Analogue-based Drug Discovery II, Volume 2

Edited by Ja´nos Fischer and C. Robin Ganellin Analogue-based Drug Discovery II

Related Titles Faller, B., Urban, L. (eds.)

Hit and Lead Profiling Identification and Optimization of Drug-like Molecules 2009 ISBN: 978-3-527-32331-9

Chorghade, M. S. (ed.)

Drug Discovery and Development 2 Volume Set 2007 ISBN: 978-0-471-39846-2

Johnson, D. S., Li, J. J. (eds.)

The Art of Drug Synthesis 2007 ISBN: 978-0-471-75215-8

IUPAC / Fischer, János / Ganellin, C. Robin (eds.)

Analogue-based Drug Discovery 2006 ISBN-13: 978-3-527-31257-3

Lednicer, Daniel

New Drug Discovery and Development 2006 ISBN-13: 978-0-470-00750-1

Edited by János Fischer and C. Robin Ganellin

Analogue-based Drug Discovery II

The Editors Prof. Dr. János Fischer Richter Plc Gyömröi ut 30 1103 Budapest Hungary Prof. Dr. C. Robin Ganellin University College London Department of Chemistry 20 Gordon Street London WC1H OAJ United Kingdom

Supported by The International Union of Pure and Applied Chemistry (IUPAC) Chemistry and Human Health Devision PO Box 13757 Research Triangle Park, NC 27709-3757 USA

All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library. Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.d-nb.de. # 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Cover Design Adam Design, Weinheim Typesetting Thomson Digital, Noida, India Printing and Binding Strauss GmbH, Mörlenbach Printed in the Federal Republic of Germany Printed on acid-free paper ISBN: 978-3-527-32549-8

V

Contents Preface XV Introduction XVII Abbreviations XXI

1

Part I

General Aspects

1

Optimizing Drug Therapy by Analogues 3 János Fischer, C. Robin Ganellin, John Proudfoot, and Erika M. Alapi Introduction 3 Pharmacodynamic Characteristics 4 Potency 4 Improving the Ratio of Main Activity and Adverse Effects 5 Improving Selectivity Through Receptor Subtypes 6 Improving Selectivity Through Unrelated Receptors 7 Improving Selectivity by Tissue Distribution 7 Improving Selectivity of Nonreceptor-Mediated Effects 10 Improving the Physicochemical Properties with Analogues 10 Analogues to Reduce the Resistance to Anti-Infective Drugs 11 Antibiotics 11 Antifungal Drugs 12 Antiviral Drugs 12 Analogue Research in Resistance to Drug Therapies in Cancer Treatment 15 Pharmacokinetic Characteristics 15 Improving Oral Bioavailability 15 Improving Absorption 16 Improving Metabolic Stability 16 Drugs with a Long Duration of Action 17 Ultrashort-Acting Drugs 18 Decreasing Interindividual Pharmacokinetic Differences 20 Decreasing Systemic Activity 21

1.1 1.2 1.2.1 1.2.2 1.2.2.1 1.2.2.2 1.2.2.3 1.2.2.4 1.2.3 1.2.4 1.2.4.1 1.2.4.2 1.2.4.3 1.2.5 1.3 1.3.1 1.3.1.1 1.3.1.2 1.3.2 1.3.3 1.3.4 1.3.5

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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Contents

1.4 1.4.1 1.4.2 1.5

Drug Interactions 22 Decreasing Drug Interactions 22 Increasing Drug Interactions 23 Summary 23 References 24

2

Standalone Drugs 29 János Fischer, C. Robin Ganellin, Arun Ganesan, and John Proudfoot Acetaminophen (Paracetamol) 30 Acetylsalicylic Acid (Aspirin) 33 Aripiprazole 35 First Generation ‘‘Typical’’ Antipsychotic Drugs (Other Names: Neuroleptics, Conventional Antipsychotics) 36 Second-Generation ‘‘Atypical’’ Antipsychotic Drugs 37 A New Approach: Aripiprazole, a Dopamine Partial Agonist 38 Bupropion 39 Ezetimibe 42 Lamotrigine 46 Metformin 47 Topiramate 49 Valproate 51 Summary 52 References 53

2.1 2.2 2.3 2.3.1 2.3.2 2.3.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10

3

3.1 3.2 3.3 3.4 3.5 3.6 3.7

4 4.1 4.2 4.3 4.4 4.5 4.6 4.7

Application of Molecular Modeling in Analogue-Based Drug Discovery 61 György G. Ferenczy Introduction 61 Cilazapril: An ACE Inhibitor 62 Atorvastatin: A HMG-CoA Reductase Inhibitor 66 PDE4 Inhibitors 70 GPIIb/IIIa Antagonists 73 HIV Protease Inhibitors 74 Epilogue 79 References 79 Issues for the Patenting of Analogues 83 Stephen C. Smith Introduction 83 Patents: Some Fundamentals 84 Patentability 85 Important Elements of the International Patent System Priority 87 Novelty 88 Inventive Step: Nonobviousness 90

86

Contents

4.8 4.9 4.10 4.11 4.12 4.12.1 4.12.2 4.12.3 4.12.4 4.12.5 4.12.6 4.12.7 4.12.8 4.12.9 4.12.10 4.13

Utility: Industrial Application 93 Selection Inventions 93 Enantiomers 94 Prodrugs and Active Metabolites 95 The Patenting Process from the Inventor’s Standpoint 97 Inventorship 98 The Priority Patent Application 98 Prior Art Disclosure 98 Patent Specification Review 99 ‘‘Best Mode’’ of Carrying Out the Invention 99 Foreign Patent Applications 99 Patent Application Publication 100 Patent Examination 100 Opposition to Grant 101 Patent Litigation 102 Pitfalls for the Unwary: Granted Versus Published Patents, Scientific Publications 102 References 105

Part II

Analogue Classes

5

Dipeptidyl Peptidase IV Inhibitors for the Treatment of Type 2 Diabetes 109 Jens-Uwe Peters and Patrizio Mattei Introduction 109 In Vitro Assays and Animal Models for the Assessment of DPP-IV Inhibitors 110 Substrate-Based DPP-IV Inhibitors 110 Sitagliptin and Analogues 119 Xanthines and Analogues 122 Pharmacological Comparison of DPP-IV Inhibitors 125 Concluding Remarks 127 References 128

5.1 5.2 5.3 5.4 5.5 5.6 5.7

6 6.1 6.2 6.2.1 6.2.2 6.2.3 6.2.4 6.3 6.3.1 6.3.2

107

Phosphodiesterase 5 Inhibitors to Treat Erectile Dysfunction 135 Nils Griebenow, Helmut Haning, and Erwin Bischoff Introduction 135 Pharmacology of Phosphodiesterases 136 The Phosphodiesterase Family 136 Pharmacological Effects of cGMP 137 PDE5: Regulation, Activation, and Structure 138 PDE5 Inhibitors and Erectile Dysfunction 143 Pyrimidinone PDE5 Inhibitors 147 Xanthines and cGMP Analogues 147 PDE5 Inhibitors Incorporating the Purinone Nucleus 150

VII

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Contents

6.3.2.1 6.3.2.2 6.3.3 6.3.3.1 6.3.3.2 6.3.4 6.3.5 6.3.6 6.3.7 6.4 6.4.1 6.5

Zaprinast 150 Purinones 150 Pyrazolopyrimidinone PDE5 Inhibitors 151 Pyrazolo[3,4-d]Pyrimidin-4-One PDE5 Inhibitors 151 1,6-Dihydro-7H-Pyrazolo[4,3-d]Pyrimidin-4-One PDE5 Inhibitors 152 Imidazotriazinone PDE5 Inhibitors 154 Imidazoquinazolinones 155 Pyrazolopyridopyrimidines 156 Miscellaneous Heterocylic-Fused Pyrimidinone PDE5 Inhibitors 156 Nonpyrimidone PDE5 Inhibitors 160 Hexahydropyrazino-Pyrido-Indole-1,4-Diones 160 Conclusions 162 References 162

7

Rifamycins, Antibacterial Antibiotics and Their New Applications 173 Enrico Selva and Giancarlo Lancini Discovery of the Pioneer Drug 173 Clinically Used Rifamycins 173 Mode of Action of Rifamycins and Structural Requirements for Activity 174 Modulation of Chemotherapeutic Properties 177 Profiles of Rifamycins Targeted at Tuberculosis Treatment 177 Rifampicin (INN), Rifampin (USAN) 178 Rifapentine 180 Rifabutin 181 Rifamycins Beyond Tuberculosis 181 Rifamycin SV and Rifamide 182 Rifaximin 182 Trials for Other Therapeutic Indications 183 Summary 183 References 184

7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 7.10 7.11 7.12 7.13

8 8.1 8.2 8.2.1 8.2.1.1 8.2.2 8.2.3 8.2.3.1 8.2.3.2 8.3 8.3.1

Monoterpenoid Indole Alkaloids, CNS and Anticancer Drugs 189 András Nemes Introduction 189 Vincamine and Derivatives: Cerebrovascular and Neuroprotective Agents 190 Medicinal Chemistry of Vincamine Derivatives 190 Structure–Activity Relationships 192 Synthesis of Vincamine Derivatives 193 Pharmacological Properties of Vincamine Derivatives 193 Mechanism of Action 193 Clinical Pharmacology 194 Antitumor Dimeric Vinca Alkaloids 195 Medicinal Chemistry of Dimeric Vinca Alkaloid Derivatives 195

Contents

8.3.1.1 8.3.2 8.3.3 8.3.3.1 8.3.3.2 8.4 8.4.1 8.4.1.1 8.4.2 8.4.3 8.4.3.1 8.4.3.2 8.5

Structure–Activity Relationships 196 Synthesis of Dimeric Vinca Alkaloid Derivatives 198 Pharmacological Properties of Dimeric Vinca Alkaloid Derivatives Mechanism of Action 199 Clinical Pharmacology 199 Antitumor Camptothecin Derivatives 201 Medicinal Chemistry of Camptothecin Derivatives 201 Structure–Activity Relationships 202 Synthesis of Camptothecin Derivatives 203 Pharmacological Properties of Camptothecin Derivatives 204 Mechanism of Action 204 Clinical Pharmacology 205 Summary and Conclusions 207 References 207

9

Anthracyclines, Optimizing Anticancer Analogues 217 Federico-Maria Arcamone Introduction: Biosynthetic Antitumor Anthracyclines 217 Analogues with Modification of the Aminosugar Moiety 219 Analogues with Modifications in the Anthraquinone Moiety 223 Analogues Modified on Ring A of the Aglycone 226 Disaccharide Analogues 229 Other Compounds 232 Summary and Final Remarks 233 References 234

9.1 9.2 9.3 9.4 9.5 9.6 9.7

10 10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9

11

11.1 11.2

Paclitaxel and Epothilone Analogues, Anticancer Drugs 243 Paul W. Erhardt and Mohammad El-Dakdouki Introduction 243 Discovery and Development of Paclitaxel 243 Clinical Success and Shortcomings of Paclitaxel 245 ABDD Leading to Docetaxel 247 Additional Structural Analogues 249 The Pursuit of Microtubule-Stabilizing Pharmacological Analogues 250 The Epothilones 252 ABDD and Development Leading to Ixabepilone 258 Conclusions 260 References 263 Selective Serotonin Reuptake Inhibitors for the Treatment of Depression 269 Wayne E. Childers Jr. and David P. Rotella Introduction 269 Neurochemistry and Mechanism of Action 270

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11.3 11.3.1 11.3.2 11.3.3 11.3.4 11.3.5 11.4 11.4.1 11.4.2 11.4.3 11.4.4 11.4.5 11.5 11.6

Preclinical Pharmacology 271 Sertraline 271 Escitalopram 272 Fluvoxamine 273 Fluoxetine 274 Paroxetine 275 Medicinal Chemistry 276 Sertraline 276 Escitalopram 278 Fluvoxamine 279 Fluoxetine 281 Paroxetine 284 Comparison of SSRIs and Other Uses 285 Summary 288 References 288

12

Muscarinic Receptor Antagonists in the Treatment of COPD 297 Matthias Grauert, Michael P. Pieper, and Paola Casarosa Introduction 297 Muscarinic Receptor Subtypes 298 Structures of Muscarinic Agonists and Antagonists 299 Muscarinic Agonists 299 Antimuscarinics 300 Discovery of Quaternary Antimuscarinics 303 Once-Daily Quaternary Antimuscarinics: Tiotropium Bromide as the Gold Standard 305 Preclinical Pharmacology: Comparison of Ipratropium and Tiotropium 309 Bronchoconstriction in Conscious Guinea Pigs According to the Method of Kallos and Pagel 310 Bronchoconstriction in Anaesthetized Dogs 310 Clinical Pharmacology 311 Antimuscarinics in Clinical Development for the Treatment of COPD 313 Summary 313 References 314

12.1 12.2 12.3 12.3.1 12.3.2 12.3.3 12.3.4 12.4 12.4.1 12.4.2 12.5 12.6 12.7

13 13.1 13.2 13.3 13.4

b-Adrenoceptor Agonists and Asthma 319 Giovanni Gaviraghi Introduction 319 First-Generation b2-Agonists: The Short-Acting Bronchodilators Second-Generation b2-Agonists: Further Derivatives of Salbutamol 321 Third-Generation b2-Agonists: The Long-Acting Bronchodilators 321

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13.5 13.6

Combination Therapy with LABA and Corticosteroids 326 Future Directions: Once-a-Day Therapy and Bifunctional Muscarinic Antagonist–b2-Agonist (MABA) 327 References 329

Part III

Case Histories

14

Liraglutide, a GLP-1 Analogue to Treat Diabetes 335 Lotte B. Knudsen Introduction 335 Discussion 338 Physiology of Native GLP-1 338 Development of Liraglutide: A GLP-1 Analogue 339 The Pharmacology of Liraglutide 346 Clinical Evidence with Liraglutide 349 Summary 350 References 351

14.1 14.2 14.2.1 14.2.2 14.2.3 14.2.4 14.3

15 15.1 15.2 15.3 15.4 15.5 15.6 15.7

16 16.1 16.1.1 16.2 16.2.1 16.2.2 16.3 16.4 16.4.1 16.5 16.6 16.7 16.8 16.9 16.9.1

333

Eplerenone: Selective Aldosterone Antagonist 359 Jaroslav Kalvoda and Marc de Gasparo Introduction 359 Development of a Specific and Selective Aldosterone Antagonist 360 Eplerenone: Selectivity and Specificity 367 Preclinical Development of Eplerenone: From Animal to Man 373 Further Development of Eplerenone 375 Conclusions 376 Epilogue 376 References 377 Clevudine, to Treat Hepatitis B Viral Infection 383 Ashoke Sharon, Ashok K. Jha, and Chung K. Chu Current Status of Anti-HBV Agents 383 Nucleoside Reverse Transcriptase Inhibitors 386 Chemical Evolution of Clevudine 387 Development of Synthetic Routes 387 Structure–Activity Relationships 388 Metabolism and Mechanism of Action 390 Pharmacokinetics 392 Woodchuck Studies 393 Clinical Studies 394 Drug Resistance 396 Toxicity and Tolerability 398 Dosage and Administration 399 Combination Therapy 399 Combination of Clevudine with Other Agents 399

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16.9.2 16.10

Combination of Clevudine with Vaccine 400 Summary 400 References 401

17

Rilpivirine, a Non-nucleoside Reverse Transcriptase Inhibitor to Treat HIV-1 409 Jerome Guillemont, Luc Geeraert, Jan Heeres, and Paul J. Lewi Introduction 409 Chemistry 412 Synthesis of TMC278 and Close Analogues 412 Modulation of the Central Heterocycle Core 418 C-5 Substitution of the Pyrimidine Core 419 Structure–Activity Relationships 420 Introduction of G Spacer Between the Aryl Ring and the Cyano Group 421 Modulation of Substituents at C-2 and C-6 on the Left Wing and of the Linker Between Left Wing and Pyrimidine Core 423 Subsitution at C-5 Position of the Pyrimidine Heterocycle 424 Modification of the Central Heterocycle Core 426 TMC278: Physicochemical Properties 429 Modeling of TMC278 and Crystal Structure 430 Pharmacokinetic and Phase II Studies of TMC278 431 Conclusions 434 References 434

17.1 17.2 17.2.1 17.2.2 17.2.3 17.3 17.3.1 17.3.2 17.3.3 17.3.4 17.4 17.5 17.6 17.7

18

18.1 18.2 18.2.1 18.3 18.3.1 18.3.2 18.3.3 18.3.3.1 18.3.3.2 18.3.3.3 18.4 18.4.1 18.4.2 18.4.3 18.5 18.6

Tipranavir, a Non-Peptidic Protease Inhibitor for Multi-drug Resistant HIV 443 Suvit Thaisrivongs, Joseph W. Strohbach, and Steve R. Turner Human Immunodeficiency Virus 443 HIV Protease 443 HIV PIs 444 Approaches to Identifying and Developing PI Leads 446 Focused Screening 446 Broad Screening for Nonpeptidic Leads 447 Structure-Based Drug Design 448 PNU-96988, A First-Generation Clinical Candidate 449 PNU-103017, A Second-Generation Clinical Candidate 450 Tipranavir, The Third Generation 453 Characteristics of Tipranavir 454 In Vitro Activity 454 Pharmacokinetics 455 Highlights of Clinical Data 456 Fragment-Based Lead Development? 457 Summary 458 References 459

Contents

19 19.1 19.2 19.3 19.4 19.4.1 19.4.2 19.4.3 19.5 19.6 19.7

20

20.1 20.2 20.3

21

21.1 21.2 21.3 21.4 21.4.1 21.4.2 21.4.3 21.4.4 21.5 21.6 21.7 21.8 21.8.1 21.8.1.1 21.8.1.2 21.8.1.3 21.8.2

Lapatinib, an Anticancer Kinase Inhibitor 465 Karen Lackey Introduction 465 Aims 467 Chemical Evolution and Proof-of-Mechanistic Approach Using Small Molecules 469 Final Set of Analogues that Led to the Discovery of Lapatinib 6-Furanyl Quinazoline Series 474 6-Thiazolylquinazoline Series 479 Alkynylpyrimidine Series 480 Final Selection Criteria and Data 482 Early Clinical Results 487 Prospects for Kinase Inhibitors 489 References 490 Dasatinib, a Kinase Inhibitor to Treat Chronic Myelogenous Leukemia 493 Jagabandhu Das and Joel C. Barrish 493 Introduction 493 Discussion 494 Clinical Findings and Summary 502 References 503 Venlafaxine and Desvenlafaxine, Selective Norepinephrine and Serotonin Reuptake Inhibitors to Treat Major Depressive Disorder 507 Magid Abou-Gharbia and Wayne E. Childers Jr. Introduction 507 Major Depressive Disorder 510 MDD Pharmacotherapy 511 The Discovery of Venlafaxine 511 Identification of an Early Lead (WY-44362) 511 Structure–Activity Relationship Studies 512 In Vivo Animal Models of Preclinical Efficacy 514 Selection of WY-45030 for Clinical Trials 514 Clinical Efficacy of Effexor1 515 An Extended Release Formulation – Effexor XR1 516 Discovery of a Second-Generation SNRI – O-Desmethylvanlafaxine 516 Effexor and Pristiq – Additional Considerations 518 Effexor 518 Onset of Action 518 Treatment of Some Anxiety Disorders 519 Painful Somatic Symptoms 519 Pristiq 519

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21.8.2.1 21.8.2.2 21.9

Anxiety and Painful Symptoms 519 Symptoms Associated with Menopause Conclusions 520 References 520 Index

525

519

XV

Preface The positive response to the first volume stimulated the editors to continue beyond the well-received book. Three very important facts supported this feeling. All copies of the book ‘‘Analogue-Based Drug Discovery’’ were sold within 18 months after its publication in February 2006. 2) The Journal of Medicinal Chemistry in its very positive review recommended the book for teaching of medicinal chemistry. 3) Last, but not the least Wiley-VCH, and, personally, Dr. Frank O. Weinreich welcomed the idea of the continuation.

1)

We started to collect new topics at the beginning of 2008. We have continued to study the general aspects of ‘‘Analogue-Based Drug Discovery’’ with the help of the chapters that describe how analogues optimize drug therapy. In a separate chapter on standalone drugs, we demonstrate that in the case of a minor number of drugs, the pioneer drug could not (or not yet) be optimized. These standalone drugs can always challenge the medicinal chemistry researchers because, as existing drugs, they can serve as starting points for researchers. We are grateful again to the IUPAC (International Union of Pure and Applied Chemistry), which supported this activity in projects. The Subcommittee for Medicinal Chemistry and Drug Development and the Division of Chemistry and Human Health provided the opportunity to the editors to discuss this work with other experts of medicinal chemistry. We are grateful for the participation of all the contributors. Many authors of the book played an important role as inventors who discovered valuable drugs, and their chapters carry a high credibility either as an analogue class study or as a case history of a drug. We are very much obliged to the helpful reviewing work done by many colleagues, whose names are as follows: Karl-H. Baringhaus, Jozsef Bódi, Derek Buckle, Mark Bunnage, Duane Burnett, Neal Castagnoli, Jonathan B. Chaires, Mukund Chorghade, Erik De Clercq, Duncan Curley, György Domány, Joelle Dubois, Andrew Fensome, Tom Heightman, Bastian Hengerer, Duy H. Hua, Robert Jones, Dale

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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Kempf, Karsten Krohn, K.H. Lee, John Lowe III, Frank C. Odds, Eckhard Ottow, Tom Perun, István Polgár, Dominick Quagliato, Waldemar Priebe, Graham Robertson, Romano Silvestri, László Szabó, Károly Tihanyi, Edwin B. Villhauer, Niels Vrang, Richard White, Michael Williams, and Puwen Zhang. All these colleagues contributed to the quality of this second volume. We express special thanks to reviewers Derek Buckle, John Lowe III, Bruce E. Maryanoff, Lester A. Mitscher, and Dominick Quagliato, who each corrected the language, and Eckhard Ottow, who corrected the structures, of a whole chapter. Some authors, besides the editors, also served as reviewers. Our thanks are due to these authors and reviewers as follows: Giovanni Gaviraghi, John Proudfoot, and David Rotella. J.F. thanks the Alexander von Humboldt Foundation (Bonn) for a fellowship in 2008 and 2009. We hope that the second volume will also be well received and that it will contribute in some way to help the experts in drug discovery and students of medicinal chemistry. October 2009 Budapest and London

János Fischer and C. Robin Ganellin

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Introduction Janos Fischer and C. Robin Ganellin Analogy plays a very important role in scientific research and especially in applied research. This is certainly true for the medicinal chemist searching for new drugs to treat diseases. The chemical structure and the similarities and differences in chemical and biological properties between compounds help guide the researcher in deciding where to position a new molecule in comparison to what is already known about other compounds. Medicinal chemistry is a relatively “young” science that spanned the whole of the twentieth century. In the first half of the century, new drug research was dominated by organic chemistry, and researchers sought improved drugs among structurally similar compounds. Full analogues (see below) dominated this kind of research. The latter half of the century saw a much greater contribution from biochemistry and pharmacology, and many pioneer drugs were discovered. This opened the way for researchers to seek to improve upon these drugs by investigating analogues. The first volume of Analogue-Based Drug Discovery focused on an important segment of medicinal chemistry, where an existing drug was selected as a lead compound and the research had, as a goal, to improve upon the lead by synthesizing and testing analogues. The chemical structure and main biological activity of such an analogue were often similar to the lead drug. Thus, the researchers generally sought a structural and pharmacological analogue (more simply called a full analogue) or if the pharmacophores were the same, a direct analogue. Usually, the aim was to achieve an improved biological activity profile, with a greater potency. The first volume included a description of many well-established analogue classes of drug that are indispensable nowadays for the treatment of peptic ulcer disease, gastroesophageal reflux disease, prevention of cardiovascular diseases (e.g., antihypertensives, cholesterol-lowering agents, calcium antagonists, and beta-adrenergic receptor blocking agents), pain (e.g., opioid analgesics), and many other diseases. The last two decades, however, have witnessed great changes in the chemical and biological methods for generating a lead compound. Combinatorial chemistry affords many more compounds than traditional synthetic methods and these are tested very rapidly by high-throughput screening (HTS) to deliver new hit and lead molecules. This procedure often paves the way for new types of structures for drug research thereby decreasing the importance of having chemical similarity. At the Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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Introduction

same time, this provides a better opportunity for novelty and therefore for patenting. This also gives rise to a greater need to compare the biological properties of these new lead compounds in order to arrive at the best pharmacological analogue. Analogue-based drug discovery (ABDD) is not a simple research method, but it is a way of thinking that, in addition to organic synthesis, uses most of the procedures that are now available to medicinal chemists, such as i. ii. iii. iv. v.

investigation of structure–activity relationships, molecular modeling, structure-based drug discovery, fragment-based drug discovery, early recognition of drug distribution properties and avoidance of potential toxicities.

Analogue-based drug discovery has the merit that the therapeutic target is already validated, but it carries the hazard of potentially losing out to competitors who may start from the same approach at about the same time. This second volume of Analogue-Based Drug Discovery has a broader scope than the first volume. The book not only contains descriptions of full analogues but also includes several pharmacological analogues. The book is divided into three parts: 1) 2) 3)

General Aspects of Analogue-Based Drug Discovery Analogue Classes Case Histories

General Aspects

The opening chapter summarizes various possibilities exemplifying how the properties of a drug may be modified to give a new drug analogue that improves patient drug therapy. There are 12 principles exemplified and within some of these principles there are several methods; hence this chapter gives a broad overview. A small number of the pioneer drugs remain without successful analogues; we describe these by the term standalone drugs. Among the most frequently used 100 drugs, 9 such standalone drugs can be identified. Their history and present situation may be used to initiate a new research activity to make their analogues. In addition to the traditional structure–activity relationship (SAR) studies, molecular modeling is the most important method that can help the medicinal chemist to find a new drug analogue. The chapter discusses several useful examples of molecular modeling in analogue research. Patenting activity is one of the basic tasks of drug research. Patents mostly concern a group of direct analogues; therefore, the first claim of a patent contains a general structure that describes this group of compounds. The chapter gives an overview of some of the issues that can affect the commercial protection of the discoveries made by medicinal chemists.

Case Histories

Analogue Classes

The discovery of dipeptidyl peptidase IV inhibitors has opened a promising chapter for the treatment of type 2 diabetes. The pioneer drug sitagliptin has been followed by several analogues in order to obtain more potent and longer acting derivatives. Serendipitous clinical observation afforded the pioneer drug sildenafil. Several analogues have been found that have optimized its properties (e.g., selectivity, duration of action). Rifamycins are antibacterial antibiotics derived from fermentation. Analoguebased drug research afforded more potent derivatives. One of the derivatives, the poorly absorbed rifaximin, has a promising application for the treatment of irritable bowel syndrome. Three analogue classes of monoterpenoid indole alkaloids are discussed: (i) vincamine derivatives, (ii) dimeric vinca alkaloid analogues, and (iii) camptothecin analogues. The successful natural product direct analogues are applied for the treatment of cerebral insufficiencies and cancer. The natural product doxorubicin is an anthracycline antibiotic used to treat a wide range of cancers, but it has a cardiotoxic adverse effect. The research into direct analogues had a goal to obtain drugs with a better therapeutic index. Paclitaxel and epothilone analogues are also examples of how natural product drugs can be used to initiate analogue-based drug research to afford new drug analogues with better properties as anticancer agents. The selective serotonin reuptake inhibitors (SSRIs) are pharmacological analogues that revolutionized antidepressant therapy. The structurally different SSRIs have different profiles for inhibiting uptake of the neurotransmitters: serotonin, dopamine, and norepinephrine. The modification of naturally occurring tropane alkaloids afforded the quaternary ammonium salts ipratropium and tiotropium, which are important drugs used for treating chronic obstructive pulmonary disease. Tiotropium, as a result of analogue-based drug discovery, has a longer duration of action that enables a once-daily dosing. The natural product adrenaline (epinephrine) was the starting point for drug research into b-adrenoreceptor agonists. From isoprenaline (isoproterenol) through the selectively acting salbutamol, and on to salmeterol, analogue research resulted in selective, more potent, and longer acting analogues with different PK profiles, which are important drugs in asthma therapy.

Case Histories

Eight case histories are described by their inventors. Liraglutide is a new antidiabetic drug, an analogue of the natural product glucagonlike peptide 1. Among the acylated GLP-1 analogues liraglutide has been developed for a once-daily treatment.

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Eplerenone is a spironolactone analogue for treating hypertension that has a greater selectivity for the mineralocorticoid receptor and reduced sexual side effects. Clevudine is a new drug for the treatment of the chronic hepatitis B virus (HBV) infection, which belongs to the class of nucleoside reverse transcriptase inhibitors. Tipranavir is a new anti-HIV agent that is a protease inhibitor. The discovery of tipranavir used structure-based and fragment-based drug design and its long discovery process started from warfarin, which is a weak HIV-1 protease inhibitor. Dasatinib can be regarded as a pharmacological analogue of imatinib. Dasatinib is more potent and it can be used in imatinib-resistant cases for the treatment of chronic myelogenous leukemia (CML). Lapatinib can be regarded as a pharmacological analogue of erlotinib. It is a tyrosine kinase inhibitor and was first approved for the treatment of solid tumors such as in breast cancer. Venlafaxine is the first marketed serotonin/norepinephrine reuptake inhibitor (SNRI) and is used for the treatment of deep depression. Its active metabolite is desvenlafaxine, which has some advantageous properties; for example, it has a more favorable metabolic profile compared to venlafaxine. Rilpivirine is a new HIV-1 nonnucleoside reverse transcriptase inhibitor (NNRTI), an analogue of etravirine. Rilpivirine is highly potent also against strains that are resistant to the first-generation NNRTI drugs. The first volume of Analogue-Based Drug Discovery discussed mostly well-established drugs. This second volume also opens the door to new drug discoveries and the editors hope that, like the first volume, all of the drugs discussed in this book will have a bright future.

XXI

Abbreviations ABC ABDD ABPM ACAT ACE ACTH ADMET AFC AIDS ALT ALL AMP cAMP ANDA a-APA APV AR ATP AUC AZT BBB Bcr-Abl BG b.i.d. BOC CBF CC50 b-CCE CGI CHB CK CL CLR

ATP binding cassette analogue-based drug discovery ambulatory blood pressure monitoring acyl-CoA:cholesterol acyltransferase angiotensin-converting enzyme adrenocorticotropic hormone absorption, distribution, metabolism, excretion and toxicity 7-amino-4-trifluoromethylcoumarin acquired immunodeficiency syndrome alanine aminotransferase acute lymphoblastic leukemia amprenavir cyclic 30 ,50 -adenosine monophosphate Abbreviated New Drug Application a-anilinophenylacetamide amprenavir androgen receptor adenosine triphosphate area under the curve azidothymidine blood-brain-barrier Breakpoint cluster region - Abelson blood glucose twice a day (from Latin bis in die) t-butoxycarbonyl cerebral blood flow 50% cytotoxic concentration ethyl b-carboline-3-carboxylate Clinical Global Impressions Scale chronic hepatitis B creatine kinase clearance renal clearance

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

XXII

Abbreviations

CLT CLV CLV-TP CML CMRglc CNS COBP COPD COX-1 COX-2 CPI/r CPT CRC CYP DA 10-DAB DAPY DATA dCK DNA cDNA cccDNA mtDNA DOC DOCA DPP-4 DSM-III EBV EC50 ED EFS EGFR EMEA EPA EPO EPS Erk ETC FAAH FBDD FDA L-FEAU FEV L-FMAU

total clearance clevudine clevudine triphosphate chronic myelogenogenous leukemia cerebral metabolic rate of glucose central nervous system chronic obstructive broncho-pneumopathies chronic obstructive pulmonary disease cyclooxygenase-1 cyclooxygenase-2 comparator protease inhibitor boosted with ritonavir camptothecin colorectal cancer cytochrome P450 isoenzyme dopamine 10-deacetyl-baccatin diarylpyrimidine diaryltriazine deoxycytidine kinase desoxyribonucleic acid complementary deoxyribonucleic acid covalently closed circular DNA mitochondrial DNA deoxycorticosterone deoxycorticosterone acetate dipeptidyl peptidase 4 Diagnostic and Statistical Manual of Mental Disorders, third edition Epstein-Barr virus effective concentration 50 erectile dysfunction electric field stimulation epidermal growth factor receptor European Medicines Agency Environmental Protection Agency European Patent Office exprapyramidal side effect extracellularly regulated kinase emtricitabine fatty acid amide hydrolase fragment-based drug design Food and Drug Administration 1-(20 -deoxy-20 -fluoro-b-L-arabinofuranosyl)-5-ethyluridine forced expiratory volume L-20 -fluoro-5-methyl-b-L-arabinofuranosyluracil

Abbreviations

GABAA GAD GI GIP GLP-1 cGMP GPIIb/IIIa HA HAART HA/ACTH HAM-A HAM-D HbA1c HBV HBcAg HBeAg HbsAg HCC HCV HDV hERG HFB HIAA HIV HIV PR HMG-CoA 5-HT 5-HTP HTS IBMX IC50 pIC50 ICS IDR IDV i.m. IND INN IOPY i.p. i.v. Ki LABA Lck hLck

gamma-aminobutyric acid A generalized anxiety disorder growth inhibition glucose-dependent insulinotropic polypeptide glucagon-like peptide-1 cyclic 30 ,50 -guanosine monophosphate glycoprotein IIb/IIIa heavy atom Highly Active Antiretroviral Therapy histamine-induced adrenocorticotropic hormone Hamilton Anxiety Taring Scale Hamilton Depression Rating Scale glycosylated haemoglobin hepatitis B virus hepatitis B core antigen hepatitis B e antigen hepatitis B surface antigen hepatocellular carcinoma hepatitis C virus hepatitis delta virus human ether-a-go-go-related gene human foreskin fibroblast 5-hydroxy-indole acetic acid human immunodeficiency virus HIV protease 3-hydroxy-3-methylglutaryl coenzyme A 5-hydroxytryptamine (serotonin) 5-hydroxytryptophan high-throughput screening isobutylmethylxanthine inhibitory concentration 50
log IC50 inhaled corticosteroids idarubicin indinavir intramuscular Investigational New Drug International Nonproprietary Name iodophenoxypyridone intraperitoneal intravenous inhibitory constant long-acting b2-agonist lymphocyte specific kinase human Lck

XXIII

XXIV

Abbreviations

mLck LDL-C LE LPV LVEF MADRS MAOI M1 MAP rMD MDD MDR MED MES MIC MR MRP MTD NAPQI NCE NCI NDA NE NMR NNRTI NO NPs NPC1L1 NRIs NRTI NSAIDs NSCLC OADs OC OCD OGTT PCA PCF PCT PDEs PDGFR PEP PGE1 PGE2 P-gp

murine Lck low-density lipoprotein-cholesterol ligand efficiency lopinavir left ventricular ejection fraction Montgomery-Asberg Depression Rating Scale monoamine oxidase inhibitor muscarinic receptor M1 subtype mitogen-activated protein restrained molecular dynamics major depressive disorder multidrug resistance minimal effective dose maximal electroshock seizure minimal inhibitory concentration mineralocorticoid receptor multidrug resistance-associated protein maximum tolerated dose N-acetyl-p-benzoquinone imine New Chemical Entity National Cancer Institute New Drug Application norepinephrine nuclear magnetic resonance nonnucleoside reverse transcriptase inhibitor nitric oxide natural products Niemann-Pick C1-Like-1 norepinephrine reuptake inhibitors nucleoside reverse transcriptase inhibitor nonsteroidal anti-inflammatory drugs non-small cell lung cancer oral antidiabetic drugs ovarian cancer obsessive-compulsive disorder oral glucose tolerance test p-chloroamphetamine plant cell fermentation Patent Cooperation Treaty phosphodiesterases platelet derived growth factor receptor prolyl endopeptidase prostaglandin E1 prostaglandin E2 permeability glyocoprotein

Abbreviations

Ph (+) PK PKG POMS PPCE PR QSAR q.d. or QD RBA RGD RNA RNApol mRNA RT RTV SAR SBDD s.c. SCID SCLC SEDDS SEF SI SIV SMC SNRI SQV Src SRI SSRIs TCR TDF TGFa TI TIBO t.i.d. TK TMPK TPV TPV/r TPT TRIPs TTP UDP

Philadelphia chromosome positive pharmakokinetic protein kinase G profile of mood state postproline cleaving enzyme progesterone receptor quantitative structure-activity relationship once a day (from Latin quaque die) relative binding affinity arginine-glycine-aspartic acid ribonucleic acid RNA polymerase messenger RNA reverse transcriptase ritonavir structure-activity relationship structure-based drug design subcutaneous severe combined immunodeficient small-cell lung cancer self-emulsifying drug delivery system sodium excreting factor selectivity index simian immunodeficiency virus smooth muscle cell serotonin/norepinephrine reuptake inhibitor saquinavir sarcoma serotonin reuptake inhibitor selective serotonin reuptake inhibitors T-cell antigen receptor tenofovir disoproxil fumarate tansforming growth factor-a tumor inhibition 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk]benzodiazepin-2 (1H)-one three times daily thymidine kinase thymidylate kinase tipranavir tipranavir/ritonavir combination topotecan Trade-related Aspects of Intellectual Property Rights time to progression uridine diphosphate

XXV

XXVI

Abbreviations

UGT USAN VEGFR VMS VSMC VSS WBC WHcAg WHsAg WHV WTO

uridine diphosphate glucuronyl transferase United States Adopted Names vascular endothelial growth factor receptor vasomotor symptoms vascular smooth muscle cell steady-state volume white blood cell woodchick hepatitis virus core antigen woodchuck hepatitis virus surface antigen woodchuck hepatitis virus World Trade Organization

Part I General Aspects

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

j3

1 Optimizing Drug Therapy by Analogues J
anos Fischer, C. Robin Ganellin, John Proudfoot, and Erika M. Alapi The medicinal chemist has a complex task to discover a useful new drug molecule. Among the different ways to accomplish this, the use of analogue research has been the most successful over the years, and most of the improvements in drugs have been obtained this way. This chapter will summarize the main aspects and demonstrate how analogues in a class of drugs are used to optimize drug therapy.

1.1 Introduction

The term analogue (from the Greek analogos) means proportionate, but in everyday terms we use it to indicate similarities between things. In medicinal chemistry, an analogue drug [1] has a chemical and/or pharmacological relationship with another drug. Structural analogues are drugs that have a similar chemical structure but quite different pharmacological properties, whereas pharmacological analogues are drugs that have a similar pharmacological activity without any discernible chemical or structural relationship. About half the drugs are analogues in both respects, and we therefore call them full analogues that are structural and pharmacological analogues. One special class of full analogues is considered to be direct analogues if they have identical pharmacophores – that is, they can be described by a general structure that includes most of the chemical skeleton. There are only a few drugs for which no successful analogues have been discovered, these we have termed standalone drugs [2]. Because of the experimental nature of structure–activity relationships (SARs), the term analogue is descriptive and not exact, nevertheless it is a useful tool in classifying the 6000 different drugs used nowadays. An analogue class is a group of drugs that displays similar in vitro and in vivo pharmacological properties; therefore, they are either pharmacological or full analogues. The first drug in the class (often termed “first in class”) used as a lead for the development of analogues can be considered a “pioneer drug.” A pioneer drug is therefore the first marketed drug in an analogue class. After the discovery of a pioneer

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

j 1 Optimizing Drug Therapy by Analogues

4

drug, there are almost always efforts to improve upon it with analogues in order to obtain new drugs with better therapeutic properties. A drug can be characterized by several chemical and pharmacological properties. Even a minor structural change can sometimes modify all these properties. In order to illustrate key concepts in the optimization of drug therapy by analogues, it is necessary to simplify this question and we will always focus on a dominant property in a given analogue class.

1.2 Pharmacodynamic Characteristics

Pharmacodynamics refers to the study of drug actions in living organisms and includes the dominant activity along with possible adverse effects. 1.2.1 Potency

The potency of a drug refers to the amount of drug required to achieve a defined biological activity. The smaller the dose required, the more potent the drug. The discovery of the histamine H2-receptor antagonist cimetidine in 1971 was a pioneer invention in the treatment and prevention of peptic ulcer and gastroesophagitis. Cimetidine is used in a daily dose of 800 mg. The application of analogue-based drug research provided ranitidine (1976), roxatidine (1979), famotidine (1979), and nizatidine (1980), which are more potent drugs and are effective at a lower dosage. Table 1.1 shows daily doses in comparison to cimetidine. Famotidine is the most potent member of the analogue class (Figure 1.1) [3]. Statins are HMG-CoA reductase inhibitors (Figure 1.2). Based on their in vitro effects, rosuvastatin is the most potent analogue and is followed in rank order by atorvastatin, simvastatin, and pravastatin [4] (Table 1.2). The same rank order of potency has been observed in clinical trials.

Table 1.1 Comparison of approximately equivalent daily doses of H2-receptor antagonists.

Drug cimetidine nizatidine ranitidine roxatidine famotidine a)

Daily dose (mg) 800 300 300 150 40

Administered as acetate hydrochloride.

Molecular weight (Da) 252 331 314 385a) 337

1.2 Pharmacodynamic Characteristics

j5

CH3

N H

NH

NH

N

S NC

CH3

H3C

CH3

N

O

NH

S

NH

N O 2N

cimetidine

ranitidine

N

S O

NH

N

O

N

S

N

H2N

NH2 O

NH2

O CH3

O

roxatidine

famotidine

CH3 H3C

N

N

NH

S

S

NH

CH3

O 2N

nizatidine Figure 1.1 Structures of H2-receptor antagonists.

1.2.2 Improving the Ratio of Main Activity and Adverse Effects

There are no drugs without some adverse effects. One important goal of analoguebased discovery approaches is to design improved drugs with a better ratio of efficacy to adverse effects.

Table 1.2 Inhibitory effects of various statins in vitro.

Drug pravastatin simvastatin atorvastatin rosuvastatin

CH3

IC50 (nM)

Molecular weight (Da)

44.1 11.2 8.2 5.4

424 419 559 482

O S

NH2

j 1 Optimizing Drug Therapy by Analogues

6

HO

O

HO

O

O O H3C

OH

O O

H3C

OH

O

H3C

H

CH3

CH3

CH3

H3C

H

CH3

HO pravastatin

simvastatin

HO

HO

OH

OH O

O

OH

OH F

F CH3

CH3 N CH3

CH3 N

N H3C O

S

N O

NH O

CH3

rosuvastatin

atorvastatin

Figure 1.2 Structures of HMG-CoA reductase inhibitors.

1.2.2.1 Improving Selectivity Through Receptor Subtypes The opportunity for improvement is clear if the mechanism of the adverse effect is known and one such example is found in the case of the adrenergic b-receptor blockers (Figure 1.3). The pioneer drug of b-receptor blockers is propranolol, invented in 1962. Subsequently, it was discovered that b-receptors occur as subtypes, for example, b1 (in the heart) and b2 (mediating smooth muscle relaxation). Propranolol blocks both b1- and b2-receptors. However, blocking b2-receptors in bronchitis and asthma can be harmful, and analogue research successfully focused on producing selective b1-blockers. The first b1-selective blocker practolol was invented in 1964, but it was withdrawn from the market due to an unusual side effect, an oculomucocutaneous reaction that can lead to blindness. This is fortunately not a class effect, and many other selective blockers were developed and used in cardiology, such as atenolol, betaxolol, metoprolol, celiprolol, nebivolol, and bisoprolol [5] as b1-selective blockers.

1.2 Pharmacodynamic Characteristics OH

OH O

NH

CH3 CH3

O H3C

CH3

NH

O

CH3

NH

practolol

propranolol

OH

OH O H3C

j7

NH

CH3 CH3

O

O

CH3

H2N

metoprolol

atenolol

OH

O

OH

CH3

NH

O

CH3

NH

O

NH

O

CH3

CH3 CH3

O O H3C

H3C

H3C

N

O

OH O

O H3C

betaxolol

bisoprolol

CH3

NH

CH3 CH3 CH3

NH

O

celiprolol

H

OH

OH NH

O H

H

F

F

nebivolol

Figure 1.3 Structures of adrenergic b-receptor blockers.

1.2.2.2 Improving Selectivity Through Unrelated Receptors Treatment with cimetidine for antiulcer therapy, the pioneer H2-receptor histamine antagonist drug, resulted in a low incidence of gynecomastia as an unwanted side effect. This was traced to a low level of antiandrogenic activity. Thus, cimetidine was shown to competitively inhibit the binding of 3 ½H
dihydrotestosterone to its cytoplasmic receptor and to decrease its specific nuclear uptake in rat ventral prostate slices [6]. Subsequent H2-receptor antagonist analogues were an improvement since they generally did not show this effect. 1.2.2.3 Improving Selectivity by Tissue Distribution Antihistamines, useful in allaying the symptoms of allergic responses such as rhinitis and itching eyes when the pollen count is high, have been in general use for over 60 years. Their use has, however, been somewhat limited by a high incidence of drowsiness or sedation. Early attempts to separate the sedative effects from the antihistaminic action were not very successful, and it took many years until it was realized that these activities were connected. In particular, the work of Schwartz and

j 1 Optimizing Drug Therapy by Analogues

8

F

HO

N

OH

NH

O

Cl

haloperidol

azacyclonol

Figure 1.4 Structures of haloperidol and azacyclonol.

his colleagues in Paris demonstrated that histamine is a neurotransmitter in histaminergic nerves in the central nervous system (CNS) [7]. Histamine, acting on the histamine H1-receptor in the brain, was shown to be a stimulant of wakefulness; blocking these receptors leads to a loss of alertness resulting in drowsiness. Some attempts were made to synthesize compounds that were selective for the peripheral versus the central H1-receptors. Although such claims were made, they were not substantiated and, indeed, the use of molecular biology has demonstrated that there is no difference between the central and peripheral histamine H1receptors. There are some differences in the H1-receptors between species but not within a species. It follows that if it is not possible to separate the activity between peripheral and central histamine H1-receptors, then it is necessary to limit the access of antihistamine drugs to the CNS. The first compound to establish itself on this basis was terfenadine, but it was not originally developed as an antihistamine. Terfenadine was developed for CNS actions (dopamine antagonism) and calcium ion-channel blocking [8]. It was chemically related (as a combination of analogues) to haloperidol and azacyclonol (Figure 1.4) but found to be restricted to peripheral systems and to act as an antihistamine; indeed, it became the first member of a new class of drugs identified as nonsedative antihistamines and was very successful. This was followed by astemizole and then by other drugs that were synthesized as analogues of previously established active but sedating antihistamines (see Table 1.3 and Figure 1.5 for some examples). Astemizole arose from a research program at Janssen Pharmaceutica reportedly aimed at antihistamines having a long duration of action and a low risk of provoking central and anticholinergic effects [9]. Some of the above-mentioned products were later withdrawn because of an unwelcome side effect on heart caused by the blockade of the hERG (human ether-a-go-go-related gene) potassium ion channel that gives rise to cardiac arrhythmias. This led to a search for new analogues. The reasons why the compounds have low concentrations in the brain are complex and not fully understood. Some important factors that determine low brain con-

1.2 Pharmacodynamic Characteristics

j9

OH Cl

O

N

N

N N OH O

O H3C OH

O H3C

R

H3C H3C

CH3

cetirizine

ebastine

R = Me, terfenadine R = COOH, fexofenadine Cl CH3

N

N O

N HO

N

O

O

loratadine

acrivastine

CH3

F

F

N

N

N

N NH

N N

H3C

N

N HN

O O

mizolastine Figure 1.5 Structures of H1-antihistamines.

CH3

astemizole

j 1 Optimizing Drug Therapy by Analogues

10

Table 1.3 H1-antihistamines that have low incidence of sedative effects.

Drug

Type

Launch

terfenadine astemizole cetirizine [10] acrivastine [11] loratadine [12] ebastine [13] fexofenadine mizolastine

pioneer, chance discovery pharmacological analogue metabolite of hydroxizine analogue of triprolidine analogue of azatidine analogue of diphenylpyraline and terfenadine metabolite of terfenadine analogue of astemizole and temelastine

1981 (withdrawn, 1997) 1983 (withdrawn, 1999) 1987 1988 1988 1990 1996 1998

centrations are (i) high binding to brain tissue proteins, (ii) poor penetration of the blood–brain barrier, and (iii) high binding to the P-glycoprotein (Pgp) efflux pump. 1.2.2.4 Improving Selectivity of Nonreceptor-Mediated Effects In many cases, the mechanisms causing side effects are not known. Platinum compounds (Figure 1.6) play a major role in oncology. Cisplatin may cause regression and control of various tumors, such as testicular, ovarian, head, neck, and colon carcinoma. However, among its side effects, renal damage can be observed. Analogues have overcome this problem; thus, for carboplatin, the nephrotoxicity is much lower [14], and oxaliplatin is devoid of nephrotoxicity [15].

O H3N H3N

Cl Pt Cl

H3N H3N

O

O Pt

Pt O O

cisplatin

O

H2 N

carboplatin

N H2

O O

oxaliplatin

Figure 1.6 Structures of platinum compounds.

1.2.3 Improving the Physicochemical Properties with Analogues

Benzylpenicillin (penicillin G) (Figure 1.7), which is acid-sensitive, is rapidly destroyed by gastric fluid at pH 2. The pioneer penicillin drug was administered by intramuscular injection to avoid this problem, but this was not convenient for the patients. Through analogues, where an electron-withdrawing substituent was introduced in the side chain, the acid sensitivity of the b-lactam ring was reduced. A range

1.2 Pharmacodynamic Characteristics

j11

NH2

NH

S

O

CH3

N

O

CH3

O

NH O R

O

OH

S N

CH3 CH3

O

OH

ampicillin, R = H amoxicillin, R = OH

benzylpenicillin

CH3

O N

H3C

NH

R1 R

O O

S N O

O

CH3

NH

S

CH3 OH

H3C

O

O

N O O

R = H, R1 = H, oxacillin R = H, R1 = Cl, cloxacillin

methicillin

R = F, R1 = Cl, flucloxacillin R = Cl, R1 = Cl, dicloxacillin Figure 1.7 Benzylpenicillin and analogues.

of such analogues proved to be resistant to acid hydrolysis and they could be given orally (e.g., ampicillin [16]). 1.2.4 Analogues to Reduce the Resistance to Anti-Infective Drugs 1.2.4.1 Antibiotics Resistance to antibiotics has become an increasing problem all over the world, and the need to find new agents continues [17]. The widespread use of penicillin G led to an alarming increase of penicillinresistant Staphylococcus aureus infections in 1960. A solution to the problem was the design of penicillinase-resistant penicillins. The first such analogue was methicillin [18]; however, it was acid-sensitive and it was inactive against Gramnegative bacteria. Methicillin is no longer used clinically because better analogues (Figure 1.7) have been discovered, such as oxacillin [19], cloxacillin [20], flucloxacillin [21], and dicloxacillin [21] that were stable to b-lactamase enzyme of S. aureus and had acid stability.

CH3 CH3 OH

j 1 Optimizing Drug Therapy by Analogues

12

OH CH3

OH N N

N N

N

N

N N

F

N

N

F

F fluconazole

F

N

F voriconazole

Figure 1.8 Structures of fluconazole and voriconazole.

1.2.4.2 Antifungal Drugs A very important subclass of antifungal drugs is the azoles. Until the 1980s, while these drugs were used only topically, no resistance was detected. Since the introduction of the systemically active azoles, resistance has emerged. Even in the case of fluconazole, which is the most potent member of this analogue class, a resistance was observed in Candida infections. In this case, voriconazole, an analogue of fluconazole (Figure 1.8), has been used with a good result [22]. 1.2.4.3 Antiviral Drugs Although the first generation of anti-AIDS drugs provided key medicines for the treatment of the disease, the emergence of HIV-1 strains resistant to various drug classes prompted efforts to discover analogues with broader activity profiles. Successful analogue design approaches have led to improved pharmacological profiles that include suppression of viral resistance for two of the three classes where multiple drugs have reached the market, the nonnucleoside reverse transcriptase inhibitors (NNRTIs) and the protease inhibitors. For the nucleoside reverse transcriptase inhibitor class, the marketed drugs were generally discovered while generating analogues of zidovudine (stavudine, didanosine, tenofovir disoproxil, abacavir, and lamivudine) (Figure 1.9) with improved therapeutic index, and benefits with regard to resistance profiles were empirically uncovered [23]. Etravirine [24] (Figure 1.10), the most recent NNRTI to reach the market, is a pharmacological analogue of the first-generation NNRTIs and displays activity against a broad panel of drug-resistant reverse transcriptase (RT) mutants. Structurally, it bears no resemblance to any of the first-generation inhibitors and it emerged by iterative design starting with the earlier alpha-APA class over a period of many years. Optimization was guided throughout by structural information. Molecular conformational flexibility is apparently a key attribute that allows the molecule to effectively bind in multiple modes to the conformationally mobile NNRTI binding site of numerous clinically relevant RTmutants. The close structural analogue, rilpivirine [25], is in phase III trials.

1.2 Pharmacodynamic Characteristics

O

O CH3

HN O

CH3

HN N

O

N

O

O OH

N3

OH stavudine

zidovudine

NH2

O N

HN

N

N

N

N

O

N

O

P

N

O

O

CH3

O O

HO

H3C tenofovir disoproxil

didanosine

NH2 HN N

N

N

O

N

N

N

O S HO HO

abacavir

lamivudine

Figure 1.9 Structures of zidovudine and analogues.

O

O

O

O

H2N

j13

O CH3

CH3 CH3

j 1 Optimizing Drug Therapy by Analogues

14

CH3

CH3 O

N

NH

N

Br CH3

NC

NH

CN

NC

N

NH N

CH3

CN

NH2

rilpivirine

etravirine Figure 1.10 Structures of etravirine and rilpivirine.

Among the HIV-1 protease inhibitors, analogue approaches specifically attempting to improve resistance profiles have also given drugs that have reached the market. Cocrystal structure information of ritonavir (Figure 1.11), a first-generation protease inhibitor, bound to HIV-1 protease, was used to design lopinavir [26]. Removal of the isopropylthiazole group of ritonavir that interacts with valine-82 led to decreased sensitivity to the protease mutants selected by ritonavir. This modification, along with the modification of the other thiazole ring in ritonavir, also substantially improved the CYP inhibition profile of lopinavir. The design of amprenavir [27] and darunavir [28] (Figure 1.12) emphasized the importance of interactions between the inhibitors and the conserved elements of the

O

N

NH

NH

NH

S

N

O

NH

O

CH3

NH

O

CH3

CH3

H3C

OH

CH3

O

O

CH3

S

H3C

O

HO

N

ritonavir

H3C

N

NH

O CH3

lopinavir

Figure 1.11 Structures of ritonavir and lopinavir.

O O

O

O NH

N OH

O

H

O

O

S O

CH3

O H

NH2

N H

N

Figure 1.12 Structures of amprenavir and darunavir.

S

OH NH 2

CH3

amprenavir

O

O

darunavir

1.3 Pharmacokinetic Characteristics

N

N Cl

N H3C

O

NH

S

N

N

NH

NH

N

NH

N

N

N

N

O

CH3

CH3

CH3

dasatinib

imatinib

CF3 N

O N

N

H3C

NH

j15

NH

N

N CH3

nilotinib Figure 1.13 Structures of imatinib, dasatinib, and nilotinib.

protein backbone of the wild-type enzyme as a means to attaining excellent potency against mutant strains resistant to the first-generation inhibitors. 1.2.5 Analogue Research in Resistance to Drug Therapies in Cancer Treatment

Imatinib (Figure 1.13) is the pioneer drug for the treatment of chronic myologenous leukemia (CML). However, a significant number of patients develop resistance to imatinib. New analogues, such as dasatinib [29] and nilotinib [30], have been introduced recently, and it is hoped that these analogues will be effective also in imatinib-resistant cases.

1.3 Pharmacokinetic Characteristics

Pharmacokinetics is the study of the metabolism of drugs with particular emphasis on the time required for absorption, duration of action, distribution in the body, and method of excretion. Through analogue design, the pharmacokinetic parameters (ADME) of a pioneer drug or a drug class can be optimized. 1.3.1 Improving Oral Bioavailability

A good oral bioavailability is necessary in most cases because the oral application of a drug is preferred to an injection therapy.

OH

j 1 Optimizing Drug Therapy by Analogues

16

HO

O

NH2 HO

CH3

O

N NH

N

NH O

O

OH

enalaprilat

O

O

OH

lisinopril

Figure 1.14 Structures of enalaprilat and lisinopril.

1.3.1.1 Improving Absorption The angiotensin-converting enzyme (ACE) inhibitor enalaprilat (Figure 1.14) is not orally absorbed but is available for intravenous administration when oral therapy is not appropriate, for example, in hypertensive emergencies. The ethyl ester prodrug, enalapril has an excellent oral bioavailability but requires hydrolysis by esterases. The analogue-based drug research afforded the lysylproline analogue, lisinopril [31], which has an acceptable bioavailability and it does not require metabolic activation. 1.3.1.2 Improving Metabolic Stability The pioneer antifungal miconazole (Figure 1.15) and its analogue tioconazole [32] are clinically effective drugs, when administered by the topical route, against fungal infections of vagina and skin. Unfortunately, tioconazole and other imidazole derivatives of that time showed only poor efficacy in animal models of fungal infection when given by either the intravenous or the oral routes. Pharmacokinetic studies indicated that these agents were very susceptible to metabolic inactivation, resulting in low oral bioavailability and low plasma levels. They were also very lipophilic and highly bound to plasma proteins, which resulted in very low circulating levels of the unbound, active form. The first orally active antifungal drug was ketoconazole [33], which was discovered at Janssen Pharmaceutica. Ketoconazole was metabolically less susceptible than earlier imidazole derivatives, resulting in good oral bioavailability; however, it was metabolized such that less than 1% of unchanged drug was excreted through urine. In addition, although ketoconazole was less lipophilic than the earlier derivatives, leading to high blood levels, it remained highly protein-bound with less than 1% in unbound form. Introducing a polar hydroxyl group and a more polar 1,2,4-triazole ring led to UK47265. It was 100 times more potent than ketoconazole when dosed via either the oral or the intravenous routes; however, its in vitro activity against fungi was modest. Unfortunately, UK-47265 proved to be hepatotoxic in mice and dogs and teratogenic in rats; therefore, it was not developed further. Researchers at Pfizer continued to

1.3 Pharmacokinetic Characteristics

j17

O CH3 N N

Cl

Cl O

Cl Cl

Cl

O

O

O

Cl

N

Cl O

N

Cl N

N

N

N

ketoconazole

miconazole

tioconazole F

Cl

F

Cl OH N

N

N

OH

N

N

N

N

N

N

N N

UK-47265

N

fluconazole

Figure 1.15 Structures of miconazole and analogues.

study further analogues, and the 2,4-difluoro derivative (fluconazole, UK-49858) showed high efficacy without any safety problem. Fluconazole [34] has a plasma half time of 5.1 h and 75% of the drug is excreted unchanged through urine. 1.3.2 Drugs with a Long Duration of Action

Captopril [35] was the first orally active ACE inhibitor (angiotensin-converting enzyme) to reach the marketplace. It is rapidly absorbed with a bioavailability of about 75%. Peak drug concentrations in plasma occur within 1 h of dosing, and the drug is then cleared rapidly (t1/2 about 2 h); therefore, a dosage regimen of two- to

Cl S

j 1 Optimizing Drug Therapy by Analogues

18

Table 1.4 Elimination half-life values of ACE inhibitors [31].

Drug captopril benazepril cilazapril enalapril fosinopril lisinopril perindopril ramipril trandolapril

Elimination half-life (h) 2 11 10 11 12 12 >24 8–14 16–24

three times daily is necessary. Since food reduces the oral bioavailability of captopril, the drug should be given 1 h before meals. Hypertension usually requires a lifelong treatment; therefore, the long-acting analogues of captopril are advantageous (Table 1.4, Figure 1.16). The long-acting once-daily dosing has practical benefits by improving patient compliance. The clinical advantage of full 24-h control of blood pressure is important to prevent cardiovascular events (e.g., myocardial infarction and stroke). The oral bioavailability of the long-acting ACE inhibitors is only slightly reduced by food. The case of the calcium channel antagonists, dihydropyridines, shows a similar picture. The pioneer drug nifedipine has a short duration of action. The long-acting analogues, such as felodipine, lacidipine, and amlodipine are more convenient for the lifelong treatment of hypertension (Table 1.5, Figure 1.17). Table 1.5 Elimination half-life values of calcium antagonist dihydropyridines [36].

Drug nifedipine felodipine lacidipine amlodipine

Elimination half-life (h) 2–4 10–15 7–18 30–50

1.3.3 Ultrashort-Acting Drugs

Esmolol [37] (Figure 1.18) is a b1-selective blocker with a very short duration of action. It is administered intravenously and used when b-blockade of short duration is desired in patients in whom adverse effects of bradycardia, heart failure, or hypotension may necessitate its rapid withdrawal. It is used in emergency situations during critical care medication.

1.3 Pharmacokinetic Characteristics

HS

H3C

O

O

N

N

H3C O

NH OH

O

O HO

captopril H3C

O

j19

O

benazepril

O

CH3

O

O

H3C

N

N

N

NH

NH

O

O

OH

O

H3C O

NH2

O

P O

OH

O

H3C

H3C

N

NH

O

O

OH

O CH3

O

OH

O

ramipril

CH3 N

NH O

N

NH

perindopril

O

O

CH3

O

H3C

O

fosinopril

lisinopril

O

O N

N

NH

O

CH3

H3C

O

O

H3C

OH

cilazapril

enalapril

HO

O

O

trandolapril Figure 1.16 Structures of ACE inhibitors.

OH

O

OH

j 1 Optimizing Drug Therapy by Analogues

20

Cl NO2 MeOOC

H3C

COOMe

N H

Cl MeOOC

CH3

H3C

nifedipine

COOEt

N H

CH3

felodipine

HO Cl

O EtOOC

H3C

COOEt

N H

MeOOC

CH3

lacidipine

COOEt

H3C

N H

O

NH2

amlodipine

Figure 1.17 Structures of nifedipine and analogues.

This special analogue type is called a soft drug. It is active only as an ester that loses its activity after metabolic hydrolysis. Esmolol’s distribution half-life is 2 min and its elimination half-life is 9 min. The same principle is used in the case of loteprednol etabonate (Figure 1.19), which is a glucocorticoid soft drug that has a good local activity after it is topically administered to the eye. Since it is rapidly deactivated after reaching the general circulation, it does not display systemic side effects. 1.3.4 Decreasing Interindividual Pharmacokinetic Differences

Interindividual pharmacokinetic differences result when a significant number of patients require a higher or multiple doses of a drug to achieve symptom relief and healing. Omeprazole [38] is the pioneer proton pump inhibitor that shows an interindividual variability in pharmacokinetics. Analogue-based drug discovery afforded pantoprazole (Figure 1.20), which possesses linear, highly predictable pharmacokinetics. It has a lower variability in pharmacokinetics compared to omeprazole,

1.3 Pharmacokinetic Characteristics

j21

OH O

NH

O

H3C

CH3

H3C O

Figure 1.18 Structure of esmolol.

HO

O

O

Cl

CH3

O

O

H

CH3 H

CH3

O H

O Figure 1.19 Structure of loteprednol etabonate.

particularly with respect to bioavailability. The pharmacokinetics of pantoprazolesodium [39] is almost the same in patients with gastrointestinal diseases and those with renal failure, and in the elderly, so that no dose adjustment is required. The drawback of omeprazole could also be overcome by using its (S)-enantiomer (esomeprazole) whose bioavailability is about double that of the racemate.

CH3 H3C

N

O

H3C

S N

O

O

O CH3

N H

esomeprazole

H3C

CH3 N

O

S N

O

N H

pantoprazole

Figure 1.20 Structures of proton pump inhibitors.

1.3.5 Decreasing Systemic Activity

In the intranasal and inhalation application of corticosteroids in the treatment of asthma and rhinitis, it is important to decrease the systemic availability of these drugs to avoid their adverse effects, such as adrenocortical insufficiency and osteoporosis. Analogue research afforded budenoside and fluticasone with low oral (systemic) bioavailability (Table 1.6, Figure 1.21).

F

O F

j 1 Optimizing Drug Therapy by Analogues

22

Table 1.6 Oral bioavailability of inhaled corticosteroids [40].

Drug

Oral (systemic) bioavailability (%)

beclomethasone budenoside fluticasone

15–20 10 <2

OH

O

O CH3

HO CH3 H

F CH3

O

HO

O

H

F

O

O

CH3

O O CH3

H

H3C

H

S

H

O F fluticasone furoate

budesonide

OH O CH3

HO CH3

CH3

H

Cl

OH

H

O beclomethasone Figure 1.21 Structures of corticosteroids.

1.4 Drug Interactions 1.4.1 Decreasing Drug Interactions

Cimetidine inhibits CYPs (e.g., CYP1A2, CYP2C9, and CYP2D6), an important class of drug-metabolizing enzymes. This interaction inhibits the metabolism of certain

1.5 Summary

drugs such as propranolol, warfarin, diazepam, and theophylline, thus producing effects equivalent to an overdose of these medicines. It is therefore advisable to avoid coadministration. These effects are avoided by analogues such as ranitidine and famotidine [41]. 1.4.2 Increasing Drug Interactions

Because of the potency of inhibition of cytochrome P-450 by ritonavir, it was found that combination of other HIV-1 protease inhibitors with ritonavir led to increased plasma levels of these drugs. Analogue research starting from ritonavir afforded the more potent lopinavir (Figure 1.11). However, lopinavir’s plasma halflife is low. In a combination of ritonavir and lopinavir, known as Kaletra, ritonavir inhibits the P-450-mediated metabolism of lopinavir and therefore the combination is remarkably effective [42]. This CYP3A4 inhibition by ritonavir has been exploited to increase the exposure of other anti-AIDS drugs that are metabolized by this particular CYP.

1.5 Summary

The chapter gives an overview of the possibilities of analogue research, illustrated with examples. Drug therapy can be optimized with the help of analogue research in the following 12 ways: . .

. . . .

. . . . . .

Increasing potency Improving the ratio of the main activity and the adverse effects: –Improving selectivity through receptor subtypes –Improving selectivity through unrelated receptors –Improving selectivity by tissue distribution –Improving selectivity of nonreceptor-mediated effects Improving the physicochemical properties with the help of analogues Decreasing resistance to anti-infective drugs Decreasing resistance to anticancer agents Improving oral bioavailability –Improving absorption –Improving metabolic stability Long-acting drugs for chronic diseases Ultrashort-acting drugs in emergency cases Decreasing interindividual pharmacokinetic differences Decreasing systemic activities Decreasing drug interactions with the help of analogues Synergistic interactions between analogues

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j 1 Optimizing Drug Therapy by Analogues

24

Acknowledgments

We thank Derek Buckle, John Lowe III, and Thomas J. Perun for carefully reading and reviewing the manuscript. J.F. thanks the Alexander von Humboldt-Stiftung (Bonn) for a fellowship in 2008 and 2009.

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Analogue-based Drug Discovery, Wiley-VCH Verlag GmbH, Weinheim, p. XXIII. Fischer, J. (2006–2008) IUPAC Project “Standalone Drugs”, www.iupac.org. Yanagisawa, I., Hirata, Y., and Ishii, Y. (1987) Studies on histamine H2 receptor antagonists. 2. Synthesis and pharmacological activities of N-sulfamoyl and N-sulfonyl amidine derivatives. J. Med. Chem., 30, 1787–1793. Stein, E.A. (2001) New statins and new doses of old statins. Curr. Atheroscler. Rep., 3, 14–18. Harting, J., Becker, K.H., Bergmann, R., Bourgois, R., Enenkel, H.J., Fuchs, A., Jonas, R., Lettenbaur, H., Minck, K.O., Schelling, P., and Schulze, E. (1986) Pharmacodynamic profile of the selective b1-adrenoceptor antagonist bisoprolol. Arzneim.-Forsch. Drug Res., 36 (1), 200–208. Winters, S.J., Banks, J.L., and Loriaux, D.L. (1979) Cimetidine is an antiandrogen in the rat. Gastroenterology, 76, 504–508. Schwartz, J.-C., Barbin, G., Duchemin, A.-M., Garbarg, M., Llorens, C., Pollard, H., Quach, T.T., and Rose, C. (1982) Pharmacology of Histamine Receptors (eds C.R. Ganellin and M.E. Parsons), Wright PSG, Bristol. Carr, A.A. and Meyer, D.R. (1982) Synthesis of terfenadine. Arzneim.-Forsch. Drug Res., 32, 1157. Janssens, F., Torremans, J., Janssen, R.A., Stokbroekx, M., Luyckx, M., and Janssen, Paul A.J. (1985) New antihistaminic N-heterocyclic 4-piperidinamines. 1. Synthesis and antihistaminic activity of N-(4-piperidinyl)-1H-benzimidazol-2amines. J. Med. Chem., 28, 1925.

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Stove, E.R. (1961) New penicillins stable towards both acid and penicillinase. Nature, 192, 1183. Doyle, F.P., Hanson, J.C., Long, A.A.W., Nayler, J.H.C., and Stove, E.R. (1963) Derivatives of 6-aminopenicillanic acid. Part VI. Penicillins from 3- and 5phenylisoxazole-4-carboxylic acids and their alkyl and halogen derivatives. J. Chem. Soc., 5838. Naylor, J.H.C. (1960) (Beecham) British Patent 905,778; Naylor, J.H.C. (1962) (Beecham) British Patent 978,299. Gothard, P. and Rogers, T.R. (2004) Voriconazole for serious fungal infections. Int. J. Clin. Pract., 58 (1), 74–80. Sluis-Cremer, N. and Ross, T. (2006) HIV-1 reverse transcriptase inhibitors: drug resistance and drug development. Curr. Pharm. Des., 12 (15), 1809–1810. Das, K., Clark, A.D., Jr., Lewi, J.J., Heeres, J., De Jonge, M.R., Koymans, L.M., Vinkers, H.M., Daeyaert, F., Ludovici, D.W., Kukla, M.J., De Corte, B., Kavash, R.W., Ho, C.Y., Lichtenstein, M.A., Andries, K., Pauwels, R., De B
ethune, M.P., Boyer, P.L., Clark, P., Hughes, S.H., Janssen, P.A.J., and Arnold, E. (2004) Roles of conformational and positional adaptability in structure-based design of TMC125-R165335 (etravirine) and related non-nucleoside reverse transcriptase inhibitors that are highly potent and effective against wild-type and drug-resistant HIV-1 variants. J. Med. Chem., 47, 2550–2560. Janssen, P.A.J., Lewi, P., Arnold, E., Daeyaert, F., De Jonge, M., Heeres, J., Koymans, L., Vinkers, M., Guillemont, J., Pasquier, E., Kukla, M., Ludovici, D., Koen, A., De B
ethune, M.P., Pauwels, R., Das, K., Clark, A.D., Jr., Volovik Frenkel, Y., Hughes, S.H., Medaer, B., De Knaep, F., Bohets, H., De Clerck, F., Lampo, A., Williams, P., and Stoffels, P. (2005) In search of a novel anti-HIV drug: multidisciplinary coordination in the discovery of 4-[[4-[[(1E)-2-cyanoethenyl]2,6-dimethylphenyl]amino]-2pyrimidinyl]-aminobenzonitrile

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(R278474, rilpivirine). J. Med. Chem., 48, 1901–1909. Sham, H.L., Kempf, D.J., Molla, A., Marsh, K.C., Kumar, G.N., Chen, C.-M., Kati, W., Stewart, K., Lal, R., Hsu, A., Betebenner, D., Korneyeva, M., Vasavanonda, S., McDonald, E., Saldivar, A., Wideburg, N., Chen, X., Niu, P., Park, C., Jayanti, V., Grabowski, B., Granneman, G.R., Sun, E., Japour, A.J., Leonard, J.M., Plattner, J.J., and Norbeck, D.W. (1998) ABT-378, a highly potent inhibitor of the human immunodeficiency virus protease. Antimicrob. Agents Chemother., 42, 3218–3224. Kim, E.E., Baker, C.T., Dwyer, M.D., Murcko, M.A., Rao, B.G., Tung, R.D., and Navia, M.A. (1995) Crystal structure of HIV-1 protease in complex with VX-478, a potent and orally bioavailable inhibitor of the enzyme. J. Am. Chem. Soc., 117, 1181–1182. Ghosh, A.K., Kincaid, J.F., Cho, W., Walters, D.E., Krishan, K., Hussain, K.A., Koo, Y., Cho, H., Rudall, C., Holland, L., and Buthod, J. (1998) Potent HIV protease inhibitors incorporating high-affinity P2-ligands and (R)-[(hydroxyethyl)amino] sulfonamide isostere. Bioorg. Med. Chem. Lett., 8, 687–690. Shah, N.P., Tran, C., Le, F.Y., Chen, P., Norris, D., and Sawyers, C.L. (2005) Overriding imatinib resistance with novel Abl kinase inhibitor. Science, 305, 399–401. Kantarjian, H., Giles, F., Wunderle, L., Bhalla, K., O’Brien, S., Wassmann, B., Tanaka, C., Manley, P., Rae, P., Mietlowski, W., Bochinski, K., Hochhaus, A., Griffin, J.D., Hoelzer, D., Albitar, M., Dugan, M., Cortes, J., Alland, L., and Ottmann, O.G. (2006) Nilotinib in imatinib-resistant CML and philadelphia chromosome-positive ALL. N. Engl. J. Med., 354, 2542–2551. Alf€oldi, S. and Fischer, J. (2006) Optimizing antihypertensive therapy by angiotensin-converting enzyme inhibitors, in Analogue-Based Drug Discovery (eds J. Fischer and C.R. Ganellin), Wiley-VCH Verlag GmbH, Weinheim. Jevons, S., Gymer, G.E., Brammer, K.W., Cox, D.A., and Leeming, M.R.G. (1979)

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Antifungal activity of tioconazole (UK20,349), a new imidazole derivative. Antimicrob. Agents Chemother., 15, 597–602. Thienpont, D., Van Cutsem, J., Van Gerven, F., Heeres, J., and Janssen, P.A.J. (1979) Ketoconazole: a new broad spectrum orally active antimycotic. Experientia, 35, 606–607. Richardson, K., Cooper, K., Marriott, M.S., Tarbit, M.H., Troke, P.F., and Whittle, P.J. (1990) Discovery of fluconazole, a novel antifungal agent. Rev. Infect. Dis., 12 (Suppl. 3), S267–S271. Ondetti, M.A., Rubin, B., and Cushman, D.W. (1977) Design of specific inhibitors of angiotensin-converting enzyme: new class of orally active antihypertensive agents. Science, 196, 441–444. Gaviraghi, G. (2006) Case study of lacidipine in the research of new calcium antagonists, in Analogue-Based Drug Discovery (eds J. Fischer and C.R. Ganellin), Wiley-VCH Verlag GmbH, Weinheim. Erhardt, P.W. (1993) Esmolol. Chron. Drug Discov., 3, 191–206.

38 Larsson, H., Carlsson, E., Junggren, U.,

39

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42

Olbe, L., Sj€ostrand, S.E.,  Ska:7pt :61ptnberg, I., and Sundell, G. (1983) Inhibition of gastric acid secretion by omeprazole in the dog and rat. Gastroenterology, 85, 900–907. Huber, R., Kohl, B., Sachs, G., SennBilfinger, J., Simon, W.A., and Sturm, E. (1995) Review article: the continuing development of proton-pump inhibitors with particular reference to pantoprazole. Aliment. Pharmacol. Ther., 9, 363–378. Miller, D.D., Brueggemeier, R.W., and Dalton, J.T. (2002) Adrenocorticoids, in Foye’s Principles of Medicinal Chemistry, 5th edn (eds D.A. Williams and T.L. Lemke), Lippincott Williams & Wilkins, Philadelphia. Hoogerwerf, W.A. and Pasricha, P.J. (2006) Pharmacotherapy of gastric acidity, peptic ulcers, and gastrooesophageal reflux disease, in Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 11th edn (eds L.L. Brunton, J.S. Lazo, and K.L. Parker), McGraw-Hill, New York. Silverman, R.B. (2004) The Organic Chemistry of Drug Design and Drug Action, 2nd edn, Elsevier/Academic Press, p. 273.

Erika M. Alapi

Richter Plc., Gyömro˝i út 19/21, 1103 Budapest, Hungary Erika M. Alapi was born in Pasztó, Hungary, in 1974. She received her MSc degree in chemistry at the Faculty of Science of E€otv€os Lor
and University in Budapest. She is working as a medicinal chemist in the field of CNS and preparing her PhD thesis “Optimizing Therapy by Analogues” under the supervision of Professor J
anos Fischer at Gedeon Richter Pharmaceutical Plc. She contributed to Analogue-Based Drug Discovery I as a coauthor.

References

J
anos Fischer

Professor, Richter Plc., Gyömro˝i út 19/21, 1103 Budapest, Hungary J
anos Fischer is a Senior Research Scientist at Richter Plc., Budapest, Hungary. He received his MSc and PhD degrees in organic chemistry from the Eotvos University of Budapest under Professor A. Kucsman. Between 1976 and 1978, he was a Humboldt Fellow at the University of Bonn under Professor W. Steglich. He has worked at Richter Plc. since 1981 where he participated in the research and development of leading cardiovascular drugs in Hungary. His main interest is analoguebased drug discovery. He is the author of some 100 patents and scientific publications. In 2004, he was elected as a Titular member of the Chemistry and Human Health Division of IUPAC. He received an honorary professorship at the Technical University of Budapest.

C. Robin Ganellin

Professor, University College London, Department of Chemistry, 20 Gordon Street, London WC1H 0AJ, UK C. Robin Ganellin studied chemistry at London University, receiving a PhD in 1958 under Professor Michael Dewar, and was a Research Associate at MIT with Arthur Cope in 1960. He then joined Smith Kline & French Laboratories in the UK and was one of the coinventors of the revolutionary drug, cimetidine (also known as Tagamet). In 1986, he was made a Fellow of the Royal Society and appointed to the SK&F Chair of Medicinal Chemistry at University College London, where he is now Professor Emeritus of Medicinal Chemistry. Professor Ganellin is coinventor of over 160 patents and has authored over 260 scientific publications. He was President of the Medicinal Chemistry Section of the IUPAC and is Chairman of the IUPAC Subcommittee on Medicinal Chemistry and Drug Development.

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John Proudfoot

Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Rd., Ridgefield, CT 06877-0368, USA John Proudfoot is a Medicinal Chemist with over 20 years of experience in drug design, ranging from fragment-based lead identification to late-stage lead optimization. He received his doctorate from the University College Dublin in Ireland, working with Professor Dervilla Donnelly. After postdoctoral studies under Professor Carl Djerassi at Stanford University and under Professor John Cashman at the University of California, San Francisco, he joined Boehringer Ingelheim Pharma in 1987. He is a coinventor of 28 issued US patents, including the one for the marketed drug, nevirapine, the first nonnucleoside HIV-1 reverse transcriptase inhibitor to receive regulatory approval.

j29

2 Standalone Drugs J
anos Fischer, C. Robin Ganellin, Arun Ganesan, and John Proudfoot

In volume I of Analogue-Based Drug Discovery we focused on analogue drugs with a double similarity, that is, those that are similar to another drug from both chemical and pharmacological viewpoints. These were referred to as structural and pharmacological analogues or, alternatively, full analogues. One special class of these analogues is considered to be direct analogues if they have identical pharmacophores – that is, they can be described by a general structure that includes most of the chemical skeleton. The book also discussed some examples of structural analogues, in which the similarity is limited to their chemical structures since they possess different pharmacological properties. We also proposed the term pharmacological analogues for drugs that have completely different chemical structures but similar pharmacological properties. Analogues are based on following a lead compound, and if this compound were a drug that opened up a new therapeutic treatment then we use the term pioneer drug. Such a drug is often referred to as “first in class.” One can subdivide pioneer drugs into those that have analogues and those that do not. Thus, the latter group provides a special subclass called “standalone drugs,”1) which are pioneer drugs with no effective analogues. An analysis of the Top 100 most important drugs based on the sales data of IMS is revealing. In 2006, the global sales of drugs amounted to US$ 638 billion. The sales of the Top 100 drugs were about 51% of the global sales, that is, US$ 326 billion. Table 2.1 shows that 84.4% of sales of the Top 100 drugs derive from small-molecule drugs, whereas the sales of macromolecular drugs and biological entities amount to 9.2% and the sales of vitamins and a hormone are 6.4%. The importance of small-molecule drugs remains very high in spite of the increasing role of macromolecular drugs (proteins) and biological entities (monoclonal antibodies).

1) In 2006, a new project of IUPAC’s Subcommittee on Medicinal Chemistry and Drug Development (Division VII, Chemistry and Human Health) was started to study the role and the importance of standalone drug. The project was chaired by J
anos Fischer with the coauthors of this chapter as project members. Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

j 2 Standalone Drugs

30

Table 2.1 Drug types of the Top 100 drugs.

Drug types Small-molecule drugs Macromolecules and biological entities Vitamins and hormone

Sales (2006) US$ (billion)

Ratio (%)

Numbers

275 30 21

84.4 9.2 6.4

80 10 10

Table 2.2 Types of small-molecule drugs among the Top 100 drugs.

Small-molecule drug type Pioneer drugs Full analogues Pharmacological analogues Structural analogues Standalone drugs

Number 10 51 8 2 9

Table 2.2 analyzes the small-molecule drugs among the Top 100 drugs according to their analogue status. It is remarkable how many (51) of full analogues are among the Top 100 drugs. The analogue drugs belong to 42 different analogue classes of drug action, as defined by the main pharmacological effect, for example, ACE inhibitors, a1-blockers, AT1 antagonists, b-lactamase inhibitors, and so on, and there are only 10 pioneer drugs (“first in class”) among the Top 100 drugs from the analogue classes. This situation derived from the fact that the pioneer drugs were improved through making analogues and then continuous optimization has afforded additional analogue drugs that are even better. The nine standalone drugs from among the Top 100 drugs (2006), acetaminophen, acetylsalicylic acid, aripiprazole, bupropion, ezetimibe, lamotrigine, metformin, topiramate, and valproate semisodium, will be discussed one by one.

2.1 Acetaminophen (Paracetamol)

Based on their structures and biological activity acetanilide, phenacetin, and paracetamol (Figure 2.1) are direct analogues; however, they also have a close metabolic relationship since paracetamol is the active metabolite of both acetanilide and phenacetin. Acetanilide (1) was discovered as an antipyretic drug after it was mistakenly used instead of naphthalene in an antiworming regimen in 1886 [1]. It was marketed under the name “Antifebrin” in 1886 by the company Kalle, then from 1908 by

2.1 Acetaminophen (Paracetamol)

HN

O

HN

O

O

acetanilide

phenacetin

HN

O

OH

paracetamol

Figure 2.1 Paracetamol and its former analogues.

Hoescht Dyeworks. Acetanilide was cheaper and easier to prepare than other antipyretics and it remained in use for many decades. In 1887, the Bayer Company introduced the 4-ethoxy derivative, phenacetin, as a less toxic analogue of acetanilide. It was a highly successful product and established the Bayer Company as a leading pharmaceutical manufacturer. Phenacetin was widely used for about 90 years until mounting concerns over carcinogenicity and kidney-damaging properties in chronic treatment [2] led to restrictions on its use in most countries. It is, however, even nowadays available in some combinations in some countries. Paracetamol (acetaminophen) was identified as an active antipyretic and analgesic agent in a clinical trial in 1893 by Joseph von Mering [3] in cooperation with the Bayer Company. However, he believed mistakenly that it produced methemoglobinemia as an adverse effect and the compound was ignored for a half century. This situation changed when Lester and Greenberg [4], and subsequently Flinn and Brodie [5], confirmed that paracetamol is the active metabolite of phenacetin and Brodie and Axelrod found that paracetamol is the main metabolite of acetanilide [6]. At the same time, Flinn and Brodie found that, even at high oral doses, paracetamol is not attended by the formation of methemoglobin. In 1953, paracetamol was marketed by the Sterling-Winthrop Company and, especially after the withdrawal of phenacetin, assumed an important role as an antipyretic and mild analgesic drug. It is used in monotherapy and in several combination preparations, which are available on the market as OTC products. In 2007, according to the IMS data, the value of paracetamol sales was greater than US$ 7.5 billion and it ranked fourth among drugs. Paracetamol has no anti-inflammatory activity and its analgesic effect is comparable to aspirin. In patients who are sensitive to aspirin, the use of paracetamol is indicated. Despite its extensive use, the mechanism of action of paracetamol remained unknown. It can be supposed that it has a primarily CNS activity. In a recent study, Hoegestaett et al. showed that paracetamol undergoes a primary deacetylation step in the liver, brain, and spinal cord that is followed by N-acylation of the resulting 4-aminophenol with arachidonic acid by FAAH to form AM404 (Figure 2.2) in the CNS [7]. The biological significance of this route was confirmed by Ottani et al.

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j 2 Standalone Drugs

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OH

O N H

Figure 2.2 N-Arachidonoyl-phenolamine (AM404).

who showed that the effect of paracetamol in pain models is prevented by cannabinoid receptor antagonists [8]. According to Hinz et al., paracetamol is a selective cyclooxygenase-2 (COX-2) inhibitor in man [9]; however, paracetamol has no significant anti-inflammatory activity. With normal use, therapeutic levels of paracetamol are considered to be safe for humans [10]; however, an overdose can cause severe liver toxicity. Paracetamol is metabolized primarily by O-glucuronidation and O-sulfation. However, a small amount is metabolized via a third metabolic pathway, in which oxidation by microsomal cytochrome P-450 gives a toxic metabolite NAPQI (N-acetyl-p-benzoquinone imine). This toxic metabolite is normally rapidly neutralized by glutathione by formation of a nontoxic adduct (Figure 2.3). The problem occurs when glutathione stores are diminished; NAPQI is no longer detoxified and covalently binds to lipid bilayer of hepatocytes, resulting in hepatotoxicity. Paracetamol toxicity is the leading cause of hospital admission for acute liver failure in the United States of America accounting for approximately 40% of all liver transplants [11]. Several direct analogues of paracetamol are summarized by Vermeulen and coworkers [12]. For example, N-methyl-paracetamol caused no hepatic necrosis in mice, rats, or hamsters in doses that caused massive hepatic necrosis in the same animals when paracetamol was administered [13]. No development is reported on these analogues. In 2007, new paracetamol analogues were described by Bazan and coworkers, in which a benzothiazolonyl moiety is linked to the p-acylaminophenol fragment [14]. In 2008, Imming and coworkers described N-(1H-indazol-5-yl)acetamide and N-(4-hydroxybenzyl)acetamide, which displayed analgesic activities comparable to paracetamol [15]. All of these analogues (Figure 2.4) try to avoid the formation of NAPQI. More time is needed to evaluate these efforts to optimize paracetamol.

HN

O

cytochrome

N

O

P450

HN

O

G-SH S-G

OH

O NAPQI

Figure 2.3 Formation of N-acetyl-p-benzoquinone imine.

glutathione

OH

2.2 Acetylsalicylic Acid (Aspirin)

O O S N N

O

HN

OH

O

O

N N H

OH Bazan (2006)

Nelson (1978)

O

HN

Imming (2008)

Figure 2.4 Some direct analogues of paracetamol (experimental phase).

2.2 Acetylsalicylic Acid (Aspirin)

Willow bark [16] and leaves have been used as antipyretic and anti-inflammatory analgesics in ancient civilizations. In 1828, Buchner isolated salicin from boiled willow bark (Figure 2.5) and in 1833 pharmacist Merck in Darmstadt used highly purified salicin as an antipyretic agent for half the price of quinine; however, salicin was poorly palatable because of its bitter taste and its structure at that time was not yet known. In 1839, Piria obtained salicylic acid from salicin by an oxidative cleavage [17]. In 1876, Maclagan [18] found that salicin and salicylic acid are equally efficacious in treating acute rheumatism, which is understandable, since salicin is a prodrug of salicylic acid. Salicylic acid became a cheap chemical through the Kolbe synthesis [19] and in 1876 it was introduced as an analgesic antirheumatic drug at the Charit
e in Berlin [20]. Despite the undoubted benefits of salicylic acid in the treatment of pain, fever, and inflammatory disorders, there were several side effects at high doses of 4–6 g per day, for example, stomach irritation, often associated with nausea and vomiting, and hearing disorders (tinnitus). In 1897, Eichengr€ un and Hoffmann at Bayer discovered acetylsalicylic acid (aspirin), which was not only as effective as sodium salicylate but also better tolerated. Since the beginning of the last century, aspirin has been the most widely used drug in the world.

OH HO HO

O

O

OH O

OH

O

HO

HO

OH

O O

salicin

salicylic acid

Figure 2.5 History of acetylsalicylic acid.

acetylsalicylic acid

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More than 60 years passed before the widely used nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, indometacin, flurbiprofen, diclofenac, naproxen, ketoprofen, piroxicam, and sulindac (Figure 2.6), were discovered. These drugs were all discovered between 1961 and 1969 without the knowledge of their biochemical mechanism of action. In 1971, Vane discovered the mechanism by which aspirin and NSAIDs exert their anti-inflammatory, analgesic, and antipyretic actions. He proved that aspirin and NSAIDs inhibit prostaglandin synthesis [21]. It was already known that PGE1 was

Cl

O N

COOH O

COOH ibuprofen (1961)

indometacin (1961)

COOH Cl H N

COOH

Cl

F

diclofenac (1965)

flurbiprofen (1964)

COOH

COOH

O O naproxen (1967)

ketoprofen (1967) O S

O

O S

N

H N

N

OH O

F COOH

piroxicam (1968)

sulindac (1969)

Figure 2.6 Nonsteroidal anti-inflammatory drugs.

2.3 Aripiprazole

O

HO

NH2 O

HO

O

O

O

OH

HO

F

HO F salicylamide

salsalate

diflunisal

Figure 2.7 Salicylic acid analogues.

a potent pyretic agent [22] and prostaglandins had also been detected in inflammatory exudates [23]. The cyclooxygenase-1 (COX-1) enzyme, which catalyzes the prostaglandin formation, was isolated in 1976 [24]. Aspirin selectively acetylates the hydroxy group of one-serine residue (Ser 530) located 70 amino acids from the C-terminus of COX-1 [25]. In 1991, the COX-2 enzyme was discovered, which produces prostaglandins, mostly PGE2, during inflammatory reactions [26]. At low concentrations, aspirin acetylates COX-1 rapidly (within minutes) and selectively. At high concentrations, over longer time periods, aspirin will also nonspecifically acetylate a variety of proteins and also COX-2 at its serine residue (Ser 516). The pharmacology of aspirin is unique because its activity derives from a reactive acetate and salicylate [27]. There is a significant difference between salicylic acid and aspirin concerning the COX-1 inhibition. In contrast to aspirin, salicylic acid prevents binding of arachidonic acid by competitive inhibition. The nonaspirin NSAIDs (Figure 2.6) also inhibit competitively the COX enzymes, which is an important distinction from aspirin effects. Another important activity of aspirin is the inhibition of thromboxane formation by blocking prostaglandin biosynthesis in platelets [28]. Aspirin is the only antiplatelet agent in clinical use with this mechanism of action. There is no successful aspirin analogue on the market. Salicylamide, salsalate, and diflunisal (Figure 2.7) are existing drugs, which are all salicylic acid analogues. Salicylamide is without anti-inflammatory activity and diflunisal has no antipyretic effect. There have been several attempts to improve aspirin by prodrug approaches, which are reviewed in a recent article by Gilmer and coworkers [29].

2.3 Aripiprazole

Schizophrenia, which affects 1–2% of the global population, is a serious brain disorder characterized by abnormal mental functions and disturbed behavior [30]. The positive symptoms include disorganized thoughts, delusions, and hallucinations, whereas the negative symptoms are characterized by social withdrawal, lack of motivation, and disturbances in basic cognitive functions. Depression and bipolar disorder are highly comorbid in schizophrenia and these are the reasons for a higher risk of suicide in this disorder.

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H3C

N

CH3 N CH3

CH3 CH3

N

N

S

S

promethazine antihistamine 1949 (launch)

Cl

chlorpromazine antipsychotic 1952 (launch)

Figure 2.8 Promethazine and chlorpromazine.

2.3.1 First Generation “Typical” Antipsychotic Drugs (Other Names: Neuroleptics, Conventional Antipsychotics)

Chlorpromazine, the first antipsychotic drug, was obtained from the antihistaminic and sedative promethazine. Charpentier et al. [31] synthesized and Courvoisier et al. investigated many analogues in order to enhance the sedative effect of promethazine [32] (Figure 2.8). Chlorpromazine was marketed by Rhône-Poulenc in 1952. Chlorpromazine initiated an analogue-based drug research, and several direct analogues were discovered and introduced as antipsychotic drugs without the knowledge of their mechanism of action. In vivo pharmacological models, such as conditioned avoidance response test, were typically used to drive these discovery efforts. A completely different approach in the Janssen Laboratories in 1958 afforded more potent drugs. The sedative effect of an analgesic pethidine derivative (R1187) was reminiscent of the sedation produced by chlorpromazine and, after exploring hundreds of derivatives, haloperidol was discovered. Haloperidol was 50–100 times potent than chlorpromazine, with a better side-effect profile (Figure 2.9) [33]. Chlorpromazine and haloperidol and their analogues helped many patients, but treatment with these first-generation antipsychotic drugs was associated with the development of extrapyramidal side effects (EPS), for example, dystonias, akathisias,

O

O

OH Cl

O

O

N

N F R1187 pethidine derivative analgesic effect Figure 2.9 Pethidine derivative and haloperidol.

haloperidol antipsychotic 1959 (launch)

2.3 Aripiprazole

and tardive dyskinesia (TD) in about 20% of patients and, as further adverse effects, elevated prolactin levels, and weight gain are observed. About 30% of patients do not respond to the first-generation antipsychotic drugs. 2.3.2 Second-Generation “Atypical” Antipsychotic Drugs

The pioneer drug clozapine was discovered in 1960 by the Wander company in Switzerland. The SAR of these tricyclic antidepressants afforded amoxapine and its 7-hydroxy metabolite had an antipsychotic activity. In the case of clozapine, the beneficial effect of the chloro substituent in position 8 and not in position 2 as in some classical tricyclic antidepressants, such as in amoxapine (Figure 2.10), was a key step in the discovery process. Clozapine was poised to revolutionize the treatment of schizophrenia because it had no EPS adverse effect in humans [34]. Clozapine was introduced in 1972 in some European countries, but agranulocytosis, a severe adverse effect, nearly terminated the use of this drug. Careful blood monitoring could decrease this severe adverse effect and clozapine has found an important place in the treatment of schizophrenia. More atypical antipsychotic drugs, such as olanzapine and quetiapine, were discovered by structural modification of clozapine, whereas haloperidol is similar to risperidone, ziprasidone, and sertindole in structure. It was proposed in 1954 that schizophrenia resulted from overactive serotonin neurotransmission [35] whereas, according to the “dopamine hypothesis of schizophrenia,” a functional hyperactivity of dopamine in the neuronal systems of the brain has a major contribution to the disease [36]. Dopamine receptors were first classified into D1 and D2 subtypes [37]. Typical and atypical antipsychotic drugs (Figure 2.11) are antagonists at D2 receptors; however, the atypical antipsychotics display an increased affinity for serotonin 5-HT2A/2C receptors. The atypical antipsychotic drugs have only a limited effect in the treatment of refractory schizophrenia. Metabolic adverse effects (obesity, diabetes) have been found in the case of clozapine and olanzapine. A prolongation of QT-interval has been observed after the use of quetiapine, ziprasidone, and sertindole.

H N

H N N

N N C

A

2 Cl

Cl 8

N A

amoxapine antidepressant

C N H

O

clozapine antipsychotic 1972 (launch)

Figure 2.10 Amoxapine and clozapine.

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O OH

CH3 N

N

N

N

N

N

N H

S

olanzapine

CH3

S

F

quetiapine S

O

N

N

O

N

N

N

N

O N

CH3 Cl

N H

Cl

risperidone

ziprasidone

N

O HN

N

F

N sertindole

Figure 2.11 Atypical antipsychotic drugs.

2.3.3 A New Approach: Aripiprazole, a Dopamine Partial Agonist

All the typical and atypical antipsychotic drugs inhibit dopamine neurotransmission by blocking the postsynaptic dopamine receptors. Unfortunately, dopamine antagonism is also responsible for the most serious side effects of these agents, for example, extrapyramidal syndrome, tardive dyskinesia, and hyperprolactinemia. Carlsson proposed that compounds stabilizing the dopaminergic system without inducing hypodopaminergia would be advantageous in the treatment of schizophrenia [38]. Researchers at Otsuka (Japan) serendipitously found among 2(1H)-quinolone derivatives new compounds with neuroleptic-like activity. The best compound was OPC-4392 (Figure 2.12), which was found to be a dopamine autoreceptor agonist and a weak postsynaptic dopamine D2 antagonist [39]. In clinical investigations, OPC-4392 proved to be active in the treatment of the negative symptoms, but it was not sufficiently potent for the treatment of positive symptoms. A more potent postsynaptic dopamine D2 antagonist was needed. Among the direct analogues of OPC-4392 (lead), aripiprazole was found to fulfill these criteria. In the apomorphine-induced stereotypy in mice, OPC-4392 had an ED50 of

2.4 Bupropion

O

N H

O

H3C

N N

CH3

OPC-4392

O

N H

O

Cl

N N

Cl

aripiprazole Figure 2.12 OPC-4392 and aripiprazole.

41 mmol/kg p.o., whereas aripiprazole showed the highest potency with an ED50 of 0.6 mmol/kg p.o. [40]. Aripiprazole proved to be a presynaptic partial agonist of dopamine D2/3 receptors. The introduction [41] of aripiprazole in 2002 was the first breakthrough in antipsychotic treatment since the discovery of clozapine and analogues. However, unmet medical needs still remain to find a more effective drug for the treatment of refractory patients, to improve treatment of negative symptoms and cognitive dysfunction [42].

2.4 Bupropion

Bupropion, also known as Amfebutamone, is an antidepressant that is also used as an anticigarette smoking treatment. Soroko et al. [43], from the Wellcome Research Laboratories (Research Triangle Park, NC, USA), describe how they sought a compound that would be active in antidepressant screening models but differ chemically and pharmacologically from the well-known tricyclic antidepressants (such as amitriptyline and imipramine), and would not be sympathomimetic, cholinolytic, or an inhibitor of monoamine oxidase. Mehta, who synthesized bupropion in the 1960s, describes [44] that they used as a screen the tetrabenazine-induced sedation [45] in mouse and rat models since most clinically useful antidepressants prevent this. As he stated [44] “optimally a compound should prevent tetrabenazine-induced sedation without itself being a stimulant at antidepressant doses.” Several observations from their earlier studies [46] of compounds related to bupropion’s chemical structure (Figure 2.13) were used in the overall design of bupropion. They were as follows: 1)

The tert-alkyl group on the nitrogen atom was found to be less susceptible to N-dealkylation than other alkyl groups.

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O H N H3C

Cl

CH3 H3C CH 3

Figure 2.13 Bupropion.

2)

The substituent in the meta position on the phenyl ring would mitigate the possibility of para-hydroxylation and consequent elimination of the drug as a glucuronide. 3) Alkoxy and alkyl substituents on the phenyl ring should be avoided since they have been found to confer antiarrhythmic activity and inhibition of free fatty acid mobilization. 4) a-Hydroxy derivatives with two-carbon side chains (e.g., isoproterenol) have usually been associated with sympathomimetic properties.

The first compound in the series of halogen substituted N-tert-butyl-2-phenylpropanolamines to show the CNS activity, using the potentiation of amphetamine as a possible screen for antidepressant activity, was 3,4-dichlorophenyl-N-tert-butylaminopropanol (Figure 2.14) [44]. Mehta continues to observe [44], “since, a priori, it was conceivable that the chemically stable aminoketones and aminoalcohols could be interconvertible in vivo, there was uncertainty as to which form was responsible for the observed activity. Thus a series of aminoketones was made and tested; bupropion was the fourth compound in this series to be designed and screened as a potential antidepressant using the modified antitetrabenazine screen developed by Sulser and Soroko” [45]. Mehta observes [44] that it was quite apparent that the aminoketones rather than the aminoalcohols were of primary interest and, as the amine moiety was tert-alkyl, bupropion was rather stable. Bupropion possessed the optimal variations in its chemical structure necessary to achieve maximum activity and therapeutic index. The drug was expected to result in vivo in relatively innocuous metabolites and to have a high degree of both water and lipid solubility, resulting in good systemic absorption after oral administration [44]. US patents protecting the compound were published in 1974 and 1975 [47], and there is an earlier German patent (1971) describing its synthesis [48]. Bupropion, like amitriptyline, produced a dose-dependent prevention of tetrabenazine-induced sedation in mice [43]. It also prevented tetrabenazine-induced

OH Cl

H N

Cl

H3C

CH3 H3C CH3

Figure 2.14 3,4-Dichlorophenyl-N-tert-butylaminopropanol.

2.4 Bupropion

blepharospasm and fall in rectal temperature, and it corrected the “hunched” posture characteristic of tetrabenazine action [43]. In addition, like amitriptyline, it markedly antagonized the 6
C drop in rectal temperature seen after reserpine, and was not a locomotor stimulant in mice [43]. Unlike amitriptyline, bupropion antagonized the intense motor activity produced by dextroamphetamine in mice [43]. Bupropion did not inhibit monoamine oxidase; it was approximately 60-fold less potent than amitriptyline as an inhibitor of norepinephrine uptake in rat hypothalamus synaptosomes (IC50 ¼ 6.5 mM) whereas it was 20-fold more potent than amitriptyline as an inhibitor of dopamine uptake in striatal synaptosomes (IC50 ¼ 3.4 mM) [43]. It had little effect on 5-HT uptake in concentrations as high as 10 mM [43]. Bupropion is chiral and it has been separated into its two enantiomers [49]. No significant differences were found [49] in the activities in the tetrabenazine-induced sedation model in mice or in the uptake of norepinephrine and dopamine into synaptosomes. However, the enantiomers are not very stable and the racemization half-life for the ()enantiomer, under physiological conditions (pH 7.6), was suggested to be approximately 44 min [49]. A phase I clinical study in 12 healthy subjects revealed no differences from lactose and it was concluded [50] that bupropion, at the doses studied (of 50 and 100 mg), was devoid of stimulant or sedative properties, or cardiovascular effects. Bupropion was approved by the US Food and Drug Administration (FDA) as an antidepressant on December 30, 1985 and marketed under the name of WellbutrinÔ. However, a significant incidence of seizures at the originally recommended dosage (400–600 mg) caused the drug to be withdrawn in 1986. Subsequently, the risk of seizures was found to be highly dose dependent, and bupropion was reintroduced to the market in 1989 with a maximum recommended dose of 450 mg/day. A sustained release formulation was approved by the FDA in 1996 and has been used clinically to treat depression. Placebo-controlled double-blind studies have confirmed the efficacy of bupropion for clinical depression [51]. Furthermore, available clinical data suggest that bupropion is an effective and generally welltolerated option in the treatment of major depressive disorder [52]. Perhaps then, bupropion should not be regarded as a “standalone” drug, although its mechanism of action appears to be different from other antidepressant drugs; like other antidepressants, its actions involve biogenic amine transmission, but it exerts a different combination of effects on biogenic amines. Bupropion is, however, the first non-nicotine medication to gain approval as an aid to smoking cessation and it was placed on the market for this purpose in 1997 under the name ZybanÔ. This use arose from finding that smokers who were taking bupropion (as Wellbutrin) as antidepressant medication found that they had a reduced desire or craving to smoke cigarettes [53]. It is administered in 150 mg doses in a sustained release formulation twice daily and reduces the severity of nicotine cravings and withdrawal symptoms. The treatment course lasts for 7 to 12 weeks with the patient halting the use of tobacco about 10 days into the course. The efficacy of bupropion is reported to be similar to that of nicotine replacement therapy [54].

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N HN N Figure 2.15 Varenicline.

Bupropion has, however, been joined by another product on the market since 2006, when Pfizer launched varenicline (Figure 2.15) as ChantixÔ in the United States and ChampixÔ in Europe. Varenicline acts by a different mechanism since it is an a4b2 nicotinic acetylcholine receptor partial agonist [55]. Both drugs, bupropion and varenicline, now have “black box” warnings from the US FDA (July 2009) [56] to highlight the risk of serious mental health side effects including depression, hostility, and suicidal thoughts. The suicide aspect seems to be much worse for varenicline that, since its approval in 2006, is reported to have been associated with 98 suicides and 188 attempts. Bupropion, since 1997, has been associated with 14 suicides and 17 attempts.

2.5 Ezetimibe

High plasma concentrations of low-density lipoprotein-cholesterol (LDL-C) are known to play an active role in the development of atherosclerotic coronary heart disease. The latter being a major cause of death and cardiovascular morbidity in the Western world. For this reason, much effort has been expended in seeking to reduce the serum cholesterol levels by drug treatment. The two significant sources for cholesterol are endogenously synthesized cholesterol and dietary cholesterol. Early discoveries found ways to reduce the equilibrium level of cholesterol. Cholestyramine and colestipol, for example, are basic anion exchange resins that sequester bile acids in the gastrointestinal tract to prevent reabsorption. The sequestered bile acids are then excreted, which results in plasma cholesterol being converted into bile acids to normalize the bile acid levels. This conversion of cholesterol lowers the plasma concentration of LDL-C. Clofibrate, and the various other fibrates that followed, reduces the plasma triglyceride concentrations. In most patients, plasma cholesterol and LDL-C concentrations also fall. The fibrates were subsequently shown to be agonists of the PPAR-a receptor (peroxisome proliferator-activated receptors that modulate carbohydrate and fat metabolism). The statins are specific inhibitors of hydroxymethylglutaryl coenzyme A (HMGCoA) reductase that were introduced into clinical practice in the 1980s. In low doses, the statins exerted a relatively selective effect on hepatic cholesterol synthesis. As the statins became established, their potencies, or dose levels, were increased in order to have a more profound effect on lowering of the LDL-C levels seen in the blood [57]. However, this gave rise to an increased incidence of such an unwelcome side effect as rhabdomyolysis especially when used in combination with fibrates.

2.5 Ezetimibe

Cholesterol absorption inhibitors have been described [58a] by scientists at Pfizer. These are synthetic saponins, tigogenin cellobiosides, tiqueside, and pharmaqueside. The latter was stated in 2002 to be in phase III clinical trials for the treatment of hypercholesterolemia, but further results do not appear to have been reported. Other efforts to inhibit the absorption of dietary cholesterol focused on the inhibition of Acyl-CoA:cholesterol acyltransferase (ACAT), the major enzyme associated with cholesterol esterification. Inhibition of this enzyme blocks absorption of intestinal cholesterol. As part of a broad chemical program to discover novel ACAT inhibitors [58b], scientists at Schering-Plough Research Institute (in Kenilworth, NJ, USA) investigated the synthesis and in vivo testing of conformationally restricted compounds related to two known chemical classes of ACAT inhibitors, namely, SA 58035 and CI 976 (Figure 2.16). Burnett et al. describe [58b] bridging of the two carbon atoms of the ethyl linkage and the nitrogen atom in SA 58035 by using 2-azetidinone containing two phenyl groups. The desired compound A was not active, but a secondary compound B, arising from acylation, did show some activity, pariculary in vivo. The latter provided an initial lead that was followed up and, by analogy with their previous work in ACAT, methoxy groups were introduced. This work eventually led to the first drug candidate, the chiral trans-azetidinone SCH 48461, for development (Figure 2.17). Surprisingly there was a strong divergence between the in vitro ACAT assay (measuring inhibition of the esterification of cholesterol with oleic acid) and the in vivo test that measured the inhibition of the rise in plasma cholesterol levels in cholesterol-fed hamsters. These results suggested the existence of another mechanism of action and further work suggested that it was novel and upstream from ACAT. Furthermore, SCH 48461 was shown to reduce serum cholesterol in human clinical trials [59]. The above findings led to abandonment of the in vitro assay. Having to rely only on an in vivo assay made it difficult to separate potential effects on intrinsic potency from effects on pharmacokinetics. This was especially difficult since SCH 48461 was extensively metabolized in vivo, making it unclear as to exactly what was the active species. Van Heeck et al. [60a] describe that an experimental protocol was devised to determine whether there were active metabolites and how they might contribute to the in vivo profile of SCH 48461. The studies revealed the rapid appearance of a complex metabolic mixture in the bile, which was more potent as an inhibitor of

O

CH3(CH2)9 Me

SiMe2(C10H21)

HN O

OMe

HN MeO

SA 58035 Figure 2.16 ACAT inhibitors.

CI 976

OMe

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PhCH2 CO

N

N

O

O

OMe

OMe

compound B

compound A

OMe

OH OH

F N

N

O

O

OMe

F

SCH 48461

SCH 58235 (ezetimibe)

Figure 2.17 Compounds leading to ezetimibe.

cholesterol absorption. Rosenblum et al. listed [60b] the putative sites of metabolism of SCH 48461, as shown in Figure 2.18, and directed their synthesis effort to making the most probable metabolites for testing. Their results led them inter alia to introduce two hydroxyl groups as metabolic products and two fluorine atoms to block metabolism sites (Figure 2.19). The result was the compound SCH 58235, ezetimibe [61], which was reported to be 50 times more potent than SCH 48461 in the

demethylation benzylic oxidation

OMe

N

demethylation

O pendant phenyl oxidation

OMe ring opening

Figure 2.18 Putative sites of metabolism of SCH 48461.

2.5 Ezetimibe

dealkylated stereoselectively oxidized

OH OH F N O F oxidation blocked

oxidation blocked Figure 2.19 Design of (SCH 58235) ezetimibe.

cholesterol-fed hamster assay. This result provides an impressive story of serendipitous discovery of a drug working by a novel mechanism, together with outstanding integration of synthetic and medicinal chemistry with metabolic and pharmacological studies, to make a potent therapeutic product. The discovery of ezetimibe led to a 10-year search for its mechanism of action. The researchers at Schering-Plough showed [62] that Niemann-Pick C1-Like 1 (NPC1L1) protein plays a critical role in the absorption of intestinal cholesterol across the plasma membrane of the intestinal enterocyte. This protein is believed to be a molecular target [63] of ezetimibe. By inhibiting cholesterol absorption at the level of the brush border of the intestine, ezetimibe reduces the amount of lipoprotein cholesterol circulated to the liver. In response to reduced cholesterol delivery, the liver reacts by upregulating LDL-C receptor that, in turn, leads to increased clearance of cholesterol from the blood [64]. Ezetimibe is marketed as a 10 mg tablet under the name ZetiaÔ and is also available in combination with simvastatin; it has also been approved for a combination with fenofibrate. Ezetimibe is a standalone product as, so far, there are no analogues yet available, probably because the mechanism of action was published only in 2005. Thus, this remarkable example of drug discovery started out as an attempt to make an analogue of leads that, though characterized pharmacologically, had not yet yielded a clinically useful product. A serendipitous discovery of a different mechanism of action led to the characterization of a previously unknown mechanism of cholesterol uptake and the clever design of a very potent candidate drug. This work represents a true breakthrough in the search for drugs that reduce the intake of dietary cholesterol and provides an outstanding example of analogue-based drug research leading to the discovery of a pioneer drug.

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2.6 Lamotrigine

Lamotrigine (Figure 2.20) is a widely used anticonvulsive agent. Its predominant indication is in the treatment of epilepsy and bipolar disorder [65, 66]. The discovery process was partly serendipitious, based on the observation that folic acid produces epileptogenic foci and that antiepileptic drugs such as phenytoin, phenobarbital, and carbamazepine have antifolate properties [67, 68]. Thus, a correlation between antiepileptic and antifolate properties was assumed. A series of aminotriazine compounds was prepared by chemists at Wellcome in the United Kingdom as potential antifolate agents. Lamotrigine was the lead from this series and was first disclosed in a 1981 patent. The patent reports significant anticonvulsant activity with an ED50 of 2.4 mg/kg in mice by oral administration [69]. Nevertheless, the compound had little effect on folate levels, and subsequent work confirmed that the original hypothesis linking folates and antiepileptics was misguided. In vitro studies demonstrated that lamotrigine primarily acts as a blocker of voltage-sensitive sodium ion channels [70]. The promising activity and tolerability of lamotrigine led to its progression to clinical studies, with phase I studies confirming its safety and potential superiority to established drugs such as phenytoin and diazepam [71]. Lamotrigine was first approved in 1994 as an antiepileptic drug for the treatment of partial seizures. Lamotrigine is the only medication besides topiramate used in the treatment of seizures associated with Lennox–Gastaut syndrome, a severe form of epilepsy. Following its widespread use as an antiepileptic, it was noticed that patients reported higher levels of happiness and mastery, or perceived internal locus of control, independent of seizure control [72]. This led to investigation of lamotrigine as an antipsychotic and it received approval in 2003 for the treatment of bipolar I disorder, the first drug to do so since lithium. In addition, off-label uses of lamotrigine include the treatment of peripheral neuropathy, migraines, borderline personality disorder, and refractory unipolar depression. Lamotrigine has virtually complete bioavailability with no first-pass effect. It is 55% bound to plasma proteins and reaches a peak plasma concentration in 2.5 h. The halflife in healthy adults is 24–35 h, with clearance primarily as the glucuronide conjugate in urine. Metabolism by uridine diphosphate glucuronyl transferases is competitive and susceptible to drug–drug interactions. However, lamotrigine does not induce cytochrome P-450 isozymes and thus does not affect plasma levels of other Cl Cl N H2N

NH2 N N

Figure 2.20 Lamotrigine.

2.7 Metformin

Cl Cl

Cl

N H2N

NH2 N

Figure 2.21 JZP-4.

medications. It is a well-tolerated drug, with skin rash being the most common side effect with an incidence of 5–10%. Other side effects include headaches, dizziness, and insomnia. The pharmacodynamics of lamotrigine may be more complex than its action on sodium channels, as its side-effect profile is different from other drugs such as oxcarbazepine that act on sodium channels [73]. Overall, lamotrigine acts as a mood stabilizer and inhibits the release of excitatory amino acid neurotransmitters such as aspartate and glutamate. The standalone status of lamotrigine has recently been challenged by a compound, elpetrigine, from GlaxoSmithKline (Figure 2.21). The switch from triazine to pyrazine and from dichloro- to trichlorophenyl results in increased potency for sodium and calcium channels, and potential reduction of the dermatological side effects [74, 75]. Elpetrigine is currently in clinical development, with phase I trials completed.

2.7 Metformin

Metformin is the most widely used oral hypoglycemic agent. It is the first-line drug for the treatment of type 2 diabetes and the only oral antidiabetic besides glibenclamide in the World Health Organization Model List of Essential Medicines [76]. Unusually for a drug molecule, it is a simple biguanide in structure and has more nitrogen than carbon atoms. The discovery can be traced back to medieval times, when extracts of the French lilac Galega officinalis were used to treat the frequent and painful urination accompanying diabetes mellitus [77]. The active constituent was later identified as galegine or isoprenylguanidine (Figure 2.22). Guanidine itself was shown by Watanabe to be elevated in the blood following removal of the parathyroid gland and suggested as the reason for decreased blood sugar levels following

HN

H N NH2 galegine

HN

NH2 NH2

guanidine

Figure 2.22 First antidiabetic guanidines.

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NH2

H N

HN

N H

NH2

Synthalin NH2

H N

HN

NH

N H

NH2

NH

Synthalin B

Figure 2.23 First marketed antidiabetic guanidine derivatives.

parathyroidectomy [78]. In supporting animal studies, rabbits injected with guanidine show a fall in blood sugar levels. Guanidine was too toxic for therapeutic use, while galegine was less so, and extracts of G. officinalis were briefly used as an antidiabetic in the early 1920s. These observations stimulated interest in the evaluation of guanidine derivatives, particularly diguanidines and biguanides. Frank at the University of Breslau prepared a series of diguanidines separated by a methylene chain. These compounds were more potent and less toxic than the monoguanidines [79]. From this series, Synthalin (Figure 2.23) progressed to clinical trials in 1926 and was marketed by Schering AG for the treatment of mild diabetes. Reports of adverse effects led to the introduction of Synthalin B with a longer methylene chain as a safer alternative. Nevertheless, both compounds were withdrawn in the 1940s due to potential liver and kidney toxicity. Meanwhile, depression of blood sugar levels was inhibitory to the reproduction of trypanosomes and Synthalin was found to have trypanocidal activity. This eventually led to the discovery of the amidine drugs stilbamidine and pentamidine, still in use for the treatment of trypanosomiasis and other protozoal infections. At the University of Vienna, Slotta and Tschesche reported a series of biguanides, including metformin, with hypoglycemic activity [80]. The compounds were nontoxic in animals, but these studies were not extended to humans and essentially forgotten for decades with the introduction of insulin. In the 1950s, Jean Sterne a physician at the Hospital Laennec and Aron Laboratories in Paris independently investigated biguanides as antidiabetic agents and selected metformin (Figure 2.24) for clinical development, naming it “Glucophage” (glucose eater) [81]. Simultaneously, the US Vitamin Corporation had examined a series of biguanides, from which phenformin emerged as the clinical candidate [82]. A year later, Mehnert in Germany reported studies with buformin [83].

NH Me

N Me

NH

N H

metformin

NH 2

NH N H

N H

NH NH 2

phenformin

Figure 2.24 Metformin and its withdrawn derivatives.

NH N H

NH

N H

buformin

NH 2

2.8 Topiramate

SAR studies with biguanides indicate that hypoglycemic activity is lost if more than one nitrogen is substituted. Among mono- and disubstituted biguanides, chains longer than five atoms were not tolerated. Despite extensive investigation, the precise mechanism of action of biguanides remains unknown. They do not increase insulin production in either the fasting or postprandial state. The primary effect of metformin is inhibition of gluconeogenesis in the liver, and increased glucose uptake across the cell membrane in peripheral tissues. A number of molecular targets have been proposed, although separating the primary effector from downstream consequences of glucose regulation is complicated. Phenformin and buformin were the first biguanides to receive clinical approval due to their greater potency than metformin [84]. Lactic acidosis is the most serious side effect of biguanides. It is more pronounced with phenformin and buformin than metformin, probably due to decreased metabolism of the alkyl chain in some patients leading to prolonged drug exposure. Both phenformin and buformin were withdrawn from the market in most countries in the 1970s, leading to renewed interest in metformin. Clinical studies demonstrated that metformin is unique in having antihyperglycemic activity without causing overt hypoglycemia or weight gain unlike sulfonylureas or insulin. Furthermore, the drug is still efficacious in cases of insulin resistance. Metformin was first marketed in France in 1979 by Lipha, now Merck, that had acquired Aron Laboratories and later received FDA approval in 1994. Metformin has 50–60% oral bioavailability with negligible binding to plasma proteins. Its plasma half-life is estimated at 1.5–4.9 h, reaching a peak concentration 1–2 h after oral dosing. Metformin does not undergo significant metabolism, and is excreted intact in urine. The most serious potential side effect is lactic acidosis, and for this reason it is contraindicated in patients with impaired liver or kidney function. Lactic acidosis is believed to be an on-target effect as metformin inhibits gluconeogenesis, the pathway by which lactate buildup is normally prevented by conversion to glucose. The most common adverse effect is gastrointestinal upset, which is rare upon prolonged steady use of the drug. In addition to antidiabetic therapy, metformin is under investigation for other diseases that feature insulin resistance such as polycystic ovary syndrome, nonalcoholic fatty liver disease, and premature puberty. The favorable effects of metformin in lowering blood pressure, serum triglycerides, total cholesterol, and low-density lipoprotein-cholesterol suggest that it is beneficial for the treatment of cardiovascular disease. Metformin has a remarkable history in terms of its discovery. The compound was first prepared in 1922 but did not receive approval for more than 50 years. Since then, it has become established as the first-line drug for the treatment of type 2 diabetes and is likely to be useful for additional therapeutic indications.

2.8 Topiramate

Topiramate (Figure 2.25) was identified in the early 1980s as an anticonvulsant agent in a pharmacological model that measures the ability of compounds to prevent

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O O S O NH2

O O

O O

O

topiramate

O O S O NH2

O O O S O O

H N

NH2 S O O

O O

RWJ 37947

H N

NH2 S O O

S JNJ-26990990

benzylsulfamide

Figure 2.25 Topiramate and related compounds.

maximal electroshock seizures in mouse [85, 86]. Structurally, it bears no resemblance to previously used anticonvulsant agents and was originally synthesized as a chemical intermediate during a program to identify inhibitors of fructose-1,6bisphosphatase as potential antidiabetic agents [87, 88]. In combination with the sulfamate functionality, which appears to be essential for activity, many variations on the scaffold are tolerated. These range from relatively simple phenylethyl groups to the rather sophisticated sugar-derived core of topiramate itself. The key publication on the discovery process emphasizes the importance of oral bioavailability, duration of action, and minimal neurotoxicity as important features in the selection of the drug for clinical development [85]. Topiramate is generally used in patients who are unresponsive to other anticonvulsant medications or is used in combination with other anticonvulsants to treat intractable seizures. Topiramate has also been approved for the prophylactic treatment of migraine headaches in some countries and this is now the most common use. Topiramate is a structurally unique anticonvulsant agent. There are no former abandoned analogues. Topiramate displays the following actions related to seizure reduction: (a) antagonism of the AMPA/kainate subtype of the glutamate receptor, (b) blockage of voltage-dependent sodium channels in nerve cells, decreasing excessive nerve-cell firing, (c) increase in the activity of the neurotransmitter gamma-aminobutyrate at some subtypes of the GABA-A receptor [89]. Topiramate also inhibits carbonic anhydrase, particularly isozymes II and IV. The relation of this activity to seizure reduction is unclear although recent patent applications covering topiramate analogues indicate that reduction of carbonic anhydrase activity could be beneficial in decreasing side effects seen with topiramate, such as paresthesia. Because there is so far no single clear mechanism of action that links to the pharmacological effects, a target-based screening approach to the identification of new pharmacological analogues is not applicable. A number of publications and patents from the original discovery group have appeared and some more potent analogues of topiramate have been disclosed [90, 91]. These efforts employed the same pharmacological models as in the original discovery. One analogue from these efforts, RWJ 37947, a cyclic sulfate analogue of topiramate, reportedly entered development, but no new information on this compound has appeared since 2002. A recent report on the design of additional topiramate analogues [92] indicates that the high potency of this compound against

2.9 Valproate

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carbonic anhydrase enzymes was thought likely to lead to undesirable side effects in certain patients and prevented its further development. Two recent patents and one publication disclose analogues, such as JNJ-26990990 (Figure 2.25), which bear some resemblance to the structural motifs in the original manuscript on topiramate [92–94]. A full report that details the optimization process leading to this clinical candidate has appeared. The switch to a sulfamide series was motivated by a desire to generate topiramate analogues with andiabetic and antiobesity effects but devoid of anticonvulsant activity. However, subsequent screening of these sulfamides with simplified structures revealed promising anticonvulsant activity. JNJ-26990990 in particular shows broad-spectrum activity in a number of seizure models and is only a very weak inhibitor of carbonic anhydrase enzymes. JNJ26990990 entered phase II clinical trials in October 2007. Somewhat analogous anticonvulsants, such as benzylsulfamide (Figure 2.25), have also been discovered from a pharmacophore-based design approach, but they appear not to have progressed to clinical development [95] (Figure 2.25).

2.9 Valproate

The usefulness of valproic acid (Figure 2.26) as an anticonvulsant was made as a serendipitous discovery [96, 97] during the investigation of khelline derivatives as potential anticonvulsants. After finding it difficult to dissolve some derivatives in common organic solvents, a suggestion led to the use of valproic acid as a solubilizer. Anticonvulsant activity was observed in all the samples and valproic acid itself was found to be the anticonvulsant agent. The initial human epilepsy trials were reported in 1964 followed by first approval in France in 1967. Valproate is a widely used anticonvulsant agent because of its activity against different types of seizures. It is also approved for the treatment of acute and chronic migraine and for mania. Valproic acid is a pioneer drug, that is, the first of its class to reach the market. There are no former or previously used analogues. Valproic acid is associated with significant side effects such as reproductive dysfunction and teratogenicity, and there is interest in developing improved analogues [98]. A number of structurally similar compounds have a valproate-like profile in animal seizure models. Valpromide, the amide derivative of valproic acid, is slightly more potent in animal models and is rapidly transformed to valproate in man,

O

O

O OH

NH2

O NH2

NH2 F

valproic acid

valpromide

Figure 2.26 Valproic acid and related compounds.

valnoctamide

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essentially acting as a prodrug. It is marketed as an antiepileptic drug and antipsychotic agent in Europe. Valnoctamide, a metabolically stable analogue of valpromide, has been marketed as an anxiolytic and sedative since the mid-1960s. It also exhibits greater anticonvulsant potency than valproate in animal models and is significantly less embryotoxic than valproic acid; however, its usefulness as an antiepileptic in man remains to be determined. Recently, a constrained cyclopropane analogue of valpromide (Figure 2.26) was reported to have superior activity in the MES models used in the discovery of vaproate and topiramate [99]. Since the broad spectrum of utility of valproic acid probably arises from its diverse actions at the molecular level, a target-based screening approach to the identification of pharmacological analogues remains problematic.

