Progress in Medicinal Chemistry 44
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Progress in Medicinal Chemistry 44 Editors: F.D. KING, B.SC., D.PHIL., C.CHEM., F.R.S.C. GlaxoSmithKline New Frontiers Science Park (North) Third Avenue Harlow, Essex CM19 5AW United Kingdom and G. LAWTON, B.SC.,
PH.D., C.CHEM.
Garden Fields Stevenage Road St. Ippolyts Herts SG4 7PE United Kingdom
AMSTERDAM BOSTON HEIDELBERG LONDON NEW YORK OXFORD PARIS SAN DIEGO SAN FRANCISCO SINGAPORE SYDNEY TOKYO
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Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK First edition 2006 Copyright r 2006 Elsevier B.V. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email:
[email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloging in Publication Data A catalogue record for this book is available from the British Library ISBN-13: 978-0-444-51737-1 ISBN-10: 0-444-51737-5 ISSN: 0079-6468 For information on all Elsevier publications visit our website at books.elsevier.com
Printed and bound in The Netherlands 06 07 08 09 10 10 9 8 7 6 5 4 3 2 1
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Preface The perceived lack of drug discovery productivity in recent times has led to much debate in the pharmaceutical/biotechnology industry as escalating R&D costs are not being matched by increased output. Few observers doubt that selecting the right targets, i.e. those, which are critical to disease pathology and are ‘druggable’, is the best starting point for improved productivity. The seven chapters of this volume describe recent progress towards drugs acting at a range of ‘druggable’ targets. One chapter addresses kinases, one covers an ion channel, two feature proteases and three of the chapters cover G-protein coupled receptors, which have historically perhaps been the most fruitful area for medicinal chemists. It is increasingly apparent that productivity depends not only on the target selection, but also on the quality of the lead compounds. Good leads produce a double effect on productivity by reducing the downstream failure rate and also the timeline of a project. Chapter 1 provides an excellent review of the use of biostructures to find high-quality leads. The emphasis is on the kinase inhibitor field where there is an abundance of available biostructures. Technology for screening by X-ray crystallography and by NMR has advanced rapidly in recent years and in silico screening methods continue to improve. All three approaches are covered in this review. The hepatitis C virus (HCV) is responsible for a world-wide epidemic with approximately 170 million people infected. It was identified only in the 1980s and since that time great efforts have been made in the search for treatments. Genetic analysis of the virus revealed coding for a serine protease (NS3) and the first clinical studies on inhibitors of the protease have recently been carried out. Chapter 2 presents a review of the medicinal chemistry approaches to this target. The major successes in treating bacterial infections that were achieved by the antibiotics discovered in the middle part of the last century are now under severe threat from the emergence of resistant strains. Very few new classes of antibacterial have been created in the past 20 years. Peptide deformylase represents a new biochemical target and clinical candidates are beginning to emerge. Chapter 3 reviews progress to date.
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PREFACE
The Transient Receptor Potential Vanilloid 1 (TRPV1) (vanilloid receptor 1) is a cation channel, which mediates the neuronal response to hot components of chilli peppers. Desensitisation using agonists provides a useful mechanism of pain control. Antagonists also show this effect and are reviewed in Chapter 4. They may find use in a range of clinical situations. Ligands acting at histamine receptors have been a valuable source of medicines. The recent cloning of the H3 receptor has led to a resurgence of interest in the field. Several neurotransmitter systems are subject to modulation by neuronal H3 receptors and it is anticipated that the antagonists will be useful in the treatment of a range of diseases including narcolepsy and attention deficit hyperactivity disorder as well as improving cognition in situations such as Alzheimer’s disease. Chapter 5 updates the medicinal chemistry of H3 receptor ligands which was last reviewed in this series in Volume 38. The cannabinoid field was reviewed in Progress in Medicinal Chemistry Volume 35. Since that time great advances have been made in our understanding of cannabinoid receptor pharmacology. Many novel ligands have been discovered and several are expected to have exciting clinical utility. Medicinal chemistry approaches to the modulation of cannabinoid receptors are extensively reviewed in Chapter 6. Preterm labour is the major cause of perinatal morbidity and mortality. Oxytocin antagonists offer an attractive approach to prevention. Chapter 7 reviews three decades of medicinal chemistry in this field. The peptide approach has resulted in valuable injectable products. Selectivity over the related vasopressin receptors and improvement in pharmacokinetic profile have been the key challenges for more recent non-peptide programmes, and these seem likely to yield orally available medicines. October 2005
Dr. F. D. King Dr. G. Lawton
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Contents Preface
v
List of Contributors
ix
1
Finding Protein Kinase Hits using Structural Information Mike Cherry, John Reader and David Williams
1
2
Blunting the Swiss Army Knife of Hepatitis C Virus: Inhibitors of NS3/4A Protease Peter W. White, Montse Llinas-Brunet and Michael Bo¨s
65
3
Peptide Deformylase Inhibitors Kelly Aubart and Magdalena Zalacain
109
4
Clinically Useful Vanilloid Receptor TRPV1 Antagonists: Just around the Corner (or too Early to Tell)? Giovanni Appendino and Arpad Szallasi
145
5
Recent Medicinal Chemistry of the Histamine H3 Receptor Michael A. Letavic, Ann J. Barbier, Curt A. Dvorak and Nicholas I. Carruthers
181
6
Recent Progress in Cannabinoid Research Julia Adam, Phillip M. Cowley, Takao Kiyoi, Angus J. Morrison and Christopher J.W. Mort
207
7
Oxytocin Antagonists as Potential Therapeutic Agents for the Treatment of Preterm Labour Michael J. Allen, David G.H. Livermore and Jacqueline E. Mordaunt
331
Subject Index
375
Author Index (Vols. 1–44)
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Subject Index (Vols. 1–44)
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List of Contributors Julia Adam Organon Research, Newhouse, Lanarkshire, ML1 5SH, Scotland, UK Michael J. Allen GlaxoSmithKline, New Frontiers Science Park (North), Third Avenue, Harlow, Essex, CM19 5AW, UK Giovanni Appendino Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita` del Piemonte Orientale, 28100 Novara, Italy Kelly Aubart Microbial, Musculoskeletal, and Proliferative Diseases CEDD, GlaxoSmithKline Pharmaceuticals, Collegeville, PA 19426, USA Ann J. Barbier Johnson and Johnson Pharmaceutical Research and Development L.L.C., San Diego, CA 92121, USA Michael Bo¨s Boehringer Ingelheim (Canada) Ltd, 2100 Cunard St., Laval, QC H7S 2G5, Canada Nicholas I. Carruthers Johnson and Johnson Pharmaceutical Research and Development L.L.C., San Diego, CA 92121, USA Mike Cherry Sareum Ltd, 2 Pampisford Park, Cambridge CB2 4EE, UK
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LIST OF CONTRIBUTORS
Phillip M. Cowley Organon Research, Newhouse, Lanarkshire, ML1 5SH, Scotland, UK Curt A. Dvorak Johnson and Johnson Pharmaceutical Research and Development L.L.C., San Diego, CA 92121, USA Takao Kiyoi Organon Research, Newhouse, Lanarkshire, ML1 5SH, Scotland, UK Michael A. Letavic Johnson and Johnson Pharmaceutical Research and Development L.L.C., San Diego, CA 92121, USA David G.H. Livermore GlaxoSmithKline, New Frontiers Science Park (North), Third Avenue, Harlow, Essex, CM19 5AW, UK Montse Llinas-Brunet Boehringer Ingelheim (Canada) Ltd, 2100 Cunard St., Laval, QC H7S 2G5, Canada Jacqueline E. Mordaunt GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK Angus J. Morrison Organon Research, Newhouse, Lanarkshire, ML1 5SH, Scotland, UK Christopher J.W. Mort Organon Research, Newhouse, Lanarkshire, ML1 5SH, Scotland, UK John Reader Sareum Ltd, 2 Pampisford Park, Cambridge CB2 4EE, UK
LIST OF CONTRIBUTORS
Arpad Szallasi Department of Pathology and Laboratory Medicine, Monmouth Medical Center, Long Branch, NJ 07740, USA Peter W. White Boehringer Ingelheim (Canada) Ltd, 2100 Cunard St., Laval, QC H7S 2G5, Canada David Williams Sareum Ltd, 2 Pampisford Park, Cambridge CB2 4EE, UK Magdalena Zalacain Microbial, Musculoskeletal, and Proliferative Diseases CEDD, GlaxoSmithKline Pharmaceuticals, Collegeville, PA 19426, USA
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Progress in Medicinal Chemistry – Vol. 44, Edited by F.D. King and G. Lawton r 2006 Elsevier B.V. All rights reserved.
1 Finding Protein Kinase Hits using Structural Information MIKE CHERRY, JOHN READER and DAVID WILLIAMS Sareum Ltd, 2 Pampisford Park, Cambridge CB2 4EE, UK
INTRODUCTION
2
SCREENING BY X-RAY CRYSTALLOGRAPHY
4
SCREENING BY NMR
15
FUNDAMENTALS OF NMR SPECTROSCOPY Using Chemical Shift Changes Nuclear-spin Relaxation Nuclear Overhauser Effects Exchange Phenomena
16 16 17 17 18
APPLICATIONS OF NMR IN SCREENING Target-based Screening Ligand-based Screening
18 18 19
EXAMPLES OF NMR METHODS FOR DISCOVERING KINASE INHIBITORS The Shapes Strategy NMR Screening by Waterlogsy Method NMR Screening of Protein Kinases by ATP–STD Method NMR Backbone Assignment of a Kinase Designing Novel Kinase Inhibitors from the NMR-based Screening of Fragments Design of Libraries for use in NMR Screening
20 20 23 24 25 27 30
IN SILICO METHODS
31
RECEPTOR-BASED SCREENING Compound Database and Receptor Preparation Protein–Ligand Docking Scoring Functions Development and Evaluation of Docking and Scoring Virtual Screening and Protein Kinases
32 33 34 36 37 41
DOI: 10.1016/S0079-6468(05)44401-X
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FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
FAULTS AND FIXES FOR VIRTUAL SCREENING Consensus Scoring Knowledge-based Screening Protein Flexibility and Induced Fit High Throughput Docking as a Virtual Screening Tool
44 44 45 48 50
ALTERNATIVES TO HIGH THROUGHPUT DOCKING
51
COMPARATIVE HOMOLOGY MODELLING
53
DE NOVO DESIGN
55
SUMMARY
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REFERENCES
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INTRODUCTION The discovery and selection of suitable compounds to start a medicinal chemistry programme is a fundamental stage in the process of drug discovery, where decisions are made that ultimately affect the probability of success for that project. Decisions are made, however, at a time in the project where data are limited, hence, where possible, multiple chemical series are taken forward on the basis that a degree of attrition is inevitable. Recent years, though, have seen a marked shift in the philosophy of finding hit compounds, returning to the idea that quality is better than quantity, as strategies that simply increase numbers, using technologies such as combinatorial chemistry and high throughput screening, have not lived up to expectation. Investment is now focused on alternative and complementary technologies that increase efficiency and productivity, and make better use of the extensive amount of information available both in-house and within the public domain. One such area of development is in the use of structural information regarding the interaction of hit compounds with a receptor. A medicinal chemist’s dream would be to immediately see every molecule synthesised matched up against the target macromolecule, giving the exact mode of binding and the degree of inhibition or stimulation. These data would then allow focused, informed design in the next phase of medicinal chemistry, not only allowing efficient use of resources, but also shortening the chemistry cycle time and therefore reducing the pre-clinical drug discovery time. Ideally, such a capability would apply to all types of target and take into account not only the flexibility of the macromolecule, but also the effect of other macromolecular-binding partners. Naturally, these data would be available at the merest touch of a button. This dream situation is
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already starting to become reality with the systematic and timely use of analytical procedures of X-ray diffraction and nuclear magnetic resonance (NMR) spectroscopy. Once thought of as a luxury often only obtained towards the end of lead optimisation and used to post-rationalise structure–activity relationship (SAR), structural data can now play a pivotal role in both discovering and selecting hit compounds. The ability to generate a wealth of structural data at the very beginning of a drug discovery project would allow the early selection of different chemical lead series based on their interactions with the target macromolecule, as well as on their intellectual property status and ease of synthesis. There are technical limitations on the speed of obtaining data from both X-ray crystallography and NMR, and it is to be expected that not all targets are amenable for these techniques. The present review, therefore, discusses three alternative approaches to finding hits using structural information; X-ray crystallography, NMR and in silico methods, which have arisen as powerful complementary techniques. The review focuses on the much studied and therapeutically important protein kinase family [1, 2] of targets, and so an introduction to the kinase structure is presented prior to discussion of X-ray crystallographic techniques. The abundance of structural information for protein kinases provides an ideal background for structure-guided drug discovery. Knighton et al. [3] first elucidated the three-dimensional (3D) structure of the catalytic domain of a protein kinase, cyclic adenosine monophosphate (cAMP)-dependent protein kinase and there are now more than 300 entries of kinase structures in the Protein Data Bank (PDB). The catalytic domain typically has 250–300 amino acids and adopts a two-lobed structure that can be further subdivided into 12 ‘subdomains I–XII’. The smaller N-terminal lobe is primarily antiparallel b-sheets with the important exception of a-helix C and constitutes subdomains I–IV. Subdomain V, a single polypeptide chain known as the linker region, connects the N-terminal lobe to the larger, predominantly helical C-terminal lobe comprising subdomains VIa–XI. A full explanation of the structure and function of each region is available elsewhere at the PKR website (pkr.sdsc.edu/html/index.shtml). The abundance of structural information has led to a significant increase in the use of structure-based methods both to identify and to optimise inhibitors of protein kinases. The focus to date has centred upon small molecule ATP-competitive inhibitors and there are numerous examples of protein–ligand complexes available to guide design strategies. ATP binds in the cleft formed between the N- and C-terminal lobes of the protein kinase, forming several key interactions conserved across the protein kinase family. The adenine moiety lies in a hydrophobic region between the b-sheet structure of subdomains I and II and residues from subdomains V and VIb. A
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large number of protein kinase inhibitors have been observed to mimic the donor–acceptor pair of hydrogen bonds made between the protein backbone and the adenine. A hydrogen bond is also observed between a residue from the linker region and the ribose group. The phosphate chain occupies a groove in the protein formed by a number of polar residues that participate in the catalytic function of the enzyme. For reference purposes, the ATPbinding site can be described by means of the ATP-binding mode as the adenine pocket, the ribose pocket and the phosphate groove. There are two additional non-conserved regions that are often noted in the development of inhibitors. The hydrophobic pocket lies directly behind the amino group of adenine (further into the cleft), the size of which is, to a large extent, determined by a single non-conserved residue called the gatekeeper residue. The selectivity profiles of chemical series have been ascribed to variations in the gatekeeper residue, where functionality exploits the hydrophobic pocket of kinases, such as the serine/threonine kinase p38. Another less apparent region, the specificity surface, lies at the opening of the cleft and has also been used to great effect in the design of selective protein kinase inhibitors. Figure 1.1 shows a schematic representation of the ATP pocket in a protein kinase.
SCREENING BY X-RAY CRYSTALLOGRAPHY In order to determine a macromolecular crystal structure, a number of processes have to be performed in a linear fashion with each stage dictating the outcome of the next. Initially, good quality protein crystals have to be made that are amenable to X-ray diffraction analysis. The routine use of cryopreservation techniques [4] over the last 10 years has meant that the required size of usable protein crystal has decreased, removing much of the burden of teasing out ordered growth conditions for making relatively large crystals. In addition, the physical damage to protein crystals from high-energy X-ray irradiation is greatly reduced by maintaining crystals frozen in conditions where ice crystal formation is prevented. This allows data sets to be collected from crystals as small as 10 mm in size, whereas previously crystals had to be larger to allow for major damage during the X-ray irradiation procedure. Advancements in the optical devices used to focus high-energy X-ray beams at the crystal have matched the advances in crystal cryopreservation meaning that good quality data sets can also be collected. Traditionally, particle accelerator facilities, known as synchrotrons, have been used as a source of very high-energy X-rays for protein crystallography. While this is still the case for allowing very rapid collection of good quality data sets, the
M. CHERRY, J. READER AND D. WILLIAMS
gatekeeper residue (MET120) hydrophobic pocket GLU121
VAL123
H
N H O
LYS72 H3N
O
TYR122
5
N
H N
N N
N
O
O O O O P O O P P O O O O
O
O
specificity surface Fig. 1.1 ATP bound to cAMP protein kinase and a schematic representation indicating the nonconserved regions (hydrophobic pocket and specificity surface) of the pocket utilised in the development of protein kinase inhibitors.
usable X-ray flux of home sources has also increased significantly as a combination of improvements that have been made to rotating anode generators, X-ray optical systems and detector systems. This improvement to in-house systems means that a good laboratory single crystal X-ray diffraction machine can now be used for relatively rapid data collection, so
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FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
that data which may have taken several days to collect on older systems can now be collected in as little as a couple of hours. The increase in productivity of in-house systems has meant that the process of putting the crystal in the beam can be a bottleneck during 24/7 operation. For this reason, automated systems have been developed to replace this manual operation, taking crystal containing cryoloops from liquid nitrogen storage and placing them in the X-ray beam prior to data collection [5]. Some synchrotron facilities now also have automated crystal-mounting systems that can be utilised in a remote fashion. In this process, a user cryopreserves many crystals on data collection loops, places them in a specialist carrier cassette, and then sends them over to the remote synchrotron in a cryostorage container by courier. The data are collected and the data sets returned by courier or via the Internet without the scientist needing to set foot outside their own laboratory. The number and quality of the software tools available, coupled with the availability of cheap computational processing, means that the data analysis part of the structure-determination process is greatly enhanced with ever decreasing timelines [6]. The events required for structural solution are the processing of collected data sets, structural solution, molecular refinement and building the structural model. The molecular replacement and model building are continued in cycles until the model is of acceptable quality to the structural biologist. On attempting to derive a novel structure, the parts of the process that can be bottlenecks are the structural solution and the model building, especially in the case of relatively large proteins of >50 kDa. Structural solution is becoming far more straightforward with the increasing numbers of proteins deposited in the public protein database (at the time of writing there were 33,000 macromolecular structures). The greater the number of available structures, the greater the probability that there is a homologous structure, which can be used as a computational search model to provide a structural solution to a novel protein by molecular replacement, a very straightforward and rapid computational process. Where no homologous structure is available, more direct experimental methods need to be performed in order to provide data for direct phase solution, which can add significant time to a project. Once a structure of the desired protein has been solved, it is a very rapid process to produce subsequent high-quality structures and, in fact, some groups have even linked various scripts together, or modified software tools to provide much more automated software aids to repeated crystal structure solution, such as when solving multiple ligand complexes of the same protein [7]. Despite the major advances in X-ray crystallography, the process of producing multiple protein–ligand complexes is not able to handle anywhere
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near the same number of compounds as high throughput compound screening (HTS) methods. Ambitious X-ray structure campaigns can produce hundreds of protein–ligand complexes in several months [8], whereas HTS can handle at least a million compounds over the same time period using in vitro screening technologies. The use of X-ray crystallography as a direct screening tool for conventional compound libraries is therefore limited, especially as corporate collections tend to range from 100,000 to several million unique compounds [9]. A method that has been developed in an attempt to enable X-ray structure determination to be used as a primary screening tool has been the use of compound cocktails in protein crystal soaking experiments [10]. This method relies on a supply of well-diffracting protein crystals amenable to ligand soaking. This means that the packing of the molecules in the crystalline matrix of the protein crystal is arranged so that ligand can easily diffuse into the active site of many of the target molecules making up the crystal, without causing the structural movements of the protein, which could reduce the crystal diffraction quality. Each compound cocktail is selected so that members are diverse in shape to facilitate identification of the ‘hit’ (Figure 1.2). Once the crystals have been soaked in compound, rapid X-ray data are collected sufficient to allow the generation of an electron density map. The difference in density between empty unsoaked crystals and those where a
Soak
Compound library cocktail
Protein crystal X-ray
Further lead optimisation chemistry (aided by structure)
Compound identified & binding mode determined
Binding site electron density solved
Fig. 1.2 Schematic diagram to show the process involved in crystallographic screening of compound cocktails.
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FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
compound has bound provides ligand electron density that allows the identification of the bound compound from the cocktail. This process is only possible due to the diverse nature of the compounds in each cocktail, which unambiguously allows the density to be assigned to a specific shape of compound. This technique filters out the most potent-binding ligands from the mixture. To identify less potent ligands in the same mixture, this compound would have to be removed from the cocktail and re-screening performed. The number of compounds in each cocktail is limited by two factors. Firstly, sufficient shape diversity to allow unambiguous identification of compound by electron density at the structural resolution must be achieved. Secondly, the concentration of each compound in the cocktail has to be high enough to allow a relatively high occupancy in the protein crystal, and that saturating concentration decreases as the number of compounds in the cocktail increases. Despite these limitations, this technique has been used to screen cocktails containing as many as 100 compounds, which potentially allows the screening of low thousands of compounds per day on some in-house systems and potentially more at synchrotron facilities. This crystallographic screening method is a tool to complement existing approaches and certainly has the potential of bringing structural data to the early phase of drug discovery. Another crystallographic screening method is one known by a number of names including needle, shape or fragment screening [11, 12] where compounds, in many cases much smaller than conventional high throughput screening compounds, are used in structure solution campaigns to provide hits for structure-based design chemistry programmes. The philosophy behind using smaller molecules is that these relatively simple chemicals can be used to probe more binding sites than conventional larger high throughput screening compounds, due to their lack of extensive functionality. For example, an inappropriate side chain, which caused steric or electronic clashes within an active site, could render an exciting chemical scaffold inactive in a screen. A simple model of ligand–receptor interactions [13] has proposed that the probability of a compound binding to a receptor decreases as the molecular weight of the compound increases. For example, the authors show from statistical modelling that for a binding site with a complexity score of 12, the probability of randomly selecting a compound with a unique binding mode is 39.9% for a ligand of complexity 3, but only 1% for a ligand with a complexity of 11. Generally, compounds of lower molecular weight and complexity are less potent binders, with activities typically between 100 mM and 10 mM, as they are usually capable of making fewer interactions with the binding site. This means that selection by screening and subsequent chemistry programmes have to be modified to accommodate a
M. CHERRY, J. READER AND D. WILLIAMS
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different approach than hits discovered by conventional high throughput screening, where compounds of less than 10 mM activity are sought. One major problem with screening compounds at high assay concentration is that they can aggregate in solution and associate and inhibit enzymes non-selectively [14]. These effects have been observed for a number of generic protein kinase inhibitors, which can form aggregates in solution, becoming promiscuous inhibitors and reduce the enzyme activity of nonATP-dependent enzymes [15]. Fortunately, this effect can be monitored and also largely prevented by running identical assays in the presence of detergent, which reduces the effect by reducing compound aggregate formation [16, 17]. The effect of light absorbing compounds can be a problem in fluorometric kinase assays, so high compound concentration kinase assays should either be radiometric or involve a compound wash out step prior to signal detection. A multi-stage screening approach was adopted for developing ATP site inhibitors of DNA gyrase [11]. In this approach, the authors first used an in silico screen using a model structure to select an initial compound screening set of 600 compounds. This set was increased to 3,000 by the addition of close analogues. Many of these compounds had an MW o300 and none were selected with an MW >400. The authors referred to these compounds as needles, because they were small enough to have been able to penetrate deep into active site pockets and clefts. An in vitro assay was then used which was selected to tolerate compound assay concentrations of 0.5 mM. The high concentration in vitro screen identified 150 hits with significant inhibition activity. The objective of the overall screening approach was to identify compounds whose mode of action was through binding in the ATP-binding pocket. For this reason the 24 kDa N-terminal fragment of DNA gyrase subunit B, which contains the ATP-binding site of the enzyme was used for further studies. These studies initially consisted of analytical centrifugation and surface plasma resonance, with the more interesting compounds also being analysed by 15N-labelled protein in NMR experiments. The use of NMR allowed the identification of residues within the active site that had a shifted N–H signal upon ligand binding and so more clearly defined how different series of compounds were binding in the ATP-binding site. For final confirmation of binding, and as the aid to structure-guided design, X-ray crystal structures were then generated with a loop-deleted version of the ATP-binding subunit fragment, which was amenable to crystallography. This approach allowed the authors to develop novel inhibitors 10-fold more potent than the clinical competitor drug using their structure-based chemistry iterations, increasing the potency of an original weak indazole needle hit by a factor of 266 (Figure 1.3).
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FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
Needle Hit
After Lead Optimisation H N
H N N
N
SBDD O
S
Ph
O
O
Me t-Bu
MNEC = 8 µg/ml
MNEC = 0.03 µg/ml
Fig. 1.3 The increase in DNA gyrase inhibition activity (MNEC ¼ maximum non-effective concentration in supercoiling assay) of weak indazole needle hit to optimised compound achieved using structure-based drug design (SBDD).
An alternative approach to generating leads for X-ray-directed drug design known as tethering has also been developed [18]. In this technique, libraries of compounds containing disulphides are used and the enzyme target either has an active site cysteine, or is engineered to contain a cysteine at the appropriate position. The enzyme is then exposed to the library compounds in reducing conditions that favour reversible interactions between the disulphides and the cysteine. Compounds that make interactions and bind in the active site drive the equilibrium towards intermolecular disulphide bond formation and the compound is covalently bound to the enzyme. Erlanson et al. [18] screened cocktails of 8–15 compounds against thymidylate synthase and detected binding by mass spectrometry. By ensuring the compound cocktails contained compounds of different molecular weight, on mass spectrometry analysis, the mass of the unmodified enzyme could be subtracted from the library-exposed enzyme to give the deconvoluted mass and thus the identity of the bound compound. A compound library of 1,200 was screened and resulted in SARs being generated for N-tosyl-D-proline. The disulphide-free version of N-tosyl-D-proline was then shown to bind to thymidylate synthase with a Ki of 1.1 mM. X-ray crystallography demonstrated that the tosyl group of N-tosyl-D-proline occupied almost the same position as the benzamidine portion of the enzyme’s natural cofactor methylenetetrahydrofolate. This suggested binding affinity
M. CHERRY, J. READER AND D. WILLIAMS
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could then be achieved by addition of glutamate, present on the natural cofactor, to the methyl group of N-tosyl-D-proline. The strategy was successful, increasing the Ki of the compound to 24 mM. Further modification of the carboxyl group, rationalised by use of the X-ray structure, took the potency up to 330 nM, resulting in an overall increase of more than 3,000-fold from the original weak-binding hit (Figure 1.4). Enzyme-adduct formation has also been successfully achieved with the Erb1 and 2 (receptor tyrosine kinases from EGFR subfamily) [19, 20], suggesting that the tethered library-screening approach would also be amenable to protein kinases. The probing of the active site of an enzyme by using multiple crystal structures containing different small molecules was originally described using different organic solvents as the probe molecules [21]. This technique showed how the information derived from the small molecules binding in Me
HO2C
HN
H
O
CO2H
S O N
CO2H
O
Ki = 1.1 mM N
O
H2N
N
HO2C
HO2C
N
HN
N H
H
H HN
CO2H
CO2H
HN
O
O
Methylenetetrahydrofolate (Natural co-factor) O
O S O N
CO2H
Ki = 24 µM
S O N
O N H
CO2H
Ki = 330 nM
Fig. 1.4 The increase in thymidylate synthase inhibition activity obtained from an initial hit identified as a disulphide-bound enzyme adduct and optimised to a reversible potent 330 nM inhibitor using crystal structure-guided design.
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FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
the active site could be used in the rational design of larger more potent binding compounds. Indeed, this approach has been heavily exploited in the NMR field, with small molecule fragment library hits being rationally expanded, or sometimes combined, to generate lead compounds for medicinal chemistry [22–24]. This approach has also been used as a lead finding tool in X-ray crystallography. Specifically, a very small focused fragment libraryscreening approach was used for the generation of a potent inhibitor of the serine/threonine kinase p38 a [25, 26]. The starting fragment set of 327 compounds was selected from available chemicals with ring systems and desired functionalities. A smaller subset of 116 kinase set compounds was then selected based on predicted binding to kinase ATP-binding sites. This kinase set was then screened using cocktails of up to four fragments at 25–200 mM soaked from 1 to 24 h into preformed apo-protein crystals. Screening was performed on a variety of equipment both in-house and at synchrotron units. After the data had been processed and structures solved, an automated method was used whereby the difference in electron density at the active site between soaked and unsoaked protein crystals was compared to ligand structures generated by in silico docking. This was used to identify which fragment from a cocktail bound to the active site (Figure 1.5). The fragments in each cocktail were selected to aid in the electron density identification process. The authors showed how a fragment with an IC50 of
Soak
Compound library cocktail
Protein crystal X-ray
Further lead optimisation chemistry (aided by structure)
Structure-based chemistry
Increase potency & selectivity
Fragment identified & binding mode determined
Binding site electron density solved
Fig. 1.5 Schematic diagram to show the process involved in crystallographic screening of compound fragment cocktails.
M. CHERRY, J. READER AND D. WILLIAMS
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Cl O
N
Ph
O
NH2
N
Cl
NH2 109 µM
1300 µM
Cl
Cl O
O
O N H N
O
O Ph
N
N H N
30 µM
0.065 µM
F
Fig. 1.6 The increase in potency of a 1.3 mM p38 a protein kinase-binding fragment inhibitor identified by X-ray screening through the major changes made to the chemical series to exploit key binding features within the active site.
1.3 mM could be rationally converted into a compound of 65 nM potency with drug-like properties (Figure 1.6). This process was initiated with an X-ray structure showing the 1.3 mM hit forming a hydrogen bond to the ATP-binding site hinge region, from the pyridyl nitrogen of the fragment to the main chain amide nitrogen of Met-109. In addition, the benzyloxy group of the fragment binds in the lipophilic specificity pocket of the protein kinase. This lipophilic pocket was exploited further by replacing the benzyl group with a 2,6-dichloro substitution to fill this space, resulting in more than a 10-fold increase in potency. Potential movement of the Phe-169 at the start of the activation loop in p38 a leads to exposure of a polar channel formed by residues Asp-168 and Glu-71 stretching from the ATP-binding site to the lipophilic pocket. This movement was exploited by substitution on the 5-position of the fragment phenyl ring with a benzamidine to bring the potency up to 30 mM and then with a large amide, resulting in the 65 nM potency. The inhibitor design was aided both by iterative crystal structures of synthesised compounds and using key information gained from solving structures of other literature-described compounds. The most X-ray intensive screening method was described by Card et al. [8] on the design of phosphodiesterase (PDE) inhibitors (Figure 1.7). The authors initially biochemically screened a 20,000 member library of small molecular weight (120–350 MW) core scaffold compounds against 5-PDE isoforms at 200 mM. Multiple isoforms of PDE were used in order to eliminate the number of false positives obtained from the screen. There were 316
14
FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
Protein crystal
Biochemical screen (hogh concentration)
or
Protein
Scaffold identified
Scaffold library
Soak or co-crystallise
X-ray Further lead optimisation chemistry (aided by structure)
Scaffold elaboration
Increase potency & selectivity
Scaffold binding mode determined
Binding site electron density solved
Fig. 1.7 Schematic diagram to show the process involved in screening and crystallographic confirmation of scaffold or template libraries.
compounds that gave enzyme inhibition of greater than 30% against three or more enzyme isoforms and these were advanced into co-crystallisation experiments. Successful co-crystallisations were achieved for 269 compounds and 107 co-crystal structures were solved, as traceable ligand electron density was not achieved with all the X-ray data sets. The rationale behind this screening approach was to identify compounds, which could act as scaffolds or templates to further chemical design and so the next step in the process was to validate the compounds as core-binding moieties. This was performed by buying and making substituted versions of the hits, determining their X-ray structures and confirming that the binding mode of the core moiety was identical between the hit and the substituted version. Once the scaffold nature of the hit was confirmed, the core’s chemical elaboration was performed. Compound libraries were first enumerated virtually and then docked in silico, while keeping the scaffold core immobile in the binding site. Compound scoring was then performed on the predicted types of favourable interaction and chemical tractability prior to synthesis. Two rounds of chemical synthesis were performed heavily backed up with co-crystal structure generation. This approach resulted in a 4,000-fold increase in potency against PDE4D for one example shown, from 82 mM as an original carboxyl pyrazole screening hit to 20 nM as a substituted 1-phenyl carboxyl pyrazole (Figure 1.8). The increase in potency was achieved with only a modest increase in molecular weight
M. CHERRY, J. READER AND D. WILLIAMS
EtO2C Me
Me
N
Me
EtO2C
N Me
N
N
H
Ph
82 µM
0.27 µM
15
Me
EtO 2C Me
N
N
Cl
0.019 µM
Fig. 1.8 The increase in potency against PDE4D of a scaffold inhibitor, identified by biochemical screening at high compound concentrations then confirmed by X-ray crystallography, through two iterations of chemistry.
from 168 to 278 Da, meaning that there was still plenty of scope for further lead optimisation to obtain the ideal biological profile. Although this approach was reported on the PDE enzyme family, a similar approach has been successfully performed on protein kinases at Sareum Plc (D. Williams, unpublished results). The use of X-ray crystallography in inhibitor screening has been quite inventive in order to provide valuable data for more rational compound design, given the overall limited throughput of this technique. The last five years have seen many advances that are starting to make the generation of protein–ligand structures much more rapid and commonplace in drug discovery. Advances in both X-ray generator technology [27, 28] and newer concepts in structure determination [29, 30] will lead to further improvements in this field, possibly ultimately resulting in structure generation throughputs only currently enjoyed in the field of high throughput assay screening. SCREENING BY NMR NMR is a valuable tool for studying both the nature and the extent of interactions between ligands and proteins. A key advantage of the method is the fact that an understanding of protein function is not necessary, allowing drug discovery even against orphan proteins. NMR techniques are being increasingly used for the discovery of weak-binding hits and their subsequent optimisation into more potent leads. Furthermore, NMR studies take place in solution, which mimics to a varying extent the natural physiological environment of proteins and allows the examination of dynamic events such as conformational changes in both protein and ligand upon binding. This is in contrast to X-ray crystallography techniques, which necessarily provide a ‘snapshot’ of protein/ligand structure.
16
FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
Several excellent recent reviews [31–35] describe the varied techniques used for determining protein structure, investigating protein–ligand binding and optimising weak interactions into high-affinity leads. It is beyond the scope of this review to give anything more than an overview of these techniques.
FUNDAMENTALS OF NMR SPECTROSCOPY In a magnetic field, certain magnetic nuclei, such as 15N, 13C or 1H, can exist in nuclear-spin states of different energy and can be induced to flip between these different energy levels by the application of radio-frequency radiation. The frequency of radiation required to effect this energy-level transition is determined by the chemical environment of the magnetic nucleus in a given applied magnetic field. The magnetic environment of a nucleus is affected through chemical bonds (spin–spin coupling) and through space (dipole– dipole coupling), and so the resonance signal for a particular nucleus is directly related to its position within a molecule. When a molecule interacts with another, for example upon ligand binding to a protein, additional intermolecular through-space interactions may become important, affecting the resonance signal for the nuclei involved in the interaction. In theory, any measurable NMR spectroscopic parameter may be used to investigate binding of a ligand to a protein, but in practice they are limited to measurements that can be determined easily and with a sufficient degree of sensitivity. There are two main approaches used in drug discovery today. The first approach examines chemical shift changes of the protein upon binding of a ligand and the second examines the NMR signals of the ligand and their change upon binding to a protein. In addition, there are also two complementary approaches that examine changes in relaxation or diffusion behaviour of a ligand upon binding to a protein [34, 35].
USING CHEMICAL SHIFT CHANGES
Upon binding of a ligand to a protein, the chemical shifts of signals for both protein and ligand will be affected. This effect, in the protein, is greatest for the residues located at the binding site and as protons are the most sensitive nuclei in NMR experiments, it is sometimes possible to detect binding of a ligand to a protein by observation of changes in the chemical shift of wellresolved protein protons. Unfortunately, it is usually extremely difficult to unambiguously assign the chemical shift changes of protein proton signals due to severe overlap, making it impossible to determine the amino acid residues involved in binding.
M. CHERRY, J. READER AND D. WILLIAMS
17
One solution to this problem is to label the target protein with the stable isotopes 15N and/or 13C. Binding of a ligand changes the resonance signals at the binding site, and these changes can be measured using 15N/1H and/or 13 C/1H correlation spectra. The 15N/1H or 13C/1H heteronuclear single quantum correlation (HSQC) spectrum of the labelled protein is acquired as a reference. Upon addition of a binding ligand followed by acquisition of a second HSQC spectrum, changes will be observed between the two spectra, particularly at the protein residues at the binding site. To simply detect binding, it is only necessary to observe changes between the two spectra. However, to determine the residues involved in binding it is necessary to assign the 15N or 13C HSQC signals to the amino acids in the protein. NUCLEAR-SPIN RELAXATION
Magnetic nuclei in a magnetic field can adopt nuclear-spin states of different energy. As a system reaches equilibrium, the population differences between the different nuclear-spin states decays exponentially through relaxation. The intensity of a resonance signal in the NMR spectrum is related to the difference in population between the two energy levels of the different nuclearspin states. This means that the rate at which this difference returns to equilibrium and relaxation has a profound effect on the sensitivity and resolution of an NMR spectrum. Longitudinal magnetisation, Mz, is restored to its equilibrium value Moz by longitudinal relaxation, characterised by the longitudinal relaxation time T1. Transverse magnetisation, Mx and My, decays by transverse relaxation characterised by the transverse relaxation time T2. Nuclear-spin relaxation is mainly modulated via through-space interactions between different nuclear spins and via Brownian rotational tumbling as the orientation of the molecule varies relative to the applied magnetic field. Slowly tumbling large molecules, such as proteins, undergo rapid transverse relaxation, which causes line broadening in the NMR spectrum. This imposes an upper limit on the size of molecules whose structures can be usefully interpreted by NMR. Small molecules tumble at high rates and have much slower relaxation rates, and therefore a sharper well-resolved NMR spectrum. NUCLEAR OVERHAUSER EFFECTS
Nuclear Overhauser effects (NOEs) cause changes in the intensity of NMR signals by through-space dipole–dipole interactions [36]. The magnitude of an observed NOE between two magnetic nuclei gives useful information on the distances between them. The observed NOE is also related to Brownian
18
FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
rotational tumbling, with small, rapidly tumbling molecules giving positive NOEs and a maximum relative signal strength of 50%, and large, slowly tumbling molecules giving negative NOEs with a maximum relative signal decrease of 100%. EXCHANGE PHENOMENA
NMR spectra are affected by dynamic processes such as the binding of a ligand to a protein. As a bound ligand will have a different NMR spectrum to the free ligand in solution, the rate constant for formation of the ligand/ protein complex, Kon, and the dissociation rate constant, Koff, will affect the NMR signals for the system at equilibrium. If the exchange process is slow, two distinct sets of signals for the ligand will be observed, but if the exchange process is rapid, a single set of NMR signals will be observed, representing the weighted average chemical shifts of the free and bound signals.
APPLICATIONS OF NMR IN SCREENING Two distinct strategies are used in NMR screening. The first strategy looks for changes in the NMR spectrum of the protein target and the second looks for changes to the NMR spectrum of the ligand. TARGET-BASED SCREENING
When a ligand binds to a protein, there is a change in the magnetic environment at the binding site and at any other residues affected by conformational changes in the protein. This results in measurable changes in the NMR spectrum of the protein and, if the affected residues are mapped onto the protein structure, it is possible to determine the ligand-binding site. There is currently, however, an upper limit on the protein molecular weight of approximately 35 kDa, as such assignments are very resource and time intensive, even for small proteins. This approach has been used in the study of ligand–protein and protein–protein interactions. The group at Abbott Laboratories has developed an extension of this method called ‘SAR by NMR’, which is used to screen collections of small molecules [22]. Using labelled protein, the resonances are assigned as far as possible and the 3D structure of the protein is solved by NMR or by X-ray crystallography. A collection of ligands is screened by comparing the 15N–1H HSQC spectrum of the protein in the absence of, and then in the presence of, a mixture of 10 or more compounds. Changes in the chemical shifts of the amide 15N and/or 1H
M. CHERRY, J. READER AND D. WILLIAMS
19
induced by the binding of a ligand can be detected and if the resonances affected have been assigned, determination of the protein residues involved in binding is straightforward. Any mixture of compounds containing a hit is then deconvoluted to find the active component. Furthermore, measuring the resonance shift changes upon ligand titration enables an estimation of the dissociation-binding constant of that ligand with the protein. 13 C–1H HSQC is based on proteins containing 13C-labelled methyl groups and is increasingly used to complement 15N–1H HSQC experiments [37]. The method is more sensitive due to the higher proton multiplicity and is suitable for screening proteins up to 40 kDa. The key binding site residues of a protein can be identified by using a known inhibitor to identify cross-peaks in the 13C–1H HSQC spectrum [38].
LIGAND-BASED SCREENING
Ligand-based NMR screening examines changes in the NMR spectrum of a ligand or a mixture of ligands in the presence and absence of the target protein or macromolecule. These methods identify which ligand binds to the target, but give no information on the binding site. The techniques require less protein, do not require labelling of protein and have no restriction on the size of the protein being studied, as they do not require assignment of the target’s resonance signals. Some experiments, such as saturation transfer difference (STD) [39] or WaterLOGSY [40] require protein concentrations of 1 mM or less meaning much less protein is required even for screening multiple mixtures of ligands. The transverse relaxation time, T2, of small molecules is usually long compared to that of proteins and therefore the resonance signals for small molecules are generally much narrower than those of proteins. However, upon binding of a ligand to a protein, T2 of the ligand will more closely resemble that of the protein, resulting in line broadening of the ligandassociated signals. Line broadening in the NMR spectrum of a ligand following addition of a macromolecule to the sample is a clear indication of binding. Furthermore, the extent of line broadening, and therefore the sensitivity of the experiment, is related to the size of the protein – with larger structures usually having a more pronounced effect on T2 of the ligand. The longitudinal relaxation time, T1, of small molecules is also altered by binding to proteins, resulting in the observation of negative NOEs for bound ligand resonances [41]. Ligand binding can therefore be detected by measuring a 2D [1H–1H]-NOESY spectrum of the ligand in the presence of a relatively low concentration of protein. Unbound small molecules generally have small, positive NOEs, but on binding to a protein, adopt the
20
FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
characteristics of that protein and can display negative (transferred) NOEs, provided that the T1 relaxation time of free ligand is long compared with 1/Koff for the complex. Practically, this means that weak-binding compounds with relatively high dissociation constants will give negative NOEs in the presence of protein, whereas tight-binding molecules may not. There are several ligand-screening techniques that look at the transfer of magnetisation from the protein to the ligand or from ligand to protein. STD NMR spectroscopy [39, 42] has been used in the past to examine tightly bound ligand/protein complexes. If there is slow exchange between the bound state and the free state, the ligand will show a pair of signals and a transfer of saturation between the free and the bound states is possible. By irradiating a signal of the free ligand, the corresponding signal of the bound ligand may be identified following saturation transfer. More common in screening, however, is the transfer of saturation from the protein to a bound ligand, which in turn, by exchange, moves into solution where it is detected. Subtracting a spectrum where the protein is saturated from one without protein saturation generates a different spectrum containing only the signals of a binding ligand. WaterLOGSY [40] is a variant of STD spectroscopy that utilises the bound water at the protein–ligand interface. Negative NOEs between water and ligand are observed due to bound water being present between the protein and the ligand, or by the presence of a shell of water molecules surrounding the ligand. This has allowed the development of experiments that allow the transfer of magnetisation from bulk water to the protein-binding site and on to a bound ligand, allowing the detection of binding of ligands to proteins. Methods based on diffusion filters have also been described by the group at Abbott. Small organic molecules diffuse about 20 times more rapidly than a protein of molecular weight 20 kDa. This difference can be used to distinguish between bound and unbound ligands in the presence of a protein. A diffusion-edited spectrum is obtained for a mixture of ligands in the absence of protein. Following addition of a small concentration of protein, the diffusion-edited spectrum is repeated and comparing the two spectra allows identification of compounds that bind to the target protein [43].
EXAMPLES OF NMR METHODS FOR DISCOVERING KINASE INHIBITORS THE SHAPES STRATEGY
An NMR screening strategy in which a small but diverse set of small molecules is screened against a potential drug target was recently described
M. CHERRY, J. READER AND D. WILLIAMS
21
by the Vertex group [44]. The set of compounds is derived from shapes commonly found in existing drugs. Detection of low-affinity binding is achieved using either differential line broadening or transferred NOE NMR techniques. The utility of this technique has been demonstrated with several enzyme targets including the serine/threonine mitogen-activated protein kinase (MAPK) p38. In selecting an NMR screening library, the objective was to acquire a set of compounds allowing optimisation of a large number of factors (e.g. cost, ease of synthesis, solubility, separation of NMR signals and diversity). The design of the library was based on earlier analyses of drugs and descriptors of their ‘drug likeness’ [45, 46]. In the study, all drug molecules were broken down into their component rings, linkers and side chains. The library was initially chosen from commonly occurring rings and linkers (collectively ‘frameworks’) and side chains from the comprehensive medicinal chemistry (CMC) database, then further filtered by predicted aqueous solubility and synthetic complexity. All library compounds were commercially available, soluble and non-aggregating in water at 1 mM, chemically and isomerically pure and contained no reactive functional groups. The compounds had an average molecular weight of 194 Da and calculated log P range of 2.2–5.5. They were also required to have a wellresolved simple 1H NMR spectrum and contain at least two protons within 5 A˚ of one another. The initially reported library contained 132 compounds from which subsets were screened depending on the pH of the screening solution. A typical protocol for NMR screening involves dissolving the protein in 500 ml of an appropriate aqueous buffer, at a concentration of 50–100 mM. Low volumes of a concentrated stock solution of ligands in deuterated dimethyl sulphoxide (DMSO) are then added, such that the final concentration of each ligand is 1 mM and the total amount of DMSO in solution could be as high as 1%. As the NMR-screening experiment may take place over a number of hours, it is important for the protein to remain stable in solution over this time period. The Vertex group routinely employs the microdrop protein screening method [47] to screen protein samples to ensure their stability under differing concentrations of DMSO, with or without stabilisers and at varying pH. To maximise throughput, it is preferable to screen the SHAPES library as mixtures. However, several problems are raised by this approach. Perhaps the most important is the fact that if the mixture contains a compound with high affinity for the protein, it will successfully compete for the majority of the protein-binding sites in the solution. This may mask the activity of a more weakly binding, but highly optimisable scaffold, resulting in the elimination of an entire compound class from further development. However, due to the inherent weak-binding
22
FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
nature of the small molecules in the SHAPES library, this has not been reported as a significant problem to date. The problem of spectral overlap has been largely overcome using automated methods that search the 1D spectra and select combinations of ligands whose spectra are generally easily distinguishable. When examining binding by following line broadening in a 1D spectrum or by analysis of transferred NOE, mixtures of 1–4 compounds are routinely used. However by using 2D tNOE spectroscopy, mixtures of up to 12 compounds have been successfully tested. Once the appropriate mixtures are selected, either the 1D, or both the 1D and 2D tNOE spectra for each mixture are recorded in the absence and presence of protein. Binding of a ligand is identified either via line broadening or by inversion of the NOE cross-peaks. These effects cannot distinguish between specific and non-specific binding, so more rigorous follow-up is needed. Looking for line broadening in the 1D spectra alone is suitable when examining binding to higher molecular weight proteins (>60 kDa) and so screening of the entire library can be completed in several hours. Smaller proteins require the acquisition of both 1D line broadening and 2D NOE spectra and this can take several days. The method has been demonstrated to have utility in hit-finding against a kinase target. It is believed that modulation of serine/threonine kinase JNK3 (c-Jun amino-terminal kinase 3) could provide therapeutic benefits in the treatment of neurodegenerative diseases such as stroke or epilepsy. After an unsuccessful high throughput screen, the SHAPES library was screened versus JNK3 using a 2D transferred nuclear Overhauser effect (tNOESY) experiment and 17 weakly active binders were discovered of which 13 were found to bind specifically in the ATP-binding site [32]. The available chemicals directory (ACD) [48] was then searched using fragment-based and similarity methods to identify analogues of the SHAPES hits, and these analogues were then virtually screened using the X-ray structure of JNK3 before a final selection of 100 high-scoring compounds were chosen. Additionally, 200 compounds from the ACD, which combined features of the SHAPES hits were selected manually. Secondary screening of the follow-up compounds found eight with potencies better than 20 mM. Three of these are shown in Figure 1.9. The thiazole and the uracil classes were found by combining the initial SHAPES hits, whereas the isoxazole was discovered by selecting analogues of the hit from the ACD directly. Four classes of compounds were selected for optimisation, affording 1 mM and 3 nM lead series. Interestingly, none of these classes of compounds had shown activity in the high throughput screen of JNK3. The Vertex group suggests that this may be because the small SHAPES compounds are more likely to bind (albeit weakly) than the more complex compounds typically found in their HTS collection.
M. CHERRY, J. READER AND D. WILLIAMS
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Follow-up compound
SHAPES hit
O
N
NH
N
N
Scaffold O Ph
Combination
Ph
S
Scaffold
Ph
Ki = 13 µM
O
Ph
N H
HN
Combination
CF3
N H
N
NH
O
N H O
NH
Ph
Ki = 0.72 µM
Ph N
H N
N Analogue
N
N N
NHAc N
Ph N
O
Me
Ki = 0.79 µM
Fig. 1.9 Analogues of JNK3 inhibitors identified using NMR screening. NMR SCREENING BY WATERLOGSY METHOD
Water is known to play a key role in ligand–protein binding, with multiple water molecules being frequently observed at the ligand–protein interface, e.g. a layer of water was detected at the lovastatin/LFA-1 interface [49]. The intermolecular water–ligand NOEs are negative, indicating a residence time of >1 ns. Saturation of water results in magnetisation transfer from bulk water to bound water located in protein cavities and in the magnetisation transfer from bulk water to the layer of water at the protein–ligand interface. The group at Pharmacia developed an NMR technique called Water–Ligand observation with gradient spectroscopy (WaterLOGSY), which is able to detect binding of ligands to proteins via the magnetisation transfer from bulk water. The method was exemplified by the selection of an indole derivative from a mixture of 10 diverse compounds, which was found to bind to cyclin-dependent kinase 2 (cdk2). The compound (Figure 1.10) is reported to have a Ki in the high micromolar range [40].
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FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
CO2 Et CO2 Et N H Fig. 1.10 Cdk2 inhibitor identified using NMR screening by the WaterLOGSY method.
NMR SCREENING OF PROTEIN KINASES BY ATP–STD METHOD
The Schering–Plough group has recently reported on the use of ATP as a site-specific marker in STD–NMR experiments for screening of kinase inhibitors [50]. ATP–STD spectra detect the NMR signals from ATP in the active site of the kinase and reduction of that signal in the presence of competitive inhibitors allows measurement of the inhibitor Ki with respect to the natural substrate. Furthermore, by adding MnCl2 to the sample, ATP is converted into a paramagnetic probe, which can be used to elucidate the distance of non-competitive inhibitors from the ATP-binding site. Four 1D experiments are performed. Initially a 1D spectrum of the kinase, ATP and TSP (trimethylsilylpropane sulfonate – as an internal reference) is recorded. Then an inhibitor (or compound of interest) is added to the sample and an additional spectrum is generated. Any compound degradation or side products are detectable by the NMR method and hydrolysis of ATP can be monitored by measuring chemical shift changes of the purine and sugar protons. In a third experiment, the ATP–STD signal is optimised. A typical STD spectrum of target-bound ATP gives peaks from the purine H8 and H2, and the sugar H10 protons. In the presence of an ATP-competitive binder, these ATP–STD signals are reduced and an inhibitor STD signal appears. This was demonstrated in an experiment in which the ATP–STD peak for the H2 proton of MgATP bound to extracellular signal-regulated kinase 2 (ERK2) was reduced by 54% by the addition of olomoucine. Olomoucine STD peaks were also observed. Competitive inhibitors can be distinguished from non-competitive inhibitors by the addition of a high-affinity inhibitor e.g. staurosporine. As staurosporine is a highly potent inhibitor and competitive with ATP for most serine/threonine kinases, its addition eliminates the STD peaks for ATP and for any other competitive inhibitors. The STD signals of non-competitive inhibitors are not affected by the addition of potent competitive inhibitors. In a fourth experiment, a paramagnetic probe can be used to determine the proximity from the ATP-binding site of non-competitive inhibitors. An MnATP probe is generated by the addition of Mn2+ ions to ATP [51]. The
M. CHERRY, J. READER AND D. WILLIAMS
25
use of the MnATP probe was demonstrated in an NMR-binding study of diarylamines to the serine/threonine kinase MEK1 (Map Erk kinase 1). The diarylamine, PD318088 is a non-ATP-competitive MEK1 inhibitor, known by X-ray crystallographic studies to bind proximally to the ATP in MEK1 [52]. NMR studies with a diarylamine analogue gave strong STD signals, indicating binding. However, these signals were not reduced by the addition of staurosporine suggesting non-competitive binding. The addition of an MnATP probe reduced the STD signals from the diarylamine compound, indicating that it was binding in close proximity to the ATP-binding site. Furthermore, proton signals on the two rings of the analogue were reduced to a different extent, allowing the orientation of the molecule to be deduced. ATP–STD NMR is described as a simple, cost-effective and robust assay that can be used to identify inhibitors of virtually any nucleotide-binding target.
NMR BACKBONE ASSIGNMENT OF A KINASE
The partial backbone assignment of the catalytic domain of protein kinase A (PKA) using a combination of NMR techniques was reported in 2004 [51]. This represented the first reported NMR assignment of any protein kinase catalytic domain. Backbone resonance assignment of the 42 kDa protein was achieved using a combination of triple-labelled (2H, 13C, 15N) protein with classical NMR assignment, back-calculation of chemical shifts from X-ray structures, the use of paramagnetic adenosine derivatives as spin-labels and selective amino acid labelling. PKA is not currently a drug target due to its broad spectrum activity and multiple physiological functions. However, it is considered to be a useful protein for studying other protein kinases [53]. Several X-ray structures of PKA, with and without bound inhibitors, have been published. The structure of the non-phosphorylated, inactive structure was, however, unknown. The authors were keen to emphasise the dynamic nature of proteins, particularly the conformational changes occurring upon activation, and upon binding of substrates and inhibitors, and point out that NMR is an ideal tool for studying motional processes by relaxation analysis. The authors expressed PKA consisting of 353 amino acids, of which eight are prolines. Resonances of 274 backbone amide peaks were visible in the spectrum, of which 191 were assigned. It was possible to assign resonances for the N- and C-terminal sequences, the majority of the N-lobe, including the glycine-rich loop, and most of the solvent-exposed residues of the C-lobe. This enabled a determination of the structure for the more flexible parts of the structure. However, many correlations were missing for the
26
FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
more rigid parts of the protein, resulting in incomplete data sets. It was only possible to correlate relatively short stretches of the protein, making it difficult to match on the sequence. To circumvent this difficulty, the authors adopted a novel approach of predicting chemical shifts for the backbone and Cb from known structures [54]. The experimental data from a correlated stretch of amino acids from a known structure was found to provide sufficient information to allow matching to the novel structure. It was possible to unambiguously assign the amino acids near to the ATP-binding site by the use of paramagnetic spin-labelled adenosine (Figure 1.11), leading to a pure distance-dependent line broadening or disappearance for those signals within approximately 20 A˚ of the spin-labelled adenosine. Selective labelling of PKA with 15N-labelled Phe, Tyr, Leu, Asp, Ile and Val was carried out allowing more facile assignment of the peaks due to those residues. For example, it was possible to assign 19 out of the 20 resonances due to valine in the protein using this approach. 13C-labelled Tyr was used in conjunction with 15N-labelled Val allowing the identification of the only Tyr–Val sequence in PKA via a 2D HNCO spectrum. The authors were able to identify the Val-123 transverse relaxation optimised spectroscopy (TROSY) peak, which had no residual correlations in the 3D spectrum. By adding the known kinase inhibitor H7, it was possible to define the binding site by examination of the chemical shift perturbations and to map the interaction surfaces. This experiment confirmed the crystallographically determined position of the ligand [55]. An examination of mutant PKA proteins was undertaken. Phosphorylation of Thr-197 is required to activate PKA and phosphorylation of Ser-338 enhances stability of the protein. Replacement of Thr-197 and/or Ser-338 by Ala was examined to determine any conformational changes in the protein. Both single substitution mutants were expressed in Escherichia coli in similar levels to wild-type protein. However, both mutants were found to be less stable, as had been previously described. The double mutant O Me Me
NH N
N
O
Me Me
N
NH2
O HO
OH
N
N
Fig. 1.11 Structure of spin-labelled adenosine (1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxylate (5-aminoadenosine)-amide).
M. CHERRY, J. READER AND D. WILLIAMS
27
Thr-197-Ala/Ser-338-Ala could also be expressed but was highly unstable and aggregated readily. To investigate folding of the Thr-197-Ala and Ser-338-Ala mutants, the proteins were 15N-labelled and their TROSY–NMR spectra were recorded. The peak pattern for the Ser-338-Ala mutant closely matched that of the wild-type protein. However, the Thr197-Ala mutant showed significant differences in the peak patterns, thus indicating conformational changes in at least one region of the protein. The ability to assign a significant percentage of the NMR signals from a kinase catalytic domain is an important prerequisite for the study of the conformational changes that the kinase catalytic domain undergoes upon activation, phosphorylation events, ATP binding and inhibitor binding. These experiments also open up the possibility of using techniques such as SAR by NMR with kinases in the future, thus potentially allowing the screening by NMR of compound libraries, and the discovery of novel and selective inhibitors of kinases. DESIGNING NOVEL KINASE INHIBITORS FROM THE NMR-BASED SCREENING OF FRAGMENTS
The Abbott group describes a method in which an existing high-affinity ligand is divided into its constituent fragments and fragments suitable for replacement are identified. The binding affinity and pose of these fragments is then examined by analysing changes in 15N–1H HSQC spectra of the 15 N-labelled protein upon addition of the fragments. NMR-based screening is then used to identify new molecules that bind in the same way as the fragment to be replaced, but which are expected to have more favourable characteristics, for example, an improved protein kinase profile. This approach is depicted in Figure 1.12 below. The method was exemplified by the discovery of novel inhibitors of adenosine kinase (AK) [56]. Compound (1) (Figure 1.13) is a novel inhibitor of AK that has been demonstrated to have potent antinociceptive activity in animal pain models. However, the molecule has solubility and side-effect profiles that could be improved. One fragment deemed suitable for replacement was the bromophenyl moiety and so a suitable core molecule was required that would allow access to the binding site, but reduce undesired binding at other areas in the active site. Core structure (2) most closely resembles the original molecule. However, the proton at the 5th position could interfere with prospective groups binding at the bromophenyl subsite. The acetylene containing compound (4) had been found to be active against AK and was deemed to contain a better core, (5), for these studies. The subset of peaks that can serve as markers for the binding of replacements were identified by comparing the HSQC spectra of 15N–AK
28
FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
(a) Summary of the fragment optimisation approach:
• The high affinity lead is divided into constituent (b)
fragments
• A replacement fragment is identified by NMR screening
• The newly identified fragment is incorporated
(c)
back into the original lead.
Fig. 1.12 Fragment optimization approach.
Br
N NH2
NH2 N
N N
N
N (1)
(4)
N
N
N
O
N O
Br NH2
N
NH2
(3)
(6) N
N
N
N
N (2)
N
(5)
N O
N
N O
Fig. 1.13 Adenosine kinase inhibitor designed using fragment optimization approach.
M. CHERRY, J. READER AND D. WILLIAMS
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complexed to (1) versus (2) and 15N–AK complexed to (4) versus (5). A subset of peaks was observed to change allowing the identification of the binding site of the bromophenyl or pyridyl moieties. Furthermore, these same peaks were observed to move upon the addition of pryridine to a solution of AK and core (5), but no movement was observed on the addition of pyridine to a solution of AK in the presence of (1) or (4). More than 2,000 potential replacements for the bromophenyl or pyridyl groups were then screened using the NMR-screening technique. Several fragments were identified which caused chemical shift changes in the desired binding site of the protein, amongst them indole (7) and 2-phenylimidazole (8) (Figure 1.14). The KDs of the fragments were then measured and found to be 3 mM in both cases. The indole linker was then incorporated back into the original lead molecule via appropriately sized linkers to generate molecules (9) and (10) (Figure 1.15). These molecules were found to have IC50s of 23 and 10 nM, respectively, compared to the 1.7 nM IC50 for (1) in an in vitro cell-free enzyme assay. Compound (10) had good activity in a cell-based assay (IC50 ¼ 370 versus 51 nM for (1)) and demonstrated promising in vivo activity with an ED50 value of 3 mmol/kg in a carrageenan-induced thermal hyperalgesia assay, compared to 0.6 mmol/kg for (1).
H N
H N Ph
N (7)
(8)
Fig. 1.14 Fragments found to cause chemical shift changes in binding site.
NH2 HN
(CH2)n
N
N H N
(9) n = 1
N
(10) n = 2 N
N O
Fig. 1.15 Fragment optimized inhibitors.
30
FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION DESIGN OF LIBRARIES FOR USE IN NMR SCREENING
The group at Vernalis describes an approach to selecting a Selection of Experimentally Exploitable Drug Startpoints (SEEDS) library for use in NMR screening [57]. The solubility requirements for the library members were defined as aqueous solubility >2 mM and solubility in DMSO of >200 mM for stock solutions. Stability of the fragments to storage and to the screening conditions in use was required. The size of the library struck a balance between chemical diversity, and the cost and practical considerations involved in handling and screening a large set of compounds. Finally, the compounds were required to contain a range of chemical functionality and to be amenable to further optimisation into drug-like candidates. The objective was to select a kinase-focused fragment library from the 1.62 M compound rCat database [58]. As the majority of known kinase inhibitors interact with at least one of the backbone carbonyls of one residue and/or the backbone NH and/or carbonyl of the residue at the +2-position, the principal constraint in the library design was to target at least one of these interactions. Combinations of pharmacophores were built by considering the conformation of hymenialdisine bound to CDK2 as shown in Figure 1.16. Each of the pharmacophore queries consisted of one donor, one acceptor and one of the two hydrophobic points indicated in Figure 1.16. The directionality of hydrogen bonds was inferred from the X-ray structure and reasonably loose tolerances of 1.5 A˚ were used for donor–acceptor distances and 2–2.5 A˚ to the hydrophobe were chosen to allow for the flexibility seen across kinase structures and to maximise the diversity amongst the identified fragments. Four pharmacophore queries were completed and the results were combined. The chosen fragments were further filtered by molecular weight (between 150 and 250), Slog P (between 2.5 and 2.5) and the presence of
H O
N O
H N
NH N
Br
NH2
Fig. 1.16 The interaction of hymenialdisine with the backbone of a kinase. Arrows mark the location and direction of hydrogen-bonding groups, the filled circles represent the hydrophobic features.
M. CHERRY, J. READER AND D. WILLIAMS
31
suitable functionality for subsequent optimisation. Compounds containing unwanted functionality such as halogens, disulphides, or bridged systems were then removed, yielding a set of 2,042 compounds. These were further analysed for diversity based on Tanimoto similarity and on a 2D, three-point pharmacophoric fingerprint method followed by an in silico solubility cut-off of 2 mM. The sets of compounds derived were pooled and inspected visually for chemical tractability, availability and cost. A total of 204 compounds were purchased, of which 174 passed QC tests. The hit-rate of the library in NMR screening was examined versus two ATP-binding proteins, heat shock protein 90 (HSP90) and cyclin-dependent kinase 2 (CDK2), and compared with other non-kinase-biased fragment libraries. A hit was generally defined as a fragment, which binds to the protein with an affinity of greater than 5 mM. The hit-rate for the kinasebiased library was found to be approximately 8.5% against CDK2, with the next best non-kinase-biased library having a hit-rate of slightly over 5%. Against HSP90, the kinase-biased library had a hit-rate of approximately 5%, with the best library having a hit-rate of 7%. Although HSP90 is an ATP-binding protein, its active site has a number of conserved water molecules that mediate interactions between the protein and active ligands. The lower activity of the kinase-biased fragment library against HSP90 compared to that for CDK2 is therefore expected. Many advances have been made in recent years in NMR instrumentation and in experimental methodology. The role and value of NMR screening has become firmly established, particularly as newer methods have a reduced requirement for protein quantity, allowing screening of fairly large compound libraries. As the capacity for screening increases, so the intelligent design of focused NMR libraries will become more important and can be expected to continually develop. Although this review has treated NMR and X-ray crystallography as two distinct methodologies, recent reviews have emphasized the synergies to be gained by combining the two techniques [59, 60].
IN SILICO METHODS Virtual screening is not a new technology, with database searching using structural fragments as queries introduced as early as the 1970s; there has since been many reviews highlighting developments [61–67]. Today in silico screening encompasses a variety of computational tools and utilizes a broad range of information to formulate a screen. Table 1.1 lists the most commonly used methodologies. At the broadest end of the spectrum, filters based on the calculation of physiochemical properties, the most obvious being the Lipinski’s rule of
32
FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION Table 1.1 IN SILICO SCREENING TECHNOLOGIES
Method
Details
High throughput docking
In silico simulation of ligand binding and ranking of datasets using scoring functions Pharmacophore hypotheses generated from the 3D structure of the receptor Pharmacophore hypotheses generated from known inhibitors Quantitative Structure–Activity Relationship–statistical models that relate biological activity to features of a molecule Comparison of molecules using molecular descriptors and a measure of similarity, for example a 2D fingerprint and the Tanimoto coefficient Exact and substructure searching
Receptor-based pharmacophore Ligand-based pharmacophore QSAR models Similarity
Structure-based searching
5 [68] or Veber’s drug-like filter [69], are now routinely used in the selection of compounds from databases and in the design of new molecules. Structure-based, high throughput docking represents the opposite end of the spectrum, where compounds are ranked and selected using 3D information regarding the predicted binding of ligand to receptor. While generating more valuable target-specific information, high throughput docking has, in practice, hit upon similar issues to experimental high throughput screening in terms of speed and accuracy. A mix and match approach can, therefore, be adopted where techniques are used in succession, thus optimizing the speed and efficiency of subsequent steps in the form of a screening cascade. A cascade typically starts with a simple drug-like filter that requires no knowledge of the enzyme structure or of inhibitor compounds. The next step in a cascade is then to incorporate information specific to the target, i.e. protein kinases, whether it is ligand-based or receptor-based data. The present review focuses on structure-based methods, primarily concentrating on high throughput docking.
RECEPTOR-BASED SCREENING The abundance of crystallographic information on the 3D structure of protein kinases, including knowledge of receptor–ligand complexes, has seen protein kinases feature consistently in datasets employed to both develop and evaluate methods for receptor-based screening. Indeed, the vast majority of literature surrounding protein kinases and receptor-based in silico
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screening concerns development and evaluation, only recently have examples emerged where receptor-based screening was used purely in application. Muegge and Enyedy [70] recently reviewed virtual screening for kinase targets, discussing the many pitfalls and areas of concern. Receptor-based virtual screening can be broken down into several distinct stages; compound database and receptor preparation, protein–ligand docking, post-docking analysis and finally compound prioritization. Recent developments have centred upon docking and scoring, but it should be stressed that each stage is crucial to overall performance and so an overview of each will now be presented.
COMPOUND DATABASE AND RECEPTOR PREPARATION
As stated above, an advantage of virtual screening is that any compound, real or virtual, can be screened and the user is not restricted to those compounds available in corporate or external collections. The technology can also be used to screen proposed libraries and even select monomers for a combinatorial library based on 3D fit to the target structure. The first stage in the preparation of the compound database is to standardize the representation of the compounds, remove additional fragments (for example salts) and fix the protonation state of ionisable groups. Docking tools do not, as yet, consider alternative stereochemistry or tautomerism and so it may also be necessary to enumerate the compounds accordingly. Filters can then be applied such as substructure searching, similarity-based methods, Quantitative Structure–Activity Relationship (QSAR) models and the drug-like filters discussed above. There are many tools available for these tasks and throughput is extremely rapid, easily handling several millions of compounds. These filters can also significantly reduce the size of a compound database as demonstrated by Baurin et al. [58] in the analysis of chemical databases from commercial suppliers. Twenty-three catalogues covering 2.3 M compounds underwent a series of filters based on 1D and 2D molecular descriptors including the Lipinski filter, the Veber filter, an aqueous solubility model and a permeability model. Of 1.6 million unique compounds only 37% passed all the filters, a significant saving in resource if screening such a dataset. Molecular recognition is a 3D event and most, if not all, docking tools call for the compounds in a suitable 3D format. For standalone docking tools there are several well-validated methods for 3D conversion [71]. Where a docking module is part of a suite of modelling tools, there is usually an associated 3D conversion utility. Most docking algorithms incorporate ligand flexibility. For those that do not, an additional stage of conformer
34
FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
generation is required, a point discussed in greater detail below. Also, depending on the docking tool there may be other factors that need to be considered including geometric isomerism, ring flips, partial charge assignment and appropriate atomic configuration. The protein kinase model, whether derived from crystallographic data or generated using comparative modelling techniques, should undergo preparation appropriate for the choice of docking algorithm. The process begins with a general evaluation, refining any potential ambiguities in the model. Decisions need to be made regarding the protonation state of ionisable groups, tautomerism, side-chain rotamers, inclusion of water molecules and possibly how to incorporate protein flexibility. The importance of having a full understanding of the application and limitations of X-ray crystallographic data were reviewed by Davis et al. [72].
PROTEIN–LIGAND DOCKING
Protein–ligand docking can be further divided into two phases. First is the sampling of protein–ligand-binding space and second is the selection of the best scoring pose(s) from the sampling process. Both phases have been approached using a variety of methods; the most popular packages are listed in Table 1.2, together with the approach employed. Sampling of the pose space, considering all possible positions, orientations and conformations of a ligand in the protein-binding site, is crucial to the success of a method. Rigid docking programs, for example Fast Rigid Exhaustive Docking (FRED) [73], require a pre-computed conformational database containing an ensemble of conformers for each ligand; these conformers are then rigidly docked into the protein active site. Rigid docking
Table 1.2 HIGH THROUGHPUT DOCKING PROGRAMS Program
Strategy
Vendor
Website
GOLD
Genetic Optimisation of Ligand Docking Incremental build Grid-Based Ligand Docking with Energetics. Flexible, exhaustive docking employing a heuristic screening process Geometric matching of ligand to receptor Fast Rigid Exhaustive Docking
CCDC
www.ccdc.cam.ac.uk
Tripos Schrodinger
www.tripos.com www.schrodinger.com
UCSF
dock.compbio.ucsf.edu
OpenEye
www.eyesopen.com
FlexX GLIDE
DOCK FRED
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benefits from advantages in speed and, once generated, the database is available for future experiments. Issues surrounding storage are less apparent with current computational resources and generating the conformational database using packages, such as Omega (OpenEye) [74, 75] and Catalyst (Accelrys), is extremely rapid. The protein environment can though favour ligand conformations that would otherwise be rejected by methods operating in the free state. McGann [76] calculated the root mean square deviation (RMSD) for conformers generated by Omega and those observed in protein–ligand complexes, and noted differences >1.5 A˚ for 2% of the ligands. The sampling, therefore, has to be exhaustive to ensure that the ensemble of conformers generated is relevant to the binding of ligand to protein. In addition to an exhaustive search of conformational space for each ligand, FRED (OpenEye) performs an exhaustive, systematic sampling of the position and orientation of each conformer in the active site. Poses are initially rejected based on steric overlap with the protein before ranking and selection, using one or more scoring functions. Grid-Based Ligand Docking with Energetics (GLIDE; Schrodinger) [77, 78] approximates a complete systematic search of the positional and orientational docking space, but incorporates ligand flexibility as part of the docking process. High throughput is attained by first performing a rough dock eliminating low-scoring poses. Surviving candidates are then subjected to an energy optimisation phase allowing full torsional freedom of the ligand before further refinement using Monte Carlo sampling. Monte Carlo and other computationally intensive methods are, at present, unfeasible for large scale screening, hence GLIDE’s approach to first reduce the dataset using a more approximate positioning and scoring phase. Monte Carlo, simulated annealing and molecular dynamics calculations have found use for smaller datasets and in the prediction of binding affinities [79–81]. A large percentage of the binding space sampled through an exhaustive search will be redundant. FRED and GLIDE attempt to circumvent the inefficiency of an exhaustive search by first performing a rough dock, reducing the dataset to a more manageable size for more detailed analysis. Alternatively, the docking phase can be site directed, only sampling binding space pertinent to the system of interest. For example, Dock (UCSF) employs a geometric matching algorithm to superimpose the ligand onto a negative image of the binding pocket. The receptor is represented using an unbiased grid of spheres that characterises the shape of the active site. Ligand atoms are matched to the spheres generating potential binding poses that are then scored using a shape and/or energy-based function. In Genetic Optimisation for Ligand Docking (GOLD; Cambridge Crystallographic Data Centre (CCDC)) [82], sampling is directed by hydrogen bond and hydrophobic fitting points. The position, orientation and conformation of
36
FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
each pose form the chromosomes used in a genetic algorithm to select the best pose using either a force field-based or empirically derived scoring function. FRED, GLIDE and GOLD all consider the molecule as a whole. Alternatively, the molecule can be divided into fragments. FlexX (Tripos) [83] employs an incremental growth strategy. First, the molecule is split into fragments, a core fragment is then placed into the active site matching the fragment to interaction sites defined for the target enzyme. The molecule is then reconstructed using knowledge of the connectivity, a database of torsion angles taken from crystallographic studies, and further interactions between ligand and receptor.
