Progress in Medicinal Chemistry, Volume 43 (Progress in Medicinal Chemistry)

v

Preface The seven chapters of this volume cover a wide range of topics which will be of interest to the drug discovery community. The HERG ion channel makes its presence felt in very many current medicinal chemistry programmes and chapter 1 provides a concise view of current knowledge. Many drug discovery projects today begin with a fluorescence-based high throughput assay of small molecules. Chapter 2 provides a thorough description of the background which will be invaluable to those who are setting up, or interpreting the results from, such screening assays. The neurokinin receptors have consumed many medicinal chemist years in the past decade and these have recently borne fruit with the approval of Aprepitant. This success has to be set against many clinical failures in this area and regular reappraisal is necessary of the importance of the receptor sub-types and whether the target for the medicinal chemist should be selective, or non-selective ligands. Chapter 3 reviews recent work in this area. Discussions of which of the five receptors to target and the relative importance of receptor sub-type selectivity are also important in the muscarinic receptor field. This is reviewed in chapter 4. The gene activation pathways which involve NFkB have been implicated in the pathology of very many diseases. The pathways are extremely complex and offer many protein targets to the drug hunter. These are described in chapter 5 and include IKK, GSK-3, NIK as well as proteasome inhibitors. The life cycle of the malaria parasite and approaches to antimalarial drugs are described in chapter 6. This disease is responsible for 2.7 million deaths each year and centuries of folk medicine have provided a rich source natural product therapeutics. The recently sequenced genomes of the plasmodium itself as well as those of the mosquito carrier and the human target are contributing to additional ways to attack the disease. Another of the world’s current major diseases is that produced by HIV. This RNA virus can rapidly mutate and the resultant moving target will continue

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PREFACE

to provide challenges for medicinal chemists. These issues may be avoided to some extent by focusing on human proteins which modulate viral transport. The approach to new therapeutics described in chapter 7 targets CCR5. This human G-protein coupled receptor is important to the viral infection process. This case history of drug discovery provides interesting insight into the parallel optimisation of multiple molecular properties in order to achieve potency, adequate absorption and clearance, and avoidance of predictors of toxicity including the HERG channel featured in chapter 1. This multidimensional optimisation is the essential heart of today’s medicinal chemistry project and its ubiquity provides comprehensive proof of the dynamic development of medicinal chemistry compared with a decade ago when the approaches were very different. We are most grateful to the authors of this volume for committing so much of their time and effort in evaluating the extensive literature that is necessary to compile these articles. We also thank the staff of the publishers for their continuing support and encouragement to the series. We send best wishes to Alec Oxford and congratulate him on his able editing of this series for the past eight years. We hope he enjoys reading this issue for the first time in print. September 2004

Dr. F. D. King Dr. G. Lawton

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List of Contributors Duncan Armour Department of Chemistry, Pfizer Global Research and Development, Sandwich Laboratories, Sandwich, Kent CT13 9NJ, UK John F. Eccleston National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK Richard M. Eglen DiscoveRx Corporation, 42501, Albrae Street, Suite 100, Fremont, CA 94538, USA Marc Gerspacher Novartis Institutes for Biomedical Research Basel, Novartis Pharma AG, CH-4002 Basel, Switzerland Burkhard Haefner Department of Inflammation, Johnson & Johnson Pharmaceutical Research and Development, A Division of Janssen Pharmaceutica, Turnhoutseweg 30, Box 6423, 2340 Beerse, Belgium Jonathan P. Hutchinson GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK David M. Jameson Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96822-2319, USA

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LIST OF CONTRIBUTORS

Vijay K. Kapoor University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India Kamal Kumar University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India Yi Li Aventis Pharmaceuticals, 1041 Route 202/206N, Bridgewater, NJ 08807, USA David Rampe Aventis Pharmaceuticals, 1041 Route 202/206N, Bridgewater, NJ 08807, USA Roy J. Vaz Aventis Pharmaceuticals, 1041 Route 202/206N, Bridgewater, NJ 08807, USA Anthony Wood Department of Chemistry, Pfizer Global Research and Development, Sandwich Laboratories, Sandwich, Kent CT13 9NJ, UK

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Contents Preface

v

List of Contributors

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1

Human Ether-a-go-go Related Gene (HERG): A Chemist’s Perspective Roy J. Vaz, Yi Li and David Rampe

1

2

Fluorescence-Based Assays John F. Eccleston, Jonathan P. Hutchinson and David M. Jameson

19

3

Selective and Combined Neurokinin Receptor Antagonists Marc Gerspacher

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4

Muscarinic Receptor Subtype Pharmacology and Physiology Richard M. Eglen

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5

The Transcription Factor NF-kB as Drug Target Burkhard Haefner

137

6

Recent Advances in the Search for Newer Antimalarial Agents Vijay K. Kapoor and Kamal Kumar

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7

The Discovery of the CCR5 Receptor Antagonist, UK-427,857, A New Agent for the Treatment of HIV Infection and AIDS Anthony Wood and Duncan Armour

239

Subject Index

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Author Index (Vols. 1– 43)

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Subject Index (Vols. 1– 43)

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Progress in Medicinal Chemistry – Vol. 43, Edited by F.D. King and G. Lawton q2005 Elsevier B.V. All rights reserved.

1 Human Ether-a-go-go Related Gene (HERG): A Chemist’s Perspective ROY J. VAZ, YI LI and DAVID RAMPE Aventis Pharmaceuticals, 1041 Route 202/206N, Bridgewater, NJ 08807, USA

INTRODUCTION

1

ION CHANNELS OF THE HEART – THE ACTION POTENTIAL

2

DRUG-INDUCED QT PROLONGATION AND THE HERG CARDIAC Kþ CHANNEL

4

ASSAY TECHNOLOGIES

6

STRUCTURAL ASPECTS OF THE HERG CHANNEL

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MODELLING OF HERG CHANNEL BLOCKERS

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EXAMPLES OF APPLICATION OF MODELS TO IMPROVE SELECTIVITY AGAINST HERG BLOCKADE

10

CONCLUSIONS

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REFERENCES

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INTRODUCTION With the recent significant labelling or withdrawals of prescription medications such as terfenadine, astemizole and grepafloxacin (Figure 1.1) from the marketplace due to their association with QT prolongation, interest in the ion channel human ether-a-go-go related gene (HERG) has increased. Pharmaceutical companies have launched tremendous efforts across the value chain in order to understand and alleviate compound interactions with this ion channel. In this review recent work in this area will be described especially as it pertains to the discovery area of the value chain. We discuss some of the biology, in vitro assays DOI: 1 0 . 1 0 1 6 / S 0 0 7 9 - 6 4 6 8 ( 0 5 ) 4 3 0 0 1 - 5

1

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HUMAN ETHER-A-GO-GO RELATED GENE (HERG): A CHEMIST’S PERSPECTIVE

Fig. 1.1 Structures of compounds referred to in the article.

and in silico models and the application of some of the models to projects in the discovery and lead optimization phase of the discovery cycle.

ION CHANNELS OF THE HEART – THE ACTION POTENTIAL Electrical activity in the human heart is controlled by the movement of ions into and out of individual cardiac cells. Voltage-dependent ion channels are the membrane-spanning proteins that allow this ionic movement to occur during each heartbeat. The summation of electrical activity occurring in the myocyte,

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a single heart cell, is known as the action potential. A schematic of an action potential and the ion channels that contribute to it are depicted in Figure 1.2A. The electrical activity of the whole human heart is usually measured in the clinic by means of an electrocardiogram (ECG). An idealized ECG trace is shown in Figure 1.2B. At rest, heart cells have a potential difference across the plasma membrane with the inside face of the membrane being more negative (by about 90 mV) than the outside face. At the beginning of each heartbeat, the first ion channels that open are the voltage-dependent Naþ channels, which allow a rapid and massive influx of positive charge, in the form of Naþ ion, into the myocyte.

Fig. 1.2 Idealized traces of the human cardiac action potential and the ECG waveform are shown. The rapid depolarization of cardiac cells, as well as the QRS wave of the ECG, is in large part controlled by voltage-dependent Naþ channels carrying sodium current (INa) into the heart. Calcium channels also carry a depolarizing current (ICa) important for setting the length of the QT interval. Several different Kþ channels act to repolarize the cell and are also major determinants of the QT interval and the T wave of the ECG. Pharmacological manipulation of any of these channels can lead to changes in the shape and/or duration of the action potential and the ECG waveforms. Inhibition of Kþ channels, most especially HERG, can result in a prolongation of the action potential duration and the QT interval.

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HUMAN ETHER-A-GO-GO RELATED GENE (HERG): A CHEMIST’S PERSPECTIVE

This influx of positive charge moves the cells away from the negative resting potential towards more positive potentials. On the cellular level, the activity of voltage-dependent Naþ channels results in the upstroke of the action potential, also known as Phase 0 depolarization (Figure 1.2A). The activity of Naþ channels is also the major determinant of the QRS wave measured on the ECG (Figure 1.2B). Voltage-dependent Naþ channels open very quickly, but they also close (or inactivate) very quickly (within a few milliseconds). However, the depolarization they leave behind serves to activate another group of depolarizing ion channels, the voltage-dependent Ca2þ channels. Ca2þ channels open and allow the flow of positive charge into the heart cells in the form of Ca2þ ion. This influx of Ca2þ serves to keep the heart cell depolarized during the heartbeat and is also the trigger for cardiac muscle contraction. The activity of Ca2þ channels contributes to the plateau phase of the cellular action potential and to the QT interval of the ECG (Figure 1.2). Once depolarization of the heart has been achieved, the process must be reversed. This task is accomplished primarily through the activity of voltagedependent Kþ channels. When voltage-dependent Kþ channels open, they allow the flow of positive charge, in the form of Kþ, out of heart cells. Unlike Naþ and Ca2þ, the concentration of Kþ is higher inside the cells than it is outside allowing for the outward flow of this ion. The activity of Kþ channels drives heart cells back toward the resting membrane potential and effectively terminates the action potential. The activity of voltage-dependent Kþ channels is important for controlling the length of the QT interval and, especially, the shape of the T wave which represents the final repolarization of the heart and the end of the ECG waveform (Figure 1.2B). Voltage-dependent Kþ channels are a very diverse group of cardiac ion channels. Indeed, several types are known to exist in the human heart and each contributes to some phase of the repolarization process (Table 1.1). DRUG-INDUCED QT PROLONGATION AND THE HERG CARDIAC Kþ CHANNEL From Figure 1.2 it should be clear that, with each beat of the heart, many ion channels must work together in a precise fashion to generate normal cardiac rhythm. Unfortunately for the drug development process, many medications can possess unwanted pharmacological activity on these ion channels. Probably, the most common electrocardiographic side effect of prescription medications is known as drug-induced (or acquired) long QT syndrome. In this case, a drug, acting on one or more cardiac ion channels, delays the repolarization of cardiac cells during the heartbeat. This activity manifests itself as a prolongation in the action potential duration on the cellular level, and as a prolongation in the QT

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Table 1.1 SOME IMPORTANT HUMAN CARDIAC Kþ CHANNELS AND THE CURRENTS THEY CARRY Channel

Current

Comment

KvLQT1/minK

Slow component of the delayed rectifier (IKs)

HERG (human ether-a-go-go related gene) Kv4.3

Rapid component of the delayed rectifier (IKr) Transient outward current (Ito) Inwardly rectifying current (IK1)

Comprised of two proteins. KvLQT1 forms a Kþ channel while minK modulates its function. Involved in late phase (Phase 3) repolarization Main molecular target for drugs that prolong QT interval Involved in earliest phase (Phase 1) of repolarization Maintains resting membrane potential, contributes to late phase repolarization

Kir 2 family

interval on the ECG. While this may be a benign condition in many patients, in certain susceptible individuals, or under certain clinical conditions, a delay in cardiac repolarization can lead to arrhythmias, most notably the life-threatening ventricular arrhythmia known as torsades de pointes. Indeed, prolongation of the QT interval is considered by many to be a biomarker for the occurrence of torsades de pointes. Drug-induced QT interval prolongation is therefore not a trivial problem for drug development. In fact, many drugs have either been withdrawn from the market, or have had their use severely curtailed, due solely to the fact that they produce some degree of QT interval prolongation. These drugs include antipsychotics (e.g. thioridazine), antihistamines (e.g. astemizole and terfenadine), antibiotics (e.g. grepafloxacin), gastric prokinetics (e.g. cisapride) and others. In principle, drugs can produce QT interval prolongation via a direct or indirect interaction with virtually any voltage-dependent ion channel in the heart. Thus, activation of Naþ or Ca2þ channels, leading to an enhancement of their activity, can result in a prolongation of the action potential duration and the QT interval. Examples of such drugs include the Naþ channel activator DPI 210106 [1] as well as the dihydropyridine and benzoylpyrrole classes of Ca2þ channel ligands [2]. Conversely, inhibition of any one of the many Kþ channels that works to repolarize the myocardium cell may also produce QT interval prolongation. Despite all these possibilities, it is now well established that

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HUMAN ETHER-A-GO-GO RELATED GENE (HERG): A CHEMIST’S PERSPECTIVE

virtually all cases of drug-induced QT prolongation and torsades de pointes arrhythmia arise from a single common mechanism, namely, selective inhibition of the HERG cardiac Kþ channel [3]. This unintentional inhibition of HERG therefore represents a major safety concern for the development of new drugs. Advances in structural biology have begun to reveal why HERG can bind so many different classes of drugs, and in silico modelling efforts are becoming increasingly important for understanding, and to aid in the removal, of this unwanted drug – channel interaction. ASSAY TECHNOLOGIES One of the challenges to the understanding of HERG channel structure–activity relationships (SAR) is what in vitro biological activity is used to measure compound interactions with the channel. There are two variables that play a role in the measurement of biological activity, the biological system in which HERG is expressed and the method used for activity measurement; both are challenging [4]. Typically, the in vitro biological system consists of the HERG channel expressed in Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells or Xenopus oocytes. Of these, the stably transfected CHO and HEK cells seem to be more reliable, since measurements in Xenopus oocytes lead to a significant underestimation of HERG potency due to partitioning of the compound into the yolk. Cell toxicity arising from high expression levels has led to alternatives to cell-based screening being investigated [5]. One such example uses ion-sensitive dyes on a solid surface coated by a membrane with the transmembrane proteins at a predefined target density and lipid composition. In terms of the system of measurement, there are three assays: binding, membrane potential and ion flux. The binding assays are based on a competitive radiolabelled ligand binding assay and do not provide information on HERG channel function. Also many molecules that do not affect the binding but affect channel function go undetected. Surface plasmon resonance is also being investigated towards a high-throughput HERG binding assay. The membrane potential assays include assays using voltage-sensitive dyes and dye pairs via fluorescence resonance energy transfer (FRET). These methods largely suffer from a lack of sensitivity. Cell-based ion-flux assays include radiolabelled (86Rb) as well as detection using atomic absorption spectroscopy of the Rb ion. Unfortunately, these assays can only pick up very potent HERG channel modulators. The most reliable method is an ion-flux assay, the patch-clamp assay [6]. This assay uses electronic negative-feedback circuits to control transcellular membrane potential and measure ionic flux (current) through open ion channels. This high information content

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measurement provides valuable information on inhibitors as well as activators. Several emerging technologies aiming to automate the voltage-clamp method appear promising [7]. Also the HERG-Lite assay [8], an antibody-based, chemiluminescent assay that monitors HERG expression on a cell surface, is promising.

STRUCTURAL ASPECTS OF THE HERG CHANNEL Potassium channels have been shown to be tetramers, and each monomer mostly consists of six transmembrane (S1 – S6) regions with the selectivity region (P region) occurring between the fifth (S5) and sixth (S6) transmembrane regions that form the pore of the channel. The simplest channels consist of merely the pore region (the S5, the P region and the S6 transmembrane sequences). The crystal structure [9] of the KcsA channel (Figure 1.3A), which consisted of only the pore, together with the structure of more complicated channels and mutagenesis work, provided the basis for understanding the SAR of HERG and other channels [10]. Two other structures, that of the open form of the pore of a ligand-gated (Caþ being the ligand) Kþ channel, the Mthk [11, 12] channel (Figure 1.3B), and that of a voltage-gated Kþ channel Kvap [13, 14], have provided insight into the open form of Kþ channels. The structure of an inward rectifying Kþ channel, KirBac1.1, has also been solved [15], which shows structural conservation between inward rectifying Kþ channels with the structures of the other solved structures. Recent work on the shaker channel

Fig. 1.3 (A) A simplified schematic of the crystal structure of KcsA with a ribbon depiction of the helices forming the pore (the S5 and S6 transmembrane helices), the front and back subunits of the tetramer being omitted for clarity and a surface depicting the space available (occupied by tetramethylammonium). (B) A simplified schematic of the crystal structure of Mthk with a ribbon depiction of the S5 and S6 helices (in grey) depicting a larger space available for blockage in the open form of this channel overlaid on the ribbon depiction of the KcsA structure (in black).

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HUMAN ETHER-A-GO-GO RELATED GENE (HERG): A CHEMIST’S PERSPECTIVE

[16] argues for an alternative picture of opening, including a smaller pore and possibly a unique hinge or swivel point. The HERG channel, like other voltage-dependent Kþ channels, has a topology of six membrane-spanning stretches, a voltage sensor (S4) and a Kþ selective pore between S5 and S6. Voltage-dependent gating, or opening and closing of the pore, is conferred by the arginine-rich fourth membrane spanning stretch (the S4 voltage sensor) present in all members of the voltage-dependent cation channel family [17]. HERG has a large intracellular C-terminal region containing a cyclic nucleotide binding domain and a large N-terminal domain, the X-ray structure of which has been solved. It has structural similarity to Per – Arnt – Sim (PAS) domains which are frequently involved in signal transduction [18]. A review of the structural aspects of the HERG channel relative to structural information of other Kþ channels has been recently published [19]. Mutagenesis work has further added to the understanding of the SAR of the HERG channel. Mutagenesis work on the HERG channel originally took the form of an alanine scanning mutagenesis of amino acids located in the S6 transmembrane domain [20]. Each mutant was tested towards the binding of three compounds, MK-499, cisapride and terfenadine. Also a homology model based on the crystal structure of KcsA was used to rationalize the data. The residues implicated in these three drugs were V625, G648 which only affected the binding of MK-499 and Y652 and F656 which were found to affect the interaction of all three compounds used in the study. Subsequent work [21] repositioning Y652 and F656 along the S6 a-helical axis showed reduced blocking sensitivity by cisapride providing evidence that these were essential interactions with this, and perhaps for other, blockers. Further work using chloroquine with mutations at only Y652 [22] suggested that this compound only blocked the channel in the open form by cation-p and p-stacking interactions with Y652 and F656, respectively. Systematic mutations at Y652 and F656 with a variety of natural amino acids [23] and measuring the effects on cisapride, MK-499 and terfenadine confirmed these interactions. In the comparison of the sequence of the S6 domain of HERG and other Kþ channels [18], it was proposed that in HERG, the sequence at 655–657, PVP was replaced by IFG making the HERG channel larger due to the lack of two proline residues. Replacement of the IFG sequence with PVP [23] also made the channel less sensitive to block by MK499, cisapride and terfenadine. All this mutational work has aided tremendously in the understanding of the determinants of drug binding to the HERG channel. MODELLING OF HERG CHANNEL BLOCKERS Models discussed within this section mainly pertain to inhibitors and blockers that block the channel from the intracellular direction. There are a few large

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peptides and molecules that block the channel from the extracellular direction; however, these are not the focus of the discussion of this section. There are two types of ligand-based molecular models that one will encounter in the literature about the HERG channel and its blockers. The first type of model arises from efforts trying to identify the potential in a molecule to block the HERG channel. These models would be used as a first pass to identify molecules as potential candidates for an in vitro assay [24]. These models could also be used for potential library design [25]. There are several models that fall in this category. The latest model [26] is a binary classification model that combines both a 2D topological similarity filter with a 3D pharmacophore ensemble procedure to discriminate between HERG actives and inactives. Another such model [27] uses a decision tree-based approach to constructing a binary classification model. The important variables that were statistically significant in describing a HERG ligand were ClogP (calculated log P) $ 3.7, a size descriptor CMR (calculated molar refractivity) in the range of 110 –176, and the molecule having a maximum pKa $ 7.3 (molecule with a positive charge). Still another model [28] used 2D substructures as well as other descriptors including 3D-Volsurf descriptors, but with tools such as PLS, self-organizing maps and neural networks. In this study, patch-clamp data were obtained from mammalian cells transfected with HERG. There were no descriptors that were pointed out as being critical or statistically deterministic in the molecules used in the study to pick out HERG binders. Keseru applied similar approach of 2D substructures via hologram QSAR methods and 3D-Volsurf descriptors, with PLS analysis on a binding data set of HERG binders [29]. Another model [30], in which the descriptors turned out to be hydrophobic surface area, number of hydrogen bond acceptors and pKa value of a piperidine moiety that was common to all structures used in the study, also suggested to the authors that initial recognition by the cell membrane was an important key step in HERG inhibition. A pharmacophore model that was proposed [31, 32] suggested four hydrophobic groups together with a positive ionizable group as being the essential elements of recognition of the HERG channel. The second type of model includes those that could be used in the lead optimization phase of a drug discovery program. The first of these, developed by Cavalli et al. [33] was a pharmacophore and 3D-QSAR, comparative molecular field analysis (CoMFA) model constructed from data gathered from the literature. Care was taken in culling the literature; Xenopus oocyte-derived data were not used. In the study, all highly active molecules possessed a basic nitrogen and so a basic nitrogen was part of the proposed pharmacophore. The pharmacophore also consisted of an ionizable nitrogen together with three hydrophobic pharmacophoric elements. The pharmacophore was further supported using CoMFA. The steric coefficients

10 HUMAN ETHER-A-GO-GO RELATED GENE (HERG): A CHEMIST’S PERSPECTIVE

obtained from the CoMFA could be used in a design to eliminate HERG activity from the molecule after deciphering how a particular active HERG molecule aligns with the pharmacophore. Another 3D-QSAR model was constructed and proposed in our labs [34, 35] and is based on a series of molecules derived from sertindole, together with a series of other drugs, most of which were tested locally. The corresponding IC50s were used to derive a comparative molecular similarity analysis (CoMSiA), which is also a 3D-QSAR method that gives similar information as the previously discussed CoMFA method. Again this model can, and has been used to design molecules with reduced HERG affinity. If a molecule with an IC50 obtained from our labs does not show activity in a ‘safe’ range, then appropriate changes can be made as suggested by the coefficient plots obtained from the model. Applications are described below. The model was further validated in the following manner. First, a homology model of the open form of the channel was constructed from the Mthk crystal structure. Then three molecules, MK499, terfenadine and cisapride, as aligned in the CoMSiA model (Figure 1.4), were docked into the homology model. It was found that the previously described mutations [20] agreed with the docked model in terms of affecting activity. The docked model is shown in Figure 1.5. Application of this model to some drug discovery programs will be discussed next. EXAMPLES OF APPLICATION OF MODELS TO IMPROVE SELECTIVITY AGAINST HERG BLOCKADE We have applied our predictive CoMSiA model successfully to alleviate HERG blockage of drug candidates during the course of lead optimization. The general strategy is to make structural modifications at or around the pharmacophoric centers of HERG blockers with minimal impact on the targeted biological activity. These pharmacophoric centers correspond to the landmarks of aromatic centroids and basic nitrogen atom used for superimposing compounds onto sertindole in our CoMSiA model. Justification for this approach is evident from the CoMSiA coefficient plots. The first strategy would involve modification of the basic or positively charged nitrogen, if it exists in the molecule, and if this modification is tolerated by the targeted activity of the molecule. The electrostatic coefficient plots validate this suggestion. Most antihistamine compounds and those targeted to the CNS have a basic nitrogen, which is essential for activity towards the target, and hence this strategy might not work. The secondary strategy is based on the assumption that decreased hydrophobicity around aromatic centers by means of heterocycle replacement or elimination of aromatic ring would lead to

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Fig. 1.4 (A) The CoMSiA steric coefficient plots showing regions (in red) where substitution would enhance selectivity against the HERG channel. (B) The CoMSiA electrostatic coefficient plots showing regions where substitution by groups that increased negative potential (in orange) would increase selectivity against the HERG channel.

diminished HERG blockage. An example where this strategy has been used is shown in Figure 1.6. Here in a series of compounds lacking a positively charged or basic nitrogen, increasing the polar nature of substituents which corresponded to the primary aromatic group in sertindole decreased HERG IC50 as measured by patch-clamping IC50s. In a different example, a judicious addition of polar groups in a position corresponding to the para position

12 HUMAN ETHER-A-GO-GO RELATED GENE (HERG): A CHEMIST’S PERSPECTIVE

Fig. 1.5 Orthogonal views of sertindole docked into the homology model of HERG obtained from the crystal structure of Mthk showing interactions with Y652 and F656.

of the benzene ring in sertindole would also decrease HERG activity. This can be seen in the example provided in Figure 1.7 where again in a series lacking a basic nitrogen, appropriate substitution decreased the patch-clamping IC50 values. In both cases discussed, represented in Figures 1.6 and 1.7, the activities towards the targets in question were not adversely affected and some of the physical and pharmacokinetic properties were affected in a positive manner. In the examples discussed below, a direct application of the model could not be done, even in a retrospective manner, due to the difference in the HERG in vitro assay used in the example and the one used in the generation of the data used to derive the model. Several examples have been reported recently that used similar approaches during lead optimizations to remove binding interactions of compounds with the HERG channel. Friesen et al. [36] at Merck reported optimization of a series of phosphodiesterase-4 (PDE4) inhibitors. Initial optimization of compound CDP-840 with respect to PDE4 inhibition, metabolism issues and pharmacokinetic properties led to the identification of tertiary alcohol shown in Figure 1.8. However, this compound was found to exhibit significant prolongation of the QT interval following intravenous administration in anesthetized dogs. Further medicinal chemistry efforts were thus carried out to optimize structure with respect to the binding affinity to the HERG channel, in addition to other pharmacological properties. By simply

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Fig. 1.6 An example showing where successful application of the CoMSiA model was used to improve selectivity against HERG. The diamonds indicate the key points of alignment on the CoMSiA model. Decrease of the hydrophobicity of the aliphatic group corresponding to the primary hydrophobic group in the pharmacophore led to improved selectivity against HERG. Activity towards the HERG channel is listed and is obtained from the same assay as described in ref. [34]. R1 is kept constant in this series only.

replacing the tertiary alcohol phenyl group with a methyl or trifluoromethyl group, they were able to arrive at compounds that exhibited lower HERG binding affinity, similar in vitro profile and improved pharmacokinetics. It is interesting to point out that no binding data were given for the original lead CDP-840, which lacks the tertiary alcohol phenyl moiety. In this case, the activity measurements were from a radiolabeled ligand displacement assay. Another example is demonstrated by the medicinal chemistry efforts devoted to minimize affinity for the HERG channel and to improve the pharmacokinetic profile of neuropeptide Y Y5-receptor antagonists (Figure 1.9A) [37]. By attaching hydrophilic functionalities to the hydrophobic center, Blum et al. found out that it was possible to modulate binding interactions of Y5 antagonists with the HERG channel. An inherently acidic

14 HUMAN ETHER-A-GO-GO RELATED GENE (HERG): A CHEMIST’S PERSPECTIVE

Fig. 1.7 An example showing where successful application of the CoMSiA model was used to improve selectivity against HERG. The diamond indicates the key point of alignment on the CoMSiA model. Subsequent substitution led to improved selectivity against HERG. Activity towards the HERG channel is listed and is obtained from the same assay as described in ref. [34]. R1 is kept constant in this series only.

acyl-sulfonamide substituent was effective in removing the HERG activity, but adversely affected the compound’s potency at the Y5 receptor. They were able to ultimately develop a potent and selective Y5-receptor antagonist with favourably weak HERG activity. Incorporation of an acidic group near the basic nitrogen is also an effective way to remove HERG activity. An example is shown in Figure 1.9B from

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Fig. 1.8 Lead optimization and alleviation of binding interactions to the HERG channel for phosphodiesterase-4 inhibitors based upon CDP-840 (8).

a report by Fraley et al. at Merck during the optimization of the indolyl quinolinone class of KDR kinase inhibitors [38]. Using the alignment depicted in Figure 1.10, our CoMSiA model was able to predict that the carboxylic acid derivative is a weaker HERG blocker by about threefold, which is consistent with the experimental observations. The HERG activity was measured in these last two examples by an inflection point in the patch-clamp measurements. We would certainly expect that further advances in structural biology and in silico modelling efforts will continue to have great impact for aiding in the removal of the unwanted drug –channel interaction. In silico models will continue to play an important role in drug discovery programs in terms of a computational tool to filter compounds in library design and screening, and to guide structural modifications during lead optimization.

16 HUMAN ETHER-A-GO-GO RELATED GENE (HERG): A CHEMIST’S PERSPECTIVE

Fig. 1.9 Lead optimization and alleviation of binding interactions to the HERG channel for (A) neuropeptide Y Y5-receptor antagonists and (B) KDR kinase inhibitors.

Fig. 1.10 Alignment of 14 (left) and 15 (right) with sertindole (grey stick) for the prediction by the CoMSiA model to rank the HERG activity.

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CONCLUSIONS In summary, we have tried to discuss the biology, the assay systems, potassium ion channel structure, models for HERG blockers and applications of these models. The intention of this chapter would be for these to be of use in projects to improve selectivity against HERG, and hence design safer drugs for the future.

REFERENCES [1] Kuhlkamp, V., Mewis, C., Bosch, R. and Seipel, L. (2003) J. Cardiovasc. Pharmacol. 42, 113–117. [2] Rampe, D., Anderson, B., Rapien-Pryor, V., Li, T. and Dage, R. (1993) J. Pharmacol. Exp. Ther. 265, 1125–1130. [3] Brown, A.M. and Rampe, D. (2000) Pharm. News 7, 15–20. [4] Bennett, P.B. and Guthrie, H.R.E. (2003) Trends Biotechnol. 21, 563– 569. [5] Noller, J., Fuchs, K., Schmitt, J. and Loffler, T. (2003) Curr. Drug Discov. July, 37–38. [6] Molleman, A. (2003) In “Patch Clamping – An Introductory Guide to Patch Clamp Electrophysiology”. Wiley, UK. [7] Kiss, L., Bennett, P.B., Uebele, V.N., Koblan, K.S., Kane, S.A., Neagle, B. and Schroeder, K. (2003) Assay Drug Dev. Technol. 1, 127–135. [8] HERGLite, Chantest web site: http://www.chantest.com/herglitemethods.html. [9] Doyle, D.A., Cabral, J.M., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., Chait, B.T. and MacKinnon, R. (1998) Science 280, 69 –77. [10] Nobel e-museum web site: http://www.nobel.se/chemistry/laureates/2003/mackinnon-lecture. html. [11] Jiang, Y., Lee, A., Chen, J., Cadene, M., Chait, B. and MacKinnon, R. (2002) Nature 417, 515–522. [12] Jiang, Y., Lee, A., Chen, J., Cadene, M., Chait, B. and MacKinnon, R. (2002) Nature 417, 523–526. [13] Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene, M., Chait, B. and MacKinnon, R. (2003) Nature 423, 33 –41. [14] Jiang, Y., Ruta, V., Chen, J., Lee, A. and MacKinnon, R. (2003) Nature 423, 42– 48. [15] Kuo, A., Gulbis, J.M., Antcliff, J.F., Rehman, T., Lowe, E.D., Zimmer, J., Cuthbertson, J., Ashcroft, F.M., Szaki, T. and Doyle, D.A. (2003) Science 300, 1922–1926. [16] Webster, S.M., Del Camino, D., Dekker, J.P. and Yellen, G. (2004) Nature 428, 864–868. [17] Morais Carbral, J.H., Lee, A., Cohen, S.L., Chait, B., Li, M. and MacKinnon, R. (1998) Cell 95, 649 –655. [18] Keating, M.T. and Sanguinetti, M.C. (2001) Cell 104, 569–580. [19] Mitcheson, J.S. and Perry, M.D. (2003) Curr. Opin. Drug Discov. Dev. 6, 667 –674. [20] Mitcheson, J.S., Chen, J., Lin, M., Culberson, C. and Sanguinetti, M.C. (2000) Proc. Natl. Acad. Sci. USA 97, 12329–12333. [21] Chen, J., Seebohm, G. and Sanguinetti, M.C. (2002) Proc. Natl. Acad. Sci. USA 99, 12461–12466. [22] Sanchez-Chapula, J.A., Navarro-Polanco, R.A., Culberson, C., Chen, J. and Sanguinetti, M.C. (2002) J. Biol. Chem. 277, 23587– 23595. [23] Fernandez, D., Ghanta, A., Kauffman, G.W. and Sanguinetti, M.C. (2004) J. Biol. Chem. 279, 10120–10127.

18 HUMAN ETHER-A-GO-GO RELATED GENE (HERG): A CHEMIST’S PERSPECTIVE [24] Biller, S.A., Custer, L., Dickinson, K.E., Durham, S.K., Gavai, A.V., Hamann, L.G., Josephs, J.L., Moulin, F., Pearl, G.M., Sanders, M. and Vaz, R. (2004) In “Pharmaceutical Profiling in Drug Discovery for Lead Selection”. Borchardt, R.T., Kerns, E.H., Lipinski, C.A., Thakker, D.R. and Wang, B. (eds), AAPS Press, Alexandria, VA. [25] Beresford, A.P., Segall, M. and Tarbit, M.H. (2004) Curr. Opin. Drug Discov. Dev. 36–42. [26] Aronov, A.M. and Goldman, B.B. (2004) Bioorg. Med. Chem. 12, 2307– 2315. [27] Buyck, C., Tollenaere, J., Engels, N. and de Clerck, F. (2002) In “14th European Symposium on Quantitative Structure– Activity Relationships, Bournemouth, UK”, poster. [28] Roche, O., Trube, G., Zuegge, J., Pfimlin, P., Alanine, A. and Schneider, G. (2002) ChemBioChem 3, 455– 459. [29] Keseru, G.M. (2003) Bioorg. Med. Chem. Lett. 13, 2773–2775. [30] Fischer, H (2003) Roche Pharmaceuticals, Personal Communication. [31] Ekins, S., Crumb, W.J., Sarazan, R.D., Wikel, J.H. and Wrighton, S.A. (2002) J. Pharmacol. Exp. Ther. 301, 427– 434. [32] Ekins, S. (2004) Drug Discov. Today 9, 276–285. [33] Cavalli, A., Poluzzi, E., De Ponti, F. and Recanatini, M. (2003) J. Med. Chem. 45, 3844– 3853. [34] Pearlstein, R.A., Vaz, R.J., Kang, J., Chen, X., Preobrazhenskaya, M., Shchekotikhin, A.E., Korolev, A.M., Lysenkova, L.N., Miroshnikova, O.V., Hendrix, J. and Rampe, D. (2003) Bioorg. Med. Chem. Lett. 13, 1829–1835. [35] Pearlstein, R., Vaz, R. and Rampe, D. (2003) J. Med. Chem. 46, 2017–2022. [36] Friesen, R.W., Ducharme, Y., Ball, R.G., Blouin, M., Boulet, L., Cote, B., Frenette, R., Girard, M., Guay, D., Huang, Z., Jones, T.R., Laliberte, F., Lynch, J.J., Mancini, J., Martins, E., Masson, P., Muise, E., Pon, D.J., Siegl, P.K., Styhler, A., Tsou, N.N., Turner, M.J., Young, R.N. and Girard, Y. (2003) J. Med. Chem. 46, 2413–2426. [37] Blum, C.A., Zheng, X. and De Lombaert, S. (2004) J. Med. Chem. 47, 2318–2325. [38] Fraley, M.E., Arrington, K.L., Buser, C.A., Ciecko, P.A., Coll, K.E., Fernandes, C., Hartman, G.D., Hoffman, W.F., Lynch, J.J., McFall, R.C., Rickert, K., Singh, R., Smith, S., Thomas, K.A. and Wong, B.K. (2004) Bioorg. Med. Chem. Lett. 14, 351–355.

Progress in Medicinal Chemistry – Vol. 43, Edited by F.D. King and G. Lawton q2005 Elsevier B.V. All rights reserved.

2 Fluorescence-Based Assays JOHN F. ECCLESTON,1 JONATHAN P. HUTCHINSON2 and DAVID M. JAMESON3 1

National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK 2 GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK 3 Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96822-2319, USA

INTRODUCTION

20

THE FLUORESCENCE PROCESS

20

FLUORESCENCE INSTRUMENTATION Light Sources Excitation Monochromator Sample Cuvette The Emission Monochromator or Filter Signal Detection Other Components of a Fluorimeter

21 22 23 24 24 25 25

FLUORESCENCE PARAMETERS Excitation Spectra Emission Spectra Quantum Yield Fluorescence Lifetime Anisotropy/polarization

26 26 27 27 27 28

FLUORESCENCE INTENSITIES

30

FLUORESCENCE RESONANCE ENERGY TRANSFER

31

FLUOROPHORES AND LABELLING STRATEGIES

32

EXAMPLES OF FLUORESCENCE ASSAYS Binding Assays Kinetic Assays

38 38 40

DOI: 1 0 . 1 0 1 6 / S 0 0 7 9 - 6 4 6 8 ( 0 5 ) 4 3 0 0 2 - 7

19

20

FLUORESCENCE-BASED ASSAYS

APPLICATION OF FLUORESCENCE TO HIGH THROUGHPUT SCREENING Detection Platforms Interference in Fluorescence Measurements Strategies to Deal with Interference

43 43 45 46

CONCLUSION

47

ACKNOWLEDGEMENTS

47

REFERENCES

47

INTRODUCTION In recent years, the use of fluorescence techniques for monitoring protein– ligand and protein– protein interactions, or for measuring enzymatic activity, has grown rapidly and has now largely replaced assays using radioactivity. This methodological shift is due to the fact that the sensitivity of fluorescence methods approaches or equals that of radioactive methods, and they avoid the problems of radioactive and scintillation fluid disposal. Also, they do not require separation of bound and free ligand, and so binding measurements are made under true equilibrium conditions (as opposed, for example, to filter binding assays). For kinetic measurements, unlike radioactive assays, a continuous record of the reaction can also be monitored. The increased use of fluorescence methods is connected with the development of a wide range of instrumentation, including multi-well plate readers, and fluorophores. Many assay kits are also now available which allow novices to readily apply fluorescence-based assays to their particular system. However, knowledge of the fundamentals of fluorescence will always allow the user to optimize the method, avoid pitfalls and recognize artefacts. This chapter aims to discuss the basics of both the fluorescence phenomenon and the instrumentation for solution studies of equilibrium and kinetic measurements.

THE FLUORESCENCE PROCESS The fluorescence process is best described by reference to the Perrin-Jabłon´ski diagram shown in Figure 2.1. Upon absorption of light, a fluorophore in the ground state (S0) is excited into higher energy singlet state levels (the level reached, i.e. S1, S2 etc., will depend upon the wavelength of the absorbed light). Rapid thermalization (which occurs in the picosecond timescale) leaves the excited molecule in the lowest vibrational level of the first excited state (S1). This excited singlet state can persist for a short time, in the order of nanoseconds,

J.F. ECCLESTON, J.P. HUTCHINSON AND D.M. JAMESON

21

Fig. 2.1 Perrin-Jabłon´ski diagram. S0 is the ground state while S1 and S2 are electronically excited states.

before the system decays back to the ground state upon emission of a photon. Since this excited to ground state transition is usually of lower energy than the excitation process (as depicted in Figure 2.1), the emission is usually at longer wavelengths than the excitation. This wavelength difference is known as the Stokes Shift, after Sir George Gabriel Stokes, who was also the person who coined the term ‘fluorescence’. Exceptions to this basic mechanism, such as emission from the S2 state, or conversion of the excited state to a triplet state (which can lead to phosphorescence), do occur but are rare and are not discussed here. Virtually all fluorescence data can be described by one of the following five parameters: the excitation spectrum, the emission spectrum, the quantum yield, the fluorescence lifetime and the anisotropy or polarization of the emission. Before discussing these parameters in detail, the instrumentation typically used for fluorescence measurements is described. FLUORESCENCE INSTRUMENTATION A basic fluorimeter consists of a light source, a means of selecting the wavelength of exciting light (monochromator or filter), a sample cell (or sample well in the case of plate readers), a means of selecting the emission wavelength (again monochromator or filter) and a detector. By using a commercial fluorimeter, as compared to homebuilt instrumentation, the user will have only

22

FLUORESCENCE-BASED ASSAYS

Fig. 2.2 Schematic representation diagram of a spectrofluorimeter indicating key components: revised with permission from commercial literature from ISS, Inc. The principle components include the Xenon arc lamp, the excitation and emission monochromators, a quartz beam splitter, a quantum counting solution (Qc), three photomultiplier tubes (PMT) and excitation (Pex) and emission (Pem) calcite prism polarizers.

marginal control of most of these components but an understanding of component parts will allow the user to optimize the measurement. The design of a basic, modern spectrofluorimeter is shown in Figure 2.2; more details on the instrumentation can be found in several recent references [1 –3]. LIGHT SOURCES

Deuterium and tungsten lamps used in absorption spectrophotometry are not often used in general purpose fluorescence instruments since they are relatively weak light sources. The most common light source used in fluorimeters are Xenon or Xenon –Mercury arc lamps. The Xenon arc lamp emits useful light over the range of around 250 – 2,000 nm (useful light down to 200 nm can be

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23

achieved if the arc lamp is not of the ‘ozone-free’ variety, which are manufactured using glass that absorbs lower UV wavelengths so as to curtail the production of ozone), although the intensity varies significantly with wavelength. The Xenon – Mercury arc lamp has the same emission but superimposed on it are sharp lines due to mercury transitions. The most intense mercury lines are at 254, 297, 302, 313, 365, 405, 436, 546 and 578 nm. Lasers as light sources are at present typically used only for specialized applications such as fluorescence-activated cell sorters (FACS), lifetime measurements or fluorescence correlation spectroscopy (FCS). The most commonly used lasers include Neodymium-YAG, Argon-ion, Krypton-ion, Helium – Cadmium, Helium –Neon and Titanium –Sapphire lasers. Light emitting diodes (LEDs) and laser diodes, which are becoming increasingly popular for instrumentation dedicated to specific excitation wavelengths, produce nearly monochromatic light and are likely to be the important light sources in the future. The characteristics of these various light sources were recently reviewed [3]. We should note that most of the light sources listed above can be operated in a continuous mode (the intensity is constant as a function of time) or in a pulsed mode (the intensity varies with time) and the choice will depend on the instrument design and function.

EXCITATION MONOCHROMATOR

In a conventional fluorimeter, monochromatic light from the arc lamp is selected using a monochromator (Figure 2.2). This approach has the advantage over optical filters (see below) of offering continuously variable wavelength selectivity. Light from the arc lamp is focussed on the inlet slit and monochromatic light, produced by the dispersion element (typically a diffraction grating) is focussed on the outlet slit. The user generally has control over these slit widths, which may be defined based on either the physical size of the slit (in mm) or the bandwidth of light passed by the monochromator (in nm). The two are related by the dispersion factor of the monochromator in terms of nm/mm. If both inlet and outlet slits of the monochromator are of the same size, the shape of the transmitted light is approximately Gaussian with a width of half-maximum based on the selected wavelength. If the monochromator dispersion is 8 nm/mm, slit widths of 2, 1 and 0.5 mm will give bandwidths of half-maximum of 16, 8 and 4 nm, respectively. It should be noted that a significant fraction of light is transmitted outside the half-maximum bandwidth, a fact that must be taken into account when selecting the observation wavelengths if scattered light is to be avoided. In addition to the selected wavelength light, grating monochromators, which operate using the principle of constructive and

24

FLUORESCENCE-BASED ASSAYS

destructive interference, also pass higher order light. For example, a monochromator set at 280 nm will also pass this wavelength when set at 560 nm (referred to as second order light) [3].

SAMPLE CUVETTE

Fluorescence measurements are generally made at 908 to the excitation direction in order to reduce the level of exciting light reaching the detector, and so fluorescence cells are generally polished on all four sides. A wide range of cells is available manufactured from various types of glass. Quartz cells have the advantage of passing light down to below 200 nm, whereas cells made from typical optical glass can only be used at wavelengths above 320 –350 nm, depending on the precise glass chemistry. Most conventional fluorimeters are designed for 4 cm £ 1 cm £ 1 cm cells and require 2 – 3 ml of solution to reach the optical path. However, cells of the same external dimensions, which can be used with much less solution volume (e.g. as little as 70 ml), are available. Also micro cells can be accommodated in a home-built metal adapter painted optical black, which allows the exciting light to pass through the sample but without passing through the sides of the cuvette or solution meniscus. If non-standard cells are used, it is important to make sure that the exciting light impinges fully on the sample and not at the meniscus. This criterion can be readily verified visually by exciting with green light (550 nm), measuring the distance from the bottom of the cell holder to this light beam and then making sure that the sample reaches this height from the bottom of the cell. If not, spacers may be used beneath the cell. Cuvettes are available from numerous vendors and information can be found on their web sites (see, e.g. www.starna.com, www.brandtech.com/cuvettes, www.oceanoptics.com, www. optiglass.com, www.scicominc.com/cuvettes.htm.

THE EMISSION MONOCHROMATOR OR FILTER

The emitted light is passed through either a second monochromator or through a filter, which ideally allows isolation of the fluorescence signal from extraneous light. The latter includes Rayleigh scattering (light at the excitation wavelength) as well as Raman scattering. Raman scattering is due to the O – H stretching mode of the water and its position is excitation wavelength dependent and can be approximated from the equation 1 1 ¼ 2 0:00034 lR lEX

ð2:1Þ

J.F. ECCLESTON, J.P. HUTCHINSON AND D.M. JAMESON

25

where lEX is the excitation wavelength and lR is the Raman wavelength. Therefore, excitation at 350 nm will give, in addition to any fluorescence emission, light at 350 nm (Rayleigh scattering of excitation light) at 397 nm (Raman scattering of water) and a band at 700 nm (the second order of the 350 nm Rayleigh scattering). In cases of excitation at visible wavelength, e.g. 490 nm for fluorescein, the Raman scatter may lie at longer wavelengths (, 588 nm) than the emission. It is therefore necessary to isolate the fluorescence signal from these other sources of light. This process is most conveniently done by using a second monochromator, which allows for facile selection of the desired wavelength range (Figure 2.2). However, emission can also be viewed through a filter which will pass a higher percentage of emitted light relative to spurious light and hence improve the signal-to-noise ratio. A wide variety of filters is available. These fall into the general categories of (a) interference filters characterized by narrow transmission bands, (b) bandpass filters which are centred at a specific wavelength (like interference filters) but which pass a relatively broad wavelength range and (c) cut-off filters which pass all light above a specific wavelength. This latter filter is often referred to as a cut-on filter depending on the viewpoint of whether the transmission commences sharply at a given wavelength (cuts-on) or equivalently if the optical density decreases sharply at that wavelength (cuts-off). Regardless, the operational principle of these types of filters, which are also known as longpass filters, is that they can be used to block any excitation light scattered towards the emission direction and then collect a large percentage of the total emission. Transmission data of a wide variety of filters can be found on web sites such as www.mellesgriot.com, www.spectra-physics.com and www.oceanoptics.com. SIGNAL DETECTION

Most modern fluorimeters use photomultiplier tubes for measuring the emitted light. In analogue systems, the output signal is dependent on the high voltage applied to the photomultiplier tube. However, the most sensitive instruments use photon counting methods which have several advantages over conventional methods [3]. The use of charge-coupled devices (CCDs) has become more common, though, especially in fluorescence microscopy, as the sensitivity of these devices has improved. OTHER COMPONENTS OF A FLUORIMETER

The components described above are the basic requirements of a fluorimeter. However, many commercial instruments have additional features (Figure 2.2).

26

FLUORESCENCE-BASED ASSAYS

For example, a quartz beam splitter, which diverts a small percentage of the excitation light to a quantum counter (typically a concentrated solution of rhodamine B in ethanol) is often used to monitor the excitation intensity. The light from the beam splitter is directed onto the quantum counter (typically placed in a triangular cuvette) and the emission is observed through a cut-off filter. By recording the ratio of the sample signal to this reference signal, one can significantly reduce any time-dependent variations in lamp intensity. The reference signal is also useful for measuring corrected excitation spectra as briefly mentioned later. Other useful accessories are polarizers. If the excitation light is polarized and the emitted light is viewed through a second polarizer, one can calculate the fluorescence polarization or anisotropy of the emission – a parameter discussed in more detail later. In the above instrumental set-ups, the emitted light is viewed at 908 by a single photomultiplier. This arrangement is conventionally described as an ‘L’ format. However, if a second monochromator/filter and photomultiplier is also at 908 to the excitation light, this gives a ‘T’ format. The T format is mainly of use for polarization measurements, but for some specialized applications it allows emitted light at two different wavelengths to be detected simultaneously.

FLUORESCENCE PARAMETERS As mentioned above, all fluorescence data can be described by five parameters.

EXCITATION SPECTRA

The wavelength of the emitted light is fixed and the wavelength of the exciting light is varied. Generally, the excitation spectrum is the same as the absorption spectrum since the number of emitted photons depends upon the number of photons absorbed (assuming that the efficiency of the emission process is invariant with excitation wavelength, which is almost always the case). However, the arc lamp light source does not give constant light intensity at different excitation wavelengths and the efficiency of the monochromator is wavelength dependent; hence, the technical or uncorrected excitation spectrum will be distorted by these factors. Uncorrected spectra can be converted to true molecular spectra by taking into account the wavelength dependence of the excitation system. This correction procedure has been described in detail elsewhere [3] and will not be discussed further because here we are generally concerned with fixed excitation and emission values.

