Acquisitions Editor: Richard Winters Developmental Editor: Erin O'Connor Manufacturing Manager: Dennis Teston Production Manager: Kathleen Bubbeo Production Editor: Eve S. Ferdman Cover Designer: Karen Quigley Indexer: Maria Coughlin ‘Composit: Maryland Composition Printer: Courier Westford © 1998, by Lippincott-Raven Publishers, All rights reserved. This book is protected by copyright. No part of it may be reproduced, stored in a retrieval system, or transmitted, in ‘any form or by any means—electronic, mechanical, photocopy, recording, or “otherwise —without the prior written consent ofthe publisher, except for brief quotations ‘embodied in critical articles and reviews. For information write Lippincott~Raven Publishers, 227 East Washington Square, Philadelphia, PA 19106-3780. ‘Materials appearing in this book prepared by individuals as part oftheir official duties as US. Government employees are not covered by the above-mentioned copyright Printed in the United States of America 9876543 Library of Congress Cataloging-in-Publication Data ‘Wilson and Gisvold’s textbook of organic medicinal and pharmaceutical chemistry.— 10th ed / edited by Jaime N. Delgado and William A. Remers : 24 contributors. pom, Includes bibliographical references and index. SBN 0-397-51583-9 (hardcover) 1. Chemistry, Pharmaceutical. 2. Chemistry, Organic, 1. Wilson, Charles Owens, 1911- . Il Gisvold, Ole, 1904. IIL Delgado, Jaime N,V, Remers, William A. (William Alan), 1932~ . V. Title: ‘Textbook of organic medicinal and pharmaceutical chemistry [DNLM: 1, Chemistry, Pharmaceutical. QV 744 W754 1998], RS403,Y43_ 1998 615'19—DC2 DNLM/DLC for Library of Congress 98-22502 cP Care has been taken to confirm the accuracy of the information presented and to describe generally accepted practices. However, the author, editors, and publisher are not responsible for errors or omissions or for any consequences from application of the information in this book and make no warranty, express or implied, with respect to the contents ofthe publication. Te authors, editors, and publisher have exerted very effort to ensure that drug selection and dosage set forth inthis text are in accordance wih current recommendations and practice at the time of publication. However, in view of ongoing research, changes in fovernment regulations, and the constant flow of information relating to drug therapy and tig reactions, the reader is urged to check the package insert foreach drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new or infrequently employed drug, ‘Some drugs and medical devices presented in this publication have Food and Drug [Adminstration (FDA) clearance for limited use in restricted research settings. Iti the ‘esponsibility of the health care provider to ascertain the FDA status of each drug or device planned or use in-theie clinical practice.CONTENTS CONTRIBUTORS xii PREFACE w CHAPTER T Introduction 1 Jaime N. Delgado and Wiliam A, Remers CHAPTER? Physicochemical Properties in Relation to Biological Action 3 John H. Block Overview 3 Drug Distribution 3 Acid-Base Properties 10 Statistical Prediction of Pharmacological Activity 18 Combinatorial Chemistry 25 Molecular Modeling (Computer-Aided Drug Design) 26 CHAPTERS Metabolic Changes of Drugs and Related Organic Compounds 43 Lawrence K. Low General Pathways of Drug Metabolism 43 Sites of Drug Biotransformation 45 Role of Cytochrome P-450 Monoxygenases in Oxidative Biotransformations 45 Oxidative Reactions 48 Reductive Reactions 81 Hyérolytic Reactions 89 Phase II, or Conjugation, Reactions 91 Factors Affecting Drug Metabolism 109 CHAPTER 4 Drug Latentiation and Prodrugs 123 Forest T. Smith and C. Randall Clark History 123 Basic Concepts _123vi CONTENTS Prodrugs of Functional Groups 125 Bioprecursor Prodrugs 132 Chemical Delivery Systems 134 CHAPTERS Biotechnology and Drug Discovery 139 John W. Regan Cloning DNA 139 Expression of Cloned DNA 142 Manipulation of DNA Sequence Information 143 New Biological Targets for Drug Development 144 Novel Drug-Screening Strategies 145 Novel Biological Agents 147 Antibodies 148 Antisense Oligonucleotide Therapy 148 Gene Therapy 149 Products 150 CHAPTER 6 Fundamentals of Immunology and Immunizing Biologicals (Vaccines and Toxoids) 153 ohn Mt. Beale Disease Prevention: An Historical Perspective 153 Fundamental Immunological Nomenclature 154 ‘The Chemical Nature of an Antigen 155 Fundamental Immunological Concepts 156 ‘Antibody Types and Reactions 162 Immunobiologicals (Vaccines and Toxoids) 164 CHAPTER? Anti-Infective Agents 173 Aros R. Martin Local Anti-Infective Agents 173 Alcohols and Related Compounds 174 Phenols and Their Derivatives 176 Oxidizing Agents 178 Halogen-Containing Compounds 178 Chlorine-Containing Compounds 179 Cationic Surfactants 180 Dyes 181 Mercury Compounds 182 Preservatives 183 Antifungal Agents 185 Antifungal Antibiotics 193 Synthetic Antibacterial Agents 196 Antitubercular Agents 204 nntitubercular Antibiotics —20'conrenrs | Vii Antiprotozoal Agents 210 Anthelmintics 216 Antiscabious and Antipedicular Agents 219 CHAPTER 8 Sulfonamides, Sulfones, and Folate Reductase Inhibitors with Antibacterial Action 223 Dwight S. Fullerton Sulfonamides and Folate Reductase Inhibitors 223 Well-Absorbed, Short-, and Intermediate-Acting Sulfonamides 229 Sulfonamides for Ophthalmic Infections 231 Sulfonamides for Burn Therapy 231 Sulfonamides for Intestinal Infections, Ulcerative Colitis, or Reduction of Bowel Flora 231 Folate Reductase Inhibitors 232 Sulfones 232 CHAPTER 9 Antimalarials 235 Dwight §.Fulerton Etiology 235 History 237 Modern Malaria Chemotherapy: An Overview 240 Development of Malaria Vaccines: Promising or a Failed Promise? 240 Quinolines and Analogues 241 Cinchona Alkaloids 244 7-Chloro-4-Aminoquinolines 246 8-Aminoquinolines 247 9-Aminoacridines 248 Mefloquine 248 Tetrahydrofolate Synthesis Inhibitors 248 Diaminopyrimidines 249 Biguanides and Dihydrotriazines 250 Sulfonamides 251 Sulfones 251 Other Antimalarials 251 CHAPTER 10 Antibacterial Antibiotics 253 ‘Aros. Marin Historical Background 253 Current Status 253, BeLactam Antibiotics 255 ‘The Aminoglycosides 291 ‘The Tetracyclines 299 ‘The Macroclides 307 The Lincomycins 312 The Polypeptides 314viii | conrenrs CHAPTER 14 Antiviral Agents 327 Arnold F Martin Properties of Viruses 327 Viral Classification 327 Prevention of Viral Infection by Chemoprophylaxis 327 Nucleoside Antimetabolites 331 Agents Under Development for HIV Infection 337 CHAPTER 12 Antineoplastic Agents 343, Wiliam A, Remors ‘Tumor Cell Properties 343 Alkylating Agents 347 Antimetabolites 356 Antibiotics 368 Plant Products 379 Miscellaneous Compounds 382 Hormones 388 Immunotherapy 391 Future Antineoplastic Agents 393 CHAPTER 13 Agents for Diagnostic Imaging 403 l8CK N. Halland Tir B. Hunter Introduction to Radiation 403 Biological Effects of Radiation 406 Radionuclides and Radiopharmaceuticals for Organ Imaging 407 Radionuclide Production 408 Technetium Radiochemistry 411 Fluorine Radiochemistry 418 Gallium Radiochemistry 418 Todine Radiochemistry 419 Indium Radiochemistry 420 Thallium Radiochemistry 422 Xenon Radiochemistry 423 Radiologic Contrast Agents 423 Paramagnetic Compounds 426 Radiologic Procedures 427 CHAPTER 14 Central Nervous System Depressants 435 Eugene |. Isaacson Some Mechanisms of Action: Anesthetics, Sedative-Hypnotics, Anxiolytics, and Anticonvulsants 435 General Anesthetics 436conTents | ix Central Nervous System Depressants Antipsychotics 449 Anticonvulsant or Antiepileptic Drugs 456 Skeletal Muscle Relaxant Properties 448 CHAPTER 15 Central Nervous System Stimulants 463 Eugene Isaacson Analeptics 463 Methylxanthines 464 Central Sympathomimetic Agents (Psychomotor Stimulants) 465 Monamine Oxidase Inhibitors 469 Tricyclic (and Mechanistically Related) Antidepressant Compounds 471 Psychedelics 474 CHAPTER 16 Adrenergic Agents 479 Rodney L Jahnson Adrenergic Neurotransmitiers 479 Adrenergic Receptors 482 Drugs Affecting Adrenergic Neurotransmission 484 Sympathomimetic Agents 485 Adrenergic Receptor Antagonists 494 CHAPTER 17 Cholinergic Drugs and Related Agents 505, George H. Cocolas Cholinergic Receptors 506 Cholinergic Neurochemistry 510 Cholinergic Agonists 511 Cholinergic Receptor Antagonists 515 Cholinergic Blocking Agents 527 Parasympathetic Postganglionic Blocking Agents 529 Solanaceous Alkaloids and Analogues 530 Synthetic Cholinergic Blocking Agents 535 Ganglionic Blocking Agents 542 Neuromuscular Blocking Agents 545 CHAPTER 18 Diuretics 553 Daniel A. Kooche! Anatomy and Physiology of the Nephron 553 Function 553 Introduction to the Diuretics 559 Site 1 Diuretics: Carbonic Anhydrase Inhibitors 561 Site 3 Diuretics: Thiazide and Thiazide-like Diuretics 563 Site 2 Diuretics: High-Ceiling or Loop Diuretics 569 Site 4 Diuretics: Potassium-Sparing Diuretics 575 Miscellancous Diuretics 579 Summary 579x | CONTENTS CHAPTER 19 Cardiovascular Agents 583 George H. Cocolas Antianginal Agents and Vasodilators 583 Antiarrhythmic Drugs 594 Antihypertensive Agents 603 Antihyperlipidemic Agents 614 Anticoagulants 620 Synthetic Hypoglycemic Agents 625 Thyroid Hormones 627 Antithyroid Drugs 628 CHAPTER 20 Local Anesthetic Agents 631 Gareth Thomas and David E. Thurston Historical Development 631 Nervous System 634 Mechanism of Action 640 Administration 642 Factors Influencing the Effectiveness of the Anesthetic Action 642 Rate of Onset and Duration of Anesthesia 644 Secondary Pharmacological Action 644 Structure-Action 645 Lexicon of Local Anesthetics 648 CHAPTER 21 Histamine and Antihistaminic Agents 657 Thomas N. Ploy and Jack DeRuiter Histamine 657 Histamine Life Cycle 657 Histamine H,-Receptor Antagonists: Antihistiminic Agents 661 Inhibition of Histamine Release 675 Histamine Hy-Receptor Antagonists 676 Histamine Hj-Receptor Ligands 683 CHAPTER 22 Analgesic Agents 687 Robert E Wilt Morphine and Related Compounds 687 Antitussive Agents 709 Anti-Inflammatory Analgesics 711 CHAPTER 23 Steroids and Therapeutically Related Compounds 727 Dwight S. Fullerton Steroid Receptors 727 Steroid Nomenclature, Stereochemistry, and Numbering 731conTents | xi Steroid Biosynthesis 735 Chemical and Physical Properties of Steroids 736 Changes to Modify Pharmacokinetic Properties of Steroids 736 Gonadotropins 736 Sex Hormones 741 Estrogens 741 Antiestrogens and Related Drugs 750 Progestins 752 Chemical Contraceptive Agents 755 Androgens and Anabolic Agents 764 Adrenal Cortex Hormones 770 Cardiac Steroids and Related Inotropic Drugs 781 Steroids with Other Activities 794 Commercial Production of Steroids 795 CHAPTER 24 Prostaglandins, Leukotrienes, and Other Eicosanoids 803 Tromas J Holmes, Jr History of Discovery 803 osanoid Biosynthesis 803 Drug Action Mediated by Eicosanoids 807 Design of Eicosanoid Drugs 808 Eicosanoids in Clinical Development for Human Treatment 810 CHAPTER 25 Carbohydrates 813 Vilas A. Prabhu and Jaime N, Delgado Classification 813 Biosynthesis 816 Stereochemical Considerations 816 Interrelationships with Lipids and Proteins 817 Sugar Alcohols 818 Sugars 819 Starch and Derivatives 822 Cellulose and Derivatives 822 Heparin 825 Glycosides 828 CHAPTER 26 Amino Acids, Proteins, Enzymes, and Peptide Hormones «aime N, Delgado and Vilas A, Prabhu Amino Acids 831 Protein Hydrolysates 835 Amino Acid Solutions 835 Protein and Protein-like Compounds 836 Enzymes 841 Hormones 846 831| conrenrs Blood Proteins 864 ‘The Impact of Biotechnology on the Development and Commercial Production of Proteins and Peptides as Pharmaceutical Products 865 Biotechnology-Derived Pharmaceutical Products 868 CHAPTER 27 Vitamins and Related Compounds 875 Gustavo F. Ortega and Jaime N. Delgado Lipid-Soluble Vitamins 875 Water-Soluble Vitamins 894 Miscellaneous Considerations 910 APPENDIX pK,s of Drugs and Reference Compounds 915 INDEX 923CONTRIBUTORS John M, Beale, Ph.0. ‘Associate Professor of Mecicinal Chemistry Director, Division of Pharmaceutical Sciences ‘St Louis College of Pharmacy 14588 Parkview Place ‘St Louis, Missoun' 69110-1088 ‘John H. Block, Ph.D. Professor of Modical Chemistry Colege of Pharmacy (regan State University £203 Pharmacy Bulging Covvalis, Oregon 97331-3507 . Randall Clark, Ph.D, Professor of Medicinal Chemistry Department of Pharmacal Sciences ‘Auburn University School of Pharmacy ‘362 Thach Avenue ‘Auburn, Alabama 36849-5501 George H. Cocolas, Ph.D. Professor of Medicinal Chemistry ‘School of Pharmacy University of North Carolina at Chapot Hil (Chapel Hill, North Carolina 27599 Jaime N. Delgado, Ph.D. Protessor of Medicinal Chemistry Jacques P. Servier Regents Professorship in Pharmacy University Distinguished Teaching Professor College of Pharmacy The University of Texas at Austin Austin, Texas 78712 ‘Jack DeRulter, Ph.D. Professor of Medicinal Chemistry Department of Pharmaceutical Sciences ‘Auburn University ‘Schoo! of Pharmacy Auburn, Alabama 36849-5501 Dwight S. Fullerton, Pho. Professor of Pharmacy (afillate) ‘School of Pharmacy University of Washington and Vice President, Pharmacy agence Blue Shield MS 519 Seat, Washington 96111-3267 Jack N. Hall, MS., RPh, BONP ‘Ginical Lecturer Department of Radiology’Nuclear Modicine College of Medicine, University of Arizona University of Arizona Health Sciences Center 1501 N. Campbell Avenue P.O, Box 245068 Tucson, Arizona 85724 Thomas J. Holmes, Jr, Ph.0. Associate Professor of Medicinal Chemistry ‘Schoo! of Pharmacy Campbell University 101 Main Street Buies Creek, North Carolina 27506-1090 Tim B, Hunter, MO. Vice-Chairman and Professor of Radiology Department of Radiology Collage of Medicine, University of Arizona University of Arizona Health Sciences Center Tucson, Arizona 85724 Eugene I. Isaacson, Ph.D. Professor of Pharmaceutical Chemistry ‘Department of Pharmaceutical Sciences College of Pharmacy Idaho State University Pocatello, Idaho 83209-8268 Rodney L. Johnson, Ph.D. Professor of Medicinal Chemistry Department of Medicinal Chermstry College of Pharmacy University of Minnesota 308 Harvard Street, Southeast ‘Minneapolis, Minnesota 55455-0343 Daniel A. Koschel, Ph.D. Professor Emeritus, Pharmacology Department of Pharmacology ‘Medical College of Ohio 3035 Arlington Avenue Toledo, Ohio 43614-5804 Lawrence K. Low, Ph.D. Group Leader Metabolism Biodegeneration Section Product Stewardship and Toxicology Mobil Business Resources Corp. £600 Bilingsport Road Paulsboro, New Jersey 08066 xiilxiv | CONTRIBUTORS Arnold R. Martin, Ph.D. Frofessor of Medicinal Chemistry College of Pharmacology University of Arizona’ Tucson, Arizona 85721 Gustavo R. Ortega, Ph.0. Professor of Medicinal Chemistry Department of Pharmaceutical Sciences ‘Schoo! of Pharmacy ‘Southwestern Oklahoma State University 100 Campus Drive Weatherford, Oklahoma 73096 Vilas A. Prabhu, PhO. Chair and Bemharat Professor of Pharmacy Department of Pharmaceutical Sciences ‘Schoo! of Pharmacy ‘Southwestern Oklahoma State University 100 Campus Drive Weatherford, Oklahoma 73096 John W. Regan, Ph.D, M.D.