2.10 Summary

Nearly every drug has one or more analogues, which are similar to the original drug both in their structures and in their pharmacological mechanism of action (“full analogues”) or their similarity is restricted to the pharmacological mechanism of action (“pharmacological analogues”). We analyzed the Top 100 most frequently used drugs based on their sales data in 2006 and, interestingly, their value amounted to 51% of the global sales of all drugs, which indicates their importance. Among these 100 drugs, 9 standalone drugs were identified, that is, compounds that are pioneer drugs for which there are no effective analogues. In this chapter, we have described the serendipitous discovery of these standalone drugs and the past and present efforts to design analogues that would improve upon their properties. Six standalone drugs, namely, acetaminophen, bupropion, lamotrigine, metformin, topiramate, and valproate, have no clear mechanism of action. They display some weak in vitro activities; however, there is no proven correlation between these in vitro activities and their pharmacological properties in animals. Acetylsalicylic acid is a selective irreversible COX-1 inhibitor, but in a higher dosage it loses its selectivity. Aripiprazole is a partial agonist of the D2/3 receptors, but other targets (5-HT2A, 5-HT2B) may also play a role in its in vivo activity. The mechanism of action of ezetimibe was discovered only recently (2005). Elucidation of the mechanism of action and serendipitous observations may lead to new useful analogues of these standalone drugs.

Acknowledgment

We thank Duane Burnett, William Greenlee, and Bruce E. Maryanoff for carefully reading and reviewing the manuscript.

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Lamotrigine in the treatment of bipolar disorder. Exp. Opin. Pharmacother., 6, 1401–1408. Reynolds, E.H., Chanarin, I., Milner, G., and Matthews, D.M. (1966) Anticonvulsant therapy, folic acid and vitamin B12 metabolism and mental symptoms. Epilepsia, 7, 261–270. Leach, M.J., Marden, C.M., and Miller, A.A. (1966) Anticonvulsant therapy, megaloblastic hematopoeisis and folic acid metabolism. Q. J. Med., 35, 521–537. Baxter, M.G., Elphick, A.R., Miller, A.A., and Sawyer, D.A. (1981) 1,2,4-Triazine derivatives, pharmaceutical compositions and intermediates utilized for their preparation. European Patent 21121. Leach, M.J., Marden, C.M., and Miller, A.A. (1986) Pharmacological studies on lamotrigine, a novel potential antiepileptic drug: II. Neurochemical studies on the mechanism of action. Epilepsia, 27, 490–497. Cohen, A.F., Ashby, L., Crowley, D., Land, G., Peck, A.W., and Miller, A.A. (1985) Lamotrigine (BW430C), a potential anticonvulsant. Effects on the central nervous system in comparison with phenytoin and diazepam. Br. J. Clin. Pharmacol., 20, 619–629. Smith, D., Baker, G., Davies, G., Dewey, M., and Chadwick, D.W. (1993) Outcomes of add-on treatment with lamotrigine in partial epilepsy. Epilepsia, 34, 312–322. Leach, M.J., Randall, A.D., Stefani, A., and Hainsworth, A.H. (2002) Lamotrigine: mechanisms of action, in Antiepleptic Drugs, 5th edn (eds R.H. Levy, R.H. Mattson, B.S. Meldrum, and E. Perucca), Lippincott Williams & Wilkins, Philadelphia, pp. 363–369. Foreman, M.M., Hanania, T., Stratton, S.C., Wilcox, K.S., White, S., Stable, J.P., and Eller, M. (2008) In vivo pharmacological effects of JZP-4, a novel anticonvulsant, in models for anticonvulsant, antimania and antidepressant activity. Pharmacol. Biochem. Behav., 89, 523–534. Foreman, M.M., Hanania, T., and Eller, M. (2009) Anxiolytic effects of lamotrigine and JZP-4 in the elevated plus maze and in

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the four plate conflict test. Eur. J. Pharmacol., 602, 316–320. Goodarzi, M.O. and Bryer-Ash, M. (2005) Metformin revisited: re-evaluation of its properties and role in the pharmacopoeia of modern antidiabetic agents. Diabetes Obesity Metab., 7, 654–665. Bailey, C.J. and Day, C. (2004) Metformin: its botanical background. Pract. Diabetes Int., 21, 115–117. Watanabe, C.K. (1918) Studies in the metabolism changes induced by administration of guanidine bases. I. Influence of injected guanidine hydrochloride upon blood sugar content. J. Biol. Chem., 33, 253–265. Frank, E., Nothmann, M., and Wagner, A. (1926) Über synthetisch dargestellte K€ orper mit insulinartiger Wirkung auf den normalen und den diabetischen organismus. Klin. Woch., 5, 2100–2107. Slotta, K.H. and Tschesche, R. (1929) Die blutzuckersenkende Wirkung der biguanide. Chem. Ber., 62, 1398–1405. Sterne, J. (1957) Du nouveau dans les antidiabetiques. La N,Ndimethylamineguanyl guanide (N.N.D.G.). Maroc Med., 36, 1295–1296. Ungar, G., Freedman, L., and Shapiro, S.L. (1957) Pharmacological studies of a new oral hypoglycaemic drug. Proc. Soc. Exp. Biol. Med., 100, 190–192. Mehnert, H. and Seitz, W. (1958) Weitere ergebnisse der diabetesbehandlung mit blutzuckersenkenden biguaniden. Munch. Med. Woch., 100, 1849–1851. Schafer, G. (1983) Biguanides: a review of history, pharmacodynamics and therapy. Diabetes Metab. Rev., 9, 148–163. Maryanoff, B.E., Nortey, S.O., Gardocki, J.F., Shank, R.P., and Dodgson, S.P. (1987) Anticonvulsant O-alkyl sulfamates. 2,3:4,5-Bis-O-(1-methylethylidene)-betaD-fructopyranose sulfamate and related compounds. J. Med. Chem., 30, 880–887. Maryanoff, B.E. and Gardocki, J.F. (1985) Anticonvulsant sulfamate derivatives, US Patent 4,513,006. Van Passel, L., Arif, H., and Hirsch, L.J. (2006) Topiramate for the treatment of epilepsy and other nervous system disorders. Exp. Rev. Neurother., 6, 19–31.

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versus models for new antiepileptic drug discovery. Epilepsy Res., 68, 22–28. Maryanoff, B.E., Costanzo, M.J., Shank, R.P., Schupsky, J.J., Ortegon, M.E., and Vaught, J.L. (1993) Anticonvulsant sugar sulfamates. Potent cyclic sulfate and cyclic sulfite analogues of topiramate. Bioorg. Med. Chem. Lett., 3, 2653–2656. Maryanoff, B.E., Costanzo, M.J., Nortey, S.O., Greco, M.N., Shank, R.P., Schupsky, J.J., Ortegon, M.E., and Vaught, J.L. (1998) Structure–activity studies on anticonvulsant sugar sulfamates related to topiramate. Enhanced potency with cyclic sulfate derivatives. J. Med. Chem., 41, 1315–1343. Parker, M.H., Smith-Swintosky, V.L., McComsey, D.F., Huang, Y., Brenneman, D., Klein, B., Malatynska, E., White, H.S., Milewski, M.E., Herb, M., Finley, M.F.A., Liu, Y., Lubin, M.L., Qin, N., Iannucci, R., Leclercq, L., Cuyckens, F., Reitz, A.B., and Maryanoff, B.E. (2009) Novel, broad-spectrum anticonvulsants containing a sulfamide group: advancement of N-((benzo[b]thien-3-yl)methyl)sulfamide (JNJ-26990990) into human clinical studies. J. Med. Chem., 52 (23), 7528–7536. Parker, M.H., Reitz, A.B., and Maryanoff, B.E., Preparation of benzo-fused heteroaryl sulfamide derivatives for the treatment of epilepsy and related disorders, WO2006023861(2006). McComsey, D.F., Parker, M.N., Reitz, A.B., and Maryanoff, B.E.,Preparation of sulfamates and sulfamides with oxygen containing ring systems useful for the treatment of epilepsy and related disorders, WO2006007435(2006). Gavernet, L., Barrios, I.A., Sella Cravero, M., and Bruno-Blanchl, L.E. (2007) Design, synthesis, and anticonvulsant activity of some sulfamides. Bioorg. Med. Chem., 15, 5604–5614. Sneader, W. (1995) Drug Prototypes and their Exploitation, John Wiley & Sons, Ltd., Chichester, p. 752.

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in clinical neuroscience. Psychopharmacol. Bull., 37 (Suppl. 2), 5–16. 98 Rogawski, M.A. (2006) Diverse mechanisms of antiepileptic drugs in the development pipeline. Epilepsy Res., 69, 273–294.

99 Pessah, N., Bialer, M., Wlodarczyk, B.,

Finnell, R.H., and Yagen, B. (2009) 1Fluoro-2,2,3,3-tetramethylcyclopropane carboxamide, a novel potent anticonvulsant derivative of a cyclic analogue of valproic acid. J. Med. Chem., 52, 2233–2242.

J
anos Fischer

Professor, Richter Plc., Gyömro˝i út 19/21, 1103 Budapest, Hungary J
anos Fischer is a Senior Research Scientist at Richter Plc., Budapest, Hungary. He received his MSc and PhD degrees in organic chemistry from the Eotvos University of Budapest under Professor A. Kucsman. Between 1976 and 1978, he was a Humboldt Fellow at the University of Bonn under Professor W. Steglich. He has worked at Richter Plc. since 1981 where he participated in the research and development of leading cardiovascular drugs in Hungary. His main interest is analoguebased drug discovery. He is the author of some 100 patents and scientific publications. In 2004, he was elected as a Titular member of the Chemistry and Human Health Division of IUPAC. He received an honorary professorship at the Technical University of Budapest.

C. Robin Ganellin

Professor, University College London, Department of Chemistry, 20 Gordon Street, London WC1H 0AJ, UK C. Robin Ganellin studied chemistry at London University, receiving a PhD in 1958 under Professor Michael Dewar, and was a Research Associate at MIT with Arthur Cope in 1960. He then joined Smith Kline & French Laboratories in the UK and was one of the coinventors of the revolutionary drug, cimetidine (also known as Tagamet). In 1986, he was made a Fellow of the Royal Society and appointed to the SK&F Chair of Medicinal Chemistry at University College London, where he is now Professor Emeritus of Medicinal Chemistry. Professor Ganellin is coinventor of over 160 patents and has authored over 260 scientific publications. He was President of the Medicinal Chemistry Section of the IUPAC and is Chairman of the IUPAC Subcommittee on Medicinal Chemistry and Drug Development.

References

Arun Ganesan

Professor, University of Southampton, School of Chemistry, Southampton S017 1BJ, UK A. Ganesan was born in Malaysia and graduated from the National University of Singapore, with a degree in chemistry and microbiology (1985) and an Honours degree in chemistry (1986). He obtained his PhD in 1992 from the University of California-Berkeley, working under the supervision of Professor Clayton H. Heathcock in the area of synthetic methodology and natural product total synthesis. Following a postdoctoral position with Professor Gregory L. Verdine at Harvard University in 1992–1993 working on DNA binding proteins, he began his independent career in 1993 as a Senior Research Chemist at the Center for Natural Product Research in Singapore. In 1996, he was promoted to Principal Investigator at the Institute, where he headed the Medicinal and Combinatorial Chemistry Group. He moved to Southampton in 1999 as a Reader to join National Combinatorial Center of Excellence. Ganesan is a Treasurer of the Royal Society of Chemistry’s High Throughput Chemistry and New Technologies subject group and a member of the Organic Division Executive. He serves on the IUPAC Subcommitee for Medicinal Chemistry and Drug Development. He is a past Chair of the Gordon Research Conference and was awarded a JSPS fellowship as visiting professor at the Tokyo Institute of Technology, Japan. He is on the advisory board of QSAR and Combinatorial Science and Current Chemical Biology. He is a cofounder and Director of Karus Therapeutics (http://www.karustherapeutics.com), a spin-off of the University of the Southampton, and a Business Fellow of the London Technology Network. John Proudfoot

Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Rd., Ridgefield, CT 06877-0368, USA John Proudfoot is a Medicinal Chemist with over 20 years of experience in drug design, ranging from fragment-based lead identification to late-stage lead optimization. He received his doctorate from the University College Dublin in Ireland, working with Professor Dervilla Donnelly. After postdoctoral studies under Professor Carl Djerassi at Stanford University and under Professor John Cashman at the University of California, San Francisco, he joined Boehringer Ingelheim Pharma in 1987. He is a coinventor of 28 issued US patents, including the one for the marketed drug, nevirapine, the first nonnucleoside HIV-1 reverse transcriptase inhibitor to receive regulatory approval.

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3 Application of Molecular Modeling in Analogue-Based Drug Discovery Gy€orgy G. Ferenczy

3.1 Introduction

Analogue-based drug discovery benefits from molecular modeling and computational chemistry in various ways. The search for new medicaments that are similar to existing ones is an approach where the knowledge accumulated for the parent molecule drives drug discovery efforts. This requires a thorough understanding of all aspects of the effect of the parent molecule and this is supported by computational studies ranging from bioinformatics to pharmacokinetics. When research is not restricted to close structural analogues, the role of molecular modeling includes the extension of the notion of analogy; important features related to, but not apparent from the structural formula, can be identified and exploited in the design of analogues. One can mention, as examples, the better appreciation of three-dimensional structures by conformational analysis, the derivation of certain properties from the electronic structure of molecules and pharmacophore models that help to distinguish between properties that are important and those that are unimportant for binding to the target. The identification of features or structural motifs that are not relevant for interaction with the target is usually also fundamental, as it helps to design molecules that are outside competitor patent space that is a critical point in analogue-based research. Moreover, the “unimportant” parts of a molecule are suitable points for modifications in order to improve selectivity profile and pharmacokinetic properties that may be crucial aspects in developing an analogue with properties superior to the original molecule. The availability of information for the original molecule is highly advantageous for modeling approaches. In fact, a critical aspect of molecular modeling is to apply it at the appropriate time when, on the one hand, enough information is available to develop predictive models and, on the other hand, the model is able to provide nonobvious information that represents a useful contribution to the research. Analogue-based discovery, by definition, is based on information gathered for the original drug molecule that can be used as input for modeling work. Structure-based approaches that exploit the atomic resolution structure of the target are efficient and increasingly applied in molecular modeling and they may also

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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be applied in analogue-based research. Experimental or homology-based enzyme/ receptor structures may make it possible to design structurally loosely related functional analogues. The above discussion already indicates that, although analogue-based drug research has some particular features, the molecular modeling approaches applied are not specific. This is all the more so since molecular modeling – and we can state that to a large extent drug discovery in general – is largely based on the concept of analogy even if research is directed to the discovery of a novel molecule rather than an analogue of an existing drug. One can mention here that lead optimization is basically the search of an improved “analogue” of the lead molecule, and even lead identification by virtual screening often exploits the information available for known ligands of a target protein to optimize the virtual screening protocol. In this chapter, the discussion is restricted to examples where the lead is a drug, drug candidate, or an endogenous ligand of the target. Perhaps, it is not fair to term some of these drugs as analogues for two reasons. One is that they deviate considerably from their parent structure and this is partly due to the involvement of molecular modeling in the research process. The other reason is that when the parent molecule is not a marketed drug that already passed all stages of the development, then the research directed toward a structural analogue has an increased risk. In the past, molecular modeling was considered to be most useful in optimizing binding to the target. However, with increasing importance of ADMET properties in the early phases of drug research, molecular modeling plays an increasingly important role in optimizing properties such as metabolism, CYP inhibition and induction, and hERG channel activity. Nevertheless, these aspects of molecular modeling are less pronounced in the following discussions mainly because these drug discovery programs started several years ago. The examples presented are based on scientific publications that describe, quote, or at leastindicatetheuseofmodelingtechniquesinthediscoveryofanalogues.Incertaincases, the discovery process has been published in detail, while in some other cases it can only be approximately recovered. Thus, the examples presented may not reflect the complete contribution of modeling techniques in the course of a particular analogue research project. Nevertheless, they illustrate the contribution of molecular modeling in this field. No attempt has been made to describe all aspects of the discovery processes; rather the focus is on molecular modeling contributions. The research that led to the drugs or drug candidates described below began several years ago and since then, structural information on some of the targets has become available. This makes it possible to reevaluate certain hypotheses and observations made in the course of the discovery and it is particularly instructive for future applications.

3.2 Cilazapril: An ACE Inhibitor

Angiotensin-converting enzyme (ACE), part of the renin–angiotensin system, is an appropriate target for antihypertensive drugs [1]. Captopril (1) of Bristol-Meyers

3.2 Cilazapril: An ACE Inhibitor

Squibb was the first marketed nonpeptidic ACE inhibitor to be launched in the early 1980s. N SH

O O

OH

1, captopril

The development of captopril was preceded by the identification of a polypeptide that was first isolated and then synthesized and was shown to inhibit ACE [2]. A schematic model of the ACE active site (vide infra) was built after the recognition of its similarity to pancreatic carboxypeptidase A [3]. The model guided structure–activity studies that led to the discovery of SQ20881, a nonapeptide, and later to captopril. The clinical investigations of these compounds proved that they are effective antihypertensives. Side effects of captopril were attributed to the mercapto function and its replacement by weaker chelating functions led to the discovery of enalapril (2, Merck) [4].

O

O N H

N O O

OH

2, enalapril

The design of another ACE inhibitor by researchers at Roche is a well-documented example where computational methods made a significant contribution. The research used as starting points the two-dimensional model of the active site [3] (Figure 3.1) and the earlier structure–activity studies of captopril and related compounds [3–6]. An analysis of the available conformations of captopril generated limits for the relative positions of the three interaction points defined by the carboxy, amide, and thiol functions [7]. Three related bicyclic systems (3, 4, 5) were chosen so that they were able to hold these functions and their functionalization was expected to predictably influence their conformations. O HS N n

N N

O O 3

OH

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O R1

N

R2

N O O

OH

4 O

R1 R2 O

COOH 5

A series of compounds was prepared, conformations were investigated by NMR, and X-ray crystallography and biological activities were measured. The collected data revealed relationships between the relative positions of the functional groups and the ACE inhibition demonstrated by the molecules. Even the best molecules were, however, less active than captopril and enalapril. This was attributed to the finding that, according to NMR and X-ray experiments, the ring systems hold the functional groups in a relative position that corresponds to a high-energy conformation of alanylproline as it was calculated by molecular mechanics [8]. Therefore, new 7,6 and 8,6-bicyclic systems (6, 7) were synthesized in accordance with molecular graphic studies that suggested conformations with appropriate orientation of the carboxyl, carbonyl, and zinc-coordinating groups.

“CARBOXYPEPTIDASE A”

Zn++

NH

R

O

CH

C

“ANGIOTENSIN–CONVERTING ENZYME”

Zn++

+

CH2 O– NH

CH

C O

NH

R2

O

CH

C –O

–O

O C

C O

NH

R1

O

CH

C

R3

O

O C

CH2 CH

C

–S

CH2 CH

CH2 O– CH2 CH

H

+

R NH CH

C O O–

N

CH

N

CH

C O O–

CH3 O C

O–

C O

Figure 3.1 Schematic representation of the binding of substrates and inhibitors at the active site of pancreatic carboxypeptidase A, and the hypothetical active site of angiotensin-converting enzyme. Reprinted with permission from Ondetti et al. [3]. Copyright (1977) AAAS.

3.2 Cilazapril: An ACE Inhibitor

Y N N R2

O

CO2 R1

6

Y N N R2

O

CO2R1

7

Among the synthesized compounds, a dicarboxylic acid derivative of 7 was found to be the most active ACE inhibitor. Its monoethylester, cilazapril (8), is a prodrug with superior biological characteristics [8] and was launched in 1990.

N O

N

O

N H

O

CO2H

8, cilazapril

Cilazapril thus contains interacting functions similar to capropril, but the chemical nature of these functions and the skeleton that holds them are significantly different. The replacement of the thiol group with homophenylalanine and the introduction of the ester group are equivalent to the modifications appearing in enalapril with respect to captopril. On the other hand, a completely new feature of cilazapril is the bicyclic skeleton that is able to present the functional groups in a favorable orientation, and owing to its rigidity offers free energy gain by decreasing the entropy penalty upon binding. The crystal structures of several ACE–ligand complexes were published later. They include the complexes of captopril and enalapril [9]. The experimental structures confirm the presence of the presumed principal interactions, and the conformations of the ligands in the complex are consistent with the results of modeling studies (Figure 3.2).

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Figure 3.2 Important interactions in the Captopril–ACE complex (PDB ID: 1UZF [9]). Captopril carbons are shown in green and carbons of interacting residues are white. N-atoms are blue, O-atoms are red, and the

S-atom is yellow. Only polar H-atoms are shown and they are colored blue green. H-bonds are indicated by dashed yellow lines and other interactions are indicated by magenta lines.

3.3 Atorvastatin: A HMG-CoA Reductase Inhibitor

The biosynthesis of cholesterol involves a key step in which 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is transformed to mevalonate by HMG-CoA reductase (HMGR). The inhibition of this process is an effective way to decrease serum cholesterol levels, and hypocholesterolemic agents decrease the risk of coronary artery disease [10]. The first HMGR inhibitors discovered were either fungal metabolites or synthetic derivatives thereof, such as compactin (9, Sankyo), lovastatin (10, Merck & Co), simvastatin (11, Merck & Co), and pravastatin (12, Sankyo). O

HO O

O O H

9, compactin

3.3 Atorvastatin: A HMG-CoA Reductase Inhibitor

O

HO O

O O

H

10, lovastatin

O

HO O

O O

H

11, simvastatin

O

OH

HO HO

O O

H

HO

12, pravastatin

Another series of HMGR inhibitors (13) with a different lipophilic group was disclosed by Merck Sharpe and Dohme [11–13].

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O

HO O

A

B Y X

13

This initiated at Parke-Davis a search for inhibitors containing a lactone and a hydrophobic moiety together with the optimal spacer in between. A series of molecules with the general formula 14 was synthesized and their cholesterol synthesis inhibition was measured [14]. O

HO O X R1

N

R2

14

First, X ¼ ethyl was identified as the optimal spacer. Then, structure–activity studies and the comparative conformational analysis of this series, compactin (9), and a molecule in the Merck series (13) led to the proposal that the X ¼ ethyl bridge in 14 (ethylene 13) adopts a nearly perpendicular position with respect to the pyrrole ring. For certain molecules, for example, Merck (13), this is the only allowed conformation, while in some other cases, the observed lower potency is attributed to the fact that this conformation is energetically disfavored. The authors of Ref. [14] also note that the calculated minimum energy conformation does not agree with the one found experimentally in the crystal of compactin [15]. The latter X-ray conformation corresponds to a secondary minimum with slightly higher energy. On the basis of the observed SAR of the series 14, it was postulated that for an optimal potency the extension of the R1 and R2 substituents cannot exceed 5.9 and
3.3 A, respectively. Further studies aimed at finding correlation between the potency and the charge distribution of the R1 group. This was prompted by the polar nature of the isobutyric ester containing fungal metabolite inhibitors (cf. molecules 9 and 10). However, it was concluded that potency is insensitive to the polarity of the R1 group. The relatively low potency of compounds of type 14 with respect to certain members of series 13 prompted the structural comparison of these molecules. The superposition of the structures revealed that a methyl group (in para position with

3.3 Atorvastatin: A HMG-CoA Reductase Inhibitor

O

HO O F F H3C

N

CH3

CH3 Figure 3.3 Overlay of HMGR inhibitor templates. Drawing from Roth [16], with permission from Elsevier. Copyright (2002).

respect to the ethylene) in 13 occupies a position that is vacant in series 14 [16] (Figure 3.3). Indeed, the 3,4-dichloro- and 3,4-dibromo-pyrrole derivatives of 14 exhibited a significant potency increase. As the dihalogen-substituted compounds were found to be toxic, additional substituents were investigated [16, 17]. Among them, the Ph and CONHPh substitutions were found to be highly potent and appear in atorvastatin calcium (15, Parke-Davis) that has been marketed since 1997. 2+

Ca

O

O HO HO F N O HN

2 15, atorvastatin calcium

The X-ray structure of HMGR-atorvastatin [18] complex makes it now possible to interpret former findings and to assess the predictions made by modeling (Figure 3.4). The binding pocket and its interactions with the ligand are in line with former observations concerning the size restriction of the R1 and R2 substituents of molecule 14, the insensitivity of the affinity to the polarity of R1 and the availability

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Figure 3.4 Atorvastatin in the binding pocket of HMGR (PDB ID: 1HWK [18]). The pocket is colored according to the electrostatic potential: blue – negative; red – positive. Annotations highlight features discussed in the text.

of space for substituents at the 3 and 4 positions of pyrrole. Moreover, the predicted nearly perpendicular orientation of the ethyl spacer with respect to the pyrrole ring was indeed observed in the experimental complex structure.

3.4 PDE4 Inhibitors

Phosphodiesterases (PDEs) are involved in the transformation of cyclic nucleotides into their corresponding 50 -monophosphates. Several subtypes of PDEs have been described. PDE4 is a cyclic 30 ,50 -adenosine monophosphate (cAMP) specific isozyme that is dominant in immune and inflammatory cells and also in airway smooth muscle. The inhibition of PDE4 is proposed for the treatment of asthma and chronic obstructive pulmonary disease (COPD) [19]. A pioneering compound in PDE4-specific inhibition is rolipram (16) developed at Schering AG. O NH

O

O 16, rolipram

This compound did not reach the market due to its undesirable side effects, but it served as a prototype of selective PDE4 inhibitors. A large number of rolipram

3.4 PDE4 Inhibitors

congeners have been synthesized and tested for PDE4 inhibition. These studies led to the identification of the features essential for PDE4 binding. This pharmacophore model helped to find inhibitors with an improved side-effect profile compared to rolipram. The structure–activity studies identified a dialkoxyphenyl group and a hydrogen bond acceptor (carbonyl of pyrrolidone in rolipram) as a minimum requirement for efficient PDE4 binding [20, 21]. It was also found that steric hindrance limits the size of the para-alkoxy substituent. The relative orientation of the alkoxy groups and the hydrogen acceptor moiety was investigated by conformational analysis of rolipram analogues and by superposition of some rigid molecules. No unambiguous assignment of a bioactive conformation was possible, but investigations suggested a basically extended conformation in which the carbonyl group points away from the para-alkoxy substituent [20, 21]. The typical distance between the dialkoxy phenyl
group and the carbonyl dipole was proposed to have a value of 8–9 A [20]. The replacement of the pyrrolidone in rolipram by a 1,4-substituted cyclohexane ring fulfilled the criteria set by the above-described pharmacophore model and allowed extension of the structure–activity studies [21]. The introduction of the cyclohexane ring offered the advantage of a more rigid structure without a chiral center. The optimization of this series led to the discovery of cilomilast (17, GlaxoSmithKline) that was subject of an approval letter by FDA. Later, however, cilomilast development was discontinued. O

N O

O

OH 17, cilomilast

In other rolipram-based series, the pyrrolidone ring was replaced by benzamide derivatives and analogues. Among them, roflumilast (18, Nycomed) is under registration [22].

O O Cl O

F

N

N H

F

Cl 18, roflumilast

The crystal structures of several PDE4 complexes have been published since 2000 [23] and it is instructive to compare these experimental complex structures with the pharmacophore model that was derived with no atomic resolution structural knowledge of the target protein. The complexes of rolipram [24–27], cilomilast [27],

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and some more molecules confirm the crucial role of the dialkoxyphenyl moiety. The two ether oxygen atoms are hydrogen bonded to the side-chain amide N-atom of a glutamine residue, and the phenyl ring is surrounded by hydrophobic residues. This part of the molecule essentially replaces the adenine part of cAMP, the natural ligand of PDE4 [28]. The role of the pyrrolidone ring is not straightforward to interpret. In all except one X-ray structure [25], it apparently does not make any direct interaction with either the metals or the enzyme residues. The pyrrolidone carbonyl group is hydrogen bonded to a water molecule that, in turn, is hydrogen bonded to other water molecules having hydrogen bonds with the enzyme. The lack of more direct interactions is somewhat unexpected as the sensitivity of the binding affinity to this structural motif of the ligand was clearly observed. On the other hand, the comparison of the structures of rolipram and cilomilast (Figure 3.5) reveals that the relative position of the hydrogen bond acceptor with respect to the dialkoxy groups is not unequivocally defined, even though the presence of both seems to be important for binding. A possible explanation is that the ligand protrudes into an ordered set of water molecules contributing to the coordination of the metal ions and residues in the pocket and then for an efficient binding, the ligand has to provide appropriately positioned functions whose interactions are able to compensate for the disturbed hydrogen bonding network. An additional difficulty in interpreting rolipram binding has also to be noted. Rolipram binds to the full-length PDE4 with two different affinities [29], but the high-affinity binding is not observed in the truncated protein containing the catalytic domain appearing in the X-ray structures. As the large PDE4 binding pocket is not fully occupied by either rolipram or cilomilast, larger molecules may also be well accommodated. However, they can bind at the expense of expelling ordered water molecules. The free energy balance of such a binding is difficult to predict, although results in Ref. [30] indicate that it may be advantageous in the case of large-enough enthalpy gain. This would open the possibility to design inhibitors that create more direct interactions with the enzyme.

Figure 3.5 Superposition of rolipram (blue; PDB ID: 1OYN [24]) and cilomilast (brown; PDB ID: 1XOM [27]) in the PDE4 binding pocket. The water molecules are removed.

3.5 GPIIb/IIIa Antagonists

3.5 GPIIb/IIIa Antagonists

GPIIb/IIIa is a membrane-associated glycoprotein belonging to the integrin family of receptors. It plays a critical role in platelet aggregation. Antagonists prevent aggregation and thus thrombosis and are potentially suitable for the treatment of cardiovascular, cerebrovascular, and peripheral vascular diseases [31]. The peptide sequence Arg-Gly-Asp (RGD) has been recognized as the adhesive motif present in macromolecules, such as fibrinogen, that mediate platelet aggregation. Fibrinogen is a covalent dimer, the two halves of which have identical sequences, and it binds to GPIIb/IIIa on adjacent platelets and cross-links them. Small RGD containing peptides inhibit fibrinogen binding to platelets and inhibit platelet aggregation [32]. Thus, small peptides, peptidomimetics, or other molecules capable of binding to the RGD recognizing motif of GPIIb/IIIa may find therapeutic applications [33], and research was focused on developing RGD analogues. SK&F-107260 (19, developed by SmithKline Beecham) is a cyclic RGD analogue that owing to its more rigid structure exhibits higher affinity for GPIIb/IIIa than for RGD itself.

S O H2N

N

H N

S HN O

H N

O OH

N H

O

NH

O

19, SK&F-107260

The synthetic peptide eptifibatide (20, jointly developed by COR Therapeutics and Schering-Plough and launched in 1998) is also an RGD analogue whose improved profile is probably also due to its relative rigidity. NH NH2 O

N H H N

HN O

OH

O

O

N H

O

HN O

S

S H2N

N H O

20, eptifibatide

O N

NH

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Structure–activity studies with peptides identified the critical structural characteristics for RGD analogues. They include a basic group (guanidino of arginine) and an
acidic group (carboxylate of aspartic acid) with a distance of 12–15 A between them. The spacer exerts an effect on the affinity by its length, rigidity, influence on the acidity and basicity of the end groups, and potentially also through direct interactions with the receptor. A large number of structural studies on the conformation of RGD containing peptides and mimics have been performed to clarify the relative orientation of the basic and acidic groups. The information accumulated suggests that the end groups are optimally linked by a linear spacer whose rigidity is a significant factor for affinity (see, e.g., Ref. [34]). Indeed, it was found that affinity is a linear function of the number of rotatable bonds with a slope of 0.7 kcal/mol (G.G. Ferenczy, unpublished), the value associated with the free energy cost of freezing a rotatable bond upon binding. Several small molecules that bind to the GPIIb/IIIa receptor and inhibit platelet aggregation in the nanomolar range have been described. An early representative, tirofiban hydrochloride [35] (21, Merck & Co), has been marketed since 1998. O S O OH

HN

O O H Cl

21, tirofiban hydrochloride

3.6 HIV Protease Inhibitors

Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). HIV protease is responsible for the cleavage of precursor polyproteins to structural and functional proteins. This aspartic protease is one of the targets of anti-HIV therapy. First-generation protease inhibitors (PIs), such as saquinavir (22), indinavir (23), ritonavir (24), nelfinavir (25), and amprenavir (26), are all transition-state analogues with a hydroxyethylene core.

O H N

N

O N H

O 22, saquinavir

NH2 O

N OH H

H N

H

3.6 HIV Protease Inhibitors

N

OH

H N

N

N O

NH

N

H N

OH

O

23, indinavir S N

OH

O N H

O

S

H N

O

N

O

24, ritonavir

HO

H N

O

S

O

N

N H

H

OH H

25, nelfinavir

H N

O O

O

OH N O

S

NH2 O

26, amprenavir

The rapid discovery and development of these peptidomimetic agents were supported by atomic resolution X-ray crystallographic structures of the HIV protease and its complexes. These first-generation PIs have several shortcomings. Most important is drug resistance that comes from mutations as the response of the virus to drug treatments. In addition, inadequate pharmacokinetics manifests itself in one or several ways such as short plasma half-life, poor aqueous solubility, high protein binding, and modest oral bioavailability.

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The development of second-generation PIs was focused on addressing the abovementioned issues. First-generation PIs were used as starting points to develop improved agents. In order to reduce the effect of mutations on the activity of inhibitors, some interactions with the side chains of HIV protease were replaced by interactions with the protein backbone. The search for improved PIs and the interpretation of their activity were supported by structural biology and molecular modeling. The analysis of the X-ray structure of HIV protease with ritonavir (24) revealed that the isopropyl group on the thiazolyl ring interacts with the isopropyl group of the Val82 side chain of the S3 pocket [36]. This is in line with the reduced Ki value of ritonavir toward proteases mutated at this position. The omission of the P3 group reduced the activity but with the advantage of low serum binding. The activity was partially regained with the introduction of conformational constraint by cyclic urea (27) [36].

OH

O N

HN

N H

O

S

H N

N

O O

27, A-1555564

Twoalternativemodelsforthepositionandinteractionsofthecyclicureamoietywereset up. One of them is hydrogen-bonded with Asp29 and the other is hydrogen-bonded with Asp30 of the HIV protease. The degree of the intramolecular van der Waals interaction between the urea and the P1 benzyl group is also different in the two models [36]. Finally, the P20 (thiazolyl)methoxycarbonyl group was replaced by the dimethylphenoxyacetyl group leading to lopinavir (28).

OH

O N

HN O

N H

H N

O O

28, lopinavir

HIV protease inhibitors with a dimethylphenoxyacetyl group were first described in Refs [37, 38]. The X-ray structures of complexes with this group showed a shift of main-chain and side-chain atoms of residues 29 and 30 revealing a new region of conformational flexibility in the HIV protease [37]. The advantage of the dimethyl with respect to the monomethyl substitution was attributed, in part, to more favorable binding entropy of the former, as it always puts one of the methyl groups in a position with a beneficial interaction. The X-ray structure also showed that the methyl group

3.6 HIV Protease Inhibitors

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Table 3.1 Similarity coefficients between parent compounds and optimized drugs.

Parent compound

Optimized compound

Similarity coefficient [41]

N O

N

O

N SH

N H

O O

O

O

0.34

O

OH cilazapril

captopril

O

HO O

O O

H

O

O

0.14

HO compactin

HO O

HO

F O

0.29

N

F

O HN Cl atorvastatin

Ref. [13] compound 100

O O Cl

O NH

O

O

F F

O

rolipram

N

N H

0.27

Cl roflumilast (Continued)

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Table 3.1 (Continued)

Parent compound

Optimized compound

Similarity coefficient [41]

NH H2N

H N

O

N H

NH2

O

O

N H

S

HO

HN

O

HO

0.43

O OH O

O

O tirofiban hydrochloride

RGD S N HN N O O

N

NH

O

O HN HN

NH

NH O

0.39

OH

OH

O

O

O

S N ritonavir

lopinavir

has an optimal size and that larger substituents on the phenyl ring probably result in clashes with protein residues [38]. The X-ray crystal structure of lopinavir in complex with the HIV protease was published in 2002 [39]. It confirmed that in line with the design strategy the interaction of lopinavir with Val82 is significantly reduced. The dimethylphenoxyacetyl group occupies a position that was anticipated. The cyclic urea binds both to the backbone and to the side chain of Asp29 and it is accompanied with a slight movement of the protein backbone near Gly48. Lopinavir is an improved analogue of ritonavir with an increased HIV protease inhibitory activity that is moderately affected by mutations of Val82. Its bioavailability is highly enhanced when coadministered with ritonavir, a potent CYP3A4 inhibitor [40]. Lopinavir, coformulated with ritonavir, was launched by Abbott first in the United States in 2000.

References

3.7 Epilogue

Five examples of analogue-based drug discovery were presented. In all these cases, molecular modeling made a substantial contribution to the introduction of significant structural modifications. An indication of the important differences between the parent compounds and the optimized drugs is their low similarity coefficients presented in Table 3.1 [41]. In all these examples, the focus was on the interaction with the target and how the information and models available for the ligand–target interaction could be used to keep or improve affinity while other, typically pharmacokinetic, characteristics were optimized. On the other hand, modeling and computational methods are more and more able to predict properties relevant to pharmacokinetics, thus providing additional support to the optimization in analogue-based drug discovery projects.

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A.W., Ferguson, E., Hoefle, M.L., Ortwine, D.F., Newton, R.S., Sekerke, C.S., Sliskovic, D.R., Stratton, C.D.,and Wilson, M.W. (1991) Inhibitors of cholesterol biosynthesis. 3. Tetrahydro-4-hydroxy-6[2-(1H-pyrrol-1-yl)ethyl]-2H-pyran 2-one inhibitors of HMG-CoA reductase. 2. Effects of introducing substituents at positions three and four of the pyrrole nucleus. J. Med. Chem., 34, 357–366. Istvan, E.S.and Deisenhofer, J. (2001) Structural mechanism for statin inhibition of HMG-CoA reductase. Science, 292, 1160–1164. Burnouf, C.and Pruniaux, M.P. (2002) Recent advances in PDE4 inhibitors as immunoregulators and antiinflammatory drugs. Curr. Pharm. Des., 8, 1255–1296. Marivet, M.C., Bourguignon, J.-J., Lugnier, C., Mann, A., Stoclet, J.-C.,and Wermuth, C.-G. (1989) Inhibition of cyclic adenosine-30 ,50 -monophosphate phosphodiesterase from vascular smooth muscle by rolipram analogs. J. Med. Chem., 32, 1450–1457. Christensen, S.B., Guider, A., Forster, C.J., Gleason, J.G., Bender, P.E., Karpinski, J.M., DeWolf, W.E., Jr., Barnette, M.S., Underwood, D.C., Griswold, D.E., Cieslinski, L.B., Burman, M., Bochnowicz, S., Osborn, R.R., Manning, C.D., Grous, M., Hillegas, L.M., Bartus, J.O., Ryan, M.D., Eggleston, D.S., Haltiwanger, R.C.,and Trophy, T.J. (1998) 1,4-Cyclohexanecarboxylates: potent and selective inhibitors of phosophodiesterase 4 for the treatment of asthma. J. Med. Chem., 41, 821–835. http://www.nycomed.com/en/Menu/ RD/Pipeline/ accessed March 2009. Xu, R.X., Hassell, A.M., Vanderwall, D., Lambert, M.H., Holmes, W.D., Luther, M.A., Rocque, W.J., Milburn, M.V., Zhao, Y., Ke, H.,and Nolte, R.T. (2000) Atomic structure of PDE4: insights into phosphodiesterase mechanism and specificity. Science, 288, 1822–1825. Huai, Q., Wang, H., Sun, Y., Kim, H.Y., Liu, Y.,and Ke, H. (2003) Threedimensional structures of PDE4D in

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complex with roliprams and implication on inhibitor selectivity. Structure, 11, 865–873. Xu, R.X., Rocque, W.J., Lambert, M.H., Vanderwall, D.E., Luther, M.A.,and Nolte, R.T. (2004) Crystal structures of the catalytic domain of phosphodiesterase 4B complexed with AMP, 8-Br-AMP, and rolipram. J. Mol. Biol., 337, 355–365. Zhang, K.Y.J., Card, G.L., Suzuki, Y., Artis, D.R., Fong, D., Gillette, S., Hsieh, D., Neiman, J., West, B.L., Zhang, C., Milburn, M.V., Kim, S.-H., Schlessinger, J.,and Bollag, G. (2004) A glutamine switch mechanism for nucleotide selectivity by phosphodiesterases. Mol. Cell, 15, 279–286. Card, G.L., England, B.P., Suzuki, Y., Fong, D., Powell, B., Lee, B., Luu, C., Tabrizizad, M., Gillette, S., Ibrahim, P.N., Artis, D.R., Bollag, G., Milburn, M.V., Kim, S.-H., Schlessinger, J.,and Zhang, K.Y.J. (2004) Structural basis for the activity of drugs that inhibit phosphodiesterases. Structure, 12, 2233–2247. Ke, H., Colicelli, J.,and Huai, Q. (2003) The crystal structure of AMP-bound PDE4 suggests a mechanism for phosphodiesterase catalysis. Biochemistry, 42, 13220–13226. Rocque, W.J., Tian, G., Wiseman, J.S., Holmes, W.D., Zajac-Thompson, I., Willard, D.H., Patel, I.R., Wisely, G.B., Clay, W.C., Kadwell, S.H., Hoffman, C.R., and Luther, M.A. (1997) Human recombinant phosphodiesterase 4B2B binds (R)-rolipram at a single site with two affinities. Biochemistry, 36, 14250–14261. Hamelberg, D.,and McCammon, J.A. (2004) Standard free energy of releasing a localized water molecule from the binding pockets of proteins: doubledecoupling method. J. Am. Chem. Soc., 126, 7683–7689. Huang, F.and Hong, E. (2004) Platelet glycoprotein IIb/IIIa inhibition and its clinical use. Curr. Med. Chem. Cardiovasc. Hematol. Agents, 2, 187–196. Plow, E.F., Pierschbacher, M.D., Ruoslahti, E., Marguerie, G.,and

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Ginsburg, M.H. (1985) The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets. Proc. Natl. Acad. Sci. USA, 82, 8057–8061. Shebuski, R.J., Berry, D.E., Bennett, D.B., Romoff, T., Storer, B.L., Ali, F.,and Samanen, J. (1989) Demonstration of Ac-Arg-Gly-Asp-Ser-NH2 as an antiaggregatory agent in the dog by intracoronary administration. Thromb. Haemost., 61, 183–188. Fischer, M.J., Bruce, G., Harms, C.S., Kline, A.D., Mullaney, J.T., Nunes, A., Scarborough, R.M., Arfsten, A.E., Skelton, M.A., Um, S.L., Utterback, B.G.,and Jakubowski, J.A. (1997) Non-peptide RGD surrogates which mimic a Gly-Asp b-turn: potent antagonists of platelet glycoprotein IIbIIIa. J. Med. Chem., 40, 2085–2101. Egbertson, M.S., Chang, C.T.-C., Duggan, M.E., Gould, R.J., Halczenko, W., Hartman, G.D., Laswell, W.L., Lynch, J.J., Jr., Lynch, R.J., Manno, P.D., Naylor, A.M., Prugh, J.D., Ramjit, D.R., Sitko, G.R., Smith, R.J., Turchi, L.M.,and Zhang, G. (1994) Non-peptide fibrinogen receptor antagonists. 2. Optimization of a tyrosine template as a mimic for Arg-Gly-Asp. J. Med. Chem., 37, 2537–2551. Sham, H.L., Kempf, D.J., Molla, A., Marsh, K.C., Kumar, G.N., Chen, C.-M., Kati, W., Stewart, K., Lal, R., Hsu, A., Betebenner, D., Korneyeva, M., Vasavanoda, S., McDonald, E., Saldivar, A., Wideburg, N., Chen, X., Niu, P., Park, C., Jayanti, V., Grabowski, B., Granneman, G.R., Sun, E., Japour, A.J., Leonard, J.M., Plattner, J.J.,and Norbecki, D.W. (1998) ABT-378, a highly potent inhibitor of the human immunodeficiency virus protease. Antimicrob. Agents Chemother., 32, 3218–3224. Tong, L., Pav, S., Mui, S., Lamarre, D., Yoakim, C., Beaulieu, P.,and Anderson, P.C. (1995) Crystal structures of HIV-2 protease in complex with inhibitors containing the hydroxyethylamine dipeptide isostere. Structure, 3, 33–40. Beaulieu, P.L., Anderson, P.C., Cameron, D.R., Croteau, G., Gorys, V., Grand-Matre,

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C., Lamarre, D., Liard, F., Paris, W., Plamondon, L., Soucy, F., Thibeault, D., Wernic, D., Yoakim, C., Pav, S.,and Tong, L. (2000) 20 ,60 -Dimethylphenoxyacetyl: a new achiral high affinity P3-P2 ligand for peptidomimetic-based HIV protease inhibitors. J. Med. Chem., 43, 1094–1108. 39 Stoll, V., Qin, W., Stewart, K.D., Jakob, C., Park, C., Walter, K., Simmer, R.L., Helfrich, R., Bussiere, D., Kao, J., Kempf, D., Sham, H.L.,and Norbeck, D.W. (2002) X-ray crystallographic structure of

ABT-378 (lopinavir) bound to HIV-1 protease. Bioorg. Med. Chem., 10, 2803–2806. 40 Chrusciel, R.A., Thalsrivongs, S.,and Nicholas, J.A. (2001) HIV protease inhibitors in early development, in Protease Inhibitors in AIDS Therapy (eds R.C. Ogdenand C.W. Flexner), Marcel Dekker, New York, pp. 119–137. 41 Tanimoto similarity coefficients calculated with FCFP_4 fingerprints by SciTegic Pipeline Pilot 7.0.1.100 from Accelrys Software Inc.

Gy€ orgy G. Ferenczy

Chinoin Zrt., Sanofi-aventis, Tó u. 1-5, 1045 Budapest, Hungary Gy€orgy G. Ferenczy received his PhD from the E€ otv€ os University in Budapest in 1988. His early career involved quantum chemical method development and applications to extended systems. He was a postdoctoral fellow at the University of Oxford, UK, and at the University of Nancy, France. He was in the Drug Design Group of Gedeon Richter Plc., Budapest, as a Research Scientist and Group Leader. In 1999, he joined Chinoin Ltd., a member of the Sanofi-Aventis group. He has authored and coauthored 50 scientific papers and patents.

j83

4 Issues for the Patenting of Analogues Stephen C. Smith

4.1 Introduction

The statement in the first edition of this book that “The most fruitful basis to discover a new drug is to start with an old drug, and this has been the most common and reliable route to new products” [1] encapsulates the main issue in patenting new drug analogues, that is, they are in fact related to existing drugs and so may attract the charge of being obvious over them. For any invention to be patentable, it has to be new in a strict formal sense over what is already known, inventive, which is not obvious over what is already known, and be useful in a broadly industrial context. Drug products are no different, but because most are in some sense analogues of a known drug, it is the requirement to be inventive over what is known that generally provides the biggest hurdle to patenting new analogues. Given the enormous expense of bringing a new drug to sale, ensuring that patent protection is obtained in most major markets and maintained for a reasonable period to recover the investment and generate profit for shareholders is critical. No company can afford to find that, shortly after launching a new product, a competitor is able to introduce the same product without their permission. Although patent attorneys and other legal professionals will do their best to provide adequate protection and ameliorate any problems that may arise, the role of the inventor and associated research staff is critical to ensuring that drug patents have the best possible foundation. Getting a patent granted by satisfying the requirements of patent offices is of course the initial objective, but having its validity survive prolonged attack from determined competitors is the ultimate aim. Any product with annual US sales of the order of US$ 80 million plus is almost certain to find the validity of its US patent challenged as part of an Abbreviated New Drug Application (ANDA) related challenge to introduce a generic version. Consequently, it is necessary to try to “challenge proof” the validity of any patent protecting a new chemical entity (NCE) by taking extra care during the application stage. Here, the inventors – who usually include one or more medicinal chemists – have a critical role to play.

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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This chapter aims to give medicinal chemists and others involved in new drug research a basic survival guide to the patent system and how it is applied, in particular to patenting drug analogues. What follows can necessarily be only a broad overview of the situation for drug analogues, but it is hoped it will give medicinal chemists and others involved in new drug research enough understanding and practical information to avoid some of the pitfalls in the patenting process and allow them to have an informed dialogue with their legal advisers.

4.2 Patents: Some Fundamentals

Patents are exclusive rights granted for inventions in all fields of technology that meet the basic criteria for patentability. They are not available for designs, artistic works, or intellectual activities for which other intellectual property (IP) rights may be available such as registered designs and copyright. Like other property rights, patents may be sold, licensed, mortgaged, or otherwise commercialized. Patents are national rights that allow the owner to stop other people from doing what is claimed in the patent without the owner’s permission during a set time within the state concerned and while renewal fees are being paid. They do not give any positive right to commercialize an invention since specific permission may be required from other rights holders or the state (e.g., marketing authorization for a drug product). A patent is a special legal instrument first developed in Europe1) in the fifteenth century to promote technological development by providing timelimited protection from competition (monopoly) in exchange for public disclosure of how to make and use the technology concerned. They require a formal application accompanied by a patent specification that sets out in detail what is protected by legal claims (typically found at the end of the patent specification) and the nature of the invention and how it can be made and used, including in the case of chemical inventions detailed examples of making and often testing of compounds falling within the legal claims. In all major countries, the specifications of unexamined patent applications are now published or made available on-line 18 months after the earliest of their filing and priority dates.2) The process of obtaining a granted patent varies (typically from 2 to 10 years) from the filing of the application during which time the patentability is examined and argued with the applicant against set criteria. Until relatively recently, many states restricted the availability of patent protection for pharmaceutical inventions, permitting only claims to processes for manufacturing an active ingredient. But now all major countries grant patents for pharmaceutical 1) The earliest known English patent for invention was granted by Henry VI to Flemish-born John of Utynam in 1449 giving him a 20-year monopoly for a method previously unknown in England for making stained glass that was needed for the windows of Eton College. 2) In the United States of America, it is still possible to prevent publication of a patent

application after 18 months by, at the time of filing, so requesting on the basis that the application will not be filed in a foreign country that itself provides for publication after 18 months. However, securing patents in at least the EU and Japan as well as the USA is the norm for analogue patenting and so this possibility is of little practical value.

4.3 Patentability

inventions with claims to chemical products, formulations, and processes for manufacture. These patents are granted for a period of at least 20 years, starting from the date when the application was filed.3) The advantage of patents that claim the chemical products themselves (“product patents”) is that they have the effect of preventing unauthorized commercial use of a claimed compound for any use (not just that found by the patentee) irrespective of how it has been made. A claim to a process for manufacturing a product by contrast only restricts the use of the process itself. This may be difficult for the patent owner to prove in practice without access to the manufacturing plant and so many patent systems particularly in Europe reverse the normal evidence requirements (“the burden of proof”) where infringement of a patented process is suspected. The alleged infringer then has to prove that the claimed process is not being used. Both granted patents and patent applications give rise to publications destroying the novelty of any of the subject matter disclosed in the specification should this be claimed in patent applications filed later. Granted patents in addition may restrict the freedom of competitors to commercialize anything within the patent claims without a license from the patent owner (“patentee”).

4.3 Patentability

The criteria that must be satisfied in deciding the patentability of an invention, that is, whether a patent may be granted or whether it should remain in force, are essentially the same in all major countries. In summary, the subject matter must be suitable material for a patent – it must not be specifically excluded by national law: thus, a method of treatment or diagnosis practiced on the human or animal body is excluded in most countries other than the United States of America; (2) novel in comparison with what is in the prior art – this includes publications of any sort (including abstracts of conference proceedings) and documented uses of the invention anywhere in the world in any language before the priority date of the application; (3) inventive over the relevant closest prior art – this includes publications and uses as in No. 2 above, but they must be relevant to the technical area and normally in relation to a single document rather than a combination of documents unless they are directly related to each other or else establish the level of general knowledge in the technical area, such as textbooks, manuals, and so on; (4) useful or capable of use primarily in an industrial context – in the United States of America “useful” has been very broadly interpreted to include methods of doing business or carrying out research, but in most other countries some technical character is needed; and (1)

3) In the United States of America, the term of drug analogue patents may be extended by up to 5 years for delays in the regulatory approval process and in Europe similar extended protection in the form of patent-like supplementary protection certificates is also available for up to 5 years.

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(5)

adequately described – there must be sufficient information in the patent specification for someone of reasonable skill in the relevant art to make and use the invention; for example, in the pharmaceutical context, the information must be sufficient for a medicinal chemist to make a compound and understand how it would be used in a drug.

For drug analogue patenting, the first criterion does not normally give problems, but satisfying the requirements of the remaining criteria, and especially that of being inventive, presents the most challenges both when patents are being written (“drafted”) and when they are being defended in litigation.

4.4 Important Elements of the International Patent System

It is important to stress at the outset that there is no such thing as a “world” patent giving rights everywhere. However, there are a number of unified patent examination and processing systems such as that provided by the European Patent Office (EPO) securing protection in up to 36 European states as designated by the applicant.4) Such systems allow a single patent application to be filed and examined in an official language (English, French, or German in the case of the EPO) leading eventually to a group of separate patents applicable in the individual nation states required. The international patent system is underpinned by three key major treaties: Paris Convention: 5) This allows a person to file a patent application in his own country, establishing a priority date for the invention, and then wait up to a year before deciding to file in other countries and still rely on the priority date of the first patent application, thus allowing opportunity to further evaluate the invention before incurring the substantial cost of foreign patent applications. Patent Cooperation Treaty (PCT): 6) This enables a person to obtain patent protection eventually in a large number of states that are signatories of the treaty 4) See EPO web site for more information: http://www.epo.org. 5) The Paris Convention for the Protection of Industrial Property, governing patents, trade marks, industrial designs, and so on, was originally signed in Paris in 1883; it has been amended several times, most recently in Stockholm in 1967 and is now acceded to by 173 states (May 2009); it requires signatories to grant reciprocal protection to a national of another signatory state and sets out basic rules that all contracting states must follow in relation to patent and other IP rights; see also World IP Organisation web site at http://www.wipo.int/treaties/en/ip/paris/. 6) The Patent Cooperation Treaty is administered by the World Intellectual Property

Organisation (WIPO) based in Geneva, Switzerland, and is open to any state that is a member of the Paris Convention; under the treaty, anyone who is a national or resident of a PCT contracting State may file an International Application either with the national patent office of the contracting State of which the applicant is a national or resident or, at the applicant’s option, with the International Bureau of WIPO in Geneva; filing a single PCT application allows protection to be obtained in all signatory states (141 with the accession of Peru and Chile in March 2009) on completion in due course of the relevant national patent requirements; see also the PCT web site at http://www.wipo.int/pct/en/.

4.5 Priority

by filing a single International Patent Application (often referred to as a PCT application). Besides reducing overall costs, the use of the PCT has the advantage of increasing by up to 18 months the time before a decision has to be made on foreign filing. This is useful where there is a continuing research activity such as on particular drug analogues where initially promising leads may disappear and the direction of research change, requiring a change in the patenting strategy. As a result, most organizations seeking patent protection internationally for pharmaceutical inventions now use the PCT as their preferred route. Agreement on Trade-related Aspects of Intellectual Property Rights (TRIPs): This was established in 1995 as part of a global open trade deal and sets out the minimum levels of protection that each government has to give to the patents and other intellectual property rights of fellow members of the World Trade Organization. This was particularly important in establishing that it was improper to discriminate against particular technologies such as pharmaceuticals or whether they were imported or locally produced. Adoption of TRIPs led to changes in the patent law of both developed and developing countries including the United States of America and India and established a minimum patent term of 20 years from the date of filing the applications.

4.5 Priority

In competitive and rapidly evolving technology areas such as drug research, it is important to secure early patent protection for inventions as soon as possible after they are made so that priority is established for the invention. This is because in all countries except the United States the patent is accorded to the first person to file a patent application. In the United States, the patent is granted to the first inventor rather than the first person to apply for a patent and an elaborate practice of patent interferences has been developed to determine the true first inventor. The Paris Convention (see above) was developed to allow a single early-filed patent application in one country to be used for up to 1 year as the basis for any number of corresponding foreign applications claiming the priority of the original national application (Figure 4.1). Everything in the second application that is literally within (or properly based on) the first application is regarded as having the same filing date as the first application – “the

1st (priority) application filed

0 months

2nd updated application filed – claiming priority for subject matter also in 1st application

12 months

Figure 4.1 Priority under the Paris convention.

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priority date” – when considering patentability over the prior art. To retain priority, the subject matter in the later application must be directly and unambiguously derivable from that in the first, the so-called “priority application.” In principle, there is no limit to the number of priority establishing applications that may be filed within the year from the first application and from which the later foreign applications may claim priority. However, they will ultimately need to be translated into the language of the state in which the priority is being asserted and so in practice for drug analogue inventions it is rare for there to be more than three priority applications. The advantage of this arrangement for analogue research is that it allows the first application to include claims amounting to a year’s research plan based on initial activity in a few lead compounds that have so far been prepared and tested. Then, as the research plan is explored and additional interesting compounds are discovered these can be either added to a new priority establishing application or else included in the foreign patent applications. For example, suppose a first application was filed claiming naphthalene derivatives having a halogen substituent based on the activity of a chloronaphthalene included in the patent specification, then a claim to halogenonaphthalenes in a second application filed up to a year later would be entitled to the priority date of the first application. This would be important if a competitor filed an application also claiming halogenonaphthalenes or if there were to be a publication of the chloronaphthalene itself some time between the first and the second patent applications since, if the priority was not maintained, then a valid patent could not be obtained by the originator.

4.6 Novelty

Deciding whether claimed subject matter is novel in relation to the prior art is critical in any assessment of validity of a patent; if novelty is not present there, then consideration of inventive step (which itself can always be argued or refuted with evidence of unexpected effects) simply is not possible. In the case of chemical inventions, it does not matter at all for what purpose, if any, the compound was used and so disclosure or use in any technical art may be considered. It is enough that the compound was made somewhere else to destroy the novelty of a later patent claim to the compound. However, it may well be that other aspects related to the known compound are still novel and inventive, such as pharmaceutical formulations or new medical uses. Thus, for example, 2,6-diisopropylphenol (Figure 4.2), the active entity of the intravenous anesthetic propofol, was first described as a stabilizer for rubber and so could not at the time when its anesthetic properties were discovered in 1977 be claimed as a compound itself but only as a special injectable formulation.7) 7) The first medical use for a known compound can now be protected under European law, that is, a product claim in the form of known substance X for use as a medicine is now permissible in Europe. Methods of medical treatment involving administration of a known substance have always been patentable in the United States.

4.6 Novelty

OH

Figure 4.2 Propofol (DiprivanÒ ).

O

O S

O H

NH2 X

O O

O O H H O (a)

CH2OSO2NHR1

R5

R2 R4

R3 (b)

Figure 4.3 (a) Topiramate (TopamaxÒ ) and (b) generic structure including topiramate.

The objective of all patent claims is to define the limits of the invention in such a way that all essential features are included and defined as generally as possible without covering embodiments (examples) that are already known or which are obvious from what is known or those that cannot be made or which simply do not work. This may be relatively straightforward for mechanical inventions but can be difficult for chemical inventions such as new drug analogues where the draftsman wants to include in the claims all of those analogues that could readily be conceived by a competitor based on the compounds actually exemplified in the patent specification. This frequently leads to the use of complex definitions in patent claims involving a generalized structural formula with individual substituents defined in lists of alternatives such as that shown above for the McNeil anticonvulsant, topiramate (Figure 4.3). Such claims8) typically include thousands or even millions of alternative compounds. Fortunately, no patent office takes the view that all possible compounds that can theoretically be listed within a general formula are implicitly described once the structure is published. What then is the effect of such a published patent? The novelty is certainly destroyed for all compounds that are specifically mentioned or listed in worked examples and the rest of a patent specification once it is published. Sometimes, subtle linguistic devices are employed by patent draftsman to indicate which compounds have actually been prepared and evaluated in bioassays and which are mere possibilities. For example, “the following compounds were obtained” implies that actual synthesis has occurred whereas “the compounds in the table may be obtained” implies that it is simply possible to make the listed compounds. In the latter case, such disclosures will still be novelty destroying unless it can be shown that such compounds cannot be made by any of the processes

8) See Appendix 4.B.

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described in the specification or other well-known means – if they can be so made the disclosure is often referred to as “enabling,” that is, there is sufficient description for a chemist with reasonable basic skills to make the compound(s) concerned.

4.7 Inventive Step: Nonobviousness

The problem with most inventions is that with hindsight they may appear to be obvious or to lack the necessary inventive step over what is already known. Consequently, showing inventive step is almost always the biggest hurdle that drug analogue patents must clear since, by definition, they start from an existing molecule already known to have useful pharmacological properties. The fact that increasing logic and theory has been applied in recent years to research processes to increase the likelihood of candidate drugs emerging quickly from drug discovery programs can make it more difficult to show that arriving at a useful analogue of an existing drug is other than predictable. From the patent perspective, so far at least the discovery process and subsequent development of clinically useful analogues of existing drugs continue to remain uncertain and regularly give rise to unexpected results. Lack of predictability and/or unexpected results in comparison with the relevant close prior art are required if inventive step is to be shown. The US Supreme Court set down in a decision in 1966 [2] (which is still followed today) the key components of obviousness determinations: “the determination of ‘nonobviousness’ is made after establishing the scope and content of prior art, the differences between the prior art and the claims at issue, and the level of ordinary skill in the pertinent art.” The Court also considered possible secondary considerations that might be taken into account as evidence of nonobviousness, such as commercial success, long-felt but unmet needs, and the lack of success of others to solve the underlying problem. So, it is necessary to decide what is the claimed invention and consider both chemical structural differences and the underlying properties of the closest compounds in the art in deciding whether an inventive step exists. The size of the inventive step is of no concern – there simply has to be one, the so-called scintilla of invention. For pharmacological analogues, where the new drug molecule has similar pharmacological activities but there are no real chemical or structural similarities with the prior art drug, then an inventive step is clearly present. However, for most drug analogues some sort of structural similarity can be discerned between the new molecule and the starting drug. This means that in virtually all applications for drug analogues patents, examiners will allege that the invention lacks an inventive step simply because of perceived structural similarity with the closest compound in the prior art (the so-called a priori or prima facie obviousness). Analogues of a natural product whose structure has not previously been published are of course a clear exception. The degree of structural or chemical closeness that makes one compound obvious from another is of course subjective and therefore arguable. The person required to

4.7 Inventive Step: Nonobviousness

judge this is the notional “person of ordinary skill in the art,” that is, for drug analogues, usually a skilled medicinal chemist. This hypothetical person is considered to be aware of all the background literature, have all the skills of a synthetic chemist at the time the application was filed, and have a general understanding of how molecules may be evaluated for potential drug use, but this person may not be in any way inventive. In complex cases, the notional “person” may be considered as a team, spanning a number of specialist skill sets such as pharmacology and formulation science. It is possible to give some rough guidance on what is likely to attract an obviousness allegation. Thus, if the relationship with the prior art is one of isomerism (positional or spatial), homology (such as propyl or ethyl for methyl), or involves replacing groups within a series (such as bromo for chloro) or generally recognized to be similar in properties (such as cyano or nitro for halogeno), then an a priori obviousness objection is likely to be made. Successfully countering (“rebutting”) such an objection by argument alone may be difficult and instead it may be necessary to use evidence of unexpected properties or difficulties in moving from the prior art to the claimed molecules. What then is the closest relevant prior art for assessing inventive step? First, this may not necessarily be the same as the known drug that was the starting point for the research. Thus, the structurally closest compounds to the claimed analogues may have a totally unrelated set of industrially useful properties (e.g., as dyestuffs) or they may have been simply published without any apparent utility in an academic paper. In both of these cases, it should be possible to counter an obviousness argument by saying that the useful pharmacological properties of the new analogues were unexpected and could not have been predicted from what was known in the prior art. In general, patent specifications only need to contain an indication of the relevant prior art without disclosing the closest piece of prior art, although applicants will usually have to get close to specifying it in order to properly set out the advantages of the invention. If it is clear from the outset that the new analogues have a very close relationship with a known drug, then the nature of the unexpected properties (such as unexpectedly superior activity, bioavailability, stability, lack of side effects or toxicity, etc.) and details of how they can be demonstrated (e.g., any special testing systems) should be set out in general terms in the patent specification. However, it is generally better to await the nature of any obviousness challenge before presenting actual evidence of the unexpected properties since these are likely to be required in comparison with what is cited as the closest prior art by the relevant patent examiner. While overcoming objections of obviousness by providing suitable evidence of unexpected effects is attractive, it may give rise to problems when a patent is the subject of litigation, especially in the United States of America. The evidence and those who produced it are then subjected to close scrutiny by the parties to the litigation examining the nature and extent of the comparisons made and the conduct of the testing. Consequently, evidence should only be offered to dispose of an obviousness objection after counterarguments have failed. Such counterarguments

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might be based on structural dissimilarities with the prior art compounds, contrary teaching in the literature such as structure–activity relationships leading away from the new analogues, a “long-felt want” for the invention indicated by false starts or failures, or difficulties in synthesizing the new analogues. However, mere evidence of the commercial success of the patented drug may not be sufficient since there may be other reasons for this such as substantial expenditure on marketing. The finding that the new analogues possess additional useful properties, even if unexpected, may not always be sufficient if the analogues exhibit similar underlying properties to those of the original drug. Thus, such properties might be seen as a bonus rather than as demonstrating an inventive step if a series of novel betareceptor antagonists structurally very similar to the known drug propranolol were found to have similar levels of antagonist properties but had greater stability. However, if the extra property was perhaps to be cardioselectivity and it was to be shown that this had previously been difficult to achieve, then this would probably have supported the presence of an inventive step. Although all the major developing countries employ similar principles when approaching assessment of inventive step to those set out by the US Supreme Court above, in practice the approaches may differ. Thus, the European Patent Office in examining an inventive step adopts the so-called “problem–solution” approach that, after determining the closest relevant prior art, establishes what it considers to be the “objective technical problem” to be solved by inspecting the patent specification and looking for the features that distinguish the invention over the prior art and then decides whether the solution – the claimed invention – would have been obvious to the notional skilled person starting from the closest prior art. In this context, when setting up the notional technical problem to be solved, it is not appropriate to use compounds from the prior art that although structurally closest to the claimed compounds would have been disregarded as irrelevant by the person skilled in the art.9) US courts have taken a similar view and held that it is not reasonable to expect that a skilled medicinal chemist would remove a structural feature known to convey superior properties to a known drug when starting to search for new improved analogues [3]. They have also rejected an attempt to suggest that one structurally analogous compound would be necessarily chosen from among the 54 in total described in an earlier patent as the starting point for improved analogues without clear teaching that it was preferred [4]. Finally, it must be mentioned that it is not usually acceptable to construct an obviousness argument based on a mosaic of parts from many different unrelated prior art documents. If several documents are used, they must be clearly linked and it must be possible to show how the skilled man would inevitably be led to combine them to reach the claimed invention.

9) European Technical Board of Appeal, decision T334/92, Eisai Benzodiazines: here the EPO was held to have improperly based the notional “problem” on a 20-year old reference that, although including structurally very similar compounds to the antiangina agents claimed, had been disregarded by other groups working in the area as a starting point for new antiangina drugs; see http://legal.europeanpatent-office.org/dg3/biblio/t920334eu1.htm.

4.9 Selection Inventions

4.8 Utility: Industrial Application

Unless compounds have some useful property that can be used in some industrial context, they cannot be validly patented. Consequently, it is important to ensure that all the molecules included in a drug analogue patent specification possess the underlying biological activity at a level that is useful. It is frequently tempting, during a large synthetic program to produce drug analogues, to include every compound made irrespective of the extent of biological activity shown in the assay systems. Yet including any significant number of such inactive compounds introduces a fatal weakness into any eventual patent since a determined competitor may well be able to discover such an area of inactivity and thus challenge the patent for lack of utility. Showing useful pharmacological properties in the target disease situation in humans or animals is normally not possible since most patents on drug analogues are applied for well before any clinical evaluation has been possible. However, data from in vitro assays or testing in an animal surrogate and which is reasonably correlated with the end disease can be used to satisfy the therapeutic utility requirement for patentability. This information correlating any assay or animal testing with the disease should be provided in the specification together with test data at least in general terms for the analogues described in the examples. Before the 1980s, in the United States of America patent applications asserting use in treating cancer in humans were regularly rejected as lacking utility because such use was considered “incredible.” However, this is fortunately no longer the case and “[Patent] Office personnel must determine if the asserted utility for the invention is credible based on the information disclosed in the application [5].”

4.9 Selection Inventions

Selection inventions, as the name implies, involve the selection of a small number of compounds previously included within a known broadly defined group of compounds (usually within the scope of the claims of an earlier patent) and finding that the selected compounds possess some unexpected useful properties such as surprisingly higher activity. However, selection inventions still have to satisfy the novelty and inventive step criteria for patentability but the resultant patents are self-standing and have an independent 20-year term. Consequently, they can be particularly useful where there is a new drug analogue research program continuing over several years and the initial patents may not contain the eventual drug development candidate. Of course, if the selection has been made from within the broad groups of compounds claimed in a patent that is still in force and that has been granted to a competitor, then commercializing one of the selected compounds will require a license under the competitor’s patent. Equally, the competitor could not commercialize a compound claimed in the selection patent without a license. Normally, such a situation would be dealt with by cross-licensing of both patents.

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Establishing novelty for a selection invention can be very difficult. None of the compounds in the selection must have been described before or suggested as “possibly obtainable” anywhere in the prior art and for any purpose. This includes not being mentioned in patent claims. As mentioned before, a typical main claim to drug analogues may nominally include hundreds of thousands of possibilities, the majority of which have not been made. Making a small selection of compounds from such a large number of possibilities is not usually a problem in terms of novelty, unless the earlier patent specification suggests that certain optional groups of compounds are preferred alternatives. However, the subsidiary claims in the patent will likely contain a general formula with short lists of alternative substituents including perhaps only a few tens or hundreds of compounds. The novelty of compounds selected from such narrow claims may thus be in doubt. It is not possible to give hard and fast rules about what size of group will be considered as novel over the prior disclosed broad group since such assessments are highly fact specific and there is considerable variation among the United States of America, Europe, and Japan. Suffice it to say that selection patents must necessarily have narrowly defined claims in terms of the number of molecules included and ideally contain only a handful. In addition to novelty, an inventive step must be shown over the prior art, that is, the known compounds from which the selection is made. This can either be based on a new and valuable property, such as the one making the compounds useful in a new disease situation, a substantial reduction in adverse properties such as toxicity or side effects, or else a substantial increase in the underlying activity of the prior art compounds, but in all cases where the new property or size of the change in properties could not have been predicted from the prior art. The nature and extent of the properties providing the inventive step and how they can be demonstrated must be properly set out in the patent specification. Also, it is even more important than in ordinary patents on drug analogues to ensure that all claimed compounds within the selection possess the unexpected and useful properties.

4.10 Enantiomers

Enantiomers of racemic drugs are probably the closest structural analogues and a patent protecting an enantiomeric form, the ultimate selection invention of one from two molecules. The first hurdle to patentability is to demonstrate novelty over the known racemic form. Fortunately, most countries take the view that an enantiomer of a known racemic form that has never been known or existed except in the presence of the other enantiomer is formally novel and move the inquiry to inventive step. At one time, it was unusual to consider separating individual enantiomers of clinically useful drugs partly because of the additional cost of manufacture, but today it is the norm and many drugs are now used as single enantiomers. It has also become easier with modern technology to produce such enantiomers on a large-scale commercial basis either by stereoselective synthesis or by physical separation

4.11 Prodrugs and Active Metabolites

F

N

O N Figure 4.4 Escitalopram (LexaproÒ ).

techniques such as chiral HPLC. Consequently, it is difficult now to argue that the skilled person would not routinely attempt to separate a racemic candidate drug molecule to establish how the useful biological property, side effects, and toxicity were distributed between the individual enantiomers. However, things that are “obvious to try” are not necessarily obvious in fact because there may be all sorts of reasons why success cannot be achieved [6]. As a result, patents claiming a single enantiomer often start with a presumption that they lack an inventive step over the known racemic form of the drug. Such a presumption can be disposed of only by concrete evidence of unexpected properties in the enantiomers. In addition, there can be difficulties arising from whether the information in the patent specification is sufficient to support a product claim to the enantiomer however prepared and without any use limitation. An interesting set of legal judgments in the English courts [7] illustrate many of the issues of patentability referred to in this chapter and involve the enantiomeric antidepressant drug escitalopram (Figure 4.4). These decisions are self-contained and present the various issues in clear terms.

4.11 Prodrugs and Active Metabolites

Both of these may be regarded as analogues of known drugs and may even be interrelated. Prodrugs are normally chemically simple derivatives or precursors of the parent drug that are not active until the drug moiety is presented in vivo and they are usually designed to overcome some deficiency in the original drug, for example, esters, ethers, oxidizable precursors, and so on of the drug molecule. Prodrugs may be difficult to patent and the issued patents susceptible to challenge when the drug itself is already known. They first need to pass the novelty hurdle that may be difficult since many simple derivatives such as alkyl esters will probably have appeared routinely together with phrases such as “or a prodrug thereof” in drug patent specifications. The prodrug concept is well known and so prodrugs of existing drugs are at face value obvious unless the added element forming the prodrug itself contains some special or unusual structural component or it can be shown that the prodrug possesses surprising or unexpected benefits, such as dramatically improved bioavailability, duration of effect, and so on.

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O

S OH N

N N

O

H

O

Figure 4.5 Hetacillin.

NH2 NH

H S N

O O

OH O Figure 4.6 Ampicillin.

One prodrug that was the subject of extensive patent litigation was hetacillin, the acetone condensate of the antibiotic ampicillin (Figures 4.5 and 4.6). This prodrug is inactive as an antibiotic until administered to humans when ampicillin is rapidly regenerated in the blood stream. As a result, its sale was eventually held in the United Kingdom to be an infringement of the existing patent on ampicillin [8]. Active metabolites may be patented insofar as their existence has not been published and it is not obvious that they will be produced from the drug itself. Since such metabolites are generally found only when the drug is administered to humans, any patent application is likely to be filed several years after the application protecting the drug itself and so will lead to later expiring patents. Consequently, there is the opportunity to maintain effective patent protection for a product even after the patent protecting the active ingredient has expired by alleging infringement of the active metabolite patent when the drug is administered to patients. Examples of patented active metabolites that resulted in litigation include the antihistamines fexofenadine, which is a carboxylic acid formed rapidly in vivo following administration of terfenadine (Figures 4.7 and 4.8) and desloratidine, which is the major metabolite of loratidine (Figures 4.9 and 4.10).

OH O

N OH OH Figure 4.7 Fexofenadine (AllegraÒ ).

4.12 The Patenting Process from the Inventor’s Standpoint

N OH OH

Figure 4.8 Terfenadine (SeldaneÒ ).

H N

N Cl Figure 4.9 Desloratidine (ClarinexÒ ).

O

O

CH3

N

N Cl Figure 4.10 Loratadine (ClaritinÒ ).

Attempts to enforce the relevant patents on these active metabolites against companies commercializing the parent drug have met with varying results [9] in different countries, demonstrating the problems particularly of novelty in patenting this type of drug analogue.

4.12 The Patenting Process from the Inventor’s Standpoint

Having a patent application filed for a drug invention is a major achievement for most medicinal chemists especially if they are one of the named inventors. However, it sets in train a series of events that can involve the inventor with the patent system for many years especially where a successful drug product results. The whole patenting process to obtain patents in the United States of America, Europe, Japan, and other commercially important countries can easily take up to 10–12 years by which time most medicinal chemists will have moved onto new projects if not new job roles.

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However, their involvement with the patents on which they are inventors may nonetheless continue, often in the following respects. 4.12.1 Inventorship

Deciding who are to be the inventors for a patent application – the inventorship – is an important legal matter affecting the ultimate validity of any patent and is resolved by the patent attorney handling the application. Inventorship is not decided like authorship of scientific papers but in the light of all the facts surrounding the invention, how it was arrived at, and who was associated with the critical steps involved. However, in the case of a new set of analogues based on an existing drug, it is probable that there will be at least one medicinal chemist inventor. 4.12.2 The Priority Patent Application

The medicinal chemist inventor is first likely to be involved when filing a priority establishing patent application – the priority application – is being considered. At this stage, a decision must be made on the extent of the compounds to be included in the claims and which synthetic methods are to be included. Then, there will be a request to supply written examples of compounds that have been made and tested. It is important at this stage to inform the patent draftsman of any compounds that are inactive so that they can be excluded from the claims either by careful construction of the substituent definitions in the general formula or by specific disclaimer.10) 4.12.3 Prior Art Disclosure

The patent draftsman must also be told about any relevant publications, seminars, or the like – prior art – that seem at all relevant to the compounds being patented. It is probable that a formal search for relevant prior art will be carried out that will pick up patent specifications and scientific journal articles, but the research team working on the invention is best placed to identify less well-documented prior art such as conference abstracts. Under the requirements of US law, the inventors and others in the research team associated with the patented invention must provide the patent attorney handling any corresponding US patent application with publications and other pertinent information so that this can be submitted to the US patent examiner who is assigned to process the application. This is a continuing obligation under US law until a US 10) Disclaimers are found at the end of the claim and are typically in the form: “what we claim is a compound of the formula A wherein the substituents R1, R2 and R3 have any of the following meanings. . .; but excluding the compounds of the formula A wherein R1 is methyl or ethyl, R2 is hydrogen and R3 is fluoro. . . .”

4.12 The Patenting Process from the Inventor’s Standpoint

patent is granted – failure to comply may lead to a charge of “fraud on the patent office” and result in the patent being unenforceable, should the patent be litigated. 4.12.4 Patent Specification Review

It is tempting for inventors to read only the examples and technical description of patent specifications, particularly because the documents tend to be repetitive and legalistic and contain seemingly standard wording about dosages, formulations, and the like – the so-called “boiler-plate” text. However, especially at the drafting stage, inventors should make sure they read all of the patent specification carefully. It is easy for “cut and paste” word-processing errors to creep in and it is very likely that the inventor will be the person on the witness stand asked to explain what he or she meant by the text! Also, it is not possible to correct errors other than those for which a correction is obvious by reading the text once patent applications have been filed. That said, it is still important to complete reviewing the draft quickly so that the application can be filed with the earliest possible date – the inventors are on the critical path to doing this and may sometimes be the rate-limiting step in the patent application process. 4.12.5 “Best Mode” of Carrying Out the Invention

A US patent application must comply with another special requirement and the specification must “set forth the best mode contemplated by the inventor of carrying out his invention.” In the case of analogue patents, this means that the best analogue known to the inventor at the time the application is filed must be included somewhere in the specification. It simply has to appear somewhere but does not have to be flagged up as the “best mode.” The penalty for not disclosing it is invalidity of the eventual US patent. It must also be present in a foreign-filed priority application if priority is to be maintained in the United States of America. 4.12.6 Foreign Patent Applications

Some nine months after the priority application has been filed, a decision has to be made whether to continue with the patenting process by filing further patent applications in foreign countries, updating the material in, and claiming priority from, the earlier application. These new filings may be made via a single PCT application and/or individual national applications. If a PCTapplication is filed, then after some degree of centralized examination of formal matters as well as a search for prior art affecting novelty and inventive step, it will lead to a series of national applications or regional applications (such as through the European Patent Office) that will then be further examined before any patents are granted. Under the Paris Convention, these applications must be filed within 12 months of the earliest priority application which is an inextensible deadline. However, since translations are often

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required, the process to prepare the updated specification for the new applications is usually started several months before the deadline. At this stage, the inventor again needs to supply further examples of analogues based on the original invention and confirm their useful biological properties and mention any inactivity of the analogues. The “best mode” requirement must also be updated by including the latest preferred analogues. The updated specification will also need to be carefully checked since this may be the final point at which it is possible to easily correct errors and omissions – new matter cannot generally be added after filing which is one reason why patent specifications tend to be so verbose–everything needs to be present that may be needed during the lifetime of the patent, which can be up to 25 years for a drug analogue patent. 4.12.7 Patent Application Publication

Assuming the priority application is maintained by filing one or more corresponding applications, the patent specification will be published more or less as originally filed 18 months after the earliest priority application, or shortly afterwards. This date is important for inventors since patent applications from competitors published after this date, apart from in the United States of America,11) must necessarily be based on later-filed priority applications and cannot then validly claim the same invention. Usually, the application is published together with an official search report of the prior art although this may be published separately. This report indicates which prior published material is considered most relevant to the novelty and inventive step of the invention and gives an early indication of how difficult it may be to obtain a granted patent. 4.12.8 Patent Examination

All patent offices have some backlog of applications and so it may be some time (perhaps as long as 2–3 years) after a patent application has been filed and examination requested before the relevant patent office will issue an official letter – sometimes referred to as an “office action” – with a fixed date for reply. The letter12) will be 11) As mentioned earlier, in the United States of America it is the person who is first to make the invention and not the first patent applicant who is awarded an eventual patent following a complex and lengthy patent interference procedure. For drug analogue patents, this procedure critically depends on being able to establish dates on which an example from the patent was first conceived and made and tested in the laboratory for its therapeutic purpose. It is most important therefore to keep proper dated laboratory documentation of the preparation and test-

ing of compounds with the pages signed and dated by the person who carried out the work and the signature corroborated by someone independent of the research work. 12) The official letters issued to patent applicants may contain some off-putting language like “all claims rejected,” “final rejection,” and the like. However, these are formal official positions that can be argued against and usually overcome by the patent attorney with technical assistance from the inventor.

4.12 The Patenting Process from the Inventor’s Standpoint

received first by the patent attorney handling the application who will usually ask for technical input from the inventors about replying to any objections about patentability over cited references. This input has to be given high priority over other work since, although extensions may be obtainable (often with payment of a fine), failure to reply on time to the patent office may lead to the patent application being considered abandoned and all rights lost. In some patent offices (such as in the United States), it is possible to meet with the examiner in person, in an attempt to resolve difficulties and the inventor may usefully also attend, as may the patent attorney. Several official letters and replies may be required before either a notice of the intention to grant a patent or confirmation of its rejection is received. In the latter situation, it is usual to appeal the rejection to the appropriate Appeal Board within the patent office or to the relevant court. In both cases, the inventor will be required to provide technical inputs to help the legal professionals argue the appeal that is likely to be critical to its eventual success. Considerable care should be exercised before any additional experimental data is generated for the purpose of overcoming objections in official letters, especially comparative test data with prior art molecules. Negative data from such testing cannot be ignored and must also be communicated to the examiner considering a US patent application, even if the testing was carried out in connection with a non-US application. 4.12.9 Opposition to Grant

In Europe, before a European patent becomes effective in the national states designated when the application was filed, it is possible for an interested party to oppose the patent grant. This must be done within 9 months of the publication date of the patent specification in the form in which it is to be granted. Thus, the end of the European opposition period is another important milestone in the patenting process. If an opposition is filed, then the inventor’s input is central to its legal defense and further time commitment can be expected for several years.13) If no opposition is filed, then the application immediately becomes a bundle of national patents that can then be enforced, albeit separately, in the countries concerned. Afterward, these patents may only be challenged before the individual national courts and so successfully opposing patent grant can be a cost-effective way of disposing of a European patent.

13) Decisions of the Opposition Division of the EPO can be appealed adding still further time until the matter is finally resolved – as an extreme example, European Patent 58481 protecting continuous release depot formulations for polypeptide drug moieties such as the anticancer agent goserelin (ZoladexÒ )

was originally opposed by 12 companies and the final appeal upholding the patent in amended form was only given on February 28, 2001 just 11 months before the patent expired at the end of its 20-year patent term on February 28, 2002!

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4.12.10 Patent Litigation

Most patent systems that used to provide opposition before patent grant (such as those in the United Kingdom, Japan, and the Republic of Korea) have now replaced this with the ability to apply to revoke the patent challenging its validity at any time during its lifetime. One important exception is India, which allows for oppositions before and after a patent is granted. In addition, in most infringement proceedings, the alleged infringer will challenge patent validity in a counterclaim for revocation or invalidation of the patent. Again, the inventor will be needed to provide important technical support to the legal action. As already mentioned, the validity of any US patent protecting a drug with significant US sales is likely to be challenged in a US Federal Court since an applicant to register a generic version for sale with the US Food and Drug Administration (FDA) must certify that the patent is invalid or else not infringed if the generic is to be sold before the patent expires.14) This may then trigger infringement proceedings against the applicant for the generic product and involvement of the inventor as a key witness over a period of several years until the litigation is resolved and any appeal concluded. Such litigation involves extensive discovery of background documents and technical papers including those held by the inventors anywhere in the world. It is most important therefore to adopt good document retention practices, discarding informal notes and draft documents, and so on (including electronic copies and e-mails) when they are superseded by final versions.

4.13 Pitfalls for the Unwary: Granted Versus Published Patents, Scientific Publications

It is important to recognize the difference between published patent applications and granted patents. Applications are published essentially as the applicant submitted them and having had only very limited formal examination, for example, to amend the title or abstract. Consequently, the claims may appear unreasonably broad. Published granted patents have been subject to full examination for patentability and the extent of the claims may well have been considerably restricted. It is these claims that need to be assessed when considering potential infringement situations. However, just because a granted patent has been published does not mean that it is necessarily still in force since it may have lapsed through failure to pay the renewal fees. 14) In the United States of America, on filing an ANDA for a generic version of a registered NCE drug, one of the following four certifications must be made: para (i) no patent is listed in the FDA’s Orange Book of patent exclusivities for the registered NCE product; para (ii) the listed patent has expired; para (iii) the listed patent will have expired before the

requested approval takes effect; and para (iv) the listed patent is invalid or will not be infringed by the generic version. The filing of a para (iv) certification is a statutory act of infringement and must be copied to the relevant patentee who then has up to 45 days to start infringement proceedings against the applicant for the generic registration.

4.13 Pitfalls for the Unwary: Granted Versus Published Patents, Scientific Publications

Most patent owners regularly review their portfolio of patents and relinquish some, so it must never be assumed that an important patent is still in force – the relevant official patent registers must be checked first.15) At the end of a drug analogue research program, the research team frequently wishes to publish its scientific findings in a major, peer-reviewed journal. However, it is important to check the proposed publication with the attorney handling the relevant patent applications before submitting the text to a journal – its publication may influence the timing of further new patent applications, especially if it includes the rationale or theory for making new analogues or their pharmacological properties.

Appendix 4.A Some Patent Jargon Terms16)

One of the problems for researchers when first venturing into the arena of patents is the specialist language and jargon used. Like all jargon, some of it may be specific to particular countries or even companies, but explanations are offered below for some of the more commonly used terms. Priority Date: The earliest date to which subject matter or claims is entitled when considering the impact of prior art: it is usually the filing date of an earlier application – the priority application – that was the first filed by the applicant for the invention anywhere in the world. Prior Art: Everything known, used, or described publicly anywhere in the world usually in documented form and pertinent to the technical area(s) with which the invention is concerned before the filing date of the patent application or the earliest priority date from which it is entitled to claim priority; it includes the general knowledge of practitioners in the art at the relevant date as documented in standard reference works. Patent: This term is used loosely with a variety of meanings depending on the context. Strictly, it refers to the legal document including a detailed final specification and claims issued (granted) by the country concerned after examination of an application. However, it is frequently used as shorthand for a patent specification alone whether in examined, final form, or in unexamined, early published form. It is also used as a verb meaning to protect an invention by filing and prosecuting a patent application to obtain a granted patent.

15) Checking the global patent status for a particular product is a specialized task best dealt with by patent attorneys, but several registers are available on-line and can be easily checked by inventors, for example, the European patent register can be searched free of charge at http://www.epoline.org/portal/ public/registerplus. 16) Much of the jargon is peculiar to the national patent system in use and so many different

glossaries of patent-related terms are available on the Internet; examples include that provided by Delphion: http://www.delphion. com/help/glossary; the European Patent Office: http://www.epo.org/help/glossary. html; the US Patent & Trademark Office: www.uspto.gov/main/glossary/index.html; and P.W. Grubb, in Patents for Chemicals, Pharmaceuticals, and Biotechnology, 4th edn, OUP 2004, ISBN: 0-19-927378-2.

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Patent Family: A group of essentially similar patents (or applications) for the same invention filed in different countries based on the same priority applications. Prosecution: Progressing a patent application from first filing to grant or rejection and involving responding to questions and objections raised by patent offices during examination. Markush Claim: A claim including a list of alternative compounds of a generally similar nature either in relation to their properties or biological activity or else having a common structural element – named after a US inventor, Eugene A. Markush, who in 1924 succeeded in getting a US patent with this type of claim.

Appendix 4.B A Typical Broad Chemical Claim: US Patent Ser. No. 4,513,006 (Topiramate)

A sulfamate of the following formula (I): X

CH2OSO2NHR1

R5

R2 R4

R3

wherein X is oxygen; R1 is hydrogen or alkyl; and R2, R3, R4, and R5 are independently hydrogen or lower alkyl, and R2 and R3 and/or R4 and R5 together may be a group of the following formula (II): R6

O C

R7

O

wherein R6 and R7 are the same or different and are hydrogen, are lower alkyl, or are alkyl and are joined to form a cyclopentyl or cyclohexyl ring.

Appendix 4.C Further Reading

P.W. Grubb, Patents for Chemicals, Pharmaceuticals and Biotechnology, 4th edn, OUP 2004, ISBN 0-19-927378-2; this book provides comprehensive background to international patenting for chemists and other scientists. The web sites of the US [http://www.uspto.gov/], European [http://www.epo.org/], and Japanese patent offices [http://www.jpo.go.jp/] are useful sources of general information about patents, trade marks, and other intellectual property rights.

References

References 1 Fischer, J. and Ganelllin, C.R. (2006)

2

3

4

5

6

Introduction to Analogue-Based Drug Discovery, IUPAC, 1st edn, Wiley-VCH Verlag GmbH, Weinheim, p. xix. Graham vs. John Deere Co., 383 U.S. 1, (1966) (US Supreme Court). This case involving agricultural plough improvements set important precedents still followed today for consideration of non-obviousness: http://supreme.justia. com/us/383/1/case.html. Eisai vs. Dr Reddy’s Labs and others, US Court of Appeals for the Federal Circuit (CAFC) (2008). In this case involving a challenge to the US Patent 5,045,552 for the antiulcer agent rabeprazole, it was alleged that the closest prior art was the drug lansoprazole that contained a 2,2,2trifluoroethoxy group known to confer excellent lipophilicity. The court held that “the record contains no reasons a skilled artisan would have considered modification of lansoprazole by removing the lipophilicity-conferring fluorinated substituent as an identifiable, predictable solution.” So, the issue of the comparability of the trifluorethoxy group of lansoprazole with the structurally similar 3methoxypropoxy group of the new antiulcer analogue rabeprazole could not arise for considering it an inventive step. Instead, the structurally more distant drug omeprazole that contains a methoxy substituent was the proper comparison drug; see www.cafc. uscourts.gov/opinions/07-1397.pdf. Takeda vs. Alphapharm, US CAFC (2007). This case concerns the antidiabetic agent pioglitazone and provides a clear summary of the US position on inventive step in relation to structural analogues and the proper choice of the prior art compound for consideration of obviousness; see http:// www.cafc.uscourts.gov/opinions/06-1329. pdf. US Manual of Patent Examining Practice (2008): http://www.uspto.gov/web/offices/ pac/mpep/documents/2100_2107_03.htm. Apotex vs. Sanofi-Sythelabo Canada (Canadian Supreme Court, 2008). The Court rejected an allegation that the enantiomeric antiplatelet agent clopidrogel

(PlavixÒ ) was anticipated by (lacked novelty over) or was obvious from the corresponding racemate that was described amongst 21 specific analogues out of more than 250 000 possible molecules within the claims of an earlier patent. In the words of the Court, “For a finding that an invention was ‘obvious to try’, there must be evidence to convince a judge on a balance of probabilities that it was more or less selfevident to try to obtain the invention. Mere possibility that something might turn up is not enough. Here, when the relevant factors are considered, the invention was not self-evident from the prior art and common general knowledge in order to satisfy the test. While there were five wellknown methods to separate this racemate into its isomers, there was no evidence that a person skilled in the art would have known which of the five known separation techniques would work with this racemate.” See http://scc.lexum. umontreal.ca/en/2008/2008scc61/ 2008scc61.html. 7 Lundbeck vs. Generics (UK) et al. EWCA Civ 311 (UK Court of Appeal) (2008); see http:// www.bailii.org/ew/cases/EWCA/Civ/ 2008/311.html. Generics (UK) et al. vs. Lundbeck UKHL 12 (House of Lords) (2009); see http://www.bailii.org/uk/cases/ UKHL/2009/12.html. 8 Beecham Group vs. Bristol Labs (UK House of Lords), see (1978). Reports of Patent Cases, 153. 9 see, for example, Merrell Dow Pharmaceuticals (now Sanofi-Aventis) vs. Norton (UK House of Lords) (1995). Here the Court held that the fenofoxadine patent was invalid over the earlier terfenadine patent since “It enabled the public to work the invention by making the acid metabolite in their livers. The fact that they would not have been able to describe the chemical reaction in these terms does mean that they were not working the invention. Whether or not a person is working a product invention is an objective fact independent of what he knows or thinks about what he is doing”; see http://www.bailii.org/uk/cases/

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UKHL/1995/14.html; see also Schering Corporation vs. Geneva Pharmaceuticals (USA: CAFC). Here the Court reaffirmed the principle that “something which would be an infringement after a patent was granted would be a novelty destroying anticipation if it occurred before grant”; it then held that the claims to the metabolite desloratidine [DCL] were invalid since the earlier patent for loratidine effectively disclosed the metabolite by describing the administration of the parent drug to patients; in particular the Court stated that “this Court has recognized that a person may infringe a claim to a metabolite if the person ingests a compound that

metabolizes to form the metabolite. . . . The [loratidine] patent discloses administering loratadine to a patient. . . . The inherent result of administering loratadine to a patient is the formation of DCL. The patent thus provides an enabling disclosure for making DCL.” However, the Court observed that there was no objection to the validity of the claims to formulations containing DCL and that similarly claims directed to a pure or isolated form of the metabolite would also have been valid; see http://www.ll.georgetown.edu/FEDERAL/ judicial/fed/opinions/02opinions/021540.html.

Stephen C. Smith

UK and European Patent Attorney NuPharm Intellectual Property, New Farm, Twemlow Green, Holmes Chapel, Cheshire CW4 8BS, UK Stephen C. Smith is Managing Director of NuPharm Intellectual Property, a company he set up in 2001 to provide IP consultancy services after he retired from AstraZeneca Plc., where he was Global Head of patents, beginning his career as a medicinal chemist. He is a member of the Board of the Intellectual Property Institute and of the newly established UK Patent Regulation and IP Regulation Boards. Steve is a UK and European Patent Attorney and a Chartered Chemist. He has a BSc (Hons) degree from the University of Nottingham and a DPhil in organic chemistry from the University of Sussex.

Part II Analogue Classes

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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5 Dipeptidyl Peptidase IV Inhibitors for the Treatment of Type 2 Diabetes Jens-Uwe Peters and Patrizio Mattei 5.1 Introduction

The symptoms of diabetes mellitus, a metabolic disorder characterized by hyperglycemia (abnormally high blood glucose) due to inadequate insulin levels, have been described since antiquity. The introduction of insulin replacement therapy for diabetes in 1922 was a major feat in the history of medicine and was awarded with the Nobel Prize in medicine in the following year. Later in the 1920s, the first oral antidiabetic drugs (OADs) were introduced. Although they were imperfect and later withdrawn, they led to the recognition that two types of diabetics exist – the juvenile type, requiring insulin therapy, and the late-onset type, which also benefits from OAD treatment [1, 2]. The late-onset form, today known as type 2 diabetes, accounts for more than 90% of all diabetic patients and affects about 4% of the world population [3]. The treatment of type 2 diabetes aims to normalize blood glucose levels by diet, exercise, and medication, and is monitored by measuring glycosylated hemoglobin (HbA1c) as a long-term marker of elevated blood glucose. The amount of HbA1c reflects the average glucose level over the last 120 days (the life span of red blood cells) and should be maintained below 7% [4]. Each percentage reduction in HbA1c leads to a 21% reduction of the risk for any diabetes-related end point [5]. Poorly controlled, chronic hyperglycemia causes microvascular damage, which affects organs with delicate capillary systems such as the eyes and kidneys, and can lead to blindness and renal failure. In addition, hyperglycemia leads to atherosclerosis of larger vessels, which increases the risk of myocardial infarction and stroke. An important complication resulting from micro- and macroangiopathy are lesions of the lower limbs (“diabetic foot”) that may ultimately require amputation. Unfortunately, the majority of diabetic patients do not reach recommended HbA1c levels and are therefore at risk of developing these disabling comorbidities. Furthermore, the prevalence of type 2 diabetes has increased over recent years, mainly due to higher life expectancies and an increasing prevalence of obesity [3]. Several classes of OADs have been introduced into clinical practice since the 1950s and are widely prescribed. However, they all come along with side effects such as hypoglycemia, weight gain, or gastrointestinal Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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problems. Moreover, they often fail to achieve sustained glycemic control. Thus, there is a critical unmet need for OADs with novel modes of action. In the late 1980s, several research groups could show that the peptidic hormone GLP-1 (glucagon-like peptide 1), which is secreted by the L-cells of the intestinal epithelium in response to food ingestion, is a potent stimulator of glucose-dependent insulin release. This finding raised hopes that exogenous GLP-1 might be used to stimulate the impaired insulin secretion in type 2 diabetic patients. Disappointingly, single subcutaneous injections of GLP-1 were ineffective in normalizing blood glucose [6]. A few years later, it was discovered that DPP-IV (dipeptidyl peptidase IV), a serine protease first isolated in 1966, rapidly cleaves and inactivates GLP-1 [7]. Several research groups recognized the implications of this finding: . . .

Inhibition of DPP-IV should prevent the rapid degradation of GLP-1 and should thus increase circulating GLP-1 levels. Increased GLP-1 levels should enhance glucose-dependent insulin secretion, leading to lower blood glucose levels. Consequently, DPP-IV inhibitors should have an antidiabetic effect.

The glucose-lowering/antidiabetic effect of DPP-IV inhibitors was soon demonstrated in animals and humans and triggered enormous research activities throughout the pharmaceutical industry in the first decade of the new millennium [8].

5.2 In Vitro Assays and Animal Models for the Assessment of DPP-IV Inhibitors

The discovery of DPP-IV inhibitors was facilitated by the availability of robust and high-throughput in vitro assays, which often rely on a simple chromogenic or fluorogenic readout. For instance, DPP-IV cleaves Ala-Pro-AFC, a peptidyl derivative of 7-amino-4-trifluoromethylcoumarin (AFC), and the green fluorescence of the cleavage product, AFC, can be distinguished from the violet-blue fluorescence of the substrate (Figure 5.1). The cleavage of Ala-Pro-AFC serves as a measure of DPP-IV activity in an in vitro assay, in which the candidate inhibitor is evaluated by its ability to suppress the formation of fluorescent AFC. Furthermore, animal models with high relevance to the human disease state were available. For instance, the oral glucose tolerance test (OGTT) in diabetic rats measures the glucose excursion, or the insulin response, after an oral ingestion of a standardized amount of glucose, and is equivalent to the OGTTused in the diagnosis of diabetes in humans. The efficaciousness of DPPIV inhibitors can be evaluated in such an animal model by their ability to reduce the glucose excursion after their administration prior to the glucose challenge.

5.3 Substrate-Based DPP-IV Inhibitors

Speculations about the relevance of DPP-IV in the processing of bioactive peptides, and its potential role in diseases such as cancer and AIDS, might have provided much

5.3 Substrate-Based DPP-IV Inhibitors

CF3 N

H2N O

O

DPP-IV O

N H

O

Ala-Pro-AFC CF3 N

H2N O

O

+ OH

Ala-Pro-OH

H2N

O

O

AFC green fluorescent

Figure 5.1 DPP-IV liberates AFC from its dipeptidyl derivative, Ala-Pro-AFC. The green fluorescence of the product is used as a readout in a DPP-IV inhibition assay.

of the impetus for DPP-IV inhibitor research in the 1980s [9]. At this time, the ACE inhibitor success story had just proven that substrate-based design is a viable approach to drug discovery, and it seems natural that this concept was also pursued in DPP-IV research. DPP-IV is an endopeptidase that releases dipeptides from the Nterminus of a wide variety of peptidic hormones, with a preference for proline at the penultimate position. This proline preference is pronounced in small substrates (such as Ala-Pro-AFC, Figure 5.1), even if the larger peptide GLP-1 (30 amino acids) is cleaved after an alanine (Figure 5.2). In the early 1990s, several academic research groups disclosed dipeptide-like DPP-IV inhibitors, in which a pyrrolidine or a thiazolidine replaces the proline, and an attached amino acid with a free amino group mimics the N-terminus of a substrate peptide (Figure 5.3). The scissile peptide bond was either omitted, as in the prototypical DPP-IV inhibitor P32/98 [10], or replaced by a functional group designed to mimic the proteolytic transition state or to covalently bind to the enzyme’s active site serine. For instance, prolineboronic acids such as 1 have been designed as transition-state analogues and are reversible, slow-binding inhibitors with activities in the low nanomolar range [11]. Phosphonates such as 2 are irreversible inhibitors, which form stable esters with DPP-IV’s catalytically active serine. However, these early types of serine-interacting inhibitors did initially not provide clear advantages over the noncovalent inhibitors, as they were too unstable, too unselective, or did not show a substantially improved activity. Nevertheless, the boronic acid dutogliptin, a DPP-IV inhibitor discovered by Phenomix, has

+

H3 N

His - Ala - Glu - Gly - Thr - Phe - ...

cleavage by DPP-IV Figure 5.2 DPP-IV cleaves GLP-1 at the penultimate position from the N-terminus.

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N

H2N

N

N H

S

O

O P32/98

N H

B(OH)2

1 IC50 = 20 nM

K i = 123 nM Demuth et al., 1991

N PO(OPh)2

O

2

Flentke et al., 1991

irreversible Powers et al., 1995

N

N

H N N H

O

H2N B(OH)2

O N

Dutogliptin

3

IC50 = 25 nM Phenomix, 2005

Ki = 2.2 nM Ferring, 1996

Figure 5.3 Early substrate-based DPP-IV inhibitors and dutogliptin.

apparently overcome these limitations and entered phase 3 clinical development in 2008 [12, 13]. In 1994, a publication demonstrated that nitriles could be used as serine-interacting motifs in inhibitors of prolyl endopeptidase (PEP), a serine protease related to DPP-IV [14]. So far, nitriles had only been known to be cysteine protease inhibitors, but were regarded unreactive to typical serine proteases. This surprising finding prompted Sherwin Wilk’s research group at the City University of New York, and researchers working with Paul D. Jenkins at Ferring Pharmaceuticals, to introduce nitriles into their substrate-based DPP-IV inhibitors [15–17]. These new cyanopyrrolidine-type DPP-IV inhibitors, for example, 3 (Figure 5.3), turned out to have an approximately 100-fold improved inhibitory potency, and additionally both a good selectivity profile and an acceptable chemical stability. Up to this point, the role of DPP-IV in glucose homeostasis was not fully recognized. Rolf Mentlein et al. from the University of Kiel had already demonstrated in 1993 that GLP-1 is a substrate of DPP-IV in vitro [7], but this did not necessarily mean that DPP-IV would be the main metabolic enzyme of GLP-1 in vivo. Actually, 3 was proposed as a potential immunomodulator, as DPP-IV is identical to CD26, a component of the T-cell receptor complex. In 1995, Jens Holst and coworkers from the University of Copenhagen concluded from their studies that DPP-IV is responsible, at least in part, for the observed rapid degradation of GLP-1 in humans and proposed that inhibition of DPP-IV could be a useful adjunct in the management of type 2 diabetes [19]. Shortly thereafter, a collaborating team of scientists working with Hans-Ulrich Demuth from the University of Halle and Christopher H.S. McIntosh and Ray A. Pederson from the University of British Columbia patented DPP-IV

5.3 Substrate-Based DPP-IV Inhibitors

inhibition as a method to lower blood glucose [20]. The patent application was disclosed in 1997 and demonstrated that DPP-IV inhibition with P32/98 did indeed improve glucose tolerance in rats. Demuth, who had spent most of his academic career working on DPP-IV, would later start the biotech company, Probiodrug, to exploit this invention and to bring P32/98 into the clinic. The improvement in glucose tolerance by P32/98 was then reproduced in human healthy volunteers and diabetic patients. P32/98 and the epimeric allo-isoleucyl-thiazolidide were licensed to Merck in late 2000. However, development of both compounds was discontinued in February 2001, after Merck had identified unacceptable toxicity profiles for both compounds. Later, insufficient selectivity over the related dipeptidases DPP-8 and/or DPP-9 was postulated to be the reason for the observed toxicities [21]. At that time, Merck had already identified fluoropyrrolidine 4 (Figure 5.4) as a potential development compound. Because the rationale for subtype selectivity was compelling, 4 was rejected on the basis of a selectivity of only 50-fold over DPP-8 and DPP-9, and medicinal chemistry focused on HTS-based DPP-IV inhibitors, which culminated in the discovery of sitagliptin (see Section 5.4). Further exploration of the substrate analogue series provided 5, with a selectivity of >10 000-fold over DPP-8/9 [22]. This compound was brought forward as a backup for sitagliptin [23]. Another potent and selective difluoropyrrolidine derivative, PF-00734200, has been discovered by Pfizer. This compound was reported to be in phase 2 clinical studies in September 2008 [24, 25]. During this time, the cyanopyrrolidines originally discovered by Sherwin Wilk and the group at Ferring had become the most popular class of DPP-IV inhibitors, as judged by the number of patent applications [18]. While the SAR around the cyanopyrrolidine ring was rather limited, a wide variety of attached amino acids with lipophilic or polar, negatively or positively charged, side chains were tolerated, which provided ample room for proprietary structures.

OCF3

N N

N

O S O NH

N

N N

N

F

O

F

H N

H2N O

4 IC50 = 35 nM Merck, 2004

N

H2N O 5 Ki = 8.8 nM Merck, 2004

Figure 5.4 Pyrrolidides without a serine-interacting motif.

F

N

F

F N H

N O

PF-00734200 IC50 = 13 nM Pfizer, 2005

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N

N H

N

H2N

O

O

N N

6 IC50 = 2 nM

7 IC50 = 64 nM

H N NC

N

N H

N O N

NVP-DPP728 IC50 = 22 nM Novartis, 1998 Figure 5.5 Scaffold change leading to N-alkylglycine DPP-IV inhibitors; NVP-DPP728 was efficacious in a proof-of-concept trial.

An important extension of this SAR was made already in 1996 by scientists at Novartis. Edwin B. Villhauer, a chemist with a long-standing interest in diabetes, was looking for a new project when Jens Holst’s paper was published in 1995. Within a few days, he and his colleagues had a DPP-IV project running. Cells that happened to express DPP-IV were just available and provided an in vitro assay. A paper from 1988, describing a DPP-IV substrate with sarcosine (N-methylglycine) as an N-terminal amino acid [26], caught Villhauer’s attention and led him to explore N-alkylglycine cyanopyrrolidines, in which the side chain of the pyrrolidine-attached amino acid is, formally, shifted to the nitrogen atom (e.g., 6 ! 7, Figure 5.5) [27]. The novel Nalkylglycine cyanopyrrolidines were amenable to resin-based chemistry, which was a very popular technology in those years, enabling the preparation of 1300 diverse compounds within 7 months. Only a few inhibitors with low nanomolar activities were identified in this campaign, one of them carrying a (5-nitro-pyridin-2-yl)aminoethyl substituent. Replacement of the nitro functionality by a nitrile then led to NVP-DPP728 (Figure 5.5) with an improved selectivity over DPP-II and PPCE (postproline cleaving enzyme), which were then standard enzymes in DPP-IV selectivity studies. Within only 9 months, the Novartis project team had identified a development compound. Clinical trials with NVP-DPP728 began in 1998. A first phase 2 trial based on the then widely held paradigm that any type 2 diabetes patient treated with a DPP-IV inhibitor should experience an immediate benefit, gave disappointing results and almost stopped the project. A detailed data analysis suggested that patients with a certain level of pancreatic beta cell activity might benefit over a longer time frame. A second trial designed with the hindsight from this analysis was a huge success: after 4 weeks of treatment, NVP-DPP728 reduced

5.3 Substrate-Based DPP-IV Inhibitors

postmeal glucose excursion, fasting glucose, and 24 h mean glucose. For the first time, it was shown that chronic DPP-IV inhibition in diabetic patients was safe and also led to a reduction in HbA1c levels [28]. NVP-DPP728’s relatively short half-life of 0.85 h was initially not seen as a disadvantage. On the contrary, the many possible physiological roles of DPP-IV made it desirable for a proof-of-concept compound that any potential adverse effects would abate quickly after a discontinuation of administration. DPP-IV cleaves, at least in vitro, not only GLP-1 but also several peptidic hormones, neurotransmitters, and chemokines. Of particular concern was initially the fact that DPP-IV is identical to CD26, a surface protein on activated T-cells, which mediates stimulatory signals; fortunately, it was found that NVP-DPP728 had no immunosuppressant effect. (Later on it was shown that the enzymatic activity of DPP-IV is not required for T-cell function.) It might have been envisioned that NVP-DPP728 could be a short-acting, meal-dependently administered drug to reduce postprandial glucose excursion. Such a treatment would allow an intermittent recovery of DPP-IV activity, and the normal regulation of other potential DPP-IV substrates, thus minimizing side effects. However, a team of Novo Nordisk researchers, collaborating with the Miami School of Medicine, demonstrated in 2001 that a 24 h infusion of GLP-1 over 7 days gave a much better outcome for diabetic patients than a 16 h infusion, indicating that a 24-h blockade of DPP-IV was needed to maximize the therapeutic effect [29]. In 2002, Ferring researchers published their results with the long-acting DPP-IV inhibitor FE 999011 (Figure 5.6), which clearly showed that full inhibition of DPP-IV over 24 h gave the best results in animal models of diabetes [30]. In the following years, most companies therefore focused on inhibitors with high metabolic stability, and today all clinically proven inhibitors show >50% plasma DPP-IV inhibition over 24 h. Apart from the demonstrated clinical efficacy and the facile synthetic access, there might be yet another reason why the N-alkylglycine inhibitors became very popular throughout the industry in the following years: it was generally perceived that they had a superior chemical stability. As already mentioned, cyanopyrrolidine DPP-IV inhibitors, and other substrate-based inhibitors with an electrophilic serine-interacting motif, are chemically unstable in solution. This solution instability is due to an intramolecular reaction between the amino function and the electrophilic motif, as depicted in Scheme 5.1. The short solution half-life typically of a few hours was

N

H2 N O

N FE 999011 Ki = 3.8 nM Ferring, 1996 Figure 5.6 Studies with FE 999011 showed that sustained inhibition of DPP-IV leads to best results in animal models of diabetes.

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R cyclization

N

H2N O

N

O

N

R

N H

NH

Scheme 5.1 The limited solution stability of cyanopyrrolidine DPP-IV inhibitors is due to an intramolecular reaction between the mandatory amino and cyano functionalities.

causing problems for formulation and was made responsible for the short in vivo halflife of some compounds. To overcome this limitation, many research groups explored N-alkylglycines with sterically hindered amines, which would undergo cyclization less readily. Early on, Novartis scientists had identified an adamantyl derivative 8 (Figure 5.7), which was one of the most potent inhibitors discovered in their program. Also, the primary metabolites of this compound were found to be highly active. Already in 1998, Villhauer synthesized one of the putative metabolites, LAF-237, which turned out to have an excellent solution stability, potent inhibitory activity, and good selectivity over related enzymes [31]. The improved pharmacokinetic profile and longer lasting pharmacodynamic effect of LAF-237 led to a replacement of Novartis’ front-runner NVP-DPP728. LAF-237 was later named vildagliptin, in reference to Villhauer, its inventor [32]. Vildagliptin has been, after sitagliptin, the second compound to obtain market approval in the European Union and other countries. In the United States, Novartis has paused its efforts to seek regulatory approval after the FDA had requested additional data to address concerns about the tolerability in patients with renal impairment and skin lesions in nonhuman primates [33] (although no skin OH N H

N O

N H

CN

8 IC50 = 3 nM

N O

CN

LAF-237, vildagliptin IC50 = 3.5 nM Novartis, 2000

HO N

H2N O

9 Ki = 7 nM

N

H2N CN

O

N

H2N CN

10 Ki = 0.9 nM

Figure 5.7 Discovery of vildagliptin and saxagliptin.

O

CN

saxagliptin Ki = 0.6 nM BMS, 2001

5.3 Substrate-Based DPP-IV Inhibitors Table 5.1 Chemical stabilities of primary amine inhibitors.

Compound

Half-lifea)

3 (Figure 5.3) FE 999011 (Figure 5.6) 9 (Figure 5.7)

5h 27 h 42 h

a)

In aqueous buffer at pH 7.2; 39.5
C.

lesions have been observed in humans during clinical trials [67]). Vildagliptin is only moderately selective over DPP-8 and DPP-9. Following the highly publicized Merck study on the potential toxicities associated with DPP-8/9 inhibition [21], Novartis undertook long-term rodent toxicity studies with vildagliptin at exposures that are high enough for complete inhibition of DPP-IV, DPP-8, and DPP-9. As vildagliptin did not display any of the toxicities observed with P32/98 and structurally related molecules, the toxicity of the compounds studied by Merck is more likely the result of unidentified off-target effects that are independent of DPP-8/9, and the relevance of isoform selectivity remains unclear [34]. Researchers at Bristol-Myers Squibb found that converting a tertiary (3, Figure 5.3) to a quaternary alpha-carbon (FE 999011, Figure 5.6) improves the solution half-life by fivefold (Table 5.1). The long-lasting pharmacodynamic effect of FE 999011 might, at least in part, be attributed to this improved solution stability. Also, the introduction of a methylene bridge into the cyanopyrrolidine ring leads to steric bulk that similarly improves the chemical stability (compare FE 999011 and 9, Table 5.1). Molecular modeling demonstrated that these effects are, in both cases, due to intramolecular van der Waals interactions. These interactions disfavor a cis conformation of the amide, which is a prerequisite for cyclization, and thereby increase stability [35]. These findings led the Bristol-Myers Squibb scientists, in striking analogy to the efforts at Novartis, to 10 with an adamantyl substituent. This compound showed an excellent plasma-DPP-IV inhibition after oral dosing in rats, despite a low bioavailability (2%). This seemed to indicate that 10 is converted into an active metabolite in vivo, which prompted the synthesis of a hydroxy analogue as a putative metabolite. Quite similar to the vildagliptin story, it was found that this metabolite, later named saxagliptin (Figure 5.7), was highly potent and had an excellent solution stability [36]. This high solution stability, together with a relatively high distribution volume, makes saxagliptin a long-acting DPP-IV inhibitor. Bristol-Myers Squibb and AstraZeneca have shared the clinical development and filed a New Drug Application in 2008 [37]. Other companies also came up quickly with N-alkylglycines with a wide variety of quaternary N-substituents. TS-021, 11, and ABT-279 (Figure 5.8) are examples of Nalkylglycines that were evaluated in clinical trials. Taisho scientists identified TS-021, which had a much higher solution stability than a previously explored primary amine and an alkylglycine analogue without a quaternary N-substituent [38]. This improved stability translated into markedly higher plasma concentrations in rats, as measured 6 h after oral administration. An oral dose of TS-021 of 0.3 mg/kg in rats almost completely inhibited plasma DPP-IV activity for 120 min and exhibited a significant

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F HO

N

N

N H

N

N

O

N H

N O N

N TS-021 IC50 = 4.6 nM Taisho, 2002

11 IC50 = 20 nM Roche, 2003

N N

N

N H

HOOC

O N

ABT-279 Ki = 1.0 nM Abbott, 2004 Figure 5.8 Various N-alkylglycine compounds in clinical development.

antihyperglycemic effect. The compound underwent phase I clinical studies in 2004, and was licensed to Eli Lilly in 2005; however, no further development was reported. Roche’s clinical compound, 11, was well tolerated in healthy volunteers up to doses of 2 g. In a multiple-dose study, the oral administration of 400 mg of 11 twice daily achieved >50% inhibition of plasma DPP-IV activity over the 12 h dose interval [39]. ABT-279 features a 5-ethynyl substituent on the cyanopyrrolidine ring, which had been demonstrated to improve selectivity over DPP-8/9 [40]. Indeed, the compound has an excellent selectivity over these enzymes as well as related peptidases and other safety-relevant targets. In healthy volunteers, ABT-279 was well tolerated up to doses of 1 g. A primary amine inhibitor with a bulky side-chain, GSK-23A (Figure 5.9), was discovered at GlaxoSmithKline [41]. A combination of steric and electronic effects

OMe

F F F

F

O S O H2N

N

N

H2N O

O

N

N GSK-23A Ki = 53 nM GSK, 2003 Figure 5.9 GSK-23A and denagliptin.

denagliptin Ki = 22 nM GSK, 2003

5.4 Sitagliptin and Analogues

might be responsible for a reduced nucleophilicity of the free amine function, which leads to an extraordinarily long half-life of 1733 h in aqueous buffer at pH 7.2 and 37
C. Denagliptin, another compound from the same company, was developed up to phase 3, but was finally put on hold in 2006 due to unfavorable data from preclinical long-term toxicology experiments [42, 43]. Today, we can look back on more than two decades of research on substrate-based DPP-IV inhibitors. These dipeptide-like compounds provided the first tools to elucidate the function of DPP-IV in vivo. Especially, P32/98 and NVP-DPP728 have played a pivotal role in establishing DPP-IV’s role in glucose homeostasis and in establishing DPP-IV as a therapeutic target for type 2 diabetes. The exciting results obtained with these and other compounds triggered a race in the pharmaceutical industry toward DPP-IV inhibitors as a novel class of antidiabetic medicines, and many companies embarked on fast-follower projects with similar substrate-based compounds. This research culminated in the discovery of vildagliptin, which has obtained market approval in several countries, and other advanced compounds undergoing clinical development. However, other important classes of DPP-IV inhibitors have also emerged more recently, as will be shown in the next sections.

5.4 Sitagliptin and Analogues

Sitagliptin has been the first DPP-IV inhibitor to be approved as a treatment for type 2 diabetes. Launched by Merck in 2006, the annual sales for 2008 have already exceeded US$ 1000 million. The medicinal chemistry team led by Ann E. Weber started in 1999 and initially focused on substrate analogue inhibitors (see Section 5.3). After the identification of unwanted off-target activity as possible reason for multiorgan toxicity, the objective became to achieve a high (>1000-fold) selectivity over related proline peptidases, especially DPP-8 and DPP-9 [23, 44, 45]. The link between activity at DPP-8/9 and toxicity remains a matter of debate, but the goal per se has successfully guided the team toward the discovery of sitagliptin. A high-throughput screening of the Merck sample library was performed in parallel with the medicinal chemistry work on substrate analogues. The screening produced only very few hits, among which the legacy compounds 12 and 13 (Figure 5.10) were followed up. At that time, no structural information of DPP-IV was available, and it was (wrongly) assumed that the pyrrolidine subunit of 13 might reside in the S1 substrate specificity pocket. As a consequence, the pyrrolidine was replaced with a thiazolidine, in analogy with substrate analogues such as P32/98. The truncated molecule 14, with a much reduced molecular weight, was roughly equipotent to 13 but left little room for structural variations. The trifluorophenyl derivative, 15, had a respectable potency but poor pharmacokinetic properties and an insufficient selectivity over DPP-8 [46]. In the meantime, the weakly active HTS hit 12 was combined with the 3-amino-4phenylbutyryl side chain of 13. The resulting hybrid molecule 16 was more than 100fold more potent. A fluorine substituent at C(2) (17) led to an additional fourfold

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Ph

Ph O

N

N

O O O S N H

O

H2N

NH

NH2

N

N

12 IC50 = 11000 nM

O O S N H

NH R

16 (R = H) IC50 = 134 nM 17 (R = F) IC50 = 34 nM

NH2 N O

O

N H

O

HN

NH2

R

O HN

N O F

Cl

18 (R = CH2Ph) IC50 = 139 nM 19 (R = H) IC50 = 3700 nM

13 IC50 = 1900 nM NH2

R N

S

R

N

R

O

NH2

N N

N

R 14 (R = H) IC50 = 3000 nM 15 (R = F) IC50 = 120 nM

F F

O F

20 (R = H) IC50 = 68 nM Sitagliptin (R = CF3 ) IC50 = 18 nM Merck, 2003

Figure 5.10 Evolution of sitagliptin from screening hits 12 and 13.

potency improvement. By removing the decoration of the piperazine, 18 and 19 were obtained. Molecule 18 was reasonably potent and selective but displayed a poor pharmacokinetic profile, which was attributed to the metabolic instability of the piperazine ring [47]. Unsubstituted piperazine 19 was only marginally active but had a low molecular weight and set the stage for further refinement. Incorporation of the 2,4,5-trifluoro substitution pattern on the phenethylamine and replacement of the piperazine by a triazolopiperazine led to a significant improvement in potency. The poor bioavailability of 20 was improved to excellent values by installation of a trifluoromethyl group in the triazole ring, resulting in sitagliptin [48]. Interestingly, triazolopiperazines (systematic name: 5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyrazine) have only recently found widespread use. The parent compound was first disclosed by Merck, as late as 2001, as an intermediate for GABAA ligands as cognition enhancers [49] and soon became a fashionable building block in various Merck projects [50, 51]. Since the public disclosure of sitagliptin as development

5.4 Sitagliptin and Analogues

compound in 2004, the trifluoromethyl-substituted triazolopiperazine has become a frequently used amine subunit across the medicinal chemistry community. Sitagliptin was discovered in the absence of biostructural information. However, as soon as Merck had determined the cocrystal structure of sitagliptin within DPP-IV, the rational design of sitagliptin analogues became feasible. The cocrystal structure shows that the trifluorophenyl group occupies the S1 pocket of the enzyme; this pocket is a central recognition motif and normally accommodates the penultimate amino acid of the substrate (Figure 5.11). The fluorine atoms at C(4) and C(5) optimally fit the hydrophobic niche in the back of the S1 pocket, whereas the fluorine at the ortho position makes a favorable electrostatic interaction with the side chains of Asn710 and Arg125 [52]. Like the class of substrate-based inhibitors, which use a pyrrolidine or thiazolidine derivative to fill the S1 pocket, this class has a rather limited SAR around the trifluorophenyl group. Accordingly, a number of sitagliptin analogues have been made, which use the 2,4,5-trifluorophenethylamine subunit for selective recognition of DPP-IV but differ in the remaining part of the molecule for additional interactions with the target and refinement of the pharmacokinetic properties. For instance, Merck has designed the cyclic analogue 21 (Figure 5.12), in which the butyryl moiety of sitagliptin is replaced by a cyclohexane. Like sitagliptin, 21 is potent and selective over DPP-8/9 but has improved pharmacokinetic properties, with lower clearance and longer half-lives across species [53]. Researchers at Abbott have adapted the major fragments of the Merck inhibitors sitagliptin and 21 to create their own DPP-IV inhibitor, ABT-341. This compound is a potent DPP-IV inhibitor, is selective over DPP-8/9, and has excellent pharmacokinetic properties, comparable to 21 [54]. Despite the similarity to sitagliptin, the binding mode of ABT-341 is different from that of sitagliptin, in that the triazolopyrazinecarbonyl subunit occupies a different part of the binding pocket and induces some conformational change at the target [55]. The compound was selected as

N N F3C

N

S1 pocket

O

F

N

-

F +

H3N

H F

HN Arg125

N H

NH2

H2N

O

Asn710 Figure 5.11 Schematic illustration of key interactions of sitagliptin with DPP-IV: the trifluorophenyl substituent resides in the lipophilic S1 pocket. The ortho-F makes favorable electrostatic interactions with Arg125

and Asn710. The protonated amine binds to a negatively charged surface of the protein (comprised of Glu205, Glu206, and Tyr662, not shown for clarity).

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N

F 3C N F 3C

N N

NH2

F

N

N

N F

NH2

N

F F

F

O F

21 IC50 = 21 nM Merck, 2006

ABT-341 K i = 1.3 nM Abbott, 2006 O O S N

N

O

H

H

NH2

F F

H O F 22 Novartis, 2007

Figure 5.12 Sitagliptin analogues with a 2,4,5-trifluorophenethylamine motif.

development candidate, but no clinical development has been reported as of December 2008. Several years after the discovery of vildagliptin, Novartis has also embarked on a DPP-IV follow-on project, using sitagliptin as seed structure. As a late entrant to the phenethylamine class, compound 22, with a bicyclic subunit, has been identified as a potent DPP-IV inhibitor [56].