SCORING FUNCTIONS
High throughput screening identifies inhibitors of the target enzyme by measuring the change in activity of the protein kinase in the presence of the chemical compound. For an ATP-competitive inhibitor, the stronger the affinity of the molecule for the enzyme-binding site the greater the inhibition. The ultimate goal of high throughput docking is to select inhibitors from the compound dataset through prediction of both binding mode and affinity. As discussed below, the prediction of binding modes has outpaced the prediction of binding affinities. Some success has been described in the use of more computationally intensive algorithms to predict binding affinities based on known experimental data [79–81]. At present, though, there is no fast and reliable method for predicting binding affinities. Considerable resource has therefore been devoted to the development of functions for scoring and ranking the large numbers of poses generated in virtual screening. There are three main types of scoring function; physical, empirical and knowledge-based. Physical, energy-based functions are cutdown versions of atomic forcefields, focusing on the calculation of the intermolecular component of the potential energy of the protein–ligand complex. In empirical functions, a model is generated from experimental observations of binding modes and affinities. Most models can be divided into terms that describe hydrogen bonding interactions, hydrophobic interactions, a ligand intramolecular strain energy and a desolvation term. Knowledge-based functions originate from the prediction of the tertiary structure of proteins. In docking, the functions are derived from a statistical analysis of known protein–ligand complexes. A pairwise potential of mean force is computed from the radial distribution function for each pair of atom types as observed in the protein–ligand complexes. Calculation of knowledge-based functions is fast and their development is facilitated by an ever
M. CHERRY, J. READER AND D. WILLIAMS
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increasing number of protein–ligand complexes, as compared to the availability of the binding affinities required to derive empirical models.
DEVELOPMENT AND EVALUATION OF DOCKING AND SCORING
There are numerous studies evaluating the performance of docking methods and scoring functions, independently and in a variety of combinations. In most docking studies, the primary measure of success is the ability to reproduce the crystallographically observed binding modes of protein– ligand complexes. As the availability of data increases, studies are also investigating the prediction of binding affinities. A comparison is usually made to other tools typically DOCK, GOLD or FlexX. These commercially available packages have been tested extensively on a wide range of receptor systems and their performances have become the benchmark by which other methods are evaluated. The starting point for any study is the construction of a suitable dataset. GOLD was initially validated on a set of 100 protein–ligand complexes and then tested against an additional 34 complexes. The set has since been expanded to 305 complexes and is available for download from the CCDC website, www.ccdc.cam.ac.uk. This set of 305 complexes was constructed from publicly available complexes taken from the RCSB PDB www.pdb.org. A series of filters was used to select the entries from several thousand complexes available in the PDB. First, an assessment of the involvement of crystallographically related protein units in ligand binding and second, the identification of bad clashes between protein side chains and the ligand, and finally, a check for structural errors and/or inconsistency of ligand placement with crystal structure electron density. The set of 305 complexes contains 10 protein kinase–ligand structures. Similarly, FlexX was validated using a set of 19 protein–ligand complexes and more recently the performance of GLIDE was tested using a dataset of 282 co-crystallized PDB structures. As the primary objective of most docking evaluations is to reproduce the 3D structure of protein–ligand complexes, all that is required is a reliable high-resolution structure. The development of empirical scoring functions, however, requires knowledge of the structure and binding affinity. Roche et al. [84] have taken the concept a step further and compiled a universal world wide web accessible database, the Ligand–Protein DataBase (LPDB), that is specifically designed to facilitate the development and evaluation of empirical scoring functions. The set of 195 complexes corresponds to 51 proteins from 21 classes of receptor. Each complex is a high-resolution structure and has an associated binding affinity for use in the parameterization of empirical functions. In addition, each complex is characterized
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FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
by a set of 1D descriptors describing features of the protein, the ligand and the complex. The properties are commonly observed features in popular scoring functions and allow a statistical analysis of the functions in relation to the protein–ligand complexes. Likewise Wang et al. [85, 86] have sourced the PDB for high-resolution complexes, where binding affinity data were available in the primary reference. They applied a rigorous set of criteria to filter the entries resulting in a set of 1,600 complexes. Of these, 900 were selected to form a refined set of particular use for the development and evaluation of docking and scoring methods. These 900 entries have been organised into a web accessible database, PDBbind, available at www.pdbbind.org. Docking methods rarely perform well across a diverse range of protein–ligand complexes, scoring functions in particular tend to show a bias towards complexes dominated by either hydrogen bonding or hydrophobic interactions. A diverse dataset is essential to ensure a fair evaluation and will also provide a better understanding of the performance of a method towards different systems. Perola et al. [87], in a detailed comparison of current docking and scoring methods, emphasised the need to formulate a test set pertinent to subsequent investigations. Their comparison was based on a highly curated test set of pharmaceutically relevant protein–ligand complexes with known binding affinities. The dataset comprised a diverse set of proteins from multiple classes of interest to drug discovery. Ligands obeyed drug/lead-like rules and the complexes satisfied constraints on resolution (o3.0 A˚) and binding mode. The set included 43 kinase entries, from 12 proteins representing tyrosine, serine/threonine and nucleotide kinases, reflecting the importance of kinases in current discovery efforts and the abundance of structural data. A rigorous procedure was followed in the preparation of the complexes; interestingly tightly bound water molecules were maintained as part of the receptor structure. Earlier studies, in particular, removed water molecules without consideration, although this can have a negative impact, and evidence permitting water molecules should be considered as a feature of the receptor structure. Three programs were tested, GOLD, GLIDE and ICM (MolSoft) with respect to their ability to reproduce crystallographic binding modes, the RMSD (A˚) between predicted and observed pose was used as a measure of success. In internal coordinate mechanics (ICM), docking was performed using stochastic sampling, including pseudo Brownian moves, and energy minimisation of a fully flexible ligand in a rigid receptor. Energy calculations were based on the ECEPP/3 force field and took into consideration the intramolecular strain of the ligand and the intermolecular interaction of the complex. Of the top ranking poses, only 61% were within 2.0 A˚ for GLIDE, 48% for GOLD and 45% for ICM. GLIDE and GOLD, however, correctly sampled the
M. CHERRY, J. READER AND D. WILLIAMS
39
crystallographically observed pose in 79 and 77% of the complexes, respectively. The results highlight one of the main difficulties of docking, that of identifying where possible the correct pose from the sampling phase. GLIDE is thought to outperform GOLD in ranking the poses, as it refines the position of each pose prior to ranking using molecular mechanics-based energy minimisation. A theory supported by the improved performance of GOLD to a level equal to that of GLIDE when an additional step is taken minimising the poses generated through sampling. One rationale for such an improvement lies in the nature of the GOLD fitness function, where a soft repulsive term is used. Soft functions are one approach to accommodating the dynamic nature of the receptor [88], discussed in more detail below, but they also lead to mis-docked molecules and a higher number of false positives. Energy minimisation counteracts the inadequacies of the soft functions, but at the price of additional computational resource. Kontoyianni et al. [89] performed a similar evaluation of docking algorithms investigating the ability of five programs, FlexX, DOCK, GOLD, GLIDE and LigandFit (Accelrys) to reproduce experimental structures of 69 protein–ligand complexes. One of the purposes of the target selection, which included several CDK2 and fibroblast growth factor receptor-1 (FGFR-1) complexes, was to explore the ability of the programs to handle conformational variations in the same receptor. Up to 60 solutions per complex were generated and accuracy was assessed by a combination of visual inspection and calculated RMSD. Applying the subjective visual inspection GOLD correctly sampled the experimental pose for 47 of the 69 complexes, next best was GLIDE with 39 close predictions. Notably, as for the Perola study [87], the close predictions were rarely in the top ranking poses, for GOLD only 10 compared to 17 high-ranking solutions for GLIDE. The results confirm that algorithms are available that can sample the binding space and reproduce experimentally observed binding modes, but scoring and ranking the solutions is altogether a more difficult task. Several studies have separated these tasks and focused solely on gaining an understanding of the capabilities and failings of scoring functions. Perez and Otiz [90] looked in detail at two functions, a molecular mechanics function based on AMBER and a knowledge-based function potentials of mean force (PMF) [91]. First, an exhaustive rigid docking was performed using the experimentally observed ligand conformation. A 7D energy landscape was constructed from the docking energies and coverage error plots (CEPs) were used to quantify the behaviour of the functions. CEPs provide a description of the extent of error incurred in recovering a given percentage of good solutions as defined by the user. The test set comprised 17 types of protein, each receptor complexed with two different, but usually related, ligands. The authors found that the molecular
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FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
mechanics function had a small but significant advantage over the statistical function in reproducing the exact crystal structure, succeeding in 27 of the 34 cases. However, when cross-docking the alternate ligand in the receptor there was no difference in performance between the methods. Three categories of energy landscape were defined; one where the experimental solution existed in a funnel-shaped energy surface; a second, where alternate docking solutions of comparable energy existed; and a third, where the native like pose was not detected on the energy surface. There is an increased probability in finding correct solutions, where a funnel-shaped surface exists and little or no chance of success in the later scenario. Statistical analysis of the results suggested that sterics are critical for both types of potential, neglecting desolvation effects and poorly defined hydrogen bonding terms adversely impact on the molecular mechanics potential, whereas a more comprehensive treatment of dispersive forces may improve the performance of PMF. Similar results were found by Ferrera et al. [92] assessing nine different scoring functions on a set of 189 protein–ligand complexes. Recognition rates up to 80% were achieved when comparing mis-docked decoys to the native pose, performance though deteriorated when crossdecoys formed part of the test set. Steric complementarity was also seen as a more important factor than electrostatics in identifying a near native pose. In a comparative evaluation of 11 scoring functions, Wang et al. [93], in addition to standard RMSD analysis, tested the ability of each function to construct a funnel-shaped energy surface. The concept of funnel-shaped energy surfaces arose from the study of protein folding and Wang postulated that an ideal scoring function would describe the ligand-docking process in such a manner. An ensemble of binding poses was computed for 100 protein–ligand complexes from an exhaustive conformational search using Autodock (Scripps Research Institute). Success rates for correctly ranking the experimental pose within an RMSD of 2.0 A˚ ranged from 76% to as little as 26%. Again, broadening the success criteria and looking at lower ranking solutions increased the probability of observing the true binding mode, but obviously at the expense of increasing the number of false positives. Correlation between RMSD and score was used as an indication of funnel-shaped behaviour. Only one function (X-score) exhibited a significant level of such behaviour, with a correlation coefficient X0.6 for 53% of the complexes and most functions demonstrated rugged energy surfaces with minimal correlation. It is clear from these studies and others that methods have evolved capable of sampling the binding space of a receptor and reproducing experimentally observed structures of protein–ligand complexes. Scoring and ranking, on the other hand, is very much a hit and miss affair dependent on factors including the nature of receptor and ligand. How then do these
M. CHERRY, J. READER AND D. WILLIAMS
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methods cope with the binding sites of protein kinases and what steps are being taken to improve the reliability of virtual screening? VIRTUAL SCREENING AND PROTEIN KINASES
In an unbiased, unconstrained attempt to find inhibitors of protein kinase B (PKB) (also known as AKT), Forino et al. [94] used FlexX to dock and rank 50,000 compounds into the crystal structure of a ternary complex (106 K) involving AMP–PNP (non-hydrolysable form of ATP). The top 2,000 compounds were then re-scored using CSCORE (Tripos), with the top 100 compounds as defined by Drugscore, the top 200 Goldscore compounds and the top 200 molecules according to Chemscore selected for testing. Only one active compound (11), Figure 1.17, was identified from the screen. The same compound was identified using GOLD when docking the top 4,000 compounds found using FlexX. Further evaluation of the top 4,000 compounds, selecting 200 common to both the Goldscore and Chemscore rankings and a visual inspection of the docked solutions, resulted in a further 100 compounds for biological testing. Of these, two additional micromolar inhibitors were found, also shown in Figure 1.16. A hit-rate of 3 in 50,000 may raise the question, have I missed anything? When presented with a set of hit compounds from an experimental high throughput screen, it is easy to forget that there will also be false negatives. PKB is recognised as a difficult target, indeed it was noted that an experimental strategy screening 270,000 compounds found only two inhibitors. For targets where chemical matter is O
Cl
Ph Me
Cl
N
O
NC
NH
O
H2N
(11)
CO2H
Me
N H CN
(12)
OH
O
O
CO2H
O (13)
Fig. 1.17 PKB inhibitors identified using in silico screening.
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FINDING PROTEIN KINASE HITS USING STRUCTURAL INFORMATION
scarce, the complementarity of virtual and experimental techniques may yield better results. Bias, constraints and consensus scoring were employed by Lyne et al. [95] to identify nanomolar inhibitors of checkpoint kinase-1. A knowledge-based strategy involving a generic physical property filter, an undesirable chemical functionality filter and a 3D pharmacophore filter reduced a subset of the AstraZeneca collection to a set of 200,000 compounds for screening. A two point pharmacophore, based on the standard donor–acceptor pair motif observed in many protein kinase inhibitors, was employed. The pharmacophore biased the set towards those compounds capable of mimicking the interactions of adenine in the receptor, thus reducing the computational resource required for the screen. However, without additional directional and spatial constraints, the pharmacophore filter alone is insufficient in generating a target-biased compound set. Docking was performed using the program FlexX-Pharm (Tripos), which allows full flexibility in the ligand while the receptor is kept rigid. Constraints were applied eliminating those compounds that did not make hydrogen bond interactions with the backbone atoms of the linker region, further reinforcing the bias towards conventional protein kinase inhibitors. Constraints are a convenient mechanism for guiding the docking process and include pharmacophore (interaction)based constraints, distance-based constraints, substructure mapping and spatial constraints. Constraints need not be compulsory and partial matches can be defined. Applying constraints does increase the risk of missing a compound acting through an alternate, potentially novel mechanism. Here, two compulsory pharmacophore constraints were applied specifying the need for the ligand to interact through hydrogen bonds with Cys-87(NH) and Glu-85(C ¼ O) of the receptor. In previous protein kinase-docking experiments, the authors had noted a tendency for ligands to be docked in the more polar regions of the ATP-binding site, namely the phosphate groove and the ribose pocket. As the objective of the screen was to identify inhibitors targeting the adenine region of the ATP pocket, the constraints were thought necessary to reduce the occurrence of mis-docked and false positive compounds resulting from the deficiencies in the scoring function. For each ligand, up to 100 poses were saved and subsequently re-scored using a consensus-scoring scheme, an approach discussed in more detail below. Briefly, multiple scoring functions are used to score each of the poses generated from the docking simulation and only those compounds that score well for each of the functions are taken forward for further investigation. There are many functions available and, as stated above, their performance can vary hugely across different receptors. So which combination is best suited to the target of interest? Lyne et al. [95] used a consensus scheme derived from a previous study of Cdk2. A database of 8,000 compounds
M. CHERRY, J. READER AND D. WILLIAMS
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containing 100 known Cdk2 inhibitors was screened and the top 300 poses for each ligand re-scored using the CSCORE module from Tripos. From an analysis of all possible function combinations, it was determined that a consensus of the FlexX and PMF functions gave the highest enrichment. The scheme was applied to the checkpoint kinase-1 poses and a score assigned to each ligand. The dataset was seeded with known checkpoint kinase-1 inhibitors and the consensus scores for these inhibitors were used as a cut-off for progressing compounds. The resulting 250 compounds were inspected visually to remove any unrealistic poses, leaving 103 compounds, which were tested in a checkpoint kinase-1 assay. Encouragingly, 36 compounds showed activity ranging from 110 nM to 68mM. Vangrevelinghe et al. [96] employed a similar approach to identify a potent and selective inhibitor of the protein kinase CK2 (caesin kinase 2). A subset of a 400,000 compound collection was selected using physical property filters and these compounds were then docked into a homology model of the human variant of CK2 using the program DOCK (UCSF). A previous systematic assessment of docking protocols indicated that the dock energy scoring function, in combination with an all atom model, should give the best possible results. Docking was constrained to the region surrounding the adenine-binding site of the ATP pocket. No other limitations were applied at the docking stage, instead a series of three post-processing treatments were used to filter the results. Compounds not forming a hydrogen bond with the linker region were removed using a pharmacophore style filter. The remaining compounds were then re-scored using an alternate function, with the top 1,592 compounds progressing to the final stage of visual inspection. From the visual inspection, 12 compounds were chosen for testing in a CK2 assay, four of which exhibited >50% inhibition at 10 mM. The indoloquinazoline compound in Figure 1.18 showed 97% inhibition at 10 mM with a resulting IC50 of 0.08 mM. The virtual screening experiment took nearly a month to complete, but with increases in computational power through parallelisation, more recent studies took
O NH N
CH2COOH
Fig. 1.18 Indoloquinazoline inhibitor of CK2.
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less than a week. Short timescales are necessary if the approach is to establish itself as a complementary tool to experimental high throughput screening. The studies discussed above demonstrate the fact that virtual screening has already been successful in selecting subsets of databases for biological testing. Scoring functions, though, are not accurate enough to reliably discriminate active from inactive compounds and so measures, such as constraints, are incorporated into a docking protocol in order to improve the chance of success. There are other measures and methods that have been devised to facilitate the use of virtual screening, the more popular of which will now be discussed.
FAULTS AND FIXES FOR VIRTUAL SCREENING CONSENSUS SCORING
Consensus scoring is a method where molecules docked into a receptor are ranked and scored using two or more different functions, and the combined score used to re-rank the compounds. The method stems from the idea that a pose scoring well across different functions is more likely to be a true positive, rather than an artifact or bias of one particular function. Charifson et al. [97] demonstrated the concept of using intersection-based consensus approach, found to enhance the discriminative power of virtual screening compared to conventional scoring and ranking. They investigated two docking methods and 13 scoring functions, both individually and in combination, on a number of receptors including the MAPK p38. A random, drug-like set of 10,000 compounds (assumed to be inactive) was seeded with approximately 500 active compounds ranging in activity from low nanomolar to 30 mM. was docked into the receptor and the top scoring pose saved for re-scoring using the remaining 12 scoring functions. Enrichment rates, the percentage of active compounds found through screening plotted against the percentage of compounds that would need to be screened, were calculated for the scoring functions individually and for combinations involving up to four functions. Analysis was also broken down into activity bands. Three functions, Chemscore, PLP and DOCK performed well on an individual basis when considering the more active compounds, recovering 50% of the low nanomolar actives in the first 10% of the test set. Performance deteriorates for the less active compounds, with a considerable increase in the false positive rate. Taking the intersection of two of the three functions dramatically reduces the false positive rate and also decreases the total number of compounds that would need to be tested.
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The intersection of two or more functions is a very simple consensus approach and a more rigorous statistical analysis may increase the robustness of any scoring scheme. Principal components analysis (PCA) was used in the derivation of a model to predict the bioactive conformation of matrix metalloproteinase (MMP) complexes [98]. Forty compounds were docked into three different receptors and the poses re-scored using eight different functions. Three of the functions were found to be of minor importance and their exclusion improved the quality of the model. When compared to a simple combination consensus scheme, the PCA model was found to select the bioactive pose more frequently for the MMP complexes. Performance was comparable on a set of 120 different receptor complexes, suggesting that consensus schemes could be optimised for different receptor families. It should also be noted that where one function performs extremely well, it is likely that the consensus scheme will see a decrease in performance. Without prior knowledge of inhibitors for a receptor it would be impossible to determine such behaviour and it is here that a scheme derived for a related enzyme, as noted in the Lyne study [95], would present the best option. Performance of a consensus scheme is also dependent upon how the top-scoring poses for re-scoring are selected and on any subsequent refinement of these poses. Ideally, each pose should be refined in a manner appropriate to the scoring function, as minor variations in pose can dramatically affect the score as noted in the investigations of energy landscapes. Indeed, simplex minimisation formed part of re-scoring for the three functions, Chemscore, PLP and DOCK score that performed well in the Charifson study [97]. Failing that, an exhaustive enumeration and scoring of the binding poses is required.
KNOWLEDGE-BASED SCREENING
Virtual screening tools are, in general, developed to cover a broad range of receptor types, although as noted previously, performance varies from system to system. What, then, can be learnt from the ever increasing number of protein kinase complexes and how can that information be used to improve the accuracy of virtual screening for protein kinase systems? Wu and Vieth [99] have developed an algorithm, SDOCKER, which can be applied to any system but relies on known protein–ligand complexes. The algorithm is based on a hybrid scoring function, combining force field energy with similarity to a predefined template bound to the receptor. CHARMm is used to calculate the force field component of the function evaluating the protein–ligand interaction energy and the ligand internal energy. The docking is then driven towards solutions where the shape of the ligand in the binding
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site matches the shape of one or more template ligands. Parameters allow the contribution of each term to be varied, ranging from a similarity-based search through to a conventional force field-based docking experiment. The hybrid function resulted in a 10% improvement in the success rate of predicting the observed binding mode for 23 ligands bound to CDK2. In a more constrained methodology, Vieth and Cummins [100] derived a method for statistically determining the Docking Mode that is Consistent with a Structure-Activity Relationship (DoMCoSAR). The method is only applicable to compounds from the same chemical class having a common core substructure, for example a combinatorial library. Two docking runs are preformed, first an unconstrained dock and secondly a constrained dock superposing the core substructure onto the poses most commonly observed in the first run. A set of descriptors is then calculated based on the interaction of each pose with the receptor. The descriptors are used to generate a QSAR model aimed at reproducing the observed SAR. For a series of oxindole compounds docked into vascular endothelial growth factor (VEGF) receptor tyrosine kinase, 11 major binding modes were predicted from the unconstrained docking run. The two most popular modes were taken forward and used to re-dock the compounds. The approach was able to identify the binding mode consistent with crystallographic data of related enzymes, but failed to generate a reliable QSAR model. Chaqui et al. [101] used a measure of similarity to score and rank compounds docked using the program FlexX. The method calculates a fingerprint for each predicted binding mode and compares that structural interaction fingerprint (SIFt) to a precomputed set of SIFts derived from 93 protein kinase complexes [102]. The SIFts essentially encode the patterns of protein–ligand interactions. Docking solutions will exhibit an interaction pattern and when that pattern matches one of the known patterns (SIFts), the docking solution is scored and ranked more highly than those that exhibit alternative interaction profiles. The protein kinases p38 and CDK2 were used as test cases to compare three different post-processing strategies; scoring and ranking using conventional technologies, scoring and ranking using SIFts and a hybrid approach where poses are filtered using the SIFt and survivors ranked using a conventional scoring function. A considerable enhancement in the enrichment curves was observed for p38 using the SIFt scoring and ranking scheme. For CDK2 hybrid schemes using any of the functions PMF, Chemscore, DScore and GScore gave enrichments better than either the SIFt or scoring function alone. The difference in performance is attributed to the different gatekeeper residues in p38 (threonine) and CDK2 (phenylalanine). The gatekeeper residue is known to play an important role in the selectivity profile of protein kinases and will therefore constitute an important feature in the SIFts. It would appear that the
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compounds docked into p38 share the ability to bind in the hydrophobic pocket and are readily identified from the fingerprint. Compounds inhibiting CDK2 bind in a more general ATP-competitive fashion, the SIFt can then eliminate alternative predictions, but the scoring function is required to discriminate between the remaining good predictions. One advantage of the SIFt scoring scheme is that it can be tailored to an individual target enzyme or the protein family as a whole. The method also provides a non-graphical, automated approach to classifying and comparing the binding modes of protein kinase inhibitors. Statistical analysis has been used in a number of studies to improve the performance of virtual screening. Vigers and Rizzi [103] employed a statistical correction factor to account for the inability of two docking tools, FlexX and GOLD, to match ligands to active sites for a series of complexes. Ligands from 15 different PDB complexes were docked into each of the 15 different receptors. Encouragingly, the 15 native structures were reproduced by both docking tools. However, in the cross-docking experiment, only three of the ligands were correctly identified as the top scoring compounds for that receptor. Certain compounds were found to score well across all the receptors, a result in the main attributed to bias in the scoring functions rather than true biological non-specificity. The authors noted that for GOLD and FlexX, a different set of ligands scored highly across all the receptors adding weight to the rationale that the promiscuity is a reflection of bias in the scoring function. An alternative scoring scheme was constructed, called multiple active site correction (MASC), based on the mean and standard deviation of the scores for each compound across all 15 receptors. The new scoring scheme increased the discriminative power of both docking tools significantly, correctly identifying 11 of the 15 ligands. In any statistical analysis, the question of validation arises. Vigers, therefore, tested the method on an additional 63 complexes taken from the GOLD test set. Results were not as good for the test set but the MASC score still showed a significant advantage over uncorrected scores. It was also found that the number of receptors required in the MASC correction factor could be reduced from the original 15 to 7 and the best results were obtained when the MASC receptors were diverse in nature. A machine-learning method was proposed by Klon et al. [104] as an alternative form of consensus scoring. The method proved unsuccessful for PKB, but showed promise for the phosphatase PTP1B (protein tyrosine phosphatase 1B). In this approach, compounds were first docked into the receptor and scored using conventional means. The top scoring compounds were then assumed to be active and used to build a naı¨ ve Bayes classification model, all compounds were subsequently re-scored and ranked using the model. The method is heavily dependent upon predicting accurate binding
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modes in the docking phase and the results for PKB appeared to be negatively affected by the presence of manganese ions in the receptor site during the docking. All the high scoring compounds for PKB were false positives found to interact with the metal ions. The classification model would then only find similar false-positive compounds. The work does illustrate how preparation is key to achieving good results and it would be interesting to see if the approach improved enrichment using an alternative model without the metal ions.
PROTEIN FLEXIBILITY AND INDUCED FIT
Preparation of the enzyme model is obviously a significant factor in obtaining accurate and reliable results from structure-based design. The evaluations discussed above have highlighted how minor changes can dramatically change results. McGovern and Shoichet [105] investigated the variation in results for virtual screening when using holo, apo and modelled conformations for 10 different enzymes. No protein kinases were included, but the lessons learned are applicable. The MDC drug data report (MDDR) database was prepared and docked into the 30 models, in the knowledge that up to 1% of the database was known to be active against each target. Enrichment factors calculated for the docking experiments showed that for seven of the targets, the holo model gave the highest enrichment, for two the apo model performed best and, finally, one homology model achieved the highest enrichment. Unsurprisingly, the holo models performed best where the active compounds in the database resembled the ligands complexed in the crystal structures, but faired worst when the compounds differed considerably. All of the experiments, though, provided some level of enrichment compared to random screening. How then can virtual screening and structure-based design cope with the conformational changes observed in enzyme structures and factors such as induced fit? Two methods, soft docking and multiple receptor conformations, are compared by Ferrari et al. [106] for their ability to select active compounds from a set of decoys. In soft docking, the steepness of the repulsive term for the scoring function is reduced, allowing greater overlap between the ligand atoms with the receptor. Here the standard 12-6 Lennard–Jones potential was substituted by a more permissive 9-6 potential. Soft functions have the advantage that they incur no additional cost in computational resource to compute, but can only cope with what would be relatively minor conformational changes in the receptor. Multiple receptor conformations involves using two or more structures either experimental or computationally generated and docking the compounds into each. Obviously, the computational
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resource for multiple receptor conformations scales linearly with the number of docking models, but has the advantage of no limit on the conformational change that can be modelled. An increase in enrichment was found for both methods, on average a 10% improvement for soft docking and 17% for multiple receptor models. Also, as the approach using multiple receptor models allows for greater movement, a wider range of inhibitors was identified compared to soft docking. For protein kinases, the multiple receptor approach would allow the user to screen against models ranging from the inactive through to the active conformation. In theory it would reduce the number of false negatives, but in practice would no doubt lead to more false-positive compounds. Barril and Morley [107] studied the use of multiple receptor models for the protein kinase CDK2. The test set comprised 49 X-ray structures exhibiting reorganisation of side chain atoms, backbone atoms and a variation in the hydration state of the enzyme with respect to the bound ligand. An inhouse docking programme, rDock, was used to independently dock a set of known inhibitors into each receptor model. The method was assessed for its ability to predict binding modes (within an RMSD of 2.0 A˚) for the ligands extracted from the X-ray structures and enrich virtual screening amidst a random set of 1,000 compounds. Two tests were performed to investigate the prediction of binding modes. First, the experimental binding mode was used as a starting point for energy minimisation using the docking function and second, docking from a random starting position. The first test determined whether the experimental pose was close to a minimum in the energy landscape for the docking function. The docking experiment determines whether the experimentally observed pose is the global minimum as defined by the docking function. The performance of the different receptor models varied significantly, ranging from 29 to 100% (average 78%) for the minimisation experiment and from 3 to 68% (average 33%) for the docking runs. The results verify that conformational variations do affect performance and that docking functions have great difficulty in discriminating the true biological pose from alternate solutions. Taking the best scoring pose predicted for each model and combining the results was found to improve performance for the docking experiment. Randomly selecting a model, the average performance for predicting the bioactive conformation was 33%. Using all the models improved the performance to 76%, though it was observed that the optimum gain occurred using 10 models after which there was a diminishing return on performance. Apart from the increased expense on computational resource, several limitations were noted for the method. In the multiple receptor method, the internal energy of the receptor was not accounted for due to uncertainties and errors in making the calculations. The docking scores were then dependent purely on the internal energy of the
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ligand and the interaction between protein and ligand. In practice, the free energy of binding incorporates the internal energy of the receptor, which is dependent on a number of factors including conformation. Another source of error lay in the choice of receptor models, as the selection sometimes had a detrimental effect on the results. A simple example would involve a model conformation, perhaps as a result of induced fit, that excluded the standard ATP-competitive mechanism of inhibition observed for many protein kinase inhibitors. Multiple receptor models can then increase the ability to predict binding poses, but how does it affect enrichment in virtual screening? On average, for CDK2 no advantage was observed when using two or more receptor models to screen a database. Any advantages gained in identifying inhibitors were offset by the inadequacies of the scoring functions, resulting in an equivalent increase in the number of false positives. A similar study investigating protein flexibility in virtual screening did report an overall improvement in enrichment for a series of protein kinases, where results from multiple models are combined using a merge and shrink procedure [108]. In the first phase of the study, 1,000 compounds seeded with co-crystallised kinase inhibitors were docked into four protein kinases cAPK, CDK2, p38 and LCK using the ICM flexible-ligand rigid receptordocking programme. For the 29 structures, all the native ligands were docked with an RMSD o1.5 A˚. As expected, cross docking the native ligands showed a decrease in success with only 70% of the ligands correctly docked. In terms of enrichment, 80% of the known inhibitors were identified in the top 1.5% of the library. The cross-docking results were then combined by extracting the top scoring result for each compound and eliminating all others. Enrichment factors using the reduced dataset showed a 1.8570.65-fold increase. For the enzymes studied here multiple crystal structures are available. This obviously is not always the case. A method, IFREDA, is reported in the study for computationally generating conformers pertinent to the holo protein. Essentially, known inhibitors are docked into the enzyme and then the resulting complex undergoes a global energy minimisation allowing both side chain and backbone atoms to move. Using the computationally generated models, rather than the X-ray structures, was found to give a comparable level of enrichment.
HIGH THROUGHPUT DOCKING AS A VIRTUAL SCREENING TOOL
It is clear from the studies discussed above that there is no universal solution to high throughput docking and methods should be tailored to the target of interest and according to what is expected from the docking experiment. Constraints, consensus scoring, knowledge-based processing and
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the incorporation of flexibility are all approaches aimed at improving the performance of virtual screening using high throughput docking. Pre- and post-processing are very important aspects of the experiment with the concept of a screening cascade already mentioned. There are also other approaches to searching compound databases for protein kinase inhibitors either as part of a cascade or as stand-alone tools.
ALTERNATIVES TO HIGH THROUGHPUT DOCKING Despite the ever-increasing number of protein kinase crystal structures available in the public domain, still only a small percentage (o10%) of the human kinome is represented. Homology modelling is discussed below as a route to generating a 3D structure of a protein kinase, where a crystal structure is unknown. There are, however, numerous well-tested methods for screening that rely on information regarding known inhibitors of the target protein as an alternative to the receptor structure. There are many reviews discussing the methodologies of ligand-based virtual screening [109–111] and so only examples, where protein kinases are the focuses of attention are presented here. The most popular methods for ligand-based virtual screening can be classified as substructure searching, similarity-based methods, QSAR modelling and pharmacophore methods. Each method requires that the molecules are characterised using a set of descriptors that are generally calculated computationally. Many descriptors have been proposed based on 1D, 2D and 3D characteristics of the molecules. 3D descriptors can be subdivided into those that are dependent on special alignment and those that are independent of the coordinate space. Pirard and Pickett [112] compared the ability of BCUTs, a 2D fingerprint and multi-pharmacophore descriptors, to classify EGFR tyrosine kinase inhibitors. BCUTs are derived from molecular graphs and have been developed to describe the bonding patterns and properties of molecules pertinent to ligand–receptor interactions including charges, polarizabilities, H-bond characteristics and surface areas. First BCUTs were calculated for a total of 770 inhibitors from five different protein kinases. The first six principal components (PCs) from a PC analysis could, for the most part, discriminate between inhibitors of the different enzymes, though overlap was found between p38 and spleen tyrosine kinase (SYK) inhibitors. Clustering using the first six PCs verified the relative separation of the different inhibitor sets. A success rate greater than 70% was obtained for all enzymes except Cdk1, in classifying the inhibitors using a five-component partial least squares discriminate analysis model (PLS DA) . On a separate test set of 52 endothelial growth factor receptor tyrosine
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kinase (EGFR TK) inhibitors, the partial least squares discriminant analysis (PLS DA) model successfully classified 48 compounds. How then did the performance of the BCUTs compare to the Daylight fingerprint and the pharmacophore analysis? The success rate in classifying the 52 novel EGFR inhibitors was low for the Daylight fingerprint, whereas the multipharmacophore analysis gave comparable classification rates to the BCUTs. The BCUTs are thought to encode more information than the 2D descriptors and are also fast to calculate, not requiring the generation of 3D databases. One downside of BCUTs is in interpretation, as for many descriptors it is not easy to place a chemical rationale behind the classification of compounds. Methods such as pharmacophore models and 3D comparative molecular field analysis (COMFA) [113] require the generation and superposition of 3D representations of molecules. Putta et al. [114] have formulated an algorithm for efficiently mining the 3D conformational space of compounds and finding alignments likely to be biologically relevant. Molecules are first superposed using subshape matching, which allows the alignment of multiple conformers of each molecule and the matching of different sized molecules. Feature maps are used to score the alignment where a high score would result from the close overlay of many, equivalent features in an alignment; typical features being hydrophobic centres and hydrogen bonding groups. The matching algorithm was validated using a set of CDK2 compounds, comparing predicted conformations of each ligand with the crystallographic observations. One of the alignments closely matched the observed binding modes, correctly identifying the standard donor–acceptor sites of interaction. The method was also able to map out other regions of the ATP-binding site, though some error was noted with regard to functionality lying near solvent space. The program CATALYST (Accelrys) generates pharmacophore models based on the superposition of inhibitors molecules. Bhattacharjee et al. [115] used CATALYST to build 3D QSAR pharmacophore models capable of identifying inhibitors of the cyclindependent kinase Pfmrk. The models were constructed by overlaying 15 structurally diverse kinase inhibitors having a range of activities. A model comprising two hydrogen bond acceptor points, an aliphatic and an aromatic pharmacophore point gave a correlation of 0.9 for the training set when calculating the activities. Cross validation using an additional 15 compounds saw a reduction in the correlation to 0.7. A further test was performed using the model to select potential inhibitors from a database of 29,000 compounds. Steric factors for the binding site were introduced through a shape-based restriction on the compounds. There are several methods for restricting the molecules in a pharmacophore screen based on sterics. Exclusion volumes prohibit any atom in the specified volume of
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space, inclusion volumes state that one atom must occupy the specified space and shape constraints state that all the atoms must fall within the defined volume. The pharmacophore screen helped to generate a shortlist of 16 compounds subsequently found to have an IC50 o25 mM. COMPARATIVE HOMOLOGY MODELLING Of the 517 protein kinases in the human kinome [116], structures are publicly available for o10% of the different enzymes. In the absence of experimental data, construction of homology models based on known structures has proven a reliable method for generating 3D information. How, then, are the models built and what are the limitations of the models especially as applied to the family of protein kinases? There are several stages to the construction of the homology model, probably the most crucial being the identification of a suitable template (or templates) and the alignment of the sequences. Template recognition is usually undertaken with programs like FASTA or BLAST, methods readily capable of identifying templates having a sequence identity >25%. Confidence in any model falls rapidly as sequence identity falls below 25% and will at best lead to an approximate reproduction of the tertiary structure. At the opposite end of the scale, errors in models built using templates where the identity >90% can be as low as the errors in the experimental crystallographic structure. Within the family of protein kinases, homology across the subfamilies generally lies in the region 25–35% ID. Homology of the residues defining the ATP-binding site is higher, as there are clusters of conserved residues that are involved in the catalytic mechanism of the enzymes. At this point, it is important to assess the template(s) that have been identified considering, among other things, the quality of the data; the activation state of the kinase structure and ligand induced conformational changes. Following the selection of one or more templates, a detailed alignment is performed based on maximising a scorings or exchange matrix and more often than not involving manual correction. A model can now be constructed, first building the backbone, then the loop regions and finally adding the side chains. There are many programs available for building homology models. Most, if not all, also incorporate the means to evaluate and refine the model after it has been constructed. Further details of the methods are presented at the CMBI website (www.cmbi.ru.nl/gvteach/ hommod/index). There are very few reported studies outlining the use of homology modelling in protein kinases. Panigrahi and Desiraju [117] performed a simple comparison of a model of the EGFR kinase domain to an experimental
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X-ray structure, citing a C-a RMSD of 1.96 A˚. McGovern and Shoichet [15] came to a number of conclusions, stating that the performance of the docking experiment is affected by the particular representation of the receptor. Perhaps, the most telling remark is that the majority of the structures including the models gave some degree of enrichment compared to random screening. Diller and Li [118] have also assessed the use of homology models in high throughput docking. Models for six protein kinases (PDGFRb, VEGFR1, EGFR, p38, SRC and FGFR-1) were built from a variety of templates using the Modeler package (Accelrys). A database of 32,000 compounds seeded with known inhibitors for each kinase was then docked into each model using the program LibDock (Accelrys) and scored with the Piecewise Linear Potential 2 (PLP2) function. A solvation term was added to the function to correct for any correlation between score and size for the molecules, a consequence of the function being based on interaction counts. Enrichment factors were calculated for each simulation and the models were also assessed for their ability to discriminate between the inhibitors, a key point of concern when building a model based on an alternative enzyme. Crystallographic data are available for four of the enzymes, allowing for a direct comparison of the docking results between experimental and computergenerated structures. The homology model of EGFR was compared to the apo crystal structure of EGFR (1M14) and achieved similar enrichment factors versus the random compounds of 4–5 and 5–6, respectively. The model for p38 performed poorly, enrichments of 1.5–2.6, compared to the crystal structure, enrichments of 7–11, mainly attributed to the fact that the crystal structure used (1A9U) is a complex containing the well-known pyridinyl-imidazole inhibitors. The situation was reversed for Src kinase, where the model achieved higher enrichments than the crystal structure. The model was based on an apo form of the related enzyme lymphocyte-specific kinase Lck, sequence identity of 66%, whereas the crystal structure was solved from protein crystals initially seeded with the ATP analogue AMP–PNP. Structures complexed with ATP or alike adopt a more closed binding site, thereby restricting access to larger ligands. For FGFR-1, neither the model nor the crystal structure performed well, again a consequence of induced fit. A second more open model built using an apo template structure faired better. The results can be summarised as follows; the variation in conformation between a model and X-ray structure can be no more than that observed between the different forms of a single protein kinase. Success is very much dependent on the conformation of the template structure and the end objective of the model. If screening for novel chemotypes, an open apo template provides the best starting point, though this will no doubt lead to an increase in false positives.
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Virtual screening formed the basis of comparison for Oshiro et al. [119] looking at enrichment factors of homology models of CDK2 versus X-ray data. MOE (Chemical Computing Group) was used to build four models of CDK2 based on templates ranging in sequence identity (ATP site residues only) from 43 to 60%. A set of 17,000 compounds containing 367 actives from 15 different scaffolds was then docked into the X-ray structure and each of the models. Compounds were docked, scored and ranked using the DOCK program. All of the screening experiments exhibited enrichment compared to random screening, with models constructed from templates greater than 50% identity consistently achieving enrichment factors equivalent to using the crystallographic structure. Fifty per cent identity was noted as a threshold value above which the C-a RMSD of model versus crystal structure was found to be, on average, of the same order as the error in the X-ray data, roughly 2 A˚, hence the comparable performance in the docking experiments. Enrichment values decreased to a 2-fold low as the sequence identity of the model templates decreased. The study further reinforces the value of homology models where crystallographic data are unavailable, provided due care and attention is paid to the generation of the model, in particular the choice of template. In an ideal scenario, crystallographic data would form part of the hit verification process, but, as noted in many of the studies discussed in this review, it is rarely the case. Selectivity is also an issue of many of the medicinal chemistry programmes, where homology modelling could play a vital role in providing comparisons between the structures of different enzymes. Homology modelling provides a rapid and cheap alternative to the significant investment required for protein crystallography. This, coupled with the fact that not all protein kinases are amenable to crystallography, means that there will always be a need to generate 3D information using alternative methods. The uncertainties of using the homology model are then often outweighed by the desire to have a degree of structural insight into the development of hit compounds.
DE NOVO DESIGN Both X-ray crystallographic and NMR methods have been discussed as approaches to finding lead compounds starting with the discovery of lower molecular weight fragments or templates. Computational de novo design methods have been in existence for some time, with the two main approaches mirroring the experimental fragment evolution and fragment linking strategies discussed by Rees et al. [120]. Fragment evolution, also referred to as seed and grow, involves docking a set of small fragments or
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templates into the active site and expanding upon the hit fragments to yield a more potent lead-like compound. Fragment linking entails joining together multiple fragment hits that are sited in proximal regions of the binding site. Initial attempts at de novo design met with limited success, as deficiencies in the methods, for example the chemical intractability of proposed compounds, limited the effectiveness of the approach. More recently, with a greater understanding of the components that constitute most drug molecules, more chemically aware strategies have evolved. Schneider et al. [121] compiled a set of 25,000 building blocks through retro-synthetic fragmentation of 36,000 known drugs. Fragmentation involved a series of chemically intelligent rules for breaking down the known drugs in a manner amenable to subsequent re-linking as part of the de novo design strategy. A ligand focused evolutionary design algorithm, TOPAS, constructs molecules from the building blocks that are similar to known inhibitors, defined by either a 2D fingerprint or 2D topological pharmacophore description of the molecules. The program Skelgen (DeNovo Design), developed by Dean and co-workers, pieces together molecules within the confines of an active site taking into account physio-chemical properties such as hydrogen-bonding, lipophilicity, electrostatic and steric parameters. The fragments that are pieced together in a dock and link approach are derived from the World Drug Index. In an investigation focused on protein kinases, Hare et al. [122] reviewed the binding modes of known kinase inhibitors extracting common molecular frameworks for guiding the generation of protein–ligand complexes. The frameworks are used as constraints in an automated docking procedure, significantly increasing the accuracy of docking and reducing the time required. Aronov and Bemis [123], in a related study, developed a novel methodology that integrates fragment-based design with virtual screening techniques. Using a previous analysis of the frameworks [45] and side chains [46] of known drugs, a set of 119 kinase inhibitors was fragmented into rings and linkers. Fragments frequently observed to form key interactions with the linker region of the enzymes were designated as scaffolds, four ring systems and eight linkers formed the remainder of the fragment set. A virtual library was constructed through an exhaustive enumeration adding the rings and linkers to the scaffold templates. The library was then docked into a test set of protein kinases comprised of Bcr–Abl (Abelson kinase), Cdk2 and tyrosine kinase Src using a constrained docking process matching the scaffold fragments to observed poses from crystallographic data. The use of experimental X-ray data to guide the docking significantly reduced the falsepositive rates associated with free docking experiments. The method, termed a minimalist approach to scaffold library design, is geared towards optimising the affinity and selectivity of ligand–receptor interactions. Knowledge
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of the interactions important to each receptor can then be extended to more novel compounds, either through adding diversity to the building blocks or more radically, designing new scaffolds. Four classes of Cdk4 inhibitor were identified by Honma et al. [124] employing the use of the homology model and a de novo design strategy. A model of Cdk4 was constructed from a crystal structure of the active form of the related enzyme Cdk2. An analysis of the ATP site and the binding of several different Cdk2 inhibitors identified a number of residues conserved in Cdk4 important to the development of small molecule inhibitors. An in-house atomistic de novo design program LEGEND identified a large number of proposed inhibitors based on the analysis of the active site of Cdk4. However, initial propositions were found to be synthetically unfeasible, a shortcoming noted previously for chemically ignorant sequential construction algorithms. Therefore an additional step comparing the core fragment of each suggested compound to known structures was introduced to ensure chemical tractability. The overall process is as follows; first, key interaction points in the enzyme are identified, a sequential growth algorithm then adds atoms in a complementary fashion to meet predefined growth criteria, finally the key components of the proposed molecule form the queries to search commercial databases. Eighteen low potency (micromolar) hits were identified from an in vitro assay after screening 382 commercially available compounds proposed by the de novo design strategy. An informer library, a target-guided library designed to probe the features of the binding site, was synthesised for one class of hit, a series of diarylurea compounds. The library yielded a 100 nM compound which was further investigated using a more detailed docking analysis taking into account the SAR derived from the library. Additional gains in potency and optimisation of the physio-chemical characteristics resulted in several low nano-molar compounds, one of which was soaked into a crystal of Cdk2 (see Figure 1.19). The predicted binding mode was in close agreement with the observed binding mode for these compounds.
O N
HN N H
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Fig. 1.19 Inhibitor of Cdk4 designed using de novo methods.
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Honma et al. [125] extended their investigation of the Cdk4 inhibitors by designing libraries to interact with residues in the binding site. An analysis of the binding mode of the lead compound, determined crystallographically, identified potential sites of substitution and the scope for substituents at each point. Two de novo programs, LUDI (Accelrys) and LEAPFROG, suggested possible additions to the scaffold based on optimising interactions with the enzyme, synthetically accessible additions were then chosen that mimicked these suggestions. The site analysis also included a comparison of the Cdk4 sequence versus other protein kinases, providing a guide to developing more selective compounds. In particular non-conserved residues in Cdk1/2 were targeted, increasing the selectivity of the inhibitors against these enzymes 100-fold. Target-focused libraries are an important application of de novo design tools, as they provide one possible resolution to the difficulties in predicting binding affinities and effects such as induced fit. Knowledge of the structure, coupled with a known scaffold, allows for an efficient exploration of the binding site mapping out key interactions and also investigating the conformational changes. Careful selection of reagents used in the library design also ensures that what is suggested computationally has a greater chance of success in the laboratory.
SUMMARY Hit identification is a crucially important stage gate in the drug discovery process. As the development candidate that emerges from pre-clinical research invariably resembles the initial hit, it is important to make as wellinformed a choice as possible when selecting which hits to progress and which to shelve. Decisions made during the hit identification process determine the chemistry direction in which a project proceeds, usually taking into account the potency of the hit, any SAR generated during the hitfinding exercise, the potential scope for generating novel and patentable chemical matter, and the synthetic tractability of the series. Over the last decade, a huge amount of financial and intellectual investment has been made in HTS to identify compounds with low micromolar IC50s, leading to major advances in combinatorial chemistry, high throughput analysis and purification, and compound handling and storage. HTS is constantly improving as the processes are refined and compound collections are improved, but overall results have been disappointing. Recently, structure-guided approaches have been used much more frequently at the hit-finding stage of drug discovery and, as this review seeks to demonstrate, these approaches have had a major impact on a number of kinase targets. The closer integration of these tools, combined with ongoing
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technological advances in computational chemistry, X-ray crystallography and NMR spectroscopy will inevitably lead to an even greater use of structural techniques for hit generation to complement or replace HTS.
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Progress in Medicinal Chemistry – Vol. 44, Edited by F.D. King and G. Lawton r 2006 Elsevier B.V. All rights reserved.
2 Blunting the Swiss Army Knife of Hepatitis C Virus: Inhibitors of NS3/4A Protease$ PETER W. WHITE, MONTSE LLINAS-BRUNET and MICHAEL BO¨S Boehringer Ingelheim (Canada) Ltd, 2100 Cunard St., Laval, QC H7S 2G5, Canada Dedicated to Professor Dr. Dieter Seebach
HEPATITIS C INFECTION Introduction The Hepatitis C Virus Current Therapies for the Treatment of HCV Infections The HCV NS3/4A Protease
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TOOLS TO STUDY NS3/4A PROTEASE INHIBITORS Enzymatic Assays Viral Replication
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INHIBITORS OF THE NS3/4A SERINE PROTEASE Non-covalent Peptidic Inhibitors Covalent Reversible Peptidic Inhibitors Non-peptidic Inhibitors
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SUMMARY AND OUTLOOK
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The title was adapted from a phrase coined by Prof. James McKerrow (University of California, San Francisco), as quoted by P. Frost in the American Chemical Society newspaper Chemistry (Spring 1999, 15–19). DOI: 10.1016/S0079-6468(05)44402-1
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BLUNTING THE SWISS ARMY KNIFE OF HEPATITIS C VIRUS
HEPATITIS C INFECTION INTRODUCTION
Liver disease resulting from Hepatitis C Virus (HCV) infection is a serious, worldwide health problem. HCV is endemic to central Africa and Southeast Asia and has probably existed for over 100,000 years in these locations [1]. However, during the 20th century it became a worldwide epidemic, currently infecting an estimated 170 million people or 3% of the world’s population [2]. HCV is a blood-borne virus, and its increased prevalence in modern times results from the increased use of blood transfusions, or the use of improperly sterilized needles, either for medical procedures such as vaccination, or for the intravenous administration of illicit drugs. However, HCV was only identified, and its genome sequenced, in the late 1980s [3]. Since that time, diagnostic tests have essentially eliminated the virus from the blood supply [4]. Furthermore, awareness of HCV, as well as of HIV, has led to decreased transmission through unsterilized needles. Intravenous drug use remains the major mode of Hepatitis C transmission, still occurring with significant frequency in certain populations, for example in prisons [5]. Acute infection by HCV is usually asymptomatic and passes unnoticed [6, 7]. The immune system brings this initial infection under control, and in some cases is able to completely eliminate the virus. However, the virus possesses several mechanisms to evade the immune response, so that approximately 80% of people become chronically infected [8]. Further progression of the disease is slow, but most chronically infected individuals eventually develop liver fibrosis, which can then lead to cirrhosis with or without progression to hepatocellular carcinoma. End-stage liver disease and liver failure resulting from HCV infection are currently the major indications for liver transplant in the U.S. and Europe [9].The progression from chronic infection to cirrhosis is poorly understood, and the extent to which pathology is caused, either directly by the virus or as a consequence of the body’s immune response, is the subject of some debate. Current therapies for HCV, as described below, work by stimulating the immune system. They are poorly tolerated and only partially effective. There is a significant need for new forms of treatment, and it has long been recognized that drugs specific for viral targets will play a critical role in combating the disease. The NS3 serine protease in particular was recognized as a prime target for intervention 12 years ago [10]. However, to this day no drugs directed against any viral target have reached the market or late-stage clinical trials. For reasons outlined below, development of anti-HCV drugs, and protease drugs in particular, has been slow and difficult. Progress against this target was last reviewed in this series in 2002 [11]. Here we will focus primarily on recently
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reported results. The last few years have seen a number of exciting developments in this field, holding out the hope that highly effective drugs directed against this target will be available in the not-too-distant future.
THE HEPATITIS C VIRUS
HCV is a small enveloped single-stranded RNA virus belonging to the Flaviviridae family and Hepacivirus genus [12]. Other Flaviviridae members include the flaviviruses such as yellow fever virus, and pestiviruses, which are responsible, for example, for bovine viral diarrhea [13]. The most closely related virus identified to date is GBV-B, which infects the tamarind, a new world monkey. GBV-B has also been tentatively classified as a Hepacivirus [12]. The HCV genome consists of approximately 9,600 bases, encoding a single polyprotein of approximately 3,000 amino acids, flanked by conserved 50 - and 30 -untranslated regions essential for replication and translation (Figure 2.1A). The genome is replicated in the cytoplasm by a virally encoded polymerase and translated by cellular machinery directed to the viral RNA by a highly structured internal ribosomal entry sequence (IRES) [13] (Figure 2.1B). The viral polyprotein comprises four structural proteins followed by six non-structural proteins. The structural proteins are: envelope glycoproteins E1 and E2, Core, and the small P7 ion channel [14, 15]. Another putative protein, F, is also encoded by an alternate reading frame in the structural protein region, but no function for the protein has been identified [16]. The non-structural proteins are designated NS2-N5B. NS2/3 possesses an essential autoprotease activity, but it is still not clear whether NS2 itself has an independent function. The bifunctional NS3 protein consists of an N-terminal 5’-UTR C E1 (IRES)
P NS2 7
NS3
NS4 A B
A
5’-UTR r EM NS2 (IRES) Neo -CV
NS3
NS4 A B
A
E2
NS5 B
3’-UTR
(A) NS5 B
3’-UTR
(B) Fig. 2.1 (A) Structure of the HCV genome, with 50 - and 30 -untranslated regions and individual proteins of the polyprotein indicated. Regions of the genome (approximately 3,000 bases) are drawn to scale. Structural protein regions are in light gray, nonstructural proteins in white, and untranslated regions in dark gray. (B) Structure of a typical replicon sequence, with the antibiotic resistance gene (Neor) in place of the structural region and the second IRES (EMCV) inserted. The NS2 sequence is often not present in the replicon.
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protease domain and a C-terminal helicase. NS4A is an essential component of the NS3 protease. The functions of NS4B and NS5A are not wellunderstood, though NS4B possesses a putative NTPase motif, which appears to play an important, though still uncharacterized, role in replication [17]. NS5A is a phospho-protein, which has been shown to interact with several host proteins and seems to play a role in subverting the host immune response. NS5B encodes the viral polymerase [13]. The junctions between structural proteins are cleaved in the lumen of the endoplasmic reticulum by cellular signal peptidases, whereas the nonstructural proteins are processed by the virally encoded proteases. The NS3 protease–NS4A cofactor complex (vide infra), cleaves all the non-structural junctions except NS2–NS3 [13]. The NS3 protease and helicase, as well as the NS5B polymerase, have significant homology to other known enzymes and were recognized early-on as potential targets for anti-viral drugs. Other possible targets include the poorly characterized NS2/3 protease activity and the interactions of the IRES or other viral proteins with host cell factors. Like most viral RNA polymerases, NS5B does not possess 30 –50 exonuclease editing capability and is relatively error prone. This results in a mutation rate of approximately 1.4 10 3 nucleotide changes per site per year [18, 19]. Combined with the estimated production of up to 1012 virions per day in an infected individual [20], HCV sequences clearly have the potential to evolve rapidly. This results in the appearance of multiple sequence variants or quasi-species in each infected individual [21]. Sequence evolution is balanced, however, by the need to maintain simultaneously both the functions of the viral proteins and also the highly ordered RNA structure found in many regions of the genome [1]. Thus longterm evolution of the virus has occurred at a much slower rate. Globally, HCV sequences have been divided into six major genotypes (1–6), which differ by 30–35% in nucleotide sequence. Within these genotypes are found over 60 subtypes, which differ from each other by 20–25% [22]. Most genotypes and subtypes are confined to the regions of Africa and Asia in which the virus has existed longest, or to recent immigrants from these regions. The worldwide outbreak of the virus has been caused almost entirely by subtypes 1a and b, 2a, b and c, and 3a. Of these the most predominant are 1a and 1b, which are responsible for approximately 70% of the infections in the Americas, Europe, and Japan [23]. Sequence variation could in principle result in differences in disease pathology, but no such link has been proven to date and the progression of the disease is generally independent of genotype. There is a strong association of particular subtypes with different modes of transmission. The high prevalence genotype 1b infection in the developed world is attributed mostly to the use of contaminated blood in transfusions. Subtypes 1a and 3a currently
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account for most new infections, which are being spread by intravenous drug use [23]. The infection of approximately 20% of the population of Egypt with the otherwise rare genotype 4 has been traced to the mass inoculation of the population with a parenteral treatment for schistosomiasis in the 1950s and 1960s, carried out with inadequately sterilized equipment [24]. It is not clear whether the predominant genotypes are in some way more efficiently transmitted, or if they owe their primary importance simply to chance, with the spread of these viruses beginning only recently with a very small number of individuals who just happened to be infected with these specific subtypes. As described below, viral genotype is an important factor in determining the effectiveness of drugs directed either at stimulating the immune system or directly against viral targets.
CURRENT THERAPIES FOR THE TREATMENT OF HCV INFECTIONS
The primary therapy for HCV infection is the intravenous administration of type-I interferon. Interferons are a group of endogenous proteins, which form part of the innate immune response [25]. Use of type I interferon-a was approved in 1990 and can reduce viral load and in some cases eliminate the virus completely. Many individuals do not respond, however, and many of those who respond initially do not clear their infections completely. The prolonged (up to 48–96 weeks) treatment course also has numerous side effects, including flu-like symptoms and psychiatric disorders [26]. Thus, in addition to those who fail to respond to treatment, many individuals are forced to discontinue therapy due to these side effects. Subsequent years have seen significant developments in interferon therapy. The most important have been the use of pegylated interferons, which have increased stability, and the introduction of combination therapy with the nucleoside analogue ribavirin [27]. Ribavirin is a broad-spectrum antiviral. The mechanism by which it acts against HCV is still controversial. However, in combination with interferon it both accelerates the initial drop in viral load and increases the probability of sustained response [28]. Ribavirin also has its own significant side effects, most importantly anemia [26]. The combination with pegylated interferon yields a sustained viral response rate of 50–60% [27]. The success rate is higher for genotypes 2 and 3 (approximately 80%) but is still less than 50% against the predominant genotype 1 [27]. Standard treatment times have been shortened to 24 or 48 weeks for genotypes 2 and 3 or genotype 1, respectively, and it may be possible to shorten treatment times even further [29]. Further improvements in immune system-boosting therapies have been described recently, including type II interferon-g or alternative forms of type I interferon, better tolerated derivatives of
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ribavirin, and other small molecules acting against a variety of host targets [30], but it remains to be seen if any of these non-virus-specific agents will have an impact on the overall effectiveness of HCV therapy. Together, these options are expected to further increase the number of individuals who can be successfully treated. However, major breakthroughs in therapy are expected only with the introduction of drugs directed specifically against viral targets such as the NS3 protease and NS5B polymerase.
THE HCV NS3/4A PROTEASE
NS3 is a 631 amino acid protein, and its first 180 amino acids encode a serine protease of the chymotrypsin family (Figure 2.2A). It has a typical chymotrypsin-family fold consisting of two b-barrels, with catalytic triad residues at the interface. His-57 and Asp-81 are contributed by the N-terminal b-barrel and Ser-139 from the C-terminal b-barrel. NS3 and closely related viral proteases are significantly smaller than other members of the chymotrypsin family, and many of the loops normally found between adjacent b-strands in trypsin proteases are truncated in NS3 [31]. Probably
Fig. 2.2 (A) Structure of full-length NS3 including the N-terminal protease domain (bottom) and C-terminal helicase domain (top). The NS4A peptide (purple) is covalently attached to the N-terminus of NS3 (see text). Within the protease domain the N- and C-terminal b-barrels are at the right and left, respectively. The zinc atom is visible at the bottom left. [98]. (B) Surface view of the NS3 protease domain showing compound (1) bound at the relatively shallow active site (See also Fig. 2.6) [42].
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as a result of its small size, in the absence of zinc, the NS3 protease is not structurally stable and requires the binding of a structural zinc, chelated by Cys-97, Cys-99, and Cys-145 and His-149, to maintain its tertiary structure [32]. The NS3 protease–Zn complex does have enzymatic activity, but full activity requires association with the 54 amino acid NS4A protein, or at least a peptide containing its central 14 amino acids [33, 34]. This peptide adopts a b-strand conformation and forms part of the N-terminal b-barrel. Only subtle changes in the positions of the protein backbone occur upon binding of the NS4A peptide, but several side chains move; in particular Asp-81 adopts an altered conformation to form a hydrogen bond to His-57 necessary for efficient catalysis [31, 35, 36]. Extensive work on the struc ture and catalytic properties of this protease has been reported over the last several years [37, 38], and only a few more recent studies will be described here. The small size of NS3, with only short interstrand loops, yields a very flat, open binding active site (Figure 2.2B). Indeed, this is the major challenge for the discovery of potent ligands for this enzyme. Early SAR studies of hexapeptide inhibitors based on C-terminal cleavage products (vide infra) identified acidic P5 and P6 residues, as well as a free acid at the C-terminus, as important for high affinity binding [39, 40]. Crystal structures with such inhibitors bound have not been reported, but modelling suggests that acidic residues at P5 and P6 could possibly form electrostatic interactions with several different positively charged residues found on the surface of the active site, including Arg-123, Arg-155, Arg-161, and Lys-165 [41]. Recently reported work using both enzymes with mutations in these positively charged residues as well as substrates with both neutral and acidic residues at P5 and P6 has revealed that charge complementarity improves affinity due to an increased on-rate, rather than a decreased off-rate. The cluster of positive charges on the active site surface accelerates binding by attracting negatively charged residues and thus increasing the initial collision rate of the protein and the inhibitor [41]. Modelling studies showed that specific ion-pairing interactions were not necessary for high affinity, and in fact were detrimental due to the high cost of desolvation. The relevance of these findings in vivo is not clear, since the affinity imparted by these acidic residues is significant only at low salt concentrations and is largely absent in physiological salt conditions [41]. In contrast, the C-terminal carboxylic acid of product-derived inhibitors plays an important role even at high ionic strength, suggesting that this group forms an ion-pair interaction with catalytic triad residues. The role of individual active site residues in inhibitor binding was investigated by using fluorescence polarization [42] or fluorescence resonance energy transfer [43] to study the binding of fluorescent inhibitors to wild-type (WT) and mutant
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enzymes. Interestingly, binding was not affected by substitution S139A, but significantly weaker binding was observed to the H57A mutant. The crystal structure of a more optimized peptidic inhibitor has confirmed that a strong hydrogen bond forms to His-57 rather than to Ser-139 (vide infra) [42]. All early work was performed with genotype 1a and 1b enzymes. Recent reports have described the catalytic and inhibitor-binding properties of sequence variants, including genotype 1 enzymes encoding resistance mutations (vide infra) and also genotype 2 and 3 proteases. Compared to genotype 1, the genotype 2 and 3 protease domains have approximately 50 and 40 amino acid differences, respectively, but only a small number of these are close to the active site. Specific differences are Val-78, Asp-79, Gln-80, and Ser-122 in genotype 1 compared to Ala, Glu, Gly, and Arg, respectively, in genotype 2, as well as Arg-123 and Asp-168 to Thr and Gln in genotype 3. In addition, residue 132 is Val in some genotype 1b sequences, Ile in genotype 1a and other 1b, and Leu in genotypes 2 and 3. This is the only active site residue not conserved between genotype 1a and 1b, and does not appear to have a significant effect on substrate or inhibitor binding. One report comparing genotype 1a, 1b, and 3a proteases found that they had almost identical catalytic efficiencies, and nearly identical affinities for hexapeptide inhibitors based on natural substrates [44]. However, introduction of unnatural amino acids at positions P4 or P5 was found to improve affinity only for genotype 1, such that these inhibitors bound five-fold more tightly to genotype 1 enzymes than to genotype 3. Mutagenesis was used to show that the sequence difference at amino acid 123 was responsible for this difference in affinity [44]. In another report the activities of both full-length NS3/4A proteins and NS3 protease domains were compared for enzymes of genotype 1, 2, and 3 [45]. This work also demonstrated that protease catalytic activity was only slightly affected by genotype differences, either in the kcat or Km parameter. The inhibitor BILN 2061, however, which was highly optimized for binding to the genotype 1 enzyme (vide infra), had 50–60-fold lower affinity for the genotype 2 and 3 enzymes. Mutagenesis studies confirmed that the active site residue differences noted above were primarily responsible for the loss of binding affinity. In contrast to the hexapeptide inhibitor study above, the single residue difference contributing most significantly to the loss of affinity for genotype 3 was at position 168 rather than at 123 [45]. A recent report has demonstrated that the proteolytic activity of NS3 plays an additional role in viral infection, beyond polyprotein processing. An important mediator of the cellular immune response is the transcription factor interferon regulatory factor 3 (IRF-3), which becomes activated on infection and then stimulates production of type-1 interferon and other antiviral genes [46]. It was found that expression of heterodimeric NS3/4A
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in cells abrogated this response [47]. Furthermore, protease activity appeared to be necessary, since the interferon response was not reduced by expression of NS3 without NS4A or a mutated NS3, and addition of a protease inhibitor also reactivated the IRF-3 response [47]. The specific molecular target of the NS3 proteolytic activity has not yet been confirmed, but the existence of this additional role for the viral protease suggests there might also be an additional advantage to treatment of HCV with protease inhibitors (or other HCV agents which inhibit viral replication and thus reduce the concentration of protease in infected cells).
TOOLS TO STUDY NS3/4A PROTEASE INHIBITORS ENZYMATIC ASSAYS
Biochemical assays to measure the activity of NS3 protease inhibitors can be performed with either the isolated protease domain or the full-length bifunctional protein. Both forms are frequently reported in the scientific and patent literature. Typically the full-length protein is expressed as a fusion with full-length NS4A, which cleaves itself during expression to yield the authentic NS3/4A heterodimer. Assays with the protease domain are carried out using an NS4A peptide of about 17 amino acids having charged residues appended at each terminus to improve solubility. The full-length heterodimeric complex is quite stable, and dissociation of the NS4A protein is not observed even at subnanomolar concentrations. In contrast, the NS4A peptide binds to the protease domain with only micromolar affinity, and it is necessary to perform assays with a large excess of the peptide as well as high concentrations of glycerol (30–50%) to stabilize the complex [48, 49]. The weakness of the peptide–protease complex is surprising, given that once bound the peptide is completely surrounded by NS3 residues [35]. However, the on-rate for binding has been found to be very slow, probably due to the significant conformational changes required in both the protein and peptide [48]. Stable complexes of the NS4A peptide with NS3 (protease domain or full-length) can be obtained by fusion of the peptide sequence to the N-terminus of NS3 [50, 51]. The design of these covalent complexes followed from the observation that the C-terminus of the NS4A peptide lies close to the N-terminus of NS3 in crystal structures [35]. The most active substrates for enzymatic assays are modelled on the NS5A/5B cleavage site spanning positions P6–P40 , and are often depsipeptides containing an ester rather than an amide at the cleavage site. Cleavage can be detected by high-performance liquid chromatography (HPLC) separation or by using substrates with a radiolabel at one end and a biotin at
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the other, so that biotinylated substrate can be separated from the radiolabelled but non-biotinylated product after the reaction [52]. However, one of the simplest assay formats uses a substrate with a fluorescent group at one end and a quencher at the other, so fluorescence increases significantly after cleavage [53]. In this case, no physical separation of substrate and product is needed. Another homogeneous format uses substrate peptides modelled on the P-side of the NS5A/5B cleavage site and modified at the C-terminus by acylation of a chromophoric alcohol such as nitrophenol [54]. Enzymatic cleavage of this ester bond yields a significant increase in absorbance. Both the fluorogenic and colorimetric formats are amenable to high-throughput, and also allow continuous monitoring of activity, making them suitable for kinetic studies. Binding assays have also been described. One method uses NS3, which is biotinylated during expression in Escherichia coli and a radiolabeled peptide pseudo substrate with a proline at P10 to prevent cleavage. The peptide–protein interaction is detected by binding the protein to streptavidincoated scintillation proximity beads [55]. In another binding assay format, interaction of a fluorescently labeled hexapeptide inhibitor with NS3 is detected by changes in fluorescence polarization [42] or fluorescence resonance energy transfer [43]. These binding assays are useful for certain mechanistic studies but have not been widely used for routine testing of inhibitors.
VIRAL REPLICATION
Cell culture models Once potent ligands for a viral protein are identified, further advancement depends on demonstrating activity in cells. Unfortunately, reproducible in vitro viral replication assays for HCV have not been reported. There are scattered reports that a very low level of genome replication, or even virus production, can be observed under certain circumstances [56]. However, recently specific sequences yielding relatively reproducible replication, at consistently detectable levels have been reported [57]. In the coming years these may allow routine assays suitable for compound evaluation to be developed, but to date drug discovery must rely on other cell culture models. The protease is uniquely suited for the development of surrogate cellular assays. Several variations of these assays have been developed in which the protease cleaves a fusion protein at an engineered NS3-specific cleavage site, directly activating a reporter gene such as secreted alkaline phosphatase (SEAP) or releasing a transcription factor which can subsequently direct reporter gene expression [58–61]. Detection of engineered target proteins by
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Western blot has also been reported [62, 63]. These assays have been widely used over the last several years but have recently been largely supplanted by the subgenomic replicon.
The replicon An important advance in HCV research was the report in 1999 that subgenomic constructs encoding only the nonstructural proteins together with an antibiotic resistance gene, flanked by authentic 50 and 30 sequences (Figure 2.1B), could replicate stably in the Huh-7 cell line isolated from human liver [64]. While monocistronic systems do exist, most replicons are bicistronic; that is, they encode two protein chains, with translation of the resistance gene directed by the HCV IRES and translation of the HCV non-structural region directed by the encephalomyocarditis virus (EMCV) IRES [56]. Replicon cell lines are typically generated by transfection of the subgenomic RNA into Huh-7 human liver cells and selection for colonies, which are able to grow in the presence of the antibiotic neomycin. The first replicons were generated using a consensus genotype 1b sequence [64] or a genotype 1a strain [65, 66]. Isolation of stable replicon cell lines was at first a long and very inefficient process, but it was soon found that the HCV sequences in stable replicon-containing cell lines had mutations, which significantly improved efficiency of replication [56, 66]. Fresh cells transfected with adapted sequences yield colonies at a frequency of 0.2–10%, whereas the original genotype 1b sequence yielded only 0.0005% [67]. The adaptive mutations map principally to the NS3 helicase domain and the NS5A sequence [56], but the mechanism by which they improve replication efficiency is still being investigated. With adapted replicons, replication efficiency is high enough so that assays do not need to be performed with stable replicon cell lines. Huh-7 cells can be transfected and the level of replication in the cell population evaluated after 24–96 h [67, 68]. Interestingly, it has been found that although a full-length genome containing the original genotype 1b replicon sequence was infectious in chimpanzees, sequences containing the adaptive mutations were not [69]. Thus, although the subgenomic replicon is believed to carry out many authentic functions of the HCV life cycle, there are significant differences in the constraints on sequences for the cell culture model and authentic infections. Very recently, a genotype 2a replicon was reported [70]. Since genotype 2 differs significantly in sequence from genotype 1, comparing the properties of this new replicon to the established genotype 1 replicons should yield new insights into HCV replication.
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Academic research using the replicon has yielded a number of insights into the biology of HCV, but it has also been widely adopted for pharmaceutical research. Compounds can by tested in 96- or 384-well plate format. RNA copy numbers can be accurately quantified using real-time polymerase chain reaction (PCR). This procedure requires that RNA be isolated from cells at the completion of the assay, and is time-consuming and expensive. Alternative detection procedures have been described in which the level of NS3 protease activity is detected after compound incubation and cell lysis [71], or in which the concentration of the neomycin resistance protein is quantified by (ELISA) [72]. Recently, replicons further engineered to contain a luciferase reporter gene in the first reading frame have been developed [68]. These allow the level of replication to be determined using a simple luminescence readout, significantly increasing throughput of the assay. With these improvements the replicon is not only used as the cell culture assay for inhibitors of defined enzymatic targets, but can also be the subject of highthroughput screening, to yield compounds which appear to target proteins of poorly understood function such as NS5A [73].
Animal models Besides humans, the only known host for HCV is the chimpanzee. A critical early development in HCV research was the discovery that specific cloned RNA genomes could be used to infect chimpanzees [74, 75]. Genomes containing point mutations in nonstructural proteins, including the NS3 protease, which abrogated activity were noninfectious, validating these proteins as essential viral targets [76]. However, for ethical as well as practical reasons the chimpanzee cannot be used routinely to test candidate drugs. To overcome this limitation, two quite elaborate animal models have been developed which use infected human liver cells grafted into severe combined immunodeficiency (SCID) mice. In the Trimera model, mice first receive lethal doses of radiation and are then given bone marrow transplants and grafts of infected human liver tissue [77]. The KMT Hepatech model uses SCID mice which express a urokinase-type plasminogen activator transgene in the liver, resulting in death of most mouse liver cells [78]. Their survival then depends on engrafted human liver cells. Replicating virus can be detected in both models, and they have further been validated using candidate antivirals: a small molecule directed against the IRES and an antibody directed against E2 in the case of the Trimera model [77] and interferon-a [79] and the protease inhibitor BILN 2061 [80] in the KMT model. However, both models use immunodeficient animals, and the duration and severity of infection is not well controlled. Thus many questions regarding the kinetics
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of viral infection or the interaction of the virus and immune system cannot be addressed. In the future, these models may prove useful in helping to validate agents directed against non-traditional targets, such as the poorly understood NS4B or NS5A, but they are not believed to be critical for the advancement of protease or polymerase inhibitors. In some cases, candidate HCV drugs have been tested in primates infected with related viruses such as GVB-B [81]. A protease-specific model has also been reported in which a replicationdefective adenovirus encoding an NS3 protease–SEAP fusion protein is injected into mouse tail veins, resulting in expression of the fusion protein in the liver [82, 83]. Protease activity can be detected both by measuring activity of liberated SEAP or by protease-induced liver damage. Protease activity was found to be reduced by administration of protease inhibitors. This model can be used to show that candidate inhibitors have adequate pharmacokinetic properties in mice to function in the intended target organ, but it is not a true disease model. A possible limitation of these mouse models is suggested by the recently recognized fact that, in addition to the liver, HCV does replicate in other cell types, including peripheral blood mononuclear cells (PBMCs) and dendritic cells [84–87]. In fact, the quasispecies distribution in non-liver cells is different, suggesting that the virus evolves distinct populations for each cell type [23, 88]. Elimination of virus from these non-liver ‘‘reservoirs’’ could possibly be the key to achieving a sustained viral response. In the long-term, full understanding of HCV will require a model in which the full virus life cycle can be studied in an immuno-competent animal. Alternatively, more intensive study of related animal viruses such as GBV-B may yield insights into the viral life cycle, which could be applied to HCV.