(Hon.) ‘Associate Professor ‘Department of Pharmacology and Toxicology College of Pharmacy Univeristy of Arizona P.O. Box 210207 Tucson, Arizona 85721-0207 Witiam A. Remers, Ph.D. Professor of Medicinal Chemistry College of Pharmacy The Univeristy of arizona Tucson, Arizona 85721 ‘Thomas N. Riley, Ph.D. Professor of Medicinal Chemistry ‘School of Pharmacy ‘Auburn Univeristy. ‘Auburn, Alabama 96849-5503 Forrest T. Smith, PhD. ‘Associate Professor of Medicinal Chemistry Department of Pharmacal Sciences ‘Aubum University ‘Schoo! of Pharmacy 1362 Thach Avenue ‘Auburn, Alabama 36649-5501 Gareth Thomas, PhO. ‘Senior Lecturer, Admissions Tutor ‘School of Pharmacy and Biomedical Sciences Portsmouth Polytechnic White Swan Road Portsmouth Hants POY 20Z, United Kingdom David E. Thurston, Ph.0. Professor of Medicinal Chemistry ‘Schoo! of Pharmacy and Biomedical Sciences Portsmouth Polytechnic White Swan Road Portsmouth Hants PO! 202, United Kingdom Robert E. Willette, Ph.D. President ‘Duo Research, Inc. 2419 East Fifth Avenue ‘Denver, Colorado 60206-4967PREFACE For almost five decades Wilson and Gisvold’s Textbook of Organic Medicinal and Pharmaceutical Chemistry has been a standard in the literature of medicinal chemistry, and the recent editions edited by Professor Robert F. Doerge have continued to receive national and international acceptance, Generations of students and faculty have depended on this textbook not only for undergraduate courses in medicinal chemistry but also to supplement graduate studies. More- over, students in other health sciences have found certain chapters useful at one time or another. It was therefore de- cided to revise the book, and the current editors and authors ‘worked on the tenth edition with the objective of continuing the tradition of a modern textbook for undergraduate stu- dents and also for graduate students who need a general review of medicinal chemistry. Since the chapters include a blend of chemical and pharmacological principles necessary for understanding structure-activity relationships and molec- ular mechanisms of drug action, the book should be useful in supporting courses in medicinal chemistry and in comple- ‘menting pharmacology courses ‘The nature and breadth of medicinal chemistry are dis- cussed inthe introduction, and the chapters on physicochem- ical properties and on drug metabolism provide the fun ‘mental theme of the conceptual approach to the study of medicinal chemistry. This theme continues as the basis for all the subsequent chapters on the classical types of therapeu- tic agents. In order to continue to integrate the basics of medicinal chemistry with recent advances that have led to contemporary phamacotherapeutics, the tenth edition has been designed to include new chapters on drug latentiation and prodrugs, immunizing biologicals, diagnostic imaging agents, and biotechnology. Moreover, the editors welcome the following new contributors to the tenth edition: Drs. Beale, Clark, DeRuiter, Hall, Hunter, Regan, Riley, Thomas, and Thurston. The chapters include summaries of current research trends that lead the reader to the original literature via bibliographies. Documentation and references continue to be an important feature of the book. ‘An important area of medicinal chemistry is the chemistry and biology of pharmaceutically important natural products, hence chapters include antibiotics, glycosides, and the study of alkaloids as prototypes that lead to synthetic medicinal. The table of contents reflects the important pharmacological types of therapeutic agents. To the extent possible each type is structurally characterized with mechanisms of action and therapeutic and pharmaceutical applications and limitations. The editors extend thanks to all the authors who have effectively cooperated in the preparation of the current edi- tion. Collectively, the authors represent many years of teach- ing and research experience in medicinal chemistry. The students also deserve acknowledgment of their critiques that directly and indirectly contributed to the quality of this edi- tion Finally, we continue to be indebted to Professors Charles, , Wilson and Ole Gisvold, the originators of the book, and to the editors and authors who worked on and stimulated the continuation of the many revisions that have led to the current edition. They have significantly contributed to the education of countless pharmacists, medicinal chemists, and other pharmaceutical scientists. Jaime N. Delgado William A. Remers xvWILSON AND GISVOLD'S Textbook of Organic Medicinal and Pharmaceutical ChemistryCHAPTER 1 Introduction Jaime N. Delgado William A, Remers The discipline of medicinal chemistry is devoted to the dis- covery and development of new agents for treating diseases. Most of this activity is directed to new natural or synthetic, ‘organic compounds. Inorganic compounds continue to be important in therapy, for example, as antacids, mineral sup- plements, and radiopharmaceuticals, but organic molecules, with increasingly specific pharmacological activities are clearly dominant. This book treats many aspects of organic rmedicinals: how they are discovered, how they act, and how they are developed into clinical agents, The process of estab- lishing a new pharmaceutical is exceedingly complex and involves the talents of people from a variety of disciplines, including chemistry, biochemistry, molecular biology, phys- ‘ology, pharmacology, pharmaceutics, and medicine, Medic- inal chemistry is concerned mainly with the organic, analyti- cal, and biochemical aspects of this process, but its scientists must interact productively with those in other disciplines. Thus, itoccupies a strategic position atthe interface of chem- istry and biology. ‘The earliest drug discoveries were probably made by ran- dom sampling of higher plants. Crude plant drugs such as opium, belladonna, and ephedrine have been important for centuries. Knowledge of crude drugs was codified into the discipline of pharmacognosy, which has held a distinguished place in the pharmaceutical sciences. Pharmacognosy has changed with time, embracing microbiology when the antibi- ties were discovered, and venturing into drug biosynthesis as biochemistry developed. In recent years, however, crude drug preparations have been largely replaced by their iso- lated and purified active components, making it unnecessary for pharmacists to verify the identity of natural materials. This factor, together with the inereasing importance of syn- thetic organic compounds, has led to reduced emphasis on pharmacognosy in the pharmacy curriculum and often to its, combination with medicinal chemistry. This textbook, while itdoes not presume to teach pharmacognosy, contains much valuable information and insight into the sources, isolation, and standardization of natural product drugs. Hundreds of thousands of new organic chemicals are pre~ pared annually throughout the world, and many of them are entered into pharmacological screens to determine whether they have useful biological activity. This process of random screening has been considered inefficient, but it has resulted in the identification of new lead compounds whose structures have been optimized to produce clinical agents. The antitu- bercular drug, ethambutol, was developed in this manner. More recently, automated high-throughput screening sys- tems utilizing cell culture systems with linked enzyme assays, and even receptor molecules derived from gene cloning have greatly increased the efficiency of random screening. It is row practical to screen enormous libraries of peptides and nuclei acids obtained from combinatorial chemistry proce- dures. ‘The opposite approach to high-volume screening, rational design, has also flourished recently. Significant advances in ray crystallography and nuclear magnetic resonance have made it possible to obtain detailed representations of en- zymes and other drug receptors. Based on these representa- tions, the techniques of molecular graphics and computa- tional chemistry have provided novel chemical structures that have led to new drugs with potent medicinal activities. The newly introduced inhibitors of HIV protease were dis- covered in this way. Even if the receptor structure is not known in detail, rational approaches based on the physico- chemical properties of lead compounds can provide new drugs. For example, the development of cimetidine as an antiuleer drug involved a careful study of the changes in antagonism of Hz histamine receptors induced by varying the physical properties of structures based on histamine, Sta- tistical methods, such as the Hansch analysis, which is based ‘on the correlation of physicochemical properties with bio- logical potency, are popular in attempts to optimize biologi- cal activity or at least to rationalize it, Significant developments in medicinal chemistry have oc- curred since the 9th edition of this textbook was written. 42 | irRODUCTION ‘They have made it desirable to add new chapters to this 10th edition. Thus, the strong focus on antiviral drug research, driven largely by the need to treat HIV infections, has prompted the creation of a separate chapter on antiviral agents, Dramatic progress in the application of molecular biology to the production of pharmaceutical agents has pro- duced such important molecules as insulin, growth hormone, granulocyte colony-stimulating factor, erythropoietin, and clotting factor VIII, all products of cloned human genes, Molecular biology has provided for the definition of new drug receptor subtypes that may be useful in designing more selective therapeutic agents, and it has been important in the development of monoclonal antibodies for diagnosis and therapy. A new chapter on biotechnology describes these exciting applications and other possibilities for therapy, in. cluding antisense oligonucleotides and gene transfer meth- ods. Biological agents are hardly new, but recent advances in understanding the immune system at the molecular level, including detailed chemical structures of immunoglobulins, make them especially interesting to medicinal chemistry, ‘Techniques of genetic engineering now allow the prepara- tion of pure surface antigens as vaccines while totally elimi- nating the pathogenic organisms from which they are de- rived. These advances are described in the new chapter on biologicals. Examples of drug latentiation have been given throughout this textbook in previous editions, but this important concept has not been treated in an organized manner. A new chapter in this edition presents the theoretical basis of drug latentia- tion and provides a carefully organized set of examples in which the therapeutic value of drugs is significantly en- hanced. Another new chapter is devoted to agents for diagnostic imaging. It includes both radiopharmaceuticals (nuclear medicine) and contrast agents, mainly iodinated organic compounds, that absorb x-rays (radiology), Fundamental concepts of radiation and its biological effects are described, and numerous practical applications in diagnosis are pro- vided. All of the chapters in the 10th edition have been revised, and those in which significant new discoveries were made have been completely rewritten. Together with the new chap- ters, they provide an outline of medicinal chemistry that is ‘contemporary and authoritative in its treatment of scientific concepts, as well as sufficiently specific about pharmaceuti- cal products to be a valuable reference for the practicing pharmacist.CHAPTER 2 Physicochemical Properties in Relation to Biological Action John H. Block Modern drug design, as compared with the classical ap- proach—Let’s make a change on an existing compound or synthesize a new structure and see what happens—has undergone a rapid evolution as an approach to solving a drug design problem. The increasing power and decreasing cost of desktop computing has had @ major impact on solving dug design problems. While drug design increasingly is tsed on modern computational chemical techniques, i also utilizes sophisticated knowledge of disease mechanisms and receptor properties. A good understanding of how the drug istransported into the body, distributed throughout the body compartments, metabolically altered by the liver and other organs, and excreted from the patient is required along with, the structural characteristics of the receptor. Acid-base chemistry is utilized to aid in formulation and biodistribu- tion. Those structural attributes and substituent patterns responsible for optimum pharmacological activity can be predicted many times by statistical techniques such as regression analysis. Computerized conformational analysis permits the medicinal chemist to predict the drug’s three- dimensional shape that is seen by the receptor. With the isolation and structural determination of specific receptors and the availability of computer software that can estimate the three-dimensional shape of the receptor, it is possible to design molecules that will show an optimum fit to the receptor. OVERVIEW A drug is a chemical molecule. Following introduction into the body, a drug must pass through many barriers, survive altemate sites of attachment and storage, and avoid signifi- cant metabolic destruction before it reaches the site of action, usually a receptor on or in a cell (Fig. 