 pages in length and detailing the synthesis of -2800 compounds, highlights the potency of the minimally substituted diary1 pyrazole (72) (p38 ICso of0.049 pM) [lo21 which is 70-fold more potent than its regioisomer (73) (p38 ICSOof 3.5 pM)[103]. Additional imidazole replacements described by SmithKline Beecham in the patent literature have a central oxazole, triazole or cyclopentene, novel variations of the imidazole or pyrazole. These additional examples are discussed in reviews of the p38 patent literature [106, 1071. F

(65)X=O

(70) L-167307

= NH (67) X = 0

(71)

(69)X=cO,Y =NH

(73) X = CH. Y = N

Other heterocyclic core structures Examples of 6-membered ring heterocycles reported in patents as core structures for the pyridyl/aryl p38 inhibitors are pyridine (74) [log], pyrimidine (75) [lo91 and pyrimidone (76) [110]. Also claimed are a number of compounds having a bicyclic heteroaromatic core. Included in this group are the indoles and azaindoles (77-81) reported by Ortho McNeil [ 1 1 11 and Amgen [ 1 121 and Fujisawa’s hydrazino pyrazoles FR 167653 (82) [113]. Beginning with an indole core and using the ICso for inhibition of TNF in LPS-stimulated human peripheral blood mononuclear cells as a surrogate for p38 inhibition, investigators at Ortho McNeil noted that potency was improved with the introduction of an electron donating group at C-6, >10pM for (77, R = H ) to 0.40pM (78, R=OMe) and 0.20pM (79, R=NH*). Since these results suggested a

J.L. ADAMS ET AL.

23

preference for polar groups in this region of the molecule, nitrogen was introduced into the indole nucleus in place of C-7. The potency of this more polar analogue (80) (ICsO= 0.037 pM) was markedly improved. Using calculated electrostatic potentials as a measure of electron density, the enhanced potency of RWJ 68354 (81) (IC50= 0.0063 pM) relative to (80) appeared to be related to an increase in electron density on the pyridyl nitrogen, a result consistent with its proposed role as a hydrogen bond acceptor. The mechanism of T N F inhibition was confirmed as p38 kinase inhibition for (81) in an immunoprecipitated p38 kinase assay (ICsOof 0.009 pM). Although Fujisawa has published extensively on the in vitro and in vivo activity of FR 167653 (82) as an inhibitor of IL-1 and T N F synthesis, there is no reference to p38 inhibition as a mechanism of cytokine inhibition [114, 1151. In conclusion, potent p38 inhibitors have been reported for compounds containing a pyrrole (70), pyrazolone (69), pyrazole (72) and 7-azaindole (81) in place of the imidazole moiety. No simple SAR has emerged which can rationalize the success of these replacements. For example, although a hydrogen bond acceptor is usually preferred adjacent to the aryl group, e.g. the pyrazole (72) and pyrazolone (69), the opposite preference for a hydrogen bond donor adjacent to the aryl group is seen for the pyrrole (64) and 7-azaindole (81). The general conclusion which can be drawn from this data is that the more polarized oxygen or nitrogen heteroatoms are preferred over carbon on the core heterocycle in the region adjacent to the aryl group.

p38 MAP KINASE

24

New structural classes of p38 inhibitors Recently, several examples of p38 inhibitors structurally dissimilar to the pyridinylimidazoles have appeared in the patent literature. Scientists at Vertex have disclosed a novel series of potent p38 inhibitors exemplified by structures (83-85) [ 1 16-1 181. The qualitative inhibition data provided in the Vertex patent are consistent with the upper right hand aryl ring in the above structures fitting into the hydrophobic pocket of p38 (see X-ray crystallography discussion) [116]. For example, aryl substituents (F and Cl), which afford potent inhibitors when substituted on the para position of 4-aryl-5-pyridinylimidazoles (16,25,27) (Table1. I) also led to potent analogues of (85) [116]. In contrast to this, replacement of the 4-F-2-methylphenyl group of (85) with larger substituents (for example 2-naphthyl) reduced potency. Similar substitutions at the imidazole C-4 of SB-203580 (33), compounds (39, 40) have also been shown to decrease potency [78]. E

(83) X = N

(a) x=w

c

(85) VX-745

Workers at Glaxo have disclosed the inhibition of p38 (and numerous other kinases) by a series of azaindolinone derivatives exemplified by (86) [ 1191. These compounds are similar to the indolinone structures previously claimed by Sugen as Tyr kinase inhibitors. Some of the Glaxo compounds were reported to inhibit p38 kinase with ICsos < 1pM. A number of companies have claimed diary1 ureas (87-89) or amides (90-92) as p38 inhibitors. The Vertex application primarily exemplifies diphenyl ureas such as (89) (p38 ICso =0.1 pM) [120], whereas the focus of the Bayer (87) [121-1241 and Boehringer Ingelheim (88) [125] applications are N-phenyl-N’-heteroaryl ureas in which the 5-membered heteroaryl ring has a t-butyl group attached to a non-adjacent position. Benzamide inhibitors have been described by Zeneca (IC50=0.040 pM for most potent example) [ 1261, Scios (9 1) (ICso = 0.065 pM) [ 1271 and SmithKline Beecham [128]. The Zeneca compound (92) (p38 IC50= 2.0 pM) originally prepared as

J.L. ADAMS ET AL.

25

a c-Raf inhibitor (c-Raf ICsoz0.070 pM) is of special interest because of recent work that implies similar binding interactions for these compounds and the pyridylimidazoles [ 1291.

(91) R' = OMe. R2 = CONHCH,CH,NMe,

(92) ZM 334372

STRUCTURAL BASIS OF KINASE SELECTIVITY

There are believed to be between 1000-2000 kinases in the human genome, all of which utilize ATP as the phosphate donor and have a similar tertiary structure and mechanism of phosphate transfer. Given the size and similarity of this protein family, the discovery of selective kinase inhibitors was initially considered an unlikely proposition. The selectivity problem which is generic to any approach to kinase inhibition appeared particularly onerous for inhibitors that target the ATP binding site, since the majority of the residues which form the ATP binding pocket are either invariant or highly conserved. Moreover, the high intracellular concentration of ATP (-3-5 mM), which is 100-1000-fold higher than the K, of ATP for most kinases, suggests that the intracellular potency of these inhibitors will be severely compromised. Nevertheless, selective and effective ATP competitive inhibitors of p38 kinase have been discovered. The following discussion begins with a summary of the available p38 kinase selectivity data and then proceeds to a discussion of recent X-ray crystallographic and protein mutagenesis results which provide a structural basis for understanding the selectivity of the pyridinylimidazole class of p38 MAP kinase inhibitors.

26

p38 MAP KINASE Table 1.3. KINASE SELECTIVITY

Kinasr.

IC,,,f i L M

88-203580 (33) [132]

L-167307 (70) [loll

p38a P3882 P38Y p38S ERK2 JNKl JNK2fll c-Raf LCK EGFR Cdc2

0.048 0.050 > 10 > 10 > 100 5.0 0.28 0.37 20 10 >50

0.005 0.008 1 42% ( d 50

Scios

(90) [I271 0.15 3.0 230

-

-

> 300

-

16

-

1.5 0.47

-

SB-239063 (99) [133, 951

Z M 336372 (92) 11291

0.044 -0.05 > 100 >I00 >I0 >50

2.0 2.0 > 50 > 50 t 50

-

-

-

-

> 500

>50 > 10 > 10

-

>500

> 50

150 -

0.07 -

-

-

Selectivity data The first report detailing the high kinase specificity of a pyridinylimidazole inhibitor of p38 was for SB 203580 (33) (Table 1.3) [81]. Interestingly p38/3, the closest homologue to p38a , is inhibited with equivalent potency but both p38y and p386 are insensitive to SB 203580 [82, 130, 1311. Subsequently, de Laszlo et al. uncovered that c-Raf and JNK (specifically the 2/3 isoform) are also sensitive to SB 203580, although some 5-10 fold less so than p38a [loll. The tyrosine kinases LCK and EGFR are the next most sensitive kinases, with 1Cs0s 100 fold higher than that of p38a [132]. All other tested kinases have ICsos for SB 203580 >I000 fold than that of p38a. High kinase selectivity for p38 appears to be characteristic of all classes of p38 kinase inhibitors; these include the pyridinylimidazoles, the pyrroloimidazoles (70) and the benzamides (90, 91). Compounds reported to have a better kinase selectivity profile than SB 203580 are the pyrrole analogue (70) [loll, SB 239063 (99) [133] and Merck’s very potent inhibitor (63) [93]. In these examples the p38p2 enzyme is inhibited with equal sensitivity. Selective inhibition of p38a versus p38p has recently been reported for SB 242325 (98) (10-fold) [134], the Scios compound (90) (20-fold) [ 1271 and a Searle pyridinylpyrazole of undisclosed structure (100-fold) [135].

-

J.L. ADAMS ET AL.

21

X-ray crystallography

Two structures of native p38cr 4136, 1371 and multiple structures of pyridinylimidazole-p38 complexes [ 138-1 401 have been solved. Important features observed in these structures are: 1) a hydrogen bond between the 4-pyridyl/pyrimidinyI nitrogen and the amide N-H of Met-109. 2) Thr-106 and Lys-53 form the sides of an aryl binding pocket which is behind the site normally occupied by the adenine ring of ATP. 3) a hydrogen bond-like interaction between Lys-53 and the unalkylated imidazole nitrogen. These features are illustrated in Figure 1.3 for S B 220025 [139]. The hydrogen bond between the nitrogen of the pyridine I pyrimidine and the N-H of Met-109 is analogous to that seen for the N-1 adenine of ATP in all available kinase crystal structures. This hydrogen bond

H s olve nt e xpos e d

u

phosphatehugar region

Figure 1.3. Illustration of Crystal Structure of SB 220025 (black) in p38cc Also shown is ATP (grey) u>ithand its expected hydrogen bonding interactions to the backbone amides ofp38.

p38 MAP KINASE

28

acceptor-donor pair is also seen in other inhibitor-protein kinase crystal structures. As illustrated for SB 220025, inhibitors having a properly positioned amino group can form a second hydrogen bond with the amide carbonyl of Met-109. This interaction occurs in preference to mimicking the hydrogen bonding pattern of adenine in which C-6 NH2 is hydrogen bonded to the amide carbonyl of His-107. An interaction of the imidazole nitrogen with Lys-53 is seen in some, but not all, structures and is unlikely to contribute significantly to selectivity. Lysine is totally conserved at this position and is required for catalytic activity. Whereas the pyridinel pyrimidine and imidazole serve chiefly as an adenine mimetic, the fluorophenyl group occupies a region of the active site not utilized by ATP and appears to be the key feature contributing to selectivity. Residues Thr-106 and Lys-53 provide the most extensive contacts with the fluorophenyl group by forming the walls of the pocket above and below the plane of the aromatic ring. Recognizing that a limited number of kinases have side-chains at position 106 the size of threonine or smaller, Tong et al. proposed a potential role of Thr-106 in determining kinase selectivity [ 1381. For example, the closely related ERK2 kinase, which has the larger glutamine side-chain at position 106, is 1000-fold less sensitive to SB 220025 (IC50=20pM) relative to p 3 8 ~[136]. As discussed below, subsequent X-ray crystallographic and mutagenesis studies have now confirmed that the relative rare occurrence of the small threonine residue at position 106 is the single most important feature of p38 that facilitates selective binding of these inhibitors to p38 relative to other protein kinases. Key aspects of the inhibitor SAR which can be explained using the inhibitor-p38 crystal structures are: 0

0

0

0

A sterically unencumbered 4-pyridyl/pyrimidinyI nitrogen is required as a hydrogen bond acceptor for the NH of Met-109; phenyl, 2- or 3-pyridyl derivatives are significantly less potent. Inhibitor potency is enhanced by substitutions which add to the electron density of the pyridine nitrogen and thereby strengthen its ability as a hydrogen bond acceptor. The hydrophobic pocket is of limited size. In the case of pyridinylimidazoles ortho- or para-substituted chloro/fluoro phenyl is optimal; cycloalkyl is too large and halogen too small. The interaction of the central heterocycle with the terminal NH2 of Lys-53 influences the relative affinity of the imidazole replacements and explains the regioselective preference for substitution of the imidazole on the nitrogen distal to Lys-53.

J.L. ADAMS ET AL. 0

29

Almost all substitutions of the imidazole at C-2 and N-1 are well tolerated. These groups extend into the sugar and phosphate binding site for ATP and also towards solvent.

The new structural classes do not have an sp2-nitrogen containing adenine mimetic which could fulfil the hydrogen bond acceptor role of the pyridine nitrogen of the pyridinylimidazoles. They do, however, possess an amide carbonyl which could serve this function. Inhibitor-protein kinase complexes in which the carbonyl of a cyclic amide is engaged in a hydrogen bonding interaction analogous to N-1 of adenine have been solved for staurosporine [141] and several oxindoles [142]. Hence, it is likely that the amide carbonyl oxygen of compounds (83-92) serve as hydrogen bond acceptors in place of the 4-pyridyl nitrogen. Furthermore, the SAR discussed above for (83-85) and the data for binding of (92) to p38 mutants [I291 suggests that a portion of these molecules bind in the hydrophobic specificity pocket of p38. Thus, it would appear that these new structural classes have many of the same binding interactions of the pyridinylimidazoles.

Mutagenesis Young et a/. explored pyridinylimidazole binding in the ATP binding pocket of p38a using scanning mutagenesis with alanine [ 1431. The findings were in agreement with the subsequent X-ray crystallographic results showing that the pyridinylimidazoles and ATP occupy different, although overlapping, regions of the ATP pocket. Guided by the X-ray crystallographic data implicating Thr- 106 as the most likely residue mediating selectivity, Wilson and co-workers prepared p38a mutants in which Thr- 106 was mutated to either Ala or Met [140]. Methionine was chosen because of its presence in p38y and p386, close family members to p38a which are insensitive to inhibition by SB 203580. As expected, the p38a T106M mutant was insensitive to inhibition by SB 203580, whereas sensitivity of the T106A mutant to SB 203580 was unchanged. Similar results were observed by Gum e t a / . and Eyers et al. for the T106M mutants of both p38a and p38b [143, 1441. The importance of residue size at position-106 of p38a was more extensively investigated by Eyers who prepared nine mutants at position-1 06. All mutants with groups larger than threonine showed reduced inhibition by SB 203580, whereas the glycine, alanine and serine mutants were more sensitive. Interestingly, the binding of (92) to p38a is also abolished for the T106M mutant [129], implying that some portion of the amide inhibitor binds into the hydrophobic pocket of p38a.

30

p38 MAP KINASE

Perhaps even more significant are the results of mutations of position-106 which converted SB 203580 insensitive kinases to ones that are inhibited by SB 203580. Thus the mutation of M106T converted p38y and p386 kinases to SB 203580 sensitive kinases [143]. The effect of this same mutation has been examined in additional MAP and SAP kinase members. The Ki for inhibition of native ERK2 by SB 203580 is >20pM; the Q106T (p38 numbering) mutation converted ERK2 into a kinase of high sensitivity (Ki= 13 nM) [96]. Native J N K l is SB 203580 insensitive and the M106T mutant gains sensitivity [143]. Further mutational studies by Gum et a/. identified additional residues proximate to the pyridinylimidiazole binding site that influence inhibitor sensitivity, albeit to a lesser extent than Thr-106. In summary, within the MAP kinase family (p38, JNK, ERK) the mutagenesis data convincingly supports the proposition that threonine 106 is a primary determinant for predicting MAP kinase sensitivity to the py r idinylimidazoles. Mutagenesis studies appear to have broad utility for the understanding of inhibitor selectivity. For example, c-Raf, LCK, EGFR all have a threonine at the position equivalent to 106 in p38a and show some sensitivity to inhibition by SB 203580. That this observation can be used in a predictive manner has been demonstrated for TGF-PI receptor kinase which has serine at the position equivalent to 106 in p38a (IC50=20pM for SB 203580), albeit the potency of inhibition is three orders of magnitude less than that for p38 [ 1441. Importantly, kinases with residues larger than threonine at position-106 are insensitive to SB 203580 >50pM). The exception to this rule are selected members of the JNK family, which are structurally and functionally close relatives to the p38 family.

PHARMACOLOGY OF p38 INHIBITORS I N VITRO

Regulation of protein synthesis The first observation suggesting involvement of p38 in cytokine biosynthesis was seen in human peripheral blood monocytes stimulated with LPS to induce pro-inflammatory cytokine expression [7, 81. Subsequently, similar observations have been seen in other cell types and with additional stimuli, such as IL-8 production in endothelial cells [145]; IL-6 and GM-CSF production in fibroblasts [ 1461; streptococci induced IL-8 release from human

J.L. ADAMS ET A L .

31

neutrophils [147]; LPS induced IL-1 expression macrophage cell lines [148]; PAF-induced RANTES production in human airway smooth muscle cells [ 1491 and beta-I integrin-induced IL-8 production in human natural killer cells [150]. The p38 MAP kinase pathway is also important for T-cell cytokine production. T N F production in T cells (HA-I .70) stimulated with superantigen or a specific antigen peptide correlates with p38 activation but is only partially p38-dependent [151]. Mice deficient in MKK3, an upstream kinase of p38, are defective in the production of IL-12 and interferon gamma by antigen-presenting cells and CD4(+) cells [152]. Taken together, inhibition of p38 leads to inhibition of cytokine production in various cell types under various stimulus conditions. Although originally characterized as a stress or inflammatory kinase, p38 MAP kinase is likely to have diverse functions besides regulation of cytokine production or action [ 1531. For example, thrombin and collagen cause p38 activation in platelets [154] and p38 is constitutively active in liver [155]. More recently, p38 has been shown to regulate cyclooxygenase-2 mRNA stability and transcription [156]. Translational regulation of cytokines

Although initially the AU rich motif in the 3’ untranslated region was thought to confer mRNA instability [157], it was later postulated that this motif might also be involved in the translational regulation of some pro-inflammatory cytokine mRNAs [ 158, 1591. Studies using chloramphenicol acetyl transferase (CAT) reporter constructs expressed in RAW264.7 cells suggested the importance of the 3’ UTR AU motif for the LPS-induced synthesis of T N F and the sensitivity of LPS-induced T N F to p38 MAP kinase inhibitors [95, 1601. In addition, this phenomenon has been further studied by the use of a tetracycline-controlled expression system to determine the half-lives of IL-6 and IL-8 [ 1611. Transcript stability was low in control cells but the stability increased when a constitutively active upstream kinase of p38 MAP kinase, MEKKl was introduced. Selective activation of the p38 MAP kinase pathway by MKK6 also leads to mRNA stabilization possibly through MAPKAPK-2 activation. Thus, it was concluded that the p38 MAP kinase pathway contributes to inducible gene expression by stabilizing mRNA through a MAPKAPK-2 and 3‘ UTR AU rich motif targetted mechanism. This hypothesis has been recently confirmed by the phenotypic changes observed in transgenic mice expressing T N F lacking the AU rich elements [162]. Interestingly, T N F 3’ UTR AU element deficient mice spontaneously

32

p38 MAP KINASE

express TNF in targeted tissues leading to pathologies similar to chronic inflammatory arthritis and Crohn’s inflammatory bowel disease. Moreover, T N F production by macrophages from these mice was no longer sensitive to p38 MAP kinase inhibitors despite normal activation of the p38 MAP kinase pathway. More recently, mice deficient in MAPKAPK-2, the downstream substrate kinase, have been shown to be resistant to LPS-induced endotoxic shock [163]. This resistance appeared to be due primarily to a marked reduction in TNF translation and not at the level or stability of T N F mRNA or secretion. This result further confirms that p38 MAP kinase is essential for the regulation of TNF biosynthesis at the post-transcriptional level through its activation of MAPKAPK-2. As indicated previously, a number of transcription factors are p38 substrates. In particular, the p38 pathway was shown to trigger c-fos gene transcription by phosphorylating and activating the transcription factors Elk1 and Sap-1 [164-1661. The transcription of c-fos in turn activates the transcription of a number of cytokines including IL-1, T N F and IL-6. Thus, p38 is also involved in the transcriptional regulation of cytokine expression [146].

p38 M A P kinase and apoptosis

Involvement of the p38 pathway in apoptosis was first observed in nerve growth factor (NGF) withdrawal induced apoptosis of rat PC12 cells [167]. It was noted that upon growth factor withdrawal, both JNK and p38 were upregulated in a sustained manner while ERK was inhibited. The sustained activation of the two kinases in apoptosis was further supported by the use of constitutively active or dominant negative mutants. However, since this first report there have been many contradictory reports suggesting that p38 MAP kinase activation is protective against apoptosis, as well as being pro-apoptotic. It appears that the role of p38 MAP kinase in apoptosis is highly dependent on the cell type and the conditions under which p38 activity is measured and is summarized below.

Hema top0 ie t ic cells

The Fas Receptor mediates a signalling cascade resulting in T cell apoptosis. Juo et al. have shown that this apoptotic event is p38 and caspase-3 mediated [ 1681. Two commonly used caspase inhibitors, ZVAD and W A D ,

J.L. ADAMS E T A L .

33

inhibited apoptosis triggered by Fas but not by other agents such as sorbitol, etoposide or UV, which also activate p38 MAP kinase in many cell types. MKK4 [169] and MKK6 activation [170] is also implicated in this model system. Hyper-phosphorylation of Rb results in the dissociation the E2F-Rb complex and increases Rb transcriptional activity. SB 203580 blocked Fas-induced hyper-phosphorylation of Rb [I 711. This effect is perhaps mediated by MNK, a down stream substrate kinase of p38 [172]. In myeloma cells, Fas-induced apoptosis is blocked by IL-6. Since IL-6 does not modulate p38 directly, it may inhibit p38 activity via the gp130 pathway [173]. In B cells lines, BCR- and Fas-induced apoptosis results in the activation of p38 and JNK which can be inhibited by the caspase-3 inhibitor, ZVAD. However, SB 203580 (33) inhibits BCR-induced, but not Fas-induced apoptosis suggesting the p38 MAP kinase pathway is critical only for BCR-induced apoptosis [173, 1741. Cross-linking of CD40 in B lymphocytes rapidly stimulates p38 MAP kinase and its downstream effector, MAPKAPK-2, resulting in NF-kB activation, cell proliferation and induction of CD54/ICAM- 1 expression. All of these effects were inhibited by the p38 MAP kinase inhibitor, SB 203580 [ 1751. Interestingly, CD40-dependent expression of CD40 and CD95/Fas and other CD40-responsive genes (clAP2, TRAFI, TRAF4ICART and DR3) were unaffected. Nemoto et al. has further expanded on these studies. At high doses, a selective p38 inhibitor, SB 202190 (2), was sufficient to induce cell death directly or potentiate apoptosis induced by Fas ligation or UV irradiation. This effect can be blocked by ZVAD and bcl-2 and most interestingly, by over expression of p38p but not p38a [176]. These seemingly contradictory results point to the complexity of the p38 signalling pathway with regard to cell proliferation and death. The engagement of both the T cell receptor and CD28 is required for the optimal activation of T cells leading to proliferation and differentiation. In murine thymocytes and splenic T cells, p38 is activated by anti-CD3 or anti-CD28 [I771 and the signal is further increased if both ligands are present. The activation of p38 correlated with T cell proliferation and production of IL-2, IL-4 and IFNy ,of these only proliferation could be blocked with a p38 inhibitor. In developing T cells, while M K K l /ERK activation is sufficient for providing selection signals, the MKK6/p38 signalling pathway is involved in negative selection of thymocytes [178]. Neutrophils constitutively undergo apoptosis, the signalling pathway for which is not clear. It is now known that in apoptotic neutrophils p38 is constitutively phosphorylated and activated [ 1791. Inhibition of p38 by

p38 MAP KINASE

34

the use of antisense or an inhibitor delayed onset of apoptosis by 24 hours. Other agents, such as catalase, N-acetylcysteine and GM-CSF altered the rate of apoptosis in a p38 independent manner. Eosinophils, similar to neutrophils, undergo spontaneous apoptosis which can be delayed or prevented by IL-5. SB 203580 (33) increased constitutive apoptosis but this could be overcome by the addition of IL-5 [180]. These results suggest that a basal p38 MAP kinase signal may regulate the survival of cytokine-deprived eosinophils through inhibition of apoptosis. Interestingly, T N F has been suggested to deliver an anti-apoptotic signal partly via the p38 pathway [181]. Erythroid growth and differentiation is regulated by growth factors, such as IL-1 and erythropoietin. TF-1 cells undergo apoptosis upon growth factor withdrawal which can be rescued by IL-1. The apoptotic signal involves activation of JNK and p38 with inactivation of ERK [182]. A p38 inhibitor (SB 203580), but not an inhibitor of the ERK pathway (PD 98059), blocks apoptosis. In another study, SKT6 cells respond to erythropoietin and transient osmotic or heat shock by undergoing erythroid differentiation. Inhibition of JNK or p38, but not ERK, inhibited stress-induced differentiation [183]. Neuronal cells In support of the initial study showing that p38 is involved in N G F withdrawal induced apoptosis of PC12 cells [ 1671,a subsequent study using foetal neurons demonstrated that insulin selectively caused a dramatic decrease in p38 MAP kinase activity and that this effect was correlated with survival [184, 1851. It is also appreciated that the up-regulation of kinases which activate p38, ASK1 or MAPKKK and MKK4/6, is pro-apoptotic [186]. Furthermore, p38 inhibitors promote the in vitro survival of sensory, sympathetic, ciliary and motor neurons in a dose-dependent fashion [187]. Activating transcription factor 2 (ATF-2), a substrate for p38u and JNK3 MAP kinases, is highly expressed in neurons where it appears to play a role in development and survival [188]. The expression and activation of ATF-2 increases following various neurodegenerative insults, such as focal ischaemia. Additionally, the activation of p38 has been linked to: 1) 2) 3) 4)

glutamate-induced apoptosis [189, 1901. hypotonic-induced apoptosis [ 1911. a neuroinflammatory mechanism in Alzheimer’s disease [192]. HIV- 1 glycoprotein (gp120) induced-injury and apoptosis [I 931.

J.L. ADAMS E T A L .

35

Cardiomyocytes Mackay and Mochly-Rosen have developed a cultured neonatal rat cardiomyocyte model to study the effect of ischaemia on p38 activation [194]. Two phases of p38 activation were observed, one at 10 min that lasted less than 1 h and the second which began at 2 h and lasted throughout the ischaemia period. In this model, a p38 inhibitor protected cardiomyocytes against extended ischaemia in a dose-dependent manner. Similar observations have been made by Ma et al. in an isolated rabbit heart model [195]. Based upon the results of experiments in isolated cardiomyocytes infected with adenovirus vectors containing either p38a or p38P and its upstream kinases, Wang et al. linked activation of p38a to cardiomyocyte apoptosis and p38p to hypertrophy [196]. Keratinocytel wound healing Exposure of human kerotinocyte cells to UVB irradiation induces apoptotic morphological changes [197]. Pretreatment of the cells with SB 203580 suppressed UVB induced apoptosis, activation of caspases-1 and -3, and cleavage of poly(ADP)ribose polymerase. The role of ASKl, an upstream kinase of p38 signalling pathway, has been studied [198]. Increased ASKl expression is seen at the healing edge of the process of wound healing where apoptotic keratinocytes are readily detectable, therefore suggesting that the activation of p38 by ASKl may be involved in epithelial apoptosis. Similar studies have been done in spinal cord injury in rats and implicate the ASK1-JNKlp38 pathways in neuronal apoptosis [199]. Hormone-induced apoptosis Adrenomedullin is a potent vasodilatory peptide that increases CAMP, decreases proliferation and increases apoptosis in cultured rat glomerular mesangial cells. It also activates JNK and p38 MAP kinase. A p38 MAP kinase inhibitor reverses the adrenomedullin mediated defect on proliferation and apoptosis while wortmannin, a PI3 kinase inhibitor, only inhibited the apoptotic response [200]. Oestrogen or the oestrogen antagonist (tamoxifen) induces apoptosis in cells expressing the oestrogen receptor. p38 MAP kinase is implicated as p38 MAP kinase activity is upregulated by the respective ligands and an inhibitor of p38 MAP kinase blocks the resulting apoptosis [201].

36

p38 MAP KINASE

Pulmonary physiology RANTES and GM-CSF are thought to play an important role in the allergic inflammation of the airway by acting as chemotactic agents for eosinophils. Similarly, an important role in airway inflammation is ascribed to IL-8, a potent neutrophil chemotactant which also attracts eosinophils and T cells. SB 203580 inhibits RANTES, GM-CSF and IL-8 production in human pulmonary vascular endothelial cells induced by either T N F or IL-1 [202,203]. The early stages of inflammatory cell adherence and diapedesis also appear to be dependent upon p38 kinase activation as suggested by inhibition of LPS- and TNF-induced ICAM- 1 expression on pulmonary microvascular endothelial cells upon treatment with SB 203580 [204]. The recent observation that IL-4-induced release of soluble CD23 by human monocytes was inhibited by SB 203580 [205] suggests that IgE synthesis might also be regulated by p38 kinase. It has been postulated that changes in airway osmolarity contribute to the production of exercise-induced bronchoconstriction and development of the late-phase airway response in asthma [206]. Recently, it has been demonstrated that exposure of bronchial epithelial cells to hyperosmolar medium induces IL-8 expression which correlates with MAP kinase phosphorylation and activation and is inhibited by SB 203580 [207]. In addition, pulmonary hypertension, commonly occurring secondary to COPD, may result from chronic hypoxia [208]. Pulmonary hypertension in response to chronic hypoxia is usually accompanied by remodelling of the pulmonary vessels. Furthermore, pulmonary artery fibroblast subjected to prolonged hypoxic conditions (up to 30 h) results in upregulation of p38 which can be reversed by reoxygenation [209]. Angiogenesis Angiogenesis involves proliferation of endothelial cells. Vascular endothelial growth factor (VEGF) is a potent endothelial selective mitogen and chemotactic agent. The increased migration of endothelial cells upon VEGF treatment results in p38 and ERK, but not JNK activation. p38 kinase appears to be involved in cell migration and microfilament reorganization, while ERK is involved in proliferation of endothelial cells [210]. The inhibition of p38 enhances HUVEC proliferation as a result of R b phosphorylation [211]. However, there are studies which show that p38 inhibitors retard basic fibroblast growth factor (FGF) mediated fibroblast proliferation [212] and tube formation by an endothelial cell line, MSS31

J.L. ADAMS ET AL.

37

[213]. A role for p38 has also been described for smooth muscle cell migration. The migration of cultured tracheal myocytes in response to platelet derived growth factor, IL- 1 and transforming growth factor beta is blocked by a p38 inhibitor [214]. Vira1 infeet ion The role of p38 in viral infection is an area which has recently attracted increased attention. The earliest report linking p38 to viral replication showed that p38 was activated in parallel with NF-tiB by a variety of agents that induced and activated the HIV-1 promoter [215]. The p38 inhibitor, SB 203580 (33), inhibited promoter activity independent of NF-KB. Further analysis of UV activation of HIV-1 suggests that while both p38 and NF-KB are important, they act independently from each other and are not sufficient to trigger a full HIV gene expression response [216]. The work by Kumar et al. was further confirmed in HIV-I infection in human T lymphocytes by the use of anti-sense oligonucleotide and the guanylhydrazone CNI- 1493 [2 171 or by using SB 203580 [218]. Subsequent studies have also shown that p38 activation can be caused by other viruses, such as influenza [219], rhinovirus [220], cytomegalovirus [221], Epstein-Barr virus [222], encephalomyocarditis virus [223], Herpes simplex virus type I [224] and Sindbis virus [225]. However, infection with respiratory syncytial virus leading to IL-8 induction was reported to involve ERK, but not p38 MAP or JNK kinase [226]. Bone biology Several studies have demonstrated that p38 inhibitors are effective in models of bone and cartilage degradation, and these data support the contention that p38 plays a central role in regulating the production of, and responsiveness to, proinflammatory cytokines in bone and cartilage. In osteoblasts, IL-1 and TNF activate p38 and this activation correlated with the induction of IL-6 production by these cells [227, 2281. The p38 inhibitor, SB 203580, inhibited both the upregulation of p38 activity and IL-6 production. In a mouse osteoblast cell line (MC3T3-E1), basic FGF and the calcium ionophore, A23 187, induced IL-6 synthesis which was dose-dependently inhibited by SB 203580 [229]. In the fetal rat bone resorption assay, SK&F 86002 (1) and related CSAIDs, but not COX inhibitors or 5-LO inhibitors (phenidone, SK&F 107469), inhibited PTH-stimulated bone resorption

38

p38 MAP KINASE

in a dose related manner (JCso of 0.5-1 pM) [230]. A role for IL-6 in bone resorption assays has been suggested, as IL-1 induced production of this cytokine was shown to be inhibited in SK&F 86002 treated calvaria cultures [231]. More recently, SB 203580 was shown to inhibit IL-1- and TNF-stimulated bone resorption in a dose related manner with an ICs0 of 0.5-1 pM [227, 2281.

Cart ilagc biology IL- 1 and TNF have been shown to upregulate p38 activity in primary bovine and human chondrocytes and in an SV40-immortalized human chondrocyte cell line TIC28A4 [227, 228, 232-2341. In in vitro bovine and human cartilage models of cytokine-induced matrix breakdown and nitric oxide (NO) production, several interesting effects have been observed following inhibition of p38. Although SB 203580 was shown to inhibit IL-1-stimulated p38 activity in bovine chondrocytes, no inhibition of IL-I -induced proteoglycan or collagen breakdown in cartilage explant cultures could be demonstrated. Neither could any effect be observed on proteoglycan synthesis in bovine cartilage explants at concentrations up to 20pM [235]. A similar lack of activity on proteoglycan breakdown and synthesis was observed with the more selective inhibitor, SB 242235 (98) [234]. In experiments with bovine nasal cartilage Ridley et al. [236] observed some inhibition of proteoglycan synthesis with SB 203580 but, as in our studies, no effects on IL-1 induced proteoglycan breakdown were observed. In an immortalized temperature sensitive chondrocyte cell line, tsT/AC62, Robbins et al. showed that IL-1 induced a rapid phosphorylation of p38 that was inhibited by SB 203580 at 20yM [237]. Although no effects were seen on proteoglycan synthesis or breakdown in bovine cartilage with the p38 kinase inhibitors [234], a potent inhibition of IL-1 -induced nitric oxide (NO) production was observed. This inhibition of NO production was shown to be the result of transcriptional regulation of iNOS gene expression [233, 234, 2381. Surprisingly, treatment of IL- I-stimulated human cartilage or chondrocytes with SB 242235 did not inhibit either IL-1-induced NO production or the induction of iNOS [234]. In other studies using human osteoarthritic cartilage, SB 203580 was shown to inhibit the spontaneous release of NO, but the effects on IL-I-induced NO were not evaluated [239]. Differences between species, cell types and stimuli with regard to inhibition of iNOS by MAP kinase inhibitors, have been reported by others. SK&F 86002 (1) had no effect on the induction of iNOS expression in mouse

J.L. ADAMS ET AL.

39

macrophages stimulated with T N F and IFN-y [240]. In RAW 264.7 murine macrophages stimulated with LPS, neither SB 203580 nor PD 98059, a selective inhibitor of the ERK2 MAPK pathway, was able to inhibit iNOS gene expression [238, 241, 2421. In contrast, Feng et al. [243] described inhibition of LPS-stimulated induction of iNOS in murine peritoneal macrophages with both PD 98059 and SB 203580. In rat endotoxin stimulated primary glial cell cultures, SB 203580 was only able to partially suppress NO and iNOS, whereas in combination with the MEK-1 inhibitor, PD 98059, an almost complete blockage of glial production of NO and iNOS gene expression was observed [244]. Similar results were observed in IL-l-stimulated rat pancreatic islets where specific p38 and ERK inhibitors individually reduced, but in combination completely blocked, IL-1 mediated NO synthesis [245]. In contrast, a p38 kinase inhibitor dramatically diminished NO and iNOS gene expression in mouse astrocyte cultures stimulated with 1 L - l ~and TNF, whereas PD 98059 showed no effect [246]. In addition to NO, several inflammatory mediators are released from chondrocytes/cartilage either spontaneously or upon exposure to IL- 1, for example prostaglandin E2 and IL-6. Studies with the selective p38 inhibitor, SB 242235 (98), showed that the compound inhibited the production of PGE2 from both bovine and human chondrocytes stimulated with IL-I [234]. The spontaneous release of PGE2 and IL-6 but not IL-8 from osteoarthritic cartilage was shown to be inhibited by SB 203580 [239]. In experiments with human synovial fibroblasts, SB 203580 was shown to inhibit IL- 1fl stimulated IL-6 gene expression and protein production [247]. Similarly, in fresh rheumatoid synovial fibroblast cultures, IL-IP and T N F induced the production of IL-6 and IL-8 concomitantly with the induction of p38 activity and SB 203580 blocked the induction of these cytokines [248]. These latter studies are of particular relevance to the findings in the adjuvant arthritic rat (see discussion to follow) where circulating levels of IL-6 are reduced by treatment with a p38 kinase inhibitor. Matrix metalloproteinases (MMPs) are responsible for excessive breakdown of connective tissue and play an important role in the pathogenesis of diseases such as rheumatoid arthritis, osteoarthritis and atherosclerosis. Fibroblasts are a rich source of MMPs and p38 MAPK pathways are involved in the regulation of MMP expression in these cells. In normal human skin fibroblasts, collagenase-3 (MMP- 13) was shown to be induced by culturing the cells in 3-dimensional collagen gels and induction of the enzyme was inhibited by SB 203580, whereas blocking the ERK pathway

40

p38 MAP KINASE

with PD 98059 augmented MMP-13 expression [249]. These authors also demonstrated that SB 203580 could inhibit MMP- 13 expression elicited by TGFB in human gingival fibroblasts [250]. In other studies with human gingival fibroblasts, SB 203580 prevented the increase in collagenase- 1 and stromelysin-1 mRNA stimulated by IL-1 [236]. There was also a dose related decrease in PGE2 and IL-6 from these cells but little inhibition of IL-8 was observed. In a human squamous cell carcinoma cell line (UM-SCC-l), phorbol ester-enhanced MMP-9 secretion and in vitro invasiveness were associated with a strong activation of p38 MAPK. Treatment of the cells with SB 203580 not only resulted in inhibition of MMP-9 secretion, but in vitro invasion was also completely blocked [2511.

ANIMAL MODELS

Inhibitors of p38 have activity in a wide variety of disease models. In all cases, p38 activation in key cell types correlates with disease initiation and progression. Thus, treatment with p38 MAP kinase inhibitors provides an attractive therapeutic strategy for a number of diseases [132, 2521.

Adjuvant-induced arthritis

A commonly utilized model of inflammatory arthritis used for the evaluation of potential antiarthritic agents is adjuvant-induced arthritis (AA) in the Lewis rat. In this model there is chronic inflammation, synovial infiltration and massive bone destruction. Until recently, most therapies for rheumatoid arthritis provided symptomatic relief but did not alter the progression of bone and cartilage destruction in the affected joints. A multitude of cytokines contribute to the overall inflammatory and bone destructive sequelae that occur in rheumatoid arthritis and it has become clear that targetting one or more of these cytokines can ameliorate the disease. Although early p38 inhibitors were active in the AA rat, the contribution of p38 inhibition to their anti-inflammatory activity was unclear as these compounds also inhibited 5-LO and COX-1 [l 1,2531. More recent compounds, such as SB 203580 (33) and SB 242235 (98), have shown excellent activity on inflammation as well as protecting the bone, cartilage and soft tissues of the joint [134, 2541. SB 242235, a more selective kinase inhibitor than SB 203580 which does not inhibit either 5-LO or COX-1, reduced paw oedema with an EDs0 of 30 mg/kg. The protective action

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of SB 242235 on joint integrity has been demonstrated utilizing new technologies such as dual X-ray absorptiometry (DEXA), magnetic resonance imaging (MRI) and micro-CT [134]. In addition to reducing LPS-induced serum T N F levels in normal rats, treatment of AA rats with SB 203580 or SB 242235 inhibited circulating levels of IL-6. Serum IL-6 in AA rats on day 21 of disease are dramatically elevated (2 ng/ml) and this is reduced by 40-70% following treatment with the inhibitors [134, 2541. Several other p38 kinase inhibitors have been described which have activity in models of rheumatoid arthritis. Researchers at Vertex Pharmaceuticals have reported VX 745 ( 8 5 ) to be active in animal models of immune-mediated arthritis [255]. Potent and selective p38 inhibitors have been described by two groups at Merck [93, 1011. L-167307 (70) is one of a series of 2,5-diaryl-3-pyridyl-pyrrolesprepared by de Laszlo et al. that was effective in the AA rat (EDSoof 7.4 mg/kg, b.i.d.) [loll. The Merck group also described a series of diarylimidazole p38 inhibitors, including the aminopyridine (63), which significantly reduced disease progression in the AA rat (EDs0 of 17.5 mg/kg) [93]. RWJ 68354 (81), synthesized at the R.W. Johnson Pharmaceutical Research Institute, is a p38 kinase inhibitor that potently inhibits T N F release in vitro and in vivo, and is active in the AA rat when dosed at 50 mg/kg [256]. Collagen- induced arthritis Collagen-induced arthritis (CIA) in mice has been used as a model to investigate the antiarthritic activity of both anti-inflammatory and disease-modifying agents. This murine model of disease is sensitive to treatment with immunosuppressive drugs and corticosteroids, but relatively insensitive to the action of NSAIDs and some antiarthritic compounds such as gold compounds and penicillamine. Several CSAIDs have been shown to alter the severity of the arthritic lesions and to reduce serum levels of the acute-phase reactant, serum amyloid P component (SAP). Early compounds that were dual inhibitors of arachidonic acid metabolism, as well as inhibitors of p38, were active in this model. These included SK&F 86002 (l), SK&F 104351 (17) and SK&F 105809 (8) [ l l , 253, 2571. More recently CSAIDs that have little or no effect on arachidonic acid metabolism but are potent inhibitors of p38 have shown therapeutic activity in CIA, for example, SB 203580 (33) [227] and SB 220025 (93) [258]. SB 203580 was effective on already established disease when administered at 50 mgl kg b.i.d. The therapeutic effects of p38 inhibitors in CIA may be due, in part.

42

p38 MAP KINASE

to their effects on angiogenesis. Chronic inflammatory diseases are often accompanied by an intense angiogenic response which supports the destructive proliferation of inflammatory tissues. Angiogenesis is a normal physiological response in wound healing, but in diseases such as RA and psoriasis it can take on a pathological role. Using a murine air pouch granuloma model, SB 220025 (93) at 30 mg/kg b.i.d. was shown to inhibit the elevated levels of T N F and IL-lP that occurred during the chronic inflammatory phase of granuloma development and there was a dose-dependent decrease in the vascular density of the granuloma [258]. Thus the inhibition of angiogenesis may contribute to the effectiveness of p38 inhibitors in inflammatory arthritic disease models. Pulmonary inflammation Ovalbumin sensitization of both mice and guinea pigs leads to activation of p38 kinase in the lungs [ 133, 173, 1741. Oral administration of SB 239063 (99), a selective p38 kinase inhibitor, to conscious guinea pigs markedly reduced ovalbumin-induced pulmonary eosinophil influx. SB 239063 also inhibited leukotriene D4 induced persistent airway eosinophilia. In addition, apoptosis of eosinophils from guinea pig bronchoalveolar lavage cells was increased by the compound in the presence of IL-5. A two component mechanism of inhibited eosinophil recruitment and increased apoptosis of eosinophils was postulated. When considered together with the previously discussed in vitro data, these results support the potential utility of p38 kinase inhibitors for the treatment of lung diseases such as asthma and chronic obstruction pulmonary disease.

Stroke The realization that brain ischaemia and trauma elicit inflammation in the brain provided the rationale for the discovery of novel therapeutic agents for stroke and neurotrauma, and for the evaluation of p38 inhibitors in stroke models [259]. Focal ischaemia is a powerful stimulus and produces significant changes in gene expression and enzyme activation that affect the development of injury. In rat models of permanent middle cerebral artery occlusion (MCAO), p38 activity was shown to be increased in the ischaemic tissue [260]. Treatment with SB 239063 (99) resulted in protection against tissue damage and reduced neurological deficit over a significant

J.L. ADAMS ET AL.

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dose and plasma concentration range [260]. Neuroprotection with SB 239063 was confirmed by histopathology, MRI and neurologic deficit analyses [261]. These findings strongly support the utility of targetting p38 for intervention in stroke.

Psoriasis

Inhibitors of p38 kinase also have anti-inflammatory activity when applied topically in murine models of dermatitis. Dose related effects on oedema and PMN infiltration were shown in the oxazolone-induced contact sensitivity model with HEP 689/SB 235699 (59). Inflammation was reduced in a TPA-induced chronic skin inflammation model and chronic oxazolone-induced dermatitis, and the expression of ear tissue IL- lp, IFN-y and IL-4 was inhibited [262]. The topical anti-inflammatory effects of HEP 689 in these murine models support the potential usefulness of p38 inhibitors as topical treatments for human skin disorders such as contact eczema, atopic dermatitis and psoriasis.

Ischaemia/reperfusion

Ischaemia/reperfusion injury in the heart involves activation of the p38 MAP kinase pathway. In studies where heart tissues were used, activation of p38 MAP kinase has been observed during ischaemia, as well as reperfusion [196,263,264]. The role of p38 MAP kinase in response to acute preconditioning stimuli and ischaemia has been studied [265]. Activation of p38 is correlated with preconditioning stimuli such as initial transient ischaemia and is suggested to be protective against lethal ischaemic insult. Inhibition of this event by a p38 inhibitor appears to abolish the preconditioning effect but is protective during the lethal ischaemic phase. This phenomenon has also been seen in isolated tissues.

Immune function

Many compounds that show anti-inflammatory and disease-modifying activity in animal models of arthritis have immunosuppressive activity. In murine T cells, the p38 MAP kinase inhibitor, SB 203580, inhibited the production of interferon-gamma by Thl cells without affecting IL-4

44

p38 MAP KINASE

production by Th2 cells [266]. SB 203580 also inhibited T cell CD28-dependent proliferation and IL-2 production [267] as well as proliferation in response to IL-2 and IL-7. In addition, p38 MAP kinase has been shown to play an important role in the intrathymic signalling of thymocytes [268]. From these studies, it would be reasonable to expect that a p38 kinase inhibitor would be immunosuppressive in vivo. This question has been addressed in murine models of immune function. SK&F 86002 (I), SK&F 104351 (17) and SK&F 105809 (8) exhibited no overt immunosuppressive activity under conditions where the macrolide rapamycin and the corticosteroid dexamethasone abrogated both cellular and humoral responses to soluble antigen [269]. Similar results have been observed when SB 203580 (33) was evaluated on antibody and cellular responses to ovalbumin. In these experiments doses as high as 60 mg/ kg delivered i.p. for 2 weeks were not immunosuppressive [227]. It has been subsequently shown that the inhibition of IL-2 driven T cell proliferation by SB 203580 is not mediated by p38 activation or p38 downstream signalling events, but instead involves a 3-phosphoinositol-dependent protein kinase 1 pathway [270]. This report and earlier reports demonstrating inhibition of JNK [271] and Raf-1 [272] by SB 203580 highlight the potential shortcomings of over reliance on SB 203580 as a tool compound for the elucidation of p38-dependent processes and propose guidelines for the use of SB 203580.

Endotoxin shock

Several of the pyridinylimidazoles have been evaluated in murine models of endotoxemia. The models represent complex physiologic states wherein a number of different mediators play important contributory roles. It is well established that products of arachidonic acid metabolism are involved and an important role has also been established for cytokines, particularly IL-1 and TNF. The effects of lipopolysaccharide (LPS) in these models is greatly enhanced by presensitizing the mice with D-galactosamine or Proprionibacterium acnes and early studies with SK&F 86002 (1) [273] and SK&F 105809 (8) [274] demonstrated that these compounds not only dramatically reduced circulating levels of T N F but also protected the animals from LPS-induced lethality. Similar results have been observed with the more selective p38 inhibitor, SB 203580, which has reduced activity on 5-LO and COX-I [227]. Thus, p38 inhibitors may represent a class of compounds that could be well suited for therapeutic intervention for indi-

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cations related to septicaemia and endotoxemia. However, more clinically relevant models would need to be investigated prior to testing this concept in man.

PRECLINICAL A N D CLINICAL DEVELOPMENT ORAL ACTIVITY

A rodent assay was developed by Griswold et al. as a measure of the in vivo effectiveness of orally administered p38 inhibitors [275]. This assay is based upon the observation that the LPS-induced increase in plasma T N F levels in mice could be blocked by the pyridinylimidazole inhibitors of p38. The LPS-induced T N F assay has moderate throughput and compound requirements (5-10 mg of compound are sufficient to determine the EDso for LPS-induced T N F in a mouse) and has been used as the initial pharmacodynamic screen to evaluate the in vivo activity of over 500 CSAIDs. This assay has also been used to access the oral activity of selected compounds in rats (Table 1.4). The LPS-TNF assay was used by Boehm et al. to discover several orally inhibiactive N-l-substituted-4-(4-fluorophenyl)-5-(pyridin-4-yl)imidazole tors of p38 with improved target selectivity, e.g. devoid of the 5-LO/COX-1 seen with SK&F 86002 and SB 203580 [276]. A preference for alpha branching or a basic group in the N-1 side-chain was found to favour oral activity in this series. One of these compounds, SB 210313 (25) (Table I.I), reduced paw oedema in the adjuvant arthritic rat, thus demonstrating p38 inhibition alone was sufficient to achieve an anti-inflammatory effect. The preferences noted above were further pursued in a series of 4-(4-fluorophenyl)-5-(2-aminopyrimidin-4-yl)imidazoles having cyclic sidechains [277]. Compounds having a piperidinyl (93, 97, 98) or transhydroxycyclohexyl side-chain (94, 99) proved to be potent p38 inhibitors with improved activity in the mouse LPS-TNF assay, whereas the corresponding cyclic oxygen (95) and sulfone (96) analogues were weak p38 inhibitors and devoid of oral activity (Table 1.4).Orally active CSAIDs have also been reported with a cyclic group at C-2 of the imidazole. These include a Merck compound (63) which has the piperidin-4-yl group [93] and a cyclic acetal(lO1) prepared at Rhone-Poulenc Rorer [278]. The ED50 of 0.6 mg/kg in the murine LPS-TNF assay determined for (63) is the most potent in vivo activity reported for any p38 inhibitor. Researchers at R.W. Johnson have reported on an orally-active alkynyl pyridinylimidazole (100) [279].

46

p38 MAP KINASE Table 1.4. ORAL ACTIVITY OF SELECTED P38 KINASE INHIBITORS

compound

p38 kinuse ICSO PM

(1) SK&F 86002 (33) SB-203580 (25) SB-210313 (53) (63) (70) L-167307 (81) RWJ 68354 (93) SB-220025 (94) (95) (96) (97) 88-226882 (98) SB-242235 (99) SB-239063 (100) RWJ 67657

0.57 0.048 1.3 0.48 0.00019 0.005 0.009 0.020 0.083 0.39 0.23 0.027 0.019 0.034 0.030

mouse EDSO

rut ED50

32 15 42 5.2 0.6 (6h pretreat) -


Reference

-

25 11 -

c25 20 -

4.4 4.0 2.6 10

EDso values are given in mgikg, p.0. for inhibition of LPS-induced TNF synthesis

(93) S B 220025 X = NH (94) X =~ux-CHOH (95) x=o (96) X = S q

(97) S B 226882

(98) S B 242235 X = NH (99) S B 239063 X = ~ux-CHOH

H

(1 00) R WJ 67657

(101) RPR200765A

Many of the patent applications report non-pyridinylimidazole p38 inhibitors. Of these the most notable are the Searle applications for

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pyridinylpyrazoles [1021and the novel structures reported by Vertex, including the clinical development compound VX-745 [106, 1161. L-167307, in which a pyrrole is substituted for the imidazole of SB 203580, has improved pharmacokinetics relative to SB 203580 [loll. Finally, the oral activity of four p38 inhibitors which have a bicyclic pyrrolo[2,3-b]pyridine in place of the core imidazole has been described by Henry ef al. of R.W. Johnson [256,280]. The most potent of these compounds was RWJ 68354 (81) which inhibited 85% of the LPS-induced T N F at a dose of 10 mg/kg compared to 33% inhibition at the same dose for SB 203580. A more lipophilic analogue of (81) with improved in vitro inhibition of cellular T N F synthesis was less active in vivo. P450 INHIBITION

The inhibition of cytochrome P450 enzymes has been observed for many members of the pyridinylimidazole class of p38 inhibitors. Since the 4-pyridyl group, which is the key element contributing to P450 inhibition, is conserved in many of the non-imidazole classes of p38 inhibitors (64-82), these compounds may also have undesirably high potency as inhibitors of hepatic P450 enzymes. Recently, successful modifications of the 4-pyridyl group have been described which afford potent and orally active p38 inhibitors free of significant P450 inhibition. These include a (2-aminopyrimidin-4-y1)imidazole SB 226882 (97) [277] and a Merck [2-(a -methyl)benzylpyridin-4-yl]imidazole(63) [93]. An alternate approach to reduce P450 inhibition has been described by McLay and coworkers at Aventis [278]. In this work the 4-pyridyl group was retained and rapid parallel synthesis was employed to identify a novel dioxane analogue, RPR 200765A (lOl), as a clinical development candidate. P450 inhibition would not be expected to be as problematic for the new structural classes of p38 inhibitors (83-92). CLINICAL STATUS

SmithKline Beecham has progressed SB 242235 (98) into clinical development as an orally administered compound for rheumatoid arthritis. Relative to SB 203580, SB 242235 is both more selective as a p38a kinase inhibitor and has improved oral activity (Table 1.4) [134]. Data from a Phase I study demonstrated SB 242235 to be readily absorbed and for doses of 25-500 mg linear pharmacokinetics were seen with a terminal elimination half-life of 15-18 h. Clear inhibitory effects on ex vivo endotoxin-induced cytokine pro-

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p38 MAP KINASE

duction (TNF, I L - l P , IL-6 and IL-8) were observed at doses 2 2 5 mg and were related to SB-242235 plasma concentration [28 11. Vertex has reported VX-745 (85) to be effective at blocking disease progression in animal models of rheumatoid arthritis and stroke. Preclinical studies have demonstrated p38 selectivity over other MAP kinases and VX-745 is reportedly free of P450-associated side-effects seen with the first generation pyridinylimidazoles. The beginning of a small Phase I1 trial in rheumatoid arthritic patients was announced in late 1999 [106]. Recently, researchers at SB and Leo Pharmaceuticals reported topical anti-inflammatory activity for HEP 689 (59) [262]. HEP 689 was originally prepared at SmithKline Beecham (SB 235699) [282] and is closely related to several other compounds described by Adams et al. [277]. The p38 co-crystal structure of HEP689 has been published by the Vertex group, who referred to the compound as VK 19911 [140]. Phase I trials have been initiated by Leo to explore the therapeutic potential of this compound (59) for the treatment of psoriasis and atopic dermatitis [106].

CONCLUSIONS AND FUTURE DEVELOPMENTS The elucidation of many of the key components of signal transduction pathways involved in the regulation of cytokine production and cytokine signalling in immune cells has provided a multitude of novel targets that can be explored to develop new therapeutic agents to treat diseases in which cytokine production or signalling is implicated. One of the first validated targets is p38a MAP kinase, the molecular target of the pyridinylimidazole inhibitors of cytokine production. Whereas the discovery of p38a was driven by the search for the molecular target of pyridinylimidazoles, the MKKl inhibitors PD 98059 [283] and U0126 [284, 2851 exemplify the purposeful targetting of a protein kinase to interrupt a signal transduction pathway. Similarly, inhibitors of JNK, PDE4 and NF-KBhave been reported to regulate cytokine synthesis. These discoveries have set the stage for the development of small molecule inhibitors as therapeutic agents to treat cytokine mediated diseases. As highly selective inhibitors of p38 MAP kinase, the pyridinylimidazoles have proven to be useful research tools for the study of signal transduction pathways and their use has increased our understanding of the complex events occurring at the molecular and cellular level. X-ray crystallography, mutagenesis and mechanistic enzymology have been used to derive a structural understanding of the interactions underlying the kinase selectivity

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of the pyridinylimidazoles which in turn has made possible the design of second generation pyridinylimidazoles, as well as new structural classes of inhibitors with improved potency and selectivity. Unlike the earlier compounds which suffered a number of shortcomings, the second generation p38 MAP kinase inhibitors have much reduced effects on cytochrome P450, improved kinase selectivity and improved in vivo activity. A large and growing literature exists on the in v i m pharmacology of p38 inhibitors. These studies have suggested an array of potential utilities for p38 kinase inhibitors. These include, rheumatoid arthritis, psoriasis and inflammatory bowel disease, plus an ever widening number of diseases which appear to have an inflammatory component, e.g. heart failure, asthma, angiogenic diseases, stroke and other neurodegenerative diseases. Importantly, a number of these suggested indications have now been validated in animal models of disease. With the development of second generation inhibitors which are now entering clinical trials, e.g. SB 242235, VX-745 and HEP 689, we should soon have a clearer picture of the therapeutic utility of p38a inhibition.

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265 Nagarkatti, D.S. and Sha’afi, R.I. (1998) J. Mol. Cell Cardiol. 30, 1651-1664. 266 Rincon, M., Whitmarsh, A,, Yang, D.D., Weiss, L., Derijard, B., Jayaraj, P., Davis, R.J. and Flavell, R.A. (1998) J. Exp. Med. 188, 1817-1830. 267 Ward, S.G., Parry. R.V., Matthews, J. and O’Neill, L. (1997) Biochem. SOC.Trans. 25, 304s. 268 Sen, J.. Kapeller, R., Fragoso, R., Sen, R., Zon, L.I. and Burakoff, S.J. (1996) J. Immunol. 156, 4535-4538. 269 Reddy, M.P., Webb, E.F., Cassatt, D., Maley, D., Lee, J.C., Griswold, D.E. and Truneh, A. (1994) Int J. Immunopharmacol. 16, 795-804. 270 Lali, F.V., Hunt, A.E., Turner, S.J. and Foxwell, B.M. (2000) J. Biol. Chem. 275, 7395-7402. 271 Whitmarsh, A.J.,Yang, S.H.. Su, M.S., Sharrocks, A.D. andDavis, R.J. (1997)Mol. Cell Biol. 17, 2360-2371. 272 Kalmes, A., Deou, J., Clowes, A.W. and Daum, G . (1999) FEBS Lett. 444, 71-74. 273 Badger, A.M., Olivera, D., Talmadge, J.E. and Hanna, N. (1989) Circ. Shock 27,51-61. 274 Olivera, D.L., Esser, K.M., Lee, J.C., Greig, R.G. and Badger, A.M. (1992) Circ. Shock 37, 301-306. 275 Griswold, D.E., Hillegass, L.M., Breton, J.J., Esser, K.M. and Adams, J.L. (1993) Drugs Exp. Clin. Res. 19, 243-248. 276 Boehm, J.C., Smietana, J.M., Sorenson, M.E., Garigipati, R.S., Gallagher, T.F., Sheldrake, P.L., Bradbeer, J., Badger, A.M. and Laydon, J.T. (1996) J. Med. Chem. 39, 3929-3937. 277 Adams, J.L., Gallagher, T.F., Boehm, J.C., Kassis, S., Gorycki, P.D., Gum, R.J., Webb, E.F., Sorenson, M.E., Smietana, J.M., Garigipati, R.S., Hall, R.F., Ayrton, A., Badger, A., Griswold, D.E., Young P.R. and Lee J.C. (1999) in Medicinal Chemistry into the Millenium (Blagbrough, I.S. and Campbell, M.M., eds.), RSC, Cambridge. 278 McLay, I.M., Collis, A., Souness, J., Ratcliffe, A,, Miller, G. and Jayyasi, Z. (1999) American Chemical Society 218th National Meeting, New Orleans, LA, U.S.A. 279 Wadsworth, S.A., Cavender, D.E., Beers, S.A., Lalan, P., Schafer, P.H., Malloy, E.A., Wu, W., Fahmy, B., Olini, G.C., Davis, J.E., Pellegrino-Gensey, J. L., Wachter, M.P. and Siekierka, J.J. (1999) J. Pharmacol. Exp. Ther. 291, 680-687. 280 Henry, J.R., Rupert, K.C., Dodd, J.H., Turchi, I.J., Wadsworth, S.A., Cavender, D.E., Schafer, P.H. and Siekierka, J.J. (1998) Bioorg. Med. Chem. Lett. 8, 3335-3340. 281 Fullerton, T., Sharma, A., Prabhakar, U., Tucci, M., Boike, S., Davis, H.. Jorkasky, D. and Williams, W. (2000) Clin. Pharmacol. Ther., 67, Abst. 01-B-4. 282 Adams, J.L., Garigipati, R.S. and Boehm, J.C. (1995) US patent 5670527; (1997) Chem. Abstr. 127, 307383. 283 Dudley, D.T., Pang, L., Decker, S.J., Bridges, A.J. and Saltiel, A.R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 7686-7689. 284 Jaffee, B. D., Manos, E. J., Collins, R. J., Czerniak, P. M., Favata, M. F., Magolda, R. L., Scherle, P. A. and Trzaskos, J. M. (2000) Biochem. Biophys. Res. Commun. 268,647-651. 285 Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J., Stradley, D. A,, Feeser, W. S., Van Dyk, D. E., Pitts, W. J., Earl, R. A,, Hobbs, F., Copeland, R. A., Magolda, R. L., Scherle, P. A. and Trzaskos, J. M. (1998) J. Biol. Chem. 273, 18623-18632. 286 Griswold, D. E., Adams, J. L., unpublished data.

Progress in Medicinal Chemistry Vol. 38, Edited by F.D. King and A.W. Oxford f 1 2001 Elsevier Science B.V. All rights reserved. ~

2 The Adenosine A3 Receptor and its Ligands JACQUELINEE. VAN MUIJLWIJK-KOEZEN', HENK TIMMERMAN' and ADRIAAN P. IJZERMAN2 Leiden/ Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Department of Pharmacochenzistry, Vrije Universiteit, De Boelelaan 1083, 1081 H V Amsterdam, The Netherlands. 2Leiden/Amsterdam Center f o r Drug Research, Division of Medicinal Chemistry, Universiteit Leiden, PO Box 9.502, 2300 R A Leiden, The Netherlands.

INTRODUCTION Adenosine receptors and their subtypes

62 62

CLONING AND MOLECULAR BIOLOGY

63

PHARMACOLOGY Characterization of the adenosine A3 receptor Species dtffrrences Binding studies Second messenger systems Proposed therapeutic application of ligands Cnrdiovascular system Immunology und respiratory system

66 66 66 69 69 71 76 11

AGONISTS Full agonists Partial agonists

79 79 85

ANTAGONISTS Xanthines Triazolonaphthpyridine and thiazolopyrimidine Flavonoids Pyridines and pyrans I ,4-Dihgdropyridines 3,5-Diaeyl-2,4-dialkylpyr idines

85 85 88 88 89 89 93

61

62

THE ADENOSINE A3 RECEPTOR AND ITS LIGANDS

3.5-Diacyl- 1.2,4- trialkylpyridinium salts Pyrans Triazoloquinazolines Isoquinolines and quinazolines Triazolopyrimidines

95 95 96 97 101

RECEPTOR MODELS

102

CONCLUSIONS

105

REFERENCES

106

INTRODUCTION ADENOSINE RECEPTORS AND THEIR SUBTYPES

The scientific history of adenosine and its receptors began in 1929 when Drury and Szent-Gyorgi described the biological actions of adenine nucleosides and nucleotides in the cardiovascular system [ 11. Four decades later, Burnstock proposed two types of receptor which could be distinguished based on their preference for binding to either adenosine or adenine nucleotides and named them PI and P2 receptors, respectively [2]. Subsequently the PI receptor was divided into Al and A2 adenosine receptors in accordance with their ability to inhibit or to stimulate CAMP production, respectively. Van Calker et al. [3] and Londos et al. [4] provided evidence for the presence of two classes of receptors using adenosine analogues such as PJA and NECA. A further subdivision of the adenosine A2 receptor into AZAand A2Bwas proposed based on high and low affinity towards 2-(4-methoxyphenylamino)adenosine (CVl674) [ 5 ] . The cloning of two different adenosine A2 receptors confirmed the existence of these subtypes [6-91. The pharmacological classification, selective ligands, characterization of the subtypes by cloning, tissue distribution and therapeutic potential of the A , and AZA receptors has been extensively reviewed [lo-141. Recently a review was also published on the adenosine A ~ receptor B ~151. The youngest member of the adenosine receptor family, the A3 receptor, was discovered in 1991 when a sequence from a rat testis cDNA library [ 161 was characterized as an adenosine receptor subtype and called A3 [ 171. The receptor gene has now also been cloned and characterized in several species: human [18, 191, sheep [20], dog [21], and rabbit [22]. This cloned

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adenosine A3 receptor was found to be different from the so-called A3 receptor postulated earlier by Ribeiro and Sebastiiio [23]. They proposed a new receptor subtype due to inconsistencies of agonist potencies in studies on atrial and neuronal tissues, which has since been questioned [24, 251. Today, the cloned receptor is regarded as the adenosine A3 receptor. An adenosine A4 receptor was postulated from binding studies with the tritiated AZA selective agonist, 2-phenylaminoadenosine (CV1808) [26]. This xanthine-insensitive A4 receptor was found in rat brain and claimed to correspond to a receptor found in the porcine coronary artery [27, 281. However, Linden and coworkers [20, 291 questioned the existence of this A4 receptor since the receptor showed comparable anomalous behaviour, depending on the temperature used during the binding assay and the nature of the ligand. Therefore the cloned and pharmacologically characterized adenosine A3 receptor remains the youngest member of the adenosine receptor family described so far and is the subject of this review. CLONING AND MOLECULAR BIOLOGY

The adenosine A3 receptor is the only adenosine receptor whose existence was not defined employing pharmacological evidence. Instead, it was discovered by cloning. The first cloned adenosine A3 receptor was obtained from a rat testis cDNA library by Meyerhof et a/. [16] in 1991, but the authors did not further characterize this G protein-coupled receptor. They noted that the novel cloned receptor showed highest homology to the canine adenosine A , and AZA receptors (47% and 42%, respectively). The cDNA fragment was named tgpcrl. It hybridized strongly and exclusively to a 1.5 kb transcript present in poly(A)+RNA from rat testis and could not be detected in other tissues. One year later, Zhou et al. [17] reported the detection of the so-called R226 clone in a rat striatal cDNA library using a PCR-amplified fragment. R226 was expressed in COS-7 and CHO cells and appeared to encode for a protein of 320 amino acids with seven lipophilic domains. After pharmacological and functional studies they claimed this protein to be a novel adenosine receptor and coined the term A3. Linden and coworkers followed up this work in 1993 with a sheep (hypophysial pars tuberalis) [20] and a human (striatal) [ 191 adenosine A3 receptor clone. About the same time, the human adenosine A3 receptor was also cloned from a heart cDNA library [18]. The rabbit adenosine A3 receptor was cloned likewise by using the PCR amplification technique

64

THE ADENOSINE A3 RECEPTOR A N D ITS LIGANDS

[22]. Initially, rabbit lung tissue was used but the mRNA was also found in liver, brain and heart. It encodes for a protein of 320 amino acids with seven transmembrane domains and highest amino acid identity with human and sheep adenosine A3 receptors, i.e. 76% and 15%, respectively. The homology between the rabbit adenosine A3 and A, receptor is only 46% which is comparable to the 50% A3/A1homology in humans. The homologies between the different cloned adenosine A3 receptors are summarized in Figures 2.1 and 2.2, showing an alignment of the amino acid sequences and a philogenetic tree. The characteristics of clones encoding adenosine A3 receptors are shown in Table 2.1. One of the columns shows the tissue distribution of mRNAs expressing adenosine A3 receptors confirming that the tissue distribution is species specific. The genes for this receptor have been identified in two species, human and mouse. The human adenosine A3 receptor gene (ADORA3) has been localized on chromosome 1 (lp13.3) and the intron/exon structure characterized [30-341. The sequence of the human adenosine A3 receptor promoter region shows many similarities to the human adenosine A, receptor promoter [35]. Both regions contain long 5’ untranslated regions and lack CAAT and TATA box sequences. The latter feature is seen in several other G protein-coupled receptors. The major difference from the human adenosine A, receptor gene is that there was no indication of alternative splicing in the 5’ untranslated region. The mouse adenosine A3 receptor gene has been mapped to chromosome 3 [36]. The mouse and human A3 genes have 71% similarity and the human chromosome lp13-p21 shows high homology with mouse chromosome 3, including the marker D3Mit 11. The existence of a rat adenosine A3 receptor variant has been reported by Sajjadi et al. [37]. The authors isolated a cDNA from rat brain which encodes for a variant form of the adenosine A3 receptor. This Aji receptor contains a seventeen amino acid insert but is otherwise identical to the adenosine A3 receptor. The rat adenosine A3 receptor gene was cloned and sequence analysis of genomic DNA identified a 1.7 kb intron that is likely to alternatively splice to produce the adenosine A3 and A3i receptors. However, stably expressed rat adenosine A3ireceptor in CHO cells resulted only in poor coupling to Gi proteins. Northern blot analysis showed restricted tissue distribution, i.e. expression in spleen and testis. In human tissues, no variant adenosine A3 receptors have been detected despite extensive PCR analysis.

J.E. VAN MUIJLWIJK-KOEZEN ET AL.

SHEEP RAT RABBIT

1 I4P.NNSTA.L M.AVNGTA.L MP.VNSTAV. MIWWTTTSA MP.DNSTTLF

SLA"Y1TM LUNVTYITV SWTSVTYITV LWLQITYITM LAIRASYIVF

HUMAN DOG SHEEP RAT RABBIT

YFIVSLALAD YFIVSLALAD YFIVSLALAD YFIVSLALAD YFIFSLALAD

51

TM2

HUMAN DOG

HUMAN DOG SHEEP RAT RABBIT

%

MSLLAIAVDR MSLLAIAVDR MSLLAIAVDR MSLLAIAVDR MSLLAIAVDR

1

TMl

EIFIGLCAIV EILIQLCAIV EILIGLCAIV EAAIGLCAW EIVIGVCAW

CNMLVIVlNvK LNRTLRTTTF ONVLVIWVIK LNPSLKTTTF

TM3 100

YLRVKLTVRY YLRVKLTVRY YLRVKLTVRY YLRVKLTVRY YLRVKLTVRY

I TM4 :SO KRVTTHRRIW LALGLCWLVS FLVGLTPMFG RRVTTQRRIW LALGLCWLVS FLVGLTPMFG RRVTMRRIW LALGLCWLVS FLVGLTPMFG RTVTTQRRIW LFLGLCWLVS FLVGLTPMFG RRVTTQRRIW LALGLCYWVS LLVGFTPMFG

v s d w SSVMRMDYMV

RSVMRMDYMV RSWOLDYMV DSVIPMEYMV

201 DIFYIIRNKL DIFWIRNKL DIFYIIRNRL DIFYIIRNKL YIFYIIRNKL

SLNLSNSKET NQNFSSSKET SQSFSGSRET SQNLTGFRET VQSFSSFKET

SHEEP RAT RABBIT

W Y p N o E INCITYFHGE INCILYFDGQ INFVSYFNVK INCVTYFKCK

jp~LvLyM01LL?!!M VPQIILYLGI LLSHANSMMN VpOnnYLGI LLSHANSMMN IPEIAMCLGT LLSHANSMMN VPDWLLVGI L L S H A "

HUMAN DOG SHEEP RAT RABBIT

301 320 ACWCHPSDS LDTSIEKNSE TYMICQSSDS LDSSTE.... ACVMCQPSKS MDPSTEQTSE ACRLCQTSDS LDSNLEQTTE ARVTCQPSDS LDPSSEQNSE

wo

QIWTAVIWVVKLNPSLQTWF

GNVLVIWVVK LNPSLQTTTF

A~WSLGITI AIVISLGITI AIVISLGVTI AIAVSLEVQM AIVISLGITI

151 m T S E m WFLsCQF DOG WNMKLTSEHQ RNVTFLSCQF SHEEP WNMKLSSA.D ENLTFLPCRF RAT W"LELS QNSSTLSCHF RABBIT WNMKPTLESA RNYSDFQCKF

DOG SHEEP RAT RABBIT

LNPS&F

IAVGVLVMPL IAVGVLVMPL IAVGVLVMPL IAVGVLVIPL IAVGFLVMPL

HuMAN

HUMAN

GMVLVIh

65

H F Y ~ L F M T CLLLIFTHASI

QFYNCLFMTC LLLIFTHASI HFYSCLFMTC' LMLIFTHASI HFYACLFMSC VLLVFTHASI GFYSCLVMSC LLLVFTHASI

TMS YFSFLTWIFI YFSFFTWILI YFSFPLWILV FFSFITWILI FFSFFTWILI

00 PL-CAIYL PLWMCAIYL PLVVMCAIYF PLVVMCIIYL PLLLMCALYV

TM6 250 I GAFYQREFKT AKSLFLVLPL FALSWLPLSI GAFYGREFKT AKSLFLVLFL FAFSWLPLSI GAFYQREFKT AKSLLLVLFL FALCWLPLSI RAFYGREFKT AKSLFLVLFL FALCWLPLSI GAFYRREFKT AKSLFLVLAL FAGCWLPLSI P I V ~ A PIVYAYKIKK PIVYAYKIKK PIVYACKIKK PIVYACKIQK

300 F~-LII LK ~ FKETYLLIFK FKETYLLILK FKETYFVILR FKETYLLIFK

Figure 2.1 Alignment of the amino acid sequences of the adenosine A J receptors of sheep, dog, humtm, rabhit, and rat [39].

THE ADENOSINE A3 RECEPTOR A N D ITS LIGANDS

66

sheep receptors

dog human

--

A1

rabbit rat 70 80 90 100 Percentage sequence identity

Figure 2.2. The philogenetic tree relating the amino arid sequences between the dqjerent adenosine receptors. On the right hand side is the udenosine A 3 receptor philogenetic tree in more detail [39].

PHARMACOLOGY CHARACTERIZATION OF THE ADENOSINE A3 RECEPTOR

The adenosine A3 receptor was characterized pharmacologically for the first time by Zhou et al. [ 171 after cloning and expression of the rat adenosine A3 receptor in COS-7 and CHO cells. The rat adenosine A3 receptor binds [12%]-APNEAwith high affinity. This binding was inhibited by adenosine receptor agonists with the affinity order of (R)-PIA = NECA > ( 9 - P I A > adenosine. The xanthine antagonists (DPCPX, XAC and isobutylmethylxanthine) did not displace the ['251]-APNEA binding [17]. Adenosine agonists inhibited forskolin-stimulated CAMP accumulation through a pertussis toxin-sensitive G protein with potency order (R)-PIA = NECA > CGS21680, indicating that the adenosine A3 receptor is coupled to Gi proteins. Species clifferences

In the initial studies, a remarkable species difference became apparent. In radioligand binding studies various adenosine receptor ligands were tested on the adenosine A3 receptor from different species. From these data and from the tissue distribution (Table 2.1) it became clear that the rat adenosine A3 receptor behaves anomalously (Table 2.2). It is remarkable

Table 2.1. CLONES ENCODING ADENOSINE A3 RECEPTORS

Species

Accession number CenBank data base / EMBL Data Librarji

cDNA library

Number of amino acids

Rat Rat

X59249 M94152

hgtl 1 testis hgtl 1 brain

320 320

Rat Sheep

X93219 -

Brain and spleen AZapII pars tuberalis

337‘b’ 317

Human Human

L22607 L20463

Striatum hZapII heart

318 319

Human

L77729 L77730

#HL1010b testis

Human Dog Rabbit

U54792

hZAP eosinophil hgtlO BRcell Lung

319 314

Tissue distribution (density)

Ref

Testis‘”’ Testis >> kidney, heart > lung > cortex, striaturn, olfactory bulb Spleen >> testis Lung, spleen > pars tuberalis, pineal gland > striatum, cerebellum, cortex, hypothalamus, testis, kidney Lung, liver > z aorta, brain > testis Placenta, lung > liver, kidney z heart > brain Corpus callosum, substantia nigra, thalamus, subthalamic nucleus, spinal cord, hippocampus adrenal cortex, adrenal medulla > spleen, intestine, aorta, eosinophils > lung Liver, lung, placenta, brain Spleen >> lung, liver heart, kidney, testis Lung, liver, brain, heart

1161 [17] [371 [28]

[19] [I81 [33]

~ 3 1 321 ,

POI 1211 . .