5.5 Xanthines and Analogues

The natural products theophylline, theobromine, and caffeine are known as xanthine alkaloids. They are among the oldest drugs, mainly exhibiting vasodilatory and stimulating effects, which can be rationalized through their actions as (nonselective) phosphodiesterase inhibitors and adenosine receptor antagonists [57]. Owing to their rich pharmacology and chemical tractability, xanthine derivatives are well represented in corporate screening libraries. After DPP-IV had emerged as an attractive target for type 2 diabetes, several companies performed a high-throughput screening to identify novel classes DPP-IV inhibitors. Compound 23 (Figure 5.13) is a commercially available “lead-like” xanthine derivative that inhibits DPP-IV in the low micromolar range. As a consequence, 23 has been discovered as a screening hit by a number of research teams. For instance, Merck invested some limited resources on substituent alterations of 23 with little success but then focused on more promising activities (see preceding sections) [44]. On the other hand, Novo Nordisk and Boehringer Ingelheim have identified

5.5 Xanthines and Analogues

R

O

R N

N 3 R

N

1

N

O

j123

7

O

O N

HN O

N N

N

N

NH

N

N

O

N

N

NH2

Xanthine R1 = R3 = R7 = H Theophylline R1 = R3 = Me, R7 = H Theobromine R1 = H, R3 = R7 = Me Caffeine R1 = R3 = R7 = Me

23 IC50 = 3900 nM

24 IC50 = 82 nM Boehringer Ingelheim, 2002

NC O

O N

N O

O

N

N

N

N

N

O

N N

N

NH2

NH2 26 IC50 = 6 nM

25 Novo Nordisk, 2003

O N

N N

O

O N

N N N

N

N

N N

N N N

NH2 Linagliptin IC50 = 1 nM Boehringer Ingelheim, 2004

NH2 27 IC50 = 1 nM Boehringer Ingelheim, 2004

Figure 5.13 Linagliptin and other DPP-IV inhibitors originating from a commercially available screening compound, 23.

a 3-aminopiperidine subunit to be a superior replacement for the piperazine moiety (compounds 24 and 25) and filed patent applications, which overlap to a significant degree [58]. Boehringer Ingelheim has best succeeded in elaborating the xanthine series: modification of the substituents at N(1) and N(7) led to 26, which was very potent on DPP-IV but had unacceptable off-target activities at the hERG channel and the muscarinic receptor M1. Replacement of the substituent at N(7) by a 2-butynyl group and installation of a quinazolylmethyl substituent in lieu of the phenacyl group gave linagliptin, in which the hERG interaction was greatly reduced and the selectivity over the M1 receptor was increased to 300-fold [59]. Comparative preclinical in vivo characterization with vildagliptin, saxagliptin, sitagliptin, and alogliptin shows that linagliptin has a superior potency and longer duration of action [60]. Linagliptin has entered phase 3 clinical trials in 2008. The X-ray crystal structure of linagliptin within DPP-IV reveals that the 2-butynyl group resides in

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CN

CN

O

O N

O N

Br

N R

CN

N

N N

N

N

N NH2

28 R = H IC50 = 13 nM 29 R = F IC50 = 4 nM

NH2 30 IC50 = 5 nM

O

NH2

Alogliptin IC50 = 7 nM Syrrx/Takeda, 2005

Figure 5.14 Structural insight led to a successful core replacement of xanthine 25, and finally to alogliptin.

the S1 pocket. The 4-methylquinazolinone group stacks on top of a tryptophan residue of the protein (Trp629); this p–p interaction [61] is not exploited by other classes of DPP-IV inhibitors and contributes to the very high affinity of linagliptin. The main binding contribution of the xanthine moiety comes from another p–p interaction, a stacking of the central uracil ring with a tyrosine side chain (Tyr547). Comparable aromatic–aromatic interactions can also be affected by a wide variety of other heterocycles [58]. For instance, Boehringer Ingelheim has reported analogue 27, in which the xanthine core has been replaced by an imidazopyridazinone. This compound is equipotent to linagliptin but has a superior selectivity over M1 (>1000-fold) and a different pharmacokinetic profile [62]. Researchers at Syrrx (now Takeda San Diego) have performed a remarkable scaffold hopping exercise, which provided interesting new classes of patentable DPP-IV inhibitors. Supported by high-throughput structural biology and molecular modeling as the company’s core expertise, they started from seed structures such as Novo Nordisk’s xanthine derivative 25 (Figure 5.14). In 25, the cyanobenzyl substituent fills the cavity of the S1 pocket. The cyano group does not engage in a covalent interaction with the enzyme (in contrast to the cyano group in the cyanopyrrolidine series) but makes a favorable electrostatic interaction with the side chains of Asn710 and Arg125, similar to that of the ortho-fluorine of sitagliptin [52]. In search for central scaffolds that could take advantage of the p–p interaction with Tyr547 like the xanthine core of 25, they identified 4-quinazolinone as a suitable heterocyclic replacement. Indeed, compound 28 was very potent. Pharmacokinetic shortcomings were amended by introducing a fluorine at the metabolically vulnerable position of the quinazolinone. Compound 29 had attractive pharmacological and pharmacokinetic properties but showed unacceptable levels of CYP3A4 and hERG inhibition. To minimize the interaction at these off-targets, more polar heterocycles were explored as quinazolinone replacements. Pyrimidinone 30 and the analogous uracil compound, later named alogliptin, retained the potency, and greatly improved the selectivity over the off-targets. Alogliptin, which is the least lipophilic in this series, showed the most favorable pharmacological profile and no evident safety issues [63, 64]. Alogliptin has progressed through clinical development very rapidly, and a New Drug Application has been filed in December 2007.

5.6 Pharmacological Comparison of DPP-IV Inhibitors

5.6 Pharmacological Comparison of DPP-IV Inhibitors

DPP-IV is a chemically very tractable target, and several DPP-IV inhibitors have progressed into clinical trials as medicines to treat type 2 diabetes. In this highly competitive field, the structural diversity is remarkable, with a primary or secondary amino group as the sole recurring motif. Nevertheless, a comparison of phase 3 clinical data at therapeutic doses shows that vildagliptin, sitagliptin, and alogliptin (as representative compounds from each structural class) have similar clinical efficacies. Thus, the average reduction of glycosylated hemoglobin (HbA1c) is 0.5–0.8% after 24 or 26 weeks of treatment at therapeutic doses (Table 5.2). It should be noted that the magnitude of the HbA1c reduction depends on the severity of the disease. For instance, vildagliptin (50 mg twice a day) achieves an HbA1c reduction of 0.6% from a baseline-HbA1c of 8% but a reduction of 1.6% from a baseline of 10% (similar patterns for HbA1c changes are reported for other classes of OADs). While the determination of meaningful changes in HbA1c requires long-term treatment of diabetic patients and a correct estimation of the therapeutic dose, DPPIV inhibition has the benefit of offering an instant-readout biomarker that can forecast the efficacy of the drug in an exploratory setting: DPP-IV activity can be easily determined in blood plasma by measuring the turnover rate of a peptidic substrate using UV spectroscopy. Thus, the notion that sustained inhibition of DPP-IV activity leads to a maximal therapeutic effect [29, 30] has been exploited by Merck in designing phase 1 clinical studies. In healthy volunteers, near-maximal (80%) DPP-IV inhibition was achieved at daily doses of 100 mg (Figure 5.15). The dose of 100 mg/day was confirmed in phase 2 studies to be therapeutically adequate in type 2 diabetic patients and later taken on to phase 3. The successful implementation of a simple pharmacodynamic readout as biomarker enabled Merck to progress sitagliptin from entry into human to phase 3 in only 2.1 years [65]. For vildagliptin, the DPP-IV inhibition after administration of 50 mg is greater than 80% over 12 h but reduced to about 20% after 24 h [66]. Accordingly, the recommended dosing regimen for vildagliptin in the majority of settings is 50 mg

Table 5.2 HbA1c changes after chronic administration of DPP-IV inhibitors (phase 3 data).

Number of subjects Duration of treatment Dose Dosing regimen HbA1c baseline Mean change from baseline HbA1c HbA1c change from placebo

Vildagliptin [67]

Sitagliptin [68]

Alogliptin [74]

90 24 weeks 50 mg Twice daily 8.6% 0.8% 0.5%a)

229 24 weeks 100 mg Once daily 8.0% 0.6% 0.8%b)

131 26 weeks 25 mg Once daily 7.90% 0.59% 0.57%c)

a) 95% confidence interval: (0.8; 0.1); p < 0.05 compared to placebo. b) 95% confidence interval: (1.0; 0.6); p < 0.001 compared to placebo. c) p < 0.001 compared to placebo.

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Placebo

Plasma DPP-IV activity [%]

100 80 60

Sitagliptin (25 mg)

40

Alogliptin (25 mg)

20

Sitagliptin (100 mg)

0 0

6

12 Time after dosing [h]

Figure 5.15 Time course of inhibition of plasma DPP-IV activity after administration of placebo [75], and multiple daily oral doses of sitagliptin (after 10 days, healthy

18

24

volunteers) [75], and alogliptin (after 14 days, type 2 diabetic patients) [69]. Adapted with permission from Excerpta Medica, Inc.: Clinical Therapeutics, copyright 2006, 2008.

twice a day [67]. Alogliptin achieves near-maximal DPP-IV inhibition over 24 h already at much lower doses – a 25 mg dose has approximately the same effect as a 100 mg dose of sitagliptin (Figure 5.15). DPP-IV inhibitors are typically hydrophilic compounds that are rapidly absorbed. Otherwise, the pharmacokinetic properties of the individual DPP-IV inhibitors are quite distinct (Table 5.3): sitagliptin has a relatively low clearance and a large volume of distribution. This translates into a long terminal half-life. Protein binding is low. Sitagliptin is predominantly excreted unchanged through the kidneys, with limited metabolic contribution through CYP3A4 and CYP2C8. Accordingly, patients with renal impairment should use lower doses [68]. In comparison, vildagliptin has a higher clearance and lower volume of distribution, which is reflected in a relatively short half-life. Protein binding is very low. CYPdependent metabolism does not occur. The major elimination pathway is hydrolytic Table 5.3 Pharmacokinetic data of DPP-IV inhibitors.

Sitagliptin [68] Dose tmax Clearance Volume of distribution Half-life Bioavailability Protein binding Renal excretion of parent

100 mg 1–4 h 350 ml/min 198 l 12.4 h 87% 38% 79%

Vildagliptin [67] 50 mg 1.7–2.5 h 680 ml/min 71 l 2 h (intravenous), 3 h (oral) 85% 9.3% 23%

5.7 Concluding Remarks

metabolism at the cyano group, followed by renal excretion of the inactive metabolite; renal excretion of parent drug accounts only for a minor fraction. Vildagliptin is not recommended for renally impaired patients due to insufficient data. Additional safety concerns are related to elevated levels of liver aminotransferases and skin lesions; therefore, monitoring for liver function and skin disorders is recommended [67]. For the less advanced DPP-IV inhibitors, only limited pharmacokinetic information is available. Alogliptin has pharmacokinetic properties similar to sitagliptin, with an apparent half-life of about 20 h and mainly renal excretion of unmetabolized drug [69]. Saxagliptin is also renally excreted, as parent and active metabolite, both of which have apparent half-lives of about 3 and 5 h, respectively [70]. The conversion of saxagliptin to its active metabolite is mediated by CYP3A4/5, a clear difference from its close structural analogue, vildagliptin [71]. Finally, linagliptin has a completely different pharmacokinetic profile in that renal excretion is only a minor elimination route. The compound is largely bound to plasma proteins, has a very long apparent terminal half-life of about 3 days, and has a bioavailability of 30% [72]. Taken together, DPP-IV inhibitors achieve an average HbA1c reduction of 0.5–0.8% after 6 months, independent of the structural class. Inhibition of DPPIV activity is a relevant biomarker for antihyperglycemic efficacy, and near-maximal inhibition over 24 h is required for an optimal effect. Besides, the individual compounds differ significantly in their mode of metabolism and excretion, which may be an important consideration for the individual patient.

5.7 Concluding Remarks

DPP-IV inhibitors represent only one of the many classes of drugs to treat patients with type 2 diabetes. The main goal of management of type 2 diabetes is to achieve glycemic levels as close to the nondiabetic range (HbA1c at 4–6%) as practicable, in order to reduce the risk of late-stage complications. A consensus algorithm of the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD) released in 2008 calls for a therapeutic intervention in cases where HbA1c exceeds 7%. In principle, most patients diagnosed with type 2 diabetes would massively benefit from weight loss and increased physical activity, but only a minority is willing and able to adhere to lifestyle changes in the long term. Therefore, medical management is the common practice, with metformin as first-line treatment. In cases where the HbA1c goal of 7% is not met with metformin alone, either insulin or a sulfonylurea should be added. Alternatively, when hypoglycemia (as frequent side effect of insulin and sulfonylureas) is particularly undesirable, pioglitazone or a GLP1 agonist can be used as an add-on to metformin. Other approved classes of drugs including DPP-IV inhibitors are not within the list of preferred agents, in part due to their limited clinical data [73].

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Sitagliptin, launched in 2006, is often used in combination with metformin. Its rapid rise in popularity is due to the favorable safety profile (no hypoglycemia, no weight gain, and no gastrointestinal side effects). The absence of competition from other DPP-IV inhibitors has also contributed to a highly successful start for this drug. Vildagliptin has been approved in several countries, and other DPP-IV inhibitors are expected to be introduced in the near future. They all lower HbA1c to a similar extent but have quite diverse pharmacokinetic properties. The result of ongoing studies, with focus on long-term benefits and safety, will determine the future role of DPP-IV inhibitors among the options to treat type 2 diabetes.

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(2007) Molecular recognition of ligands in dipeptidyl peptidase IV. Curr. Top. Med. Chem., 7, 609–619. Biftu, T., Scapin, G., Singh, S., Feng, D., Becker, J.W., Eiermann, G., He, H., Lyons, K., Patel, S., Petrov, A., Sinha-Roy, R., Zhang, B., Wu, J., Zhang, X., Doss, G.A., Thornberry, N.A., and Weber, A.E. (2007) Rational design of a novel, potent, and orally bioavailable cyclohexylamine DPP-4 inhibitor by application of molecular modeling and X-ray crystallography of sitagliptin. Bioorg. Med. Chem. Lett., 17, 3384–3387. Pei, Z., Li, X., von Geldern, T.W., Madar, D.J., Longenecker, K., Yong, H., Lubben, T.H., Stewart, K.D., Zinker, B.A., Backes, B.J., Judd, A.S., Mulhern, M., Ballaron, S.J., Stashko, M.A., Mika, A.K., Beno, D.W.A., Teinhart, G.A., Fryer, R.M., Preusser, L.C., Kempf-Grote, A.J., Sham, H.L., and Trevillyan, J.M. (2006) Discovery of ((4R,5S)-5-amino-4-(2,4,5trifluorophenyl)cyclohex-1-enyl)-(3(trifluoromethyl)-5,6-dihydro-[1,2,4] triazolo[4,3-a]pyrazin-7(8H)-yl) methanone (ABT-341), a highly potent, selective, orally efficacious, and safe dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. J. Med. Chem., 49, 6439–6442. Pei, Z. (2008) From the bench to the bedside: dipeptidyl peptidase IV inhibitors, a new class of oral antihyperglycemic agents. Curr. Opin. Drug Discov. Dev., 11, 512–532. Fei, Z., Wu, Q., Zhang, F., Cao, Y., Liu, C., Shieh, W.-C., Xue, S., McKenna, J., Prasad, K., Prashad, M., Baeschlin, D., and Namoto, K. (2008) A scalable synthesis of an azabicyclooctanyl derivative, a novel DPP-4 inhibitor. J. Org. Chem., 73, 9016–9021. Hardman, J.G., Linbird, L.L. (eds.), (2001) Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 10th edn, McGraw-Hill. Szczepankiewicz, B.G., and Kurukulasuriya, R. (2007) Aromatic heterocycle-based DPP-IV inhibitors: xanthines and related structural types. Curr. Top. Med. Chem., 569–578.

59 Eckhardt, M., Langkopf, E., Mark, M.,

60

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63

64

65

Tadayyon, M., Thomas, L., Nar, H., Pfrengle, W., Guth, B., Lotz, R., Sieger, P., Fuchs, H., and Himmelsbach, F. (2007) 8(3-(R)-Aminopiperidin-1-yl)-7-but-2-ynyl3-methyl-1-(4-methyl-quinazolin-2ylmethyl)-3,7-dihydropurine-2,6-dione (BI 1356), a highly potent, selective, longacting, and orally bioavailable DPP-4 inhibitor for the treatment of type 2 diabetes. J. Med. Chem., 50, 6450–6453. Thomas, L., Eckhardt, M., Langkopf, E., Tadayyon, M., Himmelsbach, F., and Mark, M. (2008) (R)-8-(3-Aminopiperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4methyl-quinazolin-2-ylmethyl)-3,7dihydro-purine-2,6-dione (BI 1356), a novel xanthine-based dipeptidyl peptidase 4 inhibitor, has a superior potency and longer duration of action compared with other dipeptidyl peptidase-4 inhibitors. J. Pharmacol. Exp. Ther., 325, 175–182. Meyer, E.A., Castellano, R.K., and Diederich, F. (2003) Interactions with aromatic rings in chemical and biological recognition. Angew. Chem. Int. Ed., 42, 1210–1250. Eckhardt, M., Hauel, N., Himmelsbach, F., Langkopf, E., Nar, H., Mark, M., Tadayyon, M., Thomas, L., Guth, B., and Lotz, R. (2008) 3,5-Dihydro-imidazo[4,5-d] pyridazin-4-ones: a class of potent DPP-4 inhibitors. Bioorg. Med. Chem. Lett., 18, 3158–3162. Feng, J., Zhang, Z., Wallace, M.B., Stafford, J.A., Kaldor, S.W., Kassel, D.B., Navre, M., Shi, L., Skene, R.J., Asakawa, T., Takeuchi, K., Xu, R., Webb, D.R., and Gwaltney, S.L. (2007) Discovery of alogliptin: a potent, selective, bioavailable, and efficacious inhibitor of dipeptidyl peptidase IV. J. Med. Chem., 50, 2297–2300. Gwaltney, S.L. (2008) Medicinal chemistry approaches to the inhibition of dipeptidyl peptidase IV. Curr. Top. Med. Chem., 8, 1545–1552. Krishna, R., Herman, G., and Wagner, J.A. (2008) Accelerating drug development using biomarkers: a case study with sitagliptin, a novel DPP4 inhibitor for type 2 diabetes. AAPS J., 10, 401–409.

References 66 He, Y.-L., Wang, Y., Bullock, J.M., Deacon,

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C.F., Holst, J.J., Dunning, B.E., LigherosSaylan, M., and Foley, J.E. (2007) Pharmacodynamics of vildagliptin in patients with type 2 diabetes during OGTT. J. Clin. Pharmacol., 47, 633–641. European Medicines Agency, Galvus product information (2008) http://www. emea.europa.eu/humandocs/PDFs/ EPAR/galvus/H-771-PI-en.pdf, accessed 16 December 2008. US Food and Drug Administration (2008) Januvia prescribing information, July 23. Covington, P., Christopher, R., Davenport, M., Fleck, P., Mekki, Q.A., Wann, E.R., and Karim, A. (2008) Pharmacokinetic, pharmacodynamic, and tolerability profiles of the dipeptidyl peptidase-4 inhibitor alogliptin: a randomized, double-blind, placebo-controlled, multiple-dose study in adult patients with type 2 diabetes. Clin. Ther., 30, 499–512. Boulton, D.W.and Geraldes, M. (2007) American Diabetes Association, 67th Scientific Sessions, Poster 606-P. Cole, P., Serradell, N., Bolós, J., and Castañer, R. (2008) Saxagliptin: dipeptidyl peptidase IV inhibitor antidiabetic agent. Drugs Future, 33, 577–586. H€ uttner, S., Graefe-Mody, E.U., Withopf, B., Ring, A., and Dugi, K.A. (2008) Safety, tolerability, pharmacokinetics, and pharmacodynamics of single oral doses of

BI 1356, an inhibitor of dipeptidyl peptidase 4, in healthy male volunteers. J. Clin. Pharmacol., 48, 1171–1178. 73 Nathan, D.M., Buse, J.B., Davidson, M.B., Ferrannini, E., Holman, R.R., Sherwin, R., and Zinman, B. (2009) Medical management of hyperglycemia in type 2 diabetes: a consensus algorithm for the initiation and adjustment of therapy: a consensus statement of the American Diabetes Association and the European Association for the Study of Diabetes. Diabetes Care, 32, 193–203. 74 DeFronzo, R.A., Fleck, P.R., Wilson, C.A., and Mekki, Q. (2008) Efficacy and safety of the dipeptidyl peptidase-4 inhibitor alogliptin in patients with type 2 diabetes and inadequate glycemic control: a randomized, double-blind, placebocontrolled study. Diabetes Care, 31, 2315–2317. 75 Bergman, A.J., Stevens, C., Zhou, Y., Yi, B., Laethem, M., De Smet, M., Snyder, K., Hilliard, D., Tanaka, W., Zeng, W., Tanen, M., Wang, A.Q., Chen, L., Winchell, G., Davies, M.J., Ramael, S., Wagner, J.A., and Herman, G.A. (2006) Pharmacokinetic and pharmacodynamic properties of multiple oral doses of sitagliptin, a dipeptidyl peptidase-IV inhibitor: a double-blind, randomized, placebocontrolled study in healthy male volunteers. Clin. Ther., 28, 55–72.

Patrizio Mattei

F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland Patrizio Mattei was born in Thalwil, Switzerland, in 1968. He studied chemistry at the Eidgen€ ossische Technische Hochschule in Z€ urich, where he also carried out his doctoral work under the supervision of Professor Fran¸cois Diederich. After a postdoctoral fellowship in Professor Donald Hilvert’s group at the Scripps Research Institute in La Jolla, California, he joined F. Hoffmann-La Roche Ltd. as a medicinal chemist in 1999. He is coinventor of more than 40 patents and patent applications, 5 of which are related to DPP-IV inhibitors.

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Jens-Uwe Peters

F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland Jens-Uwe Peters studied chemistry at the University of G€ottingen, where he worked with Professor A. de Meijere, and conducted his doctoral research at the Technical University of Berlin under the guidance of Professor S. Blechert. After 1 year of postdoctoral studies in the group of Professor J. Rebek at the Scripps Research Institute, La Jolla, he joined the Medicinal Chemistry group at F. Hoffmann-La Roche, Ltd. in 1999. Since then, he has been contributing to Roche’s DPPIV project and to numerous other drug discovery projects.

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6 Phosphodiesterase 5 Inhibitors to Treat Erectile Dysfunction Nils Griebenow, Helmut Haning, and Erwin Bischoff

6.1 Introduction

The second messengers, cyclic 30 ,50 -adenosine monophosphate (cAMP) and cyclic 30 ,50 -guanosine monophosphate (cGMP), play a pivotal role in mediating a plethora of functional responses to hormones and other cellular transmitters. Their intracellular concentrations are adjusted by their synthesis through adenylyl cyclase and guanylyl cyclase, and by their degradation through cyclic nucleotide phosphodiesterases to the physiologically inactive 50 -nucleoside monophosphates. Cyclic nucleotides and phosphodiesterases (PDEs) were discovered almost simultaneously in the late 1950s by Sutherland and coworkers [1–3]. Eleven members of the superfamily of phosphodiesterases are known today. They substantially differ in their tissue distribution, physicochemical properties, substrate and inhibitor specificities, and regulatory mechanisms. The sensitivity of the physiological processes regulated by cyclic nucleotides requires precise and rapid regulation of the level of these second messengers according to the requirements of the physiological status of the cell. A precise modulation of PDE function is crucial for maintaining cyclic nucleotide levels within a narrow concentration range. This is consistent with, for example, the changed phenotype of mice deficient in distinct PDEs, demonstrating the importance of finely balanced cyclic nucleotide levels [4–6]. Due to their key role in the regulation of physiological processes, inhibitors of PDEs can be used as therapeutic tools for various diseases. PDE5 is one member of the super family that specifically hydrolyzes cGMP, and a number of new PDE5 inhibitors have recently been introduced. Increasing knowledge of the molecular structure of the enzyme and the catalytic center may help gain a better understanding of the structure–activity relationship (SAR) of new inhibitors. Furthermore, potent and selective inhibitors provide pharmacological tools to investigate the physiological functions of PDE5, which may provide novel therapeutic opportunities for this class of inhibitors beyond the well-established treatment of erectile dysfunction (ED).

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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This chapter summarizes the molecular biology, regulation, biochemistry, and functions of the PDE5 isozyme. The structure–activity relationships of the known PDE5 inhibitors from distinct structural classes are discussed from the standpoint of structural diversity rather than from a pharmacological point of view. In addition, the pharmacological effects, the clinical applications, and emerging applications for PDE5 inhibitors are presented. IC50 values are quoted as reported in the original source, and no discrimination is made between IC50 values derived from PDEs of different origin.

6.2 Pharmacology of Phosphodiesterases 6.2.1 The Phosphodiesterase Family

The mammalian phosphodiesterase superfamily (type 1 PDEs) of isoenzymes today consists of 11 different subfamilies of gene products that have been characterized on the basis of their amino acid sequences, substrate specificities, intracellular and extracellular regulators, and pharmacological properties. Numerous reviews demonstrate the enormous increase in knowledge of the structural features, catalytic mechanisms, regulation, physiology, and inhibitors of this intriguing class of enzymes, although many details of their function remain unclear [7–16, 23]. Several of the isoenzymes are encoded by different genes that in addition to the presence of different splice variants brings the number of different PDE proteins up to approximately 50 in mammalian cells. The increasing knowledge of the genomic relationships, and a better understanding of the regulation and the functional characteristics of the different family members, has generated a new systematic nomenclature [17]. Under this nomenclature, a PDE family is designated by an Arabic numeral followed by a capital letter designating the gene within the respective family and the second Arabic numeral indicates the variant product derived from a single gene (e.g., PDE4A2 denominates PDE family 4, gene A, splice variant 2). The mammalian PDEs share a common structural organization, with a conserved catalytic domain located close to the C-terminus and a regulatory domain mostly close to the N-terminus of the protein. The catalytic domain contains a signature motif HD (X2)H(X4)N common to all mammalian PDEs that have consensus metal binding domains (Zn2 þ , Mg2 þ , Mn2 þ ) related to those of metal-ion phosphohydrolases [18]. Someof these PDEs(PDE2, PDE5, PDE6,and PDE10)bind cGMP with highspecificity at homologous allosteric sites that are arranged in tandem in their amino terminal domains [19]. In addition to their regulatory function of hydrolyzing cAMP and cGMP, they may also have a function as intracellular receptors or as a sink for cGMP [10, 19]. However, the physiological meaning of this latter property is not yet fully understood. Some of these PDEs are targets for many drugs that are used to treat cardiovascular diseases, asthma, erectile dysfunction, and other diseases. Different isoenzymes are characterized according to their substrate specificity, sequence homology, kinetic

6.2 Pharmacology of Phosphodiesterases Table 6.1 The superfamily of phosphodiesterases.

Name

Km (mm)

Characteristics

Number of genes

cAMP

cGMP

Ca2 þ -CaMstimulated cGMPstimulated

1–30

3

3

30–100

10–30

1

PDE3

cGMP-inhibited

0.1–0.5

0.1–0.5

2

PDE4

cAMP-specific

0.5–4

>50

4

PDE5

cGMP-specific

>40

1.5

1

PDE6 PDE7

Photoreceptor cAMP high affinity cAMP high affinity

2000 0.2

60 >1000

4 2

0.7

>100

2

>100

0.07

1

PDE10

cGMP high affinity Dual substrate

0.5

3

1

PDE11

Dual substrate

1

0.5

1

PDE1 PDE2

PDE8

PDE9

Primary tissue distribution

IBMX sensitivity

VSMC, brain, lung, heart Adrenal cortex, brain, heart, liver, corp. cav., olfact, bulbous Heart, lung, liver, immunocytes, pancreas Immunocytes, lung, brain VSMC, SMC, lung, corp. cav., platelets Retina Skeletal muscle, T-cells Widely expressed, most abundant in testis, ovary, intestine, colon Broadly expressed, liver, kidney Broadly expressed, in mice most abundant in brain, testes Testes, brain, corp. cav., skeletal muscle, prostate

þ þ

þ

þ þ

þ þ



þ

þ

VSMC: vascular smooth muscle cells; SMC: smooth muscle cells; CaM: calmodulin; corp. cav.: corpus cavernosum.

properties, and sensitivity to certain known PDE inhibitors. Table 6.1 shows these properties together with the predominant tissue expression of various PDEs. 6.2.2 Pharmacological Effects of cGMP

Nitric oxide, nitrovasodilators, and natriuretic peptides act as relaxants, regulating smooth muscle tone by direct activation of guanylyl cyclase, which leads to the

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elevation of cGMP. Thus, influencing cGMP levels by modulating PDE or guanylate cyclase activity remains an interesting pharmacological approach to regulate smooth muscle tone. Activation of cGMP-dependent protein kinase G (PKG) appears to mediate all cGMP-induced relaxant effects [20]. Disruption of the PKG I gene totally abolishes nitric oxide (NO)/cGMP-dependent relaxation of smooth muscle in mouse aorta and also causes erectile dysfunction in mice [21]. However, PKGs physiological function in the phosphorylation and activation of PDE5 has not yet been established. It is suggested that only minimal PDE5 activity is required to control the intracellular cGMP levels in close proximity to proteins involved in cGMP-induced relaxation of smooth muscle. These include myosin light-chain phosphatase and proteins associated with the regulation of intracellular Ca2 þ concentration, for example, Ca2 þ -activated K þ channel and IP3 receptor-associated cGMP kinase substrate (IRAG). In each of these cases, activation of PDE5 may provide a negative feedback regulation of cGMP and PKG when the intracellular concentration of cGMP reaches a high level. Regulation of PDE5 via dephosphorylation by myosin phosphatase might be physiologically as important as regulation of PDE5 phosphorylation by PKG in providing a relaxation contraction cycle [22]. Increased cGMP levels have also been shown to inhibit smooth muscle cell (SMC) proliferation that is a key event in the development of arteriosclerotic lesions [24, 25]. 6.2.3 PDE5: Regulation, Activation, and Structure

PDE5 catalytic activity is regulated by a plethora of mechanisms that significantly contribute to negative feedback regulation of cGMP signaling and are quite complex [26–29]. Until recently, PDE5 was known as the cGMP-binding cGMP-specific PDE because it contains allosteric sites and a catalytic site that are both highly specific for cGMP (Figure 6.1). PDE5 is a modular protein with a N-terminal R domain and a C-terminal C domain (Figure 6.1) [30]. There is significant communication between the PDE5 R domain and the C domain, and this property fashions a complex and well-integrated modulation of enzyme activity. The cGMP binding sites in the R domain and the catalytic site are not evolutionarily related, but both exhibit approximately 100-fold specificity to cGMP versus cAMP. When the C domain alone is generated as a recombinant protein, it is a monomer that retains the salient features of catalysis exhibited by PDE5 holoenzyme [31, 32]. PDE5 exhibits a Km value for cGMP of approximately 2–3 mM, a concentration that is generally considered to be well above normal physiological concentration range for cGMP. Comprehensive studies using site-directed mutagenesis of conserved amino acids in the C domain of PDE5 first identified residues that are important for contact with the critical divalent cations, those that contribute to binding of the substrate, and many of those that are key to potent interaction of inhibitors such as sildenafil, vardenafil, and tadalafil [33–35]. The N-terminal R domain of PDE5 contains a number of functional subdomains that impact PDE5 catalytic function and its interaction with inhibitors. The

6.2 Pharmacology of Phosphodiesterases

Figure 6.1 Working model of PDE5. PDE5 is a dimer of two identical approximately 100 kDa subunits. It has a C-domain located in the more carboxyl-terminal portion of the protein and an R-domain located in the more amino-terminal portion. Dimerization occurs through interactions at multiple points in the regulatory domain indicated by the black bars. The catalytic machinery is depicted as two ovals that reflect the two divalent cations that provide for catalytic hydrolysis of cGMP. Cyclic GMP is also bound to

allosteric sites in the R-domain including a highaffinity site in GAF A (indicated by the deep oval binding pocket) and perhaps in GAF B; GAF B has been shown to have numerous regulatory functions in PDE5 and may have weak cGMP binding activity as indicated by the shallow oval pocket. PDE5 can be specifically phosphorylated by PKG at a single site, Ser-102, in the R-domain, and exposure of this serine is regulated by binding of cGMP and other ligands to protein.

subdomains include two GAFs (GAF A and GAF B; GAF: cGMP-specific cGMPstimulated PDE, Adenylate cyclase, and FhlA), a single phosphorylation site (Ser-102) that is preferentially phosphorylated by PKG, and dimerization contacts [30]. Highaffinity allosteric cGMP binding is provided by the GAF cyclic GMP-hydrolyzing phosphodiesterases. High-affinity allosteric cGMP binding is provided by GAF A subdomain [36, 37], but whether GAF B can also bind cGMP is not known. In the holoenzyme, binding of cGMP to the allosteric cGMP binding sites is low, but occupation of the catalytic site by either cGMP or PDE5 inhibitors stimulates cGMP binding to the allosteric sites [38, 39]. Cyclic GMP occupation of the allosteric sites increases affinity of the catalytic site for cGMP and inhibitors. Furthermore, when the allosteric sites are occupied by cGMP, PDE5 undergoes a conformational change that exposes Ser-102 for rapid phosphorylation by PKG [39]. Phosphorylation of Ser-102 in turn increases both the affinity of the allosteric cGMP-binding sites for cGMP and the affinity of the catalytic site for substrate or inhibitors [27, 40]).

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O O N N

N

N

N

N

N

O Vardenafil (2)

O Sildenafil (1)

O O N

N

O

N

HN

O O S N H

N

N H

HN

O O S N

N

HN

O O S N

N

O

N

HN

N N O

O

Udenafil (4) N

O

N

N

O HN

O

OH N

O N H O

N

N N

Avanafil (6)

Mirodenafil (5)

N

HO

Tadalafil (3)

S O O

O O O S N

N O

HN N

N

N

O

SLX-2101 (7)

Cl

Figure 6.2 Drugs on the market and under clinical trials.

Multiple lines of evidence suggest that PDE5 exists in at least two conformations and that the distribution of the PDE5 population between these conformers can be regulated by phosphorylation/dephosphorylation, oxidation/reduction, and binding of cGMP or inhibitors. These stepwise modulations of structure and function in PDE5 are tightly controlled and regimented and bear significant consequences for feedback regulation of cGMP levels and inhibitor potency. First of all, cGMP occupation of the allosteric cGMP binding site increases the affinity of the catalytic site for cGMP, the substrate, or inhibitors such as sildenafil, vardenafil, or tadalafil. In the absence of PDE5 inhibitors, the rate of hydrolysis of cGMP is increased since the PDE5 catalytic site is usually not saturated with cGMP; with higher affinity of the catalytic site for cGMP, more will be bound to the site and hydrolyzed. As a substrate mimic, a similar event occurs for PDE5 inhibitors, that is, cGMP binding to the allosteric sites increases the affinity for the inhibitor [41]. However, the inhibitor is not degraded by PDE5 and will remain bound to the catalytic site for a longer period of time, thus representing a feed-forward process that enhances inhibitor potency. Phosphorylation of Ser-102 increases both allosteric cGMP binding and affinity of the catalytic site for cGMP or inhibitors, thereby fostering improved function of the catalytic site. The physiological and pharmacological impacts of the regulatory processes described above are clear and are likely to occur in many tissues. For example, when cGMP is elevated in vascular smooth muscle, as occurs in the penile corpus cavernosum during sexual arousal, these regulatory factors, including increased

6.2 Pharmacology of Phosphodiesterases

allosteric cGMP binding and phosphorylation of Ser-102, impact PDE5 function and the physiological process associated with the vasodilation that leads to penile erection. Activation of guanylyl cyclases to produce more cGMP will increase the activity of the catalytic site in at least three ways to degrade cGMP (Figure 6.1). First, elevation of cGMP will cause more degradation of cGMP, simply due to a greater supply of cGMP to the PDE5 catalytic site. Secondly increased cytosolic cGMP will foster cGMP binding to the allosteric cGMP-binding sites of PDE5, and this will further increase hydrolysis of cGMP at the catalytic site. Third, phosphorylation of Ser-102 on PDE5 will further increase the affinity of allosteric cGMP binding and foster greater catalytic activity. All these effects act to dampen or terminate the cGMP signaling initiated by sexual arousal and act in concert as a negative feedback regulatory process. The increased affinity of the enzyme for the substrate would also provide improved competition with inhibitors that act at the catalytic site as well, thereby blunting the potency of inhibitors to some extent. However, in a physiological setting, effects of potent PDE5 inhibitors prevail since cGMP accumulates, thereby providing vasodilation sufficient for improved penile tumescence. Negative feedback control of the cGMP pathway through sequestration by PDE5 could occur in some cells. In the corpus cavernosum, the concentration of PDE5 allosteric cGMP binding GAF A is more than 100 nM [42], higher than the concentration of cGMP (basal 20 nM). Most of this cGMP would be tightly bound to GAF A and unavailable for its target receptors such as PKG. Increased sequestration by this process would be expected to occur after cGMP elevation and stimulation of the other negative feedback systems described above. Whether or not PDE2 concentration is high enough in most cells to mediate significant sequestration of cGMP, or cAMP, has not been demonstrated. PDE6 concentration in retinal photoreceptor cells is clearly high enough to impart sequestration [43]. Some of the same sequence of events described as providing negative feedback regulation of cGMP level can also enhance the potency and efficacy of PDE inhibitors. PDE5 inhibitors occupy the catalytic site of PDE5 to block cGMP access and thereby inhibit cGMP degradation. As a result, cGMP is readily available to bind to PDE5 allosteric cGMP-binding sites, bind to and activate PKG, and foster phosphorylation of Ser-102 on PDE5 by PKG, which enhances inhibitor binding at the catalytic site and increases the cGMP level further (Figure 6.1). Increased cGMP binding to the allosteric sites in turn promotes tighter binding of inhibitors at the catalytic site that elevates cGMP even more. In the presence of the inhibitor, this regulatory process becomes a feed-forward process to promote potency and efficacy of the pharmacological action of competitive inhibitors. Furthermore, prolonged exposure of PDE5 to inhibitors causes the enzyme to undergo a conformational change that increases the affinity of the enzyme for the inhibitor. Altogether, these effects that in nature provide a powerful counter to cGMP elevation in tissues containing PDE5 also provide a powerful feed-forward process to improve potency and action of PDE5 inhibitors. Recently, the crystal structure of the catalytic domain (residues 537–860) of human PDE5 in complex with three PDE5 inhibitors, sildenafil [44], tadalafil [45], and vardenafil [46] has been published [32] (Fig. 6.2). Herein, the catalytic domain consists of three helical subdomains, an N-terminal cyclin-fold region, a linker

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region, and a C-terminal helical bundle, whereas the active site of PDE5 is located at the center of the C-terminal helical bundle domain. The substrate pocket has a total   volume of about 330 A3 and is approximately 10 A deep, with a narrow opening and a wider inner space. In detail, it is composed of four subsites, a metal binding site (M site) [29], core pocket (Q pocket), hydrophobic pocket (H pocket), and lid region (L region). The binding mode of sildenafil [47] differs from that of tadalafil (Figure 6.3). Sildenafil occupies the lid region with the N-methyl-piperazin moiety, whereas tadalafil makes no interaction with the L region of the protein. The core of sildenafil forms a bidentate hydrogen bond to the O-atom of Gln817, while the NH of tadalafil

Figure 6.3 Schematic representation of interactions made by sildenafil (a) and tadalafil (b) in complex with PDE5.

6.2 Pharmacology of Phosphodiesterases

makes a single hydrogen bond to the Gln817. The more extensive interactions with the H pocket may be one of the reasons why tadalafil maintains high affinity without binding to the L region as well as the M site. However, there is a conflicting evidence for a different binding mode of vardenafil compared to that of sildenafil [48]. 6.2.4 PDE5 Inhibitors and Erectile Dysfunction

Based on the vasorelaxing effects of cGMP, PDE5 was originally targeted for the treatment of hypertension, including pulmonary hypertension, coronary heart disease, and angina. Later on, erectile dysfunction emerged as an interesting indication for PDE5 inhibitors. Male erectile dysfunction is defined as the inability to attain and/or maintain penile erection sufficient for satisfactory sexual performance (NIH Consensus Conference 1993 [49]). ED can have a profound effect on the quality of life. Subjects frequently report anxiety, loss of self-esteem, lack of self-confidence, and difficulties in relationships with their partners. The prevalence of ED is age-related. Severe or complete ED has a prevalence of about 5% in men aged 40 and 15% in men aged 70, but less severe forms of ED are more prevalent [50]. It can also occur as a result of some defect in neurotransmission of nonadrenergic, noncholinergic (NANC) neurons in the penis associated with a variety of pre-existing factors such as hypertension, and/or coronary heart disease, diabetes, hormone levels, age, spinal cord injury, and psychological influences. Prostatectomy as a consequence of surgical treatment of prostate cancer is a further major risk factor. ED is multifactorial in etiology and frequently involves an interplay of both psychological and organic factors. Prior to the advent of sildenafil, the treatment of ED involved the use of vacuum constriction devices, penile prosthesis implantation, or intracavernosal injections with vasodilating agents. Sildenafil was the first orally active drug used for the treatment of ED that had a noteworthy commercial success. Reviews on the prevalence, pathophysiology, and risk factors associated with ED are numerous [51–54], so are summaries on its pharmacology and efficacy of treatment [55, 56]. An excellent overview of ED treatment options and compounds in development was recently published [57]. Penile erection is a hemodynamic process involving relaxation of smooth muscle in the corpus cavernosum and its associated arterioles. This relaxation results in increased blood flow into the trabecular spaces of the corpora cavernosa [53, 58]. Smooth muscle relaxation is mediated by nitric oxide that, during sexual stimulation, either directly or psychosomatically, is synthesized in the nerve terminals of parasympathetic, nonadrenergic, noncholinergic (NANC) neurons in the penis and also by the endothelial cells of the blood vessels and the lacunar spaces of the corpora cavernosa. NO activates smooth muscle cell soluble guanylate cyclase, resulting in an increased intracellular cGMP level that leads to relaxation of smooth muscle of the corpus cavernosum and of the penile arterioles [59–62]. The level of cGMP is regulated by a balance between its rate of synthesis via guanylate cyclase and its

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hydrolysis to the physiologically inactive GMP by the cGMP hydrolyzing phosphodiesterases. These are the calmodulin-stimulated PDE type 1 (PDE1), the cGMP activated PDE type 2 (PDE2), and the cGMP specific PDE type 5 (PDE5). Recently, a new cGMP specific PDE type 9 (PDE9) was described; however, its tissue distribution and physiological role are not yet known [63]. The most plentiful cGMP-PDEs in the human corpus cavernosum are PDE5 [64] (about 70%) and PDE2 (30%) [36]. Other reports describe significant amounts of PDE3, PDE4, and PDE5 [65]. Inhibition of PDE5 increases the level of cGMP after release of NO at parasympathetic nerve terminals during sexual stimulation, thus enhancing relaxation of smooth muscle. As a result, vascular tone in the arteries leading to the penis decreases. This causes increased blood flow and an enlargement of the cavernosal tissue that induce penile erection [53, 66, 67]. A prerequisite to PDE5 inhibition as a therapeutic principle is sexual stimulation. The selectivity of vasorelaxation of penile tissue is mainly based on the increased synthesis of cGMP in the cavernosal tissue during sexual stimulation. Although PDE5 is the main cGMP-metabolizing enzyme in cavernosal tissue, this unique situation of increased cGMP concentrations after nerval stimulation contributes more strongly to the selective vasodilation in cavernosal tissue than the localization of PDE5 in this tissue [68]. This is also the reason for the fact that only minor effects on systemic blood pressure have been observed in clinical studies with PDE5 inhibitors. Most of the reversible mild to moderate side effects reported for PDE5 inhibitors are mechanism related and a consequence of the abundant distribution of the enzyme in almost all smooth muscle cells. Although varying from compound to compound, the most frequent and dose-dependent side effects are headache and, to a minor extent, nasal congestion. All these effects can be interpreted as a result of the general dilation of small arterial vessels. Nonselective inhibition of PDE1 that is also a cGMP metabolizing PDE and predominantly localized in vascular smooth muscle cells (VSMCs) can also contribute to this effect. Greater selectivity could improve this profile. Dyspepsia is also often reported as a side effect, which can be explained by the high expression of PDE5 in the lower esophagus [69]. Another reported side effect is visual disturbances. These ophthalmologic effects are probably related to the inhibition of PDE6 activity present in the retina that is responsible for signal transduction in the eye [70]. Table 6.2 summarizes the existing PDE5 inhibitors on the ED market, along with compounds in clinical development for ED. Below, we would like to give a short update on the current status. First approved in 1998, sildenafil has been found to be effective in improving erections in a large numbers of men with ED resulting from a variety of causes [71–73]. Sildenafil is rapidly absorbed, with a tmax of approximately 1 h and a half-life of approximately 4 h. A number of placebo-controlled clinical trials have found sildenafil effective in treating ED resulting from both psychogenic and organic causes, such as spinal cord injury, and in other special populations such as diabetics. Several studies have shown sildenafil to improve erections in over 80% of men in the broad ED population and slightly less in the more challenging-to-treat diabetic population (between 56% [74] and 64% [75], compared to much lower placebo rates).

6.2 Pharmacology of Phosphodiesterases Table 6.2

PDE5 inhibitors on the market and under clinical development for erectile dysfunction.

Compound name

Company

Development status

Sildenafil Vardenafil Tadalafil Udenafil Mirodenafil Avanafil SLX-2101

Pfizer Bayer Schering Pharma Lilly Dong-A Pharmaceutical SK Chemicals VIVUS Surface Logix

Launched 1998 (Viagra) Launched 2003 (LevitraÒ ) Launched 2003 (CialisÒ ) Launched 2005 (ZydenaÒ ) Launched 2007 (M-vixÒ ) Phase II Phase II

Sildenafil has been generally well tolerated, with adverse events primarily associated with PDE5 inhibition in other locations resulting in flushing, headache, dyspepsia, and rhinitis. However, there have been a small percentage of men who have reported blue color vision changes, which can be linked to the inhibition of PDE6. Vardenafil was first approved by US regulators in 2003 [76]. The drug was demonstrated to have a selectivity 257-fold higher for PDE5 than PDE1, and 16fold higher for PDE5 compared to PDE6. In contrast, sildenafil was only 60-fold more selective in its blocking effect of PDE5 compared to the blockade of PDE1, and 7-fold more selective in terms of PDE6. Maximum plasma concentrations after oral administration have been reported as early as 0.7 h with a half-life of 4–5 h. In two RigiScan studies, vardenafil increased rigidity and prolonged the duration of erections compared to placebo-treated men with ED. A large phase II study of men with mild to severe ED indicated that vardenafil significantly improved erectile function (EF) in them. Efficacy was noted irrespective of etiology and age, as well as baseline severity. Phase III programs have confirmed these early results in a broader population with common comorbidities such as diabetes and hypertension, where up to 85% of men reported improved erection at 26 weeks [77]. In a specific study in men with diabetes mellitus, up to 72% of men at the highest 20 mg dose responded, compared to 13% on placebo. Physicians interviewed by Decision Resources, for example, cite vardenafil as their preferred first-line therapy for diabetics with ED [76]. An especially challenging-to-treat condition is ED following prostatectomy, and a significant increase in response with vardenafil was seen, with 65% responding compared to 13% on placebo, in men with nerve-sparing surgery [78]. Adverse effects have been generally mild to moderate and tend to decrease with time [79–81]. The most common adverse events could again be linked to generalized PDE5 inhibition and the incidence of vision disturbances has been rare. Tadalafil was launched in Europe and the United States in the first half of 2003 [82]. While high concentrations of tadalafil are seen in plasma, the tmax of the most recent formulation has been reported to be about 2 h. In contrast to sildenafil and vardenafil, tadalafil shows a prolonged half-life of 17.5 h [2, 83, 84]. Physicians have already dubbed tadalafil “the weekend pill” because its effects can last up to 36 h, whereas the other PDE inhibitors’ duration of action is generally 4–8 h. In a series of small phase

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II studies with an early formulation, clear efficacy was seen over placebo. In a phase III study with diabetic patients, improved erection in up to 64% of men was reported compared to 25% on placebo [85]. Adverse events again were PDE5-related although a consistent finding seems to be a higher incidence of back pain and myalgia than for vardenafil and sildenafil [84]. No color vision disturbances were reported, consistent with the high selectivity for PDE5 over PDE6. Udenafil, a pyrazolopyrimidinone, was developed by Dong-A Pharmaceutical Co., Ltd. and has been available in Korea in the form of 100 and 200 mg tablets since December 2005. Phase III clinical data showed that in men with mild to severe ED, the drug produced a significant improvement in erectile function score after 12 and 24 weeks of treatment. The drug was well tolerated and the adverse events were in general the same as with other PDE5 inhibitors. Biochemical potency and selectivity are similar to sildenafil. The pharmacokinetic profile (tmax of 1–1.5 h and T1/2 of 11–13 h) suggests that the drug will have a fast onset of action and a duration of possibly 24 h [86]. Mirodenafil, a pyrrolopyrimidinone structurally related to the pyrazolopyrimidinones and imidazotriazinones, entered the Korean market in 2007 [87]. In vitro potency against PDE5 (0.33 nM) is about 10 times higher compared to sildenafil. Selectivity compared to the other PDEs is similar to the marketed PDE5 inhibitors. Data from an 8-week multicenter, randomized double-blind, placebo-controlled phase II clinical study with 116 patients were presented at the International Society of Sexual Medicine 2006 conference in Cairo [88]. With dosages of 50, 100, and 150 mg, the erectile function domain score on the International Index of Erectile Function (IIEF) increased from a main baseline of approximately 13–15 to 21, 24.6, and 23, respectively. More than 50% of the patients returned to normal erectile function after 8 weeks (IIEF-EF  26) with the 100 mg dose. The recommended starting dose is 50 mg. The drug should be taken 1 h before sexual activity. A phase III study was performed with the 50 and the 100 mg dose with 516 patients in Korea. No clinical studies in other countries have been reported so far. Avanafil originated first at Tanabe Seiyaku as TA-1790 and is being developed by Vivus, Inc. Biochemically, the compound has a higher selectivity compared to sildenafil with respect to PDE1, PDE6, and PDE11, and the IC50 for PDE very close to sildenafil [89]. The half-life is rather short (T1/2 ¼ 60–90 min). A multicenter, randomized double-blind, placebo-controlled phase II clinical study included 284 with mild to moderate ED. Doses from 50 to 300 mg were taken 30 min before initiation of sexual activity. At the highest dose up to 64% were able to complete the intercourse compared to 28% in the placebo group. After treatment for a period of 12 weeks when the drug was taken 30 min before starting sexual activity, the EF of the IIEF score increased from 10.0 to 22.7. The most common adverse events were headache and flushing [90]. SLX-2101, an imidazotriazinone, is a long-acting PDE5 inhibitor. The biochemical potency IC50 (0.04 nM) is much higher than that of sildenafil. Selectivity with respect to PDE1, PDE3, and PDE6 is comparable to that of sildenafil [91], which seems to support the observation that the imidazotriazinone scaffold is especially suited for PDE5 inhibition. Phase I data were presented before the European Society of Sexual

6.3 Pyrimidinone PDE5 Inhibitors

Medicine in December 2005 [92]. RigiScan data from healthy volunteers were obtained in a dose range from 10 to 80 mg and showed erectile effects 6 h postapplication without visual sexual stimulation and up to 24–24.5 h with visual sexual stimulation. The pharmacokinetic profile showed a tmax at 1 h and a T1/2 from 9 to 14 h. The compound produces an active metabolite that has nearly the same potency and selectivity like the original drug [93]. Apart from ED, additional phase II studies on Raynaud’s disease and hypertension were also announced for 2006 and 2007, respectively.

6.3 Pyrimidinone PDE5 Inhibitors

Inhibitors incorporating the pyrimidinone heterocycle, likewise cGMP (8), the natural substrate of PDE5, constitute the largest class of PDE5 inhibitors. O N

HN H2N

N

N OH O O

cGMP (8)

O P OH O

Various natural products carrying this structural element are weak and generally nonselective PDE inhibitors, for example, caffeine (9), theophylline (10), and theobromine (11). The PDE inhibitory activity of theophylline that is used for the treatment of asthma is in the double-digit micromolar range [94]. These natural products together with the structure of cGMP itself provided a basis for the further development of more potent and/or more selective synthetic PDE5 inhibitors. O Me O

N N

O

Me N

Me

N

O

Me Caffeine (9)

O H N

N N

N

Me Theophylline (10)

HN O

Me N

N

N

Me Theobromine (11)

6.3.1 Xanthines and cGMP Analogues

IBMX (isobutylmethylxanthine (12)) is the prototypical synthetic xanthine PDE inhibitor. It shows a PDE5 inhibitory activity of 10 mM with little or no apparent

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selectivity for a panel of other PDEs (IC50 ¼ 7 mM for PDE1, 10 mM for PDE4, and >10 mM for PDE2) [95].

Me

N

O

O

O

O

N

H N

Me

N

O

N N

H N

n-Pr

N

O

N

N n-Pr

i-Bu

i-Bu

H N

N

Cl IBMX (12)

1,3-Dipropyl-8-(4-chlorbenzyl) xanthine (14)

8-(Norbornylmethyl)-IBMX (13)

A literature survey revealed a number of modified IBMX congeners [96–98]. Introduction of lipophilic groups at C8 of the IBMX framework leads to significantly improved PDE5 inhibition. 8-(Norbornylmethyl)-IBMX (13) has a nanomolar IC50 (1.5 nM) with considerably improved selectivity (IC50 PDE1 ¼ 30 nM, IC50 PDE4 > 10 mM). Overall, these compounds are characterized by a dual PDE1/PDE5 inhibition profile and a weaker inhibition of PDE2, 3, or 4. Interestingly, the introduction of a slightly larger alkyl substituent at N-2, as in 1,3-dipropyl-8-(4chlorobenzyl)xanthine (14), completely suppressed the PDE1 activity (>10 mM), an effect similar to tetracyclic guanidines. However, PDE5 activity is also reduced to 600 nM. Recently, researchers at Novartis have published novel 8-quinoline or 8-isoquinoline xanthine derivatives [99] with nanomolar PDE5 inhibitory activity as exemplified by (15) (IC50 ¼ 2 nM). However, no selectivity data are reported for these compounds. Researchers at Schering recently published xanthine PDE5 inhibitors with amino substituents in the imidazole 2-position, such as dasantafil (16) [100]. Br O N

O

O Me O

H N

N N i-Bu

N

N

N (15)

OMe

O

N

H N

OH

N

HO Dasantafil (16)

By combining structural motifs from xanthines and pyrimidinone PDE5 inhibitors (i.e., alkoxy-sulfonamidophenyl substituents) representatives of the 6-phenylpyrrolopyrimidinone series, such as (17), were synthesized [101, 102].

6.3 Pyrimidinone PDE5 Inhibitors

O Me

H N

N

On-Pr

N

O

SO2 N

R

n-Pr

N Me

(17) R = H, Cl or Br

PDE5 inhibition is in the low nanomolar range, but no selectivity data, and little SAR information, are provided. However, potency was raised by increasing the size of the pyrrolo substituent R from H to Cl or Br (14, 4.2, and 4.5 nM, respectively), but a further increase in steric bulk does not improve potency. It could be speculated that a hydrogen bond between the pyrrole NH and the alkoxy substituent on the phenyl ring serves to keep the two halves of the molecule in an almost planar arrangement. In an extension of this work, the same group from Almirall Prodesfarma reported on heterocyclic fused congeners of the above-mentioned compounds [103, 104]. Subnanomolar PDE5 inhibitors were obtained in this dihydrotriazolopurinone series and one such compound, (18), shows an IC50 of 0.34 nM.

N N H N

N O

N n-Pr

N

SO2 N

OH

N n-PrO (18)

Modified cGMP analogues have also been reported as PDE5 inhibitors [105]. Among these, the heterofused derivative (19) showed the most potent PDE5 inhibition with an IC50 of 12 nM. The analogous cGMP congener (20) was only weakly active (15 mM). OH

OH O

O N

N N H

N

HN

S

S N

N OH

H2N

N

OH O

O (19)

N

O O P OH O

(20)

O O P OH O

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6.3.2 PDE5 Inhibitors Incorporating the Purinone Nucleus 6.3.2.1 Zaprinast Although by definition not a purinone, Zaprinast (21) is mentioned here because it constitutes an intriguing starting point for the development of potent PDE5 inhibitors. Zaprinast (M&B 22948, 2-propoxyphenyl-8-azapurin-6-one) was synthesized in an approach to prepare xanthine derivatives as antiallergic compounds and was proposed as a clinical candidate for use in allergic asthma [106]. Detailed studies demonstrated the ability of Zaprinast, like other xanthines, to inhibit PDEs, especially PDE5 (Ki ¼ 130 nM) [192]. Zaprinast has been widely used to investigate the important role of cGMP as a second messenger in smooth muscle cells generated by nitric oxide and as a pharmacological tool for the evaluation of PDE5 inhibition in various conditions. O n-PrO

N

HN

X N

N 1

R 2

R

X

R1

R2

(21) Zaprinast (22) SKF 96231 (23)

N CH CH

H H n-Pr

H H

(24)

CH

NH2

-SO 2 N

N Me

-SO2 N

N Et

Zaprinast set a milestone in the further evolution of more potent PDE5 inhibitors with the introduction of the orthoalkoxyphenyl moiety at the 2-position of the pyrimidinone nucleus. It is assumed that this serves to keep the alkoxyphenyl substituent in an almost planar arrangement with respect to the rest of the molecule [107]. 6.3.2.2 Purinones Using the heterocyclic core of cGMP as a scaffold, potent and selective cGMPcompetitive PDE5 inhibitors were discovered. Incorporation of the 2-alkoxyphenyl motif from Zaprinast into purinones [108], as in SKF 96231 (22), and later by varying the substituents on the aryl ring, led to potent and selective PDE5 inhibitors, exemplified by (23) that has an IC50 of 6.4 nM and a selectivity of more than 10 000 versus PDE4 [109].

6.3 Pyrimidinone PDE5 Inhibitors

Extending the scope of this class of PDE5 inhibitors, Pfizer claimed heterocyclesubstituted purinones with essentially the same range of potency [110]. Interestingly, not only alkyl but also aryl substituents are tolerated on the imidazole ring as exemplified by (24) (IC50 < 10 nM). 6.3.3 Pyrazolopyrimidinone PDE5 Inhibitors 6.3.3.1 Pyrazolo[3,4-d]Pyrimidin-4-One PDE5 Inhibitors A large number of patents and publications appeared describing PDE5 inhibitors of this class, most of which are low nanomolar inhibitors [111–118]. Compounds that have attracted special attention are DMPPO (25), which was described as a potent and selective PDE5 inhibitor (IC50 ¼ 3 nM) with good selectivity versus PDE1, PDE2, PDE3, and PDE4 (IC50 ¼ 1000, 3000, 10 000, and 22 000 nM, respectively), and WIN 58237 (26), which was described as inhibiting PDE5 with an IC50 of 170 nM. The latter compound does not inhibit either PDE1 or PDE3 but has little selectivity for PDE4 (IC50 ¼ 300 nM) [119]. O

R

HN R

1

3

N N

N R

2

R1 (25) DMPPO MeSO2NH

(26) WIN 58237

4-Pyridyl

(27) SR265579 (28)

2-ethoxy-4-pyridyl

R2

R3

Me

Me

c-Pentyl

Me

c-Pentyl Me

Et Me

On-Pr

O N

N H

OnPr

Researchers at Sterling Winthrop investigated an analogue of WIN 58237, SR265579 (WIN 65579) (27), for the potential treatment of asthma. Introduction of the 2-alkoxy substituent in the pyridyl moiety of WIN 58237 improved PDE5 inhibitory activity to 6.4 nM (Ki) with a 14-fold selectivity for PDE4 and a 33-fold selectivity for PDE3. PDE1 and PDE2 are inhibited only at considerably higher concentrations [120]. SR265579 lowers arterial blood pressure in conscious spontaneously hypertensive rats following both intravenous and oral dosing. SR265579 also increases plasma cGMP levels and reinstates vascular responsiveness to

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nitroglycerin in conscious rats that are nitroglycerin-tolerant. No further development is reported [121]. Inhibitory activity was retained by a variety of substituents on the aromatic ring. Apart from sulfonamides, ureas are also tolerated and lead to potent inhibitors, for example, (28), IC50 ¼ 1.5 nM. In a similar way to other pyrimidinone PDE5 inhibitors, researchers at Pfizer extended the existing SAR by claiming pyrazolopyrimidines including 6-heteroaryl substitution [122]. 6.3.3.2 1,6-Dihydro-7H-Pyrazolo[4,3-d]Pyrimidin-4-One PDE5 Inhibitors As early as 1985, this heterocyclic scaffold was used for the synthesis of PDE inhibitors (e.g., by researchers at Warner-Lambert) [123]. Compound (29) showed the highest PDE1 activity (80% inhibition of PDE1 at 10 mM). Compound (30) was described as inhibiting locomotor activity in a mouse model [124]. O

1

2

N

HN R

R

N N R

3

R1

R2

R3

(29) (30)

Bn c-Pentyl

Me Et

Me Me

(31)

MeO

Et

c-Pentyl

Et

c-Pentyl

Me

c-Pentyl

On-Pr

(32)

(33) N

This structural motif was elaborated further with compounds of the type exemplified by (31), which had an IC50 of 23 nM. By incorporating the now well-established alpha alkoxy aryl moiety, single-digit nanomolar PDE5 inhibitors were obtained (IC50 ¼ 1.6 nM for (32)) [125]. Interestingly, changing the alkoxyphenyl to a quinoline moiety results in potent PDE1 inhibitors (e.g., (33) IC50 PDE1 ¼ 97 nM) [126]. Due to its clinical significance and its commercial success, sildenafil (1) is the most studied pyrazolo[4,3-d]pyrimidinone PDE5 inhibitor and has become one of the most widely recognized molecular structures within the medicinal chemistry community in much the same way as ViagraÒ is recognized globally as a brand name. Its structure was originally contained in a patent application published in 1992 [127].

6.3 Pyrimidinone PDE5 Inhibitors

The in vitro and clinical profiles of sildenafil have been extensively reviewed [128, 129, 193]. Sildenafil is a potent PDE5 inhibitor (IC50 originally reported as 3.6 nM) with good selectivity for PDE1, 2, 3, and 4 [130]. Initially, it was developed for cardiovascular indications and later found to be active in the indication of male erectile dysfunction. O R R

3

R N

HN

N

2

N R R

4

5

1

R1 (1) Sildenafil Me N (34)

HO(CH2)2 N

n-Pr

Et N

R3

R4

R5

H

OEt

Me

n-Pr

N SO2- -O(CH2)2O- Me

O

(35) (36)

N SO2-

R2

n-Pr

H

On-Pr Me

H

On-Pr O

n-Pr

NH-

N SO2-

N

n-Pr

In an approach to increase the selectivity of sildenafil against PDE6, Kim et al. reported the synthesis of modified sildenafil analogues [131]. In comparison to sildenafil (in this study, IC50 for PDE5 ¼ 1.76 nM, IC50 for PDE6 ¼ 24.6 nM), incorporating the 2-alkoxy substituent into a ring (34), IC50 for PDE5 ¼ 6.25 nM, IC50 for PDE6 ¼ 7.34 nM) or changing the sulfonamide to an amide (35), IC50 for PDE5 ¼ 0.27 nM, IC50 for PDE6 ¼ 0.43 nM) failed to increase the selectivity of the molecules, although in the case of the amide substituents the potency was markedly increased [132, 133]. Larger substituents on N-1 of the pyrazole ring are also tolerated, exemplified by the morpholino compound (36), which has an IC50 of 1.9 nM [134]. As for the purinone system, the Pfizer group also claimed heterocyclic substituted pyrazolopyrimidinones in a series of patent applications, for example, (37) IC50 for PDE5 ¼ 8.5 nM [135]. Compound (37) resembles Novartis’ xanthine series and earlier benzylated IBMX derivatives (e.g., 15). Contrary to first reports, larger substituents such as 2-pyridylmethoxy (38, IC50 for PDE5 ¼ 5.7 nM) are allowed on the 6-pyridyl substituent [136]. The 2-alkoxy substituent has also been cyclized onto the ring, but no biological data have been reported [137].

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O R

1

O

N

HN

N R N

X

R

R

2

3

4

SO2

X

R1

R2

R3

R4

(37)

N

n-Pr

Me2N(CH2)2NH-

2-Pyridyl-CH2-

n-Pr

(38)

N

2-Pyridyl-CH2-

Et N

N

Me

Et

(39)

N

n-Bu

Et N

N

Me2NCOCH2-

Et

(40)

N

MeO(CH2)2-

Et N

N

2-Pyridyl-CH2-

Et

(41)

CH

Et

Et N

N

MeO(CH2)2-

Et

In later applications, the Pfizer group claimed pyridyl pyrazolo-pyrimidinones that exhibit a high selectivity for PDE5 over PDE6, thereby overcoming one of the shortcomings of sildenafil [137–141]. Compound (39) shows potent PDE5 inhibitory activity (IC50 ¼ 0.45 nM) and an increased selectivity for PDE6 (344-fold). Inhibition of PDE6 has putatively been linked to the ophthalmic effects of sildenafil (“blue vision”). Compound (40) was a preferred compound in a patent application, indicating a high interest in the molecule. Recently, a patent application covering PDE5 inhibitors of this type for use in the treatment of diabetic ulcers has appeared, including sildenafil and compound (41), but no activity has been reported [142]. In addition to sildenafil, several other pyrazolo[4,3-d]pyrimidin-4-ones (UK 114542, UK 357903, UK 390957, UK 369003, DA-8159) have been reported to be in clinical development for the indication of erectile dysfunction [143]. 6.3.4 Imidazotriazinone PDE5 Inhibitors

Imidazo[5,1-f ][1,2,4]triazin-4(3H)-ones are purine isosteres that were first described in the patent literature as bronchodilators. Furthermore, products of their synthesis have been described as C-nucleoside isosteres and purine analogues [144–152]. Imidazotriazinones were described by the group at Bayer [153, 154] as potent and selective PDE5 inhibitors and as dual PDE1/5 inhibitors [155].

6.3 Pyrimidinone PDE5 Inhibitors

In comparison to other pyrimidinone PDE5 inhibitors, it was demonstrated that this heterocyclic class consistently yields PDE5 inhibitors with higher potency and selectivity [156]. Vardenafil (2), the N-ethyl piperazine analogue, is an especially potent PDE5 inhibitor (IC50 ¼ 0.7 nM) and is selective for PDE1 (250-fold) and PDE6 (16-fold). Vardenafil also shows potent activity in a rabbit model of erectile dysfunction [157, 158]. O EtO

Me

HN N

N

N 2

R 1

R

SO2

R1

R2

(2) Vardenafil

Et N

N

n-Pr

(42)

HO

N

n-Pr

(43) (44)

Et2NHO(CH2)2(Et)N-

n-Pr c-Pentyl

In this series, there is broad tolerance of the sulfonamide N-substituent including cyclic and noncyclic, basic and neutral moieties (compound (42) PDE5 IC50 1 ¼ nM, PDE1 IC50 ¼ 100 nM; (43) PDE5 IC50 ¼ 4 nM, PDE1 IC50 ¼ 100 nM). Branched substituents on the imidazo ring (e.g., 44) resulted in extremely potent dual inhibitors of PDE1 and PDE5 with single-digit nanomolar IC50 values for both enzymes. The group at Pfizer has filed a patent application for 2-pyrido imidazotriazinones, but no activity data have been given for these types of compounds [159]. 6.3.5 Imidazoquinazolinones

Researchers from Bristol-Myers Squibb have recently published the structure of a new series of potent PDE5 inhibitors incorporating the N-3-benzylimidazoquinazolinone skeleton. Comparing their activity and selectivity with that of sildenafil, the authors describe the new compounds as having comparable potency and improved selectivity for PDE6. Practically, all compounds of this series show a more than 1000-fold selectivity for PDE1, 2, 3, and 4 and a more than 20-fold selectivity for PDE6, compared to a reported 8-fold selectivity in case of sildenafil [160–162].

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O n-PrO

2

R N

HN N

N

1

R

R1

R2

(45)

H2NCO-

F

(46)

Et N

OMe N SO2-

Both the amide (45) and the sulfonamide analogues (46) are subnanomolar PDE5 inhibitors. Compound (45) (IC50 PDE5 ¼ 0.48 nM) shows a more than 10 000-fold selectivity for PDE1, 2, 3, and 4 and a 60-fold selectivity for PDE6. Compound (46) (IC50 PDE5 ¼ 0.62 nM) also has a comparable selectivity profile with an improved selectivity for PDE6 (90-fold). 6.3.6 Pyrazolopyridopyrimidines

In an extension of the work on imidazoquinazolinones, another BMS group reported on pyrazolopyridopyrimidinones (47), which incorporate an ortho-alkoxyphenyl substituent and show low nanomolar PDE5 inhibition (48) [163]. As shown in Table 6.3, attaching a large benzylamine substituent (49) leads to a considerable increase in potency and selectivity. This result resembles the effects of adding larger substituents to the N-2 of pyrazolopyrimidinones that also led to increased selectivity for PDE6 (cf. compound (39)). 6.3.7 Miscellaneous Heterocylic-Fused Pyrimidinone PDE5 Inhibitors

Pyrimidinones fused to six-membered heterocycles such as pyridines, pyrimidines, and triazines were originally reported by the group at Smith Kline & French, with the most potent compound showing a PDE5 inhibition of 550 nM [164]. Dumaitre et al. gave an overview of several heterocycle variations in the pyrimidinone PDE5 inhibitor series [89]. By introducing the 2-propoxyphenyl substitution pattern, they were able to show considerable differences in PDE5 inhibitory potency between the

6.3 Pyrimidinone PDE5 Inhibitors Table 6.3 PDE selectivity profile of pyrazolopyridopyrimidinone PDE5 inhibitors.

O n-PrO

R N

HN N

N

N N H

SO2 (47)

NMe2 R

(48)

PDE5 (nM)

H

Selectivity PDE1/5

PDE2/5

PDE3/5

PDE4/5

PDE6/5

2.1

400

2000

>104

3200

22

0.3

>105

>105

>105

>104

160

NH

(49)

F

different heterocycles tested. In their study, the known pyrazolopyrimidinones showed the most potent PDE5 inhibition (Table 6.4). Continuing this series of heterocycle-fused pyrimidinones, workers at SK Chemicals claimed pyrrolopyrimidinone derivatives to be PDE5 inhibitors [165]. PDE5 inhibition for the best compounds was in the subnanomolar range, the most potent compound being (64) (IC50 ¼ 0.27 nM). The methyl and n-propyl substituents on the pyrrole seemed to be optimal for this heterocyclic class, and substitution of the phenyl ring para to the alkoxy group resulted in potent PDE5 inhibition. No information was provided on the selectivity profile of this class of PDE5 inhibitors. O EtO

Me N

HN N

n-Pr

N HOOC

SO2 (64)

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Table 6.4 Miscellaneous heterocycle fused pyrimidinone PDE5 inhibitors.

O n-PrO

HN

HET N

(50) Structure (HET) (51)

PDE5 IC50 (nM) 200

N N N H

N (52)

80

N H N (53)

300

N (54)

70

N N

(55)

(56)

50

100

S

CH3 60

(57)

S

S (58)

50

S (59)

80

CH3 (60)

N N H

150

6.3 Pyrimidinone PDE5 Inhibitors Table 6.4 (Continued)

PDE5 IC50 (nM)

Structure (HET)

CH3 N

(61)

8

N CH3

CH3 (62)

100

N O CH3

(63)

30

S N

The Pfizer group also claimed quinazolinones (65) (IC50 ¼ 6.5 nM) [166] and pyridopyrimidinones (66) (IC50 ¼ 1.2 nM, IC50 PDE3 ¼ 220 nM) as PDE5 inhibitors [167]. Similarly, the group at Taisho Seiyaku demonstrated that alkoxy substituents are tolerated on the pyridopyrimidinone as exemplified by (67), which has a reported IC50 of 2.9 nM [168]. O 2

R

X

HN

O

N 3

R 1

R

X

R1

(65)

C-Me

(66)

N

HOOC

(67)

N

O

HO(CH2)2 N

N SO 2-

N SO2-

R2

R3

n-Pr

H

Et

n-Pr

n-Pr

OMe

O N N H

Further exploring the types of heterocycles fused to the pyrimidinone, researchers at Taisho Pharma claimed benzofuryls (e.g., (68), IC50 ¼ 18 nM) and both thienyl

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160

isomers (69) (IC50 ¼ 1.7 nM) and (70) (IC50 ¼ 3.5 nM) [169, 170]. O

O n-PrO

O

HN N

n-PrO

O n-PrO

N

HN N

S NH

O

NH

O N

N

NH2

(70)

(69)

(68)

S

HN

O

Overall, the variation in the right-hand side heterocycle fused to the pyrimidinone nucleus has resulted in the discovery of a variety of potent and selective PDE5 inhibitors, leaving this class of enzyme inhibitors in a very mature state.

6.4 Nonpyrimidone PDE5 Inhibitors 6.4.1 Hexahydropyrazino-Pyrido-Indole-1,4-Diones

This class of PDE5 inhibitors was elaborated from ethyl b-carboline-3-carboxylate (b-CCE (71)), which has a modest inhibitory activity toward PDE5 (IC50 ¼ 0.8 mM) [171, 172]. The most prominent member of this class, IC 351 (tadalafil, CialisÔ (3)), was originally discovered by Glaxo and Icos in a collaboration that was terminated in 1997 [173, 174]. In 1998, Eli Lilly partnered with Icos to develop tadalafil for the treatment of ED [175, 176]. O O

N O

N

N H

N

O

N H

O O

β-CCE (71)

Tadalafil (IC 351) (3)

IC 351 inhibits human recombinant PDE5 with an IC50 of 2 nM [177] and is selective against a panel of phosphodiesterases (selectivity for PDE1–PDE4 and PDE7–PDE10 >10 000; and 780 for PDE6), with the remarkable exception of PDE11 (IC50 ¼ 37 nM) [178]. In the latter study, an IC50 of 6.7 nM was reported for PDE5,

6.4 Nonpyrimidone PDE5 Inhibitors

thus indicating a selectivity ratio of about 5 for PDE11 over PDE5. Intracellular cGMP increase in rat aortic smooth muscle cells has been reported with an EC50 of 200 nM [179]. A whole series of patents covering the underlying tetrahydro-b-carboline framework have appeared [180–187]. Not only diketopiperazines such as tadalafil gave potent PDE5 inhibitors but also incorporation of five-membered hydantoins resulted in high potency (IC50 for (72) <10 nM, IC50 for ((73) ¼ 7 nM). Alkoxy substitution in the para position of the phenyl ring in the lower part of the molecule is a consistent feature of the most potent inhibitors. The last example (74) demonstrates that acyl derivatives of the tetrahydro-b-carboline skeleton can also provide potent PDE5 inhibitors [188]. Icos appears to have concentrated on tricyclic compounds of this class [189, 190]. O

O N

N

N H

O

N

N

N H

O

O O

MeO (72)

(73) O

NMe2

N

N H

O O O

(74)

The group at Ortho McNeil has disclosed related b-carboline PDE5 inhibitors in which the acyl group has been replaced by the electron-deficient heterocycles pyridine (75) (Ki ¼ 4 nM) and pyrimidine (76) (Ki ¼ 24 nM) [191].

N

N H

N

N H N

N N N N

N Me O (75) Ki=4 nM

O (76)

Ki=24 nM

Me

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6.5 Conclusions

The field of PDE5 inhibitors has reached a high degree of maturity. Common problems of medicinal chemistry such as potency, selectivity, physicochemical properties, and oral bioavailability have been successfully addressed with the known PDE5 inhibitors. The discovery of further PDE subtypes and/or the need for specific drug tissue distribution patterns may further increase the knowledge of this PDE inhibitor class. In addition, knowledge gained on different kinase inhibitor classes might be fruitful for the development of PDE inhibitors and vice versa. Since a plethora of PDEs are already known and new subtypes are still being discovered (although all family members have been discovered in the course of the human genome project), scientists in the PDE5 inhibitor field are constantly challenged to continue the search for either more selective inhibitors, compounds with a specifically desirable combination of different PDE inhibitory properties, or compounds demonstrating a very specific tissue distribution. In addition, the utility of PDE5 inhibitors beyond erectile dysfunction promises to be a fascinating area of future research.

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Erwin Bischoff

Bayer Schering Pharma AG, Cardiovascular Research, 42096 Wuppertal, Germany Erwin Bischoff studied biochemistry at the universities of T€ ubingen and Freiburg, Germany, and has been at Bayer AG since 1975. Since 1990, his research has focused on the metabolism of cyclic nucleotides, in particular the cGMPmetabolizing phosphodiesterases. Since 1995, he has investigated projects related to the treatment of erectile dysfunction. He was directly involved in the discovery of the PDE5 inhibitor vardenafil. From screening of potential drug candidates, he has guided and directed all preclinical trials on vardenafil.

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Nils Griebenow

Bayer Schering Pharma AG, Medicinal Chemistry, 42096 Wuppertal, Germany Nils Griebenow was born in Jever, Friesland, Germany, in 1964. He received his diploma and PhD degree in chemistry from the Phillips University of Marburg, working under the supervision of Professor Manfred T. Reetz at the Max Planck Institute for Coal Research, M€ ulheim. In 1995, he joined the Bayer Healthcare AG setting up combinatorial chemistry at its central research facility. In 2002, he moved to the pharmaceutical division of Bayer Healthcare AG, working in various therapeutic areas. He is coinventor of 15 patents and patent applications, and has coauthored 21 peer-reviewed papers and book chapters.

Helmut Haning

Bayer Schering Pharma AG, Medicinal Chemistry, 42096 Wuppertal, Germany Helmut Haning was born in Trier, Germany, in 1965. He studied chemistry at the Johannes Gutenberg University in Mainz and at the Max Planck Institute for Coal Research in M€ ulheim, Ruhr, where he carried out his doctoral work under Professor M.T. Reetz. After a postdoctoral assignment with Professor W. Oppolzer at the University of Geneva in the field of alkaloid synthesis, he joined Bayer in 1995. He is the coinventor of the PDE5 inhibitor, Vardenafil. At present, he is Head of the Medicinal Chemistry Department in Wuppertal, Germany.

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7 Rifamycins, Antibacterial Antibiotics and Their New Applications Enrico Selva and Giancarlo Lancini

7.1 Discovery of the Pioneer Drug

At the end of 1957, an actinomycete strain was isolated in the Lepetit Laboratories in Milan, Italy, from a soil sample collected from the Mediterranean coast near St. Rafael. This microorganism, originally classified as Streptomyces mediterranei, and finally as Amycolatopsis mediterranei, produced a complex of related antibiotics called the rifamycins. The four main components of the novel complex, called rifamycins A, C, D, and E, showed potent antimicrobial activity but poor tolerability at the site of parenteral administration [1]. A fifth acidic substance, rifamycin B, was coproduced in minor quantities by the strain. It was poorly active but became easily available when it was fortuitously observed that rifamycin B was produced preferentially when the strain culture was buffered with 2 g/l of diethyl barbituric acid (barbital). A subsequent critical observation was that the antibacterial activity of solutions of rifamycin B increased on standing, indicating its transformation into a more potent derivative(s). This spontaneous process [2] consisted of oxidation of rifamycin B to generate rifamycin O followed by mild hydrolysis yielding rifamycin S (Figure 7.1). The structures of these molecules were later elucidated by Prelog and coworkers [3]. The quinone rifamycin S showed potent antibacterial activity but suboptimal profile in vivo, with toxicity and poor tolerability. These issues were resolved by reducing rifamycin S to the corresponding hydroquinone, rifamycin SV. This antibiotic was developed and became the first marketed rifamycin, with the trade name Rifocine.

7.2 Clinically Used Rifamycins

Rifamycin SV was the archetype of a group of congeners subsequently developed for clinical use (Figure 7.2). The main therapeutic interest has been centered on their potent activity against mycobacteria, pathogenic Gram-positive cocci, and Neisseriae. Rifampicin is by far the most widely used drug, mainly for treatment of tuberculosis

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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CH3

CH3 H3C

CH3

O OH

O H3C

CH3 O

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OH OH

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H N

H3C

O

O

O

O

Rifamycin B

H N

CH3 O

O

O

H3C

O

O

O

O

O Rifamycin O

CH3

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O

CH3

OH OH

OH

H N

H3C

H N

H3C

O

O O

O

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O

O

OH

CH3

O

Rifamycin S

CH3

Figure 7.1 Transformation of rifamycin B into rifamycin S.

and leprosy. Rifapentine has been successively approved for the treatment of tuberculosis in the United States of America and Italy, and rifabutin has been approved in the United States for limited clinical use only. Other specific infections are treated with rifamycin SV, rifamide, and rifaximin. The process that led to their design, investigation, and development is typical of other antibiotics of microbial origin. It was driven by advancements in comprehension of mechanism of action, structure–activity relationships, and pharmacokinetic behavior (examined in the two subsequent sections), and was oriented both by the medical needs of the time and by the evolving strategies of the pharmaceutical industry.

7.3 Mode of Action of Rifamycins and Structural Requirements for Activity

Rifamycins are characterized by an aromatic ring spanned by an aliphatic bridge, and antibiotics with this structural feature are referred to as ansamycins (ansa in Latin means the handle of an amphora). Other known ansamycins are the streptovaricins and the tolypomycins (biologically similar to rifamycins), and also geldanamycin and maytansines (biologically quite distinct having antiblastic activity). The spatial model of rifamycin SV, shown in Figure 7.3, is derived from X-ray [4] and NMR studies [5]. Extensive investigations of the structure–activity relationships of rifamycins [6, 7] have demonstrated that the following features are essential for activity: (i) the presence of free hydroxyl groups at positions C-21 and C-23; (ii) the presence of

7.3 Mode of Action of Rifamycins and Structural Requirements for Activity

CH3

CH3 H3C

j175

CH3

O OH

O H3C

CH3 O

OH OH

OH

H N

H3C

CH3 O

O O

O

O CH3

Y=H

Y

Rifamycin SV CH3

Y = CH2CO N

OH

OH

H3C

H N

O OH

N

O

H3C

H N

O

N

O

O

Rifamide

CH3

N

N

X = CH3

Rifampicin

X=

Rifapentine

X

CH3

OH

OH

O

O

N O CH3

CH3 N Rifabutin

H N

N

O

N

O

OH

H3C

CH3

Rifaximin

CH3

CH3 Figure 7.2 Rifamycins developed to market.

a keto or hydroxyl group at position C-1 and of a free hydroxyl at position C-8; (iii) preservation of the relative spatial position of (i) and (ii) that is determined by the conformation of the ansa chain. The model in Figure 7.3 indeed shows that the critical groups at positions C-1, C-8, C-21, and C-23 are exposed on the same side of the macrocycle and are precisely positioned in space by the ansa conformation. The antibiotic inhibits at low concentrations the bacterial DNA-dependent RNA polymerase (RNApol) [8]. The inhibition is mediated by the formation of a noncovalent complex between one molecule of rifamycin and the b-subunit of a molecule of RNA polymerase. The rifamycin:enzyme complex maintains the capacity to bind the DNA template and allows the formation of the first phosphodiester bond. The subsequent

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Figure 7.3 Spatial model of rifamycin SV.

steps leading to the synthesis of long-chain RNA are, however, impaired [9]. The crystal structure of the rifampicin:RNApol complex of Thermus aquaticus [10] confirms the role critical for binding played by the groups at position C-1, C-8, C-21, and C-23. It shows also that the antibiotic binds within the DNA/RNA channel corresponding to the b-subunit region in which mutations lead to resistance. Interestingly,
the antibiotic binds in a site positioned more than 12 A apart from the catalytic center of the enzyme. Thus, it does not interfere, for instance, by mimicking the substrate or the transition states of the polymerization reaction but by inhibiting steps subsequent to the formation of the first phosphodiester bond. The active site is highly conserved between prokaryotes and eukaryotes and remains unaffected, thus explaining the selective activity against bacterial enzymes. In fact, the mammalian RNApol is not inhibited by the antibiotic [8]. Accordingly, rifamycins show low intrinsic toxicity to mammalian cells and are inactive against fungi and protozoa. They show high potency against Gram-positive pathogens (minimal inhibitory concentrations (MIC) from 0.005 to 0.1 mg/ml) and Mycobacterium tuberculosis (MICs < 0.01 mg/ml). Enteric Gram-negative pathogens are moderately sensitive. The effects of the antibiotic on bacteria depend typically on the sensitivity of their RNApol’s and on cell permeability. Except for specific mutations to resistance, the RNApol’s of Gram-positive and Gram-negative bacteria show comparable sensitivity to the antibiotic. However, permeability into cells is mediated by passive diffusion and is lower in Gram-negative than in Gram-positive cells [11]. As a result, enteric bacteria are less sensitive. Investigations of a large variety of chemical derivatives showed that modifications of the aromatic nucleus on positions 3 and/or 4 typically maintain potent activity against the enzyme, provided that the key positions and the ansa remain unaltered. The modifications at positions 3 and 4 alter, however, the physicochemical properties of the molecule and consequently their capacity to diffuse into the bacterial cell. For instance, the fermentation product rifamycin B is active against the enzyme but is

7.5 Profiles of Rifamycins Targeted at Tuberculosis Treatment

inactive against whole cells, including Gram-positives, because of permeability barriers caused by the presence of an ionized carboxylic group in its structure; the low activity mentioned earlier was due to a spontaneous degradation to the active rifamycin S. The corresponding amides (rifamide, for instance) are weakly ionized at physiological pH and show high activity [6]. Several classes of rifamycins with substitutions either at the 3 or at the 4 positions showed high antimicrobial potency [6]. In some cases, their antibacterial activity has been correlated with their lipophilicity [12]. It was observed, however, that derivatives with high lipophilic characteristics can acquire an additional mode of action, losing specificity for bacterial RNApol.

7.4 Modulation of Chemotherapeutic Properties

Rifamycins SV and rifamide were the first rifamycins to be developed. Their clinical use has been limited because of their suboptimal pharmacokinetic profiles resulting in insufficient antibiotic levels in serum and tissues. When administered orally, rifamycin SV is partially absorbed from the gastrointestinal tract. Moreover, the absorbed drug is concentrated in the liver and rapidly eliminated in the bile, failing to provide detectable blood levels [13]. Parenteral administration is also poorly effective due to quick excretion through the bile. Aiming at improving oral efficacy, and particularly for tuberculosis, the research at the Lepetit Laboratories was thus focused on synthesizing derivatives showing greater absorption from the gastrointestinal tract and slower rate of biliary excretion. It was found that modifications at position 4 yielded products with improved bacteriological activity but no better absorption and distribution, whereas derivatives substituted at position 3 were generally improved in both their in vitro activity and their pharmacokinetic characteristics [14]. The main goal was achieved by the synthesis of 3-[[(4-methyl-1-piperazinyl)imino]methyl] rifamycin SV, denoted rifampicin, obtained by condensation of 3-formyl rifamycin S with N-amino-N0 -methylpiperazine [14] (Figure 7.4).

7.5 Profiles of Rifamycins Targeted at Tuberculosis Treatment

Rifampicin resulted in one of the more effective therapeutic agents for combination therapy of tuberculosis. Subsequently, analogues were searched with longer lasting serum and tissue levels, and rifapentine was synthesized in the Lepetit Laboratories in 1975. The antibiotic was then developed to clinical use by Hoechst Marion Roussel for intermittent administration regimens in tuberculosis therapy. An additional rifamycin targeted at tuberculosis treatment, namely, rifabutin, was synthesized in 1975 in the Archifar Laboratories. Although similar to rifampicin in its antibacterial activity, it was found more active against Mycobacteria of the M. avium complex and was therefore clinically developed by Farmitalia Carlo Erba.

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CH3

CH3 H3C

OH

O H3C

CH3

O CH3 O

OH O

OH

H3C

O

OH H N

H N

H3C CH3 O

HCHO, HNEt 2 CH2

O

O O

O

O

O

O

O CH3

CH3

N CH3

CH3

MnO2

OH

OH

H2N

H N

H3C

N

O

O

OH

N

OH H N

H3C

O

CH

O

OH

N CH3

O N

CH3

N CH3

O

OH

C H

O

CH3

Figure 7.4 Synthesis of rifampicin.

7.6 Rifampicin (INN), Rifampin (USAN)

Proprietary names: Rifadin, Rimactane. In combination with Isoniazid: Rifinah, Rimactazid. In combination with Isoniazid and Pyrazinamide: Rifater. Rifampicin shows good in vitro potency [15–17] against Gram-positive cocci (Table 7.1), including methicillin-resistant Staphylococci and penicillin-resistant Streptococcus pneumoniae. Enterococci are less sensitive. Clostridium difficile, Corynebacterium spp., and Leisteria monocytogenes are highly susceptible (MIC 0.025–0.5 mg/ml). Among Gram-negatives, pathogenic Neisseria and Moraxella spp. are highly susceptible. Bacteroides fragilis is also susceptible whereas enteric Gramnegative bacteria are generally less sensitive (MIC 1–32 mg/ml). Chlamydia trachomatis and C. psittaci are inhibited at low concentrations (0.025–0.5 mg/ml). M. tuberculosis, M. kansasi, M. marinum, and M. leprae are all susceptible, with the majority of these strains inhibited by <0.01 mg/ml. M. fortuitum and members of the M. avium complex are, on the contrary, rather insensitive [18, 19]. Resistant mutants emerge with relative ease during treatment [18]. This is typical of rifamycins. It is caused by point mutations occurring in several amino acids of the RNApol that lead to various levels of resistance. The mutation rate to resistance is about 107 in Gram-positive cocci and in Escherichia coli and around 109 to 1010 in M. tuberculosis. Typically, there is no cross-resistance with any other class of antibiotics in clinical use and resistance is not transferable. During rifampicin treatment, the gastrointestinal flora rapidly mutates to resistance but reverts to susceptibility within a few weeks upon cessation of treatment.

7.6 Rifampicin (INN), Rifampin (USAN) Table 7.1 Activity of rifampicin against common pathogenic bacteria.

Strain

MIC (mg/ml)

S. aureus S. pyogenes S. pneumoniae E. faecalis M. tuberculosis N. gonorrheae N. meningitidis H. influenzae E. coli K. pneumoniae P. aeruginosa

0.008–0.06 0.03–0.1 0.06–4 1–4 0.1–1 0.06–0.5 0.01–0.5 0.25–1 8–32 16–32 32–64

From Kucers et al. [16], Parenti and Lancini [17].

Because emergence of resistant mutants is likely to occur in the course of prolonged therapies, rifampicin is used in combination with one or more unrelated drugs. Many synergistic interactions with other agents have been reported, but the principal value of combined therapy is attributed to the suppression of emergence of resistant mutants. As mentioned before, rifampicin was promoted to clinical use because of its pharmacokinetic properties [20]. When orally administered, rifampicin is well absorbed, but a high variability has been found in preparations from different manufacturers. Peak plasma levels noticeably differ between individuals and are delayed by food, which affects absorption and decreases peak levels in plasma. Food, however, does not substantially reduce the time in which effective antibacterial levels are maintained. Elimination is mainly through the bile. The process is dose dependent and the proportion passing the liver to be excreted in the urine increases with dosage. Rifampicin is principally metabolized in the liver to its desacetyl derivative, which is also excreted in the bile and urine. Intravenous and oral administration shows similar areas under the curve (AUC) and elimination half-lives. Binding to plasma protein is about 80%, but, due to its lipid solubility, rifampicin is widely distributed in the internal organs, bones, and fluids, including tears, saliva, ascitic fluid, and abscesses. The antibiotic penetrates into cells, and is active against intracellular bacteria. Low concentrations are found in the cerebrospinal fluid, but these are substantially higher when the meninges are inflamed. The most important clinical use of rifampicin is in the treatment of tuberculosis and leprosy. The drug is also recommended for the prevention of meningitis in persons who have been in contact with patients suffering from this disease [21]. Because of its importance for the treatment of tuberculosis and concerns about emergence of resistant organisms, it was recommended that the drug be reserved solely for this purpose. However, in view of evolving medical needs, the drug’s use has been proposed for the treatment of severe infections such as pneumococcal or

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staphylococcal meningitis, Staphylococcus aureus endocarditis, severe staphylococcal sepsis, and osteomyelitis [21, 22]. Rifampicin appears relatively nontoxic, even when administered for a long period.

7.7 Rifapentine

(Proprietary name: Priftin) Available in the United States of America and in some South American and Asian countries Rifapentine is structurally related to rifampicin, differing in the presence of cyclopentyl instead of methyl group on the piperazine moiety (Figure 7.2). Rifapentine shows antibacterial activity similar to rifampicin. It is, however, more active against atypical mycobacteria, especially M. avium complex (MIC < 0.06–0.5 mg/ml) [23]. The principal advantage of rifapentine in respect to rifampicin is the persistence of blood levels, which provides therapeutic levels for at least 72 h after a single administration [24] (Table 7.2), thus allowing less frequent dosing. In combination with other drugs, it is indicated for short-course therapy of tuberculosis both in the intensive phase (twice-weekly regimen) and in the continuation phase, in which once-a-week dosage is sufficient. This improves patient compliance, a critical factor in the therapy of tuberculosis [25]. Oral absorption of rifapentine is substantially increased by food. The antibiotic is well distributed in the body with concentrations in most organs exceeding plasma levels. The drug accumulates in cells providing a therapeutic advantage for inhibition of intracellular mycobacteria. In macrophages, a 24 : 1 ratio of intracellular versus extracellular concentrations was estimated [26]. In an analogy to rifampicin, rifapentine is metabolized mainly to its 25-desacetyl derivative, which is cleared more rapidly than the parent antibiotic. The main route of elimination is through the bile in the feces, with a minor fraction (17%) recovered in the urine. Rifapentine is an inducer of liver cytochromes. Other drugs concurrently administered may require adjustments in dosage because rifapentine accelerates liver metabolism. For instance, treatment with rifapentine of patients with AIDS resulted in a 70% reduction of AUC of the antiviral drug indinavir. Table 7.2

Pharmacokinetic parameters of rifampicin and rifapentine.

Dose (oral) Cmax Tmax T1/2 Distribution volume Plasma protein binding

Rifampicin

Rifapentine

300 mg 4 mg/ml 2h 2.5 h 1.5 l/kg 80%

600 mg 12 mg/ml 5h 13 h 1.5 l/kg 97%

From Parenti and Lancini [17]. Cmax: maximum plasma concentration.T1/2: half-life. Tmax: time to maximum plasma concentration

7.9 Rifamycins Beyond Tuberculosis

7.8 Rifabutin

Rifabutine; ansamycin; LM-427; ansatipine. Proprietary name: Mycobutin. Available in several countries including the United States of America and United Kingdom. Rifabutin is a semisynthetic spiropiperidyl derivative of rifamycin S approved for prevention of mycobacterial infections (Figure 7.2). In general, the antibiotic is as potent in vitro as rifampicin, but to some extent it is more active on the M. avium/intracellular complex (MIC 0.01–2 mg/ml) [27, 28]. In one study, it was found that the majority of M. avium complex strains are not inhibited by 1.0 mg/ ml of rifampicin, but approximately 87% of them are sensitive to 1.0 mg/ml of rifabutin [28]. It should be noted that M. avium complex comprises several bacterial species (29). In M. tuberculosis, M. leprae, S. aureus, and C. trachomatis, the frequency of spontaneous resistant strains is somewhat lower with rifabutin than with rifampicin, although the mechanism of resistance is the same. Oral administration of 300 mg of rifabutin achieves 0.38 mg/ml Cmax levels with 3.3 h Tmax, 16 h T1/2, and 9.3 l/kg volume of distribution [17, 30]. Organ concentrations are high, and in lungs the average concentration is 6.5 times that in plasma. Plasma protein binding is 85%. Oral absorption is rapid but incomplete, oral bioavailability being only 12–20% with considerable interpatient variation [31]. Clinical value has been demonstrated in preventing or delaying mycobacterial infections in immunocompromised patients. The drug has been approved in the United States and Italy for the prevention of M. avium/intracellular infections in AIDS patients [32]. Although some efficacy was observed in the treatment of tuberculosis, its use for this condition is not recommended. Interaction with other drugs used in AIDS treatment has been reported due to the induction of liver enzymes by the antibiotic [33].

7.9 Rifamycins Beyond Tuberculosis

When the unique potential of rifampicin in the treatment of tuberculosis was demonstrated, its use for the treatment of other infectious diseases was strongly limited by regulatory agencies on the ground that resistant Mycobecteria would emerge if patients unaware of harboring tuberculosis are treated with this antibiotic for common infections. To partially overcome these limitations, chemists at the Alfa Farmaceutici S.p.A. synthesized in 1983 a derivative of rifamycin SV, namely, rifaximin, that was not absorbed when administered orally. The antibiotic, while useful for the treatment of gastrointestinal infections, does not reach a concentration in the lung so as to select resistant Mycobacteria. Rifamycin SV and rifamide have been the rifamycins first developed and remain in use for limited and specific indications in some countries.

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7.10 Rifamycin SV and Rifamide

Proprietary names: Chibro-Rifamycin, Otofa, Rifocine Rifamycin SV is obtained by elimination of a glycolic moiety from rifamycin B and was the first rifamycin put to clinical use. Rifamide is the diethyl amide of rifamycin B (Figure 7.2). These two products share very similar biological properties and have been marketed as alternatives to one another in several countries [6, 13, 34]. Their good antimicrobial activity against Gram-positive organisms and M. tuberculosis is typical of the family. The efficacy is, however, poor in tuberculosis because of insufficient drug blood levels. As mentioned before, both drugs are absorbed orally but are rapidly eliminated through the bile. Their high concentrations in the bile is, however, effective in treating infections of the biliary tract, even when due to the generally less sensitive Gram-negative bacilli [35]. The water solubility of these molecules quickly made available formulations suitable for intramuscular injection and these preparations have had some clinical utility. However, the introduction in the United Kingdom of an injectable form of rifampicin superseded the use of rifamide, which was withdrawn from the UK market.

7.11 Rifaximin

Proprietary names: Normix, Rifacol, Fatroximin, Rifaxidin, Rifaximina (Spanish); Rifaximine (French), Ritacol, Xifaxan. Available in several European countries, the United States, and Mexico. Rifaximin is a semisynthetic derivative of rifamycin S (Figure 7.2) with a spectrum of activity similar to that of the other rifamycins. It is poorly absorbed from the gastrointestinal tract [36]. Up to 90% of the administered rifamixin is concentrated in the gut with less than 0.2% in the liver and kidney and less than 0.01% in other tissues. The antibiotic is effective against a variety of gastrointestinal complications and is extensively used in the treatment of irritable bowel disease [37], travel diarrhea [38], and diverticulitis [39]. Rifaximin has also been proposed for the treatment of chronic hepatic encephalopathy [40] and for topical treatment of bacterial vaginitis [41]. A novel interest is related to emerging gastrointestinal infections caused by C. difficile strains that are high producers of toxins. These severe infections have been reported initially in North America and recently in Europe, and are spreading in populations previously considered to be at low risk [42]. Rifaximin is highly active against this pathogen and has a distribution in the body appropriate for a specific therapy. In a recent study, a 2-week course of rifaximin following a 10–14-day course of vancomycin was effective in most patients with recurrent disease [43].

7.13 Summary

CH3

CH3 H3C

O OH

O H3C

CH3

CH3 O

OH

OH O N

H3C

CH3 O

O

O O

O

N

CH3 HO

N

CH3 N CH3

Figure 7.5 Rifalazil.

7.12 Trials for Other Therapeutic Indications

Rifalazil (Figure 7.5) is a rifamycin particularly active against Chlamydophila pneumoniae, including rifamycin-resistant strains [44]. It has been investigated for treatment of atherosclerosis. This pathology has been associated [45] with chronic inflammatory processes putatively caused by C. pneumoniae. However, a phase III study performed on patients with intermittent claudication failed to demonstrate clinically relevant results [46]. Further activities have been investigated for several rifamycins. Extensive studies have been focused on the inhibition of various polymerizing enzymes, such as eukaryotic DNA and RNApol’s and viral reverse transcriptase, but without any success [6]. Rifampicin, for instance, inhibits in vitro vaccinia viruses, but eventually it was shown to interfere with virus capsid assembly. Rifabutin has been reported to inhibit the replication of human immunodeficiency virus 1 (HIV1) in concentrations (10 mg/ml) that are not toxic to lymphoid cells, but no efficacy on HIV infections has been demonstrated. Several rifamycins have also shown some immunosuppressive effect in animals, but the clinical significance is not clear. There is also anecdotal evidence of the efficacy of topical applications of rifamycin SV in curing wounds, particularly bedsores.

7.13 Summary

The rifamycins are a family of antibacterial antibiotics acting on bacterial RNA polymerase and structurally characterized by an aromatic ring spanned by an

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aliphatic bridge (ansa). The core structure of the rifamycins used in therapy derives from rifamycin B, a fermentation product from an actinomycete first isolated in 1957. Although its antimicrobial activity was moderate, it was soon observed that oxidation followed by mild hydrolysis generated rifamycin S, a quinone molecule with potent antimicrobial activity but poor tolerability. Subsequent reduction of rifamycin S to the hydroquinone form rifamycin SV resulted in good potency and efficacy against infections by Gram-positive pathogens, and tolerability appropriate for clinical development. It was marketed for parenteral administration with the name Rifocine. A wider therapeutic window was obtained with rifamide, an amide of rifamycin B. Clinical use of these molecules was limited by lack of efficacy by oral administration and unsatisfactory pharmacokinetics. Analysis of the activity of various derivatives substituted on different regions of the molecule demonstrated that modifications of the aliphatic bridge led to inactivity. On the contrary, modifications at carbons 3 or 4 of the aromatic ring maintained or improved activity and positively modified pharmacokinetics to give oral efficacy. Several series of derivatives were then synthesized. Among them 3-[[(4-methyl-1-piperazinyl)imino] methyl]rifamycin SV, named rifampicin (INN) or rifampin (USAN) had the desired properties and was marketed worldwide. The antibiotic has unique therapeutic properties against tuberculosis and leprosy and is used in combination with unrelated antibiotics to prevent the emergence of resistant strains. Subsequently, two more analogues have been developed. Rifapentine (Priftin) that permits intermittent therapy for tuberculosis because it provides lasting blood levels. Rifabutin (Mycobutin) is targeted at infections caused by the M. avium complex and shows an appropriate organ and cellular distribution. It is approved for the prevention of mycobacterial infections in AIDS patients. In addition to tuberculosis, rifampicin is recommended for the prophylaxis of meningitis in persons exposed to patients with this infection. Because of their high antimicrobial activity and distinct mode of action (inhibition of RNA polymerase), the rifamycins may be effective in a variety of severe bacterial infections. For instance, rifaximin, which is poorly absorbed from the gut and is targeted at gastrointestinal infections, may provide a therapeutic option to treat infections caused by C. difficile, a high toxin producer, which represents an emerging medical problem.

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Determination of in vitro susceptibility of mycobacteria to ansamycin. Am. Rev. Respir. Dis., 132, 710–711. Tsukamura, M. (1987) Two groups of Mycobacterium avium complex strains determined according to the susceptibility to rifampicin and ansamycin. Microbiol. Immunol., 31, 615–623. William, J., Burman, W.J., Gallicano, K., and Peloquin, C. (2001) Comparative pharmacokinetics and pharmacodynamics of the rifamycin antibacterials. Clin. Pharmacokinet., 40, 327–341. O’Brien, R.J., Lyle, M.A., and Snider, D.E., Jr. (1987) Rifabutin (ansamycin LM 427): a new rifamycin S derivative for the treatment of mycobacterial diseases. Rev. Infect. Dis., 9, 519–530. Nightingale, S.D., Cameron, D.W., Gordin, F.M., Sullam, P.M., Cohn, D.L., Chaisson, R.E. et al. (1993) Two controlled trials of rifabutin prophylaxis against Mycobacterium avium complex infection in AIDS. N. Engl. J. Med., 329, 828–833. Kuper, J.J. and D’Aprile, M. (2000) Drug–drug interactions of clinical significance in the treatment of patients with Mycobacterium avium disease. Clin. Farmacokinet., 39, 203–204. Pallanza, R., F€ uresz, S., Timbal, M.T., and Carniti, G. (1965) In vitro bacteriological studies on rifamycin B diethylamide (rifamide). Arzneim.-Forsch., 15, 800–802. Khan, G.A. and Scott, A.G. (1967) The place of rifamycin B diethylamide in the treatment of cholangitis complicating biliary obstructions. Br. J. Pharmacol. Chemother., 31, 506–512. Corazza, G.R., Ventrucci, M., Strocchi, A., Sorge, M., Pranzo, L., Pezzilli, R. et al. (1988) Treatment of small intestine bacterial overgrowth with rifaximin, a non-absorbable rifamycin. J. Intern. Med. Res., 16, 312–316.

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Rifaximin treatment for symptoms of irritable bowel syndrome. Ann. Pharmacother., 42, 408–412. DuPont, H.L. (2005) Travelers’ diarrhea: antimicrobial therapy and chemoprevention. Nat. Clin. Pract. Gastroenterol. Hepatol., 2, 191–198. Papi, C., Koch, M., and Capurso, L. (2005) Management of diverticular disease: is there room for rifaximin? Chemotherapy, 51 (Suppl. 1), 110–114. Festi, D., Mazzella, G., Orsini, M., Sottili, S., Sangermano, A., Li Bassi, S. et al. (1993) Rifaximin in the treatment of chronic hepatic encephalopathy: results of a multicenter study of efficacy and safety. Curr. Ther. Res., 54, 598–609. Pelosini, I. and Scarpignato, C. (2005) Rifaximin, a peculiar rifamycin derivative: established and potential clinical use outside the gastrointestinal tract. Chemotherapy, 51 (Suppl. 1), 122–130. Loo, V.G., Poirier, L. et al. (2005) A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N. Engl. J. Med., 353, 2442–2449. Johnson, S., Schriever, C., Galang, M., Kelly, C.P., and Gerding, D.N. (2007) Interruption of recurrent Clostridium difficile-associated diarrhea episodes by serial therapy with vancomycin and rifaximin. Clin. Infect. Dis., 44, 846–848. Rothstein, D.M., Suchland, R.J., Xia, M., Murphy, C.K., and Stamm, W.E. (2008) Rifalazil retains activity against rifampinresistant mutants of Chlamydia pneumoniae. J. Antibiot., 61, 489–495. Becker, A.E., De Boer, O.J., and Van Der Wal, A.C. (2001) The role of inflammation and infection in coronary artery disease. Annu. Rev. Med., 52, 289–297. Powell, J.T. (2008) Three drug indications for patients with peripheral arterial disease bite the dust. Eur. J. Vasc. Endovasc. Surg., 35, 49–50.

References

Giancarlo Lancini

University of Varese, FIIRV, Via Lepetit 34, Gerenzanon (VA), Italy Giancarlo Lancini graduated in chemistry in 1956. In 1957, he joined Lepetit, where in 1966 he became Head of the Department of Antibiotics and in 1974 Director of the research laboratories. His research spans several aspects of the antibiotic screening, biosynthesis, mechanisms of action, and synthesis. He contributed to the discovery of rifamycins and teicoplanin. He taught courses on biotechnology of fermentations at the University of Pavia (1971–1982), at the University of Milan (2000–2005), and Varese (2006–present). He has authored over 100 scientific papers, and two books of which Antibiotics: An Integrated View had several editions in Italian and English and has been translated into Russian and Chinese.

Enrico Selva

University of Pavia, via Di Vittorio 23, 27027 Gropello Cairoli (PV), Italy Enrico Selva has worked on R&D of bioactive microbial products in major pharmaceutical companies and in a biotech start-up he contributed to set up and manage. His work on microbial product chemistry, screening, isolation of environmental microorganisms, fermentation, strain development, and medical microbiology contributed to the discoveries of the glycopeptide A40926 (precursor of Dalbavancin), Ramoplanin, and original antibiotics acting on bacterial protein synthesis, RNA polymerase, and cell wall biosynthesis. Four molecules are in clinical development. He is a lecturer of industrial biotechnology at the University of Pavia, Italy.

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8 Monoterpenoid Indole Alkaloids, CNS and Anticancer Drugs Andr
as Nemes

8.1 Introduction

The monoterpenoid indole alkaloids are secondary metabolites of certain flowering plants. More than 2500 isolated representatives of them are formed mainly in three plant families Apocynaceae, Loganiaceae, and Rubiaceae from two building blocks tryptamine 1 and secologanin 2 through a single precursor strictosidine 3 (Figure 8.1) [1]. Different biosynthetic pathways lead from unrearranged coryanthean-type alkaloids to the rearranged classes ibogaine and vincamine and further to bisalkaloids such as vinblastine. Quinine-type and camptothecin (CPT)-type quinoline alkaloids are also biosynthesized via strictosidine. Although the role of monoterpenoid indole alkaloids in plant biochemistry is unclear, in animals and humans they have revealed a wide spectrum of pharmacological activity that suggests considerable potential for clinical application. At present, monoterpenoid indole alkaloids are used as neuromuscular blocking agents (curare alkaloids: toxiferine 4, alcuronium 5), for the treatment of hypertension (yohimbine alkaloids: reserpine 6), addiction (ibogamine alkaloids: ibogaine 7), cerebral insufficiencies (vinca alkaloids: vincamine 8), malaria, cardiac arrhythmia (cinchona alkaloids: quinine 9, quinidine 10), and various malignancies (dimeric vinca alkaloids: vinblastine 11, vincristine 12, and Camptotheca alkaloids: camptothecin 13) (Figure 8.2). Like many other natural compounds, monoterpenoid indole alkaloids have been the subject of analogue-based drug research. No compounds with better activity have been found among the antihypertensive reserpine 6 stereoisomers, derivatives, and related molecules. The same holds in respect of the enantiomer pair quinine 9 and quinidine 10. Alcuronium 5 is the only semisynthetic derivative of active curare alkaloid toxiferine 4, which has been used clinically and marketed in Europe. Only one active analogue, the metabolite noribogaine 7, R ¼ H, is known for the antiaddictive ibogaine 7, R ¼ Me. By contrast, vincamine 8, vinblastine 11, and camptothecin 13 have all served as lead compounds for more potent or specific agents in

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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CHO H

N H 1 Tryptamine

NH2

+

H

OGlu

O

CH3O2C

NH

N H H

H H

CH3O2C 2 Secologanin

OGlu

O

3 Strictosidine

Figure 8.1 Biosynthesis of monoterpenoid alkaloid precursor.

the appointed range of activity. The numbering system of alkaloids reported throughout this chapter follows the biogenetic numbering [2].

8.2 Vincamine and Derivatives: Cerebrovascular and Neuroprotective Agents 8.2.1 Medicinal Chemistry of Vincamine Derivatives

Vincamine 8, an alkaloid of Vinca minor, was investigated by several research groups in the 1960s. Sz
asz and coworkers, for example, considered the molecule to be a potential analogue of reserpine 5 [3]. After extensive investigation, it was found, however, that 8 improved cerebral functions, rather than blood pressure. Vincamine was subsequently introduced for the treatment of cerebral insufficiencies in Europe. Research then focused on the mechanism of action of these compounds in an effort to find more potent derivatives by structural modification of the lead alkaloid molecule. Of the several thousand derivatives prepared, only three reached the market, based on their beneficial effect on brain circulation and neuronal homeostasis. A common structural feature of these molecules is the retention of the cis-fused ring system of vincamine, and an ester or carbonyl function at C-16 (Figure 8.3). Vinburnine 14, a degradation product of vincamine, which also occurs in some species of Apocynaceae as minor alkaloid, was launched in 1977 by SmithKline Beecham. The following year, it was followed by Richter’s vinpocetine 15. For vinburnine 14, demethoxycarbonylation affords a simple entry from the parent compound 8 [4]. Replacement of the hydroxy and methoxycarbonyl groups with a single carbonyl group improved the cerebral metabolic and hemodynamic effects [5]. Vinpocetine 15, produced from vincamine 8 by dehydration and transesterification [3], was significantly more active than the lead molecule in cognitive activating ability, both in preventing hypoxia-induced deficits in step-through passive avoidance retention in rats [6] and in protecting against the lethal effects of hypoxia in mice [7]. In the latter test, vinburnine 14 was almost as effective as vinpocetine. Brovincamine 16, the 11-brominated derivative of vincamine 8, was developed in the next decade and subsequently launched in Japan (1986, Sandoz) for its ability to

8.2 Vincamine and Derivatives: Cerebrovascular and Neuroprotective Agents

Figure 8.2 Structures of clinically used monoterpenoid indole alkaloids.

selectively increase cranial and coronary blood flow [8]. Brovincamine 16 also markedly increases the blood flow in the optic nerve head of albino rabbits [9] and is recommended for the treatment of ophthalmologic insufficiencies such as glaucoma (Figure 8.3).

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Structural formula

H N

N

Name

Synonymes

(-)-Eburnamonine

Vinburnine Cervoxane Vincamone

Launched (Company)

1977 (Beecham)

O 14 6

H 11

N

N 21

16

Ethyl apovincaminate

Vinpocetine Cavinton

1978 (Richter)

11-Bromovincamine

Brovincamine Sabromine

1986 (Sandoz)

20

H5C2O2C 15

H N

Br HO

N

H3CO2C 16 Figure 8.3 Structures of clinically used vincamine derivatives.

8.2.1.1 Structure–Activity Relationships In a study, performed with all possible stereoisomers of vincamine 8, vinburnine 14, and vinpocetine 15, it was established that the cerebrovascular activity strongly depends on the natural cis-(20S,21S)-eburnane skeleton [10]. 20-Epi derivatives, such as trans-(20R,21S)-vinpocetine 17, and (20R,21S)-dihydroeburnamenine-16-methanol 18 or its dinor-derivative (1S,12bS)-octahydroindoloquinolizinyl-1-methanol 19 (Vintoperol, RGH-2981) exhibit significant peripheral vasodilator effects [10, 11]. No vascular effects were found with trans-(20S, 21R) derivatives. The pharmacological profile of vindeburnol (trans-desethyl-(20S,21S,16R)-dihydroeburnamenine-16-ol, RU 24722) 20 differs from that of vincamine. In addition to improved cerebral blood flow (CBF), this compound markedly suppressed the cerebral metabolic rate during decapitation ischemia, increased the EEG resistance time, and decreased the EEG recovery time in rats under asphyxic anoxia [12]. (20S,21S)-(Nitrooxy)ethyl apovincaminate (VA-045) 21 was tested for cerebrovascular ischemia and cognitive disorders [13], while (20S,21R)-hydroxyethyl apovincaminate (RGH-10885) 22 was identified as an antioxidant and cognitive enhancer [14] (Figure 8.4).

8.2 Vincamine and Derivatives: Cerebrovascular and Neuroprotective Agents 6

9 8

10

7

5

H

1

11 12

13

N

ROOC 16

20 17 19

N

4

21

H 3

N

14 15 18

17 20S,21R R = C 2H5 21 20S,21S R = NO 2OC2H4 22 20S,21S R = HOC 2H4

H

N

R1

R2 18 20S,21R R 1 = CH2OH, R2 = C2H5 20 20S,21S R 1 = OH R2 = H

N 12b H 1 HO

N

19

Figure 8.4 Structures of pharmacologically active vincamine derivatives.

8.2.2 Synthesis of Vincamine Derivatives

As isolation of vincamine from natural sources is both expensive and limited, an efficient synthesis was needed. The biomimetic transformation of dihydrotabersonine to vincamine [15, 16] is one possible approach since tabersonine, the biogenetic precursor of vincamine 8, constitutes around 4% in the seeds of Vocanga africana. The clinical use of vincamine derivatives initiated numerous dia- and enantioselective total syntheses of this type of alkaloid [17]. The synthetic route reported in Ref. [18] affords vincamine, vinburnine, and vinpocetine from a common intermediate and was accomplished in industrial scale [18, 19]. 8.2.3 Pharmacological Properties of Vincamine Derivatives 8.2.3.1 Mechanism of Action The beneficial effect of vincamine derivatives on cerebral circulation disorders was first explained by dilatation of cerebral vessels [20], which was deduced from the common structural elements of reserpine 5 and vincamine 8. Subsequent studies, however, established that the mechanisms of action of reserpine and vincamine are different. Reserpine appears to act by inhibition of the active transport of neurohormonal amines into tissue storage sites, consequently decreasing peripheral vascular resistance to evoke a fall in blood pressure [21]. The mechanism of action of vincamine derivatives proved to be more complex. The molecular targets are the phosphodiesterase-1 (PDE-1) enzyme, voltage-dependent Na þ channels, voltageoperated Ca2 þ channels and glutamate receptors. Phosphodiesterase-1 Vinpocetine 15 noncompetitively inhibits Ca2 þ /calmodulinedependent cGMP-PDE [22] and is now considered to be a selective inhibitor of this (PDE-1) isoenzyme [23]. Inhibition of PDE-1 and increase in cAMP and cGMP levels are presumably responsible for the positive vascular effects, for the improvement in cerebral circulation, and for the beneficial effect on platelets [24].

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Na þ Channels Vinpocetine 15 inhibited Na þ currents in cortical neurons with a 44 mM K50 value [25] and reduced the Na þ load induced by toxic doses of veratridine [26]. Vinpocetine 15 selectively inhibits neurotransmitter release triggered by sodium channel activation [27]. Accumulation of Na þ in neurons during anoxia, and then oxidative stress during reperfusion, has deleterious effect, so pharmacological blockade of Na þ channels is beneficial against oxidative stress and might have a particular importance in neuroprotection [28, 29]. Ca2 þ Channels The vasodilative effects of bromovincamine 16 seem to involve inhibition of depolarization-dependent Ca2 þ slow channel [30]. Vinpocetine 15 inhibited the Ca-uptake on guinea pig forebrain synaptosomes [31] and inhibited veratridine-induced Ca influx in rat hippocampal CA1 pyramidal cells [32]. Glutamate Receptors Vipocetine 15 was able to protect neurons against the cytotoxic effect of glutamate [33] or N-methyl D-aspartate [34]. 8.2.3.2 Clinical Pharmacology Pharmacokinetics The absorption of vincamine and derivatives following oral administration is rapid. Peak plasma concentrations are reached within 1–2 h after dosing [35, 36]. A biphasic clearance with half-lives of 1 and 5 h was observed for plasma pharmacokinetics of bromovincamine 16 in humans [36]. Vinpocetine 15 acts similarly with the corresponding half-life values of 0.3 and 4.8 h [35]. The parent compound vincamine 8 can be oxidatively metabolized (CYP enzymes) to 6-hydroxy and 6-keto-vincamines and hydrolytically (hydrolase enzymes) to vincaminic acid. The latter metabolite transforms further to eburnamonine 14, by a decarboxylation–oxidation process [35]. The 6-hydroxy derivative is the main metabolite of vinburnine 14 [37]. For the ester derivatives 15 and 16, the main metabolites are apovicaminic acid [35] and bromovincaminic acid [36], respectively. Clinical Results with Vincamine Derivatives Double-blind placebo-controlled studies were accomplished to establish the effectiveness of vinburnine 14. It improved 22 of the 25 parameters measured to assess brain functions for 129 patients treated with geriatric complaints [38]. In a 12-week trial, a 60 mg daily dose vinburnine improved more frequently the symptoms of cerebrovascular disorders compared to placebo [39]. Vinburnine (40 mg i.v.) exhibited beneficial effects on blood rheology, oxygen transport, and regional circulation under normal conditions, and hypoxemia in two groups of patients (with low hemoglobin oxygen affinity and with normal values, respectively) [40]. Double-blind placebo-controlled, randomized trials have been carried out to confirm that vinpocetine is effective in the treatment of chronic cerebral diseases in the 15–30 mg/day dose range [41–44]. Positron emission tomography (PET) studies were performed on chronic ischemic stroke patients to study the pharmacological and physiological effects of vinpocetine 15. Single-dose intravenous infusion resulted in positive changes in regional

8.3 Antitumor Dimeric Vinca Alkaloids

cerebral blood flow and metabolism in both peri-stroke regions and healthy brain tissue [44]. A two-week long intravenous vinpocetine treatment increased global CBF. The highest CBF changes were observed in regions, in which the highest regional uptake of labeled vinpocetine was measured in other PETstudies. The global cerebral metabolic rate of glucose (CMRglc) did not change markedly, whereas regional CMRglc values markedly improved [45]. PET scans with brovincamine in patients with misery perfusion syndrome confirmed improved CBF but gave no evidence of favorable effects on metabolic state [46]. Oral brovincamine 16 treatments (3
20 mg daily) may retard further visual field deterioration in patients with normal-tension glaucoma, as established in a study performed with 52 patients and followed for 2 years [47]. Antitumor Monoterpenoid Indole Alkaloids A wide range of complex natural products exert antitumor activity [48], but the utility of these cytotoxic compounds in the host organism is disputed. One argument is that they are defensive agents against the hostile environment, while a more feasible argument is that they are cellular growth regulators that interfere with the cell cycle. The fact that the primary target of the most effective natural antitumor agents is DNA or tubulin makes this latter hypothesis more likely. The dimeric vinca alkaloids interfere with tubulin polymerization, while camptothecin inhibits the DNA processing topoisomerase I enzyme.

8.3 Antitumor Dimeric Vinca Alkaloids

Catharanthus roseus (formerly Vinca rosea), a Madagascan periwinkle, is a rich source of indole and bisindole alkaloids. Vinblastine 11 was first discovered in 1958 as potent cytotoxic compound by Noble et al. at the University of Western Ontario [49] and independently by Svoboda et al. at Lilly Research Laboratories [50]. Further isolations from the leaf extracts led to vincristine 12 and a series of other derivatives of vinblastine 11 [51]. Of these, 11 and 12 became clinically important antitumor agents and marketed as Velban in 1960 and Oncovin in 1963, respectively. 8.3.1 Medicinal Chemistry of Dimeric Vinca Alkaloid Derivatives

The antitumor vinca alkaloids are dimerized from a tetracyclic ibogan (“upper part”) and pentacyclic aspidosperman (“lower part”) unit. The antiproliferative activity is related to their specific interaction with tubulin, disrupting the cell division process, by destabilizing the structure of microtubules [52]. Minor structural differences between the molecules led to major differences in potency and utility. Vinblastine 11 and Vincristine 12, despite their chemical similarities, display significantly different clinical effects. Thus, vinblastine is used in the treatment of Hodgkin’s disease and metastatic testicular tumors, while vincristine is applied in combination with other antitumor drugs for the treatment of acute

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lymphocytic leukemia in children [53]. The toxicity profile of 11 and 12 is also different. Vinblastine causes severe tissue necrosis upon extravasation, while mild neurotoxicity and myelosuppression also occur. Beside the tissue necrosis, neurotoxicity is a significant potential problem with vincristine. Myelosuppression occurs to a lesser extent. Vinblastine 11 was chosen as the lead molecule to solve problems of limited therapeutic spectrum and severe toxicity associated with dimeric vinca drugs. The aspidosperman part of the molecule provides more opportunities for semisynthetic modifications. Vindesine 23, desacetyl vinblastine amide, which was selected from the modified derivatives by Lilly’s researchers, differs from the lead molecule only by deacetylation and replacing the C-16 ester group by an amide group [54]. The antitumor spectrum of 23 resembles vincristine 12, while the toxicity profile appears to be less neurotoxic. Phase I clinical trials commenced in 1977 and 23 has been used for the treatment of non-small cell lung cancer (NSCLC), lymphoblastic leukemia, and non-Hodgkin’s lymphomas. The compound was marketed in the United Kingdom, Germany, France, and Ireland as Eldisine for the treatment of acute lymphoblastic leukemia of childhood, blastic crises of chronic myeloid leukemia, and malignant melanoma in 1980 [55] (Figure 8.5). Modification of the ibogan part led to the second successful antitumor analogue of vinblastine to reach the marketplace. Navelbine or vinorelbine 24, R0 ¼ R00 ¼ H, was synthesized by Polonovsky–Potier reaction from anhydrovinblastine 25 and has become one of the most prescribed anticancer products. It has been found effective against NSCLC and less neurotoxic than vincristine. Vinorelbine 24 was discovered by Potier in 1978 at CNRS, developed and licensed to Borroughs Wellcome by Pierre Fabre (1988). First approved for NSCLC treatment in1989 in France [56], launched in the United Sates of America in 1995 (Figure 8.5). 8.3.1.1 Structure–Activity Relationships The (160 S,140 S) configuration of the ibogan part proved to be critical for anticancer activity. Structural modifications on the aspidosperman part are better tolerated. Several hundred new congeners have been synthesized in order to exploit the presence of various functional groups in the aspidosperman moiety. Some of them were selected for phase I and II clinical trials. Vindesine 23 N-alkylated analogues showed reduced antitumor activity [57]. Deacetylation at C-17, as well as acetylation of the free hydroxyl groups, inactivates the molecule. The C-17 acetoxy group of 11 was substituted by the N,N-dimethylglycinoyl group in vinglycinate 26 [56]. As a vinblastine prodrug, 26 showed improved absorption but no improvements in efficacy or toxicity [58]. Vintriptol 27, a vinblastine-23-oyl tryptophan ethyl ester, was selected from among 21 amino acid derivatives of 11 for its good antileukemic activity and diminished toxicity [59]. New spiro-fused derivatives were produced by forming an oxazolidine ring from the two substituents on C-16 with alkyl isocyanates. Vinzolidine, N-chloroethyl spirooxazolidinyl vinblastine 28 was tested in clinical studies due to its excellent activity and oral absorption [60]. No further developments were reported on the clinical use of vinblastine analogues 26–28 (Figure 8.6).

8.3 Antitumor Dimeric Vinca Alkaloids Structural formula 6'

Desacetyl vinblastine-16 carboxamide

Vindesine Eldisine

Launched (Company)

OH 18'

20'

14'

19'

15'

16' CO2CH3 5

14

N

6

20

10

CH3O 11

Synonymes

5'

N N H

Name

15 18

H

17 19

1

16

N H CH3

OH OH CO2NH2

23

1980 (Ely Lilly)

6'

N 14'

N H

18'

15' R'

R''

16' CO2CH3 5

14

N

6

20

10

CH3O 11

19' 20'

H

15 18

17 19

1

16

N H CH3

OAc OH CO2CH3

24

R' = R" = H

15',20'-Anhydro 5'-norvinblastine

j197

1989 Vinorelbine Navelbine (Pierre Fabre)

Figure 8.5 Structures of clinically used vinblastine derivatives.