2-1). At the receptor, the following equilibrium (Rx. 2-1) usually holds: Drug + Receptor = Drug-Receptor Complex Pharmacologic Response (Rx. 2-1) ‘The ideal drug molecule will show favorable binding char- acteristics to the receptor such that the equilibrium lies to the right. At the same time, the drug will be expected to dissociate from the receptor and reenter systemic circulation to be excreted. Major exceptions include the alkylating agents used in cancer chemotherapy (see Chap. 4) and a {ew inhibitors of the enzyme acetylcholinesterase (see Chap. 17). Both of these subclasses of pharmacological agents form covalent bonds with the receptor. In these cases, the cell must destroy the receptor, or, in the case of the alkylating agents, the cell would be replaced, ideally with a normal cell, In other words, the usual use of drugs in medical treat- ment calls for the drug’s effect to last for only a finite period of time. Then, ifitis tobe repeated, the drug will be adminis- tered again. Ifthe patient does not tolerate the drug well, it is even more important that the agent dissociate from the receptor and be excreted from the body. DRUG DISTRIBUTION ORAL ADMINISTRATION ‘An examination of the obstacle course (Fig. 2-1) faced by the drug will give a better understanding of what is involved in developing a commercially feasible product. Assume that the drug is administered orally. The drug must go into solu- tion in order for it to pass through the gastrointestinal mu- cosa. Even drugs administered as true solutions may not remain in solution as they enter the acidic stomach and then pass into the alkaline intestinal tract. (This will be explained further in the discussion on acid-base chemistry.) The ability 3 a4 | PHYSICOCHEMICAL PROPERTIES IN RELATION TO BIOLOGICAL ACTION Lounauisn Baye jo ewan “be “Bl suoygetnozyo oywe3eks oquy At300z"p pezeasyuyupe Bnaq ---—~ ssouezque yénoaya ssed emu Sn20 fail saurTogweaK seoed onwid~onud 30 uoTIez0%a 8300333 peatsepun 303 fa onp sz03de00u Rouptx, ] yeuyasoaur | <—— [| wsytoqezou Snap ysou 30 ays tzeA7T eta t sazxrroavsan ona sasrnowes oowa sasrrogwiay cova —-=«eaarTogwrsw awa ona NOLVINOUID OIWaLSAS — [ eHEaTY Erses awa eer ere | sarrroavian onua ona onea ona [ sa00333 | peatsea 33 sx03da20y | uorazefur | snousaeaaur | 30835 | qeuyasazuTor3se9 uotas0fur snoaueansqns zeqnosnuresqur uoTsea3s;uTWPY TeI0ofthe drug to dissolve is governed by several factors, includ- ing its chemical structure, variation in particle size and parti cle surface area, nature of the crystal form, type of tablet coating, and type of tablet matrix. By varying the dosage form and physical characteristics of the drug, itis possible tohave a drug dissolve quickly or slowly, the latter being the situation for many of the sustained action products. An example is orally administered sodium phenytoin, where Variation of both the crystal form and tablet adjuvants can significantly alter the bioavailability ofthis drug widely used inthe treatment of epilepsy. Chemical modification is also used, to a limited extent, to facilitate a drug reaching its desired target. An example isolsaazine, used in the treatment of ulcerative colitis. This «drug isa dimer of the pharmacologically active mesalamine (G-aminosalicylic acid). The latter is not effective orally be- cause itis metabolized to inactive forms before reaching the colon, The dimeric form passes through a significant portion ofthe intestinal tract before being cleaved by the intestinal bacteria to two equivalents of mesalamine. Ho0G, (coo non Olsatazine O00, Nan Mesaiamine As illustrated by olsalazine, any compound passing through the gastrointestinal tract will encounter the large umber and variety of digestive and bacterial enzymes, ‘which, in theory, can degrade the drug molecule. In practice, when a new drug entity is under investigation, it will likely be dropped from further consideration if it is found to be unable to survive in the intestinal tract. An exception would bea drug for which there is no other effective product avail- able or which provides a more effective treatment over exist- ing products and can be administered by an alternate route, including parenteral, buccal, or transdermal. In contrast, these same digestive enzymes can be used to advantage. Chloramphenicol is water-soluble enough (2.5 mg/ml) that it comes in contact with the taste receptors on the tongue, producing an unpalatable bitterness. In order to mask this intense bitter taste, the palmitic acid moiety is, aed as an ester of the chloramphenicol’s primary alcohol. ‘This reduces the parent drug’s water solubility (1.05 mg/ml) enough so that it can be formulated as a suspension that ‘pases over the bitter taste receptors on the tongue. Once in, the intestinal tract, the ester linkage is hydrolyzed by the digestive esterases to the active antibiotic chloramphenicol and the very common dietary fatty acid, palmitic aci. DRUG DISTRIBUTION | 5 a whee ow (pecs on Chloramphenicol: R= H Chloramphenicol! Palmitate: R = CO(CH,) 44CHs Olsalazine and chloramphenicol palmitate are examples of prodrugs. Most prodrugs are compounds that are inactive in their native form, but are easily metabolized to the active agent, Olsalazine and chloramphenicol palmitate are exam- ples of prodrugs that are cleaved to smaller compounds, one of which will be the active drug. Others are metabolic precur- sors to the active form. An example of this type of prodrug is menadione, a simple naphthoquinone, which is converted in the liver to vitamin K2,20, 9 or ° Menadione x Phytonadione (Vitamin K¢20)) Occasionally, the prodrug approach is used to enhance the absorption of a drug that is poorly absorbed from the gastrointestinal tract. Enalapril is the ethyl ester of enala- prilic acid, an active inhibitor of angiotensin converting en- zyme (ACE). The ester prodrug is much more readily ab- sorbed orally than the pharmacologically active carboxylic acid, em 8 T WER O Enalapril: R= CzHs Enalaprilic Acid: R Unless the drug is intended to act locally in the gastroin- testinal tract, it wll have to pass through the gastrointestinal ‘mucosa barrier into venous circulation in order to reach the site of the receptor. The drug's route involves distribution ‘partitioning between the aqueous environment ofthe ga trointestinal tract, the lipid bilayer cell membrane of the mu- ‘cosa cells, possibly the aqueous interior of the mucosa cells, the lipid bilayer membranes on the venous side of the gastro-6 | PHYSICOCHEMICAL PROPERTIES IN RELATION TO BIOLOGICAL ACTION intestinal tract, and the aqueous environment of venous cir- culation. Some very lipid-soluble drugs may follow the route of dietary lipids by becoming part of the mixed micelles, passing through the mucosa cells into the lymph ducts, serv- icing the intestines, and finally entering venous circulation via the thoracie duct The drug's passage through the mucosa cells can be pas- sive or active. As will be discussed later in this chapter, the lipid membranes are very complex with a highly ordered structure. Part of this membrane is a series of channels or tunnels that form, disappear, and reform. There are receptors that move compounds into the cell by a process called “*pino- ytosis.”* Drugs that resemble a normal metabolic precursor or intermediate may be actively transported into the cell by the same system that transports the endogenous compound. On the other hand, most drug molecules are too large to center the cell by an active transport mechanism through the passages. PARENTERAL ADMINISTRATION Many times there will be therapeutic advantages to bypass- ing the intestinal barrier using parenteral (injectable) dosage forms. This is common in patients who, because of illness, cannot tolerate or are incapable of accepting drugs orally. ‘Some drugs are so rapidly and completely metabolized to inactive products in the liver (first pass effect) that oral ad- ‘ministration is precluded. But that does not mean that the drug administered by injection is not confronted by obstacles (Fig. 2-1). Intravenous administration places the drug di- rectly into the circulatory system, where it will be rapidly 1H,0R Hy on Hse ° cy Methylprednisolone: R= H Methylprednisolone Acetate: R Methylprednisolone distributed throughout the body, including tissue depots and the liver, where most biotransformations occur (see below), in addition to the receptors. Itis possible to inject the drug directly into specific organs or areas of the body. Intraspinal and intracerebral routes will place the drug directly into the spinal fluid or brain, resp tively—bypassing a specialized epithelial tissue, the blood- brain barrier, which protects the brain from exposure to a large number of metabolites and chemicals, The blood-brain Sodium Succinate: R barrier is composed of membranes of tightly joined epithelial cells fining the cerebral capillaries. The net result is that the brain is not exposed to the same variety of compounds that other organs are. Local anesthetics are examples of adminis- tration of a drug directly onto the desired nerve. A spinal block is a form of anesthesia performed by injecting a local anesthetic directly into the spinal cord at a specific location in order to block transmission along specific neurons. Most of the injections a patient will experience in a life- time will be subcutaneous or intramuscular. These parenteral routes produce a depot in the tissues (Fig. 2-1), from which the drug must reach the blood or lymph. Once in systemic circulation, the drug will undergo the same distributive phe- nomena as orally and intravenously administered agents be- fore reaching the target receptor. In general, the same factors that control the drug’s passage through the gastrointestinal ‘mucosa will also determine the rate of movement out of the tissue depot. ‘The already described prodrug approach also can be used. to alter the solubility characteristics, which, in turn, can in- ccrease the flexibility in formulating dosage forms. The solu- bility of methylprednisolone can be altered from essentially water-insoluble methylprednisolone acetate to slightly ‘water-insoluble methylprednisolone to water-soluble meth- ylprednisolone sodium succinate. The water-soluble sodium succinate salt is used in oral, intravenous, and intramuscular dosage forms. Methylprednisolone itself is normally found in tablets. The acctate ester is found in topical ointments and. sterile aqueous suspensions for intramuscular injection. Both the succinate and acetate esters are hydrolyzed to the active methylprednisolone by the patient's own systemic hy- drolytic enzymes, ci ) Cs (¢(=0)CH,CH,C0O" Na” PROTEIN BINDING Once the drug enters the systemic circulation (Fig. 2-1) it can undergo several events. It may stay in solution, but many drugs will be bound to the serum proteins, usually albumin (Rx. 2-2), Thus, a new equilibrium must be considered. De~ pending on the equilibrium constant, the drug can remain in systemic circulation bound to albumin for a considerable period of time and not be available to the sites of biotranstor- mation, the pharmacological receptors, and excretion,Drug + Albumin = Drug-Albumin Complex. (Rx. 2-2) ‘The effec of protein binding can have a profound effect on the drug's effective solubility, biodistribution, half-life inthe body, and interaction with other drugs. A drug with such poor ‘water solubility that therapeutic concentrations of the un- bound (active) drug normally cannot be maintained still can be avery effective agent. The albumin-drug complex acts as a reservoir by providing concentrations of free drug large enough to cause a pharmacological response at the receptor. Protein binding may also limit access to certain body com partments. The placenta is able to block passage of proteins from maternal to fetal circulation. Thus, drugs that normally would be expected to cross the placental barrier and possibly harm the fetus are retained in the maternal circulation, bound to the mother's serum protein. Protein binding also can prolong the drug’s duration of ac- tion, The drug-protein complex is too large to pass through the renal glomerular membranes, preventing rapid excretion of the drug. Protein binding limits the amount of drug avail- able for biotransformation (sce below and Chap. 3) and for interaction with pecificreceptorsites. Forexample, the large, polar trypanocide suramin remains in the body inthe protein- bound form for as long as 3 months (ty2 = 50 days). The ‘maintenance dose for this drug isbased on weekly administra- tion. At frst, this might seem to be an advantage tothe patient. Iteanbe, but italso means that, should the patient have serious, adverse reactions, a significant length of time will be required before the concentration of drug falls below toxic levels The drug-protein binding phenomenon can lead to some interesting drug-drug interactions resulting when one drug, displaces another from the binding site on albumin. A large number of drugs can displace the anticoagulant warfarin {rom its albumin binding sites. This increases the effective concentration of warfarin at the receptor, leading to an in- creased prothrombin time (increased time for clot formation) and potential hemorthage. ruc oisraeuTION | 7 logically inert depots. The short-acting, lipophilic barbitu- rate thiopental disappears into tissue protein, redistributes, into body fat, and then slowly diffuses back out of the tissue depots but in concentrations too low for a pharmacological response. Thus, only the intially administered thiopental is present in high enough concentrations to combine with its receptors. The remaining thiopental diffuses out ofthe tissue depots into systemic circulation in concentrations too small to be effective (Fig. 2-1), is metabolized in the iver, and is excreted. In general, structural changes in the barbiturate series (see Chap. 14) that favor partitioning into the lipid tissue stores decrease duration of action but increase central nervous system depression, Conversely, the barbiturates with the slowest onset of action and longest duration of action contain, the more