‘“’Dixonef al. [38] have published a review of the tissue distribution of the adenosine receptors mRNAs in the rat, including the distribution of the adenosine A3 receptor. ‘b’Thefull-length receptor A3,cDNA encodes 337 amino acids and shares complete sequence identity with the rat A3 adenosine receptor, except for the presence of a seventeen amino acid insert located in the second intracellular domain.

m

m

Table 2.2. AFFINITIES OF ADENOSINE RECEPTOR LIGANDS FOR THE ADENOSINE A, RECEPTOR IN DIFFERENT SPECIES Species

Potency order agonists

Potency order antagonists

Ref:

Rat

IB-MECA > R-PIA NECA > CGS21680 > S-PIA > adenosine > ATP=ADP

Xanthines do not bind'"'

[I7, 41, 421

Sheep

IABA > NECA > R-PIA >> S-PIA >> CPA

IABOPX > BWA1433 z S P X r XAC >> DPCPX

POI

Human

IAB-MECA > AB-MECA > R-PIA > AB-MECA > CPA CCPA S-PIA > CGS21680

XAC > CGS15943 >> DPCPX > Theophylline'"

[19, 41, 43451

Rabbit

IB-MECA = IABA >> NECA > CPA

XAC > BWA1433 EE DPCPX > > SPT

[22]

Chinchilla

[I2'I] APNEA and R-PIA bind

Theophylline does not bind at 100 pM

[46]

Dog

IB-MECA > R-PIA =NECA > CGS-21680"'

IABOPX > C P X r X A C >> BWA1433 [21]

R-PIA >ABA

'"'Varani et al. [41] reported a K , value of XAC of 29 FM for displacement of specific ['251]-APNEAbinding at rat adenosine A3 receptors expressed in CHO cells. ("IUotz et al. [43] reported a lower K, value for CGS15943 than for XAC (see Table 2.3) "'The potency order for binding differs from the potency order of agonists to stimulate canine mast cell degranulation [21].

J.E. VAN MUIJLWIJK-KOEZEN ET AL.

69

that the A3-affinity order of agonists in rat [(R)-PIA = NECA > CGS21680 > (8-PIA] differed from the affinity order of the same agonists in other species [NECA > (R)-PIA > ( 8 - P I A > CGS216801. A recent study on the haemodynamic effects of A3 agonists IB-MECA and Cl-IB-MECA clearly illustrated the species differences [40]. The agonists produced different effects in rat, rabbit and pig heart. From the alignment of the amino acid sequences and the dendogram shown in Figures 2. I and 2.2 it became clear that the rat adenosine A3 receptor has a low homology with the adenosine A3 receptor of other species. These data seem to indicate that the rat adenosine A3 receptor represents a different subtype, although definitive proof is lacking. Binding studies Although various research groups have reported the same potency order of agonists and antagonists on human adenosine A3 receptors, the absolute Ki values differ from study to study. We therefore show the differences in Ki values of ligands at the human adenosine A3 receptor in Table 2.3. Ki values differed not only when different assays were used, but also when the same cell line and radioligand were used. The reasons for these discrepancies have not yet been clarified. The binding data within each of the following tables have been determined under the same circumstances. SECOND MESSENGER SYSTEMS

The adenosine A3 receptor belongs to the class of G protein-coupled receptors and activation of this receptor therefore results in stimulation or inhibition of different effectors. The adenosine A3 receptor inhibits the enzyme adenylate cyclase via Gial and Gia2 proteins [ l l , 501 and stimulates phospholipase C, probably via the Go protein. It also mediates phospholipase D activation [5 1, 521. However, some confusion exists about the identity of the G proteins coupled to the adenosine A3 receptor. Palmer et a/. [53] reported a weak coupling to the Gq, protein. Other reports [5 1, 541 showed that the NECA-induced potentiation of the antigen-stimulated Ca2+ and IP3 responses are pertussis toxin-sensitive, excluding coupling to the G, protein. Phospholipase C is activated by the adenosine A3 receptor in a G protein-dependent manner [55] and together with the pertussus toxin-sensitive PIP2 hydrolysis, it was concluded that the adenosine A3 receptor is coupled to the Go protein. In summary, activation of the adenosine A3 receptor results in a decrease in CAMP concentration via

THE ADENOSINE A3 RECEPTOR AND ITS LIGANDS

70

Table 2.3. AFFINITIES OF ANTAGONISTS AT THE HUMAN ADENOSINE A3 RECEPTOR REPORTED BY DIFFERENT RESEARCH GROUPS Compound

hA3 K i ( n M )

Reference

~

XAC

CGS15943

DPCPX

L-2493 13 'a'Displacement cells. 'b'Displacement '''Displacernent 'd'Displacernent '"'Displacement

49 f5'"' 71'6' 92"' 108 f3.0'"' 7.9 f 4.6'd' 14 f 2.4@' 30'e' 51"' 95 f9'"' 143 ic 19'"' 200 f 126'd' 759'b' 1300 & 1700 f 160'"' 3960'"' 13'"' I72 f9.l@) of specific ['251]-AB-MECA binding at A3 receptors expressed in HEK 293

of specific ['251]-ABAbinding at A3 receptors expressed in CHO cells. of specific [3H]-NECA binding at A3 receptors expressed in CHO cells. of specific [I2'I]-AB-MECA binding at A3 receptors expressed in CHO cells. of specific ['251]-ABAbinding at A3 receptors expressed in COS-7 cells [49].

the Gi protein and in an increase of IP3 concentration and thus in an increase of the intracellular Ca2+ concentration via the Go protein. This rise in calcium concentration is in some cases [56, 571 caused by a release from intracellular stores rather than by an influx of extracellular Ca2+alone [58]. Furthermore, stimulation of the adenosine A3 receptor in the human eye results in activation of chloride channels [59-621. In studies on cardiac tissue it appeared that K-ATP channels are involved in the protective function of adenosine A3 receptor agonists [63, 641. The effector mechanisms are depicted schematically in Figure 2.3. On the basis of these effectors, several in vitro cell systems have been characterized and developed. The functional effects of stimulation of the adenosine A3 receptor in primary tissues or cell cultures and in cell lines has been extensively reviewed by Jacobson [65]. Recombinant receptors, stably expressed in CHO or HEK 293 cells, have commonly been used for the purpose of screening for new ligands in either radioligand binding

J.E. VAN MU ILWIJK-KOEZEN ET AL.

71

(adenosine] \ Ca2* channel

adenylate cyclase Figure 2.3. Scheinatic representation o f t h e adenosine A3 receptor coupled to udenylute ryclasr, phospholipases C and D, and chloride and calcium channels (the latter only at high concentrations of agonist). The change in concentration of second messengers uctivates post receptor processes and leads to physiological responses. Abbreviations used: A TP, adenosine 5'-triphosphate; C A M P . cyclic adenosine 3'. 5'- monophosphate; DAG. diacylglycerol; 19,. inositol 1,4,5-1risphosphute; PIP2. phosphaiidinylitiositol-4,5-bipl~osphute;PKA, C A M P dependent protein kinase A: PKC, protein kinase C.

studies or CAMPassays [17]. Zhao r t al. [66] warned that the regulation of the adenosine A3 receptor gene expression and intracellular CAMP levels are coupled and that for this reason one should be aware of the modulation of the adenosine A3 receptor gene expression after manipulation of CAMP levels. Furthermore, ["SI-GTPy[S] binding has been used as a functional assay. Agonists stimulate [35S]-CTPy[S] binding which can be inhibited by antagonists [67-691. The GDPIGTP exchange is a measure of the intrinsic activity. A ligand's intrinsic activity can be readily determined with [35S]-GTP, which is especially useful in studying partial agonists. PROPOSED THERAPEUTIC APPLICATION OF LIGANDS

Many therapeutic applications have been suggested for ligands of adenosine receptors on the basis of the wide variety of physiological actions mediated by adenosine [70]. An overview of the potential therapeutic uses of adenosine A3 ligands is presented in Table 2 . 4 , followed by a short section

Table 2.4. PHYSIOLOGICAL EFFECTS MEDIATED BY THE ADENOSINE A3 RECEPTOR CNS actions

Species

Ligand

Brain ischaemia

Gerbil, in vitro

Agonist (22) Pretreatment

Protective

Gerbil, in vivo Gerbil, rat, in vitro Gerbil, rat, in vivo

Agonist Acute Agonist (27) Acute, high concentration Antagonist Acute or (78) pretreatment

Lethal Cell death

Administration Effect

Mechanism

Preserves microtubule associated protein 2 (MAP2) depresses NO synthase

Protective

Locomotor activity Mouse, in vivo Transmitter release Rat, in vitro

Agonist (22) Acute

Depressant

Agonist

Inhibition of synaptic transmission via mGlu Receptor

PKC-dependent

Nervous system Human, rat, in vitro development, response to trauma and ischaemia

Agonist (27) Acute

Modulation of the astrocytic cytoskeleton

Mediates cell spreading, reorganization of actin cytoskeleton and intracellular distribution of the antiapoptotic protein BCL-XL

Acute

ReJ

Table 2.4 continued. Cardiovascular actions

PHYSIOLOGICAL EFFECTS MEDIATED BY THE ADENOSINE A3 RECEPTOR

Species

Ligand

Administration Effect

Myocardial ischaemia

Blood pressure

Human, chicken, rabbit Dog, rabbit, in vitro. ex vivo

Agonist Acute (221, (27) Agonist (27) Acute

Rabbit

Agonist (20)

Chicken, in vitro Rat, in vitro

Agonist

Rat, in vivo Hamster, in viva

Pretreatment

Agonist (22) High dose. acute Agonist Acute (31, (27) Agonist (1) Acute

Rat, rabbit in vitro

Pig, in v i w Coagulation

Mechanism

ReJ

-

~~

Human, in vitro

Agonist ( 3 ) , (22), (27) Agonist (3)

Acute Acute

Protective, preconditioning Attenuated reperfusion injury

[80-841

Decreases neutrophilendothelial cell interactions Reduced myocardial K-ATP channels ischaemia & reperfusion injury Protective

u

rn

Apoptosis Hypotension Vasoconstriction

Histamine and other mediators from mast cells Histamine and thromboxane from mast cells

Coronary dilatation Histamine induced decrease in systemic pressure & increase in pulmonary pressure Rapid hypotension, died Anticoagulant Down-regulates the endothelial cell tissue factor expression

~401

Irl Y h

P

W . l

4

P

Table 2.4 continued.

PHYSIOLOGICAL EFFECTS MEDIATED BY THE ADENOSINE A3 RECEPTOR

Peripheral actions

Species

Ligand

Administration Effect

Immune system

Human, in vitro

Agonist

High concentration

Antagonist (78)

Low concentration

Human, in vitro Human , in v i m

Human, in vitro Human, in vitro Cancer

Mouse,

Agonist (27) Low concentration Agonist (27) High concentration Agonist Acute

in vitro

Mouse, in vitro

Agonist (3)

Acute

Mechanism

Apopt osis Apoptosis Apoptosis

Protects against cell death Apoptosis

Re/ r561

Pro-apoptotic Bcl-2 homology protein BAK

1941 r951

M V

4

0 icc

2

U

Inhibits MHCunrestricted killing of P8 15 tumour target cells by anti-CD3activated killer lymphocytes Inhibits the adhesion of anti-CD3activated killer lymphocytres to adenocarcinoma cells

Inflammation

Murine, in vitro

Agonist (3)

Human, in vitro

Agonist (27) Acute

Human. in vitro

Agonist (22) Acute

Acute

Mouse, in vitro

Agonist

Mouse. in vivo

Agonist (22) Pretreatment

Respiratory system Rat, in vivo Human, in vitro

Pain

Acute

Rat. in vivo

Rat, in vivo

Agonist ( 3 ) Agonist (I), (22) Agonist (12)

Acute

Acute Acute

[28,88Histamine and other mediators from mast 90, 981 cells Inhibition of [99, 1021 Interrupts the endotoxin TNF-a CD14 receptor signal transduction pathway Inhibition of inflammatory [103,104] cell chemotactic responses to PAF, LTB or RANTES Suppression of MIPor, decrease[l05, 1061 of neutrophil levels (involved in collagen-induced arthritis) Mediator release Not by CAMP, maybe ~071 calcium involvement Decreases interleukin-12 [I 081 Protects against lipopolysaccharide- and interferon-y production induced lethality Bronchospasm Mediators from mast cells [109, 1101 Inhibition of Inhibition of fMLP-triggered [11I] NADPH oxidase activity, respiratory burst calcium independent Pain enhancement, Histamine and [112-1141 5-hydroxytryptamine from oedema mast cells

?

m 4

% z

s

F

2

2

AfN 2! L

!-

16

THE ADENOSINE A3 RECEPTOR AND ITS LIGANDS

in which some pathological phenomena implicating a role for the adenosine A3 receptor are discussed. At the time of writing (this review contains the literature up to and including 1999) only agonists, partial agonists and antagonists have been described. Inverse agonists or allosteric modulators for the adenosine A3 receptor have not yet been described. The endogenous agonist adenosine fulfils a two-fold role: a homeostatic role under normal, physiological conditions and a protective role in emergency situations. High metabolic rates and insufficient oxygen supply cause an increase in ATP breakdown resulting in a high adenosine concentration.

Cardiovascular system This dual function of adenosine is clearly demonstrated by one of the first therapeutic applications proposed for ligands of the adenosine A3 receptor: protection by adenosine A3 receptor agonists in stroke. Firstly, agonists were shown to be protective in cerebral ischaemia models after chronic administration [71, 721 which is consistent with the adenosine receptor’s function during emergency situations. However, acute administration of a higher concentration of the adenosine A3 receptor agonist caused a lethal effect [71,73]. Furthermore, the use of an adenosine A3 receptor antagonist (acute administration or pretreatment) in stroke was also shown to be protective [75, 761. This paradox might be explained by the homeostatic function of adenosine. Adenosine A3 receptor activation (pathological at very high concentrations of adenosine) causes a heterologous desensitization of adenosine Al receptors which in turn limits the cerebroprotective effects of adenosine under these extreme conditions [75]. This effect is counteracted by an A3 antagonist. The paradox reported in the use of adenosine A3 receptor agonists in brain and cardiac ischaemia, i.e. protective at nanomolar and lethal at high concentrations ( B10 pM) [73], demonstrates again that toxicity always depends on the applied dose. The protective versus the lethal effects of adenosine A3 receptor activation have recently been discussed [65,115,116]. The authors suggested the existence of a tonic state of activation of the adenosine A3 receptor, possibly induced by release of endogenous adenosine, that results in cell protection. According to this theory, a blockade of the protective action of endogenous adenosine by an antagonist, or high concentrations of agonist [95], results in apoptosis, whereas the protective effects depend on the level of receptor activation and the paradigm studied. However, the mechanisms involved are not yet fully understood.

J.E. VAN MUIJLWIJK-KOEZEN ET A L .

77

There is promising therapeutic potential for adenosine A3 receptor agonists in cardiac ischaemia. Several studies showed a protective role for A3 agonists [80-85, 1171. The adenosine A3 receptor can even mediate a more sustained cardioprotective effect in ischaemic preconditioning than the adenosine A , receptor [64]. Von Lubitz, however, warned that use of A3 agonists in protecting the ischaemic heart may lead to damage to the brain [ 1 161. Recently, it was reported that the adenosine A3 receptor plays a role in the coagulation system during ischaemic or artherosclerotic processes [93]. Stimulation of the adenosine A3 receptor down-regulates the endothelial cell tissue factor expression induced by TNF-a, phorbol 12-myristate 13-acetate (PMA), or thrombin. This is the main trigger factor of clotting activation in thrombotic processes associated with coronary ischaemic disease, stroke and malignancy. Thus, in addition to its antiplatelet activity via A2 receptors [118], adenosine could act as an anticoagulant factor by stimulation of the adenosine A3 receptor.

Immunology and respiratory system The release of histamine and other mediators from mast cells by activation of the (rat) adenosine A3 receptor with synthetic agonists was extensively studied both in vitro and in vivo [87-91, 98, 1191. In vifro studies on rat and guinea pig and in vivo studies on hamster using inosine as the A3 selective agonist showed that mast cell degranulation is facilitated by calcium ion mobilization [93, 1201. The concentration of inosine is raised by intracellular uptake and deamination of adenosine. The mast cell degranulation has a number of physiological effects, such as processes of inflammation [20] and bronchospasm [109, 1101. Inflammatory mediators up-regulate adenosine A3 receptor expression in RBL-2H3 cells, possibly through activation of nuclear factor KB [121]. Selective adenosine A3 receptor antagonists were initially thought to be antiasthmatic based on the involvement of mast cell degranulation in inflammation and bronchoconstriction [20, 122, 1231. Later in vitro studies on mast cells of human and dog showed that in these species no mast cell degranulation took place after stimulation of the adenosine A3 receptor, but that the adenosine AZBreceptor appeared to be involved [21, 119, 124, 1251. However, adenosine A3 receptor antagonists may be important in respiratory illnesses other than asthma. They could be useful in the treatment

THE ADENOSINE A3 RECEPTOR A N D ITS LIGANDS

78

of allergic disease by inhibition of eosinophil transmigration or infiltration into airway lumen tissue [94, 1261. With regard to inflammatory diseases, the adenosine A3 receptor remains an important therapeutic target, because of its potential to modulate the production of pro- and anti-inflammatory cytokines. In particular, A3 agonists could be a starting point for drug development by inhibition of the production of the proinflammatory cytokine, TNF-a, by suppression of its mRNA levels [99-1021. Furthermore, activation of the adenosine A3 receptor affects interleukin-10 (IL-10) production, which in turn negatively regulates the levels of TNF-a and nitric oxide (NO') [log]. Also MIP-a and neutrophil levels are suppressed by adenosine A3 receptor activation [105, 1061 and the migration of PAF, LTB and RANTES is inhibited [103, 1041. Taken together, these results indicate that adenosine A3 receptor agonists may be useful in the treatment of acute or chronic inflammatory diseases such as septic shock or arthritis. In conclusion, the adenosine A3 receptor is an important new target for drug research because of its involvement in regulatory systems and first aid function under extreme conditions. However, a better understanding of the specific interactions in inflammatory and immunological processes and the CNS, cardiovascular and respiratory systems is needed.

8'

Compound

R'

(1) Adenosine

H NH2 H OH N H C H ~ C H Z ( ~ - N H ~H) P ~ NHCH(Me)CH2Ph H H NHCsHg CI NHC=,Hg NHPh NH2 NH(4-Me0)Ph NH2 NHCH,CH2(4-C02Et)Ph NH2

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

Inosine APNEA R,S-PIA CPA CCPA CV1808 CV1674 CGS 21680

R2

R3 CH20H CH2OH CH20H CH2OH CHzOH CHIOH CH2OH CH2OH C( = 0)NHEt

J.E. VAN MUIJLWIJK-KOEZEN ET AL.

79

AGONISTS FULL AGONISTS

The K, value of the endogenous agonist adenosine (1) for the adenosine A3 receptor is in the micromolar range [17]. This affinity is very low compared to the adenosine K , values of 10-100nM for the adenosine A, and AZA receptors [ll]. Therefore the question has been raised as to whether adenosine is indeed the endogenous agonist. Another candidate for the endogenous ligand is the adenosine metabolite inosine. In situations of metabolic stress such as inflammation or ischaemia, extracellular concentrations of inosine can be high, exactly those situations in which the adenosine A3 receptor has been suggested to play an important role. Inosine binds selectively to adenosine A3 receptors of rat and guinea pig with ICso values of 25 pM and 15 pM, respectively [ 1201. Furthermore, inosine was shown to cause dose-dependent mast cell degranulation resulting in vasoconstriction, probably via adenosine A3 receptors [92, 120, 127, 1281. To date, it is unclear whether adenosine or inosine is the endogenous agonist for the adenosine A3 receptor. All known adenosine receptor agonists are structurally closely related to adenosine itself. Modifications of the adenosine structure may lead to more or less selective agonists for the different adenosine receptors (Figure 2.4). A review on Al and A3 agonists was recently published [129]. Disubstitution of adenosine (1) at both the N 6 and 5’ position has been shown to greatly enhance affinity and selectivity for the adenosine A3 receptor [ 1301. The requirement for modifications at two sites simultaneously is illustrated by comparison of N6-benzyladenosine (lo), NECA (1 1) and N6-benzylNECA (12) (Table 2 . 5 ) . Jacobson and coworkers [42, 1311 have investigated the structure-activity as adenosine relationships of a series of N6-benzyladenosine-5’-uronamides A3 receptor agonists. In this class, 5’-substituents enhanced adenosine A3 receptor selectivity in the order N-methyl (14) > N-ethyl (1 1) = unsubstituted (1 3) > N-cyclopropyl (1 5 ) (TabZe 2.5). Recently, it was found that in the human species, a 5’-N-cyclopentyl substituent improved A? selectivity [132] although to a lesser degree than a methyl substituent. Substitution of N6-benzyl-MECA and N6-benzyl-NECA showed that meta substituted benzyl derivatives favour adenosine A? receptor potency and selectivity (Table 2.6). It was also found that positional steric effects appear to be more important than electronic effects. Substitution at the para position provided selectivity for A3 versus A ~ A

THE ADENOSINE A3 RECEPTOR AND ITS LIGANDS

80

hydrophobic moieties: elkyl,awl, cycloalkyl

OH

OH

3-substituted benzyl or cerbamoyl, methoxy

4-halogenated phenyl

\

N-alkyl carboxamlde ethyl, cyclopropyl C)

phenylethynyl, CI

8

phenylpropynyl

OH

OH

__t

N-alkyl carboxamide, OH OAcyl, vinyl, methoxy

OH

d)

Figure 2.4. The structure of adenosine and the modifications leading to ( a ) A,: ( b ) A ~ Aand ; (d) A , selectivity: and ( c ) A I R potency.

receptors. Halogen substitution at the meta position resulted in adenosine A3 selective compounds with potency order I = C1 > Br > F. These investigations led to IB-MECA (22), which is a highly potent and selective adenosine A3 receptor agonist in vitro (human [ 1331 ,chicken [80] and rabbit [134] ) as well as in vivo (gerbil [71] and mouse [lo81 ). AB-MECA (23) has been radioiodinated with the aid of a chloramine-T reaction [ 1351. The radiolabeled ['251]-AB-MECA (25), although less selective than IB-MECA (22), is widely used as high-affinity radioligand for the adenosine A3 receptor. The adenosine analogue of (25), ['251]-ABA, is also used as an A3 radioligand. Starting from IB-MECA (22), an irreversible ligand for the adenosine A3 receptor was designed [136]. This derivative (26) bears a 3-isothiocyanobenzyl group instead of a 3-iodobenzyl group and was reported to be potent and selective versus A l and A2* receptors.

J.E. VAN MUIJLWIJK-KOEZEN ET AL.

81

Table 2.5. AFFINITIES O F AGONISTS (10-15) AT RAT ADENOSINE RECEPTORS DETERMINED IN BINDING ASSAYS, EXPRESSED AS Ki (nM)

OH

OH

Cornpound

R

R'

A,

AZA

N6-Benzyladenosine (10) NECA (11) N6-BenzylNECA (12) (13) MECA (14) (15)

Benzyl H Benzyl H H H

CH20H CONHEt CONHEt CONH2 CONHMe CONHcPr

120 6.3 87 13 84 6.4

285 10 95 120 67 13

A3

120 113

6.8 1410 72 1600

ReJ [5] [5] [I301 [42] [42] [42]

Table 2.6. AFFINITIES O F O F MECA A N D NECA DERIVATIVES (16-24) AT RAT ADENOSINE RECEPTORS DETERMINED IN BINDING ASSAYS, EXPRESSED AS K, (nM)

OH

(12) (16) (17) (18) (19) (20) (21) IB-MECA (22) AB-MECA (23) IAB-MECA (24)

Et Et Et Et Me Me Me Me Me Me

H 2-OMe 3-OMe 4-OMe H 3-CI 3-Br 3-1 4-NHz 3-1-4-NHz

OH

87 52 69 209 898 916 65 54 43 1 18

95 21 38 609 597 559 64 56 I590 197

6.8 7.1 4.3 11 16 22 1.9 1.1 14 1.3

[1301 [421 ~421 [421 [421 ~421 [421 [421 ~421 ~421

THE ADENOSINE A, RECEPTOR A N D ITS LIGANDS

82

Compound

H-N

['*'I]-AB-MECA (27) 2-CI-IB-MECA

R'

R2

R3

H H CI

I251

NCS I

NH2 H H

HO OH

R (28) PENECA (29) R,S-PHPNECA HO

Ph CH(0H)Ph

OH

Further studies have shown that 2-substitution of the purine ring may enhance selectivity for the adenosine A3 receptor [ 137, 1381. 2-Methyl substitution decreased adenosine receptor affinity, but at A, and A Zmore ~ than a t A3. Bulky 2-substituents as in CGS 21680 (9) and APEC were well tolerated for binding to adenosine A3 receptors. Substitution of (22) at the 2-position with a chloro atom, leading to 2-C1-IB-MECA (27) increased adenosine A3 receptor affinity and selectivity. Compound (27) has a Ki value of 0.33 nM at rat adenosine A3 receptors and both in vitro [75] and in vivo [90] is probably the most potent and selective adenosine A3 receptor agonist available to date [139]. A series of 2-alkynyl NECA analogues has been developed by Cristalli et al. [140, 1411. A phenyl ring conjugated to a triple bond as in phenylethynylNECA (PENECA, 28) enhanced adenosine A3 receptor selectivity. Furthermore, the authors demonstrated that PHPNECA (29), although lacking an N6 substituent, shows high A3 receptor affinity (hA3 Ki = 0.42 nM). Instead of the N6-benzyl group, other N6 substituents such as carbamoyl, arylcarbamoyl and carboxamido have been investigated [69, 1421. Some substituted N6-phenylcarbamoyl-NECA derivatives showed high affinity

J.E. VAN MUIJLWIJK-KOEZEN ET AL.

83

Table 2.7. AFFINITIES O F N6 CARBAMOYL-NECA DERIVATIVES (30-39) AT RAT ADENOSINE RECEPTORS DETERMINED IN BINDING ASSAYS, EXPRESSED AS K, (nM)

EtNHCO

OH

Compound

R

OH

AI

A ~ A

Re$

A3 ~~

H 2-OMe 3-OMe 4-OMe 2-c1 3-CI 4-CI 4-SOzNHz 4-(Pyridin-2-yl-NHS02) 4-(Pyrimidin-2-yl-NHS02)

110 73 113 33 111

45 72 453 292 125

5364 1160 3856 3363 1337 420 1488 1180

740 17

39 30 42 6.6 7.4 4.4 1 I4 9.7 54 40 5

~

~421 ~421 ~421 [I421 [142] [142] [ 1421 ~ 9 1 ~691 ~ 9 1

(33-35) but only moderate selectivity (Table 2.7). In this class, an appropriate way to create higher affinity and selectivity at adenosine A3 receptors is substitution of the phenyl or benzyl group. The substituted benzyl and phenyl series showed comparable SAR. The most selective compound from this series (37) was less active than IB-MECA but showed a comparable A3 selectivity. The unsubstituted sulfonamide group of (37) seems to be selective necessary, since heteroaryl substituted derivatives (38) and the AZ,.+, agonist (39) showed decreased adenosine A3 receptor affinity. A promising new class of adenosine A3 receptor agonists has been reported by Knutsen and coworkers [143, 1441. The authors used N6-5'-disubstituted 2-chloro-adenosine analogues, in which the bulky aromatic N6 group was replaced by an N6-methoxy group and probed the structural requirements at the furanose 4'-position. In binding studies these compounds showed high affinity at the human adenosine A3 receptor (Table 2.8) and they inhibited TNF-a production in ex vivo experiments. Com-

THE ADENOSINE

84

A3

RECEPTOR AND ITS LIGANDS

Table 2.8. AFFINITIES OF 4‘-FURANOSE-MODIFIED 2-CHLORO N-METHOXYADENOSINE DERIVATIVES (4045) AT ADENOSINE RECEPTORS DETERMINED IN BINDING ASSAYS, EXPRESSED AS Ki (nM)

“r0J OH

R

Compound ~~~

~

(40) (41) (42) (43) (44) (45) (a)

OH

rAl(”’

rAZA(”

hAl”’

Ref:

280 15 74 100 1230 620

9100 980 4650 9500 63400 > 10000

6.2 10 26 4.6 20 31

[I431 [I431 [I431 [I431 [I431 [ 1441

~

CH20(CO)Me CHiOH CHiCl CH20Me CH = CH2 Isoxazole

r = rat; h =human.

pounds (40) and (43) possessed Ki values in the low nanomolar range and were 45- and 22-fold selective over A, receptors, respectively. This class of N6-methoxyadenosines proves that structural variation at the position equivalent to the ribose 5’-hydroxyl in adenosine is allowed and may even lead to increased potency.

Compound

R’

R2

(46) 1,3-Dibutylxanthine-7-ribose (47) 1,3-Dibutylxanthine-7-ribose-5’-N-methylcarboxamide (48) 3’-Deoxy-l,3-dibutylxanthine-7-ribose-5’-Nmethylcarboxamide

OH OH H

CHiOH C(=O)NHMe C(=O)NHMe

J.E. VAN MUIJLWIJK-KOEZEN ET AL.

85

PARTIAL AGONISTS

In 1990 the synthesis of adenosine agonist-antagonist hybrid structures was reported [145]. One of these xanthine 7-ribosides (46) was shown to behave as a partial agonist at the rat adenosine A3 receptor [130]. Compound (46) had a Ki value of 6 pM in a binding assay and inhibited adenylate cyclase, although with lower intrinsic activity than the full agonists (4-9). A11 compounds described in the previous section acted as full agonists in inhibiting adenylate cyclase. Structural modification in order to enhance adenosine A3 receptor affinity with a 5’-methyl uronamide substituent yielded compound (47). However, this compound behaved as a full adenosine A3 receptor agonist [146]. Modification of the ribose ring led to the remarkable compound (48), which behaved as a partial agonist at the rat adenosine A3 receptor (measured in a [35S]-GTPy[S]assay) but as an antagonist at the rat adenosine A, receptor [147]. These findings are evidence of the interesting concept that a compound may stimulate one receptor subtype while blocking another within the same species. This concept has already been demonstrated in other G-protein coupled receptors, such as the histamine H3 agonist impromidine which acts as an antagonist at the histamine H3 receptor [148, 1491. Recently, van Tilburg et al. [68, 1501 described the synthesis and biological evaluation of a new series of N6,5’-disubstituted adenosine derivatives as selective ligands for the adenosine A3 receptor (Table 2.9). These adenosine analogues were substituted with a benzyl or 3-iodobenzyl group at the N6-position, thereby showing an augmented A3 selectivity and with alkylthio groups at the 5’-position for partial agonism following similar observations for adenosine A, partial agonists [151]. These selective compounds display affinities in the nanomolar range and behave as partial agonists both in a [35S]-GTPy[S]assay and in a CAMP assay. Thus there are a number of partial agonists for the adenosine A3 receptor that are promising tools to explore the potential to induce selective effects due to differences in receptor-effector coupling in various tissues. ANTAGONISTS XANTHINES

The discovery of the adenosine A3 receptor initiated an intensive search for selective antagonists. Initially, attempts were made to characterize this

86

THE ADENOSINE A3 RECEPTOR AND ITS LIGANDS

Table 2.9. AFFINITIES OF 3-IODOBENZYL C-5’ SUBSTITUTED ADENOSINE ANALOGUES (49-52) AT ADENOSINE RECEPTORS DETERMINED IN BINDING ASSAYS, EXPRESSED AS Ki (nM) OR PERCENTAGE DISPLACEMENT AT 10 pM AND THEIR BEHAVIOUR AS PARTIAL AGONISTS [68]

Rg HN I

OH

OH

mux. [3’S/-G TPy[ S ] Compound

R

rA,(a)

rA2A(a)

hA$“)

bound

(49) (50) (51) (52)

SMe SEt S-n-Pr S-i-Pr

610 720 42%) 35%

1825 1081 42% 45%

8.8 18 44 46

41‘‘) 21 10 16

(%)(b)

(a) r

= rat; h = human. (b)Thepercentage of [35S]-GTPy[S]binding to the adenosine A3 receptors stimulated by (49-52) compared with the maximal [35S]-GTPy[S]binding caused by the full agonist NECA (100%). ‘“)EC,o=27+18nM.

receptor using different xanthine analogues, the general class of adenosine antagonists. However, the two problems of species differences and low adenosine A3 affinities of the xanthines rapidly became apparent [20, 1521. Ji et al. [ 1521 compared structurally diverse xanthines and their A3 receptor affinities (Table 2.10). The authors concluded that xanthines only weakly antagonize rat, rabbit and gerbil adenosine A3 receptors and that 8-arylxanthines showed increased affinities at the sheep and the human adenosine A3 receptor. This enhanced affinity was present for both cationic and for anionic xanthines. Efforts to substitute the xanthine structure in order to increase adenosine A3 receptor affinity of the antagonist, however, have largely failed [ 146, 1531. Therefore searches have been started for other lead structures [154]. The investigations of new lead compounds will be discussed in the following paragraphs.

J.E. VAN MUIJLWIJK-KOEZEN ET AL.

87

Table 2.10. AFFINITIES O F XANTHINES (53-56) AT ADENOSINE A3 RECEPTORS IN VARIOUS SPECIES DETERMINED IN BINDING ASSAYS, EXPRESSED AS K, (nM) [I 521

Anionic Cationic Neutral

Compound

Human

Sheep

Rat

Rabbit

Gerbil

BWA522'"' (53) BWA1433 (54) XAC ( 5 5 ) DPCPX (56)

18 55 49- 105'b' 200-3960")

3 21 180 493000

1170 15000 29000"' 5290'"'

1960 24600 20600 5270

2760 25300 2 1600 3170

'"'This compound is also called IABOPX. "'As mentioned in Table 2.3. '"Van Galen etal. [I301 reported that at rat adenosine A3 receptors expressed in CHO cells the K , value for XAC is >lo0 pM, and for DPCPX >I0 pM.

f '

(56)DPCPX

88

THE ADENOSINE A3 RECEPTOR A N D ITS LIGANDS TRIAZOLONAPHTHPYRIDINE AND THIAZOLOPYRIMIDINE

The first structures with sufficient potency and selectivity to facilitate evaluation of adenosine A3 receptors were reported by the Merck group [48,49]. The triazolonaphthpyridine L-2493 13 (57) inhibited the binding of [I2’I]-ABA to cloned human adenosine A3 receptors with a Ki value of 13 nM. It showed 308- and 1460-fold selectivity over human adenosine Al and A2* receptors, respectively. However, L-249313 had only micromolar affinity at the rat adenosine A3 receptor with a Ki value of 33-58pM [48, 651. However, in the functional assay of inhibition by an agonist of forskolin-stimulated cAMP production, it appeared that this compound binds non-competitively.

COOMe

(58) L-268605

(57) L-249313

Another Merck compound, the thiazolopyrimidine L-268605 (58) was shown to bind competitively with a Ki value of 18 nM at the human adenosine A3 receptor. At the rat adenosine A3 receptor no displacement of the radioligand was found up to a concentration of 10pM. L-268605 (58) is A3 selective, since negligible displacement was measured at 10 pM at the human A, and A ~ receptors. A In a functional assay, (58) counteracted the adenosine-induced inhibition of forskolin-stimulated cAMP production. Also, it caused a rightward shift of the agonist dose-response curve in a [35S]-GTPy[S]binding assay. FLAVONOIDS

Flavonoid derivatives were also reported to be a novel class of non-xanthine adenosine A3 receptor antagonists [155]. In a broad screening approach flavone derivatives and other phytochemicals were found to bind in the micromolar range to adenosine A,, A2* and A3 receptors. A structure-activity analysis [I 561 indicated that the hydroxyl groups of naturally occurring flavones were not essential for affinity at adenosine

J.E. VAN MUIJLWIJK-KOEZEN ET AL.

89

receptors. Galangin (60) had a Ki value of 3 pM at the human adenosine A3 receptors, but was slightly more active at both rat A, and A2A receptors ( K ,= 1 pM). Methylation (61) but not acetylation (62) of the hydroxyl groups of galangin enhanced A3 selectivity. Substitution of the phenyl ring of (61) with two methoxy groups resulted in pentamethylmorin (63), which is 15-fold selective for human adenosine A3 receptors compared to rat A, and A 2 receptors ~ (Table 2.11). Further investigations led to more selective human adenosine A3 receptor antagonists [ 1571. The hydroxyl groups of galangin (60) were alkylated and the resulting compounds (64) and (65) had increased human adenosine A3 receptor affinity. Several other morin derivatives were also synthesized and tested, e.g. (66). This tetraethyl ether of morin displayed a Ki value of 4.8pM at the human adenosine A3 receptor whilst being inactive at the rat adenosine A, and AZAreceptors. Chloro substitution of flavone (59) was also investigated. The 3-chloro and 6-chloro derivatives, (67) and (68), showed decreased adenosine affinities. However, combination of the chloro substitutions together with modifications on the 2’and 4’-position resulted in a relatively potent and selective human adenosine A3 receptor antagonist. Compound (69) showed a Ki value of 0.56pM and was -200-fold selective versus rat adenosine A , and AZA receptors. Finally, the 2-aryl group was replaced by styryl, phenylethynyl and 4-phenyl-l,3-pentadienylgroups. These compounds possessed decreased or similar human adenosine A3 receptor affinities compared to galangin (60). Different flavone analogues have been used in molecular modelling studies as described in a later section.

PYRIDINES AND PYRANS

1,4-Dihydropyridines

The 1,4-dihydropyridine blockers of L-type calcium channels are used extensively in the treatment of cardiovascular disorders [ 1581. These compounds display affinities for many, rather diverse binding sites [ 1591, including the adenosine receptors [160]. The dihydropyridines have not only been structurally optimized to behave as selective ligands for the calcium channel receptor [158], but also for the platelet activating factor (PAF) receptor [161], the C ~ receptor ~ A [162] and the nicotinic acetylcholine receptor [163]. Recently, Jacobson et al. have reported on structural modifications of dihydropyridines with selectivity for the adenosine A3 receptor [ 1601.

90

THE ADENOSINE A3 RECEPTOR AND ITS LIGANDS

Table 2.1 I. AFFINITIES O F FLAVONE ANALOGUES (59-70) AT ADENOSINE RECEPTORS DETERMINED IN BINDING ASSAYS, EXPRESSED AS K, (nM) OR PERCENTAGE DISPLACEMENT AT THE CONCENTRATION INDICATED [156,157]

5'

Compound

R

RZ

RJ

,,A,(")

Flavone (59) Galangin (60) (61) (62) Pentamethyl-morin (63) (64) (65) (66) (67) (68) (69) (70)

H 5-OH, 7-OH 5-OMe, 7-OMe 5-OAc, 7-OAc 5-OMe. 7-OMe

H H H H 2'-OMe, 4'-OMe

H OH OMe OAc OMe

3280 3450 863 966 509 6450 11600 56500 27600 46700

5-OEt, 7-OEt 5-OPr, 7-OPr S O H , 7-OEt H 6-CI 6-C1 H

H H 2'-OEt, 4'-OEt H H 2'-OiPr, 4'-Me

OEt OPr OEt

603 1100 tlO%'h' 2480 28'Ki'' 36Y0'~) 35600

(d)

c1 H CI OEt

rA2A(B)

hA

(a1

16900 3150 1210 17500 2650

3310 364 3220 317 <100/I1(~'4830 25100 11500 < IO%"' 32%'b' 19%"' 561 33800 1160

(a) r =rat; h = human. 'h'Displacement of specific binding at 100 yM. '"Displacement of specific binding at 10 y M . ' d ' T h i ~compound has a trans 2-styryl group instead of a 2-phenyl group.

Compounds (71-73) in Table 2.12 are prototypical calcium channel antagonists but they can also bind to adenosine receptors. Although nifedipine (7 1) was non-selective for the three adenosine receptor subtypes, (8-niguldipine (73) proved to be a selective adenosine A3 receptor antagonist. The 4-trans-fi-styryl analogue (74) was quite potent (&=0.67pM) and selective for the human adenosine A3 receptor but showed equal affinity at L-type calcium channels (Ki = 0.69 pM). Introduction of a 6-phenyl group, as in (75), enhanced adenosine A3 receptor selectivity versus calcium channels (Ki > 100 pM). Further studies have shown that substitution of this phenyl ring or its replacement by a 3-fury1 or 3-thienyl group decreased human adenosine A3 receptor affinity [164]. Unfortunately, the authors have not reported on the binding at calcium channels of the other compounds in Tables 2.12 and 2.13.

Table 2.12. AFFINITIES OF 1,4-DIHYDROPYRIDINE DERIVATIVES (7 1-83) AT THE HUMAN ADENOSINE A3 RECEPTOR DETERMINED IN A BINDING ASSAY, EXPRESSED AS Ki (nM) OR PERCENTAGE DISPLACEMENT AT THE CONCENTRATION INDICATED

Compound

R

__

R'

Me 2-NOlPh Me 3-NOzPh

Me

(5')-Niguldipine (73)

Me 3-NO2Ph

CH2CH&HflD

(74) (75) (76) (77)

Me Ph-CH = CHEt Ph-CH = CHEt Ph-CH = CHBz Ph-CH = CH-

r = rat; h = human.

rAJA(')

Re$

~~

Nifedipine (71) Nicardipine (72)

'a)

R3 ,A,'='

R2

CH2CH2N(CH3)CH2Ph

(trans) (trans) (rrans) (frans)

Et Et

Bz Et

Me 2890 Me 19600

FL Me

< 0% ( lop4)

Me 16100 Ph 5930 Ph 35% Ph
18200 63800

8290 3250


2800

h

49300 4770 15% 16%

670 108 58 142

b I?

Y

Table 2.12 continued. AFFINITIES OF 1,4-DIHYDROPYRIDINE DERIVATIVES (71-83) AT THE HUMAN ADENOSINE A3 RECEPTOR DETERMINED IN A BINDING ASSAY, EXPRESSED AS Ki (nM) OR PERCENTAGE DISPLACEMENT AT THE CONCENTRATION INDICATED

m P

H

Compound

R

R’

(+) MRS 1191 (78) (78a) (IS)-enantiomer (78b) (4R)-enantiomer (79) (80) (81) (82) (83)

Et Et Et Et Et Et Et Et

Ph-C CPh-C E C Ph-C = CPh-C E CPh-C E CPh-C = CPh-C CPh-C C-

(a)

r = rat; h =human

R2

R’

rAl‘a’

BZ

Ph Ph Ph Ph Ph Ph Ph Ph

40100 32% 25% 16% (lop4)
BZ

Bz CH2(2-Me)Ph CH2(3-Me)Ph CH2(4-Me)Ph CHl(3-NOz)Ph CHZ(CNO1)Ph

rAZA(a’

hA,(a’

Ref:

31, 22 21 750 112 52

[la,1671 [167] I1671 [165] [I651 [165] [I651 [165]

110

8.6 2.7

0

P

J.E. VAN MUIJLWIJK-KOEZEN ET A L .

93

Replacement of the 5-ethyl ester group by a 5-benzyl ester group (76) increased adenosine A3 subtype selectivity and affinity. The isomeric 3-benzyl, 5-ethyl diester (77) also showed adenosine A3 receptor affinity and selectivity, although to a lesser extent. Replacement of a 4-trans-P-styryl by a 4-phenylethynyl substituent resulted in the potent derivative (78) with a K, of 31nM at the human adenosine A3 receptor. In a subsequent publication, the substituent effect of the 5-benzyl ester was further explored [165]. A meta methyl substituent did not influence the adenosine A3 receptor affinity, whereas a methyl group on the ortho or para position decreased the adenosine A3 receptor affinity twofold [(79-81) in Table 2.121. However, 4-substitution with a nitro group (83) increased human adenosine A3 receptor affinity ( K , = 7 nM) with a more than 37,000-fold selectivity versus rat adenosine A, and receptors. Even at the rat adenosine A3 receptor, the 5-benzyl esters have affinities in the micromolar range (not shown). Further modifications of these dihydropyridines, such as N-R' substitution [165] or use of thioethers [166], did not result in higher human adenosine A3 receptor affinities. Most dihydropyridines are racemates because of the presence of a chiral center at C-4. Although these racemates have shown high potency and selectivity at the human adenosine A3 receptor, pharmacological information on the enantiomers is needed for quantitative SAR of this series of compounds. Jacobson's research group finally succeeded in this goal [167]. They isolated the two pure enantiomers of MRSll91 (78) via the synthesis of diastereomers using a chiral /I-keto-ester in the Hantzsch reaction, separation by HPLC, deprotection of each diastereomer and transformation of the diols into ethyl esters. The (4S)-enantiomer (78a) showed human adenosine A3 receptor affinity similar to the racemic mixture, whereas the (4R)-enantiomer (78b) showed a 35-fold decease. A possible conformational explanation of these results by molecular modelling studies will be discussed later. 3,s-Diacyl-2,4-dialkylp y r idines In vivo oxidation of 1,4-dihydropyridines results in pyridine metabolites [168] and this was one of the reasons for investigating the related pyridine derivatives as adenosine A3 receptor ligands [ 1661. Compound (84), the pyridine analogue of (75), and compound ( 8 5 ) , the pyridine analogue of MRS1191 (78), showed affinities at the adenosine receptors in the micromolar range and a loss of selectivity (Table 2.13). A methyl group at the 4-position of the pyridine did not influence adenosine

P rci

Table 2.13. AFFINITIES OF PYRIDINE (8491) AND PYRIDINIUM (92-94) DERIVATIVES AT ADENOSINE RECEPTORS DETERMINED IN BINDING ASSAYS AND EXPRESSED AS K, (nM) OR PERCENTAGE DISPLACEMENT AT THE CONCENTRATION INDICATED

m

> R

W

O

y

R

4

R*OyR4

Y H Compound

(92) (93) (94) (a)

Me

R

'R

d

R4

'R

Me Me Me Me Et Et Et

OEt OEt OEt OEt SEt SEt SEt SPr

Ph-CH = CH- (trans) Ph-C z CMe Et Et Et Et Et

Et Bz Et Et Et Et Pr Pr

Ph Ph Ph Ph Ph Ph Ph 3-CIPh

Me Et Et

SEt SEt SEt

Et Et Et

Et Et Pr

Ph Ph Ph

Me

r = rat: h = human.

H

I

rAl'a'

2490 11600 7410 3360 14800 41% 7770 4 1400 22% ( 3 1900
5 ,A,'='

hA,'"'

2400 43% 28400 3690 14900 6130
2800 2750 4470 176 42.9 20.0 8.29 7.94

20800 11600 18700

379

219 483

Ref

[I691 ~691 ~ 9 1

>

5

% b,

J.E. VAN MUIJLWIJK-KOEZEN ET AL.

95

receptor affinities (86). However, replacement of this 4-methyl by a 4-ethyl group (87) augmented adenosine A3 receptor affinity 25-fold. For this reason further analogues bear an ethyl group at the 4-position. The thioester (88) at the 3-position had a 4-fold greater affinity than its oxygen analogue (87), with a concomitant 4-fold decrease in affinity at both the adenosine A, and A Zreceptors. ~ A 2-ethyl (89) versus a 2-methyl substituent (88) afforded a twofold increase in human adenosine A3 receptor affinity whereas a 5-propyl ester (90) seemed to be twofold more potent than the corresponding 5-ethyl ester. Finally, a 3-chloro substituent on the 6-phenyl ring (91) was well tolerated. These investigations led to a potent and selective human adenosine A3 receptor antagonist (90) with a Ki value of 8 nM.

3,5-Diacyl-1,2,4-trialk~~lpyridiniu~~ salts The template approach to finding adenosine A3 receptor antagonists (calcium blockers +. 1,4-dihydropyrdines +. pyridines) was extended to a series of pyridinium salts [169]. The pyridinium salts were synthesized by alkylation of the pyridine nitrogen or by oxidation of a dihydropyridine. All salts showed a more than tenfold decrease in human adenosine A3 receptor affinities compared to the pyridine derivatives, but are still A3 selective [(92-94) versus (88-90), Tcrble 2.131. The major advantage of these pyridinium salts is their good aqueous solubility. Pyrans

The use of dihydropyridines as templates for the design of adenosine A3 receptor ligands was also extended by replacing the heterocyclic 1-amino group with an oxygen atom [ 1701. Several pyran derivatives were compared to the corresponding dihydropyridines (Table2.14).These chemically stable pyrans showed a SAR which was not completely commensurable with the SAR of the dihydropyridines. For example, the presence of a 6-phenyl substituent, which increased adenosine A3 receptor affinity in the dihydropyridine (78), either had no effect (96) or decreased (98) adenosine A3 receptor binding. Also 4-styryl pyran (97) showed adenosine A3 receptor affinity in the micromolar range whereas 4-styryl dihydropyridine (75) possessed augmented adenosine receptor affinities. Furthermore, the selectivity but not the affinity of the pyrans in binding the adenosine A3 receptor was generally enhanced compared to the corresponding dihydropyridine anaogues.

THE ADENOSINE A3 RECEPTOR AND ITS LIGANDS

96

Table 2.14. AFFINITIES OF PYRAN ANALOGUES (95-98) AT ADENOSINE RECEPTORS DETERMINED IN BINDING ASSAYS AND EXPRESSED AS Ki (nM) OR PERCENTAGE DISPLACEMENT AT 100 pM

Compound

R'

R2

R3

rAlCa1

rA2A(a)

(95) (96) (97) (98)

Ph Ph Ph-CH=CHPh-CEC-

Et Et Et BZ

Me Ph Me Ph

21900 14200

< < < <

(a) r

= rat;

< 50% < 50%

50% 50% 50% SOY"

h A?) 38 1 583 1830 20200

h = human.

TRIAZOLOQUINAZOLINES

Triazoloquinazolines as non-xanthine human adenosine A3 antagonists were also discovered by screening of compound libraries [47]. CGS 15943

CI (99)CGS 15943 R = H (100) R = Br

(101) R 1 = COEt (102)R 1 = COPh (103)MRS 1220 R1 =COB2

Cl (104) R 1 = H (105) R1= Pr

J.E. VAN MUIJLWIJK-KOEZEN ET A L .

97

(99), an A2.4 receptor antagonist [171], has been reported to be inactive at rat adenosine A3 receptors [130]. However, it was shown to have high affinity (Ki= 14 nM) at the human adenosine A3 receptor [47]. Various N-acyl derivatives were synthesized and tested. From the series of straight chain alkyl amides, it was concluded that the optimal chain length occurred with the N-propionyl derivative (101). The other compounds showed lower adenosine A3 receptor affinity than the parent compound CGS 15943 (99). On the other hand, aromatic acyl derivatives increased adenosine A, receptor affinity. The N-benzoyl derivative (1 02) displayed a Ki value of 3.0 nM, while affinities at the adenosine A , and A2Areceptors were largely decreased. The phenylacetyl derivative MRS 1220 (1 03) was also discovered as a potent and selective antagonist for the human adenosine A3 receptor, with a Ki value of 0.65 nM and 470-fold and 80-fold selectivity versus rat A, and AZAreceptors, respectively. Bromo substituents on the fury1 ring caused a decrease in human adenosine A, receptor affinity. Finally, two 5-Ox0 derivatives (104) and (105) were modified but both compounds also showed a large decrease in human adenosine A3 receptor affinity.

ISOQUINOLINES AND QUINAZOLINES

In 1998, we reported on another class of non-xanthine human adenosine A3 receptor antagonists [44, 150, 1721. A structurally diverse series of 1-substituted 3-(2-pyridinyl)isoquinoline derivatives was synthesized and

Table 2.15. AFFINITIES OF TRIAZOLOQUINAZOLINE DERIVATIVES (99-105) AT ADENOSINE RECEPTORS DETERMINED IN BINDING ASSAYS A N D EXPRESSED AS Ki (nM) [47] Compound

(a)

r = rat; h = human.

rA,")

rA2Aia'

hAj(")

21 1570 283 680 305 3950 1850

3.3 53 1 106 213 52 4380 6960

13.8 64 7.66 3.03 0.65 260 1813

THE ADENOSINE A3 RECEPTOR AND ITS LIGANDS

98

tested on binding at the human adenosine A3 receptor [44].Derivatives with electronically diverse substituents on the 1-position of the isoquinoline ring had A3 affinities in the micromolar range (106-109) (Table 2.16). For example (109), bearing a 1-phenyl group, showed low human adenosine A3 receptor affinity (Ki = 12 pM) and was slightly A , selective. However, compounds having a phenyl group coupled to the 1-position of the isoquinoline ring via a spacer, such as in (1 lo), (1 11) and (1 18) showed a large increase in human adenosine A3 receptor affinity. The requirements for A3 receptor binding are depicted in Figure 2.5.

Table 2.16. AFFINITIES O F ISOQUINOLINE DERIVATIVES (106-122) AT ADENOSINE RECEPTORS DETERMINED IN BINDING ASSAYS, EXPRESSED AS K, (nM) OR PERCENTAGE DISPLACEMENT AT 10 pM

Compound

R

(1 06) (107) (108) (109)

NH2 CI OMe Ph CH2COPh N=C(NH2)Ph N=C(NH2)-(3-Cl)Ph N=C(NH2)-(3-Me)Ph N=C(NH2)-(3-OMe)Ph N=C(NH2)-(4-Cl)Ph N=C(NH2)-(4-Me)Ph N=C(NH2)-(4-0Me)Ph NHCOPh NHCO-(3-CI)Ph NHCO-(3-OMe)Ph NHCO-(4-CI)Ph NHCO-(4-OMe)Ph

(1 10)

(111) (112) (1 13) (114) (115) ( 1 16) (117) (118)

( 1 19) (120) (121) (122) VU8504 (d)

r = rat; h = human.

hAg(a' 19% 43% 3.3 1.9 0.24 34%) 49% 43% 43% 20% 12% 18% 3.2 42% 22% 11%

14Y"

28% 2Y% 19%

31% 22% 4% I4'% 0%)

0% 7% 34% 5%1 0% 0% 0Y" 0%I 0%

17% 2600 5600 12000 660

740 7800 1300 980 2300 580 310 204 767 146 197 17

Ref:

J.E. VAN MUIJLWIJK-KOEZEN ET A L

99

F= spacer allowing conjugation

Figure 2.5. Structural requirements for isoquinoline derivatives as human udenosine A 3 receptor antagonists.

The substituent effect on the benzamidine ring was investigated (1 1 1-1 17). Meta-substitution with an electron-withdrawing substituent,

as in (112), resulted in a 10-fold decrease in potency compared to the unsubstituted benzamidine (1 1 1). Electron-donating substituents had less influence as in (1 13) and ( 1 14). An electron-withdrawing group on the para positon of the benzamidine decreased A3 affinity (1 15) whereas electron-donating groups increased this affinity, e.g. (1 16) and (1 17), compound ( 1 17) had a Ki value of 310 nM at the human adenosine A3 receptor. All benzamidine compounds showed A3 selectivity. Of all spacers tried (Figure 2.5), a carboxamide moiety proved to be optimal [172]. Analogue (1 18) showed an affinity of 200 nM at the human adenosine A3 receptor. Furthermore, the effects of substitution of the benzamide ring of (118) were investigated. A comparable SAR to the benzamidines was shown (1 18-1 22). Electron withdrawing groups on the meta orpara position of the phenyl ring showed equal or decreased affinity, whereas electron-donating groups contributed positively to adenosine A3 receptor affinity, with a para-methoxy group as optimal (122). Other modifications, such as disubstitution on the benzamide or replacement of the isoquinoline ring by a quinazoline, did not yield more potent or

I00

THE ADENOSINE A3 RECEPTOR AND ITS LIGANDS

selective human adenosine A3 receptor antagonists. These investigations led eventually to the A3-selective compound VUF8504 (122) with an affinity of 17 nM at the human adenosine A3 receptor. Recently, a further study on this class of compounds was published [173], which described 1-phenylurea-isoquinolines and 4-phen ylurea-quinazolines (Table 2.17). A structurally diverse series of 2-substituted quinazolin4-yl-phenyl urea derivatives was synthesized and tested (123-129). Compounds in which R2 is an aromatic ring and especially 3-pyridyl, as in quinazoline (127), showed the highest human adenosine A3 receptor affinity (Ki 51 nM). The influence of different substituents on the phenyl urea moiety was investigated (130-136). Substitution on the 3- or 4-position of the phenyl

Table 2.1 7. AFFINITIES OF ISOQUINOLINE DERIVATIVES (123-1 37) AT ADENOSINE RECEPTORS DETERMINED IN BINDING ASSAYS, EXPRESSED AS Ki (nM) OR PERCENTAGE DISPLACEMENT AT 10 pM [I731

H H H H H H H 4-CI 4-Me 4-OMe 3,4-diC1 2-c1 2-Me 2-OMe 2-OMe (a) r

= rat; h = human.

H tBu Ph 2-Pyridyl 3-Pyridyl 4-Pyridyl 6-Me-2-pyridyl Ph Ph Ph Ph Ph Ph Ph 3-Pyrid yl

48% 13% 36% 0% 68% 166 23% 26% 6% 30%) 3% 39% 64% 3870 52%

42% 19%) 24% 2% 42% 27% 12% 24% 3% 11YO

10% 28% 36% 22% 43%

1180

78700 287 495 51 336 264 38% 38% 40%) 40% 220 115 87 4.0

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urea yielded derivatives with modest receptor affinities, but substitution at the 2-position increased A3 affinity. A 2-methoxy substituent, as in (136), gave the maximum increase in human adenosine A3 affinity with a Ki value of 87 nM. Finally, a compound bearing both optimal substituents was tested. Thus VUF5574 (137) shows that an 'additive SAR' on the 2-position of the quinazoline ring and the phenyl urea exists. It is a potent and selective human adenosine A3 ligand with a Ki value of 4 nM. TRIAZOLOPYRIMIDINES

Recently, yet another class of adenosine A3 receptor antagonists was described, the triazolopyrimidines [1741. This class of compounds was based on a combination of the structural elements of AZA antagonists closely related to the triazoloquinazolines (e.g. 99) and the agonistic N6-substituted phenylcarbamoy1)adenosine derivatives (Table 2.7). The bulky substituents of compounds (33) and (35) were linked to the amino group of pyrazolo[4,3-e]-l,2,4-triazolo[ 1,5-~]pyrimidines(Table 2.18).

Table 2.1 8. AFFINITIES O F TRIAZOLOPYRIMIDINE DERIVATIVES (138-143) AT ADENOSINE RECEPTORS DETERMINED IN BINDING ASSAYS, EXPRESSED AS K, (nM) OR PERCENTAGE DISPLACEMENT AT 10pM [174]

(138) (1 39) (140) (141) (142) (143) (a)

Et Et Et PhCHzCHz PhCH2CH2 PhCH2CH2

r = rat; h = human.

H 4-MeO-Ph-NHCO 3-CI-Ph-NHCO H 4-MeO-Ph-NHCO 3-CI-Ph-NHCO

95 <SO%

2699 2.2 1282 1049

11

<SO% 2799 0.7 1398 1698

3579 0.28 2.09 2785 1.5 13

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THE ADENOSINE A3 RECEPTOR AND ITS LIGANDS

The N'-substituted compounds showed a substantial increase in adenosine A3 receptor affinity compared to the unsubstituted derivatives (1 38, 141). SAR studies revealed that at the N8 position a small alkyl group is optimal, leading to the N8-ethyl-N'-4-methoxyphenylcarbamoyl derivative (139) with a Ki value of 0.28 nM. This compound has the highest affinity for the human adenosine A3 receptor reported so far.

RECEPTOR MODELS The first binding site model of the rat adenosine A3 receptor was postulated by van Galen et al. in 1994 [130]. A rat adenosine A3 receptor model was built from an extensively studied rat adenosine A l receptor model [175], which in turn was based on the bacteriorhodopsin template. Ligands were docked into the model starting with the orientation of N6-cyclopentyladenosine (CPA) in the adenosine A , receptor model and minimized. The main conclusion was that the ligand binding environment for the rat adenosine A3 receptor is more constricted than for the rat adenosine A l receptor. From the docking of agonist N6-benzyladenosine (lo), a hydrophobic pocket directed toward the extracellular space was proposed. This pocket may accommodate large N6 substituents. A second hydrophobic pocket is present adjacent to C-2 capable of accommodating fairly large C-2 substituents. Some important amino acid residues in the binding pocket of the rat adenosine A3 receptor together with (10) are depicted in Figure 2.6.

hydrophobic pocket TMV ,,.Ser-249

TMVI

I Phe-184 TMV Thr-96TMII

C-2 hydrophobic pocket

Ho\.

/OH His-274 TMVll

\

~er-277 TMVll

Figure 2.6. Ligand binding site model of the rat adenosine A 3 receptor: interactions o/ N'4enzyladenosine ( 1 0 ) with side-chains of amino acid residues.

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At that time, no potent and selective adenosine A3 receptor antagonists were known and therefore the xanthines were studied. The adenosine A3 receptor lacks a His residue in transmembrane helix VI compared to the other receptor subtypes, in which this is replaced by serine, Ser-249. It was suggested that this lack of a His residue could be responsible for the inactivity of xanthines at rat adenosine A3 receptors, but the human adenosine A3 receptor also lacks this His residue while showing augmented xanthine-affinities. This amino acid residue can form a hydrogen bond with N6-H of agonists and was shown to be important for binding to A , and A ~ receptors A in mutation studies [ 1761. The model predicts that Ser-249 is too far from the ligand to be able to form a hydrogen bond. Furthermore, the model predicted that xanthine-7-ribosides should have activity at the adenosine A3 receptor, which has subsequently been demonstrated (vide supra partial agonists). Only recently, molecular modelling studies including antagonists were reported [177, 1781. A CoMFA study [178] was described for the flavonoid derivatives (Table 2. I I ) which are relatively rigid molecules and selective for the human adenosine A3 receptor. In this quantitative study models for the three receptor subtypes (human A3, rat A, and rat A2A) were derived with a high degree of (statistical) accuracy. All three CoMFA models showed the same steric and electrostatic contributions which suggest similar requirements for binding. Similarities were seen in the topology of steric and electrostatic regions with the A , and A3 receptors. The contour map analysis of the A3 CoMFA model revealed that a region of favourable bulk interaction is located around the 2’-position of the phenyl ring (on the 2-position of the flavone skeleton). Furthermore the model predicts that bulky groups around the 6-position will be tolerated and may increase A3/A, selectivity. Experimental evidence for this prediction is not yet available, except for a 6-chloro analogue (with unknown adenosine A, receptor affinity). In a second study, a homology model for the human adenosine A3 receptor model [I 771 was optimized (MacroModel and SYBY L) based on the approach described earlier [ 179, 1801. Again, bacteriorhodopsin was used as a template. Molecular orbital calculations were carried out on antagonists with affinities in the nanomolar range of different classes of compounds (54), ( 5 8 ) , (69), (89), (99) and (144). Superimposition of the geometry-optimized structures was done according to the SEAL method, using CGS 15943 (99) as a template. These superimposed antagonists were docked rigidly into the receptor model and energy minimized. A so-called cross-docking procedure was used for refinement.

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THE ADENOSINE A j RECEPTOR AND ITS LIGANDS

The major conclusion of the SEAL approach is that a common alignment was found for all antagonist structures studied, suggesting a common receptor binding site and thus common electrostatic properties of the ligands. A common feature of the antagonists is a region of negative electrostatic potential located around the nitrogen at the 6-position of the triazoloquinazoline moiety. In all ligands studied, a nitrogen or oxygen atom is present at this position and is able to form a hydrogen bond within the human adenosine A3 receptor-antagonist complex. A second common feature is a .n-system located at the furan ring of CGS 15943 (99). Furthermore, two hydrophobic regions are present in all human adenosine A3 receptor antagonists. This common pharmacophore model for binding antagonists is depicted in Figure 2.7. The SEAL method was validated using a newly synthesized symmetric 1,4-dihydropyridine (145). An affinity of 100-200 nM at the human adenosine A3 receptor was predicted for this compound. Testing of this compound revealed that the Ki value (25 1f 2 8 nM) was in good agreement with

(144) HE-EA

the predicted value. Based on the ligand-receptor model, the authors postulated an antagonist binding site surrounded by helices 111, V, VI and VII. The predicted pharmacophoric elements of the human adenosine A3 receptor may be used in the rational design of new antagonists. Recently, an example of this use has been described [167]. The enantiomers (78a) and (78b) of 5-benzyl-3-ethyl-2-methyl-6-phenyl-4-phenylethynyl-1,4dihydropyridine-3,5-dicarboxylatewere docked in the putative ligand binding site of the model described above. This study expanded on earlier defined common pharmacophoric elements for pyridine and dihydropyridine structures [166].

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Figure 2.7. Common pharmacophore modelfor binding qfantagonists at the human adenosine A3 receptor M”th CGS 15943 (99) on the left hand side (from [I151 with permission).

Unfortunately, although the models previously reported for the A , , AZA and rat A3 receptor are all based on the assumption of a common binding site, the authors of this human A3 receptor model have neither suggested an agonist binding site nor compared the human with the rat binding site model. Furthermore, a ligand model containing all classes of antagonists would be helpful in describing ligand-receptor interactions more precisely. The current model lacks the recently found classes of triazolopyrimidines, isoquinolines and quinazolines, and comparison of the different heterocyclic ring systems should be interesting. The SAR of substitution on several aromatic rings seems to run in parallel (isoquinolinebenzamidines, isoquinolinebenzamides, quinazolinephenylurea, N5-phenylcarbamoyltriazolopyrimidines) and investigation on a specific role for the aromatic methoxy group would be an interesting subject of research [58, 117, 122, 137, 1391. CONCLUSIONS The availability of cloned adenosine A3 receptors has forced the pace of the development of selective ligands. However, the pronounced species differences, e.g. between rat and man, is a cause for concern. It hinders

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THE ADENOSINE A3 RECEPTOR AND ITS LIGANDS

the traditional drug discovery process in which laboratory animal experimentation precedes clinical tests. Highly selective and potent agonists with affinities in the nanomolar range have been developed for the human adenosine A, receptor and the discovery of partial agonists is promising. The latter compounds may be useful because of their potential as tissue-selective agents. The search for selective non-xanthine adenosine A3 receptor antagonists has uncovered a huge diversity of structural classes affording high affinity ligands. Hopefully, these many new tools will provide the basis for a thorough pharmacological evaluation. Therapeutic application seems most promising for antagonists as potential anti-inflammatory drugs. REFERENCES 1 Drury, A.N. and Szent-Gyorgi, A. (1929) J. Physiol. (London) 68, 213-237. 2 Burnstock, G. (1978) in Cell Membrane Receptors for Drugs and Hormones (Bolis L. and Straub R.W., eds.), pp. 107-118, Raven, New York. 3 Van Calker, D., Muller, M. and Hamprecht, B. (1979) J. Neurochem. 33, 999-1005. 4 Londos, C., Cooper, D.M.F. and Wolff, J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77. 2551-2554. 5 Bruns, R.F., Lu, G.H. and Pugsley, T.A. (1986) Mol. Pharmacol. 29, 331-346. 6 Furlong, T.J., Pierce, K.D., Selbie, L.A. and Shine, J. (1992) Mol. Brain. Res. 15, 62-66. 7 Fink, J.S., Weaver, D.R., Rivkees, S.A., Peterfreund, R., Pollack, A.E., Adler, E.M. and Reppert, S.M. (1992) Mol. Brain Res. 14, 186-195. 8 Stehle, J.H., Rivkees, S.A., Lee, J.J., Weaver, D.R., Deeds, J.D. and Reppert, S.M. (1992) Mol. Endocrinol. 6, 384-393. 9 Rivkees, S.A. and Reppert, S.M. (1992) Mol. Endocrinol. 6, 1598-1604. 10 Palmer, T.M. and Stiles, G.L. (1995) Neuropharmacology 34, 683494. I1 Olah, M.E. and Stiles, G.L. (1995) Annu. Rev. Pharmacol. Toxicol. 35, 581-606. 12 Fredholm, B.B., Abbracchio, M.P., Burnstock, G., Daly, J.W., Harden, T.K., Jacobson, K.A., Leff, P. and Williams, M. (1994) Pharmacol. Rev. 46, 143-156. 13 Coates, J., Gurden, M.F., Harris, C., Kennedy, I., Sheehan, M.J. and Strong, P. (1994) Nucleos. Nucleot. 13, 1953-1976. 14 Jacobson, K.A., van Galen, P.J.M. and Williams, M. (1992) J. Med. Chem. 35,407-422. 15 Feoktistov, I., Polosa, R., Holgate, S.T. and Biaggioni, I. (1998) Trends Pharmacol. Sci. 19, 148-153. 16 Meyerhof, W., Muller-Brechlin, R. and Richter, D. (1991) FEBS Lett. 284, 155-160. 17 Zhou, Q.-Y., Li, C., Olah, M.E., Johnson, R.A., Stiles, G.L. and Civelli, 0. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7432-7436. 18 Sajjadi, F.G. and Firestein, G.S. (1993) Biochim. Biophys. Acta 1179, 105-107. 19 Salvatore, C.A., Jacobson, M.A., Taylor, H.E., Linden, J. and Johnson, R.G. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10365-10369. 20 Linden, J., Taylor, H.E., Robeva, AS., Tucker, A.L., Stehle, J.H., Rivkees, S.A., Fink, J.S. and Reppert, S.M. (1993) Mol. Pharmacol. 44, 524-532.

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131 Siddiqi, S.M., Jacobson, K.A., Esker, J.L., Olah, M.E., Ji, X-d., Melman, N., Tiwari, K.N., Secrist, J.A., Schneller, S.W., Cristalli, G., Stiles, G.L., Johnson, C.R. and IJzerman, A.P. (1995) J. Med. Chem. 38, 1174-1188. 132 De Zwart. M., Kourounakis, A., Kooijman, H., Spek, A.L., Link, R., von Frijtag Drabbe Kunzel, J.. IJzerman, A.P. (1999) J. Med. Chem. 42, 1384-1392. 133 Jacobson, K.A., Park, K-S., Jiang, J.L., Kim, Y.C., Olah, M.E., Stiles, G.L. and Ji, X-d. (1997) Neuropharmacology 36, 11 57-1 165. 134 Auchampach. J.A., Rizvi, A., Qiu, Y.M., Tang, X.L., Maldonado, C., Teschner, S. and Bolli, R. (1997) Circ. Res. 80, 800-809. 135 Olah. M.E., Gallo-Rodriguez, C.. Jacobson, K.A. and Stiles, G.L. (1994) Mol. Pharmacol. 45. 978-982. 136 Ji, X.-d., Gallo-Rodriguez, C. and Jacobson, K.A. (1994) Biochem. Biophys. Res. Commun. 203, 570-576. 137 Kim, H.O., Ji, X-d., Siddiqi, S.M., Olah, M.E., Stiles, G.L. and Jacobson, K.A. (1994) J. Med. Chem. 37. 36143621. 138 Franchetti, P., Cappellacci, L., Marchetti, S., Trincavelli, L.. Martini, C., Mazzoni, M.R., Lucacchini, A. and Grifantini, M. (1998) J. Med. Chem. 41, 1708-1715. 139 Alexander, S.P.H. and Peters, J.A. (1998) Trends Pharmacol. Sci. 7-8. 140 Cristalli, G., Camaioni, E.. Costanzi, S., Vittori, S., Volpini, R. and Klotz, K.N. (1998) Drug Dev. Res. 45, 176-181. 141 Klotz, K.N., Camaioni, E., Volpini, R., Kachler, S., Vittori, S. and Cristalli, G. (1999) Naunyn-Schmiedeberg’s Arch. Pharmacol. 360, 103-108. 142 Baraldi, P.G., Cacciari, B., Spalluto, G . ,Ji, X-d., Olah, M.E., Stiles, G.L., Dionisotti, S., Zocchi. C., Ongini, E. and Jacobson, K.A. (1996) J. Med. Chem. 39, 802-806. 143 Knutsen, L.J.S., Sheardown, M.J., Roberts, S.M., Mogensen, J.P., Olsen, U.B., Thomsen, C. and Bowler, A.N. (1998) Drug Dev. Res. 45, 214-221. 144 Mogensen, J.P., Roberts, S.M., Bowler, A.N., Thomsen, C. and Knutsen, L.J.S. (1998) Bioorg. Med. Chem. Lett. 8, 1767-1770. 145 Van Galen, P.J.M., IJzerman, A.P. and Soudijn, W. (1990) Nucleos. Nucleot. 9,275-291. 146 Kim, H.O., Ji, X-d., Melman, N., Olah, M.E.. Stiles, G.L. and Jacobson, K.A. (1994) J. Med. Chem. 37, 4020-4030. 147 Park, K-S., Hoffmann, C., Kim, H.O., Padgett, W.L., Daly, J.W., Brambilla, R., Motta, C., Abbracchio, M.P. and Jacobson, K.A. (1998) Drug Dev. Res. 44, 97-105. 148 Leurs, R., van der Coot, H. and Timmerman, H. (1991) in Advances in Drug Research (Testa B., ed.), vol 20. pp. 217-304, Academic Press, London. 149 Leurs, R., Smit, M.J. and Timmerman, H. (1995) Pharmac. Ther. 66, 413-463. 150 Van Tilburg, E.W., van Muijlwijk-Koezen, J.E. and IJzerman, A.P. (1998) Drug Dev. Res. 45, 182-189. 151 Van der Wenden, E.M., Carnielli, M., Roelen, H., Lorenzen, A., van Frijtag, J.K., Kunzel, D. and IJzerman, A.P. (1998) J. Med. Chem. 41, 102-108. 152 Ji, X-d., von Lubitz, D., Olah, M.E., Stiles, G.L. and Jacobson, K.A. (1994) Drug Dev. Res. 33, 51-59. 153 Kim, H.O., Ji, X-d., Melman, N., Olah, M.E., Stiles, G.L. and Jacobson, K.A. (1994) J. Med. Chem. 37, 3373-3382. 154 Siddiqi, S.M., Ji, X-d., Melman, N., Olah, M.E., Jain, R., Evans, P., Glashofer, M., Padgett, W.L., Cohen, L.A., Daly, J.W., Stiles, G.L. and Jacobson, K.A. (1996) Nucleos. Nucleot. 15, 693-717.