Resistance studies As has been observed with inhibitors of HIV, resistance to anti-HCV drugs is expected when these agents are tested in the clinic. The replicon can be used to identify and characterize potential resistance mutations. Assays to measure inhibition of replication are normally run for 24–72 h, but if cells are cultured for a longer time in the presence of high concentrations of inhibitor, colonies eventually emerge which can grow efficiently. RNA isolated from these colonies will usually contain mutations in the gene for the targeted protein. For example, several independent studies with the protease inhibitor BILN 2061, or a close analogue, have identified resistance mutations in the protease active site (vide infra). Since each isolated sequence usually contains additional mutations due to natural drift of the sequence
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on long-term culture, resistance is confirmed by engineering the specific resistance mutation into the initial sequence and confirming the decreased sensitivity to the inhibitor for the resulting replicon, as well as for the isolated enzyme containing the same mutation. However, the replicon is under fewer constraints than the full virus sequence, since it only models a part of the viral life cycle. Viruses encoding replicon resistance mutations may not be viable. Sequences encoding resistance mutations have not yet been used to infect chimpanzees, and it will probably be only after long-term clinical trials that authentic resistance mutations can be identified and compared to those that arise in replicon studies. It is encouraging that treatment with sufficiently high concentrations of protease inhibitors or interferon [89] appears to completely suppress the emergence of resistance and in fact results in ‘‘curing’’ cells of the replicon, such that no HCV replication can be detected even after removal of the drug. The replicon can also be used to evaluate combinations of inhibitors. Additivity or slight synergies have been reported for interferon–protease inhibitor [90–92], interferon–polymerase inhibitor [93], and protease– polymerase inhibitor [94] combinations. Additional advantages are found on longer-term treatment with drug combinations, since as found for HIV these combinations very effectively prevent the emergence of resistance [91, 92]. Any colonies that do form must have sequences encoding separate mutations for both drugs in the combination [94].
INHIBITORS OF THE NS3/4A SERINE PROTEASE NON-COVALENT PEPTIDIC INHIBITORS
A rather unusual feature of the NS3 protease is the presence of product inhibition [39, 95]. The initial discovery of N-terminal product inhibition of peptide substrates has been exploited in the design of potent and specific inhibitors of this protease [42, 96]. The observation that a carboxylic acid can interact very efficiently with the catalytic triad via ionic interactions, producing very potent and specific inhibitors, has led to significant research into non-covalent inhibitors [97]. The binding mode of the C-terminus of the helicase domain in the active site of the protease was revealed by the first crystal structure of the full-length NS3 protein with its N-terminus covalently linked to the NS4A peptide cofactor (Figure 2.2A) [98]. This X-ray crystal structure of a C-terminal carboxylate bound to the active site of the NS3/NS4A complex provided an atomic view of the interactions between the protease domain and the P-side product of the cis cleavage reaction. The carboxylate in the active site is stabilized via several hydrogen bonds.
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Several classes of non-covalent substrate based inhibitors have been reported, and are grouped below based on the nature of the C-terminal group interacting with the catalytic triad of the enzyme. The majority of the reported inhibitors are based on the N-terminal product of a modified substrate of the NS5A/5B cleavage side or to a lesser extent of the NS4A/4B substrate peptide. Carboxylic acid containing inhibitors After the discovery of weak inhibition by the N-terminal products of substrate peptides, researchers at Boehringer Ingelheim reported potent and specific hexapeptides providing up to a 106-fold increase in potency (Figure 2.3) [89]. Crucial to this class of inhibitors was the discovery of (1R,2S)-1-amino-2vinylcyclopropane carboxylic acid (vinyl-ACCA) as a P1 residue [99]. Initially, several reports appeared describing chemically stable replacements for the cysteine residue at the P1 position of all trans-cleaved substrates. An aliphatic side chain with two (aminobutanoic acid) or three (nor-valine) carbon atoms seemed to be an acceptable replacement, with only a moderate loss in potency and improved chemical stability [96, 100]. An improved P1 COOH O H2N
H N
N H
O
COOH
O N H
SH
N
Ki = 79,000 nM O
O
OH
N H
O
Ph
N COOH O H N O
O
H N
N H COOH
O
O N H
N O
O
N H
OH O
Fig. 2.3 Hexapeptide inhibitors of the NS3 protease.
Ki = 0.074 nM
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residue designed using a model of the cysteine bound to the S1 pocket of the NS3 protease resulted in the identification of 4,4-difluoro-aminobutanoic acid, which is an equivalent replacement for the cysteine [101]. However, Rancourt, et al. [99] reported recently SAR studies on tetrapeptide inhibitors and demonstrated that the introduction of vinyl-ACCA produces inhibitors that are 14 times more potent than those containing cysteine (Table 2.1). Vinyl-ACCA containing inhibitors are chemically stable and very specific for the NS3 protease, over other serine or cysteine proteases. Another crucial finding for these inhibitors was the discovery of the 2phenyl-4-oxoquinoline as a substituent at the 4-proline of the P2 residue (Figure 2.4) [102]. This novel substituent was designed based on NMR studies of two series of inhibitors [103]. Figure 2.4 displays a superposition of the P2 bound conformations of the two analogues. The A rings of these two inhibitors adopt very similar bound conformations, whereas their second aromatic rings (B rings) explore different regions of the conformational space and therefore occupy different binding areas. Combination of these residues led to tricyclic derivatives with an approximately 10-fold increase in inhibitory activity. Further optimization of the tetrapeptide series led to very potent inhibitors allowing for size reduction to tripeptides (Figure 2.5) [104]. Further structural studies revealed that macrocyclization can improve potency by constraining the vinyl-ACCA into the correct conformation for binding [42, 105]. In this series of macrocyclic inhibitors, the cyclopropyl P1 moiety is Table 2.1 VINYL–ACCA IN COMPARISON WITH VARIUOS P1 RESIDUES
IC50 (µM)
R SH
140 O
Ph
N H
CO2H
N H
CO2H
O H N
530
N N H
O
O
O
H
R
9.7 N H
CO2H
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Fig. 2.4 Superposition of the P2 bound conformation of two analogues.
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O
O AcHN
OMe
N
Ph
N
N H
O
IC50 = 0.002 µM
O
OH
N H
O OMe
N
Ph
IC50 = 0.025 µM O
O tBuO
N H
N O
MeO
O
N H
OH O
MeO
N
N Ph
H N
N O
O
O
S
O
O O O
N
O (1)
O OH
O
O
tBuO
H N
N OH
H N
N
N H
BILN 2061
Fig. 2.5 From tetrapeptide inhibitors to BILN 2061.
essential for potency with 2,000-fold loss in potency versus the cyclic norvaline analogue. Macrocyclic inhibitor (1) [42] is a selective inhibitor of HCV RNA replication in the replicon assay with an EC50 of 77 nM. The crystal structure of compound (1) bound to the NS3/NS4A-peptide enzyme
P.W. WHITE, M. LLINAS-BRUNET AND M. BO¨S
83
Arg 155
Asp Arg Asp155 Arg 168 123 Ala 156 Val 158 Ala Cys 157 Phe 159 Val 154 132
Asp 81
His His 57
Ser
Ala 156 Ser 139
E2 strand Lys 136
S1
Ala 157
Gly 137
Ser 138
C2 strand
Fig. 2.6 X-ray structure of Compound (1) bound to the NS3 protease. Residues of the enzyme that are in contact with the inhibitor are highlighted.
shows that the inhibitor binds in close proximity to the catalytic residues and at the interface between the N- and C-terminal subdomains (Figure 2.6). Overall, the complex can be characterized as a surface interaction with only a single, relatively shallow pocket at S1. The complex structure suggested extensive hydrogen bonding between the C-terminal carboxylate and the oxyanion hole, consisting of the amide NH’s of Ser-139, Ser-138, and Gly137. The carboxylate also appeared to hydrogen bond to the eH of His-57, whereas the side-chain of the catalytic residue Ser-139 rotated away from the P1 carboxylate (Figure 2.6) [42]. Compound (1) suffered from an unfavorable pharmacokinetic profile when studied in rats. It is cleared very rapidly from rat plasma (half-life, t1=2 ¼ 0:4 h) and is poorly bioavailable (F ¼ 2%), as reflected by the low plasma concentration (area under the plasma concentration-time curve, AUC0 N ¼ 0.2 mM h) following a single oral dose of 25 mg/kg in rats [42]. The main challenge was to further optimize this series to obtain NS3 protease inhibitors with low-nanomolar cell-based potency (EC50o10 nM) and with an adequate pharmacokinetic profile for oral absorption. BILN 2061 Structure activity studies on compound (1), focusing on optimization at the 2-position of the quinoline and at the capping group, resulted in the
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BLUNTING THE SWISS ARMY KNIFE OF HEPATITIS C VIRUS
identification of BILN 2061 [106], the first HCV-specific antiviral with proven antiviral effect in man [107]. Administration of BILN 2061 to patients infected with HCV genotype 1 for 2 days resulted in an impressive reduction of HCV RNA plasma levels (up to 3 log10 copies/mL in a substantial proportion of patients) and established proof-of-concept in humans for an HCV NS3 protease inhibitor. Virological responses were similar in interferon-a nonresponders and treatment naı¨ ve patients, as well as in patients with both high and low baseline viral RNA concentrations (Figure 2.7) [108]. In a separate clinical investigation, administration of BILN 2061 to patients infected with genotype 2 or 3 viruses yielded significant but less dramatic reductions in viral load over the course of the twoday study [109]. This result is consistent with the somewhat reduced activity of this compound against the genotype 2 and 3 proteases [45]. These short proof-of-principle clinical studies have confirmed the tremendous potential of antivirals, particularly NS3 protease inhibitors, for the treatment of HCV infection. They have also justified the general approach to compound advancement for HCV agents, in which the decision to initiate clinical trials is based on results from the replicon in combination with CMC and DMPK data. In recent toxicology studies in animals receiving high doses of BILN
Course of Virus Load / Hours, Drug naive patients vs. Non responders BILN 2061 ZW 200mg 10,000,000 Administration
Non Resp
Non Resp
Drug naive
Drug naive
Drug naive
Non Resp
Non Resp
Non Resp
Limit of linear range
Drug naive
Non Resp
Limit of linear range
Virus Load: copies RNA / m
1,000,000
100,000
10,000
1,000 0
20
40
60
80
100
120
140
160
Hours
Fig. 2.7 Virologic response of BILN 2061 (200 mg, twice a day) in pretreated and drug naı¨ve patients with minimal fibrosis. Upper and lower yellow lines represent the limits of detection of the Amplicor assay. Upper black triangles represent administration of BILN 2061.
P.W. WHITE, M. LLINAS-BRUNET AND M. BO¨S
85
2061 for 4 weeks, cardiac toxicity was identified [108]. As a consequence, no new clinical trials with BILN 2061 will be considered until the animal toxicological findings have been further investigated. Longer-term clinical trials might have resulted in the emergence of resistance to BILN 2061. Several independent reports have described resistance mutations obtained in the replicon system for BILN 2061 [91, 92, 110], or an earlier tripeptide from this series [111]. Predominant mutations were at Asp-168, as well as Arg-155 and Ala-156 [92, 110]. Mutations selected at these positions reduce the potency of BILN 2061 several hundred fold. Furthermore, activity of BILN 2061 [91, 92] or its analogue [111] against purified enzymes encoding the resistance mutations is reduced to a similar extent compared to the WT enzyme. A low level of resistance is also obtained by mutation of Gln-80 [92]. Enzymes encoding any of these mutations have similar catalytic parameters to WT NS3 [91, 92, 111], but the replication capacity of some of these mutants is reduced in transient assays [110]. As discussed above, the fitness of viruses encoding these mutations, and thus their relevance for patients, will only be determined by future longer term clinical studies. The four reported resistance mutations are clustered near the inhibitorbinding site, between the P2 side chain and the capping group (Figure 2.8). Mutation of Gln-80 or Ala-156 seems to directly affect the surface of the active site, which interacts with the P2 quinoline [42, 92]. The mutation of Arg-155 to glutamine appears to disrupt a salt bridge to the quinoline methoxy group [42, 92]. Asp-168 does not interact directly with the inhibitor, but removal of the negative charge would disrupt a network of hydrogen bonds between charged residues including Asp-168 and Arg-155, as well as Arg123. Thus mutation of this residue indirectly affects the interaction of other active site residues with the P2 quinoline [91, 92, 111]. Several different Asp168 mutants have been identified (Val, Tyr, Ala, Gly). D168 V has been most frequently identified and gives the largest level of resistance, about 500-fold. The potency of BILN 2061 is shifted 20-fold less by D168G, indicating that the size, as well as the charge, does have an effect on inhibitor binding [92]. A highly convergent route for the synthesis of BILN 2061 has also been reported [112]. The synthesis is based on the assembly of four key fragments with an overall yield of 10–15% as shown in Scheme 2.1. The crucial step is a ring closing metathesis of an acyclic precursor.
Phenethyl amide containing inhibitors In search of a replacement of the C-terminal carboxylic acid, the IRBM/ Merck group discovered that a phenethylamide group extends into the
86
BLUNTING THE SWISS ARMY KNIFE OF HEPATITIS C VIRUS
Fig. 2.8 Active site of NS3 protease with the inhibitor BILN 2061 added through molecular modelling [92], highlighting residues found to be mutated in resistance studies (Ala-156 and Asp-168, yellow; Arg-155, green; Gln-80, magenta).
prime side of the protein and interacts with Lys-136 of the enzyme surface (Figure 2.9). Mutation studies identified a lipophilic interaction between the phenyl ring and the aliphatic side chain of the Lys-136 [113]. The optimal amide was found to be 2,6-difluoro-4-carboxyl-phenethylamide [114]. Hexapeptides in this series are very potent and reversible inhibitors of the protease. Reduction in size to tripeptides, however, resulted in substantial reduction in potency (Figure 2.9). Compound (2), also displaying interesting pharmacokinetic properties in rats, is the best compound reported. Not surprisingly this additional binding is dependent on the P1 residue employed in the inhibitor backbone. In contrast to cysteine and the cysteine mimetic 2amino-4,4-difluoro-butyric acid containing inhibitors, vinyl-ACCA derived compounds are inactive [115]. Non-covalent aza-peptide inhibitors Another series of non-covalent competitive inhibitors reaching toward the P0 side has been reported by Boehringer Ingelheim researchers. Bailey, et al. [116]
P.W. WHITE, M. LLINAS-BRUNET AND M. BO¨S
2R
H2N
HO 4S a) TBTU, DIPEA, DMF
COOMe HO 4R
H 3S
+
a) HCl / dioxane
H N
N COOH b) PNBA, DEAD, N PPh3, THF Boc Boc c) LiOH (1.25 eq.), THF, water
COOMe
O
COOH
Cl
O
N O
Cl
OMe
Ru
CH2Cl2 (0.01 M) BocHN 28 h (5.5 mol%)
BocHN
OH OMe
O
DIAD, PPh3, 0 °C, to RT
MeO
MeO a) NaOH (1 eq.), THF-MeOH-water b) i-BuOCOCl, Et3N, then CH2N2 / Et2O
N COOMe O H N
N O BocHN
COOMe
O
H N
N
PCy3
O
O
N
OH
O
H N
b) TBTU, DIPEA, CH2Cl2 NHBoc
MeO OH
87
O
O OMe
c) HBr, THF d) i-PrNHCSNH2, i-PrOH, 70 °C, 1 h d) HCl / dioxane e) c-pentylOCOCl, Et3N, THF f)LiOH, THF-MeOH-water
N
S
O H N
N O O O
H N
N
O OH
O N H
Scheme 2.1 Synthesis of BILN 2061.
reported an inhibitor series that incorporates an aza-amino acyl fragment at the P1/P10 position (Table 2.2). The best compound identified contains a benzyl derivative. The choice of P1 side chain was important for providing specificity against other serine proteases. A four-carbon P1 side chain was found to be optimal (Compound (3)). NMR and SAR studies on this series of inhibitors demonstrated that they bind reversibly and non-covalently to the enzyme [116]. C-terminal acyl sulfonamide containing inhibitors The uniqueness of the non-covalent product-based inhibitors containing C-terminal carboxylic acid prompted the exploration of bioisosteric
88
BLUNTING THE SWISS ARMY KNIFE OF HEPATITIS C VIRUS
COOH
COOH O AcHN
O
H N
N H
O
COOH
H N
N H
Ph
CHF2
O
H N
N H
O
Ph
O
Ph IC50 = 2 nM
COOH SH O O
O
H N
N H
N H
O
Cl
H N O
COOH
Ki = 700 nM
O O
N H
N O
O
CHF2 N H
F
H N O
F
(2) Ki = 100 nM
N NH N N
Fig. 2.9 Phenethyl amide containing inhibitors.
replacements of this critical group [117, 118]. Initial reports on hexa- and pentapeptide derivatives concluded that bioisosteric replacement of the C-terminal carboxylic acid of the product-based inhibitors with tetrazoles, and especially with acyl sulfonamide residues, produced product-based inhibitors up to 20-fold more potent than the corresponding carboxylates (Table 2.3). The aryl acyl sulfonamide group also allows for elongation into the P0 region, as well as other structural modifications. After the initial reports of the antiviral efficacy of BILN 2061 a large number of reports disclosing macrocyclic (Figure 2.10) [71, 119–124] and acyclic (Figure 2.11) [125–135] analogues have appeared. For some of these compounds an acyl sulfonamide has been also reported at the C-terminus.
P.W. WHITE, M. LLINAS-BRUNET AND M. BO¨S
89
Table 2.2 NON-COVALENT AZAPEPTIDE INHIBITORS
IC50 (µ M)
COOH
NS3
HLE
0.60
> 1000
17.5
ND
0.099
414
Ph O
O AcHN
O
H N
N H
O
COOH
N
N H
O
O
OH
N H
O Ph
COOH O O AcHN
O
H N
N H COOH
N
N H
O
O
N H
O
N
NH2 O
COOH
Ph O
O AcHN
N H COOH
H N O
O N H
N O
O
N H
H N
N
Ph
O
(3)
Prime-side inhibitors The IRBM group combined characteristic elements of the product-based inhibitors with decapeptide inhibitors to yield inhibitors binding solely to the prime side and active site of the protease [136]. Thus, the P1 acarboxylate of the product-based inhibitors is now at the N-terminal cyclohexyl moiety, as illustrated in Figure 2.12. The peptidomimetic
90
BLUNTING THE SWISS ARMY KNIFE OF HEPATITIS C VIRUS Table 2.3 BIOISOSTERIC REPLACEMENTS OF THE C-TERMINAL CARBOXYLIC ACID
COOH
O HOOC
N H
H N
O
H N
N H
O
O P1
N H
O
O
COOH Ki (µM)
OH
H2 N 0. 22
0. 091
O
0. 16
OH O
NH
H N
H2 N
N N
O
H2 N
N
H2N
H2N
NH
0. 19
Ph
O O
0. 014
H N
N N N
S
H2 N O
S
Ph
O O 0. 004
inhibitor (4) is the result of an elegant evolution of the initial leads. However, the potencies of these prime-side inhibitors are moderate. COVALENT REVERSIBLE PEPTIDIC INHIBITORS
As an alternative to peptidic inhibitors, which display electrostatic interactions with the active site, covalent inhibitors have also been described recently. Such peptides bear a functional group that can react reversibly with the catalytic serine of the protease. These include aldehydes, a-ketoacid derivates, lactams and boronates. Research groups at Vertex and Lilly optimized inhibitors starting from pyrazinoyl capped tetrapeptidic aldehydes (Table 2.4) [137]. The main SAR
P.W. WHITE, M. LLINAS-BRUNET AND M. BO¨S
91
R N
R'
N O
S O
H N
N O
1
R
O
O O
N H
R1 = OH or Acyl sulfonamide
N H
O
N H N
N O
O
O O O S N R H
O tBuO
N H
N
S
Ph
Ph
O
N
N H N
N O
O
O tBuO
N H
O
R1
O
H N
N
O
1
R
O
O tBuO
N H R1 = OH or Acyl sulfonamide
Fig. 2.10 Recent macrocyclic tripeptide inhibitors.
work comprised substitution of the P2 proline as well as modification of the P1 residue (Table 2.5) [138–143]. These studies culminated in the development of VX-950, a a-keto-amide, which is currently undergoing clinical testing [144]. The compound binds slowly and reversibly to the protease. It
92
BLUNTING THE SWISS ARMY KNIFE OF HEPATITIS C VIRUS
R'
N
MeO
O O tBuO
MeO
N
N H
O
H N N H
O
S
R
N
O O
O
O O
R = Alkyl or Aryl R' = Heterocycle
tBuO
N
N H
H N
O
N H
O
1
R N
O
O
S
R
O O
2
R
O O tBuO
N H
Rc = OH or Acyl sulfonamide
N C
O
R O
N H
O
H N R' N
R
S
N
Rc
= OH or Acyl sulfonamide O O tBuY
N
X
Y = NH, X = C Y= O, X = NH
C
O
R O
N H
O
Fig. 2.11 Recent acyclic tripeptide inhibitors.
shows moderate replicon potency (EC50 ¼ 400 nM), good oral bioavailability and excellent liver exposure [145]. The replicon system was also used to obtain resistance mutations for this compound. The amino acid substitutions A156S [91] or A156 T [146] significantly weaken binding. Interestingly, A156S does not significantly affect the binding of the tripeptide BILN
P.W. WHITE, M. LLINAS-BRUNET AND M. BO¨S
H N HO
O
O
COOH
O N H
H N
O NH2 HO
O
Ph
O
H N
O
93
Ph
N
O COOH Br (4) IC50 = 2 µM
Fig. 2.12 Prime-side inhibitors.
Table 2.4 PEPTIDIC ALDEHYDES AS COVALENT INHIBITORS OR N O H N N N N H R' O O N O H R0
R
Ki (mM)
Benzyl
12
O
(L)
1-Naphthyl
2.1
O (D,L)
0.9
O (D,L)
N O Benzyl
O
3.1
(D,L)
F3C Benzyl (L)
O
3.8
2061, whereas the primary resistance mutations for BILN 2061, at Asp-168, do not significantly affect the potency of VX-950 [91]. The crystal structure of the inhibitory complex shows only partial overlap with the protein regions covered by BILN 2061 providing an explanation for the activity of
94
BLUNTING THE SWISS ARMY KNIFE OF HEPATITIS C VIRUS Table 2.5 PEPTIDIC KETO-AMIDES AS COVALENT INHIBITORS
Ki (µM) N O N H N
N
O
O
O
0.4
N
N H
O
O
NH
O
N H O
N
O
H N
N
N
N H
O
O
0.15
O
NH
O
N H O
N
O
H N
N
N
N H
O
O
N N
N H
H N O
O N H
(5)
0.003 N H
VX-950
O
O
NH
O
O
H N O F2 C
O
H N O
COOH
IC50=0.06
P.W. WHITE, M. LLINAS-BRUNET AND M. BO¨S
95
VX-950 against NS3 mutants that are insensitive to BILN 2061, and vice versa [91]. Scientists at Bristol-Myers Squibb used the a-ketoamide functionality to extend binding to the prime side [147]. The glycine carboxylic acid (Compound (5), Table 2.5) was identified as the most effective extension. Modelling studies, coupled with SAR, suggested that there is either hydrogen bonding or a charge complex of the acid with Lys-136 and Arg-109. The synthesis of aldehydes and ketoamides was performed on solid phase as well as in solution (Scheme 2.2). A semicarbazone linker (6) was employed for the assembly of the aldehydes on solid phase whereas the corresponding aminoalcohol was coupled in solution to the tripeptide and oxidized to the aldehyde, which produced epimeric mixtures [137]. For the synthesis of the ketoamides, hydroxyester THP resins were used as solid support ((7), Scheme 2.2) [138]. In solution the peptide bond was formed using an aminohydroxycarboxylic acid building block [138, 147]. Oxidation of the free hydroxyl group yielded the final inhibitors ((8), Scheme 2.2). The pyrrolidine-5,5-trans-lactam ring system, developed by the Glaxo group as a serine trap for a number of proteases, was applied to the NS3 protease [148, 149] (Scheme 2.3). As shown by X-ray structure analysis, the serine hydroxyl attacks the lactam carbonyl, which is activated by ring strain as well as by the N-acyl group to form a hemiketal [150]. The challenge is to find compounds, which display reasonable chemical stability and consequently favorable in vivo pharmacokinetic properties and good biochemical potency. GW0014 (Scheme 2.3) was identified as such an inhibitor and was tested in the GBV-B infected marmoset, showing a three-log virus titer reduction in this surrogate animal model [81]. The ring system is synthetically accessible via acyliminium coupling to ketene acetals followed by ring closure with tBuMgCl (Scheme 2.3) [148, 151]. Boronic acid derivatives are known to inhibit serine proteases through the formation of a reversible tight B–O bond. Bristol–Myers Squibb published a series of peptidic [152] as well as peptidomimetic (+)-pinanediol boronates [153, 154] targeting the NS3 protease (Scheme 2.4). The latter feature a bicyclic pyrimidone and pyrazinone core replacing the P2–P3 dipeptide. These heterocyclic scaffolds allow the formation of the necessary b-sheet hydrogen bonds between the carbonyl and NH of the ring system and the enzyme backbone, as well as extension into the S2 binding pocket. Inhibitors derived from these structural motifs, such as compound (9), display high enzymatic potency, but no replicon data are available. The heterocyclic scaffolds are prepared from pyroglutamic acid [154, 155]. 1-aminoalkyl boronic acid pinanediol esters are readily available through a diastereoselective homologation with dichloromethyllithium, providing (S)-a-chloroboronic esters. Aminolysis of the chloride yielded
96
BLUNTING THE SWISS ARMY KNIFE OF HEPATITIS C VIRUS O HO N
H N
N
H N
N H
H N O
(6)
O
N O N N
O
O
H N
O N
N H
O
O
O
H2N
O
O
+
OH
O
(7) N O N H N
N O
O
O N H
N O
NH
O
O O O O
N O
N H N
N O
O
O N H
N O
O
NH
O NHR
(8) O
Scheme 2.2 Synthesis of peptidic covalent inhibitors.
P.W. WHITE, M. LLINAS-BRUNET AND M. BO¨S
97
O O
H N
H N
N
N
O
O GW 0014 IC50=1µM
R TMSO
R
OEt CBZ N
CBZ
NHCOCF3
+
N
OEt
NHCOCF3
BF3.OEt2
R CBZ
R
R
COOEt K2CO3
N
NHCOCF3 EtOH
CBZ
N
R
R R
COOEt tBuMgCl NH2
CBZ THF
N
O NH
Scheme 2.3 Synthesis of pyrrolidine-5,5-trans-lactams.
(R)-aminoboronic esters, which are coupled to the pentapeptides or to the bicyclic peptidomimetics (Scheme 2.4) [152]. NON-PEPTIDIC INHIBITORS
The identification of non-peptidic lead structures remains a challenge. Screening of natural product extracts led to the identification of two polyhydroxylated biphenyls ((10a) and (10b), Figure 2.13) that show submicromolar inhibition of the viral protease [156]. A recent report discloses polyesters of glucose (11) and gallic acid (12) as micromolar inhibitors of the NS3 protease [157]. Fragment screening by NMR was applied recently in the search of nonpeptidic small molecule inhibitors. Two scaffolds (13) and (14), which bind the enzyme at the S1–S3 and the S20 binding site respectively, as shown by chemical shift perturbation, were linked together to yield competitive inhibitors such as (15) with micromolar IC50 values [158]. There have been no reports of non-peptidic inhibitors with potency and pharmacokinetics similar to the peptidic or peptidomimetic inhibitors described above.
98
BLUNTING THE SWISS ARMY KNIFE OF HEPATITIS C VIRUS
HAspGluValValPro
H N
O B O NHR
O N CF3
N H
H N
N
CF3
O
O B O
O
Cl N CF3
N H
H N
N O
O B O
O
(9) IC50=0.06µM
O B O R
Cl2CHLi
O B O
R
a)LiN(SiMe3)2 b) 4N HCl
Cl
R
O B O NH2
Scheme 2.4 Synthesis of boronates.
SUMMARY AND OUTLOOK HCV infection continues to be a serious disease with a large unmet clinical need. There has been substantial progress in HCV virology and in the development of HCV protease inhibitors, in particular, since this topic was last reviewed in this series. The replicon has become a standard tool for the evaluation of inhibitor efficacy in cells, and reports of reproducible in vitro viral replication and useful transgenic animal models for HCV disease are beginning to emerge. We must recognize, however, that progress in the
P.W. WHITE, M. LLINAS-BRUNET AND M. BO¨S
99
OH HO
OH O
O O
HO HO
O
R= O
O O
HO
R
O
Ph
OH
HO
(10a)
OH OH O
(10)
Ph OH OH
OR1 O
R5O R4O
(10b)
HO OR2
HO
OR3
O (12)
(11a), (11b) (R3,4=H,), (11c) (R4=H)
I HO
O
O
HO
I
COOH
MeO (13)
(14)
I HO
O HO
COOH
O O
I
N H
O
O (15)
Fig. 2.13 Non-peptidic inhibitors.
development of HCV protease inhibitors has been difficult and slow. Since the first crystal structures were published almost 10 years ago, it is clear that the active site, a shallow dent on the surface of the enzyme, was not an easy target for small molecule inhibitor design. Indeed, even today no effective inhibitors of this target have been reported based on high-throughput screening or de novo design approaches.
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Progress in Medicinal Chemistry – Vol. 44, Edited by F.D. King and G. Lawton r 2006 Elsevier B.V. All rights reserved.
3 Peptide Deformylase Inhibitors KELLY AUBART and MAGDALENA ZALACAIN Microbial, Musculoskeletal, and Proliferative Diseases CEDD, GlaxoSmithKline Pharmaceuticals, Collegeville, PA 19426, USA
INTRODUCTION
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PEPTIDE DEFORMYLASE AS A NOVEL ANTIBACTERIAL TARGET Function Essentiality Spectrum Selectivity
110 111 112 112 113
PROTEIN STRUCTURE OF PEPTIDE DEFORMYLASES Three-dimensional Structure Metal-binding Site Comparison with other Metalloproteases Substrate-binding Pockets
113 113 114 115 115
DESIGN AND SAR OF PEPTIDE DEFORMYLASE INHIBITORS Early Substrate-based Inhibitors Pseudopeptidic Hydroxamic Acids and N-formyl-N-hydroxylamines Non-peptidic Templates
117 117 121 127
CLINICAL CANDIDATES BB-83698 LBM-415
135 135 136
SUMMARY AND PERSPECTIVES
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ACKNOWLEDGEMENTS
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REFERENCES
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DOI: 10.1016/S0079-6468(05)44403-3
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INTRODUCTION The successful development of numerous antibiotics in the 1930s–1960s [1] created the complacent feeling in the medical and scientific communities that bacterial infections had been defeated, resulting in a decrease in academic and industrial research in the antimicrobial area. Unfortunately, bacteria adapted to their hostile new environment by developing or acquiring resistance mechanisms that eventually rendered many of these antibiotics ineffective. Currently, infectious diseases are still a leading cause of death in the world [2], while the approval of new antibacterial agents has been steadily decreasing over the last 20 years [3]. Although the development of second- and third-generation antibiotics from existing classes has improved their activity and safety, bacterial resistance to these drugs continues to increase. In order to directly address this issue, antimicrobial agents with novel mechanisms of action are needed. However, of the 11 new antibiotics approved by the Food and Drug Administration (FDA) in the last 7 years, only two act through a novel mechanism: linezolid, an oxazolidinone, and daptomycin, a cyclic lipopeptide, both of which have been approved for the treatment of Gram-positive bacterial infections only. While considerable effort is underway to develop next generation oxazolidinones covering a broader spectrum of community and hospital infections [4–6], the identification of new drugs that target previously untapped bacterial pathways will play a critical role in the development of antibiotics active against resistant pathogens. This article summarises the general characteristics of a novel antibacterial target, peptide deformylase (PDF) and reviews the design, structure–activity relationships (SAR) and properties of known PDF inhibitors, including pre-clinical and clinical data for the most advanced members of this class.
PEPTIDE DEFORMYLASE AS A NOVEL ANTIBACTERIAL TARGET A broad-spectrum antimicrobial target must be conserved across all pathogens of interest within a therapeutic product profile, essential for bacterial growth, and either absent, substantially different or non-essential in humans. PDF meets all of these criteria and is one of the most promising unexploited bacterial targets in the search for new antibiotics with a novel mode of action.
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FUNCTION
Bacterial protein synthesis initiates with formyl-methionyl-tRNA and, consequently, all newly synthesised polypeptides contain an N-formyl-methionine terminus. The N-formyl group and, in most cases, the methionine, need to be removed to provide a mature protein and these reactions are part of the methionine cycle [7] (Figure 3.1). PDF (EC 3.5.1.88) is a metalloprotease that removes the N-formyl group of the polypeptides as they emerge from the ribosome during or immediately after completion of the elongation process [8–10]. Methionine aminopeptidase (MAP; EC 3.4.11.18) then removes the N-terminal methionine depending on the nature of the second amino acid in the peptide chain [7]. Thus, deformylation plays an indispensable role in protein maturation as MAP, an essential protein for bacterial growth, cannot hydrolyse N-blocked peptides [11]. The production of N-formylated proteins is a characteristic specific to bacteria and, therefore, an obvious target for the human immune system. It has been shown that professional phagocytes, the first line of defence against invading microorganisms, express receptors that recognise N-formylated peptides [12], and that activation of these receptors mediates migration of
tRNAi Polypeptide
Met Methionyl tRNA Synthetase
Methionine Aminopeptidase
Met-tRNA
Met-polypeptide
Formyl Methionyl Transferase
Peptide Deformylase
f-Met-polypeptide
f-Met-tRNA
aa-tRNAe
tRNAe tRNAi
Ribosome Fig. 3.1 The methionine cycle.
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phagocytes to the site of infection as well as the release of antimicrobial peptides and oxidants [13]. Theoretically, inhibition of PDF would increase production of bacterial N-formylated polypeptides, possibly triggering an enhanced immune response. In fact, Fu et al. [14] have shown that exposure of Escherichia coli to subinhibitory concentrations of a PDF inhibitor increases the production of peptides that mediate chemotactic migration and activation of human neutrophils. These results indicate that PDF inhibitors could have an additional advantageous effect, potentially amplifying the human immune response to bacterial infections.
ESSENTIALITY
Deformylation of nascent polypeptides has been shown to be a function essential for growth in E. coli, Staphylococcus aureus and Streptococcus pneumoniae [15–18]. Moreover, antibacterial mode of action studies, using S. pneumoniae or S. aureus strains in which the expression of PDF is controlled by regulatable promoters, have shown that the antibacterial activity of PDF inhibitors is due to their inhibition of the PDF enzyme, as the susceptibility of the strains to these compounds is dependent on the amount of protein present in the cell [19–21]. These results further validate PDF as a target for novel antibiotics.
SPECTRUM
PDF is a ubiquitous bacterial enzyme, with at least one pdf gene present in all bacterial genomes sequenced to date [22]. Based on phylogenetic and structural analysis, PDF proteins can be subdivided into two classes that differ significantly at the C-terminus: type I, found in Gramnegative and some Gram-positive bacteria, and type II, found only in Gram-positive bacteria [22]. The active sites of both type I and type II PDFs are highly conserved, indicating that it should be possible to obtain broad-spectrum inhibitors of this enzyme [22, 23]. In fact, PDF inhibitors have been reported to have activity against a wide range of pathogens including, among others, those involved in community respiratory tract infections (S. pneumoniae, Haemophilus influenzae, Moraxella catarrhalis) [24–28], including atypical organisms (Legionella pneumophila, Chlamydia pneumoniae, Mycoplasma pneumoniae) [28–33], Gram-positive infections (S. aureus, enterococci, viridans group streptococci) [28, 34, 35], tuberculosis (Mycobacterium tuberculosis) [36], and even malaria (Plasmodium falciparum) [37].
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SELECTIVITY
As eukaryotic cytoplasmic protein synthesis does not involve N-formylation, it was generally accepted that eukaryotes did not require PDF activity. However, the recent sequencing of eukaryotic genomes has revealed the presence of PDF-like sequences in the nuclear chromosomes of parasites, plants and mammals, including humans [38]. These eukaryotic PDF enzymes contain an N-terminal exportation sequence and are located in plastids and mitochondria [39–41]. Genomes of plant and parasite organelles encode for a number of proteins that require deformylation for activity [23] and, therefore, it is not surprising to find that PDF is essential in these organisms [37, 40]. On the other hand, there is no evidence to support that this is the case in animals. In fact, it has been shown that proteins synthesised in bovine and rat mitochondria either retain their N-formyl groups or have their N-terminal signal sequences removed [23, 42], indicating that PDF is not active in mammalian mitochondria. In addition, characterisation of human mitochondrial PDF has shown that it is much less active than its bacterial counterpart [41, 43] even when N-formylated peptides corresponding to the N-terminal sequence of human mitochondrial proteins are used as substrates [41]. Finally, known PDF inhibitors are active against this enzyme in vitro, but have no effect on the growth of normal human cell lines [41]. These data suggest that PDF does not play a functional role in human mitochondria and validates PDF inhibitors as selective antibacterial agents.
PROTEIN STRUCTURE OF PEPTIDE DEFORMYLASES THREE-DIMENSIONAL STRUCTURE
X-ray crystallography data are available for numerous PDFs, including E. coli, H. influenzae, Pseudomonas aeruginosa, Thermotoga maritima and P. falciparium (type I) [44–49], and S. aureus, S. pneumoniae, Bacillus stearothermophilus and B. cereus (type II) [22, 46, 48, 50]. The sequence identity between type I and II forms of PDF is low, for example, the homology between the E. coli and S. aureus proteins is only 23% [22]. Consequently, significant tertiary structural differences are observed between the two classes (Figure 3.2). Generally, type II PDFs are larger in size due to differences at either the N- or C-termini, as well as internal insertions. In type I PDFs, the C-terminus is helical, but in type II PDFs, the C-terminus consists of a b-strand that can fold back against the enzyme to form a b-sheet. However, these differences do not significantly affect
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Fig. 3.2 Superposition of E. coli and S. aureus PDF. Comparison of the overall structure of E. coli PDF (Blue, PDB code 2A18 ) and S. aureus PDF (mangenta, PDB code 2A19). The bound nickel is shown in red and His-132, His-136 and Cys-90 from E. coli PDF are shown as ball and stick representation.
the active site regions, which are structurally quite similar between type I and II PDFs.
METAL-BINDING SITE
Fe2+-PDF While PDF was originally proposed to be a zinc-metalloprotease [51], it is now generally accepted that Fe2+ is the physiologically relevant metal ion occupying the active site in vivo [52]. The native forms of most PDF enzymes are highly unstable due to propensity to oxidation, rendering them difficult to purify [53, 54]. However, the Fe2+ can be suitably replaced by either Ni2+ or Co2+, both of which provide a stable enzyme and maintain nearly identical catalytic activity to the native Fe2+ form (Table 3.1) [45, 55]. Replacement of Fe2+ with Zn2+ is also possible, but the catalytic activity of the Zn2+ form of the enzyme is greatly diminished, presumably
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Table 3.1 COMPARISON OF THE CATALYTIC ACTIVITY OF VARIOUS METAL FORMS OF E. COLI PDF [45, 55] Protein
Substrate
Kcat/Km (M/s)
Fe2+-PDF Co2+-PDF Zn2+-PDF Fe2+-PDF Ni2+-PDF
f-ML-pNA f-ML-pNA f-ML-pNA f-MA f-MA
3.50 106 1.11 106 3.1 104 1.1 105 1.0 105
due to the tighter binding of the zinc ion to the active site metal-binding residues [56]. COMPARISON WITH OTHER METALLOPROTEASES
Although native PDFs utilise Fe2+ rather than Zn2+, the PDF metal-binding site is still very representative of a traditional zinc-metalloprotease [57], containing a conserved HEXXH motif. Crystal structures of the Fe2+, Ni2+, Co2+ and Zn2+ forms of E. coli PDF have been solved, and no significant structural differences are observed among the various metal forms [44, 56, 58]. Each metal displays a tetrahedral coordination pattern, bound to the two histidines from the HEXXH motif, as well as to a cysteine and a water molecule, although Zn2+-PDF shows a slightly smaller volume around the tetrahedron formed by the metal ligands [56]. PDF is similar to thermolysin and other zinc metalloproteases in that the conserved glutamate of the HEXXH motif is essential for catalytic activity, and presumably activates the metalbound water for hydrolysis of the formyl group from the substrate [57–59]. PDF is different, however, in that it utilises cysteine as the third metal-binding ligand along with the two conserved histidines, while thermolysin uses glutamate, and other metalloproteases use a third histidine ligand [60]. Another difference is that metalloprotease substrates contain both a prime and a nonprime side surrounding the scissile amide bond, while PDF substrates possess only a single hydrogen atom in place of a non-prime side. PDF therefore represents a unique class of metalloproteases, lacking binding pockets in the non-prime direction [61]. The rear of the metal-binding site is essentially closed and even replacing the formyl hydrogen with the methyl group of an acetyl moiety compromises activity due to steric constraints [62]. SUBSTRATE-BINDING POCKETS
In the active site of PDF proteins, three substrate-binding pockets exist along with the metal-binding site. Using standard metalloprotease nomenclature,
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S2' O O
H N
HO P3' S3'
P2'
O
O N H
H N
H
H
O
Glu-133
O O-
Me S
M++
His-132
Cys-90
His-136
S1' Fig. 3.3 Schematic representation of the PDF active site and substrate transition state. The P10 substituent is represented as methionine.
these pockets are referred to as the S10 , S20 and S30 pockets, and the corresponding positions on substrates or inhibitors are referred to as P10 , P20 and P30 (Figure 3.3). S10 pocket The S10 pocket is the binding site for the methionine side chain of the substrate, and hence it is composed of mostly hydrophobic residues. This pocket remains extremely similar in size and shape among PDFs from various Gram-positive and Gram-negative organisms, with only subtle differences noted, such as the observation that S. aureus and S. pneumoniae PDFs possess a slightly wider (1.4 A˚) pocket than H. influenzae and E. coli PDFs [46]. The only non-conservative amino acid residue substitution in this region lies on the border between the S10 and S30 pockets. This residue is a leucine in E. coli (Leu-125), H. influenzae (Leu-125), P. aeruginosa (Leu-127) and B. stearothermophilus (Leu-146), and a tyrosine in S. pneumoniae (Tyr-166), S. aureus (Tyr-147) and T. maritima (Tyr-122) [22, 46, 48]. This difference, lying on the solvent exposed edge of the pocket, seems to impart only subtle effects on binding or access to the pocket, as good enzyme inhibition can be obtained with inhibitors that bind only to the metal and into the S10 pocket in both Gram-positive and Gram-negative PDFs [46]. S20 pocket The S20 pocket is not a true ‘pocket’, but rather an open, tunnel-like space pointing out into solvent. This loosely defined region is therefore
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able to accommodate a wide variety of side chains presented by the various formylated substrates to be processed by the PDF protein [63]. There is less overall conservation among the residues lining this region [46]. Type I PDFs generally possess a larger S20 site, suggesting that substrates with large substituents at P20 may be cleaved selectively by this class of enzymes [22]. S30 pocket The S30 pocket is the least conserved region of the active site between Gramnegative and Gram-positive PDFs, due to a two residue insertion between the S10 and S20 regions in Gram-positive species (Gly-124 and Glu-125 in S. pneumoniae). Again, similar to the S20 pocket, this area is solvent-exposed and more of a surface depression than a true binding ‘pocket’ [46]. Thus, inhibitors that target specific residues within the S30 pocket are likely to be more selective for those particular PDFs.
DESIGN AND SAR OF PEPTIDE DEFORMYLASE INHIBITORS EARLY SUBSTRATE-BASED INHIBITORS
SAR from substrate specificity studies When the native form of E. coli Fe2+-PDF was successfully purified and characterised in 1997 [52] (also described in 1995 but not published until 1998 – D. Groche, PhD thesis, Universitat Heidelberg, Germany), no known inhibitors of PDF had been identified. Therefore, several groups began to investigate the possibility of designing PDF inhibitors as substrate analogues or transition-state mimetics. To this end, Pei and co-workers [63] prepared a combinatorial library of resin-bound formylated tetrapeptide substrates and evaluated which sequences were preferably deformylated by E. coli PDF. This study demonstrated that the optimal residue at P10 was, predictably, methionine (represented by norleucine in the study), with glycine and aromatic amino acids (histidine, phenylalanine and tyrosine) following at a distant second and third. At P20 , essentially no selectivity was observed, as all amino acids were selected at similar frequencies except for glycine, aspartate and glutamate, which were not selected by the enzyme as PDF substrates. At P30 , a strong preference for an aromatic residue was observed, as over 80% of the selected beads either had tyrosine, phenylalanine or histidine at this position. The final P40 position also preferred an aromatic (tyrosine) or hydrophobic residue. Thus, this study concluded that an
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PEPTIDE DEFORMYLASE INHIBITORS
optimal substrate would comprise f-MX(F/Y)Y, where X is any amino acid except Asp, Glu or Gly [63]. Around the same time, Meinnel et al. [64] were studying the substrate specificity of E. coli PDF in a more stepwise manner. Probing the minimum structural requirements necessary for a PDF substrate, they systematically removed binding elements from N-formylated Met-Ala-Ser or N-formylated Nle-Ala-Ser and examined the effects on catalytic efficiency of deformylation. This study determined that the substrate could be successfully shortened to N-formylated Nle-NH2 or N-formylated Nle-OMe, suggesting that the P20 substituent is not strongly recognised by PDF. The carbonyl of the N-terminal methionine or norleucine proved to be critical for recognition by PDF, as N-formyl Metol, possessing a hydroxyl rather than the carboxylate, displayed a Kcat/Km reduced by 2–3 orders of magnitude when compared to either N-formylated Nle-NH2 or N-formylated Met-OMe. However, N-formylated Nle itself was a very poor substrate, presumably due to intolerance of the C-terminal carboxylate. Varying the substrate residues from Met-Ala-Ser to alternative amino acids and determining binding constants of the resulting deformylated peptide derivatives led to the conclusion that a hydrophobic P10 side chain was essential, and that a basic side chain at P20 combined with a hydrophobic aromatic C-terminal blocking group enhanced PDF binding potency.
Thiols Based on this substrate specificity data, Meinnel et al. [64] designed substrate analogues with the potential for PDF inhibition. While metal-binding groups such as thiols, carboxylates, hydroxamates and phosphonates have all been successfully used to replace the scissile bond of substrates processed by thermolysin, they chose to explore thiol derivatives given their known ability to form tight-binding complexes with ferrous and nickel ions. Encouraged by the PDF inhibitory activity of the commercially available metalloprotease inhibitor thiorphan (1) (K i ¼ 189 mM), thiol-peptides with substituents predicted to have improved binding were synthesised, with HS-CH2-Nle-Arg-OMe (2) achieving low-micromolar inhibitory potency against E. coli PDF (K i ¼ 2:5 mM). In 2000, Huntington et al. [65] reported on a second generation of peptidic PDF inhibitors utilising a thiol group. These analogues had improved inhibitory potency against E. coli and B. subtilis PDF, with some examples, such as p-nitroanilide (3), showing inhibitory activity in the low nanomolar range and moderate antibacterial activity (Table 3.2A).
Table 3.2 PDF INHIBITORS WITH REPORTED ANTIBACTERIAL ACTIVITYa A. Peptidic inhibitors Cpd.
Ki (nM)b
MIC (mg/mL)
Additional comments
Reference
Thiol
(3)
19
No further data
[65]
P20 i-Pr
Actinonin
r0.23c
Unstable in vivo
[24, 72–79]
P20 t-Bu
BB-3497 BB-83698
r0.18c ND
VCR3324 VCR3375 VCR4307 LBM-415
12 3 2 ND
Efficacious in vivo (oral) Progressed to Phase I (i.v. antipneumococcal agent) Cytotoxic (3 mg/mL) Efficacious in vivo (oral) Efficacious in vivo (s.c.) Progressed to Phase I (oral community RTI agent)
[24, 72] [83, 84]
P20 Proline
E. coli (75–100 mM), B. subtilis (6.4 mM) S. a. (32), S. pn. (32), H. i. (2), E. coli (64) S. a. (4–16), S. pn. (8), H. i. (0.25) S. a. (8)d, S. pn. (0.5)d, H. i. (32–64)d S. a. (1–2), S. pn. (1), H. i. (1–4) S. a. (1–4), S. pn. (8–32), H. i. (2–4) S. a. (0.5–1), S. pn. (1), H. i. (2–4) S. a. (2)d, S. pn. (1)d, H. i. (8)d, E. f. (4)d
[27] [27] [85] [87]
B. Non-peptidic inhibitors Class
Cpd.
IC50(nM)b
MIC (mg/mL)
Additional comments
Reference
Sulfonyl Sulfinyl Benzamide Bicyclic
(24) (25) (27) (29) (31) (38)
16 100 140 49 8400 0.33c
S. a. (128), S. pn. (128), H. i. (2) S. a. (16), S. pn. (32–64), H. i. (1) H. i. (2), E. coli (2–4) H. i. (16), M. catarrhalis (4) S. a. (10) S. a. (16), S. pn. (2–4), H. i. (0.5), E. coli (12)
No further data No further data Efficacious in vivo (oral) No further data No further data Selective versus MMPs
[32] [32] [105] [106] [107] [111]
Macrocyclic
K. AUBART AND M. ZALACAIN
Class
a
MIC r16 mg/mL against at least one organism. S.a. ¼ S. aureus, S. pn. ¼ S. pneumoniae, H. i. ¼ H. influenzae, E. f. ¼ E. faecalis. E. coli PDF. c K i value; time-dependent inhibition. d MIC90 data. b
119
120
PEPTIDE DEFORMYLASE INHIBITORS
O HO
O
Ph H N
R
SH
H2N
(1)
SH O
NH
O
n-Bu
H N
N H
(2) R = OMe (3) R = HN
NO2
H-phosphonates Similarly, Pei and co-workers [66] explored the feasibility of using an H-phosphonate head group on a peptide cassette (4) to inhibit PDF activity. While inhibitor (4) was not highly potent (K i ¼ 76 mM versus E. coli Zn2+-PDF, 37 mM versus E. coli Fe2+-PDF), it was successfully used to provide co-crystals bound in the active site of both Co2+- and Zn2+-E. coli PDF [58]. These structures reveal that the H-phosphonate binds to the metal in a monodentate fashion, adopting a tetrahedral coordination state similar to that of the native resting state of the enzyme. This is in contrast to later co-crystal structures obtained with more potent hydroxamic acid or reverse hydroxamate inhibitors, which bind to the metal in a bidentate fashion (vide infra). Presumably these bidentate inhibitors mimic the true transition state of the enzyme, in which the metal centre slips to a pentacoordinate geometry in order to activate the N-formyl carbonyl of the substrate [56, 67]. i-Pr
O
H N
NO2
N H
O
(4)
n-Bu
OH O P H O
i-Pr O PhCH2O
N H
O H N O
H
n-Bu
(5)
Aldehydes Another PDF inhibitor series derived from comparisons with known metalloprotease inhibitor classes has been reported by Merck [68]. Their study investigated a small set of peptide aldehyde inhibitors, postulating that the aldehyde might bind to the metal centre in the form of a hydrate,
K. AUBART AND M. ZALACAIN
121
thus serving as a transition state mimetic. Reportedly, N-CBz-Leunorleucinal (calpeptin) (5) is a low-micromolar inhibitor of E. coli and B. subtilis Co2+-PDF, although PDF inhibitory activity requires either methional or norleucinal side chains along with the aldehyde component. This suggests that recognition of a suitable moiety in the S10 pocket is mandatory for activity, supporting the conclusions drawn from Pei and Meinnel’s studies, discussed above. PSEUDOPEPTIDIC HYDROXAMIC ACIDS AND N-FORMYL-N-HYDROXYLAMINES
Actinonin Actinonin (6), a naturally occurring peptidic hydroxamic acid, has been known as a moderate antibacterial agent since the 1960s [69], but was not reported as an inhibitor of PDF until 2000 by Versicor [70] and, shortly thereafter, by Hoffman-La Roche [32], as a result of their respective screening efforts to identify PDF inhibitors. Since then, X-ray cocrystallisation studies have depicted actinonin bound into the active site of various PDFs, such as E. coli, S. aureus, B. stearothermophilus and P. aeruginosa [22, 24, 71]. Despite minor differences in the PDF active sites and secondary structures, actinonin adopts a similar binding conformation in all of the PDF enzymes for which co-crystallisation data are available. Interestingly, the conserved residues involved in binding interactions with actinonin are either concentrated in the S10 pocket, related to or needed for metal coordination, or involved in H-bonding to the amide bonds [22]. Considering that PDF must deformylate all nascent N-formylatedmethionyl peptides, it is not surprising that the main recognition elements within the active site correlate with areas required to identify the methionine and the amide linkage, and allow for diverse substrate substitution at P20 and P30 . O
i-Pr N
N H
O
H N
OH
n-Pent O
OH (6)
Actinonin
More recent detailed kinetic investigations have revealed that actinonin is a time-dependent, essentially irreversible, inhibitor of PDF enzymatic activity [72]. This study demonstrated that the kinetics of inhibition of
122
PEPTIDE DEFORMYLASE INHIBITORS
S. aureus PDF by actinonin is consistent with a two-step binding mechanism, in which an initial encounter complex (EI) undergoes a second step to form an extremely tight-binding final complex (EI*). Although the physical nature of this transformation remains uncertain, the potency of actinonin increases by more than 2,000-fold as it progresses from the EI complex (K i ¼ 530 nM) to the EI* complex (K i o0.23 nM). While the off-rate of actinonin from the EI* complex is very slow (t1/2 for dissociation Z0.77 days), an observable recovery in activity, combined with unchanged mass spectral data after incubation with the inhibitor and denaturation, demonstrates that actinonin is not covalently bound to PDF. Furthermore, X-ray co-crystal structure analysis shows no changes in the active site upon binding to actinonin [22, 24]. While it was unknown in the 1970s that actinonin’s antibacterial activity was due to inhibition of PDF, its potential as an antibacterial drug candidate was nevertheless investigated during that time [73]. However, it was never developed as a therapeutic agent due to its lack of in vivo efficacy in animal models of infection, likely the result of actinonin’s known instability [73, 74]. Another important disadvantage of actinonin as an antibacterial agent is its potential lack of selectivity against human secondary targets, as it inhibits other classes of metalloproteases [75–79]. Nevertheless, identification of actinonin as a potent inhibitor of PDF was a key discovery in the development of newer PDF inhibitors, as it revealed the potential for superior enzyme binding through the use of a hydroxamic acid moiety as a metal-chelator.
P20 t-Butyl containing inhibitors In 2001, British Biotech (now Vernalis) reported BB-3497 (7) as a potent PDF inhibitor, identified from screening efforts against a focused collection of metalloenzyme inhibitors [24]. This screen also re-identified actinonin as a potent PDF inhibitor. BB-3497 possesses moderate, but improved, antibacterial activity versus actinonin (Table 3.2A), and demonstrates superior pharmacokinetic (PK) properties. Whether the improved oral exposure of BB-3497 is due to replacement of the hydroxamic acid with an N-formyl-Nhydroxylamine, or the result of the P20 tert-butyl group shielding the neighbouring amide bonds from hydrolytic or enzymatic cleavage is unknown. Nevertheless, BB-3497 demonstrates sufficient exposure and activity to protect S. aureus-infected mice from mortality with a single dose ED50 (the amount of antibiotic necessary to protect 50% of the infected animals from death) of 8–14 mg/kg [24].
K. AUBART AND M. ZALACAIN
O
O
O
t-Bu Me2N
N H
N n-Bu
123
H
OH
(7) BB-3497
British Biotech has described co-crystal structures of both BB-3497 and actinonin bound in the active site of E. coli PDF [24]. The metal centre (Ni2+) in both complexes adopts a pentacoordinate geometry, bound by the two oxygen atoms of the hydroxamate along with Cys-90, His-132 and His-136. This coordination pattern is consistent with the mechanism of deformylation proposed by Becker et al. [56] and Jain et al. [67], in which a pentacoordinated metal centre stabilises the transition state during hydrolysis of the formamide bond. When compared to the co-crystal structure of a substrate hydrolysis product, Met-Ala-Ser, it is clear that the side chains of these two inhibitors bind into the active site pockets similarly to the substrate [56]. Follow-up SAR studies on BB-3497 investigated alternative metalbinding groups, modifications to the methylene a to the metal-binding group, and different P10 , P20 and P30 side chains. In addition to N-formyl-Nhydroxylamine and hydroxamic acid, 12 alternative metal-binding groups were studied, including thiols, hydroxyureas, phosphonates, N-acetyl hydroxylamines, hydrazides and amidoximes, all of which caused a severe loss of potency versus the enzyme [80]. The closest replacement reported, a carboxylate, was still 200-fold less active than the N-formyl-N-hydroxylamine or hydroxamate analogues. Likewise, alterations of the methylene spacer and the P10 and P20 side chains did not produce inhibitors superior to BB-3497. Cyclohexylmethyl, cyclopentylmethyl, n-pentyl, and isobutyl all provided comparable, but not improved, activity to that of n-butyl at P10 [81]. At P20 , a wider variety of substituents were reasonably well-tolerated for enzymatic inhibitory activity, including some polar side chains such as arginine and serine, but tert-butyl was still optimal for antibacterial activity [82]. P30 was the position most tolerant to modification, as most changes still resulted in activity versus the enzyme, with some groups (e.g. benzylpiperidine) providing improved Gram-positive activity over BB-3497 [82]. The most advanced inhibitor to evolve from this class, BB-83698 (8), displays good activity versus S. pneumoniae, with an MIC90 ¼ 0:5 mg=mL against 213 recent clinical isolates including penicillin-intermediate, penicillinresistant, erythromycin-resistant and multi-drug resistant strains, but possesses weaker activity against S. aureus (MIC90 ¼ 8 mg=mL, n ¼ 154) and H. influenzae (MIC90 ¼ 32264 mg=mL, n ¼ 110) [83]. BB-83698 is efficacious
124
PEPTIDE DEFORMYLASE INHIBITORS
in vivo and is under evaluation as a novel intravenous antipneumococcal agent [84] (see Clinical Trials section). t-Bu
N N O O
O
(8)
O
O N H
N
H
OH
BB-83698
P20 Proline-derived inhibitors As part of a collaboration focused on the identification of new PDF inhibitors, Novartis and Vicuron (formerly Versicor) used a combinatorial approach similar to that of the substrate- and mechanism-based libraries pursued by earlier academic researchers (vide supra). Relying to a certain extent on information obtained with actinonin, peptidomimetic libraries were constructed using different metal-chelating groups and a fixed P10 substituent [27]. Using hydroxamate, carboxylate or thiol as the metalbinding group and n-butyl as the P10 side chain, they combined 24 amino acid building blocks at P20 with 22 amines at P30 to produce three 528membered libraries. The carboxylate and thiol libraries lacked antibacterial activity, despite having some enzymatic inhibitory activity. While most of the hydroxamates possessed antibacterial activity, many also displayed undesirable cytotoxicity to mammalian cells. From the library screening results, these researchers concluded that a proline residue at P20 provided the best antibacterial activity with the least cytotoxic liability. Production of additional, more focused arrays confirmed the general preference for proline at P20 and identified compounds such as VRC3324 (9) and VRC3375 (10) [27]. VRC3324 affords potent antibacterial activity, but still displays toxicity to mammalian cells at comparable concentrations (Table 3.2A). VRC3375, while possessing reduced antibacterial activity versus S. pneumoniae, showed reduced mammalian cytotoxicity levels. Furthermore, (10) possesses 64% oral bioavailability in the mouse and demonstrates modest in vivo efficacy in a murine S. aureus septicaemia infection model, despite its relatively short half-life (15 min). This short half-life was shown to be due to hydrolysis of the hydroxamate in vivo, as a timedependent appearance of both the carboxylic acid and the amide derivatives were observed in serum samples after dosing with (10). Moving to a series of N-alkyl urea hydroxamic acids, for example, VRC4307 (11), improved
K. AUBART AND M. ZALACAIN
125
antibacterial activity, but poor oral bioavailability in the mouse was still observed [85]. Hydrolysis of the hydroxamic acid was again theorised to be responsible for the poor oral exposure, as the compound was rapidly metabolised in vitro by mouse and rat liver microsomes (t1/2o3 min), with the major metabolites proving to be modifications of the hydroxamic acid moiety. However, (11) was reported to be more stable in human liver microsomes, with a t1/2>19 min. i-Pr H N
N H
O
O
t-BuO
O
H N
n-Bu
O
H N
N
OH
n-Bu
O
OH
O
(10) VRC3375
(9) VRC3324
H N
N Me
S
O O N
H N
N
OH
O
Me
(11) VRC4307
Perhaps to eliminate the propensity of the hydroxamic acid moiety to hydrolysis, Vicuron and Novartis also explored several series of a-substituted hydroxamic acids as PDF inhibitors [86]. In this study, only hydroxy and fluoro-substituents were tolerated or slightly superior to hydrogen, with thiol, methoxy, amino and larger a-substituents unsuitable. Replacement of the a-methylene with a nitrogen to provide an N-hydroxyurea as the metalchelating group was also of no advantage, as these analogues lost enzymatic as well as antibacterial activity. The most advanced PDF inhibitor to emerge thus far from this collaboration is LBM-415 (12) (also called NVP PDF-713 or VIC-104959), an N-formyl-N-hydroxylamine compound still containing a proline residue at P20 . The activity, PK properties, and in vivo efficacy data of (12) were presented at the 14th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) (2004) and the structure of this
126
PEPTIDE DEFORMYLASE INHIBITORS
compound was later presented at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) (2004). Testing of (12) against a large number of recent clinical isolates (2,625) provided a S. aureus MIC90 ¼ 2 mg=mL (n ¼ 537), a S. pneumoniae MIC90 ¼ 1 mg=mL (n ¼ 504), an enterococci MIC90 ¼ 4 mg=mL (n ¼ 515), and a H. influenzae MIC90 ¼ 8 mg=mL (n ¼ 501) [87]. As expected for a novel antimicrobial agent, (12) is active against drug-resistant bacteria, including MRSA, VRE and multi-drug resistant S. pneumoniae [88, 89]. Compound (12) is a bacteriostatic agent against S. aureus and bactericidal against some strains of S. pneumoniae [90–93]. This compound demonstrates 62% oral bioavailability in mice, displays non-linear absorption in rats with oral bioavailability of 22–100% at doses ranging from 12 to 436 mg/kg [94] and entered Phase I clinical trials in October 2003 (See Clinical Trials section). O +
N
H N
O
N
N F
O
O
n-Bu
H
OH
(12) LBM-415
Additional P20 proline-containing PDF inhibitors have been reported in the patent literature by Dainippon and Questcor [95, 96]. The Dainippon examples disclosed contain an N-formyl-N-hydroxylamine group and possess good antibacterial activity against S. aureus, S. pneumoniae, Streptococcus pyogenes, Enterococcus faecium and M. catarrhalis [95]. The Questcor patent application describes various proline-containing hydroxamic acid inhibitors [96]. b-Aminohydroxamic acids In 1975, SAR studies involving actinonin investigated an analogue in which the orientation of the P10 –P20 amide bond was reversed (13), but the compound was found to lack antibacterial activity. Since then, however, descriptions of some b-aminohydroxamic acids and b-amino-N-formyl-Nhydroxylamines as PDF inhibitors have appeared in the patent literature. Patent applications from Senju [97] and De Novo [98] pharmaceuticals cover P10 –P20 amides (14), ureas (15, 16) and sulfonamides (17).
K. AUBART AND M. ZALACAIN
i-Pr
OH
n-Pent O
O
O
H N
H N
N
127
OH (13)
H N
H N O
n-Bu
H N
H N
OH O
O
Z
n-Bu
(15a) Z = N(OH)CH(=O) (15b) Z = C(=O)NH(OH)
(14)
O
N H
H N
H N
N
OH
O
n-Bu
O (16)
S O
H N
H N O n-Bu
OH
O
(17)
NON-PEPTIDIC TEMPLATES
Some examples of non-actinonin-based, non-peptidic PDF inhibitors have been described, which are structurally quite different from the inhibitors detailed in the above sections. Most were identified through high-throughput screening efforts.
128
PEPTIDE DEFORMYLASE INHIBITORS
Thyropropic acid analogues In 2000, Pfizer published a report of thyropropic acid derivatives as novel, non-peptidic inhibitors of E. coli PDF [99]. Screening of the historical Parke-Davis compound collection resulted in the identification of these novel carboxylic acid PDF inhibitors, as well as peptidic hydroxamic acids similar to actinonin. Thyropropic acid analogues such as (18) inhibit E. coli PDF in the 1–20 mM range, but generally lack antibacterial activity. SAR studies indicated that the carboxylic acid is required for anti-PDF activity, as ester and amide analogues are less potent by two orders of magnitude. Presumably, the carboxylic acid group binds to the enzyme’s metal centre. GlaxoSmithKline has published a patent application covering the hydroxamic acid (19) and N-formyl-N-hydroxylamine (20) analogues of a thyropropic acid core as PDF inhibitors [100, 101]. I O
HO
I
CH2COOH
(18) 2
2
R
R
X
X
OH
O
1
R
N H (19)
OH
H
N
1
R
(20)
O
Biaryl acids Screening efforts at Merck have identified structurally novel biaryl acid analogues as PDF inhibitors (21, 22, 23) [102]. These compounds contain several aromatic rings and belong to a broad class of angiotensin II receptor antagonists. The analogues reported possess inhibitory activity versus E. coli PDF in the 1–50 mM range, with substitution at various regions of the molecule appearing feasible. All the inhibitors contain an acidic pharmacophore, including tetrazole, carboxylate, acyl sulfonamide and carboxamide. Tetrazole and acyl sulfonamide appeared to provide the best potency, although the data set was small and the differences were not dramatic. The antibacterial activity of these compounds was not reported.
K. AUBART AND M. ZALACAIN
129
N n-Pr Cl
N
N
N
N NH
N
(21)
Me N Et Me
N
N
n-BuO O
HN
O
S
O
(22) Me
Me
Et
Me N
Ph Et
Me
N
N
O
HN S
O O S
(23)
Ph
b-Sulfonyl- and b-sulfinylhydroxamic acids After screening efforts identified actinonin as a potent PDF inhibitor, scientists at Hoffman-La Roche evaluated replacing the P10 –P20 amide bond
130
PEPTIDE DEFORMYLASE INHIBITORS
with sulfonyl (24) and sulfinyl groups (25) [32]. This study produced compounds with sub-micromolar activity, with the sulfonyl-containing compounds generally demonstrating better enzyme inhibitory activity. This trend was reversed with respect to antibacterial activity, as the sulfinyl compounds displayed more potent antibacterial activity in spite of their weaker enzymatic activity (Table 3.2B). However, the spectrum of antibacterial activity was mostly limited to H. influenzae, M. catarrhalis, C. pneumoniae and M. pneumoniae. The reported Gram-positive antibacterial activity was poor. (O )n Ph
H N
S n-Bu
(24) n = 2 (25) n = 1
OH
O
Benzamides and aryl ethers GlaxoSmithKline has reported a series of aryl ether (26) and benzamide (for example SB 660618 (27)) PDF inhibitors, which contain an N-formyl-Nhydroxylamine group as the metal-binding group [46, 103–105]. These inhibitors were designed using available structural information to explore the feasibility of preparing small molecular weight, non-peptidic compounds that would bind only into the well-conserved S10 pocket. Potent enzyme activity (o100 nM, some examples o10 nM) has been demonstrated within this series and the compounds were reported to show moderate antibacterial activity. Benzamide (27) possesses excellent oral bioavailability in the rat (100%) and is orally efficacious in vivo against H. influenzae in a rat respiratory tract infection model [105]. Cl Cl
O O
N
R
OH (26)
H N
H O
O N
H
OH
(27) SB 660618
Bicyclic hydroxamic acids Researchers at Novartis, Senju and Hoffman-La Roche have reported structurally related bicyclic hydroxamic acids as potent PDF inhibitors [106–108]. In 2001, screening efforts at Hoffman-LaRoche identified hydrazide (28)
K. AUBART AND M. ZALACAIN
131
as a 27 uM inhibitor of E. coli PDF, and this molecule was subsequently co-crystallised with E. coli Ni2+-PDF. Interestingly, the urea carbonyl of (28) was not involved in any hydrogen bonding interactions with the enzyme. In attempts to improve potency, hydroxamic acid analogues of both urea (29) and sulfonylurea (30) containing compounds were prepared. While these modifications greatly improved the enzyme inhibitory activity (500-fold), antibacterial activity was poor except against M. catarrhalis (MIC ¼ 124 mg=mL) [106]. Br
Cl H N
N N H
O
N
NH2
O
H N
N H
(28)
O
OH
O
(29)
Br H N
N N
S O
OH
S N
N H
n-Pr
c-Pr (30)
O
O O
OH
(31)
At Senju Pharmaceuticals, a series of benzothiazolylidenehydroxamic acids were designed and synthesised, e.g. (31) [107]. Low micromolar inhibitory activity was obtained with analogues containing a hydrophobic group (propyl, butyl or phenyl) as the N-substituent. These compounds are reported to show very moderate antibacterial activity against S. aureus (10–100 mg/mL) [107]. More recently, screening efforts at Novartis have identified a hydroxamic acid containing a benzothiazinone ring system (32) [108]. This inhibitor is very potent versus S. aureus Ni2+-PDF (o5 nM) and displays good selectivity versus matrix metalloprotease-2 (MMP-2) and MMP-13. Unfortunately (32), and all other analogues prepared, such as carbon isosteres (33), sulfones (34), N-substituted analogues (35) and N-formyl-N-hydroxylamines (36), lacked appreciable antibacterial activity in spite of their potent enzyme inhibitory activity. Further studies performed by Novartis suggest that these molecules are unable to penetrate the outer cell membrane of E. coli, and may bind to the cell membrane of S. aureus [108].
132
PEPTIDE DEFORMYLASE INHIBITORS
O H N
X
N
O
O
S OH (32) (33) (34) (35)
X = S, R = H X = C, R = H X = SO2, R = H X = S, R = n-Pent
N H
R
N O
H
OH
(36)
Isoxazoles Researchers at Combio and Arpida have reported a series of isoxazole-3hydroxamic acids as PDF inhibitors [109]. Molecular modelling studies predict that the aryl substituent of isoxazole (37) binds into the S10 pocket and that the oxygen atom of the isoxazole is involved in a H-bonding interaction with Ile-44 in E. coli PDF, similar to the P10 carbonyl of actinonin. None of the inhibitors reported has sub-micromolar inhibitory activity against E. coli or S. aureus PDF. Not surprisingly, these moderately active inhibitors also lack antibacterial activity. O N
Cl
H N
S
OH
O (37)
Macrocycles X-ray crystal structure analysis of inhibitors bound into the active site of PDF reveals that the P10 and P30 substitutents are oriented in a manner that places them spatially close to one another. Relying upon this observation, several groups have connected the P10 and P30 substituents to generate macrocyclic PDF inhibitor structures. Pei and coworkers [110–112] synthesised the corresponding macrocyclic versions of BB-3497, expecting to improve activity and stability with respect to the acyclic inhibitor. Direct comparison of the antibacterial activity of the most potent analogue, 15membered macrocycle (38), shows no obvious improvement over acyclic BB-3497 (7) (Table 3.2A, B) [111]. Interestingly, Pei reported a potential advantage in that macrocycle (38) showed time-dependent inhibition of PDF whereas he reported that BB-3497 was a linear, competitive inhibitor [111, 113]. Since then, however, it has been demonstrated that BB-3497 is, in
K. AUBART AND M. ZALACAIN
133
fact, a time-dependent PDF inhibitor and displays non-linear kinetics [72], so it is difficult to evaluate the advantages of macrocyclic structures based on enzyme binding properties. On the other hand, Pei’s comparison of MMP inhibition data and plasma stability data reveals a clear advantage of the macrocycles, as they demonstrate greatly reduced MMP inhibition and better stability in rat plasma than an acyclic counterpart [111].
O
O
O
t-Bu
H N
N
N H
i-Pr
Me N
H
N H
O
OH
O
H N
OH
O
(39)
(38)
In the patent literature, Aventis has published a patent application covering hydroxamic acid containing macrocycles (39) with the same general template as the N-formyl-N-hydroxylamine macrocycles described above [114] and GlaxoSmithKline has published an application on macrocyclic PDF inhibitors containing a hydrazide scaffold (40) [115]. No data has been published on these inhibitors to date. Me2N
H N
N N
O
O N H
N
H
OH
(40)
Sch 382582 and Sch 382583 Schering Plough has reported the discovery of the first non-hydroxamic acid containing natural product inhibitors of PDF. Sch 382582 (41) and Sch 382583 (42) were isolated from a fermentation broth of Streptomyces sp., and the proposed structures of these compounds were derived from a combination of two-dimensional NMR studies (NOESY, HMBC and HMQCTOCSY) and X-ray crystallography studies [116]. The proposed structure
134
PEPTIDE DEFORMYLASE INHIBITORS
and stereochemical by total synthesis tides inhibit PDF weak antibacterial (MIC ¼ 32 mg=mL)
assignments of Sch 382583 have since been confirmed [117]. These carboxylic acid-containing pseudopepactivity equally with K i s ¼ 60 nM, but display only activity against a super sensitive strain of E. coli [116].