Naturally occurring vinblastine 11 congeners such as leurosidine (200 -epivinblastine), leurosine (200 -deoxy-150 ,200 -epoxyvinblastine), and other 200 -deoxy derivatives possess active antitumor properties [61]. Vincristine 12 derivatives, modified on the ibogan part as vinepidine, 200 -deoxy-200 -epivincristine 29 [62], and vinformide, 200 deoxy-150 ,200 -epoxy-200 -epivincristine 30 [63] (Figure 8.6) were more effective against lymphosarcoma and leukemia. However, neuromuscular and cardiac toxicity hindered their further clinical development. Vinflunine 24, R0 ¼ R00 ¼ F (Figure 8.5), 190 ,190 -difluoro-vinorelbine, exerted the highest level of activity against the numerous in vivo antitumor models compared to presently used vinca dimer alkaloids [64]. Clinically significant activity has been seen in phase II studies, mainly in the treatment of transitional cell carcinoma of the urothelial tract, NSCLC, and breast carcinoma [65].

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Figure 8.6 Structures of vinblastine derivatives.

8.3.2 Synthesis of Dimeric Vinca Alkaloid Derivatives

The occurrence of vinblastine 11 in the plant is rare, and the scarcity of vincristine 12 is as low as 0.03 g/kg dried material. The biological precursors catharanthine and vindoline are more frequent alkaloidal components of Catharanthus roseus. The Potier synthesis of anhydrovinblastine 25 from pentacyclic ibogan and aspidosperman halves follows a biomimetic pathway [66]. When the C16–C-21 skeletal fragmentation of the ibogan skeleton induced by the Polonovsky–Potier reaction occurs conjointly with the attack of the nucleophile vindoline, the resulting 25 bis-indole alkaloid has the natural (160 S) configuration. A considerable amount of effort has been directed toward other methods both to couple the two parts of the molecule and transform 25 to oxygenated derivatives 11 and 12 [67, 68]. The oxidation of vinblastine 11 to the less accessible vincristine 12 was established by chromic acid or sodium dichromate [69, 70]. Ammonolysis or

8.3 Antitumor Dimeric Vinca Alkaloids

hydrazinolysis of vinblastine 11 produces vindesine 18 [54]. A second Polonovsky– Potierreactionofanhydrovinblastine 25furnished C0 ring-contractednavelbine 24[71]. 8.3.3 Pharmacological Properties of Dimeric Vinca Alkaloid Derivatives 8.3.3.1 Mechanism of Action Formation of the mitotic spindle is an essential step for accurate chromosome segregation. During mitosis, the microtubules search and capture chromosomes by establishing stable connections with the kinetochore, a macromolecular complex that is assembled onto the centromere at the onset of mitosis. The microtubules are constructed by the controlled polymerization of monomeric tubulin proteins [72]. The dimeric vinca alkaloids interfere with polymerization, thus preventing cell division by preventing the formation of new microtubules. Inhibition of microtubule polymerization perturbs spindle formation, interferes with chromosome alignment, and invariably blocks cell regulators from progressing through mitosis, blocking cells in M-phase. Cancer cells proliferate more rapidly than normal cells, so disruption of cell division affects more widely the growth of the diseased cells. The parent compound vinblastine binds to the b-subunit of tubulin in its dimer in a one-to-one complex, thus preventing polymerization into microtubules. The preformed tubulin depolymerizes and the complex with vinblastine crystallizes [73]. The C-200 position appears to be particularly critical for interaction with tubulin [74]. In a study comparing the actions of vinblastine 11, vincristine 12, and vinorelbine 24 on the axonal and mitotic microtubules in mouse embryos, all three drugs produced depolymerization of mytotic interpolar microtubules and cell metaphase block at the same concentration, but only 24 gave complete depolymerization. In contrast, 24 was less active on axonal microtubules than 11 or 12 [75]. Detailed investigations evaluating microtubule dynamics have shown that vinorelbine 24, together with its difluoro derivative vinflunine, has a mode of action qualitatively different from 11 [76]. 8.3.3.2 Clinical Pharmacology Pharmacokinetics The pharmacokinetic profiles of the dimeric vinca alkaloids are remarkably similar both across species and among individual compounds; they are characterized by high plasma clearance, a large volume of distribution, and a long terminal half-life, with very limited urinary excretion [77]. A triphasic clearance curve has been observed after intravenous administration to humans with a very short distribution phase and a prolonged terminal phase with half-lives in the range 24–70 h. Urinary excretion in unchanged form is about 15%, the bulk of excretion occurs via the liver and bile after metabolism [78]. The metabolites are excreted as conjugates. Oxidative degradation by action of myeloperoxidase occurs by cleavage between C-200 and C-210 and is facilitated by the presence of a C-200 hydroxyl moiety [79]. In comparison to vinblastine or vincristine, vinorelbine has a higher clearance and a larger volume of distribution [80].

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Adverse Effects Microtubules are also major components of other cellular structures, as cilia and flagella, which are present especially in the axons of nerve cells. The inhibition of microtubule dynamics disrupts crucial physiological processes such as vesicle transport and cell shape, interphase, and axonal transport. The adverse peripheral neurological effects, such as neuropathies, as well as cardiotoxic and neuromuscular actions, can be attributed to undesirable impacts of dimeric vinca drugs on microtubules [81]. The most notable toxic side effects of vinblastine 11 are myelosuppression and tissue necrosis. Vincristine 12 causes severe neurotoxicity and tissue necrosis. Neuromuscular actions can be observed to a lesser extent for vindesine 23 and minimal for vinorelbine 24 [82]. Only mild neurotoxicity and myelosuppression occur with vinorelbine, where the most notable side effects appear to be granulocytopenia and neutropenia. Other side effects of dimeric vinca drugs are common to antitumor agents (alopecia, ulceration, nausea, and diarrhea). Resistance Acquisition of drug-resistance is a major problem for cancer patients undergoing chemotherapy. Resistance to the vinca alkaloids develops rapidly in vitro in the presence of agents. Members of the human multidrug resistance-associated protein (MRP) family are membrane proteins able to transport a wide range of anticancer drugs out of the cells. Their presence in many tumors makes them prime suspects in unexplained cases of drug resistance. MRPs are organic anion transporters, transporting anionic drugs and neutral drugs conjugated to acidic ligands, such as glutathione, glucuronate, or sulfate [83]. The permeability glycoprotein (P-gp) is an ATP binding cassette (ABC) transporter protein. The multidrug resistanceassociated proteins belong to the ABC family. In vitro, the MRP/ABC transporters can collectively confer resistance to natural product anticancer drugs and their conjugated metabolites [84]. The cross-resistance includes not only the dimeric vinca alkaloids but also, for varying degrees, other bulky natural antitumor agents such as taxanes, anthracyclines, or epipodophyllotoxins [85]. Clinical Results with Dimeric Vinca Alkaloid Derivatives Vindesine New perspectives of combined therapies with vindesine 18 as component of antitumor cocktails in treating NSCLC were examined. The concurrent and sequential treatment with radiotherapy and chemotherapy consisting of cisplatin, vindesine, and mitomycin was compared in a phase III study, performed on 320 patients, suffering from unresectable stage III NSCLC. The concurrent approach yielded a significantly increased response rate and enhanced median survival duration [86]. The neoadjuvant (preoperative) chemotherapy can significantly improve long-term survival in stage III NSCLC [87]. The results of a randomized trial with a total of 132 patients suggested the survival advantage of the three-drug regimen of vindesine, ifosfamide, and cisplatin over the two-drug combination of cisplatin and vindesine for inoperable NSCLC [88]. Vinorelbine In a large multicenter European trial for NSCLC patients, single-agent vinorelbine was compared with vinorelbine/cisplatine and vindesine/cisplatine. With regard to response rate and median survival, the vinorelbine/cisplatin

8.4 Antitumor Camptothecin Derivatives

combination was superior to the other treatment regimen. Single-agent vinorelbine was equivalent to the European standard of cisplatin/vindesine and was much less toxic [89]. Neoadjuvant vinorelbine/cisplatin therapy has improved the survival of NSCLC patients with resectable stage IIIa disease compared to surgery alone [90]. In a phase II study, performed with 50 NSCLC patients in stage IIIB/IV, a threedrug combination of gemcitabine, ifosfamide, and vinorelbine showed promising activity against NSCLC [91]. In a randomized phase III trial, two-drug vinorelbine/doxorubicin therapy gave an overall response rate of 74% in the treatment of advanced breast carcinoma [92]. Phase II studies with vinorelbine plus epirubicin [93], vinorelbine plus epirubicin and paclitaxel [94], or vinorelbine plus docetaxel [95] have yielded pathological complete remission rates of 4–20% in women with stage II/III breast cancer.

8.4 Antitumor Camptothecin Derivatives 8.4.1 Medicinal Chemistry of Camptothecin Derivatives

Camptothecin 13 was isolated from the bark of Camptotheca acuminata (Nyssaceae) by Wall and Wani in 1966 [96]. It was also found in Ervatamia heyneana (Apocynaceae) and Ophiorrhiza pumila (Rubiaceae) [97]. Camptothecin and congeners such as 10-hydroxycamptothecin 31 [98] were found potent cytotoxic agents against a wide range of experimental mammalian tumor systems. CPT itself is very insoluble; the early clinical trials were performed in the form of a water-soluble sodium salt. The trials were, however, suspended [99, 100] due to the severe toxic events. Only later was it realized that the E-ring opened form is about one-tenth as active as 13 [101]. Renewed interest occurred 15 years later, with the discovery of the unique mode of action of CPT by inhibiting nuclear mammalian topoisomerase I. Thousands of CPT derivatives have been subsequently prepared with the goal of finding a clinically useful anticancer drug, to overcome the drawbacks of CPT, notably its poor water solubility, ease of hydrolytic lactone opening to the undesirable acid form, and high serum protein binding. These analogues were obtained by the modification of the lead molecule and by total synthesis [102]. Two molecules, topotecan (Hycamtin, 32) and irinotecan (Camptosar, CPT-11, 33), gained clinical approval. Both are derivatives of 10-hydroxy-CPT 31, more stable, much more soluble in water, and less serum protein-bound than CPT (Figure 8.7). Topotecan (TPT, 32), (S)-9-dimethylaminomethyl-10-hydroxy-CPT is produced from the parent compound [103]. Compared to 13, TPT is twice less active against L1210 leukemia cells in vitro but more active in treating leukemic mice bearing L1210 and P388 leukemias. TPT 32 is also active against a number of experimental solid tumors. In vivo, it has demonstrated superior activity against Lewis lung cancer, and melanoma B16 compared to CPT [104]. TPT was launched by SmithKline Beecham for small-cell lung cancer (SCLC) treatment in 1998 in Switzerland.

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Structural formula

9

HO 10 11

7

8

A

B

12

13 N 1

O

5

6

Name

4

3

31

14

H3C

Launched (Company)

17

16

N 22 C D 2

Synonymes

E O 20

10-Hydroxycamptothecin

21

O

15

OH 19

18

N(CH3)2

O

HO

N N

RO

OH

H3C

CH3

O N

O

N

O OH

H3C

R=

C O

34,

R=

1998 (SmithKline Beecham)

O 32

33,

9-DimethylaminoethylTopotecan 10-hydroxycamptothecin Hycamtin

O

H

N

N

7-Ethyl-10-[4-(1-piperidyl)1-piperidyl]carbonyloxycamptothecin

7-Ethyl-10-hydroxycamptothecin

Irinotecan Topotecin CP-11

1994 (Daiichi Yakult Honsha)

SN-38

Figure 8.7 Structures of camptothecin derivatives.

TPT 32 has gained approval by the FDA for treatment SCLC and ovarian cancers (OC) [105]. Irinotecan (Camptosar, CPT-11 33), 7-ethyl-10-[4-(1-piperidyl)-1-piperidyl] carbonyloxycamptothecin [106] is a prodrug; its active metabolite (SN-38, 34) is 7-ethyl-10hydroxycamptothecin (Figure 8.5). Irinotecan was launched in Japan in 1994 by Daiichi and Yakult Honsha for treatment of lung, cervix, and ovarian cancers. In France, 33 was launched by Rhône-Poulenc Rorer in 1996 for the treatment of advanced colorectal cancer (CRC) refractory to standard chemotherapy. Irinotecan was approved by the FDA in 2000 as first-line treatment for advanced CRC [107]. 8.4.1.1 Structure–Activity Relationships The main structural features influencing the activity of CPT derivatives are 20(S)hydroxyl, pyridone moiety (D-ring), lactone moiety (E-ring), and planarity of the

8.4 Antitumor Camptothecin Derivatives

pentacyclic ring system. The C–D–E rings cannot be altered [97, 108]. The modifications of quinoline ring (9, 10, and 11-position of the A-ring and 7-position of the B-ring) enhance the potency of the CPT derivatives in both in vivo and in vitro studies [97, 109]. Monosubstitution at 9, 10, or 11 positions by NH2 or OH group increases the antitumor activity, while the 12-substitution greatly reduces it. Disubstitutions at 10 and 11 are unpreferable, exceptions are the 10,11-methylenedioxy and 10,11-ethylenedioxy derivatives [110]. Substitution at the C-10 position with a hydroxyl group contributes to the increased activity of TPT 32 and SN-38 34. The 11-fluoro or 11-cyano substitution increases the DNA topoisomerase inhibition [111]. Substitution at the 7-position of the B-ring resulted in an increase in water solubility [112]. The 7-ethyl position is also important for stabilizing the CPT-topoisomerase I-DNA adduct [113]. The four oxygen atoms of the D/E ring are involved in hydrogen bonding with the topo I–DNA complex. The only favorable alteration of this part of the molecule was the replacement of the a-hydroxylactone moiety with b-hydroxylactone to obtain homoCPT derivatives [114]. Several second-generation CPT derivatives are in various stages of clinical trials [115]. Rubitecan 35, a 9-nitro derivative [102], was tested for the treatment of locally advanced or metastatic pancreatic cancer [116]. 35 is metabolically converted in vivo into the equally or more potent 9-amino derivative IDEC-132 36 [117]. Lurtotecan 37, a water-soluble 7(N-methyl piperidinyl) methyl)-10,11-ethylenedioxy analogue [110] was three times more potent in vivo, compared to TPT. It has been tested for advanced or metastatic solid tumors in liposomal formulation [118]. Exatecan 38, a 10-methyl-11-fluoro derivative with an additional amino-substituted hexane ring fused on 7, 8, 9 position [119], was tested for NSCLC, CRC, and hepatocellular cancers [120]. A 7(N-methylpiperidinyl) methyl)-10-methyl-11-chloro homoCPT derivative, BN 80927 39 [121], inhibits both topoisomerase I- and topoisomerase II-mediated DNA relaxation and shows pronounced cytotoxicity against human tumor cell lines [122] (Figure 8.8). A recently published excellent SAR/QSAR study on CPT analogues helps in understanding the requirements of physicochemical properties of the substituents in key locations as well as molecules as a whole [123]. The analysis of QSAR models reveals the two most important descriptors, the hydrophobicity and molecular refractivity; the optimal log P and MR values are given. 8.4.2 Synthesis of Camptothecin Derivatives

The clinically used CPT derivatives have been obtained semisynthetically. The reduction of CPT 13, followed by oxidative acylation and hydrolysis, led to 10hydroxy-CPT 31, which was transformed to TPT 32 by Mannich reaction [102]. The reaction of CPT with propionaldehyde gives 7-ethyl-CPT. H2O2 oxidation to N-oxide and then isomerization with UV light affords 7-ethyl-10-hydroxy-CPT 40. Treatment with pyperidinopiperidine carbamoyl chloride led to CPT-11 33 [104]. Numerous convergent synthetic strategies were reported utilizing the possibilities of versatile approach of CPT [101, 124]. Comins and Nolan’s short asymmetric synthesis involves the formation of the C-ring by connecting the A/B- and D/E-

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R4

R3 R2 10 11

R1

8 7

9

5

13

N

4

16

N

B

A 12

O 6

22

E O 21

17

20 2

3

14

1

O

15

H3C

OH 19

18

R1

R2

R3

R4

E-ring

Compound

H

H

NO2

H

normal

35 Rubitecan

H

H

NH2

H

normal

36 IDEC 132

O

O

H

normal

37 Lurtotecan

F

CH 3

normal

38 Exatecan

Cl

CH 3

20-homo

39 BN 80927

CH2 N

N CH3

NH2 H

H

Figure 8.8 Selected A, B, and E ring-modified camptothecin derivatives.

fragments via N-alkylation and ring closure in an intramolecular Heck reaction [125]. Starting from commercially available materials, this method is suitable for industrial production of CPT. 8.4.3 Pharmacological Properties of Camptothecin Derivatives 8.4.3.1 Mechanism of Action The primary cellular target of CPTs is DNA topoisomerase I (topo I). The topoisomerases play an essential role in the key cellular processes of replication and transcription. DNA topoisomerases are classified into type I and type II subclasses. Type I topoisomerases change the topological state of DNA via transient enzymelinked single-strand breaks. In contrast, type II topoisomerases catalyze the strand passing reaction by transiently breaking both strands of duplex DNA [126]. The primary function of topo I is to remove both excessive positive supercoils and negative supercoils arising during DNA replication and transcription. Topo I removes the supercoils by introducing transient breaks into the DNA helix. In this reaction, DNA topo I binds noncovalently to superhelical DNA and cleaves one of the DNA strands via a nucleophilic attack of the phosphodiester bond in DNA, forming

8.4 Antitumor Camptothecin Derivatives

a covalent linkage between a tyrosine group at the active site of topo I and a 30 phosphate group along the DNA backbone [127]. The covalent intermediate is reversed when the 50 -OH of the broken strand reattacks the phosphotyrosine intermediate. Topo I dissociates from the DNA molecule and undergoes another catalytic cycle, the replication fork serves as a template for the synthesis of a new strand of DNA [128]. The rate of religation is much faster than the rate of cleavage, and the concentration of covalent binary complex remains low. CPTs bind to the normally transient cleavable DNA–topo I complex and form an enzyme–drug–DNA ternary cleavable complex. As a consequence of the formation of a cleavable complex, both the initial cleavage reaction and the religation steps are inhibited, causing collision of the replication fork, double-strand DNA breakage, and conversion of reversible cleavable complex into an irreversible complex [129]. One or more of these events generate other cellular responses that can lead to the cell cycle arrest in the G2 and S phase and to cell death [130]. Several structural models have been suggested for the interaction of CPTs with the covalent binary complex [125]. The computation models used the reported crystal structure of the binary human topo I–DNA cleavable complex [125] and made much account of hydrogen bonding. Recently, X-ray crystal structures of the ternary complexes of TPT and CPT bound to human topo I covalently joined to a DNA duplex were reported [131, 132]. The crystal structures reveal that CPTs intercalate at the site of DNA cleavage and mimic a DNA base pair. Comparing the two ternary complexes, there is a slight twist in the orientation of CPT relative to TPT. The difference can be attributed to the 7-C substituent of TPT. The increased activity of 7-, 9-, and 10-substituted CPTderivatives is also in agreement with the steric position of CPTs in the crystalline ternary complex. 8.4.3.2 Clinical Pharmacology Pharmacokinetics The different pharmacokinetics of the two clinically used CPT derivatives can be explained by their different structures and by the fact that CPT11 33, unlike TPT 32, acts as a prodrug. The terminal half-life of TPT is 3 h, while that of CPT-11 is 6.3 h, and for its active metabolite SN-38 34 11.5 h [133]. TPT is subject to alteration by esterases and the products are glucuronidated or oxidized by CYP3A4 [134]. The conversion of CPT-11 into SN-38 is accompanied with glucuronidation in the liver. The glucuronide is excreted into the bile and further deconjugated in the intestinal microflora to SN-38. CPT-11 can also undergo CYP3A4-mediated oxidative process to form a metabolite in which the first piperazine ring is oxidatively opened to an acid analogue [135]. Biliary clearance is the major route of excretion for SN-38 and CPT-11, while TPT is mainly excreted in urine. Adverse Effects The longer exposure time and higher cellular accumulation of CPT11 and its active metabolite SN-38 explain the gastrointestinal toxicities, the development of diarrhea, which is troublesome and dose limiting [136]. Myelosuppression is also a serious problem with CPTs. For TPT, neutropenia was the principal

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dose-limiting toxicity, gastrointestinal adverse effects such as diarrhea, nausea, and vomiting were successfully controlled with standard supportive care measures [137]. Resistance Resistance to CPTs has been attributed to enhanced drug efflux by ABC transporters, as breast cancer resistance protein BCRP/MXR/ABCG2 [138]. Another possible mechanism of resistance is the alteration in the structure of topo I. Crystal structures of two CPT-resistant forms of human topo I in ternary complexes with DNA and TPT were reported [139]. The alteration of Asn722 to Ser leads to elimination of a water-mediated contact between the enzyme and the TPT. A clear relationship between the carboxylesterase level and the chemosensitivity of human small-cell and non-small-cell lung cancer cell lines has been demonstrated in vitro where CPT-11 resistance was encountered in cell lines with low carboxylesterase expression. Furthermore, as SN-38 34 is inactivated to SN-38 glucuronate by glucuronidation by UDP-glucuronosyltransferase 1A1 (UGT 1A1), overexpression of UGT at the protein and mRNA levels was found to account for SN-38 resistance in a human lung cancer cell line. In addition, ABC transmembrane transporters such as P-gp and MRP also mediate resistance to CPTs and play an important role in the efflux and active excretion of CPT-11 [140]. Clinical Results with CPT Derivatives Vinca Dimer Derivatives and CPTs A critical phase III randomized trial comparing CPT-11, either alone or in combination with cisplatin, to vindesine/cisplatin demonstrated superior survival for stage IV NSCLC patients receiving irinotecan [141]. In a phase III trial, a total of 398 patients with previously untreated NSCLC were randomized to receive cisplatin þ CPT-11, cisplatin þ vindesine, or cisplatin alone. CPT-11 monotherapy is not inferior to vindesine–cisplatin in terms of survival, while the CPT-11-containing regimen is one of the most efficacious and well tolerated in the treatment of advanced NSCLC [142]. The cytotoxic interactions between vinorelbine and SN-38 on human NSCLC lines were studied in vitro. On simultaneous exposure, additive effects were observed, while sequential administrations generated antagonistic effects [143]. Vinorelbine strongly inhibits CPT-11 catabolism by CYP3A4 at clinically relevant concentrations [144]. Irinotecan (CPT-11) The combination of irinotecan/5-fluorouracil (5-FU)/leucovorin (LV) is superior to 5-FU/LV alone as first-line therapy for patients with metastatic CRC, as emerged from two prospective phase III randomized, controlled, multicenter, and multinational clinical trials [145]. A phase III study in extensive-disease SCLC compared irinotecan/cisplatin and standard etoposide/cisplatin regimens and demonstrated a significant difference in survival in the irinotecan-containing regimen [146]. A large North American phase III trial failed to confirm the abovementioned survival benefit observed with irinotecan in Japanese patients [147]. Weekly combined administration of irinotecan and cisplatin achieved a promising overall response rate, median time to tumor progression, and median survival in patients with stage IIIB/IV NSCLC [148].

References

Topotecan In an open, multicenter, randomized, and stratified phase III study in 226 advanced epithelial OC patients who had failed prior platinum-based therapy, TPT compared to paclitaxel showed a better response rate, a longer duration response, and a significant difference in median time to progression [149]. In phase III studies, TPT was shown to be equivalent in efficacy to both paclitaxel and liposomal doxorubicin as second-line therapy in patients with relapsed OC. Furthermore, non-cross-resistance between TPT and paclitaxel was demonstrated in a third-line phase III crossover study. Hematological toxicities are usually short lived, noncumulative, and manageable with dose modifications, while nonhematologic toxicities are usually mild to moderate in severity [150]. A phase II study of TPT combined with gemcitabine in the treatment of relapsed OC patients demonstrated favorable toxicity profile and efficacy of this novel combination regimen [151].

8.5 Summary and Conclusions

A wide range of pharmacologically active compounds can be found among the monoterpenoid indole alkaloids. Relatively few of them are used in clinical practice, and only three have proved as lead molecules for ABDD. To date, seven monoterpenoid indole analogues have gained clinical approval in different countries. Typically, these new drugs are direct natural product analogues. Their common structural feature is the retained natural configuration, 20S,21S for vincamine analogues, 160 S,140 S for vinblastine analogues, and 20S for camptothecins. Three of them are analogues of vincamine and used in several countries for the treatment of cerebral insufficiencies. Analogues of vinca dimer alkaloids and the modified monoterpenoid indole camptothecin are of greater importance. The spectrum of drugs, origined from ABDD of the two anticancer monoterpenoid alkaloid groups, covers the most fatal tumor species, and their share in the US$ 10 billion cytotoxic market can be estimated at more than 20%. New information obtained from studies on the molecular modes of action and the results of SAR helped to gain a better understanding of important biological processes such as cell cycle or ion transport in neurons. The discovery of analogues of the Cataranthus and Camptotheca family will continue. With regard to the next generation of cytotoxic monoterpenoid indole derivatives, members of which are in various stages of clinical trials, the future of anticancer monoterpenoid indole research is open.

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H.J., Redman, M.W., Carleton, J.E., Jett, J., Langer, C.J., Kuebler, P., Dakhi, S.R., Chansky, K., and Gandara, D.R. (2009) Phase III trial of irinotecan/cisplatin compared with etoposide/cisplatin in extensive-stage small-cell lung cancer: clinical and pharmacogenomic results from SWOG S0124. J. Clin. Oncol., 27, 2530–2535. Jagasia, M.N., Langer, C.J., Johnson, D.H., Yunus, F., Rodgers, J.S., Schlabach, L.L., Cohen, A.G., Shyr, Y., Carbone, D.P., and Devore, R.F. (2001) Weekly irinotecan and cisplatin in advanced nonsmall cell lung cancer: a multicenter phase II study. Clin. Cancer Res., 7, 68–73. Carmichel, J., Gordon, A., Malfetano, J. et al. (1996) Topotecan, a new active drug, vs paclitaxel in advanced epithelial ovarian carcinoma: International Topotecan Study Group Trial. Proc. Am. Soc. Clin. Oncol., 15, Abst. 765. Herzog, T.J. (2002) Update on role of topotecan in the treatment of recurrent ovarian cancer. Oncologist, 7 (Suppl.5), 3–10. Sehouli, J., Sttengel, D., Oskay, G., Camara, O., Hindenburg, H.J., Klare, P., Blohmer, J., Heinrich, G., Elling, D., Ledwon, P., and Lichtenegger, W. (2002) A phase II study of topotecan plus gemcitabine in the treatment of patients with relapsed ovarian cancer after failure of first-line therapy. Ann. Oncol., 13, 1749–1755.

Andr
as Nemes

Gedeon Richter Plc., Gyömro˝i u. 19-21, 1103 Budapest, Hungary Andr
as Nemes received his undergraduate education at E€ ov€ os Lór
and University, Budapest, and his PhD degree in medicinal chemistry from the Technical University in 1966. After working at the Research Institute for Medicinal Chemistry, Budapest, he joined Gedon Richter Ltd. in 1975, where his main activity was elaboration of processes for preparing heterocyclic and alkaloid origin drug substances. He won “Prominent Inventor” Award in 1970 and 1982 and Zempl
en G
eza Academic Award in 2008. He is the author of some 80 patents and scientific publications.

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9 Anthracyclines, Optimizing Anticancer Analogues Federico-Maria Arcamone

9.1 Introduction: Biosynthetic Antitumor Anthracyclines

The anthracyclines are glycosides belonging to the large family of the polyketidederived natural products that display a considerable molecular diversity [1]. In 1959, it was found that red pigments produced in the cultures of a Streptomyces spp. were active against Ehrlich carcinoma and sarcoma 180 in mice [2]. The pigments were found to be similar in structure to anthracyclines previously studied and described by different authors, notably among them Hans Brockmann [3]. A typical representative of these anthracyclines is rhodomycin A (1). Shortly afterward, an active principle that is distinctly more active in different laboratory tests for antitumor activity was independently discovered at Farmitalia Research Laboratories, Italy, in the cultures of S. peucetius [4, 5], and at Rhone-Poulenc, France, in the cultures of S. coeruleorubidus [6], and the generic name daunorubicin was attributed to the compound [7]. Characteristic structural features of the components of the fermentation broth of S. peucetius are the novel aminosugar, L-daunosamine, the 4-methoxy group in the tetracyclic aglycone, and the C-9 side chain, represented by an acetyl group in daunomycinone or a hydroxyacetyl group in adriamycinone [8]. These compounds include daunorubicin (2a), doxorubicin (2b) (originally named adriamycin) 13dihydrodaunorubicin (3), and the disaccharide derivative 4-O-a,L-daunosaminyldaunorubicin. Daunorubicin is clinically useful as an antileukemic agent [9, 10]. On the other hand, doxorubicin (since its registration as AdriamycinÒ in the United States in the early 1970s) is a component of many frequently used drug combinations in the medical treatment of lymphomas, sarcomas, breast and ovarian cancer, and other solid tumors [11]. A practical method for the industrial preparation of doxorubicin (2b) is the conversion of 2a to the 14-bromo derivative and subsequent nucleophilic substitution of the halogen atom with base [8]. Antitumor activity has been associated also with other biosynthetic anthracyclines [12].

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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O 1 4

12 5

OH OH 10 7

11 6

O

13 OH

1 D 4

C

OH

O

O

OH

R A 9 OH 14 7

B

OH OH

7

OH O

OH O

OH

OMe O

OH O

O

O

NEt2

NH2

1

OH

2a: R=H 2b: R=OH

OMe O

OH O O OH

NH2

3

Soon after the discovery of the pharmacological properties of daunorubicin, Di Marco and coworkers drew attention to the affinity of the compound for calf-thymus DNA [13] and its inhibitory properties against the reactions of DNA and RNA polymerases in cell-free systems and in cultured cells [14, 15]. The high affinity was attributed to the formation of an intercalation complex of the type already known to be formed by a number of dyes such as the acridines and other planar polycyclic aromatic molecules [16]. It was also anticipated that this type of interaction would be at the basis of the biological properties of the new compounds. The structural features of the anthracyclines, namely, the presence of a planar electron-poor chromophore and of a cationic appendage, well explained the formation of an intercalation complex. Although the micromolar range value of the association constants of the binding is inconsistent with this interaction being the sole explanation of the biological effects, since these are observed at nanomolar concentrations of the drugs, a generally accepted concept is that DNA binding is a necessary, albeit nonsufficient, requirement for bioactivity [17]. The quinone function, would, on the other hand, allow participation in redox reactions vis-a-vis appropriate electron donors or acceptors. Despite these simple chemical properties being common to all antitumor anthracyclines, it seems unlikely that it could completely explain the specific biological properties of the S. peucetius metabolites. Inhibition of the subsequently discovered mammalian topoisomerase II and its sensitivity to anthracyclines is now considered to be their main mode of action whereas intercalation and a number of other cellular disturbances brought about by these drugs is considered to be contributory. A study using cultured HeLa cells showed that daunorubicin and doxorubicin were indistinguishable in their effect both on cell mitosis and on protein and RNA synthesis, and that low doses of 50–100 ng/ml were markedly active antimitotically but had only a slight effect on RNA synthesis [18a]. On the other hand, doxorubicin is invariably more effective than daunorubicin against the growth of experimental tumors in mice [18b]. It was also demonstrated that both daunorubicin and doxorubicin reached a plasma level of around 1 mg/ml 20 min after the i.v. administration of a 10 mg/kg dose in rats, the former showing a significantly higher excretion rate and the latter a greater concentration in the

9.2 Analogues with Modification of the Aminosugar Moiety

tissues with time. Daunorubicin was extensively converted to a major metabolite (13-dihydrodaunorubicin or daunorubicinol 3) but no doxorubicin metabolite was found [18c]. These and other results suggested the importance of metabolism– tissue distribution–elimination factors for the exhibition of antitumor properties in vivo. A major dose-limiting toxic side effect of doxorubicin is the development of cardiotoxicity. This is observed at cumulative clinical dosages higher than 400 mg/m2 of body surface area. In the laboratory, the myocardium of mice treated with repeated doxorubicin administrations exhibited an increased reactivity of the sarcoplasmic reticulum with zinc iodide-osmium tetroxide reagent that allows detection of disulfide bonds derived from the oxidation of cell constituents containing sulfhydryl groups such as polypeptides, cysteine, and glutathione [19]. This might be the consequence of an oxidative stress caused by a higher than normal oxygen tension in the heart tissue [20, 21]. Excess oxygen could be related to the reduction of metabolic consumption of oxygen, a consequence of the long lasting inhibition of nucleic acid synthesis in the heart [22]. Inhibition of mitochondrial DNA transcription by doxorubicin would also lead to a diminished ATP production and consequently to cell damage in the high-energy demanding heart tissue [23]. The role of free radicals in the biological actions of the anthracyclines has been discussed [24]. With the aim of improving the pharmacological properties, synthesis of antitumor anthracyclines analogues has been carried out [8, 25a–d, 26].

9.2 Analogues with Modification of the Aminosugar Moiety

Different stereochemical variants containing 3-amino-2,3,6-trideoxy-L-hexopyranosides have been prepared and tested. The first study was concerned with the 40 epimer of 2a and 2b, involved replacement of L-daunosamine with 3-amino-2,3,6trideoxy-L-arabino-hexose, that is, L-acosamine, an aminosugar constituent, together with its 4-O-methyl derivative actinosamine, of the antibiotic actinoidin [27]. 7-O-a,LAcosaminyldaunomycinone (4a) was obtained by Koenigs Knorr coupling of daunomycinone with 3-trifluoroacetamido-4-O-trifluoroacetyl-a-L-acosaminyl chloride (5) and subsequent methanolysis, chromatographic separation, and deprotection of the resulting N,O-ditrifluoroacetylated anomeric aminoglycosides. When the coupling reaction was performed on 9-deacetyl-9-(2,20 -dimethyl-40 -methoxydioxolan-40 yl)-daunomycinone 6, chromatographic separation after methanolysis of the reaction products afforded two compounds that were treated with aqueous HCl and then with 0.1 NaOH to give, respectively, the a-anomer 4b and the b-anomer of 40 -epidoxorubicin. Compounds 4a and 4b, with an a-anomeric configuration at C-10 , are as cytotoxic as 2a and 2b in cultured cells in vitro and display antitumor effects in vivo in experimental tumors in mice at comparable doses [28], but anomers with the b configuration at C-10 show a markedly lower bioactivity. Following demonstration of a

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lower toxicity, including cardiotoxicity, when compared to 2b in vivo, compound 4b was developed, under the generic name epirubicin, to the clinical stage. A more practical process for the preparation of epirubicin (4b) was eventually developed starting from N-trifluoroacetyldaunorubicin that was oxidized at C-40 and then stereoselectively reduced with sodium borohydride. Removal or the N-trifluoroacetyl group with base and introduction of the 14-OH as described above for the conversion of 2a to 2b produced 4b [29]

O

OH

O R Cl

OH OMe O

OH O

4' NH2 4a: R=H 4b: R=OH

OH

MeO

O F3CCOO

O O OH

NHCOCF3

O HO

O

OMe O 5

OH OH 6

A quite different case was that of 30 ,40 -diepidaunorubicin 7a and of 30 ,40 -diepidoxorubicin 7b, in which the sugar moiety was L-ristosamine, the aminosugar component of the ristocetins [30], and of 7c, containing the opposite configuration at C-30 compared to the natural compounds. For the synthesis of 7a, 4-O-pnitrobenzoyl-3-N-trifluoroacetyl-ristosaminyl chloride (8a) and daunomycinone were allowed to react in the presence of silver triflate to give, after removal of the trifluoroacetyl groups, 7a that was then converted to doxorubicin stereoisomer 7b [31]. Condensation of 8b with daunomycinone and deprotection afforded L-xylo analogue 30 -epi-daunorubicin 7c [32]. Configurational analogues 7a, 7b, and 7c display lower affinity toward calf-thymus DNA and lower antitumor activity against L1210 leukemia in mice even at a 10 times higher dosage compared to the corresponding L-lyxo isomers daunorubicin (2a) and doxorubicin (2b) [25a–c]. The study was extended to include the related glycosides containing a hydroxyl group at C-60 . The new sugar moieties were obtained from intermediates 9a and 9b, by standard procedures [33]. Analogues 10a,c and 11a,c were obtained upon glycosidation of daunomycinone with the activated derivatives of the Ntrifluoroacetylated appropriate aminosugars and subsequent hydrolytic removal of the protecting groups. Doxorubicin analogues 10b, 11b, and 11d were prepared by bromination at C-14 and substitution of the halogen with base. Although 60 hydroxy analogues 10a–c display significant affinity toward calf-thymus DNA and activity in the inhibition of DNA-dependent polymerases in vitro, they are distinctly less active, on a weight basis, compared to 2a and 2b both in cultured cells and in in vivo antitumor tests. The more than one order of magnitude lower cytotoxicity exhibited in cultured cells leads to the conclusion that introduction of the additional hydroxyl group and the consequent enhanced hydrophilicity strongly

9.2 Analogues with Modification of the Aminosugar Moiety

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disfavors cellular uptake of these analogues. An even greater loss of bioactivity is observed both with compounds 11a–d and with (20 R)-hydroxydaunorubicin [25b, 34, 35].

O

OH

CF3CO Cl

O R OH

OMe O

OH

HO 4'

OH

O

4 8a: R=OpNBz 8b: as 8a, epi at C-4 8c: R=H OMe

NH2 O 3'

O Ph

O O

R 9a: R=OH 9b: R=NH2

O

OH

O

R

R

OH

R

O

7a: R=H 7b: R=OH 7c: R=OH, epi at 4'

O

NH O OMe O

OH

HO

O

O

R1 2

R

NH2

10a: R=H, R1=OH, R2=H 10b: R= R1=OH, R2=H 10c: R=R1=H, R2=OH

OH OMe O HO R1

OH O NH2 O

R2 11a: R=R2=H, R1=OH 11b: R= R1=OH, R2=H 11c: R=R1=H, R2=OH 11d: R=R2=OH, R1=H

A higher lipophilicity was expected for the analogues bearing deoxy or O-methyl modifications in the aminosugar moiety. The 40 -deoxy derivative 12a of daunorubicin was obtained starting from 4-deoxy derivative 8c that was converted to the 1chlorosugar used for the glycosidation of daunomycinone followed by standard transformations. Alternatively, the compound was prepared by deoxygenation of 4a at C-40 [29]. Glycoside 12a and related doxorubicin analogue 12b display similar behavior as regard to calf-thymus DNA complexation and the DNA-dependent polymerase reactions, but they appear one order of magnitude more potent than the natural compounds in inhibiting the colony-forming ability of cultured HeLa cells [36]. Because of a comparable or even higher in vivo activity in different experimental cancers, especially colon tumors in mice, and a lower cardiotoxicity in the animal models than doxorubicin (2b) [25b], compound 12b was submitted to phase 1 and 2 clinical trials with the generic name esorubicin. Along this line, 40 -O-methyl derivatives of daunorubicin (2a), doxorubicin (2b), and of their 40 -epimers (the actinosaminides) were also synthesized and tested. The new aminoglycosides exhibit antitumor activity in murine transplantable leukemias comparable to that of the parent compounds, with 40 -O-methyldoxorubicin 13a being the most effective [37]. Analogues bearing a 40 -C-methyl substitution of the 0 L-lyxo and L-arabino series together with the corresponding 4 -O-methyl ethers were also prepared [38]. The different analogues display 50% inhibition of HeLa cells at a concentration close to 10 nM, compounds with an equatorial C-methyl such as 13b displaying higher activity than those in which this substituent was axial. A general trend of correlation of affinity for calf-thymus DNA versus cytotoxicity was recorded. Although the C-methyl analogues exhibit antitumor activity in vivo, none shows a higher activity at the optimal doses compared to the parent compounds [39a]. However, berubicin, a 40 -O-benzyl derivative, has been recently shown to cross the blood–brain barrier, thus showing potential in the medical treatment of glioblastomas [39b].

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O

OH

O

O

OH

O OH

OH OMe O

OH

O

O NH2 12a: R=H 12b: R=OH

O

OH

O

OH

R

OMe O R2

OH

O

O NH2 R1

13a: R1=OMe, R2=H 13b: R1=OH, R2=Me 13c: R1=I, R2=H

OH

4 OMe O

OH

O

O R 14a: R=OH NH2 14b: R=H 14c: R=NH2 (demethoxy at C-4)

The pharmacological properties of epirubicin (4b) and esorubicin (12b) prompted the synthesis of other C-40 derivatives. 40 -Iodo-40 -deoxydoxorubicin (iodorubicin, 13c) was designed to improve lipophilicity at physiological pH and consequently uptake, transport, and tissue distribution of the drug by reducing the basicity of the amino group at C-30 while keeping the same group in place. The synthesis was achieved starting from N-trifluoroacetyl-40 epidaunorubicin that was converted to 13c upon treatment of the 40 -O-trifluoromethanesulfonyl derivative with tetra-n-butylammonium iodide, removal of the N-trifluoroacetyl group with base followed by the introduction of the 14-hydroxyl via the standard methodology. The compound shows a pKa value of 6.4 (2b: pKa ¼ 8.2) and a high apparent partition coefficient (Papp ¼ 31.4 between octanol and aqueous pH 7 phosphate buffer). Iodorubicin (13c) is superior to doxorubicin in terms of antitumor efficacy, particularly striking results have been obtained in doxorubicin-resistant leukemias and in metastatic Lewis lung carcinoma. It also appears devoid of cardiotoxicity as no atrial or ventricular lesions are observed in mice at dosages and schedules at which doxorubicin treatment affects 100% of the treated animals [40]. Antitumor activity of selected compounds differing in the aminosugar moiety is shown in Table 9.1. Displacement of the amino group to position 40 was carried out with the synthesis of 40 -amino derivatives 14a and 14b from which the corresponding doxorubicin analogues were also obtained [41, 42]. On the basis of their significant bioactivity, the 4-demethoxy analogues of 2a and 2b possessing an amino group in place of the hydroxyl at C-40 , that is, two amino groups on the sugar moiety, were prepared starting from 4-demethoxydaunomycinone and 2,3,4,6-tetradeoxy-3,4-ditrifluoroacetamido-L-lyxo-hexopyranosyl chloride to give 14c, from which the corresponding 14-hydroxylated compound was also obtained. The compounds did not show superior antitumor activity in the laboratory tests, but are distinctly more cytotoxic than doxorubicin on resistant cells in vitro [43]. In conclusion, epirubicin (4b), esorubicin (12b), and iodorubicin (13b) entered in this temporal succession in preclinical development and clinical evaluation. The first was demonstrated to exhibit a practically equivalent antitumor activity in different solid cancers, but a better tolerability compared to doxorubicin [44a,b]. The metabolism of epirubicin in human patients is characterized by extensive biotransformation

9.3 Analogues with Modifications in the Anthraquinone Moiety Table 9.1 Cytotoxicity in vitro on HeLa cells (LD50, ng/ml after 24 h exposure) and antitumor activity (AST%)a) against L1210 murine transplantable leukemia at optimal nontoxic doses (O.D., mg/kg body weight, treatment by the intraperitoneal route on day 1) of analogues modified in the sugar moiety.

Compound

Cytotoxicity LD50

Daunorubicin (2a) Daunorubicin, 30 ,40 -diepi- (9a) Doxorubicin (2b) Doxorubicin (2b) Doxorubicin, 40 -O-methyl- (13a) Doxorubicin, 40 -C-methyl- (13b) Epirubicin (4b) Doxorubicin, 60 -hydroxy- (10b) Iodorubicin (13b)

6.8 90 18 110b) 5.0 16.5 8.5 1000b) 2

Antitumor activity

References

O.D.

AST%

References

[31b] [31b] [40] [34] [39] [39] [39] [34] [40]

4 50 5 19c) 4.4 7.7c) 5 40 6.6c)

150 137 166 320 312 172 150 137 300

[25a] [25b] [25a] [40] [39] [39] [25a] [25b] [40]

a) Average survival time of treated animals as percentage of untreated controls. b) After 8 h exposure. c) Murine transplantable P388 leukemia, intraperitoneal treatment on day 1.

to partially or totally inactive metabolites, including a 13-dihydro derivative, epirubicinol, two 40 -glucuronides, and four aglycones. This metabolic transformation (not observed in experimental animals) might be responsible for better tolerability of this drug by patients compared to doxorubicin [45]. Epirubicin is available in oncology as PharmorubicinÒ or, in the United States, as EllenceÒ [25d]. On the other hand, esorubicin did not show, at the clinical level, the lower cardiotoxicity suggested from the results recorded in the animal experiments [46]. In the case of iodorubicin, the unexpectedly extensive metabolic transformation in the human body impaired the expression of useful pharmacological properties [47], whereas recently developed berubicin is undergoing clinical trials as a potential agent for the clinical treatment of brain tumors [39b].

9.3 Analogues with Modifications in the Anthraquinone Moiety

The first total synthesis of daunorubicin analogues provided 4-demethoxydaunorubicin (idarubicin, 15a), obtained together with the (7R),(9R) diastereomer upon glycosidation of racemic 4-demethoxydaunomycinone prepared by substantially following the procedure of Wong et al. [48]. The compound soon appeared of interest owing to its potency and antitumor activity, especially against experimental leukemia in mice, and reduced cardiotoxicity in animal models. In a subsequent study, the synthesis was carried out starting from phthalic anhydride or an appropriate derivative thereof that was allowed to react with the (R)(
) stereoisomer of 6acetyl-6-hydroxy-1,4-dimethoxytetralin to give, with a concomitant O-demethylation,

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7-deoxy-4-demethoxy-daunomycinone 16a. The introduction of the 7-hydroxyl group in the corresponding 13-ethylenedioxyketal derivative was performed by radical bromination followed by hydrolysis. Deprotection yielded 4-demethoxydaunomycinone (idarubicinone, 16b). Glycosidation was carried out using N,O-ditrifluoroacetyla-L-daunosaminyl chloride and silver triflate as the chloride acceptor [8]. Doxorubicin analogue 15b displays high cytotoxic potency but is not superior to doxorubicin in terms of antitumor activity at the optimal, nontoxic doses, in vivo. Other analogues displaying 1,4 or 2,3 substitution with methyl groups or chlorine atoms or (2,3-a)benzo-derivatives with an extended aromatic ring system are less potent and less effective than 15a. The 40 -epi, 40 -deoxy, and 40 -O-methyl derivatives of 15b were also prepared [49]. The enantiomer 15d of idarubicin has been synthesized by a similar procedure and found devoid of significant bioactivity, in agreement with the stereochemical requirements for bioactivity of antitumor anthracyclines [50a]. However, enantiomeric daunorubicin shows, interestingly, unprecedented structural selectivity as it can discriminate and selectively recognize right- and left-handed DNA [50b]. A method of idarubicinone synthesis from biosynthetic daunomycinone has also been developed for industrial purposes [51]. A study concerning the relevance of the peri substitution in the anthraquinone chromophore for antitumor activity has involved both synthetic work and isolation of novel biosynthetic compounds. It was therefore of interest to assess the role of the anthraquinone chromophoric system as a simple electron-deficient planar system responsible for the formation of intercalation complexes with suitable electron-rich acceptors or, alternatively, as a redox reagent intervening in a catalytic mechanism leading to the generation of toxic oxygen radicals [25b]. Substitution of the 6-OH or of the 11-OH or both in 2a with a methoxyl strongly reduced or abolished bioactivity and stability of the DNA intercalation complex [52, 53]. The biosynthetic analogue 4-O-demethyldaunorubicin (carminomycin, 17a), showing three hydroxyl groups peri to the quinone carbonyls, is endowed with higher cytotoxicity than 2a and exhibits comparable activity in laboratory tests at significantly lower dosages [54]. In the 4-hydroxylated series, the daunorubicin isomer with a methoxy group at C-6 shows low affinity for calf thymus DNA but still significant activity in the mouse L1210 leukemia test, whereas the corresponding 11-methylated isomer is only marginally active [55]. 4-Demethyl-6-O-methyl-doxorubicin analogue 17b is of interest because of the exhibition of activity against experimental murine tumors comparable to doxorubicin (2b), notwithstanding a lower DNA binding affinity, and because of a reduced cardiotoxicity in vivo. The presence of the methoxy group at position 6 is thought to modify the geometry of the DNA complex albeit in a way that does not affect the molecular interaction determining the tumor-inhibiting properties [56]. 11-Deoxy analogues of antitumor anthracyclines were isolated from the cultures of different Streptomyces strains related to S. peucetius. These compounds are 10 times less potent on a weight basis in vivo, but the activity of 11-deoxydaunorubicin (18a) and of 11-deoxydoxorubicin (18b) at optimal doses in the P388 mouse leukemia system is comparable to that of doxorubicin [57, 58a,b]. In the 4-demethoxy series, 11-deoxy idarubicin was prepared by Umezawa et al. [59]. Activity in vitro and in vivo of the corresponding 14-hydroxylated compound is comparable to that exhibited by

9.3 Analogues with Modifications in the Anthraquinone Moiety

j225

doxorubicin. As for the 6-deoxy analogues, the condensation of 1,4-dimethoxy-2bromonaphthalene in the presence of butyl lithium with 19, itself obtained in six reaction steps from dimethyl cyclohexenedicarboxylate, afforded 6,7-deoxyidarubicinone that was converted to 6-deoxyidarubicin 15c through the same reaction steps as used in the synthesis of 15a. The compound shows a much higher IC50 value in HeLa cells compared to idarubicin (15a) [60a]. An improved regiospecific procedure allowed preparation of 6-deoxycarminomycin 17c [60b]. O

OH

O

O

OH

O

O

4

7 O

9 OH

1

R

O O

NH2 OH 15a: R =OH, R2=H 15b: R1=OH, R2=OH 15c: R1=R2=H 15d: as 15a, epi at C-7 and C-9 1

OH O

11 6

4

R

16a: R=H 16b: R=OH

S

MeO2C

S

OHC 19

O R2

OH

4 R1

1

OH O

OH R

O

O

OH

R2

R2

OH

O

7 O

OH O

O

O

NH2

NH2

1

17a: R =OH, R2=H 17b: R1=OMe. R2=OH 17c: R1=R2=H 17d: R1=H, R2=OH

OH 18a: R1=OMe, R2=H 1 18b: R =OMe, R2=OH 18c: R1=R2=H 18d: R1=OH, R2=OH

In conclusion, modification of the anthracenequinone chromophore has produced compounds that retain the bioactivity of the parent biosynthetic compounds (Table 9.2). Compound 15 (idarubicin) was selected for further development as a useful agent for the treatment of acute leukemias. It has been registered worldwide Table 9.2 Cytotoxicity in vitro on HeLa cells (LD50, ng/ml after 24 h exposure) and antitumor activity (AST%)a) against L1210 murine transplantable leukemia at optimal nontoxic doses (O.D., mg/kg body weight, treatment by the intraperitoneal route on day 1) of analogues modified in the chromophore moiety.

Compound

Daunorubicin (2a) Daunorubicin, 11-deoxy- (18a) Daunorubicin, 4-demethyl- (27a)b) Daunorubicin, 4-demethyl-11-deoxy- (18c) Idarubicin (15a)c) Idarubicin, 7,9-diepi- (15d)d) Idarubicin, 6-deoxy- (17c) Doxorubicin (2b) Doxorubicin, 11-deoxy- (18b) Doxorubicin, 4-demethyl-6-O-methyl- (17b) Doxorubicin, 4-demethyl-11-deoxy- (18d) a) b) c) d) e) f)

Cytotoxicity

Antitumor activity

LD50

References

O.D.

AST%

References

10 70 3 14 0.15 >200 17 7 100 16 6.5

[49] [57] [54b] [58] [49] [25c] [62b] [56] [57] [56] [57]

2.9 44 0.4 1.9 1 8.0 —e) 6.6f) 66f) 4.4f) 3.4f)

144 142 133 122 150 100 —e) 190 232 177 190

[49] [57] [54b] [58] [49] [25c] —e) [56] [57] [56] [57]

Average survival time of treated animals as percentage of untreated controls. Carminomycin. 4-Demethoxydaunorubicin. Idarubicin diasteromer. Not available. Murine transplantable P388 leukemia.

9 OH

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with the trademark ZavedosÒ [61]. Unlike the other clinically useful anthracyclines, idarubicin has significant oral bioavailability (in average 28%). The oral formulation has demonstrated efficacy in advanced breast cancer, non-Hodgkin’s lymphoma, myelodysplastic syndromes, and as first-line induction therapy of acute myelogenous leukemia [25e]. Methylation or deoxygenation of the peri phenolic groups afforded compounds with reduced activity in terms of efficacy and/or of potency in respect to the respective parent compounds. However, compound 17b deserves attention as it is endowed with a distinct and potentially advantageous biochemical and pharmacological behavior. A correlation has been established between cytotoxicity and DNA breaks induced in cultured cells by the related 6-deoxy-4-demethyldoxorubicin 17d, which shows a behavior similar to doxorubicin, in agreement with the role of topoisomerase II as the target of this class of antitumor agents [62a,b].

9.4 Analogues Modified on Ring A of the Aglycone

Esterification of doxorubicin (2b) at position 14 afforded compounds exhibiting bioactivity compatible with a role as prodrugs of 2b. However, doxorubicin octanoate appeared of interest because of its reduced cardiotoxicity in the rat [8]. A prodrug behavior was also demonstrated for some derivatives at the 13-carbonyl function, such as daunorubicin benzoylhydrazone (rubidazone), which was introduced in oncology as an alternative medication to daunorubicin because of lower cardiotoxicity [63]. O

OH

O

O

OH

O

O

OH

OMe O

R OH F

H OMeO

R

OH O

O

OH

OMe O O

OMe O

OH 23

OMe O

O

OH

22

NH2

20a: R=H 20b: R=OH O

OH OH

21a: R=H 21b: R=OMe

O OH

O

O

OH

O

OMe O

OH

OH F

O R OMe O

OH OMe

O

R

O

OH O

24a: R=OH 24b: R=F

O OH

NH2

25a: R=H 25b: R=OMe

9-Deoxy derivatives 20a and 20b [64], although able to form stable intercalation complexes with DNA, display only marginal antitumor activity [65]. One good reason for this behavior is destabilization, because of the absence of a hydrogen bond between OH-7 and OH-9, of the a conformation of ring A, a specific requirement of all bioactive anthracyclines. Moreover, according to the molecular model of the

9.4 Analogues Modified on Ring A of the Aglycone

daunorubicin–DNA complex, the 9-hydroxyl group points outside the double helix and might be involved in a specific interaction such as the one determining the stabilization of the topoisomerase II–DNA–drug ternary complex [66, 67]. The introduction of a fluorine at the a-position to the said hydroxyl as a substituent on ring A at C-8 and at C-10 and at C-14 was thought of interest in order to enhance the acidity of the 9-OH (or of the 14-OH). Racemic trans-fluoridrin 21a was obtained by total synthesis whereas, more expeditiously, (8S)-fluorodaunomycinone (21b) was obtained starting from 22, the product of the reaction of daunomycinone with 2,20 -dimethoxypropane in boiling benzene in the presence of p-toluenesulfonic acid, followed by epoxidation to 23. Opening of the epoxide ring to give 24a, then fluorination to 24b, and hydrolysis afforded 21b. Glycosidation of 21a with N-trifluoroacetyl-1,4-di-O-p-nitrobenzoyl-Ldaunosaminyl chloride in the presence of trimethylsilyl triflate allowed, after separation of the diasteromeric mixture and deprotection, to obtain (8S)-fluoroidarubicin 25a, whereas 8(S)-fluorodaunorubicin 25b was obtained from 21b. Both 25a and 25b exhibit antiproliferative properties against cultured tumor cells inferior to those of the nonfluorinated parent aminoglycosides. Compound 25b was eventually converted to (8S)-fluorodoxorubicin, albeit in low yields because of low reactivity of 25b toward electrophilic bromination at C-14 [68, 69]. The significantly lower cytotoxic potency exhibited by 25a compared to unsubstituted idarubicin is consistent with the b-conformation of ring A predominating the solution of 25a over the a-conformation typical of idarubicin and the other antitumor anthracyclines [69]. This is not the case with cis-fluoridrin (8R)-fluoroidarubicin (26), the epimer of 25a, as it keeps the same conformation in solution as the biosynthetic compounds. Compound 26 was obtained starting from the product of the Diels– Alder condensation of quinizarine with 2-(1-hydroxyethyl)-1,3-butadiene and this was tautomerized and was treated with N-bromosuccinimide and tetrabutylammonium dihydrogentrifluoride to give the desired trans 8-fluoro-9-bromo derivative 27 together with the 8-bromo-9-fluoro regioisomer. Compound 27 was then converted to 9,13-epoxide 28 with base. Acid treatment of 28 and oxidation at C-13 of the resulting 9,13-diol gave ketone 29a and this was converted to 29b by protection as a 13ethylidene ketal and bromination at C-7 followed by hydrolysis. Glycosylation of 29b followed by separation of the diastereomeric glycosides and deprotection produced 26.This compound shows inhibition of the growth of cultured tumor cell lines with ID50 close to the value shown by idarubicin [69]. Moreover, (8R)-fluoroidarubicin (26) displays a significantly greater activity than that of idarubicin against doxorubicin-resistant cell lines (Table 9.3) and is more efficacious than doxorubicin and markedly more so than idarubicin in inhibiting the growth of human ovarian carcinoma xenografts in immunodepressed mice. Interestingly, the compound displays a particularly high activity in stimulating topoisomerase II DNA cleavage in vitro [70]. The compound is a candidate for further preclinical development. The synthesis of 10-fluoroderivatives was carried out from the 9,10-epoxides of 40 epidaunorubicin or 40 -epiidarubicin, which were treated with triethylamine/hydrogen fluoride to give the approximately (1 : 1) mixture of, respectively, 30a and 30c, or 30b and 30d. As deduced from the activity data against different cultured tumor cell lines

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Table 9.3 Inhibition (IC50, ng/ml) of in vitro cultured tumor cells by idarubicin (IDR) and epimeric fluoro derivatives after 24 h exposure to the drug [70].

Tumor

Compound IDR (15a)

IDR, (8S)fluoro- (25a)

IDR, (8R)fluoro- (26)

5.3 3.0 2.8 10

35 22 14 60

3.5 1.8 5.0 9.0

Cervical carcinoma A431 Lung carcinoma H480 Ovarian carcinoma A2780 Ovarian Carcinoma A2780a) a) Doxorubicin resistant cell line.

recorded at the US National Cancer Institute, compounds 30b and 30d display similar antiproliferative spectrum and potency, but both were less potent than doxorubicin [70]. O

OH

O

O

OH

OH

O

OH O

OH

OH

O

O

OH

O OH F

F O

27

O 26

OH

Br F

OH F O

O

OH

O

28

OH R

29a: R=H 29b: R=OH

NH2

Synthesis of 14-fluorodoxorubicin started from 14-bromodaunomycinone by treatment with AgBF4 and dimethylsulfoxide to give fluoroderivative 31a. This compound was brominated to 31b whose reaction with AgBF4 afforded 31c. Glycosylation of 31c with 1-p-nitrobenzoyl-3,4-N,O-diallyloxycarbonyldaunosamine and trimethylsilyltriflate and treatment of the resulting protected glycoside with the AgBF4-DMSO reagent, followed by removal of the protecting groups, produced the two epimeric forms of 14-fluorodoxorubicin 32 [71]. O

OH F 10

R

O

OH

O

O

O

O

O

OH

O

R1 OH

F OH

OH R2 OMe O

O HO

OH

NH2

30a: R=OMe 30b: R=H 30c: R=OMe, epi at C-10 30d: R=H, epi at C-10

OH OH 1 31a: R =F, R2=H 31b: R1=F, R2=Br 31c: R1=F, R2=OH

OMe O

OH

OH O O

32 OH

NH2

In conclusion, synthetic work leading to modification of alicyclic ring A of antitumor anthracyclines has established the importance of this portion of the

9.5 Disaccharide Analogues

molecule as a scaffold projecting the C-9 side chain and the C-7 sugar moiety outside the putative DNA intercalation site to interact with the topoisomerase protein, thus stabilizing the binding of the enzyme to its substrate with the consequent formation of the DNA breaks that stimulate apoptotic cell death. Reducing the stability of the a-conformation of ring A, as in the 9-deoxy derivatives 20a,b or in the (8S)-fluoro compounds 25a,b, is strongly detrimental to bioactivity. On the other hand, although no advantage has been demonstrated for the presence of the electron-withdrawing fluorine at C-10, in vicinal position to the 9-hydroxyl group at C-9 as in compounds with structure 30b or 30d, (8R)-fluoroidarubicin (26) should be considered a lead compound for the synthesis of new pharmacologically improved anthracycline analogues.

9.5 Disaccharide Analogues

The synthesis of disaccharide analogues of the antitumor anthracyclines was justified because additional stabilization of the DNA–drug–topoisomerase complex, responsible for the DNA damage leading to the apoptotic response, might be obtained through a functional enrichment of the portion of the anthracycline molecule possibly involved in the said molecular interactions [72, 73]. On the other hand, compounds not possessing an amino group in the carbohydrate moiety directly linked to the aglycon are still active, even more active than the parent compounds, albeit at much higher dosages and the addition of a daunosamine residue could bring the active dose to more reasonable values by enhancing the stability of the corresponding DNA complex. Synthesis of disaccharide derivatives of daunomycinone and of idarubicinone in which the first sugar moiety linked to the aglycon was 2-deoxy-L-fucose or 2-deoxy-Lrhamnose and the second moiety was daunosamine or a related aminosugar was achieved by glycosylation of the aglycon with the appropriate protected and activated disaccharide [74]. Disaccharide intermediate 33a, obtained upon glycosidation of pmethoxybenzyl 3-O-p-nitrobenzoyl-L-fucoside with 1-thiophenyl-3-N-trifluoroacetyl4-O-p-nitrobenzoyl-L-daunosamine in the presence of iodonium dicollidine perchlorate (IDCP) was converted to 33b with ceric ammonium nitrate and finally esterified with p-nitrobenzoyl chloride to give 33c. Reaction of the latter with the appropriate aglycon in the presence of trimethylsilyl triflate and removal of the protecting groups gave desired 34a and 34b. Similarly, intermediate 35a, obtained from p-methoxybenzyl 3-O-p-nitrobenzoyl-L-fucoside, 1,4-di-O-p-nitrobenzoyl-3-N-trifluoroacetyl-Ldaunosamine, and trimethylsilyl triflate, was converted to 35b and this to 35c as above. Finally, glycosidation of daunomycinone or idarubicinone with 35c and deacylation of the products afforded, respectively, new aminodisaccharide derivatives 36a and 36b. According to biological tests, 34b was the most interesting compound in this series [75], and therefore the synthesis of the corresponding doxorubicin analogue was carried out by reacting 14-acetoxyidarubicinone with activated disaccharide 37 according to the standard procedures, followed by removal

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of protecting groups to afford 34c, the first disaccharide analogue of doxorubicin ever obtained. Compound 34c gave rise, especially at selected DNA sites, to a more marked topoisomerase II-mediated cleavage and a very significant activity against a spectrum of human tumors, such as breast, ovary, and lung cancer; xenografted in immunodepressed mice, the compound was found markedly superior to doxorubicin in terms of tumor growth inhibition and the number of disease-free survivors among treated animals. The high activity of 34c was tentatively related to the activation of p53independent apoptosis [76]. In tumor cell cultures, 34c and doxorubicin show similar cytotoxicity notwithstanding a significantly reduced rate of cell uptake of the former. When tested on an enlarged number of experimental tumors, 34c inhibited proliferation of 11 of 16 tumor types xenografted in nude mice. This result was found superior to that of doxorubicin (doxorubicin 5/16) when directly compared for 110 days after the first treatment in two different schedules of administration. Compound 34c induced phosphorylation, and therefore inactivation, of the antiapoptotic factor bcl-2 [76]. The generic name selected for 34c by the WHO is sabarubicin [77]. Comparative in vivo activity of sabarubicin and doxorubicin is presented in Table 9.4. O

OR

O

OH

O OpNBz O O NHCOCF3 pNBzO

33a: R=pMeOBn 33b: R=OH 33c: R=pNBz

O R2

OH R

O

OH

O

O

OH

OpNBz

O

R

O

4' OH pNBzO O 35a: R=pMeOBn 35b: R=OH 35c: R=pNBz OpNBz 4'' NH 2 O OH 1 2 OCO2All 34a: R =OMe, R =H O 34b: R1=R2=H O

OH

O

O

NHCOCF3

34c: R1=H, R2=OH

O

O O

1

OH

OR

O

NH2 OH

4' OH 36a: R=OMe 36b: R=H

37

NHCO2All

pNBzO

Stability of DNA complexes formed by sabarubicin (34c) in solution is similar to those of doxorubicin [78]. X-ray diffraction data of the sabarubicin–d(CGATCG) complex indicated, similar to other antitumor anthracyclines, intercalation of two molecules at each end of the DNA duplex in the CpG steps albeit with different conformations at each of the two binding sites. Interestingly, in one of the two sites a tight interaction takes place between the amino group and a guanine residue of a second DNA molecule determining a unique crystal structure [79]. Elongation of the glycosidic moiety has therefore determined the possibility, for the daunosamine residue, to exert productive interactions with other molecular targets in close proximity with DNA.

9.5 Disaccharide Analogues Table 9.4 Activity of anthracycline drugs on human tumors xenografted in nude mice.

Tumor

Schedule

Compound Doxorubicin (2b)

MX-1 (breast) A2780 (ovary) NCI-H460 (lung)

q7d  3b) q3/4d  5d) q7d  3 q3/4d  5 q7d  3 q3/4d  5

Sabarubicin (34c)

Dose

LCKa)

Dose

LCK

10 7 7 7 8.4 5.8

1.4 1.2 2.3 2.1 0.9 1.8

5  2c) 6 5  2c) 7.2 6  2c) 6

2.3 2.4 2.1 3.1 1.4 2.4

Inhibition of in vivo tumor cell proliferation by doxorubicin (2b) and by sabarubicin (34c) given at the indicated doses (mg/kg body weight) according to two different schedules of intravenous administration [76a]. a) Drug efficacy was assessed as the logarithm of cell kill (LCK) achieved in consequence of drug treatment according to the formula LCK ¼ (T
C)/DT  3.32 where T is the mean time (days) required for the treated tumors and C for the control untreated tumors to reach an established weight, and DT is the doubling time of control tumors. b) Treatment on days 1, 8, and 15. c) Two injections with a 1 h interval. d) Treatment on days 3, 4, 10, 11, 17, 18, 24, 25, 31, and 32.

Sabarubicin (34c) shows important deviations from doxorubicin in pharmacokinetic properties. A study in human patients indicated higher plasma levels and a much smaller volume of distribution compared to doxorubicin [80]. Also, binding to serum albumin, the value of the corresponding binding constant having been measured spectrophotometrically as 1.1  105 M
1, is much stronger than that shown by doxorubicin [81]. Sabarubicin induced less severe myocyte lesions than doxorubicin when repeated equimyelotoxic doses of the two compounds where given to rats and, interestingly, functional and histological effects did not progress after the end of treatment, as is the case with doxorubicin [82]. Phase I clinical studies in patients with solid tumors, who were administered sabarubicin by a short intravenous infusion given every 3 weeks or using a weekly schedule, indicated a better tolerability of sabarubicin compared to doxorubicin [83]. Phase II studies in non-small-cell lung cancer patients with advanced disease revealed two partial responses and eight minor responses in 22 evaluable patients [84]. Seven partial responses and one stable disease were recorded in a group of 10 patients with small-cell lung cancer [85]. Sabarubicin showed significant response rates in patients with advanced resistant ovarian cancer [86] and in hormone refractory prostate cancer [87]. Following substantially similar reaction sequences, the 400 -epimer of sabarubicin, 30 -deoxy disaccharides, and a trisaccharide have also been prepared [88]. The disaccharides showed cytotoxic activity comparable to that of sabarubicin, whereas the trisaccharide, although endowed with topoisomerase II-poisoning properties in

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the cell-free enzyme test, was less active, presumably because of a lower cell uptake rate. A similar pharmacological profile is shown by nonaminated disaccharide analogues that have been synthesized more recently following the chemistry already outlined for the disaccharide derivatives reported above [89].

9.6 Other Compounds

Annamycin, 30 -deamino-4-demethoxy-30 -hydroxy-20 (R)-iodo-40 -epidoxorubicin (38), has been synthesized and has demonstrated significant activity against the proliferation of resistant human tumors implanted in mice showing that analogues of antitumor anthracyclines in which the basic amino group was substituted with a hydroxyl group were active. These compounds were equally cytotoxic as doxorubicin in cultured cells, but when tested in tumor-bearing mice the optimal dose was found to be approximately one order of magnitude higher. Their interest resided, however, in their activity against doxorubicin-resistant cell lines and in the apparently reduced cardiotoxic effects at equitoxic doses compared to 2b. Annamycin has been developed for clinical trials as a third-generation anthracycline with a liposomal formulation in order to overcome the problem of its low solubility and also to obtain more favorable access to the target tumor tissue [90]. Amrubicin (39) is a totally synthetic idarubicin analogue in which an hydroxyl group replaces the amino group at C-30 and an amino group replaces the C-9 hydroxyl [91]. The compound, as well as the 13-dihydro metabolite, stabilized topoisomerase II–DNA complex [92] and exhibited high potency in in vitro and in vivo laboratory experiments [93, 94]. Amrubicin is registered in Japan and has been proved to be a clinically useful agent in the treatment of small-cell lung cancer [95]. The semisynthetic 30 -morpholino analogues represented by structures 40a,b belong to a definitely different class of anthracycline derivatives [96]. Compounds 40a,b and related derivatives exhibit significant cytotoxicity against both doxorubicinsensitive and -resistant cell lines. Most interesting, however, is the high potency shown by these derivatives, especially by 40b, that inhibits growth of cultured cells at picomolar concentrations and whose active dose in laboratory animals is in the order of a few nanomoles per kilogram of body weight. The results of biochemical investigations have led to the conclusion that the high cytotoxicity should be related to the conversion to reactive species able to form covalent bonds with cellular DNA and to induce the formation of cross-links [97]. It was then found that when the morpholino substitution was performed on the 40 -amino derivatives such as 14a–c, the 40 -morpholino analogues did not show the intense potency of the 30 derivatives, irrespective of the stereochemistry at C-40 [98]. Among the interesting variants of 30 (2-alkoxy-4-morpholino) analogues, the methoxy derivative 40c (nemorubicin) is the most potent in P388 leukemia-bearing mice, exhibits practically the same activity when the animals are inoculated with the doxorubicin-resistant P388 tumor line, and is under clinical investigation [99, 100].

9.7 Summary and Final Remarks O

OH

O

OH

O

OH

O

OH O HO

OH

O

OH

O OH OH

NH2 O

OH

O

O

O

OHI

OH

38

O

OH

OMe O

OH O O

HO

39 1

N

R1

O

R2

2

40a: R =R =H 40b: R1=CN, R2=H 40c: R1=H, R2=OMe

Mitoxantrone is an anthracenedione derivative often referred to as a pharmacological analogue of the anthracyclines. It is used in medical oncology for the treatment of breast cancer [101]. Finally, mention should be made of the bisanthracyclines, a novel structural type characterized by improved sequence specificity and high affinity toward DNA [102].

9.7 Summary and Final Remarks

Notwithstanding the addition, in the past 30 years, of a variety chemical entities to the oncological market, the original biosynthetic anthracyclines, especially doxorubicin, are still present in many drug combinations employed in established modalities of cancer treatment. In fact, doxorubicin is still one of the most widely used drugs in cancer chemotherapy. The successful therapeutic application of doxorubicin, despite its dose-limiting cardiotoxicity and the occurrence of resistance, has stimulated a considerable effort aimed at the development of better analogues by chemical modification of the parent drug or by total synthesis of new structurally related compounds. One early approach to new analogues consisted in the synthesis of related aminosugars, differing in the stereochemistry or in the structure from daunosamine, and coupling the same to the aglycone moiety, with the aim of modifying the pharmacokinetics and/or the metabolism of the natural glycosides. This approach afforded a number of compounds with a wide variation in bioactivity, out of which epirubicin (40 -epidoxorubicin) exhibited a significantly higher chemotherapeutic index when compared to doxorubicin at the clinical level and berubicin (40 -O-benzydoxorubicin) displays activity against brain tumors. A second approach was based on totally synthetic analogues. Idarubicin (4-demethoxydaunorubicin) is an important drug that was developed to the clinical stage because of its powerful activity in experimental leukemia models and reduced cardiotoxicity. Epirubicin is characterized by the detoxification to a glucuronide, whereas idarubicin is extensively reduced to a 13-dihydro derivative showing significant antitumor activity. These drugs are characterized as the second-generation anthracyclines.

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The search for the third-generation analogues with improved activity and lower side effects has been focused on selected modifications of the aglycone moiety (deoxy and fluoro derivatives), of the sugar moiety (deoxy and iodo derivatives), or to the elongation of the latter (disaccharide derivatives). (8R)-Fluoroidarubicin was found to be as potent as idarubicin in the inhibition of the growth of tumor cell lines in vitro. Interestingly, it displayed a marked cytotoxic activity against doxorubicin-resistant cell lines. It might become the first representative of new pharmacologically important 8R-fluoro anthracycline analogues. Totally synthetic sabarubicin has gone through full preclinical development and is undergoing phase II/III clinical trials in human tumor diseases such as lung, prostate, and breast cancer. Amrubicin is a new synthetic anthracycline so successfully designed that the compound has already been on the market in Japan for a number of years and is employed in the treatment of lung cancer. Alkylating anthracyclines such as nemorubicin, characterized by a (supposedly) completely different molecular mode of action, high potency, and practically an absence of cardiotoxicity at tolerated dosages, await a final assessment as potentially manageable drugs in a field in search of medications conjugating a better tolerability with improved selectivity in terms of biochemical targets.

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35 Penco, S., Angelucci, F., and

36

37

38

39

40

41

Arcamone, F. (1978) Daunomycinderivate, Verfahren zu ihrer Herstellung und ihrer Verwendung, Ger. Offen. 2,757,057, Filed December 21, 1977 and issued July 6. Arcamone, F., Penco, S., Redaelli, S., and Hanessian, S. (1976) Synthesis and antitumor activity of 40 deoxydaunorubicin and 40 deoxyadriamycin. J. Med. Chem., 19, 1424–1425. Cassinelli, G., Ruggeri, D., and Arcamone, F. (1979) Synthesis and antitumor activity of 40 -Omethyldaunorubicin, 40 -Omethyladriamycin, and their 40 -epianalogues. J. Med. Chem., 22, 121–123. Bargiotti, A., Cassinelli, G., Penco, S., Vigevani, A., and Arcamone, F. (1982) Synthesis of branched-chain aminodeoxyhexoses related to daunosamine (3-amino-2,3,6-trideoxy-Llyxo-hexose). Carbohyd. Res., 100, 273–281. (a) Bargiotti, A., Casazza, A.M., Cassinelli, G., Di Marco, A., Penco, S., Pratesi, G., Supino, R., Zaccara, A., Zunino, F., and Arcamone, F. (1983) Synthesis, biological and biochemical properties of new anthracyclines modified in the aminosugar moiety. Cancer Chemother. Pharmacol., 10, 84–89; (b) Kazerooni, R., Conrad, C., Johansen, M.J., Sakamoto, M., Fokt, I., Schroeder, C., Thapar, N., Meyer, C., Priebe, W., and Madden, T. (2008) Hematologic pharmacodynamics linked to the pharmacokinetics of berubicin, a blood–brain barrier penetrating anthracycline active against high grade glioma, in phase I/II clinical trials. Eur. J. Cancer, 6 (12 Suppl.), 187. Barbieri, B., Giuliani, F.C., Bordoni, T., Casazza, A.M., Geroni, C., Bellini, O., Suarato, A., Gioia, B., Penco, S., and Arcamone, F. (1987) Chemical and biological characterization of 40 -iodo-40 deoxy-doxorubicin. Cancer Res., 47, 4001–4006. Bargiotti, A., Caruso, M., Suarato, A., and Giuliani, F. (1987) 30 -Deamino-30 -

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hydroxy-40 -deoxy-40 -amino doxorubicin and related daunorubicin, US Patent 4,684,629, Filed March 24, 1986 and published August 4. Bargiotti, A., Suarato, A., Zini, P., Grandi, M., and Pezzoni, G. (1991) 30 -Deamino40 -deoxy-40 -amino anthracyclines, US Patent 4,987,126, Filed January 9, 1989 and issued January 22. Bargiotti, A., Grandi, M., Suarato, A., and Giuliani, F. (1991) 4-Demethoxy-40 amino-40 -deoxy-anthracycline derivatives, US Patent 5,045,534, Filed March 30, 1990 and issued September 3. (a) Ganzina, F. (1983) 40 -Epidoxorubicin, a new analogue of doxorubicin: a preliminary overview of preclinical and clinical data. Cancer Treat Rev., 10, 1–22; (b) Plosker, G.L. and Fauld, D. (1993) Epirubicin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in cancer chemotherapy. Drugs, 45, 788–856. Robert, J. (1994) Clinical pharmacokinetics of epirubicin. Clin Pharmacokinet., 26, 428–438. Ringenberg, Q.S., Propert, K.J., Muss, H.M., Weis, R.B., Schilsky, R.L., Modeas, C., Perry, M.C., Norton, L., and Green, M. (1990) Clinical cardiotoxicity of esorubicin (40 -deoxydoxorubicin, DxDx): prospective studies with serial gated heart scans and reports of selected cases. A cancer and leukemia group B report. Invest. New Drugs, 8, 221–226. Sessa, C., Calabresi, F., Cavalli, F., Cerny, T., Liati, P., Skovsgaard, T., Sorio, R., and Kaye, S.B. (1991) Phase II studies of 40 iodo-40 -deoxydoxorubicin in advanced non-small cell lung, colon and breast cancers. Ann. Oncol., 2, 727–731. Wong, C.M., Schwenk, R., Popien, D., and Ho, T.L. (1973) The total synthesis of daunomycinone. Can. J. Chem., 51, 466–473. Penco, S., Casazza, A.M., Franchi, G., Barbieri, B., Bellini, O., Podesta, A., Savi, G., Pratesi, G., Geroni, C., Di Marco, A., and Arcamone, F. (1983) Synthesis, antitumor activity and cardiac toxicity of

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Ripamonti, M.C., Rivola, G., Arcamone, F., Di Marco, A., Casazza, A.M., Soranzo, C., and Pratesi, G. (1980) New anthracycline glycosides from Micromonospora. II. Isolation, characterization and biological properties. J. Antibiot., 33, 1468–1473. (a) Arcamone, F., Cassinelli, G., Di Matteo, F., Forenza, S., Ripamonti, M.C., Rivola, G., Vigevani, A., Clardy, J., and McCabe, T. (1980) Structures of novel anthracycline antitumor antibiotics from Micromonospora peucetica. J. Am. Chem. Soc., 102, 1462–1463;(b) Cassinelli, G., Rivola, G., Ripamonti, D., Arcamone, F., Grein, A., Merli, S., Spalla, C., Casazza, A.M., Di Marco, A., and Pratesi, G. (1982) New anthracycline glycosides: 4-O-demethyl-11deoxydoxorubicin and analogues from Streptomyces peucetius var. aureus. J. Antibiot., 35, 175–183. Umezawa, H., Takahashi, Y., Naganawa, H., Tatsuta, K., and Takeuchi, T. (1980) Synthesis of 4-demethoxy-11-deoxy analogs of daunomycin and adriamycin. J. Antibiot., 33, 1581–1985. (a) Penco, S., Angelucci, F., Arcamone, F., Ballabio, M., Barchielli, G., Franceschi, G., Suarato, A., and Vanotti, E. (1983) Regiospecific total synthesis of 6-deoxyanthracyclines: 4-demethoxy-6deoxy-daunorubicin. J. Org. Chem., 48, 405–406; (b) Angelucci, F., Arcamone, F., Barchielli, G., Suarato, A., Vanotti, E., and Penco, S. (1984) Regiospecific total synthesis of 6-deoxyanthracyclines: 6deoxycarminomycin. Chem. Commun., 530–531. (a) Cersosimo, R.J. (1992) Idarubicin: an anthracycline antineoplastic agent. Clin Pharm., 11, 152–167; (b) Buckley, M.M. and Lamb, H.M. (1997) Oral idarubicin. A review of its pharmacological properties and clinical efficacy in the treatment of haematological malignancies and advanced breast cancer. Drugs Aging, 11, 61–86. (a) Capranico, G., De Isabella, P., Penco, S., Tinelli, S., and Zunino, F. (1989) Role of DNA breakage in cytotoxicity of

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doxorubicin, 6-deoxydoxorubicin, and 4demethyl-6-deoxydoxorubicin in murine leukemia P388 cells. Cancer Res., 49, 2022–2027; (b) Arcamone, F. and Penco, S. (1989) Relationship of structure to anticancer activity and toxicity in anthracyclines. Gann Monogr. Cancer Res., 36, 81–94. Pavlovsky, S., Fernandez, I., Palau, V., Santarelli, M.T., De Bellis, R., RuizArguelles, G., Brandalise, S., and Jimenez-Fonseca, E. (1991) Short report: combination of rubidazone and cytosine arabinoside in the treatment of first relapse in acute myelocytic leukaemia. Ann. Oncol., 2, 441–442. Penco, S., Franchi, G., and Arcamone, F. (1980) Antitumor deoxy-anthracyclines and use thereof, US Patent 4,218,440, Filed September 13, 1978 and issued August 19. Arcamone, F. and Penco, S. (1987) Molecular mechanisms of carcinogenic and antitumor activity, in Pontificiae Academiae Scientiarium, Scripta Varia, vol. 70 (eds C. Chagas and B. Pullman), Adenine Press Schenectady, pp. 225–241. Penco, S., Vigevani, A., Tosi, C., Fusco, R., Borghi, D., and Arcamone, F. (1986) Conformational flexibility of ring A in a series of substituted anthracyclines: 1 H-n.m.r. and quantum mechanical studies. Anticancer Drug Des., 1, 161–165. Gresh, N., Pullman, B., Arcamone, F., Menozzi, M., and Tonani, R. (1989) Joint experimental and theoretical investigation of the comparative DNA binding affinities of intercalative anthracycline derivatives. Mol. Pharmacol., 35, 251–258. Pasqui, F., Canfarini, F., Giolitti, A., Guidi, A., Pestellini, V., and Arcamone, F. (1996) Synthesis of ring A fluorinated anthracyclines. Tetrahedron, 52, 185–198. Lombardi, P., Animati, F., Cipollone, A., Giannini, G., Monteagudo, E., and Arcamone, F. (1995) Synthesis and conformational preference of novel 8-fluoroanthracyclines. Acta Biochim. Polonica, 42, 433–444. Animati, F., Arcamone, F., Bigioni, M., Capranico, G., Caserini, C., De Cesare,

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M., Lombardi, P., Pratesi, G., Salvatore, C., Supino, R., and Zunino, F. (1996) Biochemical pharmacological activity of novel 8-fluoroanthracyclines: influence of stereochemistry and conformation. Mol. Pharmacol., 50, 603–609. Berrettoni, M., Cipollone, A., Olivieri, L., Palomba, D., Arcamone, F., Maggi, C.A., and Animati, F. (2002) Synthesis of 14-fluorodoxorubicin. Tetrahedron Lett., 43, 2867–2871. Moro, S., Beretta, G.L., Dal Ben, D., Nitiss, J., Palumbo, M., and Capranico, G. (2004) Interaction model for anthracycline activity against DNA topoisomerase II. Biochemistry, 43, 7503–7513. Arcamone, F. (1985) Properties of antitumor anthracyclines and new developments in their application, Cain Memorial Award Lecture. Cancer Res., 45, 5995–5999. Animati, F., Arcamone, F., Berettoni, M., Cipollone, A., Franciotti, M., and Lombardi, P. (1996) New anthracycline disaccharides. Synthesis of Ldaunosaminyl-a(1-4)-2-deoxy-Lrhamnosyl and of L-daunosaminyla(1-4)-2-deoxy-L-fucosyl daunorubicin analogues. J. Chem. Soc., Perkin Trans. I, 1327–1329. Arcamone, F., Animati, F., Bigioni, M., Capranico, G., Caserini, C., Cipollone, A., De Cesare, M., Ettorre, A., Guano, F., Manzini, S., Monteagudo, E., Pratesi, G., Salvatore, C., Supino, R., and Zunino, F. (1999) Configurational requirements of the sugar moiety for the pharmacological activity of anthracycline disaccharides. Biochem. Pharmacol., 57, 1133–1139. Arcamone, F., Animati, F., Berettoni, M., Bigioni, M., Capranico, G., Casazza, A.M., Caserini, C., Cipollone, A., De Cesare, M., Franciotti, M., Lombardi, P., Madami, A., Mancini, S., Monteagudo, E., Polizzi, D., Pratesi, G., Righetti, S.C., Salvatore, C., Supino, R., and Zunino, F. (1997) Doxorubicin disaccharide analogue: apoptosis-related improvement of efficacy in vivo. J. Nat. Cancer Inst., 89, 1217–1223.

77 Arcamone, F. (2008) Sabarubicin, in

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90 Priebe, W., Skibicki, P., Varela, O.,

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Neamati, N., Sznaidman, M., Dziewiszek, K., Grynkiewicz, G., Horton, D., Zou, Y., Ling, Y.-H., and Perez Soler, R. (1995) Non-cross-resistant anthracyclines with reduced basicity and increased stability of the glycoside bond, in Anthracycline Antibiotics. New Analogues, Methods of Delivery and Mechanisms of Action, ACS Symposium Series 574 (ed. W. Priebe), American Chemical Society, Washington, DC, pp. 14–46. Ishizumi, K., Ohashi, N., and Tanno, N. (1987) Stereospecific total synthesis of 9-aminoanthracyclines: ( þ )-9-amino-9deoxydaunomycin and related compounds. J. Org. Chem., 52, 4477–4485. Hanada, M., Mizuno, S., Fukushima, A., Yoshikazu, S., Noguchi, T., and Yamaoka, T. (1998) A new antitumor agent amrubicin induces cell growth inhibition by stabilizing topoisomerase II–DNA complex. Jpn. J. Cancer Res., 89, 1229–1238. Yamaoka, T., Hanada, M., Ichii, S., Morisada, S., Noguchi, T., and Yanagi, Y. (1998) Cytotoxicity of amrubicin, a novel 9-aminoanthracycline, and its active metabolite amrubicinol on human tumour cells. Jpn. J. Cancer Res., 89, 1067–1073. Morisada, S., Yanagi, Y., and Noguchi, T. (1989) Antitumor activities of a novel 9-aminoanthracycline (SM-5887) against mouse experimental tumors and human tumor xenografts. Jpn. J. Cancer Res., 80, 69–76. Yana, T., Negoro, S., Takada, M., Yokota, S., Takada, Y., Sugiura, T., Yamamoto, H., Sawa, T., Kawahara, M., Katakami, N., Ariyoshi, Y., and Fukuoka, M. (2007) Phase II study of amrubicin in previously untreated patients with extensive-disease small cell lung cancer: West Japan Thoracic Oncology Group study. Invest. New Drug., 25, 253–258. Acton, E.M., Tong, G.L., Mosher, G.W., and Wolgemuth, R.L. (1984) Intensely potent morpholinyl anthracyclines. J. Med. Chem., 27, 638–645.

References 97 Acton, E.M., Wasserman, K., and

Newman, R.A. (1988) Morpholinyl anthracyclines, in Anthracycline and Anthracenedione-Based Anticancer Agents (ed W. Lown), Elsevier, Amsterdam, pp. 55–101. 98 Suarato, A., Angelucci, F., Bargiotti, A., Caruso, M., Faiardi, D., Capolongo, L., Geroni, C., Ripamonti, M., and Grandi, M. (1995) Synthesis and study of structure–activity relationships of new classes of anthracyclines, in Anthracycline Antibiotics. New Analogues, Methods of Delivery and Mechanisms of Action, ACS Symposium Series 574 (ed. W. Priebe), American Chemical Society, Washington, DC, pp. 142–155. 99 Bakker, M., Droz, J.P., Hanauske, A.R., Verweij, J., van Oosterom, A.T., Groen, H.J., Pacciarini, M.A., Domenigoni, L., van Weissenbruch, F., Pianezzola, E., and De Vries, E.G.E. (1998) Broad phase II

and pharmacokinetic study of methoxymorpholino doxorubicin (FCE 23762MMRDX) in non-small-cell lung cancer, renal cancer and other solid tumour patients. Br. J. Cancer, 77, 139–146. 100 Broggini, M. (2008) Nemorubicin, in Anthracycline Chemistry and Biology II, Topics in Current Chemistry, vol. 283 (ed. K. Krohn), Springer, Berlin pp. 191–206. 101 Durr, F.E. (1988) Biochemical pharmacology and tumor biology of mitoxantrone and ametantrone, in Anthracycline and AnthracenedioneBased Anticancer Agents (ed. W. Lown), Elsevier, Amsterdam, pp. 163–200. 102 Portugal, J., Cashman, D.J., Trent, J.O., Ferrer-Miralles, N., Przewloka, T., Fokt, I., Priebe, W., and Chaires, J.B. (2005) A new bisintercalating anthracycline with picomolar DNA binding affinity. J. Med. Chem., 48, 8209–8219.

Federico-Maria Arcamone

Professor Dott., Naxospharma, 70 Via Giuseppe Di Vittorio, 20026 Novate (Milano), Italy Federico-Maria Arcamone, a “Scuola Normale Superiore di Pisa” chemistry licentiate, has been active in the field of natural product chemistry and antibiotic research, becoming Head of Chemical Research at Farmitalia, Milan, in 1973. He is the author of over 200 research papers and over 100 patents, and has been an active lecturer in different countries, especially in the United States as well as in Italy. He has been honored with, among others, the gold medal of the “Accademia dei XL,” the Bruce Cain Award of the American Association for Cancer Research, the “Musajo” Award of the Italian Chemical Society, and the gold medal of the Italian Federation of Chemical Industries.

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10 Paclitaxel and Epothilone Analogues, Anticancer Drugs Paul W. Erhardt and Mohammad El-Dakdouki

10.1 Introduction

As described within the first volume of this series, analogue-based drug discovery (ABDD) represents a long-standing strategy that can contribute to the generation of new chemical entities (NCE), as well as play a lead role in the continued chemical and pharmacological evolution of clinically validated therapeutic paradigms [1]. It was further noted that ABDD represents a particularly useful approach toward ligandbased drug design by virtue of the inherent drug-like properties that already are present when a successfully marketed drug is deployed as a template for elaboration into new compounds [2]. Alternatively, as summarized in a recent review article [3], the even longer standing field of small molecule natural products (NPs) offers a different type of contribution that also remains highly important for modern drug discovery. Constituting the initial source for numerous of today’s medicines [3, 4], the “seemingly limitless structural [diversity of NPs] coupled with their novel biological properties [continues to] provide enormous inspiration” [5] to medicinal chemists during the pursuit of new drugs. It has thus become a logical extension to further elaborate new NP pioneer drugs by undertaking tandem ABDD strategies. Indeed, of about 1000 NCEs approved in the United States between 1981 and 2006, nearly 50 were unaltered NPs and nearly another 250 were second-generation NP derivatives primarily created to improve clinically relevant properties such as solubility and pharmacokinetic (PK) profile [4, 6]. The present chapter will highlight just one of such NP-prompted ABDD areas, namely, that associated with the pursuit of compounds that can overstabilize microtubules and thereby cause apoptosis of human cancer cells.

10.2 Discovery and Development of Paclitaxel

Several texts and reviews have provided excellent discussions about the fascinating historical aspects that eventually led to paclitaxel’s present status as an NP pioneer

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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HO

O

AcO

OH

(a)

O

OTES

(b) HO

HO

O

O AcO

HO

AcO

HO OBz

OBz O

10-DAB

O

Ph N

(c)

Ph O

AcO O

NH

Ph

O

OTES

O

OH

10

(d)

7

AcO NH

Ph

O

OTES

O

2'

O

Ph

13

O

OH

AcO

HO

O

Ph

O

OTES HO

OBz

1 Scheme 10.1 Semisynthetic route ultimately used to prepare commercial quantities of paclitaxel shown as 1 from 10-deacetyl-baccatin III shown as 10-DAB [18]. A similar semisynthetic route can be used to prepare docetaxel (shown later) by deploying the t-

AcO OBz

butoxycarbonyl version of the benzyloxy b-lactam and by utilizing other C-13 side-chain synthons. Conditions and yields: (a) Et3SiCl/ pyridine, 86%; (b) AcCl/pyridine, 86%; (c) nBuLi/THF then b-lactam/THF, 98%; (d) HF/ pyridine, 98%.

drug [7–10]. The following account offers just a quick backdrop for our subsequent ABDD-related discussions while paying tribute to at least some of the notable scientists who played key roles in that story. First obtained from the bark of the Pacific yew about 40 years ago by Wall and Wani [11] under an NP screening program being conducted by the US National Cancer Institute, the development of paclitaxel (1 in Scheme 10.1) initially was delayed by its low yield after a tedious isolation process coupled with the misconception that its anticancer properties stemmed from a common and thus rather ordinary biological mechanism. It was not until nearly 15 years later, after Horwitz and coworkers [12] demonstrated that 1 had a unique mechanism of action derived from an overstabilization of microtubules, when excitement about this compound began to mount. The rush to then move 1 into the clinic as rapidly as possible, and the need to provide huge supplies to accommodate its immediate success, quickly led to considerable concern about the future supply of 1 because removal of a yew’s bark leads to destruction of the tree. Amid extraordinary efforts by several investigators to synthesize 1 [7–10], it was eventually demonstrated by Potier et al. that 10-deacetyl-baccatin III (10-DAB in Scheme 10.1) that is obtained from the needles of both the Pacific and the European yew in a manner that is not destructive to the source, constitutes a key intermediate for a practical semisynthesis of what had by then become an extremely precious NP [13]. Although many notable chemists, as well as a modest effort in our labs

10.3 Clinical Success and Shortcomings of Paclitaxel

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O O Ph

OH OCH3

a

O

Br

2

Ph

OCH3

3

NH

c Ph

OH

Scheme 10.2 “Acyl migration” route developed and confirmed in our labs [14] to produce the C-13 side chain via a Sharpless dihydroxylation procedure [15]. (a) AD-a mixture, t-BuOH/H2O; (b) i. PhC(OCH3)3, cat. p-TsOH; ii. AcBr,
15  C; (c) i. NaN3, DMF; ii.

Ph

O

b OCH3 OCOPh

O

Ph

OCH3 OH

H2, 10% Pd/C; overall yield ¼ 25%. Although never progressing to a competitive commercial process, this chemical strategy has recently drawn interest in the design of prodrugs having increased selectivity for cancer cells [19].

(Scheme 10.2) [14] using a Sharpless dihydroxylation procedure [15], contributed significantly to this chemistry arena, Holton’s patented semisynthesis that takes advantage of a lithium alkoxide [16, 17] eventually was adopted for converting 10-DAB to 1 via reaction with the indicated b-lactam on a commercial scale according to Scheme 10.1 [18]. Today, the production of paclitaxel can be said to have “gone green” with its principal manufacturer Bristol-Meyers Squibb having recently been recognized by the US EPA for its development of a plant cell fermentation (PCF) method that has completely replaced chemical synthesis [20]. In the PCF process, calluses of a specific taxus cell line are propagated in an aqueous medium in large fermentation tanks under ambient temperature and pressure. The final product 1 is harvested as a crude extract from the plant cell cultures, purified by column chromatography and isolated by crystallization. Compared to the semisynthetic route across a period of 5 years of commercialization, the PCF process is estimated to have avoided the concomitant production of 71 000 pounds of hazardous by-product chemicals while eliminating 10 solvents and 6 drying steps that also equates to saving a significant amount of energy [20].

10.3 Clinical Success and Shortcomings of Paclitaxel

Initially studied for the treatment of ovarian cancer [21] and shortly thereafter for the treatment of breast tumors [22], paclitaxel quickly became regarded as one of the major breakthroughs in cancer therapy during the 1990s. Since that hugely successful introduction, 1 has maintained the status of being one of the most important anticancer drugs today wherein its use in other cancers and in combination with other agents is still being optimized. As with many other drugs and for cancer chemotherapeutic agents in general, however, 1 does have some significant shortcomings relative to its deployment in the clinic. The first of these to be noted was its extremely poor aqueous solubility. Because 1 is not orally bioavailable, it must be administered by intravenous infusion (i.v.) wherein the low aqueous solubility becomes problematic. To address the solubility issue, the most widely used formulation of 1 contains 50% CremophorÒ EL and 50% dehydrated ethanol [23].

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Unfortunately, the presence of such a high level of CremophorÒ EL can frequently trigger acute hypersensitivity reactions [24], and it likely contributes to the less than ideal, nonlinear pharmacokinetic behavior of 1 observed in both mice and humans [25, 26]. A second shortcoming pertains to a very common issue among many cancer chemotherapeutic compounds. Like most agents that rely upon the rapid division of cancer cells to achieve selective toxicity relative to healthy cells, the therapeutic margin for 1 is not as large as would be desired. Thus, side-effect toxicity, particularly associated with rapidly dividing normal cells, becomes dose limiting. Finally, it was noted that upon prolonged treatment, breast cancer cells can develop resistance to the effects of 1. When this last phenomenon couples with an already small window for safe (side-effect free) therapy, further treatment at higher doses essentially becomes precluded. One of the predominant mechanisms for this resistance stems from the overexpression of the P-glycoprotein (Pgp) efflux transporter. The significance of these last two shortcomings is further exemplified by the plots displayed in Figure 10.1 that conveys data generated from our laboratories. The narrow safety margin between paclitaxel’s effects on cancer cells and its effects on rapidly dividing healthy cells is reflected by the very close proximity of the first two log dose–response curves for causing growth inhibition (GI) of cell cultures. Likewise, the profound effect that Pgp-derived resistance can have upon paclitaxel’s effects is illustrated by the third curve that has shifted to the right in such a manner that it now requires nearly three orders of magnitude higher drug concentration to cause 50% inhibition of cell growth (GI50 value). Although 1 and its related taxanes appear to be 1.2 Paclitaxel Fraction of Control Growth

1.0 0.8

NCI/ADR-RES (Pgp+/ER-)

MCF12A (Normal)

MCF7 (Pgp-/ER+)

0.6 0.4 0.2 0.0

N = 130 for most concentrations N = 5-24 for two highest and two lowest concentrations

-0.2 10-11

10-10

10-9

10-8 10-7 Concentration (M)

Figure 10.1 Log dose–response curves for the action of paclitaxel on three different human cell lines grown in cell culture. Control cultures did not receive drug. MCF-7 (red curve with circled data points) is a nonresistant breast cancer. MCF-12A (green curve with square data points) is a healthy breast epithelial cell line that is

10-6

10-5

10-4

noncancerous and nonresistant. NCI/ADR-RES (blue curve with triangular data points) is a drugresistant ovarian cancer. Pgp refers to Pglycoprotein that is either absent (
) or overexpressed ( þ ) and ER refers to estrogen receptor responsiveness ( þ ) or absence (
).

10.4 ABDD Leading to Docetaxel

exceptional substrates for Pgp [27], the far from ideal profile of activity conveyed in Figure 10.1 is a common shortcoming that is displayed by many cancer chemotherapeutic agents in general. Underscoring this situation is the fact that the Pgp component is so prevalent and is so promiscuous in its substrate structural requirements that its overexpression leads to the broadly applicable phenomenon known as “multidrug resistance” or “MDR.”

10.4 ABDD Leading to Docetaxel

In addition to the compound supply aspects that became so critical for 1’s development and progression into the clinic, the subsequently huge success of paclitaxel fueled even further by its noted shortcomings, prompted a surge in basic research on several fronts. These fronts encompassed (i) pursuit of 1’s binding site in response to the finding that its interactionwith microtubules had been shown tobe stoichiometric [28]; (ii) elaboration of classical medicinal chemistry structure–activity relationships (SARs) including detailed assessments of 1’s conformational behavior in different environments as well as production of photoaffinity label derivatives for use as receptor probes [29]; (iii) numerous efforts directed toward addressing the specific shortcomings present in its clinical profile as discussed in the previous section; and (iv) pursuit of entirely new structures that might still act via 1’s unique mechanism when it was eventually shown that an overstabilization of microtubules prompts apoptosis at the G2 to M transition stage of the cell cycle during attempted replication. While (i)–(iii) largely represent areas directed toward “structural analogues” that preserve the desired biological effect (the so-called structural and pharmacological analogues), area (iv) represents a distinct case of pursuing “pharmacological analogues” [1] wherein the latter derive from a different molecular scaffold and different three-dimensional display of pertinent functional groups, that is, the pursuit of nonobvious molecular systems even beyond scaffold-hopping according to the patent terminology and jargon of today. Aspects of some of these areas will be further discussed in the following sections after using the remainder of this section to highlight the developments leading to 1’s first clinically successful analogue derived from ABDD, namely, docetaxel. The latter is shown immediately below as 2 wherein it should be apparent that 2 is both a close structural and pharmacological analogue of 1. O

HO O

NH

O

O

OH

10 7

2'

O

Ph

2 Docetaxel

13

O

OH

AcO

HO OBz

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Responding to the critical issues associated with producing and maintaining adequate supplies of 1, Potier et al. [30–32] had become immersed in the semisynthetic methods that can take advantage of 10-DAB (Scheme 10.1) including the possibilities that this route additionally affords for the preparation of analogues modified on the C-13 position side chain. Thus, this chemistry initially was explored via the Sharpless hydroxyamination procedure [33] while utilizing tertiary-butyl-Nchloro-N-sodiocarbamate. By this approach, the resulting t-butoxycarbonyl (BOC) group can be gently removed from what becomes the 30 -nitrogen atom then poised for further derivatization, including that of acylation with benzoylchloride so as to produce the paclitaxel form of the C-13 side chain. Presumably practicing a fundamental strategy of medicinal chemistry to test both synthetic intermediates and final target compounds, Potier et al. found that their BOC version of the C-13 side chain exhibited nearly twice the potency as 1’s original benzoyl version when coupled to 10-DAB and tested in a microtubule stabilization assay [34]. This fortuitous observation occurred whether or not the C-10 position hydroxyl group was acetylated, the latter alteration in itself having little effect on 1’s activity. Further exploration of other types of groups at this position did not show any added benefit over that seen with BOC, although a para-fluorobenzoyl seemingly came the closest upon reviewing the composite of their data. Capping these initial SAR studies, the increase in potency provided by the BOC group was then demonstrated even more convincingly during cell culture and especially during in vivo studies [35, 36]. Together, these results led to the selection of this analogue, namely, 2, for further study as a preclinical development candidate [37]. As studies progressed, the enhanced potency of 2 compared to 1 was maintained and even greater differences potentially related to additional mechanisms that could contribute toward overall efficacy were uncovered, such as 2 having about 100-times greater potency than 1 toward phosphorylation of bcl-2. The latter, in turn, may lead to inactivation of this oncoprotein and a concomitant release of its braking effect upon apoptosis [38]. Although these differences in inherent potency are real and apparently served as the main incentive to complete the drive of 2 toward the clinic, they should also be viewed amid the underlying fact that 1 already had more than enough potency to serve quite well in the clinic. Thus, the more practical attribute of docetaxel over paclitaxel was yet to be fully realized and it encompasses an important alteration at another site on these molecules, namely, that at the 10-position wherein 2 no longer bears an acetyl, ester-forming moiety on the common scaffold’s hydroxyl group. Beyond producing an increase in localized hydrophilicity, it is understandable why this effect was not readily discernible within the context of the overall molecular system that remains highly lipophilic and, seemingly just like 1, essentially “water insoluble” upon gross experimental observation. Ultimately, however, the formulations for 2 during early clinical studies were able to use 100% polysorbate 80 and thereby avoided the undesirable CremophorÒ EL altogether [23]. Thus, this initially subtle difference in solubility between 1 and 2 became a very real and practical improvement in the overall profile that again can be regarded as being quite fortuitous. Not so much for the increased potency but because of these later findings, we would agree with an assessment suggested at a much earlier point in this story by

10.5 Additional Structural Analogues Table 10.1 Clinical comparison of paclitaxel (1) and docetaxel (2) [10, 23, 38, 40].

Property Formulation Administrationa) Dosing protocol Frequency PK profile Disposition Term. Half-life Excretion Metabolism Toxicityb) MDR liabilityc)

1

2

50% CremophorÒ EL þ 50% anhydrous ethanol

100% Polysorbate 80

175 mg/m2 over 3 h Repeated every 3 weeks

60–100 mg/m2 over 1 h Repeated every 3 weeks

Nonlinear with dose 20 h, highly variable Hepatic about 80% unchanged C6 and C30 -p-phenyl hydroxylation, saturable >200 mg/m2 Yes

Linear with dose 12 h, highly variable Hepatic about 80% metab. Various metabolites, nonsaturable >150 mg/m2 Yes

a) As typically deployed for treating breast carcinoma by intravenous infusion. b) Typically observed as neutropenia. c) MDR is multidrug resistance, in this case resulting largely from overexpression of Pgp transporter to the extent that treatment becomes compromised.

some of the actual players, that paclitaxel and docetaxel are “not simply two of a kind” [23]. The precisely determined solubilities of 1 and 2 in water are 0.25 and 6–7 mg/ml, respectively [39]. Some of the other clinically relevant properties for these two drugs are compared in Table 10.1 that has been collated from several sources [10, 23, 38, 40]. Perhaps as a fitting summary to the docetaxel story, it can be offered that this unique example of NP ABDD, initiated by chemical supply issues and then conducted by practicing the venerable strategy to examine chemical intermediates for both SAR purposes and the distinct possibility of serendipity, did indeed produce a clinically relevant structural and pharmacological analogue. For in the end, no two pharmacological analogues are ever truly quite the same. And moving forward from that particular period in time, the latter certainly can be restated relative to today’s pharmacogenomic trend wherein personalized medicine approaches are being pursued to improve individual therapies. Within this context, however, the variability and metabolic differences between 1 and 2 that can be noted from Table 10.1 further emphasize the need to have even more of these types of otherwise seemingly very similar compounds available to the practitioner.

10.5 Additional Structural Analogues

An intense ABDD effort has occurred over the past 15 years using the initial knowledge afforded by the discoveries of both 1 and 2 as a starting point for further design strategies. In addition to enhancing our knowledge about SAR and the

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conformational details associated with how the taxanes interact with microtubules, numerous structural analogues have been obtained for which some have improved aqueous solubility, others have less problems with multiple drug resistance, and still others exhibit increased selectivity for cancer cells compared to healthy cells. In many cases, these ABDD efforts have encompassed prodrug strategies [41] into their final structural motifs. However, even though several of these promising compounds have progressed into various stages of clinical study, none has made it to the marketplace as of this writing. Thus, their further discussion is beyond the scope of this chapter. Readers interested in this huge body of research are encouraged to see the previously cited reviews [7–10] and a broader perspective highlighted within the context of ABDD that we will be publishing elsewhere [42].

10.6 The Pursuit of Microtubule-Stabilizing Pharmacological Analogues

ABDD aimed at discovering completely different structures that have the same mechanism of action as a marketed drug generally involve a random structural survey that deploys a high-throughput screen (HTS) to serve as an effective assay for that mechanism. This approach proceeds in a truly unbiased manner with regard to structural considerations and thus precludes any hint of overlap with structure-driven ABDD. Alternatively, a detailed knowledge about the receptor or enzyme-active site associated with the desired mechanism can be used as a blueprint for the ab initio design of another series of distinctly unique structures that can also reside in such a pocket while associating with a different array of contact amino acids. Finally, it is also possible to engage an allosteric site or some other dynamic aspect inherent in the desired mechanism at the molecular level that is different from the parent drug’s interaction, providing that the same consequence then occurs within that system so as to ultimately bestow the same pharmacological end point. Note, however, that even in these less frequent strategies, the pharmacological analogue would still be working within the immediate system and it would not be working at some separate step that is upstream or downstream along the same signaling pathway. By definition, the latter would lead to a new class of drugs rather than to an analogue even if the net pharmacological end points were still essentially the same. For the taxanes, we shall see that all pharmacological analogues to date have been discovered by the more common, random screening pathway. Perhaps ironic to the semantics of the categorizations just laid out, however, whenever one or more of such analogues are discovered in this manner, it immediately becomes a challenge for a medicinal chemist to “work-in-reverse” in an attempt to discern how such pharmacological analogues may be overlapping at the molecular level with the original agent’s distinct structure that was responsible for prompting the screening campaign in the first place. Thus, after reviewing the only marketed pharmacological analogue of 1 discovered to date, this section will discuss the ongoing attempts being made to discern their common interactions with microtubules.

10.6 The Pursuit of Microtubule-Stabilizing Pharmacological Analogues

The chemotherapeutic success of 1 coupled with its unique mechanism of action prompted researchers around the world to discover new cytotoxic natural products that can promote microtubule assembly and stabilization leading to apoptosis. Recent reviews reflect the high interest in both microtubule inhibitors and stabilizers [43–46], although we will focus herein upon only stabilizers [47]. In 1993, Reinchenbach and H€ofle isolated a new class of cytotoxic compounds as secondary metabolites from the fermentations of myxobacterium Sorangium cellulosum [48]. Based on their molecular structure, they named these molecules “epothilones.” In 1995, a group at Merck Research Laboratories confirmed that the newly discovered class of molecules possesses a paclitaxel-like mechanism of action [49]. Thereafter, the “rush was on” and several other natural compounds were subsequently found to stabilize microtubules such as discodermolide 3 [50–59], eleutherobin 4 [60, 61], laulimalide 5 [62–68], and peloruside A 6 [69, 70]. These last four agents are depicted in Figure 10.2. At this point, however, only an epothilone has made it to the actual marketplace, and so only their family is further discussed in the next section.

O O

N

H O

O

OH

N

OH O

NH2

OMe

H

HO

O OAc

HO

OH O

O

OH O

4 Eleutherobin

3 Discodermolide

H HO

O H

O H

O

H

OH

H

O

O O

O

HO

HO

OCH3

HO O HO OCH3

H3CO OH

5 Laulimalide

6 Peloruside A

Figure 10.2 Chemical structures of several pharmacological analogues discovered by HTS assays designed to uncover “paclitaxel-like” activity.

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10.7 The Epothilones

Of all the new paclitaxel-like microtubule stabilizers, the epothilone family has excited the most interest and is the only one to have sent a member to the marketplace. Although epothilones A (7) and B (8) initially were described as antifungal agents [48], these 16-membered macrolides later were shown to mimic all the biological effects of 1 both biochemically and in cell culture [49]. A variety of other epothilone-related structures, such as epothilones C 9, D 10, E 11, and F 12, were also isolated from the fermentations as minor components [71–75]. In general, these compounds are reported to be 30–50 times more soluble than paclitaxel (Figure 10.3) [76]. Compounds 7 and 8 promoted microtubule polymerization, hyperstabilized polymerized microtubules, and induced microtubule bundling. Competitive studies revealed that the macrolides acted as competitive inhibitors for [3 H]1 binding to microtubules. This is consistent with the interpretation that 7 and 8 compete for the same binding site as 1. These pharmacological analogues were roughly one order of magnitude more potent in cell culture models than was 1 with IC50 values in the suband low nanomolar range. The cytotoxicities of epothilones A, B, and D, and 1 in human cancer cell lines are summarized in Table 10.2. In key contrast to 1, 7 and 8 were active both on MDR cell lines and on paclitaxel-resistant cell lines [77, 78]. The promising anticancer activity of the epothilones prompted medicinal chemists to design and synthesize hundreds of epothilone analogues with improved cytotoxic and pharmacological properties. Such syntheses led to the SAR summarized in

R

O S N

S

13 12

22

OH 15 O 1

3

O

7 R= H 8 R= CH3

OH

OH

N

7

O

5

O

O

Epothilone A Epothilone B

OH

O

9 Epothilone C

O

R

S

S OH

N

HO

OH

N O

O O

OH

O

10 Epothilone D Figure 10.3 Various epothilone natural product family members.

O

OH

O

11 R= H Epothilone E 12 R= CH3 Epothilone F

10.7 The Epothilones

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Cytotoxicity (IC50) in nM of epothilones in comparison to paclitaxel (1) in human tumor cell lines (adapted from Ref. [72]).

Table 10.2

Cell line

Epothilone A

HCT116 (colon) SW620 (colon) SW620AD (PAC-resistant colon carcinoma subline) PC-3M (prostate) A549 (lung) MCF-7 (breast) MCF-7/ADR (breast) KB-31 (epidermoid) CCRF-CEM (leukemia)

2.51 NA NA 4.27 2.67 1.49 27.50 2.10 NA

Epothilone B

Epothilone D

Paclitaxel

0.32 0.1 0.3

NA NA NA

2.79 0.2 250

0.52 0.23 0.18 2.92 0.19 0.35

NA NA 2.90 NA 2.70 9.5

4.77 3.19 1.80 9105 2.31 NA

a) NA: Data not available.

Figure 10.4. SAR studies showed that the C-1-C-8 region is highly sensitive to structural modifications where even modest changes led to diminished activity. In contrast, the C-9-C-17 portion tolerated modifications and offered greater degrees of flexibility. The C-13 and C-15 stereochemistry must be “S,” while that of C-12 can be “R” or “S.” Binding affinity was enhanced when the epoxide ring was replaced by a cyclopropane ring or by a double bond, and when the C-21 methyl was replaced by a thiomethyl moiety. Based on extensive SAR studies of epothilones, a remarkable seven analogues, both natural and synthetic, have made their way to human clinical trials. Some of these analogues are still in phase I trials, while others have advanced to phase III and, just recently, one has now made it “all the way to the market place” (see discussion in next section). Figure 10.5 depicts the structures undergoing clinical study [79]. As mentioned above, the epothilones are competitive inhibitors of 1 in terms of binding to microtubules. Several investigators have tried to identify structural overlap

- Replacement of epoxide with a cyclopropyl group INCREASES binding affinity - Replacement of epoxide with a cyclobutyl group greatly DECREASES binding affinity - The C-12 stereochemistry can be R or S, while C-13 stereochemistry MUST be S - A methyl group at C-12 ENHANCES activity

O

- Aryl analogues ACTIVE - Replacement of C-21 methyl with thiomethyl INCREASES binding when the ring is thiazole

R

S OH

N O O

OH

C-15 stereochemistry MUST be S for maximum binding affinity

Figure 10.4 Epothilones SAR. (Adapted from Refs [44] and [80].)

O

Modest structural changes DIMINISH/ABOLISH activity

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O

O S

S OH

N

S OH

N

OH

N

O

O O

OH

HN O

O

Epothilone B (Patupilone, EPO906)

OH

O

O

OH

O

Aza-epothilone B (Ixabepilone, BMS-247550)

Epothilone D (KOS-862)

O S

O

S S OH

N

N

H2N

OH

N

O

O

O

OH

O O

OH

O O

Sagopilone (ZK-epo, ZK 219477)

OH

O

OH

Amino-epothilone B (BMS-310705)

O

Dehydelone (KOS-1584)

O S S

OH

N O O

OH

O

Methylthioepothilone B (ABJ879)

Figure 10.5 Epothilone analogues that were entered into clinical trials. (Adapted from Ref. [79].) Note that aza-epothilone B has already progressed to a market launch (also see further discussion in Section 10.8).

with 1 by developing a common pharmacophore, but different conclusions were drawn [81]. One of the first attempts to find a common pharmacophore was reported by Winkler and Axelsen in 1996 when no structural information about the drug binding site was yet available [82]. In their attempt to overlap both structures, the authors relied on the SAR available for 1 and the epothilones, and used molecular mechanics software to find regions of steric and functional similarities in the conformational space of the two molecules. They developed a 3D model (Figure 10.6) that superimposed 13 of the 15 ring carbon atoms of epothilone A and most of the side chain atoms onto corresponding atoms in paclitaxel. The C-1-C-3 and C-8-C-12 fragments and the thiazole ring in epothilone A were superimposed with the C-10 -C30 , C-2 benzoyl, and C-10 acetate of 1, respectively. The authors cautioned that other plausible conformations of both molecules could be superimposed and that the steric complementarity between 1 and epothilone was not ideal. Ojima et al. proposed a pharmacophore common for 1, nonataxel, the epothilones, eleutherobin, and discodermolide [83]. Nonataxel (13 in Figure 10.7) is a paclitaxel analogue that exhibits two- to eightfold higher activity against various cancer cell lines. The authors expected that the nonaromatic groups in nonataxel would allow

10.7 The Epothilones

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Figure 10.6 Schematic 2D representation of the model proposed by Winkler and Axelsen to define the structural similarity between 1 and the epothilones. (Adapted from Ref. [82].)

better mapping of the rings of epothilones. Comparing the 3D structure of nonataxel derived from limited 2D NMR studies and restrained molecular dynamics (rMD), with that of the template-fitted epothilone B, revealed excellent topological homology between the two structures. The C-1-C-6 portion of epothilone B corresponded to the southern hydrophobic surface of nonataxel, while the thiazole side chain overlapped with the BOC group at the C-30 -N of nonataxel. These relationships are depicted in Figure 10.7. The authors claimed that the proposed model accounts for a vast SAR data associated with the structural modifications of epothilone B such as the diminished/abolished activity observed upon epimerization at the C-3 position, reduction of the C-5 carbonyl group, or deletion of any moiety in the C-3-C-8 region. Moreover, the SAR studies indicated that the C-12-C-13 epoxide is not crucial for epothilone binding to microtubules based on the fact that analogues such as 12,13desoxyepothilone B (E and Z isomers) retained excellent tubulin binding activity. In the model proposed by Ojima et al., the epoxide oxygen pointed away from the overlapping structural terrain and is thus not essential for binding. He et al. proposed an alternative common pharmacophore for 1 and the epothilones based on SAR indicating that 2-m-azidotaxol had greater activity than paclitaxel, O

A

O O O

O

OH

O

NH O

A O O

OH HO O

13 Nonataxel

AcO O

R

S OH

N O O

OH

B

O

B

Figure 10.7 Labeled boxed regions represent areas of common overlap between nonataxel and the epothilones. (Adapted from Ref. [83].)

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and that even 2-m-azidobaccatin III (shown immediately below as 14) surprisingly exhibited paclitaxel-like activity, while Baccatin III and 2-p-azidobaccatin III did not [84]. These observations highlight the specificity and significance of the m-azido substitution in enhancing biological activity. 2-m-Azidobaccatin III was cytotoxic to different cancer cell lines although the cytotoxicity was 25–45-fold less than that of 1. AcO

O

OH

HO O AcO

HO O

O

N3

14 2-m-Azidobaccatin III

Analogue 14 induced all the morphological changes in microtubules typical for paclitaxel. It promoted tubulin polymerization in the absence of GTP, stabilized the polymerized microtubules against depolymerization by cold treatments, induced microtubule bundle formation in cultured cells, and caused cell cycle arrest at mitosis. Neither baccatin III nor 2-p-azidobaccatin III demonstrated such activity. More important, 14 competitively inhibited the binding of [3 H]1 to microtubules. This observation demonstrated that 14 and 1 bind to the same or overlapping sites on the microtubules, although the binding affinity of 14 was reduced compared to that of 1. The authors conducted molecular modeling studies to rationalize the significance of the m-azido substitution. In the proposed model, the C-2 benzoyl ring was positioned into a pocket formed by His-227 and Asp-224. Placing the azido group at the meta-position kept it in close proximity to the carboxylate of Asp-224, thus forming a new salt bridge as a consequence of the electrostatic interactions and resulting in enhanced binding of the molecule to the b-tubulin. Although the C-13 side chain is normally thought to be a requisite for 1 binding, the presence of the mazido group on the C-2 benzoyl ring of baccatin III apparently can compensate for the loss of the C-13 side chain and reestablish the binding affinity of the molecule. In contrast, a repositioning of the taxane ring would be required to fit the p-azido into the binding pocket formed by His-227 and Asp-224, thus inhibiting the interaction of 1 with the microtubules. Based on the observation that the C-13 side chain is not an absolute requirement for biological activity, the authors proposed a common pharmacophore for 1 and the epothilones in which the thiazole chain of the epothilones corresponded to the C-2 side chain of 1 and the macrocyclic ring of the epothilones overlaid with the taxane core. The proposed model is distinctly

10.7 The Epothilones

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O O

O NH

O

O

OH

S

O

OH

N

O O

OH HO

O

O OBz

O

OH

O

Figure 10.8 Common overlap of 1 and epothilone B as proposed by Giannakakou et al. (Adapted from Ref. [85].)

different from the one proposed by Ojima et al. where the thiazole side chain of the epothilones was superimposed with the C-13 side chain of 1. Giannakakou et al. have proposed a common pharmacophore for 1 and the epothilones based on the data collected from molecular modeling, mutations, and cytotoxicity assays [85]. To identify tubulin residues important for epothilone binding, two epothilone-resistant human ovarian carcinoma cell lines were isolated, each with a different point mutation: 1A9/A8 (b274Thr ! Ile) and 1A9/ B10 (b282Arg ! Gln). Of the two originally proposed pharmacophores, the one in which the methylthiazole side chain of epothilone B superimposed with the C-30 phenyl ring of 1 was more clearly discernible. This result is highlighted in Figure 10.8. Mutating Thr-b-274 to Ile in clone 1A/A8 had a greater impact on the binding of epothilone than 1. The hydrogen bond that existed between the C-7 OH of epothilone B and the Thr-b-274 was disrupted, while the hydrogen bond donors or acceptors at the C-7, C-10, and C-19 positions of 1 can form alternate hydrogen bonds to compensate for the disrupted hydrogen bond. This rationale is in agreement with the cross-resistance data and the in vivo polymerization studies. In addition, the b282Arg ! Gln mutation, sitting on the M-loop, directly affected the binding of taxanes and epothilones, and disrupted the lateral contacts between protofilaments. The data collected from drug sensitivity assays against a resistant paclitaxel-selected cell line containing a b270Phe ! Val mutation provided evidence for the preference of the proposed model. In this model, the thiazole side chain was placed in close proximity to Phe-270. As expected, the pyridine-containing epothilone experienced a 10-fold change in sensitivity compared to a threefold change observed with thiazole-containing epothilone B. The proposed pharmacophore accounted for a significant amount of SAR developed for epothilones and taxanes. The oxygen atoms of the oxetane ring of 1 and the epoxide of epothilones overlapped, and are located near a cluster of polar tubulin residues (273, 275, and 276) with the Thr-274 hydroxyl group in this hydrophilic area. It is known that epothilone B, 8, is 14-fold more potent than epothilone A, 7, with the only structural difference between the two molecules being the presence of a methyl group at C-12 of epothilone B. The proposed model positioned this methyl group in the vicinity of the hydrophobic side chains of Leu-273, Leu-215, Leu-228, and Phe-270, thus stabilizing favorable hydrophobic

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interactions and accounting for the enhanced potency. Such hydrophobic interactions also accounted for the fact that replacing the epothilone’s epoxide ring by a double bond enhanced in vitro tubulin polymerization because the electron-rich p cloud of the double bond can also act as a hydrogen-bond acceptor for a nearby water molecule. Capping this effort to equate paclitaxel and the epothilones, however, the most recent report by Downing and coworkers [86] ultimately concludes “that the longstanding expectation of a common pharmacophore is not met, because each ligand exploits the tubulin-binding pocket in a unique and independent manner” [86]. These investigators combined NMR, electron crystallography, and molecular modeling across a series of analogues. In the resulting model, the epothilones occupy the same binding site as paclitaxel, but this pocket is quite expansive and displays promiscuous binding with various ligands by exploiting contacts with different residues. For example, of the five oxygen-containing polar groups present on the epothilone macrocycle, only the C7-OH falls near the similar C7-OH of paclitaxel, “making this center the only notable common non-bonded contact for the two molecules” [86]. These authors further suggest that the observed promiscuity afforded by this pocket will likely apply to the binding of other ligands that occupy the taxane site on microtubules such as the discodermolide, eleutherobin, and sarcodictyin NPs. In summary, we find it interesting to note that the studies highlighted in this section of the chapter truly come full-circle with the intended theme for the overall text, namely, that there is a very special relationship between natural product chemistry and analogue-based drug design that simply cannot be denied. Initially serving as an inspiration for medicinal chemists in their quest for novel structures having new biological properties, such NP activities are typically followed with extensive ABDD campaigns directed toward both the practical improvement of an NP-derived pioneer drug and the generation of basic medicinal chemistry principles that can be ascertained from the composite of ongoing investigations. Furthermore, when there are significant clinical benefits from these efforts, this seamless continuum of ABDD research and development activities often returns to NPs in the pursuit of additional pharmacological analogues having diverse molecular templates, exactly as is the case with our paclitaxel discourse wherein all of the “next-generation” pharmacological analogues can be seen to have come from new NP surveys.