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155 Ji, X-d., Jiang, J.-L., Olah, M.E., Stiles, G.L. and Jacobson, K.A. (1996) Drug Dev. Res. 37, 131. 156 Ji, X-d., Melman, N. and Jacobson, K.A. (1996) J. Med. Chem. 39, 781-788. I57 Karton, Y., Jiang, J.L., Ji, X-d., Melman, N.. Olah, M.E., Stiles, G.L. and Jacobson, K.A. (1996) J. Med. Chem. 39, 2293-2301. 158 Triggle, D.J. (1985) in Comprehensive Medicinal Chemistry (Emmett J.C., ed.), vol 3. pp. 1047-1099, Pergamon Press, London. 159 Coburn, R.A., Wierzba, M., Suto, M.J., Solo,A.J., Triggle, A.M. andTriggle, D.J. (1988) J. Med. Chem. 31, 2103-2107. 160 Van Rhee, A.M., Jiang, J-I., Melman, N., Olah, M.E., Stiles, G.L. and Jacobson, K.A. (1996) J. Med. Chem. 39, 2980-2989. 161 Sunkel, C.E., de Casa-Juana, M.F., Santos, L., Gomez, M.M., Villarroya, M., Gonzalez-Morales, M.A., Priego, J.G. and Ortega, M.P. (1990) J. Med. Chem. 33, 3205-3210. 162 Wetzel, J.M., Miao, S.W., Forray, C., Borden, L.A., Branchek, T.A. and Luchowski, C. (1995) J. Med. Chem. 38, 1579-1581. 163 Lopez, M.G., Fonteriz, R.I., Gandia, L., de la Fuente, M., Villaroya, M., Garcia-Sancho, J. and Garcia, A.G. (1993) Eur. J. Pharmacol. 247, 199-207. 164 Jiang, J., van Rhee, A.M., Melman, N., Ji, X-d. and Jacobson, K.A. (1996) J. Med. Chem. 39, 4667-4675. 165 Jiang, J.L., van Rhee, A.M., Chang, L., Patchornik, A., Ji, X-d., Evans, P., Melman, N. and Jacobson, K.A. (1997) J. Med. Chem. 40, 2596-2608. 166 Li, A.H., Moro, S., Melman, N., Ji, X-d. and Jacobson, K.A. (1998) J. Med. Chem. 41, 3186-320 1. 167 Jiang, J.L., Li, A.H., Jang, S.Y., Chang, L., Melman, N., Moro, S . , Ji, X-d., Lobkovsky, E.B., Clardy, J.C. and Jacobson, K.A. (1999) J. Med. Chem. 42, 3055-3065. 168 Nakabeppu, H., Asada, M., Oda, T., Shinozaki, Y. and Yajima, T. (1996) Xenobiotica 26, 229-239. 169 Xie, R.Y., Li, A.H., Ji, X-d., Melman, N., Olah, M.E., Stiles, G.L. and Jacobson, K.A. (1999) J. Med. Chem. 42, 42324238. 170 Li, A.-H., Ji, X-d., Kim, H.S., Melman, N. and Jacobson, K.A. (1999) Drug Dev. Res. 48, 171-177. 171 Francis, J.E., Cash, W.D., Psychoyos, S., Ghai, G . ,Wenk, P., Friedmann, R.C., Atkins, C., Warren, V., Furness, P., Hyun, J.L., Stone, G.A., Desai, M. and Williams, M. (1988) J. Med. Chem. 31, 1014-1020. I72 van Muijlwijk-Koezen, J.E., Timmerman, H., Link, R., van der Goot, H. and IJzerman, A.P. (1998) J. Med. Chem. 41, 399MOOO. 173 van Muijlwijk-Koezen, J.E., Timmerman, H., van der Goot, H., Menge, W.M.P.B., von Frijtag Drabbe Kiinzel, J., der Groote, M. and IJzerman, A.P. (2000) J. Med. Chem. 43, 2227-2238. 174 Baraldi, P.G., Cacciari, B., Romagnoli, R., Spalluto, G., Klotz, K.N., Leung, E., Varani, K., Gessi, S., Merighi, S, and Borea, P.A. (1999) J. Med. Chem. 42, 4473-4478. 175 Ijzerman, A.P., van Galen, P.J.M. and Jacobson, K.A. (1992) Drug Des. Discov. 9,49-68. 176 Olah, M.E., Ren, H.Z., Ostrowski, J., Jacobson, K.A. and Stiles, G.L. (1992) J. Biol. Chem. 267, 10764-10770. 177 Moro, S., Li, A.H. and Jacobson, K.A. (1998) J. Chem. Inf. Comp. Sci. 38, 1239-1248. 178 Moro, S., van Rhee, A.M., Sanders, L.H. and Jacobson, K.A. (1998) J. Med. Chem. 41, 46-52.

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Progress in Medicinal Chemistry - Vol. 38, Edited by F.D. King and A.W. Oxford 8 2001 Elsevier Science B.V. All rights reserved,

3 The Medicinal Chemistry of the P2 Receptor Family SIMON D. GUILE, FRANCIS INCE*, ANTHONY H. INGALL, NICHOLAS D. KINDON, PREMJI MEGHANI and MICHAEL P. MORTIMOREt Department of Medicinal Chemistry, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire, L E I 1 5 R H , U.K .

INTRODUCTION

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RECEPTOR HETEROGENEITY AND LIGAND STABILITY

118

P2X RECEPTORS

I I9

P2X, RECEPTOR Biological properties Structure-activity relationships P2X, Agonists P2Xl Antagonists

120 120 120 121 123

P2X2 RECEPTOR Biological properties Structure-activity relationships P2 X , Agonists P2X2 Antagonists

130 130 130 130 130

P2X3 RECEPTOR Biological properties Structure-activity relationships P2X3 Agonists P2X3 AntaRonists

130 130 131 131 131

*Author for correspondence. Present address: Vertex Pharmaceuticals (Europe) Ltd, 88 Milton Park, Abingdon, OX14 4RY. U.K. 115

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THE MEDICINAL CHEMISTRY OF T H E P2 RECEPTOR FAMILY

P2X4 RECEPTOR Biological properties Structure-activity relationships P2X4 Agonists P2X4 Antagonists

131 131 132 132 132

P2X5 RECEPTOR Biological properties Structure-activity relationships P2X5 Agonists P2Xs Antagonists

133 133 133 133

P2Xs RECEPTOR Biological properties Structure-activity relationships P2X6 Agonists P2X6 Antagonists

133 133 134 134 134

P2X7 RECEPTOR Biological properties Structure-activity relationships P2X7 Agonists P2X7 Antagonists

134 134 135 135 135

HETEROMERIC RECEPTORS P2X1/5 heteromeric receptors P2X213heteromeric receptors P2X2,3 Agonists P2X2,3 Antagonists P2X416heteromeric receptors

140 140 140 140 140 141

P2Y RECEPTORS

141

P2Y 1 RECEPTOR Biological properties Structure-activity relationships P2 Y , Agonists P2 Y , Antagonists Molecular modelling and pharmacophores Clinical

141 141 142 143 146 149 150

P2Y2 RECEPTOR Biological properties Structure-activity relationships P2Yr Agonists P2Y2 Antagonists Molecular modelling and pharmacophores

150 150 152 152 156 158

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Clinical INS-316 ( U T P ) INS-345

158 158 159

p2y3 RECEPTOR

159

P2Y4 RECEPTOR Biological properties Structure-activity relationships P2 Y, Agonists P2 Y , Antagonists

159 159 160 160 161

p2y5 RECEPTOR

161

P2Y6 RECEPTOR Biological properties Structure-activity relationships PZY, Agonists P2Y6 Antagonists

162 162 162 162 163

p2y7 RECEPTOR

164

p 2 y ~RECEPTOR

164

p2y9 RECEPTOR

164

~ 2 ~ RECEPTOR 10

164

P2Y1I Receptor Biological properties Structure-activity relationships

164 164 165

P ~ RECEPTOR T Biological properties Structure-activity relationships P2T Agonists P ~ Antagonists T Clinical

165 166 169 169 170 171

ACKNOWLEDGEMENTS

177

REFERENCES

177

INTRODUCTION The purinoreceptors are a family of receptors that are activated by nucleosides and nucleotides. In 1978 Burnstock proposed that the pur-

118

THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

inoreceptors should be divided into two families: the P1 receptors, where nucleosides are more active than nucleotides and the P2 receptors, where nucleotides are more active than nucleosides [l]. This review will concentrate on the medicinal chemistry of the P2 receptor family, with the literature being covered up to June 1999. In 1985 it was proposed that the P2 receptors be divided into two major classes, P2X and P2Y [2]. The nomenclature was further rationalized in 1996 to clarify the rather disparate designations for the P2 receptors that had emerged over the previous 20 years [3]. The P2X receptors belong to the ligand-gated cation channel family, whereas the P2Y receptors are members of the seven transmembrane G-protein coupled receptor (7-TM GPCR) A P2YAc) ~ ~ has also been family. A receptor designated as P z (~P ~ Y or identified but its position within the P2X/P2Y classes has yet to be defined (although most of its properties are consistent with those of a 7-TM GPCR). P2X and P2Y receptors are numbered sequentially according to the clone being reported. Capital letters (e.g. P2Y ,) are used to denote functional mammalian receptors or their non-mammalian homologues. Lower case (e.g. p2y3) is used to denote orphan mammalian receptors, i.e. receptors having P2 sequence homology but no identified functional response and functional non-mammalian receptors with no known mammalian homologue. This system has led to non-sequential numbering of confirmed mammalian P2 receptors and may necessitate further revision of nomenclature for this class of receptors.

RECEPTOR HETEROGENEITY AND LIGAND STABILITY

In the absence of potent and selective antagonists for most P2 receptors, agonist potency profiles have commonly been used to characterize P2 receptor subtypes. While the variously reported profiles for a particular subtype are often in broad agreement, the exact potency order can differ, sometimes significantly, between reports. The following abbreviations are used in this review: ATP, adenosine-5’-triphosphate; Bz-ATP, a nlixture of 2‘- and 3’-O-(4-benzoylbenzoyl)adenosine-5’-triphosphates; CTP, cytosine-5’triphosphate; GTP, guanosine-5‘-triphosphate;ITP, inosine-5’-triphosphate; UTP, uridine-5’triphosphate; ADP, adenosine-5’-diphosphate; CDP, cytosine-5’-diphosphate; GDP, guanosine-5’-diphosphate; IDP, inosine-5’-diphosphate; TDP, thymidine-5’-diphosphate;UDP, uridine-5’-diphosphate; AMP, adenosine-5’-monophosphate; CAMP, cyclic adenosine-3’,5’monophosphate.

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In native systems, one of the possible reasons for this discrepancy is that some of the numerous P2 subtypes are often co-expressed [4-71. Using subtype selective ligands, P2 receptor heterogeneity has now been demonstrated in a number of tissues. This may also be a problem in recombinant expression systems unless the absence of other natively expressed P2 subtypes in the system is rigorously demonstrated. The correspondence between cloned and native receptors is a significant hurdle to research in this area and has recently been reviewed [8, 91. Experimental results and conclusions regarding the SAR and pharmacology of P2 receptors must be questioned, particularly if the work predates the recognition of multiple P2 receptor expression in the tissue investigated [4]. Such data should only be taken into consideration if the specificity of the ligands used has been defined. Another major factor affecting agonist potency orders for both native and recombinant P2 receptors is the issue of ligand stability. This is particularly relevant for the natural ligands (e.g. ATP/UTP and ADP/UDP), but close analogues of the natural ligands can also be affected. Nucleotides and their derivatives can be inter-converted and these processes are catalyzed by endogenous enzymes e.g. ecto-nucleoside diphosphokinases and ectonucleotidases [lo-141. Some of these processes may be controlled by use of enzyme inhibitors. Endogenous ligands may be released during the course of the experiment and these ligands may affect the result themselves but can also lead to receptor desensitization [15, 161 or act as a partner for the phosphate exchange process described above. Until such time as a particular response is proven to be entirely due to a particular P2 receptor subtype (e.g. P2YI), it has been suggested that the pharmacology and/or receptor in question be described as ‘P2-like’ (e.g. P2Y I-like) [17]. We must await the much-needed discovery of potent, stable and selective antagonists for a number of the P2 receptor subtypes before accurate pharmacological profiling and identification of subtype tissue distribution can begin.

P2X RECEPTORS The P2X family of receptors are ATP-gated ion channels. Seven P2X receptors, P2X1-7, have been cloned and the protein sequences for these clones contain between 379 and 595 amino acids and show 36-48% sequence identity to each other [ 18, 191. Each protein has two hydrophobic regions of

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THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

around 20 amino acids each and it has been proposed that these regions form two transmembrane domains with a large extracellular loop of about 290 amino acid residues and the N- and C-termini comprising the intracellular components of the receptor. A P2X receptor is made up of several of these subunits [20]. Where more than one P2X subtype exists on a tissue, it appears that there could be a heterogeneous assembly of subunits to form receptors distinct from the homogeneous subtypes, e.g. P2Xl / 5 , P2X213 and P2X4,6 [5-71 (see section describing heteromeric receptors). Co-immunoprecipitation studies suggest that other subunit co-assemblies may also be possible [21]. P2X receptors are distributed on excitable cells such as smooth muscle cells and neurones where there is a requirement for a rapid response following activation. Activation of P2X receptors leads to rapid non-selective influx of cations (Na', Ca2+, K') across the cell membrane resulting in an increase in intracellular calcium and depolarization [22, 231. This depolarization leads to activation of voltage gated Ca2+ channels which allows a further influx of Ca2+. The response time of this process is very rapid and does not rely on the use of second messengers. Based on their propensity to undergo desensitization, P2X receptors can be divided into two groups: P2X1 and P2X3, which desensitize rapidly (within 100-300 milliseconds) and P2X2, P2X4, P2X5, P2X6 and P2X7, which desensitize slowly or not at all [18, 191. The individual P2X receptors will now be discussed in more detail.

P2X1 RECEPTOR BIOLOGICAL PROPERTIES

The P2X1 receptor has been cloned from rat vas deferens [24] and the human and mouse bladder [25]. P2Xl receptor mRNA is expressed in a variety of cell types including bladder, vas deferens, vascular smooth muscle, lung, spleen, dorsal root ganglia, spinal cord, trigeminal ganglia and coeliac ganglia [18, 241. The P2X1 receptor has also been reported to be present on platelets and megakaryocytes [26-281. When expressed in smooth muscle, the P2XI receptor elicits a contractile response in the presence of an agonist.

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STRUCTURE-ACTIVITY RELATIONSHIPS

P2Xl Agonists

The agonist potency order (EC50, intrinsic activity for partial agonists) reported for the cloned human P2X1 receptor is: Bz-ATP (1.8nM), 2-MeS-ATP ( 1 a) (54 nM), ATP (56 nM), Ap4A (2c) (180 nM), a,fl-me-ATP (3) (200nM), Ap5A (2d) (280nM), Ap6A (2e) (1.1 pM, 55'%), ATP-y-S (4) (2.3 pM, SO%), ADP-B-S (5) (2.5pM), ADP (lOpM) [29]. For the structures of the 2-substituted ATP analogues (I), see Tuble 3.1 and of the diadenosine polyphosphates (ApnA) (2), Table 3.2. The stability of different

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122

Table 3.1 STRUCTURES OF 2-SUBSTITUTED ATP ANALOGUES (la-lh) y

Conioound

R

2

Cornpound

R

Table 3.2 STRUCTURES OF DIADENOSINE POLYPHOSPHATES (ADENOSINE-pn-ADENOSINE) (2)

Compound

Ap2A Ap3A Ap4A ApSA Ap6A

(2a) (2b) (2c) (2d) (2e)

Nucleoside

Polyphosphate ( n )

Nucleosidr

Adenosine Adenosine Adenosine Adenosine Adenosine

2

Adenosine Adenosine Adenosine Adenosine Adenosine

3

4 5 6

nucleotides in the presence of ecto-nucleotidases can vary considerably,thus agonist potency orders derived from isolated tissues must be treated with caution. For example, in the guinea pig vas deferens Ap4A (2c) appears to be less metabolically stable than ApSA (2d) [12].

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P2Xl Antagonists

Suramin (6) is a non-selective, reversible P2 receptor antagonist and was first reported as a P2X receptor antagonist in the mouse vas deferens [30]. It has an ICSOof 1 pM for the P2XI receptor [18]. It is also an antagonist for other receptors such as the PDGF, GABA and glutamate receptors. Suramin is an inhibitor of ecto-nucleotidases and a number of protein kinases and proteases [31, 321.

-

SO,H

SO,H

(6)Suramin

60,H

SO,H

(7)NF023

60,H

(8)BstlOl

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THE MEDICINAL CHEMISTRY O F THE P2 RECEPTOR FAMILY

SO,H (9) NF019

HO,S

(10) XAMR0716

QO,H (11) ANTS

Five structurally similar analogues or fragments of suramin have been compared in a rat vcls deferens assay, where u,P-me-ATP (3) was used to elicit a P2X1 mediated contraction (Table 3.3) [33]. Suramin antagonized this contraction with a Kd of 4 pM. NF023 (7) and BstlOl(8) showed similar affinity whilst the antagonist potency fell off rapidly with less complex analogues. The result with (8) is significant in that it illustrates that the sulfonate groups at one end of the molecule can be removed without loss of affinity.

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Table 3.3 P2Xl ANTAGONIST ACTIVITY O F SURAMIN ANALOGUES [33] Compound

Apparent

Suramin (6) NF023 (7) BstlOl (8)

3.9

(a’

Kd

(pM)‘“’

1.0 4.8

Compound

Apparent K,, ( W L M ) ‘ ~ ’

NF019 (9) XAMR0716 (10) ANTS (11)

93.9 458 3672

rat vas deferens.

HO,S

HO,S

H

H

HO,S

(12) NF279

More recently, another analogue, NF279 (12), has been reported to be approximately 10-fold more potent as a P2X1 antagonist than either (6) or (7) (rat vas deferens, IC5,, values: (12) 2pM, (7) 13 pM and (6) 20pM) [34]. (12) was reported to show a greater selectivity for the P2X1 receptor over the P2Y I receptor and ecto-nucleotidase inhibition. In electrophysiology experiments on cloned receptors transfected into Xenopus Laevis oocytes [35], (12) was also reported to show selectivity for rat P2X1 over rat P2X2, rat P2X3, and human P2X4 receptor subtypes (ICS0 values: P2X1 19nM, P2X2 760nM, P2X3 1.6pM, P2X4 >300pM).

N o 5 4’ 0 2 3’

(13) see Table 3.4

PPADS (1 3c), (Table 3.4), first reported as a P2 receptor antagonist in the rabbit vas deferens [36], is a non-selective antagonist with ICS0values in the range 1-3pM for the recombinant P2X1, P2X2, P2X3 and P2X5 receptors

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126

Table 3.4 P2XI /P2X2 ACTIVITIES OF PPADS ANALOGUES (13a-13u) [38] ~

Compound

2’

3’

4‘

5‘

x

(1 3a)

H

H

H

H

OCH2

S03H H H H C02H H C1 H H H H H H H H H H

H S03H H H H CO2H H H H H C02H H H H H H CO2H H

H H SO3H SO3H H H H H S03H C02H H NO2 SO3H SO3H SO3H SO3H H SO,H

OCH2 OCH2 OCH2 OCH2 OCH2 OCH2 OCH2 OCH2 OCH2 OCH2 OCH2 OCH2 CH2 CH2 CH2CH2 CH2CH2 CHlCHz CH=CH

H H

H H

c1

(13t) (13~) (a’

lb)

S03H CI

PZX,@’ 55 (IGo 45 WM) 25 18 31 14 10 25 16 52 53 42 11

64 43 28 25

PZX,’b’

1.2

1.6 0.4 2.6 12

0.82 1.1

I .5

17

2.4 81 (1Cso ~OI.LM) SO3H CH2CH2CH2 22 11 SOiH CH2CH2CH2 18

P2X, (human HL-60 cells) inhibition of binding, ‘%I at 100gM. P2X2 (recombinant rat, in oocytes) inhibition of ion flux ICs0 (pM).

[18]. It is a slowly equilibrating and slowly reversible antagonist for the P2XI and P2X2 receptors. Based on site directed mutagenesis, it has been proposed that this non-competitive behaviour is due to Schiff base formation between the aldehyde moiety of PPADS and the amino group of a specific lysine residue in the extracellular domain of these two receptors [37]. The P2X3 receptor has a threonine residue in the equivalent position and PPADS shows rapid equilibration and reversibility. A number of analogues of PPADS (13a-13u) were compared in human P2X1, guinea pig P2X1, recombinant rat P2X2, and P2Y1 screens (Table 3 . 4 ) [38]. These compounds have various combinations of sulfonic acid, methoxy, chloro, and carboxylate groups substituted on the phenyl ring in place of the 2,4-disulfonic acid substituents. The simple phenyl analogue (13a) has an ICso of 45 pM, while PPADS (13c) gave an inhibition of only 18% at 100,uM in the same screen, indicating that the sulfonate moieties are

SIMON D. GUILE ET AL.

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not essential for good binding. None of the other substitution patterns offered any significant increase in affinity over the simple unsubstituted phenyl ring.

Pyridoxal-5-phosphate (P5P) (14) does not have the azophenyl group of PPADS and is 10-fold less potent than PPADS at the recombinant P2XI receptor [39]. Replacement of the phenyl group in (13a) by a substituted naphthyl group affords a compound (15) that exhibits a small increase in activity (ICso 15 pM). A number of phosphonate analogues (13n-13u) were also tested (Table 3.4) and these results show that phosphonate substitution is acceptable (13s) and gives an increase in potency over the parent phosphate (iso-PPADS, 13d).

HO,S

S03H (16 ) R = Me (Trypan Blue)

(19)R = H (NH05)

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R

$& OH

NH,

SO,H

Ho3s+

SO,H

(17) R = Me (Evans Blue)

(18) R = H (NHO1)

There are a number of polysulfonated dyes that have been reported as P2X receptor antagonists [4045]. These include Trypan Blue (16), Evans Blue (17), NHOl (18) and NHOS (19) which have an apparent Kd of 3.0, 2.5, 0.8 and 2.5pM respectively in the rat vas deferens, with (17) and (18) being >40-fold selective for P2X1 over P2Y (16) and (19) are non-selective ~411.

,.

k"

PH PH

0

0

(20a) X = HO- I-O-r-O-r-O

0

PH PHH PH

(20b)X = HO-F;-O-E+F-O 0

O H 0

SIMON D. GUILE E T AL.

I29

The 2’-(or-3’)-0-(2,4,6-trinitrophenyl)substituted nucleotides (20a, 21-23) have been reported to be potent antagonists for the recombinant human P2XI, (also rat P2X3 and P2XZj3receptors while being substantially less potent for the rat P2X2, human P2X4 and rat P2X7 receptors) (Table 3.5)[46,47]. This antagonism is non-competitive. Despite having high affinity for the P2X1 receptor, TNP-ATP (20a) has been reported to inhibit the P2X mediated constriction of rat mesenteric artery rings only at high concentration (30pM) [13]. This apparent loss in affinity is attributed to metabolic breakdown of (20a) in whole tissue and it is presumed that TNP-a$-me-ATP (20b) should be a more metabolically stable high affinity antagonist.

Table 3.5 INHIBITION OF ATP- OR c(, p-me-ATP-EVOKED CURRENTS BY (20a) AND (21-23) IN CELLS EXPRESSING P2X RECEPTORS (ICs0 nM) [46, 471 Receptor

T N P - A T P (200)

P2X, P2X* P2X3 P2X4 P2X, P2XZI3

6 2,000 0.9 15,200 >30,000 7

T N P - A M P (22)

TNP-GTP (23)

3,400 2.9

4,400 0.4

TNP-ADP (21)

-

~ ~

1,200 1.3

~

~

~

-

~

5

36

~

1.2

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THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

P2X2 RECEPTOR BIOLOGICAL PROPERTIES

The P2X2 receptor has been cloned from rat pheochromocytoma PC 12 cells [20]. P2X2 mRNA appears to be widely distributed and has been found in the brain, spinal cord, superior cervical ganglia, adrenal medulla, intestine, bladder and vas deferens [18, 201.

STRUCTURE-ACTIVITY RELATIONSHIPS

P2X2 Agonists

The agonist potency order (EC50) reported for the cloned rat P2X2 receptor is: ATP-y-S (4) (1.3 pM), ATP (1.4 pM), 2-MeS-ATP (la) (1.6 pM), Bz-ATP (5.5pM) with a,P-me-ATP (3) and Ap2A-Ap6A (2a-2e) being inactive [29]. P2X2 Antagonists

Suramin (6) is a non-selective, reversible antagonist for most P2 receptor subtypes (see P2XI antagonist section) and has an ICS0 5 pM for the P2X2 receptor [ 181. PPADS (1 3c) (see P2X1 antagonist section) is also a non-selective antagonist for the P2X2 receptor with an ICsO -2pM [18]. The results given in Table 3.4 indicate that iso-PPADS (13d) is slightly more potent than PPADS (1 3c), while the unsubstituted phenyl compound (1 3a) has similar affinity, suggesting that the sulfonate groups are not essential for binding to the receptor. The other substituent patterns offer no significant improvement in affinity [38]. The 2’-(0r-3’)-0-(2,4,6-trinitrophenyl) substituted nucleotides (20a, 21-23) are weak antagonists for this receptor [46, 471 (Table 3.5).

-

P2X3 RECEPTOR BIOLOGICAL PROPERTIES

The P2X3 receptor has been cloned from rat dorsal root ganglion [5],human heart [48] and the full-length mouse P2X3 gene has also been isolated [49].

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The P2X3 receptor appears to have a limited distribution. It has been found in a subset of rat dorsal root ganglion sensory neurones but it was not found in other tissues including neurones from the sympathetic, enteric and central nervous systems [50].

STRUCTURE-ACTIVITY RELATIONSHIPS

P2X3 Agonists

The agonist potency order (ECSo,intrinsic activity for partial agonists) for the cloned human P2X3 receptor is: Bz-ATP (79 nM, 90%), ATP (340 nM), 2-MeS-ATP (la) (360 nM), Ap4A (2c) (500nM, 64%), Ap6A (2e) (600nM, 82%), Ap5A (2d) (620nM, 81%), ATP-y-S (4) (690nM), cl,P-me-ATP (3) (740 nM), ADP-P-S ( 5 ) (8.7 pM), Ap3A (2b) (19 pM) [29]. GTP is inactive [511. P2X3 Antagonists

-

Suramin (6) is a non-selective, reversible antagonist for most P2 receptor subtypes. It has an ICso of 3 pM for the P2X3 receptor [18]. PPADS ( I 3c) is also a non-selective antagonist for the P2X3 receptor having a reported ICso of 1 ,uM [18]. The 2’-(or-3’)-0-(2,4,6-trinitrophenyl)substituted nucleotides (20a, 21-23) are potent non-competitive antagonists for the P2X3 receptor (Table 3.5) [46,47]. Removal of the p- and y-phosphates has little effect on affinity, however, deletion of the remaining a-phosphate removes all affinity. TNP-GTP (23) gave similar results to TNP-ATP (20a).

P2X4 RECEPTOR BIOLOGICAL PROPERTIES

The P2X4 receptor has been cloned from rat hippocampus [52], dorsal root ganglia [37], rat brain [53, 541, human brain [54] and rat endocrine tissue [ 5 5 ] . P2X4 receptor mRNA is expressed in brain, sensory ganglia, superior cervical ganglion, lung, bronchial epithelium, bladder, adrenal gland, testes and vas deferens [37, 521.

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THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

(24) Reactive Blue 2

H

Et

(25) Coomassie Brilliant Blue G STRUCTURE-ACTIVITY RELATIONSHIPS

PZX4 Agonists The agonist potency order (EC50, intrinsic activity for partial agonists) reported for the cloned human P2X4 receptor is: ATP (480nM), Bz-ATP (490nM, SO%), Ap4A (2c) (630nM,59%), Ap5A (2d) (1.2pM, 55%), 2-MeS-ATP (la) (2.2pM, 800/0), a,P-me-ATP (3) (8.3pM, 50%), ATP-yS (4) (1 1 pM, 61%), ADP (17pM, 29%), ADP-P-S (5) (25pM, 790/;,),Ap6A (2e) (63pM, 60%) [29]. P2X4 Antagonists

Reactive Blue 2 (Cibacron Blue) (24) (IC50 IOpM) and Coomassie Brilliant Blue G (25) (IC50130pM) are weak antagonists of the rat P2X4 receptor [56] while suramin (6), NF279 (12) (see P2X1 antagonist section) and

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PPADS (13c) are very weak antagonists [34, 351. (6) (ICs0 178pM) and PPADS (13c) (ICS0 27pM) are slightly more potent at the human P2X4 receptor [54].

P2X5 RECEPTOR BIOLOGICAL PROPERTIES

The P2X5 receptor has been cloned from rat coeliac ganglia [18] and rat heart [57] and two spliced variant cloned human P2X5 receptors have also been identified [58,59]. The P2X5 receptor has been found in the spinal cord and on sensory ganglia [IS], rat heart [57], rat kidney [60,61], rat duodenum and rat epithelial cells [62].

STRUCTURE-ACTIVITY RELATIONSHIPS

Pi?X5 Agonists The agonist potency order (EC50) reported for the cloned human P2X5 receptor is: ATP-y-S (4) (9.3 pM), ATP (1 5.4 pM), 2-MeS-ATP ( l a ) (20pM), ADP (270pM), with a$-me-ATP ( 3 ) having no effect [18]. PZX5 Antagonists Suramin (6) (30 pM) inhibited ATP-evoked currents by 94% and PPADS (13c) (10 pM) inhibited ATP-evoked currents by 8 I%, with similar inhibition being observed for 2-MeS-ATP (la) evoked currents [18].

P2X6 RECEPTOR BIOLOGICAL PROPERTIES

The P2X6 receptor has been cloned from rat superior cervical ganglion [ 181 and from rat brain [63]. The P2X6 receptor is widely expressed in the CNS and along the normal rat nephron [61].

134

THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY STRUCTURE-ACTIVITY RELATIONSHIPS

P2X6 Agonists The agonist potency order (ECSo) reported for the cloned human P2X6 receptor is: 2-MeS-ATP (la) (9 pM), 2-Cl-ATP (1b) (10 pM), ATP (12 pM), ADP (270pM). These compounds are all full agonists with ATP-y-S (4) (16pM) being a partial agonist and a,P-me-ATP (3) having no effect [18]. P2X6 Antagonists Suramin (6) is not active, however, PPADS (13c) produces a largely irreversible antagonism of the currents evoked by ATP [18].

P2X7 RECEPTOR BIOLOGICAL PROPERTIES

The P2X7 receptor, previously known as the cytolytic P2Z or ATP4receptor, has been cloned from rat brain [64], human monocytes [19] and mouse microglial cells [65,66]. The P2X7 receptor consists of 595 amino acids and is permeable to small cations. The structure of the receptor is similar to the other P2X receptors. However, it has an intracellular C-terminus that is significantly longer (230 amino acids) than that of the other P2X receptors. Activation of the P2X7 receptor causes a non-selective cation channel to open, resulting in an increase in intracellular Ca2+. Prolonged activation promotes an increase in the permeability of the cell which results in the formation of a membrane pore that allows molecules of up to 900 daltons to pass through (this size is reduced to -400 daltons in lymphocytes). The last 177 amino acids of the C-terminus appear to be important for the receptor's ability to form a membrane pore [19, 641. In HEK cells expressing the rat P2X7 receptor the size of the pore has been measured using polyethylene glycols. After activation of the P2X7 receptor by Bz-ATP (30pM) PEG 6000 prevented dye (YO-PRO) uptake and cell lysis. The smaller PEG 2000 and PEG 4000 delayed, but did not block YO-PRO uptake. These measurements suggest that the diameter of the pore is approximately 4.0 nm in size [67, 681.

SIMON D. GUILE ET AL.

13s

The endogenous receptor is found on cells of the immune system, including mast cells, monocytes, macrophages, microglial cells, lymphocytes, fibroblasts, erythrocytes, erythroleukaemia cells, hepatocytes, and parotid and salivary gland acinar cells [17], renal mesangial cells [69], bone marrow cells [70], dendritic cells [71] and neurons [72]. STRUCTURE-ACTIVITY RELATIONSHIPS

PZX7 Agonists ATP activates the cloned and endogenous P2X7 receptors, with the tetra-anion (ATP4-) being the actual agonist [67, 731. Using rat mast cells the effect of ATP analogues on the P2X7 receptor has been studied [74]. It is possible to substitute at the C-2 position of the adenine base to give 2-MeS-ATP (la), 2-Cl-ATP (1 b) and 2-EtS-ATP (lc) all being equipotent with ATP. However, substitution at the C-8 or N-6 positions affords inactive ATP analogues. Diadenosine tetraphosphate (Ap4A) (2c) is also able to activate the P2X7 receptor on rat mast cells. The unnatural L-enantiomer of ATP (26) is inactive. Phosphorothioate analogues such as ATP-c(-S (27) and ATP-B-S (28) are more potent agonists than ATP at the P2X7 receptor. Bz-ATP is the most potent agonist of P2X7 receptor presently known, being 10-100 times more potent than ATP in activating P2X7 receptors in a number of cells [17, 751, though 100-1000 times less potent than at the P2X1 and P2X3 receptors [29]. Using the cloned human receptor it has been possible to carry out a more quantitative determination: e.g. Bz-ATP (ECso 5 pM), ATP (EC50 100 pM) [29, 641. All determinations are very dependent upon receptor expression levels and upon the concentration of divalent cations in the bath medium, so meaningful comparisons between assays and receptors cannot be made. In general, the rat receptor is more sensitive to P2X7 agonists, notably Bz-ATP, than the human receptor [ 19, 701 and Bz-ATP is 20-fold more potent as an agonist than ATP.

-

P2X7 Antagonists

A monoclonal antibody specific for the human P2X7 receptor has been generated in mice. Currents caused by application of Bz-ATP were reduced in amplitude by the presence of the monoclonal antibody. In addition, preincubation of the monoclonal antibody with THP- 1 cells antagonized the ability of PIX7 agonists to induce the release of IL-lJ [76].

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THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

Hd

bH

(26)R = H (29) R = SMe

* '$ HO

OH

Using HEK293 cells stably expressing the rat P2X7receptor, Ca2+, Mg2', Zn2', Cu2' or protons all inhibited Bz-ATP-evoked currents and YO-PRO

uptake into cells in a dose dependent manner, with ICsovalues being similar for inhibition of current or inhibition of dye uptake. Copper is the most effective inhibitor (ICso0.5-3 pM) with calcium being least effective (- 50% inhibition at 2-3 mM) [77]. Using rat P2X7 receptor on the mast cell 2-MeS-L-ATP (29) was shown to reduce the rate of uptake of ethidium bromide by -50% [74]. 2',3'-Dialdehyde ATP (30), also known as oxidized ATP ([OI-ATP), is an irreversible antagonist of the P2X7 receptor (ICso 30 pM)[78]. However, it is not selective for P2X7 receptors over other P2 receptors.

SIMON D. GUILE ET AL.

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Using ethidium bromide uptake as a marker for pore formation suramin (6) 61 pM), Reactive Blue 2 (24) (I& 69pM) and 5-hexamethylene amiloride (31) (IC5,13pM) all acted as antagonists of the P2X7 receptor [79-811. The effects of ATP on rat parotid acinar cells were blocked by a series of stilbene isothiocyanate analogues [82]. HzDIDS (32) and DIDS (33) were equally effective (JC50 -35pM). SITS (34) (an analogue with only one thiocyanate group) was less effective (IC50 -125pM) and DNDS (35) (a dinitro analogue lacking the isothiocyanate groups) was inactive. The non-selective P2 antagonists PPADS (13c) and P5P (14) are also active at the P2X7 receptor [17, 751.

(33) DIDS Rl = R2 = NCS

(34)SITS R' = NCS,R2 = NHAc (35) DNDS R' = R2 = NO2

I38

THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

(36)Calmidazolium

(37)KN-62 R = Me,X = 0 (38)KN-04 R = H, X = H,

Calmidazolium (36) is a known inhibitor of some voltage-activated and cyclic-nucleotide gated ion channels. It was found to inhibit the Bz-ATP evoked currents in HEK cells expressing the rat P2X7 receptor (ICs0 13 nM); the inhibition was slowly reversible and non-competitive. Interestingly, calmidazolium (36) did not effect the Bz-ATP-induced YO-PRO uptake [77, 8 11. The calmodulin kinase I1 inhibitor KN-62 (37) was shown to be a potent inhibitor of ATP-induced cytolysis of THP-1 cells [83]. It also inhibits the ability of P2X7 receptors to activate phospholipase D in THP-1 monocytes [84]. It has been shown that (37) and KN-04 (38), an inactive analogue of (37) with respect to CaMK I1 activity, are potent antagonists

SIMON D. GUILE ET AL.

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of the P2X7 receptor on human lymphocytes. (37) antagonized ATP-stimulated Ba2+ influx into lymphocytes 13 nM) and inhibited ATP-stimulated ethidium bromide uptake (ICso 13 nM). Similar effects were observed with (38) 17nM and 37nM respectively) [85]. It has since been found that the effects of (37) and (38) are selective for the human over the rat P2X7 receptor [86]. A recent patent has disclosed a series of succinimide and thiazole-2,4diones as novel antagonists of the P2X7 receptor, exemplified by (39) [87]. Two patents have reported that simple adamantane-based anilides and benzamides are P2X7 receptor antagonists exemplified by (40) and (41) [88, 891.

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THE MEDICINAL CHEMISTRY O F T H E P2 RECEPTOR FAMILY

HETEROMERIC RECEPTORS A number of different channel subunits have been reported to co-assemble to produce heteromeric ion channels. These are described below. P2X115 HETEROMERIC RECEPTORS

P2X1 and P2X5 channel subunits have been reported to co-assemble into a heteromeric ion channel when co-expressed in Xenopus oocytes [7,90]. This P2X1/ 5 heteromeric receptor demonstrates the pharmacology of the P2Xl receptor and the kinetics of the P2X5 receptor-slowly desensitizing currents evoked by the agonist a,P-me-ATP (3) (EC50 1.1 pM) and sensitive to TNP-ATP (20a) (IC50 64nM). P2X213 HETEROMERIC RECEPTORS

When the P2X3 receptor was co-expressed in HEK293 cells with P2X2 receptors, a non-desensitizing response to ATP was observed which was not consistent with the additive effect of the agonist responses of the two P2X2 and P2X3 channels [5]. It was suggested that a heteromeric receptor was present, made up of P2X2 and P2X3 subunits. P2X2,3 Agonists The agonist potency order (ECS0,intrinsic activity for partial agonists) for the cloned rat P2X2/3 receptor is: ATP (500nM), Bz-ATP (650nM, 81%) >2-MeS-ATP (la) (1 pM), ATP-7-S (4) (2 pM), aJ-me-ATP (3) (3.4 pM, 71%)),Ap4A (2c) (48pM) [29]. GTP is inactive [51]. P2X2/3 Antagonists

Suramin (6) is a non-selective, reversible antagonist for most P2 receptor subtypes and has an ICS0of 810nM for the P2X2/3 receptor [29]. PPADS (13c) is also a non-selective antagonist for the P2X2,3 receptor having a reported ICs0 of 1.3 pM [29]. The 2’-(or 3’)-0-(2,4,6-trinitrophenyI)substituted nucleotides (20a, 21-23) are potent non-competitive antagonists for the P2X2,3 receptor (Table 3.5) [46, 471. Removal of the y-phosphate had little effect on affinity, while removal of the 8-phosphate produces a 5-fold drop in affinity and deletion of the remaining a-phosphate removes all affinity. TNP-GTP (23) gave similar results to TNP-ATP (20a).

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P2X4,b HETEROMERIC RECEPTORS

P2X4 and P2X6 channel subunits have been reported to co-assemble into a heteromeric ion channel [6]. This P2X416 heteromeric receptor shows a unique receptor phenotype with increased sensitivity to the effect of 2-MeS-ATP (la) and a,B-me-ATP (3). Suramin (6), PPADS (13c) and Reactive Blue-2 (24) were all found to be more effective antagonists against the heteromeric receptor than against either P2X4 or P2X6 homomeric receptors.

P2Y RECEPTORS The P2Y receptors are activated by purine and/or pyrimidine nucleotides and belong to the 7-TM GPCR super-family. Of the 11 currently known P2Y-like receptors found in vertebrates there are 5 having mammalian homologues that demonstrate a ‘P2Y-like’ functional response; these are P2Y1, P2Y2, P2Y4, P2Y6, P2Yll. These receptors have been cloned and the protein sequences contain between 320-377 amino acids, showing 28-52% sequence identities to each other. The individual P2Y receptors will now be described in more detail.

P2Y 1 RECEPTOR In general, references to ‘the P2Y receptor’ in the literature will equate to the P2Y receptor. The term ‘P2Y ,-like’ will be used here as explained in the introduction [17]. BIOLOGICAL PROPERTIES

The P2Yl receptor was first cloned from a chick brain cDNA library [91]. When expressed in Xenopus oocytes this receptor was found to have a ‘P2Y1-like’ pharmacological profile [92]. Homologues of this receptor have been isolated from turkey brain [16], rat ileal myocytes [93] and insuloma cells [94], bovine endothelium [95] and human HEL cells [96], placenta [97], brain [98], prostate and ovary [99]. In all cases the same approximate ‘P2Y I-like’ pharmacological profile has been found [ 171. The P2Y 1 receptor shows the 7 transmembrane regions consistent with the GPCR super-family, however, it is a relatively small protein of this class (e.g. chick 362 amino

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THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

acids, human 373 amino acids) and shows only -25% sequence identity with its closest non-P2 relative. The human homologue shows 83% sequence identity to the chick and turkey receptors and at least 95% sequence identity to the rat and bovine receptors [98]. The usual signal transduction pathway for cloned and expressed P2Y receptors and for natively expressed ‘P2Y1-like’ receptors is via the G protein, Gq/ 11. This is coupled via PLCP to IP3 formation and increase in intracellular calcium [16, 93, 991. There are numerous events downstream of IP3 formation and calcium mobilization [17]. Other natively expressed ‘P2Y I-like’ receptors are coupled, via the Gi protein, to inhibition of adenylyl cyclase. These include the ‘P2Y ,-like’ receptor on C6-2B rat glioma cells [loo] and B10 rat brain endothelial cells [loll. It has been shown, however, that these receptors are not the same as P2Y1 [ 102-1 041. P2Y I-like pharmacology has been described in various tissues [17], including guinea pig taenia coli, rat duodenum and mesenteric bed, bovine and rat aorta, bovine pulmonary artery and turkey erythrocytes. Activation in smooth muscle normally induces relaxation. P2Y 1 receptors are found in many human tissues. Two forms of P2Y I human mRNA have been found [96, 97, 991. The presence of P2Y1 receptors on platelets is discussed in the section on the P2Treceptor (vide infra). Recently, it has been shown in some systems that ATP acts as an antagonist and ADP as an agonist at the P2Y1 receptor [105]. This led to speculation that the P2Y receptor was actually the PZTreceptor (the elusive receptor found on the platelet that has a similar ligand profile). This possibility has now been discounted by conducting experiments using selective P2T and P2Y antagonists [ 1061.

STRUCTURE-ACTIVITY RELATIONSHIPS

The problems associated with ligand stability and identification of authentic, clean P2Y I receptor preparations in tissue greatly impact on the study of structure-activity relationships for the P2Y receptor. Profiles reported in the literature must be treated with caution unless these factors have been taken in to account. The most wide-ranging study of P2Y I SAR has used a variety of natural and synthetic ligands to study the P2Y1 receptor looking mainly at turkey erythrocyte response [16]. The data presented below are for this system unless otherwise stated.

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All of the P2Y I agonists described in the literature are based on a nucleotide type structure. Changes to each part of the structure will be described in turn. Agonist potencies (turkey erythrocyte) are quoted as ECSo values for inositol lipid hydrolysis [107]. In turkey erythrocytes ATP (2.8 pM) is reported to be a somewhat more potent agonist than ADP (8 pM), whilst AMP and adenosine are inactive [107, 1081. The same rank order is also found in the chick brain P2YI receptor transfected into Xenopus oocytes [92]. However, in other systems ADP is a better agonist than ATP and, in some cases, ATP has been shown to be an antagonist [I051 or a partial agonist [15, 1091. The potency of ADP in the turkey erythrocyte screen is significantly lower than that reported in other screens, e.g. turkey P2Y receptors transfected into 132 1N 1 cells (1 16 nM) and human P2Y receptors transfected into 1321N1 cells (257 nM) [98]. The methylene stabilized triphosphates a$-me-ATP (3) and j,yme-ATP (42) are inactive but the imino analogue of the latter, j,y-NH-ATP (43) (4.5pM) is similar to ATP. Amongst the thio-triphosphates, ATP-a-S (27) (8.9 pM) shows similar potency to ADP whilst ATP-11-S (4) (1.3pM) is more potent than ATP. ADP-8-S ( 5 ) (96nM) is particularly potent in this screen [108]. In general, ADP and derivatives are more potent than their ATP analogues [16, 95, 981. Several ATP derivatives having modified 2’ and/or 3’-positions have been tested and are generally much less active than ATP. 2’-DeoxyATP (44) (19pM) is more potent than 3’-deoxyATP (45) (76pM) which is about the same as 2’/ 3’-dideoxyATP (46). Several 3’-deoxy-3’-amino derivatives

(42) X = CH,

(43)X = NH

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THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

U 2 % Hd

OH

were also examined, with the 3’-amino derivative (47) being quite potent (193 nM) whilst substituted amino derivatives were inactive, suggesting a size limitation a t this position [log]. Modification of the purine is illustrated by the inactivity of UTP at the P2Y, receptor [108]. Substitution a t N-1 reduces activity, e.g., ATP-N-1-oxide (48) (17 pM) and N-1 ,N-6-etheno-ATP (49) (inactive) [I 081.

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A relatively large number of synthetic 2-substituted adenosine phosphate derivatives have been tested. In particular, a range of 2-thioethers has been prepared with varying length alkyl chains containing aryl rings and/or other interactive groups. In general, substitution at the 2-position leads to a large increase in potency at the P2Y receptor. The 2-alkylthio-ATP derivatives 2-methylthio (la) (8 nM) and 2-hexylthio (Id) (5 nM) are several hundred fold more active than ATP itself (Table 3.1). Other 2-thioethers include cyclohexylthio-ATP (le) (24 nM) and 2-[(5-hexenyl)thio]-ATP (If) (10 nM). When polar groups are added to the alkyl chain potency begins to fall, e.g. 2-[(7-aminoheptyl)thio]-ATP (lg) (73 nM). A small number of 2-phenethylthio derivatives were also prepared and showed good potency. In particular, p-aminophenethyl (1 h) (1.5 nM) appears to be one of the most potent P2YI agonists reported. 2-Substitution on ADP also gives a large increase in potency, e.g. 2-MeS-ADP (50) (6 nM) is over 1000 times more potent than ADP [107]. This was extended to a series of 2-alkylthio-AMP derivatives. Whilst AMP itself is inactive in the turkey erythrocyte screen, the 2-alkylthio derivatives had potency in the micromolar range. In this series the chain length of the alkylthio group was systematically varied from five to eleven carbons and it appears that n-hexyl (51) (59nM) was the optimum chain length. 2-Alkylthioadenosine derivatives were inactive, leading to the conclusion that at least one phosphate group is necessary for activity [110]

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Reports of agonists having substitution at the 6-position have been limited thus far to an N-6-methyl group. This substitution in the ATP series (parent or 2-substituted derivative) gives a small drop in potency, e.g. N-6-methyl-2-[(5-hexenyl)thio]-ATP (52) (26 nM). However, it was noted that substitution at N-6 removed activity at P2X receptors suggesting a way forward to design more selective ligands. N-6-Methyl-AMP derivatives are not active [107]. Substitution at the 8-position leads to reduced potency [108]. However, it appears that this position has not been widely investigated. P 2 Y, Antagonists Antagonists at the P2YI receptor fall into two main classes, the non-selective antagonists that are not based on ATP and the more selective antagonists based on the nucleotide structure. Many of the non-selective P2 antagonists described above also behave as weak antagonists at the P2Y1 receptor. These include compounds such as suramin (6), Reactive Blue 2 (24) and PPADS (13c) [l 111. As described above, ATP has been shown to act as an antagonist in some test systems [I051 and as a partial agonist in others [15, 1091. A number of adenosine-2’,5’-and 3’,5’-diphosphates and their derivatives (53-55) have been investigated (Tables 3.6-3.8). These compounds are antagonists of the response to 2-MeS-ADP (50) or 2-MeS-ATP (la) at the P2Y1 receptor, with the turkey erythrocyte system being used for most of these studies [103, 112, 1131. Adenosine 3’,5’-diphosphate (53a) and 2’,5’-diphosphate (53b), as well as their deoxy derivatives (53c) and (53d) and the 2’,3’-cyclic phosphate (53e), all act as partial agonists in the turkey erythrocyte screen with approximately the same antagonist affinity [103, 1121. The sulfate derivative

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Table 3.6 P2Y, ACTIVITIES OF COMPOUNDS (53a-53h) [103, 112, 2431

(53a) (53b) (53c) (53d) (53e) (530 (53d (53h) ") ") ")

fd) fe)

OH OP H OP cP OH OH H

OP OH OP H CP OP OH OP

OP OP OP OP OP OPS OPS OCHiP

4.19 8.46 5.76

12.7

77 75 87 81 73

-

-

11.0

~

19.7

78

2.23 1.65

0.17 0.21 -

~

-

~

~

0.83 22 I -

-

0.17 1 ~~

P = PO3H2, PS = P03HSO?H, c P = 2',3'-cyclic phosphate. Concentration to inhibit by SO'%) the effect of 10 nM 2-MeS-ATP or 2-MeS-ADP. 100 minus residual stimulation at highest antagonist concentration. Concentration at which 5O"h maximum effect achieved. Response relative to effect at maximal concentration of 2-MeSATP

A3P5PS (530 is slightly more potent. A phosphate on the 2'- or 3'-position is necessary for antagonist activity, the ASPS (53g) compound being a weak full agonist [103]. A phosphonate derivative (53h) has also been tested and this shows increased agonist activity over the phosphate analogue (53c) and has slightly reduced antagonist affinity [113]. In the cloned human P2YI receptor transfected into 1321N1 cells, compounds (53a) and (530act as antagonists of similar affinity but in this system there is no residual agonism [103]. This may be a consequence of species differences in the receptor. These compounds are selective over the other P2Y subtypes. The thio (54a) and carbocyclic (54b) analogues of the ribose compound (53c) have increased agonist activity [113], as does the 2'-methoxy analogue (54c) when compared with its 2'-hydroxy analogue (53a) (Table 3.7) 11121.

148

THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY Table 3.7 P2Yl ACTIVITIES OF (54a-54c) [I131

Compound

X

R

IC,, (@M)(')

Antagonist Effect (b)

(5 4 4 (54b)

S CH2 0

H H OMe

ND 2.53 12.4

38 73 65

( 54c)

(a),(b)Asin Table 3.6. Table 3.8 P2YI ACTIVITIES OF (55a-55j) [I131

Compound

R'

R2

Y

IC,, (pM)'"'

NHMe NHMe NHz NH2 NH2 NHMe NHMe NHEt NHPr OH

CH N N N N N N N N N

0.90 0.33 2.01 1.89 ND 0.21

0.36 1.08 -

Antagonisi Efleci'

96 99 80 78 46 95 94 100

NA NA

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A I-deaza derivative (55a) was tested and had good activity which was 3-fold less than its purine analogue (55b) MRS 2179 (Table 3.8) [113]. A number of groups have been added at the 2-position of the purine ring. Small groups, e.g. 2-C1 (55c) and 2-methylthio (55d) give a small increase in affinity over the 2-H compound (53c). The 2-(propylthio) derivative (55e) shows increased agonist activity [113]. Addition of a methyl group on the N-6 nitrogen (methylamino) gives an increase in antagonist activity over the 6-NH2 derivative and removes residual agonism, e.g. 2-H (55b, MRS 2179), 2-C1 (55f) and 2-methylthio (55g). The 2-H compound (55b, MRS 2179) has also been tested at the human receptor where it shows similar properties [114]. An ethylamino group is tolerated (55h), however, larger groups, e.g. propylamino (55) or non-nitrogen substituents, e.g. hydroxyl (55j), at the 6-position are not active. Substituents in the 8-position are not well tolerated [113].

MOLECULAR MODELLING A N D PHARMACOPHORES

Using the known primary sequence of the chick P2Y receptor and the structure of rhodopsin as a template, a model of the P2Y1 receptor has been constructed [ 1 151. This model suggests binding of the triphosphate of ATP to basic residues in TM3, TM6 and TM7. The model also predicts a hydrogen bond between the N-6 hydrogen of the purine and glutamine-296 in TM7. Using a combination of molecular modelling and site-directed mutagenesis on the human receptor, a number of important interactions have been postulated [116, 1171. The binding site is situated on the extracellular side of the TM bundle with residues on TM3, TM5, TM6 and TM7 being involved. Residues that might interact with the triphosphate, ribose and purines have been identified and interactions specific to agonists, e.g. 2-MeS-ATP (la) and antagonists, e.g. (55b) (MRS 2179) have been suggested [ 1 171. Further studies have identified a potential second or meta binding site for ATP between extracellular loop (EL) 2 and EL3. Essential disulfide bridges between Cys-124 (ELI) and Cys-202 (EL2) and between Cys-296 (EL3) and Cys-42 on the N-terminus have been found. EL2 appears to partially cover the putative binding domain of TM3, TM5, TM6 and TM7 and may form an entry channel into this region [118]. The disulfide bond between Cys-124 and Cys-202 is necessary for effective receptor trafficking to the cell surface [ 1 191.

150

THE MEDICINAL CHEMISTRY O F THE P2 RECEPTOR FAMILY CLINICAL

P2Y I receptors are present on many important cell types. Numerous suggestions have been made about the therapeutic potential for agonists/ antagonists at these receptors [ 120-1221, however, no reports of clinical studies in this area have appeared in the literature. There are patents claiming a use for P2Y antagonists in CNS neurodegenerative disorders such as Alzheimer's disease or multiple sclerosis [123], in asthma and allergy through inhibition of mast cell mediator release [124] and in diabetes through insulin secretion [125].

P2Y 2 RECEPTOR BIOLOGICAL PROPERTIES

The P2Y2 receptor has been cloned from mouse [126], rat [127-1301 and human [131-133] sources. The human P2Y2 receptor gene has been mapped to chromosome 1 1q 13.5-1 4.1 [ 1341. The existence of polymorphism of the human P2Y receptor has recently been discovered during the process of cloning the receptor from randomly taken genomic DNA samples [135]. Two allelic variants of the P2Y2 receptor were characterized, although no large pharmacological differences were observed between the two polymorphic receptors for the natural agonists tested. P2Y2 receptor mRNA is widely distributed in human tissues, with significant levels found in heart, kidney, liver, lung, testes, spleen, skeletal muscle, and placenta [126, 1311, however, very little message is found in the brain [ 1331. Cloned and expressed P2Y2 receptors from mouse, rat and human sources all exhibit the characteristic rank order agonist potency profile: UTP = ATP >> 2-MeS-ATP (la). This is identical to the original pharmacological assignment of the P2U receptor cited in the literature before 1993 [136]. However, it has recently been shown that the cloned rat P2Y4 receptor is also equally sensitive to UTP and ATP (unlike the human homologue, which is selective for UTP). Thus, the former P2U receptor, that had usually been pharmacologically characterized as the receptor exhibiting equal sensitivity towards UTP and ATP, cannot be equated with a single P2Y subtype, at least in rat tissues. Cloned and endogenous P2Y2 receptors can couple via both Gi/O and Gq/ 11 proteins to mediate Ca" mobilization

SIMON D. GUILE E T A L .

151

via PLC and IPI formation. This effect may be pertussis toxin-sensitive, -partially sensitive, or -insensitive [23]. The specific downstream involvement of a given signalling pathway appears to be partially dependent on the cell type in which the P2Y2 receptor is expressed [17, 1371. P2Y2 receptors have also been linked indirectly to the inhibition of adenylyl cyclase (via an increase in intracellular Ca’+) [I381 or an increase in CAMP (via PGE2 release) [139]. Recombinant and endogenous P2Y receptors are prone to desensitization and tachyphylaxis has been reported in a variety of cells [ 17, 140, 1411. The intracellular C-terminus of the receptor may be important because progressively larger truncations of this region of the P2Y2 receptor decreases the rate and magnitude of desensitization [ 1421. The pharmocology of P2Y2 receptors, like most of the P2Y subtypes to date, is based mainly on the rank order of agonist potencies. Functional studies show that receptors exhibiting the pharmacological properties of the P2Y2 receptor appear to be present on a wide variety of cells and tissues including: astrocytes, different types of blood cells, chromaffin cells, endothelial cells, myocytes, osteoblasts, pancreatic j-cells, pheochromocytoma PC 12 cells, pituitary cells, thyrocytes, and tumour cells [17]. In the vasculature, P2Y2 receptors are generally present on the endothelium [ 143-1461. Stimulation of P2Y2 receptors present on the endothelium results in the synthesis and release of prostacyclin and nitric oxide, leading to vasodilation. P2Y2 receptors on neutrophils stimulate degranulation, potentiate fMLP-induced superoxide formation, induce aggregation, chemotaxis and actin polymerization [147-1511. P2Y2 receptors on human nasal and lung epithelial cells are C1- secretogogues via activation of Ca3+ dependent CI- channels [ 152-1 541. Similarly, C1- secretion is activated by the receptor on intrahepatic biliary epithelial cells [155] and avian salt gland cells [156] which drives fluid secretion. P2Y2 receptors on human airway epithelial cells in vitro have been linked to increased cilia beat frequency [ 157, 1581 and on goblet cells give rise to both an increased rate and total amount of mucin secretion in vitro [158, 1591. P2Y2 receptors on human keratinocytes mediate cell proliferation indicating that the receptor may play a major role in epidermal homeostasis [160]. Sources of UTP and/or ATP have been studied under different physiological conditions. UTP and ATP have been shown to be released from endothelial cells by increased blood flow [I611 and to be released from epithelial and astrocytoma cells by aggitation of the bathing medium [162, 1631. UTP is also stored in platelets [164, 1651.

152

THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY STRUCTURE-ACTIVITY RELATIONSHIPS

P2 Y , agonists

P2Y2 receptors recognize both ATP and UTP and all synthetic agonists have been based on the modification of these two nucleotides. The purine and pyrimidine ring system, the ribose moiety, or the triphosphate group has been modified in various ways, with the aim of improving potency, enzymatic stability and/or receptor subtype selectivity. Removal of the terminal y-phosphate from ATP or UTP results in complete loss of activity [166]. Earlier literature cites UDP and ADP as moderately potent agonists at this receptor [167]. This has now been shown to be due to contamination of the nucleoside-diphosphate with the corresponding nucleoside-triphosphate [ 1661. Replacement of the bridging oxygen atoms of the triphosphate group with a methylene, dihalomethylene or imido moiety at the B,y-position in ATP or UTP results in either substantial loss of activity (>60-fold) or inactive compounds [158, 1681. Replacement of an oxygen atom on the terminal y-phosphate group with the bioisosteric sulfur atom results in compounds that retain good potency. For example ATP-y-S (4)is only slightly less active ( 3-fold) than ATP; in the case of UTP-y-S (56) it is equipotent with UTP or ATP [169]. The advantages of these changes are that they result in substantially better hydrolytic enzymatic stability compared to their natural counterparts. UTP-y-S (56) has been found to be equipotent to UTP in promoting chloride ion secretion from the epithelial cells of the cystic fibrotic

-

H

airway. If it has a longer duration of action than UTP, UTP-y-S (56) could represent an opportunity for the treatment of cystic fibrosis. Unfortunately its synthesis by enzymatic methods produces limited quantities and this remains an issue for further development [I 691.

SIMON D. GUILE E T A L .

153

Other changes involving the terminal y-phosphate group include diadenosine tetraphosphate, Ap4A (2c). This compound is reported to be a potent agonist at recombinant P2Y2 receptors with greater potency than ATP-y-S (4) and within the range of UTP and ATP, raising the possibility that it is also an endogenous regulator of these receptors [167, 1701. Ap4A (2c) is thought to be more stable towards nucleotidase-catalyzed hydrolysis compared to ATP or UTP. Bearing this in mind, further interest has been directed towards analogues of dinucleoside-polyphosphates and this is exemplified by a number of patents claiming their use in lung diseases, e.g. COPD, cystic fibrosis, and primary ciliary dyskinesia [171], ear diseases (otitis media) [172], and eye diseases (dry eye) [173]. Table 3.9 summarises the agonist activity for the most potent dinucleoside-polyphosphates (57a-57d) described in a recent patent [171]. The crossover of activity from Ap4A (2c) to the uridine series is not surprising. The 4-thiouridine moiety contained within the dinucleosidepolyphosphate (57a) is an interesting modification, suggesting that this change may be advantageous for potency. Endogenous P2Yz receptors are preferentially activated by the fully ionized forms of ATP and UTP (ATP4-, and UTP4-) in a variety of cell types such as bovine aortic endothelial cells, human neutrophils, a cultured neuroblastoma-glioma hybrid cell line, rat lactotrophs, mouse pineal gland tumour cells, and MDCK cells [17]. The UTP and ATP responses correlate with the concentration of the fully ionized form of these agonists and not with the concentration of their cation complexes or other ionized forms.

Table 3.9 P2Y2 ACTIVITY OF DINUCLEOTIDE TETRAPHOSPHATES (NUCLEOSIDE-p4-NUCLEOSIDE’)(57a-57d) [I711

Nucleoside-0-

PH l-O-fi-O-fi-O-F;-O-Nucleoside’ PH PH PH 0

0

0

0

Cotitpound

Nucltwside

Nucleosidr ’

EC.j,t ( P M )

(4-SH-U)p4(4-SH-U) (57a) Up4(dU) (57b) Up4T (57c) Up41 (57d)

4-SH-uracil Uracil Uracil Uracil

4-SH-uracil 2’-Deoxyuracil Thymidine lnosine

0.02 0.1 0.1 1 0.1 I

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THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

(58)R' = H, R2 = OH

(59)R' = OH, R2 = H

(60)R' = R2 = H

(611

All modifications to the sugar backbone of ATP and UTP reported so far have been detrimental to agonist potency. For example removal of the 2'- or 3'- hydroxyl groups to give 2'- or 3'-deoxy ATP (44), (45) or 2'- or 3'-deoxy

SIMON D. GUILE ET AL.

155

UTP ( 5 8 , 59) affords derivatives which are much less active; the 2’,3’-dideoxy (46), (60) analogues are inactive. Similarly, periodate oxidation of ATP or UTP affords the dialdehydes (30), (61) that are devoid of all activity. The arabinose analogue of UTP (62), in which the 2’-hydroxyl is inverted, is also several fold less active than UTP. Complete replacement of the ribose with an alkylene chain composed of 2-5 methylene units (63) affords compounds that are inactive [174]. It appears that the hydroxyls on the ribose sugar and an intact furanosyl moiety are important for agonist potency [ 1581. All modifications of the adenine base of ATP reported to date result in substantial loss of activity, e.g. ITP is 10-fold less potent than ATP with both 2-MeS-ATP ( l a ) and 2-Cl-ATP (1 b) being almost inactive. In contrast the pyrimidine base of UTP is more tolerant towards substitution. For example, substitution at the 5-position with small groups such as bromine decreases activity but does not eliminate it. More recently, UTP analogues with a range of substituents at the 4-position (64) have been reported to maintain good agonist activity [ I 58, 1751 (Table 3.10). 4-Thio-UTP (64a) is 5-fold more potent than UTP. This is consistent with the change noted above in the triphosphate group modifications observed in the dinucleoside-polyphosphate analogue (4-SH-U)p4(4-SH-U) (57a) (Table 3.9). With the exception of 4-thiohexyl-UTP (64b) all of the other 4-substituted compounds are substantially less active than UTP. These

-

-

Table 3.10 P2Y2 AGONIST ACTIVITY OF UTP A N D (64a-64i) [I581

Cotnpoiind

X

UTP (64a) (64b) (64~) (644

SH S-nHex S-Me Morpholino

~

EC.5,J( p M )

Con1pound

x

ECfG ( D M )

0.14 0.03 0.84 3.10 6.10

(64e) (640 (64g) (64h) (64i)

NH-nHex 0-nHex 0-Me NMeh NH-cPentyl

7.00 13.61 15.50 29.8 inactive

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THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

results suggest that, in compounds lacking the pyrimidine N-3-H, the presence of a 4-thiohexyl group does not result in a large loss of potency. Interestingly, some recent patents also claim that the etheno-CTP analogue (65) are P2Y2 agonists [172, 1731. Although no specific potency details are given, this could again be interpreted to indicate that the pyrimidine N-3-H is not an absolute requirement for agonist potency. A series of 5-substituted UTP derivatives (66) have been reported [ 1741 and tested on a cystic fibrosis epithelial airway cell line (CF/T43) as a representative of P2Y2 receptor agonist activity. These compounds are less potent than UTP and the trends showed further decreases with increasing size of the 5-substituent: Y = Me > Et > iPr > nPr > nBu.

P2 Yz antagonists

Suramin (6) and PPADS (1 3c), the non-selective P2 receptor antagonists, have been shown to have poor affinity (A2 -50yM) and inactive, respectively, at recombinant P2Y2 receptors [ 1761. Patents have been published claiming a wide variety of UTP analogues as P2Y2 antagonists for the treatment of inflammatory diseases [177-1811. The major structural changes described include replacing the oxygen atom of the

SIMON D. GUILE ET AL.

157

bridging P,y-phosphate bond with a dihalomethylene unit and/or one of the oxygen atoms on the y-phosphate by an alkoxy group. The majority of the compounds described are 4-thiouridine derivatives. In addition, there are a variety of large hydrophobic aromatic substituents at the 5-position of the uracil ring. Although stated to be antagonists, with A2 values < 100 pM, no specific data is provided. By inference, from the stated preferred compounds, it would appear that activity is improved by replacement of the bridging oxygen atom of the P,y-phosphate bond by a dichloromethylene linkage and addition of a large tricyclic aromatic ring system, such as a SH-dibenzo[a,d]cyclohepten-5-yl (67) or 4,4’-dimethylbenzhydryl (68) group at the 5-position [182]. Some of these patents have claimed uracil modified analogues as P2Y2 antagonists where it appears that the whole triphosphate group and the ribose moiety can be completely removed and replaced by a variety of alkyl, benzenoid and heterocyclic acids, amides and other acid bioisosteres [178-1811. Again no specific data is reported except that all compounds have A2 values < 100 pM. These reports suggest that substantial modifications to the ribose and triphosphate moieties appear to be more readily accommodated in the development of antagonists when compared to agonists.

158

THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY MOLECULAR MODELLING AND PHARMACOPHORES

Sequence analysis of the P2Y2 receptor and comparison with the adenosine A1 receptor indicates that there are several positively charged amino acid residues in TM3, TM6 and TM7 that are not present in the adenosine A1 receptor. It has been suggested that these residues are involved in the binding of the negatively charged triphosphate moiety [183]. This hypothesis is supported by molecular modelling and limited site-directed mutagenesis of the human P2Y2 receptor [184]. Mutation of Arg-265 to Leu and His-262 to Leu in TM6 or Arg-292 to Leu in TM7 results in a decrease in the potencies measured for ATP and UTP. These results suggest that these residues may be involved in stabilization of the agonist binding and/or receptor activation. However, the validity of this conclusion is not certain since the level of mutant receptor expression was not quantified in any of these studies. In addition, interpretation of potency estimates on the mutant receptors could not discriminate between changes related to affinity and/or efficacy of the ligands without a binding assay.

CLINICAL

INS-316 ( U T P )

A major characteristic of cystic fibrosis is a genetically defective airway epithelial chloride ion secretion. This defect is proposed to contribute to the development of dehydrated mucus that obstructs airways, provides a site for infection and severely compromises lung function. P2Y2 receptors, taken from airway epithelial cells of cystic fibrosis patients, couple to a C1secretory pathway distinct from the defective cystic fibrosis transmembrane conductance regulator (CFTR) [ 1521. Clinically there is now evidence that inhaled INS-3 16 (a proprietary formulation of UTP) can bypass the block to epithelial CI- transport and increase C1- secretion in cystic fibrosis patients [185]. UTP also stimulates lung epithelial cell cilia to beat faster resulting in more rapid clearance of mucus from the lung. Phase I1 clinical trials have shown that inhaled INS-316 alone, or in combination with amiloride, restores normal lung function to the peripheral airways of lung in cystic fibrosis patients [186]. It has demonstrated promising efficacy as an acute-use agent with rapid onset, short duration of action and limited side-effects. A Phase I11 clinical trial programme is being planned to evalu-

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ate the use of INS-316 as a lung-screening aid as it enhances the production of a quality sputum sample, which has a potential use in the diagnosis of lung cancer and respiratory infections [ 1581. INS-365

Trials of a potentially more stable analogue of UTP (INS-365, an aerosolized second generation P2Yz receptor agonist of unpublished structure) have also been reported. It is a drug candidate for chronic bronchitis, cystic fibrosis, primary ciliary dyskinesia and dry eye disease. The results from an initial Phase I clinical trial indicated that it is safe and well-tolerated. It produces a rapid increase in the quantity of sputum expectorated that is sustained for at least 1 h following a single dose [ 1871. Multicentre Phase I1 clinical trials are currently in progress in cystic fibrosis patients which will provide the first information on the potential of this compound to enhance mucociliary clearance in the disease [158]. A U.K. Phase I clinical trial using a topical opthalmic formulation of INS-365 has also been conducted. It was found to be safe and well tolerated and a U.S. and Japanese IND filing is planned in late 1999 for a trial in dry eye syndrome patients [ 1881.

p2y3 RECEPTOR The p2y3 receptor isolated from chick brain [I891 is now thought to be the homologue of the human P2Y6 receptor [I901 and will not be discussed further.

P2Y4 RECEPTOR BIOLOGICAL PROPERTIES

This uridine-nucleotide specific-receptor has been cloned from cDNA derived from human placenta [191], human chromosome X [192], and rat heart [193]. The P2Y4 receptor was originally reported to have restricted distribution with expression almost exclusively in placenta with low levels of expression in lung, and absent in most other tissues by Northern blotting experiments [ I 91, 1941. More recently, RT-PCR has allowed the detection of an expression of human P2Y4 mRNA in bone osteoblastic cell lines [ 1951,

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THE MEDICINAL CHEMISTRY O F THE P2 RECEPTOR FAMILY

retinonoblastoma, astrocytoma, glioma cell lines [ 1951, peripheral blood monocytes, lymphocytes, polymorphonuclear cells [ 1961, foetal heart [ 1971 and airway cell lines [198, 1991. The stably expressed P2Y4 receptor in 1321N1 cells has been shown to activate the IP3 signalling pathway with no effect on adenylyl cyclase activity. The human P2Y4 receptor appears to couple to two distinct G proteins; a Gi protein at the early stage and a G q l l l protein at a later stage of signalling to activate PLC and IP3 formation [200]. The IP3 response declines within minutes of stimulation of the receptor and is not readily reproducible, indicating rapid desensitization [201]. The cloned human P2Y4 receptor is highly selective for UTP over ATP whereas the cloned rat P2Y4 receptor is activated equipotently by UTP and ATP [199]. Clearly, with respect to ATP and UTP sensitivity on rat tissues, this is identical with the profile described for the P2Y2 receptor. Important implications arising from this are that some reported endogenous cell P2Y2 (or P2U) responses may in fact be mediated by a P2Y4 receptor, at least in rat tissues. Caution should therefore be exercised with interpretation of the older literature where a pyrimidine-nucleotide sensitive response was often equated to the P2Y2 receptor. It has recently been shown that the human P2Y4 receptor is expressed in a 6CFSMEo submucosal cell line by RT-PCR and Northern blotting experiments. This cell line showed a UTP-specific response which is pharmacologically similar to that seen with the human P2Y4 receptor and the effect was totally inhibited by pertussis toxin [202]. 6CFSMEo cells are representative of submucosal gland epithelial cells, which are the predominant site of CFTR expression in the human bronchi [203]. It has been proposed that the P2Y4 receptor could be a target for the treatment of cystic fibrosis by uridine-nucleotides in addition to P2Y2 and possibly P2Y6 receptors [202], although caution in interpreting these results is necessary as they are derived from an immortalized cell line. STRUCTURE-ACTIVITY RELATIONSHIPS

P2 Y4 agonists

The agonist activity (EC5")reported for the cloned human P2Y4 receptor is: UTP (2.5pM), 5-Br-UTP (69) (27pM), ATP (43pM). UDP, 5-Br-UDP (70), 2-MeS-ATP (1a) and 2-Cl-ATP (1 b) are inactive, as are the guanine nucleotides. Ap4A (2c) (7pM) and ATP (43pM) can behave as partial

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agonists showing -20-25% of the UTP response [199, 2001.

P2 Y4 antagonists Suramin (6) and PPADS (1 3c), the non-selective P2 antagonists, are essentially inactive at recombinant P2Y4 receptors expressed in I321N 1 cells [176]. No selective antagonists have been reported to act at this receptor.

0

p2y5 RECEPTOR Based on sequence homology and the binding of 2’-deoxyATP-a-S (71) it has been suggested that the orphan 6H1 receptor, found in activated chicken T cells, is a P2 receptor [204]. The turkey homologue of this receptor when

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THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

stably expressed into 1321N1 human astrocytoma cells is not activated by nucleotides. It has therefore been shown that this receptor, which had been named p2y5, is not a member of the P2Y receptor family [205].

P2Y6 RECEPTOR BIOLOGICAL PROPERTIES

This uridine-nucleotide specific receptor has been cloned from rat aortic smooth muscle [206], human placenta and spleen [207] derived cDNA libraries. P2Y6 mRNA is found abundantly in various rat tissues including placenta, thymus, lung, stomach, intestine, spleen, mesentery, and aortic smooth muscle cells [206, 2071. The receptor is less abundantly expressed in heart and kidney, and is essentially absent in brain. The P2Y6 response is pertussis toxin insensitive, indicating the involvement of Gq/ 11 proteins in stimulation of PLC and in the formation of IP3. Interestingly, the IP3 response of the human cloned P2Y6 receptor decays very slowly after stimulation, remaining above baseline for more than one hour after stimulation. This response is fully reproducible and without need for a long recovery period [201]. Recombinant P2Y6 receptors are activated by UDP but only weakly or not at all by UTP, ATP, ADP, or 2-MeS-ATP (la) [166, 2071. A receptor activated by UDP in human nasal epithelial cells, that is distinct from the P2Y2 and P2Y4 receptor, appears to be an endogenous P2Y6 receptor [208]. The receptor promotes IP3 formation and an increase in intracellular Ca2+ and C1- secretion and is present on the mucosal but not on the serosal surface. In Caco-2 human intestinal epithelial cells a receptor activated by UDP appears to be located on the apical but not on the basolateral membrane [209]. STRUCTURE-ACTIVITY RELATIONSHIPS

P2 Y6 agonists The P2Y6 receptor is stimulated most potently by nucleoside-diphosphates, such as UDP, and very weakly or not at all by nucleoside-triphosphates, such as ATP [199]. The rank order of potency of various nucleosidediphosphates at recombinant P2Y6 receptors stably expressed in 1321N I astrocytoma cells is: UDP > TDP > IDP > G D P > ADP >> CDP [20I].

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Table 3.1 I P2Yh AGONIST ACTIVITY OF (72a-72j) 12101

OH OH OH H OH OH OH OH OH OH

Phenylethynyl Mc H H H H H H H H

OH OH S-nHex OH 0-nHex 0-Me N H-cyclopentyl NH-nHex NMe2 N-morpholino

0.02 0.5 1.8 2.1 3.0 4.7 8.6 26 42 55

Some recent patents claim analogues of UDP (72) for lung disorders such as cystic fibrosis, chronic bronchitis, COPD, and primary ciliary dyskinesia [210] and Table 3. I 1 gives the P2Y6 agonist activity of 10 exemplified compounds (72). The preferred substitution on the uracil ring at the 5-position is interesting because this was also observed for modified UTP analogues claimed as P2Yz antagonists described above. This suggests some similarities in that both receptors appear to accommodate large substituents at the 5-position of the uracil ring on their respective ligands, e.g. (72a). Furthermore, it appears that modest activity is retained in 4-( 1-hexylthio) (72c), 4-( 1-hexyloxy) (72e) and 4-cyclopentylamino (72g) substituted derivatives, suggesting that, in compounds lacking the N-3-H of the uridine, the presence of large substituents at the 4-position can maintain potency [210]. P2 Y, antugonists

No selective antagonists have been reported that act at this receptor

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THE MEDICINAL CHEMISTRY O F THE P2 RECEPTOR FAMILY

p2y7 RECEPTOR A receptor showing sequence homology with known P2Y receptors was found during screening of a human erythroleukemia cDNA library. When transiently expressed into COS-7 cells this receptor bound ATP [211]. Subsequently, this receptor has been stably expressed in 1321N1 human astrocytoma cells where it is not activated by nucleotides. It has been shown that this receptor, which had been named p2y7, is not a member of the P2Y receptor family [212]; indeed the p2y7 receptor is identical to the LTB4 receptor [213]. It has also been shown that COS-7 cells natively express P2Y2 [212].

p2y8 RECEPTOR This receptor, cloned from Xenopus neural plate, is activated equipotently by purine and pyrimidine nucleotides: ATP = UTP = ITP = CTP = GTP [214]. The receptor has tentatively been named p2y8, however, as a mammalian homologue of this receptor has not been identified, its inclusion as a distinct P2Y subtype does not seem appropriate.

p2y9 RECEPTOR The human homologue of this receptor is structurally related to the chick p2y5 receptor [8]. It is now thought that this is not a member of the P2Y receptor family [ 171.