J.F. ECCLESTON, J.P. HUTCHINSON AND D.M. JAMESON

27

EMISSION SPECTRA

Here the wavelength of the exciting light is fixed and the wavelength of the emission monochromator is varied. The emission maximum is virtually always at longer wavelengths than the excitation maximum (the Stokes Shift). Again, the emission spectrum usually measured is termed a technical spectrum because both the monochromator and photomultiplier have wavelength-dependent responses, which can be corrected if necessary [3]. QUANTUM YIELD

The quantum yield ðQÞ is defined as the fraction of light absorbed by the fluorophore that is emitted as fluorescence (Q ¼ number of photons emitted/number of photons absorbed) and so can vary between 0 and 1. Q may also be defined as Q¼

kr kr þ knr

ð2:2Þ

where kr and knr are the rate constants of the radiative and non-radiative decay processes from the first excited state. Direct or absolute measurement of the quantum yield of a fluorophore is complicated. Hence, it is more common to measure the quantum yield relative to a known standard. In this case, a fluorescent standard with an excitation and emission spectrum similar to the unknown is used. Ideally, the optical density of both samples will be matched at the excitation wavelength and then the relative emission intensities (corrected for instrument response functions) can be determined. These instrument correction factors are typically provided by the manufacturer of the instrument. As discussed elsewhere [3], these correction factors can be very much dependent upon the polarization of the emission. FLUORESCENCE LIFETIME

The typical reader of this chapter may not have occasion to make lifetime measurements, as these, like quantum yield measurements, are very specialized. However, knowledge of the fluorescence lifetime may be important to the particular fluorescence assay being considered (especially polarization or anisotropy-based assays) and so some understanding is useful. Basically, the fluorescence lifetime (t) is the time that the fluorophore remains in the excited state before decaying to the ground state and is defined as 1=kr where kr is the rate constant of the decay process. The reader should remember, however, that excited state lifetimes – like radioactive decay half-lives – are parameters

28

FLUORESCENCE-BASED ASSAYS

applied to populations of molecules, i.e. the lifetime of any particular excited fluorophore cannot be predicted but rather the average behaviour of a large population of molecules can be determined (the specialized topic of singlemolecule measurements is beyond the scope of this article). More precisely, the lifetime of a fluorophore population is the time it takes for the intensity (excited by a very short light pulse) to decay to 1=e of its original intensity. Typically, fluorescence lifetimes are in the region of 0.1– 30 ns – although exceptions occur (e.g. pyrene and ruthenium derivatives can have lifetimes in the range of hundreds of nanoseconds). Fluorescence lifetimes are usually measured in one of two ways. First, with the pulse method, the fluorophore is excited with a very short pulse of light (modern lasers allow this to be in the picosecond to femtosecond range) and the time dependence of the emission process is measured. Second, in the traditional phase-modulation method, the fluorophore is continuously excited with light, which is sinusoidally modulated. Because of the finite lifetime of the excited state, the emitted light has a phase delay and is demodulated with respect to the excitation light. These two measurements allow lifetimes to be calculated. For a detailed description of these methods, the reader is referred to Refs. [1 – 4]. It should be noted that most fluorophores in biological systems do not decay with a single lifetime but show multi-exponential behaviour, which often reflects on the diverse molecular environments present. ANISOTROPY/POLARIZATION

Fluorescence anisotropy and polarization measurements are made in the same way and contain the same information, they are merely different ways of defining the measurement. The excitation light is polarized in a direction vertical to the laboratory axis and the emitted light is measured through a polarizer parallel and then perpendicular to the exciting light. (By use of a ‘T’ format the parallel and perpendicular intensities can be measured simultaneously whereas with an ‘L’ format, they are measured consecutively.) The two measurements are then used to calculate polarization (P) or anisotropy (r) by the following equations: P¼

Ik 2 I’ Ik þ I’

ð2:3Þ

r¼

Ik 2 I’ Ik þ 2I’

ð2:4Þ

where Ik and I’ are the intensities of the parallel and perpendicular polarized emission. We should note that even though these two functions differ slightly their information content is the same and the use of one or the other term is

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29

usually a matter of convenience. From these definitions one can demonstrate that: r ¼ 2P=ð3 2 PÞ: In practice, the measurement is complicated by the fact that the detection system (monochromator/filter and photomultiplier) may have different responses to light of the same intensity polarized in the parallel and perpendicular planes and so a correction must be applied. This correction is done by first exciting the fluorophore with light polarized in the direction horizontal to the laboratory axis. In this case, due to symmetry, there exists an equal chance of the emitted light being polarized in the parallel or perpendicular planes and any deviation from unity thus gives the normalization instrumental factor (usually known as the ‘G’ factor) required to obtain the correct values. The maximum values of anisotropy and polarization for fluorophores randomly oriented in solution are 0.4 and 0.5, respectively (for the derivation of these limits the reader is referred to Weber’s classic article [5]). The measured anisotropy or polarization is dependent on the ratio of the fluorescent lifetime of the fluorophore, t and its rotational rate, which can be expressed either as the Debye rotational relaxation time (r) or the rotational correlation time (tc) (we note that r ¼ 3tc ). 1 1 2 ¼ P 3




1 1 2 P0 3




3t 1þ r


 r0 t ¼ 1þ tc r



ð2:5Þ

ð2:6Þ

In these equations, r0 and P0 are the limiting anisotropy or polarization of the fluorophore and t is the lifetime. The limiting anisotropy or polarization values are those observed when the fluorophore is immobile and can be made by placing the fluorophore in anhydrous glycerol and making the measurement at a low temperature (typically below 0 8C). Although values of 0.4 and 0.5 are typically assumed for r0 and P0, respectively, care should be taken since there are many exceptions and also the limiting values are usually wavelength dependent [1 – 3, 5, 6]. From these equations it can be seen that for any given lifetime, the anisotropy/polarization of the fluorophore in solution depends on its rotational diffusion. For small fluorophores, with typical lifetimes in the nanosecond range, rapid rotation gives a low anisotropy/polarization whereas if the fluorophore is bound to a macromolecule with slow rotation the anisotropy/ polarization will be higher. To convert the observed anisotropy/polarization from a mixture of free and bound fluorophore into the fraction of bound ligand, we must understand the additivity properties of these functions. Weber explicitly derived the relationship

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FLUORESCENCE-BASED ASSAYS

governing additivity of polarizations from different species, namely ref. [7]:

 1 1 21 X 1 1 21 2 ¼ fi 2 ð2:7Þ kPl 3 Pi 3 where P is the actual polarization observed arising from i components, fi represents the fractional contribution of the ith component to the total emission intensity and Pi is the polarization of the ith component. This additivity principle was later expressed in terms of anisotropy (r) by Jabłon´ski [8] as X ð2:8Þ r0 ¼ fi ri We note that the anisotropy formulation appears to be the simpler function, but clearly the information content of the two approaches is identical and given the present day computer-assisted data analysis the difference is often moot. Perhaps, the most important consideration in such studies is the relative quantum yields of the free and bound probe. Specifically, if the quantum yield of the fluorophore changes upon binding, the fractional intensity terms in the additivity equations will alter. Although many probes (such as fluorescein) do not significantly alter their quantum yield upon interaction with proteins, others, such as methylanthraniloyl derivatives [9] can demonstrate significant enhancement of signal upon binding and one would be well advised to ascertain if such alterations occur. If the quantum yield does in fact change, one can correct the fitting equation to take this yield change into account. In terms of anisotropy the correct expression relating observed anisotropy (r) to the fraction of bound ligand (x) (where fraction bound plus fraction free equals 1), the anisotropy of bound ligand (rb), the anisotropy of free ligand (rf), and the quantum yield enhancement factor (g) is [10] r 2 rf ð2:9Þ x¼ rb 2 rf þ ðg 2 1Þðrb 2 rÞ A rigorous propagation of error treatment has explored the effect of uncertainties in various experimental parameters upon the calculated value of fraction bound and dissociation constant [11]. FLUORESCENCE INTENSITIES Although all fluorescence data can be described by the above five measurements, most readers will be more interested in fluorescence intensity measurements. It is important to realize that unlike absorbance measurements, fluorescence intensity measurements are not absolute. The absorbance (A), or optical density (OD) of a solution at a particular wavelength will be the same regardless of the spectrophotometer used since it is defined by Beer’s Law: A ¼ 1cl;

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where 1 is the molar extinction coefficient, c is the concentration and l is the path length. The fluorescence intensity of a fluorophore will depend on its intrinsic probability of absorbing light at the excitation wavelength (determined by its extinction coefficient) and the percentage of this light that is emitted as fluorescence (determined by its quantum yield). However, it also depends on the intensity of the light source, the efficiency of the optical system to transmit light, the slit widths of the monochromators, the geometry of the light collection (e.g. the apertures of the lenses), the efficiency of the photomultiplier tube (as well as the applied voltage in the case of analogue measurements) and the amplifier gain. For these reasons fluorescence intensity measurements can only be expressed in terms of arbitrary units or relative measurements. FLUORESCENCE RESONANCE ENERGY TRANSFER In the description of the fluorescence process above, it was stated that the excited state could decay with the emission of fluorescence, a radiative process, or by a non-radiative process. However, if a chromophore is in the proximity of the excited state fluorophore radiationless energy transfer may occur between the excited state fluorophore (donor) and this second chromophore (acceptor). If the acceptor is itself fluorescent, it may then emit light from its excited state and hence the observed emission will correspond to this secondary fluorophore. The efficiency of this energy transfer process can be measured by either the decrease in intensity of the donor fluorophore or the increase in intensity of the acceptor. The mechanism of fluorescence energy transfer was first proposed in 1918 by Jean Perrin and quantitative theories were developed over the next few decades. Between 1946 and 1949, T. Fo¨rster developed the most complete quantitative theory of molecular resonance energy transfer, and hence the phenomenon is often now referred to as Fo¨rster resonance energy transfer (FRET). The efficiency of transfer is given by the equation E¼

R60

R60 þ R6

ð2:10Þ

where R is the distance between the donor and acceptor and R0 is the distance between the donor and acceptor when transfer efficiency is 50%. The sixth power dependence results in a very large dependence of efficiency with distance, as shown in Figure 2.3, which depicts transfer efficiency versus R for a donor/ ˚ . R0 can be calculated from the equation acceptor pair with an R0 of 40 A R60 ¼ ð8:79 £ 10225 ÞQD k2 n24 J

ð2:11Þ

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FLUORESCENCE-BASED ASSAYS

Fig. 2.3 Depiction of the inverse sixth-power distance dependence of FRET. The transfer efficiency versus donor/acceptor distance shown is calculated for a donor/acceptor pair with an R0 of 40 A˚.

where QD is the quantum yield of the donor, k 2 is the orientation factor (related to the relative orientation of the donor and acceptor dipoles; k can vary between 0 and 4 but is usually assumed to be 2/3, the value corresponding to the case of rapid reorientation between donor and acceptor dipoles, n is the refractive index of the intervening solvent and J is the overlap integral between the emission spectra of the donor and the absorption spectrum of the acceptor. For a detailed account of this equation, see refs. [1 – 3, 12, 13]. For any pair of donor and acceptor, these parameters can be measured although some assumptions may need to be made for k2 : Thus, R0 can be calculated. However, a list for values of R0 for a range of donors and acceptors is given in ref. [13]. It can be seen from the above equations that energy transfer can be used to measure distances between donor and acceptor. However, the main use of FRET in the context of this chapter is merely to see whether or not donor and acceptor are in close proximity and hence provide a signal for binding or kinetic experiments. Choosing a pair of donor and acceptor for this purpose may be a matter of trial and error, although obviously the emission spectrum must overlap to some extent the absorption spectrum of the acceptor. However, if structural information is available, for example, of a protein– ligand complex, then the above equations can be used to choose a suitable donor/acceptor pair to optimize measurements on the protein –ligand complex. FLUOROPHORES AND LABELLING STRATEGIES Fluorophores used for fluorescence assays can be divided into intrinsic probes, which occur naturally in the system, and extrinsic probes, which need to be introduced into the system. Intrinsic probes can be part of a protein’s covalent

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structure, a co-factor or a ligand. Examples include the aromatic amino acids tryptophan and tyrosine (although the latter has relatively weak fluorescence), NADH, FAD, FMN, some porphyrins, some fatty acids and lipids, some modified nucleic acids, pyridoxal phosphate, chlorophylls and pteridines. Although intrinsic fluorophores find utility in mechanistic experiments and low throughput assays, their typically short excitation wavelengths (in the UV or mid-UV) make them less suitable for assays used in high throughput screening. Extrinsic probes have the advantage of being chosen for a particular purpose but the disadvantage is that their introduction may perturb the system. In the absence of structural information on the system of interest, their introduction is often an empirical process. The choice of which component in a system to label is determined by the nature of the process to be monitored by the assay, as discussed later. A vast variety of probes is now available with different reactive groups and lengths of spacer between the fluorophore and the reactive moiety (see, e.g. www.probes.com, www.evidenttech.com, www.intracellular.com, www.perkinelmer.com and www.amershambiosciences.com). Amino and thiol groups are those most commonly modified in biological systems, and the majority of probes are available as N-hydroxysuccinimide esters or derivatized with maleimides or iodoacetamides for this purpose. The choice of probe depends on the fluorescence parameter being measured and the intended use of the assay. Factors which should be considered include the excitation wavelength, excited state lifetime, environmental sensitivity, pH dependence of the fluorescence emission and susceptibility of the probe to photobleaching. Photobleaching is caused by irreversible chemical reaction of the excited triplet state (often via reaction with oxygen) and is a particular problem if high intensity light sources such as lasers are to be used. The fluorescence emission intensity of UV-wavelength excitable probes such as dansyl and coumarin derivatives is often highly sensitive to the polarity of their environment and can be used to monitor the binding of labelled ligands to a protein. However, these probes are generally only suitable for mechanistic evaluations and low throughput assays, since their relatively short excitation wavelength makes the measurement susceptible to test compound interference. As discussed later, interference from compounds and biological components is minimized substantially by using probes with excitation wavelengths above , 500 nm. The majority of longer wavelength probes also have much higher extinction coefficients which greatly improves sensitivity. Fluorescein (1) (lex ¼ 493 nm) has good aqueous solubility and remains a popular probe for fluorescence polarization high throughput screening assays but is quite sensitive to photobleaching and is non-fluorescent in its protonated state (pKa , 6.4). Tetramethylrhodamine (2) (lex ¼ 550 nm) is a better choice for its longer wavelength fluorescence, photostability and pH insensitivity. The CyDyee fluorophores (Amersham Biosciences), for example, Cy3 (3) (lex ¼ 550 nm)

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FLUORESCENCE-BASED ASSAYS

and Cy5 (4) ðlex ¼ 649 nmÞ; are versatile long wavelength probes which are photostable and insensitive to pH between 3 and 10. Two other classes of probe which are good for high throughput screening applications are the AlexaFluore and Bodipye (5) dyes (both from Molecular Probes). The AlexaFluors are available in a wide range of excitation wavelengths from UV to the furthest visible. They are robustly photostable, pH insensitive across a broad range, show good aqueous solubility and a high FRET efficiency. Bodipy dyes are available in a range of visible excitation wavelengths and are particularly suitable for fluorescence polarization applications because of their longer lifetimes. They have high extinction coefficients and quantum yields, and are polarity and pH insensitive. Their compact, neutral and non-polar structure is potentially less disruptive to the interaction with the binding partner.

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FLUORESCENCE-BASED ASSAYS

Labelling of small molecule ligands with the chosen probe is relatively straightforward since there may only be one reactive group present; if not, protecting group strategies can be used. If structural information is available on the interaction of interest, this can be used to guide the introduction of a probe, for example, via a group which protrudes from the binding site, and the choice of linker length. Often, however, the points available for covalent attachment of labels are limited and the process is somewhat empirical. Purification of the derivatized product is typically performed by HPLC. For example, many fluorescent nucleotide analogues have been synthesized by reacting the aminogroup of 20 -amino-20 deoxy nucleotides or 20 (30 )-O-carboxyethyl nucleotides [9]. There has been considerable success generating fluorescent ligands for active sites in proteins by labelling characterized inhibitors. These inhibitors can then be used to configure fluorescence polarization assays, which are sensitive to test compounds which compete with the probe for binding to that site. Two recent examples where known inhibitors were labelled to generate fluorescence polarization ligands, which were used for high throughput screening, are (6) and (7). (6) shows a ligand for the ATP-binding site of B-Raf kinase generated by labelling an ATP-competitive kinase inhibitor, which bears a unique amino group, with activated rhodamine green [14]. The labelled inhibitor is bound tightly by the kinase and with about a 10-fold higher affinity than the unlabelled inhibitor. (7) shows a semi-synthetic analogue of a pleuromutilin antibiotic,

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which was labelled via a spacer with the Bodipy-FL probe [15]. This fluorescent derivative binds tightly to ribosomes from a number of bacterial species (Escherichia coli Kd ¼ 7 nM) and was used in a fluorescence polarization assay to screen for compounds interacting at the pleuromutilin binding site. The configuration of these assays is discussed in more detail in a later section. For the labelling of peptides, a fluorescent amino acid can be incorporated into the synthetic procedure or fluorescent probes can be introduced at the N-terminal amino group or the 1-amino group of lysine residues within the sequence. Since the C-terminal amino acid is bound to the resin in solid phase synthesis procedures, selective N-terminal labelling is readily performed before cleavage of the chain from the resin and side chain deprotection. Labelling of proteins is more difficult because of the possibility of many different functional groups being modified. It may be that labelling of more than one functional group is not a problem. However, it is preferable to attempt to label at a single site in order to reduce the possibility that the labelling affects the biological function of the protein and increases reproducibility between preparations of a labelled protein. There are several approaches to labelling a protein with a fluorophore at a specific site. First, one may be lucky that the protein contains only one reactive cysteine residue. If this is not the case, then site-directed mutagenesis can be used to introduce a cysteine in a conserved manner. If a structure of the protein is available, then this can be used to select a residue which is on the outside of the protein and is likely to report on protein – protein or protein –ligand interactions. For example, a single cysteine was introduced into a phosphate binding protein by site-directed mutagenesis, which was then labelled with a thiol reactive coumarin derivative [16]. On binding phosphate the labelled protein shows a large enhancement of the coumarin fluorescence, and can be used as a sensor for changes in phosphate concentration. The second group often modified is the amino group. Most proteins contain many reactive lysine groups in addition to the N-terminal group. However, as the reactive species with reagents such as N-hydroxysuccinimide esters is – NH2 rather than NHþ 3 selective, use is made of the differing pKa’s of lysine (pKa , 9) and the N-terminal group (pKa , 7.9). Labelling near neutrality favours N-terminal labelling over lysine labelling. This approach also reduces the loss of the reagent by hydrolysis of the Nhydroxysuccinimide ester. Despite the widespread use of chemical modification of proteins, the problems of selective labelling and possible loss of function have led to the search for alternative methods of introducing fluorophores into proteins. The first and best known of these is to generate a fusion with one of the green fluorescent proteins [17]. However, this is a large protein (wildtype GFP from Aequorea has 238 residues) and so may affect the function of the fusion

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FLUORESCENCE-BASED ASSAYS

protein. A second approach is that of expressed protein ligation [18]. This approach uses the inducible self-cleavage activity of a protein-splicing element (an intein). The gene of the target protein is fused to a tag consisting of the intein and also a binding domain allowing affinity purification of the fusion protein, such as a chitin-binding domain. In the presence of a thiol (including fluorescent thiols) the intein undergoes specific self-cleavage which releases the fluorescently labelled target protein from the chitin bound intein tag. This method allows the introduction of one or more fluorophores at either the Nterminal or C-terminal ends of the protein or within the sequence of the protein. Another alternative to chemical modification is the C-terminal labelling of an expressed protein with a fluorescent derivative of the antibiotic puromycin [19]. Puromycin inhibits protein synthesis by competing with aminoacyl tRNA for incorporation into the growing chain. However, low concentrations of puromycin tend to incorporate only at the C-terminus of a full-length chain. Thus, the fluorescently modified group can be covalently attached to the C-terminus when mRNA lacking a stop codon is synthesized in a cell-free translation system in the presence of puromycin.

EXAMPLES OF FLUORESCENCE ASSAYS BINDING ASSAYS

As discussed previously, the anisotropy/polarization of the fluorescence emission of a probe is sensitive to its excited state lifetime and its rotational correlation time. Since the rotational motion is influenced by the size of the molecule or complex to which the probe is bound, this fluorescence parameter is particularly suitable for following binding reactions between a small labelled ligand and a macromolecular partner. Small molecules labelled with probes with lifetimes in the nanosecond range display a low anisotropy due to relatively rapid rotational motion, whereas the labelled ligand bound to a macromolecule displays a high anisotropy because of the reduced global motion. Local motion of the probe about rotatable bonds will, however, reduce the anisotropy of the bound state. The application of this method to the study of biomolecular interactions has received recent review [6, 11]. Figure 2.4 illustrates the specific use of fluorescence polarization for the mechanistic study of a protein– ligand interaction. In this experiment, 1 mM mant-GTPgS (a fluorescent, slowly hydrolysable GTP analogue) was titrated with increasing concentrations of the GTP-binding protein dynamin. The resulting binding curve was fitted with the binding Equation (1.9), which takes account of the differing quantum yields of the free and bound states of the probe, to yield the dissociation constant for

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Fig. 2.4 Example of ligand–protein binding followed by fluorescence anisotropy. 1 mM mantGTPgS (a fluorescent, slowly hydrolysable GTP analogue) was present and the concentration of the GTP-binding protein, dynamin, was varied by starting at high concentrations followed by dilution. The binding curve was fit to the anisotropy equation (in this case the yield of the fluorophore increased about 2-fold upon binding). A Kd of 8.3 mM was found. These data were obtained by Dr. Derk Binns and Dr. Joseph P. Albanesi.

the interaction (Kd ¼ 8:3 mM). More details regarding the interaction of this nucleotide with dynamin can be found in ref. [20]. Fluorescence polarization is particularly suitable as a readout in high throughput screening assays which are sensitive to antagonism of a small molecule– macromolecule interaction at equilibrium. An example was presented previously of a labelled inhibitor which is tightly bound by the B-Raf kinase. Such assays are usually configured with a macromolecule concentration similar to the dissociation constant for its interaction with the ligand (i.e. halfway up the binding curve), which gives a large polarization change but retains sensitivity to competitively binding test compounds, which cause a reduction in the observed polarization. The lower limit with which this type of assay can determine IC50 values depends on the affinity with which the labelled ligand is bound – ideally this should be ,100 nM. Further examples of this type of assay are given in ref. [21]. In clinical studies, fluorescence polarization-based immunoassays (FPIA) have become widespread since the introduction of the Abbott TDx instrument in the early 1980s [22]. In this method, a fluorescent derivative of a target molecule (e.g. a drug or metabolite) is bound to an antibody against the target molecule, which gives rise to a high polarization, and then introduction of a sample

40

FLUORESCENCE-BASED ASSAYS

containing the unlabelled target molecule (e.g. from bodily fluids) results in competitive release of the fluorophore-labelled target molecule and a reduction in the polarization. Recent examples of this specialized application are given in refs. [23, 24]. Fluorescence polarization can also be used to monitor protein –protein interactions, but in this case care must be taken to ensure that the lifetime of the fluorophore is sufficiently long so that a measurable change in polarization occurs upon dissociation of the protein oligomer. In some cases, even if the lifetime of the probe is not intrinsically long enough to expect significant changes in polarization upon dissociation of the protein complex, changes in local mobility of the fluorophore upon dissociation may occur such that significant changes in polarization are detected [25]. It is possible that a fluorophore attached nearer to the binding interface will display an environmentally induced intensity change, but such labelling attempts would require knowledge of the complex structure and risk perturbing the interaction. Another strategy is to label each component with an appropriately chosen pair of probes and use FRET measurement to detect the complex. The detection of a FRET signal is critically dependent on the distance between the probes in the complex, as discussed previously. These distance constraints can in part be relaxed by using a modification of the technique called timeresolved energy transfer (TRET). Here, long-lifetime donors based on lanthanide ion complexes transfer energy to long wavelength acceptors (e.g. allophycocyanins). The distance at which this energy transfer is effective is extended compared to conventional fluorophore pairs. This approach also allows indirect labelling strategies to be used, for example, via antibodies to a specific sequence or tag engineered into the protein. The use of pulsed excitation and time-gated detection allows short-lived background (e.g. scatter and autofluorescence) to decay before the longer lifetime emission of the probe is measured, improving the assay performance and sensitivity. TRET and allied methods are discussed in more detail in ref. [26].

KINETIC ASSAYS

Fluorescence methods are widely used for the determination of the kinetic mechanism of enzyme reactions, and are also used to configure kinetically read assays for high throughput screening. Here, we are concerned with kinetic measurements where the enzyme is at catalytic concentrations with a large excess of substrate and the steady state rate of the reaction is measured. Studies of enzyme mechanisms where the concentration of the enzyme is higher than that of the substrate resulting in the so-called single turnover conditions in which

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the formation and decay of intermediates of the mechanism are observed require rapid reaction equipment and are beyond the scope of this chapter. At the simplest level, for a fluorescence-based assay the reaction needs to proceed with either the formation of a fluorescent product from a nonfluorescent substrate or vice versa. An example of the first of these is the use of the non-fluorescent butyl ester of resorufin as a substrate in a hydrolase assay, where enzymatic cleavage of the ester bond yields highly fluorescent resorufin. An example of the latter is any enzyme reaction involving the oxidation of NAD(P)H to the non-fluorescent NAD(P)þ [27]. FRET may also be used for enzyme assays where the enzymatic reaction changes the distance or orientation of the two fluorophores in the substrate, thus changing the observed fluorescence intensity of the donor or acceptor. Using a protease reaction as an example, doubly labelled peptide substrates where the labels span the cleavage site, such as DABCYL-(Xaa)n-EDANS and Abz-(Xaa)n-3nitrotyrosine where (Xaa)n is any aminoacid sequence, have been used [28,29]. The degree of resonance energy transfer is highly dependent on the distance between donor and acceptor and this method is generally applicable for peptides of 11 –12 amino acids or less. The major aim of steady-state mechanistic measurements is to study the effect of substrate concentration on the steady-state rate of the enzymic reaction and so derive the vmax for the reaction (the rate at saturating substrate concentration) and the Km of the reaction (the substrate concentration giving half-maximum rate). Studies can then be made in the presence of potential inhibitors of the reaction in order to determine the mechanism of inhibition such as competitive, non-competitive or mixed inhibition. Analysis of such data is common to any assay method and the reader is referred to standard text books on enzymology. Before making fluorescence-based enzyme assays, it is extremely useful to record the excitation and emission spectra at the start and at the end of the reaction using the instrument conditions to be used for the kinetic measurement. Overlaying these spectra will show the optimal excitation and emission wavelengths for the kinetic measurements, i.e. the wavelengths that give the largest change in intensity and hence greatest sensitivity of the method. Such measurements will also show the presence of other components of the system, which may contribute to the fluorescence signal. For example, in the work described below with a protease from the malarial parasite, it was shown that the detergent, NP-40, contained a weakly fluorescent impurity [30]. It is then necessary to show that the intensity change is proportional to the extent of the reaction. In the case where only the substrate or product is fluorescent, this can be done by simply making a set of dilutions of the fluorophore and determining the linearity of the fluorescence intensity. In the case of energy transfer measurements, appropriate mixtures of substrate and

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FLUORESCENCE-BASED ASSAYS

product can be made to check the linearity of response. There are two reasons why the response may not be linear. The first is that at high concentrations of fluorophore, the detector becomes saturated. This problem can be easily solved by reducing the amount of light reaching the detector by either reducing the size of the slits on the excitation or emission monochromators or by using a neutral density filter. A second reason is that inner filter effects may affect the measurement if the absorbance of the solution at the excitation wavelength becomes excessive during the reaction. This problem is well illustrated in the following assay for a malarial parasite protease. A decapeptide containing the target sequence with N- and Cterminals replaced by cysteine residues was synthesized [30]. This peptide was doubly labelled by reaction with iodoacetamidotetramethylrhodamine. Normally, the intermolecular Kd for rhodamine dimerization is , 1 mM.

Fig. 2.5 (a) Sketch depicting a decapeptide labelled at both N- and C-termini with rhodamine. Enhancement of the fluorescence occurs upon proteolytic cleavage with PfSUB-1 (a subtilisin-like serine protease expressed in the merozoite stage of the human malaria parasite Plasmodium falciparum) and subsequent disruption of the weakly fluorescent ground-state rhodamine dimer. (b) Left. Absorption spectra of peptide (part A) before (solid line) and after (dotted line) proteolysis. Right: Emission spectra of peptide (part A) before (solid line) and after (dotted line) proteolysis. Dashed line shows buffer background.

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However, because of the high local concentration of rhodamine in the doubly labelled peptide, it exists as a dimer in the mM concentration range. The rhodamine dimer has very low fluorescence compared with the monomer so on cleavage of the peptide by the protease, there is a 15-fold increase in the fluorescence intensity which allows the reaction to be monitored. This scenario is depicted in Figure 2.5. However, there is a large difference in the extinction coefficient at the excitation wavelength (550 nm) between the rhodamine monomer (98,600 M21 cm21) and dimer (29,900 M21 cm21). Therefore, when a solution of 2 mM peptide (4 mM rhodamine) is cleaved by the protease, the absorbance, in a 1-cm pathlength cell, at 550 nm changes from 0.120 to 0.394. In this case, the excitation light transmitted through the 1-cm cell falls from 76 to 40% and hence the light available in the centre of the cuvette – the region typically monitored by the collection optics – to excite the fluorophore in the monomeric state is less than that in the dimer state, resulting in less than expected emitted light. This phenomenon is termed the inner filter effect and can be important when large changes in absorbance at the excitation wavelength occur during the reaction. To minimize such effects, and to conserve sample volume, these types of measurements are often carried out using smaller cuvettes, e.g. 3 mm pathlength. APPLICATION OF FLUORESCENCE TO HIGH THROUGHPUT SCREENING High throughput screening is now almost universally practised by the pharmaceutical industry in the search for small molecule leads against drug targets. Many components contribute to make a successful high throughput screening campaign – a suitable assay which tests the activity of the target being screened, a chemically diverse collection of test compounds, liquid handling equipment to transfer test compounds and assay reagents into the screening plates, a plate reader to measure the assay signal coming from each well and software to deal with the considerable amount of data generated. In the early years, assays based on radiochemical detection were typically used, but more recently the emphasis has shifted to designing assays based on fluorescence, which offers a potentially higher quality and more discriminating output, is more amenable to miniaturization and avoids the logistical problems associated with using radioactivity. DETECTION PLATFORMS

The majority of high throughput screening campaigns are run in plates of either 96, 384 or 1,536 wells. The detectors (plate readers) used to measure

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fluorescence in high throughput screens can be considered in two categories – point readers and imagers. The different detection modes (i.e. intensity, polarization, lifetime) and the information obtained from them have been discussed earlier. Point readers are photomultipier-based detectors, which measure the fluorescence signal from each well of the plate in turn, typically by moving the plate but in some cases by moving the detection head. The optical set up of a typical reader is slightly different to that of the cuvette-based fluorimeter described earlier, since the geometry of plates requires that the emitted light is monitored along the same path as the excitation light, usually from above the plate. In some instances, the detection is through the underside of glassbottomed plates but the principles are the same. Figure 2.6 shows a schematic diagram of a typical point reader. The light source is usually a Xenon arc lamp but can be a laser or pulsed source for more sophisticated measurements. The desired excitation wavelength is usually selected using a bandpass filter rather than a monochromator. The excitation light is reflected into the sample in

Fig. 2.6 Schematic diagram of a fluorimetric plate reader designed for high throughput screening.

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the plate by a dichroic mirror which is chosen to reflect light of the excitation wavelength. The longer wavelength emitted light, however, passes through the dichroic mirror thus separating it from the excitation path. A second bandpass filter appropriate to the emission wavelength of the fluorophore reduces scattered light reaching the photomultiplier which generates the signal. For the detection of fluorescence polarization, additional polarizing filters are used on the excitation and emission paths. The parallel and perpendicular components of the emission are measured in turn by exchanging the two emission polarizers (T-format measurements as discussed earlier are not possible because of the geometry of the system). For time-resolved measurements (e.g. TRET) a pulsed light source is used together with electronically time-gated detection of the emitted light. The pulsed source is obtained either using a flash lamp or a rapidly spinning disc called a chopper. Imaging readers have gained popularity in recent years, where the emitted light from the whole plate is captured on a CCD chip using a sophisticated lens array. The optical path is similar to that shown in Figure 2.6, where the CCD camera replaces the photomultiplier. Computational analysis then imposes the well structure of the plate on the image and evaluates an intensity value for each well. Polarization and time-resolved measurements are also possible with these devices. No mechanical movement of the plate is required during the measurement and the overall reading time can be greatly reduced.

INTERFERENCE IN FLUORESCENCE MEASUREMENTS

All types of assay are prone to interference – much of which originates from the test compounds themselves. In fluorescence-based assays, autofluorescence, quenching, inner-filter effects and test compound insolubility, can all perturb the measurement and an understanding of these in the context of the physical principles of fluorescence is valuable. Autofluorescence is a slightly misleading term, which refers to either a specific fluorescence signal from the test compound under the particular detection conditions or more likely a spurious signal originating from some contaminant in the sample. Quenching, which can occur by more than one mechanism, refers to a reduction in the probe’s emission intensity caused by an interaction between the quencher and the probe. The inner-filter effect is an absorbance-based phenomenon where either the excitation or emission light is attenuated by test compound or contaminant absorption (as discussed earlier). Test compound insolubility can give rise to excessive light scattering. Although this will be at the same wavelength as the excitation light, if the effect is large enough it can distort the measurement. Depending on the direction of the signal change in an assay based on fluorescence intensity, all of these effects can generate either false

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positives or false negatives. The frequency of false negatives is statistically low, since it depends on the concurrence of two low probability events (a hit and an interference phenomenon in the same well). However, the likelihood of generating false positives is very real and it is essential to bear this in mind at the analysis stage and have mechanisms in place to reject these in favour of the real hits. Furthermore, the assay should be designed so as to minimize this. The effect of compound interference on some readout modes is more specific. For example, consider a fluorescence polarization assay measuring the displacement of a small labelled ligand from a macromolecular binding partner, as discussed previously. Compound autofluorescence is likely to be of low polarization and can thus exaggerate the apparent level of ligand displacement in that well, hence generating a false positive. Conversely, the formation of aggregates between test compounds and a small labelled ligand will give rise to a high polarization state.

STRATEGIES TO DEAL WITH INTERFERENCE

A general strategy to mitigate optical interference, which should be followed whenever possible, is to use long wavelength probes. The four sources of test compound interference described above are all observed to decrease quite sharply as the excitation wavelength is increased. A striking reduction in interference is typically seen when moving from UV wavelength probes (e.g. coumarins) to visible wavelength probes. Some readout modes are more robust to interference. For example, in TRET measurements, long-lifetime donors (e.g. lanthanide ion complexes) are used in conjunction with time-gated detection of the acceptor emission. Compound autofluorescence, which is typically of very short lifetime, is allowed to decay before the longer lifetime emission from the probe is measured. A downside to this technique is that the donors used are of UV wavelength and prone to quenching. However, by monitoring the emission from both the donor and the acceptor, a correction for quench can be introduced. The direct measurement of a probe’s lifetime, which is currently in its infancy and requires sophisticated instrumentation, is another strategy to minimize interference from autofluorescence, but these measurements do suffer from quenching effects. A particular advantage of using polarization is that the total intensity (which, upon excitation with polarized light is equal to the sum of the parallel emission intensity plus twice the perpendicular emission intensity) is available as a secondary parameter, and this should be used to highlight wells displaying anomalously high or low intensity values indicative of autofluorescence or quenching respectively.

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CONCLUSION Fluorescence techniques occupy a central position in the study of protein– ligand and protein – protein interactions. Knowledge of the origins of the various fluorescence parameters described here allows sensitive and informative assays to be designed, which can be used in detailed mechanistic evaluations of interactions at equilibrium as well as the kinetics of enzyme –catalysed processes. The development of sensitive and accurate multi-well plate readers and a diversity of long wavelength fluorophores have revolutionized the use of fluorescence-based assays in miniaturized high throughput screening, and these methods now underpin the majority of early stage drug discovery programmes. ACKNOWLEDGEMENTS DMJ wishes to acknowledge support from the American Heart Association (Grant No. 0151578Z). REFERENCES [1] Valeur, B. (2002) In “Molecular Fluorescence”. Wiley-VCH, Weinheim. [2] Lakowicz, J.R. (1999) In “Principles of Fluorescence Spectroscopy”. Kluwer Academic/ Plenum Publishers, New York. [3] Jameson, D.M., Croney, J.C. and Moens, P.D. (2003) Methods Enzymol. 360, 1– 43. [4] Jameson, D.M. and Hazlett, T.L. (1991) In “Biophysical and Biochemical Aspects of Fluorescence Spectroscopy”. Dewey, G. (ed.), pp. 105–133. Plenum Press, New York. [5] Weber, G. (1966) In “Fluorescence and Phosphorescence”. Hercules, D. (ed.), pp. 217 –240. Wiley, New York. [6] Jameson, D.M. and Croney, J.C. (2003) Comb. High Throughput Chem. 6, 167 –173. [7] Weber, G. (1952) Biochem. J. 51, 145–155. [8] Jabłon´ski, A. (1960) Bull. Acad. Polon. Sci. Serie des Sci. Math. Astr. et Phys. 6, 259–264. [9] Jameson, D.M. and Eccleston, E.F. (1997) Methods Enzymol. 278, 363–390. [10] Mocz, G., Helms, M.K., Jameson, D.M. and Gibbons, I.R. (1998) Biochemistry 37, 9862– 9869. [11] Jameson, D.M. and Mocz, G. (2005) In “Protein-Ligand Interactions: Methods and Applications”. Neinhels, G.U. (ed.), Humana Press, N.J., U.S.A. [12] Clegg, R.M. (1996) Fluorescence Imaging Spectroscopy and Microscopy. Wang, X.F. and Herman, B. (eds), vol. 137. Wiley, New York. [13] Van Der Meer, B.W., Coker, G. and Chen, S.-Y.S. (1991) In “Resonance Energy Transfer. Theory and Data”. Wiley-VCH, New York. [14] Dean, D.K., Takle, A.K. and Wilson, D.M (2002) PCT Int. Appl. WO 02 24680; (2002) Chem. Abstr. 136, 279456. [15] Hunt, E. (2000) Drugs of the Future. vol. 25, 1163–1168. [16] Brune, M., Hunter, J.L., Howell, S.A., Martin, S.R., Hazlett, T.L., Corrie, J.E.T. and Webb, M.R. (1998) Biochemistry 37, 10370–10380. [17] Subramaniam, V., Hanley, Q.S., Clayton, A.H. and Jovin, T.M. (2003) Methods Enzymol. 360, 178–201.

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[18] Blaschke, U.K., Silberstein, J. and Muir, T.W. (2000) Methods Enzymol. 328, 478–496. [19] Tabuchi, I. (2003) Biochem. Biophys. Res. Comm. 305, 1–5. [20] Eccleston, J.F., Binns, D.D., Davis, C.T., Albanesi, J.P. and Jameson, D.M. (2002) Eur. Biophys. J. 31, 275– 282. [21] Turconi, S., Shea, K., Ashman, S., Fantom, K., Earnshaw, D.L., Bingham, R.P., Haupts, U.M., Brown, M.J.B. and Pope, A.J. (2001) J. Biomol. Screen. 6, 275– 290. [22] Jolley, M.E., Stroupe, S.D., Schwenzer, K.S., Wang, C.J., Lu-Steffes, M., Hill, H.D., Popelka, S.R., Holen, J.T. and Kelso, D.M. (1981) Clin. Chem. 27, 1575–1579. [23] Johnson, D.K., Combs, S.M., Parsen, J.D. and Jolley, M.E. (2002) Environ. Sci. Technol. 36, 1042–1047. [24] Lonati, S., Novembrino, C., Ippolito, S., Accinni, R., Galli, C., Troonen, H., Campolo, J., Della Noce, C., Lunghi, G. and Catena, F.B. (2004) Clin. Chem. Lab. Med. 42, 228– 234. [25] Jameson, D.M. and Seifried, S.E. (1999) Methods 19, 222–233. [26] Pope, A.J., Haupts, U.M. and Moore, K.J. (1999) Drug Discov. Today 4, 350 –362. [27] Rangachari, K., Davis, C.T., Eccleston, J.F., Hirst, E.M.A., Saldanha, J.W., Strath, M. and Wilson, R.J.M. (2002) FEBS Letts. 514, 225 –228. [28] Holskin, B.P., Bukhtiyarova, M., Dunn, B.M., Baur, P., de Chastonay, J. and Pennington, M.W. (1995) Anal. Biochem. 227, 148–155. [29] Meldal, M. and Breddam, K. (1991) Anal. Biochem. 195, 141–147. [30] Blackman, M.J., Corrie, J.E.T., Croney, J.C., Kelly, G., Eccleston, J.F. and Jameson, D.M. (2002) Biochemistry 41, 12244–12252.

Progress in Medicinal Chemistry – Vol. 43, Edited by F.D. King and G. Lawton q2005 Elsevier B.V. All rights reserved.

3 Selective and Combined Neurokinin Receptor Antagonists MARC GERSPACHER Novartis Institutes for Biomedical Research Basel, Novartis Pharma AG, CH-4002 Basel, Switzerland

INTRODUCTION Neurokinins (tachykinins) Neurokinin Receptors

50 50 51

NEUROKININ RECEPTORS AS DRUG TARGETS: POTENTIAL CLINICAL USE OF NEUROKININ ANTAGONISTS Central Nervous System Disorders Emesis Gastrointestinal and Urinary Tract Diseases, Uterus Contractions Migraine, Pain Airways Diseases

53 53 55 55 56 56

RECENT DEVELOPMENTS IN THE SEARCH FOR SELECTIVE NEUROKININ ANTAGONISTS NK1 Receptor Antagonists NK2 Receptor Antagonists NK3 Receptor Antagonists

57 59 65 68

COMBINED NEUROKININ ANTAGONISTS Dual NK1/NK2 and Triple NK1/NK2/NK3 Receptor Antagonists Dual NK2/NK3 Receptor Antagonists

68 68 93

CONCLUSIONS

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E-mail address: [email protected] (M. Gerspacher). DOI: 1 0 . 1 0 1 6 / S 0 0 7 9 - 6 4 6 8 ( 0 5 ) 4 3 0 0 3 - 9

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INTRODUCTION NEUROKININS (TACHYKININS)

Neurokinins (NKs) also known as tachykinins (TKs) are a family of small peptides with a common C-terminal sequence that consists of five amino acids Phe-X-Gly-Leu-Met-NH2, where X represents a variable amino acid (Table 3.1) [1 – 3]. The biological activity of the NKs/TKs is determined mainly by this C-terminal sequence [1]. In a widely accepted nomenclature, peptides from nonmammalian sources are referred to as tachykinins, while mammalian peptides are called neurokinins [2]. In 1931, substance P (SP) was isolated by von Euler and Gaddum as the first neurokinin [2]. In 1971, 40 years after the first isolation, the undecapeptide amino acid sequence of substance P (isolated from bovine hypothalamus extracts) was identified and also first synthesized [2]. In addition, a number of non-mammalian tachykinins like eledoisin, phyllomedusin, uperolein, physalaemin and kassinin were isolated [1, 3]. In 1984, neurokinin A (NKA) and neurokinin B (NKB) were discovered [3], and two other NKs, which are N-terminal extensions of NKA, namely neuropeptide K (NPK) and neuropeptide g (NPg) have also been isolated [3]. Recently, a novel tachykinin, hemokinin 1, was discovered [5]. The term tachykinin describes the rapid contractile action of these peptides on smooth muscle [4].

Table 3.1 STRUCTURES OF TACHYKININS AND CLOSELY RELATED ANALOGUES Name

Structure

Substance P (SP) Neurokinin A (NKA) Neurokinin B (NKB) Neuropeptide K (NPK)

Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2 Asp-Ala-Asp-Ser-Ser-Ile-Glu-Lys-Gln-Val-Ala-Leu-Leu-Lys-AlaLeu-Tyr-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-AspSer-Phe-Val-Gly-Leu-Met-NH2 Asp-Ala-Gly-His Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-SerPhe-Val-Gly-Leu-Met-NH2 Pyr-Pro-Ser-Lys-Asp-Ala Phe-Ile-Gly-Leu-Met-NH2 Pyr-Asn-Pro-Asn-Arg-Phe-Ile-Gly-Leu-Met-NH2 Pyr-Pro-Asp-Pro-Asn-Ala-Phe-Tyr-Gly-Leu-Met-NH2 Pyr-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Gly-Leu-Met-NH2 Asp-Val-Pro-Lys-Ser-Asp-Gln-Phe-Val-Gly-Leu-Met-NH2 D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-Leu-Leu-NH2 D-Lys(Nic)-Pro-Ala(3-pyridyl)-Pro-D-Phe(3,4-di Cl)-Asn-D-Trp-PheD-Trp-Leu-Nle-NH2 Suc-Asp-Phe-MePhe-Gly-Leu-Met-NH2 Arg-Ser-Arg-Thr-Arg-Gln-Phe-Tyr-Gly-Leu-Met-NH2

Neuropeptide g (NPg) Eledoisin Phyllomedusin Uperolein Physalaemin Kassinin Spantide I Spantide II Senktide Hemokinin I

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In mammals, SP, NKA, NPK and NPg are products of the gene preprotachykinin A (PPT-A, also known as PPT I). Preprotachykinin A is expressed in both the central and the peripheral nervous system. The sequence of NKB, on the other hand, is encoded by the preprotachykinin B gene (PPT-B, also known as PPT II). Preprotachykinin B is selectively expressed in the central nervous system [2, 3]. Recently another preprotachykinin gene, termed PPT-C has been described. The PPT-C gene encodes hemokinin I, a putative autocrine factor for the growth of haematopoietic cells [5 – 7] and very recently it has been shown that PPT-C gene can also generate four additional tachykinin peptides, named endokinins A, B, C and D [8]. The two PPT-A and PPT-B genes come from a common precursor gene by duplication. The precursor RNA is alternatively spliced to give three different PPT-A mRNAs (a-, b-, and g-PPT-A) comprising different exon usage within the protein coding region [2]. a-PPT-A generates SP, b-PPT-A generates SP, NKA and NPK and g-PPT-A is the producer of SP, NKA and NPg. Tachykinins are formed from their precursors by the action of specific proteases, followed by amidation of the COOH terminus [2, 3]. Amidation is important for the biological activity and is a characteristic feature of all neurokinins/tachykinins. Neurokinins/tachykinins are widely distributed in the central and peripheral nervous system. Neurons are the major source of neurokinins/tachykinins, particularly in sensory, somatic and visceral fibres with a prominent location in the peripheral endings of primary afferent capsaicin sensitive neurons. These neurons innervate many sites including airways, skin, gastrointestinal and urinary tracts [3]. Neurokinins/tachykinins are stored in granules and are released via calcium-dependent mechanisms upon sensory stimulation by irritants such as capsaicin or sulphur dioxide, or by mechanical stimulation. A mixture of peptides consisting of several neurokinins/tachykinins, calcitonin gene-related peptide (CGRP) and other peptides may be released by each neuron into the synaptic cleft [2]. The neurokinins/tachykinins exert their action on defined tachykinin or neurokinin receptors. Inactivation of neurokinins/tachykinins occurs via breakdown into fragments by the action of proteases on the cell surface [2]. Three distinct neurokinin receptors have been identified, which have been termed NK1, NK2 and NK3 receptors. Substance P displays the highest affinity for the NK1 receptors and NKA and NKB for the NK2 and NK3 receptors, respectively [9]. NEUROKININ RECEPTORS

The three receptors NK1, NK2 and NK3 belong to the family of G-protein coupled receptors (GPCR) with seven transmembrane (7TM) hydrophobic

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domains, an extra-cellular amino-terminus, and a cytoplasmic carboxy-terminus. The receptors consist of 350 –500 amino acid residues [2, 4, 10 – 12]. The human NK1 receptor cDNA has been cloned and functionally expressed in a human IM-9 lymphoblast cell line and human lung. The primary sequence consists of 407 amino acid residues and is 94.5% identical to the rat NK1 receptor (isolated from rat brain and salivary glands) [11]. Twenty-two residues differ from the rat receptor with eight each in the extracellular and intracellular domains and six residues in the membrane spanning domains [11]. In addition to the human and rat NK1 receptors, guinea pig (uterus, COS-7 cells) and mouse receptors have been isolated [11]. The genes for the human and the rat NK1 receptors have also been isolated and partially characterized. They were found to be similar in structural organization [11]. Pharmacological techniques and measurement of specific receptor mRNA in different tissues have been used to determine the distribution of the neurokinin receptors [11, 12]. High levels of NK1 receptor expression are found in both the CNS (spinal cord, striatum, hypothalamus) and the periphery (gastrointestinal system, urinary bladder, lung) [11, 12]. The NK2 receptor was the first neurokinin receptor to be cloned [11]. The isolation of the bovine NK2 receptor cDNA facilitated the subsequent isolation of NK2 receptors from other species, by homologous hybridization or by PCR using oligonucleotide primers of conserved regions. There is less homology between NK2 receptors from different species (ca. 90% or less), than between the NK1 receptors [11]. The carboxy terminal end of the human NK2 receptor is 9 –14 amino acid residues longer than the rat, mouse and bovine receptor proteins [11]. The cDNA of the human NK2 receptor has been cloned and expressed in 3T3 cells. The human NK2 receptor protein contains 398 amino acid residues with considerable homology to the bovine and the rat receptors [12]. The cloning and the characterization of the human gene encoding the NK2 receptor showed that, as in the NK1 receptor gene, there are five exons. This suggests a common origin for both receptors [11]. NK2 receptor mRNA is widely distributed in the peripheral nervous system, such as in the stomach, the urinary bladder, the gastrointestinal and the respiratory tract [4]. NK2 receptors are almost exclusively expressed in peripheral tissues, although they have also been detected in a limited number of brain regions, such as the frontal cortex and the hippocampus. However, the NK2 mRNA accounts for only around 1% of the entire neurokinin receptor mRNA in the rat brain [4]. In addition, evidence for the presence of NK2 receptors in the rat spinal cord has been disclosed. The activity of selective non-peptide NK2 antagonists in animal models of anxiety support the presence of NK2 receptors in the CNS, but the presence of NK2 receptors in human brain remains to be demonstrated [12]. The NK3 receptors have been isolated from human and rat brain and subsequently have been cloned. The human NK3 receptor consists of 465 amino acids and has a high homology (88%) with the rat NK3 receptor, containing

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seven variations in the putative TM segments [11, 12]. The rat NK3 receptor is composed of 452 amino acid residues. There is also high homology among neurokinin receptors: the human NK3 receptor sequence has 74% homology with the NK1 receptor and 68% with the NK2 receptor sequence in the same species [12]. The sequence is best conserved in the transmembrane regions, with less identity in the amino- and carboxy-terminal ends. mRNA expression studies have shown that NK3 receptor transcripts are predominantly present in the CNS, in the spinal cord, and in all brain regions including cortex, hypothalamus and cerebellum [11]. NK3 receptors are also present in peripheral tissues including kidney, placenta, lung and in the gastrointestinal system [11]. The existence of an additional neurokinin receptor proposed as NK4 receptor (or NK3B) has not yet been confirmed [13, 14]. Very recently an excellent review appeared in the literature covering in detail the latest progress in the field of tachykinins/ neurokinins and the neurokinin receptors [15]. NEUROKININ RECEPTORS AS DRUG TARGETS: POTENTIAL CLINICAL USE OF NEUROKININ ANTAGONISTS Neurokinins/tachykinins and the neurokinin/tachykinin receptors have been proposed to be involved in a number of pathological conditions including pain, arthritis, migraine, emesis, cancer, anxiety, depression, schizophrenia, asthma and airways diseases, inflammatory bowel disease and urinary incontinence, and consequently neurokinin receptor antagonists have been proposed to have potential clinical benefits in a broad range of diseases. Numerous reviews that have appeared very recently in the literature discuss and summarize the potential clinical usefulness of neurokinin/tachykinin antagonists [16 – 20, 58]. CENTRAL NERVOUS SYSTEM DISORDERS

The fact that substance P is present in brain regions associated with anxiety and depression (amygdala, hippocampus, hypothalamus and periaqueductal grey matter) led to the conclusion that substance P, via activation of its receptor, may contribute to pathological conditions of the central nervous system. In addition, evidence was found that substance P is co-located with serotonin in raphe neurones in the human brain [21]. Data from animal studies, where substance P was injected into the brains, revealed a number of behavioural changes that are comparable to the behavioural changes typically observed in anxiety and depression models. Antidepressant efficacy of the NK1 antagonist Aprepitant (MK869) (6) could be demonstrated in a placebo controlled clinical study where a dose of 300 mg (p.o., once daily) of Aprepitant was administered to patients

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Fig. 3.1 Selected selective NK1 antagonists. (Used in a number of animal and clinical studies that helped to establish the therapeutic potential of such compounds.)

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suffering from major depressive disorder for 6 weeks. In this study, Aprepitant was well tolerated and the effectiveness of the compound as an antidepressant agent was comparable with that of the serotonin uptake inhibitor paroxetine [22, 23]. In later studies, however, the antidepressant activity of Aprepitant could not be confirmed [24]. In summary, the existing experimental evidence suggest that substance P and the NK1 receptor are important players in the pathophysiology of central nervous diseases such as depression [18], however, the partially negative results with Aprepitant are contradictory to this and additional studies will be needed to get conclusive answers on the antidepressant potential of neurokinin antagonists. EMESIS

Substance P is found in the gastrointestinal afferent neurons and in the medulla of the brainstem. Experimental data generated from animal experiments suggest that NK1 receptors have a crucial role in mediating the emetic reflex, and NK1 antagonists have been proposed as potential antiemetic agents. In studies using CP-99994 (3), it has been suggested that the site where NK1 antagonists act as antiemetics is located in nucleus of the tractus solitarius [25]. In animal experiments using several species, selective NK1 antagonists, such as Aprepitant (6), CP-122,721 (4) and GR205171 could be shown to exhibit potent antiemetic effects against a number of emetic stimuli. Clinical evidence for the effectiveness of the antiemetic activity of a NK1 receptor selective antagonist could be demonstrated in several clinical trials with Aprepitant and CJ-11974 (2) [26] against cisplatin-induced emesis [27]. Aprepitant has been registered in the US for the treatment of chemotherapy-induced emesis. Improved efficacy of Aprepitant could be demonstrated when the compound was used in combination with Ondansetron (a 5-HT3 antagonist) and dexamethasone [28]. GASTROINTESTINAL AND URINARY TRACT DISEASES, UTERUS CONTRACTIONS

Tachykinins (substance P and Neurokinin A) are involved in the regulation of gastrointestinal motility, gastric secretion, gastric pain sensitivity and immune function. NK1, NK2 and NK3 receptors are expressed in enteric neurons, interstitial cells, intestinal muscle, epithelium and vasculature. Disorders of the gastrointestinal tract are related to altered expression levels of neurokinins and neurokinin receptors. From animal studies increasing evidence is available that neurokinin antagonists are able to reduce typical symptoms such as dysmotility, diarrhoea, constipation, oedema and tissue destruction. This led to the conclusion that neurokinin antagonists (especially dual NK1/NK2 and triple NK1/NK2/NK3 antagonists) may eventually be of value in the treatment

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of gastrointestinal diseases, in particular inflammatory bowel disease and irritable bowel syndrome (IBS) [29 – 31]. The organs of the lower and upper urinary tract of many species, including man, are richly innervated with primary afferent neurons as the source of neurokinins. Experimental data suggest an involvement of the neurokinins, and the NK1, NK2 and NK3 receptors, in the production of symptoms associated with urinary tract diseases [30, 31]. However, the significance of the neurokinins and their receptors with respect to the contribution to such conditions, as well as the therapeutic potential of neurokinin antagonists, has not been fully explored. Presently, one compound, TAK637 (35) [32, 33], a selective NK1 antagonist, is undergoing clinical evaluation for urinary incontinence and inflammatory bowel disease [34]. A recent paper also suggested a role for neurokinins and especially for NK2 receptors in the uterus. Further investigation will be necessary to elucidate the involvement of the neurokinins and their receptors in the pathophysiology of uterus disease conditions [35]. MIGRAINE, PAIN

Substance P was shown to be involved in mediating neurogenic inflammation, vasodilation and plasma extravasation in the dura mater. As it was proposed that inflammation of the dura mater is the source of the migraine pain, substance P antagonists (NK1 antagonist) have been suggested to exhibit antimigraine activity. In a placebo controlled clinical study, the effect of Lanepitant (8) (LY 303870, 200 mg p.o., qd, 12 weeks) on migraine prevention was evaluated. In this study Lanepitant was well tolerated but did not show any statistically significant effect in preventing migraine [36, 37]. In clinical studies performed with either GR205171 (25 mg, i.v.) or L-758,298 (up to 60 mg, i.v.) no beneficial effects over placebo could be demonstrated [38]. In recent years the results of a number of studies suggesting a role for substance P in pain transmission appeared in the literature [19, 39]. AIRWAYS DISEASES

The respiratory tract is richly innervated with sensory nerves. Activation of neurokinin containing C-fibres by different stimuli, such as capsaicin, cigarette smoke, or respiratory viral infections, leads to the release of neuropeptides (notably substance P and neurokinin A) within the lung. The release of the neuropeptides in turn causes an acute inflammatory response. This neurogenic inflammation is characterized by a number of neurokinin receptor mediated events [40 – 44]. The use of selective NK1 antagonists (e.g. CP-96345 (1) [45], CP-99994 (2) [46], FK-888 (3) [101, 102], SR-140333 (4) [47]), selective NK2

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antagonists (e.g. Saredutant (SR-48968) (5) [48], MEN-10,627 (6) [49], GR159897 (13) [50]) and dual NK1/NK2 antagonists (FK-224 (8) [101, 102], MDL105212A (10) [51], S-16474 (9) [103]) facilitated the understanding of the physiological and pathological roles of neurokinins in the airways and in airway diseases, and also helped to elucidate the roles of the involved neurokinin receptor subtypes. Microvascular leakage and vasodilation lead to protein plasma extravasation and oedema, mucus hypersecretion, recruitment of inflammatory leukocytes, mast cell degranulation (histamine release) as well as the release of cytokines from the invading leukocytes. These effects are thought to be linked to the activation of NK1 receptors, whereas bronchoconstriction, cough, and to some extent bronchial hyperresponsiveness, are believed to be triggered mainly via activation of NK2 receptors [40, 41]. The selective NK2 antagonist Saredutant (5) was capable of significantly inhibiting NKA-induced bronchoconstriction for up to 24 h after a 100 mg p.o. dose. However, the compound failed to have a beneficial effect on baseline lung function and did not improve adenosine hyperresponsiveness in asthma patients [52]. The moderately active dual NK1/NK2 antagonist FK-224 (8) showed inhibition of bradykinin-induced cough and bronchoconstriction, but the compound failed to inhibit neurokinin A-induced bronchoconstriction [53]. In another clinical trial, the dual NK1/NK2 antagonist DNK333 (78) (100 mg. p.o.) has been demonstrated to significantly inhibit NKA-induced bronchoconstriction in mild asthmatics [54]. In addition, (78) was well tolerated with no adverse effects reported. An area where neurokinin antagonists may have a positive impact is in the treatment of cough-symptoms, since the implication of neurokinins/tachykinins in the peripheral and central components of the cough reflex could be demonstrated [55]. In summary, there is some controversy concerning a significant clinical role of either selective or combined neurokinin antagonists in the treatment of airways diseases, and whether experimental evidence gained from animal experiments (guinea pigs) that supports the use of neurokinin receptor antagonists can be transferred into humans suffering from asthma [56]. Continuation of ongoing clinical trials and additional long-term clinical trials may eventually help to determine the usefulness of neurokinin antagonists in the treatment of respiratory diseases. RECENT DEVELOPMENTS IN THE SEARCH FOR SELECTIVE NEUROKININ ANTAGONISTS The search for selective neurokinin antagonists has seen tremendous progress in terms of the structural diversity of a number of potent compounds, especially

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Fig. 3.2 Selective NK2 antagonists.

in the NK1 receptor antagonists field, since the appearance of the first known potent and selective non-peptide NK1 antagonist CP-96345 (1) [45]. Shortly after the discovery of (1), the first non-peptide NK2 receptor selective compound, SR-48968 ((11), saredutant [48]) was identified, followed by the development of a large number of novel selective NK2 antagonists (Figures 3.1 and 3.2). At about the same time, the first selective NK3 antagonist SR-142801 ((14), osanetant [58, 59]) was discovered (Figure 3.3). So far, however, interest in the discovery of selective NK3 receptor antagonists appears to be limited as only a small number of novel compounds [57] in this area have appeared during recent years. The following overview on developments in the area of selective NK1, NK2 or NK3 neurokinin receptor antagonists focuses on recent and important

Fig. 3.3 Selective NK3 antagonists.