polar side chains. This latter group of barbiturates both enters and leaves the central nervous system very slowly as compared to the more lipophilic thiopental DRUG METABOLISM Al substances, including drugs, metabolites, and nutrients that are inthe circulatory system, will pass through the liver. “Most molecules absorbed from the gastrointestinal tract will ‘enter the portal vein and initially be transported tothe liver. A significant proportion ofa drug will partition or be trans- ported into the hepatocyte, where it may be metabolized by hepatic enzymes to inactive chemicals during the intial trip through the liver by what is known as the first-pass effect. Lidocaine is a classic example of the significance of the first-pass effect, Over 60% of this local anestheti antiarrhythmic agent is metabolized during its ii passage through the liver, resulting in it being impractical to administer orally. When used for cardiac arrhythmias, itis administered intravenously. This rapid metabolism of g f ft oo Nat “O38 NHC Hy HC CNH $03" Na OO * *, OO Na* “O98 - =o '803” Na* 80” Na* 03” Nat HON Suramin Sodium TISSUE DEPOTS The drug can also be stored in tissue depots. Neutral fat constitutes some 20% to 50% of body weight and constitutes, a depot of considerable importance. The more lipophilic the drug, the more likely it will concentrate in these pharmaco. lidocaine is used to advantage when stabilizing a patient with, cardiac arrhythmias. Should too much lidocaine be admin tered intravenously, toxic responses will tend to decrease because of rapid biotransformation to inactive metabolites. An understanding of the metabolic labile site on lidocaine8 | PHYSICOCHEMICAL PROPERTIES IN RELATION TO BIOLOGICAL ACTION led to the development of the primary amine analog tocai- nide. In contrast to lidocaine’s half-life of <2 hr, tocainide’s half-life is ~15 hr with 40% of the drug excreted unchanged. The development of orally active antiarrhythmic agents is discussed in more detail in Chap. 19, cis a Eats HC-CH-NHT cI CoH cH Lidocaine CH, ‘CH Tocainide A study of the metabolic fate of a drug is a requirement for all new products. Many times it is found that the ‘metabolites are also active. Indeed, sometimes the metabo- lite is the pharmacologically active molecule. These drug ‘metabolites can provide the leads for additional investiga- tions of potentially new products. Examples of where an inactive parent drug is converted to an active metabolite include the nonsteroidal anti-inflammatory agent sulindac being reduced to the active sulfide metabolite: the immuno- suppressant azathioprine being cleaved to the purine anti- ‘metabolite 6-mercaptopurine; and purine and pyrimidine antimetabolites and antiviral agents being conjugated to theit nucleotide form (acyclovir phosphorylated to acyclovir triphosphate). Many times both the parent drug and its metabolite are active, which has led to additional commercial products, instead of just one being marketed. About 75% to 80% of phenacetin (now withdrawn from the U. S. market) is converted to acetaminophen. In the tricyclic antidepressant series (see Chap. 14) imipramine and amitriptyline are N-demethylated to desipramine and nortriptyline, respectively. All four compounds have been marketed in the United States. The topic of drug metabo- lism is discussed more fully in Chap. 13. Hs (H,00;4 R = CH,8(=0) Active Sulfide Metabolite Sulindac: R= cys. captopurine HN’ N (CH,OCH,CH OR Aeyelovir: R=H Acyclovir triphosphate: R = 0-P-0-P-0-P, a A, Although a drug’s metabolism can be a source of frustra- tion for the medicinal chemist, pharmacist, and physician and lead to inconvenience and compliance problems with the patient, it is fortunate that the body has the ability to metabolize foreign molecules (xenobiotics). Otherwise, many of these substances could remain in the body for years. This has been the complaint against certain lipophilic chemi- cal pollutants, including the once very popular insecticide DDT. After entering the body, these chemicals reside in body tissues, slowly diffusing out of the depots and potentially harming the individual on a chronic basis for several years. They can also reside in tissues of commercial food animals who have been slaughtered before the drug has ‘‘washed out” of the body. EXCRETION ‘The main route of excretion of a drug and its metabolites is through the kidney. For some drugs, enterohepatic circula- tion (Fig. 2-1), where the drug reenters the intestinal tractfrom the liver through the bile duct, can be an important part afte agent’s distribution in the body and route of excretion. Either the drug or drug metabolite can reenter systemic circu lution by passing once again through the intestinal mucosa. portion of either also may be excreted in the feces. Nursing rmthers must be concerned because drugs and their metabo- le can be excreted in human milk and be ingested by the nursing infant. Itis important to keep a sense of perspective when leam- ing about drug metabolism. As will be explained in Chap. 5, dug metabolism can be conceptuatized as occurring in two tages or phases. Intermediate metabolites that are phar- rmacologically active usually are produced by Phase I reac: tins. The products from the Phase I chemistry are converted into inactive, usually water-soluble end products by the Phase Il reactions, The latter, commonly called conjugation reactions, can be thought of as synthetic reactions that in- volve addition of water-soluble substituents. Examples are lucuronic acid, sulfate, and glutathione. Obviously, drugs that are bound to serum protein or show favorable parttion- ing into tissue depots are going to be metabolized and ex- creted more slowly for the reasons already discussed. ‘This does not mean that for drugs that remain in the body for longer periods of time lower doses can be administered or the drug be taken fewer times per day by the patient. Several variables determine dosing regimens, of which the Miiity of the drug for the receptor is crucial. Reexamine Rx. 2-1 and Fig, 2-1. Ifthe equilibrium does not favor forma- tion of the drug-receptor complex, higher and usually more fiequent doses will have to be administered. Ifthe partition- ing into tissue stores, metabolic degradation, and/or excre- tion is favored, it will take more of the drug and usually ‘more frequent administration in order to maintain therapeu tie concentrations at the receptor. RECEPTOR With the possible exception of general anesthetics (see Chap. 14), the working model for a pharmacological response con- sists of a drug binding to a specific receptor. Many drug receptors actually are used by endogenously produced lig ands. Cholinergic agents interact with the same receptors as the neurotransmitter acetylcholine. Synthetic corticosteroids bind to the same receptors as cortisone and hydrocortisone Many times receptors for the same ligand will be found in «variety of tissues throughout the body. The nonsteroidal antiinflammatory agents (see Chap. 22) inhibit the prosta- slandin-forming enzyme cyclooxygenase, which is found in nearly every tissue. This class of drugs has a long list of side effects with many patient complaints. Note in Fig. 2-1 that, depending on which receptors contain bound drug, there. may be desired or undesired effects. This is because there aca variety of receptors with similar structural requirements found in several organs and tissues. Thus, the nonsteroidal ant-inflammatory drugs combine with the desired cycloxy- opus oistrIeuTION | 9 ‘genase receptors at the site of the inflammation and the unde sired receptors in the gastrointestinal mucosa, causing severe discomfort and sometimes ulceration. One of the ‘second generation’” antihistamines, terfenadine, is claimed to cause less sedation because it does not readily penetrate the blood- brain barrier. The rationale is that less of this antihistamine is available for the receptors in the central nervous system, which are responsible for the sedation response characteris- tic of antihistamines. In contrast, some antihistamines are used for their central nervous system depressant activity, which would imply that a considerable proportion of the administered dose is crossing the blood-brain barrier relative to binding to the histamine, receptors in the periphery. Although it is normal to think of side effects as undesira- ble, they sometimes can be beneficial and lead to new prod~ ucts. The successful development of oral hypoglycemic agents used in the treatment of diabetes began when it was found that certain sulfonamides had a hypoglycemic effect (see Chap. 19), Nevertheless, areal problem in drug therapy is patient compliance in taking the drug as directed. Drugs that cause serious problems and discomfort tend to be avoided by the patient. SUMMARY One of the goals is to design drugs that will interact with receptors at specific tissues. There are several ways to do this, including (a) altering the molecule, which, in tum, can change the biodistribution, (b) searching for structures that show increased specificity for the target receptor that will produce the desired pharmacological response while de- creasing the affinity for undesired receptors that produc adverse responses, and (c) the still experimental approach of attaching the drug to a monoclonal antibody that will bind to a specific tissue antigenic for the antibody. Alteration of biodistribution can be achieved by changing the drug's solubility, enhancing its ability to resist being metabolized (usually in the liver), altering the formulation or physical characteristics of the drug, and changing the route of admin- istration, If a drug molecule can be designed in such a way that its binding to the desired inced relative to the undesired receptor, and biodistribution remains favor- able, smaller doses of the drug can be administered, This, in turn, reduces the amount of drug available for binding to those receptors responsible for its side effects ‘The medicinal chemist is confronted with several chal- lenges in designing a bioactive molecule. A good fit to a specific receptor is desirable, but the drug would normally be expected eventually t0 dissociate from the receptor. The specificity for the receptor would be such that side effects ‘would be minimal, The drug would be expected to clear the body within a reasonable time. Its rate of metabolic degrada- tion should allow reasonable dosing schedules and, ideally, oral administration. Many times, the drug chosen for com- mercial sales has been selected from the hundreds of com-10 | PHYSICOCHEMICAL PROPERTIES IN RELATION TO BIOLOGICAL ACTION pounds that have been screened. It usually is a compromise product that meets a medical need while demonstrating good patient acceptance. ACID-BASE PROPERTIES ‘The majority of drugs used today can be classified as acids or bases. As will be noted shortly, a large number of drugs can behave as either acids or bases as they begin their jour- ney into the patient in different dosage forms and end up in systemic circulation. A drug’s acid-base properties can influence greatly its biodistribution and partitioning charac- teristics, Over the years, at least four major definitions of acids/bases have been developed. The model commonly used in pharmacy and biochemistry was developed inde- pendently by Lowry and Brinsted. In their definition an acid is defined as a proton donor and a base as a proton acceptor. Notice that for a base, there is no mention of the ydroxide ion. ACID-CONJUGATE BASE Representative examples of pharmaceutically important acidic drugs are listed in Table 2-1. Each acid, or proton donor, yields a conjugate base. The latter is the product produced after the proton is lost from the acid. They range from the chloride ion (reaction a), which does not accept a proton in aqueous media, to ephedrine (reaction fi), which is an excellent proton acceptor. Notice the diversity in structure of these proton donors. ‘They include the classical hydrochloric acid (reaction a), the weakly acidic dihydrogen phosphate anion (reaction 6), the ammonium cation such as is found in ammonium chloride (reaction c), the carboxylic acetic acid (reaction d), the eno- lic form of phenobarbital (reaction e), the carboxylic acid moiety of indomethacin (reaction f) the imide structure of saccharin (reaction g), and the protonated amine of ephed- rine (reaction h). Because all are proton donors, they must be treated as acids when calculating pHs of a solution or ‘percent ionization of the drug. At the same time, as will be noted shortly, there are important differences in the pharma- ceutical properties of ephedrine hydrochloride (an acid salt of an amine) as compared with indomethacin, phenobarbital, or saccharin BASE-CONJUGATE ACID The Briinsted-Lowry theory defines a base as a molecule that accepts a proton. The product resulting from the addition cof a proton tothe base is the conjugate acid. Pharmaceuti- cally important bases are listed in Table 2-2. Again, there are a variety of structures, including the easily recognizable base, sodium hydroxide (reaction a), the basic component of an important physiological buffer, sodium monohydrogen phosphate which is also the conjugate base of dihydrogen phosphate (reaction b), ammonia which is also the conjugate base of the ammonium cation (reaction c), sodium acetate Which is also the conjugate base of acetic acd (reaction d), the enolate form of phenobarbital which is also the conjugate base of phenobarbital (reaction e), the carboxylate form of indomethacin which is also the conjugate base of indometha- cin (reaction f, the imidate form of saccharin which is also the conjugate base of saccharin (reaction g), and the amine ephedrine which is also the conjugate base of ephedrine hy- OC a oS © ° é d ¢ 0 40 + an = iv + on ba" ri pi aN. a. oo JN Exot HC ands con gate base, ephedra on on, hw or val a oi, : = notre + OL On, om om, ww we eno . om Ow a OX om * The chorde anon and sodium cation are present onty to maintain charge balance, These anions play no other acc-base role14. | PHYSICOCHEMICAL PROPERTIES IN RELATION TO BIOLOGICAL ACTION ‘The modified equilibrium constant, K,, is customarily con- verted to pK, (the negative logarithm) in order to put it on the ssame scale as pH. Therefore, rewriting Bq. 2-2 in logarithmic form produces log K, = log [H,O*] + log [A~] — log [HA] = log [H30"] + log {conj. base] — log [acid] ie 25) Rearranging Eq, 2-5 gives ~log[H,0*] = —log K, + log[A~] — log [HA] = log, + log{conj. base] — log [acid] Eq. 2-6) Substituting Eqs. 2-3 and 2-4 into Eq, 2-6 produces . 