This cloned receptor, submitted to the Genbank, is not a nucleotide receptor 1171. P2Y 1 1 RECEPTOR BIOLOGICAL PROPERTIES

A P2Y receptor has been isolated from human placenta cDNA and genomic DNA libraries. It has a 1113-base pair open reading frame containing one

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intron. This receptor, named P2Y1 shows 33% sequence identity with the P2Y I receptor and has unusually long second and third extracellular loops [215]. The P2YII receptor has been expressed in 1321N1 astrocytoma and CHO-K1 cells. In each case the receptor is uniquely coupled to both the phosphoinositide and adenylyl cyclase second messenger pathways [215]. Human P2Y11mRNA has been found in spleen and HL-60 cells. STRUCTURE-ACTIVITY RELATIONSHIPS

The agonist potency order reported for the P2YI1 receptor is: ATP > 2-MeS-ATP (1 a) > > ADP = 2-MeS-ADP (50) with UTP and U D P being inactive. Thus, this appears to be the first P2Y receptor selective for adenosine triphosphates. The same potency order is found for both second messenger systems [2 1 51.

P2T

RECEPTOR

Among the P2 family of receptors the PZTsubtype is unusual, in that it has yet to be sequenced or cloned. This task is made particularly difficult by the restricted tissue occurrence of the receptor as its presence is only certain on platelets. The report of its occurrence on a glioma cell line [loo] awaits confirmation. As the platelet is anuclear, there is little mRNA present, consequently attention has been focused on its megakaryocyte progenitor. Experimental work aimed at cloning the receptor has been reviewed [2 161. While there have been a number of reports of the receptor’s apparent isolation, up to now they have all proved false. As the receptor has not been isolated, all reported SAR studies have perforce been performed on the intact platelet. Early SAR studies were founded on the premise that the P2Treceptorwas the sole P2 receptor present on the platelet and overwhelmingly made use of functional aggregatory assays [217-2211. Later studies have examined intracellular events, in particular intracellular calcium changes (as a result of influx or mobilization from stores), or CAMPlevels (as an indicator of the activity of adenylyl cyclase). These intracellular assays used in combination with P2 subtype-selective agents discovered during the past decade have now revealed the co-occurrence of the P2XI and P2Y1 receptors on the platelet as well as P2T To explain the failure to identify RNA coding for the P2T receptor protein it has been proposed that this receptor is in fact a subpopulation of the P2YI protein coupled to a different effector system and mediating different intra-

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THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

cellular events [105]. It is now clear that the Pzrreceptor is a discrete entity, co-expressed with P2Y1 and P2X1 [222]. The early literature on P2 receptor structure-activity relationships must be read with care as issues of ligand purity and stability add considerable complexity to the interpretation of the SAR of the multi-P2 receptor-platelet system.

BIOLOGICAL PROPERTIES

While the P2Treceptor has so far defied isolation, it is believed to be a member of the 7-TM GPCR class of receptors [223, 2241 and should, therefore, really be classified as a P2Y subtype of the P2 receptor family. The receptor is coupled to a Gi/2 protein and thereby down-regulates stimulated adenylyl cyclase activity [225,226]. There is little or no inhibition of the basal activity of the enzyme. Typically, investigation of the receptor function makes use of PGEl stimulation of the adenylyl cyclase. To make the nomenclature of this receptor more consistent with that of the other family members there have been proposals that it should be named P2YADp or PzYAC, recognizing the agonist or effector system and both terms may be found in the literature. Given the lack of clarity in the nomenclature, this review will continue to use the term PZT. The other reported intracellular changes occurring during platelet activation/aggregation in response to ADP, which include activation of PLC, IP3 formation and elevation of intracellular calcium, may be explained by the presence of P2YI and P2X1 receptors. On stimulation by ADP, platelets undergo shape change (from discoid to spiculated spheres), adenylyl cyclase activity is inhibited, intracellular calcium stores are mobilized and there is an influx of extracellular calcium. The contents of storage granules (which include ADP) are released, thus amplifying the initial stimulation. The cells adhere to the sub-endothelium of the blood vessel (usually at a site of damage), and begin to form a loose reversible aggregate. Subsequently, the white thrombus becomes irreversibly cross-linked by a fibrin meshwork to form an insoluble mass. The ADP that initiates the aggregatory response may be released from endothelial cells damaged by external physical insults (cuts or blows) or as a result of the rupture of atherosclerotic plaques in arterial walls. Additional amounts of ADP may arise in the breakdown of ATP and high local concentrations may be released from the contents of the platelet dense granules and from red blood cells exposed to mechanical stress (high shear).

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A rapid and sustained response to stimulation by ADP requires that all three P2 receptors expressed on the platelet (P2XI, P2Y I , and P2YAc { = P_77.])function concomitantly [222, 2231. The exact role of the P2X, receptor is as yet unclear but the P2Y I receptor mediates the shape change and intracellular calcium mobilization component of the aggregatory process [222] and is responsible for the preliminary reversible aggregation phase. Calcium influx under the control of the P2Xl receptor may assist in this part of the process. The PZTreceptor appears to be critically responsible for a sustained aggregation response.

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THE MEDICINAL CHEMISTRY O F THE P2 RECEPTOR FAMILY

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NH

STRUCTURE-ACTIVITY RELATIONSHlPS P27. agonists

ADP is the endogenous agonist of the PzTreceptor and is the key initiator of platelet aggregation in arterial thrombosis, an acute complication of atherosclerosis. I t is reported that 2-substituted analogues of ADP are more potent aggregatory agents than ADP. For example 2-Cl-ADP (73) is slightly more potent [227] while 2-MeS-ADP (50) is some 30-fold more potent [225-2281. Modification of the diphosphate, e.g. ADP-c(-S (74) and ADP-pS (9,produces partial agonists [225, 2261 or compounds only causing reversible aggregation, e.g. ADP-P-NH2 (75), [227] while modification to other sites generally leads to compounds having little or no pro-aggregatory effect, or which are antagonists. Attempts have been made to employ agonists bearing reactive centres to label the PZTreceptor and hence isolate it. Using FSBA (76), 2-BDB-TADP (77) or 2-BOP-TADP (78), the group of Colman has identified a l00kD protein they named aggregin as the PZTreceptor. However, the compounds used appear to be poorly selective among the P2 receptor subtypes, as they are pro-aggregatory at low doses

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THE MEDICINAL CHEMISTRY O F THE P2 RECEPTOR FAMILY

or short exposure times, causing elevation of intracellular calcium. After labelling the protein they block platelet aggregation in response to ADP, prevent ADP inhibiting adenylyl cyclase activity etc. [229]. As yet, the relationship of aggregin to other P2 receptors is unclear. When the effect of pro-aggregant compounds on inhibition of adenylyl cyclase is examined [227], the order of potency is 2-MeS-ADP (50) >> 2-CI-ADP (73) >ADP > ADP-fl-S (5) > tubercidin-DP (79) > NlJV”-etheno-ADP (80) >> 1-Me-ADP (8 l), which qualitatively mirrors the rank order of aggregatory activity.

PZT antagonists

Agents that have been shown to be efficacious as anti-aggregants and act via P2 receptor pathways may be divided into the following categories: pharmacological tools, e.g. suramin (6), adenosine nucleotide analogues and thienopyridines, e.g. ticlopidine (82) and clopidogrel (83). The classical pharmacological tools, suramin (6) and PPADS (13c), which have been widely used in P2 receptor research, may be quickly dismissed. Although they have been reported to be anti-aggregatory they have no effect upon adenylyl cyclase activity [227] consequently their inhibitory behaviour must be the result of their action at the other receptors. Discussion of their properties will be found in other sections of this review. Tuble 3.12 collects together data on analogues of AMP (84), ADP (85) and ATP (3), (42), (43) and (86) which have antagonist behaviour at the PZTreceptor; other nucleotide analogues derived from guanosine, inosine and uracil show no effect at the PIT receptor [227]. The only analogue of AMP for which there is published data on inhibition of the P ~ receptor T is 2-EtS-AMP (84) [230]. This compound has an affinity some 100-fold greater than the ADP analogues (85), which may be attributed to the presence of the alkylthio group, as is seen in the agonists described above. There are numerous sites at which ADP may be modified, and these will be considered in turn. Replacement of the anhydride bridge between the a- and P-phosphates by groups which raise the pKa values of the terminal phosphate group (85a)-(85c) destroys all activity. If the inethylene link is substituted by electronegative halogen atoms, restoring the pKa values to those of ADP, weak antagonists are produced (85d), (85e). In polyphosphate systems it is common to find that the first deprotonation of each phosphate moiety has a

SIMON D. GUILE E T A L .

(82) ticlopidine

(83) clopidogrel

(84) see Table 3.72

(85) see Table 3.72

(86) see Table 3.72

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THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

172

Table 3.12 P ~ ANTAGONIST T ACTIVITY OF ATP AND (3,42,43, 84, 85a-85h, 86a-864

(86i)

H H H H H H H Br H H H H H H H

SPr

NH2 NH2 NH2 NH2 NH2 NH2 CI NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2

OH

SPr

NH2

OH OH OH OH

SPr NHCH2CF3 SPr NHCH2CH20Me SPr NHCH2CH2SMe .~ SCHzCH2CFr NHCH2CH2SMe

OH OH OH OH OH OH OH OH H OH OH OH OH OH OH OH OH OH

SEt H H H H H H H H H H H H H H SMe SEt

-

-

NH CH2

-

H H

CHzCHz CCl2 CF2 0 0 0 0 CH2 0 0 0 0 0 0 0

H

0

CCl2

0

CCll CCl2

H

H H H

H

0 0 0

-

-0.5 p M Ia

La Ia 58 p M 44 p M la

~

0 0 CHz NH C F2 CCl2 CH2 50 nM

cc12 CF2

cc12 cc12

la Ia la

65 ILM Ia 340 p M 62 ~ L M 470 p M 190 p M 170 pM 320 pM"' 320 KM'"'

320 nM@' 6.9 nMla' Kh 2.2 nM 2.5 nM'"' Kh 1.3 nM 0.33 nM'"' I5 nM'"' 0.38 nM'"' 0.45 nML"'

~301 [2441

WI [i441 [244] [2441 12271 ~2271 [227] [227] [227] (2271 [227] [231] [231] 12301 [231] [231] [232] [231] (2451 [231] 12311 [231] 12311

"' ADP aggregation

-

pKa of 2 or below. The second deprotonation of a dibasic phosphate has a much higher pKa, generally close to 7. As a consequence of chemical modification, quite small shifts of this parameter can have substantial effects on the state of ionization of the system at physiological pH. In this instance, the compounds such as (85b) may be expected to exist overwhelmingly as di-anions, while ADP will be overwhelmingly a tri-anion a t pH 7.4. Binding of these simple nucleotide analogues to the receptor is clearly sensitive to the effective charge density around the terminal phosphate. In contrast to the agonism shown by ADP, the antagonist behaviour of the halo-methylene compounds (85d) and (85e) must be a reflection of more subtle effects on the preferred conformation of the phosphate chain. Modification to the adenine 2-position does not generally afford antagonists, but can modify agonist potency as discussed above.

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The adenine 6-position analogue, 6-C1-purine-5‘-diphosphate (850 is reported to be active as an antagonist. It is important to bear in mind that, in purines, the displacement of a 6-halogen is relatively easy, consequently this result must be viewed with some caution. The adenine 8-position analogue, 8-Br-ADP (85g) is inactive. The conformation of ADP about the C-l’-N-9 bond is free and there is little barrier to rotation. Substitution of the adenine nucleus at C-8 forces the molecule to adopt a syn-conformation with adenine N-3 lying over the sugar ring. Taking this data in conjunction with the data on agonists above there is strong evidence for the importance of the anti-conformation for activity. Any substituent at the 2-position larger than hydrogen demands that the conformation be the fully extended anti.

Modification of the ribose as in 2’-deoxy-ADP (85h) is surprisingly an antagonist, but with somewhat lower affinity for the receptor than ADP. Cleavage of the sugar ring of ADP to form the 2’,3’-dialdehyde (87) is reported to give rise to a compound which retains affinity, but is now an antagonist [227]. The chemical reactivity of this reagent must be borne in mind and its activity may be a consequence of covalent attachment, for the corresponding dialcohol reduction product (88) is inactive. It

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THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

may be concluded that the structural organization imposed by the intact sugar ring is essential for activity, and that the 2'-OH (at least) makes a significant binding contribution. While some general conclusions about preferred binding conformations for both agonists and antagonists may be drawn from existing data, it is apparent that the switch from agonism to antagonism is driven by more subtle factors than are presently understood. ATP has long been recognized as a (weak) antagonist of platelet aggregation (ICso 62pM) which was ascribed to its (specific) inhibition of the P2Treceptor, There is now evidence that this is in fact a non-selective effect, as both adenylyl cyclase activity and calcium mobilization are both affected in the same concentration range [222, 2271. The implication is that ATP is also an inhibitor of the P2YI subtype. The same range of modifications can be made to ATP as were discussed for the ADP derived antagonists. There is an additional complication in the investigation of ATP derived antagonists in the lability of the triphosphate side-chain (which has been responsible for many conflicting observations in SAR studies in the P2 receptor field). This is particularly significant in the present case as the first dephosphorylation of ATP gives rise to ADP, changing the agent from an antagonist into an agonist. Modification of the phosphate side-chain to stabilize the polyphosphate against breakdown by ectonucleotidases by replacement of the various anhydride oxygens by methylene groups, raises the issue of acid pKa discussed with respect to ADP analogues above. The activity of such compounds as /l,y-me-ATP (42) and the halogenated analogues is comparable to that of ATP. While alkylation of N-6 has little effect on activity on the platelet, and substituents at C-8 reduce it significantly [231], substitution in the adenine ?-position with lipophilic groups greatly enhances affinity (as seen with agonists) and selectivity. Moreover, in the presence of a 2-substituent, manipulation of the pKa of the modified polyphosphate side-chain has a significant effect on activity; the best combinations may be seen in the compounds (86e) and (860. These substances were the first reported high affinity antagonists of any of the P2 receptors, and AR-C66096 (86e) has been identified as the pharmacological tool for characterising the P2Tsubtype. It has no effect upon P2YI and P2X1 mediated processes in the platelet over its anti-aggregant concentration range [222], while activity at P2X and P2Y receptors in other tissues is only seen at concentrations some 5000-10,000-fold higher [232]. Later studies using various cloned P2 receptors have confirmed the highly selective nature of these ligands. There

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I75

has been no publication of a broad body of data specifically detailing the effects of compounds of this type on adenylyl cyclase activity in the platelet, but with such great receptor selectivity it is possible to use the anti-aggregatory data to reliably infer structure-activity relationships for the Pzr subtype. The preferred conformation for the ATP-like antagonists is the fully extended anti arrangement about the anomeric (C-l’-N-9) bond, as found for the related ADP-like compounds. Any substituent at C-2 larger than hydrogen ensures that this is the predominant form and the region of the receptor to which this substituent binds appears to be lipophilic. Alkylthio groups are the best substituents currently available with activity increasing with alkyl group size until reaching a maximum at propyl; a further increase in length has no beneficial effect. Of particular interest is the very substantial increase of affinity (ca. 100- fold) seen when the 2-ethylthio group (86d) is extended to 2-propylthio (860. Monoalkylation of N-6of the adenosine nucleus, which has no effect in the absence of a 2-substituent, contributes additional binding when the conformation is fixed. Compounds (86&(86j) illustrate some of the SAR around the 6-position. Activity tends to increase with chain length, reaching a plateau at about 4 carbon atoms. Polar groups are not tolerated in this region of the receptor [231]. The highly polar nature of compounds such as (86j) makes them unsuitable as oral drugs, for which application much less acidic, or preferably neutral, structures are desirable. Believing that it was necessary to maintain a region of high charge density corresponding to the position of the y-phosphate in ATP, a series of compounds in which the phosphate chain was replaced by carboxylic acids has been investigated. The most potent antagonist of this type was (89), whose antagonist potency was about 1% of that of (86j) but which did not show significant activity at other P2 receptors [233]. This series of compounds showed SAR trends at C-2 and N-6 that roughly parallel those seen with the phosphate compounds.

-3

HO

OH

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THE MEDICINAL CHEMISTRY OF THE P2 RECEPTOR FAMILY

(90) cs-747

A large body of work around non-phosphate compounds such as (89) and further related structures has been reported in the patent literature [234-2381, but no detailed description of the SAR in the area has appeared. The only markedly different structural class that has been found to afford selective antagonists of the PZTreceptor is the thienopyridine family, three of whose members have been investigated and reported upon: ticlopidine (82), clopidogrel (83) and (3-747 (90) [239]. N o discussion of structure-activity relationships for these compounds has been published and investigation is made particularly complex by the fact that the substances are prodrugs. In contrast to the PzT receptor antagonists described above, the onset of effect of the thienopyridines is slow, the degree of inhibition achieved is limited and the irreversible nature of the inhibition results in slow recovery of platelet function. Examination of platelets taken from human subjects treated with ticlopidine (82) or clopidogrel (83) [227] for 1 week showed that aggregation in response to ADP stimulation was inhibited, with down regulation of PGEl-stimulated adenylyl cyclase activity. There was little or no effect upon the calcium mobilization/influx pathways. The active metabolite of clopidogrel, originally described as a thiol derivative generated by oxidation to give a 2-oxo-thiophene and subsequent hydrolysis, is now reported to be (91) [240]; presumably an analogous active metabolite derives from CS-747 (90) [241]. These reactive species bind irreversibly to the PdT receptor by means of a disulfide link, and inhibit aggregation for the lifetime of the platelet.

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CLINICAL

Clopidogrel (Plavix) (83) is indicated for the reduction of atherosclerotic events (myocardial infarction, stroke, vascular death) in patients with atherosclerosis documented by recent stroke, myocardial infarction, or established peripheral arterial disease. The modified polyphosphate, AR-C69931MX (86j) is in late Phase 11 clinical development as an acute use anti-thrombotic agent and is intended for intravenous administration to patients with acute coronary syndromes (ACS) being managed with or without intervention [231].

ACKNOWLEDGEMENTS We would like to thank lain Dougall, Duncan Henderson, Bob Humphries and lan Watts for useful comments and particularly Jeanette Eldridge for her help with the organization of the references.

REFERENCES 1 Burnstock. G. (1978) in Cell Membrane Receptors for Drugs and Hormones:

2 3 4 5 6 7 8

9 10 11

12

Multidisciplinary Approach (Straub, R.W. and Bolis, L., eds.), pp. 107-1 18, Raven, New York. Burnstock. G . and Kennedy, C. (1985) Gen. Pharmacol. 16, 433440. Fredholm, B.B., Burnstock, G., Harden, T.K. and Spedding, M. (1996) Drug Dev. Res. 39, 461466. Hall, D.A. and Hourani, S.M.O. (1993) Br. J . Pharmacol. 108, 728-733. Lewis, C.J.. Neidhart, S., Holy, C., North, R.A., Buell, G.N. and Surprenant, A. (1995) Nature (London) 377, 432435. Lc, K.T., Babinski, K. and Seguela, P. (1998) J. Neuroscl. 18, 7152-7159. Torres, G.E.E., Haines, W.R.. Egan, T.M. and Voigt, M.M. (1998) Mol. Pharmacol. 54, 989-993. King, B.F., Townsend-Nicholson, A. and Burnstock, G . (1998) Trends Pharmacol. Sci. 19, 506-514. Abbracchio, M.P. and Burnstock. G. (1994) Pharmacol. Ther. 64, 445 475. Lazarowski, E.R., Homolya. L., Boucher. R.C. and Harden, T.K. (1997) J. Biol. Chem. 272, 20402-20407. Vigne, P., Breittmayer. J.P. and Frelin, C. (1998) Br. J. Pharmacol. 125, 675-680. Westfall, T.D., Mclntyre, C.A., Obeid. S , Bowes, J., Kennedy, C. and Sneddon, P. (1997) Br. J. Pharmacol. L21. 57-62.

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195 Maier, R., Glatz, A., Mosbacher, J. and Bilbe, G. (1997) Biochem. Biophys. Res. Commun. 237, 297-302. 196 Jin, J., Dasari, V.R., Sistare, F.D. and Kunapuli, S.P. (1998) Br. J. Pharmacol. 123, 789-794. 197 Bogdanov, Y., Rubino, A. and Burnstock, G. (1998) Life Sci. 62, 697-703. 198 Merten, M.D., Saleh, A., Kammouni, W., Marchand, S. and Figarella, C . (1998) Eur. J. Biochem. 251, 19-24. 199 Communi, D., Bernard, R., Janssens, R. and Parmentier, M. (1998) Drug Dev. Res. 45, 130- 134. 200 Communi, D., Motte, S., Boeynaems, J.M. and Pirotton, S. (1996) Eur. J. Pharmacol. 317, 383-389. 201 Robaye, B., Boeynaems, J.M. and Communi, D. (1997) Eur. J. Pharmacol. 329, 231236. 202 Communi, D., Paindavoine, P., Place, G.A., Parmentier, M. and Boenynaems, M. (1999) Br. J. Pharmacol. 127, 562-568. 203 Engelhardt, J.F., Yankansas, J.R., Emst, S.A., Yang, Y., Marino, C.R., Boucher, R.C., Cohn, J.A. and Wilson, J.M. (1992) Nat. Genet. 2, 240-248. 204 Webb, T.E., Kaplan, M.G. and Barnard, E.A. (1996) Biochem. Biophys. Res. Commun. 219, 105-110. 205 Li, Q., Schachter, J.B., Harden, T.K. and Nicholas, R.A. (1997) Biochem. Biophys. Res. Commun. 236, 455-460. 206 Chang, K., Hanaoka, K., Kumada, M. and Takuwa, Y. (1995) J. Biol. Chem. 270, 26 152-26 158. 207 Communi, D., Parmentier, M. and Boeynaems, J.M. (1996) Biochem. Biophys. Res. Commun. 222, 303-308. 208 Lazarowski, E.R., Paradiso, A.M., Watt, W.C., Harden, T.K. and Boucher, R.C. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2599-2603. 209 Inoue, C.N., Woo, J.S., Schwiebert, E.M., Morita, T., Hanaoka, K., Guggino, S.E. and Guggino, W.B. (1997) Am. J. Physiol. 272, C1862-Cl870. 210 Boucher, R.C., Jr., Shaver, S.R., Pendergast, W., Yerxa, B., Rideout, J.L., Dougherty, R. and Croom, D. (1999) PCT Int. Appl. WO 99 09998; (1999) Chem. Abstr. 130, 218304. 211 Akbar, G.K.M., Dasari, V.R., Webb, T.E., Ayyanathan, K., Pillarisetti, K., Sandhu, A.K., Athwal, R.S., Daniel, J.L. and Ashby, B. (1996) J. Biol. Chem. 271, 1836318367. 212 Herold, C.L., Li, Q , , Schachter, J.B., Harden, T.K. and Nicholas, R.A. (1997) Biochem. Biophys. Res. Commun. 235, 717-721. 213 Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y. and Shimizu, T. (1997) Nature (London) 387, 620-624. 214 Bogdanov, Y.D., Dale, L., King, B.F., Whittock, N. and Burnstock, G. (1997) J. Biol. Chem. 272, 12583-12590. 215 Communi, D., Govaerts, C., Parmentier, M. and Boeynaems, J.M. (1997) J. Biol. Chem. 272, 31 969-3 1973. 216 Puri, R.N. and Colman, R.W. (1998) J. Protein Chem. 17, 429451. 217 Cusack, N.J. and Hourani, S.M. (1981) Br. J. Pharmacol. 73, 405-408. 218 Cusack, N.J. and Hourani, S.M. (1981) Br. J. Pharmacol. 73, 409412. 219 Cusack, N.J. and Hourani, S.M. (1982) Br. J. Pharmacol. 75, 257-259.

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220 Cusack, N.J. and Hourani, S.M. (1982) Br. J. Pharmacol. 75, 397-400. 221 Cusack, N.J. and Hourani, S.M. (1982) Br. J. Pharmacol. 76, 221-227. 222 Daniel, J.L., Dangelmaier, C., Jin, J., Ashby, B., Smith, J.B. and Kunapuli, S.P. (1998) J. Biol. Chem. 273, 20242029. 223 Jin, J. and Kunapuli, S.P. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 807CL8074. 224 Gachet, C., Cazenave, J.P., Ohlmann, P., Hilf, G., Wieland, T. and Jakobs, K.H. (1992) Eur. J. Biochem. 207, 259-263. 225 Colman, R.W. (1991) Proc. SOC.Exp. Biol. Med. 197, 242-248. 226 Hourani, S.M. and Hall, D.A. (1996) Ciba Found. Symp. 198, 53-64. 227 Geiger, J., Honig-Liedl, P., Schanzenbacher, P. and Walter, U. (1998) Eur. J. Pharmacol. 351, 235-246. 228 Gough, G., Maguire, M.H. and Penglis, F. (1972) Mol. Pharmacol. 8, 170-177. 229 Puri, R.N., Colman, R.F. and Colman, R.W. (1997) Arch. Biochem. Biophys. 343, 14CL145. 230 Hourani, S.M.O. (1996) Drug Dev. Res. 38, 12-23. 231 Ingall, A.H., Dixon, J., Bailey, A., Coornbs, M.E., Cox, D., McInally, J.I., Hunt, S.F., Kindon, N.D., Teobald, B.J., Willis, P.A., Humphries, R.G., Leff, P., Clegg, J.A., Smith, J.A. and Tomlinson, W. (1999) J. Med. Chem. 42, 213-220. 232 Humphries, R.G., Tomlinson, W., Ingall, A.H., Cage, P.A. and Leff, P. (1994) Br. J. Pharmacol. 113, 1057-1063. 233 Ingall, A.H., Bailey, A., Coombs, M.E., Hunt, S.F., Ince, F., Teobald, B.J., Willis, P.A., Leff, P., Humphries, R.G., Nicol, A.K. and Tomlinson, W. (1998) Abstracts of XVth EFMC International Symposium on Medicinal Chemistry, Edinburgh, U.K., September 1998, Poster No. 280. 234 Brown, R., Pairaudeau, G., Springthorpe, B., Thom, S. and Willis, P. (1999) PCT Int. Appl. WO 994 1254; (1999) Chem. Abstr. 131,184960. 235 Hardern, D. and Springthorpe, B. (1999) PCT Int. Appl. WO 99 05142; (1999) Chem. Abstr. 130, 153657. 236 Brown, R. and Pairaudeau, G. (1999) PCT Int. Appl. WO 99 05144; (1999) Chem. Abstr. 130, 153658. 237 Guile, S., Ingall, A., Springthorpe, B. and Willis, P. (1999) PCT Int. Appl. WO 9905143; (1999) Chem. Abstr. 130:168386. 238 Bonnert, R., Ingall, A., Springthorpe, B. and Willis, P. (1998) PCT Int. Appl. WO 98 28300; (1998) Chem. Abstr. 129, 95506. 239 Asai, F., Sugidachi, A., Ikeda, T., Koike, H., Inoue, T., Takata, K., Iwamura, R., Kita, J . and Yoneda, K.(1998) PCT Int. Appl. EP 934928 equivalent to WO 98 08811; (1998) Chem. Abstr. 128, 217289. 240 Savi, P., Pereillo, J.M.A., Fedeli, O., Reversat, J.L., Maftouh, M., Uzabiaga, M.F., Rouchon, M.C., Combalbert, J., Picard, C., Maffrand, J.P., Pascal, M. and Herbert, J.M. (1999) Abstracts of the International Society of Thrombosis and Haemostasis XVIIth Congress, Washington D.C., U.S.A. 14-21 August 1999. 241 Asai, F., Sugidachi, A., Ogawa, T., Nagasawa, T., Shimoji, T., Suzuki, N., Kawamura, Y., Inoue, T. and Iwamura, R. (1999) Abstracts of the International Society of Thrombosis and Haemostasis XVIIth Congress, Washington D.C., U S A . 14-21 August 1999. 242 Anon. (1997) Med. Sci. Bull. (Internet Enhanced J. Pharmacol. Ther.) 20, 243 (http:/ lpharminfo.com/pubs/rnsb/clopid243.html).

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243 Nandanan, E., Camaioni, E., Jang. S.Y., Kim, Y.C., Cristalli, G., Herdewijn, P., Secrist, J.A., Tiwari, K.N., Mohanram, A,, Harden, T.K., Boyer, J.L. and Jacobson, K.A. (1999) J. Med. Chem. 42, 1625-1638. 244 Cusack, N.J. (1993) Drug Dev. Res. 28, 244-252. 245 Humphries, R.G., Tomlinson, W., Clegg, J.A., Ingall, A.H., Kindon, N.D. and Leff, P. (1995) Br. J. Pharmacol. 115, 1110-1116.

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Progress in Medicinal Chemistry - Vol. 38, Edited by F.D. King and A.W. Oxford 0 2001 Elsevier Science B.V. All rights reserved.

4 Radioligands for the Study of Brain 5-HT1A Receptors In Vivo VICTOR W. PIKE', CHRISTER HALLDIN* and HAKAN V. WIKSTROM'

'

Chemistry and Engineering Group, M R C Cyclotron Unit, Imperial College School of Medicine, Hammersmith Hospital, Ducane Road, London W12 O N N , U.K. Karolinska Institutet, Department of Clinical Neuroscience, Psychiatry Section, Karolinska Hospital, S-I 7176 Stockholm, Sweden. University Centre f o r Pharmacy, University of Groningen, A Deusinglaan 1, NL-9713 A V Groningen, The Netherlands. INTRODUCTION

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S-HT~ARECEPTORS

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Structural features Ligand binding Location in the brain Function in the brain Implication in neuropsychiatric disorders Involvement in neuropsychiatric disorder therapy Need for imaging in vivo

192 194 195 197 198 199 200

MOLECULAR IMAGING TECHNIQUES Positron emission tomography Single photon emission tomography Requirements of receptor radioligands for molecular imaging Radiochemistry techniques

20 1 20 1 202 202 203

RADIOLIGANDS FOR IMAGING BRAIN S-HTIA RECEPTORS I N VZVO Agonist radioligands Antagonist radioligands Carbon-I I labelled ligands Fluorine-I8 labelled ligands lodine-123 labelled ligands Technetium-99m labelled ligands General features of established or promising antagonist radioligands

206 206 21 1 21 1 223 232 234 235

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RADIOLIGANDS FOR THE STUDY OF BRAIN 5-HTl* RECEPTORS

A RADIOLIGANDS APPLICATIONS OF ~ - H T I RECEPTOR In clinical research In drug research

235 235 23 5

FUTURE PERSPECTIVE

238

ACKNOWLEDGEMENTS

239

REFERENCES

239

INTRODUCTION Serotonin (5-hydroxytryptamine, 5-HT, 1) is a major brain neurotransmitter that elicits a multitude of physiological functions by interactions with ~ is structurally, pharmacologivarious receptors [l]. The 5 - H T 1 receptor cally and functionally the best characterized of all known serotonin receptors (now totalling at least 15) [2, 31. There is substantial evidence, mainly from postmortem studies, to associate changes in this receptor population with several neuropsychiatric disorders, including depression and suicide [4-91, schizophrenia [lo-1 51, alcoholism [ 161 and Parkinson’s disease [ 171. Further biological, pharmacological and clinical evidence implicates this receptor in yet other conditions, especially anxiety [18]. The brain ~ - H T receptor ~A is also a functional target in both established and new drug therapies [l, 2, 19-22]. This review concerns important recent advances for the quantitative imaging of brain 5 - H T I ~ receptors in living animal and human brain, based on the use of novel radioligands, primarily with the non-invasive technique of positron emission tomography (PET) [23 and references therein], but also with single photon emission tomography (SPET) [24]. In particular, this review will address the development of the required radioligands, but it will also indicate why such imaging can be valuable in clinical research and drug development, what findings have so far been obtained and how this area might develop in the future. Before addressing these topics, it is valuable to consider the identity, structure, location and function of brain 5-HTIAreceptors, and our understanding of their molecular interactions with various ligands. ~ - H T RECEPTORS ~A Serotonin receptors belong to 7 major classes (5-HT1-7) containing various subtypes. All these receptors, except the 5-HT3 receptor, are part of the

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H Serotonin (1)

5-HT1 subfamily

5-HT2 subfamily

1

Figure 4. I , Dendrogram of the transmembrane amino acid identities between 13 serotonin receptors. The lengths of the horizontal lines are proportional to the number of transmembrane amino acid differences between the receptor sequences.

G-protein coupled receptor (GPCR) superfamily. The various human serotonin GPCRs share a high degree of amino acid sequence homology (Figure 4. I). All available evidence strongly suggests that GPCRs are folded to contain a bundle of 7 transmembrane hydrophobic a-helices (TMHs) linked by

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RADIOLIGANDS FOR THE STUDY OF BRAIN 5-HTI,4 RECEPTORS

intracellular and extracellular loops, terminating extracellularly with an amino group and intracellularly with a carboxyl group. The various serotonin GPCRs contain stretches of highly conserved amino acid sequences in several of the putative transmembrane domains, indicating a high degree of structural commonality for serotonin binding in this region. Each of these receptors is coupled to a heterotrimeric G-protein composed of a, and y-type subunits. It is generally considered that binding of the agonist (serotonin) to the GPCR causes a conformational change which releases a G-protein, causing it to dissociate into two parts, an a-subunit and a &subunit. The uncoupled receptor consequently has a lower affinity for serotonin and nearly all other agonists. The binding of the agonist, release of the G-protein and its dissociation are the first events in receptor signalling.

STRUCTURAL FEATURES

The primary sequence of the human ~ - H T receptor ~A has 422 amino acids, giving a molecular weight of -46,000 and an isoelectric point of 8.8 (Figure 4 . 2 ) [25-281. The seven hydrophobic domains, each of 20-26 amino acids that probably correspond to the transmembrane spanning regions, are linked by relatively hydrophilic sequences that probably form three intracellular and three extracellular loops. The putative second extracellular loop (e2) possesses a cysteine residue (Cys-186) that may form a disulfide bond with Cys-109, which is thought to be located at the boundary between the first extracellular loop (el) and the third transmembrane helix (TMH3). This disulfide link may play a role in stabilizing the receptor and is perhaps attacked by reducing agents known to be detrimental to the ability of the receptor to bind ligands [29, 301. The third intracellular loop (i3) is very long, consisting of 132 amino acid residues, while the C-terminal domain is short at 18 amino acids. The rat 5 - H T 1 receptor ~ also has 422 amino acid residues and shares a high degree of overall sequence homology with the human receptor (88%) and almost perfect homology in the putative transmembrane regions [28, 31, 321. This homology is far greater than the overall homology shared between the human 5-HTlAreceptor and other human serotonin receptors, such as 5-HTID(43%), 5-HT2A(19%) and 5-HT2c (18%). Hence, the rat 5 - H T 1 receptor ~ is an excellent structural model for the human receptor. The 5 - H T 1 receptor ~ also shares 45% homology with the a1-adrenoceptor

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Figure 4.2. A two dimensional represeniaiion of the human 5-HTIa recepior amino acid sequence. The bold line indicaies ihe puiaiive disuljide bridge between cysieine-109 and cysieine-187. Consensus sires f o r phosphorylarion ure labelled PKC. Asparugine-10, asparagine-1 1 and asparagine-24 are siresf o r N-glycosylaiion, The carboxyl terminal is ihoughi to be anchored to the interior.face of iheplasma membrane by one or iwopulmiioylgroups bound to cysteine-417 and cysteine-420. The entire i2 loop and the carboxyl terminal region of the i3 loop are considered to be imporiani,for binding lo G-proieins. The diagram is drawn f r o m daia obtained from: h t i p : / / w w n ~ . e . u p a s ~ ~ . c h / c g i - b i n / n i c e p r o i . p i ? 5 ~ ~ l A - H V ~ A N .

[33]. Many potent ligands for 5-HTIA receptors therefore have low selectivity versus CII -adrenoceptors; ligands with high selectivity for 5-HTIA receptors are comparatively rare. The rat 5-HTIAreceptor contains three potentially glycosylated asparagine residues (Asp- 10, Asp- 1 1 and Asp-24) within the putative extracelluar domain of the protein. Three threonine residues (Thr- 149, Thr-229, Thr-343) are within consensus sequences of the putative i2 and i3 loops for their phosphorylation by protein kinase C . These residues are involved in receptor desensitization and signal transduction [34, 351. Studies indicate that the entire i2 loop and a carboxyl terminal heptapeptide of the i3 loop are key G-protein regulatory sites [36]. There is specific evidence that Thr-149 has a key role in the rat receptor for coupling to Gpy-mediated signals [37]. These studies support a model in which the i2 loop and portions of the i3 loop form amphipathic a-helices aligned for hydrophobic interaction with a G-protein [37].

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RADIOLIGANDS FOR THE STUDY OF BRAIN 5-HTi* RECEPTORS

The 5-HTIAreceptor spans a lipid bilayer and therefore, like all GPCRs, has not been crystallized for X-ray structural analysis. Hence, concepts of the 3-dimensional structure of the 5 - H T I ~receptor were first based on molecular models derived by computer calculations, which used the X-ray determined structures of related proteins, such as bacteriorhodopsin [38], as templates. The latter had been assumed to be topologically related, although it is not a GPCR and shares only a low amino acid sequence homology. The computer calculations were performed with a variety of programmes making varied assumptions, and hence model descriptions commonly differed in details. A recent electron microscopy study of the GPCR, rhodopsin, shows previous assumptions on GPCR structure to be incorrect [39, 401. Ultimately, a clearer description of the 3-dimensional structure of the 5-HTIAreceptor should emerge. Three genetic variants of the 5-HTlA receptor are known to occur in the human population but at low frequency, specifically Gly-22-Ser (2%), Ile-28-Val (8%) and Arg-219-Leu (
The ~ - H T receptor, ~A like all other 5-HTI receptors, is pharmacologically characterized by its high affinity for the endogenous agonist, serotonin. It has high affinity for many synthetic selective agonists and antagonists [2] of which the best known are the aminotetralin, 8-OH-DPAT (selective agonist) (2), and the arylpiperazine, WAY-100635 (selective antagonist) ( 3 ) . Other important agonists are pyrimidylpiperazines, such as buspirone (4). Certain well-known P-blockers (arylpropanolamines) also have high affinity for 5-HTIA receptors, as exemplified by pindolol (5), which is usually described as an antagonist, but which exhibits weak partial agonist activity at 5-HTIA autoreceptors in the dorsal raphe nuclei [43]. Robalzotan (NAD-299,6) is a noteworthy potent and selective antagonist derived from 8-OH-DPAT [44, 451. The known genetic variants of the human 5-HTIA receptor show no differences in binding to major agonists or antagonists [461.

8-OH-DPAT (2)

WAY-1 00635 (3)

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0

Pindolol(5)

Buspirone (4)

F

Robalzotan (6)

Studies of 5-HTIAreceptors with different point mutations have revealed that different amino acids in the receptor sequence are essential for interaction with the different clases of ligand [25, 47-49] and also that the requirements for agonist binding are different to those for antagonists [50]. Several models have been proposed to describe the binding of the various classes of ligands to the 5-HTIA receptor and to account for the results of the point mutation studies [51-551. Such modelling has at times been helpful in the development of effective ligands, such as WAY-100635 [50], the first potent silent selective antagonist of 5 - H T 1 receptors ~ 155-591. However, the recent important advances in the understanding of GPCR structure, ligand binding and activation [39, 60, 611 necessitate major revision to the earlier models of ligand binding to ~ - H T receptors. ~A Hence, a detailed understanding of the interaction of the high affinity ligands with the 5-HTIAreceptor and their differences is still awaited.

LOCATION IN THE BRAIN

The human 5-HTlAreceptor is encoded by an intronless gene on chromosome 5 at the locus 5q11.2-ql3 [26]. This gene is mainly expressed in the brain, spleen and neonatal kidney. It has also been found in rat stomach [62]. mRNA coding for the ~ - H T receptor ~A in brain is found in high density

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RADIOLIGANDS FOR THE STUDY OF BRAIN S-HTIA RECEPTORS

in the dorsal raphe nucleus, septum, hippocampus, entorhinal cortex and interpendicular nucleus [31, 63-65]. There have been numerous autoradiographic studies of the binding of selective 5-HTIAreceptor radioligands to animal and human brain tissue [2,65-671. All these studies have shown that in mature rat brain the receptor is especially abundant in the gyrus dentatus, the CAI region of Ammon’s horn in the hippocampus, the lateral septum, the entorhinal and frontal cortex, and the dorsal raphe nucleus. The receptors are expressed to a low but significant extent in some thalamic and hypothalamic nuclei and are hardly detectable in striatum, substantia nigra and cerebellum. Immunostaining of the receptors with antibodies, raised to a characteristic portion of the 3i loop, revealed the same distribution as radioligand binding [68].Hence, S-HTIA receptors are not transported long distances from their site of synthesis, as represented by the correspondence of the distribution of receptors with the distribution of encoding mRNA. It should be noted that in the postnatal and developing rat brain the distribution of ~ - H T ~ A receptors and its encoding mRNA have a different distribution to that in mature brain. Especially, during the first two postnatal weeks there appear to be higher numbers of receptors in cerebellum and lower numbers in hippocampus and cerebral cortex [69-711. 5-HTIAreceptors appear to be addressed into the somatodendritic compartment of neurons. Light and electron microscopic immunocytochemistry revealed ~ - H T I A receptor immunoreactivity on neuronal cell bodies and dendrites in the dorsal raphe nuclei, and on somas and dendrites of pyramidal and granule cells in hippocampus [72]. Autoradiography of adult human brain exposed to the selective 5-HTlA receptor antagonist [3H]WAY-100635 has shown that there is a discrete distribution of 5 - H T 1receptors ~ [65] (Figure 4.3). The distribution is similar to that in rats. Thus, receptors are abundant in hippocampus, neocortex and raphe nuclei. The density in neocortex is slightly lower than in hippocampus and mainly at superficiallayers.-Other regions such as the amygdala, septum and claustrum show lower densities of receptors. Basal ganglia, such as the nucleus caudatus and putamen, and cerebellum have very low densities of receptors. In one study, estimates of receptor density obtained by computerized densitometry of the autoradiograms gave values of, for example, 128, 113,50 and 6 fmol/mg tissue for entorhinal cortex, hippocampus (CAI pyramidal layer), dorsal and median raphe, and whole cerebellum, respectively. The high density of S-HTlA receptors in the limbic forebrain is consistent with a role for these receptors in the modulation of emotion.

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FUNCTION IN THE BRAIN

The structural features of the 5-HTIA receptor, especially the long i3 loop and short C-terminal domain, are usual for receptors coupling negatively to adenylyl cyclase. It is well established that the 5-HTIAreceptor couples negatively to adenylyl cyclase through Gi or Go type G-proteins [28, 671,

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RADIOLIGANDS FOR THE STUDY OF BRAIN S-HTIA RECEPTORS

with some evidence specifically implicating Gia2 and Gia3 proteins [73, 741. The receptor also couples via a GiiGo type of protein to two ionic effects, potassium channel activation and calcium channel inhibition [28, 671. An ability of the 5 - H T 1 receptor ~ to couple to different G-proteins may account for the different effects of agonists on neuronal cells from one brain region to another [67]. For example 5 - H T 1 receptor ~ agonists inhibit forskolin-activated adenylate cyclase activity in the rat hippocampus but not in the anterior raphe nuclei. The electrophysiological effects of 5-HTIA agonists are also different in these regions [2, 671. Differences reside in the regulation of somatodendritic 5 - H T 1 ~ autoreceptors in the dorsal raphe nuclei and of postsynaptic 5-HTIA receptors in projection areas [67]. In humans, not only serotonin but also cortisol, and perhaps other hormones, are able to trigger regulatory mechanisms [28, 671. In animals, corticosterone is known to control the expression of 5-HTIA receptors negatively in the hippocampus but not in the dorsal raphe nuclei [67]. Hence 5-HTIA receptors expressed by serotoninergic neurons differ markedly in their properties from those expressed by postsynaptic receptors. The precise mechanisms underlying these differences need to be more fully understood. IMPLICATION IN NEUROPSYCHIATRIC DISORDERS

There is substantial evidence from studies of human tissue at postmortem to link abnormal levels of brain 5 - H T 1 receptors ~ to major neuropsychiatric disorders, including depression [8], schizophrenia [ 10, 131 and Parkinson’s disease [17]. The 5-HTlAreceptor has been implicated in the pathophysiology of major depression by autoradiographic studies of the brains of suicide victims and depressed subjects dying of natural causes. However, there is disagreement regarding the brain regions where receptor numbers differ between depressives and controls and the direction of such differences. For example, hippocampal 5-HTIAreceptors in suicide victims have been reported variously to be decreased [4], trending towards decreased [5], or unchanged [6]. ~ concentrations relative In the raphe of suicide victims, 5 - H T 1 receptor to those of controls were found to be reduced in one study [7] and increased in another [9]. Several laboratories have found elevated 5-HTlA receptors in the neocortex [ 10, 13, 751 and limbic regions [ 111 of schizophrenic brains at post mortem. An increase in 5-HTIAreceptor density has been found in certain Brodmann areas of brain in subjects with schizophrenia, regardless of

V.W. PIKE ET A L .

I99

antipsychotic treatment before death [76]. An abnormal persistence of cerebellar 5-HTIA receptors has been found by quantitative autoradiography in adult schizophrenic brains post morfeni [77], suggesting failure to regress in neonates and supporting neuro-developmental hypotheses of schizophrenia [78]. A significantly large increase in postsynaptic 5 - H T 1 receptor ~ concentrations has been found in the orbito-frontal (BAl I ) and temporal (BA2) cortexes of eight Parkinson’s disease subjects relative to those of controls [17]. By contrast, levels of ~ - H T Ireceptors A are found to decrease gradually with normal ageing [16], and more rapidly in Alzheimer’s disease [79, 801. INVOLVEMENT IN NEUROPSYCHIATRIC DISORDER THERAPY

The ~ - H T I receptor A is an important target for drug therapy. Selective partial agonists, such as buspirone, are clinically effective anxiolytic drugs [18]. These appear to act by activating somatodendritic 5-HTIA autoreceptors on serotoninergic cells in the dorsal raphe nuclei, so inhibiting the rate of firing of the neuron and reducing serotoninergic neurotransmission. Presynaptic ~ - H T ~receptors, A because of their greater receptor reserve, are considered to be more sensitive than postsynaptic receptors to partial agonists. There is ample evidence that a reduction in serotoninergic neurotransmission suppresses the release of hormones (e.g., ACTH corticosterone, prolactin and renin), and hence their effects, in response to stress factors. 5-HTIAreceptor agonists are also antidepressants [19, 811. Here there is convincing evidence that antidepressant action is mediated through postsynaptic 5-HTIA receptors and by implication an increase in serotoninergic transmission. Furthermore, there is now important evidence that certain 5-HTIA receptor antagonists (e.g. pindolol) accelerate the antidepressant efficacy of widely prescribed selective serotonin re-uptake inhibitors (SSRls) [20-221. In the early phase of treatment of depression with an SSRI (or M A 0 inhibitor) alone, the synaptic levels of serotonin are increased. The elevation in serotonin level activates 5 - H T I receptors ~ on the soma and dendrites in the dorsal raphe nuclei (presynaptic autoreceptors) thereby reducing the rate of firing of the neuron and the release of serotonin into projection areas. With chronic treatment, the presynaptic 5-HTIAautoreceptors become desensitized, so tempering any inhibitory effect on cell firing. Serotoninergic neurotransmission then becomes elevated due to the blockade of serotonin re-uptake sites; hence, the full antidepressant action of the SSRI is then manifest. This mechanism can

200

RADIOLIGANDS FOR THE STUDY OF BRAIN S-HTIA RECEPTORS

explain the lag before SSRIs alone take their therapeutic effect, which is typically 2-3 weeks. In this period severely depressed subjects can be at risk of suicide. Hence, a means to avoid this lag is highly desirable. Co-administered pindolol is most probably able to accelerate the antidepressive effect by rapidly blocking 5 - H T 1 autoreceptors ~ and preventing any attenuation of cell firing. This has led to the suggestion that co-administration of a pure ~ - H T I A receptor antagonist could be highly beneficial, especially in the early stage of antidepressant therapy with SSRIs. However, it should be noted that the postulated action of pindolol depends on selective interaction with the autoreceptor, since antagonist blockade of postsynaptic 5-HTIA receptors would be expected to suppress serotoninergic neurotransmission and the effect of antidepressant therapy with SSRIs [40]. Therapeutic roles for 5-HTIAagonists have also been suggested in various other conditions such as schizophrenia, chronic alcoholism and neurodegenerative disorders (Alzheimer’s disease), and for 5-HTIAantagonists in, for example, pain syndromes [2]. Finally, there is evidence that the administration of antipsychotic drugs can affect the levels of brain S-HTIAreceptors [82]. Chronic treatment of rats with haloperidol for 21 days, followed by 3 days withdrawal, reduced ~ - H T I receptors A in entorhinal cortex and increased them in cingulate cortex, whereas a similar treatment regimen with clozapine reduced receptors in frontal, parietal, temporal and entorhinal cortices, but increased them in the CA3 division of Ammon’s horn. NEED FOR IMAGING I N VIVO

An ability to image 5-HTIAreceptors in human brain in vivo is needed to assess directly the involvement of 5-HTIA receptors in neuropsychiatric diseases and their current therapies. By studying receptors in vivo, many of the uncertainties attached to studies of human brain post mortem can be avoided, such as, for example, the impact of the time lapse after death on receptor status. Measurements on human brain post mortrm are limited by the poor availability of well characterized samples. Molecular imaging in vivo would allow receptor status in the whole brain to be studied on a regional basis in well-described populations of drug-naive or drug-treated subjects and at all stages of the progress of a disease or of its treatment. This would help to resolve apparently conflicting findings from studies at post mortern on numerically small samples, and help to justify or refute current hypotheses in our understanding of neuropsychiatric disorders.

V.W. PIKE ET AL.

20 1

The quantitative imaging of 5-HTIAreceptors would also allow the study of the interactions of established and developmental drugs with these receptors. Such studies might relate either drug dose or plasma drug concentration to drug occupancy of receptor, and also measure the duration of receptor occupancy. This type of information is now proven to be useful in setting dose regimens for clinical trials.

MOLECULAR IMAGING TECHNIQUES POSITRON EMISSION TOMOGRAPHY

Positron emission tomography (PET) is an imaging technique that is able to detect positron-emitters within an animal or human body [23]. A positron emitted from an unstable neutron-deficient nuclide is externally detectable because the positron, once it has lost its kinetic energy, combines with an electron to form a transient entity, known as a positronium, which quickly annihilates. The annihilation event converts the rest mass of the positron plus the electron into two 511 keV y-rays that are emitted in virtually opposite directions (to conserve near zero linear and angular momentum). A PET camera is designed to surround the subject of interest with sensitive y-ray detectors and to record coincident pairs of y-rays. A computer algorithm can be applied to reconstruct the lines of coincidence corresponding to annihilation events. In this manner the technique can reconstruct the distribution of radioactivity within a given region, such as the brain. Modern PET cameras can perform this operation every few seconds and hence provide regional kinetic data. Collimation is effectively achieved electronically; no physical collimation is needed. Hence, the cameras are intrinsically sensitive and the attainable physical resolution is of the order of 2 mm. They can achieve truly 3D acquisition by employirfg adjacent rings of detectors over a long axial field of view (e.g. the whole human brain). True attenuation of acquired emission data can be achieved by using an external source of 511 keV photons for a transmission scan. Hence, radioactivity concentration can be measured in absolute units (e.g. Bq / mL). A major strength of the PET technique is that carbon-1 1 and fluorine-18 are almost pure positron-emitters. These positron-emitters can be incorporated into biological or drug molecules to provide biochemically specific radiopharmaceuticals, including ligands for neurotransmitter receptors. A drawback is that these radioisotopes have very short half-lives (20.3

202

RADIOLIGANDS FOR THE STUDY OF BRAIN S-HTIARECEPTORS

min for "C and 109.7 min for '*F), requiring their production, usually on a cyclotron, at or near to their site of use, and also rapid chemistry techniques for their incorporation into organic compounds [83]. SINGLE PHOTON EMISSION TOMOGRAPHY

Single photon emission tomography (SPET) is a technique for imaging low energy y-emitting radiopharmaceuticals (e.g. receptor radioligands) within an animal or human subject [24]. Physical collimation is required to acquire information on the direction of the emitted y-ray and hence the position of its source. The need for collimators limits the sensitivity and resolution of the technique. True attenuation correction is impossible, and hence accurate quantitation of regional radioactivity concentration is difficult to attain. The main radioisotopes used to label radioligands with this technique are iodine-123 ( t l I z = 13.2h), which emits a 159keV y-ray, and technetium-99m (flI2 = 6 h) which emits a 140 keV y-ray. Iodine-123 is commercially available while technetium-99m is available from commercial long-lived generators. REQUIREMENTS OF RECEPTOR RADIOLIGANDS FOR MOLECULAR IMAGING

It is generally recognized that a prospective radioligand must comply with several basic criteria to be effective for PET or SPET imaging of brain receptors in vivo [83]. Ideally, the ligand for development must show: 0

0 0 0 0

0

0

0

High selectivity for the target receptor population High affinity Antagonist action (for stable receptor-ligand interaction) Stereoselectivity (where enantiomers exist) Low or moderate lipophilicity (for brain penetration and to avoid high protein binding or high non-specific binding) Low metabolism (or metabolism to non-interfering radioactive metabolites) Amenability to labelling with a positron-emitter (generally "C or IRF) or y-emitter (preferably iodine-123) Low toxicity (acceptability for intravenous injection at low doses into human subjects).

These criteria presuppose that the target population of receptors exists at a sufficiently high concentration for measurement. Theoretically, the available concentration of receptors (the B,,,,, value) should far exceed the equi-

V.W. PIKE ET A L .

203

librium dissociation constant for the receptor-ligand complex (the K D value) for possible attainment of a useful signal (a high ratio of receptor-specific binding to non-specific binding). Hence, for 5-HTIA receptors, which appear to exist in human brain at concentrations up to about 100 fmol/mg protein (equivalent to IOpmol/mL or 10nM) the radioligand should have a K D value in the low or sub nM range). It is desirable that the radioligand should not to be metabolized by peripheral organs, such as the liver. Especially, any radioactive metabolites should not access and bind the tissue, receptor of interest or other receptors, since this may complicate or confound attempts to achieve biomathematical modelling of the acquired emission data in a PET or SPET experiment, and thus the realisation of quantitative parameters, sucl as B,,,,,, KD, or binding potential (BP = B,,,,,/ KD). The clearance of free radioligand and its radioactive metabolites must be fast enough to allow high radioactivity contrast between target receptors and blood within the time-scale of the scanning experiment (up to 5 half-lives for carbon-11 or a few hours for other radioisotopes). The final demonstration of receptor-specific binding of the radioligand in vivo should be subject to the same pharmacological criteria as in vitro. Namely, binding should be saturable, stereoselective and displaceable by selective ligands. A prospective radioligand not only has to satisfy this large array of demanding criteria, but also has to present pharmacokinetics that can be modelled biomathematically to deliver accurate binding parameters (e.g. BP) [84]. A particular administration protocol may be needed to achieve this aim, based on 1, 2 or 3 separate injections of radioligand, usually at varied specific radioactivity. If the biomathematical model requires an arterial input function, information on the appearance of radioactive metabolites in plasma may be required in each scanning experiment. RADIOCHEMISTRY TECHNIQUES

Radioligands for PET generally use carbon- 1 1 or fluorine- 18 as radiolabels [83]. Because of their short-half lives, these radioisotopes have to be produced with a nearby cyclotron, a machine for the acceleration of charged particles such as protons to high energies for use as the projectiles in the required nuclear reactions. Several cyclotrons are available commercially for dedicated application to the production of positron-emitters. The radioisotopes are only available from the cyclotron in a limited range of chemical forms (Table 4.1). The first task in the preparation of a

204

RADIOLICANDS FOR THE STUDY OF BRAIN S-HTIARECEPTORS

Table 4.1. MAJOR ROUTES TO CYCLOTRON-PRODUCED PRECURSORS FOR "C AND I8F AT HIGH SPEClFIC RADIOACTIVITY Nueleur reucfion

Target

''N(p,a)"C I4N(p,a) C "O(p,n)"F

''

L"CH3OHI

HI

"CHaI

Ag triflate

-

"CH3OSOZCF,

Figure 4.4. Some routes to usrful luhellirzg ugents from cyclotron-produced ("C]curhon dioside und ["C]tnethane. Such ugents huve been used to lube1 the "C-labelled Iigund.s discussed in tius review.

radioligand is usually to convert these simple chemical forms rapidly into a useful labelling agent at high specific radioactivity. PET radiochemistry has developed to be highly diverse and versatile, but there are a few well-established major techniques that are used for preparing most radioligands. These are "C-methylation with, for example, ["C]iodomethane or ["Clmethyl triflate, "C-acylation with various ["Clacid chlorides, "C-alkylation with higher ["Clalkyl iodides, and "C-cyanation with ["Clcyanide (Figure4.4).Generally, the labelling agents can be produced efficiently within only a few minutes and applied to deliver a purified radioligand in a form ready for intravenous administration well within an hour. Labelling reactions are usually performed over a few minutes in small volumes of solvent ( < 1 mL) with small amounts of substrate (usually a few milligrams). Radioactive product is usually separated by

V.W. PIKE ET AL.

205

single pass HPLC on a semi-preparative column, and formulated for intravenous injection by removal of solvent and dissolution in physiologically acceptable media. [''F]Fluoride can be used directly in aliphatic or aromatic nucleophilic substitution reactions to label radioligands at high specific radioactivity. For these reactions it is necessary to generate highly reactive naked [18F]fluoride from the aqueous solution of [''Flfluoride obtained from the cyclotron target. This is achieved by evaporation of the water in the presence of added acetonitrile and then dissolution of the [''F]fluoride in polar aprotic solvent (often acetonitrile or DMSO) by the addition of a bulky counterion such as tributylammonium or a potassium-cryptand complex. Reactions are usually peformed in platinum or glassy carbon vessels, using a small quantity of substrate (a few milligrams) in a small volume of solvent (< 1 mL). Techniques for separation and formulation of the labelled product are similar to those used for 'C-labelled compounds. Alternatively, electrophilic labelling agents, such as ['8F]fluoroethyl tosylate or [''F]fluoroethyl bromide, may be produced by nucleophilic substitution of one leaving group in alkanes having two terminal leaving groups (Figure 4.5). ['8F]Fluorine (i.e. ["F]F2) is rather difficult to produce at an adequately high specific radioactivity for neuroreceptor radioligands, and is seldom used for this purpose. Most of the PET radioligands mentioned later in this review have been prepared by the small range of generic techniques mentioned above. Methods for labelling radioligands for SPET are of two main kinds, namely radioiodination with iodine- 123 and chelation with technetium-99m. Iodine- 123 is commercially available as a solution of ['231]iodide. The radioiodination of ligands can be achieved by a variety

-I

lSF-

f-

APF

R'~F

RX

X = Br or OTos n=2or3

Figure 4.5. The use of cyclotron-produced [ " w u o r i d e ,for labelling organic compounds.

206

RADIOLIGANDS FOR THE STUDY OF BRAIN S-HTIARECEPTORS

of techniques, including electrophilic procedures in which the iodide is oxidised to the equivalent of I+ and used in, for example, the direct iodination or regioselective iododestannylation of arenes. Various approaches exist for the chelation of technetium-99m into receptor radioligands. The objective is to produce an uncharged chelate with high affinity for the target receptor that can adequately enter the brain and bind to the target receptor. A major difficulty is that the introduction of bulky groups for metal chelation can be highly detrimental to pharmacophore structure, ligand affinity, selectivity and lipophilicity.

RADIOLIGANDS FOR IMAGING BRAIN ~ - H T ~RECEPTORS A IN VIVO AGONIST RADIOLIGANDS

Many potent and selective ~ - H T , A receptor agonists have been labelled with tritium or radioiodine and used successfully for the autoradiography of brain 5-HTIAreceptors in vitro [2,66,671. These include ['H]8-OH-DPAT. However, attempts to develop radioligands for application in vivo based on agonists face two major hurdles. Firstly, agonists bind only to receptors that are coupled to a G-protein, which represent only a variable fraction of the receptors available for binding to antagonists. Secondly, the binding of the agonist is generally transient, since agonist binding converts the receptor into a G-protein dissociated conformational state that usually has low affinity for the agonist. An effective agonist radioligand for brain 5-HT1A receptors would nevertheless be a useful tool for selective investigation of the functional G-protein-coupled receptor, and hence there has been a significant effort in this area of radioligand development. The aminotetralin, OSU 191 (7), was the first potent 5-HTIAreceptor agonist to be labelled with carbon-11 [85]. The method was based on N-propylation of a secondary amino precursor with [' 'Clpropyl iodide; no biological evaluation of this radioligand in vivo has been reported. The potent (Ki = 5 nM) selective prototypical 5 - H T I ~receptor agonist, 8-OH-DPAT, has been labelled with carbon-1 1 by the same type of procedure [86]. An evaluation of this radioligand in vivo has also not been reported, but tritiated 8-OH-DPAT gives no receptor-specific signal in rats in vivo [Hume et ul., unpublished results]. Various other potent aminotetralin-type ~ - H T ~agonists A have been labelled with carbon- 1 I and tested as radioligands in vivo [87-891, including racemic 8-OH-

V.W. PIKE ET A L .

207

PPSMAT (8) (Ki = 0.9 nM) and 8-SMeDPAT (9) (Ki = I .2 nM). These radioligands also failed to give receptor-selective signals in rat brain in vivo. The highly selective and more potent R-isomer of 8-SMeDPAT (LY 274601) (Ki = 0.6 nM) has been labelled with carbon-1 1 and evaluated in mice in vivo [88]. This radioligand penetrated well into the brain and accumulated in regions known to have high densities of 5 - H T 1receptors, ~ such as the brain stem, including the raphe nuclei. Binding of the radioligand in receptor-rich regions was blocked, but only to a low extent, by pre-injection of 8-OH-DPAT. The radioligand was metabolized rapidly, with a half-life of 15 min.

8-OH-PPSMAT (8)

osu 191 (7) MeS

8-SMeDPAT (9)

(R)1 1 -Hydroxy- 10-methylaporphine (HYMAP) (10) is also a potent (Ki = 0.45 nM) and selective ligand for 5-HTIAreceptors [90], and has been labelled with carbon-1 1 in its N-methyl group [91]. This radioligand penetrated the brain in cynomolgus monkey to give higher uptake in 5-HTIA receptor-rich regions (e.g. raphe) than in cerebellum at 10 min after injection. There was also uptake in striatum, which is relatively poor in 5 - H T 1receptors, ~ suggesting some lack of selectivity. At 45 min the regional differences in radioactivity content had reduced to about 10% of their peak value. Pretreatment of the monkey with 8-OH-DPAT before radioligand administration abolished any regional differences in radioactivity uptake at early times. Alnespirone [(+)-S204991 (1 l), a 5-methoxy-chroman derivative, is a selective agonist with high affinity (Ki =0.36 nM) for 5-HTIAreceptors, and has been labelled in its 0-methyl group with carbon-11 [87]. This

208

RADIOLIGANDS FOR THE STUDY OF BRAIN S-HTIA RECEPTORS

Me HYMAP (10)

radioligand failed to give any receptor-selective signal in rat brain in vivo [87]. Other potent agonists, such as PNU 157760 (12) (Ki=0.50nM) and ORG 13502 (13) (Ki = 0.04 nM), have been labelled in their 0-methyl groups with carbon-1 1, and have similarly failed as radioligands in vivo [92, 931.

"81

Me0

&fry& CJ Alnespirone (1 1) PNU 157760 (12)

ORG 13502 (13)

In conclusion, no attempt to convert a potent and selective ~ - H T ~ A receptor agonist into a radioligand for the molecular imaging of G-protein A in living brain has yet succeeded, regardless coupled ~ - H T ~receptors of the potency or lipophilicity of the ligand (Table 4.2).

Table 4.2. PROPERTIES OF ESTABLISHED AND PROSPECTIVE RADIOLIGANDS FOR IMAGING BRAIN 5-HTI,4 RECEPTORS WITH PET OR SPET

Lead ligand

Icso K , KD ( n M ) (nM) ( n M ) Function'a'

LogPh' LugD'b'.'c' Label

8-OH-DPAT (2) WAY-100635 (3)

5 2.2

Agonist Antagonist

4.20 3.28

1.82 2.88

propyl-"C methyl-"C carbunyl-l'C

Failed in rat [861 Effective in rat, monkey and human [9&101] Highly effective in rat, monkey and human [65, 97, 104-1 171

Antagonist Agonist Agonist Agonist Agonist Agonist Agonist Agonist Agonist Antagonist

3.20 4.59 4.03 5.12 5.12 3.94 3.59 4.73 3.30 2.17

2.89 1.97 2.37 2.81 2.81 3.78 2.48 4.11 2.69 2.38

carbonyI-"C propyl-"~ methyl-" C methyl-"C methyl-"C methyl-llC methyl-"C methyl-"C methyl-"C carbonyl-" C

Effective in monkey and human Untested Failed in rat Failed in rat Failed in mouse

[41, 137-1393

Unpromising in monkey Failed in rat Failed in rat Failed in rat Highly effective in rat, monkey and human

[901 [g 71 [921

Antagonist

3.07

2.68

18F

Moderately effective in animal and human

[125-127, 145, 151-153, 158-1 621 [Unpublished] [1281 wnpublished] [129-131] [I 321 ~321 [133-1351 [I 33-1 351 [133, 1341

Rohalzotan (6) OSU 191 (7) 8-OH-PPSMAT (8) 8-SMeDPAT (9) R-8-MeDPAT (R-9) HYMAP (10) Alnespirone (I I ) PNU 157760 (12) ORG 13502 (1 3) Desmethyl-WAY (15)

0.37 0.3

0.98 0.17

0.6 0.84 0.6

0.17

1.2 0.9 1.2 0.6 0.45 0.19

0.36

0.50 0.04 1.4 2.1

p-MPPF (16)

3.2 3.3 1.2

p-MPPC (17)

0.8

3.39

3.00

2.4 2.6 0.51 0.33

3.60 2.53 2.53 4.53 3.85

3.21 2.14 2.14 4.14 3.45

CPC-222 (18) cis-MeOWAY (19) rmns-MeOWAY (20) 6BPWAY (21) 6FPWAY (22)

0.23

4.2

Antagonist Antagonist

Radioligand behaviour in vivo

carbonyl-' ' C methyl- I C carbonyl- " C methyl- I I C merhyl-"C methyl-"C carbonyl-"C carbonyl- C I8F

'

''

Untested Moderately effective in rat and monkey Moderately effective in rat Moderately effective in rat and human Failed in rat Small signal in rat Low signal in monkey High signal in monkey Untested

References

~ 5 1 ~871 [871 [881

[931 [118-1241

<

% z

E b Y b

P

h)

W 0

N

I

0

Table 4.2 continued PROPERTIES OF ESTABLISHED AND PROSPECTIVE RADIOLIGANDS FOR IMAGING BRAIN S-HTIA RECEPTORS WITH PET OR SPET Lead ligand MWAY (23) FEWAY (24) FPWAY (25) trans-FCWAY (26) cis-FCWAY (27) MeFBWAY (31) PyMPPF (32) p-MPPI (33) rn-MPPI (34) O-MPPI (35) RI-MPPI (36)

Icso K KD ( n M ) ( n M ) (nM) Function'a'

7.25 1.55 6.05

Antagonist Antagonist Antagonist

0.9 0.515 7.05 3.96 el

2.6

0.36

1.7 10.4 76 0.069 0.2

Antagonist

Logfib' LogD"'.'''Label 2.36 3.34 3.69 2.76 2.76 3.65 2.49 3.80

1.96 2.94 3.30 2.36 2.36 3.26 2.10 3.41

mesyl-"C

3.82 3.67 n.c.

3.43 3.28

1231

n.c.

1231

On basis of experimental evidence. from Pallas 2.1 for Windows (Compudrug) '''At pH 7.4. n.c. Not calculable. (a)

(b)Calculated

I8F

'*F '*F '8F '8F

I8F 1231

1231

Radioligand behaviour in vivo

References

Untested Moderately effective in rat Moderately effective in rat Effective in rat, monkey and human Low signal in rat Moderately effective in rat Moderately effective in rat Moderately effective in rat and monkey; Fails in human Fails in rat Untested Fails in rat

[120, 1361 [120, 1431 [120, 1431 [la1481 [132, 149, 1501 [144, 1491 [149, 1631 [164, 1651 [164, 1651 [164, 1651

I1701

m

P

ez

V.W. PIKE ET AL.

21 1

ANTAGONIST RADIOLIGANDS

Antagonists bind to both G-protein-coupled and uncoupled receptors with the same affinity and are expected not to cause major conformational changes in the receptor that might result in rapid dissociation of the antagonist. Hence, in comparison to agonists, they are in principle better leads for developing radioligands for imaging 5-HTlA receptors in vivo.

Carbon-11 labelled ligands [O-methyl-”C] W A Y-100635. Soon after the development of WAY-I 00635 as the first potent and highly selective silent antagonist of 5-HTIAreceptors in the early 1990s [58, 591, [0-methyl-3H]WAY-100635 was prepared and evaluated as a radioligand in rodents in vivo [94, 951. In the mouse there was rapid uptake of radioactivity into the brain, amounting to 1.5-1.8% of the injected dose at 5 min [94]. At 60 min the distribution of radioactivity in the brain mainly matched the distribution of 5 - H T 1receptors ~ previously determined in vitro. High levels of labelling were found in the lateral septum, gyrus dentatus and CA1 area of Ammon’s horn in the hippocampus, the dorsal raphe and entorhinal cortex. A low level of labelling was seen in striatum. No labelling was found in the substantia nigra. The ratio of the radioactivity level in hippocampus to that in cerebellum was between 7 and 8. Only ligands that have a high affinity for 5 - H T I ~receptors were able to reduce the level of radioactivity in receptor-rich regions to that in the cerebellum when given before the radioligand. The radioactivity level in cerebellum was unaffected by such pre-treatment, indicating that radioactivity in this region was non-specifically bound. Similar findings were found in rats [95]. After intravenous injection of [0-methy/-3H]WAY-100635 into rats there was rapid clearance of radioactivity from plasma but retention of radioactivity in the brain with a regional distribution similar to that for 5-HTIA receptors determined in vitro. At 60 min after radioligand injection the ratio of the radioactivity level in ~ - H T ~ receptor-rich A regions (e.g. septum, entorhinal cortex and hippocampus) to that in cerebellum reached about 16. These ratios were reduced to unity by pre-injection of a large dose of WAY-100635 and substantially reduced by pre-injection of 5 - H T I ~partial agonists, such as buspirone, while prazosin ( a , ligand), citalopram (5-HT transporter ligand) and idazoxan (a2 ligand) caused little or no reduction. Analysis

212

RADIOLIGANDS FOR THE STUDY OF BRAIN S-HTIARECEPTORS

of experiments in which increasing amounts of WAY-100635 were co-administered with the radioligand indicated B,,, values ranging from 0.3 pmol/g in striatum to 6.4 pmol/g in hippocampus. Autoradiography of human brain slices exposed to [ O-w~ethyf-~H] WAY-100635 also demonstrated high specificity for the radiolabelling of 5-HTIA receptor-rich regions of brain (Figure 4.3) [65]. Subsequently, WAY-100635 was labelled with carbon-11 in its 0-methoxy position by "C-methylation of desmethyl-WAY (15) [96-981. This radioligand gave results in rats in vivo [95-971 similar to those obtained with the tritiated radioligand and was subsequently used for the first successful imaging of 5-HTIAreceptors in rat [97], monkey [98] and human brain [99]. PET imaging of the brain with a dedicated small animal scanner in rat injected with the radioligand revealed higher radioactivity uptake in a region of interest expected to be rich in 5-HTIAreceptors (frontal cortex) relative to that in cerebellum. The kinetic data were successfully analyzed using a simple reference tissue model to provide a binding potential value in the receptor-rich region of interest [97]. After intravenous injection of radioligand into rhesus monkeys, radioactivity was found to be taken up into the brain, giving a distribution consistent with mainly specific binding to ~ - H T ~receptors. A The ratio of radioactivity level in frontal cortex to that in cerebellum reached 5 . 5 at 80 min after injection. Pretreatment of the monkeys with 8-OH-DPAT reduced this ratio to 1.4. Administration of 8-OH-DPAT at 20 min after radioligand injection also reduced this ratio [98]. Predosing of rhesus monkeys with paroxetine, a selective serotonin reuptake inhibitor, had no effect on radioligand uptake. However, pretreatment with the 5-HT releasing and reuptake-inhibiting drug, fenfluramine (8 mg/kg) at 90 min before the radioligand resulted in significant decreases (20%) in the specific binding in hypothalamus, cortex and hippocampus. Thus, the receptor binding of this radioligand appears from this study to be sensitive to large changes in endogenous 5-HT [99]. After intravenous injection of [O-methyl-"C]WAY-100635 in human male volunteers there was a rapid uptake of radioactivity into the brain [loo, 1011. Summation of the emission data obtained between 20 and 90 min after radioligand injection revealed a radioactivity distribution mainly corresponding to the distribution of ~ - H T ~receptors A determined by autoradiography in vitro. The ratio of radioactivity level in receptor-rich medial temporal cortex to that in receptor-poor cerebellum reached 3.1 at 20 min after radioligand injection, a ratio substantially lower than that seen in rats or monkeys.

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Analysis of human plasma revealed the rapid appearance of radioactive metabolites, one of which was identified as the secondary amine, ["C]WAY-100634, formed by amide hydrolysis [ 102, 1031. This radioactive metabolite was also found in monkeys, but not in rats, injected with the same radioligand [102]. WAY-100634 (14) was known to have high affinity for both 5 - H T I ~receptors and MI-adrenoceptors [I.A. Cliffe and A. Fletcher; unpublished results]. It was realized that the possible entry of the metabolite, [ I 'CIWAY-100634, into human brain, and subsequent non-specific or receptor-specific binding, might be detrimental to the quality of 5-HTIA receptor imaging with [O-merhyl-''C]WAY-100635. ["C]WAY-100634 was prepared in the laboratory as a radiotracer to investigate these possibilities. After intravenous injection of ["CIWAY- 100634 into monkey there was radioactivity uptake indicating non-specific binding in cerebellum and other regions [102]. The uptake of radioactivity was higher in frontal cortex and temporal lobe than in cerebellum, suggesting that [' 'CIWAY-100634 also binds specifically to some brain receptors. Hence. the metabolism of [0-methyl-' 'CIWAY-100635 is a severe limitation

214

RADIOLIGANDS FOR THE STUDY OF BRAIN S-HTIARECEPTORS

on its use for the possible quantitation Of ~ - H T I receptors A in human brain.