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advances in the development of such compounds. A number of reviews has appeared in the literature summarizing and documenting the progress in the search for selective NK1, NK2 and NK3 receptor antagonists during the last decade [60 –64]. NK1 RECEPTOR ANTAGONISTS

Initial Novartis efforts aiming at potent and bioavailable selective NK1 antagonists resulted in the discovery of CGP49823 (16) [65], and was followed by the discovery of NKP608 (17) (Figure 3.4). NKP608 is a potent, selective and orally bioavailable CNS active compound with an IC50 value of 2.6 nM (bovine retina NK1 receptor binding). NKP608 shows a number of activities in various disease models where NK1 receptors are involved. In particular, anxiolytic-like and anti-depressant effects could be demonstrated in various animal species after oral administration of the compound. These findings suggest NKP608 as a potential therapeutic agent having beneficial effects in psychiatric disorders such as anxiety and/or depression [66, 67]. Additional piperidine and quinuclidine derivatives as selective NK1 antagonists derived from CP-96345 (1) and CP-122721 (4) have been identified in the laboratories of Pfizer [68, 69]. Derived from Aprepitant (6), (18) has properties in terms of potency (IC50 of 0.19 nM in inhibiting 125I-substance P binding to the hNK1 receptor) and pharmacokinetic behaviour on a par with Aprepitant. Additionally this compound is suitably soluble in aqueous formulations (water solubility of the hydrochloride of (18) . 100 mg/ml) that can be used for i.v. administrations [70]. Based on evidence that an intra-molecular p – p

Fig. 3.4 Selective NK1 antagonists (Novartis).

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interaction between the two aromatic rings of NK1 antagonists such as (6) is important for stabilizing the bioactive conformation, a hypothesis was generated that appropriately substituted spirocyclic ketals might be used to freeze the conformation needed for NK1 receptor binding. The results of this effort revealed that [5,5]-spiroketals (e.g. (19) (Figure 3.5), 1.1 nM), and in particular [4,5]-spiroketal derivatives such as (20) (0.1 nM), indeed represent NK1 antagonists exhibiting potent receptor affinities (inhibition of SP binding to hNK1 receptors in CHO cells) and also excellent CNS penetration. Later [4,5]-spiro-piperidine derivatives such as compound (21) (0.11 nM) with favourable in vitro and in vivo properties have been discovered [71, 72].

Fig. 3.5 Selective NK1 antagonists (Merck).

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Fig. 3.6 Selective NK1 antagonists (Merck).

Screening combinatorial chemistry libraries in the laboratories of Merck led to the identification of 2-aryl-indole derivatives exhibiting potent affinities to NK1 receptors (Figure 3.6). Lead optimization efforts led to the discovery of 2-aryl-indoles and 2-aryl-azaindoles, for example (22) –(24) as highly potent NK1 antagonists (IC50 values of 0.16, 0.15 and 0.12 nM, respectively; displacement of labelled SP from cloned hNK1 receptors expressed in CHO cells). Pharmacokinetic and pharmacological experiments performed with (22) and (23) revealed that the compounds have good absorption properties and good central activity, but the compounds appear to be subject to a high first pass metabolism that is responsible for rather low bioavailabilities [73 –75]. Roche has entered the neurokinin antagonist field only rather recently, but appears to be very active in this field as numerous patent applications have been published within the last 2 –3 years covering phenyl-pyridine and phenylpyrimidine derivatives (Figure 3.7). In a hNK1 receptor binding assay, the phenyl-pyridine derivatives (25) and (26) were shown to exhibit pKi values of 8.9 [76] and 9.29 [77], respectively, and the phenyl-pyrimidine derivative (27) exhibited a pKi value of 8.45 [78]. In addition, patent applications claiming triazaspirodecanone, oxa-diazaspiroundecanone derivatives and piperidine derivatives exemplified by compounds (28) – (30) as potent NK1 antagonists (h NK1, pKi values of 8 –9) have been claimed by Roche [79 –81]. In a patent application filed by GlaxoSmithKline (GSK) 2-phenylpiperazine1-carboxylic acid benzylamide derivatives containing a novel piperazino-urea structural element are claimed as potent and in vivo active NK1 antagonists (Figure 3.8). Selected examples, e.g. (31) (S, S-isomer) and (32) (S,R-isomer) exhibit potent affinities to the NK1 receptor with pKi values of 9.27 and 9.81, respectively. (31) and (32) inhibited NK1 agonist induced foot tapping in gerbils

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SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

Fig. 3.7 Selective NK1 antagonists (Roche).

after oral administration with ED50 values of 0.04 and 0.05 mg/kg, respectively [82]. Replacement of the piperazine ring by a piperidine ring and introduction of an acyl-piperazine residue at position 4 of the piperidine resulted in the derivative (33), a compound that contains three chiral centres which exhibited a pKi of 9.36 and an ED50 of 0.05 mg/kg in the gerbil foot tapping model [83]. TAK-637 (35) has been identified from a series of cyclized analogues of the naphthyridine derivative (34). It was shown that an 8-membered ring system

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Fig. 3.8 Selective NK1 antagonists (GSK).

gave the most potent compounds, which lock the amide in the transconfiguration. In addition to the chiral centre caused by the methyl-group at position 9 of the 8-membered ring system, an additional chiral centre is caused by the fact that the carbonyl oxygen can be above or below the ring system (atropisomerism). Nevertheless, researchers at Takeda managed to work out a synthesis for the specific preparation of this compound [84] (Figure 3.9). TAK637 [(aR,9R)-isomer] exhibits highly potent NK1 receptor affinity (IC50 ¼ 0.45 nM, inhibition of SP binding to human IM-9 cells) and shows beneficial effects on bladder function in animal experiments [85]. The compound has entered clinical trials for a number of indications [86]. Another highly potent selective NK1 antagonist has been reported by Sanofi. SSR240600 ((36), R-enantiomer) inhibited the binding of radioactive

Fig. 3.9 Selective NK1 antagonists (Takeda).

64

SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

Fig. 3.10 A selective NK1 antagonist (Sanofi).

substance P to tachykinin NK1 receptors in human lymphoblastic IM9 cells (Ki ¼ 0.0061 nM), human astrocytoma U373MG cells (Ki ¼ 0.10 nM), and human brain cortex (IC50 ¼ 0.017 nM). In vivo SSR240600 exhibited potent activity against a number of NK1 receptor agonist induced peripheral and central effects after intravenous and intraperitoneal administration [87, 88] (Figure 3.10). In a combinatorial chemistry effort to overcome the suboptimal oral bioavailability properties of their earlier NK1 antagonist LY-303870 (8), researchers at Lilly identified non-basic side chain replacements of the piperidinyl-piperidine moiety (Figure 3.11). The resulting derivatives exemplified by (37) (IC50 ¼ 0.43 nM) and (38) (IC50 ¼ 0.98 nM) have been reported to exhibit favourable PK properties after oral administration [89].

Fig. 3.11 Selective NK1 antagonists (Lilly).

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NK2 RECEPTOR ANTAGONISTS

Significant progress in the search for potent and bioavailable selective NK2 receptor antagonists has been reported recently from the laboratories of Pfizer (Figure 3.12). Their strategy to improve metabolic stability of the original lead compound (39) was to incorporate the metabolically labile amide N-methyl group into a ring system and to transpose the carbonyl group into the ring, leading to lactam derivatives such as (40) which displayed an improved metabolic stability. In human liver microsomes (39) was rapidly degraded ðT1=2 , 10 minÞ whereas (40) was significantly more stable ðT1=2 , 30 minÞ: Another metabolic weak point, i.e. N-benzyl oxidation, could be addressed

Fig. 3.12 Selective NK2 antagonists (Pfizer).

66

SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

by replacement of the benzyl substituent with a cyclopropyl-methyl group. Further lead optimization aiming at reduction of the overall lipohilicity of the compounds led to the identification of UK-224,671 (41) (IC50 ¼ 4 nM, NK2 receptor binding, T1=2 ¼ 120 min). UK-224,671 entered clinical studies for the treatment of urinary incontinence, where unfortunately a low bioavailability in humans was observed. A potential follow-up compound, UK-290,795 (42) (IC50 ¼ 0.4 nM, NK2 receptor binding) has been identified as exhibiting increased potency and an improved oral bioavailability, as well as a prolonged duration of action. Piperidine derivatives such as (43) (IC50 ¼ 7.5 nM, NK2 receptor binding), represent another version of conformationally constrained analogues which have also been published recently [90 –92]. The morpholine derivative SR-144190 (45) (Sanofi-Synthelabo) [93] was described as a highly potent and selective NK2 receptor antagonist, exhibiting a similar pharmacological profile as saredutant (11) from which it is derived structurally. From the laboratories of AstraZeneca, the highly selective NK2 receptor antagonists ZD7944 (44) with an ethyl-dihydro-isoindolone moiety has been reported as a compound exhibiting a Ki value of about 1 nM (inhibition of NKA binding to human NK2 receptors) [94] (Figure 3.13). In a study originally aimed at the discovery of NK3 antagonists based on the structure of osanetant (14), researchers at Merck obtained structurally novel potent NK2 antagonists via transposition of the carbonyl-group into the piperidine ring resulting in a lactam series of compounds. This represents a remarkable switch of receptor selectivity from NK3 to NK2 receptor affinity (Figure 3.14). Compounds exemplified by (46) and (47) exhibited NK2 receptor IC50 values of 6.4 and 2.5 nM, respectively in displacing NKA from human NK2 receptors and the selectivity of (46) and (47) versus the NK3 receptor was 84 and 51 fold, respectively [95].

Fig. 3.13 Selective NK2 antagonists (Astra Zeneca, Sanofi).

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Fig. 3.14 Selective NK2 antagonists (Merck).

A series of novel cyclized pseudopeptides containing the –Trp-Phe-(D)-PheC-CH2-NH-sequence has been reported recently by Menarini (Figure 3.15). The primary goal of the studies was to discover cyclic pseudopeptide structures as NK2 receptor antagonists that, in comparison with the earlier compounds such as MEN10627 (12) (pKi ¼ 9.2), are equipotent but have a reduced molecular weight and contain fewer chiral centres. (48) (MEN11558), (49) and (50) exhibit potent pKi values (inhibition of NKA binding to NK2 receptors in CHO cells) of 8.7, 10.3 and 9.2, respectively [96, 97].

Fig. 3.15 Selective NK2 antagonists (Menarini).

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SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

Fig. 3.16 Selective NK3 antagonists (Sanofi).

NK3 RECEPTOR ANTAGONISTS

In the area of selective NK3 receptor antagonists apparently only Sanofi have reported on further developments in recent years. Modifications at the 4-piperidine substituents, i.e. replacement of the N-methyl-acetamide residue present in (14) with a 1,1,3-trimethyl-urea substituent led to the development of SSR-146977 (51) (Figure 3.16). This compound acts as a potent and selective antagonist of the tachykinin NK3 receptor showing inhibition of the NKB binding to NK3 receptors (Ki of 0.26 nM), as well as potent inhibition of NKB induced guinea pig ileum contractions (pA2 ¼ 9.07). SSR146977 exhibits peripheral and central in vivo activity against a variety of neurokinin B induced or mediated effects in animal experiments (e.g. blockade of bronchial hyperresponsiveness, inhibition of citric acid induced cough, prevention of locomotor activity decrease) after intraperitoneal administration [98]. COMBINED NEUROKININ ANTAGONISTS DUAL NK1/NK2 AND TRIPLE NK1/NK2/NK3 RECEPTOR ANTAGONISTS

In the early days of neurokinin receptor antagonist research, pharmaceutical companies focused on selective antagonists, in particular selective NK1 antagonists and selective NK2 antagonists. Results from studies performed with selective NK antagonists led to the conclusion that NK1 and NK2 receptors (and possibly NK3 receptors) may be involved in the pathogenesis and progression of many diseases where neurokinins and their receptors are involved [99]. The involvement of more than one neurokinin receptor has been hypothesized for airways diseases such as asthma [99]. As a consequence, many pharmaceutical companies have shifted their efforts aiming at selective NK-antagonists towards the discovery of dual NK1/NK2 antagonists and

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Fig. 3.17 Peptidic dual NK1/NK2 antagonist.

triple NK1/NK2/NK3 antagonists [99, 100]. Initially with the moderately active FK-224 (52) [101, 102] and S-16474 (53) [103], only peptidic dual NK1/ NK2 receptor antagonists were available (Figure 3.17). Aventis The first really potent non-peptidic dual NK1/NK2 antagonist MDL-105,212A (54) [104], was identified in the laboratories of Merrell Dow (now Aventis). This compound is structurally related to SR-48968 (11) and was designed based on comparative modelling studies with the structures of (11) and NK1 antagonists. In an attempt to improve pharmacokinetic properties, MDL-105172A (55) [105] was identified by introduction of a morpholine residue (Figure 3.18). (55) exhibited a slightly improved oral activity in guinea pigs. In addition to NK1 and NK2 receptor affinity, (55) also exhibits potent NK3 receptor affinity. Aimed at improved potency and balanced NK1/NK2 receptor affinity, additional compounds have been prepared derived from this series of compounds [106, 107]. Yamanouchi Based on the structure of SR-48968 (11), researchers at Yamanouchi discovered YM-38336 (56), which exhibits binding affinities to the NK2 and NK1 receptors with IC50 values of 8.9 and 680 nM, respectively. Introduction of methoxy

70

SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

Fig. 3.18 The structures of MDL-105,212A and MDL-105,172A.

substituents into the benzoyl-residue, together with modifications at the spiropiperidine ring system, led to an improvement of the NK1 affinity of the resulting compound (57) (Figure 3.19). YM-44778 (57) exhibits a balanced affinity to guinea pig urinary bladder NK1 receptors (IC50 ¼ 18 nM, inhibition of [125I]-substance P binding) and hamster urinary bladder NK2 receptors (IC50 ¼ 16 nM, inhibition of [125I]-NKA binding) [108, 109].

Fig. 3.19 The structures of YM-38336 (56) and YM-44778 (57).

Merck Design efforts based on the structure of (11) in the laboratories of Merck led to the preparation of a variety of spiropiperidine derivatives exemplified by (58), a dual NK1/NK2 antagonist that was reported to exhibit IC50 values of 0.2 and 1.5 nM to cloned human NK1 and NK2 receptors, respectively. In addition, (58) also showed oral activity in guinea pigs (inhibition of resinaferatoxin induced PPE, ED50 ¼ 0.3 mg/kg, p.o.). [110, 111]. In a closely related series of compounds, the spiropiperidine residue was replaced with substituted aryl piperazine moieties, e.g. (2-fluoroethyl)-2-methoxypurinyl)piperazine

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Fig. 3.20 The structures of (58)–(60).

(Figure 3.20). These modifications led to the discovery of (59), a compound that has IC50 values of 0.45, 9 and 25 nM (cloned human NK1, NK2 and NK3 receptors, respectively) [112]. Starting from the NK1 selective neurokinin antagonists FK888 (5), (60) has also been discovered. This compound shows balanced and fairly potent affinities to the NK1 and NK2 receptor, with IC50 values of 20 and 12 nM, respectively [113]. Takeda, Pfizer, Lilly According to patent applications filed in the mid to late 1990s claiming compounds with dual NK1/NK2 antagonistic properties, additional companies (e.g. Lilly, Pfizer, Takeda) apparently have been engaged in the search for dual antagonists. The design of these compounds had also been based on the structures of their proprietary selective NK1 antagonists and/or on the structure of the selective NK2 antagonist (11) [114–119]. However, no recent developments in terms of combined neurokinin receptor antagonists have been reported from these companies. Novartis At Novartis, efforts aiming at the discovery of dual NK1/NK2 antagonists as potential anti-asthma agents led to the discovery of a series of N-[(E)-3-carbamoyl1-(4-chloro-benzyl)-allyl]-N-methyl-3,5-bis-trifluoro-methyl-benzamides, as dual

72

SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

NK1/NK2 antagonists (Figure 3.21). In a series of piperidine carboxamides that have been derived originally from the selective NK1 antagonists CGP49823 (16) and CGP60829 (17) [67, 120], a number of compounds with moderate NK2 receptor affinity (in addition to potent NK1 receptor affinity) were identified (Figure 3.21). It was hypothesized that opening of the piperidine ring should allow for a more flexible structure with an increased chance of binding additionally to the NK2 receptor. Formal elimination of a CH2-group from the piperidine ring led to 5-aryl-4benzoyl-amino-pent-2-ene-carboxamides, a new series of ‘open chain’ neurokinin receptor antagonists. In general, compounds from this structural class exhibited high affinity to the NK1 receptor (inhibition of 3H-sar9-substance P binding to bovine retinal membranes) and a number of compounds exhibited an additional affinity to the NK2 receptor (inhibition of 125I-NKA binding to

Fig. 3.21 The discovery of 5-aryl-4-benzoyl-amino-pent-2-ene-carboxamides.

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human NK2-CHO-cells). It was found that the NK1 affinity was not very dependent on the nature of the terminal amide substituent; primary as well as secondary amides are tolerated the only exception being anilides, where reduced NK1 binding affinities were found. However, in order to achieve NK2 affinity, terminal amide substituents containing at least one additional heteroatom appear to be essential. In particular, substituents containing an additional amide residue such as the caprolactam-moiety led to compounds (67) – (69) exhibiting good NK2 affinities in addition to their highly potent NK1 affinities (Table 3.2). The caprolactam derivative (68) was originally prepared and tested as a mixture of four stereoisomers [120]. In order to find out which of the isomers are responsible for the affinity of the compound to the different neurokinin receptors, all four isomers have been prepared as described in the synthesis scheme (Scheme 3.1). Coupling of the racemic acid (71), prepared in three synthetic steps from 4-chloro-phenylalanine, with either enantiomerically pure D (R)- or L (S)-a-amino-1-caprolactam followed by deprotection and acylation with 3,5bistrifluorobenzoyl-chloride led in both cases to a mixture of two diastereoisomers which could easily be separated on silica gel. In order to assign the absolute stereochemistry of the chiral centre in the carbon chain of the four isomers, a stereoselective synthesis to prepare specifically one isomer as an enantiomerically pure compound has been identified. In the 4-amino-piperidine series of selective NK1 antagonists, e.g. CGP49823 (16), CGP60829 (17), it was shown that the compounds possessing the S-configuration at C-2 exhibited higher affinity to the NK1 receptor in comparison to the corresponding R-enantiomers. Therefore, the efforts were focused on the stereoselective preparation of an isomer that possessed the same stereochemistry at the corresponding chiral centre. Using the same synthetic procedure as just outlined for the preparation of the four diastereoisomers (72) – (75), the R,R-isomer was prepared starting from commercially available (R)-4-chlorophenylalanine methylester which, after BOC-protection followed by N-methylation, was reduced to the aldehyde. Chain elongation using trimethylsilyl-P,P-diethyl-phosphonoacetate and an acidic work up procedure led to the (R) carboxylic acid (71). Introduction of D -a-amino-1-caprolactam in the presence of EDC, removal of the BOC protecting group and subsequent acylation of the nitrogen with 3,5-bistrifluoromethylbenzoyl chloride led to the final product in an overall yield of 20% after recrystallization from CH2Cl2/n-pentane (ee ¼ 95% before and ee ¼ 98.5% after recrystallization). During the synthesis starting with (R)-4-chlorophenylalanine methylester ee . 97% no significant epimerization of the chiral centre occurred. On the basis of the chromatographic behaviour of (73) (R,R-isomer), the absolute configuration of the stereocentres of all the isomers produced starting from (71) could be assigned

74

SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

Table 3.2 NK1 AND NK2 AFFINITIES OF 5-ARYL-4-BENZOYL-AMINO-PENT-2-ENECARBOXAMIDES

R (compound #)

NK2 NK1 binding binding IC50, nM IC50, nM

R

NK1 NK2 binding binding IC50, nM IC50, nM

20% n.d. @50 nM

0.8

368

9

inactive

1

66

8.5

inactive

0.7

55

7.2

inactive

10

49

2.8

460

12

470

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Scheme 3.1 Synthesis of diastereoisomers (72)–(75) and stereospecific preparation of the (R,R)isomer (72).

and all four compounds were tested for their binding affinities in the NKreceptor assays. As can be seen from the NK1 and NK2-binding values in Table 3.3, the R,Risomer (72) clearly exhibits the highest affinity for both the NK1 and the NK2 receptor. Changing the stereochemistry of the backbone chiral centre from R to S, resulting in the S,R-isomer, (73), leads to a decrease in binding affinities

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SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

Table 3.3 IN VITRO BINDING AFFINITIES OF COMPOUNDS (68) AND (72)– (75) TO NK1- AND NK2-RECEPTORS

NK1 binding IC50, nM NK2 binding IC50, nM

(68) (mixture of four stereoisomers)

(72) (R,R)

(73) (S,R)

(74) (S,S)

(75) (R,S)

0.76 55

0.5 24

16 123

86 300

3.6 120

to the NK1 and NK2 receptors by a factor of 30 and 5, respectively. Inversion of the stereochemistry at the chiral centre of the caprolactam moiety leading to the R,S-isomer (75) results in reduced binding affinities to the NK1 and NK2 receptors by a factor of 7 and 5, respectively. In comparison to the R,R-isomer (72), its enantiomer the S,S-isomer (74) exhibits a 170 times lower binding affinity to the NK1 and a 12 times lower affinity to the NK2 receptor [121]. Although (72) in terms of NK1/NK2 antagonistic activity is very promising, additional compounds with a variety of aryl substituents were prepared. Compounds with 4-chlorophenyl (72), dichlorothienyl (76), 3,4-dichlorophenyl (78), 2-naphthyl (79), and 3-(1-methyl)indolyl (80) substituents exhibit binding affinities to the NK1 receptor in the low nanomolar range. In contrast to this the dibromothiophene derivative (77) exhibits a somewhat decreased binding affinity to the NK1 receptor. NK2 receptor affinities are between 17 –60 nM, except for DNK333 (78), where the additional chlorine leads to a five times higher affinity to the NK2 receptor in comparison with the monochloro-derivative (72). Both DNK333 and the di-bromothiophene derivative (77) exhibit balanced binding affinities to both the NK1 and the NK2 receptors (Table 3.4) [122, 123]. DNK333 (78), based on its favourable receptor affinity properties, was chosen for further profiling. In in vitro functional tests the compound demonstrated blockade of bronchoconstrictor responses induced by selective NK1 (pA2 ¼ 7.93) and selective NK2 (pA2 ¼ 7.27) agonists in the guinea pig isolated trachea model. DNK333, intravenously administered, was capable of antagonizing either sar9-substance P (a selective and stable NK1 receptor agonist)or (Ala5, b-Ala8)-a-NKA (4 – 10) (a selective and stable NK2 receptor agonist)-induced bronchoconstriction in anaesthetized guinea pigs. The relative

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Table 3.4 IN VITRO BINDING AFFINITIES TO NK1- AND NK2-RECEPTORS

Ar

NK1 binding IC50, nM

NK2 binding IC50, nM

Ar

NK1 binding IC50, nM

NK2 binding IC50, nM

0.5

24

4.8

3.6

17

1.1

60

26

0.5

24

20

5.5

potency was ca. 6-fold in favour of NK1-blockade, a finding that is consistent with the in vitro relative antagonist potency in a guinea pig isolated trachea model. DNK333, orally administered, inhibited b-ala8-NKA induced bronchoconstriction with an ED50 value of 1 mg/kg (airways resistance) and with an ED50 value of 2.8 mg/kg (dynamic compliance) in anaesthetized squirrel monkeys. In pharmacokinetic experiments, DNK333, after oral administration of 10 mg/kg to guinea pigs, shows an AUC0 – 24 h of 6378 ng h/ml and a bioavailability of approximately 41% when the compound was applied formulated in a microemulsion [124]. In a phase I clinical study, a single 100 mg oral dose of DNK333 was able to significantly inhibit neurokinin A induced bronchoconstriction in mild asthma patients [125].

78

SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

Fig. 3.22 In vitro binding affinities of DNK333 (78) and (81) to NK1- and NK2-receptors.

A possible way to simplify the structure of DNK333 proved to be the elimination of the backbone chiral centre via replacement of the stereogenic carbon by a nitrogen. The resulting compound, a hydrazide derivative (81), in terms of potency and selectivity exhibits a similar profile to DNK333 (Figure 3.22). As first reported by researchers from Pfizer and Merck, as well as confirmed by other companies [126, 127], the common feature of structures binding to NK1 receptors is a pair of closely spaced, staggered aromatic rings. In an effort to simplify the structures of compounds such as DNK333 it was hypothesized that it is possible to freeze such a conformation via replacing the four carbons of the butenyl fragment by a phenyl-ring. A molecular modelling experiment analysing energy minimized conformations of CGP49823 (16), (72) and a proposed biphenyl derivative (82) suggested that a suitably substituted biphenyl derivative does indeed place the two phenyl rings in the required orientation for NK1 receptor binding. In the biphenyl derivatives one chiral centre could in addition be eliminated. This makes this type of compound amenable to a less complex synthetic access and allows for rapid derivatization in order to optimize the biological profile (Figure 3.23). As in the 5-aryl-4-benzoyl-amino-pent-2-ene-carboxamide series of compounds (results see Tables 3.2 and 3.4) NK1 affinity of the biphenyl derivatives (Table 3.5) is largely unaffected by the nature of the amide substituent, but NK2 affinity was very dependent on the nature of the amide substituent and on the degree of chlorination of this aryl-group, with NK2 affinities ranging from 560 nM for a compound with no chlorine (84) down to 43 nM for the 3,4dichloro compound (83). Apparently a caprolactam residue as R5 is not essential for dual activity, as the dimethylamino-propyl substituted compound (85) is equipotent to the corresponding caprolactam derivative (82) [128].

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Fig. 3.23 Overlap of (16) (grey), (72) (white) and (82) (dark grey).

Sankyo Based upon information derived from an analysis of the structure of MDL 105,212 (54) researchers at Sankyo designed structurally closely related but novel 1-{2-[(5R)-(3,4-dichlorophenyl)-3-(3,4,5-trimethoxybenzoyl)oxazolidine-5-yl]ethyl}piperidine derivatives, such as the compounds shown in Table 3.6. It proved possible to optimise binding affinities to both the NK1and to the NK2 receptor to nanomolar levels via the introduction of a variety of spiro-substituted piperidine residues. (86) in addition to fairly potent and balanced receptor affinities, has also been reported to exhibit inhibitory effects in vivo on SP-induced increases in vascular permeability and on NKA-induced bronchoconstriction in guinea pigs after i.v. administration [129]. Following the discovery of the oxazolidine derivatives, preparation of the closely related morpholine derivatives was undertaken (Table 3.7). R-113281 (90) and (91), in addition to their affinities to NK1 and NK2 receptors, possess potent affinity to NK3 receptors. R-113281 was reported to exhibit potent antagonism toward SP-induced tracheal vascular hyperpermeability and NKA-, NKB- and capsaicin-induced bronchoconstriction in guinea pigs [130, 131].

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SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

Table 3.5 IN VITRO BINDING AFFINITIES OF BIPHENYL DERIVATIVES TO NK1- AND NK2-RECEPTORS

R5

NK1 binding IC50, nM

NK2 binding IC50, nM

Cpd.

R1

R2

83

3,5-(CF3)2

3,4-(Cl)2

0.8

43

82

3,5-(CF3)2

4-Cl

2.7

70

84

3,5-(CF3)2

4-H

1.5

560

85

3,5-(CF3)2

4-Cl

1

108

Schering-Plough Researchers at Schering-Plough based one of their approaches towards dual NK1/NK2 antagonists on the 1-phenyl-ethylenediamine fragment that is present in the selective NK1 antagonist CP-99994 (3). The 1-phenyl-ethylenediamine was incorporated into a substituted benzoyl-piperazine ring system in order to allow the two aryl rings to be oriented in a similar way to that in CP-99994.

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Table 3.6 IN VITRO BINDING AFFINITIES OF (86)–(89) TO NK1- AND NK2-RECEPTORS

R

NK1 binding IC50, nM 6.7

23

NK2 binding IC50, nM 7.5

19

R

NK1 binding IC50, nM 5.9

12

NK2 binding IC50, nM 7.3

3

Hydrogen or chloro-substituents were chosen as substituents A and B at the piperazine phenyl ring. The additional R1/R2 substituted amino acetic acid residues at the other piperazine nitrogen resemble those present in NK1 and NK2 antagonists known at that time (Figure 3.24). This approach led to the identification of compounds (92) and (93) (as racemates in both cases), which appeared to exhibit good potencies at both the NK1 and the NK2 receptors (Table 3.8). To investigate the influence of

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SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

Table 3.7 IN VITRO BINDING AFFINITIES OF (90) AND (91) TO NK1-, NK2- AND NK3RECEPTORS

R

NK1 binding IC50, nM

NK2 binding IC50, nM

NK3 binding IC50, nM

21

3.1

0.72

40

6.8

2.7

the absolute stereochemistry on the activity, (92) and (93) were separated into their corresponding enantiomers by means of chiral HPLC. Interestingly the S-enantiomers are highly potent NK1 antagonists exhibiting a 150-fold reduced potency at NK2 receptors, whereas the R-enantiomers are potent NK2 antagonists with a somewhat reduced potency at NK1 receptors. The absolute stereochemistry at the chiral centres of the compounds was determined by preparation of a synthetic precursor as an enantiomerically pure compound, followed by X-ray structure determination [132].

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Fig. 3.24 Design of piperazine derivatives as neurokinin antagonists.

Table 3.8 BINDING AFFINITIES OF RACEMATES (92) AND (93) AND THE CORRESPONDING ENANTIOMERICALLY PURE PIPERAZINE DERIVATIVES

Compound

R

NK1 Ki (nM)

NK2 Ki (nM)

(92) (racemate) (S-92) (S-isomer) (R-92) (R-isomer) (93) (racemate)a (S-93) (S-isomer) (R-93) (R-isomer)

CF3 CF3 CF3 Me Me Me

5.3 1.8 48 1.8 0.9 25

17.2 270 8.3 4.9 219 3.4

a

(SCH62373)

84

SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

In another approach towards the design of dual NK1/NK2 antagonists, scientists at Schering-Plough proposed a combination of structural elements of the selective NK2 antagonist SR 48968 (11) with structural elements of the potent NK1 antagonist (94) [133], leading to a novel class of branched dual antagonists such as the proposed hypothetical structure (95) [134]. Actual synthetic preparation of such a compound (containing a modified piperidine residue: hydroxyl versus acetamino) followed by separation of diastereoisomers and enantiomers surprisingly led to the identification of the R, R-isomer (97) as the dual antagonist with Ki values of 9 and 34 nM, respectively. In contrast the S,S-isomer (96), retaining the same absolute sterochemistry at the chiral centres as (11) and (94), resulted in a potent selective NK1 antagonist (Ki value of 5 nM), exhibiting only very weak affinity to NK2 receptors (Figure 3.25). The racemic trans diamino compound (98) with a Ki value of 5 nM, also proved to be a potent, but selective NK1 antagonist. During the work aimed at a feasible synthetic pathway leading to compounds such as (97), researchers at Schering-Plough discovered (Z)-oximes (synthetic intermediates; e.g. (99) and (100)) as an additional sub-series of interesting dual NK1/NK2 antagonists. An intense lead optimization effort focussed mainly on the optimization of the piperidine and the benzylic ether regions of such compounds (Figure 3.26). An extended SAR study introducing various piperidine residues showed that this region of the molecule appears to be quite tolerant to structural alterations. It was demonstrated that the 4-hydroxy-4-phenyl substituent present in the original lead structure can be replaced by piperidine, pyrrolidine, piperazine or piperidone residues [135, 136]. Modifications at the benzylic ether region revealed that this part of the molecule can also contribute to more potent dual NK1/NK2 antagonistic properties of the resulting compounds. The optimal linker between the terminal aryl group and the rest of the molecule appeared to be an amide rather than the ether that was present in the original lead structure, demonstrated by the improvement in affinity to both receptors of (107) in comparison to (103) (Table 3.9) [137]. From the many highly potent and balanced dual NK1/NK2 antagonists, SCH 206272 (108) was identified for further profiling. This compound, in addition to potent NK1 and NK2 receptor binding affinities, also exhibits potent binding affinity to the NK3 receptor (Ki ¼ 0.3 nM) [138]. SCH206272 was also shown to be active after oral administration in inhibiting substance P induced airway microvascular leakage and in inhibiting neurokinin A induced bronchoconstriction in guinea pigs. In pharmacokinetic studies performed in dogs, oral administration of a 3 mg/kg dose led to the observation of an AUC0 – 24 h of 13.6 mg h/ml and a bioavailability of 66% [139]. Furthermore (108), also inhibited [Met – O – Me] substance P induced relaxation of the human pulmonary artery (pKB ¼ 7.7) and neurokinin A induced contractions of

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Fig. 3.25 The design of novel NK1/NK2 antagonists.

the human bronchus (pKB ¼ 8.2). In guinea pig models involving endogenously released neurokinins, SCH206272, administered orally, inhibited hyperventilation-induced bronchoconstrictions, capsaicin-induced cough and nebulized hypertonic saline-induced airways microvascular leakage. From this data, it was concluded that the potent and orally active SCH206272 may have beneficial effects in human diseases thought to be mediated by neurokinins and their receptors [140]. Modifications at the oxime residue of this series of compounds resulted in the discovery of (109), another highly potent dual antagonist with Ki values of 0.4 (NK1) and 0.5 nM (NK2) (Figure 3.27). This compound also proved to be potent in vivo in inhibiting substance P induced airways microvascular permeability and in inhibiting neurokinin A induced bronchoconstriction in guinea pigs after oral administration [141].

86

SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

Fig. 3.26 Oxime ether containing dual NK1/NK2 antagonists.

AstraZeneca Based on the structure of (11) and on the structure of AstraZeneca’s selective NK2 antagonist ZD7944 (44), a novel structural class of dual NK1/NK2 antagonists exhibiting equipotent activities at both NK1 and NK2 receptors was designed in the laboratories of AstraZeneca. An array of compounds (110) – (113) (Table 3.10) that were derived from the aforesaid lead structures was prepared, from which the naphthoyl substituent was identified as an optimal substituent at R1 together with a variety of substituted piperidine residues at R2. The most active compound in this series was the methane sulphinyl derivative (110) which contains the same piperidine substituent as the selective NK2 antagonist (44). Apparently the naphthoyl substituent at the R1 position appeared to be mainly responsible for the additional high NK1 receptor affinity. Not surprisingly, in the further optimization work, introduction of substituents to the naphthalene ring was undertaken (Table 3.11). As in many NK1 antagonists [142], an electron-withdrawing substituent was introduced at the meta-position (position 3) of the naphthalene ring system. Introduction of a cyano group at position 3 led to the identification of ZD6021 (116), which

Table 3.9 IN VITRO BINDING AFFINITIES OF OXIME ETHER DERIVATIVES (101)–(108) TO NK1- AND NK2-RECEPTORS

NK2 binding IC50, nM

(101) R/S

13

10

(102) R/S

9

7

(103) R/S

18

4

Piperidine region

Benzylic ether region

M. GERSPACHER

NK1 binding IC50, nM

(Comp-No.) Stereochem.

87 (Continued )

88

Table 3.9 CONTINUED NK2 binding IC50, nM

(104) R/S

14

13

(105) R/S

1

2

(106) R/S

4

9

SCH205528 (107) R

1

0.7

Piperidine region

Benzylic ether region

SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

NK1 binding IC50, nM

(Comp-No.) Stereochem.

M. GERSPACHER

89

Fig. 3.27 The structure of (109). Table 3.10

R2-N

FUNCTIONAL ACTIVITY (pK B) OF DUAL NK1/NK2 ANTAGONISTS (110)– (113) DETERMINED ON RABBIT PULMONARY ARTERY

pKB NK1

pKB NK2

8.18

7.81

7.55

7.92

7.69

7.82

pKB NK1

pKB NK2

7.89

7.34

hR2-N

90

SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

Table 3.11 FUNCTIONAL ACTIVITY (pKB) OF DUAL NK1/NK2 ANTAGONISTS (110) AND (114)– (116) DETERMINED ON RABBIT PULMONARY ARTERY

Cpd.

R

pKB NK1

pKB NK2

(110) (114) (115) (116) (ZD6021)

H NO2 Br CN

7.89 8.16 8.15 8.98

8.18 9.03 7.67 8.26

proved to be most potent and balanced dual NK1/NK2 antagonist from this series of compounds. Position 3 was shown to be the optimal position of the CN-group, as moving the cyano-group to positions 4, 6 or 7 resulted in compounds which in terms of potency and balance between NK1 and NK2 activity, were less active in comparison to ZD6021. ZD6021 possesses the (S)-stereochemistries at the aryl-methine and at the sulfoxide chiral centres. Change of the stereochemistry at the sulfoxide chiral centre from (S) to (R) led to a slight decrease in NK1 binding affinity and to a very slight increase in NK2 affinity, whereas changing the stereochemistry at the aryl-methine position led to a dramatic decrease of NK2 affinity. ZD6021 was further profiled in in vitro functional assays and in in vivo animal models, against the effects of neurokinin receptor agonists and was selected based on its favourable properties for further detailed preclinical evaluation [143]. Orally administered ZD6021 to guinea pigs led to a dose dependent inhibition of ASMsubstance P-induced plasma protein extravasation and [b-Ala8]-neurokinin A-induced bronchoconstriction. Pharmacokinetic studies in rats and dogs revealed bioavailabilities of 9 and 18%, respectively [144]. Further lead optimization work on ZD6021 led to a number of derivatives having additional substituents at the naphthyl residue and altered piperidine

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substituents. The, in some cases, subtle alterations in comparison with ZD6021 led to compounds with substantially altered binding properties for NK1 and NK2 receptors. Addition of a 4-methoxy or a 4-hydroxy group to the aryl-piperidine moiety led to (117) and (118), where good NK1 affinity is maintained while the NK2 affinities are increased. Introduction of a methoxy substituent at position 2 of the naphthyl residue, on the other hand, led to a significant increase in binding affinity of the resulting compound (119) to the NK1 receptor and to a decrease in binding affinity to the NK2 receptor. Interestingly (119), in comparison to ZD6021 (116), exhibits a three times improved oral bioavailability in dogs (51% versus 18%). Replacement of the methanesulfinyl-phenyl-piperidine residue by an oxo-tetrahydropyrimidine-piperidinyl-carboxamide or an oxobipiperidinyl-carboxamide substituent led to compounds such as (120) or (121) where in addition to potent NK1 affinity, the NK2 affinity could be restored to a potent level (Table 3.12). (121) showed in vivo activity against NK1- and NK2-receptor agonist induced effects after oral administration to guinea pigs; bioavailability in dogs (15%) remained essentially on the same fairly low level as that observed for ZD6021 [145]. A number of recent patent applications demonstrates an ongoing interest for AstraZeneca in the search for novel N-(2-phenyl)-substituted propyl- and butyl-1-naphthamide derivatives as dual NK1/NK2 receptor antagonists [146 – 148]. Another patent application filed by AstraZeneca covers a novel series of [1, 5] benzoxazocinones as NK1 receptor antagonists (Figure 3.28). From the patent application it appears that the compounds claimed may also additionally possess antagonist activity at NK2 and NK3 receptors [149]. Fujisawa In a series of recent patent applications, Fujisawa have claimed benzhydryl derivatives that are useful for the treatment of tachykinin mediated diseases and act as substance P, Neurokinin A and Neurokinin B antagonists (Figure 3.29). Compounds (122) –(124) have been shown to exhibit anti-emetic activity in vivo in dogs [150 – 152]. Ortho-McNeil In a patent application Ortho-McNeil claims a series of piperidine and piperazine acetamides as nervous system agents that apparently exert their therapeutic effects via modulation of NK1/NK2/NK3 receptors [153] (Figure 3.30).

92

SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

Table 3.12

R1-N

FUNCTIONAL ACTIVITY (pK B) OF DUAL NK1/NK2 ANTAGONISTS (116)– (121) DETERMINED ON RABBIT PULMONARY ARTERY

pKB NK1 pKB NK2 R1-N

pKB NK1 pKB NK2

8.98

8.26

9.50

7.50

8.93

8.34

8.14

8.11

8.20

8.70

8.70

8.20

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Fig. 3.28 [1,5] Benzoxazocin-6-one derivatives.

DUAL NK2/NK3 RECEPTOR ANTAGONISTS

The NK3 receptor is located on the ganglia of parasympathetic nerves, potentially influencing the release of several neurotransmitters, whereas the NK2 receptor is found in peripheral organs (lung, GI tract, bladder). A hypothesis

Fig. 3.29 Benzhydryl derivatives.

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SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

Fig. 3.30 Piperidine- and piperazine acetamides.

proposing a synergistic effect from dual NK2/NK3 receptor antagonists in the treatment of diseases that possess a significant neuronal component such as pulmonary or GI-tract disorders may be admissible [154]. GSK By stepwise modification of the structure of the NK3 selective antagonist talnetant (15), in particular by the introduction of a variety of substituents at the 3-position of the quinoline moiety of the molecule, researchers at GSK have been able to obtain highly potent dual NK2/NK3 antagonists. Docking studies using 3D models of the hNK2 and hNK3 receptors helped to design the appropriate substitution pattern needed for potent affinity to both the NK2 and the NK3 receptor. This effort finally led to the identification of SB-400238 (128), a highly potent compound with Ki values of 0.8 nM at both the NK2 and the NK3 receptor. In this SAR-study, it could also be demonstrated that, depending on the nature of the substituents at positions 3 and 4 of the quinoline moiety, selective NK2 antagonists such as (129) can also be obtained (Table 3.13). Sanofi In a recently published patent application, Sanofi-Synthelabo claims a series of piperidine-carboxamide derivatives derived from the structures of the NK2 and NK3 selective antagonists (11) and (14) as potent dual NK2/NK3 antagonists (Figure 3.31). Primary site of structural modifications appears to be the substituents at the position 4 of the piperidine ring. From the patent

M. GERSPACHER Table 3.13

95

BINDING AFFINITIES OF DUAL NK2/NK3 ANTAGONISTS (15) AND (125)– (129) AT CLONED hNK2 AND hNK3 RECEPTORS

R2

Ki, hNK2 (IC50, nM)

(15) (talnetant)

–OH

144

(125)

–CH2-NMe2

Compound

R1

Ki, hNK3 (IC50, nM) 1.4

51.6

3.3

(126)

1.5

1.2

(127)

0.6

1.7

SB-400238 (128)

0.8

0.8

(129)

1.0

200

application covering this series of compounds, one example (130) exhibits highly potent NK2 receptor affinity with a Ki value of 0.04 nM. No information, however, is available on the NK3 receptor affinity of this compound [155].

96

SELECTIVE AND COMBINED NEUROKININ RECEPTOR ANTAGONISTS

Fig. 3.31 The structure (130), a dual NK2/NK3 antagonist.

CONCLUSIONS Currently an exceedingly large number of structurally highly diverse potent and orally bioavailable selective NK1, NK2 and NK3 receptor antagonists, as well as combined neurokinin receptor antagonists are available. Almost every major player in the pharmaceutical industry has been, or still is, engaged in research projects aimed at the identification of such compounds. Based on evidence generated in numerous in vitro and animal experiments using mainly selective neurokinin antagonists great expectations have been generated for a therapeutic value of neurokinin antagonists in many central as well as peripheral diseases. As a consequence, a number of clinical studies have been performed during recent years. The early clinical studies mostly used selective NK1 antagonists for the potential treatment of central disorders such as chemotherapy induced emesis, anxiety and depression and confirmed the usefulness of these agents, at least as anti-emetic agents. Aprepitant was recently approved by the FDA for prevention of chemotherapy induced nausea and vomiting. On the other hand, despite initial positive results in anxiety and depression trials with compounds such as Aprepitant, in follow-up clinical studies no significant beneficial effects could be shown. Clinical studies with a number of other NK1-selective compounds such as Lanepitant, GR205171 and L-758,298 (prodrug of Aprepitant) for the treatment of migraine (and pain) have also been disappointingly unsuccessful. With respect to peripheral diseases, initial clinical experience in the treatment of airways diseases with potent neurokinin antagonists has been generated with SR-48968, MEN-11420 (both NK2 selective), FK-888 and CP-99994 (both NK1 selective). In summary, so far the outcome of the clinical studies (with mainly selective neurokinin antagonists) in the asthma area was considered to be mainly negative. On the other hand DNK333 (a dual NK1/NK2 receptor antagonist) has successfully demonstrated its ability to antagonize NKA-induced bronchoconstriction in asthmatics after oral administration. In studies testing the ability of neurokinin

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antagonists against cough, CP99994 was ineffective, however the (weakly active) dual NK1/NK2 antagonists FK224 demonstrated effectiveness in a cough trial. Therefore, this indication may be an area where potent combined neurokinin antagonists may be of significant clinical value. Currently many companies are shifting their efforts towards finding a potential application of neurokinin antagonists in diseases of the gastrointestinal and the urinary tract. The selective NK1 antagonist TAK 637 is currently in clinical trials for urinary incontinence and IBS. Last but not least, it should be noted that the clinical data so far accumulated indicates that the neurokinin antagonists (selective as well as combined neurokinin antagonists) are very well tolerated in humans, with no major findings in terms of side effects. In conclusion, it is fair to say that the promise for clinical effectiveness in a number of diseases which neurokinin antagonists made, based on their favourable pharmacological behaviour in animal studies, has not been realized. The disappointing outcome of a number of clinical trials has also led to the speculation that insufficient bioavailability, together with the fact that in many diseases two or all three neurokinin receptors are involved, results in the need for combined rather than selective neurokinin antagonists in such conditions. Additional clinical (including long term) studies in appropriately selected patient population with highly potent, selective and combined neurokinin antagonists exhibiting optimized pharmacokinetic properties may be needed to answer the question whether neurokinin antagonists can hold their promise as being of value in a number of human diseases.

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Progress in Medicinal Chemistry – Vol. 43, Edited by F.D. King and G. Lawton q2005 Elsevier B.V. All rights reserved.

4 Muscarinic Receptor Subtype Pharmacology and Physiology RICHARD M. EGLEN DiscoveRx Corporation, 42501, Albrae Street, Suite 100, Fremont, CA 94538, USA

INTRODUCTION

105

MOLECULAR BIOLOGY AND BIOCHEMISTRY OF MUSCARINIC RECEPTORS

108

MUSCARINIC M1 RECEPTORS

112

MUSCARINIC M2 RECEPTORS

118

MUSCARINIC M3 RECEPTORS

122

MUSCARINIC M4 RECEPTORS

128

MUSCARINIC M5 RECEPTORS

130

CONCLUSION

131

ACKNOWLEDGEMENTS

132

REFERENCES

132

INTRODUCTION Muscarinic and nicotinic cholinergic receptors mediate metabotropic and ionotropic effects of acetylcholine (1), respectively, on the central and peripheral nervous systems. Muscarinic receptors, a group of class I heptahelical G protein coupled receptors (GPCRs) [1], comprise five distinct subtypes, denoted as muscarinic M1, M2, M3, M4 and M5 receptors [2]. Each subtype has a unique distribution in DOI: 1 0 . 1 0 1 6 / S 0 0 7 9 - 6 4 6 8 ( 0 5 ) 4 3 0 0 4 - 0

105

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MUSCARINIC RECEPTOR SUBTYPE PHARMACOLOGY AND PHYSIOLOGY

central and peripheral nervous systems, and several are expressed both pre- and postjunctionally, the specific location varying between tissues [3]. In the central nervous system, muscarinic receptors are involved in a variety of vegetative, sensory, cognitive and motor functions. The most prominent actions in the peripheral nervous system are on the parasympathetic system where acetylcholine causes slowing of the heart rate and stimulates glandular secretion and smooth muscle contraction. Given these key physiological roles, extensive efforts have been made to develop therapeutics that selectively agonize or antagonize each muscarinic receptor subtype.

Naturally occurring products, including the agonists, muscarine (2) (a toxin from the mushroom Aminita muscaria and from which the receptor family derives its name), pilocarpine (3) (from the rutaceae plant family), or

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107

antagonists, such as atropine (4) or (– )-hyoscine (5) (from the solanaceae plant family), were initially used with limited clinical benefit in central and peripheral nervous system disorders [4, 5]. These compounds, and many subsequent derivatives, while selective for muscarinic receptors over other GPCRs, lacked intra subtype selectivity, and exhibit several side effects that restricted their therapeutic use [6]. Although pharmacological studies dating from the 1950s suggested at least three subtypes [1, 4, 7] it was not until the early 1990s, when all five subtypes were cloned, that the diversity in the muscarinic receptor family was fully appreciated. The expression of these receptors in clonal cell lines, devoid of any endogenous muscarinic receptors, subsequently led to the unambiguous delineation of muscarinic receptor biochemistry and pharmacology. These properties are in agreement with the pharmacology of endogenous receptors and provide robust tools to characterize novel agonist and antagonists. Over the succeeding decade, several medicinal chemistry programs identified compounds with modest selectivity for muscarinic receptor subtypes, resulting in novel therapeutics [8] for clinical evaluation (Tables 4.1 and 4.2). In the last 5 years or so, the pharmacological literature using these and other compounds has been extended by data from phenotypic studies in transgenic mice (generated by homologous recombination methods) lacking muscarinic receptors [9, 10] (Table 4.3). Although the physiology of muscarinic receptors is now better

Table 4.1

MUSCARINIC RECEPTOR AGONISTS AND ANTAGONISTS UNDER DEVELOPMENT FOR COGNITIVE DISORDERS

Compound

Receptor selectivity

Indication

Status

Alvameline (8)

M1 agonist/ M2 antagonist Non-selective M1 agonist

AD

Phase III discontinued

AD Sjo¨gren’s disease AD AD AD

Phase II, discontinued Approved

AD AD AD

Preclinical/Phase I Phase I discontinued Phase III, GI side effects, product under investigation Phase II

Arecoline (14) Cevimeline (18)

SCH-217443 (27), etc SDZ-210-086 (16) Xanomeline transdermal

M1 agonist M1/M2 agonist M1 agonist/ M2 partial agonist M2 antagonists M1 agonist M1 agonist

YM 796 (17)

M1 agonist

LY 593093 Milameline (9) Sabcomeline (10)

AD

Phase I Phase III discontinued Phase III discontinued

AD, Alzheimer’s disease. At the time of writing, the structure of LY 593093 has not been published.