1A] {con}. base] pH = pK + log ffpgj~ PKe + los facia] 2 Equation 2-7 is more commonly called the Henderson- Hasselbalch equation and isthe basis for most calculations involving weak acids and bases. It is used to calculate the ‘pH of solutions of weak acids, weak bases, and buffers con- sisting of weak acids and their conjugate bases or weak bases and their conjugate acids. Because the pK, is a modified equilibrium constant, it corrects for the fact that weak acids do not completely react with water. ‘Avery similar set of equations would be obtained from the reaction of a protonated amine, BH", in water. The reaction would be: Conj. Con}. Acid Base Acid Base BH? + H,0=H,0'+ B (Rx.2-4) The equilibrium constant, K.y, is defined as: {H,0°1[B] |BH*][H,0] {conj. acid]{conj. base] Kew Tacidjfbase] (Eq. 2-8) Notice that Eq, 2-8 is identical to Eq. 2-1 when the general {conj. acid]{conj. base] representation is used. Therefore, using the same simplifying assumption that water will re- ‘main at a constant concentration of 95.5 M in dilute solu- tions, Eq. 2-8 can be rewritten as: (H,0")[B} [BH") [conj. acidj[conj. base] [acid] (Eq, 2-9) Rearranging Eq. 2-9 into logarithmic form and substituting Ky = Ko(55.5) = the relationships expressed in Eqs. 2-3 and 2-4 yields the same Henderson-Hasselbalch equation (Eq. 2-10), {conj. base} [acid] (Eq, 2-10) Rather than trying to remember the specific form of the Henderson-Hasselbalch equation for an HA or BH acid, it is simpler to use the general form of the equation (Eq. 2- 11) expressed in both Eqs. 2-7 and 2-10, [conj. base] [ With this version ofthe equation, there is no need to remem- bber whether the species in the numerator/denominator is ion- ized or un-ionized. The molar concentration of the proton acceptor is the term in the numerator and the molar concen- tration of the proton donator is the denominator term. What about weak bases such as amines? In aqueous solu tions, water will function as the proton donor or acid (Rx. 5), producing the familiar hydroxide anion (conjugate base). Traditionally, a modified equilibrium constant called the pK was derived following the same steps that produced Eq, 2. Itis now more common to express the basicity ofa che cal in terms of the pK, using the relationship in Bq. 2-1: pK, + log pH = pK, + log (Eq. 211) Conj. Con Acid Base Acid Base H,0 + B = BHt+OH” (Rx.25) PK, = pKy — 14 (Eq, 2-12) ‘Waring! Itisimportant torecognize that a pK, forabaseis in reality the pK, of the conjugate acd (acid donor or protonated form) ofthe base. The pK. forephedrine is listed inthe Appen- dix as9.6 and for ammonia 9,3. Inreality, thisisthe pK. of the protonated form such as ephedrine hydrochloride (reaction m in Table 2-3) and ammonium chloride (reaction ¢ in Table 2- 3), respectively. This is confusing to students, pharmacists, clinicians, and experienced scientists, It is crucial that the chemistry ofthe drug be understood when interpreting a pK value, When reading tables of pX, values, such as those found in the Appendix, itis essential to realize that the listed value is forthe proton donor form of the molecule, no matter what form is indicated by the name. See Table 2-4 for several ‘worked examples as to how the pX, is used to calculate pHs of solutions, required ratios of {conjugate baseJ/Lacid), and percent ionization at specific pHs. Just how strong, or weak, are the acids whose reactions in water are illustrated in Table 2-3? Bear in mind that the K,s or pK,s are modified equilibrium constants that will indicate whether the acid’s reaction in water (proton donor ‘or base) tends to go to completion, resulting inthe formation of large amounts of conjugate acid and conjugate base, or whether the base accepts few protons from water, forming little conjugate acid and conjugate base. Refer back to Eq. 2-2 and, using the K, values in Table 2- 5, substitute the K, term foreach ofthe acids, Forhydrochloric1. Whats the rao of ephecrine to ephedrine HCl (pK, 9.6) in the inesinalvact at pH 8 0? Use Equation 2-11 (oonecino) 8 +190 Tephedrne HO [ephedrine] Tephedine HO] 02s The eumber whose log is ~1.6 is 0.026, mearing thal there are 25 pats ephedine for every 1000 parts ephedrine HC! in the testinal rat whose envionment is pH 8°. Whats the pH of abutter containing 0.1 M acete acs (pK, 4.8) sd 0.08 M sodium acetate? Use Equation 211 atthe oo 201M cic sé ok Ute oni tain okay te pa seen Coa ater Re aoa og ai ham ee) g Whats the pH of a 0.08 Mf sodium acetate solution? Remember, teen hough tis isthe conjugate base of acatio acid, the pK, wil sil be used. The px, term inthe following equation carecs for the fact that a protow acceptor lactate anion) is prosent in the salu. The equation for calculating the pH of a solution conta ing ether an A” or 8 base is Ky + pK, + log (base) rs Whats the DH of an ammonium acetate soluton? The pK, ofthe mmonism (NH,*) cation is 9.3, Aways bear in mind thatthe aK teers to he abilty of the proton donor form ta release the proton ito water to form H;0". Since this isthe salt of a weak acd (NH,") and the conugate base of a weak acid (acetate anor), the foloving equation is used. Note that molar concentration Is no a vatabie inthis calculation, PK, + PK, P+ Ph oy 2 be \atis he percentage fonizaton of ephectine HCI (pK, 9.6) in an tiesinal rat buffered al pH 8.0 (see example 1)? Use Equation 28 because this is a BH acid % ionization = 976% Terra” Cniy 24% of ephedkine is present as the unionized conjugate ease Whats the percentage ionization of indomethacin (pK, 45) in an ‘esina rat butlered at pH 807 Use Equation 2-13 because ths s an HA aod 99.97% Fer all practical purposes indomethacin is present only as the monic conjugate base n that region of the intestine butered at HBO. ACID-BASE PROPERTIES | 15 acid, it should be obvious that a K, of 1.26 10 means that the numerator term containing the concentrations of the con- {gate acid and conjugate base products is huge relative tothe denominator term representing the concentration of the reac- tants. In other words, there essentially is no unreacted HCl left in an aqueous solution of hydrochloric acid. At the other extreme is ephedrine HCI with a pK, of 9.6 or K, of 2.51 X 107", Here, the denominator representing the concentration ‘of ephedrine HCI greatly predominates over that of the prod ucts, which, in this example, would be ephedrine (conjugate base) and HO" (conjugate acid). In other words, the proton- ated form of ephedrine is a very poor proton donor. It holds ‘onto the proton. Indeed, free ephedrine (the conjugate base in this reaction) isan excellent proton acceptor. ‘A general rule for determining whether a chemical is strong or weak acid or base is pK, <2: strong acid; essentially no basic properties in water pK, 4-6: weak acid; very weak conjugate base PK, 8-10: very weak acid; weak conjugate base pK, >12: essentially no acidic properties in water; strong conjugate base tis important to realize that this delineation is only approxi- mate. Other properties also become important when consid ering cautions in handling acids and bases. Phenol has a pK. of 9.9, slightly less than that of ephedrine HCl. Why is phenol considered corrosive tothe skin, whereas ephedrine HCI or free ephedrine is considered innocuous when applied to the skin? Phenol has the ability to partition through the normally protective lipid layers of the skin. Due to this prop- erty, this extremely weak acid has carried the name carbolic acid, Thus, the pX, simply tells a person the acid properties of the protonated form of the chemical. It does not represent anything else concerning other potential toxicities. PERCENT IONIZATION Using the drug’s p, the formulation pharmacist can adjust the pH in order to insure maximum water solubility (ionic form of the drug) or maximum solubility in nonpolar media (nonionic form). This is where the understanding of the drug's acid-base chemistry becomes important. Note Rxs. 2-6 and 2 REPRESENTATIVE K, AND pK, VALUES FROM THE REACTIONS LISTED IN TABLE'2.3 (SEE THE APPENDIX) Hydrochloric acid 126 x 108 =61 Dinyckagen phosphate 631 x 10-8 72 ‘Ammons (ammoniun) 501 x 107 93 ‘oatic acid 158 x 107 48 Phenobarbital 316 x 10-8 75 Sacchatin 251 x 10-2 16 Indomethacin 316 x 10-8 45 Ephedrine (as the HC! sat) 2st x 197" 9616 | PHYSICOCHEMICAL PROPERTIES IN RELATION TO BIOLOGICAL ACTION Conj. Con}. Acid Base Acid Base HAdnionzes) + HO = Hy0° + Agency) (RK. 2-6) Conj. Con ‘Acid Base Acid Base BH Gonizeay + HzO = H30° + Bounionizeay) (RX. 2-7) ‘Acids can be divided into two types, HA and BH”, based ‘on the ionic form of the acid (or conjugate base). HA acids 0 from nonionized acids to ionized conjugate bases (Rx. 2-6). Incontrast, BH" acids go from an ionized (polar) acid to nonionized (nonpolar) conjugate base (Rx. 2-7). In general, ‘pharmaceutically important HA acids include the inorganic acids (¢g., HCI, H,S0,), enols (c.g, barbiturates, hydan- toins) carboxylic acids (¢g., low molecular weight organic acids, arylacetic acids, N-aryl anthranilic acids, salicylic acids), and amides and imides (eg., saccharin, sulfon- amides). The chemistry is simpler for the pharmaceutically ‘important BH* acids: they are all protonated amines. A poly- functional drug can have several pX,s (e.g., amoxicillin). ‘The latter's ionic state is based on amoxicilin’s ionic state at physiological pH 7.4 (see the following di percent ionization). Pas = 9.6 f 6 rO—NH-CH-CH SCH St So Nis? _Z0—Ngh cry ot ao PKyg = 7.4 ° PKs Amoxil itin ‘The percent ionization of a drug is calculated using Eqs. 2-13 for HA acids and 2-14 for BH’ acids. 100 Siowization = Nope (Ba. 213) {ionization = Tras Ea. 2-14) A plot of percent ionization versus pH is illustrative of hhow the degree of ionization can be shifted significantly with small changes in pH. The curves for an HA acid (indometha- cin) and BH" (protonated ephedrine) are shown in Fig. 2-2. First, note that when pH = pK, the compound is 50% ion- ied (or 50% un-ionized). In other words, when the pK, is equal to the pH, the molar concentration of the acid equals the molar concentration of its conjugate base. In the Henderson-Hasselbalch equation, the pk, = pH when log {conj. base/[acid] = 1. An increase of | pH unit from the PK, (increase in alkalinity) causes an HA acid (indometha- cin) to become 90.9% in the ionized conjugate base form but results in a BH” acid (ephedrine HCl) decreasing its percent ionization to only 9.1%. An increase of 2 pH units indeernecin Ephesrine phen 4 hee 96 FIG.2:2._ Percentage ionized versus pH fer indomethacin (cK, 4.5) ‘and ephedrine (ok, 86) essentially shifis an HA acid to complete ionization (99%) and a BH’ acid to the nonionic conjugate base form (0.99%) Just the opposite is seen when the media is made more acidic relative to the drug’s pK, value, Increasing the hydro- gen ion concentration (decreasing the pH) will shift the equi- librium to the left, thereby increasing the concentration of| the acid and decreasing the concentration of conjugate base. In the case of indomethacin, a decrease of | pH unit below the pK, will increase the concentration of un-ionized (pro- tonated) indomethacin to 9.1%. Similarly, a decrease of 2 ‘PH units results in only 0.99% of the indomethacin being Present in the ionized conjugate base form. The opposite is seen for the BH” acids. The percent of ephedrine present as the ionized (protonated) acid at 1 pH unit below the pk, is 90.9% and 2 pH units below the pK, is 99.0%. These results are summarized in Table 2-6. With this knowledge in mind, retum to the drawing of amoxicillin. At physiological pH, the carboxylic acid (HA acid: pK,1 2.4) will be in the ionized carboxylate form, the primary amine (BH acid; pK,» 7.4) will be 50% protonated and 50% in the free amine forms, and the phenol (HA acid; pK,s 9.6) will be in the nonionized protonated form. A knowledge of percent ionization makes it easier to explain and predict why problems and discomfort due to pH ex- TABLE 26 PERCENTAGE IONIZATION RELATIVE TO THE pK, Tontzation (16) HA Acids BH Acids pK, —2 pH unis 099 980 Ky — 1 pH unt Eh 909 pk, =pH 500 500 pk, +1 pH unt 09 ar pk, +2 pH unis 580 099rT ttemes can occur with the use of some preparations. Phenyt- cin (HA acid; pK, 8.3) injection must be adjusted to pH 12 with om hydroxide in order to insure complete ionization and maximize water solubility. In theory, a pH of 10.3 will result 99.0% of the rug being inthe anionic water-soluble conjugate base, To reduce even further that insoluble 1% in the acd form and maintain excess alkalinity, the pH is raised to 12to obtain 99.98% of the drug inthe ionized form. Even then, acosolvent system of 40% propylene glycol, 10% ethyl sleohol, and 50% water for injection is used to insure a complete solution. This highly alkaline solution is irritating tothe patient and generally cannot be administered as an admixture with other intravenous fluids that are buffered smote closely at physiological pH 74. This decrease in pH will sult in the parent un-ionized phenytoin precipitating oat of solution, Phenytoin Sodium Aajustments in pH to maintain water solubility can some- times lead to chemical stability problems. An example is indomethacin (HA acid; pK, 4.5), which is unstable in alka- lie media, Therefor, the preferred oral liquid dosage form is suspension buffered at pHl 4 to 5. Because this is near the drug's pK, only 50% will be inthe water-soluble form, There is a medical indication requiring the intravenous ad- ‘inisration of indomethacin to premature infants. The intra venous dosage form is the lyophilized (Ireeze- FIG, 23. Passage of HA acids through lipid barons. BH +H,0 —2H,0" +8 | FIG. 2.4. Passage of BH" acide