WAY-1 00634 (14)

[CarbonyI-"C] W A Y-100635. WAY-I00635 has been labelled with carbon-11 in its carbonyl position by acylation of WAY-100634 with ["C]cyclohexanecarbony1 chloride [65, 97, 1041. This has provided a new radioligand (now simply known as WAY) that avoids the formation of ["C]WAY-I00634 as a radioactive metabolite. In human brain, WAY provides several-fold greater signal contrast (ratio of radioactivity level in a 5-HTIA receptor-rich region to that in cerebellum) than [O-rnethyl-"C]WAY-100635 [105]. Raphe nuclei are clearly delineated by this radioligand [Figure 4.61. This dramatic improvement in signal contrast is due to lower non-specific binding of radioactive compounds in all brain regions [ 1051. Analysis of blood after injection of WAY into human subjects revealed the rapid clearance of radioactivity and the rapid appearance of more polar radioactive metabolites; only 10% of the radioactivity in plasma at 10 min is unchanged WAY. The least polar radioactive metabolite was identified as the predicted [ I 'C]cyclohexanecarboxylic acid formed by acid hydrolysis [106, 1071 (Figure 4.7). This metabolite has been prepared in the laboratory and studied in monkey with PET; it enters brain only to a low extent [106, 1081. Biomathematical models have been developed to extract information on regional binding potential from the PET data acquired with WAY [109, 1 101. It has been concluded that a simplified reference tissue (cerebellum) receptor binding, either in terms model can be used to quantity ~ - H T I A of region of interest data or as parametric images [110]. Also it has been shown that the high signals in neocortex and raphe nuclei could be described using a kinetic analysis with a metabolite-corrected arterial input function [ 1091. Furthermore, an anatomic standardization technique for the description of regional 5-HTlAreceptor binding potential in human brain has been developed from experiments with WAY in six human subjects [ l l 1, 1121. Hence, WAY is currently the preferred radioligand for application in clini-

-

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x

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RADIOLIGANDS FOR THE STUDY OF BRAIN ~ - H T I RECEPTORS A

cal research and drug development. Usually the simplified reference tissue model is used since this avoids the need for arterial cannulation of the subject under study. Estimates of binding potential from this model are highly reproducible [ 1 131. WAY has also been used successfully for PET experiments in monkeys to assess the effects of various pharmacological challenges on ~ - H T Ireceptor A occupancy [114]. More recently it has been successfully applied to similar PET studies in small animals. WAY binding to 5 - H T I ~receptors in rat brain may be susceptible to competitive displacement by dramatically elevated levels of serotonin (e.g. in response to fenfluramine challenge) [115]. However, similar susceptibility in human brain has not yet been demonstrated. The preparation of WAY from air-sensitive reagents is rather demanding. The need to produce WAY on a regular basis for PET experimentation has given rise to modifications [I 16, 1171 to the original radiosynthesis [ 1041. [Carbonyl-l'C]Desmethyl- W A Y-1OO635. Demethylation of WAY appears to be a very minor route of metabolism in human subjects [ 1061. The resultant metabolite, [carbonyl-"Cldesmethyl-WAY (now known as DWAY), has been prepared in the laboratory by acylation of desmethyl-WAY100634 with [l'C]cyclohexanecarbonyl chloride [I 181, a procedure which is directly analogous to the preparation of WAY. This has provided a potential radioligand, since desmethyl-WAY (15) is known to be a potent and selective antagonist for 5-HTIAreceptors [I 19, 1201, with lipophilicity, as indexed by calculated logP or logD, somewhat lower than that of WAY-100635 (Table 4 . 2 ) .

Desmethyl-WAY (1 5)

DWAY was initially evaluated in animals in vivo [ I 18, 1211. After intravenous injection of the radioligand into rats there was brain uptake of radioactivity. At 60 min after injection, the ratios of radioactivity in each brain region to that in cerebellum correlated with previous measures of 5 - H T I ~receptor density in v i m and in vivo. The highest ratio was about

V.W. PIKE ET A L .

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22 in the hippocampus. After intravenous injection of the radioligand into the cynomolgus monkey there was avid uptake of radioactivity into the brain (5.7% of the injected dose at 5 min). The image acquired between 9 and 90 min after radioligand injection showed high radioactivity uptake in brain regions rich in 5-HTIAreceptors (e.g. frontal cortex and neocotex), moderate uptake in raphe nuclei and low uptake in cerebellum. A transient equilibrium was achieved in cortical regions at about 60 min, when the ratio of radioactivity concentration in frontal cortex to that in cerebellum reached 6. The corresponding ratio for raphe nuclei was about 3 . More recently DWAY has been compared to WAY by PET experiments in human volunteers [122-1241. PET scans acquired with each radioligand in the same subject were very similar. Curves for radioactivity uptake into brain regions were also similar for each radioligand. The maximal ratio of specific binding in cortex to nonspecific binding in cerebellum was slightly higher for DWAY and was reached slightly later at 30 min, compared to 18 min for WAY. DWAY also appears to be metabolized in the same manner as WAY and at a similar rate, as judged by analysis of radioactive species in plasma. This is consistent with amide hydrolysis being the primary route of metabolism. However, the kinetic behaviour of DWAY differs from that of WAY in one important respect, the uptake of radioactivity into brain per unit dose. DWAY gives about twice the level of radioactivity in all brain regions (Figure 4.8). This feature of DWAY has potentially important advantages over WAY, since it may facilitate precision in biomathematical modelling, and also permit a repeat experiment in a human subject or a single experiment in a child with greater radiation safety. Comparative modelling of DWAY and WAY is now in progress.

"C-Labelled nnalogues qf WA Y-100635 with varied a c j ~ groups. l WAY and DWAY are labelled by "C-acylations of amine precursors with ["C]cyclohexanecarbony1 chloride [65, 97, 1041. The labelling agent is produced by C-carboxylation of a Grignard reagent followed by chlorination with thionyl chloride. This is a generic process, since the Grignard reagent can be varied to give any one of a variety of "C-labelled acid chlorides (Figure 4 . 4 ) . This opens up the possibility of preparing [carbonyl-"Clacyl analogues of WAY that are known or expected to have altered but high affinity for 5-HT,* receptors and also modified lipophilicity (Table 4 . 2 ) . Analogues of WAY in which the acyl group is p-fluoro-benzoyl (p-MPPF, 16) or p-chlorobenzoyl @-MPPC, 17) (Table 4 . 2 ) have been labelled with carbon-1 1 in this manner [McCarron et al., unpublished results].

WAY

DWAY

800

=

800,

-cl"s"lale -Hippocampus

700)

7001

--cCmpules

-.a

500

-

400

-Cerebellum

C

%

8 s e

.-6 P a

4 FrmW

I

coriex

Rlyhe nuclei

-p

500

8

400

C

.I

5

e

300

300

3

200

E zoo U

100

100

E

0

0

0

10

20

30 Time (min)

40

50

60

70

0

10

20

30

40

50

60

70

Time (min)

Figure 4.8. Comparative regional brain uptake curves for WA Y and D WA Y in the same human subject. The data are normalizedfor the same injected dose of radioactivity ( b y courtesy of Dr Bengt AndreC, Karolinska Instifutet).

V.W. PIKE ET A L

219

X = F: p-MPPF (16) X= CI: pMPPC (17)

p-MPPF is a potent antagonist at the ~ - H T I receptor A (Table 4 . 2 ) [125]. "F-labelled p-MPPF has shown some promise as a radioligand for ~ - H T I A receptors in primates and humans, but is inferior to WAY or DWAY with respect to signal contrast [126, 1271 (see later discussion). Hence, ["Cb-MPPF has not been further developed. (Table 4 . 2 ) . At p-MPPC is also a potent ligand for the ~ - H T receptor ~A 60 min after injection of carbonyl-"C-labelled p-MPPC into rats there was evidence of specific binding to ~ - H T I receptor-rich A structures, such as the hippocampus. However, the signal (ratio of radioactivity concentration to that in cerebellum) was small compared to that achievable with WAY (Hume et al, unpublished results). The same ligand has been labelled with carbon-1 1 in the 0-methyl group [128]. [O-methyl-l'Clp-MPPC gave a substantially lower specific signal than WAY in the brain after injection into the rat or monkey, though levels of non-specific binding were similar. I t was suggested that this radioligand might be susceptible to competition from endogenous serotonin for binding to the ~ - H T I receptor A because of an apparently fast off rate. In this regard, the administration of fenfluramine is known to raise synaptic 5-HT levels by inhibiting 5-HT reuptake through the presynaptic serotonin transporter. Predosing rats with high doses of fenfluramine (2.5 mg/kg) at 20 min before injection of [carbonyl-I' Clp-MPPC was not found to reduce the receptor-specific signal in the brain (Hume et a f . , unpublished results). The rapid clearance of WAY from plasma and its rapid metabolism are disadvantages for successful quantitation of ~ - H T receptors ~A in the brain, since they limit the input of radioligand into the brain and may give rise to radioactive metabolites which contaminate the signal in the receptor-rich and reference region (cerebellum). An ideal radioligand would not be metabolized at all. The main route of metabolism of WAY is amide hydrolysis. Hence, attempts to restrict metabolism have focussed on changing the ligand structure in the region of the amide bond. A wide ranging set of acyl analogues of WAY-100635 has been prepared and tested for binding affinity to ~ - H T I A receptors in vitro [129, 1301.

220

RADIOLIGANDS FOR THE STUDY OF BRAIN ~ - H T ~RECEPTORS A

The more potent compounds have been assessed for susceptibility to metabolism using a metabolic model of the human liver (cytosol and microsomal preparations). None of the selected compounds were metabolized in cytosolic media. They were also more slowly metabolized than WAY- 100635 in microsomal preparations, all by amide hydrolysis. One of these potent compounds, having a bulky 2-bicyclo[2,2,2]octanyl group in place of the cyclohexyl group in WAY-100635 (CPC-222,lS) (Tahle4.2), was labelled with carbon-11 in the methoxy group and found to penetrate rat brain to give a regional distribution of radioactivity mainly consistent with binding to ~ - H T receptors. ~A At 45 min the ratio of radioactivity concentration in hippocampus to that in cerebellum was about 10. The level of radioactivity in this and other S - H T ~ A receptor-rich regions could be reduced to that in cerebellum by pre-injection of WAY-100635 or CPC-222 but not ketanserin (5-HT2 ligand) or raclopride (D2 ligand).

CPC-222 (1 8)

["C]CPC-222 was subsequently evaluated in human volunteers [I 3 I]. After radioligand injection, there was a high accumulation of radioactivity A The peak corin brain regions having a high density of ~ - H T I receptors. tical concentration was 1-2.5'%1of the injected does per litre. The ratio of radioactivity in receptor-rich regions to that in cerebellum reached 2.5-4 at 45 min after radioligand injection. No ["C]WAY-100634 was found in plasma, as predicted from the previous studies in vitro. Hence, ['I CICPC-222 appears to be an effective metabolically stable radioligand, but inferior to WAY or DWAY, with respect to signal in vivo. Analogues of WAY-100635 bearing either a cis or truns 4-methoxy group on the cyclohexyl ring have been prepared [cis-MeOWAY (19) or trans-MeOWAY (20), respectively] [132]. The trans-isomer (Ki = 2.6 nM) was found to be more potent than the cis-isomer (Ki = 24 nM) for binding to ~ - H T I receptors A in vitro (Tuble 4.2).These ligands were easily labelled with carbon-1 1 in their 4-methoxy groups to give radioligands that were expected to avoid giving pharmacologically active radioactive metabolites in vivo by amide hydrolysis [132]. Only the trans-isomer gave a signal in

V.W. PIKE ET A L .

22 1

the brain after intravenous injection into rats, as represented by a modest maximum hippocampus to cerebellum radioactivity ratio of 2.2 at 5 min after radioligand injection [ 1321.

OMe

X = cis-OMe: cis-MeO-WAY(19) X = trans-0Me:transMeO-WAY (20)

[CLirbonyl-"C] (pyridinyl-6-halo) analogues of WA Y. Analogues of WAY100635 bearing a halogen (Br or F) in the 6-position of the pyridinyl ring have been prepared as part of a strategy for developing new IXFand "31-labelled radioligands for 5-HTlA receptors [ 1331. These compounds bind strongly to ~ - H T I receptors A with Ki values close to that of WAY100635 itself (Table 4 . 2 ) [133]. The syntheses start from the 2,6-dihalopyridines and lead to the descyclohexanecarbonyl compounds as intermediates. This has provided an opportunity to evaluate the 6-halo analogues as l'C-labelled compounds in monkeys, since the available amines are readily acylated with [ I C]cyclohexanecarbonyl chloride. The 6-bromo compound (6BPWAY, 21) has been labelled with carbon-l 1 and assessed as a radioligand by PET experiments in cynomolgus monkey [ 1341. This radioligand enters the brain, giving a maximal radioactivity uptake of 5.5% of the injected dose at 6min. The uptake of radioactivity was greater in 5-HTl,4 receptor-rich brain regions, such as temporal cortex, frontal cortex and raphe nuclei than in receptor-devoid cerebellum. Radioactivity cleared slowly from all regions. In an experiment where the monkey was injected with WAY-I00635 (0.5 mg/kg) at 20 min before radioligand injection there was a significantly lower uptake of radioactivity in ~ - H T I A receptor-rich regions. Analysis of plasma revealed polar radioactive metabolites, including the ["C]cyclohexanecarboxylic acid expected from amide hydrolysis. However, parent radioligand was found to disappear less rapidly from monkey plasma than WAY or DWAY. The apparently slower amide hydrolysis may be due to steric hindrance of the amide bond by the bulky bromine atom or perhaps, a difference in the leaving group ability

222

RADIOLIGANDS FOR THE STUDY OF BRAIN 5-HTlh RECEPTORS

of the amine. These results indicate that halo-substitution of the pyridinyl 6-position in WAY is well tolerated for retention of potent binding to in vivo, as confirmed by assay in vitro (Tuble 4.2). ~ - H T I receptors A

X = Br: GBPWAY (21) X = F: GFPWAY (22)

The results obtained with 6BPWAY also suggested that the 6-pyridinyl position of WAY would be a good site for the introduction of fluorine-18. In order to understand how (pyridinyl-6-fluoro)WAY (6FPWAY, 22) might behave in vivo, this ligand was labelled by "C-acylation of the appropriate amine precursor for PET experiments in cynomolgus monkeys [134]. ["C]6FPWAY was found to enter the brain easily with maximal uptake representing about 8% of the injected dose. Compared to the radioactivity level in the cerebellum, the maximal ratio of radioactivity in the cortex was about 5.5 and in raphe about 3.5-4. Equilibrium was reached at about 20-25 min after radioligand injection. Pretreatment of a monkey with WAY- 100635 before radioligand injection reduced the radioactivity uptake in all brain regions to that in the cerebellum, so indicating the selectivity of the radioligand for brain ~ - H T I receptors. A The same radioligand has also been used successfully for the selective delineation of ~ - H T Ireceptors A in human post mortem brain tissue [135]. ["CIO-Mesyll-Analogue of W A Y. The 0-mesyl analogue of WAY-100635 (MWAY, 23) has been shown to be an antagonist at S-HTIAreceptors, with only slightly lower affinity than WAY-I00635 (Table 4.2) [120]. This compound is also calculated to have lower lipophilicity than WAY or DWAY (Tuble 4 . 2 ) . MWAY has recently been labelled with carbon-11 in high radiochemical yield by treating desmethyl-WAY- 100635 with a new labelling agent, ["Clmesyl chloride [136]. This new IC-labelled ligand now awaits evaluation in vivo.

'

["C]Robulzoran. Robalzotan (NAD-299, 6 ) is a highly potent (KD = 0.16 nM), moderately lipophilic and selective antagonist of ~ - H T I A

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MWAY (23)

receptors (Table 4.2) [41], and also a putative drug with potential for the treatment of depression. This compound has been tritiated and in this form has been shown to bind to both G-protein and uncoupled S-HTIAreceptors in rat brain (hippocampus) [42]. After intravenous injection of [3H]robalzotan into mice, there was substantial accumulation of radioactivity in the brain, with a discrete distribution at 30 min consistent with a high proportion of specific binding to 5-HT,* receptors [137]. More than 90% of the radioactivity in hippocampus was due to intact radioligand at 15 min after injection. The receptor-specific signal was saturable with high doses of ligand. Robalzotan has recently been labelled with carbon-1 1 in its carboxamido group by palladium-mediated [ "Clcyanide for triflate displacement followed by peroxide-mediated oxidation. Post mortem human brain slices have been incubated with this radioligand for subsequent autoradiography, which showed specific binding of the radioligand to 5-HTIA receptors [138]. This radioligand has also been studied in the monkey [ 1391. After intravenous injection of the radioligand there was rapid accumulation of radioactivity in the brain (3.5% at 4min). Uptake of radioactivity was high in neocortex and raphe and low in cerebellum. The ratios of radioactivity level in the neocortex and raphe to that in cerebellum at transient equilibrium (1 5-20 min) were 3 and 2, respectively. Administration of WAY-100635 before or at 20 min after radioligand injection reduced radioactivity levels in all regions to that of the cerebellum. At 20 min after injection 50% of the total radioactivity in plasma represents parent radioligand 11401. Two of the major radioactive metabolites are more polar than the parent radioligand, and one minor radioactive metabolite is less polar. This radioligand therefore appears quite promising, but has not yet been studied in human subjects.

Fluorine-18 labelled ligands An effective ligand for ~ - H T I receptors A that can be easily labelled with fluorine-1 8 ( t l l z = 109.7min) would be transportable over considerable dis-

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RADIOLIGANDS FOR T H E STUDY O F BRAIN S-HTIARECEPTORS

tances and therefore valuable to centres having positron scanners but no cyclotron (so-called 'satellite' PET centres). Fluorine-1 8 may also have an advantage in giving better count statistics in the final stage of a PET scan and in any accompanying analysis of radioactive metabolites in plasma. Moreover, in tissue the average distance between positron emission and annihilation is significantly lower for fluorine-1 8 than for carbon-1 1, so reducing this constraint on the resolution achievable by new high resolution scanners [23]. Fluorine-18 has only one major disadvantage, which is that only a low proportion of the injected radioactive dose will contribute to the acquired emission data for acceptably short PET experiments in human subjects. There has therefore been considerable interest in the development of IXF-labelled analogues of WAY as radioligands for ~ - H T I A receptors. ['8F]Fluoroalkyloxy analogues qf WA Y-100635. Consideration of the A predicts probable binding mode of WAY-100635 to the ~ - H T I receptor that the binding domain can tolerate bulkier 0-alkyl groups in the 2-position of the ligand. One approach to the production of an "F-labelled ligand for the ~ - H T I receptor A has therefore been to replace the 0-methyl group of WAY-100635 with an [lXF]w-fluoroalkoxygroup. This has been achieved either by reaction of desmethyl-WAY- 100635 with ['*F]wfluoroethyl or [18F]w-fluoropropylbromide [141, 1421 or by direct substitution of a tosylate group with ['8F]fluoride in the w-tosyloxyethyl analogue O f WAY-100635 [143]. The affinity of the fluoroethyl analogue (FEWAY, 24) for rat brain ~ - H T ~receptors A (Ki = 0.9nM) is similar to that of WAY-100635 in the same assay (Table 4 . 2 ) [128]. The fluoropropyl analogue (FPWAY, 25) also has a high affinity for ~ - H T I A receptors (Table 4.2) [120]. Both FEWAY and FPWAY have been shown to be antagonists at ~ - H T I A receptors in functional tests [ 1201.

n = 2: FEWAY (24) n = 3: FPWAY (25)

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Radiofluorinated (24) and (25) have been evaluated in rats in vivo [143]. After intravenous injection of each radioligand, radioactivity was avidly taken up into the brain with the highest uptake seen in ~ - H T Ireceptor-rich A regions. The ratios of the radioactivity level in hippocampus to that in cerebellum at 60min were 5.5 and 7.5 for ["FIFEWAY and ["FIFPWAY, respectively. For each radioligand, the regional distribution of radioactivity in the brain was abolished by pretreatment with WAY-I00635 or FPWAY, but not by ketanserin (5-HT2 receptor ligand), sulpiride ( DZreceptor ligand) or SCH 23390 (Dl receptor ligand). Although these results were moderately encouraging, [IXF]FEWAYand ['*F]FPWAY have never been assessed as radioligands in primates. This is probably because the results obtained with [O-~nerhyl-"CIWAY- 100635 suggest that [" FIFEWAY and ["FIFPWAY will be metabolized by amide hydrolysis to give radioactive compounds that are pharmacologically active and able to penetrate the brain. ['"FjFluoro~~vclohexa~ie analogues of WA Y-100635. One strategy that has been pursued for the development of an "F-labelled ~ - H T I receptor A ligand has been to introduce fluorine- 18 into the cyclohexyl group in the expectation that this will only slightly perturb the structure of the ligand and its ability to bind selectively to the ~ - H T I A receptor. An analogue of WAY- 100635, bearing a trans 4-flUOrO group (truns-FCWAY, 26), has been prepared and shown to bind to 5 - H l - l ~receptors with almost identical affinity to that of WAY-I00635 in the same assay (Table 4.2) [144]. The lipophilicity of FCWAY is calculated to be marginally less than that of WAY (Tuble 4.2). The selectivity of the ligand in vitro has not, however, been reported in detail. ["FITrans-FCWAY has been prepared by a four-step radiochemical procedure, involving the three-step preparation of ['XF]4-fluorocyclohexanecarbonyl chloride and reaction of this with the secondary amine, WAY- 100634 [ 1441. The overall radiochemical yield was quite low (-20%).

X = trans-F: trans-FCWAY (26) X = cis-F: cis-FCWAY (27)

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RADIOLIGANDS FOR THE STUDY O F BRAIN ~ - H T I RECEPTORS A

In rats, ['xF]truns-FCWAYwas found to give a high ratio of radioactivity level in hippocampus to that in cerebellum (1 9.3), a value higher than that for WAY (15.9) [145]. This ratio was reduced to 11.8 and 0.6 by co-administration of 50 and 200 nmol of WAY-100635, respectively. Reduction of the ratio to unity for WAY is achievable with only 50nmol of WAY-100635. The reason for this difference is not yet clear. ["F] Trans-FCWAY was otherwise found to have similar pharmacokinetics to WAY. ["FITrans-FCWAY has also been evaluated with PET in rhesus monkeys [146, 1471. The radioligand penetrated the brain to give high ratios of radioactivity level in ~ - H T I receptor-rich A regions to that in cerebellum (e.g. 9.9 for frontal cortex) (Figure 4 . 9 ) . Hence the distribution of ~ - H T receptors ~A is well delineated (Figure 4.10). The ratio for receptor-poor basal ganglia was 3.6 and higher than that in the comparatively receptor-rich thalamus (2.1), but this may be an artefact due to 'partial volume' effects in the imaging of a small monkey brain. Transient equilibrium of specific binding was achieved between 120 and 150min. These ratios were substantially reduced by pre-injection of WAY-100635. The plasma-free fraction was found to be 49% compared to 31% for WAY. ["F]Trans-FCWAY gave a similar metabolic profile to WAY. Thus, the radioactivity in plasma represented by unchanged radioligand was 37%) at 15mjn, 21% at 50min

h

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Time (min) Figure 4.9. Tinie-rrrLiiouctivity curvesfbr [''F]FC W A Y in u conirol monkey study showing un uprtrkr puttern consisient wiih binding to 5 - H T l , receptors. The dutcr are Jiom rcferencr 147 (courtesy of Dr Riehurd Curson, N I H , m d with pernzission,fronithil publishers of Nuclear Medicine and Biology).

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and 16'%,at 90min. The main radioactive metabolite was found to be ['sF]4-fluorocyclohexanecarboxylic acid, formed by amide hydrolysis. Defluorination also occurred. ['8F]4-Fluorocyclohexanecarboxylic acid plus ['8F]fluoride probably accounted for up to 30% of the extravascular radioactivity in cerebellum between 60 and 120 min after radioligand injection. ["F]Trans-FCWAY has now been studied in human volunteers with PET [148]. Brain uptake of radioactivity after radioligand injection is slower than in monkeys, possibly due to a lower plasma free fraction ( 6%). Peripheral metabolism was faster than in monkeys, with 12, 5 and 3% of the radioactivity in plasma representing unchanged radioligand at 12, 40 and 90min, respectively. As in monkeys, the main metabolites were ['8F]4-flu~rocyclohexanecarboxylic acid and [ I 8 Flfluoride. At 30 min, brain radioactivity uptake was highest in medial temporal and frontal regions, low in basal ganglia and thalamus and lowest in cerebellum. At later times there was substantial skull uptake of [18F]fluoride, which at 60min amounted to twice the whole brain average. Even so, it was concluded that this radioligand can give quantitative measurements of ~ - H T I receptors A in the human brain. Thus, [18F]trans-FCWAY,because of the magnitude and selectivity of its signal, appears to be the most promising "F-labelled ligand for PET imaging of 5-HTl* receptors. The cis-isomer of FCWAY (27) has been labelled by multistage radiochemistry [ 1431 and also by single-step nucleophilic substitution of a trans-tosylate group with [I8F]fluoride [McCarron et al., unpublished results]. The latter radiochemistry is low yielding (
-

['YF]p-Fl~orophenyl analogues of WA Y-100635. A second approach to introduce an easily radiofluoridated group into WAY-1 00635 has been to replace the cyclohexyl group with a phenyl group bearing a good leaving group for nucleophilic displacement with ["Flfluoride. The phenyl analogue of WAY (MPP, 28) is a potent 5-HT1* receptor ligand (Table 4 . 2 ) [151]. It

228

RADIOLIGANDS FOR THE STUDY OF BRAIN 5 - H T I RECEPTORS

Figure 4.10. Coronril itnuges urquired 40-60 niin ,following it 2 tnCi Bolus injei,tion of' [l'flFC WA Y. Imuges ure displuyed on N common scule ofO-I000nCi/cc and roomed 10 show oni,v the brain region. Highest uptuke is seen in,frontul cortex und temporal cortex with lowest upruke in cerebellum ,followed by tlzalamus and basal gangliu. The images m e from reference 147 (courtesy i?f Dr Richurd Curson. N1 H , und with permissionfrom the publishers of Nuclear Medicine and Biology).

was demonstrated that the nitro group in a p-nitrophenyl analogue of WAY-100635 (MPPN) can be replaced with ['8F]fluoride to give a prospective radioligand, ["Flp-MPPF, albeit in low radiochemical yield (lo'%,, decay-corrected) [126]. Later, it was shown that the radiochemical yield could be usefully increased to 40% by microwave heating of the reaction [152, 1531. Analogous attempts to prepare the rn- and o-isomers still gave only very low radiochemical yields (c3Y0 decay-corrected) [ 1521. Non-radioactive [ 1261 and tritiated p-MPPF [ 154, 1551 have been prepared by other routes. y-MPPF (29) has been found to be a potent and selective antagonist at ~ - H T Ireceptors A [125] (Table 4.2).The o-isomer, o-MPPF (30), is four-fold less potent [152]. [3Hlp-MPPF was found to bind to both the high and low affinity states of the 5-HTl,4 receptor in vitro [12, 155, 1561, again indicating

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MPP (28)

the ligand to be an antagonist. Autoradiography of rat brain exposed to ['HIp-MPPF revealed intense labelling in ~ - H T I receptor-rich A regions, such as lateral septa1 nucleus, entorhinal cortex, dorsal raphe and fields CA1-2 of Ammon's horn [155].

OMe

Y X = F , Y = H : p-MPPF(29) X = H, Y = F : CE MPPF (30)

After intravenous injection of VHIp-MPPF into the mouse, there was avid uptake of radioactivity into the brain. At 10 min after intravenous injection, the ratio of radioactivity bound specifically to ~ - H T I receptors A in the cortex to that bound non-specifically by radioligand was five [ 1571. Pre-injection of 8-OH-DPAT, WAY- 100635, pindolol or idazoxan (a?-ligand with moderate affinity for ~ - H T I receptors) A inhibited cortical radioactivity uptake in a dose-dependent manner, whereas pre-injection with fluoxetine (5-HT transporter ligand), paroxetine (5-HT transporter ligand), SCH 23390, haloperidol ( DZ receptor ligand) or prazosin had no effect. The behaviour of ["FFIp-MPPF has been studied in rats in vivo [126, 1271. Initial brain uptake of radioactivity was high (l'% at 2 min) and then declined (to 0.06'%1at 60 min). At 30 min the ratio of radioactivity in hippocampus to that in cerebellum was 4 [I261 or 5.6 [127]. This ratio was reduced to unity by pre-injection of WAY-100635, indicating a high proportion of receptor-specific binding in the hippocampus. Similar findings have been reported by others [ 1581. Radioactivity did not accumulate in the femur, suggesting that ['XF]fluoridewas not a major metabolite [ 1271.

230

RADIOLIGANDS FOR THE STUDY OF BRAIN 5-HTIA RECEPTORS

[18Flp-MPPF has also been studied in unanaesthetized rats [158]. The ratio of radioactivity level in hippocampus to that in cerebellum was 4.2 at 30 min after radioligand injection, and this was reduced to unity by injection of WAY-100635. Pre-injection of prazosin or altanserin (5-HT1 receptor ligand) had no effect on the uptake in the hippocampus. HPLC analysis showed that 75-95'%1 of the radioactivity in the cortex, striatum, hippocampus and cerebellum was due to unchanged radioligand. At 60 min about 30% of the radioactivity in plasma was unchanged radioligand. After intravenous injection of ['XFlp-MPPFinto the cat, autoradiography of the brain revealed the same distribution of radioactivity as for ['H]8-OH-DPAT, with high labelling of the hippocampal formation, (CAI-CA3, dentate gyrus), the entorhinal cortex, the amygdala, the lateral septum and the dorsal raphe [ 1591. [I8Flp-MPPF has also been further evaluated as a radioligand in cats in vivo [152, 153, 1601. After intravenous injection of the radioligand there was rapid uptake of radioactivity in the brain (4% at 4min). The dorsal, lateral and ventral hippocampus [ 1531 and cingulate cortex [ 1601 became labelled. The ratio of radioactivity in hippocampus to that in cerebellum reached 5 at 30 min [ I 521. The corresponding ratio for cerebral cortex was 3.8 [152]. These signals were abolished by injection of a large dose of WAY-100635 at 80min after [I531 or 30min before the radioligand [152, 1531, thereby showing the selectivity and reversibility of radioligand binding. Receptor-specific binding in the hippocampus was also markedly reduced by pindolol but unaffected by prazosin, ketanserin or fluoxetine [ 1601. ['*Flp-MPPF has been studied in a monkey with PET [I 271. After intravenous injection of the radioligand, there was uptake and retention of radioactivity in the brain, giving a ratio of radioactivity level in hippocampus to that in cerebellum of 3 at 30 min. This ratio was reduced to unity by administration of 8-OH-DPAT at 23 min after radioligand injection. Analysis of plasma revealed that 20% of the radioactivity was unchanged radioligand at 30 min. In view of the encouraging results obtained in rodents, cats and monkeys, [I8Flp-MPPF has been further evaluated as a radioligand in human volunteers [161, 1621. The radioligand was rapidly cleared from plasma and was also rapidly metabolized, with only about 12% of the radioactivity representing parent radioligand at 10 min after intravenous injection. The brain distribution of radioactivity up to 70min after radioligand injecA with tion appeared to match the known distribution of ~ - H T I receptors, conspicuously high radioactivity uptake in medial temporal cortex, and

V.W. PIKE ET AL.

23 1

low radioactivity in basal ganglia and cerebellum. However, the expected uptake of radioactivity in raphe nuclei has not yet been described. About 40% of the radioactivity uptake in cortical areas was blocked by pre-administration of pindolol. Binding potentials, derived from biomathematical modelling of the acquired PET data, are about 4-6 times lower than achievable with the more potent WAY. Hence, this radioligand, because of its rather low signal, appears to have limited utility for wideA in human subjects. spread PET scanning of ~ - H T I receptors An analogue of [l8Fb-MPPF, bearing a 3-methyl group (["F] MeFBWAY, 31) has also been prepared [144, 1491. This is a potent ~ - H T I A receptor ligand (Ki = 3.96 nM) and an antagonist in functional tests (Tublr 4 . 2 ) [144]. In rats in vivo, this radioligand gave a ratio of radioactivity level in the hippocampus to that in cerebellum of 4.9, a slightly higher value than for [lXFlp-MPPF in the same test (4.7). Co-injection of 50nmol of WAY-100635 reduced this ratio to 0.1 [145]. MeFBWAY is less selective than p-MPPF for binding to 5-HT1* receptors in vitro; in particular MeFBWAY has quite high affinity for CLI -adrenoceptors (Ki = 13.2 f 3.7 nM). This clearly emphasizes the subtle dependence of ~-HTIA receptor selectivity on the structure of the cycloalkyl/arylcarboxamido group in WAY- 100634 analogues.

OMe

Me

MeFBWAY (31)

Other analogues of ['*Flp-MPPF have been prepared in which the 2-pyridinyl group has been replaced with a 5-methylpyridino, pyrimidino, pyrazino, phenyl, 4-pyridino, 5-chloropyridino or 3-pyridino group [ 149, 1631. Five of these radioligands were evaluated in rats in vivo. The potent pyrimidino analogue (PyMPPF, 32) was found to give the highest ratio (6.7) of radioactivity in hippocampus to that in cerebellum, a value exceeding that PPF of ~ F ~ ~ - M (5.7). ('"F]( Pyridin)~l-6-jhoro) W A Y-10063.5. As discussed earlier, analogues of WAY, bearing either a bromo or fluoro group in the 6-position of the 2-pyridinyl moiety, have been prepared [I 33, 1341. Encouraging results

232

RADIOLIGANDS FOR T H E STUDY OF BRAIN 5-HTIA RECEPTORS

PyMPPF (32)

were obtained in monkey with a "C-labelled version of 6FPWAY (22) [134, 1351. It was therefore of interest to see if the 6-[I8F]fluoro analogue could be prepared as a prospective radioligand. Nucleophilic substitution with [I8F]fluoride in the 6-bromo compound, (21), gave the desired radioligand in 90'%~radiochemical yield (decay-corrected) [ 133, 1341. This radioligand in vitro and has a lipophilicity simishows high affinity for ~ - H T Ireceptors A lar to that of WAY-100635 (Table 4 . 2 ) . However, it may be expected to metabolize by amide hydrolysis to give a radioactive compound that penetrates the brain to bind strongly to S-HTIAand al-adrenoceptors. This new radioligand has yet to be evaluated in vivo.

Iodine-123 labelled ligand,s

There is great interest in being able to study brain ~ - H T I receptors A with the widespread SPET technique. WAY- 100635 was quickly recognized as an important lead for such radioligand development, but it is not particularly easy to carry out the site-selective introduction of radioiodine. Zhuang et ul. developed a strategy of replacing the cyclohexane ring with a phenyl ring to enable radioiodine to be introduced more readily [164]. Radioiodine has been introduced into this ring at the ortho, meta or para position by regioselective iododestannylation of an appropriate precursor [ 164, 1651. All three ligands (33-35) show high affinity for the S-HTIA receptor in vitro [164, 1651 (Table 4.2). p-MPPI (33), was shown to be a ~ - H T I A receptor antagonist, but with significantly high affinity for c11-adrenoceptors (35nM) and dopamine D2 receptors (Ki = 19nM) [166]. [O-methyl-3HHlp-MPPI gave higher non-specific binding in v i m than the fluoro analogue, ['Hlp-MPPF [ 1541, presumably because of its higher lipophilicity (Table 4 . 2 ) . Iodine-I 25 labelled p-MPPI was used successfully for the in v i m autoradiography of rat brain ~ - H T I Areceptors, and appeared to bind to G-protein coupled and uncoupled receptors [167], so indicating its antagonist action.

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X = p-I: pMPPl (33) X = ml: m-MPPI (34) X = 0-1: &MPPI (35)

["'lp-MPPI was found to be a promising radioligand in rats in vivo, giving good brain penetration (1.2% of dose at 2 min) and a maximum ratio of radioactivity level in hippocampus to that in cerebellum of 3.3 at 30min after radioligand injection [ 1681. This signal was reduced by pre-injection of the rats with WAY or 8-OH-DPAT, but not ketanserin or haloperidol. However, the similarly potent m-isomer failed to give any signal. This difference in behaviour remains unexplained, but may be due to differences in metabolism or even intrinsic efficacy. SPET experiments of ['2311p-MPPIinjected into monkey showed uptake of radioactivity into the brain to give a maximal ratio of radioactivity level in hippocampus to that in cerebellum of 3 at 50min after injection. High uptake of radioactivity was also observed in other ~ - H T I receptor-rich A regions (e.g. frontal cortex) and a high proportion of this could be displaced by subsequent administration of high doses of 8-OH-DPAT or p-MPPI [ 1691. Despite these encouraging results in vitro and in animals, this radioligand failed to give a receptor-selective signal in a SPET experiment on a human volunteer. A species difference in metabolism may account for this result, since the biodistribution of radioactivity after intravenous injection of the radioligand into a human subject was consistent with rapid metabolism by amide hydrolysis to ['2311p-iodobenzoic acid and its conjugates [ 1691. In an attempt to improve on ['2311pMPPI as a radioligand several analogues with cyclized amido systems have been prepared to resist metabolism [170]. Potent ligands for the S-HTIAreceptor were found in this set and labelled with iodine-I25 (e.g. FU-MPPI, 36). However, none of these radioligands was found to be a successful radioligand in rats in vivo. Hence, no successful radioligand is yet available for SPET.

234

RADIOLIGANDS FOR THE STUDY OF BRAIN 5-HTt* RECEPTORS

FU-MPPI (36)

Technetium-99m lubrlled ligands

Attempts have been made to develop technetium-99m labelled 5-HTl~receptorligands for application with SPET, based on WAY-100635 or p-MPPI as the lead compound. Two approaches have been taken, a ‘pendant chelation group’ approach and an ‘integrated chelation group’ approach. In the pendant group approach, a technetium complexing moiety replaces the iodo group in p-MPPI. In the integrated group approach the chelating moiety replaces the piperazine ring in p-MPP [171] (e.g. 37), or alternatively replaces the cyclohexanecarbonyl and 2-pyridinyl groups in WAY-100635 [172] (e.g. 38). So far these and similar approaches have produced uncharged neutral ligands, but have failed to achieve the necessary receptor and combination of high affinity and selectivity for the ~ - H T , A good ability to penetrate the blood-brain barrier.

G”34,

f2 m N om f

s

s

(37)

OMe

V.W. PIKE ET A L .

235

Generul feutures of established or promising antagonist rudioligands

Comparison of the properties and behaviour of ~ - H T Ireceptor A antagonists that have been proposed and tested as radioligands in vivo, shows that the most effective (3, 6, 15, 16, 26) and promising radioligands (18, 22) for studies in humans have low nanomolar or sub-nanomolar affinity with moderate lipophilicity (2.76 < calculated log P < 3.85; 2.36 < calculated log D < 3.45) (Table 4.2).

APPLICATIONS OF 5-HT1A RECEPTOR RADlOLIGANDS IN CLINICAL RESEARCH

Brain ~ - H T I A receptor density has been investigated in 15 drug-free depressed patients, 20 SSRI-treated patients and 18 normal volunteers with W A Y and PET [173, 1741. Depressive patients, regardless of whether on or off treatment or in a depressed state or not, were found to have moderately but significantly reduced receptor densities in nearly all brain regions (Figure 4.11). WAY has also been used with PET to measure regional S-HTIAreceptor densities in healthy controls and in depressed subjects with primary, recurrent, familial mood disorders [175, 1761. The mean receptor binding potential was reduced 42%0 in the midbrain raphe and 25-33% in limbic and cortical areas in the mesiotemporal, occipital and parietal cortex. These abnormalities were greatest in bipolar depressives and in unipolar depressives who had bipolar relatives. The results are consistent with other evidence that ~ - H T ~receptor A sensitivity is reduced in major depressive disorder and post rnorteni data showing a widespread deficit of ~ H T I A receptor expression in primary mood disorders. IN DRUG RESEARCH

The results of a clinical trial of a combination of pindolol and an SSRI for the accelerated treatment of depression have been equivocal, and question the supposed action of pindolol at 5 - H T I autoreceptors ~ in the dorsal raphe nuclei. This mechanism has been tested in monkeys and humans using WAY and PET. In cynomolgus monkey, pindolol, given intravenously at 1 mgi kg induced 60% occupancy of brain ~ - H T I receptors. A A 10mg oral dose of pindolol in healthy males inhibited central ~ - H T ~receptors A at 2 h,

236

RADIOLIGANDS FOR THE STUDY O F BRAIN S-HTIA RECEPTORS

'

Figure 4. 11. Biiirhg potmtiul i ~ u l u e s f ~[carbonyl-' r c] WA Y b i d i n g t o S-HT,, receptors tiispluyed,for controls, drug-free depressed .suI?j~~ts und drug-treuted depressed .rubjects. Depressed subjects. whether drug-free or drug-treated, show u mode.rr1te hut s i g i i i f i c u ~reduction ~~ qf'hiriding potentiul in tnoSt ofthe 20 exumitzed bruin regions (Figure reprinted with uutlior undpuhli.slzer '.r perniissiorr ,f).onrGrusbj,, P. (1999) Journal of Psychopharmacology, 13. 316-351).

measured as 7-290/;1 by a simple ratio model and 40% by a simplified reference tissue model [177]. In another study [178], 10 male volunteers were scanned with WAY on two occasions, the first occasion at baseline in the absence of pindolol and in the second after a single oral dose of pindolol ( 5 , 10 or 20 mg). PET data were analyzed using the reference tissue model. A 20 mg dose was found to cause a 40% occupancy of 5-HT,* receptors at both pre- and post-synaptic receptors. A 10 mg dose produced a 40% occupancy of pre-synaptic but not post-synaptic receptors, while a 5 mg dose produced no significant occupancy at all (Figure 4.12). It was concluded that the standard clinical dose of pindolol(2.5 mg t.d.s.) may not be sufficient for reliable occupancy of 5-HT,,4 receptors, and that this may explain the variable response to combined pindolol-SSRI therapy in depressive subjects. The preference for pindolol, when given at moderate doses resulting in plasma concentrations of 1-20 ng/ml, to occupy the autoreceptor rather than

V.W. PIKE ET AL.

237

*

5 mg (n = 3)

10 mg (n = 4)

20 mg (n= 3)

Dose of Pindolol Figure 4.12. Occupancy of pre- (open columns) and post-synuptic (solid columns) 5-HT,,, receptors by various doses ofpindolol in humun subjects. bused on data in reference 178. (Figure courtesy of Dr Eugenii A. Rabiner. M R C Cyclotron Unit).

the post-synaptic receptor may be an important component of its action in accelerating depression therapy with SSRIs [179]. The occupancy of human brain ~ - H T I receptors A by doses of other B-blockers, such as penbutolol [179, 1801 and tertatolol [180] is now also being studied. The recently developed 5-HTl* receptor antagonist and putative antidepressant, robalzotan (6) [41, 421, when given intravenously to cynomolgus monkey in the dose range 2-100mg/kg, occupied brain ~ - H T I A receptors in a dose-dependent and saturable manner with a maximal occupancy calculated at 70-80”/0 in vivo. The relationship between drug plasma concentration and receptor occupancy was best described by a hyperbolic function that was used to select appropriate doses in man [181]. Similar results were subsequently obtained in PET experiments in human subjects [ 1821. These results illustrate and reaffirm that quantitative molecular imaging is useful for the evaluation of the receptor occupancy of new drugs and for setting doses for clinical trials. WAY now finds extensive application for this purpose. For example, the occupancy of human brain S-HTIA receptors by the developmental drugs, S15535 (39), a partial agonist, and by EMD 68843 (40), an agonist at the autoreceptor and an antagonist at the postsynaptic receptor, are now being studied using WAY with PET [179].

238

RADIOLIGANDS FOR THE STUDY O F BRAIN S-HTIA RECEPTORS

EMD 68843 (40)

FUTURE PERSPECTIVE WAY is proving immensely useful as a radioligand for 5-HTl* receptors in current clinical research PET programmes that are investigating neuropsychiatric disorders and drug action. There is scope to use WAY-I00635 as a lead compound for the development of new radioligands with improved properties for specific purposes. Radioligands are required that are additionally: i) highly brain-penetrant ii) poorly metabolized, and iii) amenable to labelling by a simple reliable technique. An extension of these aims might be to discover a radioligand that is also susceptible to displacement by elevated levels of endogenous serotonin, arising as a result of imposed psychological or pharmacological challenges, by analogy with the successful use of ["Clraclopride for the detection of dopamine release [183, 1841. Such a radioligand would provide important insight into brain serotoninergic function. Here, the precise characteristics required of the radioligand are not yet wholly clear. The preparation and evaluation of analogues of WAY, with perhaps modified receptor binding kinetics, might be useful to achieving the aim of detecting elevated serotonin levels in brain. Finally, there is still a need for an effective SPET radioligand.

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239

DWAY appears to have some advantages over WAY as a radioligand for PET but has not yet entered wide scale application. ['8F]trans-FCWAY is currently the most promising l8F-labe1led ligand.

ACKNOWLEDGEMENTS The authors are extremely grateful to the Human Frontier Science Programme Organisation for major support to this project (grant no RG0235/1998-B). The authors also thank the EC for support through COST B3, COST B12 and a COST STUDY (COST-STY-98-4022). The contributions of our many co-workers, especially Professor Lars Farde, Professor Paul Grasby, Dr Bengt Andree, Dr Eugenii Rabiner, Dr Johan Sandell, Dr Camilla Lundqvist, Dr Sandrine Marchais, Dr Julie McCarron, Dr Sue Hume, Dr Roger Gunn, Dr Carl-Gunnar Swahn, Ms Safiye Osman and Mr Bartek Nowicki, to cited publications and to material in this review, is gratefully appreciated. Dr Hikan Hall is especially thanked for provision of autoradiographic data, and Dr Sandrine Marchais for lipophilicity calculations and reading of the manuscript. Finally the authors are grateful to Dr Richard Carson (NIH) for kindly providing data on FCWAY.

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RADIOLIGANDS FOR THE STUDY O F BRAIN 5-HTIA RECEPTORS

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153 Le Bars, D., Ginovart, N., Lemaire, C., Plenevaux, A., Aerts, J., Pujol, J.F., Mekhsian, P., Weissmann, D., Leviel, V., Luxen, A. and Comar, D. (1997) J. Labelled Compd. Radiopharm. 40, 238-239 (Abstract). 154 Zhuang, Z.-P., Hurt, S.D., Stevenson, D.A., Kung, M.-P. and Kung, H.F. (1994) J. Labelled Compd. Radiopharm. 37, 297-298. (Abstract). 155 Kung, H.F., Stevenson, D.A., Zhuang, Z.P., Kung, M.R., Frederick, D. and Hurt, S.D. (1996) Synapse 23, 344-346. 156 Price, G.W., Ho, W., Scott, J.V., Selkirk, J.V. and Brown, A.M. (1998) Br. J. Pharmacol. 25, 55P. (Abstract). 157 Barton, C.L., Jay, M.T. and Hutson, P.H. (1998) Br. J. Pharmacol. 25, 55P. (Abstract). 158 Plenevaux, A., Aerts, J., Lemaire, C., Brihaye, C., Lebars, D., Mekhsian, P., Weissmann, D., Pujol, J.F., Comar, D. and Luxen, A. (1997) J. Labelled Compd. Radiopharm. 40, 148-150. (Abstract). 159 Lemaire, C., Cantineau, R., Guillaume, M., Plenevaux, A. and Christiaens, L. (1991) J. Nucl. Med. 32, 2266. 160 Le Bars, D., Ginovart, N., Hassoun, W., Weissmann, D., Lemaire, C., Plenevaux, A,, Aerts, J., Luxen, A., Pujol, J.F. and Leviel, V. (1999) J. Labelled Compd. Radiopharm. 42 (Suppl. I), S3953396. (Abstract). 161 Plenevaux, A., Lemaire, C., Salmon, E., Laureys, S., Fuchs, S., Maquet, P., Degueldre, C., Aerts, J., Brihaye, C., Damhaut, P., Biver, F., Goldman, S., Le Bars, D., Ginovart, N., Cinotti, L., Pujol, J.-F. and Luxen, A. (1999) J. Labelled Compd. Radiopharm. 42 (Suppl. I), S60-S62. (Abstract). 162 Passchier, J, van Waarde, A,, Pieterman, R.M., Elsinga, P.H., Pruim, J., Hendrikse, N.H., Willemsen. A.T.M. and Vaalburg, W. (2000) Nucl. Med. Biol. 27, 473476. 163 Lang, L., Jagoda, E. and Eckelman, W.C. (1999) J. Nucl. Med. 40, 37P. (Abstract 149). 164 Zhuang, Z.-P., Kung, M.-P. and Kung, H.F. (1994) J. Med. Chem. 37, 1406-1407. 165 Kung, H.F. (1995) PCT Int. Appl. WO 95/24218; Chem. Abstr. 124, 117345rn. 166 Kung, H.F., Kung, M.-P., Clarke, W., Maayani, S. and Zhuang, Z.-P (1994) Life Sci., 55, 1459-1 462. 167 Kung, M.-P., Frederick, D., Zhuang, Z.-P. and Kung, H.F. (1995) J. Exp. Pharmacol.. Exp. Ther. 272, 429-437. 168 Kung, M.-P., Zhuang, Z.-P., Frederick, D. and Kung, H.K. (1994) Synapse 18,359-366. 169 Kung, H.F., Frederick, D., Kim, H.-J., McElgin, W., Kung, M.-P., Mu, M., Mozley, P.D., Vessotskie, J.M., Stevenson, D.A., Kushner, S.A. and Zhaung, Z.-P. (1996) Synapse 24, 273-281. 170 Zhuang, Z.-P., Kung, M.P., Mu, M. and Kung, H.F. (1997) J. Labelled Compd. Radiopharm. 40,88-90. (Abstract). 171 Plossl, K, Zhuang, Z.-P., Meegalla S.K., Bradshaw, J., Frederick, D., Stevenson, D.A., Kung, M.-P. and Kung, H.F. (1995) J. Labelled Compd. Radiopharm. 37, 306308. (Abstract). 172 Papagiannopoulou, D., Mallo I., Papadopoulos, M., Pirmettis, T., Maina, B., Nock, A,, Leon, A. and Chiotellis, E. (1999) Eur. J. Nucl. Med. 989, 0 s 115. (Abstract). 173 Sargent, P.A., Rabiner, E.A., Kjaer, K.H., Messa, C., Cowen, P.J., Bench, C.J. and Grasby, P.M. (1999) J. Cereb. Blood Flow Metab. 19 (Suppl. l), S318 (Abstract). 174 Sargent, P.A., Kjaer, K.H., Bench, C.J., Rabiner, E.A., Messa, C., Meyer, J., Gunn, R.N., Grasby, P.M. and Cowen, P.J. (2000) Arch. Gen. Psychiatry, 57, 174-180. 175 Drevets, W.C., Frank, E, Price, J.C., Kupfer, D.J., Holt, D, Greer, P.J., Huang, H., Gautier, C. and Mathis, C. (1999) Biol. Psychiatry 46, 1375-1387.

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176 Drevets, W.C., Frank, E., Price, J.C., Kupfer, D.J., Greer, P.J. and Mathis. C. (2000) Nucl. Med. Biol. 27. 499-507. 177 Andree, B., Thorberg, S.O., Halldin, C. and Farde, L. (1999) Psychopharmacology, 144, 303-305. 178 Rabiner, E.A., Sargent, P.A., Gunn, R.N., Bench, C.J., Cowen, P.J. and Grasby, P.M. (1998). lnt. J. Neuropsychopharmacol. 1 (Suppl I), S65. 179 Rabiner, E.A., Gunn, R.N., Wilkins, M.R., Sargent, P.A., Mocaer, E., Sedman, E., Cowen, P.J. and Grasby, P.M. (2000) Nucl. Med. Biol. 27, 509-513. 180 Rabiner, E.A., Gunn, R.N., Castro, M.E., Sargent, P.A., Cowen, P.J., Koepp, M.J., Meyer, J.H., Bench, C.J., Harrison. P.J., Pazos, A., Sharp, T. and Grasby, P.M. (2000). Neuropsychopharmacology 23, 285-293. 181 Farde, L., Andree, B., Ginovdrt, N., Halldin. C. and Thorberg, S.-0. (2000) Neuropsychopharmacology, 22, 422-429. 182 Andree, B., Halldin, C.. Thorberg, S.-O.,Sandell, J. and Farde, L. (2000) Nucl. Med. Biol. 27, 515-521. 183 Volkow, N.D., Wang, G.-J., Fowler, J.S., Logan, J., Schlyer, D., Hitzemann, R., Lieberman, J., Angrist, B., Pappas, N., MacGregor, R., Burr, G., Cooper, T. and Wolf, A.P. (1994) Synapse 16, 255-262. 184 Koepp, M.J., Gunn, R.N., Lawrence, A.D., Cunningham, V.A., Dagher, A, Jones T., Brooks, D.J., Bench. C.J. and Grasby, P.M. (1998) Nature (London) 393, 266-268.

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Progress in Medicinal Chemistry - Vol. 38, Edited by F.D. King and A.W. Oxford 0 2001 Elsevier Science B.V. All rights reserved.

5 Discovery of Montelukast: a Once-a-Day Oral Antagonist of Leukotriene D4 for the Treatment of Chronic Asthma ROBERT N. YOUNG Merck Frosst Centre f o r Therapeutic Research, P. 0. Box 1005, Pointe Claire - Dorval, QuPbec, Canada H 9 R 4P8

INTRODUCTION

249

LEUKOTRIENE RECEPTORS

252

FIRST GENERATION LEUKOTRIENE D4 ANTAGONISTS In vitro assays In vivo assays

253 253 254

LEUOKOTRIENE ANALOGUES

255

ANALOGUES OF FPL-55712

256

SECOND GENERATION LEUKOTRIENE D4 ANTAGONISTS

259

REFERENCES

212

INTRODUCTION In the late 1930s, two Australian physiologists, Feldberg and Kellaway [l], described a substance released in perfused lungs of guinea pigs when treated with cobra venom. The perfusate was shown to cause a slow and sustained contraction of smooth muscle tissue (as opposed to histamine which causes 249

DISCOVERY OF MONTELUKAST

250

a rapid and reversible contraction). They proposed that the perfusate contained an unknown material for which they coined the term ‘slow reacting substance’ (or SRS). Two years later, Kellaway and Trethewie [2] reported that perfused lungs from sensitized guinea pigs, when challenged with the sensitizing antigen, released a similar substance which was given the name ‘slow reacting substance of anaphylaxis’ (SRS-A) [3]. Structural characterization of this substance (or substances) proved to be very difficult. However, studies suggested that SRS-A was produced from mast cells [4, 51, polymorphonuclear leukocytes (PMNs) [6] and later studies showed that human lung and nasal polyps were able to generate SRS-A [7]. Over the years, the general consensus grew that SRS-A may play an important role in the pathophysiology of human asthma but the very limited availability of biologically derived material and its instability severely hampered its study. A number of pharmaceutical companies were nonetheless stimulated to investigate SRS-A as a target for a novel asthma therapy. Researchers at the Merck Frosst Centre for Therapeutic Research used biological SRS-A to screen for lead structures in the 1970s. Needless to say, such assay development was heroic and allowed little throughput and difficult reproducibility. Also using biologically derived SRS-A and screening on guinea pig ileum, workers at Fisons in England discovered FPL-55712 (I), derived from their research on analogues of chromoglycate, that antagonized the smooth muscle contracting activity of naturally derived SRS-A [8]. This then represented the first described putative antagonist of SRS-A.

%0HO

Pr

0 - OH P

qj Pr

(1) FPL-55712

(2)‘Leukotriene C‘

COOH

ROBERT N. YOUNG

25 1

(3)'Leukotriene A'

In 1979, as the culmination of many years of research, Bengt Samuelsson of the Karolinska Institute proposed a structure for SRS-A. Samuelsson proposed the structure which he termed leukotriene C (2), as a peptido-lipid conjugate derived from the addition of cysteine or a cysteine-containing peptide to a tetraene epoxide (3) (which he termed leukotriene A) which itself was derived from oxidation of arachidonic acid [9, lo]. In the original proposal, the exact nature of the cysteine-containing amino acid or peptide was not definitively defined, nor was the absolute stereochemistry about the chiral centres in the molecule or the relative geometry of several of the double bonds in the triene chromophore. The complete structure assignment of SRS-A was determined via total synthesis and comparison of synthetic material with naturally derived SRS-A. As the result of a significant body of work in several laboratories around the world, SRS-A was shown to be a variable mixture of three substances designated as leukotriene C4, leukotriene D4 and leukotriene E4 (LTC4, LTD4 and LTE4), differing by the nature of the cysteine-containing peptide attached at C-6 of the molecule [l 11 (Figure 5.1). It was subsequently shown that LTC4 is formed in cells and transformed by a y-glutamyl transpeptidase to LTD4 which is, in turn, converted by a dipeptidase to LTE4. The availability of synthetic leukotrienes enabled scientists to study their actions in a variety of biochemical and physiological conditions. It was determined that LTD4 is the predominant component of SRS-A and exhibited potent contractile activity on smooth muscle [12, 131 and, thus, subsequent research was directed toward the study of the effects of LTD4. These studies indicated that LTD4 exhibited a variety of potent activities in the lung including induction of bronchoconstriction and changes in vascular permeability [14, 151. LTD4 has been shown to induce hyperreactivity of human lung tissue [16, 171, affect mucociliary clearance [IS, 191 and has been implicated in hyperplasia of smooth muscle in the lung [20]. These and other studies led to the strengthening of the hypothesis that a blocker of LTD4 activity might yield a novel and effective therapy for asthma.

DISCOVERY OF MONTELUKAST

252

Arachidonic acid

Leukotriene D4

hH -

S(S)-HPETE

Leukotriene E4

Figure 5.1. Biosynthesis of' cysteinylleukotriences.

LEUKOTRIENE RECEPTORS LTDQwas found to be an extremely potent spasmogenic mediator of respiratory smooth muscle, being up to 10,000 times more potent than histamine in eliciting contraction of human lung tissue [21]. In addition, studies on synthetic analogues and isomers of LTD4 indicated that this activity was highly stereospecific [22, 231 and that it could be inhibited by the putative SRS-A antagonist, FPL-55712 [8]. The development of synthetic routes to LTD4 allowed the preparation of ['H]-LTD4 with high specific activity [24,25]which then enabled the investigation of specific binding sites for [3H]-LTD4. These studies demonstrated specific binding to a variety of guinea pig and human tissues [26-301. Further studies demon-

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strated modulation of binding by GTP [31] and thus it was proposed that LTD4 exhibited much of its effects through interaction with a stereospecific G-protein coupled receptor (GPCR) transducing its signal via cyclic-AMP [32]. Other studies indicated a heterogeneity of leukotriene receptors, particularly in guinea pig lung [33], with some indication that there are separate receptors which recognized LTC4 and LTD4. Studies in human lung were complicated by the binding of LTC4 to a glutathione transferase [34] and its rapid conversion to leukotriene D4 by endogenous enzymes present in many preparations. The inhibition of this transformation by serine-borate complex [35] allowed the study of the true effects of LTC4 and LTD4 on a variety of tissues and subsequently it was shown that, unlike guinea pig, in human lung the majority of the effects of leukotrienes are mediated through the LTD4 receptor alone. This receptor has subsequently been termed the CysLT-1 receptor [36]. A second receptor (CysLT-2) [37] has been defined pharmacologically, but its significance in humans is not known. It should be noted that all drug discovery efforts between the elucidation of the structures of leukotrienes in 1979 and subsequent marketing of LTD4 receptor antagonists in the mid-to-late 1990s took place utilizing tissue binding assays to an otherwise uncharacterized receptor molecule. Only very recently have photoaffinity probes identified a putative 45 KD, glycosylated receptor [38,39] and molecular cloning of orphan receptors has finally identified the CysLT-1 receptor [40].

FIRST GENERATION LTD4 ANTAGONISTS With the availability of synthetic and radiolabelled LTD4, it became possible to set up higher throughput in vitro screening assays, evaluate the in vivo effects of LTD4 and show the effects of compounds. In our laboratories, a number of in vitro and in vivo assays were set up to support screening and development of leukotriene D4 antagonists. I N VITRO ASSAYS

Initial screening assays were developed, including [3H]-LTD4 binding to guinea pig lung membrane and inhibition of LTD4-induced contraction of guinea pig ileum. A compound inhibiting LTD4 binding and contractions was subsequently tested for specificity by assessing its ability to inhibit histamine-induced contractions of guinea pig ileum. Selective compounds

254

DISCOVERY OF MONTELUKAST

were confirmed to be of interest by testing their ability to inhibit [3H]-LTD4 binding to human lung membrane. Activities could be further quantified by determination of -logKB or pA2 values versus LTD4 on isolated guinea pig trachea or human tracheal tissue. Further specificity tests involved assays evaluating effects of compounds on contractions induced by a variety of other contractile mediators such as serotonin, acetylcholine and prostaglandin F2u.Finally, compounds were tested for their ability to inhibit antigen-induced contractions of tracheal tissues from sensitized guinea pigs or human tissue challenged with goat anti-human IGE antibody.

IN VIVO ASSAYS

A number of in vivo assays were developed or adopted to assay the ability of putative LTD4 antagonists to inhibit LTD4 activity or antigen-induced bronchoconstriction in vivo. The compounds deemed to be suitably potent and specific LTD4 antagonists in in vitro testing were evaluated for their ability to inhibit leukotriene D4-induced bronchoconstriction in animals. In anaesthetized, artifically ventilated guinea pigs, the compounds could be tested either by intravenous or intraduodenal administration. A second in vivo assay used in the earlier stage of the drug discovery process was based on a locally developed inbred colony of rats which had been shown to be hyperreactive to a variety of mediators. Thus, compounds were tested (usually by oral administration) for their ability to inhibit antigen-induced dyspnoea in hyperreactive rats that had been pretreated with methysergide [41]. However, this assay, proved to be capricious and it proved to be difficult to maintain a stable colony of rats. Thus, the assay was abandoned in later years of the discovery effort. Another in vivo assay that proved important in the discovery process was the testing of compounds for their ability to inhibit bronchoconstriction induced by aerosolized LTD4 or ascaris antigen in conscious squirrel monkeys [42]. Leukotrienes or antigen could be administered by aerosol through a specially-fitted mask and drugs could be administered either by aerosol, intravenously or by oral dosing. Testing against antigen-induced bronchoconstriction, however, was limited by the small number of ascaris sensitive squirrel monkeys available. Finally, the compounds were evaluated for their ability to inhibit either a LTD4- or antigen-induced early or late-phase bronchoconstriction in allergic sheep [43]. This model proved to be particularly useful for drugs administered by continuous infusion to maintain a definable blood drug concentration and to correlate such concentrations with efficacy.

ROBERT N.YOUNG

25 5

LEUKOTRIENE ANALOGUES Early efforts to design LTD4 antagonists were focused on compounds derived from structural analogues of LTD4 and from the putative receptor antagonist, FPL-57712 (I). Attempts to design LTD4 analogues by replacing the cysteinyl-glycine moiety with either simple mimetics, such as thioproprionic acid, or heterocyclic analogues such as 7-thiochromone-2-carboxylic acid, led to the discovery of moderately potent antagonists (e.g. compound 4) [44] that lacked suitable pharmaceutical properties, apparently due to the metabolic and chemical instability of the polyene chain. Replacements of the polyene chain with substituted aryl mimetics also led to moderately active compounds, but these compounds generally lacked the required pharmaceutical properties of good oral absorption and half-life suitable for further evaluation. Researchers from Smith Kline and French identified a leukotriene mimetic SK&F-I 02922 (5) [45] that was further refined to provide the orally active SK&F-’I04353 (6) [46] which subsequently entered clinical trials where it demonstrated efficacy [47] but was eventually dropped from development [48]. Researchers at Ciba Geigy pursued similar compounds including CGP-457 15A (iralukast) (7) [49] which entered clinical development but was also eventually dropped from development.

0

C ,-O ..O -,H

/

(5) SK&F-102922

DISCOVERY OF MONTELUKAST

256

(6) SK&F-104353

-

% oH J 0 F 3 c HO Pr

swcooH

(7) CGP-45715A (iralukast)

0

ANALOGUES OF FPL-55712 In previous studies, FPL-557 12 exhibited moderately potent LTD4 antagonist activity [50]but its usefulness was limited because it was orally inactive and displayed an extremely short half-life after intravenous administration [511. Attempts were made to circumvent these pharmacokinetic problems by administering FPL-55712 as an aerosol and the compound entered limited clinical trials where it showed some efficacy. In particular, FPL-557 12 prevented antigen-induced decreases in tracheal mucus velocity when aerosolized in ragweed-sensitive patients [52]. Aerosolized FPL-557 12 was also shown to abolish the cough response and bronchoconstriction induced by inhalation of LTD4 [53]. FPL-557 12 improved forced expiratory volume in one second (FEVJ in two out of four chronic asthmatic patients [541.

ROBERT N. YOUNG

257

Early screening efforts at Merck Frosst identified 4-(4-nonylphenyl)4-ketobutanoic acid (8), which showed significant activity against LTD4-induced contraction of guinea pig ileum. Considering structural similarities between this compound and FPL-557 12, it was suspected that the chromone ring moiety was not necessary for activity. It was thought that the chromone might also be the source of the metabolic instability of FPL-55712 and thus a series of analogues were prepared replacing the chromone ring with a phenylketobutanoic acid moiety. Structureactivity studies indicated that a thioether analogue (9) demonstrated oral absorption with a moderate half-life [ 5 5 ] . Metabolism studies indicated that the ketobutanoic acid moiety was rapidly metabolized in dogs and rats by reduction to the corresponding alcohol (10) and P-oxidation to the thiophenyl acetic acid (1 1) (Figure 5 . 2 ) . While both metabolites retained intrinsic biological activity, it was thought that if blockade of 0-oxidation

'reductase'

%0

HO

0-s (10)

Pr

'p-oxidation'

0-s

HO Pr

(11)

Figure 5.2. In vivo metabolism of arylketobutanoic acids.

DISCOVERY OF MONTELUKAST

258

could be achieved, a compound with a more uniform oral bioavailability and enhanced half-life could be derived. Eventually, such an analogue, L-649923 (12), was identified which showed superior activity when dosed by intravenous, intraduodenal or oral administration. L-649923 inhibited [3H]-LTD4 binding in guinea pig lung membrane with a Ki of 400nM. L-649923 showed intraduodenal and oral activity in guinea pig, rat and squirrel monkey at doses from 1-lOmg/kg. Notably, in the squirrel monkey, 1 hr pretreatment of a 5 mg/kg oral dose of L-649923, inhibited airway resistance and compliance changes induced by inhaled ascaris antigen [56]. An analogous compound, L-648051 (13), was identified which, despite showing poor oral bioavailability, appeared to exhibit superior properties on isolated tissues [57]. This compound was shown to effectively block airway resistance and dynamic compliance changes induced by aerosolized LTD4 in squirrel monkeys when dosed as a O.l'% nebulized solution. It was therefore decided that L-649923 would be developed as an orally active agent and L-648051 would be developed as an aerosol agent [58, 591. Subsequently, both compounds entered clinical trials. In clinical studies, L-649923 was shown to shift an LTD4-induced bronchoconstriction dose response curve 3.8-fold at a dose of 1000 mg [60]. At this dose, it had no effect on histamine-induced bronchoconstriction, thus indicating specificity. In an antigen challenge study [61], L-649923 as a single, 1000 mg oral dose had a marginal effect on early phase changes in FEV, and peak exploratory flow rate, and no effect on the late response. The small degree of protection provided by L-649923 in an antigen challenge model was not judged to be clinically relevant.

Pr

(12) L-649923

0

HO Pr

ROBERT N. YOUNG

259

Pr

(14) LY-171883

L-64805 1 was evaluated in three clinical studies as a powder aerosol given at doses up to 12 mg just before and during allergen challenge [62-641 where it showed either no effect or marginal effects that attained statistical, but not clinical, significance. Researchers at Eli Lilly and Co. also attempted to improve on the basic structure of FPL-55712. Their efforts yielded LY-171883 (14) in which the chromone ring was replaced by a tetrazole [65]. This compound showed potency and oral activity comparable to that observed for L-649923. It was also shown to block early- and late-phase responses to antigen in allergic sheep when dosed orally at 30 mg/kg, 2 h before challenge [66]. LY-171883 eventually entered clinical trials where it was shown to have a significant effect on LTD4-induced bronchoconstriction in normal patients [67] at oral doses up to 400 mg. In a second study at 400 mg, a non-significant effect was demonstrated for blockade of LTD4-induced fall in forced expiratory flow and a small but statistically significant reduction of the early-phase, but not late-phase, bronchoconstriction response to antigen [68]. In a third study, evaluated at 600 mg twice daily, the compound exhibited a small effect on cold air-induced bronchoconstriction in asthmatic patients [69]. In toxicology studies, LY-171883 showed indications of hepatotoxicity in rats [70] and mice [71]. Moreover, the compound was shown to induce peroxisomal enzyme activity in rats and mice and this activity was associated with increased incidence of hepatocellular carcinomas in carcinogenicity studies in mice [72]. The rather weak effects observed during clinical trials on FPL-55712, L-649923, L-648051 and LY-171883 led to a waning of enthusiasm in the pharmaceutical industry for LTD4 antagonists and to a questioning of the importance of LTD4 in the aetiology of asthma.

SECOND GENERATION LEUKOTRIENE D4 ANTAGONISTS In the Merck laboratories, the clinical results obtained with L-649923 and L-648051 was also met with disappointment. However, due to the relatively

DISCOVERY OF MONTELUKAST

260

small shifts in the dose response curve for LTD4 observed in humans, it was considered that the compounds were not really potent enough to properly test the hypothesis of the role of leukotrienes in asthma. Therefore, efforts were redoubled to find compounds with enhanced potency and, preferably, with pharmacokinetic properties to allow twice daily or once daily dosing. It must be noted that a substantial, parallel effort was underway to discover inhibitors of leukotriene biosynthesis. These efforts have recently been reviewed [73]. Based on the observed potency of the bis-acidic leukotriene analogue, SKF 102922 ( 5 ) [45], and considering the fact that LTD4 contains two acidic functionalities, efforts were made to incorporate a second acidic functionality into analogues of L-649923 [74]. Although these efforts yielded some compounds with increased potency, they generally lacked the desired pharmacokinetic properties and work in this area was eventually abandoned.

+ N’

(15)

(17) REV-5901

CI

(18) L-660711 (MK-0571)

(20)ICI-204219 (zafirlukast)

26 1

ROBERT N. YOUNG Table 5.1

R

R 3-Pyridyl (15 ) 4-Pyridyl 2-Pyridyl Phenyl 3-Hydroxyphenyl (16 ) 4-Hydroxyphenyl 2-H ydroxyphenyl 3 4 1 -Hydroxyhexyl)phenyl

[3HJ-LTD,binding ( G . P . ) ,IC,, W M

6 3.3 > 50

6 0.56 50 150

0.51

Meanwhile, an intensive screening effort was proceeding, evaluating compounds from the Merck sample collection in both the LTD4 binding assay and for their ability to inhibit LTD4-induced contraction of guinea pig ileum strips in organ culture. A lead structure identified from this effort was 2-(2-(3-pyridyl)ethenyl)quinoline (15). Not only did this compound have reasonable activity in the binding assay (ICso= 6.0 pM) but it also exhibited activity in the hyperreactive rat model when dosed orally, with an EDso of 3 mg/kg. A hypothetical model of the leukotriene D4 receptor had been previously derived [58] which postulated that the receptor recognized the flat 7c system of LTD4 as well as the 2 polar acidic chains. Noting that the styrylquinoline analogue (Table 5 . I ) had similar potency (ICso = 6 f2 pM), it was postulated that this extended conjugated system could interact with the LTD4-receptor in a manner similar to that postulated for LTD4. Therefore, studies were initiated to derive structure-activity relationships to determine where one might best add polar and acidic functionality in these molecules to further enhance the activity [75]. Evaluation of a series of phenolic analogues indicated that the 3-hydroxy analogue (16) had enhanced potency (ICso= 0.56pM) (Table 5. I ) . This preferred 3-substitution was subsequently supported by activity reported for the LTD4 receptor antagonist REV-5901 (RG-5901) (17) [76]. Notably, although (16) was 10 times more potent than the 3-pyridyl analogue (15) in vitro, it was less potent in vivo in the rat model. Analysis of blood samples of dosed rats indicated that (16) was rapidly converted to a glucuronide, thus explaining the lower oral activity. Structure-activity studies indicated that

262

DISCOVERY OF MONTELUKAST Table 5.2.

-

R'

R2

['H]-LTD4 binding ( G . P . ) ,IC,, p M

H 6-Hexenyl 6-Methyl H 5-Chloro 6-Chloro 7-Chloro 6,7-Dichloro 7-Chloro

OCHzCOOMe OCH2COOMe OCH2COOMe O(CH2)3COOH O(CH2)3COOH O(CH2)3COOH O(CH2)3COOH O(CH2)3COOH CH(SCH2CH$OOH)*

1.o 12% AT 1 p M 0.93 0.48 1.4 0.27 0.039 0.054 0.003

homologation of the phenolic function to incorporate a carboxylic acid moiety led to compounds with improved pharmacokinetic properties. Optimization of the lipophilic binding of the quinoline component of the molecule led to the discovery that a 7-halogen substituent enhanced activity dramatically (Table 5.2). A substantial boost in activity was observed when a second acidic functionality was incorporated into the molecule. This was achieved by incorporating two thiopropionic acid moieties via a dithioacetal in a manner similar to that which had been employed previously in SKF 102922 ( 5 ) [45]. However, compounds incorporating two acidic functions were found to exhibit sub-optimal oral activity, particularly in monkey models of LTD4-induced bronchoconstriction. This was considered probably to relate to a high degree of plasma protein binding. Transformation of one of the carboxylic acid functions to an amide led to compounds with further enhanced potency in v i m and in vivo. After careful study, the mono-dimethylamide (L-660711, MK-0571) (18) was identified. MK-057 1 not only exhibited sub-nanomolar potency for inhibition of LTD4 binding to guinea pig lung membrane (ICso= 0.8 nM), but it also showed superior potency in rat (EDSo= 0.07 mg/kg) and monkey models [77]. In squirrel monkeys, MK-0571 was shown to effectively block LTD4-induced bronchoconstriction 4 h following an orally administered dose of 0.1 mgikg. MK-057 1 also blocked bronchoconstriction induced by ascaris antigen in antigen-sensitive monkeys 2 h following oral administration of a 0.5 mg/kg dose. In addition, MK-0571 was shown to

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effectively block both early- and late-phase responses in ascaris-challenged allergic sheep when dosed intravenously by infusion at 8 pg/kg/min [78]. MK-0571 is a racemic compound and both enantiomers were found to be biologically active, with only small differences in their biological profiles [79]. The decision was made to develop the racemic compound (MK-057 1) and evaluate its efficacy in the treatment of human asthma. MK-0571 entered clinical studies in 1989. Initially, the compound was evaluated for its ability to antagonize aerosolized LTD4 when administered as a continuous intravenous infusion [80]. Results of these tests were gratifying in that infusions of either 28 mg or 277 mg resulted in shifts in the dose-response curve to aerosolized LTD4 of 3 44-fold and 3 84-fold, respectively. MK-057 I was further evaluated by the intravenous route of administration for its ability to block antigen-induced bronchoconstriction in asthmatic patients [81, 821. The infusion dose of 450 mg administered over 8 h elicited profound blockade of both early- and late-phase bronchoconstriction (88% and 63'!h3 respectively). Further studies [83] indicated a very effective block of exercise-induced asthma when a dose of 160 mg was administered by infusion (the decrease in lung function, as measured by FEV,, was inhibited by 69.5%). Most important for understanding the role of leukotrienes in asthma was the observation that intraveous infusions of MK-0571 produced bronchodilatation [84] in asthmatic patients. No such bronchodilatation was observed in non-asthmatic patients. These findings suggested that there was a continuous release of leukotrienes in certain asthmatic patients and suggested that LTD4 played an important role in the disease process. MK-0571 was subsequently studied in a 6 week study in mild-to-moderate asthmatic patients where it showed efficacy as measured by improvement in lung capacity (FEVI), beta-agonist usage and symptom scores [85]. In these studies, the compound was dosed orally at 150 mg three times daily. In the course of ongoing concurrent safety assessment studies, it was found that MK-0571 caused liver-weight changes in rats a t high doses. Subsequently, it was shown that these liver-weight changes were associated with hepatic peroxisome proliferation [86]. Considering the earlier experience with LY-I 71 883 that had shown a correlation with peroxisome proliferation, liver-weight changes in rodents and an increased incidence of hepatic tumours in safety assessment studies, the decision was made to halt the development of MK-0571. At this point, however, the enantiomers were re-examined. The biological profile of the two enantiomers were very similar. However, when they were evaluated for their ability to induce peroxisome proliferation, it was found that all of the peroxisome

264

DISCOVERY OF MONTELUKAST

proliferator activity was associated with the S-enantiomer, L-668018, and that the R-enantiomer, L-668019 (MK-0679, verlukast) (19) was free of such activity [86]. It was considered that such activity was likely mediated through interaction with a specific nuclear receptor (peroxisome proliferation activated receptor) (PPAR) [87]. An efficient stereospecific synthesis of verlukast was devised, utilizing an enzymatic resolution of the prochiral diester as the key step (Figure 5.3) [88].

1

HS---COOEt

BF3'EtpO

!