O H N
R
O
i-Pr
O OH
N O
NH
(41) R=SMe Sch 382582 (42) R=H Sch 382583
i-Pr
Other patent activity Other patent reports covering PDF inhibitors from structural classes different from those discussed above include: hydrazides (43) [118], aryl-substituted pyrrolidines (44) [119], benzimidazoles (45, 46) [120–121], hydantoins (47) [122] and oxo-pyrrolidines (48) [123]. A prodrug approach utilising PDF has been published by Pei, and patents have been published by NewBiotics, but in this case the compounds of interest are used as substrates rather than inhibitors (49) [124–126].
H N
N N
N
N H
N
O
O
OH
O
H
H N
N
n-Pent OH
n-Bu
OH
O
CF3 (43)
t-Bu H N
(44)
N
O
O
NH
N
N H
N
H
Cl
(45)
O
H N
OH
N
O
OH (46)
H
K. AUBART AND M. ZALACAIN
O Ph O
O
O
N
N
H
Ph
O N
N
H
OH
N n-Pent OH H
n-Bu (48)
(47)
1
R Toxin
135
O O
R
2
O
O
N H
N H
H
MeS
(49)
CLINICAL CANDIDATES Two PDF inhibitors, BB-83698 (8) and LBM-415 (12), have been investigated in Phase I clinical trials, and these studies represent the most advanced status that any PDF inhibitor has attained. BB-83968 was investigated for intravenous administration, while LBM-415 was studied following oral administration. BB-83698
BB-83698 represents the first PDF inhibitor to enter clinical trials. This pseudopeptidic inhibitor shows potent activity versus S. pneumoniae, with an MIC90 ¼ 0:5 mg=mL, but moderate antibacterial potency against S. aureus (MIC90 ¼ 8 mg=mL) and little activity versus H. influenzae (MIC90 ¼ 32264 mg=mL) [83]. Subcutaneous efficacy of BB-83698 has been demonstrated in a murine model of acute pneumonia [127] and, using a murine thigh infection model, Craig and Andes have shown that AUC/MIC is the parameter that best correlates with in vivo efficacy [84]. In October 2002, studies were initiated to investigate BB-83698 as an antipneumococcal i.v. agent for hospitalised patients. The Phase I study explored i.v. administration of BB-83698 at single dose-escalating levels up to 475 mg and systemic exposures were reportedly linear in both animals and humans [84]. While dose-limiting CNS effects
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were observed in dogs, no significant adverse effects were noted in humans. Using an MIC of 0.25 mg/mL for S. pneumoniae, an AUC/MIC ratio of 184 was calculated from the 475 mg dose, which is predicted to be within the efficacious range by the PK/PD studies [84]. However, the compound was reportedly terminated (for undisclosed reasons) when British Biotech became Vernalis (Financial Times, September 30, 2003). Since then, the PDF portfolio has been transferred to Oscient Phamaceuticals, but it is unknown whether BB-83698 will ever be progressed to a Phase II study.
LBM-415
LBM-415 (12) possesses a broader spectrum of activity than BB-83698, with good potency against S. aureus (MIC90 ¼ 2 mg=mL), S. pneumoniae (MIC90 ¼ 1 mg=mL) and enterococci (MIC90 ¼ 4 mg=mL), and moderate activity against H. influenzae (MIC90 ¼ 8 mg=mL) [87]. This compound demonstrates good oral bioavailability in mice (62%) and rats (22–100%) [94], and as with BB-83698, pre-clinical studies by Craig and Andes [128] have shown that AUC/MIC is the PK/PD parameter that best correlates with in vivo efficacy. In October 2003, Phase I clinical trials were initiated in Europe to explore LBM-415 as a potential oral agent for community respiratory tract infections. At single oral doses ranging from 100 mg to 3000 mg, linear pharmacokinetics were observed and the mean terminal half-life (t1/2) ranged from 2 to 4.2 h [129]. Evaluation of food effects revealed that while the Tmax was delayed and the Cmax was lowered, there were no differences in overall systemic exposure. A multiple dose study (250, 500 and 1,000 mg twice daily for 11 days) showed no accumulation after 11 days, and the overall profile supported a twice-daily dosing regimen [129]. Although no adverse effects were noted up to the single 3 g oral dose [130], Novartis and Vicuron have since suspended development of this PDF inhibitor (www.vicuron.com; news release 4, March 2004). The reasons for termination were undisclosed. Vicuron has recently been acquired by Pfizer (June 2005).
SUMMARY AND PERSPECTIVES The continuous increase of antibiotic resistance, including the emergence of multidrug resistance in common pathogens, has created an urgent need for antibiotics with novel mechanisms of action. PDF, an essential metalloprotease that removes the N-formyl group of all newly synthesised bacterial
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polypeptides, is one of the best characterised and validated novel targets in the search for new agents to cover broad-spectrum community and hospital infections. Given the nature of the PDF substrate, it is not surprising to find that initial efforts focused on the design of peptide-like PDF inhibitors. This line of research was supported by the discovery that actinonin, a naturally occurring peptidic hydroxamic acid, inhibits PDF activity. Generally, these types of inhibitors show excellent potency against the enzyme and moderate antibacterial activity, but concerns persist regarding potential toxicity due to poor selectivity versus human metalloproteases. Also, hydroxamic acids are rather notorious for poor pharmacokinetic properties, although some pseudopeptidic hydroxamic acid PDF inhibitors containing a P20 proline residue have shown oral efficacy in animal models of infection. Some of the initially poor PK of the actinonin-like molecules seems to be resolved by the use of an N-formyl-N-hydroxylamine in place of a hydroxamate. Indeed, the two compounds that have entered clinical trials, BB-83698 (8) and LBM-415 (12), contain an N-formyl-N-hydroxylamine as the metal- chelator. The fact that these inhibitors have reached Phase I clinical trials is very encouraging, and although it seems they will not be further developed (possibly due to insufficient exposure), they showed no adverse effects in humans, providing an exciting result for this class of inhibitors. Thus far, less serious attention seems to have been focused on alternative templates, and only one non-peptidic PDF inhibitor, benzamide SB 660618 (27), has been reported to demonstrate in vivo efficacy in an animal model of infection. However, non-peptidic inhibitors may potentially be superior drug candidates, as incorporation of good PK properties and selectivity against MMPs may be easier to achieve. With the abundance of PDF structural information available, the design of additional classes of non-peptidic inhibitors with suitable developability characteristics is likely to continue. Nevertheless, it is still relatively early in the search for clinically relevant PDF inhibitors, and the fact that two compounds have already reached Phase I clinical trials highlights the rapid progress made since PDF was identified as a valid antibacterial target in the late 1990s. Based upon the encouraging results obtained thus far, we believe it is only a matter of time before a novel PDF inhibitor is approved for clinical use.
ACKNOWLEDGEMENTS The authors would like to extend special thanks to Dr. Siegfried B. Christensen for many helpful discussions and to Dr. Kathrine Smith for providing the figure of the PDF crystal structures.
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[103] Aubart, K.M., Christensen, S.B. and Briand, J. (2001) PCT Int. Appl. WO 01 085160; (2001) Chem. Abstr. 135, 352763. [104] Christensen, S.B., Cummings, M.D. and Karpinski, J.M. (2002) PCT Int. Appl. WO 02 098901; (2002) Chem. Abstr. 138, 11391. [105] Karpinski, J.M., Christensen, S.B., Aubart, K.M., Van Aller, G.S., DeMarsh, P.L., Lewandowski, T.F., Rittenhouse, S., Kulkarni, S.G., McIntyre, T.A., Woods, L.C., Lonetto, M.A., Pearson, S.C., Smith, K.J., Bhat, A., Cummings, M. and Saylers, K. (2005) 229th ACS National Meeting, San Diego, CA, USA, Abstracts of Papers. [106] Apfel, C., Banner, D.W., Bur, D., Dietz, M., Hubschwerlen, C., Locher, H., Marlin, F., Masciadri, R., Pirson, W. and Stalder, H. (2001) J. Med. Chem. 44, 1847–1852. [107] Takayama, W., Shirasaki, Y., Sakai, Y., Nakajima, E., Fujita, S., Sakamoto-Mizutani, K. and Inoue, J. (2003) Bioorg. Med. Chem. Lett. 13, 3273–3276. [108] Molteni, V., He, X., Nabakka, J., Yang, K., Kreusch, A., Gordon, P., Bursulaya, B., Warner, I., Shin, T., Biorac, T., Ryder, N.S., Goldberg, R., Doughty, J. and He, Y. (2004) Bioorg. Med. Chem. Lett. 14, 1477–1481. [109] Cali, P., Naerum, L., Mukhija, S. and Hjelmencrantz, A. (2004) Bioorg. Med. Chem. Lett. 14, 5997–6000. [110] Hu, X., Nguyen, K.T., Verlinde, C.L.M.J., Hol, W.G.J. and Pei, D. (2003) J. Med. Chem. 46, 3771–3774. [111] Hu, X., Nguyen, K.T., Jiang, V.C., Lofland, D., Moser, H.E. and Pei, D. (2004) J. Med. Chem. 47, 4941–4949. [112] Pei, D. (2005) US Pat. Appl. Publ. US 05 026821; (2005) Chem. Abstr. 142, 198353. [113] Nguyen, K.T., Hu, X. and Pei, D. (2004) Bioorg. Chem. 32, 178–191. [114] Capet, M., Genevois-Borella, A., Martin, J.P., Mikol, V., Carrez, C. and Wentzler, S. (2001) PCT Int. Appl. WO 01 040198; (2001) Chem. Abstr. 135, 33496. [115] Aubart, K.M., Benowitz, A.B., Christensen, S.B., Lee, J. and Silva, D.J. (2005) PCT Int. Appl. WO 05 017124; (2005) Chem. Abstr. 142, 235497. [116] Chu, M., Mierzwa, R., He, L., Xu, L., Gentile, F., Terracciano, J., Patel, M., Miesel, L., Bohanon, S., Kravec, C., Cramer, C., Fischman, T.O., Hruza, A., Ramanathan, L., Shipkova, P. and Chan, T.M. (2001) Tet. Lett. 42, 3549–3551. [117] Coats, R.A., Lee, S., Davis, K.A., Patel, K.M., Rhoads, E.K. and Howard, M.H. (2004) J. Org. Chem. 69, 1734–1737. [118] Aubart, K.M., Benowitz, A.B., Christensen, S.B., IV, Karpinski, J.M., Lee, J. and Silva, D.J. (2003) PCT Int. Appl. WO 03 101442; (2003) Chem. Abstr. 140, 41828. [119] Patel, D.V., Yuan, Z., Jain, R.K., Lewis, J.G. and Jacobs, J. (2002) PCT Int. Appl. WO 02 102791; (2002) Chem. Abstr. 138, 55864. [120] Beckett, P.R., Launchbury, S., Pain, G. and Pratt, L.M. (2002) PCT Int. Appl. WO 02 041886; (2002) Chem. Abstr. 136, 401760. [121] Karpinski, J.M., Aubart, K.M. and Christensen, S.B., IV. (2003) PCT Int. Appl. WO 03 104209; (2003) Chem. Abstr. 140, 42176. [122] Aubart, K.M., Bhat, A., Christensen, S.B., IV, Leber, J.D. and Liao, X. (2003) PCT Int. Appl. WO 03 077913; (2003) Chem. Abstr. 139, 255322. [123] Aubart, K.M., Xiang, J., Christensen, S.B., IV, Liao, X. and Cummings, M.D. (2002) PCT Int. Appl. WO 02 070540; (2002) Chem. Abstr. 137, 226582. [124] Wei, Y. and Pei, D. (2000) Bioorg. Med. Chem. Lett. 10, 1073–1076. [125] Sergeeva, M.V. and Doppalapudi, V.R. (2002) PCT Int. Appl. WO 02 089739; (2002) Chem. Abstr. 137, 358150. [126] Sergeeva, M.V. and Doppalapudi, V.R. (2003) PCT Int. Appl. WO 03 088913; (2003) Chem. Abstr. 139, 354460.
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Progress in Medicinal Chemistry – Vol. 44, Edited by F.D. King and G. Lawton r 2006 Elsevier B.V. All rights reserved.
4 Clinically Useful Vanilloid Receptor TRPV1 Antagonists: Just around the Corner (or too Early to Tell)? GIOVANNI APPENDINO1 and ARPAD SZALLASI2 1
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita` del Piemonte Orientale, 28100 Novara, Italy 2 Department of Pathology and Laboratory Medicine, Monmouth Medical Center, Long Branch, NJ 07740, USA
INTRODUCTION
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THE THEORY: CAN TRPV1 ANTAGONISTS FUNCTION AS CLINICALLY USEFUL DRUGS? (THE PROS AND THE CONS)
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TRPV1 ANTAGONISTS: AN OVERVIEW OF CHEMISTRY AND PHARMACOLOGY Iodinated Capsaicinoids and Resiniferonoids 4-Heteroarylpiperazine-1-Carboxyarylamides N-Arylcinnamides N-(Aza)naphthyl-N0 -aryl(benzyl)ureas N-Aryl-N0 -Alkylaminocarbonyl Ethylendiamines N, N0 -Dibenzylthioureas Miscellaneous Natural and Endogenous Products Miscellaneous Structures from the Proprietary Literature
151 152 154 158 160 161 162 163 164
POTENTIAL CLINICAL INDICATIONS FOR TRPV1 BLOCKADE Chronic, Intractable Pain (Neuropathic, Cancer, AIDS, etc.); Vulvodynia Faecal and Urinary Incontinence Inflammatory Bowel Disease and Motility Disorders Chronic Arthritis Acute Pancreatitis Human Hair Growth Control (Alopecia) and Dermatologic Disorders Antitussive Activity Immunoregulation
170 170 170 171 171 171 171 172 172
DOI: 10.1016/S0079-6468(05)44404-5
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CONCLUDING REMARKS: TRPV1 AGONISTS VERSUS ANTAGONISTS – WHICH WAY TO GO?
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REFERENCES
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INTRODUCTION A subset of sensory nerves (those with un-myelinated axons) is characterized by a unique sensitivity to capsaicin (1), the piquant ingredient in hot chilli peppers. The excitation of these nerves is followed by a lasting refractory state, traditionally referred to as desensitization, or, under certain conditions such as neonatal treatment, by gross neurotoxicity [1]. Capsaicin-sensitive nerves function as polymodal nociceptors (their activators include noxious heat, inflammatory ‘soup’ and pungent compounds such as capsaicin, piperine, zingerone, but not mechanical pressure) and convey pain into the central nervous system (CNS) [1, 2]. Moreover, these nerves are involved in various reflex responses (micturition and cough being prominent examples) as well as local neurogenic inflammatory and vasoregulator functions [2]. These latter functions are mediated by sensory neuropeptides, such as substance P (SP) and calcitonin gene-related peptide (CGRP), stored in and released from capsaicin-sensitive nerves [3]. Desensitization to capsaicin has a clear therapeutical potential. In fact, capsaicin-containing creams (e.g. Zostrix and Axsain) have been in clinical use for decades for indications such as diabetic neuropathy, postmastectomy pain syndrome and postherpetic neuralgia [1]. In 1990, specific binding of [3H]resiniferatoxin (2, RTX), an ultrapotent capsaicin analogue isolated from the latex of the cactus-like plant Euphorbia resinifera, provided the first direct proof for the existence of a distinct capsaicin receptor [4]. Based on the chemical motif (a vanillyl moiety) shared by (1) and (2), this receptor was termed the vanilloid receptor VR1 [1]. The molecular cloning of the rat capsaicin receptor [5], subsequently renamed as the transient receptor potential vanilloid receptor 1 (TRPV1) [6], has fuelled intensive research into its physiology and resulted in the discovery of small molecule TRPV1 antagonists [7, 8]. This breakthrough discovery was followed by the cloning and pharmacological characterization of the human [9, 10], guinea pig [11], rabbit [12], avian [13] and mouse [14] homologues of TRPV1. Studies with TRPV1-deficient animals confirmed the pivotal role that this receptor plays in the development of postinflammatory hyperalgesia [15, 16]. Now there is good evidence that TRPV1 expression is regulated in humans. As of 2005, diseases with up-regulated TRPV1 include inflammatory bowel
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disease (IBD) [17], irritable bowel syndrome [18], vulvar allodynia [19] and reflux oesophagitis [20], but this list is expected to grow in the foreseeable future. These findings lend further support to the notion that TRPV1 ligands could be clinically useful analgesic, anti-inflammatory drugs. At present, two conceptually different (but not mutually exclusive) therapeutic strategies are being pursued in the vanilloid field: one is to use optimized TRPV1 agonists to ‘desensitize’ (in practice, defunctionalize) capsaicin-sensitive nerves [1, 21] and the other is to employ small molecule antagonists for the pharmacological blockade of TRPV1 [7, 8]. The first approach is time-proven, but is riddled by known side-effects such as pain [1], as well as emerging concerns such as impaired control of cancerous growth [22]. The therapeutic potential of TRPV1 agonists has recently been subject to excellent and exhaustive reviews [21, 23, 24] and, consequently, will not be dealt within this paper. Instead, we focus on the proliferating number of small molecule TRPV1 antagonists. Although the therapeutic utility of TRPV1 antagonists was recently discussed both by us [7] and others [8], the rapid advances in this field necessitate frequent re-evaluation. Chronic neuropathic pain is debilitating to patients and its management is frustrating for physicians. The recent withdrawal of COX-2 inhibitors from the market has further emphasized the need for new classes of safe and effective analgesic, anti-inflammatory drugs. Whether TRPV1 antagonists can be such drugs, this is the question we attempt to answer here.
THE THEORY: CAN TRPV1 ANTAGONISTS FUNCTION AS CLINICALLY USEFUL DRUGS? (THE PROS AND THE CONS) TRPV1 functions as a non-selective cation channel with a limited selectivity for Ca2+ [5]. Interestingly, TRPV1 also causes intracellular acidification at neutral pH via a ‘proton-hopping permeation mechanism’ [25]. In a much simplified way, TRPV1 can be thought of as a membrane ‘sensor’ that is gated by heat. Noxious heat (>44 1C) opens the channel on its own power, whereas some ingredients in inflammatory ‘soup’ (e.g. acidification, nerve growth factor [NGF] and arachidonic acid metabolites such as 12-HPETE) act in concert to lower the heat activation threshold of TRPV1 to (below) body temperature [26]. This latter effect is mimicked by piquant agents like capsaicin (chilli pepper) [5] and piperine (black pepper) [27] that explains why we feel these spices as ‘hot’. Capsaicin acts as a ‘gating modifier’ that shifts activation curves towards physiological membrane potentials [28]. Surprisingly, ethanol is also able to activate TRPV1 [29, 30].
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In reality, the regulation of TRPV1 is mind-bogglingly complex. Under resting conditions, multiple mechanisms act in unison to keep TRPV1 in a closed (inactive) state. Possibly most important, TRPV1 is under the inhibitory control of phosphoinositol phosphate PIP2 [31]: hence, it can be liberated under this control by phospholipase C, an enzyme that cleaves PIP2, and which is coupled to such important pain receptors like bradykinin B2. The inhibitory control of PIP2 is facilitated by adenosine [32]. TRPV1 appears to be mostly sequestered in intracellular compartments where it exists in a homomeric complex, most likely as a tetramer [33]: in fact, a tetramerization domain is present in the C-terminus of TRPV1 [34]. Of note, in the mouse a splice variant of TRPV1 was recently identified that can act as a dominant negative modulator [35]. Moreover, TRPV1 is believed to be associated with cytoplasmic proteins that can further fine-tune its activity. An example of this phenomenon is b-tubulin for which TRPV1 carries a binding domain on its C-terminus [36]. Upon activation, neurons begin trafficking TRPV1 to the membrane where the receptors become activated, desensitized and then ‘recycled’ to the intracellular compartments. Translocation of TRPV1 to the cell membrane occurs via SNARE (snapin and synaptotagmin IX)-mediated exocytosis [37]. Broadly speaking, activation involves phosphorylation by protein kinases (most notably, protein kinase A [PKA] and C [PKC]) and desensitization involves de-phosphorylation by phosphatases (e.g. calcineurin) [38]. Among PKC isozymes, PKCm seems to be of particular importance [39]. These findings provide new insights into the mechanisms that underlie observations that have long puzzled scientists, such as the strikingly different structure–activity relations for vanilloid-induced Ca2+-uptake versus inhibition of [3H]RTX binding [1]. Recently, Blumberg and colleagues [40] identified no fewer than five parameters (potency, maximal response, latency of response, variability in latency and desensitization) in which TRPV1 agonists differ even in a simple in vitro assay like calcium response in CHO cells transfected with rTRPV1. For some ligands the binding site on TRPV1 is extracellular, whereas for others it is located on one of the intracellular loops. An intriguing example of the latter group is anandamide [38], an endogenous activator of the cannabinoid CB1 receptor. Consequently, the apparent activity of anandamide is also a function of a transporter that facilitates its transport to the intracellular milieu where anandamide can bind to TRPV1 [38]. As CB1 receptors are co-expressed with TRPV1 on some sensory neurons where they mediate opposite actions, anandamide can both excite (via TRPV1) and inhibit (through CB1) the very same nerves depending on its concentration and the state of the transporter [38]. At present, ‘endovanilloids’ [41] (i.e. endogenous ligands of TRPV1) include anandamide [41], N-arachidonyldopamine
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E600
149
E648
T704
S502
S800 T370 CaM
PIP2
S116 Capsaicin binding
pH sensitivity
Ankyrin domain
PKA, PKC, CaMKII
PKA PKA
PKA, CaMKII PKC
Fig. 4.1 Topological organization of the vanilloid receptor TRPV1. Highlighted are the molecular determinants of TRPV1 regulation, such as recognition (binding) domains for capsaicin and acids, and phosphorylation sites for protein kinases. Numbers designate the key amino acid residues deduced from the rTRPV1 primary sequence. Adapted from Ferrer-Montaniel, A. et al. (2004) Eur. J. Biochem. 271, 1820–1826.
(NADA) [42], N-oleoyldopamine (OLDA) [43] and 12-HPETE [44] (some also consider protons as endovanilloids). The mapping of pharmacologically ‘hot’ points in TRPV1 is in progress (Figure 4.1). For example, Thr-370 is the key residue that is phosphorylated by PKC (sensitization) and de-phosphorylated by calcineurin (desensitization) [45]. Other residues phosphorylated by PKC include Ser-502 and Ser800 [46]. High-affinity [3H]RTX binding has been linked to a single residue, Met-547, in the S4 membrane domain, which is believed to form a ‘binding pocket’ with Tyr-511 in the S3 domain [47]. In capsaicin-sensitive neurons, TRPV1 is co-expressed with various receptors involved in pain perception (e.g. bradykinin B2, purine P2X3). There is important ‘cross-talk’ among these receptors. For example, stimulation
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of B2 receptors recruits TRPV1 via activation of phospholipase C [48]. By contrast, activation of TRPV1 down-regulates voltage-gated calcium channels through Ca2+-dependent de-phosphorylation by calcineurin [49]. TRPV1 also plays a central role in intercellular pro-inflammatory feedback loops. An important example is mast cells and sensory nerves. Mast cells release tryptase that, in turn, activates the protease-activated receptor PAR-2; activation of PAR-2 then opens TRPV1 via PKC [50]. In keeping with this, PAR-2 agonists reduce the heat activation threshold of TRPV1 from 42 1C to below body temperature [51]. Excited nerve endings release SP that, as a positive feedback, binds to neurokinin NK1 receptors on mast cells. Mast cells also express TRPV1 [52]. Consequently, endovanilloids can act in concert to stimulate mast cells and activate capsaicin-sensitive nerve endings. Of relevance is the finding that PAR-2 is up-regulated in the bladder during experimental cystitis [53]. PKC is central to TRPV1 regulation, inasmuch that it couples an array of receptors to TRPV1. Some examples have been discussed above. Other notable examples include the chemokine receptor CCR1 [54], the metabotropic purine receptor P2Y [55] and the prostaglandin receptors EP1 and IP [56]. PKA and PKC are, however, not the only kinases to regulate TRPV1. The Ca2+/calmodulin-dependent kinase II (CaMKII) sensitizes TRPV1 by phosphorylation [57, 58], as does phophatidylinositol 3-kinase (PI3 K) via its downstream target AKT [59]. This latter finding links TRPV1 to the ERK (extracellular signal-regulated protein kinase) pathway. The non-receptor tyrosine kinase Src likewise potentiates capsaicin-induced currents [60]. Given the central place of TRPV1 in pro-inflammatory and algesic vicious circles, it is a reasonable assumption that blockade by antagonists of TRPV1 should disrupt these circles. In keeping with this, animals whose TRPV1 had been disrupted ( / ) were shown to display reduced inflammatory hyperalgesia [15, 16]. Moreover, agents (e.g. capsaicin and RTX) that silence (desensitize) TRPV1 are powerful analgesic-antiphlogistic drugs in experimental animals [1]. Other findings, however, necessitate caution when extrapolating these results to humans. TRPV1 is ‘a’, and not ‘the’, heat and proton receptor and the relative contribution of TRPV1 versus other relevant heat and/or proton receptors can be very different in animal species and man. Consequently, unlike TRPV1 agonists that have proven clinical value by ‘defunctionalizing’ human sensory nerves, in humans TRPV1 antagonists may be only minimally effective on their own merit. Another caveat is our incomplete understanding of nociception. For example, most recently a close relative of TRPV1, TRPV4, was demonstrated to play an important role in inflammationinduced thermal hyperalgesia [61]. Moreover, a functional human
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TRPV1 splice variant (termed hTRPV1b) was identified that is activated by heat, but not by vanilloids or protons [62]. TRPV1 ANTAGONISTS: AN OVERVIEW OF CHEMISTRY AND PHARMACOLOGY Natural products have provided a host of lead structures to inspire the synthesis of vanilloid ligands. The first vanilloid ligands were TRPV1 agonists, analogues of the natural products capsaicin (a vanillamide, 1) and RTX (a homovanillyl ester, 2a), and were accordingly classified as capsaicinoids and resiniferonoids [1, 2, 63, 64]. As resiniferonol, the diterpene skeleton of RTX, is expensive and difficult to work with, phorbol was also introduced as a diterpene moiety on which to build phorboid vanilloids [1, 63]. The capsaicinoid, resiniferanoid and phorboid chemotypes are structurally well characterized and can accommodate many of the countless structural variations that natural products have inspired; however, RTX remains the most potent vanilloid described to date. By contrast, most TRPV1 antagonists emerged from the random screening of chemical libraries. Consequently, they show little, if any, resemblance to capsaicin and/or RTX, and their structural heterogeneity makes it impossible to define a common antagonist chemotype. Indeed, given the bewildering structural diversity of TRPV1 antagonists, it is reasonable to assume that these compounds are also heterogeneous in the spectrum of pharmacological actions: some may block most TRPV1 agonists, whereas others may selectively antagonize certain agonists. The first vanilloid receptor antagonist was Ruthenium Red (RR), an inorganic dye [1, 2]. Commercial RR is a mixture of compounds [65] and it remains to be understood (a) exactly what the active ingredient is and (b) how it antagonizes TRPV1. Nevertheless, RR is clearly a non-competitive antagonist that blocks the channel pore [2] with no effect on [3H]RTX binding [4]. RR was useful in dissecting responses mediated by capsaicin-sensitive nerves in vitro, but its use in animals was severely restricted by the toxicity (e.g. convulsive activity) that it caused [1]. The first competitive TRPV1 antagonist is capsazepine (3), a conformationally constrained O-demethyl thiourea analogue of capsaicin that emerged from a programme aimed at the optimization of the agonistic properties of capsaicinoids [66]. The second competitive antagonist to be
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discovered was 5-iodoRTX (2b) and is even more closely related to its natural product lead [67]. Almost a decade elapsed between the discovery of capsazepine and 5-iodoRTX, but the renewed interest in TRPV1 antagonists by the pharmaceutical industry has since greatly accelerated the rate at which novel antagonists are discovered. In the next sections, the pharmacodynamic and pharmacokinetics properties of the most important TRPV1 antagonists will be briefly discussed, focusing on those whose pharmacology has already been characterized. Me
O N H
Me
iPr
H
O
Ph
H
HO
Me
OMe
O
(1) O HO Cl
S HO
O O
N
O 2
R N H
1
R
HO
(3)
(2a) R1 = R2 = H (2b) R1 = I; R2 = H (2c) R1 = H; R2 = I
OMe OH
IODINATED CAPSAICINOIDS AND RESINIFERONOIDS
The recognition that the introduction of an iodine atom in the (homo) vanillic moiety of RTX (2a) [67] and nonivamide (4a) [68] reverses the agonistic activity of these TRPV1 ligands is a remarkable example of how small structural changes can modulate the activity of a lead structure. The molecular basis of this dramatic ‘iodine effect’ is unknown, but other examples of vanilloid activity swap induced by changes of aromatic substitution have been reported. For instance, replacement of the methoxyl group by a fluorine atom in thiourea mimics of RTX could convert an agonist (5a) into an antagonist (5b) [69]. Furthermore, in a series of AMG 9810-related cinnamides, the vanillyl substitution pattern (3-methoxy-4hydroxyphenyl) (6a) was associated with agonism, and the isovanillyl pattern (3-hydroxy-4-methoxyphenyl) (6b) with antagonism [70]. Also worth mentioning is the observation that benzo-homologation of the 3-pyridylbenzamide (7) to the 5-isoquinolylbenzamide (8) resulted in a similar antagonist switch [71].
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Me 3
R
O
Me
2
R
N H
n-C8H17
NHSO2Me H N
1
R
HO
t- Bu
O
H N
R
OMe O
(4a) (4b) (4c) (4d)
S
R 1 = R2 = R3 = H R1 = R2 = H; R3 = I R1 = R3 = H; R2 = I R1 = I; R2 = R3 = H;
(5a) R = OMe (agonist) (5b) R = F (antagonist)
n-C5H11 H N
t-Bu H N N
O
(7) agonist
O
2
R
1
R
(6a) R1 = OMe; R2 = OH agonist (6b) R1 = OH; R2 = OMe antagonist
n-C5H11
N H N O
(8) antagonist
In nonivamide and RTX derivatives, the antagonist activity depends on the location of iodine, with antagonism being maximal at C-50 in RTX (2b) and at C-60 in nonivamide (4b). Complementary iodination at C-50 in nonivamide (4c) could still reverse activity [68], but iodination of RTX at C-60 (2c) yielded only a partial agonist [72]. Detailed structure–activity studies on the effect of aromatic substitution were carried out with nonivamide (4a), a compound structurally simpler and more easily available than RTX [73]. While iodination at C-2’ (4d) led to the loss of affinity for TRPV1, iodine was better than bromine or chlorine for the reversal of activity, both at C-50 and at C-60 . Within C-60 substituted nonivamides, iodine was also better than alkyl (alkenyl, alkynyl) groups for the reversal of activity. Finally, comparison of potency among a series of agonist capsaicinoids and their corresponding 60 -iodinated antagonists showed a poor correlation. For example, 60 -iodophenylacetylrinvanil (PhAR, 9b) showed very potent antagonist activity (IC50 ¼ 6 nM), in keeping with the agonist ultrapotency of phenylacetylrinvanyl (PhAR, 9a) [74]. In contrast, olvanil (10a) and arvanil (11a), both more potent agonists than nonivamide [75], yielded 60 -iododerivatives (compounds 10b and 11b, respectively) less potent as antagonists than 60 -iodononivamide, and retvanil
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(12a), another potent vanilloid agonist [75] gave a virtually inactive 60 iododerivative (12b) [73]. Taken together, these observations imply that capsaicinoids and their 60 -iododerivatives bind to TRPV1 in a substantially different manner, although molecular details still await clarification. H N
(C7H14) O
R
OMe OH
H N
n-C6H13 O
(9a) R = H (9b) R = I
R
O
(C7H14)
(C8H17)
O
OMe
Ph OH
(10a) R = H (10b) R = I
Me H N 4
R
OMe OH
H N
n-C5H11
O
O
R
(11a) R = H (11b) R = I
Me
OMe OH
Me
Me
Me
(12a) R = H (12b) R = I
Iodinated capsaicinoids are easily available by synthesis [68, 73] and can function as potent TRPV1 antagonists in in vitro functional assays. On the other hand, little is known about their activity in vivo, or their metabolic stability and bioavailability. Since capsaicin itself is poorly absorbed after oral ingestion or from skin, and then is rapidly metabolized [1], these issues are of great relevance for further pharmaceutical development. 4-HETEROARYLPIPERAZINE-1-CARBOXYARYLAMIDES
Owing to a curious overlap of interest in different laboratories, this class of vanilloid antagonists is better investigated than others with regards to both structure–activity relationships and in vivo profile of activities. BCTC The discovery that the 4-heteroarylpiperazine-1-carboxyanilide template can generate potent TRPV1 antagonists was independently reported by three groups [76–79]. Most of the published work on this class of compounds was carried out by Purdue Pharma, and is focused on the optimization of (13a), a lead compound that had emerged from random screening of a chemical
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library. Compound (13a) was reported to inhibit rTRPV1 activation by capsaicin and acids with IC50 values of 58 nM and 39 nM, respectively; unfortunately, it was not orally bioavailable in rats [79]. Since the modular structure of (13a) is easily amenable to parallel synthesis, extensive structure–activity studies were carried out with this compound. This generated not only a large body of structure–activity data, but also led to a remarkable optimization of the metabolic and pharmacokinetic properties of (13a).
HN N
N
1
R
N
HN N
N N
(13a) R1 = iPr; R2 = CF3 (13b) R1 = t-Bu; R2 = Cl (13c) R1 = NO2; R2 = CF3
Me
O Me
2
R
O
N
Cl N
S
HN N
N
N N
N X
N
(14) F
R
O
CF3
(15a) R = CF3; X = N (15b) R = H; X = CH (15c) R = Me; X = CH
From these studies, BCTC (13b, N-(4-tertbutylphenyl)-4-(3-chloropyridin2-yl)tetrahydropyrazine-1-(2 H)-carboxamide) emerged as a first generation lead [79]. BCTC differs from (13a) in the substitution pattern of the aryl and heteroaryl moieties, and was more potent than (13a) in inhibiting rTRPV1 activation by both acids (IC50 ¼ 4.8 nM versus 39 nM) and capsaicin (IC50 ¼ 35 nM versus 58 nM). Importantly, BCTC also showed a certain degree of oral bioavailability. At 30 mg/kg p.o., BCTC was active in rat models of acute inflammatory and neuropathic pain [80] with significant penetration into the CNS [81]. BCTC also prevented rTRPV1 activation by acids, and, when compared to capsazepine, it showed a better selectivity profile against a panel of molecular targets (ion channels, receptors, enzymes, transporters) of clinical relevance. On the other hand, further pre-clinical investigations highlighted significant shortcomings. Thus, BCTC showed poor aqueous solubility, limited metabolic stability and complex pharmacokinetics. Even worse, BCTC potently inhibited hERG (human ether-a-go-go related gene) channels, with potentially harmful cardiovascular effects such as prolongation of the cardiac QT interval, ventricular arrhythmias and fibrillation [82]. (Parenthetically, capsaicin also interacts at erg receptors in the rat glioma cell line NG108-15 [83], and it is tempting to speculate that a similar interaction at heart erg receptors may be at least partially responsible for the wellknown cardiotoxic actions of high-dose capsaicin administration [1]).
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CLINICALLY USEFUL VANILLOID RECEPTOR TRPV1 ANTAGONISTS
Subsequently, a second series of analogues was prepared, based on the observation that the introduction of an alkyl group into the piperazine moiety can lead to a marked increase in potency. These changes are sufficient to compensate for the aryl and heteroaryl moieties that are per se neutral or detrimental for TRPV1 inhibition and are also beneficial in terms of overall pharmaceutical profile [82]. By coupling changes on the aryl and heteroaryl moieties (6-fluoro-2-benzothiazolyl in place of the 4-tertbutylphenyl; and 4-methyl-6-chloro-2-pyridazinyl in place of the 3-chloro-2pyridyl) with the introduction of a methyl group in R-configuration on the piperazinyl moiety, the second generation lead (14) was discovered. This compound was less potent than BCTC in functional assays of hTRPV1 inhibition (EC50 ¼ 103 nM versus 34 nM for capsaicin stimulation, and 226 nM versus 30 nM for acid stimulation), but had a decreased affinity for hERG, a much longer half-life, and an improved aqueous solubility and oral bioavailability. Compared to other classes of vanilloid antagonists, the 4-heteroarylpiperazine-1-carboxamides are characterized by the presence of a mildly basic nitrogenous polar head. Based on the published studies [79, 82], the structure–activity relationships of this class of vanilloid antagonists can be summarized as follows: 1. The TRPV1 inhibiting potency depends on the nature of (a) the piperazine N-1 group (carbonyl better than thiocarbonyl), (b) the substituent at the aniline p-position (branched alkyl groups are better than the linear ones; polar groups are detrimental for activity), (c) the substituent at the N-4 heteroaryl group, with small, relatively non-polar substituents such as chlorine being optimal in the pyridyl series and the 4,6-disubstitution being critical for activity in the piperazinyl series and (d) the presence of an alkyl substituent on the piperazine 2-position, with a 2R methyl being optimal for activity. 2. The hERG inhibiting activity depends on the nature and substitution pattern of the piperazinyl 4-substituent, with the pyridazinyl group being significantly less potent than the pyridyl group, but also being associated with an overall better metabolic stability. 3. The nature of the carboxamide aryl substituent has a dramatic effect on the pharmacokinetic properties, with the 2-benzothiazolyl group leading to a longer half-life compared to a phenyl group. The discovery of the clinical drug candidate (14) highlights the challenges that face the development of TRPV1 inhibitors (Table 4.1). The general
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Table 4.1 FUNCTIONAL POTENCIES OF TRPV1 INHIBITORS Compound
IC50 (nM)
TRPV1
Reference
I-RTX (2b) Capsazepine (3) (4b) (5b) I-PhAR (9b) BCTC (13b) (15) AMG9810 (16) (17b) (20) (22b)
0.4 56 10 8 6 35 65 17 0.4 4 3
Human Human Human Rat Human Rat Human Human Human Human Human
[73] [73] [73] [69] [74] [79] [78] [70] [70] [85] [71]
lipophilic nature of these compounds translates into a complex pharmacokinetic profile, compounded by a combination of erratic oral bioavailability and overall poor water solubility. Potent compounds (EC50 in the range of 10 to 200 nM) could be obtained relatively easily in this class, but pre-clinical optimization requires a delicate balance of potency, pharmacokinetics and target specificity. Similar considerations might well apply to other classes of TRPV1 antagonists. (At present, it is unclear if interaction at hERG plagues also other types of TRPV1 antagonists, but the reported inhibition of erg by both capsaicin and arvanil [83] implies that this might be the case.) Johnson & Johnson piperazine carboxamides The piperazine-1-carboxamide (13c) [77, 78] exemplifies one of the major problems plaguing synthetic libraries, namely the overall low chemical diversity. Indeed, compound (13c) is very closely related to the Purdue Pharma hit (13a). In contrast to the finding that polar p-substituents at the anilyl moiety are detrimental for the activity of (13a) [79], (13c) showed potent inhibitory activity (EC50 ¼ 74 nM) against capsaicin-induced activation of hTRPV1 [77, 78]. The published structure–activity relationships for (13c) nicely complement those for (13a), especially with regard to the diamine core. Thus, the cyclic 1,3-diamine motif was essential for activity, with ring expansion, conformational constraint as well as extrusion of one nuclear nitrogen to attain a 3-aminopyrrolidine or a 4-aminopiperidine core, being all detrimental for activity [78]. The biological profile of the optimized lead structure emerging from these studies, compound (15a), was extensively investigated both in vitro and in vivo. The in vitro pattern of activity was in general excellent inasmuch
158
CLINICALLY USEFUL VANILLOID RECEPTOR TRPV1 ANTAGONISTS
as (15a) could inhibit TRPV1 activation by a variety of stimuli, including reactive oxygen species and phorbol myristoyl acetate (PMA)-induced phosphorylation. Also, oral bioavailability and metabolic stability were acceptable. On the other hand, some intriguing observations were made in a series of animal experiments. For example, (15a) could potently and completely inhibit the inflammatory and painful responses induced by capsaicin. By contrast, it could only partially reverse capsaicin-induced hypothermia [78]. These observations suggest that the blockage by TRPV1 antagonists might be biological end-point specific. Importantly, (15a) also caused mild hyperthermia, a side-effect that might well arise with other TRPV1-antagonists, and that presumably stems from the inactivation of a constitutionally active endogenous vanilloid pathway. This model is at variance with knock-out (k.o.) TRPV1 mice that have normal temperature. Animals born without a certain receptor, however, frequently adapt by developing alternative pathways. Clearly, these observations are worth further investigation for example by using conditional TRPV1 k.o. animals. Another interesting finding was that two structurally close analogues of (15a), namely compounds (15b) and (15c), showed marked species-related differences in activity: they behaved as weak antagonists towards hTRPV1, and weak agonists towards rTRPV1 [78]. This observation indicates that great caution should be taken when extrapolating TRPV1 actions from animal models to humans!
N-ARYLCINNAMIDES
AMG 9810 The cinnamide (16, AMG-9810) was identified as a potent TRPV1 antagonist by random screening of a synthetic, proprietary library [70]. (16) was reported to block hTRPV1 activated by various agonists (e.g. capsaicin, acids, heat and endovanilloids), with EC50 values in the hundred nM range, and showed excellent selectivity when assayed against a panel of over 80 targets of pharmaceutical relevance. Given i.p., (16) showed potent activity in rat models of inflammatory injury as well as thermal, and mechanical hyperalgesia. On the other hand (and just like so many others TRPV1 antagonists identified by random screening), (16) showed poor oral absorption and metabolic stability in the rat. To counter these shortcomings, a detailed structure–activity study was undertaken. The lead structure was divided into three sections (benzodioxan-2-yl system, acrylamide core, aryl group) that were independently optimized. Again, the structural simplicity
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of the hit compound made it possible to generate a large body of structure–activity information. While the acrylamide core could not be modified without detrimental effects on the activity, changes in the two aryl moieties were adequately tolerated. The 7-quinolinyl group was associated with a significant increase in potency, especially in the pH stimulation assay, and the 2-(4-morpholino)-6-(trifluoromethyl)pyridin-3-yl and 2-(piperidin1-yl)-6-(trifluoromethyl)pyrimidin-3-yl systems proved acceptable replacement for the 4-tert-butylphenyl group in the lead structure. These changes were next combined to generate the analogues (17a) and (17b) that outperformed (16) both in the capsaicin- (IC50 ¼ 2 and 0.4 nM, respectively, versus 17 nM) and the pH-induced assays (IC50 ¼ 1.3 and 1.0 nM, respectively, versus 15 nM). The pharmacokinetic profile of (16) and its two analogues were investigated in Sprague-Dawley rats. Removal of the metabolically labile tertbutyl group on the aryl moiety slowed metabolism and the rate of clearance. However, the overall half-life of (17a) was unaffected because of a lower volume of distribution. On the other hand, (17b) showed an increased halflife (ca. 3 h versus 1 h) compared to (16) and (17a). While the oral bioavailability of (16) was negligible, (17a) and (17b) were better absorbed, with bioavailability values of 39% and 17%, respectively. While undoubtedly improved in terms of pharmacokinetics compared to (16), the bioactivity of (17a) and (17b) awaits validation in vivo. X
O
O
O O
N H t-Bu
N
N H
(16) O MeO
(17a) X = O (17b) X = CH2
N N CF3
N H
(18)
Cl
SB-366791 SB-366791 (18) emerged from screening an in-house library as a potent competitive inhibitor of both hTRPV1 and rTRPV1, endowed with superior target selectivity compared to capsazepine [84]. Structure–activity relationships of SB-366791 remain to be reported.
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CLINICALLY USEFUL VANILLOID RECEPTOR TRPV1 ANTAGONISTS N-(AZA)NAPHTHYL-N0 -ARYL(BENZYL)UREAS
Abbott lead The 7-hydroxynaphthalenyl urea (19) was identified by high-throughput screening (HTS) of a chemical library as a potent (EC50 ¼ 22 nM) inhibitor of hTRPV1 [85]. This compound, however, was inactive in animal models of inflammatory pain, and was not orally available [85]. A structure–activity study was therefore undertaken, capitalizing on the assumption that the phenolic hydroxyl, though critical for activity, was also providing a potential site for metabolism, and should therefore be replaced with heterocyclic fragments having a similar charge distribution. Eight hetero(bi)cyclic aromatics were evaluated as 7-hydroxynaphthyl-equivalents, with the 5isoquinolyl giving the most active analogue. Furthermore, the replacement of the N0 -aryl group with an N0 -benzyl group enhanced activity, and the pCF3 group was discovered to be an excellent replacement for the m-methylthio substituent, apparently another site of metabolic lability. Compound (20) eventually emerged as an optimized lead (EC50 ¼ 4 nM) with satisfactory pharmacokinetic and solubility properties as well as potent activity in animal models of visceral and chronic inflammatory pain [85]. O
O HN
n-C5H11
O 1
N H
HN
SMe
N H
R
HN
N H
2
HO
R N
(19)
N
(20) R1 = H; R2 = CF3 (22a) R1 = CF3; R2 = Cl (22b) R1 = H; R2 = t-Bu
(21)
Johnson & Johnson leads The benzamide (8) and urea (21) emerged as hTRPV1 antagonists from efforts aimed at the optimization of the agonist activity of the pyridin-2ylcarboxamide (7) [71]. Compounds (8) and (21) showed submicromolar affinity for TRPV1 (as measured by displacement of [3H]RTX from hTRPV1), but only modest antagonist potency (IC50 ¼ 24 mM for 8 and 0.49 mM for 21). Therefore, structural modification was pursued using both affinity – (displacement of RTX binding) and functional assays (antagonism of capsaicinevoked Ca2+ influx). The correlation between the Ki and the IC50 values suggested a common binding site for these compounds and the agonists.
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Similar to capsaicin, (8) and (21) showed a recognizable polar head, a linking group and a lipophilic tail, and these regions were independently explored. Not unexpectedly, the results showed that the proper positioning of the heterocyclic moiety and the aryl moiety containing the ‘cannabinoidlike’ n-pentyl chain were critical for activity, while N-methylation of the amide and urea nitrogens was detrimental. The n-pentyl chain of the distal urea (benzamide) end could be replaced by a CF3 group, but not by polar groups or hydrogen, in accordance with the critical role of added lipophilicity. Based on these observations, compounds (22a) and (22b) endowed with subnanomolar binding affinity (0.8 and 0.5 nM, respectively) and low nanomolar functional activity were eventually discovered. It is interesting to note that the final lead structure (22a) showed a vanillyl-type ‘lipophilic’ tail, blurring the distinction between the hydrophilic and the lipophilic endings of the hit structure, and leading to an overall similarity to capsaicin [71]. N-ARYL-N0 -ALKYLAMINOCARBONYL ETHYLENDIAMINES
The hit, SB-452533, (23a) was discovered by HTS of an in-house library at GSK [86]. Compound (23a) could potently and reversibly inhibit activation of TRPV1 by capsaicin, low pH and heat. Quaternization of (23a) by N-methylation afforded (24), unlikely to cross the cell membranes, but that could nevertheless retain a certain degree of inhibitory activity on TRPV1 activation. When (24) was applied intracellularly, no activity could be detected. The binding of capsaicin to TRPV1 has traditionally been considered intracellular, but this and other observations actually suggest a more complex situation. When assayed in HEK293 cells transfected with the cloned human, rat and guinea pig TRPV1, (23a) showed similar potencies. Not unexpectedly, (23a) showed poor metabolic stability and a structure–activity study to optimize potency and drug-like properties was initiated. Modification on the left-handed N-aryl section showed that: 1. the order of potency was 2-Br> 3-Br> 4-Br and 2. the 2-Br group could be replaced by a 2,3- and a 2,5-dichloro substituent. In the right-hand section, N-ethylation of the aniline nitrogen was optimal, while shifting the methyl to the 4-position increased activity. This, combined with fluorination at the m-position, afforded (23b) as the most potent compound of this series. Since the pharmacokinetic properties of (23b) were not disclosed, it is not clear if manipulation of the hit compound led, apart from an increase in potency, to a corresponding improvement of the pharmacological profile.
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CLINICALLY USEFUL VANILLOID RECEPTOR TRPV1 ANTAGONISTS 1
Me
R
2
R
Br
H N
H N
Br
H N
Me
H N
N+
N Et
O
Et
O
(24)
(23a) R1 = Me; R2 = H (23b) R1 = F; R2 = Me
N, N0 -DIBENZYLTHIOUREAS
The N-3-acyloxy-2-benzylpropyl-N0 -benzyl thiourea template (25) emerged as the first structurally simplified RTX-mimic, with the N0 -benzyl moiety corresponding to the vanillyl moiety of RTX, the thiourea linker to the C-20 ester group, and the 3-acyloxy- and the 2-benzyl moieties to the C-3 keto group and the phenyl ring of the ortho-phenylacetate sites of the natural product [69]. Remarkably, isosteric substitution of the phenolic hydroxyl with a sulfonamide group coupled to replacement of the 3-methoxyl with a fluorine atom switched the vanilloid profile from agonism to antagonism [87, 88]: while the 3-fluoro-4-methoxy derivative (5a) was a potent agonist (EC50 ¼ 22 nM), replacement of the methoxyl with a fluorine atom generated the potent antagonist (5b) (IC50 ¼ 8 nM) [69]. These considerations yielded the potent antagonist IBTU (26). C1
A
B R
I OH t-Bu
O O C2
H N
H N
OMe
Cl
OH H N
H N
S
S
(25)
(26)
OMe
The structure–activity relationships of this class of RTX-mimics were investigated in detail [87, 88], establishing that: (a) The thiourea linker was better than its amine or urea versions. (b) The substitution pattern of the benzylamine A region determines the agonist/antagonist activity, with a 3-methoxy and a 4-sulfonamido groups favouring agonism and antagonism, respectively.
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(c) N-hydroxylation of the left-handed urea nitrogen apparently mimics the 4-hydroxyl of RTX, but its relevance for in vivo activity was not clear. (d) The spatial relationship between the critical A, B, C1 and C2 regions is optimal as expressed in the original template and its modification was detrimental for activity. (e) Lipophilic appendices are necessary at the C1 and C2 regions to confer potency. In these investigations, compounds were assayed for binding affinity and antagonist activity, and a generally poor correlation was found [89]. This is hardly surprising as a similarly poor correlation was described previously for TRPV1 agonists [1]. Apparently, [3H]RTX binding and Ca2+-uptake detect two distinct populations of TRPV1: it is believed that binding detects mostly inactive receptors sequestered in intracellular compartments whereas the Ca2+-uptake response reflects activated TRPV1, transported into the membrane. As these receptor populations are in different subcellular milieu, their pharmacology can be distinct.
MISCELLANEOUS NATURAL AND ENDOGENOUS PRODUCTS
Since natural products are essentially chemical weapons that plants use for self-defence, it is not surprising that only a few weak vanilloid antagonists have been discovered in the natural products pool. These plant secondary metabolites include ginsenosides from ginseng [90], the flavonoid galangin [91] and the sesquiterpene lactone thapsigargin [92]. These compounds are weak (mM level) TRPV1 inhibitors, and their affinity for TRPV1 is marginal compared to other targets (e.g. SERCAs for thapsigargin). Several endogenous steroids can also bind and inhibit TRPV [93]. For example, dehydroepiandrosterone (27a, DHEA), a major blood steroid, can reversibly inhibit capsaicin-induced currents in dorsal root ganglion neurons with an EC50 value 6.7 mM, which is close to the physiological concentration of this compound. Since DHEA levels climax in the mid-20s and then decrease with age, elderly people might be more sensitive to capsaicin than young adults. Even more interesting, distinct structure–activity relationships were discovered for steroids: for instance, 3-epiDHEA (27b) could potentiate, and not inhibit, capsaicin responses, raising the possibility that the steroid framework might provide an interesting platform for the discovery of new TRPV1 antagonists. At a molecular level, it is not clear if steroids are allosteric modulators of TRPV1 or if they bind directly to the capsaicinbinding domain of the receptor.
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CLINICALLY USEFUL VANILLOID RECEPTOR TRPV1 ANTAGONISTS
Another intriguing observation is that the female sex-hormone 17bestradiol (28) could dramatically potentiate capsaicin responses, whereas the male hormone testosterone had marginal inhibitory activity. Sex differences in pain responses have long been known, with women being more sensitive to capsaicin-induced pain than men [94]. The differential modulation of capsaicin responses by female and male hormones might provide a rationale to explain this observation. Me O Me 3
Me OH
H H
HO
H H
H
H
HO
(27a) 3α-OH (27b) 3β-OH
(28)
MISCELLANEOUS STRUCTURES FROM THE PROPRIETARY LITERATURE
Research on the identification of vanilloid antagonists has been pursued more intensively in industry than in academia. Thus, a SciFinder search for new chemical entities endowed with this type of activity pulled out 34 entries from the proprietary literature, and only 14 from journal articles during the period January 2004–June 2006. The patent literature can be difficult to evaluate and compare with the published data. Bioactivity is often not disclosed (or commented), and activity can be broadly claimed for a series of lead structures without specifying their optimal substitution. On the other hand, analysis of the patent literature does not only complement the published data, but also offers a preview of information that will be eventually disclosed and detailed in journals. Given the relevance of proprietary literature in the realm of vanilloids research, the main trends emerging from its analysis will be briefly summarized. The most intriguing finding emerging from the patent literature is the possibility of obtaining vanilloid antagonism in structures that lack a carbonyl group. The presence of an amide, ester, urea or thiourea moiety is the single major structural feature shared by the antagonists discussed in the previous sections. Nevertheless, research groups at Merck, Neurogen and Amgen have discovered antagonists within several classes of heterocyclic derivatives where the carbonyl or thiocarbonyl group is replaced by an imino group embedded into a heterocyclic framework. Compound (29) from Merck exemplifies this type of structure [95]. Related compounds are
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165
represented by the Merck leads (30) [96] and (31) [97]. These structures share a framework of three aromatic moieties, which are joined by an aryl–aryl bond and by a nitrogen link. The trifluoromethyl and tert-butyl substituents are related to the more mainstream carbonyl-type leads (32) [98] and (33) [99]. A similar framework is also present in the Neurogen lead (34) [100], and, with an oxygen link, in the Amgen lead (35) [101], although the bioisosteric replacement of an amide is less obvious for (35). Related compounds from Amgen (36) [102] and Neurogen (37) [103] feature four cyclic elements. Compound (36) has a piperazine moiety linking the pendant aryl groups, and is somewhat reminiscent of the 4-heteroarylpiperazione-1carboxamides (13)–(15). Additional heterocyclic-containing antagonist from Neurogen features a benzoisothiazole S,S-dioxide moiety (38) [104] and a pyrido[3,2-d]pyrimidine core structure (39) [105]. This class of non-carbonyl vanilloid ligands is undoubtedly of great interest, but nothing is known on their pharmacokinetics and metabolic fate. N F3 C
MeO
CF3
N
N N
N
N H
CF3
N H Me
N Me
30
29
O
F N
N
N H
N N H
N
CF3
31
O
N
32
H N
N N
CF3 Cl N H
CF3
N H
33 34
t-Bu
166
CLINICALLY USEFUL VANILLOID RECEPTOR TRPV1 ANTAGONISTS
O S
F3C
F3C
N N
N N H
CF3
N N
N
NH2
36 35
H N
F3C F3C O
N N
N
S
H N
N
N O
O
38
t-Bu
N
F3C
MeO N
N Me 37
t-Bu
N
N
N O Me
HN N
O Me
Me
39
The remaining lead structures from the patent literature are mostly modifications of frameworks described previously, with, however, some interesting changes. For example, the N-hydroxythiourea moiety of (40) (Digital Biotech) [106] is a compound related to the simplified RTX analogues of general formula (25), and the inclusion of the amide bond into various heterocylic systems (benzopyrazolone, pyrido[3,2-d]pyrimidinone, quinoxalinone, (41)–(43), respectively [107–109]).
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Me Me OH
OH t-Bu
O
H N
N
O
OMe
S (40)
F3 C H N
N
N O
CF3 (41)
F
N
i-Pr
NH
S
N N
(42)
O
Cl
N O
Me
O
c-Pr
Cl (43)
The 4-heteroarylpiperazinone-1-carboxamide moiety is a common feature in the recent patent literature, further complicating the legal state of these compounds that were already independently discovered and patented by three different laboratories (see the section on 4-Heteroarylpiperazine-1carboxyarylamides). Thus, (36) from Amgen is closely related to the BCT analogue (14) by Purdue Pharma, as are the ureas (44) (Euro-Celtique) [110] and (45) (Janssen) [111]. Compounds (46) [112] and (47) [113] from EuroCeltique represent interesting variations, the basic piperazine nitrogen is replaced by a double bond in (46) and by a hydroxymethylene in (47). In a similar vain, the isoquinolyl ureas (48) [114] and (49) [115] are closely related to the Abbott naphthyl urea (19), just as the Astra-Zeneca N-arylglycinamide (50) [116] is related to the Abbott amide (20).
168
CLINICALLY USEFUL VANILLOID RECEPTOR TRPV1 ANTAGONISTS F3 C
O O Ph(CH2)2HN
HN N
N
N
N
HN N N
N
N
Cl
O
N
F3 C
N
N N CF3
t-Bu
Me
O
O
N
HN N
S
46
45
44
HN
N H
OH N
t-Bu
N
48
47 F
OMe
O
O HN H2NO2 SO
HN OMe
N H
49
HO
O H N
N H
t-Bu
50
The remaining compounds from the patent literature can be broadly identified as amide and the urea/thiourea types. Within antagonists of the amide type, a considerable and surprising variation in the nature of the carboxylic moiety is observed. Thus, while amides (51) (Vertex) [117], (52) (AstraZeneca) [118] and (53) (Renovis) [119] are derivatives of non-polar acids, the acyl moiety of (54) (Yamanouchi) [120], (55) (Ajinomoto) [121] and (56) (Bayer) [122] bears a polar amine group, potentially susceptible to protonation, which probably enhances their solubility. Within the urea derivatives, the GlaxoSmithKline compound (57) [123], an analogue of the lead structure (23), shows an interesting conformational constraint of the ethylendiamine moiety. Finally, the N,N0 -diaryl aminoamide motif present in (58) (AstraZeneca) [124] can be considered as an homologation of the urea pharmacophore.
G. APPENDINO AND A. SZALLASI O
N
N H
O N H
t-Bu
S
O
H N
N H
169
OH
Br
51
52
O OMe
N H t-Bu 53
N
i-Pr
O
N
S
O
N N H
N
N H
Cl
CF3
Ph 55
54
O F3C
N N H
Cl 56
HO
Me N
O
Br
N H
O
N N
57
H N
HN CF3
HO t-Bu
58
Given the structural diversity of these structures, and the variety of the biological end-points used to identify them, it is possible that the capsaicinbinding site is not the only recognition element of TRPV1 targeted by these compounds. Despite this limitation, some general considerations can be made regarding aromatic (heteroaromatic) substitution. Thus, the most
170
CLINICALLY USEFUL VANILLOID RECEPTOR TRPV1 ANTAGONISTS
popular (and presumably optimal) lipophilic substituents are the tert-butyl and the trifluoromethyl, while, within halogen substituents, bromine and chlorine are apparently more frequent than iodine, despite the remarkable ‘iodine effect’ observed on the aromatic component of capsaicinoids and resiniferonoids noted earlier. POTENTIAL CLINICAL INDICATIONS FOR TRPV1 BLOCKADE Generally speaking, the theoretical foundation of TRPV1 antagonist therapy is based on three tenets: (a) TRPV1 expression is up-regulated in conditions of inflammatory hyperalgesia, (b) production of endogenous TRPV1 activators is increased during inflammatory hyperalgesia and (c) inflammatory hyperalgesia is reduced in animals with disrupted TRPV1 gene ( / ). Importantly, physiological temperature regulation is not affected in TRPV1 ( / ) mice [125, 126]. CHRONIC, INTRACTABLE PAIN (NEUROPATHIC, CANCER, AIDS, ETC.); VULVODYNIA
In diabetic rats, TRPV1 is enhanced on myelinated fibres and is hyperphosphorylated by PKC [127]. In accordance with these findings, anti-TRPV1 antiserum was shown to ameliorate pain in a murine model of diabetic neuropathy [128]. In humans, the density of TRPV1-positive nerve fibres is increased in women with chronic breast pain [129] and with vulvodynia [19]. Disruption of TRPV1 gene causes attenuation of bone cancer pain in mice [130]. Pharmacological blockade of TRPV1 by agonists relieved pain in AIDS patients [131]. FAECAL AND URINARY INCONTINENCE
Capsaicin-sensitive nerves sense bladder fullness and form the afferent limb of the micturition reflex [132]. In conditions of bladder hypersensitivity, TRPV1 is up-regulated on these nerves (presumably via NGF [133, 134]) and ‘deafferentation’ of the bladder by intravesical capsaicin or RTX was proven beneficial [135–137]. Parenthetically, intravesical RTX is also an effective analgesic agent during experimental cystitis in the rat [138]. By
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171
contrast, intravesical RTX is without therapeutic benefit in patients with interstitial cystitis [139]. This is an important reminder that efficacy in animal models does not necessarily translate into benefit in patient! The expression of TRPV1 in the bladder is, however, not restricted to afferent nerves: urothelium, detrusor muscle and fibroblasts also express TRPV1 in the human bladder [140]. The implication of these findings for intravesical vanilloid therapy is unclear [141], but the increase in TRPV1 immunoreactivity in the urothelium in patients with neurogenic detrusor overactivity (that occurs in concert with increased TRPV1 in bladder afferents) is a very intriguing finding [142]. In the male urogenital system, TRPV1 is also present in testicles, prostate and scrotal skin [143], and it was postulated that TRPV1 ligands may be beneficial in the treatment of benign prostatic hyperplasia [144]. It is very likely that similar mechanisms also play a central role in the development of faecal incontinence. In fact, enhanced TRPV1 expression was demonstrated in patients with faecal incontinence [18]. INFLAMMATORY BOWEL DISEASE AND MOTILITY DISORDERS
Enhanced TRPV1 expression was demonstrated in IBD in man [17] and capsazepine was shown to attenuate disease severity in experimental colitis both in mice [145] and rats [146]. These findings confer a therapeutic potential for TRPV1 antagonists in IBD and motility disorders [147, 148]. A related finding is the demonstration of increased TRPV1 in the inflamed human oesophagus secondary to gastro-oesophageal reflux disorder [20]. CHRONIC ARTHRITIS
Compared to wild-type mice, in TRPV1 gene-deleted ( / ) mice, complete Freund’s adjuvant evokes significantly less oedema, hyperalgesia and arthritis score [149]. ACUTE PANCREATITIS
Capsazepine attenuates interstitial oedema and neutrophil infiltration in the rat pancreas following infusion of the secretagogue cerulein [150]. HUMAN HAIR GROWTH CONTROL (ALOPECIA) AND DERMATOLOGIC DISORDERS
Functional TRPV1 is expressed in keratinocytes, hair follicles, sebocytes and sweat gland cells [151, 152]. In organ cultures, activation by capsaicin of
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TRPV1 results in premature hair follicle regression and apoptosis [153]. This finding confers a therapeutic potential for TRPV1 antagonists in the treatment of alopecia. In prurigo nodularis, enhanced TRPV1 expression was found that normalized during capsaicin treatment [154]. ANTITUSSIVE ACTIVITY
TRPV1 is involved in the cough reflex [155] and 5-iodo-RTX was shown to function as a potent antitussive drug in guinea pigs [156]. IMMUNOREGULATION
The role of TRPV1 in (neuro)immunoregulation has been detailed recently [157]. Since then, TRPV1 expression was also demonstrated in dendritic cells [158].
CONCLUDING REMARKS: TRPV1 AGONISTS VERSUS ANTAGONISTS – WHICH WAY TO GO? Vanilloid agonists like capsaicin and RTX are clinically proven drugs, though their use is plagued by a combination of undesired side-effects such as pain and limited bioavailability. Some of these limitations can be circumvented by the use of local (e.g. perineural [159] or intraganglionic [160]) administration. Recently, RTX given directly to trigeminal ganglia of Rhesus monkeys was shown to induce powerful analgesic action with no neurological deficit or signs of toxicity [160]. Other potential side-effects are, however, more worrisome. For instance, denervation by capsaicin promoted breast cancer metastasis to lung and heart in adult mice injected with 4T1 mammary carcinoma cells [22]. This observation is, however, at variance with the antitumour effects of TRPV1 agonists against uterine cervix carcinoma cells [161]. Another worrisome report is the development of a paradoxical neuralgic state in rats following RTX administration [162]. In principle, TRPV1 antagonists should be devoid of the undesirable sideeffects of agonists. Vanilloid antagonists, however, also have potential complications. For instance, it was speculated that capsaicin-sensitive nerves are instrumental in maintaining normal blood pressure [163]. If so, prolonged use of TRPV1 antagonists may be hypertensive. Even worse, interfering with TRPV1 was shown to aggravate experimental cardiomyopathy in rats [164] and TRPV1 antagonists were found to mask afferent responses (equivalent of chest pain) to myocardial ischemia in ferrets [165]. If these
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findings also hold true in humans, they would preclude the use of TRPV1 antagonist in patients with hypertension and cardiovascular disorders. These potential cardiac side-effects are especially concerning given the demonstrated ability of some TRPV1 antagonists to directly interfere with hERG receptors in the heart [82]. But the list of potential complications associated with the clinical use of TRPV1 antagonists does not stop here. Expression of TRPV1 is up-regulated in the rat stomach after chemical (HCl) injury: this mechanism is supposed to be protective against mucosal damage [166]. In fact, capsazepine was shown to aggravate HCl-induced gastric ulcers [167]. This phenomenon may prevent the oral use of TRPV1 antagonists that are absorbed in the stomach. Furthermore, TRPV1 is present on rat islet beta cells, where it plays a role in insulin secretion [168]. TRPV1 is also expressed in human brain endothelium where its function remains to be determined [169]. Last, an area of special concern is the enigmatic presence of TRPV1 throughout the whole neuroaxis of the rat [170]. Recently, it was suggested that TRPV1 antagonists may impact on behaviours including anxiety and affect [171]. In summary, the current interest in developing TRPV1 ligands into clinically useful drugs is heavily biased towards antagonists. This is hardly unexpected given the minimal clinical success (some would say failure) of pharmaceutical companies like Novartis (then Sandoz), Proctor and Gamble, and Eli Lilly with capsaicinoids in the past. However, despite intensive research, as of today there is no unequivocal evidence to suggest that TRPV1 antagonists are superior to agonists. Thus, it appears to be a fair statement that both TRPV1 agonists and antagonists may find a future niche in clinical practice.
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Progress in Medicinal Chemistry – Vol. 44, Edited by F.D. King and G. Lawton r 2006 Elsevier B.V. All rights reserved.
5 Recent Medicinal Chemistry of the Histamine H3 Receptor MICHAEL A. LETAVIC, ANN J. BARBIER, CURT A. DVORAK and NICHOLAS I. CARRUTHERS Johnson and Johnson Pharmaceutical Research and Development L.L.C., San Diego, CA 92121, USA
INTRODUCTION
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NEW BIOLOGICAL INSIGHTS
182
CLINICAL APPLICATIONS
188
HISTAMINE H3 RECEPTOR ANTAGONISTS/INVERSE AGONISTS Imidazole-based Ligands Non-imidazole-based Ligands Ligands with Multiple Modes of Action
188 188 190 197
HISTAMINE H3 RECEPTOR AGONISTS
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MOLECULAR MODELLING
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CONCLUSION
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REFERENCES
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INTRODUCTION The biology of the histamine H3 receptor has been detailed in several recent reviews and will not be discussed in great detail here [1–4]. Rather, we intend to draw attention to the major new biological and medicinal chemistry insights that have emerged in the histamine H3 research community over the last couple of years. The key to an understanding of the biology of the histamine H3 receptor is the fact that it is an inhibitory auto- and heteroreceptor. Activation of the H3 DOI: 10.1016/S0079-6468(05)44405-7
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receptor decreases the release of the neurotransmitter of the neuron on which the H3 receptor is located. Several neurotransmitter systems are subject to modulation by the H3 receptor. First and foremost, the H3 receptor, when activated, decreases the synthesis [5] and release [5, 6] of histamine. Antagonism (or inverse agonism, as described below) of the H3 receptor thus leads to a general activation of histaminergic neurotransmission in the brain. The major effects associated with this are an increase in waking, improved cognition, and suppression of food intake [7]. Indeed, histamine is one of the major neurotransmitters involved in the maintenance of the waking state, an effect largely mediated through histamine H1 receptors. By increasing the amount of histamine released from neurons, thereby promoting activation of H1 receptors, H3 antagonists increase waking, as has been extensively illustrated in several animal models [8–10]. Interestingly, in contrast to classical stimulants this increased waking is not accompanied by locomotor activation [8]. The therapeutic hope is that H3 antagonists will provide the alertnessinducing benefit of stimulants without their addictive liability or locomotor effects for a number of disorders, including narcolepsy, attention deficit hyperactivity disorder (ADHD), and fibromyalgia. Table 5.1 contains a list of potential therapeutic indications for H3 antagonists and the animal experiments that support these claims. It is hard to separate histamine’s effect on waking from its effect on cognition, since the former is a prerequisite for the latter. The observation that H3 antagonists improve cognitive function in a variety of models [11], possibly via an increase of acetylcholine release, opens up the possibility that H3 antagonists will be useful in the treatment of Alzheimer’s disease, ADHD, and other cognitive dysfunctions [3, 4]. As heteroreceptors, H3 receptors also modulate several other neurotransmitter systems. The literature describes effects on noradrenaline [12], serotonin [13], g-aminobutyric acid [14], glutamate [15], dopamine [16], and acetylcholine [17]. Since these neurotransmitters play important roles in various disease states, it is clear that the effects of H3 antagonists on these systems, if demonstrated in vivo in human, could be harnessed for a number of additional therapeutic indications, examples of which are listed in Table 5.1. For instance, increases in noradrenaline and serotonin release could lead to anti-depressant effects.
NEW BIOLOGICAL INSIGHTS Although the basic function of the H3 receptor has been known for over two decades, recent years have witnessed a spate of new biological insights, some of which are of particular interest to the medicinal chemist.