through the pid barr BHY +H,0 == H,0° + first encounter the acidic stomach, where the pH can range from 2 to 6 depending on the presence of food. HA acids ‘with pX,s of 4 to 5 will tend to be nonionic and be absorbed partially through the gastric mucosa, (The main reason most, acidic drugs are absorbed from the intestinal tract rather than the stomach is that the microvilli of the intestinal mucosa Provide a huge surface area relative to what is found in the gastric mucosa of the stomach.) In contrast, amines (pK, 9 to 10) will be protonated (BH* acids) and usually will not be absorbed until reaching the mildly alkaline intestinal tract (DH = 8). Even here only a portion of the amine-containing drugs willbe in their nonpolar conjugate base form. When- ‘ever a nonpolar form of either an HA acid (as the acid) or a BH” acid (as the conjugate base) passes the lipid barrier, the equilibrium conjugate base/acid (as determined by the pH of the environment) must be maintained. For example, once in systemic circulation, the plasma pH ‘of 74 will be one ofthe determinants as to whether the drug will tend to remain in the aqueous environment ofthe blood ‘or partition across lipid membranes into hepatic tissue to be ‘metabolized, into the kidney for excretion, into tissue depots, ‘or tothe receptor tissue. A useful exercise is to calculate the [eonj. basel/facid) ratio using the Henderson-Hasselbalch equation (Eq. 2-11) or percent ionization for ephedrine (pK, 9.6; Bq, 2-14) and indomethacin (pK, 4.5; Eq, 2-13) at pH 3.5 (stomach), pH 8.0 (intestine), and pH 7.4 (plasma) (see ‘examples I, 6, and 7 in Table 2-4), OF course, the previously discussed effect of protein binding can greatly alter any pre diction of biodistribution based solely on pK,18 | PHYSICOCHEMICAL PROPERTIES IN RELATION TO BIOLOGICAL ACTION STATISTICAL PREDICTION OF PHARMACOLOGICAL ACTIVITY Just as mathematical modeling is used to explain and model ‘many chemical processes, it has been the goal of medicinal chemists to quantify the effect of a structural change on a defined pharmacological response. This would meet three sgoals in drug design: to (a) predict biological activity in ‘untested compounds; (b) define the structural requirements required for a good fit between the drug molecule and the receptor, and (c) design a test set of compounds in order to ‘maximize the amount of information concerning structural requirements for activity from a minimum number of com- pounds tested. This aspect of medicinal chemistry com- monly is referred to as quantitative structure activity rela- tionships (QSAR). ‘The goals of QSAR studies were first proposed around 1865 to 1870 by Crum-Brown and Fraser, who showed that the gradual chemical modification in the molecular structure of a series of poisons produced some important differences in their action.' They postulated that the physiological ac- tion, & of a molecule is some function of its chemical consti- tution, C. This can be expressed as follows: = 20 (Eq, 2-15) Equation 2-15 states that a defined change in chemical struc- ture would result in a predictable change in physiological action. The problem now becomes one of numerically dfin- ing chemical structure. It still is a fertile area of research. ‘What has been found is that biological response can be pre- dicted from physical chemical properties such as vapor pres- sure, water solubility, electronic parameters, steric descrip- tors, and panition coefficient (Eq. 2-16). Today, the partition coefficient has become the single most important physical chemical measurement for QSAR studies. Note that Eq. 2- 16 is the equation for a straight line (Y = mx + 6), Log BR = a(physical chemical property) + ¢ (Eq. 2-16) where BR =a defined pharmacological response usually ex- pressed in millimoles such as the effective dose in ‘50% of the subjects (EDs), the lethal dose in 50% of the subjects (LDso), or the minimum inhibitory concentration (MIC). tis common to express the bio- logical response as a reciprocal, 1/BR or 1/C «a = the regression coefficient or slope of the straight line = the intercept term on the ¥ axis (when the physical chemical property equals zero) The log value of the dependent variable (concentration necessary to obtain a defined biological response) is used to linearize the data, As will be shown shortly, QSARs are not always linear. Nevertheless, using logarithms is an accept- able statistical technique. Now, why is the biological re- sponse usually expressed as a reciprocal? Sometimes, one obtains a statistically more valid relationship. More impor- tantly, expressing the biological response as a reciprocal usu- ally produces a positive slope (Fig. 2-6). Let us examine a hypothetical example, Assume that BR is the EDs. Physicochemical eR 18R Compound property (mmole) __ (mmole) A 010 a 0.25 8 018 03 033 c 020 02 oS DB 025 oa 10 Notice that it requires four times as much compound A. relative to compound D to produce the same defined biologi- cal response in 50% of the test subjects. Also notice that the physicochemical property is increasing as the potency is decreasing. In other words, thee isan inverse relationship between physicochemical property and biological potency. Incontrast, there is a positive relationship between the recip- +ocal of the biological response (1/BR) and physicochemical property because I/BR is increasing as the physicochemical property is increasing. ‘As has been emphasized previously, the drug will go through a series of partitioning steps: (a) leaving the aque ous extracellular fluids, (b) passing through lipid mem- branes, and (c) entering other aqueous environments before reaching the receptor. In this sense, a drug is undergoing the same partitioning phenomena that happens to any chemical in a separatory funnel containing water and a nonpolar solvent such as hexane, chloroform, or ether. The difference between the separatory funnel mode! and what actually occurs in the body is that the partitioning in the funnel will reach an equilibrium where the rate of chemical leaving the aqueous phase and entering the or- ganic phase will equal the rate of chemical moving from the organic phase to the aqueous phase. This is not the physiological situation, Refer to Fig. 2-1, and note that dynamic changes are occurring to the drug such as it being metabolized, bound to serum albumin, excreted from the body, and bound to receptors. The circumstance for the drug is not static. Upon administration, the drug will bbe pushed through the membranes because of the high concentration of drug in the extracellular fluids relative to the concentration in the intracellular compartments. In an attempt to maintain equilibrium ratios, the flow of the drug will be from systemic circulation through the membranes onto the receptors. As the drug is metabolized and excreted from the body, it will be pulled back across the membranes and the concentration of drug at the recep- tors will decrease. Because much of the time the drug’s movement acrossD> ml ‘STATISTICAL PREDICTION OF PHARMACOLOGICAL ACTIVITY | 19 membranes isa partitioning process, the patition coefficient has become the most common physicochemical property. The question that now must be asked is what immiscible nonpolar solvent system best mimics the aqucous-lipid membrane bari found inthe body’ It is now realized that the n-octanol water system is an excellent estimator of drug pattoning in biological systems. Indeed, one could argue that was fortuitous that n-octanol was available in reasona- bie purity forthe early partition coefficient determinations. To appreciate why this is so, it is necessary to understand the chemical nature of the lipid membranes. These membranes are not anhydrous fatty or oily struc- tures. Asa first approximation, they ean be considered as tilayers composed of lipids consisting of a polar cap and large hydrophobic tail, Phosphoglycerides are major compo- nents of lipid bilayers. Other groups of bifunctional lipids include the sphingomyelins, gatactocerebrosides, and plas- mmologens. The hydrophobic portion is composed largely of unsaturated fatty acids, mostly with cis double bonds. In Addition, there is a considerable amount of cholesterol esters, protein, and charged mucopolysaccharides inthe lipid mem- branes. The final result is that these membranes are highly organized strctures composed of channels for transport of ‘important molecules such as metabolites, chemical regula- tors (hormones), amino acids, glucose, and fatty acids into the cell and removal of waste products and biochemically produced products out of the cell. The cellular membranes ae dynamic, with the channels forming and disappearing «depending on the cell's and body’s needs. Hydrophobic Tail | SBR \ Yuu ‘Oo FIG. 28. Schematic representation of the coll membrane. complex, highly organized, dynamically functioning struc- ture. For purposes of the partitioning phenomenon, picture the cellular membranes as two layers of lipids (Fig. 2-5). The two outer layers, one facing the interior of the cell and the other the exterior, wll consist of the polar ends of the bifunc~ tional lipids. Keep in mind that these surfaces are exposed to an aqueous polar environment. The polar ends ofthe charged phospholipids and other bifunctional lipids will be solvated by the water molecules. There also will be considerable amounts of charged proteins and mucopolysaccharides pres- ‘ent on the surface. In contrast, the interior of the membrane will be populated by the hydrophobic aliphatic chains from the fatty acid esters PARTITION COEFFICIENT With this representation in mind, a partial explanation can be presented as to why the n-octanol/water partitioning system ° a OH (CHy) g-O-CH_ 0 CHOC (CHa) Cy CHO—P—O-R +— Hydrophilic Head ° Lecithin: Cephalin: In addition, the membranes on the surface of nucleated cellshave specific antigenic markers, major histocompatibil- ity complex (MHC), by which the immune system monitors thecell’s status, There are receptors on the cell surface where hormones such as epinephrine and insulin bind, setting off aseres of biochemical events within the cell. Some of these revepfrs are used by viruses to gain entrance into the cells ‘were the virus reproduces. AS newer instrumental tech. rigues are developed, and genetic cloning permits isolation ofthe genetic material responsible for forming and regulat- ing the structures onthe cell surface, the image of a passive lipid membrane has disappeared to be replaced by a very R= OCH,CH)N*(CHs) 9 R= OCH,CH.NHy* seems to mimic the lipid membranes/water systems found in the body. It turns out that n-octanol is not as nonpolar as initially might be predicted. Water-saturated octanol con- tains 2.3 M water, whereas n-octanol-saturated water con- tains little of the organic phase. The water in the n-octanol phase apparently approximates the polar properties of the lipid bilayer, whereas the lack of octanol in the water phase ‘mimics the physiological aqueous compartments, which are relatively free of nonpolar components. In contrast, parti- tioning systems such as hexane/water and chloroform/water contain so little water inthe organic phase that they are poor20 | PHYSICOCHEMICAL PROPERTIES IN RELATION TO BIOLOGICAL ACTION ‘models forthe lipid bilayer/water system found in the body. ‘At the same time, it must be remembered that the n- ‘ctanol/water system is only an approximation of the actual environment found in the interface between the cellular ‘membranes and the extracellularintracelluar fluids. ‘The basic procedure for obtaining a partition coefficient is to shake a weighed amount of chemical in a flask contain- ing a measured amount of water-saturated octanol and octa- nol-saturated water. Many times, the aqueous phase will be buffered with a phosphate buffer at pH 7.4 to reflect physio logical pH. This corrects for the ratio of {conjugate ‘base|/facid] found in vivo, The amount of chemical in one or both ofthe phases is determined by an appropriate analytical technique and the partition coefficient calculated from Eq, 2-17. The octanol/water partition coefficient has been deter- mined for thousands of compounds, including drugs, agricul- tural chemicals, biochemical intermediates and metabolites, ‘and common chemicals. Many of these determinations have been obtained in several other onganic solvent/aqueous sys- tems such as ether, chloroform, triolein, and hexane. [chemical ox ‘Tehemical ln, ‘The determination of partition coefficients is tedious and time consuming. Some chemicals are too unstable and either degrade during the procedure, which can take several hours, or cannot be obtained in sufficient purity for an accurate determination. This has led to attempts at approximating the partition coefficient, Perhaps the most popular approach has been high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC). In each case, the support phase is nonpolar, either by permanent bonding (usually oc- tadecylsilane) ora coating of octanol, mineral oils, or related materials, The mobile phase usually contains some water miscible organic solvent in order to hold enough of the chemical, whose partition coefficient is being determined, in solution. Sometimes the partion coefficient is calculated from the retention data by regression analysis using Eq. 18. The “a” and ““c” terms have the same uses as in Eq 2-16, P (Eq. 2-17) log P = a(log retention) + ¢ (Eq. 2-18) ‘This model has at least two Limitations. First, in order to obtain valid numerical values for “‘a’” and “‘c”” in Eq. 2- 16, partition coefficients must be obtained initially by the classical shake flask method, their retention times obtained in the identical chromatographic system that will be used for the new compounds, and the results analyzed using Eq. 18. Second, chromatographic determinations of the partition coefficients usually only work when determining the rete tion times of chemicals ofthe same chemical class and sit lar substitution patterns. Because of these limitations ‘common to use directly the retention data in the prediction of biological response (Eq. 2-19). A chemical’s retention on a chromatographic