Pseudornonas lipase

Me2AINMe2

L-668018

1) HNMe2,DCI 2) NaOH, H+

( 19) L-668019; (MK-0679) (verlukast)

Figure 5.3.Synthesis of MK-0679 (verlukast)

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In 1990, verlukast entered clinical studies where it was shown to be well-tolerated and bioavailable [89]. Clinical studies demonstrated that verlukast and MK-057 1 were comparably potent LTD4 receptor antagonists in asthmatic patients [90]. Verlukast was shown to elicit significant bronchodilation in asthmatic patients which was additive with beta-agonists [91, 921. In chronic asthma studies, verlukast was shown to improve a number of parameters including FEVl measurements, symptom scores and beta-agonist inhaler use [93] when dosed orally in a twice daily regime (2x250 mg) over a 6-week period. Unfortunately, these results were countered by the observation of a small but meaningful incidence of liver enzyme elevation with long-term use of the drug. In keeping with our goal to develop a new therapy which was not only effective but would find an important role in the treatment of all classes of asthmatics, including pediatric asthma, the decision was made to suspend development of verlukast. It was notable that no such liver effects had been observed in long-term safety studies at very high doses in rats and monkeys and all efforts to ascertain the mechanism of this toxicity proved fruitless. Nonetheless, the studies with MK-0571 and verlukast and other results published on the unrelated antagonist, ICI-204219 (zafirlukast) (20) [94] had proven the important potential for LTD4 receptor (CYsLT1) blockade in the treatment of asthma. The goal, therefore, became to discover compounds free of the liver effects found with MK-0571 or verlukast, and preferably with greater potency and longer duration which would allow once-daily dosing. While verlukast was being evaluated in clinical studies, efforts were ongoing to identify possible backup structures with significant structural diversity. Particular efforts were made to remove the dithioacetal from the structure since this moiety was shown to be a site of oxidative metabolism for both MK-0571 and verlukast [95]. It was also considered desirable to replace the styryl double bond in these structures with another linking unit due to the observed photoisomerization of MK-0571 and verlukast. Replacement of the ethylene linkage in these thioethers with a variety of 2-atom linkages generally led to approximately 10-fold loss of activity (Table 5.3). Replacement of the dithioacetal by a thioether was shown to yield compounds with comparable intrinsic potency and incorporation of a branching alkyl group was shown to yield improved potency (Table 5.3). Further studies indicated that incorporation of an aromatic ring into the alkyl side-chain yielded compounds with potency comparable to that of MK-0571 on the CysLTl receptor [96]. Incorporation of an alkyl group into the thiopropionic acid chain yielded an enhancement in potency and,

266

DISCOVERY OF MONTELUKAST Table 5.3.

X

R’

R2

[ j H ] - L TDd binding (G.P.), ICs0 nM

CH20 CH2CH2 CH2S CH2O CH2O CH20

H H H H H Me

(CH2)2CHMeCH2CONMeZ SCH2CH2CONMe2 SCH2CH2CONMe2 SCH2CH2CONMe2 CH2CH2Ph-2-CONMe2 CH2CH2Ph-2-CONMe2(21)

8.6 31 15 14 0.95 0.52

finally, an optimized compound (21) was evaluated for its pharmacokinetic properties and oral activity. These studies indicated that the compound had a half-life substantially shorter than that of verlukast in the rat. Metabolic studies indicated that the amide moiety of compound (21) was transformed through hydrolysis and oxidative metabolism and therefore further efforts were initiated to replace the amide functionality. A careful study evaluating a variety of substituents for effects on intrinsic potency and clearance indicated that many polar and non-polar substituents were tolerated and the dimethylcarbinol moiety was found to have superior potency and improved clearance values (Table 5.4). The optimized compound (22) consisted of four diastereomers and these were prepared and evaluated (Table 5.5). As had been observed in the case of MK-0571, the intrinsically more potent isomers were associated with a propensity to induce peroxisomal enzymes in vivo in the mouse while neither of the non-inducing isomers had profiles superior to that of verlukast. It was therefore decided to re-evaluate the analogues of (22), which reincorporated the ethylene linking unit in place of the ether. An optimized analogue, L-695499 (23), and its isomer (24), were evaluated. Once again, peroxisomal enzyme-induction activity resided in the compound (24) bearing the R configuration of the benzylic center. The 5’-isomer (23) was free of peroxisomal enzyme-induction activity but exhibited some degree of toxicity in mice at high doses. Further optimization yielded a compound L-699392 (25),

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Table 5.4.

R'

R'

Clearance'"'

[ j H ] - L TD4 binding ( G . P . ) ,IC,, n M

H Et Et Et H Et Me Me Me

CONMe2 COOH CONHEt CONH2 H SOzMe NO2 CHMq C(OH)Me2 (22)

64 8.8 106 58 2.9 35 12 2.2 12

0.95 2.0 0.45 0.47 6.5 0.8 0.4 6.3 0.7

'"'ml/min/kg estimated from AUC from dosing rats at 5 mg/kg.

Table 5.5. Me-COOH

Isomer

FACO activity'a'

(S)SCH2 CH(R)MeCOOH (S)SCH2 CH(S)MeCOOH (R)SCH2 CH(R)MeCOOH (R)SCH2 CH(S)MeCOOH

+10%) +128%

-I'Xr

+155%

[-'H]-LTD4binding ( G . P . ) ,IC.5" n M

6 18 3 0.15

'aflncrease in fatty acyl Co-A oxidase (FACO) activity in mice after 4 days dosing at 400mg/kg p.0.

DISCOVERY OF MONTELUKAST

268

which appeared to have the best overall profile in the series [97]. However, compounds such as (24) were tantalizingly superior, both in vitro and in vivo. Studies were initiated to determine whether it was possible to modify the structure in this series so as to remove the peroxisomal enzyme induction activity while retaining the superior intrinsic and in vivo potency of the R-isomer series.

MeyCOOH

EtC,O , OH

CI

Et -COOH

CI

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CI

(25) L-699392

Published studies indicated that the induction of peroxisomal enzyme activity in rodents was mediated by PPAR [98, 991. It seemed probable that compounds in the leukotriene receptor antagonist series interacted with this receptor as well as the LTD4 receptor. It also seemed probable that the elements of receptor recognition for PPAR would not necessarily parallel those for the LTD4 receptor. Therefore, a variety of compounds were examined incorporating modified thioalkanoic acid moieties with particular emphasis on incorporation of steric bulk into this side-chain. Numerous analogues were made and evaluated, both for their LTD4 binding activity and for their effect on the induction of liver-weight increase and peroxisomal enzymes in mice. These studies indicated an important correlation between alkyl substitution on the side-chain and peroxisomal enzyme induction. While most of these compounds retained a high degree of intrinsic potency at the LTD4 receptor (Table 5.6), those with a-alkyl groups tended to show liver effects. However, P,P-dialkyl substituted analogues were generally free of such effects while retaining LTD4 antagonist potency. Optimization of in vivo properties was carried out on this j,P-dialkyl substituted series and a useful technique to assess the effects of plasma proteins on potency was developed as a guide to compound selection. Thus, addition of 0.05% human serum albumin or 1% pooled human plasma, or 1% squirrel monkey plasma to the competition binding assays allowed compound comparisons. This allowed selection of an optimized compound (26), incorporating a cyclopropyl unit into the beta position (Table 5.7) [IOO, 1011 which exhibited the best overall profile of potency, freedom from liver-weight changes and peroxisome proliferation and superior pharmacokinetics in a variety of animal species. This compound (L-70663 1, MK-0476, montelukast) (26) was selected for development in 1991. Subsequently, studies identified excellent backup candidates (27) [ 1021 and (28) [103], but these compounds were not developed further in light of the success of montelukast.

DISCOVERY OF MONTELUKAST

270

Table 5.6. CORRELATION OF CHAIN SUBSTITUTION AND PEROXISOMAL ENZYME INDUCTION

CI

LTD4 binding (G.P. lung)

GO (nM)

Thioether chain SR

(u-S)SCH,CHEtCOOH (B-R,S)SCHMeCH2COOH (B-R,S)SCHEtCH2COOH (B-S)SCHMeCH2COOH (B-R)SCHMeCH2COOH

0.1 0.8 0.6 0.3 0.4 (a-S,R)(B-S,R)SCHMeCHMeCOOH0.4 (a-R)(B-R)SCHMeCHMeCOOH 0.3 (a-s)(p-S)SCHMeCHMeCOOH 2.3 SCHMe2CH2COOH 0.7 (R, S)SCH2CHMeCH2COOH 0.4 SCH2CMe2CH2COOH 0.3 SCH2C(CH2CH2)CH2COOH (25) 0.5 'a)

Liver weight increase'a)

FACO activity'a'

+63% +5% +9% +11'% 12% +27% +10% 32% +3% +lo% +2% +0%

+608% +22% +inn% +24% +109% +673'%, +9 1% +393% - 10%

+ +

+I no/u +

+25% 16%

Average increase after dosing 8 mice at 400 mg/kg p.0. for 4 days,

Table 5.7. EFFECT OF PROTEIN ON THE K, VALUES OF MONTELUKAST AND MK-0571 FOR COMPETITION FOR BINDING OF [3H]-LTD4 ON G.P. LUNG MEMBRANE Ki (nMJ Compound

No additive

0.05% HSA

1% Human plasma

1% Squirrel monkey plasma

Montelukast MK-0571

0.18 f 0.03 0.57 f 0.23

0.21 f 0.08 9.2 f 2.1

0.19 f 0.01 1.9 f 0.6

0.26 f 0.02 1.8 0.42

(26)L-706631(MK-0476) (rnontelukast)

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Montelukast sodium entered clinical studies and was shown to be orally absorbed, well tolerated at doses from 2 to 800 mg and to have an excellent half-life of 4-5 h in man [104]. The compound was shown to be an effective LTD4 antagonist and to significantly block LTD4-induced bronchoconstriction. Thus, montelukast, orally administered as a 40 mg dose once daily for two days inhibited LTD4- induced bronchoconstriction 20-24 h after the last dose, causing shifts of 18- to >IOO-fold in the LTD4 bronchoconstriction dose response curve [105]. In other studies, under a similar paradigm, montelukast at 0.4-100 mg once daily dosing caused significant inhibition of exercise-induced bronchoconstriction [ 106, 1071. Other studies indicated efficacy in the treatment of chronic asthma [log, 1091. Dose ranging studies indicated that montelukast improved the signs and symptoms of asthma in chronic asthmatic patients with optimal effects observed at doses as low as 10 mg once daily [110, 1 1 11. No dose-limiting toxicity was observed in any of these dose response studies up to 200 mg once daily. Furthermore, because of the important need for new therapies to treat childhood asthma, a clinical development programme was undertaken to evaluate montelukast in children 6-14 years old. In important studies, pediatric patients receiving a 5 mg chewable montelukast tablet or placebo once daily at bedtime were compared. Patients receiving montelukast demonstrated significant improvements in FEV,, total daily ‘as needed’ beta agonist use, incidence of asthma exacerbations and improvement in asthma-specific quality of life [112]. Montelukast was also shown to provide inhibition of exercise-induced bronchoconstriction in pediatric patients when administered 5 mg once daily at bedtime [113]. Other Phase I11 clinical

272

DISCOVERY OF MONTELUKAST

efficacy studies in adults utilizing montelukast, a 10 mg dose administered once daily at bedtime, provided statistically and clinically meaningful benefits in a variety of parameters of asthma control including FEV1, daytime asthma symptoms, as needed inhaled beta-agonist usage and daytime and night-time asthma symptoms [114, 1151. Other studies demonstrated that with once daily treatment with montelukast, patients stably controlled with inhaled corticosteroids could be significantly tapered to reduce daily corticosteroid usage, on average by 46.7% (relative to 30.3% in patients treated with placebo) [116, 1171. The culmination of all these efforts was the filing in 1997 of a New Drug Application and Worldwide Marketing Application for montelukast sodium (Singulair@) for the treatment of both adult and pediatric asthma. In the summer of 1997, monteklast was first approved in Mexico and then a few months later, in Finland, more than 18 years after the start of this programme. The drug is now approved in 76 countries around the world and it and other leukotriene modifiers such as zafirlukast (Accolate@) are now recognized as the first important advance in asthma therapy in the last 25 years.

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62 Bel, E.H., T i m e r s , M.C., Dijkman, J.H., Stahl, E.G. and Sterk, P.J. (1990) J. Allergy Clin. Immunol. 85, 1067-1075. 63 Rasmussen, J.B., Eriksson, L.O. and Andersson, K.E. (1991) Allergy 46, 266-273. 64 Rasmussen, J.B., Eriksson, L.O., Tagari, P., Stahl, E.G. and Andersson, K.E. (1992) AIlergy 47, 604-609. 65 Marshall, W.S., Goodson, T., Cullinan, G.J., Swanson-Bean, D., Haisch, K.D., Rinkema, L.E. and Fleisch, J.H. (1987) J. Med. Chem. 30, 682-689. 66 Abraham, W.M., Wanner, A., Stevenson, J.S. and Chapman, G.A. (1986) Prostaglandins 31, 457467. 67 Phillips, G.D., Rafferty, P., Robinson, C. and Holgate, S.T. (1988) J. Pharm. Exp. Ther. 246, 732-738. 68 Fuller, R.W., Black, P.N. and Dollery, C.T. (1989) J. Allergy Clin. Immunol. 83,939-944. 69 Israel, E., Juniper, E.F., Callaghan, J.T., Mathur, P.N., Morris, M.M., Dowell, A.R., Enas, G.G., Hargreave, F.E. and Drazen, J.M. (1989) Am. Rev. Respir. Dis. 140, 1348-1353. 70 Hoover, D.M., Bendele, A.M., Hoffman, W.P., Foxworthy, P.S. and Eacho, P.I. (1990) Fundam. Appl. Toxicol. 14, 123-130. 71 Bendele, A.M., Hoover, D.M., van Lier, R.B., Foxworthy, P.S. and Eacho, P.I. (1990) Fundam. Appl. Toxicol. 15, 676-682. 72 Eacho, P.I., Foxworthy, P.S., Dillard, R.D. Whitesitt, C.A., Herron, D.K. and Marshall, W.S. (1989) Toxicol. Appl. Pharmacol. 100, 177-184. 73 Young, R.N. (1999) Eur. J. Med. Chem. 34, 671-685. 74 Young, R.N., Gauthier, J.Y. and Frenette, R. (1991) U.S. Patent 4990526. See EP206741A2; Chem. Abstr. 107, 58842~. 75 Zamboni, R., Belley, M., Champion, E., Charette, L., DeHaven, R., Frenette, R., Gauthier, J.Y., Jones, T.R., Leger, S., Masson, P., McFarlane, C.S., Metters, K., Pong, S.-S., Piechuta, H., Rokach, J., Therien, M., Williams, H.W.R. and Young, R.N. (1992) J. Med. Chem. 35, 3832-3844. 76 Van Inwegen, R.G., Khandwala, A,. Gordon, R., Sonnino, P., Coutts, S. and Jolly, S. (1987) J. Pharmacol. Exp. Ther. 241, 117-124. 77 Jones, T.R., Zamboni, R., Belley, M., Champion, E., Charette, L., Ford-Hutchinson, A.W., Frenette, R., Gauthier, J.Y., Leger, S., Masson, P., McFarlane, C.S., Piechuta, H., Rokach, J., Williams, H., Young, R.N., DeHaven, R.N. and Pong, S.S. (1989) Can. J. Physiol. Pharmacol. 67, 17-28. 78 Abraham, W.M. and Stevenson, J.F. (1988) FASEB J. 2, A1056, Abstract 4425. 79 Gauthier, J.Y., Jones, T., Champion, E., Charette, L., DeHaven, R., Ford-Hutchinson, A.W., Hoogsteen, K., Lord, A,, Masson, P., Piechuta, H., Pong, S.-S., Springer, J.P., Therien, M., Zamboni, R. and Young, R.N. (1990) J. Med. Chem. 33, 2841-2845. 80 Kips, J.C., Joos, G.F., DeLepeleire, I., Margolskee, D., Buntinx, A., Pauwels, R.A. and Van der Straeten, M.E. (1991) Am. Rev. Respir. Dis. 144, 617-21. 81 Hendeles, L., Davison, D., Blake, K., Harman, E., Cooper, R. and Margolskee, D. (1990) J. Allergy Clin. Immunol. 85, 197, Abstract 213. 82 Rasmussen, J.B., Eriksson, L.O., Margolskee, D., Tagari, P., Williams, V.C. and Andersson, K.E. (1992) J. Allergy Clin. Immunol. 90, 193-201. 83 Manning, P.J., Watson, R.M., Margolskee, D.J., Williams, V.C., Schwartz, J.I. and O’Byrne, P.M. (1990) N. Engl. J. Med. 323, 1736-1739. 84 Caddy, J.N., Margolskee, D.J., Bush, R.K., Williams, V.C. and Busse, W.W. (1992) Am. Rev. Respir. Dis. 146, 358-363.

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85 Margolskee, D., Bodman, S., Dockhorn, R., Isreal, E., Kemp, J., Mansmann, H., Minotti, D.A., Spector, S., Stricker, W., Tinkelman, D., Townley, R., Winder, J. and Williams, V. (1991) J. Allergy Clin. Immunol. 87, 309, Abstract 677. 86 Grossman, S.J., Delucca, J.G., Zamboni, R.J., Keenan, K.P., Patrick, D.H., Herold, E.G., VanZwieten, M.J. and Zacchei, A.G. (1992) Toxicol. Appl. Pharmacol. 116, 21 7-224. 87 Boie, Y., Adam, M., Rushmore, T.H. and Kennedy, B.P. (1993) J. Biol. Chem. 268, 5530-5534. 88 Bhupathy, M., Hughes, D.L., Amato, J.S., Bergan, J.J., Leazer, J.L., Lovelace, T.C., McNamara, J.M., Reamer, R.A., Sidler, D.R., Grabowski, E.J.J., Reider, P.J. and Shinkai, I. (1992) Pure Appl. Chem. 64, 1939-1944. 89 Schwartz, J.I., Margolskee, D.J., Bjornsson, T.D., Williams, V.C., Hsieh, J.Y., Lin, C.C. and Rogers, J.D. (1993) Br. J. Clin. Pharmacol. 36, 464-466. 90 Kips, J., Margolskee, D., Delepeleire, I., Joos, G., Williams, V., Buntinx, A. and Pauwels, R. (1991) Am. Rev. Respir. Dis. 143, A599. 91 Impens, N., Reiss, T.F., Teahan, J.A., Desmet, M., Rossing, T.H., Shingo, S., Zhang, J., Schandevyl, W., Verbesselt, R. and Dupont, A.G. (1993) Am. Rev. Respir. Dis. 147, 1442-1 446. 92 Lammers, J.W., VanDaele, P., Van den Elshout, F.M., Decramer, M., Buntinx, A., DeLepeleire, I. and Friedman, B. (1992) Pulmon. Pharmacol. 5, 121-125. 93 Margolskee, D., Friedman, B., Williams, V., Botto, A., Noonan, N., Altman, L., Bone, R., Dockhorn, R., Ellis, E., Eschenbacher, W., Findlay, S., Grant, J., Grossman, J., Hendles, L., Izrael, E., Kemp, J., Nelson, H., Pearlman, D., Stricker, W., Tinkelman, D., Townley R., Vandewalker, M., Wanderer, A,, Weisberg, G. and Winder, J. (1992) 8th Inter. Conf. Prostaglandins, Rel. Comps., July 26-31, Montreal, 163, Abstract 639. 94 For an extensive review, see Adkins, J.C. and Brogden, R.N. (1998) Drugs, 55, 121-144. 95 Nicoll-Griffith, D., Yergey, J., Trimble, L., Williams, H., Rasori, R. and Zamboni, R. (1992) Drug Metab. Dispos. 20, 383-389. 96 Labelle, M., Prasit, P., Belley, M., Blouin, M., Champion, E., Charette, L., DeLuca, J.G., Dufresne, C., Frenette, R., Gauthier, J.Y., Grimm, E., Grossman, S.J., Guay, D., Herold, E.G., Jones, T.R.,Lau, C.K., Leblanc, Y., Leger, S., Lord, A., McAuliffe, M., McFarlane, C., Masson, P., Metters, K.M., Ouimet, N., Patrick, D.H., Perrier, H., Pickett, C.B., Piechuta, H., Roy, P., Williams, H., Wang, Z., Xiang, Y.B., Zamboni, R.J., Ford-Hutchinson, A.W. and Young, R.N. (1992) Bioorg. Med. Chem. Lett. 2, 1141-1 146. 97 Labelle, M., Belley, M., Champion, E., Gordon, R., Hoogsteen, K., Jones, T.R., Leblanc, Y., Lord, A., McAuliffe, M., McFarlane, C., Masson, P., Metters, K.M., Nicoll-Griffith, D., Ouimet, N., Piechuta, H., Rochette, C., Sawyer, N., Xiang, Y.B., Yergey, J., Ford-Hutchinson, A.W., Pickett, C.B., Zamboni, R.J. and Young, R.N. (1994) Bioorg. Med. Chem. Lett. 4, 463468. 98 Green, S. (1992) Biochem. Pharmacol. 43, 393-401. 99 Zhu, Y., Alvares, K., Huang, Q., Rao, M.S. and Reddy, J.K. (1993) J. Biol. Chem. 268, 26817-26820. 100 Labelle, M., Belley, M., Gareau, Y., Gauthier, J.Y., Guay, D., Gordon, R., Grossman, S.G., Jones, T.R., Leblanc, Y., McAuliffe, M., McFarlane, C., Masson, P., Metters, K.M., Ouimet, N., Patrick, D.H., Piechuta, H., Rochette, C., Sawyer, N., Xiang, Y.B., Pickett, C.B., Ford-Hutchinson, A.W., Zamboni, R.J. and Young, R.N. (1995) Bioorg. Med. Chem. Lett. 5 , 283-288.

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101 Jones, T.R., Labelle, M., Belley, M., Champion, E., Charette, L., Evans, J., Ford-Hutchinson, A. W., Gauthier, J.Y., Lord-A,, Masson, P.. McAuliffe, M., McFarlane, C.S., Metters, K.M., Pickett, C., Piechuta, H., Rochette, C., Rodger, I.W., Sawyer, N., Young, R.N., Zamboni, R. and Abraham, W.M. (1995) Can. J. Physiol. Pharmacol. 73, 191-201. 102 Lau, C.K., Dufresne, C., Gareau, Y., Zamboni, R., Labelle, M., Young, R.N., Metters, K.M., Rochette, C., Sawyer, N., Slipetz, D.M., Charette, L., Jones, T., McAuliffe, M., McFarlane, C. and Ford-Hutchinson, A.W. (1995) Bioorg. Med. Chem. Lett. 5, 1615-1620. 103 Labelle, M., Gareau, Y., Dufresne, C., Lau, C.K., Belley, M., Jones, T.R., Leblanc, Y., McAuliffe, M., McFarlane, C.S., Metters, K.M., Ouimet, N., Perrier, H., Rochette, C., Sawyer, N., Slipetz, D., Xiang, Y.B., Wang, Z., Pickett, C.B., Ford-Hutchinson, A.W., Young, R.N. and Zamboni, R.J. (1995) Bioorg. Med. Chem. Lett. 2551-2556. 104 Schoors, D.F., DeSmet, M., Reiss, T., Margolskee, D., Cheng, H., Larson, P., Amin, R. and Somers, G . (1995) Br. J. Clin. Pharmacol. 40, 277-280. 105 DeLepeleire, I., Reiss, T.F., Rochette, F., Botto, A,, Zhang, J., Kundu, S. and Decramer, M. (1997) Clin. Pharmacol. Ther. 61, 83-92. 106 Reiss, T.F., Hill, J.B., Harman, E., Zhang, J., Tanaka, W.K., Bronsky, E., Guerreiro, D. and Hendeles, L. (1997) Thorax 52, 1030-1035. 107 Bronsky, E.A., Kemp, J.P., Zhang, J., Guerreiro, D. and Reiss, T.F. (1997) Clin. Pharmacol. Ther. 62, 556-561. 108 Reiss, T.F., Altman, L.C., Chervinsky, P., Bewtra, A,, Stricker, W.E., Noonan, G.P., Kundu, S. and Zhang, J. (1996) J. Allergy Clin. Immunol. 98. 528-534. 109 Reiss, T.F., Sorkness, C.A., Stricker, W., Botto, A., Busse, W.W., Kundu, S. and Zhang, J. (1997) Thorax, 52, 45-58. 110 Altman, L.C., Munk, Z., Seltzer, J., Noonan, N., Shingo, S., Zhang, J. and Reiss, T.F. (1998) J. Allergy Clin. Immunol. 102, 50-56. 1 11 Noonan, M.J., Chervinsky, P., Brandon, M., Zhang, J., Kundu, S., McBurney, J. and Reiss, T.F. (1998) Eur. Resp. J. 11, 1232-1239. 112 Knorr, B.A., Matz, J., Bernstein, J., Becker, A., Reiss, T.F., Friedman, S., Erickson, U. and Nguyen, H.H. (1997) Am. J. Respir. Crit. Care Med., 155, A664. 113 Kemp, J.P., Dockhorn, R.J., Shapiro, G.G., Nguyen, H.H., Reiss, T.F., Seidenberg, B.C. and Knorr, B. (1998) J. Pediatr. 133, 424-428. 114 Reiss, T.F., Chervinsky, P., Edwards, T., Dockhorn, R., Nayak, A,, Hess, J., Zhang, J. and Shingo, S. (1996) Eur. Resp. J. 9 (23), 273S, Abstract 1734. 115 Reiss, T.F., Chervinsky, P., Edwards, T., Dockhorn, R., Nayak, A,, Hess, J., Zhang, J. and Shingo, S. (1997) Am. J. Respir. Crit. Care Med. 155, A2662. 116 Kundu, S., Noonan, N., Friedman, B.S., Reiss, T.F. and Leff, J.A. (1997) Am. J. Respir. Crit. Care Med. 155, A352. 117 Leff, J.A., Israel, E., Noonan, M.J., Finn, A.F., Godard, P., Lofdahl, C.G., Friedman. B.S., Connors, L., Weinland, D.E., Reiss, T.F. and Kundu, S. (1997) Am. J. Respir. Crit. Care Med. 155. A976.

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Progress in Medicinal Chemistry - Vol. 38, Edited by F.D. King and A.W. Oxford 2001 Elsevier Science B.V. All rights reserved

c)

6 The Histamine Ligands

H3

Receptor and its

HOLGER STARK', JEAN-MICHEL ARRANG2, XAVIER LIGNEAU', MONIQUE GARBARG', C. ROBIN GANELLIN4, JEAN-CHARLES SCHWARTZ2 and WALTER SCHUNACK'* I Freie Universitut Berlin, Institut f u r Phurmazie, Konigin-Luise-Strasse 24-4, 14195 Berlin, Germany. Unit6 de Neurobiologie et Pharmacologie Mol6culaire ( U . 109), Centre Paul Broca de I'INSERM, 2 ter rue d'Albia, 75014 Paris, France. Laboratoire Bioprojet, 30 rue des Francs-Bourgeois, 75013 Paris, France. Department of Chemistry, Christopher Ingold Laboratories, University College London, 20 Gordon Street, London W C I H OAJ, U.K.

INTRODUCTION

280

TRANSDUCTION

280

RESPONSES

28 I

BIOLOGICAL ASSESSMENT O F H3-RECEPTOR LIGANDS

283

LIGANDS Aminergic agonists Partial agonists Antagonists Imidaiole-containing compounds Non-imiriuzole compounds Radioligands

284 284 289 290 290 296 291

POTENTIAL CLINICAL APPLICATIONS OF H3-RECEPTOR LIGANDS 299

REFERENCES

* Correspondence to be addressed to

Professor Schunack. 279

280

THE HISTAMINE H3 RECEPTOR AND ITS LIGANDS

INTRODUCTION Ironically, the detection of the histamine H3 receptor was first performed with burimamide (1) [l], an Hz/H3-receptor antagonist formerly used for the first differentiation between histamine H I and H2 receptors [2] (for review see [3]). In 1983Arrang et al. showed that the H3 receptor, in contrast to the previously described H I and H2 receptors, is located presynaptically and acts as an autoreceptor inhibiting histamine synthesis from L-histidine and histamine release from vesicles in histaminergic neurons [ 11. Later, the existence of H3 receptors was proven with selective agonists and antagonists using classical pharmacological analysis [4].The recent cloning of human [ 5 ] and rodent [6,7] histamine H3 receptors and the identification of two splice variants [6] has brought fresh impetus into this research field. In the meantime, many pharmacological auto- and heteroreceptor functions have been demonstrated in characterizing the H3 receptor in the central nervous system and in peripheral tissues with a number of structurally different ligands (for reviews see [3, 8-13]). Unlike H1 and H2-receptor antagonists, however, which are widely used for the therapy of allergic and acid related diseases, no selective ligands for H3 receptors so far have been introduced for therapeutic use, although some promising candidates have reached clinical phases and are currently under further development.*

TRANSDUCTION The H3 receptor, like other histamine receptors, belongs to the superfamily of heptahelical receptors coupled to G proteins. Its coupling to effector systems via a G protein was first indicated by studies of [3H]-labelled H3-receptor agonist binding and shown to be regulated by guanine nucleotides [1416] (Figure 6.I ) . Moreover, a direct G protein activation by H3-receptor agonists was evidenced by measurement of [35S]-GTPyS binding in rat cerebral cortical membranes. The sensitivity of this activation to pertussis toxin strongly suggests that H3 receptors mainly couple to GiIG, proteins in mammalian brain [17, 181. *The following abbreviations are used in this article: ADHD, Attention-deficit hyperactivity disorder; HMT, Histamine N-methyltransferase; HVOR, Horizontal vestibular-ocular reflex; 5-HT, 5-Hydroxytryptamine, serotonin; Ia, Intrinsic activity; PET, Positron emission tomography; SPECT, Single photon emission computed tomography.

HOLGER STARK ET AL.

28 1

[ L-Histidine nerve ending

e--; Histamine

I

j

-+--: G protein

Figure 6.1. Schematic histamine neurone uctivity (ACh, acetylcholine; 5-HT, serotonin; D A , dopamine; N A , noradrenalin; IPj,inositol frisphosphate; DG, diucylclycerol: C A M P , cyclic adenosine monophosphate)

The main effector system coupled to H3 receptors is not clear. Native H3 receptors in human gastric [19] or mouse pituitary (Morisset et al., unpublished) cell lines are negatively coupled to phosphoinositide turnover. The Ca2+ dependence of the presynaptic inhibition mediated by H3 autoreceptors also suggested a restrictrion of extracellular Ca2+ influx [20]. Such restriction of Ca2+ access into the nerve terminal, associated with H3-receptor activation, was shown to involve N-type Ca2+ channels in rat tissues [21, 221 and guinea-pig duodenum [23]. Like other Gi-protein coupled receptors the human H3 receptor exposed in a variety of transfected cell lines (e.g. rat C6 glioma, human embryonic kidney 293, human SIC-N-MC neuroblastoma) inhibited forskolin-stimulated CAMPformation in response to histamine [24].

RESPONSES Since its discovery as an inhibitory autoreceptor in the central histaminergic neuronal pathways [ l , 251, the H3 receptor has been found to negatively modulate transmitter release from peripheral cholinergic, adrenergic and non-adrenergic non-cholinergic (NANC) nerve endings [I 3, 261. In the brain, functional studies have also provided evidence for inhibitory H3 receptors on nerve terminals of noradrenergic [27-301, serotonergic [3 11,

282

THE HISTAMINE H1 RECEPTOR AND ITS LIGANDS

dopaminergic [32, 331, and peptidergic [34-371 neurons (Figure 6.1). In rat entorhinal cortex, acetylcholine release is also modulated by H3 receptors [38], but the latter are presumably not located on cholinergic axon terminals [39] (Figure 6. 1).Whereas H3 autoreceptors seem tonically activated in vivo under basal conditions [8, 401, HAheteroreceptors may be activated only under some particular physiological conditions, since H3 antagonists generally do not facilitate the release of the corresponding transmitter. Whether or not this response is associated with splice variants with long or short forms of the H3 receptor still remains unclear [6]. Blockade of H3 autoreceptors enhances histamine turnover [8] and release in vivo, as shown by microdialysis [41,42], and thioperamide (2), a reference H3-receptor antagonist, has been widely used to further clarify or unravel neurochemical and behavioural effects of endogenous histamine [40]. For example, the role previously suggested of histaminergic neurons in arousal has been further established with H3-receptor ligands [43, 441.

The improvement by thioperamide and other H3-receptor antagonists of attention, learning and memory suggests a role for endogenous histamine in cognition [4549]. In the hypothalamus, thioperamide induces c-fos expression through endogenous histamine release within paraventricular and supraoptic nuclei [50].Endogenous histamine release may be involved in the secretory responses of prolactin, corticotrophin and a-endorphin

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283

induced by stress or endotoxin, since this effect is largely prevented by (R)-a-methylhistamine (6) (Table 6.1), a reference agonist [51, 521. Suppression of food intake by H3-receptor antagonists is also likely to be due to endogenous histamine release from neurons projecting to the paraventricular and ventromedial hypothalamic nuclei [53-551. The involvement of central histaminergic neurons in the inhibition of seizures is supported by the anticonvulsant effects of H3-receptor antagonists [56-591. Another beneficial effect of H3-receptor antagonists is suggested for vertigo. Systemic administration of thioperamide in the guinea-pig demonstrated its significant vestibuloplegic effect, since it decreased the horizontal vestibular-ocular reflex (HVOR) gain [60]. Betahistine (3), a mixed H,-receptor agonist/H3-receptor antagonist of low to moderate affinity [6 11, currently used for the symptomatic treatment of vestibular-related syndromes (Meniere’s disease), has been shown to depress the HVOR gain [62] and to facilitate vestibular compensation in the cat [63] and guinea-pig [64].

BIOLOGICAL ASSESSMENT OF H3-RECEPTOR LIGANDS The functional assay which allowed the discovery of H3 receptors is an autoreceptor system which involves evaluating the inhibition of [3H]-histamine synthesis or release from Kf-depolarized brain slices from rat [l, 41 or other species including man [65]. Labelling of the endogenous pool by using the [3H]-precursor is mandatory. The test was modified thereafter using electrical stimulation [66] or, for convenient screening purposes, synaptosomes [67]. H3 heteroreceptor systems in the brain, i.e. on noradrenergic [27], serotonergic [311 and cholinergic [38] neurons were also used. In the case of cholinergic neurons, the response seems too complicated for use in compound screening [39]. The inhibition of the twitch response of guinea-pig ileum in the presence of mepyramine (H,-receptor antagonist) reflects the presence of presynaptic H3 receptors on cholinergic neurons [68,69]. The apparent affinity of ligands can be determined by competition with the various radioligands, but with these assays it is not easy to distinguish between agonists of different intrinsic activities (ia) and antagonists, particulary in view of the complex modulatory actions of guanyl nucleotides [ 14, 161. This situation has changed dramatically since the availability of cloned H3 receptors of different species, which also permit a functional determination.

284

THE HISTAMINE H3 RECEPTOR AND ITS LIGANDS

A re-evaluation of most H3-receptor ligands seems necessary as very recently it has been shown that many of them display partial agonist or inverse agonist properties [70-721. Ligands may be assessed in vivo through evaluation of their effects in [3H]-histamine synthesis [4]or W-methylhistamine levels in brain [73]. Despite the fact that other factors can also influence the N-methylhistamine level [74, 751 this is a robust and reliable in vivo screening system. Ex vivo binding competition [76] or a radio-receptor assay may assist in assessing bioavailability, pharmacokinetics and brain penetration of H3-receptor ligands.

LIGANDS AMINERGIC AGONISTS

The endogenous ligand histamine (4)showed higher affinity for H3 receptors than for other histamine receptor subtypes (histamine pD2 values H I , 6.7; H2, 6.0; H3, 7.2). This observation was the starting point for a large number of structural variations on this molecule [77]. Alkylation of histamine at the imidazole nucleus led to a dramatic loss in affinity [l 11. Replacement of the imidazole ring by other heterocycles also resulted in compounds without agonist activity [ 111. Only side-chain alkylation of the ethanamine moiety is acceptable for maintaining H3-receptor activity (Table 6.1) [3]. Alkylation with longer space-filling alkyl groups in the N"-position led to antagonists [78]. A pyrrolidino moiety like the W-group led to a partial agonist (18) [4],whereas monomethylation gave an agonist with increased affinity, i.e. N*-methylhistamine (5) (Table 6.1) [l]. Thus enhancement of the van der Waals volume of the W-substituent over methyl diminishes agonist properties and increases antagonist activity. W-Methylhistamine is about three times more potent than histamine at H3 receptors. Unfortunately, the affinity to other histamine receptor subtypes is still high [79]. In this respect, it is of interest that Helicobacter pylori is able to produce N"-methylhistamine (5) in the gut [80, 811. The compound showing an improved selectivity is (R)-a-methylhistamine (6) [4]. It possesses much lower affinity for H I and H2 receptors and high affinity for H3 receptors (pD2 8.2). In addition, the low affinity of the (9-distomer shows that H3 receptors discriminate between enantiomers even though histamine itself is not chiral. Furthermore, this proves the usefulness of (R)-cr-methylhistamine as a pharmacological tool and a

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285

Table 6.1. RELATIVE POTENCIES OF HISTAMINE H3-RECEPTOR AGONISTS. Compound

(4)

HI

(TNH2 N

100

H2

100

H3

100

H

(rN'Me H

(5)

12

74

270

1

1550

E

0.5 H

286

THE HISTAMINE H3 RECEPTOR A N D ITS LIGANDS

reference agonist. The chiral dimethylated derivative (Ra,SP)-a,B-dimethylhistamine (7) is slightly more potent than (R)-a-methylhistamine (Table 6.1) and shows even higher selectivity (ratio 130,000:1, H3 versus Hl/H2 potency) and striking stereoselective discrimination properties [82]. All these methylated histamine derivatives are highly polar and strongly basic compounds capable of penetrating the blood-brain barrier only to a limited extent. These ligands are also found to be substrates for histamine N-methyltransferase (HMT, E.C. 2.1.1.8), the major histamine-catabolizing enzyme in man [83]. Therefore these agonists are likely to enter the brain only in very low concentrations and to have a short half-life and poor bioavailability in man (Figure 6. I ) . Novel azomethine prodrugs were designed in which (R)-amethylhistamine ( 6 ) is bioreversibly linked to 2-hydroxybenzophenone or related derivatives in order to prevent recognition by HMT [84-881. These prodrugs containing a hydrolyzable azomethine double bond are characterized by reduced basicity and increased lipophilicity leading to improved bioavailability (Figure 6.2) [88, 891. The phenolic hydroxyl group stabilizes the Schiff s base by an intramolecular hydrogen bond as shown by X-ray investigations [84, 881. The penetration through the blood-brain barrier strongly depends on the substitution pattern and the aryl structure (Figure 6.2). Furthermore, these prodrugs are no longer substrates for the inactivating enzyme thereby leading to a prolonged duration of action in man [85]. Depending on the structure of the pro-moiety it is possible to realize drug targetting into central or peripheral tissues (Figure 6.2). The lead structure of these prodrugs is BP 2.94 (9) (FUB 94). The utility of this prodrug approach has been demonstrated by testing BP 2.94 (9)

Figure 6.2. Prodrug concepl f o r histumine Hj-receptor agonists

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287

in man after oral administration [85]; BP 2.94 is currently under clinical investigation in a Phase I1 trial [90]. Whereas BP 2.94 (9) acts mainly in the periphery [84-861, derivatization into the related dichloro derivative (1 0) led to high concentration of (6) in the central nervous system [84, 871. The pyrrolo derivative (1 1) possesses a prolonged drug release leading to a retarded prodrug acting over a long period [88]. Consideration of the likely conformations of (R)-a-methylhistamine and a$-dimethylhistamine led to the synthesis and testing of conformationally constrained H3-receptor agonists. The 2-methylpyrrolidin-3-yl analogue immepyr (12) (which has 2R,3S chirality) was shown to be an active H3-receptor agonist (pD2 7.1) [91]. In this compound the side-chain amino and p-methyl groups of a,/?-dimethylhistamine have been incorporated together into a 5-membered ring to give a secondary amine. It has been suggested that the special attribute of immepyr is its very high selectivity in vivo (H3:H1>> 550) as demonstrated in the guinea-pig (inhibition of sympathetic hypertensive responses versus augmentation of neural cholinergic bronchospasm) [91]. However, it is not clear, whether immepyr is a full agonist and a substrate for HMT. The analogous compound Sch 50971 (13), which has 3R,4R chirality, shows comparable activity (pD2 7.5) [92-941.

A homologous ring structure, immepip (14) (pD2 8.0), in which the amino group is incorporated into a 6-membered ring to give a piperidine derivative has been found to be as potent as (R)-a-methylhistamine ( 6 ) [95]. It is a full agonist on the guinea-pig jejunum. Immepip is not a chiral compound and the secondary amino group is at a distance of 4-methylene groups from the imidazole ring. The pyrrolidino analogue (i.e. the secondary amino is in

THE HISTAMINE H3 RECEPTOR A N D ITS LIGANDS

288

a 5-membered ring) is suggested to be a partial agonist (pD2 7.3, ia 0.8) [96]. These are intriguing compounds. The corresponding open chain analogues [n= 3 (15) or 4 (16)] (Table 6.2) were originally described as antagonists [97] (see also impentamine (17) n = 5), but they were subsequently shown to act also as partial agonists, i.e. depending on the test system [98]. Even oxygen-containing heterocyclic compounds like the tetrahydrofuran derivative imifuramine (1 8) possess agonist H3-receptor activity [99]. The tolerance of the H3 receptor to modifications of the histamine side-chain and the initial observation [11 that some H2-receptor ligands display high affinity at H3 receptors led several laboratories to identify imetit (S-[2-(~idazol-4-yl)ghyl]jso~hiourea) (19) as a highly potent and selective H3 agonist [67, 100-1021. Imetit (19) is a full agonist and is 4 times more potent than (R)-a-methylhistamine (6) and approximately 60 times more potent than histamine (4) itself. In imetit, the separation between the ring and the charged side-chain N atoms is a 4-atom chain as in immepip, but this direct comparison is too simplistic. The sulfur atom in imetit is not critically important and may be replaced by 0 or CH2. Indeed, the isourea (X = 0) (20) is equipotent [I031 with imetit and the carboxamidine (X=CH2) (21) is slightly more potent (Table 6.2) [101, 1031. When the structural similarities of these different histamine H3-receptor agonists were taken together and fitted to the imidazole nucleus and the basic moiety in the side-chain it was shown that all these compounds have a single common pharmacophore, which led to a pseudoreceptor model [ 104-1 061.

Table 6.2. HISTAMINE HOMOLOGUES AND ANALOGUES AS H3-RECEPTOR AGONISTS.

NPNH2

4N 1 H

(4) (15) (16) (17)

n

pD2

ia

2

1.2 6.1 8.5

1 0.3 0.6 0.3

3 4 5

8.2

(19)

s

(20) (17)

0

CH2

2.8 2.7 1.2

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PARTIAL AGONISTS

Compounds like impentamine (1 7) [97] (Table 6.2) and the pyrrolidine derivative (8) [4] are related to agonists like histamine (4) or, in the case of the sulfonamide derivative (22) [ 1071, like (R)-cc-methylhistamine (6), and their partial agonist activity is not particularly surprising. Despite the similarity of these aminergic agonists, partial agonism was also found in some test models for the ether derivative iodoproxyfan (23) [ 1081 suggesting that binding behaviour is more complex than shown before.

0

(N HT O A ! % (26)

Me

H

(27a) R = H (27b) R = M e

Investigation of the structural demands for these compounds on agonist properties showed that the basic aminergic moiety is not essential. In this respect some rn-substituted 4-(w-(phenoxy)alkyl)-1H-imidazoles show striking in vitro and in vivo behaviour regarding partial or full agonism [ 109, 1101. Trifluproxim (UCL 1470) (24) is one example of this compound class showing partial agonism in vitro (ia 0.4) and full agonism in vivo (EDs0 0.6 mg/kg, ia 1.0) [110]. Furthermore, novel developments of leads in the carbamate FUB 316 (25) (in vitro ia 0.2) [72], FUB 475 (26) (in vitro ia 0.15) and the ether series FUB 373 (27a) (in vitro ia 0.22), FUB 407 (27b) (in vitro ia 0.55) [70] resulted in compounds with partial activity in vitro and for some compounds like FUB 373 (27a) (ED50 0.51 mg/kg, ia 1 .O) and FUB

290

THE HISTAMINE

H3

RECEPTOR A N D ITS LIGANDS

407 (27b) (EDSo0.29 mg/kg, ia 1.0) full agonist activity in vivo after oral administration [70]. The position and the volume of substituents with high steric demands seem critical in these series for inducing a change in receptor conformation necessary for activation as slight variations of the methyl group in aliphatic ethers [70] or aryl substitution on diphenylmethyl ethers resulted in high affinity antagonists [72]. ANTAGONISTS

Imidazole-containing compounds The first compounds used for the characterization of histamine H3 receptors were selected from the pool of H I - and H2-receptor ligands. H2-receptor agonist impromidine and H2-receptor antagonist burimamide (1) (-logKi 7.2) had the same stimulating effect on the release of histamine from histaminergic neurons, thus acting as H3-receptor antagonists by inhibiting the negative feed back mechanism caused by histamine’s receptor activation [ 11. Histamine H3-receptor antagonist activities are given in the figures based on the use of comparable test systems. Thioperamide (2) (-logKi 8.4), one of the reference drugs, was the first potent and selective H3-receptor antagonist [4]. The imidazolylpiperidine moiety has been a useful starting point for many derivatives wherein the thiourea-containing pharmacophore of thioperamide (2) has been varied. Carboperamide (28) (-log& 7.7) [ l l 11, G T 2016 (29) (-logKi 7.4) [112], AQ 0145 (30) (-1ogKi 7.4) [113-1151, a benzothiazole (31) (-1ogKi 6.6) [116], and UCL 1283 (32) (-log& 7.4) [117] are examples of successful substitutions of the piperidine nitrogen with different

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29 1

variations of polar functionality and lipophilic moiety. However, the rigid piperidino moiety is not an essential element of H3-receptor antagonists. Amide [ 1181 and sulfonamide [107, 119, 1201 derivatives of histamine or of higher homologues of histamine are also moderate to potent antagonists. Recently, activators of carbonic anhydrase enzymes of related sulfonamide or urea structures have been described without mentioning any H3-receptor potency [ 121, 1221. Structure-activity patterns for analogues of burimamide have also been described [123]. Even lipophilic groups can be omitted as shown partly by impentamine (1 7), the homologue of histamine containing a pentamethylene chain [97]. Knowledge of these and other antagonists led to the development of a general construction pattern [lo]: a heterocyclic ring is connected by a chain to a polar group which may be connected by another chain to a lipophilic moiety [ll]. Whereas the first part seems to be crucial for H3-receptor activity, the last one can be omitted although it is generally affinity enhancing. Most attempts to substitute or replace the imidazole nucleus have led to compounds with drastically reduced affinity for H3 receptors

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THE HISTAMINE H3 RECEPTOR AND ITS LIGANDS

[117, 124-1271. Despite this uniformity in the imidazole heterocycle a large number of variations can be made in the polar moiety with retention of antagonist affinity. In particular, highly hydrophilic functionalities like guanidine FUB 136 (33) (-log& 9.1) [128], amidine (34) (-logKi 8.2) [129, 1301, isothiourea (clobenpropit, (35), -logKi 9.3) [12, 1001, urea [131], carbamate FUB 120 (36) (-logKi 8.0) [70, 132-1351, sulfonamide [107], sulfamide (37) (-logKi 8.6) [I 191, esters [136], ketones [136], imidazole [137], and imidazoline [ 1381 mostly provide potent antagonists. Even silyl-containing derivatives have been described [ 1391. A recent investigation on recombinant cells showed that clobenpropit (35) showed comparable affinities for histamine H3 receptors of rat and man whereas thioperamide (2) was surprisingly tenfold less active at human than at rat H3 receptors [71. NH

0

NH

Many guanidines and isothioureas have been described, and their affinity can be varied by structural variations of the lipophilic moieties. One of the most potent ligands in these series is the isothiourea derivative clobenpropit (35) with subnanomolar in vitro activity [loo]. Unfortunately, these polar compounds display some pharmacokinetic disadvantages. They cross biological membranes only to a small extent and consequently have poor brain 26 mg/kg) [ 1321. Therefore penetration after peroral application (ED50 in vivo activity is lower than that attained by thioperamide (2) (ED50 N

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1.0 mg/kg) although the latter shows lower in vitro affinity than clobenpropit [ 1321. Lipophilicity is an important factor for in vivo activity. Compounds optimized for in vivo activity often do not have high affinity in vitro, but they might be highly selective and potent compounds in vivo. The polar group in H3-receptor antagonists may also be a heterocycle. A series of histamine analogues belonging to the general construction pattern have been studied [ 1 16, 1 171 where the polar group is NH or S directly connected to the lipophilic moiety which can be various nitrogen containing heterocycles. Examples are the nitropyridine UCL 1199 (38) (-logKi 8.3) [117] and the benzothiazole (39) (-logKi 8.0) [116]. The oxadiazole derivatives FUB 217 (40) (-logKi 7.6) [I081 and GR 175737 (41) (-logKi 8.2) [1401 are related heterocyclic compounds in which the oxadiazole element

seems to mimic an alternative structure for the polar functionality. In another development, replacement of the heterocycle or the first spacer by phenyl has furnished a series of potent antagonists, some of which are quite active in vivo, especially when the polar group changed to an ether functionality (see above) [141]. Carbamates, for example, FUB 120 (36) (-logKi 8.0) [132, 1421 and ether derivatives (42-48) [108, 141-1431 are antagonists showing relatively high affinity in vitro, high potency in vivo and frequently high selectivity for histamine H3 receptors. The selectivity of these novel classes of histamine H3-receptor antagonists is of special importance because thioperamide (2), clobenpropit (39, iodophenpropit (49), and other structurally related antagonists have been reported to possess high affinity for 5-HT3 receptors

294

THE HISTAMINE H3 RECEPTOR AND ITS LIGANDS

[ 1441. Whereas the diphenylmethyl derivative FUB 322 (42) (-logKi 7.6) possesses a combined H1-/H3-receptorantagonism [142], the other compounds in that series show low or negligible affinity to related neurotransmitter receptors tested. UCL 1390 (44) (-logKi 7.9) was the first compound in this series reported and shows high in vivo potency (EDSo0.53 mg/kg) [141]. Further optimization or variation led to FUB 181 (43) (-logKi 7.9) [49, 1451 or compounds derived from the endogeneous precursor L-histidine for example, (45) (-logKi 7.3) [146, 1471. Compounds with high in vivo activity additional to their high affinity and selectivity are the cyclopropyl ketone ciproxifan (46) (-logKi 9.3) [ 148-1 501 and the related oxime derivative imoproxifan (47) (-logKi 9.6) [151]. Ciproxifan was one of the first compounds reported to possess an EDso value below 1 mg/kg

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29 5

with oral administration to mice (EDSo0.14 mg/kg) [148]. Imoproxifan and a related o-fluorinated derivative are orally active even below 0.1 mg/kg (ED50 0.034 mg/kg and 0.032 mg/kg, respectively) [151]. Due to their selectivity and extraordinary potency, these compounds are among the most useful tools for investigations on H3 receptors. The high lipophilicity of the ether derivatives containing an imidazolylpropoxy moiety, the proxifan series, is even surpassed by compounds containing a purely hydrocarbon substituent on the imidazole ring. Whereas the double bond of GT-2227 (50) (-logKi 8.4) [147] or the triple bond of GT-233 1 ( 5 1) (-logKi 9.9) (PerceptinTM)[ 152-1 541 may be regarded as a polar group according to the general construction pattern of H3-receptor antagonists, these elements are totally missing in compounds (48) (-logKi 6.4) and (52) (-logKi 7.1) [155]. This change of the paradigm

in H3-receptor antagonist structures was firstly described in 1998 [ 1551 and later confirmed by another group [156]. GT-2331 (51) (EDs0 0.08 mg/kg i.p.) [153] is at the moment one of the most interesting antagonists because this compound was reported to be under development in Phase 11 clinical trial for the indication of attention-deficit hyperactivity disorder (ADHD) [157, 1581.

296

THE HISTAMINE H j RECEPTOR AND ITS LIGANDS

Non-imidazole compounds Marketed compounds such as betahistine (3), used for the treatment of vestibular disturbances like Menikre's disease [ 1591, the typical neuroleptic haloperidol [160], the atypical neuroleptic clozapine (53) [161-1631 and the P-receptor antagonist sabeluzole (54) [ 1641 show low to moderate H3-receptor antagonist potency and lack strong structural similarities to H3-receptor antagonists described above, as they are devoid of the imidazole nucleus. Subsequent development [165] of these leads led to compound (56) of moderate potency [166,167]. Compounds with higher affinity were developed by replacement of imidazole with piperidino or pyrrolidino moieties as shown with (55) and (57) [168-1711. A common feature of these compounds is that the side-chain on the nonaromatic containing N-heterocycle is closely related to former highly potent antagonists in the imidazole series (cf. (44),

(55)

(33), respectively). Similar replacement of the imidazole moiety can be performed in some other antagonist series maintaining H3-receptor affinity [172]. Despite some speculations, up to now a final conclusion for the reason

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of this striking case of restricted bioisosterism could not be given. Studies with point-mutated H3 receptors may provide further evidence for the kind of ligand-receptor interactions of these non-imidazole compounds. The therapeutic use of compounds like betahistine (3) [ 1731 and clozapine (53), or their observed side-effects, may be taken as reliable hints for the potential therapeutic value of antagonists for histamine H3 receptors.

RADIOLIGANDS

As H3-receptor agonists, tritium-labelled commercially available [3H](R)-a-methylhistamine [cf. (6)) and [3H]-Na-methylhistamine (cf. (5)] are commonly used as radioligands. Despite the widespread use of the N"-methylated histamine derivative, which seems to be related to its higher specific radioactivity, it should be emphasized that the (R)-a-methylated derivative is the agonist radioligand of choice because of its higher H3-receptor affinity and selectivity.

In the antagonist field initial experiments were performed with moderate success on the S-[3H]-methylated thioperamide derivative (cf. (2)) [ 1741 and also with S-[' 'C]-methylated derivatives [ 1751.A tritium-labelled antag10) [176]. Compound (58) is a onist is [3H]-GR168320 (58) (-log&, guanidino analogue of thioperamide (2) and displays relatively high non-specific binding. The antagonists mostly used are ['251]-iodophenpropit (49) (-logKi 9.0) [177, 1781 and ['251]-iodoproxyfan (23) (-logKi 9.7) [179, 1801. Both compounds display high affinity binding for H3 receptors in which (49) also displays some affinity for other receptors, especially 5-HT3 receptors [178, 1811, whereas (23) shows high selectivity and low nonspecific binding, but partial agonism in some test models [108]. Attempts to conduct PET or SPECT studies on radioligands from various classes of H3 antagonists so far have met with limited success 1182-1871. Further approaches are currently under investigation.

298

THE HISTAMINE

H3

RECEPTOR AND ITS LIGANDS

POTENTIAL CLINICAL APPLlCATIONS OF H3-RECEPTOR LIGANDS In contrast to the widely used HI- and H2-receptor ligands no H3-receptor ligand has so far been approved for regular therapeutic use. Clinical observations with agents displaying nonselective activity at H3 receptors together with preclinical data obtained with selective H3-receptor agonists or antagonists might both be taken into account to predict applications of the latter agents [188, 1891. H3-receptor agonists inhibit the release of proinflammatory tachykinins and calcitonin-gene-related peptide (CGRP) from sensory C-fibres in a large variety of tissues, indirectly depress mast cell activity, and display ‘peripheral’ antinociceptive activity [34, 35, 86, 190, 1911. From these actions at the level of the airways [26, 1921, potential applications in asthma can be envisaged. In animal models of migraine consisting of electrical stimulation of the trigeminal nerve or capsaicin administration, plasma protein extravasation within the meninges is reduced by H3-receptor agonist administration to the same extent as with administration of sumatriptan, a 5-HT1-receptor agonist of established antimigraine activity [34, 193, 1941. Initial experiments have already been performed on migraine patients using a low S.C. dose of histamine to activate H3 receptors [195]. The antiinflammatory activity of H3-receptor agonists has been demonstrated in a large variety of tissues associated with antinociceptive effects and on some tests mostly connected to sensory C-fibres [90, 1961 showing a gastric mucosal protective effect [ 197-1991. This constitutes a promising pattern for a novel class of anti-inflammatory agents. Taken these effects together with the observed significant decrease of gastric acid secretion it may also be a new approach for the therapy of gastric oesophageal reflux disease. All these properties are demonstrated in a single agent, BP 2.94, which poorly enters the brain thus reducing the risk of side-effects, displays excellent oral bioavailability and has a long half-life in humans [86]. Ongoing Phase I1 clinical trials with BP 2.94 will allow assessment of these various hypotheses. For brain-penetrating H3-receptor agonists, ‘sedative’ properties in humans can be predicted from their actions on monoaminergic (including histaminergic) neuron activity as well as behavioural changes in cats and rats [13, 40, 43, 441. Due to their influence on cardiac H3 receptors and on noradrenaline release, agonists may offer a novel approach to myocardial ischemia [194, 200, 2011.

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The most sustained behavioural effects of H3-receptor antagonists, apparently occurring through the enhancement of endogenous histamine, of arousal [43, 441, improved learning ability in aged rodents [45] and improvement in attentional tasks [148], all suggest a potential utility in the treatment of mental disorders, such as the treatment of Alzheimer’s disease symptomatology [202, 2031. This conviction is reinforced by at least two sets of observations: tuberomammillary neurons seem relatively spared in Alzheimer patients [204] and tacrine is more active at inhibiting histamine than acetylcholine degradation in mouse brain in vivo [205]. Another related application for younger patients is ADHD for which GT-233 1 is currently in Phase I1 clinical trials [158]. The indication of obesity is still a matter of debate [206]. The presence of H3 receptors in vestibular nuclei or the modulation of dynamic vestibular functions in thioperamide-treated guinea-pigs suggest the utility of H3-receptor antagonists as antivertigo or antimotion-sickness drugs devoid of sedating properties [60, 2071. The anticonvulsant activity of a variety of H3-receptor antagonists on a mouse model suggests their utility in epilepsia [56, 57, 208, 2091. Finally, the increased level of histamine metabolites in the cerebrospinal fluid of schizophrenic patients [210], the dense distribution of H3 receptors in limbic brain areas possibly involved in schizophrenia and the significant H3-receptor antagonist potency of clozapine, an atypical antipsychotic compound with a poorly defined mechanism of action, all suggest that selective H3 receptor antagonists may represent a novel class of antipsychotic or antidepressive agents [74, 21 I]. We hope that such therapeutic applications now anticipated to occur during the next few years will at the same time clarify new aspects of physiological and pathophysiological effects of histamine neuron activity.

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178 Leurs, R., Tulp, M.T.M., Menge, W.M.P.B., Adolfs, M.J.P., Zuiderfeld, O.P. and Timmerman, H. (1995) Br. J. Pharmacol. 116, 2315-2321. 179 Ligneau, X., Garbarg, M., Vizuette, M.L., Diaz, J., Purand, K., Stark, H., Schunack, W. and Schwartz, J.-C. (1994) J. Pharmacol. Exp. Ther. 271, 452459. 180 Stark, H., Purand, K., Huh, A., Ligneau, X., Garbarg, M., Schwartz, J.-C. and Schunack, W. (1996) J. Med. Chem. 39, 1220-1226. 181 Allen, M. (1998) Eur. J. Pharmacol. 361, 261-268. 182 Ponchat, M., Demphel, S., Fuseau, C., Coulomb, C., Bottlaender, M., Schwartz, J.-C., Stark, H., Schunack, W., Athmani, S., Ganellin, C.R. and Crouzel, C. (1997) 12th International Symposium on Radiopharmaceutical Chemistry, Uppsala, Sweden, Proceedings pp. 605-608. 183 Windhorst, A.D., Timmerman, H., Klok, R.P., Custers, F.G.J., Menge, W.M.P.B., Leurs, R., Stark, H., Schunack, W., Gielen, E.G.J., van Kronenburgh, M.J.P.G. and Herscheid, J.D.M. (1999) Nucl. Med. Biol. 26, 651-659. 184 Windhorst, A.D., Timmerman, H., Menge, W.M.P.B., Leurs, R. and Herscheid, J.D.M. (1999) J. Labelled Comp. Radiopharm. 42, 293-307. 185 Windhorst, A.D., Timmerman, H., Klok, R.P., Menge, W.M.P.B., Leurs, R. and Herscheid, J.D.M. (1999) Bioorg. Med. Chem. 7, 1761-1767. 186 Airaksinen, A., Vahatalo, J.K., Kovalainen, J.T., Kuikka, J., Christiaans, J.A.M., Kauppinen, T., Lotjonen, S., Vepsalainen, J., Hiltunen, J., Gynther, J., Tuomisto, L. and Bergstrom, K.A. (1999) J. Labelled Comp. Radiopharm. 42 (Suppl. I), S403S405. 187 Iwata, R., Hovath, Yanai, K., Pascali, C., Kovacs, G.Z. and Ido, T. (1999) J. Labelled Comp. Radiopham. 42 (Suppl. I), S3874388. 188 Leurs, R.. Blandina, P., Tedford, C. and Timmerman, H. (1998) Trends Pharmacol. Sci. 19, 177-183. 189 Onodera, K. and Miyazaki, S. (1999) Nippon Yakurigaku Zasshi 114, 89-106. 190 Dimitriadou, V., Rouleau, A,, Trung Tuong, M.D., Newlands, G.F.J., Miller, H.R.P., Luffau, G., Schwartz, J.-C. and Garbarg, M. (1994) Clin. Sci. 87, 151-163. 191 Imamura, M., Smith, N.C.E., Garbarg, M. and Levi, R. (1996) Circ. Res. 78, 863-869. 192 Ichinose, M. Belvesi, M.G. and Barnes, P.J. (1990) J. Appl. Physiol. 68, 21-25. 193 Buzzi, M.G., Dimitriadou, V., Theoharides, T.C. and Moskowitz, M.A. (1992) Brain Res. 583. 137-149. 194 Malinowska, B., Godlewski, G. and Schlicker, E. (1998) J. Physiol. Pharmacol. 49, 191-21 1. 195 Guerrero, R.O.M., Cardenas, M.A.I., Ocampo, A.A. and Pacheco, M.F. (1 999) Headache 39, 576-580. 196 Seyedi, N., Maruyama, R. and Levi, R. (1999) J. Pharmacol. Exp. Ther. 290, 656-663. 197 Bertaccini, G. and Coruzzi, G. (1995) Dig. Dis. Sci. 40, 2052-2063. 198 Belcheva, A,, Marazova, K., Lozeva, V. and Schunack, W. (1997) Inflamm. Res. 46 (SUPPI. l), S113-Sl14. 199 Morini, G., Grandi, D., Krause, M. and Schunack, W. (1997) Inflamm. Res. 46 (Suppl. I), SI 0 I-s 102. 200 Gothert, M., Garbarg, M., Hey, J.A., Schlicker, E., Schwartz, J.-C. and Levi, R. (1995) Can. J. Physiol. Pharmacol. 73, 558-564. 201 Levi, R. and Smith, N.C.E. (2000) J. Pharmacol. Exp. Ther. 292, 825-830. 202 Panula, P., Rinne, J., Kuokkanen, K., Eriksson, K.S., Sallmen, T., Kalimo, H. and Relja, M. (1998) Neuroscience, 82, 993-997.

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203 Goivannini, M.G., Bartolini, L., Bacciottini, L., Greco, L. and Blandina, P. (1999) Behav. Brain Res. 104, 147-155. 204 Airaksinen, M.S., Paetau, A,, Paljarvi, L., Reinikainen, K., Riekkinen, P., Suomalainen R. and Panula, P. (1991) Neuroscience, 44, 465431. 205 Morisset, S., Traiffort, E. and Schwartz, J.-C. (1996) Eur. J. Pharmacol. 315, RILR2. 206 Itoh, E., Fujimiya, M. and Inui, A. (1999) Biol. Psychiatry 45, 475481. 207 O’Neill, A.B., Pan, J.-B., Sullivan, J.P. and Brioni, J.D. (1999) Meth. Find. Exp. Clin. Pharmacol. 21, 285-289. 208 Fischer, W. and van der Goot, H. (1998) J. Neural. Transm. 105, 587-599. 209 Kakinoki, H., Ishizawa, K., Fukunaga, M., Fuji, Y.and Kamei, C. (1998) Brain Res. Bull. 46, 461465. 210 Prell, G.D., Green, J.P., Kaufmann, C.A., Khandelwal, J.K., Morrishow, A.M., Kirch, D.G., Linnoila, M. and Wyatt, R.J. (1995) Schizophrenia Res. 14, 93-104. 21 1 Pkrez-Garcia, C., Morales, L., Cano, M.V., Sancho, I. and Alguacil, L.F. (1999) Psychopharmacology 142, 21 5-220.

Progress in Medicinal Chemistry - Vol. 38, Edited by F.D. King and A.W. Oxford Published by Elsevier Science B.V.

7 Isothermal Titration Calorimetry in Drug Discovery WALTER H.J. WARD and GEOFFREY A. HOLDGATE AstraZeneca, R & D Mereside, Alderley Park, Macclesfield, Cheshire, SKlO 4TG, U.K.

SUMMARY

310

INTRODUCTION

31 1

EXPERIMENTAL MEASUREMENT O F BINDING THERMODYNAMICS Parameters used to characterize thermodynamics of binding Description of an isothermal titration calorimeter Collection and analysis of experimental data Range and uncertainty in thermodynamic parameters Characterization of compounds from medicinal chemistry Comparison with other methods to measure affinity Indirect estimation of AHo without ITC van't Hoff analysis

314 314 317 317 320 32 1 323 325

CHARACTERIZATION O F TARGET PROTEINS AND TEST COMPOUNDS The significance of enthalpy-driven versus entropy-driven binding Enthalpy-entropy compensation decreases changes in affinity Solvation changes are important in the thermodynamics of binding Changed interactions are more likely to effect AH' than A G Assessment of protein preparations Evaluation of assays Characterization of protein constructs Following enzyme-catalysed reactions without the need for model substrates Measurement of critical micelle concentration and partitioning into lipids Identification and characterization of target proteins Elucidation of the intermolecular complex which gives biological activity Further characterization of active compounds Thermodynamic discrimination by receptors

327 327 328 328 329 332 333 333 334 335 335 336 337 338

MOLECULAR RECOGNITION AND LIGAND DESIGN Interpretation of binding thermodynamics Starting to understand affinity

339 339 340

~

309

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ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

Affinity, enthalpy, entropy and interaction type Hydrophobic interactions Hydrogen bonds and ion-pairs Water molecules localized at protein-ligand interfaces Changes in protonation upon binding Cooperative binding Energetic contributions from non-interfacial residues Context-dependent influences upon binding Global versus local effects on energetics of association Insight into biomolecular interactions from studies of protein folding Structure-based thermodynamic design Further examples of ITC in molecular recognition and ligand design Design of ligands for streptavidin Hydrophobic interactions in a cavity engineered info lysozyme Recognition of S-peptide hy ribonuclease S-protein

342 342 343 345 349 351 351 352 353 354 356 358 358 359 359

STRUCTURAL AND THERMODYNAMIC STUDIES TO HELP OPTIMIZATION OF DNA GYRASE INHIBITORS ITC shows small inactive fragments are valid models for intact gyrase Structural and thermodynamic basis of resistance to novobiocin Characteristics of inhibition by triazines A large change in AH'' correlates with a different binding mode for triazines

360 360 362 363 366

FUTURE PROSPECTS

370

REFERENCES

370

SUMMARY Isothermal titration calorimetry (ITC) follows the heat change when a test compound binds to a target protein. It allows precise measurement of affinity. The method is direct, making interpretation facile, because there is no requirement for competing molecules. Titration in the presence of other ligands rapidly provides information on the mechanism of action of the test compound, identifying the intermolecular complexes that are relevant for structure-based design. Calorimetry allows measurement of stoichiometry and so evaluation of the proportion of the sample that is functional. ITC can characterize protein fragments and catalytically inactive mutant enzymes. It is the only technique which directly measures the enthalpy of binding (AH"). Interpretation of AHo and its temperature dependence (ACp) is usually qualitative, not quantitative. This is because of complicated contributions from linked equilibria and a single change in structure giving modification of several physicochemical properties.

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31 1

Measured AH" values allow characterization of proton movement linked to the association of protein and ligand, giving information on the ionization of groups involved in binding. Biochemical systems characteristically exhibit enthalpy-entropy compensation where increased bonding is offset by an entropic penalty, reducing the magnitude of change in affinity. This also causes a lack of correlation between the free energy of binding (AGO) and AH". When characterizing structure-activity relationships (SAR), most groups involved in binding can be detected as contributing to AH", but not to affinity. Large enthalpy changes may reflect a modified binding mode, or protein conformation changes. Thus, AH" values may highlight a potential discontinuity in SAR, so that experimental structural data are likely to be particularly valuable in molecular design.*

INTRODUCTION Isothermal titration calorimetry (ITC) is a uniquely powerful tool for characterization of the thermodynamics of test compounds binding to target proteins. It helps to improve understanding of biomolecular recognition which assists in the invention of improved compounds. In ITC, a solution of a test compound usually is injected into a sample of target protein. Interaction between the compound and protein leads to release, or uptake, of tiny amounts of heat (z5 pcal in a modern instrument) whilst the mixture is held at a close approximation to constant temperature. Monitoring of the relationship between dose of test compound and magnitude of heat change allows direct calculation of affinity (which * The following abbreviations and symbols have been used in this review: 1 A = 0.1 nm; I cal = 4.184 J; ADPNP, adenylyl-B-7-imidodiphosphate;B, crystallographic temperature factor; c,

slope factor for thermogram; cmc, critical micelle concentration; AAap, change in apolar accessible surface area; AAp, change in polar accessible surface area; ACp, change in heat capacity (specific heat); AG ', standard Gibbs free energy change; AGE, coupling free energy; AH', standard enthalpy change; AH",,1, standard calorimetric enthalpy change; AH"i, enthalpy of ionization; AH",b,, observed enthalpy change; AHUvh, standard van't Hoff enthalpy change; A S , standard entropy change; DSC, differential scanning calorimetry; ICso, concentration giving 50% inhibition; FKBP, FKS06 binding protein; ITC, isothermal titration calorimetry; K,, equilibrium association constant; k,,,, catalytic constant (mole product formed/mole enzymeis); Kd. equilibrium dissociation constant; K,, Michaelis constant; n, stoichiometry (mole ligand/mole protein); np, number of protons released; NMR, nuclear magnetic resonance; pK,, -log K,; R, Gas Constant (1.987 cal/K/mol); SAR, structure-activity relationship; SE, standard error; T, absolute temperature.

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ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

may be expressed as equilibrium dissociation constant, Kd), standard enthalpy change (AHO) and stoichiometry (n). Kd corresponds to the free ligand concentration when 50% of the binding sites are occupied. ITC requires no specialized reagents and the same detection method is used for all target proteins, Accordingly, the time required for assay development is minimal which is a valuable attribute in the pharmaceutical industry. There have been many informative reviews on the thermodynamics of biomolecular interactions and ITC [ 1-81. Here, we focus on its application in drug discovery. The technique has a number of useful characteristics. Measurements are performed in solution, requiring no immobilization of either partner, nor any chemical modification of the reactants. The direct nature of ITC means that interpretation of measured affinities is relatively straightforward, when compared to enzyme kinetics and displacement assays which are complicated by the effects of substrates, intermediates, products or reporter ligands. Direct detection also helps to identify the partners in the molecular complex with the target protein. For example, ITC allows determination of whether prior binding of substrate is required before association with an inhibitor. This information helps to ensure that relevant 3-D structures are used in molecular design. Kinetic equivalence occurs in competition assays involving interactions between macromolecules, since binding of the compound to either partner produces exactly the same dose-response relationship. This characteristic can confuse the interpretation of data on protein kinases, proteolytic enzymes, regulatory proteins, transcription factors, signalling pathways and protein phosphatases. ITC avoids kinetic equivalence because it is possible to monitor binding to each partner individually. There is no requirement for small model compounds which are often used as (perhaps misleading) models for one of the macromolecular partners. ITC data have a high degree of precision (between experiments, Kd values generally agree within a factor of two). The stoichiometry of interaction reflects the purity and functional integrity of the protein preparation. This is useful for evaluating engineered protein fragments and protocols for purification and storage. The ability of ITC to monitor binding directly means that it can characterize protein fragments which do not have native biochemical activity. This is particularly useful when recombinant DNA technology is unable to generate sufficient quantities of complete proteins (such as transmembrane or multisubunit proteins). ITC can be used to assess the validity of using engineered protein fragments, fusion proteins or tagged proteins in assays

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and structural studies to support molecular design. The technique has similar utility in assessing whether target protein function becomes artefactual following addition of sequences to aid purification such as fusion proteins (e.g. glutathione transferase or maltose binding protein) or tags (e.g. hexahistidine, biotin or the c-myc antigen). ITC measures a similar range of affinities to other assay methods (AAG" = 4 kcal/mol), but the range of observed enthalpy changes is much larger (AAH" = 25 kcal/mol). When modifying the structures of interacting molecules, increased bonding (favourable enthalpy change, AH") tends to be offset by increased order (unfavourable entropy change, AS"), leading to only small changes in AG", Kd or IC50. Such enthalpy-entropy compensation appears to be widespread in aqueous systems around physiological temperatures. In the absence of 3-D structural data, it is difficult to determine whether a change in structure that has little effect on AGO does so because the modified group does not interact with the target protein, or because it interacts equally well with solvent. ITC is unique in being able to investigate this question by directly measuring the contribution of AH" to AGO and thereby estimating TAS". Changes in AH" may indicate that the modified group is at the molecular interface. There are considerable synergies between ITC (measuring affinity and suggesting when a change in binding mode occurs) and structural biology (providing 3-D coordinates for the binding mode, but giving no information on the magnitude or direction of any affinity changes). ITC in different buffers allows measurement of net proton movement during binding (numbers and direction) which is useful because X-ray crystallography only rarely detects protons directly, although they may be important in molecular recognition. The principal limitation of ITC is the requirement for test protein (for each titration, current instrumentation requires around 10-25 nmol which is 0.5 mg for M, = 20,000), ideally with aqueous ligand solubility of 100 pM and affinity in the range K d = l O n M to 10pM. The technique does not destroy the protein sample and indirect approaches can extend the usable ranges of ligand affinities and solubilities. The interaction under investigation must produce a measurable heat change, although the experimental conditions usually can be manipulated to satisfy this requirement. ITC is not well-suited to characterization of binding where there are cooperative interactions between multiple binding sites, because of difficulties in deconvoluting the heat signal. At present, ITC is not a high throughput technique. Slow heat flow in the instrument means that each titration takes more than 1 h.

3 I4

ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

As thermodynamic and associated 3-D structural data accumulate, there will be improvement in the understanding and the prediction of affinity. This will help in molecular design. There have been attempts to predict the thermodynamics of interactions, based upon changes in polar and apolar solvent accessible surface areas. These efforts have stimulated research and understanding. At present, we regard such methods as suitable only in restricted cases. This is because the magnitude of the observed AHo is highly dependent upon multiple equilibria which are linked to binding (e.g. protein conformation changes, displacement of water molecules and ions). Enthalpy-entropy compensation means that predictions of affinity are more reliable than expected AH" values. Below, we aim to provide an overview of the range of applications and potential of ITC in drug discovery. We give our opinions on strengths and weaknesses. The depth of our own knowledge and understanding varies across these diverse fields. We encourage readers to consult the original references and to form their own views.