10.8 ABDD and Development Leading to Ixabepilone

As previously discussed, epothilones A and B are novel cytotoxic macrolides obtained from bacterial fermentation that represent pharmacological analogues of 1 by virtue of having deployed an assay for the latter’s microtubule-stabilizing properties to identify their interesting biological properties. Their mechanism is essentially the same as that of 1 except that they seem to interact with microtubules at different

10.8 ABDD and Development Leading to Ixabepilone O

O

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S OH

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a

S OH

N N3

O O

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8 Scheme 10.3 Synthesis of ixabepilone (15) from epothilone B (8) [86]. (a) Pd(PPh3)4, 10 mol%, NaN3, degassed THF-H2O, 45  C, 1 h, 65–70%; (b) PPh3, THF, 45  C for 14 h, then 28% NH4OH, H2O, 45  C for 4 h, or H2, EtOH,

OH

COOH

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c

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N NH

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15 PtO2, 50 wt%, 10 h, then an additional 25 wt% of PtO2, 10 h, or PMe3, THF-H2O, 25  C, 2 h, 53–89%; (c) DPPA, NaHCO3, DMF (2.5 mM), 4  C, 24 h or EDCl, HOBt, MeCN (0.03 M), 25–65%.

contact sites within what appears to be a rather promiscuous receptor for the taxanes and various other ligands. In contrast to 1, the epothilones are subject to different drug-induced resistance mechanisms including sensitivity to b-tubulin mutations. However, while they exhibit potent in vitro activity including action against MDR cancer cells, their in vivo activity was disappointing because of pharmacokinetic related issues. The latter involved poor metabolic stability and a short half-life due in part to the presence of the lactone moiety within the parent NP scaffold. Ixabepilone, 15, is an analogue of epothilone B, 8, wherein the lactone has been replaced by a lactam that is much less susceptible to hydrolytic metabolic pathways. As shown in Scheme 10.3, this ABDD-related transformation can be accomplished via a semisynthesis in three steps [87]. Compound 15 is comparable to 8 in all biochemical and in vitro tests including activity against three different MDR cancer cell lines. Like 1 and 2 (Table 10.1), it is a parenteral agent with a recommended dosage protocol of 40 mg/m2 administered via intravenous infusion over 3 h every 3 weeks [88]. Although its PK profile was considerably improved, 15 is subject to gradual metabolism primarily by CYP3A that can potentially lead to drug–drug interactions. It is mostly eliminated as various metabolites in the feces (65%) and urine (21%) after a terminal elimination half-life of 52 h [88]. Compound 15 is used to treat metastatic or locally advanced breast cancer. It is specifically indicated either for use in combination with capecitabine in patients who have failed treatment with an anthracycline such as doxorubicin and a taxane such as 1 or 2, or for use as a monotherapy in patients whose tumors are refractory (resistant) to anthracyclines, taxanes, and capecitabine. The most common adverse reactions (20% or higher) are peripheral neuropathy, myalgia, alopecia, and several other related conditions. The most common hematologic abnormalities (40% or higher) are neutropenia, anemia, and so on. Thus, the side-effect toxicity profile is not too dissimilar from that of 1 and 2 (Table 10.1). In summary for this section, it can be offered that ixabepilone (15) appears to “stand out” most over paclitaxel and docetaxel (1 and 2) by its ability to be deployed in MDR tumors, thus supplying another very useful weapon to the armamentarium that is needed to combat cancer, particularly in the latter’s advanced onslaughts. Interestingly, 15’s development reflects a case where an NP “pioneer drug” (namely,

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paclitaxel) served to prompt a “pharmacological-ABDD” campaign that eventually led to another NP family (namely, the epothilones) that then required additional ligandbased drug design to address a significant PK issue.

10.9 Conclusions

Figure 10.9 attempts to capture the breadth of ABDD activities and their associated contributions that have stemmed from the initial discovery of paclitaxel, 1. Starting with the US National Cancer Institute (NCI) Plant Program’s survey wherein Taxus brevifolia was first collected in 1962 and from which 1 was isolated by Wall and Wani at the Research Triangle Institute (RTI) in 1966, it was Horwitz et al.’s later demonstration in 1979 showing 1 to possess a unique mechanism of action involving stabilization of microtubules that finally ignited the preclinical development of this compound that, in turn, led to an approved US Investigational New Drug (IND) application/phase I clinical study in 1984 and an eventual market launch in 1993. Contributing to this flow of events was Potier et al.’s key finding that 10-deacetyl-baccatin III could be isolated from natural sources without the latter’s depletion and then used in an effective semisynthesis strategy so as to meet the critical supply issues associated with taxane compounds. A similar contribution came from Holton et al. for coupling a b-lactam version of the C-13-position side chain to 10-DAB in high yield by deploying a specific lithium salt form of the latter. Potier’s contributions also went on to produce the first ABDD-derived analogue of 1 to reach the market, namely, docetaxel, 2, after a European phase I study in 1990 that quickly led to a successful launch in 1995. Although 2 essentially has the same pharmacological profile as 1 with a bit more potency, it does have enough of an increase in aqueous solubility so that a less toxic formulation can be advantageously deployed in the clinic. Importantly and as also shown in Figure 10.9 across the middle of the panel, all aforementioned events came together during the 1990s to inspire a barrage of ABDD campaigns that, even today, are still contributing very significantly to the evolution of basic medicinal chemistry principles across several drug design constructs. The latter encompass contemporary topics such as (i) altering a drug candidate’s lipophilicity so as to provide a better PK profile or formulation partner, particularly by taking advantage of highly polar prodrug strategies; (ii) the interdisciplinary pursuit of efficacy-related SAR and ligand-receptor (active site) binding details using a composite of medicinal chemistry, molecular biology, computational chemistry, and X-ray and NMR spectroscopy approaches; (iii) delineation of these same types of details for Pgp using the same composite of interdisciplinary approaches, particularly with practical end points that can be directed toward improving a drug candidate’s oral bioavailability and distribution profile wherein the latter includes avoidance of Pgpderived MDR during chemotherapy and modulation for or against passage across the blood–brain barrier (BBB) depending upon what is desired; and (iv) enhancing a drug

1962 NCI 1966 Wall et al. 1979 Horwitz et al.

HO

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HO HO

OTES

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1989, 1992 Holton et al. semisynthesis

1980 Potier et al. semisynthesis O

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Paclitaxel 1984 IND/ Phase I 1993 Market Launch

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Docetaxel 1990 Eur./ Phase I 1995 Market Launch

(i) Water Solubilizing Prodrug Principles

(ii) SAR & Microtubule Binding Studies

(iii) Pgp SAR

Pharmacological Analog Studies

Improved Clinical Candidates

Improved Clinical Candidates

Novel Chemosensitizers

(iv) Selectivity Targeting Principles

Improved Clinical Candidates

O S OH

N HN O

OH

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Ixabepilone 2007 Market launch

Figure 10.9 Summary of significant events leading to the discovery of paclitaxel and for the subsequent ABDD activities that led to the immediate discovery of docetaxel and eventual discovery of ixabepilone. Also highlighted are the significant contributions from the ABDD efforts toward establishing general principles associated with (i) designing water soluble

prodrug moieties; (ii) understanding SAR and ligand binding that causes stabilization of microtubules; (iii) understanding SAR and substrate binding that causes inhibition or avoidance of Pgp; and (iv) designing simple and increasingly complex prodrug systems that can be used to achieve selectivity.

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candidate’s selectivity of actions and reducing side-effect toxicity by again using a composite of interdisciplinary approaches for which the derived molecular constructs are rapidly growing in sophistication and complexity as a workable knowledge about such things is expanded and again by particularly taking advantage of prodrug (conjugate) strategies. Despite this enormous amount of impressive efforts in basic research activities that have resulted in numerous clinical candidates, as well as in ongoing clinical trials for several selected agents, it becomes disappointing to discern that there is an already significant and still growing gap in time for the “second-generation” taxoids to truly arrive. We reserve this phrase to mean the next round of compounds that actually become launched into the marketplace after the arrival of the “first-generation” compounds, rather than using it for what we instead refer to as still being “drug wannabes” [89] wherein no matter how impressive their perceived attributes may be, the latter are only on route to the market. In this same context, we prefer to consider both 1 and 2 to represent a pair of first-generation agents with 1 constituting the true “pioneer drug” and “first-in-its-class.” Alternatively, given the theme of “NP-derived ABDD” for this book, it is certainly worthwhile to note that the emphasis placed upon screening for microtubule stabilizing agents as an integral part of the ABDD activities directly stemming from 1 has next led to yet another structurally novel NP that has indeed been entered into the marketplace. In our view, the latter is shown appropriately in Figure 10.9 as flowing directly from the ABDD-driven receptor binding studies (ii) so as to lead to what can be called a “pharmacological analogue.” From such a prestigious lineage it has ultimately taken the form of 15 and gone on to successfully achieve a market launch in 2007. Nevertheless, while considering 15 to thus represent a “second-generation microtubule stabilizer” (for which there could soon be additional NP arrivals also flowing down this same pathway of activities) and without purposefully trying to further confuse the semantics of this situation, we still very much look forward to seeing the arrival of the “second structural generation taxoids” into the marketplace (and perhaps a third and maybe even a fourth as well).

Acknowledgments

We thank Dr. Laurie Mauro, RPh, PharmD, for her assistance in obtaining the clinical related data, particularly the up-to-date information pertaining to the most recently marketed agent. We likewise thank both of the editors and the several reviewers for their useful comments that eventually led to the present, abbreviated version of our submission, particularly Drs. K.-H. Lee (University of North Carolina, USA) and Jo€elle Dubois (ISCN-CNRS, France). Finally, we thank the technical assistance of Mrs. Nicole R. Bearss, during the culling of this huge body of literature and, in particular, for her efforts during the assembly of the reference section.

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epothilones. Curr. Med. Chem. Anticancer Agents, 2, 123–148. Altmann, K.H. (2003) Epothilone B and its analogs: a new family of anticancer agents. Mini Rev. Med. Chem., 3, 149–158. Stachel, S.J., Biswas, K., and Danishefsky, S.J. (2001) The epothilones, eleutherobins, and related types of molecules. Curr. Pharm. Des., 7, 1277–1290. Fumoleau, P., Coudert, B., Isambert, N., and Ferrant, E. (2007) Novel tubulintargeting agents: anticancer activity and pharmacologic profile of epothilones and related analogues. Ann. Oncol., 18, 9–15. Altmann, K.H. (2005) Recent developments in the chemical biology of epothilones. Curr. Pharm. Des., 11, 1595–1613. Goodin, S., Kane, M.P., and Rubin, E.H. (2004) Epothilones: mechanism of action and biologic activity. J. Clin. Oncol., 22, 2015–2025. Kingston, D.G.I. (2004) What makes epothilones stick? Chem. Biol., 11, 153–155. Jimenez-Barbero, J., Amat-Guerri, F., and Snyder, J.P. (2002) The solid state, solution and tubulin-bound conformations of agents that promote microtubule stabilization. Curr. Med. Chem. Anticancer Agents, 2, 91–122. Winkler, J.D. and Axelsen, P.H. (1996) A model for the taxol (paclitaxel)/epothilone pharmacophore. Bioorg. Med. Chem. Lett., 6, 2963–2966. Ojima, I., Chakravarty, S., Inoue, T., Lin, S., He, L., Horwitz, S.B., Kuduk, S.D., and Danishefsky, S.J. (1999) A common pharmacophore for cytotoxic natural products that stabilize microtubules. Proc. Natl. Acad. Sci. USA, 96, 4256–4261. He, L., Jagtap, P.G., Kingston, D.G.I., Shen, H.J., Orr, G.A., and Horwitz, S.B. (2000) A common pharmacophore for Taxol and the epothilones based on the biological activity of a taxane molecule lacking a C-13 side chain. Biochemistry, 39, 3972–3978. Giannakakou, P., Gussio, R., Nogales, E., Downing, K.H., Zaharevitz, D., Bollbuck, B., Poy, G., Sackett, D., Nicolaou, K.C., and Fojo, T. (2000) A common pharmacophore for epothilone and taxanes: molecular basis for drug resistance conferred by

References tubulin mutations in human cancer cells. Proc. Natl. Acad. Sci. USA, 97, 2904–2909. 86 Nettles, J.H., Li, H., Cornett, B., Krahn, J.M., Snyder, J.P., and Downing, K.H. (2004) The binding mode of epothilone A on a,b-tubulin by electron crystallography. Science, 305, 866–869. 87 Borzilleri, R.M., Zheng, Z., Schmidt, R.J., Johnson, J.A., Kim, S.H., DiMarco, J.D., Fairchild, C.R., Gougoutas, J.Z., Lee, F.Y.F., Long, B.H., and Vite, G.D. (2000) A novel application of a Pd(0)-catalyzed

nucleophilic substitution reaction to the regio- and stereoselective synthesis of lactam analogues of the epothilone natural products. J. Am. Chem. Soc., 122, 8890–8897. 88 Hegde, S. and Schmidt, M. (2008) To market, to market-2007. Ann. Rep. Med. Chem., 43, 455–497. 89 Erhardt, P.W., Drug discovery, in Advanced Pharmacology (eds K.A. Bachmann, M. Hacker, and W. Messer), Elsevier, Oxford, UK, p. 175, (2009).

Mohammad El-Dakdouki

The University of Toledo College of Pharmacy, Center for Drug Design and Development, 2801 West Bancroft Street, Toledo, OH 43606-3390, USA and Research Associate, Michigan State University, Department of Chemistry, East Lansing, MI 48824-1322, USA Mohammad El-Dakdouki obtained a BS in chemistry from the Lebanese University in 2001 and an MS in chemistry from the American University of Beirut (AUB) in 2004. He then joined the Center for Drug Design and Development at the University of Toledo where he acquired a PhD in medicinal chemistry in 2009 for his work on the synthesis of agents targeting cancer cells while reducing MDR liability. At present, he is working as a Research Associate in the Department of Chemistry at the Michigan State University where his main interest is in the development of carbohydrate-based cancer vaccines. Paul W. Erhardt

Professor Medicinal Chemistry, Director, The University of Toledo College of Pharmacy, Center for Drug Design and Development, 2801 West Bancroft Street, Toledo, OH 43606-3390, USA Paul W. Erhardt received a PhD in medicinal chemistry from the University of Minnesota and did postdoctoral research at the University of Texas at Austin. He then worked for nearly 20 years in the pharmaceutical industry where he is credited with the discovery of esmolol. For the past 15 years, he has been at the University of Toledo where he is a Professor of Medicinal Chemistry in the College of Pharmacy and is serving as Director of the Center for Drug Design and Development.

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11 Selective Serotonin Reuptake Inhibitors for the Treatment of Depression Wayne E. Childers Jr. and David P. Rotella

11.1 Introduction

Selective serotonin reuptake inhibitors (SSRIs) are most widely used for the treatment of depression. Approved agents in this class include fluoxetine (1, ProzacÒ ), sertraline (2, ZoloftÒ ), paroxetine (3, PaxilÒ ), escitalopram (4, LexaproÒ ), the active isomer of racemic citalopram that was approved initially (see Section 11.3.2), and fluvoxamine (5, LuvoxÒ , approved in the EU only) (Figure 11.1). These compounds act by elevating levels of the neurotransmitter serotonin in regions of the brain that influence mood and represent a potentially useful symptomatic approach for the treatment of depression. Compounds in this class were introduced to clinical practice in the late 1980s and have remained important tools for pharmacological treatment of depression. SSRIs are recognized to have an improved side-effect profile compared to tricyclic antidepressants such as imipramine or amitriptyline [1]. SSRIs act by binding to the serotonin reuptake transporter on neurons to prevent the reuptake of serotonin from the synapse. This elevates levels of serotonin and produces beneficial effects on mood, among other actions. Even though all these compounds share a common mechanism of action, there are features about specific members of the group that allow researchers and clinicians distinguish between them. Paroxetine, fluvoxamine, and fluoxetine are relatively more potent inhibitors of the important drug-metabolizing enzyme CYP2D6, compared to escitalopram [2]. The potency and secondary receptor selectivity profiles of these agents are also different and can play a role in efficacy and adverse event profiles. Clinical studies have shown that patients who do not respond to the maximally tolerated dose of fluoxetine may respond to citalopram [3]. The array of different serotonin receptors, their role in the expression of SSRI activity, and the selectivity profile of each SSRI at these receptors differ [4]. Paroxetine and fluoxetine have activity at the norepinephrine transporter, and sertraline shows some dopamine transporter activity. In contrast, escitalopram has negligible affinity for other transporters. The affinity of SSRIs for other receptors, such as histamine, muscarinic, and 5-HT2C, is known to differ and may be expressed in the adverse event profile of the drug [4].

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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NHCH3

O

NHCH3

F3C Cl Cl 1 fluoxetine O

2 sertraline

NC

O

O

NH

F 3 paroxetine

O N(CH3)2

F 4 escitalopram

N

O

F3C

NH2

OCH3 5 fluvoxamine

Figure 11.1 Approved SSRIs.

The serotonin reuptake transporter is a protein with a comparatively broad tolerance for structural variation of inhibitors, as evidenced by the structural diversity of compounds in this class. As a result, and as shown in Figure 11.1, a wide range of structures show activity at this site. The opportunity for wide structural variation, coupled with the unmet medical need in the treatment of depression, has stimulated a substantial medicinal chemistry effort to discover compounds with this activity, as well as novel organic chemistry approaches to these important molecules. This chapter will summarize the published structure–activity relationships for each of the commercially available SSRIs, mechanism of action studies, preclinical and clinical pharmacology, and a survey of other potential uses for SSRIs.

11.2 Neurochemistry and Mechanism of Action

The exact mechanism of action for the antidepressant activity seen with SSRIs remains somewhat uncertain, but a number of biochemical events associated with SSRI treatment have been demonstrated [5]. As described above, SSRIs bind competitively to the 5-HT transporter and prevent the reuptake of 5-HT from the synapse. The acute result is an increase in synaptic 5-HT, which would be expected to exert beneficial effects in depression where there is significant evidence for serotonergic deficiencies. However, the increase in serotonin results in enhanced stimulation of presynaptic 5-HT autoreceptors and an acute decrease in the firing rate of serotonergic neurons, thus leading to a reduction in the secretion of 5-HT from presynaptic terminals. Ultimately, these presynaptic autoreceptors become

11.3 Preclinical Pharmacology

desensitized. Desensitization of terminal 5-HT1B/5-HT1D autoreceptors causes a greater amount of 5-HT to be released per impulse, while desensitization of somatodendritic 5-HT1A autoreceptors on the cell body leads to an enhanced firing rate by the neuron in general. Long-term administration of SSRIs also causes downregulation of the 5-HT transporter (but not the norepinephrine transporter). All these compensatory responses contribute to an eventual enhancement in serotonergic activity. The time needed for these compensatory responses to manifest themselves may, in part, explain the delayed onset of action in the clinical efficacy seen with SSRIs [6].

11.3 Preclinical Pharmacology 11.3.1 Sertraline

In the seminal paper describing preclinical characterization of sertraline, Koe and colleagues showed that the compound has an IC50 value of 58 nM as an inhibitor of serotonin reuptake in rat brain synaptosomal preparations and shows approximately 20-fold selectivity versus dopamine and norepinephrine transporters [7]. Dosedependent antagonism of the 5-HT depleting activity of p-chloroamphetamine (PCA), a compound that requires uptake into serotinergic neurons for activity, was demonstrated with an ED50 of 0.68 mmol/kg. This activity is six times more potent than chlorimipramine and 60-fold more active than amitriptyline. When 2 was acutely administered to rats at a dose of 32 mmol/kg subcutaneously (s.c.), brain levels of 5-HT were unchanged after 1 and 4 h, and a small increase in 5-HIAA (5-hydroxyindole acetic acid) levels was measured. Elevation of this serotonin metabolite suggested a decrease in 5-HT turnover as a result of inhibition of serotonin reuptake. Sertraline was 3–10 times more potent (depending on the end point) than fluoxetine or fluvoxamine in a serotonin potentiation model, and was much more potent than chlorimipramine. The magnitude of these effects was time dependent. At a dose of 5.6 mg/kg orally, maximal activity was observed at 1–3 h, with less of an effect using a 30 min pretreatment period, and no activity was observed with a 4 h pretreatment protocol. In a mouse forced swim test, sertraline demonstrated dosedependent reduction in immobility over a dose range of 3.2–56 mg/kg (s.c.). For comparison, in the same assay, fluvoxamine was inactive over a dose range of 3.2–100 mg/kg s.c. and fluoxetine reduced immobility at a dose of 56 mg/kg s.c. Lower doses of fluoxetine (down to 10 mg/kg) and doses up to 100 mg/kg were inactive. Sertraline did not reverse reserpine-induced hypothermia in mice. This activity is associated with norepinephrine and is consistent with monoaminergic reuptake selectivity exhibited by sertraline and some other SSRIs. In addition, at doses up to 32 mg/kg s.c., sertraline did not prevent oxotremorine-induced symptoms in mice. Some tricyclic antidepressants, such as amitriptyline, do ameliorate

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tremors, salivation, and diarrhea, consistent with known anticholinergic properties of the compound. Later work showed that over 4 days, sertraline downregulates b-receptors in rat brain, lowering receptor expression without changing affinity [8]. This property has been investigated as a preclinical biomarker for antidepressant activity [9]. 11.3.2 Escitalopram

The initial characterization of escitalopram was carried out on the racemic compound, citalopram [10, 11]. In a rabbit blood platelet model of serotonin reuptake inhibition, citalopram gave an IC50 of 14 nM and reversed serotonin depletion in rat brain with an ED50 (s.c.) of 0.8 mg/kg. It was essentially inactive as an inhibitor of norepinephrine uptake in vitro and in vivo. Like sertraline, citalopram was active in a mouse 5-hydroxytryptophan (5-HTP) potentiation model, with an oral ED50 of 1.2 mg/kg. In rats, citalopram potentiated tryptophan activity in the presence of an MAO inhibitor, at doses as low as 0.5 mg/kg orally. In this model, the tricyclic antidepressant chlorimipramine was less potent. Citalopram administration to MAO inhibitor-treated rabbits and dogs resulted in a dose-dependent, rapid, and substantial increase in body temperature. Serotonin-induced contraction of isolated rat fundus strips was potentiated, as shown by leftward shift in dose–response curves in the presence of a fixed concentration of the SSRI. Compared to several tricyclic antidepressants, citalopram showed significantly less potent anticholinergic and antihistaminergic activity in isolated guinea pig ileum tissue strips. For example, chlorimipramine showed an IC50 of 0.62 mM for anticholinergic activity, while citalopram showed an IC50 of 7.2 mM. When the separate enantiomers of citalopram were studied, it was observed that the (S)-enantiomer possessed essentially all of the serotonin reuptake inhibition activity of the racemate [12]. The (S)-isomer has an IC50 of 1.5 nM and the (R)-isomer shows an IC50 of 250 nM for SSRI activity in vitro using a rat brain synaptosome preparation, and the (S)-isomer (eutomer) retained the selective monoamine transporter profile observed with the racemate. In this same assay, the racemate has an IC50 of 1.8 nM. In a 5-HTP model, the (R)-isomer (distomer) was inactive and escitalopram showed an ED50 (s.c.) of 0.55 mg/kg in mice. This value is within twofold of the oral ED50 of the racemate in the same assay (vide supra). Subsequently, it was shown that the racemate and (S)-enantiomer show approximately twofold different potencies for inhibition of binding of ½125
I-RTI-55, a cocaine analogue employed to study binding to the serotonin transporter [13]. These and other observations stimulated more detailed studies of the potential effect of the (R)-isomer on the activity of escitalopram. Mørk and coworkers demonstrated that the (R)-isomer inhibited the increase in extracellular serotonin in the frontal cortex of freely moving rats induced by escitalopram as measured by microdialysis. Escitalopram (dose range 1–3.9 mg/kg s.c.) produced a greater maximal increase in extracellular 5-HT levels, compared to the racemate (2–8 mg/kg s.c.)

11.3 Preclinical Pharmacology

in the same brain region. The (R)-isomer was inactive in this assay. A pharmacokinetic interaction was ruled out as a potential explanation, based on measurement of comparable levels of escitalopram administered with and without the (R)-isomer [14]. At the receptor level, it was shown that escitalopram binds with high affinity to an allosteric site on the serotonin transporter and reduces the dissociation rate of [3 H]-escitalopram as a function of concentration. The (R)-isomer affects the binding of [3 H]-escitalopram in a similar manner but with an approximately threefold less effect on dissociation rate. This suggests the distomer has appreciable affinity for the allosteric site and may influence the binding of escitalopram when the racemate is administered [15]. In a variety of behavioral models, citalopram and escitalopram differ by approximately fourfold in potency [16]. In a rat resident intruder model, citalopram and escitalopram show ED50s (s.c.) of 1.3 and 0.3 mg/kg, respectively, and in a mouse black/white two compartment box model, the minimally effective dose (MED) (s.c.) of escitalopram is 0.5 mg/kg, while the racemate showed an MED of approximately 2 mg/kg. This difference in preclinical models extends to potency in humans (see Section 11.5). 11.3.3 Fluvoxamine

The first formal publication on fluovoxamine (as a maleate salt) compared the compound’s in vitro and in vivo effects with those exhibited by tricyclic antidepressant agents [17]. Fluvoxamine was less potent at inhibiting serotonin uptake into guinea pig platelets (IC50 400 nM) than chlorimipramine (IC50 6.3 nM) but essentially equipotent with imipramine (IC50 1.6 mM) and more potent than desmethylimipramine (IC50 12.5 mM). However, the compound was significantly more potent at inhibiting serotonin uptake into rat synaptosomes (Ki 84 nM) than either imipramine or desmethylimipramine (Ki 230 and 1200 nM, respectively). These comparisons were later extended to other tricyclic agents and serotonin uptake inhibitors (such as fluoxetine, femoxetine, and zimelidine), where fluvoxamine was equipotent to or more potent than the various drugs examined at inhibiting serotonin uptake into rat synaptosomes [18], with the exception of paroxetine, which was more potent than fluvoxamine [19]. In vitro, fluvoxamine showed around 180-fold selectivity for serotonin uptake over norepinephrine and dopamine uptake [18, 19]. In comparison, imipramine and fluoxetine were less selective (NE/5-HT 0.65 and 120, respectively), while paroxetine was more selective (NE/5-HT 320). Fluvoxamine was over 30 times less potent than imipramine at antagonizing norepinephrine-induced contractions in isolated rat vas deferens. The compound shows little or no affinity for adrenergic a1, a2, and b receptors, serotonin 5-HT2 receptors, dopamine D2 receptors, and muscarinic cholinergic receptors [19]. In vivo, fluvoxamine demonstrated effects that are associated with serotonin uptake [17]. The compound inhibited serotonin uptake into rat hypothalamus synaptosomes ex vivo after oral administration (ED50 23 mg/kg) and maintained its

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selectivity for serotonin uptake versus norepinephrine uptake (ED50, norepinephrine uptake >30 mg/kg). At a dose of 25 mg/kg, i.m. twice daily, it significantly inhibited serotonin release induced by either H75/12 or H77/77, as well as probenecidinduced serotonin turnover in rat brain. Fluvoxamine was 2–10 times more potent than imipramine, chlorimipramine, and desmethylimipramine at potentiating the serotonin-like effects of 5-hydroxytrpytophan in mice, and it also potentiated the serotonin-like effects of pargyline, a property not shared by those tricyclic antidepressants. A 40 mg/kg oral dose of fluvoxamine was also able to inhibit the reserpineinduced lowering of the pentylenetetrazole seizure threshold by approximately 50%. In contrast, fluvoxamine was inactive or only weakly active at blocking tetrabenazine-induced ptosis in mice and tetrabenazine-induced ptosis and compulsive hyperactivity in rats [18], activities thought to be associated with norepinephrine uptake [20–22]. Following the initial report on fluvoxamine, a number of other publications have appeared that support the compound’s profile as a selective serotonin uptake inhibitor. These data were reviewed by Palmer and Benfield in 1994 [23]. 11.3.4 Fluoxetine

Fluoxetine first appeared in the literature in 1974 as LY110140 (in the form of a hydrochloride salt) [24]. The compound inhibited serotonin uptake into rat synaptosomes (Ki ¼ 52 nM) and was nearly 200-fold selective for serotonin compared to the uptake of norepinephrine or dopamine. Fluoxetine displayed similar potency and selectivity for serotonin to that seen with chlorimipramine and was more potent and selective than imipramine and nortriptyline. In ex vivo studies, a 28.5 mmol/kg i.p. dose of fluoxetine inhibited uptake of serotonin into rat synaptosome preparations by 56%. That dose of fluoxetine did not significantly alter norepinephrine uptake either in brain or in heart preparations. Since that initial report, a number of papers have appeared in which fluoxetine has been compared with various antidepressant compounds. These data have been summarized in a 1995 review by Lilly researchers [25]. Compared to other SSRIs, fluoxetine was found to be roughly as selective for serotonin versus norepinephrine uptake as femoxetine and zimelidine, and less selective than paroxetine, citalopram, and indalpine. Fluoxetine also inhibits the uptake of serotonin in rat and human platelets both in vitro (Ki ¼ 70 nM and 54 nM, respectively) and ex vivo [26]. In fact, ex vivo uptake into human platelets was used as a biomarker for fluoxetine in phase I clinical studies [27]. Fluoxetine binds reversibly to a recognition site on the serotonin transporter. Saturable binding to rat cortical membranes occurred at concentrations between 0.5 and 1 nM of [3 H]-fluoxetine, with a Kd value of 2.9 nM and Bmax of 800 fmol/mg protein [28]. The binding is sodium-dependent, with optimal binding occurring at the physiological concentration of sodium (120 mM). The binding of fluoxetine and other SSRIs correlated with serotonin uptake (r ¼ 0.88; P < 0.001) [25]. The correlation between binding and norepinephrine uptake for these compounds was poor (r ¼ 0.21). Treatment of rats with the selective serotonergic neurotoxin p-

11.3 Preclinical Pharmacology

chloroamphetamine produced a parallel reduction in serotonin uptake and fluoxetine binding in various rat brain regions [29]. Fluoxetine has little affinity for a number of neurotransmitter binding sites, including a1-, a2-, and b-adrenergic, histamine H1, muscarinic cholinergic, opiate, and dopaminergic [30]. Similarly, fluoxetine was selective for the serotonin transporter over serotonergic receptors, including 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, and 5-HT3 [31]. Fluoxetine did show some affinity for rat 5-HT2C receptors (Ki ¼ 270 nM), although it has been reported that the compound is a more potent ligand for cloned rat receptors than for cloned human receptors [32]. Both agonist [33] and antagonist [34, 35] activities have been reported for fluoxetine in native (astrocyte) and clonal cellular systems. A growing body of evidence implicates the 5-HT2C receptor in depression [36, 37]. However, the role of fluoxetine’s 5-HT2C affinity in its antidepressant activity, if any, remains unclear. Fluoxetine was found to be active in a number of models used to predict antidepressant activity in humans [38]. Many of these models have been developed to introduce environmental factors that are thought to be similar to those associated with depression in humans, such as helplessness, despair, and isolation. Other models, such as olfactory bulbectomy-induced hyperactivity, simply introduce behavioral changes that are modified by administration of known antidepressant agents. A summary of fluoxetine’s activity in various animal models of depression is presented in Table 11.1. 11.3.5 Paroxetine

The preclinical pharmacology of paroxetine has been extensively reviewed [42–44]. Paroxetine remains one of the most potent SSRIs described to date and its tritiated form is often used as a standard ligand in serotonin transporter binding studies. One of the original papers on paroxetine’s preclinical pharmacology [45] demonstrated the compound’s ability to potentiate the hypermotility and anticonvulsant effects of 5-HTP, a precursor to serotonin. Paroxetine was more potent than either fluoxetine or zimelidine in these models; the tricyclic agents imipramine, chlorimipramine, and protriptylene were poorly active or inactive at oral doses up to 100 mg/kg. Paroxetine’s selectivity for 5-HT uptake over NE uptake was confirmed in a second report by demonstrating its ability to reverse p-chloroamphetamine-induced hypermotility (a 5-HT uptake-associated effect). The compound had no effect on H77/77-induced hypermotility, an effect associated with NE uptake [46]. Paroxetine demonstrated

Table 11.1 Activity of fluoxetine in animal models of depression.

Animal model Learned helplessness Forced swim Olfactory bulbectomy

Species

Effective dose(s) mg/kg

References

Rat Rat Rat

2, 4 (i.p.) 80 (i.p.) 10, 30

[39] [40] [41]

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potent inhibition of 5-HTuptake into rat brain synaptosomes (Ki 1.1 nM) and roughly 300-fold selectivity for 5-HT uptake versus NE uptake [47]. According to these data, paroxetine is more selective than fluvoxamine, fluoxetine, and zimelidine but less selective than citalopram. This kind of potency and selectivity were originally demonstrated in binding studies in rat cortex preparations [48] but have since been confirmed in numerous rat and human in vitro and ex vivo systems [42]. Paroxetine inhibits uptake of 5-HT into blood platelets of rhesus monkeys, healthy human volunteers, and, more recently, patients with treatment-resistant depression, although no significant correlation was seen between changes in platelet serotonin levels and Hamilton depression rating scale scores [43]. Like most SSRIs, paroxetine has little affinity for most postsynaptic neurotransmitter receptors, including adrenergic, D2-, 5-HT1-, 5-HT2-, and H1-receptors. It does possess more muscarinic cholinergic affinity than other SSRIs (Ki for displacement of [3 H]-quinuclidinyl benzilate 89 nM). This activity did not seem to translate into overt cholinergic side effects in the clinic [49], although there is some evidence that paroxetine may possess a greater potential for cholinergic liability than other SSRIs [50]. Paroxetine has a prolonged effect on serotonin uptake in vivo. In mice, 75% inhibition of 5-HT uptake was still present 24 h after a single intraperitoneal dose of 10 mg/kg [51]. In that study, the selectivity for 5-HT uptake versus uptake of NE and DA was 500-fold and 2500-fold, respectively (based on plasma concentrations). There was no significant effect on NE uptake in humans [52]. Paroxetine caused no significant alterations in response to administration of tyramine. In contrast, a 50 mg/kg dose of amitriptyline resulted in a significant increase in the dose of tyramine required to elicit a response. Paroxetine demonstrated efficacy in some animal models of depression. It reduced the number of escape failures in a rat learned helplessness model when given orally at 15 or 30 mg/kg on 3 consecutive days prior to testing [53]. Paroxetine reduced hyperactivity and rearing behavior in olfactory bulbectomized rats [54]. Conflicting reports have appeared in the literature concerning paroxetine’s activity in the forced swim test, with it being described as both active [55] and inactive, despite attenuating stress-induced 5-HT turnover in both amygdala and cortex [56]. In a few reports, paroxetine reduced immobility in the tail suspension test in gerbils [57] and mice, although species differences may affect the murine response [58].

11.4 Medicinal Chemistry 11.4.1 Sertraline

By the mid 1970s, a diverse range of structures had been studied for their ability to inhibit reuptake of neurotransmitters in the brain [59–61]. As part of an effort to better understand molecular properties that contribute to this activity, a series of conformationally restrained compounds was tested as reuptake inhibitors of cate-

11.4 Medicinal Chemistry

NHCH3

H3C

CH3

*

O

j277

OCH3 *

O NHCH3

*

N CH3 8

*

6

7

NH2

Figure 11.2 Early analogues of monoamine reuptake inhibitors.

cholamines and serotonin in rat brain synaptosomal preparations [62]. This group of compounds included fluoxetine and analogues of sertraline (6), escitalopram (7), and paroxetine (8) (Figure 11.2). All these molecules demonstrated submicromolar activity as serotonin reuptake inhibitors, with the exception of 7, which showed a preference for norepinephrine (IC50 0.011 mM) and weaker activity as a 5-HT reuptake inhibitor (IC50 7.4 mM). The sertraline derivative 6 inhibited dopamine (IC50 0.15 mM), serotonin (IC50 0.84 mM), and norepinephrine (IC50 0.018 mM) reuptake. The phenylpiperidine template 8 related to paroxetine demonstrated stronger inhibition of 5-HT reuptake (IC50 0.22 mM) compared to dopamine or norepinephrine. These results led Koe to suggest that the distance and relative orientation between the basic nitrogen atom and phenyl group (highlighted with  in Figure 11.2) are key determinants of serotonin reuptake inhibition. Other structural features such as added steric bulk and stereochemistry modulate potency and can influence selectivity. Furthermore, if all these properties are within acceptable limits, increasing rigidity can result in improved potency as a serotonin reuptake inhibitor, as exemplified by 9, with an IC50 of 0.038 mM. A more detailed study of the effect of tetralin ring stereochemistry, aromatic and amine substituents, and neurotransmitter reuptake inhibition was reported by Welch et al. [63]. This study evaluated a series of aminotetralin analogues containing a variety of substituents on both aromatic rings (Figure 11.3). Halogens were preferred, compared to hydrogen, trifluoromethyl, and alkoxy analogues. 3,4-Dichloro substitution provided more potent 5-HT activity relative to either monochloro analogue. In this series, secondary and tertiary amines showed comparable activity, and both were R1

N

R2

R1

N

R2

R3

R3

R4 R5 10 Figure 11.3 Sertraline analogues.

R4 R5 11

R1, R2=H, CH3 R3-5=H, halogen, O-alkyl, CF3

9

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more potent than the corresponding primary amino derivatives. This feature of structure–activity was investigated because it was recognized that N-dealkylation was a potential metabolic pathway for compounds in this series. Stereochemistry on the tetralin ring plays a key role in neurotransmitter uptake inhibition selectivity. trans isomers (10), regardless of aromatic or amine substitution, consistently demonstrate a preference for the norepinephrine site. Selectivity for serotonin reuptake was demonstrated by some but not all cis isomers (11). Resolution of 10 and 11 into pure diastereomers revealed that dextrorotatory isomers of 10 displayed potent neurotransmitter uptake inhibition at dopamine, norepinephrine, and serotonin, while levorotatory isomers were effectively inactive as reuptake blockers. In contrast, resolution of 11, which ultimately resulted in the identification of sertraline, showed that dextrorotatory isomers showed increased selectivity for serotonin reuptake inhibition, while the levorotatory isomers demonstrated improved selectivity for dopamine [64]. Prior work that focused on norepinephrine reuptake in rats established that the 4position was preferred for the phenyl substituent on the tetralin ring. Furthermore, exploration of a small set of amines (pyrrolidine, N-methyl piperazine, isopropylamine, and cyclopropylamine) revealed that the N-methyl derivatives studied above were superior. These conclusions were established using in vivo models that evaluated NE reuptake in cardiac tissue and epinephrine-induced hyperthermia [66]. 11.4.2 Escitalopram

As noted above, phthalans such as citalopram analogue 7 showed a preference for inhibition of NE reuptake [62]. Removal of the alkyl substituents at C3 of the phthalan ring system provided a series (12, Figure 11.4) that demonstrated a preference for serotonin reuptake inhibition [65]. Racemic analogues with substituents in both aromatic rings were evaluated for in vitro serotonin reuptake inhibition in blood platelets and in vivo in a 5-HT potentiation model. 5- or 6-substituted fluoro, trifluoromethyl, and chloro analogues in ring A, with ring B substituted or unsubstituted increased potency, relative to hydrogen in both the blood platelet assay and the in vivo screen. There was not a large difference in vitro between these substituted analogues. Addition of a 30 or 40 moiety to ring B increased in vitro activity, compared

R1 5 6

NC

A

O

O

N(CH3)2

N(CH3)2

B 4' R 3' 2

12 Figure 11.4 Citalopram analogues.

Cl 13

11.4 Medicinal Chemistry

to the B ring unsubstituted analogues, with many derivatives showing IC50 values less than 1 nM. A limited number of monomethyl amine analogues were prepared and, in each case, the secondary amine was less active than the tertiary amine, unlike the results obtained in the tetralin series. Quantitative structure–activity analysis revealed that there was not a direct correlation between in vitro and in vivo activity. This QSAR study did highlight the electronic effect of substituents and lipophilicity as important factors that contributed to SSRI activity. The electronic effects A-ring substituents at C-5 were hypothesized to exert their influence on interactions at the receptor, while B-ring substituents were hypothesized to interact with the binding site primarily by lipophilic interactions. A homology model, constructed using the crystal structure of the bacterial leucine transporter, suggested the serotonin binding site is formed by transmembrane domains 1, 3, 6, and 8 [66]. In addition, specific residues in transmembrane domains 1 and 3 (Tyr-95 and Ile-172, respectively) were identified as key residues for SSRI binding, including citalopram [67]. Very recently, results were reported that highlight the role of serine-438 on the serotonin reuptake channel [68]. Mutation of this residue to threonine decreases affinity of citalopram and tricyclic antidepressants up to 175-fold. Serine-438 is located within the serotonin binding site on the channel. Structural analysis of the bacterial protein and alignment with  the serotonin transporter suggested that Ser-438 is approximately 4 A from Asp-98, a key amino acid that interacts directly with a sodium ion, as well as the basic amino group found in all SSRIs. Interestingly, when citalopram derivative 13 was tested on the mutant receptor, there was less than a twofold difference in Ki values between the wild-type and mutant receptor. This suggests a direct steric interaction between citalopram and the serine residue on the transporter. N-Methyl and bis-des-methyl citalopram showed 5.8-fold and 1.7-fold differences, respectively, for binding to the wild type and Ser-438-Thr mutant, further supporting the steric hypothesis. Citalopram is a racemic mixture, and evaluation of the respective (R)- and (S)enantiomers revealed that essentially all the serotonin reuptake activity is expressed by the (S)-isomer, escitalopram, 4 (Figure 11.1). In rat brain synaptosomes, 4 had a Ki value of 1.5 nM and the (R)-isomer had a Ki value of 250 nM [12, 69]. Details on the biological and pharmacological differences between these enantiomers are outlined in Sections 11.3.2 and 11.5. 11.4.3 Fluvoxamine

Fluvoxamine was one of the first SSRIs approved for use as an antidepressant. Relatively little information on the medicinal chemistry leading to the discovery of fluvoxamine has been published. Fluvoxamine, a monocyclic derivative, is structurally distinct from most SSRIs. It also possesses an oxime moiety, which is bioisosteric for the vinylalkylamine found in many tricyclic antidepressants such as amitryptyline. This class appears in an early publication from Philips-Duphar describing their efforts at identifying platelet aggregation inhibitors [70]. Most of the

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R1

R6

R5

R

N O

N H

N O

R3

R2

H

R = C4H9, OC4H9

R4 14

NH2

15

Figure 11.5 Fluvoxamine analogues.

structure–activity information for fluvoxamine comes from the patent literature and is based on in vivo tests. The original patent on this series disclosed a series of aminoalkyl ethers of substituted benzyl oximes (14, Figure 11.5) [71]. The authors alluded to the possibility that some of the examples might display monoamine oxidase inhibitory activity but no data were presented. Antidepressant efficacy was measured by the ability of the compound to block tetrabenazine-induced ptosis [72], an activity shared by tricyclic antidepressants and thought to be caused by blockade of norepinephrine reuptake [73–75]. The compounds were classified into two general categories: “active” and “not active.” The authors stated that compounds where both R1 and R2 were a hydrogen atom had strong activity. A wide variety of alkyl and cycloalkyl groups at R3 were tolerated. Likewise, substitution on the aryl ring was well tolerated, with groups of various sizes and electronic properties leading to active compounds. In fact, the only examples that were classified as “not active” were the ones where R3 was a hydrogen atom and the aryl ring was substituted in the 2-position by a bulky n-butyl or n-butoxy group (15,Figure 11.5). Bicyclic systems such as quinoline, naphthyl, and benzothienyl were also suitable replacements for phenyl. Many of the compounds also possessed sedative and anticonvulsive properties. Subsequent patents around this general class of compounds disclose subgenera that potentiate the effects of serotonin and, to varying degrees, norepinephrine without inhibiting monoamine oxidase. The noradrenergic activity was measured using the previously described tetrabenazine test while the serotonergic properties were evaluated by the ability of compounds to potentiate the behavioral effects induced by administration of the serotonin precursor 5-hydroxytryptophan [76]. The basic genus exemplified by compound described by these patents is shown in Figure 11.6. A series of alkyl, alkoxy, and alkyl nitrile groups was exemplified and compared for the position occupied by R2 [77–79]. These were compared with standard analogues from the earlier patent where R2 is H or methyl that displayed monoamine oxidase inhibitory activity. In general, analogues where R2 possessed a C4–C5 alkyl chain (see, for example, 17a–17c, Figure 11.6) were two- to fivefold more potent at potentiating serotonin than norepinephrine, with ED50 values in the range N O R1

NH2

N O R1

R2

R2

16

17

Figure 11.6 Alkylated fluvoxamine analogues.

NH2

R1 17a -(CH2)3CH3 17b -(CH2)4OCH3 17c -(CH2)4CN

11.4 Medicinal Chemistry

j281

Cl N O O F

N OH

N O

18

haloperidol

NH2

R1

N O

(CH2)4OCH3 F

N

primaperone

NR2R3

R1

O

fluvoxamine

NR2R3

R1

N

R4 R5

19

Figure 11.7 Amine analogues of fluvoxamine.

of 5–75 mg/kg, i.p. This trend held for compounds possessing a variety of substituents in the R1 position, including methylthio, methylsulfonyl, and trifluoromethyl. The exception to this trend was seen when the R1 substituent was chloro, bromo, or 3,4-dichloro [77, 79]. The examples disclosed in those patent applications were all more potent at potentiating norepinephrine. A somewhat more potent series of compounds was disclosed in a 1980 patent [80] in which the primary substituent in the R1 position (in either the 3- or the 4-position) was a nitro moiety. These analogues displayed a variety of norepinephrine/serotonin selectivities, depending on the nature of the R1 substitution pattern and the nature of R2. Interestingly, a few examples from this patent possessed a methyl group on the carbon adjacent to the terminal amine. In two cases, this substitution pattern abolished noradrenergic activity. More recently, a group from the University of Ankara prepared and examined a series of aryloxime ethers (Figure 11.7) that combined the structural features of fluvoxamine with those of the sigma-1 ligands haloperidol and primaperone [81]. They examined compounds for their ability to inhibit freezing in the mouse behavioral despair test. The most active compound (18, R1 ¼ H; R2 ¼ H; R3 ¼ C2H5) reduced freezing by over 96% at a dose of 10 mg/kg, i.p. Of the compounds that possessed haloperidol-like structures, the most potent (19, R ¼ F; R2, R3 ¼ H; R4 ¼ OH; R5 ¼ 4-Cl-phenyl) reduced freezing by 86% at the same dose. The butyrophenone-derived analogues (19) were shown by spectroscopy to be mixtures of syn and anti isomers. 11.4.4 Fluoxetine

The history of the medicinal chemistry that led to the discovery of fluoxetine has been described in a 1995 review by Lilly researchers [82]. It was observed that diphenhydramine and other antihistamines enhanced blood pressure and heart rate responses to norepinephrine and inhibited uptake of monoamines. In addition, diphenhydramine showed comparable potency to imipramine and amitryptyline in

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NHCH3 O

N(CH3)2

O OCH3

diphenhydramine

NHCH3

nisoxetine

O F3C

fluoxetine

Figure 11.8 Design approach for fluoxetine.

the tetrabenazine test. Reversing the positions of one of the phenyl groups and the oxygen moiety of diphenhydramine (Figure 11.8) led to a series of phenoxyphenylpropylamines. One member of that series, LY94939 (nisoxetine) reversed hypothermia in mice induced by both apomorphine and reserpine. It was later shown to inhibit norepinephrine uptake in brain synaptosomes but was not a potent blocker of serotonin or dopamine uptake. Information on SAR of the fluoxetine series is scattered throughout the literature within publications often describing the pharmacology of a single compound. However, the 1995 review from Lilly researchers provides a summary of their results with the phenoxyphenylpropylamine series [82]. Table 11.2 shows data from representative examples. The primary changes described involved substitution on the phenoxy ring. The parent compound (R ¼ H) displayed moderate potency as a serotonin uptake inhibitor, with little selectivity for serotonin over norepinephrine. In general, substitution in the 2-position of the phenoxy ring increased potency for norepinephrine uptake. Substitution in the 3- and 4-positions increased potency for serotonin uptake, with the best selectivity for serotonin over norepinephrine being seen in the 4-substituted analogues. The 4-trifluoromethyl substitution pattern was particularly effective at inducing 5-HTuptake selectivity. This compound (LY110140) ultimately became fluoxetine. Modeling studies suggest that the dramatic reversal in selectivity seen between 2-substituted analogues such as nisoxetine and 4-substituted derivatives such as fluoxetine may indeed be derived primarily from the differences in phenoxy ring torsion angle since the low-energy conformations of the rest of the scaffold are very similar [83]. Two naphthyloxy analogues were described in the 1995 review (Table 11.2). Dapoxetine displayed potency and selectivity for uptake inhibition that was similar to that described for fluoxetine. However, duloxetine, a thienyl analogue, displayed significantly less selectivity for serotonin uptake. The enantiomers of fluoxetine and norfluoxetine were also prepared and examined. The (R)- and (S)-enantiomers of fluoxetine displayed similar potency at inhibiting serotonin uptake and were roughly equipotent compared to the racemic mixture. Interestingly, the (R)-enantiomer of the primary amine analogue norfluoxetine was 14-fold less potent at inhibiting serotonin uptake than the (S)-isomer. Changes to the amine moiety of the phenoxyalkylamine series can have profound effects on the pharmacology of the series. Fluoxetine is a secondary alkyl amine. Removal of the pendant methyl group (to give norfluoxetine) had little effect on either potency or relative selectivity for serotonin and norepinephrine uptake compared to

11.4 Medicinal Chemistry Table 11.2 Monoamine reuptake activity of fluoxetine analogues.

NHCH3 O R

R

H 2-F 2-CH3 2-CF3 2-OCH3 3-CF3 4-F 4-Cl 4-CH3 4-CF3 (fluoxetine) 4-OCH3 4-CF3, -NH2 (norfluoxetine)

NHCH3

Inhibition of serotonin uptakea) (Ki, nM)

Inhibition of norepinephrine uptakea) (Ki, nM)

102 898 390 1498 1371 166 638 142 95 17 71 17

200 5.3 3.4 4467 2.4 1328 1276 568 570 2703 1207 2176

8

1000

5

16

O dapoxetine

NHCH3 O S a)

duloxetine

Inhibition of monoamines into rat synaptosomes.

fluoxetine. Dimethyl substitution on the amine is also tolerated; however, more dramatic changes reduce uptake activity [84]. For example, a recent paper by Orjales et al. describes efforts to build upon the fluoxetine scaffold to generate compounds with both 5-HT1A antagonist and serotonin uptake inhibitory properties [85]. Appending amine groups known to be present in potent serotonin 5-HT1A

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O

H3C

O O O

O

OCH3

O O

O

O

H NCH3

F

NH

O

NH

NHCH3

MDL28618A

Femoxetine

Paroxetine

Reboxetine

Figure 11.9 Paroxetine analogues.

ligands to fluoxetine analogues not only imparted 5-HT1A and 5-HT2A affinity but also eliminated serotonin transporter affinity (as measured by displacement of [3 H]-paroxetine from rat cerebral cortex preparations). Substitution on the 3- and 4-positions of the aryl rings is better tolerated. In their efforts to identify compounds with dual activity as serotonin uptake inhibitors and histamine H3 antagonists, one group appended groups known to impart H3 affinity to the aryl rings of nisoxetine [86]. With few exceptions, the compounds generally displayed potent affinity for rat and human serotonin transporters. Constraint within the alkyl chain has also been examined [87]. Although analogues such as MDL28618A and femoxetine (Figure 11.9) retained selectivity for serotonin transporters, they suffered a loss in potency for that target (approximately 10-fold, compared to fluoxetine). Removal of the methyl group on femoxetine to give the secondary amine analogue resulted in a 10-fold increase in serotonin transporter potency. Many of the known serotonin uptake inhibitors are semiconstrained phenoxyalkylamine analogues (e.g., paroxetine and reboxetine). 11.4.5 Paroxetine

The SAR of paroxetine and related analogues has been reviewed [87, 88]. The paroxetine scaffold first appeared in a US patent, which disclosed its racemic form along with its ( þ )- and ()-enantiomers and a small number of N-methyl analogues and fluorine positional isomers [89]. The compounds were shown to antagonize p-chloroamphetamine-induced depletion of serotonin in rat ex vivo studies. Paroxetine can be considered a constrained analogue of fluoxetine in which the aminoalkyl chain has been incorporated into a piperidine moiety. The racemic mixture contains two chiral centers, and paroxetine is the (3S,4R)()-isomer. Both bulky groups are in the equatorial position. Paroxetine is a very potent ligand for rat and human serotonin transporters. The compound displays subnanomolar Ki values for both rat and human serotonin transporters (rat Ki 0.1 nM; human Ki 0.9 nM [90, 91]). This high affinity correlates with the compound’s significant potency as an inhibitor of serotonin uptake into rat synaptosomes (IC50 1.9 nM). The (3S,4R)-isomer possesses 60–100 times greater affinity for the rat serotonin transporter than its antipode or the cis isomers of the paroxetine scaffold.

11.5 Comparison of SSRIs and Other Uses

The para-fluoro group is not essential for serotonin transporter affinity [92]. However, substitution of fluorine with other groups such as methyl in either aromatic ring reduces affinity by as much as an order of magnitude. ortho-Substitution on either aromatic ring (especially the phenoxy ring) particularly affects affinity, suggesting that there are stereochemical requirements for optimal binding [93]. Conversion of the secondary amine to a tertiary amine reduces affinity by 100-fold. Likewise, substitution of the methylenedioxy group with phenyl analogues (e.g., 4-methoxyphenyl, 3-hydroxy-4-methoxyphenyl, etc.) also reduces serotonin transporter affinity dramatically. Restriction of the piperidine ring with an ethylene bridge to give tropane-like molecules is unfavorable, giving compounds with at least 100-fold less affinity [94]. Paroxetine is over 300-fold selective for the serotonin transporter versus the norepinephrine transporter, in terms of both affinity [91] and in vitro inhibition of neurotransmitter transport into rat brain synaptosomes [95]. The compound also shows little affinity or inhibitory activity for the dopamine transporter. Formal structure–activity studies for the 5-HT/NE transporter selectivity of the paroxetine scaffold have not appeared in the literature. Restriction of the piperidine ring into a tropane-like scaffold, however, reduced selectivity for the serotonin transporter versus the dopamine transporter [94].

11.5 Comparison of SSRIs and Other Uses

Table 11.3 lists a variety of properties of SSRIs, including monoamine selectivity, pharmacokinetic and metabolic properties. SSRIs represent a significant improvement over other available treatments for depression because of their reduced side-effect profile. It is important to recognize that SSRIs have not been shown to have improved efficacy, relative to tricyclic antidepressants [96]. Instead, their improved pharmacologic receptor and cardiac

Table 11.3 Comparison of SSRIs.

Parameter

Fluoxetine

Paroxetine Fluvoxamine

Sertraline

Citalopram

SRI KD (nM) [122] NE KD (nM) [122] DAT KD (nM) [122] 1 active metabolite(s) [87] Plasma t1/2 (h) [87] Elimination t1/2 (h) [87] 1 CYP metabolizing enzyme(s) [87]

0.81 240 3600 O-Desmethyl, norfluoxetine 24–96 50

0.13 40 490 None

2.2 1300 9200 None 7–63 15–20

0.29 420 25 N-Desmethyl sertraline 22–35 19–37

1.16 4070 28 100 Desmethyl escitalopram 23–75 27–32

24 22

2D6

2D6

2D6

3A4

2C19, 2D6, 3A4

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safety profiles confer tolerability advantages that have resulted in greater acceptance by clinicians and patients. In addition to demonstrated efficacy in mood disorders, SSRIs have also been administered for the treatment of obsessive-compulsive disorder (OCD), panic, social anxiety, and post-traumatic stress disorders, bulimia nervosa, and premenstrual dysphoric disorder [4]. It is now well recognized that SSRI efficacy in the treatment of depression is not immediate and may require several weeks before measurable mood changes are apparent (see Section 11.2 for details). Attempts have been made to analyze the comparative efficacy of selected agents. Definitive conclusions based on the data reported are scant because of variations in patient populations in the trials, the number of participating centers, and inclusion criteria [96, 97]. Reported differences in adverse event profiles and/or frequency tend to be small and nonreproducible. The mean response rate for citalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline was 50% in one analysis [98]. As noted in Sections 11.3.2 and 11.4.2, escitalopram was shown preclinically to possess all of the antidepressant activity associated with citalopram. This observation translated into clinical practice in terms of an efficacious dose for the eutomer. For example, patients that were initially treated with 20–40 mg daily of citalopram can be treated equally well with 10–20 mg escitalopram [99]. Recently, differences in serotonin reuptake transporter binding constants between citalopram and escitalopram were compared in a receptor occupancy study in patients. Multiple (but not single) doses of the distomer reduced occupancy of the serotonin transporter by the (S)-isomer. These circumstances were intended to mimic the steady-state concentration of the individual isomers, and it was shown that the (R)-isomer was cleared more slowly than the (S)-isomer, lending further credence to the data obtained by Moore and coworkers regarding the comparative efficacy of the two agents [100]. The properties and clinical effects associated with individual SSRIs are related to their pharmacological profiles. When a patient begins SSRI therapy, elevated serotonin leads to activation of postsynaptic 5-HT2A and 5-HT2C receptors. This leads to reduced norepinephrine and dopamine levels and can result in “low energy” and apathy experienced by patients in the initial phase of treatment [101]. Patients treated initially with fluoxetine and sertraline may experience more immediate relief of these symptoms because of the 5-HT2C affinity associated with fluoxetine and dopamine reuptake effect associated with sertraline [102, 103]. Many patients suffering from depression have other accompanying mood disorders. Recent estimates suggest that up to 70% of patients diagnosed with depression also meet diagnostic criteria for anxiety [104]. Among SSRIs, fluvoxamine and paroxetine have demonstrated the ability to relieve symptoms of anxiety, which can include agitation and insomnia. These properties may be associated with the norepinephrine reuptake inhibition associated with paroxetine and the s1 affinity demonstrated by fluvoxamine [105]. Sexual dysfunction is an adverse event associated with SSRI administration. As a group, there is little difference in frequency of this side effect; however, paroxetine exhibits the greatest reduction in delayed orgasm, libido, and erectile dysfunction compared to others [106]. These properties may be associated with paroxetine’s known anticholinergic and nitric acid synthase inhibitory activity [107].

11.5 Comparison of SSRIs and Other Uses

Each SSRI has a unique cytochrome P450 inhibition and metabolism profile [108]. Escitalopram is a comparatively weak inhibitor of the primary CYPs, 2D6, 2C19, and 3A4, while fluvoxamine and fluoxetine show more potent inhibition of 2D6, and to a lesser extent, 2C19. Paroxetine is a more potent inhibitor of CYP2D6, compared to sertraline. Escitalopram is metabolized at similar rates by at least three CYP isoforms (2D6, 3A4, 2C19). Consequently, metabolic polymorphism or coadministration of an inhibitor of one of these three CYPs is unlikely to lead to a clinically significant drug–drug interaction [109]. However, inhibition of 2 CYPs can result in a substantial increase in plasma drug concentration [110]. Fluoxetine, as the first SSRI to enter the market, has been studied more extensively than other agents in this group. Because it is a substrate for CYP2D6, patients who lack this enzyme or those taking other drugs that are also metabolized by 2D6 can experience higher plasma concentrations than usual and require dosage adjustment and blood level monitoring [111]. Fluvoxamine is unique among SSRIs in its interactions with the inducible CYP450, CYP1A2 [112]. The ability of fluvoxamine to inhibit several P450 enzymes contributed to a hypothesis that suggested the compound, or a metabolite, interacts with the heme cofactor, rather than at a substrate binding site [111]. Paroxetine has been shown to be a potent 2D6 inhibitor, with a Ki of approximately 150 nM [113]. This property may lead to inhibition of metabolism of the drug and may contribute to nonlinear pharmacokinetic properties as well [114]. In addition to their use in the treatment of depression, SSRIs have been reported to be used in the treatment of obsessive-compulsive disorder, panic disorder, eating disorders, as adjunctive agents in the treatment of the negative symptoms associated with schizophrenia, dementia, premenstrual dysphoric disorder, and chronic pain syndromes. An overview of selected results from these studies is given below. Fluoxetine, paroxetine, sertraline, and fluvoxamine are approved for the treatment of OCD. These agents are not used alone. Rather, they are a part of a more comprehensive treatment program that includes behavior modification and counseling. Efficacy was established in double-blind, placebo-controlled studies using doses similar to those employed in the treatment of depression [115, 116]. Fluoxetine, sertraline, paroxetine, and fluvoxamine have also been reported to show efficacy in clinical trials in patients with panic disorders [117, 118]. Among trials of SSRIs for treatment of eating disorders, one recent study suggests that different agents may be preferred for patients with distinct symptoms associated with bulimia. In this study, citalopram and fluoxetine were compared in a small number of patients. In patients whose bulimia was associated with depression, citalopram appeared to give favorable results, while in patients who expressed a greater degree of anger, fluoxetine was preferred. Both compounds provided comparable and improved control of dietary psychopathology [119]. Schizophrenia is a psychiatric disorder that involves positive (e.g., hallucinations) and negative (e.g., social withdrawal) symptoms that can also include comorbid depression. SSRIs have been investigated as adjunctive additions to pharmacotherapy for the disease, with the objective of improving mood and negative symptomatology [120]. When used in combination with first-generation (typical) antipsychotics such as haloperidol, variable results were obtained in small trials where fluoxetine, sertraline, citalopram, and fluvoxamine were used. In some

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cases, improvements in negative symptoms were observed, while in others, no change was detected. These studies used different inclusion and exclusion criteria, and were carried out for variable periods of time (5–12 weeks). In most of these studies, patients with depressive symptoms showed measurable improvement based on the Hamilton psychiatric rating scale for depression (HAM-D). Addition of an SSRI did not exacerbate positive symptoms or adverse events associated with the antipsychotic agent (e.g., extrapyramidal syndrome) in patients. SSRIs have been less well studied in patients being treated with atypical antipsychotics. In patients being treated with antipsychotic agents, potential drug–drug interactions should be closely monitored. Examples have been reported that lead to increased plasma concentrations of clozapine, olanzapine, and haloperidol. The use of SSRIs in patients suffering from dementia has also been reported, and in general, SSRIs improve mood symptoms in patients suffering from Alzheimer’s disease [121].

11.6 Summary

Selective serotonin reuptake inhibitors revolutionized pharmacotherapy of depression. These compounds provided important tools to better understand the neurochemistry and neurobiology of this complex disorder, and in doing so, laid the groundwork for other agents, in particular dual serotonin/norepinephrine reuptake inhibitors, that may extend the therapeutic benefits offered by SSRIs in patients with depression. SSRIs are useful in the treatment of mood disorders other than depression, and are being explored for potential utility in a variety of others. The structural diversity in this group of compounds provided fertile ground for medicinal chemists to explore not only structure–activity relationships associated with neurotransmitter reuptake but also structural features in the receptor that interact with these compounds. Furthermore, although it is beyond the scope of this chapter, a variety of new, useful methodologies have been developed in organic chemistry to synthesize these structurally complex molecules and continue to be of interest to the field. Application of this new technology to other medicinally interesting structures is ongoing.

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Wayne E. Childers Jr

Principal Research Scientist III, Wyeth Research, Chemical Sciences, 865 Ridge Road, Monmouth Junction, NJ 08852, USA Wayne E. Childers is a Principal Research Scientist for Wyeth Research, Inc. He has been working with Wyeth for 22 years as a medicinal chemist. Wayne received his BA (1979) degree from Vanderbilt University in chemistry and PhD (1984) in organic chemistry from the University of Georgia under the direction of Dr. Harold Pinnick. He served as an assistant adjunct professor at Bucknell University before accepting a position as a postdoctoral fellow at the Johns Hopkins University School of Medicine in the laboratories of Dr. Cecil Robinson. Wayne joined Wyeth in 1987. Over the past 22 years, Wayne has worked in and made contributions to numerous therapeutic areas, including psychiatric diseases, stroke, and Alzheimer’s disease, and the treatment of chronic pain. David Rotella

Principal Research Scientist III, Wyeth Research, Chemical Sciences, 865 Ridge Road, Monmouth Junction, NJ 08852, USA David Rotella is a principal research scientist for Wyeth Research, where he has focused on CNS projects. He earned a BS Pharm degree from the University of Pittsburgh (1981) and a PhD (1985) from the Ohio State University under Donald. T. Witiak. After postdoctoral studies in organic chemistry at the Penn State University under Ken S. Feldman, he served as an Assistant Professor at the University of Mississippi. David has worked with Cephalon, Bristol-Myers, and Lexicon, on projects in neurodegeneration, schizophrenia, and cardiovascular and metabolic diseases.

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12 Muscarinic Receptor Antagonists in the Treatment of COPD Matthias Grauert, Michael P. Pieper, and Paola Casarosa

12.1 Introduction

Acetylcholine is one of the most important signaling molecules in mammalian nervous systems. It is almost ubiquitously expressed in biological systems and can be identified in many human cell types. Besides its function as a neurotransmitter of the parasympathetic nervous system, there is strong evidence that acetylcholine also exerts functions in non-neuronal cells. Acetylcholine mediates its effects through nicotinic receptors, which belong to the family of ligand-gated ion channels, and via G-protein-coupled muscarinic receptors. Both endogenous acetylcholine and a series of synthetic receptor agonists, termed cholinomimetics, trigger a diverse array of signaling pathways via nicotinic ion channels and muscarinic receptors [1, 2]. Compounds that block the effects of acetylcholine at the muscarinic receptors are termed antimuscarinics. Naturally occurring antimuscarinics are ingredients of a number of plants, in particular of the night shadow family, Solanaceae. The use of night shadow herb preparations has a long history. They induce effects in the central nervous system, and due to its hallucinogenic effects, smoke of herb mixtures was inhaled during sacred ceremonies in different cultures. Papyrus records dated back to the second millennium BC describe the use of herbs containing antimuscarinic alkaloids. The first specific description of the therapeutic use of datura preparations can be found in Ayurvedic medicine from India in the seventeenth century for the treatment of asthmatic disorders. These herbal medications were prepared for inhalation in the form of medical smoke [3]. To avoid the severe side effects on the central nervous system, Boehringer Ingelheim developed several quaternary antimuscarinics for clinical use. Quaternary compounds are poorly absorbed from mucosal surfaces and do not penetrate the blood–brain barrier to any relevant extent. These compounds are used for the treatment of abdominal pain or chronic obstructive pulmonary disease (COPD). COPD is a respiratory disease characterized by shortness of breath due to slow and progressive airflow obstruction. The major risk factor is cigarette smoke inducing chronic inflammatory processes as well as airway remodeling. COPD is one of the

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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most important health burdens in society today, with steadily increasing morbidity, mortality, and health care costs [4].

12.2 Muscarinic Receptor Subtypes

In pharmacological studies using Boehringer Ingelheim’s anticholinergic pirenzepine 1 (Figure 12.1), the compound was shown to discriminate between subclasses of muscarinic binding sites [5]. These investigations set the basis for the discovery of muscarinic receptor subtypes. Molecular cloning approaches have since demonstrated the existence of five muscarinic receptor genes (M1–M5) [6]. The muscarinic receptor subtypes have significant homology and are very similar across mammalian species. So far only very few agonists and antagonists are available with any selectivity toward one of these receptor subtypes. As a typical antagonist, atropine binds to all different muscarinic receptor subtypes with subnanomolar affinity, but with less than 10-fold selectivity [7]. The lack of subtype selective compounds, together with the fact that most native mammalian tissues coexpress different muscarinic receptor subtypes, has limited the characterization of the physiological role of each subtype for a long time [8, 9]. Knockout mice lacking the respective muscarinic receptor subtypes M1–M5 have been established, which permitted significant insight into the physiological role of the different muscarinic receptors [10, 11]. The mRNAs of all muscarinic receptor subtypes are found in the brain. However, the M1 and M4 mRNAs are the most abundant subtypes in CNS (about 40–50% expression content of the total muscarinic receptor population) whereas the M5 mRNA represents less than 2% of the whole brain population. In peripheral tissues, mRNA of M1 is found in salivary glands and in sympathetic ganglia, and mRNA of M2 is identified in cardiac muscle and smooth muscle. The mRNA of M3 has been detected in exocrine glands, the smooth muscle of the conducting airways, gastrointestinal and urinary tracts, and in the eye, while

O

H N

N

N O

N N 1 pirenzepine Figure 12.1 Structure of pirenzepine.

12.3 Structures of Muscarinic Agonists and Antagonists Table 12.1 Receptor subtypes mediated pharmacological action of antimuscarinics.

Bronchodilatation Reduced saliva secretion Reduced secretion from sweat glands Reduced gastric secretion/GI motility Reduced contraction of urinary bladder Increased heart rate (tachycardia) Dilatation of the papillary muscle CNS effects: excitation, ataxia

M3/(M1) M1/M3 M3 M1/M3 M2/M3 M2/(M3) M3 M1/M4

the M4 mRNA is found in the lung, salivary glands, and ileum. The M2 and M4 also serve as autoreceptors located on presynaptic terminals that act as a negative feedback loop leading to a reduction in acetylcholine neurotransmitter release, whereas the M1 and M3 receptors are located postsynaptically. Present knowledge suggests that in the lung M1 receptors mediate ganglionic neurotransmission and enhance constriction of airway smooth muscles [12]. The M2 receptor subtype, although being abundantly expressed in smooth muscle cells, only plays a modulatory role in acetylcholinemediated bronchoconstriction. Finally, activation of pulmonary M3 receptors results in bronchoconstriction and mucus gland hypersecretion, major symptoms of obstructive airway diseases such as asthma and chronic obstructive pulmonary disease. Based on the distribution of the muscarinic receptors, main pharmacological actions of anticholinergic compounds can be attributed to different receptor subtypes. These effects and the corresponding receptor subtypes are summarized in Table 12.1 [13].

12.3 Structures of Muscarinic Agonists and Antagonists 12.3.1 Muscarinic Agonists

The structure of muscarinic cholinergic agonists can be divided into two groups. The first group is synthetic analogues of acetylcholine 2 in which the ester functionality is stabilized against rapid cleavage by acetylcholinesterase. Examples are methacholine 3, where an alpha-methyl group protects the ester functionality, and carbachol 4, where the ester group is exchanged by a carbamate group. The S-( þ )-enantiomer of methacholine is more than 700 times more potent than the R-(
)-enantiomer [14]. While acetylcholine and carbachol bind to muscarinic and nicotinic receptors, methacholine has a preference for the muscarinic receptor. The second group is cholinomimetic alkaloids such as muscarine 5, pilocarpine 6, or arecoline 7. They bind to the same site as the synthetic analogues of acetylcholine. Muscarine gives the name of the whole receptor class and acts almost exclusively on muscarinic receptors. It was first isolated from the mushroom Amanita muscaria.

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O

O

+

N

+

N

O

2 acetylcholine

O

3 methacholine

O N

N

+

O

5 muscarine

NH2

O

4 carbachol

N

OH N

O +

N

O 6 pilocarpine

O

O 7 arecoline

Figure 12.2 Structure of acetylcholine, synthetic analogues, and cholinomimetic alkaloids.

The structures of these compounds mimic that of acetylcholine. Like acetylcholine and the synthetic congeners, muscarine possesses a quaternary ammonium group, and the ester functionality is imitated by the hydroxylated tetrahydrofuran system. In contrast, arecoline is a tertiary amine derivative incorporated in a ring system. Under physiological conditions, the amine will be protonated and interacts with the receptor in the ionic form. It has been shown that an aspartic acid in the third transmembrane helix of the muscarinic receptors (Asp 3.32) forms an ionic bond with the cationic nitrogen of acetylcholine and other muscarinic compounds [15]. The ester group of arecoline is located at a similar distance to the cationic nitrogen as in acetylcholine; however, the ester is inverted. The methyl-imidazole system in pilocarpine has a pKa of about 7.2 and will be partially protonated at physiological pH. Therefore, it is reasonable to assume that this moiety mimics the trimethyl ammonium group of acetylcholine and interacts with Asp 3.32. However, modeling studies indicate that the lactam ring binds differently (Figure 12.2) [16]. 12.3.2 Antimuscarinics

Atropine 8 and scopolamine 10 are tropane alkaloids that show prominent antimuscarinic activity. They are esters formed by a combination of tropic acid and a-tropine or scopine. Atropine is a mixture of both enantiomers in which the tropic acid is racemized during extraction. The more potent enantiomer is called hyoscyamine 9 and contains S-tropic acid. The tropane alkaloids occur naturally in many members of the Solanaceae plant family. The most common plants containing anticholinergic alkaloids are Atropa belladonna, Hyoscamus niger, Mandragora officinarum, and different datura and Brugmansia species. Due to their higher content of alkaloids, Duboisea species also play an important role as a source of alkaloid starting material for industrial drug production [17]. Preparations from these plants were used for many centuries as anesthetic or for their psychostimulating action. Atropine was isolated by Geiger and Hess in

12.3 Structures of Muscarinic Agonists and Antagonists

1833 [18]. They showed the beneficial effect of atropine on airway diseases and opened this field for targeted drug development. The isolated alkaloids still have a prominent use in medicine. Atropine and scopolamine are topically used in ophthalmology to temporarily induce mydriasis by blocking the contraction of the circular papillary sphincter muscle. In addition, they block the ciliary muscle of the lens resulting in dilatation of the pupil and paralysis of accommodation. Furthermore, atropine is used in cardiac arrest and in the treatment of bradycardia by blocking the vagus nerve of the parasympathetic system that is responsible for decreasing heart rate. Due to a better penetration of the blood–brain barrier, scopolamine shows more prominent CNS effects than atropine. Therefore, scopolamine has long been used in Parkinsonism and for the treatment of antipsychoticinduced extrapyramidal side effects, and it is still in use as a preanesthetic. For many years, atropine was used to provide symptomatic relief from various gastrointestinal disorders including spasm, peptic ulcers, irritable bowel syndrome, and pancreatitis. However, the discovery of M1-selective muscarinic antagonists such as pirenzepine, selective H2-histamine antagonists, and especially proton-pump inhibitors have replaced the use of conventional antimuscarinics in gastric secretion disorders [19]. Finally, atropine and scopolamine are used as antidotes for poisoning by organophoshate insecticides or organophosphate chemical weapons (Figure 12.3). Structures of these antimuscarinic alkaloids resemble those of cholinergic agonists. They also possess a tertiary amine that will be protonated under physiological conditions and bear an ester group at a similar location as seen in the agonistic compounds. Indeed, it has been shown that both parts of the molecules, the tropane moiety and the tropic acid, are important for activity. N-Methyscopolamine 11 can be displaced by cholinergic agonists in a competitive manner. Site-directed mutagenesis studies on the human M2-receptor [20] and on the M3-receptor binding site [21, 22] indicated that tropane alkaloids and muscarinic agonists should have overlapping, but not identical, binding sites. The bulky lipophilic portion attached to the ester group and the sterically more demanding amino group are the major differences between the tropane alkaloids and cholinergic agonists. These parts of the molecule significantly enlarge the structure of the antagonists and point to a region of the receptor that is not contacted by agonistic compounds. Additional interactions of these parts of the molecule with the receptor

N

N

N O

OH O O

8 atropine

OH

OH

O

O

O

9 hyoscyamine

O

10 scopolamine

Figure 12.3 Structure of atropine, hyoscyamine, and scopolamine.

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Figure 12.4 Superimposition of Nmethylscopolamine and acetylcholine in a muscarinic receptor model (nonactivated mode). N-Methylscopolamine (colored in yellow), acetylcholine (colored in magenta), and

the amino acids of the receptor involved in the binding (labeled in the Ballesteros–Weinstein numbering [23]) are depicted. Red balls indicate water molecules involved in acetylcholine binding. Part of the receptor surface is shown.

may account for the higher affinity of tropane alkaloids compared to cholinergic agonists. This is visualized by superimposition of N-methylscopolamine and acetylcholine in an M3 receptor model (Figure 12.4): it has been demonstrated that Asp 3.32, Tyr 7.39, and Tyr 7.43 interact with the ionic part of both molecules. Asn 6.52 is involved in the binding of the hydroxyl group of N-methylscopolamine but not in the binding of acetylcholine [16]. The aromaticity of Tyr 6.51 is crucial for the binding of N-methylscopolamine [24]. Therefore, it is likely that Tyr 6.51 interacts with the tropane system. In contrast, the ester group of acetylcholine seems to interact with the hydroxyl group of Thr 5.42 and of Tyr 6.51 [25]. This might be possible only when water molecules are involved in a hydrogen bonding network because acetylcholine itself is too small to span the whole receptor and to make direct contacts with all amino acid residues that have been shown to be involved in binding. It has been demonstrated that binding of acetylcholine to the M3 muscarinic receptor results in a conformational change of the transmembrane segments III and VII that might be responsible for agonistic activity [26]. Increasing the size of the ammonium head group of acetylcholine by three ethyl moieties resulted in compound 12 that still bound to the muscarinic receptor but failed to induce this conformational change and therefore behaved as an antagonist. Furthermore, it has been demonstrated that S-(
)-tropinoylcholine 13 is a competitive antagonist of acetylcholine [27]. Therefore, it is reasonable to speculate that muscarinic antagonists cannot induce conformational changes at the receptor responsible for the agonistic trigger due to steric hindrance caused by the increased size of the molecule (Figure 12.5).

12.3 Structures of Muscarinic Agonists and Antagonists +

N O

OH

+

OH

N

+

O

N

O

O

O

O

O

12 acetyltriethylcholine

11 N-methyscopolamine

13 S-(–)-tropinoylcholine

Figure 12.5 Structure of N-methylscopolamine, acetyltriethylcholine and S-(
)-tropinoylcholine.

12.3.3 Discovery of Quaternary Antimuscarinics

As outlined in Section 12.2, muscarinic receptors have important physiological functions in several organs. The difficulty in generating receptor subtype-selective compounds and the fact that especially the muscarinic M3 receptor subtype is so broadly expressed limit the therapeutic use of tertiary muscarinic antagonists because of an unfavorable side-effect profile. To overcome this shortcoming, quaternary antimuscarinics such as scopolamine butyl bromide 14, ipratropium bromide 15, and oxitropium bromide 16 have been developed (Figure 12.6). These compounds act topically, are poorly absorbed, and do not penetrate the blood–brain barrier to any relevant extent. Quaternization of nitrogen leads to a new stereocenter. Alkylation of atropine or scopolamine almost exclusively gave isomers with the new alkyl substituent in the equatorial position as in scopolamine butyl bromide. To obtain the isomers with a larger alkyl substituent in the axial position, the alkyl group has to be introduced first, followed by quaternization with methyl bromide. Ipratropium bromide was obtained from N-isopropyl nortropine 17 by transesterification with methyl a-formylphenylacetate, subsequent reduction of the formyl group with sodium borohydride, and quaternization with methyl bromide (Figure 12.7) [28]. Direct quaternization of

+

N

Br

+

N

Br

+

N

O

Br

O OH O O

14 scopolamine butyl bromide

OH O O

15 ipratropium bromide

OH O O

16 oxitropium bromide

Figure 12.6 Structure scopolamine butyl bromide, ipratropium bromide, and oxitropium bromide.

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O O O

N

N

N CH3Br

O HO

OH

O

O

O

17

O

19

18

N

+

N OH O

Br

+

N NaBH4

OH O O

15 ipratropium bromide

Br

(CH3)2CHBr

OH O

O

O

8 atropine

20

Figure 12.7 Syntheses of ipratropium bromide and the geometrical isomer 20.

atropine with isopropyl bromide yielded the geometrical isomer 20. For the synthesis of oxitropium bromide, scopolamine was demethylated with phosgene or by oxidation with potassium permanganate. The derived norscopolamine was then alkylated with ethyl bromide and subsequently methylated with methyl bromide [29]. Scopolamine butyl bromide is used as an antispasmodic drug for the treatment of abdominal pain caused by gastrointestinal spasms and menstrual cramps. It was first registered in Germany in 1951 and has since become available worldwide both as a prescription drug and as an over-the-counter medicine in many countries. After oral administration, the bioavailability of scopolamine butyl bromide was estimated to be less than 1%. However, scopolamine butyl bromide is available at the lumen of the intestine where it exerts its effects through high-affinity binding to muscarinic receptors present locally. To treat bile or kidney cramps, scopolamine butyl bromide has to be parenterally administered [30]. Ipratropium bromide and oxitropium bromide have been developed as bronchospasmolytic compounds. After inhalation, these compounds act as very effective bronchodilators in the management of COPD and reduce airway mucus secretion [31]. At therapeutic doses, they have no other significant pharmacological effect because their systemic absorption is rather low. The bronchospasmolytic potency of both compounds is higher compared to atropine. The onset of action is slightly delayed and the duration of action is significantly prolonged [32, 33]. This is indeed a general trend: quaternization of atropine with a methyl group resulted in a compound with improved spasmolytic activity whereas compounds with a larger alkyl substituent in the equatorial position in general showed reduced spasmolytic activity [34]. In contrast, quaternary antimuscarinics bearing the larger alkyl group

12.3 Structures of Muscarinic Agonists and Antagonists

in the axial position are significantly more potent than their stereoisomers [29, 35]. Competitive binding experiments have shown that ipratropium bromide displaces 3 H-atropine about two orders of magnitude more potently than its geometrical isomer 20 with the isopropyl group in the equatorial position [36]. Ipratropium bromide and oxitropium bromide are nonselective antimuscarinics that block M1, M2, and M3 receptors with similar potency. As mentioned above, the M2 receptor is a presynaptically located autoreceptor and controls the release of acetylcholine by a negative feedback mechanism. Blockade of this receptor in the airway may increase the release of acetylcholine and can thereby, in theory, reduce the degree of bronchodilatation or the duration of action [37]. Therefore, the development of antimuscarinics that are selective for the M1 and M3 receptors may have therapeutic advantages. 12.3.4 Once-Daily Quaternary Antimuscarinics: Tiotropium Bromide as the Gold Standard

Compared to atropine, ipratropium bromide showed a prolonged duration of action as a bronchospasmolytic agent after inhaled administration. The reason for this extended effect is probably an increased residence time in the bronchial tissue due to the reduced penetration of the quaternized structure. However, the duration of action still requires three to four doses daily. Therefore, a further prolongation of the duration of action would be desirable to continuously block the muscarinic receptors. Once-daily dosing would significantly improve quality of life and compliance of chronically treated patients. To improve the duration of action, a further prolonged retardation in bronchial tissue has to be accomplished or, alternatively, the dissociation kinetics from the receptor has to be prolonged. Therefore, a program was initiated to evaluate receptor off-rate kinetics of quaternary antimuscarinics. Tritium-labeled ipratropium bromide showed a dissociation half-life of 0.26 h from the M3 receptor [38]. To evaluate newly synthesized compounds, the receptor affinity and the dissociation kinetic parameters have been evaluated [39]. As shown in Table 12.2, N-methylscopolamine 11 exhibits a dissociation half-life of 0.77 h from the human M3 receptor. This is in the same range as the receptor dissociation of ipratropium bromide. Exchange of the hydroxyl methylene group by a hydroxyl group in N-methylscopolamine (11) resulted in a 10-fold loss in potency for the M3 receptor (compound 21). The corresponding thiophene derivative 22 has almost the same potency as the phenyl derivative 21. In tiotropium bromide (23), an additional thiophene group was incorporated into the molecule that improved the potency by a factor of more than 100. In fact, tiotropium bromide is the most potent antimuscarinic synthesized so far. Enlargement of the axial N-alkyl-substituent, from methyl- through ethyl-, and isopropyl-, to n-propyl, steadily reduced the M3 receptor affinity as seen in compounds 24, 25, and 26, respectively. Furthermore, both methylation of the hydroxyl group in the ester moiety (compound 27) and removal of the hydroxyl group (compound 30) caused a significant loss in potency. Also, the exchange of the

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Table 12.2 pKi values and dissociation half-life at the M3 receptor for tiotropium analogues.

R3 +

N R2 R2

O . Compound

R1

R1

O

R2

R3

pKi h M3

t1/2 (h)

O

Me

10.2

0.77

O

Me

8.98

O

Me

8.93

O

Me

11.2

O

Et

10.77

O

iPr

9.77

OH 11

*

OH 21

*

OH 22

*

S

0.04

S 23

OH *

27.0

S

S 24

25

OH *

S

S OH *

S

4.45

12.3 Structures of Muscarinic Agonists and Antagonists Table 12.2 (Continued)

Compound

R1

t1/2 (h)

R2

R3

pKi h M3

O

nPr

8.95

O

Me

8.90

O

Me

10.0

0.76

O

Me

10.6

0.92

O

Me

10.3

0.32

O

Me

10.0

O

Me

9.9

S 26

27

OH *

S

S O *

0.17

S

S 28

OH S

*

S 29

*

S

S 30

31

32

*

S

OH *

*

S

0.12

(Continued)

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Table 12.2 (Continued)

Compound

R1

R2

R3

pKi h M3

t1/2 (h)

—

Me

10.56

3.3

S 33

OH *

S

pKi h M3: affinity at h M3 receptor; t1/2: dissociation half-life at the M3 receptor; mean of two independent experiments.

hydroxyl group by a methyl substituent (compound 29) or by a hydroxyl methylene group (compound 28) attenuated the potency. Finally, replacement of one or both thiophene groups by a cyclo-pentyl ring was detrimental for the potency of these compounds (compound 31 and 32). This indicated that the hydroxyl-dithienyl-acetyl group is perfectly designed to gain an optimal interaction with the M3 receptor. Ultimately, it has been demonstrated that the epoxy ring of scopolamine contributes to the potency of tiotropium. In fact, the corresponding compound 33 with the a-tropine moiety is approximately four times less potent than tiotropium. Comparison of the receptor off-rate kinetics indicates that compounds with a pKi below 10 (22, 27, and 32) have rather fast off-rate kinetics whereas the receptor offrate kinetics of compounds with a pKi above 10 increases significantly. Although an O

O

O

O

36 BrMg

S OH

O N

S

S

O

O

+

N

N

O HO

37

CH3Br O O

34

35 Figure 12.8 Synthesis of tiotropium bromide.

S OH S

–

Br

O O O

S OH S

23 tiotropium bromide

12.4 Preclinical Pharmacology: Comparison of Ipratropium and Tiotropium

exact correlation of both parameters might not be possible, it is obvious that increasing affinity results in a more prolonged receptor off-rate kinetic. Indeed, tiotropium showed the slowest receptor off-rate and the highest affinity (KD: 0.33 nM [38]). Comparison of the dissociation half-life from the M1–M3 receptor subtypes revealed that tiotropium exerts much longer receptor occupancy at the M3 receptor compared to the other muscarinic receptors [38]. Therefore, it can be speculated that the inhibition of M3-mediated bronchoconstriction will last much longer than side effects mediated by blockade of other muscarinic receptors. The synthesis of tiotropium bromide is shown in Figure 12.8. Starting with oxalic acid dimethyl ester, two equivalents of thiophene magnesium bromide were introduced. The hydroxyl-dithienyl-acetic acid methyl ester was then transesterified with scopine. Subsequently, the amino function was quaternized with methyl bromide.

12.4 Preclinical Pharmacology: Comparison of Ipratropium and Tiotropium

Tiotropium bromide and ipratropium bromide display a high affinity toward the muscarinic receptor subtypes M1–M3. While ipratropium bromide dissociates from the M3 receptor subtype with a half-life of 0.26 h, tiotropium shows a much longer dissociation half-life at the M3 receptor (27 h), thereby inducing long-lasting bronchodilation. At the molecular level, tiotropium binds to the M3 receptor much longer than to the M2 and M1 receptors with dissociation half-lives of 2.6 and 10.5 h, respectively [49]. Comparable results were obtained in earlier studies [38]. The sustained occupancy of the M3 receptor clearly explains the long-lasting bronchodilation of tiotropium bromide resulting in a once-daily dose regimen in patients. In contrast, the binding half-life at the M2 receptor that could induce unwanted effects is 10 times shorter. This phenomenon results in a “kinetic selectivity” of tiotropium toward the M3 receptor subtype, the pharmacological target responsible for bronchoconstriction and mucus hypersecretion in the pathophysiology of chronic airway diseases. The duration of action of the different antimuscarinics was monitored in preclinical in vitro studies using electric field stimulation (EFS) of isolated human and guinea pig conducting airways. In this model, electrical stimulation induces a release of endogenous acetylcholine resulting in constrictive responses of isolated conducting airways. The functional potency of the different antimuscarinics was tested in this model for their ability to inhibit the EFS effect. To measure the antagonist duration of action, unbound compound was removed from the system. EFS again resulted in constriction of the organs soon after removal of atropine or ipratropium from the organ bath (t1/2 [offset]: 81.2 min), as predicted on the basis of their short dissociation half-lives from the M3 receptor. In contrast, tiotropium bromide induced a long-lasting protection from EFS-induced constrictions of the isolated airways (t1/2 [offset]: 540 min), again in line with its sustained residence time at the M3 receptor [40].

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Increased cholinergic tone appears to be the major reversible component of COPD, inducing bronchoconstriction as a major disease symptom. Conclusively, animal models have been established inducing mild bronchoconstriction by topically administering acetylcholine to the airways (by inhalation) or systemically (by intravenous administration). The effects of the short-acting ipratropium bromide (15) and the long-acting anticholinergic tiotropium bromide (23) are described in two in vivo bronchoconstriction models that are conducted according to German and European animal welfare regulations. 12.4.1 Bronchoconstriction in Conscious Guinea Pigs According to the Method of Kallos and Pagel

Conscious guinea pigs are exposed to aerosolized ACh in an inhalation box. Untreated animals respond with bronchoconstriction to this stimulus followed by a respiratory collapse due to the hypoxaemic condition. The guinea pigs are removed from the ACh exposure immediately and they recover within a very short time [41]. Antimuscarinics are administered as solutions of different concentrations aerosolized into the inhalation boxes. When animals were treated with ipratropium bromide, a dose-dependent inhibition of the acetylcholine-induced bronchoconstriction could be observed. At a concentration of 1 mg/ml, 40% of the animals were protected from an acetylcholine-induced collapse whereas at 3 mg/ml all animals were protected, initially (Figure 12.9). Bronchoprotection attenuated over time, and 12 h after inhalation of the drug no effect could be observed any more. Tiotropium bromide showed a steep dose–response curve at concentrations of 0.1–1 mg/ml. While 0.1 and 0.3 mg/ml did not induce any detectable bronchoprotection, the concentration of 1 mg/ml protected 100% of the guinea pigs from bronchospasm. Even 24 h after inhalation of tiotropium bromide, 60% of the animals were protected and did not respond to ACh challenge. 12.4.2 Bronchoconstriction in Anaesthetized Dogs

Repeatedly administered intravenous acetylcholine induces mild and transient increases in respiratory resistance in anaesthetized dogs that can be measured using a pneumotachograph. Predrug values of acetylcholine-induced increases in respiratory resistance were compared with values obtained after inhalation of the test compounds. Inhalative treatment with 25 mg ipratropium bromide or 4 mg tiotropium bromide initially caused 80% protection of the acetylcholine-induced respiratory resistance (Figure 12.10). The bronchoprotective effect of ipratropium bromide rapidly attenuated over time. In contrast, tiotropium induced a long-lasting effect. More than 40% of the effect still remained 24 h after administration of tiotropium bromide.

12.5 Clinical Pharmacology

(b) 100

50 3.0 mg/ml 1.0 mg/ml

0 0

6

12 time [h]

18

24

bronchoprotection [%]

bronchoprotection [%]

(a)

j311

100

1.0 mg/ml 0.3 mg/ml 0.1 mg/ml

50

0 0

6

12

18

time [h]

Figure 12.9 In vivo investigation of ipratropium bromide (a) and tiotropium bromide (b) in a guinea pig model of ACh-induced bronchoconstriction.

12.5 Clinical Pharmacology

For clinical trials, a dry powder formulation of tiotropium bromide was prepared using the HandiHalerÒ device. Clinical studies in COPD patients usually measure the forced expiratory volume in 1 s, FEV1, as lung function parameter. This parameter measures the air volume of a maximal exhalation of a patient during 1 s. Once-daily inhalations of tiotropium bromide showed a clear dose-dependent bronchodilation with a long duration of action in the first placebo-controlled clinical trial in COPD patients [42]. In particular, the trough value, i.e., the bronchoprotection measured before administration of the next daily dose of tiotropium, showed significantly improved lung function compared to ipratropium. The significantly longer duration of action compared to ipratropium bromide found in preclinical studies was confirmed in phase II clinical studies over 13 weeks in 228 COPD patients. Tiotropium bromide at 18 mg administered with a dry powder inhaler system (HandiHalerÒ, Boehringer Ingelheim) once-daily was more effective than ipratropium at 40 mg four times a day with a comparable safety profile (Figure 12.11) [43]. Tiotropium induced a long-lasting bronchodilation over 24 h with a trough value exceeding that of ipratropium bromide. Both the efficacy and the long duration of action of tiotropium bromide have since been confirmed in numerous clinical studies showing beneficial effects on lung function, COPD exacerbations, and quality of life of COPD patients [44–47]. The favorable safety profile of tiotropium bromide dry powder inhalation has been confirmed in a large 4-year trial in 5993 COPD patients (UPLIFT trial). This study showed that the therapy with tiotropium was associated with improvements in lung function, quality of life, and COPD exacerbations. The decline in lung function over 4 years was, however, not influenced. Tiotropium bromide reduced respiratory morbidity and cardiac morbidity in COPD patients in this clinical landmark trial [48].

24

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100 tiotropium 4 µg

bronchoprotection [%]

80

ipratropium 25 µg

60 40 20 0 0.5

6

12

24

time [h] Figure 12.10 In vivo investigation of ipratropium bromide and tiotropium bromide in a dog model of ACh-induced bronchoconstriction.

Figure 12.11 Bronchodilation of tiotropium versus ipratropium in COPD patients. Reproduced from Ref. [43] with permission from BMJ Publishing Group Ltd.

12.7 Summary

Br +

N

O

+

OH

N

O

Br

O

S OH

O

S

O 38 glycopyrrolate (NVA 237)

39 aclidinium (LAS 34273)

Figure 12.12 Structure of glycopyrrolate and aclidinium.

12.6 Antimuscarinics in Clinical Development for the Treatment of COPD

Two other antimuscarinics are under late-stage development for the treatment of COPD. Glycopyrrolate has been marketed for years (RobinulÒ ) as premedication for general anesthesia as well as for treatment of peptic ulcers. Now this molecule is under development with inhaled administration for the treatment of COPD (NVA 237, Novartis). The molecule is an ester formed by combination of 2-hydroxy-N,Ndimethylpyrrolidine and 2-cyclopentyl-2-hydroxyl-pheylacetic acid. Aclidinium has been developed at Almirall Prodesfarma and is in phase III clinical development. The molecule bears the same acid moiety as tiotropium attached to a quaternized hydroxyl-quinuclidine base. Both antimuscarinics have been compared in preclinical models with tiotropium bromide. Tiotropium showed the highest potency at the human M3 receptor. Furthermore, the dissociation half-life was significantly longer for tiotropium (27 h) compared to glycopyrrolate (6.1 h) and aclidinium (10.7 h). In agreement with these in vitro findings, tiotropium provided the best bronchoprotection in vivo over a 24 h period in a dog model (Figure 12.12) [49].

12.7 Summary

The discovery of modern antimuscarinics is mainly based on modifications of naturally occurring tropane alkaloids. Starting from atropine and scopolamine, quaternization of the amino group led to compounds with an improved safety profile by limiting the systemic distribution after inhaled administration, which made these compounds very useful as bronchodilators in the treatment of COPD. Furthermore, quaternization of the amine also improved the duration of action due to prolonged retardation in the bronchial tissue. Further optimization of potency and receptor offrate kinetics by modifying the ester part of the molecule eventually led to tiotropium bromide. The duration of action of this drug enables once-daily dosing, inducing sustained bronchodilation and improving quality of life of chronically treated COPD patients. All these modifications can be judged as analogy-based drug discovery and other molecules under development for the treatment of COPD follow this principle.

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Acknowledgment

We are very grateful to Christopher Tautermann for the helpful discussion of the muscarinic receptor model data and the superimposition of N-methylscopolamine and acetylcholine in this model.

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Matthiesen, S. (2006) Control by cholinergic mechanism. Eur. J. Pharmacol., 533, 57–68. Wessler, I.K. and Kirkpatrick, C.J. (2001) The non-neuronal cholinergic system: an emerging drug target in the airways. Pulm. Pharmacol. Ther., 14, 423–434. Chapmann, K.R. (1993) History of anticholinergic treatment in airways diseases, in Anticholinergic Therapy in Obstructive Airways Diseases (ed. N.J. Gross), Franklin Scientific Publications, pp. 9–18. Rabe, K.F., Hurd, S., Anzueto, A., Barnes, P.J., Buist, S.A., Calverley, P., Fukuchi, Y., Jenkins, C., RodriguezRoisin, R., van Weel, C., and Zielinski, J. (2007) Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: gold executive summary. Am. J. Respir. Crit. Care Med., 176, 532–555. Hammer, R., Berrie, C.P., Bridsall, N.J.M., Burgen, A.S.V., and Hulme, E.C. (1980) Pirenzepine distinguishes between different subclasses of muscarinic receptors. Nature, 283, 90–92. Bonner, T.I., Buckley, N.J., Young, A.C., and Brann, M.R. (1987) Identification of a family of muscarinic receptor genes. Science, 237, 527–532. Buckley, N.J., Bonner, T.I., Buckley, C.M., and Brann, R. (1989) Antagonist binding properties of five cloned muscarinic receptors expressed in CHO-K1 cells. Mol. Pharmacol., 35, 469–476. Hulme, E.C., Birdsall, N.J.M., and Buckley, N.J. (1990) Muscarinic receptor

9

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16

subtypes. Annu. Rev. Pharmacol. Toxicol., 30, 633–673. Eglen, R.M. and Nahorski, S.R. (2000) The muscarinic M5 receptor: a silent or emerging subtype? Br. J. Pharmacol., 130, 13–21. Bymaster, F.P., McKinzie, D.L., Felder, C.C., and Wess, J. (2003) Use of M1–M5 muscarinic receptor knockout mice as novel tools to delineate the physiological roles of the muscarinic cholinergic system. Neurochem. Res., 28, 437–442. Wess, J., Eglen, R.M., and Gautam, D. (2007) Muscarinic acetylcholine receptors: mutant mice provide new insights for drug development. Nat. Rev. Drug Discov., 6, 721–733. Lee, A.M., Jacoby, D.B., and Fryer, A.D. (2001) Selective muscarinic receptor antagonists for airway diseases. Curr. Opin. Pharmacol., 1, 223–229. Rang, H.P., Dale, M.M., Ritter, J.M., and Flower, R.J. (2008) Cholinergic transmission, in Rang and Dale’s Pharmacology, 6th edn, Chirchill Livingstone Elsevier, pp. 144–167. Ringdahl, B. (1986) Dissociation constant and relative efficacies of acetylcholine, ( þ )- and (
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)S- and L( þ )Rtropinoylcholines at cholinergic sites. J. Pharmacol. Exp. Ther., 202, 105–115. Schulz, W., Banholzer, R., and Pook, K.-H. (1976) A new method for technical synthesis of tertiary and quaternary D,L-tropic acid esters of some Nsubstituted nortropan- and granatan3-ols. Drug Res., 26, 960–974. Banholzer, R. and Pook, K.H. (1985) Synthesis of anticholinergically active N-alkylnorscopolamines and their quaternary salts with particular consideration of the bronchospasmolytic compound (
)-N-ethylnorscopolamine methobromide (Ba 253 BR). Drug Res., 35, 217–228. Tytgat, G.N. (2007) Hyoscine butylbromide: a review of its use in the treatment of abdominal cramping and pain. Drugs, 67, 1343–1357. Barnes, P.J. (2004) The role of anticholinergics in chronic obstructive pulmonary disease. Am. J. Med., 117, 24S–32S. Bauer, R., P€ uschmann, S., and Wick, H. (1976) The effect of (8r)-3a-hydroxy-8isopropyl-1aH,5aH-tropaniumbromide()-tropate (ipratropium bromide) on tracheobronchial spasm, bronchial and salivary secretions, EKG and heart rate. Drug Res., 26, 981–985. Bauer, R. (1985) Pharmacology of oxitropium bromide, an anticholinergic bronchodilator. Drug Res., 35, 435–440.

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Relations between constitution and pharmacological activity in tropeinens and their quaternary derivatives, especially N-octylatropinium bromide. Drug Res., 7, 217–222. Banholzer, R., Pook, K.H., and Stiasni, M. (1986) Synthesis of the bronchospasmolytic agent flutropium bromide and of some homologous and configuration isomeric compounds. Drug Res., 36, 1161–1166. Beld, A.J., Van Den Hoven, S., Wouterse, A.C., and Zegers, M.A.P. (1975) Are muscarinic receptors in the central and peripheral nervous system different? Eur. J. Pharmacol., 30, 360–363. Patel, H.J., Barnes, P.J., Takahashi, T., Tadjkarimi, S., Yacoub, M.H., and Belvisi, M.G. (1995) Evidence for prejunctional muscarinic autoreceptors in human and guinea pig trachea. Am. J. Respir. Crit. Care Med., 152, 872–878. Disse, B., Reichl, R., Speck, G., Traunecker, W., Rominger, K.L., and Hammer, R. (1993) BA 679 BR, a novel long-acting anticholinergic bronchodilator. Life Sci., 52, 537–544. Motulsky, H.J. and Mahan, L.C. (1984) The kinetics of competitive radioligand binding predicted by the law of mass action. Mol. Pharmacol., 25, 1–9. Takahashi, T., Belvisi, M.G., Patel, H., Ward, J.K., Tadjkarimi, S., Yacoub, M.H., and Barnes, P.J. (1994) Effect of Ba 679 BR, a novel long-acting anticholinergic agent, on cholinergic neurotransmission in guinea pig and human airways. Am. J. Respir. Crit. Care Med., 150, 1640–1645. Kallos, P. and Pagel, W. (1937) Experimental investigations on asthma bronchale. Acta Med. Scand., 91, 292–305. Maesen, F.P., Smeets, J.J., Sledsens, T.J., Wald, F.D., and Cornelissen, P.J. (1995) Tiotropium bromide, a new long-acting antimuscarinic bronchodilator: a pharmacodynamic study in patients with chronic obstructive pulmonary

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disease (COPD). Dutch Study Group. Eur. Respir. J., 8 (9), 1506–1513. van Noord, J.A., Bantje, T.A., Eland, M.E., Korducki, L., and Cornelissen, P.J. (2000) A randomised controlled comparison of tiotropium and ipratropium in the treatment of chronic obstructive pulmonary disease. The Dutch Tiotropium Study Group. Thorax, 55, 289–294. Casaburi, R., Mahler, D.A., Jones, P.W., Wanner, A., San, P.G., ZuWallack, R.L., Menjoge, S.S., Serby, C.W., and Witek, T. (2002) A long-term evaluation of once-daily inhaled tiotropium in chronic obstructive pulmonary disease. Eur. Respir. J., 19, 217–224. Casaburi, R. and Conoscenti, C.S. (2004) Lung function improvements with once-daily tiotropium in chronic obstructive pulmonary disease. Am. J. Med., 117 (Suppl. 12A), 33S–40S. Niewoehner, D.E., Rice, K., Cote, C., Paulson, D., Cooper, J.A., Jr., Korducki, L., Cassino, C., and Kesten, S. (2005) Prevention of exacerbations of chronic obstructive pulmonary disease with tiotropium, a once-daily inhaled anticholinergic bronchodilator: a randomized trial. Ann. Intern. Med., 143, 317–326. Tonnel, A.B., Perez, T., Grosbois, J.M., Verkindre, C., Bravo, M.L., and Brun, M. (2008) TIPHON study group. Effect of tiotropium on health-related quality of life as a primary efficacy endpoint in COPD. Int. J. Chron. Obstruct. Pulmon. Dis., 3, 301–310. Tashkin, D.P., Celli, B., Senn, S., Burkhart, D., Kesten, S., Menjoge, S., and Decramer, M. (2008) UPLIFT Study investigators. A 4-year trial of tiotropium in chronic obstructive pulmonary disease. N. Engl. J. Med., 359 (15), 1543–1554. Casarosa, P., Bouyssou, T., Schnapp, A., Gantner, F., and Pieper, M. (2009) Preclinical evaluation of long acting anticholinergics (LAAC): comparison of tiotropium and investigationaldrugs. J. Pharmacol. Exp. Ther., 330, 660–668.

References

Paola Casarosa

Boehringer Ingelheim Pharma GmbH & Co. KG, Pulmonary Diseases Research, 88397 Biberach an der Riss, Germany Paola Casarosa received a master’s degree in medicinal chemistry at the University of Torino, Italy. Afterward, she joined the group of Professors H. Timmermann and R. Leurs at the Vrije Universiteit in Amsterdam, The Netherlands, where she received a PhD in molecular pharmacology, with a thesis on viralencoded GPCRs. After a postdoctoral experience at Bichat Hospital in Paris, where she worked on the role of viral chemokine receptors in HIV proliferation, she joined Organon NV, a Dutch pharmaceutical company, as lab head in the therapeutic area of rheumatoid arthritis. Since 2007, Paola has been working in the Department of Pulmonary Diseases Research at Boehringer Ingelheim Pharma GmbH & Co. KG, Germany.

Matthias Grauert

Boehringer Ingelheim Pharma GmbH & Co. KG, Diseases Chemical Research, 88397 Biberach an der Riss, Germany Matthias Grauert studied chemistry at the Georg-August University of G€ottingen, Germany. There he joined the group of Professor U. Sch€ollkopf and received his diploma degree and PhD in organic chemistry with a thesis on asymmetric synthesis. In 1987, he started to work as lab head in the Department of Chemical Research at Boehringer Ingelheim KG, Ingelheim, Germany. In 1994, he took a sabbatical to work in the lab of Dr. Michael A. Rogawski, at the National Institute of Neurological Disorders and Stroke, NIH, Bethesda, Maryland, USA. Since 1998, he has been Group Leader in the Department of Chemical Research at Boehringer Ingelheim Pharma GmbH & Co. KG, in Biberach, Germany. He has been working on different drug discovery projects on CNS, Oncology, Pulmonary Disease, and Metabolic Disease.

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Michael P. Pieper

Boehringer Ingelheim Pharma GmbH & Co. KG, Pulmonary Diseases Research, 88397 Biberach an der Riss, Germany Michael Pieper studied veterinary medicine at the University of Veterinary Medicine, Hanover, Germany. He received a DVM for his research on LTB4-activated granulocytes conducted in collaboration with Professor Wolfgang L€ oscher, Department of Pharmacology, Toxicology & Pharmacy, University of Veterinary Medicine, Hanover, and Boehringer Ingelheim KG in 1994. After a postdoctoral period at the same university, he headed a pharmacology lab at General Pharmacology and subsequently Pulmonary Diseases Research at Boehringer Ingelheim. Here, he led the team of pharmacology for the Boehringer Ingelheim anticholinergics program. He is a board-certified Veterinarian for Pharmacology and Toxicology. As senior principal scientist, he is currently responsible for the Product and Pipeline Scientific Support for Bronchodilators at Boehringer Ingelheim.

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13 b-Adrenoceptor Agonists and Asthma Giovanni Gaviraghi

13.1 Introduction

Bronchial asthma is recognized as a complex disease characterized by bronchial constriction, airway smooth muscle hyperreactivity, and inflammation. In the early twentieth century, it was discovered that adrenaline 1, a natural neurotransmitter, was able to control severe acute asthma by relaxing airway smooth muscle [1]. Classification of adrenergic receptors by Ahlquist [2] into a- and b-types explained the bronchodilator effect of adrenaline 1, as a b-adrenoceptor agonist, and paved the way for its use as an antiasthma drug. However, the subsequent further classification of b-receptors into b1 and b2 assigned the bronchial dilatation to the b2-subtype and opened the way for finding new agonists endowed as potent and more selective antiasthma drugs [3]. In addition, the short-acting effect of adrenaline 1 identified the need for b2-agonists with longer duration of action. Interestingly, a synthetic analogue of adrenaline ephedrine 2 was identified as the antiasthmatic component of an old Chinese drug and although less potent and selective than adrenaline, it showed that synthetic modification of the adrenaline molecule was possible and potentially useful for improving its biological and pharmacokinetic properties [4].

13.2 First-Generation b2-Agonists: The Short-Acting Bronchodilators

The first step toward better compounds was the synthesis of isoprenaline 3, a selective b2-agonist devoid of vasopressor activity but producing a marked hypotension and cardiac stimulation. Structure–activity relationship studies on the isoprenaline molecule indicated that the cardiac stimulating effect could be reduced simply by introducing an a-substituent in the lateral chain of the isoprenaline 3 molecule. Thus, isoetarine 4 was identified as a better drug. Its blood pressure reduction side effect was partially reduced by inhaled route administration to the asthmatic patient. Isoetarine 4 has been the first effective inhaled antiasthmatic drug introduced in the

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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Chemical structures of some b-adrenoceptor agonists with an isopropyl group attached to the nitrogen atom.

Table 13.1

OH R2

H N

R1

R3 R4

(±)

Compound

R1

R2

R3

R4

Isoprenaline 3 Isoetarine 4 Orciprenaline 5 Soterenol Dichloroisoprenaline

H C2H5 H H H

HO HO HO CH3
SO2
NH Cl

HO HO H H Cl

H H HO HO H

Compounds are racemic with the relative stereochemistry as indicated for orciprenaline diastereoisomers.

therapeutic armamentarium. And although vascular and cardiac effects were still present even with topical administration to the lungs, isoetarine 4 was clearly showing the way forward for optimization in this field (Table 13.1). Final classification of b1- and b2-adrenoceptors in the 1960s prompted a number of investigators to synthesize more selective derivatives in the hope of avoiding the cardiac stimulant effects of the known b-agonists. Also, the short duration of action, due to fast metabolism of the catechol moiety, represented a big limiting factor in their therapeutic use. Orciprenaline 5, where the catechol hydroxy groups have been put in the meta position, was the first compound resistant to catechol methyl transferase and thus endowed with a more persistent effect. With all this information derived from the SAR studies, the medicinal chemistry background of the b2 class was ready for a further improvement. This was achieved by introducing the tert-butyl group on the nitrogen atom of the side chain (Table 13.2). Both terbutaline 6 and salbutamol 7 emerged as the most selective drugs, with reduced cardiac stimulating effect. Salbutamol, moreover, showed the best pharmacokinetic profile as one catechol hydroxy group has been replaced by a CH2-OH, thus reducing the drug oxidation and methylation by catechol O-methyl transferase. Salbutamol 7 was the real breakthrough, being able to possess both potency and selectivity along with an acceptable duration of action; its wide use for relieving acute bronchoconstriction episodes in the last 40 years clearly demonstrates its good profile. In line with the other compounds described in Figure 13.1, salbutamol was developed as a racemic mixture, although the pharmacologically active eutomer is R-salbutamol. Interestingly, the R-enantiomer is more susceptible to first-pass metabolism than the S-distomer that is therefore enriched at steady state [5].

13.4 Third-Generation b2-Agonists: The Long-Acting Bronchodilators Chemical structures of some b2-adrenoceptor agonists with a tert-butyl group attached to the nitrogen atom.

Table 13.2

R1

OH

R2

H N

R3 R4 Compound

R1

R2

R3

R4

Colterol Terbutaline 6 Salbutamol 7 Carbuterol Sulfonterol Clenbuterol 11 Tulobuterol

H H H H H H Cl

HO HO HO
CH2 H2N
CO
NH CH3
SO2
CH2 Cl H

HO H HO HO HO H2N H

H HO H H H Cl H

13.3 Second-Generation b2-Agonists: Further Derivatives of Salbutamol

Results obtained with salbutamol 7 and terbutaline 6 prompted significant synthetic activities in various research groups. An early example of these efforts was fenoterol 10, bearing a 4-hydroxyphenyl ring on the terminal methyl group of orciprenaline 5. Initially developed as a bronchodilating b-adrenoreceptor agonist in 1964 [6], a better picture of its b2 selectivity emerged following disclosure of the properties of salbutamol 7 some years later [7]. Another molecule identified before the full disclosure of salbutamol and terbutaline 6 is clenbuterol 11 [8]. This compound has been found to have significant anabolic effects on animals [9]; however, it has found application in equine medicine [10]. Other early compounds included formoterol 15 [11], which will be discussed in more detail later, and the heterocyclic derivatives pirbuterol 8 [12], broxaterol 9 [13], and procaterol 12 [14]. Procaterol, despite its potency and selectivity for the b2-receptor, offered no clinical advantage over salbutamol [15]. The same lack of clinical advantage was true for all b2-agonists that immediately followed salbutamol with the exception of formoterol.

13.4 Third-Generation b2-Agonists: The Long-Acting Bronchodilators

The excellent clinical results obtained with salbutamol 7 prompted the search for a compound behaving as a long-acting b2-agonist (LABA), for the management of severe persistent asthma. This work, initiated in 1980, was based on the idea of

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OH

OH

H N

HO

OH

H N

HO

HO

H N

HO Adrenaline 1 OH

HO

Isoprenaline 3

Ephedrine 2 OH

H N

HO

OH

H N

HO

H N

HO OH Orciprenaline 5

Isoetarine 4 OH HO

OH

H N

N

HO

HO

OH

H N

N O Broxaterol 9

HO Pirbuterol 8 OH HO

OH H N

Cl OH

OH

OH

HN

Cl Clenbuterol 11 OH H N

N

H N

HO

HO Procaterol (±)12 OH

H N

H2N

Fenoterol (±)10

O

H N

Br

Salbutamol 7

HO

OH Terbutaline 6

Quinprenaline 13 OH

H N

O

HO

H N OMe

HO Salmeterol 14

NH

H O

Formoterol (±)15

Figure 13.1 Chemical structure of beta agonists. With the exception of adrenaline and ephedrine, all compounds are racemic mixtures. Where diastereomers are possible, the relative stereochemistry is indicated.

13.4 Third-Generation b2-Agonists: The Long-Acting Bronchodilators

OH HO

H N

HO A

B

Figure 13.2 Adding a lipophilic moiety to a saligenin ethanolamine core to increase duration of action.

designing compounds able to exploit an increased specific lipophilic binding in an area close to the b2-adrenoceptor [16, 17]. Thus located, the molecule would be able to continue to act at the receptor active site over a longer timescale [18]. The strategy adopted was the introduction of lipophilic substituents (B) at pharmacophorically tolerated positions on a saligenin ethanolamine core (A). In this way, affinity and selectivity for the receptor could be maintained or increased while exploiting lipophilic exo-receptor binding sites to give longer duration of action (Figure 13.2). Compounds were evaluated in vitro using superfused, isolated guinea pig tracheal strips. Contraction was provoked initially with agents such as PGF2a as spasmogen, but these were superseded over time with the more reproducible electrical stimulus [19]. Preliminary results obtained are summarized in Table 13.3. A compound synthesized previously, salmefamol 16, gave weight to the hypothesis of a lipophilic substituent on a saligenin core improving the duration of action. It was also found to be longer acting (6 h) than salbutamol in man [20]. Using calculated log P as a guide [16], further modifications to salmefamol were made, leading to compounds 18

Table 13.3 Structure–activity and structure–duration of action relationships for a range of 4-aryl substituents with varying degrees of lipophilicity.

OH HO

H N X

HO Compound

6 16 17 18 19 a)

X

% b2 potencya) (isoprenaline 3 ¼ 100%)

Duration of actiona) (min)

clog P

Salbutamol OCH3 (salmefamol) O(CH2)2OC2H5 O(CH2)4CH3 OPh

50% 37% 48% 3% 9%

3.7 7.3 6.7 >50 22.5

0.66 1.68 1.85 3.96 3.96

PGF2a-contracted guinea pig trachea.

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Table 13.4 Structure–activity and structure–duration of action relationships for a range of hexyloxy spaced substituents.

OH

H N

HO

O

( )n

HO Compound

n

% b2 potencya) (isoprenaline 3 ¼ 100%)

Duration of actiona) (min)

clog P

20 21 22

2 3 4

200% 83% 111%

189 >400 >420–>720

2.8 3.35 3.88

a)

Electrically stimulated guinea pig trachea preparation.

and 19 that demonstrated further improvements to the duration of action, albeit at the expense of potency at the b2-receptor. Subsequent structure–activity studies explored the hexyloxy spacer group between the saligenin core and the aromatic ring. These compounds, summarized in Table 13.4, were able to recoup the levels of b2-receptor activity seen with salbutamol while maintaining the long duration of action. Examination of the effects of simultaneous variations to the alkyl chain length either side of the ether oxygen atom was also carried out, as shown in Table 13.5 [16]. In terms of maintaining potency, the m chain needed to contain five or six methylene groups with two–four methylenes in the n chain. Duration of action followed a general calculated log P trend, with 3.3–4.5 being optimal. Maintaining the total m þ n count at 10 methylenes, the optimal positioning of the ether oxygen was investigated and the results are shown in Table 13.6. Significant variation was found in the duration of action in this series, in which the clog P remained constant. Salmeterol 14 proved to have an optimal duration of action and potency. In the same period, further investigations were carried out on the formulation and route of administration of formoterol 15. An oral formulation gave no evidence of an increased duration of action compared to salbutamol [21]. Significant differences were, however, noted in an inhaled formulation, with a duration of action of at least 8 h [22]. Subsequent comparative studies have demonstrated that both salmeterol 14 and formoterol 15 have a duration of action of at least 12 h [23–25]. The exact mechanism responsible for the different pharmacodynamic effects observed for salbutamol, fomoterol, and salmeterol 14 has been the subject of considerable study. A number of hypotheses, essentially based on the influence of physicochemical properties on membrane interactions, have been developed to account for the variation in both onset and duration of action [4, 16]. Salbutamol, with its fast onset and short duration, is generally used to relieve symptoms. Salmeterol 14 has a slow onset and long duration and is applied for maintenance

13.4 Third-Generation b2-Agonists: The Long-Acting Bronchodilators Table 13.5 Structure–activity and structure–duration of action relationships of varying chain lengths either side of the ether oxygen atom.

OH HO

H N

O ( )m ( )n

HO Compound

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

m

n

clog P

% b2 potencya) (isoprenaline 3 ¼ 100%)

Duration of action (min)

4 4 4 4 5 5 5 5 6 6 6 6 6 6 7

3 4 5 6 2 3 4 5 1 2 3 4 5 6 3

2.3 2.83 3.3 3.88 2.3 2.83 3.35 3.88 2.3 2.83 3.35 3.88 4.41 4.8 3.88

1% 6% 10% 15% 83% 27% 200% 71% 10% 91% 42% 111% 18% 13% 11%

6.8 20.5 >30 >60 5.0 >14 >30 >400 6.5 10.7 >35.0 >720 >300 290 >300

treatment. Fomoterol, however, has a fast onset and long duration and can therefore potentially be used in both acute and chronic settings. The hydrophilic nature of salbutamol ensures that it remains in the aqueous extracellular environment. It can therefore directly access the b-adrenoceptor active Table 13.6 Structure–duration of action relationships of scanning the oxygen atom position at fixed chain length (m þ n ¼ 10).

OH HO

H N

O ( )m ( )n

HO Compound

38 39 40 41 14 (Salmeterol) 42

m

n

clog P

Duration of action (min)

4 5 2 8 6 9

6 5 8 2 4 1

3.88 3.88 3.88 3.88 3.88 3.88

>60.0 >400.0 2.7 >30 >720 47.6

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Figure 13.3 Physicochemical properties modulating the partitioning behavior of formoterol 14, salbutamol 6, and salmeterol 13. (Adapted from Ref. [16].)

site in a rapidly equilibrating manner and thus have rapid onset and short duration of action. Salmeterol, on the other hand, is believed to rapidly partition into the outer phospholipid monolayer, before diffusing slowly through the membrane to the b-adrenoceptor active site. The fast onset of action and long duration of formoterol 15 has been explained by the compound not only being able to exploit lipophilic membrane association but also being able to directly access the b-adrenoceptor active site, without slow lateral diffusion through the phospholipid (Figure 13.3). This hypothesis was used to guide the design of longer acting compounds.

13.5 Combination Therapy with LABA and Corticosteroids

The beneficial bronchodilating activity of b2-receptor agonists in the control of bronchoconstriction induced by asthma and chronic obstructive broncho-pneumopathies (COBP) has been widely proved in therapy over the past 40 years. Short-acting b2-agonists such as salbutamol can be used for relieving the bronchoconstriction in the acute asthma attack while the LABA by virtue of their long lasting activity can control the constriction in chronic persistent asthma patients. Many recent studies have demonstrated that adding inhaled corticosteroids (ICS) such as fluticasone to LABA increases the clinical benefits in the therapy of moderate and severe asthma. The rationale for this combination therapy lies in the synergistic

13.6 Future Directions: Once-a-Day Therapy and Bifunctional Muscarinic

mechanisms shown by the two drugs: steroids reduce bronchial inflammation and increase the transcription of b2-receptors, thus reducing the downregulation of b2-receptors produced by chronic exposure [26]. This strong synergy led to the development of inhaled fixed dose combinations of LABA and ICS such as salmeterol/fluticasone and formoterol/budesonide that in turn increase patient compliance to the chronic asthma therapy. In conclusion, with the availability of short-acting b2-agonists, ICS, and LABA in the modern therapeutic armamentarium, both episodic asthma attacks and chronic persistent asthma can be fully controlled by inhaled short-acting b2-agonists and by regular administration of inhaled ICS and LABA fixed combination, respectively.

13.6 Future Directions: Once-a-Day Therapy and Bifunctional Muscarinic Antagonist–b2-Agonist (MABA)

Results obtained with the long-acting b2-adrenoceptor agonists salmeterol 14 and formoterol 15 have encouraged research into even longer acting agents, able to be administered once a day, possibly in combination with a once-a-day glucocorticoid agonist [27, 28]. An example of such a compound is milveterol 43 (Figure 13.4), which is in phase II clinical trials for asthma and COPD [29, 30]. Indacaterol 44, another b2-adrenoceptor with potential for once-daily administration, has also been the subject of recent clinical investigations [31]. It has been shown to provide sustained bronchodilation in asthma patients [32] and for COPD [33].

OH

O

H N

HN N H

HO HN

H O

44 Indacaterol

H N

H N O

HO

H N

HO

OH

43 Milveterol

OH

OH

OH

NHSO2Me 45 PF-610,355 Figure 13.4 The long-acting b2-adrenoceptor agonists milveterol, indacaterol, and PF-610,355.

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N OH O Me

H N

HO

S N O H

OH 46

O S

OH

HN

N

O

H

H N O

HO

O

S

47

S OH

H N

O O HN HO

OH

O H N

H N Cl

N

O

O

48

Figure 13.5 Examples of bifunctional muscarinic antagonist–b2-agonists.

Another LABA, PF-610,355 45, is also undergoing phase II clinical trials for asthma and COPD [34]. The potential benefits of coadministering a b2-adrenoceptor agonist with a muscarinic antagonist such as ipratropium have been known for some time and this has been an effective approach for the management of acute asthma [35, 36]. At present, a number of research groups are investigating the possibility of a single molecule with ability to modulate both the b2 adrenergic and the muscarinic systems (Figure 13.5). Generally, this approach has been based upon incorporating a system able to interact with the muscarinic receptor into the extended side chain common to longacting b2-adrenoceptor agonists such as salmeterol and formoterol. Examples of this approach include compounds 46 [37, 38], 47 [39], and 48 [40]. At least one such compound GSK961081, the structure of which has yet to be disclosed, is undergoing phase II clinical trials. This approach, should it prove successful, could represent a significant step forward in the management of asthma and COPD.

References

Acknowledgments

I thank Dr. G. Recchia, Dr. D. Micheli, and Dr. S. Magnoni (GSK) for their valuable scientific support and discussion and Dr. R. Thomas (Siena Biotech) for his technical contribution to the preparation of this manuscript.

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10 Erichsen, D.F., Aviad, A.D., Schultz, R.H.,

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and Kennedy, T.J. (1994) Clinical efficacy and safety of clenbuterol HCl when administered to effect in horses with chronic obstructive pulmonary disease (COPD). Equine Vet. J., 26, 331–336. (a) Ida, H. (1976) Comparison of the action of BD 40A and some other b-adrenoceptor stimulants on the isolated trachea and atria of the guinea pig. Arzneim.-Forsch., 26, 839–842;(b) (1976) Cardiorespiratory activities of 3-formylamino-4-hydroxya-(N-1-methyl-2-p-methoxyphenethyl aminomethyl)-benzylalcoholhemifumarate (BD 40A) and some other b-adrenoceptor stimulants in conscious guinea pigs. Arzneim.-Forsch., 26, 1337–1340. Moore, P.F., Constantine, J.W., and Barth, W. (1978) Pirbuterol, a selective b2 adrenergic bronchodilator. J. Pharmacol. Exp. Ther., 207, 410–418. Chiarino, D., Fantucci, M., Carenzi, A., Della Bella, D., Frigeni, V., and Sala, R. (1986) New isoxazole derivatives with a potent and selective b2-adrenergic activity. Pharmaco. Ed. Sci., 41, 440–453. Yabuuchi, Y. (1977) The b-adrenoceptor stimulant properties of OPC-2009 on guinea-pig isolated tracheal, right atrial and left atrial preparations. Br. J. Pharmacol., 61, 513–521. Crowe, M.J., Counihan, H.E., and O’Malley, K. (1985) A comparative study of a new selective b2-adrenoceptor agonist, procaterol and salbutamol in asthma. Br. J. Clin. Pharmacol., 19, 787–791. Johnson, M. (1995) Salbutamol. Med. Res. Rev., 15, 225–256, and references cited therein. Brittain, R.T. (1990) Approaches to a long-acting, selective b2-adrenoceptor stimulant. Lung, 168, 111–114.