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Table 5.1 POTENTIAL THERAPEUTIC APPLICATIONS FOR H3 ANTAGONISTS Indication
Model/experiment
Effect of H3 antagonist
Reference
Narcolepsy Sleepiness ADHD
Narcoleptic Doberman Wakefulness state in rats Passive avoidance in spontaneously hypertensive rats Various cognition models
Decreases cataplexy Increases waking Improves cognitive performance
[55] [8] [57]
Improves cognitive performance in most, but not all models Decreases food intake H3 KO mouse is heavier than wild type mice
[3] [65] [11] [1] [61] but see also [62] [58]
Alzheimer’s disease Obesity
Food intake H3 knockout mouse
Diabetes Alcohol abuse
Impaired insulin tolerance in high-fat diet-fed mice Alcohol-preferring rat
Pain Allergic rhinitis
Mouse hot-plate test Nasal congestion model
Meniere’s disease Epilepsy
Labyrinthectomy Electrically evoked convulsions in mice Prepulse inhibition
Schizophrenia
Normalizes blood glucose levels Decreases alcohol consumption Induces nociception Decreases congestion (together with H1 antagonist) Attenuates body rotations Inhibits convulsions Improves prepulse inhibition
[60] [46] [59] [63], [64] [66] [56]
At the basic molecular biology level, the existence of multiple receptor isoforms may introduce another level of complexity to the H3 receptor story, as they may display differential affinities for ligands and even couple to distinct signalling pathways [2, 18]. For instance, Drutel et al. [19] compared the coupling of several rat H3 receptor isoforms to the cAMP and mitogen activated protein kinase (MAPK) pathways, and found differences of up to 5- to 10-fold between the longest and shortest isoforms. Wellendorph et al. [20] also described functional differences between human H3 receptor isoforms. The significance of these findings is unclear, as several groups have disputed the existence of human isoforms [21, 22]. No isoforms have been reported in the mouse [23]. The activity level of many G-protein coupled receptors (GPCRs) is regulated through the formation of multimeric complexes. The multimerization state of the receptor can affect downstream effects such as coupling to intracellular signalling, trafficking, and receptor desensitization [24]. Dimerization of H3 receptors is just beginning to be investigated [25] while heterodimerization with other receptors has not yet been described. It is entirely
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conceivable that species-, tissue-, and isoform-specific multimerization effects may lie at the root of some of the complex pharmacology of this receptor. The opportunities and challenges for medicinal chemistry can readily be imagined. The discussion of molecular biology leads quite naturally to a consideration of new pharmacological insights. Recent years have seen a refinement, and in some cases a revision, of the classification of H3 receptor ligands. Antagonists are now considered inverse agonists or partial agonists. Agonists turn out to be antagonists as well. There is a bewildering fluidity in the classification of compounds like proxyfan (1). The first consideration here has to be species specificity. For instance, chloroproxyfan (2) behaved as a full agonist only at the human H3 receptor and as a partial agonist at the rat, mouse, and guinea-pig H3 receptor [23]. Esbenshade et al. [26] found that GT-2331 (11) had much lower affinity for the human H3 receptor than for the rat H3 receptor and that it was also a partial agonist. A direct comparison of binding affinities of several compounds at the human, dog, monkey, rat, and guinea-pig cortex revealed that many compounds have a lower affinity for the human H3 receptor versus that of other species [27]. These findings underline the critical importance of selecting the appropriate species for screening and modelling. Additionally, the behaviour of a ligand can depend on the degree of constitutive activity in a particular model and it has become increasingly clear that the histamine H3 receptor is constitutively active in many experimental systems and probably also in vivo [28–31]. Thus, compounds that were classified as antagonists may actually decrease the constitutive activity of the H3 receptor and be more properly known as inverse agonists. If the system has a low degree of constitutive activity, agonists will push the receptor to a higher level of activity. To make matters even more interesting, some ligands will set the system at a specific level of activity. If the system is quiescent, the compound will appear to be an agonist. If the system’s constitutive activity is high, that same compound will behave like an antagonist or inverse agonist. Such compounds are called protean agonists [32], and it has been shown that some H3 ligands belong to that category [31]. Thus, the medicinal chemist is faced with the intriguing prospect that a given H3 ligand may behave very differently in distinct brain areas, depending on the level of constitutive H3 receptor activity in those anatomical regions. The possible permutations are endless: provided that optimal procognitive efficacy be achieved by a compound that is an inverse agonist in the nucleus basalis magnocellularis (to increase acetylcholine release in the frontal cortex) but an agonist in the amygdala? Do neutral H3 antagonists induce an anorexic effect, or can that only be obtained by inverse agonism, as some groups have claimed [1]? Inverse agonists have their own disadvantages. For instance, chronic inverse agonism can lead to
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upregulation of the receptor and toleration to chronic antagonism [32]. Only careful pharmacological dissection of the effects of known H3 ligands, as well as a precise delineation of the extent of constitutive activity of the H3 receptor in the human, will be able to answer these questions. N
O
Ph
N HN
HN
Cl
(1)
S
N
O
NH NH2
HN (3)
(2)
Although most of the medicinal chemistry effort in the H3 receptor field has been focused on the development of antagonists, there is some interest in agonists as well. Histamine H3 receptor agonists decrease the release of histamine in the central and peripheral nervous system and lead to a weakened histaminergic tone. In the brain, their effects will therefore be comparable to those of H1 receptor antagonists, with sedation and induction of sleep as a prominent observation. Indeed, H3 agonists such as the imidazoles (4) (BP 2.94) or (5) (Sch 50971) induce significant increases of slow-wave sleep or induce sedation in animal models [10, 33]. Potent and selective brain-penetrating H3 agonists could provide a new therapeutic option for the treatment of insomnia. HO N
N HN
Me (4)
Me NH N NH (5)
In conditions of myocardial ischaemia, excessive release of noradrenaline from sympathetic nerve endings in the heart is a major contributor to the development of potentially life-threatening arrhythmias. H3 receptors are present on sympathetic nerves in the human heart [34], and there is in vitro evidence that the H3 agonist imetit (3) decreases the release of noradrenaline from human myocardium in anoxic, but not in normoxic conditions [35]. However, in an isolated guinea-pig heart ischaemia model, the H3 agonist Ra-methylhistamine did not influence the release of noradrenaline, possibly because a high increase in histamine release led to the H3 receptors being fully saturated with their endogenous ligand [36]. The same group reported in a later paper that when the ischaemia conditions were maintained for 20 min instead of 10 min, the H3 receptor agonist imetit was then able to
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decrease both noradrenaline overflow and the incidence of ventricular fibrillation, which seemed to indicate that the H3 receptor was not fully activated by endogenous ligand in these conditions [37]. Hearts obtained from H3 receptor knockout mice displayed a higher incidence of arrhythmias in ischaemic conditions, as well as a higher release of noradrenaline, than hearts from wild-type mice [38]. Thus, the evidence seems to indicate that activation of the H3 receptor during ischaemia may afford some protection against noradrenaline-influenced arrhythmias. Depending on whether the H3 receptors in the human heart are fully activated during ischaemic conditions, H3 agonists may be cardioprotective and anti-arrhythmic agents. Interestingly, although the appetite-suppressant effects of H3 antagonists are a focus of much research and speculation, as described above, and although centrally acting H1 antagonists are known to increase food intake in animals and humans, relatively little information is available about possible appetite-stimulant effects of H3 agonists. It has been shown that H3 agonists such as imetit and R-a-methylhistamine can block the satiety-inducing effects of bombesin [39] or the cholecystokinin-induced reduction of food intake [40], but R-a-methylhistamine does not seem to have a direct appetiteinducing effect [39, 40], even when given i.c.v. [41]. This could be due to methodological issues, or it could indicate that the baseline H3 receptor occupation during these studies was already so high that further stimulation with an H3 agonist did not cause a measurable behaviour. This area merits further research, as stimulators of appetite could conceivably be put to good use in wasting diseases. Finally, there are some reports that H3 agonists may decrease inflammation in various tissues [33, 42]. There has been some interest in capitalizing on this property by using H3 agonists as clinical therapeutics for asthma [43] or migraine [44]. In an asthma study with R-a-methylhistamine, no antibronchoconstrictive effects could be observed [43]. The migraine trial reported that low subcutaneous doses of Na-methylhistamine (1–3 ng) were effective for migraine prophylaxis, whereas the higher dose caused intense headaches, possibly because of cross reactivity with the H1 receptor [44]. Finally, a review of the medicinal chemistry of the H3 receptor would not be complete without a mention of pain and itch. For both phenomena, there is conflicting evidence with both agonists and antagonists being proposed as therapeutic agents. For instance, there are some indications that H3 agonists may be useful in some types of pain [42]. The H3 agonist immepip (69), when administered systemically or intrathecally, has some analgesic effects in a model for mechanical pain, but not in a model of thermal pain [45]. These effects were absent in H3 receptor knockout mice, indicating that spinal H3 receptors play a role in pain perception [45]. In contrast, Farzin et al. [46] found that the H3 antagonist thioperamide (7) had anti-nociceptive effects
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in the mouse hot-plate test and the writhing test. A Japanese group investigated a large number of H3 antagonists and found that several of them were effective in models of neuropathic pain [47]. The reason for the discrepancy between these results is not known. It is possible that cross reactivity of certain compounds with the H4 receptor may cloud the interpretation of the results in specific models. Indeed, the H4 receptor is involved in inflammatory processes [48], and could thus conceivably be involved in the development of certain pain states. S N H
N
NH2
N
N
HN
HN (7)
(6)
O NH N
N
O
S
HN
HN
N H Cl
(9)
(8)
NH N
S
HN
N H I
(10)
The H3 antagonists clobenpropit (9) and iodophenpropit (10), when injected intradermally, cause scratching behaviour in wild-type and mast-cell deficient mice, whereas the H3 agonist R-a-methylhistamine has no effect by itself but blocked the thioperamide-induced scratching [49, 50]. In contrast, another group found that imetit- and histamine-induced scratching in BalbC mice could be blocked by thioperamide [51, 52]. Recent evidence suggests that the histamine receptor involved in pruritus may actually be the H4 receptor [53], which is related to the H3 receptor but is mainly expressed in immune cells [54]. Some of the apparent contradictions in the data may be explained by the use of mouse strains with different sensitivities to histamine and by the fact that the pharmacological tools used in these studies,
188 RECENT MEDICINAL CHEMISTRY OF THE HISTAMINE H3 RECEPTOR
like thioperamide and clobenpropit, are not ideally suited to distinguish between the pharmacology of the H3 and the H4 receptor [53, 54]. Further experiments with selective H3 and H4 agonists and antagonists will be needed to answer these questions.
CLINICAL APPLICATIONS The definitive identification of a therapeutic raison d’eˆtre for H3 antagonists will happen in the clinic. A handful of H3 ligands are reported to have entered clinical testing. ABT-834 entered Phase-I trials for the indication of cognitive disorders in May 2003 but no news has been reported since that time. GT-2331 (11) was approved for Phase-II clinical trials in 1999 but no news has been reported since then [4]. At this point, there are no data available to assess the therapeutic potential in human disease (See Table 5.1). In conclusion, H3 ligands offer the attractive vista of multiple applications in various disorders, but the ultimate definition of their therapeutic utility will have to await clinical trial results. Future work will determine whether inverse agonists, neutral antagonists, or protean agonists will constitute the more useful pharmacological intervention.
HISTAMINE H3 RECEPTOR ANTAGONISTS/INVERSE AGONISTS Numerous pharmaceutical companies and academic laboratories have published data on a wide variety of small molecule H3 antagonists/inverse agonists. Highlights of the data reported since the last review in this book series [67] will be reported here.
IMIDAZOLE-BASED LIGANDS
There are many reports on the use of imidazole-based H3 antagonists. Many of the earlier compounds are essentially histamine (6) analogues. For example, thioperamide (7), ciproxifan (8), and clobenpropit (9) are three widely studied imidazole-based H3 antagonists. These histamine analogues have been discussed in earlier reviews on H3 antagonists [65, 67, 68]. One should note that the earlier functional characterization of numerous histamine-based ligands is now confounded by the knowledge gained from the more recent pharmacological investigations using the cloned human receptor. Additionally, the more recent characterization of the fourth histamine receptor has demonstrated that several such ligands, including the
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prototypical H3 antagonist thioperamide (7), exhibit affinity for the H4 receptor [54]. Recently disclosed imidazole-based compounds include GT-2331 (11) and the carbamate (12). These compounds have been characterized as inverse agonists and have been shown to reduce food intake in rats [69–71]. The absolute configuration of these ligands has been disputed, however, it appears that the absolute configuration of (11) is as shown [72]. According to published data (11) is one of the first H3 antagonists to be taken into clinical trials in humans [4]. A team at Schering–Plough has also reported on imidazole-based H3 antagonists. Their efforts have resulted in the discovery of SCH 79687 (13), a potent and selective H3 antagonist (Ki ¼ 1.9 nM in the rat) [73, 74]. The compound has good pharmacokinetics in rat and monkey. The authors have demonstrated in vivo decongestant activity for SCH 79687 (13) when dosed in combination with an H1 antagonist. H
HN
H N
N H
HN
t-Bu
HN
(12)
H N
N
O
N
(11)
O
Cl
H N
Cl
O
O HN
S O
N
Cl
(13)
H N
(14)
Cl H N HN
O
N
S O
(15)
Several series of sulphonamide, sulphamide, and guanidine H3 antagonists are under investigation by the group at The James Black Foundation [75–77]. The sulphonamide (14), which is reported to have a pKi of 8.58 in guinea-pig cortex homogenate, is representative of the sulphonamide series [78]. A group at Schering–Plough has disclosed structurally very similar sulphonamides. These include the sulphonamide (15), reported to have a
190 RECENT MEDICINAL CHEMISTRY OF THE HISTAMINE H3 RECEPTOR
Ki of 9 nM in guinea-pig brain membranes [79]. The compound was also reported to have an ED50 of 2.2 mg/kg in a CNS hypertension model. NON-IMIDAZOLE-BASED LIGANDS
The collaboration between several academic laboratories has shown that the imidazole moiety on some of the earlier H3 antagonists can be replaced with a piperidine. For example, the ciproxifan analogue (16) has a pKi of 8.4 in rat cerebral cortex [80]. This compound showed in vivo efficacy after p.o. administration to a mouse. Other piperidines studied by these researchers include (17) (H3 pA2 ¼ 7.21 in guinea-pig ileum) [81] and (18) (human Ki ¼ 2.8 nM, pA2 ¼ 7.42 in guinea-pig ileum) [82]. The authors have also found that benzyl ethers such as (19) are somewhat weaker H3 antagonists (pA2 ¼ 6.3 in guinea-pig ileum) [83]. O Ph N
N
O (16)
(17)
N O
N O
N (18)
(19)
Scientists at Abbott have published extensively on their H3 medicinal chemistry efforts. Early publications reported on structure–activity relationships starting with A-923 (20), a lead identified from high-throughput screening [84, 85]. A-923 was shown to be a potent rat H3 antagonist (Ki ¼ 2 nM), however the compound was found to have poor bioavailability and poor selectivity over other receptors. Lead optimization efforts led to the discovery of compound (21), a potent H3 antagonist (Ki ¼ 1.6 nM) with good selectivity over the H1 and H2 receptors [86]. This research also led to the discovery of biphenyl compounds, including A-331440 (22), which is reported to bind to both the human H3 (pKi ¼ 8.56) and rat H3 receptors (pKi ¼ 7.87) with good selectivity over the H1 and H2 receptors [87]. The authors demonstrated that A-331440 is a competitive antagonist and a potent inverse agonist of the H3 receptor. A-331440 was also shown to reduce
M. A. LETAVIC ET AL.
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body weight in obese mice. In this study, A-331440 lowered overall body weight and had positive effects on insulin tolerance and fat levels [58]. A-331440 was found to give a positive result in an in vitro micronucleus assay and this group subsequently reported that analogues of A-331440, including A-417022 (23), were not active in the micronucleus assay [88]. (23) was also active in obesity models. O
N Et
O O H S N O
N
N
O
O
N
F
O
(20)
(21)
CN
CN
N
F NH N
O
N
Me2N
O N
Me2N
(24)
(23)
(22)
O N O
Me N
O Me
(25)
Another biphenyl compound, A-349821 (25), was shown to have good affinity at both the rat and human H3 receptors and was efficacious in a dipsogenia model (demonstrating reversal of an H3 agonist effect by an antagonist) [89] and in models of cognition [90]. The authors also reported that (25) has good bioavailability and low clearance in dog and monkey. High-throughput screening efforts in the same group have led to the discovery of tetrahydrocyclohepta[b] quinoline H3 ligands. For example, compound (24) was found to have a human H3 pKi of 8.76 [91].
192 RECENT MEDICINAL CHEMISTRY OF THE HISTAMINE H3 RECEPTOR
Abbott has also demonstrated in vivo efficacy with two piperazine amides, A-304121 (26) and A-317920 (27). Both (26) and (27) bind to the rat H3 receptor with high affinity (pKi ¼ 9.15 and 8.6, respectively) [92] and are active in several in vivo models, including the acute dipsogenia model and models of cognitive performance and inhibitory avoidance [93]. Unfortunately, these compounds showed markedly reduced affinity for the human H3 receptor, reinforcing the need to screen against the human receptor. O
O
N
Me
O
O
N
H2N
O
O
Me N H
(26)
O
N N
O
(27)
N O CN
N O N H Me2N
N
Me
O
F
Me
N N
O
-L-tartrate
O (28)
(29)
Further modifications of (26) led to the oxadiazoles as depicted by (28) [94]. Some of the oxadiazoles have improved human H3 affinity and (28) is efficacious in the dipsogenia model after i.p. administration. However, the molecular weight and log P of these H3 ligands is rather high. Most recently, the Abbott group disclosed a series of benzofurans with potent affinity for the human and rat receptors. ABT-239 (29) is one of the more extensively profiled members of this series. (29) has a Ki of 0.45 nM at the human receptor [95], good rat pharmacokinetics and demonstrated efficacy in models of cognition [96]. Recently, a scaleable synthesis of ABT239 has been reported [97] and the researchers have published detailed accounts on the pre-clinical pharmacokinetics and efficacy of this compound [98, 99], indicating continued interest in this series of H3 antagonists. The group at Johnson and Johnson has published several reports on their efforts to find potent H3 antagonists. High-throughput screening using the recombinant human receptor identified the imidazopyridine RWJ-20085 (30) as a weak H3 receptor ligand (Ki ¼ 4 mM). Medicinal chemistry efforts then led to the discovery of the piperidine propyloxy compound (31) [100]. This imidazopyridine has a Ki at the human H3 receptor of 2 nM and
M. A. LETAVIC ET AL.
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is selective over the histamine H1, H2, and H4 receptors. The compound demonstrated high brain penetration following i.p. administration and had an ED50 of 0.2 mg/kg s.c. in brain receptor occupancy studies using ex vivo autoradiography. The compound also has good pharmacokinetics in rat. N
N O
N
N
N
N
O
N
(31)
(30)
H O
N
O
N
H
N
(32)
(33)
O
N
O
O
N
N
N (34)
i-Pr
(35)
In efforts designed to replace the imidazopyridine ring system, indolizidines such as (32) [101] (human H3 Ki ¼ 13 nM), and related heterocycles [102–104] were identified as potent H3 antagonists. Indolizidine (32) suffered from rapid metabolism in human liver microsomes, however, substitution on the indolizidine ring suppresses this liability. Octahydroindolizidines including (33) (Ki ¼ 0.2 nM) were also shown to be H3 antagonists [105]. Additional work at Johnson and Johnson revealed that the bis-piperidine JNJ-5207852 (34) is a potent and selective H3 antagonist (pKi ¼ 9.24, pA2 ¼ 9.84) [106, 107]. This H3 antagonist has in vivo activity in rodent models of arousal, decreases slow-wave sleep, and has no effect on locomotor activity [8]. The authors also demonstrated that (34) had no effect on wakefulness or sleep in H3 knockout mice. It is interesting to note that when mice were treated daily with (34) for four weeks no effect on body weight was observed. Pharmacokinetic studies in rats showed that (34) has high bioavailability and extensive brain penetration. These researchers also found that conformationally restricted analogues of (34) were ligands for the H3 receptor [108, 109]. For example, compound (35)
194 RECENT MEDICINAL CHEMISTRY OF THE HISTAMINE H3 RECEPTOR
has a human pKi of 8.67 and a pA2 of 9.32, is highly selective over other related receptors, and showed efficacy in a rat EEG model of wakefulness at 1 mg/kg s.c [109]. The group at Johnson and Johnson has also disclosed m- and parasubstituted alkynes, as represented by (36) (Ki ¼ 0.8 nM) and (37) (Ki ¼ 5.5 nM) [110], related phenylpiperidines (for example (38), Ki ¼ 0.6 nM) [111] and piperazine amides such as (39) (Ki ¼ 0.7 nM) [112]. All of these compounds are quite potent H3 antagonists. No in vivo data have been reported for the latter analogues. This team also identified a series of 2alkoxyimidazoles that are H3 antagonists [113]. One example is the isopropylpiperidine (40), which has a human Ki of 3.7 nM. Modifications of the original synthesis of (40) led to a report on a 3-step synthesis of large quantities of this H3 antagonist [114], suggesting significant interest in this series. N
N O N
N
N
O N
N
(38)
(37)
(36)
O
O N
N
N
i-Pr
Me N
N i-Pr O
N
Cl
(40)
(39)
Researchers at GlaxoSmithKline have numerous recent patent applications describing several series of H3 antagonists including the benzazapines (41) [115] and (42) [116], quinolizidines (43) [117], isoindolines (44) [118], and the piperidine amides (45) [119] and (46) [120]. All of these structures were disclosed as functional H3 antagonists. O O
Et2N N
O
N MeNH
(41)
N
N O
(42)
M. A. LETAVIC ET AL.
195
F N
O
Cl
N
O
N O
N O
(43)
(44)
O N
F3C
N
NC
N
N
i-Pr
i-Pr
N
N N
O O (45)
(46)
In addition to the imidazole-based compounds detailed earlier, more recently the Schering–Plough group reported on several new series of H3 antagonists including oximes (47) [121], benzimidazoles (48) [122], benzimidazolones (49) [123], and indoles (50) [124]. All of the compounds shown have Kis of less than 10 nM in guinea-pig brain. N
OMe
S
N
N
N
N
N
N
N NH2
N
F
N
O
N O
(47)
NH 2
(48)
F F
N O
N
N
N F
N N
N O
(49)
N
NH2 F
NH2
N N
N O (50)
Researchers at Novo Nordisk have found that certain acyl piperazines are potent H3 antagonists [125, 126]. Examples include structures (51) and (52), both of which have a Ki of 1.2 nM against human H3. The group
196 RECENT MEDICINAL CHEMISTRY OF THE HISTAMINE H3 RECEPTOR
at Novo Nordisk has also discovered a class of cinnamides that are H3 antagonists [127, 128]. Some members of this series, represented by (53) (H3 Ki ¼ 4.7 nM), were counter screened at the H1, H2, and H4 receptors and were not active. The authors reported that, in some cases, these compounds were cytochrome P450 inhibitors in vitro, however this problem could be overcome by varying the substituent on the aromatic ring. Cl
O
Cl
O N
N O
N
N
O N
O
N
CF3
(51)
(53)
(52)
O N
S N
N
N N
N (54)
N
N n-Pr (55)
N
N (56)
More recently, the same group disclosed two series of quinolines that are H3 antagonists [129, 130]. The quinolines (54) and (55) are potent H3 antagonists (Ki ¼ 2.9 and 1.8 nM, respectively). The authors focused on the cyclopropyl derivative (55) because cyclopropyl amines are less basic than other alkylamines. These cyclopropyl amines are reported to be the least basic non-imidazole H3 antagonists known. It appears that the group at Novo Nordisk is primarily interested in H3 antagonists for the treatment of obesity. Other recently published piperazine-based H3 antagonists include compound (56), which is reported to have an H3 pA2 of 7.25 in the guinea-pig jejunum [131]. Researchers at Lilly have prepared a series of alkylamine H3 antagonists. Examples include the amide (57), which has a Ki of 1.05 nM and the tetrahydroisoquinoline (58), which has a Ki of 0.37 nM [132]. Both compounds are inactive at the H1, H2, and H4 receptors. This same group also disclosed a series of azepines, represented by (59) (H3 Ki ¼ 0.85 nM) and (60) [133]. Compound (60) is reported to have 100% bioavailability and a 12.4 h halflife in rat. Related dihydroindoles such as (61) (Ki ¼ 0.5 nM) and tetrahydroquinolines were also shown to be H3 antagonists [134].
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N
O
N
N N
N
O
Et
O (58)
(57) NH
N Ac N
N
O
O (60)
(59)
N N
N Me
O (61)
LIGANDS WITH MULTIPLE MODES OF ACTION
In addition to the work described earlier, the group at Schering–Plough has also reported on dual H1/H3 antagonists. These compounds, conceptually prepared by linking a chlorpheniramine structure to imidazole-based alkylamines, are expected to be useful for the treatment of allergic rhinitis and nasal congestion. One example is compound (62), a potent dual H1/H3 antagonist (Kis ¼ 7 and 15 nM, respectively) [135, 136]. Me N HN
N
N
N (62)
HN
H N
N
(63)
Cl
H N
Me N
N
N N
(64)
N
O
(65)
198 RECENT MEDICINAL CHEMISTRY OF THE HISTAMINE H3 RECEPTOR
Collaboration between several academic laboratories has identified dual H3 antagonists/histamine N-methyltransferase inhibitors. Compound (63) is representative of this class. The compound has a pKi of 10.0 at the human H3 receptor and an IC50 of 51 nM at histamine N-methyltransferase [137]. Related imidazoles such as (64) (human H3 Ki ¼ 4.1 nM, rat histamine N-methyltransferase IC50 ¼ 24 nM) [138] and piperidines (65) (human H3 Ki ¼ 67 nM, rat histamine N-methyltransferase IC50 ¼ 61 nM) [139] are also dual acting agents. It is thought that these dual acting agents will further increase the amount of extracellular histamine since histamine N-methyltransferase degrades histamine.
HISTAMINE H3 RECEPTOR AGONISTS Histamine H3 agonists have also been the subject of a recent review [140]. These compounds are typically based on the endogenous ligand histamine (6). The imidazole ring is an essential element that confers agonism at the H3 receptor. What differentiates H3 agonists from each other are modifications of the side chain to improve potency and selectivity, especially selectivity over the roughly 40% homologous H4 receptor. Indeed, the addition of a methyl group onto the aminoethyl side chain of histamine provides the prototypical H3 agonist R-a-methylhistamine (66). R-a-methylhistamine is the more potent eutomer (pEC50 9.17) being 10-fold more potent than the endogenous ligand at the human H3 receptor. The compound was subsequently found to have good selectivity over H4 (pEC50 5.95). H
H NH2
N
Me
N H
NH2
N H
N H
N H
N
N
N NH
N H (69)
NH2 (68)
(67)
(66)
H
N
N
N H (70)
N
N H
Me
(71)
Researchers at the Vrije Universiteit of Amsterdam in collaboration with others [141] have investigated conformationally restricted forms of histamine. For example, constraining the side chain with a cyclopropyl group (67)
M. A. LETAVIC ET AL.
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having the (S,S) configuration, effectively tying up a-methylhistamine, had a pEC50 8.23 at the human receptor. In addition, investigators at Hokkaido University reported that they could obtain comparable activity by incorporating a cis-cyclopropyl moiety while extending out the basic amine (68) [142]. While exploring derivatives of immepip (69) (pEC50 9.88), the Vrije Universiteit of Amsterdam group also found that the piperidine could be replaced with pyridine and only a methyl group is tolerated on the piperidine nitrogen. The pyridine analogue (70) had pEC50s of 9.74 and 6.04 at the human H3 and H4 receptors, respectively. The compound behaves as a full agonist, even with the less basic pyridine heterocycle, and induces a concentration-dependent decrease of electrically induced twitch contraction of guinea-pig ileal myenteric plexus with a pD2 of 7.45 [143]. The N-methyl derivative (71) has comparable potency at the human H3 receptor as (69) with improved selectivity over H4 (pKi 7.7 versus 5.7). In vivo microdialysis experiments have shown that (71) reduced the level of histamine in a rat brain by 75% following i.p. administration. Moreover, this effect could be blocked with co-infusion of clobenpropit (9) via the microdialysis probe [144].
MOLECULAR MODELLING The application of molecular modelling to develop pharmacophore models for both H3 agonists and antagonists has received only limited attention recently. Studies have led to a proposal for the stabilization of the active state of the receptor via a proton relay process for a small series of histamine-like agonists, which were in turn considered to adopt a folded conformation [145]. A more exhaustive molecular modelling approach using the program SLATE has provided a greater understanding of the bioactive conformation of a range of imidazole containing antagonists [146]. However, this model does not appear to account for the behaviour of the many non-imidazole containing antagonists discovered over the last several years, suggesting the possibility of alternative binding sites.
CONCLUSION During the last four years, following the cloning of the histamine H3 receptor cDNA, the level of activity amongst both academic and pharmaceutical company laboratories has increased enormously. This activity has provided a greater understanding of the basic biology of the target, answering many questions about the potential therapeutic roles for histamine H3 receptor ligands but also raising some more fundamental questions
200 RECENT MEDICINAL CHEMISTRY OF THE HISTAMINE H3 RECEPTOR
about the nature of the receptor system. At the same time, the medicinal chemistry of the various ligands has also changed, primarily owing to the involvement of several pharmaceutical companies who have exploited highthroughput screening techniques to find novel templates for drug design. One consequence of these efforts is the discovery of numerous nonimidazole H3 antagonists capable of addressing the shortcomings of the earlier imidazole-based compounds. Several of the newer structures appear to have acceptable drug-like properties with several advancing into the clinic and thus the role of these agents as therapeutics should soon be established.
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206 RECENT MEDICINAL CHEMISTRY OF THE HISTAMINE H3 RECEPTOR [137] Apelt, J., Ligneau, X., Pertz, H.H., Arrang, J.-M., Ganellin, C.R., Schwartz, J.-C., Schunack, W. and Stark, H. (2002) J. Med. Chem. 45, 1128–1141. [138] Grassmann, S., Apelt, J., Sippl, W., Ligneau, X., Pertz, H.H., Zhao, Y.H., Arrang, J.-M., Ganellin, C.R., Schwartz, J.-C., Schunack, W. and Stark, H. (2003) Bioorg. Med. Chem. 11, 2163–2174. [139] Grassmann, S., Apelt, J., Ligneau, X., Pertz, H.H., Arrang, J.-M., Ganellin, C.R., Schwartz, J.-C., Schunack, W. and Stark, H. (2004) Arch. Pharm. (Weinheim, Ger.) 337, 533–545. [140] de Esch, I.J.P. and Belzar, K.J. (2004) Mini-Rev. Med. Chem. 4, 955–963. [141] Kitbunnadaj, R., Zuiderveld, O.P., De Esch, I.J.P., Vollinga, R.C., Bakker, R., Lutz, M., Spek, A.L., Cavoy, E., Deltent, M.-F., Menge, W.M.P.B., Timmerman, H. and Leurs, R. (2003) J. Med. Chem. 46, 5445–5457. [142] Kazuta, Y., Hirano, K., Natsume, K., Yamada, S., Kimura, R., Matsumoto, S., Furuichi, K., Matsuda, A. and Shuto, S. (2003) J. Med. Chem. 46, 1980–1988. [143] Kitbunnadaj, R., Zuiderveld, O.P., Christophe, B., Hulscher, S., Menge, W.M.P.B., Gelens, E., Snip, E., Bakker, R.A., Celanire, S., Gillard, M., Talaga, P., Timmerman, H. and Leurs, R. (2004) J. Med. Chem. 47, 2414–2417. [144] Kitbunnadaj, R., Hashimoto, T., Poli, E., Zuiderveld, O.P., Menozzi, A., Hidaka, R., de Esch, I.J.P., Bakker, R.A., Menge, W.M.P.B., Yamatodani, A., Coruzzi, G., Timmerman, H. and Leurs, R. (2005) J. Med. Chem. 48, 2100–2107. [145] Kovalainen, J.T., Christiaans, J.A.M., Ropponen, R., Poso, A., Peraekylae, M., Vepsaelaeinen, J., Laatikainen, R. and Gynther, J. (2000) J. Am. Chem. Soc. 122, 6989–6996. [146] de Esch, I.J.P., Mills, J.E.J., Perkins, T.D.J., Romeo, G., Hoffmann, M., Wieland, K., Leurs, R., Menge, W.M.P.B., Nederkoorn, P.H.J., Dean, P.M. and Timmerman, H. (2001) J. Med. Chem. 44, 1666–1674.
Progress in Medicinal Chemistry – Vol. 44, Edited by F.D. King and G. Lawton r 2006 Elsevier B.V. All rights reserved.
6 Recent Progress in Cannabinoid Research JULIA ADAM, PHILLIP M. COWLEY, TAKAO KIYOI, ANGUS J. MORRISON and CHRISTOPHER J.W. MORT Organon Research, Newhouse, Lanarkshire, ML1 5SH, Scotland, UK
INTRODUCTION
208
CANNABINOID RECEPTORS AND ENDOCANNABINOIDS
208
ANANDAMIDE TRANSPORT INHIBITORS The Hydrophobic Chain The Carboxamide/Carboxylate Group The Polar Head Group
210 211 211 211
FATTY AMIDE ACID HYDROLASE INHIBITORS Organosulfonate and Organophosphonate Analogues Trifluoromethylketones a-Ketoheterocycles Carbamates Other Structures
212 213 215 216 217 219
CANNABINOID RECEPTOR AGONISTS Classical Cannabinoids Cannabidiol Derivatives (Resorcinols) Non-classical Cannabinoids Endocannabinoid Derivatives Indole and its Derivatives
220 220 233 235 237 247
CB2 AGONISTS CB2 Selective Classical Cannabinoids Indoles and Indazoles Resorcinol Derivatives Benzo[c]Chromen-6-one Derivatives
259 260 262 266 268
Other Heterocyclic CB2 Agonists
269
DOI: 10.1016/S0079-6468(05)44406-9
207
208
RECENT PROGRESS IN CANNABINOID RESEARCH
THERAPEUTIC APPLICATIONS OF CANNABINOID AGONISTS
270
CB1 RECEPTOR ANTAGONISTS 1,5-Diaryl-pyrazoles 4,5-Dihydro-1H-pyrazole Derivatives Imidazole-, Thiazole-, Pyrrole- and Triazole-based CB1 Receptor Antagonists Pyridine-, Phenyl-, Pyrimidine- and Pyrazole-based CB1 Receptor Antagonists Azetidine-based CB1 Receptor Antagonists Substituted Amide-based CB1 Receptor Antagonists Hydantoin-based CB1 Receptor Analogues Recent CB1 Receptor Antagonists
272 273 282 285 295 301 303 304 307
THERAPEUTIC APPLICATIONS OF CB1 RECEPTOR ANTAGONISTS
308
CB2 RECEPTOR ANTAGONISTS
310
SUMMARY AND FUTURE PROSPECTS
313
REFERENCES
313
INTRODUCTION This article aims to update the previous review of the cannabinoids in this series, which was written in 1998 by Mechoulam et al. [1]. The intervening 7 year period has seen a great deal of activity in the area that has both expanded our knowledge of cannabinoid pharmacology and delivered novel ligands and drug candidates. Cannabinoid ligands and their pharmacology have also been the topic of a number of recent review articles [2–7]. CANNABINOID RECEPTORS AND ENDOCANNABINOIDS The previous review article in this series described the discovery and cloning of cannabinoid CB1 and CB2 receptors and a classification of these receptors was provided in 2002 [1, 6]. Cannabinoid CB1 receptors are expressed primarily within the central nervous system (CNS), where they are widely distributed. The CB1 receptor is also present in some peripheral tissues. The CB2 receptor is found mainly in cells of the immune system and exhibits 48% homology with the CB1 receptor. Both receptors are coupled to Gi/o proteins. Evidence exists for the presence of additional cannabinoid receptor subtypes, as covered by a recent review article [8]. Furthermore, a recent patent application described high-affinity binding of a number of cannabinoid ligands to GPR55, suggesting that this might be one of the additional cannabinoid receptors responsible for the pharmacological observations [9].
J. ADAM ET AL.
209
Since the cloning of the cannabinoid receptors, their endogenous ligands, the endocannabinoids, have received a great deal of research interest. A number of recent review articles have extensively covered the endocannabinoid system [10–12], so the coverage in this article will be brief. The best studied of the endocannabinoids are anandamide (N-arachidonylethanolamine, AEA)(1) and 2-arachidonylglycerol (2-AG)(2). Anandamide was first identified from porcine brain extracts by Devane and co-workers in 1992 [13], while 2-AG was first reported in 1995 to have been isolated from canine gut [14] and rat brain [15]. More recently, noladin ether (2-arachidonylglyceryl ether, 2-AGE)(3) [16], virodhamine (O-arachidonyl-ethanolamine)(4) [17] and N-arachidonyl-dopamine (NADA)(5) [18] were proposed as endogenous ligands for the cannabinoid receptors. In a subsequent publication, the authors failed to detect noladin ether in mammalian brains and questioned the relevance of this compound as an endocannabinoid [19]. Anandamide, noladin ether and NADA have functional selectivity for CB1 receptors, virodhamine is CB2 selective and 2-AG is essentially non-selective. (1) Anandamide R = CONH(CH2)2OH n-C4H9
(2) 2-Arachidonylglycerol R = CO2CH(CH2OH)2 4
(CH2)3 R
(3) Noladin ether R = CH2OCH(CH2OH)2 (4) Virodhamine R = CO2(CH2)2NH2 (5) N-Arachidonyl-dopamine R = CONH(CH2)2-3,4-(OH)2-Ph
The biosyntheses of anandamide and 2-AG have been studied in depth [10]. These compounds appear to be synthesised on demand in response to certain stimuli, rather than being stored in cells. Little is known regarding the biosynthesis of noladin ether, virodhamine or NADA. The mechanism of release of endocannabinoids from cells and their subsequent re-uptake remain as points of discussion. Several groups have proposed the existence of an anandamide transporter that can selectively release and remove endocannabinoids from their site of action [20–22]. Indeed, a number of compounds have been proposed as inhibitors of the putative anandamide transporter as will be outlined in the following section. An alternative suggestion is that the release and re-uptake of endocannabinoids is a simple diffusion process, with concentrations and hence diffusion being driven by the enzyme fatty acid amide hydrolase (FAAH) [23]. This suggestion has recently been refuted by Fegley and co-workers [24], who investigated anandamide internalisation and activity in wild-type and FAAH knock-out mice treated with anandamide transport (ANT) inhibitors, concluding that anandamide uptake was independent of FAAH activity. The discussions will only be concluded once the putative anandamide transporter is identified and cloned.
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RECENT PROGRESS IN CANNABINOID RESEARCH
FAAH was originally purified and cloned from rat liver microsomes and is able to catalyse the hydrolysis of anandamide and 2-AG, in addition to other long-chain fatty acid amides [25]. Studies into the structure and role of this enzyme have generated interest in the potential therapeutic applications of FAAH inhibitors [26–28]. FAAH knock-out mouse brains contained 15fold higher levels of anandamide than their wild-type counterparts and these animals have also been shown to be more responsive to exogenously administered anandamide [29]. These animals also showed a reduced response to painful stimuli, supporting the hypothesis that FAAH inhibition may provide novel analgesics. Levels of 2-AG were not elevated in the FAAH knock-out animals, apparently due to the existence of alternative metabolic fates for this compound [30].
ANANDAMIDE TRANSPORT INHIBITORS To date the ANT inhibitors reported in the literature are all derivatives of longchain fatty acids. These long-chain fatty acids can be split into three regions: the hydrophobic chain, the carboxamide/carboxylate group and the polar head group. These regions are highlighted for anandamide (1) (Figure 6.1). Comprehensive reviews of ANT inhibitors and their potential as drugs for CNSrelated disorders have been recently reported [31, 32], therefore the following discussion will only highlight some of the structure–affinity relationships (SAR) established. The interested reader is directed to the review of Lo´pez-Rodrı´ guez et al. [31] for a more detailed discussion.
carboxamide/carboxylate group
hydrophobic chain O N H
n-C5H11
OH
polar head group (1) Anandamide
Fig. 6.1 Structure of anandamide.
J. ADAM ET AL.
211
Table 6.1 SAR OF THE HYDROPHOBIC CHAIN [33] R-CONH(CH2)2OH Cpd.
R
IC50 (mM)
a
15.1
(1)
n-C4H9
(6)
4 (CH2 )3-
10.6
n-C6H13 2
(CH2 )7-
(7)
10.5
n-C8 H17 (8) (9) a
n-C8H17 n-C17H35
(CH2 )7(CH2)7-
>100 >100
Anandamide.
THE HYDROPHOBIC CHAIN
A variety of long-chain hydrocarbons have been found to be tolerated in this position as well as the parent hydrocarbon. In general, a minimum of one double bond located near or at the mid-point of the carbon chain is required for activity. In addition, the double bond must be cis configured (Table 6.1) [33].
THE CARBOXAMIDE/CARBOXYLATE GROUP
A wide range of groups are tolerated in this position including esters, amides, thioesters, ureas, acyl hydrazines and carbamates exhibiting a wide range of potencies and microsomal stabilities (Table 6.2) [24, 33–36].
THE POLAR HEAD GROUP
The head group has been extensively studied and a diverse range of groups are tolerated including aliphatic, cyclic, aromatic and heteroaromatic groups [33, 34, 37]. In general, the presence of a polar head group is favoured but not essential. The presence of aromatic/heteroaromatic moieties is well tolerated; in addition, bulky groups can also be accommodated (Table 6.3). The furan derivative (23) has been tested in vivo in tests of motor activity (open-field test) and antinociception (hot-plate test) as well as its capacity to enhance the hypokinetic and/or analgesic actions of subeffective doses of
212
RECENT PROGRESS IN CANNABINOID RESEARCH Table 6.2 SAR OF THE CARBOXAMIDE/CARBOXYLATE GROUP n-C4H9
IC50 Ref. Cpd. R (mM)
Cpd. R
(1)
4 (CH2)3 R
–CONH(CH2)2OH
15.1
IC50 Ref. (mM)
[33] (14) H N
1.78 [35]
Me
H N
OH
O
(10)
–CO2(CH2)2OH
6.7
[33] (15)
O (11)
–CONH2
9
7.1
H N
O
OH Me
[33] (16)
3.29 [35]
Me
H N
[35]
O
OH
O
(12)
25
-CON
[33] (17)
OH
O
O
N H (13)
-C (O)SCH2
O
9
H N
[34] (18)
17.7
[36]
2.5
[24]
OMe OH
H N O
anandamide [38]. It was found that (23) was mostly inactive when administered alone, but was able to potentiate the action of an exogenous dose of anandamide that produced no effect by itself.
FATTY AMIDE ACID HYDROLASE INHIBITORS The enzyme FAAH was first identified in the 1980s [39], cloned in the 1990s [25] and more recently, the crystal structure [40] of FAAH bound to methyl arachidonyl phosphonate has been published. The FAAH enzyme has wide substrate specificity capable of catalysing the hydrolysis of a wide variety of
J. ADAM ET AL.
213
Table 6.3 SAR OF THE POLAR HEAD GROUP n-C4H9
Cpd.
R
(1)
–(CH2)2OH
4 (CH2 )3 CONHR
IC50 (mM)
Ref.
Cpd.
15.1
[33]
(22)
R
IC50 (mM)
Ref.
2.2
[33]
0.8
[34]
16.5
[37]
OH (19)
–Et
48.5
[33]
(23)
10.4
[33]
(24)
37.7
[33]
O (20)
Me
-( CH2)2 O
O
OH
(21)
Me OH
bioactive fatty acid amides and esters including the endogenous cannabinoid ligands anandamide and 2-AG. Several other long-chain fatty acids and amides also act as substrates including palmitoylethanolamide, stearoylethanolamide, oleoylethanolamide and linoleoylethanolamide [41–44]. It has been postulated that selective FAAH inhibitors may elevate the levels of endogenous cannabinoids while avoiding the unwanted behavioural effects associated with the CB1 agonists. A number of comprehensive reviews on the pharmacology, possible therapeutic uses and SAR of FAAH inhibitors have been published recently [26, 45, 46]. Therefore, the following discussion will focus on highlighting the types of structures that act as FAAH inhibitors.
ORGANOSULFONATE AND ORGANOPHOSPHONATE ANALOGUES
The first compound to find widespread use as an inhibitor of AEA breakdown was phenylmethylsulfone (PMSF) (25). This non-selective serine protease inhibitor has been found to act as an irreversible inhibitor of FAAH preventing AEA breakdown [41, 47], and is commonly added to binding
214
RECENT PROGRESS IN CANNABINOID RESEARCH
assays to improve the metabolic stability of AEA [48, 49]. PMSF has been found to increase the potency of AEA in inhibiting contractions in the guinea pig myenteric plexus [50], and to potentiate the effects of anandamide in vivo [51, 52]. However, the low potency (IC50 ¼ 290 nM) [53] and poor selectivity of this ligand has led to the development of more potent and selective sulfonyl fluoride inhibitors of FAAH. A number of fatty acid sulfonyl fluorides with improved potencies have been reported [53, 54], in particular, palmitylsulfonyl fluoride (26) and arachidonylsulfonyl fluoride (27, AM374). Both these derivatives can inhibit FAAH with IC50s in the low nanomolar range (IC50 values of 7 and 0.11 nM, respectively). In addition, both these compounds have good selectivity over the CB1 receptor (IC50 values of 520 and 304 nM, respectively). Furthermore, AM374 (27) has been found to produce a significant inhibition of [3H]-acetylcholine release from hippocampal brain slices [55] and to enhance the behavioural effects of low doses of anandamide in vivo [56]. PhCH2SO2F
n-C16H33SO2F
(25)
(26)
n-C4H9 2
(30)
n-C4H9
4 (CH2 )4 R
n-C12H25PO(OMe)F (29)
(27) R = SO2F (28) R = PO(OMe)F
(CH2 )10 PO(OMe)F n-C8H17PO(OMe)F (31)
n-C3H7
4 (CH2 )3 COCF3
(32)
A number of organophosphate compounds have been found to irreversibly inhibit FAAH, the most studied being methylarachidonyl fluorophosphonate, MAFP (28) [57]. MAFP was initially designed and developed as an inactivator of cytosolic phospholipase A2 (cPLA2) [58, 59]. It was found to be a highly potent and selective inhibitor of FAAH (relative to other amide hydrolytic enzymes). However, it also bound irreversibly to the CB1 receptor and prevented the subsequent binding of CP 55,940 [60]. A detailed study on modifications to the alkyl chain of (28) has recently been carried out [61]. Both saturated (29) and unsaturated (30) analogues inhibit FAAH. In particular, the fully saturated C-12 derivative (29), gave a highly potent inhibitor of FAAH (IC50 ¼ 3 nM) and the CB1 receptor (K D ¼ 2:5 nM). Shortening of the alkyl chain to the C-8 derivative (31), O-1887, gave a slight drop in FAAH affinity (IC50 ¼ 15 nM) but importantly, (31) failed to interact with the CB1 receptor (KD>10,000 nM).
J. ADAM ET AL.
215
TRIFLUOROMETHYLKETONES
The first substrate analogue inhibitors of FAAH were reported in 1994. The anandamide analogues prepared represented three classes of putative transition-state inhibitors: a-trifluoromethyl ketones, a-ketoesters and a-ketoamides [62]. In the initial screening studies, it was found that the trifluoromethyl ketone compounds tested were effective inhibitors of AEA hydrolysis. A selected set of a-keto esters also inhibited hydrolysis, while a-keto amides were ineffective. In particular, arachidonyl trifluoromethyl ketone (32), gave almost 100% inhibition of anandamide hydrolysis. A detailed investigation of the structural requirements for FAAH inhibition with a-trifluoromethyl ketones has been carried out by Boger and co-workers [63]. The electrophilic carbonyl is essential for activity; replacement with a methyl ketone results in complete loss of activity (Table 6.4, (33) and (34)). Saturated alkyl chains are tolerated but must be at least seven carbons long, shortening the chain length further results in dramatic losses of potency (Table 6.4, (36)–(39)). In contrast, extending the chain from seven through seventeen carbons has little effect on potency (Table 6.4, (36) and (38)). Incorporation of the oleamide D9,10 cis double bond is favoured with an almost 2-fold increase in potency compared to its saturated congener (cf. (33) and (36)), the trans isomer is also tolerated (Table 6.4, (35)). Replacement of the oleamide D9,10 cis double bond and alkyl tail with a phenyl group (Table 6.4, (40)) gives a further enhancement in affinity.
Table 6.4 FAAH INHIBITORS – TRIFLUOROMETHYL KETONES [63]
O R
Cpd. (33) (34)
(36) a
IC50 (mM)a
R
n-C8H17
(CH2)7-
n-C8 H17
(CH2 )7 COMe
(35) n-C8H17 n-C17H35-
CF3
(CH2)7-
IC50 (mM)a
Cpd.
R
(37)
n-C9H19-
0.13
(38)
n-C7H15-
1.2
0.78
(39)
n-C5H11-
52.8
0.24
(40)
Ph(CH2)7-
0.46 >100
Inhibition of [14C]-oleamide hydrolysis in rat liver plasma membranes.
0.12
216
RECENT PROGRESS IN CANNABINOID RESEARCH a-KETOHETEROCYCLES
The Boger group [64–66] has extensively studied the use of a-ketoheterocycles as FAAH inhibitors. In their initial studies, a range of a-ketoheterocycles based on oleic acid was synthesised. A range of five- and six-membered monocyclic heterocycles and three bicyclic heterocycles (benzothiazole, benzimidazole and benzoxazole) was examined. Although many of the compounds tested were found to inhibit FAAH activity with micromolar affinities, the best results were obtained with heterocycles that incorporated a weakly basic nitrogen a – to the heterocycle (Table 6.5) [64]. Following on from this, the benzoxazole scaffold was examined in detail and it was found that incorporation of a second weakly basic nitrogen to give the a-keto oxalopyridone (47) led to a >150-fold enhancement in binding affinity (Table 6.5). Using this head group, modifications to the oleyl side chain were investigated and found to parallel the SAR found with the trifluoromethyl ketones previously studied by Boger [63] (see above), the best results being obtained with saturated chain lengths of C12–C18. A further enhancement in potency was seen when a phenyl ring was introduced into an optimally long side chain. The best compound identified from this series was the oxalopyridone (48) with Ki values of 94 and 200 pM in human and rat, respectively. However, when tested in vivo, this compound failed to potentiate the effects of anandamide [67]. Table 6.5 FAAH INHIBITORS – a-KETOHETEROCYCLES [64] n-C8H17
Cpd.
R
(33)
–CF3
Ki (mM) 0.082
(41)
N
(42)
S N
(43)
O N
>100
0.017
(CH2 )7 COR
Cpd.
R
Ki (mM)
(44)
N
(45)
O N
(46)
N
0.37
>100
0.11 N
N
0.065
N N Me
(47)
N O
N
0.0023
J. ADAM ET AL.
217
Following on from these studies, Boger and co-workers [65, 66] disclosed a series of substituted oleyl a ketooxazoles exploring the introduction of substituents in the 4 and 5 positions. Analogous to the observations made in the benzoxazole series, incorporation of a second heterocyclic ring containing a weakly basic nitrogen proximal to the oxazole substantially increased FAAH inhibition. Replacement of the oleyl side chain with the optimal side chain found in the trifluoromethyl ketone and benzoxazole series gave a further enhancement in FAAH inhibitiory activity. In particular, the pyridyl oxazole derivative (49) had a Ki of 7 nM and was found to have good selectivity over a panel of serine hydrolases. In addition, the compound failed to bind to the CB1 or CB2 receptor [67]. Compound (49) has been tested in vivo in mice and found to augment not only exogenously administered AEA but also the activity of endogenously produced fatty acid amide in models of antinociception. These effects could be inhibited by pretreatment with a CB1 receptor antagonist. Importantly, the antinociceptive properties of (49) occurred in the absence of any significant effects on motility or motor coordination that typically accompany the global activation of CB1 receptors by direct agonists [67]. Boger and co-workers [68] have recently extended their studies to encompass heterocyclic sulfoxides and sulfones. In general, these compounds were less potent (up to 1,000 fold) than their a-keto heterocycle congeners. The best results were obtained with the benzoxazole sulfoxide (50) with a Ki of 2.6 mM. O Ph(CH2)5
N (48)
O
O
O
O
Ph(CH2 )5 N
N (49)
Ph(CH2 )5
S
O N
N (50)
CARBAMATES
Tarzia et al. [69, 70] have recently reported the FAAH inhibitory activity of a series of alkylcarbamic acid aryl esters. The starting point for their studies was the known serine hydrolase inhibitor carbamyl (51) that had no activity at FAAH. Replacement of the small methyl group of carbamyl (51) with more lipophilic groups and, in particular, bulky lipophilic groups resulted in increased affinity at FAAH (Table 6.6). Exploration of replacements of the naphthyl moiety revealed that replacement with a biphenyl group resulted in improved affinity and in particular, the 3-biphenylyl group proved effective
218
RECENT PROGRESS IN CANNABINOID RESEARCH Table 6.6 SAR OF CARBAMYL DERIVATIVES [69, 70] R1NHCOOR2
Compound
R1
R2
IC50 (nM)
(51) (52) (53) (54) (55)
Me Et Cyclohexyl Cyclohexyl Cyclohexyl
1-Naphthyl p-Tolyl 2-Naphthyl 4-Biphenylyl 3-Biphenylyl
>30,000 >30,000 324 2,297 63
(Table 6.6). The most potent compound identified in this series was the cyclohexyl 3-biphenylyl carbamate (55) with an IC50 of 63 nM. Following on from these initial studies, a systematic exploration of phenyl ring substitution on the distal phenyl ring was carried out [70]. A range of groups were tolerated giving good inhibitory activity in the nanomolar range. The compound giving best potency in this series was the amido biphenyl derivative (56) with an IC50 value of 4.6 nM. In addition, this compound was selective against three other serine hydrolases, had no effect on the AEA transporter and failed to bind to the CB1 and CB2 receptors [71]. When tested in vivo, (56) augmented the levels of endogenous AEA and produced CB1dependent anxiolytic and antinociceptive properties. Importantly, as with the a-keto heterocycle derivative (49), the carbamate inhibitor (56) did not induce any of the common side effects associated with direct CB agonists. CONH2
MeNH
O O
H N
O O
(51)
(56)
Bristol-Myers Squibb has recently disclosed two different series of carbamate-based FAAH inhibitors. The first of these is a series of 4,5-diarylimidazoles in which 30 compounds are specifically claimed, an example being compound (57). This compound is reported to have an IC50 value of o10 nM. In addition, (57) was also active in vivo in rodent models of chemoinduced, thermal and neuropathic pain [72]. The second series of compounds is based on oxime carbamoyl FAAH inhibitors such as (58). Compound (58) is reported to have an IC50 value of o10 nM and activity in rodent models of inflammatory pain, thermal pain and inflammatory oedema [73].
J. ADAM ET AL.
219
A series of dioxane carbamate inhibitors has recently been disclosed by Sanofi-Aventis. No biological data are provided for specific compounds, although most compounds are described as having IC50 values for FAAH inhibition ranging from 0.005–1 mM. Compound (59) is one of over 25 compounds specifically claimed [74]. Sanofi-Aventis has disclosed a series of piperidine- and piperazine-alkyl carbamates as cannabinoid and/or FAAH modulators. No compounds are specifically claimed in the patent. Compound (60) is reported to have an IC50 value of 85 mM and is active in a mouse pain model [75]. Ph N N
H N
F H N
Ph (CH2 )6
n-BuO
O (57)
Ph
(58)
NHCO2 CH2 CONH2 O
Me
N
O
Me
O
O
O
F
NHCO2 CH2 CONHMe N
N (60)
(59)
OTHER STRUCTURES
A number of arachidonic acid substrate analogues have been found to inhibit FAAH. Hillard and co-workers [37] found during their studies into the SAR of ANT inhibitors (see above) that these ligands also inhibit FAAH. From these studies, the most potent FAAH inhibitor identified was chloromethylarachidonyl amide (61) with an IC50 value of 14 nM. This ligand also bound to the CB1 receptor in the presence and absence of PMSF but with different binding affinities (84 nM and 2.6 mM, respectively) suggesting that it may also be acting as a substrate at FAAH. In addition, the archetypal AEA transport inhibitor AM 404 (22) was also an excellent inhibitor of FAAH (IC50 ¼ 0:5 mM) more than 5-fold lower than its binding affinity value for AEA uptake (IC50 ¼ 3:4 mM) [37]. Another arachidonyl-based inhibitor of FAAH is arachidonyl serotonin (62), which was reported to inhibit FAAH from rat basophilic leukaemia cells with an IC50 value of 5.6 mM, and with very little affinity at CB1 receptors [76].
220
RECENT PROGRESS IN CANNABINOID RESEARCH
A series of palmitoylethanolamine-derived inhibitors has been described in the literature as FAAH inhibitors [77, 78]. This study explored the effect of shortening the chain length and replacement of the ethanolamine head group with primary, secondary and tertiary amide alternatives. Of the compounds synthesised and tested, two compounds gave reasonable affinities for FAAH inhibition, palmitoyl-isopropylamide (63) (IC50 ¼ 13 mM) and palmitoyl-allylamide (64) (IC50 ¼ 3:4 mM). Both these compounds had little affinity for either CB1 or CB2 receptors. Finally, derivatives of the endogenous compound 2-octyl-g-bromoacetate (65) have been reported as FAAH inhibitors [79]. In a limited SAR study, it was found that replacement of the bromine with a chlorine atom had little effect on affinity. The replacement of the alkyl chain with oleyl-chain mimics resulted in an increase of affinity for FAAH (approximately 5-fold). The removal of the halogen and replacement with either a proton or methyl resulted in inactive compounds. The most potent compound identified in this series was compound (66) with an IC50 value of 0.6 mM [79]. (61) R = Cl n-C4H9
4 (CH2)3 CONH(CH2 )2 R
OH
(62) R = N H
n-C15H31CONHallyl
n-C15H31CONHs-Bu (63)
O
Me n-C6H13
O (65)
(64)
O
O Br
n-C8H17
(CH2)4
O
O Br
(66)
CANNABINOID RECEPTOR AGONISTS CLASSICAL CANNABINOIDS
Before the discovery of specific cannabinoid receptors, the term ‘cannabinoid’ was used to describe the biologically active constituents of the Cannabis sativa plant, including D9-THC (67), cannabidiol (68) and their analogues and derivatives, many of which have characteristic pharmacological effects.
J. ADAM ET AL.
221
Table 6.7 D9-THC AND CANNABIDIOL Mouse ED50 (mmol/kg)a Cpd.
CB1 Ki (nM)
(67) D9-THC
CB2 Ki (nM)
41 21
(68) Cannabidiol
SA
TF
3.2
RT
4.5
Ref.
4.5
[80] [81]
36
4,350 1,265
108
483
58
[80] [82] [81]
230 2,860
a
General procedures for mouse behavioural experiments are described in Reference [141]. SA ¼ spontaneous activity; TF ¼ tail flick; RT ¼ rectal temperature.
D9-THC, the main psychoactive component of cannabis, is a moderately potent partial agonist of the CB1 and CB2 receptors, while cannabidiol has little affinity for either receptor (Table 6.7). The term ‘classical cannabinoids’ is used to describe cannabinoid receptor modulators structurally related to (67), which have a tricyclic dibenzopyran core. While several other structural types of cannabinoid receptor modulators have been discovered in recent years, the classical cannabinoids are still by far the most extensively studied group in terms of SAR and pharmacology. 11
7 Me
Me
9
1
10
OH 10a 1
8 7
2'
(67)
4
3
3
5
2
6a Me O Me 6 5
2
6
1'
4
4' 3'
Me 10
5'
8
2'
OH 3' 4'
9
HO
1'
5'
(68) Cannabidiol
∆9-THC 11 9 8
Me 10
OH
10a
1
7
2
6a Me O Me 6 5
(73) ∆8-THC
2' 4
3
1'
4' 3'
5'
1''
2''
4'' 3''
5''
222
RECENT PROGRESS IN CANNABINOID RESEARCH
The naturally occurring isomers (67) and (73) have very similar pharmacological profiles. Owing to the greater chemical stability of (73) and its analogues, SAR around this template has been much more widely studied than those around (67). While caution must be exercised in comparing data obtained by different laboratories and using different assay formats, the general trends that emerge provide a qualitative picture of the structural requirements for activity. Three pharmacophoric elements around the classical cannabinoid template have been shown to be important for CB1 receptor binding and/or activation: the hydrocarbon side chain at C3, the substituent at C9 and the phenolic hydroxyl group at C1 [83–85]. The effects of alterations to the tricyclic core have also been explored. Quantitative SAR (QSAR) pharmacophore models have been used to rationalise the observed SAR and to predict the activity of novel compounds [86–89].
C3 Side-chain modifications During the 1940s, more than 15 years before the structure of (67) was elucidated as the major psychoactive constituent of marijuana [90] and 40 years before the identification of specific cannabinoid receptors, Adams and co-workers [91, 92] demonstrated that a 10 ,20 -dimethylheptyl (10 ,20 -DMH) side chain was the optimal 3-alkyl substituent for cannabinoid activity in vivo in the D6a,10a-THC series. In this early research, the ataxia test in dogs was used as a standard measure of psychoactive cannabinoid activity. The isomeric 10 ,10 -DMH side chain was found to be less potent than the 10 ,20 DMH one in the D6a,10a-THC series, but the two side chains have similar binding affinity and potency in the D8-THC series. The 10 ,10 -DMH side chain has the advantage of not containing any chiral centres and has therefore found its place as a widely used replacement for the pentyl side chain of (67) and (73) in synthetic analogues. Tables 6.8–6.11 illustrate the wide range of C3 side-chain modified D8THC analogues that have been reported in the literature, together with associated in vitro and in vivo data. The affinity of classical cannabinoid analogues for the CB1 receptor has been shown to correlate with depression of spontaneous activity and the production of antinociception, hypothermia and catalepsy in mice, and with psychomimetic activity in humans [93]. However, in some cases, there were unexplained differences between the observed trends in binding affinity and the trends in activity in mouse behavioural models. This may point to differences in efficacy among full agonists, partial agonists and antagonists/inverse agonists, or may reflect differences in in vivo metabolism or blood–brain barrier penetration or a combination of these factors.
J. ADAM ET AL.
223
Table 6.8 D8-THC ALKYL SIDE CHAINS Me
OH
Me Me
O
R
Mouse ED50 (mmol/kg) Cpd. R (69) (70) (71) (72) (73) (74) (75) (76) (77) (78) (79) (80) (81) (82) (83) (84) (85) (86) (87) (88) (89) (90) (91) (92) (93) (94) (95) a b
CB1 Ki (nM) CB2 Ki (nM) SA
14 14 65 11 44 29 1R-Me-pentyl 7.6 1S-Me-pentyl 20 2R-Me-pentyl 19 2S-Me-pentyl 11 3R-Me-pentyl 38 3S-Me-pentyl 53 4-Me-pentyl 141 1,1-Dimethylpentyl 3.9 n-Hexyl 41 1,1-Dimethylhexyl 2.7 n-Heptyl 22 0.43 1,1-Dimethylheptyl 0.77 0.83 1S,2R-DMH 0.46 1R,2S-DMH 0.6 1R,2R-DMH 0.84 1S,2S-DMH 0.81 n-Octyl 8.5 1,1-Dimethyloctyl 0.09 1,1-Dimethylnonyl 1.6 1,1-Dimethyldecyl 6.1 1,1-Dimethylundecyl 26 1,1-Dimethyldodecyl 126
a
1,1-Dimethylethyl 1,1-Dimethylpropyl n-Butyl 1,1-Dimethylbutyl n-Pentyl (D8-THC)
a a
9.0 2.9
TF
RT
Ref.
>100 25 10 1.9 4.8
a
[96] [96] [98] [96] [80] [97] [94] [94] [94] [94] [94] [94] [94] [96] [98] [96] [98] [99] [80] [100] [101] [101] [101] [101] [98] [96] [96] [96] [96] [96]
a
6.3 4.2 4.5
25 6.1 0.3 0.6 0.6 10 3 3 1.1 1.2 0.17 0.14
1.5 4.8 2.1 6.1 5.2 1.2 1.0 0.4 1.8 0.21 0.61
2.4 4.8 2.1 1.5 12 4.8 30 1.5 0.10 0.11 0.16
0.27
0.14
0.15
0.03 0.04 0.45 0.12 0.39 0.24 2.1
0.03 0.10 0.42 0.18 0.34 0.30 5.1 38
0.07 0.07 0.63 0.19 0.24 1.2 3.2 9.4
0.39 0.49
b b
b
Inactive
Effects not dose dependent. Partial effect reported (SA/TF max o80%; RT max decrease o4 1C).
b
224
RECENT PROGRESS IN CANNABINOID RESEARCH
Table 6.9 D8-THC CONSTRAINED, UNSATURATED AND AROMATIC HYDROCARBON SIDE CHAINS Me OH
Me Me
O
R
Mouse ED50 (mmol/kg) Cpd.
R
(96) (97) (98) (99) (100) (101) (102) (103)
(1-Hexyl)c-propyl (1-Hexyl)c-pentyl Adamantyl (1-c-Pentyl-1-methyl)ethyl (1-c-Hexyl-1-methyl)ethyl (1-c-Heptyl-1-methyl)ethyl But-3-ynyl Hex-2-ynyl
(104) (105) (106) (107) (108) (109) (110)
Hept-1-ynyl 1,6-Heptadiynyl cis-Hept-1-enyl 1-Methyleneheptyl Oct-2-ynyl 2,7-Octadiynyl cis-Oct-2-enyl
(111) (112)
Oct-3-ynyl cis-Oct-3-enyl
(113)
Oct-4-ynyl
(114) (115)
cis-Oct-4-enyl Non-2-ynyl
(116) (117)
Benzyl 1,1-Dimethylbenzyl
a
CB1 Ki CB2 Ki (nM) (nM) 0.44 0.45 7 0.34 0.57 0.94 367 11 11 36 0.65 172 460 0.86 2.2 4.9 5 4.7 3.2 4.6 9.0 3.4 6.9 19 21 11 3.7 4 68 12
SA
TF
RT
0.86 1.92 0.39 0.65 0.22
a
a
a
a
a
148
9.6 3.3
28.7 3.0 a
a
a
3.1 a
a
0.09
0.09
0.13
0.51 1.4
0.03 0.088
0.05
0.11
2.1 2.8 1.1 0.07
a
1.5 0.16
3.3 a
0.09
a
a
0.19
a
3.3
4.2
0.22 2.2 6.6
0.12 0.31 0.41
0.57 1.9 3.8
86 0.91
Partial effect reported (SA/TF max o80%; RT max decrease o4 1C).
Ref.
[82] [102] [103] [97] [97] [97] [98] [98] [104] [98] [105] [107] [98] [98] [105] [98] [104] [98] [98] [107] [98] [98] [107] [98] [107] [98] [98] [104] [109] [109]
J. ADAM ET AL.
225
Table 6.10 D8-THC SIDE CHAINS FUNCTIONALISED IN THE 10 -POSITION Me
OH
Me Me
O
R
Cpd.
R
CB1 Ki (nM)
CB2 Ki (nM)
Ref.
(118) (119) (120) (121) (122) (123) (124) (125) (126) (127) (128) (129) (130) (131) (132)
1-Oxoheptyl 1-Benzoyl 1-Hydroxyheptyl (2-Hexyl)-1,3-dioxolane-2-yl 1,3-Dithiolane-2-yl (2-Pentyl)-1,3-dithiolane-2-yl (2-Hexyl)-1,3-dithiolane-2-yl (2-c-Pentyl)-1,3-dithiolane-2-yl (2-c-Hexyl)-1,3-dithiolane-2-yl (2-c-Heptyl)-1,3-dithiolane-2-yl (2-Phenyl)-1,3-dithiolane-2-yl (2-Hexyl)-4,5-dimethyl-1,3-dithiolane-2-yl (2-Hexyl)-1,3-benzodithiolane-2-yl 2-Hexyl-1,3-dithiane-2-yl 1-Iodoheptyl
22 297 86 0.52 168 0.85 0.32 9.5 1.9 1.8 17 32 57 1.8 328
84 24 66 0.22 103 0.58 0.52 2.7 1.1 6.6 18 20 257 3.6
[105] [109] [105] [102] [102] [97] [105] [97] [97] [97] [109] [102] [102] [102] [110]
Table 6.8 shows the simple alkyl side-chain modifications of (67). The binding affinity of these analogues increases with increasing side-chain length, from butyl up to octyl. Methyl substituents in the 10 - and 20 -positions of the side chain have a beneficial effect on CB1 receptor affinity and in vivo potency, as previously shown by Adams. Huffman et al. [94] further demonstrated that methyl substitution in the 30 -position resulted in compounds with comparable binding affinity to the parent compound, while methyl substitution in the 40 -position decreased binding affinity. A trend similar to that seen for the pentyl side chain was observed for monomethyl substitution on the heptyl side chain (data not shown) [95]. Reduced CB1 binding affinity was seen for compounds with 10 ,10 -dimethyl-substituted side chains longer than octyl (92–95). These compounds also showed reduced, or no in vivo activity [96]. Compounds with conformationally restrained side chains, unsaturated side chains and aromatic side chains have been synthesised in order to better define the steric requirements of the binding site in the side-chain region. These are shown in Table 6.9. On average, the conformationally restrained
226
Table 6.11 OTHER FUNCTIONALISED D8-THC SIDE CHAINS Me
Me Me
O
R
Mouse ED50 (mmol/kg) Cpd.
R
(73)
Pentyl (D8-THC)
(133)
5-Fluoropentyl
CB1 Ki CB2 Ki (nM) (nM) 44
SA
a
2.9
12
TF
RT
4.8 4.8 6.3
Ref.
4.5 49 31
57 9 (134)
5,5,5-Trifluoropentyl
0.82
0.82
a
25 30 (135)
5-Bromopentyl
a
0.51
1.0
0.23
0.90
1.8
0.08
0.05
0.09
0.13
0.13
0.19
7.6 (136)
5-Iodopentyl 7.8
(137)
5-Bromo-1,1-dimethylpentyl 0.43
(138)
7-Bromo-1,1-dimethylheptyl 1.3
[80] [112] [112] [110] [81] [112] [110] [81] [112] [110] [112] [110] [80] [110] [80] [110]
RECENT PROGRESS IN CANNABINOID RESEARCH
OH
(139) (140) (141) (142) (143) (144) (145) (146) (147)
(149) (150) (151) (152) (153) (154)
a
6-Cyano-1,1-dimethylhexyl 5-Cyano-5-hydroxy-1,1-dimethylpentyl 6-Cyano-6-phenyl-1,1-dimethylhexyl 5-(p-Cyanophenoxy)-1,1-dimethylpentyl 5-Carboxy-1,1-dimethylpentyl 5-(N,N-dimethyl carboxamido)-1,1-dimethylpentyl 5-(N,N-diethyl carboxamido)-1,1-dimethylpentyl 5-[N-(piperidin-1-yl)carboxamido]-1,1-dimethylpentyl 5-[N-(p-chlorophenyl)carboxamido]-1,1-dimethylpentyl 5-[N-(o,p-dichlorophenyl)carbox amido]-1,1-dimethylpentyl 5-[N-(p-aminosulfonylphenyl) carboxamido]-1,1-dimethylpentyl 5-{N-[(p-aminosulfonylphenyl) methyl]carboxamido}-1,1-dimethylpentyl 5-{N-[(p-aminosulfonylphenyl)ethyl] carboxamido}-1,1-dimethylpentyl
1.3 0.71 0.43 1.8 0.6 0.19 13 21 9.2 18 3 1.5 222 0.86 2.5 13 24 1.2 4.5 187 395 18 41 19 42 60 180 29 246
0.29 1.0 0.02
0.01
0.52
0.02
0.03
0.01
1.4
0.29
3.9
3.1
9.2
0.22
2.5
9.2
0.004
0.075
0.29
0.20
0.23
0.91
0.09
0.34
0.36
[82] [82] [113]
1.1 2.9 3.2 14 5.8 1.1 4.0 2.0 2.5 3.2 11
a
a
a
a
26
7.0
37 3.8 10 2.3
5.9
56
a
a
>176
3.1 67 >176
[113] [107] [113] [107] [113] [107] [113] [107] [107] [113] [107] [113] [107] [113] [107] [113] [107] [113] [107] [113] [107] [113] [107] [113] [107]
J. ADAM ET AL.
(148)
(1-Hexyl)-2,2-dichlorocyclopropyl (1-Hexyl)-2,2-dibromocyclohexyl 5-Cyano-1,1-dimethylpentyl
Partial effect reported (SA/TF max o80%; RT max decrease o4 1C).
227
228
RECENT PROGRESS IN CANNABINOID RESEARCH
10 ,10 -dimethyl cycloalkyl side-chain analogues of (99–101) were found to have in vitro activity similar to their straight-chain analogues (83), (85) and (91), respectively. However, there was no trend of increasing CB1 receptorbinding affinity with increasing ring size to mirror the effect of increasing chain length in the open-chain series [97]. Compounds with cis double bonds in the side chain were in general found to be more potent and efficacious than their triple-bond congeners, both in in vivo and in in vitro functional assays [98, 106, 107]. QSAR models have been generated for the compounds with unsaturated [108] and 10 ,10 -dimethyl [96] side chains to determine more precisely the pharmacophoric requirements of the receptor. It is postulated that for optimum potency, the side chain must be of a suitable length and flexibility to have the ability to loop back so that its terminus is in proximity to the phenolic ring. The widely used, potency enhancing 10 - and 20 -methyl substituents would be expected to increase the tendency of the side chain to adopt a looped back, rather than an extended conformation. Aromatic rings are also tolerated in the side chain, as demonstrated by Krishnamurthy et al. [109]. The 10 ,10 -dimethylbenzyl compound (117) showed higher binding affinity than the simple benzyl compound (116). Compound (117) also showed around 13-fold selectivity for CB2 over CB1 binding. A range of different functional groups has been introduced into the 10 position of the C3 side chain, as shown in Table 6.10. A number of different functionalities were shown to be tolerated in this position, with lipophilic groups such as methyl and dithiolane being preferred over polar groups such as ketones and alcohols. Ketone (118) had similar CB1 receptor affinity to the unfunctionalised n-heptyl compound (84), while alcohol (120) had lower affinity. The phenyl ketone (119) had lower CB1 receptor affinity than the simple benzyl-substituted compound (116), but higher CB2 affinity, with about 12-fold selectivity for CB2 over CB1. A dithiolane group in the 10 -position has been shown to be at least as effective as the 10 ,10 -dimethyl group in enhancing the binding affinity of the classical cannabinoids, as can be seen by comparing compounds (123) and (124) with compounds (83) and (85). However, the constrained dithiolane compounds (125–127) showed decreased activity compared to their 10 ,10 dimethyl analogues (99–101). In contrast to its 10 ,10 -dimethyl and ketone analogues, (117) and (119), the phenyl dithiolane compound (128) does not exhibit any CB2 selectivity. Surprisingly, an iodo substituent in the 10 -position was not well tolerated, suggesting that this substituent had an unfavourable interaction with the receptor or a detrimental effect on the conformation of the side chain [110].
J. ADAM ET AL.
229
A range of 10 -substituted D8-THC, D9-THC and D6a,10a-THC derivatives, including many of those in Tables 6.9 and 6.10 has been disclosed in a patent application by Moore et al. [111]. The compounds are described as either agonists or antagonists of the CB1 and/or CB2 receptors. The in vivo activity of (1-cyclohexyl-1-methyl) ethyl compound (100) in a rat haemorrhagic shock model and the in vitro cytotoxic effects of the 10 ,10 -dimethylbenzyl compound (117) against glioma cells are described. Halogen, cyano, carboxylic acid and amide functional groups have been introduced at the terminus of the C3 side chain, as shown in Table 6.11. This area of the molecule seems to be tolerant to both lipophilic and more polar groups. Halogen substitution at the terminal carbon of the side chain led to enhancement of affinity, with the bulkier halogens, bromine and iodine, producing the largest effects [110, 112]. The less lipophilic 5-cyano-10 , 10 -dimethylpentyl and 5-(N,N-dimethyl-carboxamido)-10 ,10 -dimethylpentyl compounds (141) and (147) showed high affinity and in vivo potency similar to the 5-bromo-10 ,10 -dimethylpentyl compound (137). However, the cyanohydrin compound (143) showed decreased affinity and potency. This may indicate less tolerance for a hydrogen bond donor in this position. The corresponding carboxylic acid (146) showed very low binding affinity at CB1, but retained affinity at CB2. The piperidine hydrazide compound (149) exhibited high binding affinity despite the presence of a hydrogen bond donor. In this case, the hydrogen bond donor was separated by a longer linker group from the tricyclic core. Chloro- and sulfonamide-substituted aromatic amides showed decreased binding affinity and in vivo potency compared to the simple aliphatic amides. Side chains with an additional (CH2)1-2 linker between the amide and the phenylsulfonamide group resulted in partial or absent in vivo effects [113]. The (CH2)-linked compound, (153), showed around 80-fold selectivity for CB2 over CB1 binding [107]. A number of compounds have been prepared that contain both a double or triple bond and a terminal functional group in the side chain [98, 107, 114]. In general, the combined modifications reinforced the SAR trends seen for the individual modifications.
C9 Substituent modifications A major route of metabolism for (67) and (73) is oxidation at the C9 position to form hydroxymethyl and carboxyl metabolites. The hydroxymethyl metabolites are potent CB1 agonists with pharmacological profiles similar to the parent compounds, while the carboxy metabolites have reduced activity [115]; the 9-carboxy analogue of (73) does not bind to the CB1 receptor [93].