EXPERIMENTAL MEASUREMENT OF BINDING THERMODYNAMICS PARAMETERS USED TO CHARACTERIZE THERMODYNAMICS OF BINDING

An experiment to measure interactions between a test compound and its target protein constitutes a closed thermodynamic system. This is because the interacting molecules are materially contained, although they can freely exchange energy with their surroundings. A closed system together with its surroundings constitute an isolated thermodynamic system [9]. The laws of thermodynamics might be interpreted erroneously as dictating in biochemical experiments that AG is zero and AS is positive. However, the laws apply to isolated, not closed, systems so that measured values of AG, AH and AS may be positive, negative or zero. ITC is the only technique that is able to measure directly the values of AH", Kd and n, in a single experiment. The magnitudes of AS' and the standard Gibbs free energy change (AGO) are also obtained for the binding process which are related by the following expression: RT In Kd = AGO = AH' - TAS"

6)

where R is the gas constant and T is the experimental temperature. Note that

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we use Kd which is the equilibrium constant for dissociation, whereas the other thermodynamic parameters relate to association. Kd can be more easily understood than its inverse, the association constant K,, because it gives a measure of the free ligand concentration required for half-saturation and so can be related to the concentration required for 50% inhibition (ICs0). The logarithmic nature of the relationship between affinity and AGO, AH" or TAS" should be emphasized. A change of 1 kcal/mol in AGO corresponds to a factor of approximately 5-fold in Kd near physiological temperature. The statistical uncertainty in measured affinities is within a certain multiple of Kd, so that this becomes a linear function of A G . This means that the error distribution in AGO (but not Kd) is Gaussian (or normal), so that it is valid to use standard statistical criteria (such as standard error, or Student's t-test) in order to determine whether a change is significant. This is particularly useful when considering error propagation in structure-activity relationships (SAR). Calculated values for A G , AH" and AS" relate to standard conditions which usually are 298 K, at 1 atmosphere pressure and 1 M activity for each of the reactants, apart from hydrogen ion concentration (pH 7.0 may be used) [4,91. It is important to remember that the values of thermodynamic parameters may vary according to the concentrations of reactants in the titration and that ideal solution behaviour is assumed (that is, the definition of Kd describes the only interaction between solutes). Each of the measured thermodynamic parameters reflects the attribute of the products minus that of the reactants. For example, a negative AAG" indicates that the products have less Gibbs free energy than the reactants. Binding is favoured under standard conditions only if AGO is negative (that is Kd < 1 M). A change in ligand structure which makes AG" less negative (AAG" positive) disfavours binding. Negative values of AHo and positive values of ASo are favourable (see equation (i)). AH" can be considered as reflecting changes in bonding. For example, a negative value indicates that the products form more, or stronger bonds. There is little or no correlation between the magnitude of AHo and that of AGO (for a specific example, see Figure 7.11 and [3]). A 9 may be thought of as a measure of disorder in the system. Increased bonding tends to decrease disorder. Thus, AH" and AS" tend to make opposing contributions to affinity, giving enthalpy-entropy compensation which masks effects on A G (equation (i)). Binding is described as exothermic if AH" is negative (heat is released) and endothermic if it is positive (heat is taken up). A process with a negative AGO is described as exergonic, whereas a positive value characterizes an endergonic change.

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ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

ITC allows measurement of a further parameter, the change in heat capacity (or specific heat) upon binding, ACp. It is the differential of AH" with respect to temperature, AC,, = dAHo/dT

(ii)

Experimental determination of ACp usually assumes the relationship ACp = (AH;* - AH+,)/(T2 - T I )

(iii)

where T1 and T2 refer to different temperatures. Calculation may be complicated because ACp itself changes with temperature [lo, 111. Measurement of ACp is not always undertaken because it requires several titrations, giving higher costs in terms of samples and time. Furthermore, it is not the most informative thermodynamic parameter because it varies over a relatively narrow range and is difficult to relate to the mechanism of binding. From a fundamental perspective, however, ACp is an important parameter because it governs the magnitudes of AHo and AS", as shown in the relationships:

0

T

0

Like the other parameters, ACp, is defined in terms of products minus reactants. Thus, a negative value indicates that the products have a lower heat capacity than the reactants. A lower heat capacity indicates that input of a smaller amount of energy is required to increase the temperature. The physical factors which modulate heat capacity in biochemical systems are complex, so that it is impossible to determine by ITC alone the magnitude of individual contributions [ 12, 131. For example, bonding may increase heat capacity, but old bonds are broken and new bonds are formed between many atoms during binding and calorimetry measures only the overall difference between products and reactants.

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DESCRIPTION OF A N ISOTHERMAL TITRATION CALORIMETER

Several excellent publications give experimental details on the application of ITC to protein-ligand interactions [4-6, 8, 11, 14-18]. Here, we give only an outline of the technique, highlighting some aspects which are important in drug discovery. Until relatively recently, the application of calorimetric methods in drug discovery has been limited, mainly due to a lack of instrument sensitivity and insufficient quantities of purified proteins. Now, however, commercially available instruments with high sensitivity, coupled with advances in recombinant DNA technology and protein purification more often allow characterization of interactions of biological significance. Isothermal calorimeters monitor the heat change induced by titration at approximately constant temperature. The most sensitive instruments are capable of measuring the heat effects produced by association of about 10nmol of reactants (http:/ /www.calorimetry.com/ or http: / / www.calorimetrysciences.com/or http: / / www.thermometric.com/). The most common types of differential power compensation isothermal titration calorimeters in general use have two cells (one sample and one reference) which are enclosed in an adiabatic jacket (Figure 7. I). The cells and jacket are connected to independent heaters with devices measuring the temperature difference between the two cells and the jacket. During an experiment, the reference cell is heated by a very small constant power and a variable power is applied to the sample cell in order to maintain a fixed temperature difference relative to the reference. The voltage applied to the sample cell is the experimentally measured quantity which is related to the heat of the reaction. Exothermic reactions generate a negative signal, whereas endothermic reactions produce a positive applied voltage. Integration of this power with respect to time gives the amount of heat produced, or absorbed, by the reaction. In order to work below room temperature, coolants must be circulated around the jacket. Reactions can be studied over a range, typically from around 2-80°C, given sufficient stability of reagents. As a consequence of the feedback control, the temperature of the system actually increases slightly (less than O.l"C/h). COLLECTION A N D ANALYSIS OF EXPERIMENTAL DATA

In a typical titration, the observed heat effect is composed of three major components, the heat of complex formation, the heat of dilution (of protein and ligand) and the heat of mixing. Control measurements are required in

3 18

ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY stepper motor

4

A

Figure 7 . 1 . Schematic representation of a titration calorimeter.

order to extract the heat of complex formation [ S , 161. In practice, the heat of dilution of the protein usually is estimated from the data towards the end of the titration (monitoring heat changes on injection of ligand after binding is saturated) and the heat of dilution of ligand is measured by titration into buffer containing no protein. It is important to use very similar (preferably identical) buffers in the sample chamber and the injection syringe. This is because any differences give a heat signal during the titration which compromises the signal to noise ratio. In practice, the protein is usually dialysed before the titration and then the solid test compound is dissolved in the buffer from the dialysis. Phosphate buffer often is used because it has a relatively small enthalpy of ionization (0.8 kcal/mol for pK, = 7.2, [4]). This means that any proton movement linked to the binding equilibrium has a relatively small effect on the observed enthalpy change. ITC data are analyzed by estimating the variable parameters, usually n, K, ( = 1/Kd) and AH", by fitting to either the cumulative heat of complex formation throughout the titration, or, more commonly, the individual heats associated with each injection (for derivations and further information, see [6, 8, 1 1, 14-1 61). Often, a simple single site binding model can be fitted to these data. A critique on the application of this relationship is given by Indyk and Fisher [19]. Systems involving cooperative interactions between multiple binding sites are difficult to characterize by ITC.

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Calorimetry data on weak interactions allow estimation of Kd, but not n and so require an independent measure of the concentration of functional binding sites in order to calculate AH'. Cumulative heat tends to be more informative than individual heats for direct characterization of weak interactions. During a titration, binding causes a heat change in the sample cell and power is applied to give the desired temperature difference with respect to the reference cell. The power against time plot (top panel in Figure 7.2) shows a peak (because there is a heat change after the injection) and then returns to baseline during the restoration of the desired temperature difference. The area under the peak is integrated to give a measure of the heat change and then the parameter values for the best fit line are determined by non-linear regression, giving a plot that is sometimes known as a thermogram (bottom panel in Figure 7.2). In a typical experiment, most of the added ligand is bound in the initial injections, due to the large excess of free protein. These injections give the largest heat changes which allow estimation of AH". The heat changes become smaller as the titration proceeds and the concentration of free protein falls, so that some of the injected ligand remains free. These data allow estimation of affinity and stoichiometry. The stoichiometry is calculated from the depletion of total lime (min) 50

0

I

!:I I-

-8 t 0

0

Y

-10

'

I

100

.

l

.

200

150

I

'

I

'

250

l

I

320

ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

ligand, giving a lower free ligand concentration, in a similar fashion to tight binding inhibition in dose-response analysis of enzyme kinetic data [20, 211. Towards the end of the titration, the protein is saturated with ligand and further injections reflect the heat of dilution. The parameters that can be measured depend upon the concentration of protein required in order to give a measurable signal and the affinity of the test compound. This is because the shape of the thermogram is determined by a unitless parameter c, which is the concentration of binding sites divided by the dissociation constant [14, 161. For very tight binding (c + GO), all of the added ligand is bound until saturation occurs and the isotherm approaches a step shape, allowing estimation of AH" and the stoichiometry of binding. For weak binding (c 5 0.1) the curve approaches a horizontal trace. Both of these examples provide little information on the value of Kd (setting only maximum and minimum limits respectively). Values of c within the range 1-1000 usually allow deconvolution of the binding isotherm in order to estimate the magnitude of Kd. The sample concentration should be chosen to give a suitable value of c. The ligand injection schedule (number, volume, ligand concentration and timing) also must be designed in such a way as to yield information on AH", Kd and the heats of dilution and mixing. RANGE A N D UNCERTAINTY IN THERMODYNAMIC PARAMETERS

In order to provide information on binding, the extent of variation of a measured parameter between compounds must be much greater than the uncertainty in its magnitude. We have investigated this point by considering our own unpublished results and surveying published data [lo, 22-29]. This has allowed tentative identification of typical ranges for thermodynamic parameters for binding of compounds that are likely to be of interest in medicinal chemistry (Table 7. Z). Values outside these typical ranges are more common for ligands which are unlikely to become drugs, for example interactions between two proteins, or binding of ions. A G has the lowest signal to noise ratio because it must fall in a relatively narrow range, corresponding to Kd values of 0.01-10pM. The information content of AGO, however, is high because it measures affinity, whereas AHo and TAS" only influence affinity. Indeed, compounds may bind even when AH" or TAS" are unfavourable. Compared to other experimental methods, ITC gives a more precise measure of Kd values. AH" has a larger signal to noise ratio than does AGO. TAS" is less precise because errors are compounded when it is calculated as the difference between AH"

WALTER H.J. WARD ET AL.

32 I

Table 7.1. TYPICAL VALUES OF THERMODYNAMIC PARAMETERS FOR DRUG-LIKE MOLECULES BINDING TO PROTEINS Parameter

Typical Range

Typical SE

Range/SE

AG AH' TAS" ACP

-7 to - 1 1 kcal/mol -20 to +5 kcalimol -15 to +10 kcalirnol -400 t o +lo0 cal/(K mol)

0.2 kcalimol 0.2 kcal/mol 0.3 kcal imol 1 OY"

20 125 83 25

and AGO. The relative range of ACp values is smaller than that of AHo or TAS". The observed stoichiornetry for a 1:l interaction is usually between 0.9 and 1.1. Thus, the following changes are usually significant: 5-fold in Kd, >1 kcal/mol in AAG" or AAH", >2 kcal/mol in TAAS".

CHARACTERIZATION OF COMPOUNDS FROM MEDICINAL CHEMISTRY

The range of affinities that can be measured by direct titration is dependent upon two factors. The sensitivity of the instrument sets a lower limit on the concentration of protein that can be used to characterize tight binding compounds, whereas the measurement of weak binding affinities is limited by the available protein concentration. However, it is possible to extend the range of measurable affinities by several methods, as described below. Competition or displacement experiments [30-321 can be used when limited solubility prevents the use of high ligand concentrations, or when the affinity is too high, or too low, to measure directly by ITC. This method does require ligands with suitable characteristics (affinity and a different AH") in order to perform the displacement and the indirect measurement of thermodynamic parameters is associated with a higher level of uncertainty than is a direct approach. Differential scanning calorimetry allows measurement of very high affinities by following the melting temperature of the protein at different ligand concentrations [ 3 3 ] . However, further studies are required in order to estimate the affinity at physiological temperature from that measured at the melting temperature. In order to extend the range of measurable binding affinities, it is also possible to exploit the fact that free energy changes are defined by the initial and final states of the system, regardless of the pathway connecting them. If the affinity for a particular interaction cannot be measured at the target pH

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or temperature, it may be possible to measure it under different conditions and then adjust the value according to the effects of the shift in conditions. For example, it may be possible to measure affinity at an experimentally tractable pH. Proton movement at the target pH and relevant pK, values can be characterized. These data then can be used to calculate the magnitude of Kd at the target pH [34, 351. A similar approach can be used where Kd is measured at a suitable (usually higher) temperature and then the value is corrected to physiological temperature using a measured ACp value [36]. Alternative approaches to extend the range of affinities measured by ITC include coupling binding to a system which gives a heat signal. For example, binding which involves uptake or release of protons may be followed more easily in buffers that have large enthalpies of ionization (such as tris), displacement or uptake of divalent metal ions may be detected using ethylenediaminetetraacetic acid, or changed exposure of thiols may be observed in the presence of reducing agents [4]. In order to facilitate ITC on compounds with limited solubility in aqueous buffer, it is possible to employ organic solvents such as ethanol or dimethyl sulfoxide. However, dilution of organic solvents generally leads to significant heat effects, so that the matching of the solvent concentration between sample and injectant solutions is important in order to maintain a suitable signal to noise ratio. Care should be taken when interpreting the thermodynamic parameters obtained, since there may be perturbations arising from the change in hydrophobicity of the solution. The solubility of compounds may be increased by adjusting the pH of the buffer in order to increase the degree of ionization. A weakly buffered ligand solution may be prepared at a pH which confers sufficient solubility and then titrated into a more strongly buffered protein solution at the pH of interest. The dilution of ligand and binding to the macromolecule leads to a low free ligand concentration in the sample cell, so that the compound remains in solution at the required pH. Control titrations have to be undertaken to allow for the heat change due to the shift in pH of the ligand solution. This technique has been used successfully to study the binding of pepstatin A to endothiapepsin [37] and a carboxylated triazine (17) to a fragment of DNA gyrase (see below). If the ligand has only limited solubility, it may be advantageous to place the ligand solution into the cell, rather than the syringe, since the starting concentration of ligand need not be so high. However, this approach does require that the protein availability, solubility and stability is adequate for high concentrations to be used in the syringe.

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COMPARISON WITH OTHER METHODS TO MEASURE AFFINITY

There are many well-established methods to measure binding affinities in protein-ligand interactions (Table 7.2). Different techniques have specific requirements such as protein quantity, catalytic activity, molecular weight, need for immobilization and special reagents (for example fluorescent labels or tagged protein). The data generated vary with respect to precision and information content. For example, all techniques may allow determination of a Kd value and some may provide extra information relating to concentration of binding sites, rate constants for association and dissociation, thermodynamic parameters, or an indication of possible conformational changes upon binding. Arguably, ITC provides the most information of these techniques, being the only approach to allow direct measurement of AH". It is also the least demanding in terms of time required for assay development. Measured AHo values can be informative, as described in this review. However, ITC has the greatest cost in terms of the quantities of protein required, especially if the affinity of the ligand is low. Nevertheless, sufficient protein is often available in a project which also involves high throughput screening or protein structural studies. Under favourable circumstances, a protein sample may be dialyzed after ITC in order to allow repeated use for several measurements. Monitoring binding by using an optical biosensor (for example the instruments supplied by BIAcore or IAsys) has increased rapidly over recent years [38,39]. This method has the advantage of using much less protein than ITC. It has been claimed to facilitate measurement of association and dissociation rate constants. However, caution must be exercised since the estimated magnitudes often are quite different to those in solution. A variety of reasons may lead to the simple binding model being invalidated. It is necessary to perform tests in order to check for consistency of kinetic parameter values during data analysis [40]. It may be required to have special reagents, such as biotinylated protein which may be immobilized on a streptavidin chip. Although the sensitivity decreases with molecular weight, the latest instruments are reported to detect binding of molecules with molecular weights as low as 200 Daltons which would be low enough for many pharmaceutical applications. An advantage of ITC and optical biosensors over enzyme kinetics is that they can follow binding directly, in the absence of competing substrates, intermediates and products. The requirement for catalysis in enzyme kinetics leads to complexities in the interpretation of data. It is also necess-

Table 7.2. COMPARISON OF METHODS TO MEASURE BINDING AFFINITIES

Method

Special ReaKents

Protein usage (pmol)

Assay time (hours)

Running cost

Capital cost

Data generated

None

20,000

1-3

Low

High

I&, AH”, AS”, ACp, n

One partner immobilized 5

I

High, costly sensor chips

Very hg h

&. Optimization may be timeconsuming

May require labelled substrate

2

Low

Low

ITC

Optical Biosensor

Enzyme Kinetics

20

Kd. Interpretationmay be complex and optimization may be timeconsuming

Equilibrium Dialysis, Gel filtration, Filter Binding, Ultrafltration, Fluorescence

May require labelled ligand

5-20

2

Low to medium

Low to medium

Ki

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ary to have active protein and a suitable assay in order to determine affinity by kinetics, whereas both ITC and optical biosensors are capable of providing this information with inactive protein fragments. Direct binding also avoids kinetic equivalence, where the target molecule cannot be identified because association with either partner would follow the same dose-response relationship (see above). ITC is unlike many methods in that optical clarity of the solutions is not needed and there are no restrictions on the molecular weights of the reactants. Several additional methods, some of which are shown in the Table 7.2, may be used to rank compounds in terms of affinity for the target protein. However, it is through a combination of methods that understanding of ligand binding may be achieved.

INDIRECT ESTIMATION OF AHo WITHOUT ITC-VANT HOFF ANALYSIS

Equation (i) is an integrated form of the van't Hoff relationship which suggests that measured values for the temperature dependence of Kd should allow estimation of the magnitudes of AHo and AS". This method is potentially more attractive than ITC because it consumes smaller quantities of reagents, does not require a calorimeter and it may be possible to use impure preparations of target protein. Thus, it is particularly attractive for membrane proteins, such as receptors. However, there are potential problems with this approach. Firstly, it assumes that AHo is constant over the experimental temperature range which is rarely true. Secondly, even if the temperature dependence of AH" is taken into account (by considering ACp), there is enthalpy-entropy compensation, so that AGO often changes little with temperature, giving a poor signal to noise ratio. Thirdly, the magnitudes of the estimated thermodynamic parameters are correlated with each other and cannot be determined independently. We now consider these points in more detail. ACp is rarely zero [24, 41, 421, so that the magnitude of AH" usually changes markedly with temperature. A typical ACp % -250 cal/(K mol), over 4-37°C gives a AAH' of 8.25 kcal/mol which is significant compared with a typical AHo of -10 kcal/mol at 27°C. Thus, unlike equation (i), the relationship for data analysis should include ACp as follows

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where the subscripts 1 and 2 relate to different temperatures and the values for K are dissociation constants, The relationship becomes more complex if ACp changes over the required temperature range as seen when oligosaccharides bind to a monoclonal antibody [ 11, 431 and when peptides bind to RNAseA [lo]. An important problem with this application of van't Hoff analysis is that it attempts to derive two parameter values (AH" and ACp) from changes in AGO which are only small over the accessible temperature range (this gives uncertainty in the ratio KI / K Zin equation (vi)). Low temperatures are precluded by freezing and high temperatures are inaccessible due to protein instability, Thus, for example, when novobiocin binds to a 24 kDa fragment from DNA gyrase, AGO changes by less than 0.1 kcallmol between 9 and 37°C [26]. Furthermore, the estimated parameter values are correlated with each other and are not independent. This contrasts with ITC which directly gives an independent measure of AH'. Thus, parameters derived from van't Hoff analysis including a ACp term are subject to a larger degree of uncertainty than those from equivalent ITC experiments. In the absence of cooperative effects, there must be equivalence between calorimetric and van? Hoff enthalpies (AHocal= AHovh),because both approaches reflect the same closed thermodynamic system [4, 71. However, there are reports of differences between these parameters. It has been suggested that this may be due to equilibria linked to the binding process, such as displacement of small molecules and ions from the surfaces of associating solutes [41-451. These factors alone cannot account for the lack of agreement [7]. The discrepancies may reflect cooperativity, lack of precision in measured dH",h values, non-ideal behaviour and use of different concentrations of solutes, or artefacts from assay methods (e.g. immobilization of the protein). We have calculated AH",h from Kd values determined by calorimetry, allowing direct comparison with AHocalvalues obtained from the same experiment. Best fit values of AHoVhmay differ from AHoca,by over I kcal/mol, but the standard error for AHovhis large, so that these differences are unlikely to be significant. Studies on ligands associating with the maltodextrin binding protein of E. coli [27] provide an interesting example. In the range 7-35"C, AH",al/AH"vh for P-cyclodextrin is 0.63-0.69, with the difference from one perhaps reflecting limited precision. For maltose, the ratio is reported to vary from 1.17 to 25.1. However, several factors may complicate measurement of the energetics for binding [27]. Maltose solutions contain M and 8-anomers which could bind with different energetics and the equilibrium between them may change over the time-scale of ITC. Conversely, P-cyclodextrin does not have an anomeric equilibrium

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and there is a relatively constant relationship between AH;a, and the estimated AH;h. An integrated form of the van't Hoff equation, containing ACp as a variable has been used in several systems, such as inhibitors binding to thrombin [46] and peptides binding to an Fv fragment of an antibody [47]. Interesting applications have been made in the binding of ligands to receptors (see below). CHARACTERIZATION OF TARGET PROTEINS AND TEST COMPOUNDS THE SIGNIFICANCE OF ENTHALPY-DRIVEN VERSUS ENTROPY -DRIVEN BINDING

Association of a protein and ligand is often described as enthalpy-driven or entropy-driven. Below, we consider the reliability and information content of such a classification. As the experimental temperature is increased, the association of biomolecules may change from entropically favourable to unfavourable. This behaviour is exemplified by the antibiotic novobiocin binding to a fragment of its target protein, DNA gyrase (TAS" = 1.1 kcal/mol at 9°C and -7.3 kcal/mol at 37°C) [26]. Similar changes are seen in the magnitude of AH", giving enthalpy-entropy compensation (AAG" < 0.1 kcal/mol over this range of temperature). The changes in AH" and TAS" arise from commonly observed negative values of ACp. (It follows that associations may be more difficult to detect by ITC at low experimental temperatures, where AHo may be closer to zero). Accordingly, there are potential pitfalls in the terms enthalpy-driven, or entropy-driven, binding. This characteristic may change according to the assay conditions such as temperature, or the nature of the buffer. For example, proton movement upon binding in tris buffer may give a large enthalpic signal which is not seen in a different buffer at the same pH. In addition to the experimental temperature and identity of buffer, many other factors such as ions and osmotic strength may influence the observed energetics. Thus, any interpretation must be tentative. It is reasonable to suggest that entropy-driven binding tends to be more hydrophobic in character than does enthalpy-driven association. In practice, AH" is negative for most biomolecular associations. The term entropy-driven tends to be used whenever ASo is positive. As a generalization, a lead compound whose binding is enthalpy-driven may be more attractive than a compound

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ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

of similar affinity with entropy-driven binding. This is because there is more scope for increasing affinity by increasing hydrophobicity of the enthalpy-driven lead, without encountering compromised activity in vivo due to limited solubility or an inappropriate log P value. ENTHALPY-ENTROPY COMPENSATION DECREASES CHANGES IN AFFINITY

Consider a complex between a target protein and a test compound. The structure of the compound is modified in order to give new interactions with the protein. This makes AHo more negative. There is also increased order in the complex as a result of this change, giving a negative contribution to AS". Experimental data indicate that these two contributions to AGO tend to be of similar magnitude in aqueous systems at physiological temperature (see later, especially Figure 7.11). This is perhaps not surprising, given that AH' and ASo are dependent upon ACp (equations (iv) and (v)). Theoretical This enthalpyconsiderations are consistent with these observations [48,49]. entropy compensation means that changing the structure of the test compound tends to have a larger effect on AH" than on AGO, Kd or ICso values. This has many implications for medicinal chemistry. There may be differences in AH", but not AGO, for related compounds which form different interactions with the target protein. Alanine scanning mutagenesis may alter residues in the binding site of a protein, giving a change in AH", but not AGO. Enthalpy-entropy compensation represents an important challenge for the medicinal chemist who is trying to improve the affinity of compounds for the target protein. Compensation is a global phenomenon in that it reflects the overall properties of the entire molecules which associate. There may be local increases in entropy upon binding, as shown by NMR on mouse major urinary binding protein I and topoisomerase I when they respectively bind 2-sec-butyl-4,5-dihydrothiazoleand DNA [50]. The importance of these local changes in the overall entropy of binding is not clear. Enthalpy-entropy reinforcement has been reported for peptides binding to RNase S and pp6OC-"'" SH2 domain [51]. However, we question this interpretation because most of the values of AAH" and TAAS" are small relative to the degree of uncertainty in the measured parameter values. SOLVATION CHANGES ARE IMPORTANT IN THE THERMODYNAMICS OF BINDING

Consider a free protein, Pr and ligand, L, solvated by different numbers of water molecules, respectively a and b (equation vii). When Pr and L form

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a complex, solvated by c water molecules, then (a + b - c) waters are released to bulk, where they can form new interactions with each other. (vii) It should be noted that the different water molecules under consideration do not all make identical contributions to the energetics, because they have varying degrees of association with the groups that they solvate. The model illustrates that interactions with many water molecules always change during association in biological systems. Thus, water makes a major contribution to the observed thermodynamic parameters. Binding involves, not only the formation of new interactions, but also the loss of old ones. ITC measures the overall effects of many changes. It is difficult to deconvolute the magnitude of any of these contributions, except for proton movement (see below). The changed bonding and entropy of a water molecule can be seen from the different volumes occupied according to its location, estimated as 22.9 A3 when buried, 24.5 A3 when at the protein surface and 29.7 A3 when in bulk [52]. These volumes, derived in part from crystal structures, reflect changes in heat capacity and other thermodynamic parameters. Thus, reorganization of water molecules between solvation shell and bulk solvent seems to play a key role in enthalpy-entropy compensation [49, 53, 541. CHANGED INTERACTIONS ARE MORE LIKELY TO EFFECT AH' than AGO

We have seen how enthalpy-entropy compensation masks the effects of changed interactions on AGO. Thus, the SAR of measured AH" values can highlight groups involved in binding which make similar contributions to the Gibbs energy of the complex and that of the free partners [17]. This information is particularly useful when there are no 3-D structures available for the complex, because these groups would not be detected in assays which reflect only AGO. In alanine scanning mutagenesis, affinity is measured before and after changing protein residues in an attempt to identify the interface of a complex. There are many cases where AHo reveals interfacial groups that are difficult to detect in AGO [55]. Examples include mutants at the interface between the proteins barstar and barnase [56], human growth hormone and its receptor [57] and lysozyme and HyHELlO antibody [58]. There are many more examples where AH" has not been measured, but mutagenesis of residues known to be at the interface has only a minor effect on AGO. When

330

ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

12 interfacial residues are changed on lysozyme, only 4 give AAG" > 1 kcal/mol for the interaction with the D1.3 antibody [59]. In a survey of 2325 alanine mutants at protein interfaces, there is a poor correlation (regression coefficient = 0.44) between the effects of mutations on AAG" and the buried surface area of those side-chains [60]. Similar trends are seen when the structure of the ligand is modified, as in medicinal chemistry. Enthalpy-entropy compensation masks the effects of interfacial groups on AW for different peptides binding to the periplasmic transporter protein, OppA [61] and for tryptophan analogues binding the trp repressor [31]. The ammonium group of bound tryptophan (Kd = 56 pM) appears to form hydrogen bonds with three main-chain carbony1 groups of the repressor protein, suggesting that it may be important in binding. However, replacement of the ammonium by a hydrogen atom gives indole 3-propionic acid which binds more tightly (Kd = 29 pM). ITC reveals that both AHo and TAS" are more positive for the binding of Trp, respectively by 2.9 and 2.5 kcal/mol. Without 3-D structural data, the SAR of Kd would have suggested incorrectly that the ammonium group does not interact with the protein. The measured AH" values show that it is involved in binding, but forms interactions of similar strength with solvent, giving only a small contribution to AGO. Changes in AH" can also highlight different modes of binding to a target protein. These changes may not be detectable as a change in affinity. We detect large changes in AHo with small changes in AGO when triazines bind in two different orientations to a fragment of DNA gyrase (Figure 7.12 and Table 7.6). In studies by a group at Pharmacia and Upjohn, representatives from two structural classes of stromelysin inhibitors (1-3) show small differences in affinity (AAG" = 0.2-0.5 kcal/mol) and larger shifts in AHo (3.0-3.5 kcal/mol) which correlate with different modes of binding (Table 7.3) [29].

(1) R = CH3 (2) R = CH2-CF3

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33 1

Table 7.3. STRUCTURAL AND THERMODYNAMIC ASPECTS OF INHIBITORS BINDING TO STROMELYSIN

Inhibitor

(nM)

AG' (krul/niol)

AW (kcal/mol)

Zn2+ Co-ordination

(1) PNU-99533 (2) PNU-143677 (3) PNU-143988

240 53 196

-9.4 -10.1 -9.6

-4.7 -4.2 -7.7

Amide-hydroxamate Amide-hydroxamate Thiadiazole

Kd

Subsites

S,' to S3' S,' to S3'

s, to s,

Data from [29]

(3)

The numbers of compounds in these studies are small and it is always difficult to assign changes in thermodynamics to specific modifications in structure. However, a change in AH" <2 kcal/mol suggests that a modified binding mode is unlikely, whereas a change of >3 kcal/mol implies that there may be a different binding mode. This helps to identify where there may be a discontinuity in SAR which is valuable information for medicinal chemistry. Accordingly, we suggest the following model for exploitation of structural and thermodynamic data in molecular design (Figure 7.3). First, experimental data on the structure of a protein-ligand complex are used to design a new compound and to predict the magnitude of AH". The actual value of AHo then is measured by ITC. If there is good agreement with the predicted value of AH" and AAH" is small ( t 2 kcal/mol), then there is confidence to build a homology model for the 3-D structure of the complex between the protein and the new ligand. This model can then be used in a new cycle of molecular design. Conversely, if there is poor agreement between observed and predicted AH' values, or AAH' is large (>3 kcal /mol), then homology modelling is associated with a higher degree of uncertainty. The complex between the new ligand and the protein would be a strong candidate for experimental 3-D structure determination in order to support the next cycle of molecular design. We consider that this approach is most useful when it is difficult to obtain experimental 3-D struc-

332

ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY 3-D structure of complex with existing compound

Use structure to -Try desian new comDound

- -

Model structure of Predict interactions new complex and

t

0bseNedMH"small and as expected

ITC

to explain ITC data

t

Determine structure of new complex

t

Observed M H " large or unexpected

Figure 7.3. Proposed exploitation of structural and thermodynamic data in iterative molecular design.

tures for many new protein-ligand complexes. For example, when co-crystallization is required because the inhibitors cannot be soaked into crystals of apo-protein.

ASSESSMENT OF PROTEIN PREPARATIONS

Expression, purification and supply of target protein is critical to modern drug discovery. It is important that protein preparations satisfy functional criteria prior to their use in functional assays, screening, or 3-D structure determination. ITC is a powerful tool in quality assessment of protein preparations, due to its precision and ability to measure not only affinity, but also stoichiometry of ligand binding. Protein preparations from different purification and storage protocols can be assessed by ITC. This can give information which is not available from other widely used methods such as polyacrylamide gel electrophoresis, amino-acid analysis, mass spectrometry, binding assays, or measurement of enzyme activity. For example, preparations containing a mixture of correctly and incorrectly folded protein may have the desired sequence and molecular weight. They may bind ligands or turn over substrates as well as any previous preparation. However, the stoichiometry of ligand binding measured by ITC may reveal the presence of non-functional protein. We have demonstrated that use of fully functional protein reduces the risk of detecting compounds with artefactual activity.

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EVALUATION OF ASSAYS

The high degree of precision and direct nature of ITC means that it provides reliable data which may be used in order to assess the validity of other protocols. This is particularly important in the pharmaceutical industry where the need for high throughput and minimal consumption of reagents may provide an impetus towards less rigorous assay methods. Thus, ITC can be used as a standard to evaluate data from enzyme kinetic assays and binding assays involving immobilized proteins (such as scintillation proximity assays, ELISA assays, or BIAcore). Many assays use peptides as models to mimic the behaviour of full-length proteins. Compared to proteins, peptides are often easier to prepare and purify. Peptides also offer greater feasibility for specific chemical labelling in order to assist in assay configuration. For example, it is difficult to modify a protein at a single location with a fluorescent reporter, or by biotinylation. During assays, the high affinity of the biotin-streptavidin system is often exploited in immobilization, or detection of peptides. ITC can test the reliability of peptides as models of proteins. In a thorough study, a 14 residue Pro-containing peptide from the p85 subunit of PI-3 kinase was compared with full-length p85 in terms of the thermodynamics of binding to the Fyn SH3 domain [62]. The peptide and protein have similar affinities (Kd values respectively 16 and 3pM) and very different thermodynamic profiles. Association of the peptide has AH@= -12.3 kcal/mol, compared with +10.6 kcal/mol for the protein. The difference seems to reflect contributions from a large favourable AH@and unfavourable ASo as the peptide shifts from having little secondary structure when free in solution towards being helical after complexation with the SH3 domain.

CHARACTERIZATION OF PROTEIN CONSTRUCTS

Recombinant DNA technology is often used to generate protein samples for testing during drug discovery. Validity of the approach requires that ligand binding to authentic and recombinant proteins follows the same SAR. Divergent SAR is more likely when the authentic protein has post-translational modifications (such as proteolytic processing, phosphorylation or glycosylation). Tags are often added to recombinant proteins in order to facilitate purification (such as fusion proteins with glutathione transferase, maltose binding protein or hexahistidine, or epitopes to allow

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ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

purification on antibody affinity columns). Again, the tagged protein should follow the same SAR as its native counterpart. It is particularly difficult to produce large quantities of membrane proteins by recombinant methods. Proteins composed of different types of subunits also represent a challenge. Thus, it is often necessary, or expeditious, to assay isolated domains or fragments of proteins, or individual components of macromolecular assemblies. Such protein preparations should be characterized in order to ensure that the results are not misleading. ITC is one of a number of techniques which can be used to evaluate protein constructs. As highlighted above, it is particularly useful because it is rapid, has high precision, allows measurement of stoichiometry and does not require catalytic activity. FOLLOWING ENZYME-CATALYSED REACTIONS WITHOUT THE NEED FOR MODEL SUBSTRATES

Measurement of enzyme kinetic parameters may be undertaken by ITC because the latest instruments are sufficiently sensitive and rapid in response. The approach involves following the rate of heat change during a reaction. This method can be employed when there are no other readily observable signals produced by the reaction of interest and hence there may be no feasible alternative method for measurement of the kinetic parameters. Usually, in these situations, model or derivatized substrates producing an observable change (for example an optical change), or coupled assays would be employed. An advantage of ITC is that the natural substrate can be used in a continuous assay, as well as giving information relating to the enthalpy change of the reaction. ITC, therefore, avoids potential pitfalls when model substrates (such as peptides) could follow different kinetics (or mechanisms) when compared to natural substrates (such as proteins) which cannot readily be assayed by other techniques. For example, hydrolysis of amides by a-chymotrypsin tends to be rate limited by formation of the acyl enzyme intermediate, whereas hydrolysis of esters usually is rate limited by deacylation [63]. Similarly, some hirudin analogues bind at a distance from the catalytic centre of thrombin where they inhibit proteolytic activation of fibrinogen, but not hydrolysis of a peptidyl coumarin ~41. ITC has been used to measure kinetic parameters for HIV-1 protease [65], p38 mitogen activated kinase [66], yeast cytochrome c oxidase [67], acylase I and subtilisin BPN’ [68]. The method involves initiating the reaction by the

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addition of enzyme to the substrate solution in the cell and then monitoring the resulting heat flow. The plot of power versus time is calibrated using the enthalpy of reaction which is estimated from the area under the curve after the reaction has reached completion, for a known substrate concentration. MEASUREMENT OF CRITICAL MICELLE CONCENTRATION A N D PARTITIONING INTO LIPIDS

Determination of the critical micelle concentration (cmc) by ITC involves the measurement of heat changes upon dilution of aliquots of a solution which is initially well above its cmc. As aliquots are injected into the cell, the micelles dissociate, usually with a measurable heat change, allowing the molar enthalpy of micelle dissociation as well as the cmc to be determined (after correction for the effects of dilution etc.). As the concentration in the cell increases, the measured heat signal decreases until the concentration is at the cmc, when no further dissociation of micelles occurs and the heat signal reaches a lower level. ITC also has been used to characterize the partitioning of compounds into membranes [69] which may have implications for the biological activity of therapeutic agents. Binding of magainin peptides to membranes has negative values of AHo and AS" and a positive ACp, all of which are the opposite sign to that expected for a hydrophobic effect [70]. Circular dichroism measurements show that the peptides fold from a random coil to an a-helix on penetrating the membrane. This is known to be an exothermic process which seems to contribute to the observed thermodynamics. IDENTIFICATION A N D CHARACTERIZATION OF TARGET PROTEINS

ITC has given new information on protein-protein interactions which are involved in the regulation of cell physiology. For example, some studies on signal transduction pathways have been particularly informative because ITC is a direct binding technique. Test compounds do not have to perturb interactions between macromolecules, thus avoiding kinetic equivalence and allowing definitive identification of the target. The technique has been employed in the characterization of cooperativity and receptor oligomerization for basic and acidic fibroblast growth factor (FGF), the FGF receptor and heparin [71, 721. ITC has been applied extensively in order to study SH2-mediated signalling pathways and phosphotyrosine analogues [73-801. The approach also has allowed characterization of other

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proteins of pharmaceutical interest, such as human group I1 phospholipase A2-factor Xa [81], BAG1-Hsc70 [82], calmodulin [83, 841 and myosin light chain kinase [84]. When the stoichiometry of a protein-ligand interaction is known, ITC can allow measurement of the concentration of functional protein. This allows calculation of the turnover number (moles of substrate per mole of active sites per second, kcat)from the rate at saturating substrate (VmaX).The parameter k,,,/ K, is important for characterization of substrate specificity and the energetics of enzyme catalysis [63].

ELUCIDATION OF THE INTERMOLECULAR COMPLEX WHICH GIVES BIOLOGICAL ACTIVITY

The direct nature of calorimetry makes it particularly informative as a method to characterize the molecular mechanism of active compounds. Thus, ITC can give information on whether another ligand influences the biological activity of the test compound. A second ligand may be required for binding, to compete (directly or indirectly) with the test compound, or to have no effect. This may give insight into the validity of structural overlays and factors which influence activity in vivo. This

rn

information also is essential to ensure that the 3-D structures of only relevant intermolecular complexes are used in ligand design. For example, inhibition of enoyl (acyl carrier protein) reductase by triclosan requires prior binding of NAD' [85]. Crystallography shows that the bound coenzyme forms part of the binding site for the inhibitor. However, crystals of enzyme-inhibitor complex can be obtained in the absence of NAD+.

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The 3-D structure in such crystals would not be relevant for understanding the biological activity of triclosan, nor for the design of improved analogues. Similarly, ITC demonstrated that myristoyl CoA binds to N-myristoyl transferase in the absence of a peptide to act as an acceptor substrate [23]. The inhibitor, SG58272 (4) was shown to bind to enzyme-myristoyl CoA complex, but not free enzyme [86]. This result indicates that a crystal structure of enzyme-SG58272 complex would give misleading information for structure-based drug design. Likewise, tricyclic farnesyl protein transferase inhibitors, such as a Schering compound (5) have been shown to have higher affinity for enzyme-farnesyl pyrophosphate complex than for free enzyme [87].

0 Y

FURTHER CHARACTERIZATION OF ACTIVE COMPOUNDS

ITC has been used to elucidate the mechanism of interaction of b-lactamase inhibitors with their target protein [88]. This study suggested that the free compounds do not bind Zn2+. Mechanistic information also emerged from an ITC study on cationic porphyrins binding to oligodeoxynucleotides [89]. Measured stoichiometries showed formation of 1:1, 1:2 and 1.3 complexes. These correspond to the numbers of tetrad planes in the target DNA multiplexes. Such multiplexes are thought to be formed when telomerase adds repeat sequences to DNA, rendering cancer cells resistant to apoptosis. Thus, ITC suggests that the porphyrins may follow a mechanism which could be exploited in agents to treat cancer.

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ITC has been employed in characterization of ligands identified in the application of SAR by NMR [90-921. This exciting technique involves use of NMR to screen for ligands which bind simultaneously to the same protein. There is potential for a large entropic advantage when the two ligands are linked by a covalent bond prior to binding the target protein. There is a complementarity between ITC and NMR, because they can measure affinity in different ranges. NMR is suitable only for weak leads (Kd > 100 pM), whereas ITC typically requires affinities below 10 pM for direct measurement. Further drug-target interactions which have been characterized by ITC include FdUMP-thymidylate synthase [93] and captopril and lisinopril interacting with angiotensin converting enzyme [94, 951.

THERMODYNAMIC DISCRIMINATION BY RECEPTORS

The thermodynamics of ligand binding to receptors has been characterized by various forms of van't Hoff analysis. However, some parameter values may be questionable because analysis assumes ACp is zero [96]. These and other studies suggest that there may be thermodynamic discrimination between binding of agonists and antagonists (Table 7 . 4 ) . Thus, the sign of ASo appears discriminatory for j-receptors and the sign of AH" seems discriminatory for nicotinic and A, receptors (Tuble 7.4). However, the directionality is reversed for A, receptors.

Table 7.4. REPORTED THERMODYNAMIC DISCRIMINATION OF AGONISTS FROM ANTAGONISTS Receptor

Agon ists

An tugonisis

AHo ASo

Negative Negative

Negative or positive Positive

Neuronul nicotinic' AH" AS'

Negative Negative or positive

Positive Positive

Adenosine A t AH AS"

Positive Positive

Negative Negative or positive

P-receptor

Refrrencr [971

P6, 991

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It has been suggested that thermodynamic discrimination arises because agonists tend to be more polar, binding in an oriented fashion with a relatively large entropic cost. Conversely, the receptor is proposed to retain more flexibility when complexed to an antagonist, because it is not constrained to the signalling conformation, characteristically giving a more positive entropy change [loo]. In our view, this attractive hypothesis remains untested by current experimental data. In addition to the questions around the precision of reported values, the observed parameters are influenced by further equilibria coupled to the binding process (such as movement of protons or localized interfacial water molecules) and by differences in physical properties (especially hydrophobicity) between agonists and antagonists which may be quite large, especially if they come from different chemical classes. These complexities may explain the observed reversal of thermodynamic discrimination between classes of receptor as seen in a comprehensive review [98]. If feasible, direct measurement of AHo by ITC, rather than current values obtained indirectly by van’t Hoff analysis could help to test the hypothesis of thermodynamic discrimination.

MOLECULAR RECOGNITION AND LIGAND DESIGN

INTERPRETATION OF BINDING THERMODYNAMICS

We have already highlighted the importance of considering the differences between the products and the reactants rather than only the products. The situation becomes more complicated when the structure of the ligand is changed, as in medicinal chemistry. Figure 7.4 shows a scheme where a protein Pr binds a ligand L with a Gibbs free energy change AGO which is the difference between the Go value of the products (Pr.L complex) and the reactants (Pr and L). Binding of a modified compound L’ gives a new Gibbs free energy change AGO’ which reflects the difference between Pr.L‘ and the reactants Pr and L’. In structure-based drug design, the change in affinity (AAG“) sometimes is assigned to modified interactions between the ligand and the protein (AGoPr,L).However, inspection of Figure 7.4 shows that this often is not correct, because AAGo also contains a contribution from a ‘reorganisation term’ [2] which reflects the altered Gibbs free energy on moving from free

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ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

AGO,

= Gar - GoL

6GOPr.L

-

= GOP,.L' GoPr.L

L to free L' (AGOL)

AAG" = (AG" - AG") = (AG;,, - AG; )

(viii)

The magnitude of AGOL may be important. Similar considerations apply to the other thermodynamic parameters, AH", AS" and ACp. The thermodynamic consequences of changing the structure of a free ligand may be important in medicinal chemistry. For example, consider the situation where L' makes an additional hydrogen bond with the protein. This may be detected in a crystal structure and assumed to be an improvement in molecular design. However, measurement of affinity may show that this group interacts equally well (or even better) with solvent water and so makes no contribution to (or even decreases) affinity. STARTING TO UNDERSTAND AFFINITY

ITC provides an important link between the understanding of molecular structure and function. Prediction of affinity from the 3-D structure of a complex is widely regarded as unreliable. Measurement by calorimetry gives a definitive value. Affinity and its enthalpic and entropic components, are dependent upon differences between free and bound partners and their interaction with solvent and solutes. Accordingly, the 3-D structure of the complex reveals only some of the factors which influence affinity. Experimental structural data do not allow full characterization of the energetics of the complex. Multiple bonds between ligand and protein suggest that formation of the complex is exothermic, but there is little correlation between AH" and AGO (Figure

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34 1

7.11). Experimental data on molecular dynamics is tenuous. Protein crystal structures struggle to discriminate between some atoms, such as amide nitrogen or oxygen. This can make it difficult to identify acceptors and donors in hydrogen bonds. Crystallography and NMR also have limited ability to detect water molecules which may be important in biomolecular recognition [loll. NMR detects only slowly exchanging water molecules, whereas crystallography detects water molecules that are located in sites at high occupancy. These water molecules may be difficult to detect by NMR, because they exchange quickly or vibrate. The resolution of experimental 3-D structures is rarely sufficient to allow accurate estimation of interaction strengths from interatomic distances. Although NMR detects hydrogen atoms, they are revealed by X-ray data only if resolution is high. This adds to problems in characterizing hydrogen bonds. Failure to detect hydrogen atoms also leads to uncertainty over protonation states. ITC offers complementary data, because it is readily able to detect changes in protonation upon binding. On the other side of the thermodynamic difference equation, free test compounds usually have multiple conformations which make it very difficult to assign structure and energetics. Furthermore, the conformational distribution of the target protein when free may be quite different to that of the protein in the complex. Thus, in order to begin understanding the thermodynamic contributions of specific chemical groups, high resolution 3-D structural information is required in addition to ITC. Computational approaches, especially molecular dynamics, also help to improve our ability to explain and predict relationships between structure and affinity. Experimental measurement of molecular dynamics by NMR also is enlightening. Together, ITC and 3-D structural data can support computational chemistry in drug discovery [ 102-1 081. When considering SAR in drug discovery, difficulties arise because there are many factors which contribute to the observed thermodynamics of binding and ITC measures the overall sum of these effects. There is limited scope to deconvolute the individual components. Thus, the most reliable information comes from studies where small changes are made to the structures of the partners and then ITC is combined with high resolution information on the free partners and the resulting complex. Such favourable systems are rare. Even in these circumstances, it is challenging to assign effects to specific chemical groups, because changing the structure of a partner always changes several physical properties and a number of linked equilibria, such as interactions with solvent.

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There are many papers which attempt to partition the observed thermodynamics into various contributing factors, such as polar and apolar surface area changes, protonation, conformational, rotational and translational entropy and solvent effects [37, 48, 78, 87, 103, 1091. In some cases, these contributions have been assigned to specific chemical groups. In our view, some of these studies overstate the current level of understanding of biomolecular recognition. These analyses do, however, highlight some important factors and suggest how they may contribute to binding. AFFINITY, ENTHALPY, ENTROPY A N D INTERACTION TYPE

Different types of interaction, including hydrophobic, hydrogen bond, ionic, van der Waals and covalent, make contributions to AH", AS" and ACp. However, linked equilibria and the interdependence of different physical properties make it very difficult to deconvolute the contributory factors. Thus, different experimental systems suggest various magnitudes and signs of contribution [2,3,7,63,110, 1 1 11. When following small changes in structure in well-defined systems, measured thermodynamic parameters sometimes give an indication of the nature of interactions. A framework for understanding ACp, AH" and AS" in biomolecular recognition was described in a key paper by Sturtevant [12]. Water molecules which cannot be detected in structural data, appear to play a major role in governing the thermodynamics of both polar and non-polar interactions. Energetic consequences may also occur when association leads to immobilization or displacement of structurally localized water molecules. Molecular vibrations also are influential [12, 131. Precise prediction of affinity would be an outstanding achievement in the application of binding thermodynamics to medicinal chemistry. Insight into the structural basis of affinity has been gained from thermodynamic studies of protein unfolding. However, it is our view that current methods have limited scope to predict binding thermodynamics. It may be possible to predict Kd within two orders of magnitude, but the precision in expected AH" and AS" values often is lower than that in AGO. This level of accuracy may help in the identification of compounds that bind, but tends to be misleading when trying to rank compounds on the basis of affinity.

Hydrophobic interactions Unpaired hydrogen bonding groups are energetically disfavoured. Non-polar solutes cannot satisfy the hydrogen bonding potential of solvent

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water. Accordingly, under many conditions, water molecules surrounding a hydrophobic solute may make stronger hydrogen bonds with each other than do bulk water molecules. This could be because water molecules at the interface are not constrained by interactions with the hydrophobic solute, so that there is more scope to form optimal bonds with other waters. Thus, the shell of waters around non-polar solutes can be more rigid than bulk water and water solvating polar solutes. Burial of apolar surface area on formation of a hydrophobic interaction releases these waters, allowing them to move into bulk where solvent-solvent interactions are weaker. This tends to be endothermic and entropically favourable [l, 121. Close proximity between atoms in hydrophobic interactions also generates induced dipoles (van der Waal's interactions). The enthalpic and entropic consequences of this effect may be opposite in sign and smaller in magnitude, relative to the contributions from solvent reorganization 1631. Overall, hydrophobic interactions usually make a positive contribution to ASo and often also have a small positive effect on AH" (equations (xiv)-(xvi)) [l-3, 12, 48, 631. Hydrophobic interactions seem to make a negative contribution to ACp. This is because the rigid shell of water around the free partners becomes weaker at higher temperatures. Thus, AH" becomes less positive as temperature increases (see equation (iii)). A single methyl group contributes up to 3 kcal/mol to AGO (around 120-fold in Kd) which is more than might be expected from solvent transfer experiments, because the protein binding site has an entropic advantage arising from being ordered prior to ligand binding [63, 1121. Hydrogen bonds and ion-pairs In general, electrostatic charges or hydrogen bonding groups on free solutes form interactions with solvent which are exchanged for solute-solute and solvent-solvent upon formation of the complex (equation (vii)). The entropic gain from release of water of solvation into bulk makes a favourable contribution to affinity. Compilation of hydrogen bond inventories illustrates that addition or removal of hydrogen bonding groups tends to have a similar effect on both sides of the binding equation [63]. Thus, there is a relatively small overall contribution to the thermodynamics of complex formation which represents the difference between the products and the reactants. This contribution may not even favour binding, as illustrated by SAR on tryptophan analogues binding to the repressor protein [31] and by triazines associating with a fragment of DNA gyrase. An optimal uncharged hydrogen bond, therefore, contributes a factor of up

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to approximately 10-fold to affinity (AAGO = -1.5 kcal/mol) and tends to be linked with negative values of AAH" and AAS" (see equations (xiv)-(xvi)) [2, 3, 12, 48, 63, 1111. However, many hydrogen bonding groups may have effects that are quite different. Progressively larger contributions are seen where one partner is charged, or in ion-pairs. They may contribute 5,000-fold or more to affinity ( A A W = -5 kcal/mol) 1631. This may reflect a large energetic penalty when deletion of a group from a ligand means that a charged group on the protein has no partner in the complex. There are examples where a single hydrogen bonding group can contribute over 10,000-fold to Kd. This is seen for a hydroxyl group on triclosan binding to enoyl (acyl carrier protein) reductase [85], 6-hydroxy-1, 6-dihydropurine ribonucleoside associating with adenosine deaminase [113, 1141 and tyrosine binding to tyrosyl tRNA synthetase [115, 1161. Several factors appear to contribute. For the des-hydroxy ligands, there may be a penalty for desolvation of one or more charged groups which are components of the binding site on the protein. Another factor may arise because each of these hydroxyl groups forms more than one hydrogen bond. Formation of the first hydrogen bond is linked with an entropic penalty and an enthalpic gain. The entropic consequences of formation of a further bond from the same group are smaller, but the enthalpic gain potentially remains similar. Thus, the second hydrogen bond may make a larger contribution to affinity [1171. A detailed study of a range of ligands illustrates a correlation between affinity and selectivity [I 181. This follows from high affinity requiring complementarity between the test compound and target protein. It is a common view that ligand hydrophobicity gives affinity, whereas hydrogen bonding gives specificity [631. This perception perhaps arises because hydrophobic interactions tend to be easier to design than hydrogen bonds, because their principal requirements are exclusion of water and a lack of steric clashes. The limited range of distances and permitted angles for hydrogen bonds makes them more directional. Of course, ionic interactions are less discriminatory. Selectivity also may be generated by different numbers (or strengths) of hydrogen bonds being formed in complexes with different proteins. It is estimated that the strength of a single hydrogen bond is 3-9 kcal/mol [63]. As explained above, such a large contribution is not usually seen during binding because there is hydrogen bond exchange. Now, consider a situation where a hydrogen bonding group becomes located in a region where it cannot interact with any polar groups upon binding. In such cases, the hydrogen bonding group often (perhaps partially) satisfies its requirements by trapping a water molecule at the interface, as seen by crystallographic

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studies in ligand SAR and in protein engineering [61, 63, 1191. There is an entropic cost for immobilization of a new water molecule at the interface. Also, there may remain a penalty for incomplete satisfaction of the hydrogen bonding requirements of the interfacial water molecule. Now, consider a situation where there is insufficient space to allow an interfacial water molecule. Binding of the ligand becomes associated with a large penalty which may approach the energy of formation of one, or more, hydrogen bonds. Binding is no longer a process of hydrogen bond exchange. There is a net loss of hydrogen bonds which gives a large penalty for binding to the wrong protein. Thus, individual hydrogen bonding groups often make a small contribution to affinity for the correct protein, due to hydrogen bond exchange. There may be a much larger contribution to selectivity, because of the magnitude of the penalty for desolvating a polar group. WATER MOLECULES LOCALIZED AT PROTEIN-LIGAND INTERFACES

The majority of water molecules that interact with the free partners and with the complex cannot be located in experimental 3-D structures [loll. These exchangeable water molecules are large in number and so may have a major influence on ligand affinity, as revealed by shifts in energetics when binding is followed in D20 [25]. Conversely, some water molecules may be more localized and so be detectable by techniques such as NMR and X-ray crystallography. Mass spectrometry may also be able to measure the number of tightly bound water molecules [120, 1211. These ordered, or structural, water molecules raise questions for the medicinal chemist. Is it best to treat these waters as part of the structure of the target protein, with a ligand designed to hydrogen bond to the water? Or, should the aim be to gain entropy from displacement of interfacial waters, by introducing a hydrogen bonding group onto the ligand in order to interact directly with the protein? Many factors influence the answers to these questions. Below, we describe how a combination of ITC and structural studies has given information to provide some assistance in selection of the best strategy. Water molecules are small, highly polarizable and capable of accepting or donating multiple hydrogen bonds. Thus, polarizable water is ideally suited to bind at the interface in such a way as to maximize the complementarity between the target protein and test compound. This capability is illustrated in studies of peptides binding to the OppA protein [61, 119, 1221. Here, different numbers of water molecules are immobilized at the interface in order to accommodate ligands of various sizes at similar affinities.

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ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

Several informative studies on the thermodynamics of interfacial water molecules have been published by Connelly and colleagues. Initial investigations focussed on the large macrocyclic immunosuppressive agents tacrolimus (6) (also known as FK506) and rapamycin (7) binding to FK506 binding protein (FKBP). X-ray crystal structures show that the hydroxyl group of Tyr-82 in free bovine FKBP is complexed to two water molecules. Crystallographic temperature factors show that one is bound more tightly than the other (respectively, B = 5.7 and 28 A2). These waters are displaced when the Tyr-82-OH forms a hydrogen bond with a carbonyl oxygen atom on the bound tacrolimus or rapamycin. The significance of these water molecules has been characterized by ITC and X-ray crystallography on bound and free forms of wild-type FKBP and the Tyr-82+ Phe mutant human protein [22]. The change in species does not appear to be significant. This is a particularly suitable system for study because the effects of the mutation are localized in this region of the protein structure. The two water molecules solvating the side-chain of residue 82 are absent from the free mutant protein. Crystallography also does not detect a new interfacial water near this position in the complex of the Phe-82 protein with tacrolimus. This may be either because the carbonyl oxygen is not solvated in the complex, which seems unlikely as there is no evidence for a large energetic penalty, or it may be solvated by a water molecule which is not detected crystallographically, perhaps because it is not tightly bound or can occupy alternative positions [loll. Displacement of two fewer ordered water molecules upon binding to the mutant protein has a negative effect on AH" (AAH"= -3.0 to -4.3 kcal/ mol) and TAS", overall making AGO more positive (AAG" f0.6 to

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+0.8 kcal/mol) [22] (Figure 7.5). ACp is around 100 cal/(K mol) more negative for binding to the mutant. A survey of protein-ligand complexes suggests that making a negative contribution to ACp, AHo and TAS" is a general property of interfacial water molecules [26]. Thus, a medicinal chemistry strategy to displace the waters bound to Tyr-82 of free FKBP could be linked with favourable changes in affinity and entropy, in spite of an enthalpic penalty. The observed energetics for binding to FKBP vary according to whether measurements are made in water or D20, but even in this well-defined system it is difficult to interpret the consequences of this shift [22, 1231. These investigations have been extended into a simple model system, where interpretation is less difficult. On average, the dissociation of one bound water molecule from various inorganic crystalline hydrates is linked with thermodynamic parameters of ACp=O to 9 cal/(K mol), AHo=O to +4 kcal/mol, TAS" = 0 to +2 kcal/mol and AGO = 0 to +4 kcal/mol (Figure 7.5) [ 124, 1251. Linked equilibria and conformational changes may cause proteins to behave quite differently to these figures. The changes when immobilizing an additional water molecule on binding of novobiocin to the Arg-136 -+ His mutant of DNA gyrase B fragment are much larger [26]. A A H is -5.4 kcallmol, AAGo=+2.2 kcal/mol and AACp -159 cal/(K mol) (Figure 7.5). These parameters seem to reflect changes in addition to the immobilization of one more water molecule. Conversely, much smaller effects are seen when comparing the binding of Lys-Trp-Lys and Lys-Ala-Lys peptides to OppA. Association of the Ala peptide involves immobilization of 3 more water molecules, with AAG" and AAH" changing

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ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

respectively by -0.4 and -0.3 kcal/mol (Figure 7.5) [61, 1191. These results illustrate the potential of medicinal chemistry which aims to interact with, rather than displace, interfacial waters. The thermodynamics of interfacial water molecules and their potential exploitation in molecular design are reviewed by Ladbury [126]. These studies illustrate the wide range in consequences of displacing interfacial water molecules (Figure 7.5) and give some insight into the factors which influence where a specific system is likely to fall within this range. Indeed, the data in Figure 7.5 have not been standardized on the effects of one average water molecule because specific waters have different effects. Water molecules are localized only if their association with the protein is favourable. Therefore molecular design to displace such water molecules has always to pay a penalty which corresponds to the free energy of binding of the interfacial water molecule. A new group on the ligand should not only displace the water, but also satisfy the hydrogen bonding needs of the protein. It may not make stronger hydrogen bonds than the interfacial water because it is more constrained by attachment to the rest of the ligand. However, the new group on the ligand pays a smaller entropic penalty than the water molecule when it becomes immobilized

Gyrase OPPA Rapamycin Tacrolimus Crystalline hydrates -6

-4

-2

0

2

4

MG'or M H " (kcaVmol), or number of waters Figure 7.5. Diversity in energeticsfor immobilization of interfaciul water molecules in different systems. Hatched bars: AAG". White bars: AAW. Black burs: Change in number of interfacial water molecules. Gyrase: Effecfs of Arg-I36 --t His mutation on binding of novobiocin. OppA: Comparison of binding of Lys-Trp-Lys and Lys-Ala-Lys. Rapamycin and tacrolimus: Effects of T y r - 8 2 4 Phe mutation in FKBP on binding of named ligand. Limiting values are shown for crystalline hydrates.

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at the interface. This is because the substituent is linked to a ligand which is already at or near the correct position. This entropic factor appears important for the success of gaining increased affinity. It has been suggested that crystallographic B-values above 20 A* may identify water molecules that are candidates for replacement by hydrogen bonding groups on the ligand, because the penalty for displacement is relatively small [ 1271. Displacement of interfacial water has been exploited successfully in the optimization of HIV-1 protease inhibitors, where the carbonyl oxygen of a cyclic urea displaces an ordered water molecule which is detected by X-ray crystallography [ 1281.

CHANGES IN PROTONATION UPON BINDING

In line with the importance of making small changes in order to obtain interpretable thermodynamic parameters, measurement of proton movement linked with binding is a valuable application of ITC. It is possible to measure the number of protons released to, or taken up from, the buffer during a binding reaction. This measurement is made by performing several titrations where the only change in conditions is the enthalpy of ionization of the buffer. Consider the equilibrium M+L

t 'M.L+npH+

0x1

with an associated heat effect AHo and where np is the number of protons released. The protons freed upon binding are taken up by the buffer, with an additional heat effect which is -npAHi, as shown: npHf

+ n,B

I _ npBH+

(x)

where AHi is the enthalpy of ionization of the buffer. The observed enthalpy change is therefore the sum of the two heat effects = AHo - n,AH,

(xi)

This relationship can be fitted to measured values AH,b, at various values of AHoi.The gradient of the straight line gives an estimate of the number of protons released from the binding reaction to the buffer. The intercept (where AHoi= 0) reflects the enthalpy change for the reaction of interest [35]. Buffers with different values of AHi are required for such studies and are listed elsewhere [4, 1291.

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ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

This analysis has been applied in several systems, including elastase interacting with an ovomucoid domain [130], octenoyl CoA binding to acyl CoA dehydrogenase [ 1311 and pepstatin associating with plasmepsin [ 1321. ITC also has been used in order to characterize proton movement upon binding of inhibitors to stromelysin (matrix metalloprotease 3 which has been implicated in arthritis and cancer [28]). The compounds investigated were Galardin (8) (where a hydroxamic acid interacts with the active site Zn2+ and Glu-202), PD166793 (9) and PD180557 (10) (where, in each case,

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35 1

a carboxylate interacts with the Zn2+ and Glu-202). At pH 6.7, binding of Galardin (hydroxamic acid pK, = 8.7) is shown to result in one extra proton becoming associated with the enzyme-inhibitor complex. Binding of the Parke-Davis compounds (pK, = 4.7) leads to association of two extra protons. These results imply that a second proton is located between the carboxylate of these inhibitors and that of Glu-202. His-224 is located in the S,' subsite of stromelysin. Mutagenesis to Asn-224 leads to no additional protons being linked with binding of the hydroxamic acid and one extra proton for the carboxylates. This suggests that, in the wild-type enzyme, His-224 becomes protonated on binding of each of the inhibitors. Crystallography and modelling suggests that this subsite is occupied in the complexes with PD166793 and PD180557, but not with Galardin. This study illustrates the complementarity between ITC and structural studies. COOPERATIVE BINDING

We have highlighted that the ability to measure AH' gives insight which cannot be obtained from AGO. In addition to reflecting changes at the interface between the target protein and the test compound, AH' is also influenced by changes throughout the structures of the interacting partners. Thus, it reflects conformational changes and cooperative effects. This is illustrated in studies of the immunosuppressive agent, mycophenolic acid, binding to inosine monophosphate dehydrogenase [ 1331 and substrates binding to glutamate dehydrogenase [ 1341. However, it rapidly becomes very difficult to deconvolute thermograms in order to estimate the interdependent parameter values in cooperative systems, especially when different steps have opposite signs for AH" [19]. ENERGETIC CONTRIBUTIONS FROM NON-INTERFACIAL RESIDUES

Conformational changes in proteins upon ligand binding can give large signals in AH" and ACp which highlight the potential for pitfalls in the prediction of 3-D structures and so the potential value of experimental determination of 3-D structure. It has been suggested that the unusually large ACp [over -900 cal/(K mol)] when hirudin binds to thrombin is due to the ordering of loops [103]. Similarly, the Ala-77+Val mutation of the trp repressor increases the stability of the free protein and reduces the magnitude of AHo for Trp binding by over 3 kcal/mol, whilst changing

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AGO by less than 0.5 kcal/mol[135]. It appears that Trp binding reduces the number of available conformations for the wild-type protein. This effect is smaller for the more stable mutant. Residues which are not located at the interface between partners often make important contributions to the energetics of binding. In 75 protein-protein complexes, only 50% of the atoms that lose solvent accessibility on binding are located at the interface [136]. The Tyr-42+Ala and Arg-46 +Ala mutants of the protease inhibitor C12 each give a similar decrease in affinity for subtilisin (respectively 2.2 and 3.1 kcal/mol) [137]. However, only Tyr-42 is at the molecular interface. Similarly, directed evolution has been used to change the substrate specificity of aspartate aminotransferase from Asp to Val (giving a 2x106 fold increase in kcar/Km) [138]. This generated 17 mutations, only one of which is in contact with the substrate and with several being more than 10 I$ from the active site. We have already seen how interacting groups may be changed without a large effect on AGO. These data illustrate the converse, how a change in AGO can be seen on modifying the groups which do not interact directly.

CONTEXT-DEPENDENT INFLUENCES UPON BINDING

The contributions of individual groups to binding are not always additive. The presence of one group often enhances, or reduces, the effects of another. Characterization of such context-dependent, or coupling, effects enhances understanding of SAR and so is of value in medicinal chemistry. ITC is a potentially valuable technique to measure coupling because it allows precise measurement of affinity and also reveals enthalpic and entropic effects. Figure 7.6 illustrates a conceptual approach to detect such effects that are transmitted from one part of a molecule to another upon binding. AWAF, AWAB,and AWA,B,represent the free energy changes upon association of pairs of solutes, where one partner remains constant and the other is varied at two positions (A to A’ and B to B’). In medicinal chemistry, the protein is usually the unchanged partner. The coupling energy is given by the equation (xii) where (AG,’&B, - AGiB,)is the change in free energy of binding on moving from A to A’ in the presence of B’ and (AGi,B- A G A B ) is the difference

WALTER H.J. WARD ET AL.

AB

-

A G O A B

AB' AG OAB'

353

A'B AGO,,

c

A'B' AGOKw

Figure 7.6. Analysis of context-dependent influences upon binding. The roupling energy is memured as the difference between the effect of the change from A to A' in the presence of B (top side of square) and that in the presence of B' (bottom side of squure).

in the presence of B. Equation (xii) re-arranges to: (xiii) A value of AG,"= 0 indicates no coupling. A negative AGE demonstrates that coupling enhances binding, whereas a positive AGp shows that coupling hinders association. A similar method of analysis can be applied to other thermodynamic parameters, not only AGO. Limitations in the analysis occur because apparent additivity may arise from cancelling out of the effects of changes (more likely for AGO than AH"), or could be due to group B being located at the wrong position to detect an effect experienced elsewhere in the molecule. This informative analysis was applied to the effects of mutations on AGO for binding and catalysis by tyrosyl tRNA synthetase [139]. ITC demonstrates coupling in AH", but not AGO, between residues in the Glu-Glu-Ile sequence of phosphoTyr peptides binding to the SH2 domain of Src kinase [75]. Likewise, many enthalpic coupling effects are seen for mutants in the interaction between barnase and its protein inhibitor, barstar [56].The significance of coupling in AH" is more difficult to interpret than coupling in AG". GLOBAL VERSUS LOCAL EFFECTS ON ENERGETICS OF ASSOCIATION

1TC can measure the effects of many factors on biomolecular interactions. Characterization of proton movement upon binding is described above.

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By changing the concentration of salt in different titrations, the technique has been used to investigate the importance of ionic interactions [ 140-1431, Binding at different osmotic strengths has been employed to probe the importance of solvation changes [ 144-1 461, with consistent results being obtained for different solutes suggesting that the data are not unduly perturbed by the solutes interacting with the molecules of interest. Solvent reorganization on binding has also been probed by measuring thermodynamic solvent isotope effects. Here, ITC in water is compared with that in D20 [25]. Each of these approaches has the important limitation of measuring the global effect which may represent the overall result of many changes to the interacting system. Each binding process involves many water molecules and ions which vary considerably in the magnitude of their contribution. It is often impossible to interpret the global measured parameters in terms of individual water molecules or ions. It is this local information that is required for exploitation in molecular design. The thermodynamic contribution of individual water molecules has been shown to be variable, depending upon the nature of the local environment (see above).

INSIGHT INTO BIOMOLECULAR INTERACTIONS FROM STUDIES O F PROTEIN FOLDING

There is an extensive literature on the thermodynamics of protein unfolding studied by DSC. The accumulated data have been used to derive empirical relationships aimed at prediction of ACp, AHo and TAS" from changes in polar and apolar accessible surface area [147-1511. Some relationships also include a factor which reflects the packing density of atoms [152]. The reliability of these predictions has been questioned on the basis that insufficient consideration is given to either the effects of the experimental conditions [153] or to the residual structure in the unfolded state [105]. Typical relationships are those of Gomez and Freire [37] AH" = 0.0403 AAp - 0.0229AAap TAS" = 0.02019 AAp - 0.03285AAap AGO = 0.0201 1 AAp 0.00995AAap

+

(xiv) (xv) (xvi)

where the temperature is 300K, predictions are in kcal/mol and AAp and AAap are respectively the polar and apolar accessible surface area changes in A2 (products minus reactants and so usually negative in sign). For

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the purpose of these calculations, serine and threonine are treated as apolar, perhaps reflecting the effects of water of solvation in the unfolded state. These relationships suggest that for protein folding, contributions from polar interactions are enthalpically favourable and entropically unfavourable. Conversely, apolar interactions are enthalpically unfavourable and entropically favourable. In terms of AGO, both polar and apolar interactions are favourable, with polar interactions making around a 2-fold larger contribution per pi2 buried. These approaches have been extended to thermodynamic mapping, where the energetic contributions to binding are assigned to specific groups in the structure of the partners [37, 1301. We consider this to be unreliable when applied to medicinal chemistry. This is because the relationships derived from protein unfolding relate to intramolecular interactions, with large changes in solvent accessible surface area. The empirical parameters are averaged across peptide chemistry which is divided into only two categories, polar and apolar. Current methods in thermodynamic mapping attempt to exploit these average values by assigning contributions to individual groups without taking into account their local environment. The problems become more acute in medicinal chemistry, because the surface area changes are smaller so that behavioural outliers make a relatively greater contribution. Furthermore, the empirical relationships are parameterized on peptide chemistry not on the more diverse range of medicinal chemistry. Use of these algorithms to predict affinity is also compromised because they are not parameterized on intermolecular, but on intramolecular interactions. This perhaps contributes to their greater reliability for predicting AAG" rather than AGO. The energetic contribution of groups that are not at the interface between the protein and test compound is another factor which needs to be taken into account when predicting affinity. This question recently has been addressed. Theoretical methods suggest conformational changes which are consistent with previously obtained experimental data. This agreement is seen with NMR hydrogen exchange data on association of lysozyme with a monoclonal antibody [ 1541and with immobilization of residues in HIV-1 protease upon binding of acetyl pepstatin [ 1551. In our view, it remains impossible to evaluate fully the success of thermodynamic mapping. Agreement with ITC data may be due to errors in individual interactions which compensate in the measured overall parameters. Accordingly, thermodynamic mapping stimulates thinking about molecular design, but should be used with caution (see above).

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Structure-based thermodynamic design Having mapped out thermodynamic contributions in macromolecular interactions, the next step was to predict the energetics of interactions between proteins and small molecule inhibitors, which has been described as structure-based thermodynamic design. This approach appears reasonably accurate for the binding of peptides, presumably reflecting the origins of the empirical relationships in protein folding [156, 1571. In the design of two peptides, empirical thermodynamic algorithms were employed using surface area changes from the structure of the pepstatin A-endothiapepsin complex [I 581. AG" was predicted within 0.5 kcal/mol. Good agreement between observed and predicted AG" was obtained (within f l . 1 kcal/mol) for 13 peptidic HIV-1 protease inhibitors [159]. Thermodynamic mapping is also consistent with the observed reduction in affinity for the Val-82 +Ala mutant which is resistant to inhibitors. However, many authors do not obtain such good agreement between observed and predicted parameter values. Examples include interactions between antigen and antibody [160, 1611, growth hormone mutants and the receptor [57], the enzyme barnase and its protein inhibitor barstar [56], ligands and acyl CoA dehydrogenase [162], and peptides and the OppA transporter protein [ 1221. The uncertainty in predictions is highlighted by different algorithms failing to agree for the same interaction [7], such as that between an antibody and cytochrome c [163]. In one study, there is a striking level of disagreement between observed and predicted values of AH" and TAS" for 9 different protein-ligand complexes [26]. However, many of these predictions required the assumption that bound and free partners remained in the same conformations, because structural data were available only for the complexes. There tends to be enthalpy-entropy compensation in these discrepancies so that predicted AG" values are more accurate than the other predicted parameters. Overall, AG" was predicted to within f 3 . 6 kcal/mol (equivalent to 440-fold in Kd) which is not sufficiently accurate for use in molecular design. However, it should be considered that the structures of both partners were changed in this comparison. Medicinal chemistry can benefit from the prediction of SAR, where the structure of only one partner is changed. For the single case in this data set where only one partner was modified, the change in binding affinity for novobiocin in the Arg- 136 +His mutant of DNA gyrase B was predicted to within 1.4 kcal/mol [26].

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Progress in medicinal chemistry would be transformed if it were possible to use empirical relationships to predict precise values for binding energetics from modelled structures. The first step is to predict the location and orientation of binding (for example using the program GOLD, see [164]). Here, an experimental 3-D structure of a related complex is invaluable [I071 and ITC data can suggest whether the binding mode changes in between the experimental and modelled structures. Having modelled the binding mode and obtained indirect evidence for its validity, the next step is prediction of affinity. This represents a challenge, because it remains difficult to understand observed (not predicted) thermodynamic parameters, even when experimental (not modelled) structures are available [ 104, 1051. These challenges have been addressed by Murphy and colleagues [157, 1651, who highlight factors such as lack of structural data on free partners, contributions from linked equilibria (especially changes in protein conformations and protonation upon binding) and chemical groups that follow behaviour that is distant from the average. These critiques emphasize the need for more thermodynamic data on the binding of drug-like molecules. Several authors highlight how predicted AGO values are more accurate than those for other thermodynamic parameters. This is to be expected given enthalpy-entropy compensation and the sensitivity of AHo and TAS" to the experimental conditions. Of course, this limitation does not invalidate application in medicinal chemistry, because it is AGO which has overall control of affinity. However, inaccurate prediction of AHodoes highlight limitations in the theoretical basis of the approach [165]. Empirical relationships based on protein unfolding data are no more accurate than other computational approaches to predict binding affinity [107]. It is reasonable to expect precision and reliability to improve when more data are available on drug-like compounds. This will require more attention to the structures and interactions (including those with solvent) of the free partners and the local environment of each atom in the complex, rather than a typical contribution averaged across a wide and diverse range. This is likely to be achieved, not by placing atoms in certain classes (such as a hydrogen bond donor), but rather by interpretation of structural data in terms of physical properties including electrostatics, polarization and solvation effects. For example, genetic algorithms may have the potential to predict how different atoms influence the properties of each other.