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Table 4.2

MUSCARINIC RECEPTOR ANTAGONISTS IN THE DEVELOPMENT FOR SMOOTH MUSCLE DISORDERS

Compound

Receptor selectivity

Indication

Status

Tolterodine (Detrusitol; Detrol) (6) Detrol LA (oral controlled release tolterodine) Ditropan XL (oral controlled release oxybutynin) (37) Oxybutynin (transdermal patch) S-Oxybutynin Darifenacin (Enablex) (35) Solefenacin (Vesicare) (36) NS-21 (Temiverine)

Non-selective Non-selective

OAB OAB

Approved Approved

Non-selective

OAB

Phase III

Non-selective Non-selective M3-selective M3-selective M3-selective/ Ca channel blocker Non-selective Non-selective Non-selectivea

OAB OAB OAB OAB OAB

Phase III Phase II Phase III Phase III Phase III

OAB OAB COPD

Phase III Phase III Approved

Vamicamide (Urocut) Trospium Tiotropium (38)

Abbreviations: COPD – chronic obstructive pulmonary disease; OAB – overactive bladder. a Preferential slow-off rate from M3 receptors.

understood, the most successful muscarinic antagonist launched in the last decade remains tolterodine (6) (Detrol; Detrusitol); a compound with little in vitro selectivity between the five subtypes and for which the mechanism for selectivity in vivo is unknown [11]. This chapter assesses the current resurgence of interest in muscarinic receptor physiology and pharmacology, and the development of novel therapeutics (Table 4.4). Given the extensive literature on this receptor family, most literature cited is from the last 2 years, with the preceding years being covered in several reviews [1, 2, 4, 5, 8 –10]. Each of these reviews has extensive bibliographies to which the reader is referred for more information. MOLECULAR BIOLOGY AND BIOCHEMISTRY OF MUSCARINIC RECEPTORS Muscarinic receptors are an archetypal GPCR glycoprotein family encoded by five distinct, but intronless genes [1]. All five receptors have been cloned from several species including human, cow, pig, rat and mouse, and exhibit a high degree of homology across species. Ligands for most Class I GPCRs, such as the muscarinic receptor family, bind to highly conserved pocket deep within the transmembrane regions, causing activation via transmembrane domains TM3, TM5, TM6 and TM7. Delineation of the critical residues for muscarinic ligand

R.M. EGLEN Table 4.3

109

SUMMARY OF MOUSE PHENOTYPES IN ANIMALS WITH DISRUPTED MUSCARINIC RECEPTOR GENES

Genetic knock-out model

Functional responses to muscarinic agonist

Other phenotypic effects

Muscarinic M1 knockout

Selective impairment in nonmatching-to-sample working memory and consolidation Impaired pilocarpine-induced seizures Preserved pilocarpine-induced salivation Preserved pilocarpine-induced tremor

Absence of slow, voltageindependent N and P/Q-type calcium currents in sympathetic ganglia Absence of M current potassium channel modulation in sympathetic neurons

Muscarinic M2 knockout

Impaired oxotremorine induced tremor Impaired oxotremorine-induced analgesia Reduced oxotremorine-induced hypothermia Attenuated muscarinic regulation of heart rate Preserved oxotremorine-induced salivation Attenuated muscarinic agonist induced increases in serum corticosterone levels Impaired performance in passive avoidance test

Slight reduction in potency of muscarinic agonists inducing ileal smooth muscle contraction Absence of fast, voltageindependent N and P/Q-type calcium currents in sympathetic ganglia

Muscarinic M3 knockout

Attenuated contractility of isolated ileum and urinary bladder to carbachol Impaired salivation response to pilocarpine Impaired pupillary constriction response to pilocarpine Reduced body weight, mass of peripheral fat deposits and food intake

Retardation of growth neonatal growth. Enhanced pupil size. Preservation of intestinal motility in vivo Marked reduction in urinary bladder voiding (more marked in males than females) Preserved reproductive abilities

Muscarinic M4 knockout

Absence of oxotremorinestimulated [3H] dopamine outflow in potassium stimulated striatal slices Preserved hypothermia, salivation, tremor, analgesia induced by oxotremorine

Increased locomotor activity after D1 agonist administration Impaired migration of epidermal keratinocytes (Continued )

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Genetic knock-out model

Functional responses to muscarinic agonist

Other phenotypic effects

Lack of muscarinic agonist induced mediated analgesic responses in mice lacking both M2 and M4 receptors Muscarinic M5 knockout

Reduced sensitivity to the rewarding effects of morphine and cocaine and reduced severity of drug withdrawal symptoms Impaired maintenance phase of secretory response to pilocarpine

Lack of acetylcholine induced dilation of cerebral arteries and arterioles

The phenotypes were obtained by classical gene targeting techniques, in which individual muscarinic receptor genes were disrupted in mouse embryonic stem (ES) cells using targeting vectors. In general, disrupting muscarinic receptor genes does not result in gross physiological abnormalities, nor do compensatory changes occur in the expression of other muscarinic receptor subtypes.

binding, established by site directed mutagenesis studies or cloning of chimeric receptor constructs, has provided better understanding of the binding sites and domains responsible for G protein coupling [12]. Acetylcholine is predicted to bind to amino acids on the outer regions of the binding pocket with a critical asparagine (Asp105) residue involved in the binding of the positively charged headgroup. Although Asp105 is conserved in many Class I GPCRs, five other key residues are unique to the muscarinic receptor family, i.e. Thr231, Thr234, Tyr148, Tyr506, Tyr529 and Tyr533. This similarity in ligand-binding sites across all five subtypes is the principal reason why the identification of subtype selective ligands has been historically difficult. It is evident that, in addition to the agonistbinding site, muscarinic receptors possess allosteric sites at which synthetic compounds can modulate agonist activation [13]. The nature of these sites differs from the typical agonist-binding site and also varies between the five subtypes. In contrast to most agonists identified to date, therefore, allosteric modulators of muscarinic receptors may be subtype-selective, providing the opportunity for identification of specific modulation of subtype activation [14]. Several emerging concepts of GPCR function have raised complexities in the design of novel muscarinic therapeutics. First, muscarinic receptors can function as dimers of either homomeric or heterodimeric assemblies [15]. This is most pronounced when muscarinic receptors are expressed at high levels in systems such as Drosophila Sf9 cells [16], although the degree to which this occurs

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Table 4.4 SUMMARY OF THE THERAPEUTIC POTENTIAL OF SELECTIVE MUSCARINIC RECEPTOR AGONISTS AND ANTAGONISTS Muscarinic receptor subtype

Selective ligand

M1

Agonist

M1 M2

Agonist Antagonist Antagonist

M3 M4 M4 M5

Antagonist Agonist Antagonist Antagonist

Therapeutic indication Alzheimer’s disease, cognitive impairment Sjo¨gren’s disease Gastric acid secretiona Alzheimer’s diseaseb, cognitive impairmentb, schizophreniab, cardiac arrhythmiasc OA, COPD, IBSb Schizophreniab, antinociceptionb Parkinson’s diseaseb Cerebral ischemiab, addiction and withdrawalb

Several non-selective muscarinic antagonists, such as scopolamine, are used to treat motion sickness, glaucoma, muscarine poisoning. Some are also used in surgical procedures where fluid secretion needs to be reduced or to counter the effects of neuromuscular blocking agents. No uses have yet been proposed for selective agonists at the muscarinic M2, M3 or M5 receptors. Abbreviations: OA, overactive bladder, COPD, chronic obstructive pulmonary disease, IBS, irritable bowel syndrome. a The approved use of the muscarinic M1 antagonist, pirenzepine (Gastrozepin), is as a gastric antisecretory agent. b Preclinical observations that await clinical validation. c The approved use of AF-DX116 (Otenzepad) (32) is for cardiac arrhythmias.

in vivo is unclear. However, dimeric assemblies influence ligand pharmacology and govern the cellular pathway activated. Muscarinic M3 receptors, for example, require co-activation of the dimer in order to signal via the ERK1/2 pathway [17]. Second, muscarinic receptors can be constitutively active, arising from receptor mutation, over expression or over expression of the G proteins to which they couple. The influence of pathology on constitutive activity is unclear, although the phenomenon clearly affects ligand action. Several ‘silent’ antagonists (atropine, for example) act as inverse agonists and partial agonists (pilocarpine, McN A 343 (7) for example) express full agonism at constitutively activated muscarinic receptors [2]. Muscarinic receptors couple to heterotrimeric guanine nucleotide binding proteins (G proteins) and mobilize several second messengers. In general, muscarinic M2 and M4 receptors preferentially couple to Gai, and muscarinic M1, M3 and M5 subtype to Gaq [2]. The cellular effector of the subtypes

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principally depends upon the Ga subunit mobilized. In some cases, the Gb/g subunits play a key role in cellular signaling and provide a mechanism by which the M2 receptor activates phospholipase Cb. The receptor/G protein interface can also be a site of drug action – the general anesthetic, propofol, for example, selectively disrupts the interaction between the M1 receptor and the G protein [18]. In neuronal and non-neuronal tissues, muscarinic M2 and M4 receptors inhibit elevated adenylyl cyclase activity [2], as well as prolonging potassium channel [19] or non-selective cation (TRP) channel opening [20]. Muscarinic M1, M3 and M5 receptors mobilize inositol phosphoinositides, notably inositol (1,4,5) trisphosphate (Ins P3) and 1,2-diacylglycerol, via activation of phosphoinositide-specific phospholipase Cb, thereby increasing intracellular calcium [2]. These three subtypes also activate other cellular messengers such as nitric oxide or phospholipase A2, although these effects are secondary to elevations in intracellular calcium [19]. There are additional complexities in muscarinic receptor signaling. In isolated cells, for example, muscarinic receptors couple to Gaq proteins and protect the cell against apoptosis [21], suggesting more long-term effects of activation on programmed cell death pathways. Muscarinic receptor signaling can also ‘channel’, in that one subtype may preferentially couple to a signaling pathway, even though more than one subtype couples to the same G protein, often in a tissue or cell-dependent fashion. Muscarinic M3 receptors, for example, couple more efficiently than M5 receptors to phospholipase Cb, even though both mobilize Gaq [22]. The precise molecular mechanism of this phenomenon is unclear but it does raise the possibility that novel muscarinic agonists may have a tissue selective action. In the following sections, each muscarinic receptor subtype is reviewed individually, with emphasis placed on the physiology of the receptor subtype and pharmacology of small molecule ligands. This division is somewhat arbitrary since most tissues express more than one subtype and the overall physiological response to acetylcholine reflects the participation of each. A summary of the therapeutic status of muscarinic agonists and antagonists can be found in Tables 4.1, 4.2 and 4.4. A summary of the phenotypic effects of deleting each subtype in transgenic mice can be found in Table 4.3. MUSCARINIC M1 RECEPTORS Muscarinic M1 receptors are abundantly expressed in all major forebrain areas including the cerebral cortex, hippocampus and striatum [1, 3, 23]. Consistent with this distribution, muscarinic M1 receptors are implicated in learning and memory processes [24]. Enhanced cholinergic receptor activation, either by the use of acetylcholinesterases or muscarinic agonists, ameliorates cognitive

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decline [24]. Currently, the only approved therapies for the disease are a group of acetylcholinesterase inhibitors. Selective M1 agonism has been suggested for many years as an approach to retard the cognitive decline in dementias, such as those seen in Alzheimer’s disease, age-associated memory impairment or cognitive impairments associated with schizophrenia. The ‘cholinergic hypothesis’ of dementia [25] is fundamentally based on observations that the presynaptic muscarinic M2 receptor (as well as 5-HT2 and nicotinic a4b2 receptors) population selectively declines and the postsynaptic M1 receptor population is preferentially preserved in Alzheimer’s disease [26]. Extensive pharmacological data supporting a role for the M1 receptor in cognition are supported by studies with transgenic mice lacking the M1 receptor, in which memory consolidation processes are impaired [27]. This effect is surprisingly modest given the pronounced effect of centrally acting muscarinic antagonists on cognition, suggesting either a complex role of muscarinic M1 receptors or that more than one subtype is involved [27]. It has been suggested that the M1 receptors are not, in fact, essential for memory formation, but are important for memory processes involving interactions between the cerebral cortex and hippocampus [28]. Nonetheless, selective M1 agonism remains a therapeutic approach to Alzheimer’s disease, age-associated memory impairment or cognitive impairments associated with schizophrenia, potentially resulting in compounds to improve cognition with few side effects [26]. The overproduction of amyloid b peptide and its subsequent deposition as insoluble amyloid plaques is a key pathophysiological lesion leading to Alzheimer’s disease. Consequently, reducing the production of this protein may slow disease progression [24]. In isolated tissues, muscarinic M1 agonism augments the release of the amino terminal form of amyloid precursor protein [29]. Although the biochemical pathway underlying this effect is unclear, increases in Ab protein promotes activation of protein kinase C and calcium/ calmodulin dependent kinase II: a process counteracted by M1 receptors. This finding has been subsequently confirmed [30] in Alzheimer’s disease patients using the muscarinic M1 receptor agonist, cevimeline (18), where Ab levels declined after chronic treatment. Similar observations in Alzheimer’s disease patients are seen with the muscarinic M1 agonists, alvameline (8), milameline (9), sabcomeline (10), RS 86 (11), talsaclidine (12) and xanomeline (13), suggesting that M1 agonists, in general, lower Ab (particularly Ab42) levels [31]. Long-term Phase III clinical trials have not, however, been conducted to assess the potential of these agents to retard progression of the disease. Indeed, current trials using muscarinic agonists in Alzheimer’s disease are usually applied only to mild cognitive impairment and it thus is difficult to distinguish between symptomatic and disease-slowing effects of these compounds in most current trial designs [32].

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Collectively, muscarinic M1 agonism represents a novel therapeutic approach to Alzheimer’s disease with two potential benefits, i.e. moderate reversal of cognitive impairment and decreased amyloid plaque formation [24]. An established medicinal chemistry goal is to identify centrally acting, potent and selective agonists for use in this disorder (Table 4.1). Early clinical studies with muscarinic agonist, such as arecoline (14), pilocarpine or oxotremorine (15) and (11), were disappointing due to their low efficacy and high side effect potential. Over the last decade additional compounds, such as (8), sabcomeline and xanomeline or spiropiperidines and spiroquinuclidines such as SDZ 210-086 (16) and YM-796 (17), were identified and clinically evaluated, again with

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disappointing results and early discontinuation of clinical trials [33]. An exception is cevimeline (18), currently approved for the autoimmune condition, Sjo¨gren’s disease [30].

The failure of these compounds in trials in dementia patients probably relates to the ‘receptor reserve’ associated with muscarinic M1 receptor function in Alzheimer pathology [4, 32]. As discussed above, when the M1 receptor selectivity of an agonist is marginal, and the receptor reserve high, responses at several muscarinic receptor subtypes occurs. This is most relevant to novel muscarinic agonists of low intrinsic efficacy (such as partial agonists), with which expression of agonism is critically dependent on the prevailing receptor reserve [4]. It is thus likely that most muscarinic M1 agonists [26] possess ‘functional’, rather than absolute, receptor subtype selectivity [4]. As such, prediction of agonism in a clinical therapeutic setting is problematic. One example of this phenomenon is the pharmacology of the agonist, xanomeline (13), which, although selective in muscarinic M1 binding assays, is functional at muscarinic M1, M2 and M4 receptors. Although, the compound has a desirable profile in preclinical in vitro and in vivo studies, data from both healthy volunteers and Alzheimer’s patients shows several side effects, resulting in a high discontinuation rate [33]. In contrast, a series of selective M1 partial agonists, originally identified from a combinatorial library in a high throughput screening campaign, have now been reported from the Lilly group. These compounds include the agonist, LY 593093, which possesses good

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bioavailability and exhibits higher selectivity and efficacy in comparison to previous clinical candidates, such as xanomeline [34]. An alternative approach to the design of selective M1 agonists is to exploit ectopic sites on the muscarinic M1 receptor that allosterically regulate agonist function [35]. This region is not conserved amongst other muscarinic receptor subtypes and highly selective compounds could be synthesized that augment existing activation by endogenous acetylcholine. Moreover, identification of allosteric compounds, particularly those with positive allosteric properties, would act to ‘fine tune’ the activation of classical agonists. WIN 62577 (19), a neurokinin NK1 receptor antagonist, is an allosteric muscarinic M3 receptor enhancer with micromolar affinity, although attempts to modify the compound to produce potent and selective M1 allosteric enhancers were unsuccessful [36]. However, muscarinic allosteric agonists of exceptional selectivity, including AC-42 (20), have been reported from the Acadia group [37]. The high selectivity of (20) may emanate from an ectopicbinding site in the upper portions of transmembrane domains TM1 and TM7; a domain of the muscarinic M1 receptor that markedly diverges within the five subtypes [37]. Additional compounds from this group with a similar mode of action reportedly improve cognitive performance in rodents without inducing cholinergic side effects, although few details are presently available [38].

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The muscarinic M1 receptor also plays a role in other CNS disorders including schizophrenia. Muscarinic receptors have been a compelling target for the treatment of psychosis, since muscarinic antagonists produce symptoms in humans similar to the positive and negative behaviors associated with the disease. Genetic polymorphisms of the muscarinic M1 receptor are also associated with schizophrenia [39]. Xanomeline [4] exhibits antipsychotic activity in both preclinical and clinical studies. However, as the compound is a mixed M1/M4 agonist, the antidopaminergic effects may also be mediated via the muscarinic M4 receptor (see later). The antipsychotic drug, clozapine (21), exhibits limited muscarinic M1 agonist activity, and these characteristics are more pronounced in a metabolite, N-desmethyl clozapine (22) [40]. Both compounds modulate M1 receptor activity via an allosteric site that partially overlaps with the orthostericbinding site for acetylcholine [40]. Furthermore, N-desmethyl clozapine augments hippocampal N-methyl-D -aspartate receptor currents, suggesting that agonists with both M1 allosteric activity and N-methyl-D -aspartate activity could provide novel antipsychotic therapies.

A limited literature suggests that acetylcholine may have a hormonal, i.e. extraneuronal, action [41] in order to regulate immune system function. Muscarinic M1 and M2 receptors are expressed in human lymphocytes and appear to mediate the autocoid effects of acetylcholine [42]. Although this aspect of muscarinic M1 receptor function has not been extensively pursued, initial studies suggested that M1 receptors on lymphocytes may act as a marker for disease progression in CNS disorders [43]. In lymphocytes from bronchial asthmatic patients, muscarinic M2 receptor expression is also increased [44].

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The immune function of acetylcholine is not well established, but emerging data suggest a direct relationship between muscarinic M1 receptor activation and interleukin 2 production [45].

MUSCARINIC M2 RECEPTORS Muscarinic M2 receptors are widely expressed in both central and peripheral nervous systems [1]. Selective muscarinic M2 antagonism increases cholinergic overflow by reducing autoreceptor function in both the brain and the periphery [46]. However, studies in mice deficient in both M2 and M4 receptors suggest a role for both subtypes in modulating hippocampal cholinergic function [47]. Genetic variants in the human M2 receptor gene also correlate with differences in cognitive performance [48]. Several workers [32] have suggested that either selective M2 receptor antagonism or compounds with mixed M2 antagonism and M1 agonism is a therapeutic approach to increase cholinergic function in Alzheimer’s disease, particularly at a stage where cholinergic tone is not completely lost (Table 4.1). The muscarinic M2 antagonists, SCH 57790 (23) or the pyridobenzodiazepinone, BIBN-99 (24), improve cognitive performance in preclinical models [49, 50]. Bilateral infusions of muscarinic M2 antagonists into the dorsolateral striatum of cognitively impaired rats also enhance memory performance [51]. A series of piperidines, synthesized in the Schering-Plough group, are potent and selective M2 receptor antagonists, with clinical studies in dementia underway using compounds such as SCH 72788 (25), SCH 211803 (26), SCH 217443 (27) and related compounds [32, 52, 53]. These compounds have improved oral

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bioavailability over SCH 57790, although clinical data have not been reported (Table 4.1). The potential utility of muscarinic M2 receptor antagonism in treatment of cognitive decline [52] has spurred patenting of several other chemical series, notably from the piperidine alkaloids, originally derived from

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the bark of Galbulimima baccata, of which (þ ) himbacine (28) is a prototypic example. Derivatives include epihimandravine and himbacine analogues containing ring substituted decahydro naphthofurans [54], hydroisobenzofuran-1 (3H)-ones [55], benzylidene ketals [56] and dimenthindene derivatives [57]. Finally, compounds with mixed histamine H3 agonists/muscarinic M2 antagonists may also be useful in the treatment of cognitive disorders [58]. In the caudate putamen, muscarinic M2 receptors act as inhibitory heteroreceptors on dopaminergic terminals. Consequently, selective muscarinic

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M2 receptor blockade may provide a therapeutic approach to schizophrenia, a disease associated with excessive dopamine transmission [59]. BuTAC (29) is a partial agonist at muscarinic M2 and M4 receptors, and an antagonist at muscarinic M1, M3 and M5 receptors. In rodents, the partial agonist BuTAC exhibits antipsychotic behavior, resembling clozapine and olanzapine, and induces a reduction in dopamine cell firing in the limbic ventral tegmental area, possibly by an M2 antagonist action [60]. However, mutated mice lacking the muscarinic M4 receptor also display supersensivity of dopamine D1 receptors, indicating that the muscarinic M4, as opposed to the M2 receptor is also important in this respect [61]. Muscarinic M2 receptors may play a role in adult depressive disorders. Serum cortisol levels are elevated in major depressive disorders, notably in adult women [62]. Females possessing a thymidine at nucleotide 1890 in the 30 untranslated region of the human M2 receptor gene have an elevated predisposition for major depression [63]. In mice, muscarinic M2 receptors mediate agonist-induced activation of the hypothalamic –pituitary –adrenocortical axis, as animals deficient in this subtype do not show enhanced release of serum corticosterone in response to muscarinic agonists [64]. Moreover, centrally active muscarinic agonists stimulate the hypothalamic – pituitary– adrenocortical axis via the release of corticotrophin-releasing hormone [10]. However, it is unclear from the transgenic mice work where the locus of action of M2 activation occurs (central vs. peripheral nervous systems) [10]. These data, collectively, may implicate activation of muscarinic M2 receptors in the effects of cortisol-induced depressive disorders, although this concept has yet to be clinically investigated.

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In the periphery, the muscarinic M2 receptor is expressed in the myocardium, and mediates well-known negative chronotropic and inotropic effects of acetylcholine [2]. Accordingly, in mice lacking the M2 receptor, the bradycardic effects of muscarinic agonists are completely abolished [65]. Emerging mRNA analysis and pharmacological data show that other muscarinic subtypes are expressed in this tissue, acting to modulate ion channel activity and thus heart function [65, 66]. In the myocardium, the muscarinic M2 receptor is sensitive to changes in membrane voltage, probably at a site in the vicinity of the receptor– G protein interface [67]. However, the activation of inward rectifying potassium currents in isolated cells is sensitive to prevailing culture conditions, suggesting caution in the interpretation of data relating to muscarinic receptor function and ion channel activation [68]. The relationship of the muscarinic M2 receptor to myocardial ion channel activation is affected in diseases such as Chagas’ disease, a parasitic infectious disease associated with long-term cardiac malfunction. Here, circulating M2 receptor autoantibodies attenuate muscarinic M2 function [69], and in idiopathic dilated cardiomyopathy, muscarinic M2 autoantibodies are also elevated [70]. In an animal model of cardiomyopathy, cardiac remodeling is also associated with an increase in circulating M2 receptor autoantibodies, suggesting a similar autoimmune reaction to that seen in the disease [71]. The Trypanosoma cruzi antigen, cruzipain, also induces antibodies against the M2 receptor, directly implicating the receptor in the etiology of Chagas’ disease [72]. In designing compounds to modulate muscarinic M2 receptor, the role of an allosteric site has been emphasized, with several studies indicating a markedly different structure activity relationship from the classical agonist-binding site [73]. The allosteric site modulates agonism in either a negative or positive allosteric fashion. Site-directed mutagenesis studies have shown that two amino acid residues in the muscarinic M2 receptor entirely account for the allosteric selectivity of compounds, such as curacurine V, alcuronium, gallamine, caracurine V, bis (ammonio) alkanes and bisquaternary dimers of strychnine and brucine, as well as their associated derivatives [74]. The exploitation of this site in the design of novel therapeutics has not been extensively studied. MUSCARINIC M3 RECEPTORS The muscarinic M3 receptor is widely distributed in the CNS, although at lower levels than other muscarinic receptor subtypes [1, 9, 10]. Muscarinic M3deficient mice are hypophagic and lean, suggesting a central role for this subtype in regulating food intake [75]. Although this may involve modulation of hypothalamic melanin concentrating hormone levels, deficits in salivary flow could also contribute to the hypophagic phenotype [10]. The physiology

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is complex, however, as the muscarinic M3 receptor may not be required for the basal flow of salivary fluid and it is highly likely that other muscarinic receptor subtypes, such as the muscarinic M5 receptor, may be involved [10]. Initial indications of muscarinic receptor heterogeneity, developed in the 1960s and 1970s, stemmed from pharmacological studies using isolated myocardium and smooth muscle tissue [1]. In these experiments, the muscarinic receptor subtype mediating negative inotropy pharmacologically differed from the subtype mediating smooth muscle contraction (i.e. muscarinic M2 vs. M3 receptors, respectively) [7]. These differences provided simple bioassays that identified several, structurally diverse antagonists with marked muscarinic M2 or M3 selectivity [1, 4, 5]. The most important compounds identified in this way were 4-DAMP (30) and pFHSiD (31), both preferential for the muscarinic M3 over the M2 receptor. AF-DX 116 (32) (Otenzepad), AF-DX 384 (33) methoctramine (34), and tripitramine, conversely, were shown to be preferential for the M2 over the M3 receptor [1, 4, 5]. It is now evident that these early bioassays are far more complex in terms of the muscarinic receptors involved. The myocardium, for example, expresses other muscarinic receptors than the M2 receptor, even though the predominant effect of acetylcholine is mediated by this subtype (see above). In rat isolated myocytes, for example, M3 receptors augment inositol phosphate accumulation, resulting in positive inotropic effects [76, 77]. Contractile responses in smooth muscle also reflects participation of more than the muscarinic M3 receptor, as most smooth muscle tissues express both muscarinic M2 and M3 receptors in a ratio of about 4:1 [78]. The pharmacology of the contractile action of muscarinic agonists generally reflects muscarinic M3 receptors alone [79], although the developmental stage is important; contractile responses in the fetal ovine ileum, for example, are mediated via the M2 and not M3 receptors [80]. Studies in tissues from several species, including human

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[81, 82], show that both receptors are involved in the control of muscle motility and can be revealed under discrete experimental conditions [78, 79]. These data have implications for drug discovery programs aimed at identifying muscarinic antagonists for the treatment of smooth muscle dysfunction [4, 78, 79], specifically in the design of the pharmacological profile required in the compound (Table 4.2) [83]. Several lines of data show that muscarinic M2 receptors act synergistically with the M3 receptor to modulate contraction [78]. Activation of muscarinic M2

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receptors, for example, opposes elevations in smooth muscle myocyte adenylate cyclase activity, thereby abrogating muscle relaxation [79]. Heterologous desensitization of responses to other contractile agents in gastrointestinal smooth muscle also requires activation of both subtypes [84]. Muscarinic M2 receptors open a non-selective cation channel, thereby augmenting entry of extracellular sodium ions – a major mechanism for cholinergic excitation of smooth muscle [85] since cation channel opening may be the predominant mediator of smooth muscle contractile activity [86 –88]. The current model is that muscarinic M3 receptors exert a permissive role over the M2 mediated cation current activation via a process that involves elevations in intracellular calcium, possibly independent of Ins P3 mobilization [89]. Nonetheless, many functional studies suggest a modest role for muscarinic M2 receptor activation in smooth muscle contraction, unless specialized experimental (or pathophysiological) conditions (e.g. elevated adenylate cyclase levels, heterologous desensitization, aging, elevated insulin levels) prevail [90]. Transgenic mice studies show that smooth muscle contraction is not dependent upon muscarinic M2 receptor and support a major role for M3 receptor [91]. Importantly, this phenotype varies between smooth muscles. In the M3 knockout mice, isolated gastrointestinal smooth muscle motility to muscarinic agonists is impaired by approximately 77% and the residual contraction is mediated by muscarinic M2 receptors. Qualitatively similar data are seen in isolated urinary detrusor muscle, but the residual M2 receptor-mediated component is much less than in gastrointestinal tissue [91]. In vivo data from these mice reveal that gastrointestinal function is unimpaired, arguing that the muscarinic system per se does not control gut function. In contrast, enhanced urinary retention is evident in these animals, suggesting that muscarinic control over the bladder is critical to urinary bladder voiding [91]. Mutant mice lacking both M2 and M3 receptors exhibit marked distension of the urinary bladder, although there are no intestinal complications [92]. Collectively, these data suggest that muscarinic M3 blockade alone is useful for treating urinary tract disorders. Treatment of gastrointestinal motility disorders may require concurrent blockade of muscarinic M2 and M3 receptors. Several muscarinic antagonists have, in fact, been developed as therapeutics for hyperactive smooth muscle disorders (Table 4.2), including overactive bladder (OAB), irritable bowel syndrome (IBS) and chronic obstructive pulmonary disease (COPD) [4, 77]. Of these, tolterodine (6) is a potent muscarinic antagonist developed for the treatment of OAB that possesses equivalent muscarinic M2 and M3 receptor affinities [11] but exhibits selective actions in vivo. Several controlled clinical studies demonstrate a low propensity for dry mouth or alterations in pupillary accommodation at doses of tolterodine that modulates OAB [93 –95]. However, as the compound in vitro lacks selectivity between muscarinic receptor subtypes, its mechanism of selective action in vivo is unclear [11].

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Darifenacin (Enablex) (35), in contrast, is a selective muscarinic M3 antagonist [96] and preliminary data show inhibition of bladder responsiveness at doses that do not affect salivation [97, 98]. The compound also reduces OAB; specifically, the time between the first sensation of urgency and urination. Solifenacin (Vesicare) (36) is also a compound with selective M3 antagonist actions with a longer duration of action than darifenacin [99], but with comparable efficacy to tolterodine. These two compounds, together with tolterodine, are potential front line therapies for OAB having efficacy at doses accompanied by reduced anticholinergic side effects. In some elderly patients, however, with all these agents dry mouth remains a notable compliance problem [100].

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Alternative approaches to reducing anticholinergic side effects in the treatment of OAB have, therefore, included optimizing the pharmacokinetic profile by modifying the formulation. For example, Ditropan XL is a transdermal formulation of the non-selective antagonist, oxybutynin (Oxytrol) (37) under advanced clinical evaluation, reportedly with a reduced incidence of side effects and improved compliance, and providing a once-daily treatment for OAB. In a similar fashion, Detrol LA is a formulation of tolterodine that also provides a once-daily treatment regimen. Trospium (Regurin) is also a muscarinic antagonist in clinical evaluation, but has little selectivity between receptors [101]. However, a different formulation of the drug is in development in order to provide once-daily therapy and improved pharmacokinetics. Unlike darifenacin or solifenacin, trospium is a quaternary amine that does not cross the blood – brain barrier and over 3 days of dosing improves OAB. Since it is secreted in the urine unchanged, it may exert a local action in the bladder as it concentrates in the urine. Indeed, an unexplored area of research is the locus of action of muscarinic antagonists at the urothelium, a urinary bladder tissue that expresses muscarinic receptors and that mediates the release of a diffusible factor to induce relaxation of the underlying smooth muscle layer [102]. Muscarinic receptor blockade may also be a useful approach for the treatment of IBS, with several approaches directly antagonizing muscarinic receptors or indirectly modulating cholinergic transmission via the 5-HT3 or 5-HT4 receptors [103]. Muscarinic receptor antagonists identified for use in this disease, such as otilonium [104], are non-selective antagonists, with additional properties of calcium channel blockade and antagonism of other neurotransmitter receptors, all of which contribute to an antispasmolytic action [105]. A second issue in the treatment of IBS is to reduce the pain associated with the disease. Muscarinic agonists are well-known antinociceptive agents, with potencies approaching those of the opiates [106]. A compound with a mixed muscarinic agonist/ antagonist action may be an optimal approach, in that motility would both be attenuated and nociception reduced [4]. Cholinergic-mediated constriction of airways involves activation of postjunctional M3 receptors, as well as prejunctional M2 receptors [107]. Studies using knockout mice reveal a complex interplay of muscarinic M2 and M3 receptors in peripheral airways [108], with M1 receptors counteracting cholinergic bronchoconstriction, and neuronal M2 receptors inhibiting acetylcholine release from parasympathetic nerves. The function of these autoreceptors is selectively abrogated by several agents including parainfluenza infection, double stranded RNA, ozone exposure, ovalbumin sensitization and vitamin A deficiency [109], resulting in increased cholinergic overflow and enhanced airway smooth muscle contraction. These neuronal effects probably underlie the paradoxical bronchospasm seen with several muscarinic antagonists, such as rapacuronium, in the treatment of asthmatic bronchoconstriction [110]. Localization of eosinophils to

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airway nerves, via an interaction with specific adhesion molecules, in asthmatics may also attenuate muscarinic M2 receptor function [111]. Collectively, these data indicate that muscarinic antagonism per se is an inappropriate option for the treatment of asthma. In the treatment of COPD, by contrast, short acting muscarinic antagonists such as iprotropium and oxitropium, have been used as effective bronchodilator therapies [112], since they reverse airway constriction and reduce bronchial fluid secretion Tiotropium (Spriva) (38) has been suggested as a novel first line therapeutic approach with once-a-day dosing efficacy superior to ipratropium (39), accompanied by an improved side effect profile [113]. Tiotropium functionally acts an antagonist preferential for the muscarinic M1 and M3 receptors, by virtue of the preferential slow dissociation kinetics from these receptors [114]. Prolonged treatment of COPD patients with the drug does not cause tolerance and is well tolerated, although dry mouth is evident in some patients [115].

MUSCARINIC M4 RECEPTORS In the central nervous system, muscarinic M4 receptors are distributed in the corpus striatum, being co-localized with dopamine receptors on striatal projecting neurons [116]. In the periphery, the subtype is present on various prejunctional nerve endings, where it acts to inhibit parasympathetic and sympathetic transmission [117]. The muscarinic M4 receptor may play a role in psychosis, with the mixed M1/M4 agonist xanomeline (13) having antipsychotic effects (see above). This compound, even after acute administration, selectively inhibits mesolimbic firing of dopamine cells, suggesting that muscarinic agonists could have a faster onset of action than current antipsychotics, with fewer side effects [118]. Mice lacking the muscarinic M4 receptor also display an increased sensitivity to the disruptive effect of phencyclidine on pre-pulse inhibition [119].

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This preclinical effect is a model of psychosis and the data support the contention that the M4 receptor is a suitable target for the treatment of schizophrenia. Parkinson’s disease is a neurodegenerative characterized by slow movements, muscular rigidity, tremor and balance disturbances. These symptoms arise from the loss of dopaminergic neurons projecting to the striatum, causing an imbalance between the cholinergic and dopaminergic systems, such that the former predominates. Non-selective muscarinic antagonists are effective in treating the disease, although side effects limit their use. Transgenic mice lacking the M4 receptor show increased locomotor activity and an enhancement of dopamine D1 receptor-mediated effects [10]. It is likely that the striatal M4 receptors exert an inhibitory action on dopamine D1 receptor function. Consequently, selective M4 antagonists have been developed for the treatment of Parkinson’s disease, including benzoxazines such as PD 0298029 (40), the latter of which has a favorable pharmacokinetic profile and good bioavailability in the clinic [120]. Activation of the muscarinic M4 receptor is also affected by an allosteric site, with the compound, thiochrome recently shown to selectively enhance agonist function [121], although this aspect of the receptor has not been exploited in the design of novel therapeutics.

Activation of central muscarinic receptors leads to potent antinociception [9, 106, 121], although the precise nature of the muscarinic receptor subtype(s) mediating the response is unclear. In mice, this analgesic response induced by muscarinic agonists, CMI-936 (41) or CMI-1145 (42) is pertussis toxin-sensitive [122]; a finding consistent with involvement of either muscarinic M2 or M4 receptors [123]. Transgenic mice deficient in muscarinic M2 receptors also show a striking reduction in muscarinic-dependent antinociceptive responses [10]. The highly selective muscarinic M4 antagonist, MT-3 (isolated from the venom of the African green mamba, Dendroapsis augusticeps), also antagonizes these responses [123] suggesting that

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muscarinic M4 receptors also mediate antinociceptive effects. However, the phenotype of the M4-deficient mouse indicates no change in the antinociceptive action of muscarinic agonists [124], probably due to residual presence of muscarinic M2 receptors. Indeed, in mice lacking both M2 and M4 receptors, muscarinic agonists are devoid of analgesic activity [125, 126]. It is probable that the muscarinic M2 receptor plays a predominant role in antinociception and the effect of the M4 receptor is minor by comparison. Selective agonism of the latter will not, however, result in major effects on the cardiovascular system yet may provide a viable approach to analgesia [10]. MUSCARINIC M5 RECEPTORS The muscarinic M5 receptor is the only muscarinic subtype expressed by the dopamine containing neurons of the substantia nigra pars compacta, a structure that provides the principal dopamine innervation to the striatum [127]. Activation of muscarinic M5 receptors thus facilitates striatal dopamine release – although it is likely that other muscarinic receptors, including the M4 receptor, are involved [128]. The muscarinic M5 receptor is also the predominant subtype expressed in the ventral tegmental area, a tissue that provides major dopamineric innervation to the nucleus accumbens and other limbic areas [129]. These brain areas play a major role in the rewarding effects of several drugs of abuse. In muscarinic M5 deficient mice, stimulation of the laterodorsal tegmental area, which provides major cholinergic input to the ventral tegmental area dopamineric neurons, is markedly disrupted [130]. A preliminary report from the Lilly group using an antibody retrieval method [131] suggests in fact that the muscarinic M5 receptor has a distribution more widespread than previously thought, but with a distribution highly consistent with a role in mediating dopaminergic transmission. The potential of selective muscarinic M5 blockade as an approach to narcotic addiction corroborates to the extensive use of scopolamine and extracted alkaloids in the detoxification of heroin addicts [132]. Overall, antagonism of muscarinic M5 receptors may be an important approach to novel therapeutics in both schizophrenia and compound addiction. In the periphery, the muscarinic M5 receptor is expressed at low levels in the iris, oesophagus and lymphocytes, although the function in these tissues, if any, is unclear [133]. Brain microvasculature expresses muscarinic receptors, with endothelial cells expressing both muscarinic M2 and M5 receptors, and vascular smooth muscle cells expressing all subtypes excepting M4 receptors [134]. Neuronally released acetylcholine regulates cortical perfusion and blood – brain barrier permeability via changes in local blood flow involving the muscarinic receptor-induced release of nitric oxide [135]. The pharmacological profile of the muscarinic receptor subtype mediating cerebral vascular dilation corresponds

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best with the M5 subtype [135]. Indeed, in the rat, muscarinic M5 receptors have been localized to the circle of Willis and pial arteries [136]. In mice lacking M5 receptors, cholinergic dilatation of basilar and pail arties is lost, supporting this suggestion [137]. Therapeutically, deficits in cholinergic-induced vasodilatation may be involved in the aetiology of Alzheimer’s disease, or stroke-induced dementia, suggesting that selective muscarinic M5 antagonism is a useful approach in these pathologies. Finally, dry mouth is a frequent anticholinergic side effect and can be dose limiting in the elderly (see above). Consequently, delineation of the subtypes involved is important in the design of novel muscarinic antagonists. The role of the muscarinic M5 receptor in modulating salivary gland function is unclear, however, although mice with a truncated M5 receptor have increased drinking behaviour and reduced salivation in comparison to the wild type [138]. Mice deficient in M5 receptors also secrete less saliva during the late phase of the salivation response, although other subtypes are clearly involved in the effects of muscarinic agonists on salivary secretion, including the muscarinic M3 receptor [10]. Against this suggestion is the recent finding that the pharmacological profile of the guinea-pig submandibular gland is consistent with a homogeneous muscarinic M3 receptor population [139]. Interestingly, the antagonists, oxybutynin, tolterodine and darifenacin exhibited lower affinities in this tissue in comparison to the guinea-pig urinary bladder, raising the possibility that the cellular background of the M3 receptor influences its pharmacology [139]. These findings may provide a mechanistic basis for a ‘bladder selective’ action of these antagonists in vivo, as discussed above.

CONCLUSION The role of muscarinic receptors is highly complex and, although not completely understood, is becoming clearer. The recent pharmacological evidence with novel compounds, together with data from transgenic mice, suggests that all five subtypes have a defined function in both peripheral and central nervous systems. Several novel agonists and antagonists have now been identified with authentic subtype specificity in vitro and in vivo, and additional opportunities for selective subtype modulation may arise from development of selective allosteric regulators. Several clinical development programmes are currently underway with selective M1 agonists, M1 allosteric regulators or M2 antagonists for the treatment of Alzheimer’s disease, as well as M3 antagonists for the treatment of OAB or COPD (Table 4.4). These compounds may provide novel approaches for a variety of disorders, as well as advancing the current understanding of the metabotropic role of acetylcholine.

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ACKNOWLEDGEMENTS The author wishes to thank Dr C.C. Felder, Lilly Corporate Center, Indianapolis, IN, USA, for his insightful comments on the manuscript and for providing the preliminary data on the muscarinic M5 receptor distribution. The author also wishes to thank Drs N.J.M. Birdsall and S. Lazareno, Medical Research Council, Mill Hill, UK, for their thorough reading of the manuscript and thoughtful input.

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Progress in Medicinal Chemistry – Vol. 43, Edited by F.D. King and G. Lawton q2005 Elsevier B.V. All rights reserved.

5 The Transcription Factor NF-kB as Drug Target BURKHARD HAEFNER Department of Inflammation, Johnson & Johnson Pharmaceutical Research and Development, A Division of Janssen Pharmaceutica, Turnhoutseweg 30, Box 6423, 2340 Beerse, Belgium INTRODUCTION

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THE ROLE OF NF-kB IN DEVELOPMENT AND DEFENCE NF-kB in Development: of Flies, Mice, and Men NF-kB from Innate to Adaptive Immunity NF-kB Helping Cells Handle Stress

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NF-kB AS CRUCIAL FACTOR IN THE PATHOGENESIS OF HUMAN DISEASE NF-kB Dysregulation as a Causative Factor in Inflammatory Disease NF-kB as a Major Culprit in Cancer NF-kB Activation at the Heart of Cardiovascular Disease NF-kB in the Pathogenesis of Obesity and Diet-induced Insulin Resistance – not a Fat Chance? NF-kB Hitting the Nerve in Neurological Disorders? Parasitic Subversion of NF-kB Signalling in Viral Diseases

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THE NF-kB SIGNAL TRANSDUCTION NETWORK: POTENTIAL POINTS OF PHARMACOLOGICAL INTERVENTION NF-kB Activation – Canonical and Otherwise Reactive Oxygen Species as Second Messengers in NF-kB Activation – True or False? Drug Targets within the NF-kB Signalling Network – Old and New

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DISCOVERY AND DEVELOPMENT OF SMALL MOLECULE NF-kB INHIBITORS Inhibitors of Protein Kinases Involved in NF-kB Activation Inhibitors of NF-kB DNA Binding NF-kB Transactivation Inhibitors with Unknown or Undisclosed Mechanism of Action Proteasome Inhibitors Potential for NF-kB Inhibition as Adjunctive Anticancer Therapy Possible Mechanism-based Toxicity of NF-kB Inhibition The Future for NF-kB Inhibitors as Drug Candidates

158 158 170 171 174 179 180 181

REFERENCES

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INTRODUCTION Nuclear factor kB (NF-kB) is recognized to have a crucial role in the regulation of genes involved in pathological processes such as chronic inflammation and tumourigenesis. The pathways leading to the activation of this transcription factor have generated considerable interest within the pharmaceutical industry as providers of targets for drug discovery. NF-kB was identified almost 20 years ago as a regulator of immunoglobulin gene expression [1]. Originally thought of as restricted to the B-cell lineage, NF-kB has since been found to be ubiquitously expressed and to be a critical component of regulatory networks controlling cell survival, proliferation, and differentiation within as well as outside the immune system. NF-kB consists of a pair of proteins of the Rel family (Figure 5.1) which have combined in a specific way to form a homo- or heterodimer [2]. Rel family proteins are characterized by the possession of a Rel homology domain (RHD), which contains two immunoglobulin-like subdomains and shows close to 50% sequence similarity across the family. It mediates DNA binding via its Nterminal subdomain, dimerization via its C-terminal one, and nuclear translocation via at least one nuclear localization sequence. The two classes of Rel proteins are distinguished by the absence (class I) or presence (class II) of a transcriptional activation domain. Class I proteins are synthesized as longer precursor proteins (NF-kB1, shown, and NF-kB2) which are processed to yield p50 and p52, respectively. The C-terminal portions of these precursors, which are removed by proteolytic processing, resemble the IkB proteins, e.g. IkBa, IkBb, and IkB1, by possessing a series of ankyrin repeats, which mediate their interaction with Rel protein dimers, as well as a PEST (proline-, glutamate-, serine-, and threonine-rich) domain, involved in the regulation of stability. Indeed, that of NF-kB1 is identical to IkBg which is encoded by a separate

Fig. 5.1 Domain structure of class I and class II Rel proteins. AR: ankyrin repeats; GRR: glycinerich region; LZ: leucine zipper; N: nuclear localization sequence; RHD: Rel homology domain; TAD: transactivation domain. Arrowhead indicates cleavage site.

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gene [3]. A glycine-rich region in class I proteins functions as a processing signal. While NF-kB1 has only one nuclear localization sequence, NF-kB2 has two. Characteristic for class I Rel proteins is the possession of an insert in the RHD of 32 amino acids in p50 and 18 amino acids in p52. Class II Rel proteins are c-Rel, RelA (p65) and RelB (shown). In contrast to the other Rel proteins, RelB has a leucine zipper near its N-terminus but lacks a PKA phosphorylation site in the RHD. A highly simplified, canonical pathway (Figure 5.2) of NF-kB activation by certain extracellular stimuli (Table 5.1), such as cytokine binding to a receptor at the plasma membrane, involves phosphorylation followed by polyubiquitinylation, i.e. the attachment of a chain of the 76-amino acid protein ubiquitin, and proteolytic degradation of inhibitor-of-NF-kB (IkB) which is associated

Fig. 5.2 Pathways of NF-kB activation. Ub: ubiquitin; UbL: ubiquitin ligase. Open arrows indicate progression along the pathway. Closed arrows indicate a modification.

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THE TRANSCRIPTION FACTOR NF-kB AS DRUG TARGET Table 5.1 CLASSES OF NF-kB ACTIVATORS

Activator class

Selected examples

Viral gene products

Epstein-Barr Virus EBNA-2, Hepatitis B virus HBx, HIV-I Tat, HTLV-I Tax Helicobacter pylori membrane proteins, Staphylococcus enterotoxins, lipopolysaccharide IL-1, IL-12, IL-18, TNFa Epidermal growth factor, nerve growth factor, insulin Hydrogen peroxide, singlet oxygen, superoxide anion Asbestos fibres, cigarette smoke, heavy metal, okadaic acid, PCB77 Cisplatin, haloperidol, paclitaxel, vinblastine

Bacterial components Cytokines Growth factors and hormones Reactive oxygen species Environmental hazards and toxins Therapeutic drugs For an exhaustive list see ref. [214].

with the transcription factor. Release from IkB results in the accumulation of NF-kB in the nucleus where it binds to specific DNA sequences (consensus sequence of the kB sites: GGGRNYYYCC where R is a purine, Y a pyrimidine, and N any nucleotide) in the regulatory elements of the genes it controls (Table 5.2) [2]. For maximum stimulation of its transactivation function, covalent modification of the Rel proteins themselves by phosphorylation and acetylation is required. Central to the phosphorylation of IkB in the canonical

Table 5.2 CLASSES OF NF-kB TARGET GENE PRODUCTS Activator class

Selected examples

Cytokines and chemokines Growth factors and hormones

IL-1, IL-6, IL-8, TNFa, IP-10, RANTES Granulocyte macrophage colony-stimulating factor, platelet-derived growth factor B chain, angiotensin II TNF receptor, IL-2 receptor a chain, CCR5 TLR 2, bradykinin B1 receptor, platelet activating factor receptor Cyclin D1, cyclin D3 Fas ligand, c-IAP-1 and 2, Bcl-2 Ig k light chain, Ig 1 heavy chain, T-cell receptor b chain ICAM-1, P-selectin, tenascin C Cyclooxygenase 2, matrix metalloproteinase 9, glucose-6-phosphate dehydrogenase JunB, NF-kB1 and 2, IRF-1, c-Rel, RelB, c-Myc IkBa, apolipoprotein C III, fibronectin HIV-1, cytomegalovirus, herpes simplex virus

Cytokine and chemokine receptors Other cell surface receptors Cell cycle regulators Regulators of cell survival/apoptosis Immunoglobulins and related proteins Cell adhesion molecules Enzymes Transcription factors Other non-catalytic proteins Viruses For an exhaustive list see ref. [214].

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pathway is IkB kinase (IKK) whose two isoforms, IKKa and IKKb, are present in a large multiprotein complex. IKKa and b share 52% of their amino acid residues, an N-terminal catalytic domain, a C-terminal helix – loop – helix domain modulating catalytic activity, and a central leucine zipper motif mediating homoas well as heterodimer formation. An unrelated regulatory protein, IKKg, interacts with these dimers by binding to a specific domain in IKKb. A zinc finger motif at its C terminus serves to link the IKK complex to upstream activators. Complexes of p50/p65 heterodimers and IkBa but not IkBb or IkB1 constitutively shuttle between cytoplasm and nucleus, where stimulus-induced ubiquitination (and possibly also proteasomal degradation) of IkBa takes place. This is because, unlike the other two isoforms, IkBa only incompletely masks the p50 nuclear localization sequence. Expulsion from the nucleus is stimulated by nuclear export sequences in IkBa and p65. In unstimulated cells, the movement of these complexes is in a dynamic equilibrium with the majority of complexes being present in the cytoplasm at any one moment. Apart from the canonical pathway, there exists at least one other route to NF-kB activation [4]. This non-canonical pathway does not involve IkB and neither depends on IKKb nor IKKg (Figure 5.2). It is activated by a much smaller set of stimuli comprising B-cell activating factor of the TNF family (BAFF), CD40 ligand (CD40L), and lymphotoxin a/b. The latter two molecules also activate the canonical pathway albeit less efficiently. The mechanism of interaction between the two pathways is still unclear. It could be that some of the IKKa molecules activated in the non-canonical pathway join the canonical one. Alternatively, proteins upstream of IKKa in the canonical pathway, such as adaptor proteins interacting with transmembrane receptors, might behave promiscuously when stimulated and associate with receptors in the canonical pathway. Phosphorylation of Rel proteins has recently been emerging as yet another mechanism for NF-kB regulation. While mitogen- and stress-activated kinase 1 (MSK1) has been described as a nuclear RelA kinase, the enzyme phosphorylating RelB on serine 368, thus regulating its binding to other Rel proteins as well as p100 processing [5], has so far not been identified.

THE ROLE OF NF-kB IN DEVELOPMENT AND DEFENCE NF-kB IN DEVELOPMENT: OF FLIES, MICE, AND MEN

Following from results of genetic screens in Drosophila or gene targeting experiments in mice, and from the identification of the cause of a rare human tumour, NF-kB has been increasingly recognized as a transcription factor regulating not only immune and inflammatory responses but also developmental processes in fruit fly embryos as well as mammalian tissues ranging from

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the immune to the nervous system [6, 7]. A fundamental role for the rel gene Dorsal in Drosophila embryonic pattern formation was established a few years before the discovery of NF-kB by a screen for maternal effect mutations. These are mutations in genes whose products are translated from maternal mRNA accumulated in the egg prior to fertilization. A gradient of nuclear Dorsal increasing from the dorsal to the ventral side of the early embryo is involved in the generation of dorsoventral polarity [6]. NF-kB was originally identified as a nuclear factor which binds to a conserved sequence motif in the immunoglobulin Ck light chain gene enhancer suggesting a role in B-cell maturation. This was later confirmed by gene knockout analysis in mice [8 –10]. Only animals in which both nfkb1 and nfkb2 or rela had been knocked out showed markedly impaired B-cell maturation indicating functional overlap among these rel genes in B-cell development. Loss of both nfkb1 and nfkb2 gene function results in a developmental block at the immature IgMþIgD2 stage while foetal liver cells from nfkb1/rela doubleknockout mice transplanted into lethally irradiated wildtype animals already failed to produce B220þ B-cell precursor cells. The differentiation of T lymphocytes and dendritic cells was also shown, by gene-targeting experiments, to depend on NF-kB. In addition to impaired B-cell maturation, nfkb1/nfkb2 double-knockout mice show osteopetrosis due to a block in osteoclast differentiation. NF-kB can, therefore, also be assigned a role in bone development. Furthermore, knockout studies have also demonstrated a role for NF-kB in liver development. Mice lacking functional rela die in utero due to massive liver apoptosis. This is a consequence of increased sensitivity of their hepatocytes to the proapoptotic effect of tumour necrosis factor a (TNFa) as shown by the viability of double-knockout mice in which the gene coding for this cytokine was also inactivated [11]. Mice in which ikba has been knocked out show elevated TNFa mRNA levels in the epidermis and develop severe dermatitis. Their skin shows abnormal morphology including excessive cell proliferation in the basal layer [12]. Surprisingly, a similar hyperplastic phenotype was observed in the skin of transgenic mice in which NF-kB activation in the basal layer was inhibited by tissue-specific expression of a constitutively active, transdominant negative mutant of IkBa. While the phenotype of the knockout mice appears to be caused by an abnormal inflammatory response, the skin hyperplasia of the transgenic mice is the result of a defect in epidermal morphogenesis. The same developmental defect can be induced by pharmacological inhibition of NF-kB in the skin after topical application of pyrrolidine dithiocarbamate. Activation of NF-kB in the epidermal basal layer of mice by tissue-specific expression of constitutively nuclear p50 or p65, in contrast, results in reduced keratinocyte proliferation and consequently an abnormally thin skin [13]. Based on these and further observations, a model for the role of NF-kB in skin development

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in which activation of the transcription factor during terminal keratinocyte differentiation promotes cell cycle arrest and survival has been proposed [14]. In addition to epidermal hyperplasia, transgenic mice expressing transdominant negative IkBa show defective development of hair follicles, exocrine glands, and teeth as a consequence of increased apoptosis resulting from reduced NF-kB activation [15]. Familial cylindromatosis, or turban tumour syndrome, is a rare human autosomal dominant predisposition to develop mostly benign tumours, which arise in sweat and scent glands as well as hair follicles due to abnormal survival, proliferation, and differentiation of epidermal stem cell-derived progenitor cells. Tumour growth is driven by excessive activation of NF-kB after loss of function of its negative regulator CYLD by somatic mutation of the second copy of the gene coding for this deubiquinating enzyme [16, 17]. These observations clearly demonstrate a requirement for NF-kB in the morphogenesis of the skin appendices in mice as well as humans. Despite the observation that the earliest activation of NF-kB in mouse embryos occurs in the brain, knockout analysis of rel genes initially provided only limited evidence for a function of NF-kB in murine brain development. Subsequent gene-targeting studies, however, found NF-kB to be involved in neuronal cell survival in the developing as well as neuroprotection and synaptic plasticity in the adult brain [7]. NF-kB activity has recently been shown to also be important for the development of brain structures other than neuronal tissue. Rat Schwann cell differentiation and axon myelination in cocultures was found to be severely impaired when NF-kB activation was blocked and myelin synthesis was much reduced in Schwann cells from rela knockout mice [18]. According to another recent study, NF-kB functions in synaptic signalling affecting the learning behaviour of mice. Mice in which rela had been knocked out lack synaptic NF-kB and show a learning deficit when trained in a radial arm maze providing extra-maze spatial cues. The transcription factor thus appears to have a role not only in the structural but also the functional development of the brain [19].

NF-kB FROM INNATE TO ADAPTIVE IMMUNITY

Multicellular organisms, with their much larger and more complex bodies, are an ideal home for a host of potentially pathogenic microbes. It is, therefore, not surprising that evolution has equipped not only all metazoans but also higher plants with sophisticated means of defence against these invaders culminating in the adaptive immune system of the vertebrates. Innate immunity is the evolutionarily most ancient type of host defence. It relies on a limited number of germ-line encoded receptors, which recognize pathogen-associated molecular patterns and activate the synthesis of an arsenal

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of antimicrobial substances. Among these effector molecules are antimicrobial peptides. An important family of pattern recognition receptors are the Toll-like receptors (TLRs) [20 – 22]. The first TLR to be identified was Drosophila Toll (dToll). dToll lies upstream of Dorsal and was discovered in the same genetic screen for maternal effect mutants as this Drosophila Rel protein. Later on, it was realized that dToll is not only involved in dorsoventral axis formation in the embryo but also in innate immune responses in the adult fly. In this latter function, dToll signals via Dif, one of the two other Drosophila Rel family members. The third Drosophila Rel protein, Relish, closely related to mammalian p100 and p105, is also a regulator of fruit fly immune gene expression but is activated by a different, as yet unidentified receptor and appears to be critical for the expression of a different subset of defence genes. Like their Drosophila homologues, mammalian TLRs, found in all tissues, activate NF-kB to induce the expression of genes of the innate immune response such as those encoding cytokines. Cytokines, such as the interleukins, are intercellular signalling proteins which regulate the function and behaviour of cells involved in immune and inflammatory responses [23]. These are not just cells of the innate immune system, such as macrophages, but also non-immune cells like endothelial cells, or even cells of the adaptive immune system, i.e. T and/or B lymphocytes. Thus, cytokines help co-ordinate first-line, innate and second-line, adaptive immune responses. They achieve this by binding to specific receptors in the plasma membrane, many of which then activate signal transduction networks that have NF-kB as a central component. Hence, Toll-like proteins are not the only receptors which signal through NF-kB. One of the hallmarks of the adaptive immune system is the generation of antigen-specific receptors by DNA-rearrangement in B or T cells [24, 25]. These proteins are the B-cell receptors (BCRs), which in their secreted forms are known as antibodies, and the T-cell antigen receptors (TCRs), respectively. The BCRs as well as the TCRs belong to the immunoglobulin superfamily and, like the TLRs and many cytokine receptors, activate NF-kB. This results in the transduction of signals in the nucleus in B- and T-cell activation and effector function [26]. The stimulation of NF-kB activity by these receptors occurs via the same upstream signalling component as the one induced by Toll-like or cytokine receptors, i.e., IKK. As mentioned before, NF-kB was first identified as a transcription factor regulating immunoglobulin gene expression, and thus as a protein involved in adaptive immune responses. However, with time and further research, it became clear that the role of this transcription factor in the adaptive immune system is more diverse. As was shown by knockout experiments described above, NF-kB is crucial for B-cell survival and development. More recent work has demonstrated that the generation of mature T cells is similarly impaired when the activation of NF-kB is prevented by lineage-specific deletion of the gene for IKKb [27].