support is the result of a combination of its paritioning, steric, and electronic properties. Because these same physical chemical properties are important vat ables in determining a drug’s biological response, excellent correlations have been obtained between chromatographic retention parameters and biological response. While the model represented by Eq. 2-19 is useful in predicting biolog- ical response, itis not as definitive as the models presented below (Eqs. 2-20 102-22) because the precise physical chem. ical properties are combined into one chromatographic reten- tion term. In other words, it is not possible to determine the relative importance of lipophilicity, electronic effects, or steric influence on the biological response when using Eq. 219. Log BR = a(log retention) + ¢ (Eq. 2-19) Most recently, there has been a concentrated effort to cal- culate the partition coefficient based on the atomic compo- nents of the molecule, Each atom type is assumed to contrib- ute a fixed amount to the chemical’s partition coefficient Because this assumption breaks down quickly, several cor- rection factors are used. Cyclohexene will serve as an ex- ample. Log P = 6(carbon atoms) + 12(hydrogen atoms) + (n = I)bonds + double bond correction Log P = 6(0.20) + 12(0.23) + 5(-0.09) + (~0.55) = 2.96 For purposes of comparison, the observed octanol/water parition coefficient (expressed as a logarithm) is 2.86. Be- cause of the correction factors, these calculations become so complex that they must be done by a computer program that analyzes the structure and identifies those structural at tributes requiring correction factors. Convenient as the cal culation method may be, it must be realized that its accu- racy is dependent on frst determining experimental partition coefficients of chemical exhibiting very similar chemistry. Is from these experimentally determined partition coeffi- cients that the correction factors are derived ‘There are several commercial drug design software pack- ages that contains modiles that estimate a chemical’ pati- tion coefficient. Some use the method described in the previ- ‘ous paragraph, Others use quantum chemical parameters. In all cases, it is important that the algorithm be validated against test sets of diverse chemical structures whose part- tion coefficients have been determined by the classical shake flask method. ‘There are simpler methods for estimating lipophilicity that will give reasonably correct results. These are based on the additive effect on the partition coefficient that is seen when varying a series substituents on the same molecule. Over the years, fairly extensive tables have been developed that contain the contribution (7) ofa wide variety of substituents to the panition coefficient. The method can be illustrated for chlorobenzene. The log P for benzene is 2.13 and for chlorobenzene is 2.84. The value forthe chlorine substitu-cat is obtained by subtracting the log P values for benzene and chlorobenzene, Tey = OR Pesioberene ~ 1OE Prenzene fe = 284 ~ 213 = 0.71 While the 7 substituent method has its limitations, paticu- lly when there are significant resonance and inductive ef- fects due tothe presence of multiple substituents, itcan work well fora series of compounds that have similar substitution patterns. OTHER PHYSICOCHEMICAL PARAMETERS ‘Theresa series of other constants that measure the contribu- tion by substituents to the molecule’s total physicochemical properties. These include Hammett’s o constant; Taft's ste~ ric parameter, Ey; Charton’s steric parameter, 1; Verloop's multidimensional steric parameters, L, By, Bs; and molar reactivity, MR. The latter has become the second most useful physicochemical parameter used in classical QSAR. modeling. It is a complex term based on the mole- cale’s refractive index, molecular weight, and density, and can be considered @ measure of the molecule’s bulk and electronic character. One reason for its popularity is that it iseasy to calculate from tables of atoms using a minimum of cretion factors. Of the listed physicochemical parameters g10up, is most easy to locate values for 7, 0, E,, and MR. [Arepresentative list can be found in Table 2-7. Table 2-7s illustrative of several items that must be kept inmind when selecting substituents to be evaluated in terms. TABLE_2.7 SAMPLING OF PHYSICOCHEMICAL PARAMETERS USED IN QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIPS INVESTIGATIONS ‘Substtuent Group = Omen Spurn wa =a 000000000 «000 103, tH, 058-007-017 -126 565, =cH;CH, 102-007-015 151 10.0, ~OHSCHICH, = 155 007-013-160 1496, CiCHs)e 183-007 015-171 1496 00H 002 012-027-085 787 “123-016 086-061 542 O18 034 006-045 92 O71 037-023, 097608 08 = 039023 1.16 a8 i203) 018-140 13.94 088 04s 054-240 S02 067 012-097-085 25 055 038050 1118 -097 021000 1493 -08 = O71 078252738 -057 088068 0516.0, From Hansch,C..Le0, A. Substitent Constants or Correlation ‘Analy in Chemistry and Biology. New York, John Wiley & Sons, ‘73? STATISTICAL PREDICTION OF PHARMACOLOGICAL ACTIVITY | 21 of the type of factors that influence a biological response. For electronic parameters such as 0, the location on an aro- matic ring is important due to resonance versus inductive effects, Notice the twofold differences seen between de and Ga for the three aliphatic substituents and iodo, and severalfold difference for methoxy, amino, fluoro, and phe- nolic hydroxyl Selection of substituents from a certain chemical class may not really test the influence of a parameter on biological activity. There is little numerical difference among the dea (oF Gara Values for the four aliphatic groups or the four halo- gens. Itis nt uncommon to go to the tables and find missing parameters such as the E, values for acetyl and N-acyl. Nevertheless, medicinal chemists can use information from extensive tables of physicochemical parameters in order to minimize the number of substituents required to find out if the biological response is sensitive to electronic, steric, and/or partitioning effects.” This is done by selecting substituents in each of the numerical ranges for the different parameters. In Table 2-7, there are three ranges of 7 values, (1.2310 =0.55), (0.28 100.56), and (0.71 to 1.55); three ranges of MR values, (0.92 to 2.85), (5.02 to 8.88), and (10.30 to 14.96); and two main clusters of values, one for the aliphatic substituents and the other for the halogens. In the ideal situation, substituents will be selected from each ofthe clusters in order to determine the dependence of the biological response over the largest possible variable space. Depending on the biological responses obtained from testing the new compounds, itis possible to determine if lipophilic- ity (partitioning), steric bulk (molar refraction), or electron ‘withdrawing/donating properties are important determinants of the desired biological response. QSAR MODELS Currently, there are three models or equations seen in QSAR analysis using physicochemical parameters, represented by Eqs. 2-20, 2-21, and 2-22. These three equations are ilus- trated (Fig. 2-6) using the logarithm of the partition coeffi- cient (log P) as the physical chemical parameter. First there is the linear model (Eq. 2-20). Because plots indicated a nonlinear relationship between biological response and the Partition coefficient, @ parabolic model was developed (Ea. 2-21), Examination of Fig. -6 shows an optimum log P (log, P.), where maximum biological activity will be obtained before a decrease in activity is seen. One explanation for this phenomenon is that hydrophilic drugs will tend to stay in the aqueous phase whereas lipophilic chemicals will pre fer the lipid bilayer. In both cases, less drug is being trans- ported to the receptor resulting in the actual concentration of receptor bound drug being decreased. In other words, the ‘equilibrium seen in Rx. 2-1 shifts to the left. There will be a group of drugs whose log P will place them near the top hydrophilic balance ofthe parabola such that their lipophi22 | PHYSICOCHEMICAL PROPERTIES IN RELATION TO BIOLOGICAL ACTION Log BR = 076 toy P+ 1.30 Log Biological Response Leg BR = 072 Log P~ 025 (ag F)* + 1.88 * og BF = 112 Lag P= 138 Logie + 1) + 140 peor | Lg Porttion Coefficient FIG. 2-6. Lag biologic response versus og parition coeficont using ner, parabole, and bilinear models will permit them to penetrate both aqueous and lipid barriers and reach the receptor. Log 1/C = a (log P) + ¢ (Eq, 2-20) Log 1/C = a (log P) ~ b (log PP + ¢ (Eq, 2-21) Log 1/C = a (log P) ~ b log(6P + 1) + ¢ (Eq. 2-22) ‘The third QSAR equation in current use is the bilinear model (Eq. 2-22). It consists of two straight tines: one ascending and one descending. The B-term connects the two lines. There are several interpretations for the B term. One explanation is based on the ratio of the rate constant for diffusion out of the octanol layer into the aqueous environment being different from the rate of diffusion out of the aqueous layer into the octanol. In other words, what may be simulated with the bilinear model is recognition that the rate of diffusion from the extracellular fluids into the lipid bilayer is different from the rate of diffusion out of the lipid bilayer into the intracellular environment ‘Another interpretation is recognition that the kinetics of partitioning through the lipid bilayer will be different from the kinetics of binding to the receptor. A third explanation takes into account the different volumes of the aqueous and lipid bilayers in the biological system. With this background in mind, three examples of QSAR equations taken from the medicinal chemistry literature will be presented. One will show a linear relationship (Eq. 2-23) and the others a parabolic (Eq, 2-24) and a bilinear (Eq. 2- 25) correlation. A study ofa group of griseofulvin analogues showed that there isa linear relationship (Eq. 2-23) between the biological response and both lipophilicity (log P) and clectronic character (a). It was suggested thatthe antibiotic activity may depend on the enone system facilitating the addition of griseofulvin to a nucleophilic group such as the SH moiety in a fungal enzyme, Griseorvivin: R= By ayers Xam Log BR = (0S6)log P + 2.19)0, ~ 1.32 (Eq, 223) AA parabolic relationship (Eq. 2-24) was reported for a series of substituted acetylated salicylates (substituted aspi- rns) tested for antiinflammatory activity * There is anontin- ear relationship between the biological response and li philicity and a significant detrimental steric effect seen with substituents at position 4. The two sterimol parameters used in this equation were L, defined as the length ofthe subst ent along the axis of the bond between the first atom of the substituent and the parent molecule and B2, defined as a Width parameter. Steric effects were not considered signifi- cant at position 3 because the sterimol parameters for substit- uents at position 3 are not part of Eq. 2-24. The optimal partition coefficient (Log Pg) for the substituted aspirins in this assay was 2.6. Oy OH _-CHy ¢ fi ¥ Aspirin: X Log I/EDy = 1.03 log P ~ 0.20(log P)? ~ 0.05 Ls = 0.24 Byy + 2.29 Eq. 2-24) Related to these mathematical QSAR models based on biological responses is QSAR used to analyze pharmacoki- netic activity. One example of this (Eq. 2-25) isa simulation of barbiturate absorption, which leads to the bilinear model.5* Loghoirr = 0.949|ogP ~ 1.238log(BP + 1) ~3.131 (Bq, 225) where log 8 = —1.271; log Py = 1.79; and kouse = diffe sion rate constant. At this point, itis appropriate to ask the question: Are all the determinations of partition coefficients and compilation of physical chemical parameters useful only where a statistically valid QSAR model is obtained? The answer is a firm *‘no.”” One of the most useful spinoffs from the field of QSAR has been the application of experimental design to the selection of new compounds to be synthesized and tested. Let's assume that a new series of drug molecules is to be synthesized based on the following structure. The goal is to test the effect of the 16 substituents in Table 2-7_ STATISTICAL PREDICTION OF PHARMACOLOGICAL ACTIVITY | 23 teach of the three positions on our new series. The number of possible analogues is equal to 16°, or 4.096, compounds, assuming that all three positions will always be substituted with one of the substituent from Table 2-7. If hydrogen is included when a position is not substituted, there are 17°, 4913, different combinations. The problem is to select a small numberof substituents that will represent the different ranges or clusters of values for lipophilicity, electronic influ- ace, and bulk. An initial design set could include the methyl and propyl from the aliphatic, fluorine and chlorine from the halogens, N-acetyl and phenol from the substituents showing tygrophiicity, and a range of electronic and bulk values, Including hydrogen, there will be 7°, or 343, different combi- rations. Obviously, that is too many for an initial evaluation, Instead, certain rules have been devised to maximize the information obtained from a minimum number of com- pounds, These include the following: 1. Each substituent must occur more than once at each position on which it is found, 2. The number of times that each substituent a a particular position appears should be approximately equal. 