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for asthma and COPD? Drugs, 67, 503–515. Moran, E.J., Griffin, J.H., and Choi, S. (2001) Preparation of 4-(arylhydroxyethylaminoethyl) phenylamino hydroxyethylbenzenes and related compounds as b2 adrenergic receptor agonists and partial agonists. WO2001042193. Pahl, A. and Szelenyi, I. (2007) Chronic obstructive pulmonary disease (COPD). Drugs Fut., 32, 799–807. Sturton, R.G., Trifilieff, A., Nicholson, A.G., and Barnes, P.J. (2008) Pharmacological characterization of indacaterol, a novel once daily inhaled b2 adrenoceptor agonist, on small airways in human and rat precision-cut lung slices. J. Pharmacol. Exp. Ther., 324, 270–275. Beeh, K.M., Derom, E., Kanniess, F., Cameron, R., Higgins, M., and van As, A. (2007) Indacaterol, a novel inhaled b2-agonist, provides sustained 24-h bronchodilation in asthma. Eur. Respir. J., 29, 871–878. Beier, J., Chanez, P., Martinot, J.-B., Schreurs, A.J.M., Tk
acov
a, R., Bao, W., Jack, D., and Higgins, M. (2007) Safety, tolerability and efficacy of indacaterol, a novel once-daily b2-agonist, in patients with COPD: a 28-day randomised, placebo controlled clinical trial. Pulm. Pharmacol. Ther., 20, 740–749. Brown, A.D., Bunnage, M.E., Glossop, P.A., James, K., Lane, C.A.L., Lewthwaite, R.A., Moses, I.B., Price, D.A., and Thomson, N.M. (2005) A preparation of sulfonamide of (aminoethyl)phenols, useful for the treatment of allergic and respiratory diseases. US2005182091. Garrett, J.E., Town, G.I., Rodwell, P., and Kelly, A.M. (1997) Nebulized salbutamol with and without ipratropium bromide in the treatment of acute asthma. J. Allergy. Clin. Immunol., 100, 165–170. Fitzgerald, M.F. and Fox, J.C. (2007) Emerging trends in the therapy of COPD: bronchodilators as mono- and combination therapies. Drug Discov. Today, 12, 472–478.

References 37 James, K., Jones, L.H., and Price, D.A.

(2007) Preparation of phenol amines as b2-adrenergic agonists and muscarinic antagonists for disease treatment. WO2007107828. 38 Norman, P. (2008) Pfizer’s dual-acting b2 agonists/muscarinic M3 antagonists. Exp. Opin. Ther. Patents, 18, 1091–1096. 39 Alcaraz, L., Kindon, N., and Sutton, J.M. (2008) Bicyclo[2.2.1]hept-2-ylamine

derivatives as M3 muscarinic and b2 adrenergic modulators, their preparation, pharmaceutical compositions, and use in therapy. WO2008149110 40 Mammen, M., Dunham, S., Hughes, A., Lee, T.W., Husfeld, C., and Stangeland, E. (2004) Preparation of biphenyl derivatives as b2-adrenergic agonists and muscarinic antagonists for pulmonary disorders. US2004167167

Giovanni Gaviraghi

Siena Biotech SpA, 35, Strada del Petriccio e Belriguardo, 53100 Siena, Italy Giovanni Gaviraghi, Dr. C Chem MD, is CEO of Siena Biotech, a company working on CNS diseases. Before, he spent more than 20 years at Glaxo where he was R&D Director of the Verona Center. His research interest includes CNS (stroke, pain, and depression), infectious (betalactams, quinolones, and macrolides), and cardiovascular diseases (hypertension, arteriosclerosis, and cardiac ischemia). He was involved in the discovery and development of Lacidipine, a potent calcium channel blocker for the treatment of hypertension. Before joining Glaxo, he was the Head of Lab at ISF and a Lecturer at Polytechnic of Milan.

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Part III Case Histories

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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14 Liraglutide, a GLP-1 Analogue to Treat Diabetes Lotte B. Knudsen 14.1 Introduction

The therapeutic potential of glucagon-like peptides (GLPs), particularly glucagon-like peptide-1 (GLP-1), is only now beginning to be realized at the start of the twenty-first century. It was, however, the discovery of secretin in 1902 by Bayliss and Starling [1] that initiated interest in the endocrine function of the gut and pancreas. These scientists speculated that signals arising from the gut could elicit an endocrine response affecting carbohydrate disposal. In 1929, Zunz and LaBarre described an intestinal extract that could produce hypoglycemia [2], and in a separate paper, LaBarre used the term “incretin” to describe activity in the gut that might stimulate pancreatic endocrine secretions [3]. Despite initial interest in “incretin,” research virtually stopped in this area due to the outbreak of World War II and the publication of several negative papers by Ivy and colleagues [4–6]. Twenty-five years later, McIntyre suggested that a humoral substance was released from the jejunum during glucose absorption, acting in concert with glucose to stimulate insulin release from pancreatic b-cells [7]. In 1969, Unger and Eisentraut referred to the gut–pancreas association as the enteroinsular axis [8], and this axis was subsequently described as involving nutrient, neural, and hormonal signals from the gut to the pancreatic islet cells. To be termed an “incretin,” any substance acting on this pathway must be secreted in response to nutrient stimuli and must stimulate glucose-dependent insulin secretion [9]. A second incretin hormone, following the discovery of glucose-dependent insulinotropic polypeptide (GIP), was postulated to exist as a consequence of the cloning of cDNAs encoding the preproglucagon gene in anglerfish pancreas [10–12]. Habener and colleagues conducted some of the very early work to characterize preproglucagon, but it was Bell who first identified GLP-1(1-37) [13]. In 1986/1987, it was discovered that the truncated forms, GLP-1(7-37) and (7-36)amide, were the active insulinotropic isoforms of GLP-1 [14, 15]. Lowering of blood glucose with GLP1 was first shown in three studies by Nathan, Nauck, and Kreymann [16–18], with the

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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Figure 14.1 Differential post-translational processing of proglucagon in the pancreas and in the gut and brain. The numbers indicate amino acid positions in the 160-amino acid proglucagon sequence. The vertical lines

indicate positions of basic amino acid residues, typical cleavage sites. GRPP, glicentin-related pancreatic polypeptide; IP-1, intervening peptide-1; IP-2, intervening peptide-2. Reproduced with permission from Holst et al. [19].

therapeutic application of GLP-1 realized in 1993 with the observation that GLP-1 could normalize blood glucose levels in patients suffering from type 2 diabetes [17]. The GLP-1(7-37) isoforms, together with the GLP-1(7-36)amide, are the insulinotropic peptides derived from the preproglucagon gene, products of the post-translational processing of proglucagon (Figure 14.1). The amino acid sequence of GLP-1 is highly conserved across animal species [20]. This shows not only how important this hormone is but also how vital the particular amino acid sequence is (Figure 14.2).

Figure 14.2 Amino acid sequence of native, truncated GLP-1(7-37).

14.1 Introduction

The binding affinity and biological activity are particularly affected by His on position 7, Gly on position 10, Phe on position 12, Thr on position 13, Asp on position 15 (all directly involved in receptor interaction), Phe on position 28, and Ile on position 29 [21]. The conformation of GLP-1 includes an N-terminal random coil and two helical segments joined by a linker region; this closely resembles the structure of glucagon [22]. The preproglucagon gene is expressed in several cell types in the body. The pancreas contains a- and b-cells: a-cells process proglucagon and therefore secrete glucagon. Only small quantities of GLP-1 have been found secreted from pancreatic a-cells [23]. Proglucagon and its fragments are furthermore secreted in the small and large bowels. Intestinal L-cells that process proglucagon are the major source of GLP1; these are mainly situated in the distal jejunum and ileum and also throughout the whole intestine. Proglucagon processing occurs in the central nervous system: GLP-1 is therefore an important neurotransmitter in the brain [24]. GLP-1 activity is mediated by the GLP-1 receptor, a class 2, G-protein-coupled receptor [25]. This receptor is found in many organs including the pancreas, stomach, intestines, and parts of the peripheral and central nervous systems, and these are the main therapeutic targets [26, 27]. GLP-1 receptors have been found in other tissues that may be relevant for its therapeutic effect, namely, the kidneys, endothelium, small blood vessels, and heart [28–31]. Due to the organ systems that GLP-1 acts on, it is an attractive therapeutic target for type 2 diabetes mellitus and obesity. For example, in pancreatic b-cells, GLP-1 receptor activation increases adenylate cyclase activity that leads to glucose-dependent insulin secretion [14, 18]; in addition, GLP-1 exerts a glucose-dependent, suppressive effect on glucagon secretion [32]. Both effects act to lower blood glucose levels. Indeed, glucagon antagonism has long been suggested as a treatment for both type 1 and type 2 diabetes [33]. Together with glucose, GLP-1 acts on the b-cell to promote insulin gene transcription and therefore promotes insulin synthesis [34]; furthermore, GLP-1 can restore the b-cell’s sensitivity to glucose, thereby improving b-cell function [35]. In addition, there are animal studies that support a “protective” b-cell effect of GLP-1, with apoptosis suppression, neogenesis and proliferation stimulation, and increases in b-cell mass [36, 37]. Furthermore, GLP-1 has advantageous gastrointestinal effects: gastric emptying is slowed following meal consumption [38] and it reduces gastric acid secretion [39], thus enabling food to be processed slowly. Studies have shown that GLP-1 infusion can reduce hunger sensations [40]. Consequently, a beneficial effect on body weight may be expected with any therapeutic application of GLP-1 [41]. An additional effect of GLP-1 that is of potential therapeutic interest is its actions on the cardiovascular system. Mostly based on animal data, it is thought that GLP-1 improves myocardial and endothelial function [42, 43] and GLP-1 may also directly protect the myocardium [29]. With these above effects, it is obvious to see why research in type 2 diabetes mellitus has focused on GLP-1 as an exciting therapeutic possibility (Figure 14.3).

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Figure 14.3 GLP-1 actions in peripheral tissues. GLP-1 acts directly on the endocrine pancreas, heart, stomach, and brain, whereas the actions on liver and muscle are indirect. Reproduced with permission from Drucker [44].

14.2 Discussion 14.2.1 Physiology of Native GLP-1

GLP-1 is secreted, primarily in response to food intake, throughout the distal small intestine and colon [45]. However, it is only oral glucose ingestion that stimulates the secretion of GLP-1 [46]; intravenous infusion of glucose in humans does not result in an increase in basal GLP-1 levels [46]. GLP-1 is secreted in a biphasic pattern: the first phase occurs within 15 min of eating, with peak GLP-1 levels seen around 90 min after eating in healthy individuals [47]. It is thought that the vagus nerve has a role to play in the first phase of GLP-1 secretion, as the majority of L-cells are located in the distal small intestine, secretion cannot be stimulated purely by the contact of food here [48]. The second, longer phase of GLP-1 secretion is most likely caused by foodstimulating intestinal L-cells [49]. GLP-1 is rapidly metabolized into GLP-1(9-37) and GLP-1(9-36)NH2 by the enzyme dipeptidyl peptidase-4 (DPP-4) [50, 51] and has a half-life of approximately 2 min [50]. DPP-4 is an ubiquitous enzyme and is found circulating as well as in many organs including the kidneys, lungs, pancreas, and liver. Furthermore, it is expressed in endothelial cells that are located very close to intestinal L-cells, thereby inactivating almost half the GLP-1 that enters from the portal circulation before it enters the systemic circulation [52].

14.2 Discussion

GLP-1 and its metabolites are mainly cleared via the kidney through glomerular filtration and renal catabolism [53]. This renal elimination does mean that in patients with renal failure or insufficiency, accumulation of GLP-1 levels can occur [54, 55]. Fasting plasma levels of GLP-1 appear to range between 5 and 10 pmol/l, and in healthy individuals increase to approximately 30 pmol/l after food intake [47]. The postprandial increase in GLP-1 levels depends on the size of the meal [56]. Some data suggest GLP-1 secretion in patients with type 2 diabetes is impaired compared to healthy individuals [47, 56]: healthy individuals have a significantly greater and more prolonged GLP-1 response, with levels peaking at around 90–120 min [47]. However, the effect is small and contradicted in other studies, so this probably does not have much significance. Much more important is that the other main incretin, GIP, only induces a very small release of insulin in patients with diabetes compared to healthy individuals in whom GLP-1 and GIP have more equal effects [57]. Thus, in patients there is a lack of insulin secretion from GIP that pharmacological levels of GLP-1 may restore. 14.2.2 Development of Liraglutide: A GLP-1 Analogue

Given the pharmacokinetic profile of endogenous GLP-1, it is clear that any GLP-1 therapy would need to approach the relevant drug discovery process in two ways: prolongation of action of pharmacologically administered GLP-1 or a GLP-1 analogue, either by administering a sustained release formulation or by making longacting analogues. On top of the rapid degradation of GLP-1 by DPP-4 and swift renal clearance, several other hurdles had to be overcome in developing a viable GLP-1based therapy. GLP-1 is a peptide and therefore cannot be administered orally; it has to be delivered by injection or through another nonoral route. GLP-1 also has a tendency to fibrillate, making it difficult to handle in solution. These problems have been documented [58]. Novo Nordisk began research for developing a GLP-1-based therapy in 1991. A study by Nauck and colleagues, published in 1993, demonstrated the viability of normalizing fasting plasma glucose concentrations with exogenous administration of GLP-1 [17] and thus underpinned the research efforts. Novo Nordisk’s first approach was to produce a natural GLP-1 drug either from GLP-1(7-36)amide or GLP-1(7-37). Research focused on a subcutaneous formulation, and as part of this effort a clinical study was conducted to compare GLP-1 infusion over 16 h with infusion over 24 h [59]. This study, which was not published until 2001, found that GLP-1 had to be continuously infused to obtain the most optimal glycemic control [59]; the authors concluded that, “. . .Because of the short plasma half-life of native GLP-1, long-acting derivatives should be developed to make GLP-1 treatment clinically relevant.” Consequently, a sustained release formulation was entered into preclinical development in 1994. However, it was early in 1996 that this formulation was withdrawn due to the high incidence of injection site reactions (in pigs). During this time, Novo Nordisk conducted structure–activity relationship studies on GLP-1, publishing a systematic survey of a series of GLP-1 analogues using an

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alanine scan [21]. This study investigated the replacement of each amino acid of GLP1 with L-alanine in order to identify the side chain functional groups involved in the interaction of the GLP-1 receptor. The analogues that were identified to have the lowest receptor binding affinity were studied further, with far-UV CD spectroscopy, to identify if conformational changes were induced at these positions. The study demonstrated that the amino acids at positions 7, 10, 12, 13, and 15 of the GLP-1 molecule were involved in receptor binding while those at positions 28 and 29 were involved in mediating the correct confirmation for binding. With the realization that GLP-1 was metabolically unstable, Novo Nordisk carried out work on DPP-4 stabilized simple amino acid analogues of GLP-1 throughout 1994/1995 [60]: four GLP-1 analogues with N-terminal substitutions of threonine (Thr), glycine (Gly), serine (Ser), or a-aminoisobutyric acid (Aib) were synthesized and tested for stability. All analogues bound to the cloned human pancreatic GLP-1 receptor but with differing affinities (as measured by IC50): the Aib and Gly analogues bound with an affinity (0.45
0.05 and 2.8
0.42 nmol/l, respectively) in the same range as GLP-1(7-36)amide (0.78
0.29 nmol/l), but the Thr and Ser analogues had lower binding affinities (49
3.7 and 9.0
1.9, respectively, both p < 0.001 versus GLP-1(7-36)amide). Probably due to the presence of the polar hydroxyl group in the serine and threonine residues, binding was impaired. Stability of these peptides was measured by the group of Jens Holst at the University of Copenhagen by incubating the analogues in porcine plasma at 37  C. GLP-1(7-36)amide had a t1/2 of 28.1
1.2 min, but all analogues had a significantly prolonged t1/2 in comparison: Gly, 159
12 min; Ser, 174
12 min; Thr, 197
14 min, all p < 0.0001. Pharmacokinetic parameters of these peptides were tested in vivo: i.v. infusion of the four analogues in anesthetized pigs resulted in N-terminal plasma t1/2 values of between 3.3 and 3.9 min and C-terminal plasma t1/2 values of between 3.5 and 4.4 min (Table 14.1), values that were significantly greater than for GLP-1(736)amide [60]. The ability of these analogues to stimulate insulin secretion and/or inhibit glucagon secretion was tested in an isolated perfused pancreas. The Aib analogue was at least as potent as GLP-1(7-36)amide in stimulating insulin and inhibiting glucagon secretion, and was also the most potent (p < 0.05) of all the analogues in reducing glucagon output. The Gly analogue was not significantly different from GLP-1(7
36)amide in stimulating insulin or inhibiting glucagon secretion, but it was more potent (p < 0.05) than the Ser and Thr analogues in inhibiting glucagon release. This study demonstrated that small alterations in the N-terminus of GLP-1 conferred resistance to the action of DPP-4, but with retained biological activity and an improved metabolic stability [60]. However, the t1/2 of these analogues in pigs was approximately 4 min, still making them unsuitable drug candidates. Consequently, further research into extending the duration of action of the analogues was required. Alongside the successful development at Novo Nordisk of a prolonged acting insulin analogue, modified with the addition of a fatty acid side chain, research for a viable GLP-1 therapy turned toward acylated analogues. The attachment of a fatty acid side chain to this subcutaneously administered insulin analogue was shown to significantly protract the action of insulin, by facilitating serum albumin binding [61–63].

14.2 Discussion Table 14.1 In vivo plasma t1/2 for GLP-1(7-36)amide and N-terminally modified analogues calculated using N- and C-terminally directed RIAs.

Group

Thr8-GLP-1(7-37) Gly8-GLP-1(7-37) Ser8-GLP-1(7-36)amide Aib8-GLP-1(7-36)amide

Analogue t1/2 (min)

GLP-1(7-36)amide t1/2 (min)

N-terminal

C-terminal

N-terminal

C-terminal

3.9
0.2a),b) 3.3
0.4a),b) 3.7
0.4a),b) NDL

4.2
0.4c) 3.5
0.7c) 4.2
0.2c) 4.4
0.2c)

0.7
0.05d) 0.9
0.03d) 0.9
0.06d) 1.1
0.07d)

4.0
0.1 4.3
0.5 4.1
0.2 4.3
0.3

Values are mean
SEM; n ¼ 4. NDL: not determined due to lack of cross-reactivity. Reproduced with permission from Deacon et al. [60]. a) NS, p > 0.05 versus C-terminal t1/2 for analogue. b) p < 0.01 versus N-terminal t1/2 for GLP-1(7-36)amide. c) NS, p > 0.05 versus C-terminal t1/2 for GLP-1(7-36)amide. d) p < 0.01 versus C-terminal t1/2 for GLP-1(7-36)amide.

Consequently, a series of potent fatty acid derivatives of GLP-1 were investigated for their structure–activity relationships and pharmacokinetic properties [64]. Fatty acids or fatty diacids were optionally extended with a “spacer” between the e-amino group of the lysine side chain and the carboxyl group of the fatty acid. Acylation with simple fatty acids increased the net negative charge of the resulting molecule by 1, whereas peptides acylated with a L-glutamoyl spacer or with diacids provided a net increase of the negative charge by 2. This was expected to increase the albumin binding affinity and to provide a higher solubility of the compound at physiological pH. The compounds investigated in this study and their potency for the cloned human GLP-1 receptor expressed in BHK (baby hamster kidney) cells are shown in Table 14.2. All compounds acylated with a fatty acid equal to or longer than 12 carbon atoms had a considerably longer half-life than GLP-1 (after s.c. administration, t1/2 was 1.2 h) [64]. The t1/2 values of some of the most potent analogues are described in Table 14.3. This study demonstrated that long fatty acids could be attached to a variety of positions along the analogue at the C-terminal without loss of potency. It was interesting to observe that attaching a fatty acid to position 8 (at the N-terminal) resulted in a loss of potency, supporting the earlier research that amino acids at positions 7, 10, 12, 13, and 15 of the GLP-1 molecule are involved in receptor binding, while those at positions 28 and 29 are involved in the conformational binding of the molecule [21]. Altogether, this study identified several potent compounds with plasma half-lives of over 10 h that made them suitable for once-daily administration. From these compounds, liraglutide was selected for clinical development. Another study was undertaken to look at the structure–activity relationships in a series of compounds based around liraglutide. In all compounds, acylation on position 26 was explored, using the GLP-1 analogue R34-GLP-1(7-37) [65]. The amino acid sequences of these compounds were very similar to that of native GLP-1, with only one substitution of Lys34 to Arg. This substitution was chosen with the

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Table 14.2

Potencies of the compounds tested for the cloned human GLP-1 receptor expressed in

BHK cells. Parent peptide

Acyl site

Acyl substituent

Potency (EC50, pM)

GLP-1(7-37) K8R26,34-GLP-1(7-37) K18R26,34-GLP-1(7-37) K23R26,34-GLP-1(7-37) R34-GLP-1(7-37) (liraglutide) K27R26,34-GLP-1(7-37) R26-GLP-1(7-37) K36R26,34-GLP-1(7-36) R26,34-GLP-1(7-38) GLP-1(7-37) GLP-1(7-37) GLP-1(7-37) GLP-1(7-37) R34-GLP-1(7-37) R34-GLP-1(7-37) R34-GLP-1(7-37) R34-GLP-1(7-37) R34-GLP-1(7-37) Desamino-H7R34-GLP-1(7-37) R34-GLP-1(7-37) R34-GLP-1(7-37) R34-GLP-1(7-37) Desamino-H7R26-GLP-1(7-37) Desamino-H7R26-GLP-1(7-37) Desamino-H7R26-GLP-1(7-37) K36R26,34-GLP-1(7-36) K36R26,34-GLP-1(7-36) K36R26,34-GLP-1(7-36) R26,34-GLP-1(7-38) R26,34-GLP-1(7-38) R26,34-GLP-1(7-38) R26,34-GLP-1(7-38) G8R26,34-GLP-1(7-38) E37R26,34-GLP-1(7-38) E37G8R26,34-GLP-1(7-38) E37G8R26,34-GLP-1(7-38)

K8 K18 K23 K26 K27 K34 K36 K38 K26,34 K26,34 K26,34 K26,34 K26 K26 K26 K26 K26 K26 K26 K26 K26 K34 K34 K34 K36 K36 K36 K38 K38 K38 K38 K38 K38 K38 K38

None c-Glu-C16 c-Glu-C16 c-Glu-C16 c-Glu-C16 c-Glu-C16 c-Glu-C16 c-Glu-C16 c-Glu-C16 bis-C16-diacid bis-c-Glu-C16 bis-c-Glu-C14 bis-C12-diacid C16-diacid C14-diacid c-Glu-C18 c-Glu-C14 c-Glu-C12 c-Glu-C16 GABA-C16 b-Ala-C16 Iso-Nip-C16 c-Glu-C16 C8 c-Glu-C8 C20-diacid C16-diacid c-Glu-C18 C16-diacid C12-diacid c-Glu-C18 c-Glu-C14 c-Glu-C16 c-Glu-C16 c-Glu-C16 c-Glu-C18

55
19 1260
210 35.2
6.2 30.1
3.3 61.0
7.1 36.3
0.3 121
26 36.4
2.1 53.0
2.8 7000
7 16 700
3 700 3050
350 177
52 154
66 72
0.7 194
24 22.0
7.1 27.3
8.4 687
129 84.4
22.1 113
3 410
120 2360
370 236
66 169
1 210
14 7.89
1.21 116
3 5.6
3.5 4.19
0.98 115
21 54
1 328
14 27.2
0.1 135
7 213
30

K: lysine; R: arginine; E: glutamic acid; G: glycine. Abbreviations used for acyl groups in lysine Ne-acylated peptides: c-Glu-C8 ¼ c-L-glutamoyl(Naoctanoyl); c-Glu-C14 ¼ c-L-glutamoyl(Na-tetradecanoyl); c-Glu-C16 ¼ c-L-glutamoyl(Nahexadecanoyl); c-Glu-C18 ¼ c-L-glutamoyl(Na-octadecanoyl); C8 ¼ octanoyl; C12-diacid ¼ vcarboxyundecanoyl; C16-diacid ¼ v-carboxypentadecanoyl; C20-diacid ¼ v-carboxynonadecanoyl; GABA-C16 ¼ c-aminobutyroyl(Nc-hexadecanoyl); iso-Nip-C16 ¼ 1-(hexadecanoyl)piperidyl-4carboxy. Data are given as mean
SD of two individual experiments with triplicate samples. Reproduced with permission from Knudsen et al. [64].

14.2 Discussion Table 14.3 Plasma half-lives in pigs of GLP-1 and selected potent acylated compounds.

Compound

Plasma t1/2 (h) mean
SD

GLP-1(7-37) K23 R26,34-GLP-1(7-37) (acyl substituent: c-Glu-C16) R34-GLP-1(7-37) (acyl substituent: c-Glu-C16) (liraglutide) R26-GLP-1(7-37) (acyl substituent: c-Glu-C16) K36R26,34-GLP-1(7-36) (acyl substituent: c-Glu-C16) R34-GLP-1(7-37) (acyl substituent: c-Glu-C12) R34-GLP-1(7-37) (acyl substituent: GABA-C16) R34-GLP-1(7-37) (acyl substituent: b-Ala-C16) K36R26,34-GLP-1(7-36) (acyl substituent: C16-diacid) E37G8R26,34-GLP-1(7-38) (acyl substituent: c-Glu-C16)

1.2 20
2 14
2 13 12
1 15
3 31
4 8.8
1 13
4 11
1

Reproduced with permission from Knudsen et al. [64].

original selection of R34-GLP-1(7-37) (acyl substituent: c-Glu-C16) to allow monoacylation. The compounds, potencies, and t1/2 values from this study are presented in Table 14.4. It appears that the chain length of the fatty acid affects the potency for the cloned receptor only if the length is longer than 16 carbon atoms: compound 6 had a potency of 170 pM compared to that of 39, 66, 29, 21, and 61 pM, respectively, for compounds 1–5. However, the length of the fatty acid does appear to be closely related to protraction in vivo. Compound 1 with a C10 fatty acid has a half-life of 0.8 h, compound 2 with a C11 fatty acid has a half-life of 5.1 h, compound 5 with a C16 fatty acid has a half-life of 16 h, and compound 6 with a C18 fatty acid has a half-life of 21 h. Figure 14.4 shows the pharmacokinetic profiles of compounds 2, 3, 5, and 6 in pigs. The same relationship between fatty acid chain length and protraction in vivo was seen in compounds 7–10 (these are similar to compounds 1–6, but the spacer is different: c-aminobutyric acid) and compounds 11–13 (b-alanine as the spacer). For the same length fatty acid, the c-Glu spacer resulted in the longest half-life in vivo (compound 2 ¼ 5.1 h, compared to compound 8 ¼ 1.7 h, and compound 11 ¼ 1.2 h). This study demonstrated that there was a straightforward relationship between structure and potency, with only very long fatty acids decreasing potency slightly, whereas protraction correlated directly with the length of the fatty acid chain within the same series. The spacer component was important for potency but did not affect protraction of the pharmacokinetic profile in pigs. In contrast, introducing hydrophilicity at the omega terminal of the fatty acid moiety did not affect protraction but tended to decrease potency. From these compounds, R34-GLP-1(7-37) (acyl substituent: c-Glu-C16), or liraglutide, was first synthesized in February 1997 and entered preclinical development in August the same year. Alongside the development of liraglutide, it should be mentioned that there are other incretin-based therapies either already available or close to market. Another candidate GLP-1 analogue has been developed separately liraglutide: exenatide. It is a synthetic version of a component of the saliva of the gila monster lizard

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Table 14.4 Compounds, their potency on human cloned GLP-1 receptor, and in vivo protraction in

pigs. Compound Acyl group

GLP-1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

— -CO-(CH2)8CH3 -CO-(CH2)9CH3 -CO-(CH2)10CH3 -CO-(CH2)12CH3 -CO-(CH2)14CH3 (liraglutide) -CO-(CH2)16CH3 -CO-(CH2)8CH3 -CO-(CH2)9CH3 -CO-(CH2)10CH3 -CO-(CH2)12CH3 -CO-(CH2)9CH3 -CO-(CH2)10CH3 -CO-(CH2)12CH3 -CO-(CH2)14CH3 -CO-(CH2)14CH3 -CO-(CH2)14CH3 -CO-(CH2)14CH3 -SO2-(CH2)11CH3 -SO2-(CH2)11CH3 -CO-(CH2)7CH¼CH2 -CO-(CH2)10NHCO-CH3 -CO-(CH2)10NH2 -CO-(CH2)11NH2 -CO-(CH2)11OH -CO-(CH2)15OH -CO-(CH2)10SO3H -CO-CH2O-(CH2)8CH3 -CO-(CH2)2O(CH2)9CH3 -CO-CH2O(CH2)2O(CH2)2O(CH2)2CH3 -CO-(CH2)8(OH)CH2(OH)(CH2)6OH -CO-(CH2)6CH3 -CO-(CH2)6CH3 -CO-(CH2)6CH3 -CO-(CH2)4NHCO-(CH2)4CH3 -CO-(CH2)10NHCO-(CH2)2CH3

Spacer

Potency (pmol/l)

Half-life (h)

— c-Glu c-Glu c-Glu c-Glu c-Glu c-Glu Gaba Gaba Gaba Gaba b-Ala b-Ala b-Ala D-c-Glu a-Glu — Triethylenglycol — b-Ala c-Glu c-Glu c-Glu b-Ala c-Glu b-Ala b-Ala c-Glu c-Glu c-Glu c-Glu 8-Aminooctanoyl 9-Aminononanoyl 10-Aminodecanoyl 5-Aminopentanoyl b-Ala

55
19 39
17 66
23 29
7 27
2 61
7 170
40 28
5 8.6
3.6 19
1 35
11 37
6 27
3 55
15 74
32 76
1 4440
440 1570
60 2110
210 350
20 36
0.4 160
30 140
30 110
20 45
8 65
7 110
50 81
19 70
20 320
150 210
20 48
4 64
12 39
12 74
35 48
2

1.2 0.8 5.1 7.6 9.0 16 21 1.6 1.7 2.4 4.6 1.2 2.8 6.5 22 12 16 13 15 9.4 1.9 2.0 1.1 2.0 2.6 4.6 2.5 1.7 3.1 0.2 2.3 2.5 4.5 4.3 2.0 3.4

c-Glu

400
100

18

c-Glu c-Glu

31
14 73
1

O

36

37 38

-CO-(CH2)9phenyl -CO-(CH2)4cyclohexyl

2.4 2.0

14.2 Discussion Table 14.4 (Continued)

Compound Acyl group

Spacer

Potency (pmol/l)

c-Glu

26
2

—

380

41

c-Glu

260
80

6.0

42

c-Glu

18
10

6.8

43

b-Ala

360
150

8.8

O

39

S

Half-life (h)

3.0

R

O O

40

S

R

14

O

The receptor potency data are given as mean
SD of two or three individual experiments with triplicate samples. Protraction is expressed as the terminal half-life after subcutaneous administration. Reproduced with permission from Madsen et al. [65].

(Heloderma suspectum) and has been approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMEA). Exenatide is a 39-amino acid peptide amide. The amino acid sequence of exenatide is H-His-Gly-Glu-Gly-ThrPhe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-GluTrp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2. It was originally identified as exendin-4 from gila monster venom, sharing 53% sequence homology with mammalian GLP-1; it acts as a high-potency agonist on the GLP-1 receptor [66]. In addition, there are DPP-4 inhibitors: sitagliptin and vildagliptin prevent the degradation of GLP-1, thereby increasing plasma levels of endogenous GLP-1 [67].

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Figure 14.4 Pharmacokinetic profiles of compounds 2 (-CO-(CH2)8CH3 [acyl spacer c-Glu]), 3 (-CO-(CH2)10CH3 [acyl spacer c-Glu]), 5 (-CO-(CH2)14CH3 (liraglutide) [acyl spacer c-Glu]), and 6 (-CO-(CH2)16CH3 [acyl spacer c-Glu]) in pigs. Reproduced with permission from Madsen et al. [65].

Sitagliptin has been approved by FDA and EMEA, and vildagliptin has been approved by EMEA. The main difference between GLP-1 analogues and DPP-4 inhibitors is that the analogues have to be injected whereas the inhibitors are orally available. However, the GLP-1 analogues are also more efficacious, especially regarding body weight reduction. 14.2.3 The Pharmacology of Liraglutide

As outlined above, liraglutide has a molecular structure that is very similar to human GLP-1, with only two structural modifications: substitution of Lys34 to Arg and the attachment of a 16C fatty acid side chain to Lys26 via a glutamic acid linker (Figure 14.5). As a result, the amino acid sequence of liraglutide has 97% homology with native human GLP-1 [64].

Figure 14.5 Amino acid structure of liraglutide.

14.2 Discussion

Acylation of a molecule with a fatty acid has been shown to prolong the duration of action of a basal insulin analogue, insulin detemir, by causing reversible albumin binding, as well as by the molecule forming a hexamer [68]. The protraction mechanism for liraglutide is a result of albumin binding and self-association into heptamers. Liraglutide binds to albumin [69], resulting in a long plasma half-life, also following i.v. administration. The hydrophobic palmitate residues interact to form the core of the seven-molecule complex, resulting in slow dissociation and the inability to pass easily through capillary membranes following subcutaneous injection [70]. Furthermore, it is probable that some of the protraction in absorption of liraglutide is caused by reduced susceptibility to DPP-4 degradation. The in vitro potency of liraglutide was confirmed in a study using plasma membranes from a cell line expressing the cloned human GLP-1 receptor: liraglutide was a full and potent agonist with an EC50 of 61
7 pM [64]. Liraglutide is a selective agonist and has shown no cross-reactivity to 114 different receptors and ion channels, including the closely related glucagon receptor. In perifused mouse islets, liraglutide stimulated insulin secretion in a glucose-dependent and dose-dependent fashion with potency comparable to that of native GLP-1. Other in vitro testing of liraglutide has found that it not only prevents cytokine and free fatty acid-induced apoptosis in isolated rat pancreatic islets but also stimulates primary rat b-cell proliferation [37, 71]. The in vivo effects of liraglutide were explored in a series of studies investigating subchronic dosing regimens in different animal models of diabetes and obesity [72–76]. In diabetic ob/ob and db/db mice, liraglutide lowered blood glucose dosedependently compared to a vehicle group [72]. In addition, an increase in pancreatic b-cell mass was seen [72]. Exendin-4 (exenatide) was also tested in this study: like liraglutide, exendin-4 decreased blood glucose levels compared to vehicle, although liraglutide displayed a longer duration of action. Furthermore, exendin-4 significantly increased the b-cell proliferation rate in the db/db mice; however, a statistically significant effect on b-cell mass was not seen in this study. Increased b-cell mass and insulin release were seen following liraglutide administration in Zucker diabetic fatty rats [73]. Blood glucose levels were approximately 12 mM lower compared to vehicle animals (p < 0.0002) and plasma insulin levels were up to three times higher over 24 h (p < 0.001). This study showed that part of the blood glucose lowering effect was mediated by a reduction in food intake. Liraglutide was studied extensively in pigs as a model in which pharmacokinetic parameters usually correlate well with humans. One month of treatment with oncedaily liraglutide in streptozotocin-induced b-cell-reduced diabetic Goettingen minipigs led to a significant lowering of blood glucose. Hyperglycemic clamp studies showed increased glucose utilization and insulin release and decreased plasma glucagon levels following 2 mg/kg i.v. administration of liraglutide [74]. Both the insulin release and the glucagon-lowering effect were glucosedependent. Liraglutide lowered body weight in Sprague Dawley rats given access to candy and normal rat fodder [75]. Candy and fodder feeding increased body weight, fat mass, and feeding-associated energy expenditure. Twelve weeks of liraglutide treatment reduced body weight to the level of normal-weight control rats. The weight lost was fat mass. The DPP-4 inhibitor, vildagliptin, was used as an active comparator, but this

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drug had no effect on body weight, food intake, or energy expenditure. Despite weight loss, liraglutide-treated rats did not experience a decrease in energy expenditure compared to candy-fed controls. Most interestingly, liraglutide led to an altered food preference. The animals treated with liraglutide selectively had a lower candy intake and a higher chow intake. Obese pigs are a good model of human obesity as pigs have eating patterns like humans. They eat meals during daylight and sleep during darkness. Liraglutide markedly reduced food intake from 18.4
0.6 to 7.3
0.3 MJ/day in obese Goettingen minipigs [76]. Body weight (mean loss of  4.3
1.2 kg, 4–5% after 7 weeks of treatment) was reduced following liraglutide treatment. The newest studies support the role of liraglutide in cardiovascular protection. A study in mice showed liraglutide-treated animals had better survival rates after an induced myocardial infarct [77]. Another study using a human endothelial cell-line showed liraglutide reduced both cytokine and glucose-induced inflammatory responses [78]. Clinical pharmacology studies have supported and developed further the preclinical results. After subcutaneous injection, liraglutide is absorbed slowly into the human circulation: Tmax occurs between 9 and 12 h following dosing and the mean elimination half-life is between 11 and 15 h [79]. There appears to be no single organ responsible for primary degradation or clearance of liraglutide. Liraglutide can be degraded in vitro by endogenous peptidases, including DPP-4 [80]. However, in the body this does not appear to be an important pathway for liraglutide clearance, in contrast to GLP-1 degradation. Consequently, liraglutide has no renal or hepatic metabolism or excretion. This does suggest that in patients with renal or hepatic impairment there will be a low risk of accumulation and/or drug interactions via that route with liraglutide [81, 82]. Of the other GLP-1 analogues, exenatide is dosed twice daily; it reaches peak plasma concentrations in approximately 2 h and has a terminal half-life of approximately 2.5 h in human subjects [83]. Nonclinical studies have shown that exenatide is predominantly eliminated by glomerular filtration, with subsequent proteolytic degradation [84–86]. The pharmacokinetic profile of liraglutide, measured in a randomized, doubleblind study of 64 healthy men, demonstrated detection in plasma over 24 h, therefore suggesting suitability for once-daily dosing [79]. Steady state was reached after 3 days of once-daily dosing of liraglutide (1.25–12.5 mg/kg) in a study of 30 healthy volunteers [87]. A placebo-controlled, crossover study in 13 patients with type 2 diabetes confirmed the once-daily dosing of liraglutide with decreased plasma glucose concentrations over 24 h following subcutaneous administration [88]. A 14-week double-blind, placebo-controlled study in 165 patients with type 2 diabetes has confirmed the blood glucose-lowering efficacy of liraglutide (administered once daily at 0.65, 1.25, or 1.9 mg as monotherapy) [89]. Significant reductions in HbA1c of 0.98, 1.40, and 1.45% from baseline were seen in the three dose groups, respectively, whereas HbA1c increased by þ 0.29% with placebo (p < 0.0001 against all liraglutide treatment groups). Other measurements of glycemic control were significantly reduced with liraglutide treatment compared to placebo. In all

14.2 Discussion

treatment groups, body weight decreased, with the greatest loss (3 kg) seen in the 1.9 mg dose group (difference of 1.21 kg versus placebo, p ¼ 0.039). Liraglutide was well tolerated, with no reported hypoglycemic episodes. Data on cardiovascular outcomes and b-cell function from the same study have also been reported [90, 91]. Liraglutide treatment improved certain biomarkers associated with increased cardiovascular risk [90]; there were also improvements in first- and second-phase insulin secretion, following liraglutide administration, together with improvements in arginine-stimulated insulin secretion during hyperglycemia [91]. 14.2.4 Clinical Evidence with Liraglutide

Following the success of liraglutide in phase II clinical testing, a series of phase III placebo-controlled clinical trials (the LEAD, Liraglutide Effect and Action in Diabetes, program) were conducted to investigate the efficacy and safety of liraglutide administered as monotherapy or in combination with other oral antidiabetic drugs. Ranging in duration from 26 to 52 weeks, six trials examined liraglutide dosed at 1.2 or 1.8 mg daily to patients with type 2 diabetes as follows: LEAD 1, in combination with sulfonylurea versus thiazolidinedione plus sulfonylurea; LEAD 2, in combination with metformin versus sulfonylurea plus metformin; LEAD 3, monotherapy versus sulfonylurea; LEAD 4, in combination with metformin and thiazolidinedione versus metformin plus thiazolidinedione; LEAD 5, in combination with metformin and sulfonylurea versus insulin glargine plus metformin and sulfonylurea; and LEAD 6, in combination with metformin and/or sulfonylurea versus exenatide with metformin and/or sulfonylurea [92–97]. In this series of clinical trials, HbA1c reductions of up to 1.6% were seen with liraglutide treatment, significantly greater than the reductions seen in the comparison groups. For example, HbA1c was reduced by 1.6% from baseline when used as monotherapy in patients previously treated only with diet and exercise and no antidiabetic medication. HbA1c in these patients was reduced to below 7.0% and sustained at this level for the 52-week duration of the study. This was a significantly greater reduction than achieved with sulfonylurea monotherapy (0.88%, p < 0.0001) [94]. Similar improvements in other parameters of glycemic control such as fasting plasma glucose and postprandial plasma glucose were also seen across studies. In these studies, major hypoglycemia was a rare occurrence with liraglutide treatment (6 from 2505 patients experienced a major episode, with only 1 patient requiring medical assistance) as a result of its glucose-dependent mechanism of action [92, 96]. The incidence of minor hypoglycemic events was also low (0.03–1.93 events per patient per year) [92, 94, 95, 97]. The beneficial effect on body weight seen in the animal studies has been realized in the clinical trials: as monotherapy, liraglutide treatment has resulted in a weight loss of around 2.5 kg. In the same study, patients treated with glimepiride gained þ 1.12 kg, leading to a difference in weight between liraglutide and glimepiride treatment of 3.6 kg (p < 0.0001) [94]. Greater body weight reductions of almost 3 kg have been seen when liraglutide was administered in combination with

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metformin [93]. Nausea and other similar side effects have been reported with liraglutide, presumably related to its delay in gastric emptying: transient nausea has been experienced by 10.5–40% of patients [92–97]. Nausea, however, disappears over 4 weeks in most patients [94] and is considered mild with a Visual Analogue Score of 1.5 on a scale of 1 to 7 [98]. Advantageous effects on b-cell function and reductions in systolic blood pressure (of up to 6.7 mmHg) have been noted in patients treated with liraglutide in these trials [97, 99]. Liraglutide (1.8 mg/day dosed once daily) has been compared with exenatide (2  10 mg dosed twice daily) in a clinical trial over 26 weeks in 464 patients with type 2 diabetes [97]. Both GLP-1 agonists were administered with metformin and/or sulfonylureas. HbA1c reductions were significantly greater with liraglutide than with exenatide (1.12% versus 0.79%, p < 0.0001), and both treatments reduced body weight in patients from baseline (3.24 kg with liraglutide in comparison to 2.87 kg with exenatide). With liraglutide, the percentage of patients reporting nausea was 10% after 5 weeks, 4% after 10 weeks, and 2.5% after 26 weeks, whereas the frequency with exenatide was 18, 13, and 8.6%, respectively. An additional clinical benefit of liraglutide over exenatide is related to their amino acid sequences: liraglutide has 97% homology with human GLP-1 whereas exenatide has only 53% homology. Rates of antibody formation against liraglutide of between 0 and 12.7% have been reported in the clinical trials described above [92, 93, 96], with no apparent effect on glycemic control. However, antibody formation against exenatide appears to be higher with approximately 50% of patients developing antibodies in clinical trials [100–102]. In a subset of patients on exenatide (3%), the antibodies demonstrated neutralizing effects and there was no blood glucose lowering [103]. For liraglutide, there has been no association between antibodies and clinical response.

14.3 Summary

It has taken over 100 years since the initial discovery of “secretin” for the therapeutic potential of GLP-1-based treatments to be realized. It was in the 1980s that research into the preproglucagon gene resulted in the discovery of GLP-1(7-37), the insulinotropic form of GLP-1. Studies looking into the effects of GLP-1 infusion in patients with diabetes demonstrated the therapeutic promise of such a related treatment, and research into developing GLP-1 analogues with suitable binding affinities and biological activities began. One of the major obstacles that had to be overcome was rapid degradation of native GLP-1 by DPP-4 enzymes in the circulation. Novo Nordisk undertook research into acylated GLP-1 analogues. From the resulting compounds that had increased self-association, albumin binding, and resistance to DPP-4 degradation, liraglutide, a once-daily human GLP-1 analogue, was developed. Preclinical pharmacology investigations demonstrated that liraglutide treatment has many of the beneficial effects of human GLP-1: improved glycemic control, reduced

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Lotte B. Knudsen

Senior Principal Scientist, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark Lotte Bjerre Knudsen is a Senior Principal Scientist at the Diabetes Research Unit of Novo Nordisk. Lotte Bjerre Knudsen has worked for Novo Nordisk for 20 years and has been responsible for several inventions and publications in the GLP-1 area. Lotte Bjerre Knudsen has held various positions at Novo Nordisk both as Scientist, Department Head, and now Senior Principal Scientist, mostly in molecular pharmacology and in vivo pharmacology.

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15 Eplerenone: Selective Aldosterone Antagonist Jaroslav Kalvoda and Marc de Gasparo

15.1 Introduction

Aldosterone, the most potent natural mineralocorticoid, was isolated in 1952 by Simpson and Tait in an amorphous form, in minute amount, from adrenal extracts [1]. It was crystallized [2] in 1953 by Reichstein, and its structure (Figure 15.1) was deduced in collaboration by the UK-group (Simpson and Tait) and Reichstein’s and Ciba Laboratories in Basel [3, 4]. The first total synthesis of the racemic compound was performed by the research group of Ciba [5–7] and the partial synthesis of the optically active hormone was accomplished by Ciba and the laboratory of Jeger at the ETH in Z€ urich [8, 9]. Aldosterone (originally named electrocortin) is essentially produced by the adrenal glomerulosa (endocrine role), but a local synthesis also occurs in brain, heart, and vessels (paracrine role). It has both epithelial and nonepithelial effects. The classical epithelial effect is responsible for fluid and electrolyte balance (Na þ reabsorption, K þ and Mg þ þ excretion, and H þ secretion) and in turn left ventricular and vascular remodeling and blood pressure regulation. The nonepithelial effect of aldosterone occurs at a variety of sites and is responsible for the inflammatory reaction, endothelial dysfunction, impaired nitric oxide synthesis, collagen formation, and fibrosis. All these effects of aldosterone are due to its binding to a cytosolic receptor and its translocation to the nucleus where it exerts its genomic effect [10–12]. A more recently described nongenomic action of aldosterone occurring through stimulation of a membrane receptor is characterized by a rapid activation of Na/K exchange through Na/K ATPase stimulation, independent of the mineralocorticoid receptor (MR). This effect is insensitive to inhibitors of transcription or translation. The nongenomic effect of aldosterone includes coronary vasoconstriction, negative inotropic response, and potentiation of the vasoconstrictor effect of angiotensin [13, 14].

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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OH

OH 21

O HO

H

18 17

H O

OH

O

OH H

O

O

O

OH

11

H3C

H H

H3C H

H

O

H3C

H

H H

H

H

O

O

Aldosterone Figure 15.1 Tautomeric equilibria of aldosterone.

15.2 Development of a Specific and Selective Aldosterone Antagonist

At the end of the 1950s, a large number of analogues of aldosterone were synthesized at Ciba. All these compounds showed, however, either little activity compared to aldosterone or no mineralocorticoid or antimineralocorticoid effect at all. The excessive salt loss clinically observed in the adrenogenital syndrome, which is not due to insufficient aldosterone excretion but due to a deficit of cortisol synthesis and compensatory hypersecretion of ACTH, prompted the Ciba steroid unit in 1958 to search for an adrenal sodium excreting factor (SEF). The first series of extractions were done with urine from female patients with congenital adrenogenital syndrome and later with hog adrenals. A compound with pronounced sodium excretion was identified as 3b,16a-dihydroxy-5a-pregnan-20-one [15] (Figure 15.2). Since other 16a-hydroxylated pregnane metabolites had been isolated from adrenals, this seemed to indicate that 16-hydroxylation was connected to an important physiological property and could be involved in maintaining mineral balance. A number of 16-hydroxylated pregnanes [16] were therefore prepared and tested in Desaulles’ laboratories at Ciba. An increased sodium excretion can be induced in intact rats with a variety of agents. The natriuretic effect of SEF, however, was not observed in adrenalectomized rats. Moreover, in renal hypertonic rats, SEF significantly decreased blood pressure [17]. CH3 O CH3 CH3

H

H

3

16

H

5

HO H

SEF

Figure 15.2 Sodium excreting factor.

OH

15.2 Development of a Specific and Selective Aldosterone Antagonist

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The unspecific sodium excreting property of SEF was, however, not considered of interest and was not further investigated. Already in the early 1950s, G.D. Searle had started a program to develop new antihypertensive compounds. The hypertensive DOCA (deoxycorticosterone acetate) salt-rat was used to evaluate the effect of the novel spirolactones to block the sodiumretaining action of DOCA and aldosterone [18–20]. After the preclinical and later clinical testing of various representatives of the spirolactone series, for example SC 5233 (the unsubstituted parent compound), the most interesting analogue [18, 21–27], SC 9420 (Figure 15.3), was finally marketed under the name spironolactone (AldactoneÒ ). It has been extensively used to treat hypertension, primary and secondary hyperaldosteronism, hypokalemia, liver cirrhosis, and nephrotic syndrome [28]. Unfortunately, this first commercial mineralocorticoid receptor antagonist causes hormone-related side effects such as menstrual irregularity, gynecomastia, and impotence due to its low receptor selectivity [29, 30]. More than 20 years later, Ciba-Geigy started again a program to search for novel aldosterone antagonists with a primary goal to identify structures free from the unwanted secondary effects of spironolactone [31–33]. It was generally accepted that O CH2OR O

CH3 CH3 H

H3C O H3C

H

H H

H

O

H

O DOC : R = H DOCA : R = Ac

SC 5233 O

H3C O H3C

H H

O

H S

H3C

O

Spironolactone SC 9420 Figure 15.3 Structural formulae of deoxycorticosterone (DOC), its acetate (DOCA), the unsubstituted parent spirolactone (SC 5233), and the marketed diuretic spironolactone (SC 9420).

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these sexual disturbances were mediated mainly by the interaction of the aldosterone antagonist with gestagen (GR) and androgen (AR) receptors [34, 35]. During the past few decades, more detailed knowledge concerning both the primary structure and the mechanism of action of members of the steroid hormone receptors family has accumulated. Cloning of the genes coding for these intracellular proteins that mediate complex effects on development, growth, and physiological homeostasis by selective modulation of gene transcription has allowed their detailed biochemical characterization. All these molecules share, besides the rather specific ligand binding area, a highly conserved cystein-rich region that functions as the DNA binding domain. In spite of this overwhelming progress, which would have been considered a science fiction at the time the project was started, one is still far away from a real “rational” design of a steroid drug and one had to resort to various “semirational” methods. The final solution of the defined task was therefore based upon a rather classical approach. Using the structure of DOCA and, in a later phase of the project, that of spironolactone itself (Figure 15.3) as a starting point, U. Joss at Ciba-Geigy studied the binding of more than 100 modified steroids to corticoid and sex hormone receptors. Hydroxy groups as potential sites for hydrogen bonds, oxygen or carbon bridges, and halogen atoms were introduced into the parent structures (Figure 15.4). Of special interest, however, were molecules containing additional double bonds, cyclopropyl (see also the Schering AG project [36–39]), or epoxy groups in specific positions of the steroid skeleton, which permitted a subtle manipulation of the overall conformation of various parent compounds. As a consequence, flattening of the molecular shape or changes from a more convex to a more concave structure, as well as distances between anchorage points of the molecule to a putative receptor, could be checked. In this regard, there could be studied the influence of the introduction of double bonds and of an oxirane function in position 9(11) on the special arrangement of the molecules, on their binding to the mineralocorticoid (MR), androgen (AR), and progesterone (PR) receptors, and on their in vivo activity.

O

OH

CH3

O

H3C O

11

CH3

CH3

9

O

O

Figure 15.4 Basic structures of DOC (deoxycorticosterone) and spirolactones. The arrows indicate positions that have been used for introduction of various substituents. (From Ref. [33].)

15.2 Development of a Specific and Selective Aldosterone Antagonist

Prior to the synthesis of the various structures, the respective Dreiding models were examined. Later, the most stable conformations of the various deoxycorticosterone (DOC) and spironolactone analogues were estimated using the MM2 force field energy minimization program of ChemOffice (Cambridge Soft) or a molecular modeling program developed in-house by N.C. Cohen and P. Furet. The distance between the two angular methyl groups (C-18 and C-19) and between the oxygen in position 3 and 17, respectively, was used as a crude measure of the bending of the steroid skeleton. The flatness of the B-C-D portion of the skeleton of the parent DOC derivative or
spirolactone was expressed by the distance of about 4.8 A between the two angular methyl groups. Unsaturation in position 9(11) induces a more concave structure and consequently
a reduction in the C(18)–C(19) distance to about 4.5 A (Figures 15.5 and 15.6). A similar effect is observed after the introduction of a double bond into the 6(7) position (Figure 15.6). The presence of both double bonds leads to the same distance (i.e.,
about 4.6 A). Finally, an additional double bond in position 1(2) flattened even more the ring AB area and adjusted the distance of the two methyl groups to the original value of about
4.8 A (Figure 15.6). The influence of additional double bonds on the relative binding affinity (RBA) of DOC analogues to the mineralocorticoid, androgen, and progesterone receptor is shown in Table 15.1.

Figure 15.5 Distance between C(18) and C(19) of DOC and 9(11)-dehydro-DOC.

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Figure 15.6 Distance between C(18) and C(19) in unsaturated DOCA analogues. The arrows in the 2D formulae show positions of the additional double bonds.

15.2 Development of a Specific and Selective Aldosterone Antagonist Table 15.1 Relative binding of deoxycorticosterone acetate and its unsaturated analogues to the mineralocorticoid (MR), androgen (AR), and progesterone (PR) receptors.

Compound

MR, aldosterone ¼ 1

AR, R 1881 ¼ 1

PR, progesterone ¼ 1

0.6

0.015

0.2

0.2

0.003

0.03

0.4–1

n.d.

0.06

0.0006

0.008

0.025

n.d.

0.001

CH2OAc O

CH3 CH3

H

H

H

DOCA

O

CH2OAc O

CH3 CH3

H

H

H

O CH2OAc CH3 CH3

O

H

<0.03

H O

CH2OAc CH3 CH3

O

H H

O CH2OAc CH3 CH3

H H

O

O

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Conformational changes, especially induced by the introduction of a single additional double bond, as well as possible electronic effects, did not influence substantially the interaction of the steroid with the MR, but it significantly reduced the binding to the sex hormone receptors. The best profile was shown to occur with compounds having the most concave structure, that is, the shortest distance between the methyl groups, namely, the 9(11)-dehydro-DOC. A quite similar situation was encountered in the spirolactone series. Analyzing the influence of an epoxide function in the 9(11) position on the spatial arrangement of the two standard structures (DOC and spironolactone), one can observe, for example, that, as expected, the two isomeric oxiranes (a- and b-epoxide) considerably differ in respect to their 3D structure (Figure 15.7). In the b-epoxide series, the two methyl groups were forced apart to reach a distance
of about 5.3 A. The molecule adopted a convex form. On the other hand, in the a-epoxide group, the distance between the two centers reached a minimum, about
4.2 A, and a more concave shape was generated (Figure 15.7). Introduction of an additional double bound into position 6(7) partially reverted the above-mentioned effect of the 9a,11-epoxide. As the first compounds of the oxirane series, the two stereoisomeric epoxides of 9(11)-dehydro-DOC were synthesized and subjected to the standard receptor tests. The known b-epoxide (Figure 15.7) obtained from “anhydro-corticosterone” exhibited only a very low affinity toward the MR. The a-epoxide, on the contrary,

Figure 15.7 Distances between C(18) and C(19) in isomeric 9,11-epoxides of deoxycorticosterone. (Modified from Ref. [33].)

15.3 Eplerenone: Selectivity and Specificity

showed a relatively good binding to the MR and a highly reduced affinity to the AR. These results stimulated the preparation of various 9a,11-epoxy derivatives of known active aldosterone agonists or antagonists. Binding of 12 pairs (parent unsaturated compounds and the corresponding 9a,11-epoxides) to the MR, AR, and PR are summarized in Figures 15.8 and 15.9. As one can see, the introduction of a 9a,11-epoxy function into the parent structure always significantly reduces the affinity of the compound to the sex hormone receptors. A much smaller, sometimes irrelevant, effect was observed regarding the binding to the MR. Because of these encouraging findings, the interest of the scientists of Ciba-Geigy concentrated on the synthesis and in vivo testing [40– 41] of the three analogues of the most active compounds of Searle, namely, 9a,11-epoxy-spironolactone, 9a,11-epoxyprorenone, and 9a,11-epoxy-mexrenone (eplerenone) (Figure 15.10 and Table 15.2). The first synthesis [31–33] of the most promising compound of the novel epoxy analogues of spironolactone, CGP 30 083/epoxy-mexrenone/eplerenone (see Table 15.2), is described in a short version in Scheme 15.1. The starting material containing a 9(11)-double bond was available by multiple step sequences either from hydrocortisone or by microbiological hydroxylation of intermediates already containing the spirolactone ring, followed by dehydration. Better approaches to eplerenone have been developed in the R&D group of CibaGeigy and later by the chemists of G.D. Searle. The overall structure of eplerenone was confirmed by X-ray analysis. The a-side of the molecule is shielded by the ester group, which might preclude a direct interaction of the epoxide with proteins. The distance between the two methyl groups on the
b-face of the molecule corresponds to the estimated value of less than 4.2 A. Superposition of the rings A and B of the X-ray structure of eplerenone (¼CGP) with that of spironolactone (¼Spiro) shows (Figure 15.11) that the rest of theepoxy derivative is bent
upward. As a result, the lactone carbonyl O-atoms are separated by about 2.7 A. As spironolactone and, for example, 17a-methyl-testosterone are both highly planar, the above-mentioned bending might be one of the reasons for eplerenones’ lower affinity for the AR in vitro. This effect seems to be rather general, as even 17a-methyltestosterone loses its affinity to AR after introduction of a 9a, 11-epoxid function.

15.3 Eplerenone: Selectivity and Specificity

The relative affinity of spironolactone and eplerenone for the steroid receptors was determined in a binding assay by comparing the concentration of test compounds required to produce 50% inhibition (IC50 test) with that of the standard compound without the 9–11 epoxy group (IC50 standard) [40]. While the relative binding affinity of eplerenone for the mineralocorticoid receptor was 20 times lower than that of spironolactone, it was 1000- and 10 000-fold lower for the progesterone and androgen receptors, respectively [41] (Figure 15.12). Interestingly, while the introduction of an

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Figure 15.8 Effect of 9a,11-epoxy substitution on the affinity of six spirolactones for the mineralocorticoid receptor (column 1), androgen receptor (column 2), and the progesterone receptor (column 3). Affinities are expressed relative to aldosterone (MR), to R 1881 (AR), and progesterone (PR). The

effect of 9a,11-epoxy substitution is visualized by plotting the affinities of the epoxy derivatives at the right side of each column and that of the unsubstituted compound on the left, and connecting the points by a straight line. (From Ref. [33].)

15.3 Eplerenone: Selectivity and Specificity

Figure 15.9 Effect of 9a,11-epoxy substitution on the affinity of spironolactone (pair G), three 7a-COOR-substituted spirolactones (pairs H, J, and K: —), one K-salt (pair L: – –), and one agonist (pair M: ---). (From Ref. [33].)

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O

O

H3C O

H3C O 11

O

CH3

CH3

O

9

H O

7

H

H O

S

H H

H H3C

O

9α,11-epoxy-soironolactone (CGP 33033)

9α,11-epoxy-prorenone (CGP 29245) O

H3C O CH3 H

O H O

O O CH3 9α,11-epoxy-mexrenone (CGP 30383)

Eplerenone Figure 15.10 9a,11-Epoxy-spirolactones showing the best biological profile.

epoxy group in spironolactone reduces only marginally the affinity for the MR, it markedly affects the affinity for the androgen and progesterone receptors. Despite a lower binding affinity of eplerenone in vitro, its affinity in vivo to kidney receptor of adrenalectomized rat measured with tritiated aldosterone was twice as high indicating a greater oral potency than spironolactone (ED50 0.8 mg/kg versus 1.7 mg/kg) [40, 41]. In fact, a bolus oral dose of 10 mg eplerenone blocked 92% of aldosterone binding in rat kidney. This discrepancy between in vitro and in vivo binding affinity appears to be linked to the minimal a1-acid glycoprotein binding of eplerenone (9%) compared to spironolactone (90%) [42]. The antimineralocorticoid activity was measured in adrenalectomized rat loaded with saline and treated with the test compound 30 min before aldosterone injection, according to Kagawa et al. [18] (Table 15.2). Eplerenone significantly reverses the effect of aldosterone and increases Na excretion and the Na/K ratio with a twofold greater efficacy at 3 mg/kg compared to the same dosage of spironolactone or epoxyspironolactone (CGP 33033) [40].

15.3 Eplerenone: Selectivity and Specificity Table 15.2 In vivo activity of selected 9a,11-epoxy-spirolactones ED50 (mg/kg p.o.) (Ø ¼ no

activity). Compound

Antimineral (Kagawa)

Gestagenic (McGinty)

[0,4-5] Antiandrogenic Prostate

Sem. ves.

O

H3C O CH3

H

H

5

100

35

20

5–10

Ø (100)

Ø (180)

Ø (180)

3

100

Ø (180)

60

H S

O O

CH3

SPIRONOLACTONE O

H3C O CH3

O

H

H S

O O

CH3

(CGP 33033) O

H3C O CH3 H

O H H

O H

(CGP 29245) (Continued)

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Table 15.2 (Continued)

Compound

Antimineral (Kagawa)

Gestagenic (McGinty)

[0,4-5] Antiandrogenic Prostate

Sem. ves.

Ø (180)

Ø (180)

O Eplerenone H3C O O

CH3

3

H

Ø (100)

H O

O O CH3 (CGP 30083) From Ref. [33].

O

O

O

O

O

O

11

HCN/AlEt3

H

DIBAH

H

H

9 7

H

H

H

6

O

O

CN

H

H2O2 Cl3C-CN

O CH2N2

H H

H COOCH3

O

O

O

O

CHO

CrO3

O

O

O

O

O

Eplerenone (=CGP 30083) RELATIVE BINDING AFFINITY (MR): 0.005 RELATIVE BINDING AFFINITY (AR): 0.000007 (!) RELATIVE BINDING AFFINITY (PR): <0.00005 (!)

Scheme 15.1 Synthesis of eplerenone.

COOCH3

H H

O

COOH

15.4 Preclinical Development of Eplerenone: From Animal to Man

Figure 15.11 Superposition of X-ray structures of eplerenone and spironolactone. (From Ref. [33].)

The lower affinity of the epoxy derivatives for the nonmineralocorticoid receptors was reflected in in vivo studies. There was a 3–10-fold decrease in the antiandrogenic and progestogenic effect compared to spironolactone without disturbance of the vaginal or ovulatory cycle [40] (Table 15.2). This suggests a decreased probability of the occurrence of side effects in humans after the introduction of the 9a,11-epoxy group. Indeed, the superiority of eplerenone over spironolactone in preventing sexual side effects was confirmed in humans [41, 43, 44]. Thus, these early preclinical in vitro and in vivo studies indicated a greater specificity and selectivity of eplerenone with higher efficacy for blockade of the epithelial effect of aldosterone in the kidney and decreased potential side effects.

15.4 Preclinical Development of Eplerenone: From Animal to Man

Having shown the antimineralocorticoid effect of eplerenone in adrenalectomized and aldosterone pretreated rats, it was desirable to assess its activity in a relevant animal procedure testable in healthy volunteers [45]. Conscious and well-trained mongrel dogs were perfused with aldosterone (0.6 mg/ kg/h) administered intravenously over 7 h. After 2 h of infusion, the aldosterone antagonist was orally administered. Blood samples for plasma aldosterone and urine samples for Na and K content were collected hourly. Essentially the same procedure

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Figure 15.12 Relative binding affinities of spironolactone (S) (solid square) and 9a,11-epoxyderivatives (E): epoxy-spironolactone (¼CGP 33033, see Figure 15.10) (triangle) and eplerenone (circle). (From Ref. [41].)

was used in man. In this setting, plasma aldosterone was nearly threefold increased and plateaued around 150 and 200 pg/ml in dog and in man, respectively. Oral administration of spironolactone and eplerenone caused a significant increase in urinary Na excretion but had only a marginal effect on K in dog like in man. Three doses (25, 50, and 75 mg) of an unformulated eplerenone were also tested in 10 normal healthy volunteers receiving 9a-fluorhydrocortisone (FlorinefÒ ) and compared with the commercial formulation of spironolactone (50 mg). This placebo-controlled, crossover, randomized, and balanced study showed a positive correlation between the effect on urinary Na excretion and the dose of eplerenone, with the 50 mg doses being equivalent to 50 mg of commercial spironolactone [41]. Therefore, “9a,11-epoxymexrenone fulfills the expectations of an improvement in this therapeutic line, since its specificity for the aldosterone receptor has been greatly improved and its effects on urinary sodium and potassium have been detected at a low dose” [41]. At this stage, the development of the compound was stopped in 1987 at Ciba-Geigy as “steroids are not fashionable anymore” (sic). The 45-year story of the antialdosterones and the development of eplerenone was previously reported by M
enard [46].

15.5 Further Development of Eplerenone

15.5 Further Development of Eplerenone

In 1992, a license for eplerenone was secured by Searle and the compound was further developed by Searle, Monsanto, Pharmacia, and finally Pfizer. Several reviews have been published on the preclinical and clinical studies with eplerenone [42, 47–53]. In various animal models of hypertension, myocardial infarction, or heart failure, eplerenone reduced blood pressure, myocardial injury, reactive fibrosis, collagen deposition, and tissue necrosis, and it inhibits both the oxidative stress and the inflammatory cascade [47, 54–58]. Eplerenone also limits glomerulosclerosis and tubulointerstitial fibrosis in strokeprone hypertensive rats [59]. It reduces proteinuria and improves glomerular and arteriolar injuries in models of severe nephrosclerosis [60, 61]. Eplerenone has high bioavailability (98%) that is not affected by food. Plasma protein binding is moderate (30–60%) but concentration dependent. It is metabolized by CYP450-3A4 to 6b and 21-hydroxy eplerenone, which are inactive. The effective half-life is short, 4–5 h, compared to that of spironolactone (13–17 h), and its maximal plasma concentration (Tmax) reaches after 1–2 h. The area under the curve (AUC) is not dose-proportional. It is excreted in urine (2/3) and in the feces (1/3), and less than 1.7% of the dose is excreted unchanged in the urine [62, 63]. In patients with mild-to-moderate hypertension, 100 mg eplerenone reduced blood pressure by 75% compared to a similar dosage of spironolactone and the through-to-peak ABPM ratios showed a 24 h antihypertensive control [49]. It was effective both in low renin hypertensive Afro-American patients and in hypertensive diabetics [50]. There was no antiandrogenic or progestagenic effect and the incidence rate of adverse events was similar to placebo. The antihypertensive efficacy of eplerenone was confirmed in a number of clinical trials either as monotherapy or in combination with other antihypertensive agents. It was well tolerated in these studied populations [51–53]. The large clinical trial EPHESUS in patients with heart failure [64] showed that eplerenone titrated to a maximum of 50 mg/day reduced morbidity and mortality in patients with acute myocardial infarction complicated with left ventricular dysfunction (LVEF < 40%) and heart failure [65]. Addition of eplerenone to the standard therapy (ACE inhibitor, angiotensin receptor blocker (ARB), b blocker) resulted in a 15% decrease in total mortality over a 16-month average followup. There was also a 13% reduction in mortality caused by cardiovascular events. The relative risk of cardiac sudden death was also significantly decreased by 33%. Finally, eplerenone also appears beneficial in renal diseases. Selective aldosterone blockade with eplerenone (200 mg/day) significantly reduced proteinuria (62%) in type 2 diabetes with mild to moderate hypertension and this effect was greater than with enalapril (45%) despite a similar fall in blood pressure [66]. Eplerenone within the therapeutic range is well tolerated and has placebo-like side effects (diarrhea, abdominal pain, albuminuria, cough, dizziness, and fatigue).

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In placebo-controlled studies for hypertension, the overall rates of adverse events were 47% with eplerenone and 45% with placebo. Eplerenone dose-dependently increases plasma aldosterone and renin levels (inhibition of the negative regulatory feedback of aldosterone on renin secretion by the juxtaglomerular cells) without any significant increase in serum K as observed in the Ephesus trial (5.5% versus 3.9% in placebo) [64, 65]. Hyperkalemia (>6 mEq/l) and increased creatinine, however, may occur more frequently in renal insufficiency or diabetes with albuminuria. Potassium plasma levels should, however, be regularly monitored. Increased creatinine occurs in 2.4% compared to 1.5% in placebo [67]. Sex steroid complications are rare (gynecomastia, 0.4%; mastodynia, 0.1%; and abnormal vaginal bleeding, 0.4% as anticipated from the receptor binding data [67]. On the basis of preclinical and clinical data, eplerenone was approved by the FDA for the treatment of hypertension, alone or in combination with other antihypertensive agents, and to improve survival of stable patients with left ventricular systolic dysfunction and clinical evidence of congestive heart failure after an acute myocardial infarction. It is also licensed in Europe for treatment of heart failure.

15.6 Conclusions

Eplerenone has advantages over spironolactone because of its greater selectivity for the mineralocorticoid receptor and its dramatically reduced sexual side effects. The beneficial effects of eplerenone were investigated in numerous preclinical studies using various animal models. Eplerenone decreases blood pressure, reverses the effect of aldosterone on Na/K, and prevents aldosterone-mediated arterial stiffness. It improves endothelial function and reduces inflammation. It has renal protective effect and decreases glomerular hyperfiltration. It also decreases fibrosis as it activates matrix metalloproteinases. Eplerenone also decreases left ventricular mass and increases left ventricular ejection fraction. The convincing results in animal models have been translated to humans especially for heart failure syndrome. Despite the limited indications recognized at the moment by the health authorities (hypertension and heart failure), there are potentials for additional applications in conditions such as nephropathy, nephrotic syndrome, cirrhosis with ascitis, hypokalemia, Conn syndrome, all indications recognized for spironolactone and where eplerenone could be superior due to its better tolerability.

15.7 Epilogue

Do we need a third-generation MR antagonist as potent as spironolactone, as selective as eplerenone, nonsteroidal, cheap to manufacture, with long patent life, and extend renal tubule sparing properties especially to patients with primary aldosteronism, as suggested by Funder [68]? Plasma glucocorticoid levels are more than 1000-fold

References

higher than those of aldosterone and occupy the epithelial and extraepithelial MR in pathological circumstances depending on the intracellular redox state and the NAD levels. In this condition, MR activation is accompanied by vascular inflammation and oxidative stress triggering endothelial dysfunction and vascular remodeling as observed in primary aldosteronism. So far, eplerenone has been shown to have the required anti-inflammatory properties [60, 69], but a more systematic exploration may be required to extend its indication outside the aldosterone/salt excess diseases. Taking into account the role of glucocorticoid binding to the MR [68], aldosterone receptor blockade would remain a more appropriate approach compared to aldosterone synthase CYP11 B2 inhibitor presently under investigation.

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6

quantitative method for the bioassay of the effect of adrenal cortical steroids on mineral metabolism. Endocrinology, 50, 150–161. Simpson, S.A., Tait, J.F., Wettstein, A., Neher, R., Von Euw, J., and Reichstein, T. (1953) Isolation from the adrenals of a new crystalline hormone with especially high effectiveness on mineral metabolism. Experientia, 9, 333–335. Simpson, S.A., Tait, J.F., Wettstein, A., Neher, R., Von Euw, J., Schindler, J., and Reichstein, T. (1954) Aldosteron. Isolierung und eigenschaften. Über bestandteile der nebennierenrinde und verwandte Stoffe. 91. Mitteilung. Helv. Chim. Acta, 37, 1163–1200. Simpson, S.A., Tait, J.F., Wettstein, A., Neher, R., Von Euw, J., Schindler, J., and Reichstein, T. (1954) Die konstitution des aldosterons. Über bestandteile der nebennierenrinde und verwandte stoffe. 92. Mitteilung. Helv. Chim. Acta, 37, 1200–1223. Schmidlin, J., Anner, G., Billeter, J.R., and Wettstein, A. (1955) Synthesis in aldosterone-series. I. Total synthesis of racemic aldosterone. Experientia, 11, 365–368. Schmidlin, J., Anner, G., Billeter, J.R., Heusler, K., Wieland, P., and Wettstein, A. (1957) Totalsynthese des aldosterons. A. (214)-Lacton der D,L-8a-1-oxo-2-methallyl4-hydroxy-4b-methyl-7-€athylendioxy-4a, 10a-dodecahydro-phenanthren-2carbons€aure. Über steroide, 147.

7

8

9

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11

12

Mitteilung. Helv. Chim. Acta, 40, 1034–1051. Schmidlin, J., Anner, G., Billeter, J.R., Heusler, K., Ueberwasser, H., Wieland, P., and Wettstein, A. (1957) Totalsynthese des aldosterons. B. (18 11)€ Lacton der D,L-5-3-Athylendioxy-11hydroxy-20-oxo-pregnen-18-s€aure. Über steroide, 148. Mitteilung. Helv. Chim. Acta, 40, 1438–1455. Heusler, K., Wieland, P., Cainelli, G., Kalvoda, J., Anner, G., Arigoni, D., Meystre, C., Wettstein, A., and Jeger, O. (1960) Partial synthesis of aldosterone. Experientia, 16, 21–24. Heusler, K., Kalvoda, J., Meystre, Ch., Wieland, P., Anner, G., Wettstein, A., Cainelli, G., Arigoni, D., and Jeger, O. (1961) Partialsynthese von aldosteron. Über steroide, 177. Mitteilung. Helv. Chim. Acta, 44, 502–517. Hills, A.G., Chalmers, T.M., Webster, G.D., Jr., and Rosenthal, O. (1953) Adrenal regulation of the distribution of water and electrolytes in the human body. J. Clin. Invest., 32, 1236–1247. Laragh, J.H. (1960) The role of aldosterone in man. Evidence for regulation of electrolyte balance and arterial pressure by a renal-adrenal system which may be involved in malignant hypertension. JAMA, 174, 293–295. Williams, J.S. and Williams, G.H. (2003) 50th anniversary of aldosterone. J. Clin. Endocrinol. Metab., 88, 2364–2372.

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hydroxyandrosten-3-one lactones, US Patent 3,013,012 to G.D. Searle & Co. Atwater, N.W., Bible, R.H., Brown, E.A., Burtner, R.R., Mihina, J., Nysted, L.N., and Sollman, P.R. (1961) Steroidal aldosterone antagonists IV. J. Org. Chem., 26, 3077–3083. Colussi, G., Rombola, G., De Ferrari, M.E., Macaluso, M., and Minetti, L. (1994) Correction of hypokalemia with antialdosterone therapy in Gitelman’s syndrome. Am. J. Nephrol., 14, 127–135. Jeunemaitre, X., Chatellier, G., Kreft-Jais, C., Charru, A., DeVries, C., Plouin, P.F., Corvol, P., and Ménard, J. (1987) Efficacy and tolerance of spironolactone in essential hypertension. Am. J. Cardiol., 60, 820–825. Corvol, P., Michaud, A., Ménard, J., Freifeld, M., and Mahoudeau, J. (1975) Antiandrogenic effect of spirolactones: mechanism of action. Endocrinology, 97, 52–58. Grob, J. and Kalvoda, J. (1985) US Patent 4,559,332, Ciba-Geigy Ltd. Biollaz, M. (1987) US Patent 4,670,551, Ciba-Geigy Ltd. Grob, J., Biollaz, M., Schmidlin, J., Wehrli, H., Wieland, P., Fuhrer, H., Rihs, G., Joss, U., de Gasparo, M., Haenni, H., Ramjoue, H.P., Whitebread, S.E., and Kalvoda, J. (1997) Steroidal, aldosterone antagonists: increased selectivity of 9a,11epoxy derivatives. Helv. Chim. Acta, 80, 566–585. Cutler, G.B., Jr., Pita, J.C., Jr., Rifka, S.M., Menard, R.H., Sauer, M.A., and Loriaux, D.L. (1978) SC 25152: a potent mineralocorticoid antagonist with reduced affinity for the 5 alphadihydrotestosterone receptor of human and rat prostate. J. Clin. Endocrinol. Metab., 47, 171–175. Huffman, D.H., Kampmann, J.P., Hignite, C.E., and Azarnoff, D.L. (1978) Gynecomastia induced in normal males by spironolactone. Clin. Pharmacol. Ther., 24, 465–473. Nickisch, K., Bittler, D., Casals-Stenzel, J., Laurent, H., Nickolson, R., Nishino, Y., Petzoldt, K., and Wiechert, R. (1985) Aldosterone antagonists. 1. Synthesis and activities of 6 beta, 7 beta:15 beta,

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References

Marc de Gasparo

MG Consulting Co., Es Planches 5, 2842 Rossemaison, Switzerland Marc de Gasparo, a Swiss citizen, received his medical degree at the Catholic University of Louvain (Belgium). He was a Research Fellow at the Medical Clinic at the University Hospital and had clinical training at the Gestational Endocrinopathy Outpatient Clinic. After working in the Department of Biochemistry at UC, San Francisco, he joined the Research Department of Ciba-Geigy, Basel. He led the team involved in the development of the selective aldosterone receptor antagonist, eplerenone, reported the existence of two angiotensin II receptor subtypes, and developed valsartan, a nonpeptidic angiotensin II receptor antagonist. He is the author and coauthor of more than 200 publications.

Jaroslav Kalvoda

Leimgrubenweg 21, 4102 Binningen, Switzerland Jaroslav Kalvoda was born in Bratislava (SK), completed his undergraduate and graduate studies (PhD thesis: Absolute Configuration of Morphine) at the ETH Z€ urich. After 3 years working as a supervisor of PhD students (natural products), he joined Ciba Ltd., Basel, as a Research Chemist (steroids), took over the position of a group and section leader, became head of the unit “Special Chemistry” (leucotrienes and b-lactams), and was nominated Fellow of Ciba-Geigy Research. Later on, he became responsible, as section leader, for chemistry in the unit “Exploratory Research and Services” (peptides, peptidomimetics, enzyme inhibitors, and nucleotides). He has authored or coauthored 105 papers/reviews.

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16 Clevudine, to Treat Hepatitis B Viral Infection Ashoke Sharon, Ashok K. Jha, and Chung K. Chu

16.1 Current Status of Anti-HBV Agents

Hepatitis B virus (HBV) infection is still a major global health problem despite marked progress in public health intervention programs. It is estimated that more than 2 billion individuals have been infected. Of these, approximately 350 million are chronically infected and at risk of serious illness and death from cirrhosis and hepatocellular carcinoma (HCC), diseases that are estimated to cause 600 000 deaths each year worldwide [1]. HBV affects 1.25 million persons in the United States and HBV infection accounts annually for 4000–5500 deaths in the United States [1–4]. Chronic hepatitis B (CHB) is an important cause of cirrhosis and liver failure and a common cause of HCC. Hepatitis B virus-related HCC is the third most common cause of cancer death in the Asia–Pacific region. According to serological criteria, high prevalence of chronic HBV infection is found in areas of sub-Saharan Africa, Southeast Asia, the Eastern Mediterranean countries, south and western Pacific islands, the interior of the Amazon basin and in certain parts of the Caribbean [5]. The availability of safe and effective vaccines has significantly reduced burden of the diseases. Although many countries have a selective vaccination policy, no uniform approach for identifying risk groups exists among countries. HBV is a double-stranded DNA virus of a hepadnaviridae family, which also infects ducks, ground squirrels, and woodchucks. Hepadnaviruses have a strong preference for infecting liver cells, but small amounts of hepadnaviral DNA can also be found in kidney, pancreas, and mononuclear cells [6–9]. However, infection at these sites is not linked to extrahepatic disease. The virus is a spherical particle with a diameter of 42 nm with an outer shell (or envelope) composed of several proteins collectively known as hepatitis B surface antigen (HBsAg) proteins. This outer HBsAg surrounds an inner protein shell, composed of hepatitis B core antigen proteins (HBcAg) referred to as the core particle or capsid. The core particles surround the viral DNA and an enzyme that has DNA polymerase as well as reverse transcriptase functions.

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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The core gene produces hepatitis B e antigen (HBeAg) that is responsible for active viral replication. The primary routes of transmission are vertical (mother to child), blood and bloodproduct exposure, and sexual intercourse. Once the virus passes the immune system, it attaches to a susceptible liver cell (hepatocyte) and the viral genome enters the nucleus by nuclear import to start the replication cycle that can be divided into three parts: RNA synthesis, reverse transcription, and DNA replication (Figure 16.1). In the nucleus, the viral genomic DNA is repaired by viral polymerase to form a covalently closed circular form called covalently closed circular DNA (cccDNA) [10–12]. The cccDNA is the template for the transcription by cellular RNA polymerase II for the formation of genomic and subgenomic RNA. All the viral RNA is then transported to cytoplasm, where its translation produces the viral envelope, core and polymerase proteins. Next, assembly of nucleocapsids is initiated, and after priming the genomic RNA is incorporated into the assembling viral core [13, 14]. Synthesis of the (
)-strand DNA is then initiated by reverse transcriptase with simultaneous degradation of the genomic RNA. A short oligoribonucleotide from the genomic RNA hybridizes to the (
)-strand DNA and primes the synthesis of the ( þ )-strand DNA by

Figure 16.1 Schematic diagram for HBV life cycle. HBV virions bind to surface receptors and are internalized. Viral core particles migrate to the hepatocyte nucleus, where their genomes are repaired to form a covalently closed circular DNA that is the template for viral messenger RNA (mRNA) transcription. The viral mRNA that results is translated in the cytoplasm to produce the viral surface, core, polymerase, and

X proteins. There, progeny viral capsids assemble, incorporating genomic viral RNA (RNA packaging). This RNA is reversetranscribed into viral DNA. The resulting cores can either bud into the endoplasmic reticulum to be enveloped and exported from the cell or recycle their genomes into the nucleus for conversion to cccDNA.

16.1 Current Status of Anti-HBV Agents

DNA polymerase. Some viral cores bearing the mature genome are transported back to the nucleus, where their newly minted DNA genomes can be converted to cccDNA to maintain a stable intranuclear pool of transcriptional templates [15]. The remaining viral cores are transported into the endoplasmic reticulum (ER) and after viral assembly and budding, the virus is released into the blood stream [2]. HBV infection spontaneously resolves in 90–95% of immunocompetent adults [16]; however, 5–10% develop chronic HBV infection that is characterized by persistence of circulating hepatitis B surface antigen in the blood. The prevalence of chronic hepatitis B infection continues to be highly variable, ranging from 10% in some Asian and western Pacific countries to under 0.5% in the United States and northern European countries [17]. These individuals with CHB are at risk for progression to cirrhosis and/or hepatocellular carcinoma. Depending on the presence of hepatitis B e antigen and its antibody (anti-HBe), CHB can be divided into two categories: hepatitis B e antigen-positive (HBeAg ( þ )) and hepatitis B e antigen-negative (HBeAg (
)). Classical hybridization techniques [18] can be used to detect serum HBV DNA replication in both types of CHB. It is believed that the HBeAg (
) CHB is associated with certain precore or core mutants [18, 19]. A recent study showed that people who are positive for both HBsAg and HBeAg were six times more likely to progress to hepatocellular carcinoma compared to those with HBsAg alone [20]. Similarly, the possibility of hepatocellular carcinoma is four times greater in those with detectable serum HBV DNA level in comparison to those with an undetectable level. Although the management of both types of CHB is a clinical challenge, the HBeAg (
) patient faces more difficulties [21, 22]. The majority of patients with CHB require long-term antiviral therapy, which may often result in the emergence of drug resistance. The reverse transcriptase of HBV does not have the proofreading ability and produces 1012–1013 virions per day, which can lead to every possible mutation [23]. However, due to the overlapping open reading frames, many of the spontaneous mutations may not be viable and would not be propagated. Nevertheless, some spontaneously occurring mutations may persist and small numbers of potentially resistant HBV mutants may be present before exposure to any nucleoside anti-HBV agent [24]. The development of drug-resistant mutants is dependent on (i) long-term persistence of infected cells along with the HBV genome as cccDNA, (ii) viral mutation frequency, (iii) mutability of the antiviral target site, (iv) selective pressure exerted by the drug, (v) rate of virus replication along with the immune incompetence, and (vi) the fitness of the resistant virus. Hepatitis B vaccination is the most important strategy to prevent CHB. However, with the introduction of anti-HBV agents, there is renewed hope that the development of cirrhosis and its complications in people with established CHB can be avoided. Approved antiviral regimens have been shown to improve the short-term outcome of the disease in some patients; however, they do not provide a cure or durable remission in most patients who are chronically infected with HBV [25]. Drugs, which are approved by the US FDA for the treatment of chronic HBV, consist of two groups: immunomodulators (interferons) and direct antiviral agents (nucleoside analogues).

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16.1.1 Nucleoside Reverse Transcriptase Inhibitors

Nucleoside analogues suppress HBV-DNA replication mainly by inhibiting the viral polymerase/reverse transcriptase as triphosphates. A number of nucleoside analogues have previously been tested for antiviral efficacy on patients with chronic hepatitis B. The nucleoside analogues target the viral polymerase that is a multifunctional enzyme of HBV, serving as both DNA-dependent DNA polymerase and reverse transcriptase. Lamivudine, adefovir dipivoxil, entecavir, telbivudine, and tenofovir disoproxil are FDA-approved nucleoside reverse transcriptase inhibitors (NRTIs) for the treatment of CHB. Clevudine, valtorcitabine, and pradefovir are under various phases of clinical trials (Figure 16.2). Clevudine also known as L-20 -Fluoro-5-methyl-b-L-arabinofuranosyluracil (LFMAU) is an L-thymidine analogue possessing potent antiviral activity against HBV, hepatitis delta virus (HDV), and Epstein–Barr virus (EBV) [26]. Bukwang Pharmaceutical Co. in South Korea received the NDA approval for anti-HBV from the South Korean regulator in November 2006. This chapter describes the brief discovery, preclinical, and clinical accounts of clevudine.

Figure 16.2 Structures and current status of nucleoside analogues of anti-HBV agents.

16.2 Chemical Evolution of Clevudine

16.2 Chemical Evolution of Clevudine

Clevudine is unique with respect to other presently used anti-HBV agents as it has a fluoro group at 20 -b position [27–30]. The presence of a fluorine in a nucleoside has unique effects of its chemical, biological, and structural properties [31, 32]. The Dform of clevudine (FMAU) was synthesized earlier and evaluated as a potent antiviral agent, but it caused myelosuppression and neurotoxicity [33, 34]. Several nucleosides with the unnatural L-configuration were shown to be potent chemotherapeutic agents against human immunodeficiency virus (HIV), hepatitis B virus, and certain forms of cancer [32, 35]. This information provided a rationale to Chu and coworkers to discover L-FMAU (clevudine) to evaluate its therapeutic potential [36]. 16.2.1 Development of Synthetic Routes

Initially L-FMAU was synthesized by Chu and coworkers using L-ribose as a starting material [26]. Subsequently, a practical synthetic procedure for the large-scale synthesis of L-ribose derivative 1 from L-xylose was developed (Scheme 16.1) [37]. L-Xylose was converted to L-ribose derivative 1 in five steps following oxidation–reduction of 30 -OH, utilizing pyridinium dichromate (PDC), NaBH4, and benzoylation [38]. Compound 1 was then successively treated with HCl to deprotect acetonide followed by benzoylation with benzoyl chloride (BzCl)-pyridine and then acetic anhydride-acetic acid-H2SO4 to obtain 1-O-acetyl-2,3,5-tri-O-benzoyl-b-L-ribofuranose (2). Treatment of 2 with HCl in DCM followed by hydrolysis yielded 3, which was treated with SO2Cl2 and imidazole in DMF-DCM to obtain the imidazolyl sulfonate 4. Fluorination of 4 using KHF2 yielded 5, which was then brominated with

Scheme 16.1 Synthesis of L-FMAU from Lxylose. Reagents and condition: (i) BzCl, py; (ii) 1% HCl/CH3OH; (iii) Ac2O, AcOH, concentrated H2SO4; (iv) (1) HCl (g), AcCl, DCM, 0  C; (2) H2O, CH3CN; (v) SO2Cl2, DMF,

DCM,
15  C to rt, imidazole; (vi) KHF2, 2,3butanediol, 48% HF/H2O, reflux; (vii) HBr/ AcOH, DCM, rt; (viii) silylated thymine, CHCl3, reflux; and (ix) methanolic ammonia.

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Scheme 16.2 Synthetic procedure of L-FMAU from L-arabinose. Reagent and condition: (i) BnOH, HCl (gas), 94%; (ii) DMP, p-TSOH, acetone; (iii) PCC, Ac2O, DCM; (iv) NaBH4, methanol (53% from 9); (v) 4% aqueous TFA, reflux; (vi) 1% HCl in methanol, rt; (vii) benzoyl chloride, pyridine; (viii) acetic anhydride, acetic

acid, conc. H2SO4 (40% from compound 12); (ix) HCl (g), AcCl, DCM, 63.7%; (x) SO2Cl2, DCM:DMF, imidazole; (xi) Et3N3HF, EtOAc; (xii) HBr/AcOH (45% w/v), DCM; (xiii) (a) silylated thymine, CHCl3, reflux (69.5% from 3); (b) methanolic ammonia, 88.2%.

HBr-AcOH to obtain the bromo derivative 6. The coupling of 6 with silylated thymine in 1,2-dichloroethane (DCE) yielded the b-isomer 7 as the major product with a trace amount of the a-anomer. The a-isomer was easily removed by silica gel column chromatography and recrystallization. The free nucleoside 8 (L-FMAU) was obtained by the deprotection using NH3-CH3OH. Later, a modified practical synthetic procedure starting from L-arabinose was also developed by Chu and coworkers [39] as shown in Scheme 16.2. L-Arabinose was converted to L-ribose 13 in five steps, which was used for the synthesis of key intermediate 6 to complete the synthesis of clevudine. In addition, Sznaidman et al. [40] have developed another protocol for the synthesis of L-FMAU from L-arabinose. The key step is the introduction of a fluoro group using selectfluor with glycal 16 as shown in Scheme 16.3. 16.2.2 Structure–Activity Relationships L-2

0

-Fluoro-5-methyl-b-L-arabinofuranosyluracil has been shown to be a potent antiHBV agent in vitro as well as an inhibitor of EBV [26, 41–43]. In comparison to the corresponding D-enantiomer, L-FMAU exhibits more potent anti-HBV activity in vitro without any significant cytotoxicity in a variety of cell lines such as CEM, 2.2.15, H1, and bone marrow progenitor cells (Table 16.1). In addition, L-FMAU does not

16.2 Chemical Evolution of Clevudine

Scheme 16.3 Synthesis of L-FMAU from Larabinose. Reagent and condition: (i) Ac2O, pyridine; (ii) HBr/AcOH, Ac2O, 57%; (iii) Zn dust, CuSO4, NaOAc, AcOH, 60%; (iv) selectfluor, nitromethane:water, 70%; (v)

NaOMe, MeOH, 100%; (vi) H2SO4, MeOH, reflux, 80%; (vii) BzCl, pyridine, 44%; (viii) HBr/ AcOH, DCM; (ix) (a) silylated thymine, CHCl3, reflux 42%; (b) n-butyl amine, methanol, reflux, 3 h, 82%.

interfere with mitochondrial function, which has been the major concern for some antiviral nucleosides such as FIAU (1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-5-iodouracil) and DDC (Zalcitabine) [44–46]. A series of 1-(2-deoxy-2-fluoro-b-L-arabinofuranosyl)pyrimidine nucleosides have been synthesized to study the structure–activity relationships of related nucleosides and evaluated for their antiviral activity against HBV in 2.2.15 cells. The replacement of a methyl at 5-position of uracil by ethyl [(1-(20 -deoxy-20 -fluoro-b-L-arabinofuranosyl)-5-ethyluridine, L-FEAU)], halides (F, Cl, Br, I), CF3, ethynyl, or a Z-bromovinyl group leads to complete loss of activity (Figure 16.3). Among the compounds synthesized, the lead compound, L-FMAU, exhibited the most potent anti-HBV Table 16.1 Anti-HBV and anti-EBV activities, growth inhibitions, and selectivity indices of L-FMAU and its analogues [26].

Compound

L-FMAU D-FMAU L-FEAU D-FEAU

Anti-EBV Anti-HBV activitya) in activityb) 2.2.15 cells in H1 cells (EC50, mM) (EC90, mM)

0.1 2.0 >5.0 5.0

5  0.8 0.1  0.02 >20 1  0.05

Growth inhibition (ID50, mM)c) with the following cells:

MT2

CEM

H1

100 8–9 100 100

>100 913  70 0 <10 >100 >400 90 >400

Selectivityc)

2.2.15d) 2.2.15-HBV H1-EBV >200 50 NDe) ND

>2000 25 ND ND

183 >100 >90 >90

a) EC50, 50% effective concentration. b) H1 cells were treated for 5 days, and the 90% inhibitory dose was determined by bioassay. c) For growth inhibition assays, triplicates were evaluated for each concentration of the drug. ID50, 50% inhibitory dose. d) The toxicity to 2.2.15 cells was ascertained by using a growth inhibition assay in the presence of the drug for a period of 72 h. e) ND, not determined. FMAU ¼ 20 -fluoro-5-methyl-b-L-arabinofuranosyluracil; FEAU ¼ 20 -fluoro5-ethyl-b-L-arabinofuranosyluracil.

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Figure 16.3 Structure–activity relationship of L-FMAU. aEC50 concentration of nucleosides in HBV 2.2.15 cells.

activity (EC50 0.1 mM). None of the other uracil derivatives showed significant antiHBV activity up to 10 mM. Among the cytosine analogues, cytosine itself and 5-iodocytosine showed moderately potent anti-HBV activity (EC50 1.4 and 5 mM, respectively). The cytotoxicity of these nucleoside analogues has also been assessed both in 2.2.15 cells and in CEM cells. None of these compounds displayed any toxicity up to 200 mM in 2.2.15 cells [38]. Extensive structure–activity relationship studies have also been carried out among purine nucleosides. Among them the adenine and hypoxanthine derivatives exhibit good in vitro anti-HBV activity (EC50 1.5 and 8 mM, respectively) without significant toxicity up to 200 mM [47]. From the structure–activity studies, the thymine analogue appeared to be the most promising compound in terms of potency and in vitro toxicity. For these reasons, L-FMAU (clevudine) was selected as a preclinical candidate for further development.

16.3 Metabolism and Mechanism of Action

Clevudine requires a step-wise biotransformation to its triphosphate form to exert its pharmacological activities (Figure 16.4) [48]. The 50 -mono phosphorylation is cata-

16.3 Metabolism and Mechanism of Action O H3C

O

O H 3C

NH N F

O O

T K or dC K OH

OH L-FMAU

j391

H 3C

NH N F

O O

T MPK O

P

NH N F

O O

O

P

P

OH

OH L-FMAU-MP

L-FMAU-DP 3-Phosphoglycerate kinase O H 3C

No incorporation into growing DNA

L-FMAU-TP-HBVPolymerase Complex

HBV Polymerase

NH N F

O O

O

P

P

OH L-FMAU-TP

Figure 16.4 Metabolic pathway of L-FMAU as a HBV polymerase inhibitor [54].

lyzed by thymidine kinase (TK) along with deoxycytidine kinase (dCK) in human primary hepatocytes. The diphosphorylation may be associated with thymidylate kinase (TMPK) activity and this conversion of 50 -monophosphate to the corresponding 50 -diphosphate is the rate-limiting step in clevudine phosphorylation. Finally, the active triphosphate formation is carried out by 3-phosphoglycerate kinase [49–51]. Clevudine is known to preferentially inhibit HBV pol DNA-directed DNA synthesis during viral replication [42, 49]. It has been reported that clevudine triphosphate (CLV-TP), unlike other nucleoside inhibitors, is not incorporated into DNA by HBV DNA polymerase [52]. This has been rationalized using molecular dynamics study [53], in which L-FMAU-TP binds to the active site of HBV polymerase, the sugar moiety of L-FMAU-TP separates from the 30 -end of the primer strand by the conformational change, and the a-phosphate atom also moves out from the 30 -OH   group of the primer end by more than 2 A (5.7 A) [53]. Therefore, the incorporation of L-FMAU-TP into the growing viral DNA chain to act as a chain terminator would not take place easily because of this deformed polymerization geometry, which may be the reason that L-FMAU is not incorporated into the HBV DNA (Figure 16.5). Using an endogenous HBV polymerase assay, the mode of inhibition for CLV-TP was determined to be noncompetitive [42]. Since several active site mutations are known to confer resistance to clevudine in vitro, CLV-TP may be binding at or near the active site of the HBV polymerase without being utilized as a substrate. Clevudine enters cells through both facilitated nucleoside transport and nonfacilitated passive diffusion and is a substrate for three intracellular kinases (thymidine kinase, deoxycytidine kinase, and mitochondrial pyrimidine kinase), which are responsible for its phosphorylation [48–50]. These features suggest that relatively low plasma levels of clevudine may provide therapeutic levels of clevudine triphosphate in target cells. High levels of clevudine triphosphate were formed in primary human hepatocytes and the major CLV metabolite was the monophosphate. Maximum triphosphate levels were achieved with exogenous CLV concentrations of

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Figure 16.5 Comparison of polymerization geometry of HBV [53].

approximately 10 mM. The level of phosphorylation of CLV was dependent upon exogenous drug concentration and exposure time. When cells were exposed to extracellular concentrations of CLV (0.5–1.0 mM) that approximated the human plasma Cmax for the 30 mg dose, high concentrations of CLV triphosphate were detected. This intracellular concentration of CLV triphosphate was approximately 300- to 500-fold higher than the inhibition constant (Ki ¼ 0.12 mM) reported for the HBV polymerase. Exogenously added nucleosides, thymidine, deoxycytidine, lamivudine, zidovudine, and emtricitabine, which are likely to compete with clevudine for phosphorylation, did not inhibit the phosphorylation of CLV [48]. The clevudine triphosphate mainly inhibits the viral ( þ )-strand DNA synthesis without being incorporated into DNA [42] and shows little effect on (
)-strand DNA synthesis [55, 56]. This property makes clevudine unique in contrast to other nucleoside analogues. In addition, clevudine does not inhibit human cellular polymerases and does not affect mitochondrial function or host DNA synthetic machinery [38, 42]. Interestingly, the sustained antiviral effect of clevudine after discontinuation of the drug was observed for at least 6 months or even 1 year after treatment, which has not been achieved by other anti-HBV agents. Researchers have suggested that a significant reduction in cccDNA and/or immunomodulatory effects may be responsible for the sustained antiviral effect after drug withdrawal [57].

16.4 Pharmacokinetics

The pharmacokinetic studies of clevudine in rats [58, 59] and woodchucks [60] demonstrate that there were no significant differences in the pharmacokinetic parameters between the doses (a < 0.05) with the exception of slower elimination rate in woodchuck. Thus, the disposition of L-FMAU was linear over the dosage of 10–50 mg/kg. The half-life (t1/2) following oral administration was greater than the intravenous administration in both rats and woodchucks, which could be partly due to the prolonged oral absorption. After intravenous administration, the steady-state

16.4 Pharmacokinetics Table 16.2 Pharmacokinetic parameters of clevudine at day 28 [57].

Parameters mean (% CV)

n

Cmax (mg/ml)a)

Cmin (mg/ml)b)

tmax (h)

AUC0 ! t (h mg/ml)c)

t1/2 (h)

CL/F (ml/min)d)

50 mg group 100 mg group 200 mg group

10 10 6

0.4 (19) 0.8 (24) 1.6 (30)

0.07 (27) 0.12 (12) 0.21 (19)

1.5 (68) 1.1 (52) 1.7 (24)

3.0 (20) 5.7 (13) 12.3 (12)

61.0 (29) 43.6 (16) 50.8 (29)

285 (22) 296 (13) 276 (13)

a) Cmax ¼ maximum clevudine concentration at steady state. b) Cmin ¼ minimum plasma clevudine concentration at steady state. c) AUC0 ! t ¼ area under the plasma clevudine concentration–time over a dosing interval at steady state. d) CL/F ¼ apparent total body clearance.

volume (VSS) averaged 0.99  0.17 l/kg, greater than the total volume of water in the body of a woodchuck, indicating that the compound was distributed intracellularly. The intravenous administration indicates the total clearance (CLT ¼ 1.15  0.28 l/h/ kg) in rat is faster than that of woodchucks (CLT ¼ 0.23  0.07 l/h/kg). The renal clearance (CLR) was 60% of the total clearance (CLT) in both animal models. The bioavailability of clevudine in rat and woodchuck is about 60 and 20%, respectively. In phase II dose-escalating trials of clevudine in patients, after the 10 mg dose, clevudine concentrations were low and close to the limit of assay detection, thus preventing reliable pharmacokinetic evaluations. Mean (% coefficient of variation) pharmacokinetic parameters at steady state on day 28 for the 50, 100, and 200 mg cohort patients are summarized in Table 16.2 [60]. 16.4.1 Woodchuck Studies

Woodchuck hepatitis virus (WHV) and its natural host, the Eastern woodchuck (Marmota monax), constitute a useful model of HBV-induced disease, including hepatocellular carcinoma [61–63]. Four weeks of L-FMAU therapy was well tolerated by chronically infected woodchucks and inhibited WHV replication in a dosedependent manner [64]. Dramatic reductions in all measured serologic and intracellular markers of viral replication and viral gene expression were observed at the highest doses of L-FMAU used in this study. No evidence of lactic acidosis, a characteristic of the patterns of toxicity induced by the D-isomers of this family of nucleosides (filauridine, D-FEAU, and D-FMAU), was observed in any of the treated animals [64]. Doses of 3.0 and 10 mg/kg appeared to be similar in effectiveness with respect to maximal suppression of viremia and intrahepatic WHV replication by the end of the treatment period (Figure 16.6). However, treatment with 10 mg/kg conferred additional antiviral benefit in the form of a more sustained reduction in WHV replication, serum WHsAg, and intrahepatic WHcAg expression following drug withdrawal. A preliminary analysis of cccDNA in animals treated with 10 mg/kg L-FMAU in this study demonstrated an average reduction of cccDNA levels of at least 10-fold after 4 weeks of therapy. In this study, the relative levels of cccDNA in

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Figure 16.6 Mean serum WHV DNA of woodchucks treated with varying doses of clevudine for 28 days (n ¼ 4/group) [64].

individual animals at the end of 4 weeks of therapy with 10 mg/kg L-FMAU are inversely related to the duration of the suppression of WHV replication following drug withdrawal [64]. In a small controlled study, clevudine suppressed hepatitis delta virus viremia in chronically infected woodchucks [65]. Suppression was correlated with the marked reduction of woodchuck hepatitis virus surface antigen in individual animals, consistent with the concept that repression of surface antigen expression may be a useful antiviral strategy for HDV. This study indicates that the WHV/woodchuck model of experimental chronic hepatitis infection could be applied to therapeutic studies of chronic HDV superinfection. Nevertheless, the relatively rapid progression to hepatocellular carcinoma in WHV-infected woodchucks does pose challenges for the use of this model for evaluating drug efficacy against chronic HDV disease.

16.5 Clinical Studies

In phase I/II dose-escalating clinical study [57], randomized phase II clinical trial [66], 4-week and 12-week clevudine therapy produced potent viral suppression during treatment along with induced prolonged post-treatment antiviral suppression effects. Recently, a phase II, multicenter, randomized study comparing 10, 30, and 50 mg clevudine once daily for 12 weeks, with 24 weeks off-treatment follow-up, was carried out to evaluate the safety, pharmacokinetics, and antiviral activity [67]. Nucleosidena€ıve patients with chronic hepatitis B and without coinfection with HIV/HCV were registered. A total of 31 patients were enrolled into the 10 mg (n ¼ 10), 30 mg (n ¼ 11), and 50 mg (n ¼ 10) groups. At week 12, one of 10, five of 11, and two of 10 patients had viral load below the assay lower limit of detection. Clevudine was well tolerated and did not show any severe adverse events. The mean plasma half-life of clevudine was 70 h and consequently is not the cause of the delayed viral rebound seen in some patients. Through modeling, 97% of the maximal treatment effect was reached with a 30 mg daily dose.

16.5 Clinical Studies

Figure 16.7 Clevudine therapy 30 mg treatment for 24 weeks: median HBV DNA and ALT values over time [68].

Another clinical study concluded that clevudine 30 mg treatment for 24 weeks was well tolerated and exhibited more potent antiviral activity and a higher ALT normalization rate than the 12-week treatment with durable efficacy at week 24 off therapy (Figure 16.7) [68]. In a double-blind, randomized, placebo-controlled phase III study [69, 70] at 31 centers in South Korea, clevudine 30 mg or placebo once a day for 24 weeks, and followup for additional 24 weeks, was evaluated for the antiviral effect in HBeAg (
) and HBeAg ( þ ) patients without coinfection with HIV/HCV or evidence of hepatocellular carcinoma (Figures 16.8 and 16.9). Clevudine treatment for 24 weeks produced prompt and profound viral suppression. Median serum HBV DNA reductions from baseline at week 24 were 5.10 log10 and 0.27 log10 copies/ml in the clevudine and placebo groups, respectively. Viral suppression in the clevudine group was sustained even after withdrawal of treatment with 3.73 log10 reduction at week 34 and 2.02 log10 reduction at week 48, compared to 0.51 log10 reduction at week 34 and 0.68 log10 reduction at week 48 in the placebo group. The proportion of patients who achieved normalization of ALT levels was 68.2% in the clevudine group and 17.5% in the placebo group at week 24. Interestingly, there was no emergence of

Figure 16.8 Double-blind, randomized, placebo-controlled phase III study [70]: clevudine is highly efficacious in hepatitis B e antigen-negative (HBeAg
) chronic hepatitis B with durable off-therapy viral suppression.

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Figure 16.9 Double-blind, randomized, placebo-controlled phase III study [69]: 24-week clevudine therapy showed potent and sustained antiviral activity in HBeAg-positive chronic hepatitis B.

resistance noted in both studies. In an in vitro study reported by Pharmasset [71], clevudine demonstrated no antiviral effects on HIV. Therefore, it provides a suitable treatment strategy for HIV/HBV coinfected patients and cross-resistance with antiHIV agents can be avoided. A recent study [72] involving seven patients, who had developed muscle weakness during long-term clevudine therapy for hepatitis B were enrolled in this study, indicates that a long-term therapy can cause depletion of mtDNA and lead to a muscle myopathy characterized by mitochondrial dysfunction and myonecrosis in a minority of the patients. Therefore, it is advised that careful clinical observation with respect to muscle-related symptoms should be made and regular measurements of serum CK levels and lactate levels should be performed on chronic hepatitis B patients who are taking clevudine for more than 32 weeks. However, the observed depletion of the mtDNA was probably caused by the disruption of the other cellular metabolism, not by the inhibition of DNA polymerase c or incorporation of CLV-TP into the mitochondrial DNA c, as it has previously been shown during the preclinical studies that there was no increase of lactic acid production by clevudine [42].

16.6 Drug Resistance

Clevudine was tested in different cell lines containing existing drug-resistant mutants (Table 16.3) [56, 73–77]. Although there were some conflicts, a majority of the data show that lamivudine-associated dual mutants (M204I þ L180M or M204V þ L180M) and M204I single mutant cause cross-resistance to clevudine in vitro. The M204V or L180M mutant had only reduced susceptibility to clevudine. A recent molecular modeling study also supports the above resistance profile [54]. The modeling studies clearly suggest the binding mode of clevudine triphosphate describes the backward and upward shifting of L-sugar ring in the active site without

16.6 Drug Resistance Table 16.3 Antiviral activity and fold resistance (FR)a) of anti-HBV agents against wild-type and 3TCresistant HBV polymerases [54, 77–80].

HBV strain

Wild type L180M M204V M204I L180M–M204Vc)

3TC

ADV

ETV

LdT

L-FMAU

IC50

FR

IC50

FR

IC50

FR

IC50

FR

IC50

FR

0.6 0.8 8.5 >50 >50

1.0 1.7 18 >100 >100

3.9 2.0 2.8 2.7 0.6

1 0.5 0.7 0.7 0.2

0.8 na na na 5.0

1 1b) 10b) nab) 6

0.17 1.96 na 39.5 22.2

1 >10 na >100 >100

0.1 >100 1.5 >100 >100

1 >100 15 >100 >100

na: Not available. a) Fold resistance ¼ (mutant IC50)/(wt IC50). b) Separate study on ETV shows that M204V mutation had 10-fold reduction in the potency of ETV, while L180M had no significant effect [81]. c) Dual mutant 3TC-resistant strain (L180M–M204V and L180M–M204I) having similar pattern of resistance profile against most of the potential compounds [82, 83].

affecting the neighboring amino acid residue, which makes it suitable for wild-type HBV-polymerase (Figure 16.10). A careful examination of binding mode shows the unfavorable stacking interaction of the thymine base of clevudine with the base of DNA-primer, resulting further in a distorted base pairing interaction with DNA-template. These studies support the fact that clevudine is not a perfect mimic of natural substrate. This also might provide an explanation for the high genetic barrier, which lowers the chance for the emergence of viral resistance during clevudine therapy. In L180M mutation (leucine to methionine), the lack of methyl group in the backside hydrophobic pocket

Figure 16.10 Binding mode of L-FMAU-TP in wild-type HBV showing the backward–upward shifting of sugar ring in comparison to TTP (only the mainframe of the triphosphate (Pa-O-Pb-O-Pc) shown for the sake of clarity) [54].

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Figure 16.11 (a) Binding mode of L-FMAU-TP along with mutant M204V residue showing negligible steric clash. (b) Binding mode of LFMAU-TP along with M204I and L180M residues (dual mutant L180M þ M204I HBV) showing the hydrophobic methyl group of

M204I is oriented toward hydrophilic group (30 OH) of L-FMAU-TP. In addition, the absence of one methyl group in the hydrophobic pocket due to L180M mutation (only mainframe of the triphosphate (Pa-O-Pb-O-Pc) shown for the sake of clarity) [54].

decreases binding affinity of clevudine. However, M204 residue orients toward the lower hydrophobic pocket as clevudine binds to the active site with the upward shifting of ribose ring. Therefore, M204 mutation may not significantly affect in terms of steric clash (Figure 16.11a). Further studies of the proposed binding mode reveal a steric clash in the case of M204I mutation in HBV due to the unfavorable position and orientation of the methyl group of the M204I residue (Figure 16.11b). This could be the reason for the reduced susceptibility of clevudine both in M204I and in dual mutant L180M/M204I. In the case of dual mutation L180M þ M204V, there is a lack of two methyl groups from the backside hydrophobic pocket, and consequently, clevudine is no longer active in this dual mutant HBV strain.

16.7 Toxicity and Tolerability

According to the available clinical trial data, clevudine was well tolerated in the patients and demonstrated potent antiviral activity [57, 61, 66, 67]. Both in vitro and in vivo studies have demonstrated the favorable toxicological profile of clevudine [26, 38, 58, 67]. Clevudine had an excellent safety profile with no marked dose-related adverse events or laboratory abnormalities during treatment or within 2-month followup period. The general adverse events included mild headache, asthenia, dizziness, abdominal pain, pharyngitis, and diarrhea. However, despite the potent anti-HBV activity characterized by sustained antiviral activity even after discontinuation of the

16.9 Combination Therapy

drug, concerns regarding possible long-term toxicity remain because the underlying reason of muscle myopathy, although it is reversible, is still unclear [57]. Hence, data from longer dosing studies may be needed to evaluate the ultimate safety of clevudine. A recent study indicates that long-term therapy (more than 32 weeks) with clevudine can cause depletion of mtDNA and lead to a myopathy characterized by mitochondrial dysfunction and myonecrosis in a minority of the patients [72]. Modification of the dosage and/or the treatment schedule, such as a drug vacation after 1-year treatment for a period of 6 months or 1 year for those who achieve the sustained antiviral effects, or using an additional agent to mitigate the muscle myopathy, may be seriously considered in the future clinical trials to assess and to manage the long-term side effects of clevudine.

16.8 Dosage and Administration

Based on phase II [57, 69] and phase III [69, 70] clinical trials, clevudine 30 mg once daily was safe, most efficacious and well tolerated. Bukwang, a pharmaceutical company of Korea which has been developing the drug, launched clevudine as a 30 mg once-daily capsule on the Korean market in November 2006 under the brand name Levovir.

16.9 Combination Therapy 16.9.1 Combination of Clevudine with Other Agents

In general, all orally available anti-HBV nucleosides target the viral polymerase, but each compound may have slightly different mode of action on overall viral replication. Lamivudine, adefovir, tenofovir, telbivudine, and emtricitabine (ETC) inhibit the reverse transcriptase and ultimately DNA synthesis [84]. Clevudine has moderate effects on the priming and (
)-strand DNA synthesis but has potent inhibitory activity on ( þ )-strand DNA synthesis [56]. Entecavir inhibits all three activities of HBV polymerase: priming, reverse transcriptase, and DNA synthesis. Based on these distinct modes of action, the combination of drugs targeting different steps of viral genome replication may lead to additive or synergistic effects. The antiviral effects of clevudine plus emtricitabine (ETC) or emtricitabine alone have also been studied in both HBeAg ( þ ) and HBeAg (
) patients in two separate 24-week studies [85, 86]. In a double-blind, multicenter study, patients with chronic hepatitis B, who had completed a phase III study of emtricitabine (ETC), were randomized 1 : 1 to 200 mg ETC once daily (QD) plus 10 mg CLV QD or 200 mg ETC QD plus placebo for 24 weeks with 24 weeks of followup. After 24 weeks of treatment, 74% (ETC-CLV) versus 65% (ETC alone) had serum HBV DNA levels of <4700

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copies/ml (P ¼ 0.114). Twenty-four weeks post-treatment, the mean change in serum HBV DNA levels from baseline was 1.25 log10 copies/ml (ETC þ CLV), 40% had undetectable viremia (versus 23% for ETC alone), and 63% had normal ALT levels (versus 42% for ETC alone) (P < 0.025 for all end points). There was no significant difference between arms observed, but significantly greater virologic and biochemical response 24 weeks post-treatment in the ETC þ CLV arm. Studies also showed that the resistance rate in treatment-na€ıve individuals is lower than those previously treated with antiviral nucleosides. 16.9.2 Combination of Clevudine with Vaccine

The recovery from HBV infection involves significant clearance of viral load and development of protective immunological processes that reduce the lifetime risk of serious liver disease [87]. Thus, immunotherapies that elicit humoral and cellular response patterns resembling to the recovery from HBV could provide an additive strategy [88]. Rational approach for the therapy of chronic HBV infection therefore involves the reduction of viral load combined with appropriately timed immune modulation/stimulation. Combination of clevudine and vaccine therapy had benefits based on sustained reduction of viraemia, antigenaemia, and hepatic WHV DNA and RNA, inhibition of progression of chronic hepatitis, reduced frequency of chronic liver injury, and delayed onset of hepatocellular carcinoma. This study demonstrates enhanced benefits of combination chemoimmunotherapy against viral load and disease progression in chronic hepadnaviral infection and provides a platform for further development of such treatment regimens [61, 89]. Additional studies are needed to develop optimal stimulation of immune response relative to drug therapy in order to induce stable production of neutralizing antibody and to more fully eradicate residual viral replication.

16.10 Summary

Clevudine is a potent and unique anti-HBV agent, differentiated from all other available nucleoside anti-HBV agents as listed below: 1) Clevudine is phosphorylated by cytosolic deoxycytidine kinase and thymidine kinase as well as mitochondrial pyrimidine kinase. Therefore, the antiviral activity of clevudine is not limited by the loss or alteration of these enzymes. 2) Clevudine is proposed to have a unique binding mode at the triphosphate catalytic binding site, thus lack of incorporation of clevudine-TP to the growing HBV DNA chain and thereby lack of selective pressure, resulting in higher genetic barrier for resistance. This character may be related to the low viral breakthrough observed during the long-term treatment periods. 3) Clevudine demonstrated sustained antiviral activity with ALT normalization and no rebound after discontinuation in woodchuck and human up to 6 months to

References

1 year possibly due to the reduction of cccDNA. Furthermore, ALT has been normalized throughout the treatment periods, which was sustained for 6 months to 1 year even after discontinuation of the drug. 4) Clevudine has sustained suppression of both serum HBsAg and serum HBV DNA after discontinuation of therapy in some patients. In some of the good responders (5–6% of patients), clevudine induced a steady decline and eventual clearance of HBsAg after cessation of therapy. 5) In general, clevudine is safe and well tolerated, but during the long-term use (48 weeks), a minority of the patients experienced muscle myopathy, which is reversible after discontinuation of the drug. Therefore, patients should be carefully monitored for the level of creatine kinase in future clinical studies. Furthermore, in order to mitigate the side effects, modification of both the dosage and the treatment schedule, such as drug vacation after 1 year of treatment, for 6 months or 1 year for those who achieve the sustained antiviral, should be considered. Acknowledgments

Authors would like to gratefully acknowledge the following collaborators of their scientific and clinical studies on clevudine: Professor Yung-Chi Cheng of Yale University School of Medicine; Professor Bud C. Tennant of Cornell University College of Veterinary Medicine; Professor Brent E. Korba of Georgetown University School of Medicine; Professor H. S. Lee of Seoul National University School of Medicine; Professor B.C. Yoo of Samsung Hospital; Bukwang Pharmaceuticals as well as supports from National Institute of Allergy and Infectious Diseases, National Institutes of Health grants (AI-33655 and AI-25899).

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References

Chung K. Chu

The University of Georgia, College of Pharmacy, Athens, GA 30602, USA Chung K. Chu “David” received his PhD in medicinal chemistry from the State University of New York/Buffalo in 1974. He then joined Memorial Sloan-Kettering Cancer Center, New York, with Drs J.J. Fox and K. Watanabe. In1982, he joined the University of Georgia as an assistant professor. He is presently a Distinguished Research Professor (Emeritus) and Director of Drug Discovery Group at the College of Pharmacy, University of Georgia. Professor Chu has received numerous awards, including NIH Merit Award, 2001–2011, and Inventor of the Year Award (University of Georgia), 2002. He discovered nucleoside analogues LODDC (Troxatyl), clevudine (approved for Hepatitis B in S. Korea as Levovir), and DOT (an anti-HIV agent), which is in various stages of clinical trials. His main research interests lie in the discovery and the development of nucleosides as antiviral and anticancer agents.

Ashok K. Jha

The University of Georgia, College of Pharmacy, Athens, GA 30602, USA Ashok Kumar Jha was born in Bihar, India, in 1976. He completed his master’s in chemistry from Delhi University, New Delhi, India, in 1997. He earned his PhD in chemistry from Central Drug Research Institute, Lucknow, India, in 2004, working on design and synthesis of nonsteroidal molecules as SERMs (selective estrogen receptor modulators). Thereafter, he joined Jubilant Chemsys Ltd., Noida, India, as a research scientist. In 2006, he joined Drug Discovery Group, College of Pharmacy, University of Georgia, as a postdoctoral associate with Professor C.K. Chu, working on the synthesis of carbocyclic nucleosides as potential antiviral and anticancer agents.

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Ashoke Sharon

The University of Georgia, College of Pharmacy, Athens, GA 30602, USA Ashoke Sharon (born in 1975 in Bihar, India), after receiving a PhD from CDRI, Lucknow, joined the Drug Discovery Group, University of Georgia, USA, in 2004 for postdoctoral research on antiviral drug discovery under the guidance of Professor C. K. Chu. In 2009, he returned to India to join the Birla Institute of Technology, Mesra, as a Senior Lecturer and continue his antiviral drug discovery with his own research group. His research interest includes molecular modeling and synthesis of nucleoside/nonnucleoside analogues toward antiviral drug discovery.

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17 Rilpivirine, a Non-nucleoside Reverse Transcriptase Inhibitor to Treat HIV-1 Jerome Guillemont, Luc Geeraert, Jan Heeres, and Paul J. Lewi 17.1 Introduction

Acquired immune deficiency syndrome (AIDS) is an illness caused by a chronic infection with human immunodeficiency virus (HIV). The breakdown of the immune system resulting from HIV infection leads to increasing susceptibility to other infections and immune disorders. Today, more than 33 million people are infected worldwide with HIV [1]. It was demonstrated in 1983 that HIV was the causative agent of AIDS [2, 3]. From that moment on, strategies were developed to find agents with activity against this virus. Four years after chemical library screening programs were initiated, zidovudine was approved by the Food and Drug Administration (FDA) [4] and introduced as the first treatment for HIV-1 infection. This nucleoside analogue inhibits the viral reverse transcriptase (RT), a polymerase responsible for the conversion of viral single-stranded RNA into double-stranded DNA. Hence, zidovudine belongs to the class of nucleoside analogue reverse transcriptase inhibitors (NRTIs). As RT plays an essential role in the HIV replication cycle, inhibition of this enzyme prevents virus replication. Unfortunately, soon after zidovudine was introduced into the market resistance of HIV against the drug emerged. Further screening programs demonstrated that it was possible to inhibit RT via an alternative, nonnucleosidic, approach. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are able to induce their own binding pocket in the vicinity of the RT active site. Binding of an NNRTI in this allosteric site causes major changes in the RT conformation resulting in the inhibition of the enzyme. This class is active only against HIV-1 but not against other retroviruses. The first NNRTIs discovered were the TIBO series at Janssen Pharmaceutica [5–7] and the nevirapine-like compounds at Boehringer Ingelheim. In 1996, nevirapine was introduced to the market as the first NNRTI for treating patients infected with HIV-1 virus [8–10]. As with the nucleoside inhibitors, resistance against nevirapine very soon emerged. Delavirdine was approved in 1997, followed by efavirenz in 1998 [11]. For a long time, efavirenz has been considered as the gold standard among the NNRTIs because of its excellent in vivo potency and ease of administration (once a day). At the time of its introduction, Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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nevirapine was given twice a day. In addition, the safety profile of efavirenz was better than that of nevirapine in terms of rash and hepatotoxicity. The HIV-1 RTenzyme is a heterodimer consisting of a p66 and a p51 subunit. Both subunits are translated from the same mRNA but differently processed at their Cterminal after translation. As a result, the structural similarity of the subunits is limited. The p66 subunit folds in a shape resembling a right hand with five domains: fingers, palm, thumb, connection, and RNase H. The p51 subunit supports this conformation of p66. NNRTIs are noncompetitive inhibitors inducing their own hydrophobic binding pocket within the p66 palm domain of RT, close to the catalytic site. This pocket contains sidechains of several aromatic (Y181, Y188, F227, Y232), hydrophobic (P59, L100, V106, V179, L234, P236), and hydrophilic residues (K101, K103, S105, D132, E224) of the p66 subunit and two residues of the p51 subunit (I135 and E138). In the absence of inhibitor, the pocket is filled with the side chains of the Y181 and Y188 residues of p66. Upon binding of an NNRTI, the three-stranded b-sheet that makes up the wall of the pocket is repositioned and the side chains of Y181 and Y188 that are part of this b-sheet move upward. As a consequence, the primer grip region is deformed and the binding pocket appears. Moreover, binding of an NNRTI completely disrupts the conformation of the catalytic triad. The resulting reduction of the flexibility of both the binding pocket region and the thumb domain in the p66 subunit leads to the inhibition of the polymerization activityofRT.TherearetwomaintheoriesabouttheworkingmechanismofNNRTIs[12]. The molecular arthritis theory postulates that binding of an NNRTI leads to a more rigid and hence inactive enzyme. In the second hypothesis, a distortion of the catalytic site upon NNRTI binding is deemed responsible for RT inhibition. Mutations associated with NNRTI resistance are clustered in the hydrophobic NNRTI-binding pocket. Key NNRTI mutations are found at position 100, 103, 106, 108, 181, 188, 190, 225, and 236 of HIV-1 RT [13]. Mutations K103N and Y181C were the first to be identified [14–16] and to date are amongst the most prevalent mutations observed in the clinic [17]. Single mutations may cause severe decrease in viral susceptibility to first-generation NNRTIs (nevirapine, delavirdine, and efavirenz). Moreover, some mutations such as K103N and Y181C dramatically limit the sequential use of first-generation NNRTIs, but newer generation NNRTIs such as etravirine can easily cope with single RT mutations [18, 19]. In 1994, the search for new NNRTIs was scaled back at Janssen Pharmaceutica because of some disappointments with the TIBO [20, 21] and the a-anilinophenylacetamide (a-APA) series of compounds [22, 23]. S HN

Cl

N N Cl TIBO

NH2

O

N H Cl

O

α-APA

Inthesameyear,thefirstmemberofthediaryltriazine(DATA)series[23]wasprepared and compared with a-APA, virological testing revealed high activity against wild-type virus and activity against different viral mutants resistant to other treatment regimens.