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Thus, NF-kB plays a central role in the workings of the innate immune system, the co-ordination of innate and adaptive immune responses and the development and function of the adaptive immune system. Consequently, aberrant activation of this transcription factor in the immune system is often a major contributor to the pathogenesis of autoimmune disease, chronic inflammation, and leukaemia. NF-kB HELPING CELLS HANDLE STRESS

NF-kB is not only crucially involved in the defence against pathogenic microorganisms but also helps to protect cells from the proapoptotic effects of cellular stressors such as ionizing radiation and toxic chemicals [28]. Reactive oxygen species (ROS), e.g. hydrogen peroxide and singlet oxygen, produced in response to these noxious stimuli, are thought to act as second messengers in stress-induced activation of the transcription factor. In certain cell types, at least, such oxygen intermediates appear to also have a role in NF-kB activation by stimuli for which specific plasma membrane receptors are known. For example, interleukin-1 (IL-1) was found to activate NF-kB via ROS generation in monocytic and lymphoid but not epithelial cells [29]. Activation of NF-kB by ROS can occur via the canonical signalling pathway but evidence for an IkBdegradation-independent mechanism involving tyrosine phosphorylation of IkB by Src family kinases has also been found (see below). IKK activation by ROS was recently reported to be mediated by protein kinase D, formerly known as protein kinase Cm [30].

NF-kB AS CRUCIAL FACTOR IN THE PATHOGENESIS OF HUMAN DISEASE A survey of the vast literature on NF-kB shows that this transcription factor has been implicated in the pathogenesis of the majority of diseases on which the pharmaceutical industry has been concentrating its efforts including the biggest killers in the developed world, heart disease and cancer [31]. A common link between most if not all of these diseases appears to be the involvement of inflammatory processes in which the dysregulation of NF-kB plays a major causative role. NF-kB DYSREGULATION AS A CAUSATIVE FACTOR IN INFLAMMATORY DISEASE

The aetiology of most chronic inflammatory diseases is still poorly understood. However, analysis of biopsies taken from patients and of animal models for such

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diseases points to abnormally high NF-kB activation as a common and critical factor, which is regularly accompanied by the expression of adhesion molecule genes in affected tissues, the consequent recruitment of immune cells to sites of inflammation, and the production of proinflammatory cytokines such as IL-1, IL-6, IL-8, and TNFa by these cells [32]. Many of these cytokines, themselves, stimulate NF-kB activation and in chronic inflammation, this appears to result in a self-perpetuating, runaway process of NF-kB activation, immune cell recruitment, cytokine production and so on. Why, in contrast to non-pathological inflammatory processes, which are a normal part of host defence, these events go out of control in chronic inflammatory disease, is a question to which there is as yet no conclusive answer. Luckily for the patients, knowing the answer to this question does not appear to be a prerequisite for the effective treatment of chronic inflammation. The findings that NF-kB inhibition during the resolution of inflammation can protract the inflammatory response [33] and that some patients with Crohn’s disease, a type of inflammatory bowel disease, have a mutation in the NOD2 gene which encodes an activator of NF-kB suggest that NF-kB not only has a role in the initiation but also in the resolution of inflammation [34]. This is because under non-pathological conditions, the transcription factor induces the expression of not only proinflammatory but, with a certain delay, also anti-inflammatory genes. However, in chronic inflammation, the response is obviously never terminated in the first place. Therefore, it does not appear logical to conclude from these results that treatment of patients with NF-kB inhibitors may have adverse rather than beneficial consequences. That this is not so is borne out by the fact that a common effect of the nonsteroidal anti-inflammatory drugs aspirin (acetyl salicylate), sulfasalazine, mesalamine, and sulindac is inhibition of NF-kB [35, 36].

NF-kB AS A MAJOR CULPRIT IN CANCER

Rheumatoid arthritis has been described as a locally invasive tumour due to the characteristic hyperplasia of the synovial lining, extensive angiogenesis, and progressive joint invasion as well as destruction associated with the disease [37]. This is not as far-fetched as it may seem since synoviocytes from arthritic joints show anchorage-independent growth and there are reports of p53 mutant synovial cells in the disease. Indeed, it is increasingly recognized that there exists a connection between inflammation and the pathogenesis of many types of cancer [38]. Chronic irritation, caused, for example, by asbestos fibres in the lung, or chronic infection by the stomach bacterium Helicobacter pylori can lead to inflammatory conditions which may eventually progress to cancer [39]. Moreover, certain types of chronic inflammatory disease frequently give rise to neoplasms. Inflammatory bowel disease developing into colon cancer is one

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example where a link between inflammation and tumourigenesis has been firmly established. Inflammatory cells in the tumour microenvironment promote cancer cell proliferation, survival, migration, and metastasis as well as tumour angiogenesis by releasing chemokines and proteases. Tumour cells in turn may also secrete chemokines which attract additional immune cells into the microenvironment and use the same homing mechanisms as those involving chemokine receptors and adhesion molecules when they disseminate and form metastases [40]. NF-kB is activated in many haematological and solid cancers due to gene amplification or over-expression. In the case of Hodgkin’s lymphoma, NF-kB activation may be caused by mutation of ikba. Chromosomal translocations involving nfkb2 or bcl3 (encodes an IkB protein) have been found in certain types of leukaemia and in non-Hodgkin lymphomas. Activation of the transcription factors helps cancer cells survive stress induced by hypoxic conditions, ionizing radiation, or cytotoxic drugs, because it stimulates the expression of genes promoting tumour angiogenesis or cell survival. Furthermore, NF-kB activation may drive cancer cell proliferation by inducing the expression of genes involved in cell cycle progression as well as genes encoding cytokines and other growth factors. By activating protease, chemokine, and adhesion molecule genes, the transcription factor may promote metastasis. Thus, its activation confers a number of selective advantages to tumour cells and may be an important step in cancer progression [41, 42].

NF-kB ACTIVATION AT THE HEART OF CARDIOVASCULAR DISEASE

NF-kB is activated in a wide variety of cardiovascular diseases including atherosclerosis, myocardial infarction, and heart failure [43]. In some of them, such as myocardial infarction, NF-kB appears to be consequence rather than cause of the pathological processes leading to the disease, and may be seen as a cellular response to stress or injury in an attempt to prevent further damage. In others, such as atherosclerosis and also heart failure, however, NF-kB activation and consequent expression of cytokine and/or adhesion molecule genes appear to be part of inflammatory processes which are central to the pathogenesis of these diseases. Various drugs used to treat heart failure were found to modulate cytokine production and one of them at least, Pimobendan, has been reported to inhibit NF-kB activation [44]. In atherosclerosis, NF-kB activation in arterial smooth muscle cells at sites of chronic injury or stress results in their proliferation as well as in the expression of adhesion molecule and chemokine genes. This attracts monocytes, which infiltrate the intima, where they participate in initial lesion development and eventually plaque formation after differentiation into macrophages, intracellular lipid accumulation, and

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conversion into foam cells. The transcription factor has, therefore, been identified as a therapeutic target of major importance in the treatment of this leading cause of morbidity and mortality in the developed world. NF-kB IN THE PATHOGENESIS OF OBESITY AND DIET-INDUCED INSULIN RESISTANCE – NOT A FAT CHANCE?

It has long been known that high-dose salicylate can reduce the symptoms of diabetes. Insulin resistance is the hallmark of type 2 diabetes mellitus, and a characteristic feature of obesity as well as cardiovascular disease. Recent findings suggest a link between insulin-resistance induced by a high-fat diet and IKKb, one of the molecular targets of salicylates [45, 46]. The first evidence for a role for IKKb in insulin signal transduction was the finding that this kinase can phosphorylate the insulin receptor substrate IRS-1 [47]. This has been proposed to result in impaired activation of downstream signalling component phosphoinositide (PI) 3-kinase by this docking protein and to contribute to fat-induced insulin resistance. IKKb knockout mice, and mice treated with a high salicylate dose known to inhibit IKKb did not show the reduction in glycolysis and glycogen synthesis, IRS-1 tyrosine phosphorylation and IRS-1 associated PI 3-kinase activity observed in skeletal muscle of control mice after intravenous lipid infusion. Moreover, type 2 diabetes patients on high-dose aspirin therapy showed reduced plasma glucose, fatty acid, and lipid levels. However, in a more recent study, mice in which IKKb was conditionally inactivated in adult myocytes showed no difference in obesity-induced insulin resistance when compared to control animals [48]. Independent of IKKb being a valid drug target for the treatment of type 2 diabetes or not, these results neither prove nor disprove a connection between NF-kB and the disease. Once again, the common link appears to be inflammation. Both obesity and type 2 diabetes have been described as chronic inflammatory conditions due to increased plasma concentrations of the proinflammatory cytokine IL-6 and other markers of inflammation [49, 50]. Since NF-kB is known to be critically involved in their production, a role for this transcription factor in the pathogenesis of these conditions appears likely. NF-kB HITTING THE NERVE IN NEUROLOGICAL DISORDERS?

Activation of NF-kB, thought to be induced by ROS, has been observed in the common neurodegenerative disorders Alzheimer’s and Parkinson’s disease, as well as in ischaemic brain injury [51, 52]. If NF-kB activation in neurodegenerative disease and stroke is involved in the induction of neuronal damage, or if it is part of a neuroprotective response to it, still appears to be a matter of debate. Although an increasing body of evidence points to the latter possibility, in ischaemic brain injury, at least, activation of the transcription factor has been

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linked to inflammation, which occurs in the injured brain region in the wake of a stroke and which is regarded as detrimental. PARASITIC SUBVERSION OF NF-kB SIGNALLING IN VIRAL DISEASES

Many highly pathogenic viruses are known to hijack components of signal transduction pathways which activate NF-kB [53, 54]. Among them are the two notorious human retroviruses: human immunodeficiency virus (HIV) and human T-cell leukaemia virus (HTLV). NF-kB is essential for the transcription of HIV genes, the long terminal repeat (LTR) of the virus containing binding sites for the transcription factor [55]. NF-kB activation by the HIV Tat protein has been implicated in neurological disease induced by the virus [56]. Tat appears to stimulate NF-kB activity by activating the protein tyrosine kinase Lck [57]. HTLV Tax protein associates with and activates IKK resulting in persistent activation of NF-kB [58, 59]. This is thought to promote survival of infected T cells and thus to contribute to their neoplastic transformation. THE NF-kB SIGNAL TRANSDUCTION NETWORK: POTENTIAL POINTS OF PHARMACOLOGICAL INTERVENTION When the long-sought IkB kinase, for which numerous candidates had been proposed, was finally identified [60], the key steps in the signalling pathway from cytokine receptor activation at the plasma membrane to NF-kB translocation into the nucleus were thought to have been finally worked out. However, with the discovery that IKK exists as two catalytically active isoforms [61, 62], the question immediately arose whether these two kinases had distinct functions in NF-kB activation. This turned out to be the case and it was realized that there exist more pathways to NF-kB than just the canonical one [63]. After their initial focus on serine phosphorylation of IkB, researchers working in the field of NF-kB signalling began to look for other forms of post-translational, covalent modification which are employed to regulate NF-kB activity. They have since discovered a role, not only for serine but also tyrosine phosphorylation, one for acetylation, as well as a novel function for ubiquitinylation, thus turning the linear canonical NF-kB signal transduction pathway into an intricate signalling network and starting to reveal the complex regulatory circuitry which controls this important transcription factor. NF-kB ACTIVATION – CANONICAL AND OTHERWISE

The finding that there is more to NF-kB signalling than the canonical pathway should not have come as a surprise. Even in Drosophila, there are two pathways

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to NF-kB activation [64, 65]. Two Drosophila Rel proteins, Dif and Dorsal, are activated by the Toll pathway while the third, Relish, is activated by a different signalling cascade called IMD pathway after the immune deficiency (imd) mutation, which renders mutant flies susceptible to infection by gram-negative bacteria. Toll mutants, in contrast, succumb to fungal or gram-positive bacterial infections. This is because mutant flies are unable to mount an adaptive immune response, which consists of the synthesis of pathogen-specific antimicrobial peptides. Non-canonical pathways In mammals, a non-canonical pathway was found to be involved in the activation of p100, the product of the nfkb2 gene, by proteolytic removal of the C-terminal IkB-like sequence to release p52 [66]. This pathway is activated by stimuli involved in lymphocyte development or effector functions, i.e. BAFF, CD40L, and lymphotoxin a/b, and involves activation of IKKa by the NF-kB-inducing kinase NIK [67 – 69]. IKKa which is not essential for the canonical pathway as shown by knockout analysis, is phosphorylated by NIK on serine 176 in the activation loop [70]. IKKa in turn, phosphorylates p100 thus triggering its ubiquitinylation and proteolytic processing. This function is similar to that of Drosophila IKK, which does not phosphorylate the Ikb homologue Cactus but rather the p105 homologue Relish inducing its processing and consequent activation. Moreover, IKKa has a critical function in the activation of NF-kB-dependent gene expression independent of the phosphorylation of IkB or Rel proteins. The kinase has been shown to phosphorylate promoter-associated histone H3 in the nucleus on serine residue 10 leading to the acetylation of this nucleosomal protein and consequent chromatin remodelling, facilitating gene expression [71, 72]. IKK is not the only kinase, which can catalyse this reaction. Protein kinase A (PKA) and MSK1, which like IKK can regulate NF-kB DNA binding and transactivation activity by phosphorylating p65 (see below), also phosphorylate histone H3 on this serine residue. Histone – DNA interactions and NF-kB activity at promoters regulated by this transcription factor, therefore, appear to be co-ordinately controlled. Similar to p100, p105, the product of nfkb1, is proteolytically processed to produce an NF-kB subunit. This appears to be regulated by glycogen synthase kinase 3b (GSK-3b), which has been shown by knockout analysis to be required for NF-kB activation (see below). A model has been proposed in which GSK-3b phosphorylates serines 903 and 907 in the IkB-like C-terminal domain of p105 allowing IKK, activated by TNFa via the PI 3-kinase signalling pathway, to phosphorylate serines 927 and 932, thus triggering the degradation of p105.

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According to this model, degradation of p105 is blocked by inactivation of GSK3b through phosphorylation of serine 9 by Akt downstream of PI 3-kinase. As a consequence, default processing of p105 to p50 occurs [73]. Another serine/ threonine protein kinase reported to stimulate p105 processing by C-terminal phosphorylation is the NIK homologue Tpl-2 [74]. This enzyme is the product of the proto-oncogene Tpl-2 (tumour progression locus 2) in the rat or Cot (cancer Osaka thyroid) in humans. Cot is overexpressed in a variety of human tumours including mammary carcinomas [75]. In the absence of an activating stimulus, Tpl-2 is associated with p105 and its kinase activity is inhibited. Upon activation, the enzyme dissociates from p105, phosphorylates its target proteins, and is subsequently degraded [76]. Akt has been reported to activate Tpl-2 by phosphorylation on serine 400 [77]. Phosphorylation of this residue is required for IKK activation by Tpl-2 via phosphorylation of NIK, which in turn phosphorylates IKK. There is also evidence for tyrosine phosphorylation of IKK. The protein tyrosine kinase Src has been found to associate with IKK and an inhibitor highly selective for Src family kinases, i.e. PP2, blocks tyrosine phosphorylation of IKK as well as TNFa-induced expression of intercellular adhesion molecule 1 (ICAM-1) and cyclooxygenase 2 in A549 and NCI-H292 human lung carcinoma cells, respectively [78, 79]. IkB phosphorylation on serines 32 and 36 (IkBa) or 19 and 23 (IkBb) by IKK is seen as a crucial step in the activation of NF-kB given that most stimuli activating the non-canonical pathway also activate the canonical one (BAFF being the exception). There is also increasing evidence that tyrosine phosphorylation of IkB can release NF-kB from its inhibitor, independently of IKK. Oxidative stress, the protein tyrosine phosphatase inhibitor pervanadate, nerve growth factor, TNFa in murine bone marrow macrophages, and hepatitis C virus protein NS5A have all been found to induce NF-kB activation by stimulating phosphorylation of IkB on tyrosine 42 [80 – 84]. Src and Syk family protein tyrosine kinases have been implicated in the catalysis of this reaction. The PI 3-kinase subunit p85, which itself associates with Src, as well as the Src family kinase Lck have been shown to bind IkB after tyrosine phosphorylation via their SH2 domain providing a potential mechanism for the removal of the tyrosine-phosphorylated inhibitor from NF-kB [85, 86]. Earlier results indicated that tyrosine phosphorylation of IkB does not lead to its proteolytic degradation [87] but oxidative stress and NS5A, which have been shown to also induce phosphorylation of tyrosine residues in the IkB PEST domain, as well as pervanadate, stimulate IkB degradation [80, 84, 88]. In all of these cases, the protease calpain appears to be involved. PEST domain serine phosphorylation by casein kinase 2 (CK2) induces proteasome-dependent IkB degradation and consequently NF-kB activation in response to UV light [89]. Serine and tyrosine phosphorylation of IkB need not be mutually exclusive events controlling NFkB activation, but may combine to efficiently dissociate the inhibitor from

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the transcription factor. However, certain NF-kB activating agents, such as the anticancer drug doxorubicin, appear to induce IkB degradation by the proteasome independently of both IKKa and b, whose genes had been knocked out, and of the IkB PEST domain, which had been deleted in this study [90]. Moreover, the tumour suppressor protein p53 has lately been reported to induce NF-kB activation by an IKK-independent mechanism involving p65 phosphorylation on serine 536 by ribosomal S6 kinase 1 (Rsk1), a member of the MAPKAPK (mitogen-activated protein kinase-activated protein kinase) family [91]. This serine/threonine kinase has also been reported to be required in certain cell types for NF-kB activation induced by TPA (tetradecanoyl phorbol acetate ¼ phorbol myristate acetate (PMA)) [92, 93]. It has recently been proposed that Fas-associated factor 1 (FAF1), a binding protein of the death receptor Fas, can inhibit NF-kB activity by physically interfering with p65 nuclear translocation stimulated by TNFa, IL-1, and bacterial lipopolysaccharide (LPS) [94]. If correct, this may yet be another mechanism for regulation of NF-kB nuclear translocation independent of IKK but since this result was obtained with cells overexpressing FAF1, it has to be viewed with some caution. Along the same lines, kB-Ras, a Ras-like small G-protein, has recently been found to prevent IkB degradation and consequently NF-kB activation by forming a ternary complex with the transcription factor and the inhibitor and blocking phosphorylation of the latter. As a consequence, additional NF-kB-activating signals which induce the removal of kB-Ras from this complex have been proposed to exist [95]. The role of protein phosphorylation Protein phosphorylation plays a critical role in the regulation of NF-kB activation, not only at the level of nuclear translocation, but also of DNA binding and transactivation in the nucleus. Multiple protein serine/threonine kinases, activated by different stimuli, phosphorylate NF-kB. This is thought to modulate the magnitude and kinetics of its transcriptional response. They are cAMPdependent protein kinase, also known as PKA, CK2, IKK, PKCz, MSK1, NF-kB activating kinase (NAK alias TBK1 or T2K), and cGMP-dependent kinase (PKG) [96 – 103]. PKA binds to NF-kB and increases DNA-binding of p65 by phosphorylating serine 276 in the RHD thereby disrupting its interaction with an inhibitory region at the C terminus. Moreover, upon phosphorylation, serine 276 becomes available to bind one of the two highly homologous transcriptional coactivators and acetyltransferases p300 and CBP (CREB binding protein). Such complexes appear to be able to efficiently displace p50 homodimers in association with histone deacetylase 1 (HDAC1) bound to many NFkB-regulated genes in the absence of activating stimuli. MSK1 phosphorylates

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the same residue. Phosphorylation of the equivalent residue in the RHD of p50, i.e. serine 337, which lies in a consensus PKA phosphorylation site, has been shown to be critical for DNA binding of this NF-kB subunit. CK2 phosphorylates p65 on serine 529 whereas IKK and NAK phosphorylate serine 536. These phosphorylations in the transactivation domain result in an increase in p65-mediated transcriptional activation and in the case of serine 536 phosphorylation, at least, a reduction in affinity for IkBa [91]. Which isoform of IKK catalyses this reaction appears to depend on the activating stimulus [104]. PKCz downstream of Ras and PI 3-kinase has been implicated in the phosphorylation of both p65 and c-Rel [99, 105]. TBK1 has been put forward as an IKK kinase [106] but results of genetic studies suggest that it acts downstream rather than upstream of IKK [107]. Indeed, a complex of TBK1 with NAK-associated protein 1 has been found to phosphorylate p65 on serine 536 in response to TNFa [105]. PKG phosphorylates p65, p52, and p50, thus increasing transactivation by NF-kB. GSK-3b has also been implicated in p65 phosphorylation [108] but the evidence is still sketchy as is the evidence for tyrosine phosphorylation of NF-kB. The roles of acetylation and ubiquitinylation Apart from phosphorylation, acetylation and ubiquitinylation have been demonstrated to be employed in the control of NF-kB activity [2, 104]. Both p50 and p65 are reversibly acetylated on multiple lysine residues by p300 and CBP. Deacetylation is catalysed by HDAC3. Acetylation of p65 lysine residues regulates DNA binding (lysine 221) and transactivation (lysine 310) positively, and association with IkB and nuclear export negatively (lysines 218 and 221). Similarly, acetylation of lysines 431, 440 and 441 in p50 has been found to increase DNA-binding and transactivation by heterodimeric NF-kB. Acetylation of lysines 122 and 123, in contrast, has been reported to diminish p65 DNA binding. Apparently, ubiquitin in the NF-kB signal transduction pathway can be more than just a tag marking IkB for degradation by the proteasome. Ubiquitinylation of the non-catalytic, regulatory subunit IKKg in response to TNFa has been reported to be important for IKK activation by the cytokine and to be mediated by c-IAP1, an inhibitor-of-apoptosis protein which acts as ubiquitin protein ligase, and whose gene is regulated by NF-kB [109]. In B and T lymphocytes, antigenic stimulation has also been found to result in IKKg ubiquitinylation required for NF-kB activation. This is mediated by the Bcl10 protein, which is known to interact with TNF-receptor-associated factors (TRAFs) and c-IAPs [110]. A similar mechanism involving transient sumoylation, the attachment of the ubiquitin-like SUMO-1 protein, triggering nuclear translocation

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and ubiquitinylation of IKKg, has been reported to exist in genotoxic stressinduced IKK activation [111]. These, as well as similar findings, demonstrate that ubiquitinylation can control not only protein fate, by marking proteins for degradation by the proteasome or the lysosome, but also protein function. Nuclear IKKg has been found to have a function in the regulation of NF-kBdependent gene expression [112]. It competes with p65 for binding to CBP thus inhibiting p65-mediated transactivation. In addition to IKKg, IKKb is ubiquitinylated in response to TNFa, and also to HTLV Tax [113]. A single ubiquitin molecule appears to be attached after phosphorylation of the kinase on serines 177 and 182 in the activation loop of the catalytic domain. This monoubiquitinylation of IKKb is dependent on the presence of IKKg and is thought to regulate the activity of the kinase. Polyubiquitinylation of IkB as well as IKKg and monoubiquitinylation of IKKb are not the only ubiquitinylation events which play a role in the activation of NF-kB. The catalytic activity of TAK1, a MAP3K (mitogen-activated protein kinase kinase kinase) family protein serine/threonine kinase upstream of IKK in IL-1/Toll-like and TNFa receptor signalling, is ubiquitinylation dependent [114, 115]. However, in this case, it does not appear to be the kinase itself but TRAF6, TAB2, and TAB3, a ubiquitin ligase and two adaptor proteins, respectively, linking the kinase to the receptors, which are ubiquitinylated [116]. Suppression of TAK1 expression by siRNA only incompletely inhibits IL-1 and TNFa-induced NF-kB activation suggesting that other kinases may be able to compensate for the loss of TAK1 [115, 117]. Indeed, two other MAP3Ks, MEKK2, and MEKK3, have been found to activate IKK in response to IL-1 and TNFa [118]. This is a further example of redundancy in the network of kinases employed in NF-kB activation. Multiple kinases appear to co-operate in the activation of IKK, and IKK itself is not the only kinase acting to induce IkB degradation. While MEKK3 stimulates IkBa phosphorylation by IKK resulting in rapid but transient NF-kB activation, MEKK2 promotes IKK-induced IkBb degradation producing delayed but persistent activation of the transcription factor. These two kinases, therefore, appear to be instrumental in bringing about the biphasic response observed for NF-kB activated by stimuli such as proinflammatory cytokines or LPS.

REACTIVE OXYGEN SPECIES AS SECOND MESSENGERS IN NF-kB ACTIVATION – TRUE OR FALSE?

Results of a recent study have rekindled a long-standing debate as to whether ROS have a second messenger function in NF-kB activation or not [119]. The argument has been made that it is difficult to envisage how any signalling protein in the NF-kB pathway could be activated by the highly reactive and nonspecific putative ROS second messengers. This alone is, of course, insufficient to

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criticize the notion that ROS activate NF-kB since a similar argument can be made for ionizing radiation as inducer of NF-kB activation. However, the authors have demonstrated that two antioxidant compounds, N-acetyl-L -cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), used previously to establish if ROS have a role in NF-kB activation, exert their effect on the transcription factor by a different, ROS-independent mechanism of action. Moreover, evidence has been put forward suggesting that ROS produced by NADPH oxidase in response to TNFa or TPA are not required for the activation of the transcription factor – at least in the HeLa cells used in this study, which do not belong to the monocytic cells for which NF-kB activation by NADPH-generated ROS had previously been observed [29]. Instead, HeLa cells are epithelial cells which have been reported not to possess this mechanism of NF-kB activation. This, together with the fact that not all evidence for a role for ROS in NF-kB activation is derived from experiments in which NAC and PDTC were employed, makes it appear unlikely that these results will settle the debate around ROS in NF-kB signalling. In this context, it is intriguing that expression of xanthine oxidoreductase, a housekeeping enzyme of purine catabolism catalyzing the synthesis of uric acid, which as an antioxidant helps to protect cells from the damaging effects of reactive oxygen and nitrogen species, has been proposed to be regulated by NF-kB [120]. It appears that the most appropriate stimulus for activating inducible synthesis of an enzyme involved in the detoxification of oxygen radicals would be those radicals themselves.

DRUG TARGETS WITHIN THE NF-kB SIGNALLING NETWORK – OLD AND NEW

Currently, among all the potential drug targets within the signal transduction pathways leading to NF-kB activation, IKKb appears to be the first and foremost choice of the pharmaceutical industry [121, 122]. This is because IkB degradation, triggered through phosphorylation by this kinase, has long been regarded as the critical step in the activation of the transcription factor. Proteasome inhibitors targeting the next step in NF-kB activation, i.e. the proteolytic degradation of IkB, are highly likely to exert other effects contributing to their therapeutic efficacy. Until very encouraging clinical trials results had been released for Velcade (bortezomib, PS-341) (36) [123], many drug discovery scientists were highly sceptical about the safety of such compounds because of predicted pleiotropic effects of proteasome inhibition. Inhibitors of GSK-3b which are being investigated at many pharmaceutical companies [124], may in the future also be developed as anti-inflammatory drugs because this kinase is now known to be required for TNFa-induced NF-kB activation, but they are not primarily seen as compounds for the inhibition of this transcription factor.

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Another strategy which some pharmaceutical companies are pursuing is the discovery of inhibitors of NF-kB transactivation using cellular assays for NFkB-dependent reporter gene expression. Compounds active in these assays, may, of course, target any one, or even several, of the signalling proteins involved in NF-kB activation. In fact, it appears quite possible that the best strategy for the inhibition of NF-kB with respect to potential unwanted side effects may be the targeting of more than one step in the activation of the transcription factor with small molecules showing limited potency against each individual target. This is because IKKb is required for the phosphorylation of proteins outside the canonical NF-kB signal transduction pathway such as IRS-1, and recent findings suggest that phosphorylation of IkB on serine residues by IKK alone may neither be sufficient, nor even necessary in all situations, for the degradation of IkB (see above). Highly potent inhibition of IKKb may, therefore, have unwanted side effects and may only inhibit IkB degradation to a limited extent. A similar argument concerning an a priori, mechanistic limitation of the maximum possible efficacy of specific inhibitors applies to the targeting of one of the three MAP3Ks: TAK1, MEKK2, and MEKK3, acting upstream of IKK in IL-1 and TNFa-induced NF-kB activation. However, it might be therapeutically desirable to inhibit say MEKK2 rather than MEKK3 and consequently persistent instead of transient activation of NF-kB. This is because certain target genes, such as the one encoding the chemokine RANTES appear to require persistent activation of the transcription factor for their expression [125]. Rapid and transient activation is bound to induce a subset of NF-kB-regulated genes needed for an immediate response to transient stress exposure, whereas persistent activation of the transcription factor most likely stimulates expression of genes specifically involved in dealing with more permanent stress, such as infection, or of genes involved in NF-kB-driven cell differentiation [126]. Compounds which act on multiple protein kinases involved in NF-kB activation, upstream and/or downstream of IKKb, with relatively modest potency may produce less side effects, but may still inhibit NF-kB very potently due to amplification of their effects along the pathway from the NF-kB-activating signal at the plasma membrane to the transcription factor in the nucleus. Extensive profiling of compounds identified in NF-kB transactivation screens with respect to kinase inhibition may help identify such promising combinations of target kinases and molecular modelling may aid in the optimization of interesting compounds by chemical modification provided that the structures of the relevant kinases can be obtained. An additional reason for looking for inhibitors of protein kinases within NF-kB signalling pathways other than IKK, is the potential for the identification of compounds which do not affect the expression of a majority of NF-kB-dependent genes, but only that of a specific, desired subset. This may be especially true for kinases acting downstream of IKK, which are thought to play a part in

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the differential regulation of the DNA-binding and/or transactivation activity of different Rel family members, since it has become clear that the various Rel proteins possess distinct expression patterns, responses to signals, and target genes. Furthermore, their specificity for certain promoters has been found not to be determined by the binding site sequence alone [127]. Differential modulation of the activities of these kinases may thus provide a basis for greater specificity of drug action. In this context, it is very interesting that MSK1, another member of the MAPKAPK family, regulated by the MAP kinases ERK (extracellular signalregulated kinase) and p38, has been proposed to specifically promote the expression of the proinflammatory subset of NF-kB-regulated genes [101, 128]. There is also the possibility that inhibition of protein kinases acting upstream of IKK may result in the inhibition of NF-kB activation in response only to specific stimuli and/or only in specific cell types. PKCu, an nPKC (novel PKC) isoform highly expressed in T cells, for example, has been found to be specifically involved in TCR-induced IKK activation [129]. T cells from a PKCu-deficient mouse strain show complete disruption of TCR-mediated NF-kB activation, while TNFa or IL-1 induced stimulation of the transcription factor remains unimpaired [130]. However, another strain of PKCu knockout mice, which was generated by using a different targeting vector, show only very limited reduction in TCR-induced NF-kB activity while activation of NFAT in response to TCR stimulation is completely blocked in the T cells of these mice. As a possible explanation, generation of a truncated form of PKCu acting as a dominant negative protein in the first mouse strain and blocking signal transduction via this, as well as via other isoforms of PKC, has been suggested [131]. In the second strain, other PKC isoforms may be able to compensate for the lack of PKCu in TCR-induced NF-kB but not NF-AT activation. While this issue has not been resolved, targeting of PKCu for the specific inhibition of NF-kB in activated T cells remains a strategy with an uncertain chance of success. Two other upstream kinases, IKK1 and TBK1, which are being considered as drug targets by some scientists, have been shown to be critical not only for NF-kB but also for interferon regulatory factor 3 activation required for interferon b expression and consequently antiviral immune responses [132, 133]. Systemic inhibition of these kinases is, therefore, feared to allow pathogenic viruses to replicate and spread throughout the patient’s body. Another class of drug targets with potential application in the inhibition of NF-kB, which is already being actively pursued by three biotech companies, i.e. Rigel Pharmaceuticals, Celgene, and Proteologics, are the E3 ubiquitin ligases [134, 135]. The human genome contains several hundred genes for such enzymes, each of which ubiquitinylates a specific, limited set of substrates, thus providing an opportunity for the discovery of relatively specific drugs inhibiting only a limited number of ubiquitinylation events.

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Targeting SCF(b-TrCP), the ligase which ubiquitinylates IkB [136], or those as yet unknown E3 enzymes which ubiquitinylate IKKg or IKKb would be an obvious strategy for NF-kB inhibition. Greater specificity in terms of inhibition of NF-kB activation in response to specific stimuli may be achievable by aiming at TRAF6 and other ligases catalyzing the ubiquitinylation of the adaptor proteins that link TAK1 to the different Toll-like and cytokine receptors which signal through this kinase. However, since other protein kinases can compensate for IKK or TAK1 inhibition, the potential of these strategies seems limited. Although there are already reports of impending clinical trials, no chemical structures of the small molecule E3 ubiquitin ligase inhibitors have so far been released. One concern with inhibition of the E3 ligase ubiquitinylating IkB is that this may promote neoplastic transformation, since this enzyme also appears to be involved in the proteolytic degradation of b-catenin [137]. The same applies to the targeting of GSK-3 (see below) and, in essence, also to the inhibition of TAK1 because this kinase, although not involved in b-catenin degradation, negatively regulates b-catenin transactivation activity [138]. DISCOVERY AND DEVELOPMENT OF SMALL MOLECULE NF-kB INHIBITORS INHIBITORS OF PROTEIN KINASES INVOLVED IN NF-kB ACTIVATION

Inhibitors of protein serine/threonine kinases came into focus within the pharmaceutical industry in the 1980s after the bacterial indolocarbazole staurosporine was discovered to inhibit PKC at nanomolar concentrations. When the involvement of activated or overexpressed growth factor receptor protein tyrosine kinases in many forms of cancer was recognized around the same time, the pharmaceutical industry started to seriously consider protein kinases as drug targets and the first tyrosine kinase inhibitors, natural products such as genistein as well as synthetic benzene malononitriles, were soon identified. While the latter compounds are competitive with the substrate, most tyrosine kinase inhibitors synthesized later are ATP-competitive. These molecules belong to chemical classes including anilido phthalimides, pyrazolo pyrimidines, pyrido pyrimidines, pyrrolo isatins, quinoxalines, and quinazolines. While staurosporine was subsequently found to be unspecific, later indolocarbazoles, such as 7-hydroxystaurosporine (Kyowa Hakko UCN-01) (1) have progressed to the clinical phase of development [139]. Other classes of serine/ threonine kinase inhibitors in development, which are also almost exclusively ATP-competitive, include imidazoles, bisindolylmaleimides, pyrazolo triazines, quinazolines, and quinolines [140].

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Despite there now being a whole host of protein kinases which have been identified as regulators of NF-kB activation, and which, therefore, constitute potential drug targets, drug discovery research has so far been focusing on IKK [121, 122, 141]. It is true that GSK-3b is also being hotly pursued, but not primarily with treatment of NF-kB-related pathologies in mind. Following the approval of the first kinase inhibitor drugs, Fasudil, rapamycin, and Gleevec (2), the question has been raised if protein kinases may become the major class of drug targets in the 21st century [142]. Although there is, indeed, an increasing number of protein kinase inhibitors in clinical trials, major obstacles still need to be overcome. The problem is not, as originally thought, that kinase inhibitors with sufficient selectivity cannot be developed because protein kinases show conservation of the amino acid residues critical for their catalytic activity. Instead, it is becoming more and more clear that absolute specificity, which appears unlikely to be achievable, anyway, may not really be desirable. This is because inhibition of kinases other than the one which was originally targeted may open up avenues for clinical development for other indications, such as in the case of the Bcr – Abl inhibitor Gleevec (2) which was originally aimed at the platelet-derived growth factor receptor and whose inhibition of the closely related c-Kit makes it a promising drug for the treatment of gastrointestinal stromal tumours [143]. Moreover, similar to some long-established drugs such as aspirin, the clinical efficacy of certain kinase inhibitors, such as UCN-01, is most likely due to the inhibition of multiple targets, not necessarily all kinases. A major problem in the discovery of kinase inhibitors with cellular and in vivo activity is that most compounds coming out of high-throughput screening campaigns using assays for the inhibition of in vitro activity of recombinant enzyme, often against a substrate which is not the natural one, are ATP competitive. De novo designed inhibitors are also usually based on the ATP binding site. This means that once inside a cell, if indeed they are cell permeable, they most likely have to compete with an ATP concentration significantly above the Km for ATP of their target because for many protein kinases, this value lies in

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the low micromolar range while cellular ATP concentrations are roughly a thousand times higher.

Since many kinases are organized into cascades of sequential activation by phosphorylation leading to an amplification of the signal, inhibition of multiple kinases at different points in the pathway by a small molecule with modest selectivity and potency may have a result comparable to the specific inhibition of only one kinase with high potency due to an additive or even superadditive, synergistic effect. How inhibitors of just the right set of kinases may be identified in a rational rather than a serendipitous fashion is as yet difficult to envisage unless these kinases all happen to belong to the same family. It may, however, in the future be possible to design such compounds once the three-dimensional structures of all the 500 plus kinases in the human kinome [144], and a complete and accurate wiring diagram of the cellular regulatory circuitry, have been obtained. This is still science fiction, though. In the case of Gleevec, the Novartis researchers who discovered the drug were fortunate that, while being ATPcompetitive, it binds in a way that induces the Bcr–Abl kinase domain to adopt an inactive conformation resulting in increased specificity and potency [145]. Another compound, the Boehringer Ingelheim p38 MAP kinase inhibitor BIRB796 (doramapimod) (3), has been reported to act in a similar way by binding to both an allosteric site outside, and to amino acid residues inside the ATP binding pocket [146]. The Parke-Davis compound PD98059 (4), which is not ATPcompetitive, binds to the inactive form of MAP kinase kinase 1, thus preventing its conversion to the active form induced by phosphorylation [147]. Moving to an assay format in which inhibition of conversion to an active conformation rather than inhibition of an already active enzyme is assayed may allow the discovery of more kinase inhibitors with this mechanism of action. However, such compounds are unlikely to work if the target is constitutively active as is the case for many oncogenic kinases. The aim of inhibiting IKK, and consequently NF-kB, in inflammatory disease, is to reduce production of proinflammatory cytokines. It is true that p38 inhibitors [148, 149], which are being actively pursued by numerous pharmaceutical companies, can do the same job. They block the production of such cytokines apparently by negatively affecting both the transcription of their

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genes, most likely via reduced MSK1 and, as a result, NF-kB activation (Guy Haegeman, personal communication), and the translation of their messenger RNAs. However, despite having been investigated for more than a decade and many compounds having entered clinical trials, no approvable p38 inhibitor drugs have so far been forthcoming [150]. Consequently, inhibitors of kinases activating NF-kB are still well worth pursuing.

IKK inhibitors Derivatives of the alkaloid b-carboline were among the first IKK inhibitors to be discovered. Starting from the methylated derivative 5-bromo-6-methoxy-bcarboline (5), which had been identified as a non-specific inhibitor of IKK, Millennium Pharmaceuticals, in collaboration with Aventis, has investigated the structure –activity relationship (SAR) of b-carbolines with the aim to optimize potency and selectivity [151]. By varying the substituents of the A ring, they found that electrophilic groups in position 6 increased potency. 6,8-Dichloro-bcarboline, a by-product of the synthesis of the monochlorinated analogue, showed a 5-fold increase in potency and a more than 20-fold improved selectivity over the original compound but was poorly soluble in water preventing its evaluation in cellular assays. To achieve greater solubility, ether groups were introduced at

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position 7 resulting in more soluble, potent, and selective IKK inhibitors, with the most potent compound in this series showing greater than 200-fold selectivity for IKK versus CK2, PKA, and PKC. This data, however, is too limited to allow the conclusion that the compound is specific for IKK since b-carbolines have been described as inhibitors of other enzymes including monoamine oxidase A [152], CDKs [153], and, more worryingly, cytochrome P450 [154] at nanomolar concentrations. 6-Chloro b-carboline derivatives substituted at position 8 with amides, sulfonamides, ureas, carbamates or substituted amines were also synthesized. With the exception of anilines, these compounds showed much reduced activity towards the other three kinases suggesting that hydrophobic groups in positions 7 or 8 conferred selectivity for IKK. One of these analogues, named PS-1145 (6), was tested in cellular assays [155, 156]. Immune complex kinases assays performed on lysates of TNFa-stimulated HeLa cells showed inhibition of endogenous IKK (IC50 ¼ 150 nM). Moreover, IkB phosphorylation and NF-kB DNA binding in these cells were found to be inhibited in a dose-dependent manner. In vivo, the compound reduced LPSinduced TNFa production in mice by 60% at 5 h post-challenge when given orally at 50 mg/kg 1 h prior to LPS administration. In multiple myeloma cells (MMCs), (6) inhibited TNFa-induced phosphorylation of IkB, NF-kB activation, and ICAM-1 expression. The compound also inhibited proliferation and promoted TNFa-induced apoptosis in these cells. Furthermore, (6) reduced secretion of IL-6, a growth factor for MMCs, by bone marrow stromal cells (BMSCs) in co-cultures of the two cell types. (6) was also investigated for the treatment of transplant rejection in a mouse heterotopic cardiac transplant model in which oral administration at 25 mg/kg/day for 14 days delayed graft rejection from 7 to 30 days. Millennium has since reported to be pursuing other, so far undisclosed structures, designated MLN-120A and B [155], active in cellular assays for cytokine production and a rat model of adjuvant-induced arthritis.

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Aventis, in collaboration with pharmacologists at Frankfurt University, has recently published that the nanomolar IKK inhibitor (IC50 ¼ 10 nM) S1627, whose structure has not been disclosed, reduces hyperalgesia in rat models of inflammation and neuropathic pain [157]. The compound was found to prevent zymosan-induced nuclear translocation of NF-kB in the spinal cord. The authors conclude that IKK may be a promising target for the treatment of pathological pain. Bayer Yakuhin identified 2-amino-3-cyano-4-aryl-6-(2-hydroxyphenyl)pyridines as potent and selective inhibitors of IKKb [155, 156, 158]. Their lead compound (7) inhibits IKKa and a few other tested kinases to the same extent as IKKb (IC50 ¼ 1.5 mM) only at more than 10 times higher concentrations. Moreover, this molecule inhibited NF-kB-dependent gene expression in cellular assays at low micromolar concentrations. The compound was optimized during SAR studies in which, initially, the phenyl group in position 4 of the pyridine moiety was substituted with hydrophilic groups to increase aqueous solubility. Introduction of a basic amino or a carboxylic acid function at the 20 or 30 position increased inhibition of the isolated, recombinant enzyme but most compounds carrying the latter substituent lost activity in a cellular assay for TNFa-induced RANTES production. If the 40 position was modified, cellular activity was lost. Modification of the phenol group in position 6, as well as replacement or substitution of the amino group in position 2 of the pyridine, resulted in loss of either or both of the monitored activities. While the original lead lacked oral availability in LPS-induced TNFa production in mice, the more hydrophilic analogues showed high potency when administered either orally or intraperitoneally. A piperidinyl analogue (8), which was the most active compound in vivo while maintaining selectivity for IKKb, was selected for further investigation. Replacement of the substituted phenyl at position 4 of the pyridine ring with piperidine (9) considerably increased inhibition of IKKb and cellular cytokine production, but bioavailability was poor [159, 160]. Modification of

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the ortho-phenol group of this 4-piperidin-3-yl-pyridine derivative by synthesis of 60 -alkoxy analogues produced unstable compounds. However, when a more stable 4-piperidin-4yl-pyridine analogue (10), which showed only modest inhibition of IKKb but potent cellular activity, was modified in the same way, activity against the kinase and cellular potency was markedly increased. A cyclopropyl analogue (11) was potently active in assays for NF-kB (IC50 ¼ 147 nM) but not AP-1 or NF-AT-dependent reporter gene expression and in arachidonic acid-induced ear oedema formation in mice at 1 mg/kg (p.o.). This is seen as evidence for selective inhibition of NF-kB transactivation activity by inhibition of IKKb. However, the 6’-hydroxy derivative of the 4-piperidin-3ylpyridine (12) was reported to have similar potency against the kinase but 5-fold lower potency in TNFa-induced RANTES production in A549 cells. Simulation results suggest that this compound shows comparable permeability (Mark Noppe, personal communication). Therefore, one could also argue that the increased cellular activity of the 4-piperidin-4-yl-pyridine analogue (11) may be a consequence of the inhibition of additional components of the NF-kB signal transduction pathway.

Apart from the compounds described in some detail above, there is a much larger number of IKK inhibitors for which no SAR data have been released. Many of them are described in a recently published review article by Michael Karin, one of the leading investigators of NF-kB regulation, and scientists at Lilly Research Laboratories [122, 155, 156]. These compounds will only be mentioned briefly here. Among the most interesting of these is Bristol-Myers Squibb’s BMS-345541 (13), an imidazoquinoxaline derivative which inhibits IKK by binding to an allosteric site outside the ATP-binding pocket and which is, therefore, not ATP-competitive. This compound reduces LPSinduced cytokine production as well as collagen-induced joint inflammation and destruction in mice.

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Scientists at Signal Pharmaceuticals, now a subsidiary of Celgene, were among the first to purify the IKK complex and to identify its two principal components IKKa and b. Soon after this, they began to identify IKK inhibitors in collaboration with Serono. SPC-839 (14), a quinazoline analogue, also known as AS-602868, which was derived from an earlier compound, SPC-495, is over 200 times more active against IKKb (IC50 ¼ 62 nM) than a, inhibits NF-kB transactivation and cytokine production in Jurkat cells as well as TNFa production and paw oedema formation in rats. The compound also has significant activity against Jun N-terminal kinase 2 (JNK2) (IC50 ¼ 600 nM). Moreover, it has recently been reported to alleviate cachexia and kidney dysfunction in a murine model of human acquired immune deficiency syndrome (AIDS). Another series of IKK inhibitors, they have been investigating are 4-(pyrimidine-2-ylamino)benzamide derivatives (15) which are also active against JNK2 albeit with lower potency.

GlaxoSmithKline and AstraZeneca have both been investigating 2-aminothiophene 3-carboxamide derivatives (16). They have claimed virtually

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identical molecules. Compounds in this class are selective for IKKb, inhibit cytokine production in human cells and mice as well as paw oedema formation in rats. Furthermore, GlaxoSmithKline has found structurally similar 4-aminoimidazole carboxamide IKK inhibitors (17). AstraZeneca has recently disclosed the results of their SAR analysis of similar 3-aminothiophene 2-carboxamide derivative IKK inhibitors (18) [161]. Alkylation of urea or amide nitrogens caused significant reduction of activity. Replacement of the thiophene core with imidazole or pyrazine had the same effect. Substitution of the phenyl substituent resulted in no improvement in activity against the isolated kinase or in a cellular assay for TNFa production. The metabolically more stable and orally active 4-fluorophenyl derivative was selected as starting point for lead optimization.

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In addition to the diarylpyridine derivatives described above, Bayer has claimed optically active pyridooxazinones (19) one of which inhibits IKKb in vitro with an IC50 value of 4 nM and reduces cytokine production in cells, mice and rats. Compounds in this class have also been reported to be active in assays for LPS-induced TNFa production in mice. Aventis has disclosed indole (20) as well as benzimidazole carboxamide derivative IKK inhibitors. The most active ones have nanomolar IC50 values. Tularik, now Amgen, in collaboration with Roche, has discovered imidazolylquinolinecarboxaldehyde semicarbazones (21) selective for IKKb with anti-inflammatory activities in mice. Pharmacia, now part of Pfizer, has discovered pyrazoloquinoline derivatives with high nanomolar activity against IKK and Leo has identified orally available pyridyl cyanoguanidine antitumour compounds which were later

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shown to inhibit IKK (IC50 ¼ 8 nM) [162]. One of these compounds, CHS-828 (22), is in phase I/II clinical trials for the treatment of solid tumours. Following are some more recently disclosed IKK inhibitors [155, 156]. Roche has found substituted pyridines and pyrimidines (23) with high nanomolar activity against isolated recombinant IKK. A further product of the Tularik (Amgen)/Roche collaboration is a novel series of quinoline (24) and quinazoline derivative IKK inhibitors. Pfizer has described substituted indazoles (25) as well as tricyclic pyrazole derivatives (26) which selectively inhibit IKKb GlaxoSmithKline has disclosed novel tricyclic aminothiophene inhibitors of IKKb (27). Boehringer has reported a series of 3-aminothieno[2,3b ]pyridine-2-carboxamide derivatives (28).

GSK-3 inhibitors First identified in 1980 as one of several cytoplasmic serine/threonine kinases able to phosphorylate and inactivate glycogen synthase, GSK-3 has since been found to be much more than just another kinase regulating glycogen synthesis [163 – 165]. This enzyme, which exists as two isoforms, a and b, is now known as a key player in the control of numerous transcription factors, NF-kB being the most recent addition to the list [166]. With the discovery that GSK-3b is required for the activation of NF-kB by TNFa came the realization that the kinase, already hotly pursued in antineurodegenerative and antidiabetic drug discovery, may also serve as target for anti-inflammatory drugs [167]. In this context, it is especially interesting that, unlike in Wnt/b-catenin signalling, GSK-3a, is unable to compensate for a loss of GSK-3b activity in TNFa-induced NF-kB activation. Thus, provided that an inhibitor highly selective for the b isoform of GSK-3 can be found, targeting this kinase will not invariably be fraught with the danger of promoting tumourigenesis by interfering with b-catenin degradation. However, the high conservation of the ATP binding site between the two isoforms (identity of 53 out of 54 surrounding amino acid residues) makes it appear unlikely that this region of the kinase can serve as target for such still hypothetical isoform-specific GSK-3 inhibitors. If they can be found, there is, at least in principle, a chance that specific inhibitors of GSK-3b may yield the sought-after small molecule inhibitor of TNFa given that the proinflammatory effects of this cytokine are thought to largely depend on NF-kB activation. In this context, it is interesting that it has been possible to find compounds, which inhibit GSK-3 but not CDKs whose catalytic domains are very similar. Moreover, while a number of GSK-3 inhibitors have indeed been shown to induce b-catenin stabilization in transformed cell lines, one of the most potent and specific compounds

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identified so far failed to do so when tested in rats, suggesting that this may only occur in cells with certain pre-existing genetic abnormalities [164]. Pharmaceutical companies which have reported extensive GSK-3 inhibitor discovery programs are Chiron, Novo Nordisk, Sanofi in collaboration with Mitsubishi Pharma, Vertex, and the aptly named GSK (GlaxoSmithKline) [155, 156]. In addition, there are several academic research laboratories which are looking for inhibitors of this kinase and the compounds which they have identified include natural products such as indirubins, which have been recognized as the active ingredient of a traditional Chinese medicine used to treat CML [168], and the marine natural product hymenialdisine [169]. The large majority of the GSK-3 inhibitors which have been studied are ATPcompetitive but at least one series of compounds, 2,4-disubstituted thiadiazolidinones, have been claimed to inhibit the kinase by an ATP-uncompetitive mechanism [170]. Since GSK-3 inhibitors are currently being developed primarily for the treatment of Alzheimer’s disease and type 2 diabetes mellitus, little information on their behaviour in models of inflammatory disease is as yet publicly available. It is, therefore, difficult to say if they have the expected inhibitory effect on the activation of NF-kB by TNFa and, if indeed they do, whether this will result in unacceptable adverse effects caused by the proapoptotic pathway activated by this cytokine becoming dominant. It is quite possible that in order for a small molecule to be able to safely block TNFa-induced inflammatory processes to the same extent as the anti-TNFa antibodies currently used in the treatment of rheumatoid arthritis, it will have to block both the antiapoptotic, proinflammatory TNFa signal dependent on NF-kB and the proapoptotic signal leading to caspase 8 activation. It remains to be seen if a GSK-3b inhibitor, which does not significantly affect GSK-3a activity, and which does not promote TNFa-induced apoptosis, will be discovered. So far, such a compound is not in sight. NIK inhibitors NIK is a MAP3K necessary for IKKa activation by stimuli such as BAFF (see above) which regulates B-cell survival and maturation. It has been suggested that selective inhibition of this IKK isoform may, therefore, be beneficial in lupus erythematodes, in which BAFF levels have been found to be elevated, and related B-cell mediated autoimmune diseases. However, so far, no selective IKKa inhibitors have been reported. Inhibition of NIK can be expected to have a significant and fairly specific effect on IKKa activity, B-cell maturation, and progression of lupus-like autoimmune diseases because, although this MAP3K has been reported to also activate IKKb gene disruption analysis has shown

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that it is not required in the canonical pathway. Until the recent disclosure by Aventis of a series of pyrazolo[4,3-c ]isoquinoline NIK inhibitors (29), only Signal Pharmaceuticals, in collaboration with Serono, had announced their interest in this kinase as a drug target, but no structures have been disclosed by them or any further development reported [155, 156]. Compound (29) has been found to inhibit LPS-induced TNFa, IL-6, and IL-1 release from human peripheral lymphocytes as well as TNFa and IL-1 production in whole blood with half-maximum effects at low micromolar concentrations without causing significant cytotoxicity.

INHIBITORS OF NF-kB DNA BINDING

As a matter of course, one way of effectively inhibiting NF-kB is preventing its binding to DNA. Although, so far, no NF-kB DNA binding inhibitors have been reported to be in development at pharmaceutical or biotech companies, such compounds have been identified by academic research groups to be among the active ingredients of alcoholic medicinal plant extracts used in traditional Mexican Indian medicine for the treatment of inflammatory disease [171]. Sesquiterpene lactones, such as helenalin (30) and parthenolide (31), have been shown to covalently modify p65 by alkylating cysteine 38, consequently inhibiting its ability to bind to DNA. Complete inhibition of p65 DNA binding was observed at concentrations of 10 and 20 mM, respectively, in electrophoretic mobility shift assays [172]. Moreover, (30) has been shown to potently inhibit the transcription of genes encoding cytokines including IL-6 and TNFa at this concentration [173]. (31) was originally reported to exert its anti-inflammatory activity via inhibition of IKK [174] but this observation could not be confirmed [172]. Closely related helenanolide-type sesquiterpene lactones, such as chamissonolide, which do not interfere with NF-kB DNA binding, also inhibit cytokine gene expression, albeit less potently. It, therefore, appears that additional activities contribute to the anti-inflammatory effect of this class of compounds. Complete inhibition of NF-kB DNA binding has also been found to

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occur upon addition of selenite at a concentration of 7 mM to human T and adenocarcinoma cells [175]. This is thought to be caused by adduct formation with thiol groups of the transcription factor. This reaction, together with the activation of selenium-dependent glutathione peroxidase, which converts NFkB-activating hydrogen peroxide to water, is thought to be the mechanism by which selenite prevents eosinophil infiltration in a mouse model of asthma. As part of an effort to find inhibitors of LTR-directed HIV gene expression, metalchelating compounds have been identified as inhibitors of NF-kB DNA binding [176]. The most potent one among them, active at micromolar concentrations, is aurine tricarboxylic acid (32).