230
RECENT PROGRESS IN CANNABINOID RESEARCH Table 6.12 D8-THC C9 SUBSTITUENTS R
OH
Me Me
O
n-C5H11
Mouse ED50 (mmol/kg) Cpd. (73)
CB1 Ki (nM) CB2 Ki (nM) SA
R 8
Methyl (D -THC)
44 126
TF
2.9
4.8
a
4.8 23
(155) Hydroxymethyl (156) Carboxyl
55 26 >10,000
(161) (162) (163) (164)
1-Hydroxypropyl Phenyl Benzyl 2-Phenylethyl
49 a
b
15
107 (158) Isopropyl (159) n-Butyl (160) 1-Hydroxyethyl
4.5
7.4 a
(157) Fluoromethyl
RT
76 90 78 109 178 82 106
21 a c a
a a a a a a
Ref. [80] [93] [112] [116] [93] [117] [93] [115] [112] [93] [116] [116] [116] [118] [116] [116] [116] [116] [118]
a
Partial effect reported (SA/TF max o80%; RT max decrease o4 1C). Inactive. c See text. b
Some alternative substituents have also been introduced in the C9 position, as shown in Table 6.12. Of the direct methyl group replacements reported, small groups such as hydroxymethyl and fluoromethyl are tolerated, while larger groups in this position result in compounds with reduced in vivo potency and efficacy. The n-butyl derivative (159) was the only C9 methyl replacement analogue that showed a marked effect on hypothermia (5 1C decrease at 168 mmol/kg; ED50 not calculated) [116].
J. ADAM ET AL.
231
Table 6.13 D8-THC-DMH C9 SUBSTITUENTS Compound
CB1 Ki (nM) 8
(85) Methyl (D -THC-DMH)
0.77 0.83 0.10 0.23 32
(165) Hydroxymethyl (HU-210) (166) Carboxyl (CT-3)
CB2 Ki (nM) 0.49 0.17 171
Ref. [80] [100] [117] [120] [117]
The CB1 binding affinity of the hydroxymethyl and carboxyl analogues can be increased by substituting the C3 pentyl side chain for a dimethylheptyl side chain (Table 6.13). 11-Hydroxy-10 ,10 -DMH D8-THC, HU-210 (165), is an extremely potent cannabinoid agonist that has been widely used as a pharmacological tool [119]. Its (+) enantiomer, HU-211 (dexanabinol), which is in clinical development for the treatment of cognitive disorders, does not have high affinity for CB1 receptors [120]. The dimethylheptyl side-chain analogue of 9-carboxy-D8-THC, ajulemic acid, CT-3, (166) is currently in clinical development for treatment of pain and inflammation [121, 122]. Compound (166) does show some affinity for CB1 and CB2 receptors, but may also exert anti-inflammatory and analgesic effects through other mechanisms. It does not appear to be psychoactive in humans [123]. R OH (165) HU-210 R = CH2OH (166) CT-3 Me Me
R = COOH
n-C6H13
O Me
Me
C1 modifications In general, modification or deletion of the C1 phenolic hydroxyl group results in significantly reduced CB1 receptor affinity [93]. A number of 1-deoxy and 1-alkoxy D8-THC analogues have been shown to be selective ligands for the CB2 receptor (see below).
232
RECENT PROGRESS IN CANNABINOID RESEARCH
Core modifications There are limited examples in the literature where substituents have been introduced in other positions on the tricyclic core (Table 6.14). Substitution at the C2 position of the aryl ring with an iodo substituent resulted in only a slight drop in binding affinity. However, a nitro substituent in the 2-position was not tolerated. Molecular modelling studies suggested that this may be attributed to dramatic reductions in the electron density around the phenolic hydroxyl group and the oxygen in the pyran ring. Substitution at the C4 position with either a bromo or a nitro group resulted in loss of affinity at CB1, perhaps as the result of an unfavourable steric interaction in the binding site. The 2,4-diiodo and 2,4-dinitro compounds were also inactive [80]. Tetracyclic compounds in which the C3 side chain is conformationally restricted by linking to either the C2 or the C4 position have been described by Khanolkar et al. [99]. All the tetracyclic compounds had lower CB1 and CB2 affinity than the analogous non-constrained compounds (84). The besttolerated constraint was the ‘southbound’ constraint in compound (173). Table 6.14 D8-THC SUBSTITUTIONS AT C2 AND C4 Me OH
Me Me
2 R O 4
n-C5H11
Mouse ED50 (mmol/kg) Cpd.
R
(167) (168)
4-Br 2-I
(169) (170) (171) (172) (173) (174) (175)
2,4-di-I 2-NO2 4-NO2 2,4-di-NO2 (Tetracyclic) (Tetracyclic) (Tetracyclic)
a
CB1 Ki (nM)
CB2 Ki (nM)
5,250 89 >10,000 >10,000 1,630 >10,000 22 402 542
SA
TF
RT
Ref.
>250
>250 0.68
>250 20
>175 29 >275 >75
>175
>175 101 40 >75
[80] [112] [80] [80] [80] [80] [80] [99] [99] [99]
a
a
>275 >75
59 162 456
Partial effect reported (SA/TF max o80%; RT max decrease o4 1C).
J. ADAM ET AL.
233
Me
Me
OH
OH
Me Me
1
R
O
Me Me
O
n-C6H13
2
R
(173) R1 = H, R2 = n-C6H13 (174) R1 = n-C5H11, R2 = H
(175)
CANNABIDIOL DERIVATIVES (RESORCINOLS)
Cannabidiol has low affinity for CB1 and CB2 receptors and is not psychoactive, but has nevertheless shown a number of pharmacological activities of its own including anti-inflammatory and neuroprotective effects. Some side chain-modified cannabidiol derivatives have also been evaluated for cannabinoid receptor affinity and these are shown in Table 6.15. Bisogno et al. [124] have shown that, in contrast to classical cannabinoid derivatives, the unnatural (+) enantiomers of cannabidiol derivatives have higher affinity for CB1 and CB2 receptors than the natural ( ) enantiomers, as shown in Table 6.16. Within both the ( ) series and the (+) series, the SAR showed some similarity to those in the classical cannabinoid series, with extended and halogenated side chains tending to increase binding affinity. The 100 ,100 -dimethylheptyl, 7-hydroxy (+)-cannabidiol analogue (187) exhibited particularly high CB1 binding affinity. In the same study, cannabidiol and some of its analogues were also found to be weak agonists of VR1 receptors and weak inhibitors of anandamide uptake and degradation [124]. Further (+)-cannabidiol analogues were prepared and evaluated by Fride et al. [125], who showed that carboxylic acid derivatives (189) and (191) also have high CB1 receptor affinity [126]. In vivo activity was assessed in locomotion, rearing, catalepsy, analgesia (hot plate), hypothermia and inhibition of intestinal motility in mice as a percentage of the maximum possible effect at a single dose. The (+)-cannabidiol analogues were shown to inhibit defecation and reduce arachidonic acid-induced ear swelling in mice. Few central cannabinoid effects were observed [125]. The carboxylic acid and alcohol derivatives have been described in a patent application by the same group, as therapeutically useful modulators/regulators of the immune system and gastrointestinal tract [127].
234
RECENT PROGRESS IN CANNABINOID RESEARCH Table 6.15 CANNABIDIOL SIDE-CHAIN MODIFICATIONS Me OH
Me
HO
R
Mouse ED50 (mmol/kg) CB2 SA CB1 Ki (nM) Ki (nM)
Cpd. R
(68) (176) (177) (178) (179) (180) (181)
Pentyl (cannabidiol, CBD)
TF
4,350 108 1,265 230 >10,000 >10,000 1,1-Dimethylheptyl (CBD-DMH) >10,000 1,800 (1-Hexyl)cyclopropyl 59 99 5,5,5-Trifluoropentyl 1,480 >75 (2-Hexyl)-1,3-dithiolan-2-yl 136 50 (1-Hexyl)-2,2-dichlorocyclopropyl 665 33 (1-Hexyl)-2,2-dibromocyclohexyl 189 63
RT
483
58
27
>75
Ref.
[80] [82] [124] [124] [82] [80] [105] [82] [82]
Table 6.16 ENANTIOMERS OF CANNABIDIOL DERIVATIVES Compound
CB1 Ki (nM)
CB2 Ki (nM)
Ref.
(68) ( )-Cannabidiol (CBD) (182) (+)-CBD (176) ( )-CBD-DMH (183) (+)-CBD-DMH (184) ( )-7-OH-CBD (185) (+)-7-OH-CBD (186) ( )-7-OH-CBD-DMH (187) (+)-7-OH-CBD-DMH (188) ( )-1-CO2H-CBD (189) (+)-1-CO2H-CBD (190) ( )-1-CO2H-CBD-DMH (191) (+)-1-CO2H-CBD-DMH
>10,000 842 >10,000 17 >10,000 5.3 4,400 2.5 >10,000 13 1,900 5.8
>10,000 203 1,800 211 >10,000 101 671 44 >10,000 322 5,000 156
[124] [124] [124] [124] [124] [125] [124] [124] [124] [125] [124] [125]
J. ADAM ET AL.
235
1
R
(185) R1 = CH2OH, R2 = n-C5H11 (187) R1 = CH2OH, R2 = C(Me)2n-C6H13 (189) R1 = CO2H, R2 = n-C5H11 (191) R1 = CO2H, R2 = C(Me)2n-C6H13
OH
Me
R
HO
2
Oxidation of the phenol ring of cannabidiol (73) or cannabinol to a quinone ring has been shown to afford compounds with anti-tumour activity. However, these compounds do not bind to the CB1 receptor and their mechanism of action is unclear [128]. NON-CLASSICAL CANNABINOIDS
The term ‘non-classical cannabinoids’ is applied to a group of bicyclic compounds identified by researchers at Pfizer in the 1980s [129]. These compounds lack the pyran ring of the classical cannabinoids and the second phenolic hydroxyl group of the cannabidiols, resulting in a simplified substructure represented by CP 47,497 (192) [130, 131]. The non-classical cannabinoids still retain the three main pharmacophoric elements described above for the classical cannabinoids and the SAR in these regions parallels that of the classical cannabinoids [132]. A fourth important pharmacophoric element was established for the nonclassical cannabinoid series in the form of a southern aliphatic hydroxyl group. Addition of this group to (192) resulted in the high-affinity CB1 and CB2 receptor full agonist CP 55,940 (193) [129, 133], the tritiated form of which was used to first demonstrate specific cannabinoid binding sites in brain tissue [134]. Its enantiomer, CP 56,667 (194) has lower affinity for the CB1 receptor (Table 6.17). OH
OH OH
OH
1
R
n-C6H13
n-C6H13 Me
Me
Me HO
(192) CP 47,497 R1 = H (193) CP 55,940 R1 = (CH2)3OH
(194) CP 56,667
Me
236
RECENT PROGRESS IN CANNABINOID RESEARCH Table 6.17 NON-CLASSICAL CANNABINOIDS Ki (nM)
Compound
Mouse ED50 (mmol/kg) CB2
CB1
(192) CP 47,497
9.5
(193) CP 55,940
SA
TF
RT
a
0.94
4.1
0.11
0.24
1.1
8.0
a
a
Ref. [93] [135] [93] [81] [135] [93] [81] [135] [136] [136] [137] [137]
0.92 0.69
(194) CP 56,667
62 24
(195) (196) (197) (198) a
(-)CP 55,244 (+)CP 55,244 (Hybrid) (Hybrid)
0.11 >1,000 71 1,353
Partial effect reported (SA/TF max o80%; RT max decrease o4 1C).
Melvin et al. [136] have shown that the positioning of the southern aliphatic hydroxyl group is critical for optimal binding. The constrained analogue of (193), (-)-CP 55,244 (195) showed very high CB1 binding affinity and complete enantioselectivity. Conformational studies with diastereoisomers of (195) have suggested that the ability to form an intramolecular hydrogen bond between the phenolic hydroxyl group and the southern aliphatic hydroxyl group may be important for receptor binding [138]. However, molecular docking of these compounds in a homology model based on the bovine rhodopsin X-ray crystal structure has suggested that these two hydroxyl groups could form intermolecular hydrogen bonds with different residues in the receptor-binding site [139]. OH
OH
OH
OH
n-C6H13
n-C6H13 Me
Me
Me
Me
HO
HO (195) (-)-CP 55,244
(196) (+)-CP 55,244
Classical/non-classical hybrid cannabinoids, such as (197) and (198), have been described by Tius et al. [137, 140]. In these compounds, a southern
J. ADAM ET AL.
237
aliphatic hydroxyl group was added to the classical cannabinoid template. Addition of a C-6a hydroxyethyl substituent resulted in a loss of binding affinity, while the introduction of C-6b hydroxyethyl or hydroxypropyl groups resulted in increased affinity compared to the corresponding ethyl or propyl analogues. OH
OH OH
OH
Me n-C5H11
O
HO Me
O
n-C5H11
HO (197)
(198)
ENDOCANNABINOID DERIVATIVES
The development of SAR for endocannabinoid-derived structures has primarily focused on the anandamide skeleton (1) with a large number of publications addressing the requirements for activity and stability of this scaffold. More recently, some SAR has begun to emerge for the other endocannabinoids, in particular 2-AG (2). The following discussion will focus on highlighting some of the main features that contribute to affinity and/or stability; each endocannabinoid will be treated separately. A number of detailed reviews on this subject have been published [142–146]. A number of protocols are available for measuring cannabinoid-binding affinity and as such there is a variation in reported Ki values for endocannabinoids across labs. For this reason, wherever possible, the relative affinity compared to AEA (measured in that protocol) will be given in an attempt to provide a benchmark for comparison. Anandamide derivatives As outlined earlier, anandamide was the first among the endogenous cannabinoid receptor agonists to be identified. It exhibits higher binding affinity for the CB1 receptor (K i ¼ 89 nM) than for the CB2 receptor (Ki ¼ 371 nM) [81]. Anandamide has typical cannabinoid activities including decreased spontaneous motor activity, immobility and production of hypothermia and analgesia [147, 148]. However, this action in vivo is of shorter duration than
238
RECENT PROGRESS IN CANNABINOID RESEARCH
that of some of the classical cannabinoids. The low potency and shortlasting activity in vivo was recognised to be due to AEA susceptibility to enzymatic hydrolysis by FAAH (see above). Anandamide SAR studies can be split into three categories; modifications to the fatty acid chain, modifications to the carboxamide group and modifications to the polar head group.
The fatty acid chain In general for the C20 series, maximal activity is achieved with amides of arachidonic acid (1) [81], mead acid (199) [149], and dihomo-g-linoleic acid (200) [150] (see Table 6.18). Decreasing the unsaturation (201), (202), or abolishment of the n-pentyl chain (203) [150] led to less active or inactive compounds. Variable results were seen with longer chains. The C22:4 n-6 analogue (204) is as active as AEA (1) whereas the C22:6 n-3 analogue (205) is less active than the C20:5 n-3 analogue (203) [150]. Replacement of the double bonds with triple bonds (206) resulted in loss of activity [150] (see Table 6.18). Forcing the fatty acid chain into a hairpin conformation by cyclisation (207) also resulted in inactive compounds [151]. In a study looking at oxygenated metabolites of AEA and their interaction with the cannabinoid system, a series of hydroxylated alkyl chains was prepared using different lipoxygenases as biocatalysts [152–154]. Of the seven AEA derivatives prepared, only the 5R-hydroxy (208), 12S-hydroxy (209) and 15S-hydroxy (210) derivatives had any affinity for the CB1 receptor. Interestingly, the 13S-hydroxy compound (211) that was inactive at the CB1 receptor displayed some affinity for the CB2 receptor [152] (see Table 6.18). To improve the metabolic stability of AEA analogues, a number of modifications to the C2 site of AEA have been explored [150, 155, 156]. Introduction of a methyl (212) or dimethyl group (213) in this position gave a modest increase in binding affinity and enhanced metabolic stability (cf. with and without PMSF). In contrast, use of an ethyl (214) or isopropyl group (215) resulted in a drop in binding affinity. Introduction of a chiral methyl group into the C-2 position had little effect on binding affinity but the R-isomer (216) had improved metabolic stability compared to its enantiomer (217) [157]. In addition, both (216) and (217) retained their selectivity over the CB2 receptor (see Table 6.18). Several groups have suggested that the C16–C20 portion of AEA and the C-3 pentyl side chain of (67) may play a similar role in the binding site of the CB receptor [158–161]. To explore this, a number of groups have substituted the C16–C20 pentyl side chain with a 10 ,10 -dimethylheptyl chain, a widely
Table 6.18 AEA DERIVATIVES – THE FATTY ACID CHAIN SAR RCONH(CH2)2OH Cpd.
(1) (199) (200)
R
CB1 (nM) +PMSF 89
n-C4H9 n-C7H15
3
(CH2 )6-
2
Me
1,810
1
[81]
0.96
[149]
1.4
[150]
1,500
38
[150]
>1,000
>26
[150]
162
4
[150]
34
0.9
[150]
324
8.3
[150]
(CH2 )9(CH2 )35
n-C4H9
4 (CH2 )5-
Me 6
(CH2 )2-
(206)
(CH2 )3n-C4H9
753
Ref.
(CH2 )9-
(204) (205)
371
53
n-C4H9
(203)
5,400
Relative CB1 Ki (Ki/KiAEA)
J. ADAM ET AL.
3
(CH2 )3-
n-C4H9
n-C8H17
CB2 (nM)
4 (CH2 )3-
(201) (202)
CB1 (nM) PMSF
No activity
[150]
4
(Continued ) 239
240
Table 6.18 CONTINUED R
(207)
O
CB1 (nM) +PMSF
CB1 (nM) PMSF
CB2 (nM)
Relative CB1 Ki (Ki/KiAEA)
No activity
Ref.
[151]
(CH2 )3n-Bu OH
(208)
HO
680
710
7.6
[152]
150
500
1.7
[152]
600
>1,000
6.7
[152]
(CH2 )3-
n-C4H9 3
(209)
n-C5H11
(CH2 )32
OH
(210)
n-C4H9
(CH2)33
OH
RECENT PROGRESS IN CANNABINOID RESEARCH
Cpd.
(211)
n-C5H11
>1,000
(CH2)7-
600
>11
[152]
2
OH
(212)
53
137
0.6
[155]
47
41
0.5
[155]
461
285
5.2
[155]
4,030
2,920
54
457
4,905
0.7
[157]
35
3,941
4,259
0.45
[157]
0.08
[158]
n-C4H9 4
Me
(213) Me
n-C4H9 4
Me
(214) n-C4H9 4
Et
45
[155]
n-C4H9 4
i-Pr
(216) n-C4H9 4
Me
(217)
J. ADAM ET AL.
(215)
n-C4H9 4
(218)
Me
7
Me (CH2 )3-
n-C6H13 4
Me
241
242
RECENT PROGRESS IN CANNABINOID RESEARCH
used replacement in classical cannabinoids (see above). While the replacement of this portion of the molecule did not result in as great an enhancement as with the classical cannabinoids, the dimethylheptyl derivative (218) (Table 6.18) did produce a 13-fold enhancement in CB1 binding affinity compared with AEA [158]. In summary, the receptor recognises unsaturated and partially saturated fatty acyl chains 20–22 carbons long. In addition to the receptor-binding studies detailed here, a large body of work examining the conformational requirements and potential folding arrangements of the fatty acid chain via a number of different computational methods have been published [162].
The carboxamide group A limited series of amide replacements have been examined including thioamides (219) [163], reverse amides (220) [164], heterocycles (221) [164], ethers (222) [165], carbamates (223), (224) [165], ureas (225) [165] and ketones (226) [166] (see Table 6.19). In general, replacement of the amide results in a loss of binding affinity compared to AEA. In some cases, replacement of the amide results in increased stability with regard to hydrolysis by FAAH [164, 165].
Table 6.19 AEA DERIVATIVES – THE CARBOXAMIDE GROUP SAR n-C4H9
Cpd.
R
CB1 (nM) CB1 (nM) CB2 (nM) +PMSF – PMSF
(1) –CONH(CH2)2OH 89 (219) –CSNH(CH2)2OH 1,380 (220) –CH2NHCO(CH2)2OH 115 (221) O
(222) (223) (224) (225) (226)
Me N –CH2O(CH2)2OH –CH2OCONHn-Pr –NHCO2(CH2)2F –NHCONH(CH2)2F –CO(CH2)2OH
4 (CH2)3 R
797 471 331 55 360
5,400 134 679
52
Relative CB1 Ki Ref. (Ki/Ki AEA)
371 1 6,300 17.7 3,540 1.9 6.8 106 0.11
[81] [163] [164] [164]
8.95 5.3 3.7 0.6 4
[165] [165] [165] [165] [166]
J. ADAM ET AL.
243
The polar head group The most extensive investigation of SAR has been carried out in this region of AEA. The following SAR has been established. In general, a secondary amide is optimal. The primary amide is inactive (227) and tertiary amides are less active than AEA or inactive (228), (229) [37, 150, 163]. The hydroxyl group is not required for activity and a wide range of replacements are tolerated such as hydrogen (230), small alkyl (231), halogen (232), (233), ether (234), phosphate (235) or aromatic (236) (see Table 6.20). In particular, the chloroethyl derivative (232) was shown to have 11-fold improved affinity compared to AEA. The introduction of a methyl into the head group at the C10 -position and in particular the R-isomer (237) resulted in 4-fold higher affinity than AEA, while the S- isomer (238) had 2-fold lower affinity than AEA [48, 157], the R-isomer (237) was also resistant to enzymatic hydrolysis [157]. However, introduction of larger alkyl groups into the C10 position such as the i-butyl analogue (239) leads to a large drop in activity [155]. Methylation of the C20 position also leads to improved affinity compared to AEA. In particular, the R-isomer (240) has 4-fold enhanced affinity; this derivative is also stable to enzymatic hydrolysis by FAAH [48] (see Table 6.20). Extension of the head group by insertion of a methylene group is tolerated, the N-propanol derivative (241) having a slight increase in binding affinity compared to AEA. Further extension of the carbon chain, such as the butyl derivative (242), led to a decrease in activity [150, 151]. Replacement of the ethanolamine head group is also well tolerated. Substitution with a cyclopropyl (243) [37], allyl (244) or propargyl group (245) [164] all led to an increase in binding affinity compared to AEA. Replacement of the head group with aromatics is also allowed. The phenyl derivative (246) retains affinity at the CB1 receptor [37], whereas the 2-substituted N-methyl pyrrole (247) has a 2-fold improved affinity compared to AEA [167]. Interestingly, the 3-substituted furan derivative (23) that has micromolar affinity for the AEA transporter (see above) does not bind to the CB1 receptor, but has good affinity for the CB2 receptor [167]. These results are summarised in Table 6.20. Overall, these results suggest that the hydroxyl group is not vital for binding and that both hydrophobic and hydrophilic head groups can be accommodated. The cavity in which the head group binds is relatively small as only modest variations in this position lead to high-affinity ligands. With regard to CB2 selectivity, very few reports have looked in detail at the requirements for CB2 binding in AEA derivatives and most ligands synthesised to date have tended to be relatively selective for the CB1 receptor.
244
n-C4H9
Cpd.
R
CB1 (nM) +PMSF
(1) (227) (228) (229) (230) (231) (232) (233) (234) (235) (236)
–NH(CH2)2OH –NH2 –NEt2 –N[(CH2)2OH]2 –NHEt –NHn-Pr –NH(CH2)2Cl –NH(CH2)2F –NH(CH2)2OMe –NH(CH2)2OP(O)(OH)2
89
(237)
CH3 -NH
CH2OH
CB1 (nM) - PMSF 5,400 >1,000 >1,000
CB2 (nM) 371
174 34 7.1 5.29 26.7 163
-NH(CH2)2
4 (CH2)3 COR
3,400 4,640 85.2 190.8 250
195 908
28.3
868
Relative CB1 Ki (Ki/Ki
AEA)
Ref.
1 >25 >25 2.2 0.87 0.05 0.09 0.43 2 4.9 1.8
[81] [150] [150] [163] [150] [37] [164] [164] [150] [150] [156]
0.26
[157]
SO2NH2
20.6
RECENT PROGRESS IN CANNABINOID RESEARCH
Table 6.20 AEA DERIVATIVES – THE POLAR HEAD GROUP SAR
(238)
CH3 -NH
(239)
268
8,216
2.2
[157]
5,420
>10,000
20
28
0.26
[48]
29.9 497.4 193 2,980 4,900
226 290
124
70
0.76 12.7 0.015 0.16 0.18 0.76 0.43
[150] [150] [37] [164] [164] [37] [167]
>1,000
67
CH2OH i-Bu
-NH
(240)
[155]
CH2OH
CH3 -NHCH2 OH NH(CH2)3OH NH(CH2)4OH NHc–Pr NHCH2CHQCH2 NHCH2CRCH NHPh
-NHCH2
61
2.2 9.91 10.8 109
N Me
(23)
-NHCH2
>3.5
[167]
J. ADAM ET AL.
(241) (242) (243) (244) (245) (246) (247)
173
O
245
246
RECENT PROGRESS IN CANNABINOID RESEARCH
2-Arachidonylglycerol 2-AG (2) was first isolated in 1995 from canine gut [14] and rat brains [15] and shown to be an endogenous CB ligand [14]. (2) is present in the rat brain in amounts 170–1,000 times greater than AEA [15, 168] and has been shown to be a potent full agonist at both the CB1 and CB2 receptors (CB1: K i ¼ 472 nM and CB2: K i ¼ 1; 400 nM) [14, 169]. In addition, this endocannabinoid has typical cannabinoid-like activities including decreased spontaneous motor activity, immobility and production of hypothermia and analgesia [14]. The FAAH enzyme has been reported to hydrolyse (2) four times faster than its hydrolysis of AEA [170]. In addition, it has been proposed that the major enzymatic hydrolysis pathway of (2) occurs via the monoacylglycerol lipase rather than FAAH [171, 172]. Compound (2) has been reported to induce a rapid transient increase of intracellular free Ca2+ concentration in NG108-15 and HL-60 cells through a cannabinoid receptor-dependent mechanism [173, 174]. Furthermore, the Ca2+ increase in HL-60 cells appeared to be CB2 mediated as the effect could be blocked with a CB2 antagonist but not a CB1 antagonist [174]. Very little work has been carried out on the SAR of this endocannabinoid. In general, for the CB1 receptor, arachidonic acid was found to be the preferred fatty acid moiety, although some partially saturated structures had almost comparable activities [173]. It appears that the presence of a double bond at the D5-position is crucial for activity. With respect to the head group, the 2-glycerol isomer is preferable over both the 1- and 3-analogues. The ester moiety can be replaced by a ketone but agonistic activity in NG108-15 cells drops approximately 100-fold [175]. With regard to the CB2 receptor, much of the SAR overlaps with the results obtained for the CB1 receptor with the exception that glycerol esters of C22 fatty acids do not show appreciable activity in the CB1 assay, but do show appreciable activity in the CB2 assay [173].
Noladin ether Noladin ether (3) was recently isolated from porcine brain [16] and found to bind to the CB1 receptor (K i ¼ 21:2 nM), to bind weakly to the CB2 receptor (Ki>3 mM) and it causes typical cannabinoid-like effects such as sedation, hypothermia, intestinal immobility and mild antinociception in mice [16]. This endocannabinoid had previously been synthesised independently by both Mechoulam and co-workers [176] and Sugiura et al. [173]. SAR studies of this endocannabinoid are lacking in the literature, however, a recent publication highlighted the importance of the tetra-unsaturated C20 chain
J. ADAM ET AL.
247
for high affinity at the CB1 receptor, increased saturation or replacement of the double bonds (with, for example, a cyclopropyl) had a detrimental effect on binding affinity [177].
INDOLE AND ITS DERIVATIVES
Indoles The discovery and cloning of the CB1 and CB2 receptors has opened the way for the pharmaceutical industry to identify cannabinoid agonists with completely new templates. However, the first novel class of cannabinoid agonists (i.e. those not derived from THC) was discovered accidentally by the Sterling-Winthrop Research Group while studying conformationally restrained analogues of pravadoline (248) [178]. These pravadoline analogues displayed reduced ability to behave as non-steroidal anti-inflammatory agents that inhibit cyclooxygenase but increased ability to bind to the CB1 receptor.
OMe
O
Me
O
OH Me
Me N
O
N N
O
O N
Me Me
N
O
C5H11 (248) Pravadoline
(254a) (R)-(+) WIN 55,212-2
(270)
In this initial study they found that the 4-methoxyphenyl ring could be replaced by a number of other aromatic rings, in particular, the 1-naphthyl ring was found to impart good potency. In addition, small substituents in the indole 2-position were favoured and importantly that potency resided in only one enantiomer of the aminoalkyl indole (AAI) (see Table 6.21). One compound of particular importance synthesised at this time is WIN 55,212-2 (254a), a potent agonist at both the CB1 and CB2 receptors. Although this compound was not developed as a drug, it has been widely used as a pharmacological tool in cannabinoid research. The Sterling-Winthrop research group [179] went on to carry out an extensive SAR study on unconstrained aminoalkyl indoles with over 100 analogues synthesised and tested, and
248
RECENT PROGRESS IN CANNABINOID RESEARCH
Table 6.21 AMINOALKYL INDOLES – SAR OF THE BENZOXAZINE CORE [178]
O
Ar
R N
O N
O
Cpd.
Ar
R
MVD activity IC50 (nM)a
CB1 IC50 (nM)
(249) (250) (251) (252) (253) (254) (255) (R)-(+)-(254a) (S)-( )-(254b)
4-MeO-C6H4 4-MeO-C6H4 4-MeO-C6H4 2-F-C6H4 1-Naphthyl 1-Naphthyl 5-Quinolinyl
H Me Et Me H Me Me
44.5 123 28% @ 10 mM 76 2 6 21 0.43 >10,000
249 152 27% @ 1 mM 1,426 7.37 5.56 132.5 2.77 8,002
a
Mouse vas deferens inhibitory activity.
extended their previously reported SAR to include variations in the nature of the aminoalkyl substituent, the substituents on the naphthyl group and substituents on the indole nucleus. In general, similar trends were seen with regard to the key structural features required for cannabinoid activity i.e. an aromatic (bi)aroyl ring in the 3-position, a small substituent in the 2-position and a cyclic aminoethyl substituent in the 1-position. Following on from this they demonstrated that the aminoethyl side chain could also be replaced (see Table 6.22) [180], the most potent ligands bearing a naphthoyl moiety. In particular, the piperidinyl analogue (258) was found to have a high affinity in the binding assay and potency in the mouse vas deferens assay. In an attempt to rationalise these results with a pharmacophore model that would fit both the classical cannabinoids and AAIs, Huffmann and coworkers [181] suggested that the ketonic oxygen of WIN 55,212-2 was aligned with the phenolic hydroxyl of (67). In this arrangement, the naphthyl moiety of the AAIs would overlay the cyclohexyl and pyran rings of the classical cannabinoid structure. The indole nitrogen and substituent attached to it would then be placed in a corresponding position to the alkyl chain on C3. To test this hypothesis, Huffman designed a number of AAIs in which the aminoethyl chain was replaced with other substituents and in
J. ADAM ET AL.
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Table 6.22 AMINOALKYL INDOLES – SAR OF THE N-1 SUBSTITUENT [180] O
N R
Cpd.
R
(256) -CH2
R'
R0
MVD activity IC50 (nM)
CB1 IC50 (nM)
Me
2.4
6.5
Me
1.2
5.4
H
0.47
1.2
N Me
(257) -CH2
N Me
(258) -CH2
N Me
particular, carbon chains. Short side chains resulted in inactive compounds, whereas chains with four to six carbons produced best results [181] (see Table 6.23). In particular, the n-pentyl substituent (263) was optimal exhibiting 2-fold greater affinity than WIN 55,212-2 and 4-fold greater affinity than (67) in the in vitro binding assay. In addition, (263) also displayed potent in vivo activity. Following on from this, and to further exemplify this pharmacophore model, Huffman [182] described a novel hybrid structure that combined the hydroxydibenzopyran ring of THC and the indole moiety of the AAIs into one molecule. It was found that the hybrid molecule (270) had a similar affinity (19 nM) for the CB1 receptor in vitro as (67) (41 nM). The compound was also active in vivo in the mouse tetrad model of cannabimimetic activity and had comparable potency to (67) [182]. An alternative binding model has been proposed by Xie et al. [183] using 2D-NMR spectroscopy and molecular modelling with (254a) and 9-nor-9bOH-hexahydrocannabinol (HHC). In this case it is suggested that the ketonic group of (254a) corresponds to the phenolic hydroxy group, the naphthyl moiety to the C3 alkyl side chain and the nitrogen of the morpholinyl group to the C9 hydroxyl of HHC. A similar structural alignment has
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RECENT PROGRESS IN CANNABINOID RESEARCH
Table 6.23 AMINOALKYL INDOLES – N-1 ALKYL CHAIN SUBSTITUENTS [181] O
N R
Me
Mouse ED50 (mmol/kg) Cpd.
R
CB1 Ki (nM)
SA
TF
RT
(259) (260) (261) (262) (263) (264) (265) (266) (267) (268) (269) (254a)b (67)c
Me Et n-Pr n-Bu n-Pentyl n-Hexyl n-Heptyl 4-Pentenyl 2-Phenylethyl c-Hexylethyl c-Propylmethyl
>10,000 1,180 164 22 9.5 48 >10,000 38 1,250 46 140 24 41
18.7 2.6 0.7 o2.7 117 3.5 No maxa 55.4 6.5 0.19 0.92
84.7 0.23 0.25 9.5 >261 0.34 No maxa 58.7 7.1 1.4 2.7
99.1 4.1 4.3 17.1 >261 5.6 No maxa 69.7 36.9 1.5 2.5
a
‘‘No max’’ indicates that the compound produced only slightly greater than 50% of the presumed maximal effect. b WIN 55,212-2. c 9 D -THC.
been proposed by Razdan and co-workers [184]. To test the hypothesis, a series of 4-substituted indoles was prepared containing the key structural features of the AAIs except that the naphthyl substituent was transposed from position 3 to position 4 via an oxygen linker. A total of nine compounds were synthesised and tested. Alkyl chain-substituted compounds failed to show any affinity either in vitro or in vivo (see Table 6.24 (271)–(273)). On the other hand, introduction of a naphthyl moiety either directly or via a number of different linkers (274)–(279), in particular, a ketomethylene group (277), led to compounds with moderate potency in vitro and in vivo [184]. The validity of pharmacophore models that overlay the classical cannabinoids and AAIs has been brought into question by recent mutation work on the CB1 receptor. It was found that mutation of Lys-192 in the third
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Table 6.24 AMINOALKYL INDOLES – SAR 4-SUBSTITUTED INDOLES [184] OR
N
O N
Mouse ED50 (mg/kg) Cpd.
R
CB1 Ki (nM)
SA
TF
RT
(271) (272) (273) (274) (275) (276) (277) (278) (279) (254a)a (67)b
CH(CH3)C5H11 (CH2)8CH3 CH(CH3)(CH2)3Ph CH2-1-Naphthyl CH2-2-Naphthyl CH2CO-1-Naphthyl CH2COCH2-1-Naphthyl (CH2)3-1-Naphthyl (CH2)4-1-Naphthyl
>10,000 >10,000 >10,000 221 1,300 287 127 2,220 3,390 8.7 41
>100 >100 >100 3.5 >30 4.6 6.1 >30 55 0.1 1.0
>100 >100 >100 3.3 >30 8.4 1.7 >30 78 0.4 1.4
>100 >100 >100 12.3 >30 >30 18.7 >30 79 12 1.4
a b
WIN 55,212-2. D9-THC.
transmembrane domain to an Ala resulted in a complete loss of binding for the classical cannabinoid (165), in contrast to this; binding of (254a) was only slightly affected [185]. These results would suggest that the AAIs interact with the CB1 receptor in a somewhat different fashion from the dibenzopyran cannabinoids. One alternative that has been proposed is that the main binding arrangement of the AAIs to the receptor is through aromatic stacking. To explore this proposal a number of naphthyl methanes have been prepared [186]. These compounds lack the ketone functionality that is thought to play the same role as the phenoxy group in the classical cannabinoids. Hence there is no viable possibility for hydrogen-bonding interactions between the receptor and the ligand at this position. Substitutions in both the 4-position of the naphthyl and the 2-position of the indole were explored (see Table 6.25). In the case of the unsubstituted indole, binding affinity was maintained with only a small drop in binding affinity being observed compared to their naphthoyl congeners (280)–(282). In contrast to this, substitution in the
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RECENT PROGRESS IN CANNABINOID RESEARCH
Table 6.25 AMINOALKYL INDOLES – SAR OF SUBSTITUTED NAPHTHYLMETHANES [186] R'
R N C5H11
Cpd.
R
R0
CB1 Ki (nM)
Cpd.
R
R0
CB1 Ki (nM)
(280) (281) (282) (283)
H H H Me
H Me OMe H
22 23 17 151
(284) (285) (254a)a (67)b
Me Me
Me OMe
127 323 9.9 41
a b
WIN 55,212-2. D9-THC.
2-position of the indole had a marked effect on binding, with affinity dropping up to 19-fold (cf. (282) and (285)). The high binding affinities obtained for the unsubstituted indole compounds thus support the hypothesis that H-bonding may not be the main binding feature of the AAIs and potentially aromatic stacking may be the dominant interaction. To explain the sudden drop in potency for the 2-methyl substituted indoles, the authors initiated a molecular modelling and receptor-docking study. From this study, they conclude that the loss in binding affinity is due to the loss of an aromatic stacking interaction [186]. More recently, the utility of the indole group as a scaffold for cannabinoid agonists has been demonstrated by a number of new patent applications appearing in the literature (286)–(290) [187–190]. Of particular note is compound (286) that is reported to have 18-fold selectivity for the CB1 receptor (CB1: K i ¼ 0:08 nM; CB2: K i ¼ 1:44 nM). In addition to the indole scaffold, a number of patent applications by AstraZeneca claim indole-like scaffolds such as benzimidazoles (289) [191–193] and azaindoles (290) [194]. Although these compounds bind to both CB1 and CB2 receptors, the inventors claim that they may be useful in treating diseases without the associated CNS side effects.
J. ADAM ET AL.
Ph
Ph
OMe
I O
O
253
N
O
N H
N O
O N
N
(CH2)5 F
N
N
N
MeO (288)
(287)
(286)
OEt O N
(F3 CCH2)2N
t-Bu
t-BuCONH
N N
N Ph
(289)
(290)
Pyrrole and its derivatives In Huffman’s [181] overlay model of the classical cannabinoids and AAIs, the benzenoid portion of the indole ring did not participate in the overlap and only the pyrrole ring was involved. In order to investigate this, a homologous series of pyrroles was prepared and their pharmacology examined (see Table 6.26). In analogy to the indoles, the best results were obtained with the n-pentyl analogue (295), although this compound was approximately 10-fold less potent than its indole analogue. Following on from this Tarzia et al. [195] designed and synthesised a range of pyrrole compounds based on the Huffman overlay. A range of substituents was explored around the pyrrole ring, the selections are detailed in Table 6.27. As seen with the AAIs, replacement of the naphthyl moiety with a monocyclic aromatic ring was detrimental to affinity. In particular, replacement with a phenyl ring resulted in complete loss of activity (299). A similar result was seen when the naphthyl moiety was replaced with an aliphatic chain (301). Replacement of the ketone group with an amide is tolerated, albeit with a loss in binding affinity. In addition, depending on the
254
RECENT PROGRESS IN CANNABINOID RESEARCH
Table 6.26 PYRROLE DERIVATIVES – N-1 ALKYL CHAIN SUBSTITUENTS [181] O
N R
Mouse ED50 (mg/kg) Cpd.
R
CB1 Ki (nM)
SA
TF
RT
(291) (292) (293) (294) (295) (296) (297) (254a)b (67)c
Me Et n-Pr n-Bu n-Pentyl n-Hexyl n-Heptyl
>10,000 >10,000 >10,000 666 87 399 309 24 41
106 84 86 No maxa 3.6 8.8 11 0.19 0.92
No maxa No maxa 81.8 No maxa 1.2 9.6 9.7 1.4 2.7
53.3 77.2 90.1 >108 78.8 62.8 52.1 1.5 2.5
a
‘‘No max’’ indicates that the compound produced only slightly greater than 50% of the presumed maximal effect. b WIN 55,212-2. c 9 D -THC.
Table 6.27 PYRROLE DERIVATIVES – SAR OF SUBSTITUTED PYRROLES [195] R
Me
1
2
COR
N
Me
n-C5H11
Cpd.
R1
R2
CB1 Ki (nM)
CB2 EC50 (nM)
(298) (299) (300) (301) (302) (303)
1-Naphthyl Ph 1-Naphthyl HO(CH2)3 o-(Ac)C6H4NH c-HexylNH
H H Br H H H
45 >1,000 13 >3,000 367 415
10 >1,000 6.8 >10,000 >1,000 483
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255
amide substitution an interesting selectivity profile was seen, for example, the substituted aniline compound (302) gave a moderate binding affinity at the CB1 receptor (367 nM) but lost all affinity at the CB2 receptor (>1,000 nM). In contrast, the aminocyclohexyl amide (303) retained affinity at both receptors albeit reduced compared to the naphthyl analogues. The best results were obtained with the 4-bromo substituted compound (300) giving good affinity at both the CB1 (13 nM) and CB2 (6.8 nM) receptor. The substituted pyrrole (300) was also tested for intrinsic activity at the CB1 receptor using [35S]-GTPgS binding in rat cerebellar membranes and found to behave as a full agonist with an EC50 value of 140 nM. The pyrazole scaffold is usually associated with cannabinoid antagonists/ inverse agonists and in particular, rimonabant (382) (see below). However, recent work examining a series of (382) analogues for antagonist activity gave rise to a number of compounds that also displayed cannabimimetic effects in vivo [196]. In particular, replacement of the N-piperazine in (382) with carbon chains that more directly correspond to the lipophilic side chain of (67) gave compounds exhibiting agonistic properties in the mouse triad model of cannabimimetic activity (see Table 6.28). The best results were obtained with 2-fluoroethyl amide (306) that had a relatively low binding affinity (852 nM), but had a moderate potency in vivo [approximately 5-fold less active than (67)].
Indenes In the early clinical trials of Pravadoline (248), the Sterling-Winthrop Research group observed CNS side effects. In an effort to circumvent these issues, indenes were explored as an alternative core [197]. The rationale behind this approach came from the observation that sulindac (308), an indene analogue of indomethacin (309), had anti-inflammatory properties comparable to (309) but without the CNS-associated side effects. CO2H
CO2H
F
MeO Me
Me N SOMe
(308) Sulindac
O (309) Indomethacin
Cl
256
RECENT PROGRESS IN CANNABINOID RESEARCH
Table 6.28 PYRROLE DERIVATIVES – SAR OF SUBSTITUTED PYRAZOLES [196] Me
CONHR N N
Cl
Cl
Cl
Mouse ED50 (mg/kg) Cpd.
R
CB1 Ki (nM)
SA
TF
RT
(304) (305) (306) (307) (382)b (67)c
n-Pentyl n-Heptyl (CH2)2F n-Pr
32 48 852 167 6.2 41
11 27 7a 9 >30 0.92
21 20 13 24 >30 2.7
11 12 8 10 >30 2.5
a
Estimated ED50 – non-linear dose–effect curve. Rimonabant. c 9 D -THC. b
A series of 11 compounds was prepared with and without a 2-Me substituent. Owing to the method of preparation, the 2-Me substituted compounds were prepared as a mixture of stereoisomers (4:1; E:Z), whereas the unsubstituted compounds had predominantly E configuration (>95%). It was found that, as with the AAIs, naphthyl-substituted derivatives were potent agonists in vitro and in vivo (see Table 6.29). Following on from this Reggio et al. [198] extended this work by preparing the stereo-defined indenes in a bid to identify the bioactive conformation of the AAIs. Through conformational analysis it had been established that the indene E-isomer mimics the s-trans conformation of (254a) while the Z-isomer mimics the s-cis conformation (Figure 6.2). Measurement in CB1/CB2 binding assays revealed that the E-naphthylindene isomer had significantly higher binding affinity than the Z-isomer (see Table 6.29). This would then suggest that the s-trans conformer of (254a) and related AAIs is the bioactive conformer at the cannabinoid receptors.
J. ADAM ET AL.
257
O
O
N
N
O
O N
N
N
N
O
O
O
O E-isomer
Z-isomer
s-trans
s-cis
Fig. 6.2 Conformational analysis of indene isomers.
Table 6.29 INDENES – SAR OF SUBSTITUTED INDENES
R
R' O N
Cpd.
R
R0
CB1 IC50 (nM)
(310) (311) (312) (313) (314) (315) (316) (254a)a
Ph 1-Naphthyl 1-Naphthyl 1-Naphthyl 1-Naphthyl 1-Naphthyl 1-Naphthyl
H, >95% E H, >95% E Me, 4:1 E:Z H, 100% E H, 100% Z Me, 100% E Me, 100% Z
33%@1 mM 1 10 2.72 148 2.89 1945 5.56 2.48
a
WIN 55,212-2.
CB2 Ki (nM)
2.72 132 2.05 658 0.28
Ref. [197] [197] [197] [198] [198] [198] [198] [197] [198]
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RECENT PROGRESS IN CANNABINOID RESEARCH
Other structures A number of new scaffolds unrelated to the classical/non-classical cannabinoids and AAIs have been reported in the literature to deliver cannabinoid agonists. Bayer [199–203] has claimed in a series of patents a number of aryl sulfonyl esters as cannabinoid agonists for the treatment and prophylaxis of neurodegenerative diseases. Following on from this a number of publications detailing the in vitro and in vivo profiles on two of these compounds, BAY 38-7271 (317) and BAY 59-3074 (318), have been published. Compound (317) was found to have a high affinity for rat and human CB1 receptors (K i ¼ 0:46 and 1.09 nM, respectively), as well as for recombinant human CB1 and CB2 receptors (K i ¼ 1:85 and 5.96 nM, respectively) and also exhibited full agonist activity using the [35S]-GTPgS technique with an EC50 value at human CB1 receptors of 15.8 nM (no data reported for the CB2 receptor). (317) also displayed typical cannabinoid-like behaviour in the rat hypothermia model and drug discrimination assays which could be reversed by the selective CB1 antagonist, rimonabant (382). In addition, (317) also proved effective in a rat traumatic brain injury model reducing infarct volume by 70% at 100 ng/kg/h infusion starting immediately after subdural haematoma. It also reduced infarct volume (27% at 1,000 ng/kg/h) in a rat model of focal cerebral ischaemia. Phase I studies concluded that (317) was safe and well tolerated when administered intravenously for either 1 or 24 h in humans and (317) is currently being developed for the treatment of traumatic brain injury and cerebral ischaemia [204, 205]. The structurally related compound BAY 59-3074 (318) also has high affinity at human CB1/CB2 receptors (K i ¼ 48:3 and 45.5 nM, respectively). However, and in contrast to (317), (318) exhibited partial agonist activity at hCB1 using the [35S]-GTPgS technique with an EC50 of 142.7 nM [206, 207]. The compound also behaved as a partial agonist at hCB2 receptors with an EC50 of 15.8 nM. BAY 59-3074 (318) also displayed typical cannabinoid activity in vivo in rat hypothermia and drug discrimination assays that could be reversed by the selective CB1 antagonist rimonabant (382). At doses of 0.3–3 mg/kg p.o., (318) induced antihyperalgesic and antiallodynic effects against thermal or mechanical stimuli in rat models of chronic neuropathy and inflammatory pain. The antiallodynic efficacy was maintained for 2 weeks of daily administration, and no withdrawal symptoms were seen during the 2 days after the last dosing regimen [206, 207]. Novartis AG has filed a patent application on novel naphthalene derivatives as potent cannabinoid agonists, especially at the CB1 receptor [208]. One compound was specifically claimed, the naphthalene derivative (319), which exhibited CB1 binding with a Ki value of 15 nM. This compound was also active in an in vivo model of neuropathic pain, reversing hyperalgesia
J. ADAM ET AL.
259
caused by partial ligation of the sciatic nerve in Wistar rats after oral dosing with an ED50 of 0.18 mg/kg. In addition, Novartis filed a patent application on a series of quinazolines as cannabinoid agonists [209]. Compound (320) is one of the two compounds specifically claimed and exhibited CB1 and CB2 binding with Ki values of 34 and 11 nM, respectively. It was shown to be a full agonist at the CB1 receptor with an EC50 of 132 nM (no functional data for the CB2 receptor). Compound (320) was also active in the neuropathic pain model described above with an ED50 of 0.5 mg/kg after oral dosing. AstraZeneca has filed a patent application on novel bis-aryl compounds as CB1/CB2 agonists that lack CNS penetration and thus avoid the unwanted side effects associated with activation of central CB1 receptors [210]. Over 100 compounds are specifically claimed e.g. (321). Compounds were tested in receptor binding assays using human CB1 and CB2 receptor preparations. Respective Ki values were in the ranges 50–5,000 and 15–2,800 nM, although no specific data were presented. Shionogi [211] has filed a patent application on a series of thiazine derivatives; no compounds are specifically claimed. The thiazine derivative (322) was active in vivo in the formalin-induced licking-and-biting model in ICR mice with an ED50 of 1.5 mg/kg after oral dosing. HO CN F3C
O
OSO2(CH2)3CF3
OSO2(CH2)3CF3
O
(318) BAY 59-3074
(317) BAY 38-7271
Me
O
EtO2C
N N
(320)
O O
(319)
Ph
Me OH SO2NHMe
N Me HO(CH2)2NH
On-C5H11 O
S
N N
Me
SMe S
(321)
(322)
CB2 AGONISTS Receptor subtype selective CB2 agonists are seen as potential candidates for the treatment of a variety of diseases, including pain-related indications. The promise of useful therapeutic effects without unwanted CNS side effects makes the development of CB2 selective compounds a particularly attractive
260
RECENT PROGRESS IN CANNABINOID RESEARCH
proposition. Several classes of CB2 selective agonists, including classical cannabinoids, resorcinols, indoles, indazoles and other heterocyclic derivatives and their SAR studies have been reported. CB2 SELECTIVE CLASSICAL CANNABINOIDS
Of the three main pharmacophoric elements in the classical cannabinoid template discussed previously, the phenolic hydroxyl group at C1 has been shown to be the most important for CB2 selectivity. In 1996, a group at Merck Frosst [212] and the Huffman group [213] independently demonstrated that removal of this hydroxyl group in the 10 ,10 -dimethylheptyl series leads to compounds with high CB2 receptor affinity and between 8- and 40fold selectivity for CB2 over CB1 binding affinity (Table 6.30). The Merck Frosst paper further describes modification of the phenol to a methoxy group, resulting in higher CB2 selectivity [212]. 1
R
R
Me Me
2
(323) R1 = Me, R2 = H (324) R1 = CH2OH, R2 = H (325) R1 = Me, R2 = OMe n-C6H13
O Me
Me
The CB2 receptor has been shown to tolerate shorter C3 side chains than the CB1 receptor and the Huffman group has exploited this in combination with C1 modifications to develop highly selective CB2 receptor ligands (Tables 6.31 and 6.32) [214, 215]. The 11-hydroxy analogues of the 10 10 -dimethyl compounds in Tables 6.31 and 6.32 were also prepared by the Huffman group. In general, these showed higher affinity for both CB1 and CB2 receptors than the simple methyl analogues, but reduced CB2 selectivity (data not shown) [215]. Table 6.30 1-DEOXY AND 1-METHOXY D8-THC DERIVATIVES Cpd. 8
(85) D -THC-DMH (323) 1-Deoxy-D8-THC-DMH (324) 1-Deoxy-11-hydroxy-D8-THC-DMH (325) 1-Methoxy-D8-THC-DMH
CB1 Ki (nM)
CB2 Ki (nM)
CB1/CB2
Ref.
0.83 250 23 1.2 15,850 924
0.49 21 2.9 0.032 20 65
1.7 12 8 38 793 14
[212] [212] [213] [213] [212] [214]
J. ADAM ET AL.
261
Table 6.31 1-DEOXY D8-THC C3 SIDE-CHAIN ANALOGUES Me
Me Me
O
R
Cpd.
R
CB1 Ki (nM)
CB2 Ki (nM)
CB1/CB2
Ref.
(326) (327) (328) (329) (330) (331) (332) (333) (323) (334) (335)
1,1-Dimethylethyl 1,1-Dimethylpropyl n-Bu 1,1-Dimethylbutyl n-Pentyl (1-Deoxy-D8-THC) 1,1-Dimethylpentyl n-Hexyl 1,1-Dimethylhexyl 1,1-Dimethylheptyl (DMH) 1,1-Dimethyloctyl 1,1-Dimethylnonyl
2,150 2,290 2,790 677 >10,000 399 1,610 295 23 51 178
58 14 54 3.4 32 10 273 19 2.9 76 449
37 164 52 199 >312 40 6 16 8 0.7 0.4
[214] [214] [214] [214] [214] [214] [214] [214] [213] [214] [214]
Table 6.32 1-METHOXY D8-THC C3 SIDE-CHAIN ANALOGUES [215] Me OMe
Me Me
O
R
Cpd.
R
CB1 Ki (nM)
CB2 Ki (nM)
CB1/CB2
(336) (337) (338) (339) (340) (325)
1,1-Dimethylethyl 1,1-Dimethylpropyl 1,1-Dimethylbutyl 1,1-Dimethylpentyl 1,1-Dimethylhexyl 1,1-Dimethylheptyl (DMH)
>10,000 >10,000 >10,000 4,001 3,134 713
1,867 1,404 325 43 18 57
>5.4 >7.1 >31 93 174 12
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RECENT PROGRESS IN CANNABINOID RESEARCH
Compounds where the C1 hydroxyl group is linked to the C2 position by a cyclic ether link, with a range of different groups in the C9 position, were prepared by Reggio et al. [216]. These compounds showed only modest CB2 selectivity. Compound (341) was the most potent and selective with a binding affinity (Ki) of 5.8 nM at CB2 and 26 nM at CB1. A patent application from Merck Frosst, published in 1997, discloses CB2 selective compounds with modified core structures where the pyran oxygen is replaced by nitrogen, such as (342), which has a Ki of around 64 nM at CB2 and around 25,000 nM at CB1 [217]. OH
Me O
Me Me
O
n-C5H11
Me Me
N
OMe
Me (341)
(342)
INDOLES AND INDAZOLES
Indomethacin morpholinyl amide (343) was found as a weak but CB2 selective agonist through a topological similarity search using WIN 55,212-2 (254a) as the template [218]. The binding affinity (Ki) of (343) was 435 and >20,000 nM for hCB2 and hCB1, respectively. A subsequent SAR study revealed that N-(2,3-dichlorobenzoyl) and N-naphthoyl indole derivatives showed improved activity. Further optimisation of the substituents on the indole C3-position was also performed, and the more selective and potent CB2 agonists, L-768,242 (344) and L-759,787 (345) were discovered. These compounds showed >100-fold CB2 selectivity with significant potency (Ki of 12 and 8.5 nM, respectively). X N
O
(343) X = -CH2CO-, R1 = OMe, R2 = 4-Cl-phenyl
1
R
Me N 2
R
O
(344) L-768,242 X = -(CH2)2-, R1 = OMe, R2 = 2,3-diCl-phenyl Ki(hCB1) = 1917nM, Ki(hCB2) = 12nM (345) L-759,787 X = -CH2-, R1 = H, R2 = 1-naphthyl Ki(hCB1) = 877nM, Ki(hCB2) = 8.5nM
J. ADAM ET AL.
263
During the study of N-alkylindole derivatives as cannabinoid ligands, JWH-015 (346) was found as a potent CB2 selective agonist, Ki (CB2) 13.8 nM with modest selectivity, ðCB1 Þ : ðCB2 Þ ¼ 28 [81]. For the optimisation study of (346), more than 40 indoles were prepared and their CB1 and CB2 receptor affinities were determined [219]. In most cases, N-pentyl indoles showed significant activity but low receptor subtype selectivity, whereas N-propyl derivatives had better selectivity for CB2. The SAR of the substituents on the naphthyl ring was also studied, and it is noteworthy that introduction of a methoxy group in the 2-position of the naphthyl ring reduced the binding affinity only for the CB1 receptor, even though the compound bears a pentyl group on the indole ring. As a result, three CB2 selective compounds, JWH-120 (347), JWH-151 (348) and JWH-267 (349) were found; JWH-151 was a full agonist whereas JWH-120 and JWH-267 exhibited partial activity in GTPgS binding assay. 4
R
(346) JWH-015 R1 = n-Pr, R2 = R3 = R4 = H Ki(CB1) = 383nM, Ki(CB2) = 13.8nM 3
R O 2
R Me N 1
R
(347) JWH-120 R1 = n-Pr, R2 = R4 = H, R3 = Me Ki(CB1) = 1054nM, Ki(CB2) = 6.1nM (348) JWH-151 R1 = n-Pr, R2 = R3 = H, R4 = OMe Ki(CB1) >10,000nM, Ki(CB2) = 30nM (349) JWH-267 R1 = n-C5H11, R2 = OMe, R3 = R4 = H Ki(CB1) = 381nM, Ki(CB2) = 7.2nM
The group of Bristol-Myers Squibb found a C3 amido-indole (350) as a lead compound for their CB2 agonist program [220]. Compound (350) demonstrated moderate binding affinity for CB2 (Ki 250 nM), which was improved dramatically to a Ki of 8 nM by the introduction of methoxy group on the C7 of the indole core. The 7-methoxy indole (351) showed good receptor subtype selectivity, CB1 : CB2 ¼ 500, however, the methyl ester on the phenylalanine moiety was susceptible to microsomal hydrolysis. In an effort to discover non-ester CB2 agonists, compound (352), having a (1S)-fenchyl group instead of the phenylalanine methyl ester, was prepared. The introduction of a larger alkyl group onto the C2 position of the indole ring decreased the CB2 binding affinity (H>Me>Et>n-Pr; Table 6.33). The non-substituted compound (353) showed dose-dependent inhibitory activity in an in vivo anti-inflammatory model [lipopolysaccharide (LPS)induced TNF-a production in mice] after i.v. administration with an ED50 of 5 mg/kg. Unfortunately, (353) was inactive after oral administration, and
264
RECENT PROGRESS IN CANNABINOID RESEARCH Table 6.33 1-AMINOALKYLINDOLE ANALOGUES Me Me O
Me
N H R
N OMe
O N
Cpd.
R
CB1 Ki (nM)
CB2 Ki (nM)
CB1/CB2
Ref.
(352) (353) (354) (355)
Me H Et n-Pr
10,000 245
30 11 103 6% @ 100 nM
333 22
[220] [221] [220] [220]
the poor oral efficacy was thought to be caused by metabolical instability, e.g. de-alkylation at the N1 position.
Ph CO2Me
O N H
(350) R = H, Ki(CB2) = 250 nM
Me N R
(351) R = OMe, Ki(CB2) = 8nM, CB1/CB2 = 500 O
N
It was hypothesised that the increased activity of indoles bearing smaller groups at the C2 position described above would be derived from the conformational preference of conformer A over conformer B as a result of steric
J. ADAM ET AL.
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Me Me
Me Me O
Me
N H
HN
O R
R N
N
O N
OMe
Me
O N
OMe
Amide conformer A
Amide conformer B
Fig. 6.3 Structures of amide conformers A and B.
effects (Figure 6.3) [221]. Based on this hypothesis, indazoles and indolopyridones, such as (356) and (357) were designed. For (356), it was predicted that conformer A would be preferred over conformer B, as the result of an intramolecular stereoelectronic interaction. Actually, (356) was more potent than all of the indole derivatives mentioned above. The conformationally constrained indolopyridone (357) had the most potent activity in the series [K i ðCB2 Þ ¼ 1:0 nM]. In the in vivo model, (357) exhibited inhibition activity against LPS-induced TNF-a release both after i.v. and oral administration. CB2-selective indazole derivatives, such as (358), were also reported from the research group of the University of Connecticut [222]. The Ki values of (358) for CB2 and CB1 were 0.15 and 6.84 nM, respectively. Another type of C3 amido-indole, exemplified by (359), was disclosed by Sanofi Synthelabo [223]. Me Me O
Me Me O
Me
N
N
Me
H N O
N OMe
N
(356) Ki(CB2) = 2 nM
O
N OMe
N
(357) Ki(CB2) = 1 nM
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RECENT PROGRESS IN CANNABINOID RESEARCH
Me Me Me
O
O
NH
N H N
Me
O N
N N
(CH2)3NHSO2Me
Cl
(359)
(358)
NO2
Me NHSO2Me
O
I S O
N
N O N
O
S
O
Me
F
(360) AM1241
(361)
AM1241 (360) exhibited high affinity and selectivity for CB2 [K i ðCB1 Þ ¼ 280 nM, Ki ðCB2 Þ ¼ 3:4 nM]. (360) Dose dependently inhibited experimental neuropathic pain in a spinal nerve ligation-induced tactile and thermal hypersensitivity model [224]. Other indole derivatives bearing sulfonamide moieties on the side chain, such as compound (361), were disclosed [225]. Though 67 derivatives including pyridyl and other heteroaromatics instead of the indole core were listed, no specific biological data were shown.
RESORCINOL DERIVATIVES
As previously discussed, phenol derivatives bearing a six-membered carbocyclic ring next to the hydroxy group of the phenol represent a major class of cannabinoid ligands. These compounds correspond to pyran ring-opened analogues of (67). Several efforts have been made to find CB2 selective agonists among the series.
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CH2OH OMe
Me Me
n-C6H13
MeO Me
Me
HU-308 (362)
One of the conspicuous resorcinols is HU-308 (362), which is a CB2specific agonist; this compound does not bind to CB1 (Ki>10 mM), but has a significant affinity for CB2 (K i ¼ 22:7 nM) [226]. HU-308 elicited analgesic activity in a formalin-induced peripheral pain model and an anti-inflammatory effect on arachidonic acid-induced ear inflammation, though it showed no activity in a tetrad of behavioural tests, which are linked to CNS activity (Table 6.34). In order to study the SAR of the resorcinol derivatives, about 40 compounds were synthesised and tested in biological assays [227]. The 2cyclohexylresorcinol derivative, O-1422 (363), showed binding affinity for both CB1 and CB2 receptors, with a moderate CB2 selectivity [K i ðCB1 Þ ¼ 11 nM and K i ðCB2 Þ ¼ 1:5 nM]. Reducing the ring size of the carbon ring to cyclopentyl (364) decreased CB1 affinity by 9-fold and CB2 affinity by 5-fold, though increasing the ring size to cycloheptyl (365) did not affect the binding affinity. Introduction of a methyl group at the 2- or 3-position of Table 6.34 RESORCINOL DERIVATIVES [227] OH R n-C6H13
HO Me
Cpd. (363) (364) (365) (366) (367) (368)
O-1422 O-1424 O-1656 O-1658 O-1657 O-1659
Me
R
CB1 Ki (nM)
CB2 Ki (nM)
CB1/CB2
c-Hexyl c-Pentyl c-Heptyl 2-Methyl c-hexyl 3-Methyl c-hexyl 4-Methyl c-hexyl
11 95 18 16 14 45
1.5 7 2 1 0.8 5
7.3 14 9.0 16 18 12
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RECENT PROGRESS IN CANNABINOID RESEARCH Table 6.35 CYCLOHEXYL RESORCINOL DERIVATIVES [227] OMe Me
R n-C6H13
MeO Me
Me
Cpd.
R
CB1 Ki (nM)
CB2 Ki (nM)
CB1/CB2
(369) O-1999 (370) O-1966A
H OH
>10,000 5,055
466 23
>21 220
the cyclohexyl ring did not dramatically alter the affinity. However, moving the methyl group to the 4-position (368) decreased affinity for both cannabinoid receptors. Conversion of the two phenolic hydroxy groups of (367) to methoxy groups (369) reduced both CB1 and CB2 binding activity, CB1 affinity was decreased more than 700-fold. Further efforts to find more CB2 selective agonists in the dimethoxyphenyl series resulted in discovery of O1966A (370), which has good affinity and selectivity for CB2 [K i ðCB1 Þ ¼ 5; 055 nM and K i ðCB2 Þ ¼ 23 nM, respectively] (Table 6.35). O
Me
OH
Me (CH2 )4
HO Me
Me
AM1703 (371)
Another resorcinol derivative, AM 1703 (371), which has a carbon–carbon triple bond at the end of the alkyl chain, was reported to be a highly potent and selective CB2 agonist (K i ðCB2 Þ ¼ 0:59 pM, with 500-fold selectivity against CB1) [228]. BENZO[C]CHROMEN-6-ONE DERIVATIVES
Benzo[c]chromen-6-one derivatives, such as AM 1710 (372), were found to be CB2 selective agonists [229]. Although these compounds are structurally similar to the THC derivatives, the dimethyl group on 6-position of the
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269
THCs is converted to a carbonyl group and the C-ring is aromatised. Species differences in CB2 binding activities of these compounds were observed: (372) and AM 1714 (373) showed 10- to 15-fold higher affinities at rat CB2 than human CB2 [230]. OR (372) AM1710 R = Me Ki = 28nM (humanCB2) Ki = 2.0nM (ratCB2)
OH
O
n-C6H13
O
(373) AM1714 R = H Ki = 29nM (humanCB2) Ki = 1.9nM (ratCB2)
Me Me
OTHER HETEROCYCLIC CB2 AGONISTS
Benzimidazole-5-carboxamide-based agonists have been reported [231], for example, compound (374) showed good activity and about 100-fold selectivity for CB2 [K i ðCB1 Þ ¼ 84:8 nM, K i ðCB2 Þ ¼ 0:8 nM]. Thirty-five related compounds were also disclosed along with (374). Tetrahydroquinolone (375a) also exhibited CB2 agonist activity [232] and the CB2 binding affinity was 17-fold enhanced by the conversion of the six-membered carbon ring to the corresponding eight-membered ring (375b). Tetrahydroisoquinoline (376) and its derivatives were reported as CB2 agonists [233]. To estimate the CB2 agonist activity of (376), the inhibition activity on forskolin-stimulated cyclic AMP production was tested and the IC50 was 0.4 nM. O
OEt
NH(CH2)2 Ph
O X
N
(CF3 CH2)2N
N
O
n-Bu N c-Hex (374)
(375a) X = CH2 Ki(CB1) = 2851nM Ki(CB2) = 28nM (375b) X = (CH2)3
Me Me NH O c-Hep
Ki(CB1) = 908nM Ki(CB2) = 1.6nM
(376) IC50(CB2) = 0.4nM
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RECENT PROGRESS IN CANNABINOID RESEARCH
Several types of CB2 agonists with monocyclic core structures have been reported, these compounds being structurally distinct from other series of CB2 agonists. Tetrazine derivative (377) exhibited 90-fold selectivity for CB2 and 2-imino-1,3-thiazolidine (378) showed more than 500-fold selectivity [234, 235]. A pyridone analogue (379) of a bicyclic CB1 agonist, CP 47,497 (192) showed CB2 selective activity [K i ðCB1 Þ ¼ 973 nM, K i ðCB2 Þ ¼ 56 nM)] [236], though the origin of the selectivity was unclear. The pyridine derivatives such as (380) were disclosed by Glaxo; (380) was more than 100 times selective for CB2 over CB1 receptors [237]. CO2 Et Ph
S N
OH
i-Pr
O
N
N
N
N
N
N
Ph O
CO2 Et
SEt
n-C5H11 (379)
(377)
(378)
Ki(CB1) = 1672nM
Ki(CB1) = >5000nM
Ki(CB2) = 19nM
Ki(CB2) = 9nM
N N
n-Pr
H N
Cl
H N O
CF3
(380)
THERAPEUTIC APPLICATIONS OF CANNABINOID AGONISTS Standardised preparations of cannabinoid agonists are available for therapeutic use in some countries [238]. Dronabinol (MarinolTM), an oral preparation of D9-THC (67), is used clinically as an appetite stimulant in AIDS patients and an antiemetic in cancer chemotherapy. A synthetic analogue of (67), nabilone (CesametTM), (381), is also used to suppress nausea and vomiting in cancer chemotherapy. There have been a number of studies to evaluate the therapeutic effect of cannabinoids against spastic disorders, including multiple sclerosis and spinal cord injury. For example, a randomised placebo-controlled trial in more than
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271
600 multiple sclerosis patients with oral cannabis extract or (67) was performed and an objective improvement in mobility was observed in the study [239]. O
CH2OH OH
Me Me
OH
n-C6H13
O Me (381) Nabilone
Me
Me Me
n-C6H13
O Me
Me
(165) HU-210
Though cannabinoids exhibit a wide range of antinociceptive activities in a variety of animal models, e.g. neuropathic pain [240], hyperalgesia [241] and inflammatory pain [242], there are a limited number of reports in human trials on the use of cannabinoids against pain disorders. One of the early clinical trials was a preliminary double-blind study of oral (67) in patients experiencing cancer pain, which was published in 1975 [243]. While pain relief was demonstrated to be significantly superior to placebo after administration of 15 and 20 mg of (67), some CNS side effects including substantial sedation and mental clouding were observed. A more recent clinical study revealed that the maximally tolerated dose of cannabis extract in 40 cancer patients was 0.15 mg/kg [238]. There have been very few clinical trials of cannabinoids for the treatment of neuropathic pain and it was reported that an overall benefit of oral dronabinol was not obtained on refractory neuropathic pain because of the CNS side effects [244]. One of the potential uses of cannabinoids is to reduce the dosage of morphine administered to treat chronic pain disorders. Holdcroft et al. [245] reported that cannabis extract decreased the requirement of morphine in a familial Mediterranean fever patient. The CNS side effects of cannabinoid agonists are thought to be caused by CB1 receptor activation in brain. Recently, it was reported that CB2 selective agonists showed antinociceptive effects in various pre-clinical pain models including models of hyperalgesia and neuropathic pain without any CNS cannabinoid effects [246]. To date, the mechanism of inhibition of pain responses by CB2 agonists has not been resolved completely, however, an indirect action on inflammatory processes and stimulation of the release of endogenous opioids have been proposed [247]. An alternative approach is topical administration of a cannabinoid agonist. Analgesic and anti-hyperalgesic effects of a topically applied CB1/CB2 agonist HU-210 (165) to human skin were reported [248] and no psychotomimetic side effects were observed.
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RECENT PROGRESS IN CANNABINOID RESEARCH
It has been revealed that cannabinoids exhibit neuroprotectant activities in both in vitro and in vivo models [249]. The neuroprotective effects are mainly based on regulation of transmitter release, modulation of calcium homeostasis, anti-oxidant properties and modulation of immune responses. A number of neurological disorders, including brain trauma, cerebral ischaemia, Parkinson’s disease and Alzheimer’s disease represent possible therapeutic areas for cannabinoids with neuroprotective properties. Cannabinoids are also suggested to have potential against glaucoma due to their neuroprotective nature and lowering of intraocular pressure [250]. Other therapeutic uses of cannabinoid agonists have been reported. The potential of cannabinoids as a treatment for asthma is supported experimentally. A CB1 agonist, (R)-methanandamide (21), inhibited nerve growth factor (NGF)-induced airway hyperresponsiveness in vivo [251]. The antipruritic effect of cannabinoids has been reported, the action being mediated by both CB1 and CB2 pathways [252]. Treatment with cannabis extract improved urinary tract symptoms of multiple sclerosis patients significantly in an open-label pilot study [253]. CB2 selective agonists are thought to be promising agents against a variety of disorders related to the immune system, because of their immunomodulatory properties and lack of psychotropic effects. Myocardial ischaemia-reperfusion injury [254], atherosclerosis [255], glioma [256], leukaemia/lymphoma [257] and osteoporosis [258] are suggested as target diseases for CB2 agonists. Cough reflex was also inhibited by a CB2 agonist JWH-133 (329) in a guinea pig model of cough [259]. Me
O n-C4H9 4
(CH2)3
Me N H
OH Me Me
n-Pr
O Me
(21) R-Methanandamide
Me
(329) JWH-133
CB1 RECEPTOR ANTAGONISTS In recent years pre-clinical testing and, in particular, clinical data on SR141716A (Rimonabant, Acomplia) (382) from Sanofi-Aventis (previously
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273
Sanofi-Synthelabo) has stimulated a great deal of interest in the discovery of cannabinoid CB1 receptor antagonists. This section aims to cover the structures and therapeutic applications of CB1 antagonists published to date. Reviews of CB1 receptor antagonists and their therapeutic applications have recently appeared in the literature [260–262].
1,5-DIARYL-PYRAZOLES
The most important class of CB1 receptor antagonists identified to date are the 1,5-diaryl-pyrazoles. This class includes rimonabant (382), first described in patent applications from Sanofi just over a decade ago [263, 264]. Rimonabant has proved invaluable in the elucidation of cannabinoid receptor pharmacology, as described in the section on therapeutic applications below. Several groups have published on structural analogues of (382), one of the earliest being the disclosure of CP 272871 (383) from Pfizer, which displays lower affinity for the CB1 receptor than (382), in addition to reduced selectivity over the CB2 receptor subtype. Both (382) and (383) have been shown to act as inverse agonists rather than neutral antagonists in vitro [265]. A recently published patent application from Sanofi-Aventis claims a series of 4-cyanopyrazole analogues of (382), with 42 specific examples [266]. Makriyannis and co-workers [267] have published on SAR within the 1,5diaryl-pyrazole class of compounds. These studies indicated a requirement for a para-substituted phenyl ring at the 5-position of the pyrazole, iodophenyl proving optimal in terms of potency. Retention of the 3-carboxamide moiety was important, as was retention of the 2,4-dichlorophenyl substituent at the 1-position. The most potent compound in the series, AM 251 (384), showed slightly higher affinity for the CB1 receptor than rimonabant coupled with excellent selectivity over the CB2 receptor. Furthermore, the presence of the iodophenyl moiety allows for use of this compound as a single photon emission computed tomography (SPECT) ligand for imaging studies [268]. SR147778 (385) is a close structural analogue of (382), first described in a patent application from Sanofi-Synthelabo [269]. In this application, (385) is claimed to have an improved duration of action over (382) following oral dosing in mice. This was demonstrated through receptor occupancy studies and the ability of the compounds to reverse cannabinoid agonist-induced hypothermia. Compound (385) also displayed slightly higher affinity for CB1 receptors than (382) (Ki values of 5.4 and 34 nM, respectively) while retaining excellent selectivity over CB2 receptors. A subsequent publication from Sanofi-Synthelabo further described the in vitro and in vivo profiles of (385) [270]. The compound reversed WIN 55,212-2-mediated effects
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RECENT PROGRESS IN CANNABINOID RESEARCH
(hypothermia, analgesia and gastrointestinal transit) in mice following both i.p. and p.o. administration. (385) also reduced ethanol or sucrose consumption in mice and rats and food intake in fasted and non-deprived rats. The compound is currently in Phase II clinical trials.