17.1 Introduction

In 1995, Dr. Paul Janssen founded the Center for Molecular Design. The HIV project was revitalized and the first DATA compound was selected as a lead compound for further chemical optimization. The available modeling capacity combined with crystal structures of the RT binding site complexed with different NNRTIs, including the first DATA compounds, enabled new research proposals. These proposals in turn led to new medicinal chemistry approaches at Johnson & Johnson in Spring House (PA) to further optimize the DATA series. After 2 years of disappointments, notwithstanding progress in antiviral activity, dapivirine (TMC120) was discovered. This was the first interesting diarylpyrimidine (DAPY) compound [25]. N

Cl

Cl N N

N

N

HN

H

N

N NH2

N

H

N TMC120-dapivirine DAPY

DATA

Dapivirine displayed activity in therapy-na€ıve patients. The compound is now under development as a vaginal microbicide, in collaboration between Tibotec Pharmaceuticals Ltd. and International Partnership for Microbicides (IPM), intended to decrease the rate of sexual transmission of HIV-1 [26–28]. Dapivirine is not substituted in the 5or 6-position of the pyrimidine ring. Therefore, it was decided to elaborate on these positions for further optimization. After some attempts, a bromine atom and an amino group were introduced to the 5- and 6-position, respectively. CN

CN

CN

O H

N

NH

N

NH

N N

Br

N NH2

dapivirine

TMC125-etravirine

The result was etravirine (INTELENCEÔ, TMC125), which received regulatory approval in 2008 [29–32]. Etravirine was developed because of its high potency against wild-type and NNRTI-resistant HIV-1. It is a relevant new option for treatmentexperienced patients with NNRTI resistance and improves patients’ quality of life [33a,33b]. Research was pursued to find a next generation of DAPY compounds combining the broad-spectrum activity of etravirine with an improved pharmacokinetic profile. In this new program, R152929 was considered as the new molecular starting point [34, 35]. The compound displayed broad-spectrum activity against HIV-1, not only against the wild type and different single mutants but also against double and

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412

multiple mutants of clinical relevance. Moreover, the compound was highly bioavailable both in rats and in dogs. The structure–activity relationship (SAR) of compounds with other substituents instead of the methyl (dapivirine) or cyano (R152929) in the para-position of the left phenyl wing was very encouraging. Even compounds with a t-butyl or a cyclohexyl were active, indicating that many substituent patterns were allowed, which was confirmed by molecular modeling. On the basis of the molecular modeling [36], priority was given to this para-substituent followed by modifications on the core scaffold. As an additional strategy, it was decided to capitalize on the combined scientific expertise acquired within the iodo phenoxy pyridone series (IOPY) [37–39] and DAPY series [40] to introduce the most interesting substitution combinations especially on the left wing of the DAPY scaffold. O H

I

N

X

28

H

N

NH

N N

CN

CN

CN

H

N

NH

N N

R3 R2

R1

CN

R1

R2 H

N

R3 N N

R4

dapivirine

R152929

NH

R5

The purpose of this chapter is to provide an overview of the Tibotec strategy developed to discover TMC278 (rilpivirine or R278474) [41]. We will discuss synthesis pathways of the next-generation DAPY compounds, the SAR, the physicochemical properties, and clinical trials performed with TMC278.

17.2 Chemistry 17.2.1 Synthesis of TMC278 and Close Analogues

TMC278 (rilpivirine or R278474) can be prepared according to different synthesis routes (see Schemes 17.1–17.8). These synthesis pathways have also been used to develop the SAR of the nextgeneration DAPYcompounds. Historically, DAPY [24] derivatives 8 were prepared by fusion between the 4-(4-chloropyrimidin-2-ylamino)-benzonitrile 6 and substituted aniline intermediates 7 as depicted in Scheme 17.1.

17.2 Chemistry

CN

CN

S

N

O

4

S

N

O a

N

N

H

N

H

H

H

b

N

N

O

H

N

3

2

j413

5 CN

L

L R1

R2

c H

7

Cl

N

N

H

N

R2

H

N

H

d

N

CN

R1 N

N

H

N 6

8

Scheme 17.1 Synthesis of DAPY derivatives. Reagents and conditions: (a) NaOH, MeI, 60
C, 3 h, Y ¼ 90%; (b) melting, 150
C, Y ¼ 70%; (c) POCl3, reflux, 1 h, Y ¼ 80%; (d) melting, 150
C, 1–2 h, Y ¼ 10–60%.

The chloropyrimidine intermediate 6 was synthesized in three steps from 2thiouracil 2. The methylsulfanyl intermediate 3 was prepared from methyl iodide and the commercially available thiouracil 2 in the presence of sodium hydroxide after heating at 60
C for 3 h. Treatment of 4-aminobenzonitrile 4 with the thiopyrimidine H N

O

6

H

6

+

H

CN

N

O

O

+ H

H

N

H

7a

CN

7f

O

H CN

+

6

6

N H 7e

N

N

N

N

H

H

N

I

+

7b

Br

TMC 278, 1a 6

6

+ H

N

H

7d

Scheme 17.2 Retrosynthetic plan to prepare TMC278 and DAPY close analogues.

+ H

N 7c

N

H

H

j 17 Rilpivirine, a Non-nucleoside Reverse Transcriptase Inhibitor to Treat HIV-1

414

H

N

a

O

N

+

b

O

N

H 7g

9

7c

O

H

Br

Br

N 7h

N

N

CN

CN

CN

N

N

CN

Cl

CN

N c

6

d N

H

N

e

H

H

N

N

N

H

N

N 7e

1a

7i

Scheme 17.3 First synthetic route for TMC278 and close analogues. Reagents and conditions: (a) 120
C, 48 h; (b) DMF, n-BuLi, THF, 70 to 0
C; (c) (EtO)2P(O)CH2CN, t-BuOK, THF, RT, 3 h; (d) ZnCl2, EtOH, 80
C, 20 h; (e) melting, 150
C, 1 h.

precursor 3 at 150
C for 2 h delivered compound 5, which in turn was treated with POCl3 at reflux for 1 h giving the key intermediate 6. This intermediate 6 was used to modify the left-wing side in order to synthesize a variety of different series of TMC278 analogues [35]. These DAPY derivatives of the CN

7e

CN

CN

CN

ZnCl2 EtOH

N Cl Zn Cl

N H

O

N O H Cl Zn N

Et

Et H

+

O Et

N

Cl

H

CN

CN

ZnCl2 OZnCl H H H

O

Et

H

10

Cl CN

N

N O

N

N

OZnCl

H OEt

Scheme 17.4 Mechanistic pathway to generate the intermediate 7i.

H

N

7i

H

17.2 Chemistry

R1

a R1

+

R2 H

N

H

Cl

N

N

O

O

CN

O

O

N

H

7a

6 O

O

H

H

R2 N

N

H

CN CN

d R1

N

N

CN

c

b R1

R2 N

11

CN

H

CN

N

N

H

j415

H

H

R1

R2 N

N

N

H

H

R2 N

N

12

H

N

N

N

N

13

1

Scheme 17.5 Second synthetic route for TMC278 and close analogues. Reagents and conditions: (a) melting, 150
C, 2 h; (b) LiAlH4, THF, 5
C, 12 h; (c) MnO2, CH2Cl2, RT, 20 h; (d) (EtO)2P(O) CH2CN, t-BuOK, THF, RT, 2 h.

next generation were synthesized according to the six main pathways that were based on classical reactions such as the Heck reaction, Wittig/Wadsworth–Emmons reaction conditions, and halogen/metal exchange reactions, as described in the retrosynthetic plan proposed in Scheme 17.2. The first synthesis pathway, which has been used for the preparation of TMC278 and its analogues, started from the 3,5-dimethyl-4-amino-benzaldehyde 7b. Protection of the aniline group by a formamidine function made it possible to functionalize position 4 of the aniline intermediate easily. This protection was very useful to introduce more complicated specific substituents such as the acrylonitrile moiety to generate cinnamic acid derivatives. The N,N dimethylformamidine dimethyl acetal 9 reacts with anilines such as 7c to give the desired dimethylformamidine derivative 7g. The halogen/metal

CN

CN

O

H

CN

O

H

b

a + H

N

H

7b

H

N

N

Cl H

N

N

N

N N

6

CN

H

H

N

13

Scheme 17.6 Third synthetic route for TMC278 and close analogues. Reagents and conditions: (a) melting, 150
C, 1 h; (b) (EtO)2P(O)CH2CN, t-BuOK, THF, RT, 2 h.

N

N N

1a

H

j 17 Rilpivirine, a Non-nucleoside Reverse Transcriptase Inhibitor to Treat HIV-1

416

CN

CONH2

CN a

H

N

Br

N

H

c

b

N

H

H

7f

H

Cl

N

N

CN

N

7i

CN

6

CN

CN

7c

H

H

N

H

N

N

H

N d

N

H

1a

f

e

H

H

7i

N

H .HCl

7j

150
C, 1 h; (d) acrylonitrile, Pd(OAc)2, P(o-Tol)3, NEt3, CH3CN, 150
C, 12 h; (e) HCl/ EtOH, Et2O, 60
C, 30 min; (f) CH3CN, 80
C.

Scheme 17.7 Fourth synthetic route for TMC278. Reagents and conditions: (a) acrylamide, Pd(OAc)2, P(o-Tol)3, NEt3, CH3CN, 70
C, 15 h; (b) POCl3, 25
C, 12 h; (c) melting, CN

CN

I a

H

N

b

H

H

N

c

H

H

H

H

N

E/Z= 80/20 CN

CN CN

7m

+

N

N

Cl

e H

N

N

N

6

CN

CN

d H

.HCl

H

7m

7i

7l

7k

N

N N

1a

H

H

N

N

N N

H

.HCl

1a salt

Scheme 17.8 Synthesis of TMC278. Reagents and conditions (a) BTMAICl2, MeOH, CH2Cl2, CaCO3; (b) acrylonitrile (1.5 eq.), Pd/C 0.5%, NaOAc (1.2 eq.), DMA, 140
C, Y ¼ 80%; (c) EtOH, HCl 6 N in iPrOH, 60
C, Y ¼ 70%; (d) NMP, HBr/AcOH, reflux, Y ¼ 80%; (e) HClaq, AcOH.

17.2 Chemistry

exchange was followed by treatment with dimethyl formamide, which led to the desired key aldehyde 7h and was used to obtain the acrylonitrile derivative 7e by Wadsworth–Emmons reaction in a good yield. The deprotection to this type of amidine was described by Toste et al. [42] using zinc chloride in the presence of ethanol as solvent. According to the reaction mechanism, the N-formyl derivative 10 is the main intermediate, which is further deprotected to the corresponding aniline derivative 7i in a good yield by refluxing in alcoholic zinc chloride solution (see Scheme 17.4). Another route (Scheme 17.5) has been developed using the ethyl 4-amino-3,5dimethylbenzoate 7a as starting material and via this route, the cyanovinyl group was introduced in the final step leading to the target compound TMC278. This method could also be applied to modify the substituent patterns in the 2-position and 6position by using other amino-benzoic acids. The ester 11 was prepared by mixing the aniline ester 7a and chlorpyrimidine derivative 6, followed by heating the mixture at 150
C for 2 h. In the next step, the carboxylic ester 11 was reduced to alcohol 12 with LiAlH4 without affecting the cyano function present on the scaffold. After the reduction, a necessary oxidation by MnO2 of alcohol 12 furnished, in a high yield, the corresponding aldehyde 13. The intermediate 13 was needed to introduce the acrylonitrile moiety by a Wittig reaction with diethyl cyanomethyl phosphonate in the presence of potassium tert-butoxide (t-BuOK), with tetrahydrofuran (THF) as solvent. This second synthesis approach gave TMC278 1a with an E-stereoselectivity and was also applied to modify substitutions on the trisubstituted phenyl ring. The next alternative synthesis route (Scheme 17.6) was a variant of the second approach. Indeed, it was possible to directly prepare the aldehyde 13 by heating the chloropyrimidine 6 with the 4-amino-3,5-dimethylbenzaldehyde 7b. In an attempt to simplify the reaction conditions, exploration experiments were started by using Heck reaction conditions aiming at a direct introduction of the acrylonitrile group into the aromatic system. It was necessary to use palladium(II) diacetate as a catalyst in the presence of triethylamine in acetonitrile as solvent. Finally, compound 1a was obtained with a ratio E/Z of 96/4. In order to find a preparation procedure in a large scale, the Heck reaction [43–45] has been extensively studied. Several approaches have been considered, attempted, and further optimized as described in Schemes 17.7 and 17.8. Finally, the synthetic process for the production of TMC278 involved the chloropyrimidine 6 and the 3-(4-amino-3,5-dimethylphenyl)acrylonitrile 7i as key building blocks [46]. A robust and high-yield Heck reaction procedure was developed to prepare the intermediate 7l. With the iodoaniline derivative 7l instead of the bromoaniline 7c, the Heck coupling was performed without a costly ligand and the loading of Pd/C was dramatically reduced (0.5 mol%), which made the purification and isolation of the target compound less complicated and the procedure greener. In the final optimized process, TMC278 1a was synthesized in five high-yield reaction steps. The pure E-isomer was isolated as a slightly yellow crystalline powder.

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j 17 Rilpivirine, a Non-nucleoside Reverse Transcriptase Inhibitor to Treat HIV-1

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Then, TMC278 hydrochloride salt 1a was prepared from the free base in a mixture of aqueous hydrogen chloride with aqueous acetic acid as a solvent. 17.2.2 Modulation of the Central Heterocycle Core

Chemical exploration made it relatively simple to synthesize pyrrolidino and pyrrolopyrimidine derivatives 18 and 19 in five steps by a convergent synthesis route as outlined in Scheme 17.9. The starting material 2-acetylbutyrolactone reacted with guanidine 15 at 100
C in a diglyme/water mixture to form the dihydroxy pyrimidine derivative 16, which was chlorinated to 17 with POCl3 at 60
C for 2 h. Conversion of the dichloro pyrimidine derivative 17 in the melt with 4-acrylonitrile-2,6-dimethyl-aniline 7i led to the pyrrolidinopyrimidine derivative 18. Oxidation of compound 18 with 2,3dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) provided the desired pyrrolopyrimidine compound 19. The central pyrimidine heterocyclic ring within TMC278 was also replaced by other bicyclic heterocycles such as purines and triazolopyrimidine. Both new classes of compounds were obtained in four steps from 2,4-dichloro-5nitropyrimidine 20, commercially available as a starting material (Scheme 17.10). After the introduction first of the 4-acrylonitrile-2,6-dimethyl-aniline 7i and then of the p-cyano aniline, 5-nitropyrimidine 22 was prepared that was reduced by using stannous chloride to afford the desired 5-aminopyrimidine 23a compound. This key intermediate 23a treated with functionalized aldehydes or orthoformates gave access OH

NC

NC

NC

a

,HCl

,HCl NH

NH2

15

14

HN

OH

N

b N H

NH2

N

16 CN

CN

CN CN

Cl c

Cl

CN

N N

NH2

d

Scheme 17.9 Pyrrolidino and pyrrolopyrimidines series. Reagents and conditions: (a) H2N-CN, H2O, diglyme, 100
C, 72 h, Y ¼ 55%; (b) 2-acetylbutyrolactone, Et3N,

e

N

18

NH

N

N

N H

17

NH

N

N

CN

N

19

EtOH, reflux, 12 h, Y ¼ 88%; (c) POCl3, 60
C, 12 h, Y ¼ 30%; (d) 4-(2-cyanoethenyl)-2,6dimethylphenylamine, 180
C, 20 h, Y ¼ 13%; (e) DDQ, 1,4-dioxane, reflux, Y ¼ 7%.

17.2 Chemistry

j419

CN

CN

CN

O2N

HN

Cl

N

Cl

N

20

b

N

O2N

NH

N

HN

Cl

N

a

N

O2N

c

21

22 CN

CN

CN

CN

24b R=H 24c R=Et 24d R=Ph 24e R=2-Furan 24f R=2-Pyridine

N R N

d

NH

N

CN

CN

HN

N H2N

NH

N

e

N H2N

N

N

23a

24a

CN

CN

CN

CN f

N

N

N H

25 Scheme 17.10 Purine and triazolopyrimidine series. Reagents and conditions: (a) 7i, 140
C, neat, 45 min, Y ¼ 35%; (b) 14, 300
C, 5 min, Y ¼ 95%; (c) SnCl2, H2O, EtOH, 70
C, 12 h, Y ¼ 87%; (d) RC(OEt)3 or R-CHO,

NH

N

S

g

N

NH

N

N

N

N

26

nitrobenzene, 100 or 180
C, 20 h, Y ¼ 23–76%; (e) BrCN, EtOH, THF, RT, Y ¼ 48%; (f) thiophosgene, 1,4-dioxane, RT, 12 h, Y ¼ 47%; (g) NaNO2, AcOH, H2O, RT, 5 h, Y ¼ 53%.

to substituted purine 24 compounds. The treatment of the intermediate 23a with sodium nitrite in water and acetic acid as solvent led to the triazolopyrimidine 26 derivative with a good yield. 17.2.3 C-5 Substitution of the Pyrimidine Core

To gain more insight into the SAR, it was necessary to introduce a variety of substituents into the 5-position of the central pyrimidine ring. Starting from TMC278 1a, a SAR study was launched by introducing various substituents at position 5 of the pyrimidine ring. The main objective of this work was to assess the influence of a 5-substitution on activity, with the TMC278 level of activity as a reference (Scheme 17.11).

NH

N N

j 17 Rilpivirine, a Non-nucleoside Reverse Transcriptase Inhibitor to Treat HIV-1

420 CN

CN

CN

CN

c)

NH

N

HN

N

I

HN

N

23f

b)

Br

HN HO2C

23b

1a

CN

f)

NH

N N

NH

N N

23c

d)

CN

HN R'RNO2C

NH

N N

23d-e

CN CN

HN

CN

e)

NH

N

CN

CN

a)

HN

CN

CN

CN

NH

N

HN

N

N

Ar,Het

23g

NH

N

23h-i CN CN

CN

CN CN

CN

g)

h) HN R

NH

N

N R'

23j-m

N

HN H2N

HN

NH

N

N

N

N

23n

CN

23a

NH

N

CN i)

O R"

HN N H

NH

N N

23o-p

Scheme 17.11 Chemical investigations around position 5 of the pyrimidine ring. Reagents and conditions: (a) ICl (in CH2Cl2), CaCO3, EtOH, H2O, RT, 24 h, Y ¼ 78%; (b) Me4Sn, PdCl2(PPh3)2, DMF, 70
C in autoclave, 24 h, Y ¼ 26%; (c) NBS, CH3CN, RT, 4 h, Y ¼ 86%; (d) Ar ¼ Ph: PhB(OH)2, Pd(PPh3)4, K2CO3 2 N, DME, MeOH, 100
C, 6 h, Y ¼ 40%, Het ¼ 3-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl) pyridine, Pd(PPh3)4, K2CO3 2 N, DME, MeOH, 100
C, 20 h, Y ¼ 65%; (e) (i) PdCl2(PPh3)2, NEt3, EtOH, P(CO) 15 bar, 100
C, 72 h, Y ¼ 73%, (ii) LiOHH2O, THF, H2O, 50
C,

overnight, Y ¼ 95%; (f) R ¼ R0 ¼ H: (i) SOCl2, reflux, 1 h, (ii) NH4OH 30%, 0
C, 1 h, Y ¼ 47% (2 steps), R ¼ R0 ¼ Me: HOBt, EDCI, dimethylamine, RT, 24 h, Y ¼ 28%; (g) 2,5dimethoxytetrahydrofuran, AcOH, 90
C, 50 min, 64% yield; (h) R ¼ H, R0 ¼ Me: HCHO, NaBH3CN, AcOH cat., CH3CN, RT, 2 h, 27% yield, R ¼ Ph, 2-pyridyl or 2-furanyl, R0 ¼ H: PhCHO, 2-pyridyl-CHO or 2-furanyl-CHO, NaBH3CN, AcOH cat., CH3CN, RT, 40 h, 25–33% yield; (i) R00 ¼ H: HCO2H, HCO2Et, reflux, 4 h, 55% yield, R00 ¼ Me: CH3COCl, NEt3, CH2Cl2:THF (1 : 1), 55% yield.

17.3 Structure–Activity Relationships

R152929 (compound 27a) was used and considered as a reference compound [34] before the advent of TMC278 (1a).

17.3 Structure–Activity Relationships

As a general remark, R152929 (27a) is a very potent broad-spectrum HIV-1 inhibitor with subnanomolar activity against wild-type (IIIB) and nanomolar activity against single mutants such as K103N, Y181C, and Y188L (EC50 values below 10 nM). Against the single-mutant L100I and the double-mutant K103N þ Y181C, the compound is clearly less active (EC50 values of 31 nM). Against the double-mutant L100I þ K103N, the compound displays the lowest activity (EC50 ¼ 100 nM) (Table 17.1). On the basis of SAR studies with some other left-wing 4-substituted phenyl derivatives and modeling studies, it was concluded that it was optimal to have a cyano group in the 4-position. Indeed, other attempted 4-substituted analogues did not generate more potent compounds. 17.3.1 Introduction of G Spacer Between the Aryl Ring and the Cyano Group

The first aim was to introduce a spacer between the cyano group and the substituted phenyl ring of the left wing of the DAPY scaffold [34] (see Table 17.1). Table 17.1 Modulation of the G spacer between the phenyl ring and the cyano group.

H N NC

H N

N N

G

CN

27 Compound

G-CN

27a -CN 1a -CH¼CH-CN 27b -CH¼CH-CN 27c -CH¼C(Me)-CN 27d -CH¼C(Me)-CN 27e -CH¼C(CH2-CN)-CN 27f -C(Me)¼CH-CN 27g -CH¼CH-CH2-CN 27h -C(Cl)¼CH-CN 27i -CH2-CH2-CN 27j -CH2-CH(Me)-CN 27k -CH(Me)-CH2-CN 27l -2-furyl-5-CN 27m -CH2-CN 27n -CH2-O-(CH2)2-CN Efavirenz

E/Z

— 100/0 0/100 100/0 0/100 12/88 100/0 100/0 70/30 — — — — — — —

EC50c) (nM) IIIB

SIa)

0.4 0.5 0.6 0.8 0.8 0.4 1 1 4 0.6 0.6 4 0.8 0.6 1 1.6

12 500 60 000 475 25 000 3750 5012 30 000 10 000 6310 3981 1995 3162 12 589 19 953 10 000 32 315

L K Y Y L100I þ K103N þ 100I 103N 181C 188L K103N Y181C 31 0.4 6.3 0.6 3.1 31 0.8 40 2.5 0.8 1 32 40 25 40 31.0

2 0.3 1.6 0.6 1 6 1 2.5 2 0.4 0.6 8 8 3 40 46.1

6.3 7.9 1.3 2 5 31 1.6 3.1 4 31 6 31 2.5 4 31 20 6.3 8 1.3 20 2 50 40 32 25 250 32 40 2 160 3.9 55.0

a) SI ¼ CC50/EC50 (Selectivity index). b) nd ¼ not determined. c) EC50 ¼ 50% effective concentration for inhibition of viral cytopathicity.

100 8 8 31 >10 ndb) 25 250 31 32 130 500 nd nd >10 1459

31 1 4 2.5 20 nd 2.5 1 6 2.5 5 nd nd nd 40 55.1

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Elongation with one carbon generated compound 27m that displayed a lower activity, not only against wild type but also against the single mutants Y181C and Y188L, whereas the activity against the single-mutant L100I was higher. Further elongation of the carbon chain led to 27i that remained surprisingly active: the activity against wild-type (IIIB) remained at subnanomolar level. For the first time, a slightly superior activity against the single K103N mutant compared to wild type was observed, whereas the activity against the single-mutant L100I improved to subnanomolar level. Furthermore, there was a significant increase in activity against both double mutants L100I þ K103N and K103N þ Y181C. There was a decrease in activity against the single-mutant Y188L. In the 4-arylthio and 4-aryloxy-3-iodopyridin-2(1H)-one 28 classes (IOPY), a SAR study led to the identification of very promising compounds. O

O H

H

I

N

N

O I

H

X

N

I O

R3 R2

CN

R1

28a

28

CN

28b IC50 (nM)

IIIB K103N Y181C Y188L

0.4 1.6 20 126

0.8 0.6 1.2 5.0

From the optimization of these IOPY molecules, cyanovinyl substitution was shown to generate highly potent RT inhibitors against HIV-1. Unfortunately, the poor solubility and bioavailability of the best derivatives led the team to stop the IOPY program. Introduction of this cyanovinyl group instead of the cyano moiety in the DAPYclass generated the compound 1a with subnanomolar activity against wild-type (IIIB) and the single mutants such as L100I and K103N. Against single mutants such as Y181C and Y188L and the double mutants L100I þ K103N and K103N þ Y181C, nanomolar activities were observed. The geometry of the molecule was important because the highest activity resided in the E-configuration. On the other hand, compound 27b with the Z-configuration still had a convincing activity against wild type and the mutants tested in the panel. Introduction of a methyl group in the a- or b-position of the cyanovinyl moiety delivered highly potent compounds (27c, d, and f) although their activity was lower than that of 1a. Introduction of a chlorine atom in the b-position of the cyanovinyl group gave compound 27h (E/Z 70/30), which was less active than 27f. Saturation of the double bond in 27c and 27f to 27j and 27k, respectively, did not have a beneficial effect on activity. Substitution of the alpha hydrogen of the cyanovinyl group with a cyanomethyl moiety to compound 27c (E/Z 12/88) generated high activity against wild-type HIV-1(IIIB), but the activity against the

17.3 Structure–Activity Relationships

different single mutants was not impressive. Compounds such as TMC278 (1a) were clearly superior in activity compared to efavirenz, not only with regard to wild-type HIV-1 (IIIB) but also against the tested single and double mutants within the panel. Within the series, TMC278 (1a) is the most potent compound and was selected as a candidate for clinical development. It is a compound with high potency, which can be further associated with a high bioavailability in rats and dogs. 17.3.2 Modulation of Substituents at C-2 and C-6 on the Left Wing and of the Linker Between Left Wing and Pyrimidine Core

In the following approach, the effect of different substituents on activity in the 2- and 6-position on the left wing was evaluated [35] (see Table 17.2). Omitting the methyl groups in the 2- and 6-positions (8c, R1 ¼ R2 ¼ H, E/Z 80/20) strongly decreased activity against wild-type HIV-1. The effect was even more pronounced against the single mutants and the double mutants. Introduction of

Table 17.2 Substitution at C2 and C6-positions on the left wing.

CN CN

R2 N

R1 X

NH N

Compound

X

R1

R2

E/Z

EC50 (nM) IIIB

8a 8b 8c 8d 8e 8f 8g 8h 8i a)

NH NH NH NMe NH NH NH NH NH

Me H H H Me Me Me Me Cl

Me Me H Me Et iPr OMe Cl OMe

SI ¼ CC50/EC50.

100/0 100/0 80/20 80/20 85/15 96/04 91/09 100/0 100/0

0.5 0.9 33.9 1.0 3.0 4.0 0.7 0.8 0.1

SIa)

L100I K103N Y181C Y188L L100I þ K103N þ K103N Y181C

60 000 0.4 0.3 1.3 2 >29 372 9.2 0.6 13.4 43.6 >737 585.1 57.7 1523 303.3 11 351 2.5 0.7 12.0 16.8 841 0.9 0.5 4.0 2.6 2947 3.1 1.3 11.1 3.8 846 0.5 <0.1 1.9 0.9 3316 0.4 0.1 2.3 2.3 3981 0.2 0.3 1.0 2.5

8 478.7 6440 103.8 8.2 38.3 21.1 6.6 15.8

1 89.6 3952 160.3 5.3 10.6 4.3 4.1 2.0

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a methyl group (8b, R1 ¼ H, R2 ¼ Me) in the 2-position of 8c of the left-wing phenyl increased activity against wild type, the single and double mutants of HIV-1 within the whole panel. Substitution of one methyl group with an ethyl group (8e, R2 ¼ Et) had a decreasing effect on activity against wild type, and the single and the double mutants of HIV-1. A further decrease in activity was observed when the ethyl moiety in 8e was replaced by a more bulky i-propyl group (8f, R2 ¼ i-Pr). Substitution of the ethyl group in 8e with a methoxy moiety (8g, R2 ¼ OMe) improved activity against all HIV-1 strains with the exception of the double-mutant L100I þ K103N; the activity against the mutant strains L100I and K103N was even higher than against the wild-type IIIB HIV-1 strain. Replacing the methyl moiety in 8g with a chlorine atom delivered compound 8i (R1 ¼ Cl, R2 ¼ OMe), which displayed the highest activity in the series against wild-type HIV-1. Furthermore, compound 8i demonstrated subnanomolar activity against the single L100I and K103N mutants of HIV-1 and nanomolar activity against the other single and double mutants of HIV-1. Although replacement of the NH-linker in 8b with an N-methyl linker (8d, X ¼ NMe, E/Z 80/20) gave a similar activity against wild-type HIV-1 and the single mutants K103N and Y181C, it increased activity against the single mutants L100I and Y188L and double-mutant L100I þ K103N. Decreased activity was observed against the double-mutant K103N þ Y181C of HIV-1. 17.3.3 Subsitution at C-5 Position of the Pyrimidine Heterocycle

Next, different substituents were assessed in the 5-position of the pyrimidine ring within the TMC278 scaffold (see Table 17.3). Compound 23c (R ¼ C(¼O)OH) demonstrated the weakest activity against HIV-1. This was in contrast with the potent activity of compound 23d (R ¼ C(¼O)NH2) that showed nanomolar activity against wild-type HIV-1, the single mutants K103N, Y181C, and Y188L and the double mutants L100I þ K103N and K103N þ Y181C, whereas subnanomolar activity was seen against the single mutant L100I. Double methylation of the carboxamide moiety in compound 23e (R ¼ C(¼O)NMe2) decreased activity not only against wild-type HIV-1 but also against the single and double mutants within the panel. Introduction of relatively large substituents, such as phenyl (for 23h), 3-pyridyl (23i), 1-pyrrolyl (23n), and benzyl-amino (23k), and the corresponding 2-pyridyl- (23l) and 2-furanyl-analogues (23m) was tolerated, generating compounds with high potency not only against wild-type HIV-1 but also against the single and double mutants within the panel. Introduction of a small methyl substituent 23f (R ¼ Me) had a marginal effect on activity compared to TMC278 1a. Replacement of the apolar methyl group with a polar nitro group in compound 22 (R ¼ NO2) in particular had a strong decreasing effect on activity against the single mutants L100I, Y181C, and Y188L and the double mutants L100I þ 103N and K103N þ Y181C, respectively. Reduction of the nitro group to an amino moiety in 23a (R ¼ NH2) had a favorable effect on activity, giving

17.3 Structure–Activity Relationships Table 17.3 Substitution at C5-position on the central heterocyclic core.

CN CN

HN

NH

N N

R

Compound R

EC50 (nM) IIIB

1a 23f 22 23a 23j 23o 23p 23c 23d 23e

H Me NO2 NH2 NMe2 NHC(¼O)H NHC(¼O)Me C(¼O)OH C(¼O)NH2 C(¼O)NMe2

L100I K103N Y181C

Y188L

L100I þ K103N þ K103N Y181C

0.5 0.4 0.3 1.3 2 8 0.5 0.4 0.4 1.25 2.0 1.6 0.6 2.0 0.8 10 19.95 15.85 0.5 0.8 0.6 3.2 4.0 4.0 0.8 0.5 0.6 5.0 2.5 0.1 0.4 0.6 0.6 2.5 3.2 6.3 0.6 6.3 1.25 5.0 12.6 63.1 251.2 398 398 1995 1995 1995 2.0 0.5 1.25 3.2 3.2 1.6 4.0 31.6 8.0 25.1 50.1 199.5

1 0.6 39.8 3.2 2.5 4.0 25.1 3162 1.6 79.4

3.2

4.0

3.2

25.1

39.8

50.1

7.0

0.6

2.0

0.6

5.0

4.0

12.6

31.6

0.1

0.1

0.8

6.3

4.0

8.0

N H

0.8

1.6

1.6

5.0

12.6

N H

0.6

0.6

0.1

2.5

2.0

2.5

1.6

N H

0.5

0.5

0.5

2.0

1.6

1.6

0.1

23h

23i N

N

23n

23k N

23l

23m

O

10

10

6.3

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subnanomolar activity against wild-type HIV-1 and against the single mutants L100I and K103N, whereas nanomolar activity was observed for the other single and double mutants within the panel. Conversion of the amino group to the dimethyl amino moiety in 23j (R ¼ NMe2) in general had a marginal effect on activity with an exception against the double-mutant K103N þ Y181C that could be associated with a 40-fold increase in activity. Acylation of 23a (R ¼ NH2) with a formyl moiety (23o: R ¼ NHC(¼O)H) or an acetyl group in (23p: R ¼ NHC(¼O)Me) had a marginal effect on wild-type and singlemutant strain activities, although a lower activity against the double-mutant strains was observed. 17.3.4 Modification of the Central Heterocycle Core

By introducing a bridge between the amino-linker in the 4-position and the 5-position of the pyrimidine ring, a new generation of bicyclic pyrimidine derivatives was produced (see Table 17.4). All bicyclic pyrimidines derived from TMC278 (1a) exhibited activity against wildtype HIV-1 in subnanomolar or nanomolar ranges and their overall antiviral profiles were better than the profile of FDA-approved reference NNRTIs. The influence of 4,5-cyclization on the activity against wild-type HIV-1 and mutant strains can be evaluated considering the pyrrolidinopyrimidine and pyrrolopyrimidine derivatives 18 and 19. Annulation at positions 4 and 5 of the central pyrimidine core resulted in a good overall profile of 18 and 19, with the compounds showing single-digit nanomolar activity on wild-type HIV-1 and on most single and double mutants. Nevertheless, compounds 18 and 19 demonstrated a slightly lower activity profile than the nonannulated compound 1a (TMC278). Similar to TMC278, these compounds showed no loss of activity on mutants bearing the L100I or K103N mutation compared to the wild type. Moreover, compound 19 showed no decrease in activity against the double mutants L100I þ K103N, K101E þ K103N, and V106 þ F227L. To better understand the ability of these compounds to maintain their wild-type potency on L100I and K103N mutants, a model of the NNRTI binding pocket was constructed and used to dock compounds 1a, 18, and 19. In this model, the additional methyl substituent at the central pyrimidine core in derivatives 18 and 19 resides close to amino acids L100, K101, K103, and V179 in the p66 subunit and E138 in the p51 subunit of the enzyme. The introduction of an annulated ring on the central pyrimidine core and the subsequent loss of one rotatable bond results in reduced flexibility of the compounds in the area where amino acids V179, Y181, Y188, and E138 of the NNRTI pocket reside. Therefore, these compounds are more vulnerable to mutations Y181C and Y188L than TMC278. The imidazopyrimidine compound 24b, which has no methyl substituent on the pyrimidine core, was also evaluated. This compound showed similar anti-HIV activity against TMC278 with subnanomolar activity on wild type and single-digit nanomolar activity on mutants bearing Y181C or a combination of K103N and K101I.

17.3 Structure–Activity Relationships Table 17.4 Bicyclic heterocycles and their inhibition against the wild-type, single-, and double-

mutant strains.

CN

CN

CN CN

N

N

N N

N

N

R

24a-f EC50 (nM)

IIIB

1a / / 18b) 19 / 24a NH2 24b H 24c Ph 24d 2-Furyl 24e SH 24f / Efavirenz

N

N

18-19

N

N

R

N

26 Compounda)

N

N

N

N

CN

CN

SI

L100I K103N Y181C Y188L L100I þ K101E þ K103N þ K103N K103N Y181C

0.6 5799 0.4 1.3 7833 1.6 2.9 7665 2.5 2.5 >25 000 8.0 0.2 14 469 0.6 9.1 3850 50.2 3.4 10 133 14.8 1.1 >25 000 2.6 0.8 >25 000 5.3 1.6 32 315 31.0

0.5 1.1 2.9 13.2 0.6 33.8 4.6 2.8 1.6 46.1

2.0 7.2 13.6 20.4 2.8 78.5 18.7 12.2 17.8 3.9

1.6 7.9 19.9 71.7 16.0 30.3 45.4 84.3 22.0 55.0

4.6 4.3 3.3 85.5 8.1 625.6 172.2 422.9 81.4 1459

1.2 6.2 3.8 17.6 1.4 155.7 14.4 8.3 4.4 122.7

2.1 11.6 17.7 26.9 44.2 411.9 55.4 40.1 ndc) 55.1

a) Displaying E-stereochemistry. b) E/Z ¼ 83/17. c) nd ¼ not determined.

This compound is, however, less active against those mutants than TMC278. A series of substituted imidazopyrimidines was designed to define the role of modulations in this region. Introduction of substituents on the five-membered ring (24a–e) resulted in an overall lower activity against wild-type and mutant viruses compared to 24b. The furyl (24d) and amino derivatives (24a) presented a better overall antiviral profile than the phenyl (24c) derivative. Molecular modeling studies showed that for substituted imidazopyrimidines, the central pyrimidine core is significantly displaced to accommodate the extra aliphatic or aromatic side chains compared to their counterpart 24b. Hence, the left aryl ring is considerably displaced in the pocket, resulting in a weaker interaction of those compounds with amino acid Y188. For the furyl derivative, the left phenyl ring and the acrylonitrile side chain overlap very well with TMC278, the nonannulated structure. This is illustrated in Figure 17.1, which shows an overlap of

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Figure 17.1 Superposition of 1a (gray) with 24c (orange) and 24d (green). The Y181 and Y188 side chains of the TMC278 model are shown. Hydrogens have been removed for clarity.

TMC278, 24c and 24d as prototypes. For clarity only, the Y181 and Y188 side chains of the complex with TMC278 are shown, but the position of these side chains in the two other complexes is the same. Although the figure does not show the full NNRTI binding pocket, it is clear that the interactions with the enzyme will be different, not only with Y181 and Y188 but also in the right-hand part of the pocket, where the cyano-phenyl group is displaced. The nonsaturated sulfur-substituted bicyclic compound 24e had nanomolar activities against wild-type HIV-1 and the mutants L100I, K103N, and K101E þ K103N, which is a better antiviral profile than all imidazo-substituted pyrimidines. For viruses harboring Y181C, Y188L, K103N, and K101E/Y181C mutations, the profile of this compound is similar to the furyl derivative. Finally, the triazolopyrimidine 24f was evaluated. The compound showed a subnanomolar activity against wild-type HIV-1 and the mutant harboring mutation similar to TMC278 (1a) and to wild-type data for 24b. For the remaining mutants, the triazole derivative 24f displayed lower activity. Molecular modeling showed that after mutation, the isoleucine side chain at position 100 includes a methyl group pointing toward compound 24f. As a consequence, the compound is pushed downward in the NNRTI pocket, as illustrated in Figure 17.2. This results in a weaker hydrogen-bond interaction with the backbone of K101 due to a longer distance between the H of the inhibitor’s NH group and the

carbonyl O of the K101 backbone (3.28 A for the L100I mutant compared to 2.20 A with wild type). For the double-mutant L100I þ K103N, a reduction in antiviral activity was seen for 24f as for most compounds. This could be explained as a combined effect of the L100I and K103N mutations that change the electronic nature of the bottom of the pocket from hydrophobic to polar. Indeed, in the wild-type model, the lysine side chain at position 103 makes primarily hydrophobic interactions because the end of the side chain is pointing away from the pocket. However, the mutant asparagine side chain is significantly shorter and the polar amide group contacts the binding pocket.

17.4 TMC278: Physicochemical Properties

Figure 17.2 Superposition of 24f in wild-type (green) and in L100I (gray) model. For clarity, only amino acid 100 and the backbone of K101 are shown. The distance of the hydrogen bond between 24f and the backbone C¼O is also indicated.

17.4 TMC278: Physicochemical Properties

TMC278 (1a) is a small molecule without stereogenic center and possesses a double bond with an E-configuration. In general, the physicochemical properties of TMC278 are comparable to those obtained for the closely related DAPY R152929. The compound is highly lipophilic and ionizable in aqueous solution. Its solubility in water is low and depends on pH. On the other hand, TMC278 is readily soluble in dimethylsulfoxide and moderately soluble in PEG-400. Studies performed on the chemical stability of TMC278 displayed no degradation and proved that it is stable under all investigated storage conditions. Chemical name: 4-[4-[4-[(E)-2-Cyanovinyl]-2,6-dimethylphenylamino]pyrimidin2-ylamino]benzonitrile. Molecular formula: C22H18N6

CN

Molecular weight:

366.4

Melting point:

250 °C

Appearance:

crystalline product nonhygroscopic

CN

Stereochemistry:

no stereogenic center (double bond) E-form

H

N

N

N N

H

pKa :

5.6

clog P:

(pH = 8) 4.8

Solubility: water ( pH = 7)

=> 20ng/ml

20% HPβCD water => 8ng/ml PEG-400 Stability (in dry state):

=> 40mg/ml stable

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17.5 Modeling of TMC278 and Crystal Structure

The HIV-1 RT computer model of the Center for Molecular Design was based on crystal structures [47, 48] of the first-generation DATA and DAPY compounds. Modeling of TMC278 [32] pointed out that the cyano group in the 4-position of the right-wing phenyl group gave a strong dipole–dipole interaction with the carbonyl group of H235. Moreover, the amino linker between the central pyrimidine ring and the right-wing phenyl is responsible for the formation of a strong hydrogen bond with the carbonyl backbone of amino acid K101 of RT. On the other hand, the backbone NH-group of amino acid K101 generates a second hydrogen bond with 1N of the central pyrimidine ring. The lipophylic left wing produces hydrophobic interactions with the tyrosine amino acids 181 and 188 of RT. Furthermore, the cyanovinyl moiety is located in a new subpocket generated by the amino acids W229 and F227. Presumably, the high activity of TMC278 is due to the high flexibility and adaptability of the molecule to bind to the NNRTI binding pocket of mutated viruses (Figure 17.3). The crystal structure has recently been published [49]. In general, the data generated within the model agreed with that of the crystal structure. However, there were some new important data generated by the crystal structure model. The side chain of amino acid K101 forms an important bridge with the side chain of amino acid E138 within RT, when TMC278 is present on its binding site, which makes it very difficult for TMC278 to escape from the enzyme. The presence of the bridge makes it look like a closed door. When the bridge (long chain of K101 can be considered as a door to open and to close) is open, the door is open and the compound can escape from the enzyme. Furthermore, TMC278 forms a hydrogen bridge via a water

Figure 17.3 Structure of HIV-1 in complex with TMC278 [49]. (a) Overall structure of the wildtype HIV-1 RT/TMC278 complex determined at
1.8 A resolution. (b) The position and

conformation of TMC278 were defined by the
difference in electron density calculated at 1.8 A resolution (3.5 contours).

17.6 Pharmacokinetic and Phase II Studies of TMC278

molecule and the NH-linker on the 4-position of the pyrimidine ring with amino acid E138. Overall, TMC278 undergoes many strong interactions with the enzyme.

17.6 Pharmacokinetic and Phase II Studies of TMC278

The pharmacokinetics of rilpivirine in healthy volunteers indicated its suitability for a once-daily dosing regimen. In phase I trials, the compound had a terminal elimination half-life (t1/2) of 45 h on average, was well absorbed after oral administration as a tablet formulation, and the time to maximum plasma concentration (Cmax) was consistently around 4 h across studies and doses (12.5–300 mg). Rilpivirine is eliminated via the feces, with a very minor elimination route via renal clearance [50]. The single- and multiple-dose pharmacokinetics of rilpivirine when administered as an oral tablet were dose-proportional over the range 25–150 mg. Steady-state conditions were reached within 11 days of commencing once-daily oral administration [51]. Intake of rilpivirine without food reduced the exposure by approximately 40–50%; therefore, the compound has to be administered with food [52]. In HIV-1-infected patients, the exposure to rilpivirine increased less than proportional to the dose, and the steady-state exposure was a little lower compared to healthy volunteers. The average exposure (area under the plasma concentration–time curve from time of administration to 24 h after dosing, AUC24h) to rilpivirine at 25, 75, and 150 mg once daily (for 96 weeks) in HIV-1-infected patients was 2767, 5906, and 10 281 ng h/ml, respectively. Rilpivirine has a mild CYP3A-inducing effect at higher doses, which appears to be dose-related [53]. A weak induction of CYP2C19 activity was observed after repeated administration of 150 mg rilpivirine once daily [54]. However, rilpivirine did not induce clinically relevant changes in the pharmacokinetics of other coadministered drugs. As to the effects of other drugs on rilpivirine, the compound is a CYP3A substrate, and its pharmacokinetics can therefore be affected by inhibitors and inducers of this enzyme. The CYP3A inducers rifabutin [55] and rifampin [56] reduced the steady-state exposure (AUC24h) to rilpivirine, while the CYP3A inhibitors ketoconazole [56], ritonavir-boosted lopinavir [57], and ritonavir-boosted darunavir [58] all enhanced its steady-state exposure. Given the low solubility of rilpivirine, especially at higher pH, the proton-pump inhibitor omeprazole [54] or the H2antagonist famotidine [59], drugs that increase the gastric pH, influence the solubility and hence the absorption of rilpivirine 150 mg once daily. Based on the metabolic profiles and elimination routes, an interaction of rilpivirine with drugs of the NRTI class is unlikely. Indeed, the modest increase in tenofovir disoproxil fumarate (TDF) exposure (AUC24h) by rilpivirine 150 mg once daily was considered not clinically relevant, and TDF did not affect the exposure to rilpivirine [60]. In a phase IIa proof-of-concept study (TMC278-C201), 47 treatment-na€ıve patients were randomized to receive either rilpivirine (25, 50, 100, or 150 mg once daily; 9 patients per group) or placebo (11 patients) for 7 days [61]. All dosages of rilpivirine gave statistically significant (p < 0.01) reductions in plasma viral load from baseline to

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day 8 (median reduction: 1.1 to 1.3 log10 HIV-1 RNA copies/ml), without an apparent dose relationship for antiviral activity. The majority (78%) of patients receiving rilpivirine had a plasma HIV-1 RNA decrease of more than 1 log10, and in four patients (12%) the virus titer fell below 400 HIV-1 RNA copies/ml on day 8. No viral load rebounds or NNRTI resistance were seen during therapy. In this trial, rilpivirine was well tolerated with no relevant differences in tolerability profiles between active treatment and placebo. The phase IIb TMC278-C204 trial (ClinicalTrials.gov identifier: NCT00110305) is an ongoing, multinational (54 centers in 14 countries), randomized, active-controlled, dose-ranging study in treatment-na€ıve patients with HIV-1 infection. Patients had a median baseline plasma viral load of 4.85 log10 HIV-1 RNA copies/ml and a median CD4 count of 203 cells/ml. Three blinded once-daily doses of rilpivirine (25, 75, or 150 mg) were investigated for 96 weeks (primary analysis at week 48) for their dose–response relationships of efficacy and safety, relative to an open-label control therapy with efavirenz 600 mg once daily. For all groups, two NRTIs (zidovudine/lamivudine or TDF/emtricitabine) were selected as background therapy. Other classes of HIV inhibitors, drugs that could affect exposure to rilpivirine, and immunomodulators were not allowed, although within-class substitutions and dosage adjustments for tolerability reasons were allowed for NRTIs. The primary objective of this study was to evaluate the dose-efficacy relationship for rilpivirine at week 48, the primary efficacy parameter being the proportion of patients attaining a confirmed plasma viral load below 50 HIV-1 RNA copies/ml [62, 63]. Secondary end points included plasma viral loads (<50 at other study visits, and <400 HIV-1 RNA copies/ml) and changes in viral load and CD4 counts from baseline. Safety and tolerability, immunologic and pharmacokinetic analyses, and a resistance analysis were secondary objectives. The trial has now been extended to 5 years. At week 48, high response rates were seen in all rilpivirine and efavirenz groups [62]. Mean changes from baseline to week 48 in viral load were similar between groups and ranged from 2.63 to 2.65 log10 HIV-1 RNA copies/ml (Figure 17.4). These response rates were well maintained across groups from week 48 to week 96, and differences in efficacy were not statistically significant [64, 65]. Rates of virologic failure at either week 48 or week 96 were low and not statistically significantly different between groups. Of note, mean CD4 cell counts increased further from baseline over the period from week 48 to week 96. The limited number of virologic failures in this study did not enable determination of the definitive resistance profile for rilpivirine, and resistance findings will be explored further in phase III trials [64, 65]. Eight NNRTI resistance-associated mutations (RAMs) (L100I, K101E, K103N, V108I, E138K, E138R, Y181C, and M230L) were noted in nine (of 36) patients receiving rilpivirine, while in three (of eleven) patients receiving efavirenz, the NNRTI RAMs K103N and V106M were observed. The most frequent NNRTI RAM observed in patients receiving rilpivirine was E138K, whose role in resistance is unclear because in one patient with an E138K NNRTI RAM at baseline the viral load remained below 50 HIV-1 RNA copies/ml at week 96 with rilpivirine treatment.

17.6 Pharmacokinetic and Phase II Studies of TMC278

Figure 17.4 Virologic response rates over time in study TMC278-C204 [64, 65].

Rilpivirine was generally well tolerated [64, 65]. Overall, the incidence of any grade 2–4 adverse event that was at least possibly related to rilpivirine was 20% in the rilpivirine combined group, which was significantly (p < 0.01) lower than the rate of 37% with efavirenz. There were no relevant changes in types or incidence of adverse events between weeks 48 and 96. Discontinuation due to adverse events and rash appeared more frequent in the two higher dose groups than in the 25 mg group, but the overall rate of serious adverse events was similar for all drug-dosed groups, without consistent patterns or apparent dose relationships. Five (of thirty-six) patients reported serious events possibly related to rilpivirine (hepatic enzyme increases or cytolytic hepatitis, blood amylase increase, and abdominal pain/constipation in the rilpivirine 25 mg once-daily group; suicide attempt and anemia in the rilpivirine 150 mg once-daily group), and one (of eleven) was reported with efavirenz (arthralgia). Incidences of grade 3–4 events that were possibly related to study medication did not statistically significantly differ among rilpivirine groups and compared with efavirenz, occurring in 5% of patients in the rilpivirine combined group and in 8% of efavirenz-treated patients. Both neurologic and psychiatric adverse events were generally less frequent in the rilpivirine groups than in the efavirenz group, with no indication of relationship with dosage. All rashes observed in the study were of grade 1 or 2 severity, resolving with continuing treatment except for one case of grade 3 rash, associated with dapsone treatment, in the rilpivirine 75 mg once-daily group at approximately 24 weeks of treatment. This was the only patient in the rilpivirine groups who discontinued because of rash. Two patients in the efavirenz group withdrew because of skin disorders. Rashes were more common in the rilpivirine 150 and 75 mg once-daily groups than in the 25 mg once-daily group. Mean changes from baseline at week 96 in the levels of total cholesterol, lowdensity lipoprotein-cholesterol, high-density lipoprotein-cholesterol, and triglycer-

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ides were lower in the rilpivirine groups than in the efavirenz group, with no rilpivirine dose relationships for mean changes in lipid parameters. The rilpivirine 25 mg once-daily dose was selected for further clinical development in treatment-na€ıve HIV-1-infected patients because it offered the best benefit–risk balance. Two fully enrolled ongoing phase III clinical studies in treatment-na€ıve patients, TMC278-TiDP6-C209 (ECHO; ClinicalTrials.gov identifier NCT00540449) and TMC278-TiDP6-C215 (THRIVE; ClinicalTrials.gov identifier NCT00543725) are designed to demonstrate noninferiority of rilpivirine to efavirenz with regard to the proportion of patients achieving a confirmed plasma viral load below 50 HIV-1 RNA copies/ml at week 48.

17.7 Conclusions

NNRTIs are very potent and usually well-tolerated unique inhibitors of HIV-1 RT. For this reason, they are a key component of HAART for treatment-na€ıve patients. However, due to their low genetic barrier, resistance to first-generation NNRTIs emerges faster than to most boosted HIV-1 protease inhibitors, and their tolerability is not always ideal. For these reasons in the development of TMC278, we focused our efforts not only on improving NNRTI potency and bioavailability but also on creating an antiretroviral with an improved resistance profile and better tolerability. Development of the next DAPY generation led to highly potent and selective NNRTIs against a broad spectrum of HIV-1 variants, including strains that are resistant to the first-generation NNRTI drugs. Rilpivirine has been selected for clinical advancement because of its demonstrated optimal characteristics with regard to resistance profile, antiviral efficacy, and tolerability. At present, TMC278 is in phase III clinical trials in treatment-na€ıve patients.

Acknowledgments

We thank Professor Eric De Clercq and Dr. John Proudfoot for their critical review of this manuscript, Kalyan Das for the X-ray crystallography pictures and Anik Peeters for the docking illustrations. Last but not least, special thanks go to Marie-Pierre de B
ethune for her very helpful suggestions.

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Williams, P., and Boven, K. (2008) Efficacy and safety of TMC278 in treatmentna€ıve, HIV-infected patients: week 96 data from TMC278-C204. 9th International Congress on Drug Therapy in HIV Infection, Glasgow, UK, Poster P002.

Luc Geeraert

Tibotec BVBA, Gen De Wittelaan L 11B 3, 2800 Mechelen, Belgium Luc Geeraert graduated as a master of Bioscience Engineering at the University of Leuven, Belgium, in 1991. He obtained additional masters in Industrial Engineering and Teaching and a degree in Journalism. After 2 years of biochemical research at the University of Leuven, he joined the Competitive Intelligence Department of Janssen Pharmaceutica (Johnson & Johnson) in 1997. Since 2005, he has been a scientific writer for antiinfective research at Tibotec and Virco (Johnson & Johnson).

Jerome Guillemont

Janssen-Cilag/Tibotec, Campus de Maigremont BP315, 27430 Val de Reuil, France Jerome Guillemont obtained his PhD in 1989 at the University of Rouen, France, in organic chemistry. He joined JanssenCilag the same year where he has contributed with his team to the success of the Johnson and Johnson Pharmaceutical Research and Development and Tibotec organizations. He discovered, with his team, Rilpivirine as a new nonnucleoside reverse transcriptase inhibitor for HIV and TMC207 as a new antituberculosis agent, respectively, under phase III and II clinical trials. He has coauthored more than 50 patents and 27 publications. He is a Research Fellow at Tibotec, leading medicinal chemistry teams for the Antimicrobial Research Group.

References

Jan Heeres

Professor, University of Antwerp, Leemskuilen 18, B2350 Vosselaar, Belgium Jan Heeres received a master’s degree in chemistry. He is inventor and coinventor of azole antimycotics miconazole, econazole, and isoconazole, the first oral broad-spectrum antimycotic ketoconazole, itraconazole, plant- and wood-protecting agents such as propiconazole and azaconazole, and mitratapide, used for the treatment of obese dogs. In Dr. Paul Janssen’s Centrum for Molecular Design (CMD), three nonnucleoside reverse transcriptase inhibitors (NNRTIs, HIV-1), dapivirine, etravirine, and rilpivirine were discovered. He received the J&J medal, the UIPAC-Richter Prize, was a Distinguished Research Fellow, and is a guest Professor at the University of Antwerp.

Paul J. Lewi

Visiting Professor, Pater Van Mierlostraat 18, 2300 Turnhout, Belgium Paul J. Lewi, PhD, Eng., is a Visiting Professor from Belgium. Employed with Janssen Pharmaceutica (Beerse) from1962 to 2005, he served as vice-president Center for Molecular Design. After retiring, he was appointed Visiting Professor at the Universities of Leuven (KUL), Brussels (VUB), and Antwerp (UA); took part in founding Chemometrics; Designed Spectral Map Analysis, a multivariate statistical method for the analysis of pharmacological data; and was coinventor of the DAPYclass of HIV-nonnucleoside reverse transcriptase inhibitors.

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18 Tipranavir, a Non-Peptidic Protease Inhibitor for Multi-drug Resistant HIV Suvit Thaisrivongs, Joseph W. Strohbach, and Steve R. Turner

18.1 Human Immunodeficiency Virus

The human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS), has been one of the most studied infectious agents of the past 25 years [1–4]. The tremendous scientific endeavor to deconvolute the HIV viral machinery has led to identification of numerous successful targets for therapeutic intervention. Today, there are more than 20 approved drugs in three established classes: nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs), as well as leading examples in three developing classes – entry, fusion, and integrase inhibitors – available for use in the treatment of HIV infection. These medications have had an undeniable positive impact on the course of care for millions of individuals.

18.2 HIV Protease

The HIV protease (PR) is a member of the aspartyl family of proteolytic enzymes that include human pepsin and renin. The PR is required in the late stages of the viral life cycle to process retroviral polyproteins necessary for viral particle maturation. The active enzyme – perhaps out of evolutionary necessity – is a C2 symmetrical homodimer formed by the association of two relatively small (99 amino acids), identical monomeric chains [5]. The substrate binding site is rather large; the protease is promiscuous with respect to cleavage recognition, presenting multiple interaction points along the active site cleft. This motif may have significant implications for the development of PI-resistant variants and related viral viability. The catalytic aspartic acid residues, typically denoted as Asp25 and 250 – corresponding to their positions within each of the monomer peptide sequences – lie at the floor of the dimeric complex proteolytic region. Additional critical components are

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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Figure 18.1 X-ray crystal structure of HIV-1 protease containing a bound benzopyranone ligand. Hydrogen bonding interactions between the 4-hydroxy group and the catalytic aspartic

acid residues on the floor of the active site, and between the lactone oxygens and the isoleucine residues (partially obscured) of the flap region are depicted as dotted lines.

isoleucine residues 50 and 500 (similar nomenclature) of the so-called “flap” region – a flexible loop of approximately six amino acids from each monomer – that forms the ceiling of the active site when occupied by substrate or inhibitors (see Figure 18.1). Also of significance is a water molecule frequently found bridging the enzyme’s isoleucine residues and peptide-derived substrate; this “conserved” water molecule is replaced in select nonpeptidic inhibitor-protease crystal structures (vide infra). Because catalytically competent protease is indispensable to the viral life cycle, inhibition of protease has long been regarded, and more recently clinically validated, as an attractive strategy for therapeutic intervention in AIDS. 18.2.1 HIV PIs

There have been 10 PIs, including one PI prodrug, approved by the US Food and Drug Administration (FDA) for the treatment of HIV infection: saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, lopinavir, fosamprenavir, atazanavir, tipranavir, and darunavir (Figure 18.2). The protease inhibitor class of antiretroviral agents, and their use in management of HIV infection, has been extensively reviewed [6–8]. A vast majority of PIs have been designed based on the basis of a modified peptidomimetic motif wherein the scissile bond of a peptidic substrate is replaced by a noncleavable transition-state isostere. Multiple additional alterations and structural refinements of these compounds through repetitive drug design cycles have afforded

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18.2 HIV Protease H 2N

H

O O N

OH

H N

N H

OH

N

H

N

N O

O

N H

Saquinavir SQV 1995

O

O

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H N

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Indinavir IDV 1996 H

O N

N H

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S

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H N OH

O

N H

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S HO N

O

H N

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S

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S O O

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O

HN

Amprenavir APV 1999

O

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O Lopinavir LPV 2000 Ca2+

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NH2

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O

Fosamprenavir fAPV 2003

N

O

O P

H N

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N H

Atazanavir ATV 2003

OH

O

O

OH

O

H N

O CF3

O HN

Tipranavir TPV 2005

H

N

Nelfinavir NFV 1997

Ritonavir RTV 1996

O

OH

S O O

N

O

O

O Darunavir DRV 2006

Figure 18.2 Approved HIV protease inhibitors and their year of US approval.

H N

O O

NH2

OH N

S O O

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clinical agents and eventually commercial medicines. A tremendous amount of medicinal chemistry has gone into the development of this class starting from the native substrate and peptidic lead structures [9, 10]. A parallel approach, utilizing a fragment/structure-based drug design campaign to transform a broad screening hit and peptidomimetic leads into three clinical agents, is described in the next section.

18.3 Approaches to Identifying and Developing PI Leads 18.3.1 Focused Screening

Analogue-based drug design can be approached from multiple directions. Two notable avenues are (1) novel applications of known compounds for related therapeutic targets and (2) the utility of proven drug motifs as potential leads for new disease targets [11]. As an example of the former approach, a focused screening of an existing collection of proprietary renin inhibitors was undertaken to identify lead structures for their evaluation as potential inhibitors of HIV protease. A primary motivation for this approach was the recognition of the familial relationship between the new target, HIV protease, and the aspartyl protease renin. From this work, a previous renin inhibitor clinical candidate, U-71038, was shown to possess unexpectedly potent activity against the PR (Ki 10 nM, Figure 18.3). Another renin inhibitor, U-75875, was identified with improved PR binding affinity (10
) and decreased renin inhibition activity (10
), resulting in an 100-fold improvement in selectivity over U-71038. More important, while U-71038 did not exhibit significant anti-HIV activity in vitro, U-75875 was shown to arrest viral maturation in vitro [12]. It

N

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O

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H N

N

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N O N H

N

N H

U-71038 IC50 0.26 nM (human renin) Ki 10 nM (HIV-1)

NH H N

O

O

H N O

O

O

O

NH

OH

OH O

H N

O N H

N

Figure 18.3 Peptidomimetic aspartyl protease inhibitors.

U-75875 IC50 2.3 nM (human renin) Ki 1 nM (HIV-1)

18.3 Approaches to Identifying and Developing PI Leads

is also worth noting that U-75875, which showed potent inhibition of simian immunodeficiency virus (SIV) protease, was demonstrated to exhibit activity in a nonhuman primate model (SIV-infected rhesus monkey) [13]. While more efforts were made to further optimize this class of peptidomimetic compounds, the known challenges of low aqueous solubility, poor permeability, limited metabolic stability, low oral bioavailability, and potentially high cost of goods were never truly overcome in a single compound. During these studies, however, efforts were also underway to identify nonpeptidic lead matter through broad screening. The resulting discovery of compounds displaying pharmacologically relevant activity against a dissimilar target afforded an opportunity to utilize an established chemical class as a template for new disease indications. 18.3.2 Broad Screening for Nonpeptidic Leads

A medium throughput screen on a dissimilarity set of an existing historical compound file identified warfarin as a weak inhibitor of HIV-1 protease (IC50  30 mM, Figure 18.4) [14]. Compared to the binding affinity results from the focused screening of renin inhibitory compounds, a 30 mM lead may not seem as attractive. However, the simplicity of the structure and the recognition that the coumarin class has a demonstrated history of therapeutic utility fueled additional interest. Warfarin itself is an important, commercially available, oral anticoagulant still prescribed nearly 50 years after its introduction. It is also noteworthy that scientists at ParkeDavis independently identified similar substrate through related screening efforts [15]. A focused screen of compounds similar to the warfarin hit, conducted on the same compound collection, resulted in identification of phenprocoumon – also a known, orally active anticoagulant – as a micromolar inhibitor of both HIV-1 and HIV-2 proteases (Figure 18.4). Perhaps of greater import were two additional critical factors: phenprocoumon also showed weak antiviral activity in a cellular assay and a cocrystal structure of the inhibitor–enzyme complex was solved. The crystallographic data showed the inhibitor’s C4 hydroxyl group to be interacting with the catalytic aspartic acid residues,

O

OH

O

O

OH

O

O

Warfarin

Phenprocoumon

IC50 ~30 µM HIV-1

Ki ~1 µM HIV-1 and HIV-2

Figure 18.4 Nonpeptidic broad screening leads.

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N O O

N H

NH H N

O

MW Ki HIV-1 #HA LE

OH

OH O

H N

O

OH

N H

N O

U-75875

Phenprocoumon

812 <1 nM 59 ~0.21

280 ~1 µM 21 ~0.40

O

Figure 18.5 Lead substrate profiles.

while the lactone oxygens were directly interacting with the amides of enzymatic flap isoleucine residues. In a binding pose that was later found to be highly conserved across several subsequent iterations of structural modification, the working pharmacophore was characterized by this ceiling-to-floor occupancy of the protease active site, with a key absence of the flap region-bound water molecule commonly observed in cocrystal structures of peptidomimetic inhibitors (see Figure 18.1). Finally, clear differentiation between the peptidomimetic and coumarin lead structures can be recognized in terms of the recently adopted ligand efficiency (LE) metric [16]. The LE descriptor is a powerful tool to help evaluate lead structural motifs and guide series selection. The term provides a means to “normalize” an agent’s potency in relation to its size and reflects the averaged contribution made by each heavy atom (HA) to total binding affinity. Generally speaking, lead compounds with larger LE values tend to be more tractable in the drug discovery process. The peptidomimetic lead was very large and exhibited nanomolar binding affinity, while the coumarin screening lead was relatively small and only modestly active. The LE term (Figure 18.5) suggests a clear preference toward the benzopyranone core, albeit with the caveat that comparing structures of widely disparate molecular weights can be complex [17]. Accordingly, the path that seemed to hold the greatest promise was to build upon the coumarin pharmacophore (LE value 0.4), with structure-based design direction derived from the early peptidomimetic structures (LE value 0.2). Within the framework of these guiding principles, the benzopyran template was selected as the launching point for medchem optimization. 18.3.3 Structure-Based Drug Design

The four components of a drug design cycle – rapid crystallographic determination, advanced molecular modeling capability, readily modified chemical lead matter, and critical and robust biological assays – were in place. An important factor contributing

18.3 Approaches to Identifying and Developing PI Leads

Figure 18.6 Representation of coumarin (dark) and pyrone (light) ligands, illustrating differential positioning of substituents made possible by the pyrone C6-a branch point.

to the development of new analogues was the ability to design based on two distinct lead structure profiles – peptidomimetic and coumarin. Thus, binding interactions and enzymatic subsite information derived from examination of the peptidomimetic crystal structure were used to inform and guide modifications to the coumarin template. 18.3.3.1 PNU-96988, A First-Generation Clinical Candidate Significant improvement in binding affinity of the coumarin ligand could be realized by scaffold modifications in the region of the fused phenyl ring. It was observed that the planar coumarin scaffold imposed a severe limitation on positioning of substituents, in terms of allowing optimized access to multiple enzymatic binding pockets. A greater degree of flexibility could be achieved by paring away the fused phenyl ring and by directly appending functionality to the remaining pyrone nucleus (Figure 18.6). The modification thus conceived corresponds to the 6-substituted a-pyrone system, exemplified by compound 1 (Figure 18.7). Compound 1 showed greatly enhanced binding affinity to HIV-1 protease (Ki 38 nM, 32 nM against HIV-2 PR), as well as antiviral activity in cell culture, with an ED50 value of 3 mM [14]. This compound, which has an MW of 362, can be prepared as a mixture of diastereomers in only two steps from readily available starting materials. An asymmetric synthesis

OH

O 1

OH O

Ki = 38 nM U-96988

O 2

OH

O

Ki = 75 nM

O

O

3 Ki = 15 nM

Figure 18.7 Identification of the first clinical candidate and cycloalkylpyrone-based inhibitors.

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has also been reported [18]. Its pharmacokinetic behavior has been evaluated in rats and dogs, where it showed high oral bioavailability (76 and 45%, respectively), very low clearance (0.8 and 0.7 ml/min/kg), and half-life consistent with maintenance of therapeutic levels for 6 h. As U-96988, compound 1 became the first clinical protease inhibitor candidate from this effort. Development of this first-generation PI was later discontinued in favor of subsequent analogues with superior potency. 18.3.3.2 PNU-103017, A Second-Generation Clinical Candidate Returning to phenprocoumon, an alternative approach to enhanced occupancy of additional enzymatic subsites was envisioned via replacement of the coumarin phenyl ring with a more flexible cycloalkyl ring (Figure 18.8). A number of these cycloalkylpyrones were prepared and found to be potent protease inhibitors, with compound 2 (Figure 18.7), for example, showing a Ki value (75 nM) more than 10-fold improved from the corresponding coumarin analogue [19]. Additional SAR refinement showed that replacement of the C3-a ethyl group with cyclopropyl provided a further boost in activity, leading to compound 3, which approaches two orders of magnitude improvement in binding affinity relative to the coumarin lead structure. Compounds such as 2 and 3 are available in three synthetic steps from commercial reagents and hold an advantage over inhibitors of the motif exemplified by 1 in containing only a single stereogenic center. Key insights gained from additional consideration of structural data were to give rise to perhaps the least obvious but most significant modification of the program. In

Figure 18.8 Overlay of coumarin (dark) and cycloalkylpyrone (light) scaffolds, illustrating potential for additional binding site occupancy resulting from greater flexibility of the cycloalkyl ring.

18.3 Approaches to Identifying and Developing PI Leads

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Figure 18.9 Overlay of cyclooctylpyranone ligand with the peptidomimetic U-75875, suggesting substitution at the meta-position of pyrone phenyl ring.

overlays of pyrone ligands related to compound 2 with the potent peptidomimetic inhibitor U-75875 (Figure 18.9), it was noted that the meta-position of the pyrone phenyl ring was located in close proximity to the alpha carbon of the peptidomimetic inhibitor’s P2 amino acid residue (histidine). This suggested that substituents placed at the meta-position of the C3-a phenyl ring of pyrone inhibitors might follow a trajectory similar to that described by the peptidomimetic compound, thus enabling access to additional enzymatic binding pockets. In addition, functionality present within the appended substituent might be expected to form hydrogen bonding interactions with the enzyme similar to those made by the amide backbone of the peptidomimetic inhibitor. The execution of this concept within the coumarin and pyrone series of ligands is exemplified by compounds 4–6 (Figure 18.10). These analogues contain Boc-protected forms of glycine, histidine, and beta-alanine, respectively, linked to the metaposition of the P2 phenyl ring by an amide functional group. This modification was found to improve binding affinity up to 10-fold [20]. Crystallographic analysis of these

OH

OH

OH O O

NHBoc

O HN

O HN O

4 Ki = 160 nM

N H

Boc

O

H N

HN O

5 Ki = 4 nM

O

O

NH N

Figure 18.10 Amide substitution at the meta-position of side-chain phenyl ring.

6 Ki = 4 nM

Boc

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compounds confirmed new interactions made by the Boc group within the S3 pocket and by the amide carbonyl group with protein backbone residues including Asp29. It was also demonstrated that enhanced activity could be imparted by moieties other than amino acid residues (data not shown). The engagement of Asp29 by the amide functional group is noteworthy, as interaction with this residue has been implicated as potentially important for design of inhibitors that retain high activity against PI-resistant PR mutants [21]. A further refinement was substitution of the m-carboxamide linkage by a sulfonamide tether, a modification again suggested by molecular modeling, which indicated the potential for an additional hydrogen bonding interaction with the second oxygen of the sulfonamide. This concept led to molecules exemplified by compound 7 (PNU-103017) [22]. Cyclooctylpyrone 7 exhibited a subnanomolar Ki value against HIV-1 protease and, more significantly, improved antiviral activity (IC50 1.5 mM) relative to similar carboxamides [23]. In addition, compound 7 was shown to be selective for HIV proteases over other aspartyl proteases, and pharmacokinetically well-behaved in rats and dogs, with low clearance, oral bioavailability of 42 and 77%, respectively, and high achievable blood levels. Crystallographic analysis once again confirmed the highly conserved binding pose of pyrone inhibitors, with the lactone forming hydrogen bonds to the flap region isoleucines, and the 4-hydroxy moiety associated with the active site aspartate residues (Figure 18.11). Enzymatic subsites ranging from S3 to S10 were occupied by ligand substituents. The favorable attributes of compound 7 led to its acceptance as a second-generation clinical candidate. Largely due to a very high degree of protein binding, the potency of compound 7 was still modest, particularly in comparison to the most active contemporary

Figure 18.11 Cycloalkylpyrone inhibitors: second clinical candidate and binding conformation.

18.3 Approaches to Identifying and Developing PI Leads

peptidomimetic inhibitors. The PK and safety profiles derived from early clinical studies of PNU-103107 and its predecessor PNU-96988 did not project sufficient confidence that these compounds could achieve efficacious outcome in HIV-infected patients, and these two early compounds were eventually discontinued. 18.3.3.3 Tipranavir, The Third Generation Additional design efforts continued to focus on optimizing occupancy of the enzyme’s S10 and S20 binding subsites. It was proposed that transforming the pyrone ring system to a dihydropyrone, and thereby converting the C6 center to sp3-hybridization, could present two different substituents to the enzyme, potentially filling both subsites (Figure 18.12). In practice, the dihydropyrone scaffold was found to provide reasonably active HIV protease inhibitors, showing Ki values ranging down to 15 nM after suitable optimization of the C6 moieties [24]. With the addition of sulfonamide-linked substituents in the meta-position of the C3-a side chain phenyl ring, as described previously, exquisitely potent analogues with Ki values in the single-digit picomolar range were realized [25]. Following significant SAR exploration of sulfonamide moieties, compound 8 (PNU-140690) was selected as the third-generation clinical candidate. In addition to extremely high binding affinity (Ki 8 pM) to HIV-1 protease, PNU-140690 also showed an excellent antiviral IC50 value of 30 nM and broad-spectrum activity against drug-resistant clinical strains of HIV (vide infra). A crystal structure of compound 8 bound to HIV-1 protease was determined (Figure 18.13), and served to confirm both absolute stereochemistry and important inhibitor–protein interactions. In accordance with previous examples, hydrogen bonding interactions were found between the C4 hydroxyl and the catalytic aspartate residues (Asp25 and Asp250 ), and between the lactone carbonyl and both NH groups

Figure 18.12 Comparison of pyrone (dark) and dihydropyrone (light) ligands, showing variation in disposition of C6 region substituents.

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Figure 18.13 Compound 8 (PNU-140690) bound to HIV-1 protease. Important interactions with active site residues are shown as dotted lines. The ligand is oriented with the C6 phenethyl and propyl substituents to the right and the lactone carbonyl pointed upward.

of the flap isoleucine residues Ile50 and Ile500 , the latter association also serving to displace the bound water molecule usually found in this region. The C6 phenethyl and propyl substituents were located in the S20 and S10 subsites, and the C3-a ethyl and phenyl groups projected into the S1 and S2 subsites, respectively. The pyridyl ring of the sulfonamide occupied the S3 subsite, and the sulfonamide group itself formed two hydrogen bonds to Asp30 at the active site. The backbone NH group of the Asp29 residue formed interactions with the pyridine nitrogen and an oxygen of the sulfonamide. PNU-140690 subsequently became known as tipranavir (TPV) [26]. As AptivusÒ , the compound was granted full FDA approval for treatment of HIV infection in the United States in June 2005, and the European Union approval followed in October 2005.

18.4 Characteristics of Tipranavir 18.4.1 In Vitro Activity

In addition to its 8 pM inhibition of HIV-1 protease, tipranavir also shows excellent binding affinity (Ki < 1 nM) to HIV-2 protease and is active against protease variants containing the V82A and V82F/I84V mutations closely associated with PI resistance [26]. Selectivity for HIV protease over other aspartyl proteases was demonstrated by micromolar level Ki values against human pepsin and cathepsins D and E.

18.4 Characteristics of Tipranavir

Early data on TPV suggested utility in combating PI-resistant virus that had rapidly begun to emerge in response to anti-HIV chemotherapy. In H9 cell culture, tipranavir inhibited replication of HIV-1IIIB with an IC90 value of 160 nM and displayed additivity to synergistic effects in combination with reverse transcriptase inhibitors. Importantly, potent antiviral activity was preserved across a number of drug-resistant strains of virus, including clinical isolates resistant to other protease inhibitors and to AZT. This remarkable retention of activity against strains of virus resistant to other PIs represents a distinguishing feature of TPV relative to earlier peptidomimetic PIs [27, 28]. Previously, viral strains that developed resistance against a particular peptidomimetic PI commonly showed cross-resistance to other peptidomimetic PIs. In a particularly striking study of 105 clinical HIV isolates having a more than 10-fold reduced susceptibility to at least three available PIs, all but two (98%) remained susceptible to TPV within 10-fold of wild type, 90% remained within threefold, and a significant proportion actually showed enhanced susceptibility to TPV [29]. Although resistance to tipranavir is known to develop, the process involves a relatively high genetic barrier and requires accumulation of multiple mutations, each of which affects sensitivity only incrementally. This tenacious retention of high activity in the face of multiple amino acid residue mutations has been attributed both to a mix of enthalpic and entropic binding components and to the compound’s network of hydrogen bonding interactions to highly conserved regions of the protease [30]. 18.4.2 Pharmacokinetics

The pharmacokinetic behavior of TPV is complicated by its interactions with CYP 3A4, of which it is both a substrate and inducer, and its apparent induction of Pglycoprotein. These effects contribute to decreasing trough concentrations over time, reaching steady state after about 7 days. When TPV is dosed alone, trough concentrations of >1 mM cannot be reliably attained even with 1000 mg b.i.d. dosing, and exposures obtained at higher dosages are nonproportional and somewhat variable. The use of ritonavir to enhance or “boost” blood levels of protease inhibitors is a well-accepted strategy [31, 32]. Coadministration of 200 mg ritonavir with TPV (the combination being commonly referred to as “TPV/r”) dramatically improves the PK profile of TPV, acting to decrease clearance and increase half-life, AUC, and plasma concentrations. Although the drug interaction is complex due to the opposing effects of CYP induction by TPV and CYP inhibition by RTV, the net effect of the drug combination is CYP 3A4 inhibition. Thus, the “boosted” combination acts to minimize the amount of TPV that must be administered to elicit the desired pharmacodynamic effect. The reduction in dosage in turn allows a diminished pill burden, which in general acts to enhance regimen compliance. In addition to RTV coadministration, the second important innovation for improved bioavailability was the use of a self-emulsifying drug delivery system (SEDDS). The SEDDS formulation, designed to dissolve lipophilic compounds and

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subsequently promote their dissolution in gastric milieu, in this case resulted in a two- to threefold improvement in bioavailability relative to the hard capsule formulation. With a dosage of 500/200 mg TPV/r b.i.d., a steady-state plasma trough TPV concentration of 26 mM, or greater than 10-fold the antiviral IC50 value, could be maintained. This value represents an increase in TPV plasma levels of greater than 20-fold relative to that achieved by the unboosted regimen [33, 34]. The 500/200 b.i.d. TPV/r dosage was selected as achieving the optimal balance between efficacy and tolerability, and was carried forth into phase III trials. 18.4.3 Highlights of Clinical Data

The phase III clinical trials for TPV were termed RESIST (Randomized Evaluation of Strategic Intervention in multidrug reSistant patients with Tipranavir). These trials, RESIST-1 in North America and Australia and RESIST-2 in Europe and Latin America, compared ritonavir-boosted tipranavir with four boosted comparator protease inhibitors (APV, IDV, LPV, and SQV). In all cases, the selected protease inhibitor was coadministered with a background regimen containing reverse transcriptase inhibitors, with or without the fusion inhibitor enfuvirtide. Prior to randomization, each patient was assigned by a physician to the comparator protease inhibitor and background regimen most appropriate to the patient’s individual viral genotype and treatment history, in accordance with contemporary best practice. Each treatment arm was then randomized, with half the patients in each arm receiving TPV in place of the selected comparator PI. Patients accepted for study enrollment were treatment-experienced, with viral loads >1000 RNA copies/ml of a viral genotype associated with resistance to protease inhibitors, as indicated by one or more mutations at selected protease codons. The 1509 patients enrolled in the two studies were divided equally between the tipranavir (TPV/r) arm and the comparator (CPI/r) arm. Data analysis at 24 weeks showed clear superiority of TPV/r over each of the four comparator regimens, as measured by reductions in viral load. Although the response rates in all cases varied inversely with the number of protease-associated mutations, the loss in sensitivity to TPV with increasing number of mutations was relatively modest (Figure 18.14). Thus, for viral genotypes showing three to four mutations, the response rate for TPV/r was nearly fourfold the response seen with the comparator regimens. The viral load reductions, as well as increases in CD4 þ counts that were also observed, were of magnitudes associated with a decreased risk of progression to AIDS [35, 36]. At 48 weeks, all major virological markers continued to indicate significant superiority of TPV therapy over comparator regimens, with approximately twofold improvements seen in several parameters: number of patients with >1 log reduction in viral load, number of patients achieving viral load of <50 HIV-1 RNA copies/ml, mean reduction in viral load, and mean increase in CD4 þ count. Virological failures in the TPV arm were less than half that seen in the comparator arm. A large increase

50

60

40

50

30 20 10 TPV/r

0 1-2

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Figure 18.14 Viral response rate versus number of primary mutations and individual CPI (RESIST 24 week data). Because patients were stratified according to comparator

Response rate

Response rate

18.5 Fragment-Based Lead Development?

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0 LPV

SQV

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regimen initially selected on the basis of current best practice and physician judgment, distribution is nonuniform and response rates in TPV/r arms vary.

was also noted in median time to virological failure. Not surprisingly, improved treatment response was also associated with enfuvirtide use and with more susceptible viral phenotypes. Long-term durability of response to TPV treatment has been evaluated from extended follow-up of a phase II study (BI 1182.2). At 80 weeks, 74% of patients on TPV/r (500/100 mg b.i.d.) had completed the study, with a median viral load reduction of 2.55 log10 copies/ml. Furthermore, 39% of these patients continued to derive meaningful virological benefit after 5 years of TPV/r-based antiretroviral therapy [37].

18.5 Fragment-Based Lead Development?

In a retrospective analysis, the phenprocoumon lead may be viewed as a “fragment” starting point for subsequent elaboration within the context of fragment-based drug design [38, 39]. Critical to the identification of the warfarin “hit” was the development of a robust screening assay with sufficient sensitivity to detect modestly potent chemical matter. With the ability to rapidly collect crystallographic information on select enzyme–inhibitor complexes, further fragment growth was aided by modeling overlays with previous high-affinity binding peptidomimetic structures. A direct comparison with the “Rule of 3” guidelines for lead development [40, 41] – molecular weight 300, clog P  3, number of hydrogen bond donors 3, and number of rotatable bonds 3 – suggests that phenprocoumon possesses most of the properties necessary for lead expansion (Figure 18.15). Using this convention, the pyrone core fragment was “grown” or “expanded” using lead optimization cycles into clinical agents and finally a commercial product.

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Phenprocoumon MW

OH

O

O

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< 300

clog P *

4.7

<3

# HBD

1

<3

# rot bonds

3

<3

(*clog D @ pH 6.5 2.8) Figure 18.15 Phenprocoumon and the “Rule of 3.”

The development of pyrone-based inhibitors from the original screening hit through the marketed drug tipranavir was accomplished largely without the benefit of high-volume predictors of ADME performance. Advancement decisions were largely driven by cell potency, with subsequent pharmacokinetic evaluation of selected compounds. A more modern paradigm might seek to include higher throughput data, such as from in vitro microsomal stability assays, to help guide compound design. Similarly, in silico models might be constructed and validated, and used to ameliorate potential liabilities associated with the structural series under investigation.

18.6 Summary

The development of tipranavir, which commenced over 20 years ago, provides an early and highly successful example of iterative structure-based design. Beginning with a small, weakly active ligand with desirable pharmaceutical properties, identified by medium-throughput screening, iterative design cycles provided >105-fold improvement in target affinity (Table 18.1). Initially, the phenyl component of the benzopyranone lead structure was excised to provide the pyrone core; alternatively, the phenyl ring was saturated and expanded to afford the cycloalkylpyrone class. The final core modification was transformation of the C6 center to an sp3 carbon, allowing a differential positioning of two substituents in this region. One of the most significant structurally driven innovations was appendage of functionality at the meta-position of the pyrone benzyl group. Initial substituents chosen for this position were carboxamide-based components similar to those found in peptidomimetic examples, with the final refinement being the use of heteroaromatic sulfonamides. These structural improvements were rationally guided by information provided by rapid throughput X-ray crystallography, in concert with binding motifs elucidated from overlays with a high-affinity peptidomimetic ligand. This process of refinement can also be understood in terms of fragment-based drug design, in which a small, weakly bound initial structure is progressively elaborated in a fashion that allows

References Table 18.1 Evolution of tipranavir.

Structure

Kia)

MW

#HA

280

21

1000

0.40

362

27

38

0.38

503

36

0.8

0.35

600

42

0.008

0.37

LE

OH

O

O

Phenprocoumon

OH

O

O

U-96988

OH

O

CN

O HN

PNU-103017

S O O

OH

O

CF3

O HN

Tipranavir a)

N S O O

In nM, against HIV-1 protease spacers.

occupancy of additional enzymatic binding pockets, while preserving critical binding interactions at the catalytic site. The drug discovery process described in this chapter not only serves as an illustration of these design concepts but also culminated in a useful marketed treatment for HIV-infected individuals, many of whom have faced significant medical challenges due to failure of first-line anti-HIV therapy.

References 1 Knoll, B., Lassmann, B., and Temesgen,

Z. (2007) Current status of HIV infection: a review for non-HIV-treating physicians. Int. J. Dermatol., 46 (12), 1219–1228.

2 Ho, D.D. and Bieniasz, P.D. (2008) HIV-1 at

25. Cell (Cambridge, MA), 133 (4), 561–565. 3 Waters, L. (2008) HIV therapy: past,

present and future. Drug Discov. World, Summer, 11–18.

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5

6

7

8

9

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11

12

compounds approved within 25 years after the discovery of HIV. Int. J. Antimicrob. Agents, 33 (4), 307–320. Tomasselli, A.G. and Heinrikson, R.L. (2000) Targeting the HIV-protease in AIDS therapy: a current clinical perspective. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol., 1477 (1–2), 189–214. Mastrolorenzo, A., Rusconi, S., Scozzafava, A., Barbaro, G., and Supuran, C.T. (2007) Inhibitors of HIV-1 protease: current state of the art 10 years after their introduction. From antiretroviral drugs to antifungal, antibacterial and antitumor agents based on aspartic protease inhibitors. Curr. Med. Chem., 14 (26), 2734–2748. Martinez-Cajas, J.L. and Wainberg, M.A. (2007) Protease inhibitor resistance in HIV-infected patients: molecular and clinical perspectives. Antivir. Res., 76 (3), 203–221. Mitsuya, H., Maeda, K., Das, D., and Ghosh, A.K. (2008) Development of protease inhibitors and the fight with drug-resistant HIV-I variants, in HIV-1: Molecular Biology and Pathogenesis: Clinical Applications, Advances in Pharmacology, vol. 56, 2nd edn, Academic Press, pp. 169–197. Abdel-Rahman, H.M., Al-Karamany, G.S., El-Koussi, N.A., Youssef, A.F., and Kiso, Y. (2002) HIV protease inhibitors: peptidomimetic drugs and future perspectives. Curr. Med. Chem., 9 (21), 1905–1922. Randolph, J.T. and DeGoey, D.A. (2004) Peptidomimetic inhibitors of HIV protease. Curr. Top. Med. Chem., 4 (10), 1079–1095. Wermuth, C.G. (2006) Analogues as a means of discovering new drugs. In Analogue-Based Drug Discovrg, WileyVCH, pp. 3–23. Thaisrivongs, S., Tomasselli, A.G., Moon, J.B., Hui, J., McQuade, T.J., Turner, S.R., Strohbach, J.W., Howe, W.J., Tarpley, W.G., and Heinrikson, R.L. (1991) Inhibitors of the protease from human immunodeficiency virus: design and modeling of a compound containing

13

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16

17

18

19

a dihydroxyethylene isostere insert with high binding affinity and effective antiviral activity. J. Med. Chem., 34 (8), 2344–2356. Martin, L.N., Soike, K.F., Murphey-Corb, M., Bohm, R.P., Roberts, E.D., Kakuk, T.J., Thaisrivongs, S., Vidmar, T.J., Ruwart, M.J., Davio, S.R., and Tarpley, W.G. (1994) Effects of U-75875, a peptidomimetic inhibitor of retroviral proteases, on simian immunodeficiency virus infection in rhesus monkeys. Antimicrob. Agents Chemother., 38 (6), 1277–1283. Thaisrivongs, S., Tomich, P.K., Watenpaugh, K.D., Chong, K.-T., Howe, W.J., Yang, C.-P., Strohbach, J.W., Turner, S.R., McGrath, J.P., Bohanon, M.J., Lynn, J.C., Mulichak, A.M., Spinelli, P.A., Hinshaw, R.R., Pagano, P.J., Moon, J.B., Ruwart, M.J., Wilkinson, K.F., Rush, B.D., Zipp, G.L., Dalga, R.J., Schwende, F.J., Howard, G.M., Padbury, G.E., Toth, L.N., Zhao, Z., Koeplinger, K.A., Kakuk, T.J., Cole, S.L., Zaya, R.M., Piper, R.C., and Jeffrey, P. (1994) Structure-based design of HIV protease inhibitors: 4hydroxycoumarins and 4-hydroxy-2pyrones as non-peptidic inhibitors. J. Med. Chem., 37 (20), 3200–3204. Hagen, S., Prasad, J.V.N.V., and Tait, B.D. (2000) Nonpeptide inhibitors of HIV protease. Adv. Med. Chem., 5, 159–195. Hopkins, A.L., Groom, C.R., and Alex, A. (2004) Ligand efficiency: a useful metric for lead selection. Drug Discov. Today, 9 (10), 430–431. Reynolds, C.H., Tounge, B.A., and Bembenek, S.D. (2008) Ligand binding efficiency: trends, physical basis, and implications. J. Med. Chem., 51 (8), 2432–2438. Gammill, R.B., Judge, T.M., Phillips, G., Zhang, Q., Sowell, C.G., Cheney, B.V., Mizsak, S.A., Dolak, L.A., and Seest, E.P. (1994) Asymmetric synthesis of an HIV protease inhibitor via a novel a-oxoketene/ ketene [4 þ 2] cycloaddition reaction. J. Am. Chem. Soc., 116 (26), 12113–12114. Romines, K.R., Watenpaugh, K.D., Tomich, P.K., Howe, W.J., Morris, J.K., Lovasz, K.D., Mulichak, A.M., Finzel, B.C., Lynn, J.C., Horng, M.-M., Schwende, F.J., Ruwart, J.J., Zipp, G.L., Chong, K.-T.,

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Johnson, P.D., Aristoff, P.A., Tomich, P.K., Lynn, J.C., Horng, M.M., Chong, K.T., Hinshaw, R.R., Howe, W.J., Finzel, B.C., and Watenpaugh, K.K. (1996) Structure-based design of HIV protease inhibitors: 5,6-dihydro-4-hydroxy-2pyrones as effective, nonpeptidic inhibitors. J. Med. Chem., 39 (23), 4630–4642. Thaisrivongs, S., Skulnick, H.I., Turner, S.R., Strohbach, J.W., Tommasi, R.A., Johnson, P.D., Aristoff, P.A., Judge, T.M., Gammill, R.B., Morris, J.K., Romines, K.R., Chrusciel, R.A., Hinshaw, R.R., Chong, K.T., Tarpley, W.G., Poppe, S.M., Slade, D.E., Lynn, J.C., Horng, M.M., Tomich, P.K., Seest, E.P., Dolak, L.A., Howe, W.J., Howard, G.M., Schwende, F.J., Toth, L.N., Padbury, G.E., Wilson, G.J., Shiou, L., Zipp, G.L., Wilkinson, K.F., Rush, B.D., Ruwart, M.J., Koeplinger, K.A., Zhao, Z., Cole, S., Zaya, R.M., Kakuk, T.J., Janakiraman, M.N., and Watenpaugh, K.K. (1996) Structure-based design of HIV protease inhibitors: sulfonamide-containing 5,6-dihydro-4hydroxy-2-pyrones as non-peptidic inhibitors. J. Med. Chem., 39 (22), 4349–4353. Turner, S.R., Strohbach, J.W., Tommasi, R.A., Aristoff, P.A., Johnson, P.D., Skulnick, H.I., Dolak, L.A., Seest, E.P., Tomich, P.K., Bohanon, M.J., Horng, M.M., Lynn, J.C., Chong, K.-T., Hinshaw, R.R., Watenpaugh, K.D., Janakiraman, M.N., and Thaisrivongs, S. (1998) Tipranavir (PNU-140690): a potent, orally bioavailable nonpeptidic HIV protease inhibitor of the 5,6-dihydro-4-hydroxy-2pyrone sulfonamide class. J. Med. Chem., 41 (18), 3467–3476. Poppe, S.M., Slade, D.E., Chong, K.-T., Hinshaw, R.R., Pagano, P.J., Markowitz, M., Ho, D.D., Mo, H., Gorman, R.I., Dueweke, T.J., Thaisrivongs, S., and Tarpley, W.G. (1997) Antiviral activity of the dihydropyrone PNU-140690, a new nonpeptidic human immunodeficiency virus protease inhibitor. Antimicrob. Agents Chemother., 41 (5), 1058–1063. Rusconi, S., La Seta Catamancio, S., Citterio, P., Kurtagic, S., Violin, M., Balotta, C., Moroni, M., Galli, M., and

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References

Joseph W. Strohbach

Pfizer Inc., AA2I/AA243, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA Joseph W. Strohbach studied at the Marquette University (BS) and the University of Notre Dame (MS). He began his pharmaceutical career with Upjohn Company. During the course of the past 20 years, he has pursued small molecules and chemical technologies for a variety of targets in multiple therapeutic areas. He is a Senior Principal Scientist in medicinal chemistry for Pfizer in St. Louis, Missouri, USA.

Suvit Thaisrivongs

Pfizer Inc., BB4D/BB484, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA Suvit Thaisrivongs is the Head of Chemistry at Pfizer, St. Louis, in Missouri, USA. He is a synthetic organic chemist by training, with a bachelor’s degree from the Harvard College, a doctoral degree from the California Institute of Technology, and a postdoctoral research at the Swiss Federal Institute. He has over 25 years of human health pharmaceutical research experience spanning a wide range of therapeutic areas with a number of legacy pharmaceutical companies.

Steve R. Turner

Pfizer Inc., BB2G/BB216, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA Steve R. Turner received degrees in chemistry from UCLA (BS, 1981) and California Institute of Technology (MS, 1983). In 1984, he joined research efforts at Upjohn Company in Kalamazoo, Michigan, and over the subsequent 25 years has pursued small-molecule intervention in human diseases spanning numerous therapeutic areas. At present, he is a Senior Principal Scientist in medicinal chemistry for Pfizer in St. Louis, Missouri, USA.

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19 Lapatinib, an Anticancer Kinase Inhibitor Karen Lackey

19.1 Introduction

All the cells in the human body use a system of communication that connects the messages from outside the cells to the nucleus that tell the cell to grow, stop growing, differentiate, or signal to other cells. There are over 500 different protein kinases that transfer the terminal phosphate from ATP to a specific protein substrate in a signal cascade that involves complexes of signaling and scaffold proteins to convey these messages. The kinases are classified as receptor and nonreceptor, and by the amino acid that they phosphorylate, tyrosine or serine/threonine. These proteins have several standard regions, such as the ATP pocket, substrate binding region, regulatory domain, alternate nonregulatory sites, SH2 and SH3 domains. Aberrant signaling occurs in many different disease pathologies, and for cancer, hyperactivation or overexpression can lead to tumor formation. While all of the regions of a signaling protein offer potential drug intervention points to modulate the kinase activity, the discovery of lapatinib focused on creating unique interactions within the ATP binding site of two important receptor tyrosine kinases: EGFR and erbB-2. The challenge for the drug discovery effort was to achieve the necessary selectivity for these two kinases without inhibiting the remaining members of the kinome plus building in the required properties to be effective in patients as an orally administered drug. For this chapter, the analogues that led to the discovery of lapatinib demonstrate that a highly selective and effective compound could be created for use in cancer therapy for solid tumors that overexpressed these aberrant signaling proteins. Approximately 20 years ago, the early kinase inhibitors that were ATP competitive were proven to be potent and effective at blocking cellular signaling, but they were generally found to be nonselective. Popular examples are staurosporine analogues and flavone/isoflavone derivatives. These early kinase inhibitors were important in establishing the principle that the inhibition of cellular signaling could lead to apoptosis, growth inhibition, and in vivo effects. Second-generation kinase inhibitors were equally as potent but had attained interesting selectivity improvements.

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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The landmark publication in 1994 by Parke-Davis researchers essentially proved that it was feasible to inhibit kinase signaling through inhibitor binding in the ATP binding site of EGFR and that this approach was effective at blocking specific pathways [1]. The compound was not optimized for drug characteristics, and many scientific groups synthesized more elaborately substituted quinazolines over the next 15 years. Figure 19.1 shows representative enzyme data to demonstrate the contrast in kinase inhibition profiles for staurosporine and PD153035. The data are generated in pIC50 format, and the maximum concentration tested was at a pIC50 of 5.0 (10 mM). Data were generated in GlaxoSmithKline assays. Third-generation kinase inhibitors and beyond have been discovered using a plethora of heterocyclic scaffolds created by designing specific interactions within the ATP binding site, and have proven effective in the disease setting. Here are just a few of the many high-quality compounds (Figure 19.2): imatinib (Gleevec) was designed to inhibit bcr-abl but also had activity against c-kit and PDGFR that appear to improve the effectiveness and broaden the patient population to cancers that are driven by c-kit (e.g., gastric) [2]. Sorafenib (Nexavar) was originally designed as a cRaf1 inhibitor, but it also possessed potency for VEGFR2 that appears to improve the clinical effectiveness [3]. Gefitinib (Iressa) was the first small-molecule inhibitor of EGFR TK and was approved for use in lung cancer [4]. Sunitinib (Sutent) was originally discovered and developed as an orally bioavailable VEGFR and PDGFR tyrosine kinase inhibitor [5]. It was found highly effective in many preclinical models of efficacy and in

H N

R

PI

Staurosporine

A

8

Au

ro

ra

K3

p3

IK

K2 SK 3b csr c VE G FR 2 JN K1

FR

D

C

G

p3 8 IK K3 Au ro ra A

O N

3K a C K1 AL K5 EG FR C D K G 2 SK 3b csr c VE G FR 2 JN K1

N

10.5 9.5 8.5 7.5 6.5 5.5 4.5 O

O

Staurosporine pIC50 values

N H

K5

Representative Kinase Assays

PD153035

O

EG

K1

O R

PI

N

MeO

C

3K

a

N

AL

Br

HN MeO

pIC50 values

PD153035 10.5 9.5 8.5 7.5 6.5 5.5 4.5

Representative Kinase Assays

Figure 19.1 Structures and some representative kinase inhibition data for PD153035 and staurosporine.

19.2 Aims

Cl N HN

HN N

N Me

O

CF3 NH

HN

O

O

N

O

Me

NHMe

N

N

Imatinib (Gleevac)

Sorafenib (Nexavar) NEt2

N

NH

H3C HN

O O

O

F

O

N N

Gefitinib (Iressa)

CH3

Cl N H

F O N H

Sunitinib (Sutent)

Figure 19.2 Examples of clinically used kinase inhibitors.

mechanism of action (MoA) studies for these and other primary targets. More recently, sunitinib was evaluated for its potential usefulness in Ret/PTC-positive thyroid cancers [6]. Lapatinib (also known as GW2016, GW572016, Tykerb) is a potent dual inhibitor of erbB-2 and EGFR TK, and is virtually inactive in over 150 other kinases tested [7]. After many years of generating derivatives to achieve the activity and selectivity profile, and to ensure drug-like properties, lapatinib entered clinical trials in 2001 [8]. This chapter will describe the journey of the modifications to the quinazoline series that led to an effective cancer drug approved for use in breast cancer in over 20 countries.

19.2 Aims

Tyrosine kinase receptors are membrane-spanning receptors with a general structure consisting of a cysteine-rich extracellular ligand binding domain, a hydrophobic membrane-spanning region, and an intracellular domain containing the kinase function [9]. Peptide ligands, such as epidermal growth factor (EGF) and transforming growth factor-a (TGFa), bind to the extracellular region of EGFR and regulate erbB receptor signaling that results in either homo- or heterodimerization of the four

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family members of these receptors (EGFR, erbB-2, erbB-3, and erbB-4). This dimerization of erbB receptors is essential for their function and leads to the activation of tyrosine kinase activity via autophosphorylation, heterodimerization, and transactivation-autophosphorylation of erbB-2 since no soluble ligand has been identified. The formation of the erbB family dimers activates downstream Erk1/2 MAP kinases and PI3K/AKT kinase survival pathways. Various types of molecules are being generated and studied for modulation of the erbB family targets that include antibodies to the extracellular domain, vaccines, and kinase inhibitors either alone or in combination [10]. Overexpression of either EGFR or erbB-2 in a solid tumor correlates with a poorer clinical outcome in a variety of malignancies such as breast, ovarian, head and neck, and gastric cancers [11, 12]. The class 1 receptor tyrosine kinase pathway is hyperactivated or disregulated by a number of mechanisms: overproduction of ligands, overproduction of receptors, and/or constitutive activation of receptors. By blocking the kinase activity of both EGFR and erbB-2, it was expected that the signal would be blocked from all these methods of activation, thus representing an ideal drug intervention point in a disease with a high unmet medical need. Well-known examples of potent, relatively selective EGFR TK inhibitors (but not dual erbB family TK inhibitors) used in the clinic for anticancer therapy are Iressa and Tarceva. Small changes in kinase inhibitor structure result in dramatic selectivity, potency, and chemical property differences. Many of the molecules appear similar in structure (e.g., quinazoline analogues), but the potential effectiveness of the molecules as drugs differs due to their broader kinase inhibition profiles and physicochemical properties. Potent, unique erbB family inhibitors have been made, but the biological effectiveness of these compounds varies more dramatically between structural classes. For example, Lichtner and colleagues compared the cellular effects of two classes of potent EGFR TK inhibitors: quinazolines and 4,5-dianilino-phthalimides [13]. The quinazoline inhibitors affected the ligand binding properties by stabilizing the ligand/receptor/inhibitor complex resulting in potent cellular activity, while the dianilinothalimides did not. This observation was a novel mode of action for the quinazoline analogues only even, though both classes of compounds bind in the ATP site with similar potency. Such complexity in inhibitor/receptor interactions provided justification for extensive SAR exploration of erbB family tyrosine kinase inhibitors. Our aim was to discover a dual erbB-2/EGFR tyrosine kinase inhibitor that caused tumor growth inhibition in relevant preclinical models of cancer with direct evidence for the link between efficacy and the desired mechanism of action. We used a panel of cell lines that included two control lines, with parallel xenograft mouse models, allowing us to measure both phenotype and the reduction in phosphorylation levels of the inhibited signal. As is typical in most medicinal chemistry efforts, we used physicochemical measurements and pharmacokinetic studies to ensure our lead compounds had oral bioavailability for an intended single, daily, oral dose in patients. The most important aim was to discover a cancer therapy with a large therapeutic index by strictly adhering to the concept of targeting tumor cells over normal cells, and selecting drug candidates that demonstrated little or no toxicity.

19.3 Chemical Evolution and Proof-of-Mechanistic Approach Using Small Molecules

O HN O

Br

HN

N

O

O

N

O

PD153035 potent EGFR TK inhibitor

H N

O

S

N Dual erbB2/EGFR TK Inhibition achieved

O HN

N

F

N N

Cl HN N

O O

N Lapatinib potent dual erbB-2/EGFR TK inhibitor approved for Breast cancer in March 2007

N

N N

N GW2974 Mechanism of action linked with efficacy in preclinical models

Figure 19.3 Analogue progression from EGFR selective to dual inhibition to drug candidate quality.

19.3 Chemical Evolution and Proof-of-Mechanistic Approach Using Small Molecules

As mentioned earlier, the substituted 4-anilinoquinazoline (PD153035) had been reported as a potent, relatively selective EGFR TK inhibitor [1]. In Figure 19.3, the analogue progression is summarized for the drug discovery program. The erbB-2 activity was critical to build into the quinazolines series and was accomplished with large substituents appended to the 4-position of the aniline portion of the molecule. There were no crystal structures at the time to explain this observed activity, but several large substitutions conveyed the same dual EGFR/erbB-2 inhibitory activity. A considerable amount of SAR was developed and followed by the nomination of GW2974 as a drug candidate for development. Unfortunately, GW2974 did not possess an acceptable therapeutic index, so more work was needed. However, GW2974 and closely related analogues did offer the proof-of-mechanism and link to efficacy. The data strongly suggested that a dual inhibition profile was feasible and offered clear benefits for a potential cancer therapy [14]. In this section, the analogues that led to each of these milestones will be discussed. Compounds listed in Table 19.1 demonstrate the diversity of tolerated groups for maintaining dual inhibition [13, 15]. Substitutions in the 4-anilino portion of the molecule such as N1-benzylindazolyl, 4-benzyloxyphenyl, and phenylsulfonylphenyl all conferred dual inhibition. There was no significant difference in enzyme potency

j469

GW5289

GW0277

GW2974

Table 19.1

N

O

N N

O S O

N

N

N

N

O

N

N

N

N

N

N N

Structure

N

N

N

N

N

F

O

0.032

0.026

0.025

0.007

0.016

0.028

EGFR inhibition IC50 (mM)

ErbB-2 inhibition IC50 (mM)

Representative quinazoline analogues that established dual EGFR and erbB-2 activity in preclinical models.

HFF BT474 N87 HN5 HB4a r4.1 HB4a c5.2 HFF BT474 N87 HN5 HB4a r4.1 HB4a c5.2

HFF BT474 N87 HN5 HB4a r4.1 HB4a c5.2

Cell line

>9


>13


>75


>30 0.4 0.4 0.3 >25 0.5 >7.5 0.6 0.6 0.4 >7.3 0.94 >10 1.2 1.2 0.8 >10 0.88

Selectivity

IC50 (mM)

470

j 19 Lapatinib, an Anticancer Kinase Inhibitor

Tarceva OSI-774

GW9525

GW5945

N H

S

O

O O

S O O

O

N

O

O

O

O

N

N

N

N

N

N

N

O O S

N

HN

F

N N

1.05

0.030

0.024

0.017

0.029

0.022

HFF BT474 N87 HN5 HB4a r4.1 HB4a c5.2 HFF BT474 N87 HN5

HFF BT474 N87 HN5 HB4a r4.1 HB4a c5.2

>23 0.6 0.8 1.1 12.5 1.7 >8.7 4.2 5.1 0.18

23 0.4 0.6 0.6 19 1.4

47


19.3 Chemical Evolution and Proof-of-Mechanistic Approach Using Small Molecules

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between the quinazoline and the pyridopyrimidine series. A wide range of substituents were introduced at the 6-position to improve compound properties while having very little effect on the enzyme activity including disubstituted amines, aminomethyl heterocycles, oxadiazoles, and amino alkyl ethers. No substituents were tolerated in the 5- and 8-positions of the quinazoline or pyridopyrimidine scaffolds for dual erbB-2 and EGFR inhibitory activity. The compounds were more than 50-fold selective for ErbB-2 and EGFR versus other proliferative kinases that were investigated including cRaf-1, CDK1, CDK2, c-Src, ERK2, MEK, p38, Tie2, VEGFR2, and c-Fms. Tarceva (OSI-774) is reported to be EGFR selective and was included in Table 19.1 for comparison. It was approximately 40-fold more potent on EGFR than ErbB-2 (17 and 680 nM, respectively) in our in vitro enzyme assays. The efficacy of these dual inhibitors on human tumor cells was assessed in a cellbased proliferation assay using protein staining to estimate the relative cell number. The cell-based assay included the EGFR overexpressing cell line, HN5, the ErbB-2 overexpressing cell line, BT474, and a cell line overexpressing both receptors, N87 [8]. Human foreskin fibroblast (HFF) cells, which are from normal tissue and express low levels of EGFR and ErbB-2, were included as a control cell line for nonspecific toxicity. The compounds’ activities ranged from 16- to >75-fold potent on the tumor cells than the normal fibroblasts. The type I receptors transduce a mitogenic signal after autophosphorylation by activating the downstream effector Ras, which then signals through Raf, MEK1, and ERK2, resulting in translation of response genes and then cell division [16]. A cell line that overexpresses activated Ras, therefore, should be resistant to the antiproliferative effects of a selective EGFR or ErbB-2 inhibitor. A transfected cell system was created that contained either erbB-2 (HB4a c5.2) or the valine 12 mutant of Ha-ras (HB4a r4.2) to determine if the antiproliferative effects of the compounds were due to inhibiting the receptor kinase as opposed to targeting downstream effectors of cell proliferation. In this system, the ideal cellular profile was potent inhibition of the tumor lines (e.g., IC50 values <0.25 mM) N87, HN5, BT474 HB4a c5.2, and inactivity in the control lines (e.g., IC50 values >30 mM) HFF and HB4a r4.2. In addition to signaling through the Ras pathway, the type I receptors also mediate cell survival pathways downstream of PI3K [17]. Perturbation of EGFR or ErbB-2 signaling cascades may inhibit these survival pathways and could result in selective cell death regardless of the ras status of a tumor. So, HB4a r4.2 was a suitable control for the cell-based assays, but it may not be a reliable predictor of the effects of mutated ras oncogenes in EGFR- or ErbB-2positive tumors in the clinical setting. Results for two representative compounds evaluated for subcutaneous human tumor xenograft growth in mice are shown in Figure 19.4. Both GW2974 and GW0277 caused significant inhibition of tumor growth in both the BT474 (erbB-2 driven) and the HN5 (EGFR driven) models in a dose-dependent manner [14]. Body weight was the ex vivo measure of tolerability. GW2974 produced no weight differences between treated and control, whereas the GW0277-treated group experienced modest weight reduction in the treated group. Inhibition of receptor autophosphorylation in the tumor implants was verified by Western blot analysis following treatment of mice with tumors from the HN5 and BT474 cell lines with five

19.3 Chemical Evolution and Proof-of-Mechanistic Approach Using Small Molecules

Figure 19.4 Tumor xenograft growth inhibition for GW2974 and GW0277. Vehicle control *, 10 mg/kg ~, or 30 mg/kg & of the compound. Panels a and b: BT474 (erbB-2 þ þ þ ) tumor implants treated with GW2974

and GW0277 compared with control. Panels c and d: HN5 (EGFR þ þ þ ) tumor implants treated with GW2974 and GW0277 compared with control [14].

oral doses of GW2974, 30 mg/kg, b.i.d. or vehicle control. Tumors were excised and analyzed for EGFR (HN5) or ErbB-2 (BT474) expression and receptor autophosphorylation. Phosphorylation of both receptors was inhibited by approximately 90% with treatment, while the expression level of both the ErbB-2 and EGFR protein remained unchanged. In conclusion, five representative compounds (out of more than 2500 compounds generated in the pyridopyrimidine and quinazolines series) inhibited both the EGFR and the ErbB-2 kinase domain and also proved potent on cells overexpressing either receptor singly or both receptors together. The most potent and selective compound, GW2974, was well tolerated in the human xenograft murine assays and showed strong evidence that the efficacy was due to the desired mechanism of action. Progression into development was halted due to unforeseen toxicity presumably unrelated to the desired mechanism for selecting and treating patients. The chemical diversity displayed in these compounds suggested that opportunities for modulating compound properties such as potency, selectivity, and drug-like properties existed. Preclinically, the aim to create dual inhibitors of EGFR/erbB-2 for the treatment of

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large R groups tolerated however cellular in vitro potency is decreased

optimal chain length

R O S O

large substitution important for ErbB-2 tyrosine kinase inhibition furan and thiazole were the best 5-membered ring heterocycles

Z O, N, NR retain enzyme potency

X

HN

substitutions decrease enzyme potency

O Y

N W

N

Cl, Br are best, substitutions effect cellular activity

small substitutions preferred, F is best

N and C are equally good in enzyme and cell

Figure 19.5 SAR summary for quinazolines and pyridopyrimidine analogues.

solid tumors overexpressing either or both of these receptors had been proven feasible.

19.4 Final Set of Analogues that Led to the Discovery of Lapatinib

The overall summary of the SAR from an analysis of all the compounds generated in the project is shown in Figure 19.5. In this section, the analogues with two principal cores will be discussed, along with one example of trying to transfer the SAR to a new core scaffold. 19.4.1 6-Furanyl Quinazoline Series

The strategy to generate SAR for the core 6-furanyl quinazolines series used a mixand-match approach combining 6-position side-chain modifications and 4-anilino modifications. Several trends were observed. For example, the orientation of the substituents on the furan ring affected the cellular activity. The side-chain substituents were used to optimize the cellular activity of the series with modifications in chain length, heteroatom linker, and terminal R-group changes. In this section, analogues with the 4-benzyloxyaniline moiety are described, while the combinations with the other preferred anilines are covered in the next section to avoid redundant SAR discussions. The regiochemistry of the linking furan ring with the side-chain substituent turned out to be quite important. Compounds 1–4 in Table 19.2 offer a comparison of each connection to the furan ring plus an example with no side chain appended. While substitution at the 40 and 50 position of furan (3 and 2, respectively) appeared to be similarly potent at inhibiting the EGFR and ErbB-2 enzymes, the 50 -substituted

19.4 Final Set of Analogues that Led to the Discovery of Lapatinib Table 19.2 Type I receptor inhibition activity and cellular efficacy results for regiochemical furanylquinazoline analogues.

O

H3C

S O O

H N

4'

3'

HN 2'

5'

N

O

. #

Position

1 2 3 4

None 50 40 30

N

Average ErbB-2/ EGFR IC50 mM

Average tumor HN5/ BT474/N87 IC50 mM

0.074 0.027 0.023 0.068

2.98 0.60 2.68 3.81

analogue 2 was four to five times more potent against the tumor cell lines than either the 30 or 40 regioisomer (3, 4). Further analogues synthesized in the series, therefore, retained the 5-substituted-2-furanyl quinazolines as the core scaffold. Well over 1000 compounds were generated with the 4-benzyloxyaniline group or a substituted analogue thereof on a quinazoline core. It was determined that the 3-anilino (X) and 3-benzyloxy (Y) positions were optimal for dual EGFR and ErbB-2 inhibition as designated in the structure for Table 19.3. Compounds with substitutions in the 2- and 6-position of the aniline ring had diminished erbB family enzyme inhibition, thus the work focused only on combining 3- and 4-position substituents. The average standard deviation of the mean for the enzyme results described in Tables 19.2–19.8 is equal to 0.2 units of the log of the IC50 value (EGFR and ErbB-2), which is important when interpreting differences in enzyme potencies. Using a fixed 5-methylsulfonylethylamino-2-furanyl group as the 6-position side chain, many combinations of substituents were explored, and some representative examples are listed in Table 19.3. Chlorine and bromine at position X afforded insignificant increases in enzyme potency but appeared to offer improvements in cellular efficacy (9, 10 versus 5). Larger groups in position X (8) generally diminished activity. In combination with suitable X substituents, fluorine at position Y yielded compounds with greater potency in the cellular assay panel. Larger groups at Y also reduced activities. There was no ligand-bound crystal structure available at the time these analogues were being generated, so work was guided by the SAR in enzyme and cellular assays. The exploration of the terminal substitution on the 6-position side chain was performed to determine if changes would significantly affect the enzyme and tumor cell inhibition potency. Enzyme inhibition IC50 values for most of the analogues were in the range of 10–35 nM for both EGFR and ErbB-2, as shown for representative examples in Table 19.4. The assumption was that the side chain was pointing toward

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Table 19.3 Type I receptor inhibition activity and cellular efficacy results for representative examples of the modified benzyloxyaniline analogues. O

O

S O HN HN

Y

X N

O N

. #

X

Y

ErbB-2 IC50 mM

ErbB-1 IC50 mM

HN5 IC50 mM

BT474 IC50 mM

N87 IC50 mM

1 2 3 4 5 6 7 8 9 10

Cl H H H H Cl Br MeO CF3 CF3

F H F Br CF3 H H F H F

0.010 0.026 0.031 0.10 0.36 0.022 0.025 0.09 0.20 0.22

0.012 0.027 0.024 0.043 0.091 0.018 0.025 0.10 0.16 0.08

0.12 0.65 0.84 2.56 6.9 0.25 0.26 3.33 5.90 1.94

0.08 0.28 0.77 2.13 4.2 0.25 0.27 2.07 10.14 4.49

0.08 0.87 0.86 3.46 5.2 0.28 0.37 1.90 6.50 2.89

Table 19.4 Type I receptor inhibition activity and cellular efficacy results for representative examples of terminal side-chain modifications. R O S O

O HN

HN

F

Cl N

O N

. #

Lapatinib 14 15 16 17 18

R

ErbB-2 IC50 mM

EGFR IC50 mM

HN5 IC50 mM

BT474 IC50 mM

N87 IC50 mM

-CH3 n-Pr i-Pr -Ph 2-pyridyl

0.010 0.026 0.024 0.030 0.030 0.026

0.022 0.021 0.018 0.019 0.019 0.017

0.12 1.6 0.28 1.07 1.55 0.50

0.08 3.14 0.20 1.04 0.97 0.39

0.08 8.66 0.13 0.87 0.79 0.23

N N CH3

19.4 Final Set of Analogues that Led to the Discovery of Lapatinib Table 19.5 Type I receptor inhibition activity and cellular efficacy results for representative examples of side-chain modifications. R O S O

O HN

Y

Cl

Z

N

O N

. #

R

Y

Z

19 20 21 22 23 24

Me Ph Ph Me Me Me

F F H F F F

-O-O-NCH2CH2-N-(n-Pr) -N-(CH2CN) -N-(Bn)

ErbB-2 IC50 mM

ErbB-1 IC50 mM

0.017 0.028 0.038 0.02 0.022 0.141

0.026 0.025 0.050 0.085 0.027 0.090

BT474 IC50 mM 0.17 NT NT 0.25 0.067 0.61

NT, not tested.

and extending into the protein/solvent interface. While little or no change in potency was observed for the enzyme assay, the inhibition of tumor cell proliferation significantly varied (15, 18 versus 14, 16, 17). No substituent proved to be superior to the simple methyl sulfone. Linker SAR for the 6-position side chain showed that modifications were well tolerated in the kinase enzyme inhibition assays. It is interesting to note that there is virtually no difference in the enzyme potency of a small linkage group, oxygen, and larger groups of n-propylamino or benzylamine as shown in Table 19.5. The ether 19 and N-CH2CN 23 analogues were relatively potent in the cellular assays. To keep the SAR discussion straightforward, only BT474 cellular assay data are shown in Table 19.5. However, the values in the HN5 and N87 cell lines were comparable. No advantage was observed in relative potency in the in vitro assay systems of the tertiary amines (e.g., 21–24) compared to the secondary amino linker (e.g., 1, 12), except possibly improved selectivity for tumor over normal cells (see discussion below). The extra synthetic steps and increased molecular weight were deemed nonbeneficial for these alternate side chains. A cyclic version of the side chain was investigated for potential value since the disubstituted amine was tolerated in the terminal substituent investigation. Some examples of those derivatives are shown in Table 19.6. Similar trends in activity were observed in the enzyme and cellular assays for the substitution of the benzyloxy aniline moiety in this series, but some plasma stability issues were also identified with this side chain. Table 19.7 contains representative examples from each of the groups of modifications to the 6-furanyl-quinazoline template discussed thus far. The average of the

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Table 19.6 Type I receptor inhibition activity and cellular efficacy results for representative examples of thiomorpholine side chain with benzyloxyaniline changes. O O S

HN N

Y

X N

O N

. #

X, Y

ErbB-2 IC50 mM

EGFR IC50 mM

HN5 IC50 mM

BT474 IC50 mM

N87 IC50 mM

25 26 27 28

CF3, F Cl, F F, H Cl, H

0.141 0.018 0.035 0.023

0.089 0.028 0.016 0.012

1.11 0.06 1.0 0.32

3.00 0.10 1.20 0.42

2.09 0.08 1.13 0.46

tumor cell IC50 values was used to assess the dual EGFR and erbB-2 inhibitory properties in assays that are run under similar conditions. The therapeutic potential of this series was considered very high because greater than 100-fold selectivity was observed for all these analogues. The 6-furanyl-4-(4-benzyloxyanilino)-quinazoline scaffold afforded the necessary drug-like properties and dual ErbB-2 and EGFR tyrosine kinase inhibition to discover a potential anticancer therapeutic agent. The halogen substitution on the benzyloxyanilino group was key to improving the enzyme/cell ratio of activity, with 4-(3-fluorobenzyloxy)-3-chloroanilino providing the most promising cellular efficacy. The 2,5-orientation of the substitutions on the furanyl ring was important to achieve the desired cellular activity, while there was no apparent difference in the enzyme activity. Quite a diverse amine substitutions were tolerated, presumably due to the binding mode of these inhibitors where the aniline is tucked into the back of the ATP binding pocket, and the side chain on the furanyl portion extends out toward solvent. Table 19.7 Cellular activity and selectivity for representative substituted 6-furanyl-quinazolines.

# Lapatinib 15 19 23 26

Average tumora IC50 mM

HFF IC50 mM

Selectivity N/T

0.09 0.20 0.16 0.11 0.08

9.9 >30 >30 >30 8.18

111 >150 >188 >273 102

a) Average IC50 values for HN5, N87, and BT474 combined.

19.4 Final Set of Analogues that Led to the Discovery of Lapatinib

19.4.2 6-Thiazolylquinazoline Series

While the tolerance for substitution in the 6-position of the quinazoline was generally large for potent enzyme inhibition, the SAR from the cellular activity suggested that it was best to retain the 2-(methylsulfonyl)ethyl amino group side-chain substitution linked via a methylene unit to a heterocyclic ring. The 6-thiazole ring was investigated as a linking heterocycle by combining the “best” anilines for dual inhibition with the optimized side chain linked via a thiazole ring. The results are listed in Table 19.8. To simplify the SAR discussion, the average IC50 value is used for the cellular efficacy in the three tumor lines: HN5, BT474, and N87. Table 19.8 Catalytic enzyme assay results of 6-thiazolylquinazoline aniline derivatives.

O

H N

S O

HN

S

N

N N

. Entry

R

EGFR HFF ErbB2 IC50, mM IC50, mM IC50, mM

Aniline group

29

Average N/T tumora selectivity IC50, mM

0.048

0.082

>18

1.3

>14

0.028

0.071

12

0.09

138

0.55

0.54

>30

0.014

0.008

>30

0.29

>103

0.014

0.010

>23

0.11

>211

O HN

30

N N HN

31

13

>2.3

F

O

F

F HN

GW2764

Cl O HN

GW3340

Cl O

F

HN

a)

Average IC50 values for HN5, N87, and BT474 combined.

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A good correlation is observed between the catalytic enzyme activity profile and the cellularefficacyofthesederivatives. Forexample,30 isapotentdualEGFRand erbB-2TK inhibitor and shows a good average tumor IC50 value compared to 31 that is significantly less effective in both assay systems. The best overall cellular activity is observed with compounds 30 (N-1-benzylindazole) and GW3340 (4-[3-fluorobenzyloxy]-3-chloroaniline), and the most promising 6-thiazolylquinazoline compounds (30, GW2764, and GW3340) exceed 100-fold selectivity for tumor cells over normal cells. Compounds were progressed for further study provided the IC50 values for cellular efficacy were below 300 nM and the selectivity ratio exceeded 20-fold for tumor versus normal cells. Pharmacokinetic parameters were generated in mice by treating them with a single i.v. or oral dose of compound at 10 mg/kg. The data are compiled in Table 19.9 and demonstrate a range of plasma exposures for these compounds with oral bioavailability (%F) ranging from 11 to 40%. The compounds were also administered orally at 100 mg/kg b.i.d. for 21 days in the subcutaneous xenograft studies for both BT474 (erbB-2 þþþ ) and NH5 (EGFR þþþ ) human tumor cell lines. The tumor inhibition (%TI), which was recorded for the final day of the study, is listed in Table 19.9 and shown in a graph in Figure 19.6 [18]. The control line represents a vehicle-treated group of animals and approximately two tumor size doublings occur during the treatment period. The data illustrate that compound GW3340 was the most efficacious compound in this 6-thiazoylquinazoline series, displaying approximately 80% tumor inhibition in both xenograft models. A dose-related response was seen for 0, 30, 100 mg/kg b.i.d. oral doses of compound GW3340 and the lack of significant body weight loss suggested a good therapeutic index. 19.4.3 Alkynylpyrimidine Series

An example of analogue work where the quinazoline scaffold was replaced by a pyrimidine while retaining the key “large” aniline afforded the parent 5-phenylakynyl-4-anilinopyrimidine 33 with modest dual inhibition of EGFR and ErbB-2 (Table 19.10) [19]. The analogue without the 4-substituent (32) was considerably less active in the erbB-2 enzyme assay. The 3-acetamidophenyl alkynyl 34 had potent kinase inhibition and was among the most potent dual inhibitors in the series. The 3-acetamide group appeared to occupy the same region of the ATP binding pocket as the amine in the preferred side chain of the quinazoline series (methylsulfonylethylamine). A heteroatom in this position was important for

Table 19.9 Representative murine in vivo data.

Compound#

IV AUC

PO AUC

F (%)

% TIa HN5

% TIa BT474

30 GW2764 GW3340

2489 14 722 19 040

274 2439 7520

11 16.6 39.5

65 84 81

0 59 83

a) TI ¼ tumar growth inhibition.

19.4 Final Set of Analogues that Led to the Discovery of Lapatinib

Inhibition of BT474 xenograft growth by GW3340

700

1600

600

1400

500

1200

Tumor Weight (mg)

Tumor Weight (mg)

Inhibition of HN% xenograft growth using GW3340

400 300 200 100

1000 800 600 400

0

200 0

Control

4

8

12 Days

16

20

24

0 0

(6) @ 30 mg/kg

(6) @ 100 mg/kg

Control

4

8

12 Days

(6) @ 30 mg/kg

16

Table 19.10 Examples of SAR for alkynylpyrimidines.

F O HN R

Compound

Cl N

N

. HFF EGFR IC50 ErbB-2 IC50 Average tumora (mM) (mM) IC50 (mM) IC50 (mM)

R

H Ph O

— — 17.6

— — >30

0.32 0.079 0.012

21.4 0.78 0.013

0.031

0.029

3.86

22.4

0.033

0.047

1.48

15.8

0.040

0.013

5.34

>30

0.015

0.009

0.80

>30

N

35

O

O

N

O

36 O

OH

37 N

38 N

OH

H N

H N O

a)

O S O

average IC50 values for HN5 and BT474 combined.

20

24

(6) @ 100 mg/kg

Figure 19.6 Inhibition tumor growth activity in the xenograft model.