NF-kB TRANSACTIVATION INHIBITORS WITH UNKNOWN OR UNDISCLOSED MECHANISM OF ACTION

Since NF-kB is a transcription factor, an obvious way of screening for inhibitors of its activity are assays for NF-kB-dependent reporter gene expression. With the recent introduction of high-throughput screening platforms for eukaryotic cells [177], chemical libraries containing several hundred thousand small molecules can be screened in such cellular assays almost as rapidly as in activity assays for individual, isolated target proteins. One of the advantages of such NFkB transactivation assays is the potential for yielding inhibitors of all of the different druggable targets within the NF-kB signal transduction network. The major disadvantage is that it is not known a priori against which one of these targets a given hit is active, and that identification of the molecular target(s) of such a compound often requires considerable additional effort. However, hits which came out of HTS campaigns against isolated targets frequently show cellular and/or in vivo activities which cannot be explained by the predicted mechanism of action, and therefore, also require further investigation. Moreover, it is becoming increasingly clear that for pathological processes, which are governed by complex intracellular events, targeting of a single cell component

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with a small molecule may not be sufficient for achieving maximum therapeutic efficacy. Cellular screening assays provide an opportunity for the discovery of compounds which act on multiple disease-relevant proteins. Scientists at Sumitomo Pharmaceuticals have identified 6-aminoquinazoline derivatives as a novel class of inhibitors of NF-kB dependent gene expression showing low nanomolar potency [155, 156, 178]. Compounds in this series were found to be highly active in a reporter gene transactivation assay (luciferase gene expression from an artificial promoter containing five consecutive NF-kB binding sites in Jurkat cells stimulated with a combination of PMA and phytohemagglutinin (PHA)), in an assay for LPS-induced TNFa production in mouse splenocytes, and in a rat model of rheumatoid arthritis. Extensive SAR analysis of this class of compounds was performed. Variation of the length of the spacer between the quinazoline ring and the 4-chlorophenyl group in position C-4 has shown that inhibition of NF-kB-dependent reporter gene expression or TNFa production is maximized when the spacer is the ethylene chain. To determine the contribution of the quinazoline ring, it was replaced with quinoline, isoquinoline, or phthalazine. All of these analogues exhibit much reduced potency. Investigation of the effect of C-6 and C-7 substituents on the quinazoline ring has shown that the presence of a basic nitrogen in position C-6 was required for maximum inhibitory activity. Replacement of the hydrogen in position C-7 with a chloro, amino or nitro group resulted in reduced activity. Since replacement of the terminal phenyl ring in position C-4 of the quinazoline diminished activity, further lead optimization was achieved by its substitution with alkoxy groups in the para position. This led to the n-pentyloxy analogue (33) as the most potent compound, with IC50 values of 2 and 3 nM in the transactivation and the TNFa production assay, respectively. Earlier efforts at Signal Pharmaceuticals to identify inhibitors of both NF-kBand AP1-driven gene expression in Jurkat cells have yielded several pyrimidinebased molecules with similar nanomolar activities in both assays [179 –182]. Both transcription factors, either alone or in combination, regulate the expression of a large number of proinflammatory genes, and, therefore, AP-1 is also a potential target for anti-inflammatory drugs [183]. In a more recent publication, they describe quinazoline analogues in collaboration with Tanabe [155, 156, 184]. Extensive SAR analyses to increase potency and achieve oral availability have been performed on these compounds and published. Only the latest results will be reviewed here. Interestingly, the quinazoline analogues include SPC-839 (14), which has previously been described as an inhibitor of IKK (see above). Until the recent publication of a mechanism for NF-kBdependent AP-1 activation stimulated by the non-physiological doxycycline or, to a lesser extent, serum (20%) [185], it was difficult to envisage how the activity of this compound on AP-1-stimulated transcription could be due to IKK

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inhibition. However, inhibition of AP-1-dependent reporter gene expression, usually induced in these experiments with PMA or PMA/PHA, is not a common feature of potent and supposedly selective IKKb inhibitors suggesting that inhibition of this kinase alone cannot explain this activity of (14). Moreover, the fact that inhibition of the activity of both transcription factors apparently occurs with identical potency (IC50 ¼ 8 nM) suggests that there is another cellular target of this compound. It is true that JNK-2, which SPC-839 has been reported to inhibit in vitro, albeit 10 times less potently than IKKb, is a protein kinase involved in AP-1 activation, but it is difficult to envisage how this activity of the compound could produce the much more potent inhibition of AP-1-dependent reporter gene expression observed. Likely cellular targets, whose inhibition could be responsible for this effect, are kinases of the MEKK family, which can activate both MAP kinase pathways leading to AP-1 activation as well as IKK, and thus NF-kB.

The quinazoline analogues studied were derived from a substituted pyrimidine-5-carboxylate lead compound, which lacked oral availability and showed poor cell permeability, by fusing a phenyl ring onto the pyrimidine obtaining a quinazoline ring. Initially, the effects of a 20 -thienyl, phenyl, and trifluoromethyl substitution at position 2 of the quinazoline ring was tested in the transactivation assays. The thienyl derivative was the most active compound and was further modified by introduction of different substituents on the phenyl moiety, including methoxy groups at positions 5, 6, 7, and/or 8. The 5- and 6-methoxy derivatives proved to be between 10 and 100-fold more active than

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the other analogues on the Jurkat cells. The 2-trifluoromethyl analogue behaved in a similar fashion when substituted on the phenyl moiety, with the 5- rather than the 6-methoxy derivative being the most active compound in this series, but even these two derivatives inhibited transactivation at least 10-fold less than the corresponding methoxy analogues of the thienyl series. In the CaCO-2 cell permeability and the rat adjuvant-induced arthritis assays, of all analogues tested, only (14) showed acceptable cell permeability and significant reduction of paw swelling. Daiichi Suntory have identified the benzoquinone derivative SUN-C8079 (34) as an inhibitor of IL-1 and TNFa-induced NF-kB-dependent transcription in A549 human lung carcinoma cells and of LPS and TNFa-stimulated inducible nitric oxide synthase gene expression in murine macrophages. In mice, (34) protected against LPS-induced mortality (50% protection at 30 mg/kg) [155, 156]. ChemGenex Therapeutics has recently released information on a series of novel imidazoline NF-kB inhibitors (35) with nanomolar IC50 values. These compounds are being studied as potential chemosensitizing drugs in anticancer combination therapy. They are reported to enhance antitumour activity of the DNA-damaging compound cisplatin and the topoisomerase I inhibitor camptothecin in mice bearing RIF-1 murine fibrosarcoma [155, 156]. PROTEASOME INHIBITORS

Although there is some evidence for proteasome-independent NF-kB activation (see above), IkB degradation by proteasomes still appears to be a critical event in response to most stimuli activating the transcription factor. Moreover, generation of the Rel proteins p50 and p52 is proteasome-dependent. Proteasome inhibition has been shown to suppress NF-kB activity and to enhance chemo- and radiotherapy induced apoptosis in cancer cell lines and tumour models. Therefore, inhibition of these proteolytic multiprotein complexes remains a valid strategy for targeting NF-kB in cancer and other diseases. This is borne out by the success of the Millennium proteasome inhibitor Velcade (36) in the treatment of multiple myeloma, a B-cell cancer in which chronic NF-kB activation has a major causative role [186 –188].

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The 20S proteasome was discovered as a cation-sensitive neutral endopeptidase from bovine pituitary with an apparent molecular weight of 700 kDa [189, 190]. It received its name after the discovery of its identity with the 20S ‘prosome’ particles assumed to regulate mRNA translation [191]. The 20S proteasome is the catalytic core of the 26S proteasome, the key enzyme complex of non-lysosomal, ATP-dependent protein degradation catalyzing the rapid turnover of many regulatory proteins such as IkB and cyclins. Most of these proteins are marked for proteasome-mediated degradation by phosphorylation-induced polyubiquitinylation [192]. Proteasomes are also essential for the elimination of abnormal, misfolded proteins arising by gene mutation or posttranslational damage, as well as for antigen-processing and even for the much slower bulk protein turnover. In addition to the 20S particle, the 26S complex contains over 20 additional proteins, including ATPases, located in a distinct complex called the PA700 proteasome activator or the 19S complex. These proteins are thought to play a role in determining substrate specificity and in the unfolding of the substrates prior to insertion into the barrel-shaped 20S catalytic core. A second activator is known as PA28 or the 11S regulator. It modulates proteasome-catalysed processing of certain antigens for presentation to the immune system. Proteasome complexes involved in antigenic peptide generation have been described as immunoproteasomes. They are formed by replacement of certain subunits of so-called constitutive proteasomes in cells stimulated with interferon g [193, 194]. Like the 19S complex, the 11S regulator caps the 20S proteolytic core for which five catalytic activities have been described: (1) a chymotrypsin-like activity which cleaves after large hydrophobic amino acid residues, (2) a trypsin-like activity which cleaves after basic residues, (3) a post-glutamyl peptide hydrolase-like activity (PGPH) which cleaves after acidic residues, (4) one which cleaves preferentially after branched-chain amino acids (BrAAP activity), and (5) one cleaving after small neutral amino acids (SNAAP activity).

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A number of pharmaceutical and biotech companies have proteasome inhibitors in development [155, 156]. Most of these compounds are peptide derivatives. Target diseases are inflammation, cancer, neurodegenerative disorders, and protozoal infections. A joint venture between Kyorin and Nisshin has yielded a peptidyl boronic acid (37), which has been reported to inhibit immunoproteasomes from bovine spleen (IC50 ¼ 68 ng/ml) more potently than constitutive proteasomes (IC50 ¼ 390 ng/ml) from bovine kidney. In a rat model of inflammatory bowel disease, the compound was found to be more effective than the immunosuppressant azathioprine. Eisai has found antitumour activity of epoxyketones derived from the microbial proteasome inhibitors epoxomicin (38) and eponemycin (39) in a xenograft model of human breast cancer [155, 156, 195, 196]. Novartis is investigating boronate dipeptide derivatives, which are selective covalent inhibitors of the chymotrypsin-like activity of the 20S proteasome (IC50 ¼ 0.3 nM). Introduction of methoxy groups into the phenyl ring of the first amino acid residue increased the inhibitory activity of these compounds towards the trypsin-like and the PGPH activity while leaving inhibition of the chymotrypsin-like activity unaffected [155, 156]. Compounds in this series were reported to inhibit cancer cell proliferation with ED50 values between 5 and 50 nM, and to induce apoptosis in the cell lines tested. Dose-dependent antitumour activity was seen in a human PC3 prostate cancer xenograft and a syngeneic Lewis lung carcinoma mouse model at doses close to the maximum tolerated dose. Previously, Novartis had described a series of 2-aminobenzylstatine derivatives with a maximum IC50 value of 7 nM [155, 156, 197]. ARIAD has also disclosed boronate peptide analogue proteasome inhibitors (40) but no information on their efficacy is as yet available. Cephalon and Novuspharma, now part of Cell Therapeutics, have released data on their dipeptidyl proteasome inhibitor CEP-1612 (41) [155, 156] showing that treatment of A-549 human lung adenocarcinoma cells with the compound induces accumulation of the CDK inhibitors p21Waf1 and p27Kip1 followed by apoptosis. Moreover, significant inhibition of tumour growth was observed in nude mice bearing A-549 tumours which received CEP-1612 at 10 mg/kg/day for 31 days. 4SC has claimed N,N0 diarylurea proteasome inhibitors for the treatment of protozoal infections including malaria trypanosomiasis and leishmaniasis [155, 156]. One compound (42) inhibits the 20S proteasome with an IC50 value of 1 mM and is active against both multiresistant and chloroquine-sensitive malaria parasite (Plasmodium falciparum) strains. Nereus have released data on a novel marine natural product proteasome inhibitor, salinosporamide A (43) resembling MLN-519 (44) (see below) [155, 156, 198]. The compound is orally available and shows pico- to nanomolar activity against isolated human proteasome, inhibits IkB degradation, induces apoptosis in PC-3 human prostate cancer cells as well as human MMCs, and is well tolerated by mice after a single dose of 0.25 mg/kg or five daily doses of 0.1 mg/kg. Millennium, which already has the first-in-class proteasome inhibitor

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Velcade on the market in the US for the treatment of relapsed and refractory multiple myeloma, was developing MLN-519, derived from lactacystin (45), a blactone irreversible proteasome inhibitor from Streptomyces lactacystinaeus [199], for stroke. For this purpose, a co-development agreement with PAION was signed in 2002, when phase I trials in which the compound was well-tolerated had been completed. However, this contract has since been terminated by PAION and no further development has been reported [155, 156]. Velcade [200, 201] is a dipeptidyl boronic acid (36). Peptidyl boronates are more potent and selective inhibitors of the 20S proteasome chymotrypsin-like activity than the peptidyl aldehydes investigated earlier. These compounds also inhibit serine proteases, cysteine proteases, and calpain. The boronate group interacts with an active site threonine residue critical for catalytic activity. In cell culture and xenograft models of human cancers, (36) increases sensitivity to conventional chemotherapeutics and, as a consequence, overcomes chemoresistance. This observation has led to phase I and II combination trials. The drug not only inhibits growth in isolated human MMCs but, like PS-1145, also interferes with their survival-promoting interaction with BMSCs in co-cultures by blocking adhesion molecule expression, suppresses BMSC production of IL-6, and inhibits angiogenesis in xenografts. Velcade has been approved by the Food and Drug Administration, and more recently also by the European Commission, for the treatment of multiple myeloma which has progressed despite administration of at least two prior therapies. Of 193 evaluated patients treated in the phase II registration trial, 10% showed a complete or near-complete, 18% a partial, and 7% a minimal response. Designation as complete response required 100% disappearance of M protein (a monoclonal immunoglobulin secreted by the myeloma cells and used as biological marker for tumour burden) as determined by immunofixation, less than 5% plasma cells in the bone marrow, no increase in size or number of lytic bone lesions, and disappearance of soft tissue tumours (plasmacytomas). Median response duration was 12 months. Common adverse events included fatigue, nausea, diarrhoea, constipation, thrombocytopenia and peripheral neuropathy. The control arm of a current multicenter phase III study receiving high-dose dexamethasone standard therapy was halted on the recommendation of an independent monitoring committee, due to statistically significant improvement in time to progression as determined by interim data analysis, and the patients were given the option to be put on Velcade. Unlike the phase II study, this trial included patients who had received only one prior therapy and its results are expected to lead to the expansion of the patient population which may be treated with the drug. Clearly, proteasome inhibition does not only affect NF-kB. The more specific inhibitor of NF-kB activation PS-1145 inhibits MMC proliferation much less potently than Velcade [186]. Thus, although inhibition

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of the transcription factor is thought to be critical for the efficacy of the drug in multiple myeloma, additional effects such as accumulation of CDK inhibitors are likely to contribute. This may open up avenues for the development of the drug for other types of cancer in which NF-kB activation may play a lesser role. Clinical trials are already under way in which Velcade is tested on patients with mantle cell lymphoma, another B-cell cancer, or solid tumours including colorectal carcinoma as a single agent or in combination with standard chemotherapy [155, 156]. Further studies for the treatment of other haematological and solid cancers are ongoing or planned by Millennium and Johnson & Johnson who co-develop the drug. However, a phase II metastatic colon cancer trial of Velcade as single agent, or in combination with the topoisomerase I inhibitor irinotecan, was recently stopped for lack of efficacy.

POTENTIAL FOR NF-kB INHIBITION AS ADJUNCTIVE ANTICANCER THERAPY

Activation of NF-kB is an important step in the response of cells to a large variety of toxic chemicals which include common cancer chemotherapeutics, as well as many novel, more specific cancer drugs currently in development such as HDAC inhibitors [202]. NF-kB activation induced by these drugs, or by radiotherapy, appears to protect cells from the damaging effects of these therapies and is, therefore, likely to put a limit on their efficacy against cancer cells, especially since many tumours show increased NF-kB activity already prior to treatment. HDAC inhibitors, for example, have recently been demonstrated to be limited in their ability to induce apoptosis in cancer cells by activating NF-kB via the PI 3-kinase/Akt pathway [203]. Cells in which this pathway, or NF-kB itself, is inhibited before exposure to HDAC inhibitors show sensitization to apoptosis induction by these compounds suggesting that a combination of HDAC and NF-kB inhibitors may result in increased cancer cell killing in patients. As described above, the proteasome inhibitor Velcade, whose therapeutic effect on cancer cells is thought to be largely due to inhibition of IkB degradation, and consequently of NF-kB activation, has been shown to synergize with other cancer drugs. One example is its potentiation of HDAC inhibitor-induced killing of Bcr –Abl positive chronic myelogenous leukaemia (CML) cells resistant to Gleevec [204]. Given that IkB degradation in response to anticancer drug treatment can occur independently of IKK, inhibitors of the proteases catalyzing this proteolytic reaction appear much more suitable for use as adjuncts in anticancer chemotherapy than compounds inhibiting IKK. Combination of NF-kB inhibitors with other, more established drugs may not only become a successful strategy in the treatment of cancer but also of viral diseases such as AIDS [176].

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Selection of proteins essential for NF-kB activation as targets for drug discovery has raised concerns that, owing to the fundamental role of this transcription factor in immune cell differentiation and function, unwanted side effects of NFkB inhibitors might be unacceptable, at least in the treatment of chronic, non-life threatening disease such as rheumatoid arthritis [121, 205]. Moreover, the observations that knocking out genes coding for such proteins results in massive liver apoptosis in mouse embryos [8, 206], and that NF-kB inhibition in combination with expression of a Ras oncogene induces human epidermal neoplasia in a mouse model, suggest that there may be a danger of liver damage and carcinogenesis caused by drugs targeting NF-kB [207]. With respect to the possibility of NF-kB inhibition causing skin cancer, several points have to be made. First, human skin does not consist of keratinocytes alone, but also contains immune cells, unlike in the model used. Second, human beings are not normally immune-deficient, as were the mice employed in this model. Third, small molecule inhibitors are rarely as potent in inhibiting NF-kB as is the degradation resistant IkB used in these experiments. Fourth, NF-kB inhibitors would not be given long term when used in cancer therapy. Based on recent findings showing that NF-kB activation can under certain circumstances promote rather than suppress apoptosis [208], the possibility that inhibition of the transcription factor may result in abnormal cell survival eventually leading to malignancy will have to be taken into consideration when developing NF-kB inhibitors for conditions which require long-term drug administration. As to the potential for liver damage caused by NF-kB inhibitors, it has recently been shown that the lethal embryonic liver apoptosis observed in the knockout mice is caused, not by circulating, but by plasma-membrane-bound TNFa, which is not present in healthy human livers [209]. While this observation suggests that liver damage caused by induction of apoptosis is not likely to occur in humans treated with an inhibitor of NF-kB, massive loss of B and T cells [7, 210], which have both been shown to depend on IKK-mediated activation of the transcription factor for their development and survival, is a more serious concern. However, this dependence has been concluded from the results of experiments with cells in which IKK activity was completely absent due to gene targeting. As was said before, pharmacological inhibition is bound to be incomplete and, therefore, unlikely to have the same effect. There can be no doubt that an effective inhibitor of NF-kB activation will suppress immune functions which, indeed, is exactly what it is intended to do in the treatment of inflammatory disease. Whether such a compound will do more harm than good with respect to the immune system is likely to depend on the particular target or combination of targets on which it acts, the Rel protein or combination of Rel proteins this action affects, and the extent to which they are inhibited. Also, the problem of general immune

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suppression resulting in susceptibility to opportunistic infections will be of much greater concern if the compound is to be taken over longer periods of time as in the treatment of chronic inflammatory disease, than if it is administered to treat an acute condition. Judging from the surprisingly limited side effects of the proteasome inhibitor Velcade, which inhibits IkB degradation and consequently NF-kB activation with great efficacy, there currently appears to be no reason to reject NF-kB as drug target on the basis of potential adverse effects which might be induced by inhibition of this transcription factor.

THE FUTURE FOR NF-kB INHIBITORS AS DRUG CANDIDATES

Inhibition of NF-kB is a strategy that has great potential for delivering drugs which will result in noticeable improvements in the treatment of inflammatory disease, cancer, and other illnesses, be it as single agents or in combination with other medications or therapies, and thus to bring considerable benefit to millions of patients. However, it appears more uncertain that the NF-kB signalling components, other than the proteasome, which are currently being targeted by the pharmaceutical industry are the best drug targets that can be found within the regulatory network controlling NF-kB, and that inhibitors of these proteins will make it to the market. Claims that highly specific inhibitors of IKK, for example, show great efficacy in animal models of human disease may have to be taken with a pinch of salt, since none of these compounds appear to have been tested against all other kinases now known to be involved in NF-kB activation. It should not come as a surprise if some of these molecules, at least, will be found to also act on one or more of these other NF-kB-activating kinases and that their inhibition significantly contributes to the efficacy of the compound. After all, an absolutely specific kinase inhibitor is something that still needs to be demonstrated. Recent findings, which have shown that degradation of IkB does not exclusively depend on phosphorylation by IKK, indicates that there may be a limit to the degree of NF-kB inhibition that can be achieved by a compound targeting just this one kinase. Moreover, the IKK inhibitor SPC-839 (14) has been found to inhibit both NF-kB- as well as AP-1-dependent reporter gene expression with practically identical IC50 values, strongly suggesting that it has a cellular target upstream of both of these transcription factors, possibly a kinase of the MEKK family, in addition to its in vitro target IKK. This compound may, therefore, turn out to be an example of a molecule inhibiting multiple kinases in the NF-kB pathway. The recent large-scale mapping of the protein – protein interactions that form the regulatory network controlling NF-kB activation by TNFa, and the identification of 80 new interacting partners [211], among which are 10 previously unknown modulators of the transcription factor, may lead to the identification of novel

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promising drug target candidates, such as TRAFs. Analysis and modelling of this network is already under way [212, 213]. A question that may be raised when considering NF-kB inhibitors for the treatment of rheumatoid arthritis is whether there is really an unmet medical need, because very effective therapy in the form of anti-TNFa antibodies is already available. The development of these biopharmaceuticals was indeed a big step forward in the treatment of this painful and debilitating disease, but although lack of oral availability may not be an issue, high production costs and consequently high price certainly is. Thus, it is not by accident that pharmaceutical companies which market such antibodies are still looking for small molecule drugs to treat rheumatoid arthritis and other inflammatory diseases. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]

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Progress in Medicinal Chemistry – Vol. 43, Edited by F.D. King and G. Lawton q2005 Elsevier B.V. All rights reserved.

6 Recent Advances in the Search for Newer Antimalarial Agents VIJAY K. KAPOOR and KAMAL KUMAR University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India

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ANTIMALARIAL DRUGS FROM NATURAL PRODUCTS Alkaloids Terpenoids Coumarins and Related Compounds Flavonoids and Isoflavonoids Naphthoquinones and Anthraquinones Ellagic Acid and a Phenolic Glycoside Lignans Fungal Products Marine Derived Natural Products

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CHEMICAL MODIFICATIONS OF NATURAL PRODUCTS Artemisinin Analogues Febrifugine Analogues Bruceolide Derivatives

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TOTALLY SYNTHETICS Quinolines Acridines Biguanides Peroxides Chalcones Miscellaneous Chemical Systems Synthetics Based on Biochemical Approach Synthetics as Chemosensitizers

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INTRODUCTION Malaria is a protozoal disease endemic to many tropical and subtropical regions of the world. The protozoan parasite Plasmodium is the cause of the disease, which is transmitted through the bite of an infected female mosquito of the genus Anopheles. Out of the four species of Plasmodium infecting humans, Plasmodium falciparum is the most dangerous malarial parasite responsible for approximately 95% of the mortality cases; and is reportedly [1] expected to spread to the central or northern regions of Europe and North America within a few decades. It is estimated that there are 300 –500 million new cases of malaria throughout the world and up to 2.7 million deaths per year [2]. Among the antimalarial drugs, chloroquine has been one of the most successful because of its high efficacy and affordable price for developing countries where malaria is most prevalent. Unfortunately, the emergence of multidrug-resistant strains of P. falciparum has reduced the efficacy of chloroquine for prophylaxis and treatment of the disease. This has stimulated the search for new and better antimalarial drugs, both from natural and synthetic sources. New targets for antimalarial therapy are being identified, and the effectiveness of new chemical entities targeted specifically against these targets is being explored. In man, the infective protozoa are P. falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. P. falciparum causes malignant tertian malaria and this is the most dangerous form of human malaria. P. vivax is the cause of benign tertian malaria, in which the clinical attacks are milder than those of P. falciparum. P. malariae causes quartan malaria, an infection that is common in localized areas of the tropics. P. ovale is the cause of a rare malarial infection with a periodicity like that of P. vivax, but it is milder and more rapidly cured. The Plasmodium life cycle in man begins with the bite of an infected female mosquito, which injects sporozoites into the circulation. The sporozoites get lodged in the parenchymal cells of the liver, where they multiply and develop into tissue schizonts. This constitutes the primary exoerythrocytic or pre-erythrocytic phase of infection, and it lasts for 5– 16 days, depending on the species of Plasmodium. The tissue schizonts then rupture, each releasing thousands of merozoites. The merozoites enter the circulation and invade

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erythrocytes to start the erythrocytic phase or blood cycle. A portion of these parasites infect more liver cells, and this is termed the secondary exoerythrocytic phase. In P. vivax and P. ovale infections, but not in P. falciparum and P. malariae infections, some tissue parasites may remain dormant (latent forms or hypnozoites), which may result in relapse months or years later in the infected patient. In erythrocytes, most parasites undergo asexual development through trophozoites and finally mature to schizonts. There is rupture of the schizontcontaining erythrocytes, each releasing between 6 and 24 merozoites. It is when the erythrocytes burst that a feeling of chill and fever follows, and liberated merozoites infect more red blood cells and start the cycle afresh. The cycle continues until death of the host, modulation by drug or acquired immunity. The periodicity of fever in tertian or quartan malaria is thus based on the timing of schizogony of a generation of erythrocytic parasites. Some of the merozoites differentiate into male and female parasites known as gametocytes. These gametocytes can undergo sporogony (sexual cycle) in the gut of a female mosquito. The resulting zygote develops in the gut wall as an oocyst and ultimately gives rise to the infective sporozoite, which invades the salivary gland of the mosquito. The events of the malarial parasite life cycle are summarized in Figure 6.1. A brief mention may be made about sites of the action by the antimalarial drugs. There is no drug available which acts as a sporozoitocide. In the absence

Fig. 6.1 The life cycle of a malarial parasite.

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of such drugs the term casual prophylactic is used for drugs acting on pre-erythrocytic forms. Such tissue schizontocides are used for casual prophylaxis. Pyrimethamine is extensively employed for casual prophylaxis of falciparum malaria. Primaquine also possesses casual prophylaxis activity, but is not recommended for this purpose because of its toxicity. Tissue schizontocides act against secondary exoerythrocytic forms (latent or hypnozoite forms) of P. vivax and P. ovale in the liver, and are used to prevent relapse. Such agents, along with a suitable blood schizontocide, can achieve a radical cure of P. vivax and P. ovale infections. Primaquine is an antirelapse drug, and pyrimethamine also shows some of this type of activity against P. vivax. Schizontocides (blood schizontocides) act on the asexual erythrocytic forms of all species of Plasmodium, and are of interest for use to achieve clinical or suppressive cure. Chloroquine, quinine and mefloquine are typical fast-acting schizontocides. Pyrimethamine, sulphonamides and sulphones also possess schizontocidal activity, but the action is slow. Gametocytocides destroy sexual erythrocytic forms of plasmodia and thus prevent transmission of malaria to the mosquito. Primaquine has this activity, particularly against P. falciparum. Chloroquine, quinine and mepacrine possess such activity against P. vivax and P. malariae, but not against P. falciparum. Sporontocides, such as proguanil, prevent or inhibit the formation of malarial oocysts and sporozoites in infected mosquitoes. ANTIMALARIAL DRUGS FROM NATURAL PRODUCTS Among the natural products used for the treatment of malaria, quinine (1) occupies a significant status. An alkaloid found in the bark of the South American Cinchona tree, quinine has been used as a medicinal agent for treating malaria for more than 350 years. Despite its declining use because of its potential toxicity, quinine is still the drug of choice for treating chloroquine- and multidrug-resistant falciparum malaria. It has served as a prototype in the development of synthetic antimalarials. The second breakthrough in the use of natural products in the treatment of malaria was in 1979, when artemisinin (2), a sesquiterpene lactone endoperoxide isolated from Artemisia annua (a Chinese weed qing hao), was reported as a rapidly acting, effective and safe drug for the treatment of P. vivax and P. falciparum infections [3]. Artemisinin could be used to treat patients with cerebral malaria, an otherwise fatal condition, as well as those infected with drugresistant strains of P. falciparum. This discovery prompted a search for new natural products with improved antimalarial activity. The search has been

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carried out by screening higher plants, fungi and marine sources. A variety of compounds belonging to the categories of alkaloids, terpenoids, coumarins and related compounds, flavonoids and isoflavonoids, naphthoquinones and anthraquinones, and lignans having antimalarial activity have been isolated. These are discussed next. Fungal and marine derived natural products are discussed separately.

ALKALOIDS

A number of alkaloids have been reported to possess antimalarial activity. The majority of the alkaloids with such activity contain an isoquinoline moiety, either in the free form or fused with other homo- or heterocyclic rings. Naphthylisoquinoline alkaloids, derived from tropical vines of the families Ancistrocladaceae and Dioncophyllaceae, form a new and promising class with pronounced growth inhibiting activities against P. falciparum and Plasmodium berghei in vitro. These alkaloids are structurally interesting due to the presence of a biaryl axis linking the naphthalene and isoquinoline moieties. The presence of large ortho substituents confers atropisomerism. In a structure – activity relationship study carried out by Francois et al. [4] dioncophylline C (3), isolated from Triphyophyllum peltatum [5], displayed maximum activity (IC50 ¼ 0.014 mg/ml) against P. falciparum, followed by dioncophylline A atropisomer, 7-epi-dioncophylline A (4) [6] (IC50 ¼ 0.19 mg/ml). Other naphthylisoquinoline alkaloids, dioncolactone A (5) [7], 50 -O-demethyl-8-O-methyl-7-epi-dioncophylline A (6) [6] and hamatine (7), isolated from Ancristrocladus hamatus [8], displayed IC50s in the range of 1 – 4 mg/ml. The SAR study also revealed that, for maximum activity of dioncophylline A (8), a free NH and OH is a prerequisite [4]. An interesting observation was made with regard to increasing methylation of the nitrogen of the quinoline ring of (8). Whereas the mono-N-methyl derivative, (9), had a significant decrease [9] in antiplasmodial activity towards asexual erythrocytic forms of P. falciparum, the quaternary salt,

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N,N-dimethyldioncophyllinium A iodide (10), by contrast, showed an increased activity, even compared with the parent compound [10].

Ancristrogriffithine A and ancristrogriffine A, new monomeric naphthylisoquinoline alkaloids isolated from Ancristrocladius griffithii, have shown activity

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against P. falciparum [11]. A new naphthylisoquinoline alkaloid, 8-Omethyldioncophyllindol B, isolated from Triphyophyllum peltatum, has been found to exhibit good antiplasmodial activity [12]. Among the three bisbenzylisoquinoline alkaloids isolated from the bark of Abuta grandifolia, krukovine (11) exhibited potent antiplasmodial activity, with IC50 values of 0.44 and 0.022 mg/ml against K1 (chloroquine-resistant) and T9-96 (chloroquine-sensitive) P. falciparum, respectively [13]. Angerhofer et al. [14] have tested 53 bisbenzylisoquinoline alkaloids isolated from different plant sources for antiplasmodial activity against chloroquine-sensitive and chloroquine-resistant clones of P. falciparum, in comparison with their cytotoxicity against human KB cells. No clear general conclusion could be drawn with respect to configuration of the chiral centre or modification of substituents on changes in cytotoxicity and antiplasmodial activity. However, it was observed that both quaternization and N-oxide formation resulted in a loss of antimalarial activity, and that a decrease in lipophilicity contributed to a lower toxicity.

Six new phenolic aporphine-benzylisoquinoline alkaloids isolated from the roots of Thalictrum faberi exhibited potent antimalarial activity [15]. The 6a,7-dehydroaporphine alkaloids, dehydrostephanine (12) and dehydrocrebanine (13), isolated from the fresh tubers of Stephania venosa, showed potent activity with IC50 values of 40 and 70 ng/ml, respectively, against T9/94 strain of P. falciparum [16]. Moderate activity has been reported for two new bis-dehydroaporphine alkaloids isolated from roots of Polyalthia debilis [17]. Another aporphine alkaloid (2 )-roemrefidine (14), isolated from Sparattanthelium amazonum, has also been found to be active against both resistant and sensitive strains of P. falciparum in vitro, and against P. berghei in mice [18].

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Antimalarial activity and structure –activity relationships of 39 tertiary and quaternary protoberberine alkaloids [19] and 17 protoberberinium salts [20] have been investigated. In vitro activities against P. falciparum (multidrugresistant strain K1) for 21 monomeric isoquinoline alkaloids have been assessed and structure – activity relationships drawn [21]. Crotsparine (15), isolated from Uvaria klaineana, showed antiplasmodial activity against chloroquine-sensitive and chloroquine-resistant K1 and FCB1 strains of P. falciparum [22].

The alkaloid nitidine (16), which has isoquinoline heterocycle fused with 2,3methylenedioxynaphthalene, has been found to be the major antimalarial component of Toddalia asiatica, a plant used by the Pokot tribe of Kenya to treat fevers [23]. Certain 2-substituted quinolines isolated from the infused stem bark of Galipea longiflora have shown activity against mice infected with P. vinckei petteri, justifying the traditional use of the plant in the treatment of malaria [24]. Antimalarial activity has been reported of four tetrahydroquinoline alkaloids from trunk bark of Galipea officinalis [25]. A new benzoquinolizidine alkaloid klugine (17) and the known alkaloid cephaeline isolated from Psychotria kulgii exhibited potent antimalarial activity against P. falciparum clones W2 and DC [26].

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Amongst the four acridone alkaloids isolated from the stem bark of Swinglea glutinosa, 1,3,5-trihydroxy-4-methoxy-2-(3-methylbut-2-enyl)-10-methyl-9acridone (18) was found to be the most active when assessed for antiplasmodial activity on a Nigerian chloroquine-sensitive strain and the chloroquine-resistant strain FeM29 of P. falciparum [27]. Normelicopicine and arborinine, two acridone alkaloids isolated from Teclea trichocarpa, have shown limited in vitro activities against P. falciparum strains of HB3 and K1 [28]. I-Azaaporphinoidal alkaloids hadranthine, sampangine and 3-methoxysampangine, isolated from Duguetia hadrantha, demonstrated in vitro antimalarial activity against P. falciparum (W-2 clone) [29]. Lycorine and 1,2di-O-acetyllycorine, isolated from the bulbs of Brunsvigia littoralis, exhibited antimalarial activity against two strains of cultured P. falciparum [30]. Two tertiary, quasi-symmetric bisindole alkaloids, named strychnogucines A and B, isolated from the roots of Strychnos icaja, showed antiplasmodial activity. Strychnogucine B (19) was more active against a chloroquine-resistant strain than against chloroquine-sensitive one (best IC50, 80 nM against W2 strain) [31]. 18-Hydroxy-isosungucine, isolated earlier from the same plant, has also shown antiplasmodial activity [32]. Recently, the in vitro antiplasmodial activities of 69 alkaloids isolated from various Strychnos species have been evaluated against chloroquine-resistant and chloroquine-sensitive lines of P. falciparum, and structure – activity relationships have been discussed [33]. The indole alkaloids cryptolepine, 11-hydroxycryptolepine and neocryptolepine, isolated from root bark of Cryptolepis sanguinolenta, showed a strong antiplasmodial activity against P. falciparum chloroquine-resistant strains, the highest activity residing with cryptolepine. In vitro tests on infected mice showed that the hydrochloride salt of cryptolepine exhibited a significant chemosuppressive effect against P. berghei yeolii and P. berghei berghei [34]. A carbazole alkaloid heptaphylline isolated from Clausena harmandiana

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possessed antiplasmodial activity with an IC50 value of 3.2 –6.4 mg/ml [35]. Monomeric (20) and dimeric (21) carbazoles, isolated from Murraya euchrestifolia, showed promising activity (IC50 7.62 and 10.8 mg/ml, respectively) against P. falciparum [36].

A new aminosteroid sarachine (22), 3b-amino-22,26-epiminocholest-5-ene, isolated from leaves of Saracha punctata, has shown potent in vitro antiplasmodial activity, with an IC50 of 25 nM [37].

The spermine alkaloids budmunchiamines L4 and L5, isolated from stem bark and leaves of Albizia adinocephala, were found to inhibit the malarial enzyme plasmepsin II [38]. Isolated from Phyllanthus fraternus, a plant used in Ghanian traditional medicine to treat malaria, two alkylamides E,E-2,4octadienamide and E,Z-2,4-octadienamide have shown moderate antiplasmodial activity [39].

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TERPENOIDS

Antiplasmodial activity has been displayed by sesqui-, di- and tri-terpenoids, which are non-nitrogenous constituents of plants. Amongst the sesquiterpenoids isolated from the fruits of Reneilmia cincinnati, the hydroxylated germacradienes (23) and (24) showed potent antiplasmodial activity. The finding supported the use of the powdered fruits of the plant in traditional practice of treating fevers [40]. Other sesquiterpenoids reported to have antiplasmodial activity are tagitinin C, isolated from Tithonia diversifolia [41] and kudtriol, isolated from Jasonia glotinossa [42].

Xantholides isolated from aerial parts of Xanthium strumarium have shown antimalarial activity against chloroquine-resistant P. falciparum strain K1 [43]. The quininoid diterpenes with a nor-abietane skeleton, such as cryptotanshinone (25), isolated from an Iranian plant Perovskia abrotanoides, inhibited the growth of cultured 3D7 strain of P. falciparum [44]. Antiplasmodial activity has also been demonstrated by scopadulcic acid A found in Scoparia dulcis [45]. The diterpene ajugarin-1, isolated from the aerial parts of Ajuga remota, the most frequently used medicinal herb for malaria treatment in Kenya, was found to be moderately active when evaluated for in vitro antiplasmodial activity. However, ergosterol-5,8-endoperoxide, occurring in the same plant, was found to be three times more potent [46]. Labdanes isolated from Aframomum latifolium and Aframomum sceptrum were also found active [47]. Limonoids, which are tetracyclic nortriterpenoids, have been found to possess antimalarial activity. Gedunin (26), a limonoid obtained from the bark and seeds of Khaya grandifolia, has shown moderate activity against P. falciparum in vitro. Furthermore, gedunin has shown an additive effect in combination with chloroquine [48]. Quassinoids, which are degradation and rearrangement products of triterpenoids, have also shown interesting biological activities. The samaderines, isolated from the stems of the Indonesian plant Quassia indica, were shown to exhibit significant growth inhibitory activity against cultured chloroquine-resistant K1 strain of P. falciparum. Samaderine X (27a)

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was found to have IC50 0.014 mm [49]. The quassinoids isolated from Hannoa chlorantha and Hannoa klaineana, such as chaparrinone (27b) exhibited moderate to high antiplasmodial activity [50]. Structure – activity relationship showed that a hydroxyl function at C14 was unfavourable for antiplasmodial activity, and the keto function at C2 for a related compound was of crucial importance for high activity.

The triterpenoid ester, E-p-coumaroylalphitolic acid (28), isolated from the roots of Cochlospermum tinctorium, has shown interesting antiplasmodial activity [51]. The commonly occurring triterpenoid, lupeol, was reported to inhibit the P. falciparum growth by 45% when tested at 25 mg/ml [52].

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COUMARINS AND RELATED COMPOUNDS

Of several 4-phenylcoumarins isolated from the stem bark of Exostema mexicanum, 40 ,5,7,8-tetramethoxy-4-phenylcoumarin (29) exhibited the most potent in vitro activity against a chloroquine-sensitive strain (PoW) and a chloroquine-resistant strain (Dd2) of P. falciparum, with IC50 values 3.6 and 1.6 mg/ml, respectively [53]. The stem bark of the plant is reported to be used in Latin American folk medicine as a quinine substitute for malaria treatment [53]. Coumarins isolated from bark of Kayea assamica [54] and roots of Toddalia asiatica [55] were also found to be active antiplasmodial agents. In vitro antimalarial activity has also been reported for five xanthones isolated from the bark of Garcinia cowa, which is used in Thailand as a traditional medicine for fevers [56]. A new trans-hexahydrodibenzopyran derivative, named machaeriol B (30), isolated from stem bark of Machaerium multiflorum, has demonstrated in vitro activity (IC50 120 ng/ml) against the P. falciparum W-2 clone [57].

FLAVONOIDS AND ISOFLAVONOIDS

The flavonol glycosides isolated from Hydrangeae dulcisfolium showed characteristic proliferation inhibition of P. falciparum at low concentrations [58]. Flavonoids and isoflavonoids obtained from the root bark of Erythrina abyssinica also showed antiplasmodial activity [59]. Moderate in vitro activity displayed by the extracts of stem and leaves of Andira inermis has been attributed to its isoflavonoid constituents [60]. 8-Prenylmucronulatol (31), isolated from Smirnowia iranica, has shown moderate in vitro antiplasmodial activity [61]. A new rotenoid (32), along with known flavonoids, was found to be the antiplasmodial principles of stem bark of Millettia usaramensis subspecies usaramensis [62].

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NAPHTHOQUINONES AND ANTHRAQUINONES

Antiplasmodial activity has been reported for some isofuranonaphthoquinones isolated from roots of Bulbine capitata [63]. The plant is claimed to have antipyretic properties. The tropical tree, Morinda lucida, which is used in ethnomedicine in certain West African countries for the treatment of fever, has yielded some anthraquinones showing good activity against both chloroquine-susceptible (3D7) and chloroquine-resistant (Dd2) strains of P. falciparum; compound (33) was the most active (IC50 21.4 mM) [64]. The root bark of Stereospermum kunthianum is reported to be a valuable remedy for certain tribes in Uganda to treat fever. Antiplasmodial activity has been reported for the naphthoquinones and anthraquinones isolated from this plant [65].

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ELLAGIC ACID AND A PHENOLIC GLYCOSIDE

Ellagic acid (34), a dilactone from gallic acid, is reported to have antimutagenic and tumour chemopreventive activities. Recently, it has been reported to inhibit the growth of a chloroquine-sensitive and chloroquine-resistant clone of P. falciparum, with an IC50 of 0.5 mM. Ellagic acid, along with a phenolic glycoside 3,4,5-trimethoxyphenyl-(60 -O-galloyl-O-b-D -glucopyranoside (35), was found responsible for the antiplasmodial effects of the extracts of leaves and bark of Tristaniopsis calobuxus, with an IC50 of 3.2 mM [66]. LIGNANS

Antiplasmodial activity against both the chloroquine-sensitive POW and chloroquine-resistant clone Dd2 of P. falciparum has been reported for the sesquilignans and sesquineolignans isolated from the aerial parts of Bonamia spectabilis, a climbing shrub occurring in Madagascar and in tropical East Africa [67]. A new lignan, dehydrodiconiferyl dibenzoate, isolated from the roots of the palm Euterpe precatoria, has shown moderate antiplasmodial activity [68]. Mention may be made of machaeridiol B (36), a 1,3-catechol isolated from stem bark of Machaerium multiflorum, which has shown activity against D6 and W2 clones of P. falciparum [69].

FUNGAL PRODUCTS

Certain naphthoquinones isolated from insect pathogenic fungus Cordyceps unilateralis BCC 1869 have shown antimalarial activity against

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P. falciparum [70]. Cordytropolone (37), isolated from Cordyceps sp. BCC 1681, showed in vitro activity against K1 strain of P. falciparum with IC50 value of 2.2 mg/ml [71]. Moderate activity has also been reported for preussomerins, isolated from lichenicolous fungus Microsphaeropsis species [72].

MARINE DERIVED NATURAL PRODUCTS

Several novel natural products have been isolated from marine organisms, and many of these have shown interesting biological activities. A variety of compounds, such as alkaloids and terpenoids isolated from marine sources, have exhibited antimalarial activity. Manzamines are complex polycyclic marine-derived alkaloids first reported in 1986 from the Okinawan sponge genus Haliclona [73]. These alkaloids have previously been reported to exhibit a diverse range of bioactivities, including antimalarial activity [74]. Recently, novel manzamine alkaloids have been isolated from an undescribed genus of Indo-Pacific sponge of family Peterosiidae [75]. In an in vivo assay against P. berghei, the manzamines (38) – (40) were found to be more potent and less toxic than the currently available antimalarial drugs, artemisinin and chloroquine. These manzamines have been reported to be valuable candidates for further investigation and for development as promising leads in the treatment of malaria [75].

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The alkaloids fascaplysin (41) and homofascaplysin (42), isolated from the sponge Hyrtios cf. erecta collected from Fiji, have been found to have potent in vitro activity against P. falciparum [76]. High antiplasmodial activity has been reported for a series of marine pyridoacridone alkoloids related to ascididemin [77].

Some new diterpenes of the eunicellin class, isolated from the Gorgonian coral Briareum polyanthes, were reported to be active against P. falciparum [78]. A series of diterpene isonitriles and isothiocyanates isolated from the tropical marine sponge Cymbastela hooperi were reported in a short review [79]

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to have antiplasmodial activity. Recently, 3D-QSAR and receptor modelling studies performed with these isonitriles have generated a pharmacophore hypothesis consistent with the experimentally derived biological activities [80]. These studies suggested that the active isonitrile compounds, like the quinoline antimalarials, exert their antiplasmodial activity by preventing FP detoxification. Norsesterterpene peroxide acids, isolated from several genera of marine sponges, have displayed a wide range of bioactivity including antimalarial activity [81, 82]. Recently, sigmosceptrellin-B (43), isolated from the Red Sea sponge Diacarnus erythraeanus along with other norsesterterpene peroxide acids, has shown in vitro antimalarial activity against P. falciparum (D6 and W2 clones) with IC50 values of 1,200 and 3,400 ng/ml, respectively [83]. Sigmosceptrellin-B had also previously been isolated from Sigmosceptrella laevis [84].

Another peroxide moiety-containing compound plakortide F (44), isolated from Caribbean sponge Plakortis spp., has also shown significant activity against P. falciparum in vitro [85]. A related endoperoxide (45), isolated from Plakortis halichondrioides collected in Puerto Rico, has displayed potent antiplasmodial activity [86]. The findings suggested that the peroxide system is necessary for antimalarial activity. CHEMICAL MODIFICATIONS OF NATURAL PRODUCTS ARTEMISININ ANALOGUES

Artemisinin (2) (qinghaosu) is a cadinane sesquiterpine endoperoxide isolated from A. annua, a plant used in traditional Chinese system of medicine for the treatment of fever and malaria [3]. Artemisinin and its semisynthetic derivatives are highly efficacious in the treatment of chloroquine-, mefloquine- or multidrug-resistant P. falciparum infection. The generally accepted mode of action of artemisinin as an antimalarial is attributed to its endoperoxide moiety.

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It is suggested that once inside the Plasmodium, the peroxide function reacts with intraparasitic Fe(II) haem produced by proteolysis of the host haemoglobin. The scission of the peroxide bond leads to an intermediate oxyradical, which then abstracts a hydrogen atom from C-4 position to form a more stable carbon radical [87, 88]. Though an effective antimalarial, the need for chemical modification of artemisinin arose because of its high rate of recrudescence, poor solubility in oil and water, short plasma half-life and poor oral activity. The chemical modification of artemisinin resulted in emergence of arteether (46) and artemether (47) with increased solubility in oil; and sodium artelinate (48) and sodium artesunate (49). Although the analogues showed higher potency than artemisinin, and are in clinical use, they too suffer from drawbacks. Both arteether and artemether are reported to have short plasma half-lives and have displayed severe central nervous system toxicity in rats and dogs. Sodium artesunate is associated with a problem of instability in aqueous solution, high rate of recrudescence and extremely short plasma half-life [89]. Attempts to improve the therapeutic profile of artemisinin derivatives through other chemical modifications continue. Excellent accounts on the developments on the chemistry and biological activity of artemisinin and its derivatives have appeared earlier [90 – 93]. Some of the recent advances made in this direction are covered here under the classified sub-titles.

Analogues with different peripheral substituents C-10 substituents. The earlier C-10 substituted derivatives of artemisinin, arteether, artemether and artesunic acid were synthesized to overcome the problem of low solubility of artemisinin in both oil and water. The pharmacokinetic studies carried out on these derivatives in vivo or in liver homogenates demonstrated that these compounds undergo a rapid hydroxylation by cytochrome P-450 enzyme, generating a hemiketal intermediate which decomposes to produce dihydroartemisinin (50). As dihydroartemisinin itself is

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a highly active antimalarial, and its derivatives get converted in vivo to dihydroartemisinin, the derivatives are considered as prodrugs of dihydroartemisinin. These derivatives suffer from a high rate of recrudescence and low rate of radical cure which, in general, may be related to their short plasma half-lives. The short half-life, in turn, may be attributed to the fast conversion of these derivatives to dihydroartemisinin (50). Taking into consideration these metabolic steps, a number of other C-10 substituted derivatives of artemisinin were designed with the approach that the newer derivatives are poorer substrates for cytochrome P-450 and hence would have longer half-lives. Based on the approach of decreasing the rate of oxidative dealkylation of the target compounds and to increase the lipophilicity of the molecule, Lin and Miller [94] synthesized a series of dihydroartemisinin a-alkylbenzylic ethers, of which all the compounds exhibited at least equal or better in vitro antimalarial activity than artelinic acid when assessed against two clones (D-6 and W-2) of P. falciparum. Derivatives having a small methyl substituent at the a-methylene group showed weaker activity than derivatives with a larger carbethoxyalkyl substituent, indicating that the lipophilicity and the steric effect at this part of the molecule play an important role in their antimalarial activity. A series of stereoisomeric derivatives (51) of dihydroartemisinin with a 4-( p-substituted phenyl)butyric acid as both the free acid and as its methyl ester displayed a 2–10 fold increase in in vitro activity against D-6 and W-2 clones of P. falciparum over artemisinin or artelinic acid [89]. In general, R-diastereomers were found to be more potent than the corresponding S-diastereomers. The study also suggested electronic effects may also play a role in determining the efficacy of the compounds.

In a study to determine the effect of the presence of fluorine atom(s) on alkyl or aryl groups at C-10, fluorinated artemisinin derivatives were prepared and evaluated for antimalarial activity [95]. A 2 –5 fold increase in activity was

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observed upon introduction of fluorine in 9a- and 9b-hydroxyarteether. Several fluoroalkyl ethers of dihydroartemisinin were found to have only moderate activity on in vitro evaluation against P. falciparum W-2 asiatic strain, but showed excellent in vivo activity against P. berghei (NT 173) [96]. Recently, adopting the approach to increase the metabolic stability of artemisinin derivatives, O’Neill et al. [97] prepared a number of C-10 phenoxy derivatives of dihydroartemisinin, of which all the compounds were found to have potent in vitro activity against K-1 chloroquine-resistant strain of P. falciparum. In general, the a-series exhibited similar biological activity to their b-diastereoisomers. The b-p-trifluoromethylphenoxy derivative (52) demonstrated excellent antimalarial activity on in vivo evaluation against P. berghei in the mouse, with an ED50 of 2.12 mg/kg, equal to that of dihydroartemisinin and superior to that of artemether (ED50 of 6.02 mg/kg). Metabolic studies carried out in rats revealed that the compound was not metabolized to dihydroartemisinin, suggesting that it has a longer half-life and potentially lower toxicity. The C-10 phenoxy derivatives are thus worthy of further investigation as next-generation lead compounds. Based on the consideration that the analogue should have moderate lipophilicity ðlog P ¼ 0:6 – 1:8Þ, several glucuronide conjugates of dihydroartemisinin and hydroxylated metabolites of b-arteether were prepared [98]. Among the C-10 glucuronides of dihydroartemisinin, the glucuronide of b-dihydroartemisinin (53) was 20 times less active than its aglycone, while the a-isomer glucuronide was virtually inactive. A number of benzylamino (54) and alkylamino (55) derivatives of dihydroartemisinin were prepared, taking into consideration that such chemical modification would enhance the drug activity by increasing the cellular accumulation within the acidic parasite food vacuole by ion trapping [99].

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On the basis of similar reasoning, two new series of artemisinin derivatives that incorporate two basic nitrogens in the form of piperazine moiety were prepared [100]. The first series (56) contained a metabolically susceptible C-10 ether linkage, while the second series consisted of C-10 carba linked amino analogues, such as (57), which had the advantage of resisting P450-catalysed oxidation to form dihydroartemisinin. Antimalarial assessment demonstrated that these compounds were 4-fold more potent than artemisinin and about 2-fold more potent than artemether in vitro against chloroquine-resistant strains. Both the series of analogues have the added advantage of being available as watersoluble salt preparations.

Removing the ether function at C-10, Pu and Ziffer [101] had also earlier synthesized several 10b-alkyldeoxoartemisinins and tested them in vitro against two drug resistant strains of P. falciparum. In in vitro testing 10bpropyldeoxoartemisinin (58) was shown to be as active as arteether. 10Deoxoartemisinin (59) is also a potent antimalarial, and several of C-3 and C-9 analogues of 10-deoxoartemisinin were reported to be much more active than the natural (þ )-artemisinin or 10-deoxoartemisinin [102]. C-9 substituents. Of a number of artemisinin analogues where b-methyl at C-9 was differently modified, only the 9b-nitroethyl substituted derivative (60a) was shown to have antimalarial activity comparable to artemisinin [103]. Analogues of 10-deoxoartemisinin (60b; R ¼ methyl) with substitution at C-9 of R ¼ H, ethyl, propyl, butyl, pentyl, 3-phenylpropyl, 3-( p-chlorophenyl)propyl were found to be active [102]. The potency against the W2 clone of P. falciparum was found to increase steadily to propyl (5-fold artemisinin), but butyl showed a large increase in potency, at about 21 times that of artemisinin, while pentyl was much less potent (1.5 times artemisinin). The 3-arylpropyl was similar to the butyl, being 25 –70 fold more potent than artemisinin.