3. Notwo substituents should be present in a constant com- bination 4. When combinations of substituents are a necessity, they should not occur more frequently than any other combi- nation fs \ ro Ry. CH Ry Following these guidelines, the initial test set can be re- duced to 24 to 26 compounds. Depending on the precision ofthe biological tess, it will be possible to see if the data will fit QSAR model. Even an approximate model usually wil indicate the types of substituents to test further and what Positions on the molecules are sensitive to substitution and, if sensitive, to what degree variation in lipophilic, electronic, or bulk character are important, Just to be assured that the model is valid, itis a good idea to synthesize a couple of compounds that the model predicts would be inactive. As each group of new compounds is tested, the QSAR model isrefined until the investigators have a pretty good idea as to ‘what substituent patterns are important for desired activity. It must be emphasized that these same techniques used to de- velop potent compounds with desired activity also can be sed fo evaluate the influence of substituent patterns on un- ested toxic effects and pharmacokinetic properties. In their pure form, the rules listed above can be used to select a minimum number of compounds for a test set using what are known as identity variables. No physicochemical parameters are required. In its simplest form, the equation takes the form outlined in Eq. 2-26. This approach has been known as a Free-Wilson analysis.” Log BR = Y (substituent contibutions + contribution from the base molecule (4,226) ‘An example is a small set of phosphorous containing ace- tylcholinesterase inhibitors that were selected using the rules for designing a test set and evaluated as possible insecti- cides." The result is a complex equation that produces a coefficient for each substituent, They are summarized in Table 2-8 Examination of this table shows that ethyl and ethoxy at Ry, ethoxy and isopropoxy at Rz,and oxo at Ry have minimal influence on biological activity. In contrast, methyl and iso- propoxy at R, methoxy, propoxy, and butoxy at Ra all three nitrophenoxy substituents at Ry, and thio at Ry significantly influence the biological response. The predicted Log 1/BR for the compound, where R, = methyl, R> = propoxy, Rs = 4-nitrophenoxy, and Ry = thio, would be calculated from Bq, 2-26: Log BR = Ry + Ry + Ry + Ry + Base Molecule = 0.729 + 0.543 + 0.611 — 1.673 + 5.143 = 5353 One of the newer QSAR methods combines statistical techniques with molecular modeling and has been refered to as three-dimensional QSAR (3D-QSAR). The independent variables include spatial distances among and between phar- ‘macophores and physicochemical parameters located at spe- cific distances from the mofecule. Because 3D-QSAR is de- pendent on the molecular modeling algorithms, it will be discussed later in the molecular modeling section of this chapter. CLASSIFICATION METHODS ‘There are other statistical techniques besides regression anal- ysis that are used in drug design. These fit under the classifi- cation of multivariate statistics and include discriminant analysis, principal component analysis, and pattern recogni- tion. The latter can consist of a mixture of statistical and nonstatistical methodologies. The goal usually is to try to ascertain those physicochemical parameters and structural attributes that contribute toa class or type of biological activ. ity. Then the chemicals are classified into groupings such as ‘carcinogenic/noncarcinogenic, sweeUbitter, active/inactive, and depressant/stimulant. ‘The term ‘multivariate’ used because of the wide vari ety and number of independent or descriptor variables that ‘may be used. The same physicochemical parameters seen in QSAR analyses are used, but, in addition, the software in24 | TABLE 28 (COEFFICIENTS FOR SUBSTITUENTS IN A SET OF ACETYLCHOLINESTERASE INHIBITORS (8) iy = CHa, CsH, OCH, OCsHs, OCsHy, OC(CH)e Re = OCHs, OC:Hs, OCsHr, OC(CHs)e, OCyHy PHYSICOCHEMICAL PROPERTIES IN RELATION TO BIOLOGICAL ACTION a = —O(CeH,)(2-NOz), —O(CeH.)(3—NO.), —O(CeHy)(4¢—NO3) y= 0,8 a & & me Methy! 0729 Methoxy — -0.598 2Nittophenoxy 0.856 Or 0.052 Etry! 0167 Ethoxy 0.186 3.Nttophenoxy — ~ 1.134 Tio ~ 1.673, Ethoy — -0.168 Propoy 0.543 ‘ENttoptenoxy 0811 Propoxy — -0.405 Isopropory —0.164 Isopropony —1.267 Butoxy 0766 Contribution from the base molecule = 5.148, the computer programs “breaks” the molecule down into substructures, These structural fragments also become vari- ables. Examples of the typical substructures used include carbonyls, enones, conjugation, rings of different sizes and types, N-substitution patterns, and aliphatic substitution pat- tems such as 1,3- or 1,2-disubstituted, The end result is that for even a moderate size molecule typical of most drugs, there can be 50 to 100 variables. ‘The technique is to develop a large set of chemicals well characterized in terms of the biological activity that is going to be predicted. This is known as the training set. Ideally, it should contain hundreds, if not thousands, of compounds, divided into active and inactive types. In reality, sets smaller than 100 are studied. Most of these investigations are retro- spective ones in which the investigator locates large data sets from several sources. This means that the biological testing likely followed different protocols. That is why clas- sification techniques tend to avoid using continuous vari- ables such as EDso, LDso, and MIC. Instead, arbitrary end- points such as active or inactive, stimulant or depressant, sweet oF Sour, etc, are used. Once the training set is established, the multivariate tech- nigue is carried out. The algorithms are designed to group the underlying commonalities and select those variables hav- ing the greatest influence on biological activity. The predic~ tive ability is then tested with a test set of compounds that have been put through the same biological tests used for the training set. For the classification model to be valid, it is important forthe investigator to select datasets whose results are not intuitively obvious and could not be classified by 4 trained medicinal chemist. Properly done, classification ‘methods can identify structural and physicochemical de- scriptors that can be powerful predictors and determinants of biological activity. ‘There are several examples of successful applications of this technique.” One study consisted of a diverse group of 140 tranquilizers and 79 sedatives subjected to a two-way classification study (tranquilizers versus sedatives). The ring types included phenothiazines, indoles, benzodiazepines, barbiturates, diphenylmethanes, and a variety of heterocy- cles. Sixty-nine descriptors were used intially to character- ize the molecules. Eleven of these descriptors were crucial to the classification, 54 had intermediate use and were de~ pendent on the composition of the training set, and four descriptors were of litle use. The overall range of prediction accuracy was 88% to 92%. The results with the 54 descrip- tors indicate an important limitation when large numbers of descriptors are used. The inclusion or exclusion of descrip- tors and parameters can be dependent on the composition of the training set. It is important that the training set be representative of the population of chemicals that are going to be evaluated. Indeed, repeating the study on different ran- domly selected training sets is important. Classification techniques lend themselves to studies lack- ing quantitative data. An interesting classification problem involved olfactory stimulants in which the goal was to select chemicals that had a musk odor. A group of 300 unique ‘compounds was selected from a group of odorants that in- cluded 60 musk odorants plus 49 camphor, 44 floral, 32 ‘ethereal, 41 mint, 51 pungent, and 23 putrid odorants. Ini- tially, 68 descriptors were evaluated. Depending on the ap- proach, the number of descriptors was reduced to 1 to 16, consisting mostly of bond types. Using this small number, the 60 musk odorants could be selected from the remaining 240 compounds, with an accuracy of 95% to 97%. ‘The use of classification techniques in medicinal chemis- try has matured over years of general use. The types of de- scriptors have expanded to spatial measurements in three dimensional space similar to those used in 3D-QSAR (see below). Increasingly, databases of existing compounds are scanned looking for molecules that possess what appear to be the desired parameters. If successful, compounds that areDoe predicted tobe active provide the starting point for synthesiz~ ing new compounds for testing. One can see parallels be- tween the search of chemical databases and screening plant, animl, and microbial sources for new compounds. Although the statistical and pattern recognition methodologies have teen in use for a very long time, there still needs to be considerable research ito their proper use, and further test- ing of ther predictive power is needed. The goal of scanning databases of aleady synthesized compounds in order to se- lect compounds for pharmacological evaluation will require considerable additional development inthe various multivar- inte techniques COMBINATORIAL CHEMISTRY lepant a the statistical techniques described above are, the goal remains to synthesize large numbers of compounds in order not to miss promising marketable products, At the same time, traditional synthetic and biological testing are ‘very costly. This has led to the technique called “‘combinato- ‘ial chemistry.” The latter employs libraries of chemical ‘oietes that react with a parent or base molecule in a small umber of defined synthetic steps. Return to the two exam- ples presented withthe discussion ofthe Free-Wilson analy sis (see above). As the number of different substituents is considered, literally >10,000 compounds are possible. Re- ‘member thatthe medicinal chemist can select subsets of substituents that vary in lipophilicity, steric bulk, induction, and resonance effects, and use the four rules for the placing and uilzation ofthe substituents, Properly done, a relatively small numier of compounds will be obtained that will show the dual importance of each of the physicochemical param- ‘ets being evaluated at each position on the molecule and the effect of specific moieties at each position. This “*ra- tional" approach to drug design assumes that there is some understanding ofthe target receptor and that there is lead molecule, commonly called the ‘prototype molecule.”” A. dlasic example is the dihydrofolate reductase inhibitor, methotrexate, which has been one of the prototypes that lab- ‘ontories have used to synthesize and test new inhibitors Another example is the benzodiazepine structure, It has a defined structure whose activity varies with the substituents What about the situation where little is known about the ‘mechanisms causing the disease process? Until recently, this has heen the normal situation when searching for new mole- cules with the desired pharmacological response. With the discovery of penicillin came the realization that microbial organisms produced “‘antibiotcs.”” This started screenings cof microbial products looking for new antibiotics. In a simi- far manner, thousands of synthetic compounds and plant e tracts have been screened for anticancer activity. Some have called this “irational"” drug design, but ithas produced most ofthe drugs curently prescribed. This approach also is very expensive, particularly when one realizes the cost to synthe- siz, isolate, and test each new compound plus the time and COMBINATORIAL CHEMISTRY | 25 expense it takes to take an active compound through the efficacy and safety testing before its release to the general public is approved by government regulatory agencies. Combinatorial chemistry is one method of reducing the cost of drug discovery in which the goal isto find new leads ‘or prototype compounds or optimizing and refining the struc- ture activity relationships.'*!" Libraries of “reactive chemical moieties provide the chemical diversity of products that will be screened for activity. The chemistry is elegant, but relatively simple in that the same few reactions are re quired to make thousands of compounds in a particular se- ries. The reactions must be clean and reproducible and have high yields. Often, solid-state synthetic methods are used in which compounds are “grown” onto polymer support. Robotics can be used to reduce further the cost of synthesis Biological testing can also be automated in a process called “high through put screening," which can test tens to hundreds of structures at atime, Many times itis possible to take advantage of gene cloning techniques, clone the desired receptor, and measure the binding of the newly synthesized ‘compounds to the cloned receptor. In order to maintain some focus on the needed structures, information theory has been used to construct the libraries of substituents, These libraries tend to maximize chemical diversity in terms of physicochemical parameters. Many of these libraries are sold commercially by firms specializing in this technique. The synthetic methodologies cover the spectrum of producing thousands of relatively pure com- pounds to mixtures of compounds that are tested as mixtures Of course, mixtures can be difficult to classify and it can be difficult to determine which products in the mixture are ac- tive and which ones are inactive. Elegant methods have been developed that chemically ““tag’” each compound with a small peptide, nucleotide, or other small molecule that is pharmacologically inert. When mixtures of products are ob- tained, they are screened for activity. Only those mixtures that are biologically active are retained. In a process called “deconvolution,” the synthesis is repeated in an iterative ‘manner producing smaller and sometimes overlapping mix tures. The sereening is repeated until the active compounds are identified. Examine Table 2-9, This simplified outline shows how four steps will identify the three active compo- nents in a 20-compound investigation. (Keep in mind that the actual combinatorial process will produce hundreds or thousands of compounds for testing.) Assume that the project calls for synthesizing 20 com- pounds A to T. Rather than carry out 20 distinct syntheses followed by 20 separate screening experiments, all of which can take weeks, four combinatorial syntheses are carried out such that four mixtures containing five compounds each are obtained. Only the three mixtures that test positive in the screening assay are retained. The synthesis is repeated pro- ducing five mixtures of three components each and the test- ing is repeated. Six more syntheses are carried out this time producing overlapping two-component mixtures, and the as- says are repeated. It is now possible to determine that com- fe