32 33 34

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successful dual kinase inhibition in these compounds, an interesting finding since, in the quinazoline series, most of the dual activity was derived from the aniline substitution. Many analogues were generated that had diverse, elongated side chains. The potency of 35 with the extended substitution of the amide connection is an example of tolerance in the binding site. In addition to phenyl-substituted alkynes, many heterocyclic analogues were evaluated. A few illustrative examples are included in Table 19.10. The hydroxymethyl furan 36 and the hydroxymethylpyridine 37 retained dual enzyme activity. Heterocycles with a urea methylene linker, as in sulfone analogue 38, were quite potent with IC50 values below 0.015 mM in the EGFR and ErbB-2 enzyme assays. Compounds 34–38 were more than 100-fold selective against a panel of non-EGFR family kinases, including CDK2, GSK3, SRC, and VEGFR2. These compounds were also tested in cellular proliferation assays and the data are included in Table 19.10. The IC50 values were averaged for just the tumor cell lines BT474 (erbB-2 driven) and HN5 (EGFR driven), and the HFF line was used as the control comparison. Despite being potent enzyme inhibitors, the compounds did not exhibit acceptable cellular potency. The attributes of the quinazoline series SAR were incorporated and much of the design was based on the then available ligand-bound crystal structure. However, after many analogues were generated in the arylalkynylpyrimidine series, the conclusion was that the unique qualities of the entire lapatinib molecule could not be attained in this chemical series.

19.5 Final Selection Criteria and Data

Within the series of 6-substituted-4 anilino-quinazolines, a lack of correlation was observed between the DMPK in vitro assays and the pharmacokinetic in vivo profiles; that is, there was no straightforward way to predict which analogues had the most likely chance of being orally available. Compounds were screened in several in vitro developability assays: solubility in multiple solvents (neutral aqueous, simulated gastric fluid, and simulated intestinal fluid), Caco-2 permeability, p450 enzyme assays, and microsomal metabolism studies. It was confirmed that there was no chemical class effect in the p450 enzyme assays. A facile in vivo assessment for PK was developed using a truncated protocol of four time points and two animals per time point to predict the full pharmacokinetic profile for oral dosing. A benchmark compound was established based on demonstration of reproducible oral bioavailability of 60% in mice. All the best compounds were compared for oral bioavailability and the calculated values for the area under the curve (AUC) relative to the benchmark were used to rank the most promising analogues. Some substitution patterns for improved oral bioavailability emerged from the data, which suggested that furan and thiazole were superior 6-position substitutions as compared with linker substituents including pyridine, numerous other heterocycles, ethers, and amines. Because GW2974 failed due to toxicity, the kinase selectivity and toxicity assessment were key to the final selection of the drug candidate. Six highly functionalized

19.5 Final Selection Criteria and Data

O H N O O

S

H N

N

N

S

O

N

O

S

O

N

O N

S

N

N

GW4049

O

O O

Cl

HN

H N

N GW2016

O

O

F

Cl

HN O

S

O

N GW2764

O

O

N

N

N

O

S

N GW4783

F

HN

H N

Cl

HN

S

GW3340

H N

O

O

F

Cl

HN

j483

N N

S

HN

N

N

O N

GW8295

Figure 19.7 Top six derivatives evaluated for candidate selection.

quinazolines and pyrido-pyrimidine analogues, shown in Figure 19.7, were progressed in parallel into early toxicity studies. Despite the structural similarity, 22 distinguishing criteria were used to make our decision to progress the best dual erbB2/EGFR TK inhibitor to development and clinical trials. The selection criteria were efficacy parameters (cellular and in vivo), biometabolism parameters (time of drug exposure over IC50 or IC90 levels, percent oral bioavailability, p450 enzymes), toxicity measurements (cellular, cardiovascular, 7-day rat studies, Ames test), and chemical issues (cost of goods, scalability) [20]. Comparing GW2016 with GW3340, where the structural difference is the fivemembered heteroaryl linking ring, furan versus thiazole, the oral bioavailability of GW3340 was approximately 40% in rats, approximately 40% in mice, and approximately 6% in dog, while GW2016 was best in the dog (85%), then in the mouse (40%), and worst in the rat (18%). The fluorine substitution seemed to have an effect on cellular activity and exposure parameters. Comparing GW2016 with GW4783, and GW2764 with GW3340, the oral bioavailability was approximately twofold higher in the mouse, and the cellular potency IC50 tumor cell average was consistently below 0.15 mM for the 3-fluorine substituted analogue, and the IC50 value was consistently above 0.25 mM for the nonfluorinated analogues. The halogenated benzyloxyaniline derivatives were superior to the benzylindazole derivatives in the duration of drug exposure above the average cellular IC50 and IC90 values. Blood samples were collected at the end of 21-day efficacy studies from all the animals receiving 100 mg/kg b.i.d. of compound and were analyzed for the following clinical chemistry parameters: hemolysis, albumin, alkaline phosphatase, serum glutamic-oxaloacetic transaminase, blood urea nitrogen, cholesterol, total protein, glucose, sodium, potassium, and chloride. While the general appearance and lack of body weight loss demonstrated that these top six compounds were well tolerated, varied results were obtained in their clinical chemistry analyses. A summary of the data for lapatinib evaluated in a range of efficacy assays is provided in Table 19.11. Lapatinib has IC50 values against erbB-2 and EGFR receptor tyrosine kinases of 9 and 11 nM, respectively, with greater than an order of magnitude loss in activity for the other family member, erbB-4. A small molecule–kinase

F

Summary of lapatinib preclinical efficacy data [20].

HN5, 30 mg/kg, b.i.d. HN5, 100 mg/kg, b.i.d. BT474, 30 mg/kg, b.i.d. BT474, 100 mg/kg, b.i.d.

HN5 BT474 In vivo human xenograft antitumor activity

BT474 (EGFR þ , erbB-2 þ þ þ ) N87 (EGFR þ þ þ , erbB-2 þ ) CaLu-3 (erbB-2 þ þ þ ) Cellular downstream phosphosignal inhibition pAKT and pErk levels (HN5) pAKT and pErk levels (HN5) In vivo xenograft downstream phosphosignal inhibition pAKT and pErk levels (HN5) pAKT and pErk levels (HN5) Cell outgrowth

Catalytic enzyme erbB-2 EGFR Cell growth inhibition HFF (EGFR þ ) HN5 (EGFR þ þ þ , erbB-2 þ )

Assay

Table 19.11

34% (32) 101% (40) 42% (32) 94% (40)

30 mM 1.1 mM

30 mg/kg 30 mg/kg

>90% at 5 mM >90% at 1 mM

100 nM 90 nM 130 nM

12 000 nM 120 nM

9.2 nM 10.8 nM

Efficacy

Values are growth inhibition percent relative to control animals receiving vehicle alone. Number of replicates in average is given in parentheses.

Cells were treated with lapatinib for 3 days and after removal of compound, the concentration where no outgrowth occurred is listed

Same tumor implant biopsied pre- and post-treatment

Inhibition also occurred in the presence of EGF

Expression levels are noted in parentheses on a relative scale. No detectable levels were found where the receptor is omitted

>10 000 nM for c-raf1, MEK, ERK, c-Fms, CDK1, CDK2, p38, IC50 values

IC50 values

Comment

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19.5 Final Selection Criteria and Data

Figure 19.8 A view through the N-terminal lobe of EGFR for the bound conformation of lapatinib as determined by X-ray crystallography. Image created with Maestro from Schrodinger, LLC.

interaction map was created for lapatinib (GW2016) by workers at Ambit using an ATP site-dependent competition binding assay in a panel of 119 kinases and demonstrated a very clean profile as well [21]. The ligand-bound EGFR TK crystal structure was solved quite late in the project and made an impact on understanding why these compounds were so effective (discussions below). In Figure 19.8, lapatinib is shown bound in the ATP pocket of EGFR. The costructure demonstrates that the benzyloxy aniline moiety binds in a well-defined lipophilic pocket, the quinazoline makes a hydrogen bond at the hinge region, and the methylsulfonylethylamino group binds at the solvent interface. The costructure of lapatinib bound to EGFR revealed an inactive-like conformation in contrast to the published active-like structure with Tarceva [22, 23], characterized by several differences. The shape of the ATP site was considered closed in the Tarceva/EGFR interactions versus an open conformation for lapatinib/EGFR. The typical hydrogenbonding pattern with the quinazoline scaffold involves with Thr766 that was also identified as the critical mutation leading to reduced sensitivity in the clinic setting [24]. Lapatinib does not have this interaction but rather a water-mediated interaction with Thr830. The position of the C helix can form an intact Glu738-Lys721 salt bridge or a large back pocket as is seen in the lapatinib crystal structure. The conformation of the COOH-terminal tail was also different, in one case partially blocking the ATP cleft as compared with being poorly defined in the other. The conformation of the activation loop demonstrates an A-loop similar to those found in inactive structures versus those found in active structures. More recently, Qui and colleagues reported ligand-bound protein crystal structures for erbB-4 in both an active form and an inactive form [25]. They demonstrated that lapatinib binds to an erbB-4 inactive form with the same

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contact residues as for EGFR, making it seem as though this type of small-molecule “slow off-rate” kinase inhibitor can be designed to interact specifically with an inactive form of these receptor proteins. A panel of tumor cell lines incorporated varying levels of type I receptor expression, and lapatinib was found equally effective in lines overexpressing either or both EGFR and erbB-2. The average selectivity for the tumor cell lines tested over normal lines was 100-fold. Cell proliferation was examined after 3 days of compound treatment followed by removal of the compound and media, followed by the addition of fresh media and continued observation of cell growth (called an outgrowth assay) to measure the cytotoxicity as compared with cytostasis. Concentrations were reached where no outgrowth was observed, and lapatinib appeared more effective in the erbB-2 overexpressing lines. Detailed enzyme kinetics coupled with crystal structure studies provided insight into why lapatinib was more effective in signal inhibition than other potent dual inhibitors [22]. The differences in the off-rates observed for lapatinib and Tarceva suggested that the two compounds were binding to EGFR in different ways, as summarized in Table 19.12. The inhibitor off-rates were evaluated using an EGFR enzyme reactivation procedure and Tarceva was found to have a rapid off-rate (halflife <10 min) whereas lapatinib had a much slower off-rate (half-life ¼ 300 min). A similar dissociation rate was observed with lapatinib using the erbB-2 enzyme reactivation procedure with no evidence for the irreversible covalent interaction that is observed with CI-1033. When HN5 tumor cells were treated with Tarceva and lapatinib, >85% reduction in tyrosine phosphorylation was observed without any reduction in protein levels. HN5 tumor cells were treated for 4 h with an inhibitor, and the receptor phosphorylation was analyzed at multiple time points after inhibitor washout to determine if the inhibitor binding kinetics affected the receptor tyrosine phosphorylation. The slow off-rate found for lapatinib in the purified intracellular domain enzyme reaction correlated with the observed, prolonged signal inhibition in tumor cells. At doses of 30 and 100 mg/kg b.i.d., lapatinib demonstrated tumor growth inhibition of 34 and 101%, respectively, in the HN5 xenograft model with regression (defined as >25% reduction in tumor volume) in 33% of the treated animals [8]. In the BT474 model, inhibition of 42 and 94%, respectively, was observed and regressions occurred in 10% of the treated animals. It was important to relate the antitumor activity to the inhibition of receptor phosphorylation. The level of erbB-2

Table 19.12 Data summarizing binding kinetics of a selective EGFR TK inhibitor, Tarceva, compared

with the dual EGFR/erbB-2 TK inhibitor, lapatinib [19]. Compound

erbB-2 Ki (nM)

EGFR Ki (nM)

Dissociation rates

Lapatinib Tarceva

13  1 870  90

3.0  0.2 0.4  0.1

T1/2 ¼ 300 min T1/2 < 10 min

EGFR activity postcompound washout 15% at 72 h 100% at 24 h

Tumor Weight (mg)

19.6 Early Clinical Results 1500

> 90% Inhibition of ErbB-2 p-Tyr

1000

500

0

5

10

15

20

Days of Therapy

Control

Treated 100 mg/kg

Average Control Average GW572016 30 mg/kg Average GW572016 100 mg/kg

Figure 19.9 The BT474 model of efficacy correlated with mechanism of action for lapatinib.

phosphotyrosine in the tumor excised after therapy in the 100 mg/kg treatment was reduced by 93% in the BT474 model and 85% in the HN5 model. The general appearance of the study animals, lack of body weight loss, and their normal clinical chemistry suggested that lapatinib was not toxic at this dose. Figure 19.9 shows the graph of antitumor activity in BT474 xenograft model at two doses, with the phosphotyrosine Western blots demonstrating the signal inhibition. The activity and safety profiles observed in the BT474 model suggested that lapatinib could also be safely combined with standard chemotherapy, potentially offering improved clinical treatment. Effects of lapatinib on proliferation, survival, and downstream proteins in signaling cascades reveal more details on the mechanism of action and potential biomarkers for determining the effective dose and patient population. Lapatinib inhibits the activity of both erbB-2 and EGFR, and interrupts downstream activation of Erk1/2 MAP kinases and AKT. Normal erbB-2 signaling activates the PI3K/AKTpathway that is involved in regulating cell survival, and constitutive activation of AKT in tumors has been found in chemotherapy resistance [26]. The inhibition of AKT by lapatinib was associated with a 23-fold increase in apoptosis compared to vehicle controls, consistent with the findings of the outgrowth study.

19.6 Early Clinical Results

Translational research studies comparing results in preclinical models with phase I clinical trial data demonstrated encouraging results in combination therapy of lapatinib and topotecan, a topoisomerase I inhibitor [27]. The authors use the rationale that survival signaling and transporter mechanisms disrupt the effectiveness of topotecan.

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They found that the combination enhances topotecan accumulation and toxicity both in relevant tissue culture and in xenograft mouse models in a mechanistically consistent manner. The phase I data suggested that the combination of lapatinib (1250 mg/day for 28 days) and topotecan (3.2 mg/m2 i.v. on days 1, 8, and 15) is safe and tolerable. It appears that the reduction of topotecan below the accepted MTD of 4.0 mg/m2 when dosed alone was due to the observation that lapatinib reduced the clearance of topotecan through transporter mechanisms. Stable disease was observed in 46% of the patients, and the authors report that the results warranted a phase II trial of topotecan and lapatinib combination in ovarian cancer. Another important comparison has been made by Ulhoa-Cintra, Greenberg, and Geyer in their review of the emerging role of small molecule-based therapies after the success of trastuzumab, a revolutionary antibody drug that targets the extracellular domain of erbB-2 [28]. Even with trastuzumab’s successes, resistance occurs in a large majority of advanced breast cancers, making complementary mechanisms (e.g., the intracellular kinase domain) an important component of potential cancer therapies. A dose range of 500–1600 mg/day of lapatinib was evaluated in patients with heavily pretreated metastatic solid tumors, where partial responses were observed in patients with trastuzumab-resistant metastatic breast cancer. Data summarized by Medina and Goodin provided a good overall picture of the in vivo effects of lapatinib [29]. The standard, FDA-approved dose for lapatinib is 1250 mg/day that affords a steady-state Cmax of 2.4 mg/ml and an AUC of 36.2 mg h/ml, and the exposure appeared to nearly double when administered with food. Lapatinib seemed to have similar pharmacokinetics in pediatric patients, although a much smaller patient number was evaluated. Lapatinib interacts with CYP3A4, and coadministration with ketoconazole increased the levels of lapatinib by over threefold. A small sampling of the early clinical trial data is provided in Table 19.13. The phase III clinical trial evaluating the efficacy of the combination of lapatinib þ capecitabine in advanced breast cancer patients who failed several prior regimens of cancer therapy demonstrated a significant advantage over capecitabine-alone therapy by the interim analysis of the clinical plans. Specifically, the time to progression (TTP) Table 19.13 Examples of lapatinib clinical trial data [27–29].

Treatment

Phase

Patients included

Lapatinib þ capecitabine

Phase I

45 advanced solid tumors

Lapatinib þ trastuzumab

Phase I

48 HER2 þ breast cancer

Lapatinib

Phase II

138 HER2 þ breast cancer

CR, complete response; PR, partial response; SD, stable disease.

Summary of responses 1 CR 4 PR 6 SD 1 CR 5 PR 10 SD 0 CR 33 PR 71 SD

19.7 Prospects for Kinase Inhibitors

was 4.4 months for capecitabine alone and 8.4 months in the combination therapy suggesting a 51% improvement. The trial was halted and patients were offered the combination therapy. Further followup data from the trial showed an overall response rate of 24% for patients on lapatinib þ capecitabine and 14% for those treated with capecitabine alone. The dominant side effects were usually diarrhea and rash [28]. In a phase II study of lapatinib monotherapy in breast cancer patients who had brain metastases, >20% reduction in CNS tumor volume occurred in 46 out of 241 patients and >50% reduction occurred in 19 out of 241 patients. These results are encouraging for the potential of lapatinib in combination therapies for this unmet medical need. Tykerb (trade name for lapatinib) was first approved for use in breast cancer in March 2007 in the United States, and approvals followed in more than 20 countries over the next 2 years. Ongoing clinical trials in multiple cancers in multiple combinations seek to define the patients who will most benefit from a dual EGFR and erbB-2 tyrosine kinase inhibitor [30].

19.7 Prospects for Kinase Inhibitors

The present structure-based design methods for creating potent, selective kinase inhibitors are based on available crystal structures and sophisticated protein homology modeling paired with cheminformatic approaches. It is entirely feasible that the kinome of approximately 500 kinases could someday have ligands identified for every potential kinase drug target or combination of targets. Furthermore, the ligandbound protein crystal structures offer insights beyond binding pocket potency enhancements. The compound designs can determine ways to bind to and stabilize different protein conformations or to overcome and avoid a variety of resistance mechanisms. Screening technologies have also made enormous progress in understanding selectivity issues and ways to use SAR to design selective compounds or combinations with a desired kinase inhibition profile. Being able to screen over 150 kinases provides many lead-hopping opportunities as well as SAR to design in or out a particular activity. Measuring the substrate reversible phosphorylation in cellular systems and correlating it with the disease pathology can provide a means to do proofof-mechanism studies in preclinical and clinical settings. The kinase target class has been proven druggable, and it remains to be seen how many more effective kinase drugs can be generated for multiple diseases with an unmet medical need [31].

Acknowledgments

The scientists who contributed to the discovery of Tykerb are Karen Affleck, Patricia Allen, Krystal Alligood, Perry Brignola, Zongwei Cai, Malcolm Carter, Stuart Cockerill, Renae Crosby, Scott Dickerson, Stephen Frye, Micheal Gaul, Cassandra Gauthier,

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Tona Gilmer, Bob Griffin, Steve Guntrip, Yu Guo, Robert Harris, Nelson Johnson, Amanda Jowett, Barry Keith, Blaine Knight, Michael Luzzio, Robert Mook, Robert Mullin, Doris Murray, Martin Page, Kim Petrov, James Onori, Nelson Rhodes, David Rusnak, Robert Shaw, Lisa Shewchuk, Archie Sinhababu, Debby Smith, Kathryn Smith, Jeremy Stables, Colin Stubberfield, Neil Spector, Sarva Tadepalli, Peter Topley, Dana Vanderwall, Jim Veal, Ann Walker, Paul Wissel, Edgar Wood, Yue-Mei Zhang.

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29 Medina, P.J. and Goodin, S. (2008)

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Karen Lackey

Vice President, GlaxoSmithKline, Molecular Discovery Research, Discovery Medicinal Chemistry, 5 Moore Drive, Research Triangle Park, NC 27709, USA Karen Lackey is vice-president of Molecular Discovery Research Chemistry at GlaxoSmithKline, responsible for exploratory chemistry, compound collection enhancement, and new technology with research groups in the UK and United States. In her previous role as international medicinal chemistry director, her team advanced more than 55 early stage programs in multiple therapeutic areas. Karen has over 75 published articles, patents, and book chapters to her credit covering various aspects of drug discovery with an emphasis on oncology, infectious diseases, and cell signaling research.

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20 Dasatinib, a Kinase Inhibitor to Treat Chronic Myelogenous Leukemia Jagabandhu Das and Joel C. Barrish

20.1 Introduction

Over the last quarter century, the scientific understanding of protein kinases and their inhibitors has undergone a major evolution. The discovery of the Src kinase oncogene in 1978 initiated an explosion of cross-disciplinary research that helped elucidate and clarify signaling mechanisms in numerous cell types [1]. The corresponding search for inhibitors as tools to understand the role of protein kinase inhibitors in disease was made more challenging by the sheer number of structurally similar kinases as revealed in the past decade by the sequencing of the human genome [2]. Ultimately, validation of the key role of kinases in disease came from the identification, characterization, and regulatory approval of the first marketed kinase inhibitor imatinib (GleevecÒ ) for the treatment of chronic myelogenous leukemia (CML) [3]. CML is a myeloproliferative disorder characterized by hypoproliferation of stem cells, followed by their differentiation into peripheral white blood cells (WBC). This disorder is characterized by the presence of the Philadelphia chromosome (Ph þ ) that arises from the translocation of the abl kinase domain on chromosome 9 with a specific breakpoint cluster region on chromosome 22 resulting in a constitutively active kinase. Imatinib is a potent inhibitor of Bcr-Abl that is used as frontline therapy in all stages of Ph þ CML where it works to normalize peripheral WBC counts (hematological response) and reduces the Ph þ clone in bone marrow stem cells (cytogenetic response). Five-year response rates for patients on first line therapy with imatinib are high: 97% of patients experience a complete hematologic response, 89% a major cytogenetic response, and 82% a complete cytogenetic response [4]. However, in addition to some patients who display a primary resistance to imatinib, others acquire resistance during treatment. The percentage of patients who acquire resistance to imatinib varies depending on the disease state: 22, 32, and 41%, respectively, for chronic, accelerated, and blast-phase patients [5].

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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There are several mechanisms that appear to play a role in the acquisition of imatinib resistance: amplification of the Bcr-Abl gene, Bcr-Abl independence possibly related to reliance on other kinases (e.g. Src-family kinases), and drug efflux (e.g. P-gp) [6]. However, a significant percentage of imatinib resistance develops due to specific mutations in the Bcr-Abl kinase domain. These mutations prevent imatinib binding either by removing specific interactions with the enzyme or by destabilizing the requisite inactive conformation. The numbers of imatinib-resistant mutations identified have increased with time. While acquired resistance can be overcome in some cases by increasing the imatinib dose, it has become clear that there is a significant need for additional agents both for frontline therapy and to treat patients who develop imatinib resistance [7].

20.2 Discussion

Dasatinib (30) originated as a lead compound from an immunology drug discovery program directed toward the identification of a selective inhibitor of the lymphocyte specific kinase (Lck). Lck, a member of the Src family of nonreceptor tyrosine kinases, is expressed primarily in T-cells and natural killer cells, and is required for T-cell activation, development, and initiation of T-cell antigen receptor-mediated (TCR) signal transduction pathways [8]. An inhibitor of Lck was anticipated to have potential utility as an immunosuppressive agent, for example, in the treatment of T-cellmediated autoimmune and inflammatory diseases including rheumatoid arthritis, solid organ transplant, psoriasis, and multiple sclerosis [9]. In search of a novel structural lead, we screened our internal compound collection using a medium-throughput filter binding assay. Aminothiazole 1, identified as a weak ATP-competitive inhibitor of both murine (mLck) and human Lck (hLck) with little activity in cells, provided a unique template for optimization of both biochemical and cellular potencies. A systematic SAR optimization approach varying different fragments of the aminothiazole lead 1 was thus initiated [10]. SAR investigation of the carboxanilide side chain on the tert-butylcarbamate analogue 2 revealed some of the key structural requirements for optimal Lck inhibitory activity. Substitution on the phenyl ring was of paramount importance since the unsubstituted benzamide analogue 3 was significantly less potent. In contrast, the 2,6-dimethyl substituted analogue 4 retained most of the activity and substitution of one of the ortho-methyl groups with a small group (5) was allowed. Replacement of one or both of the ortho-methyl substituents with sterically demanding group(s) was less preferred. The para-position of the phenyl ring accommodated a wide variety of substituents (6), while substitution at the meta-position (7) was suboptimal for potency (Figure 20.1) [10]. We postulated that small substiuents at the ortho-positions are required to orient the anilide ring out of the plane formed by the anilide and thiazole groups so that it can fit into a narrow but deep hydrophobic pocket of the enzyme. This hydrophobic region of the protein otherwise known as the

20.2 Discussion

N H2N S

CH3 H N

CH3 H 3C H 3C

O H3C

CH3

CH3 H N

N

H N

O

S CH3 O

O H3C

1 mLCK IC50 = 6.6 µM hLCK IC50 = 5 µM T-cell IC50 >10 µM

H 3C H 3C

O

H N

N S

CH3 O

CH3

R

2, R = CH3, mLCK IC50 = 3 µM 6, R = Br, mLCK IC50 = 2.4 µM CH3 H N

R2

O R1

3, R1, R2 = H, mLCK IC50 > 50 µM 7, R1, R2 = CH3, mLCK IC50 = 27.4 µM

H 3C H 3C

O

H N

N S

CH3 O

CH3 H N

CH3

O R

4, R = CH3, mLCK IC50 = 7.5 µM 5, R = Cl, mLCK IC50 = 9.6 µM

Figure 20.1 Activities of thiazoles 1–7.

kinase specificity pocket is located close to the gatekeeper residue (Thr316 in Lck) and is not occupied by ATP [11]. A more dramatic enhancement of the Lck inhibitory potency was observed upon functionalization of the 2-amino substitution on the thiazole ring. In general, derivatization of the amino group led to improved Lck inhibitory potency. Methyl carbamate (8), acetamide (9), and methyl urea (10) were identified to be roughly 7–20fold more potent than the lead analogue 1. Aromatic and heteroaromatic carboxamide (12 and 13) modifications also led to significant increases in biochemical potency. Through iterative SAR modification, n-butyl urea analogue 11 was identified to be the most potent analogue with an IC50 value of 30 nM against human Lck. Attempts to further increase the biochemical potency in this series were not fruitful (Figure 20.2) [10]. SAR modification was now directed toward the central thiazole ring. Attempts to replace the thiazole core of 1 with either a five- or a six-membered heteroaromatic ring were in general unsuccessful for activity. We next turned to substitution at the 4position of the thiazole ring. In the tert-butylcarbamate series, replacement of the 4methyl substituent with either hydrogen (16), ethyl (17), trifluoromethyl (18), or phenyl (19) resulted in a significant loss of potency (hLck IC0 50s > 30 mM). At this stage, earlier SAR findings in a related series prompted us to reinvestigate modifications at C4. In particular, a cyclopropyl amide modification at the 2-position of a benzothiazole led to a highly potent Lck inhibitor 20 (IC50 ¼ 9 nM) [12]. To our pleasant surprise, the corresponding cyclopropyl amide off the 4-unsubstituted thiazole core led to analogue 21 that was approximately 40-fold more potent than the corresponding 4-methylthiazole analogue 14. This observation is at odds with our earlier findings in the tert-butyl carbamate series in which replacement of 4-Me by 4H (2 and 16, respectively) resulted in a dramatic loss in potency. Analogue 21 also

j495

j 20 Dasatinib, a Kinase Inhibitor to Treat Chronic Myelogenous Leukemia

496

CH3 H N

N

H N

R

S O

CH3

S O

O H3C

O

H N

CH3

O

CH3

Cl

8, R = OCH3, hLCK IC50 = 0.7 µM 9, R = CH3, hLCK IC50 = 0.48 µM 10, R = NHCH3, hLCK IC50 = 0.24 µM 11, R = NHn-Bu, hLCK IC50 = 0.03 µM

H3C H3C

CH3 H N

N

H N

R

12, R = 3-thienyl, hLCK IC50 = 0.39 µM 13, R = 2-thienyl, hLCK IC50 = 0.13 µM 14, R = c-propyl, hLCK IC50 = 1.4 µM 15, R = NHn-Bu, hLCK IC50 = 0.62 µM

N 2

CH3 O

S

R 4

H N

CH3

O H3C

CH3

16, R = H, hLCK IC50 > 30 µM 17, R = Et, hLCK IC50 > 30 µM 18, R = CF3, hLCK IC50 > 30 µM 19, R = Ph, hLCK IC50 > 30 µM Figure 20.2 Activities of thiazoles 8–19.

potently inhibited anti-CD3 and anti-CD28 stimulated proliferation in a T-cell proliferation assay (IC50 ¼ 880 nM). Further SAR optimization on the 4-unsubstituted thiazole analogue 21 proved elusive. Minor structural changes led to dramatic drops in activity and as such a very narrow SAR pattern with no further improvement in activity was observed in this series (Figure 20.3) [10]. Parallel SAR studies with benzothiazoles demonstrated that C2-carboxamide of 20 could be replaced with a heteroaromatic amine with a profound increase in biochemical potency [13]. Docking of 20 to a model of the Lck kinase active site suggested that the cyclopropyl amide carbonyl group does not participate in any binding interaction with the enzyme and therefore can be replaced with a heteroaromatic amine that can function as a constrained “amide mimetic.” Such modifications in the benzothiazole series led to a profound increase in biochemical potency. Further enhancement in both biochemical and cellular potency was achieved by the H N

N 2

H N

S

O

CH3

N

4

H N

S O

O 20

H N

O

Cl

hLCK IC 50 = 9 nM T-cell IC 50 = 4.7 µM Figure 20.3 Activities of benzothiazole 20 and thiazole 21.

CH3

Cl 21 hLCK IC 50 = 35 nM T-cell IC 50 = 880 nM

20.2 Discussion H N

H3 C

N H N

S N

O

H N

H 3C

N H N

S

N NH

CH3 N

O 22

CH3

hLCK IC50 = 1 nM hLCK Ki = 540 pM T-cell IC50 = 262 nM

CH3

O

Cl 23

Cl

hLCK IC50 = 4 nM T-cell IC50 = 140 nM N

H N

H3 C

H N

S N

N CH3

CH3

O 24

j497

Cl

hLCK IC50 = 1 nM hLCK Ki = 130 pM T-cell IC50 = 80 nM

Figure 20.4 Activities of benzothiazole 22 and thiazoles 23–24.

introduction of a polar substituent onto the heteroaryl ring. Compound 22 was identified as a highly potent Lck inhibitor. Fortunately, similar structural modifications in the 4-unsubstituted thiazole series also provided a significant boost in potency. In general, the cyclopropyl amide moiety can be replaced with various five or six-membered heteroaryl amines. However, the optimal potency was achieved with the pyridyl and pyrimidinyl amine substitution (e.g., 23 and 24). On the basis of its exquisite potency, pyrimidinyl analogue 24 (hLck Ki ¼ 130 pM, T-cell proliferation IC50 ¼ 80 nM) was selected for further characterization (Figure 20.4) [14]. Thiazole 24 was evaluated for its selectivity against a panel of in-house kinases. In addition to Lck, 24 is a potent inhibitor of all Src family kinases (e.g., Src, Ki ¼ 96 pM) and Bcr-Abl kinase (IC50 < 1 nM). In rats, 24 was orally bioavailable (F% 65) with an acceptable plasma half-life (t1/2 ¼ 4 h), large volume of distribution (Vss ¼ 12 l/kg), and moderate clearance (CL ¼ 29 ml/min/kg). Oral efficacy of 24 was demonstrated in a chronic model of adjuvant arthritis in rats with established disease upon oral administration at doses of 0.3 and 3 mg/kg b.i.d. [14]. In addition to its potent Src family kinase and Bcr-Abl kinase activities, thiazole 24 possessed significant antiproliferative activities against the PC3 human prostate tumor (IC50 ¼ 67 nM) and K562 human blast-phase CML cell lines (IC50 ¼ 1.6 nM) [15]. These results generated substantial interest in the potential of this class of inhibitors as anticancer agents, and thus a highly collaborative effort between the Immunology and Oncology departments at Bristol-Myers Squibb was initiated. As with the benzothiazoles, further improvements in both biochemical and cellular activities with the pyridine and pyrimidine-based thiazoles were achieved through appending polar functional substituents to the heteroaromatic ring. All these analogues were potent inhibitors of the Src family and Bcr-Abl kinases, with IC50 values in the nanomolar to subnanomolar range, and demonstrated robust antiproliferative activity in K562, a human blast-phase CML cell line. To further differentiate among these analogues, they were evaluated against a panel of solid

j 20 Dasatinib, a Kinase Inhibitor to Treat Chronic Myelogenous Leukemia

498

tumor cell lines, and screened in a 4 h mouse exposure assay upon administration of a single oral dose of 50 mg/kg. Selected analogues with acceptable pharmacokinetic profiles were then evaluated for in vivo efficacy in mice in a human PC3 prostate tumor xenograft model; a partial summary of these data is given in Table 20.1. In the pyridine series, introduction of a hydroxyethylamine side chain led to analogue 25 with potent activities in solid tumor cell assays. However, in the 4 h mouse coarse exposure study, 25 suffered from an apparent high clearance, as evidenced from a large difference between the peak (7 mM) and 4 h (304 nM) plasma exposures. In contrast, analogue 26 with a morpholinopropyl amine side chain had a high peak plasma concentration (8.6 mM) and a sustained level in excess of 1 mM during the 4 h of the assay. Despite its excellent potency and PK profile, analogue 26 failed to show tumor growth inhibition in the mouse PC3 tumor xenograft model. In analogy with the findings in the pyridine series, morpholino-pyrimidine analogues 27 and 28 maintained both high peak and sustained plasma levels, but compound 27 was also not efficacious in the PC3 tumor xenograft model. Only the piperazinopyrimidine analogue 30 (dasatinib) displayed the right balance of tumor cell potencies and acceptable plasma exposure upon oral dosing [15]. Thus, in vivo antitumor activity with dasatinib was demonstrated in a PC3 prostate tumor xenograft assay in mice. Upon oral administration twice daily for 2 weeks, dasatinib inhibited growth of the established tumor in a dose-dependent manner with 64 and 76% tumor growth inhibition at oral doses of 14 and 35 mg/kg, respectively. The selectivity profile of dasatinib was assessed in a kinase panel represented by an appropriate mix of kinase families. Dasatinib is an exquisitely potent inhibitor of the Src and Bcr-Abl kinase with Ki values of 16 and 30 pM, respectively. In addition, profound inhibition of other Src family kinases including Lck, Yes, and Fyn (IC50 ¼ 200–500 pM) was observed. Dasatinib also demonstrated significant activity against c-kit (IC50 ¼ 5 nM), ephrin (EPH) receptor A2, and PDGFRb (IC50 ¼ 28 nM). More than 1000-fold selectivity was observed over other receptor, and non-receptor tyrosine kinases and serine-threonine kinases [15]. Dasatinib can be synthesized from commercially available 2-chlorothiazole 31 in six steps with an overall 61% yield (Scheme 20.1). Lithiation of 2-chlorothiazole 31 with n-butyllithium in THF at
78  C followed by addition of 2-chloro-6-methylphenyl isocyanate formed carboxanilide 32 in 86% yield. Protection of the carboxamide as its p-methoxybenzyl (PMB) derivative 33 followed by its amination with 4amino-6-chloro-2-methylpyrimidine in refluxing THF in the presence of sodium hydride and subsequent deprotection of the PMB group afforded 34 in 78% overall yield in three steps. Reaction of 34 with 1-(2-hydroxyethyl)piperazine in dioxane under reflux provided 30 that was isolated as its hydrochloride salt after treatment with methanolic hydrogen chloride [14, 15]. The oral bioavailability and pharmacokinetics of dasatinib were investigated in several animal species. Dasatinib displayed an intermediate systemic plasma clearance (25–62 ml/min/kg), extensive tissue distribution (>3 l/kg), and high plasma binding (>90%) in mouse, rat, dog, and monkey. The oral bioavailability ranged from 14% in mouse to 34% in dog. The low oral bioavailability is attributed to its poor absorption and/or rapid first-pass metabolism [16].

CH

CH

N

N

N

N

25

26

27

28

29

30

NT: Not tested.

X

CH3

CH3

CH3

H

N H

N H N

OH

R1

O

N

N

N H

N H

H

H

N

N

R2

O

OH

OH

N

O

R2 X O Cl

H N

CH3

12

9

23

170

34

5

9

11

14

82

10

13

PC3 (prostate)

Antiproliferative activity, IC50 (nM)

.

S

N

MDA-MB-231 (breast)

R1

N

H N

Antiproliferative activities and 4 h plasma exposures of pyridinyl and pyrimidinyl analogues.

Compound

Table 20.1

4806

3468

11375

17088

13298

7195

AUC0–4 h (nM h)

2723

2502

2809

9612

8582

7000

Cmax (nM)

Coarse oral exposure (mouse)

700

180

3119

1919

1076

304

4 h conc. (nM)

Active

NT

NT

Inactive

Inactive

NT

In vivo PC3

20.2 Discussion

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j 20 Dasatinib, a Kinase Inhibitor to Treat Chronic Myelogenous Leukemia

500

CH3 N C O

N

n-BuLi, Cl

S

CH3

Cl

N

H N

NaH, p-methoxybenzyl chloride Cl

S

THF, –78 oC, 86% Cl

31

THF, 95%

O

32

OCH3

CH3

N N Cl

S

CH3

1. NaH, THF, ∆, 4-amino6-chloro-2-methyl-pyrimidine, 83%

CH3

S

Cl

O

2. TfOH, TFA-CH2Cl2 (1:1), 99%

Cl

N

N

H N

N H

N Cl

O

34 33 CH3

1. 1-(2-hydroxyethyl)piperazine 1,4-dioxane, DIEA, ∆, 12 h 2. 2 N HCl, Et 2O-MeOH, 0.5 h 91% (two-steps)

CH3

S Cl

N

N

H N

N H

N N N

O

OH

30 (Dasatinib)

Scheme 20.1 Synthesis of compound 30 (dasatinib).

The X-ray crystal structure of dasatinib (30) bound to Abl kinase highlighted some of the key H-bond interactions [17] (Figure 20.5). Dasatinib occupied the ATP binding site of the enzyme. The thiazole ring nitrogen and the amine hydrogen of 30 were engaged in a pair of H-bond interactions with Met318 located in the hinge region of the ATP binding site of Abl. In addition, the carboxamide NH of dasatinib formed a

Figure 20.5 Structure of dasatinib–Abl complex (left panel) and location of the imatinib-resistant mutations (right panel).

20.2 Discussion

H-bond with the hydroxyl group of Thr315. The 2-chloro-6-methyl-phenyl ring of dasatinib is orthogonal to the thiazole carboxamide and occupied the deep, angular but narrow hydrophobic pocket beyond the gatekeeper residue Thr315. The hydroxyethylpiperazine linker of dasatinib extends into the solvent-exposed surface of the protein and does not appear to be engaged in any H-bond interaction. In contrast to imatinib (35), dasatinib binds to the active conformation and numerous intermediate conformations of the protein. In the dasatinib-bound conformation of Abl, the activation loop is phosphorylated at Tyr393 and adopts an extended (DFG-in) conformation similar to the one found in the activated Lck structure. The phosphate binding region or the P-loop is only slightly disordered suggesting that dasatinib does not engage in any productive interaction with the P-loop amino acid residues. The most prominent structural differences in the bindings of imatinib (35) and dasatinib (30) lie at the DFG motif of the activation loop. The published crystal structure of imatinib–Abl complex [18] shows that the imatinib-bound form of Abl requires the activation loop, to fold back onto the P-loop, and the inhibitor, leading to an inactive (DFG-out) and inhibitory kinase conformation of the protein. Dasatinib (30) is reported to be significantly more potent than imatinib (35) in both biochemical and cellular assays [19]. The biochemical assay with dephosphorylated wild-type glutathione-S-tranferase-fused Abl kinase demonstrated more than 600fold increased potency for dasatinib compared to imatinib. A 325-fold increased potency was also observed in the cellular proliferation assay with Ba/F3 cells expressing wild-type Bcr-Abl. The cytotoxic activity of both dasatinib and imatinib correlated well with the inhibition of Bcr-Abl tyrosine phosphorylation in Ba/F3 cells expressing wild-type Bcr-Abl. In vivo efficacy of dasatinib was demonstrated in a K562 human CML xenograft model in severe combined immunodeficient (SCID) mice. In this model, a partial tumor regression was observed after administration of once-a-day dose of either 5 mg or 50 mg/kg orally on a 5 days on and 2 days off schedule. Complete disappearance of tumor mass was achieved at the end of two treatment cycles. No gross overt toxicity was observed at either dose level [15]. Since its discovery and approval, imatinib (35) has been first line of therapy for the treatment of CML in newly diagnosed patients. Despite its proven clinical efficacy, emergence of resistance to imatinib, primarily due to point mutations in Bcr-Abl kinase domain, has been a major concern. Taking into account the assumption that a dual Src-Abl kinase inhibitor such as dasatinib (30) may have less stringent structural requirements for binding to Abl, Shah et al. [20] evaluated its activity against some of the clinically most relevant Bcr-Abl mutants. Dasatinib was roughly 450- and 325-fold more potent, respectively, than imatinib in inhibiting wild-type Bcr-Abl activities in the biochemical and cellular proliferation assays. Dasatinib also inhibited kinase activities of 14 of the initial 15 identified imatinib-resistant isoforms at low nanomolar concentrations. In addition, dasatinib was shown to inhibit the growth of 14 cell lines at a similar concentration range. Only the Ba/F3 cell line expressing the T315I mutant was resistant to dasatinib treatment, even at high micromolar concentration. In addition to cellular potency, in vivo efficacy with dasatinib treatment was demonstrated in a mouse model of imatinib-resistant Bcr-Abl-driven disease.

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j 20 Dasatinib, a Kinase Inhibitor to Treat Chronic Myelogenous Leukemia

502

CH3

N

H N S Cl

N

O

N H

N H3C

N

N

H N

H N

N

CH3

N

CH3

O

N N

30 (Dasatinib) Bcr-Abl IC50 = 0.6 nM Bcr-Abl cell prolif. IC 50 = 0.8 nM

OH

N

35 (Imatinib) Bcr-Abl IC 50 = 280 nM Bcr-Abl cell prolif. IC 50 = 260 nM

Figure 20.6 Bcr-Abl activities of dasatinib (30) and imatinib (35).

Oral administration of dasatinib at a dose of 10 mg/kg, twice daily for 14 days, to SCID mice pretreated with Ba/F3 cells transfected with nonmutated Bcr-Abl or its M315T isoform significantly reduced the severity of the disease as measured by bioluminescence imaging and prolonged survival time. Consistent with in vitro data, mice harboring the T315I mutant failed to respond to dasatinib treatment. Analysis of the dasatinib-bound conformation of Abl kinase [17] (Figure 20.5) can offer some insight into its favorable activity against the clinically most relevant mutants. A majority of the imatinib-resistant Bcr-Abl mutants are located in the Ploop region (Figure 20.5, right panel) that is highly distorted in the imatinib-bound inactive conformation of Abl. P-loop interactions do not appear to be critical for the binding of dasatinib to Abl in its active conformation. Several other mutations at the catalytic domain, activation loop, and the carboxy terminal loop may play a critical role to stabilize a specific imatinib-bound conformation of Abl. Dasatinib makes a critical H-bond interaction with T315 at the gatekeeper region of the protein and therefore is exquisitely sensitive to T315I mutation, presumably due to the loss of a key hydrogen bond interaction and increased steric bulk that interferes with the van der Waals interaction between the 2-chloro-6-methyl phenyl ring and the side-chain methyl group of the amino acid residue.

20.3 Clinical Findings and Summary

Dasatinib was evaluated in a dose-escalating phase I study in patients in various stages of CML, as well as with Ph-positive ALL, who were intolerant or resistant to imatinib [21]. A complete hematologic response was observed in most chronic-phase CML patients (93%); the rate for major cytogentic responses was 45%. Major hematologic responses were seen in 70% of patients with accelerated phase CML, CML with blast crisis, or Ph-positive ALL; the major cytogenetic response rate for these patients was 25%. Phase II studies also showed a significant number of imatinib-resistant or intolerant patients achieving complete hematologic and cytogenetic responses in chronic phase [22] (90 and 52%, respectively) and accelerated phase [23] (39 and 24%, respectively) CML. In myeloid and lymphoid blast crisis, major hematologic responses were 34 and 31%, respectively, and major cytogenetic

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17

18

19

20

21

22

of dasatinib (BMS-354825): a potent oral multi-targeted kinase inhibitor Src and Bcr-Abl. Cancer Chemother. Pharmacol., 61, 365–376. Tokarski, J.S., Newitt, J.A., Chang, C.Y.J., Cheng, J.D., Wittekind, M., Kiefer, S.E., Kish, K., Lee, F.Y.F., Borzilleri, R., Lombardo, L.J., Xie, D., Zhang, Y., and Klei, H.E. (2006) The structure of dasatinib (BMS-354825) bound to activated Abl kinase domain elucidates its inhibitory activity against imatinib-resistant Abl mutants. Cancer Res., 66, 5790–5797. Nagar, B., Bornmann, W.G., Pellicena, P., Schindler, T., Veach, D.R., Miller, W.T., Clarkson, B., and Kuriyan, J. (2002) Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI571). Cancer Res., 62, 4236–4243. O’Hare, T., Walters, D.K., Stoffregen, E.P., Jia, T., Manley, P.W., Mestan, J., CowanJacob, S.W., Lee, F.Y., Heinrich, M.C., Deininger, M.W.N., and Druker, B.J. (2005) In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant imatinib-resistant Abl kinase domain mutants. Cancer Res., 65, 4500–4505. Shah, N.P., Tran, C., Lee, F.Y., Chen, P., Norris, D., and Sawyers, C.L. (2005) Overriding imatinib resistance with a novel Abl kinase inhibitor. Science, 305, 399–401. Moshe, T., Shah, N.P., Kantarjian, H., Donato, N., Nicoll, J., Paquette, R., Cortes, J., O’Brien, S., Nicaise, C., Bleickardt, E., Blackwood-Chirchir, M.A., Iyer, V., Chen, T.-T., Huang, F., Decillis, A.P., and Sawyers, C.L. (2006) Dasatinib in imatinib-resistant Philadelphia chromosome-positive leukemias. N. Engl. J. Med., 354, 2531–2541. Hochhaus, A., Kantarjian, H.M., Baccarani, M., Lipton, J.H., Apperley, J.F., Druker, B.J., Facon, T., Goldberg, S.L., Cervantes, F., Niederwieser, D., Silver, R.T., Stone, R.M., Hughes, T.P., Muller, M.C., Ezzeddine, R., Countouriotis, A.M., and Shah, N.P. (2007) Dasatinib induces notable hematologic and cytogenetic responses in chronic-phase chronic

23

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25

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myeloid leukemia after failure of imatinib therapy. Blood, 109, 2303–2309. Guilhot, F., Apperley, J., Kim, D.-W., Bullorsky, E.O., Baccarani, M., Roboz, G.J., Amadori, S., de Souza, C.A., Lipton, J.H., Hochhaus, A., Heim, D., Larson, R.A., Branford, S., Muller, M.C., Agarwal, P., Gollerkeri, A., and Talpaz, M. (2007) Dasatinib induces significant hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in accelerated phase. Blood, 109, 4143–4150. Cortes, J., Rousselot, P., Kim, D.-W., Ritchie, E., Hamerschlak, N., Coutre, S., Hochhaus, A., Guilhot, F., Saglio, G., Apperley, J., Ottmann, O., Shah, N., Erben, P., Branford, S., Agarwal, P., Gollerkeri, A., and Baccarani, M. (2007) Dasatinib induces complete hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in blast crisis. Blood, 109, 3207–3213. Kantarjian, H., Pasquini, R., Hamerschlak, N., Rousselot, P., Holowiecki, J., Jootar, S., Robak, T., Khoroshko, N., Masszi, T., Skotnicki, A., Hellmann, A., Zaritsky, A., Golenkov, A., Radich, J., Hughes, T., Countouriotis, A., and Shah, N. (2007) Dasatinib or highdose imatinib for chronic-phase chronic myeloid leukemia after failure of first-line imatinib: a randomized phase 2 trial. Blood, 109, 5143–5150. Soverini, S., Colarossi, S., Gnani, A., Castagnetti, F., Rosti, G., Bosi, C., Paolini, S., Rondoni, M., Piccaluga, P.P., Palandri, F., Giannoulia, P., Marzocchi, G., Luatti, S., Testoni, N., Iacobucci, I., Cilloni, D., Saglio, G., Baccarani, M., and Martinelli, G. (2007) Resistance to dasatinib in Philadelphia-positive leukemia patients and the presence or the selection of mutations at residues 315 and 317 in the BCR-ABL kinase domain. Haematologica, 92, 401–404. Finn, R.S., Dering, J., Ginther, C., Wilson, C.A., Glaspy, P., Tchekmedyian, N., and Slamon, D.J. (2007) Dasatinib, an orally active small molecule inhibitor of both the src and abl kinases, selectively inhibits growth of basal-type/“triple-negative”

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breast cancer cell lines growing in vitro. Breast Cancer Res. Treat., 105, 319–326. 28 Huang, F., Reeves, K., Han, X., Fairchild, C., Platero, S., Wong, T.W., Lee, F., Shaw,

P., and Clark, E. (2007) Identification of candidate molecular markers predicting sensitivity in solid tumors to dasatinib: rationale for patient selection. Cancer Res., 67, 2226–2238.

Joel C. Barrish

Vice President, Discovery Chemistry, Bristol-Myers Squibb Research and Development, Route 206 & Province Line Road, P. O. Box 4000, Princeton, NJ 08543-4000, USA Joel C. Barrish received his doctorate in 1982 in organic chemistry from Columbia University working with Professor W. Clark Still. After 5 years at Hoffmann-LaRoche, he joined Bristol-Myers Squibb in 1988 where he has carried out research in several therapeutic areas. He has led teams that have advanced multiple compounds into clinical development, including SPRYCELÒ of which he is a coinventor. He is VicePresident of Immunology and Metabolic Diseases Chemistry.

Jagabandhu Das

Research Fellow, Discovery Chemistry, Bristol-Myers Squibb Research and Development, Route 206 & Province Line Road, P. O. Box 4000, Princeton, NJ 08543-4000, USA Jagabandhu Das received his PhD in chemistry (1978) from the University of New Brunswick under the supervision of Professor Zdenek Valenta. After a postdoctoral tenure with Professor E.J. Corey at Harvard University, he joined the Squibb Institute for Medical Research in 1982. At present, he is a Research Fellow in the Department of Immunology, Discovery Chemistry, at the Bristol-Myers Squibb. His research interests include design and synthesis of biologically active molecules. He is a coinventor of dasatinib (SprycelÒ ), coauthor of 49 publications, and coinventor of 66 issued US patents. He is the recipient of the Bristol-Myers Squibb Ondetti and Cushman Award for Scientific Innovation (2007) and Thomas Alva Edison Patent Award (2008) from Research and Development Council of New Jersey.

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21 Venlafaxine and Desvenlafaxine, Selective Norepinephrine and Serotonin Reuptake Inhibitors to Treat Major Depressive Disorder Magid Abou-Gharbia and Wayne E. Childers Jr.

21.1 Introduction

The history of antidepressant pharmaceuticals dates back to the early 1950s when Irving Selikoff and Edward Robitzek [1] noticed that clinical trial patients treated with the experimental antituberculosis agent iproniazid (1, Figure 21.1) displayed gentle stimulation and renewed vigor. This observation was further extended by a number of researchers, most notably Nathan Kline who publicized iproniazid as a “psychic energizer” [2]. Data from Ernst Zeller’s group suggested that the mechanism of action for iproniazid was inhibition of monoamine oxidase [3, 4]. Following those discoveries, several monoamine oxidase inhibitors (MAOIs) were identified and used as antidepressant agents. However, over the years their use has been limited by the potential for severe cardiovascular side effects, the need for dietary restrictions and the risk of drug–drug interactions. At roughly the same time, Roland Kuhn discovered that an experimental tricyclic compound, imipramine (2, Figure 21.1), had a significant antidepressant effect [5]. Subsequent research identified several other tricyclic derivatives. Like the MAOIs, these “first-generation” antidepressant agents displayed efficacy in the clinic, but they also had undesirable side effects. Over the years, the biogenic amines have provided medicinal chemists with solid starting points for the design and discovery of innovative therapeutics for the treatment of a variety of mood and anxiety spectrum disorders (Figure 21.2). Evidence from numerous groups established that deficiencies in serotonin (5-HT) and norepinephrine (NE) contribute to the development of major depressive disorder (MDD), commonly referred to as “depression” [6, 7]. These observations prompted a flurry of research activities aimed at discovering small-molecule antidepressants that would modulate 5-HT and NE deficiencies. One approach to that end is to increase synaptic neurotransmitter levels by inhibiting reuptake of biogenic amines through their presynaptic transporters (Figure 21.3) [8, 9]. Indeed, the observation that imipramine and other tricyclic antidepressants block NE reuptake spurred scientists to more fully explore the potential of reuptake inhibitors. Early work focused on designing selective reuptake inhibitors of 5-HT and NE. Efforts targeting

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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N

HN O NH N N 1 iproniazid

2 imipramine

Figure 21.1 Structures of iproniazid (1) and imipramine (2).

serotonin-selective reuptake inhibitors (SSRIs) led to the development of many currently prescribed antidepressant drugs and tool compounds (Figure 21.4) such as ProzacÒ (fluoxetine, 3), PaxilÒ (paroxetine, 4), and ZoloftÒ (sertraline, 5), while research aimed at identifying selective norepinephrine inhibitors (NRIs) produced agents such as nisoxetine (6), DeprileptÒ (maprotiline, 7), and EdronaxÒ (reboxetine, 8) [13–15]. These agents were perceived to possess a more favorable side-effect profile compared to MAOIs and tricyclic antidepressants, which often demonstrated cardiovascular and anticholinergic liabilities [10]. However, like MAOIs and tricyclics, SSRIs and NRIs demonstrated a time lag between initiation of treatment and the onset of antidepressant action [11, 12]. And while more recent clinical data suggest that some agents may induce improvement in a few measures of depression earlier than others (especially in severely depressed patients) [13], the goal of a definitive and robust rapid onset of action remains elusive. The empirical observation that reserpine produced depression-like symptoms in man [14] led to what was arguably the first paradigm used to screen antidepressant activity, namely, the inhibition of reserpine-induced behavioral effects in rodents. Limited by the state of technology at the time, researchers in the 1950s and 1960s relied on behavioral observations or in vivo functional measures to develop a battery of assays that they hoped would predict antidepressant activity [15]. These assays were Norepinephrine

Serotonin

Energy Interest

Anxiety Irritability Impulsivity Mood, emotion, cognitive function

Motivation

Sex Appetite Aggression

Drive Dopamine Figure 21.2 Biogenic amines as starting points for drug discovery.

21.1 Introduction

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Presynaptic Neuron

Vesicles

Adrenergic Autoreceptor

Serotonergic Autoreceptor

NE Transporter 5-HT Transporter

5-HT

NE

5-HT NE Postsynapatic Receptors

Postsynaptic Neuron Figure 21.3 Schematic of 5-HT and NE neurotransmission and reuptake.

ones in which known clinically effective antidepressants elicited a response and compounds were screened for their ability to mimic the response of these standards. At first, the mechanisms by which these effects were elicited and modulated were poorly understood (see Kelwala et al. [16] for a discussion of these early behavioral

HN

O

CH3

NH O

CF3

H H H3C N

H

Cl

O

O

Cl F

3 fluoxetine

4 paroxetine

CH3 O

H N

H3C O

CH3 O

O N H 6 nisoxetine

5 sertraline

CH3 O 7 maprotiline

Figure 21.4 Structures of known SSRIs and NRIs.

8 reboxetine

NH

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models), but as the catecholamine hypothesis of depression began to emerge it became evident that many of these models had at least some basis in brain 5-HTand/ or NE levels and that 5-HTand/or NE uptake inhibitors could be identified with these models. For example, reserpine-induced effects were ultimately shown to result from depletion of monoamines. Another early “antidepressant” model, tetrabenazineinduced ptosis, was later attributed to depletion of NE. NE uptake was also assessed by examining a compound’s ability to potentiate NE-induced cardiovascular effects. 5-HTuptake could be assessed by evaluating the compound’s ability to potentiate the behavioral effects caused by administration of the 5-HT precursor 5-hydroxytryptophan (5-HTP) or the MAO inhibitor pargyline, which inhibits metabolism of 5-HT. By the 1970s drug discovery researchers were targeting selective SSRIs and NRIs. In vitro technology had advanced to a practical point. Biologists were using various techniques to study the underlying mechanisms of depression and drug discovery researchers had embraced in vitro assays as more efficient means of screening compounds. The uptake of radiolabeled 5-HT could be measured using blood platelets and both 5-HT and NE uptake could be examined using radiolabeled neurotransmitters and synaptosomes prepared from brain. In vivo uptake inhibition was now being examined. Compounds administered in vivo were examined for their ability to inhibit the 5-HTand NE depletion induced by the tyramine derivatives H75/ 12 and H77/77 in isolated brain tissue. Another consequence of 5-HT uptake inhibition is a decrease in 5-HT turnover. This activity could also be measured in isolated tissue following in vivo administration of a test compound by measuring levels of the 5-HT metabolite 5-hydroxyindole acetic acid. In the 1970s and early 1980s radiolabeled binding studies took the place of in vitro assays as the first line screen for potential reuptake inhibitors, prompted by the observation that many clinically effective antidepressants displaced imipramine from its binding site(s) in rat brain tissue. It was in this environment that venlafaxine and desmethylvenlafaxine were first identified, as discussed later in this chapter. But there was not a perfect correlation between imipramine displacement and monoamine uptake inhibition. Those mechanisms had to be confirmed using the in vitro uptake assays. By the mid-1990s, the rat and human 5-HT and NE transporters had been cloned and binding assays had been developed using specific radiolabeled ligands (½3 H
-paroxetine for 5-HT; ½3 H
-nisoxetine for NE). These binding assays have since evolved to provide efficient high-throughput platforms for recent drug discovery efforts. Venlafaxine [17] and desvenlafaxine (see below) were later confirmed to have affinity for 5-HT and NE transporters.

21.2 Major Depressive Disorder

MDD represents a serious medical condition affecting over 150 million people worldwide [18, 19]. Patients who suffer from MDD present with a spectrum of emotional and physical symptoms [20]. Emotional symptoms may include depressed mood, anhedonia, sadness, low self-esteem, anxiety, feelings of guilt, feelings of

21.4 The Discovery of Venlafaxine

worthlessness, difficulty in concentrating or making decisions and recurrent thoughts of death or suicide. Physical symptoms can include fatigue, headache, disturbed sleep, dizziness, restlessness, changes in weight, gastrointestinal complaints, and sexual apathy or dysfunction. MDD affects men and women of all ages, races, and economic levels, with higher prevalence among women (nearly twofold higher than men). MDD is usually diagnosed by a health care professional as being present if a patient demonstrates at least five of the common emotional and physical symptoms for more than two consecutive weeks [9]. 21.3 MDD Pharmacotherapy

Nearly 50% of depressed patients recover within 6 months of being diagnosed with and treated for MDD. Over 90% of depressed patients respond to therapy with either a single drug or a combination of agents [21, 22]. However, the long-term outcome of current depression therapy is less encouraging. Nearly 80% of patients will relapse into depression within 18 months after initial response to drug treatment, with upward of 17% of patients categorized as having a very poor outcome. In addition, patient noncompliance due to a delayed onset of action, side effects, and the continued presence of comorbid symptoms such as anxiety and somatic pain results in 48% discontinuation of treatment within the first 3 months of drug therapy [21, 22]. Although side effects vary by class of drug, they include stimulation, lethargy, dizziness, gastrointestinal effects, weight changes, and sexual dysfunction [10]. Thus, there remains an unmet medical need for newer antidepressant drugs that demonstrate a shorter onset of action, efficacy in a broad range of genetically diverse patient populations, and achieve a higher rate of patient compliance and remission [23]. 21.4 The Discovery of Venlafaxine N(CH3)2 OH HCl H3CO

Venlafaxine hydrochloride WY-45030 21.4.1 Identification of an Early Lead (WY-44362)

With the goal of identifying an antidepressant drug with a faster onset of action we initiated a medicinal chemistry program to identify reuptake inhibitors of both 5-HT and NE (serotonin/norepinephrine reuptake inhibitors, SNRIs). The program

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(H3C)2N

OH

9 ciramadol - 3 chiral centers - 8 possible isomers

hydroxyl translocation

N(CH3)2

(H3C)2N

10 WY-44053 - 1 chiral center - no analgesic activity

(H3C)2N OH

OH

gamfexine

11 WY-44362 - weak NRI and SRI - weak antidepressant activity

Figure 21.5 Evolution of early lead 11 (WY-44362).

evolved from investigations into the mixed opiate agonist–antagonist analgesic ciramadol (9, Figure 21.5) [24]. In an effort to simplify the structure of ciramadol by removing two of its chiral centers, we translocated the hydroxyl group to give WY44053 (10). This compound displayed no analgesic activity in vivo. Insertion of a methylene group to yield WY-44362 (11) likewise resulted in a compound with no analgesic activity. However, structural similarities between 11 and a weak antidepressant agent, gamfexine, suggested evaluation of 11 for antidepressant activity. This compound displayed both weak SRI and NRI activity in vitro and weak antidepressant activity in vivo and became the lead structure for optimization. 21.4.2 Structure–Activity Relationship Studies

Inhibition of binding of ½3 H
-imipramine to rat brain homogenates was used as a preliminary screen for antidepressant activity (Table 21.1). While this property is not universal among second generation antidepressants, imipramine binding had been correlated with the ability of agents to inhibit monoamine uptake [25, 26]. This screening effortidentifiedthegem-dimethylsubstitutionpatternonthebasicaminemoietyasbeing optimal. In addition, imipramine receptor affinity was highest for compounds with a six-membered cycloalkanol ring and an aryl moiety with halogen or methoxy in the 3and4-positions,butnotthe2-position.Bis-substitutiononthearylringwasadvantageous in some cases (e.g., compound 26) and not in others (e.g., compounds 28 and 29). While the imipramine binding assay served to prioritize compounds, selection of compounds for in vivo testing was more heavily based on the ability to inhibit the uptake of tritiated 5-HT and NE into rat brain synaptosomes. As in the imipramine binding assay, compounds with a chloro, bromo or methoxy group in the 4-position of the aryl ring provided the most potent reuptake inhibitors and the cycloalkanol moiety proved to be optimal (Table 21.2). The majority of compounds tested showed similar potencies for inhibiting both 5-HTand NE transport. The exception was the ( þ )-enantiomer of 12, which was over 30-fold more potent at blocking 5-HT reuptake. The profile of the ()-enantiomer of 12 was similar to the one seen for the racemate, where potency for blockade of 5-HT reuptake was around threefold more than the potency for blocking NE reuptake. Interestingly, some members of the series (e.g., compound 28) showed weaker affinity for the imipramine receptor but strong inhibition of 5-HTreuptake, a result that is contrary to the general belief that a relationship exists between the imipramine binding site and the 5-HT reuptake mechanism [27].

21.4 The Discovery of Venlafaxine Table 21.1 Imipramine binding of selected analogues [24].

NR1R2 OH R (CH2)n

Compound 11 12 (venlafaxine) ( þ )-12 ()-12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 2 (imipramine)

R

NR1R2

n

Imipramine binding, IC50 (nM)

H 4-OCH3 4-OCH3 4-OCH3 4-OCH3 4-OCH3 4-OH 4-Cl 4-Br 4-CH3 4-CF3 4-NH2 2-Cl 3-OCH3 3-Cl 3-Br 4-OCH3 4-OCH3 3-Cl, 4-Cl 3-Br, 4-OCH3 3-OCH3, 4-OCH3

N(CH3)2 N(CH3)2 N(CH3)2 N(CH3)2 NH2 NHCH3 N(CH3)2 N(CH3)2 N(CH3)2 N(CH3)2 N(CH3)2 N(CH3)2 N(CH3)2 N(CH3)2 N(CH3)2 N(CH3)2 N(CH3)2 N(CH3)2 N(CH3)2 N(CH3)2 N(CH3)2

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 3 2 2 2

300 90 109 140 900 300 60 100 62 205 88 >10 000 526 150 130 52 126 300 37 190 700 1.7

Table 21.2 Inhibition of NE and 5-HT reuptake in rat synaptosomes by selected analogues [24].

Compound

12 (venlafaxine) ( þ )-12 ()-12 16 17 27 28 2 (imipramine)

IC50 (mM), synaptosomal uptake inhibition NE

5-HT

0.64 3.14 0.76 0.30 0.21 0.07 0.14 0.26

0.21 0.10 0.19 0.18 0.11 0.08 0.2 0.12

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21.4.3 In Vivo Animal Models of Preclinical Efficacy

The primary in vivo model employed to assay potential antidepressant activity was the antagonism of reserpine-induced hypothermia. Numerous compounds from the aforementioned showed significant activity in this model but were less potent than desipramine, a standard tricyclic antidepressant [24]. Several compounds from this series also antagonized histamine-induced adrenocorticotropic hormone release, an activity shared by classical antidepressants. Compounds were finally assessed for their ability to downregulate b-adrenergic responsiveness in the rat pineal gland. Several compounds, including 12, 27, and 28, were found to significantly reduce responsiveness of the adenylate cyclase-coupled b-adrenergic system both on singledose treatment and on repeated administration (Table 21.3). This result was in contrast to that obtained with many known antidepressants, including desipramine, which reduced b-adrenergic responsiveness only after repeated administration. 21.4.4 Selection of WY-45030 for Clinical Trials

Initial interest in the development of an antidepressant candidate centered on compound 27 (WY-45881) based on its favorable in vitro and in vivo preclinical profiles [24]. However, a positive finding with 27 in the Ames test [24] refocused attention on 12 (venlafaxine). The racemate of 12 was chosen because neither enantiomer distinguished itself in the in vivo models. Subsequent examination of compound 12 found it to have very weak affinities for a-adrenergic, serotonergic 5-HT1A and 5-HT2, dopamine D2, opiate, H1, muscarinic cholinergic, and benzoTable 21.3 In vivo results with selected analogues [24].

Compound

Antagonism of reserpine-induced hypothermia; MEDa), mg/kg i.p.

Antagonism of HA/ACTHb); % decrease at 10 mg/kg i.p.

% Change of NEc) responsiveness at 10 mg/kg i.p.

Single treatment

12 (venlafaxine) ( þ )-12 ()-12 27 28 Desipramine

10 30 3 3 1 0.4

26 54 37 55 34 52

51 No significant effect 53 51 65 No effect

a) MED ¼ mimimal effective dose. b) HA/ACTH ¼ histamine-induced adrenocorticotropic hormone release. c) NE ¼ norepinephrine.

Chronic treatment 5 days, b.i.d. 43 No significant effect 51 74 82 81

21.5 Clinical Efficacy of Effexor

diazepine binding sites [28]. Many antidepressants have been shown to downregulate cortical b-adrenergic and/or 5-HT2 receptors following repeated administration to rats. Compound 12 was without effect on these receptor densities following a 14-day administration [24]. Compound 12 displayed neurochemical properties that correlated with classical antidepressant activity, including a reduction in noradrenergic neuronal firing in the rat locus coeruleus [29] and the rat dorsal raphe [30]. These data together with pragmatic considerations such as ease of synthesis contributed to the selection of 12 as a clinical candidate. Compound 12 was later shown to have positive activity in numerous animal behavioral models used to predict potential antidepressant activity, such as the resident intruder social interaction paradigm [31], the rat olfactory bulbectomy model [32], and the tail suspension and forced swim tests [33]. Reviews on the pharmacology of compound 12 have been published [34, 35].

21.5 Clinical Efficacy of EffexorÒ

Compound 12 advanced to clinical trials for depression and was ultimately approved in 1993. The compound was given the generic name venlafaxine and marketed as a hydrochloride salt in an immediate release formulation under the trade name Effexor. The controlled clinical trial results with Effexor have been published and widely reviewed [35–38]. Controlled clinical studies of Effexor (first publications appearing in 1988) included patients meeting the criteria of the Diagnostic and Statistical Manual of Mental Disorders, third edition (DSM-III), for major depression. Patients meeting study criteria had to present a baseline score of 20 on the 21-item Hamilton Depression Rating Scale (HAM-D). Most studies used a two times daily (b.i.d.) or three times daily (t.i.d.) dosing regimen of Effexor and were carried out for 6 weeks with a 4–10-day placebo/washout period. The majority of trials used both the HAM-D and Montgomery-Asberg Depression Rating Scale (MADRS) as the primary efficacy variable, and the Clinical Global Impressions Scale (CGI) was used as a global assessment of efficacy. A response to treatment was usually defined as an improvement in either the HAM-D or MADRS score of at least 50% from baseline score. Effexor demonstrated superior clinical efficacy compared to placebo in numerous open-label and double-blind, placebo-controlled studies in doses ranging from 10 to 375 mg in the short-term treatment of depression [35, 36]. In most instances, 75–225 mg/day was adequate to produce a clinical response, with 75 mg/day being the usual recommended starting dose [36]. The long-term efficacy of Effexor has also been assessed. An open-label study involving 327 patients suggested that the antidepressant action of Effexor (mean dose 143–186 mg/day) was maintained over a 12-month period [39]. Meta-analysis of four double-blind, placebo-controlled studies involving 304 patients suggested that patients treated with Effexor (50–300 mg/day) for an additional 12 months after responding to the first 6 weeks of therapy displayed a significantly lower cumulative relapse rate compared to those treated with placebo [40]. Some clinical data suggest that Effexor may show positive effects in the treatment of resistant depression, although it is not approved or

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recommended for this use. Seventy patients who had failed to respond to treatment with at least three antidepressant drugs from at least two classes (MAOI, tricyclic, SSRI, atypical) were treated with 170–375 mg/day of Effexor. One-third of them experienced a partial or complete response after 12 weeks of treatment and half of the complete responders maintained their response for at least 3 months [41].

21.6 An Extended Release Formulation – Effexor XRÒ

Effexor was initially marketed as an immediate release formulation that required twice-daily dosing. A more convenient microencapsulated extended release formulation was marketed in 1997 as Effexor XR [42]. This formulation allowed once-daily dosing. Like Effexor, Effexor XR displayed efficacy in double-blind, placebo-controlled clinical trials in patients diagnosed with MDD [43]. Single-dose pharmacokinetic studies revealed that Effexor XR produced a lower maximum concentration (Cmax) and a longer half-life for both venlafaxine and the active metabolite Odesmethylvenlafaxine. In multiple dose studies, the immediate release and extended release formulations were found to exhibit bioequivalence.

21.7 Discovery of a Second-Generation SNRI – O-Desmethylvanlafaxine N(CH3)2 OH HO

CO2H CO2H

O-Desmethylvenlafaxine succinate WY-45233 The clinical efficacy of Effexor and Effexor XR prompted the search for secondgeneration SNRIs by Wyeth and others. Duloxetine was marketed as CymbaltaÒ in 2004 and is reported (on the basis of animal data) to have similar affinities for the 5-HT and NE transporters [44]. In Wyeth’s search for new SNRIs, the Wyeth researchers investigated venlafaxine’s metabolism. Several oxidative metabolites were identified in several species [45]. Three major metabolites (Figure 21.6) were identified in humans: N,O-didesmethylvenlafaxine (30), N-desmethylvenlafaxine (31), and O-desmethylvenlafaxine (32) (Figure 21.6). These metabolites were profiled for their pharmacological properties [31]. The major human oxidative metabolite, 32 (WY-45233, formed by oxidation of Effexor primarily by the 2D6 isozyme of cytochrome P450), was found to be an inhibitor of 5-HT transporters and NE transporters (Table 21.4). Like venlafaxine, compound 32 showed no appreciable affinity for dopamine D2, a-1 adrenergic, muscarinic cholinergic, 5-HT1A, 5-HT2, H1, and opiate receptors [31]. A later study confirmed the in vitro

21.7 Discovery of a Second-Generation SNRI – O-Desmethylvanlafaxine (H2C)3N OH

H3CO

12

H 3CHN

H3CHN

(H 3C) 2N OH

OH H3CO

HO

30

OH

HO

31

32 WY-45233

Figure 21.6 Major human oxidative metabolites of Effexor [41].

SNRI profile for 32 and provided comparisons with a number of known 5-HTand NE reuptake inhibitors [46]. That study also showed that 32 demonstrated no significant affinity for over 90 receptors, enzymes, second messenger systems, and ion channel binding sites. Table 21.4 Monoamine uptake inhibition of Effexor and major human metabolites [45].

Uptake inhibition, IC50 (mM)

Compound

12 30 31 32

5-HTa)

NEb)

DAc)

0.21 2.8 1.6 0.18

0.64 >10 4.7 1.16

2.8 >30 21.1 13.4

a) 5-HT ¼ 5-hydroxytryptamine (serotonin). b) NE ¼ norepinephrine. c) DA ¼ dopamine.

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Compound 32 demonstrated a good antidepressant profile in vivo (reviewed by Sobera et al. [47]). The compound reversed reserpine-induced hypothermia and induced pineal b-adrenergic subsensitivity. It also inhibited the firing of 5-HTneurons in the rat dorsal raphe and the firing of NE neurons in the rat locus coeruleus. Results from pharmacokinetic and clinical efficacy trials with 32 administered as a succinate salt began to appear in the literature in 2005 and have been extensively reviewed [47– 50]. The succinate salt of compound 32 (desvenlafaxine) was approved by the US Food and Drug Administration (FDA) for the treatment of MDD in February 2008 and was marketed in a sustained release once-daily formulation under the trade name PristiqÒ . One of the major advantages of Pristiq cited by reviewers as compared to venlafaxine is the lack of CYP2D6 (2D6 isozyme of cytochrome P450) oxidative metabolism [49, 51]. This advantage results in a favorable metabolic profile compared to venlafaxine in individuals who are poor CYP2D6 metabolizers (PMs) or are cotreated with drugs that act as CYP2D6 inhibitors. Owing to genetic, ethnic, and drug-induced variability in the rate of CYP2D6 drug metabolism, Pristiq, which is independent of CYP2D6 metabolism, can be expected to produce reliable and predictable plasma concentrations of Odesmethylvenlafaxine in a wide genetically diverse patient population [52]. The lack of significant metabolism might result in more consistent intraindividual and interindividual pharmacokinetic profiles, eliminating the need for extensive titration to achieve a therapeutic dose.

21.8 Effexor and Pristiq – Additional Considerations 21.8.1 Effexor 21.8.1.1 Onset of Action A continuing shortcoming of antidepressant drugs is their delayed onset of clinical effect. In most trials, noticeable relief from symptoms requires 2–4 weeks of continued dosing [13]. Such delay can expose the patient to continued impairment, a higher risk of discontinuation of treatment, and an increased risk of suicide [53]. The delay in onset of action is thought to correlate with the time required for monoamine processes to adapt to drug treatment [54]. Drugs that influence the NE neuronal pathway might have an advantage in terms of onset of action because, unlike serotonergic receptors, the a2-adrenoreceptor does not become desensitized following sustained NE reuptake blockade [54] (although tricyclic antidepressants block NE uptake yet require several weeks to see a clinical effect). Some clinical data suggest that the beneficial effects of Effexor may be seen within 1–2 weeks of initiation of treatment, at least with aggressive dosing (200–300 mg/day), although no trials specifically designed to differentiate between monoamine reuptake inhibitors based on onset of clinical activity have been reported [37, 54]. However, these high doses increase the risk of side effects.

21.8 Effexor and Pristiq – Additional Considerations

21.8.1.2 Treatment of Some Anxiety Disorders Effexor XR was the first antidepressant to be approved by the US FDA for the treatment of generalized anxiety disorder (GAD) in 1999. The clinical data for Effexor XR supporting its role as efficacious anxiolytic have been extensively reviewed [51, 55, 56]. Effexor XR demonstrated efficacy in GAD, social anxiety disorder (SAD), and panic disorder (PD). Anxiety disorders are often comorbid in patients who suffer from MDD and other depression disorders [57]. Effexor XR improved the signs and symptoms of comorbid anxiety in depressed patients as measured by the Hamilton Anxiety Rating Scale (HAM-A) [56]. 21.8.1.3 Painful Somatic Symptoms Painful somatic symptoms of MDD are also common [58]. Effexor XR has established its effectiveness in open-label [59] and double-blind placebo-controlled [60] clinical trials for depressed patients with a number of painful somatic symptoms, although Effexor and Effexor XR are not approved for this use and Wyeth does not recommend using them for this indication. Tricyclic and monoamine reuptake inhibitor antidepressants are frequently prescribed to treat chronic pain conditions outside of depression [61]. Effexor XR has been evaluated clinically in a number of pain conditions (reviewed by Jann and Slade [62] and Grothe et al. [63]) and improved pain scores in some of these trials. However, Effexor and Effexor XR are not approved for the treatment of chronic pain or other painful disorders and Wyeth does not recommend that use. 21.8.2 Pristiq 21.8.2.1 Anxiety and Painful Symptoms Pooled clinical data suggest that Pristiq is effective in treating anxiety patients with MDD (reviewed by Pae [50]). At least two clinical studies were initiated in which Pristiq was examined in fibromyalgia patients [64], but the results of those studies have not been published. Pristiq is not approved for the treatment of anxiety disorders or pain and Wyeth does not recommend it for these uses. 21.8.2.2 Symptoms Associated with Menopause Animal studies [65] and clinical trials [66–69] have suggested that Pristiq may be effective in relieving the vasomotor symptoms (VMS) associated with menopause. VMS or hot flushes remain an unapproved condition for Pristiq in the United States, and Wyeth cannot recommend that use in the United States. In Mexico and the Philippines, however, Pristiq has both the MDD and VMS approvals. Phase III clinical trials data presented by Wyeth at recent scientific meetings showed that Pristiq significantly improved mood in women with higher than average profile of mood state (POMS) scores [50].

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21.9 Conclusions

Effexor was the first SNRI marketed in the United States in 1993 and has proved its value clinically. Millions of depressed patients have benefited from the use of Effexor and Effexor XR. The discovery of Effexor provides a lesson in how a successful drug targeting a particular disease state can unexpectedly spring from a totally unrelated program. Medicinal chemists must remain watchful and ready to capitalize on these serendipitous events when they present themselves. Effexor XR represents a useful, convenient-to-use addition to the physician’s antidepressant arsenal. Many patients with MDD who did not respond to tricyclic antidepressants and SSRIs responded favorably to Effexor XR, albeit in open-label clinical trials [38, 41]. In addition, preclinical and clinical data seem to indicate that engaging the NE system in addition to the 5-HT system provides another mechanism for treating patients with MDD. Pristiq was discovered and developed as the major active metabolite of venlafaxine with the advantages over Effexor that include lack of need for initial drug titration and a role in the treatment of patients who are poor CYP2D6 metabolizers or are taking other medications that inhibit the 2D6 isozyme. Since the fortuitous clinical observations and discoveries of the 1950s, great strides have been made in the treatment of depression. However, there is still much room for improvement. All approved antidepressants are monoamine-based. Engaging new mechanisms may open the door to new antidepressants in the future. Side effects and a delayed onset of action still account for noncompliance among patients, and some patients remain refractory to antidepressant treatment. Medicinal chemists continue to pursue the quest for additional antidepressant agents. Part of this effort has taken them deeper into the realm of monoamine reuptake inhibitors [70], while others are exploring new molecular targets [71] that they hope will provide more dynamic drugs. The path forward is challenging [72], but a better understanding of the underlying disease mechanisms and the etiology of the disease states [73] will undoubtedly lead to more effective treatments for this devastating disorder.

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Magid Abou-Gharbia

Associate Dean for Research, Professor of Medicinal Chemistry, Director of Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, 3307 North Broad Street, Philadelphia, PA 19104, USA Magid Abou-Gharbia, PhD, FRSC, is Associate Dean for Research, Professor of Medicinal Chemistry, and Director of the Moulder Center for Drug Discovery Research (MCDDR) at the School of Pharmacy, Temple University, Philadelphia, PA. Before joining the Temple University in September 2008, Magid worked for 26 years at Wyeth in many therapeutic areas where he and his research team of 500 scientists made numerous contributions to medicinal chemistry that resulted in 100 US patents and over 350 worldwide patents and the discovery of several innovative marketed drugs, 2 of which are described in this chapter.

Wayne E. Childers Jr

Principal Research Scientist III, Wyeth Research, Chemical Sciences, 865 Ridge Road, Monmouth Junction, NJ 08852, USA Wayne E. Childers is a Principal Research Scientist for Wyeth Research, Inc. He has been working with Wyeth for 22 years as a medicinal chemist. Wayne received his BA (1979) degree from Vanderbilt University in chemistry and PhD (1984) in organic chemistry from the University of Georgia under the direction of Dr. Harold Pinnick. He served as an assistant adjunct professor at Bucknell University before accepting a position as a postdoctoral fellow at the Johns Hopkins University School of Medicine in the laboratories of Dr. Cecil Robinson. Wayne joined Wyeth in 1987. Over the past 22 years, Wayne has worked in and made contributions to numerous therapeutic areas, including psychiatric diseases, stroke, and Alzheimer’s disease, and the treatment of chronic pain.

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Index a abacavir 12, 13 ABDD (analogue-based drug discovery) XVII–XX, 4, 20, 61, 78, 189, 207, 243, 247, 250, 258, 260, 313, 444 ABC (ATP binding cassette) transporter protein 200, 206 ABPM (ambulatory blood pressure monitoring) 375 absorption 16, 126, 194, 196 ACAT (acyl-CoA:cholesterol acyltransferase) inhibitors 43 ACE (angiotensin-converting enzyme) 16, 62 ACE inhibitors 30 acetaminophen (paracetamol) 30, 31 acetanilide 30, 31 acetylcholine 297, 299, 302 acetylcholine-induced bronchoconstriction 309 acetylcholinesterase 299 acetylsalicylic acid (aspirin) 31, 33, 34 acetyltriethylcholine 303 aclidinium 313 acrivastine 9 ACTH (adrenocorticotropic hormone) 360, 514 active metabolite XX, 30, 31, 96, 97, 117, 127, 147, 202, 516, 520 active site 63, 64 acute asthma attack 326, 328 acute myelogenous leukemia 226 adefovir dipivoxil 386, 399 ADME (absorption, distribution, metabolism and excretion) 15 ADMET (absorption, distribution, metabolism, excretion and toxicity) 62 adrenaline see epinephrine adrenergic autoreceptor 509

adrenergic a and b-receptor 319 adrenergic b-receptor blockers 5 adrenergic b1 and b2 receptor subtypes 5, 319 b2-adrenoreceptor agonists XIX, 319 Adriamycin Ò see doxorubicin adverse effect 5 AFC (7-amino-4-trifluoromethylcoumarin) 110, 111 agranulocytosis 37 AIDS (acquired immunodeficiency syndrome) 110, 181, 184, 409, 443, 444, 456 alcuronium 189, 191 Aldactone Ò see spironolactone aldosterone 359, 360 aldosterone antagonists 359, 362 Allegra Ò see fexofenadine allosteric site 409 alogliptin 123–126 ALT (alanine aminotransferase) 395, 400, 401 ALL (acute lymphoblastic leukemia) 502 Alzheimer’s disease 288 AM404 see N-arachidonoyl-phenolamine Amanita muscaria 299 Ames test 483, 514 amfebutamone see bupropion Amicolatopsis mediterranei 173 amino-epothilone B 254 amitriptyline 39, 41, 269, 271, 276, 279, 281 amlodipine 18, 20 amoxapine 37 amoxicillin 11 AMP see amprenavir cAMP (cyclic 30 ,50 -adenosine monophosphate) 135 ampicillin 9, 11, 96 amprenavir 14, 15, 74, 75, 444, 445–457 amrubicin 232, 234

Analogue-based Drug Discovery II. Edited by János Fischer and C. Robin Ganellin Copyright Ó 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32549-8

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analgesics 31, 512 analogue class 3, 4, 11, 30 analogue drug 3 analogy XVII ANDA (Abbreviated New Drug Application) 83, 102 animal welfare regulations 309 annamycin 232 ansa chain 175, 184 ansamycins 174 anthracyclines 217 anti-AIDS drugs 23 antiandrogenic activity 7 antibody formation 350 anticholinergic activity 272, 286, 298 anticoagulant 447 anticonvulsive agent 46, 49, 51, 89 antidiabetic effect 110 antidotes 301 Antifebrin Ò see acetanilide anti-inflammatory activity 31–33 antimuscarinics 297, 300, 305, 309, 313 – nonselective 304 antioxidant 192 antiplatelet effect 35 antipyretics 30, 33 antituberculosis agent 507 antitumor vinca alkaloids 195 anxiety 286 a-APA (a-anilinophenylacetamide) 12, 410 apomorhine-induced hypothermia 282 apomorphine-induced stereotypy 38 apoptosis 251 Aptivus Ò see tipranavir APV see amprenavir aqueous solubility 201, 245, 250, 447 AR (androgen receptor) 362, 365 L-arabinose 388 N-arachidonoyl-phenolamine 31, 32 arecoline 299, 300 aripiprazole 35, 39 aspirin see acetylsalicylic acid astemizole 8, 9 AT1 antagonists 30 atazanavir 444, 445 atenolol 6, 7 atorvastatin 4, 66 ATP (adenosine triphosphate) 219, 465, 494, 495 Atropa belladonna 300 atropine 298, 300, 301, 304, 313 “atypical” antipsychotic drugs 27 AUC (area under the curve) 179, 180, 375, 431

AUC24h (steady-state exposure) 431 avanafil 140, 146 azacyclonol 8 aza-epothilone B 254, 259 2-m-azodobaccatin III 256 2-p-azidobaccatin 256 2-m-azidotaxol 255 AZT (azidothymidine) see zidovudine

b baccatin III 256 Bacteriodes fragilis 178 BBB (blood-brain-barrier) 221, 260, 297, 301 Bcr-Abl (Breakpoint cluster region - Abelson) kinase 493, 494, 497, 501 beclomethasone 22 benazepril 18, 19 benzylpenicillin (penicillin G) 9, 11 berubicin 221, 223, 233 “best mode” requirement 99, 100 betaxolol 6, 7 BG see blood glucose BHK (baby hamster kidney) 341 b.i.d. or bid or BID twice a day (from Latin bis in die) 455, 456, 480 bile cramps 304 binding pocket 70 bioavailability 14, 18, 46, 50, 95, 117, 127, 181, 226, 245, 304, 375, 393, 422, 423, 434, 447 452, 455, 480, 497 biomarker 272, 274, 349 bipolar disorder 46 bisoprolol 6, 7 a1-blockers 30 blood-brain barrier see BBB blood glucose (BG) 109, 110, 335, 347, 348 BOC (t-butoxycarbonyl) protecting group 248 body weight reduction 346–351 brain circulation 190 brain tumors 223 breast cancer XX, 217, 226, 259, 467, 488, 489 bromovincamine see brovincamine bronchial asthma 5, 70, 299, 319, 327, 328 bronchitis 5 bronchodilator 304, 313 bronchospasmolytics 304 brovincamine 190, 191, 194, 195 broxaterol 321 budenoside 21, 22 buformin 48, 49 bulimia 287 bupropion 39–41

Index

c caco-2 permeability 482 caffeine 122, 123, 147 calcium channels 193, 194 Camptotheca acuminata 201 Camptosar Ò see irinotecan camptothecin (CPT) and derivatives XIX, 189, 191, 201, 203–205, 207 Candida infections 12 capecitabine 259, 488 captopril 17–19, 62 carbachol 299, 300 carbamazepine 46 carbonic anhydrase 51 carboplatin 9 carbuterol 321 carcinogenicity 31 cardiotoxicity 219, 222, 223, 233, 234 carminomycin 224, 225 Catharantus roseus 195, 198 cathechol O-methyl transferase 320 cathepsin D and E 454 cavernosal tissue 144 CBF (cerebral blood flow) 192, 194 CC50 (50 % cytotoxic concentration) 421 b-CCE (ethyl b-carboline-3-carboxylate) 160 CD4 (cluster of differentiation 4) glycoprotein 432 – cell counts 432, 456 celiprolol 6, 7 cell death 205, 243 cell permeability 176 cerebral circulation 193 cerebral insufficiency 190, 207 cervical carcinoma 228 cetirizine 9 CGI (Clinical Global Impressions Scale) 515 Champix Ò see varenicline Chantix Ò see varenicline CHB (chronic hepatitis B) 383, 385, 386, 396 chemical stability 115 – 117 chemoimmunotherapy 400 Chlamydia difficile 182, 184 Chlamydia trachomatis 178 Chlamydia psittaci 178 Chlamydophila pneumoniae 183 chlorimipramine 271, 272, 274, 275 chlorpromazine 36 cholesterol absorption 43–45 cholesterol-fed hamster assay 45 cholestyramine 42 cholinergic agonists 301

chronic hepatitis B see CHB chronic model of adjuvant arthritis 497 chronic myelogenous leukemia (CML) XX, 14, 196, 493 chronic obstructive pulmonary disease see COPD Cialis Ò see tadalafil cilazapril 18, 19, 62 cilomilast 71, 72 cimetidine 4, 5, 22 cirrhosis 383, 385 cisplatin 9 citalopram 269, 272, 274, 279, 285 – 287 citramadol 512 CK (creatine kinase) 396, 401 c-kit tyrosine kinase 466 Clarinex Ò see desloratadine Claritin Ò see loratadine class effect 6 clenbuterol 321, 322 clevudine (CLV, L-FMAU) XX, 383, 386, 387, 389, 390, 392–396, 400 – clinical studies 394 – mechanism of action 390 – metabolism 390, 391 – PK 392, 394 – resistance 400 – synthesis 387, 388 – toxicity 398, 399 clofibrate 42 Clostridium difficile 178 cloxacillin 10, 11 clozapine 37, 288 CL (clearance) 497 CLR (renal clearance) 393 CLT (total clearance) 393 CLV see clevudine CLV-TP (clevudine triphosphate) 391, 392, 396 CML (chronic myelogenogenous leukemia) 493, 501 CML xenograft model 501 CMRglc (cerebral metabolic rate of glucose) 195 CNS (central nervous system) 8 COBP (chronic obstructive bronchopneumopathies) 326 cognitive enhancer 192 colestipol 42 colterol 321 compactin 66, 68 conditioned avoidance response test 35 congeners 71, 148, 149, 173, 196, 201, 300

j527

j Index

528

constrained analogue 284 COPD (chronic obstructive pulmonary disease) XIX, 70, 297, 299, 304, 311–313, 327, 328 corticosteroids 21, 22, 326 Corynebacterium spp. 178 CPI/r (comparator protease inhibitor boosted with ritonavir) 456, 457 CPT see camptothecin 189 CPT-11 see irinotecan cRaf1 inhibitor 466 CRC (colorectal cancer) 203, 206 creatine kinase see CK Cremophor Ò EL 245, 246, 248 cross-licensing 93 cyclooxygenase-1 (COX-1) enzyme 34 cyclooxygenase-2 (COX-2) enzyme 34 cyclooxygenase-2 (COX-2) inhibitors 32 Cymbalta Ò see duloxetine CYP (cytochrome P450 isoenzyme) inhibition 62, 124, 287, 482 CYP3A4 205, 206, 287, 455, 488 CYP2C19 287, 431 CYP2D6 269, 287, 518

d DA (dopamine) 508, 517 10-DAB see 10-deacetyl-baccatin III dapivirine 411, 412 dapoxetine 282 dapsone 433 DAPY (diarylpyrimidine) derivatives 411–414, 421, 422, 430 darunavir 14, 15, 431, 444, 445 dasantafil 148 dasatinib XX, 14, 15, 493, 498, 501 – clinical trials 502, 503 – first-pass metabolism 498 – oral bioavailibility 498 – PK 498 – plasma clearance 498 – resistance 501 – synthesis 500 – X-ray structure of dasatinib-Abl complex 500 DATA (diaryltriazine) derivatives 410, 411, 430 daunorubicin 217 – 219, 223, 225 daunorubicinol see 13-dihydrodaunorubicin daunosamine 217, 229, 230, 233 dCK (deoxycytidine kinase) 391 10-deacetyl-baccatin III (10-DAB) 244, 248 dehydelone 254

delavirdine 409, 410 denagliptin 118, 119 deoxycytidine 392 depression XX, 41, 269 Deprilept Ò see maprotiline desipramine 514 desloratadine 96, 97 desmethylimipramine 273, 274 N-desmethylvenlafaxine 516 O-desmethylvenlafaxine (desvenlafaxine) 516 desvenlafaxine XX, 507, 510, 520 – succinate salt 518 – lack of CYP2D6 interaction 518 diabetes, type 2 47, 109, 112, 125, 128, 336, 349–351, 375 diarrhea 205 diazepam 46 dichloroisoprenaline 320 diclofenac 34 dicloxacillin 10, 11 didanosine 12, 13 N,O-didesmethylvenlafaxine 516 diflunisal 35 13-dihydrodaunorubicin 217 dimeric vinca alkaloid analogues XIX diphenhydramine 281, 282 Diprivan Ò see propofol direct analogues XVII–XIX, 3, 29, 30, 32 disclaimer 98 discodermolide 251, 254, 258 dissociation half-life 309 distribution volume 180, 199, 231 diverticulitis 182 DNA (deoxyribonucleic acid) 204–206, 224, 226, 230, 233, 362, 383, 391, 392 DNA polymerases 383, 384, 386, 391, 396 DNA replication 384 cDNA (complementary desoxyribonucleic acid) 335 cccDNA (covalently closed circular DNA) 384, 385, 392, 393, 401 mtDNA (mitochondrial DNA) 396, 399 DOC (deoxycorticosterone) 361, 362 DOCA (deoxycorticosterone acetate) 361, 362 docetaxel 244, 247, 248, 259–261 dopamine XIX dopamine D1 and D2 receptors 37 dopamine receptor partial agonist 38 dopamine transporter 269, 285 dopamine uptake inhibition 517 doxorubicin XIX, 217, 218, 222, 223, 225, 226, 228, 230, 231, 233, 259

Index DPP-4 or DPP-IV (dipeptidyl peptidase 4) inhibitors XIX, 109–111, 125, 338, 340, 345–348 Dreiding models 363 drowsiness 7 drug blood level 182 drug candidate 43, 44, 90 drug-drug interactions 22, 23, 46, 180, 206, 259, 287, 288, 455, 507 DSM-III (Diagnostic and Statistical Manual of Mental Disorders, third edition) 515 dulogliptin 112 duloxetine 516 duration of action 18, 95, 324 – 326, 347

e ebastine 9 eburnamonine see vinburnine EBV (Epstein-Barr virus) 386, 388 EC50 (effective concentration 50) 161, 347, 390, 421, 449 ED (erectile dysfunction) 135, 138, 143–145, 147, 154, 160, 286 Edronax Ò see reboxetine efavirenz 409, 410, 421, 422, 432, 433 Effexor Ò see venlafaxine Effexor XR Ò 516, 519, 520 efficacy 5, 182, 285 EFS (electric field stimulation) 309 EGFR (epidermal growth factor receptor) tyrosine kinase 465–468 – overexpression 468 – inhibitors 468, 469 Eldisine Ò see vindesine electrically stimulated guinea pig trachea 324 electrolyte balance 359 eleutherobin 251, 254, 258 elimination half-life 18 Ellence Ò see epirubicin elpetrigine 47 EMEA (European Medicines Agency) 345, 346 emtricitabine (ETC) 392, 399, 432 enalapril 16, 18, 19, 63 enantiomer 94 endocarditis 180 enelaprilat 16 enfuvirtide 456 entecavir 386, 399 EPA (Environmental Protection Agency, USA) 245 ephedrine 319, 322 EPHESUS trial 375, 376 epilepsy 46

epinephrine (adrenaline) XIX, 319, 322 epirubicin 220, 222, 223, 233 eplerenone XX, 359, 367 – synthesis 367, 372 EPO (European Patent Office) 86, 92 epothilone analogues XIX, 243, 251, 252 epothilone A - F 252 epothilone B 255, 257, 258 EPS see exprapyramidal side effect eptifibatide 73 erbB-2 tyrosine kinase 465, 467 – overexpression 468 erbB-2/EGFR dual inhibitor 468, 472 erbB-3 tyrosine kinase 468 erbB-4 tyrosine kinase 468 Erk 1/2 MAP kinases 468 erlotinib 468, 471, 472, 485 Escherichia coli 178 escitalopram 95, 269, 270, 272, 277, 286 esmolol 18, 21 esomeprazole 21 esorubicin ETC 221–223 ETC see emtricitabine etravirine XX, 12, 13, 410, 411 exenatide 343, 345, 347–350 exendin-4 345 exatecan 203, 204 extrapyramidal side effect (EPS) 36, 301 ezetimibe 42, 44

f FAAH (fatty acid amide hydrolase) 31 famotidine 4, 5, 431 FBDD (fragment-based drug design) XVIII, XX, 446, 457, 458 FDA (Food and Drug Administration) 102, 345, 346, 376, 386, 409, 426, 444, 454, 518 FDA’s Orange Book 102 felodipine 18, 20 femoxetine 273, 274, 284 fenofibrate 45 fenoterol 321, 322 L-FEAU (1-(20 -deoxy-20 -fluoro-b-Larabinofuranosyl)-5-ethyluridine) 389 D-FEAU 393 FEV1 (forced expiratory volume in 1 s) 311 fexofenadine 9, 96 fibrinogen 73 filauridine 393 fixed dose combinations 327 flucloxacillin 10, 11 fluconazole 12, 17

j529

j Index

530

fluoxetine 269 – 271, 274, 275, 281–287, 508, 509 flurbiprofen 34 fluticasone 21, 22, 326 fluvoxamine 269 – 271, 273, 274, 279 – 281, 285, 286 L-FMAU (L-20 -fluoro-5-methyl-b-Larabinofuranosyluracil) see clevudine D-FMAU (or FMAU) 387, 393 forced swim test 271, 275, 276, 515 formoterol 321, 322, 324 – 327 fosamprenavir 444, 445 fosinopril 18, 19 fragment-based drug design see FBDD full analogues XVII, 3, 29, 30, 52, 247

g GABAA (gamma-aminobutyric acid A) ligands 120 GAD (generalized anxiety disorder) 519 Galega officinalis 47 galegine 47 gamfexine 512 gastroesophagitis 4 gefitinib 466–468 GIP (glucose-dependent insulinotropic polypeptide) 335, 339 glaucoma 191 Gleevec Ò see imatinib glia monster venom 345 glibenclamide 47 glimepiride 349 glioblastomas 221 glomerular filtration 338, 348 GLP-1 (glucagon-like peptide-1) XIX, 110, 111, 335, 346, 350 – truncated GLP-1 (7-37) 336 – gastric emtying 337 GLP-1 receptor 337, 347 glucagon receptor 347 Glucophage Ò see metformin glutamate receptors 193, 194 glutathione 32 glycemic control 349, 350 glycopyrrolate 313 cGMP (cyclic 30 ,50 -guanosine monophosphate) 135, 137, 138, 143, 147, 161, 193 goserelin 101 GPIIb/IIIa (glycoprotein IIb/IIIa) antagonists 73 GR (gestagen receptor) 362 Gram-negative pathogens 176, 182 Gram-positive pathogens 176, 178, 184

growth inhibition (GI) of cell cultures 246 guanidine 47 guanylyl cyclase 137, 143 gynecomastia 7, 361, 376

h HA (heavy atom) 448 HAART (Highly Active Antiretroviral Therapy) 434 half-life 126, 127, 146, 179, 180, 194, 199, 259, 338, 343, 348, 375, 392, 431, 497, 516 Haloderma suspectum 345 haloperidol 8, 36, 281, 288 HA/ACTH (histamine-induced adrenocorticotropic hormone) release 514 HAM-A (Hamilton Anxiety Taring Scale) 519 HAM -D (Hamilton Depression Rating Scale) 288, 515 HandiHaler Ò device 311 HbA1c (glycosylated haemoglobin) 109, 115, 125, 348–350 HBV (hepatitis B virus) XX, 383, 386–388 – life cycle 384 – wild-type 397 HBcAg (hepatitis B core antigen) protein 383 HBeAg (hepatitis B e antigen) 384, 385 HbsAg (hepatitis B surface antigen) 383, 385, 401 HCC (hepatocellular carcinoma) 203, 383, 385 HCV (hepatitis C virus) 395 HDV (hepatitis delta virus) 386, 394 helplessness model, learned 275, 276 hepatitis B virus see HBV hepatitis C virus see HCV hepatitid delta virus see HDV hepatocellular carcinoma see HCC hepatotoxicity 410 hERG (human ether-a-go-go-related gene) channel 9, 62, 123 hetacillin 96 HIAA (5-hydroxy-indole acetic acid) 271, 510 histamine H1 receptors 8 histamine H2-receptor antagonist 4, 7 histamine H3-receptor antagonist 284 histaminergic nerves 8 hit 447, 457 HIV (human immunodeficiency virus) 387, 396, 409, 424, 443 HIV PR (HIV protease) XX, 74–76, 443, 444 – active site 444 – X-ray crytal structure 444 – inhibitor 447, 453 HIV-1 PR inhibitor 14, 74, 409, 411, 421, 447

Index HIV-2 PR inhibitor 447 HIV-1 RT enzyme 410, 434 HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors 4, 42, 66 Hodgkin’s disease 195 5-HT (5-hydroxytryptamine) see serotonin 5-HTP (5-hydroxytryptophan) potentiation model 272, 275, 277, 510 HTS (high-throughput screening) 113, 119, 122, 250, 251 human foreskin fibroblast (HFB) 472 HycamtinÒ see topotecan hydrocortisone 367 Hyoscamus niger 300 hyoscyamine 300 hyperaldosteronism 361 hyperglycemia 109 hyperkalemia 376 hyperprolactinemia 38 hypertension 18, 143, 361, 375, 376 hypoglycemia 349 hypoglycemic agent 47, 109 hypokalemia 361

i IBMX (isobutylmethylxanthine) 147 ibogaine 189, 191 ibuprofen 34 ICS (inhaled corticosteroids) 326 idarubicin 223, 225, 227, 228, 233, 234 IDR see idarubicin idarubicinone 224 IDV see indinavir i.m. (intramuscular) 274 imatinib XX, 14, 15, 466, 467, 493, 501 – resistance 493, 501 – resistant Bcr-Abl mutants 502 imipramine 39, 269, 273–275, 281, 507, 508 [3H]-imipramine binding assay 512, 513 immunomodulator 112, 385 impotence 135, 361 incretin 335, 339 indacaterol 327 indalpine 274 indinavir 74, 75, 180, 444, 445, 456 indomethacin 34 infringement 85, 102 IND (Investigational New Drug) USA, FDA 260 INN (International Nonproprietary Name) 184 insulin 48, 109, 110, 127 insulin detemir 347 insulin glargine 349

Intelence Ò see etravirine 411 intercalation complex 218, 224, 226 interferons 385 interindividual pharmacokinetic variability 20 invention 85, 86 inventive step 88, 90, 92 – 94 inventorship 98 iodorubicin 222, 223 IOPY (iodo phenoxy pyridone) 412, 422 i.p. (intraperitoneal) 281 ipratropium bromide XIX, 303–305, 309 – 312, 328 – synthesis 308 iproniazid 507, 508 Iressa Ò see gefitinib irinotecan 201–203, 205, 206 irreversible inhibitor 111 isoetarine 319, 320, 322 isoprenaline (isoproterenol) XIX, 319, 320, 322, 324 isoproterenol 40 isosteres 154 itching eyes 7 i.v. (intravenous) administration 218, 245, 340, 347 Ixabepilone Ò see aza-epothilone B

k

Kaletra Ò (combination of ritonavir and lopinavir) 23 ketoconazole 16, 17, 431, 488 ketoprofen 34 Ki (inhibitory constant) 446, 449, 450, 452, 453, 497, 498 kidney cramps 304 kidney toxicity 31, 48

l LABA (long-acting b2-agonist) 321, 326 lacidipine 18, 20 b-lactamase inhibitors 30 lactic acidosis 49, 393 lamivudine 12, 13, 386, 392, 399, 432 lamotrigine 46 lapatinib 465, 467, 482 – preclinical efficacy 483, 484 – EGFR, X-ray crystallography 485 – binding kinetics 486 – clinical results 487–489 – combination with capecitabine laulimalide 251 Lck (lymphocyte specific kinase) 494 – inhibitors 495, 497

j531

j Index

532

– active site 496 hLck (human Lck) 494 mLck (murine Lck) 494 LDL-C (low-density lipoproteincholesterol) 42 LE (ligand efficiency) 448 lead compound 38, 43, 62, 88, 122, 189, 196, 207, 448, 468 LEAD (Liraglutide Effect and Action in Diabetes) 349 left ventricular systolic dysfunction 376 Leisteria monocytogenes 178 Lennox-Gastaut syndrome 46 leprosy 174, 179, 184 leukemia 196, 201, 217, 225 Levitra Ò see vardenafil Levovir Ò see clevudine Lexapro Ò see escitalopram license 85, 93 linagliptin 123, 124, 127 lipophilicity 222 liposomal formulation 203, 232 liraglutide XIX, 335, 339, 341, 346, 349–351 lisinopril 16, 18, 19 lithium 46 liver cell (hepatocyte) 384 liver toxicity 32, 48 long-acting b2-agonist see LABA lopinavir 14, 23, 76, 78, 431, 444, 445, 456, 457 loratadine 9, 96, 97 loteprednol etabonate 18, 21 lovastatin 66, 67 LPV see lopinavir lung cancer 201, 202, 228 lurtotecan 203, 204 Luvox Ò see fluvoxamine LVEF (left ventricular ejection fraction) 375, 376 lymphoma 217

m MADRS (Montgomery-Asberg Depression Rating Scale) 515 Mandragora officinarum 300 MAOI (monoamine oxidase inhibitor) 272, 280, 507, 508, 510 M1 (muscarinic receptor M1 subtype) 123, 124 macromolecular drugs 29 major depressive disorder (MDD) 507, 510, 511, 515, 516, 518–520 maprotiline 508, 509 Markush claim 104

rMD (restrained molecular dynamics) 255 MDD see major depressive disorder MED (minimal effective dose) 273 medical use 88 melanoma 196, 201 meningitis 179, 184 menstrual irregularity 361 MES (maximal electroshock seizure) model 50 metabolic inactivation 16, 44 metabolic stability 44, 115, 447 methacholine 299, 300 metformin 47 – 49, 127, 128, 349, 350 methemoglobinemia 31 methicillin 10, 11 N-methyl-paracetamol 32 N-methyl-scopolamine 301–303, 305 methylthioepothilone B 254 metoprolol 6, 7 MIC (minimal inhibitory concentration) 176, 178, 180 miconazole 16, 17 microdialysis 272 migraine 50, 51 mirodenafil 140, 146 microsomal metabolism studies 482 microtubules 199, 200, 243, 244, 247, 248, 251, 252, 255, 256, 260 milveterol 327 mitoxantrone 233 mizolastine 9 Moraxella spp. 178 molecular modeling XVIII, 61, 62, 76, 78, 117, 226, 254, 256, 257, 282, 300, 363, 396, 397, 412, 452, 496 monoclonal antibodies 29, 30 monopoly 84 monoterpenoid indole alkaloids 189, 207 MR (mineralocorticoid receptor) 359, 365 MRP (multidrug resistance-associated protein) 200, 206 MTD (maximum tolerated dose) 488 multidrug resistance (MDR) tumors 247, 250, 259 multiple sclerosis 494 muscarine 299, 300 muscarinic M2 receptor subtype 304 muscarinic M3 receptor subtype 303, 305, 309 muscarinic receptors (M1-M5) 298, 299, 301 muscarinic receptor antagonists 297 muscle myopathy 396, 399, 401 mycobacterial infections 181 Mycobacterium avium 180, 181, 184

Index Mycobacterium kansasi 178 Mycobacterium leprae 178 Mycobacterium marinum 178 Mycobacterium tuberculosis 178, 182 Mycobutin Ò see rifabutin mydriasis 301 myelosuppression 205, 387 myxobacterium 251

n NAPQI (N-acetyl-p-benzoquinone imine) 32 naproxen 34 nausea 206 Navelbine Ò see vinorelbine NCE (New Chemical Entity) 83, 102, 243 NCI (National Cancer Institute, USA) 228, 244, 260 NDA (New Drug Application) 117, 124, 386 NE see (norepinephrine) nebivolol 6, 7 Neisseria spp. 178 nelfinavir 74, 75, 444, 445 nemorubicin 232, 234 nephrotoxicity 9 neuromuscular blocking agents 189 neuronal homeostasis 190 neurotoxicity 387 neurotransmitters XIX neutropenia 205 nevirapine 409, 410 Nexavar Ò see sorafinib nicotinic receptors 299 nifedipine 18, 20 nilotinib 14, 15 nisoxetine 282, 284, 508, 509 nizatidine 4, 5 NMR (nuclear magnetic resonance) spectroscopy 255 NNRTI (nonnucleoside reverse transcriptase inhibitor) XX, 12, 409, 430, 433, 443 NO (nitric oxide) 143, 150 nonataxel 254, 255 non-Hodgkin’s lymphoma 196, 226 nonobviousness 90 norepinephrine (NE, noradrenaline) XIX, 271, 507, 508 norepinephrine transporter 269, 274, 276, 277, 509, 510, 516 norepinephrine uptake inhibition 283, 517 norfluoxetine 282 noribagoine 189, 191 Normix Ò see rifaximin nortriptyline 274

novelty 85, 88, 89, 93, 94, 97 NPs (natural products) 243, 244, 258 NPC1L1 (Niemann-Pick C1-Like-1) protein 45 NRIs (norepinephrine reuptake inhibitors) 508, 512, 513 NRTI (nucleoside reverse transcriptase inhibitor) 12, 386, 443 NSAIDs (nonsteroidal anti-inflammatory drugs) 34 NSCLC (non-small cell lung cancer) 196, 200, 201, 203, 206, 231 nucleoside analogues 385, 386, 409

o OADs (oral antidiabetic drugs) 109, 125 obesity 109, 351 OC (ovarian cancer) 202, 207, 217, 228, 231, 245, 488 OCD (obsessive-compulsive disorder) 286, 287 oculomucocutaneous reaction 6 OGTT (oral glucose tolerance test) 110 olanzapine 37, 38, 288 olfactory bulbectomy 275, 276 omeprazole 20, 431 Oncovin Ò see vincristine onset of action 271, 324–326, 508, 511 OPC-4392 38 orciprenaline 320–322 osteomyelitis 180 ovarian cancer see OC oxacillin 10, 11 oxaliplatin 9 oxitropium bromide 303, 304 oxcarbazepine 47

p paclitaxel 207, 243 – 246, 248, 254, 256, 259–261 paclitaxel analogues XIX pain models 32 pancreatic cancer 203 panic disorders 287 pantoprazole 20, 21 paracetamol see acetaminophen parathyroidectomy 48 paresthesia 50 pargyline 274, 510 Paris Convention 86, 87, 99 Parkinsonism 301 paroxetine 269, 270, 273–275, 277, 284–286, 508, 509 patent 103

j533

j Index

534

patent application publication 100 patent claims 89 patent family 104 patent litigation 102 patentability 84, 85, 93, 95, 101 patentee 85 patenting of analogues 83 Paxil Ò see paroxetine PC3 prostate tumor xenograft assay 498 PCA (p-chloroamphetamine) 271 PCF (plant cell fermentation) 245 PCT (Patent Cooperation Treaty) 86 PD153035 466 PDEs (phosphodiesterases) 135, 137 PDE1 (phosphodiesterase 1) enzyme 193 PDE4 (phosphodiesterase 4) enzyme inhibitors 70 PDE5 (phosphodiesterase 5) enzyme inhibitors 135, 143, 147, 149, 162 PDE6 (phosphodiesterase 6) enzyme 144 PDGFR (platelet derived growth factor receptor) tyrosine kinase 466 peloruside A 251 penicillinase-resistant penicillins 9 pentamidine 48 PEP (prolyl endopeptidase) inhibitor 112 pepsin 443, 454 peptic ulcer 4 peptidomimetic agents 75 perindopril 18, 19 PGE1 (prostaglandin E1) 34 PGE2 (prostaglandin E2) 35 PGF2a - contracted guinea pig trachea 323 P-gp (permeability glyocoprotein) 200, 206, 246, 455, 494 Ph (þ) Philadelphia chromosome positive 493 pharmaceutical formulation 88 pharmacodynamics 4 pharmacokinetics 14, 205 pharmacological analogues XVIII–XX, 3, 29, 30, 52, 90, 234, 247, 251, 252, 258, 262 pharmacophore models 61, 71, 254–257 Pharmorubicin Ò see epirubicin phenacetin 30, 31 phenformin 48, 49 phenobarbital 46 phenprocoumon 447, 457, 459 phenytoin 46 Philadelphia chromosome (Ph þ) 493, 502 3-phosphoglycerate kinase 391 PI3K/Akt kinase 468 pilocarpine 299, 300 pioglitazone 127

pioneer drugs XVII, XVIII, 3, 5, 14, 18, 20, 29, 30, 51, 52, 63, 243, 259 p – p interaction 124 pirbuterol 321, 322 pirenzepine 298, 301 piroxicam 34 PK (pharmakokinetic) properties 15, 146, 147, 177, 180, 231, 246, 498 PKG (protein kinase G) 138 plant cell fermentation see PCF plasma clearance 199 plasma protein binding 180, 201 platelet aggregation 73 platinum compounds 9 POMS (profile of mood state) scores 519 PNU-75875 451 PNU-96988 449, 459 PNU-103017 452, 459 PNU-140690 see tipranavir pollen count 7 potency 4, 68, 69, 119, 124, 140, 141, 146, 154, 203, 223, 232, 248, 252, 258, 269, 271, 273 274, 305, 313, 343, 347, 370, 411, 424, 434, 495, 497, 501 PPCE (postproline cleaving enzyme) 114 PR (progesterone receptor) 365 practolol 5, 7 pradefovir 386 pravastatin 4, 5, 66, 67 Priftin Ò see rifapentine primaperone 281 prior art disclosure 98, 103 priority dates 84–87, 103 Pristiq Ò see desvenlafaxine procaterol 321, 322 prodrug 33, 65, 95, 196, 202, 226, 262 proglucagon 336 promethazine 36 propofol 88, 89 propranolol 5, 7 prosecution 104 prostate cancer 231 protein binding 452 protein kinases 465 proton pump inhibitor 20 protozoal infections 48 protriptylene 275 Prozac Ò see fluoxetine psoriasis 494 pyretic agent 35

q QSAR (quantitative structure-activity relationship) 203

Index q.d. or QD once a day (from Latin quaque die) 399 quetiapine 37, 38 quinidine 189, 191 quinine 189, 191 quinprenaline 322

r R152929 412, 420, 421 ramipril 18, 19 ranitidine 4, 5 rash 410, 433 rat olfactory bulbectomy model 515 rat resident intruder model 273 RBA (relative binding affinity) 363, 365 reactive acetate 35 reboxetine 284, 508, 509 receptor subtype 5 renin 376, 443, 444 – inhibitors 446 renin-angiotensin system 62 reserpine 189 – 191, 193 reserpine-induced behavioral effects in rhodents 508 reserpine-induced hypothermia in mice 271, 512 resident intruder social interaction paradigm 515 RESIST (Randomized Evaluation of Strategic Intervention in multidrug Resistant Patients with tipranavir) clinical trials 456 resistance – to antibiotics 9, 176, 178, 181, 183, 184 – to antifungal drugs 12 – to antiviral drugs 12, 385, 391, 396, 397, 400, 409–411, 434, 455 – to anticancer drugs 14, 200, 206, 222, 232, 233, 246, 259, 488, 493, 501 Ret/PTC-positive thyroid ancer 467 reverse transcription 384 RGD (arginine-glycine-aspartic acid) analogues 73 rhabdomyolysis 42 rheumatoid arthritis 494 rhinitis 7 rhodomycin A 217 L-ribose 387, 388 rifabutin 174, 177, 181, 431 Rifactane Ò see rifampicin Rifadin Ò see rifampicin rifalazil 183 rifamide 174, 176, 177, 181, 184 rifampicin 173, 177–179, 184, 431 rifampin see rifampicin

rifamycins XIX, 173, 174, 176 rifamycin B 174, 176, 182, 184 rifamycin O 174 rifamycin S 174, 176, 181, 182, 184 rifamycin SV 173, 177, 181, 182, 184 – spatial model 174, 175 rifapentine 174, 177, 180, 184 rifaximin XIX, 174, 181, 182 Rifocine Ò see rifamycin SV rilpivirine (TMC278) XX, 12, 13, 409, 412, 419, 420, 422, 424, 427, 431 – clinical studies 432 – molecular modeling 427, 428 – physicochemical properties 429 – PK 431 – synthesis 414–418 risperidone 37, 38 ritonavir 14, 23, 75, 76, 444, 445, 455 RNA (ribonucleic acid) synthesis 384 RNApol (RNA polymerase) enzyme 175, 176, 178, 184, 384 mRNA (messenger RNA) 384, 410 Robinul Ò see glycopyrrolate roflumilast 71 rolipram 70–72 rosuvastatin 4 roxatidine 4, 5 RT (reverse transcriptase) 383, 384, 386, 409, 410 RT mutations 410 RTV see ritonavir rubidazone 226 rubitecan 203, 204 “Rule of 3” guidelines for lead development 457, 458

s sabarubicin 230, 231, 234 sagopilone 254 salbutamol XIX, 320 – 323, 325, 326 salicin 33 salicylamide 35 salicylic acid 33, 35 saligenin ethanolamine 323 salmefamol 323 salmeterol XIX, 322, 324–327 salsalate 35 saquinavir 74, 444, 445, 456, 457 SAR (structure-activity relationship) XVIII, 3, 68, 113, 114, 135, 152, 174, 192, 202, 247, 253, 255, 257, 270, 282, 319, 339, 388, 412, 419, 421 422 – 427, 450, 468, 469, 474, 479, 494, 495 sarcodictyin 258

j535

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536

sarcoma 217 saxagliptin 116, 117, 123, 127 SBDD (structure-based drug design) XVIII, XX, 458 s.c. (subcutaneous) 271, 339, 341 scaffold hopping 124, 247 scalability 483 schizophrenia 35, 287 SCID (severe combined immunodeficient) mice 501, 502 SCLC (small-cell lung cancer) 201, 206, 231, 232 scopolamine 300, 301, 304, 313 scopolamine butyl bromide 303 secologanin 189, 190 secretin 335, 350 sedation 7 SEDDS (self-emulsifying drug delivery system) 455 SEF (sodium excreting factor) 360, 361 seizure 41 Seldane Ò see terfenadine selection invention 93, 94 selective b1 blockers 5, 18 selectivity 5, 113, 117–119, 124, 145, 146, 150, 154, 156, 157, 160, 250, 269, 273, 274, 276, 285, 367, 434, 480, 498 serotonergic autoreceptor 509 serotonin XIX, 37, 269, 271, 507, 509 serotonin transporter 270–272, 274, 276, 284, 285, 509, 510, 516 – allosteric site 272 serotonin 5-HT1A autoreceptor 271 serotonin 5-HT1A antagonist 282 serotonin 5-HT1B/1D autoreceptor 271 serotonin 5-HT2A/2C receptors 37 serotonin potentiation model 271 serotonin uptake inhibition (SRI) 283, 284, 517 sertindole 37, 38 sertraline 269 – 271, 276, 277, 285, 286, 508, 509 SI (selectivity index: ratio of CC50/EC50) 421, 423 sildenafil XIX, 138, 140, 142–145, 152–154 simvastatin 4, 45, 66, 67 SIV (simian immunodeficiency virus) 447 sitagliptin 113, 120, 121, 123, 125, 126, 128, 345, 346 small-cell lung cancer see LCLC small-molecule drugs 29, 30 smoking cessation 41 SMC (smooth muscle cell) 137, 150 smooth muscle relaxation 5

SN-38 202, 205, 206 SNRI (serotonin/norepinephrine reuptake inhibitor) XX, 507, 511, 520 sodium ion channels 46, 193, 194 soft drug 18 solid organ transplant 494 solid tumors 231, 503 solubility 422 solubilizer 51 sorafenib 466, 467 Sorangium cellulosum 251 soterenol 320 spironolactone XX, 361, 362 SQV see saquinavir Src (sarcoma) family kinases 493, 494, 497 Src-Abl kinase inhibitor 501 SRI (serotonin reuptake inhibitor) 510, 512, 513 SSRIs (selective serotonin reuptake inhibitors) XIX, 269, 277, 508 standalone drug XVIII, 3, 29, 30, 52 Staphylococcus aureus 9 statins 4 staurosporine 465, 466 stavudine 12, 13 stilbamidine 48 Streptococcus pneumoniae 178 Streptomyces spp. 217 Streptomyces coeruleorubidus 217 Streptomyces peucetius 217, 218, 224 strictosidine 189, 190 structural analogue 3, 29, 30 structural and pharmacological analogues see full analogues structure-based drug design see SBDD substrate-based inhibitors 121 sulfonterol 321 sulfonylurea 349 sulindac 34 sunitinib 466, 467 Sutent Ò see sunitinib synergistic interaction 179, 326, 327, 455 Synthalin A 48 Synthalin B 48 systemic activity 21 systolic blood pressure 350, 351

t tabersonine 193 tadalafil 138, 140, 142, 143, 145, 160 tail suspension test 515 Tarceva Ò see erlotinib tardive dyskinesia 37, 38 Taxus brevifolia 260

Index T-cell proliferation assay 494 TCR (T-cell antigen receptor) 494 TCR-mediated signal transduction pathways 494 TDF see tenofovir disoproxil fumarate telbivudine 386, 399 tenofovir disoproxil fumarate (TDF) 12, 13, 386, 399, 432 teratogenicity 51 terbutaline 320–322 terfenadine 8, 9, 96, 97 tetrabenazine-induced ptosis 280, 510 tetrabenazine-induced sedation 39 TGFa (tansforming growth factor-a 467 theobromine 122, 123, 147 theophylline 122, 123, 147 therapeutic index 12, 246, 468 therapeutic margin see therapeutic index Thermus aquaticus 176 thiazolidinedione 349 thymidine 392 TI (tumor inhibition) 480 tinnitus 33 TIBO (4,5,6,7-tetrahydro-5-methylimidazo [4,5,1-jk]benzodiazepin-2(1H)-one) 409, 410 t.i.d. (three times daily) 515 tioconazole 16, 17 tiotropium bromide XIX, 304, 305, 310–313 – synthesis 308, 310 – selectivity 454 tipranavir (TPV) XX, 443, 444, 453, 455, 458, 459 – binding to HIV-1 and HIV-2 PR 454 – resistance 455 – PK 455 – clinical data 456 tirofiban hydrochloride 74 TK (thymidine kinase) 391 TMC278 see rilpivirine TMPK (thymidylate kinase) 391 Topamax Ò see topiramate topiramate 49, 50, 89 topoisomerase I 195, 201, 203, 204, 206, 487 topoisomerase II 218, 226, 227, 228, 231, 232 topotecan (TPT) 201, 203, 205 – 207, 487 toxiferine 189, 191 TPV see tipranavir TPV/r (tipranavir/ritonavir combination) 455–457 TPT see topotecan trandolapril 18, 19 transition-state analogues/isostere 74, 111, 444

trastuzumab 488 travel diarrhea 182 tricyclic antidepressants 269, 507, 508, 514 TRIPs (Trade-related Aspects of Intellectual Property Rights) 87 tropane alkaloids XIX, 300, 302, 313 S-(-)-tropinoylcholine 303 trypanosomiasis 48 tryptamine 189, 190 TTP (time to progression) 488 “typical” antipsychotic drugs 36 tuberculosis 173, 174, 177, 179, 180, 184 tubulin polymerization 195, 199 tulobuterol 321 Tykerb Ò see lapatinib tyrosine kinase receptors 467

u udenafil 140, 146 UDP (uridine diphosphate) 206 UGT 1A1 (UDP-glucuronosyltransferase 1A1) 206 UK-47265 ultrashort-acting drugs 18 unmet medical need 270 UPLIFT trial utility 93 USAN (United States Adopted Names) 184

v valnoctamide 51, 52 valproate 51 valpromide 51 valtorcitabine 386 van der Waals interaction 117 vancomycin 182 vardenafil 138, 140, 142, 143, 145, 155 varenicline 42 VEGFR2 (vascular endothelial growth factor receptor 2) 466 Velban Ò see vinblastine venlafaxine XX, 507, 510, 511, 514, 520 – active metabolite 516 – clinical studies 515, 516 – CYP2D6 interaction 518 – extended release formulation 516 – metabolism 516, 517 – onset of action 518 – pharmacology 515 Viagra Ò see sildenafil vildagliptin 116, 122, 123, 125–127, 345, 347 vinblastine and analogues 189, 191, 195, 196, 198 – 200, 207 vinburnine 190, 192, 194

j537

j Index

538

Vinca minor 190 Vinca rosea see Catharantus roseus vincamine and analogues XIX, 189–194, 207 vincristine 189, 191, 195, 198–200 vindeburnol 192, 193 vindesine 196, 200, 206 vinepidine 197, 198 vinflunine 197 vinformide 197, 198 vinglycinate 196, 198 vinorelbine 196, 199–201, 206 vinpocetine 190, 192, 194 vintoperol 192 vintriptol 196, 198 vinzolidine 196, 198 viral life cycle 443, 444 viral polymerase 386 virion 385 vitamins 30 VMS (vasomotor symptoms) 519 Vocanga africana 193 vomiting 206 voriconazole 12 VSMC (vascular smooth muscle cell) 137 VSS (steady-state volume) 393, 497

w warfarin 447, 457 WBC (white blood cell)

Wellbutrin Ò see bupropion WHcAg (woodchick hepatitis virus core antigen) 393 WHsAg (woodchuck hepatitis virus surface antigen) 393 WHV (woodchuck hepatitis virus) 393 willow bark 33 WTO (World Trade Organization) 87

x xanthine alkaloids 122 X-ray crystallography 458 L-xylose 387

y yew’s bark 244

z zalcitabine 389 zaprinast 150 Zavedos Ò see idarubicin ZetiaÒ see ezetimibe zidovudine 12, 13, 392, 409, 432, 455 zimelidine 273–275 ziprasidone 37, 38 Zoladex Ò see goserelin Zoloft Ò see sertraline Zyban Ò see bupropion