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C-7 substituents. The geminal difluoro derivative, 7,7-difluoroarteether, was found to be more active than the parent compound [95]. Among the glucuronides of hydroxylated metabolites of arteether, 7b-hydroxyarteetherglucuronide was found to be the most potent [98]. C-6 substituents. One of the metabolites of arteether, with the C-6 methyl hydroxylated, was converted to its glucuronide [98]. An analogue of arteether, where the C-6 methyl was converted to difluoromethyl, was also reported to have an increased activity [95]. C-5 substituent. Glucuronide of 5a-hydroxyarteether, a metabolite of arteether, has been prepared [98]. C-5a substituent. 5a-Hydroxyarteether was obtained as a novel microbial transformation product when arteether was subjected to microbial metabolism using Cunninghamella elegans [104]. C-4 substituent. (4R)-4-Acetoxyartemisinin was synthesized, and on in vitro testing against P. falciparum strains D6 (Sierra Leone) and W2 (Indo-China) showed lower activity than artemisinin, reflecting the importance of proton abstraction for the mode of action of artemisinin [103]. C-3 substituents. Novel C-3 substituted analogues of 10-deoxoartemisinin were prepared and tested in vitro against W-2 and D-6 strains of P. falciparum [102]. Whereas the 3-ethyl was 10-fold less potent than 10-deoxoartemisinin, the 3-propyl was 7-fold more potent. Arylalkyl substituents also resulted in a reduced potency and the conclusion was reached that antimalarial potency cannot be explained solely on the basis of hydrophobicity.

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Dimers, trimers and tetramers Dimers of artemisinin linked through the deoxy C-10 position were prepared. The benzoylmethylene (61), aryl (62) and furan (63) dimers showed antimalarial activity better than artemisinin [105]. Various C-9 linked artemisinin dimers, trimers and tetramers have shown antimalarial activity better than, or comparable to, artemisinin when subjected to in vitro screening system against K1 multidrug-resistant strain of P. falciparum [106].

11-aza and 13-carba analogues A series of novel N-substituted 11-aza-artemisinin derivatives have been prepared [107 –109]. The most active derivative, N-(20 -acetaldehydo)-11-azaartemisinin (64), was found to be 26 times more potent in vitro and 4 times more potent in vivo than artemisinin [107]. In another study, among a series of N-alkyl-11-aza-9-desmethylartemisinin, the N-methyl analogue (65) was found to be . 5 times more potent than artemisinin against W-2 strain of P. falciparum [110]. These results demonstrate that replacement of the lactone moiety of artemisinin by a lactam does not reduce its biological activity.

Replacing the non-peroxide trioxane oxygen atom of artemisinin by carbon, Avery et al. [111, 112] synthesized (þ )-13-carba-artemisinin (66) and related

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structures. It was interesting to note that whereas (66) and (þ )-10-deoxo-13carba-artemisinin (67) did not display substantial antimalarial potency in vitro against P. falciparum, the diastereomeric 10-deoxo-13-carba-artemisinin (68) possessed good antimalarial potency in vitro. This mitigates to some extent the argument that 1,2,4-trioxane moiety is essential for good antimalarial potency. Ring-contracted analogues A number of novel ring-contracted artemisinin derivatives were synthesized and tested for antimalarial activity [113]. The antimalarial activity of the ringcontracted analogue (69) was found to be comparable to that of arteether when tested in mice infected with P. berghei (K-173 strain) and was also found to be active against chloroquine-resistant NS strain infection in mice. Diastereoisomeric derivatives of dihydroartemisinin, having the ring-contracted artemisinin analogue attached through ether linkage at C-10, also showed a similar biological profile.

Tricyclic analogues Encouraged by the display of excellent antimalarial activity of tricyclic 1,2,4trioxanes, Posner et al. [114] synthesized a series of benzylic ethers (70) as potential next-generation antimalarials. p-Fluorobenzyl tricyclic trioxane (71) was found to be the most active in the series. It was considerably more active than artemisinin in the W-2 clone and especially in the African Sierra Leone D-6 clone of P. falciparum malarial parasite that is resistant to mefloquine, but sensitive to chloroquine. It also had considerable activity in mice infected with P. berghei, and had 10 times higher activity than artemisinin in killing immature P. falciparum gametocytes. New artemisinin tricyclic analogues

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bearing a methyl group at C-5a were synthesized [115], but this substitution was found to be detrimental to the antimalarial activity of these trioxanes, thus reinforcing the hypothesis that tight haemin – trioxane complexes are involved in the activation phase of these compounds. Tricyclic trioxanes bearing a 1,2-dioxacyclohexane cycle instead of 1,2dioxacycloheptane as in artemisinin were found to have no significant antimalarial activity [116]. Synthesis of a new type of tricyclic 1,2,4-trioxanes (72) has been reported [117], but no biological activity was reported.

FEBRIFUGINE ANALOGUES

The roots of Dichroa febrifuga, a saxifrageous plant, have been traditionally used in China for the treatment of malaria fevers for centuries without any reported parasitic resistance [118]. Febrifugine (73) and isofebrifugine (74) were isolated as the active alkaloids against malaria [119, 120]. Febrifugine could not attain the status of a chemotherapeutic agent against malaria because of its powerful emetic side effect. However, the antimalarial potency of febrifugine attracted the attention of medicinal chemists to use it as a lead to synthesize its various analogues to develop novel antimalarial drugs. Pyridine analogues [121] and methylenedioxy analogues [122] of febrifugine were synthesized and screened for antimalarial activity. Analogues having a modified or unmodified 4-quinazolinone ring were found to be active, while those produced through the modification of the side chain attached to the N-3 position of the 4-quinazolinone ring were ineffective.

Recently, new types of analogues of febrifugine and isofebrifugine have been synthesized which exhibit excellent antimalarial activities with high selectivity

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against malarial parasite [118, 123]. The keto analogue (75) of febrifugine was found to have EC50 2.0 £ 1028 M against P. falciparum in vitro. The analogues (76) – (78) also showed potent antimalarial activities. The structure – activity relationship study demonstrated that the 4-quinazolinone and C-20 and C-300 oxygens play an important role in activity.

BRUCEOLIDE DERIVATIVES

Some plants of the family Simaroubaceae have been traditionally used as a cure for malaria. Quassinoids, which form the major constituents of such plants, have been found to inhibit proliferation of P. falciparum in vitro [124]. Among the antimalarial active quassinoids, bruceolide (79) is one of the representative compounds with a common core skeleton; the C-15 esters of this parent alcohol, occurring in the genus Brucea, are known as bruceolides. Several semisynthetic derivatives of bruceolide have been reported to have antimalarial activity.

Using bruceolide as the starting material, 3,15-O-diacetylbruceolide (80) was prepared, which showed potent in vitro antimalarial activity [125]. It was also found to possess potent in vivo antimalarial activity against P. berghei infected mice [126]. Recently, several carbonate derivatives of bruceolide were synthesized and screened for antimalarial activity [127]. 3,15-Dimethyl (81) and 3,15-diethyl (82) carbonates of bruceolide have been found to have promising in vivo antimalarial potency.

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TOTALLY SYNTHETICS Among the totally synthetics, a number of chemical categories have been investigated, resulting in the development of clinically useful drugs for the treatment of malaria. These include quinolinemethanols, 8-aminoquinolines, 4-aminoquinolines, 9-aminoacridines, biguanides and aminopyrimidines. In terms of clinical success, 4-aminoquinolines, represented by chloroquine, have emerged as the most important class. Chloroquine has remained an affordable and effective antimalarial drug for more than five decades. Its effectiveness, however, has been eroded by the evolution of malarial parasites resistant to chloroquine. The problem of chloroquine-resistant malaria has prompted the search for novel alternative synthetic drugs effective against resistant strains. Some of the recent developments in this area are discussed in this section. QUINOLINES

Following the success of chloroquine (83) as an effective antimalarial drug, investigations centred around 4-aminoquinolines and other quinoline derivatives. Several chloroquine analogues were prepared and tested to find new antimalarial agents with improved efficacy that can be used successfully against chloroquine- and multidrug-resistant strains of P. falciparum. A series of novel short chain chloroquine derivatives have been synthesized, some of which were found to be significantly more potent than chloroquine against a chloroquineresistant strain of P. falciparum in vitro [128]. This study also concluded that the ability to accumulate at higher concentrations within the food vacuole of the parasite is an important parameter that dictates their potency against chloroquine-sensitive and chloroquine-resistant K1 P. falciparum. Structure – activity relationships for chloroquine and closely related 4-aminoquinoline antiplasmodial compounds have been studied [129 – 131]. New modular molecules, such as (84) with a trioxane skeleton linked to 4-aminoquinoline, have also been reported [132].

Another 4-aminoquinoline, amodiaquine (85), was found to be effective against certain chloroquine-resistant strains of P. falciparum. Amodiaquine has a 4-hydroxyanilino moiety, which has been found to be responsible for the toxicity,

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as this moiety is believed to undergo enzyme-catalysed oxidation to a quinoneimine, followed by nucleophilic addition to proteins.

In a series of amodiaquine analogues, tebuquine (86) was shown to be the most potent; significantly more active than amodiaquine and chloroquine both in vitro and in vivo. As tebuquine also contained 4-hydroxyanilino moiety, it would be expected to give a toxic quinoneimine in a manner similar to amodiaquine. Replacement of the 4-hydroxy function in amodiaquine with isosteric fluorine gave an analogue which was active against chloroquine-resistant and sensitive P. falciparum [133]. On similar lines, the 4-hydroxy function of tebuquine was replaced with either fluorine or hydrogen. In both cases, however, there was a reduction in antimalarial activity [134]. A new series of 4-aminoquinolines lacking the hydroxyl group responsible for toxicity has been synthesized. On in vitro evaluation against both chloroquine-sensitive and resistant strains of P. falciparum several compounds were found to be active in low nanomolar range; a morpholino derivative cured mice infected by P. berghei and displayed lower toxicity than amodiaquine upon mouse macrophage [135]. A number of double drugs containing a labile linker have recently been synthesized, based on the hypothesis that an elevation of glutathione content in parasites leads to the increased resistance to chloroquine, while glutathione depletion in resistant P. falciparum strains is expected to restore the sensitivity to chloroquine. Higher intracellular glutathione levels depend inter alia on the efficient reduction of glutathione disulphide by glutathione reductase. Following this approach, double drugs were synthesized where a 4-aminoquinoline was joined with a glutathione reductase inhibitor through metabolically labile ester bond. The most active double drug of the series was where a 4-aminoquinoline alcohol (87) was linked with the most efficient glutathione reductase inhibitor (88) [136]. The biological results were encouraging.

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Based on the excellent profile of the Mannich base drug pyronaridine (89), developed in China in 1970, 12 new quinoline di-Mannich base compounds were synthesized [137]. Some of the resulting compounds, such as (90), were found to be more active than amodiaquine, chloroquine or pyronaridine.

New trifluoromethyl- and bis(trifluoromethyl) quinoline derivatives having a keto function at position 4, represented by (91), have been synthesized [138]. Compounds having 2,8-bis(trifluoromethyl)quinoline scaffold were found to have much lower in vitro antimalarial activity than chloroquine, but were twice as active as those with either the 2- or 8-trifluoromethylquinolines.

A series of indolo[3,2-c ]quinolines represented by the general structure (92) has been synthesized, with a basic R group being essential for antimalarial

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activity. Structure – activity relationships have shown that there is a poor correlation between antimalarial activity and hydrophobicity, that larger R groups are detrimental to activity, but that the greater the charge on the basic ˚ from the quinoline nitrogen, the better the nitrogen located 10 – 11 A antimalarial activity, suggesting that this nitrogen is protonated and functions as an H-bond donor in the drug-receptor interaction [139]. Using the rationale that the bulky bisquinoline structure may be less efficiently extruded by chloroquine-resistant P. falciparum, a number of bisquinolines have been synthesized and tested. A new series, where the 4-aminoquinoline part of chloroquine is retained and the two units are joined by bisamide links from carbocyclic ring, has been synthesized and screened against chloroquine-sensitive and resistant strains of P. falciparum in vitro [140, 141]. The resistant indices for all the compounds were found to be lower than for chloroquine. The position of attachment and length of the linker chain had marked effect on the activity. Compound (93) was found to be the most active, with an IC50 120 nM against chloroquine-resistant FAC8 strain.

The antimalarial activity of the bisquinoline, trans-N1,N2-bis(7-chloroquinolin-4-yl)cyclohexane-1,2-diamine [142], and alkylidene-linked chloroquine dimers [143] has been reported. Antiplasmodial activity of bis-, tris- and tetraquinolines with linear or cyclic amino linkers has also been reported [144]. Recently, three series of monoquinolines consisting of a 1,4-bis(3-aminopropyl)piperazine linker and a large variety of terminal groups have been synthesized [145]. The replacement of a quinoline moiety by different substituents, aromatic and aliphatic, was undertaken to explore the influence of the terminal group on the biological profiles of the new molecules. From a structure –activity relationship of bisquinolines, a binding model for the putative quinoline drug receptor was postulated which suggested that there are two (or more) anionic sites as well as a p-electron rich, flat area positioned parallel to the quinoline ring [146]. The role of bisquinolines in malaria chemotherapy has been reviewed [147].

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8-Aminoquinolines represent a class of typical tissue-schizontocidal agents. Primaquine (94) is the widely used drug of this class which exerts its action against the primary and secondary tissue forms of the Plasmodium. Primaquine has been shown to possess efficacy against the drug-resistant strains of P. falciparum. Unfortunately, the drug suffers from the most serious side effect of causing haemolytic anaemia that is substantially enhanced in individuals who are genetically deficient in the glucose-6-phosphate dehydrogenase (G6PD) enzyme. Thus, there is pressing need to search for alternative drugs in this category which do not have this liability. Recently, a series of 8-(4-amino-1methylbutylamino)-5-alkoxy-4-ethyl-6-methoxyquinolines have been prepared and tested in vitro and in vivo for blood-schizontocidal activities against drugresistant and drug-sensitive Plasmodium strains [148]. The compounds (95) and (96) exhibited activity superior to that of chloroquine. ACRIDINES

The class of acridines is represented by quinacrine (mepacrine) (97), which was discovered in 1932, as the first antimalarial drug with blood schizontocidal activity. Later, it was replaced by chloroquine. Recently, new bisacridines have been synthesized where two quinacrine moieties are joined by alkanediamines, polyamines or polyamines substituted by side chains. All the compounds were tested for their antimalarial activity against the chloroquine-resistant strain FCBIR of P. falciparum [149, 150]. Compound (98), which displayed a high order of activity against different strains, was suggested as the lead compound from the series.

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BIGUANIDES

In the category of biguanides, proguanil (99) is a widely used antimalarial. It is a prodrug, as in humans proguanil is metabolized to the active cycloguanil (100), which acts as a blood schizontocide by inhibiting plasmodial dihydrofolate reductase. A similar compound PS-15 (101) was synthesized by Canfield et al. [151], which was shown to be three times as potent in the form of its triazine metabolite.

Recently, 34 analogues of PS-15 have been prepared; several of them maintain or exceed the in vivo activity of PS-15 [152]. A wide range of electron donating and electron withdrawing substituents on the aromatic ring, linker length and hetero-atom replacement were tolerated against the W2 strain of P. falciparum but good activity against the K-1 strain was retained only with the arylO- or arylS-propyl linkers.

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The 1,2,4-trioxane moiety, present in naturally occurring antimalarial artemisinin and its semisynthetic derivatives, has been the basis for the synthesis of potential antimalarial peroxides. Posner et al. [153, 154] have prepared a number of cyclic peroxy ketals, of general structure (102), where aryl group was differently substituted phenyl and R,R varied in ring size from cyclobutyl to cycloheptyl. Some of the compounds were found to be 1/4 – 1/10 as potent (31 – 85 nM) as artemisinin (IC50 ¼ 8.4 nM) against P. falciparum. A series of 3-aryltrioxanes (103) were shown to be efficacious in vivo as potent antimalarials even when administered orally to rodents [155]. A series of monocyclic and spirocyclic 1,2,4trioxanes with significant antimalarial activity have been prepared from chiral allylic alcohol 4-methyl-3-penten-2-ol [156]. Compounds with a trioxane skeleton linked with 4-aminoquinoline have also been prepared which showed low nM inhibitory potency against three strains of P. falciparum [132].

The 1,2,4,5-tetraoxacyclohexane (tetraoxane) moiety became an interesting pharmacophore since the antimalarial activity of dispiro-1,2,4,5-tetraoxane (104) (WR148999) was found to be very similar to that of the 1,2,4-trioxanes [157]. Several tetraoxanes with antimalarial activity have been reported [157 – 161]. Out of 16 alkyl-substituted dispiro-1,2,4,5-tetraoxanes three, for example (105), cured between 40 and 60% of the mice infected with P. berghei on oral administration [161]. In order to explore the influence of steroid carrier on the antimalarial activity in vitro, cholic acid-derived 1,2,4,5-tetraoxanes were synthesized [162, 163]. The cholic acid derived carrier was envisaged to render solubility under physiological conditions and to enhance cell membrane permeability because of its amphiphilic character. The compounds of cis-series were found to be about two times more potent than the trans isomers against P. falciparum D6 and W2 clones.

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Several acyclic endoperoxides have also been prepared with a variety of functional groups, of which 1,1-bis(methyldioxy)cyclododecane (106) showed the most notable antimalarial activity, particularly in vivo [164]. Recently, novel 1,2,5,6-tetraoxacycloalkanes and 1,2,5-trioxacycloalkanes have been prepared; compounds exemplified by (107) and (108) showed substantial antimalarial activity in vitro, with (108), in particular, having activity comparable to that of artemisinin [165].

CHALCONES

Chalcones, 1,3-diphenyl-2-propen-1-ones, drew the attention of medicinal chemists when licochalcone A, a natural product isolated from Chinese liquorice roots, was reported to exhibit potent in vivo and in vitro antimalarial activity [166]. A series of chalcones and their derivatives were synthesized and identified as novel potential antimalarial agents using both molecular modelling and in vitro testing against the intact parasite [167]. The 2,4-dimethoxy-40 -butoxychalcone (109) was reported to have outstanding antimalarial activity [168].

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Recently, chalcones with 20 ,30 ,40 -trimethoxy, 20 ,40 -dimethoxy, 40 -methoxy, 40 ethoxy, 20 ,40 -dihydroxy and 40 -hydroxy groups on ring B were synthesized and evaluated in vitro against P. falciparum (K1) in a [3H]-hypoxanthine uptake assay [169]. Predictive structure – activity relationships were also obtained. Antimalarial chalcones are generally thought to act against malarial cysteine protease, an enzyme used by the parasite for the degradation of host haemoglobin for its nutritional purposes. MISCELLANEOUS CHEMICAL SYSTEMS

Besides the above-mentioned chemical categories, a number of compounds with potential antimalarial activities among varied and unrelated chemical systems have been synthesized. These are discussed in this section. Halofantrine is a phenanthrene system-containing antimalarial, which has been used as an alternative to quinine and mefloquine. Because of certain side effects, its use is generally not recommended. Recently, several diaza-analogues of phenanthrene were synthesized and evaluated for their antiplasmodial activity [170]. All derivatives showed moderate to good activity in vitro, both on a Nigerian chloroquine-sensitive strains and on the chloroquine-resistant FCB1Columbia and FCM29 strains. The best results were obtained with 1,10phenanthroline skeleton. Phenyl b-methoxyacrylates have been identified as new antimalarial agents [171]. These are believed to exert their activity by inhibition of mitochondrial electron transport at the cytochrome bc1 complex. 2,5-Bisacylaminobenzophenone (110a) has been described as a lead structure for a novel class of antimalarials active against multi-resistant P. falciparum Dd2 [172]. Replacement of the 3phenylpropionyl moiety of the lead structure by a 4-propoxycinnamic acid residue (110b) resulted in significant improvement in antimalarial activity [173].

Compounds represented by the general structure (111), with oxazine as the heterocycle, were prepared from purpurogallin through a hetero Diels –Alder reaction, and have been suggested as novel class of potential antimalarials [174].

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Other systems which have been investigated and found to be active as antimalarials include thiochromones [175], polyhydroxyphenyl and hydroxamic acid derivatives [176], a steroid 16a-bromoepiandrosterone [177], ring substituted imidazoles [178], tert-butylperoxyamines [179], ethyl 5-phenyl-6oxa-1-azabicyclo[3.1.0]hexane-2-carboxylate derivatives [180] and substituted pyrazoles [181]. SYNTHETICS BASED ON BIOCHEMICAL APPROACH

New promising targets based on biochemical approach for antimalarial chemotherapy have been recognized, such as enzymes involved in the degradation of haemoglobin by intraerythrocytic malaria parasite. The parasites degrade haemoglobin in an acidic food vacuole to provide free amino acids for parasite protein synthesis. The cysteine protease (falcipain) and aspartic protease (plasmepsin) are two of the many potential targets. Selective inhibitors of these parasitic proteases as potential antimalarial agents have been synthesized. A number of vinyl sulphones were found to be inhibitors of falcipain, and their antimalarial effect correlated with their inhibition of falcipain [182, 183]. This suggests that such compounds may have promise as antimalarial drugs. A series of plasmepsin I and plasmepsin II inhibitors has been prepared [184]. A few active compounds have been shown to inhibit parasite growth in cultured infected human parasites. A novel class of pyrimidinyl peptidomimetics, the core structure of which comprised a peptidomimetic segment for recognition and binding to the target enzyme, has been synthesized [185]. Although no significant inhibitory activity against plasmepsin was observed, the compounds exhibited potent in vitro growth inhibitory activity (IC50 ¼ 10 – 30 ng/ml) against both chloroquinesensitive (D-6) and chloroquine-resistant (W-2) P. falciparum clones. Compound (112) was found to be the most active of the class, the antimalarial efficacy of which was comparable to that of chloroquine.

Cyclin dependent protein kinases (CDKs) play an important role in the development of the parasite and have become attractive targets to combat malaria. PfPK5 and Pfmrk are two plasmodial CDKs that have been well

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characterized. In a recent study, several oxindole-based compounds have been identified as effective inhibitors of Pfmrk [186]. P. falciparum dihydrofolate reductase (PfDHFR) is an important target for antimalarial chemotherapy. Drugs like pyrimethamine and cycloguanil act through inhibition of PfDHFR. Unfortunately, emergence of resistant parasites has significantly reduced the efficacy of these drugs. Resistance of these parasites to such drugs has been attributed to mutations of DHFR, principally Ser108Asn (S108N) and others. A number of 2,4-diaminopyrimidine analogues of general structure (113) have been prepared which show high binding affinity with wild type and mutant DHRFs [187]. Some of these compounds have shown good antimalarial activities against pyrimethamine-resistant P. falciparum containing mutant DHFRs with low cytotoxicity to three mammalian cell lines.

Recently, an approach towards molecular docking of the structures contained in Available Chemical Directory (ACD) database to search for novel inhibitors of PfDHFRs has been described [188]. Twelve new compounds whose structures are completely unrelated to known antifolates are reported to be identified. These have been found to inhibit, at the micromolar level, the wild type and resistant mutant PfDHFRs. Another biochemical approach to search for new antimalarials involves interference with the phospholipid metabolism by competition or substitution of phospholipid polar head analogues. Around 85% of malarial phospholipid consist of phosphatidylcholine and phosphatidylethanolamine. A series of compounds, primary, secondary and tertiary amines and quaternary ammonium and bisammonium salts, most of them synthesized as potential choline or ethanolamine analogues were tested against the in vitro growth of P. falciparum [189]. They were active over 1023 –1028 M concentration range, the most potent compound being nPr3NþC12H25Br2 with an IC50 of 33 nM. A principal component analysis, ascending hierarchical classification and stepwise discriminant analysis showed that lipophilicity and electronegativity distribution in the molecular space was essential for antimalarial potency. SYNTHETICS AS CHEMOSENSITIZERS

The problem of resistance in P. falciparum to chloroquine has also been addressed to through a different strategy. It has been observed that

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chloroquine-resistant P. falciparum strains exhibit reduced drug accumulation as compared with the susceptible ones [190], and addition of structurally different compounds in drug regimen can restore chloroquine efficiency. Such compounds are known as chemosensitizers. Several drugs representing different pharmacological groups, such as verapamil [191] (a calcium channel blocker), desipramine [192] and fluoxetine [193] (both antidepressants), and antihistaminic drugs [194, 195] have demonstrated reversal of chloroquine resistance in malaria parasite. The pharmacological actions of these drugs, however, reduce the clinical value of these chemosensitizers. A series of new chemosensitizers from aromatic amine ring systems – phenothiazine, iminodibenzyl, iminostilbene and diphenylamine have been examined for their drug-resistance reversing efficacy [196]. Based on the model of maprotiline (114), which has shown reversal of resistance, a number of 9,10-dihydro-9,10-ethano- and ethenoanthracene derivatives have been synthesized and evaluated for their effect on chloroquine susceptibility in P. falciparum strains [197].

GENOMIC ADVANCES The sequencing of the human genome has opened a new vista for the discovery of molecular targets associated with various disease types such as cardiovascular diseases, diabetes, immune disorders and cancer. There are now 30,000– 40,000 known genes which create enormous opportunity for drug discovery. In 2002, significant genomic advances were published, which form a major contribution to efforts in the fight against malaria. The 278-megabase genome sequence of Anopheles gambiae, the major mosquito vector of the parasite P. falciparum has been determined [198]. Additionally, the complete genome sequence of P. falciparum, the parasite which causes the deadliest form of malaria in humans, has been reported [199 – 201]. The triad of genome sequences of the vector, the parasite and of the human offers unprecedented opportunities to develop new antimalarial drugs, potential vaccine and new insecticides for vector control. The advances would help in unravelling the pathophysiological relationship between the human host, the parasite and the vector. The Anopheles genome sequence provides an architectural scaffold for mapping, identifying, selecting and exploiting desirable insect vector genes; and

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will promote our understanding of mosquito biochemistry, physiology and behaviour as well as of malaria epidemiology [202]. Molecular mechanisms of insecticide resistance would also be understood promoting development of a new generation of insecticides. The genome sequence would also accelerate the engineering of mosquitoes refractory to the Plasmodium. Genes involved in the mosquito’s sense of smell and taste have been identified, which could be blocked to keep the insects from zeroing on humans [203]. P. falciparum is the first eukaryotic parasite for which a complete genome is known. The genome sequence provides the foundation for future studies of this organism. It will accelerate the search for new drugs and vaccines to combat malaria. A high-throughput proteomics approach has been applied to identify potential drug and vaccine targets [204]. A high-accuracy mass spectrometric proteome analysis of P. falciparum has been carried out [205]. A study has shown that P. falciparum became resistant to chloroquine through mutations in a single gene [206].

CONCLUSION Malaria still remains a serious global health problem affecting around 500 million people with over two million deaths per year. P. falciparum is the most lethal of the four species of Plasmodium that infects humans. The emergence of multidrug-resistant strain of P. falciparum, and insecticidal resistance in the mosquito further aggravate the problem. The situation thus makes it imperative to discover new antimalarial drugs and the search goes on to find new leads from natural products, exploring plants, fungi and marine sources; semisynthetics and synthetics. Newer molecules with better potential than the existing antimalarial drugs have been discovered. Bisquinolines, bisacridines, 8-aminoquinolines and endoperoxides hold promise in this direction. Several new biochemical targets have been identified. The concept of chemosensitization, that is addition of structurally variant compounds representing different pharmacological groups in the antimalarial drug regimen, is being explored. The significant genomic advances resulting in genome sequencing of A. gambiae, the major mosquito vector, and of P. falciparum, the parasite for deadliest form of malaria, have opened new hopes towards control of malaria.

ADDENDUM During the preparation of this chapter a recent review on medicinal chemistry perspective on artemisinin and related endoperoxides has appeared [207].

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Progress in Medicinal Chemistry – Vol. 43, Edited by F.D. King and G. Lawton q2005 Elsevier B.V. All rights reserved.

7 The Discovery of the CCR5 Receptor Antagonist, UK-427,857, A New Agent for the Treatment of HIV Infection and AIDS ANTHONY WOOD and DUNCAN ARMOUR Department of Chemistry, Pfizer Global Research and Development, Sandwich Laboratories, Sandwich, Kent CT13 9NJ, UK

INTRODUCTION

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HIGH-THROUGHPUT SCREENING AND ASSAYS

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HIT-TO-LEAD STUDIES

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BEYOND HIT-TO-LEAD

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THE IDENTIFICATION OF UK-427,857

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OTHER CCR5 ANTAGONISTS

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CONCLUSION

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INTRODUCTION Since the identification of the human immunodeficiency virus (HIV) as the causative agent of AIDS in 1983 [1, 2], there have been intense efforts to develop effective treatment and control measures. Notwithstanding this, by 2003 the Joint United Nations Programme on HIV/AIDS estimated that 42 million people DOI: 1 0 . 1 0 1 6 / S 0 0 7 9 - 6 4 6 8 ( 0 5 ) 4 3 0 0 7 - 6

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THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

worldwide were infected with HIV, and that the virus had claimed the lives of more than 20 million people since the start of the epidemic [3, 4]. Furthermore, the rate of infection remains on the increase in the developed and developing world. Following the introduction of highly active antiretroviral therapy (HAART) regimes, using combinations of nucleoside/nucleotide reverse transcriptase and protease inhibitors, HIV has increasingly been considered to be a manageable, chronic disease [5]. However, there is still a need for new therapeutic agents with improved dosing regimes that are better tolerated with a reduced side effect burden [6]. Side effects and complicated dosing regimes have led to reduced patient compliance [7, 8] and contributed to the emergence of viral resistance. This is leading to a rise in numbers of those who have detectable viraemia during treatment with HAART, and those who are infected with HIV strains which are resistant to all three classes of drugs in the HAART regimes (estimated to be 5– 10% of patients undergoing therapy) [9]. It is also leading to a rise in the numbers of newly infected, treatment-naı¨ve patients who are already carrying virally resistant strains (estimated to be up to 20%) [9 – 11]. Consequently, therapies based on new mechanisms of action are particularly desirable. HIV gains entry into cells by fusing the lipid membrane of the virus with the host cell membrane, an event that is triggered by the interaction of proteins between the HIV envelope and cell surface receptors. The virus binds with its gp120 protein to the CD4 receptor forming a complex that undergoes a conformational change creating a co-receptor binding site for a chemokine receptor [12]. For M-tropic strains, which predominate during the initial phase of the disease and during transmission, this co-receptor is CCR5. T-tropic virus strains, which tend to predominate in the late stages of the disease, use the CXCR4 chemokine receptor (although approximately 50% of individuals remain infected with strains that maintain their dependence for CCR5 even in late stage disease). Chemokine receptor binding then triggers further conformational changes in the viral gp41 fusion protein unmasking the fusion peptide and facilitating its insertion into the host cell lipid bilayer and subsequent viral entry. Targeting the viral fusion process has become a new focus of research for the next generation of HIV antiretroviral therapies [13]. In contrast to existing antiretroviral agents that work after viral entry, these should have an advantage in that they will not need to access intracellular compartments. Other groups have focussed on blocking the interaction of the gp120 protein with CD4, e.g. BMS-806 [14], on developing CXCR4 receptor antagonists such as the bicyclam AMD3100 [15], or on blocking the formation of the necessary rearrangement of the gp41 protein [16]. This latter approach resulted in the development of the 36-amino-acid peptide Enfuvirtide (T-20), which was approved by the FDA in March 2003. Although this drug validates the viral

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241

fusion process as a viable target clinically for the treatment of HIV/AIDS, a complicated manufacturing process results in a high cost (approximately US$20,000 per patient year) and twice-daily subcutaneous injections cause a very high incidence (98% of patients) of local site irritation, both of which will probably limit its clinical utility [17]. When our project was initiated, we chose instead to target the CCR5 receptor, as genetic evidence had just become available to underpin the biological rationale for CCR5 blockade leading to an antiviral effect. A section of the human population has a 32 base pair deletion in the CCR5 coding region (D32). In homozygotes this results in a failure to express the receptor on the cell surface, and there is evidence that this group of people are resistant to infection with M-tropic HIV-1 [18]. More recently it has been shown that individuals who are heterozygous for the D32 gene show significantly longer progression times to the symptomatic stages of infection and appear to respond better to HAART treatment [19]. Moreover, both heterozygous and homozygous carriers of the D32 allele are apparently fully immunocompetent with no other obvious abnormalities, suggesting that the absence of CCR5 function may not be detrimental and that a CCR5 receptor antagonist should be well tolerated. This chapter describes the drug discovery programme that led to the identification of UK-427,857, a prototype CCR5 antagonist with excellent potency against lab-adapted and primary HIV-1 isolates, as a clinical candidate for the treatment of HIV. In particular, it deals with strategies for minimizing cardiac toxicity whilst maintaining ADME properties commensurate with low dose. HIGH-THROUGHPUT SCREENING AND ASSAYS The CCR5 receptor is a member of the family of G-protein coupled receptors (GPCRs) and is predicted to have a typical seven transmembrane structure [20]. This class of proteins is heavily represented in the ‘druggable genome’ [21], which gave us some encouragement that a drug-like ligand should be identifiable. The receptor binds the chemotactic chemokines, MIP-1a, MIP-1b and RANTES. Whilst these have been used to produce synthetic peptides such as AOP-RANTES [22], which have been useful for the validation of CCR5 as a target for HIV therapy, we did not believe that these offered a viable starting point for a low-molecular weight, orally bioavailable agent. Nor did we wish to pursue an antibody-based approach [23]. We, therefore, elected to use a high-throughput screen (HTS) to identify novel starting points for our programme. However, the selection of an appropriate screen was problematic. Initially, we did not have purified viral proteins available to us that would have been

242

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

of sufficient quality to construct a robust assay. We, therefore, chose to use a screen based on the inhibition of binding of radiolabelled MIP-1b to the CCR5 receptor stably expressed in HEK-293 cells [24] – a similar approach using MIP-1a has also been described by workers at Merck [25]. We could not be certain at the time that a compound that inhibited the binding of an endogenous chemokine would necessarily block the binding of the HIV gp120 protein. Indeed, more recent work using chimeric CCR5 receptors indicates that the binding domains of HIV gp-120 and MIP-1a are in fact distinct and separate [26, 27], although MIP-1a can act as an allosteric antagonist of gp120 binding. Furthermore, although the monoclonal antibody PRO140 blocks the binding of HIV-1 to the CCR5 receptor, it does not alter the binding of the natural chemokine ligands [23], again suggesting that the natural agonist and the viral proteins have different binding sites. However, since most small-molecule ligands of peptidergic GPCRs are not thought to function by disrupting the large surface area interactions of the protein –protein complex between the natural agonist and receptor, but rather to act allosterically to stabilize receptor conformations that bind the natural agonist less effectively, we believed that it should be possible to identify ligands that could prevent the binding of both the endogenous ligands and the HIV gp-120 protein. This turned out to be the case, although throughout our programme we did sometimes encounter discrepancies between the MIP-1b binding assay and our antiviral assays. Similar observations have been noted by others [28]. The HTS identified a number of hits which were then triaged [29]. One frequent criticism of the HTS approach is that it tends to produce large, lipophilic hits which prove difficult to optimise, particularly as medicinal chemistry programmes tend to increase the size of compound along the path from lead to drug [30, 31]. Indeed this has resulted in some questioning the value of the whole HTS approach to drug discovery. Apart from filtering out compounds which fell outside of Lipinski’s ‘Rule of Five’ [32] we also took into account target affinity and ligand efficiency (LE), as measured by heavy atom contributions to binding [33]. This latter criterion is particularly useful, as it allows the comparison of compounds with quite different affinities, and tends to favour smaller, low-molecular weight compounds. This reduces the risk of picking a starting point just based on its headline affinity, when, of all the properties that can be manipulated by the medicinal chemist, affinity is often the easiest to improve. A further compound was eliminated from consideration due to concerns over toxicity associated with a nitropyridine group, leaving us with four hits, two of which UK-107,543 (1) (MIP-1b IC50 0.4 mM, LE 0.29 kcal/mol/non-H atom), and UK-179,645 (2) (MIP-1b IC50 1.1 mM, LE 0.20 kcal/mol/non-H atom) formed the basis of the programme reviewed here.

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243

Both of these hits contain structural elements that have been termed ‘privileged structures’ [34 – 36] due to the frequency with which they are observed in drug discovery programmes. The origin of this effect has not been determined, although it has been hypothesized that it may reflect the existence of complementary binding sites within the proteins [37]. It may also be due to the effective display of functional groups that these templates inherently have due to conformational effects. Alternatively, it may be due to the composition of the compound screening collections that historically have been biased towards compounds from old discovery programmes. For the medicinal chemist, hits from privileged structures have the advantage of well-precedented chemistry, information on the potential liabilities of the template, but the potential disadvantage of polypharmacology and patentability concerns. Workers at Takeda [38], Merck [25] and Schering [39, 40] have reported isolating hits from their own independent HTS efforts which similarly also contained known GPCR pharmacophores. Neither of our hits, (1) and (2) could be considered to be ideal, having high molecular weight and lipophilicity, polypharmacology, weak binding affinity and no measurable antiviral activity. With considerations of the clinical need for a potent, durable and therefore, convenient and safe agent in mind, we established project goals aiming for a potent antiviral agent with an antiviral

244

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

IC90 similar to or better than, that of existing protease inhibitors, at least 100-fold selectivity over related targets, and pharmacokinetic properties commensurate with at worst b.i.d. dosing, with no CYP450 inhibition. We knew, therefore, that we would have to improve the quality of our starting point, to have any chance of delivering a high-quality compound with a good probability of surviving through preclinical and clinical studies. Therefore, the goal of our initial hit-to-lead studies was to optimise hits (1) and (2) by combining their most attractive features to produce novel, selective antagonists with enhanced ligand efficiency [33] and measurable antiviral activity. HIT-TO-LEAD STUDIES Most drug discovery groups define a hit-to-lead phase post-HTS when the viability of the hits is assessed through limited SAR studies, ideally performed with some elements of parallel/combinatorial chemistry [41]. This process uses less resource than a full medicinal chemistry programme, and aims to evaluate the potential of hits with a goal of reducing late-stage attrition. Our first goal was the replacement of the imidazopyridine in (1) as we believed that this was the cause of the profound type II cytochrome P450 2D6 inhibition [42] observed with this compound (CYP 2D6 IC50 40 nM). The inhibition of this enzyme can cause variable drug levels and serious safety concerns in combination therapy. Type II inhibitors generally contain a nitrogen heterocycle which can coordinate directly to the iron atom in the haeme unit of the enzyme, resulting in a large increase in the redox potential of the P450 and high occupation of the substrate binding site, leading to a dramatic reduction in turnover rates for the enzyme [43]. Modelling [44] indicated that the pyridine nitrogen in (1) was probably directly ligating to the haeme iron, (Figure 7.1), suggesting that the carbon analogue (3) would be preferable. The resultant benzimidazole (3), was a potent inhibitor of MIP-1b binding (MIP-1b IC50 4 nM), albeit still antivirally inactive, and was now a much weaker type I inhibitor of the 2D6 enzyme (CYP 2D6 IC50 710 nM). While still a concern, we felt that the resolution of this issue could wait while we searched for an antivirally active analogue. Another concern for us was the high lipophilicity of (3) – we introduced the amide which featured in (2), to increase the polarity of the template whilst keeping the molecular weight low. We reasoned that one of the phenyl rings of the benzhydryl group restricts the conformational space of the other, an effect known as hydrophobic collapse [45, 46], and that introduction of a more polar linker should not interfere with potency. Indeed, the benzamide (4) exhibited good chemokine receptor binding (MIP-1b IC50 45 nM), albeit slightly less potently than (3). Most encouragingly though, promising levels of antiviral activity could be measured, see Table 7.1. Subsequently structure (4) became the focus of our SAR investigations.

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Fig. 7.1 Modelling of the binding mode of (1) to cytochrome P450 2D6.

245

246

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

Table 7.1 MIP-1b INHIBITORY ACTIVITY AND ANTIVIRAL ACTIVITY OF SELECTED AMIDE ANALOGUES

R

MIP-1b a IC50 (nM)

AV b IC50 (nM)

(4)

45

210

(5)

100

740

(6)

820

9250

(7)

270

7110

(8)

50

700

(9)

430

.10000

40

75

(10)

a b

The concentration required to inhibit binding of [125I]MIP-1b by 50%. The concentration required to inhibit replication of HIVBaL into PM-1 cells by 50%.

A. WOOD AND D. ARMOUR

247

Initially, we investigated the SAR of different amide substituents, using parallel array chemistry. All such compounds were purified by either reversephase HPLC or semi-automated flash chromatography, and characterized by LC – MS [47]. Selected compounds were characterized further by NMR and microanalysis. Biological data for several representative compounds are summarized in Table 7.1. This set of analogues identified the benzamide (4), isopropylamide (8) and cyclobutyl amide (10) as the most potent analogues. Compounds with additional polarity, e.g. (6) and (9), showed a sharp decrease in potency suggesting that the amide substituent interacts with a predominantly lipophilic binding site on the CCR5 receptor. More bulky substituents such as the phenacetylamide (5) had decreased potency. The smallest ligand, acetamide (7), appeared to be less efficient for binding to CCR5. A homochiral synthesis of the two enantiomers unambiguously established that the activity resided with the ðSÞ enantiomer of the benzamide (11) (MIP-1b IC50 13 nM, AV IC50 190 nM), with the ðRÞ enantiomer (13) have much reduced affinity (MIP-1b IC50 580 nM), and no measurable antiviral activity (AV IC50 . 10 mM). Similarly, the homochiral synthesis of the ðSÞ enantiomer of the cyclobutyl amide (12), established that this was the active isomer (MIP-1b IC50 20 nM, AV IC50 73 nM).

248

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

The encouraging activity of (12) prompted us to continue our SAR investigations keeping the cyclobutyl substituent constant. However, (12) was still a Type I CYP2D6 inhibitor [42] when tested against a panel of cytochrome P450 enzymes. As CYP2D6 levels are polymorphic, being absent in 5 –9% of the Caucasian population [48], metabolic clearance can be variable for drugs with this interaction and drug – drug interactions are more common. ˚ away from a phenyl ring A pharmacophore that contains a basic amine 5 –7 A is common for both CYP2D6 inhibitors and substrates [49]. The basic centre is believed to interact with a key residue, Asp301, that is an important determinant for binding [50]. The lead (12) was readily docked into a model of the enzyme [44] (Figure 7.2). Believing that we could reduce the interaction of the key Asp301 residue in the 2D6 enzyme either by sterically encumbering the basic piperidine nitrogen or by reducing the basicity of the piperidine nitrogen, we designed a series of analogues which varied the central piperidine core (Table 7.2). Consistent with the steric encumbrance theory, both the tropanone-derived azacyclo-octyl derivatives (14) (the exo isomer) and (15) (the endo isomer), were devoid of 2D6 activity, and both proved to be highly potent inhibitors of viral replication. While we were initially surprised that both isomers were so similar in activity, 1H NMR analysis shows that the benzimidazole forces the endo substituted bridged piperidine ring in (15) into a boat conformation. The resulting structure overlaps well with that of the exo substituted analogue (14), as shown by molecular modelling (Figure 7.3). The bonus of enhanced binding affinity and improved antiviral activity for the tropanes (14) and (15), may be due to either an improved fit of the ligands

Fig. 7.2 Modelling of (12) bound to CYP2D6.

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Table 7.2 MIP-1b INHIBITORY ACTIVITY AND ANTIVIRAL ACTIVITY OF PIPERIDINE ANALOGUES

R

MIP-1b a IC50 (nM)

AV b IC90 (nM)

(14)

2

13

(15)

6

3

(16)

.1000

(17)

17

(18)

1.2

n.t.

.1000

140

(Continued)

250

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857 Table 7.2 CONTINUED MIP-1b a IC50 (nM)

AV b IC90 (nM)

(19)

21.5

n.t.

(20)

39.7

n.t.

(21)

9

n.t.

(22)

8

R

53

n.t.: not tested. a The concentration required to inhibit binding of [125I]MIP-1b by 50%. b The concentration required to inhibit replication of HIVBaL into PM-1.

to the receptor, or possibly due to the enhanced rigidity of the 2-phenylpropylamine side chain as a consequence of gþ g2 (syn pentane) interactions [51]. The smaller azetidine (16) lost all binding affinity to CCR5. The thiagranatane (19) and endo-oxogranatane analogue (20) had reduced affinity when compared to the tropanes (14) and (15), and so appeared to offer little advantage. The piperidine derivatives (17) and (18), exo-oxogranatane (21) and 2,6 dimethyl piperidine (22) were potent inhibitors of chemokine binding. However, translation into antiviral activity was relatively poor, presumably for the same reasons that we have discussed earlier.

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Fig. 7.3 Overlap between exo and endo analogues (14) and (15).

Having established for (15), potent antiviral activity against a lab-adapted HIV strain (BaL) propagated using a CCR5 clone expressing cell line, we felt that it was important to confirm that the antiviral effect we observed was also translated to low passage primary origin viral isolates cultured in peripheral blood lymphocytes (PBLs). These assays are labour-intensive but may provide a more clinically relevant insight into the antiviral profile of the compound in light of the viral isolate and the host cell being of direct ex vivo origin, therefore, more closely mimicking the biological interactions that exist for pathogenesis in HIVinfected patients. Also, since the gp120 protein is a known variable epitope of HIV, it was also possible that sequence changes might result in a loss of antiviral activity. Encouragingly, (15) showed potent antiviral activity against a range of primary origin CCR5-tropic HIV isolates in PBLs, with IC50s ranging from 0.9 to 9.6 nM. Having established that we could achieve our primary pharmacological goals the hit-to-lead stage of the project was successfully concluded, resource was increased, and (15) was profiled more extensively. BEYOND HIT-TO-LEAD As compound (15) progressed through our safety screens, it became apparent that it was also a potent inhibitor of the human ether a-go-go-related gene (HERG) potassium channel [52], (99% inhibition at 1 mM). The function of HERG channels is to conduct the rapidly activating delayed rectifier potassium current (IKr), which has a key role in the control of cardiac rhythm [53]. HERG channel inhibition has risen to prominence recently in the drug discovery process as the predominant cause of acquired long QTc interval prolongation. A number of previously approved drugs which are associated with a prolongation of the QTc interval due to effects on IKr, have now been given black box warnings

252

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

or indeed have been withdrawn, because of a link to sudden cardiac death and ventricular arrythmia, a condition known as Torsades de Pointes [54] (see also chapter 1). Indeed, QTc prolongation has been reported to have caused issues during the clinical development of other CCR5 receptor antagonists such as SCH351,125 [55]. HERG channel inhibition has also been reported to be a problem for some analogues in the Merck series [56]. We were particularly concerned about this undesirable ion channel activity, as drugs to treat HIV are not given in isolation, but rather as cocktails of agents, to prevent the emergence of viral resistance. Many of the agents that are used in a clinical setting have interactions with cytochrome P450 enzymes that may profoundly affect the levels of drugs observed in the systemic circulation. This, combined with our desire to maintain a high plasma concentration of drug above the antiviral IC90 at all times, results in the demand for a large safety window. Thus, achieving good selectivity with respect to IKr blockade became a prime project objective. Unfortunately, all of the analogues in Table 7.2 proved to be unsuitable for further progression, whether due to a lack of antiviral efficacy or undesired HERG activity. Recent progress in the solution of several ion channel structures has greatly increased our understanding of the molecular determinants of binding of ligands to ion channels [57]. Although no crystal structures are available of the HERG channel itself, homology models based on the bacterial KcsA channel, coupled with site-directed mutagenesis studies [58], computational pharmacophore models [59], and a detailed analysis of the structure activity relationships of ligand series, have allowed us to build realistic homology models of the HERG channel, that can be used to predict how compounds might bind [60]. The HERG channel exists as a transmembrane spanning tetramer. By analogy with the crystal structure of the related KcsA channel, the protein consists of a large water filled pore that functions as a gateway from the intracellular side of the membrane to a set of helices that function as an ion selectivity filter by stripping the hydration shell from potassium ions and replacing these with backbone interactions, allowing the passage of ions to the extracellular side at diffusion limited rates. Basic compounds such as dofetilide are believed to bind in the channel, attracted by the negatively charged neck of the filter, making specific interactions with the aromatic amino acid residues lining the pore. Docking of both compounds into a model of the HERG ion channel [60] suggested that the introduction of polar groups into the amide substituent could increase selectivity against the HERG channel as more polar substituents would be less well accommodated by the hydrophobic amino acid residues that line the channel in the proximity of the aqueous pore, (Figure 7.4). The azetidine acetamide (23) (Table 7.3) had the desired effect substantially reducing channel affinity without impacting on antiviral potency. Further polar amide substituents are shown in Table 7.3. All analogues feature the endo substituted azabicyclo-octane as it was the most potent piperidine

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Fig. 7.4 Models of the HERG Channel. (a) HERG pharmacophore superimposed on the channel model, lipophilic binding areas shown as light grey rings, basic centre shown as a dark ball. Side chains are only shown for two of the four subunits of the channel protein for ease of viewing. (b) Dock of (15) into the HERG channel model.

replacement. Potassium channel activity could be avoided most effectively with strong hydrogen bonding groups, such as amides (23) and (24), and acid (25). However, the cell permeability as measured by apical to basal flux rate through a monolayer of Caco-2 cells [61] was compromised, predicting poor oral absorption in man. It is sometimes assumed that compounds which are fully ‘rule of five’ compliant [32], such as UK-395,859 (23) must have good permeability. However, for transcellular passive diffusion across membranes, although log P is an important consideration (and is incorporated in the rule of five), it can be more useful to treat this composite parameter in terms of its contributing factors – molecular weight and hydrogen bonding potential. As molecular weight increases one can achieve apparently acceptable lipophilicity by increasing hydrogen bonding potential. Unfortunately, this strategy has a major negative impact on permeability. For our series, hydrogen bonding potential, more than the overall log P; appears to determine the permeability [62, 63], and there is a clear relationship between cell permeability and the calculated polar surface area (PSA) of these molecules with the exception of compounds (26) and (27) (Table 7.3). ˚ 2 have Generally it is accepted that compounds with a PSA which exceeds 120 A 2 ˚ poor permeability, whereas compounds with PSA below 60 A have very good permeability [62, 63]. As this relationship is sigmoidal the transition from nonpermeable to permeable can be very sharp. It can be seen that, for this series of compounds, the transition from permeable to non-permeable is probably around ˚ 2, values in agreement with the work of others on other compound series 60 –80 A [64, 65]. Although the PSA of (26) and (27) are beyond this cut-off, cell

254

AVa IC90 (nM)

Kþ channel inhibitionb

CaCo-2 fluxc Papp (cm/s)

Polar surface area (A˚2)

(15)

3

80%, 300 nM

n.t.

45.1

(23)

1

0%, 300 nM

,1 £ 1026

76.9

(24)

1

0%, 300 nM

1.5 £ 1026

76.1

R

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

Table 7.3 ANTIVIRAL ACTIVITY, POTASSIUM CHANNEL ACTIVITY AND CACO-2 CELL ABSORPTION OF AMIDE SUBSTITUENTS WITH POLAR GROUPS

6

0%, 1 mM

,1 £ 1026

134.6

(26)

5

25%, 300 nM

10 £ 1026

122.8

(27)

3

10%, 300 nM

11 £ 1026

114.6

(28)

1

0%, 300 nM

4.9 £ 1026

66.0

(29)

3

16%, 1 nM

7 £ 1026

61.7

(30)

0.6

0%, 100 nM

23 £ 1026

58.9

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(25)

a

The concentration required to inhibit replication of HIVBaL into PM-1 cells by 90%. Percentage inhibition of tritium labelled dofetilide binding to HERG stably expressed on HEK-293 cells at different concentrations. c Apical to basal flux rate of compound through a monolayer of Caco-2 cells at 25 mM, pH 7.4. b

255

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THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

penetration does occur, presumably because of overestimation of the polarity of the tetrazole and nitrile groups (aided too perhaps, by the low molecular weight of (27)). We designed compounds with reduced hydrogen bonding potential in two ways. Firstly by relying on proximity effects for polar groups one can reduce the overall hydrogen bonding potential by isomerization, e.g. compounds (28) and (29), or alternatively, an aliphatic ether can be substituted for the amide function, thus exploiting an SAR element which sometimes improves the permeability of peptide isosteres [66 –68], e.g. compound (30). Both of these approaches improved permeability in line with our expectations and maintained good selectivity with respect to HERG binding. This compound (30) was profiled extensively. It is a potent inhibitor of binding of the chemokines MIP-1a, MIP-1b and RANTES, to CCR5. Interestingly, kinetic binding studies with radiolabelled (30) showed that the compound has a rapid onset rate onto the receptor, but has a slow offset from the receptor, with t1=2 ¼ 3:5 h [69]. It is highly selective for CCR5, is a functional antagonist and does not induce neutrophil chemotaxis to chemokines such as GRO, PAF, NAP-2, IL5 or C5A at concentrations up to 25 mM, highlighting its selectivity for CCR5 and the absence of the involvement of this receptor in these immune parameters. It is a potent inhibitor of HIVBaL in PM-1 cells and in peripheral blood mononuclear cells (PBMCs, IC90 ¼ 2 nM). This compound was devoid of cytotoxicity in parallel assays (up to 1 mM tested) and had no activity against the replication of the CXCR4-tropic isolate HIVIIIB in PBMCs, thereby supporting our conclusion that the antiviral activity of this compound was entirely attributable to blockade of CCR5. The compound was also clean in AMES. Unfortunately, when pharmacokinetic experiments on (30) were performed, the data was less favourable. Dog and human hepatocyte clearance was high, 27 and 14 ml/min/kg, respectively. In vivo, dog clearance was approaching liver blood flow and oral bioavailability was , 10% due to extensive first pass metabolism, which was predicted to be similar in man. Our attention turned to trying to define the lipophilicity window that would allow us to find compounds with adequate flux in combination with reasonable metabolic stability. Analysis of the data that we had on our series to date identified a narrow window of lipophilicity centred around a log D , 2:0; where compounds with both appropriate human liver microsomal stability and Caco-2 flux (apical to basolateral membrane) were found (Figure 7.5). It should be emphasized that focusing on this region does not guarantee success but merely increase the probability relative to other parts of the distribution where no solution is likely to be found. Our problem however, was even more multidimensional. We also needed to maintain antiviral potency and minimize affinity for the HERG channel. To address these issues, we decided to look back at some of our early SAR in search of compounds that were intrinsically more

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257

Fig. 7.5 A graph of human liver microsome stability and Caco-2 flux against log D for the benzimidazole series indicating our key target window for log D:

active than our benzimidazoles, in the hope that these would allow us to reduce molecular weight and hence reduce lipophilicity, without having to increase the hydrogen bonding potential. In the course of our early programme we had looked at alternative heterocycles as replacements for the benzimidazole moiety on the original piperidine ring system (Table 7.4). We were particularly intrigued by the 1,2,4triazole (36), a compound that achieved good CCR5 affinity despite its low molecular weight (, 400). Initially, lack of antiviral activity for this compound had limited our interest. However, we now felt that the dramatically reduced lipophilicity of the triazole gave us greater flexibility to modify and optimize potential hydrophobic interactions with the receptor to improve antiviral activity. We felt that this reduced lipophilicity also gave the triazole (36) an advantage over the essentially equipotent oxadiazoles (31) and (32). Transfering our learning from the benzimidazole series, we synthesized the isomeric exo and endo tropane isomers of the triazoles for direct comparison. These compounds were now also prepared as single ðSÞ enantiomers, and to ensure that we explored our target lipophilicity window properly, we prepared both 3,5-dimethyl and 3-isopropyl-5-methyl substituted triazole analogues (Table 7.5). The 3,5-dimethyltriazoles (37) and (38) display similar potencies. However, increasing the steric demands of the substituents of the triazole with the 3-isopropyl-5-methyltriazoles (39) and (40), results in a clear separation of activity between the exo and endo series. From NMR studies it is again apparent

258

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

Table 7.4 MIP-1b INHIBITORY ACTIVITY AND ANTIVIRAL ACTIVITY OF PIPERIDINE ANALOGUES

R

MIP-1b a IC50 (nM)

AV b IC50 (nM)

cLog P

(13)

40

75

3.2

(31)

90

2920

1.4

(32)

70

(33)

n.t.