O Me
O
N N H
NC
NHPh
N 1
N
N
R
N MeO
Cl
Cl
Cl (382) SR141716A (Rimonabant) R1 = Cl
(383) CP-272871
(384) AM 251 R1 = I
R
O Et
N N H
N N Br
Cl
Cl (385) R = H, SR147778 (386) R = OH
A close analogue of (385), the 4-hydroxypiperidine analogue (386) was recently described in a patent application from Sanofi-Synthelabo [271] and perhaps formed as a metabolite of the parent compound. Martin and co-workers [196] have also published on the discovery and SAR of pyrazole cannabinoids as described in the CB1 agonist section. The analogues were tested for CB1 receptor binding affinity and in a battery of in
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275
vivo tests, including hypomobility, antinociception and hypothermia. Variations of the pyrazole substitution pattern in positions 1, 4 and 5 led to compounds with varying affinity that consistently behaved as antagonists in vivo. Introduction of ether moieties into the 3-position of the pyrazole core led to the identification of both partial agonists and antagonists. For example, compound O-1043 (387) retained a Ki of 53 nM and demonstrated antagonism in vivo. An analogue, in which the amide was replaced with a ketone (O-1271) (388), retained a Ki of 82 nM and again demonstrated antagonism in vivo. Makriyannis and Liu claimed a series of pyrazole analogues in a patent application published in 2003 [272]. Of the 29 compounds specifically exemplified in the patent application, compound (389) was demonstrated to reduce lever presses when administered to rats that were trained to expect delivery of a food pellet as the outcome. It was proposed that the reduction in lever pressing was the result of decreased appetite brought about by CB1 receptor antagonism.
Me
O
R
1
R
N
N
N Cl
N N H
Cl
Cl (387) O-1043 R = OCH2-2,4-diF-phenyl (388) O-1271 R = CO(CH2)4Me
2
R
N Cl
Cl (389) R1 = CH2OH, R2 = 3-pyridyl (390) NIDA-41020 R1 = Me, R2 = OMe
Horti and co-workers [273] published on their studies to produce analogues of rimonabant with reduced lipophilicity. The aim of the work was to produce ligands for positron emission tomography (PET) studies in humans. Highly lipophilic ligands were noted to be a problem due to resulting high levels of non-specific binding. An additional limiting factor in the work was the need to be able to introduce an appropriate atom for a PET labelling study (18F, 76Br or 11C). NIDA-41020 (390) retained high CB1 receptor affinity (K i ¼ 4:1 nM, (382) K i ¼ 1:8 nM in the same assay format) and a reduced lipophilicity in comparison with (382) (e log Doct values of 4.78 and 5.36, respectively).
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RECENT PROGRESS IN CANNABINOID RESEARCH
Several studies have been reported on the application of conformational restraint to the 1,5-diaryl-pyrazole series in an attempt to provide compounds with modified properties. In one approach, a Sanofi-Synthelabo patent application claimed a series of conformationally restrained compounds, exemplified by compound (391). Compounds of the invention were stated to be CB1 receptor antagonists with Ki values below 5 10 7 M and selectivity over CB2 receptors of at least 10-fold [274]. Lange and co-workers [275] published on conformationally restrained analogues of rimonabant (382), again constraining the pyrazole 4-position and the 5-aryl substituent into a ring system. The highest affinity compound within the series was compound (392), which displayed a pKi value of 7.2 ((382) pKi 7.6 in the same assay). This compound (392) was found to be slightly more potent than (382) in a cell-based functional assay for CB1 antagonism (pA2 values of 8.8 and 8.6, respectively). In a subsequent publication, Ruiu et al. [276] synthesised and characterised NESS-0327 (393). This compound had also been prepared and tested in the publication from Lange et al. (393) was reported to show higher affinity for the CB1 receptor than (382) (Ki values of 350 fM and 1.8 nM, respectively), in addition to higher affinity for the CB2 receptor (Ki values of 21 and 514 nM, respectively). Selectivity for the CB1 receptor over the CB2 receptor was calculated at more than 60,000-fold for (393). Potent antagonism was demonstrated in a variety of in vitro and in vivo paradigms for (393). X O S
O
N N H
(391)
N Cl
R
N N H
N
N Cl
Cl
Me
N
N N Cl
O
N N H
Cl
Cl (392) X = (CH2)2, R = NO2 (393) NESS-0327 X = CH2, R = Cl
Cl (394)
Thomas and co-workers [277] reported that irradiation of (382) with a 450 W high-pressure mercury lamp brought about photocyclisation to a constrained analogue (394). The structure of the product was elucidated through NMR and X-ray diffraction analysis. The compound retained high affinity for the CB1 receptor (K i ¼ 48 nM) and good selectivity over the CB2 receptor (K i ¼ 3; 340 nM).
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A series of 21 compounds, differing from (382) in the amide region, has been synthesised by Franciso et al. [278] and their biological properties studied. The biological activity of the compounds was assessed in two binding assays using [3H]-CP 55,940 (see Table 6.36) and [3H]-rimonabant (data not shown), and their functional activity determined using a GTPgS assay. Upon Table 6.36 MODIFIED AMIDE SUBSTITUENT ANALOGUES OF RIMONABANT (382) – IN VITRO DATA
Me
CONHR N N
Cl
Cl
Cl
Cpd
R
(382) (395) (396) (397) (398) (399) (400) (401) (402) (403) (404) (405) (406) (407) (408) (409) (410) (411) (412) (413) (414) (415)
Piperidine 6.18 Et 46.3 n-Pr 29.9 i-Pr 29.4 n-Bu 13.4 i-Bu 11.5 n-Pentyl 11.4 n-Hexyl 18.1 c-Hexyl 2.46 Morpholinyl 22.9 OH 1,690 (CH2)2OH 385 (CH2)3OH 160 154 (CH2)4OH 117 (S)-(+) CH(CH3)CH2OH (R)-( ) CH(CH3)CH2OH 117 374 NH2 NHMe 555 NHEt 143 NHn-Pr 74.8 NHn-Bu 50.9 NHi-Bu 41.8
a
CB1 Ki (nM) CB2 Ki (nM) [ 313a 3,110 2,960 1,740 1,600 704 1,110 6,870a 228 2,400 7,820a 4,270 1,250 5,720a 5,900 1,770 121,200 6,660 6,061 2,620 2,850 2,190
Value greater than the highest point on the displacement curve.
35
S] GTP–gS EC50 (nM)
56,300 30,300 11,100 16,000 8,450 7,540 5,270 29,400 26,000a 38,900 1.67 106a 241,000 304,000 294,000 292,000 4.19 105a 1.2 107a 4.95 105a 105,000 128,000 73,600
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RECENT PROGRESS IN CANNABINOID RESEARCH
increasing the length of the alkyl chain, the CB1 binding affinity increases with i-butyl and n-pentyl analogues (399) (400) showing the highest affinity. Increasing the chain length beyond this (e.g. n-hexyl (401)) resulted in a decrease in affinity compared with (399) and (400). In the hydroxyalkylamide series (404)–(409) and the alkylhydrazide series, this trend was also evident. The introduction of a hydroxyl group led to a marked decrease in affinity (cf. (395) and (405), (396) and (406)). There was little difference in binding affinity observed between the two hydroxymethylethyl enantiomers (408) and (409) with the incorporation of the hydroxyl group again proving to have a deleterious effect on CB1 binding. The morpholinyl analogue of rimonabant (403) again had comparatively low affinity (K i ¼ 22:9 nM), confirming that the presence of a polar oxygen was not particularly well tolerated. Within the series, the most potent side chain was found to be cyclohexyl (402). The affinity of (402) was only slightly improved compared with (382), implying that the piperidine nitrogen is not likely to be involved in any crucial electrostatic or hydrogen-bond formation with the receptor. Throughout the series, the CB2 receptor affinity was modest with CB1/CB2 selectivity ranging from 5-fold (404) to 378-fold (401), and all the compounds were found to be inverse agonists. Five compounds with reasonably high CB1 receptor affinities (399)–(403) were investigated for their ability to antagonise the CB1 agonistmediated inhibition of electrically evoked contraction of mouse vas deferens tissue. All the compounds tested were found to produce antagonism. In line with the earlier obtained binding data, (402) was found to be the most potent in the vas deferens experiment. Compounds (400), (402) and (403) also displayed a noticeable inverse effect, enhancing the amplitude of the twitch prior to addition of the cannabinoid agonist WIN 55,212-2, an effect not shown by (399) and (401). A QSAR study was performed to understand the pharmacophoric requirements of the aminopiperidine region. A number of conformations of each molecule were generated using a quenched molecular dynamics approach and the resultant conformers overlaid by superimposing their pyrazole rings using a root mean square minimisation procedure. A comparative molecular field analysis (CoMFA) approach was then used to derive a QSAR and allow visualisation of the regions where changes in steric or electrostatic properties resulted in changes in measured biological property (CB1, CB2 and GTPgS binding). From this, it was observed that a side chain no longer than 3 A˚ in length could be tolerated in the aminopiperidine region. Furthermore, increased affinity and potency were predicted for compounds having a substituent bearing a positive charge density in this region. CoMFA has also been used by Shim and co-workers [279] to help understand the interaction between rimonabant (382) and the CB1 receptor.
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Conformational analysis of (382) was first performed using the AM1 calculation method. Four distinct low-energy conformers (Tg, Ts, Cg, Cs) were identified corresponding to either trans (T) or cis (C) geometry around the amide bond and either skew (s) or gauche (g) depending on their torsion angle around the {(CQO)–N–N–C} bond in the aminopiperidine region. In the unprotonated form of (382), the Tg conformer was found to be the most energetically favourable followed by the Cg, Ts and Cs forms, which was in agreement with the AM1 calculated values. In the protonated state, the most stable conformation was Ts, followed by Tg, which was more stable than Cs and Cg. Both protomeric states were examined in the subsequent QSAR model since the extensive conjugation of the molecule made it uncertain as to which form would predominate under physiological conditions. A DISCO-derived pharmacophore model was developed using a previously published superposition model [280] of cannabinoid agonists CP 55,244 (196) and WIN 55,212-2 (254a). Construction of a 3D-QSAR CoMFA model for (382) and a series of 31 closely related structural analogues was performed using binding affinities determined from displacement of [3H]-CP 55,940 in rat brain membranes. The Tg, Cg and Ts conformers only were considered for the neutral species, while the Ts, Tg and Cs conformers in the protonated species were used in the model. The values obtained for the r2 and cross-validated r2 were indicative of statistical robustness and predictivity in all cases except for the protonated Tg conformer where the r2cv value fell to 0.36, below the accepted threshold for internal predictive ability. The data obtained from the studies suggested a crucial role for the N1 aromatic ring in forming a hydrophobic interaction with the CB1 receptor. It was also suggested that the second aryl ring may prevent the change in receptor conformation necessary for agonist activity. In addition, it was postulated that the C3 amide substituent might interact with the Lys-192 residue, believed to be participating in a specific ligand-binding interaction, thereby prohibiting agonist activity and also inducing or stabilising the receptor conformation required for inverse agonism. This latter theme was subsequently explored by Hurst et al. [281]. Receptor models of the R and R* forms of CB1 were constructed using the 2.8 A˚ crystal structure of bovine rhodopsin. Rimonabant (382) and a conformationally constrained analogue (VCHSR), in which the (N-piperidinyl)-3-carboxamide moiety of rimonabant was replaced by 3-[(E)-2-cyclohexylethenyl], were evaluated in these models, and in vitro, in both the wild-type CB1 receptor and in a mutant in which the Lys residue at position 192 had been mutated to Ala. The two ligands were docked into the aromatic transmembrane helix (TMH) 3-4-5-6 regions of the active and inactive TMH bundle, and the energy miminised using the AMBER force field. In the inactive R complex, (382) was observed to form a hydrogen bond with Lys-192 (TM3.28) which is part
280
RECENT PROGRESS IN CANNABINOID RESEARCH
of a salt bridge with Asp-366 (TM6.58). In addition, aromatic stacking interactions were formed with Trp-279 (TM5.43) with the dichlorophenyl ring interacting with Phe-200 (TM3.36) and the monochlorophenyl ring interacting with Tyr-275 (TM5.39). In the active state, the relative positions of key TMH3 and TMH6 residues Lys-192 and Asp-366 were observed to have changed, breaking their salt bridges and preventing the interaction between Lys-192 and the C3-substituent of (382). In the VCHSR/CB1 R complex, the modelled interactions were very similar to those of (382), but lacked the hydrogen-bonding interaction between Lys-192 and the C3 substituent. Analysis of the VCHSR/CB1 R* complex suggested that VCHSR should have approximately equal affinity for both the R and R* states, conferring the property of neutral antagonism. Consistent with this hypothesis, it was found that VCHSR behaved as a neutral antagonist at the wild-type CB1 receptor, as witnessed by the attenuation of the inhibition of the calcium current by (254a) in superior cervical ganglion neurons. It was therefore proposed that the interaction between the Lys-192 residue and the C3 substituent of (382) was the principal determinant in the high affinity for the inactive R state and hence the experimentally observed inverse agonism. Molecular modelling and automated docking studies by Salo et al. [282] also confirmed that Lys-192 played a key role in the binding mode of HU210 (165), CP 55,940 (193), rimonabant (382), WIN 55,212-2 (254a) and 2-AG. Further mutagenesis and molecular modelling work [283] compared the interaction of (382), (254a) and anandamide (1) with CB1 wild type and F3.25A, F3.36A, W5.43 and W6.48 mutants. The binding of (382) and (254a) was found to be affected by the F3.36A, W5.43 and W6.48 mutations, implying their presence in the binding site of these two ligands. In contrast, only the F3.25A mutation was found to influence the binding of (1), suggesting the existence of a distinct binding site. It was therefore suggested that an aromatic micro domain in the TMH3-4-5-6 region comprising F3.36A, W4.64, Y5.39, W5.43 and W6.48 was responsible for binding both (382) and (254a), but that the endocannabinoid-binding pocket may only partially overlap with the former lipophilic domain. Patent applications from Pfizer disclosed 1,5-diaryl-pyrazoles bearing bioisosteric replacements for the 3-carboxamide moiety. One application showed that the amide could be replaced by a-aminoketones as exemplified by compound (416) [284]. The corresponding alcohols and their ethers were also described, including compounds that allowed the amine substituent and ether to form a ring system, such as a morpholine unit. This application also allowed for the replacement of the 1,5-diaryl-pyrazole by a 1,2-diarylimidazole bearing a 3-carbonyl substituent, as exemplified by compound (417). A further patent application from Pfizer claims compounds in which imidazoles replace the 3-carboxamide moiety in the 1,5-diaryl-pyrazole
J. ADAM ET AL.
281
series [285]. Compound (418), one of the compounds specifically claimed in the patent, showed a CB1 receptor affinity of 371 nM. O O
N
N
N
N
Me
Me Ph N
N N
N
N
Cl
Cl
N
Cl
Cl
Cl
Cl
Cl (416)
(417)
(418)
In addition, Pfizer has identified a number of related, fused bicyclic pyrazole analogues [286–292]. These compounds, of which structures (419–425) are specified examples, are claimed to be of use in the treatment of a number of diseases including alcoholism, psychosis, tobacco abuse, Parkinson’s disease and obesity. Me N O
Cl N Cl
N
N
N
Cl
Ph
N N
N
N
Cl
N
Cl (419)
Cl
Cl
O
(420)
Cl
N
N
N
N i-Pr
N
N i-Pr O
F F
F
(421)
Cl
(422)
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RECENT PROGRESS IN CANNABINOID RESEARCH
Me H2NCO
N
N
Cl N
N
Cl N
X
N
NHi-Pr
N
N
N
N
Cl
(425)
F
(423) X = CH (424) X = N
4,5-DIHYDRO-1H-PYRAZOLE DERIVATIVES
Solvay Pharmaceuticals have claimed 4,5-dihydro-1H-pyrazole derivatives in a series of patent applications [293–296], compounds (426–429) represent examples of the structures involved. O
O N
Cl
N
Cl
N
S
N
Ph
N
NHMe
NEt2
N
O
O
S
R
SMe
(428) O
O
Ph
N
Cl
S
N
(426) R = Cl (SLV-319) (427) R = CF3 (SLV-326)
N
NH
Cl
N Ph (429)
Solvay subsequently published in vitro, in vivo, computational and X-ray diffraction data relating to this series of 3,4-diarylpyrazolines [297]. In this
J. ADAM ET AL.
283
publication, a number of compounds were evaluated, in the first instance in vitro at the human CB1 and CB2 receptors, expressed into Chinese hamster ovary (CHO) cells by radioligand binding. In addition, the CB1 functional activity was measured via an arachidonic acid release functional assay. Results for a number of the compounds are shown in Table 6.37. For compounds (431–434), a significant improvement in binding affinity was observed when the 4-Me group in (430) was substituted with either Cl (431), 2,4,6-trimethyl (433) or F (434). Replacement of 4-Me with 4-OMe (432) failed to improve the affinity or functional activity. In the amidinesubstituted series (435)–(447), dimethyl compounds (435) and (436) both Table 6.37 PYRAZOLINE DERIVATIVES AND RIMONABANT (382) – IN VITRO DATA [297]
Cl O N
S
N
O
N R2
R1
N R3
Cpd.
R1
R2
R3
CB1 Ki (nM)
CB1 pA2
CB2 Ki (nM)
(382) (430) (431) (432) (433) (434) (435) (436) (437) (438) (439) (440) (441) (442) (443) (444) (445) (446) (447)
4-Me 4-Cl 4-OMe 2,4,6-Me3 4-F 4-Cl 4-F 2-Cl 3-Cl 4-CF3 4-Cl H 4-F 4-Me 3-CF3 2,4,6-Me3 4-OMe 3,4-Benzo
H H H H H Me Me H H H H H H H H H H H
H H H H H Me Me Me Me Me Me Me Me Me Me Me Me Me
25 197 16.1 196 24.2 52.6 280 >1,000 75.4 13.9 221 25.2 170 338 119 36.5 54.2 22.9 21.8
8.6 8.4 9.5 8.3 9.4 9.0 8.5 o7.5 8.3 8.6 9.3 8.7 7.5 8.5 8.6 9.1 9.4 8.0 8.5
1,580 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000 >1,000
284
RECENT PROGRESS IN CANNABINOID RESEARCH
show reduced affinity compared with their unsubstituted counterparts (431) and (434), respectively. The affinity of the amidine-NH2 analogues was broadly similar to that of the amidine-NHMe compounds. 3-Chloro (438) and 4-chloro (440) were approximately equipotent, however, there was some loss of activity witnessed when the substituent was moved to the 2-position (437). In contrast to (432), the 4-methoxy analogue (446) in the amidineNHMe series was found to have excellent CB1 affinity (22.9 nM) while trifluoromethyl substitution at the 3-position (444) also yielded a more potent CB1 ligand than the 4-CF3 congener (439). A bulky 2-naphthyl-substituted analogue also proved to have excellent affinity. The amidine functionality was shown to be crucial for good CB1 affinity, since replacement by an amide drastically reduced affinity, whereas the substitution of the amidineNH2 group for SMe, was more tolerated. Racemic (439) and (440) were resolved and each of the enantiomers tested for their CB1 affinity. In each case, the levorotatory (4S-) eutomers were found to be more potent than their dextrorotatory equivalents; 4S-(439) (427) showed an affinity of 35.9 nM and 4S-(440) (426) had an affinity of 7.8 nM. Throughout the series, good CB1/CB2 selectivity was evident. Table 6.38 describes some of the key in vivo data for the significant pyrazoline compounds in a CB1 agonist-induced hypotension rat model and hypothermia mouse model, compared with the Sanofi-Aventis CB1 antagonist, rimonabant (382). From the initial disappointing discovery that progenitors (430) and (431) were inactive when administered orally in both assays, it was found that (435), the amidine-NMe2 analogue of (431) did exhibit in vivo effects. Subsequently, (437)–(439) were also found to have in vivo efficacy in both assays when administered orally. In common with the in vitro data, the 4S-analogues of (439) and (440) were found to be active in vivo, while the 4R-enantiomers failed to display CB1 activity. Table 6.38 PYRAZOLINE DERIVATIVES AND RIMONABANT (382) – IN VIVO DATA Cpd.
Rat hypotension ED50a
Mouse hypothermia Cpd. LEDb
Rat hypotension ED50a
Mouse hypothermia LEDb
(382) (430) (431) (435)
3.2 >30 >30 >30
3 >30 >30 10
8.9 15 2.0 5.5
3 3 1 3
(439) (440) (427) (426)
a Antagonism of CB agonist (CP 55,940)-induced hypotension in rat, expressed as ED50 (mg/kg, p.o.). b Antagonism of CB agonist (WIN 55,212-2)-induced hypothermia in mouse, expressed as least effective dose (LED) (mg/kg, p.o.).
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285
An X-ray diffraction study of (426) revealed the existence of a hydrogen bond between the N-atom at the 2-position of the dihydropyrazole ring and the hydrogen atom of the amidine. A series of Confort-generated conformers of (426) were generated and minimised (MOPAC, PM3) and then compared with the favoured trans-gauche Tg conformer [279] of rimonabant (382). The conformer of (426) which overlapped best with the Tg conformer of (382) was then docked into a reconstructed model of the CB1 receptor [281]. It was found that hydrogen bond was formed between one of the oxygen atoms of SO2 and the Asp-366:Lys-192 salt bridge. An additional H-bond was also observed between Ser-383 and the remaining SO2 oxygen atom. A stacking interaction could be envisaged between the 4-Cl-phenyl ring of (426) and the Phe-170 residue. Two hydrophobic pockets, defined by Trp-279, Phe-200, Trp-356 and Tyr-275, Trp-255, Phe-278 bind the two 4-Cl-phenyl rings. Investigation of the differences in crystal packing between (431) and (426) from comparison of their respective X-ray structures, revealed that (431) was more tightly packed than (442), reflected in their respective melting points of 235 and 170 1C. It was postulated that the absence of in vivo activity for (431) may be explained by the resultant reduction in water solubility and dissolution rate compared with (426). The comparatively high calculated polar surface area of (431) (122.5 A˚2) compared with (426) (89.3 A˚2) was also proposed as a factor influencing the marked difference in bioavailability between the two related compounds. Compound (426) (SLV319) is currently being developed with Bristol-Myers Squibb for the potential treatment of obesity and other metabolic disorders. Phase I trials for obesity were started in April 2004. Earlier Phase I clinical trials for the treatment of schizophrenia and psychosis, which commenced in April 2002, appear to have been abandoned.
IMIDAZOLE-, THIAZOLE-, PYRROLE- AND TRIAZOLE-BASED CB1 RECEPTOR ANTAGONISTS
4,5-Diarylimidazole derivatives, exemplified by (448) have been claimed by Merck as either antagonists or inverse agonists of the CB1 receptor [298]. In this series of compounds, the central imidazole motif can be considered a bioisostere of the pyrazole core of rimonabant. A similar approach was utilised by Solvay Pharmaceuticals [299] in a patent application published shortly after that of Merck. The isomeric 1,2-diaryl-4-carboxamide nucleus (449) can again be considered as a structural isostere, serving to orient the two substituted aryl groups and carboxamide group in geometry analogous to that of (382). An alternative methylimidazole core, 1,2-diaryl-5-methylimidazole (450), was also
286
RECENT PROGRESS IN CANNABINOID RESEARCH
utilised by Merck [300]. More recently, Pfizer has disclosed a series of novel imidazole derivatives useful in the treatment of obesity, alcoholism and depression [301]. The specified compound (451) was stated to have CB1 binding affinity in the range of 1–10 nM.
Me
Cl
O
Cl O
N
N N H
N
N H
N
N
Cl Cl Cl Cl
(448)
(449)
N H
Cl N
i-PrNH
N
N
Cl
O
O
Cl
Me
(450)
N Cl
N Cl
(451)
Novel 6-aminopurine compounds, for example, (452) have been claimed by Pfizer as particularly useful in the treatment of obesity and alcohol abuse [302]. In conjunction with an opioid receptor antagonist, the related compounds such as (453) are claimed as a combination therapy for treating alcohol, cocaine or tobacco addiction or dependence, reducing alcohol withdrawal symptoms and aiding in the cessation or lessening of alcohol abuse, substance abuse or behavioural dependency, including gambling [303]. Compound (453) has also been claimed to have utility in combination with a nicotinic acetylcholine agonist in the treatment of obesity or compulsive overeating [304] and compulsive gambling, nicotine and drug dependence [305].
J. ADAM ET AL.
N
CONH2
EtNH
N
N
N
Cl
Cl N
N
Cl
O
Cl
N N
287
X
S Cl
N H N
N
N
N
(453)
(452)
(454) X = CH (455) X = N
Cl Cl
Cl
Thiazole-based cannabinoid CB1 modulators were claimed in two patent applications by AstraZeneca [306, 307] where the strategy of bioisosteric pyrazole-replacement is evidenced by the specified compounds (454) and (455), respectively. No specific biological data were presented but the specified compounds were stated to have IC50 values o200 nM. A central thiazole motif was also utilised by Solvay Pharmaceuticals [308]. In vivo antagonism was evaluated using CP 55,940-induced hypotension in rats, however no specific results were given. Compound (455) was shown to have broadly similar affinity at both CB1 and CB2 receptor subtypes with pKi values of 7.8 and 8.1, respectively. A thiazole core has also been utilised by Solvay Pharmaceuticals [309] in the search for a novel bioisosteric replacement of the rimonabant (382) pyrazole core. The affinity of several compounds for the human CB1 receptor was determined in transfected CHO cells using tritium-labelled CP 55940. Antagonism was determined in the same cell line by WIN 552122-induced release of arachidonic acid. The pKi of (456) was found to be 6.9, while the pA2 value was measured as 8.7. A series of six 1,2,4-triazole analogues has been prepared by Jagerovic et al. [310], via their corresponding N-acylbenzamides (Table 6.39). O
Cl N
H N H
O
H
S H
N H (456)
N
N
Ph Cl
N n-C5H11
(457)
Cl
288
RECENT PROGRESS IN CANNABINOID RESEARCH Table 6.39 STRUCTURES OF 1,2,4-TRIAZOLE ANALOGUES OF RIMONABANT (382)
N
2
R
(CH2 )nMe
N N
1
R
Cpd.
R1
R2
n
Cpd.
R1
R2
n
(458) (459) (460)
4-Cl 4-Cl 4-Cl
H 4-Cl 4-Cl
1 1 5
(461) (462) (463)
4-Cl 2,4-di-Cl H
2,4-di-Cl 2,4-di-Cl H
5 5 6
The biological activity of the compounds was examined by assessing their ability to antagonise WIN 55,212-2 induced inhibition of electrically induced contractions in mouse vas deferens and guinea pig ileum. Compounds (459)–(463) were not seen to induce modification of the contractile response in either the vas deferens or ileum tissues when administered alone, effectively ruling out the possibility that these compounds behaved as agonists. Compound (458) was found to inhibit contractile activity in the guinea pig ileum, an effect that was not antagonised by (382), thus precluding cannabinoid agonist activity, however the mechanism underlying the observed inhibition was not investigated further. Upon incubation of (458–463) in the presence of WIN 55,212-2, analogue (461), bearing the familiar (4-chlorophenyl)-(2,4-dichlorophenyl) substitution, induced a significant and dosedependent decrease of the inhibition produced by the aminoalkylindole agonist in both tissues. The binding affinity of (461) in rat cerebellular membranes using [3H]-rimonabant and [3H]-WIN 55,212-2 was found to be moderate (K i ¼ 855:6 and 748 nM, respectively) compared with rimonabant (382) (K i ¼ 4 nM). The effects of (461) in vivo were also assessed in the cannabinoid tetrad behavioural tests (nociception, temperature, spontaneous motility and catalepsy). Compound (461), administered intraperitoneally (1 mg/kg) was found to behave as a cannabinoid antagonist, reversing the effects of WIN 55,212-2 (1.5 mg/kg, i.p.). In addition to triazole-based biosteres of (382), thiazoles and imidazoles have also been investigated by Lange et al. [311]. The three series of compounds were assessed on their CB1 binding affinity and functional activity
J. ADAM ET AL.
289
and their selectivity over the CB2 receptor in CHO cells stably transfected with human CB1 and CB2 receptor. Comparison of thiazole-based regioisomers (464) and (465) has revealed a marked preference for the phenyl substitution of the former, a trend also observed in the subsequent imidazole series (data not shown), but not in the triazole series as evident from the data presented for (466) and (467) (Table 6.40). The thiazole (464) was approximately 8-fold less potent than the corresponding imidazole (470) (Table 6.41) suggesting that the seemingly subtle change of the central scaffold imparts a marked change in CB1 receptor recognition. Ethyl substitution at the imidazole 5-position (469) was found to increase potency over the unsubstituted analogue (468), while methyl substitution (470) had a slightly deleterious effect on binding (Table 6.41). Chloro (491), bromo (492), cyano (493) and fluoromethyl (494) substitution at this position were all well tolerated (Table 6.43). Introduction of a chloro-substituted pyridine (475) in place of the more usual p-chlorophenyl group (470) resulted in a slight loss of affinity for the CB1 receptor, as did replacement of the pchloro group of (470) with bromo (471), fluoro (472) and in particular, methoxy (473). Trifluoromethyl substitution (474) however, was well tolerated. The effect of replacing the carboxamide N-piperidinyl group of (470) was also investigated. Reducing the ring of the piperidinyl group by one carbon Table 6.40 THIAZOLE- AND TRIAZOLE-BASED BIOISOSTERES OF RIMONABANT (382) – IN VITRO DATA [311] 1
4
R
R O
Cl
O
Cl
N
N N
N
N
S
N
2
3
R
R
Cl
Cpd. (382) (464) (465) (466) (467)
N
N
Cl
R1
R2
Cl H
H Cl
R3
Cl H
R4
CB1 Ki (nM)
CB1 pA2
CB2 Ki (nM)
H Cl
25 227 >1,000 356 382
8.6 8.1 7.2 8.3 7.6
1,580 5,841 4,668 3,562 5,444
290
RECENT PROGRESS IN CANNABINOID RESEARCH
Table 6.41 IMIDAZOLE-BASED BIOISOSTERES OF RIMONABANT (382) – IN VITRO DATA [311] 3
R
O
Cl N
N
N
N 2
R X 1
R
Cpd.
R1
R2
R3
X
CB1 Ki (nM)
CB1 pA2
CB2 Ki (nM)
(468) (469) (470) (471) (472) (473) (474) (475)
Cl Cl Cl Br F OMe CF3 Cl
H Et Me Me Me Me Me Me
Cl Cl Cl Cl Cl Cl Cl Cl
C C C C C C C N
23 14 30 60 52 106 29 55
8.2 9.0 8.6 8.5 7.7 8.7 8.6 8.4
542 430 608 489 765 326 634 758
(476) yielded a compound approximately equipotent with (470) while increasing the ring (477) or introducing a morpholine group (478) reduced the binding affinity (Table 6.42). Larger bicyclic substituents were well tolerated, as evidenced by (479), (485) and (486). Cyclopentylamine (480), cyclohexylamine (481) and cycloheptylamine (482) substitutents were also accommodated in the lipophilic binding pocket. However, the presence of a hydroxyl group resulted in a marked decrease in affinity, for example, (483) and (484). The CB1 functional activity of a number of the imidazole-based compounds reported exceeds that measured for rimonabant (382) (Table 6.43). While marked CB1/CB2 selectivity is witnessed throughout the series, the selectivity of direct (382) analogue (470) was found to be approximately 3fold lower than for (382) itself (Table 6.44). The in vivo properties of compounds (464), (466) and (468)–(470) relative to (382) were investigated in two animal models: a CB1 agonist-induced hypotension rat model and a CB1 agonist-induced hypothermia mouse model. Shown to be only moderately active in the in vitro screens, thiazole (464) and triazole (466) analogues failed to demonstrate convincing oral activity. Compound (468) was active in the hypotension model but showed
J. ADAM ET AL.
291
Table 6.42 IMIDAZOLE-BASED BIOISOSTERES OF RIMONABANT (382) – IN VITRO DATA [311]
Cl O
Cl N
R N Me
Cl
Cpd. (476) (477)
(478) (479) (480) (481) (482) (483)
R
CB1 Ki (nM)
H N
N
H N
N
H N
N
H N
N
CB2 Ki (nM)
27
8.2
774
64
8.7
505
197
7.5
3,297
40
9.8
1,412
33 35 35 399
8.9 9.1 9.1 7.3
357 160 349 3,469
172
o7.5
3,959
34
8.4
696
19
9.1
54
333 171
8.2 8.9
242 1,984
828 94
7.4 8.6
2,520 815
O
-NHc-Pentyl -NHc-Hexyl -NHc-Heptyl H N
CB1 pA2
OH
(484) N
OH
(485) N
(486)
H N
(487) (488)
-NHOt-Bu
(489) (490)
-NEt2 -Ot-Bu
N H CF3
292
RECENT PROGRESS IN CANNABINOID RESEARCH
Table 6.43 IMIDAZOLE-BASED BIOISOSTERES OF RIMONABANT (382) – IN VITRO DATA [311] 3
R
O
Cl N
N
N
N 2
R X 1
R
Cpd.
R1
R2
R3
X
CB1 Ki (nM)
CB1 pA2
CB2 Ki (nM)
(491) (492) (493) (494)
Cl Cl Cl Cl
Cl Br CN CH2F
Cl Cl Cl Cl
C C C C
27 23 30 36
8.5 8.4 8.6 8.9
823 746 1,590 906
Table 6.44 IMIDAZOLE DERIVATIVES AND RIMONABANT (382) – IN VIVO DATA [311] Cpd.
Rat hypotension ED50a
Mouse hypothermia Cpd. LEDb
Rat hypotension ED50a
Mouse hypothermia LEDb
(382) (464) (466)
3.2 >30 23.6
3 Not determined >30
11.7 15.8 2.4
>30 30 10
(468) (469) (470)
a Antagonism of CB agonist (CP 55,940)-induced hypotension in rat, expressed as ED50 (mg/kg, p.o.). b Antagonism of CB agonist (WIN 55,212-2)-induced hypothermia in mouse, expressed as least effective dose (LED) (mg/kg, p.o.).
no activity in the mouse hypothermia model. The 5-ethyl (469) and in particular, the 5-methyl (470) analogues showed in vivo efficacy in both models, strongly suggesting that the 5-methylimidazole scaffold behaves as a true bioisostere of the pyrazole ring of (382). Reconstruction [297] of the CB1 homology model reported by Hurst et al. [281] was carried out, and ligands (464), (466) and (470) docked into the receptor along with rimonabant (382). A perfect 3D overlap of (382) and (470) was observed. In the case of the thiazole (464) and triazole (466), the
J. ADAM ET AL.
293
carboxamide and aryl rings were found to overlap well with (382), although the central heterocycle deviated slightly from the orientation adopted by (382) and (470). The importance of the substitution patterns on the phenyl rings was also investigated. It was been reported that (382) binds into the active site of the CB1 protein in its most stable so-called transdichlorophenyl (TDC) conformation, in which the dichlorophenyl ring is oriented trans with respect to the carbonyl of the carboxamide group. It has been proposed that the salt bridge formed between residues Lys-192 and Asp-366 is stabilised by a hydrogen-bonding interaction with the carboxamide carbonyl of (382) and is crucial for conferring inverse agonism [281]. The 4-chlorophenyl ring was observed to participate in direct stacking interactions with Trp-255, Tyr-275, Phe-278, and Trp-279 forms a stacking interaction with both aromatic rings of the ligand. In addition, the 2,4-dichloro ring is located in the proximity of further two aromatic residues, Trp-356 and Phe-200, where a small lipophilic pocket enhances the binding interaction with the 2-chloro substituent. It was proposed that the TDC conformation of (382) is stabilised by a weak hydrogen-bond interaction between the carboxamide NH and the proximal nitrogen atom in the pyrazole core. It was postulated that the TDC conformation of (472) is also stabilised by such an interaction, favouring this conformation over the alternative cisdichlorophenyl (CDC) conformer, which would potentially give rise to steric occlusion between the ortho-chloro substituent on the 2,4dichlorophenyl ring and the residues Phe-278 or Val-364. A series of 4,5-diarylimidazole-2-carboxamides has been reported as human CB1 inverse agonists [312]. The initial HTS hit, (495), based on an analogous scaffold to (382), was found to have moderate affinity for the CB1 receptor (7,000 nM).
Ph
H N
S N
Ph
Ac Ph
(495)
The relatively low affinity was ascribed to the absence of substitution on the phenyl rings and the absence of an amide group to form an interaction with the receptor. Among the compounds synthesised during the hit optimisation program were (496–507) (Table 6.45). The trichloro derivative corresponding most closely to rimonabant (382), (496) was found to have excellent CB1 affinity (IC50 ¼ 6:1 nM) (cf. (382):
294
RECENT PROGRESS IN CANNABINOID RESEARCH Table 6.45 4,5-DIARYLIMIDAZOLE DERIVATIVES – IN VITRO DATA [312] R2 Cl
R4
O
N NHR1 N R3 Cl
Cpd.
R1
R2
R3
R4
CB1 IC50 (nM)
(496) (497) (498) (499) (500) (501) (502) (503) (504) (505) (506) (507)
N-Piperidinyl c-Hexyl N-Morpholinyl c-Pentyl c-Heptyl c-Hexyl Ph c-Hexyl N-Piperidinyl c-Hexyl N-Piperidinyl c-Hexyl
H H H H H H H H Cl Cl Cl Cl
Cl Cl Cl Cl Cl Cl Cl Cl H H Cl Cl
Me Me Me Me Me Me Me Et Me Me Me Me
6.1 4.0 170 17 8.0 70 60 4.1 190 46 16 5.2
K i ¼ 6 nM). The closely related cyclohexyl analogue (497) was found to be slightly more potent than both. However, as witnessed in the analogous series by Lange et al. [311], replacement of the N-piperidinyl group by N-morpholinyl was detrimental to the CB1 binding affinity, suggesting that polar functionality was not well tolerated into the amidoalkyl region. A cycloheptyl group at this position was relatively well tolerated (500), with a more substantial decrease in affinity reported upon reduction of the cycloalkyl ring to cyclopentyl (499). The isomer of (496) in which the phenyl substitution was swapped between the two aryl rings (504) was found to be considerably less potent than (496) and indeed less potent than either of the tetrachloro analogues (506) and (507). Replacement of the imidazole N-Me group by N-Et (503) resulted in a broadly equipotent analogue. A number of oxazole derivatives were also synthesised, of which (508) was among the most potent (IC50 ¼ 80 nM). In general however, the binding affinities reported for the oxazole compounds were at best modest, and comparable with the related imidazole NH series (data not shown).
J. ADAM ET AL.
295
O
Cl O
NHc-Hex N Cl Cl
(508)
In vivo studies on the leading compounds (496) and (497) were carried out. Preliminary rat PK studies (1 mg/kg i.v., 2 mg/kg p.o.) for (496) indicated a good profile for i.v. administration (AUCnom ¼ 1:32 mM h kg=mg; Clp ¼ 27:5 ml=min=kg; Vdss ¼ 3:7 L=kg; t1=2 ¼ 2:4 h) and oral absorption (F ¼ 50%), in addition to a high brain:plasma ratio (B:P ¼ 2.99–3.40 between 0.25 and 4 h). An immediate, dose-dependent and prolonged reduction in food intake was subsequently observed in a body weight loss study carried out using diet-induced obese rats. At doses of 1, 3 and 10 mg/kg p.o., the reduction was reported to be 13.4%, 30.6% and 67.5% relative to the vehicle-treated animals. At 18 h after dosing of (496) at 10 mg/kg, a cumulative, statistically significant dose-dependent weight loss of 13 g was observed, compared with a 6 g gain for the vehicle-treated animals. A poorer result was obtained with (497), with a net weight loss of 2 g versus a 10 g weight gain for the vehicle-treated animals. This result was, however, in line with a poorer PK profile, resulting in lower bioavailability (F ¼ 30%) and slower brain penetration (B : P ¼ 0:8422:61 between 0.25 and 4 h). Stated to be particularly useful in the treatment of obesity, a series of 1,5diaryl-pyrrole-based compounds from AstraZeneca was claimed in a recent patent application [313], including compound (509). Although no specific biological data were presented, the CB1 receptor affinities are claimed to be less than 1 mM and preferably less than 200 nM. PYRIDINE-, PHENYL-, PYRIMIDINE- AND PYRAZOLE-BASED CB1 RECEPTOR ANTAGONISTS
Six-membered heterocycles have also been extensively used to mimic the central pyrazole scaffold of rimonabant (382). Merck and Co. has utilised pyridines (510) [314] , and pyrimidines (511) [315] in this capacity. Sanofi-Aventis has also claimed a series of pyridine-based analogues [316], as exemplified by (512) and additionally the non-heteroatom containing terphenyl (513) [317]. Pfizer too, has filed a patent application to protect a series of closely related pyrimidine-2-carboxamides [318]. Although no compounds are specifically claimed, the examples include compound (514) in which the chloro-substituted
296
RECENT PROGRESS IN CANNABINOID RESEARCH
diaryl motif is once again present. The affinity of this analogue in the CB1 GTPgS binding assay was claimed to be less than 20 nM. O
O Ph
NHPh
Ph
OMe N
Me
N Ph
OCH2 Ph
Cl (510)
(509)
F O
N N
Cl
Cl
(511)
Cl
Cl
Cl
Cl
O
O N H
N
N
N
X Cl
Ph
N Ac Cl
Cl (512) X = N (513) X = CH
(514)
In a recent communication from Merck [319], the synthesis and biological activities of several 5,6-diarylpyridine derivatives as CB1 inverse agonists were described. From the initial HTS screening on the Merck sample collection was identified (515) which was found to have moderate activity (IC50 ¼ 530 nM) at the human CB1 receptor. It was proposed that the pyridine ring behaved as an alternative scaffold to the pyrazole ring of (382), with the pyridine nitrogen overlapping with the N2 atom of the pyrazole. In addition, it was speculated that the 3-cyano group might act as a bioisostere
J. ADAM ET AL.
297
for the carboxamide of (382). Investigation of the phenyl substitution of this series yielded significant improvement in CB1 potency (Table 6.46). CN O N MeO
NMe2 (515)
The initial 4-Cl analogue (516) was found to exhibit only modest affinity, however derivative (517) showed CB1 inverse agonist activity comparable with (382) while also demonstrating good selectivity over the CB2 receptor.
Table 6.46 5,6-DIARYLPYRIDINE DERIVATIVES – IN VITRO BINDING DATA [319]
CN
4
R
O N 2
R
1
R
3
R
Cpd. R1 R2 R3 R4
CB1 IC50 (nM) Cpd. R1
R2 R3 R4
(382) (516) (517) (518) (519) (520) (521)
6.2 2,800 11 3.1 1.8 2.7 1.3
Cl Cl H Cl Cl Cl F
H Cl Cl Cl Cl Cl
H Cl Cl Cl Cl Cl
Cl Cl Cl Cl Cl Cl
H H 4-F 3,5-di-F 2,4-di-F 3,4-di-F
(522) (523) (524) (525) (526) (527) (528)
Cl Cl Cl Cl F Me F
Cl Cl Cl H Cl Cl F
CB1 IC50 (nM)
3-Cl 5.8 3.4 4-CF3 3,4-di-F 18 3,4-di-F 8 3,4-di-F 7.2 3,4-di-F 6.3 3,4-di-F 66
298
RECENT PROGRESS IN CANNABINOID RESEARCH
The 4-fluoro (518), 4-trifluoromethyl (523) and 3-chloro derivatives (522) also showed improved binding. 3,5-Disubstitution was also well tolerated (519), however the 3,4-difluoro compound (521) had the most potent inverse agonism of the series with an EC50 of 8 nM, with an average 110% maximal response and 400-fold selectivity over CB2. In addition to the crucial role of the 4-chloro substituent on the 5-phenyl ring, the 2,4-dichloro substitution on the 6-phenyl ring, which mimics that of (382), was also found to be optimal. Replacement of the 3-cyano functionality was also investigated with a series of 3-amido derivatives (Table 6.47). Of these, the 3,4-difluorobenzyl analogues were the most potent. Modification, including lengthening and branching the R2 and R3 alkyl groups have comparatively little effect on potency (cf. (529)–(531)). Replacement of the 3,4-difluorobenzyl motif with n-butyl did however considerably reduce the CB1 binding affinity. Cyclohexylmethyl did appear to be tolerated in this position with compounds approximately equipotent with their benzyl counterparts (cf. (529) and (537)) suggesting that this group binds in a lipophilic pocket rather than making a direct aromatic interaction.
Table 6.47 5,6-DIARYL-3-AMIDOPYRIDINE DERIVATIVES – IN VITRO BINDING DATA [319] 2
NHR
O
1
OR N Cl
Cl
Cl
Cpd.
R1
R2
CB1 IC50 (nM) Cpd.
R1
R2
(529) (530) (531) (532) (533)
3,4-di-F-Bn 3,4-di-F-Bn 3,4-di-F-Bn 3,4-di-F-Bn 3,4-di-F-Bn
Me Et n-Pr (CH2)2F i-Pr
3.1 1.9 1.7 1.5 1.8
n-Bu n-Bu n-Bu CH2(c-Hexyl) CH2(c-Hexyl)
H 32 Me 17 n-Pr 21 Me 3.4 n-Pr 5.2
(534) (535) (536) (537) (538)
CB1 IC50 (nM)
J. ADAM ET AL.
299
Table 6.48 5,6-DIARYL 2-AMINOPYRIDINE DERIVATIVES – IN VITRO BINDING DATA [319]
O NR1R2 N Cl
Cl
Cl
Cpd.
R1
R2
CB1 IC50 (nM)
Cpd.
R1
R2
CB1 C50 (nM)
(539) (540) (541) (542) (543)
–(CH2)5– H H H H
n-Hexyl n-Hept-4-yl c-Pentyl c-Hexyl
340 43 32 53 95
(544) (545) (546) (547)
H H H H
c-Heptyl Piperidin-1-yl Ph Bn
35 56 210 31
Investigation of a 2-amido series was also carried out by Meurer et al. (Table 6.48). These compounds were found to be generally less potent than the 2-alkoxy-3-amido series with (545), the closest analogue of (382), among the most potent. In addition, the n-hexyl (540) and 4-heptyl (541) analogues were found to have comparable affinity, as was the benzyl congener (547). The relatively poor binding of the 2- and 3-amido pyridine series compared with (382) was ascribed to either the difference in ring size between pyridine and pyrazole resulting in a slight change in relative orientation of substituents, and/or the basicity of the nitrogen and finally the absence of a neighbouring methyl group to force the amide group of rimonabant into a position perpendicular to the pyrazole ring. In vivo studies were also performed on those compounds deemed to have the most favourable binding and functional assay data, (521) and (531). Pharmacokinetic studies carried out on Sprague-Dawley rats (1 mg/kg i.v., 2 mg/kg p.o.) using compound (521) suggested favourable i.v. PK properties (AUCnom ¼ 9:3 mM h kg=mg; Clp ¼ 3:6 ml=min=kg; Vdss ¼ 0:8 L=kg; t1=2 ¼ 3:6 h), moderate oral absorption (F ¼ 27%) but slow brain penetration and a low brain:plasma ratio (B : P ¼ 0:0320:26 at 0.25–4 h). Compound (521) was subsequently investigated in a food intake and body weight loss
300
RECENT PROGRESS IN CANNABINOID RESEARCH
study using diet-induced obese rats. In contrast to (382), oral administration of (521) and 1, 3 and 10 mg/kg did not lead to an immediate reduction in food intake at any of the doses, however after 18 h after dosing at 10 mg/kg, a cumulative but non-significant (p >0.05) 22% reduction in food intake and a dose-dependent weight loss (1 g compared with an 8 g gain for the vehicle-treated animals) was observed. It was postulated that the slow CNS penetration witnessed in the earlier PK study was preventing an immediate effect on the food intake. Pyrazines have been utilised first by AstraZeneca [320–325] and subsequently by Bristol-Myers Squibb [326]. Although no specific biological data were presented in the initial two AstraZeneca patent applications, compounds including the specified (548) are claimed to have IC50 values less than 1 mM with the most preferred compounds having an IC50 value of less than 200 nM. Nine compounds are specifically claimed in the later AstraZeneca application [322] including (549) in which the N-piperidin-1-yl carboxamide of (382) is replaced with the bioisosteric piperidin-1-yl-oxycarbonyl. Subsequently, a tetrazole-containing 5,6-bis(4-methylphenyl)pyrazine derivative (550) was reported to have an IC50 of 1.4 nM [323]. A related tetrasubstituted compound, (551), which had been disclosed generically in an earlier AstraZeneca application [321], has been specifically claimed [324]. The affinity of this compound for the CB1 receptor was described only as less than 1 mM. Finally, from AstraZeneca, a third series of tetrasubstituted pyrazines was revealed [327], of which compound (552) was stated to have excellent CB1 affinity (1.8 nM). The now-familiar diarylpyrazine carboxamides have more recently been the subject of a patent application from Bristol-Myers Squibb [326]. Of the several compounds claimed, (553) shows that a hydroxymethyl functionality is tolerated adjacent to the carboxamide nitrogen but no specific data were presented in the application. 1
R
1
R
N
X
N
N
N
N R
1
O
O
1
(548) R = H, X = NH
R
1
N H R
N
2
2
(550) R1 = Me, R =
N
N N
1
(549) R = Cl, X = O
(551) R1 = Cl, R2 =
O N
N
J. ADAM ET AL.
Cl
Me
O N
OH
O N
Ot-Bu N
N
Me Me
N H
N
O
Cl
301
Me
(552)
(553)
Merck has recently utilised a furo[2,3-b]pyridine core (554) as a bioisosteric replacement for the pyrazole scaffold of rimonabant (382) [328]. The same basic pharmacophore, that of two halo-substituted aryl groups and a third hydrophobic motif proximal to a hydrogen-bond acceptor, can be witnessed in the benzodioxole-based compounds, such as (555), disclosed by Roche [329]. Ph
H2N
F O
O
O
Cl
O
N
O S
N
O Cl Cl
(554)
Cl Cl
(555)
AZETIDINE-BASED CB1 RECEPTOR ANTAGONISTS
A number of azetidine-based compounds have been disclosed in patent applications from Aventis Pharma for CB1-modulated treatment of diseases such as obesity, Parkinson’s disease, schizophrenia, respiratory and neurological diseases [330–334]. Compound (556) was specifically claimed for use in two formulation patent applications [330, 331] for a stable semi-solid composition and oral emulsion composition, respectively. The optional coadministration of an agent that activates norepinephrinergic and serotoninergic neurotransmission (for example, sibutramine) or dopaminergic neurotransmission was also claimed for the treatment of obesity. The optional use of a dopamine agonist (for example, levodopa) was claimed
302
RECENT PROGRESS IN CANNABINOID RESEARCH
additionally for the reduction of dyskinesia in such neurological diseases as Parkinson’s disease. Earlier Aventis patent applications disclose related azetidine derivatives such as (557) [334], (558) [333] and (559) [332]. The structural similarity to (382), of the three exemplified compounds shown, is less overt than the scaffold-hopping approaches discussed above, however the p-chloro-substituted diaryl motif is preserved. The sulfonamide oxygen atoms may be performing the same hydrogen-bond acceptor role as the carbonyl in rimonabant. F O
O MeO2 S
MeO2 S
F
N
S
S
R
N
N
N
Cl
Cl Cl
Cl
Cl
Cl
(557) R = 3,5-diF-phenyl (559) R = 5-Cl-2-pyridyl
(556)
(558)
O N
O N H
N O
Cl (560)
N N
O
Cl
NH
Cl
BOC
Cl (561)
An azetidine motif was also present in two series of CB1 antagonist compounds disclosed by Vernalis Research [335, 336]. In the former, compound (560) was claimed to have an affinity of 285 nM in transfected HEK293 cells using tritium-labelled (382). Among the preferred indications were psychosis, schizophrenia, smoking cessation and eating disorders associated with excessive food intake. Compound (561) was claimed to have an affinity of 0.8 nM in the same binding assay [336].
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303
In two related applications from Roche, a pyrrole/imidazole [337] or 2methylpyrrole [338] perform the role of central scaffold. Among the compounds specifically claimed are (562) and (563), stated to be antagonists or inverse agonists. Interestingly, a cyclohexylmethyl group is present where one might expect the more usual halo-substituted phenyl, and in this respect, may share a related pharmacophore to the pyrrole-based CB1 agonists described by Wiley et al. [181] in which the most potent analogues bear an extended pyrrole N-alkyl substituent. O O N O
N S N
N N
O O
(562)
(563)
SUBSTITUTED AMIDE-BASED CB1 RECEPTOR ANTAGONISTS
A number of distinct CB1 receptor modulators have been claimed by Merck for indications including eating disorder, obesity, psychosis and drug dependence. In the earliest [339], the familiar biaryl pharmacophore is preserved, although a substituted acyclic propyl chain, rather than a heterocyclic one, serves to orient the phenyl rings and carboxamide group in the required configuration (564). Two closely related series were also described in applications from Merck [340, 341]. Again, no specific biological data were presented, however the benzofuran (565) and benzodioxan analogues (566) typify the series. More recently, Merck has claimed a further two series of CB1 antagonists [342, 343] of which (567) and (568) were specifically claimed in each of the series, respectively. O
Me
Me Et
N H
R
Cl
(565) R = O
O
N H
Cl
H N
O
(566) R = O
NC (564)
Cl
304
RECENT PROGRESS IN CANNABINOID RESEARCH Me
Cl
Ph
O N H
Me Me
Me
O
HO
O
N H Cl Cl
Me O Me
N
CF3
(568)
(567) CN
HYDANTOIN-BASED CB1 RECEPTOR ANALOGUES
A series of 29 3-alkyl 5-arylimidazolidinones, or hydantoins, active at the CB1 receptor has been published by Kanyonyo et al. [344] with a subsequent publication describing the relationship between the experimentally derived lipophilicity and proposed modes of binding for non-polar and polar hydantoins derivatives [345] (Table 6.49). The binding data for compounds (569)–(592) were obtained by screening at a concentration of 10 mM in a competitive displacement experiment using tritium-labelled (382) in CHO membranes transfected with the human CB1 receptor. Within the series, affinity was seen to improve as the alkyl chain length, and hence lipophilicity was increased. Compounds (588) and (589) were, however, surprisingly active for their calculated lipophilicity. The lipophilicity of the hydantoin analogues was measured by reverse-phase HPLC and found to correlate most closely with the values calculated by the CLIP [346] rather than CLOGP method [347]. In addition, para-substitution of the two phenyl rings improved potency, with 5,5’-bis(4-bromophenyl) substitution, compounds (588)–(592), being optimal. For the non-polar hydantoins, the potency at the CB1 receptor decreased Br>OMe>F>Me>H, whereas no such relationship was evident for the non-polar compounds. Although certain polar groups, for example, morpholine (588) and hydroxyl (589) were well tolerated, the more basic amine group in compounds (570) and (571) had a strongly negative influence on binding. Molecular modelling studies comparing the hydantoins with the non-classical cannabinoid HHC utilised molecular lipophilic potential and hydogen bond potential to generate a superposition model. Two distinct binding modes, for polar and nonpolar hydantoins, were identified. The three most potent compounds (588), (589) and (591) were further characterised using a [35S]-GTPgS binding assay [348]. This assay allowed the test ligands to be defined as agonists (positive intrinsic activity), partial agonists (partial positive intrinsic activity), antagonist (no intrinsic activity) or inverse agonists (negative intrinsic activity). All three hydantoin ligands were found to behave as neutral
J. ADAM ET AL.
305
Table 6.49 3-ALKYL-5,50 -DIPHENYLIMIDAZOLIDINEDIONES – IN VITRO DATA [344,345] 1
R
O
(CH2)n R
2
N N H
O
1
R
Cpd.
R1
R2
n
c log Pa
% Displacement at 10 mMb
(569) (570) (571) (572) (573) (574) (575) (576) (577) (578) (579) (580) (581) (582) (583) (584) (585) (586) (587) (588) (589) (590) (591) (592)
H H H H H H H H H H Me Me Me OMe OMe F F F F Br Br Br Br Br
N-morpholine N-piperidine NMe2 Me Me Me Me Me Ph i-Pr N-morpholine Me Me N-morpholine Me N-morpholine Me Me Me N-morpholine OH Me Me Me
2 2 2 2 3 4 5 7 1 0 2 5 6 2 5 2 5 6 7 2 3 5 6 7
2.16 3.83 2.83 3.84 4.26 4.8 5.37 6.50 4.11 2.89 3.49 6.48 7.04 2.73 5.81 2.81 5.81 6.42 6.94 3.86 3.76 6.87 7.45 7.99
o5 o15 o5 o20 25.1 35.4 35.6 61.2 40.6 o5 23.9 46.8 51.3 21.7 66.6 30.3 40.6 51.4 62.5 91.2 88.4 72.1 89.2 80.0
a
Lipophilicity calculated using the CLIP method. Results expressed as the percentages of the displaced specific binding of [ 3H]-SR-141716A (mean7SEM, n ¼ 325). b
antagonists with no intrinsic activity, but competitively inhibiting HU210induced [35S]-GTPgS binding in rat cerebellum homogenates. A series of closely related thiohydantoins was subsequently investigated by Muccioli et al. [349]. The key data are summarised in Table 6.50.
306
RECENT PROGRESS IN CANNABINOID RESEARCH
Replacement of the oxygen in the 2-position of the hydantoin ring with sulfur was found to increase CB1 receptor potency between 2- and 4-fold. As in the preceding series, para-substitution of the phenyl rings was found to increase potency, with iodo-susbstitution, e.g. (602), showing greater affinity than Br-substitution, e.g. (598), which in turn was more potent than the corresponding chloro analogue (593) (R2 ¼ iBu). Substitution on the N3position was also found to have a pronounced influence on CB1 binding affinity. Increasing the chain length to over six carbon atoms resulted in a marked loss of activity. Benzyl substitution (594) was better tolerated than the corresponding phenyl ethyl congener (595), while the truncated allyl group, typified by analogues (600) and (603), was found to be optimal in this series. The most potent thiohydantoin analogue identified was found to be considerably less potent than rimonabant (382) (K i ¼ 589 and 5.4 nM, respectively), however, the compounds were selective over the CB2 receptor. Compounds (593), (594) and (600)–(602) were examined in the [35S]-GTPgS binding assay and, like the related hydantoin series, found to behave as inverse agonists at the CB1 receptor.
Table 6.50 3-ALKYL-5,50 -DIPHENYLTHIOXOIMIDAZOLIDIN-4-ONES – IN VITRO DATA [349] 1
R
2
O
R N N H
S
1
R
Cpd.
R1
R2
CB1 Ki (nM)a
Cpd.
R1
R2
CB1 Ki (nM)a
(593) (594) (595) (596) (597) (598) (599)
Cl Cl Cl Br Br Br Br
i-Bu CH2Ph (CH2)2Ph Et i-Pr n-Bu i-Bu
2,089 2,188 3,801 2,193 3,630 1,412 1,778
(600) (601) (602) (603) (382)a (254a)b (165)c
Br Br I I
Allyl CH2Ph n-Bu Allyl
871 993 724 589 5.4 3,802 18.6
a
Rimonabant. WIN55,212-2. c HU-210. b
J. ADAM ET AL.
307
An analogue of (-)-cannabidiol, O-2654 (604) has recently been disclosed as a high-affinity CB1 antagonist ligand [350]. Compound (604) was found to be significantly more potent (K i ¼ 114 nM) than cannabidiol (K i ¼ 4:9 mM) in the displacement of tritium-labelled CP 55,940 in mouse brain tissues. The value obtained for (604), while less potent than that reported for (382) (1.98–12.3 nM), is comparable with the early Lilly antagonist LY320135 (K i ¼ 141 nM) [351]. In addition, (604) was found to antagonise WIN 55,2122 by competing with the agonist for the CB1 receptors. Unlike (382), the cannabidiol analogue is thought to behave as a neutral antagonist.
RECENT CB1 RECEPTOR ANTAGONISTS
A recent patent application from Roche [352] described a 2-aminobenzothiazole series. Roche claimed that compound (605) exhibited an IC50 value of 0.73 mM at CB1, and showed in excess of 10-fold selectivity over the CB2 receptor. The compounds were described as being of potential use in the treatment of a range of diseases, including CNS and psychiatric disorders, type-2 diabetes, gastrointestinal diseases, cardiovascular disorders, infertility disorders, inflammation, cancer, atherosclerosis, cerebral vascular incidents and cranial trauma. A series of 12 tetrahydroquinoline compounds, functioning as antagonists or inverse agonists of the CB1 receptor, has been disclosed [353]. The requisite three hydrophobic groups, in the example (606), three unsubstituted phenyl groups are present, as is an adjacent hydrogen-bond acceptor (either the 2-oxo group of the central heterocycle, or the sulfonamide oxygen). A sulfonamide moiety was also present in a patent application, which at the time of writing is the most recent disclosure from Merck and Co. [354]. Again, a branched butyl chain forms the backbone from which two aryl groups are appended (607). CB1 functional activity for the compounds was claimed to be lower than 1 mM while the CB2 functional activity was reported to be higher than 1 mM. Me
Cl
Me
S
OH N (CH2)3N3 Me
O HO
(604)
Cl
Cl
(605)
308
RECENT PROGRESS IN CANNABINOID RESEARCH
Ph Me N
O NHSO2 Et
Ph
NHSO2 Ph
Cl
(606)
(607) Cl
THERAPEUTIC APPLICATIONS OF CB1 RECEPTOR ANTAGONISTS Our current understanding of the potential therapeutic applications of CB1 antagonists owes a great deal to the discovery of rimonabant (382). Indeed, clinical data demonstrating the efficacy of (382) in the treatment of obesity and nicotine addiction has provided a substantial driving force for the expanding research effort into this approach. The increase in appetite resulting from cannabis use has long been recognised and has also been observed in animals that have been administered cannabinoid agonists [355]. The endocannabinoid system also appears to play a key role in the regulation of appetite [356]. Endocannabinoid levels in the limbic forebrain have been shown to increase with food deprivation, while 2-AG levels in the hypothalamus reduce during feeding [357]. Anandamide (1) administration into the ventromedial hypothalamus has also been shown to stimulate appetite in rats [358]. The hypothalamus uses leptin as the primary signal to modulate food intake and energy balance and there is evidence linking leptin and endocannabinoids in the regulation of food intake [359]. Di Marzo and co-workers associated defective leptin signalling with elevated hypothalamic levels of endocannabinoids in obese db/db and ob/ob mice and Zucker rats, cerebellar endocannabinoid levels were not affected. Acute leptin treatment of normal rats and ob/ob mice reduced (1) and 2-AG levels in the hypothalamus. In additon to the central role of endocannabinoids in the regulation of feeding behaviour, a peripheral role has also been described. Gomez and coworkers [360] reported that food deprivation produced a 7-fold reduction in (1) levels in the small intestine of rats, but not in the brain or stomach. Intestinal (1) levels returned to normal when feeding resumed. The authors also showed that peripheral, but not central administration of (382) reduced food intake. The endocannabinoid system has also been reported to regulate peripheral lipogenesis [361].
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The role of the endocannabinoids and exogenously administered agonists outlined above led to an interest in testing the effects of selective CB1 receptor antagonists on feeding behaviour [261, 362, 363]. Rimonabant (382) and SR147778 (385) have been shown to reduce food intake and weight gain in ordinary rats without altering water intake [270, 364–366]. Rimonabant (382) has also been shown to cause a transient decrease in food intake accompanied by a sustained decrease in bodyweight and adiposity in dietary-induced obese mice [367]. Leptin, insulin and glucose levels were all normalised in these mice following (382) treatment, together with an increase in serum adiponectin levels [367, 368]. While (382) did not modify HDL-cholesterol and had modest effects on total cholesterol, it significantly reduced triglycerides and LDLcholesterol and increased the HDL/LDL-cholesterol ratio. CB1 knock-out mice exhibit resistance to dietary-induced obesity in terms of food intake, weight gain, body fat composition and the development of dietary-induced insulin resistance [369]. The effects of (382) on feeding behaviour and body weight are not observed in these animals, confirming that these are mediated through the CB1 receptor [359, 367]. CB1 knock-out mice do not demonstrate a decrease in food intake under free feeding conditions, but food intake is reduced following food deprivation. Clinical trials with (382) have provided the most exciting evidence for the utility of CB1 antagonists in the treatment of obesity, results of the Rimonabant in Obesity (RIO)-Europe trial having been published very recently [370, 371]. In this trial, 1,507 patients with body-mass index of 30 kg/m2 or greater, or body-mass index greater than 27 kg/m2 with treated or untreated dyslipidaemia, hypertension or both, were randomised to receive doubleblind treatment with placebo, 5 mg (382) or 20 mg (382) once daily in addition to a mild hypocaloric diet (600 kcal/day deficit). The primary efficacy endpoint was weight change from baseline after 1-year treatment in the intention-to-treat population. Weight loss at 1 year was significantly greater in patients treated with (382) 5 mg (mean 3.4 kg) and 20 mg ( 6.6 kg) compared to placebo ( 1.8 kg). Significantly more patients treated with (382) 20 mg than placebo achieved weight loss of 5% or greater (50.9% versus 19.2%) and 10% or greater (27.4% versus 7.3%). Rimonabant (382), 20 mg, produced significantly greater improvements than placebo in waist circumference, HDL-cholesterol, triglycerides, insulin resistance and prevalence of metabolic syndrome. The effects of (382) 5 mg were of less significance. (382) was generally well tolerated with mild and transient side effects including nausea, dizziness, diarrhoea and vomiting. Rimonabant (382) has also shown promise in pre-clinical and clinical studies as an aid to smoking cessation. (382) decreases nicotine self-administration in rats and nicotine-induced dopamine release in nucleus acumbens [372], and also reversed nicotine-seeking behaviour in rats several weeks
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after nicotine withdrawal [373]. In a 10-week, placebo-controlled trial, (382) was shown to prolong abstinence rates from tobacco during the final 4 weeks of the treatment period [370]. Furthermore, while patients in placebo groups in several studies gained weight, (382) patients lost weight or experienced less weight gain than those on placebo. Weight gain following nicotine withdrawal is a major factor in reducing abstinence rates. In addition to their utility as aids to smoking cessation, CB1 receptor antagonists have shown potential pre-clinically in the treatment of addiction to other substances including morphine, heroin and alcohol [374–377]. Rimonabant (382) was also included in a clinical study to assess the safety and efficacy of four novel compounds for the treatment of schizophrenia and psychoaffective disorder [378]. The other compounds included in the trial were a neurokinin NK3 antagonist, a serotonin 2A/2C antagonist and a neurotensin NTS1 antagonist. Haloperidol and placebo groups were used as controls in the study. Sixty-nine patients received (382) (20 mg once per day), which failed to demonstrate efficacy in this trial. The reasons for the lack of efficacy may be due to inadequate dosing or an indication that CB1 antagonism is not appropriate in the treatment of this condition. Pre-clinical data support the potential application of CB1 antagonists in the treatment of various other conditions. These include memory disorders [379], sexual dysfunction [380], neuro-inflammation [381] and asthma [382].
CB2 RECEPTOR ANTAGONISTS CB2 receptor antagonists have received much less attention than their CB1 counterparts, with only a relatively small number of compounds available and less clarity on their potential therapeutic role. A selection of the available compounds that have been shown to act as antagonists of the CB2 receptor and their suggested utilities will be covered in this section. As observed in the CB1 antagonist section, pyrazole derivatives again form an important class of antagonists for the CB2 receptor. A structural analogue of rimonabant (382), SR144528 (608), is a potent CB2 antagonist/ inverse agonist identified by Sanofi [383]. This compound has proved to be a useful tool in determining the function of CB2 receptors. The compound has subnanomolar affinity for CB2 receptors with 700-fold selectivity over CB1 receptors. Following oral administration, (608) totally displaced the ex vivo [3H]-CP 55,940 binding to mouse spleen membranes without interacting with CB1 receptors in the brain. Conformationally restricted pyrazole-derived CB2 selective antagonists were described in a patent application from Sanofi-Synthelabo [384]. Compounds included in the application, exemplified by compound (609) are
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claimed to act as antagonists at CB2 receptors (Kio5 10 7 M) with selectivity of at least 10-fold over CB1 receptors. Moving away from pyrazole-derived compounds, an aminoalkyl-indole, AM630 (610), acts as a CB2 receptor antagonist/inverse agonist with a CB2 Ki value of 31.2 nM and 165-fold selectivity over the CB1 receptor subtype [385]. It is interesting to note that this compound has been shown to act as a lowaffinity partial agonist, antagonist or inverse agonist at CB1 receptors [6]. Iwamura and Ueda [386] described compound (611) as a CB2 selective inverse agonist in a patent application. The potential therapeutic roles of CB2 antagonists are not clearly defined at the moment, although roles in regulation of the immune system and inflammation have been widely proposed. This patent application describes that activity of compound (611) in a mouse model of asthma, in which the compound suppressed immediate and late-phase asthmatic response and airway hyper-responsiveness. O
O N H N
N
N
N
Cl
Cl
N N H
S
Cl
Cl (609)
(608) SR144528 MeO
O
I
O N H
N O O
N H
O
N O (610) AM630
(611)
O O
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A series of tricyclic pyrazoles has been synthesised and their CB1 and CB2 affinity determined in mouse cerebellum membranes and mouse spleen homogenate, respectively [387]. Table 6.51 describes some of the pertinent data. Despite the close structural similarity between rimonabant (382) and the fused pyrazole analogues described by Mussinu and co-workers [276], the majority of the latter show a moderate-to-strong preference for the CB2 receptor. In particular, (612), the closest homologue to (382) within the series, shows very low nanomolar affinity (K i ¼ 0:34 nM) at CB2, while only comparatively modest CB1 affinity (2,050 nM). 6-Fluoro (613) and 6-bromo (614) analogues were also potent and selective CB2 ligands, while substitution of the larger 6-iodo group (615) resulted in a decrease in both affinity and selectivity. The unsubstituted analogue (618) exhibited a profile similar Table 6.51 TRICYCLIC PYRAZOLE DERIVATIVES – CB1 AND CB2 BINDING DATA [387] O 1
4
R
NHR N
N
3
R R
2
Cpd.
R1
R2/R3
R4
(382)a (612) (613) (614) (615) (616) (617) (618) (619) (620) (621) (622) (623) (624) (625) (626)
6-Cl 6-F 6-Br 6-I 5-Cl 7-Cl H 6-Me 6-OMe 6-Cl 6-Cl 6-Cl 6-Cl 6-Cl 6-Cl
2,4-di-Cl 2,4-di-Cl 2,4-di-Cl 2,4-di-Cl 2,4-di-Cl 2,4-di-Cl 2,4-di-Cl 2,4-di-Cl 2,4-di-Cl 4-Cl H 4-OMe 2,4-di-Cl 2,4-di-Cl 2,4-di-Cl
1.8 N-piperidine 2,050 N-piperidine 1,268 N-piperidine 1,570 N-piperidine 333 N-piperidine 8.25 N-piperidine 723 N-piperidine 1152 N-piperidine 363 N-piperidine 399 N-piperidine 1,787 N-piperidine >5,000 N-piperidine 3,035 N-pyrrolidine 798 1,881 N(Me)2 2,183 NH2
a
Rimonabant.
CB1 Ki (nM)ax CB2 Ki (nM)ay CB1 Ki/CB2 Ki 514 0.34 0.225 0.27 5.5 0.23 6.79 0.385 0.04 12.3 0.9 48 120 9.9 144 455
0.0035 6,029 5,635 5,814 60 3,587 105 2,992 9,810 32 1,985 104 25 81 13 5
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to that of the 6-Cl congener (612). Movement of the 6-Cl substituent to C5 slightly increased CB2 affinity, while the 7-Cl analogue (617) witnessed a slight decrease in CB2 binding. The 6-Me analogue (619) was found to be the most potent and selective ligand, eclipsing the 250-fold CB2-selective SanofiAventis ligand, SR144528 (608).
SUMMARY AND FUTURE PROSPECTS It is hoped that the scope of this review article has given the reader a good impression of the rate at which cannabinoid research is progressing. The last decade has seen a rapid growth in cannabinoid research, which looks set to continue. The therapeutic potential of cannabinoid agonists has long been acknowledged through the use of cannabis for medicinal purposes. However, the accompanying psychotropic effects have restricted the usefulness of this approach. We are now starting to observe that at least some of the benefits of cannabinoids can be realised without CNS side effects through strategies such as peripheral restriction of compounds, CB2 subtype selectivity and subtle modulation of the endocannabinoid system. Improved drug delivery systems are also proving beneficial in this regard, although beyond the scope of this review. Antagonism of cannabinoid receptors is now attracting a huge amount of attention, following on the successful rimonabant (382) clinical trials. At the time of writing, a launch date in 2006 is anticipated for (382) and it will be intriguing to observe the future of this mechanistic approach in the treatment of various conditions, particularly obesity. With an ever-growing knowledge of the endocannabinoid system, and the possible identification of further receptor subtypes, the future of cannabinoid research seems very bright indeed. REFERENCES [1] [2] [3] [4] [5] [6]
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Progress in Medicinal Chemistry – Vol. 44, Edited by F.D. King and G. Lawton r 2006 Elsevier B.V. All rights reserved.