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FURTHER EXAMPLES OF ITC IN MOLECULAR RECOGNITION AND LIGAND DESIGN

Design of ligands f o r streptavidin This is a potentially informative system to study relationships between structure and binding energetics. This is because biotin has a very high affinity for streptavidin (Kd=40 fM) [166]. Investigation of ligand SAR may reveal strategies to reduce enthalpy-entropy compensation in ligand design. Initial efforts by Weber and colleagues focused on crystallographic and thermodynamic characterization of the binding of natural and synthetic ligands [166]. This approach was extended to the design and characterization of azobenzene ligands [ 1671. These studies give insight into the thermodynamic consequences of displacing water molecules from the interface between the ligand and the protein. There is some correlation between the magnitude of the temperature factors (B-values) derived from X-ray crystallography and the apparent entropic contribution in formation of protein-ligand complexes. However, caution is required because entropic factors (such as flexibility and vibration) are not the only influences that can increase B-factors. Accordingly, interpretation is more reliable when comparing B-values for different atoms in the same structure, rather than between structures. A different class of ligand also has been investigated [168]. A heptapeptide binds with similar affinity to the azobenzenes, but association of the peptide is exothermic (like biotin), in contrast to endothermic binding of azobenzenes. This expected relationship correlates with the relative importance of polar (exothermic) and hydrophobic (endothermic) interactions. It appears that high affinity interactions, such as that with biotin, often involve electrostatic polarization of the ligand to enhance interactions with the binding site, rather like stabilization of the very tightly bound transition state in enzyme catalysis. These studies show that similar hydrogen bonding for different ligands gives little indication of affinity, nor of whether binding is enthalpy or entropy driven. Residual disorder of the ligand after binding, or displacement of bound water molecules both appear to make favourable entropic contributions. It is suggested that lead compounds may be discovered by screening with flexible ligands (such as peptides), followed by experimental determination of bound conformation and then making ligands which are restricted to the bound conformation in order to obtain an entropic advantage. Increasing rigidity, of course, is a common approach to increase affinity in medicinal chemistry. The degree of success depends on many factors,

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including the complementarity of the fixed ligand conformation for the target protein and the extent to which rigidification compromises the rate constant for association of the protein-ligand complex. Hydrophobic interactions in a cavity engineered into lysozyme The binding of 91 nonpolar compounds into a cavity engineered in T4 lysozyme has been characterized [ 1691. Sixteen ligands were studied by ITC. Experimental data were interpreted in terms of desolvation, immobilization and atomic packing. A poor correlation was found between affinity and AGO for solvent transfer, underlining the importance of shape in hydrophobic interactions. The analysis also suggests that interaction strength is highly dependent upon induced fit. The energetics could not be predicted with precision. Recognition of S-peptide by ribonuclease S-protein Studies by Sturtevant and colleagues have proved particularly informative, highlighting the limitations of applying empirical relationships to predict the consequences of structural changes [170]. This is a favourable system for investigation, because there is a wealth of 3-D structures and calorimetric data. Furthermore, the ligands are peptidic which should minimize difficulties in applying empirical relationships derived from protein unfolding. These studies have analogy to medicinal chemistry in that the changes made to ligand structure are small, as they are in lead optimization during drug discovery. Ribonuclease A consists of an S-peptide (residues 1-20) which reversibly docks onto the S-protein (residues 21-1 24) to produce the catalytically active enzyme. Residues 16-20 of the S-peptide are not important for binding, allowing study using a truncated peptide consisting of the first 15 residues. X-ray crystallography shows that Met-1 3 is almost fully buried upon complex formation. In an investigation of hydrophobic interactions, this residue was substituted with 7 other amino-acids ranging in size from glycine to phenylalanine and then binding was characterized by ITC [ 1701. Again, there is an inconsistent relationship between AAGO for binding and that for solvent transfer. Subsequent studies show a lack of correlation between measured ACp values and the change in solvent accessible surface area upon binding [lo]. On modifying the structure of the ligand, the observed changes in ACp are of similar magnitude to the degree of uncertainty in the parameter value.

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AACp varies from -50 to +270 cal/(K mol) for Met-13-tAla and Met-13 +Val respectively. The estimated standard error of each AACp is 180 and 130 cal/(K mol) respectively. These values were published in 1992 and more modern calorimeters have increased sensitivity which may improve the signal to noise ratio. Crystal structures were determined for each complex. The analysis implies that there is no clear relationship between AAap and ACp for such small changes in structure. It is suggested that low frequency vibrational modes in the complex may account for some of the discrepancies. Further investigation was made of a very small change in structure in order to increase the possibility of interpretable data [171]. A sulfur atom was changed to a methylene group in the Met-13 to norleucine peptide. There was a striking enthalpy-entropy compensation (AAG" = +0.8 kcal/mol, AAH" = +7.9 kcal/mol) and a high resolution (1.85 A) crystal structure was determined. Atoms of the ligand residue at position 13 in the complex have high B-values, suggesting that residual entropy may contribute to the large favourable TAAS" (+7.1 kcal/mol). The difference in binding appears to be largely due to the shift in chemical and dynamic behaviour moving from -S- to -CH2- and not differences in the geometry of the bound ligand nor its binding site. These studies reveal how subtle changes can have large effects on thermodynamic parameters which may be masked in AGO. Conversely, large changes in AH" do not necessarily reflect a major change in binding mode.

STRUCTURAL AND THERMODYNAMIC STUDIES TO HELP OPTIMIZATION OF DNA GYRASE INHIBITORS ITC SHOWS SMALL INACTIVE FRAGMENTS ARE VALID MODELS FOR INTACT GYRASE

The enzyme DNA gyrase catalyses the ATP-dependent supercoiling of DNA and is a target for antibacterial agents. In E. coli, it consists of two subunit types, A and B, of molecular masses 97 and 90 kDa respectively, in an A2B2tetramer. The coumarin antibiotics, such as novobiocin, function by inhibiting the ATPase activity of the B subunit [172]. The gyrase B subunit contains a 43 kDa N-terminal domain which includes the binding site for ATP. The crystal structure of this 43 kDa fragment complexed with a hydrolysis-resistant ATP-analogue, adenylyl-B-y-imidodiphosphate (ADPNP), has been determined [ 1731. The compound promotes dimeriza-

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Asn-46 ASP-73

Hp0

Tyr-S

4”” 0 I

HN

Figure 7.7.Purt of the crystal structure ofthe complex between ADPNP anda 43 kDa,fragment from the B subunit of DNA gyrase [I 731. Tyr-5’ belongs to the opposite subunit of the dimer. Hydrogen bond.7 are shown us dushed lines and a hydrophobic interaction is indicated by an arc.

tion of the fragment in solution [174, 1751 and the protein-ligand complex crystallizes as a dimer containing one bound ADPNP molecule per monomer. The adenine N-3 nitrogen accepts a hydrogen bond across the subunit interface from the hydroxyl group of Tyr-5’ from the N-terminal arm of the other monomer (Figure 7.7). The main chain carbonyl group of this residue also forms a hydrogen bond across the dimer interface with guanidinium group of Arg- 136. Each 43 kDa fragment contains two sub-domains: an N-terminal (24 kDa) and a C-terminal domain. Attempts to co-crystallize the 43 kDa fragment with various inhibitors have not been successful. However, crystal structures of the 24 kDa fragment complexed with novobiocin, or other antibiotics, have been reported [176, 1771. ITC on the catalytically inactive 43 and 24 kDa fragments [26] shows that the Kd values of novobiocin are similar to the inhibition constant for intact DNA gyrase in assays of

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ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

DNA supercoiling and ATPase activity [178]. These data suggest that the fragments are valid models of intact gyrase for structural and thermodynamic studies.

STRUCTURAL AND THERMODYNAMIC BASIS OF RESISTANCE TO NOVOBIOCIN

Novobiocin (1 1) prevents dimerization of the 43 kDa fragment in solution [174, 1751. Similarly, the crystal structure of the novobiocin liganded 24 kDa fragment is monomeric [176]. However, many of the amino-acid residues that bind the adenine moiety of ADPNP to the 43 kDa fragment also interact with novobiocin in the complex with the 24 kDa fragment. The coumarin ring of novobiocin replaces the absent Tyr-5’ in a direct interaction with the guanidinium group of Arg-136 (Figure 7.8). This residue appears important in coumarin binding, since resistant isolates of E. coli have mutations of Arg-136 (to His, Ser or Cys [179]). Site-directed mutagenesis has been used to prepare an Arg-136 +His 24 kDa fragment [26]. This change was selected because it is the naturally occurring variant that is most closely related to the wild-type in terms of side-chain character.

He-94

I

HN

Figure 7.8. Part o f t h e crystul structure of the complex between novobiocin (1 1) and a 24 kDu frugment from the B subunit of D N A gyruse [26]. Hydrogen bonds are shown us duslied lines and LI hydrophobic interuction is indicated by an arc.

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ITC has been used to characterize the binding of novobiocin to wild-type and His-136 mutant 24 kDa fragments [26]. On mutation, the Kd increases from 32 to 1200nM. The enthalpy of binding is more favourable for the mutant (AHo shifts from -12.1 to -17.5 kcal/mol) and the entropy

1

of binding is much more unfavourable (TAS" changes from -1.8 to -9.4 kcalimol). Both of these changes are in the opposite direction to that expected if loss of the Arg residue reduces hydrogen bonding. The observed change in ACp, from -295 to -454 cal/(mol K) cannot be attributed to a common cause which is increased burial of the hydrophobic surface. The crystal structure of a complex between the His-I36 24 kDa fragment and novobiocin [26] shows the sequestration of a water molecule into the volume vacated by removal of the guanidinium group (Figure 7.9). This water molecule appears to make a favourable enthalpic contribution by forming hydrogen bonds which link novobiocin to the mutant protein. Immobilization of the water leads to an entropic penalty and a negative contribution to ACp (see above). These studies explain the importance of mutation of Arg-136 in antibiotic resistance. CHARACTERISTICS OF INHIBITION BY TRIAZINES

The binding of a second class of inhibitors, the triazines, to the 24 kDa fragment of DNA gyrase from E. coli also has been investigated [180]. As expected for this more hydrophobic series, binding tends to be both entropy and enthalpy-driven, in contrast to the enthalpy-driven association of novobiocin (Table 7.5). For a tris(m-fluoroanilino) triazine with a solubilizing, charged p-quaternary amine derivative on one fluoroaniline (12), X-ray crystallography indicates that the anilines occupy three subsites, respectively containing Asp-73, Ile-94 and Arg-136 (Figure 7. lo). The symmetrical

364

ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY Glv-77

HN

Arg-136

Thr-80

R2

OH Gly-7i Arg-76

R1

Figure 7.9. Part ofthe crystal structure of the complex of novobiocin ( I I ) with (upper) wild-type 24 k D a fragment f r o m the B subunit of D N A gyrase and (lower) an Arg-I36 -+His antibiotic resistant mutant [26]. Hydrogen bonds are shown as dashed lines.

Table 7.5. THERMODYNAMICS OF BINDING TO A 24 kDa FRAGMENT OF DNA GYRASE AT 300K

Compound

K,I ( p h i )

(kcal/mol)

AH" (kcal/mol)

TAS" (kcal/mol)

Novobiocin ( 1 1) Triazine (12)

0.032 0.67

-10.3 -8.4

-12.1 -1.2

-1.8 +1.2

AGO

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lle-94

Ala-47

'

Arg-76

HNY \o

Asp-73

Arg-136

~d

Figure 7.10. Part of the crystal structure of the complex between a triazine (12) and a 24 kDa fragment from the B subunit of D N A gyrase [180]. Hydrogen bonds are shown as dashed lines and hydrophobic interactions are indicaied by arcs.

aspects of these inhibitors raise the possibility that changes in structure could either cause local modifications in interactions with the target protein, or modify the mode of binding. Reorientation of the inhibitor would cause a discontinuity in SAR which is likely to mislead medicinal chemistry. Structural and thermodynamic studies provide a level of understanding which assists in the optimization of the triazines.

(12) R' = R2 = F, R3 = NH. (13) R' = H, R2 = F, R3 = NH.

The binding of 1 1 triazines to the 24 kDa fragment has been characterized by ITC. There is a marked enthalpy-entropy compensation (Figure 7.11, the slope of the best fit line for AH" versus TAS" is 0.95zt0.02), with AH"

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2

8

-6

-10

-18

-16

-14

-12

-10

-8

-6

-4

AHo (kcaVmol) Figure 7 . 1 1 . Thermodynamics of inhibitors binding to the 24 kDa fragment of D N A gyrase at 300K. Open symbols: T A P ; filled symbols: AG"; circles: triazines; squares: coumarins. Best f i r lines are shown for the triuzine data.

varying between -5.6 and -17.2 kcal/mol, whilst AGO remains relatively constant between -7.4 and -8.8 kcal/mol. There is very little correlation between AH' and AGO (the slope of best fit line is 0.05 d~0.02).These data highlight how enthalpy-entropy compensation generates a challenge for the medicinal chemist who is aiming to improve affinity. Only three coumarins have been analyzed, so that the data are less conclusive. The results are again consistent with enthalpy-entropy compensation, although the coumarins tend to have higher affinity than the triazines. A LARGE CHANGE IN AH' CORRELATES WITH A DIFFERENT BINDING MODE FOR TRIAZINES

Five different modifications to any one of the aniline substituents of the triazines give only small changes in binding energetics (relative to structure (12), AAH" = -1.4 to +1.6 kcal/mol and AAG" = -0.3 to +1.0 kcal/mol) [ 1801(Figure 7.12). Crystallography (inhibitors 12-14) or NMR (17) on four of these complexes indicates that, in all cases, the charged substituent reaches solvent via the Ile-94 subsite (Table 7.6). The small changes in energetics reflect modified hydrogen bonding, where the overall shifts in differences between bound and free partners are small. This appears

WALTER H.J. WARD ET AL.

/o\ A N A N D A

F

H N

N

H

(15) R' = F, R2 = H, R 3 = NH. (16) R' = F, R2 = H, R3 = 0.

F

H

H

R'

361

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ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

-8

-6

-4

-2

0

2

M G " or M H " (kcallrnol)

Figure 7.12. Binding energetics relative to compound (12)f o r inhibitors associating with a 24 k D a fragment of D N A gyrase [180]. Hatched bars. BAG"; White bars. AAH".

Table 7.6. SOME KEY FEATURES O F COMPLEXES BETWEEN TRIAZINES AND A 24 kDa FRAGMENT FROM DNA GYRASE

Compound

H-bond acceptor f o r Arg-136

Solubilizing group and route to solveni

Fluoroanilino F None from ligand Cyclic urea 0 As (12) As (12) As (12) Pyridine N-oxide

Quaternary amine via Ile-94 subsite As (12) As (12) As (12) As (12) Hexanoate via Ile-94 subsite Propanoate via Arg-136 subsite

From reference [NO]. Complexes containing (12-14) were characterized by X-ray crystallography, Those with (17) and (18) were studied by measuring nuclear Overhauser effects in NMR. The other structures were predicted using molecular modelling.

common for changes in hydrogen bonding groups (see above). Molecular modelling suggests that the bridging -0-(R3)in (16) acts as a hydrogen bond acceptor from the side-chain amide of Asn-46, whereas the bridging -NH- in compounds (12-15) and (17) seems to donate a hydrogen bond to the same amide. The changes in energetics on moving between several different triazines (12-17) are all smaller than the shift in moving from these triazines to the coumarin, novobiocin (1 1) (Figure 7.12). However, another triazine, (18), shows an even larger change in energetics [180]. In this com-

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.. HN+ Asp-73

do

Arg-136

vo I HN

Figure 7.13. Part of thesoluiion siruciure of the complex between a iriazinepyridine N-oxide (1 8 ) and a 24 kDa fragmeni f r o m the B subunit of D N A gyrase [180]. Hydrogen bonds are shown us dashed lines and hydrophobic interaciions are indicated by arcs.

pound, the major changes are replacement of two of the m-fluoroanilines by a primary amine and a m-pyridine N-oxide. This 5-propanoate pyridine N-oxide triazine binds with a large change in enthalpy (AAHO = -8.6 kcal/mol) relative to the original tris(m-fluoroanilino) triazine (1 2). This is not linked with a large shift in affinity (AAG" = -0.4 kcal/mol, a 1.7-fold change in Kd) (Figure 7.12). NMR suggests that the charged propanoate reaches solvent via the Arg-136 subsite, not the expected Ile-94 subsite [180] (Table 7.6 and Figure 7.13).Many factors may contribute to the large shift in AH" for (18) compared to the other triazines. However, these data do suggest that an altered binding mode may be a major contributor to the large AAH", as seen for occupancy of alternative subsites by stromelysin inhibitors (Table 7.3 and [29]). Changes in enthalpy or binding mode may not be detectable in Kd. Measured AH" values, therefore, can highlight discontinuities in SAR which increase errors in structure-based design from related protein-inhibitor complexes. Observation of large changes in AHo (more than 2 kcal/mol) suggest that drug design should be based on an experimentally determined structure, rather than extrapolating from a related complex (Figure 7.3). Thus, ITC could be particularly informative in projects where there is some symmetry in the structure of the inhibitors, or when the target protein exists in multiple conformations (such as basal and activated kinases), or has multiple binding sites (multisubstrate and allosteric enzymes).

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ISOTHERMAL TITRATION CALORIMETRY IN DRUG DISCOVERY

FUTURE PROSPECTS Calorimetry is already a powerful technique in medicinal chemistry. Improvements in technology will widen further the application of calorimetric methods. Recently launched ITC instruments have improved sensitivity (Microcal VP-ITC), or automation (Calorimetry Sciences Corporation JTC) which allows increased throughput. Increased sensitivity brings the possibility of monitoring smaller heat changes, measuring tighter binding affinities, reducing the use of protein and avoiding some consequences of limited ligand solubility. This increases the potential for ITC to characterize binding interactions that are outside the scope of older machines. Integrated circuit calorimeters have been manufactured [ 18 I] which could lead to ITC in small volumes on silicon chips in 96-well plates. At present, the sensitivity is not sufficient to allow routine characterization of biomolecular interactions. Overcoming this limitation could take calorimetry into the realm of high throughput screening. Combination of ITC data with experimentally determined 3-D structures and a wide range of computational approaches will improve our understanding of molecular recognition. A key advance will be the ability to reduce the extent of enthalpy-entropy compensation when designing new compounds. Detailed studies on ligands that exhibit much higher affinities than close analogues should reveal strategies to achieve this goal. ITC will increase efficiency and creativity when medicinal chemistry is applied to the optimization of lead compounds.

REFERENCES 1 Eftink, M. and Biltonen, M. (1980) in Biological Microcalorimetry (Beezer, A.E., ed.), pp. 343-412, Academic Press, London. 2 Connelly, P.R. (1994) Curr. Opin. Biotechnol. 5, 381-388. 3 Sturtevant, J.M. (1994) Curr. Opin. Biotechnol. 4, 69-78. 4 Cooper A. and Johnson, C.M. (1994) in Methods in Molecular Biology, Vol. 22: Microscopy, Optical Spectroscopy and Macroscopic Techniques (Jones, C., Mulloy, B. and Thomas, A.H., eds), pp. 109-124, Humdna Press, Totowa, N.J. 5 Cooper A. and Johnson, C.M. (1994) in Methods in Molecular Biology, Vol. 22: Microscopy, Optical Spectroscopy and Macroscopic Techniques (Jones, C., Mulloy, B. and Thomas, A.H., eds), pp. 137-1 SO, Humana Press, Totowa, N.J. 6 Ladbury, J.E. and Chowdhry, B.Z. (1996) Chem. Biol. 3, 791-801. 7 Cooper, A. (1999) Curr. Opin. Chem. Biol. 3, 5577563. 8 Jelesarov, I. and Bosshard, H.R. (1999) J. Mol. Recognit. 12, 3-18.

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Subject Index AB-MECA, 80, 81 ['*'I]-AB-MECA, 80, 82 Adenosine, 78, 79 Adenosine A2* agonists, 63 Adenosine A3 agonists, 79-85 fl-benzyladenosine-5'-uronamides,79, 81 N6-carbamoyl-NECA derivatives, 83 cardiac ischaemia and, 76, 77 N6-S-disubstituted 2-chloro-adenosines, 83, 84 4'-furanose modified-2-chloro N-methoxy adenosines, 84 histamine release by, 77 inhibition of pro-inflammatory cytokine production, 78 MECA and NECA derivatives, 81 stroke and, 76 structural effects on selectivity, 80 structure-activity relationships of, 79-85 suppression of neutrophil levels, 78 Adenosine A3 antagonists, allergic diseases and, 78 1,4-dihydropyridines, 91, 92, 104 flavanoids, 88, 90, 10 isoquinolines and quinazolines, 97- 101 pharmacophore models for, 104 pyrans, 95, 96 pyridinium derivatives, 93-95 receptor models, 102-105 triazolonaphthpyridines and thiazolopyrimidines, 88 triazolopyrimidines, 101 triazoloquinazolines, 96-10 1 xanthine derivatives, 85-87 Adenosine A, partial agonists, 85, 86 fl,S'-disubstituted adenosines, 85, 86 xanthine-7-ribosides, 85 Adenosine A3 receptors, amino acid sequences, 65 antagonist affinities, 70, 68 cDNA libraries, 63 clones encoding for, 67

functional assays, 71 mechanism of activation, 69, 70 pharmacology, 66-79 philogenetic tree, 65 physiological effects mediated by, 72-75 species differences, 66-69 therapeutic applications of ligands, 71-78 Adenosine receptors, agonist selectivity, 79 molecular biology, 63 philogenetic tree, 66 subtypes, 62 Adenyl-8, y-imidodiphosphate (ADPNP), binding to DNA gyrase, 360, 361 Alnespirone, 207, 208, 209 Angiogenesis, role of p38 MAP kinase in, 36, 37 ANTS, I24 Ap4A, 121, 153 APNEA, 78 Apoptosis, role of p38 MAP kinase in, 32-35 A3P5PS. 147 AQ 0145, 290 AR-C69931MX, 177 Arylbutanoic acids, metabolism, 257 ATP, 2-substituted analogues, 22, 121 Azomethine derivatives of lustamine H3 agonists, 286, 287 fl-Benzyladenosine, 79, 81, 102 fl-BenzylNECA, 79 Betahistine, 283, 296 Binding thermodynamics, interpretation of, 339 measurement of, 314-327 parameters for drug-like molecules binding to proteins, 321 BP 2.94, 286, 287, 298 6BPWAY, 209, 222 BstlOl, 123, 125 Burimamide, 280 Buspirone, 195

378

SUBJECT INDEX

BWA 1433, 87 BWA 522/IABOPX, 87 Calmidazolium, 138 Carboperamide, 290 CCPA, 78 CGP 45715A (iralukast), 255 CGS 15943, 70, 96, 104, 105 CGS 15943, 97, 103-105 CGS 21680, 78 Ciproxifan, 294 2-CI-IB-MECA, 82 Clobenproprit, 291, 292 Clopidogrel, 171, 177 Clozapine, 296 CNI-1493, 37 Coomassie brilliant blue G, 132 CPA (N6-cyclopentyl adenosine), 78, 102 CPC-222, 209, 220 [“C]CPC-222, 220 CS-747, 176 CV 1674, 62, 78 CV 1808, 63, 78 Cysteinylleukotrienes, biosynthesis, 252 Cytochrome P450, inhibition by pyridinylimidazoles, 18-21, 47 Cytokine production, inhibition by pyridinylimidazoles, 4, 17-21 Cytokine-supressive binding proteins (CSBP), 5, 6 Desmethyl-WAY (DWAY), 209, 216 [Carbonyl-’ CIDesmethyl-WAY- 100635, 2 16 Diadenosine polyphosphates, 122 DIDS, 137 a, b-Dimethylhistamine, 286, 287 Dinucleotide tetraphosphates, 153 DNA gyrase inhibitors, optimization by ITC, 360-369 DNDS, 137 DPCPX, 70, 87 EMD 66843,237 Evans blue, 128 cis-FCWAY, 210, 225, 228 trans-FCWAY, 210, 225-228 FEWAY, 210, 224

FK 506 (tacrolimus), 346 Flumizole, 16 FPL-55712, 250, 252 analogues, 256-259 FPWAY, 224 6FPWAY. 209, 222 FR 167653, 22, 23 FUB 120, 291, 293 FUB 136, 291 FUB 181, 294 FUB 217, 293 FUB 316, 289 FUB 322, 293 FUB 373, 290 FUB 407, 290 FUB 475, 289 FU-MPPI, 210, 233, 234 Galangin, 89 Galardin, 350 [3H]-GR 168320, 297 GR 175737, 293 G T 2016, 290 G T 2227, 295 G T 2331 (perceptin), 295. 299 HzDIDS, 137 H3 heteroreceptor, 280 HE-EA, 103 HEP 689 (SB 235699), 21, 43, 48 Histamine H3 agonists, 284-289 Histamine H3 autoreceptors, 280-282 Histamine H3 ligands, assays for, 283-284 potential clinical applications, 298, 299 Histamine H3 receptor agonists, 284-289 functional responses, 298 pharmacophore model, 288 transduction mechanism, 280 Histamine H3 receptor antagonists, 290-297 behavioural effects, 299 enhancement of histamine turnover, 282 imidazole derivatives, 290-296 non-imidazole derivatives, 296, 297 structure-activity relationships, 29 1-297 Histamine H3 receptor partial agonists, 289, 290 Histamine H3 receptor radioligands, 297 PET and SPECT studies with, 298

SUBJECT INDEX Histamine turnover, enhancement by H3 antagonists, 282 HYMAP, 207, 208, 209 IAB-MECA, 81 IB-MECA, 80, 81 IB-MECA, 80, 81 ICI-204219 (zafirlukast), 260, 265 Imetit, 288 Imifuramine, 288 Immepip, 287 Immepyr, 287 Imoproxifan, 295 Impentamine, 289 Inosine, 78,79 INS-316 (UTP), 155, 158 INS-365, 159 Interfacial water molecules, 345-349 Iodoproxyfan, 289, 297 lralukast (CGP 45715A), 255 Isothermal titration calorimeters, 317, 318, 370 Isothermal titration calorimetry (ITC), applications, 314 characterization of active compounds by, 337 characterization of binding, 336-338 characterization of target proteins and their ligands, 327-339 collection of data, 317-320 in molecular recognition and ligand design, 339-360 limitations of, 313 measurement of binding thermodynamics, 3 14-3 17 measurement of critical micelle concentration and partitioning into lipids, 335 protein preparations for, 332-334 technique o f , 311-314 thermodynamic parameters, 320 KN-04, 138 KN-62, 138 L-167307, 22, 26, 41, 46, 47 L-249313, 70, 88 L-268605, 88

379

L-64805 1, 258 L-649923, 258 L-660711 ( M K 4 5 7 I ) , 260, 262, 263, 265 L-668018, 264 L-668019 (MK-0679, verlukast), 264, 265 L-669392, 266 L-695499, 266 L-706631 (MK-0476, montelukast), 269-272 Leukotriene A, 251 Leukotriene A4. 252 Leukotriene C, 250 Leukotriene C4, (LTC4), 251 Leukotriene D4 (LTD4), 251, 265 [3H]-LTD4,252 Leukotriene D4 antagonists, 253-272 clinical studies, 256, 258-260, 263, 265, 271, 272 first generation, 253-259 FPL-55712 analogues, 256-259 in vitro assays, 253 in vivo assays, 254 leukotriene analogues, 255 second generation, 259-272 Leukotriene D4 receptor (CysLT-I), 253 Leukotriene E4 (LTE4), 251 Leukotriene receptors, 252 Levamisole, 16 LPS (lipopolysaccharide-stimulated IL-I and TNF) production, inhibitors of, 4 LY-171883, 259 MECA, 81 MeFBWAY, 210, 231 cis-MeO-WAY, 209, 221 trans-MeO-WAY, 209, 220, 221 N"-Methylhistamine, 255, 284 (R)-u-Methylhistamine, 283-286 Mitogen activating protein kinase kinase (MAPKK), 8, 10 MK-0571 (L-66071 I ) , 260, 262, 263, 265 Montelukast (MK-0476), 269-272 MPP, 228, 229 p-MPPC, 209, 219 0-MPPF, 228, 229 p-MPPF, 209, 219, 228-231 m-MPPI, 210 o-MPPI, 210 p-MPPI, 210, 233

380

SUBJECT INDEX

MPPN, 228 MRS 1191, 92, 93 MRS 1220, 96, 97 MRS 2179, 148, 149 MWAY, 210, 223 NECA, 78, 79, 81 NF019, 124, 125 NF023, 123, 125 NF279, 125 NHO1, 128 NH05, 127, 128 Nicardipine, 91 Nifedipine, 91 (S)-Niguldipine, 91 Novobiocin, binding to DNA gyrase, 362 Nucleotides, 2’-0-2,4,6-trinitrophenyl substituted, 129 8-OH-DPAT, 194, 209 [3H]8-OH-DPAT, 206 8-OH-PPSMAT, 207, 209 O R G 13502, 208, 209 OSU 191, 206, 209 P2 receptor modelling, 2PYI, 149 2PY2, 158 P2 receptors, nomenclature, 118 P2T receptors and their ligands, 165-177 P2X receptors and their ligands, 119 heteromeric, 140-141 P2X,, 120-130 P2X2, 130 P2X3, 130-131 P2X4, 131-133 P2Xs, 133 P2X6, 133-134 P2X7, 134-1 39 P2Y receptors and their ligands, 141 P2Y1, 141-150 PY2, 150-159 P2Y3, 159 P2Y.4, 159-161 P~Ys,161 P2Y.5, 162-163 P2Y7.10, 164 P2Yl1, 164-165 P5P, 137

p38 MAP kinase inhibitors, animal models, 40-45 clinical studies and therapeutic potential, 45-48 effect on bone and cartilage degradation, 37-40 mutagenesis studies, 29, 30 non-imidazoles, 2 1-25 oral activity of, 46 pharmacology, 3 0 4 5 pyridinylimidazoles, 13-2 1 structural basis of kinase selectivity, 25-29 p38 MAP kinases, 5 activation by viruses, 37 inhibitors, 15-30 isoforms, 6-8, 10, 26, 29 kinase substrates of, 10-12 pathways, 10 role in apoptosis, 32-35 sequence alignment of homologues, 6 transcription factor substrates, 12 upstream activators, 8 p38 MAP kinase pathways, 10 in regulation of metalloproteinases, 39 p38a kinase activity, inhibition by pridinylimidazoles, 13-1 5 p38~1,mutagenesis studies, 29 PD 98059, 39, 40, 48 PD 166793, 350 PD 180557, 350 PENECA, 82 Pentamethyl-morin, 90 Perceptin (GT-2331), 295, 299 Peroxisomal enzyme, induction by LTD4 antagonists, 266-270 Peroxisome proliferation activated receptor (PPAR), 264, 269 R,5’-PHPNECA, 82 R,S-PIA, 78 Pindolol, 195, 236, 237 PNU-99533, 331 PNU-143677, 331 PNU-143988, 331 PNU 157760, 208, 209 PPADS, 125, 161, 170 analogues, 126 Prodrugs, azomethine derivatives of H3 agonists, 286, 287

SUBJECT INDEX Pro-inflammatory cytokines, 2 Proton emission tomography (PET), 201,202 PyMPPF, 210, 231 Pyridinylimidazoles, inhibition of cytochrome P450 by, 18-21, 47 inhibitors of cytokine biosynthesis, 17-21 structural basis for kinase selectivity, 25-29 synthesis, 18, 20 X-ray crystal structure of complexes with p38, 27-29 Radioligands for PET, properties, 209, 210, 238 requirements for molecular imaging, 202 synthesis, 203-205 Radioligands for SPET. 205, 206 properties, 209, 210 requirements for molecular imaging, 202 Rapamycin, 346 Reactive blue 2, 132 REV-5901, 260, 261 Robalzotan, 195, 209 RPR 200765, 46, 47 RWJ 67657, 46 RWJ 68354, 23, 41, 46, 47

S 15535, 237 Sabeluzole, 296 SB 202190, 4, 5, 33 SB 203580, 10, 12-14, 19, 26, 30, 33-40, 4 3 4 7 , 49 SB 206718, 4 [1251]-SB206718, 5 SB 210313, 19, 45, 46 SB 220025, 27, 28, 41, 42, 46 SB 226882, 46, 47 SB 235699 (HEP 689, VK 19911), 21, 43, 48 SB 238039, 21 SB 239063, 26, 42, 46 SB 242235, 38-41, 4 6 4 8 SB 242325, 26 Sch 50971, 287 Serotonin 5-HTIAagonist radioligands, 206 Serotonin 5-HTIAagonists, therapeutic role for, 200 Serotonin ~ - H T I A antagonist radioligands, 21 1 "C-labelled, 21 1-223

38 1

L231-labelled,232-234 '*F-labelled, 223-232 in clinical research, 235 in drug research, 235-238 properties of, 209, 210, 238 Serotonin 5-HTIAreceptors, function in the brain, 197 imaging in vivo, 196, 200, 212 implication in psychiatric disease, 198-199 involvement in neuropsychiatric disorder therapy, 199 ligand binding, 194-195 location in the brain, 195 structural features, 192-194, 196, 197 Serotonin receptors, 190-192 SG 58272, 337 Single photon emission tomography (SPET), 202 SITS, 137 SK&F 81114, 15, 16 SK&F 86002, 4, 16, 37, 38, 41, 44-46 SK&F 86055, 16 SK&F 102922, 255, 260 SK&F 104351, 19, 41, 44 SK&F 104353, 255 SK&F 105809, 16, 19, 41, 44 Slow reacting substance of anaphylaxis (SRS-A), 250, 251 8-SMeDPAT, 207, 209 (R)%SMeDPAT (LY 24601), 207, 209 SSRIs, potentiation of antidepressant effect by 5-HTIAantagonists, 199, 235-238 Streptaviridin, ligand design by ITC, 358 Stromolysin inhibitors, binding to DNA gyrase, 330, 331 Suramin, 121, 123, 125, 131, 161, 170 analogues, 125 Thioperamide, 282, 292 Ticlopidine, 171 1,3,5-Triazines. binding to DNA gyrase, 363-369 Trifluproxim, (UCL 1470), 289 Trypan blue, 127, 128 U 0126, 48 UCL 1199, 293 UCL 1283, 290

382 UCL 1390, 294 UCL 1470 (trifluproxim), 289 Verlukast (MK-0679), 264, 265 VK 19911 (HEP 689), 21, 48 VU 8504, 98 VUF 5574, 100 VX 745, 24, 41, 47, 48 WAY 100634, 213, [llC]WAY-l00634, 213, 215 WAY 100635 (WAY), 194, 209, 238 "C-labelled analogues, 217-223 '231 -labelled analogues, 232, 233

SUBJECT INDEX "F-labelled analogues, 223-232 metabolism, 215 technetium-99m labelled ligands, 234 [Carbonyl-"CIWAY- 100635, 214, 21 5 [3H]WAY-100635, in autoradiography, 196, 197 [O-methyL1'C]WAY-100635, 213 XAC, 70, 87 XAMR0716, 124 Zafirlukast (ICI-204219), 264, 265 ZM 336372, 25, 26

Cumulative Index of Authors for Volumes 1-38 The volume number. (year of publication) and puge number ure given in rhat order.

Adams, J.L., 38 (2001) 1 Adams, S.S., 5 (1967) 59 Agrawal, K.C., 15 (1978) 321 Albrecht, W.J., 18 (1981) 135 Allain, H., 34 (1997) 1 Allen, N.A., 32 (1995) 157 Allender, C.J.. 36 (1999) 235 Andrews, P.R., 23 (1986) 91 Ankier, S.I., 23 (1986) 121 Arrang, J.-M., 38 (2001) 279 Badger, A.M., 38 (2001) 1 Bailey, E., 11 (1975) 193 Ballesta, J.P.G., 23 (1986) 219 Banting, L., 26 (1989) 253; 33 (1996) 147 Barker, G., 9 (1973) 65 Barnes, J.M., 4 (1965) 18 Barnett, M.I., 28 (1991) 175 Batt, D.G., 29 (1992) 1 Beaumont, D.. 18 (1981) 45 Beckett, A.H., 2 (1962) 43; 4 (1965) 171 Beckman, M.J., 35 (1998) 1 Beddell, C.R., 17 (1980) 1 Beedham, C., 24 (1987) 85 Beeley, L.J., 37 (2000) 1 Beisler, J.A.. 19 (1975) 247 Bell, J.A., 29 (1992) 239 Belliard, S., 34 (1997) 1 Benfey, B.G., 12 (1975) 293 Bentue-Ferrer, D., 34 (1997) 1 Bernstein, P.R., 31 (1994) 59 Binnie, A,, 37 (2000) 83 Black, M.E., I I (1975) 67 Blandina, P., 22 (1985) 267 Bond, P.A., 11 (1975) 193 Bonta, I.L., 17 (1980) 185 Booth, A.G., 26 (1989) 323 Boreham, P.F.I., 13 (1976) 159 Bowman, W.C., 2 (1962) 88

Bradner, W.T., 24 (1987) 129 Bragt, P.C., 17 (1980) 185 Brain, K.R., 36 (1999) 235 Branch, S.K., 26 (1989) 355 Braquet. P.. 27 (1990) 325 Brezina, M., 12 (1975) 247 Brooks, B.A., 11 (1975) 193 Brown, J.R., 15 (1978) 125 Brunelleschi, S., 22 (1985) 267 Bruni, A,, 19 (1982) 111 Buckingham, J.C., 15 (1978) 165 Bulman, R.A., 20 (1983) 225 Carman-Krzan, M., 23 (1986) 41 Cassells, A.C., 20 (1983) 119 Casy, A.F.,2(1962)43;4(1965) 171; 7(1970) 229; 11 (1975) I; 26 (1989) 355 Casy, G., 34 (1997) 203 Caton, M.P.L., 8 (1971) 217; 15 (1978) 357 Chambers, M.S., 37 (2000) 45 Chang, J., 22 (1985) 293 Chappel, C.I., 3 (1963) 89 Chatterjee, S., 28 (1991) 1 Chawla, A.S., 17 (1980) 151; 22(1985) 243 Cheng, C.C., 6 (1969) 67; 7 (1970) 285; 8 (1971) 61; 13 (1976) 303; 19 (1982) 269; 20 (1983) 83; 25 (1988) 35 Clark, R.D., 23 (1986) 1 Cobb, R., 5 (1967) 59 Cochrane, D.E., 27 (1990) 143 Coulton, S., 31 (1994) 297; 33 (1996) 99 Cox, B., 37 (2000) 83 Crossland, J., 5 (1967) 251 Crowshaw, K.,15 (1978) 357 Cushman, D.W., 17 (1980) 41 Cuthbert, A.W., 14 (1977) 1 Dabrowiak, J.C., 24 (1987) 129 Daly, M.J., 20 (1983) 337

384

CUMULATIVE AUTHOR INDEX

D’Arcy, P.F., 1 (1961) 220 Daves, G.D., 13 (1976) 303; 22 (1985) 1 Davies, G.E., 2 (1962) 176 Davies, R.V., 32 (1995) 115 De Clercq, E., 23 (1986) 187 De Gregorio, M., 21 (1984) 111 De Luca, H.F., 35 (1998) 1 De, A,, 18 (1981) 117 Demeter, D.A., 36 (1999) 169 Denyer, J.C., 37 (2000) 83 Derouesnt, C., 34 (1997) 1 Dimitrakoudi, M., 11 (1975) 193 Donnelly, M.C., 37 (2000) 83 Draffan, G.H., 12 (1975) 1 Drewe, J.A., 33 (1996) 233 Dubinsky, B., 36 (1999) 169 Duckworth, D.M., 37 (2000) 1 Duffield, J.R., 28 (1991) 175 Durant, G.J., 7 (1970) 124 Edwards, D.I., 18 (1981) 87 Edwards, P.D., 31 (1994) 59 Eldred, C.D., 36 (1999) 29 Ellis, G.P.,6(1969)266;9 (1973)65; lO(1974) 245 Evans, B., 37 (2000) 83 Evans, J.M., 31 (1994) 409 Falch, E., 22 (1985) 67 Fantozzi, R., 22 (1985) 267 Feigenbaum, J.J., 24 (1987) 159 Feuer, G., 10 (1974) 85 Finberg, J.P.M., 21 (1984) 137 Fletcher, S.R., 37 (2000) 45 Floyd, C.D., 36 (1999) 91 FranGois, I., 31 (1994) 297 Frank, H., 27 (1990) 1 Freeman, S., 34 (1997) 11 1 Fride, E., 35 (1998) 199 Gale, J.B., 30 (1993) 1 Ganellin, C.R., 38 (2001) 279 Garbarg, M., 38 (2001) 279 Garratt, C.J., 17 (1980) 105 Gill, E.W., 4 (1965) 39 Ginsburg, M., 1 (1961) 132 Goldberg, D.M., 13 (1976) 1 Gould, J., 24 (1987) 1

Graham, J.D.P., 2 (1962) 132 Green, A.L., 7 (1970) 124 Green, D.V.S., 37 (2000) 83 Greenhill, J.V., 27 (1990) 51; 30 (1993) 206 Griffin, R.J., 31 (1994) 121 Griffiths, D., 24 (1987) 1 Griffiths, K., 26 (1989) 299 Groenewegen, W.A., 29 (1992) 217 Groundwater, P.W., 33 (1996) 233 Guile, S.D., 38 (2001) 115 Gunda, E.T., 12 (1975) 395; 14 (1977) 181 Gylys, J.A., 27 (1990) 297 Hacksell, U., 22 (1985) I Hall, A.D., 28 (1991) 41 Hall, S.B., 28 (1991) 175 Halldin, C . , 38 (2001) 189 Halliday, D., 15 (1978) 1 Hammond, S.M., 14(1977) 105; 16(1979) 223 Hamor, T.A., 20 (1983) 157 Hanson, P.J.,28 (1991) 201 Hanus, L., 35 (1998) 199 Hargreaves, R.B., 31 (1994) 369 Harris, J.B., 21 (1984) 63 Hartley, A.J., 10 (1974) 1 Hartog, J., 15 (1978) 261 Heacock, R.A., 9 (1973) 275; 11 (1975) 91 Heard, C.M., 36 (1999) 235 Heinisch, G., 27 (1990) I; 29 (1992) 141 Heller, H., 1 (1961) 132 Heptinstall, S., 29 (1992) 217 Herling, A.W., 31 (1994) 233 Hider, R.C., 28 (1991) 41 Hill, S.J., 24 (1987) 30 Hillen, F.C., 15 (1978) 261 Hino, K., 27 (1990) 123 Hjeds, H., 22 (1985) 67 Holdgate, G.A., 38 (2001) 309 Hooper, M., 20 (1983) 1 Hopwood, D., 13 (1976) 271 Hosford, D., 27 (1990) 325 Hubbard, R.E., 17 (1980) 105 Hughes, R.E., 14 (1977) 285 Hugo, W.B., 31 (1994) 349 Hulin, B., 31 (1994) 1 Humber, L.G., 24 (1987) 299 Hunt, E., 33 (1996) 99

CUMULATIVE AUTHOR INDEX Ijzerman, A.P., 38 (2001) 61 Imam, S.H., 21 (1984) 169 Ince, F., 38 (2001) 115 Ingall, A.H., 38 (2001) 115 Ireland, S.J., 29 (1992) 239 Jacques, L.B., 5 (1967) 139 James, K.C., 10 (1974) 203 Jaszberenyi, J.C., 12(1975)395; 14(1977) 181 Jenner, F.D., 11 (1975) 193 Jewers, K., 9 (1973) 1 Jindal, D.P., 28 (1991) 233 Jones, D.W., 10 (1974) 159 Judd, A,, 11 (1975) 193 Judkins, B.D., 36 (1999) 29 Kadow, J.F., 32 (1995) 289 Kapoor, V.K., 16(1979) 35; 17(1980) 151; 22 (1985) 243 Kawato, Y., 34 (1997) 69 Kelly, M.J., 25 (1988) 249 Kendall, H.E., 24 (1987) 249 Kennis, L.E.J., 33 (1996) 185 Khan, M.A., 9 (1973) 117 Kiefel, M.J., 36 (1999) 1 Kilpatrick, G.J., 29 (1992) 239 Kindon, N.D., 38, (2001) 115 Kirst, H. A,, 30 (1993) 57; 31 (1994) 265 Kitteringham, G.R., 6 (1969) 1 Knight, D.W., 29 (1992) 217 Kobayashi, Y., 9 (1973) 133 Koch, H.P., 22 (1985) 165 Kopelent-Frank, H., 29 (1992) 141 Kramer, M.J., 18 (1981) 1 Krogsgaard-Larsen, P., 22 (1985) 67 Kulkarni, S.K., 37 (2000) 135 Kumar, M., 28 (1991) 233 Kumar, S., 38 (2001) I Lambert, P.A., 15 (1978) 87 Launchbury, A.P., 7 (1970) 1 Law, H.D., 4 (1965) 86 Lawen, A,, 33 (1996) 53 Lawson, A.M, 12 (1975) 1 Leblanc, C., 36 (1999) 91 Lee, C.R., 11 (1975) 193 Lee, J.C., 38 (2001) 1 Lenton, E.A., 11 (1975) 193

385

Levin, R.H., 18 (1981) 135 Lewis, A.J., 19 (1982) I; 22 (1985) 293 Lewis, D.A., 28 (1991) 201 Lewis, J.A. 37 (2000) 83 Lien, E.L., 24 (1987) 209 Ligneau, X., 38 (2001) 279 Lin, T.-S., 32 1995) 1 Liu, M.-C., 32 (1995) 1 Lloyd, E.J., 23 (1986) 91 Lockhart, I.M., I5 (1978) 1 Lord, J.M., 24 (1987) 1 Lowe, LA., 17 (1980) 1 Lucas, R.A., 3 (1963) 146 Lue, P., 30 (1993) 206 Luscombe, D.K., 24 (1987) 249 Mackay, D., 5 (1 967) 199 Main, B.G., 22 (1985) 121 Malhotra, R.K., 17 (1980) 151 Manchanda, A.H, 9 (1973) 1 Mander, T.H., 37 (2000) 83 Mannaioni, P.F., 22 (1985) 267 Martin, I.L., 20 (1983) 157 Martin, J.A., 32 (1995) 239 Masini, F., 22 (1985) 267 Matsumoto, J., 27 (1990) 123 Matthews, R.S., 10 (1974) 159 Maudsley, D.V., 9 (1973) 133 May, P.M., 20 (1983) 225 McCague, R., 34 (1997) 203 McLelland, M.A., 27 (1990) 51 McNeil, S., 11 (1975) 193 Mechoulam, R., 24 (1987) 159; 35 (1998) 199 Meggens, A.A.H.P., 33 (1996) 185 Megges, R., 30 (1993) 135 Meghani, P., 38, (2001) 115 Merritt, A.T., 37 (2000) 83 Michel, A.D., 23 (1986) 1 Miura, K., 5 (1967) 320 Moncada, S., 21 (1984) 237 Monkovic, I., 27 (1990) 297 Montgomery, J.A., 7 (1970) 69 Moody, G.J., 14 (1977) 51 Morris, A., 8 (1971) 39; 12 (1975) 333 Mortimore, M.P., 38 (2001) 11 5 Munawar, M.A., 33 (1996) 233 Murphy, F., 2 (1962) 1; 16 (1979) 1 Musallan, H.A., 28 (1991) 1

386

CUMULATIVE AUTHOR INDEX

Musser, J.H., 22 (1985) 293 Natoff, I.L., 8 (1971) 1 Neidle, S., 16 (1979) 151 Nicholls, P.J., 26 (1989) 253 Nodiff, E.A., 28 (1991) 1 Nordlind, K., 27 (1990) 189 Nortey, S.O., 36 (1999) 169 O’Hare, M., 24 (1987) 1 Ondetti, M.A., 17 (1980) 41 Ottenheijm, H.C.J., 23 (1986) 219 Oxford, A.W., 29 (1992) 239 Paget, G.E., 4 (1965) 18 Palatini, P., 19 (1982) 111 Palazzo, G., 21 (1984) 111 Palfreyman, M.N., 33 (1996) 1 Palmer, D.C., 25 (1988) 85 Parkes, M.W., 1 (1961) 72 Parnham, M.J., 17 (1980) 185 Parratt, J.R., 6 (1969) 11 Patel, A,, 30 (1993) 327 Paul, D., 16 (1979) 35; 17 (1980) 151 Pearce, F.L., 19 (1982) 59 Peart, W.S., 7 (1970) 215 Petrow, V., 8 (1971) 171 Pike, V.W., 38 (2001) 189 Pinder, R.M., 8 (1971) 231; 9 (1973) 191 Ponnudurai, T.B., 17 (1980) 105 Powell, W.S., 9 (1973) 275 Power, E.G.M., 34 (1997) 149 Price, B.J., 20 (1983) 337 Prior, B., 24 (1987) 1 Procopiou, P.A., 33 (1996) 331 Purohit, M.G., 20 (1983) 1 Ram, S., 25 (1988) 233 Reckendorf, H.K., 5 (1967) 320 Reddy, D.S. 37 (2000) 135 Redshaw, S., 32 (1995) 239 Rees, D.C., 29 (1992) 109 Reitz, A.B., 36 (1999) 169 Repke, K. R. H., 30 (1993) 135 Richards, W.G., 11 (1975) 67 Richardson, P.T., 24 (1987) 1 Roberts, L.M., 24 (1987) 1 Roe, A.M., 7 (1970) 124

Rose, H.M., 9 (1973) 1 Rosen, T., 27 (1990) 235 Rosenberg, S.H., 32 (1995) 37 Ross, K.C., 34 (1997) 1I 1 Roth, B., 7 (1970) 285; 8 (1971) 61; 19 (1982) 269 Russell, A.D., 6 (1969) 135; 8 (1971) 39; 13 (1976) 271; 31 (1994) 349; 35 (1998) 133 Ruthven, C.R.J., 6 (1969) 200 Sadler, P.J., 12 (1975) 159 Sampson, G.A., I 1 (1975) 193 Sandler, M., 6 (1969) 200 Sarges, R., 18 (1981) 191 Sartorelli, A.C., 15 (1978) 321; 32 (1995) 1 Schiller, P. W., 28 (1991) 301 Schmidhammer, H., 35 (1998) 83 Schon, R., 30 (1993) 135 Schunack, W., 38 (2001) 279 Schwartz, J.-C., 38 (2001) 279 Schwartz, M.A., 29 (1992) 271 Scott, M.K., 36 (1999) 169 Sewell, R.D.E., 14 (1977) 249; 30 (1993) 327 Shank, R.P., 36 (1999) 169 Shaw, M.A., 26 (1989) 253 Sheard, P., 21 (1984) 1 Shepherd, D.M., 5 (1967) 199 Silver, P.J., 22 (1985) 293 Silvestrini, B., 21 (1984) 111 Singh, H., 16 (1979) 35; 17 (1980) 151; 22 (1985) 243; 28 (1991) 233 Skotnicki, J.S., 25 (1988) 85 Slater, J.D.H., 1 (1961) 187 Smith, H.J., 26 (1989) 253; 30 (1993) 327 Smith, R.C., 12 (1975) 105 Smith, W.G., 1 (1961) I; 10 (1974) 11 Solomons, K.R.H., 33 (1996) 233 Sorenson, J.R.J., 15 (1978) 21 I ; 26 (1989) 437 Souness, J.E., 33 (1996) 1 Southan, C., 37 (2000) I Spencer, P.S.J., 4 (1965) I ; 14 (1977) 249 Spinks, A,, 3 (1963) 261 Stlhle, L., 25 (1988) 291 Stark, H., 38 (2001) 279 Steiner, K.E., 24 (1987) 209 Stenlake, J.B., 3 (1963) I ; 16 (1979) 257 Stevens, M.F.G., 13 (1976) 205 Stewart, G.A., 3 (1963) 187

CUMULATIVE AUTHOR INDEX Studer, R.O., 5 (1963) 1 Sullivan, M.E., 29 (1992) 65 Suschitzky, J.L., 21 (1984) 1 Swain, C.J., 35 (1998) 57 Swallow, D.L., 8 (1971) 119 Sykes, R.B., 12 (1975) 333 Talky, J.J., 36 (1999) 201 Taylor, E.C., 25 (1988) 85 Taylor, E.P., 1 (1961) 220 Taylor, S G., 31 (1994) 409 Tegner, C., 3 (1963) 332 Terasawa, H., 34 (1997) 69 Thomas, G.J., 32 (1995) 239 Thomas, I.L., 10 (1974) 245 Thomas, J.D.R., 14 (1977) 51 Thompson, E.A., 1 1 (1975) 193 Thompson, M., 37 (2000) 177 Tilley, J.W., 18 (1981) 1 Timmerman, H., 38 (2001) 61 Traber, R., 25 (1988) 1 Tucker, H., 22 (1985) 121 Tyers, M.B., 29 (1992) 239 Upton, N., 37 (2000) 177 Valler, M.J., 37 (2000) 83 Van den Broek, L.A.G.M., 23 (1986) 219 Van Dijk, J., 15 (1978) 261 Van Muijlwijk-Koezen, J.E., 38 (2001) 61 Van Wart, H.E., 29 (1992) 271 Vincent, J.E., 17 (1980) 185 Volke, J., 12 (1975) 247 Von Itzstein, M., 36 (1999) 1 Von Seeman, C., 3 (1963) 89 Von Wartburg, A,, 25 (1988) 1 Vyas, D.M., 32 (1995) 289 Waigh, R.D., 18 (1981) 45 Wajsbort, J., 21 (1984) 137

Walker, R.T., 23 (1986) 187 Walls, L.P., 3 (1963) 52 Walz, D.T., 19 (1982) 1 Ward, W.H.J., 38 (2001) 309 Waring, W.S., 3 (1963) 261 Watson, N.S., 33 (1996) 331 Watson, S.P., 37 (2000) 83 Wedler, F. C., 30 (1993) 89 Weidmann, K., 31 (1994) 233 Weiland, J., 30 (1993) 135 West, G.B., 4 (1965) 1 Whiting, R.L., 23 (1986) 1 Whittaker, M., 36 (1999) 91 Whittle, B.J.R., 21 (1984) 237 Wiedling, S., 3 (1963) 332 Wien, R., l(1961) 34 Wikstrom, H., 29 (1992) 185 Wikstrom, H.V., 38 (2001) 189 Wilkinson, S., 17 (1980) 1 Williams, D.R., 28 (1991) 175 Williams, J.C., 31 (1994) 59 Williams, K.W., 12 (1975) 105 Williams-Smith, D.L., 12 (1975) 191 Wilson, C., 31 (1994) 369 Wilson, H.K., 14 (1977) 285 Witte, E.C., 11 (1975) 119 Wold, S., 25 (1989) 291 Wood, E.J., 26 (1989) 323 Wright, I.G., 13 (1976) 159 Wyard, S.J., 12 (1975) 191 Yadav, M.R., 28 (1991) 233 Yates, D.B., 32 (1995) 115 Youdim, M.B.H., 21 (1984) 137 Young, P A . , 3 (1963) 187 Young, R.N., 38 (2001) 249 Zee-Cheng, R.K.Y., 20 (1983) 83 Zon, G., 19 (1982) 205 Zylicz, Z., 23 (1986) 219

387

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Cumulative Index of Subjects for Volumes 1-38 The volume number,(year of publication) and page number are given in that order.

Adamantane, amino derivatives, 18 (1981) 1 Adenosine A3 receptor ligands, 38 (2001) 61 Adenosine triphosphate, 16 (1979) 223 Adenylate cyclase, 12 (1975) 293 Adipose tissue, 17 (1980) 105 Adrenergic blockers, a-, 23 (1986) 1 p-, 22 (1985) 121 a2-Adrenoceptors, antagonists, 23 (1986) 1 Adrenochrome derivatives, 9 (1973) 275 Adriamycin, 15 (1978) 125; 21 (1984) 169 AIDS, drugs for, 31 (1994) 121 Aldehyde thiosemicarbazones as antitumour agents, 15 (1978) 321; 32 (1995) 1 Aldehydes as biocides, 34 (1997) 149 Aldose reductase inhibitors, 24 (1987) 299 Alzheimer’s disease, chemotherapy of, 34 (1997) I ; 36 (1999) 201 Allergy, chemotherapy of, 21 (1984) I ; 22 (1985) 293 Amidines and guanidines, 30 (1993) 203 Aminoadamantane derivatives, 18 (1 98 1) 1 Aminopterins as antitumour agents, 25 (1988) 85 8-Aminoquinolines as antimalarial drugs, 28 (1991) 1 Analgesic drugs, 2 (1962) 43; 4 (1965) 171; 7 (1970) 229; 14 (1977) 249 Anaphylactic reactions, 2 (1962) 176 Angiotensin, 17 (1980) 41; 32 (1995) 37 Anthraquinones, antineoplastic, 20 (1983) 83 Antiallergic drugs, 21 (1984) 1; 22 (1985) 293; 27 (1990) 34 Antiarrhythmic drugs, 29 (1992) 65 Anti-arthritic agents, 15 (1978) 21 1; 19 (1982) 1; 36 (1999) 201 Antibacterial agents, 6 (1969) 135; 12 (1975) 333; 19 (1982) 269; 27 (1990) 235; 30 (1993) 203; 31 (1994) 349; 34 (1997) resistance to, 32 (1995) 157; 35 (1998) 133

Antibiotics, antitumour, 19 (1982) 247; 23 (1986) 219 carbapenem, 33 (1996) 99 p-lactam, 12 (1975) 395; 14 (1977) 181; 31 (1994) 297; 33 (1996) 99 macrolide, 30 (1993) 57; 32 (1995) 157 mechanisms of resistance, 35 (1998) 133 polyene, 14 (1977) 105; 32 (1995) 157 resistance to, 31 (1994) 297; 32 (1995) 157; 35 (1998) 133 Anticancer agents see Antibiotics, Antitumour agents Anticonvulsant drugs, 3 (1963) 261; 37 (2000) 177 Antidepressant drugs, 15 (1978) 261; 23 (1986) 121 Antiemetic drugs, 27 (1990) 297; 29 (1992) 239 Antiemetic action of 5-HT3 antagonists, 27 (1990) 297; 29 (1992) 239 Antiepileptic drugs, 37 (2000) 177 Antifilarial benzimidazoles, 25 (1988) 233 Antifolates as anticancer agents, 25 (1988) 85; 26 ( 1989) 1 Antifungal agents, I (1961) 220 Antihyperlipidaemic agents, 11 (1975) 119 Anti-inflammatory action of cyclooxygenase-2 (COX-2) inhibitors, 36 (1999) 201 of thalidomide, 22 (1985) 165 of 5-lipoxygenase inhibitors, 29 (1992) 1 of p38 MAP kinase inhibitors, 38 (2001) 1 Anti-inflammatory agents, 5 (1967) 59; 36 (1999) 201; 38 (2001) 1 Antimalarial 8-aminoquinolines, 28 (199 1) 1 Antimicrobial agents for sterilization, 34 (1997) 149 Antineoplastic agents, a new approach, 25 (1988) 35

390

CUMULATIVE SUBJECT INDEX

anthraquinones as, 20 (1983) 83 Antipsychotic drugs, 33 (1996) 185 Anti-rheumatic drugs, 17 (1980) 185; 19 (1982) I; 36 (1999) 201 Antisecretory agents, 37 (2000) 45 Antithrombotic agents, 36 (1999) 29 Antitumour agents, 9 (1973) 1; 19 (1982) 247; 20 (1983) 83; 23 (1986) 219; 24 (1987) 1; 24 (1987) 129; 25 (1988) 35; 25 (1988) 85; 26 (1989) 253; 26 (1989) 299; 30 (1993) 1; 32 (1995) 1; 32 (1995) 289; 34 (1997) 69 Antitussive drugs, 3 (1963) 89 Anti-ulcer drugs, of plant origin, 28 (1991) 20 1 ranitidine, 20 (1983) 67 synthetic, 30 (1993) 203 Antiviral agents, 8 (1971) 119; 23 (1986) 187; 36 (1999) I Anxiolytic agents, pyrido[ 1,2-a]benzimidazoles as, 36 (1999) 169 Anxiolytic agents, CCK-B antagonists as, 37 (2000) 45 Aromatase inhibition and breast cancer, 26 (1989) 253; 33 (1996) 147 Aspartic proteinase inhibitors, 32 (1995) 37; 32 (1995) 239 Asthma, drugs for, 21 (1984) I; 31 (1994) 369; 31 (1994) 409; 33 (1996) 1; 38 (2001) 249 ATPase inhibitors, gastric, H+/Kf- 31 (1994) 233 Azides, 31 (1994) 121 Bacteria, mechanisms of resistance to antibiotics and biocides, 35 (1998) 133 Bacterial and mammalian collagenases: their inhibition, 29 (1992) 271 1 -Benzazepines, medicinal chemistry of, 27 (1990) 123 Benzimidazole carbamates, antifilarial, 25 (1988) 233 Benzisothiazole derivatives, 18 (1981) 117 Benzodiazepines, 20 (1983) 157; 36 (1999) 169 Benzo[b]pyranol derivatives, 37 (2000) 177 Biocides, aldehydes, 34 (1997) 149 mechanisms of resistance, 35 (1998) 133 British Pharmacopoeia Commission, 6 (1969) 1

Bronchodilator and antiallergic therapy, 22 (1985) 293 Calcium and histamine secretion from mast cells, 19 (1982) 59 Calcium channel blocking drugs, 24 (1987) 249 Camptothecin and its analogues, 34 (1997) 69 Cancer, aromatase inhibition and breast, 26 (1989) 253 azides and, 3 1 (1 994) 121 camptothecin derivatives, 34 (1997) 69 endocrine treatment of prostate, 26 (1989) 299 retinoids in chemotherapy, 30 (1993) 1 Cannabinoid drugs, 24 (1987) 159; 35 (1998) 199 Carbapenem antibiotics, 33 (1996) 99 Carcinogenicity of polycyclic hydrocarbons, 10 (1974) 159 Cardiotonic steroids, 30 (1993) 135 Cardiovascular system, effect of azides, 31 (1994) 121 effect of endothelin, 31 (1994) 369 4-quinolones as antihypertensives, 32 (1995) 115 renin inhibitors as antihypertensive agents, 32 (1995) 37 Catecholamines, 6 (1969) 200 CCK-B antagonists, 37 (2000) 45 Cell membrane transfer, 14 (1977) 1 Central nervous system, drugs, transmitters and peptides, 23 (1986) 91 Centrally acting dopamine D2 receptor agonists, 29 (1992) 185 Chartreusin, 19 (1982) 247 Chelating agents, 20 (1983) 225 tripositive elements as, 28 (1991) 41 Chemotherapy of herpes virus, 23 (1985) 67 Chemotopography of digitalis recognition matrix, 30 (1993) 135 Chiral synthesis, 34 (1997) Cholesterol-lowering agents, 33 (1996) 331 Cholinergic receptors, 16 (1976) 257 Chromatography, 12 (1975) 1; 12 (1975) 105 Chromone carboxylic acids, 9 (1973) 65 Clinical enzymology, 13 (1 976) 1

CUMULATIVE SUBJECT INDEX Collagenases, synthetic inhibitors, 29 (1992) 27 1 Column chromatography, 12 (1975) 105 Combinatorial chemistry, 36 (1999) 91 Computers in biomedical education, 26 (1989) 323 Medlars information retrieval, 10 (1974) 1 Copper complexes, 15 (1978) 211; 26 (1989) 437 Coronary circulation, 6 (1969) I 1 Coumarins, metabolism and biological actions, 10 (1974) 85 Cyclic AMP, 12 (1975) 293 Cyclooxygenase-2 (COX-2) inhibitors, 36 (1999) 201 Cyclophosphamide analogues, 19 (1982) 205 Cyclosporins as immunosuppressants, 25 (1988) I ; 33 (1996) 53 Data analysis in biomedical research, 25 (1988) 291 Diaminopyrimidines, 19 (1982) 269 Digitalis recognition matrix, 30 (1993) 135 Diuretic drugs, 1 (1961) 132 DNA-binding drugs, 16 (1979) 151 Dopamine Dz receptor agonists, 29 (1992) 185 Doxorubicin, 15 (1978) 125; 21 (1984) 169 Drug-receptor interactions, 4 (1965) 39 Drugs, transmitters and peptides, 23 (1986) 91 Elastase, inhibition, 31 (1994) 59 Electron spin resonance, 12 (1975) 191 Electrophysiological (Class 111) agents for arrhythmia, 29 (1992) 65 Enantiomers, synthesis of, 34 (1997) 203 Endorphins, 17 (1980) 1 Endothelin inhibition, 31 (1994) 369 Enkephalin-degrading enzymes, 30 (1993) 327 Enkephalins, 17 (1980) 1 Enzymes, inhibitors of, 16 (1979) 223; 26 (1989) 253; 29 (1992) 271; 30 (1993) 327; 31 (1994) 59; 31 (1994)297; 32 (1995) 37; 32(1995)239;36(1999) I ; 36(1999)201; 38 (2001) 1

39 1

Enzymology, clinical use of, 10 (1976) 1 in pharmacology and toxicology, 10 (1974) I 1 Erythromycin and its derivatives, 30 (1993) 57; 31 (1994) 265 Feverfew, medicinal chemistry of the herb, 29 (1992) 217 Fibrinogen antagonists, as antithrombotic agents, 36 (1999) 29 Flavonoids, physiological and nutritional aspects, 14 (1977) 285 Fluoroquinolone antibacterial agents, 27 (1990) 235 mechanism of resistance to, 32 (1995) 157 Folic acid and analogues, 25 (1988) 85; 26 (1989) 1 Formaldehyde, biocidal action, 34 (1997) 149 Free energy, biological action and linear, 10 (1 974) 205 GABA, heterocyclic analogues, 22 (1985) 67 GABAA receptor ligands, 36 (1999) 169 Gastric H+/K+-ATPase inhibitors, 31 (1994) 233 Gas-liquid chromatography and mass spectrometry, 12 (1975) 1 Genomics, impact on drug discovery, 37 (2000) 1 Glutaraldehyde, biological uses, 13 (1976) 27 1 as sterilizing agent, 34 (1997) 149 Gold, immunopharmacology of, 19 (1982) I Guanidines, 7 (1970) 124; 30 (1993) 203 Halogenoalkylamines, 2 (1 962) 132 Heparin and heparinoids, 5 (1967) 139 Herpes virus, chemotherapy, 23 (1985) 67 Heterocyclic analogues of GABA, 22 (1985) 67 Heterocyclic carboxaldehyde thiosemicarbazones, 16 (1979) 35; 32 (1995) I Heterosteroids, 16 (1979) 35; 28 (1991) 233 High-throughput screening techniques, 37 (2000) 83 Histamine, H3 ligands, 38 (2001) 279

392

CUMULATIVE SUBJECT INDEX

Hz-antagonists, 20 (1983) 337 receptors, 24 (1987) 30; 38 (2001) 279 release, 22 (1985) 26 secretion, calcium and, 19 (1982) 59 5-HTIA receptors, radioligands for in vivo studies, 38 (2001) 189 Histidine decarboxylases, 5 (1967) 199 HIV proteinase inhibitors, 32 (1995) 239 Hydrocarbons, carcinogenicity of, 10 (1974) 159 Hypersensitivity reactions, 4 (1965) 1 Hypoglycaemic drugs, 1 (1961) 187; 18 (1981) 191; 24 (1987) 209; 30 (1993) 203; 31 (1994) 1 Hypotensive agents, 1 (1961) 34; 30 (1993) 203; 31 (1994) 409; 32 (1995) 37; 32 (1995) 115 Immunopharmacology of gold, 19 (1982) 1 Immunosuppressant cyclosporins, 25 (1988) 1 India, medicinal research in, 22 (1985) 243 Influenza virus sialidase, inhibitors of, 36 (1999) 1 Information retrieval, 10 (1974) 1 Inotropic steroids, design of, 30 (1993) 135 Insulin, obesity and, 17 (1980) 105 Ion-selective membrane electrodes, 14 (1977) 51 Ion transfer, 14 (1977) 1 Irinotecan, anticancer agent, 34 (1997) 68 Isothermal titration calorimetry, in drug design, 38 (2001) 309 Isotopes, in drug metabolism, 9 (1973) 133 stable, 15 (1978) 1 Kappa opioid non-peptide ligands, 29 (1992) 109; 35 (1998) 83 Lactam antibiotics, 12 (1975) 395; 14 (1977) 181 /I-Lactamase inhibitors, 31 (1994) 297 Leprosy, chemotherapy, 20 (1983) 1 Leukocyte elastase inhibition, 31 (1994) 59 Leukotriene D4 antagonists, 38 (2001) 249 Ligand-receptor binding, 23 (1 986) 41 Linear free energy, 10 (1974) 205 5-Lipoxygenase inhibitors and their anti-inflammatory activities, 29 (1992) 1

Literature of medicinal chemistry, 6 (1969) 266 Lithium, medicinal use of, 11 (1975) 193 Local anaesthetics, 3 (1963) 332 Lonidamine and related compounds, 21 (1984) 111 Macrolide antibiotics, 30 (1993) 57; 31 (1994) 265 Malaria, drugs for, 8 (1971) 231; 19 (1982) 269; 28 (1991) 1 Manganese, biological significance, 30 (1993) 89 Manufacture of enantiomers of drugs, 34 (1997) 203 Mass spectrometry and glc, 12 (1975) 1 Mast cells, calcium and histamine secretion, 19 (1982) 59 cholinergic histamine release, 22 (1985) 267 peptide regulation of, 27 (1990) 143 Medicinal chemistry, literature of, 6 (1969) 266 Medlars computer information retrieval, 10 (1974) 1 Membrane receptors, 23 (1986) 41 Membranes, 14 (1977) 1; 15 (1978) 87; 16 (1979) 223 Mercury (11) chloride, biological effects, 27 (1990) 189 Methotrexate analogues as anticancer drugs, 25 (1988) 85; 26 (1989) 1 Microcomputers in biomedical education, 26 (1989) 323 Molecularly imprinted polymers, preparation and use of, 36 (1999) 235 Molybdenum hydroxylases, 24 (1987) 85 Monoamine oxidase inhibitors, 21 (1984) 137 Montelukast and related leukotriene D4 antagonists, 38 (2001) 249 Multivariate data analysis and experimental design, 25 (1988) 291 Neuraminidase inhibitors, 36 (1999) I Neurokinin receptor antagonists, 35 (1998) 57 Neuromuscular blockade, 2 (1962) 88; 3 (1963) I; 16 (1979) 257 Neurokinin receptor antagonists, 35 (1 998) 57

CUMULATIVE SUBJECT INDEX Neurosteroids, as psychotropic drugs, 37 (2000) 135 Next decade [the 197O’s], drugs for, 7 (1970) 215 Nickel(I1) chloride and sulphate, biological effects, 27 (1990) 189 Nitriles, synthesis of, 10 (1974) 245 Nitrofurans, 5 (1967) 320 Nitroimidazoles, cytotoxicity of, 18 (1981) 87 NMR spectroscopy, 12 (1975) 159 high-field, 26 (1989) 355 Non-steroidal anti-inflammatory drugs, 5 (1967) 59; 36 (1999) 201 Non-tricyclic antidepressants, 15 (1978) 39 C-Nucleosides, 13 (1976) 303; 22 (1985) 1 Nutrition, total parenteral, 28 (1991) 175 Obesity and insulin, 17 (1980) 105 Ondansetron and related 5-HT3 antagonists, 29 (1992) 239 Opioid peptides, 17 (1980) I receptor antagonists, 35 (1998) 83 receptor-specific analogues, 28 (1991) 301 Opioid receptor antagonists, 35 (1998) 83 Organophosphorus pesticides, pharmacology of, 8 (1971) 1 Oxopyranoazines and oxopyranoazoles, 9 (1973) 117 P2 Purinoreceptor ligands, 38 (2001) 115 p38 MAP Kinase inhibitors, 38 (2001) 1 Paclitaxel, anticancer agent, 32 (1995) 289 Parasitic infections. 13 (1 976) 159; 30 ( I 993) 203 Parasympathomimetics, 1 1 (1975) 1 Parenteral nutrition, 28 (1991) 175 Parkinsonism, pharmacotherapy of, 9 (1973) 191; 21 (1984) 137 Patenting of drugs, 2 (1962) I; 16 (1979) 1 Peptides, antibiotics, 5 (1967) 1 enzymic, 31 (1994) 59 hypoglycaemic, 31 (1994) 1 mast cell regulators, 27 (1990) 143 opioid, 17 (1980) 1 Pharmacology of Alzheimer’s disease, 34 (1997) 1 Pharmacology of Vitamin E, 25 (1988) 249

393

Phosphates and phosphonates as prodrugs, 34 (1997) 111 Phospholipids, 19 (1982) 111 Photodecomposition of drugs, 27 (1990) 51 Platelet-aggregating factor, antagonists, 27 (1990) 325 Platelet aggregration, inhibitors of, 36 (1999) 29 Platinum antitumour agents, 24 (1987) 129 Polarography, 12 (1975) 247 Polycyclic hydrocarbons, 10 (1974) 159 Polyene antibiotics, 14 (1977) 105 Polypeptide antibiotics, 5 (1 967) 1 Polypeptides, 4 (1965) 86 from snake venom, 21 (1984) 63 Positron emission tomography (PET), 38 (2001) 189 Prodrugs based on phosphates and phosphonates, 34 (1997) 11 1 Prostacyclins, 21 (1984) 237 Prostaglandins, 8 (1971) 317; 15 (1978) 357 Proteinases, inhibitors of, 31 (1994) 59; 32 (1995) 37; 32 (1995) 239 Pseudornonas aeruginosa, resistance of, 12 (1975) 333; 32 (1995) 157 Psychotomimetics, 11 (1975) 91 Psychotropic drugs, 5 (1967) 251; 37 (2000) 135 Purines, 7 (1970) 69 Pyridazines, pharmacological actions of, 27 (1990) I ; 29 (1992) 141 Pyrimidines, 6 (1969) 67; 7 (1970) 285; 8 (1971) 61; 19 (1982) 269 Quantum chemistry, 11 (1975) 67 Quinolines, 8-amino-, as antimalarial agents, 28 (1991) 1 4-Quinolones as antibacterial agents, 27 (1990) 235 as potential cardiovascular agents, 32 (1995) 115 Radioligand-receptor binding, 23 (1986) 417 Ranitidine and H2-antagonists, 20 (1983) 337 Rauwolfia alkaloids, 3 (1963) 146

394

CUMULATIVE SUBJECT INDEX

Recent drugs, 7 (1970) 1 Receptors, adenosine, 38 (2001) 61 adrenergic, 22 (1985) 121; 23 (1986) 1 cholecystokinin, 37 (2000) 45 fibrinogen, 36 ( 1 999) 29 histamine, 24 (1987) 29; 38 (2001) 279 neurokinin, 35 (1998) 57 opioid, 35 (1998) 83 purino, 38 (2001) 115 Renin inhibitors, 32 (1995) 37 Ricin, 24 (1987) 1 Single photon emission tomography (SPET), 38 (2001) 189 Screening tests, 1 (1961) 1 Serine protease inhibitors, 31 (1994) 59 Serotonin 5-HTIAradioligands, 38 (2001) 189 Snake venoms, neuroactive, 21 (1984) 63 Sodium cromoglycate analogues, 21 (1984) 1 Sparsomycin, 23 (1986) 219 Spectroscopy in biology, 12 (1975) 159; 12 (1975) 191; 26 (1989) 355 Statistics in biological screening, 3 (1963) 187; 25 (1988) 291 Sterilization with aldehydes, 34 (1997) 149 Steroids, hetero-, 16 (1979) 35; 28 (1991) 233 design of inotropic, 30 (1993) 135 Synthesis of enantiomers of drugs, 34 (1997) 203

Tetrahydroisoquinolines, 8-adrenomimetic activity, 18 (1981) 45 Tctrahydronaphthalenes, 8-adrenomimetic activity, 18 (1981) 45 Tetrazoles, 17 (1980) 151 Thalidomide as anti-inflammatory agent, 22 (1985) 165 Thiosemicarbazones, biological action, 15 (1978) 321; 32 (1995) 1 Thromboxanes, 15 (1978) 357 Tilorone and related compounds, 18 (I98 1) 135 Toxic actions, mechanisms of, 4 (1965) I8 Trdnquillizers, I (1961) 72 1,2,3-Triazines, medicinal chemistry of, 13 (1976) 205 Tripositive elements, chelation of, 28 (1991) 41 Trypanosomiasis, 3 (1963) 52 Venoms, neuroactive snake, 21 (1984) 63 Virus diseases of plants, 20 (1983) 119 Viruses, chemotherapy of, 8 (1971) 119; 23 (1986) 187; 32 (1995) 239; 36 (1999) 1 Vitamin D3 and its medical uses, 35 (1998) 1 Vitamin E. pharmacology of, 25 (1988) 249