1.4

730

.10000

0.5

(34)

540

.10000

0.8

(35)

900

.10000

1.1

(Continued)

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259

Table 7.4 CONTINUED R (36)

MIP-1b a IC50 (nM)

AV b IC50 (nM)

cLog P

49

.10000

0.8

n.t.: not tested. a The concentration required to inhibit binding of [125I]MIP-1b by 50%. b The concentration required to inhibit replication of HIVBaL into PM-1.

that to minimize 1,3-diaxial strain between the triazole and the carbon bridge of the tropane, in the endo series the tropane adopts a pseudo-boat conformation, rather than the normally preferred chair conformation, as adopted by exo series. As a consequence of these conformational effects the triazole lies in approximately the same geometric position for both the exo and endo series. As such, it is difficult to rationalize the dramatically deleterious effect observed for isopropyl substitution in the endo series, other than to postulate that the greater steric crowding for (40) results in a subtle conformational difference that is less well tolerated by the receptor. From the antiviral data alone there was little to discriminate between compounds (38) and (39), so both were progressed further (Table 7.6). Table 7.5 ANTIVIRAL ACTIVITY OF TRIAZOLE TROPANE ANALOGUES Exo

Endo

(37) R ¼ Me AV IC90 ¼ 13 nMa (38) R ¼ i-Pr AV IC90 ¼ 8 nM

(39) R ¼ Me AV IC90 ¼ 6 nM (40) R ¼ i-Pr AV IC90 ¼ 101 nM

a

The concentration required to inhibit replication of HIVBaL into PM-1.

260

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857 Table 7.6 IN VITRO DATA FOR COMPOUNDS (38) AND (39)

(38) (39) a

HLM (min)

Kþ channel inhibitiona

Log D

Caco-2 ( £ 1026 cm/s)

Polar surface area (A˚2)

55 45

30%@ 300 nM 33%@ 300 nM

1.6 1.3

4.5 3.9

75.5 87.8

Percentage inhibition of tritium labelled dofetilide binding to HERG stably expressed on HEK-293 cells at different concentrations.

Calculated polar surface areas indicated that both compounds lie in the range that we had previously observed as being in the transition zone from permeable to non-permeable, and this was confirmed by the moderate Caco-2 flux values that were obtained for both. Although compound (38) showed some inhibition of the HERG potassium channel in vitro, it was assessed further as a potential candidate for clinical

Fig. 7.6 Graph of percentage change in inhibition of dofetilide binding or purkinje fibre APD prolongation for (38) versus concentration of drug, expressed as a multiple of the predicted clinical concentration (based on a AV IC90 8 nM for (38)), with comparison curves for terfenadine, terfenadine and ketoconazole, and an ‘ideal’ candidate profile.

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development. The compound was potent and stable and showed acceptable absorption and dose predictions [70] based on dog (t1=2 ; 7 h; Clou 172 ml/min/kg, F 43%, absorption . 80%, estimated human dose 100 mg b.i.d.) and rat pharmacokinetics (absorption 20%, estimated human dose 350 mg b.i.d.). The dofetilide binding assay that we use routinely as a high-throughput screen to measure inhibition of the HERG channel, while effective as a screening tool, is limited by being based on a single channel type, which has been cloned and expressed to a high level in a cell line. Therefore, (38) was tested in dog Purkinje fibre to better assess the QTc risk potential. This has the advantage of giving a view of the functional relevance of any and all ion channel inhibition for a compound. Unfortunately, (38) showed a terfenadine-like selectivity window between our predicted free plasma levels needed to maintain antiviral coverage and QTc threshold effects (Figure 7.6). This meant that drug –drug interactions with CYP450 inhibitors could lead to clinical QTc prolongation, allowing for substantial variation in total exposure and peak to trough concentrations when co-administered with other agents such as Ritonavir, frequently used as a component of a HAART regime. Despite this setback we were encouraged that a modest increase in the antiviral potency of (38) to , 1 nM levels whilst maintaining the other properties at a similar level, would deliver a candidate with an acceptable therapeutic window. This optimism, however, had to be tempered with the knowledge that antiviral potency appeared to be strongly linked to lipophilicity. For example, homologation of the cyclobutyl amide (38) to the cyclopentyl amide (41) (AV IC90 ¼ 1.6 nM, Log D 2.1, HLM 21 min, Caco-2 5.6 £ 1026 cm/s) gave both a predictable increase in potency and a predictable reduction in stability.

THE IDENTIFICATION OF UK-427,857 We decided to focus firstly on making further small changes to the triazole heterocycle (Table 7.7). As can be seen for analogues (42 –46), simply varying the triazole substituent was generally deleterious for antiviral activity. Furthermore, inhibition of the HERG channel was moderately increased for

262

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857 Table 7.7 ANTIVIRAL ACTIVITY OF TRIAZOLE ANALOGUES

R

AVa IC90 (nM)

HLM (min)

Log D

HERG Channel (%I @ 300 nM)

(38)

8

55

1.6

30%

(42)

9

n.t.

1.1

34%

(43)

60

19

2.2

53%

(44)

.100

22

2.3

46%

(45)

n.t.

32

2.0

48%

98

1.0

42%

(46)

77

(Continued)

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Table 7.7 CONTINUED AVa IC90 (nM)

HLM (min)

Log D

HERG Channel (%I @ 300 nM)

(47)

29

56

2.1

65%

(48)

1

13

2.3

85%

R

n.t. not tested. a The concentration required to inhibit replication of HIVBaL into PM-1.

all of these analogues, generally tracking with increased lipophilicity (but not always, see, for example, compound (46). Replacement of the 1,2,4-triazole with a 1,3,4-triazole gave compound (47), which possessed dramatically improved cell permeability (Caco-2 30 £ 1026 cm/s, apical –basal) for a compound with an identical molecular weight. This is presumably due to the smaller dipole moment of the 1,3,4-triazole (3.0 Debye) compared to the 1,2,4-triazole (6.1 Debye) resulting in overall weaker hydration for compound (47). However, HERG channel inhibition was also increased and antiviral activity reduced. The polarity of the triazole may be important for reducing the level of interaction with the lipophilic aromatic residues that line the channel mouth. Certainly, the triazole moiety lies in a region in our HERG pharmacophore model where lipophilicity is preferred, and so may have a similar effect to the polar amide groups described earlier. The imidazole analogue (48) possessed improved antiviral potency. However, both reduced microsomal stability and ion channel effects limited our interest in this series. We adjusted our focus to modifying the amide moiety within the series and our synthesis was such that the amide coupling could be undertaken as the final step. This again enabled us to use parallel chemistry techniques to assess the potential chemical space of this region as rapidly as possible. Within Table 7.8 we have described a small number of analogues that demonstrate our key SAR learning from these modifications. As we noted before, replacement of the cyclobutyl amide in (38) with the cyclopentyl amide (41), gave an increase in antiviral potency and cell permeability; however, microsomal stability decreased. This can all be attributed to an increase in lipophilicity. Excitingly though, when we tested (41)

264

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857 Table 7.8 ANTIVIRAL ACTIVITY OF AMIDE ANALOGUES

R

MIP1b (nM)

AVa IC90 (nM)

HLM (min)

Log D

HERG Channel (%I @ 300 nM)

(49)

5

n.t.

43

1.9

n.t.

(50)

3

n.t.

95

1.5

n.t.

(51)

15

n.t.

.120

n.t.

n.t.

(52)

5

14

.120

1.8

14%

(53)

26.5

125

n.t.

n.t.

0%

(54)

2

2.1

0%

1.0

51

n.t. not tested. a The concentration required to inhibit replication of HIVBaL into PM-1.

in the dofetilide binding assay it appeared that the ion channel effects were decreasing, presumably due to a steric clash with the channel. Mass spectrometry data on the products of microsomal metabolism of (41) suggested that metabolism was primarily occurring on the left-hand side amide, so the trifluoropropionoyl amide (51) and trifluorobutanoyl amide (52) were prepared as blocking groups. Although showing relatively poor antiviral potency, (52) displayed excellent microsomal stability and again improved ion channel effects compared to (38). It was apparent that modification of the amide could

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simultaneously limit oxidative metabolism and increase steric demands to generate selectivity over the HERG potassium channel. Using this knowledge gave the impetus to prepare the 4,40 -difluorocyclohexylamide, UK-427,857 (54) which possessed excellent antiviral potency and reasonable microsomal stability combined with good selectivity over potential ion channel effects. Subsequently (54) was tested at 1,000 nM and showed no significant binding to the HERG potassium channel. The compound also possessed the required levels of aqueous solubility and could be crystallized as the free base (Figure 7.7). With these promising biological and physiochemical properties, the compound was progressed to in vivo pharmacokinetic profiling. Predictions to man suggested that an oral 100 mg dose twice daily would provide a free drug concentration of greater than the antiviral IC90 throughout the dosing regime. UK-427,857 (54) was also found to show no significant inhibition (IC50 . 100 mM) of any of the major P450 isoforms tested (1A2, 2C9, 3A4) and , 50 mM against CYP2D6. This is . 30,000-fold the target free plasma concentration, suggesting that the drug will not affect metabolism of coadministered agents. This would be a key benefit when compared to currently marketed therapies [71]. The potency of UK-427,857 (54) was encouraging when tested against HIVBaL in peripheral blood monocytes (PBMCs) where the receptor is expressed under its native conformations (IC90 ¼ 6 nM), but was even more exciting when extensively tested against primary origin HIV isolates. In a PBMC assay, UK-427,857 showed potent cross-clade antiviral activity against all CCR5-tropic isolates with a geometric mean IC90 of 2.03 nM. UK-427,857 (54) possesses high selectivity over a range of other chemokine receptors, particularly CCR2b which is the most closely related chemokine receptor genetically [72], as well as a range of pharmacologically relevant targets [73]. As with previous compounds in this series, no cytotoxicity or activity against CXCR4-tropic HIV

Fig. 7.7 X-ray crystal structure of UK-427,857 (54).

266

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

isolates was observed, supporting the conclusion that antiviral activity was entirely attributable to CCR5 blockade [74, 75]. Interestingly, UK-427,857 activity seemed insensitive to changes in multiplicity of infection (MOI) in our antiviral assays and showed no variation in potency against MIP-1b binding to CCR5 at varying chemokine concentrations. Receptor offset studies using tritiated UK-427,857 indicated that the non-competitive behaviour is probably a consequence of slow receptor offset kinetics ðt1=2 . 8 hÞ [75]. This profile was achieved with some trade-off in our permeability goals leading to a predicted bioavailability of 10% driven by 20% permeability and 50% first pass loss. We rationalized this parameter as the most appropriate one to compromise on for a number of reasons. Firstly, absorption could be readily evaluated early in the clinical development programme. In addition to this, whilst UK-427,857-like Caco-2 flux is likely to give incomplete absorption, it does not guarantee it! This is especially true for low dose, soluble, stable compounds, that are not significant Pgp substrates, as under these conditions slow absorption can be achieved without significant gut wall metabolismmediated first pass loss [32, 76]. Since, UK-427,857 has good metabolic stability, is only a moderate Pgp substrate (as shown by knock-out mouse exposure studies) and is readily soluble we were confident that the molecule possessed at least the right profile to mitigate against low permeability. UK-427,857 (54) was, therefore, progressed as a clinical candidate for the treatment of HIV infected individuals. We were delighted when Phase I human data confirmed that at a clinical exposure of 100 mg we achieved our desired objective of maintaining plasma levels above the IC90 at trough within the window provided by a b.i.d. regime. Not too surprisingly, given the slow absorption profile the Tmax is relatively late and we do see food effects with respect to the pharmacokinetic profile [77]. Phase II monotherapy studies with UK-427,857 resulted in impressive efficacy (, 1.42 log reduction in circulatory HIV RNA copy number) following 10 days monotherapy at 100 mg b.i.d. [78]. Further details of the clinical evaluation of this compound will be reported in due course. OTHER CCR5 ANTAGONISTS A number of other approaches to CCR5 antagonists have been reported in the literature. It is not our intention to review all of these here as such reviews already exist [79, 80], but rather to briefly highlight a few key compounds of interest which have undergone or are undergoing clinical investigation. Workers at Takeda have described TAK-779 (55), a quaternary ammonium compound intended for subcutaneous delivery [38, 81]. Although potent in vitro, clinical development appears to have been curtailed, at least partly due to adverse reactions associated with the route of administration.

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Several development compounds have been reported by workers at ScheringPlough including Sch-351125/Sch-C (56) [39, 40] including some preliminary clinical data [82], and Sch-417690/Sch-D (57) [83]. Sch-D has been reported to be more potent in vitro, have reduced HERG affinity and similarly, also to be under clinical investigation.

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Workers at Ono have discovered the spirodiketopiperazine AK602/ONO4128/GW873140 (58), a compound which is now in Phase II clinical trials [84]. Although very little data has been reported to date on this compound, it is a development from a series that this group has described previously [85]. CONCLUSION The path from the HTS hits (1) and (2) to the development candidate UK427,857 (54) proved to be challenging, taking two and a half years and 965 analogues. At times it appeared impossible to achieve the delicate balance of antiviral activity, metabolic stability, absorption and ion channel activity that UK-427,857 represents. A large number of colleagues too numerous to list here from an array of disciplines were instrumental in the identification of UK427,857 through their contributions to the HTS, chemical synthesis, biological, pharmacokinetic and safety screening, and many more colleagues have contributed in the subsequent development and clinical phases of the programme. We acknowledge all of their efforts, and hope that UK-427,857 or another CCR5 receptor antagonist eventually finds its way into the armoury of therapeutic agents for the treatment of HIV and AIDS. REFERENCES [1] Barre-Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W. and Montagnier, L. (1983) Science 220, 868 –871. [2] Popovic, M., Sarin, P.S., Robert-Gurroff, M., Kalyanaraman, V.S., Mann, D., Minowada, J. and Gallo, R.C. (1983) Science 219, 856 –859. [3] http://www.unaids.org. [4] Fauci, A.S. (2003) Nat. Med. 9, 839 –843. [5] Pomerantz, R.J. and Horn, D.L. (2003) Nat. Med. 9, 867–873. [6] Carr, A. (2003) Nat. Rev. Drug. Disc. 2, 624–634. [7] Duran, S., Save`s, M., Spire, B., Cailleton, V., Sobel, A., Carrieri, P., Salmon, D., Moatti, J.-P. and Leport, C. (2001) AIDS 15, 2441–2444.

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[59] Cavalli, A., Poluzzi, E., De Ponti, F. and Recanatini, M. (2002) J. Med. Chem. 45, 3844– 3853. [60] Perry, M., de Groot, M.J., Helliwell, R., Leishman, D., Tristani-Firouzi, M., Sanguinetti, M.C. and Mitcheson, J. (2004) Mol. Pharmacol. 66, 240–249. [61] Artursson, P., Palm, K. and Luthman, K. (1996) Adv. Drug Deliv. Rev. 12, 67–84. [62] Conradi, R.A., Hilgers, A.R., Ho, N.F.H. and Burton, P.S. (1991) Pharm. Res. 8, 1453– 1460. [63] Van de Waterbeemd, H. and Jones, B. (2003) Prog. Med. Chem. 41, 1–59. [64] Palm, K., Stenberg, P., Luthman, K. and Artursson, P. (1997) Pharm. Res. 14, 568–571. [65] Kelder, J., Grootenhuis, P.D.J., Bayada, D.M., Delbressine, L.P.C. and Ploemen, J.P. (1999) Pharm. Res. 16, 1514–1519. [66] Szelke, M., Jones, D.M. and Hallett, A. (1982) Eur. Pat. Appl. EP 45665. [67] Holladay, M.W. and Rich, D.H. (1983) Tetrahedron Lett. 24, 4401–4404. [68] Holladay, M.W., Salituro, F.G. and Rich, D.H. (1987) J. Med. Chem. 30, 374 –383. [69] Dorr, P., Dobbs, S., Rickett, G., Lewis, B., Macartney, M., Westby, M. and Perros, M. (2003) 43rd Annual InterScience Conference on Antimicrobial Agents and Chemotherapy, Chicago, USA, Poster. 2003 F1462. [70] Van de Waterbeemd, H., Smith, D.A., Beaumont, K. and Walker, D.K. (2001) J. Med. Chem. 1313–1333. [71] Piscitelli, S.C. and Gallicano, K.D. (2001) N. Engl. J. Med. 344, 984 –996. [72] Premack, B.A. and Schall, T.J. (1996) Nat. Med. 2, 1174–1178. [73] Napier, C., Dorr, P., Gladue, R., Haliday, R., Leishman, D., Machin, I., Mitchell, R., Nedderman, A., Perros, M., Roffey, S., Walker, D. and Webster, R. (2003) 10th Conference on Retroviruses and Opportunistic Infections, Boston, USA. [74] Macartney, M.J., Dorr, P.K., Smith-Burchnell, C., Mori, J., Westby, M., Hitchcock, C. and Perros, M. (2003) 43rd Annual InterScience Conference on Antimicrobial Agents and Chemotherapy, Chicago, USA, Poster H-875. [75] Dorr, P., Macartney, M., Rickett, G., Smith-Burchnell, C., Dobbs, S., Mori, J., Griffin, P., Lok, J., Irvine, R., Westby, M., Hitchcock, C., Stammen, B., Price, D., Armour, D., Wood, A. and Perros, M. (2003) 10th Conference on Retroviruses and Opportunistic Infections, Boston, USA. [76] Curatolo, W. (1998) Pharm. Sci. Technol. Today 1, 387–393. [77] Abel, S., Van der Ryst, E., Muirehead, G., Rosario, M., Edgington, A. and Weissgerber, G. (2003) 10th Conference on Retroviruses and Opportunistic Infections, Boston, USA. [78] Pozniac, A.L., Fatkenheuer, G., Johnson, M., Hoepelman, I.M., Rockstroh, J., Goebel, F., Abel, S., James, I., Rosario, M., Medhurst, C., Sullivan, J., Youle, E. and Van der Ryst, E. (2003) 43rd Annual InterScience Conference on Antimicrobial Agents and Chemotherapy, Chicago, USA, Slide Session E354A. [79] Mills, S.G. and DeMartino, J.A. (2004) Curr. Top. Med. Chem. 4, 1017–1033. [80] Seibert, C. and Sakmar, T.P. (2004) Curr. Pharm. Des. 10, 2041–2062. [81] Baba, M., Nishimura, O., Kanzaki, N., Okamoto, M., Sawada, H., Iizawa, Y., Shiraishi, M., Aramaki, Y., Okonogi, K., Ogawa, Y., Meguro, K. and Fujino, M. (1999) Proc. Natl. Acad. Sci. 96, 5698–5703. [82] Reynes, J., Rouzier, R., Kanouni, T., Baillat, V., Baroudy, B., Keung, A., Hogan, C., Markowitz, M. and Laughlin, M. (2002) AIDS-14th International Conference, Barcelona, Spain. [83] Tagat, J.R., McCombie, S.W., Nazareno, D., Labroli, M.A., Xiao, Y., Steensma, R.W., Strizki, J.M., Baroudy, B.M., Cox, K., Lachowicz, J., Varty, G. and Watkins, R. (2004) J. Med. Chem. 47, 2405–2408. [84] Maeda, K., Ogata, H., Harada, S., Miyakawa, T., Nakata, H., Koh, Y., Tojo, Y., Shibayama, S., Takaoka, Y., Sagawa, K., Daikichi, F., Moravek, J., Arnold, E. and Mitsuya, H. (2004) AIDS15th International Conference, Bangkok, Thailand, Poster 540. [85] Maeda, M., Yoshimura, K., Shibayama, S., Habashita, H., Tada, H., Sagawa, K., Miyakawa, T., Aoki, M., Fukushima, D. and Mitsuya, H. (2001) J. Biol. Chem. 276, 35194–35200.

Subject Index Bortezomib, 155 B-raf kinase, 36 BodipyTM, 34 Bruceolides, 215 Carbonates, 215 3, 15-O-diacetyl, 215 Budmunchiamines L4 & L5, 198

AC-42, 116 (4R)-4-Acetoxyartemesinin, 211 Acridines, 220 Acridone alkaloids, 197 AF-DX 116, 123 AF-DX 384, 123 Ajugarin-1, 199 AlexaFluorTM, 34 Alvameline, 113 Alzheimer’s disease M1 agonists in, 113 8-Aminoquinolines, 220 Amodiaquine, 216 Ancristogriffine A, 194 Ancristogriffithine A, 194 Anopheles gambiae genome sequence, 227 Anthraquinones, 202 Anxiety neurokinin receptors in, 53 Aprepitant, 53, 54, 96 Arborinine, 197 Arteether, 207 Artelinate sodium, 207 Artesunate sodium, 207 Artemether, 207 Artemisinin, 192, 206 11-aza, 212 13-carba, 212 Dihydro, 207 Dimers, 212 Ring contracted, 213 Arthritis neurokinin receptors in, 53 AS-602868, 165 Astemizole, 1 Asthma neurokinin receptors in, 53 Atropine, 107 Autofluorescence, 45 Azabicyclooctane, 252

CaCo2 flux, 254, 263 Carbazoles, 198 Casual prophylactics, 192 CCR5 Receptor, 239 Antagonists, 266 Assay, 241 CDP-840, 12 CEP-1612, 177 CGP-49823, 54, 72, 78 CGP-60829, 72, 73 Chalcones, 223 Chaparrinone, 200 Chemosensitizers, 226 Chloroquine, 192, 216 CHS-828, 168 Cisapride, 5 CJ-11974, 54 Cordytropolone, 204 Coumarins, 201 E-p-Coumaroylalphitolic acid, 200 CP-122721, 54, 57 CP-96345, 54, 57 CP-99994, 54, 80, 96 Crotsparine, 196 Cryptolepine, 197 Cryptotanshinone, 199 Cuvettes, 24 Cyclin dependent protein kinases (CDKs), 225 Cycloguanil, 221 CyDyeTM, 33 Cytochrome P450 2D6, 244, 248, 265

Beer’s law, 30 BIBN-99, 118 BIRB-796, 160 Bisacridines, 220 Bisquinolines, 219 BMS-345541, 164

4-DAMP, 123 Darifenacin, 126, 131 Dehydrodiconiferyl dibenzoate, 203 273

274

SUBJECT INDEX

Dehydrocrebanine, 195 Dehydrostephanine, 195 Depression neurokinin receptors in, 53 10-Deoxyartemesinin, 210 Detrusitol, 108 Dihydroartemisinin, 207 7, 7-Difluoroarteether, 211 Dioncophylline A, 193 Dioncophylline C, 193 DNK-333, 57, 76 Dorsal, 142 Double drugs, 217 DPI210-106, 5 Electrocardiogram (ECG), 3 Ellagic acid, 203 Emesis neurokinin receptors in, 53, 54 Ergosterol-5, 8-endoperoxide, 199 Eunicellins, 205 Excited state lifetime, 27 Falcipain, 225 Fascaplysin, 205 Febrifugine, 214 FK-224, 57, 69 FK-888, 56, 96 Flavonoids, 201 Fluorescence Absorbance, 30 Anisotropy, 28 Assays, 19 Binding assays, 38 Kinetic, 40 Polarization-based immunoassays (FPIA), 39 Cells, 24 Emission spectra, 27 Excitation spectra, 26 Intensity, 30 Lifetime, 27 Polarization, 28 Quantum yield, 27 Quenching, 45 Polarization, 39 Resonance energy transfer, 31 Signal detection, 25 Fluoroscein, 33 Fluorophores, 32 Fo˝rster resonance energy transfer (FRET), 31

Gametocytes, 191 Gametocytocides, 192 Gedunin, 199 Genome sequence, 227 Germacradienes, 199 Glucose-6-phosphate dehydrogenase (G6PD), 220 Glutathione reductase inhibitor, 217 Glycogen synthase kinase (GSK), 150, 159 Inhibitors, 168 GR-159897, 57 GR-205171, 55, 56, 96 Grepafloxacin, 1 Hadranthanine, 197 Halofantrine, 224 Hemokinin 1, 50 Heptaphylline, 197 trans-Hexahydrodibenzopyran derivative, 201 High throughput screening, 43 Himbacine, 120 HIV, 239 Homofascaplysin, 205 Human Ether-a-go-go (HERG), 1 Binding assay, 6 Blockers, 8, 252, 260 Blockers: pharmacophore model, 9 Structure, 7 Hyoscine, 107 IkB, 138, 151 IKK, 140, 149, 156 Inhibitors, 161 Imaging readers, 45 Indolo[3, 2-c]quinolines, 218 Isofebrifugine, 214 Isoflavanoids, 201 KDR kinase inhibitor, 15 Klugine, 196 Krukovine, 194 Kudtriol, 199 L-758298, 54, 96 Lanepetant, 56, 96 Ligand efficiency, 242 Lignans, 203 Limonoids, 199

SUBJECT INDEX Lupeol, 200 LY-303870, 54, 64 LY-593093, 115 Lycorine, 197 Machaeridiol B, 203 Machaeriol B, 201 Malaria, 189 Manzamines, 204 Maprotiline, 227 MDL-105172A, 69 MDL-105212A, 56, 69 Mefloquine, 192 MEN-10627, 57, 67 MEN-11420, 96 MEN-11558, 67 Mepacrine, 192, 220 Merozoites, 191 Methoctramine, 123 Migraine neurokinin receptors in, 53, 56 Milameline, 113 MK499, 10 MK 869, 54 MLN-120A, 162 Monochromator, 23 Muscarinic Receptors, 105 M1 Subtype, 112 Allosteric regulation, 116 M2 Subtype, 118 Allosteric regulation, 122 M3 Subtype, 122 M4 Subtype, 128 M5 Subtype, 130 Naphthquinones, 202 Neurokinin Receptor Antagonists, 49 Neuropeptide K, 50 Neuropeptide Y5 receptor antagonist, 13 NFkB, 137 In development, 141 In immunity, 143 In cancer, 146 NIK inhibitors, 169 Nitidine, 196 NKP-608, 59 Normelicopicine, 197 Norsesterterpene peroxide acids, 206

275

ONO-4128, 268 Optical density, 30 Osanetant, 58 Otenzepad, 123 Oxybutynin, 127, 131 Pain neurokinin receptors in, 53, 56 PD 0298029, 129 PD 98059, 160 PDE4 inhibitor, 12 Peptidomimetics, pyrimidyl, 225 Peroxides, 222 Peroxy ketals, 222 Perrin-Jabl⁄ on´ski diagram, 20 pFHSiD, 123 Photomultiplier tubes, 25 Pilocarpine, 106 Pimobendan, 147 Plakortide F, 206 Plasmepsin, 225 Plasmodium, 190 Plasmodium flaciparum dihydrofolate reductase (PfDHFR), 226 Plasmodium flaciparum gene sequence, 227 Pleuromutilin antibiotic, 36 Point readers, 44 Polar Surface Area (PSA), 253 8-Prenylmucronulatol, 201 Preussomerins, 204 Primaquine, 192, 220 Proguanil, 192, 221 Propofol, 112 10b-Propyldeoxoartemesinin, 210 Proteosome inhibitors, 155, 175 Protoberbine alkaloids, 196 PS-15, 221 Puromycin antibiotic, 38 Pyrimethamine, 192 Pyronaridine, 218 Quassinoids, 199 Quinacrine, 220 Quinine, 192 Quinoid diterpenes, 199 QT interval, 4 R-113281, 79 Rayleigh scattering, 24

276

SUBJECT INDEX

Raman scattering, 24 Reactive oxygen species, 154 Rel, 138 Roemrefidine (-), 195 Rotenoid, 201 RS 86, 113

SR-48968, 57, 96 SSR-146977, 68 SSR-240600, 63 Stokes shift, 21 Strychnogucine A & B, 197 SUN-C8079, 174

S-16474, 57, 69 Sabcomeline, 113 Samaderine X, 199 Sampangine, 197 Sarachine, 198 Saredutant, 57 SB-400238, 94 SB-452466, 36 SB-477790, 35 SCH-206272, 85 SCH-211803, 118 SCH-217443, 118 SCH-351125, 252, 267 SCH-417690, 267 SCH-57790, 118 SCH-72788, 118 Schizonts, 190 Schizontocides, 192 Schizophrenia Neurokinin receptors in, 53 M1 agonists in, 113, 117 M2 antagonists in, 121 M4 antagonists in, 129 Scopadulcic acid, 199 SDZ-210086, 114 Sertindole, 11 Sesquilignans, 203 Sesquineolignans, 203 Sigmosceptrellin-B, 206 Solifenacin, 126 SPC-495, 166 SPC-839, 166, 174 Spectrofluorimeter, 22 Sporonticides, 192 Sporozoites, 190 SR-140333, 54 SR-142801, 58 SR-144190, 66

Tachykinins, 50 Tagitinin C, 199 TAK-637, 56, 62, 97 TAK-779, 266 Talsaclidine, 113 Tebuquine, 217 Terfenadine, 1 Tetramethylrhodamine, 33 Tetraoxane, 222 Time resolved energu transfer (TRET), 40 Tolterodine, 108, 125, 131 Toll-like receptors, 144 Triazole antivirals, 259, 262 1, 2, 4-trioxanes, tricyclic, 213 Trospium, 127 Ubiquitinylation, 153 UCN-01, 159 UK-224671, 66 UK-290795, 66 UK-395859, 253 UK-427857, 239 Velcade, 155, 176, 178 WIN 62577, 116 WR148999, 222 Xanomeline, 113, 117 Xantholides, 199 YM-38336, 69 YM-44778, 69 YM-796, 114 ZD-6021, 91 ZD7944, 64, 86

Cumulative Index of Authors for Volumes 1– 43 The volume number, (year of publication) and page number are given in that order. Bischoff, E., 41 (2003) 249 Black, M.E., 11 (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

Aboul-Ela, F., 39 (2002) 73 Adams, J.L., 38 (2001) 1 Adams, S.S., 5 (1967) 59 Afshar, M., 39 (2002) 73 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 Altmann, K.-H., 42 (2004) 171 Andrews, P.R., 23 (1986) 91 Ankersen, M., 39 (2002) 173 Ankier, S.I., 23 (1986) 121 Arrang, J.-M., 38 (2001) 279 Armour, D., 43 (2005) 239 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 Beher, D., 41 (2003) 99 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

Camaioni, E., 42 (2004) 125 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) 1; 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 Clitherow, J.W., 41 (2003) 129 277

278

CUMULATIVE AUTHOR INDEX

Cobb, R., 5 (1967) 59 Cochrane, D.E., 27 (1990) 143 Corbett, J.W., 40 (2002) 63 Costantino, G., 42 (2004) 125 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

Ferguson, D.M., 40 (2002) 107 Feuer, G., 10 (1974) 85 Finberg, J.P.M., 21 (1984) 137 Fletcher, S.R., 37 (2000) 45 Flo¨rsheimer, A., 42 (2004) 171 Floyd, C.D., 36 (1999) 91 Franc¸ois, I., 31 (1994) 297 Frank, H., 27 (1990) 1 Freeman, S., 34 (1997) 111 Fride, E., 35 (1998) 199

Dabrowiak, J.C., 24 (1987) 129 Daly, M.J., 20 (1983) 337 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 Deaton, D.N., 42 (2004) 245 Demeter, D.A., 36 (1999) 169 Denyer, J.C., 37 (2000) 83 Derouesne´, 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 Drysdale, M.J., 39 (2002) 73 Dubinsky, B., 36 (1999) 169 Duckworth, D.M., 37 (2000) 1 Duffield, J.R., 28 (1991) 175 Durant, G.J., 7 (1970) 124

Gale, J.B., 30 (1993) 1 Ganellin, C.R., 38 (2001) 279 Garbarg, M., 38 (2001) 279 Garratt, C.J., 17 (1980) 105 Gerspacher, M., 43 (2005) 49 Gill, E.W., 4 (1965) 39 Ginsburg, M., 1 (1961) 132 Glennon, R.A., 42 (2004) 55 Goldberg, D.M., 13 (1976) 1 Gould, J., 24 (1987) 1 Graczyk, P.P., 39 (2002) 1 Graham, J.D.P., 2 (1962) 132 Green, A.L., 7 (1970) 124 Green, D.V.S., 37 (2000) 83 Green, D.V.S., 41 (2003) 61 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

Eccleston, J.F., 43 (2005) 19 Edwards, D.I., 18 (1981) 87 Edwards, P.D., 31 (1994) 59 Eglen, R.M., 43 (2005) 105 Eldred, C.D., 36 (1999) 29 Ellis, G.P., 6 (1969) 266; 9 (1973) 65; 10 (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

Hacksell, U., 22 (1985) 1 Haefner, B., 43 (2005) 137 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 Haning, H., 41 (2003) 249 Hanson, P.J., 28 (1991) 201 Hanus, L., 35 (1998) 199 Hargreaves, R.B., 31 (1994) 369

CUMULATIVE AUTHOR INDEX Harris, J.B., 21 (1984) 63 Harrison, T., 41 (2003) 99 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) 1; 29 (1992) 141 Heller, H., 1 (1961) 132 Henke, B.R., 42 (2004) 1 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 Hu, B., 41 (2003) 167 Hubbard, R.E., 17 (1980) 105 Hudkins, R.L., 40 (2002) 23 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 Hutchinson, J.P., 43 (2005) 19 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 Jameson, D.M., 43 (2005) 19 Ja´szbere´nyi, J.C., 12 (1975) 395; 14 (1977) 181 Jenner, F.D., 11 (1975) 193 Jennings, L.L., 41 (2003) 167 Jewers, K., 9 (1973) 1 Jindal, D.P., 28 (1991) 233 Jones, B.C., 41 (2003) 1 Jones, D.W., 10 (1974) 159 Jorvig, E., 40 (2002) 107

279

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; 43 (2005) 189 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 King, F.D., 41 (2003) 129 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 Krause, B.R. (2002) 121 KrogsgaardLarsen, P., 22 (1985) 67 Kulkarni, S.K., 37 (2000) 135 Kumar, K., 43 (2005) 189 Kumar, M., 28 (1991) 233 Kumar, S., 38 (2001) 1 Kumar, S., 42 (2004) 245 Kwong, A.D., 39 (2002) 215 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 Lentzen, G., 39 (2002) 73 Levin, R.H., 18 (1981) 135 Lewis, A.J., 19 (1982) 1; 22 (1985) 293 Lewis, D.A., 28 (1991) 201 Lewis, J.A. 37 (2000) 83 Li, Y., 43 (2005) 1 Lien, E.L., 24 (1987) 209 Ligneau, X., 38 (2001) 279

280

CUMULATIVE AUTHOR INDEX

Lin, T.-S., 32 1995) 1 Liu, M.-C., 32 (1995) 1 Lloyd, E.J., 23 (1986) 91 Lockhart, I.M., 15 (1978) 1 Lord, J.M., 24 (1987) 1 Lowe, I.A., 17 (1980) 1 Lucas, R.A., 3 (1963) 146 Lue, P., 30 (1993) 206 Luscombe, D.K., 24 (1987) 249 Mackay, D., 5 (1967) 199 Main, B.G., 22 (1985) 121 Malhotra, R.K., 17 (1980) 151 Malmstro¨m, R.E., 42 (2004) 207 Manchanda, A.H., 9 (1973) 1 Mander, T.H., 37 (2000) 83 Mannaioni, P.F., 22 (1985) 267 Maroney, A.C., 40 (2002) 23 Martin, I.L., 20 (1983) 157 Martin, J.A., 32 (1995) 239 Masini, F., 22 (1985) 267 Matassova, N., 39 (2002) 73 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 McFadyen, I., 40 (2002) 107 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 Metzger, T., 40 (2002) 107 Michel, A.D., 23 (1986) 1 Middlemiss, D.N., 41 (2003) 129 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) 115 Munawar, M.A., 33 (1996) 233 Murchie, A.I.H., 39 (2002) 73 Murphy, F., 2 (1962) 1; 16 (1979) 1

Musallan, H.A., 28 (1991) 1 Musser, J.H., 22 (1985) 293 Natoff, I.L., 8 (1971) 1 Neidle, S., 16 (1979) 151 Nicholls, P.J., 26 (1989) 253 Niewo¨hner, U., 41 (2003) 249 Nodiff, E.A., 28 (1991) 1 Nordlind, K., 27 (1990) 189 Nortey, S.O., 36 (1999) 169 O’Hare, M., 24 (1987) 1 O’Reilly, T., 42 (2004) 171 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 Pellicciari, R., 42 (2004) 125 Perni, R.B., 39 (2002) 215 Petrow, V., 8 (1971) 171 Picard, J.A., 39 (2002) 121 Pike, V.W., 38 (2001) 189 Pinder, R.M., 8 (1971) 231; 9 (1973) 191 Poda, G., 40 (2002) 107 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 Rampe, D., 43 (2005) 1 Reckendorf, H.K., 5 (1967) 320 Reddy, D.S., 37 (2000) 135 Redshaw, S., 32 (1995) 239

CUMULATIVE AUTHOR INDEX 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 Rodgers, J.D., 40 (2002) 63 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) 111 Roth, B., 7 (1970) 285; 8 (1971) 61; 19 (1982) 269 Roth, B.D., 40 (2002) 1 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., 11 (1975) 193 Sandler, M., 6 (1969) 200 Saporito, M.S., 40 (2002) 23 Sarges, R., 18 (1981) 191 Sartorelli, A.C., 15 (1978) 321; 32 (1995) 1 Saunders, J., 41 (2003) 195 Schiller, P. W., 28 (1991) 301 Schmidhammer, H., 35 (1998) 83 Scho¨n, 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 Sliskovic, D.R., 39 (2002) 121 Smith, H.J., 26 (1989) 253; 30 (1993) 327 Smith, R.C., 12 (1975) 105 Smith, W.G., 1 (1961) 1; 10 (1974) 11

Solomons, K.R.H., 33 (1996) 233 Sorenson, J.R.J., 15 (1978) 211; 26 (1989) 437 Souness, J.E., 33 (1996) 1 Southan, C., 37 (2000) 1 Spencer, P.S.J., 4 (1965) 1; 14 (1977) 249 Spinks, A., 3 (1963) 261 Sta˚hle, L., 25 (1988) 291 Stark, H., 38 (2001) 279 Steiner, K.E., 24 (1987) 209 Stenlake, J.B., 3 (1963) 1; 16 (1979) 257 Stevens, M.F.G., 13 (1976) 205 Stewart, G.A., 3 (1963) 187 Studer, R.O., 5 (1963) 1 Subramanian, G., 40 (2002) 107 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 Talley, J.J., 36 (1999) 201 Taylor, E.C., 25 (1988) 85 Taylor, E.P., 1 (1961) 220 Taylor, S G., 31 (1994) 409 Tegne´r, 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., 11 (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 de Waterbeemd, H., 41 (2003) 1 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 Vaz, R.J., 43 (2005) 1 Vincent, J.E., 17 (1980) 185

281

282 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 Wartmann, M., 42 (2004) 171 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., 1 (1961) 34 Wikstro¨m, H., 29 (1992) 185

CUMULATIVE AUTHOR INDEX Wikstro¨m, H.V., 38 (2001) 189 Wilkinson, S., 17 (1980) 1 Williams, D.R., 28 (1991) 175 Williams, J., 41 (2003) 195 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, A., 43 (2005) 239 Wood, E.J., 26 (1989) 323 Wright, I.G., 13 (1976) 159 Wyard, S.J., 12 (1975) 191 Wyman, P.A., 41 (2003) 129 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

Cumulative Index of Subjects for Volumes 1 – 43 The volume number, (year of publication) and page number are given in that order. Antiarthritic agents, 15 (1978) 211; 19 (1982) 1; 36 (1999) 201 Anti-atherosclerotic agents, 39 (2002) 121 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 ß-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 Antidiabetic agents, 41 (2003) 167; 42 (2004) 1 Antiemetic action of 5-HT3 antagonists, 27 (1990) 297; 29 (1992) 239 Antiemetic drugs, 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, 1 (1961) 220 Antihyperlipidemic agents, 11 (1975) 119

ACAT inhibitors, 39 (2002) 121 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 agonists, b3-, 41 (2003) 167 Adrenergic blockers, a-, 23 (1986) 1 ß-, 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 Allergy, chemotherapy of, 21 (1984) 1; 22 (1985) 293 Alzheimer’s disease, chemotherapy of, 34 (1997) 1; 36 (1999) 201 M1 agonists in, 43 (2005) 113 Amidines and guanidines, 30 (1993) 203 Aminoadamantane derivatives, 18 (1981) 1 Aminopterins as antitumour agents, 25 (1988) 85 8-Aminoquinolines as antimalarial drugs, 28 (1991) 1; 43 (2005) 220 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 Antiapoptotic agents, 39 (2002) 1 Antiarrhythmic drugs, 29 (1992) 65 283

284

CUMULATIVE SUBJECT INDEX

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; 39 (2002) 1 Antimalarial agents, 43 (2005) 189 Antimalarial 8-aminoquinolines, 28 (1991) 1 Antimicrobial agents for sterilization, 34 (1997) 149 Antineoplastic agents, a new approach, 25 (1988) 35 anthraquinones as, 20 (1983) 83 Anti-osteoporosis drugs, 42 (2004) 245 Antipsychotic drugs, 33 (1996) 185 Anti-rheumatic drugs, 17 (1980) 185; 19 (1982) 1; 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, 129; 25 (1988) 35, 85; 26 (1989) 253, 299; 30 (1993) 1; 32 (1995) 1, 289; 34 (1997) 69; 42 (2004) 171 Antitussive drugs, 3 (1963) 89 Anti-ulcer drugs, of plant origin, 28 (1991) 201 ranitidine, 20 (1983) 67 synthetic, 30 (1993) 203 Antiviral agents, 8 (1971) 119; 23 (1986) 187; 36 (1999) 1; 39 (2002) 215 Anxiety neurokinin receptors in, 43 (2005) 53 Anxiolytic agents, CCK-B antagonists as, 37 (2000) 45 Anxiolytic agents, pyrido[1,2-a]benzimidazoles as, 36 (1999) 169 Aromatase inhibition and breast cancer, 26 (1989) 253; 33 (1996) 147 Arthritis neurokinin receptors in, 43 (2005) 53 Aspartic proteinase inhibitors, 32 (1995) 37, 239 Asthma, drugs for, 21 (1984) 1; 31 (1994) 369, 409; 33 (1996) 1; 38 (2001) 249 neurokinin receptors in, 43 (2005) 53 Atorvastatin, hypolipidemic agent, 40 (2002) 1 ATPase inhibitors, gastric, H+/K+-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, 31 (1994) 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 Caspase inhibitors, 39 (2002) 1 Catecholamines, 6 (1969) 200 Cathepsin K inhibitors, 42 (2004) 245 CCK-B antagonists, 37 (2000) 45 CCR5 Receptor antagonists, 43 (2005) 239 Cell membrane transfer, 14 (1977) 1

CUMULATIVE SUBJECT INDEX Central nervous system, drugs, transmitters and peptides, 23 (1986) 91 Centrally acting dopamine D2 receptor agonists, 29 (1992) 185 CEP-1347/KT-7515, inhibitor of the stress activated protein kinase signalling pathway (JNK/SAPK), 40 (2002) 23 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; 40 (2002) 1 Cholinergic receptors, 16 (1976) 257 Chromatography, 12 (1975) 1, 105 Chromone carboxylic acids, 9 (1973) 65 Clinical enzymology, 13 (1976) 1 Collagenases, synthetic inhibitors, 29 (1992) 271 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) 11 Corticotropin releasing factor receptor antagonists, 41 (2003) 195 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) 1; 33 (1996) 53

Data analysis in biomedical research, 25 (1988) 291 Depression neurokinin receptors in, 43 (2005) 53 Diaminopyrimidines, 19 (1982) 269 Digitalis recognition matrix, 30 (1993) 135

285

Diuretic drugs, 1 (1961) 132 DNA-binding drugs, 16 (1979) 151 Dopamine D2 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 III) agents for arrhythmia, 29 (1992) 65 Emesis neurokinin receptors in, 43 (2005) 53 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, 297; 32 (1995) 37, 239; 33 (1996) 1; 36 (1999) 1, 201; 38 (2001) 1; 39 (2002) 1, 121, 215; 40 (2002) 1, 23, 63; 41 (2003) 99, 249; 42 (2004) 125, 245 Enzymology, clinical use of, 10 (1976) 1 in pharmacology and toxicology, 10 (1974) 11 Epothilones A and B and derivatives as anticancer agents, 42 (2004) 171 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 Fluorescence-based assays, 43 (2005) 19 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 (1974) 205

286

CUMULATIVE SUBJECT INDEX

GABA, heterocyclic analogues, 22 (1985) 67 GABAA receptor ligands, 36 (1999) 169 Gas-liquid chromatography and mass spectrometry, 12 (1975) 1 Gastric H+/K+-ATPase inhibitors, 31 (1994) 233 Genomics, impact on drug discovery, 37 (2000) 1 Glutaraldehyde, biological uses, 13 (1976) 271 as sterilizing agent, 34 (1997) 149 Gold, immunopharmacology of, 19 (1982) 1 Growth hormone secretagogues 39 (2002) 173 Guanidines, 7 (1970) 124; 30 (1993) 203 Halogenoalkylamines, 2 (1962) 132 Heparin and heparinoids, 5 (1967) 139 Hepatitis C virus NS3·4 protease, inhibitors of, 39 (2002) 215 Herpes virus, chemotherapy, 23 (1985) 67 Heterocyclic analogues of GABA, 22 (1985) 67 Heterocyclic carboxaldehyde thiosemicarbazones, 16 (1979) 35; 32 (1995) 1 Heterosteroids, 16 (1979) 35; 28 (1991) 233 High-throughput screening techniques, 37 (2000) 83; 43 (2005) 43 Histamine, H3 ligands, 38 (2001) 279 H2-antagonists, 20 (1983) 337 receptors, 24 (1987) 30; 38 (2001) 279 release, 22 (1985) 26 secretion, calcium and, 19 (1982) 59 5-HT1A receptors, radioligands for in vivo studies, 38 (2001) 189 Histidine decarboxylases, 5 (1967) 199 HIV CCR5 antagonists in, 43 (2005) 239 proteinase inhibitors, 32 (1995) 239 HMG-CoA reductase inhibitors, 40 (2002) 1 Human Ether-a-go-go (HERG), 43 (2005) 1 Hydrocarbons, carcinogenicity of, 10 (1974) 159 Hypersensitivity reactions, 4 (1965) 1 Hypocholesterolemic agents, 39 (2002) 121; 40 (2002) 1 Hypoglycaemic drugs, 1 (1961) 187; 18 (1981) 191; 24 (1987) 209; 30 (1993) 203; 31 (1994) 1 Hypolipidemic agents, 40 (2002) 1

Hypotensive agents, 1 (1961) 34; 30 (1993) 203; 31 (1994) 409; 32 (1995) 37, 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 ß-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 (1986) 41 Linear free energy, 10 (1974) 205 Lipid-lowering agents, 40 (2002) 1 5-Lipoxygenase inhibitors and their antiinflammatory 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; 43 (2005) 189 Manganese, biological significance, 30 (1993) 89 Manufacture of enantiomers of drugs, 34 (1997) 203

CUMULATIVE SUBJECT INDEX 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 (II) chloride, biological effects, 27 (1990) 189 Methotrexate analogues as anticancer drugs, 25 (1988) 85; 26 (1989) 1 Microcomputers in biomedical education, 26 (1989) 323 Migraine neurokinin receptors in, 43 (2005) 53 Molecular modelling of opioid receptor-ligand complexes, 40 (2002) 107 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 Muscarinic Receptors, 43 (2005) 105 Neuraminidase inhibitors, 36 (1999) 1 Neurokinin receptor antagonists, 35 (1998) 57; 43 (2005) 49 Neuromuscular blockade, 2 (1962) 88; 3 (1963) 1; 16 (1979) 257 Neuropeptide Y receptor ligands, 42 (2004) 207 Neurosteroids, as psychotropic drugs, 37 (2000) 135 Next decade [the 1970’s], drugs for, 7 (1970) 215 NFkB, 43 (2005) 137 Nickel(II) chloride and sulphate, biological effects, 27 (1990) 189 Nicotinic cholinergic receptor ligands, a4b2, 42 (2004) 55

287

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) 1 receptor antagonists, 35 (1998) 83 receptor-specific analogues, 28 (1991) 301 receptor-ligand complexes, modelling of, 40 (2002) 107 Oral absorption and bioavailability, prediction of, 41 (2003) 1 Organophosphorus pesticides, pharmacology of, 8 (1971) 1 Oxopyranoazines and oxopyranoazoles, 9 (1973) 117 Poly(ADP-ribose)polymerase (PARP) inhibitors, 42 (2004) 125 P2 Purinoreceptor ligands, 38 (2001) 115 p38 MAP kinase inhibitors, 38 (2001) 1 Paclitaxel, anticancer agent, 32 (1995) 289 Pain neurokinin receptors in, 43 (2005) 53, 55 Parasitic infections, 13 (1976) 159; 30 (1993) 203 Parasympathomimetics, 11 (1975) 1 Parenteral nutrition, 28 (1991) 175 Parkinsonism, pharmacotherapy of, 9 (1973) 191; 21 (1984) 137 Patenting of drugs, 2 (1962) 1; 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 Peroxisome proliferator-activated receptor gamma (PPARg) ligands, 42 (2004) 1

288

CUMULATIVE SUBJECT INDEX

Pharmacology of Alzheimer’s disease, 34 (1997) 1 Pharmacology of Vitamin E, 25 (1988) 249 Phosphates and phosphonates as prodrugs, 34 (1997) 111 Phosphodiesterase type 4 (PDE4) inhibitors, 33 (1996) 1 Phosphodiesterase type 5 (PDE5) inhibitors, 41 (2003) 249 Phospholipids, 19 (1982) 111 Photodecomposition of drugs, 27 (1990) 51 Plasmodium, 43 (2005) 190 Plasmodium flaciparum dihydrofolate reductase (PfDHFR), 43 (2005) 226 Platelet-aggregating factor, antagonists, 27 (1990) 325 Platinum antitumour agents, 24 (1987) 129 Platelet aggregration, inhibitors of, 36 (1999) 29 Polarography, 12 (1975) 247 Polycyclic hydrocarbons, 10 (1974) 159 Polyene antibiotics, 14 (1977) 105 Polypeptide antibiotics, 5 (1967) 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) 111 Prostacyclins, 21 (1984) 237 Prostaglandins, 8 (1971) 317; 15 (1978) 357 Proteinases, inhibitors of, 31 (1994) 59; 32 (1995) 37, 239 Proteosome inhibitors, 43 (2005) 155 Pseudomonas 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) 1; 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 QT interval, 43 (2005) 4 Radioligand-receptor binding, 23 (1986) 417 Ranitidine and H2-antagonists, 20 (1983) 337 Rauwolfia alkaloids, 3 (1963) 146 Recent drugs, 7 (1970) 1 Receptors, adenosine, 38 (2001) 61 adrenergic, 22 (1985) 121; 23 (1986) 1; 41 (2003) 167 cholecystokinin, 37 (2000) 45 corticotropin releasing factor, 41 (2003) 195 fibrinogen, 36 (1999) 29 histamine, 24 (1987) 29; 38 (2001) 279 neurokinin, 35 (1998) 57 neuropeptide Y, 42 (2004) 207 nicotinic cholinergic, 42 (2004) 55 opioid, 35 (1998) 83 peroxisome proliferator-activated receptor gamma (PPARg), 42 (2004) 1 purino, 38 (2001) 115 Renin inhibitors, 32 (1995) 37 Reverse transcriptase inhibitors of HIV-1, 40 (2002) 63 Serotonin, 41 (2003) 129 Ricin, 24 (1987) 1 RNA as a drug target, 39 (2002) 73 Schizophrenia Neurokinin receptors in, 43 (2005) 53 M1 agonists in, 43 (2005) 113, 117 M2 antagonists in, 43 (2005) 121 M4 antagonists in, 43 (2005) 129 Screening tests, 1 (1961) 1 Secretase inhibitors, g-, 41 (2003) 99 Serine protease inhibitors, 31 (1994) 59 Serotonin 5-HT1A radioligands, 38 (2001) 189 Serotonin (5-HT)-terminal autoreceptor antagonists, 41 (2003) 129 Single photon emission tomography (SPET), 38 (2001) 189 Snake venoms, neuroactive, 21 (1984) 63 Sodium cromoglycate analogues, 21 (1984) 1

CUMULATIVE SUBJECT INDEX

289

Sparsomycin, 23 (1986) 219 Spectroscopy in biology, 12 (1975) 159, 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 Stress activated protein kinase inhibitors, 40 (2002) 23 Synthesis of enantiomers of drugs, 34 (1997) 203

Tilorone and related compounds, 18 (1981) 135 Time resolved energy transfer (TRET), 43 (2005) 40 Toxic actions, mechanisms of, 4 (1965) 18 Tranquillizers, 1 (1961) 72 1,2,3-Triazines, medicinal chemistry of, 13 (1976) 205 Tripositive elements, chelation of, 28 (1991) 41 Trypanosomiasis, 3 (1963) 52

Tachykinins, 43 (2005) 50 Tetrahydroisoquinolines, ß-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

Venoms, neuroactive snake, 21 (1984) 63 Virtual screening of virtual libraries, 41 (2003) 61 Virus diseases of plants, 20 (1983) 119 Viruses, chemotherapy of, 8 (1971) 119; 23 (1986) 187; 32 (1995) 239; 36 (1999) 1; 39 (2002) 215 Vitamin D3 and its medical uses, 35 (1998) 1 Vitamin E, pharmacology of, 25 (1988) 249

Ubiquitinylation, 43 (2005) 153