7 Oxytocin Antagonists as Potential Therapeutic Agents for the Treatment of Preterm Labour MICHAEL J. ALLEN,1 DAVID G.H. LIVERMORE1 and JACQUELINE E. MORDAUNT2 1
GlaxoSmithKline, New Frontiers Science Park (North), Third Avenue, Harlow, Essex, CM19 5AW, UK 2 GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
INTRODUCTION
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EPIDEMIOLOGY AND THE IMPACT OF PRETERM LABOUR
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THE ANATOMY OF THE UTERUS
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TREATMENT OF PRETERM LABOUR The Role of Oxytocin in Labour Regulation of Oxytocin during Labour Regulation of the Oxytocin Receptor Experiences with Oxytocin Antagonists Selective OT or Mixed OT/V1A Blockade? Extra Uterine Roles of Oxytocin
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METHODS USED TO MEASURE OXYTOCIN ANTAGONIST ACTIVITY Competition Binding Functional Responses Quantified by Second Messenger Measurement Functional Responses Quantified by Smooth Muscle Contraction
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PEPTIDE ANTAGONISTS Atosiban
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NON-PEPTIDE ANTAGONISTS
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OXYTOCIN AGONISTS
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DOI: 10.1016/S0079-6468(05)44407-0
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STRUCTURAL STUDIES OF OXYTOCIN RECEPTOR
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CONCLUSION
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REFERENCES
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INTRODUCTION The natural hormone, oxytocin, is a well-known, clinically proven inducer of labour in pregnant women. It is generated in the hypothalamus and locally in the uterus, where its pivotal role in the generation of uterine contractions has been established. In the last decade, the concept of an oxytocin antagonist as an inhibitor of uterine contractions for the treatment of preterm labour has been established. An intravenously administered drug, atosiban (TractocileTM), is approved in Europe for this indication. In this report, these developments and the discovery of new potent and selective, orally bioavailable non-peptidic antagonists are reviewed, covering the literature to the end of 2004. The common abbreviations used in this review are defined below.1 A general review of the condition of preterm labour and treatment options was published in 2003 [1]. A review of the oxytocin receptor, focussing on expression, signalling and function was also published in 2003 [2].
EPIDEMIOLOGY AND THE IMPACT OF PRETERM LABOUR Preterm birth is defined by the World Health Organisation as birth occurring prior to 37 weeks gestation (normal gestation is 40 weeks) and is the cause of the majority of perinatal morbidity or mortality. By this definition the incidence of preterm birth is 5–10% in the western world [3]. Predicting preterm delivery is extremely difficult, though there are known risk factors that 1 Abbreviations: cAMP, cyclic 30 ,50 adenosine monophosphate; ELISA, enzymelinked immunosorbent assay; FLIPR, fluorescence imaging plate reader; OT, oxytocin receptor; V1a, vasopressin V1a receptor; V1b, vasopressin V1b receptor; V2, vasopressin V2 receptor; cys, cysteine; pro, proline; tyr, tyrosine; ile, isoleucine; thr, threonine; orn, ornithine; gly, glycine; phe, phenylalanine; asp, aspartate; asn, asparagine; trp, tryptophan; arg, arginine; p.o., oral; i.v., intravenous; CHO, Chinese hamster ovary; HEK, Human embryonic kidney; pmp, pentamethylenepropionic acid; tic, tetrahydroisoquinoline-3-carboxylic acid; 7TM, seven transmembrane; GPCR, G-protein coupled receptor; PSA, polar surface area. IC50, Ki, pKi, AD50, ID50, pA2, pKB and DR10 are defined in the section ‘Methods used to measure oxytocin antagonist potency’.
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significantly increase the risk of a preterm delivery. These include multiple pregnancy, previous preterm delivery, vaginal infection, low socio-economic class, intravenous drug use and multiple sexual partners. Recently a diagnostic/ prognostic test using foetal fibronectin has become available, though sensitivity and specificity is not high enough to provide a conclusive diagnosis [4]. Clinical outcome is associated with the degree of prematurity. Those infants born before 32 weeks gestation (about 25% of the total preterm births) have a significantly higher risk of death or disability. The majority of infants born at 32 weeks have some clinical morbidity [3]. Mortality drops from about 80% at 23 weeks to less than 10% at 30 weeks gestation. Long-term disability can range from severe (such as cerebral palsy or blindness) to milder affects such as below-average school achievement or mild respiratory problems. The acute financial costs to a hospital are also dependent on the gestational age of the infant. The average cost of a term birth was $1,100 in a US hospital in 2003 [5]. At 36 weeks gestation, the cost was $2,600 per infant which rose to $203,000 at 25 weeks gestation. The long-term costs for care of those that have been disabled by preterm labour have not been estimated, but will almost certainly be significantly higher than the acute costs. For example, the lifetime costs of caring for an individual with cerebral palsy have been estimated as being close to $1 million [6]. THE ANATOMY OF THE UTERUS The pregnant uterus is a complex organ consisting of both maternal and foetal tissues. The foetus is held within the amniotic cavity, bathed in amniotic fluid, which is produced by the foetus. Surrounding the amniotic fluid are two sets of membranes, both foetal in origin. The thinner amnion lines the amniotic cavity and beyond this is the chorion. The chorion is adjacent to the maternal tissue lining the uterus, the decidua. The foetal membranes and the decidua are largely composed of connective tissue, endothelium, epithelium and immune cells. Surrounding the decidua is the major mass of the uterus, the myometrium. The myometrium comprises mostly smooth muscle cells held together by connective tissue. During labour, the myometrium forcefully contracts to expel the foetus through the cervix. It is beyond the scope of this chapter to describe all the changes that lead up to labour, though the specific role of oxytocin will be discussed below. TREATMENT OF PRETERM LABOUR The only drug that has been licensed for the treatment of preterm labour in the US is ritodrine, a b-2 adrenoceptor agonist. b-2 Adrenoceptors are
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present on the myometrium and on activation can stimulate relaxation of the muscle. The effectiveness, however, is limited to extending pregnancy by approximately 48 h and there are significant maternal side effects such as tremor, tachycardia, arrhythmias and, in very rare cases, death. A delay in delivery of 48 h is useful as it allows the administration of steroids to the mother, which accelerates lung development of the foetus. In addition to ritodrine, other b-2 agonists have been used off-label in both the US and Europe, the most commonly prescribed being terbutaline. During recent years, the use of b-2 agonists has declined following cautions on the side effects and ritodrine has now been withdrawn in the US. Various non-licensed drugs are used in both the US and Europe. In the US magnesium sulphate is commonly used. Though there is significant doubt of clinical efficacy [7], magnesium sulphate is safe if used carefully. The calcium channel blocker nifedipine is used in Europe, though as yet there is very limited data on clinical efficacy and safety. Non-steroidal antiinflammatory drugs have also been shown to have clear efficacy. For example, indomethacin has been demonstrated to extend gestation by >1 week. Use of these drugs, however, is limited by significant foetal side effects such as constriction of the ductus arteriosus, renal impairment and intraventricular haemorrhage [7]. More recently, a novel form of therapy in the form of oxytocin antagonists has become available. Though currently only an intravenous formulation for acute therapy is available in Europe (but not in the US), this class of drugs offers hope for a more effective treatment for preterm labour. THE ROLE OF OXYTOCIN IN LABOUR
The precise mechanisms triggering the onset of labour are unknown. There are many changes that occur while preparing the uterus and foetus for the onset of labour. However, it is clear that regulation of both oxytocin and the oxytocin receptor plays an important role in childbirth. Oxytocin is well established as an inducer of labour and is frequently used clinically for this purpose. Oxytocin acts by both direct and indirect mechanisms to stimulate uterine contraction. Firstly, it acts directly on the myometrium to cause uterine contractions and secondly, oxytocin acts to stimulate the production of other mediators of uterine contractions, most notably prostaglandins [8]. REGULATION OF OXYTOCIN DURING LABOUR
It is well established that the concentration of oxytocin in plasma is increased in a pulsatile manner prior to, and during, parturition in a wide
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range of species including monkeys [9]. Studies in women have been less conclusive, as they are generally conducted in a less well-controlled manner and are often not sampled fast enough to look for pulsatile release, though an increase in frequency during labour has been observed [10]. Significant additional interest in the potential role of oxytocin in labour developed from 1993, when it was reported for the first time that oxytocin, which had previously been thought to only be produced in the hypothalamus, may also be synthesised locally in the uterus as indicated by the presence of mRNA encoding for oxytocin [11]. This observation has recently been confirmed by using immunohistochemistry and mass spectrometry to confirm the presence of local oxytocin peptide in the uterus. These studies localised oxytocin to the chorionic trophoblast, decidual stroma and glandular epithelium, and demonstrated that local uterine concentrations of oxytocin increased with the onset of labour [12]. REGULATION OF THE OXYTOCIN RECEPTOR
The oxytocin receptor is one of the most dramatically regulated of all G-protein coupled receptors. The receptor is virtually undetectable in the non-pregnant and early pregnant uterus, but increases steadily in the last trimester with a final increase at the onset of labour [13]. Even in the absence of changes in the level of oxytocin peptide, these changes in receptor density could lead to uterine contractions in response to normal concentrations of oxytocin. The regulation of oxytocin receptors occurs in both the myometrium, where oxytocin directly stimulates contractions, and in the amnion and deciduas, where oxytocin may be acting indirectly by the production of prostaglandins [12]. EXPERIENCES WITH OXYTOCIN ANTAGONISTS
Due to the large body of evidence that oxytocin and the oxytocin receptor play an important role in labour, there has been interest in identifying and developing oxytocin receptor antagonists for the treatment of preterm labour. Studies with these antagonists have helped to establish the role of oxytocin in labour and have driven the wish to develop an orally active oxytocin antagonist that may be given over a prolonged period. Nearly all the clinical data comes from the use of atosiban (see Peptide Antagonists), a peptide oxytocin antagonist that is licensed in Europe for acute (48 h) treatment of preterm labour. Early clinical studies demonstrated the ability of atosiban to inhibit uterine contractions associated with labour [14]. Following these successful phase II trials, full phase III trials were
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conducted with atosiban treating, for 48 h, women with threatened preterm labour [15–18]. These trials showed that a 48 h treatment with atosiban was at least as safe and as effective as treatment with b2 agonists (the only treatment previously licensed by the Food and Drug Administration (FDA) for the treatment of threatened preterm labour). Compared to placebo, women in preterm labour after 28 weeks gestation were significantly less likely to deliver within 7 days. Additionally, a study investigating the use of a continuous low dose subcutaneous infusion of atosiban showed that maintenance therapy delayed the re-occurrence of labour compared to placebo [19], giving more confidence that an orally active oxytocin antagonist would be beneficial.
SELECTIVE OT OR MIXED OT/V1A BLOCKADE?
Oxytocin and vasopressin are closely related peptides sharing seven out of nine amino acids. Vasopressin is able to bind to all vasopressin and oxytocin receptors with nanomolar affinity, whereas oxytocin has greater affinity for the oxytocin receptor than the vasopressin receptors. The affinity of the peptides for the receptors is shown in Table 7.1 (data from Mouillac et al. [20]). Atosiban, rather than being a selective oxytocin antagonist, has high affinity for both OT and V1a receptors, indeed it has higher affinity for the V1a receptor [21]. V1a receptors are also known to be present on the uterine myometrium leading some authors to speculate that V1a antagonist activity may be beneficial in the treatment of preterm labour [22]. However, there is little data linking V1a receptors to oxytocin-induced uterine contractions. Oxytocin has lower affinity for the V1a receptor. It has been shown that oxytocin-induced contractions postpartum can be inhibited by a selective oxytocin antagonist, L-368,899 (27) [23]. Additionally, the in vitro spontaneous contractions of uterine strips taken from women at term are inhibited more strongly by the more potent and selective oxytocin antagonist L-371,257 (29) than by atosiban [24]. There are also no reports of local vasopressin production within the uterus, no upregulation of the vasopressin Table 7.1 AFFINITY OF OXYTOCIN AND VASOPRESSIN LIGANDS FOR THE OT, V1A, V1B AND V2 RECEPTORS Compound
OT Ki (nM)
V1a Ki (nM)
V1b Ki (nM)
V2 Ki (nM)
Oxytocin Vasopressin
1.0 1.7
78 3.2
250 3.2
89 0.4
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receptor during pregnancy and no changes in plasma vasopressin levels associated with labour. It therefore appears that a selective oxytocin antagonist would have the same efficacy as a mixed OT/V1a antagonist. Though the potential drawbacks of having V1a antagonist activity are limited, blockade of this receptor does appear to compromise an animal’s ability to cope with hypovolaemia, such as might occur with haemorrhage or trauma [25]. In the absence of a clear advantage of V1a blockade, it is therefore prudent to avoid such activity.
EXTRA UTERINE ROLES OF OXYTOCIN
Outside the uterus, oxytocin has long been known to be essential for milk secretion. In recent years, however, there has also been a growing body of evidence that oxytocin plays a role in the brain in modulation of maternal, sexual and social behaviour. Oxytocin is required for lactation. Milk is initially secreted into small sacs (alveoli) within the mammary gland. These alveoli are surrounded by smooth muscle, which contract to eject the milk. The oxytocin receptor is the key receptor that mediates this ejection of the milk. The central role of oxytocin has led to it being described as the ‘love hormone’, and recently Insel et al. [26] postulated that oxytocin is the molecular basis of monogamy. In their laboratory, they examined the behaviour of prairie voles, which form strong pair bonds after mating with prolonged, repeated bouts of copulation. It was found that central administration of an oxytocin antagonist inhibited the pair bond formation, whereas central infusion of oxytocin stimulated pair bond formation in the absence of mating. Since oxytocin is released during sexual arousal and orgasm in both men and women [27], it is possible that a similar role may play a part in the formation of pair bonds in humans. Similarly to the formation of pair bonds between adult animals, it has been demonstrated that oxytocin is also required for the onset of maternal behaviour in rodents [28].
METHODS USED TO MEASURE OXYTOCIN ANTAGONIST ACTIVITY COMPETITION BINDING
In competition binding, the affinity of a compound is measured by its ability to compete with labelled oxytocin (or an analogue). Human receptors are
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usually used and expressed most commonly in CHO or HEK cells. Traditionally, a radiolabel (3H or 125I) is most commonly used to measure the amount bound – either by filtration (separating out bound from free label) or by a homogenous technique such as scintillation proximity assay (SPA). The affinity of the competing agent may then be calculated by applying the Cheng–Prussof equation where Ki ¼ IC50/(([Ligand]/Ligand Kd)+1). This is usually conveniently reported as a pKi – the negative logarithm of the Ki. We have also used a non-radiometric-binding approach based on fluorescence polarisation [29], where a fluorescent label is used in place of a radiolabel. As the fluorescently tagged oxytocin binds to the receptor, its rotational velocity is reduced and the polarisation of the fluorophore increases. The displacement of the ligand may be measured by a decrease in polarisation. FUNCTIONAL RESPONSES QUANTIFIED BY SECOND MESSENGER MEASUREMENT
The potency of an antagonist may also be measured in functional assays. In the case of OT, V1a and V1b receptors, stimulation leads to an increase in cellular inositol 1,4,5-trisphosphate and intracellular calcium ion concentration, [Ca2+]. Both these second messengers may be measured, though it is more common to measure [Ca2+] for drug screening, as it is more compatible with high-throughput screening techniques. Cells are loaded with a calcium-sensitive dye (commonly Fluo-3 or Fluo-4). As intracellular [Ca2+] increases, the fluorescence output of the dye changes. These changes may be measured using a laser-based imaging system such as FLIPR. Antagonist potency over a range of inhibitor concentrations may be measured by inhibiting a single concentration of oxytocin, giving an IC50 – the concentration causing 50% inhibition of the control response. This should be corrected to a functional pKi according to the equation fpKi ¼ IC50/ (([agonist]/agonist EC50)+1), as the IC50 will vary depending on the concentration of the agonist. A more robust (but considerably lower throughput) approach is to examine the effect of increasing antagonist concentration on a whole agonist dose–response curve. This approach, a Schild analysis, demonstrates whether a compound is acting competitively or not, and produces a pKB which is the most robust measurement of functional antagonist potency. The pKB is the negative logarithm of the equilibrium dissociation constant. In the case of V2 receptors, similar analytical methods may be used but the second messenger that is measured is cAMP, the concentration of which is measured by biochemical techniques such as ELISA. Functional antagonism is sometimes reported as a pA2, being the negative logarithm of the molar concentration of an antagonist producing a twofold shift to the right of an agonist dose–response curve.
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FUNCTIONAL RESPONSES QUANTIFIED BY SMOOTH MUSCLE CONTRACTION
The potency of oxytocin antagonists at inhibiting uterine contractility in vitro and in vivo can be measured. As with second messengers, the potency in vitro may be measured either as an IC50 or fpKi, or by the more robust Schild analysis yielding a pKB. In vivo potency has been quantified in a variety of ways: AD50 or ID50 [30], which is the dose of a compound (usually given in mg/kg) that causes 50% inhibition of the control response to a fixed dose of oxytocin. This is a simple and reliable method, but as with IC50 determination in vitro, it is dependent upon both the dose of oxytocin used for the control response and the sensitivity of that animal to oxytocin (AD50 will increase as either the dose of oxytocin or the sensitivity to oxytocin is increased). The best method for controlling sensitivity to oxytocin is to remove the ovaries of the animal (to remove endogenous steroids) and then to give a fixed dose of estrogen, which stimulates up-regulation of the oxytocin receptor. More commonly, though, estrogen is given to nonovarectomised animals to simplify the experimental process. The IC50 is similar to AD50 or ID50 experiments, except that plasma concentration of the antagonist is measured during the experiment and the potency is expressed as the plasma concentration of the antagonist causing 50% inhibition of the oxytocin response. An alternative assessment of potency is the DR10: in this experiment a simplified (2–3 point) dose response curve to oxytocin is conducted. A low dose of antagonist is given and the oxytocin dose–response curve repeated. A higher dose of antagonist is then given followed by another oxytocin dose–response curve. This cycle is then repeated at further antagonist doses (usually 3–4 antagonist doses are given). At each antagonist concentration, a dose ratio is calculated as the dose of oxytocin required to give a specified response in the presence of the antagonist divided by the dose of oxytocin required to stimulate that same response in the absence of oxytocin. The data may then be plotted (as log dose ratio-1 against log antagonist dose) and the dose that would cause a 10-fold shift (reduction) in oxytocin potency estimated. This is a more complex experimental design than an AD50 experiment, but expresses potency in a way that is independent of the oxytocin dose used or the sensitivity of a particular animal to oxytocin.
PEPTIDE ANTAGONISTS The pivotal role played by oxytocin and its receptor during parturition has prompted many research groups to seek an effective antagonist of these effects. The starting point for these endeavours was the endogenous agonist,
340
OXYTOCIN ANTAGONISTS AS POTENTIAL THERAPEUTIC AGENTS
the cyclic peptide oxytocin (1) and the closely related vasopressin (2). The numbering sequence which is used in this review for related cyclic peptide antagonists is also shown in (1). It was soon found that simple modifications of these structures led to potent peptidic antagonists. In this early work, before the era of recombinant receptors, agonist and antagonist receptor activity against oxytocin and vasopressin receptors was determined typically by measuring oxytocic and vasopressor responses in isolated tissue, usually rat. Tissue containing just oxytocin or vasopressin receptors was not available and given the close relationship between the endogenous agonists, it is not easy to gauge the absolute level of selectivity and potency of these molecules at their various receptors. OH O H2N
O
N H
S
1
NH O
HN
O
O
O
Tyr2
Ile3
Asn5
Gln4
HN
R
Cys6
HN
O
Pro7
S N
Cys1
(1)
O
NH2 NH
O
Leu8 Gly9
NH2
2
R NH
O H2N
O
R1
(1) = (S)CH(Me)Et, R2 = CH2CH(Me)2 Oxytocin 1 (2) R = CH2Ph, R2 = (CH2)3NH(=NH)NH2 Vasopressin
One of the first discoveries, from du Vigneaud’s laboratories at Cornell University, was that the replacement of cysteine with L-penicillamine at the 1-position of oxytocin (1) gave an analogue (3), which antagonised the contractile effects of oxytocin on rat uterine tissue in vivo following intravenous infusion [31]. Further elaboration of this template led to the discovery of more potent antagonists based on desaminooxytocin and containing a cyclic spiro substituent in place of the gem dimethyl group in (3). Thus, the cyclohexyl compound, [1-b-mercapto-b,b-pentamethylenepropionic acid]oxytocin (pmp-oxytocin) (4) was prepared and had enhanced oxytocin antagonist activity (pA2 7.4 in a rat uterine assay) [32]. These compounds also antagonised the effects of oxytocin on the related
M.J. ALLEN, D.G.H. LIVERMORE AND J.E. MORDAUNT
341
vasopressin receptor (pA2 8.3 in an avian vasopressor assay). The pmp modification has continued to be a popular strategy for conferring antagonist activity into cyclic peptidic oxytocin analogues, particularly in the research groups of Manning at the Medical College, Ohio [33, 34] and of Flouret at Northwestern University Medical School, Chicago [35]. Manning’s group initially applied the 1-desaminopenicillamine modification to an oxytocin analogue in which the glutamine at the 4-position was replaced with threonine. These compounds showed improved selectivity for oxytocic antagonist activity over vasopressor activity in the rat. Thus, [l-deaminopenicillamine-4-threonine]oxytocin (5) had a pA2 of 7.5 against oxytocin-induced rat uterine contractions, with an anti-vasopressor pA2 of 6.7 [36]. Increased potency was achieved by replacing the gem dimethyl group in (5) with a gem diethyl group [37] or especially the spirocyclohexane pmp group [38]. Other modifications which enhanced oxytocin antagonist activity and selectivity include introduction of an ornithine at position 8 and capping of the 2-tyrosine hydroxyl group as a methyl or ethyl ether. Many permutations of these modifications have been prepared. One of the most potent compounds to come out of this work is the methoxy derivative (6), with a pA2 of 9.1[38]. Similar modifications starting from argininevasopressin have led to mixed antagonists of the vasopressor and oxytocic responses of vasopressin in rat tissue [39]. 5
OH
OR
O
O
3
R
O
N Me H HN
Me
S S N HN O
NH O
O
O
O
S
O
HN N
4
R NH
O
NH2 NH
O NH2
2
NH
O
(3) R3 = NH2; R4 = (CH2)2CONH2 (5) R3 = H; R4 = (R)CH(Me)OH
O
O
O
R
NH
O
NH O
HN O NH2
H2N
HN
S HN
O
H N
O H2N
O
(4) R5 = H; R2 = CH2CH(Me)2 (6) R5 = Me; R2 = (CH2)3NH(=NH)NH2
A similar strategy has been employed by Flouret’s group [40]. Systematic substitution with L-tryptophan at each of the nine residues in [1-pmp-2D-trp-8-arg]oxytocin (7) was carried out [41]. This modification was
342
OXYTOCIN ANTAGONISTS AS POTENTIAL THERAPEUTIC AGENTS
particularly effective in the proline–arginine–glycinamide tail; tryptophan substitution in the ring gave analogues of lower potency. Thus, replacement of the arginine residue at the 8-position with tryptophan removes a positive charge in the molecule and yields a compound (8) with a pA2 of 8.2 against oxytocin-induced contractions in rat uterus and with no measurable antidiuretic activity in a rat arginine-vasopressin assay. A similar exercise replacing all available amino acid residues in [1-pmp-2-D-trp-8-arg]oxytocin with D-cysteine has also been reported [42]. As with the L-tryptophan scan, introduction of D-cysteine at the ring residues was poorly tolerated. Unexpectedly, potent antagonism was, however, observed with replacement at the 6-position, giving a compound (9) with a three-fold increase in oxytocin antagonist activity (pA2 8.3). This modification has generally been poorly tolerated in the oxytocin and vasopressin series of peptides. Indeed, 6-D-cysoxytocin is virtually devoid of any oxytocic activity [43]. Antagonist activity was also maintained following substitution with D-proline at the 7-position or D-arginine at the 8-position, confirming the opportunities for unnatural amino acid substitution in the tail region of the peptide. One compound from this series, (10), has been tested in vitro in human myometrium tissue obtained at term following caesarean section and shown to inhibit contractions induced by oxytocin [44] with a pA2 of 7.6. This is one of the first direct indications that the use of an oxytocin antagonist may be of benefit in the treatment of preterm labour in humans. This compound has been extensively studied in the near-term baboon and has been shown to inhibit nocturnal and near-term contractions following an intravenous bolus injection [45]. Further studies on the effect of oxytocin antagonism in the weeks leading up to delivery in the baboon have also been published [46].
NH
NH O
O O
H N HN
S
S
HN
* NH O
HN
O
O
O
O
O
N
NH2 NH
O NH2
R
HN
O
O
O
O
NH2 NH
O NH2
2
R NH
O
O O
NH O
HN O
NH H2N
1
HN
S
S N
O
H N
HN HN
H2N
NH2 (7) * (R)
(9) * (S)
= =
O
(8) R1 = (R)CH(Me)Et; R2 = HN
(10) R1 = CH2Ph; R2 = (CH2)3NH(=NH)NH2
M.J. ALLEN, D.G.H. LIVERMORE AND J.E. MORDAUNT
343
Recently, introduction of a 4-sulphur atom into the spirocyclohexane ring has been reported, along with replacement of 6-Cys with the more constrained penicillamine to give potent oxytocin antagonists, e.g. (11) [47, 48]. Introduction of a fluoresceinyl group, linked through an ornithine or lysine residue, into oxytocin-derived peptides has led to some potent fluorescent agonists and antagonists [49]. Thus (12) (mixture of 5- and 6substituted fluorescein derivatives) had a Ki of 6 nM against the human oxytocin receptor, 217 nM against human V1a and is inactive against the V1b and V2 receptors. The authors hope that fluorescent antagonists of this kind may prove useful as pharmacological tools for studying the function of oxytocin and vasopressin receptors. OMe O NH O
H N
N HN
Me O
Me
HN HN
O
O
O HN HN
NH2
HN O
NH
NH O
O NH2
NH O
O
O
O NH
O
O NH2
H2N
N
NH O
O
S
S
S
HN
S
HN
S
O
H N
O
OH NH2
NH
H N
HO H2N
O
OH
O O HO
(11)
O
O
(12) O
Another strategy for the introduction of antagonism into oxytocin and vasopressin cyclic peptides is the incorporation of the conformationally constrained tetrahydroisoquinoline-3-carboxylic acid group (tic) [50]. Substitution at a number of positions in the oxytocin/vasopressin sequence was investigated. In general, this strategy proved more successful for the design of vasopressin antagonists and usually led to a fall in oxytocin antagonist potency. The strategy of introducing non-natural aminoacids into the oxytocin peptide skeleton in order to make antagonists has also been exploited by Havaas et al. [51], who replaced the proline at the 7-position with sarcosine and modified the tyrosine residue at the 2-position to introduce further conformational constraint. A representative example is shown, (13), with a
344
OXYTOCIN ANTAGONISTS AS POTENTIAL THERAPEUTIC AGENTS
Ki of 6 nM against a guinea-pig uterine oxytocin preparation and at least 100-fold selectivity over rat vasopressin tissue preparations. These molecules show a pseudo-irreversible pharmacology, with an extended action against spontaneous contractibility of rat uterus 24 h post partum. Agonism and antagonism has also been proposed to be correlated with the conformation of the proline residue at the 7-position. The trans-amide has been associated with an agonist profile and the cis-amide conformation with antagonism. Introduction of a tert-butyl group at the 5-position of the pyrrolidine ring has been shown by proton nuclear magnetic resonance studies to augment the proportion of cis-amide present in solution. Consistent with this, 5-tert-butyl proline substituted oxytocin derivatives, such as (14), are potent antagonists (pA2 about 8) with a tenfold reduced agonist response compared to the unsubstituted analogue [52]. Me
OH
Me O
O
HN
N
NH O
HN
O
O
O
S HN
O
O
N
NH2 NH
HN
S tBu
S
O
N H
HN
S Me
H2N
O
H N
O
HN O
NH O
O
O
NH2 NH
NH O
O O
O NH2
NH2 NH H2N
O
O
HN
H2 N
NH2
O
(14)
(13)
HN
The conformational requirements for oxytocin agonism and antagonism have been extensively researched in the laboratories of Hruby at the University of Arizona, with a strong focus on structural studies. This group reported on the crystal structure (Figure 7.1) of the oxytocin agonist, desaminoxytocin, (15) [53, 54]. Conformationally constrained bicyclic analogues of oxytocin have been described with potent oxytocin antagonistic activity. A representative compound is (16), which has a pA2 of 8.2 in an isolated rat uterus assay [55]. The corresponding penicillamine derivative (17) was even more potent, with a pA2 of 8.7 in the rat uterus assay and 400fold selectivity for the rat oxytocin receptor over the rat vasopressin receptor [56]. The corresponding compound without the cyclic constraint between the glutamine and lysine residues was a very weak agonist, having less than one thousandth of the potency of oxytocin itself. These results lend credence to
M.J. ALLEN, D.G.H. LIVERMORE AND J.E. MORDAUNT
345
Fig. 7.1 Structure of desaminooxytocin (15).
the concept that the bicyclic constraint facilitated binding to the receptor, but prevented a conformational change required to elicit an agonist response. Further information was obtained from an NMR and molecular dynamics study of (17) [57], which provided a model of the bioactive conformation of oxytocin peptide antagonists. This model identifies a b-turn between residues 2-Tyr and 3-Ile and a cis-amide bond between 6-Cys and 7-Pro as the key structural requirements for antagonism. A further conformational constraint was introduced with the preparation of four stereoisomers of a 20 ,60 -dimethyl-40 -methoxytyrosine-substituted oxytocin derivative (18) and the study of their pharmacological properties [58]. It was observed that the (2S,3S) isomer was a very potent oxytocin antagonist (pA2 8.3), while the (2R,3R) isomer was somewhat less potent (pA2 7.6), and the (2S,3R) and (2R,3S) diastereomers were virtually inactive. These diastereomers have highly constrained side-chain conformations, which enabled their use to determine the topographical requirements of the aromatic group for antagonist activity. The active (2R,3R) and (2S,3S) isomers adopt a gauche conformation of the aryl group and amide nitrogen atom when viewed along the axis of the b and a carbon atoms of the tyrosine sidechain, while the inactive diastereomers stabilise a trans arrangement (Figure 7.2).
346
OXYTOCIN ANTAGONISTS AS POTENTIAL THERAPEUTIC AGENTS Me H2 N CO
MeO Me Me H
H CH3
Ar
CO
H2N
H
H CH3
Ar
CO
H2N
Ar
H3 C
Ar
H3 C
CO H N 2
H2N
H
H
H
(2R,3R) gauche
(2S,3S) gauche
(2R,3S) gauche
CO H
0
(2S,3R) gauche
0
0
Fig. 7.2 Stable conformations of diastereomers of b-methyl-2 ,6 -dimethyl-4 -methoxytyrosine. OH
OH O
O O
N H HN
S
S
N
NH O
O
O
N
NH2 HN
O
NH
O
O
NH O
O
NH O H2N
(15)
O
(16)
OH
Me
O O
N Me H
HN
S
Me
S HN
HN
NH O
O
O HN
Me
O
O
HN N
O
O NH
NH
HN O
NH O
O
O
O
NH2
O
O H2 N
(17)
NH
NH
NH
O
O
(18)
O
O
NH2 NH H2N
Me
S
N
O
OMe
O
N Me H S
NH
NH
O
O O
O
O
NH2
NH2 NH H2N
O
HN
O
HN
HN
HN
S
S
O
O
N H
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347
A tetrahydroisoquinoline carboxylic acid (tic) group has been used to provide further conformational rigidity and insights into agonist and antagonist function [59]. Compounds such as (19) were devoid of agonist activity, but were more potent as antagonists than the corresponding phenylalanine analogues, suggesting stabilisation of a favourable antagonistbinding conformation.
OH O N NH2 O
O
N H HN
S S
HN N HN O
NH O
O
O
O
O
NH2 NH
O NH2
NH O H2N
O
(19)
Researchers at the Merck laboratories adopted a different approach. Instead of modifying the oxytocin peptide structure, they identified a novel class of peptidic antagonist from screening of natural products. The first compound reported was L-156,373 (20), a cyclic hexapeptide isolated from Streptomyces silvensis [60]. This compound has a Ki of 150 nM in a rat uterine oxytocin-binding assay, with some selectivity for oxytocin over vasopressin. Structural modification around this lead molecule led to the identification of a more potent compound, L-365,209 (21) [61], with a Ki of 1.7 nM against the rat oxytocin receptor. This was the first compound with reported in vivo activity in an anaesthetised rat model of oxytocin-induced uterine contractions. It was active at 0.46 mg/kg following intravenous administration.
348
OXYTOCIN ANTAGONISTS AS POTENTIAL THERAPEUTIC AGENTS
Ph
O
N O
N
N H O N Me
O
OH
O
O
N
H N
N H
H N
N Me
NH O
(20) L-156,373
N O N
O
O
N
O
O
N
N N
(21) L-365,209
ATOSIBAN
Atosiban, [1-deamino-D-2-tyr(OEt)-4-thr-8-orn]-oxytocin, (22) is the only oxytocin antagonist currently available for prescription by obstetricians in Europe. It is a cyclic peptide derivative, based on the structure of oxytocin and vasopressin and contains an O-ethyl tyrosine residue, which confers antagonist activity on this template [62]. It was initially prepared in the research laboratories of Ferring and was selected from a series of 17 analogues based on 1-desaminoxytocin [63]. It demonstrated antiuterotonic activity in vitro and in vivo in rats and was also active in a human myometrial tissue preparation. It showed fewer side effects on blood pressure and diuresis than the other compounds described. Using recombinant receptors expressing human oxytocin and vasopressin, it was subsequently shown to be a mixed antagonist of OT and V1a receptors. Its use has been approved in Europe for the treatment of preterm labour, but is not licensed for use in the United States. This peptide must be administered intravenously, as unsurprisingly it is not orally bioavailable. It is marketed under the brand name of TractocileTM. Clinical experience [18, 19] with this compound, which is discussed elsewhere in this review, has attested to its effectiveness in at-risk patients. Strategies for Phase IV clinical studies to further elucidate the role of atosiban as a treatment for preterm labour have been published [64]. Other compounds from this series have been described by Ferring with improved pharmacokinetic properties (longer half-life) and increased human selectivity for oxytocin over vasopressin receptors [65, 66] and much of this work has been patented [67].
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OEt O O
H N HN
S S
O
HN N HN O
NH O
O
O
O
Me NH
OH NH2
NH O H2N
O
NH2
(22) Atosiban
NON-PEPTIDE ANTAGONISTS The search for an effective non-peptide oxytocin antagonist has become a major goal of a number of pharmaceutical companies because of the poor pharmacokinetic properties and especially the lack of oral bioavailability associated with peptidic antagonists. Early research in this field was dominated by Merck, but in recent years significant research efforts at GlaxoSmithKline and Serono have been published. A number of other companies, notably Sanofi-Aventis, Yamanouchi and Wyeth, have had a major presence in vasopressin receptor research and oxytocin is frequently included in patent claims for the molecules. Occasionally, oxytocin-selective compounds have been reported, usually derived by adaptation of the vasopressin antagonist template. The first non-peptide oxytocin antagonists, based on a spiropiperidine template, were described by Merck in 1992 [68–70]. The binding affinity data for key compounds from this series are summarised in Table 7.2. The initial screening hit, L-342,643, (23), had modest (4 mM) affinity for rat uterine oxytocin receptors and very little vasopressin selectivity [71]. A structure activity relationship (SAR) study was carried out around this template, focussing on the toluenesulphonamide group. This work led to the identification of bulky lipophilic substitution as key to improved oxytocin potency, while the introduction of a carboxylic acid group led to improved
350
OXYTOCIN ANTAGONISTS AS POTENTIAL THERAPEUTIC AGENTS Table 7.2 BINDING AFFINITIES OF SPIROPIPERIDINE OXYTOCIN ANTAGONISTS
Compound
Rat OT
Rat V1A
(23) (24) (25) (26) (27)
1600a 780d 52d 12d 8.9d
9800b 89000e 5600e 610e 370e
Rat V2 >40000c 83000f 17000f 1300f 570f
Reference [70] [67] [71] [72] [73]
Binding affinity inhibition constant Ki (nM) versus [3H]oxytocin in rat uterine tissue. Binding affinity inhibition constant Ki (nM) versus [3H]arginine vasopressin in rat liver tissue. c Binding affinity inhibition constant Ki (nM) versus [3H] arginine vasopressin in rat kidney tissue. d Binding affinity IC50 (nM), concentration required for half-maximal inhibition of binding of [3H]oxytocin to rat uterine tissue. e Binding affinity IC50 (nM), concentration required for half-maximal inhibition of binding of [3H]arginine vasopressin to rat liver tissue. f Binding affinity IC50 (nM), concentration required for half-maximal inhibition of binding of [3H] arginine vasopressin to rat kidney tissue. a b
aqueous solubility, an essential requirement for intravenous administration. A preferred compound from this work was the camphor sulphonamide, L-366,509 (24), which has improved potency against the rat, rhesus monkey and human oxytocin receptors, but still retains some vasopressin activity. A significant finding with (24) was the observation that it was a competitive antagonist (pA2 7.3) of oxytocin in a rat isolated uterus model and showed prolonged inhibition of oxytocin-stimulated rat uterine activity when administered intravenously or intraduodenally. This demonstration of oxytocin inhibitory activity in vivo led to a significant programme of lead optimisation round this molecule at the Merck laboratories, with the aim of increasing potency at least tenfold. More potent compounds, e.g. the spiroindane L-367,773 (25), were indeed found [72]. This compound was orally bioavailable and active in a rat isolated uterus model, with a pA2 of 7.9. Even more potent compounds were subsequently identified, e.g. the aminosuccinimide derivative (26) [73]. A further breakthrough came with the replacement of the spiroindanepiperidine group with ortho-tolylpiperazine, leading to compounds with improved pharmacokinetic properties [74]. One of the compounds from this series, L-368,899 (27), became the first non-peptidic oxytocin antagonist to enter Phase I clinical trials. This compound was a potent oxytocin antagonist at the rat and human receptors, with a pA2 of 8.9 in the rat isolated uterus model. Although (27) was selected from the series on the basis of its good oral bioavailability in rats and dogs, it proved to have very low (o1%)
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351
bioavailability in rhesus monkeys, which was attributed to a high rate of hepatic metabolism in this species [75].
N O
N
N
S O
O
S O
O
S O Me
Me H
HO
HN Me
Me Me
COOH O
(24) L-366,509
(23) L-342,643
N
NH
(25) L-367,773
Me N N O
S
N O
O
S
O
Me
Me
O N O H2N
(26)
O
Me MeSO2
N H
Me
NH2
(27) L-368,899
Work on this series of non-peptide oxytocin antagonists was then terminated at Merck, in favour of a promising new template. Binding affinity data for key compounds in this new series are summarised in Table 7.3. Some years ago, dihydroquinolinones such as OPC-21268, (28), had been disclosed as vasopressin V1 antagonists. This compound underwent clinical evaluation by Otsuka for hypertension and cardiac failure [76], but was
352
OXYTOCIN ANTAGONISTS AS POTENTIAL THERAPEUTIC AGENTS Table 7.3 BINDING AFFINITIES OF BENZOXAZINONE OXYTOCIN ANTAGONISTS
Compound
Rat OT
Rat V1A
(28) (29) (30) (31) (33) (34) (35) (36) (37) (38) (39)
230a 32b 19a 3.7b 15a 0.71a
Rat V2
Human OT Human V1A Human V2 Reference
>30000c 170d >30000c 4.6d 4.1g 1.4g 38g 10g 58g 6.1g 2.0g 10g 10g
52000e 3200e
>81000f 37000f
[77] [77] [78] [79] [81] [81] [81] [81] [82] [82] [82]
Binding affinity inhibition constant Ki (nM) versus [3H]oxytocin in rat uterine tissue. Binding affinity inhibition constant Ki (nM) versus [3H]arginine vasopressin in rat liver tissue. c Binding affinity inhibition constant Ki (nM) versus [3H] arginine vasopressin in rat kidney tissue. d Binding affinity inhibition constant Ki (nM) versus [3H]oxytocin in human uterine tissue. e Binding affinity inhibition constant Ki (nM) versus [3H]arginine vasopressin in human platelet tissue. f Binding affinity inhibition constant Ki (nM) versus [3H] arginine vasopressin in human kidney tissue. g Binding affinity inhibition constant Ki (nM) versus [3H]oxytocin in human CHO cells. a b
found to have poor affinity for the human V1a receptor compared to rat [77]. However, this compound was shown by the Merck researchers to have modest but significant affinity for both the rat and human oxytocin receptors [78]. This series evidently offered potential for a selective human oxytocin antagonist, though the non-selective vasopressin pharmacology in rat presented some challenges. Replacement of the dihydroquinolinone moiety with quinazolinone led to a loss of potency, but the benzoxazinone was found to retain the activity shown by (28). A breakthrough came when the acetamidopropyl substituent was constrained into a piperidine ring, leading to an oxytocin antagonist L-371,257, (29), with much improved potency. This compound was highly active as an oxytocin antagonist in human uterine tissue and showed around a 1,000-fold selectivity compared to a human V1a preparation. However, it retained the poor selectivity in rat, where it was a potent V1a antagonist. It was active in vivo, blocking oxytocin-induced uterine contractions in the rat after intravenous or intraduodenal administration. Further optimisation of the pharmacokinetic properties of this series has been achieved through the incorporation of a pyridine N-oxide group. A preferred compound is
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353
L-372,662, (30), which retained excellent in vitro and in vivo potency, with an AD50 0.7 mg/kg following intravenous administration in rat [79]. It also showed very high (>90%) oral bioavailability in both rat and dog and has excellent aqueous solubility suitable for intravenous administration. Enhancement of in vitro potency in this series was achieved through substitution with a fluorine atom on the phenyl ring of the benzoxazinone moiety and through incorporation of additional lipophilic substituents on the terminal pyridine ring. However, these modifications were generally associated with inferior pharmacokinetic or metabolic properties, although the introduction of a 4-trifluoromethyl group on the pyridine ring offered some protection from P450-mediated metabolism. O N
O
N
N
O
N
O
O O
(28) OPC-21268
NHAc
MeO
N
Ac
O
(29) L-371,257
Further work around the benzoxazinone template led to the identification of a series of ortho-trifluoroethoxyphenylacetamides, exemplified by L-374,943, (31) [80]. This compound offered significant advantages compared to the original series. Thus, the compound was extremely potent in a functional assay against rat uterine tissue (pA2 9.2) and was active in vivo after intraduodenal administration to rats, with an AD50 of 0.6 mg/kg, making it one of the most potent compounds in this assay. Rat oral bioavailability was 19%. This series showed the same species differences with respect to V1a activity as that observed in the original series. Despite attempts to exploit some SAR differences observed with the terminal piperidine substituent, it appears not to have proved possible to achieve good in vivo activity as well as acceptable pharmacokinetic properties. A related analogue, (32), containing a quinolinone in place of the usual benzoxazinone, was described recently [81], but the focus of this publication has shifted onto the use of 35S radiolabelling for use as a pharmacological tool.
354
OXYTOCIN ANTAGONISTS AS POTENTIAL THERAPEUTIC AGENTS
O
O N
N O
+ O N
N
O
N
Me
MeO
O
N
O
N
O
Ac
OCH2CF3
O (30) L-372,662
(31) L-374,943
No other work has appeared from the Merck group for a number of years and oxytocin receptor antagonism appears to be no longer an active research area for the company. Scientists at GlaxoSmithKline have published work around a similar template [82]. The binding affinities for representative compounds from this series are included in Table 7.3. Novel left and right portions of the molecule were sought by preparing libraries from fragments derived from cleavage of the central amide bond in the benzoxazinone (29). No alternatives were found for the benzoxazinone moiety, but several heterocyclic acids were identified as replacements for the methoxy benzoic acid core, which would also allow further elaboration to probe the area occupied by the acetyl piperidine moiety. Additional libraries around thiazole and pyrimidine cores yielded a number of moderately active compounds, but none had sufficient potency to progress. Investigation of a related indole template, however, yielded potent compounds, as exemplified by the sulphonamide derivative (33). Activity was improved further by introducing steric constraints to the sidechain and introduction of a 7-methyl substituent on the indole ring, leading to compound (34) [82]. Derivatives generally possessed only moderate pharmacokinetic properties however (clearance: 25–45 ml/min/kg in dog), which was attributed to metabolic vulnerability of the indole (C2–C3) double bond. Attempts to block metabolism by C2, C3 di-methyl substitution resulted in the loss of oxytocin activity. O N
N
O
O
N
N N
O
O
SO2Me
O
OCH2CF3
(32)
N (33)
Me N SO Me 2
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Parallel SAR investigation around benzofuran as the benzoic acid core replacement yielded 2-substituted derivatives with moderate potency improvements over the unsubstituted core, but generally much better pharmacokinetic profiles, as exemplified by compound (35) [82] (dog clearance 8 ml/min/kg). Extensive library work to expand the diversity of derivatives and improve potency within this template led to the identification of a pyridone derivative (36). However, this compound possessed moderate pharmacokinetic properties (dog clearance 34 ml/min/kg). In an attempt to address this issue and explore the SAR of the pyridone fragment further, a range of substituted pyridones was made [83]. From this series, 5-trifluoromethyl substitution was found to give high potency compounds with improved pharmacokinetic properties. A preferred compound, (37), with a dog clearance of 8 ml/min/kg, was progressed further to in vivo studies. O N
O
O N
O
N N
O
NHAc
O
SO2Me
N
N
Me
O
(34)
(35) O N
O
N H N
O
O O N
O R
(36) R = H (37) R = CF3
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OXYTOCIN ANTAGONISTS AS POTENTIAL THERAPEUTIC AGENTS
However, potency in the oxytocin-induced rat uterine contraction model was much lower (IC50 3 mM) than predicted from its in vitro potency at the rat oxytocin receptor (IC50 2 nM). This lower potency was attributed to high protein binding (96%) and substantial shifts in in vitro oxytocin-binding affinity were observed when compounds from this series were tested in the presence of physiologically relevant concentrations of serum albumin. In an attempt to improve the in vivo properties of molecules in this series, a strategy of reducing the PSA and incorporating a basic moiety was employed [84]. From previous SAR investigations, the amide linker between the benzofuran core and the pyridone moiety was believed to be non-essential for binding. A pharmacophore approach was used to identify alternative linkers with reduced PSA between the 2-position of the benzofuran ring and the pyridone nitrogen atom, based on low energy conformations of (37). Although high potency compounds (38) and (39) with lower PSA were identified, the compounds were still highly protein bound. It was concluded that the structural characteristics of this series, rather than just physicochemical properties, were responsible for the high protein binding. This series was therefore abandoned in favour of alternative novel templates. O
O N
N
O
O
O N
N
N N
O
CF3
O
O
H O
O
N
N CF3
(38)
(39)
A benzodiazepine template was also reported by researchers at GlaxoSmithKline [85]. The lead molecule GW405212, (40), was identified from a 1,296-member library of 1,4-benzodiazepines prepared on Tentagel beads and screened initially in pools of 30 against CHO cells expressing the human oxytocin receptor. It is a highly potent inhibitor of oxytocin binding with a Ki of 8 nM [86]. However, all attempts to improve the pharmacokinetic properties of this molecule were unsuccessful. It appears that the functionality responsible for the oxytocin activity is distributed around the periphery
M.J. ALLEN, D.G.H. LIVERMORE AND J.E. MORDAUNT
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of this molecule. Removal of any of these groups led to a loss of potency. It was concluded that the high molecular weight and amide bond count in this molecule constituted a poor starting point for a drug discovery programme. Me H N
Cl H2N
Cl
O O
O
H N
N
O O
N Ph
(40) GW405212
Another series disclosed by researchers at GlaxoSmithKline is based on the diketopiperazine template, which is the subject of recent patent applications [87,88]. The molecules claimed to have the generic structure (41). A recent publication [89] describes some of the initial SAR in this series. Compounds containing an indanyl group (R1), a phenyl group (R3) and small-branched aliphatic groups (R2 and R4) were identified as having high potency, with all stereocentres having the R configuration. A representative compound from this series is (42), with a Ki of 4 nM. O R1
R3
HN
O
R4
N R2
Ph NHiPr
N
O
O
HN
O O
(41)
iPr
(42)
There is extensive patent literature claiming oxytocin antagonists. In many of these examples, the principal focus appears to be on vasopressin, with oxytocin as a secondary claim and where biological data are included, oxytocin antagonist activity is often quite weak. A number of companies do, however, have substantive oxytocin antagonist patents, including SanofiAventis, Serono, Wyeth and Yamanouchi. Sanofi-Aventis, through the research activities of the heritage company Sanofi-Synthelabo, have long had an interest in vasopressin antagonists, including the V1a-selective indoline SR49059, (43). The role of V1a in the female reproductive system
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OXYTOCIN ANTAGONISTS AS POTENTIAL THERAPEUTIC AGENTS
falls outside the scope of this review. The reader interested in the pharmacology of this molecule is referred to one of the recent review articles, which have appeared in this area, e.g. [90,91]. However, the related indolin-2-ones are the subject of a specific oxytocin patent [92]. A preferred compound is SSR126768, (44) [93], which is very active at rat and human oxytocin receptors (Ki 0.44 nM), with good selectivity against vasopressin receptors. Moreover, the compound inhibits oxytocin-induced uterine contractions in both rat (pA2 8.5) and human isolated myometrium, is orally efficacious at 3 mg/kg in inhibiting oxytocin-induced uterine contractions in the conscious rat and delays parturition in rats following a 30 mg/kg orally administered dose. O Cl OH
Cl
N O
MeO
S
Et NH2
N O
O
N
N
Cl Me
Cl
O
O N
OMe
(43) SR49059
MeO
OMe
(44) SSR126768
Serono have become increasingly active in this area in recent years and have a number of recent patents assigned to their subsidiary, Applied Research Systems. Following a high-throughput screen, interesting oxytocin activity was discovered in a series of pyrrolidine oximes. The first compounds to be disclosed were amides such as (45) [94]. The pharmacology of (45) has been described in some detail [95]. It had a binding Ki of 28 nM in human CHO cells, was somewhat less active against the rat receptor (135 nM) and some modest V1a selectivity (170 nM). It was also active in inhibiting contractions in isolated rat uterine strips (pA2 7.8) and is active in vivo. It inhibited oxytocin-induced contractions in the non-pregnant rat with an ED50 of 3.5 mg/kg after intravenous dosing and 89 mg/kg after oral administration. Furthermore, it showed some inhibition of spontaneous uterine contractions in the pregnant rat. In an attempt to identify compounds with better pharmacokinetic properties, the amide group was replaced by ester [96], oxadiazole and
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thiadiazole [97] groups, with retention of oxytocin antagonist activity [98]. A particularly interesting modification is replacement with a hydroxymethyl group [99], the SAR round this series having recently been presented as a poster communication [100]. Potent activity was observed with a number of hydroxymethyl, hydroxyethyl and methoxymethyl derivatives, but aminomethyl substitution was poorly tolerated. The Z isomer of the methoxime was always around threefold more potent than the E isomer. A possible issue with this series is the potential isomerisation of the oxime group. However, most pharmacokinetic and pharmacological properties for the lead compound (46) appear favourable. It had a Ki of 95 nM against human oxytocin, with good rat activity (Ki 210 nM) and again modest vasopressin selectivity (V1a 330 nM). It had good pharmacokinetic properties in rat, being 55% orally bioavailable, with low clearance (0.34 l/kg/h) and exhibits low levels of human serum protein binding (71%). Consistent with these drug-like properties, the compound was active in a number of in vivo models. At 30 mg/kg p.o., it inhibited oxytocin-induced contractions in non-pregnant rats by 51% and spontaneous contractions in pregnant rats were inhibited in a dose-dependent manner (around 40% inhibition observed with a 60 mg/kg oral dose). Further preclinical studies are reported to be ongoing. Two other series of oxytocin antagonists are reported by Serono in the patent literature. A series of sulphanilide compounds with extremely potent (low nanomolar to subnanomolar) activity was reported in 2002 [101]. A lead compound appears to be (47), which gave Ki values of 0.65 nM and 0.67 nM, respectively, at human and rat oxytocin receptors, and was reported to be highly selective versus vasopressin V1a receptors. Moreover, it proved to have good metabolic stability in human and rat studies. (47) inhibited spontaneous contractions in late-term pregnant rats by up to 45% at a dose of 60 mg/kg p.o. and it also inhibited oxytocin-induced contractions by 35% at an oral dose of 30 mg/kg (ED50 ¼ 1.4 mg/kg i.v.) [102]. MeO N
MeO N H N
N Me
O
O
(45)
OH
N
OH Me
O
(46)
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OXYTOCIN ANTAGONISTS AS POTENTIAL THERAPEUTIC AGENTS
A series of 1,3,4-triazoles has also been patented by Serono/Applied Research Systems [103]. The 2-benzylthio compound, (48), had a Ki of 45 nM in a human oxytocin-binding assay. Cl OMe
N H
O O
H N
N S
O O
O
NH N N N
N S
OMe
Me2N N H
O
(47)
Cl
(48)
Researchers at the Yamanouchi Pharmaceutical Co. have published extensively on vasopressin antagonists with a benzazepine template. Although most of their work was directed towards the V1a and V2 receptor subtypes, a number of compounds also had significant oxytocin antagonist activity. An example from the patent literature is the gem-difluorobenzazepine, (49) [104]. An important dual V1a/V2 antagonist, conivaptan, YM-087, (50) also has significant oxytocin antagonist activity (rat uterus preparation Ki 44 nM) along with potent V1a (1.2 nM) and V2 (2.2 nM) activity [105]. This compound was under development by Yamanouchi for heart failure, oedema and hyponatremia [106]. Oxytocin activity has also been reported in a series of 1,5-benzodiazepines, (51) [107]. These compounds were generally around 10–100-fold less potent at OT compared to V1a and V2 in binding assays using rat tissue preparations. Given the close relationship between the oxytocin and vasopressin receptors, it must be assumed that many other molecules of this type also possess dual oxytocin–vasopressin antagonist activity. However, with the benzodiazepine series, the major thrust of research work has been directed towards the vasopressin receptor.
M.J. ALLEN, D.G.H. LIVERMORE AND J.E. MORDAUNT
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Me O
R
HN N F
N
N
OH N
N
F
N
N
O
O
O
NH
S Me
Ph Ph
N
O
NH Ph O
(49)
(50) YM-087 conivaptan
(51)
Wyeth has a number of patents claiming oxytocin-selective benzodiazepine antagonists, which have evolved from their extensive research with the vasopressin receptor [108, 109]. These oxytocin-selective compounds are based on their vasopressin V2 antagonist clinical candidate, lixivaptan, (52) [110, 111] and related molecules, e.g. (53) and generally contain a biarylcarbonyl or cyclohexenylphenylcarbonyl moiety attached to a tricyclic pyrrolobenzodiazepine template, (54) and (55). Some potent compounds have been identified in this series. Thus, at 100 nM concentration, compound (55) inhibited oxytocin binding in CHO cells by 56%. The corresponding figures for V1a and V2 are 2% and 13%, respectively [112]. The 4-pyridinylpiperazine analogue has a reported IC50 of 11 nM against human oxytocin receptor expressed in CHO cells [113], while the bishydroxyethylamino derivative (56) was particularly potent with an IC50 of 1.4 nM [114]. Me N
N N
N
S N
O NH
Cl
F
O
NH
O
(52) lixivaptan
O
Me
(53)
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OXYTOCIN ANTAGONISTS AS POTENTIAL THERAPEUTIC AGENTS
O O CF3
N
N N N
O
O
Me
Me
N NR2
(55)
(54)
N BOC
Although mostly amide derivatives have been exemplified, acyl amidines are also claimed, e.g. the dimethylaminoethylidene (57), with an IC50 of 4.4 nM against OT and 20-fold and 67-fold selectivity over V1a and V2 in a binding assay [115]. O
O
N
N
CF3
N Me
N
O N(CH2 CH2OH)2
N
(57)
(56)
O
Me NMe2
The benzazepine template has also been extensively exploited by researchers at Otsuka. Again, much of this work has been directed at the vasopressin receptor, although claims for oxytocin antagonist activity are occasionally made, but without substantive biological data. Some of these compounds, e.g. OPC-31260 (58) appear to be related to the Yamanouchi template, (50) [116]. However, in an oxytocin-binding assay developed using human uterine smooth muscle cells, (58) was shown to have much lower potency (Ki 2,500 nM) than either the Yamanouchi antagonist (50) (Ki 30 nM) or the Merck antagonist (29) (Ki 2 nM) [117]. OXYTOCIN AGONISTS Although not strictly within the scope of this review, it is interesting to note that Ferring has recently reported on the properties of a non-peptidic series of oxytocin agonists. The authors envisage that the compounds may be of value in providing an oral alternative to the current practice of
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intravenously administered oxytocin for the induction of labour and may also have a role in the treatment of male erectile dysfunction, presumably based on the part oxytocin is believed to play in the regulation of male and female sexual activity. Selectivity for oxytocin over vasopressin V2 has been a problem with this series, but success was obtained with compound (59), which has an oxytocin agonist EC50 of 33 nM and a 25-fold selectivity versus vasopressin V2 [118, 119]. Related compounds from this series have been claimed in a recent patent and are reported to be oxytocin agonists and vasopressin V1a antagonists [120, 121]. Me Me2N
Me
Me
N
O
Me N H
N
H N
O
(58) OPC-31260
N N
N
N
N
H N O
S
O
(59)
STRUCTURAL STUDIES OF OXYTOCIN RECEPTOR The human oxytocin receptor gene was isolated and characterised in 1994 [122], heralding the development of modern cloned receptor screening. The oxytocin receptor belongs to the Family A series of G-protein coupled 7-transmembrane receptors (GPCRs). A schematic representation of the generic structure of 7TM receptors is shown in Figure 7.3. From amino acid residue analysis, the position of the seven hydrophobic transmembrane regions can be elucidated. The single letter amino acid representation for oxytocin and vasopressin receptors and the predicted transmembrane regions based on the human oxytocin sequence using the published transmembrane hidden Markov model (TMHMM) method [123] is indicated in Table 7.4 [124]. Substantial homology is apparent across these receptors, especially in the transmembrane regions (box highlighted) where non-peptide antagonists are believed to bind. The different human/rat selectivity of OPC-21268 (28) has been attributed to the replacement of the glycine residue in transmembrane domain region VII of the human V1a receptor to alanine in the corresponding rat receptor and in human and rat OT [125].
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OXYTOCIN ANTAGONISTS AS POTENTIAL THERAPEUTIC AGENTS
N-terminus
extracellular
transmembrane
transmembrane
intracellular
C-terminus
Fig. 7.3 7TM structure (adapted from [2]).
In common with many other receptors in this family, structural homology models have been built based on the photoreceptors bacteriorhodopsin and rhodopsin. These models are built by identification of seven a-helical hydrophobic regions in the receptor sequence, having regard to certain common structural features within the GPCR family. These a-helices are then compared with the published crystal structure of the rhodopsin receptor, with in silico replacement of the amino acids with those obtained for the sequenced receptor. Although this approach is somewhat hit-and-miss, examples of its successful application to the design of receptor antagonists have been reported. Pertinent to this review is the three-dimensional model of vasopressin V1a receptor, which has been described and validated through a combination of site-directed mutagenesis and the design of irreversible legends based on the non-peptide antagonist, SR49059 (43), through the incorporation of cysteine sulphydryl-reactive isothiocyanate or a-chloroacetamide moieties on the phenylsulphonyl group [126]. From this study, it was concluded that Phe-225 on transmembrane region V was directly involved in the binding of this non-peptidic antagonist. In the case of the oxytocin receptor, a model has been built and used to dock oxytocin, a peptide antagonist, L-366,948, (60) [127] and a peptide agonist [4-thr, 7-gly]oxytocin (61) [128, 129]. The same group has recently published the docking of the peptide antagonist, atosiban (22), to the oxytocin receptor [130]. No studies on the docking of non-peptide antagonists, or on the preparation and characterisation of irreversible ligands, which might provide insight into their binding sites, had appeared by the end of 2004.
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Table 7.4 SEQUENCE AND ALIGNMENT FOR HUMAN AND RAT OT, V1A, V1B AND V2 RECEPTORS
366
OXYTOCIN ANTAGONISTS AS POTENTIAL THERAPEUTIC AGENTS OH O H2N
H N N H
S
O O
N HN
O N H
N H
O
NH O
O
O
HN
H N
O
N N
HN
S
O
O
N H
O
O
Me NH
OH NH2
N O
NH O
(60) L-366,948
H2N
O
(61)
The molecular pharmacology of oxytocin and vasopressin agonist and antagonist binding has been extensively studied by Wheatley’s group. Sitedirected mutagenesis studies have been used to elucidate the amino acid residues critical for ligand recognition and receptor–effector coupling. Thus, Asp-85 situated on transmembrane region II on mutation to asparagine led to the loss of oxytocin agonist binding and oxytocin-stimulated phosphoinositide hydrolysis [131], but retained antagonist-binding properties. In contrast, Gly-107 (extracellular domain) and Phe-293 (transmembrane VI), on mutation to alanine and isoleucine respectively, retained oxytocin agonist binding and phosphoinositide hydrolysis. The vasopressin V1a receptor sequence from Glu-37 to Asn-47 is an absolute requirement for arginine vasopressin agonist binding, but this subdomain did not affect antagonist binding, using either peptide or non-peptide antagonists [132]. Substitution into the V1a receptor with the entire N-terminus of the oxytocin receptor did however yield a chimeric receptor, which displayed high affinity agonist binding and indeed functioned essentially as a working V1a receptor. The reverse receptor, in which the N-terminus of the oxytocin receptor was substituted with the corresponding sequence from the vasopressin V1a receptor, has also been studied [133]. In this case, the vasopressin sequence restored full pharmacological function to a dysfunctional N-terminus truncated oxytocin receptor. Although this truncated receptor ([D2-35]OT) has a marked 2,000-fold reduction in affinity for oxytocin, peptide antagonists and the non-peptide camphor-based tolylpiperazine antagonist L-368,899 (27) bound to this receptor with similar affinity to that seen with the wild-type receptor. It would be interesting to study this and other chimeric receptors based on the oxytocin receptor, with spliced-in sections of the vasopressin V1a receptor
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in order to compare the pharmacology of the various oxytocin peptide and non-peptide antagonists. This would be of particular interest with regard to the Merck benzoxazinone series of antagonists, where significant speciesdifferences between human and rat V1a activity were found. Recent work has identified a critical role for a conserved arginine Arg-34 in the N-terminus for agonist binding and receptor activation, loss of which leads to a dysfunctional receptor [134]. A substantial review of the molecular pharmacology of human vasopressin receptors (including oxytocin) and peptidic agonists and antagonists has appeared [135].
CONCLUSION The work described above demonstrates the major advances that have been made in the oxytocin field over the last 15 years. Modification of the natural hormone has led to peptide antagonists suitable for short-term intravenous administration in the acute treatment of preterm labour. One of these molecules, atosiban, has been approved in Europe for this indication. Recently, non-peptidic antagonists with generally improved selectivity for the oxytocin receptor over the related vasopressin receptors and improved pharmacokinetic properties, including oral bioavailability, have been discovered. These molecules offer the prospect of much-improved therapeutic options for the distressing condition of preterm labour, which too frequently leads to the birth of very immature babies with very limited life expectancy or with major disabilities. Treatment of the mother in preterm labour with an oxytocin antagonist to achieve acute tocolysis and then as maintenance for the critical last trimester of pregnancy may make a major difference to these parents and their offspring.
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374
Subject Index A-331440, 190 ABT-239, 192 ABT-834, 188 Actinonin, 121 Alopecia, 172 AM1241, 266 AM 251, 274 AM630, 311 AMG 9810, 158 Analytical centrifugation, 9 Anandamide, 148 Anandamide transport inhibitors, 210 Antitussive, 172 N-arachidonyldopamine, 149 Arthritis, 172 Arvanil, 153 Atisoban, 336, 340, 353 Axsain, 146
Databases Ligand-protein, 38 rCAT database, 30 Dehydroepiandrosterone (DHEA), 163 De novo design, 56 Fragment evolution, 56 LEAPFROG, 58 LEGEND, 57 LUDI, 58 Skelgen, 56 TOPAS, 56 Docking High throughput, 51 IFREDA, 50 Induced fit, 48 Programs, 34 Scoring, 37 SDOCKER, 46 MASC, 47 DoMCoSAR, 46 DNA gyrase, 9
BAY 59-3074, 258 BB-3497, 122 BB-83698, 123, 136 BCTC, 154 BILN 2061, 72, 76, 82, 83 Boronic acid derivatives, 95 BP 2.94, 185
Endocannabinoids, 209 Endovanilloids, 148 17b-Estradiol, 164 Fatty acid amide hydrolase inhibitors, 212 GT-2331, 184, 188 GW0014, 95 GW405212, 363
Cannabidiol, 221, 233 Cannabinoid receptors, 208 Capsaicin, 146 Capsazepine, 151 CB1 antagonists, 272 CB2 agonists, 259 CB2 antagonists, 310 Chloroproxyfan, 184 Clobenpropit, 187 Comparative homology modelling, 53 Conivaptan, 367 Consensus scoring, 44 CP 272871, 273 CP 55244, 236
Hepatitis C Virus, 66 Histamine receptor (H3) antagonists, 188, 198 Hymenialdisine, 31 IBTU, 162 Imetit, 185 Immunoregulation, 172 Incontinence, 170 Indomethacin, 338 375
376
SUBJECT INDEX
Inflammatory bowel disease, 171 Iodophenpropit, 187 JNJ-5207852, 193 L-156,373, 353 L-342,643, 355 L-365,209, 353 L-366,509, 355 L-366,948, 371 L-367,773, 355 L-368,899, 356 L-371,257, 357 L-372,662, 357 L-374,943, 359 Lactation, 341 LBM-415, 125, 136 Lixivaptan, 368 Methionine aminopeptidase, 111 MMP complexes, 45 Multiple sclerosis, 271 NESS-0327, 276 Nifedipine, 338 NMR ATP-STD, 24 Fragment screening, 27 Heteronuclear single quantum correlation (HSQC), 17 Kinase inhibitors, 21 Nuclear Overhauser effects (NOE), 18 Nuclear-spin relaxation, 17 SAR by NMR, 19 Saturation transfer difference, 19 Shapes strategy, 21 WaterLOGSY, 19, 23 Noladin ether, 246 Nonivamide, 152 NS3-NS4A Protease, 66 NVP PDF-713, 125 O-1043, 275 Obesity, 309 Olvanil, 153 N-Oleoyldopamine, 149 OPC-21268, 357, 370
OPC-31260, 369 Oxytocin , 335 Pmp-, 345 1-Pmp-2-D-trp-8-arg, 346 4-Thr, 7-gly, 371 Pain, 170, 271 Pancreatitis, 171 PAR2, 150 PD318088, 25 Peptide deformylase, 109 Structure, 113 Peptide deformylase inhibitors Aldehyde, 119 Biaryl acids, 128 N-Formyl-N-hydroxylamine, 121, 130 Hydroxamic acid, 121, 130 H-Phosphonate, 119 Thiol inhibitors, 118 Thyropropic acid, 128 Phenylacetylrinvanyl, 153 Phorboid vanilloids, 151 Phosphodiesterase inhibitors (PDE), 13 Piperine, 147 Pravadoline, 247 Preterm labour, 336 Protease-activated receptor, 150 Protein ligand docking, 34 Protein kinases, 1 AK, 27 AKT, 41 cAPK, 50 ATP binding site, 5 Bcr-Abl, 57 Catalytic domain, 3 CDK2, 31, 43, 47, 49, 50, 52, 55, 57 CDK4, 57 Checkpoint kinase-1, 42 CK2, 43 EGFR , 51, 52, 54 Erb1, 11 FGFR-1, 54 JNK3, 22 LCK, 50 MEK1, 25 P38, 12, 21, 44, 47, 50, 54 PDGFRb, 54 Pfmrk, 53
SUBJECT INDEX PKA, 25 PKB, 41 SRC, 54, 57 SYK, 52 VEGFR , 46, 54 Web site, 3 Protein tyrosine phosphatase 1B (PTP1B), 48
Smoking cessation, 309 SNARE, 148 SR141716A, 272 SR144528, 310 SR49059, 364, 371 SSR126768, 364 Surface plasma resonance, 9
Retvanil, 153 Rimonabant, 272, 309 Ritodrine, 338 RTX, 151 Ruthenium red, 151 RWJ-20085, 192
Tethering, 10 Thioperamide, 186 Thymidylate synthase, 10 Tractocile, 336 TRPV1, 145 Uterus, 337
SAR by NMR, 19 SB-366791, 159 SB-452533, 161 SB 660618, 130 Sch 382582, 134 Sch 382583, 134 Sch 50971, 185 Sch 79687, 189 Screening Fragment, 8, 27 Knowledge-based, 45 Ligand based NMR, 19 Needle, 8 NMR, 15, 18 Shape, 8 Virtual, 41 Selection of experimentally exploitable drug startpoints (SEEDS), 30 SIFt, 46
Vanilloid receptor, 145 Vasopressin, 340 VIC-104959, 125 Vinyl-ACCA, 79 VRC3324, 124 VRC3375, 124 VRC4307, 124 VX-950, 91, 94 WaterLOGSY, 19, 23 Websites CMBI, 54 Protein kinase, 3 X-ray crystallography, 5 YM-087, 367
377
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378
Cumulative Index of Authors for Volumes 1– 44 The volume number, (year of publication) and page number are given in that order. 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 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 Bo¨s, M., 44 (2006) 65 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 Adam, J., 44 (2006) 209 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, M.J., 44 (2006) 335 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 Appendino, G., 44 (2006) 145 Arrang, J.-M., 38 (2001) 279 Armour, D., 43 (2005) 239 Aubart, K., 44 (2006) 109 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 Barbier, A.J., 44 (2006) 181 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
Camaioni, E., 42 (2004) 125 Carman-Krzan, M., 23 (1986) 41 Carruthers, N.I., 44 (2006) 181 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) 89 379
380
CUMULATIVE AUTHOR INDEX
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 Cherry, M., 44 (2006) 1 Clark, R.D., 23 (1986) 1 Clitherow, J.W., 41 (2003) 129 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 Cowley, P.M., 44 (2006) 209 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
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 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 Dvorak, C.A., 44 (2006) 181
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; 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
Hacksell, U., 22 (1985) 1 Haefner, B., 43 (2005) 137 Hall, A.D., 28 (1991) 41
CUMULATIVE AUTHOR INDEX 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 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
381
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 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 Kiyoi, T., 44 (2006) 209 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., 39 (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; 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
382
CUMULATIVE AUTHOR INDEX
Lee, J.C., 38 (2001) 1 Lenton, E.A., 11 (1975) 193 Lentzen, G., 39 (2002) 73 Letavic, M.A., 44 (2006) 181 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 Lin, T.-S., 32 (1995) 1 Liu, M.-C., 32 (1995) 1 Livermore, D.G.H., 44 (2006) 335 Llinas-Brunet, M., 44 (2006) 65 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 Mordaunt, J.E., 44 (2006) 335 Morris, A., 8 (1971) 39; 12 (1975) 333 Morrison, A.J., 44 (2006) 209 Mort, C.J.W., 44 (2006) 209 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
CUMULATIVE AUTHOR INDEX 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 Reader, J., 44 (2006) 1 Reckendorf, H.K., 5 (1967) 320 Reddy, D.S., 37 (2000) 135 Redshaw, S., 32 (1995) 239 Rees, D.C., 29 (1992) 109 Reitz, A.B., 36 (1999) 169 Repke, K.R.H., 30 (1993) 135 Richards, W.G., 11 (1975) 67 Richardson, P.T., 24 (1987) 1 Roberts, L.M., 24 (1987) 1 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
383
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 Szallasi, A., 44 (2006) 145 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
384
CUMULATIVE AUTHOR INDEX
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 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 White, P.W., 44 (2006) 65 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 Wikstro¨m, H.V., 38 (2001) 189 Wilkinson, S., 17 (1980) 1 Williams, D., 44 (2006) 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 Zalacain, M., 44 (2006) 109 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– 44 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 X-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 X-, 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) 65 385
386
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 Ami-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[l,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; 44 (2006) 207 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
CUMULATIVE SUBJECT INDEX 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 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
387
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 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
388
CUMULATIVE SUBJECT INDEX
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 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 Hepatitis C virus NS3/NS4A protease inhibitors, 44 (2006) 65 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; 44 (2006) 181 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 X-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
CUMULATIVE SUBJECT INDEX 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 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 receptorligand 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
389
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 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 Oxytocin antagonists, 44 (2006) 331
390
CUMULATIVE SUBJECT INDEX
Poly(ADP-ribose)polyrmerase (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 Peptide deformylase inhibitors, 44 (2006) 109 Peroxisome proliferator-acrtvated receptor gamma (PPARg) ligands, 42 (2004) 1 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
CUMULATIVE SUBJECT INDEX peroxisome proliferator-activated receptor gamma (PPARg), 42 (2004) 1 purino, 38 (2001) 115 Rerin 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 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
391
Structure-based lead generation, 44 (2006) 1 Synthesis of enantiomers of drugs, 34 (1997) 203 Tachykinins, 43 (2005) 50 Tetrahydroisoquinolines, X-adrenomimetic activity, 18 (1981) 45 Tetrazoles, 17 (1980) 151 Thalidomide as anti-inflammatory agent, 22 (1985) 165 Thiosemicarbazones, biological action, 15 (1978) 321; 32 (1995) 1 Thromboxanes, 15 (1978) 357 Tilorone and related compounds, 18 (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 Ubiquitinylation, 43 (2005) 153 Vanilloid receptors, TRPV1 antagonists, 44 (2006) 145 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
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